DEMANDE OU BREVET VOLUMINEUX
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PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
~~ TTENANT LES PAGES 1 A 415
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
COMPOSITION FOR THE TREATMENT OF DAMAGED TISSUE
FIELD OF INVENTION
The present invention relates to a composition, in particular a pharmaceutical
composition. The present invention also relates to uses of that composition -
in
particular in the treatment of damaged tissue.
BACKGROUND ART
It is desirable to be able to treat damaged tissue, such as in wounds, more in
particular in chronic wounds. Examples of chronic wounds include chronic
dermal
ulceration.
~ 5 Chronic dermal ulcers are a major cause of morbidity in the ageing
population, and
represent a significant economic burden on .healthcare systems. Recent figures
for
chronic dermal ulcers, including pressure sores, diabetic and venous ulcers,
indicate
a total of about 3.75 million and 12 million patients in the US .and world-
wide,
respectively (Wound Healing Technological Innovations and Market Overview
(1998)
2o Technology Catalysts International Corporation, VA, USA). Of these
patients,
approximately 70% are classified as moderate to severe. Despite~recent
advances in
their treatment, the healing of these ulcers remains slow (typically 16 weeks
for a
venous ulcer with best care) and agents which are efficacious in reducing the
time to
closure will bring medical and commercial benefit.
The present invention seeks to overcome these problems.
SUMMARY ASPECTS OF THE PRESENT INVENTION
3o In accordance with the present invention, damaged tissue, such as wounds
(in
particular chronic wounds), can be treated more effectively if a combination
of a
growth factor and an inhibitor agent is used. The inhibitor agent used is, or
is
derivable from or is based on, a protease inhibitor. In more detail, the
inhibitor agent
inhibits the action of specific proteins that are upregulated in a wound
environment
wherein those proteins have an adverse effect in the wound environment. Here,
typically the adverse effect is a deleterious effect on wound heating.
Typically these
1
CA 02395487 2002-06-26
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adverse proteins are adverse proteases that are upregulated in a wound
environment. Hence, the inhibitor agent is a specific inhibitor agent.
Thus, one aspect of the present invention concerns a composition for use in or
as a
pharmaceutical (otherwise called a medicament), wherein said composition
comprises an inhibitor agent that inhibits the action of at least one specific
protease
protein that is upreguiated in a wound environment.
in one preferred aspect, the present invention concerns a composition for use
in or
to as a pharmaceutical (otherwise called a medicament), wherein said
composition
comprises an inhibitor agent that inhibits the action of a specific protease
protein that
is upregulated in a wound environment.
The combination of the protease inhibitor and the growth factor results in a
beneficial
t5 additive effect, which in some cases is synergistic.
We believe that, in use, the protease inhibitor agent of the present invention
protects
the growth factor in the damaged tissue environment and to such an extent that
the
degradation of the growth factor is hindered, delayed, reduced or even
eliminated.
The use of an inhibitor agent that inhibits the action of one or more specific
adverse
proteins - in particular one or more specific proteases - that are upregulated
in a
wound environment is in direct contrast to the teachings, of workers who have
used
non-selective inhibitors. By way of example, reference may be made to Kiyohara
Yoshifumi ef al (Database Biosis Database Accession No. PREV199497178695
XP002139251 reporting on Biological & Pharmaceutical Bulletin 1993 vol 16
pages
1146-1149); Wlaschek et al (British Journal of Dermatology 1997 137{4) page
646);
Witte et al (Surgery (St Louis) 1998 vol 124 (2) pages 464-470); Ryou et al
(Arch
Pharmacal Res 1997 vol 20 (1) pages 34-38); Singer et al (New England Journal
of
Medicine Sept 2 1999 vol 341 (10) pages 738-746); Chen Chin et al (Wound
Repair
and Regeneration vol 7 (6) pages 486-494); and US-A-5290762.
DETAILED ASPECTS OF THE PRESENT INVENTION
According to one aspect, the present invention provides a pharmaceutical for
use (or
when in use) in the treatment (e.g. healing) of damaged tissue (such as
damaged
tissue in a wound); the pharmaceutical comprising a composition, which
composition
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comprises: (a) a growth factor; and (b) an inhibitor agent; and optionally (c)
a
pharmaceutically acceptable carrier, diluent or excipient; wherein the
inhibitor agent
can inhibit the action of at least one specific adverse protein (e.g. a
specific protease)
that is upregulated in a damaged tissue environment.
In accordance with the present invention, the growth factor is sometimes
referred to
as "component (a); the inhibitor agent is sometimes referred to as "component
(b)";
and the pharmaceutically acceptable carrier, diluent or excipient is sometimes
referred to as "component (c)".
Typically, for topical mixtures or locally injected mixtures, the relative
ratio of inhibitor
agent to growth factor may be between 1000:1 and 1:1 (ori a mg:mg or a %:%
basis).
TypicaNy, for a systemically administered inhibitor agent with a topical or
locally
t5 injected growth factor, the relative ratio of inhibitor agent to growth
factor may be
between 10,000:1 and 10:1 (on a mg:mg basis).
According to another aspect, the present invention provides a composition
according
to the present invention for use in medicine.
According to another aspect, the present invention provides the use of a
composition
according to the present invention in the manufacture of a pharmaceutical to
treat
damaged tissue, such as wounds.
According to another aspect, the present invention provides the use of a
composition
according to.the present invention in the manufacture of a pharmaceutical to
treat
chronic damaged tissue, such as chronic wounds.
According to another aspect, the present invention provides the use of a
composition
3o according to the present invention in the manufacture of a pharmaceutical
to treat a
chronic dermal ulcer.
According to another aspect, the present invention provides a method of
therapy,
said method comprising administering to a subject a composition according to
the
present invention and in an amount to treat (e.g. heal) damaged tissue, such
as a
wound.
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According to another aspect, the present invention provides a process for
preparing a
composition according to the present invention; said process comprising the
steps of
admixing one or more of said agents) according to the present invention with a
growth factor and optionally a pharmaceutically acceptable carrier, diluent or
excipient.
According to another aspect, the present invention provides a process; said
process
comprising the steps of: (a) admixing one or more of said agents) according to
the
present invention with a growth factor and optionally a pharmaceutically
acceptable
~U carrier, diluent or excipient; (ii) administering said composition to a
subject in need of
same.
According to another aspect, the present invention provides performing an
assay to
identify one or more agents that are capable of acting as an inhibitor agent
according
~5 to the present invention.
According to another aspect, the present invention provides a process for
preparing a
composition according to the present invention; said process comprising the
steps of:
(i) performing an assay to identify one or more agents that are capable of
acting as
2o an inhibitor agent according to the present invention; (ii) admixing one or
more of
said agents) with a growth factor and optionally a pharmaceutically acceptable
carrier, diluent or excipient.
According to another aspect, the present invention provides a process; said
process
2s comprising the steps of: (i) performing an assay to identify one or more
agents that
are capable of acting as an inhibitor agent according to the present
invention; (ii)
admixing one or more of said agents) with a growth factor and optionally a
pharmaceutically acceptable carrier, diluent or excipient; (iii) administering
said
composition to a subject in need of same.
It is to be understood that components (a) and (b) may be present in the same
admixture for administration to a subject or they may be administered to a
subject
sequentially or simultaneously, and in doing so they may be applied by similar
or
different techniques. Thus, the components may be administered together, such
as
3s in the same admixture. In the alternative, one of the components may be
administered orally, systemically, topically or by injection and the other of
the
components may be taken by a similar route (e.g. one of orally, systemically,
topically
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
or by injection) or by a different route (e.g. a different one of orally,
systemically,
topically or by injection). In one preferred embodiment of the present
invention, one
component is applied topically and the other component is applied
systemically. In
another preferred embodiment of the present invention, one component is
applied
s topically and the other component is applied topically.
Thus, according to one aspect, the present invention provides a pack for use
in the
treatment (e.g. healing) of damaged tissue, such as a wound; the pack
comprising at
least two compartments; wherein first of said compartments houses a growth
factor;
~o and wherein second of said compartments houses an inhibitor .agent, wherein
the
inhibitor agent can inhibit the action of at least one specific adverse
protein (e.g. a
specific protease) that is upregulated in a damaged tissue, such as a wound,
environment. In the pack of the present invention, the growth factor andlor
the
inhibitor agent may be admixed with a pharmaceutically acceptable carrier,
diluent or
~5 excipient. In addition, or in the alternative, the pack of the present
.invention
comprises a third compartment, which third compartment houses a
pharmaceutically
acceptable carrier, diluent or excipient.
With the present invention, such as the pack of the present invention, the
growth
2o factor and the inhibitor agent may be in different forms. By way of
example, one may
be a solution or tablet and the other may be a cream. In one preferred
embodiment
of the present invention, one component of the pack is to be applied topically
and the
other component of the pack is to be applied systemically. It is to be
understood that
the pack could contain extra compartments.
According to one aspect of the present invention, there is provided a process
for
preparing a pharmaceutical for use in damaged tissue, such as wound, treatment
(e.g. healing); the process comprising forming a composition by admixing (a) a
growth factor with (b) an inhibitor agent; and optionally with (c) a
pharmaceutically
3o acceptable carrier, diluent or excipient; wherein the inhibitor agent can
inhibit the
action of at least one specific adverse protein (e.g. a specific protease)
that is
upregulated in a damaged tissue, such as a wound, environment.
According to one aspect of the present invention, there is provided the use of
a
growth factor according to the present invention in the manufacture of a
pharmaceutical to treat a subject that is being treated with an inhibitor
agent
according to the present invention.
s
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
According to one aspect of the present invention, there is provided the use of
an
inhibitor agent according to the present invention in the manufacture of a
pharmaceutical to treat a subject that is being treated with a growth factor
according
to the present invention.
According to one aspect of the present invention, there is provided a method
of
therapy, said method comprising administering to a subject a composition
according
to the present invention and in an amount to treat (e.g. heal) damaged tissue,
such
l0 as a wound. Here, all or some (preferably all) of said growth factor
according to the
present invention may be administered by a different route than all or some
(preferably all) of said inhibitor agent according to the present invention.
However,
preferably at least the inhibitor and/or the growth factor is applied
topically. In one
preferred aspect, both the inhibitor and the growth factor are applied
topically. In
I S another preferred aspect, the inhibitor is applied orally and the growth
factor is
applied topically.
According to one aspect of the present invention, there is provided the use of
a
composition according to the present invention in the manufacture of a
2o pharmaceutical to treat chronic damaged tissue, such as chronic damaged
wounds.
Here, all or some (preferably all) of said growth factor according to the
present
invention may be administered by a different route than all or some
(preferably all) of
said inhibitor agent according to the present invention. However, preferably
at least
the inhibitor andlor the growth factor is applied topically. In a preferred
aspect, both
25 the inhibitor and the growth factor are applied topically. In another
preferred aspect,
the inhibitor is applied orally and the growth factor is applied topically.
According to one aspect of the present invention, there is provided the use of
a
growth factor according to the present invention in the manufacture of a
3o pharmaceutical to treat a subject that is being treated with an inhibitor
agent
according to the present invention. Here, all or some (preferably all) of said
growth
factor according to the present invention may be administered by a different
route
than all or some (preferably all) of said inhibitor agent according to the
present
invention. However, preferably at least the inhibitor andlor the growth factor
is
35 applied topically. In a preferred aspect, both the inhibitor and the growth
factor are
applied topically. In another preferred aspect, the inhibitor is applied
orally and the
growth factor is applied topically.
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
According to one aspect of the present invention, there is provided the use of
an
inhibitor agent according to the present invention in the manufacture of a
pharmaceutical to treat a subject that is being treated with a growth factor
according
to the present invention. Here, all or some (preferably ali) of said growth
factor
according to the present invention may be administered by a different route
than all
or some (preferably all) of said inhibitor agent according to the present
invention.
However, preferably at least the inhibitor and/or the growth factor is applied
topically.
!n a preferred aspect, both the inhibitor and the growth factor are applied
topically. In
another preferred aspect, the inhibitor is applied orally and the growth
factor is
applied topically.
According to one aspect of the present invention there is provided a
pharmaceutical
comprising:
(a) a growth factor;
(b) an i:UPA andlor an iMMP; and optionally
(c) a pharmaceutically acceptable carrier, diluent or excipient;
2o wherein the iUPA and/or the iMMP can inhibit the action of at least one
specific
adverse protein (e.g. a specific protease) that is upregulated in a damaged
tissue,
such as a wound, environment.
With this embodiment, the growth factor may be endogeneous growth factor.
Here, all or some (preferably all) of said growth factor according to the
present
invention may be administered by a different route than all or some
(preferably all) of
said inhibitor agent according to the present invention. However, preferably
at least
the inhibitor and/or the growth factor is applied topically. In a preferred
aspect, both
3o the inhibitor and the growth factor are applied topically. In another
preferred aspect,
the inhibitor is applied orally and the growth factor is applied topically.
According to one aspect of the present invention there is provided the use of
a
pharmaceutical comprising:
(a) a growth factor;
(b) an i:UPA and/or an iMMP; and optionally
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
(c) a pharmaceutically acceptable carrier, diluent or excipient;
wherein the iUPA and/or the iMMP can inhibit the action of at least one
specific adverse protein (e.g. a specific protease) that is upregulated in a
damaged
tissue, such as a wound, environment
to treat damaged tissue, such as wound.
With this embodiment, the growth factor may be endogeneous growth factor.
f0
Here, all or some (preferably all) of said growth factor according to the
present
invention may be administered by a different route than all or some
(preferably all) of
said inhibitor agent according to the present invention. However, preferably
at least
the inhibitor and/or the growth factor is applied topically. In a preferred
aspect, both
t 5 the inhibitor and the growth factor are applied topically. In another
preferred aspect,
the inhibitor is applied orally and the growth factor is applied topically.
According to one aspect of the present invention there is provided a
pharmaceutical
composition comprising:
(i) an i:UPA
(ii) an iMMP; and optionally
(iii) a pharmaceutically acceptable carrier, diluent or excipient;
wherein the iUPA and/or the iMMP can inhibit the action of at least one
specific
adverse protein (e.g. a specific protease) that is upregulated in a damaged
tissue,
such as a wound, environment.
For ease of reference, these and further aspects of the present invention are
now
3o discussed under appropriate section headings. However, the teachings under
each
section are not necessarily limited to each particular section.
PREFERABLE ASPECTS
Preferably said growth factor is selected from one or more of: PDGF (platelet
derived
growth factor), FGF (fibroblast growth factor), CTGF (connective tissue
derived
growth factor), KGF (keratinocyte-derived growth factor), TGF (transforming
growth
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
factor), CSF (colony stimulating factor), VEGF (vascular endothelial growth
factor),
EGF (epidermal growth factor), Chrysalin, or active variants, homologues,
derivatives
or fragments of any thereof.
Preferably said growth factor is selected from one or more of VEGF, EGF, PDGF,
FGF, CTGF-like, KGF-2, TGF-Vii, GM-CSF (granulocyte/macrophage stimulating
factor) , Chrysalin, or active variants, homologues, derivatives or fragments
thereof.
Preferably said growth factor is at least PDGF, or an active variant,
homologue,
derivative or fragment thereof. Examples of fragments include the PDGF A-chain
and the PDGF B-chain.
Typically, the protein that is upregulated in a damaged tissue, such as a
wound
environment, is a protease.
Preferably said inhibitor agent is an inhibitor of urokinase-type plasminogen
activator
(otherwise referred to as an I:uPA - sometimes written as i:UPA or as I:UPA)
andlor
an inhibitor of a matrix metalloproteinase (otherwise referred to as an I:MMP -
sometimes written as i:MMP)
Preferably said damaged tissue is a wound.
Preferably said wound is a chronic wound.
Preferably said wound is a dermal ulcer.
Preferably said routes) of administration is(are) selected from at least one
or more
of: oral administration, injection (such as direct injection), topically,
inhalation,
parenteral administration, mucosal administration, intramuscular
administration,
3o intravenous administration, subcutaneous administration, intraocular
administration
or transdermal administration.
Preferably said routes) of administration is(are) oral administration andlor
topical
administration.
Preferably at least a part (preferably all) of said inhibitor is administered
(delivered)
by topical administration and so is formulated for such an administration
route.
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WO 01/49309 PCT/IB00/01935
Preferably at least a part (preferably all) of said growth factor is
administered topically
and so is formulated for such an administration route.
Preferably, the inhibitor is at least an i:UPA. In an alternative embodiment,
or in
addition, preferably the inhibitor is at least an i:MMP; wherein said MMP is
MMP'3
and/or MMP 13.
INHIBIT THE ACTION OF AT LEAST ONE SPECIFIC ADVERSE PROTEIN (E.G. A
1o SPECIFIC PROTEASE) THAT IS UPREGULATED IN A DAMAGED TISSUE
The term "inhibit the action of at least one specific adverse protein (e.g. a
specific
protease) that is upregulated in a damaged tissue" means that the inhibitor
agent of
the present invention does not have an activity profile over a broad number of
proteins. Instead, the inhibitor agent is capable of substantially selectively
acting on
a specific adverse protein (e.g. a specific protease) that is upregulated in a
damaged
tissue. In some circumstances, the inhibitor agent may act on a few specific
profeins
that are upregufated in a damaged tissue. However, preferably, the inhibitor
agent is
capable of selectively acting on one specific adverse protein (e.g. a specific
2o protease) that is upregulated in a damaged tissue. Alternatively expressed
in a
highly preferred aspect, the inhibitor agent of the present invention is an
agent that
limits the specific proteolytic degradation effects) of at least one specific
adverse
protease that has a deleterious effect on wound healing.
Preferably, the inhibitor agent is selective - for example being at least
about 50-fold,
more preferably at least about 75-fold, more preferably at least about 100-
fold, in
terms of relative Ki measured using purified enzymes - over other proteases
found in
the damaged tissue, such as wound, environment. Depending on the selection of
inhibitor agent, examples of other protease proteins may include one or more
of:
3o MMPs, tPA, plasmin and neutrophil elastase, some of which have a beneficial
effect
on would healing.
For some applications, preferably the agent has a K; value against a
particular
desired protein target of less than about 100 nM, preferably less than about
75 nM,
preferably less Than about 50 nM, preferably less than about 25 nM, preferably
less
than about 20 nM, preferably less than about 15 nM, preferably less than about
10
nM, preferably less than about 5 nM.
to
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For some applications, preferably the agent has at least about a 100 fold
selectivity
to a particular desired target, preferably at least about a 150 fold
selectivity to the
desired target, preferably at least about a 200 fold selectivity to the
desired target,
preferably at least about a 250 fold selectivity to the desired target,
preferably at least
about a 300 fold selectivity to the desired target, preferably at least, about
a 350 fold
selectivity to the desired target, preferably at least about a 400 fold
selectivity to the
desired target; preferably at least about a 450 fold selectivity to the
desired target,
preferably at least about a 500 fold selectivity to the desired target,
preferably at least
1 o about a 600 fold selectivity to the desired target, preferably at least
about a 700 fold
selectivity to the desired target, preferably at least about an 800 fold
selectivity to the
desired target, preferably at least about a 900 fold selectivity to the
desired target,
preferably at least about a 1000 fold selectivity to the desired target.
~ 5 For some applications, preferably the inhibitor agent of the present
invention has a K;
value of less than about 100 nM, preferably less than about 75 nM, preferably
less
than about 50 nM, preferably less than about 25 nM, preferably less than about
20
nM, preferably less than about 15 nM, preferably less than about 10 nM,
preferably
less than about 5 nM.
For some embodiments of the present invention, preferably the agents of the
present
invention have a log D of -2 to +4, more preferably -1 to +2. The log D can be
determined by standard procedures known in the art such as described in J.
Pharm.
Pharmacol. 1990, 42:144.
'
In addition, or in the alternative, for some embodiments preferably the agents
of the
present invention have a caco-2 flux of greater than 2x10-ficms'', more
preferably
greater than 5x10~scms''. The caco flux value can be determined by standard.
procedures known in the art such as described in J. Pharm. Sci 79, 7, p595-600
(1990), and Pharm. Res. vol 14, no. 6 (1997).
TREATMENT
It is to be appreciated that all references herein to treatment include one or
more of
curative, palliative and prophylactic treatment. Preferably, the term
treatment
includes at least curative treatment and/or palliative treatment.
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The treatment may be of one or more of chronic dermal ulceration, diabetic
ulcers,
decubitus ulcers (or pressure sores), venous insufficiency ulcers, venous
stasis
ulcers, burns, corneal ulceration or melts.
The treatment may be for treating conditions associated with impaired damaged
tissue, such as wound, healing, where impairment is due to diabetes, age,
cancer or
its treatment (including radiotherapy), neuropathy, nutritional deficiency or
chronic
disease.
to AMINO ACID SEQUENCE
Aspects of the present invention concern the use of amino acid sequences.
These
amino acid sequences may be a component of the composition of the present
invention - such as the growth factor component. In another embodiment, the
amino
t 5 acid sequences may be used as a target to identify suitable inhibitor
agents for use in
the composition of the present invention. In another embodiment, the amino
acid
sequences may be used as a target to verify that an agent may be used as an
inhibitor agent in the composition of the present invention.
20 As used herein, the term "amino acid sequence" is synonymous with the term
"polypeptide" and/or the term "protein". In some instances, the term "amino
acid
sequence" is synonymous with the term "peptide". In some instances, the term
"amino acid sequence" is synonymous with the term "protein". In some
instances,
the term protein is a protease.
The amino acid sequence may be prepared isolated from a suitable source, or it
may
be made synthetically or it may be prepared by use of recombinant DNA
techniques.
In one aspect, the present invention provides an amino acid sequence that is
used as
3o a component of the composition of the present invention.
In another aspect, the present invention provides an amino acid sequence that
is
capable of acting as a target in an assay for the identification of one or
more agents
and/or derivatives thereof capable of acting as an inhibitor of said amino
acid.
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NUCLEOTIDE SEQUENCE
Aspects of the present invention concern the use of nucleotide sequences.
These
nucleotide sequences may be used to express amino acid sequences that may be
used as a component of the composition of the present invention - such as the
growth factor component. In another embodiment, the nucleotide sequences may
be
used as a target to identify suitable inhibitor agents for use in the
composition of the
present invention. In another embodiment, the nucleotide sequences may be used
as
a target to verify that an agent may be used as an inhibitor~agent in the
composition
( 0 of the, present invention.
As used herein, the term "nucleotide sequence" is synonymous with the term
"polynucleotide".
(5 The nucleotide sequence may be DNA or RNA of genomic or synthetic or of
recombinant origin. The nucleotide sequence may be double-stranded or single-
stranded whether representing the sense or antisense strand or combinations
thereof.
2o For some applications, preferably, the nucleotide sequence is DNA,
For some applications, preferably, the nucleotide sequence is prepared by use
of
recombinant DNA techniques (e.g. recombinant DNA).
25 For some applications, preferably, the nucleotide sequence is cDNA.
For some applications, preferably, the nucleotide sequence may be the same as
the
naturally occurring form.
30 In one aspect, the present invention provides a nucleotide sequence
encoding a
substance capable of acting as a target in an assay .(such as a yeast two
hybrid
assay) for the identification of one or more agents andlor derivatives thereof
capable
of acting as an inhibitor of said nucleotide sequence (or the amino acid
encoded
thereby).
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VARIANTSIHOMOLOGUES/DERIVATIVES
In addition to the specific amino acid sequences and nucleotide sequences
mentioned herein, the present invention also encompasses the use of variants,
homologues and derivatives of any thereof. Here, the term "homaiogue" means an
entity having a certain homology with the subject amino acid sequences and the
subject nucleotide sequences. Here, the term "homology" can be equated with
"identity".
In the present context, an homologous sequence is taken to include an amino
acid
sequence which may be at least 75, 85 or 90% identical, preferably at least 95
or
98% identical to the subject sequence.. Typically, the homologues will
comprise the
same active sites etc. as the subject amino acid sequence. Although homology
can
also be considered in terms of similarity (i.e. amino acid residues having
similar
~5 chemical properties/functions), in the context of the present invention it
is preferred to
express homology in terms of sequence identity.
In the present context, an homologous sequence is taken to include a
nucleotide
sequence which may be at least 75, 85 or 90% identical, preferably at least 95
or
2o 98% identical to the subject sequence. Typically, the homologues will
comprise the
same sequences that code for the active sites etc. as the subject sequence.
Although
homology can also be considered in terms of similarity (i.e. amino acid
residues
having similar chemical propertieslfunctions), in the context of the present
invention it
is preferred to express homology in terms of sequence identity.
Homology comparisons can be conducted by eye, or more usually, with the aid of
readily available sequence comparison programs. These commercially available
computer programs can calculate % homology between two or more sequences.
% homology may be calculated aver contiguous sequences, i.e. one sequence is
aligned with the other sequence and each amino acid in one sequence is
directly
compared with the corresponding amino acid in the other sequence, one residue
at a
time. This is called an "ungapped" alignment. Typically, such ungapped
alignments
are performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into
consideration that, for example, in an otherwise identical pair of sequences,
one
14
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WO 01/49309 PCT/IB00/01935
insertion or deletion will cause the following amino acid residues to be put
out of
alignment, thus potentially resulting in a large reduction in % homology when
a global
alignment is performed. Consequently, most sequence comparison methods are
designed to produce optimal alignments that take into consideration possible
insertions and deletions without penalising unduly the overall homology score.
This
is achieved by inserting "gaps" in the sequence alignment to try to maximise
local
homology.
However, these more complex methods assign "gap penalties" fo each gap that
to occurs in the alignment so that, for the same number of identical amino
acids, a
sequence alignment with as few gaps as possible - reflecting higher
relatedness
between the two compared sequences - will achieve a higher score than one with
many gaps. "Affine gap costs" are typically used that charge a relatively high
cost for
the existence of a gap and a smaller penalty for each subsequent residue in
the gap.
is This is the most commonly used gap scoring system. High gap penalties will
of
course produce optimised alignments with fewer gaps. Most alignment programs
allow the gap penalties to be modified. However, it is preferred to use the
default
values when using such software for sequence comparisons. For example when
using the GCG Wisconsin Bestfit package the default gap penalty for amino acid
2o sequences is -12 for a gap and -4 for each extension.
Calculation of maximum % homology therefore firstly requires the production of
an
optimal. alignment, taking into consideration gap penalties. A suitable
computer
program for carrying out such an alignment is the GCG Wisconsin Bestfit
package
25 (University of Wisconsin, U.S.A.; Devereux et al.,~ 1984, Nucleic Acids
Research
12:387). Examples of other software than can perform sequence comparisons
include, but are not limited to, the BLAST package (see Ausubel et al., 1999
ibid -
Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the
GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for
30 offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to
7-60).
However, for some applications, it is preferred to use the GCG Bestfit
program. A
new tool, called BLAST 2 Sequences is also available for comparing protein and
nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS
Microbiol
Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).
.i 5
Although the final % homology can be measured in terms of identity, the
alignment
process itself is typically not based on an' all-or-nothing pair comparison.
Instead, a
is
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scaled similarity score matrix is generally used that assigns scores to each
pairwise
comparison based on chemical similarity or evolutionary distance. An example
of
such a matrix commonly used is the BLOSUM62 matrix - the default matrix for
the
BLAST suite of programs. GCG Wisconsin programs generally use either the
public
default values or a custom symbol comparison table if supplied (see user
manual for
further details). For some applications, it is preferred to use the public
default values
for the GCG package, or in the case of other software,.the default matrix,
such as
BLOSUM62.
Once the software has produced an optimal alignment, it is possible to
calculate
homology, preferably % sequence identity. The software typically does this as
part of
the sequence comparison and generates a numerical result.
The sequences may also have deletions, insertions or substitutions of amino
acid
residues which produce a silent change and result in a functionally equivalent
substance. Deliberate amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the
amphipathic nature of the residues as long as the secondary binding activity
of the
substance is retained. For example, negatively charged amino acids include
aspartic
2o acid and glutamic acid; positively charged amino acids include lysine and
arginine;
and amino acids with uncharged polar head groups having similar hydrophilicity
values include leucine, isoleucine, valine, glycine, alanine, asparagine,
glutamine,
serine, threonine, phenylalanine, and tyrosine.
Conservative substitutions may be made, for example according to the Table
below.
Amino acids in the same block in the second column and preferably in the same
line
in the third column may be substituted for each other:
ALIPHATIC Non-polar G A P
I Ly --.
Polar - uncharged C S T M
NQ
Poiar - charged D E
KR
AROMATIC H F W Y
;o The present invention also encompasses homologous substitution
(substitution and
replacement are both used herein to mean the interchange of an existing amino
acid
16
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WO 01/49309 PCT/IB00/01935
residue, with an alternative residue) may occur i.e. like-for-like
substitution such as
basic for basic, acidic for acidic, polar for polar etc. Non-homologous
substitution
may also occur i.e. from one class of residue to another or alternatively
involving the
inclusion of unnatural amino acids such as ornithine (hereinafter referred to
as Z),
diaminobutyric acid ornithine (hereinafter referred to as B), norleucine
ornithine
(hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine
and
phenylglycine.
Replacements may also be made by unnatural amino acids ,include; alpha* and
alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide
derivatives
of natural amino acids such as trifluorotyrosine*, p-CI-phenylaianine*, p-Br-
phenylaianine*, p-I-phenylalanine*, L-allyl-glycine*,' f3-alanine*, L-c:c-
amino butyric
acid*, L-~-amino butyric acid*, L-cx-am.ino isobutyric acid*, L-~;-amino
caproic acid#, 7-
amino heptanoic acid*, L-methionine sulfone"', L-norleucine*, L-norvaline*, p-
nitro-L-
i5 phenylalanine*, L-hydroxyproline#, L-thioproline*, methyl derivatives of
phenylalanine
(Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino), L-Tyr
(methyl)*,
L-Phe (4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*,
L-
diaminopropionic acid " and L-Phe (4-benzyl)*. The notation * has been
utilised for
the purpose of the discussion above (relating to homologous or non-homologous
20 substitution), to indicate the hydrophobic nature of the derivative whereas
# has been
utilised to indicate the hydrophilic nature of the derivative, #* indicates
amphipathic
characteristics.
Variant amino acid sequences may include suitable spacer groups that may be
25 inserted between any two amino acid residues of the sequence including
alkyl groups
such as methyl, ethyl or propyl groups in addition to amino acid spacers such
as
glycine or ~i-alanine residues. A further form of variation, involves the
presence of
one or more amino acid residues in peptoid form, will be well understood by
those
skilled in the art. For the avoidance of doubt, "the peptoid form" is used to
refer to
3o variant amino acid residues wherein the a-carbon substituent group is on
the
residue's nitrogen atom rather than the a-carbon. Processes for preparing
peptides
in the peptoid form are known in the art, for example Simon RJ et ai., PNAS
(1992)
89(20), 9367-9371 and Horweil DC, Trends Biotechnol. (1.995) 13(4), 132-134.
35 The nucleotide sequences for use in the present invention may include
within them
synthetic or modified nucleotides. A number of different types of modification
to
1~
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WO 01/49309 PCT/IB00/01935
oligonucleotides are known in the art. These include methylphosphonate and .
phosphorothioate backbones and/or the addition of acridine or polylysine
chains at
the 3' and/or 5' ends of the molecule. For the purposes of the present
invention, it is
to be understood that the nucleotide sequences described herein may be
modified by
any method available in the art. Such modifications may be carried out in to
enhance
the in vivo activity or life span of nucleotide sequences of the present
invention.
The present invention also encompasses the use of nucleotide sequences that
are
complementary to the sequences presented herein, or any derivative, fragment
or
1o derivative thereof. If the sequence is complementary to a fragment thereof
then that
sequence can be used a probe to identify similar coding sequences in other
organisms etc.
HYBRIDISATION
The present invention also encompasses the use of nucleotide sequences that
are
capable of hybridising to the sequences presented herein, or any derivative,
fragment
or derivative thereof -such as if the agent is an anti-sense sequence.
2o The term "hybridization" as used herein shall include "the process by which
a strand
of nucleic acid joins with a complementary strand through base pairing" as
well as
the process of amplification as carried out in polymerase chain reaction (PCR)
technologies.
The present invention also encompasses the use of nucleotide sequences that
are
capable of hybridising to the sequences that are complementary to the
sequences
presented herein, or any derivative, fragment or derivative thereof.
The term "variant" also encompasses sequences that are complementary to
sequences that are capable of hydridising to the nucleotide sequences
presented
herein.
Preferably, the term "variant" encompasses sequences that are complementary to
sequences that are capable of hydridising under stringent conditions (e.g.
50°C and
0.2xSSC {1 xSSC = 0.15 M NaCI, 0.015 M Na3citrate pH 7.0}) to the nucleotide
sequences presented herein.
is
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More preferably, the term "variant" encompasses sequences that are
complementary
to sequences that are capable of hydridising under high stringent conditions
(e.g.
65°C and 0.1 xSSC {1 xSSC = 0.15 M NaCI, 0.015 M Na3citrate pH 7.0}) to
the
nucleotide sequences presented herein.
The present invention also relates to nucleotide sequences that can hybridise
to the
nucleotide sequences of the present invention (including complementary
sequences
of those presented herein).
a o The present invention also relates to nucleotide sequences that are
complementary
to sequences that can hybridise to the nucleotide sequences of the present
invention
(including complementary sequences of those presented herein).
Also included within the scope of the present invention are polynucleotide
sequences
that are capable of hybridising to the nucleotide sequences presented herein
under
conditions of intermediate to maximal stringency.
In a preferred aspect, the present invention covers nucleotide sequences that
can
hybridise to the nucleotide sequence of the present invention, or the
complement
thereof, under stringent conditions (e.g. 50°C and 0.2xSSC).
!n a more preferred aspect, the present invention covers nucleotide sequences
that
can hybridise to the nucleotide sequence of the present invention, or the
complement
thereof, under high stringent conditions (e.g. 65°C and 0.1 xSSC).
REGULATORY SEQUENCES
In some applications, the polynucleotide for use in the present invention is
operably
finked to a regulatory sequence which is capable of providing for the
expression of
3o the coding sequence, such as by the chosen host cell. By way of example,
the
present invention covers a vector comprising the polynucleotide of the present
invention operably linked to such a regulatory sequence, i.e. the vector is an
expression vector.
The term "operably linked" refers to a juxtaposition wherein the components
described are in a relationship permitting them to function in their intended
manner.
A regulatory sequence "operably linked" to a coding sequence is ligated in
such a
19
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WO 01/49309 PCT/IB00/01935
way that expression ofi the coding sequence is achieved under condition
compatible
with the control sequences.
The term "regulatory sequences" includes promoters and enhancers and other
expression regulation signals.
The term "promoter" is used in the normal sense of the art, e.g. an RNA
polymerase
binding site.
1 o Enhanced expression of the polynucleotide encoding the polypeptide of the
present
invention may also be achieved by the selection of heterologous regulatory
regions,
e.g. promoter, secretion leader and terminator regions, which serve to
increase
expression and, if desired, secretion levels of the protein of interest from
the chosen
expression host and/or to provide for the inducible control of the expression
of the
polypeptide of the present invention
Preferably, the nucleotide sequence ofi the present invention may be operably
linked to
at least a promoter.
2o Aside firom the promoter native to the gene encoding the polypeptide of the
present
invention, other promoters may be used to direct expression of the polypeptide
of the
present invention. The promoter may be selected for ifs efficiency in
directing the
expression of the polypeptide of the present invention in the desired
expression host.
In another embodiment, a constitutive promoter may be selected to direct the
expression of the desired polypeptide of the present invention. Such an
expression
construct may provide additional advantages since it circumvents the need to
culture
the expression hosts on a medium containing an inducing substrate.
3o Examples of strong constitutive and/or inducible promoters which are
preferred for
use in fungal expression hosts are those which are obtainable from the fiungal
genes
for xylanase (xlnA), phytase, ATP-synthetase, subunit 9 (oliC), triose
phosphate
isomerase (tpi~, alcohol dehydrogenase (AdhA), a-amylase (amy),
amyloglucosidase
(AG - from the glaA gene), acetamidase (amdS) and glyceraldehyde-3-phosphate
dehydrogenase (gpd) promoters.
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Examples of strong yeast promoters are those obtainable from the genes for
alcohol.
dehydrogenase, lactase, 3-phosphoglycerate kinase and triosephosphate
isomerase.
Examples of strong bacterial promoters are the a-amylase and SP02 promoters as
well as promoters from extracellular protease genes.
Hybrid promoters may also be used to improve inducible regulation of the
expression
construct.
1 o The promoter can additionally include features to ensure or to increase
expression in a
suitable host. For example, the features can be conserved regions such as a
Pribnow
Box or a TATA box. The promoter may even contain other sequences to affect
(such
as to maintain, enhance, decrease) the levels of expression of the nucleotide
sequence
of the present invention. For example, suitable other sequences include the
Sh1-intron
I5 or an ADH intron. Other sequences include inducible elements - such as
temperature,
chemical, light or stress inducible elements. Also, suitable elements to
enhance
transcription or translation may be present. An example of the latter element
is the TMV
5' signal sequence (see Sleat Gene 217 [19871 217-225; and Dawson Plant Mol.
Biol.
23 [1993] 97).
2o
SECRETION
Often, it is desirable for a polypeptide for use in the present invention to
be secreted
from the expression host into the culture medium from where the polypeptide of
the
25 present invention may be more easily recovered. According to the present
invention,
the secretion leader sequence may be selected on the basis of the desired
expression host. Hybrid signal sequences may also be used with the context of
the
present invention.
3o Typical examples of heterologous secretion leader sequences are those
originating
from the fungal amylogiucosidase (AG) gene (glaA - both 18 and 24 amino acid
versions e.g. from Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces
and
Kluyveromyces) or the a-amylase gene (Bacillus).
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CONSTRUCTS
The term "construct" - which is synonymous with terms such as "conjugate",
"cassette"
and "hybrid" - includes a nucleotide sequence for use according to the present
invention
directly or indirectly attached to a promoter. An example of an indirect
attachment is the
provision of a suitable spacer group such as an intron sequence, such as the
Sh1-intron
or the ADH intron, intermediate the promoter and the nucleotide sequence of
the
present invention. The same is true for the term "fused" in relation to the
present
invention which includes direct or indirect attachment. fn some cases, the
terms do not
t o cover the natural combination of the nucleotide sequence . coding for the
protein
ordinarily associated with the wild type gene promoter and when they are both
in their
natural environment.
The construct may even contain or express a marker which allows for the
selection of
t 5 the genetic construct in, for example, a bacterium, preferably of the
genus Bacillus,
such as Bacillus subtilis, or plants into which it has been transferred.
Various markers
exist which may be used, such as for example those encoding mannose-6-
phosphate
isomerase (especially for plants) or those markers that provide for antibiotic
resistance -
e.g. resistance to 6418, hygromycin, bleomycin, kanamycin and gentamycin.
For some applications, preferably the construct of the present invention
comprises at
least the nucleotide sequence of the present invention operably linked to a
promoter.
VECTORS
The term "vector" includes expression vectors and transformation vectors and
shuttle
vectors.
The term "expression vector" means a construct capable of in vivo or in vitro
3o expression.
The term "transformation vector" means a construct capable of being
transferred from
one entity to another entity - which may be of the species or may be of a
different
species. If the construct is capable of being transferred from one species to
another -
such as from an E. coli plasmid to a bacterium, such as of the genus Bacillus,
then the
transformation vector is sometimes called a "shuttle vector". It may even be a
construct
capable of being transferred from an E. coli plasmid to an Agrobacterium to a
plant.
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WO 01/49309 PCT/IB00/01935
The vectors of the present invention may be transformed into a suitable host
cell as
described below to provide for expression of a polypeptide of the present
invention.
Thus, in a further aspect the invention provides a process for preparing
polypeptides
for use according to the present invention which comprises cultivating a host
cell
transformed or transfected with an expression vector as described above under
conditions to provide for expression by the vector of a coding sequence
encoding the
polypeptides, and recovering the expressed polypeptides.
to The vectors may be for example, plasmid, virus or phage vectors provided
with an
origin of replication, optionally a promoter for the expression of the said
polynucleotide and optionally a regulator of the promoter
The vectors of the present invention may contain one or more selectable marker
t5 genes. The most suitable selection systems for industrial micro-organisms
are those
formed by the group of selection markers which do not require a mutation in
the host
organism. Examples of fungal selection markers are the genes for acetamidase
(amdS), ATP synthetase, subunit 9 (oliC), orotidine-5'-phosphate-decarboxylase
(pvrA), phleomycin and benomyl resistance (benA). Examples of non-fungal
2o selection markers are the bacterial 6418 resistance gene (this may also be
used in
yeast, but not in filamentous fungi), the ampicillin resistance gene (E. coh),
the
neomycin resistance gene (Bacillus) and the E. coil uidA gene, coding for
~3-glucuronidase (GUS).
25 Vectors may be used in vitro, for example for the production of RNA or used
to
transfect or transform a host cell. ,
Thus, polynucleotides for use according to the present invention can be
incorporated
into a recombinant vector (typically a replicable vector), for example a
cloning or
3o expression vector. The vector may be used to replicate the nucleic acid in
a
compatible host cell. Thus in a further embodiment, the invention provides a
method
of making polynucleotides of the present invention by introducing a
polynucleotide of
the present invention into a replicable vector, introducing the vector into a
compatible
host cell, and growing the host cell under conditions which bring about
replication of
3s the vector. The vector may be recovered from the host cell. Suitable host
cells are
described below in connection with expression vectors.
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WO 01/49309 PCT/IB00/01935
The present invention also relates to the use of genetically engineered host
cells
expressing an amino acid sequence (or variant, homologue, fragment or
derivative
thereof) according to the present invention in screening methods for the
identification
of inhibitors and antagonists of said amino acid sequence. Such genetically
engineered host cells could be used to screen peptide libraries or organic
molecules.
Antagonists and inhibitors of said amino acid sequence, such as antibodies,
peptides
or small organic molecules will provide the basis for pharmaceutical
compositions for
the treatment of damaged tissue, such as wounds. Such inhibitors or
antagonists
can be administered alone or in combination with other therapeutics for the
treatment
I o of such diseases.
The present invention also relates to expression vectors and host cells
comprising a
polynucleotide sequences encoding said amino acid sequence, or variant,
homologue, fragment or derivative thereof for to screen for agents that can
inhibit or
~ 5 antagonise said amino acid sequence.
EXPRESSION VECTORS
The nucleotide sequence for use in the present invention can be incorporated
into a
2o recombinant replicable vector. The vector may be used to replicate and
express the
nucleotide sequence in and/or from a compatible host cell. Expression may be
controlled using control sequences which include promoters/enhancers and other
expression regulation signals. Prokaryotic promoters and promoters functional
in
eukaryotic cells may be used. Tissue specific or stimuli specific promoters
may be
?5 used. Chimeric promoters may also be used comprising sequence elements from
two or more different promoters described above.
The protein produced by a host recombinant cell by expression of the
nucleotide
sequence may be secreted or may be contained intracellularly depending on the
3o sequence and/or the vector used. The coding sequences can be designed with
signal sequences which direct secretion of the substance coding sequences
through
a particular prokaryotic or eukaryotic cell membrane.
FUSION PROTEINS
The amino acid sequence of the present invention may be produced as a fusion
protein, for example to aid in extraction and purification. Examples of fusion
protein
24
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WO 01/49309 PCT/IB00/01935
partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding
and/or
transcriptional activation domains) and (j3-galactosidase. It may also be
convenient
to include a proteolytic cleavage site between the fusion protein partner and
the
protein sequence of interest to allow removal of fusion protein sequences.
Preferably
the fusion protein will not hinder the activity of the protein sequence.
The fusion protein may comprise an antigen or an antigenic determinant fused
to the
substance of the present invention. In this embodiment, the fusion protein may
be a
non-naturally occurring fusion protein comprising a substance which may act as
an
adjuvant in the sense of providing a generalised stimulation of the immune
system.
The antigen or antigenic determinant may be attached to either the amino or
carboxy
terminus of the substance.
In another embodiment of the invention, the amino acid sequence may be ligated
to a
~5 heterologous sequence to encode a fusion protein. For example, for
screening of
peptide libraries for agents capable of affecting the substance activity, it
may be
useful to encode a chimeric substance expressing a heterologous epitope that
is
recognized by a commercially available antibody.
2o GROWTH FACTOR
An essential component of the composition of the present invention is the
presence
and/or use of one or more growth factor(s). The growth factor may be an
endogeneous growth factor and/or an exogeneously applied growth factor, which
25 exogeneously applied growth factor may be the same as or similar to an
endogeneous growth factor.
In accordance with the present invention, the growth factor may be one or more
growth factors) that is(are) capable of being efficacious in enhancing damaged
3o tissue, such as wound, healing.
As used herein, the term "growth factor" means a substance (typically a
peptidic or
proteinacious substance) which stimulates the growth and/or migration of cells
that
are involved in the damaged tissue, such as wound, healing process, including
35 fibrobiasts, keratinocytes and/or endothelial cells. Such a substance may
be (or be
homologous to or derived from) a protein or peptide produced by cells within
the
body, in which case it is an endogenous growth factor. In the alternative, it
may be
2s
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WO 01/49309 PCT/IB00/01935
have been discovered from libraries of peptidic or proteinacious substances
foreign
to the human body.
By way of background information, growth factors are discussed in Molecular
Biology
of The Cell (2"d ed., 1989; Alberts, B., Bray, D., Lewis, J., Raff, M.,
Roberts, K. &
Watson, J.D., eds.), wherein it is stated:
"The conditions that must be satisfied before a cell will grow and divide are
considerably
more complex for an animal cell than for yeast. If vertebrate cells in a
standard artificial
culture medium are completely deprived of serum, they normally will not pass
the restriction
point, even though all the obvious nutrients are present; and they will halt
their growth as well
as their progress through the chromosome cycle. Painstaking analyses have
revealed that the
essential components of serum are highly specific proteins, mostly present in
very low
concentrations (in the order of 10-9 to 10-1 1 M). Different types of cells
require different sets
l.i of these proteins. Some of these proteins in serum are directly and
specifically involved in
stimulating cell division and are called growth factors. One example is
platelet-derived
growth factor, or PDGF."
Growth factors are also discussed in WO-A-99159614.
2o
!n cell biology experiments, many growth factors enhance the proliferation
and/or
motility of the major cell types involved in dermal wound healing, principally
keratinocytes and dermal fibroblasts {Singer, A.J. & Clark; R.A.F. {1999) New
Engl.
J. Med. 341, 738-746). Pharmaceutical preparations of many growth factors have
25 been examined for their efficacy in chronic dermal ulcers. For example,
platelet
derived growth factor {PDGF), fibroblast growth factor (FGF), transforming
growth
factor (33 (TGFj33), keratinocyte-derived growth factor-2 (KGF-2), epidermal
growth
factor {EGF) and granulocyte macrophage colony stimulating factor (GM-CSF)
have
all been taken to the clinic to evaluate their efficacy as wound healing
agents for
3o chronic dermal ulceration. Whilst these agents have given some encouraging
results
in animal models of wound healing. only recombinant PDGF (Regranex) has so far
demonstrated sufficient efficacy in the clinic to justify its use in the
therapy of chronic
dermal ulceration.
35 The reason for the failure of these growth factors to provide pronounced
clinical
efficacy has been open to much speculation. For example, it has been suggested
that the complexity of the wound healing system, involving multiple
interacting cell
26
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WO 01/49309 PCT/IB00/01935
types, and growth factors having actions at distinct temporal phases during
the
wound healing process, explains why growth factor therapy has not
revolutionised
wound healing therapy (Borel, J.P. & Maquart, F.X. (1998) Ann. Biol. Clin.
(Paris) 56,
11-19).' In addition, the half life of growth factors in the wound environment
is known
to be short, limiting the time available for pharmacological effect. For
example, the
half life of TGF(33 after injection into venous ulcers was reported to be
approximately
30 minutes.
One hypothesis which explains the short half fife of growth factors in chronic
dermal
1o ulcers, and their limited clinical efficacy, is that chronic derma( ulcers
represent a
protease rich environment and that these proteases degrade both growth factors
and/or their receptors.
Many proteases have been shown to be over-expressed andlor over-activated in
chronic dermal ulcers compared to normal, acute healing wounds. For example,
using a variety of biochemical and histological techniques (such as fluid
phase
protease assays, immunohistochemistry, gel and in situ zymography and ELISAs)
matrix metalloproteinases (MMPs), including MMP-13 and MMP-3 (Saarialho-Kere
U.K. (1998) Arch. Dermatol. Res. 290, S47-54), neutrophil elastase (Herrick,
S.,
2o Ashcroft, G., Ireland,.G., Horan, M., McCollum, C. & Ferguson, M. (1998)
Lab. Invest.
77, 281-8), uPA (Rogers, A.A., Burnett, S., Lindholm, C., Bjellerup, M.,
Christensen,
O.B., Zederfeldt, B., Peschen, M. & Chen, W.Y. (1999) Vasa 28, 101-5) and
plasmin
(Palolahti, .M, Lauharanta, J, Stephens, RW, Kuusela, P, Vaheri. (1993) Exp.
Dermatol.2, 29-37), have all been shown to be present in high quantities in
either
wound fluid from chronic dermal ulcers, or in sections of wound tissue from
the same.
In addition, it has been shown that when growth factors are added to wound
fluid
from chronic dermal ulcers, they are proteolytically degraded in vitro (Lauer,
G.,
Flamme, I., Kreig, T., Sollberg, S. & Eming, S. (1998) J. Invest. Dermatol.
110, 528,
abstract 338), and when wound fluid is added to cells in culture, they Jose
their
3o responsiveness to growth factors.
It is also to be noted that up until now no one had identified which
protease(s) is/are
responsible for this degradation. This was largely attributable to the fact
that up until
now accurate modelling of the effects of protease inhibitors on growth factors
and
their receptors had been impossible to perform. In this regard, many proteases
-
which are from divergent structural and mechanistic classes and which are over-
expressed and over-active in chronic dermal ulceration - activate one another
via a
2~
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
network of interacting and circular pathways. A(so, some proteases are
essential for
ce(( migration and co((agen deposition, critical components of normal wound
healing,
which indicates that unless appropriate selectivity is achieved in protease
inhibitors,
wound healing would be expected to be impaired (Pitcher, B.K., Wang, M., Qin,
X.J.,
Parks, W.C., Senior, R.M., Weigus, H.G. (1999) Ann. N.Y. Acad. Sci. 878, 12-
24). In
addition, the level of endogenous inhibitors of proteases (such as Tissue
Inhibitors of
Metalloproteinases [TIMPs] and plasminogen activator inhibitors [PAIs]) is
also
altered in chronic dermal ulcers, which adds to the complexity and
unpredictability of
the pathology (Itoh, Y. & Nagase, H. (1995) J. Blot. Chem. 270, 16518-16521;
Knauper, V., Lopez-Otin, C., Smith, B., Knight, G. & Murphy, G. (1996) J.
Blot. Chem.
271, 1544-1550). Hence, overall, the effects of specific inhibition of
particular
proteases on growth factor preservation and function in chronic dermal
ulceration up
until now were unknown.
~5 In accordance with the present invention, we believe that limiting specific
proteolytic
degradation affects the efficacy of a variety of growth factors (both
endogenous and
therapeutically applied) in chronic dermal ulcers. The composition of the
present
invention therefore concerns specific protease inhibitors, which are used in
combination with one or more growth factors. The composition of the present
2o invention overcomes the problem(s) associated with the prior art therapies.
.
If the inhibitor agent is a protein, then it may be applied topically or
orally or
intraveneously as that protein (in any formulation). In addition, or in the
alternative,
the DNA encoding that protein may 'be applied to the damaged tissue, such as a
25 wound, such as when incorporated into a suitable vector, such as by using a
device,
such as by way of example a gene gun (e.g. Lu, B., Scott, G. & Goldsmith, L.A.
(1996) Proc. Assoc. Am. Physicians 108, 168-172).
The growth factor of the present invention may be applied topically as a
protein (in
3o any formulation). In addition, or in the alternative, the DNA encoding the
growth
factor may be applied to the damaged tissue, such as a wound, such as when
incorporated into a suitable vector, such as by using a device, such as by way
of
example a gene gun (e.g. Lu, B., Scott, G. & Goldsmith, L.A. (1996) Proc.
Assoc.
Am. Physicians 108, 168-172).
Examples of growth factors for use in the present invention include one or
more of
PDGF, FGF, CTGF (in particular CTGF-like), KGF (in particular KGF-2), TGF (in
2s
CA 02395487 2002-06-26
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particular TGF-(3), CSF (in particular GM-CSF), VEGF, EGF, Chrysalin. Details
on
these growth factors are presented below.
VEGF
A growth factor for use in the composition of the present invention may be
VEGF.
Background teachings on this growth factor have been presented by Victor A.
McKusick ei' al on http://www.ncbi.nlm.nih.gov/Omim. For ease of reference,
the
to following information has been extracted from that source.
Many po(ypeptide mitogens such as basic fibroblast growth factor and platelet-
derived growth
factors are active on a wide range of different cell types. In contrast,
vascular endothelial
growth factor is a mitogen primarily for vascular endothelial cells.
It is, however, structurally related to platelet-derived growth factor.
Tischer et al. (1991) demonstrated that VEGF, also called vascular
permeability factor (VPF),
is produced by cultured vascular smooth muscle cells. By analysis of
transcripts from these
cells by PCR and cDNA cloning, they demonstrated 3 different forms of the VEGF
coding
region. These cDNAs had predicted products of 189, 165, and 121 amino acids.
They found
that the VEGF gene is split among 8 exons and that the various VEGF coding
region forms
arise through alternative splicing: the i65-amino acid form is missing the
residues encoded by
exon 6, whereas the 121-amino acid form is missing the residues encoded by
exons 6 and 7.
VEGF, a homodimeric glycoprotein of relative molecular mass 45,000, is the
only mitogen
that specifically acts on endothelial cells. It may be a major regulator of
tumor angiogenesis
in vivo.
Millauer et al. (1994) observed in mouse that its expression was upregulated
by hypoxia and
its cell-surface receptor, FIkI is exclusively expressed in endothelial cells.
Folkman (1995) noted the importance of VEGF and its receptor system in tumor
growth and
suggested that intervention in this system may provide promising approaches fo
cancer
therapy.
VEGF and placental growth factor constitute a family of regulatory peptides
capable of
controlling blood vessel formation and permeability by interacting with 2
endothelial tyrosine
kinase receptors, FLTI and KDR/FLK1. See also VEGFB. A third member of this
family
may be the ligand of the related FLT4 receptor involved
in lymphatic vessel development.
Soker et al. (1998) described the purification and the expression cloning from
tumor cells of a
VEGF receptor that binds VEGF165 but not VEGF121. This isoform-specific VEGF
receptor
(VEGF165R) is identical to human neuropilin-1 a receptor for the
collapsin/semaphorin
family that mediates neuronal cell guidance. When coexpressed in cells with
KDR,
29
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neuropilin-1 enhances the binding of VEGF165 to KDR and VEGF165-mediated
chemotaxis.
Conversely, inhibition of VEGF16~ binding to neuropilin-I inhibits its binding
to KDR and
its mitogenic activity for endothelial cells. Soker et al. (i998) proposed
that neuropilin-1 is a
VEGF receptor that modulates VEGF binding to KDR and subsequent bioactivity
and
p therefore may regulate VEGF-induced angiogenesis. '
Mattei et al. (1996) used radioactive in situ hybridization to map VEGF to
6p21-p12. Wei et
al. (1996) reported the localization of the VEGF gene to chromosome 6p12 by
fluorescence in
situ hybridization.
To explore the possibility that VEGF and angiopoietins collaborate during
tumor
angiogenesis, Holash et al. ( 1999) analyzed several different murine and
human tumor
models. Holash et al. (1999) noted that angiopoietin-1 was antiapoptotic
for.cultured
endothelial cells and expression of its antagonist angiopoietin-2 was induced
in the
endothelium of co-opted tumor vessels before their regression. In contrast,
marked induction
of VEGF expression occurred much Later in tumor progression, in the hypoxic
periphery of
1 ~ tumor cells surrounding the few remaining internal vessels, as well as
adjacent to the robust
plexus of vessels at the tumor margin. Expression of Ang2 in the few surviving
internal
vessels and in the angiogenic vessels at the tumor margin suggested that the
destabilizing
action of angiopoietin-2 facilitates the angiogenic action of VEGF at the
tumor rim. Holash et
al. (1999) implanted rat RBA mammary adenocarcinoma cells into rat brains.
Tumor cells
rapidly associated with and migrated along cerebral blood vessels. There was
minimal
upregulation of VEGF. Holash et al. ( 1999) suggested that a subset of tumors
rapidly co-opts
existing host vessels to form an initially well vascularized tumor mass.
Perhaps as part of a
host defense mechanism there is widespread regression of these initially co-
opted vessels,
leading to a secondarily avascular tumor and a massive tumor cell loss.
However, the
remaining tumor is ultimately rescued by robust angiogenesis at the tumor
margin.
Carmellet et al. (1996) and Ferrara et al. (1996) observed the effects of
targeted disruption of
the Vegf gene in mice. They found that formation of blood vessels was abnormal
but not
abolished in heterozygous VEGF-deficient embryos and even more impaired in
homozygous
VEGF-deficient embryos, resulting in death at mid-gestation. Similar
phenotypes were
observed in F(1) heterozygous embryos generated by germline transmission. They
interpreted
their results as indicating a tight dose-dependent regulation of embryonic
vessel development
by VEGF. Mice homozygous for mutations that inactivate either of the 2 VEGF
receptors
also die in utero. However, 1 or more ligands other than VEGF might activate
such receptors.
Ferrara et al. likewise reported the unexpected finding that loss of a single
VEGF allele is
lethal
in a mouse embryo between days 11 and 12. Angiogenesis and blood-island
formation were
impaired, resulting in several developmental anomalies. Furthermore, VEGF-null
embryonic
stem cells exhibited a dramatically reduced ability to form tumors in nude
mice.
Springer et al. (1998) investigated the effects of long-term stable production
of the VEGF
protein by myoblast-mediated gene transfer. Myoblasts were transduced with a
retrovirus
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
carrying a murine VEGF164 cDNA and injected into mouse leg muscles. Continuous
VEGF
delivery resulted in hemangiomas containing localized networks of vascular
channels.
Springer et al. (1998) demonstrated that myoblast-mediated VEGF gene delivery
can lead to
complex tissues of multiple cell types in normal adults. Exogenous VEGF gene
expression at
high levels or of long duration can also have deleterious effects. A
physiologic response to
VEGF was observed in nonischemic muscle; the response in the adult did not
appear to occur
via angiogenesis and may have involved a mechanism related to vasculogenesis,
or de
novo vessel development. Springer et al. (1998) proposed that VEGF may have
different
effects at different concentrations: angiogenesis or vasculogenesis.
Fukumura et al. (1998) established a line of transgenic mice expressing the
green fluorescent
protein (GFP) under the control of the promoter for VEGF. Mice bearing the
transgene
showed green cellular fluorescence around the healing margins and throughout
the granulation
tissue of superficial ulcerative wounds. Implantation of solid tumors in the
transgenic mice
led to an accumulation of green fluorescence resulting from tumor induction of
host VEGF
I ~ promoter activity. With time, the fluorescent cells invaded the tumor and
could be seen
throughout the tumor mass. Spontaneous mammary tumors induced by oncogene
expression
in the VEGF-GFP mouse showed,strong stromai, but not tumor, expression of GFP.
In both
wound and tumor models, the predominant GFP-positive cells were fibroblasts.
To determine the role of VEGF in endochondral bone formation, Gerber et al.
(1999)
inactivated VEGF through the systemic administration of a soluble receptor
chimeric protein
in 24-day-old mice. Blood vessel invasion was almost completely suppressed,
concomitant
with impaired trabecular bone formation and expansion of the hypertrophic
chondrocyte zone.
Recruitment and/or differentiation of chondroclasts, which express gelatinase
B/matrix
metalloproteinase-9, and resorption of terminal chondrocytes decreased.
Although
proliferation, differentiation, and maturation of chondrocytes were apparently
normal,
resorption was inhibited. Cessation of the anti-VEGF treatment was followed by
capillary
invasion, restoration of bone growth, resorption of the hypertrophic
cartilage, and
normalization of the growth plate architecture. These findings indicated to
Gerber et al.
(1999) that VEGF-mediated capillary invasion is an essential signal that
regulates growth
plate morphogenesis and triggers cartilage remodeling. Gerber et al. (I999)
concluded that
VEGF is an essential coordinator of chondrocyte death, chondroc(ast function,
extracellular
matrix remodeling, angiogenesis, and bone formation in the growth plate.
An appropriate amino acid sequence and an appropriate nucleotide sequence are
presented in a later section herein.
EGF
A growth factor for use in the composition of the present invention may be
EGF.
31
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WO 01/49309 PCT/IB00/01935
Background teachings on this growth factor have been presented by Victor A.
McKusick et al on http:l/www.ncbi.nfm.nih.govlUmim. For ease of reference, the
following information has been extracted from that source.
What is now known as epidec~nal growth factor was first described by Cohen
(1962).
Epidermal growth factor has a profound effect on the differentiation of
specific cells in vivo
and is a potent mitogenic factor for a variety of cultured cells of both
ectodermal and
mesodermal origin (Carpenter and Cohen, 1979).
Gray et al. (1983) presented the sequence of a mouse EGF cDNA clone, which
suggested that
!0 EGF is synthesized as a large protein precursor of 1,168 amino acids.
Mature EGF is a single-chain polypeptide consisting of 53 amino acids and
having a
molecular mass of about 6.000. Urdea et al. ( 1983) synthesized the gene for
human EGF.
Smith et al. (1982) synthesized and cloned the gene for human (3-urogastrone.
Urogastrone is a polypeptide hormone found predominantly in the duodenum and
in the
I5 salivary glands. It is a potent inhibitor of gastric acid secretion and
also promotes
epithelial cell proliferation. (3-urogastrone contains a single polypeptide
chain of 53 amino
acids, while gamma-urogastrone has the same sequence of amino acids 1-52 but
lacks the
carboxyterminal arginine of the (3 form. Sequence comparison indicates that
urogastrone is
identical to EGF
20 EGF is produced in abundance by the mouse submandibular ;land. Tsutsumi et
al. (1986)
found that sialoadenectomy decreased circulating EGF to levels below detection
but did not
affect testosterone or FSH levels. At the same time a decrease in spermatids
in the testis and
mature sperm in the epididymis decreased. The changes were corrected by
administration of
EGF. A role of EGF in some cases of human male infertility, particularly those
with
25 unexplained oligospermia, was proposed.
During the immediate-early response of mammalian cells to mitogens, histone H3
is rapidly
and transiently phosphorylated by one or more kinases. Sassone-Corsi et al.
(1999)
demonstrated that EGF-stimulated phosphorylation of H3 requires RSK2, a member
of the
pp90(RSK) family of kinases implicated in growth control.
30 By the study of human-rodent somatic cell hybrids with a genomic DNA probe,
Brissenden et
al. (1984) mapped the EGF locus to 4q21-4qter, possibly near TCGF, the locus
coding for T-
cell growth factor.
Both nerve growth factor and epidermal growth factor are on mouse chromosome 3
but they
are on different chromosomes in man: 1p and 4, respectively (Zabel et al.,
1985). Zabel et al.
35 (1985) pointed out that mouse chromosome 3 has one segment with rather
extensive
homology to distal Ip of man and a second with homology to proximal Ip of man.
By in situ
hybridization, Morton et al. (1986) assigned EGF to 4q25-q27. The receptor for
EGF is on
chromosome 7.
32
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An appropriate amino acid sequence and an appropriate nucleotide sequence are
presented in a later section herein.
PDGF
A growth factor for use in the composition of the present invention may be
PDGF.
Teachings on PDGF may be found in WO-A-09713857, WO-A-09108761, WO-A-
0931671, US-A-05034375 and WO-A-09201716.
to
An appropriate amino acid sequence and an appropriate nucleotide sequence for
PDGF A-chain are presented in a later section herein.
An appropriate amino acid sequence and an appropriate nucleotide sequence for
l5 PDGF B-chain are presented in a later section herein.
FGF
A growth factor for use in the composition of the present invention may be
FGF.
25
Background teachings on this growth factor are presented by Galzie, Z.,
Kinsella,
A.R. & Smith, J.A. (1997) Fibroblast growth factors and their receptors,
Biochem. Cell
Biol. 75, 669-685. Another review is by Werner, S. (1998) Cytokine & Growth
Factor
Reviews 9, 153-165.
An appropriate amino acid sequence and an appropriate nucleotide sequence are
presented in a later section herein.
CTGF
A growth factor for use in the composition of the present invention may be
CTGF.
Background teachings on this growth factor have been presented by Victor A.
McKusick et a! on http:/Iwww.ncbi.nlm.nih.gov/Omim. For ease of reference, the
following information has been extracted from that source.
"Bradham et al. (I991) described a new mitogen produced by human umbilical
vein
33
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
endothelial cells, which they termed connective tissue growth factor. The
protein, related to
platelet-derived growth factor, was predicted from its cDNA to be a 349-amino
acid, 38-kD
cysteine-rich secreted protein. Martinerie et al. ( 1992) identified a locus
sharing homology
with the nov protooncogene overexpressed in avian nephroblastoma and
corresponding to the
CTGF gene. They assigned the CTGF gene to 6q23.1 by a combination of study of
mouse/human somatic cell hybrids and fluorescence in situ hybridization. They
showed that
CTGF is situated proximal to MYB.
By analysis of Northern blots, Kim et al. (1997) found that CTGF is expressed
as a 2.4-kb
mRNA in a broad spectrum of human tissues. Sequence comparison revealed that
CTGF
belongs to a group known as the immediate-early genes, which are expressed
after induction
by growth~factors or certain oncogenes. The immediate-early genes have
significant sequence
homology to the insulin-like growth factor-binding proteins (IGFBPs) and
contain the
conserved N-terminal IGFBP motif (see IGFBP7). CTGF shares 28 to 38% amino
acid
identity with IGFBPs I-6. Kim et al. (1997) demonstrated that CTGF
specifically bound
IS insulin-like growth factors (IGFs), although with relatively low affinity.
They proposed that
the immediate-early genes, together with IGFBP7, constitute a subfamily of
iGFBP genes
whose products bind IGFs with low affinity."
An appropriate amino acid sequence and an appropriate nucleotide sequence are
2o presented in a later section herein.
CTGF-LIKE
A growth factor for use in the composition of the present invention may be
CTGF-like.
25 This growth factor is sometimes referred to as CT58 and WISP-2. It has the
following accession numbers: AF074604, AF083500, AF100780, 076076.
Background teachings on this growth factor have been presented by Victor A.
McKusick et al on http://www.ncbi.nlm.nih.gov/Omim. For ease of reference, the
3o following information has been extracted from that source.
Pennica et al. (Pennica, D.; Swanson, T. A.; Welsh, J. W.; Roy, M. A.;
Lawrence, D. A.; Lee,
J.; Brush, J.; Taneyhill, L. A.; Deuel, B.; Lew, M.; Watanabe, C.; Cohen, R.
L.; Melhem, M.
F.; Finley, G. G.; Quirke, P.; Goddard, A. D.; Hillan, K. J.; Gurney, A. L.;
Botstein, D.;
35 Levine, A. J. : WISP genes are members of the connective tissue growth
factor family that are
up-regulated in Wnt-I-transformed cells and aberrantly expressed in human
colon tumors.
Proc. Nat. Acad. Sci. 95: 14717-14722, 1998) cloned and characterized 3 genes
downstream
in the Wnt signaling pathway that are relevant to malignant transformation:
WISP1, WISP2,
and WISP3. The WISP2 cDNA encodes a 250-amino acid protein that is 73%
identical to the
34
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
mouse protein. The authors found that WISP2 RNA expression was reduced in 79%
of
human colon tumors, in contrast to WISPI and WISP3, which were overexpressed
in colon
tumors. By use of radiation hybrid mapping panels, Pennica et al. (1998)
mapped the WISP2
gene to 20q 12-q I 3.
An appropriate amino acid sequence and an appropriate nucleotide sequence are
presented in a later section herein.
KGF
to
A growth factor for use in the composition of the present invention may be
KGF, in
particular KGF-2.
Background teachings on this growth factor have been presented by Victor A.
McKusick et al on http:I/www.ncbi.nlm.nih.gov/Omim. For ease of reference, the
following information has been extracted from that source.
"Rubin et al. (1989) identified a growth factor specific for epithelial cells
in conditioned
medium of a human embryonic lung fibroblast cell line. Because of its
predominant activity in
keratinocytes, it was referred to as keratinocyte growth factor. KGF was found
to consist of a
single polypeptide chain of about 28 kD. It was a potent mitogen for
epithelial cells but lacked
mitogenic activity on either fibroblasts or endothelial cells. Microsequencing
showed an
amino-terminal sequence containing no significant homology to any known
protein. The
release of this growth factor by human embryonic fibroblasts raised the
possibility that KGF
2S may play a role in mesenchymal stimulation of normal epithelial cell
proliferation. In an
addendum, Rubin et al. (1989) noted that by use of all the nucleotide probes
based on the N-
terminal sequence reported in their paper, they had isolated clones encoding
KGF and had
found significant structural homology between KGF and the other 5 known
members of the
fibroblast growth factor (FGF) family.
Werner et al. (1994) assessed the function of KGF in normal and wounded skin
by expression
of a dominant-negative KGF receptor (176943) in basal keratinocytes. The skin
of transgenic
mice was characterized by epidermal atrophy, abnormalities in the hair
follicles, and dermal
hyperthickening. Upon skin injury, inhibition of KGF receptor sib aling
reduced the
proliferation rate of epidermal keratinocytes at the wound edge, resulting in
substantially
delayed reepithelialization of the wound.
Mattei et al. (1995) used isotopic in situ hybridization to map Fgf7 to region
F-G of mouse
chromosome 2. By analysis of DNA from human-rodent somatic cell hybrids with
an exon 1
probe, Kelley et al. (1992) found that FGF7 is located on human chromosome 15.
Mouse
chromosome 2 presents a conserved region of synteny with 15q13-q22. Thus, the
human
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
mutation may reside at this site. Using the murine Fgf7 probe for in situ
hybridization to
human metaphase chromosomes, Mattel et al. (1995) found signals on chromosome
i5.
Kelley et al. (1992) found a portion of the KGF gene (comprised of exons 2 and
3, the intron
between them, and a 3-prime noncoding segment) that was amplified to
approximately l6
S copies in the human genome and distributed to multiple chromosomes.
Using a cosmid probe encodin~~ KGF exon 1 for fluorescence in situ
hybridization, Zimonjic
et al. (1997) assigned the KGF7 gene to 1Sq15-q2l.l. In addition, copies of
KGF-like
sequences hybridizing only with a cosmid probe encoding exons 2 and 3 were
localized to
dispersed sites on chromosome 2q21, 9p 1 l, 9g l2-q 13, 18p 11, 18q 1 1, 21 q
1 l, and 21q21.1.
The distribution of KGF-like sequences suggested a role for alphoid DNA in
their
amplification and dispersion. In chimpanzee, KGF-like sequences were observed
at
chromosomal sites, which were each homologous to sites in human, while in
gorilla a subset
of 4 of these homologous sites was identified. In orangutan 2 sites were
identified, while
gibbon exhibited only a single site. The chromosomal localization of KGF
sequences in
1 S human and great ape genomes indicated that amplification and dispersion
occurred in multiple
discrete steps, with initial KGF gene duplication and dispersion occurring in
multiple discrete
steps, with initial KGF gene duplication and dispersion taking place in gibbon
and involving
loci corresponding to human chromosomes 15 and 21. The Endings of Zimonjic et
al. (1997)
supported the concept of a closer evolutionary relationship of human with
chimpanzee and
with primates and a possible selective pressure for KGF dispersion during the
evolution of
higher primates."
An appropriate amino acid sequence and an appropriate nucleotide sequence are
presented in a later section herein.
TGF
A growth factor for use in the composition of the present invention may be
TGF, in
particular TGF-Vii.
Background teachings on this growth factor have been presented by Victor A.
McKusick et al on http:Ilwww.ncbi.nlm.nih.govlOmim. For ease of reference, the
following information has been extracted from that source.
3S "TGF(3 is a multifunctional peptide that controls proliferation,
differentiation, and other
functions in many cell types. It was first identified by its ability to cause
phenotypic
transformation of rat fibroblasts. TGF(3 is chemically distinct from TGFa. It
has essentially no
sequence homology with TGFa or with epidermal growth factor, of which TGFa is
an analog.
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
Members of the same gene family as TGFj3 include inhibin, which inhibits
pituitary secretion
of follicle stimulating hormone, and Mullerian inhibitory substance, which is
produced by the
testis and is responsible for regression of the Mullerian ducts (anlagen of
the female
reproductive system) in the male embryo. Many cells synthesize TGF(3 and
almost al( of them
have specific receptors for this peptide. a and (3 TGFs are classes of
transforming growth
factors, TGF(3 acts synergistically with TGFa in inducing transformation. It
also acts as a
negative autocrine growth factor. By somatic cell hybridization and in situ
hybridization, Fujii
et al. (1985, 1986) assigned TGF(3 to 19q13.1-q13.3 in man and to chromosome 7
in the
mouse. Dickinson et al. (1990) mapped the Tgf(3-1 gene to mouse chromosome 7.
Marquardt
i0 et al. (1987) determined the complete amino acid sequence. Dickinson et al.
(1990) pointed
out that high levels of TGF(31 mRNA and/or protein have been localized in
developing
cartilage, endochondral and membrane bone, and skin, suggesting a role in the
growth and
differentiation of these tissues.
Heldin et al. (1997) discussed new developments in the understanding of the
mechanisms
I S used by members of the TGF-~3 family to elicit their effects on target
cells.
SMAD proteins mediate TGF(3 signaling to regulate cell growth and
differentiation.
Stroschein et al. ( 1999) proposed a model of regulation of TGF(3 signaling by
SnoN in which
SnoN maintains the repressed state of TGF(3 target genes in the absence of
ligand and
participates in the negative feedback regulation of TGFj3 signaling. To
initiate a negative
20 feedback mechanism that permits a precise and timely regulation of TGF(3
signaling, TGF~3
also induces an increased expression of SnoN at a cater stage, which in turn
binds to SMAD
heteromeric complexes and shuts off T'GF(3 signaling.
Using quantitative PCR in 15 cases of Duchenne muscular dystrophy (DMD) and.13
cases of
Becker muscular dystrophy (BMD, as well as 11 spinal muscular atrophy patients
(SMA) and
25 16 controls, Bernasconi et al. (1995) found that TGF~iI expression as
measured by mRNA
was greater in DMD and BMD patients than in controls. Fibrosis was
significantly more
prominent in DMD than in BMD, SMA, or controls. The proportion of connective
tissue
biopsies increased progressively with age in DMD patients, while TGF(31 levels
peaked at 2
and 6 years of age. Bernasconi et al. (1995) concluded that expression of
TGF(31 in the early
30 stages of DMD may be critical in initiating muscle fibrosis, and suggested
that antifibrosis
treatment might slow progression of the disease, increasing the utility of
gene therapy.
Although transforming growth factor-(3 plays a central role in tissue repair,
this cytokine is, as
pointed out by Border and Noble (1995), a double-edged sword with both
therapeutic and
pathologic potential. TGF-(3 has been implicated also in the pathogenesis of
adult respiratory
35 distress syndrome (Shenkar et al., 1994), and the kidney seems to be
particularly sensitive to
TGF-(3-induced fibrogenesis. TGF-(3 has been implicated as a cause of fibrosis
in most forms
of experimental and human kidney disease (Border and Noble, 1994).
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TGF-(3 plays an important role in wound healing. A number of pathologic
conditions, such as
idiopathic pulmonary fibrosis, scleroderma, and keloids, which share the
characteristic of
fibrosis, are associated with increased TGF-~i-1 expression. To evaluate the
role of TGF-~i-1
in the pathogenesis of fibrosis, Clouthier et al. (1997) used a transgenic
approach. They
S targeted the expression ofa constitutively active TGF-(3-1 molecule to
liver, kidney, and white
and brown adipose tissue using the regulatory sequences of the rat
phosphoenolpyruvate
carboxykinase gene. In multiple lines, targeted expression of the transgene
caused severe
fibrotic disease. Fibrosis of the liver occurred with varying degrees in
severity depending
upon the level of expression of the TGF(31 gene. Overexpression of the
transgene in kidney
also resulted in fibrosis and giomerular disease, eventually leading to
complete loss of renal
function. Severe obstructive uropathy (hydronephrosis) was also observed in a
number of
animals. Expression in adipose tissue resulted in a dramatic reduction in
total body white
adipose tissue and a marked, though less severe, reduction in brown adipose
tissue, producing
a lipodystrophy-like syndrome. Introduction of the transgene into the ob/ob
background
IS suppressed the obesity characteristic of this mutation; however, transgenic
mutant mice
developed severe hepato- and splenomegaly. Clouthier et al. (1997) noted that
the family of
rare conditions known collectively as the lipodystrophies are accompanied in
almost all forms
by other abnormalities, including fatty liver and cardiomegaly. Metabolic and
endocrine
abnormalities include either mild or severe insulin resistance,
hypertriglyceridemia, and a
hypermetabolic state
In a study of l70 pairs of female twins (average age 57.7 years), Grainger et
al. (1999)
showed that the concentration of active plus acid-activatable latent TGF(31 is
predominantly
under genetic control (heritability estimate O.S4). SSCP mapping of the TGF[31
gene promoter
identified 2 single-base substitution polymorphisms. The 2 polymorphisms (G to
A at position
-800 by and C to T at position -S09 bp) are in linkage disequiIibrium. The -
509C-T
polymorphism was significantly associated with plasma concentration of active
plus acid-
activatable latent TGF(31, which explained 8.2% of the additive genetic
variance in the
concentration. Grainger et al. (1999) suggested, therefore, that
predisposition to
atherosclerosis, bone diseases, or various forms of cancer may be correlated
with the presence
of particular alleles at the TGF(31 Locus.
Crawford et al. (1998) showed that thrombospondin-1 is responsible for a
significant
proportion of the activation of TGF(31 in vivo. Histologic abnormalities in
young TGF~iI null
and thrombospondin-1 null mice were strikingly similar in 9 organ systems.
Lung and
pancreas pathologies similar to those observed in T GF(31 null animals could
be induced in
3S wildtype pups by systemic treatment with a peptide that blocked the
activation of TGF(31 by
thrombospondin-1. Although these organs produced little active TGF(31 in
thrombospondin-1
null mice, when pups were treated with a peptide derived from thrombospondin-1
that could
activate TGF(31, active cytokine was detected in situ, and the lung and
pancreatic
38
CA 02395487 2002-06-26
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abnormalities reverted toward wildtype.
Dubois et al. (1995) demonstrated in vitro that pro-TGF~3I was cleaved by
furin to produce a
biologically active TGF~i i protein. Expression of pro-TGF(31 in furin-
deficient cells produced
no TGF(31, while coexpression of pro-TGF~31 and Turin led to processing of the
precursor.
Blanchette et al. ( 1997) showed that furin mRNA levels were increased in rat
synovial cells
by the addition of TGF(3l. This effect was eliminated by pretreatment with
actinomycin-D,
suggesting to them that regulation was at the gene transcription level.
Treatment of rat
synoviocytes and kidney fibroblasts with TGF(31 or TGF(32 resulted in
increased pro-TGF(31
processing, as evidenced by the appearance of a 40-kD immunoreactive band
corresponding to
f 0 the TGF(31 amino-terminal pro-region. Treatment of these cells with TGF(32
resulted in a
significant increase in extracellular mature TGF(31. Blanchette et al. (1997)
concluded that
TGF(31 upregulates gene expression of its own converting enzyme."
An appropriate amino acid sequence and an appropriate nucleotide sequence are
l5 presented in a later section herein.
CSF
A growth factor for use in the composition of the present invention may be
GSF, in
2o particular GM-CSF.
.t
Background teachings on this growth factor have been presented by Victor A.
McKusick et al on http:I/www.ncbi.nim.nih.gov/Omim. For ease of reference, the
following information has been extracted from that source.
"Colony-stimulating factors (CSFs) are proteins necessary for the survival,
proliferation, and
differentiation of hematopoietic progenitor cells. They are named by the cells
they stimulate.
Macrophage CSF is known as CSF. Granutocyte-macrophage CSF (CSF2; also
symbolized
GMCSF) stimulates both cell types. Multi-CSF is known as interleukin-3 (IL3).
The CSF in
human urine, active in stimulating granulocyte-macrophage colony formation by
murine cells,
was the first CSF to be purified to homogeneity. It is a glycoprotein of MW
45,000 and is a
homodimer. Wong et al. (1985) isolated cDNA clones for human GMCSF. Huebner et
al.
(1985) assigned the GMCSF locus to Sq21-q32 by somatic cell hybrid analysis
and in situ
hybridization. This is the same region as that involved in interstitial
deletions in the 5q-
syndrome and acute myelogenous leukemia. They found a partially deleted GMCSF
allele and
a 5q- marker chromosome in a human promyelocytic leukemia cell Line. The
truncated
GMCSF gene appeared to lie at the rejoining point for the interstitial
deletion. By in situ
hybridization, Le Beau et al. (1986) assigned FMS to 5q33 and GMCSF to 5q23-
q31. Both
39
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genes were deleted in the Sq- chromosome from bone marrow cells of 2 patients
with
refractory anemia and del(S)(q I Sq33.3). From study of other cases they
concluded that FMS is
located in band 5q33.2 or Sq33.3 rather than Sq34-q3S as reported earlier.
Pettenati et al.
(1987) concluded that the order of loci from the centromere toward Sqter is
CSF2, CSFI, and
S FMS ( 164770). By long range mapping, Yang et al. ( 1988) demonstrated that
the GMCSF
and IL3 genes are separated by about 9 kilobases of DNA. They are tandemly
arranged head
to tail with IL3 on the S-prime side of GMCSF. Frolova et al. (1991)
identified 2 RFLPs in a
70-kb segment of genomic DNA that includes these 2 genes as well as flanking
sequences.
Using these markers in studies of the panel from the Centre d'Etude du
Polymorphisme
Humain (CEPH), they studied linkage with a number of other expressed genes on
chromosome ~. Thangavelu et al. (1992) presented a physical and genetic
linkage map that
encompassed 14 expressed genes and several markers located in the distal half
of the long arm
of chromosome S. By fluorescence in situ hybridization, Le Beau et al. (1993)
mapped the
CSF2 gene to Sq3 l. 1.
I S Group B streptococcus (GBS) is the most common bacterial infection causing
pneumonia and
sepsis in newborn infants. Host responses to GBS include activation of both
alveolar
macrophages and polymorphonuclear leukocytes. Phagocytosis and killing of GBS
in the
lungs is enhanced by surfactant protein A, which increases phagocytosis and
reactive oxygen
species-mediated killing. Because macrophage function is strongly influenced
by GMCFS,
LeVine et al. ( 1999) tested whether GBS clearance from the lungs was
intluenced by GMCFS
in vivo. Mice homozygous for a knockout of the Cfs2 gene cleared group B
streptococcus
from the lungs more slowly than wildtype mice. Expression of GMCSF in the
respiratory
epithelium of homozygous deficient mice improved bacterial clearance to levels
greater than
that in wildtype mice. Acute aerosolization of GMCSF to wildtype mice
significantly
2S enhanced clearance of GBS at 24 hours. In the homozygous knockout mice, GBS
infection
was associated with increased neutrophilic infiltration in lungs, while
macrophage infiltrates
predominated in wildtype mice, suggesting an abnormality in macrophage
clearance of
bacteria in the absence of GMCSF. While phagocytosis of GBS was unaltered,
production of
superoxide radicals and hydrogen peroxide was markedly deficient in
macrophages from
homozygous knockout mice."
An appropriate amino acid sequence and an appropriate nucleotide sequence are
presented in a later section herein.
CHRYSALIN
A growth factor for use in the composition of the present invention may be
Chrysafin.
Chrysaiin is being developed by Chrysalis Biotechnology Inc. Chrysalis is a
small
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
(12 residue) peptide derived from the sequence of thrombin. Chrysalin is
described
in EP-A-0328552.
TISSUE DAMAGE UPREGULATED PROTEINS
In accordance with the present invention, use is made of selective inhibitors
of
adverse proteins (in particular adverse proteases that have a deleterious
effect on
wound healing) that are upregulated in a damaged tissue, such as a wound,
environment.
to
The damaged tissue environment for treatment may be a chronic wound, such as a
chronic dermal ulcer.
In addition, or in the alternative, the damaged tissue environment for
treatment may
t 5 be one or more those associated with age-related macular degeneration,
corneal
ulceration, corneal melting, irritable bowel syndromeldisorderldisease,
gastric
ulceration, renal failure, peripheral neuropathies (e.g. diabetic
retinopathy),
neurodegenerative diseases, bone diseases or injury, cartilage diseases or
injury,
muscle diseases or injury, tendon diseases or injury, ischaemic damage,
peridontal
20 disease, psoriasis, bullous pemphigoid, epidermolysis bullosa, spinal cord
disease or
injury.
Preferably said damaged tissue is a wound, more preferably a chronic wound,
such
as a chronic dermal ulcer.
In particular, use is made of selective inhibitors of proteases that are
upreguiated in a
damaged tissue, such as a wound, environment, in particular a chronic wound
environment, such as chronic dermal ulcers. In this respect, the composition
of the
present invention comprises an agent that targets one or more of said proteins
in
order to act as an inhibitor against said protein.
In another embodiment, one or more of said proteins are used in an assay to
screen
for agents that are capable of inhibiting said proteins. The identified agents
are then
used to prepare a composition according to the present invention.
Examples of protease proteins that are upregulated in a damaged tissue, such
as a
wound, environment, in particular a chronic wound environment, such as chronic
41
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dermal ulcers, are plasminogen activators and certain matrix
metalloproteinases. A
particular example of a suitable plasminogen activator is urokinase-type
plasminogen
activator. Particular examples of matrix metalloproteinases are matrix
metalloproteinase 1, matrix metalloproteinase 2, matrix metalloproteinase 3,
matrix
metalloproteinase 7, matrix metalloproteinase 8, matrix metalloproteinase 9,
matrix
metalioproteinase 10, matrix metalioproteinase 11, matrix metalloproteinase
12,
matrix metalloproteinase 13, matrix metalloproteinase 14, matrix
metalloproteinase
15, matrix metalloproteinase 16, matrix metalloproteinase 17, matrix
metalloproteinase 19, matrix metalloproteinase 20, matrix metalloproteinase
21,
Io matrix metalloproteinase 24, and matrix metalloproteinase FMF. Details on
some of
these proteins are presented below.
UROKINASE
In accordance with the present invention, a target for the inhibitor agent of
the
present invention - or a putative inhibitor agent in an assay of the present
invention -
may be urokinase-type plasminogen activator (uPA).
Urokinase (urinary-type plasminogen activator or uPA; International Union of
Biochemistry classification number EC.3.4.21.31 ) is a serine protease
produced by a
large variety of cell types (smooth muscle cells, fibroblasts, endothelial
cells,
macrophages and tumour cells). It has been implicated as playing a key role in
cellular invasion and tissue remodelling. A principal substrate for uPA is
plasminogen which is converted by cell surface-bound uPA to yield the serine
?5 protease plasmin. Locally produced high plasmin concentrations mediate cell
invasion by breaking down the extracellular matrix. Important processes
involving
cellular invasion and tissue remodelling include wound repair, bone
remodelling,
angiogenesis, tumour. invasiveness and spread of metastases.
3o In particular, uPA is one of the proteases which is over-expressed in
chronic dermal
ulcers. uPA is a serine protease produced by a large variety of cell types
(smooth
muscle cells, fibroblasts, endothelia! cells, macrophages and tumour cells).
It has
been implicated as playing a key role in cellular invasion and tissue
remodelling. A
principal substrate for uPA is plasminogen which is converted by cell surFace-
bound
35 uPA to yield the serine protease plasmin.
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Beneficial effects of urokinase inhibitors have been reported using anti-
urokinase
monoclonal antibodies and certain other known urokinase inhibitors. For
instance,
anti-urokinase monoclonal antibodies have been reported to block tumour cell
invasiveness in vitro (W.Hollas, et al, Cancer Res. 51:3690; A.Meissauer, et
al,
Exp. Cell Res. 192:453 (1991 ); tumour metastases and invasion in vivo
(L.Ossowski,
J. Cell Biol. 107:2437 (1988)); L.Ossowski, et al, Cancer Res. 51:274 (1991))
and
angiogenesis in vivo (J.A.Jerdan et al, J. Cell Biol. 115[3 Pt 2]:402a (1991).
Also,
AmilorideT"", a known urokinase inhibitor of only moderate potency, has been
reported to inhibit tumour metastasis in vivo (J.A.Keilen et al, Anticancer
Res.,
8:1373 (1988)) and angiogenesis I capillary network formation in vitro
(M.A.Alliegro et
al, J. Cell Biol. 115[3 Pt 2]:402a).
Conditions of particular interest for treatment by urokinase inhibitors
include chronic
dermal ulcers (including venous ulcers, diabetic ulcers and pressure sores),
which
~5 are a major cause of morbidity in the ageing population and cause a
significant
economic burden on healthcare systems. Chronic derma! ulcers are characterised
by excessive uncontrolled proteolytic degradation resulting in ulcer
extension, loss of
functional matrix molecules (e.g. fibronectin) and retardation of
epithelisation and
ulcer healing. A number of groups have investigated the enzymes responsible
for
2o the excessive degradation in the wound environment, and the role of
plasminogen
activators has been highlighted (M.C. Stacey ef al., Br. J. Surgery, 80, 596;
M.
Palolahti et al., Exp. Dermafol., 2, 29, 1993; A.A. Rogers et al., Wound
Repair and
Regen., 3, 273, 1995). Normal human skin demonstrates low levels of
plasminogen
activators which are localised to blood vessels and identified as tissue type
25 plasminogen activator (tPA). In marked contrast, chronic ulcers demonstrate
high
levels of urokinase type plasminogen activator (uPA) localised diffusely
throughout
the ulcer periphery and the lesion, and readily detectable in wound fluids.
uPA could affect wound healing in several ways. Plasmin, produced by
activation of
3o plasminogen, can produce breakdown of extracellular matrix by both indirect
(via
activation of matrix metalloproteases) and direct means. Plasmin has been
shown to
degrade several extracellular matrix components, including gelatin,
fibronectin,
proteoglycan core proteins as well as its major substrate, fibrin. Whilst
activation of
matrix metalloproteases (MMPs) can be performed by a number of inflammatory
cell
35 proteases (e.g. eiastase and cathepsin G), the uPAlplasmin cascade has been
implicated in the activation of MMPs in situ, providing a broad capacity for
degrading
all components of the extracellular matrix. Furthermore, and in addition to
its effect
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on production of plasmin, uPA has been shown to catalyse direct cleavage of
fibronectin yielding antiproliferative peptides. Thus, over-expression of uPA
in the
wound environment has the potential to promote uncontrolled matrix degradation
and
inhibition of tissue repair. Inhibitors of the enzyme thus have the potential
to promote
healing of chronic wounds.
Further background teachings on uPA have been presented by Victor A. McKusick
et
al on http://www.ncbi.nlm.nih.govlOmim. For ease of reference, the following
information has been extracted from that source.
to
"Urokinase is the urinary plasminogen activator. (Tissue plasminogen activator
is a second
type; it has a single polypeptide chain of 70,000 daltons and is unrelated to
urokinase
immunologically.) Urokinase is a protein that has a molecular weight of about
54,000 daltons
and is composed of 2 disulfide-linked chains, A and B, of molecular weights
18,000 and
I ~ 33,000, respectively. Salerno et al. ( 1984) developed separate monoclonal
antibodies for the A
and B chains and by using them identified a single-chain biosynthetic
precursor in a rabbit
reticulocyte cell-free protein-synthesizing system directed by human kidney
total
polyadenylated RNA. Thus, the precursor must be cleaved in a way that the
insulin precursor
is cleaved.
20 By combined somatic cell genetics, in situ hybridization, and Southern
hybridization, Tripputi
et al. (1985) localized the human urokinase gene to IOq24-qter. By use of
specific cDNA
probes in the study of human-mouse somatic cell hybrids, Rajput et al. (1985)
mapped the
human plasminogen activator and urokinase genes to chromosomes 8 and 10,
respectively. By
Southern blot analysis of DNA from mouse-Chinese hamster and mouse-rat somatic
cell
?5 hybrids, Rajput et al. (1987) assigned the mouse equivalent (Plau) to mouse
chromosome 14.
Urokinase may occur as a single-chain form or as a 2-chain derivative, which
is generated by
cleavage of the peptide bond between lys(158) and ile(159) in the single-chain
form by
plasmin. Lijnen et al. (1988) produced site-specific mutation in position 158
(lys-to-glu).
Studies of the enzymatic properties of the mutant form, which was resistant to
plasmin,
30 indicated that the amino acid in position 158 is a main determinant of the
functional properties
of the single-chain form, but not of the 2-chain form.°
An appropriate amino acid sequence and an appropriate nucleotide sequence are
presented in a later section herein.
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MMP
In accordance with the present invention, a target for the inhibitor agent of
the
present invention - or a putative inhibitor agent in an assay of the present
invention
may be one or more matrix metalloproteinases (MMPs) wherein said MMP has a
deleterious effect on wound healing in damaged tissue.
MMPs constitute a family of structurally similar zinc-containing
metalloproteases,
which are involved in the remodelling, repair and degradation of extracelluiar
matrix
proteins, both as part of normal physiological processes and in pathological
conditions. At least 18 members of the human family have been sequenced.
Since they have high destructive .potential, the MMPs are usually under close
regulation, and failure to maintain MMP regulation has been implicated as a
t5 component of a number of conditions. Examples of conditions where MMPs are
thought to be important are those involving bone restructuring, embryo
implantation
in the uterus, infiltration of immune cells into inflammatory sites,
ovulation,
spermatogenesis, tissue remodelling during wound repair and organ
differentiation
such as such as in venous and diabetic ulcers, pressure sores, colon ulcers
for
2o example ulcerative colitis and Crohn's disease, duodenal ulcers, fibrosis,
local
invasion of tumours into adjacent areas, metastatic spread of tumour cells
from
primary to secondary sites, and tissue destruction in arthritis, skin
disorders such as
dystrophic epidermolysis bulosa, dermatitis herpetiformis, or conditions
caused by or
complicated by embolic phenomena, such as chronic or acute cardiac or cerebral
25 infarctions.
Substrates for the MMPs are diverse - and sometimes include other members of
the
gene family. For example, MMP-14 is known to digest and activate proMMP-2 and
both MMP-3 and MMP-9 can digest and activate proMMP-1. Some MMP substrates
3o are also matrix components - such as collagen which is digested, for
example by
MMP-1 (also known as collagenase-1), denatured collagen or gelatin which is
digested for example, by MMP-2 (also known as gelatinase-A), fibronectin which
is
digested for example by MMP-3 (allso known as stromelysin-1) and
glycosaminoglycans which is digested for example by MMP-3.
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CA 02395487 2002-06-26
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For recent reviews of MMPs, see Zask et al, Current Pharmaceutical Design,
1996,.
2, 624-661; Beckett, Exp. Opin. Ther. Patents, 1996, 6, 1305-1315; and Beckett
et al,
Drug Discovery Today, vol 1 (no.1 ), 1996, 16-26.
Alternative names for various MMPs and substrates acted on by these are shown
in
the table below (Zask et al, supra).
Enzyme Other names Preferred substrates
MMP-1 Collagenase-1, interstitial Collagens I, II, III,
collagenase VII, X,
elatins
MMP-2 Gelatinase A, 72kDa gelatinaseGelatins, collagens
IV, V, VII,
X, elastin, fibronectin;
activates ro-MMP-13
MMP-3 Stromelysin-1 Proteoglycans, laminin,
fibronectin, elatins.
MMP-7 Pump, Matrilysin Proteoglycans, laminin,
fibronectin, gelatins,
collagen
IV, elastin, activates
pro-MMP-
1 and -2 .
MMP-8 Colla enase-2, neutro hil Colla ens I, II, III
colla enase
MMP-9 Gelatinase B, 92 kDa gelatinaseGelatins, collagens
IV, V,
eiastin
MMP-12 Macrophage metalloelastase Elastin, collagen IV,
fibronectin, activates
pro-
MMP-2 & 3.
MMP-13 Colla enase-3 Colla ens I, II, III,
elatins
MMP-14 MT-MMP-1 Activates pro-MMP-2
& 13,
elatins
MMP-15 MT-MMP-2
MMP-16 MT-MMP-3 Activates ro-MMP-2
_
MMP-17 MT-MMP-4
1o Examples of
suitable MMP
targets) for
the inhibitor
agent of the
present invention
-
or for a putative tion
inhibitor agent - may
in an assay be any
of the present
inven
suitable member of one I (MMP1 matrix
or more ),
of: matrix
metalloproteinase
metalloproteinase2 (MMP2), matrix metalloproteinase(MMP3), matrix
3
metalloproteinase7 (MMP7), matrix metalloproteinase(MMPB), matrix
8
metalloproteinase9 (MMP9), matrix metalloproteinase(MMP10),matrix
10
metalloproteinase11 {MMP11),matrix metalloproteinase(MMP12),matrix
12
metalloproteinase13 {MMP13),matrix metalloproteinase{MMP14),matrix
14
metalloproteinase15 {MMP15),matrix metalloproteinase(MMP16),matrix
16
metalloproteinase17 {MMP17),matrix metalloproteinase(MMP19),matrix
19
2o metalloproteinase20 (MMP20),matrix metalloproteinase(MMP21),matrix
21
metalloproteinase24 (MMP24), {MMPFMF).
and matrix
metalloproteinase
FMF
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Some of these targets are discussed in slightly more detail. In addition,
appropriate
amino acid sequences and appropriate nucleotide sequences are presented in a
later
section herein.
For some embodiments of the present invention, preferably the target for the
inhibitor
agent of the present invention may be MMP13 and/or MMP3.
MMP1
to
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMP1.
Background teachings on matrix metalloproteinase I (MMP1) have been presented
1S by Victor A. McKusick et a! on http://www.ncbi.nlm.nih.govlOmim: For ease
of
reference, the following information has been extracted from that source.
"Brinckerhoff et al. ( 1987) identified a cDNA clone of human collagenase (EC
3.4.23.7 ). The
clone identified a single collagenase gene of about 17 kb from blots of human
genomic DNA.
20 Restriction enzyme analysis and DNA sequence data indicated that the cDNA
clone was full
length and that it was identical to that described for human skin fbroblast
collagenase.
Collagenase is the only enzyme able to initiate breakdown of the interstitial
collagens, types I,
II, and III. The fact that the collagens are the most abundant proteins in the
body means that
collagenase plays a key role in the remodeling that occurs constantly in both
normal and
ZS diseased conditions. The identity of human skin and synovial cell
collagenase and the ubiquity
of this enzyme and of its substrates, collagens I, II, and III, imply that the
common mechanism
controlling collagenolysis throughout the body may be operative in both normal
and disease
states. Gerhard et al. (1987) confirmed the assignment of the collagenase gene
to chromosome
11 by the use of a, DNA probe for Southern analysis of somatic cell hybrids.
Analysis of cell
30 lines with rearrangements involving chromosome I I indicated that the gene
is in the region
Ilqll-q23. Church et al. (1983) had used somatic cell hybrids between mouse
cells and
human normal skin and corneal fibroblasts and recessive dystrophic
epidermolysis bullosa
(RDEB) skin fibroblasts to assign the human structural gene for collagenase to
chromosome
11. Production of collagenase was measured by a specific radioimmunoassay. It
appeared that
35 both the normal and the RDEB collagenase gene mapped to chromosome 1 I.
This was earlier
taken to indicate that the abnormal collagenase produced by RDEB cells
represented a
mutation of the structural gene. . Later work indicated that both the
autosomal dominant
(131750) and autosomal recessive forms of dystrophic epidermolysis bullosa are
due to
-47
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
mutations in the type VII collagen gene (COL7A1; 120120). The excessive
formation of
co(lagenase must represent a secondary phenomenon, not the primary defect. It
should be
noted that fibroblasts from patients with the Werner syndrome also express
high constitutive
levels of collagenase In vitro (Bauer et al., 1986).
Pendas et al. (1996) isolated a l.~-Mb YAC clone mapping to 1 1q22. Detailed
analysis of this
nonchimeric YAC clone ordered 7 MMP genes as follows: cen--MMPB --MMP10 --MMPI-
-
MMP3 --MMP12 --MMP7 --MMP13 --tel.
Note on nomenclature: In reporting on the nomenclature of the matrix
metalloproteinases,
Nagase et al. (i992) referred to interstitial collagenase as MMPI."
l0
MMP2
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMP2.
Background teachings on matrix metalloproteinase 2 (MMP2) have been presented
by Victor A. McKusick et al on http://www.ncbi.nlm.nih.gov/Omim. For ease of
reference, the following information has been extracted from that source.
"Type IV collagenase is a metalloproteinase that specifically cleaves type IV
collagen, the
major structural component of basement membranes. The metastatic potential of
tumor cells
has been found to correlate with the activity of this enzyme.
Huhtala et al. (1990) determined that the CLG4A gene is 17 kb long with I3
exons varying in
size from. I 10 to 901 by and 12 introns ranging from 175 to 4,350 bp.
Alignment of introns
showed that introns 1 to 4 and 8 to 12 of the type IV collagenase gene
coincide with intron
locations in the interstitial collagenase and stromelysin genes, indicating a
close structural
relationship of these metalloproteinase genes. Devarajan et al. (1992)
reported on the structure
and expression of 78-kD gelatinase, which they referred to as neutrophil
gelatinase.
Type IV collagenase, 72-kD, is o~cially designated matrix metalloproteinase-2
(MMP2). It is
also known as gelatinase, 72-kD (Nagase et al., 1992).
Irwin et al. (1996) presented evidence that MMP2 is a likely effector of
endometrial menstrual
breakdown. They cultured human endometrial stromal cells in the presence of
progesterone
and found an augmentation of proteinase production after withdrawal of
proteinase: the same
results were achieved by the addition of the P receptor antagonist RU486.
Characterization of
3~ the enzyme by Western blotting revealed it to be MMP2. Northern blot
analysis showed
differential expression of MMP2 mRNA ,in late secretory phase endometrium.
Angiogenesis depends on both cell adhesion and proteolytic mechanisms. Matrix
metalloproteinase-2 and integrin a-V/(3-3 are functionally associated on the
surface of
angiogenic blood vessels. Brooks et al. (1998) found that a fragment of MMP2,
which
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comprises the C-terminal hemopexin-like domain (amino acids 445-635) and is
termed PEX.
prevents this enzyme from binding to a-V/(3-3 and blocks cell surface
collagenolytic activity
in melanoma and endothelial cells. PEX blocks MMP2 activity on, the chick
chorioallantoic
membrane where it disrupts angiogenesis and tumor growth. Brooks et al. (1998)
also found
that a naturally occurring Form of PEX can be detected in vivo in conjunction
with a-V/(3-3
expression in tumors and during developmental retinal neovascularization.
Levels of PEX in
these vascularized tissues suggest that it interacts with endothelial cell a-
V/(3-3 where it
serves as a natural inhibitor of MMP2 activity, thereby regulating the
invasive behavior of
new blood vessels. The authors concluded that recombinant PEX may provide a
potentially
novel therapeutic approach For diseases associated with neovascularization.
By hybridization to a panel of DNAs from human-mouse cell hybrids and by in
situ
hybridization using a gene probe, Fan et al. ((989) assigned the CLG4 gene to
16q21; see
Huhtala et al. (1990). By hybridization to somatic cell hybrid DNAs, Collier
et al. (1991)
assigned both CLG4A and CLG4B to chromosome 16. Chen et al. (1991) mapped l2
genes
on the long arm of chromosome 16 by the use of 14 mouse/human hybrid cell
lines and the
fragile site FRA I6B. The breakpoints in the hybrids, in conjunction with the
fragile site,
divided the long arm into 14 regions. They concluded that CLG4 is in band 16q
13.
Morgunova et al. (1999) reported the crystal structure of the.full-length
proform of human
MMP2. The crystal structure revealed how the propeptide shields the catalytic
cleft and that
the cysteine switch may operate through cleavage of loops essential for
propeptide stability.
Becker-Fol(mann et al. (1997) created a high-resolution map of the linkage
group on mouse
chromosome 8 that is conserved on human 16q. The map extended from the homolog
of the
MMP2 locus on 16q 13 (the most centromeric locus) to CTRB on 16q23.2-q23.3."
MMP3
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMP3.
3o Thus, according to this embodiment, the present invention provides a
pharmaceutical
for use in damaged tissue, such as wound, treatment (e.g. healing); the
pharmaceutical comprising a composition which comprises: (a) a growth factor;
and
an inhibitor agent; and optionally c) a pharmaceutically acceptable carrier,
diluent or
excipient; wherein the inhibitor agent can inhibit the action of at least one
specific
adverse protein (e.g. a specific protease) that is upregulated in a damaged
tissue,
such as a~wound,environment; wherein said specific protein is MMP3.
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
Background teachings on matrix metalloproteinase 3 (MMP3) have been presented
by Victor A. McKusick et al on http:l/www.ncbi.nlm.nih.gov/Omim. For ease of
reference, the following information has been extracted from that source.
"Human fibroblast stromelysin {also called transin or matrix metalloproteinase-
3) is a
proteoglycanase closely related to collagenase (MMPI) with a wide range of
substrate
specificities. It is a secreted metalloprotease produced predominantly by
connective tissue
cells. Together with other metalloproteases, it can synergistically degrade
the major
components of the extracellular matrix (Sellers and Murphy, 1981). Stromelysin
is capable of
degrading proteogiycan. Fbronectin, laminin, and type IV collagen, but not
interstitial type I
collagen. Whitham et al. (1986) found that the amino acid sequences predicted
from the
cDNAs of collagenase and stromelysin indicate that they are closely related
enzymes, with a
particularly well-conserved region of 14 amino acids, that shares significant
homology with
the zinc-chelating region of the bacterial metalloprotease
thermo(ysin.(Matthews et al., 1974).
l~ Wilhelm et al. (1987) purified and determined the complete primary
structure of human
stromelysin. It is synthesized in a preproenzyme form with a calculated size
of X3,977 Da and
a l7-amino acid long signal peptide. A comparison of primary structures
suggested that
stromelysin is the human analogs of rat transin. Saus et al. (1988) determined
the complete
primary structure of human matrix metalloproteinase-3 (MMP3), which has 477
amino acid
residues, including a 17-residue signal peptide. The findings indicated that
MMP3 is identical
to stromelysin. MMP3 and collagenase were found to be 54% identical in
sequence,
suggesting a common evolutionary origin of the 2 proteinases. Furthermore,
MMP3 and
collagenase expression appeared to be coordinately modulated in synovial
fibroblast cultures.
Levels of mRNA for both proteins are induced by interleukin-t-~i and
suppressed by retinoic
acid or dexamethasone. Koklitis et al. (1991) purifed 2 forms of recombinant
human
prostromelysin.
By somatic cell hybridization and in situ hybridization, Spurr et al. (1988)
mapped the
stromelysin locus to I 1q and confirmed the location of the collagenase gene
on chromosome
I 1, specifically on I 1q. Gatti et al. (I989) placed the STMY locus in the I
1q22-q23 region by
linkage analysis with markers in that area, including ataxia-telangiectasia.
By pulsed field gel
electrophoresis, Formstone et al. (1993) showed that a cluster of
metalloproteinase genes--
stromelysin I, fibroblast collagenase (MMP1), and stromelysin II (MMP10)--are
located in a
135-kb region of chromosome 11. The physical proximity of these 3 genes,
together with the
DNA marker DIIS385, was confirmed using 2 YAC clones, and their relative order
determined. This information, combined with the pattern of marker
representation in a panel
of radiation-reduced chromosome 11 hybrids, suggested that the order was cen--
STMY2--
CLG-STMYI--DI1S385--ter. Pendas et al. (1996) noted that the family of human
MMPs was
composed of 14 members at the time of their report. MMP genes have been mapped
to
chromosomes 11, 14 (MMP14, 16 (MMP2, 20 (MMP9), and 22 (MMP11), with several
clustered within the long arm of chromosome 11. Pendas et al. (1996) isolated
a I.5-Mb YAC
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
clone mapping to 1 1q22. Detailed analysis of this nonchimeric YAC clone
ordered 7 MMP
genes as follows: cen--MMPB --MMP 10--MMP 1--MMP3--MMP12 --MMP7 --MMP 13 tel.
Kerr et al. (1988) examined the role of FOS (164810) in growth-factor
stimulation of transin,
a matrix-degrading secreted metalloproteinase. The stimulatory effect of both
platelet-derived
growth factor ( 190040) and epidermal growth factor on transin transcription
involved factors
recognizing the sequence TGAGTCA, which is found in the transin promoter and
is a binding
site for the transcriptional factor JUN/APl and for associated FOS and FOS-
related
complexes.
Wound repair involves cell migration and tissue remodeling, and these ordered
and regulated
processes are facilitated by matrix-degrading proteases. Saarialho-Kere et al,
(199?) found
that interstitial collagenase is invariantly expressed by basal keratinocytes
at the migrating
front of healing epidermis. Because the substrate specificity of collagenase
is limited
principally to interstitial tibrillar collagens, other enzymes must also be
produced in the
wound environment to restructure tissues effectively with a complex matrix
composition. The
l~. stromelysins can degrade many noncollagenous connective tissue
macromolecules. Using in
situ hybridization and immunohistochemistry, Saarialho-Kere et al. ( 1994)
found that both
stromelysin I and stromelysin II are produced by distinct populations of
keratinocytes in a
variety of chronic ulcers. Stromelysin I mRNA and protein were detected in
basal
keratinocytes adjacent to but distal from the wound edge in what probably
represented the
30 sites of proliferating epidermis. In contrast, stromelysin II mRNA was seen
only in basal
keratinocytes at the migrating front, in the same epidermal cell population
that expressed
collagenase. Stromelysin I producing keratinocytes resided on the basement
membrane,
whereas stromelysin II producing keratinocytes were in contact with the dermal
matrix.
Furthermore, stromelysin I expression was prominent in dermal fcbroblasts,
whereas no signal
25 for stromelysin II was seen in any dermal cell: These findings demonstrated
that the 2
stromelysins are produced by different populations of basal keratinocytes in'
response to
wounding and suggested that they serve distinct roles in tissue repair.
Using immunofluorescence staining, RT-PCR, and in situ hybridization, Lu et
al. (1999)
localized stromelysin I to the epithelial layers of unwounded and wounded
corneas. They
30 found stromelysin I in the deep stromal layer in the frst 3 days after
wounding and in the area
of newly synthesized stromal matrix 1 week after surgery. They stated that
stromelysin I
activates matrilysin (MMP7) (Imai et al., 1995) and that stromelysin I and
matrilysin interact
during tissue remodeling. They concluded that stromelysin I may be involved in
the reparative
process in the wound bed after excimer keratectomy, whereas matrilysin may
play a role in
35 epithelial wound remodeling not only in the migration phase but also in the
subsequent
proliferation phase.
There is a common polymorphism in the promoter sequence of the STMY1 gene,
with 1 allele
containing a run of 6 adenosines (6A) and the other 5 adenosines (5A). Ye et
al. (1996)
followed up on a previously reported 3-year study by Richardson et al. (1989)
of patients with
40 coronary atherosclerosis which indicated that those patients who were
homozygous for the 6A
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CA 02395487 2002-06-26
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allele showed a more rapid progression of both global and focal
atherosclerotic stenoses. This
observation supported the ftndings by others that the metalloproteinases are
involved in
connective tissue remodeling during atherogenesis. Ye et al. (1996)
investigated whether the
5A/6A promoter polymorphism plays a role in the regulation of STMYI gene
expression. In
transient expression experiments, a STMYI promoter construct with 6A at the
polymorphic
site was found to express less of the reporter gene than a construct
containing SA. Binding of
a nuclear protein factor was more readily detectable with an oligonucleotide
probe
corresponding to the 6A allele as compared with a probe corresponding to the
5A allele. Thus,
Ye et al. (1996) found that the 5A/6A polymorphism appears to play an
important role in
regulating STMYI expression. In a study by Quinones et al. (1989), the
frequency of the 2
alleles (5A/6A) was found to be 0.51/0.d9 in a sample of 35~ healthy
individuals from the
UK.
Sternlicht et al. (1999) examined how MMP3, or STR1, affects tumor progression
using 3
genetic approaches: phenotypically normal mammary epithelial cells that
express STRI in a
tetracycline-regulated manner, and an STRI transgene targeted to mouse mammary
glands by
the mouse 'whey acidic protein' (WAP) gene promoter. Phenotypically normal
mammary
epithelial cells with tetracycline-regulated expression of STR1 formed
epithelial glandular
structures in vivo without STRI but formed invasive mesenchymal-like tumors
with STRI.
Once initiated, the tumors became independent of continued STRI expression.
STR1 also
promoted spontaneous premalignant changes and malignant conversion in mammary
glands of
transgenic mice. These changes were blocked by coexpression of a TIMPI
(305370)
transgene. The premalignant and malignant lesions had stereotyped genomic
changes unlike
those seen in other murine mammary cancer models. These data indicated that
STR1
in#luences tumor initiation and alters neoplastic risk."
MMP7
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMP7.
Background teachings on this matrix metalloproteinase have been presented by
Victor A. McKusick et al on http://www.ncbi.nlm.nih.govlOmim. For ease of
reference,
the following information has been extracted from that source.
The putative metalloproteinase I (PUMPI) gene was identified through studies
of collagenase-
related connective-tissue-degrading metalloproteinases produced by human
tumors. Muller et
al. (Muller, D.; Quantin, B.; Gesnel, M.-C.; Milton-Collard, R.; Abecassis,
J.; Breathnach, R.
The co(lajenase gene family in humans consists of at least four members.
Biochem. J. 253:
187-192, 1988) found that the PUMP protein has 267 amino acids and is
significantly shorter
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
than stromelysin or collagenase (477 and 469 amino acids, respectively).
Putative
metalloproteinase I was later called matrilysin or matrix metalloproteinase-7
(MMP7).
MMP8
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMPB.
Background teachings on this matrix metalloproteinase have been presented by
to Victor A. McKusick et al on http://www.ncbi.nlm.nih.gov/Omim. For ease of
reference,
the following information has been extracted from that source.
Neutrophil collagenase, a member of the family of matrix metalloproteinases;
is distinct from
the collagenase of skin fibroblasts and synovial cells in substrate
specificity and immunologic
crossreactivity. Hasty et al. (Hasty, K. A.; Pourmotabbed, T. F.; Goldberg, G.
L; Thompson,
J. P.; Spinella, D. G.; Stevens, R. M.; Mainardi, C. L. : Human neutrophil
collagenase: a
distinct gene product with homology to other matrix metalloproteinases. J.
Biol. Chem. 26~:
I 1421-11424, 1990.) cloned and sequenced a cDNA encoding human neutrophil
eollagenase
using a lambda-gtl I cDNA library constructed from mRNA extracted from the
peripheral
leukocytes of a patient with chronic granulocytic leukemia. The coding
sequence predicts a
467-amino acid protein. It hybridized to a 3.3-kb mRNA from human bone marrow.
Other
features of the primary structure confirmed that neutrophil collagenase is a
member of the
family of matrix metalloproteinases (e.g., MMPI) but distinct from other
members of the
family. Neutrophil collagenase shows a preference for type I collagen in
contrast with the
greater susceptibility of type III collagen to digestion by fibroblast
collagenase. Devarajan et
al. (Devarajan, P.; Mookhtiar, K.; Van Wart, H.; Berliner, N. : Structure and
expression of the
cDNA encoding human neutrophil collagenase. Blood 77: 2731-2738, 1991)
isolated a 2.4-kb
cDNA clone encoding human neutrophil collagenase. From its sequence, it was
shown to
encode a 467-residue protein which exhibited 58% homology to human fibroblast
collagenase
and had the same domain structure.
MMP9
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMP9.
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Background teachings on matrix metalloproteinase 9 (MMP9) have been presented
by Victor A. McKusick et al on http://www.ncbi.nim.nih.govlOmim. For ease of
reference, the following information has been, extracted from that source.
"The 72- and 92-kD type IV collagenases are members of a group of secreted
zinc
metalloproteases which, in mammals, degrade the collagens of the extracellular
matrix. Other
members of this group include interstitial collagenase and stromelysin. The 72-
kD type IV
collagenase is secreted from normal skin fibroblasts, whereas the 92-kD
collagenase (CLG4B)
is produced by normal alveolar macrophages and granulocytes. Both CLG and STMY
have i0
exons of virtually identical length, are located on 1 Iq, and are regulated in
a coordinate
fashion. By hybridization to somatic cell hybrid DNAs, Collier et al. ( 1991 )
demonstrated that
both CLG4A and CLG4B are situated on chromosome 16. However, St Jean et al.
(1990
assigned CLG4B to chromosome 20. 'They did. linkage mapping of the CLG4B locus
in 10
CEPH reference pedigrees using a polymorphic dinucleotide repeat in the 5-
prime flanking
l ~ region of the gene. St Jean et al. ( 1990 observed lod scores of between
10.45 and 20.29 with
markers spanning chromosome region 20q11.2-q13.1. Further support for
assignment of
CLG4B to chromosome 20 was provided by analysis of human/rodent somatic cell
hybrids.
Both CLG4A and CLG4B have 13 exons and similar intron locations (Huhtala et
al., 1991).
Due to these similarities, the CLG4B cDNA clone used in the mapping to
chromosome l6
may have hybridized to CLG4A rather than to CLG4B on chromosome 20.
The I3 exons of both CLG4A and CLG4B are 3 more than have been found in other
members
of this gene family. The extra exons. encode the amino acids of the fbronectin-
tike domain
which has been found only in the 72- and 92-kD type IV collagenases. The 92-kD
type IV
collagenase is also known as 92-kD gelatinase, type -V collagenase, or matrix
metalloproteinase 9 (MMP9); see the glossary of matrix metalloproteinases
provided by
Nagase et al. (1992).
Linn et al. (1996) reassigned MMP9 (referred to as ~ CLG4B by them) to
chromosome 20
based on 3 different lines of evidence: screening of a somatic cell hybrid
mapping panel,
fluorescence in situ hybridization, and linkage analysis using a newly
identified
polymorphism. They also mapped mouse Clg4b to mouse chromosome 2, which has no
known homology to human chromosome 16 but large regions of homology with human
chromosome 20.
By targeted disruption in embryonic stem cells, Vu et al. (1998) created
homozygous mice
with a null mutation in the MMP9/gelatinase B gene. These mice exhibited an
abnormal
pattern of skeletal growth plate vascularization and ossification. Although
hyperirophic
chondrocytes developed normally, apoptosis, vascularization, and ossification
were delayed,
resulting in progressive lengthening of the growth plate to about 8 times
normal. After 3
weeks postnatal, aberrant apoptosis, vascularization, and ossification
compensated to remodel
the enlarged growth plate and ultimately produced an axial skeleton of normal
appearance.
Transplantation of wildtype bone marrow cells rescued vascularization and
ossification in
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
MMP9-null growth plates, indicating that these processes are mediated by MMP9-
expressing
cells of bone marrow origin, designated chondroclasts. Growth plates from MMP9-
null mice
in culture showed a delayed release of an angiogenic activator, establishing a
role for this
proteinase in controlling angiogenesis.
MMP10
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMP10.
l0
Background teachings on this matrix metalloproteinase have been presented by
Victor A. McKusick et al on http://www.ncbi.nlm..nih.govlOmim. For ease of
reference,
the following information has been extracted from that source.
~ Stromelysin is a metalloproteinase related to collagenase (there is about
55% similarity in
their amino acid sequences) whose substrates include proteoglycans and
fibronectin, but not
type I collagen. Stromelysin ll is also called matrix metalloproteinase-lu, or
Motrm. otuuer
et al. (Mullet, D.; Quantin, B.; Gesnel, M.-C.; Milton-Collard, R.; Abecassis,
J.; Breathnach,
R. : The collagenase gene family in humans consists of at least four members.
Biochem. J.
20 253: 187-192, 1988) detected RNAs capable of hybridizing to a rat
stromelysin cDNA in 11
of 69 human tumors tested. These studies were undertaken because of the strong
likelihood
that tumor invasion and metastasis require enzymic degradation of a host
interstitial matrix, a
concept that is supported by reports of increased proteolytic activities in
tumor cells. By
molecular cloning of cDNAs to these RNAs, Mullet et al. (1988) identified them
as a mixture
25 of stromelysin RNA and a transcript of a hitherto undescribed related gene,
that of stromelysin
II. They also isolated cDNAs corresponding to a more distantly related human
gene, the
PUMP1 gene.
MMP11
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMP11.
Background teachings on this matrix metalloproteinase have been presented by
Victor A. McKusick et al on http:/Iwww.ncbi.nlm.nih.gov/Omim. For ease of
reference,
the following information has been extracted from that source.
ss
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WO 01/49309 PCT/IB00/01935
The family of matrix metalloproteinases appears to be involved in physiologic
and pathologic
processes associated with extracellu(ar matrix remodeling such as those that
occur in
embryonic development, tissue repair, and tumor progression. Matrisian,
Stromelysin III, a
member of this gene family, is overexpressed in the stromal cells of invasive
breast
carcinomas but not in the stromal cells surrounding benign breast
fibroadenomas. By in situ
hybridization, Levy et al. (Levy, A.; Zucman, J.; Delattre, O.; Mattel, M.-G.;
Rio, M.-C.:
Basset, P. : Assignment of the human stromelysin 3 (STMY3) gene to the q11.2
region of
chromosome 22. Genomics l3: ss 1-883, 1992.) assigned the STMY3 gene to 22q.
Using a
panel of somatic cell hybrids containing different segments of 22q, they
demonstrated that the
STMY3 gene is in band 22q11.2, in close proximity to the BCR gene involved in
chronic
myeloid leukemia. Both STMY 1 and STMY2 are located on chromosome 11.
Stromelysin III
is also called matrix metalloproteinase-11, or MMPlI. The nomenclature of the
matrix
metalloproteinases, together with symbols and EC numbers, was provided by
Nagase et al.
(Nagase, H.; Barrett, A. J.; Woessner, J. F., Jr. : Nomenclature and glossary
of the matrix
I ~ metalloproteinases. Matrix Suppl. 1: 42 l-424, 1992).
MMP12
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMP12.
Background teachings on this matrix metalloproteinase have been presented by
Victor A. Mcl<usick et a! on http:llwww.ncbi.nlm.nih.govlOmim. For ease of
reference,
the following information has been extracted from that source.
The matrix metalloproteases (MMPs) are a family of related matrix-degrading
enzymes that
are important in tissue remodeling and repair during development and
inflammation.
Abnormal expression is associated with various diseases such as tumor
invasiveness, arthritis,
and atherosclerosis. MMP activity may also be related to cigarette-induced
pulmonary
emphysema. Belaaouaj et al. (Belaaouaj, A.; Shipley, J. M.; Kobayashi, D. K.;
Zimonjic, D.
B.; Popescu, N.; Silverman, G. A.; Shapiro, S. D. : Human macrophage
metalloelastase:
genomic organization, chromosomal location, gene linkage, and tissue-specific
expression. J.
Biol. Chem.270: 14568-14675, 1995) described the genomic organization of the
HME gene
(also symbolized MMP12). The 13-kb gene is composed of 10 exons and shares the
highly
conserved intron-exon borders of other MMPs. The authors also demonstrated
tissue-specific
expression in macrophages and stromal cells. They localized the gene to
11q22.2-q22.3 by
fluorescence in situ hybridization.
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MMP13
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMP13.
Thus, according to this embodiment, the present invention provides a
pharmaceutical
for use in damaged tissue, such as wound, treatment (e.g. healing); the
pharmaceutical comprising a composition which comprises: (a) a growth factor;
and
an inhibitor agent; and optionally c) a pharmaceutically acceptable carrier,
diluent or
to excipient; wherein the inhibitor agent can inhibit the action of at least
one specific
adverse protein (e.g. a specific protease) that is upregulated in a damaged
tissue,
such as a wound, environment; wherein said specific protein is MMP13.
Background teachings on matrix metalloproteinase 13 (MMP13) have been
I5 presented by Victor A. McKusick et al on http:Ilwww.ncbi.nlm.nih.gov/Omim.
For
ease of reference, the following information has been extracted from that
source.
"Freije et al. ( 1994) cloned a cDNA coding for a 'new' human matrix
metalloproteinase
(MMP) from a cDNA library derived from a breast tumor. The isolated cDNA
contains an
20 open reading frame coding for a polypeptide of 471 amino acids. The
predicted protein
sequence displays extensive similarity to previously known MMPs and presented
all the
structural features characteristic of this protein family, including the well-
conserved
PRCGXPD motif. In addition, it contains in its amino acid sequence several
residues speciftc
to the collagenase subfamily (tyr214, asp235, and g1y237) and lacks the 9-
residue insertion
25 present in the stromelysins. Because of the structural characteristics,
Freije et al. (1994) called
the new MMP collagenase-3, since it represented the third member of this
family, composed
of fibroblast (MMP1) and neutrophil (MMPB) collagenases.
Pendas et al. (1997) reported that the MMP13 gene contains 10 exons and spans
approximately 12.5 kb. The overall gene organization is similar to those of
other MMP genes,
30 including MMP1, MMP7, and MMPi2.
Freije et al. (i994) expressed the CLG3 cDNA in a vaccinia virus system and
found that the
recombinant protein was able to degrade fibrillar collagens, providing support
to the idea that
the isolated cDNA codes for an authentic collagenase. Northern blot analysis
of RNA from
normal and pathologic tissues demonstrated the existence in breast tumors of 3
different
35 mRNA species, which seemed to be the result of utilization of different
polyadenylation sites
present in the 3-prime noncoding region of the gene. By contrast, no CLG3 mRNA
was
detected either by Northern blot or RNA polymerase chain reaction analysis
with RNA from
other human tissues, including normal breast, mammary fibroadenomas, liver,
placenta, ovary,
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uterus, prostate, and parotid land. A possible role for this metalloproteinase
in the tumoral
process was proposed.
By fluorescence in situ hybridization, Pendas et al. (1995) localized the CLG3
gene (also
symbolized MMP13) to 1Iq32.3. Physical mapping of a YAC clone containing CLG3
revealed that this gene is tightly linked to those genes encoding other matrix
metalioproteinases, including.: Fbrobiast collagenase (MMPI), stromelysin-1
(MMP3), and
stromelysin-2 (MMP10). Further chapping of this region using pulsed field gel
electrophoresis
showed that the CLG3 gene is located on the telomeric side of the matrix
metalloproteinase
cluster. Pendas et al. (1995) found the relative order of the loci to be cen--
STMY2--CLGI--
!0 STMY1--CLG3--tel. Pendas et al. (1996) isolated a 1.5-Mb YAC clone mapping
to I Iq22.
Detailed analysis of this nonchimeric YAC clone ordered 7 MMP genes as
follows: cen--
MMPB--MMP t 0--MMP 1--MMP3--MMP 12--MMP7--MMP 13--tel.
Mitchell et at. (1996) concluded that the expression of MMP13 in
osteoarthritic cartilage and
its activity against type II colla~~en indicates that the enzyme plays a
significant role in
I 5 cartilage collagen degradation and must, therefore, form part of a complex
target for proposed
therapeutic interventions based on collagenase inhibition. Reboul et al.
(1996) likewise
presented data on collagenase-3 expression and synthesis in human cartilage
cells and
suggested its involvement in human osteoarthritis cartilage pathophysiology."
20 MMP14
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMP14.
25 Background teachings on matrix metalioproteinase' 14 (MMP14) have been
presented by Alan Scott et al on http:l/www.ncbi.nlm.nih.gov/Omim. For ease of
reference, the following information has been extracted from that source.
"Matrix metalloproteinases (MMPs) are Zn(2+)-binding endopeptidases that
degrade various
30 components of the extracellular matrix (ECM). The MMPs are enzymes
implicated in normal
and pathologic tissue remodeling processes, wound healing, angiogenesis, and
tumor invasion.
MMPs have different substrate specificities and are encoded by different
genes. Sato et al.
(1994) cloned a cDNA for the human gene from a placenta cDNA library (they
called the gene
MMP-X1 and the gene product membrane-type metalloproteinase). The authors
noted that the
35 protein was expressed at the surface of invasive tumor cells. Using
degenerate PCR, Takino et
al. (1995) cloned the entire genomic sequence of this member of the MMP
superfamily (see
MMPI). The cDNA identified codes for a 582-amino acid protein which shared
conserved
sequence and a similar domain structure to other MMPs. They noted that the
cDNA, termed
MMP-X 1 by them, had a unique transmembrane domain at the C terminus. Thus,
they
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predicted that MMP-X 1 was a membrane spanning protein rather than a secretory
protein like
the other MMPs. Northern blots showed that MMP-X! expression was present at
varying
intensity in almost all tissues examined, but was highest in the placenta.
Mignon et al. (1995) tabulated 11 members of the matrix metalloproteinase
family and their
S chromosomal locations; with I exception, the genes encoding them had been
mapped. Six of
them, including 3 collagenases and ? stromelysins, had been assigned to I 1 q.
Membrane-type
matrix metalloproteinase (MMPl4),may be an activator of pro-gelatinase A and
is expressed
in fibroblast cells during both wound healing and human cancer progression. By
isotopic in
situ hybridization, Mignowet al. ((995) mapped the MMP14 gene to 14q1 t-q12.
By gene targeting, Holmbeck et crl. (1999) generated mice deficient in the
Mmpl4 gene,
which they called MTl-MMP. Mmpl~4 deficiency caused craniofacial dysmorphism,
arthritis,
osteopenia, dwarfism, and fibrosis of soft tissues due to ablation of a
collagenolytic activity
that is essential for modeling of skeletal and extraskeletal connective
tissues. These findings
demonstrated the pivotal function of MMP l4 in connective tissue metabolism
and illustrated
IS that modeling of the soft connective tissue matrix by resident cells is
essential for the
development and maintenance of the hard tissues of the skeleton."
MMP15
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMP15.
Background teachings on this matrix metalloproteinase have been presented by
Victor A. McKusick et al on http:I/www.ncbi.nlm.nih.gov/Omim. For ease of
reference,
the following information has been extracted from that source.
Will and Hinzmann (Will, H.; Hinzmann, B. : cDNA sequence and mRNA tissue
distribution
of a novel human matrix metalloproteinase with a potential transmembrane
segment. Europ. J.
Biochem. 231: 602-608, 1995) isolated a cDNA encoding a novel MMP (MMP15) from
a
human°lung cDNA library. The MMP15 cDNA encodes a 669-amino acid
protein that has the
typical structural features of an MMP. In addition, it contains a predicted
transmembrane
segment at the C terminus. MMP15 shares 73.9% sequence similarity with MMP14,
a
membrane-localized MMP that also contains a C-terminal transmembrane segment.
MMP16
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMP16.
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WO 01/49309 PCT/IB00/01935
Background teachings on this matrix metalloproteinase have been presented by
Victor A. McKusick et al on http:I/www.ncbi.nlm.nih.govlOmim. For ease of
reference,
the following information has been extracted from that source.
Takino et al. (Takino, T.; Sato, H:; Shinagawa, A.; Seiki, M. : Identification
of the second
membrane-type matrix metalloproteinase (MT-MMP-2) gene from a human placenta
cDNA
library: MT-MMPs form a unique membrane-type subclass in the MMP family. J.
Biol.
Chem.270: 23013-23020, 1995) isolated a novel MMP cDNA (MMP 16) from a human
placenta cDNA library. The MMP16 protein consists of 604 amino acids and has a
characteristic MMP domain structure. Additionally, MMP16 has a C-terminal
extension
containing a potential transmembrane domain, similar to MMP14, MMPIS, and
MMP17.
MMP17
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMP17.
Background teachings on this matrix metalloproteinase have been presented by
Victor A. McKusick et al on http:I/www.ncbi.nlm.nih.gov/Omim. For ease of
reference,
2o the following information has been extracted from that source.
Puente et al. (Puente, X. S.; Pendas, A. M.; Llano, E.; Velasco, G.; Lopez-
Otin, C. : Molecular
cloning of a novel membrane-type matrix metalloproteinase from a human breast
carcinoma.
Cancer Res.56: 944-949, 1996.) cloned a cDNA encoding matrix metalloproteinase-
17
(MMP17) from a human breast carcinoma cDNA library using degenerate PCR.
MMP17,
named MT4-MMP by the authors, is a 518-amino acid protein that has a domain
organization
characteristic of the MMP family, including a prodomain with an activation
locus, a zinc
binding site, and a hemopexin domain. MMPI7 also has a C-terminal extension
that contains
a putative transmembrane domain, indicating that it is a member of the
membrane-type MMP
subclass (see MMP14, MMP15, MMP16).
MMP19
For some embodiments of the present invention, the target for the inhibitor
agent of
the present invention may be MMP19.
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
. _. . .~ _ " . .. a
Background teachings on this matrix metalloproteinase have been presented by
Victor A. McKusick et al on http:/Iwww.ncbi.nlm.nih.gov/Omim. For ease of
reference,
the following information has been extracted from that source.
Using an MMP similarity search of the EST database, Cossins et al. (Cossins,
J.; Dudgeon, T.
J.; Catlin, G.; Gearing, A J. H.; Clements, J. M. : Identification of MMP-18,
a putative novel
human matrix metalloproteinase. Biochem. Biophys. Res. Commun. 228: 494-498,
1996)
identified a partial cDNA clone that encodes the 3-prime end of a putative
MMP, which they
called MMP18 but which has officially designated MMP19. They PCR-amplified the
5-prime
end and cloned and sequenced the full-length cDNA. MMP19 contains an open
reading frame
of 508 amino acids with a predicted molecular weight of 57,238 and has all the
characteristic
features of the MMP family. MMP18 contains a putative signal sequence,
followed by a
prodomain with a conserved 'cysteine switch' region. Expression of a single
transcript of 2.7
kb was detected in placenta, lung, pancreas, ovary, small intestine, spleen,
thymus, and
prostate, and at much lower levels in testis, colon, and heart. No MMP19 mRNA
was
detected in brain, skeletal muscle, liver, kidney, or peripheral blood
leukocytes.
INHIBITOR AGENT
2o An essential component of the composition of the present invention is an
inhibitor
agent. The inhibitor agent may be any suitable agent that can act as an
inhibitor of a
respective protein (e.g. protease) that is upregulated in a damaged tissue,
such as a
wound, environment - wherein the protein (protease) has an adverse
(deleterious)
effect on the healing of damaged tissue.
The term "inhibitor" as used herein with respect to the agent of the present
invention
means an agent that can reduce and/or eliminate and/or mask and/or prevent the
action of a respective protein (e.g. protease) that is upregulated in a
damaged tissue,
such as a wound, environment - wherein the protein (proteases) has an adverse
(deleterious) effect on the healing of damaged tissue.
Particular inhibitor agents include one or more suitable members of: an
inhibitor of
uPA (I:uPA), an inhibitor of MMP1 (I:MMP1), an inhibitor of MMP2 (I:MMP2), an
inhibitor of MMP3 (i:MMP3), an inhibitor of MMP7 (I:MMP7), an inhibitor of
MMPB
(I:MMPB), an inhibitor of MMP9 (I:MMP9), an inhibitor of MMP10 (I:MMP10), an
inhibitor of MMP11 (I:MMP11), an inhibitor of MMP12 (I:MMP12), an inhibitor of
MMP13 (I:MMP13), an inhibitor of MMP14 (I:MMP14), an inhibitor of MMP9
(I:MMP15), an inhibitor of MMP16 (I:MMP16), an inhibitor of MMP17 (I:MMP17),
an
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inhibitor of MMP19 (I:MMP19) an inhibitor of MMP20 (I:MMP20), an inhibitor of
MMP21 (I:MMP21 ), an inhibitor of MMP24 (I:MMP24), an inhibitor of
MMPFMF(I:MMPFMF).
The inhibitor agent can be an amino acid sequence or a chemical derivative
thereof.
The substance may even be an organic compound or other chemical. The agent
may even be a nucleotide sequence - which may be a sense sequence or an anti-
sense sequence. The agent may be an antibody. For some applications,
preferably,
the inhibitor agent is a synthetic organic molecule.
l0
Thus, the term "inhibitor" includes, but is not limited to, a compound which
may be
obtainable from or produced by any suitable source, whether natural or not.
The inhibitor may be designed or obtained from a library of compounds which
may
I5 comprise peptides, as well as other compounds, such as small organic
molecules,
such as lead compounds.
By way of example, the inhibitor may be a natural substance, a biological
macromolecule, or an extract made from biological materials such as bacteria,
fungi,
20 or animal (particularly mammalian) cells or tissues, an organic or an
inorganic
molecule, a synthetic agent, a semi-synthetic agent, a structural or
functional
mimetic, a peptide, a peptidomimetics, a derivatised agent, a peptide cleaved
from a
whole protein, or a peptides synthesised synthetically (such as, by way of,
example,
either using a peptide synthesizer br by recombinant techniques or
combinations
25 thereof, a recombinant agent, an antibody, a natural or a non-natural
agent, a fusion
protein or equivalent thereof and mutants, derivatives or combinations
thereof.
As used herein, the term "inhibitor" may be a single entity or it may be a
combination
of agents. Hence, the inhibitor agent of the composition of the present
invention may
3o be two or more agents that are capable of inhibiting the action of one or
more
proteins that are upregulated in a damaged tissue, such as a wound,
environment.
Thus, the composition of the present invention may comprise an I:uPA and an
I:MMP. In another embodiment, the composition of the present invention may
comprise an I:uPA and an I:MMP1 and/or an I:MMP2 and/or an I:MMP3 andlor an
35 I:MMP7 and/or an I:MMP8 and/or an I:MMP9 andlor an I:MMP10 and/or an
I:MMP11
and/or an I:MMP12 and/or an I:MMP13 and/or an I:MMP14 andlor an I:MMP15
and/or an I:MMP16 and/or an I:MMP17 and/or an I:MMP19 and/or an I:MMP20
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WO 01/49309 PCT/IB00/01935
and/or an I:MMP21 and/or an I:MMP24 and/or an I:MMPFMF. In another
embodiment, the composition of the present invention may comprise a first
I:uPA and
a second I:uPA and/or a first I:MMP and/or a second I:MMP.
The inhibitor agent of the composition of the present invention may comprise
one
agent that is capable of inhibiting the action of two or more proteins that
are
upregulated in a damaged tissue, such as a wound, environment. Thus, the
composition of the present invention may comprise an agent that is capable of
acting
as an I:uPA and an I:MMP. In another embodiment, the composition of the
present
to invention may comprise an agent that is capable of acting as an I:uPA and
an
I:MMP1 and/or an I:MMP2 andlor an I:MMP3 and/or an I:MMP7 and/or an I:MMP8
and/or an I:MMP9 andlor an I:MMP10 andlor an I:MMP11 and/or an f:MMP12 and/or
an I:MMP13 and/or an I:MMP14 andlor an I:MMP15 and/or an (:MMP16 and/or an
I:MMP17 andlor an I:MMP19 and/or an (:MMP20 andlor an I:MMP21 and/or an
I:MMP24 and/or an (:MMPFMF.
The inhibitor agent of the present invention may even be capable of displaying
other
therapeutic properties:
2o The inhibitor agent may be used in combination with one or more other
pharmaceutically active agents.
If a combination of active agents are administered, then they may be
administered
simultaneously, separately or sequentially.
STEREO AND GEOMETRIC ISOMERS
Some of the specific inhibitor agents andlor growth factors may exist as
stereoisomers and/or geometric isomers - e.g. they may possess one or more
3o asymmetric andlor geometric centres and so may exist in two or more
stereoisomeric
and/or geometric forms. The present invention contemplates the use of all the
individual stereoisomers and geometric isomers of those inhibitor agents, and
mixtures thereof. The terms used in the claims encompass these forms, provided
said forms retain the appropriate functional activity (though not necessarily
to the
same degree).
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WO 01/49309 PCT/IB00/01935
PHARMACEUTICAL SALT
The inhibitor agent of the present invention - and possibly the growth factor
of the
present invention - may be administered in the form of a pharmaceutically
acceptable
salt.
Pharmaceutically-acceptable salts are well known to those skilled in the art,
and for
example include those mentioned by Berge et al, in J.Pharm.Sci., 66, 1-19
(1977).
Suitable acid addition salts are formed from acids which form non-toxic salts
and
include the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate,
bisulphate,
phosphate, hydrogenphosphate, acetate, trifluoroacetate, gluconate, lactate,
salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate,
gluconate,
formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate and
p-toiuenesulphonate salts.
When one or more acidic moieties are present, suitable pharmaceutically
acceptable
base addition salts can be formed from bases which form non-toxic salts and
include
the aluminium, calcium, lithium, magnesium, potassium, sodium, zinc, and
pharmaceutically-active amines such as diethanolamine, salts.
zo
A pharmaceutically acceptable salt of an inhibitor agent of the present
invention may
be readily prepared by mixing together solutions of the agent and the desired
acid or
base, as appropriate. The . salt may precipitate from solution and be
collected by
filtration or may be recovered by evaporation of the solvent.
The inhibitor agent of the present invention may exisit in polymorphic form.
The inhibitor agent of the present invention may contain one or more
asymmetric
carbon atoms and therefore exists in two or more stereoisomeric forms. Where
an
3o agent contains an alkenyl or alkenylene group, cis (E) and trans (Z)
isomerism may
also occur. The present invention includes the individual stereoisomers of the
agent
and, where appropriate, the individual tautomeric forms thereof, together with
mixtures thereof.
Separation of diastereoisomers or cis and traps isomers may be achieved by
conventional techniques, e.g. by fractional crystallisation, chromatography or
H.P.L.C. of a stereoisomeric mixture of the agent or a suitable salt or
derivative
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
thereof. An individual enantiomer of the agent may also be prepared from a
corresponding optically pure intermediate or by resolution, such as by
H.P.L.C. of the
corresponding racemate using a suitable chiral support or by fractional
crystallisation
of the diastereoisomeric salts formed by reaction of the corresponding
racemate with
a suitable optically active acid or base, as appropriate.
The present invention also includes all suitable isotopic variations of the
agent or a
pharmaceutically acceptable salt thereof. An isotopic variation of an agent of
the
present invention or a pharmaceutically acceptable salt thereof is defined as
one in
to which at least one atom is replaced by an atom having the same atomic
number but
an atomic mass different from the atomic mass usually found in nature.
Examples of
isotopes that can be incorporated into the agent and pharmaceutically
acceptable
salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen,
phosphorus,
sulphur, fluorine and chlorine such as 2H, 3H,~'3C, '4C, '5N, "O, 'g0, 3'P,
32p, ass, '8F
~ 5 and 36C1, respectively. Certain isotopic variations of the agent and
pharmaceutically
acceptable salts thereof, for example, those in which a radioactive isotope
such as
3H or '4C is incorporated, are useful in drug andlor substrate tissue
distribution
studies. Tritiated, i.e., 3H, and carbon-14, i.e., '4C, isotopes are
particularly preferred
for their ease of preparation and detectability. Further, substitution with
isotopes
2o such as deuterium, i.e., ZH, may afford certain therapeutic advantages
resulting from
greater metabolic tability, for example, increased in vivo half-life or
reduced dosage
requirements and hence may be preferred in some circumstances. Isotopic
variations of the agent of the present invention and pharmaceutically
acceptable salts
thereof of this invention can generally be prepared by conventional procedures
using
25 appropriate isotopic variations of suitable reagents.
It will be appreciated by those skilled in the art that the agent of the
present invention
may be derived from a prodrug. Examples of prodrugs include entities that have
certain protected groups) and which may not possess pharmacological activity
as
3o such, but may, in certain instances, be administered (such as orally or
parenterally)
and thereafter metabolised in the body to form the agent of the present
invention
which are pharmacologically active.
It will be further appreciated that certain moieties known as "pro-moieties",
for
35 example as described in "Design of Prodrugs" by H. Bundgaard, Elsevier,
1985 (the
disclosured of which is hereby incorporated by reference), may be placed on
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
appropriate functionalities of the agents. Such prodrugs are also included
within the
scope of the invention.
The present invention also includes (wherever appropriate) the use of
zwitterionic
forms of the inhibitor agent of the present invention - and possibly the
growth factor
of the present invention.
The terms used in the claims encompass one or more of the forms just
mentioned.
SOLVATES
The present invention also includes the use of solvate forms of the inhibitor
agent of
the present invention - and wherever applicable the growth factor of the
present
invention, The terms used in the claims encompass these forms.
PRO-DRUG
As indicated, the present invention also includes the use of pro-drug forms of
the
inhibitor agent of the present invention - and wherever applicable the growth
factor of
the present invention. The terms used in the claims encompass these forms.
CHEMICAL SYNTHESIS METHODS
Typically the inhibitor agent of the present invention will be prepared by
chemical
synthesis techniques.
It will be apparent to those skilled in fihe art that sensitive functional
groups may need to
be protected and deprotected during synthesis of a compound of the invention.
This
may be achieved by conventional techniques, for example as described in
"Protective
Groups in Organic Synthesis" by T W Greene and P G M Wuts, John Wiley and Sons
3o Inc. (1991), and by P.J.Kocienski, in "Protecting Groups", Georg Thieme
Verlag (1994).
It is possible during some of the reactions that any stereocentres present
could, under
certain conditions, be racemised, for example if a base is used in a reaction
with a
substrate having an having an optical centre comprising a base-sensitive
group. This is
possible during e.g. a guanylation step. ft should be possible to circumvent
potential
problems such as this by choice of reaction sequence, conditions, reagents,
protection/deprotection regimes, etc. as is well-known in the art.
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WO 01/49309 PCT/IB00/01935
The compounds and salts of the invention may be separated and purified by
conventional methods.
Separation of diastereomers may be achieved by conventional techniques, e.g.
by
fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric
mixture of a
compound of formula (I) or a suitable salt or derivative thereof. An
individual
. enantiomer of a compound of formula (I) may also be prepared from a
corresponding
optically pure intermediate or by resolution, such as by H.P.L.C. of the
corresponding
to racemate using a suitable chiral support or by fractional crystallisation
of the
diastereomeric salts formed by reaction of the corresponding racemate with a
suitably optically active acid or base.
The inhibitor agent or growth factor of the present invention or variants,
homologues,
~ 5 derivatives, fragments or mimetics thereof may be produced using chemical
methods
to synthesize the agent in whole or in part. For example, if they are
peptides, then
peptides can be synthesized by solid phase techniques, cleaved from the resin,
and
purified by preparative high performance liquid chromatography (e.g.,
Creighton
(1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New
2o York NY). The composition of the synthetic peptides may be confirmed by
amino
acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton,
supra).
Syntesis of peptide inhibitor agents or of the growth factors (or variants,
homologues,
25 derivatives, fragments or mimetics thereof) can be performed using various
solid-
phase techniques (Roberge JY et al (1995) Science 269: 202-204) and automated
synthesis may be achieved, for example, using the ABI 43 1 A Peptide
Synthesizer
(Perkin Elmer) in accordance with the instructions provided by the
manufacturer.
Additionally, the amino acid sequences comprising the agent or any part
thereof, may
3o be altered during direct synthesis and/or combined using chemical methods
with a
sequence from other subunits, or any part thereof, to produce a variant agent
or
growth factor.
In an alternative embodiment of the invention, the coding sequence of a
peptide
35 inhibitor agent or growth factor (or variants, homologues, derivatives,
fragments or
mimetics thereof) may be synthesized, in whole or in part, using chemical
methods
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WO 01/49309 PCT/IB00/01935
we!! known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser
215-
23, Horn T ei~ al (1980) Nuc Acids Res Symp Ser 225-232).
MIMET)C
As used herein, the term "mimetic" relates to any chemical which includes, but
is not
limited to, a peptide, poiypeptide, antibody or other organic chemical which
has the
same qualitative activity or effect as a reference agent
1o CHEMICAL DERIVATIVE
The term "derivative" or "derivatised" as used herein includes chemical
modification
of an agent. Illustrative of such chemical modifications would be replacement
of
hydrogen by a halo group, an alkyl group, an acyl group or an amino group.
CHEMICAL MODIFICATION
In one embodiment of the present invention, the inhibitor agent may be a
chemically
modified inhibitor agent.
The chemical modification of an agent of the present invention may either
enhance or
reduce hydrogen bonding interaction, charge interaction, hydrophobic
interaction,
Van Der Waals interaction or dipole interaction between the agent and the
target.
In one aspect, the identified agent may act as a model (for example, a
template) for
the development of other compounds.
RECOMBINANT METHODS
The growth factor of the present invention may be prepared by recombinant DNA
techniques.
UROKINASE INHIBITOR
A component of the composition of the present invention may be an inhibitor of
urokinase-type plasminogen activator. Typically, the I:uPA will be capable of
being
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
identified as being an I:uPA by a uPA assay - such as the assay protocol
presented
herein.
Thus, in one aspect, the present invention relates to a method of enhancing
the
healing of chronic dermal ulcers, including venous stasis ulcers, diabetic
ulcers and
decubitus ulcers (or pressure sores), by treating the patient with a
combination of a
selective inhibitor of uPA and a growth factor. This combination therapy is
more
effective than treatment with the individual agents.
to The inhibitors of uPA can either be applied topically or administered
orally,
depending on the properties of the inhibitor and the way in which they are
formulated.
Thus, according to one aspect of the present invention, the composition may
comprise an I: uPA - such as a selective uPA inhibitor - and a growth factor.
With the
is co-administration of these two components a more profound efficacy can be
achieved than by administration of either a growth factor or a uPA inhibitor
alone.
Here, efficacy may be measured by the standard of the FDA in this area - such
as
the time to closure of chronic dermal ulcers under conditions of best care and
compared to best care alone.
In one preferred aspect, topical formulations of selective. uPA inhibitors can
be co-
administered with topically administered growth factors, such as PDGF, either
by
physically mixing the substances and using a formulation which releases both
substances into the damaged tissue, such as a wound, environment, or by
applying
one substance at a time and using a treatment protocol which separates
application
of'the agents. Alternatively, combined treatment can be achieved using an
orally
administered uPA inhibitor with topical application of a growth factor.
We believe that the use of I:uPA when co-administered with growth factors is
very
3o advantageous and was, also, unexpected and unpredictable. In this respect,
many
literature reports show that uPA is required as part of the signalling cascade
downstream from growth factor receptors. We have determined that, whilst this
may
be the case, the protective effects of a selective uPA inhibitor on growth
factors, and
cellular responses to growth factors, predominates.
In accordance with the present invention, the I:uPA may be applied topically
mixed
with the growth factor or the I:uPA may be applied topically but at a
different time to
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
the growth factor or the I:uPA may be administered orally and the growth
factor may
be applied topically.
The I:uPA may be naturally occurring or it may be a synthetic entity.
A number of I:uPAs are known. For example, reference may be made to C. Magi!!
ef
al. Emerg. Therap. Targets 1999, 3(1), 109-133, and H. Yang et al.
Fibrinolysis 1992,
6 (Suppl 1), 31-34.
to Examples of naturally occurring proteinacious inhibitors include
plasminogen
activator inhibitor proteins PAI-1 and PAI-2 (see Antalis, T.M., Clark, M.A.,
Barnes,
T., Lehrbach, P.R., Devine, P.L., Schevzov, G., Goss, N.H., Stephens, R.W. 8~
Tostoshev, P. (1988) Proc. Nat!. Acad. Sci. U.S.A. 85, 985-999). Reference may
also be made to WO 99!49887. Another naturally occurring proteinacious
inhibitor is
~ s a-antitrypsin.
Other naturally naturally occurring inhibitors include E-Aminocaproic acid (s-
aca) -
which is a weak inhibitor. Vitamin E (oc-tocopherol) is an irreversible
inhibitor of
urokinase which acts via an unknown mechanism. Natural catechols isolated from
2o green tea such as epigallocathechin-3 gallate (EGCG) inhibit urokinase. The
nortriterpenoid demethylzeylasteral (TZ-93) isolated from Tripterygium
wilfordii
inhibits urokinase activity. The protein aprotinin is a weak inhibitor of
urokinase but
not t-PA.
~o
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
Me
Me / ~ Me Me Me
HZN COZH Me \ 0 ~ Me
Me
Me
E-aca a-tocopherol
OH
OH
HO / O '' ~ I OH
~~~ O
OH O ~ OH
OH
OH
EGCG TZ-93
nnP rO2H
In addition, synthetic inhibitors of uPA exist. These synthetic inhibitors
will typically
be organic compounds. Typically the organic compounds will comprise a
guanidine
group (i.e. -N=C(NHZ)(NH~)) and one or more hydrocarbyl groups. Here, the term
"hydrocarbyl group" means a group comprising at least C and H and may
optionally
comprise one or more other suitable substituents. Examples of such
substituents
may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic grQUp etc. In
addition to the
1o possibility of the substituents being a cyclic group, a combination of
substituents may
form a cyclic group. If the hydrocarbyl group comprises more than one C then
those
carbons need not necessarily be linked to each other. For example, at least
two of
the carbons may be linked via a suitable element or group. Thus, the
hydrocarbyl
group may contain hetero atoms. Suitable hetero atoms will be apparent to
those
skilled in the art and include, for instance, sulphur, nitrogen and oxygen.
For some
applications, preferably the agent comprises at least one cyclic group,
wherein that
cyclic group is a polycyclic group, preferably being a fused polycyclic group -
such as
an isoquinoline group. For some applications, preferably the guanidine group
is
attached to said hydrocarbyl group. For some applications, the agent comprises
at
least the one of said cyclic groups linked to another hydrocarbyl group, which
other
hydrocarbyl group has an ester group, an acid group or an alkoxy group
thereon.
The agent may contain halo groups. Here, "halo" means fluoro, chloro, bromo or
iodo.
~1
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
The agent may contain one or more of alkyl, alkoxy, alkenyl, alkylene and
alkenylene
groups - which may be unbranched- or branched-chain.
The agent may be in the form of a pharmaceutically acceptable salt - such as
an
acid addition salt or a base salt - or a solvate thereof, including a hydrate
thereof.
For a review on suitable salts see Berge et al, J. Pharm. Sci., 1977, 66, 1-
19.
The I:uPAs may have a reversible or irreversible action.
t0
Reported irreversible inhibitors generally rely on forming a covalent bond
with the
active site serine (Ser-195) which forms part of the catalytic triad of
urokinase.
Camostat (FOY-05) and its more plasma stable metabolite (FOY-251 ) are potent
trypsin inhibitors which were found to inhibit urokinase irreversibly at
nanomolar
concentrations. Arginyl chloromethylketones also bind and inactivate urokinase
with
Glu-Gly-Arg-CH~CI being the best inhibitor. Cyclic peptide
(methyl)phenylsulfonium
(1) inhibits urokinase along with bovine trypsin and, to a lesser degree t-PA.
HzN\ /N / HzN\ /N
~NH \ I O ,, O Me ~NH \ I O /
I
O \ , O~N~Me O \ I COzF
I IO
FOY-305 FOY-251
0
Me~s~ I \ Gly)4
/ /
\ I HN NH
N
N-
H N
1
H
Hz
The benzothiazole ketone MOL-174 is a potent inhibitor of thrombin which also
demonstrates affinity for urokinase. The peptidic boronate (2) is a
competitive
inhibitor of urokinase. Phenylalanine derived structues (e.g. 3) were also
shown to
inhibit urokinase. CVS-3083 is a potent inhibitor of urokinase. CVS-3083 is an
~2
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
arginyl aldehyde which acts as a transition state mimic by forming a
reversible
covalent bond with Ser-195. Plasma kallikrein selective inhibitor (PKSI-527)
weakly
inhibits urokinase.
H O O OHO~B~C7H
H''° N S H
N~ ~ ~ / O N N '~~~N~S~
O O N~ ~ H
NH2
NH
H~N~NH
MOL-174 2
HZN
!OH O
O
S~ ~N~ N NH
H lpl Me H H OH
3 CVS-3083
w
H
N
N
H2N H O I / C02H
PI~SI-527
Following the discovery of E-aca, a number of aromatic and heterocyclic
amidines
were reported as urokinase inhibitors (e.g. 4-9). Bis-(5-amidino-
1o benzimidazolyl)methane (BABIM; 8) was one of the more potent, but was
poorly
selective over other trypsin-like serine proteases.
Another inhibitor that may be used is Nafamostat (FUT-175) which can inhibit
various
serine proteases, including urokinase. However, for some embodiments the
inhibitor
is not Nafamostat since the selectivity may not be great as desired for some
applications.
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NH NH
HzN / I HzN / /
\ NH \ \ O \
z
4 5
NH NH NH
H N / I / I ~NHz HZN / / I OH
\ \ \ \ O~O \
O ~ / NHz
NH
6 7
H H NH
HN ~ ~ N N N N ~ ~ NH HzN ~/' 1
HZN NHz
g 9
Aromatic guanidines have also been reported as urokinase inhibitors. The
diuretic
drug amilorideT"" is an inhibitor of urokinase. Simple phenyl guanidines such
as 4-
chloro and 4-(trifluoromethyl)phenylguanidine (10 and 11 respectively) are
selective
inhibitors of urokinase.
O NH HzN- -N / HzN\ rN
CI N
tNi NHz NH \ I NH
w CI CF5
HZN N NHz
Amiloride - 10 11
Bridges et al. reported a series of benzothiophenes and thienothiophenes as
to urokinase inhibitors [see EP-A-0568289j. Compounds of formula I were
mentioned,
e.g. B-428 (la) and B-623 (Ib).
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WO 01/49309 PCT/IB00/01935
R2 R1 I
R3 N-R6 NH
\ i ~ ~ \
R4 / S~ 'N-R~ / S NHz
R5
I B-4?8 (Ia) B-62~ (Ib)
Specific examples are: 4-iodobenxo[b]thiophene-2-carboxamidine (la); 4-[5-(4-
carboxamidinophenyl)fur-2-yl]benzo[b]thiophene-2-carboxamidine; 4-[EIZ-2-
(benzo-
1,3-dioxolan-5-yl)ethenyl]benzo[b]thiophene-2-carboxamidine (/b); and 4-
[(benzo-
1,3-dioxolan-5-yl)ethynyl]benzo[b]thiophene-2-carboxamidine.
Tanaka et al. reported a series of 4,5,6,7-tetrahydrobenzo[b]thiophenes
as.urokinase
inhibitors [see WO-A-98/11089]. Compounds of the Formula II, e.g. /la, were
mentioned.
l0
R1 Me
I
A~X \ NH NH
~~N-R3 NH
R2 Y 'S z
II IIa
A specific example is: 2-amidino-4-n-butyl-4,5,6,7-tetrahydrobenzo[b]thiophene
(/la).
Greyer et al. reported a series of 2-amidinonaphthalenes as urokinase
inhibitors [see
WO-A-99105096]. Compounds of formula III were mentioned, e.g. Illa.
i ~N
C NH \N- 'NH NH
W NHz I ~ ~ NHz
N / /
A z
H2N ~ / O
III e.g. IIIa
~s
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WO 01/49309 PCT/IB00/01935
Specific examples are: 6-(aminoiminomethyl)-N-[4-(aminomethyl)phenyl]-4-(2-
pyrimidinylamino)-2-naphthalenecarboxamide (/!la); 6-(aminoiminomethyl)-N-[4-
(hydroxymethyl)phenyl]-4-(2-pyrimidinylamino)-2-naphthalenecarboxamide; 6-
(aminoiminomethyl)-N-phenyl-4-(2-pyrimidinylamino)-2-naphthalenecarboxamide;
and methyl [7-(aminoiminomethyl)-3-[[[4-(aminomethy!)phenyl]amino]carbonyl]-1-
naphthalenyl]carbamate.
Illig et al. reported heteroaryl amidines, methylamidines and guanidines as
protease
to inhibitors, in particular as urokinase inhibitors jsee WO-A-99!40088].
Compounds of
the general formula IV, e.g. IVa, were mentioned.
R1 R~
NRQ ' \
z"Y X R3 NH
Fi~N
IV e.g. IVa
Specific examples are: 4-[4-(2,5-dimethoxyphenyl)(1,3-thiazol-2-yl)]-5-
methylthiothiophene-2-carboxamidine (IVa); 2-{3-[2-(5-amidino-2-methylthio-3-
thienyl)-1,3-thiazol-4-yl]phenoxy}acetic acid; and 5-methylthio-4-{4-[3-(2-oxo-
2-
piperazinylethoxy)phenyl](1,3-thiazol-2-yl)}thiophene-2-carboxamidine.
Schirlin et al. reported ketone bearing peptidase inhibitors for inhibiting
e.g. urokinase
[see US-A-5849866]. Ketone-bearing inhibitors of generic formula V are new.
Specific urokinase inhibitors include Va.
R1NH-CHR2-C(O)-X H-Glu-Gly-Arg-COOH
V Va
Barber et al. reported isoquinolines as urokinase inhibitors jsee WO-A-
99/20608].
Compounds of formula VI were disclosed, e.g. Vla.
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WO 01/49309 PCT/IB00/01935
R4 R3
R5 \ ~ R2
~N
R6 ~ ~'
R7 R1
VI e.g. VIa
In more detail, the compounds of WO-A-99/20608 are isoquinolinylguanidine
derivatives of formula (I) :-
3
Rs R'
/ / ~ (I)
a \ \ N
R
or a pharmaceutically acceptable salt thereof, wherein
one of R' and R~ is H and the other is N=C(NHz)2 or NHC(=NH)NH2,
.
R3 is H, halogen, C,_6 alkyl optionally substituted by one or more halogen, or
C~_6
alkoxy optionally substituted by one or more halogen,
R4, R5, R6 and R' are each independently H, OH, halogen, C,_6 alkyl optionally
~s substituted by one or more substituents independently selected from halogen
or OH,
C,_6 alkoxy optionally substituted by one or more halogen, CN, CO(C,_6 alkyl
optionally substituted by one or more halogen), (Cm - alkylene)COZRB, (C~ -
alkylene)CN, O(C~ - alkylene)CN, O(Cn- alkylene)COZRB, (Cm -
alkylene)CONR9R'°,
(Cm - alkylene)NR9COR'°, O(C~ - alkylene)CONR9R'°, (Cm -
alkylene)NR9SOzR",
(Cm - alkylene)S(O)PR", (Cm - alkylene)SO~NR9R'°, CH=CHCORe,
CH=CHCONR9R'°, CH=CHS02R8, CH=CHS02NR9R'°, CH=CHS02aryl, or
a group of formula X-aryl or X-het,
or, where two of R4, R5, R6 and R' are attached to adjacent carbon atoms, they
can
be taken together to form an -O(C~ - alkylene)O- moiety,
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WO 01/49309 PCT/IB00/01935
R$ is H, C~_6 alkyl optionally substituted by one or more halogen, or
aryl(C,_6
alkylene),
R9 and R'° are each independently H, C,_6 alkyl optionally substituted
by one or more
halogen, aryl(C,_6 alkylene), aryl, heteroaryl or heteroaryl(C,_6 alkylene),
or R9 and R'° may be linked together by an alkylene moiety to form,
with the atoms to
which they are attached, a 4- to 7-membered ring optionally incorporating an
additional hetero-group selected from an O or S atom or a NR'Z group,
to R" is aryl, heteroaryl, or C,_6 alkyl optionally substituted by one or more
halogen,
R'2 is H, C,_6 alkyl optionally substituted by one or more halogen, or CO(C,_6
alkyl
optionally substituted by one or more halogen),
l5 X is a direct link, C~ - alkylene, O, (C~ - alkylene)O, O(C~ - alkylene),
CH(OH), C(C,_6
alkyl)OH, CO, S(O)P(Cm - alkylene), (Cm - alkylene)S(O)P, CH=CH, or C--_C,
"aryl" is phenyl or naphthyl optionally substituted by one or more
substituents
independently selected from halogen, C,_6 alkyl optionally substituted by one
or more
2o substituents independently selected from halogen and OH, C~_6 alkoxy
optionally
substituted by one or more halogen, CN, O(C~- alkylene)CN, (C~- alkylene)CN,
CO(C,_6 alkyl optionally substituted by one or more halogen), (Cm -
alkylene)COaR'3,0(C~- alkylene)CO~R'3, (Cm - alkylene)CONR'4R'S, (Cm -
alkylene)NR'4COR'S, O(C~ - alkylene)CONR'4R'S, (Cm - alkylene)S(O)PR'3, (Cm -
25~ alkylene)SO2NR'4R'S, (Cm - alkylene)NR'4S02R's, CH=CHSOZR'3,
CH=CHSOZNR'4R'S, CH=CHSOZaryl', CH=CHCOR'3, and CH=CHCONR'~R'S,
"heteroaryl" is an optionally benzo-fused 5- or 6-membered heterocyclic group
linked
by any available atom in the heterocyclic or benzo-ring (if present), which
30 heterocyclic group is selected from dioxolyl, furyl, thienyl, pyrrolyl,
oxazolyl, thiazolyl,
isoxazolyl, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, thiadiazolyl,
triazolyl,
tetrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl and pyranyl,
said "heteroaryl" group being optionally substituted by one or more
substituents
35 independently selected from halogen, C,_6 alkyl optionally substituted by
one or more
substituents independently selected from halogen or OH, C,_g alkoxy optionally
substituted by one or more halogen, CN, O(C~- alkylene)CN, (C"- alkylene)CN,
~s
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WO 01/49309 PCT/IB00/01935
CO(C,_6 alkyl optionally substituted by one or more halogen), (Cm -
alkylene)C02R'3,
O(C~ - alkylene)COaR'3, (Cm - alkylene)CONR'4R'S, (Cm - alkylene)NR'4COR'S,
O(C
- alkylene)CONR'4R'S, (Cm - alkylene)NR'4SOzR'6, (Cm - alkylene)S(O)PR'3, (Cm -
alkylene)SOZNR'4R'S, CH=CHCOR'3, CH=CHCONR'4R'S, CH=CHS02R'3,
CH=CHS02NR'4R'S, or CH=CHSOZaryl',
"het" is an optionally benzo-fused 5- or 6-membered heterocyciic group linked
to the
"X" moiety by any available atom in the heterocyclic or benzo-ring (if
present), which
heterocyclic group is selected from dioxolyl, dioxolanyl, furyl, thienyl,
pyrrolyl,
oxazolyl, oxazinyl, thiazinyl, thiazolyf, isoxazolyl, isothiazolyl,
imidazolyl, pyrazo(yI,
oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl,
pyridazinyl, pyrazinyl
and pyranyl,
or a fully unsaturated, partially or fully saturated analogue thereof,
such "het" group being optionally substituted by one or more substituents
independently selected from halogen, C,_6 alkyl optionally substituted by one
or more
substituents independently selected from halogen and OH, C,_6 alkoxy
optionally
substituted by one or more halogen, CN, O(C~- alkylene)CN, (C~- alkylene)CN,
2o CO(C~_6 alkyl optionally substituted by .one or more halogen), (Cm -
alkylene)COzR'3,
O(C~- alkylene)CO~R'3,(Cm - alkylene)CONR'4R'S, (Cm - alkylene)NR'4COR'S, O(C
alkylene)CONR'4R'S, (Cm - alkylene)NR'4S02R'6, (Cm - alkylene)S(O)PR'3, (Cm -
alkylene)SOZNR'4R'S, CH=CHCOR'3, CH=CHCONR'4R'S, CH=CHS02R'3, .
CH=CHS02NR'4R'S, and CH=CHSO~aryl',
"aryl'" is phenyl or naphthyl optionally substituted by one or more
substituents
independently selected from halogen, C~_6 alkyl optionally substituted by one
or more
substituents independently selected from halogen or OH, C~_6 alkoxy optionally
substituted by one or more halogen, CN, O(C~- alkylene)CN, (C~- alkylene)CN,
3o CO(C,_6 alkyl optionally substituted by one or more halogen), (Cm -
alkylene)COZR'3,
O(C~- alkylene)C02R'3, (Cm - alkylene)CONR'4R'S, (Cm - alkylene)NR'4COR'S, O(C
- alkylene)CONR'4R'S, (Cm - alkylene)S(O)pR'3, (Cm - alkylene)S02NR'4R'S, (Cm -
alkylene)NR'4SO~R'6, CH=CHS02R'3, CH=CHSOZNR'4R'S, CH=CHCOR'3, and
CH=CHCONR'4R'S,
R'3 is H, C,_6 alkyl optionally substituted by one or more halogen, or
aryl2{C,_6
alkylene),
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R'4 and R'S are each independently H, C,_6 alkyl optionally substituted by one
or
more halogen, arylz(C,_6 alkylene), aryh, heteroaryl' or heteroaryl'~(C,_6
alkylene),
or R9 and R'° may be linked together by an alkylene moiety to form,
with the atoms to
which they are attached, a 4- to 7-membered ring optionally incorporating an
additional hetero-group selected from an O or S atom or a NR's group,
R'6 is arylz, heteroaryl', or C,_s alkyl optionally substituted by one or more
halogen,
to "aryl2" is phenyl or naphthyl optionally substituted by one or more
substituents
independently selected from halogen, C,_6 alkyl optionally substituted by one
or more
substituents independently selected from halogen or OH, C,_6 alkoxy optionally
substituted by one or more halogen, CN, O(C~- alkylene)CN, (C~- alkylene)CN,
or
CO(C,_6 alkyl optionally substituted by one or more halogen),
t5
"heteroaryl'" is an optionally benzo-fused 5- or 6-membered heterocyclic group
linked
by any available atom in the heterocyclic or benzo-ring (if present), which
heterocyclic group is selected from dioxolyl, furyl, thienyl, pyrrolyl,
oxazolyl, thiazolyl,
isoxazolyl, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, thiadiazolyl,
triazolyl,
2o tetrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl and pyranyl,
said "heteroaryl'" group being optionally substituted by one or more
substituents
independently selected from halogen, C,_6 alkyl optionally substituted by one
or more
substituents independently selected from halogen or OH, C,_6 alkoxy optionally
25 substituted by one or more halogen, CN, O(C~ - aikylene)CN, (C~ -
alkylene)CN, or
CO(C,_6 alkyl optionally substituted by one or more halogen),
wherein the "C-alkylene" linking groups in the definitions above are linear or
branched, and are optionally substituted by one or more (C,_6 alkyl optionally
30 substituted by one or more halogen) groups,
m is an integer from 0 to 3,
n is an integer from 1 to 3,
and
35 p is an integer from 0 to 2.
so
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WO 01/49309 PCT/IB00/01935
The most preferred compounds are selected from:
(4-chioro-7-(2-methoxyphenyl)isoquinolin-1-yi}guanidine (Vla);
(4-chlore-7-(3-methoxyphenyi)isoquinolin-1-yl)guanidine;
(4-chlore-7-(4-methoxyphenyl)isoquinolin-1-yl)guanidine;
(4-bromo-7-(3-methoxyphenyl)isoquinolin-1-yl)guanidine;
(4-bromo-7-(4-methoxyphenyl)isoquinolin-1-yl)guanidine;
(4-chlore-7-(a-hydroxybenzyl)isoquinolin-1-yl)guanidine;
(4-chlore-7-(3-carboxyphenyl)isoquinolin-1-yl)guanidine;
1-guanidine-7-sulphamoylisoquinoline;
1-guanidine-7-phenylsulphamoyiisoquinoline;
4-chlore-1-guanidine-7-sulphamoylisoquinoline;
4-chioro-7-cyclopentyisuiphamoyl-1-guanidinoisoquinoline;
4-chlore-1-guanidine-7-(1-pyrrolidinosulphonyl)isoquinoline;
4-chlore-1-guanidine-7-morpholinosulphonylisoquinoline;
~5 4-chlore-1-guanidine-7-[(N-methyipiperazino)sulphonyl]isoquinoline;
4-chlore-1-guanidine-7-(phenylsulphanyl}isoquinoline; and
4-chlore-1-guanidine-7-(phenylsulphonyl)isoquinoline.
Another preferred compound disclosed in WO-A-99/20608 for use in the present
invention is (4-chloro-7-(2, 6-dimethoxyphenyl)isoquinolin-1-yl)guanidine -
viz:
m
oMe I w w
rN
I ~ OMe NYNH=
INH2
which can be prepared by the method reported in WO-A-99!20608 (see
Example 39).
Another preferred compound disclosed in WO-A-99120608 for use in the present
invention is [7-(3-Carboxyphenyl)- 4-chloroisoquinolin-1-yl]guanidine - viz:
i
~1
/ iN
I / N\'NHZ
COZH TNH2
which can be prepared by the method reported in WO-A-99/20608 (see
Example 55).
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Suitable I:uPA compounds for use in the present invention are disclosed in PCT
patent application No. PCT/IB99/01289 (incorporated herein by reference),
which
was filed on 15 July 1999 (published as WO-A-00/05214). Claiming priority
dates of
24 July 1998 and 16 April 1999 Some relevant teachings of that patent
application
are provided herein (see the section titled "PCS9494 Compounds")
Preferred compounds from WO-A-00/05214 are presented as Examples 32b therein
(hereinafter referred to as "compound 5214". The formula for Compound 5214 is
presented in the Examples section. Another preferred compound from WO-A
00!05214 is Example 34b therein.
Other suitable I:uPA compounds for use in the present invention are disclosed
in GB
patent application No. 9908410.5 which was ~file'd on 13 April 1999
(incorporated
herein by reference) and in US patent application No. 09/546410 (incorporated
t5 herein by reference) and European patent application No. 00302778.6
(incorporated
herein by reference) and in Japanese patent application No. 2000-104725
(incorporated herein by reference). Some relevant teachings of those patent
applications are provided herein (see the section titled "PCS9482 Compounds").
UROKINASE INHIBITOR ASSAY PROTOCOL
The following presents a protocol for identifying one or more agents capable
of acting
as an I:uPA that would be suitable for use in the composition of the present
invention.
Materials
uPA (urokinase type plasminogen activator). High molecular weight human
urokinase from urine, 3000 IU/vial (Calbiochem, 672081) reconstituted in HZO
to
give 30000 IU/ml stock and stored frozen (-18°C). S-2444, chromogenic
urokinase
3o substrate, 25 mg/vial (Quadratech, 820357) was reconstituted in H20 to give
3 mM
stock and stored at 4°C. Human tPA stimulator (Chromogenix 822130-63/9)
was
reconstituted to 1 mg/ml in buffer; and used fresh. Human tPA (one chain) 10
p.g/vial
(Chromogenix, 821157-039/0) was reconstituted to 4 p.g/ml in buffer and used
fresh.
S-2288, chromogenic substrate for serine proteases, 25 mg/vial (Chromogenix,
820852-39) was reconstituted in HZO to give 10 mM stock and stored at
4°C. Human
plasmin, 2 mg/vial (Quadratech, 810665) was reconstituted to 1 mg/ml in buffer
and
s2
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WO 01/49309 PCT/IB00/01935
stored frozen (-18°C). Chromozym-PL (Boehringer Mannheim, 378 461), 1
mM stock
in buffer prepared fresh.
Methods
Chromogenic assays are performed to measure uPA, tPA and plasmin activity and
inhibition of this activity by serine protease inhibitors.
IC;o and K; values for compounds are calculated by incubation of 33 IU/ml uPA
with
0 0.18mM 52444 (substrate) and various compound concentrations, all diluted in
uPA
assay buffer (75 mM Tris, pH 8.1, 50 mM NaCI). A pre-incubation of compound
with enzyme is carried out for I S minutes at 37°C, followed by
substrate addition and
further incubation for 30 minutes at the same temperature. The final assay
volume is
200p,1. Absorbance is read at 405nM following pre-incubation (background, time
t5 zero measurement) and following the 30 minute incubation with substrate
using a
SPECTRAMax microplate reader (Molecular Devices Corporation. Background
values, are subtracted from the final absorbance values. Percentage inhibition
is
calculated and plotted against compound concentration to generate IC;o values.
The
enzymatic K; is calculated from the known Km of the substrate, 90 pM, using
the
2o equation K;=IC;o/((1+([S]/Km)).
The method for analysis of tPA inhibition is similar to that for uPA
inhibition. The
assay utilises f nal concentrations of tPA of 0.4 ~g/ml with 0.1 mg/ml tPA
stimulator,
0.4 mM S2288 (substrate) and various concentrations of inhibitors, made up in
uPA
25 assay buffer. Pre-incubation is carried out with compound, enzyme and
enzyme
stimulator, as for uPA, prior to the incubation with substrate. Incubation
time is 60
minutes at performed at 37°C. Data analysis is identical to that
described above for
uPA, using a known Km for tPA of 250 ~M.
3o Plasmin inhibition is assayed by incubating human plasmin at 0.7 p,g/ml
with 0.2 mM
Chromozym-PL (substrate) and various concentrations of inhibitors in uPA assay
buffer. Pre-incubation is carried out as for uPA and the incubation is
performed at
37°C for 30mins. Data manipulation and percentage inhibition is
calculated as for
uPA, using a known Km for plasmin of 200~,M.
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Analysis
The following Table presents numerical values as to what would constitute an
agent
that would not work as an i:uPA in accordance with the present invention (i.e.
a "fail")
and what would constitute an agent that would work as an I:uPA in accordance
with
the present invention (i.e. a "pass"). In addition, the following Table
presents
numerical values as to what would constitute an agent that would work very
well as
an I:uPA in accordance with the present invention (i.e. a "very good").
to
K; for uPA Selectivity over
inhibition of tPA
and
plasmin
Pass <100 nM AND >300-fold
Fail >100 nM OR <300-fold
Very good <40 nM AND >1,000-fold,
preferably >1,500-fold,
preferably >2,000-fold,
preferably >2,500-fold
MMP INHIBITOR
A component of the composition of the present invention may be an inhibitor of
an
MMP that has a deleterious effect on wound healing of damaged tissue.
Typically,
the I:MMP will be capable of being identified as being an I:MMP by an MMP
assay -
such as the assay protocol presented herein
2o Thus, in one aspect, the present invention relates to a method of enhancing
the
healing of chronic dermal ulcers, including venous stasis ulcers, diabetic
ulcers and
decubitus ulcers (or pressure sores), by treating the patient with a
combination of a
selective inhibitor of particular MMPs and a growth factor. This combination
therapy
is more effective than treatment with the individual agents.
The inhibitors of the adverse MMP can either be applied topically or
administered
orally, depending on the properties of the inhibitor and the way in which they
are
formulated.
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Thus, according to one aspect of the present invention, the composition may
comprise an I:MMP - such as a selective MMP inhibitor - and a growth factor;
wherein said MMP has an adverse effect on wound healing in damaged tissue.
With
the co-administration of these two components a more profound efficacy can be
achieved than by administration of either a growth factor or a MMP inhibitor
alone.
Here, efficacy may be measured by the standard of the FDA in this area, namely
the
time to closure of chronic dermal ulcers under conditions of best care and
compared
to best care alone.
(n one preferred aspect, topical formulations of selective MMP inhibitors can
be co-
administered with topically administered growth factors, such as PDGF, either
by
physically mixing the substances and using a formulation which releases both
substances into the damaged tissue, such as the wound, environment, or by
applying
~5 one substance at a time and using a treatment protocol which separates
application
of the agents. Alternatively, combined treatment can be achieved using an
orally
administered MMP inhibitor with topical application of a growth factor.
We believe that the use of certain I:MMP when co-administered with growth
factors is
2o very advantageous and was, also, unexpected and unpredictable. In this
respect,
there are many literature reports show that MMPs are required as part of the
cellular
response downstream from growth factor receptors. We have determined that,
whilst
this may be the case, the protective effects of a selective MMP inhibitor on
growth
factors predominates and this provides the scientific basis for the invention.
In accordance with the present invention, the I:MMP may be applied topically
mixed
with the growth factor or the I:MMP may be applied topically but at a
different time to
the growth factor or the I:MMP may be administered orally and the growth
factor may
be applied topically.
A number of I:MMPs are known.
By way of example, naturally occurring proteinacious inhibitors that exist
include
Tissue Inhibitors of Metalloproteinases (TIMPs) - see Bode, W., Fernandez-
Catalan,
C., Grams, F., Gomis-Ruth, F.X., Nagase, H., Tschesche, H., Maskos, K. (1999)
Ann.N.Y. Acad. Sci. 878, 73-91 and Vaalamo, M., Leivo, T., Saarialho-Kere, U.
(1999) Human Pathology 30 (7), 795-802. These include TIMP-1, TIMP-2, TIMP-3
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
and TIMP-4.
In addition, synthetic inhibitors of MMP exist. These synthetic inhibitors
will typically
be organic compounds. Typically the organic compounds will comprise two
hydrocarbyl groups linked by a -C(O)N(H)- group. Here, the term "hydrocarbyl
group" means a group comprising at least C and H and may optionally comprise
one
or more other suitable substituents. Examples of such substituents may include
halo-, alkoxy-, vitro-, an alkyl group, a cyclic group etc. In addition to the
possibility
of the substituents being a cyclic group, a combination of substituents may
form a
to cyclic group. If the hydrocarbyl group comprises more than one C then those
carbons need not necessarily be linked to each other. For example, at least
two of
the carbons may be linked via a suitable element or group. Thus, the
hydrocarbyl
group may contain hetero atoms. Suitable hetero atoms will be apparent to
those
skilled in the art and include, for instance; sulphur, nitrogen and oxygen.
For some
is applications, preferably the agent comprises at least one cyclic group,
wherein that
cyclic group is a polycyclic group, preferably not being a fused polycyclic
group.
The agent may contain halo groups. Here, "halo" means fluoro, chloro, bromo or
iodo.
The agent may contain one or more of alkyl, alkoxy, alkenyl, alkylene and
alkenylene
groups - which may be unbranched- or branched-chain.
The agent may be in the form of a pharmaceutically acceptable salt - such as
an
?5 acid addition salt or a base salt - or a solvate thereof, including a
hydrate thereof.
For a review on suitable salts see Berge et al, J. Pharm. Sci., 1977, 66, 1-
19.
Preferably the I:MMP inhibits MMP-3 and/or MMP-13. More preferably, the I:MMP
is
selective vs MMP-1 andlor MMP-2 andlor MMP-9 and/or MMP-14.
Some known MMP inhibitors conform to the following general formula:
o P,' o
NH~ , P3'
X ~! ~ - NH
A O pi'
"GENMMP"
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WO 01/49309 PCT/IB00/01935
wherein "A" is known as the "alpha" group and XCO is is a zinc-binding group
such as a carboxylic acid or hydroxamic acid moiety.
In addition, or in the alternative, a Large number of known synthetic
inhibitors of
MMPs generally conform to one of the generic structures in Scheme presented
below, and.contain a zinc-binding group (ZBG) which co-ordinates with the
catalytic
zinc atom of the MMP active site. The ZBG can typically be carboxylic acids,
hydroxamic acids, thiols, phosphiriates and phosphonates. Reference can be
made
to to recent reviews for examples of these classes (see Whittaker, M.; Floyd,
C.D.;
Brown, P.; Gearing, A.J.H. Design and Therapeutic Application of Matrix
Metalloproteinase Inhibitors. Chem. Rev. 1999, 99, 2735-2776; and Michaelides,
M.R.; Curtin, M.L. Recent Advances.in Matrix Metalloproteinase Inhibitor
Research.
Current Pharmaceutical Design, 1999, 5, 787-819).
SCHEME
R3 i5 O R3
R4 R1 R3 i4 R4
ZBG N~ ZBG ,R4 RZ N ZBG RS
R1 R R1 N ZBG \R5 R1
R2 O R2 R3 O R2 O
R3 R3 R1 R1 R3
R4 R2 R2 I
ZBG N~S\R4 ZBG S,RS ZBG~S~R3 ZBG~g\N~R4
R1~ I~\O R1 110 II~O !l O
R2 O R2 O O O
HO~ ~O HO ,O
ZBG~ - ~ HST /?~ jF~
HO NHOH HO R
Examples of such suitable I:MMPs are mentioned in WO-A-90/05719, WO-A-
99135124, WO-A-99129667, WO-A-96!27583, WO-A-99/07675, and WO-A-98/33768.
Preferred inhibitors for use in the present invention are described in WO-A-
90/05719,
WO-A-99/35124, WO-A-99129667 and PCTIIB00/00667 filed 18 May 2000.
A preferred compound from WO-A-90/05719 is compound 5719 - the structural
formula for which is presented in the Examples section.
s~
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A preferred compound from WO-A-99/29667 is that presented as Example 66
therein
("compound 9470"). The structural formula of Compound 9470 is presented in the
Examples section.
A preferred compound from WO-A-99/35124 is that presented as Example 15
therein
("compound 9454") - the structural formula for which is presented in the
Examples
section.
l0 Another preferred compound is Example 14 of WO-A-99/35124.
Other preferred compounds are disclosed in PCTlIB00/00667 - in particular
Example
1, Example 2 and Example 3. A very preferred compound from PCTlIB00/00667 is
Example 1.
~5
The inhibitor compounds of WO-A-99/35124 may be presented by the following
general formula:
R~ ~Ar
y
Rs
Rs
O O
NH~
NH
R~ O Rz
cn
and pharmaceutically acceptable salts thereof, wherein
R' is H, OH, C,~ alkyl, C,~ alkoxy, or CZ~ alkenyl,
Rz is C,_6 alkyl optionally substituted by fluoro, indolyl, imidazolyl,
S02(C~.~ alkyl), C5_7
cycloalkyl, '
or by an optionally protected OH, SH, CONH2, COZH, NH2 or NHC(=NH)NHa group,
CS_~ cycioalkyi optionally substituted by Ci_6 alkyl,
ss
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WO 01/49309 PCT/IB00/01935
or is benzyl optionally substituted by optionally protected OH, C,_6 alkoxy,
benzyloxy
or benzylthio,
wherein the optional protecting groups for said OH, SH, CONHa, NHZ and
NHC(=NH)NHZ groups are selected from C,_6 alkyl, benzyl, C,_6 alkanoyl,
and where the optional protecting groups for said CO~H is selected from C,_s
alkyl or
benzyl,
R3, RS and R6 are each independently selected from H and F,
R4 is CH3, CI or F,
X is HO or HONH,
Y is a direct link or O,
l5
Z is either a group of formula (a):
Rio
' JH
R"
where R'° is C,_4 alkyl, C,~ alkoxymethyl, hydroxy(C2~ alkyl),
carboxy(C,~ alkyl) or
20 (amino or dimethylamino)C2~ alkyl,
and R" is phenyl, naphthyl or pyridyl
optionally substituted by up to three substituents indeperidently selected
from halo
and methyl;
or (b)
_ / Ria
H
R'4 is H, OH, CH3 or halo,
Ar is a group of formula (c), (d) or (e):
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Ra
R' S Ria R~ S Ris
~A
"' R~ Ra /"y~ .R9
R9
(c) (d) (e)
wherein
A is N or CR'a,
B is N or CR'3,
provided that A and B are not both N,
R' and R9 are each independently H or F,
Ra, R'2 and R'3 are each independently H, CN, C~_6 alkyl, hydroxy(C~_6 alkyl),
1o hydroxy(C~_6)alkoxy, C,_6 alkoxy(C~_6)alkoxy,(amino or dimethylamino)C~_6
alkyl,
CONH2, OH, halo, C~_6 alkoxy, (C,_6 alkoxy)methyl, piperazinylcarbonyl,
piperidinyl,
C(NH2)=NOH or C(=NH)NHOH, with the proviso that at least two of R8, R'2 and
R'3
are H.
As indicated preferred compounds from WO-A-99/35124 are Example 15
(hereinafter
referred to as "compound 9454") and Example 14 therein. The formula for
Compound 9454 is presented in the Examples section.
Suitable I:MMP compounds for use in the present invention are also disclosed
in GB
2o patent application No. 9912961 which was filed on 3 June 1999 (incorporated
herein
by reference), US patent application No. 60/169578 filed on 8 December 1999
(incorporated herein by reference) and PCT patent application No.
PCT/IB00/00667
filed on 18 May 2000 (incorporated herein by reference). Some relevant
teachings of
those patent applications are provided herein (see the section titled
"PCS10322
Compounds").
Examples of preferred inhibitors for use in the present invention are shown
below.
Inhibitors of MMPs can either be applied topically or administered orally,
depending
on the properties of the inhibitor and the way in which they are formulated.
CA 02395487 2002-06-26
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Ex. Name Structure S nthesis
1 (3R)-3-({[(1 S)-2,2-Dimethyl-1-~H a See
'
I
({[(1R)-1- a Example
w_ 1
phenylethyl]amino}carbonyl)propyl]I of
amino}carbonyl)-6-[(3-methyl-4-~ WO-A-
"
phenyl)phenyl] hexanoic O H O 99/35124
acid ~ CHI
II
N~
HO
H I
O /rCH a
H
C
5
CHI
2 N1-[(1S)-2,2-Dimethyl-1-({[(1R)-1-cH a See
'
I
phenylethyl]amino}carbonyl)propyl]-, Example
w 3
v
(N4-hydroxy)-(2R)-2-{3-[3-methyl-~ I of
"
(4- WO-A-
phenyl)phenyl]propyl}butanediamid~ H OI' CH3 99/35124
N~
B. HONH
H
O ~CH a
H
C
~
O
CH
3 (3R)-3-({[(1S)-2,2-Dimethyl-1-cH~ a See
I
({[(1 S)-2-methoxy-1- , ~ Example
phenylethyl]amino}carbonyl)-~ I 14 of
propyl]amino}carbonyl)-6-[(3- WO-A-
methy!-4-pheny!)pheny!] o H o[[ cHzocH, 99/35124
hexanoic N~
~
acid I ~
H
HO
O e)'CH a
C
H
a
CHI
4 (3R)-3-({[(1 S)-2,2-Dimethyl-1-cH a See
3
I
({[(1S)-2-methoXy-1- ~ ple
ocH, E
phenylethyl]amino}carbonyl)propyl]\ 1 5 of
amino}carbonyl)-6-(3'-methoxy-2- WO-A-
methylbiphen-4-yl)hexanoicO H OII CHZOCH3 99/35124
acid N~
'
HO
I \
H
H C ~ CHa a
(2R)-N1-[(1S)-2,2-Dimethyl-1-cH a See
'
I
({[(1S)-2-methoxy-1- , Example
~
phenylethyl]amino}carbonyl)propyl]-~J 16 of
2-{3-[(3-methyl-4- WO-A-
phenyl)phenyl]propyl}-(N4-o H oII ~HZOCH~ 99/35124
N
.
hydroxy)butanediamide. HOHN
~H
I \
~C C CHy
6 N-Hydroxy 2-[(4-{4-[6-(2-Me aII See
hydroxyethoxy)pyridin-2-yl]-3-w ~N~O~..OH Example
1
methylphenyl}piperidin-1-I a of
yl)sulphonyl]-2-methylpropanamide~SN 0667//B00/0
HoHN
M
e Me
7 N-Hydroxy 2-{[4-(4-{6-[2-Me ' ~ See
N~0~OMe Example
2
(methoxy)ethoxy]pyridin-2-yl}-3-r ~ of
methylphenyl)piperidin-1-HoH ~sN PCTIIB00/0
~ne the 0667
yl]sulphonyl}-2-
methylpropanamide
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8 N-Hydroxy 4-{[4-(4-{6-[2- See
",~ ~l Example 3
hydroxyethoxy]pyridin-2-yl}-3- ~ ~ ~N~o'~o" pCT/IB00/0
methylphenyl)piperidin-1-
o ,N 0667
yl]sulphonyl}tetrahydro-2H-pyran-
4-carboxamide o0
(Ex. = Example)
MMP INHIBITOR ASSAY PROTOCOL
The following presents a protocol for identifying one or more agents capable
of acting
as an I:MMP that would be suitable for use in the composition of the present
invention.
t0 Materials
Enzymes
All of the following enzymes were made by standard techniques in the art:
Human MMP-l,catalytic domain, initial stock concentration 1 pM
Human MMP-2, catalytic domain, initial stock concentration 6.94 p,M
Human MMP-3, catalytic domain, initial stock concentration 36 p,M
Human MMP-9, catalytic domain, initial stock concentration 4.565 ~M
Human MMP-14, catalytic domain, initial stock concentration 10 p.M.
Substrates
MMP-1 substrate (Bachem; Cat.No.M-2055) reconstituted in dimethylsulphoxide
(DMSO) to give a 1 mM stock and stored frozen (-18°C). MMP-2, MMP-3,
MMP-9
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substrate (Neosystem Laboratories; Cat.No.SP970853) reconstituted in DMSO to
give
a 1 mM stock and stored frozen (-18°C). MMP-14 substrate (Bachem; Cat.
No. M-
1895) reconstituted in DMSO to give a l mM stock and stored frozen (-
18°C).
Assay Buffers
For MMP-1 the assay buffer used is 50 mM Tris, 200 mM NaCI, 5mM CaCl2, 20 ACM
ZnCl2, 0.05% (w/v) Brij 35, pH 7.5. For MMP-2, MMP-3 and MMP-9 the assay
buffer used is 100 mM Tris, 100 mM NaCI, 10 mM CaCl2, 0.05% (w/v) Brij 35, pH
7.5. For MMP-14 the buffer used is 50 mM Tris, 100 mM NaCI, 10 mM CaCh,
0:25%(w/v) Brij 35, pH 7.5.
Other Materials
t5 APMA (Sigma; Cat.No. A-9563) reconstituted in DMSO to give a 20 mM stock
and
stored at 4oC. Trypsin (Sigma;T-1426) reconstituted in assay buffer (50 mM
Tris, pH
7.5, 100 mM NaCI, 10 mM CaCl2, 0.25% Brij 35) to give a 0.1 p.g/ml stock.
Trypsin-
chymotrypsin inhibitor, 100mg/vial (Sigma;T-9777) reconstituted in assay
buffer to
give a 0.5p,g/ml stock.
Mefihods
Enzyme Activation
All enzymes are pre-activated at 37°C with aminophenylmercuric acetate
(APMA) or
trypsin before being made up to the final concentrations used in the assay.
MMP-1
(30 nM) is activated with 0.93 mM APMA for 20 minutes, MMP-2 (30 nM) is
activated with I.32 mM APMA for I hour, MMP-3 (I010 nM) is activated with 1.81
mM APMA fox 3 hours, MMP-9 (100 nM) is activated with 2 mM APMA for 2 hours
3o and MMP-14 (900nM) is activated with 0.9 ng/ml trypsin for 25 minutes after
which
4.5 ng/ml trypsin inhibitor is added.
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MMP Assav Protocol
All assays are carried out in black 96-well plates with a final volume of 100
p.1 in each
well. Compounds are dissolved in dimethylsulphoYide (DMSO) to 1 mM. Solutions
are then serially diluted in buffer to give the final concentrations shown.
The addition
of substrate is preceded by an initial pre-incubation of enzyme and inhibitor
at 37°C
for IS minutes. For MMP-2, MMP-3, MMP-9 and MMP-I4 fluorescence is read
every ? minutes at 328nm 7~°~ and 393nm 7~°", for 1 hour using a
Fluorostar
tluorimeter (BMG) with BIOLISE software. For MMP-1 assays the filters used are
t o , 3 ~ Snm ~,e;~ and 440nm ~.°",; fluorescence is read every 2
minutes for 1 hour.
Analysis
The following Table presents numerical values as to what would constitute an
agent
~5 that would not work as an I:MMP3 in accordance with the present invention
(i.e. a
"fail") and what would constitute an agent that would work as an I:MMP in
accordance with the present invention (i.e. a "pass"). In addition, the
following Table
presents numerical values as to what would constitute an agent that would work
very
well as an I:MMP3 in accordance with the present invention (i.e. a "very
good").
K; for MMP of Selectivity over
other
interest MMPs thought to
be
essential for damaged
tissue, such as
wound,
healing processes
Pass <100 nM AND >100-fold
Fail >100 nM OR <100-fold
Very good <40 nM AND >200-fold
preferably >300-fold,
preferably >400-fold,
preferably >450-fold
The above assay protocol may be adapted for other MMP targets.
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OTHER ACTIVE COMPONENTS
The composition of the present invention may also comprise other therapeutic
substances in addition to the growth factor and the inhibitor agent.
ANTIBODY
As indicated, the inhibitor agent for use in the composition of the present
invention
may be one or more antibodies.
to
The "antibody" as used herein includes but is not limited to, polyclonal,
monoclonal,
chimeric, single chain, Fab fragments and fragments produced by a Fab
expression
library. Such fragments include fragments of whole antibodies which retain
their
binding activity for a target substance, Fv, F(ab') and F(ab')2 fragments, as
well as
~5 single chain antibodies (scFv), fusion proteins and other synthetic
proteins which
comprise the antigen-binding site of the antibody. Furthermore, the antibodies
and
fragments thereof may be humanised antibodies, for example as described in US-
A-
239400. Neutralizing antibodies, i.e., those which inhibit biological activity
of the
substance polypeptides, are especially preferred for diagnostics and
therapeutics.
Antibodies may be produced by standard techniques, such as by immunisation
with
the substance of the invention or by using a phage display library.
If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit,
goat,
horse, etc.) is immunised with an immunogenic polypeptide bearing a epitope(s)
obtainable from an identified agent and/or substance of the present invention.
Depending on the host species, various adjuvants may be used to increase
immunological response. Such adjuvants include, but are not limited to,
Freund's,
mineral gels such as aluminium hydroxide, and surface active substances such
as
lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet
hemocyanin, and dinitrophenol. BCG (Bacilli Calmette-Guerin) and
Corynebacterium
parvum are potentially useful human adjuvants which may be employed if
purified the
substance polypeptide is administered to immunologically compromised
individuals
for the purpose of stimulating systemic defence.
Serum from the immunised anima! is collected and treated according to known
procedures. if serum containing polyclonai antibodies to an epitope obtainable
from
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an identifed agent and/or substance of the present invention contains
antibodies to
other antigens, the polyclonal antibodies can be purified by immunoaffinity
chromatography. Techniques for producing and processing polyclonal antisera
are
known in the art. in order that such antibodies may be made, the invention
also
provides polypeptides of the invention or fragments thereof haptenised to
another
polypeptide for use as immunogens in animals or humans.
Monoclonal antibodies directed against particular epitopes can also be readily
produced by one skilled in the art. The general methodology for making
monoclonal
antibodies by hybridomas is well known. Immortal antibody-producing cell lines
can
be created by cell fusion, and also by other techniques such as direct
transformation
of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.
Panels of monoclonal antibodies produced against orbit epitopes can be
screened for
various properties; i.e., for isotype and epitope affinity.
Monoclonal antibodies may be prepared using any technique which provides for
the
production of antibody molecules by continuous cell lines in culture. These
include,
but are not limited to, the hybridoma technique originally described by
Koehler and
Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique
(Kosbor
et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-
2030) and the EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies
and Cancer Therapy, Alan R Liss Inc, pp 77-96). In addition, techniques
developed
for the production of "chimeric antibodies", the splicing of mouse antibody
genes to
human antibody genes to obtain a molecule with appropriate antigen specificity
and
biological activity can be used (Morrison et a! (1984) Proc Natl Acad Sci
81:6851-
6855; Neuberger et al (1984) Nature 312:604-608; Takeda et al (1985) Nature
314:452-454). Alternatively, techniques described for the production of single
chain
antibodies (US Patent No. 4,946,779) can be adapted to produce the substance
specific single chain antibodies.
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening recombinant immunoglobulin libraries or panels of
highly
specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad
Sci 86:
3833-3837), and Winter G and Milstein C (1991; Nature 349:293-299).
Antibody fragments which contain specific binding sites for the substance may
also
be generated. For example, such fragments include, but are not limited to, the
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F(ab')2. fragments which can be produced by pepsin digestion of the antibody
molecule and the Fab fragments which can be generated by reducing the
disulfide
bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may
be
constructed to allow rapid and easy identification of monoclonal Fab fragments
with
the desired specificity (Huse WD et al (1989) Science 256:1275-128 1 ).
GENERAL ASSAY TECHNIQUES
Any one or more of appropriate targets - such as an amino acid sequence and/or
Io nucleotide sequence for a protein that is upregulated in a damaged tissue,
such as a
wound, environment - may be used for identifying an agent capable of
inhibiting the
action of said protein.
The target employed in such a test may be free in solution, affixed to a solid
support,
~ 5 borne on a cell surface, or located intracellularly. The abolition of
target activity or
the formation of binding complexes between the target and the agent being
tested
may be measured.
The assay of the present invention may be a screen, whereby a number of agents
2o are tested. In one. aspect, the assay method of the present invention is a
high
through put screen.
Techniques for drug screening may be based on the method described in Geysen,
European Patent Application 84/03564, published on September 13, 1984. In
25 summary, large numbers of different small peptide test compounds are
synthesized
on a solid substrate, such as plastic pins or some other surface. The peptide
test
compounds are reacted with a suitable target or fragment thereof and washed.
Bound entities are then detected - such as by appropriately adapting methods
well
known in the art. A purified target can also be coated directly onto plates
for use in a
3o drug screening techniques. Alternatively, non-neutralising antibodies can
be used to
capture the peptide and immobilise it on a solid support.
This invention also contemplates the use of competitive drug screening assays
in
which neutralising antibodies capable of binding a target specifically compete
with a
35 test compound for binding to a target.
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Another technique for screening provides for high throughput screening (HTS)
of
agents having suitable binding affinity to the substances and is based upon
the
method described in detail in WO 84!03564.
It is expected that the assay methods of the present invention will be
suitable for both
small and large-scale screening of test compounds as well as in quantitative
assays.
In one preferred aspect, the present invention relates to a method of
identifying
agents that selectively inhibit one or more protease proteins that are
upregulated in a
t o damaged tissue, such as a wound, environment.
REPORTERS
A wide variety of reporters may be used in the assay methods (as well as
screens) of
l5 the present invention with preferred reporters providing conveniently
detectable
signals (eg. by spectroscopy). By way of example, a number of companies such
as
Pharmacia Biotech (Piscataway, NJ), Promega (Madison, WI), and US Biochemical
Corp (Cleveland, OH) supply commercial kits and protocols for assay
procedures.
Suitable reporter molecules or labels include those radionuclides, enzymes,
20 fluorescent, chemiluminescent, or chromogenic agents as well as substrates,
cofactors, inhibitors, magnetic particles and the like. Patents teaching the
use of
such labels include US-A-3817837; US-A-3850752; US-A=3939350; US-A-3996345;
US-A-4277437; US-A-4275149 and US-A-4366241.
25 HOST CELLS
The term "host cell" - in relation to the present invention includes any cell
that could
comprise the target for the agent of the present invention.
3o Thus, a further embodiment of the present invention provides host cells
transformed
or transfected with a polynucleotide that is or expresses the target of the
present
invention. Preferably said polynucleotide is carried in a vector for the
replication and
expression of polynucleotides that are to be the target or are to express the
target.
The cells will be chosen to be compatible with the said vector and may for
example
35 be prokaryotic (for example bacterial), fungal, yeast or plant cells.
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The gram negative bacterium E. coli is widely used as a host for heterologous
gene
expression. However, large amounts of heterologous protein tend to accumulate
inside the cell. Subsequent purification of the desired protein from the bulk
of E. coli
intracellular proteins can sometimes be difficult.
In contrast to E. coli, bacteria from the genus Bacillus are very suitable as
heterologous hosts because of their capability to secrete proteins into the
culture
medium. Other bacteria suitable as hosts are those from the genera
Streptomyces
and Pseudomonas.
Depending on the nature of the polynucleotide encoding the polypeptide of the
present invention, and/or the desirability for further processing of the
expressed
protein, eul<aryotic hosts such as yeasts or other fungi may be preferred. In
general,
yeast cells are preferred over fungal cells because they are easier to
manipulate.
However, some proteins are either poorly secreted from the yeast cell, or in
some
cases are not processed properly (e.g. hyperglycosylation in yeast). In these
instances, a different fungal host organism should be selected.
Examples of suitable expression hosts within the scope of the present
invention are
fungi such as Aspergillus species (such as those described in EP-A-0184438 and
EP-A-0284603) and Trichoderma species; bacteria such as Bacillus species (such
as
those described in EP-A-0134048 and EP-A-0253455), Streptomyces species and
Pseudomonas species; and yeasts such as Kluyveromyces species (such as those
described in EP-A-0096430 and EP-A-0301670) and Saccharomyces species. By
way of example, typical expression hosts may be selected from Aspergillus
niger,
Aspergillus niger var. tubigenis, Aspergillus niger var. awamori, Aspergillus
aculeatis,
Aspergillus nidulans, Aspergillus orvzae, Trichoderma reesei, Bacillus'
subtilis,
Bacillus licheniformis, Bacillus amylolipuefaciens, Kluyveromyces lactis and
Saccharomyces cerevisiae.
The use of suitable host cells - such as yeast, fungal and plant host cells -
may
provide for post-translational modifications (e.g. myristoylation,
glycosylation,
truncation, lapidation and tyrosine, serine or threonine phosphorylation) as
may be
needed to confer optimal biological activity on recombinant expression
products of
the present invention.
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ORGANISM
The term "organism" in relation to the present invention includes any organism
that
could comprise the target according to the present invention and/or products
obtained therefrom. Examples of organisms may include a fungus, yeast or a
plant.
The term "transgenic organism" in relation to the present invention includes
any
organism that comprises the target according to the present invention and/or
products obtained.
TRANSFORMATION OF HOST CELLS/HOST ORGANISMS
As indicated earlier, the host organism can be a prokaryotic or a eukaryotic
organism. Examples of suitable prokaryotic hosts include E. coli and Bacillus
subtilis.
Teachings on the transformation of prokaryotic hosts is well documented .in
the art,
for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd
edition, .1989, Cold Spring Harbor Laboratory Press) and Ausubel et al.,
Current
Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.
2o If a prokaryotic host is used then the nucleotide sequence may need to be
suitably
modified before transformation - such as by removal of introns.
In another embodiment the transgenic organism can be a yeast. In this regard,
yeast
have also been widely used as a vehicle for heterologous gene expression. The
species Saccharomyces cerevisiae has a long history of industrial use,
including its
use for heterologous gene expression. Expression of heterologous genes in
Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast
Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and
by
King ef al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T
3o Yarronton, eds, pp 107-133, Blackie, Glasgow).
For several reasons Saccharomyces cerevisiae is well suited for heterologous
gene
expression. First, it is non-pathogenic to humans and it is incapable of
producing
certain endotoxins. Second, it has a long history of safe use following
centuries of
commercial exploitation for various purposes. This has led to wide public
acceptability. Third, the extensive commercial use and research devoted to the
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organism has resulted in a wealth of knowledge about the genetics and
physiology
as well as large-scale fermentation characteristics of Saccharomyces
cerevisiae.
A review of the principles of heterologous gene expression in Saccharomyces
cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny
(1993,
"Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol 5,
Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
Several types of yeast vectors are available, including integrative vectors,
which
to require recombination with the host genome for their maintenance, and
autonomously replicating plasmid vectors.
in order to prepare the transgenic Saccharomyces, expression constructs are
prepared by inserting the nucleotide sequence of the present invention into a
is construct designed for expression in yeast. Several types of constructs
used for
heterologous expression have been developed. The constructs contain a promoter
active in yeast fused to the nucleotide sequence of the present invention,
usually a
promoter of yeast origin, such as the GAL1 promoter, is used. Usually a signal
sequence of yeast origin, such as the sequence encoding the SUC2 signal
peptide,
20 is used. A terminator active in yeast ends the expression system.
For the transformation of yeast several transformation protocols have been
developed. For example, a transgenic Saccharomyces according to the present
invention can be prepared by following the teachings of Hinnen et al (1978,
?5 Proceedings of the National Academy of Sciences of the USA 75, 1929);
Beggs, J D
(1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153,
163-
168).
The transformed yeast cells are selected using various selective markers.
Among
3o the markers used for transformation are a number of auxotrophic markers
such as
LEU2, HIS4 and TRP1, and dominant antibiotic resistance markers such as
aminoglycoside antibiotic markers, eg G418.
Another host organism is a plant. The basic principle in the construction of
35 genetically modified plants is to insert genetic information in the plant
genome so as
to obtain a stable maintenance of the inserted genetic material. Several
techniques
exist for inserting the genetic information, the two main principles being
direct
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introduction of the genetic information and introduction of the genetic
information by
use of a vector system. A review of the general techniques may be found in
articles
by Potrykus (Anna Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and
Christou
(Agro-Food-Industry Hi-Tech March/Aprif 1994 17-27). Further teachings on
plant
transformation may be found in EP-A-0449375.
Thus, the present invention also provides a method of transforming a host cell
with a
nucleotide sequence that is to be the target or is to express the target. Host
cells
transformed with the nucleotide sequence may be cultured under conditions
suitable
to for the expression of the encoded protein. The protein produced by a
recombinant
cell may be displayed on the surface of the cell. If desired, and as will be
understood
by those of skill in the art, expression vectors containing coding sequences
can be
designed with signal sequences which direct secretion of the coding sequences
through a particular prokaryotic or eukaryotic cell membrane. Other
recombinant
I5 constructions may join the coding sequence to nucleotide sequence encoding
a
polypeptide domain which will facilitate purification of soluble proteins
(Kroll DJ ef al
(1993) DNA Cell Biol 12:441-53).
THERAPY
The agents identified by the assay method of the present invention may be used
as
therapeutic agents - i.e. in therapy applications.
As with the term "treatment", the term "therapy" includes curative effects,
alleviation
effects, and prophylactic effects.
The therapy may be on humans or animals.
The therapy can include the treatment of'one or more of chronic dermal
ulceration,
3o diabetic ulcers, decubitus ulcers (or pressure sores), venous insufficiency
ulcers,
venous stasis ulcers, burns, corneal ulceration or melts.
The therapy may be for treating conditions associated with impaired damaged
tissue,
such as wound, healing, where impairment is due to diabetes, age, cancer or
its
treatment (including radiotherapy), neuropathy, nutritional deficiency or
chronic
disease.
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PHARMACEUTICAL COMPOSITIONS
The present invention also provides a pharmaceutical composition comprising a
therapeutically effective amount of the agents) and/or growth factor of the
present
invention and a pharmaceutically acceptable carrier, diluent or excipient
(including
combinations thereof).
The pharmaceutical compositions may be for human or anima! usage in human and
veterinary medicine and will typically comprise any one or more of a
pharmaceutically
to acceptable diluent, carrier, or excipient. Acceptable carriers or diluents
for
therapeutic use are well known in the pharmaceutical art, and are described,
for
example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R.
Gennaro edit. 1985). The choice of. pharmaceutical carrier, excipient or
diluent can
be selected with regard to the intended route of administration and standard
!5 pharmaceutical practice. The pharmaceutical compositions may comprise as -
or in
addition to - the carrier, excipient or diluent any suitable binder(s),
lubricant(s),
suspending agent(s), coating agent(s), solubilising agent(s).
Preservatives, stabilizers, dyes and even flavoring agents may be provided in
the
20 pharmaceutical composition. Examples of preservatives include sodium
benzoate,
sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending
agents may be also used.
There may be , different composition/formulation requirements dependent on the
25 different delivery systems. By way of example, the pharmaceutical
composition of
the present invention may be formulated to be administered using a mini-pump
or by
a mucosal route, for example, as a nasal spray or aerosol for inhalation or
ingestable
solution, or parenterally in which the composition is formulated by an
injectable form,
for delivery, by, for example, an intravenous, intramuscular or subcutaneous
route.
3o Alternatively, the formulation may be designed to be administered by a
number of
routes.
Where the agent is to be administered mucosally through the gastrointestinal
mucosa, it should be able to remain stable during transit though the
gastrointestinal
35 tract; for example, it should be resistant to proteolytic degradation,
stable at acid pH
and resistant to the detergent effects of bile.
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Where appropriate, the pharmaceutical compositions can be administered by .
inhalation, in the form of a suppository or pessary, topically in the form of
a lotion,
solution, cream, ointment or dusting powder, by use of a skin patch, orally in
the form
of tablets containing excipients such as starch or lactose, or in capsules or
ovules
either alone or in admixture with excipients, or in the form of elixirs,
solutions or
suspensions containing flavouring or colouring agents, or they can be injected
parenterally, for example intravenously, intramuscularly or subcutaneously.
For
parenteral administration, the compositions may be best used in the form of a
sterile
aqueous solution which may contain other substances, for example enough salts
or
monosaccharides to make the solution isotonic with blood. For buccal or
sublingual
administration the compositions may be administered in the form of tablets or
lozenges which can be formulated in a conventional manner.
For some embodiments, the agents and/or growth factors of the present
invention
~ 5 may also be used in combination with a cyclodextrin. Cyclodextrins are
known to
form inclusion and non-inclusion complexes with drug molecules. Formation of a
drug-cyclodextrin complex may modify the solubility, dissolution rate,
bioavailability
and/or stability property of a drug molecule. Drug-cyclodextrin complexes are
generally useful for most dosage forms and administration routes. As an
alternative
2o to direct complexation with the drug the cyclodextrin may be used as an
auxiliary
additive, e.g. as a carrier, diluent or solubiliser. Alpha-, beta- and gamma-
cyclodextrins are most commonly used and suitable examples are described in WO-
A-91 /11172, WO-A-94/02518 and WO-A-98/55148.
25 If the growth factor andlor the inhibitor agent is a protein, then said
protein may be
prepared in situ. in the subject being treated. In this respect, nucleotide
sequences
encoding said protein may be delivered by use of non-viral techniques (e.g. by
use of
liposomes) and/or viral techniques (e.g. by use of retroviral vectors) such
that the
said protein is expressed from said nucleotide sequence.
In a preferred embodiment, the pharmaceutical of the present invention is
administered topically.
Hence, preferably the pharmaceutical is in a form that is suitable for topical
delivery.
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ADMINISTRATION
The term "administered" includes delivery by viral or non-viral techniques.
Viral delivery
mechanisms include but are not limited to adenoviral vectors, adeno-associated
viral
(AAV) vectos, herpes viral vectors, retroviral vectors, lentivirai vectors,
and baculoviral
vectors. Non-viral delivery mechanisms include lipid mediated transfection,
liposomes,
immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and
combinations
thereof.
1 o The components of the present invention may be administered alone but will
generally be administered as a pharmaceutical composition - e.g. when the
components are is in admixture with a suitable pharmaceutical excipient,
diluent or
carrier selected with regard to the intended route of administration and
standard
pharmaceutical practice.
For example, the components can be administered (e.g. orally or topically) in
the
form of tablets, capsules, ovules, elixirs, solutions or suspensions, which
may contain
flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-
,
pulsed- or controlled-release applications.
If the pharmaceutical is a tablet, then the tablet may contain excipients such
as
microcrystalline cellulose, lactose, sodium citrate, calcium carbonate,
dibasic calcium
phosphate and glycine, disintegrants such as starch. (preferably corn, potato
or
tapioca starch), sodium starch glycollate, croscarmellose sodium and certain
complex silicates, and granulation binders such as polyvinylpyrrolidone,
hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose,
gelatin and acacia. Additionally, lubricating agents such as magnesium
stearate,
stearic acid, glyceryl behenate and talc may be included.
3o Solid compositions of a similar type may also be employed as fillers in
gelatin
capsules. Preferred excipients in this regard include lactose, starch, a
cellulose, milk
sugar or high molecular weight polyethylene glycols. For aqueous suspensions
and/or elixirs, the agent may be combined with various sweetening or
flavouring
agents, colouring matter or dyes, with emulsifying and/or suspending agents
and with
diluents such as water, ethanol, propylene glycol and glycerin, and
combinations
thereof.
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The routes for administration (delivery) include, but are not limited to, one
or more of:
oral (e.g. as a tablet, capsule, or as an ingestable solution), topical,
mucosal (e.g. as
a nasal spray or aerosol for inhalation), nasal, parenteral (e.g. by an
injectabie form),
gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous,
intrauterine,
intraocular, intradermal, intracranial, intratracheal, intravaginal,
intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including
intravitreal
or intracameral), transdermal, rectal, buccal, vaginal, epidural, sublingual.
In a preferred aspect, the pharmaceutical composition is delivered topically.
(0
Preferably, the composition of the present invention is administered topically
for
treating chronic dermal ulcers.
It is to be understood that. not all of the components of the pharmaceutical
need be
administered by the same route. Likewise, if the composition comprises more
than
one active component, then those components may be administered by different
routes.
If a component of the present invention is administered parenteraliy, then
examples
of such administration include one or more of: intravenously, intra-
arterially,
intraperitoneaily, intrathecaliy, intraventricularly, intraurethrally,
intrasternally,
intracranially, intramuscularly or subcutaneously administering the component;
andlor by using infusion techniques.
For parenteral administration, the component is best used in the form of a
sterile
aqueous solution which may contain other substances, for example, enough salts
or
glucose to make the solution isotonic with blood. The aqueous solutions should
be
suitably buffered (preferably to a pH of from 3 to 9), if necessary. The
preparation of
suitable parenteral formulations under sterile conditions is readily
accomplished by
3o standard pharmaceutical techniques well-known to those skilled in the art.
As indicated, the components) of the present invention can be administered
intranasally or by inhalation and is conveniently delivered in the form of a
dry powder
inhaler or an aerosol spray presentation from a pressurised container, pump,
spray
or nebuliser with the use of a suitable propellant, e.g.
dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as
1,1,1,2-tetrafluoroethane (HFA 134AT"") or 1,1,1,2,3,3,3-heptafluoropropane
(HFA
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227EAT""), carbon dioxide or other suitable gas. In the case of a pressurised
aerosol,
the dosage unit may be determined by providing a valve to deliver a metered
amount. The pressurised container, pump, spray or nebuliser may contain a
solution
or suspension of the active compound, e.g. using a mixture of ethanol and the
s propellant as the solvent, which may additionally contain a lubricant, e.g.
sorbitan
trioleate. Capsules and cartridges (made, for example, from gelatin) for use
in an
inhaler or insufflator may be formulated to contain a powder mix of the agent
and a
suitable powder base such as lactose or starch.
Alternatively, the components) of the present invention can be administered in
the
form of a suppository or pessary, or it may be applied topically in the form
of a gel,
hydrogel, lotion, solution, cream, ointment or dusting powder. The
component{s) of
the present invention may also be dermally or transdermally administered, for
example, by the use of a skin patch. They may also be administered by the
~ s pulmonary or rectal routes. They may also be administered by the ocular
route. For
ophthalmic use, the compounds can be formulated as micronised suspensions in
isotonic, pH adjusted, sterile saline, or, preferably, as . solutions in
isotonic, pH
adjusted, sterile saline, optionally in combination with a preservative such
as a
benzylalkonium chloride. Alternatively, they may be formulated in an ointment
such
20 as petrolatum.
For application topically to the skin, the component{s) of the present
invention can be
formulated as a suitable ointment containing the active compound suspended or
dissolved in, for example, a mixture with one or more of the following:
mineral oil,
2s liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene
polyoxypropylene compound, emulsifying wax and water. Alternatively, it can be
formulated as a suitable lotion or cream, suspended or dissolved in, for
example, a
mixture of one or more of the following: mineral oil, sorbitan monostearate, a
polyethylene glycol, liquid paraffin, po(ysorbate 60, cetyl esters wax,
cetearyl alcohol,
30 2-octyldodecanol, benzyl alcohol and water.
DOSE LEVELS
Typically, a physician will determine the actual dosage which will be most
suitable for
3s an individual subject. The specific dose level and frequency of dosage for
any
particular patient may be varied and will depend upon a variety of factors
including
the activity of the specific compound employed, the metabolic stability and
length of
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action of that compound, the age, body weight, general health, sex, diet, mode
and
time of administration, rate of excretion, drug combination, the severity of
the
particular condition, and the individual undergoing therapy.
Depending upon the need, the agent may be administered at a dose of from 0.01
to
30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1
to 1
mg/kg body. weight.
If the composition is applied topically, then typical doses may be in the
order of about
t0 1 to 50 mg/cm2 of damaged tissue, such as wound, area.
FORMULATION
The components) of the present invention may be formulated into a
pharmaceutical
is composition, such as by mixing with one or more of a suitable carrier,
diluent or
excipient, by using techniques that are known in the art.
PHARMACEUTICALLY ACTIVE SALT
2o The agent of the present invention may be administered as a
pharmaceutically
acceptable salt. Typically, a pharmaceutically acceptable salt may be readily
prepared by using a desired acid or base, as appropriate. The salt may
precipitate
from solution and be collected by filtration or may be recovered by
evaporation of the
solvent.
ANIMAL TEST MODELS
In vivo models may be used to investigate and/or design therapies or
therapeutic
agents to treat chronic wounds. The models could be used to investigate the
effect of
3o various tools/lead compounds on a variety of paramaters which are
implicated in the
development of ro treatment of a chronic wound. These animal test models can
be
used as, or in, the assay of the present invention. The animal test model will
be a
non-human animal test model.
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GENERAL RECOMBINANT DNA METHODOLOGY TECHNIQUES
Although in general the techniques mentioned herein are well known in the art,
reference may be made in particular to Sambrook et al., Molecular Cloning, A
Laboratory Manual (1989) and Ausubel et al., Short Protocols in Molecular
Biology
(1999) 4t" Ed, John Wiley & Sons, Inc. PCR is described in US-A-4683195, US-A-
4800195 and US-A-4965188.
SUMMARY
t0
In summation, the present invention relates to a pharmaceutical for use in
damaged
tissue, such as wound, treatment (e.g. healing); the pharmaceutical comprising
a
composition which comprises: (a) a growth factor; and (b) an inhibitor agent;
and
optionally (c) a pharmaceutically acceptable carrier, diluent or excipient;
wherein the
inhibitor agent can inhibit the action of at least one specific protease
protein that is
upregulated in a damaged tissue, such as a wound, environment.
The present invention also relates to uses of said composition, as well as to
process
for making same.
Otherwise expressed, the present invention relates to a pharmaceutical for use
in
damaged tissue, such as wound, treatment (e.g. healing); the pharmaceutical
comprising a composition which comprises: (a) a growth factor; and (b) an
inhibitor
agent; and optionally (c) a pharmaceutically acceptable carrier, diluent or
excipient;
wherein the inhibitor agent can inhibit the action of at least one specific
protease
protein that is upregulated in a damaged tissue, such as a wound, environment;
and
wherein said protease protein would otherwise be capable of detrimentally
degrading
said growth factor.
3o EXAMPLES
The present invention will now be described only by way of example.
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Test 1
Biochemical determination of protection ctrowth factor degradation by protease
inhibitors
Experiments are designed to assess the potential of uPA inhibitors and MMP
inhibitors to.protect growth factors from degradation by individual protease
enzymes.
To assess the susceptibility of a growth factor to degradation by a protease,
to individual growth factors are incubated with a range of protease enzymes
(including
uPA, tPA, plasmin or MMPs-1, -2, -3, -9, -13 or 14) at 37°C, for times
ranging from 15
minutes to 48 hours. The effect of uPA on growth factor degradation is
assessed in
both the presence and absence of plasminogen.
~ 5 Degradation of a particular growth factor by individual proteases is then
assessed by
either quantifying the reduction in growth factor levels or measuring the
presence of
peptide degradation products.
Biological techniques suitable for the quantification of growth factor
degradation
20 include: HPLC detection, Western blots analysis using specific growth
factor
antibodies and the use of radiolabelled growth factors.
In instances where individual proteases are found to result in measurable
growth
factor degradation during the incubation period, then protease inhibitor
compounds
25 are evaluated for their protective activity against this degradation.
Compounds are pre-incubated (for 15minutes) and degradation is assessed by one
of the methods as described above. All compounds are tested at concentrations
previously shown to inhibit the activity of individual proteases as measured
against a
3o fluorescent substrate. The vehicle (DMSO) used does not effect growth
factor
stability.
These experiments demonstrate the potential of I:uPAs (such as those mentioned
above) or certain I:MMPs (such as those mentioned above) to protect growth
factors
35 from degradation and therefore the clinical potential of treatments
involving co-
administration with these agents with growth factors.
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Test 2
Functional enhancement of growth factor activity in cell biology experiments
Migration
s
Experiments are conducted with primary human dermal cells such as fibroblasts,
keratinocytes and endothelial cells. Control studies measure the migratory
capacity
of cells through or over a suitable physiological matrix (e.g. collagen,
fibronectin,
MatrigelT""). Individual growth factors are tested for their ability to
enhance the
migration of cells over a given time, and the optimum concentration of growth
factor
is thus determined for future experiments. To assess the effect of individual
proteases on cell migration, various concentrations of purified human
proteases are
pre-incubated with the appropriate growth factor(s). Following this treatment,
growth
factors are re-tested for their ability to enhance cell migration over this
altered matrix.
15 If cell migration is reduced under these circumstances then it was
concluded that the
protease tested is capable of degrading the matrix over which the cells are
migrating.
To assess the functional protective effect of protease inhibitors, compounds
are
added to the matrix prior to addition of the purified protease.
2o Proliferation
Experiments are conducted with primary human dermal cells such as fibroblasts,
keratinocytes and endothelial cells. The endpoint of these studies is cell
proliferation
as measured by standard methods such as thymidine incorporation or cell
number.
25 Individual growth factors are tested for their ability to enhance the
proliferation of
cells over a given time, and the optimum concentration of growth factor is
thus
determined for future experiments. Protease inhibitors alone are also tested
for their
ability to enhance cell proliferation. Combination experiments involve
assessing the
proliferative effect of growth factors following pre-treatment of the growth
factor with a
3o specific protease. To assess the functional protective effect of protease
inhibitors,
growth factors are pre-incubated with the protease inhibitor compounds prior
to
addition of the purified protease. Cell proliferation is then determined as
described
above.
35 These experiments demonstrate that I:uPAs (such as those mentioned above)
and
I:MMPs (such as those mentioned above) can protect growth factors and/or
growth
factor receptors to give an additive and/or synergistic effect on cell
function,
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demonstrating the clinical potential of co-administration of these inhibitors
with
growth factors.
E~cample 1: The effect of human uPA, Plasmin, MMP-3 and MMP-13 and their
inhibitors on Growth Factors in vitro
Materials and methods
Materials
l0
Human recombinant TGF-(32 and KGF-2 were obtained from R&D Systems. Human
recombinant VEGF was obtained from Pharmingen. Trypsin, APMA, Trypsin-
Chymotrypsin
inhibitor, human recombinant PDGF-BB, aprotinin, Tween-20 and goat anti-VEGF
antibody,
were obtained from Sigma. Antibodies to TGF-(32, KGF-2 and PDGF-BB were
obtained from
f5 Santa Cruz Biotechnology Inc. Plasmin, human tPA stimulator, S-2288 and S-
2444
chromogenic serine and urokinase substrates respectively were obtained from
Quadratech.
uPA was obtained from Calbiochem. Chromozym-PL was from Boehringer Mannheim.
MMP-1, MMP-2, MMP-3, MMP-9, MMP-13 and MMP-14 were cloned, expressed and
purified by standard techniques. MMP-13 assay substrate DNP-Pro-Cha-Gly-
Cys(Me)-His-
20 Ala-Lys(NMA)NHZ was obtained from Peptides International Inc. MMP-1
substrate, Dnp-
Pro-~3-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys(N-Me-Ala)-NHZ and MMP-14
substrate,
Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NHz were obtained from Bachem. MMP-2, MMP-3,
MMP-9 substrate, Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NHZ was
obtained
from Neosystem Laboratories. compound 5719, compound 5214, compound 9470 and
25 compound 9454 were synthesised by standard techniques and prepared as a 10
mM stock
solutions in DMSO. All electrophoresis and Western blotting reagents were from
Invitrogen
(NOVEX). Blocking reagent (SuperBlock) was from Pierce and TBS (Tris-buffered
saline)
was obtained from Bio-Rad. Western blotting development reagents were obtained
from
Vector Laboratories. All chemicals were reagent grade.
Methods
i) Inhibition of enzymes by synthetic compounds
MMP assays
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Enzyme activation
All enzymes were pre-activated at 37°C with aminophenylmercuric acetate
(APMA) or
trypsin before being made up to the final concentrations used in the assay.
MMP-1 (30 nM)
was activated with 0.93 mM APMA for 20 minutes, MMP-2 (30 nM) was activated
with 1.32
mM APMA for 1 hour, MMP-3 ( I O I 0 nM) was activated with 1.8 f mM APMA for 3
hours or
heat activated at 55°C for three hours, MMP-9 ( 100 nM) was activated
with 2 mM APMA for
2 hours, human MMP-l3 ( 100 nM) was activated with 2 mM APMA for 2 hours, and
MMP-
14 (900 nM) was activated with 0.9 ng/ml trypsin for 25 minutes, followed by
the addition of
4.5ng/ml trypsin inhibitor.
Assay Buffers
For MMP-1, the assay buffer used was 50 mM Tris, 200 mM NaCI, 5 mM CaCh, 20
p.M
ZnCh, 0.05% (w/v) Brij 35, pH 7.5, For MMP-2, MMP-3 and MMP-9, the assay
buffer used
was 100 mM Tris, 100 mM NaCI, 10 mM CaCl2, 0.05% (wlv) Brij 35, pH 7.5. For
MMP-13,
the assay buffer used was 50 mM Tris, pH 7.5, 200 mM NaCI, 5 mM CaCh, 20mM Zn
Ch
and 0.02% (wlv) Brij 35. For MMP-14, the assay buffer used was SO mM Tris, 100
mM
NaCI, 10 mM CaCh, 0.25% (w/v) Brij 35, pH 7.5.
K; determinations
MMP-1 inhibition was assayed by incubating activated catalytic domain human
MMP-1 at 1
nM in assay buffer with 10 p,M Dnp-Pro-(3-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-
Lys(N-Me-
Ala)-NHz and six concentrations of inhibitors. The incubation was performed at
37°C for 60
minutes. The mean velocity between 0 and 60 minutes, which was linear with
time, was used
to calculate the K;.
MMP-2, MMP-3 and MMP-9 inhibition was assayed by incubating activated
catalytic domain
of human MMP-2, MMP-3 and MMP-9 at 1 nM in assay buffer with 5 ~M substrate
Mca-
Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NHz, and six different
concentrations of
inhibitor. The incubation was performed at 37°C for 60 minutes. The
mean velocity between 0
and 60 minutes, which was linear with time, was then used to calculate the K;.
MMP-14 inhibition was assayed by incubating activated catalytic domain human
MMP-14 at
1 nM in assay buffer with 10 p.M Mca-Pro-Leu-G!y-Leu-Dpa-Ala-Arg-NH2 and six
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concentrations of inhibitor. The incubation was performed at 37°C for
60 minutes. The mean
velocity between 0 and 60 minutes, which was linear with time, was then used
to calculate the
K;.
Compound and Substrate Concentrations
The final assay concentrations of inhibitors used in the MMP-1 assays to
determine K; were
50, 40, 30, 20, 10 and S ~tM. For MMP-2, the final assay concentrations of
inhibitors used
were 1000, 800, 600 400, 200 and 100 nM. For MMP-3, the final assay
concentrations of
inhibitors used were 5, 4, 3, ?, l and 0.5 nM. For MMP-9 and MMP-14, the final
assay
concentrations of inhibitors used were 5, 4, 3, 2, 1 and 0.5 uM.
MMP-l, -2, -3, -9 and -14 assay protocol
I S All assays were carried out in a black 96-well plate with a final volume
of 100 p1 in each
well. Inhibitors were dissolved in dimethylsulphoxide (DMSO) to 1 mM.
Solutions were then
serially diluted in buffer to give the final concentrations shown. The
addition of substrate was
preceded by an initial pre-incubation of enzyme and inhibitor at 37°C
for 15 minutes. For
MMP-2, MMP-3, MMP-9 and MMP-14, fluorescence was read every 2 minutes at 328nm
~,e,~
and 393nm ~.em for 1 hour using a Fluorostar fluorimeter (BMG) with BIOLISE
software. For
MMP-1 assays, the filters used were 355nm ~,e;~ and 440nm ~.°m;
fluorescence was read every
2 minutes for I hour.
MMP-13 assays
The ICso for MMP-I3 was determined by incubating activated enzyme at a final
concentration
of 60 ng/ml (1 nM) in MMP-13 assay buffer, with 10 1xM DNP-Pro-Cha-Gly-Cys(Me)-
His-
Ala-Lys(NMA)NHz substrate and varying concentrations of inhibitors (30, 3,
0.3, 0.03, 0.003
and 0.0003 p.M) in a final assay volume of 100 ~I. Assays were carried out in
96-well
microfluor plates. All incubations were performed at 37°C and
fluorescence readings
determined at 360nm ~.ex and 450nm ~.em.
For the assay, the fluorescence values at time zero were subtracted from those
determined at
IS or 20 minutes. The % response was then calculated by comparison to positive
controls
(enzyme, buffer and substrate in the absence of inhibitor). ICSO values were
then determined
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using FitCurve (Excel Tessella Stats add-in). Outliers were determined using
the Grubbs test
(Barnet & Lewis, 1994).
Calculation of K; values
These were estimated using the following equation:
ICso = (K; * 1+(S/K,n),
l0 where S is the substrate concentration and K", the Michaelis-Mentors
coefficient.
Serine protease assays
uPA (urokinase type plasminogen activator) inhibition was assayed by
incubating human uPA
IS at 33 IU/ml in 75 mM Tris, pH 8.1, 50 mM NaCI with 180 p,M S2444
(substrate) and various
concentrations of inhibitors. For the primary screen results, the incubation
was performed at
37°C for 30 minutes. Percentage inhibition was calculated and then
plotted against compound
concentration using the Excel add-in Fit Curve to give the ICso and a K; was
calculated from
the known Km of the substrate, 90 pM.
tPA (tissue type plasminogen activator) inhibition was assayed by incubating
human tPA at
0.4 pg/ml with 0.1 mg/ml tPA stimulator in 75 mM Tris, pH 8.1, 50 mM NaCI with
0.4 mM
52288 (substrate) and various concentrations of inhibitors. The incubation was
performed at
37°C for 60 mins. Percentage inhibition was calculated.
Plasmin inhibition was assayed by incubating human plasmin at 0.7 ~g/ml in 75
mM Tris, pH
8.1, 50 mM NaCi with 0.2 mM Chromozym-PL (substrate) and various
concentrations of
inhibitors. The incubation was performed at 37°C for 30mins. Percentage
inhibition was
calculated.
These assays were carried out in a 96-well plate.. The uPA and plasmin assays
had a final
volume of 200 p1 and the tPA assay has a final volume of 100 p.1. Inhibitors
were dissolved
in DMSO to 0.4 mM and then serially diluted to give the final concentrations
100, 30, 10, 3,
1, 0.3, O.land 0.03 p.M. The incubation was performed after an initial pre-
incubation at 37°C
for 15 mins and absorbance was read at 405 nM at 0 mins and at the end of the
incubation on
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a SPECTRAMax microplate reader (Molecular Devices Corporation), using
SOFTMaxPRO
software.
ii) Growth factor incubation conditions
The extent of proteolysis of the growth factors was assayed by incubating TGF-
[3,, VEGF,
PDGF-BB and KGF-2 with the proteases uPA, p(asmin, MMP-3 and MMP-13 in assay
buffer
(either uPAlplasmin buffer, 50 mM tris-HCI, pH 7.4 or MMP assay buffer, 100mM
Tris,
iOmM NaCI, IOmM CaCi~, 0.05% (w/v) Brij 35, pH 7.5). The choice of buffers had
no effect
on proteolysis during this work. The growth Factors were added to the
incubation mixture at a
Fnal concentration of 7.9 mg/ml, unless otherwise stated.
The effects of uPA were determined by incubation at a typical final
concentration of 25 ug/ml
( 1500 U/ml) with each growth factor. The effects of plasmin were determined
at a typical
IS final concentration of O.lmg/ml by incubation with the individual growth
factors in assay
buffer. MMP-3 and -13 were incubated at a typical final concentration of 10 nM
with the
growth factors in assay buffer. Dual protease assays carried out with uPA and
MMP-3
together were performed in J OOmM Tris, l OmM NaCI, IOmM CaClz, 0.05% (w/v)
Brij 35,
pH 7.5. All incubations were performed in siliconised tubes (Sigma Aldrich,
UK).
The inhibitors used in these experiments were compound 9454, compound 9470 and
compound 5214. These were dissolved in DMSO at a concentration of 10 mM.
Typical final
concentrations for these inhibitors were in the range of 100 wM to 10 nM.
Aprotinin was
dissolved in the Tris buffer at 10 mg/ml and used at a typical concentration
of 10 pg/ml.
All assays were carried out at 37°C and enzymes were pre-incubated for
15 minutes with or
without inhibitor as appropriate, prior to addition of growth factors. After
the addition of
growth factor, the incubation mixtures were divided into aliquots in
siliconised tubes for each
time point used. Incubations were carried out over a time course typically of
24 hours, unless
otherwise stated. They were stopped by the addition of an equal volume of 2X
Novex
reducing loading buffer (final concentration 1.09 M glycerol, 141 mM Tris-
base, 106 mM
Tris-HC1, 73 mM lithium dodecyl sulphate (LDS), 0.SlmM
ethylenediaminetetraacetic acid,
0.22 mM Serva Blue 6250, 0.175 mM Phenol Red, pH 8.5) and samples prepared for
electrophoresis by incubating at 70°C for 10 minutes.
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iii) Electrophoresis
LDS-PAGE was performed using the NOVEX Xcell II Mini-Cell gel apparatus
(Groningen, Holland) using a variation on the method of Laemmli (1970). Equal
volumes of samples were loaded onto NuPage 4-12% Bis-Iris gels with molecular
weight markers (SeeBlue PIus2 Pre-stained Standards). Molecular weight
determination was performed by comparison of bands with markers of molecular
weight 3, 6, 14, 17, 28, 38, 49, 62, 98 and 188 kDa. 79 ng of growth factor
was
loaded per lane and samples were resolved by vertical slab electrophoresis at
200V
for 35 minutes, using running buffer (50 mM 2-(N-morpholino) propane sulphonic
acid, 50 mM Tris-base, 3.5mM sodium dodecyl sulphate, 1 mM EDTA, pH 7.3)
containing 0.25% NuPAGE Antioxidant in the upper cathodic chamber. Following
electrophoresis Western blotting was carried out or gels were stained using
SifverXpress kit from NOVEX.
l5
iv) Western Blotting
Samples were separated under reduced and denaturing conditions and
electrophoretically transferred to nitrocellulose membranes using the XCell II
blot
20 module. Transfer was carried out at 25 V for 60 minutes using NOVEX
transfer
buffer (20% Methanol, 25 mM bicine, 25 mM Bis-Tris, 1.0 mM EDTA, 0.1% (v/v)
antioxidant, pH 7.3). After blotting, membranes were blocked for either 1 or
24 hours
using SuperBlock. The membranes were incubated in primary antibody (primary
antibodies were at a dilution of 1:400 in TTBS (Tween-20 Tris-buffered saline,
20 mM
25 Tris-HCI, pH 7.4, 500 mM NaCI, 0.1% Tween-20) for one hour. Membranes were
then washed and visualisation was performed using the Vector system of
peroxidase
conjugated secondary antibody; peroxidase was visualised by Nova-Red substrate
kit.
30 v) Quantitation
Analysis of immunoblotted and developed membranes was performed using a GS-
700 Imaging Densitometer (Bio-Rad, UK) and SystemOne v4.1.1 software.
Inhibitor
studies were analysed by quantitation of the loss of parent protein on the
blotted
35 membrane over the time course of the experiment. Percentage loss of protein
was
calculated using the following equations:
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= Ucontrol - Upost-protease
and
% inhibition = ~~ ~~ - ~Vpost-protease plus inhibitor ~ D
where D is the degradation value and V is the trace volume of parent growth
factor
band.
Results
1. Catculated values of IC; for inhibitors of Piasmin, uPA and tPA
Table 1 gives data showing the potency of compound 5214 as a selective
inhibitor of uPA.
The results show that compound 5214 is a potent inhibitor of uPA. Full
inhibition of tPA and
plasmin could not be achieved within the solubility limit of the compound. As
tCso values
could not be produced against these enzymes, it was not possible to calculate
a l~; against
either tPA or plasmin. Hence results show the percentage inhibition of the
compound at 100
p.M.
By contrast, aprotinin is a selective inhibitor of plasmin: data from the
literature as shown in
Table 2 to support this statement.
Data in Table 3 snows compound 5719 to be a non-selective inhibitor of MMPs,
compound
9454 to be a selective MMP-3 inhibitor and compound 9470 to be a selective
dual inhibitor
of MMP-3 and MMP-13.
2. Growth factor proteolysis
Table 4 indicates that proteases are able to digest growth factors that are
relevant to wound
healing either because the growth factors are endogenously present in normal
healing wounds
or because they may be added exogenously as pharmaceutical agents to chronic
dermal
ulcers.
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3. Ability of enzyme inhibitors to reduce growth factor degradation
The ability of selective protease inhibitors to reduce the digestion of growth
factors by
proteases is shown in Tables 5 to 8. (The apparent loss of potency of these
compounds
compared to e:cperiments where synthetic substrates are used appears to be due
to the protein-
binding properties of the agents reducing their free concentration within the
incubation with
growth factors.)
Under appropriate conditions, addition of two inhibitors is able to protect
growth factors from
degradation more than either of the inhibitors used at the same concentration
(Table 9).
Table 1.
Summary of compound 52 i 4 potency determinations against uPA, tPA and plasmin
IS
Protease Calculated IC; (nM)
UPA 9.6
" 7.8
' 11.0
" 11.0
Mean t sem
9.9 t 0.76
inhibition at 100
~M
TPA 38
47
50
Mean f sem
45.0 ~ 3.61
Plasmin 31
" 33
" 28
" 27
Mean t sem
29.8 t 1.38
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Table 2.
Summary of K; values for aprotinin against plasmin, uPA and tPA
Enzyme Calculated K; Reference
(nM)
Plasmin 1.0 Wiman (1980)
uPA 27000 Lottenberg et al, (
1988)
tPA >500000~ Lottenberg et al, (1988)
~No inhibition seen of tPA by aprotinin at the highest inhibitor concentration
of
S00 p,M.
Table 3.
Inhibition of MMP-1, -2, -3, -9, -13 and -14 by various synthetic compounds.
Compound Calculated
K; (nM)
,
MMP-I MMP-2 MMP-3 MMP-9 MMP-13 MMP-14
compound 0.61 0.73 0.58 0.47 1.52 3.68
5719
compound >19392 35215 44 52396 857 35481
9454
compound 1785 269 1 406 0.95 1710
9470
" limited by compound solubility
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Table 4.
Proteolytic digestion of growth factors by purified proteases*
uPA Plasrrtin MMP-3 MMP-13
PDGF-BB ++ +++ + (+)
TGF-(32 + ++ (+) +
VEGF ++ +++ + (+)
KGF-2 ++ +++ +++ ++
*The e:ctent of hydrolysis is represented by a score from significant
(represented as '+') to
major (represented as '+++'). Reduction of parent growth factor not
accompanied by the
appearance of degradation products is represented by (+).
Table 5.
Reduction of uPA-catalysed degradation of PDGF-BB by compound 5214
Inhibitor concentrationPercentage inhibition
(p.M) of
proteolysis
0.1 17
I 76
10 83
100 91
~In this case the degradation products at ll.SkDa were compared
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Tabte 6.
Reduction of MMP-3-catalysed degradation of KGF-2 by compound 9454
Inhibitor concentrationPercentage inhibition
(p.M) of
proteolysis
0. I 45
1 ~0
62
100 68
Table 7.
Reduction of MMP-3-catalysed degradation of KGF-2 by compound 9470
Inhibitor concentrationPercentage inhibition
(yM) of
proteolysis
0.01 24.5
0. I 64.5
I 72.2
10 91.1
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Table $.
Reduction of MMP-13-catalysed degradation of KGF-2 by compound 9454
Inhibitor concentrationPercentage inhibition
(~tM) of
proteolysis
0.1 1,60
1 10.0
23.7
100 82.9
5 Table 9.
Inhibition of uPA and MMP-3-mediated KGF-2 degradation by compound 5214 and
compound 9454 used either alone or in combination.
Inhibitors used (100Percentage inhibition
p.M) of
proteolysis
compound 5214 38.6
compound 9454 16.3
compound 5214 and 49.3
compound 9454 combined
10 References
Barnet, V and Lewis, T. (1994) in Outliers in Statistical Data, p.223, Wiley,
Chichester, UK.
Laemmli, U. K. (1970) Nature, 227, 680-685.
Lottenberg, R., Sjak-Shie, N., Fazleabas, A. T. & Roberts, R. M. (1988)
Thrombosis
Research, 49: 549-556.
Wiman, B. (1980) Thromb. Res. 17, 143-152.
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Example 2: Non-selective protease inhibitors perturb normal wound healing irr
vivo
Materials and Methods
Test article and vehicle
The test article was compound 5719 (0.3% wlv formulation in CMC hydrogel) and
the
vehicle was CMC hydrogel,
The test article and the vehicle were stored at room temperature in the dark.
Animals
The experiment was performed in 3 female SPF pigs (crossbreed of Danish
country, Duroc
and Yorkshire). At start of the acclimatisation period the body weight of the
animals was
about 30 kg.
An acclimatisation period of one week was allowed during which the animals
were observed
daily in order to reject an animal presenting a poor condition.
Housing
The study took place in an animal room provided with filtered air at a
temperature of 21°C ~
3°C and relative humidity of 55% ~ 15%. The room was designed to give
10 air changes per
hour. The room was illuminated to give a cycle of 12 hours light and 12 hours
darkness.
Light was on from 0600 to 1800 h.
The animals were housed individually in pens.
Bedding
The bedding was softwood sawdust "L1GNOCEL 3-4" from Hahn & Co, D-24796
Bredenbek-Kronsburg. Regular analyses for relevant possible contaminants were
performed.
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Diet
A commercially available pig diet, ''Altromin 9033" from Chr. Petersen A/S, DK-
4100
Ringsted was offered (about 700 g twice daily). Analyses for major nutritive
components and
relevant possible contaminants were performed regularly.
Drinking water
l0 Twice daily the ar2imals were oftered domestic quality drinking Water.
Analyses For relevant
possible contaminants were performed regularly.
Animal and pen identification
The pigs were identified by an eartag with study number and animal number. The
pens were
identified by a card marked with study number, and anima! number.
Surgery
The lesions were established on day 1. The animals were anaesthetised with
Stresnil~ Vet.
Janssen, Belgium {40 mg azaperone/ml, 1 ml/lOkg), and Atropin DAIC, Denmark (I
mg
atropine/ml, 0.05 ml/kg), given as a single intramuscular injection Followed
by i.v. injection
of Hypnodil~ Janssen, Belgium (50 mg metomidate/ml, I-2 ml).
An area dorso-laterally on either side of the back of the animal were shaved,
washed with
soap and water, disinfected with 70% ethanol which was rinsed off with sterile
saline, and
finally dried with sterile gauze.
Eight circular full thickness lesions (diameter 20 mm) were made on the
prepared area, four
on each side of the spine. The lesions were numbered I (most cranial) to 4
(most caudal) on
the left side of the animal, and 5 (most cranial) to 8 (most caudal) on the
right side of the
animal.
Coagulated blood was removed with sterile gauze.
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Just before surgery, about 8 hours termination of surgery and whenever
necessary, thereafter,
the animals were given an intramuscular injection of 0.01 mg buprenorphine/kg
(Anorfin~,
0.033 m(/kg, A/S GEA, Denmark).
Dosing
After surgery and daily thereafter, the test articles were applied as follows:
Animal
No.
1 2 3
LocalisationLeft Right Left Right Left Right
Cranial A B B A A B
B A A B B A
A B B A A - B
Caudal B A A B B A
IO A = compound 5719 (0.3% w/v formulation in CMC hydrogel)
B = CMC hydrogel (vehicle)
The dosing volume ofeach dosing was I,ml.
Dressing
The dressings were covered with a gauze bandage fixed by Fixomul~. The
dressings, the
gauze and the Fixomul~ were retained by a netlike body-stocking, Bend-a-rete~
(Tesval,
Italy).
The dressings were changed on a daily basis.
Prior to each changing the animals were anaesthetised with an intramuscular
injection in the
neck (1.0 m1/10 kg body weight) of a mixture of Zoletil 50~ Vet., Virbac,
France (125 mg
tiletamine and 125 mg zolazepam in 5 ml solvent, 5 ml), Rompun~ Vet., Bayer,
Germany (20
mg xylazine/mI, 6.5 ml), Ketaminol~ Vet., Veterinaria AG, Switzerland (I00 mg
ketamine/ml, 1.5 ml) and Methadon~ DAK, Nycomed DAK, Denmark (10 mg
methadon/ml,
2.5 ml).
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Observations
Each lesion was observed daily. The outlines of the wound edge and the
epithelial edge will
be drawn on sterile transparent sheets, and the areas contained inside the
edges were
measured planimetrically. The measurement of areas was performed by Scan Beam
ApS,
N~rregade 10, DK-9560 Hadsund.
Statistics
IO Data were processed to give group mean values and standard deviations where
appropriate.
Possible outliers were identified, too. Each variable was tested for normality
by the Shapiro-
Wilk method. In case of normal distribution, two=way analysis of variance was
carried out
for the variable with the factor: animal and treatment, and if significant
difference were
detected, possible intergroup differences were assessed using the least-
squares means.
IS Otherwise the possible intergroup differences were identified with Wilcoxon
Rank-Sum test.
The statistical analyses were made with SAS ~t procedures (version 6.12)
described in
"SAS/STAT~ User's Guide, Version 6, Fourth Edition, Vol. 1+2", 1989, SAS
Institute Inc.,
Cary, North Carolina 275 t 3, USA.
Results
Treatment Non-epithelialised
area
DAY 8 DAY
9
MEAN S.D. N p MEAN S.D. N p
compound 5719 294.0 41.1 12 248.0 23.2 12
CMC hydrogel 188.0 4I.7 I2 * 114.8 24.8 12 **
DAY IO DAY
I I
MEAN S.D. N p MEAN S.D. N p
compound 5719 210.1 25.6 12 148.9 74.5 12
CMC hydrogel 44.0 22.3 12 * 13.9 10.5 I2 **
** means p<0.01
S.D. = standard deviation N = number of wounds
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The Table shows that a non-selective MMP inhibitor perturbs wound healing.
Studies using
selective MMP inhibitors (in particular MMP-3 inhibitors) showed no effect on
normal
wound healing.
Similarly for serine proteases, published studies with knock-out mice
(Carmeliet et al., 1994)
show that in uPA -/- mice, a relatively mild phenotype is apparent, whilst in
mice that are
uPA -/- and tPA -/-, a more severe phenotype is apparent. The double knock-
out, which is the
genetic equivalent of using a non-selective serine protease inhibitor, shows
increased
incidence (in terms of mice and organs affected) and extent of spontaneous
fibrin deposition,
reduced fertility. and life span, and obliterated fbrinolysis. It is therefore
reasonable to
conclude that a selective inhibitor of uPA will be a far more effective wound
healing product
than a non-selective agent.
Reference
Carmeliet, P., Schoonjans, L., Kieckens, L., Ream, B., Degen, J., Bronson, R.,
De Vos, R.,
van den Oord, J.J., Collen, D. & Mulligan, R.C. (1994) Nature 368:419-424.
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PCS9494 Compounds
As indicated above, suitable inhibitor compounds (agents) for use in the
present
invention are disclosed in PCT/IB99101289 (WO-A-00/05214). It is to be
understood
that if the following teachings refer to further statements of inventions and
preferred
aspects then those statements and preferred aspects have to be read in
conjunction
with the aforementioned statements and preferred aspects - viz pharmaceutical
compositions either comprising an iUPA and/or an iMMP and a growth factor (as
well
as the uses thereof) or comprising an iUPA and an iMMP and an optional growth
factor (as well as the uses thereof).
The PCS9494 compounds are isoquinolines that are useful as urokinase
inhibitors, and are in
particular isoquinolinyljuanidines useful as urokinase inhibitors. In
particular the
isoquinolinyl5uanidine compounds are of the formula (I) :-
R'-'C'-NRZ-'C
(I)
and the pharmaceutically acceptable salts thereof, wherein:
G is N=C(NHz)~ or NHC(=NH)NH~;
Rl is H or halo;
X is CO, CHZ or SO2;
RZ is H, aryl, heteroaryl, C3.~ cycloalkyl or C~_6 alkyl each of which C3a
cycloalkyl and Cl~
alkyl is optionally substituted by one or more substituents independently
selected from halo,
aryl, het, C3.7 cycloalkyl, Csa cycloalkenyl, OH, C,_s alkoxy, O-hetl, C~_3
alkyl, COzR' and
NRdRS;
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X~ is arylene, C,_6 alkylene optionally substituted by one or more RG group,
or cyclo(Ca_
~)alkylene optionally substituted by R6, which cyclo(Cd_~)alkylene ring can
optionally contain
a hetero moiety selected from O, S(O)P or NR';
or R' and X' can be taken together with the N atom to which they are attached
to form an
azetidine, pyrrolidine, piperidine or homopiperidine ring;
R3 is CO~R', CH~OH, CONRsR9 or CH~NR~R9;
or, when X~ is taken independently from R' and is methylene optionally
substituted by one or
more RG group, or is a l,l-cyclo(C.,_~)alkylene optionally containing a hetero
moiety selected
from O, S(O)P or NR' and optionally substituted by R~,
then R'' and R3 can be taken together with the N and X~ groups to which they
are attached, as
I S a group of formula (IA) or (IB):
~IY~X~ O .~I~X~ O
12.
X O X NR
(IA) (I$)
wherein X'' is ethylene, n-propylene or n-butylene;
R4 and RS are each independently H, aryl or Ci.b alkyl optionally substituted
by aryl;
R6 is halo, OH, C,_6 alkoxy, C,_6 alkylthio, C3.7 cycloalkyl, SH, aryl, COZR',
CONHRB, or Ci_6
alkyl optionally substituted by aryl, C~_6 alkoxy, COZH, OH, CONR8R9 or by
NR$R9;
R' is H or C,.6 alkyl;
R8 and R9 are either each independently H, or Ci_6 alkyl optionally
substituted by OH, COZR',
C,_6 alkoxy or by NR4R5;
or R8 and R9 can be taken together with the N atom to which they are attached,
to form a 4- to
7-membered ring optionally incorporating an additional hetero- group selected
from O, S and
NR';
p is 0, l or 2;
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"aryl" is phenyl optionally substituted by one or more substituents
independently selected
from C,_6 alkyl, C,_6 alkoxy, or halo;
"het" is a saturated or partly or fully unsaturated 5- to 7-membered
heterocycle containing up
to 3 hetero-atoms independently selected from O, N and S, and which is
optionally substituted
by one or more substituents independently selected from C,_6 alkyl, C,_6
alkoxy, COzR' or
halo;
''heteroaryl" is a fully unsaturated 5- to 7-membered heterocycle containing
up to 3 hetero-
atoms independently selected from O, N and S, and which is optionally
substituted by one or
more substituents independently selected from C,_~ alkyl, C,_s alkoxy, CO~R'
or halo;
"heti" is tetrahydropyran-2-yl {2-THP); .
and "arylene" is phenylene optionally substituted by one or more substituents
independently
selected from Ci_~ alkyl, C,_~ alkoxy, COzR' or halo.
''Alkyl" groups can be straight or branched chain. "Halo" in the definitions
above refers to F,
C1 or Br.
"Cycloalkylene" groups in the definition of the X~ linker moiety which
optionally contains a
hetero moiety selected from O, S(O)P or NR' and is optionally substituted by
R6, can be linked
via any available atoms. "1,1-Cycloalkylene" groups in the definition of the
XI linker moiety
which optionally contains a hetero moiety selected from O, S(O)p or NR' and is
optionally
substituted by R6, means the linkage is via a common quaternary centre at one
position in the
ring, viz. for example: 1,1-cyclobutylene and 4,4-tetrahydropyranylene are to
be regarded as
both belonging to the same genus of "1,1-cycloalkylene" groups optionally
containing a
hetero moiety selected from O, S(O)P or NR' and optionally substituted by R6.
The two definitions given For the "G" moiety in compounds of formula (I) are
of course
tautomeric. The skilled man will realise that in certain circumstances one
tautomer will
prevail, and in other circumstances a mixture of tautomers will be present. It
is to be
understood that all tautomeric forms of the substances and mixtures thereof
are covered.
Preferably G is N=C(NHz)z.
Preferably R' is halo.
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More preferably R~ is chloro or bromo.
Most preferably R~ is chloro.
Preferably X is SO,.
Preferably R'' is H, C3_~ cycloalkyl or C,_6 alkyl each of which C3_~
cycloalkyl and Ci_6 alkyl is
optionally substituted by aryl, het, C3_~ cycloalkyl, OH, Ohet~, C,_6 alkoxy,
COZH, COZ(C~_6
alkyl) or by NR''R' , or R'' and X~ can be taken together with the N atom to
which they are
attached to form an azetidine, pyrrolidine, piperidine or homopiperidine ring.
More preferably R' is H, C~_~ alkyl optionally substituted by aryl or by
optionally substituted
pyridyl or by NR''RS or by HO or by Ohet~, or R' and X~ can be taken together
with the N
l5 atom to which they are attached to form an azetidine, pyrrolidine,
piperidine or
homopiperidine ring.
Further more preferably R' is H, CH~CH~N(CH3)~, CH3, CH~CH~OH, CH~CHzO(2-THP),
pyridinylmethyl, benzyl or methoxybenzyl, or Rz and X~ can be taken together
with the N
atom to which they are attached to form an azetidine, pyrrolidine, piperidine
or
homopiperidine ring linked to the R3 moiety via the 2-position of said ring.
Most preferably RZ is H, CH~CHZN(CH3)z, CH3, CHzCHZOH, CHzCH20(2-THP) or RZ
and
X' are taken together with the N atom to which they are attached to form a
pyrrolidine ring
linked to the R3 moiety via the 2-position.
Preferably X~ is phenyiene optionally substituted by one or two substituents
independently
selected from methoxy and halo, or is Ci_3 alkylene optionally substituted by
one or more
group selected from aryl or (Ci_6 alkyl optionally substituted by aryl, C~_6
alkoxy, CO~H, OH,
NHZ or CONHZ), or is cyclo(C4_~)alkylene optionally contain a hetero moiety
selected from O
or NR', which ring is optionally substituted by R6, or is taken together with
R' and with the N
atom to which they are attached to form an azetidine, pyrrolidine, piperidine
or
homopiperidine ring.
More preferably, X~ is methylene optionally substituted by one or more group
selected from
aryl or (C,~ alkyl optionally substituted by OH, NHZ or GONHZ),
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or is cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene,
tetrahydropyranylene,
piperidinylene substituted by R',
or is taken together with R'' and with the N atom to which they are attached
to form an
azetidine, pyrrolidine, piperidine or homopiperidine ring.
Yet more preferably X~ is C(CH3)~, l, L-cyclopentylene, 4,4-
tetrahydropyranylene, N methyl-
4,4-piperidinylene, CHI, CH(CH(CH3)~), CH(CH~)~NHz, CH(CHZ)3NH~, CH(CH~)CONH,,
I,l-cyclobutylene, l,l-cyciopentylene, 1,1-cyclohexylene, I,I-cycloheptylene,
N-methyl-4,4-
piperidinylene, 4,4-tetrahydropyranylene, or is taken together with R' and
with the N atom to
which they are attached to form an azetidine, pyrrolidine, piperidine or
homopiperidine ring
t0 linked to the R3 moiety via the 2-position.
Most preferably X~ is C(CH;)~, l, l-cyclopentylene, 4,4-tetrahydropyranylene,
N methyl-4,4-
piperidinylene, or is taken together with R' and with the N atom to which they
are attached to
form an azetidine, pyrrolidine, piperidine ring (inked to the R3 moiety via
the 2-position.
1S
Preferably R3 is CO~R' or CONR~R''.
More preferably R3 is CO~H, CONH~, CON(CH3)(CH~)~OH, CON(CH3)(CH~)~NHCH3,
COz(C,_;alkyl), CONH(CH~)~OH, CONH(CHZ)~OCH3, (morpholino)CO or (4-
20 methylpiperazino)C0.
Most preferably R3 is CO~H.
A preferred group of substances (a) are the compounds where X is SO~ in which
the R3-X~-NR'-
2S moiety is, where X~ is taken independently from Rz and is methylene
optionally substituted by
one or more R6 group, or is a 1,1-cyclo(C4_~)alkylene optionally containing a
hetero moiety
selected from O, S(O)P or NR' and optionally substituted by R6,
and RZ and R3 can be taken together with the N and X~ groups to which they are
attached, as a
group of formula (IA) or (IB):
~tV~X' O ~liX~ O
X O X NR
(IA) (IB)
wherein X'' is ethylene, n-propylene or n-butylene.
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In this group of substances (a) X~ is preferably C(CH3)z, 1,1-cyclobutylene,
1,1-
cyclopentylene, 1,1-cyclohexy(ene, 4,4-tetrahydropyranylene or N methyl-4,4-
piperidinylene,
most preferably I,1-cyclopentylene.
In this group of substances (a) X'' is preferably ethylene.
A preferred group of substances are the compounds in which the substituent R~
has the values as
described by the Examples below, and the salts thereof.
A preferred group of substances are the compounds in which the substituent X
has the values as
described by the Examples below, and the salts thereof.
A preferred group of substances are the compounds in which the substituent R'
has the values as
described by the Examples below, and the salts thereof.
A preferred group of substances are the compounds in which the substituent X~
has the values as
described by the Examples below, and the salts thereof.
A preferred group of substances are the compounds in which the substituent R3
has the values as
described by the Examples below, and the salts thereof.
Another preferred group of substances are the compounds in which each of the
substituents Rl,
X, RZ Xt and R3 have the values as described by the Examples below, and the
salts thexeof.
A preferred group of substances are the compounds where R' is chloro or bromo;
X is SOZ;
Rz is H, CHzCH2N(CH3)2, CH3, CHZCHZOH, CHzCHzO(2-THP), pyridinylmethyl, benzyl
or
methoxybenzyl, or RZ and X1 can be taken together with the N atom to which
they are
attached to form an azetidine, pyrrolidine, piperidine or homopiperidine ring
linked to the R3
moiety via the 2-position of said ring;
X' is C(CH3)z, 1,1-cyclopentylene, 4,4-tetrahydropyranylene, N methyl-4,4-
piperidinylene,
CHI, CH(CH(CH3)2), CH(CHz) 4NHz, CH(CH~) 3NH~, CH(CHa)CONH2, 1,1-
cyclobutylene,
l,l-cyclopentylene, 1,1-cyclohexylene, l,l-cycloheptylene, N-methyl-4,4-
piperidinylene,
4,4-tetrahydropyranylene, or is taken together with Rz and with the N atom to
which they are
attached to form an azetidine, pyrrolidine, piperidine or homopiperidine ring
linked to the R3
moiety via the 2-position;
R3 is CO~H, CONHZ, CON(CH3)(CHZ)20H, CON(CH3)(CHZ)ZNHCH3, COZ(C,_3alkyl),
CONH(CHZ)ZOH, CONH(CHZ)zOCH3, (morpholino)CO or (4-methylpiperazino)CO;
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and the salts thereof.
Another preferred group of substances are those in which R~ is chloro; X is
SO~;
R'' is H, CH~CHZN(CH3)Z, CH3, CH~CH~OH, CH~CH~O(2-THP) or R'' and X~ are taken
together with the N atom to which they are attached to form a pyrrolidine ring
linked to the R'
moiety via the 2-position;
Xi is C(CH3)~, l, l-cyclopentylene, 4,4-tetrahydropyranylene, N methyl-4,4-
piperidinylene, or
is taken together with R'' and with the N atom to which they are attached to
form an azetidine,
pyrrolidine, piperidine ring linked to the R3 moiety via the 2-position;
and R3 is CO~H;
and the salts thereof.
Another preferred group of substances are the compounds of the Examples below
and the
salts thereof. More preferred within this group are the compounds of Examples
32(b), 34(b),
IS 36(b), 37(b), 38, 39(a and b), 41(b), 43(b), 44(b), 71, 75, 76, 78, 79,
84(b), and 87(b and c)
and the alts thereof.
Preferred compounds or salts are selected from:
N [(4-chloro-I-guanidine-7-isoquinolinyl)sulphonyl]-D-proline;
2-{[(4-chloro-I-guanidine-7-isoquinolinyl)sulphonyl]amino}isobutyric acid;
1-{[(4-chioro-1-guanidine-7-
isoquinolinyl)sulphonyi]amino}cyclobutanecarboxylic acid;
N [(4-chloro-I-guanidine-7-isoquinoiinyl)sulphonyi]cycloleucine;
N [(4-chloro-I-guanidine-7-isoquinolinyl)sulphonyl]cycloleucine;
1-{[(4-chlore-I-guanidine-7-isoquinolinyl)sulphonyl]amino}-N (2-
hydroxyethyl)cyclopentanecarboxamine;
I-{[(4-chloro-I-guanidine-7-isoquinolinyl)sulphonyl]amino}-N [2-
(dimethylamino)ethyl]cyclopentanecarboxamine;
I-{[(4-chloro-I-guanidine-7-isoquinolinyl)sulphonyl]amino}-N [2-
(dimethylamino)ethyl]cyclopentanecarboxamine;
N [(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-N [2-
(dimethylamino)ethyl]cycloleucine;
1-{[(4-chloro-I-guanidine-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylic acid;
4-{[(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl)amino}tetrahydro-2H pyran-
4-
carboxylic acid;
tent-butyl (2R)-1-({4-chloro-1-guanidine-7-isoquinolinyl}sulphonyl)-2-
piperidinecarboxylate;
(2R)-I-({4-chlore-I-guanidine-7-isoquinolinyl}sulphonyl)-2-
piperidinecarboxylic acid;
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1-[({4-chloro-I-guanidino-7-isoquinolinyl}sulphonyl)amino]-N (2-hydroxyethyl)-
N
methylcyclopentanecarboxamide;
1-[({4-chloro-I-guanidino-7-isoquinolinyl} sulphonyl)amino]-N (2-
methoxyethyl)cyclopentanecarboxam ide;
4-chloro-I-guanidino-N [1-(morpholinocarbonyl)cyclopenty(]-7-
isoquinolinesulphonamide;
4-chloro-I-guanidino-N { l-[(4-methylpiperazino)carbonyl]cyclopentyl}-7-
isoquinolinesulphonamide;
N ({4-bromo-1-guanidino-7-isoquinoiinyl}suiphonyl)-N [2-
(dimethylamino)ethyl]cycloleucine;
t0 1-{[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl][2-(tetrahydro-2H pyran-
2-
yloxy)ethyl]amino}cyclopentanecarboxylic acid; and
N'-{4-chloro-7-[(10-oxo-9-oxa-6-azaspiro[4.5]dec-6-yl)sulphony(]-(-
isoquinolinyl}guanidine;
and the pharmaceutically acceptable salts thereof.
The invention further provides Methods for the production of substances of the
invention, which
are described below and in the Examples. The skilled man will appreciate that
the substances of
the invention could be made by methods other than those herein described, by
adaptation of the
methods herein described in the sections below and/or adaptation thereof, and
of methods known
in the art.
In the Methods below, unless otherwise specified, the substituents are as
defined above with
reference to the compounds of formula (I) above.
Method I
Compounds of formula (I) can be obtained from the corresponding 1-
aminoisoquinoline
derivative (II):
(II)
R'-JCS-NRi
by reaction with cyanamide (NHzCN) or a reagent which acts as a "NHC+=NH"
synthon such
as carboxamidine derivatives, e.g. IH-pyrazole-I-carboxamidine (M. S.
Bernatowicz, Y. Wu,
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G. R. Matsueda, J. Org. Chem., 1992, ~7, 2497), the 3,5-dimethylpyrazole
analogue thereof
(M.A.Brimble et al, J.Chem.Soc.Perkin Trans.I (1990)311), simple O-
alkylthiouronium salts
or S-alkylisothiouronium salts such as O-methylisothiourea (F.EI-Fehail et al,
J.Med.Chem.(1986), 29, 984), S-methylisothiouronium sulphate (S.Botros et al,
J.Med.Chem.(1986)29,874; P. S. Chauhan et al, Ind. J. Chem., 1993, 32B, 858)
or S-
ethylisothiouronium bromide (M.L.Pedersen et al, J.Org.Chem.(1993) 58, 6966).
Alternatively aminoiminomethanesu(phinic acid, or aminoiminomethanesulphonic
acid may
be used (A.E.Miller et al, Synthesis (1986) 777; K.Kim et al, Tet.Lett.(1988)
29,3183).
1O Other methods for this transformation are known to those skilled in the art
(see for example,
"Comprehensive Organic Functional Group Transformations", 1995, Pergamon
Press, Vol 6
p639, T. L. Gilchrist (Ed.); Patai's "Chemistry of Functional Groups", Vol. 2.
"The
Chemistry of Amidines and Imidates", 1991, 488).
IS Aminoisoquinolines (II) may be prepared by standard published methods (see
for example,
"The Chemistry of Heterocyclic Compounds" Vol. 38 Pt. 2 John Wiley & Sons, Ed.
F. G.
Kathawala, G. M. Coppolq, H. F. Schuster) including, for example, by
rearrangement from
the corresponding carboxy-derivative (Hoffmann, Curtius, Lossen, Schmidt-type
rearrangements) and subsequent deprotection.
Aminoisoquinolines (II) may alternatively be prepared by direct displacement
of a leaving
group such as Cl or Br with a nitrogen nucleophile such as azide (followed by
reduction), or
by ammonia, or through Pd-catalysis with a suitable protected amine (such as
benzylamine)
followed by deprotection using standard conditions well-known in the art.
Haloisoquinolines are commercially available or can alternatively be prepared
by various
methods, for example those described in : Goldschmidt, Chem.Ber.(1895)28,1532;
Brown
and Plasz, J.Het.Chem.(1971)6,303; US Patent 3,930,837; Hall et al,
Can.J.Chem.(1966)44,2473; White, J.Org.Chem.(1967)32,2689; and Ban,
Chem.Pharm.Bull.(1964)12,1296.
1,4-(Dichloro- or dibromo)isoquinolines can be prepared by the method
described by
M.Robison et al in J.Org.Chem.(1958)23,1071, by reaction ofthe corresponding
isocarbostyryl compound with PCIS or PBrS.
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Method 2
Compounds of formula (I) can be obtained From the corresponding
aminoisoquinoline
derivative (II) as defined in Method 1 above, via reaction with a reagent
which acts as a
protected amidine(2+) synthon (III),
z+
PN~NHP1
(III)
such as a compound PNHC(=X)NHP,, PN=CXNHP, or PNHCX=NP,, where X is a leaving
group such as C(, Br, I, mesylate, tosylate, alkyloxy, etc., and where P and
P, may be the
same or different and are N-protecting groups such as are well-known in the
art, such as t-
butoxycarbonyl, benzyloxycarbonyl, arylsulphonyl such as toluenesulphonyl,
vitro, etc.
l5 Examples of reagents that act as synthons (III) include N, N'-protected-S-
alkylthiouronium
derivatives such as N, N'-bis(t-butoxycarbonyl)-S-Me-isothiourea, N, N'-
bis(benzyloxycarbonyl)-S-methylisothiourea, or sulphonic acid derivatives of
these (J. Org.
Chem. 1986, 51, 1882), or S-arylthiouronium derivatives such as N, N'-bis(t-
butoxycarbonyl)-S-(2,4-dinitrobenzene) (S. G. Lammin, B. L. Pedgrift, A. J.
Ratcliffe, Tet.
Lett. 1996, 37, 6815), or mono-protected analogues such as [(4-methoxy-2,3,6-
trimethylphenyl)sulphonyl]-carbamiriiidothioic acid methyl ester or the
corresponding
2,2,5,7,8-pentamethylchroman-6-sulphonyl analogue (D. R. Kent, W. L. Cody, A.
M.
Doherty, Tet. Lett., 1996, 37, 8711), or S-methyl-N-nitroisothiourea
(L.Fishbein et al,
J.Am.Chem.Soc. (1954) 76, 1877) or various substituted thioureas such as N, N'-
bis(t-
butoxycarbonyl)thiourea (C. Levallet, J. Lerpiniere, S. Y. Ko, Tet. 1997, 53,
5291) with or
without the presence of a promoter such as a Mukaiyama's reagent (Yong, Y.F.;
Kowalski,
J.A.; Lipton, M.A. J. Org. Chem., 1997, 62, 1540), or copper, mercury or
silver salts,
particularly with mercury (II) chloride. Suitably N-protected O-alkylisoureas
may also be
used such as O-methyl-N-nitroisourea (N.Heyboer et al, Rec.Chim.Trav.Pays-Bas
( 1962)81,69). Alternatively other guanylation agents known to those skilled
in the art such as
1-H-pyrazole-1-[N,N'-bis(t-butoxycarbonyl)]carboxamidine, the corresponding
bis-Cbz
derivative (M. S. Bernatowicz, Y. Wu, G. R. Matsueda, Tet. Lett. 1993, 34,
3389) or
monoBoc or mono-Cbz derivatives may be used (B. Drake.. Synthesis, 1994, 579,
M. S.
Bernatowicz.. Tet. Lett. 1993, 34, 3389). Similarly, 3,5-dimethyl-1-
nitroguanylpyrazole may
be used (T.Wakayima et al, Tet.Lett.(1986)29,2143).
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The reaction can conveniently be carried out using a suitable solvent such as
dichloromethane, N,N-dimethylformamide (DMF), methanol.
The reaction is also conveniently carried out by adding mercury (II) chloride
to a mixture of
the aminoisoquinoline (II) and a thiourea derivative of type (III) in a
suitable base / solvent
mixture such as triethylamine / dichloromethane.
R'-X'-NRZ-X
(IV)
The product of this reaction is the protected isoquinolinylguanidine (IV),
where G~ is a
protected guanidine moiety N=C(NHP)(NHP,) or tautomer thereof, where P and P,
are
nitrogen-protecting groups such as t-butoxycarbonyl ("Boc"), benzyl,
benzy(oxycarbonyl,
etc., which can conveniently be deprotected to give (I) or a salt thereof.
For example, if the protecting group P and/or P, is t-butoxycarbonyl,
conveniently the
deprotection is carried out using an acid such as trifluoroacetic acid (TFA)
or hydrochloric
acid, in a suitable solvent such as dichloromethane, to give the
bistrifluoroacetate salt of (I).
If P and/or P~ is a hydrogenolysable group, such as benzyloxycarbonyl, the
deprotection could
be performed by hydrogeno(ysis.
Other protection / deprotection regimes include : nitro (K.Suzuki et al,
Chem.Pharm.Bull.
(1985)33,1528, Nencioni et al, J.Med.Chem.(1991)34,3373, B.T.Golding et al,
J.C.S.Chem.Comm.(1994)2613; p-toluenesulphonyl (J.F.Callaghan et al,
Tetrahedron (1993)
49 3479; mesitylsulphonyl (Shiori et al, Chem.Pharm.Bull.(1987)35,2698,
ibid.(1987)35,2561, ibid., (1989)37,3432, ibid., (1987)35,3880, ibid.,
(1987)35,1076; 2-
adamantoyloxycarbonyl (Iuchi et al, ibid., (1987) 35, 4307; and
methylsulphonylethoxycarbonyl (Filippov et al, Syn.Lett.(1994)922)
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It will be apparent to those skilled in the art that other protection and
subsequent deprotection
regimes during synthesis of a compound of the invention may be achieved by
various other
conventional techniques, for example as described in "Protective Groups in
Organic Synthesis"
by T W Greene and P G M Wuts, John Wiley and Sons Inc. (1991), and by
P.J.Kocienski, in
"Protecting Groups", Georg Thieme Verlag ( 1994).
Method 3
Compounds of the formula (1) can be obtained from compounds of formula (V)
R~
(V)
R'-\'-NRz X
where Z is a suitable leaving group such as C1, Br or OPh, by displacement of
the leaving
group by the free base of guanidine.
Compounds of formula (V) are available as mentioned above in the section on
preparation of
compounds of formula (II) in Method 1, and routine variation thereof.
The free base of guanidine may conveniently be generated in situ from a
suitable salt, such as
the hydrochloride, carbonate, nitrate, or sulphate with a suitable base such
as sodium hydride,
potassium hydride, or another alkali metal base, preferably in a dry non-
erotic solvent such as
tetrahydrofuran (THF), DMSO, N,N-dimethylformamide~(DMF), ethylene glycol
dimethyl
ether (DME), N,N-dimethyl acetamide (DMA), toluene or mixtures thereof.
Alternatively it
can be generated from a suitable salt using an alkoxide in an alcohol solvent
such as
potassium t-butoxide in t-butanol, or in a non-erotic solvent as above.
The thus formed free guanidine can be combined with the I-isoquinoline
derivative (V), and
the reaction to form compounds of formula (I) can be carried out at from room
temperature to
200°C, preferably from about 50°C to 150°C, preferably
for between 4 hours and 6 days.
It will be clear to those skilled in the art, that some of the functionality
in the R3, RZ and/or X~
groups may need to be either protected and released subsequent to guanylation
or added, or
generated after the guanidine moiety had been added to the substrate.
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For example, an acid group could be carried through the guanylation stage
while protected as
an ester and subsequently hydrolyseded. Base-catalysed hydrolysis of an ethyl
ester and acid-
catalysed hydrolysis of a t-butyl ester are two such suitable examples of
this. In another
example, an alcohol may be protected with groups well documented in the
literature such as a
2-tetrahydropyranyl ether (2-THP) and subsequently removed by treatment with
acid.
The addition of new functionality after the guanidine moiety has been
installed is also
encorr~passed by the invention. For example, alkylation of the sulphonamido NH
(i.e. "X-
NR'" is SO,NH) with an alkyl halide may be performed in the presence of a base
such as
potassium carbonate and optionally in the presence of a promoter such as KI.
In another
example, an acid group may be converted to an amide through a range of
coupling conditions
known to those skilled in the art, or conveniently though the acid chloride
while in the
presence of a free or protected guanidine. Alternatively an ester group can be
directly reacted
with an amine to generate an amide; if this occurs in an intramolecular
process, a lactam may
IS be formed. Using similar methodology esters and lactones may be prepared.
Additional
functionality could have been present in a protected form at this stage and
subsequently.
revealed - such as an amino group which could be protected by groups well
documented in
the literature, e.g. a Boc group and subsequently removed under standard
conditions, such as
treatment with a strong base such as HCI or TFA.
Method 4
Compounds of the invention where one or more substituent is or contains a
carboxylic acid
group or carbamoyl group can be made from the corresponding compound where the
corresponding substituent is a nitrile by full or partial hydrolysis.
Compounds of the invention
where one or more substituent is or contains a carboxylic acid group can be
made from the
corresponding compound where the corresponding substituent is a carbamoyl
moiety, by
hydrolysis.
The hydrolysis can be carried out by methods well-known in the art, for
example those
mentioned in "Advanced Organic Chemistry" by J.March, 3rd edition (Wiley-
Interscience)
chapter 6-5, and references therein. Conveniently the hydrolysis is carried
out using
concentrated hydrochloric acid, at elevated temperatures, and the product
forms the
hydrochloride salt.
Method 5
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Where desired or necessary the compound of formula (I) is converted into a
pharmaceutically
acceptable salt thereof. A pharmaceutically acceptable salt of a compound of
formula (I) may be
conveniently be prepared by mixing together solutions of a compound of formula
(I) and the
desired acid or base, as appropriate. The salt may be precipitated from
solution and collected by
filtration, or may be collected by other means such as by evaporation of the
solvent.
Other Methods
Compounds of the forri~ula (I) where one or more substituent is or contains C1
or Br may be
l0 dehalogenated to give the corresponding hydrido compounds of formula (I) by
hydrogenolysis, suitably using a palladium on charcoal catalyst, in a suitable
solvent such as
ethanol at about 20°C and at elevated pressure.
Compounds of formula (I) where one or more substituent is or contains a
carboxy group may
15 be prepared from a compound with a group hydrolysable to give a carboxy
moiety, for
example a corresponding nitrite or ester, by hydrolysis, for example by acidic
hydrolysis with
e.g. conc: aq. HC! at reflux. Other hydrolysis methods are well known in the
art.
Compounds of formula (I) in which one or more substituent is or contains an
amide moiety
20 may 6e made via reaction of an optionally protected corresponding carboxy
compund, either
by direct coupling with the amine of choice, or via initial formation of the
corresponding acid
chloride or mixed anhydride, and subsequent reaction with the amine, followed
by
deprotection if appropriate. Such transformations are well-known in the art.
25 Certain of the compounds of formula (I) which have an electrophilic group
attached to an
aromatic ring can be made by reaction of the corresponding hydrido compound
with an
electrophilic reagent.
For example sulphonylation of the aromatic ring using standard reagents and
methods, such
30 as fuming sulphuric acid, gives a corresponding sulphonic acid. This can
then be optionally
converted into the corresponding sulphonamide by methods known in the art, for
example by
firstly converting to the acid chloride followed by reaction with an amine.
Certain of the compounds of the invention can be made by cross-coupling
techniques such as by
35 reaction of a compound containing a bromo-substituent attached to e.g. an
aromatic ring, with
e.g. a boronic acid derivative, an olefin or a tin derivative by methods well-
known in the art, for
example by the methods described in certain of the Preparations below.
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Certain of the compounds of the invention having an electrophilic substituent
can be made via
ha(ogen/metal exchange followed be reaction with an electrophilic reagent. For
example a
bromo-substituent may react with a lithiating reagent such as n-butyllithium
and subsequently an
electrophilic reagent such as CO,, an aldehyde or ketone, to give respectively
an acid or an
alcohol.
Compounds of the invention are available by either the methods described
herein in the
Methods and Examples or suitable adaptation thereof using methods known in the
art. It is to
be understood that the synthetic transformation methods mentioned herein may
be carried out
in various different sequences in order that the desired compounds can be
efficiently
assembled. The skilled chemist will exercise his judgement and skill as to the
most efficient
sequence of reactions for synthesis of a given targetcompound.
EXPERIMENTAL SECTION
GENERAL DETAILS
Melting points (mp) were determined using either Gallenkamp or Electrothermal
melting
point apparatus and are uncorrected. Proton nuclear magnetic resonance (~H
NMR) data were
obtained using a Varian Unity 300 or a Varian Inova 400. Low resolution mass
spectral
(LRMS) data were recorded on a Fisons Instruments Trio 1000 (thermaspray) or a
Finnigan
Mat. TSQ 7000 (APCI). Elemental combustion analyses (Anal.) were determined by
Exeter
Analytical UK. Ltd.
Column chromatography was performed using Merck silica gel 60 (0.040-0.063
mm).
Reverse phase column chromatography was performed using Mitsubishi MCI gel
(CHP 20P).
The following abbreviations were used: ammonia solution sp. gr. 0.880
(0.880NH3); diethyl
azodicarboxylate (DEAD); 1,2-dimethoxyethane (DME); N,N dimethylacetamide
(DMA);
N,N dimethylformamide (DMF); dimethylsulphoxide (DMSO); tetrahydrofuran (THF);
trifluoroacetic acid (TFA); toluene (PhMe); methanol (MeOH); ethyl acetate
(EtOAc)
propanol (PrOH). Other abbreviations are used according to standard chemical
practice.
Some nomenclature has been allocated using the IUPAC NamePro software
available from
Advanced Chemical Development Inc. It was noted following some preparations
involving
guanylation of intermediates containing a quaternary centre adjacent to a base-
sensitive group
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e.g. an ester, that some racemisation had occurred, so in such cases there may
be a mixture of
enantiomers produced.
EXAMPLES
Example 1:
(a) tert-Butyl 2-{((4-chloro-1-guanidine-7-
isoquinolinyl)sulphonyl]amino}benzoate
(b) 2-{[(4-Chloro-I-guanidine-7-isoquinolinyl)sulphonyl]amino}benzoic acid
hydrochloride
c1 a c1
/ CO;tBu \ \ / CO_t8u \ \ / COzH \ \
_ _ a~
\ I ~ I / iN ~ \ I ~ I / iN \ I N-S I / iN
H 0 CI H 0 N~NH_ 0 NYNHi
NHZ NHi
Guanidine hydrochloride (60 mg, 0.63 mmol) was added in one portion to a
suspension of
NaH (i8 mg, 80% dispersion by wt in mineral oil, 0.6 mmol) in DMSO (3.0 mL)
and the
mixture was heated at 60 °C under N~ for 30 min. tert-Butyl 2-{[(1,4-
dichloro-7-
isoquinolinyl)sulphonyi]amino}benzoate (110 mg, 0.24 mmol) was added and the
mixture
heated at 100 °C for 24 h. The cooled mixture was poured into water and
extracted with
EtOAc (x3) and the combined organic phase was then washed with brine and
evaporated in
vacuo. The residue was purified by column chromatography upon silica gel,.
using CH2CI2-
MeOH-0.880NH3 (97:3:0.3 to 95:5:0.5) as eluant to give a yellow resin (36 mg).
This resin
was suspended in water and extracted with ether (x3). The combined organic
phase was
washed with brine and evaporated in.vacuo to give tent-butyl 2-{[(4-chloro-1-
guanidine-7-
isoquinolinyl)sulphonyl]amino}benzoate (30 mg, 0.063 mmot) as a brown solid.
TLC Rf0.60 (CH~CIz-MeOH-0.880NH3, 90:10:1).
' H (CD30D, 400 MHz) 8 I .4 (9H, s), 7.1 ( 1 H, dd), 7.5 ( 1 H, dd), 7.7 ( 1
H, d), 7.8 ( I H, d), 7.9
(1H, d), 8.0 (IH, d), 8.1 (1H, s), 9.1 (1H, s) ppm.
LRMS 475 (MH+).
The silica gel column was then eluted with MeOH and the combined washings were
concentrated in vacuo to give an off white solid. This was dissolved in a
solution of EtOH
saturated with HCI gas and the mixture stirred at room temperature. The
solvents were
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evaporated in vacuo and the residue was then dissolved in EtOAc-MeOH, filtered
and again
evaporated in vacuo. The solid was triturated with water and then dried to
give 2-{[(4-chloro-
1-guanidino-7-isoquinolinyl)sulphonyl]amino}benzoic acid hydrochloride (11.8
mg, 0.02
mmol) as a pale yellow solid.
mp >280 °C (dec).
~ H (CD30D, 400 MHz) 8 7.0 ( l H, dd), 7.3 ( 1 H, dd), 7.65 ( 1 H, d), 7.8 ( 1
H, d), 8.1 ( 1 H, d), 8.2
(IH,d),8.3(lH,s),8.9(iH,s)ppm.
l0
LRMS 420, 422 (M+), 42 l (MH+).
Anal. Found: C, 43.58; H, 3.37; N, 14.65. Calc for
C,~Hi,~CIN504S~I.OHCI~0.7H20: C, 43.54;
H, 3.53; N, 14.94.
Example 2:
(a) tart-Butyl 3-{((4-chloro-1-buanidino-7-
isoquinolinyl)sulphonyl]amino}benzoate
(b) 3-{((4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl)amino}benzoic acid
trifluoroacetate
COztBu CI COztBu CI COZH CI
/ I o I \ \ ~ ,~ I a I \ \ ~ I N-S ~ ,
\ N_5 / l N N_S / l N
H 0 CI H 0 NYNHz H 0 N\/NH2
NHZ TNHz
Guanidine hydrochloride (140 mg, 1.47 mmol) was added in one portion to a
suspension of
NaH (44 mg, 80% dispersion by wt in mineral oil, 1.47 mmol) in DMSO (4.0 mL)
and the
mixture was heated at 60 °C under N2 for 30 min. A solution of tart-
butyl 3-{[(1,4-dichloro-7-
isoquinolinyl)-sulphonyl]amino}benzoate (280 mg, 0.59 mmol) in DMSO (2.0 mL)
was
added and the mixture heated at 90 °C for 18 h. The cooled mixture was
poured into water (50
mL), extracted with EtOAc (x3) and the combined organic phase was then
evaporated in
vacuo. The residue was purified by column chromatography upon silica gel using
CH~CIz-
MeOH-0.880NH3 (97:3:0.3 to 95:5:0.5) as eluant to give tart-butyl 3-{[(4-
chloro-1-
guanidino-7-isoquinolinyl)sulphonyl]amino}benzoate (64 mg, 0.13 mmol) as a tan
solid.
mp >142 °C (dec).
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IH (CD30D, 400 MHz) 8 1.5 (9H, s), 7.25-7.35 (2H, m), 7.65-7.7 (2H, m), 7.95
(1H, d), 8.05
( 1 H, d), 8.1 ( 1 H, s), 9.1 ( 1 H, s) ppm.
LRMS 475 (MH+).
Anal. Found: C, 51.07; H, 4.55; N, 13.94. Calc for C~,H~~CINSO,,S~0.23CH~C12:
C, 51.46; H,
4.57; N, 14.13.
ter-t-Butyl 3-{[(4-chloro-1-~~uanidino-7-
isoquinolinyl)sulphonyl]amino}benzoate (30 mg,
l0 0.063 mmol) was dissolved in CF,CO,H ( l.0 mL) and the mixture stirred at
room temperature
for 1 h. The mixture was diluted with PhMe and the solvents were evaporated in
vaeuo. The
residue was triturated with Et,O and then azeotroped with CH~C1~ to give 3-
{[(4-chloro-1-
guanidino-7-isoquinolinyl)sulphonyl]amino}-benzoic acid trifluoroacetate (29
mg, 0.055
mmol) as an off white solid.
IS
mp > 180 °C (dec).
1H (CD30D, 400 MHz) 8 7.2-7.35 (2H, m), 7.55 ( 1 H, d), 7.65 ( 1 H, s), 8.15 (
1 H, d), 8.3 ( 1 H,
d), 8.3 S ( 1 H, s), 8.85 ( 1 H, s) ppm.
LRMS 419, 421 (MH~).
Anal. Found: C, 42.51; H, 3.07; N, 13.19. Calc for C,~H,4CIN504S~1.OCF3COZH:
C, 42.75; H,
2.83; N, 13.12.
2~
Example 3:
(a) Methyl 3-{[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]amino}-4-
methoxybenzoate
(b) 3-{[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]amino}-4-
methoxybenzoic acid
hydrochloride
C02Me CI COzMe CI COZH CI
\ , N_S I / iN ~ \ I N_S I / iN \ I H_~5 I / iN
OMe H O Ct OMs H 0 NYNHZ OMe 0 N \/NHZ
NH2 TNHZ
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Guanidine hydrochloride ( 179.8 mg, 1.88 mmol) was added in one portion to a
suspension of
NaH (54.9 mg, 80% dispersion by wt in mineral oil, 1.83 mmol) in DMSO (10 mL)
and the
mixture was heated at 60 °C under Nw for 20 min. Methyl 3-{[(1,4-
dichloro-7-
isoquinolinyl)sulphonyl]amino}-4-metho~ybenzoate (238.6 mg, 0.541 mmol) was
added and
the mixture heated at 90 °C for 2=1 h. The solvents were evaporated in
vacuo and the residue
was purified by. column chromatography upon silica gel using CHZC1~-MeOH-
0.880NH3
(97:3:0.3 to 90: i0:1) as eluant to give methyl 3-{[(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]amino}-4-methoxybenzoate (203.2 mg, 0.43 mmol) as a
pale yellow
solid.
mp 134-137 °C (dec).
~ H (DMSO-dr, 300 MHz) c5 3.45 (3 H, s), 3.8 (3 H, s), 6.95 ( 1 H, d), 7.05-
7.4 (4H, br s), 7.7
( 1 H, d), .7.8 ( 1 H, s), 8.0 (2 H, s), 8.1 ( I H, s), 9.05 ( 1 H, s), 9.9 (
1 H, br s) ppm.
IS
LRMS 464, 466 (MH+)
Anal. Found: C, 48.37; H, 3.81; N, 14.75. Calc for C,aH,8CIN505S~O.15CH~Cl2:
C, 48.26; H,
3.87; N, 14.69.
An aqueous solution of NaOH (0.7 mL, I.0 M, 0.7 mmol) was added slowly to a
stirred
solution of methyl 3-{[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]amino}-
4-
methoxybenzoate (52.2 mg, 0.113 mmol) in dioxane (2.5 mL).and the mixture
stirred at room
temperature for 1.5 h, and then at 70 °C for 3 h. The mixture was
cooled to room temperature,
dilute HCl (2 mL, 2 N) was added, the solvents were evaporated in vacuo and
the residue was
dried by azeotroping with i-PrOH (x3). The solid was extracted with hot i-PrOH
(x4), the
combined organic extracts were filtered, and the solvents were evaporated in
vacuo. The
residue was triturated with Et~O to give 3-{[(4-chloro-1-guanidino-7-
isoquinolinyi)sulphonyi]amino}-4-methoxybenzoic acid hydrochloride (29 mg,
0.055 mmol)
as a solid.
mp 258 °C (dec).
~H (DMSO-d6, 300 MHz) b 3.45 (3H, s), 6.95 (1H, d), 7.7 (1H, d), 7.8 (1H, s),
8.3-8.7 (4H, br
s), 8.3 (1H, d), 8.4 (1H, d), 8.45 (1H, s), 8.9 (1H, s), 10.05 (1H, br s),
10.9 (1H, br s), 12.75
(1H, br s) ppm.
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LRMS 450 (MH+).
Anal. Found: C, 44.50; H, 4.60; N, 12.17. Calc for
C,8H,6C1N505S~i.OHCI~1.0(CH3)zCHOH~I.OHzO: C, 44.b9; H, 4.82; N, 12.41.
Example 4:
(a) N [(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]glycine t-butyl ester
hydrochloride
(b) N ((4-Chloro-1-guanidine-7-isoquinolinyl)sulphonyl]glycine
trifluoroacetate
a c1 c1
w w ~ ~ .
/ iN n ~ I / iN ~ ~ I / iN
tBu02C~H-S tBuO;C H-S HOC H-S
0 CI 0 N\'NH, 0 N' /NH=
'N~H, ~N'H2
NaH (29 mg, 80% dispersion by wt in mineral oil, 0.97 mmol) was added in one
portion to a
stirred solution of guanidine hydrochloride ( 146 mg, 1.52 mmol) in DMSO (2.0
mL) and the
mixture was heated at 60 °C under Nz for 30 min. N (( 1,4-Dichloro-7-
isoquinolinyl)sulphonyl]glycine t-butyl ester (150 mg, 0.38 mmol) was added
and the mixture
heated at 90 °C for 9 h. The cooled mixture was diluted with water, (30
mL), extracted with
EtOAc (4x20 mL) and the combined organic extracts were washed with water,
brine, dried
(NazS04) and evaporated in vacuo. The residue was dissolved in EtzO and a
solution of HCl
in EtzO (1 M) was added to give a sticky precipitate . The EtzO was decanted
and the residue
triturated with EtOAc to give a white solid. Filtration with EtOAc and EtzO
washing gave N
j(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]glycine t-butyl ester
hydrochloride (68 mg,
0.14 mmol).
mp 172-175 °C.
'H (DMSO-d6, 300 MHz) 8 1.2 (9H, s), 3.75 (2H, s), 8.3 (1H, d), 8.35-8.4 (2H,
m), 8.5 (1H,
s), 8.5-8.9 (4H, br), 9.1 ( 1 H, s), 11.3 (1 H, br s) ppm.
LRMS 414, 416 (MH+)
Anal. Found: C, 42.45; H, 4.92; N, 14.76. Calc for
C,6HzoCIN50dS~1.OHC1~0.33H20~0.2EtOAc: C, 42.58; H, 4.95; N, 14.78.
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N-[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]glycine t-butyl ester
hydrochloride (50
mg, 0.11 mmol) was dissolved in CF3CO~H ( 1.0 mL) and the mixture stirred at
room
temperature for 1.5 h. The mixture was diluted with PhMe and the solvents were
evaporated
in vacuo. The residue was triturated with Et~O and EtOAc to give N [(4-chloro-
1-guanidino-
7-isoquinolinyl)sulphonyl]glycine trifluoroacetate (36 mg, 0.073 mmol) as a
white powder.
tH (CF3CO,D, 400 MHz) 8 4.1 (2H, s), 8.25 (1H, d), 8.3 (1H, s), 8.55 (1H, d),
9.0 (1H, s)
ppm.
LRMS 358 (MH+), 715 (MPH+)
Anal. Found: C, 36.25; H, 2.86; N, 14.28. Calc for
C,ZHi~ClN50aS~I.OCF3CO~H~0.2Et0Ac:
C, 36.32; H, 3.01; N, 14.31.
IS
Example 5:
(a) N [(4-chloro-1-guanidino-7-isoquinoiinyl)sulphonylj-(3-alanine t butyl
ester
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonylj-(3-alanine
trifluoroacetate
I c1 c1
t8u0ZC~N-~ l / sN tBu02C~N_5 I / .~N -- HOzC~N-S I / iN
H 0 CI H 0 N' /NH2 H 0 N\'NHZ
'N~HZ 'N~H,
Guanidine hydrochloride (140 mg, 1.46 mmoi was added in one portion to a
stirred
suspension of NaH (35 mg, 80% dispersion by wt in mineral oil, 1.17 mmol) in
DME (8.0
mL) and the mixture was heated at 30 °C under Nz for 30 min. N [( 1,4-
Dichloro-7-
isoquinolinyl)sulphonyl]-(3-alanine t-butyl ester (150 mg, 0.37 mmol) was
added and the
mixture heated at 90 °C for 18 h. The cooled mixture was diluted with
EtOAc, washed with
water, brine, dried (MgS04) and evaporated in vacuo. The residue was purified
by column
chromatography upon silica gel using CH~CIZ-MeOH-0.880NH3 (97:3:0.3 to
95:5:0.5) as
eluant to give N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-(3-alanine
t-butyl ester (75
mg, 0.175 mmol) as a yellow foam
mp >180 °C (dec).
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tH (DMSO-dh, 300 MHz) 8 1.35 (9H, s), 2.3 (2H, t), 2.9 (2H, dt), 7. i-7.4 (4H,
br), 7.8 ( 1 H, br
t), 8.05 (2H, s), 8.1 ( 1 H, s), 9.1 ( 1 H, s) ppm.
LRMS 428 (MH+).
Anal. Found: C, 47.32; H, 5.24; N, 16.02. Calc for C,~H»C1N;O4S~0.2H~0: C,
47.32; H,
5.23; N, 16.23.
N-[(4-Chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-(3-alanine t-butyl ester
(30 mg, 0.07
l0 mmol) was dissolved in CF3CO~H (1.0 mL) and the mixture stirred at room
temperature for 1
h. The mixture was evaporated in vacate, azeotroping with PhMe, MeOH and then
CH,Ch, to
give N-[(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-(3-alanine
trilluoroacetate (32 mg,
0.066 mmol) as a white solid.
mp >200 °C (dec).
iH (DMSO-dh + DSO, 400 MHz) cS 2.35 (2H, t), 3.0 (2H, t), 8.2 ( 1 H, d),8.3 (
1 H, d), 8.4 ( 1 H,
s), 9.1 ( 1 H, s) ppm.
LRMS 372 (MH+).
Anal. Found: C, 37.38; H, 3.11; N, 14.52. Calc for C,3H,4C1N504S~I.OCF3CO2H:
C, 37.08; H,
3.11; N, 14.42.
Example 6:
(a) N [(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-N methylglycine t-
butyl ester
(b) N [(4-Chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-N methylglycine
bis(trifluoroacetafe)
I c1 I
0
tBuOzC~N-S ~ ~ N tBuOzC~N-S ~ ~ HO,C~N-S ~ ~ N
I II I II I II
Me 0 CI Me O N \'NH; Me 0 NYNHZ
'N~HZ NH2
Guanidine hydrochloride (286 mg, 2.99 mmol was added in one portion to a
stirred
suspension of NaH (77.5 mg, 80% dispersion by wt in mineral oil, 2.58 mmol) in
DME (2.0
mL) and the mixture was heated at 50 °C under Nz for 20 min. A solution
of N [(1,4-dichloro-
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7-isoquinolinyl)sulphonyl]-N methylglycine t-butyl ester (393 mg, 0.97 mmol)
in DME (10
mL) was added and the mixture heated at 90 °C for 2 h. The solvents
were evaporated in
vacuo and the residue was puritied by column chromatography upon silica gel
using CH~CI,-
MeOH-0.880NH3 (97:3:0.3) as eluant to give N [(4-chioro-1-guanidino-7-
isoquinolinyl)sulphonyl]-N methylglycine t-butyl ester (260 mg, 0.607 mmol) as
an off white
foam
mp 84 °C.
~H (DMSO-dh, 300 MHz) b 1.3 (9H, s), 2.85 (3H, s), 3.95 (2H, s), 7.0-7.5 (4H,
br), 8.0 (IH,
d), 8.05 ( I H, d), 8.1 ( 1 H, s), 9.05 ( 1 H, s) ppm.
LRMS 427 (MH+), 855 (MPH+).
IS Anal. Found: C, 47.92; H, 5.38; N, 15.07. Calc for C,~HZZCINSO~S: C, 47.72;
H, 5.18; N,
I 6.3 7.
N [(4-Chloro-I-guanidino-7-isoquinolinyl)sulphony(]-N methylglycine t-butt'(
ester (255 mg,
5.96 mmol) was dissolved in CF3COaH (4.0 mL) and CHzCIz (2.0 mL), and the
mixture
stirred at room temperature for I h. The mixture was diluted with PhMe and the
solvents were
evaporated in vacuo to give N [(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]-N
methylglycine bis(trifluoroacetate) (349 mg, 0.56 mmol) as a white powder.
mp 240-242 °C (dec).
~H (DMSO-d6, 300 MHz) 8 2.9 (3H, s), 4.05 (2H, s), 8.3 (1H, d), 8.4 (1H, d),
8.4-8.7 (4H, br),
8.5(lH,s),8.9(IH,s)ppm.
LRMS 372, 374 (MH~), 744 (MZH+).
Anal. Found: C, 36.26; H, 3.10; N, 11.04. Calc for
C,3H,QCINSOdS~2.OCF3COzH~0.3PhMe: C,
36.56; H, 2.96; N, 11.16.
Example 7:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N phenylglycine t-
butyl ester
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(b) N ((4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl)-N phenylglycine
trifluoroacetate
c1 ct a
0 l ~ ~1 o I
..\ II / ,N ~ a / iN ,\ II / ,N
tBu02C N-S tBuO_C N-S HOZC N-S
0 CI 0 NYNH2 0 N\'NH2
\ I \ I NHZ \ I 'N~HZ
NaH (32 mg, 80% dispersion by wt in mineral oil, 1.07 mmol) was added in one
portion to a
stirred suspension of guanidine hydrochloride (164 mg, 1.71 mmol) in DME (5.0
mL) and
the mixture was heated at 60 °C under N, for 30 min. N [(1,4-Dichloro-7-
isoquinolinyl)sulphonyl]-N phenylglycine t-butt'( ester (200 mg, 0.43 mmol)
was added and
the mixture heated at 95 °C for 6 h. The solvents were evaporated in
vaczro and the residue
was purified by column chromatography upon silica gel using CH~CI,-MeOH-
0.880NH3
(97:3:0.3 to 95:5:0.5) as eluant to give N [(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]-
N-phenylglycine t-butyl ester (28 mg, O.OS7 mmol) as a yellow resin.
tH (DMSO-dh, 300 MHz) 8 1.3 (9H, s), 4.45 (2H, s), 7.2-7.3 (2H, m), 7.2-7.4
(4H, br), 7.3-7.4
(3H,m),7.9(lH,d),8.0(lH,d),8.1 (IH,s),8.95(lH,s)ppm.
LRMS 490, 492 (MH+), 981 (MPH+)
N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N phenylglycine t-butyl
ester (25 mg,
0.05 mmol) was dissolved in CF3CO~H (1.0 mL) and the mixture stirred at room
temperature
for 2 h. The mixture was concentrated in vacuo, azeotroping with PhMe, and the
residue
triturated with BtzO to give N j(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]-N
pheny(glycine trifluoroacetate (13 mg, 0.23 mmol) as a pale yellow powder.
mp 218-223 °C.
'H (DMSO-dh, 300 MHz) 8 4.5 (2H, s), 7.1-7.2 (2H, d), 7.25-7.4 (3H, m), 7.8-
8.4 (4H, br),
8.0(lH,d),8.2(lH,d),8.35(lH,s),8.9(lH,s)ppm.
LRMS 434, 436 (MH+), 744 (MPH+).
Anal. Found: C, 42.5S; H, 3.39; N, 11.90. Calc for
C,8H,6C1N504S~1.OCF~COZH~Hz0~0.2Etz0: C, 42.74; H, 3.52; N, 12.22.
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Example 8:
(a) N ((4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-N (cyelopenfyimethyi)-
glycine t-
butyl ester
(b) N [(4-Chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-N
(cyclopentylmethyl)glycine
CI CI CI
II ~ /N ~ 1 / rN ~ II / /N
teu02C~N-S iBuO;C N-S HOZC N-5
0 CI ~ 0 N\ /NH; ~ 0 N~NHx
TNH= 'N~Hz
Guanidine hydrochloride (96 mg, i.00 mmol was added in one portion to a
stirred suspension
l0 ofNaH (19 mg, 80% dispersion by wt in mineral oil, 0.63 mmol) in DME (5.0
mL) and the
mixture was heated at 60 °C under N~ for 30 min. A solution of N [( 1,4-
dichloro-7-
isoquinolinyl)sulphonyl]-~V (cyclopentylmethyl)glycine t-butyl ester ( 120 mg,
0.25 mmol) in
DME (5.0 mL) was added and the mixture heated at 90 °C for 3 h. The
solvents were
evaporated in vacuo, the residue was dissolved with EtOAc (200 mL), and washed
with
l~ aqueous NHdCI (I50 mL), dried (MbSOd) and evaporated in vacuo. The residue
was purified
by column chromatography upon silica gel using pentane-EtOAc (100:0 to 40:60)
as eluant to
give N [(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-N (cyclopentylmethyl)-
glycine t-
butyl ester (60 mg, 0.12 mmol).
20 IH (CDCl3, 400 MHz) b 1.1-1.25 (2H, m), 1.35 (9H, s), 1.45-1.7 (4H, m), 1.7-
1.8 (2H, m), 2.1
(lH,m),3.25(2H,d),4.0(2H,s), 8.OS(lH,d),8.1(lH,d),8.15(lH,s),9.2(lH,s)ppm.
LRMS 496 (MH+)
25 Anal. Found: C, 52.99; H, 6.07; N, 13.82. Calc for Cz~H3oC1N504S: C, 53.38;
H, 5.90; N,
14.15.
A solution of HC1 (2 mL, 2 M, 4 mmol) was added to a solution of N [(4-chloro-
1-guanidino-
7-isoquinolinyl)sulphonyl]-N (cyclopentylmethyl)glyeine t-butyl ester (50 mg,
0.10 mmol) in
30 dioxane (4.0 mL) and the mixture was heated at 60 °C for 24 h. The
solvents were evaporated
in vacuo, and the residue triturated with dichloromethane to give N [(4-chloro-
1-guanidine-7-
isoquinolinyl)sulphonyl]-N (cyclopentylmethyl)glycine hydrochloride (40 mg,
0.080 mmol)
as a white solid.
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mp 139-142 °C.
~H (CD30D, 400 MHz) 8 1.2-1.3 (2H, m), 1.5-1.7 (4H, m), 1.7-1.8 (2H, m), 2.2
(1H, m), 3.65
(2H, d), 4.2 (2H, s), 8.35 ( 1 H, d), 8.45 ( 1 H, s), 8.45 ( I H, d), 8.9 ( I
H, s) ppm.
LRMS 440 (MH+).
Anal. Found: C, 43.48; H. 5.32; N, 12.91. Calc for
C,BH»CIN;O.~S~I.OHCI~I.OH~O~0.05CH~C1~~0.05 dioxane: C, 43.17; H, 5.1 l; N,
13.92.
Example 9:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N
(cyclohexylmethyl)glycine t-
butyl ester
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N
(cyclohexylmethyl)glycine
l5 hydrochloride
c1 c1 c1
op 1 oi~ ~ op 1
II / ,N -~ ~ II / ,N ---~ ~ It / /N
t8°OZC N-S ieuO:C N-S HOZC N-S
O CI O NYNH2 O N\/NHx
NHZ NTH=
Guanidine hydrochloride (125 mg, 1.31 mmol was added in one portion to a
stirred
suspension ofNaH (25 mg, 80% dispersion by wt in mineral oil, 0.82 mmol) in
DME (10
mL) and the mixture was heated at 60 °C under NZ for 30 min. lV [(1,4-
Dichloro-7-
isoquinolinyl)sulphonyl]-N (cyclohexylmethyl)-glycine t=butyl ester (160 mg,
0.33 mmol)
was added and the mixture heated at 80-90 °C for 2.5 h. The solvents
were evaporated in
vacuo, the residue was dissolved with EtOAc (200 mL), and washed with aqueous
NH~CI
( 150 mL), dried (MgS04) and evaporated in vacuo. The residue was purified by
column
chromatography upon silica gel using pentane-EtOAc (100:0 to 40:60) as eluant
to give N
[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyi]-N (cyclohexylmethyl)glycine
t-butyl ester
(65 mg, 0.127 mmo() as an off white foam.
1H (CDC13, 400 MHz) b 0.8-0.95 (2H, m), 1.1-1.25 (3H, m), 1.3 (9H, s), 1.6-1.8
(6H, m), 3.1
(2H, d), 4.0 (2H, s), 8.0 (1H, d), 8.1 (1H, d), 8.15 (1H, s), 9.2 (1H, s) ppm.
LRMS 510 (MH+).
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Anal. Found: C, 54.21; H, 6.46; N, 13.46. Calc for C23H3zCIN5O4S: C, 54.16; H,
6.32; N,
13.73.
A solution oFHCI (2 mL, 2 M, 4 mmol) was added to a solution ofN [(4-chloro-1-
guanidino-
7-isoquinolinyl)sulphonyl]-N-(cyclohexylmethyl)glycine t-butyl ester (53 mg,
0.10 mmol) in
dioxane (4.0 mL). The mixture was stirred at 23 °C for 18 h and then
heated at 50-60 °C for
16 h. On cooling, a white precipitate crashed out of solution. The solid was
collected by
filtration, triturated with EtOAc and then dried under vacuum to give N [(4-
chloro-1-
guanidino-7-isoquinolinyl)sulphonylJ-N (cyclohexylmethyl)glycine hydrochloride
(26 mg,
0.057 mmol).
1H (CDC13, 400 MHz) 8 0.8-1.0 (2H, m), I.1-1.3 (3'H, m), 1.55-l.8 (6H, m), 3.2
(2H, d), 4.1~
(2H, s), 8.3 ( 1 H, d), 8.45 ( 1 H, d), 8.45 ( I H, s), 8.9 ( 1 H, s) ppm.
IS LRMS 454, 456 (MH+)
Anal. Found: C, 44.70; H, 5.1 S; N, t3.56. Caic for C~3H3,CINSO4S~HCI~H,O: C,
44.89; H,
5.36; N, 13.77.
Example 10:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N: benzylglycine t-
butyl ester
(b) N ((4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N benzylglycine
trifluoroacetate
c1 a c1
w w w w w w
/ iN ~ ~ I / iN --~ ~ ~ I / iN
tBuOzC N-S tBuO=C N-S HO,C N-S
0 CI I ~ 0 N\'NHz I ~ 0 N\'NH,
ZS / / NNHx / 'N~H=
Guanidine hydrochloride (180 mg, 1.88 mmol) was added in one portion to a
suspension of
NaH (45 mg, 80% dispersion by wt in mineral oil, 1.5 mmol). in DME (I l mL)
and the
mixture was heated at 60 °C under Nz for 30 min. N [(1,4-Dichloro-7-
isoquinolinyl)sulphonyl]-N benzylglycine t-butyl ester (225 mg, 0.467 mmol)
was added and
the mixture heated at 90 °C for 18 h. The cooled mixture was poured
into water, extracted
with EtOAc (x3) and the combined organic phase was then washed with water,
dried
(Na2S04) and evaporated in vacuo. The residue was purified by column
chromatography upon
silica gel using CH~CIz-MeOH-0.880NH3 (97:3:0.3) as eluant to give N [(4-
chloro-1-
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guanidino-7-isoquinolinyl)sulphonyl]-N benzylglycine t-butyl ester ( 172 mg,
0.34 mmol) as a
yellow foam.
mp > 150 °C (dec).
~H (DMSO-d~, 400 MHz) 8 1.2 (9H, s), 3.8 (2H, s), 4.45 (2H, s), 7. f-7.4 (4H,
br), 7.2-7.35
(~ H, m), 8.0 ( I H, d), 8. I ( 1 H, d), 8.1 (s, 1 H), 9.1 ( 1 H, s) ppm.
LRMS 504, 506 (MH+).
Anal. Found: C, 55.19; H, 5.55; N, 13.23. Calc for C,3H~~CIN5O4S~O.lC6Hi,,: C,
55.30; H,
5.39; N, 13.66.
N-[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N-benzylglycine t-butyl
ester (50 mg,
0.10 mmol) was dissolved in CF~CO~H (1.0 mL) and the mixture stirred at room
temperature
for 1 h. The mixture was diluted with PhMe and the solvents were evaporated iu
waczrv. The
residue was azeotroped with PhMe and then CH~C1~ to give N [(4-chloro-1-
guanidino-7-
isoquinolinyl)sulphonyl]-N benzylglycine trifluoroacetate (52 mg, 0.10 mmol)
as a white
solid.
mp 274 °C (dec).
~H (DMSO-d6, 400 MHz) b 3.95 (2H, s), 4.5 (2H, s), 7.2-7.35 (5H, m), 8.3 (IH,
d), 8.35 (1H,
d), 8.4-8.6 (4H, br), 8.45 ( 1 H, s), 8.9 ( 1 H, s), 10.6 ( 1 H, br), 12.7 ( 1
H, br) ppm.
LRMS 448, 450 (MH+), 497 (MzH~)
Anal. Found: C, 43.96; H, 3.39; N, 11.87. Calc for
C,9H,$CIN504S~1.OCF3COzH~0.5H~0: C,
44.I8; H, 3.53; N, 12.27.
Example 11:
(a) N ((4-chtoro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (2-
methylbenzyl)glycine t
butyl ester
(b) N ((4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (2-
methylbenzyl)glycine
trifluoroacetate
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c1 c1 c1
w w w w w w
o J 1 _ o J ~ o J
/~ I I / i N ---- /~ I I / i N ~ I I / ~ N
tBuO,C N-S tBu07C N-S HOzC N-S
0 CI I ~ 0 N \/NH= I ~ 0 NYNH2
/ Me / Me TN z / Me NHS
Guanidine hydrochloride ( i 20 mg; 1.26 mmoi) was added in one portion to a
suspension of
NaH (32 mg, 80% dispersion by wt in mineral oil, l.Od~mmol) in DME (l0 mL) and
the
mixture was heated at 60 °C under N, for 30 min. N [( 1,4-Dichloro-7-
isoquinolinyl)sulphonyl]-N (2-methylbenzyl)glycine t-butyl ester (200 mg,
0.405 mmol) was
added and the mixture heated at 90 "C for 2 h. The cooled mixture was diluted
with EtOAc,
washed with water, brine, dried (Na,SOa) and evaporated in vacuo. The residue
was purified
by column chromatography upon silica gel using pentane-CH~CI~ (50:50), then
CH~CI~, and
l0 finally CHZCh-MeOH-0.880NH3 (95:5:0.0 as eluarit to give N [(4-chloro-1-
guanidino-7-
isoquinolinyl)sulphonyl]-N (2-methylbenzyl)glycine t-butyl ester (94 mg, 0. l
8 mmol) as an
off white solid.
mp > I 10 °C (dec).
t~
~H (CDC13, 400 MHz) 8 1.25 (9H, s), 2.3 (3H, s), 3.8 (2H, s), 4.6 (2H, s), 7.1-
7:2 {4H, m),
8.05 ( 1 H, d), 8.1 ( 1 H, d), 8.15 (s, 1 H), 9.3 ( 1 H, s) ppm.
LRMS 518, 520 (MH+).
zo
Anal. Found: C, 56.21; H, 5.83; N, 12.57. Calc for
Cz4H28C1N504S~0.3Hz0~0.25C6H,4: C,
56.20; H, 5.94; N, 12.85.
N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (2-methylbenzyl)glycine
t-butyl
25 ester (30 mg, 0.058 mmol) was dissolved in CF3CO~H (1.0 mL) and the mixture
stirred at
room temperature for 1 h. The mixture was diluted with PhMe and the solvents
were
evaporated fn vacuo. The residue was azeotroped with PhMe and then Et20 to
give N [(4-
chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (2-methylbenzyl)glycine
trifluoroacetate
(29 mg, 0.05 mmol) as an off white solid.
mp >150 °C (dec).
~H (CD30D, 400 MHz) 8 2.3 (3H, s), 3.95 (2H, s), 4.7 (2H, s), 7.05-7.2 (4H,
m), 8.35 (1H, d),
8.45 ( 1 H, s), 8.45 ( 1 H, d), 8.9 ( 1 H, s) ppm.
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LRMS 462, 464 (MH+).
Anal. Found: C, 45.51; H, 3.95; N, 11.36. Calc for
C~~H~oCIN50.~S~1.OCF3CO~H~1.OH~0~O.IPhMe: C, 45.20; H, 3.98; N, 1 1.61.
Example 12:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (2-
methoxybenzyl)glycine t-
butyl ester trifluoroacetate
IO (b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (2-
methoxybenzyl)glycine
trifluoroacetate
ci ci c1
/ iN ~ ~ I / iN ~ ~ I / iN
t8u02C~N-S tBuO_C N-S HO=C N-S
O CI I ~ 0 N"NH= I ~ O N \ /NHZ
/ OMe / OMe 'NH' / OMe NTH,
15 Guanidine hydrochloride (225 mg, 2.36 mmol) was added in one portion to a
stirred
suspension of NaH (44 mg, 80% dispersion by wt in mineral oil, 1.47 mmol) in
DME (20
mL) and the mixture was heated at 60 °C under N~ for 30 min. N [(1,4-
Dichloro-7-
isoquinolinyl)sulphonyi]-N (2-methoxybenzyl)glycine t-butyl ester (300 mg,
0.59 mmol) was
added and the mixture heated at 90 °C for 2 h. The cooled mixture was
poured into water and
20 extracted with EtOAc (x3). The combined organic extracts were then washed
with water,
brine, dried (Na~S04) and evaporated in vacuo. The residue was purified by
column
chromatography upon silica gel using hexane-EtOAc (80:20), and then CHZCIZ-
MeOH-
0.880NH3 (95:5:0.5 to 90:10:1) as eluant to give the product as a yellow semi-
solid. This
semi-solid was dissolved in EtOAc, a solution of TFA (35 p,L) in EtOAc ~(25
mL) was added
25 and the solvents were evaporated in vacuo, azeotrop,ing with PhMe. The
residue was triturated
with i-PrzO, the resulting white solid was collected by filtration, and then
dried to give N [(4-
chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-IV (2-methoxybenzyl)glycine t-
butyl ester
trifluoroacetate (219 mg, 0.338 mmol).
30 mp >197 °C (dec).
1H (DMSO-dh, 400 MHz) i; 1.25 (9H, s), 3.6 (3H, s), 4.0 (2H, s), 4.45 (2H, s),
6.8-6.9 (2H,
m), 7.1-7.2 (2H, m), 8.3 (2H, s), 8.4-8.6 (4H, br s), 8.5 (s, 1 H), 8.8 ( 1 H,
s) ppm.
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LRMS 534, 536 (MH+)
Anal. Found: C, 48.33; H, 4.55; N, 10.52. Caic for Co4H2gCIN5O5S~i.OCF3C0,_H:
C, 48.18; H,
4.51; N, 10.81.
N-[(4-chloro-I-guanidino-7-isoquinolinyl)sulphonyl]-N (2-methoxybenzyl)glycine
t-butyl
ester trifluoroacetate (I50 mg, 0.231 mmol) was dissolved in CF3CO~H (1.0 mL)
and the
mixture stirred at room temperature for 40 min. The mixture was diluted with
PhMe,
concentrated in vacuoazeotroping with PhMe, and the residue triturated with i-
Pr,O to give
l0 1V [(4-chloro-i-guanidino-7-isoquinolinyl)sulphonyl]-N(2-
methoxybenzyl)glycine
trifluoroacetate (122 mg, 0.206 mmol) as a white solid.
mp > 165 °C (dec).
i 5 ~ H (DMSO-d~, 400 MHz) 8 3.6 (3 H, s), 4.0 (2H, s), 4.5 (2H, s), 6.8 ( l
H, d), 6.85 ( 1 H, dd),
7.1-7.2 (2H, m), 8.3 (2H, s), 8.35-8.5 (4H, br s), 8.5 (s, 1 H), 8.8 ( 1 H, s)
ppm.
LRMS 478, 480 (MH+).
20 Anal. Found: C, 44.64; H, 3.58; N, 11.83. Calc for
CzoH~oClN505S~1.OCF3COZH: C, 44.69; H,
3.68; N, 11.63.
Example 13:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (3-
methoxybenzyl)glycine t-
25 butyl ester hydrochloride
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (3-
methoxybenzyl)glycine
ci
' '1
HO C~N-5 I ~ ~ N
x
' 0 N\'NH2
. ~ ~ ~Hz
OMe
30 Guanidine hydrochloride (149 mg, 1.55 mmol) was added in one portion to a
suspension of
NaH (35 mg, 80% dispersion by wt in mineral oil, I.I6 mmol) in DME (10 mL) and
the
mixture was heated at 60 °C under N~ for 30 min. N [(1,4-Dichloro-7-
isoquinolinyl)sulphonyl]-N (3-methoxybenzyl)glycine t-butyl ester (200 mg,
0.39 mmol) was
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added and the mixture heated at 90 °C for 2 h. The cooled mixture was
poured into water,
extracted with EtOAc (x3), and the combined organic extracts were washed with
brine, dried
(Na~SOa) and evaporated in vacuo. The residue was dissolved in Et~O-EtOAc and
a solution
of HCI in Et~O (0.5 M) was added to give a precipitate. The solid was
collected by filtration,
triturated with EtOAc and then dried to give N [(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]-N-(3-methoxybenzyl)glycine t-butyl ester
hydrochloride (124 mg,
0.21 mmol) as a white solid.
mp 203-20S °C.
l0
'H (DMSO-d~, 300 MHz) cS 1.2 (9H, s), 3.65 (3H, s), 4.05 (2H, s), 4.5 (2H, s),
6.7 (1H, s),
6.75-6.85 (2 H,, .m), 7.2 ( 1 H, dd), 8.3 ( 1 H, d), 8.35 ( I H, d), 8.S (s, I
H), 9.3 ( 1 H, s), 11.6 ( 1 H,
br s) ppm.
IS ' I,RMS 534, S36 (MH+), 1069 (M,H+).
Anal. Found: C, 50.22; H, S.l l; N, 12.23. Calc for Cz4H,~CIN505S~I.OHCI: C,
S6.S2; H, 5.12;
N, 12.28.
20 N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (3-
methoxybenzyl)glycine t-butyl
ester hydrochloride (95 mg, 0.167 mmoi) was dissolved in CF3CO~H (1.0 mL) and
the
mixture stirred at room temperature for 1 h. The mixture was diluted with PhMe
and the
solvents were.evaporated in vacuo. The residue was dissolved in EtOAc and
stirred at room
temperature for 1 h. The resulting precipitate was co((ected by filtration,
washed with EtzO
25 and dried to give N [(4-chloro-1-guanidino-7-isoquinoliny!)sulphonyl]-N (3-
methoxybenzyl)glycine (6S mg, 0.128 mmol) as a white powder.
mp 290 °C (dec).
30 'H (CF3COzD, 400 MHz) 8 3.9 (3H, s), 4.3 (2H, s), 4.6 (2H, s), 6.9-7.0 (3H,
m), 7.3 (1H, dd),
8.35 (1H, d), 8.45 (1H, s), 8.6 (1H, d), 8.95 (1H, s) ppm.
LRMS 477, 479 (MH+), 9SS (MZH+).
35 Anal. Found: C, 48.67; H, 4.09; N, 13.88. Calc for
CZOHZOCiN505S~0.25CF3CO~H: C, 48.62;
H, 4.03; N, 13.83.
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Example 14:
(a) N ((4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-N (3-
chlorobenzyl)glycine t-
butyl ester hydrochloride
(b) N [(4-Chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-N (3-
chlorobenzyl)glycine
trifluoroacetate
c1
~ i ~N
~e~ ie~o:c~N-s
O N\/NH2
NNH,
CI
. NaH (35 mg, 80% dispersion by wt in mineral oil, 1.16 mmo() was added in one
portion to a
suspension of guanidine hydrochloride ( 150 mg, 1.55 mmol) in DME ( 10 mL) and
the
mixture was heated at 60 °C under N~ for 30 min. N [( i,4-Dichioro-7-
isoquinolinyl)sulphonyl]-N (3-chlorobenzyl)glycine t-butyl ester ( 185 mg,
0.36 mmol) was
added and the mixture heated at 90 °C for 5 h. The cooled mixture was
diluted with Et~O,
washed with water, dried (Na~S04) and evaporated in vaczro. The residue was
dissolved in
EtzO and a solution of HCl in Et20 (1 M) was added to give a precipitate. The
solvents were
evaporated in vacuo, and the white solid triturated,with EtOAc and then dried
to give N [(4-
chloro-1-guanidine-7-isoquinolinyl)sulphony(J-N (3-chlorobenzyl)glycine t-
butyl ester
hydrochloride (85 mg, 0.145 mmol).
mp 203-205 °C.
~H (DMSO-d6, 300 MHz) 8 1.2 (9H, s), 4.1 (2H, s), 4.55 (2H, s), 7.2-7.35.(4H,
m), 8.3 (1H,
d), 8.35 (1H, d), 8.5 (s, 1H), 9.3 (1H, s), 11.55 (1H, br s) ppm.
LRMS 538, 540 (MH+), 1076 (M~H~)
Anal. Found: C, 47.04; H, 4.53; N, 11.82. Calc for
C~3H~SChN504S~I.OHCI~O.SH~O: C,
47.31; H, 4.66; N, 11.99.
N [(4-Chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-N (3-chlorobenzyl)glycine
t-butyl ester
hydrochloride (60 mg, 0.104 mmol) was dissolved in CF3COZH (0.5 mL) and the
mixture
stirred at room temperature for 1 h. The mixture was diluted with PhMe and the
solvents were
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evaporated in vacuo. The residue was dissolved in EtzO and stirred at room
temperature for 1
h. The resulting precipitate was collected by filtration, washed with Et~O and
dried to give N-
[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (3-chlorobenzyl)glycine
trifluoroacetate
(31 mg, 0.052 mmol) as a white solid.
mp 306-308 °C.
~H (CF3CO,D, 400 MHz) 8 4.3 (2H, s), 4.55 (2H, s), 7.0-7.1 (2H, m), 7.1-7.15
(2H, m), 8.25
( 1 H, d), 8.4 ( 1 H, s), 8.5 ( 1 H, d), 8.8 ( 1 H, s) ppm.
LRMS 482,484 (MH+), 496, 498 (MH+ of corresponding methyl ester).
Anal. Found: C, 42.60; H, 3.04; N, 12.03. Calc for C19H17ChN5OQS'I.OCF3CO~H:
C, 42.29;
H, 3.04; N, 11.74
Example 15:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonylj-N (4-
methoxybenzyl)glycine t-
butyl ester hydrochloride
(b) N ((4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (4-
methoxybenzyl)glycine
c1 a c1
o I w w o ~ w w " o ~ w w
II / ,N ~ 11 / iN ~ II / iN
tBuOZC N-S tBuO=C N-S HO=C N-S
0 CI ~ 0 N\'NHz ~ 0 NYNH=
Me0 I / Mep l / , 'N~Hz Me0 I / NHz
Guanidine hydrochloride (118 mg, 1.24 mmol) was added in one portion to a
stirred
suspension of NaH (23 mg, 80% dispersion by wt in mineral oil, 0.78 mmol) in
DME (10
inl,) and the mixture was heated at 60 °C under Nz for 30 min. N [(1,4-
Dichloro-7-
isoquinolinyl)sulphonyl]-N (4-methoxybenzyl)glycine t-butyl ester (155 mg,
0.31 mmol) was
added and the mixture heated at 90 °C for 1 h. The cooled mixture was
poured into water and
extracted with EtOAc (x3). The combined organic extracts were then washed with
water,
brine, dried (Na~S04) and evaporated in vacuo. The residue was purified by
column
chromatography upon silica gei using hexane-EtOAc (80:20), and then CHzCl2-
MeOH-
0.880NH3 (95:5:0.5 to 90:10:1) as eluant to give a yellow gum. Trituration
with i-Pr20 gave
N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (4-methoxybenzyl)glycine
t-butyl
ester (80 mg, 0.15 mmol) as a sticky yellow solid. A small sample ( 10-15 mg)
was dissolved
in EtOAc, a solution of HCl in EtzO was added and the solvents were evaporated
in vacuo, to
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give N [(4-chloro-1-guanidino-7-isoquinolinyi)sulphonyi]-N (4-
methoxybenzyl)glycine t-
butyl ester hydrochloride ( 18 mg) as a solid. (All characterisation data is
for the HCI salt).
mp > 192 °C (dec).
~H (DMSO-dh, 400 MHz) cS 1.2 (9H, s), 3.7 (3H, s), 4.0 (2H, s), 4.4 (2H, s),
6.8 (2H, d), 7.1
(2H, d), 8.3 ( 1 H, d), 8.3 ( I H, d), 8.4-8.9 (4H, br s), 8.5 (s, I H), 8.2 (
( H, s) ppm.
LRMS 534, 536 (MH+)
Anal. Found: C, 51.36; H, 5.53; N, l 1.23. Calc for
C,.~H~$CINSOSS~1.OHC1~0.28i-Pr~O: C,
51.48; H, 5.54; N, 1 1.69.
~V [(4-chloro-I-guanidino-7-isoquinolinyl)sulplzonyi]-N (4-
methoxybenzyl)glycine t-butyl
ester (65 mg, 0.122 mmol) was dissolved in CF3CO~H ( 1.0 mL) and the mixture
stirred at
room temperature for 40 min. The mixture was diluted with PhMe, concentrated
ir7 vacuo, and
the residue purified by column chromatography upon silica gel using CHZCh-MeOH-
0.880NH3 (83:15:3) as eluant to give N [(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]-N
(4-methoxybenzyl)glycine ( 11 mg, 0.023 mmol) as a white solid.
mp >293 °C (dec).
'H (DMSO-d6, 400 MHz) i; 3.7 (3H, s), 3.8 (2H, s), 4.4 (2H, s), 6.85 (2H, d),
7.15 (2H, d),
7.2-7.5 (4H, br s), 8.0 ( 1 H, d), 8.1 ( 1 H, d), 8.15 (s, 1 H), 9.1 ( 1 H, s)
ppm.
Anal. Found: C, 48.44; H, 4.47; N, 14.12. Calc for CZOH~oCIN505S~ 1.OH~O: C,
48.34; H,
4.27; N, 14.28.
Example 16:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonylJ-N (2-
pyridylmethyl)glycine t-
butyl ester
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (2-
pyridylmethyl)glycine
dihydrochloride
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ci ci ci
o i~ ~1 0 l~ ~1 0 l
rw ~~ r ~N , ~ n r ~N ~ n r ~N
tBu02C N-S ~ tBuOZC N-S H02C N-S
0 CI I ~ O N\'NHZ I ~ 0 NYNH=
iN iN ~N'H2 ,N NH,
Guanidine hydrochloride (293 m~~, 3.07 mmoi was added in one portion to a
stirred
suspension of NaH (57 mg, 80% dispersion by wt in mineral oil, I .92 mmol) in
DME (I0
mL) and the mixture was heated at 60 °C under N, for 30 min. A solution
of N [(1,4-dichloro-
7-isoquinolinyl)sulphonyl]-N (2-pyridylmethyl)glycine t-butyl ester (370 mg,
0.78 mmol) in
DME (10 mL) was added and the mixture heated at 90 °C for 1 h. The
solvents were
evaporated in vacuo, the residue was dissolved with EtOAc (200 mL), and washed
with
aqueous NH.~C1 ( 150 mL), dried (MgSOa) and evaporated in vacuo. The residue
was purified
by column chromatography upon silica gel using pentane-EtOAc ( 100:0 to 20:80)
as eluant to
give N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (2-
pyridylmethyl)glycine l-butyl
ester ( 120 mg, 0.24 mmol) as a pale yellow foam.
I H (CDC13, 400 MHz) 8 1.3 (9H, s), 4.1 (2 H, s), 4.65 (2 H, s), 7.2 ( 1 H,
m), 7.5 ( 1 H, d), 7.65
( I H, dd), 8.05 ( I H, d), 8. I ( 1 H, d), 8. I ( I H, s), 8.45 ( 1 H, d),
9.25 ( I H, s) ppm.
LRMS 505 (MH+)
Anal. Found: C, 51.93; H, 5.03; N, 15.45. Calc for
CZZH~SC1N604S~O.lHzO~0.2Et0Ae: C,
52.24; H, S.IB; N, 15.89.
A solution of HC1 (3 mL, 2 M, 6 mmol) was 'added to a solution of N [(4-chloro-
I-guanidino
7-isoquinolinyl)sulphonyl]-N (2-pyridylmethyl)glycine t-butyl ester (1 I S mg,
0.23 mmol) in
dioxane (5.0 mL) and the mixture was heated at 60 °C for I8 h. The
solvents were evaporated
in vacuo and the residue triturated with hot EtOAc to give N [(4-chloro-1-
guanidino-7
isoquinolinyl)sulphonyl]-N (2-pyridylmethyi)glycine dihydrochioride (95 mg,
0.167 mmol)
as an off white solid.
mp 216-220 °C.
'H (CD30D, 400 MHz) 8 4.4 (2H, s), 5.1 (2H, s), 8.05 (1H, m), 8.3 (1H, d), 8.4
(IH, d), 8.5
(lH,s),8.5(lH,d),8.6(lH,dd),8.85(lH,d),9.3(lH,s)ppm.
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Anal. Found: C, 39.01; H, 4.01; N, 14.14. Calc for
C,8H,7CIN604S~2.OHCi~2.OH20~0.12dioxane: C, 39.05; H, 4.25; N, I4.78.
Example 17:
(a) N [(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-N (3-
pyridylmethyl)glycine t-
butyl ester
(b) N ((4-Chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-N (3-
pyridylmethyl)glycine
dihydrochloride
a a c1
n ~ ~ / iN n ~ ~ / iN ~ 101 ~ / iN
t8u0=C N-S tBuO.C N-S HOiC N-S
I N ~ CI I N O N\/NFix. I N O N\/NH,
i i 'N~Hz i TNH=
~0
Guanidine hydrochloride (317 mg, 3.32 mmol was added in one portion to a
stirred
suspension ofNaH (62.3 mg, 80% dispersion by wt in mineral oil, 2.08 mmol) in
DME (10
mL) and the mixture was heated at 60 "C underN~ for 30 min. A solution ofN
[(1,4-dichloro-
l5 7-isoquinolinyl)suiphonyl]-N (3-pyridylmethyl)glycine t-butyl ester (400
mg, 0.83 mmol) in
DME (10 mL) was added and the mixture heated at 80 °C for 4 h. The
solvents were
evaporated in vacuo, the residue was dissolved with EtOAc (200 mL), and washed
with
aqueous NHøCl (200 mL), dried (MgSOa) and evaporated in vacuo. The residue was
purified
by column chromatography upon silica gel using (i) pentane-EtOAc (70:30 to
50:50) and then
20 (ii) CHzCiz-MeOH-0.880NH3 (95:5:0.5 to 90:101) as eluant to give N [(4-
chloro-1-guanidino-
7-isoquinolinyl)sulphonyl]-N (3-pyridylrilethyl)glycine t-butyl ester (104 mg,
0.21 mmol) as
a pale yellow solid.
'H (CDCl3, 400 MHz) 8 1.3 (9H, s), 3.8 (2H, s), 4.5 (2H, s), 6.4-6.8 (4H, br),
7.2 (1H, m), 7.6
25 (lH,d),8.0(lH,d),8.05(lH,s),8.05(lH,d),8.4(lH,s),8.5(lH,d),9.3(lH,s)ppm.
LRMS 505, 507 (MI-i").
Anal. Found: C, 51.95; H, 5.02; N, 16.25. Calc for C»H~SC1N604S: C, 52.33; H,
4.99; N,
30 16.64.
CF3CO~H ( 1.0 mL) was added to a stirred solution of N [(4-chloro-1-guanidine-
7-
isoquinolinyl)sulphonyl]-N (3-pyridylmethyl)glycine t butyl ester (100 mg,
0.20 mmol) in
CHZCIZ (1.0 mL) and the mixture was stirred at 23 °C for 3.5 h. The
solvents were evaporated
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in vacuo, azeotroping with PhMe and CH~CI~. The oily residue was dissolved in
EtOAc and a
solution of EtOAc saturated with HCI (3.0 mL) was added which gave a
precipitate. The
white solid was collected by filtration and dried to give N [(4-chloro-I-
guanidino-7-
isoquinolinyl)sulphonyl]-N (3-pyridyimethyi)glycine dihydrochioride (48 mg,
0.086 mmol).
I H (CD30D, 400 MHz) 8 4.25 (? H, s), 4.9 (2H, s), 8.05 ( 1 H, dd), 8.4 ( ( H,
d), 8.45 ( 1 H, s),
8.5(lH,d),8.7(lH,d),8.8(lH,d),9.0(lH,s),9.2(lH,s)ppm.
Anal. Found: C, 39.32; H, 4.07; N, 15.07. Calc for
t0 C,8Hi7CIN~0aS~2.OHC1~1.5H~0~O.OSEtOAc~O.OS CH~CI~: C, 39.19; H, 3.72; N,
14.64.
Example 18:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (4-
pyridylmethyl)glycine t-
butyl ester
1 ~ (b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonylj-N (4-
pyridylmethyl)glycine
dihydrochloride
c1 c1 ca
o I ~ ~ o I ~ ~ o I
ri / ~N ~ a / ~N rr / ~N
tBuOZC~N-S'~~, t8u0,C N-S HOzC~N-S
O CI O N ~ NHZ 0 N ~ NH=
NI / NI / z NI / a
20 Guanidine hydrochloride (300 mg, 3.14 mmol was added in one portion to a
stirred
suspension ofNaH (59 mg, 80% dispersion by wt in mineral oil, 1.97 mmol) in
DME (10
mL) and the mixture was heated at 60 °C under NZ for 30 min. A solution
of N [( I,4-dichloro-
7-isoquinolinyl)sulphonyl]-N (4-pyridylmethyl)glycine t-butyl ester (379 mg,
0.79 mmol) in
DME (10 mL) was added and the mixture heated at 80 °C for 4 h. The
solvents were
25 evaporated in vacuo, the residue was dissolved with EtOAc (200 mL), and
washed with
aqueous NH4C1 (150 mL), dried (MgS04) and evaporated in vacuo. The residue was
purified
by repeated column chromatography upon silica gel using (i) pentane-EtOAc
(70:30 to 50:50)
and then with (ii) CHZCh-MeOH-0.880NH3 (95:5:0.5 to 90:101) as eluant to give
N [(4-
chloro-I-guanidino-7-isoquinolinyl)sulphonyl]-N (4-pyridylmethyl)glycine t-
butyl ester (96
30 mg, 0.19 mmol).
1H (CDCl3, 400 MHz) 8 1.3 (9H, s), 3.9 (2H, s), 4.5S (2H, s), 7.25 (2H, d),
8.05 (1H, d), 8.1
(lH,d),8.15(lH,s),8.6(2H,d),9.3(lH,s)ppm.
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L1ZMS SOS, 507 (MH+)
Anal. Found: C, 52.63; H, 5.09; N, 16.18. Calc for CZ,H~SC1N604S: C, 52.33; H,
4.99; N,
16.64.
CF3CO~H ( 1.0 mL) was added to a stirred solution of N [(4-chloro-1-guanidino-
7-
isoquinolinyl)sulphonyl]-N (4-pyridylmethyl)glycine t-butyl ester (88 mg, 0.17
mmol) in
CH,Ch (1.0 mL) and the mixture was stirred at 23 °C for 3.S h. The
solvents were evaporated
in vacuo, azeotroping with CH~C1~. The oily residue was dissolved in CH~C12-
MeOH (1.0
l0 mL, 9:1) and a solution of EtOAc saturated with HCI (3.0 mL) was added
which gave a
precipitate. The white solid was collected by filtration and dried to give N
[(4-chloro-1-
guanidino-7-isoquinolinyl)sulphonyl]-N (4-pyridylmethyl)glycine
dihydrochloride (18 mg,
0.033 mmol).
t H (CD30D, 400 MHz) 8 4.3 (2H, s), 5.0 (2H, s), 8.2 (2H, d), 8.4 ( I H, d),
8.5 ( 1 H, s), 8.55
( I H, d), 8.8 (2H, d), 9.1 ( 1 H, s) ppm.
Anal. Found: C, 39.57; H, 4.12; N, 14.85. Calc for
CixH,~CIN60aS~2.OHC1~I.SH~O: C, 39.39;
H, 4.04; N, 15.39.
Example 19:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N ((1R)-1-
phenylethyl]glycine t-
butyl ester
(b) N ((4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N [(1R)-1-
phenylethyl]glycine
hydrochloride
ct c! c1
ol~ ~1 oy y oy y
II / ,iN ~ II / iN ~ II / ,N
tBuO,C N-S tBuOZC N-S HOZC N-S
R O Ct R O N w NH= R 0 N w NHZ
~Me ~Me ~ ~Me
I / . I / NHZ I / NHz
NaH (30 mg, 80% dispersion by wt in mineral oil, 1.01 mmol) was added in one
portion to a
stirred suspension of guanidine hydrochloride (1S4 mg, 1.61 mmol) in DME (6.0
mL) and
the mixture was heated at 60 °C under NZ for 30 min. A solution of N
[(1,4-dichloro-7-
isoquinolinyl)sulphonyl]-N [(1R)-1-phenylethyl]glycine t-butyl ester (200 mg,
0.40 mmol) in
DME (3.0 mL) was added and the mixture heated at 9S °C for S h. The
solvents were
evaporated in vacuo and the residue was purified by column chromatography upon
silica gel
using pentane-EtOAc (SO:SO to 33:66) as eluant to give N [(4-chloro-1-
guanidino-7-
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isoquinolinyl)sulphonyl]-N [(iR)-t-phenylethyl]glycine t-butyl ester (125 mg,
0.23 mmol) as
pale yellow foam after repeated evaporation from CHZCh.
mp 106-111 °C.
'H (DMSO-d~, 300 MHz) b 1.2 (9H, s), 1.3 (3H, d), 3.7 (1H, d), 3.95 (IH, d),
S.OS (1H, q),
7.1-7.4 (4H, br), 7.2-7.3 (5 H, m), 8.0 ( 1 H, d), 8. I ( 1 H, s), 8.2 ( 1 H,
d), 9.15 ( I H, s) ppm.
LRMS 518, 520 (MH+), 1035 (MPH+)
Anal. Found: C, 55.15; H, 5.55; N, 12.84. Calc for
C,dHZBC(NSO;~S~0.2EtOAc~O.ICH,CI~: C,
54.96; H, 5.52; N, 12.87.
N [(4-Chloro-I-guanidino-7-isoquinolinyl)sulphonyl]-N [(IR)-I-
phenylethyl]glycine t-butyl
l ~ ester ( 100 mg, 0.19 mmol) was dissolved in a solution of EtOAc saturated
with HCI (7.0 mL)
and the mirture stirred at room temperature for 4 h. The mi.cture was
concentrated in vacuo
and the residue triturated with EtOAc to give N [(4-Chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]-N [(1R)-1-phenylethyl]glycine hydrochloride (75 mg,
O.I4 mmol) as
a white powder.
mp 185-190 °C.
'H (DMSO-d6, 300 MHz) 8 1.35 (3H, d), 3.85 (1H, d), 4.15 (1H, d), 5.3 (1H, q),
7.15 (5H, br
s), 8.3 ( 1 H, d), 8.4-8.8 (4H, br), 8.4 ( 1 H, d), 8.5 ( 1 H, s), 9.1 ( 1 H,
s), 11.3 ( 1 H, br), 12.5 ( 1 H,
br) ppm.
Anal. Found: C, 47.42; H, 4.40; N, 13.54. Calc for
C~oHZOC1N50,,S~I.OHCI~O,SHaO~0.2EtOAc: C, 47.59; H, 4.53; N, 13.34.
Example 20:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N [(1ST-1-
phenyfethyl]gIycine t-
butyl ester
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N [(1,5~-1-
phenylethyl]glycine
hydrochloride
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a cc c1
cy ~ oy y 0y y
tBuO CAN-S / ~N tBuO CAN-S / ~N ~ HO CAN-S ~ ~N
0 CI S O N~ NHz 5 0 Nw NHx
Me Ma ~ Me
I2 / I~ / NH2 I~ / NH,
NaH (30 mg, 80% dispersion by wt in mineral oil, 1.01 mmol) was added in one
portion to a
stirred suspension of guanidine hydrochloride ( 154 mg, 1.61 mmol) in DME (6.0
mL) and
5 the mixture was heated at 60 °C under Na for 30 min. A solution of N
[( 1,4-dichloro-7-
isoquinolinyl)sulphonyl]-N [(I,S')-1-phenylethyl]glycine t-butyl ester (200
mg, 0.40 mmol) in
DME (3.0 mL) was added and the mixture heated at 95 °C for 5 h. The
solvents were
evaporated in vacuo and the residue was purified by column chromatography upon
silica gel
using pentane-EtOAc (50:50 to 33:66) as eluant to give N j(4-chloro-I-
guanidino-7-
isoquinolinyl)sulphonyl]-N [(1.5~-l-phenylethyl]glycine t-butyl ester (128 mg,
0.23 mmol) as
pale yellow foam after repeated evaporation from CH~CI~.
mp 109-1 I S °C.
t 5 I H (DMSO-dh, 300 MHz) 8 1.2 (9H, s), l .3 (3 H, d), 3.7 ( 1 H, d), 3.95 (
1 H, d), 5.05 ( 1 H, q),
7.1-7.45 (4H, br), 7.2-7.3 (5H, m), 8.0 (1H, d), 8.1 (1H, s), 8.2 (1H, d),
9.15 (1H, s) ppm.
LRMS 518, 520 {MH+), 1035 (MPH+)
Anal. Found: C, 55.26; H, 5.56; N, 12.86. Calc for
C~,,H~8C1N504S~O.IEtOAc~O.OSCH~C12: C,
55.28; H, 5.54; N, 12.97.
N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N [(1ST-1-
phenylethyl]glycine t-butyl
ester (100 mg, 0.19 mmol) was dissolved in a solution of EtOAc saturated with
HCI (4.0 mL)
and the mixture stirred at room temperature for 4 h. The mixture was
concentrated in vacuo
and the residue triturated with EtOAc to give N [(4-Chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]-N [(1ST-1-phenylethyl]glycine hydrochloride (72 mg,
0.14 mmol) as
a white powder.
mp 196-200 °C.
1H (DMSO-d", 300 MHz) 8 1.35 (3H, d), 3.85 (1H, d), 4.15 (1H, d), 5.3 (1H, q),
7.15 (5H, br
s), 8.3 ( 1 H, d), 8.4-8.8 (4H, br), 8.4 ( 1 H, d), 8.5 ( 1 H, s), 9. t ( 1 H,
s), 11.3 ( 1 H, br), 12.4 ( 1 H,
br) ppm.
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Anal. Found: C, 47.42; H, 4.30; N, 13.51. Calc for
CzoH~oC1N50dS~1.OHC(~I.OHzO~O.IEtOAc: C, 47.47; H, 4.45; N, 13.57.
Example 21:
(a) N benzyl-N [(4-chloro-1-guanidino-7-isoquinolinyl)suiphonyl]-L-alanine t-
butyl ester
(b) N Benzyl-N [(4-chloro-1-guanidino-7-isoquinotinyl)sulphonyl]-L-alanine
hydrochloride
CI CI CI
Me 0 I \ ~1 Me \ \
Me \ \
II / iN j~ ~ I / rN j~ ~ I / iN
tBuO,C ~ N-S iBuOZC ~ N-S HO,C ~ N-S
0 CI 0 N~NH= 0 NYNH,
I ~ TNHz I / NH2
~0
NaH (30 mg, 80% dispersion by wt in mineral oil, 1.0I mmo() was added in one
portion to a
stirred suspension of guanidine hydrochloride ( 154 mg, 1.61 mmol) in DME (5.0
mL) and
I S the mixture was heated at 60 °C under NZ for 45 min. A solution of
N benzyl-N [( 1,4-
dichloro-7-isoquinolinyl)sulphonyl]-L-alanine t-butyl ester (200 mg, 0.40
mmol) in DME (2.0
mL) was added and the mi:cture heated at 95 °C for 4 h. The solvents
were evaporated in .
vacuo and the residue was purified by column chromatography upon silica gel
using pentane-
EtOAc (50:50 to 20:80) as eluant to give N benzyl-N [(4-chloro-1-guanidino-7-
20 isoquinolinyl)sulphonyl]-L-alanine t-butyl ester (120 mg, 0.225 mmol) as
pale yellow foam
after repeated evaporation from CHzCIz,
~H (DMSO-d6, 300 MHz) 8 1.1 (9H, s), 1.15 (3H, d), 4.35 (1H, d), 4.5 (1H, q),
4.7 (1H, d),
7.1-7.45 (4H, br), 7.2-7.4 (5H, m), 8.0 (1H, d), 8.1 (1H, d), 8.15 (1H, s),
9.1 (1H, s) ppm.
LRMS 518, 520 (MH+).
Anal. Found: C, 55.33; H, 5.55; N, 12.82. Calc for
CZaH~sC1N504S~O.IEtOAc~O.OSCH~CIz: C,
55.30; H, 5.48; N, 13.19.
N Benzyl-tV [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-L-aianine t-
butyl ester (100
mg, 0.19 mmol) was dissolved in a solution of EtOAc saturated with HCI (5.0
mL) and the
mixture stirred at room temperature for 18 h. The mixture was concentrated in
vacuo,
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azeotroping with EtOAc, to give N benzyl-N [(4-chloro-I-guanidino-7-
isoquinolinyl)sulphonyl]-L-alanine hydrochloride (77 mg, 0.15 mmol) as a white
powder.
mp 256-262 °C.
~H (DMSO-dh, 300 MHz) b 1.2 (3H, d), 4.35 (1H, d), 4.7 (1H, q), 4.8 (1H, d),
7.1-7.4 (5H,
m), 8.3 (2H, s), 8.4-8.7 (4H, br), 8.5 ( 1 H, s), 9.05 ( 1 H, s), 11.2 ( 1 H,
br), 12.7 ( 1 H, br) ppm.
LRMS 461, 463 (MH+).
l0
Anal. Found: C, 48.02; H, 4.38; N, 13.33. Calc for
C~aHzoCIN50~S~1.OHCI-0.25H~0~O.IEtOAc: C, 47.88; H, 4.39; N, 13.69.
Example 22:
I S (a) N (t butoxycarbonylmethyt)-N [(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl)glycine t-butyl ester
(b) N (Carboxymethyl)-N [(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]glycine
hydrochloride
ci ci ci
W ~ ~ ~1
0
i ~N ~ a ~ i ~N --- ~ ° s .N
tBuO C N-S tBuOzC N-S HO C N-S
0 N\/NH2 tBu02CJ 0 NHZ HOZCJ- 0 NYNHz
2O NH NHx NHx
z
Anhydrous K~C03 (88 mg, 0.64 mmol) and then t-butyl bromoacetate (56 pL, 0.38
mmol)
were added to a stirred solution of N [(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]glycine t-butyl ester (132 mg, 0.33 mmol) in DMF (2.0
mL) and the
25 mixture was stirred at 23 °C for 18 h. The mixture was diluted with
EtOAc (300 mL), washed
with brine (150 mL), water (200 mL), dried (MgS04) and evaporated in vacuo.
The residue
was purified by column chromatography upon silica gel using pentane-EtOAc
(80:20 to
50:50) as eluant to give N (t-butoxycarbonylmethyl)-N [(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]glycine t-butyl ester (101 mg, 0.19 mmol) as a pale
yellow foam.
~H (CDCl3, 400 MHz) 8 1.4 (18H, s), 4.1 (4H, s), 8.0 (1H, d), 8.1 (1H, d),
8.15 (1H, s), 9.25
(1H, s) ppm.
LRMS 528 (MI-I+).
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Anal. Found: C, 49.57; H, 5.78; N, 12.73. Calc for
CZZHsoCINsO6S~O.lHzO~O.lEtOAc: C,
49.95; H, 5.80; N, 13.00.
A solution of HCl (3 mL, 2 M, 6 mmol) was added to a solution of N [(4-chloro-
1-guanidino-
7-isoquinolinyl)sulphonyl]-N (t-butoxycarbonylmethyl)glycine t-butyl ester (90
mg, 0. I7
mmol) in dioxane (4.0 mL). The mixture was stirred at 23 °C for 18 h
and then heated at 70
°C. The solvents were evaporated in vactro and the residue dried to
give N (carboxymethyl)-
N [(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]glycine hydrochloride (61
mg, 0.127
mmol) as a white solid.
mp 296-300 °C (dec).
~ H (DMSO-d~, 400 MHz) 8 4.05 (4H, s), 7.9-8.3 (4H, 6r), 8.2 ( 1 H, d), 8.25 (
I H, d), 8.35 ( 1 H,
s), 9.0 ( 1 H, s) ppm.
Anal. Found: C, 38.29; H, 3.58; N, 14:13. Calc for
C,.~Hi.~C1N506S~1.OHC1~O.1H~0~0.3dio:cane: C, 37.99; H, 3.69; N, 14.57.
Example 23:
(a) N [(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-L-alanine t-butyl
ester
(b) N [(4-Chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-L-alanine
trifluoroacetate
ct ct c1
Me ~ Me ~ Me
/ iN ~ ' IOI I / iN ~ 101 I / iN
tBuOxC ~ H-S ~ tBuOzC ~ H-S ' HOZC ~ N-S
0 CI 0 NYNH2 H 0 N\/NHZ
NHZ TNHi
NaH (37 mg, 80% dispersion by wt in mineral oil, 1.23 mmol) was added in one
portion to a
stirred solution of guanidine hydrochloride (189 mg, I.97 mmol) in DME (6 mL)
and the
mixture was heated at 60 °C under NZ for 30 min. 1-{[(1,4-Dichloro-7-
isoquinolinyl)sulphonyl]amino}-L-alanine t-butyl ester (200 mg, 0.49 mmol) was
added and
the mixture heated at 90 °C for 7 h: The cooled mixture was
concentrated in vacuo, the
residue suspended in water and extracted with EtOAc (3x30 mL). The combined
organic
extracts were dried (MgS04) and the solvents evaporated in vacuo. The residue
was purified
by column chromatography upon silica gel using CHZCIz-MeOH-0.880NH3 (95:5:0.5)
as
eluant to give N [(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-L-alanine t-
butyl ester
(160 mg, 0.37 mmol) as a white powder.
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'H (DMSO-d~, 300 MHz) 8 1.1 (9H, s), 1.15 (3H, d), 3.8 (1H, dq), 7.1-7.4 (4H,
br), 8.0 (1H,
d), 8.05 { 1 H, d), 8.1 ( 1 H, s), 8.3 ( 1 H, d), 9.05 ( 1 H, s) ppm.
CF3CO~H ( 1.0 mL) was added to a stirred solution of N [(4-chloro-1-guanidino-
7-
isoquinolinyl)sulphonyl]-L-alanine t-butyl ester (ca. 150 mg, 0.35 mmol) in
CHzCh (3.0 mL)
and the mixture stirred at room temperature for 2 h. The mixture was
evaporated in vacuo,
azeotroping with PhMe and CH~CI~, and then triturated with Et,O to give N [(4-
chloro-1-
guanidino-7-isoquinolinyl)sulphonyl]-L-alanine trifluoroacetate (62 mg, 0.126
mmol) as a
white powder.
mp >250 °C.
' H (CD30D + TFA-d, 300 MHz) 8 1.35 (3 H, d), 4.05 { 1 H, q), 8.3 ( 1 H, d),
8.4 ( 1 H, s), 8.45
( 1 H, d), 8.9 ( 1 H, s) ppm.
LRMS 389, 391 (MNHa+)
Anal. Found: C, 36.66; H, 3.1 1; N, 14.00. Calc for
Ci3H,aCIN504S~1.OCF3COZH~0.3Hz0: C,
36.64; H, 3.21; N, 14.24.
Example 24:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-D-alanine methyl ester
(b) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-D-alanine
hydrochloride
c1 c1 r
Me 0 I \ \
II / iN II / iN ~ II / iN
MeOzC ° N-S Me02C ° N-S HO,C ° N-S
H O CI H O N \'NH2 H O N\ /NH2
TNHi TNH=
NaH (35 mg, 80% dispersion by wt in mineral oil, 1.17 mmol) was added in one
portion to a
stirred solution of guanidine hydrochloride (179 mg, 1.87 mmol) in DMSO (5 mL)
and the
mixture was heated at 60 °C underNz for 45 min. 1-{[(1,4-Dichloro-7-
isoquinolinyl)sulphonyl]amino}-D-alanine methyl ester (170 mg, 0.47 mmol) was
added and
the mixture heated at 90 °C for 4 h. The cooled mixture was poured into
water and extracted
with EtOAc (3x30 mL). The combined organic extracts were dried (MgS04) and the
solvents
evaporated in vacuo. The residue was purified by column chromatography upon
silica gel
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using pentane-EtOAc (66:33 to 0: t 00) as eluant to give N [(4-chloro-1-
guanidine-7-
isoquinolinyl)sulphonyl]-D-alanine methyl ester (22 mg, 0.057 mmol) as a
yellow foam/oil.
t H (CD30D, 300 MHz) 8 1.3 (3 H, d), 3.4 (3 H, s), 4.1 ( 1 H, q), 8.1 ( 1 H,
d), 8.1 ( 1 H, d), 8.15
S ( 1 H, s), 9.1 ( 1 H, s) ppm.
LRMS 386, 388 (MH+).
A solution of NaOH ( 1 mL, 2 M, 2 mmol) was added to a solution of N [(4-
chloro-1-
guanidine-7-isoquinolinyl)sulphonyi]-D-alanine methyl ester (17 mg, 0.044
mmol) in MeOH
(3 mL) and the mixture was heated at 60 °C for 18 h. The cooled mixture
was neutrilised with
dilute HCl (2 M), the MeOH was evaporated in vaczro, and the residue
triturated with water
(10 mL). The solid was collected by filtration, with water washing, and dried
under high
vacuum to give N [(4-chloro-i-guanidine-7-isoquinolinyl)sulphonyl]-D-alanine
I S hydrochloride (9 mg, 0.021 mmol) as an off white powder.
t H (DMSO-d~, 300 MHz) 8 1.2 (3 H, d), 3.8 ( I H, dq), 7.2-7.6 (4I-I, br),
8.05 ( 1 H, d), 8. I ( I H,
d), 8.15 ( 1 H, s), 8.2 ( 1 H, m), 9. I ( 1 H, s) ppm.
Anal. Found: C, 37.56; H, 3.98; N, 15.74. Calc for
Ct3Ht4C1N504S~1.OHCl~O.SH20: C, 37.42;
H, 3.86; N, 16.78.
Example 25:
(a) 1-{[(4-chloro-1=guanidine-7-isoquinolinyl)sulphonyl]amino}-L-valine t-
butyl ester
(b) N [(4-Chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-L-valine
trifluoroacetate
i ci ct
Me~Me ~ ~ Me~Me ~ ~ Me~Me
tBuOiC i~N-S I ~ ~ N ~ tBu02C~N-S I ~ ~ N ~ NOZC~N-S I ~ ~ N
H 0 CI H 0 N\/NHZ H 0 N\'NNZ
TNHz ~NHx
NaH (35 mg, 80% dispersion by wt in mineral oil, 1.17 mmot) was added in one
portion to a
stirred solution of guanidine hydrochloride ( 176 mg, 1.84 mmol) in DMA (4 mL)
under N
and the mixture was heated at 60 °C for 30 min. 1-{[(1,4-Dichloro-7-
isoquinolinyl)sulphonyl]amino}-L-valine t-butyl ester (161 mg, 0.43 mmol) was
added in one
portion and the mixture heated at 80 °C for 18 h. The cooled mixture
was poured into water
(50 mL), extracted with EtOAc (2x20 mL) and the combined organic extracts were
washed
with brine, dried (NazS04) and evaporated in vacuo. The residue was dissolved
EtZO and a
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solution of HC1 in EtzO ( 1 M) was added which gave a white precipitate. The
EtzO was
decanted and the solid residue dissolved in MaCN and the solution cooled to
ca. 0 °C which
gave a precipate. This solid was collected by filtration and then dried to
give 1-{[(4-chloro-I-
guanidino-7-isoquinolinyl)sulphonyl]amino}-L-valine t-butyl ester
hydrochloride (36 mg,
0.072 mmol) as a white solid. Evaporation of the combined organic mother
liquors gave a
gum which was purified by column chromatography upon silica gel using CH~C1~-
MeOH-
0.880NH3 (90:10:1) as eluant to give 1-{[(4-chloro-1-guanidine-7-
isoquinolinyl)sulphonyl]amino}-L-valine t-butyl ester (104 mg, 0.228 mmol).
(The sample
was characterised as the hydrochloride salt.)
mp 192-194 °C (dec).
'H (DMSO-d~, 300 MHz) ij 0.8 (3H, d), 0.85 (3H, d), 1.05 (9H, s), 2.0 (1H,
sept), 3.7 (1H,
dd), 8.3 ( I H, d), 8.4 ( t H, d), 8.4 ( i H, d), 8.45 ( i H, s), 8.5-8.7 (4H,
br), 9.05 ( 1 H, s), 11.3
( I H, br), ppm.
LIZMS 456, 458 (MH+).
Anal. Found: C, 45.67; H, 5.54; N, 13.97. Calc for
C,9HZ6C1N504S~I.OHCI~O.SH~O: C, 45.51;
H, 5.63; N, 13.97.
1-{[(4-Chloro-1-guanidine-7-isoquinolinyl)sulphonyl]amino}-L-valine t-butyl
ester (104 mg,
0.228 mmol) was dissolved in CF3CO~H (1.0 mL) and the mixture stirred at room
temperature
for 1 h. The mixture was diluted with PhMe (25 mL) and concentrated in vacuo.
The residue
was crystallised with Et~O. containing a small amount of EfiOAc to give a
white solid. This
solid was then triturated with water and dried to give 1-{[(4-chloro-1-
guanidine-7
isoquinolinyl)sulphonyl]amino}-L-valine trifluoroacetate (39 mg, 0.084 mmol).
mp >300 °C.
' H (TFA-d, 400 MHz) 8 0.95 (3 H, d), 1.0 (3 H, d), 2.25 ( 1 H, sept), 4.0 ( 1
H, d), 8.3 ( 1 H, d),
8.4 ( 1 H, s), 8.5 5 ( 1 H, d), 9.0 ( 1 H, s) ppm.
LRMS 400, 402 (MH+).
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Anal. Found: C, 41.29; H, 4.37; N, 14.99. Calc for
C,SH,$C1N504S~O.SCF3CO~H~0.3Hz0: C.
41.57; H, 4.16; N, 15.15.
Example 26:
(a) 1-{[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]amino}-D-valine t-
butyl ester
hydrochloride
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonylj-D-valine hydrochloride
c1 c1 c1
Me Me ~ ~ Me Me ~ ~ Me Me
/ /N ~ ~ I / iN ~ ~ I / .~N
tBuO;C ° H-S i8u0=C ° H-S HOzC ° H-5
0 CI 0 NYNHz 0 NYNH=
NHZ NH-
l0
NaH (35 mg, 80% dispersion by wt in mineral oii, I. I7 mmol) was added in one
portion to a
stirred solution of guanidine hydrochloride ( 176 mg, 1.84 mmol) in DMSO (2.5
mL) under N
and the mixture was heated at 23 °C for 30 min. I-{[(1,4-Dichloro-7-
isoquinolinyl)sulphonyl]amino}-D-valine t-butyl ester (200 mg, 0.46 mmol) was
added in one
portion and the mixture heated at 90 °C for 3 h. The cooled mixture was
poured into water,
extracted with EtOAc and the combined organic extracts were washed with brine,
dried
(MgS04) and evaporated in vacuo. The residue was dissolved Et,O and a solution
of HCI in
EtzO (0.5 mL, 1 M) was added which gave a white precipitate. Purification by
column
chromatography upon silica gei using CHzCI,-MeOH-0.880NH3 (95:5:0.5) as eluant
furnished
the product which was again treated with a solution of HCI in Et,O (1 M) to
give 1-.{[(4-
chloro-1-guanidino-7-isoquinolinyl)sulphonyl]amino}-D-valine t-butyl ester
hydrochloride
(76.6 mg, 0.151 mmol).
mp I24-125 °C (dec).
~H (DMSO-dh, 300 MHz) 8 0.8 (3H, d), 0.85 (3H, d), 1.05 (9H, s), 2.0 (1H,
sept), 3.7 (1H,
dd), 8.3 ( I H, d), 8.4 ( 1 H, d), 8.4 ( I H, d), 8.45 ( 1 H, s), 8.4-8.8 (4H,
br), 9.05 ( 1 H, s), I I .2
(1H, br) ppm.
LRMS 456, 458 (MH+), 478, 480 MNa~).
Anai. Found: C, 46.07; H, 5.67; N, 13.50. Calc for
C,9H~6C1NSO4S~I.OHC1~O.SMeOH: C,
46.07; H, 5.75; N, I3.77.
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i-{[(4-Chloro-I-guanidino-7-isoquinoiinyl)sulphonyl]amino}-D-valine t-butyl
ester
hydrochloride (61 mg, 0.12 mmol) was dissolved in a solution of EtOAc
saturated with HCl
(I O mL) at 0 °C, and the mixture stirred at room temperature for 4 h.
The mixture was
concentrated in vaczzo, the residue extracted with hot EtOAc, and the organic
solution was
then concentrated irz vaczzo and dried to give 1-{[(4-chloro-i-guanidino-7-
isoquinolinyl)sulphony!]amino}-D-valine hydrochloride (24.3 mg, 0.050 mmol) as
a pale
yellow solid.
mp > 190 °C (dec).
~ H (TFA-d, 400 MHz) b 0.95 (3 H, br s), 1.0 (3 H, br s), 2.3 ( 1 H, br s),
4.05 ( 1 H, br s), 8.35
( 1 H, br s), 8.4 ( 1 H, br s), 8.55 ( 1 H, 6r s), 9.1 ( 1 H, br s) ppm.
LRMS 400 (MH~), 417 (MNHQ+)
,
Anal. Found: C, 41.29; H, 4.76; N, 14.16. Calc for
C,SHI~CINSO,~S~I.OHCI~0.7H~0~0.4EtOAc: C, 41.18; H, 4.91; N', 14.46.
Example 27:
(a) 1-{[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl)amino}-D-tent-leucine
t-butyl
ester hydrochloride
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-D-tent-leucine
hydrochloride
c1 c1 c1
Me . Me Me
Me Me ~ ~ Me Me ~ ~ Me Me
/ iN ~ 101 I / iN --- ~ ~ I / iN
t8u0~C o N-S tBuOzC ° N-S HO=C ° N-S
H 0 CI H 0 NYNHZ H 0 NYNHz
NH= NH2
NaH (58 mg, 80% dispersion by wt in mineral oil, 1.27 mmol) was added in one
portion to a
stirred solution of guanidine hydrochloride (191 mg, 2.0 mmol) in DMSO (5.0
mL) under N
and the mixture was heated at 23 °C for 30 min. A solution of 1-{[(1,4-
dichloro-7-
isoquinolinyl)sulphonyl]amino}-D-tert-leucine t-butyl ester (225 mg, 0.50
mmol) in DMSO
(3.0 mL) was added in one portion and the mixture heated at 90 °C for 9
h. A second portion
of guanidine (0.67 mmol)[prepared from guanidine hydrochloride (100 mg) and
NaH (20
mg)] in DMSO (1.0 mL) was added and the mixture heated at 90 °C for an
additional 8 h.
The cooled mixture was poured into water, extracted with EtOAc and the
combined organic
extracts were washed with water, brine, dried (MgS04) and evaporated in vacuo.
The residue
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was dissolved EtzO and a solution of HCl in EtzO ( 1.5 mL, I M) was added
which gave a
white precipitate. The solvents were evaporated in vacuo and the residue
triturated with Et~O
to give 1-{[(4-chloro-1-guanidine-7-isoquino(inyl)sulphonyl]amino}-D-tent-
Ieucine t-butyl
ester hydrochloride (222 mg, 0.43 mmol).
mp 187-189 °C.
H (DMSO-dh, 400 MHz) 8 0.9 (9H, s), 0.95 (9H, s), 3.6 ( 1 H, d), 8.3 ( I H,
d), 8.4 ( 1 H, d), 8.4-
8.8 (4H, br), 8.5 ( 1 H, s), 9.0 ( 1 H, s), 1 l .15 ( 1 H, br) ppm
l0
LRMS 470, 472 (MH+).
Anal. Found: C, 46.55; H, 5.78; N, 13.46. Calc for
C~oH~8CIN50~S~I.OHCI~0.5HZ0: C, 46.60:
H, 5.87; N, 13.59.
l5
1-{[(4-Chloro-1-guanidine-7-isoquinolinyl)sulphonyl]amino}-D-tert-leucine t-
butyl ester
hydrochloride (188 mg, 0.36 mmol) was dissolved in a solution of EtOAc
saturated with HCl
(30 mL) and the mixture stirred at room temperature for 5 h. The mixture was
concentrated in
vacuo and the residue heated with EtOAc to give a white solid. The hot organic
solution was
20 decanted and the solid dried in vacuo to give 1-{[(4-chloro-I-guanidine-7-
isoquinolinyl)sulphonyl]amino}-D-tert-leucine hydrochloride (109.3 mg, 0.24
mmol) as a
white solid.
mp 234-236 °C (dec).
~ H (TFA-d, 400 MHz) b 1.1 (9H, s), 3 .9 ( 1 H, s), 8.3 S ( I H, d), 8.5 ( 1
H, s), 8.6 ( I H, d), 9.1
(IH, s) ppm.
LRMS 414, 416 (MH+).
Anal. Found: C, 41.70; H, 4.86; N, 15.01. Calc for
C~6HZOC1N504S~1.OHCI~0.5Hz0: C, 41.84;
H, 4.83; N, 15.25.
Example 28:
(a) N [(4-chloro-1-guanidine-7-isoquinotinyl)sulphonyl]-L-phenylalanine t
butyl ester
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(b) N ((4-Chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-L-phenylalanine
trifluoroacetate
c1 \ ~ c1 \ I ci
lBuOzC~i N-O I ~ ~ N tBuO,C~N-S I ~ ~ N HO-C~N-S I ~ ~ N
H II ' H II H II
O CI 0 N"NH: O N"NH:
~N'Hi ~N'HZ
NaH (22 mg, 80% dispersion by wt in mineral oil, 0.73 mmol) was added in one
portion to a
stirred suspension of guanidine hydrochloride (76.7 mg, 0.80 mmol) in DMSO
(5.0 mL) and
the mixture was heated at 60 °C under N~ for 20 min. N [(1,4-Dichloro-7-
isoquinolinyl)sulphonyl]-L-phenylalanine t-butyl ester (103 mg, 0.21 mmol) was
added and
the mixture heated at 95 °C for 17 h. The solvents were evaporated in
vacuo and the residue
was purified by column chromatography upon silica gel using CH~CIZ-MeOH-
0.880NH3
(95:5:0.5 to 80;20:2) as eluant to give N [(4-chloro-1-guanidine-7-
isoquinolinyl)sulphonyl]-
L-phenylalanine t-butyl ester (34.7 mg, 0.069 mmol) as a yellow resin.
I H (DMSO-dh, 300 MHz) 8 1.0 (9H, s), 2.7 ( 1 H, dd), 2.8 ( 1 H, dd), 3.9 ( 1
H, dd), 7.1-7.2 (5H,
m), 7.1-7.3 (4H, br s), 7.9 ( 1 H, d), 7.95 ( i H, d), 8.1 (s, 1 H), 8.5 ( 1
H, br d), 8.95 ( 1 H, s) ppm.
LRMS 504, 506 (MH*)
N [(4-Chlore-1-guanidine-7-isoquinolinyl)sulphonyl]-L-phenylalanine t-butyl
ester (30 mg,
0.060 mmol) was dissolved in CF3COZH (2.5 mL) and the mixture stirred at room
temperature
for 2.5 h. The mixture was diluted with CHZCh and PhMe, concentrated in vacuo,
azeotroping
with PhMe, and the residue triturated with EtzO to give N [(4-chloro-1-
guanidine-7-
isoquinolinyl)sulphonyl]-L-phenylalanine trifluoroacetate (24.4 mg, 0.42 mmol)
as a white
solid.
mp 306 °C (dec).
'H (DMSO-ds, 300 MHz) 8 2.7 (1H, dd), 3.0 (1H, dd), 3.95 (1H, m), 6.9-7.1 (5H,
m), 7.8-8.4
(4H, br ), 7.9 (1H, d), 8.05 (1H, d), 8.3 (s, 1H), 8.6 (1H, br s), 8.8 (1H, s)
ppm.
LRMS 448 (MH+)
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Anal. Found: C, 44.35; H, 3.78; N, 11.38. Calc for
C,9H,8CINSO~S~1.OCF3CO~H~0.5H20~0.12Etz0: C, 44.50; H, 3.69; N, 12.08.
Example 29:
(a) 1-{[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]amino}-O-methyl-D-
serine t-
butyl ester hydrochloride
(b) N [(4-Chloro-1-guanidino-7-isoquinolmyl)sulphonyl]-O-methyl-D-serine
hydrochloride
r c1 I
Me0 ~ ~ Me0 ~ ~ Me0
I~I ~ / / N - ~ I~t ~ / / N ~ ICI ~ / r N
t8u0 C o N-S tBuO:C o N-S HO C ° N-S
= H 0 CI H 0 N \/NHz Z H O N \ /NHz
TNHz ~N'H2
NaH (50 mg, 80% dispersion by wt in mineral oil, 1.66 mmol) was added in one
portion to a
stirred solution of guanidine hydrochloride (260 mg, 2.72 mmol) in DMSO (4 mL)
under N
and the mixture was heated at 50 °C for 30 min. I-{[(I,4-Dichloro-7-
15 isoquinolinyl)sulphony(]amino}-O-methyl-D-serine t-butyl ester (300 mg,
0.689 mmol) was
added in one portion and the mixture heated at 90 °C for 8 h. The
cooled mixture was poured
into water (50 mL), the aqueous solution was extracted with EtOAc (x2) and the
combined
organic extracts were washed with water, brine, dried (MgSO~). The solvents
were evaporated
in vacuo and the residue purified by column chromatography upon silica get
using CHZCl2-
20 MeOH-0.880NH3 (90:10: I) as eluant to give the desired product. This
material was treated
with a solution of HC1 in Et20 (1.0 mL, 1 M), the solvents evaporated in
vacuo, and the
residue triturated with EtZO (x2) to give I-{[(4-chloro-I-guanidino-7-
isoquinolinyl)sulphony(]amino}-O-methyl-D-serine t-butyl ester hydrochloride
(18 mg, 0.036
mmol) as a white solid.
1H (d4-MeOH, 300 MHz) 8 1.2 (9H,s), 3.2 (3H,s), 3.5-3.6 (lH,m), 3.6-3.7
(lH,m), 4.1-4.2
( 1 H,m), 8.3 5-8.5 (3 H,m), 8.9 ( I H,s) ppm.
LRMS 458 (MH).
1-{[(4-Chloro-1-guanidino-7-isoquinolinyl)su(phony(]amino}-O-methyl-D-serine t-
butyl
ester hydrochloride (18 mg, 0.036 mmol) was dissolved in a solution of EtOAc
saturated with
HCI (5 mL) and the mixture stirred at room temperature for 3 h. The mixture
was
concentrated in vacuo and the residue triturated with EtOAc (x3) to give 1-
{[(4-chloro-1-
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guanidine-7-isoquinolinyl)sulphonyl]amino}-L-tent-leucine hydrochloride (9 mg,
0.02 mmol)
as an off white solid.
t H (d-TFA, 400MHz) 3.6 (3 H,s), 4.0-4.2 (2H,m), 4.65 ( 1 H, br s), 8.4 (I
H,d), 8.5 ( 1 H,s), 8.6~
1 H,d), 9.1 ( I H,s) ppm.
LRMS 402 (.MH).
Example 30:
(a) N [(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-D-aspartic acid di-t-
butyl ester
(b) N [(4-Chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-D-aspartic acid
hydrochloride
i a ct
tBuOzC ~ ~ tBuOxC ~ ~ HO,C
0 ~ / /N ~ ~ O
tBuO,C o N-S tBuO,C ° N-S HOxC o N-S
H 0 CI H 0 N\'NH, H 0 N\'NH,
~NHs 'N~H,
Guanidine hydrochloride ( 190 mg, 2.0 mmol) was added in one portion to a
stirred
suspension ofNaH (47 mg, 80% dispersion by wt in mineral oil, 1.57 mmol) in
DME (7 mL)
and the mixture was heated at 60 °C under N~ for 30 min. I-{[(I,4-
Dichloro-7-
isoquinolinyl)sulphonyl]amino}-D-aspartic acid di-t-butyl ester (250 mg, 0.50
mmol) was
added and the mixture heated at reflux for I8 h. The cooled mixture was
diluted with EtOAc,
washed with water, brine, dried (IVIgS04) and the solvents evaporated in
vacuo. The residue
was purified by column chromatography upon silica gel using CHzCl2-MeOH-
0.880NH3
(97:3:0.3) as eluant to give N [(4-chloro-I-guanidine-7-
isoquinolinyl)sulphonyl]-D-aspartic
acid di-t-butyl ester (50 mg, 0.095 mmo() as a yellow solid.
~H (CDC13, 400 MHz) 8 I.2 (9H, s), 1.4 (9H, s), 2.7 (1H, dd), 2.8 (1H, dd),
4.1 (1H, br t), 8.05
(IH,d),8.1(lH,d),8.I5(lH,s),9.3(lH,s)ppm.
LRMS 528, 530 (MH+)
N [(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]-D-aspartic acid di-t-butyl
ester (50 mg,.
0.095 mmol) was dissolved in a solution of EtOAc saturated with HCI (10 mL)
and the
mixture stirred at room temperature for 4 h. The mixture was concentrated in
vacuo and the
residue triturated with PhMe and then Et20 to give N [(4-chloro-I-guanidine-7-
isoquinolinyl)sulphonyl]-D-aspartic acid hydrochloride (29 mg, 0.062 mmol) as
an off white
solid.
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~ H (CD30D, 400 MHz) b 2.7 ( 1 H, dd), 2.8 ( 1 H, dd), 4.4 ( 1 H, br t), 8.35
( 1 H, d), 8.45 ( 1 H, s),
8.45 ( 1 H, d), 8.9 ( 1 H, s) ppm.
LRMS 415 (M+)
Anal. Found: C, 36.05; H, 3.72; N, 13.62. Calc for
C,,~H,,~CINSO6S~1.OHCl~0.8H~0: C, 36.03;
H, 3.59; N, 15.01.
Example 31:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-L-protine t-butyl
ester
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-L-proline
hydrochloride
ci a ci
~N_S I / iN ~N~S I / iN ~N-~ I / iN
m O CI ~, O N w NHi y 0 N ~ NH=
COZtBu CO,tBu Y C02H
NH, NHZ
NaH (35 mg, 80% dispersion by wt in mineral oil, 1.16 mmol) was added in one
portion to a
stirred solution of guanidine hydrochloride (I77 mg, 1.85 mmol) in DME (S mL)
and the
mixture was heated at 60 °C under N~ for 45 min. A solution of 1-{[(1,4-
dichloro-7-
isoquinolinyl)sulphonyl]amino}-L-proline t-butyl ester (200 mg, 0.46 mmol).in
DME (2 mL)
was added and the mixture heated at 95 °C for 4 h. The solvents were
evaporated in vacuo and
the residue was purified by column chromatography upon silica gel using
pentane-EtOAc
(80:20 to 0:100) as eluant, followed by azeotroping with CH~CIz, to give N [(4-
chloro-I-
guanidino-7-isoquinolinyl)sulphonyl]-L-proline t-butyl ester (153 mg, 0.32
mmol) as a pale
yellow foam.
1H (DMSO-dh, 300 MHz) 8 1.35 (9H, s), 1.6-1.7 (1H, m), 1.7-1.9 (2H, m), 1.9-
2.0 (1H, m),
3 .15-3.25 ( 1 H, m), 3 .3 5-3 .5 ( 1 H, m), 4.1 ( 1 H, dd), 7.15-7.4 (4H,
br), 8.05 ( 1 H, d), 8.1 ( 1 H, d),
8.1 ( 1 H, s), 9.05 ( 1 H, s) ppm.
LRMS 454, 456 (MH~), 907 (MZH+).
Anal. Found: C, 50.02; H, 5.41; N, 14.84. Calc for
C,9H~QC1N504S~O.IEtOAc~O.OSCHzCI2: C,
50.02; H, 5.37; N, 15.00.
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N-[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-L-proline t-butyl ester
(60 mg, 0.13
mmol) was dissolved in a solution of EtOAc saturated with HC1 (5.0 mL) and the
mixture
stirred at room temperature for 1 h. The mixture was concentrated in vacuo ,
azeotroping with
EtOAc, and the residue triturated with CH~CIa to give N [(4-chloro-1-guanidino-
7-
isoquinolinyl)sulphonyl]-L-proline hydrochloride (44 mg, 0.095 mmol) as a
white powder.
mp 185-189 °C.
1H (DMSO-d,;, 300 MHz) 8 1.5-1.7 (1H, m), l.7-2.0 (3H, m), 3.2 -3.5 (2H, m),
4.2 (1H, dd),
I O 8.3-8.8 (4H, br), 8.2 (2 H, s), 8.5 ( I H, s), 8. i ( 1 H, s), 9.05 ( I H,
s), 1 I .2 ( I H, br) ppm. .
Anal. Found: C, 39.89; H, 4.06; N, 14.93. Calc for
C,SHI6CINSOaS~I.OHCI~I.OH~O~O.IEtOAc: C, 40.1 l; H, 4.33; N, 15.19
Example 32:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-D-proline t-butyl
ester
(b) N ((4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-D-proline
hydrochloride
c1 ct c1
o ~~ ~1 0
_u i ~N - _u i ~N -- _u a iN
~N 0 CI ~N 0 Nw NH2 ~N O Nw NHx
COZtBu CO=t8u Y COzH
NHZ NH=
Guanidine hydrochloride (220 mg, 2.3 mmol) was added in one portion to a
stirred
suspension of NaH (55 mg, 80% dispersion by wt in mineral oil, 1.83 mmol) in
DME (8 mL)
and the mixture was heated at 60 °C under NZ for 30 min. 1-[[(1,4-
Dichloro-7-
isoquinolinyl)sulphonyl]amino}-D-proline t-butyl ester (250 mg, 0.58 mmol) was
added and
the mixture heated at reflux for 5 h. The cooled mixture was diluted with
EtOAc, washed with
water, brine, dried (MgS04) and the solvents evaporated in vacuo. The residue
was purified
by column chromatography upon silica gel using CHZC12-MeOH-0.880NH3 (97:3:0.3)
as
eluant to give N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-D-proline t-
butyl ester
(200 mg, 0.44 mmol) as a yellow solid.
mp >170 °C (dec).
1H (CDC13, 400 MHz) 8 1.45 (9H, s), 1.7-1.8 (1H, m), 1.8-2.05 (3H, m), 3.3-
3.45 (1H, m),
3.5-3.6 (1H, m), 4.3 (1H, dd), 6.3-6.6 (4H, br), 8.05 (1H, d), 8.1 (1H, d),
8.1 (1H, s), 9.2 (1H,
s) ppm.
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LRMS 454, 456 (MH+)
Anal. Found: C, 49.57; H, x.27: N, I4.9~. Calc for
C,~H,4C(NSOaS~0.2H~0~0.04CH~C1~: C,
49.61; H, 5.3 5 : N, 15 .19.
N-[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-D-proline t-butyl ester
(50 mg, 0.11
mmol) was dissolved in a solution ofEtOAc saturated with HCI (10 mL) and the
mixture
stirred at room temperature for 2.5 h. The mixture was concentrated in vacz~o,
azeotroping
with CH~CI~, to give N-[(4-chloro-l-guanidino-7-isoquinolinyl)sulphonyl]-D-
proline
hydrochloride (40 mg, 0.09? mmoi) as a white powder.
mp >200°C (dec).
~ H (CD30D, 400 MHz) 8 I .7-1.85 ( 1 H, m), 1.9-2.2 (3 H, m), 3.4 -3.5 ( 1 H,
m), 3.5-3.6 ( 1 H,
m), 4.4 ( 1 H, dd), 8.4 ( 1 H, d), 8.45 ( 1 H, s), 8.5 ( 1 H, d), 8.9 ( 1 H,
s) ppm.
LRMS 397, 399 (MH+)
Anal. Found: C, 40.22; H, 3.92; N, 14.88. Calc for
C,SH~6ClN50dS~1.OHC1~0.2H~0~0.25CH~C1~: C, 39.89; H, 3.93; N, 15.25.
It was noted that some racemisation had occurred during repetition of the
above preparation
in some conditions. An alternative route to Example 32(b) was developed,
reversing the
guanylation/hydrolysis sequence, as exemplified below:
1. Hydrolysis
0
o vs~ _
\ ~O OH
CI ~ / O
CI ~ / OO
N CI
NCI
tent-Butyl (2S7-1-[(1,4-dichloro-7-isoquinolinyl)sulfonyl]-2-
pyrrolidinecarboxylate (50.0 g,
0.116 mol) was dissolved in conc. HCI (12 M, 200 ml) and stirred for 3.5 h.
Water (200 ml)
was added over 30 minutes and the resultant white precipitate stirred for a
further 0.5 h,
filtered and washed with water (3 x 100 ml). Drying under vacuum gave (2S~-1-
[(1,4-
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dichloro-7-isoquinolinyl)sulfonyl]-2-pyrrolidinecarboxylic acid as a white
solid (42.9 g, 0.1 14
mol).
1H (db-DMSO, 300 MHz) 8 1.6-1.95 (3H, m), 1.95-2.1 (IH, m), 3.25-3.35 (1H, m),
3.35-3.45
S ( 1 H, m), 4.3 ( 1 H, dd), 8.3 S (2H, s), 8.6 ~( 1 H, s), 8.65 ( 1 H, s)
ppm.
Chiral analysis was performed using capillary electrophoresis, showing an
enantiomeric
purity of 97.41 %.
2. Guanylation of free acid
N
\S/~
OH
\O /rOH -
CI ~ / 0
N"CI
Potassium t-butoxide (49.0 g, .0437 mol) and guanidine.HC! (42.8 g, 0.448 mol)
in DME
(210 ml) was heated to reflex under nitrogen for 20 min. (2S~-1-[(1,4-dichloro-
7-
isoquinolinyl)sulfonyl]-2-pyrrolidinecarboxylic acid (42,0 g, 0.112 mot) was
added and
heating continued at reflex for 5.5 h. Water (420 ml) was added and the
mixture acidified
with c. HCI to pH = S giving a solid which was removed by filtration, washed
with aq. DME
(1:1, 2 x 75 ml) and water (2 x 75 ml) and dried to yield the title compound
(b) as a yellow
solid (40.71 g, 0.102 mot).
'H (d6-DMSO, 300 MHz) b 1.5-1.65 (1H, m), 1.7-2.0 (3H, m), 3.1-3.25 (1H, m),
3.35-4.05
(1H, m), 4.2 (1H, dd), 7.2-7.7 (4H, br s), 8.0 (1H, d), 8.1-8.2 (2H, m), 9.0S
(1H, d).
Chiral analysis was performed using capillary electrophoresis, showing an
enantiomeric
purity of 99.76% (n=2).
Example 33:
4-Chloro-1-guanidino-7-{[(2R)-(hydroxymethyl)-1-
pyrrolidinyl]sulphonyl}isoquinoline
hydrochloride
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a c1
a
N_S ' / iN N_S I / iN
R 0 CI ~, 0 N\'NH=
HO HO 'N~H2
NaH (26 mg, 80% dispersion by wt in mineral oil, 0.87 mmol) was added in one
portion to a
stirred solution of guanidine hydrochloride (126 mg, 1.32 mmol) in DMSO (2 mL)
and the
mixture was heated at 50 °C under N, for 20 min. A solution of 1,4-
dichloro-7-{[(2R)-
(hydroxymethyl)-1-pyrrolidinyl]suiphonyl}isoquinoline (120 mg, 0.33 mmol) in
DMSO (3
mL) was added in one portion and the mixture heated at 80-90 °C for, l
h. The cooled mixture
was poured into water, extracted with EtOAc (2x) and the combined organic
extracts were
then washed with water (x3), brine, dried (MgS04) and evaporated in vacuo. The
residue was
purified by column chromatography upon silica gel using CH~C(a-MeOH-0.880NH3
(95:5:0.
to 80:20:5) as eluant to give the desired product as an off white, sticky
solid. This material
was dissolved in MeOH, a solution of HCI in Et,O (I M) was added and the
solvents were
evaporated in vacuo. The residue was recrystallised from MeOH to give 4-chloro-
1-
guanidino-7-{[(2R)-(hydroxymethyl)-1-pyrrolidinyl]sulphonyl}isoquinoline
hydrochloride
(43 mg, 0.10 mmol) as a white solid.
mp 275-276.5 °C.
'H (CD30D, 400 MHz) S I.S-I.6S (2H, m), 1.8-I.9S (2H, m), 3.25 -3.35 (2H, m),
3.45-3.SS
( 1 H, m), 3.6-3.65 ( 1 H, m), 3 .7-3 .85 (2H, m), 8.4 ( 1 H, d), 8.45 ( 1 H,
s), 8.5 ( 1 H, d), 8.9 ( 1 H, s)
ppm.
LRMS 383 (MH+), 405 (MNa+), 767 (MPH+).
Anal. Found: C, 42.36; H, 4.54; N, 16.14. Calc for
C,SH,$C1N503S~1.OHC1~0.25Hz0: C,
42.41; H, 4.63; N, 16.49.
Example 34:
(a) 1-{[(4-chioro-I-guanidino-7-isoquinolinyl)sulphonyl]amino}isobutyric acid
methyl
ester
(b) 2-{[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]amino}isobutyric acid
hydrochloride
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ct c1 I
Me Me O I ~ ~ Me Me 0 I \ \ Me Me 0 I
II / ,N --~ x II / iN ~ II ~ iN
MeO,C H-S MeOzC H-S H02C H-S
0 CI O N\/NH= 0 N\'NH,
TNHi TNHz
NaH (32 mg, 80% dispersion by wt in mineral oil, 1.07 mmol) was added in one
portion to a
stirred solution of guanidine hydrochloride ( 167 mg, 1.7 mmol) in DMSO (5 mL)
and the
mixture was heated at 50 °C under N, for 20 min. 1-{[(1,4-Dichloro-7-
isoquinolinyl)sulphonyl]amino} isobutyric acid methyl ester ( 161 mg, 0.43
mmol) was added
in one portion and the mixture heated at 80 °C for 6.5 h. The cooled
mixture was poured into
water (~0 mL), extracted with EtOAc (2x100, 2x25 mL) and the combined organic
extracts
were washed with water, brine, dried (Na,SOa) and evaporated in vacuo. The
residue was.
purified by repeated column chromatography upon silica gel using (i) CH~CI~-
MeOH-
0.880NH3 (95:5:0.5), (ii) hexane-EtOAc (70:30) and then (iii) CH~CI~-MeOH-
0.880NH3
(90:10:01), as eluant to give the product as a yellow oil. Trituration with
Et~O gave 1-{[(4-
chloro-1-guanidino-7-isoquinolinyl)sulphonyl]amino} isobutyric acid methyl
ester (23 mg,
0.054 mmol) as yellow solid.
mp > 170 °C (dec).
~H (CD30D, 300 MHz) S 1.4 (6H, s), 3.5 (3H, s), 8.15-8.25 (3H, m), 9.1 (1H, s)
ppm.
LRMS 400, 402 (MH+).
Anal. Found: C, 44.02; H, 4.65; N, 16.29. Calc for CiSH,$C1N504S~0.9HZ0~O.li-
Pr~O: C,
43.95; H, 5.01; N, 16.43.
A solution ofNaOH (1 mL, 2 M, 2 mmol) was added to a solution of 1-{[(4-chloro-
1-
guanidino-7-isoquinolinyl)sulphonyl]amino} isobutyric acid methyl ester ( 16.5
mg, 0.041
mmol) in MeOH (0.5 mL) and the mixture was heated at 40-SO °C for 16 h.
The cooled
mixture was neutrilised with dilute HCl (0.5 mL, 2 M) to give a precipitate.
The solid was
collected by filtration, with copious water washing, and then dissolved in
conc. HCI. The
solvents were evaporated in vacuo azeptroping with PhMe, and then dried under
high vacuum
to give 1-{[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-amino}isobutyric
acid
hydrochloride (12 mg, 0.026 mmol) as a pale cream solid.
mp 258 °C (dec)
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~ H (CD30D, 400 MHz) cS t .45 (6H, s), 8.4 ( 1 H, d), 8.4 ( 1 H, s), 8.45 ( 1
H, d), 8.9 ( t H, s) ppm.
LRMS 386, 388 (MH+)
Anal. Found: C, 37.89; H, 4.33; N, 15.18. Calc for
C,aH,~CIN;O,~S~I.OHCI~1.5H~0~O.OSEt~O:
C, 37.65; H, 4.56; N, 15.46.
Example 3~:
2-{[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]amino}-2-methylpropanamide
hydrochloride
c1
Me Me 0 I \ ~ Me Me 0 I
II / iN / iN
H,N-~N-5 H=N--~N-S
0 H 0 CI 0 H 0 N \'NH2
'N~H2
NaH (41 mg, 80% dispersion by wt in mineral oil, 1.36 mmol) was added in one
portion to a
stirred solution of guanidine hydrochloride (210 mg, 2.2 mmol) in DMSO ( 10
mL) under N,
and the mixture was heated at 23 °C for 30 min. Z-{[(1,4-Dichloro-7
isoquinolinyl)sulphonyl]amino}-2-methylpropanamide (225 mg, 0.50 mmol) was
added in
one portion and the mixture heated at 90 °C for 8 h. The cooled mixture
was partially
concentrated in vacuo and the residue poured into water. The aqueous, solution
was extracted
with EtOAc (x4) and the combined organic extracts were washed with water,
brine, dried
(MgS04). The solvents were evaporated in vacuo and the residue purifed by
column
chromatography upon silica gel using CH~CIz-MeOH-0.880NH3 (90:10:1) as
eluantto give
the desired product. This material was dissolved in MeOH and treated with a
solution of HC1 '
in Et20 (1.0 mL, 1 M) to furnish 2-{[(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]amino}-2-methylpropanamide hydrochloride (86 mg, 0.188
mmol) as
an off white powder.
mp 279-281 °C.
1H (TFA-d, 400 MHz) S 1.6 (6H, s), 8.35 (1H, br s), 8.4 (1H, s), 8.55 (1H, s),
9.1 (1H, br s)
ppm.
LRMS 385, 387 (MH+).
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Anal. Found: C, 39.68; H, 4.81; N, 18.18. Calc for C,,~H,~CIN603S~1.OHC1~1.2
MeOH: C,
39.71; H, 5.00; N, 18.28.
Example 36:
(a) Ethyl 1-{[(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl~amino}cyclobutanecarboxylate
(b) 1-{[(4-Chloro-1-guanidino-7-
isoquinolinyl)sulphony(]amino}cyclobutanecarboxylic
acid hydrochloride
c1 c1 c1
' ~ ~ '1
i ~N ~ ~ i ~N ~ ~ s ~N
Et0 C N-S Et0 C N-S HO_C N-S
H O CI 2 H 0 N~NH_ H O N\'NNz
NH, ~NH2
NaH (37 mg, 80% dispersion by wt in mineral oil, 1.24 mmol) was added in one
portion to a
stirred solution of guanidine hydrochloride (189 mg, 1.98 mmol) in DMSO (6 mL)
and the
mixture was heated at 60 °C under N~ for 30 min. Ethyl 1-{[(1,4-
dichloro-7-
IS isoquinolinyl)sulphonyl]amino}-cyclobutanecarboxylate (200 mg, 0.50 mmol)
was added in
one portion and the mixture heated at 80 °C for 10 h. The cooled
mixture was poured into
water, extracted with EtOAc (2x50 mL) and the combined organic extracts were
dried
(MgS04) and evaporated in vacuo. The residue was purified by column
chromatography upon
silica gel using pentane-EtOAc (50:50 to 0:100) as eluant to give ethyl 1-{[(4-
chloro-1-
20 guanidino-7-isoquinolinyl)sulphonyl]amino}cyclobutanecarboxylate (150 mg,
0.34 mmol) as
a yellow powder.
mp 165-169 °C
25 ~H (DMSO-dh, 300 MHz) 8 1.0 (3H, t), 1.6-1.8 (2H, m), 2.05-2.2 (2H, m),
2.25-2.4 (2H, m),
3.8 (2H, q), 7.0-7.4 (4H, br), 8.05 (2H, s), 8. I ( 1 H, s), 8.6 ( 1 H, s),
9.05 ( 1 H, s) ppm.
LRMS 426, 428 (MH+).
30 Anal. Found: C, 46.62; H, 4.62; N, 15.82. Calc for C«H~oClN504S~0.2SCHZCIz:
C, 46.45; H,
4.63; N, 15.70.
A solution of NaOH (5 mL, 2 M, 10 mmol) was added to a solution of ethyl 1-
{[(4-chloro-1-
guanidino-7-isoquinolinyl)sulphonyl]amino}cyclobutanecarboxylate (100 mg, 0.23
mmol) in
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MeOH (5 mL) and the mixture was heated at SS °C for6 h. The cooled
mixture was
neutrilised with dilute HCl (5 mL, 2 M) to give a precipitate and the MeOH was
evaporated ifr
vacuo. The solid was collected by filtration, with copious water washing, and
dried under
high vacuum to give 1-{[(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]amino}cyclobutanecarboxylic acid hydrochloride (15 mg,
0.033
mmol).
~H (DMSO-dh, 400 MHz) c~ (.65-1.8 (2H, m), 2.05-2.2 (2H, m), 2.25-2.4 (2H, m),
8.3 (1H, d),
8.3 5-8.7 (4H, br), 8.4 ( 1 H, d), 8.5 ( l H, s), 8.7 ( 1 H, s), 8.95 ( 1 H,
s), 11.0 ( 1 H, br), 12.5 ( 1 H,
br) ppm.
Anal. Found: C, 40.06; H, 4.34; N, 15.09. Calc for
Ci;H,6C1N;O,~S~1.OHC1~1.OH~0: C, 39.83;
H, 4.23; N, 15.48.
Example 37:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]cyclo-leucine ethyl
ester
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]cycloleucine
(c) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]cycloleucine
trifluoroacetate
c1 c1 c1
/ iN -Y ~ 101 I / iN ~ 0 , / iN
Et02C N-S EtO2C H-S HOZC H-S
0 CI 0 NYNHZ 0 N~NHx
2O NHZ TNHZ
NaH (1.12 g, 80% dispersion by wt in mineral oil, 37.3 nimol) was added
portionwise to a
stirred suspension of guanidine hydrochloride (5.85 g, 59.4 mmol) in DMSO (320
mL) and
the mixture was heated at 30-50 °C under Nz for 30 min. N [(1,4-
Dichloro-1-guanidino-7-
isoquinolinyl)sulphonyl]-cycloleucine ethyl ester (6.2 g, 14.9 mmol) was added
in one portion
and the mixture heated at 80 °C for 8 h. The cooled mixture
concentrated in vacuo to ca. 160
mL and poured into water (800 mL). The aqueous mixture was extracted with
EtOAc (4x150
mL) and the combined organic extracts were then washed with water, brine,
dried (MgS04)
and evaporated in vacuo. The residue was purified by column chromatography
upon silica gel
using CH~CIz-MeOH-0.880NH3 (95:5:0.5 to 90:10:1) as eluant and then
recrystallised from
EtOAc to give N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]cyclo-leucine
ethyl ester
(1.43 g, 3.25 mmol) as a yellow solid.
mp 225-226 °C
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~H (DMSO-dh, 300 MHz) S I.1 (3H, t), 1.35-1.45 (2H, m), 1.45-1.5 (2H, m), 1.85-
1.95 (4H,
br), 3.9 (2H, q), 7.1-7.35 (4H, 6r), 8.0 ( I H, d), 8.05 ( 1 H, d), 8.1 ( 1 H,
s), 9.1 ( 1 H, s) ppm.
LRMS 440, 442 (MH+).
Anal. Found: C, 49.02; H, 4.97; N, 15.61. Calc for C~$H»C1N504S: C, 49.14; H,
5.04; N,
15.92.
A solution of NaOH (75 mL, 2 M, I50 mmol) was added to a solution of N [(4-
chloro-1-
guanidino-7-isoquinolinyl)sulphonyl]cycloleucine ethyl ester (1.39 g, 3.16
mmol) in MeOH
(75 mL) and the mixture heated at 40-50 °C for 24 h. The cooled mixture
was neutrilised with
dilute HCI (75 mL, 2 M) to give a precipitate and the MeOH was evaporated in
vacuo. The
solid was collected by filtration, with copious water washing, and dried under
high vacuum to
t5 give N [(4-chloro-1-guanidino-7-isoquinolinyl)su(phony(]cycloleucine (1.27
g, 3.08 mmol) as
a white powder.
Anal. Found: C, 46.40; H, 4.39; N, 16.66. Calc for C,6H,$CIN504S: C, 46.66; H,
4.41; N,
17.00.
N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]cycloleucine (8 mg) was
dissolved in
CF3COZH (ca. 1.0 mL) and the mixture was evaporated in vacuo, azeotroping with
PhMe. The
residue was triturated with i-Pr~O and Et~O to give a white solid. The solid
was dissolved in
MeOH, filtered and the filtrate evaporated in vacuo to give N [(4-chloro-1-
guanidino-7-
isoquinolinyl)suIphonylJcycloleucine trifluoroacetate (12 mg).
mp >178 °C (dec).
~H (DMSO-d~, 400 MHz) 8 1.3-1.45 (2H, m), 1.45-1.55 (2H, m), I.85-1.95 (4H,
br), 8.25-8.6
(4H, br), 8.3 (1H, d), 8.4 (IH, d), 8.5 (IH, s), 8.85 (1H, s), 10.8 (IH, br),
12.4 (1H, br) ppm.
LRMS 412, 414 (MH+).
Anal. Found: C, 39.50; H, 3.62; N, I 1.50. Calc for
C,6H,8CIN504S~1.OCF3COZH~I.OHZO: C,
39.75; H, 3.89; N, 12.88.
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Example 38:
1-{[{4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]amino}-N (2-
hydroxyethyl)cyclopentanecarboxamine hydrochloride
ci ci
HO:C N-g I ~ iN HO~N~N-S I ~ iN
H 0 N~NHZ IOI H O N"NHz
NH2 ~N'HZ
(COCI), (60 uL, 0.67 mmol) and then DMF (3 drops) were added to a stirred
suspension of
N-[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]cycloleucine hydrochloride
(150 mg,
0.334 mmol) in CH~C1~ ( 15 mL) and the mixture was stirred at 23 °C for
30 min. The solvents
i0 were evaporated in vacuo, azeotroping with PhMe, to give the corresponding
acid
ch(oride.This material was redissolved in CH~CI~ (15 mL) and added to a
stirred solution of3-
hydroxyethylamine (400 pL) in CH~CI, (15 mL) and the mixture stirred for l h.
The solvents
were evaporated in vacuo and the residue was purified by column chromatography
upon silica
gel using CH~CI~-MeOH-0.880NH3 (90:10:1) as eluant to give l-{[(4-chloro-1-
guanidino-7-
t5 isoquinolinyl)sulphonyl]amino}-N (2-hydroxyethyl)cyclopentanecarboxamine.
This material
was dissolved in EtOAc-EtOH and a solution of HC1 in Et~O (1 M) was added
which gave a
precipitate. The solvents were decanted and the solid was triturated with
Et~O, collected by
filtration and dried to give 1-{[(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]amino}-N
(2-hydroxyethyl)cyclopentanecarboxamine hydrochloride (77 mg, 0.155 mmol) as a
white
20 solid.
mp 244-246 °C.
~H (CD30D, 300 MHz) 8 1.35-1.5 (2H, m), 1.5-1.65 (2H, m), 1.85-2.0 (2H, m),
2.0-2.15 (2H,
25 m), 3.1-3.2 (2H, m), 3.S-3 .65 (2H, m), 8.4 ( 1 H, d), 8.4S ( 1 H, s), 8.5
( 1 H, d), 8.95 ( 1 H, s) ppm.
LRMS 455 (MH+), 477 (MNa+).
Anal. Found: C, 43.63; H, 5.03; N, 16.65. Calc for C,8Hz3C1N604S~1.OHC1~0.25
HzO: C,
30 43.60; H, 4.98; N, 16.95.
Example 39:
(a) 1-{[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonylJamino}-N [Z-
(dimethylamino)ethyl]cyclopentanecarboxamine
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(b) 1-{((4-Chlore-1-guanidine-7-isoquinolinyl)sulphonylJamino}-N (2-
(dimethylamino)ethyl]cyclopentanecarboxamine dihydrochloride
c~ c~
~[ iN ~N ~ I / iN
HOZC~H-S Me:N ~H-S
0 N\/NH= 0 0 N\/NHx
NNHZ ' TNHZ
A solution HCI in Et~O (0.S mL, 1 M) was added to a stirred solution ofN [(4-
chioro-1-
guanidine-7-isoquinolinyl)sulphonylJcycloleucine (100 mg, 0.243 mmol) in MeOH.
The
solvents were evaporated in vacuo and the residue azeotroped with PhMe to give
the
corresponding hydrochloride salt.
i0
(COCI)z (42 p,L, 0.48 mmol) and then DMF (2 drops) were added to a stirred
solution of N
[(4-chloro-I-guanidine-7-isoquinolinyl)sulphonyl]cycloleucine hydrochloride
(0.243 mmol)
in CH~CI~ (5 mL) and the mixture was stirred at 23 °C for 18 h. The
solvents were evaporated
in vacuo, the residue redissolved in CI-(,C1, (5 mL), and 2-
(dimethylamino)ethylamine (60
15 p.L, 0.48 mmol) was added and the mixture stirred for 3 h. The solvents
were evaporated in
vacuo and the residue partioned between EtOAc and aqueous NaHC03 ( 10 %). The
organic
phase was dried and evaporated. The residue was purified by column
chromatography upon
silica gel using CHaCh-MeOH-0.880NH3 (95:5:0.5 to 90:10:1) as eluaiit to give
1-{[(4-
chloro-1-guanidine-7-isoquinolinyl)sulphonyl]amino}-N [2-
20 (dimethylamino)ethyl]cyclopentanecarboxamine.
LRMS 482 (MH+)
This material was dissolved in EtOAc, a solution of HCI in Et~O (1 M) was
added and the
25 solvents were evaporated in vacuo to give 1-{[(4-chloro-1-guanidine-7-
isoquinolinyl)sulphonyl]amino}-N [2-
(dimethylamino)ethyl]cyclopentanecarboxamine
dihydrochloride (28 mg, 0.048 mmol) as a white solid.
IH (TFA-d, 400 MHz) 8 1.5 (2H, br s), 1.7 (2H, br s), 2.1 (4H, br s), 3.2 (6H,
s), 3.7 (2H, br
30 s),4.0(2H,brs),7.8(lH,brs),8.45(lH,d),8.5(lH,s),8.6(lH,d),9.5(lH,s)ppm.
LRMS 482 (MH+).
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Anal. Found: C, 41.25; H, 5.63; N, 16.59. Calc for
C~oH~sCIN~03S~2.OHC1~1.SH~0: C, 41.28;
H, 5.72; N, 16.85.
Example 40:
4-Chloro-1-guanidino-N (1-(hydroxymethyl)cyclopentyl]-7-
isoquinolinesulphonamide
hydrochloride
ci ci
HO~N'~ I / iN HON~~ I / iN
H p y H 0 N\'NH:
'N~H2
NaH (30 mg, 80% dispersion by wt in mineral oil, 1.O mmol) was added in one
portion to a
stirred solution of guanidine hydrochloride ( 157 mg, 1.6 mmol) in DMSO (5 mL)
and the
mixture was heated at 60 °C under N~ for 20 min. 1,4-Dichloro-N [ 1-.
(hydroxymethyi)cyclopentyl]-7-isoquinolinesulphonamide (150 mg, 0.40 mmol) was
added in
one portion and the mixture heated at 80 °C for 4 h. A second portion
of guanidine (0.40
IS . mmol)[prepared from guanidine hydrochloride (38 mg) and NaH (12 mg)] in
DMSO (1 mL)
was added and the mixture heated at 80 °C for an additional 6 h. The
cooled mixture was
poured into water (80 mL), extracted with EtOAc (2x50 mL) and the combined
organic
extracts were then washed with brine, dried (MgS04) and evaporated in vacuo.
The residue
was purified by column chromatography upon silica gel using CHZCIz-MeOH-
0.880NH3
(97.5:2.5:0.25 to 80:20:5) as eluant to give the partially purified product
(90 mg). This
material was converted to the corresponding hydrochloride salt by treatment
with a solution
of HCl in EtzO (1 M) and then recrystallised from EtOH to give 4-chloro-I-
guanidino-N [I-
(hydroxymethyl)cyclopentyl]-7-isoquinolinesulphonamide hydrochloride (16 mg,
0.040
mmol) as a white solid.
mp 245-247 °C.
'H (CD30D, 400 MHz) 8 1.4-1.55 (4H, m), 1.55-1.7 (2H, m), 1.8-1.9 (2H, m), 3.5
(2H, s), 8.4
(IH, d), 8.45 (1H, s), 8.45 (IH, d), 8.9 (1H, s) ppm.
LRMS 398, 400 (MH+).
Anal. Found: C, 44.17; H, 4.84; N, 15.88. Calc for C,6HZOC1N5O3S~I.OHCI: C,
44.24; H, 4.87;
N, 16.12.
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Example 4I:
(a) N ((4-chloro-1-guanidino-7-isoquinolinyl)sulphonyi]-N [2-
(dimethylamino)ethyl]eycloleucine ethyl ester dihydrochloride
(h) N ((4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N (2-
(dimethylamino)ethyl]cycloleucine dihydrochloride
c1 c1 a
w w w w w w
i ~N ~ ~ I s ~N ~ ~ i ~N
Et02C N-S EtO,C N-S HO=C N-S
0 CI ~ 0 N~NH2 ~ 0 NYNHZ
NMez NMei ~NHz NMei NHZ
l0 NaH (32 mg, 80% dispersion by wt in mineral oil, 1.05 mmol) was added in
one portion to a
stirred solution of guanidine hydrochloride ( 145 mg, 1.52 mmol) in DMSO (4
mL) and the
mixture was heated at 50 °C under N~ for 20 min. N [( 1,4-Dichloro-7-
isoquinolinyl)sulphonyl]-N [2-(dimethylamino)ethyl]cycloleucine ethyl ester
hydrochloride
( 160 mg, 0.305 mmol) was added in one portion and the mixture heated at 90
°C for I h. The
IS cooled mixture was poured into water, extracted with EtOAc (2x20 mL) and
the combined
organic extracts were then washed with brine, dried (Na,S04) and evaporated in
vaeuo. The
residue was dissolved in EtzO, filtered, and~a solution of HCl in EtzO (I M)
was added which
gave a precipitate. The solvents were evaporated in vacuo and the residue
recrystallised from
hot EtOH to give N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N [2-
20 (dimethylamino)ethyl]cycloleucine ethyl ester dihydrochloride (123 mg, 0.20
mmoI) as a pale
yellow solid.
mp 228-230°C.
25 ~H (TFA-d, 400 MHz) S 1.45 (3H, t), 1.7 (2H, br s), 1.9 (2H, br s), 2.2
(2H, br s), 2.5 (2H, br
s), 3.3 (6H, s), 3.75 (2H, br s), 4.3 (2H, br s), 4.4 (2H, q), 8.15 (IH, br
s), 8.4 (1H, d), 8.5 (1H,
s), 8.65 (1H, d), 9.35 (1H, s) ppm.
LRMS 511, 513 (MH+).
Anal. Found: C, 43.74; H, 5.88; N, 13.75. Calc for C»H3,C1N604S~2.OHC1~1.OH20:
C, 43.90;
H, 5.86; N, 13.96.
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A solution ofNaOH (5 mL, 5 M) was added to a solution ofN [(4-chloro-1-
guanidino-7-
isoquinolinyl)sulphonyl]-N [2-(dimethylamino)ethyl]cycloleucine ethyl ester
dihydrochloride
(75 mg, 0.128 mmol) in dioxane (5 mL) and the mixture was heated at 80
°C for 30 h. The
cooled mixture was diluted with water (20 mL), the dioxane was evaporated in
vacuo, and the
aqueous residue neutrilised with dilute HCI (2 M) to pH 6. The precipitate was
collected by
filtration with water washing, and then dissolved in MeOH, filtered and
evaporated in vacuo.
The residue was purified by column chromatography upon silica gel using CH~Ch-
MeOH-
0.880NH3 (90:10:1 to 80:20:5) as eiuant to give to give the desired product.
This material was
dissolved in MeOH-EtOAc, a solution of HCl in Et~O ( 1 M) Was added and the
solvents were
ID evaporated in vaczro. The residue was triturated with EtOAc to give N [(4-
chloro-1
guanidino-7-isoquinolinyl)sulphonyl]-N [2-(dimethylamino)ethyl]cycloleucine
dihydrochloride (15.4 mg, 0.025 mmol).
iH (TFA-d, 400 MHz) S 1.7 (2H, br s), 1.9 (2H, br s), 2.2 (2H, br s), 2.6 (2H,
6r s), 3.25 (6H,
. s),3.8(2H,brs),4.3(2H,brs),8.1
(IH,brs),8.4(IH,d),8.5(lH,s},8.65(IH,d),9.4(tH,
s) ppm.
LRMS 483 (MH+).
Anal. Found: C, 39.03; H, 5.60; N, 14.02. Calc for C~oHz7C1N60aS~2HC1~3H20: C,
39.38; H,
5.78; N, 13.78.
Example 42:
N (t-Butoxycarbonylmethyl)-N ((4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]cycloleucine ethyl ester
ct ct
o l w w ~ o ~ w w
EtOzC H-& ~ ~ N EtO2C N-& ~ ~ N
0 N w NHz J 0 N ~ NHZ
tBuOsC
NHZ Nfiz
Anhydrous K~C03 (34 mg, 0.25 mmol) and t-butyl bromoacetate (44 p,L, 0.30
mmol) were
added to a stirred solution ofN [(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]cycloleucine ethyl ester (110 mg, 0.25 mmol) in DMF
(1.0 mL) and
the mixture was stirred at 23 °C for 18 h. The mixture was diluted with
EtOAc (60 mL),
washed with water (3x100 mL), dried (MgS04) and evaporated in vacuo. The
residue was
purified by column chromatography upon silica gel using pentane-EtOAc (100:0
to 20:80) as
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eluant to give N (t-butoxycarbonylmethyl)-N [{4-chloro-1-guanidine-7-
isoquinolinyl)sulphonyl]cycloleucine ethyl ester (95 mg, 0.17 mmol) as a white
solid.
~H {CDC13, 400 MHz) 8 1.3 {3H, t), 1.45 (9H, s), 1.6-1.7 (4H, m), 1.85-1.95
(2H, br), 2.25-
2.35{2H,m),4.2(2H,q),4.5(2H,s),8.1 (lH,d),8.15(IH,s),8.3(lH,dd),9.3(lH,d)ppm.
LRMS 554 (MHt)
Anal. Found: C, 52.31; H, 5.94; N, 13.33. Calc for C~aH3~C1N506S: C, 52.03; H,
5.82; N,
( 0 12.64.
Example 43:
(a) Methyl I-{[(4-chloro-I-guanidine-7-
isoquinoliny()sulphonyl]amino}cyclohexanecarboxylate
I S (b) I-{((4-Chloro-I-guanidine-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylic
acid hydrochloride
c1 c1 I
w w w w ~ v w
/ iN Q ~ I / iN 101 / iN
Me02C H S Me02C H-S HO=C N-S
O CI 0 NYNH2 H 0 NYNHi
NH: NHZ
20 NaH (22.3 mg, 80% dispersion by wt in mineral oil, 0.743 mmo() was added in
one portion to
a stirred solution of guanidine hydrochloride ( I l7 mg, 1.98 mmol) in DMSO (5
mL) and the
mixture was heated at 50-70 °C under N~ for 25 min. Methyl I-{[(1,4-
dichloro-7-
isoquinolinyl)sulphonyl]amino}-cyclohexanecarboxytate (I24 mg, 0.30 mmol) was
added in
one portion and the mixture heated at 80 °C for 8 h. The cooled mixture
vas poured into
25 water (50 mL), extracted with EtOAc (2x50 mL) and the combined organic
extracts were
washed with water, brine, dried (MgS04) and evaporated in vacuo. The residue
was
crystallised from a minimum of hot EtOAc to give methyl I-{[(4-chloro-I-
guanidine-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate (12 mg, 0.043 mmol) as
yellow
solid. Evaporation of the mother liquors and trituration of the residue with
Et20 gave a second
30 crop (7 mg).
mp >220 °C (dec).
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~H (DMSO-d,~, 400 MHz) S 1. i-1.35 (6H, m), i.65-1.75 (2H, m), 1.75-1.85 (2H,
m), 3.35
(3 H, s), 7.1-7.4 (4H, br), 8.0 ( I H, d), 8.05 ( 1 H, d), 8.1 ( 1 H, s), 8. I
5 ( 1 H, s), 9.0 ( 1 H, s) ppm.
LRMS 440, 442 (MH+)
Anal. Found: C, 48.55; H, x.12; N, 15.73. Calc for C,$H~~CIN;O,~S~0.3H~0: C,
49.14; H,
5.04; N, 15.92.
A solution ofNaOH (1 mL, 2 M, 2 mmol) was added to a solution of methyl 1-{[(4-
chloro-1
guanidino-7-isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate (12 mg, 0.027
mmol) in
MeOH (4 mL) and the mixture was heated at 50-60 °C for 4 d. The cooled
mixture was
neutrilised witli dilute HC1 (1 mL, 2 M) to Give a precipitate. The solid was
collected by
tiltration, with copious water washing, and then triturated with EtOAc. The
solid was
dissolved in conc. HCI, the solvents were evaporated in vacuo azeptroping with
PhMe, and
l5 then dried under high vacuum to give 1-{[(4-chloro-I-ouanidino-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylic acid hydrochloride (1 l
mg, 0.021
mmol).
mp 194 °C (dec)
~H (DMSO-d~, 400 MHz) 8 1. l-1.4 (6H, m), 1.6-1.8 (2H, m), 1.8-1.95 (2H, m),
8.15-8.7 (4H,
br), 8.2 ( I H, s), 8.3 ( 1 H, d), 8.4 ( I H, d), 8.45 ( I H, s), 8.9 ( 1 H,
s), 10.9 ( 1 H, br), 12.4 ( 1 H, br)
ppm.
LRMS 426 (MII'~).
Anal. Found: C, 39.87; H, 5.05; N, 13.16. Calc for
C,~HZOCIN504S~l.OHCI~3.OHz0: C, 39.54;
H, 5.27; N, 13.56.
Example 44:
(a) Methyl 4-{((4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]amino}tetrahydro-2H
pyran-4-carboxylate
(b) 4-{[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]amino}tetrahydro-2H
pyran-4-
carboxylic acid hydrochloride
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o ci o ci o ci
~ o ~ ~l ~ o ~ ~ ~ o
MeOzC' _N-S I ~ ~ N ~ MeO,C"N-S I ~ ~ N HOzC' _N-S I ~ ~ N
H 0 CI H 0 NYNH2 H O N\/NH,
NH= ~N'HZ
NaH (33.5 mg, 80% dispersion by wt in mineral oil, 1.12 mmol) was added in one
portion to
a stirred solution of guanidine hydrochloride (176 mg, i.84 mmo!) in DMSO (3.0
mL) under
S N, and the mixture was heated at SO °C For 15 min. Methyl 4-{[(1,4-
dichloro-7-
isoquinolinyl)sulphonyl]amino} tetrahydro-2H pyran-4-carboxylate (187 mg,
0.446 mmol)
was added in one portion and the mixture heated at 80 °C for 8 h. A
second portion of
guanidine (0.45 mmol)[prepared from guanidine hydrochloride and NaH] in DMSO
(I.0 mL)
was added and the mixture heated at 90 °C for an additional 4 h. The
cooled mixture was
l0 poured into water (100 mL) , extracted with EtOAc~(3x50 mL) and the
combined organic
extracts were washed with brine, dried (Na~SO~). The solvents were evaporated
in vacuo and
the residue purified by column chromatography upon silica gel using CHaCI~-
MeOH-
0.880NH3 (9S:S:O.S) as eluant, and then crystallised with EtOAc, to give to
give methyl 4-
{[(4-chloro-I-guanidino-7-isoquinolinyl)sulphonyl]amino}tetrahydro-2H pyran-4-
15 carboxylate (83 mg, 0.186 mmol) as a yellow solid.
mp 24S-247 °C.
'H (CDC13, 400 MHz) b 3.3 (3H, s), 3.35-3.45 (8H, m), 7.1-7.4 (4H, br), 8.0S
(2H, s), 8.1
20 (lH,s),8.4(IH,s),9.0(lH,s)ppm.
LRMS 442, 444 (MH+).
Anal. Found: C, 46.18; H, 4.56; N, 15.32. Cale for Ci~H~oC1N505S~0.2H20: C,
45.83; H,
25 4.62; N, 15.72.
A solution ofNaOH (I mL, 2 M, 2 mmol) was added to a solution of methyl 4-{j(4-
chloro-I-
guanidino-7-isoquinoiinyl)sulphonyl]amino}tetrahydro-2H pyran-4-carboxylate
(68 mg,
0.153 mmol) in MeOH (12 mL) and the mixture was heated at reflux for 30 h. The
cooled
30 mixture was neutrilised with dilute HCl (1 mL, 2 M), partially concentrated
by evaporation in
vacuo to give a precipitate which was collected by filtration, with water
washing. The solid
was extracted with warm cone. HCI, the solution decanted from insoluble
material and the
solvents were evaporated in vacuo. The solid residue was azeptroped with PhMe
and then
dried under high vacuum to give 4-{[(4-ehloro-1-guanidino-7-
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isoquinolinyl)sulphonyl]amino}tetrahydro-2H pyran-4-carboxylate acid
hydrochloride (30
mg, 0.062 mmol) as a white solid.
mp 190-2l0 °C (dec).
' H (DMSO-d~, 400 MHz) 8 3.2-3.5 (8H, m), 8.2-8.7 (4H, br), 8.3 ( 1 H, d), 8.4
( 1 H, d), 8.45
( 1 H, s), 8.95 ( 1 H, s), 1 l .0 ( 1 H, br s), I 2.6 ( I H, br s) ppm.
Anal. Found: C, 39.76; H, 4.33; N, 14.12. Calc For
C,6H,8CINSOSS~1.OHCl~1.1H~0: C, 39.69;
H, 4.41; N, 14.47.
Example 45:
(a) t-Butyl (~)-cis-2-{[(:~-chloro-1-guanidine-7-
isoquinolinyl)sulphonyl]amino}-
cyclohexanecarboxylate
(b) (t)-cis-2-{[(4-Chloro-1-buanidino-7-
isoquinolinyl)sulphonyl[amino}cyclohexanecarboxylic acid hydrochloride
c1 c1 c1
COstBU ~ ~ COZtBu ~ ~ CO,H
0 ~ / iN a _~ I / iN a I01 I / iN
0 CI H 0 NYNH2 H 0 N \'NH2
NH2 ~N'H=
Guanidine hydrochloride (325 mg, 3.4 mmol) was added in one portion to a
stirred
suspension of NaH (89 mg, 80% dispersion by wt in mineral oil, 2.97 mmol) in
DME (5 mL)
and the mixture was heated at 60 °C under NZ for 30 min. A solution of
t-butyl (~)-cis-2-
{[(1,4-dichloro-7-isoquinolinyl)sulphenyl]amino}cyclohexanecarboxylate (391
mg, 0.85
mmol) in DME (5 mL) was added and the mixture heated at 90 °C for 6 h.
The solvents were
evaporated irz vacuo, the residue was dissolved with EtOAc, washed with
aqueous NH4Cl,
dried (MgS04) and evaporated in vacuo. The residue was purified by column
chromatography
upon silica gel using using toluene-i-PrOH-0.880NH3 (100:0:0 to 90:10:0.05) as
eluant to
give t-butyl (~)-cis-2-{[(4-chloro-1-guanidine-7-
isoquinolinyl)sulphenyl]amino}cyclohexanecarboxylate (75 mg, 0.15 mmol) as a
white solid.
'H (CDC13, 400 MHz) S 1.1-1.8 (7H, mm), 1.4 (9H, s), 1.95 (1H, m), 2.55 (1H,
dd), 3.45 (1H,
br s), 5.9 ( 1 H, d), 6.0-6.5 (4H, br), 8.05 ( 1 H, d), 8.1 ( 1 H, d), 8.15 (
1 H, s), 9.3 ( 1 H, s) ppm.
LRMS 482, 484 (MF-I~).
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CF3COZH (3.0 mL) was added to a stirred solution of t-butyl (t)-cis-2-{[(4-
chloro-1-
guanidino-7-isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate (66 mg, 0.14
inmol) in
CH,CIZ (3.0 mL) and the mixture was stirred at 23 °C for 6 h. The
solvents were evaporated iu
vacuo, azeotroping CHaCh (x3). The residue was dissolved in EtOAc and a
solution of HCI in
Et~O (200 ~L, 1.0 M) was added which gave a precipitate. The white solid was
collected by
filtration and dried to give (~)-cis-2-{[(4-chloro-I-guanidino-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylic acid hydrochloride (35 mQ,
0.069
mmol).
to
mp 220-223 °C (dec).
1H (DMSO-d~, 400 MHz) 8 1.l-1.3 (3H, m), 1.4-1.6 (4H, m), 1.7-1.8 (1H, m), 2.5
(1H, m),
3.75 ( l H, br s), 8.0 ( 1 H, d), 8.25-8.6 (4H, br), 8.35 (2H, s), 8.45 ( 1 H,
s), 8.95 ( l H, s) ppm.
l~
Anal. Found: C, 42.95; H, 4.96; N, 13.79. Calc for
C,~H,oClN504S~I.OHCI~1.2SH~0~0.3Et~0:
C, 43.11; H, 5.27; N, 13.81.
Example 46:
20 Ethyl (~)-traps-2-{[(4-chloro-I-guanidino-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate
ci c1
co,e~ ~ ~ cazef ~
-S ~ / iN N_S ~ / iN
H O CI H 0 NYNH=
NHT
Guanidine hydrochloride (458 mg, 4.8 mmol) was added in one portion to a
stirred
25 suspension of NaH (90 mg, 80% dispersion by wt in mineral oil, 2.97 mmol)
in DME ( I O
mL) and the mixture was heated at 60 °C under N2 for 30 min. A solution
of ethyl (~)-cis-2-
{[(1,4-dichloro-7-isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate (377
mg, 0.87
mmol) in DMA (5 mL) was added and the mixture heated at 90 °C for 4 h.
The solvents were
evaporated iu vacuo, the residue Was dissolved with EtOAc (200 mL), washed
with aqueous
30 NH4C1 (20 mL), then with water (500 mL), and the combined aqueous washings
were
extracted with EtOAc (2x50 mL). The combined EtOAc extracts were washed with
water
(4x100 mL), dried (MgS04) and evaporated in vacuo. The residue was purified by
column
chromatography upon silica gel using using toluene-i-PrOH-0.880NH3 (100:0:0 to
90:10:0.05) as eluant to give ethyl (~)-traps-2-{[(4-chloro-I-guanidino-7-
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isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate (65 mg, 0.14 mmol) as a
white solid.
[A small amount of ethyl (~)-cis-2-{[(4-chloro-1-guanidino-7- .
isoquinoiinyl)sulphonyl]amino}cyclohexanecarboxylate (<20 mg) was also
isolated.]
tH (CDC13, 400 MHz) b I.l-1.8 (6H, mm), 1.1 (3H, t), 1.9 (1H, m), 2.0 (1H, m),
2.25 (1H,
td), 3.45 ( 1 H, m), 3.8-4.0 (2 H, m), 8.05 ( 1 H, d), 8.1 ( 1 H, d), 8.15 ( 1
H, s), 9.3 ( 1 H, s) ppm.
LRMS 454, 456 (MH+).
Example 47:
(a) t-Butyl cis-4-{[(4-chloro-1-;uanidino-7-
isoquinolinyl)sulphonyl]amino}cyclohexane-
carboxylate
(b) t-butyl traits-4-{((4-chloro-1-buanidino-7-
isoquinolinyl)sulphonyl]amino}cyclohexane-carboxylate
I~ (c) cis-4-{[(4-Chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylic acid hydrochloride
ci ci ct
tBuO C ~ ~ t8u0 C ~ ~ HO=C
O~/rN 2~I~I~/rN ~I~I~//N
0 CI H 0 N\'NHx H 0 NYNH2
'N~H2 NN2
Guanidine hydrochloride (286 mg, 3.0 mmol) was added in one portion to a
stirred
suspension of NaH (56 mg, 80% dispersion by wt in mineral oil, 1.82 mmol) in
DME (5 mL)
and the mixture was heated at 60 °C under N~ for 30 min. A solution of
t-butyl cis-4-{[( 1,4-
dichloro-7-isoquinolinyl)sulphonyl]amino}cyc(ohexanecarboxylate (346 mg, 0.75
mmol) in
DME ( I S mL) was added and the mixture heated at 90 °C for 2 h. A
second portion of
guanidine (0.75 mmol)[prepared from guanidine hydrochloride (72 mg) and NaH
(22 mg)] in
DME (5 mL) was added and the mixture heated at 90 °C for 1 h. DMA ( 10
mL) was then
added to the heterogeneous reaction mixture and the now homogeneous mixture
heated for an
additional 6 h. The solvents were evaporated in vacuo, the residue was
quenched aqueous
NH,,CI (10 mL), diluted with water (150 mL) and extracted with EtOAc (2x150
mL). The
combined organic extracts were washed with water ( I00 mL), dried (MgSOd) and
evaporated
in vacuo. The residue was purified by repeated column chromatography upon
silica gel using
(i), pentane-EtOAc ( 100:0 to 25:75) and then (ii), PhMe-EtOAc (50:50 to
0:100) as eluant to
give t-butyl cis-4-{[(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate (247 mg, 0.51 mmol). [A
small
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amount of t-butyl traps-4-{[(4-chloro-l-guanidino-7-
isoquinolinyl)suiphonyl]amino}cyclohexanecarboxylate (20 mg) was also
isolated.]
'H (CDCl3, 400 MHz) 8 1.4 (9H, s), 1.5-1.8 (8H, mm), 2.3 (1H, m), 3.4 (1 H,
m), 4.8-4.9 (1H,
br), 6.1-6.~ 5 (4H, br), 8.05 { 1 H, d), 8.1 { 1 H, d), 8.15 ( 1 H, s), 9.3 (
1 H, s) ppm.
LRMS 482 (MHt), 963 (MPH+)
Anal. Found: C, X2.14; H, 5.92; N, 14.19. Calc for C,iHz$CINSOdS: C, 52,33; H,
5.86; N,
14.53.
t-Butyl cis-4-{[(4-chloro-I-guanidino-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate (55 mg, 0.121 mmol) was
suspended
in a solution of EtOAc saturated with HCl (50 mL) and the mi~cture heated at
ref(uY. The
mixture was cooled, the white solid was collected by filtration, with EtOAc
washing, and then
dried to give cis-4-{[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]amino}-
cycloheranecarbo:cyfic acid hydrochloride (1 10 mg, 0.236 mmol).
mp 287-289 °C.
'H (CDC13, 400 MHz) 8 1.5-1.6 (6H, m), 1.8-1.9 (2H, m), 2.35 (IH, m), 3.4 (1H,
m), 8.35
(lH,d),8.45(lH,s),8.5(lH,d),8.9(IH,s)ppm
Anal. Found: C, 43.88; H, 4.61; N, 14.69. Calc for
C,~H~oClN504S~1.OHC1~0.2H20: C, 43.82;
H, 4.63; N, 15.03.
Example 48:
(a) Ethyl traps-4-{[(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]amino}cyclohexane-
carboxylate
(b) traps-4-{[(4-Chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylic acid hydrochloride
t c! c!
EtOzC~.,, ~ ~ EtO2C~.", ~ ~ HOzC
~N-S I / i N ~N_S I / i N ~ ...~N-S I / i N
H 0 Ct H 0 NYNHz H 0 N \/NHz
NHz 1NHZ
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Guanidine hydrochloride (273 mg, 2.86 mmol) was added in one portion to a
stirred
suspension of NaH (55 mg, 80% dispersion by wt in mineral oil, 1.82 mmol) in
DME ( 10
mL) and the mixture was heated at 60 °C under N~ For 30 min. A solution
of ethyl traps-4-
{[(1,4-dichloro-7-isoquinolinyl)sulphonyl)amino}cyclohexanecarboxylate (370
mg, 0.78
mmol) in DMA (10 mL) was added and the mixture heated at 90 °C for 3 h.
The solvents
were evaporated in vacuo, the residue was partitioned between Et~O (100 mL),
aqueous
NHaCI ( I 0 mL), and water ( 150 mL). The separated aqueous phase was
extracted with Et~O
(3x100 mL) and the combined organic extracts were dried (MgSO.~) and
evaporated in vacaro.
The residue was purified by column chromatography upon silica gel using
toluene-i-PrOH-
l0 0.880NH3 (100:0:0 to 90:10:0.05) as eluant to give ethyl traps-4-{[(4-
chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate (70 mg, 0.15 mmol).
~H (CDC13, 400 MHz) 8 1.1 (3H, s), I.1-1.3 (4H, mm), 1.6 (2H, 6r d), 1.8 (2H,
br d), 2.l (1 H,
m), 2.9 ( t H, m), 3.95 (2H, q), 7. l-7.4 (4H, br), 7.8 ( 1 H, d), 8.0 ( 1 H,
d), 8.1 ( 1 H, d), 8. t ( 1 H,
I 5 s), 9.1 ( I H, s) ppm.
LRMS 454, 456 (MH+).
Anal. Found: C, 50.27; H, 5.56; N, 14.92. Calc for C,9H~dC1N504S: C, 50.27; H,
5.32; N,
20 15.43.
A solution of HCl (5 mL, 2 M, 10 mmol) was added to a solution of ethyl traps-
4-{[(4-
chloro-1-guanidino-7-isoquinolinyl)sulphonylJamino}cyclohexanecarboxylate (55
mg, 0.121
mmol) in dioxane (5.0 mL) and the mixture was heated at reflux for 2 h. The
solvents were
25 evaporated irr vacuo and the residue was purifed by column chromatography
upon MCI gel
(CHP 20P) using water-MeOH (100:0 to 20:80) as e(uant to give traps-4-{[(4-
chloro-1-
guanidino-7-isoquinolinyl)sulphonyl]amino}-cyclohexanecarboxylic acid. This
material was
dissolved in dilute HCl (20 mL, 0.1 M), the solvents were evaporated in vacuo,
and the
residue triturated with EtZO to give traps-4-{[(4-chloro-1-guanidino-7-
30 isoquinolinyl)sulphony(]amino}cyclohexanecarboxylic acid hydrochloride (35
mg, 0.067
mmol) as a white solid.
mp >205 °C (dec).
35 1H (CD30D, 400 MHz) 8 1.2-I.4 (4H, mm), 1.8 (2H, br d), 1.9 (2H, br d), 2.1
(1H, m), 3.1
(lH,m),8.3(lH,d),8.45(IH,s),8.5(lH,d),8.9(IH,s)ppm.
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Anal. Found: C, 42.75; H, 5.04; N, 13.35. Calc for
C,~HZOCIN50aS~1.OHC1~1.SH~0~0.4Et~0:
C, 43.04; H, 5.44; N, 13.49.
S Example 49:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]glycine t-butyl ester
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)carbonyl]glycine trifluoroacetate
cI cI cI
I ~ v I w v I w v
tBuO_C~N ~ ~N ~ t8u0,C~N ~ N ~ HO.C~N ~ N
H 0 CI H 0 NYNH; H 0 N\/NHZ
NH~ 'N~H,
l0
NaH (34 mg, 60% dispersion in mineral oil, 0.85 mmol) was added to a stirred
solution of
guandine hydrochloride (80 mg, 0.84 mmol) in DMSO (2 mL) at 23 °C .
After 30 min, N
[(1,4-dichloro-7-isoquinolinyl)carbonyl]glycine t-butyl ester (120 mg, 0.34
mmol) was added
and the resultant solution heated at 90 °C for 21 h. After cooling, the
mixture was poured into
water (30 mL), extracted with EtOAc, and then with CH~C12, and the combined
organic
extracts were dried (Na~SOa) and evaporated in vacuo. The residue was purified
by column
chromatography on silica gel using CH~C1~-MeOH-0.880NH3 (90:10:1) as eluant to
give N
[(4-chloro-I-guanidino-7-isoquinolinyl)carbonyl]glycine t-butyl ester (25 mg,
0.07 mmol) as
a yellord gum.
LRMS 378 (MH+), 7S6 (MZH+).
A solution of N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]glycine t-
butyl ester (24 mg,
0.06 mmol) in CF3COzH (0.5 ml) was stirred at 0 °C for 1.S h. The
reaction mixture was
diluted with PhMe, evaporated in vacuo, azeotroping with PhMe, and the residue
triturated
with EtzO to give N [(4-chloro-I-guanidino-7-isoquinolinyl)carbonyl]glycine
trifluoroacetate
(21 mg, 0.05 mmol) as a white solid.
mp > 300 °C.
1H (TFA-d, 400 MHz) ~ 4.6 (2H, s), 8.4 ( 1 H, d), 8.45 ( 1 H, s), 8.6 ( 1 H,
d), 9.3 ( 1 H, s) ppm.
LRMS 322 (MH+).
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Anal. Found: C, 40.60; H, 2.91; N, 15.47. Calc for Ci3H,~CIN503~CF3COZH: C,
40.58; H,
2.93; N, 15.46.
Example ~0:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-(3-alanine t-butyl
ester
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)carbonyl]-(3-alanine
c1 ci ci
w ~ ~ I y
tBuO;C~N , / i N tBuO;C~N I / i N HO,C~N / i N
H
H 0 CI H O NYNHi 0 NYNH,
NH2 NH:
NaH (114 mg, 60% dispersion in mineral oil, 2.85 mmol) was added portionwise
to a stirred
solution of guanidine hydrochloride (272 mg, 2.85 mmol) in DMSO (8 rnL) and
the solution
was heated at 80 °C for 20 min. N [( 1,4-Dichloro-7-
isoquinolinyl)carbonyl]-[3-alanine l-butyl
ester (420 mg, I. t4 mmol) was added and the mixture heated at 90 °C
overnight. The cooled
mixture was poured into water, extracted with EtOAc, and the combined organic
extracts
l5 were washed with water, saturated brine, dried (Na~S04) and evaporayted in
vacuo. The
residue was crystallised from i-Pr,O-CHZCh to give N [(4-chloro-1-guanidino-7-
isoquinolinyl)carbonyl]-[3-alanine t-butyl ester (190 mg, 0.48 mmol).
mp 224-226 °C.
'H (DMSO-d6, 400 MHz) 8 1.4 (9H, s), 2.55-2.5 (2H, m), 3.5 (2H, dt), 7.0-7.3
(4H, br s), 7.85
( 1 H, d), 8.0 ( 1 H, s), 8.1 ( 1 H, d), 8.65 ( 1 H, t), 9. I ( 1 H, s) ppm.
LRMS 392 (MH+), 783 (MZH+)
2~
Anal. Found: C, 54.89; H, 5.68; N, 17.94. Ca(c for C,BHZZCIN503: C, 55.17; H,
5.66; N,
' 17.87.
A solution of N j(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-(3-alanine t-
butyl ester (145
mg, 0.37 mmol) in CF3CO~H ( 1.5 mL) was stirred at 0 °C for 30 min, and
then at room
temperature for I h. PhMe (IS mL) was added, the mixture evaporated in vacuo,
and the
residue triturated with EtOAc and Et~O to give N [(4-Chloro-1-guanidino-7-
isoquinolinyl)carbonyl]-(3-alanine (117 mg, 0.26 mmol) as a white solid.
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mp 235-236 °C.
~H (DMSO-dh, 300 MHz) 8 2.6 (2H, t), 3.55 (2H, dt), 8.25 (1H, d), 8.35-8.4
(2H, m), 8.5 (4H,
br s), 8.8-8.9 (2H, m) ppm.
LRMS 336 (MH+).
Anal. Found: C, 42.72; H, 3.56; N, 14.5S. Calc for CidH,,,CIN50~~0.25Et0Ac: C,
42.75; H,
3 .5 7; N, 14.49.
Example S1:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl[cyctoleucine ethyl
esfier
(b) N ((4-Chloro-1-guanidino-7-isoquinolinyl)carbonyl[cycloleucine
c1 c1 c1
w w w w w w
EIOZC~H I / l N Et02C- _H I / l N Ii0 C"H I / l N
0 CI 0 N\/NH, 0 N\/NHZ
'N~HZ ~N'H2
NaH (45 mg, 60% dispersion in mineral oil, 1.13 mmoi) was added to t-BuOH and
the
mixture heated at 50 °C for 15 min. Guanidine hydrochloride (I05 mg,
1.10 mmoi) was added
and the mixture heated at SO °C for an additional 15 min. N [(1,4-
Dichloro-7-
isoquinolinyl)carbonyl]cycloleucine ethyl ester (350 mg, 0.92 mmol) was added
and the
mixture heated at reflux for 17 h. The solvents were evaporated in vacuo and
the residue
purified by column chromatography on silica gel using CHZC12-MeOH-0.880NH3
(90:10:1) as
eluant, followed by trituration with CHZCIz-i-PrzO, to give N [(4-chloro-1-
guanidino-7-
isoquinolinyl)carbonyl]cycloleucine ethyl ester (55 mg, 0.14 mmol) as a pale
yellow powder.
~H (CDC13, 300 MHz) 8 1.0 (3H, t), 1.5-1.65 (4H, m), 1.8-2.0 (2H, m), 2.0-2.15
(2H, m), 3.9
(2H,q),6.7(4H,brs),7.5(lH,s),7.7(lH,d),7.8(lH,s),7.9(lH,d),8.95(lH,s)ppm.
LRMS 404 (MHT).
Anal. Found: C, 55.94; H, 5.42; N, 16.94. Ca(c for Cl9HzzC1N503~0.25 HzO: C,
55.87; H,
S.SS; N, 17.14.
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A partly heterogeneous solution of N [(4-chloro-1-guanidino-7-
isoquinolinyl)carbonyl]cycloleucine ethyl ester (45 mg, 0.11 mmol) in dioxane
(1.5 mL) was
stirred with aqueous NaOH (I mL, 2 M) for 2.5 h at 23 °C. Dilute HCI (1
mL, 2 M) was
added to give a cream suspension. The solid was collected by filtration and
dried irr vacuo to
yield ~V [(4-chloro-I-guanidino-7-isoquinolinyl)carbonyl]cycioleucine (40 mg,
0.1 i mmol).
mp > 275 °C.
~ H (TFA-d, 400 MHz) 8 1.9-2.1 (4H, m), 2.2-2.4 (2H, m), 2.5-2.7 (2H, m), 8.3
( 1 H, d), 8.35
( l H, s), 8.45 ( 1 H, d), 9.25 ( 1 H, s) ppm.
LRMS 376 (MH+), 751 (MPH+).
Anal. Found: C, 51.67; H, 4.92; N, 17.39. Calc for C,7H,8CIN503~H~O: C, 51.84;
H, 5.1 1; N.
17.78.
Example 52:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-phenylglycine P-
butyl ester
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-phenylglycine
trifluoroacetate
a ~ ci ~ ci
I ~ w w ~ ~ w w 1 ~ w w
1
tBuO C N ~ ~ N ~ 18u0.C N r ~ N HO C N ~ N
H 0 C~ 0 N\'NHz x H 0 NYNH=
'N~H= NH2
A mixture of guanidine hydrochloride (326 mg, 3.41 mmol) and NaH (137 mg, 60%
dispersion in oil, 3.43 mmol) in DMSO (5 mL) was heated to 70 °C, a
solution of N [(1,4-
dichloro-7-isoquinolinyl)carbonyl]-DL-phenylglycine t-butyl ester (590 mg,
1.37 mmol) in
DMSO (3 mL) was added, and the mixture heated at 80-90 °C overnight.
After cooling, the
reaction mixture was poured into water (50 mL) and extracted with EtOAc (3x30
mL). The
combined organic extracts were washed with water, dried (Na2S04), and
evaporated in vacuo.
Purification of the residue by column chromatography on silica gel using
CHZCIz-MeOH-
0.880NH3 (90:10:I) as eluant, followed by crystallisation from i-Pr~O, gave N
[(4-chloro-1-
guanidino-7-isoquinolinyl)carbonyl]-DL-phenylglycine t-butyl ester (110 mg,
0.24 mmol) as
a pale yellow solid.
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mp 158 °C (foam), 170 °C (dec).
1H (CDC13, 300 MHz) i5 l.4 (9H, s), 5.7 (1 H, d), 6.5 (4H, br s), 7.25-7.4
(3H, m), 7.4-7.5 (3H,
m), 8.05 ( 1 H, d), 8.10 ( 1 H, s), 8.15 ( I H, d), 9.2 ( I H, d) ppm.
LRMS 454 (MH+)
Anal. Found: C, 61.53; H, 5.96; N, 14.37. Calc for C~3H~~CIN503~0.3i-Pr~O: C,
61.3; H,
5.92; N, 14.27.
A solution ofN [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl-DL-
phenylglycine t-butyl
ester ( 100 mg, 0.22 mmol) in CF3CO~H ( 1.~ mL) was stirred at 0 °C for
30 min, and then at
33 °C for 1 h. The reaction mixture was.diluted with PhMe (15 mL) and
evaporated in vacz~o.
The residual jum was triturated with EtOAc, and then Et~O, and the resulting
white solid was
dried irz vaczro to give N [(4-chloro-1-guanidine-7-isoquinolinyl)carbonyl]-DL-
phenylglycine
trifluoroacetate (50 mg, 0.10 mmof).
tH (DMSO-dh, 300 MHz) 8 5.6 ( 1 H, d), 7.3-7.45 (3H, m), 7.55 (2H, d), 8.2 ( 1
H, d), 8.2-8.4
(5H, m), 8.45 ( 1 H, d), 8.95 ( 1 H, s), 9.4 ( 1 H, d) ppm.
LRMS 398 (MH+).
Anal. Found: C, 49.72; H, 3.68; N, 14.04. Calc for Ci9H,6C1N503~0.95CF3COZH:
C, 49.27; H,
3.35; N, 13.68.
Example 53:
(a) N [(4-chloro-1-guanidine-7-isoquinolinyl)carbonyl]-L-phenylglycine t-butyl
ester
(b) N [(4-Chloro-1-guanidine-7-isoquinolinyl)carbonyl]-L-phenylglycine
trifluoroacetate
c1 I w ct I w ct
/ w w / w w / w w
tBuOzC~L N I / ~ N ~ tBuOzC~N I / ~ N ~ NOzC~i N I / i N
H 0 CI H 0 N\'NHZ H 0 N\/NHz
~N'Hz 'N~HZ
NaH (38 mg, 80% dispersion in mineral oil, 1.27 mmol) was added to a stirred
solution of
guanidine hydrochloride (121 mg, 1.27 mmol) in DMSO (4 mL) at 23 °C,
and the mixture
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heated at 80-85 °C for I S min. N [( 1,4.-Dichloro-7-
isoquinolinyl)carbonyl]-L-phenylglycine t-
butyl ester (218 mg, 0.51 mmol) was added and the mixture heated at 85
°C for 4 h. The
cooled solution was poured into water and extracted with EtOAc (x3). The
combined organics
were washed with saturated brine, dried (Na~S04) and evaporated in vacuo. The
residue was
S crystallised with i-Pr~O to give N [(4-chloro-I-guanidino-7-
isoquinoiinyl)carbonyl]-L-
phenylglycine t-butyl ester (SS mg, 0.12 mmol) as a pale yellow solid.
mp 147 °C (dec).
~H (CDC13, 400 MHz) b 1.4 (9H, s), S.7 ( 1 H, d), 6.2-6.8 (4H, br s), 7.3-7.4
(3H, m), 7.45-7.5
(3 H, m), 8.0-8. I (2 H, m), 8. I S-8.2 ( I H, d), 9.2 ( I H, s) ppm.
LRMS 4S4 (MH~), 907 (MPH+).
Anal. Found: C, 61.22; H, 6.01; N, 13.91. Calc for Ca3H24CIN5O3'O.4r-Pr?O: C,
61.21; H,
6.07; N, 14.0S.
A solution ofN [(4-chloro-I-guanidino-7-isoquinolinyl)carbonyl]-L-
phenylglycine t-butyl
ester (40 mg, 0.09 mmol) in CF3CO~H ( i mL) was stirred at room temperature
for 1 h. The
reaction mixture was diluted with PhMe, evaporated in vacuo, and the residue
triturated with
EtOAc to give N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-L-
phenylglycine
trifluoroacetate (32 mg, 0.06 mmol) as a white powder.
mp 163 °C (shrinks), > 200 °C (dec).
tH (TFA-d, 400 MHz) 8 5.85 ( 1 H, s), 7.3 S-7.4 (3 H, m), 7.4-7.45 (2H, m),
8.25 ( 1 H, d), 8.3
(lH,s),8.4(lH,d),9.15(lH,s)ppm.
LRMS 398 (MH'~), 79S (MZH+)
Anal. Found: C, 48.28; H, 3.74; N, 13.57. Calc for
C,9H,6CIN503~CF3COZH~O.SH~O: C,
48.43; H, 3.48; N, 13.45.
Example 54:
(a) N ((4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-D-phenylglycine t-butyl
ester
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(b) N ((4-Chloro-1-guanidine-7-isoquinolinyl)carbonyl-D-phenylglycine
trifluoroacetate
c1 L ~ ci I ~ ci
/ w w ~ w w /
I ~ _ I ~ I
tBuOZC ° N / ~ N tBuO,C o N / ~ N HO C ° N / i N
H 0 CI H 0 NYNHz H 0 N"NH;
NH: ~NHZ
NaH (30 mg, 80% dispersion in mineral oil, 1.0 mmol) was added to a solution
of guanidine
hydrochloride (97 mg, I .0 mmol) in DMSO (3 mL) and the solution heated to 80
°C for 30
min. N [(1,4-Dichloro-7-isoquinolinyl)carbonyl]-D-phenylglycine t-butyl ester
(17~ mg, 0.41
mmol) was added, the mixture heated at 85 °C for 3.5 h, and then at 23
°C overnight. The
mixture was poured into water (25 mL), extracted with EtOAc (3x20 mL), and the
combined
i0 organics washed with brine, dried (MbSOa), and evaporated in vaczro. The
reside was purified
by column chromatography on silica gel using CH~CI,-MeOH-0.880NH3 (95:5:0.5)
as eluant,
followed by crystallisation from CH~CI,-i-Pr~O, to give N [(4-chloro-1-
guanidine-7-
isoquinolinyl)carbonyl]-D-phenylglycine t-butyl ester (37 mg, 0.08 mmol) as a
solid.
mp 154-156 °C (dec).
IH (CDC13, 400 MHz) 8 1.4 (9H, s), 5.7 (1H, d), 7.3-7.4 (3H, m), 7.4-7.5 (3H,
m), 8.05 (1H,
d), 8.05 ( 1 H, s), 8.15 ( 1 H, d), 9.2 ( I H, s) ppm.
LRMS 454 (MH+), 907 (MZH+).
Anal. Found: C, 61.15; H, 6.00; N, 13'.87. Calc for C23HzaCIN503~0.45i-
PrzO~0.2 HBO: C,
61.31;H,6.15;N,13.91.
A solution of N [(4-chloro-1-guanidine-7-isoquinolinyl)carbonyl]-D-
phenylglycine t-butyl
ester (40 mg, 0.09 mmol) in CF3CO~H (0.5 mL) was stirred at room temperature
for I h. The
solution was diluted with PhMe, evaporated in vacuo, and the residue was
triturated with EtzO
to give N [(4-chloro-1-guanidine-7-isoquinolinyl)carbonyl]-D-phenylglycine
trifluoroacetate
(21 mg, 0.04 mmol) as a white powder.
mp 222 °C (dec).
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~ H (TFA-d, 400 MHz) S 5.9 ( 1 H, s), 7.4-7.5 (3 H, m), 7.5-7.55 (2H, m), 8. 3
( I H, d), 8.35 ( I H,
s), 8.4 ( 1 H, d), 9.2 ( 1 H, s) ppm.
LRMS 398 (MH+), 795 (MPH+).
Anal. Found: C, 49.02; H, 3.42; N, 13.26. Calc for
C,9H,6C1N503.CF3CO~H~0.25H~0: C,
48.85; H, 3.42; N, 13.56.
Example ~~:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-valine t-butyl ester
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-valine
trifluoroacetate
cr cr cr
Me Me ~ ~ Me Me ~ ~ ' Me Me
1 _
lBuO C N ~ ~ N t8u0_C N ~ ~ N HO C N ~ N
H 0 CI H 0 NYNH2 = H 0 N\/NHz
NHx 'N~H=
IS NaH (88 mg, 60% dispersion in mineral oil, 2.2 mmol) was added to a stirred
solution of
guanidine hydrochloride (210 mg, 2.2 mmol) in DMSO (5 mL) at 70 °C and
the solution
stirred for 30 min. N [(1,4-Dichloro-7-isoquinolinyl)carbonyl]-DL-valine t-
butyl ester (350
mg, 0.88 mmol) was added and the solution heated at 80-90 °C overnight.
The cooled
mixture was poured into water, extracted with EtOAc (3x20 mL), and the
combined organic
extracts were dried (MgS04) and evaporated in vacuo. The residue was
crystallised, with
CH~CI~-i-Pr~O to give N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-
valine t-butyl
ester (285 mg, 0.68 mmol) as a yellow solid.
mp 178-180 °C (dec).
'H (CDC13, 300 MHz) shows 1:1 mixture of rotamers, 8 1.0 (1/2 of 6H, d), 1.05
(1/2 of 6H,
d), 1.5 (9H, s), 2.2-2.4 ( 1 H, m), 4.7 ( 1 /2 of 1 H, d), 4.75 ( 1 /2 of 1 H,
d), 6.2-6.8 (4H, br s), 6.9
( 1 H, d), 8.05 ( 1 H, d), 8.1 ( 1 H, s), 8.15 ( 1 H, d), 9.1 ( 1 H, s) ppm.
LRMS 420 (MH+), 839 (MzH+).
Anal. Found: C, 56.00; H, 6.35; N, 16.33. Calc for CZOH26CIN503~O.SHzO: C,
55.71; H, 6.36;
N, 16.32.
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A solution ofN [(4-Ch(ore-I-guanidine-7-isoquinolinyl)carbonyl]-DL-valise t-
butyl ester
(200 mg, 0.48 mmol) in CF3COZH (1.5 mL) was stirred at 0°C for 30 min,
and at 23 °C for 1
h. The reaction mixture was diluted with PhMe, evaporated in vacuo, and the
residue
triturated with EtOAc to give N [(4-chloro-1-guanidine-7-
isoquinolinyl)carbonyl]-DL-valise
trifluoroacetate ( 170 mg, 0.36 mmol) as a white solid.
mp 243-245°C (dec).
1 H (DMSO-ci~, 300 MHz) shows a 1:1 mixture of rotamers, 8 0.95 (1/2 of 6H,
d), 1.0 (1/2 of
6H, d), 2.15-2.3 ( 1 H, m), 4.35 ( 1 H, t), 8.25 ( 1 H, d), 8.4 ( 1 H, s),
8.45 ( 1 H, d), 8.4-8.6 (4H, br
s), 8.85 ( 1 H, d), 8.9 ( 1 H, s) ppm.
LRMS 364 (MH+). .
Anal. Found: C, 44.96; H, 3.95; N, 14.56. Calc for C,6H,8CIN503~CF3CO~H: C,
45.24; H,
4.01; N, 14.65.
Example 56:
(a) N [(4-chloro-1-guanidine-7-isoquinolinyl)carbonyl]-DL-proline t-butyl
ester
(b) N [(4-Chloro-1-guanidine-7-isoquinolinyl)carbonyl]-DL-proline
trifluoroacetate
ci 1
w w w w w w
nN I / i N ---~ ~N I / i N ~ N I / i N
~''~~, O CI [~' 0 Nw' NHz ~ O NY~ NHz
COztBu COztBu Y;,,, CO H ;,,,
"'~x z "'~z
NaH (65 mg, 60% dispersion in mineral oil, 1.63 mmol) was added to a stirred
solution of
guanidine hydrochloride (154 mg, 1.61 mmol) in DMSO (5 mL) at 50 °C and
the solution
stirred for 15 min. N [(1,4-Dichloro-7-isoquinolinyl)carbonyl]-DL-proline t-
butyl ester (253
mg, 0.64 mmol) was added and the mixture was heated at 80 °C overnight.
The mixture was
poured into water (20 mL) and extracted with EtOAc (x2). The combined organic
extracts
were washed with water, brine, dried over (MgSOd), and evaporated in vacuo.
The residue
was crystallised with CHZC12-i-PrzO to give N [(4-chloro-1-guanidine-7-
isoquinolinyl)carbonyl]-DL-proline t-butyl ester (241 mg, 0.58 mmol).
mp 147-149°C (dec).
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~H (CDC13, 300 MHz) shows 1:3 mixture of rotamers, 8 1.55 (9H, s), 1.8-2.1
(3H, m), 2.15-
2.45 ( 1 H, m), 3.55-3.65 ( 1 H, m), 3.75-3.85 ( 1 H, m), 4.3 5-4.45 ( 1 H,
m), 6.5-7.2 (4H, br m),
7.7 ( 1/4 of I H, d), 7.85 (3/4 of I H, d), 7.9-8. I (2H, m), 8.85 ( l l4 of 1
H, s), 8.95 (3/4 of I H, s)
ppm.
LRMS 418 (MH+), 835 (MPH+).
Anal. Found: C, 58.46; H, 6.49; N, 14.95. Calc for C~oH~,,CIN503~0.4i-Pr~O: C,
58.65; H,
6.50; N, ( 5.27.
A solution of N [(4-Chloro-(-guanidino-7-isoquinolinyl)carbonyl]-DL-proline t-
butyl ester.
( 175 mg, 0.42 mmol) in CF;CO,H ( 1 mL) was stirred at room temperature for 1
h. The
solution was diluted with PhMe, evaporated in vacuo, and the residue was
triturated with Et~O
to hive N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-proline
trifluoroacetate (156
mg, 0.33 mmol) as a white solid.
mp 185 °C (dec).
~H (DMSO-dh + I drop TFA-d, 300 M Hz) 8 1.8-2.1 (3 H, m), 2.25-2.4 ( 1 H, m),
3.45-3.7 (2H,
m), 4.4-4.5 (1H, m), 8.0-8.6 (4H, m) ppm.
LRMS 362 (MH+).
Anal. Found: C, 45.65; H, 3.84; N, 14.43. Calc for C,6H,6CINSO3~CF3COzH: C,
45.43; H,
3.60; N, 14.72.
Example 57:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-phenylalanine t-
butyl ester
(b) N-[(4-Chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-phenylalanine
trifluoroacetate
c1 I w c1 I w c1
tBuO C N I ~ ~ N ~ tBuO C N I ~ ~ N HO C N I ~ ~ N
H 0 CI = H 0 N\'NHz 2 H 0 N\'NHZ
~N'H2 TNHz
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NaH (78 mg, 60% dispersion in mineral oil, 1.95 mmol) was added to a solution
of guanidine
hydrochloride (188 mg, 1.97 mmol) in DMSO (6 mL) at 50 °C and the
solution was stirred for
IS min. N [(1,4-Dichloro-7-isoquinolinyl)carbonyl]-DL-phenylalanine t-butyl
ester (350 mg,
0.79 mmol) was added and the mixture heated at 80 °C overnight. The
cooled mixture was
poured into water (50 mL) and extracted with EtOAc (2x25 mL). The combined
organics
were washed with brine, dried (Na,SO.~) and evaporated in vacuo. The residue
was
crystallised with CH,Ch-i-Pr~O to give N [(4-chloro-1-guanidino-7-
isoquinolinyl)carbonyl]-
DL-phenylalanine t-butyl ester (172 mg, 0.37 mmol) as a cream coloured solid.
mp 201-203 °C (dec).
~H (CDC13, 300 MHz) b 1.45 (9H, s), 1.5-1.8 (1 H, br m), 3.25 (2H, d), 5.0 (1
H, dt), 6.0-6.8
(3H, br s), 6.9 (1H, d), 7.15-7.35 (5H, m), 8.0-8.1. (3H, m), 9.1 (1H, s) ppm.
LRMS 468 (MH+), 935 (MPH+)
Anal. Found: C, 61.60; H, 5.60; N, 14.97. Calc for CadH26C1N503: C, 61.60; H,
5.76; N,
14.68.
A solution of N [(4-Chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-
phenyla(anine t-butyl
ester (210 mg, 0.48 mmol) in CF3CO~H (l~mL) was stirred at room temperature
for 1 h. The
solution was diluted with PhMe, evaporated in vacuo, and the residue was
triturated with EtaO
to give N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-phenylalanine
trifluoroacetate (196 mg, 0.37 mmol).
mp 192 °C (dec).
~H (DMSO-dh + 1 drop TFA-d, 300 MHz) 8 3.1 ( 1 H, dd), 3.25 ( 1 H, dd), 4.7 (
1 H, dd), 7.1-
7.35(SH,m),8.25(lH,d),8.35(lH,s),8.35(lH,d),8.9(lH,s),9.15(1/2H,dpartially
exchanged amide NH) ppm.
LRMS 412 (MH+).
Anal. Found: C, 50.92; H, 3.81; N, 13.57. Calc for CZOH,8C1N503~0.9CF3COzH: C,
50.90; H,
3.70; N, 13.61.
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Example 58:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-leucine t-butyl
ester
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-leucine
trifluoroacetate
Me CI Me CI Me CI
Me ~ ~ Me ~ ~ Me
tBuO C- 'N I / ~ N t8u0_,~N I / ~ N HO,~_ N I / ~ N
H 0 . CI H 0 NYNH; H 0 N\'NH2
NHi ~N'HZ
NaH (73 mg, 60% dispersion in mineral oil, 1.83 mmol) was added to a stirred
solution of
guanidine hydrochloride (174mg, 1.82 mmol) in DMSO (6 mL) at 50°C and
the solution
stirred for 15 min. ~V [(1,4-Dichloro-7-isoquinolinyl)carbonyl]-DL-leucine t-
butyl ester (300
mg, 0.73 mmol) was added and the solution heated at 80 °C overnight.
The cooled mi:cture
was poured into water (50 mL), extracted with EtOAc (2:c25 mL) and the
combined organic
e:ctracts were washed with brine, dried (Na~S04) and evaporated ift vacuo. The
residue was
crystallised with CH~Ch-i-Pr~O to give N [(4-chloro-1-guanidino-7-
isoquinolinyl)carbonyl]-
DL-leucine t-butyl ester ( 185 mg, 0.43 mmol).
mp 210-212°C (dec).
'H (CDC13, 300 MHz) 8 0.9-1.0 (6H, m), 1.5 (9H, s), 1.6-1.8 (3H, m), 4.7-4.8
(1H, m), 6.4-
7.0 (4H, br s), 6.85 ( 1 H, d), 8.05 ( 1 H, d), 8.05 ( 1 H, s), 8.1 S ( 1 H,
d), 9.15 ( 1 H, s) ppm.
LRMS 434 (MH+), 866 (MPH+)
Anal. Found: C, 58.35; H, 6.75; N, 15.51. Calc for CZ,Hz$C1N503~O.lSi-Pr2O: C,
58.55; H,
6.75; N, 15.59.
A solution of N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-leucine t-
butyl ester
(184 mg, 0.57 mmol) in CF3COZH (1 mL) was stirred at room temperature for 1 h.
The
solution was diluted with PhMe, evaporated in vacuo, and the residue was
triturated with Et20
to give N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-leucine
trifluoroacetate (183
mg, 0.37 mmol).
mp 249 °C.
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tH (DMSO-dh, 300 MHz) 1:1 mixture of rotamers, 8 0.9 (1/2 of 6H, d), 0.95 (1/2
of 6H, d),
1.6-1.8 (3 H, m), 4.45-4.5 ( 1 H, m), 8.35 ( 1 H, d), 8.4 ( 1 H, s), 8.4 ( 1
H, d), 8.3-8.6 (4H, br s),
8.95 ( 1 H, s), 9.0 ( 1 H, d) ppm.
LRMS 378 (MH+).
Anal. Found: C, 46.3 I; H, 4.27; N, 14.08. Calc for C,7H,oCIN503~CF3CO~H: C,
46:39; H,
4.3 0; N, 14.24.
Example ~9:
(a) t-butyl DL-3-{[(4-chtoro-1-guanidino-7-isoquinolinyl)carbonyl]amino}-3-
phenylpropanoate
(b) DL-3-{[(4-Chloro-1-guanidino-7-isoquinolinyl)carbonyl]amino}-3-
phenylpropanoic
acid trifluoroacetate
c1 I ~ c1 I ~ I
tBuO:C N I / i N t8u0,C / N I / , N ~ HO=C I / i N
N
0 CI H 0 NYNHZ H 0 N\/NHx
NHz ~NHZ
NaH (67 mg, 60% dispersion in oil, 1.68 mmol) was added to a solution of
guanidine
hydrochloride (161 mg, 1.69 mmol) in DMSO (6 mL) and the solution was heated
to SO °C
for 15 mins. t-Butyl DL-3-[(1,4-dichloro-7-isoquinolinyl)carbonyl]amino}-3-
phenylpropanoate (300 mg, 0.67 mmol) was added and the mixture heated at 80
° C overnight.
The cooled mixture was.poured into water (50 mL) and extracted with EtOAc
(2x25 mL).
The combined organic extracts were washed with brine, dried (Na2SO4) and
evaporated in
vacuo. The residue was crystallised with i-Pr~O to give t-butyl DL-3-{[(4-
chloro-1-guanidino-
7-isoquinolinyl)carbonyl]amino}-3-phenylpropanoate (55 mg, 0.12 mmol) as a
yellow solid.
mp 227 °C (dec).
tH (CDC13 + drop of DMSO-dh, 300 MHz) 8 1.25 (9H, s), 2.75 (1H, dd), 2.85 (1H,
dd), 5.5
(1H, ddd), 6.4-6.8 (4H, br s), 7.1-7.35 (5H, m), 7.8 (1H, d), 7.9 (1H, d),
7.95 (1H, s), 8.05
( 1 H, d), 9.05 ( 1 H, s) ppm.
LRMS 468 (Ml->~).
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Anal. Found: C, 61.48; H, 5.62; N, 14.70. Calc for CZdHZ6CIN5O3: C, 61.60; H,
5.60; N,
14.97.
A solution oft-butyl DL-3-{[(4-chloro-I-guanidino-7-
isoquinolinyl)carbonyl]amino;-3-
phenyipropanoate (153 mg, 0.33 mmol) in CF3CO~H (1 mL) was stirred at room
temperature
for 1 h. The solution was diluted with PhMe, evaporated in vacuo, and the
residue was
triturated with Et~O to give DL-3-{[(4-chloro-1-guanidino-7-
isoquinolinyl)carbonyl]amino;-
3-phenylpropanoic acid trifluoroacetate ( 132 mg, 0.25 mmol).
mp. 241-244°C.
I H (DMSO-d~ + 1 drop TFA-cl, 300 MHz) 8 2.8 ( 1 H, dd), 2.95 ( I H, dd), 5.5-
~.6 ( 1 H, m), 7.2-
7.35 (3H, m), 7.4 (2H, d), 8.25 ( I H, d), 8.35 ( 1 H, s), 8.4 ( 1 H, d), 8.9
( 1 H, s) ppm.
LRMS 412 (MH+).
Anal. Found: C, 49.95; H, 3.64; N, 13.03. Calc for CzoH,8CIN503~CF3CO~H: C,
50.25; H,
3.45; N, 13.32.
. ..
Example 60:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-aspartic acid a,(3-
di-t-butyl
ester
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-aspartic acid
25 trifluoroacetate
I c1 I
t8u0 C ~ ~ t8u0 C ~ ~ ' HOzC
t8u0ZC"H I ~ i N ~ tBuOZC- 'H I ~ i N N02C~H I ~ i N
0 CI 0 N\'NH= . 0 N\'NH=
'N~H= TNHZ
NaH (53 mg, 80% dispersion in mineral oil, 1.77 mmol) was added to a solution
of guanidine
30 hydrochloride (168 mg, 1.76 mmol) in DMSO (6 mL) and the solution ws heated
to 50 °C for
30 min. N [(1,4-Dichloro-7-isoquinolinyl)carbonyl]-DL-aspartic acid a,(3-di-t-
butyl ester (330
mg, 0.70 mmol) was added and the mixture heated at 80-90 °C overnight.
The cooled mixture
was poured into water (50 mL) and extracted with EtOAc extract (5x20 mL). The
combined
organic extracts were washed with water, brine, dried (Na~S04) and evaporated
in vacuo. The
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residue was purified by (i), trituration with i-Pr~O (ii), column
chromatography on silica gel
using CHZCIZ-MeOH-0.880NH3 (95:5:0.5) as eluant, and (iii), crystallisation
from i-PrzO, to
give N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-aspartic acid a,(3-
di-t-butyl ester
( 14~ mg, 0.29 mmol) as a yellow solid.
mp 165-167 °C.
~H (CDC13, 300 MHz) 8 1.45 (9H, s), 1.5 (9H, s), 2.9 (1H, dd), 3.0 (1H, dd),
4.95-~.0 (1H, m),
7.5 ( 1 H, d), 7.95 ( I H, s), 8.0 ( 1 H, d), 8.15 ( 1 H, d), 9.2 ( 1 H, s)
ppm.
LRMS 492 (MH+), 983 (M,Hr).
Anal. Found: C, 56.06; H, 6.28; N, 13.9?. Calc for C,3H?~CIN;OS: C, 56.15; H,
6.15; N,
14.24.
l5
A solution ofN [(4-chloro-I-guanidino-7-isoquinolinyl)carbonyl]-DL-aspartic
acid oc,~3-di-t-
butyl ester ( 120 mg, 0.24 mmol) in CF3CO~H ( 1 mL) was stirred at room
temperature for 1 h.
The solution was diluted with PhMe, evaporated in vacuv, and the residue was
triturated with
Et20 to give N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-aspartic
acid
trifluoroacetate (60 mg, 0.12 mmol).
mp 125 °C (dec).
~H (TFA-d, 400 MHz) 8 3.3-3.4 (2H, m), 5.35-5.4 (1H, m), 8.25 (1H, d), 8.3
(1H, s), 8.45
(1H, d), 9.2 (1H, s) ppm.
LRMS 380 (MIA), 758 (MZH+)
Anal. Found: C, 43.22; H, 3.75; N, 14.31. Calc for
C1;H,4CIN505~0.8CF3COzH~0.25Et~0: C,
43.19; H, 3.56; N, 14.31.
Example ' 61:
(a) O-t-butyl-N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-serine t-
butyl ester
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-serine
trifluoroacetate
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ci ct ct
teuo~ I ~ ~ ceuo~ I ~ ~ Ho~
tBuO_C N ~ ~ N IBuO,C N / N HO=C N
H 0 Ct H 0 N \'NH2 H 0 N \'NNZ
~N'Hz ~N'HZ
NaH (54 mg, 80% dispersion in mineral oil, 1.80 mmol) was added to a solution
of guanidine
hydrochloride ( 173 mg, 1.81 mmol) in DMSO (6 mL) and the solution was heated
to 80 °C
for 30 min. O-t-Butyl-N [(1,4-dichloro-7-isoquinolinyl)carbonyl]-DL-serine -
butyl ester (330
mg, 0.70 mmol) was added and the mixture heated at 80 °C for 3 h. The
cooled mixture was
poured into water (50 mL) and extracted with EtOAc. The combined organic
extracts were
washed with water, brine, dried (Na~SO,~) and evaporated in vacuo. The residue
was
crystallised with i-Pr~O to give O-t-butyl-N [(4-chloro-I=guanidine-7-
isoquinolinyl)carbonyl]-DL-serine t-butyl ester (138 mg, 0.30 mmol) as a
yellow solid.
mp 215-219 °C.
~ H (CDC13, 300 MHz) 8 1.2 (9I-I, s), t .5 (9H, s), I .5-1.7 ( 1 H, br s),
3.75 ( 1 H, dd), 3.95 ( I H,
t 5 dd), 4.8-4.9 ( 1 H, m), 6.2-6.8 (3 H, br s), 7.25-7.3 ( I H, m), 8.0 ( 1
H, s), 8.05 ( 1 H, d), 8.15 ( I H,
d), 9.2 ( 1 H, s) ppm.
LRMS 464 (MH+), 927 (MPH+).
Anal. Found: C, 56.88; H, 6.65; N, 15.10. Calc for CZZH3oC1N504~0.25Hz0~0.2i-
Pr~O: C,
57.00; H, 6.87; N, 14.32.
A solution of O-t-butyl-N [(4-chloro-1-guanidine-7-isoquinoliny()carbonyl]-DL-
serine t-butyl
ester in CF3COZH (1 mL) was stirredat room temperature for 1 h. The solution
was diluted
with PhMe, evaporated in vacuo, and the residue was recystatlised twice from
MeOH-EtOAc
to give N [(4-chloro-1-guanidine-7-isoquinoliny!)carbonyl]-DL-serine
trifluoroacetate (68
mg, 0.19 mmol) as a white solid.
mp 203 °C (dec).
'H (TFA-d, 400 MHz) b 4.4 (1H, dd), 4.5 (1H, dd), 5.2-5.25 (1H, m), 8.35 (1H,
s), 8.4 (1H,
d), 8.5 (1H, d), 9.2 (1H, s) ppm.
LRMS 352 (MH+), 703 (MZH+).
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Anal. Found: C, 42.48; H, 3.69; N, 14.21. Calc for
C,,~H,4CIN504~CF3CO~H~0.4EtOAc: C,
42.19; H, 3.66; N, 13.98.
Example 62:
(a) N [(=1-chloro-1-guanidino-7-isoquinolinyl)carbonylj-DL-a-
cyclopentylgiycine t-butyl
ester
(b) N [(4-Chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-a-cyclopentylglycine
trifluoroacetate
c1 c1 c1
iN I / iN I / /N
t8u0,C H v tBuO;C H v HO:C H v 1'
0 CI 0 N\'NH: 0 N\'NN2
'N~HZ 'N~H:
NaH (30 mg, 80% dispersion in mineral oil, 1.00 mmol) was added to a solution
of guanidine
hydrochloride (96 mg, 1.01 mmol) in DMSO (3 mL) and the solution was heated at
75-80 °C.
N [(1,4-Dichloro-7-isoquinolinyl)carbonyl]-a-cyclopentylglycine t-butyl ester
(170 mg, 0.40
mmol) was added and the mixture heated at 80 °C for 4.5 h. The cooled
mixture was poured
into water (25 mL) and extracted with EtOAc (3x20 mL). The combined organic
extracts
were washed with water, brine, dried (NaZSOd) and evaporated in vacuo to give
N [(4-chloro-
1-guanidino-7-isoquinolinyl)carbonyl]-DL-a-cyclopentylglycine t-butyl ester
(105 mg, 0.23
mmol) as a yellow solid.
An analytical sample was prepared as follows: this yellow solid was extracted
with hot i-Pr~O
(3x20 mL), the hot solution was filtered, and on cooling gave the title
compound as a pale
yellow solid (40 mg) which was collected by filtration and dried in vacuo.
mp 219-221 °C (dec).
~H (CDCl3, 300 MHz) 8 1.4-1.8 (18H, m), 2.25-2.4 (1H, m), 4.7 (1H, dd), 6.2-
6.9 (3H, br s),
6.95 (1H, d), 8.05 (1H, d), 8.1 (1H, s), 8.15 (1H, d), 9.15 (1H, s) ppm.
LRMS 446 (MH+), 891 (MZH+)
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Anal. Found: C, 58.83; H; 6.39; N, 15.34. Calc for CZZHzsCIN;03~0.2H~0: C,
58.78; H, 6.37;
N, 15.30.
A solution of~V [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]-DL-a-
cyclopentylglycine
t-butyl ester (66 mg. 0.15 mmol) in CF3CO~H (0.6 mL) was stirred at room
temperature for 1
h. . The solution was diluted with PhMe, evaporated in vacuo, and the residue
was crystallised
with EtOAc. This solid was then triturated with Et~O to give N [(4-chloro-I-
guanidino-7-
isoquinolinyl)carbonyl]-DL-a-cyclopentylglycine trifluoroacetate (62 mg, 0.10
mmol) as
white powder.
mp 235 °C (dec).
I H (TFA-d, 400 MHz) & I .4-1.8 (6H, m), 1.85-2.0 (2H, m), 2.4-2.55 ( 1 H, m),
4.8 ( 1 H, d),
8.25 ( 1 H, d), 8.3 S ( 1 H, s), 8.45 ( I H, d), 9.16 ( 1 H, s) ppm.
LRMS 390 (MH+), 779 (M~H'~)
Anal. Found: C, 47.34; H, 4.36; N, 13.60. Calc for C,sH2oC1N;O3~CF3COZH: C,
47.67; H,
4.20; N, 13.90.
Example 63:
(~) N Benzyl-N [(4-chloro-1-guanidino-7-isoquinolinyl)carbonyl]glycine
hydrochloride
(b) N Benzyl-N [(4-chloro-1-guanidine-7-isoquinolinyl)carbonyl]glycine
hydrochloride
c1 c1 c1
tBuO=C~N I / ~ N tBuOzC~N I / ~ N H0 CAN I / i N
0 CI 0 NYNHZ 0 N\/NNZ
NHZ I / NH=
NaH (16 mg, 80% dispersion in mineral oil, 0.53 mmol) was added to a solution
of guanidine
hydrochloride (82 mg, 0.86 mmol) in DME (4 mL) and the mixture was heated at
60 °C for
min. A solution of N benzyl-N [(1,4-dichloro-7-isoquinolinyl)carbonyl]glycine
t-butyl
30 ester (96 mg, 0.21 mmol) in DME (2 mL) was added and the mixture was heated
at 90 °C for
4 h. The cooled mixture was partioned between Et20 and water, and the combined
organic
extracts were dried and evaporated in vacuo. The residue was dissolved in Et20
and a
solution of HCI in EtzO (1 M) was added to give a precipitate ofN benyl-N [(4-
chloro-I-
guanidino-7-isoquinolinyl)carbonyl]glycine hydrochloride. Evaporation of the
ethereal
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mother liquors gave recovered, unreacted N benzyl-N [(1,4-dichloro-7-
isoquinolinyl)carbonyl]glycine t-butyl ester which was again reacted with
guanidine (as
above) to give a second batch. Total yield: 70 mg, 0.15 mmol.
mp 130 °C (dec).
~ H (DMSO-d~, 400 MHz) 5:6 mixture of rotamers, 8 1.2 (6/11 of 9H, s), I .4
(5/11 of 9H, s),
4.0 (6/( 1 of 2H, s), 4.05 (5/1 I of 2H, s), 4.5 (5/1 I of 2H, s), 4.75 (6/1 1
of 2H, s), 7.2-7.5 (5H,
m), 7.9-8.0 ( I H, m), 8.2-8.3 ( i H, m), 8.3 5 ( 1 H, s), 8.75 (5/ l I of 1
H, s), 8.85 (6/1 I of I H, s)
ppm.
LRMS 468 (MH+), 934 (MPH+)
Anal. Found: C, 56.98; H, 5.71; N, 13.01. Calc for
C~4H~~CIN503~HCI~0.5H~0~0.2i-Pr~O: C,
56.70; H, 5.82; N, 13.12
A solution ofN benzyl-N [(4-chloro-1-guanidino-7-
isoquinolinyl)carbonyl]glycine
hydrochloride (50 mg, 0.10 mmo() in CF3CO~H ( I mL) was stirred at room
temperature for 1
h. The solution was diluted with PhMe, evaporated in vacuo, and the residue
was triturated
with Et~O to afford a white solid (41 mg). This solid was dissolved in EtOAc
and a solution
of HC1 in Et~O was added which gave a precipitate. The mother liquors were
decanted and
the solid triturated with MeCN to give N benyl-N [(4-chloro-1-guanidino-7-
isoquinolinyl)carbonyl]glycine hydrochloride (l6 mg, 0.04 mmol) as an off
white powder.
~H (TFA-d, 400 MHz) 1:4 mixture of rotamers, 8 4.2 (1/5 of 2H, s), 4.45 (4/5
of 2H, s), 4.7
(4/5 of 2H, s), 4.95 (1/5 of 2H, s), 7.2 (2H, d), 7.3-7.4 (3H, m), 8.15 (1/5
of 1H, d), 8.2 (415 of
lH,d),8.4(IH,s),8.45(4/5oflH,d),8.5(ll5oflH,d),8.7(1/SoflH,s),8.8(4/SoflH,s)
ppm.
LRMS 412 (MH+), 823 (MZH+), 845 (M~Na+).
Anal. Found: C, 52.55; H, 4.33; N, 15.10. Calc for C~oH,gClN503~HCl~0.5H20: C,
52.52; H,
4.41; N, 15.32.
Example 64:
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(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)methyl]-N methyl-DL-phenyl;lycine
t-butyl
ester
(b) N: [(4-chloro-1-guanidino-7-isoquinolinyl)methyl]-N methyl-DL-
phenylglycine t-butyl
ester dihydrochloride
(c) N [(4-Chloro-1-guanidino-7-isoquinolinyl)methyl]-N methyl-DL-phenylglycine
trifluoroacetate
c~ ~ c1 ~ c1
w w ~ ~ w w ~ ~ w v
I ~ i ~ i
I8u0iC N ~ N ~ tBuO:C N / HO:C N / N
Me CI Me N"NH, Me N\'NH,
TNHZ 'N~Hi
NaH (21 mg, 80% dispersion in mineral oil, 0.7 mmol) was added to t-BuOH (2.5
ml) and
heated at 50 °C for IS min. Guanidine hydrochloride (68 mg, 0.71 mmol)
was added and
heated at 50 °C for an additional 15 min. N [(1,4-Dichloro-7-
isoquinolinyl)methyl]-N methyl-
DL-phenylglycine t-butyl ester (102 mg, 0.24 mmol) was added and the mixture
heated at 95
°C for 9.5 h. The cooled mixture was evaporated in vacuo and the
residue was purified by
l5 column chromatography on silica gel using hexane-EtOAc (9:1), and then
CH,Ch-MeOH-
0.880NH3 (90:10:1 ) as eluant to give N [(4-chloro-1-guanidino-7-
isoquinolinyl)methyl]-N
methyl-DL-phenylglycine t-butyl ester (26 mg, 0.06 mmol) as a yellow gum. A
portion of this
material was dissolved in Et~O, a solution of HCl in Et~O was added and the
resultant
precipitate was triturated with hexane and then i-Pr~O to give the
corresponding
dihydrochloride salt.
~H (CD30D, 400 MHz) free base, 8 1.4 (9H, s), 2.2 (3H, s), 3.7 ( 1 H, d), 3.8
( 1 H, d), 4.2 ( 1 H,
s), 7.3-7.4 (3H, m), 7.5 (2H, d), 7.9 (1H, d), 8.05 (IH, d), 8.05 (IH, s),
8.35 (IH, s) ppm.
LRMS 454 (MH+)
Anal. Found: C, 51.89; H, 6.01; N, 12.42. Calc for CzaHasC1N502~2HC1~1.SH~0:
C, 52.04; H,
6.01; N, 12.64.
A solution of N [(4-chloro-1-guanidino-7-isoquinolinyl)methyl]-N methyl-DL-
phenylglycine
t-butyl ester (20 mg, 0.44 mmol) in CHZC1~ (2 mL) was stirred with CF3COZH (2
mL) at room
temperature for 4 h. The solvents were evaporated in vacuo, and the residue
was triturated
with Et~O and then EtOAc to give N [(4-chloro-1-guanidino-7-
isoquinolinyl)methyl]-N
methyl-DL-phenylglycine trifluoroacetate (6.5 mg, 0.02 mmol) as a white solid.
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WO 01/49309 PCT/IB00/01935
mp 180-182 °C.
1H (TFA-d, 400 MHz) 3:~ mixture of rotamers, 8 2.7 (5/8 of 3H, s), 3.05 (3/8
of 3H, s), 3.95-
4.05 (3/8 of 1 H, m), 4.55-4.7 (5/8 of I H, m), 4.95-5.1 ( 1 H, m), 5.35 (5/8
of l H, s), 5.45 (3/8
of 1 H, s), 7.4-7.7 (5H, m), 7.95 (3!8 of l H, d), 8.1 (5/8 of 1 H, d), 8.3 5
( 1 H, s), 8.4-8.65 (2H,
m) ppm.
LRMS 400 (MH+).
l0
Ana(. Found: C, 50.10; H, 4.27; N, 12.90. Calc for CZOH~oC1N50~~CF3COZH ~H~O:
C, 49.87;
H, 4.37; N, 13.22.
Example 6~:
(a) N benzyl-N [(4-chloro-1-guanidino-7-isoquinolinyl)methylJglycine t-butyl
ester
(b) N Benzyl-N [(4-chloro-1-guanidino-7-isoquinolinyl)methyl]glycine
bistrifluoroacetate
c1 c1 c1
i~ 1 i~ ~1 i
t8uOZC~N ~ ~ N ~ tBuOzC~N ~ HO=C~N ~ N
CI I ~ N"NHz I ~ N\'NH2
/ / ~N'H= / ~N'Hx
NaH (48.6 mg; 80% dispersion in mineral oil, 1.62 mmol) was added to t-BuOH (5
mL) and
heated to 50 °C for 15 min. Guanidine hydrocloride (155 mg, 1.62 mmol)
was added and
heated at 50 °C for an additional 20 min. N Benzyl-N [(1,4-dichloro-7-
isoquinolinyl)methyl]glycine t-butyl ester (40 mg, 0.09 mmol) added and the
mixture was
then heated at 95 °C for 20 h. The cooled mixture was evaporated in
vacuo and the residue
purified by column chromatography on silica gel using CHZCIz-MeOH-0.880NH3
(95:5:0.5),
followed by trituration with hexane and crystallisation with i-PrzO, to give N
benzyl-N [(4-
chloro-1-guanidino-7-isoquinolinyl)methyl]glycine t-butyl ester (5 mg, 0.01
mmol) as a white
solid.
IH (CD30D, 400 MHz) 8 1.45 (9H, s), 3.15 (2H, s), 3.8 (2H, s), 3.95 (2H, s),
7.2-7.4 (5H, m),
7.85-7.95 (1H, m), 8.0-8.1 (2H, m), 8.5-8.55 (1H, m) ppm.
LRMS 454 (MH+), 907 (MZH+)
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Anal. Found: C, 62.57; H, 6.13; N, 15.17. Calc for CZdHzaCIN50z~0.4H20: C,
62.51; H, 6.29;
N, 15.19.
A solution of N benzyl-N [(4-chloro-I-i=uanidino-7-
isoquinolinyl)methyl]glycine t-butyl ester
( 16 mg, 0.04 mmol) in CF3CO,H ( 1 mL) was stirred for at room temperature 1.5
h. The
solution was diluted with PhMe, evaporated in vacuo, and the residue was
triturated with EtzO
to give N benzyl-N [(4-chloro-1-guanidino-7-isoquinolinyl)methyl]glycine
bistrifluoroacetate
(6 mg, 0.02 mmol) as a white solid.
mp 199 °C dec.
I H (TFA-d, 400 MHz) b 4.2 (2 H, s), 4.6 ( 1 H, d), 4.75 ( 1 H, d), 4.85 ( 1
H, d), 4.95 ( I H, d), 7.3-
7.5 (5H, m), 8.0 ( I H, d), 8.3 ( 1 H, s), 8.45 ( I H, d), 8.55 ( I H, s).
ppm.
IS
LRMS 398 (MH+).
Anal. Found: C, 44.50; H, 3.81; N, 10.80. Calc for
C~oH~oCIN50z~2CF3CO,H~1.2H~0: C,
44.52; H, 3.80; N, 10.82.
Example 66:
(a) Na-((4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-Ns-tent
butyloxycarbonyl-L-
lysine tart butyl ester
(b) Na-[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-L-lysine
dihydrochloride.
NHBoc NHBoc NHZ
CI CI I
\ \' \ \ \ \
IOI I / iN 101 I / iN --~ 101 I / iN
cB~oZc ~ p-s ~BuoZc ~ ~-s Ho2c ~ p-s
O CI 0 N\'NHZ O N\/NHZ
'N~H2 'N~HZ
NaH (44 mg, 80% dispersion in mineral oil, 1.47 mmol) was added in a single
portion to a
solution of guanidine hydrochloride (224 mg, 2.35 mmol) in DMSO (5 ml) and
stirred at
. room temperature until solution occurred. Na [(1,4-dichloro-7-
isoquinolinyl)sulphonyl]-Ns-
tert-butyloxycarbonyl-L-lysine -tart-butyl ester (330 mg, 0.59 mmol) was added
and the
solution stirred at 100°C for'6 h. After cooling, the reaction mixture
was quenched with water
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WO 01/49309 PCT/IB00/01935
(3.0 ml), extracted with EtOAc (3 :c 20 ml) and the combined organic e:ctracts
washed with
water and brine. The organic solution was evaporated in vacuo and the residue
purified by
column chromatography upon silica gel using CH~C12-MeOH-0.880 NH3 (90:10: I)
as eluant
to give Na-[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-NE-tert-
butylo:cycarbonyl-L-
~ lysine tent-butyl ester (152 mg, 0.26 mmol). An analytical sample was
obtained by
crystallisation from i-Pr~O.
~H (CDC13, 300 MHz) 8 l.l~ (9H, s), 1.3-I.S (13H, m), 1.5-1.8 (2H, m), 3.0-3.1
(2H, m), 3.8-
3.9 ( l H, m), 4.5-4.6 ( 1 H, m), 5.2-5.4 ( I H, m), 6.25-6.6 (3 H, m), 8.0 (
1 H, d), 8.05 ( 1 H, d), 8.1
( 1 H, s), 9.1 ( 1 H, s) ppm.
LRMS 585 (MH+).
Anal. Found: C, 51.02; H, 6.32; N, 14.12. Calc for Cz5H37CIN~OGS: C, 51.32; H,
6.37; N,
14.36.
Na-[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyt]-NE-tent-butylo:cycarbonyl-
L-lysine
tert-butyl ester (119 mg, 0.20 mmol) was dissolved in EtOAc (10 m() and
saturated with
gaseous HCI. After 20 min, the resultant white precipitate was obtained by
filtration and
recrystallised from EtOH to give Na-[(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]-L-
lysine (13 mg, 0.03 mmol).
'H (DMSO-db + CF3CO~D, 300 MHz) S 1.1-1.7 (6H, m), 2.65-2.75 (2H, m), 3.75-
3.80 (1H,
m), 8.25 ( 1 H, d), 8.3 5 ( 1 H, d), 8.25 ( 1 H, s), 8.9 ( 1 H, s) ppm.
LRMS 429 (MH+)
Anal. Found: C, 37.00; H, 4.93; N, 15.72. Calc for C,6H~iCIN604S~2HC1
H~O~0.15 EtOH: C, 37.15; H, 4.95; N, 15.97.
Example 67:
Na-[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-D-lysine dihydrochloride
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WO 01/49309 PCT/IB00/01935
NHBoc NhIBoc NHZ
CI CI I
I \ I \ \ . O I \ \
II / ~N ~ j\ II / ,N ~ II / iN
tBUO~C o ~-S tBuOzC ° ~-S HOzC ° p-S
O CI O N \ /NHZ O N \ /NH,
~N'Hz 'N~H2
NaH (33 mg, 80% dispersion in mineral oil, 1.1 mmol) was added to a stirred
solution of
guanidine hydrochloride ( 170 mg, I :78 mmol) in DMSO (3 ml) at 50°C.
After 30 min, Na-
[(1,4-dichloro-7-isoquinolinyl)sulphonyl]-Ns-tart-butyloxycarbonyl-D-lysine
tart-butyl ester
(250 mg, 0.44 mmol) was added and the solution stirred at 90°C for 8 h.
The cooled mixture
was poured into water and the precipitate extracted into Et~O (4 x 15 ml). The
combined
organic extracts were washed with brine, dried (Na~S04) and treated with IN
ethereal HCI.
The solution was concentrated in vacz~o, and the residue triturated with Et~O
and then EtOAc-
EtOH to give ~Va-[(4-chloro-l-guanidino-7-isoquinolinyl)sulphonyl]-D-lysine
dihydrochloride (90 mg, 0.18 mmol).
1H (DMSO-db, 400 MHz) ~ l.2-l.4 (2H, m), 1.4-1.7 (4H, m), 2.6-2.75 (2H, m),
3.9-4.0 (1H,
m), 7.75-7.85 (3 H, br s), 8.3 ( 1 H, d), 8.35 ( 1 H, d), 8.4 ( 1 H, d), 8.4 (
1 H, s), 8.2-9.0 (3 H, br m),
9.1 ( 1 H, s) ppm.
LRMS 429 (MH+)
Anal. Found: C, 36.15; H, 5.10; N, 15.06. Calc for Cl6HzICIN604S~
?0 2HC1~2H~0~0.13 EtOAc: C, 36.18; H, 5.16; N, 15.25.
Example 68:
(a) N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-L-glutamine tart-butyl
ester
(b) N [(4-Chloro-1-guanidino-7-isoquino(inyl)sulphonyl]-L-glutamine
trifluoroacetate
CONHZ CI ONHZ CI CONHZ CI
O I \ \ O I \ \ O I \ \
II / ,N ~ II / iN ~\ II / iN
tBu02C ~ ~-S t8u02C a ~-S HOZC ~ ~-S
O CI O N\/NHz 0 N\/NHZ
~N'HZ ~N'HZ
NaH (25 mg, 80% dispersion in mineral oil, 0.83 mmol) was added to a solution
of guanidine
hydrochloride (128 mg, 1.34 mmol) in DMSO (2 ml) and stirred at 50°C
for 1 h. N [(1,4-
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Dichloro-7-isoquinolinyl)sulphonyl]-L-glutamine tert-butyl ester (150 mg, 0.32
mmol) was
added and the resultant solution stirred at 100°C for 6 h, allowed to
cool and then poured into
water. The aqueous mixture was extracted with EtOAc (3 x 30 ml) and
concentrated irr
vacuo. _The residue was purified by column chromatography upon silica gel
using CH~CI,-
MeOH-0.880 NH3 (90:10:1) as eluant to give N [(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]-L-glutamine tcjrt-butyl ester (30 mg, 0.06 mmol) as a
buff coloured
powder
~H (DMSO-d," 300 MHz) 8 1.0-l.2 (9H, s), 1.6-1.75 (1H, m), 1.75-1.9 (1H, m),
2.05-2.15
(2 H, m), 3.26-3.8 ( I H, m), 6.65-6.75 ( 1 H, br s), 7.0-7.45 (S H, br m),
7.95-8.1 (3 H, m), 8.35
( 1 H, d), 9.0 ( I H, s) ppm.
LRMS 485 (MH~~).
l~ N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-L-glutamine tert-butyl
ester (15 mg,
0.03 mmol) was dissolved in trifluoroacetic acid (1 ml) and the resultant
solution stirred at
room temperature for 1 h, diluted with toluene and concentrated to.a residue.
Trituration with
Et~O gave a powder to which was added MeOH and the suspension filtered. The
filtrate was
concentrated and then triturated with EtOAc to give N [(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]-L-glutamine trifluoroacetate (9 mg, 0.02 mmol).
'H (DMSO-d6+TFA-d, 300 MHz) cS 1.6-1.75 (1H, m), 1.8-2.0 (1H, m), 2.0-2.15
(2H, m),
3 .8-3.9 ( 1 H, m), 8.3 ( 1 H, d), 8.3 5 ( 1 H, d), 8.4 ( I H, s), 8.8 ( 1 H,
s) ppm.
LRMS 429 (MH+)
Example 69:
(2R)-1-({4-Chloro-1-guanidino-7-isoquinolinyl}sulphonyl)-2-
pyrrolidinecarboxamide
CI CI
O
N-~ / ~ N N-S ~ / i N
0 H N iN II
O N \ /NHZ
COZH ~ CONH '~Z
NHZ NHZ
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Oxalyl chloride (136 p.1, 1.56 mmol) was added to a solution ofN-[(4-chloro-I-
guanidino-7-
isoquinolinyl)sulphonyl]-D-proline (339 mg, 0.78 mmol) in CHzCh (30 ml),
followed by
DMF (100 p.1), and the reaction stirred at room temperature for 10 min. The
mixture was
evaporated in vacuo and azeotroped with toluene, to give an off white solid.
This was
suspended in CH~CI~ (15 ml), 0.880 NH3 (760 p.1, 7.8 mmol) added, and the
reaction stirred at
room temperature for 18 h. The mixture was partitioned between CH~CI~ and
water, and the
layers separated. The aqueous phase was extracted with CH~Ch, the combined
organic
solutions dried (MgSO.~) and evaporated in vaczro. The crude roduct was
purified by column
chromatography upon silica gel using an elution gradient of CH~CI~-MeOH-0.880
NH3
l0 (100:0:0 to 95:5:0.1) to afford (2R)-1-({4-chloro-I-guanidino-7-
isoquinolinyl}sulphonyl)-2-
pyrrolidinecarboxamide ( 102mg, 0.26mmol) as a pale yellow solid.
~ H (d:,-MeOH, 400 MHz) 8 I .5-1.6 ( 1 H, m),. l .7-2.0 (3H, m), 3.3-3.4 ( I
H, m), 3.>j-3.65 ( I H,
m), 4.1-4.2 (1H, m), 8.1-8.2 (3H, m), 9.15 (1H, s) ppm.
l~
LRMS 397 (MH+), 419 (MNa)~.
Anal. Found: C, 44.05; H, 4.42; N, 20.14. Calc for C,SH,~C1N603S + 0.15
CHZCI,: C, 44.43;
H, 4.26; N, 20.52.
Example 70:
(2R)-1-({4-Chloro-1-guanidino-7-isoquinolinyl}sulphonyl)-N (2-hydroxyethyl)-2-
pyrrolidinecarboxamide.
c1
' '1
N-~ ~ / i N dH2
I I
O HZN~N f
C02 '~H
NHZ ~OH
Oxalyl chloride (40 p.1, 0.46 mmol) was added to a solution of N-[(4-chloro-1-
guanidino-7-
isoquinolinyl)sulphonyl]-D-proline (100 mg, 0.23 mmol) in CHZCl2 (10 ml),
followed by
DMF (1 drop), and the reaction stirred at room temperature for 30 min. The
mixture was
evaporated in vacuo and azeotroped with toluene. The residue was dissolved in
CHZCIZ (5
ml), and added to a solution of ethanolamine (17 p,1, 0.28 mmol) in CHzCl2 (5
ml), the
reaction stirred at room temperature for 2 h, then concentrated in vacuo. The
crude product
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was purified by column chromatography upon silica gel using an elution
gradient of CH~C1~-
MeOH-0.880 NH3 (95:5:0.5 to 90:10:1) to afford (2R)-1-({4-chloro-1-guanidino-7-
isoquinolinyl}sulphonyl)-N (2-hydroxyethyl)-2-pyrrolidinecarboxamide (65 mg,
0.147 mmol)
as a yellow foam
'H (DMSO-d~, 300 MHz) b 1.45-l.8 (4H, m), 3.15 (3H, m), 3.35-3.55 (3H, m), 4.1
(1H, m),
4.65 ( 1 H, m), 7.9 ( 1 H, m), 8.0 ( 1 H, d), 8.1 S (2 H, m), 9.1 ( 1 H, s)
ppm.
LRMS 441, 443 (MHO)
Anal. Found: C, 43.96; H, 4.89; H; 17.47. Calc. for C,~H~,C1N60,~S~0.4CH~C1~:
C, 44.01; H.
4.63; N, 17.70%.
Example 71:
(a) tort-butyl (2R)-1-({4-chloro-1-guanidino-7-isoquinolinyl}sulphonyl)-2-
piperidinecarboxylate
(b) (2R)-1-({4-Chloro-1-guanidino-7-isoquinolinyl}sulphonyl)-2-
piperidinecarboxylic
acid hydrochloride
ci c1 c1
w o ~ ~ ~ o I y
O I I II / iN II / iN
I I / i N N-$ ~ N
Q OI ~ ~ O HN NHz ~ O HN NHz
O a CO,Bu CO,H
'B NH NH
Guanidine hydrochloride (128 mg, 1.34 mmol) was added to a solution ofNaH (32
mg, 80°/B
dispersion in mineral oil, 1.07 mmol) in DME (5 ml), and the mixture stirred
at 60°C, for 30
min. tert-Butyl (2R)-1-[(1,4-dichloro-7-isoquinolinyl)sulphonyl]-2-
piperidinecarboxylate
(150 mg, 0.34 mmol) was added and the reaction heated under reflux for 7 h,
and stirred for a
further 18 h at room temperature. The mixture was diluted with EtOAc, washed
with water,
brine, dried (MgSOd), and evaporated in vacuo. The residual yellow gum was
purified by
column chromatography upon silica gel using CH~Ch-MeOH-0.880 NH3 (97:3:0.3) as
eluant
to give tent-butyl (2R)-1-({4-chloro-1-guanidino-7-isoquinolinyl}sulphonyl)-2-
piperidinecarboxylate, as a yellow solid (126 mg, 0.27 mmol).
mp 157-158°C
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IH (CDC13, 400MHz) 8 1.3 (9H, s), 1.4 (1H, m), 1.6-1.8 (4H, m), 2.15 (1 H, m),
3.3 (I, m),
3.85(lH,m),4.75(lH,m),8.0(IH,d),8.1(lH,d),8.15(lH,s),9.2(IH,s)ppm.
LRMS 468 (MH+)
Anal. Found: C, 51.23; H, 5.68; N, 14.51. Calc. for C~oHZ6CIN;O.~S: C, 51.33;
H, 5.60; N,
14.97%.
A solution of tort-butyl (2R)-1-({4-chloro-I-guanidino-7-
isoquinolinyl}sulphonyl)-2-
piperidinecarboxylate (50 mg, 0.107 mmol) in EtOAc saturated with HC1 (10 ml),
was stirred
at room temperature for 2 h. The solution was concentrated in vacuo, and
azeotroped several
times with CH~CI~, to give (2R)-1-({4-chloro-1-guanidino-7-
isoquinolinyl}sulphonyl)-2-
piperidinecarboxylic acid hydrochloride (37 mg, 0.083 mmol) as a white solid.
mp dec>220°C
~H (CD30D, 400MHz) 8 1.35 (1H, m), 1.5 (1H, m), 1.65-1.8 (3H, m), 2.2 (IH, m),
3.2-3.3
(2H, m), 3.95 ( 1 H, m), 8.3 ( 1 H, d), 8.45 (2H, m), 8.9 ( 1 H, s) ppm.
LRMS 412, 414 (MH+)
Example 72:
(a) Methyl 4-[({4-chloro-1-guanidino-7-isoquinolinyl}sulphonyl)amino]-1-methyl-
4-
piperidinecarboxylate
(b) 4-[({4-Chloro-1-guanidino-7-isoquinolinyl}sulphonyl)aminoJ-1-methyl-4-
piperidinecarboxylic acid hydrochloride
~i
c1
w y
O ~ ~ H ~ I / iN
/ i N - ~ N-S
-N II -N~ O HN' /NHZ
O CI Coz ~H
COZMe
NH
Guanidine hydrochloride (270 mg, 2.83 mmol) was added to a solution of NaH (65
mg, 80%
dispersion in mineral oil, 2.16 mmol) in DMSO (6 ml), and the solution stirred
at 60°C for 30
min. Methyl 4-{[(1,4-dichloro-7-isoquinolinyl)sulphonyl]amino}-1-methyl-4-
piperidinecarboxylate (300 mg, 0.7 mmol) was added and the reaction stirred at
80°C for 5 h.
Additional NaH (30 mg, 1 mmol), and guanidine hydrochloride (135 mg, 1.4 mmol)
in
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DMSO (1 ml) were added, and the reaction heated for a further 2 % h. The
cooled mixture
was poured into water, and extracted with EtOAc. The combined organic extracts
were
washed with brine, dried (Na,SOa) and evaporated in vacuo. The residual yellow
solid was
purified by column chromatography upon silica gel using an elution gradient of
CH~C1~-
MeOH-0.880 NHS (95:5:0.5 to 90:10:1) to afford methyl 4-[({4-chloro-1-
guanidino-7-
isoquinolinyl}sulphonyl)amino]-I-methyl-4-piperidinecarboxylate (232 mg, 0.51
mmol).
mp dec>205°C
~H (CD~OD, 400MHz) 8 2.05 (4H, m), 2.15 (3H, s), 2.25 (2H, m), 2.4 (2H, m),
3.4 (3H, s),
8.05-8.15 (3 H, m), 9.1 ( 1 H, s) ppm.
LRMS 455 (MH+)
IS Anal. Found: C, 47.17; H, 5.02; N, 17.96. Calc. for C,8H~3CIN604S~0.25H~0:
C, 47.06; H,
5:16; N, 18.29%.
A solution of methyl 4-[({4-chloro-1-guanidino-7-
isoquinolinyl}sulpionyl)amino]-I-methyl-
4-piperidinecarboxylate (100 mg, 0.22 mmol) in aqueous NaOH (2 ml, 2M, 4mmol)
and
MeOH (5 ml) was stirred at 60°C for 42 h. The cooled solution was
neutralised using 2M
HC1, and the mixture concentrated in vaczro, until precipitation occurred. The
solid was
filtered, dried and dissolved in concentrated HCI, and the solution evaporated
in vacuo. The
resulting solid was triturated with Et~O, then i-PrOH, and dried under vacuum
, to give 4-
[({4-chloro-1-guanidino-7-isoquinolinyl}sulphonyl)amino]-1-methyl-4-
piperidinecarboxylic
acid hydrochloride (18 mg, 0.035 mmol).
~H (DMSO-d~, 400MHz) b 2.1 (2H, m), 2.3 (2H, m), 2.7 (3H, s), 2.8-3.0 (2H, m),
3.3 (2H,
m), 8.25-8.75 (7H, m), 9.1 (1H, s) ppm.
LRMS 441 (MH+)
Example 73:
(a) tert-butyl N [(1-guanidino-7-isoquinolinyl)sulphonylJ-D-prolinecarboxylate
(b) N [(1-Guanidino-7-isoquinolinyl)sulphonylJ-D-proline hydrochloride
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0
'~ ~N~
~ \~
° oho
i
°,n ~~~, N IN
H=N~NH,
A mixture of tort-butyl N [(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-D-
prolinecarboxylate (~00 mg, 0,44 mmol) and 5% palladium on charcoal (150 mg)
in EtOH
(30 ml) was hydrogenated at 50psi and 50°C for 24 h. The cooled mixture
was filtered
through Arbocel~, and the filter pad washed well with EtOH. The combined
filtrates were
concentrated in vacuo and the residue puritied by column chromatography upon
silica gel
using an elution gradient of CH~Ch-MeOH-0.880 NH3 (97:3:0.3 to 95:5:0.5) to
afford tert-
butyl N [(1-guanidino-7-isoquinolinyl)sulphonyl]-D-prolinecarboxylate (143 mg,
0.34 mmol)
IO as an off white solid.
~ H (CDC 13, 400MHz) 8 I .45 (9H, s), 1.75 ( 1 H, m), 1.95 (3 H, m), 3.4 ( I
H, m), 3.55 ( 1 H, m),
4.3(lH,m),7.1 (lH,d),7.75(IH,d),8.0(lH,d),8.15(lH,d),9.25(lH,s)ppm.
LRMS 420 (MH+)
A solution of tert-butyl N [(1-guanidino-7-isoquinolinyl)sulphonyl]-D-
prolinecarboxylate
(130 mg, 0.31 mmol) in EtOAc saturated with HCl (7 ml) was stirred at room
temperature for
1 h. The reaction mixture was evaporated in vacuo and azeotroped with CHZCh,
to give N
[(1-guanidino-7-isoquinolinyl)sulphonyl]-D-proline hydrochloride (118 mg,
0.295 mmol) as a
white solid.
mp dec>250°C
'H (DMSO-d6, 400MHz) b 1.6 (1H, m), 1.75-1.95 (3H, m), 3.2 (1H, m), 3.4 (1H,
m), 4.4 (1H,
m), 7.7 (1H, m), 8.2 (2H, m), 8.3 (1H, m), 9.05 (1H, s) ppm.
LRMS 364 (MH+)
Example 74:
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1-(({4-Chloro-1-guanidine-7-isoquinolinyl}sulphonyl)amino]-N methyl-N [2-
(methylamino)ethyl]cyclopentanecarboxamide hydrochloride
c1
c1 c1
w w ~ ~ H o, ~ I
O I ~ O I N S / /N
N-S / ~ N ~ N-S / ~ N ~ 'O HN NH
O Z
0 HN' /NHz 001 HN\ /NHZ -N
HO ~NH CI ~NH
~NHMe
DMF (5 drops) was added to a suspension of 1-{[(I-guanidine-4-chloro-7-
isoquinolinyl)sulphonyl]amino}cyclopentanecarboxylic acid hydrochloride (1.l
g, 2.46
mmol) in CH~CI~ (100 ml), followed by oxalyl chloride (319 p.1, 3.68 mmol),
and the reaction
stirred at room temperature for 45 min. Additional oxalyl chloride ( 106 p.1,
1.23 mmol) was
added, and stirring continued for a further 30 min. The mixture was evaporated
in vaeuo,
triturated with CH~C1~ and the residue then dissolved in CH~C1~ (100 ml).
This solution of acid chloride ( 10 ml) vas added to a solution of N,1V'-
dimethylethylenediamine (500 p.1, 4.7 mmol) in CHaCh (20 ml) and the resultant
solution
stirred at room temperature for 1 h. After evaporation to dryness, the residue
was partitioned
IS between water and CH~Ch, the aqueous layer separated and extracted with
EtOAc. The
combined organic extracts were dried (Na~SOd), evaporated to a gum and
purified by column
chromatography upon silica gel eluting with CHzCl2-MeOH-0.880 NH3 (90:10:1) as
eluant, to
give an oil. This,was dissolved in EtOAc, treated with ethereal HC1 (1N), and
the white
precipitate, filtered and triturated with Et~O, i-Pr~O, and EtOH to yield the
title compound (28
mg, 0.058 mmol).
mp 206°C (foams).
'H (DMSO-d6, 400 MHz) 8 1.35 (4H, m), 1.7 (2H, m), 2.0 (2H, m), 2.6 (3H, s),
3.05 (2H, m),
3.2 (3 H, s), 3 .4 (2H, m), 3.5 (2H, m), 8.3 5 ( 1 H, d), 8.4 ( 1 H, d), 8.45
( 1 H, s), 8.6-8.8 (4H, m),
9.2 (1H, s) ppm.
LRMS 482, 484 (MH+).
Example 75:
1-[( f 4-Chloro-1-guanidine-7-isoquinolinyl}sulphonyl)amino]-N (2-
hydroxyethyl)-N
methylcyclopentanecarboxamide hydrochloride
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CI CI
/ ~ N ~ N-g / ~ N
II II
00 HN\ /NHZ ~e0 HN\ /NHZ
~H -N ~N H
~OH
A suspension of t-{[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]amino}
cyclopentanecarbonyl chloride (1 10 mg, 0.245 mmol) in CH~CI~ (10 ml)
(prepared as
described in example 76) was added over a minute to a solution of N-
methylethanolamine
(500 ~l, 6.25 mmol) in CH~CI~ (10 ml), and the resulting yellow solution
stirred at room
temperature for 72 h. The reaction mixture was evaporated in vaczro and the
residue purified
by column chromatography upon silica gel using CHzCh-MeOH-0.880 NHS (90: I0:1)
as
eluant to give a clear gum. This was dissolved in EtOAc, ethereal HCI ( I N)
added, .the
mixture evaporated in vaczro and triturated with EtOAc. The resulting solid
was filtered and
.dried under vacuum at 50°C to give 1-[({4-chloro-1-guanidino-7-
isoquinolinyl}sulphonyl)amino]-N (2-hydroxyethyl)-N
methylcyclopentanecarboxamide
hydrochloride.
'H (DMSO-d6, 400MHz) 8 1.4 (4H, m), 1.8 (2H, m), 2.0 (2H, m), 2.6 (1H, m),
3.05-3.2 (4H,
m), 3 .3 5-3 .6 (4H, m), 8.3 ( 1 H, d), 8.4 ( 1 H, d), 8.45 ( 1 H, s), 8.5 5
(4H, m), 9 .0 ( 1 H, s), 11.0
( 1 H, s) ppm.
LRMS 468, 471 (MH+)
Anal. Found: C, 41.87; H, 5.55; N, 15.40. Calc. for C,9H~SCIN60dS~HCl~2H~0: C,
42.15; H,
5.58; N, 1.52%.
Example 76:
1-[({4-Chloro-1-guanidino-7-isoquinolinyl}sulphonyl)amino]-N (2-
methoxyethyl)cyclopentanecarboxamide hydrochloride
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~NHz
1-[({4-Chloro-1-guanidino-7-isoquinolinyl}sulphonyl)amino]-N (2-
methoxyethyl)cyclopentanecarboxamide was prepared from 2-methoxyethylamine and
1-
{[(4-chloro-I-guanidino-7-isoquinolinyl)sulphonyl]amino} cyclopentanecarbonyl
chloride,
following the same procedure described in example 76. This product was treated
with ethereal
HCI (1N) and the mixture evaporated in vacaro. The residual solid was
dissolved in EtOH,
water ( 1 drop) added, the solution concentrated in vacuo until precipitation
occured, and the
resulting solid filtered, washed with Et~O, and dried under vacuum, at
50°C, to afford 1-[({4-
chloro-1-guanidino-7-isoquinolinyl}sulphonyl)amino]-N (2-
methoxyethyl)cyclopentanecarboxamide hydrochloride (35 mg, 28%).
~H (DMSO-dh, 300MHz) 8 1.3-1.5 (4H, m), 1.9 (4H, m), 2.95 (2H, m), 3.2 (5H,
m), 7.55
( 1 H, t), 8.2 ( 1 H, s), 8.35 (2H, m), 8.45 ( 1 H, s), 8.6 (4H, m), 9.1 ( 1
H, s) ppm.,
LRMS 469, 471 (MH+)
Anal. Found: C, 43.33; H, 5.38; N, 15.82. Calc.for CigHz5CIN6O4S~HC1~1.2H20:
C, 43.30; H,
5.43; N, 15.95%.
Example 77:
(a) N (2-tent butyl aminoethylcarbamate)-1-[({4-chloro-1-guanidino-7-
isoquinolinyl~sulphonyl)amino]cyclopentanecarboxamide
(b) N (2-Aminoethyl)-1-[({4-chloro-1-guanidino-7-
isoquinolinyl}sulphonyl)amino]cyclopentane-carboxamide dihydrochloride
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cl
c1 c1
' '1
_ i'
O H II ~ / iN O
H II ~ / /N N S N-ISI / iN
N-S 00 HN\ /NHZ O HN NHZ
O HN~NHz ~ O
O HN~ NH
CI NH HN' NH
N 1I'H
NHZ
O O
A suspension of 1-{[(4-chloro-I-guanidino-7-isoquinolinyl)sulphonyl]amino}
cyclopentanecarbonyl chloride (220 mg, 0.49 mmol) was added to a solution of
tort-butoxy 2-
aminoethylcarbamate (250 mg, 1.56 mmol) in CH~CI~ (10 ml), and the reaction
stirred at
room temperature for 18 h. The mixture was evaporated in vacuo and the residue
purified by
column chromatograpliy,upon silica gel using CH~CIa-MeOH-0.880 NHS (90:10:1 )
as eluant
to give a yellow oil. This product was crystallised From MeOH-i-Pr~O to afford
N-(2-tert-
f0 butyl aminoethylcarbamate)-I-[({4-chloro-1-guanidino-7-
isoquinolinyl}sulphonyl)amino]cyclopentanecarboxamide (27 mg, 0.05 mmol) as a
pale
yellow solid.
1H (CDCl3, 300MHz) b 1.3 (1 1H, m), 1.4 (2H, m), 1.8 (2H, m), 1.9 (2H, m),
2.45 (2H, m),
15 3.0 ~ (4H, m), 5.65 ( 1 H, m), 6.8 (4H, m), 7.1 ( I H, m), 7.2 ( 1 H, m),
7.9 (3 H, m), 9.1 ( 1 H, s)
ppm.
LRMS 576 (MNa+)
20 A solution of~V (2-tert-butyl aminoethylcarbamate)-I-[({4-chloro-1-
guanidino-7-
isoquinolinyl}sulphonyl)amino]cyclopentanecarboxamide (20 mg, 0.036 mmol) in
ethereal
HCl (I ml, IN) was stirred at room temperature for 2 h. The reaction mixture
was diluted with
MeOH, concentrated in vacuo , and the residue triturated with Et~O, then i-
Pr~O, and dried, to
give N (2-aminoethyl)-1-[({4-chloro-1-guanidino-7-
25 isoquinolinyl}sulphonyl)amino]cyclopentanecarboxamide dihydrochloride (16
mg, 0.30
mmol) as an off white powder
1H (DMSO-d~, 400MHz) 8 1.6 (4H, m), 1.85 (2H, m), 1.9 (2H, m), 2.8 (2H, m),
3.2 (2H, m),
5.4 ( I H, br s), 7.9 (2H, br s), 8.05 ( 1 H, m), 8.2 ( I H, s), 8.4 ( 1 H,
m), 8.45 ( I H, s), 8.55-8.75
30 (4H, m), 9.25 ( I H, s) ppm.
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LEZMS 454 (MH+)
Example 78:
4-Chloro-1-guanidino-N [1-(morpholinoearbonyl)cyclopentyl]-7-
isoquinolinesulphonamide hydrochloride
CI
O O
N_s N_s ~ o ~ N
n 1 T
o z ~oo HN\ /NHZ
c1 N NH
0
The title compound was prepared from 1-{[(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl]amino} cyclopentanecarbonyl chloride, and morpholine,
following a
similar procedure to that described in example 74.
~H (DMSO-d~, 300MHz) 8 1.35 (4H, m), 1.7 (2H, m), 2.0 (2H, m), 3.4-3.65 (8H,
m), 8.35-
8.65 (8H, m), 8.95 ( 1 H, s) ppm.
LRMS 480, 482 (MH+)
Example 79:
4-Chloro-1-guanidino-N {1-[(4-methylpiperazino)carbonyl]cyclopentyl}-7-
isoquinolinesulplzonamide dihydrochloride
c1
c1
w y
w w o
/ i N N-g ~ /
I I NHz
CI-S p CI
O CI O
CNJ
Triethylamine (1.36 ml, 10.0 mmol) was added to a solution of (1-
aminocyclopentyl)(4
methyl-1-piperazinyl)methanone dihydrochloride (567 mg, 2.0 mmol) and 1,4-
dichloro-7
isoquinolinesulphonyl chloride (592 mg, 2.0 mmol) in CHzCl2 (25 ml), and the
reaction
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stirred at room temperature For 18 h. The mixture was concentrated in vacuo
and the residue
partitioned between EtOAc and water, and the layers separated. The organic
phase was
washed with water, extracted with HCI (2N), and these combined acidic extracts
washed with
EtOAc, and re-basified using Na,C03. This aqueous solution was extracted with
EtOAc, the
combined organic extracts washed with brine, dried (Na~SO~) and evaporated in
vacuo to ~~ive
a foam. This was crystallised from CH~C1,-i-Pr~O to afford 1,4-dichloro-N { 1-
[(4-methyl-I-
piperazinyl)carbonyl]cyclopentyl}-7-isoquinolinesulphonamide (153 mg, 0.33
mmol) as a
solid.
l0 'H (CDC1;, 300MHz) 8 1.5-1.75 (6H, m), 2.25-2.45 (9H, m), 3.6 (4H, m), 5.1
(1H, s), 8.25
(lH,d),8.35(IH,d),8.5(lH,s),8.9(IH,s)ppm.
Anal. _Found: C, 49.12; H, 5.02; N, f.06. Calc. for C,oH~,~CI~Na03S~0.3CH,C1~:
C, 49.07; H,
4.99; N, 11.28%.
NaH (22 mg, 80% dispersion in mineral oil, 0.73 mmol) was added to a solution
of guanidine
hydrochloride (142 mg, 1.49 mmol) in DMSO (2 ml), and the solution stirred at
50°C for 30
min. 1,4-Dichloro-N-{ 1-[(4-methyl-1-piperazinyl)carbonyl]cyclopentyl}-7-
isoquinolinesulphonamide (140 mg, 0.28 mmol) was added and the reaction
stirred at 90°C
for 5 h. The cooled reaction was poured into water, the mixture extracted with
EtOAc, and the
combined extracts washed with brine, dried (NazSOd) and evaporated in vacuo.
The residual
yellow foam was dissolved in i-PrOH, ethereal HCI (1N) was added, the solution
evaporated
in vacuo and the product suspended in ethanol. This mixture was filtered, the
filtrate cooled in
an ice-bath, and the resulting solid filtered, washed with EtOH, and dried, to
give 4-chloro-I-
guanidino-N { 1-[(4-methyl-1-piperazinyl)carbonyl]cyclopentyl}-7-
isoquinolinesulphonamide
dihydrochloride (68 mg, 0.12 mmol).
'H (DMSO-dh, 300MHz) 8 1.35 (4H, m), 1.7 (2H, m), 2.0 (2H, m), 2.75 (3H, s),
3.0 (2H, m),
3.25-3 .45 (4H, m), 4.4 (2H, m), 8.3 ( 1 H, d), 8.4 ( 1 H, d), 8.45 ( 1 H, s),
8.6 (4H, m), 8.7 ( 1 H, s),
9.1 (1H, s), 11.15 (2H, br s) ppm.
LRMS 494, 496 (MH+)
Example 80:
(a) N-[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonylJ-N-(methyl)cycloleucine
ethyl
ester
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(b) N-[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N-(methyl)cycloleucine
hydrochloride
CI c1 c1
iN ~ ~ ~ , ~ ~-~ I / iN
O CI N-S II
0 0o HN NHZ ~o HN"NHZ
0 0' NH Ho NH
NaH (31 mg, 80% dispersion in mineral oil, 1.04 mmol) was added to a solution
of guanidine
hydrochloride (164 mg, 1.67 mmol) in DMSO (4 ml), and the solution heated at
50°C for 1 h.
N-[(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N-(methyl)cycloleucine ethyl ester
(180 mg,
0.42 mmol) in DMSO (2 ml) was added, and the reaction heated at 80°C
for 3 h. The cooled
reaction mixture was poured into water, and extracted with EtOAc. The combined
organic
extracts were washed with brine, dried (MgSOd), and evaporated iu vacuo. The
residual
yellow oil was purified by column chromatography upon silica gel using CH~Ch-
MeOH-
0.880 NH3 (90:10:1) as eluant, and recrystallised from EtOAc to afford N-[(4-
chloro-I-
guanidino-7-isoquinolinyl)sulphonyl]-N-(methyl)cycloleucine ethyl ester (105
mg, 0.23
mmol) as a yellow solid.
mp 186-188°C
'H (DMSO-d6, 400MHz) 8 1.1 (3H, t), 1.55 (4H, m), 2.0 (2H, m), 2.2 (2H, m),
2:95 (3H, s),
4.0 (2H, q), 7.2-7.4 (4H, 6r s), 8.05 (2H, m), 8.15 ( 1 H, s), 9.1 ( 1 H, s)
ppm.
LRMS 454, 456 (MH+)
Anal. Found: C, 50.04; H, 5.38; N, 15.31. Calc. for C,9H~4CINSOdS~0.2Hz0: C,
49.88; H,
5.38; N, 15.31%.
A solution ofN-[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-N-
(methyl)cycloleucine
ethyl ester (80 mg, 0.176 mmol) in NaOH (1m1, 2N) and MeOH (10 ml) was stirred
at 70°C
for 18 h. The cooled mixture was neutralised using HC( (2N), and the MeOH was
removed in
vacuo. The resulting precipitate was filtered off, washed with water and re-
dissolved in
concentrated HCI. This solution was evaporated in vacuo, azeotroped with
toluene, the
residue dissolved in EtOH and filtered. The filtrate was evaporated in vacuo
and the resultimg
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solid recrystallised from i-PrOH, to give N-[(4-chloro-I-guanidino-7-
isoquinolinyl)sulphonyl]-N-(methyl)cycloleucine hydrochloride (18 mg, 0.039
mmol) as a
yellow solid.
mp 225°C (dec.).
I H (DMSO-d~ + TFA-d, 400 MHz) S I .4-1.6 (4H, m), 1.95-2.0 (2H, m), 2.15-2.25
(2H, m),
3.0 (3 H, s), 8.3 ( 1 H, d), 8.3 S ( 1 H, d), 8.45 ( 1 H, s), 8.95 ( I H, s)
ppm.
l0 LRMS 426, 428 (MH+)
Anal. Found: C, 41.50; H, 4.79; N, 13.82. Calc for C,~H~oCIN;04S~HCI~ 1.8H~0:
C, 41.27; H, S.O 1; N, 14.15.
Example 81:
(a) N [(4-Bromo-1-guanidino-7-isoquinolinyl)sulphonylJ-D-proline tort-butyl
ester
hydrochloride
(b) N [(4-Bromo-1-guanidino-7-isoquinolinyl)sulphonyl]-D-proline hydrochloride
Br
w y
/ iN
0 CI NHZ
CO,Bu'
NaH (48 mg, 80% disperson in mineral oil, 1.6 mmol) was added to a solution of
guanidine
hydrochloride (233 mg, 2.43 mmol) in DMSO (8 ml) and the solution stirred at
room
temperature for 30 min. N [(4-Bromo-1-chloro-7-isoquinolinyl)sulphonyl]-D-
proline tert-
butyl ester (290 mg, 0.61 mmol) , was added and the reaction stirred at
60°C for 2 h, and
allowed to cool to room temperature overnight. The mixture was poured into
water, and
extracted with EtOAc. The combined organic extracts were washed with brine,
dried
(MgS04) and evaporated in vacuo. The residual yellow oil was purified by
column
chromatography upon silica gel using CH~CIz-MeOH-0.880 NH3 (97.5:2.5:0.25) as
eluant, to
give a yellow foam. This was dissolved in EtZO, treated with ethereal HCI, the
mixture
evaporated in vacuo and the residue triturated with Et20 to give N [(4-bromo-1-
guanidino-7-
isoquinolinyl)sulphonyl]-D-proline tert-butyl ester hydrochloride (166 mg,
0.31 mmol) as a
white solid.
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mp. 203°C
~ H (DMSO-d~, 300MHz) ~ 1.4 (9H, s), 1.65 ( I H, m), I .8 (2H, m), 2.0 ( 1 H,
m), 3.35 ( I H, m),
3.45 ( I H, m), 8.3 5 (2 H, m), 8.5-8.8 (5 H, m), 9.1 ( 1 H, s), 11.4 ( 1 H,
s) ppm.
LRMS 497, 499 (MH+)
Anal. Found: C, 41.96: H, 4.65; N, 12.65. Calc. for C,~H~aBrN50,,S~HCI~O.SH~O:
C, 41.96:, 4.82; N, 12.88%.
N [(4-Bromo-1-guanidino-7-isoquinolinyl)sulphonyl]-D-proline tart-butyl ester
hydrochloride (I50 mg, 0.28 mmol) was treated with an ice-cold solution of HCI
in EtOAc
(20 ml), and the reaction allowed to warm to room temperature, and stirred for
4 h. The
solution was concentrated in vacuo and the crude product purified by column
chromatography
upon silica gel using CH~Ch-MeOH-0.880 NH3 (90:10:1) as eluant. The product
was treated
with ethereal HCI, the resulting precipitate filtered, washed with Et~O and
dried to afford N
[(4-bromo-1-guanidino-7-isoquinolinyl)sulphonyl]-D-proline hydrochloride (75
mg, 0.156
mmol) as a white powder.
1H (DMSO-d6, 300MHz) 8 1.6 (1H, m), 1.7-2.0 (3H, m), 3.2-3.45 (2H, m), 4.4
(1H, m), 8.3
(2H, m), 8.5-8.85 (5H, m), 9.15. ( 1 H, s) ppm.
LRMS 443 (MH+)
Anal. Found: C, 35.56; H, 3.54; N, 13.52. Calc. for C15H,6BrN504S~HCl~ 1.SH~O:
C, 35.62: H, 3.99; N, 13.85%.
Example 82:
(ZR)-1-({4-Chloro-1-guanidino-7-isoquinolinyl]sulphonyl)-N [2-
(dimethylamino)ethyl]-
2-pyrrolidinecarboxamide
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CI
' '1
N-~ ~ / i N NH
I I
O HN' /NHZ
Oi OH ~ H
NH
N [(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl]-L-proline hydrochloride
(300 mg, 0.69
mmol) was suspended in a solution of DMF (5 drops) and CH~CI~ (IS ml), and
oxalyl
chloride ( 1 ~0 ~1, 1.72 mmol) added dropwise. The reaction was stirred at
room temperature
5. for 3 h, then concentrated in vacuo and azeotroped with toluene. The
residue was dissolved in
CH~CI~ (15 ml), N-(2-aminoethyl)-N,N-dimethylamine (1 ml, 0.9 mmol) added and
the
reaction stirred at room temperature for 2 h. The mixture was evaporated in
vcrczio, the residue
partitioned between EtOAc and Na,C03 solution, the layers separated, and the
organic phase
washed with brine, dried (Na~SOa) and evaporated in vacuo. The residual yellow
solid was
l0 purified by column chromato~~raphy upon silica gel using an elution
gradient of CH~CIa-
MeOH-0.880 NH3 (95:x:0.5 to 90:10:1) to give (2R)-1-({4-chloro-1-guanidino-7-
isoquinolinyl}sulphonyl)-N [2-(dimethylamino)ethyl]-2-pyrrolidinecarboxamide
(195 mga
0.42 mmol) as a yellow solid.
15 ~ H (DMSO-dh, 400 MHz) ~ I .5 ~ ( 1 H, m), 1.65 ( 1 H, m), 1.7 (2H, m),
2.15 (6H, s), 2.25 (2H,
t), 3.2 (3 H, m), 3.~ ( 1 H, m), 4.1 ( I H, dd), 7.2-7.4 (4H, br s), 7.8 ( 1
H, m), 8.0 ( 1 H, d), 8.15
(2H, m), 9.1 (1H, s) ppm.
Anal. Found: C, 47.67; H, 5.61; N, 20.31. Calc. for C,9H~6CIN~03S~O.SHzO:
20 C, 47.84; H, 5.71; N, 20.56%.
Example 83:
1-{({4-Chloro-1-guanidine-7-isoquinolinyl}sulphonyl)(2-
(dimethylamino)ethyl]amino}-
N (2-hydroxyethyl)-N methylcyclopentanecarboxamide dihydrochloride
ci
i i
O\\ \ \ IN N~\S\\ \ \
HO N \ N\
I iS\O N~\ ~ O ~ O ~N~
O r--NH=
H,N
/N\ H N OH ~N\
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N [(4-chloro-I-guanidino-7-isoquinolinyl)sulphonyl]-N [2-(dimethylamino)ethyl]
cycloleucine dihydrochloride ( 170 mg, 0.3 I mmmol) was dissolved in DMF ( 10
p.1) and
CH~Ch (IS ml). Oxalyl chloride {100 p.1, 1.15 mmol) was added and the mixture
stirred at
room temperature for 3 h. The solvent was removed in vacuo, replaced with
fresh CH~Ch, N
methylethanolamine (230 ~I, 2.86 mmol) in CH,CI~ (10 ml) added, and the
reaction stirred for
2 h. The solvent was removed in vacuo and the resultant gum extracted with
Et~O and
EtOAc. These combined organic extracts were concentrated in vacuo , and the
crude product
purified by column chromatography upon silica gel eluting with CH~CI~-MeOH-
0.880 NH3 .
(90:10: I). The resulting yellow oil was dissolved in EtOAc, and acidified
with ethereal HCl
f0 (1N) to give the title compound as a cream solid (17 mg, 0.03 mmol):
~H (DMSO-dh+TFA-d, 300MHz) 8 1.55 (4H, m), 2.0 (2H, m), 2.4 (2H, m), 2.6 (3H,
s), 2.9
(6H, s), 3.35 (2H, m), 3.5 (3H, m), 3.95 (2H, m), 4.3 (2H, t), 8.4 (3H, m),
8.5 (1 H, s), 9.35
(I H, s) PPm.
l5
LRMS 540, 542 (MH+)
Example 84:
(a) Ethyl N [(4-bromo-1-guanidino-7-isoquinolinyl)sulphonyl[-N [2-
20 (dimethylamino)ethyl]-cycloleucine dihydrochloride
(b) N ({4-Bromo-1-guanidino-7-isoquinolinyl}sulphonyl)-N [2-
(dimethylamino)ethyl]cycloleucine dihydrochloride
A mixture ofNaH (28 mg, 80% in mineral oil, 0.93 mmol) and guanidine
hydrochloride (126
mg, 1.32 mmol) in dry DMSO (3 ml) was heated at 50°C for 30 min. N [(4-
Bromo-1-chloro-
7-isoquinolinyl)sulphonyl]-N [2-(dimethylamino)ethyl]cycloleucine
hydrochloride (150 mg,
0.26 mmol) was added and the mixture heated to 90°C for I h, cooled,
poured into water and
extracted with EtOAc (3 x). The combined organic extracts were washed with
water and
brine, dried (Na~S04) and concentrated in vacuo to a yellow gum. After column
chromatography on silica gel eluting with CH~Ch- MeOH-0.880 NH3 (95 : 5: 0.5),
the residue
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was dissolved in EtOAc and acidified with ethereal HCI (IN) to afford a white
precipitate.
This was filtered, dried and recrystallised from EtOH to give a white solid
(20 mg, 0. 04
mmol). Concentration of the mother liquors afforded a second crop (95 mg, 0.17
mmol) of
ethyl N [(4-bromo-I-guanidino-7-isoquinolinyl)sulphonyl]-N [2-
(dimethylamino)ethyl]cycloleucine dihydrochloride.
~H (DMSO-d~, 300MHz) 8 1.15 (3H, t), 1.6 (4H, m), 2.0 (2H, m), 2.3 (2H, m),
2.9 (6H, s),
3.5 (2 H, m), 3 .95 (2 H, m), 4.0 (2 H, q), 8.34 (2 H, s), 8.6 ( I H, s), 9.4
( 1 H, s), I 1.6 ( 1 H, br s)
ppm.
LRMS 555, 557 (MH+).
Anal. Found: C, 39.67; H, x.61; N,.12.51. Calc. For C~~H3,BrN60.,S~2HC1~2H,0:
C, 39.77; H, 5.61; N, 12.65%.
l5
Ethyl N-[(4-Bromo-1-guanidino-7-isoquinolinyl)sulphonyl]-N-[2-
(dimethylamino)ethyl]cycloleucine dihydrochloride (95 mg, 0.17 mmol) in EtOH
(3 ml) was
treated with NaOH (4N, 8 ml) and the solution stirred at 60°C for 5 h
and allowed to stand
for 60 h at room temperature. The reaction mixture was acidified using 2N HCI,
concentrated
in vacuo and the residue azeotroped with i-PrOH to give an off white solid.
This was
extracted into MeOH, the solution evaporated in vacuo and the residue purified
by column
chromatography upon silica gel using CHZCh- MeOH-0.880 NH3 ($0 : 20: 5) as
eluant. The
product was suspended in EtOAc, treated with ethereal HCI, the mixture
evaporated in vacuo
and the product triturated with EtOAc to afford N ({4-bromo-1-guanidino-7-
isoquinolinyl}sulphonyl)-N [2-(dimethylamino)ethyl]cycloleucine
dihydrochloride (IS mg,
0.027 mmol) as a pale yellow solid.
~H (DMSO-d6, 300MHz) b 1.45-1.6 (4H, m), 1.95 (2H, m), 2.2 (2H, m), 2.6 (6H,
s), 3.1 (2H,
m), 3.7 (2H, t), 7.35-7.6 (4H, br s), 8.0 (1H, d), 8.15 (1H, d), 8.25 (1H, s),
9.15 (1H, s) ppm.
LRMS 527, 529 (MH+)
Anal. Found : C, 41.31; H, 5.35; N, 14.14. Calc. for C~oHz~BrN604S~HC1~H~O:
C, 41.27; H, 5.19; N, 14.44%.
Example 85:
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(a) Ethyl 3-{[(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]amino}-2,2-
dimethylpropanoate hydrochloride
(b) N ({4-Chloro-1-guanidine-7-isoquinolinyt}sulphonyl)-2,2-dimethyl-(3-
alanine
hydrochloride
S
0 0 0
o~~o °\//
S N oIIII o ~ S H~~~~o~ I ~\S~H~~~~~oH
H CI / CI /
CI I
N N ~ N N
N CI
H;N NHZ HZN NN,
Ethyl 3-{ [(4-chloro-1-guanidine-7-isoquinol inyl)sulphonyl]amino}-2,2-
dimethylpropanoate
hydrochloride was prepared (29%) as a white solid, from ethyl 3-{[(1,4-
dichloro-7-
isoquinolinyl)sulphonyl]amino}-2,2-dimethylpropanoate, following a similar
procedure to
that described in example 83.
mp. 183-187°C
IS
~H (DMSO-dh, 300MHz) 6 I.1 (6H, s), 1.15 (3H, t), 2.95 (2H, d), 4.0 (2H, q),
7.95 (1H, t),
8.35 ( 1 H, m), 8.4 ( 1 H, m), 8.45 ( I H, s), 8.5-8.65 (3 H, br s), 9.1 ( 1
H, s), 11.2 ( I H, s).
LRMS 428 (MH+)
Anal. Found: C, 43.99; H, 5.01; N, 14.69. Calc. for C,~HZ~C1N504S~HC1:
C, 43.97; H, 4.99; N, 15.08%.
A solution of ethyl 3-{[(4-chloro-1-guanidine-7-isoquinolinyl)sulphonyl]amino}-
2,2-
dimethylpropanoate hydrochloride (28 mg, 0.06 mmol) in NaOH solution (2N,
O.SmI), and
MeOH (1 ml), was stirred at 75°C for 24 h. The cooled mixture was
acidified to pH 6 using
HCl (2N), concentrated in vacuo to remove the MeOH, and the resulting
precipitate filtered,
washed with water and dried. The solid was suspended in a MeOH/EtOAc solution,
ethereal
HCI added, and the mixture evaporated in vacuo to afford N ({4-chloro-1-
guanidine-7-
isoquinolinyl}sulphonyl)-2,2-dimethyl-(3-a(anine hydrochloride as a white
solid (22 mg, 0.05
mmol).
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mp. Dec>304°C
~H (DMSO-d~, 300MHz) 8 1.05 (6H, s), 2.9 (2H, d), 7.9 (1H, t), 8.3-8.6 (6H,
m), 9.05 (IH, s)
PP~n.
Example 86:
(a) 1-[({4-Chloro-1-guanidino-7-isoquinolinyl}sulphonyl)amino]-N,1V
dimethylcyclopentanecarboxamide
(b) 1-{[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl][2-(tetrahydro-2H
pyran-2-
yloxy)ethyl]amino}-NNdimethylcyclopentanecarboxamide
(c) 1-[({4-Chloro-1-guanidino-7-isoquinolinyl}sulphonyl)(2-hydroxyethyl)amino]-
N,N
dimethylcyclopentanecarboxamide hydrochloride
HZN~ ~NN=
Oxalyl chloride (3.5 ml, 4.0 mmol) was added to a suspension ofN ({4-chloro-1-
guanidino-7-
isoquinolinyl}sulphonyl)cycloleucine hydrochloride (870 mg, 1.94 mmol) in
CHzCh (100
ml), followed by DMF (5 drops), and the reaction stirred at room temperature
for 2 h. The
solution was concentrated in vacuo and azeotroped with toluene to give a
yellow gum. This
was dissolved in CH~C12 (100 ml), the solution cooled to -20°C, and
cooled N,N-
dimethylamine ( 10 ml) added. The reaction was allowed to warm to room
temperature with
stirring, over 30 min, then concentrated in vacuo, and the residue azeotroped
with toluene.
The crude product was purified by column chromatography upon silica gel using
CH2Ch-
MeOH-0.880 NH3 (95 : 5: O.S) as eluant, and crystallised from MeOH to afford
to afford 1-
[({4-chloro-1-guanidino-7-isoquinolinyl}sulphonyl)amino]-N,N
dimethylcyclopentanecarboxamide (302 mg, 0.69 mmol) as a yellow solid.
mp. 264-268°C.
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~H (DMSO-d~, 400MHz) b 1.35 (4H, m), 2.0 (2H, m), 2.2 (2H, m), 3.1 (6H, s),
8.35 (2H, m).
8.4-8.7 (2H, m), 9.1 (1H, s) ppm.
LRMS 439, 441 (MH+)
Anal. Found: C, 49.07; H, 5.27; N, 18.51. Calc. for C,8H~3CIN603S~0.3H~0:
C, 48.66; H, 5.35; N, 18.91%.
KaC03 (113 mg, 0.82 mmol) was added to a solution of 1-[({4-chloro-I-guanidino-
7-
I 0 isoquinolinyl } sulphonyl)amino]-N,N dimethylcyclopentanecarboxamide ( 1
SO mg, 0.34
mmol) in DMF (2.5 ml), and the mixture heated to 75°C. 2-(2-
Bromoethoxy)tetrahydro-2H-
pyran (J.C.S. 1948; 4187) ( 150 mg, 0.72 mmol) and sodium iodide (3 mg) were
then added
and the reaction stirred at 75°C for 3 days. The cooled reaction
mixture was poured into
water, and extracted with EtOAc. The combined organic ectracts were washed
with brine,
I~ dried (Na,SO,,) and evaporated in vacuo. The residual yellow oil was
purified by column
chromatography upon silica gel using EtOAc as eluant, and triturated with a
hexane-EtOAc
(20:1) solution, to give 1-{[(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl][2-(tetrahydro-
2H pyran-2-yloxy)ethyl]amino}-N N dimethylcyclopentanecarboxamide (56 mg,
0.099
mmol).
'H (CDC13, 400MHz) 8 1.45-1.85 (?H, m), 2.9-3.2 (6H, m), 3.35-3.6 (4H, m),
3.95 (2H, m),
4.1 (1H, m), 4.65 (1H, s), 8.1 (3H, m), 9.25 (1H, s) ppm.
Ethereal HC1 was added dropwise to a solution of 1-{[(4-chloro-1-guanidino-7-
isoquinolinyl)sulphonyl][2-(tetrahydro-2H pyran-2-yloxy)ethyl]amino}-N N
dimethylcyclopentanecarboxamide (37 mg, 0.065 mmol) in EtOAc (1.5 ml); until
no further
precipitation occurred. The resulting suspension was stirred at room
temperature for 20 min,
and then evaporated in vacuo. The residue was purified by column
chromatography upon
silica gel using CHZCIZ-MeOH-0.880 NH3 (95:5:0.5) as eluant, and azeotroped
with toluene.
This product was dissolved in a MeOH-CH~CIz solution, ethereal HC1 added (5
ml), and the
mixture evaporated in vacuo, and triturated with Et20 to afford 1-[({4-chloro-
1-guanidino-7-
isoquinolinyl}sulphonyl)(2-hydroxyethyl)amino]-N,N
dimethylcyclopentanecarboxamide
hydrochloride (9mg, 0.017mmo1) as a cream/white solid.
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~H (DMSO-dh+ TFA-d, 300MHz) 8 1.25-1.45 (4H, m), 1.7 (2H, m), 2.25 (2H, m),
2.8-3.0
(6H, m), 3.3 (2 H, m), 3.7 (2H, t), 8.35 ( I H, d), 8.4 ( 1 H, d), 8.S ( 1 H,
s), 8.6 ( I H, br s), 9.0 ( 1 H,
s) ppm.
LRMS 483 (MH+)
Example 87:
(a) Ethyl 1-{[(~-chloro-1-guanidino-7-isoquinolinyl)sulphonyl](2-(tetrahydro-
2H pyran-
2-yloxy)ethyl]amino}cyclopentanecarboxylate
(b) 1-{((4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl][2-(tetruhydro-2hl
pyran-2-
yloxy)ethyl]amino}cyclopen,tanecarboxylic acid
~ N'-{4-Chloro-7-[(10-oxo-9-oxa-6-azaspiro[4.5]dec-6-yl)sulphonyl]-1-
isoquinolinyl}guanidine hydrochloride
\ S\N O
CI ~ / ~ O
O
NCI O
Ov ~O
v i
\ \N O~ \ S\N OH
CI / ~ O CI /
O O ~ O O
N /N N /N
H N FI=N
I S NH N"=
NaH (4S mg, 80% dispersion in mineral oil, 1.S mmol) was added to a solution
of guanidine
hydrochloride (231 mg, 2.4 mmol) in DMSO (S ml), and the solution stirred at
SO°C for 20
min. Ethyl 1-{[(1,4-dichloro-7-isoquinolinyl)sulphonyl][2-(tetrahydro-2H pyran-
2-
yloxy)ethyl]amino}cyclopentanecarboxylate (330 mg, 0.6 mmol) was added and the
reaction
stirred at 70°C for 2'h h. The cooled reaction was poured into water,
extracted with EtOAc,
and the combined organic extracts washed with brine, dried (MgS04) and
evaporated in
vacuo. The residual yellow gum was purified by column chromatography upon
silica gel
2S using CHZCIz-MeOH-0.880 NH3 (9S:S:O.S) as eluant to give ethyl 1-{[(4-
chloro-1-guanidino-
7-isoquinolinyl)sulphonyl][2-(tetrahydro-2H pyran-2-
yloxy)ethyl]amino}cyclopentanecarboxylate as an orange oil.
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~H (CDC13, 400MHz) 8 1.25 (3H, t), 1.45-1.75 (14H, m), 2.1 (2H, m), 2.35 (2H,
m), 3.5 (1 H.
m), .3.75-3.9 (4H, m), 4.0 ( 1 H, m), 4.2 (2 H, q), 4.61 ( 1 H, s), 8.05-8.15
(3 H, m), 9.25 ( 1 H, s)
ppm.
LRMS 568 (M~)
A solution of ethyl 1-{[(4-chloro-1-guanidino-7-isoquinolinyl)sulphonyl][2-
(tetrahydro-2H
pyran ~-yloxy)ethyl]amino}cyclopentanecarboxylate in MeOH (5 ml), was heated
to 75°C,
NaOH solution ( 1 ml, 2N, 2 mmol) added, and the reaction stirred at
50°C for 48 h. The
cooled reaction mixture was concentrated in vacuo , to remove the MeOH, and
the remaining
aqueous solution acidifed to pH 6 using 1N HCI. The resulting precipitate was
filtered,
washed with water, and the filtrate extracted with EtOAc. The combined organic
extracts
were dried (MgSO.,), and evaporated in vaczzo to give 1-{[(4-chloro-1-
guanidino-7-
isoquinolinyl)sulphonyl][2-(tetrahydro-2H pyran-2-
yloxy)ethyl]amino}cyclopentanecarboxylic acid (9 mg, 0.017 mmol) as a pale
yellow solid.
~H.(CDC13, 300MHz) 8 1.4 (4H, m), 1.55 (4H, m), 2.0 (2H, m), 2.2 (2H, m), 3.35
(3H, m),
3.45-3.75 (5H, m), 4.5 ( 1 H, m), 8.0 ( 1 H, d), 8.15 (2H, m), 9.15 ( 1 H, s)
ppm.
Anal. Found: C, 49.50; H, 5.50; N, 12.26. Calc. for C~3H3pCIN5O6S~HZO:
C, 49.50; H, 5.78; N, 12.55%.
1-{[(4-Chloro-1-guanidino-7-isoquinolinyl)sulphonyl][2-(tetrahydro-2H pyran-2-
yloxy)ethyl]amino}cyclopentanecarboxylic acid (20 mg, 0.037 mmol) was
dissolved in
EtOAc (20 ml), ethereal HC1 (10 ml) added, and the reaction stirred at room
temperature for
18 h. The resulting precipitate was filtered, washed with EtOAc and dried
under vacuum to
give N'-{4-Chloro-7-[(10-oxo-9-oxa-6-azaspiro[4.5]dec-6-yl)sulphonyl]-1-
isoquinolinyl}guanidine hydrochloride (17 mg, 0.36 mmol).
'H (CDC13, 300MHz) 8 1.6-1.8 (4H, m), 2.25 (4H, m), 3.95 (2H, t), 4.4 (2H, t),
8.35 (2H, m),
8.45 (1H, s), 9.25 (1H, s), 11.5 (1H, s) ppm.
LRMS 437 (M+)
Anal. Found: C, 44.04; H, 4.58; N, 14.17. Calc. for, C,BHzoC1N504S~HCl~H~O:
C, 43 .91; H, 4.71; N, 14.22%.
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Example 88:
(a) N-[(4-chloro-1-guanidino-7-isoquinolinyl)methyl]cycloleucine methyl ester
(b) N ({4-Chloro-1-guanidino-7-isoquinolinyl}methyl)cycloleucine
dihydrochloride
OMe / N OMe / N OH
/ I H H I _H
CI ~ 0 OI ~ ~ O CI ~ O
~N I ~N
N CI
H2N~NH2 HZN~NHZ
NaH (52 mg, 80% dispersion in mineral oil, 1.73 mmol) was added to a slurry
oFguanidine
hydrochloride (265 mg, 2.77 mmol) in DMSO (2.5 ml) and the mixture heated to
50°C for 20
mins. N-[(1,4-Dichloro-7-isoquinolinyl)methyl]cycloleucine methyl ester (245
mg, 0.69
mmol) in DMSO (2.5 ml) was added and after heating at 90°C for 4 % h,
the solution was
poured into water (50 ml). The mixture was extracted with EtOAc (2 x), the
combined
organic extracts washed~with water, brine and then dried (Na~S04). The residue
was purified
by column chromatography upon silica gel eluting with CH~C1~-MeOH -0.880 NH3
(90 : 10
1) to give a yellow solid. This was dissolved in a CH~C(~-MeOH solution and
acidified with
ethereal HCI ( 1N), concentrated in vacuo and the crude product recrystallised
from EtOH to
give N-[(4-chloro-1-guanidino-7-isoquinolinyl)methyl]cycloleucine methyl ester
(30 mg, 0.08
mmol) as a cream solid.
mp.271-275°C
'H (DMSO-d6, 300MHz) ~ 1.25 (3H, t), 1.75 (2H, m), 1.9 (2H, m), 2.1-2.3 (4H,
m), 4.25 (2H,
q), 4.35 (2H, m), 8.25 (3H, m), 8.4 (1H, s), 9.3 (1H, s), 11.7 (1H, s) ppm.
LRMS 390 (MH+)
Anal. Found: C, 49.09; H, 5.74; N, 14.71. Calc. For C,9Hz4C1N50z~2HCl~0.2H20:
C, 48.93;
H, 5.71; N, 15.02%.
N-[(4-Chloro-1-guanidino-7-isoquinolinyl)methyl]cycloleucine methyl ester (100
mg, 0.27
mmol) was dissolved in methanol (4 ml) at 50°C, NaOH (2N, 1 ml) was
added, and the
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reaction mixture heated for 2 days at 50°C. The cooled mixture was
basified to pH 6 with
NaOH (2N) to give a precipitate which was filtered off and washed with water.
The solid was
dissolved in MeOH/EtOAc, acidified with ethereal HCl (IN) and triturated with
i-PrZO to
give the title compound (b) as a pale yellow solid (10 mg, 0.03 mmol).
mp 281-289°C
~H (DMSO-cl~+ TFA-d, 300MHz) 8 1.8 (2H, m), 1.85 (2H, m), 2.1~ (2H, m), 2.25
(2H, m),
4.4 (2 H, s), 8.2 ( l H, d), 8.3 ( I H, d), 8.4 ( I H, s), 9. I 5 ( 1 H, s)
ppm.
LRMS 362 (MH+)
PREPARATIONS
Preparation l:
f5 7-Bromo-1,4-dichloroisoquinoline
/COxH / ~ COCI ~ CON
I i
Br ~ Br Br ~ I
A solution of 4-bromocinnamic acid (5.03 g, 22.2 mmol) in SOCK (IS mL) was
stirred at 23
°C for 16 h, and then heated at reflux for a further 2 h. The solvents
were evaporated in vacuo
and the residue azeotroped with PhMe (x3) to yield 4-bromocinnamoyl chloride
(22 mmol) as
an orange-brown solid.
'H NMR (CDC13, 300 MHz) 8 6.65 (1H, d), 7.4 (2H, d), 7.6 (2H, d), 7.8 (1H, d)
ppm.
A solution of NaN3 (2.2 g, 33.8 mmol) in water (7.5 mL) was added dropwise
over 5 min to a
stirred solution of 4-bromocinnamoyl chloride (22 mmol) in acetone (22 mL) at -
10 °C. The
heterogeneous mixture was stirred at 0°C for 1 h and diluted with water
(25 mL). The
precipitate was collected by filtration and dried in vacuo over Pz05 to give 4-
bromocinnamoyl
azide (5.22 g, 20.7 mmol) as a golden-coloured solid.
'H NMR (CDCl3, 300 MHz) b 6.4 (1H, d), 7.4 (2H, d), 7.5 (2H, d), 7.65 (1H, d)
ppm.
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~~ /coN,
Br \ \ I NH
Br
R' O
A warm solution of 4-bromocinnamoyl azide (5.22 g, 20.7 mmol) in Ph~O (25 mL)
was added
dropwise over l5 min to stirred Ph,O ( 10 mL) at 270 °C. [CAUTION:
Potentially explosive
use a blast screen.] The mixture was heated at 270 °C for 1.5 h, cooled
to 23 °C and then
poured into hexanes (400 mL). The precipitate was collected by filtration,
with hexanes
(''x100 mL) rinsing, and purified by column chromatography upon silica gel
using hexanes-
EtOAc (6:4 to 0:100) as eluantto give 7-bromo-1(2f~-isoquinolone (1.64 g, 7.3
mmol) as a
white solid.
' H NMR (DMSO-dr" 300 MHz) 8 6.55 ( 1 H, d), 7.25-7.15 ( 1 H, m), 7.6 ( 1 H,
d), 7.8 ( 1 H, d),
8.25 ( l H, s), 11.4 ( 1 H; br s) ppm.
c1
\
\ I NH \ iN
8r
O CI
A mixture of 7-bromo-l(2f~-isoquinolone (1.28 g, 5.69 mmol) and PCIS (2.04 g,
9.80 mmol)
was heated at 140 °C for S h. The cooled mixture was quenched with ice
(50 g) and 0.880NH3
was added until alkaline by litmus paper. The aqueous mixture was extracted
with CHZCIz
(3x50 mL) and the combined organic phases were dried (MgS04) and evaporated in
vacuo.
The residue was purified by column chromatography upon silica gel using
hexanes-EtOAc
(97:3 to 95:5) as eluant to give 7-bromo-1,4-dichloroisoquinoline (1.13 g,
4.08 mmol) as a
white solid.
mp 133.5-135 °C.
'H (CDCl3, 300 MHz) 8 7.9 (1H, d), 8.1 (1H, d), 8.35 (1H, s), 8.5 (1H, s).
LRMS 276, 278 (MH+).
Anal. Found: C, 39.04; H, 1.32; N, 5.06. Calc for C9H.,BrCIZN: C, 39.03; H,
1.46; N, 5.06.
Preparation 2:
t-Butyl 2-aminobenzoate
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\ coa \ coaeu \ co,teu
I NOz I N02 I NHZ
A mixture of 2-nitrobenzoyl chloride ( 1 S mL, 110 mmol) and t-BuOH ( I 00 mL)
were heated
at reflux for 3 h. The cooled mixture was poured onto ice-water, basified with
Na~C03 and
extracted with CH~Ch (x2). The combined organic extracts were washed with
brine, the
solvents evaporated in vacuo and the residue was purified by column
chromatography upon
silica gel using hexanes-EtOAc (9S:S) as eluant to give t-butyl 2-
nitrobenzoate (4.9 g, 22
mmol) as a yellow oil.
I H (CDCI3, 300 MHz) 8 1.6 (9H, s), 7.S ( 1 H, dd), 7.6 ( 1 H, dd), 7.7 ( 1 H,
d), 7.8 ( 1 H, d) ppm.
LRMS 240 (MNH,,T).
A solution of t-butyl, 2-nitrobenzoate (4.9 g, 22 mmol) in EtOH ( 160 mL) was
stirred with
1S 10°I° palladium-carbon (700 mg) under an atmosphere of H~ (60
psi) at 23 "C. After 4 h, the
mixture was filtered and evaporated in vacuo to give t-butyl 2-arriinobenzoate
(4.0 g, 20.7
mmol) as a yellow oil.
~H (CDC13, 300 MHz) b 1.6 (9H, s), S.6-S.8 (2H, br s), 6.6 (1H, dd), 6.6 (1H,
d), 7.2 (1H, dd),
7.8 ( 1 H, d) ppm.
LRMS 194 (MH+).
Preparation 3:
2S t-Butyl2-{[(1,4-dichloro-7-isoquinolinyl)sulphonyl]amino}benzoate
c1 1
/ co,teu ~ ~ / coZtau
\ I NH tar I / iN ~ ~ I N-S I / iN
H II
CI 0 CI
n-Butyllithium (0.88 mL, 2.5 M in hexanes, 2.2 mmol) was added dropwise to a
stirred
solution of 7-bromo-1,4-dichloroisoquinoline (S70 mg, 2.0 mmol) in THF-Et20
(10 mL, 1:1)
under N2 at -78 °C. After S min, the mixture was added to a solution of
SO~CIz (0.35 mL, 4.35
mmol) in hexane (10 mL) at -78 °C under Nz, and the mixture was slowly
warmed to 23 °C
and then stirred for 4.S h. The solvents were evaporated in vacuo, azeotroping
with CH~C1
and PhMe, the residue was suspended in CH~Ch (12 mL) containingNEt3 (1.15 mL,
8.25
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WO 01/49309 PCT/IB00/01935
mmol) and t-butyl 2-aminobenzoate (520 mg, 2.7 mmol) was added. The mixture
was stirred
at room temperature for 3 d and then heated at reflux for 6 h. The cooled
mixture was diluted
with CH~CI~, washed with aqueous HG1 (2 M), brine, and then evaporated in
vacuo. The
residue was purified by column chromatography upon silica gel using hexanes-
EtOAc (97:3
to 95:5) as eluant to give, initially, 1,4,7-trichloroisoquinoline (200 mg)
followed by t-butyl ?-
{[(1,4-dichloro-7-isoquinolinyl)sulphonyl]amino}benzoate (120 mg, 0.26 mmol)
as ayellow
resin
~ H (CDC13, 400 MHz) 8 1.5 (9H, s), 7.05 ( 1 H, dd), 7.5 ( 1 H, dd), 7.7 ( I
H, d), 7.8 ( 1 H, d), 8.2
l0 ( 1 H, d), 8.3 ( I H, d), 8.4 ( 1 H, s), 8.8 ( 1 H, s), I 0.0 ( I H, s)
ppm.
LRMS 454 (MH+).
Preparation 4
t 5 t-Butyl 3-aminobenzoate
COZH CO~t8u COZtBu
N02 NOz NH2
A mixture of 3-nitrobenzoic acid (5 g, 30 mmol), di-tert-butyl dicarbonate (20
g, 92 mmol),
20 and DMAP (0.84 g, 6.9 mmol) in THF (60 mL) was stirred at 23 °C for
2 d. The mixture was
poured onto ice-water, basified with Na~C03 and extracted with CH~C12 (x3).
The combined
organic extracts were washed with brine, the solvents evaporated in vacuo and
the residue
. was purified by column chromatography upon silica gel using hexanes-EtOAc
(95:5) as
eluant to give t-butyl 3-nitrobenzoate (5.4,g, 24 mmol) as a colourless oil.
~H (CDC13, 400 MHz) 8 1.4 (9H, s), 7.6 (1H, dd), 8.3 (1H, d), 8.4 (1H, d), 8.8
(1H, s) ppm.
A solution of t-butyl 3-nitrobenzoate (5.8 g, 26 mmol) in EtOH (260 mL) was
stirred with
10% palladium-carbon (I.0 g) under an atmosphere of H~ (60 psi) at 23
°C. After 4 h, the
mixture was filtered and evaporated in vacuo to~give t-butyl 3-aminobenzoate
(4.0 g, 20.7
mmol) as a white solid.
~H (CDCl3, 400 MHz) 8 1.6 (9H, s), 3.6-3.9 (2H, br s), 6.8 (1H, d), 7.2 (IH,
dd), 7.3 (1H, s),
7.4 ( I H, d) ppm.
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LRMS 194 (MH~), 387 (MPH+)
Preparation 5:
t-Butyl 3-{[( 1,4-dichloro-7-isoquinolinyl)sulphonyl]amino}benzoate
S
co,~e° a coyer c1
/ w w /
\ I NH Br I / iN \ I N_~ I / iN
H II
CI 0 CI
n-Butyllithium (0.88 mL, 2.5 M in hexanes, 2.2 mmol) was added dropwise to a
stirred
solution of 7-bromo-1,4-dichloroisoquinoline (570 mg, 2.0 mmol) in THF-Et~O
(10 mL, 1:1)
under N~ at -78 °C. After 5 min, the mixture was added to a solution of
SO~C1~ (0.35 mL, 4.35
mmol) in hexane ( 10 mL) at -78 °C under N~, and the mixture was slowly
warmed to 23 °C
and then stirred for 4.5 h. The solvents were evaporated in vacuo, azeotroping
with PhMe, the
residue was suspended in CH~Ch (12 mL) and t-butyl 3-aminobenzoate (520 mg,
2.7 mmol)
followed by NEt3 (1.15 mL, 8.25 mmol) were added. The mixture was stirred at
room
1S temperature for 4 d and evaporated in vaczrn. The residue was purified by
column
chromatography upon silica gel using hexanes-EtOAc (90:10 to 50:50) as eluant
to give,
initially, 14,7-trichloroisoquinoline (150 mg) followed by t-butyl 2-{[(1,4-
dich(oro-7-
isoquinolinyl)sulphonyl]amino}benzoate (289 mg, 0.63 mmol) as a brown solid
which was
used without further purification.
~H (CDC13, 400 MHz) selected data: 8 1.5 (9H, s), 7.20-7.25 (IH, m), 7.3-7.45
(lH,,m), 7.5
(lH,dd),7.6(IH,s),8.45(lH,d),8.5(lH,d),8.6(lH,s),8.9(IH,s)ppm.
LRMS 454 (MH+):
Preparation 6:
1,4-Dichloro-7-isoquinolinesulphonyl chloride
I
~ w w ~ ~ w w
~NH / NH
4 0
A solution of N chlorosuccinimide (9.66 g, 72 mmol) in MeCN (80 mL) was added
dropwise
to a stirred solution of 1-(2I~-isoquinolone (10 g, 69 mmol) in MeCN (250 mL)
which was
being heated under reflux. The mixture was heated under reflux for an
additional 1.5 h and
then cooled to room temperature. The resulting precipitate was collected by
filtration, with
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MeCN rinsing, and then dried in vacuo to give 4-chloro-1(2I~-isoquinolone (1
l.3 ~~. 62.9
mmol) as a pale pink solid.
' H (DMSO-dh, 300 MHz) cS 7.5 ( 1 H, s), 7.6 ( 1 H, dd), 7.8-7.9 (2 H, m),
8.25 ( 1 H, d), I I .5 ( 1 H.
br s), ppm.
LRIvIS 180, t.82 (MH~), 359, 361, 363 (M~Hr)
c1 c1
NH I / NH
CISO,
0 0
4-Chloro-1-(21~-isoquinolone (20.62 g, I t5 mmol) was added portionwise to
stirred
chlorosulphonic acid (61 mL, 918 mmol) at 0 °C. The mixture was heated
at l00 °C for 3.5 d
and then cooled to room temperature. The reaction mixture was added in small
portions onto
ice-water [CAUTION] and the resulting precipitate was collected by filtration.
The solid was
l~ washed with water, triturated with MeCN and then dried in vaczro to give 4-
chloro-1-oxo-l,2-
dihydro-7-isoquinolinesulphonyl chloride (18.75 g, 67.4 mmol) as a cream
solid.
'H (DMSO-d~, 400 MHz) 8 7.45 ( 1 H, s), 7.8 ( 1 H, d), 8.0 ( 1 H, d), 8.5 ( 1
H, s), 11.5 ( 1 H, br s)
ppm.
Anal. Found: C, 39.37; H, 2.09; N, 4.94. Calc for C9HSChN03S: C, 38.87; H,
1.81; N, 5.04.
c1 c1
w w
CISO= I ~ NH ~ CIS02 I ~ ~ N
0 CI
POC13 (9.65 mL, 103.5 mmol) was added to a stirred suspension of 4-chloro-1-
oxo-1,2-
dihydro-7-isoquinolinesulphonyl chloride (22.1 g, 79.6 mmol) in MeCN (500 mL)
at room
temperature and the mixture was then heated at reflux for 15 h: On cooling,
the MeCN
solution was decanted from the insoluble sludge and evaporated in vacuo. The
residue was
extracted with hot EtOAc and evaporated to leave a solid which was stirred
with Et~O (1.2 L)
at room temperature overnight. The ethereal solution was decanted from the
insoluble
material and evaporated in vacuo to give 1,4-dichloro-7-isoquinolinesulphonyl
chloride (20 g,
67 mmol) as a pale yellow solid.
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IH (DMSO-d~, 400 MHz) 8 8.2 (2H, s), 8.5 ( 1 H, s), 8.55 ( 1 H, s) ppm.
Anal. Found: C, 37.19; H, 1.34; N, 4.77. Calc for C9H,~CI~NO~S: C, 36.45; H,
1.36; N, 4.72.
Preparation 7:
Methyl 3-t[(1,4-dichloro-7-isoquinolinyl)sulphonyl]amino}-4-methoxybenzoate
CI CO.,Me CI
\ / \ \
0
CISO I / i N \ I N-S I / i N
H Il
CI OMe 0 CI
Methyl 3-amino-4-methoxybenzoate (212 mg, I .17 mmol) was added to a stirred
solution of
1,4- _dichToro-7-isoquinolinesulphonyl chloride (342 mg, 1.15 mmol) in CH~CI,
(10 mL)
containing 2,6-lutidine (0.135 mL, 1. l6 mmol) under N~ at 0 °C. After
5 min, the mixture was
warmed to room temperature and stirred for 22 h. The solvents were evaporated
in vaczro and
the residue was suspended in EtOAc (50 mL), and then washed with water, brine,
dried
t~ (MgSO,~) and evaporated in vercuo. The residue was purified by column
chromatography upon
silica gel using hexanes-EtOAc (80:20 to 20:80) as eluant to give methyl 3-{[(
1,4-dichloro-7-
isoquinolinyl)sulphonyl]amino}-4-methoxybenzoate (365 mg, 0.83 mmol) as an off
white
solid.
1H (CDCl3, 300 MHz) 8 3.7 (3H, s), 3.9 (3H, s), 6.75 (1H; d), 7.2 (1H, s), 7.8
(1'H, dd), 8.15
(lH,dd),8.25(lH,s),8.3(lH,d),8.5(s, 1H),8.85(lH,s)ppm.
LRMS 441 (MH+), 458 (MNH4+).
Preparation 8:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]glycine t-butyl ester
c1 c1
0
I\ ~ I\
CIS02 / ~ N tBu02C~H-IS / i N
CI 0 CI
NEt3 (0.59 mI:, 4.24 mmol) was added to a stirred solution of glycine t-butyl
ester
hydrochloride (340 mg, 2.02 mmol) and 1,4-dichloro-7-isoquinolinesulphonyl
chloride (500
mg, 1.68 mmol) in CH~CIz (25 mL) under N~ and the mixture was stirred at room
temperature for 18 h. The mixture was diluted with CHZC12 (25 mL), washed with
dilute HCI
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(x2, 1 M), saturated aqueous NaHC03, brine, dried (MgS04) and evaporated in
vacuo. The
solid was triturated with EtOAc, collected by filtration and dried to give N
[(1,4-dichloro-7-
isoquinolinyl)sulphonyl]glycine t-butyl ester (435 mg, 1.11 mmol) as a white
solid.
mp 194-196 °C.
~H(CDC13,300MHz)81.3(9H,s),3.8(2H,d),5.3(IH,brt),8.25(lH,d),8.4(lH,d),8.5
( 1 H, s), 8.9 ( 1 H, s) ppm.
LRMS 391 (MH'), 408, 410 (MNHa*)
Anal. Found: C, 45.58; H, 4.03; N, 7.03. Calc for C,;H,6CI,N~OaS: C, 46.04; H,
4.12; N, 7.16.
Preparation 9:
N [( 1,4-Dichloro-7-isoquinolinyl)sulphonyl]-(3-alanine t-butyl ester
c1 c1
y ~~ y y
CISO ~ ~N t8u0,C~N_S / iN
H II
CI 0 CI
NEt3 (0.60 mL, 4.3 mmol) was added to a stirred solution of [3-alanine t-butyl
ester
hydrochloride (331 mg, 1.82 mmol) and 1,4-dichloro-7-isoquinolinesulphonyl
chloride (510
mg, 1.72 mmol) in CH~Ch (10 mL) under N~ and the mixture was stirred at room
temperature for 22 h. The mixture was diluted with CHzCIz (50 mL), washed with
half
saturated brine, dried (MgS04) and. evaporated in vacuo. The residue was
purified by column
chromatography upon silica gel using pentane-EtOAc (90:10 to 60:40) as eluant
to give N
[(1,4-dichloro-7-isoquinolinyl)sulphonyl]-(3-alanine t-butyl ester (580 mg,
1.43 mmol) as a
white solid.
~H (CDC13, 300 MHz) b 1.4 (9H, s), 2.5 (2H, t), 3.25 (2H, dt), 5.5 (1H, br t),
8.25 (1H, d), 8.4
(lH,d),8.5(lH,s),8.9(lH,s)ppm.
LRMS 405, 407 (MH+), 422 (MNH4~).
Anal. Found: C, 47.41; H, 4.46; N, 6.80. Calc for C,6Hi8CIzNz04S: C, 47.42; H,
4.48; N, 6.91.
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Preparation 10:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N methylglycine t-butyl ester
c1 c1
w w
I ° ~ ~ ,N
CISOi ~ ~ N tBuO;C~N-S
CI Me 0 CI
N Methylglycine t-butyl ester hydrochloride (264 mg, 1.45 mmol) was added to a
stirred
solution of 1,4-dichloro-7-isoquinolinesulphonyl chloride (376 mg, 1.27 mmol)
in CH~C1~ (25
mL) containing NEt; (0.44 mL, 3.16 mmol) under N~ at 0 °C, and the
mixture was then stirred
at room temperature for 22 h. The solvents were evaporated in vaczro, the
residue dissolved in
EtOAc (50 mL), washed with water, brine, dried (MgSO~) and evaporated in
vaczro. The
residue was purified by column chromatography upon silica gel using pentanes-
EtOAc
(80:20) as eluant to give N-[(1,4-dichloro-7-isoquinolinyl)sulphonyl]-N
methylglycine t-butyl
ester (485 mg, 1.20 mmol) as a white solid.
IS ~H (CDC13, 300 MHz) 8 1.35 (9H, s), 3.0 (3H, s), 4.05 (2H, d), 8.2 (1H, d),
8.35 (1H, d), 8.5
( 1 H, s), 8.85 ( 1 H, s) ppm.
LRMS 709 (MZH+).
Anal. Found: C, 47.37; H, 4.43; N, 6.79. Calc for C,6H,8C1~N~OdS: C, 47.42; H,
4.48; N, 6.91.
Preparation 11:
N Phenylglycine t-butyl ester
NHZ tBu02C~NH
tBuOxC~Br
..5
t-Butyl chloroacetate (10 g, 66.3 mmol) was added dropwise to a stirred
solution of aniline
(11.3 g, 120 mmol) in NEt3 (10 mL), and the mixture was stirred at to room
temperature for
24 h and then at 60 °C for 18 h. The cooled mixture was diluted with
Et20 (100 mL), filtered
with Et20 rinsing, and the filtrate was then washed with water, brine, dried
(MgSO~) and
evaporated in vacuo. The residue was purified by column chromatography upon
silica gel
using hexanes-EtOAc (98:2 to 92:8) as eluant to give N phenylglycine t-butyl
ester (6.56 g,
31.6mmol) as an oil.
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H (CDC13, 400 MHz) 8 I .5 (9H, s), 3.8 (2H, s), 4.45 ( 1 H, br s), 6.6 (2H,
d), 6.7 ( 1 H, t), 7.2
(2H, dd) ppm.
LRMS 208 (MH+), 415 (MPH+).
Preparation 12:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N phenylglycine t-butyl ester
c1 c1
I w v I w v
/ iN
tBuO;C~NH CISO, / ~N tBuO:C~N-S
CI 0 CI
~ I
1,4-DichToro-7-isoquinolinesulphonyl chloride (300 mg, 1.01 mmol) was added to
a stirred
solution of N phenylglycine t-butyl ester (228 mg, 1.10 mmol) in CH~CI~ (5.0
mL) containing
NEt3 (0.35 mL, 2.5 mmol) under N~ at room temperature, and the mixture stirred
for 5 d. The
mixture was diluted with CH~CIZ (50 mL), washed with dilute HCl (20 mL, 1 M),
saturated
aqueous NaHC03, dried (MgSO,,) and evaporated in vacuo. The residue was
purified by
column chromatography upon silica gel using hexanes-EtOAc (90:10 to 60:40) as
eluant to
give N [(1,4-dichloro-7-isoquinolinyl)sulphonyl]-N phenylglycine t-butyl ester
(485 mg, 1.20
mmol) as a white solid.
'H (CDC13, 300 MHz) 8 1.4 (9H, s), 4.4 (2H, d), 7.2-7.4 (5H, m), 8.05 (1H, d),
8.3 (1H, d),
8.45 (1H, s), 8.7 (1H, s) ppm.
LRMS 467 (MHO).
Preparation 13:
N (Cyclopentylmethyl)-N [( 1,4-dichloro-7-isoquinolinyl)sulphonyl]glycine t-
butyl ester
c1 c1
I / ~N
18u0 C~N-S I / ~N tBuO,C~N-S
H 0 CI 0 CI
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PPh3 (243 mg, I .5 mmol) and then a solution of DEAD (236 p.L, 1.5 mmol) in
THF (2 mL)
were added to a stirred solution ofN [(i,4-dichloro-7-
isoquinolinyl)sulphonyl]glycine t-butyl
ester (391 mg, 1.00 mmol) and cyclopentanemethanol (130 p.L, 1.2 mmol) in THF
(3 mL)
under N, at 0 °C, and the mixture was stirred at room temperature for
18 h. An additional
S portion of cyclopentanemethanol ( 1.2 mmol), PPh3 ( i .5 mmol), and DEAD
(1.5 mmol) were
added and the mixture stirred at room temerature for a further 2 d. The
solvents were
evaporated in vaczzo and the residue was purified by column chromatography
upon silica gel
using pentane-EtOAc ( 100:0 to 9~:5) as eluant to give N (cyclopentylmethyl)-N
[(1,4-
dichloro-7-isoquinolinyl)sulphonyl]glycine t-butyl ester (144 ma, 0.30 mmol)
as a white
l0 solid.
1H (CDCi;, 400 MHz) b 1.1~-1.4 (3H, m), 1.3 (9H, s), 1.5-1.7 (3H, m), 1.7-1.8
(2H, m), 2.1
( 1 H, m), 3.2~ (2 H, d), 4.1 (2H, s), 8.2~ ( 1 H, d), 8.35 ( 1 H, d), 8.5 ( 1
H, s), 8.85 ( I H, s) ppm.
1S LRMS 473 (MH+), 490, 492 (MNH~+).
Anal. Found: C, 53.23; H, 5.~8; N, 5.86. Calc for C~, H~~CI~N,O.,S: C, 53.28;
H, 5.54; N, 5.92.
Preparation 14:
20 N (Cyclohexylmethyl)-N [( 1,4-dichloro-7-isoquinolinyl)sulphonyl]glycine, t-
butyl ester
ct c1
I o
0 ~ II I / ,N
tBu02C~H-S ~ ~ N tBuOzC N-S
0 CI 0 CI
Cyclohexylmethyl bromide (209 p,L, 1.5 mmol) was added to a stirred solution
ofN [(1,4-
2S dichloro-7-isoquinolinyl)sulphony(]glycine t-butyl ester (391 mg, 1.00
mmol) and anhydrous
IC~C03 (276 mg, 2.0 mmol) in DMF (5 mL) under NZ at 23 °C. The mixture
was stirred for 2
h and then heated at SO-60 °C for 6 h. The cooled mixture was diluted
with EtOAc (200 mL),
washed with water (250 mL), dried (MgS04), and the solvents were evaporated
irz vacuo. The
residue was purified by column chromatography upon silica gel using pentane-
EtOAc (100:0
30 to 95:5) as eluant to give N (cyclohexylmethyl)-N [(1,4-dichloro-7-
isoquinolinyl)sulphonyl]glycine t-butyl ester (320 mg, 0.66 mmol_).
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tH (CDC13, 400 MHz) cS 1.1~-1.3 (3H, m), 1.3 (9H, s), 1.5-1.8 (8H, m), 3.15
(2H, d), 4.05
(2H,s),8.2(lH,d),8.35(lH,d),8.45(lH,s),8.85(IH,s)ppm.
LRMS 487 (MH+), 504, 506, 508 (MNHa+).
Preparation l~:
N Benzylglycine t-butyl ester
t8u0 C~8r NNx 18u0xC~NH
x
\ \
I
A solution oft-butyl bromoacetate (1.5 mL, 10.1 minol) in CH~CI, (10 mL) was
added
dropwise to a stirred solution of benzylamine ( 10.9 mL, 100 mmol) in CH~Ch
(40 mL) at 0
°C, the mixture was stirred for I h and then warmed to room temperature
and stirred for an
additional 3 d. The mixture was washed with water (3x50 mL), dilute HC1 (1 N)
and the
combined aqueous washings were extracted with Et~O. The organic phase was
washed with
saturated aqueous NaHC03, dried (Na~S04) and evaporated in vacuo. The residue
was
dissolved in Et~O, treated with a solution of HCI in ether (0.5 M) and the
resulting precipitate
was collected and dissolved in EtOAc. This solution was filtered through
hyflo, and partially
evaporated in vacuo to give a thick slurry. The solid was collected by
filtration, washed with
Et~O and then dried to give N benzylglycine t-butyl ester hydrochloride (1.03
g, 4.00 mmol)
as a white solid.
~H (CDC13, 300 MHz) S 1.4 (9H, s), 3.5 (2H, s), 4.4 (2H, s), 7.3-7.4 (3H, m),
7.55-7.65 (2H,
m), 10.2-10.3 (2H, br s).
LRMS 222, (MI~-I~), 443 (MZH+).
Preparation 16:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N benzylglycine t-butyl ester
c1 c1
tBuOxC~NH I \ ~ ~ 0 I \
I \ CISOx / ~N tBuOxC~N-S ~ ~N
CI ~ 0 CI
I
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1,4-Dichloro-7-isoquinolinesulphonyl chloride (300 mg, 1.01 mmol) was added to
a stirred
solution of N benzylglyeine t-butyl ester (310 mg, 1.20 mmol) in CHZCh (20 mL)
containing
NEt3 (0.35 mL, 2.5 mmol) under N~ and the mixture was stirred at room
temperature for 3 d.
The mixture was diluted with CH,Ch and washed with dilute HCI (2 M), saturated
aqueous
NaHC03, brine, dried (Na~SO.~) then and evaporated in vacuo. The residue was
purified by
column chromatography upon silica gel using hexanes-EtOAc (90:10) as eluant to
give N
[( 1,4-dichloro-7-isoquinolinyl)sulphonyl]-N benzylglycine t-butyl ester (290
mg, 0.60 mmol)
as an off white solid.
(0 mp 134-136 °C.
1H (CDCI~, 400 MHz) 8 1.3 (9H, s), 3.9 (2H,~s), 4.55 (2H, s), 7.25-7.4 (5H,
m), 8.25 (1H, d),
8.4 ( 1 H, d), 8.5 ( I H, s), 8.9 ( I H, s) ppm.
t5 LRMS 481 (MH~), 498 (MNH~~).
Anal. Found: C, 54.52; H, 4.50; N, 5.77. Calc for C>?H»ChN~O~S: C, 54.89; H,
4.61; N, 5.82.
Preparation 17:
20 N (2-Methylbenzyl)glycine t-butyl ester
t8u0xG~GI NHx tBuOxC~NH
\ \
Me ~Me.
t-Butyl chloroacetate (2.13 g, 14.1 mmol) was added to a stirred solution of 2-
25 methylbenzylamine ( 1.71 g, 14.1 mmol) in CHZCIZ (20 mL) containing NEt3
(2.95 mL, 21.2
mmol) under NZ and the mixture was stirred at room temperature for 17 h. The
solvents were
evaporated in vacuo, the residue suspended in EtOAc and and washed with water,
brine, dried
(MgS04) then and evaporated in vacuo. The residue was purified by column
chromatography
upon silica gel using pentanes-EtOAc (95:5 to 80:20) as eluant to give N (2-
30 methylbenzyl)glycine t-butyl ester (1.29 g, 5.48 mmol).
~H (CDCl3, 300 MHz) 8 1.5 (9H, s), 2.35 (3H, s), 3.3 (2H, s), 3.8 (2H, s), 7.1-
7.2 (3H, m),
7.25-7.3 (1H, m) ppm.
35 LRMS 236 (MH+), 471 (MZH+).
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Preparation 18:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N (2-methylbenzyl)glycine t-butyl
ester
c1 c1
tBuO,C~NH ~ ~ I / eN
CISOZ ~ ~ N iBuO:C~N-S
I CI ~ 0 CI
~Me I
~Me
J
1,4-Dichloro-7-isoquinolinesulphonyl chloride (400 mg, 1.35 mmol) was added to
a stirred
solution of N (2-methylbenzyl)glycine t-butyl ester (380 mg, 1.61 mmol) in
CH~CI~ (20 mL)
containing NEt3 (0.28 mL, 2.5 mmol) under N~ and the mixture was stirred at
room
l0 temperature For 18 h. The mixture was diluted with CH,Ch and washed with
dilute HCI (2
M), saturated aqueous NaHC03, brine, dried (MbSOa) then and evaporated in
vacaro. The
residue was purified by column chromatography upon silica gel using pentane-
EtOAc (100:0
to 90:10) as eluant to give N [(1,4-dichloro-7-isoquinolinyl)sulphonyl]-N (2-
methylbenzyl)glycine t-butyl ester (480 mg, 0.97 mmol) as a white solid.
mp 96-98 °C.
tH (CDC13, 400 MHz) 8 1.25 (9H, s), 2.3 (3H, s), 3.9 (2H, s), 4.6 (2H, s), 7.1-
7.25 (4H, m),
8.3 ( 1 H, d), 8.4 ( 1 H, d), 8.5 ( I H, s), 8.9 ( I H, s) ppm.
LRMS 495 (MH+), 512 (MNHd+)
Anal. Found: C, 55.70; H, 4.86; N, 5.63. Calc for Cz3HzdCl2NZO4S: C, 55.76; H,
4.88; N, 5.65.
Preparation 19:
N (2-Methoxybenzyl)glycine t-butyl ester
tBuOzC~Br NHz tBuO=C~NH
I ~ OMe I ~ OMe
A solution of t-butyl bromooacetate (1.5 mL, 10.2 mmol) in CH~CIz (30 mL) was
added to a
stirred solution of 2-methoxybenzylamine (6.88 g, 50.2 mmol) in CHzCl2 (70 mL)
under NZ at
0 °C, and the mixture was then stirred at room temperature for 1 h. The
mixture was
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thoroughly washed with dilute HCI (30 ml, 1 M) and the separated aqueous phase
was
extracted with in CH~CI~. The combined organic extracts were washed with
saturated
NaHC03, brine, dried (Na~SOa) then and evaporated in vacuo. The residue was
purified by
column chromatography upon silica gel using in CH~Ct~-MeOH (99:1 to 95:5) as
eluant to
give ~V (2-methoxybenzyl)glycine t-butyl ester (0.90 g, 3.58 mmo() as a pale
yellow oil.
~H (CDC13, 400 MHz) 8 1.25 (9H, s), 2.0 (1H, br s), 3.3 (2H, s), 3.8 (2H, s),
3.85 (3H, s), 6.85
( 1 H, d), 6.9 ( 1 H, dd), 7.2-7.3 (2H, m) ppm.
LRMS 2S2 (MH+), S03 (M,H+), S2S (M~Na+).
Anal. Found: C. 66.52; H, 8.54; N, S.S4. Calc for C,4H~,N03: C, 66.91; H,
8.42; N, S.S7.
Preparation 20:
N [( 1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N (2-methoxybenzyl)glycine t-
butyl ester
a c1
z o
tBuO \ NH OISO: I ~ ~ N ~ t8u0=C~N-S I ~ ~ N
~I Ct ~ 0 CI
_OMe ~
home
1,4-Dichloro-7-isoquinolinesulphonyl chloride (500 mg, 1.69 mmol) was added to
a stirred
solution of N (2-methoxybenzyl)glycine t-butyl ester (508 mg, .2.02 mmol) in
CHZCIz (30
ml) containing NEt3 (0.35 ml, 2.S mmol) under N~ and the mixture was stirred
at room
temperature for 21 h. The mixture was diluted with CH~C1~ and washed with
dilute HCl (2
M), saturated aqueous NaHC03, brine, dried (NaZS04) then and evaporated in
vacuo. The
residue was purified by column chromatography upon silica gel using hexane-
EtOAc (95:5 to
90:10) as eluant and then triturated with hexane-i-PrzO to give N [(1,4-
dichloro-7-
isoquinolinyl)sulphonyl]-N (2-methoxybenzyl)glycine t-butyl ester (501 mg,
1.02 mmol) as a
yellow solid.
mp 106-108 °C.
'H(CDC13,400MHz)b1.3(9H,s),3.7(3H,s),4.0(2H,s),4.6(2H,s),6.8(lH,d),6.9(1H,
dd), 7.2 ( 1 H, dd), 7.3 ( 1 H, d), 8.2 ( 1 H, d), 8.3 ( 1 H, d), 8.45 ( 1 H,
s), 8.8 ( 1 H, s) ppm.
LRMS 511, S 13 (MH+), 528 (MNH4~)
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Anal. Found: C, 54.09; H, 4.78; N, 5.33. Catc for Cz3HzaClzNzOSS: C, 54.01; H,
4.73; N, 5.48.
Preparation 21:
N (3-Methoxybenzyl)glycine t-butyl ester
NH; t8u0,C~NH
tBuOZC~Br
/ /
OMe OMe
A solution of t-butyl bromoacetate ( I.~ mL, 10.1 mmol) in CHzCIz (30 mL) was
added
dropwise to a stirred solution of 3-methoxybenzylamine (6.86 g, 50 mmol) in
CHzCh (20 mL)
at 0 °C, and the mixture was then warmed to room temperature and
stirred for 1.5 h. Dilute
HC1 (30 mL, I M) was added and the mixture stirred for IS min. The aqueous
phase was
extracted with CH,CIz and the combined organic extracts were washed with
water, brine,
saturated aqueous NaHC03, dried (MgSO,~) then and evaporated in vacuo. The
residue was
1~ purified by column chromatography upon silica gel using CHzCIz-MeOH (99:1
to 90:10) as
eluant to give the required amine as a colourless oil. Treatment with a
solution of HCI in ether
(1 M) gave N (3-methoxybenzyl)glycine t-butyl ester hydrochloride (0.83 g,
2.88 mmol) as a
white solid.
mp 141-142 °C.
'H (CDC13, 300 MHz) S 1.45 (9H, s), 3.5 (2H, s), 3.85 (3H, s), 4.35 (2H, s),
6.9 (1H, d), 7.1
(1H, d), 7.3 (1H, s), 73-7.35 (1H, m), 10.3 (2H, br s) ppm.
LRMS 252 (MH+), 503 (MzH~).
Anal. Found: C, 58.37; H, 7.75; N, 4.83. Calc for C,dHz,N03~HC1: C, 58.43; H,
7.71; N, 4.87.
Preparation 22:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N (3-methoxybenzyl)glycine t-butyl
ester
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c1
I ' '1
tf3u02C~N CIS02 ~ iN
,\ CI
I
OMe
NEt3 (0.59 mL, 4.24 mmol) and then 1,4-dichToro-7-isoquinolinesulphonyl
chloride (500 mg,
1.68 mmol) were added to a stirred solution of N (3-metho:cybenzyl)glycine t-
butyl ester
hydrochloride (582 mg, 2.02 mmol) in CHzCIz (25 mL) under Nz and the mixture
was stirred
at room temperature for 18 h. The mixture was diluted with CHzCIz (25 mL),
washed with
dilute HCI (x2, I M), saturated aqueous NaHC03, brine, dried (MgSOa) and
evaporated in
vacuo. The residue was extracted with i-PrzO which gave a precipitate on
standing. The white
solid was collected by filtration and dried to give N [( 1,4-dichloro-7-
isoquinolinyl)sulphonyl]-N (3-metho:cybenzyl)glycine t-butyl ester (262 mg,
0.51 mmol). A
second batch (165 mg, 0.32 mmot) was obtained by evaporation of the mother
liquors and
purification of the residue by column chromatography upon silica gel using
hexane-EtOAc
(80:20).
mp 129-131 °C.
~H (CDC13, 300 MHz) 8 1.3 (9H, s), 3.75 (3H, s), 3.9 (2H, s), 4.55 (2H, s),
6.8-6.9 (2H, m),
6.85(lH,s),7.25(lH,m),8.3(lH,d),8.4(l H,d),8.5(lH,s),8.9(lH,s)ppm.
LRMS 511 (MH+), 528 (MNHQ+)
Anal. Found: C, 54.03; H, 4.79; N, 5.34. Calc for Cz3HzaCIzNzOSS: C, 54.01; H,
4.73; N, 5.48.
Preparation 23:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N (3-chlorobenzyl)glycine t-butyl
ester
c1
0
I ~ '~
tBu02C~H-S ~ i N
0 CI
3-Chlorobenzyl chloride (0.063 mL, 0.50 mmol) was added to a stirred solution
ofN [(1,4-
dichloro-7-isoquinolinyl)sulphonyl]glycine t-butyl ester (195.5 mg, 0.50 mmol)
in DMF (5
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mL) containing K~C03 (83 mg, 0.60 mmol) and the mixture was stirred at room
temperature
for 18 h. The mixture was diluted with water (SO mL), extracted with Et20
(3x30 mL) and
with EtOAc (3x30 mL), and the combined organic extracts were then washed with
water,
brine, dried (Na,SO.~) and evaporated in vacuo. The solid was triturated with
hexanes,
S collected by filtration and dried to give N [(1,4-dichloro-7-
isoquinolinyl)sulphonyl]-N (3-
chlorobenzyl)g(ycine t-butyl ester (212 mg, 0.41 mmol) as a pale yellow solid.
nip 141-143 °C.
t0 ~H (CDC13, 400 MHz) S 1.3 (9H, s), 3.95 (2H, d), 4.5 (2H, s), 7.15-7.3 (4H,
m), 8.25 (1H, d),
8.35 ( 1 H, d), 8.~ ( 1 H, s), 8.85 ( 1 H, s) ppm.
LRMS 515, 517 (MH+), 532, 534 (MNH4+)
t5 Ana(. Found: C, 51.14; H, 4.14; N, 5.31. Calc for C22H,,C13N,OdS: C, 51.22;
H, 4.10; N, 5.43.
Preparation 24:
N (4-Methoxybenzyl)glycine t-butyl ester
NH, tBu02C~NH
tBuOZC~Br
I
MeO
20 Meo
A solution of t-butyl bromoacetate ( 1.5 mL, 10.2 mmol) in CH~C1~ (30 mL) was
added
dropwise to a stirred solution of 4-methoxybenzylamine (6.89 g, 50.2 mmol) in
CHZCh (70
mL) at 0 °C, and the mixture was then warmed to room temperature and
stirred for 1 h. Dilute
25 HCl (30 mL, 1 M) was added and the mixture stirred for 10 min. The aqueous
phase was
extracted with CH~CIZ and the combined organic extracts were washed with
saturated aqueous
NaHC03, brine, dried (Na~S04) then and evaporated in vacuo. The residue was
purified by
column chromatography upon silica gel using CHZC12-MeOH (99:1 to 90:10) as
eluant to give
the required amine as a colourless oil. Treatment with a solution of HC1 in
ether (1 M)
30 followed by trituration with Et~O gave N (4-methoxybenzyl)glycine t-butyl
ester
hydrochloride (148 mg, 0.51 mmol) as an orange solid.
mp 133-134 °C.
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tH (CDC13, 400 MHz) 8 1.45 (9H, s), 3.5 (2H, s), 3.8 (3H, s), 4.3 (2H, s), 6.9
(2H, d), 7.5 (2H,
d), 10.2 (2H, br s) ppm.
LRMS 252 (MH+), 503 (MzH+), 525 (MzNa+).
Anal. Found: C, 58.08; H, 7.71; N, 4.80. Calc for C,4Hz,N03~HCI: C, 58.42; H,
7.71; N, 4.87.
Preparation 25:
N [(1,4-Dichloro-7-isoquinolinyl)sulphony(]-N(4-methoxybenzyl)glycine t-butyl
ester
a t
0
I~ ~1 I~
tBuO;C~NH CISO= ~ ~ tBuO,C~N-S ~ ~ ~N
I ~ CI I ~ 0 CI
Me0 ~ Me0
NEt~ (0.25 mL, 1.78 mmol) and then 1,4-dichloro-7-isoquinolinesulphonyl
chloride (210 mg,
0.71 mmol) were added to a stirred solution of N (4-methoxybenzyl)glycine t-
butyl ester
hydrochloride (245 mg, 0.85 mmol) in CHzCIz (20 mL) under Nz and the mixture
was stirred
at room temperature for 18 h. The mixture was diluted with CHzCIz, washed with
dilute HC1
(2 M), saturated aqueous NaHC03, brine, dried (NazS04) and evaporated in
vacuo. The
residue was purified by column chromatography upon silica gel using hexane-
EtOAc (95:5 to
90:10) as eluant and then triturated with hexane-i-PrzO to give N [(1,4-
dichloro-7-
isoquinolinyl)sulphonyl]-N (4-methoxybenzyl)glycine t-butyl ester (160 mg,
0.31 mmol) as a
white solid.
mp 117-118 °C.
tH (CDC13, 300 MHz) 8 1.3 (9H, s), 3.8 (3H, s), 3.9 (2H, s), 4.5 (2H, s), 6.85
(2H, d), 7.2 (2H,
d),8.3(lH,d),8.35(lH,d),8.5(lH,s),8.9(lH,s)ppm.
LRMS 511 (MHO), 528 (MNH4~)
Anal. Found: C, 53.90; H, 4.59; N, 5.34. Calc for Cz3HzaCIzNzOsS: C, 54.01; H,
4.73; N, 5.48.
Preparation 26:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N (2-pyridylmethyl)glycine t-butyl
ester
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a c1
I' 1 I' '~
° ° i .N
tBuO C~N-S ~ ~N tt3u0=C~N-S
H 0 CI ' O CI
I iN
2-(Chloromethyl)pyridine hydrochloride (246 mg, 1.5 mmol) was added to a
stirred solution
of N [( l,4-dichloro-7-isoquinolinyl)sulphonyl]glycine t-butyl ester (391 mg,
I .0 mmol) and
anhydrous K~CO; (415 mg, 3.0 mmol) in DMF (5 mL) under N, at 23 °C and
the mixture was
stirred for 18 h. The cooled mixture was azeotroped with xylene, diluted with
EtOAc, washed
with water, and the organic extracts were then dried (MgSOa) and evaporated in
vacuo. The
residue was purified by column chromatography upon silica gel using pentane-
EtOAc (100:0
to 50:50) as eluant to give N-[(1,4-dichloro-7-isoquinolinyl)sulphonyl]-N (2-
pyridyhnethyl)glycine t-butyl ester (400 mg, 0.83 mmol) as a white solid.
1 H (CDC13, 400 MHz) S I .3 (9H, s),.4.1 (2H, s), 4.7 (2H, s), 7.1 ( 1 H, m),
7.5 ( I H, d), 7.7 ( l H,
dd), 8.25 ( 1 H, d), 8.35 ( I H, d), 8.45 ( 1 H, m), 8.5 ( 1 H, s), 8.9 ( 1 H,
s) ppm.
LRMS 482, 484 (MH+)
Preparation 27:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N (3-pyridylmethyl)glycine t-butyl
ester
c, c1
' ' '
° I ° I i ~N
tBu02C~N-S ~ ~N tBuOzC~N-S
H 0 CI O CI
I i
"
3-(Chloromethyl)pyridine hydrochloride (246 mg, 1.5 mmol) was added to a
stirred solution
of N [(1,4-dichloro-7-isoquinolinyl)sulphonyl]glycine t-butyl ester (391 mg,
1.0 mmol) and
anhydrous KZC03 (416 mg, 3.0 mmol) in DMF (5 mL) under NZ at 23 °C and
the mixture was
stirred for 18 h. The cooled mixture was azeotroped with xylene, diluted with
EtOAc, washed
with water, and the organic extracts were then dried (MgS04) and evaporated in
vacuo. The
residue was purified by column chromatography upon silica gel using pentane-
EtOAc (100:0
to 50:50) as eluant to give N [(1,4-dichloro-7-isoquinolinyl)sulphonyl]-N (3-
pyridylmethyl)glycine t-butyl ester (400 mg, 0.83 mmol) as a white solid.
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iH (CDCl3, 400 MHz) b 1.3 (9H, s), 4.1 (2H, d), 4.7 (2H, s), 7.1 (1H, m), 7.5
(I H, d), 7.7 (1H.
dd), 8.25 ( 1 H, d), 8.3 5 ( 1 H, d), 8.45 ( I H, m), 8.5 ( 1 H, s), 8.9 ( 1
H, s) ppm.
LRMS 482, 484 (MH+).
Preparation 28:
N [( 1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N (4-pyridylmethyl)glycine t-
butyl ester
c1 a
\ \
~ ~ ,N
teu0 CAN-S ~ ~N t8u0=CAN-S
H 0 CI \ 0 CI
NI
4-(Chloromethyl)pyridine hydrochloride (246 mg, 1.5 mmol) was added to a
stirred solution
of N [( 1,4-dichloro-7-isoquinolinyl)sulphonyl]glycine t-butyl ester (391 mg,
I .0 mmol) and
anhydrous K~C03 (416 mg, 3.0 mmol) in DME (5 mL) under N~ at 23 °C and
the mixture was
stirred for 18 h. The cooled mixture was azeotroped with xylene, diluted with
EtOAc, washed
with water, and the organic extracts were then dried (MgS04) and evaporated in
vacuo. The
residue was purified by column chromatography upon silica gel using pentane-
EtOAc (100:0
to 50:50) as eluant to give N [(1,4-dichloro-7-isoquinolinyl)sulphonyl]-N (4-
pyridylmethyl)glycine t-butyl ester (397 mg, 0.82 mmol) as a white solid.
~H (CDCl3, 400 MHz) 8 1.3 (9H, s), 4.0 (2H, d), 4.6 (2H, s), 7.3 (2H, d), 8.25
(1H, dd), 8.4
(lH,d),8.5(lH,s),8.6(2H,d),8.9(lH,d)ppm.
LRMS 482, 484 (MH+).
Preparation 29:
N [(1R)-1-Phenylethyl)]glycine t-butyl ester
NHZ tBuO,C~N
R
tBu0xC~8r I \ R''Me I \ "Me
A solution of t-butyl bromoacetate (5.0 g, 25.6 mmol) in CHzCIz (5 mL) was
added dropwise
to a stirred solution of (+)-(R)-a-methylbenzylamine (4.65 g, 38.5 mmol) in
CH~C12 (40 mL)
at 0 °C, and the mixture was then warmed to room temperature and
stirred for 18 h. The
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mixture was diluted with CHZCh, washed with water, with dilute HCI (1 M) and
then dried
(MgSO.~). The solvents were evaporated in vacuo to give N [(1R)-1-
phenylethyl)]glycine t-
butyl ester (3.l S g, 13.4 mmol) as a white powder.
mp 193-197 °C.
' H (CDC13, 300 MHz) 8 1.4 (9H, s), 1.95 (3 H, d), 3.3 ( 1 H, d), 3.6 ( 1 H,
d), 4.6 ( 1 H, q), 5.3
(1H, s), 7.3-7.45 (3H, m), 7.5-7.6~ (2H, m).
LRMS 236 (MH+).
Preparation 30:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N-[(1R)-1-phenylethyl)]glycine l-
butyl ester
cI I
w y , I w y
0
t8u0.C~NH CISO; ~ ~N tBuO:C~N-S
CI I ~ R , O ' CI
1$ ~Me Me
A mixture ofNEt3 (0.59 mL, 4.21 mmol), 1,4-dichloro-7-isoquinolinesulphonyl
chloride (500
mg, 1.69 mmol) and N [(1R)-1-phenylethyl)]glycine t-butyl ester (476 mg, 2.02
mmol) in
CH~Ch (8 mL) were stirred under N~ at room temperature for 18 h. The mixture
was diluted
with CHZCIa (50 mL), washed with dilute HCl (2 M), saturated aqueous NaHC03,
brine, dried
(Na2S0.~) and evaporated in vacuo. The residue was purified by column
chromatography upon
silica gel using pentane-EtOAc (90:10) as eluant to give lV [(1,4-dichloro-7-
isoquinolinyl)sulphonyl]-N [(1R)-1-phenylethyl)]glycine t-butyl ester (490 mg,
0.99 mmol) as
a colourless oil.
'H (CDC13, 300 MHz) 8 1.3 (9H, s), 1.4 (3H, d), 3.9 (1H, d), 4.1 (1H, d), 5.15
(1H, q), 7.1-
7.25(SH,m),8.4(lH,d),8.5(lH,d),8.65(lH,s),8.7(lH,d)ppm.
LRMS 495 (MH+), 512 (MNH,~+).
Preparation 31:
N [(1ST-1-Phenylethyl)]glycine t-butyl ester
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NH= tBu02C~NH
s
tBuOxC~Br I ~ s Me ~ I ~ Me
A solution of t-butyl bromoacetate (5.0 g, 25.6 mmol) in CH~C12 (5 mL) was
added dropwise
to a stirred solution of (-)-(S~-a-methylbenzylamine (4.65 g, 38.5 mmol) in
CHzCIZ (40 mL)
at 0 °C, and the mixture was then warmed to room temperature and
stirred for 18 h, The
mixture was diluted with CH~CIZ, washed with water, with dilute HC( (1 M) and
then dried
(MgSO,~). The solvents were evaporated in vacuo to give N [(1ST-1-
phenylethyl)]glycine t-
butyl ester (2.02 g, 8.6 mmol) as a white powder.
~ mp 197-202 °C.
~H (CDC13, 300 MHz) 8 1.4 (9H, s), 1.9 (3H, d), 3.3 (1H, d), 3.55 (1H, d), 4.5
(1H, q), 5.3
( I H, s), 7.3-7.45 (3 H, m), 7.5-7.6 (2H, m) ppm.
l5 LRMS 236 (MH+)
Preparation 32:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N [(1ST-1-phenylethyl)]glycine t-
butyl ester
c1 c1
l
0
tBu02C~NH CIS02 ~ ~N tBuOxC~N-S
s CI ' ~ s 0 CI
Me
A mixture of NEt3 (0.59 mL, 4.21 mmol), 1,4-dichloro-7-isoquinolinesulphonyl
chloride (500
mg, 1.69 mmol) and N [(15~-1-phenylethyl)]glycine t-butyl ester (476 mg, 2.02
mmol) in
CHZC1~ (8 mL) were stirred under NZ at room temperature for 24 h. The mixture
was diluted
with CHZCh (50 mL), washed with dilute HC( (2 M), saturated aqueous NaHC03,
brine, dried
(NaZS04) and evaporated in vacuo. The residue was purified by column
chromatography upon
silica gel using pentane-EtOAc (90:10) as eluant to give N [(1,4-dichloro-7-
isoquinolinyl)sulphonyl]-N [(1ST-1-phenylethyl)]glycine t-butyl ester (420 mg,
0.85 mmol) as
a colourless oil.
IH (CDC13, 300 MHz) 8 1.3 (9H, s), 1.4 (3H, d), 3.9 (1H, d), 4.1 (1H, d), 5.15
(1H, q), 7.1-
7.25(SH,m),8.4(lH,d),8.5(lH,d),8.65(lH,s),8.7(lH,d)ppm.
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LRMS 495 (MH~), 512 (MNHa+).
Preparation 33:
N Benzyl-L-alanine t-butyl ester
Ma Me
tBuO;C~NH= ~ CHO tBuOzC~NH
\ \
Benzaldehyde (2.69 ml, 26.4 mmol) was added to a stirred slurry of L-alanine t-
butyl ester
(4.0 g, 22.0 mmol) and NEt3 (3.07 ml, 22.0 inmol) in CH~CIz (70 ml) at 23
°C and the
mixture was stirred for 10 min. NaBH(OAc)3 (6.44 g, 30.4 mmol) was added
portionwise and
the mixture stirred at 23 °C for 24 h. The mixture was washed with
water, dried (MaSOa) and
the solvents were evaporated in vacuo. The residue was purified by column
chromatography
upon silica gel using CH~CI~-MeOH (99:1 to 95:5) as eluant to give to give N
benzyl-L-
I S alanine t-butyl ester (3.97 g, 16.9 mmol) as a colourless oil.
tH (CDCl3, 300 MHz) ~ 1.3 (3 H, d), 1.5 (9H, s), 2.1 ( 1 H, s), 3.25 ( 1 H,
q), 3.7 ( 1 H, d), 3.8
( 1 H, d), 7.2-7.4 (5H, m) ppm.
LRMS 236 (MH+), 258 (MNa+).
Preparation 34: .
N Benzyl-N [(1,4-dichloro-7-isoquinolinyl)sulphonyl]-L-alanine t-butyl ester
cI c1
Me I \ \1 Ma I \ \~
0
tBuOZC~NH CISOx ~ ~N t8u0ZC~N-S
CI I ~ 0 CI
A solution of 1,4-dichloro-7-isoquinolinesulphonyl chloride (600 mg, 2.02
mmol) in CHZC12
(3 ml) was added to a stirred solution of N benzyl-L-alanine t-butyl ester
(571 mg, 2.43
mmol) and NEt3 (0.70 ml, 5.06 mmol) in CHZCIz (3 ml) and the mixture was
stirred at room
temperature for 24 h. The mixture was diluted with CH~CIz (50 ml), washed with
dilute HCI
(2 M), saturated aqueous NaHC03, brine, dried (NazSOa) and evaporated in
vacuo. The
residue was purified by column chromatography upon silica gel using pentane-
EtOAc (95:5
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to 85:15) as eluant to give N benzyl-N [(1,4-dichloro-7-
isoquinolinyl)sulphonyl]-L-alanine t-
butyl ester (470 mg, 0.95 mmol) as a colourless solid.
mp 92-96 °C.
'H .(CDC13, 300 MHz) 8 1.3 (9H, s), 1.35 (3H, d), 4.4 (1H, d), 4.7 (1H, q),
4.8 (1H, d), 7.1-7.3
(3 H, m), 7.3-7.4 (2 H, m), 8.15 ( 1 H, d), 8.3 ( 1 H, d), 8.45 ( 1 H, s), 8.7
( 1 H, s) ppm.
LRMS 495 (MH+)
Preparation 35:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-L-alanine t-butyl ester
c1
c1
Me ~ \ \
\ \ ~ . 0
CISO ~~ ~ N t8u0,C~H-~S ~ ~ N
CI 0 . CI
A solution of 1,4-dichloro-7-isoquinolinesulphonyl chloride (500 mg, 1.69
mmol) in CHzC(Z
(3 mL) was added to a stirred solution of L-alanine t-butyl ester (322 mg,
1.77 mmol) and
NEt3 (0.82 mL, 5.9 mmol) in CH~CIz (6 mL) and the mixture was stirred at 23
°C for 17 h.
The mixture was diluted with CH~CI~, washed with dilute HCI (2 M), saturated
aqueous
NaHC03, brine, dried (MgS04) and evaporated in vacuo. The residue was purified
by column
chromatography upon silica gel using pentane-EtOAc (90:10 to 50:50) as eluant
to give N
[(1,4-dichloro-7-isoquinolinyl)sulphonyl]-L-alanine t-butyl ester (500 mg,
1.23 mmol) as a
white powder.
mp 115-119 °C.
'H (CDC13, 300 MHz) 8 1.2 (9H, s), 1.4 (3H, d), 4.0 (IH, dq), 5.4 (1H, d),
8.25 (1H, d), 8.4
(1H, d), 8.5 (1H, s), 8.9 (1H, s) ppm.
LRMS 405 (MH+).
Anal. Found: C, 47.57; H, 4.39; N, 6.72. Calc for C,6H~$C12NZOdS: C, 47.42; H,
4.48; N, 6.91.
Preparation 36:
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N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-D-alanine methyl ester
a °'
\ \ Me I \ \
I ~ o
CISO ~ ~ N Me02C' o 'N-S ~ ~ N
0 CI
CI
A solution of 1,4-dichloro-7-isoquinolinesulphonyl chloride (500 mg, 1.69
mmol) in CHzCIz
(3 mL) was added to a stirred solution of D-alanine methyl ester (247 mg, 1.77
mmol) and
NEt3 (0.82 mL, 5.9 mmol) in CH~Ch (6 mL) and the mixture was stirred at 23
°C for 16 h.
The mixture was diluted with CH~Ch, washed with dilute HCI (2 M), saturated
aqueous
NaHC03, brine, dried (MgS04) and evaporated in vacuo. The residue was purified
by, column
chromatography upon silica gel using pentane-EtOAc (90:10 to 50:50) as eluant
to give N
[(1,4-dichloro-7-isoquinolinyl)sulphonyl]-D-alanine methyl ester (420 mg, 1.16
mmol) as a
white powder.
mp 150-152 °C.
I H (CDC13, 300 MHz) b 1.45 (3 H, d), 3.55 (3 H, s), 4.15 ( I H, dq), 5.4 ( I
H, d), 8.2 ( I H, d), 8.4
(1H, d), 8.5 (1H, s), 8.9 (1H, s) ppm.
LRMS 363, 365 (MH+).
Anal. Found: C, 42.97; H, 3.29; N, 7.42. Calc for C~3H12CIzN204S: C, 42.99; H,
3.33; N, 7.71.
Preparation 37:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-L-valine t-butyl ester
c1 c1
Me~Me \ \
\ \ 0 I
CISO I ~ ~ N tBuOzC~N-S ~ ~ N
0 CI
CI
NEt3 (0.59 mL, 4.2 mmol) was added to a stirred mixture of 1,4-dichloro-7-
isoquinolinesulphonyl chloride (500 mg, 1.69 mmol) and L-valine t-butyl ester
(354 mg, 1.69
mmol) and in CHZCIz (25 mL) and the mixture was stirred at 23 °C for 3
d. The mixture was
washed with dilute HCI (2x20 mL, 1 M), saturated aqueous NaHC03, brine, dried
(MgS04)
and evaporated in vacuo. The residue was extracted with hexane, which
crystallised on
standing, to give N [( 1,4-dichloro-7-isoquinolinyl)sulphonyl]-L-valine t-
butyl ester (463 mg,
1.07 mmol) as a white solid.
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mp 127-129 °C.
I H (CDC13, 300 MHz) 8 0.9 (3 H, d), 1.0 (3H, d), 1.1 (9H, s), 2.0-2.2 ( 1 H,
m), 3.8 ( 1 H, dd),
5.25 ( 1 H, d), 8.2 ( l H, d), 8.35 ( I H, d), 8.5 ( 1 H, s), 8.9 ( 1 H, s)
ppm.
LRMS 433, 435 (MH+), 450, 4~2 (MNH~+)
Anal. Found: C, 49.86; H, 5.13; N, 6.40. Calc for C,gH>?C1~N~O,~S: C, 49.89;
H, 5.18; N, 6.46.
(0
Preparation 38: ,
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-D-valine t-butyl ester
c1
c1
Mew Me ~
~ ICI ~ / i N
CISO / ~N IHuO,C' o'H-S
0 CI
CI
D-Valine t-butyl ester has been prepared previously, see: Shepel, E. N.;
Iodanov, S.;
Ryabova, I. D.; Miroshnikov, A. L; Ivanov, V. T.; Ovchinnikov, Yu A. Bioorg.
Khim. 1972,
2, 581-593.
D-Valine t-butyl ester (354 mg, 1.69 mmol) and then NEt3 (0.59 mL, 4.2 mmol)
were added
to a stirred solution of 1,4-dichToro-7-isoquinolinesulphonyl chloride (500
mg, 1.69 mmol)
and in CH~CIZ (20 mL) and the mixture was stirred at 23 °C, for 16 h.
The mixture was diluted
with CHzCh (50 mL), washed with saturated aqueous NaHC03, water, aqueous
citric acid (1
M)~ water, brine, dried (MgSO~) and evaporated in vacuo. The residue was
dissolved in i-
Pr~O and hexane was added which gave a precipitate. The solvents were
evaporated in vacuo
and the solid was triturated with hexane to give N [(1,4-dichloro-7-
isoquinolinyl)sulphonyl]-
D-valine t-butyl ester (532 mg, 1.22 mmol) as a white solid. An analytical
sample was
obtained by recrystallisation from hexane.
mp 117-119 °C.
'H (CDCl3, 400 MHz) 8 0.9 (3H, d), 1.0 (3H, d), 1.1 (9H, s), 2.0-2.2 (1H, m),
3.8 (1H, dd),
5.3(lH,d),8.2(lH,d),8.35(lH,d),8.5(lH,s),8.9(lH,s)ppm.
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LRMS 433, 435 (MH+).
Anal. Found: C, 49.99; H, 5.28; N, 6.34. Calc for C,BH~~ChN20,~S: C, 49.89; H,
5.12; N, 6.46.
Preparation 39:
rV [( 1,4-Dichloro-7-isoquinolinyl)sulphonyl]-D-tert-leucine t-butyl ester
c1
CI Me
Me~Me \ \
I \ \
ICI ~ / i N
CISO: / ~ N tBuO=C o H-S
CI 0 CI
A mixture of D-tert-leucine t-butyl ester hydrochloride (250 mg, 1.12 mmol),
NEt~ (0.40 mL,
2.87 mmol) and 1,4-dichloro-7-isoquinolinesulphonyl chloride (330 mg, 1.1 l
mmol) in
CH~CI~ (20 mL) was stirred at 23 °C for 16 h. The mixture was diluted
with CH~CI, (50 mL),
washed with water, aqueous citric acid (1 M), water, saturated aqueous NaHC03,
brine, dried
(MgSOd) and evaporated in vacaro. The residue was purified by column
chromatography upon
t5 silica gel using hexane-EtOAc (90:10) as eluant to give N [(1,4-dichloro-7-
isoquinolinyl)sulphonyl]-D-tert-leucine t-butyl ester (250 mg, 0.56 mmol) as a
white foam.
mp 140-142 °C.
~H (CDC13, 400 MHz) 8 1.0 (9H, s), 1.05 (9H, s), 3.6 (1H, d), 5.35 (1H, d),
8.2 (1H, d), 8.35
(lH,d),8.45(lH,s),8.85(lH,s).
LRMS 447, 449, 451 (MHO).
Anal. Found: C, 51.03; H, 5.41; N, 6.13. Calc for C,9Hz4ClZNz04S: C, 51.01; H,
5.41; N, 6.26.
Preparation 40:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-L-phenylalanine t-butyl ester
/ I c1
c1
\ \ \
C1S0 I / ~ N ~ tBuOiC~N-S / ~ N
0 CI
CI
280
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A mixture of L-phenylalanine t-butyl ester (352 mg, 1.37 mmol), NEt3 (0.41 mL,
2.97 mmol)
and 1,4-dichloro-7-isoquinolinesulphonyl chloride (399 mg, 1.35 mmol) in
CH~CI~ (10 mL)
was stirred at 23 °C for 20 h. The solvents were evaporated in vacuo
and the residue
suspended in EtOAc. This solution was washed with water, brine, dried (MgSOa)
and
evaporated in vacue~. The residue was puriFed by column chromatography upon
silica gel
using pentane-EtOAc (90:10 to 70:30) as eluant to give N [(1,4-dichloro-7-
isoquinolinyl)sulphonyl]-L-phenylalanine t-butyl ester (450 mg, 0.94 mmol) as
a white
crystallised foam.
1 o ' H (C DC I;, 300 vlHz) cS I .2 (9H, s), 2.95 ( 1 H, dd), 3.1 ( 1 H, dd),
4. I ( 1 H, m), 5.3 ( 1 H, d), 7.0-
7.2 (~ H, m), 8. I ( 1 H, d), 8.25 ( 1 H, d), 8.5 ( 1 H, s), 8.75 ( 1 H, d)
ppm.
LRMS 48l (MH'), 498 (MNH~~).
I S Preparation 41:
N (Benzyloxycarbonyl)-O-methyl-D-serine t-butyl ester
MeO~ MeO
0 0
HOxC o H"O I ~ tBuOzC o H 0 I W
20 Condensed isobutylene gas (35 mL) was added to a solution of N
(benzyloxycarbonyl)-O-
methyl-D-serine dicyclohexlamine salt (2.5 g, 5.76 mmol) in CHZCl2 (35 mL) at -
78 °C in a
steel bomb. Conc. H~S04 (0.5 mL) was added, the vessel was sealed and the
mixture allowed
to warm to 23 °C [CAUTION: Pressure]. The mixture was stirred at 23
°C for 6 d, the vessel
was vented and excess isobutylene was allowed to evaporate. The mixture then
poured into
25 aqueous NaHC03 (30 mL, 10 %), extracted with CHzCI2 (3x30 mL), and the
combined
organic extracts were dried (NazS04) and evaporated in vacuo. The residue was
purified by
column chromatography upon silica gel using hexane-EtOAc (80:20) as eluant to
give N
(benzyloxycarbonyl)-O-methyl-D-serine t-butyl ester (1.2 g, 3.88 mmol) as a
colorless oil.
30 'H (CDC13, 400 MHz) 8 1.45 (9H, s), 3.35 (3H, s), 3.6 (1H, dd), 3.75 (1H,
dd), 4.35 (1H, br
d), 5.1 (2H, s), 5.6 (1H, br d), 8.4-8.9 (5H, m) ppm.
LRMS 310 (MH+), 327 (MNH4+)
35 Preparation 42:
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O-Methyl-D-serine t-butyl ester
MeO~ 0 MeO
tBuOZC o H- -O I ~ t8u0ZC o NHx
A solution of N {benzyloxycarbonyl)-O-methyl-D-serine t-butyl ester ( I .1 ~
~, 3.72 mmol) in
MeOH (20 rriL) was hydrogenated over I 0% Pd/C ( 150 mg) under an atmosphere
of H~ (15
psi) at 23 °C for 18 h. The mixture was filtered and the filtrate
evaporated h~ vacuo. The
residue was dissolved in Et,O, a solution of HCI in Et~O (1 M) was added, the
solvents were
evaporated in vacuo to give a white solid and this material was triturated
with hexane to give
O-methyl-D-serine t-butyl ester hydrochloride (0.62 g, 2.90 mmol).
mp 167-169 °C (dec).
i H (CDC13, 400 MHz) 8 I .5 (9H, s), I .8-2.2 ( I H, br s), 3.4 (3H, s), 3.9 (
1 H. dd), 4.0 ( 1 H, dd),
I S 4.2 ( I H, t), 8.4-8.9 (3 H, br s) ppm.
LRMS 176 (MH+)
Anal. Found: C, 45.26; H, 8.59; N, 6.39. Calc for C8H,7N03~HCI: C, 45.39; H,
8.57; N, 6.62.
Preparation 43:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-O-methyl-D-serine t-butyl ester
c1
c1
Me0\ MeO
I / iN
//ll~~
/ i N tBuO C o N-S
tBu02C o NH= CISO= 2 H p CI
CI
A mixture of O-methyl-D-serine t-butyl ester hydrochloride (300 mg, 1.42
mmol), NEt3 (0.50
mL, 3.6 mmol) and 1,4-dichloro-7-isoquinolinesulphonyl chloride (420 mg, 1.42
mmol) in
CHZCIZ (20 mL) was stirred at 23 °C for 3 d. The mixture was diluted
with CH~CIz (30 mL),
washed with water, aqueous citric acid (1 M), water, saturated aqueous NaHC03,
brine, dried
(MgSOa) and evaporated in vacuo. The residue was purified by column
chromatography upon
silica gel using hexane-EtOAc (80:20) as eluant to give N [(1,4-dichloro-7-
isoquinolinyl)sulphonyl]-O-methyl-D-serine t-butyl ester (356 mg, 0.82 mmol)
as a white
solid.
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mp 135-137 °C.
I H (CDC13, 400 MHz) b 1.25 (9 H, s), 3.3 (3 H, s), 3.6 ( 1 H, dd), 3.7 ( 1 H,
dd), 4.1 ( t H, br s),
5.6(lH,brd),8.25(lH,d),8.35(IH,d),8.5(lH,s),8.9(lH,s)ppm.
LRMS 435, 437 (MH+), 452, 454 (MNH.,+).
Anal. Found: C, 47.04; H, 4.62; N, 6.42. Calc for Ct7H~oCI~N~OSS: C, 46.90; H,
4.63; N, 6.44.
Preparation 44:
N [(1,4-Diciloro-7-isoquinolinyl)sulphonyl]-D-aspartic acid di-t-butyl ester
ct c1
to°o,c~
tBu02C~
O
/ i N ieu0_C o N-S ~ ~ N
tBuO~C o NH= CISO, H p CI
CI
A mixture of D-aspartic acid di-t-butyl ester (462 mg, 1.64 mmol), NEt3 (0.50
mL, 3.6 mmol)
and 1,4-dichloro-7-isoquinolinesulphonyl chloride (400 mg, 1.35 mmol) in CHZCh
(30 mL)
was stirred at 23 °C for 18 h. The mixture was diluted with CHzCIz (30
mL), washed with
dilute HC1 (2 M), saturated aqueous NaHC03, brine, dried (MgS04) and
evaporated in vacuo
to give N [(1,4-dichloro-7-isoquinolinyl)sulphonyl]-D-aspartic acid di-t-butyl
ester (520 mg,
1.03 mmol) as a white solid.
mp 106-110 °C.
t H (CDC13, 400 MHz) 8 1.2 (9H, s), 1.4 (9H, s), 2.7-2.8 ( 1 H, dd), 2.8-2.9 (
1 H, dd), 4.15 ( 1 H,
m),8.2(lH,d),8.4(lH,d),8.5(lH,s),8.9(lH,s)ppm.
LRMS 507 (MH+)
Preparation 45:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-L-proline t-butyl ester
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c1 cI
w w w w
/ iN n ~ I / iN
CISO= ~N-S
CI L~. O CI
COzIBu
A mixture of L-proline c-butyl ester hydrochloride (335 mg, 1.61 mmol), NEt3
(0.53 mL, 3.78
mmol) and 1,4-dichloro-7-isoquinolinesulphonyl chloride (449 mg, 1.51 mmol) in
CH~CIz (10
mL) was stirred at 23 °C for 20 h. The solvents were evaporated irT
vacuo and the residue
suspended in EtOAc. This solution was washed with water, brine, dried (MgSOa)
and
evaporated iii vacuo. The residue was purified by column chromatography upon
silica gel
using pentane-EtOAc (90: (0 to 70:30) as eluant to give N [(1,4-dicliloro-7-
isoquinolinyl)sulphonyl]-L-proline t-butyl ester (543 mg, 1.26 mmol) as a
white solid.
'H (CDC13, 300 MHz) 8 1.45 (9H, s), 1.8-2.1 (3H, m), 2.1-2.3 (1H, m), 3.4-3.6
(2H, m), 4.4
( 1 H, dd), 8.3 ( 1 H, d), 8.4 ( 1 H, d), 8.5 ( 1 H, s),.8.9 ( I H, d) ppm.
L1ZMS 431 (MH+), 448, 450 (MNHa~).
Anal. Found: C, 50.09; H, 4.62; N, 6.37. Calc for C,BHZOCI~N~04S: C, 50.12; H,
4.67; N, 6.49.
Preparation 46:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-D-proline t-butyl ester
c1
w w
y 1 ~ _° ~ / , N
clso2 ° N o c1
c' co,te°
A mixture of D-proline t-butyl ester hydrochloride (340 mg, 1.64 mmol), NEt3
(0.50 mL, 3.6
mmol) and 1,4-dichToro-7-isoquinolinesulphonyl chloride (400 mg, 1.35 mmol) in
CH~C1~ (30
mL) was stirred at 23 °C for 20 h. The mixture was diluted with CHZCIz
(50 mL), washed
with dilute HCI (2 M), saturated aqueous NaHC03, brine, dried (MgS04) and
evaporated in
vacuo to give N [(1,4-dichloro-7-isoquinolinyl)sulphonyl]-D-proline t-butyl
ester (550 mg,
1.28 mmol) as a white solid.
mp 80-82 °C.
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~H (CDC13, 400 MHz) b 1.4 (9H, s), 1.9-2.0 (3H, m), 2.2 (I H, m), 3.4-3.6 (2H,
m), 4.4 (1 H,
m), 8.3 ( I H, d), 8.4 ( 1 H, d), 8.5 ( 1 H, s), 8.9 ( I H, s) ppm.
LRMS 431 (MH~), 448 (MNH,,+)
Anal. Found: C, 49.76; H, 4.75; N, 6.39. Calc for C~BH~oChN~O.~S: C, 50.12; H,
4.67; N, 6.49.
Preparation 47:
1,4-Dichloro-7-{[(2R)-(hydroxymethyl)-1-pyrrolidinyl]sulphonyl} isoquinoline
c1 c'
0
NH I / iN N_S I / iN
R CISO: II
O CI
HO CL
HO
A mixture of (R)-2-pyrrolidinemethanol (1.l mL, 11.0 mmol), NEt3 (I.5 mL, 20
mmol) and
1,4-dichloro-7-isoquinolinesulphonyl chloride (3.0 g, 10 mmol) in CH~CI~ (50
mL) was
IS stirred at 23 °C for 30 min. The mixture was diluted with CH~CI~ (50
mL), washed with
aqueous citric acid ( 1 N), water, brine, dried (MgS04) and evaporated in
vaca~o to give 1,4-
dichloro-7-{[(2R)-(hydroxymethyl)-1-pyrrolidinyl]sulphonyl}isoquinoline (4.0
g, 11 mmol)
as a white solid.
mp 167.5-168.5 °C.
~H (CDC13, 400 MHz) s 1.5-1.55 (1H, m), 1.6-2.0 (3H, m), 2.5 (1H, br t), 3.3-
3.4 (IH, m),
3.5-3.6 (1H, m), 3.7-3.8 (3H, m), 8.25 (1H, d), 8.4 (1H, d), 8.5 (1H, s), 8.9
(1H, s) ppm.
LRMS 361, 363 (MH+), 378 (MNH4+), 383 (MNa+).
Anal. Found: C, 46.65; H, 3.91; N, 7.61. Calc for C,4H,dC12NZ03S: C, 46.55; H,
3.91; N, 7.75.
Preparation 48:
Methyl2-{[(1,4-dichloro-7-isoquinolinyl)sulphonyl]amino}isobutyrate
c1 c'
Me Me ~ ~ Me Me 0 I
MeOzC~NHZ CISO= I / ~ N MeOzC"H-S / ~ N
CI 0 CI
2g5
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A mixture of methyl 2-aminoisobutyrate (310 mg, 2.02 mmol), NEt3 (0.70 mL,
5.0~ mmol)
and 1,4-dichloro-7-isoquinolinesulphonyl chloride (500 mg, 1.69 mmol) in
CH~CI~ (30 mL)
was stirred at 23 °C for 17 h. The mixture was diluted with CH~CI, (50
mL). washed with
dilute HCI (2 M), saturated aqueous NaHC03, brine, dried (Na~SOa) and
evaporated in vcrcz~o
The residue was purified by column chromatography upon silica gel using hexane-
EtOAc
(70:30) as eluant to give methyl 2-{[(1,4-dichloro-7-
isoquinolinyl)sulphonyl]amino}isobutyrate (210 mg, 0.56 mmol) as a white
solid.
mp 159.x-l61 °C.
1H (CDC13, 400 MHz) ~ 1.5 (6H, s), 3.7 (3H, s), 5.55 (1H, s), 8.25 (1H, d),
8.35 (1H, d), 8.5
( 1 H, s), 8.9 ( 1 H, s) ppm.
LRMS 377 (MH~)
Anal. Found: C, 44.24; H, 3.72; N, 7.29. Calc for C,.~HIdCI~N,O,~S: C, 44.57:
H, 3.74; N, 7.43.
Preparation 49:
2-{[(1,4-Dichloro-7-isoquinolinyl)sulphonyl]amino}-2-methylpropanamide
cI cI
\ \ ~ Me Me O \ \
Me Me I II I / iN
H=N-~NHz CISO; ~ ~ H2N~H 0 CI
0 CI
A mixture of 2-amino-2-methylpropanamide (200 mg, 1.96 mmol), NEt3 (0.69 mL,
5.0 mmol)
and 1,4-dichloro-7-isoquinolinesulphonyl chloride (580 mg, 1.96 mmol) in
CH~CIa (20 mL)
was stirred at 23 °C for 17 h. The mixture was diluted with CHZCh (50
mL), washed with
water, aqueous citric acid (1 N), water, brine, dried (MgSOd) and evaporated
in vacuo The
residue was purified by column chromatography upon silica gel using CHZCIZ-
MeOH-
0.880NH3 (90:10:1) as eluant to give 2-{[(1,4-dichloro-7-
isoquinolinyl)sulphonyl]amino}-2-
methylpropanamide (228 mg, 0.62 mmol) as a white solid.
mp 220-222 °C.
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t H (d,-MeOH, 400 MHz) 8 I .4 (6H, s), 3.3 (2 H, s), 8.4 ( 1 H, dd), 8.45 ( 1
H, d), 8.55 ( I H, d),
8.9 ( 1 H, s).
LRMS 362, 364 (MH+), 379, 381 (MNH4+).
Anal. Found: C, 42.81; H, 3.70; N, 1 I.15. Calc for C~3H,3CIZN303S~0.25H~0: C,
42.58; H,
3.71; N, 11.46.
Preparation 50:
Ethyl I-aminocyclobutanecarboxylate
H02C~NHz Et02C~NH,
A solution 1-aminocyclobutanecarboxylic acid (500 mg, 4.34 mmol) in EtOH ( l0
mL) was
saturated with HC1 gas, and the mixture was stirred at 23 °C for 4 d.
The solvents were
evaporated in vacuo, azeotroping with PhMe and CH~CI~, to give ethyl 1-
aminocyclobutanecarboxylate hydrochloride (754 mg, 4.20 mmol) as an off white
solid.
'H (DMSO-dh, 300 MHz) 8 1.25 (3H, t), 1.9-2.1 (2H, m), 2.3-2.5 (4H, m), 4.2
(2H, q), 8.8
(2H, br s) ppm.
LRMS 287 (MPH+).
Preparation 51:
Ethyll-{[(1,4-dichloro-7-isoquinolinyl)sulphonyl]amino}cyclobutanecarboxylate
c1 c1
0
EtO2C_ 'NHZ CISO= I / ~ N Et0=C H-S ~ ~ N
CI O CI
A mixture of ethyl 1-aminocyclobutanecarboxylate hydrochloride (382 mg, 2.12
mmol), NEt3
(1.04 mL, 7.43 mmol) and 1,4-dichloro-7-isoquinolinesulphonyl chloride (630
mg, 2.12
mmol) in CHzCl2 (8 mL) was stirred at 23 °C for 18 h. The mixture was
diluted with CHzCl2,
washed with dilute HCl (2 M), saturated aqueous NaHC03, brine, dried (MgS04)
and
evaporated in vacuo. The residue was purified by column chromatography upon
silica gel
using pentane-EtOAc (90:10 to 80:20) as eluant to give ethyl 1-{[(1,4-dichloro-
7-
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isoquinolinyl)su(phonyl]amino}cyclobutanecarboxylate (480 mg, 1.19 mmol) as a
white
powder.
mp 123-125 °C.
~H (CDC13, 300 MHz) 8 1.2 (3H, t), 1.9 ?.l (2H, m), 2.4-2.6 (4H, m), 4.0 (2H,
q), 5.5 (1H, br
s),8.25(lH,d),8.4(IH,d),8.5(lH,s),8.9(lH,s)ppm.
LRMS 403, 405 (MH+), 420 (MNHd+)
Preparation 52:
Cycloleucine ethyl ester
HO,C~NH, EtO,C~NH,
A solution of cycloleucine (8.94 g, 69.2 mmol) in EtOH ( 100 mL) was saturated
with HCI
gas, and the mixture was stirred at 23 °C for 2 d. The solvents were
evaporated in vacuo, the
residue was dissolved in water (200 mL) and the solution basified with solid
NaHC03. The
aqueous solution was extracted with EtOAc (3x100 mL) and the combined extracts
were
washed with brine, dried (MgS04) and evaporated in vacuo. The residue was
dissolved in
hexane-Et~O (I :1) and a solution of HCI in Et~O-dioxane (0.5 M, 1:1) was
added which gave
a precipitate. This off white solid was collected by filtration and dried to
give cycloleucine
ethyl ester hydrochloride (6.57 g, 33.9 mmol).
'H (db-DMSO, 400 MHz) 8 1.2 (3H, t), 1.6-1.8 (2H, m), 1.8-2.0 (4H, m), 2.05-
2.15 (2H, m),
4.15 (2H, q), 8.6-8.7 (3 H, br s) ppm.
Preparation 53:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]cycloleucine ethyl ester
a °I
Q 0
E102C~NHZ CISOZ I ~ ~ N ~ ESO,C~H-IS ~ ~ N
3o CI 0 CI
A mixture of cycloleucine ethyl ester hydrochloride (5.56 g, 28.7 mmol), NEt3
(9.9 mL, 72
mmol) and 1,4-dichloro-7-isoquinolinesulphonyl chloride (7.10 g, 24.0 mmol) in
CHZCh (480
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mL) was stirred at 23 °C for 3 d. The mixture was diluted with CH~C1~,
washed with dilute
HCI (2 M), saturated aqueous NaHC03, brine, dried (NazS04) and evaporated in
vaeuo. The
residue was purified by column chromatography upon silica gel using pentane-
EtOAc (80:20
to 70:30) as eluant to give N [(1,4-dichloro-7-
isoquinolinyl)sulphonyl]cycloleucine ethyl ester
(6.36 g, I5.2 mmol) as a white solid.
mp 127-129 °C.
~H (CDC13, 400 MHz) 8 1.2 (3H, t), 1.6-1.8 (4H, m), 1.9-2.0 (2H, m), 2.1-2.2
(2H, m), 4.1
(2H,q),5.25(lH,s),8.25(IH,d),8.35(IH,d),8.5(lH,s),8.9(IH,s)ppm.
LIZMS 417, 419 (MH+).
Anal. Found: C, 48.57; H, 4.35; N, 6.58. Calc for Ci~H,8ChN30aS: C, 48.93; H,
4.35; N, 6.71.
IS
Preparation 54:
1,4-Dichloro-N [1-(hydroxymethyl)cyclopentyl]-7-isoquinolinesulphonamide
CI CI
\ \ ( ~ \ \
H0~ I / . ~N NON-g I / iN
NHZ CISOx v H II
0 CI
A mixture of 1-amino-1-cyclopentylmethanol (559 mg, 4.86 mmol), NEt3 (0.85 mL,
6.0
mmol) and 1,4-dichloro-7-isoquinolinesulphonyl chloride (1.2 g, 4.05 mmol) in
CH~CIZ (80
mL) was stirred at 23 °C for 16 h. The mixture was diluted with CHZC1~
(50 mL), washed
with dilute HC1 (2 M), saturated aqueous NaHC03, brine, dried (Na2S04) and
evaporated in
vacuo. The residue was purified by column chromatography upon silica gel using
CH~C1Z-
MeOH-0.880NH3 (95:5:0.5) as eluant, followed by trituration with Et~O, to give
to give 1,4-
dichloro-N [1-(hydroxymethyl)cyclopentyl]-7-isoquinolinesulphonamide (0.62 g,
1.65 mmol)
as a white solid.
mp 148-150 °C.
~H (CDCl3, 400 MHz) 8 1.5-1.6 (4H, m), 1.6-1.7 (2H, m), 1.7-1.8 (2H, m), 2.2
(1H, br t), 3.65
(2H,d),5.1(lH,s),8.3(lH,d),8.35(lH,d),8.5(lH,s),8.9(lH,s)ppm.
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LRMS 375 (MH+)
Preparation 55:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N [2-
(dimethylamino)ethyl]cycloleucine ethyl
ester
c1 c1
v w I w w
I / iN ~ / iN
EtOZC H-S Et0_C N-S
0 CI ~ O CI
NMe=
2-(Dimethylamino)ethyl chloride (140 mg, 1.3 mmol) was added to a stirred
solution ofN
l0 [( 1,4-dichloro-7-isoquinolinyl)sulphonyl]cycloleucirie ethyl ester (200
mg, 0.48 mmol) and
anhydrous K,CO~ (80 mg, 0.58 mmol) in DMF (4 mL) under N~ at 23 °C and
the mixture was
stirred for 21 h. The cooled mixture was diluted with EtOAc, washed with water
, dried
(Na~SOa), and the solvents were evaporated in vaearo. The residue was
dissolved in Et~O and
a solution of HCI in Et~O ( I M) was added which gave a precipitate. This oFf
white solid was
collected by filtration and dried to give to give N [(1,4-dichloro-7-
isoquinolinyl)sulphonyl]-
N [2-(dimethylamino)ethyl]cycloleucine ethyl ester (170 mg, 0.32 mmol).
mp 238-240 °C.
IH (DMSO-d6, 300 MHz) b 1.15 (3H, t), 1.55-1.7 (4H, m), 2.0-2.1 (2H, m), 2.2-
2.35 (2H, m),
2.8 (6H, s), 3.35-3.45 (2H, m), 3.75-3.85 (2H, m), 4.0 (2H, q), 8.45 ( 1 H,
d), 8.5 ( 1 H, d), 8.7
( 1 H, s), 8.7 ( 1 H, s) ppm.
LRMS 488, 490 (MIA).
Anal. Found: C, 47.53; H, 5.37; N, 7.96. Calc for CZ,HZ~C1zN30~S~0.25H20: C,
47.65; H,
5.43; N, 7.94.
Preparation 56:
Methyll-{[(1,4-dichloro-7-isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate
cI c1
0
I / i N Me02C~N-S / ~ N
MeOzC NHZ CIS02 v 1' H ~ CI
CI
290
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Methyl I-aminocyclohexanecarboxylate has been prepared previously, see:
Didier, E.;
Horwell, D. C.; Pritchard, M. C. Tetrahedron, 1992, ~t8, 8471-8490.
A mixture of methyl 1-aminocyclohexanecarboxylate (325 mg, 1.68 mmol), NEt3
(0.49 mL,
3.5 mmol) and 1,4-dichloro-7-isoquinolinesulphonyl chloride (415 mg, 1.40
mmol) in CH,Ch
(30 mL) was stirred at 23 °C for 16 h. The mixture was diluted with
CH~Ch, washed with
dilute HCl (2 M), saturated aqueous NaHC03, brine, dried (Na~SOa) and
evaporated ifT vaczro.
The residue was purified by column chromatography upon silica gel using hexane-
EtOAc
(80:20 to 70:30) as eluant, followed by trituration with i-Pr~O, to give to
give methyl 1-{[(1,4-
dichloro-7-isoquinolinyl)sulphony)]amino}-cyclohexanecarboxylate (132 mg, 0.32
mmol) as
a white solid.
mp 185-186 °C.
(5
~H (CDC13, 300 MHz) 8 1.2-1.5 (6H, m), 1.8-2.0 (4H, m), 3.6 (3H, s), 4.95 (1
H, s), 8.25 (I H,
d), 8.4 ( 1 H, d), 8.5 ( 1 H, s), 8.9 ( l H, s) ppm.
LRMS 418 (MH+)
Anal. Found: C, 48.94; H, 4.43; N, 6.42. Calc for C,~H,BChN~OdS: C, 48.93; H,
4.35; N, 6.71.
Preparation 57:
Methyl 4-aminotetrahydro-2H pyran-4-carboxylate
HO C~NH
z MeOxC NHZ
4-Aminotetrahydro-2H pyran-4-carboxylic acid has been prepared previously,
see: Palacin,
S.; Chin, D. N.; Simanek, E. E.; MacDonald, J. C.; Whitesides, G. M.; McBride,
M. T.;
Palmore, G. J. Am. Chenz Soc., 1997, 119, 11807-11816. '
A solution 4-aminotetrahydro-2H pyran-4-carboxylic acid (0.50 g, 3.4 mmol) in
MeOH (10
mL) was saturated with HCl gas at 0-5 °C, and the mixture was then
heated at reflux for 3.5 h.
The solvents were evaporated in vacuo, the residue was dissolved in saturated
aqueous
NaHC03 and the aqueous solution was extracted with CHZCIZ (2x50 mL). The
combined
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extracts were dried (MgSOa) and evaporated in vacuo to give methyl 4-
aminotetrahydro-2H
pyran-4-carboxylate (410 mg, 2.58 mmol).
'H (CDC13, 300 MHz) S 1.4-l.6 (4H, m), 2.05-2.2 (2H, m), 3.6-3.7 (2H, m), 3.75
(3H, s), 3.8-
3.9 (2H, m) ppm.
LRMS 160 (MH~).
Preparation 58:
Methyl4-{[(1,4-dichloro-7-isoquinolinyl)sulphonyl]amino}tetrahydro-2Hpyran-4-
carboxylate
c1 o c'
0
][ I o
C ~ II / iN
MeO~C~NH; CISO_ ~ ~N ~ MeO,C H-S
CI ~ CI
A mixture of methyl 4-aminotetrahydro-2H pyran-4-carboxylate (400 mg, 2.51
mmol), NEt3
(0.44 mL, 3.14 mmol) and 1,4-dichloro-7-isoquinolinesulphonyl chloride (621
mg, 2.09
mmol) in CH~CIa (30 mL) was stirred at 23 °C for 20 h. The mixture was
diluted with CHZC1~,
washed with dilute HCI (2 M), saturated aqueous NaHC03, brine, dried (Na~S04)
and
evaporated in vacuo. The residue was purified by column chromatography upon
silica gel
using hexane-EtOAc (80:20) and then CHzCh-MeOH-0.880NH3 (95:5:0.5) as eluant,
followed by trituration with i-Pr~O, to give to give methyl 4-{[(1,4-dichloro-
7-
isoquinolinyl)sulphonyl]amino}tetrahydro-2H pyran-4-carboxylate (197 mg, 0.47
mmol) as a
white solid.
mp 168-170 °C.
'H (CDC13, 400 MHz) 8 1.8-1.95 (2H, m), 2.1-2.2 (2H, m), 3.5 (3H, s), 3.5-3.7
(4H, m), 5.4
(lH,s),8.25(lH,d),8.4(lH,d),8.5(lH,s),8.9(lH,s)ppm.
LRMS 419 (MH~).
Anal. Found: C, 45.97; H, 3.85; N, 6.36. Calc for C,6H~sChN205S: C, 45.83; H,
3.85; N, 6.68.
Preparation 59:
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t-Butyl (~)-cis-2-{[(1,4-dichloro-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate
a c1
co,te° \ \ co,le° ~ \
~ ~ 0
v 'NH; CISO: I / ~N ~H_S I / iN
CI 0 CI
t-Butyl (~)-cis-2-aminocyclohexanecarboxylate has been prepared previously,
see: Xie, J.;
Soleilhac, J. M.; Renwart, N.; Peyroux, J.; Roques, B. P.; Fournie-Zaluski, M.
C. Int. J. Pept.
Pl'OIG'131 Res 1989, 3-~, 246 ?55.
A mixture of t-butyl (~)-cis-2-aminocyclohexanecarboxylate hydrochloride (282
mg, 1.20
mmol), NEt; (0.33 mL, 2.37 mmol) and 1,4-dichloro-7-isoquinolinesulphonyl
chloride (282
Illg, 0.95 mmol) in CHZCIZ ( 10 mL) was stirred at 23 °C for 1 h. The
solvents were evaporated
in vaczro and the residue suspended in EtOAc (100 mL). This solution was
washed with dilute
HCI ( 10 mL, I M), water, dried (MgSOd) and evaporated in vaearo to give t-
butyl (~)-cis-2-
{[(1,4-dichloro-7-isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate (395
mg, 0.86
mmol) as a white solid.
1H (CDCl3, 300 MHz) 8 1.l-1.8 (16H, m), 1.95-2.1 (1H, m), 2.5-2.6 (1H, m), 3.4-
3.55 (1H,
m),6.1 (lH,d),8.25(IH,d),8.35(lH,d),8.45(lH,s),8.9(lH,s).
LRMS 459, 461 (MH+)
Anal. Found: C, 51.99; H, 5.28; N, 6.01. Calc for C~oH~4CI2N~O,~S: C, 52.29;
H, 5.27; N, 6.10.
Preparation 60:
Ethyl (~)-cis-2-{[(1,4-dichloro-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate
c1 I
CO,Et \ \ COiEt
\ \
'"NHz CIS02 I / i N ~ aH_S I / i N
CI 0 CI
A mixture of ethyl (~)-cis-2-aminocyclohexanecarboxylate hydrochloride (251
mg, 1.20
mmol), NEt3 (0.33 mL, 2.4 mmol) and 1,4-dichloro-7-isoquinolinesulphonyl
chloride (296
mg, 1.00 mmol) in CHZCIZ (10 mL) were stirred at 23 °C for 1 h. The
mixture was diluted
with CHZCIz (100 mL), washed with dilute HCl (30 mL, 1 M), water, dried
(MgS04) and
evaporated in vacuo to give ethyl (~)-cis-2-{[(1,4-dichloro-7-
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isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate (385 mg, 0.89 mmol) as a
white
solid.
1H (CDC13, 400 MHz) ~ 1.2 (3H, t), 1.2-l.4 (3H, m), 1.4-1.7 (3H, m), 1.75-1.85
(1H, m), 2.0-
2. l ( 1 H, m), 2.65 ( 1 H, q), 3.5-3.6 ( 1 H, m), 3.95-4.0 ( 1 H, m), 4.05-
4.15 ( 1 H, m), 5.9 ( 1 H, d),
8.2 ( 1 H, d), 8.3 5 ( I H, d), 8.5 ( 1 H, s), 8.9 ( I H, s).
LRMS 431, 433 (MH+).
Anal. Found: C, 50.45; H, 4.79; N, 6.31. Calc for C,xHzoClzNzOxS: C, 50.12; H,
4.67; N, 6.49.
Preparation 61:
t-Butyl cis-4-{[(1,4-dichloro-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate
c1 c1
tBuO;C~ ~ ~ tBu02C
I~ ~I ~[j0
v 'NH; CI502 I ~ ~ N ~H-S I / i N
I S CI 0 CI
t-Butyl cis-4-aminocyclohexanecarboxylate has been prepared previously, see:
Barnish, I. T.;
James, IC.; Terrett, N. IC.; Danilewicz, J. C.; Samuels, G. M. R.; Wythes, M.
J. Eur. Patent,
1988, EP 274234.
A mixture of t-butyl cis-4-aminocyclohexanecarboxylate (282 mg, 1.20 mmol),
NEt3 (0.33
mL, 2.37 mmol) and 1,4-dichloro-7-isoquinolinesulphonyl chloride (296 mg, 1.00
mmol) in
CHzCIz (10 mL) was stirred at 0 °C for 1 h. The mixture was diluted
with CHzCIz (150 mL),
was washed with dilute HCl (30 mL, 1 M), water, dried (MgS04) and evaporated
in vacuo.
The residue was purified by column chromatography upon silica gel using
pentane-EtOAc
(100:0 to 75:25) to give t-butyl cis-4-{[(1,4-dichloro-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboxylate (360 mg, 0.78 mmol) as a
white
solid.
~H (CDCl3, 400 MHz) 8 1.4 (9H, s), 1.5-1.65 (6H, m), 1.75-1.85 (2H, m), 2.3
(1H, m), 3.45
(IH,m),4.75(IH,d),8.25(IH,d),8.4(lH,d),8.5(lH,s),8.9(IH,s)ppm.
LRMS 459, 461 (MH+), 476 (MNH4+).
Anal. Found: C, 52.34; H, 5.28; N, 5.98. Calc for CzoHzaClzNzO4S: C, 52.29; H,
5.27; N, 6.10.
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Preparation 62:
Ethyl traps-4-{[(1,4-diciloro-7-
isoquinolinyl)sulphonyl]amino}cyclohexanecarboYylate
c1 c1
EtO,C.",/~ ~ ~ EtOZC.,,
~ 0
'"NHZ CISO; I ~ i N ~H-5 I ~ i N
CI 0 CI
Ethyl traps-4-aminocyclohexanecarboxylate has been prepared previously, see:
Skaric, V.;
Kovacevic, M.; Skaric, D. J. C'lrc~nr. Soc., I'erkin Trcrns.l 1976, 1 199-
1201.
A mixture of ethyl trurrs-4-aminocyclohexanecarboxylate ( 168 mg, 0.81 mmol),
NEt3 (0.22
mL, 1.6 mmol) and 1,4-dichloro-7-isoquinolinesulphonyl chloride (200 mg, 0.67
mmol) in
CH~CI~ (8 mL) was stirred at 0 °C for 1 h. The mixture was diluted with
CH~CI~ (100 mL),
was washed with dilute HCI (50 mL, 1 M), water, dried (MgSOa) and evaporated
in vaczro to
give ethyl trams-4-{[( 1,4-dichloro-7-
isoquino(inyl)sulphonyl]amino}cyclohexanecarboxylate
(232 mg, 0.54 mmol) as a white solid.
~H (CDC13, 400 MHz) 8 1.15-1.3 (5H, m), 1.4-1.55 (2H, m), 1.9-2.0 (4H, m), 2.1-
2.2 (1H, m),
3.2-3.3(lH,m),4.1 (2H,t),4.55(lH,d),8.25(lH,d),8.35(lH,d),8.5(lH,s),8.9(lH,s)
LRMS 431 (MH+).
Preparation 63:
1,4-DichToro-7-isoquinolinecarbonyl. chloride
c1
w w
Br'~NH I ~ NH
Br
75 0 0
A solution of N chlorosuccinimide (4.13 g, 31 mmol) in MeCN (50 mL) was added
dropwise
to a stirred solution of 7-bromo-1-(21~-isoquinolone (6.6 g, 29.5 mmol) in
MeCN (150 mL)
which was heating under reflux. The mixture was heated under reflux for an
additional 3 h
and then cooled to room temperature. The resulting precipitate was collected
by filtration,
with MeCN rinsing, and then dried in vacuo to give 7-bromo-4-chloro-1(2I~-
isoquinolone
(6.72 g, 26.0 mmol) as a white solid.
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mp 241-243 °C.
1 H (DMSO-d~, 300 MHz) 8 7.5 ( 1 H, s), 7.73 ( 1 H, d), 7.8 ( 1 H, dd), 8.3 (
I H, s) ppm.
LRMS 259 (MH+), 5l7 (MPH+).
Anal. Found: C, 41.69; H, 1.90; N, 5.37. Calc for C9H;BrCINO: C, 41.80; H,
1.95; N, 5.42.
ci c1
I w a I ~ .w
Br ~ N / N
ElO,C
0 0
A mixture of 7-bromo-4-chloro-1(21-isoquinolone (1.0 g, 3.87 mmol) and
bis(triphenylphosphine) palladium (II) chloride (100 mg, 0.14 mmol) in EtOH
(15 mL) and
NEt3 (2 mL) was heated to 100 °C in a pressure vessel under an
atmosphere of CO ( I 00 psi)
for 48 h. After cooling and venting the vessel, the catalyst was removed by
filtration, and the
filtrate was evaporated in vacuo. The residue was purified by column
chromatography upon
silica gel using hexane-EtOAc (50:50) as eluant, and then by crystallisation
from i-Pr~O. This
material was combined with CH~CI~ washings ofthe catalyst residues to give
ethyl 4-chloro-
1-oxo-1,2-dihydro-7-isoquinolinecarboxylate (743 mg, 2.95 mmol) as a white
solid.
mp 184-186 °C.
IH (CDC13, 300 MHz) 8 1.45 (2H, t), 4.45 (2H, q), 7.4 (1H, s), 7.95 (1H, d),
8.4 (1H, d), 9.05
( 1 H, s) ppm.
LRMS 252 (MH+), 269 (MNH4+), 503 (M~H~).
Anal. Found: C, 57.02; H, 3.99; N, 5.53. Calc for C,~H,oC1N03: C, 57.27; H,
4.01; N, 5.57.
c1 ci
~ ~ ~1
EtOZC ~ NH EtOZC I ~ ~ N
0 CI
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Ethyl 4-chloro-l-oxo-1,2-dihydro-7-isoquinolinecarboxylate (500 mg, 1.99 mmol)
was
warmed in POC13 (3 mL) until a clear solution formed, and was then allowed to
stand at 23 °C
for 18 h. The reaction mixture was poured into warm water, extracted with
EtOAc (3x20
mL), and the combined organic extracts washed with water and saturated brine,
dried
S (MgSO.~), and evaporated lit VQCllO. The residue was purified by column
chromatography
upon silica gel using hexane-EtOAc (90:10) as eluant followed by
crystallisation from i-Pr~O
to give ethyl 1,4-dichloro-7-isoquinolinecarboxylate (377 mg, 1.40 mmol) as a
pale pink
solid.
mp 92-94 "C.
~H (CDC13, 300 MHz) b 1.45 (2H, t), 4.45 (2H, q), 8.25 (1H, d), 8.4-8.45 (2H,
m), 9.05 (1H,
s) ppm.
l5 LRMS 270 (MH+)
Anal. Found: C, X3.27; H, 3.48; N, 5.14. Calc for C,,H~Cf,NO~: C, 53.36; H,
3.36; N, 5.19.
c~ c~
~ ~ ~1
e~o2c ° ~ N Ho c I
z
c~ ci
Ethyl 1,4-dichloro-7-isoquinolinecarboxylate (500 mg, 1.85 mmol) in THF (2 mL)
was
treated with an aqueous solution of NaOH (3.7 mL, 1 M) and EtOH (few drops)
added to give
a single phase mixture. After stirring at room temperature overnight, HCl (3.7
mL, 1 M) was
added to give a thick slurry which was filtered off, washed with water, and
crystallised from
i-PrOH. The fluffy white crystalline solid was triturated with hexane and
dried to afford 1,4-
dichloro-7-isoquinolinecarboxylic acid (240 mg, 0.99 mmol).
mp 226-228 °C.
~H (DMSO-d~, 300 MHz) 8 8.3 (1H, d), 8.4 (1H, d), 8.55 (1H, s), 8.8 (1H, s)
ppm.
LRMS 242 (MH+).
Anal. Found: C, 49.59; H, 2.08; N, 5.74. Calc for C,oHSCIZN02: C, 49.62; H,
2.08; N, 5.78.
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c1 c1
w y
HO C I ~ ~ N CI I ~ i N
CI O CI
Oxalyl chloride (144 pL, 1.65 mmol) was added to a suspension of 1,4-dichloro-
7-
isoquinolinecarboxylic acid (200 mg, 0.83 mmol) at room temperature in CH~CI~
(10 mL),
followed by DMF ( I drop). After 30 min the resultant clear solution was
evaporated in vacz~o
to afford 1,4-dichloro-7-isoquinolinecarbonyl chloride which was used without
further
purification.
f0
Preparation 64:
N [(1,4-Dichloro-7-isoquinolinyl)carbonyl]glycine t-butyl ester
c1 c1
Iw ~1 Iw v
CI ~ ~ tBuO.,C~N ~ N
' H
O CI 0 CI
A solution of 1,4-dichloro-7-isoquinolinecarbonyl chloride (213 mg, 0.8 mmol)
in CH~C1~ (10
mL) was added to a stirred suspension of glycine t-butyl ester hydrochloride
(166 mg, 0.99
mmol) and NEt3 (253 pL, 1.82 mmol) in CH~C12 (5 mL). The reaction mixture was
stirred at
room temperature overnight, quenched with a drop of water and then evapourated
in vacuo.
The residue was purified by column chromatography upon silica gel using hexane-
EtOAc
(70:30) as eluant to give N [(1,4-dichloro-7-isoquinolinyl)carbonyl]glycine t-
butyl ester (140
mg, 0.39 mmol). An analytical sample was prepared by crystallisation from i-
Pr20-CHZC1~.
mp 162-164 °C.
'H (CDC13, 300 MHz) 8 1.5 (9H, s), 4.15-4.2 (2H, m), 6.9 (1H, s), 8.25-8.3
(2H, m), 8.4 (1H,
s), 8.75 (1H, s) ppm.
LRMS 355 (MH~).
Anal. Found: C, 53.98; H, 4.36; N, 7.83. Calc for Ci6HI6ChNaO3: C, 54.10; H,
4.54; N, 7.89.
Preparation 65:
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N [(1,4-Dichloro-7-isoquinolinyl)carbonyl]-(3-alanine t-butyl ester
c1 c1
I~ ~1 I~ ~1
CI / ~N tBu02C~ / iN
N
0 CI H 0 CI
A solution of 1,4-dichloro-7-isoquinolinecarbonyl chloride (450 mg, 1.7 mmol)
in CH~C1~ (20
mL) was added to a stirred solution of (3-alanine t-butyl ester hydrochloride
(376 mg, 2.07
mmol) and NEt3 (530 pL, 3.81 mmol) in CH,C1~ (10 mL) and the mixture was
stirred at room
temperature for 3 h. The mixture was washed with HCI (2x30mL, 1 M), aqueous
NaHC03
( 10%, 30 mL), dried (Na~SO.,), and evaporated in vacuo. The residue was
crystallised from i-
Pr~O to give N [( 1,4-dichloro-7-isoquinolinyl)carbonyl]-(3-alanine t-butyl
ester (440 mg, 1.19
mmol) as a white solid.
mp 131-133 °C.
1H (CDC13, 400 MHz) a 1.5 (9H, s), 2.6 (2H, t), 3.7-3.8 (2H, m), 7.15 (l I-I,
br s), 8.2-8.3 (2H,
m), 8.4 ( I H, s), 8.65 ( 1 H, s) ppm.
LRMS 369 (MH+), 740 (M,H+).
Anal. Found: C, 55.11; H, 4.88; N, 7.48. Calc for C,~H,BClzN~03: C, 55.29; H,
4.91; N, 7.59.
Preparation 66:
N [(1,4-Dichloro-7-isoquinolinyl)carbonyl]cycloleucine ethyl ester
c1 c1
w w w w
CI I / ~ N ElOiC H I / i N
75 0 CI 0 CI
A solution of 1,4-dichloro-7-isoquinolinecarbonyl chloride (270 mg, 1.04 mmol)
in CH~C12
(12 mL) was added to a stirred solution of cycloleucine ethyl ester
hydrochloride (300 mg,
1.55 mmol) and NEt3 (415 p.L, 2.98 mmol) in CH~Ch (20 mL) and the mixture was
stirred at
room temperature for 1h. The mixture was washed with dilute HCI (2 M), aqueous
NaHC03
(10 %), dried (Na~S04), and evaporated in vacuo. The residue was crystallised
from i-Pr~O to
give N [(1,4-dichloro-7-isoquinolinyl)carbonyl]cycloleucine ethyl ester (372
mg, 0.98 mmol)
as a white solid.
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mp 178-180 °C.
1H (CDC1;, 300 MHz) ~ 1.3 (3H, t), 1.8-2.05 (4H, m), 2.1-2.3 (2H, m), 2.3-2.45
(2H, m), 4.25
(2 H, q), 6.95 ( 1 H, br s), 8.2-8.25 (2 H, m), 8.4 ( 1 H, s), 8.7( 1 H, s)
ppm.
LRMS 382 (MH'), 398 (MNH~~), 763 (MzH+).
Anal. Found: C, 56.71; H, 4.77; N, 7.27. Calc for C,BH,sCI~N~03: C, 56.70; H,
4.76; N, 7.35.
Preparation 67:
N [(1,4-Dichloro-7-isoquinolinyl)carbonyl]-DL-phenylglycine t-butyl ester
c1 I ~ a
w w / w w
CI I / ~ N ~ tBuO:C H I / i N
0 CI 0 CI
A solution of 1,4-dichloro-7-isoquinolinecarbonyl chloride (450 mg, 1.73 mmol)
in CHZCh
(20 mL) was added to a stirred solution of DL-phenylglycine t-butyl ester
hydrochloride (505
mg, 2.07 mmol) and NEt3 (530 p.L, 3.81 mmol) in CHZCIz (30 mL) and the mixture
was
stirred at room temperature for 3 h. The mixture was washed with dilute HCl
(2x30 mL, 1
M), aqueous NaHC03 ( 10%), dried (NaZS04), and evaporated in vaeuo to give N
[(1,4-
dichloro-7-isoquinolinyl)carbonyl]-DL-phenylglycine t-butyl ester (600 mg,
1.39 mmol) as a
waxy solid. An analytical sample was prepared by the slow evaporation of a
solution in
CH~CIz to give a fluffy white solid.
mp 146-149 °C.
1H (CDC13, 300 MHz) 8 1.5 (9H, s), 5.7 (1H, d), 7.3-7.5 (6H, m), 8.2-8.3 (2H,
m), 8.4 (1H, s),
8.8 ( 1 H, s) ppm.
LRMS 431 (MH+), 861 (MPH+).
Anal. Found: C, 60.57; H, 4.76; N, 6.42. Calc for CZZH~oCIZN203~0.25H~0: C,
60.63; H, 4.74;
N, 6.43
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Preparation 68:
N [(1,4-Dichloro-7-isoquinolinyl)carbonyl]-L-phenylglycine t-butyl ester
c1 ~ ~ c1
w w / w w
CI-~ ~ / ~N tBuO;C~H ~ / iN
O CI O C!
J
A solution of 1,4-dichloro-7-isoquinolinecarbonyl chloride (148 mg, 0.57 mmol)
in CH~CI~ (6
mL) was added to a stirred solution ofS-(+)-phenylglycine t-butyl ester
hydrochloride (138
mg, 0.57 mmol) and NEt3 (200 pL, 1.44 mmol) in CH~Ch (5 mL), and the mixture
was stirred
at room temperature overnight. The mixture was diluted with CHZCh (25 mL),
washed with
dilute HCl (0.5 M), aqueous NaHC03 (10%), brine, dried (Na~S04), and
evaporated in vaca~o
to give N-[( 1,4-dichloro-7-isoquinolinyl)carbonyl]-L-phenylglycine t-butyl
ester (218 mg,
0.51 cnmol) as a gum. An analytical sample was prepared by trituration with
hexane yielding
a solid.
mp 173-175 °C.
1H (CDC13, 300 MHz) 8 1.45 (9H, s), 5.7 (1H, d), 7.3-7.5 (6H, m), 8.25 (2H,
s), 8.4 (1H, s),
8.8 ( 1 H, s) ppm.
LRMS 431 (MH+), 448 (MNH4+), 861 (MzH+), 883 (MZNa+).
Anal. Found: C, 58.83; H, 4.88; N, 5.90. Calc for CZZH2oC12Nz03~Hz0: C, 58.80;
H, 4.93; N,
6.23
Preparation 69:
N-[(1,4-Dichloro-7-isoquinolinyl)carbonyl]-D-phenylglycine t-butyl ester
c1 ~ ~ c1
/
CI I / i N t8u0zC ° H I / i N
0 CI 0 CI
A solution of 1,4-dichloro-7-isoquinolinecarbonyl chloride (148 mg, 0.57 mmol)
in CH~C12 (6
mL) was added to a stirred solution of R-(+)-pheny(glycine t-butyl ester
hydrochloride (138
mg, 0.57 mmol) and NEt3 (200 ~L, 1.44 mmol) in CH~C1~ (5 mL), and the mixture
was stirred
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at room temperature overnight. The mixture was diluted with CH~CI~ (25 mL),
washed witty
dilute HCl (0.5 M), aqueous NaHC03 (10 %), brine, dried (NazSO~), and
evaporated in vacuo.
Trituration of the residue with hexane gave N [( 1,4-dichloro-7-
isoquinolinyl)carbonyl]-D-
phenylglycine t-butyl ester (203 mg, 0.47 mmol) as a white solid.
t H (CDC13, 300 MHz) eS 1.4 (9H, s), 5.7 ( 1 H, d), 7.3-7.5 (6H, m), 8.25 (2H,
s), 8.4 ( 1 H, s), 8.8
( t H, s) ppm.
LRMS 431 (MH+), 448 (MNH.~~), 861 (MPH*), 883 (M~Na+).
Anal. Found: C, 61.17; H, 4.70; N, 6.37. Calc for C~~H~oCIZN~O~: C, 61.26; H,
4.67: N, 6.50
Preparation 70:
N [(1,4-Dichloro-7-isoquinolinyl)carbonyl]-DL-valine t-butyl ester
c1 c1
Me"Ma
I I ~ I I
CI ~ ~ tBuO,C N
0 CI H 0 CI
A solution of 1,4-dichloro-7-isoquinolinecarbonyl chloride (450 mg, 1.73 mmol)
in CHzCl2
(20 mL) was added to a stirred solution of DL-valine t-butyl ester
hydrochloride (435 mg,
2.07 mmol) and NEt3 (530 pL, 3.81 mmol) in CHzCl2 (10 mL) and the mixture was
stirred at
room temperature for 3 h. The mixture was washed with dilute HCl (1 M),
aqueous NaHC03
(10%), dried (Na~S04), and evaporated in vacuo. The residue was crystallised
with i-Pr~O to
give N [(1,4-dichloro-7-isoquinolinyl)carbonyl]-DL-valine t-butyl ester (390
mg, 0.98 mmol)
as a white solid.
tH (CDC13, 400 MHz) 8 1.0-1.05 (6H, m), 1.5 (9H, s), 2.3-2.4 (1H, m), 4.7-4.8
(1H, m), 6.85
(1H, d), 8.25-8.3 (2H, m), 8.4 (1H, s), 8.75 (1H, s) ppm.
LRMS 397 (MH+), 793 (MZH~)
Anal. Found: C, 57.20; H, 5.53; N, 6.99. Calc for C,9H2?ClzN2O3: C, 57.44; H,
5.58; N, 7.05.
Preparation 71:
N [(1,4-Dichloro-7-isoquinolinyl)carbonyl]-DL-proline t-butyl ester
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c( I
I~ ~~ I~
CI / iN N / iN
0 CI ~ 0 CI
CO,tBu
DL-Proline t-butyl ester hydrochloride (320 mg, 1.54 mmol) and then NEt3 (513
ltL, 3.69
mmol) were added to a stirred solution of 1,4-dichloro-7-isoquinolinecarbonyl
chloride (270
mg, 1.04 mniol) in CH~CI~ (32 mL) and the cloudy solution was then stirred at
room
temperature for 4 h. The mixture was diluted with CH~CI~ (20 mL), washed with
dilute HCl
( I M), saturated brine, dried (Na,SO~), and evaporated in vacuo. The residue
was crystallised
with i-Pr~O to give N [(I,4-dichloro-7-isoquinolinyl)carbonyl]-DL-proline t-
butyl ester (395
mg, 1.00 mmol) as a white solid.
mp 144-146 °C.
I H (CDC13, 300 MHz) shows a 3: I mixture of rotamers 8 1.15 ( 1/4 of 9H, s),
I .55 (3/4 of 9H,
s), 1.8-2.15 (3H, m), 2.2-2.4 ( 1 H, m), 3.45-3.9 (2H, m), 4.2-4.3 ( I/4 of 1
H, m), 4.6-4.7 (3/4 of
t 5 1 H, m), 7.9 ( 1/4 of 1 H, d), 8.05 (3/4 of 1 H, d), 8.2-8.3 ( 1 H, m),
8.4 ( I H, s), 8.~5 ( 1 H, s) ppm.
LRMS 395 (MH+), 789 (M~H~).
Anal. Found: C, 57.79; H, 5.11; N, 6.97. Calc for Ci~H~oCl~N~03: C, 57.73; H,
5.10; N, 7.09.
Preparation 72:
N [(1,4-Dichloro-7-isoquinoliny()carbonyl]-DL-phenylalanine t-butyl ester
a / I c(
CI I / i N ~ tBu02C H I / i N
0 CI 0 CI
A mixture of NEt3 (330 p.L, 2.37 mmol), DL-phenylalanine t-butyl ester
hydrochloride (293
mg, 1.14 mmol) and 1,4-dichloro-7-isoquinolinecarbonyl chloride (247 mg, 0.95
mmol) in
CHZCh (20 mL) was stirred at room temperature for 18 h. The solvents were
evaporated in
vacuo and the residue partioned between dilute HCl (1M) and EtOAc. The organic
phase was
washed with brine, dried (NazS04) and evaporated in vacuo. The residue was
crystallised with
i-PrzO to give N [(1,4-dichloro-7-isoquinoliny()carbonyl]-DL-phenylalanine t-
butyl ester (384
mg, 0.86 mmol) as a white solid.
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mp 156-157 °C.
1H (CDC13, 300 MHz) 8 1.5 (9H, s), 3.2-3.3 (2H, m), 5.0 (1H, dt), 6.8 (1H, d),
7.2-7.49 (5H,
m), 8.2 ( 1 H, d), 8.25 ( 1 H, d), 8.4 ( 1 H, s), 8.6 ( 1 H, s) ppm.
LRMS 445 (MH+)
Anal. Found: C, 62.02; H, 4.98; N, 6.28. Calc for C~3H~~C1~Na03: C, 62.03; H,
4.98; N, 6.29.
Preparation 73:.
N [( l,4-Dichloro-7-isoquinolinyl)carbonyl]-DL-leucine t-butyl ester
CI Ma CI
\ \ Me~ I \ \
i
CI ~ ~ N t8u0,C N / N
0 CI H 0 CI
A solution of 1,4-dichloro-7-isoquinolinecarbonyl chloride (247 mg, 0.95 mmol)
in CH~CI
(10 mL) was added to a solution of DL-leucine t-butyl ester hydrochloride (255
mg, 1.14
mmol) and NEt3 (330 pL, 2.37 mmol) in CH~CIZ (10 mL) and the mixture was
stirred at room
temperature overnight. The solvents were evaporated in vacuo and the residue
was partioned
between dilute HCl (1 M) and EtOAc. The organic phase was washed with brine,
dried
(NaZS04) and evaporated in vacuo. The residue was crystallised with i-PrzO to
give N [(1,4-
dichloro-7-isoquinolinyl)carbonyl]-DL-leucine t-butyl ester (285 mg, 0.69
mmol).
mp 183-184 °C.
1H (CDC13, 300 MHz) 8 1.0-1.1 (6H, m), 1.5 (9H; s), 1.65-1.85 (3H, m), 4.75-
4.85 (1H, m),
6.8(lH,d),8.2(2H,s),8.4(lH,s),8.7(lH,s)ppm.
LRMS 411 (MH+)
Anal. Found: C, 58.39; H, 5.84; N, 6.76. Calc for CZpHZdCI2N~O3: C, 58.40; H,
5.88; N, 6.81.
Preparation 74:
t-Butyl DL-3-{[(1,4-dichloro-7-isoquinolinyl)carbonyl]amino}-3-
phenylpropanoate
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c1 I ~ c1
/
CI I / iN tBuO:C N I / sN
H
0 CI O CI
A solution of 1,4-dichloro-7-isoquinolinecarbonyl chloride (247 mg, 0.95 mmol)
in CH~CI,
( 10 mL) was added to a solution of DL-3-amino-3-phenylpropionic acid t-butyl
ester (252
mg, 1.14 mmol) and NEt3 (260 pL, 1.87 mmol) in CH?Cl, (10 mL) and.the mixture
was
stirred at room temperature overnight. The solvents were evaporated in vacuo
and the residue
was partioned between dilute HCI (1 M) and EtOAc. The organic phase was washed
with
brine, dried (Na~SO~) and evaporated in vaczro to give t-butyl DL-3-{[(1,4-
dichloro-7-
isoquinolinyl)carbonyl]amino;-3-phenylpropanoate (323 mg, 0.73 mmol). An
analytical
sample was prepared by crystallisation with i-Pr~O-hexane to yield a white
powder.
mp 153-155 °C.
IH (CDC13, 300 MHz) & 1.4 (9H, m), 2.9-3.05 (2H, m), 5.6 (1H, dt), 7.2-7.4
(5H, m), 7.9 (1H,
l S d), 8.2 (2 H, s), 8.4 ( I H, s), 8.7 ( 1 H, s) ppm.
LRMS 445 (MH+).
Anal. Found: C, 61.99; H, 5.07; N, 6.15. Calc for Cz3H??ChNZO3: C, 62.03; H,
4.98; N, 6.29.
Preparation 75:
N [(1,4-Dichloro-7-isoquinolinyl)carbonyl~-DL-aspartic acid a,(3-di-t-butyl
ester
c1 cI
teuozc~
CI ~ / i N ~ tBuO2C H ~ / i N
0 CI 0 CI
A solution of 1,4-dichloro-7-isoquinolinecarbonyl chloride (247 mg, 0.95 mmol)
in CHzCh
( 10 mL) was added to a solution of aspartic acid a,(3-di-t-butyl ester
hydrochloride (321 mg,
1.14 mmol) and NEt3 (330 pL, 2.37 mmol) in CH~CI~ (10 mL) and the mixture was
stirred at
room temperature overnight. The mixture was diluted with CHZC12 (30 mL),
washed with
dilute HCI (3x30 mL, 1 M), saturated aqueous Na~C03, brine, dried (MgS04) and
evaporated
in vacuo. The residue was crystallised from hexane to give, in two crops, N
[(1,4-dichloro-7-
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isoquinolinyl)carbonyl]-DL-aspartic acid a,(3-di-t-butyl ester (298 + 88 mg,
0.63 + 0.19
mmol) as a fluffy white solid.
mp I 12-114 °C.
IH (CDC13, 300 MHz) 8 1.45 (9H, m), 1.55 (9H, m), 2.9 (1H, dd), 3.05 (1H, dd),
4.9-5.0 (1H,
m), 7.45 ( 1 H, d), 8.25-8.35 (2 H, m), 8.45 ( 1 H, s), 8.75 ( 1 H, s) ppm.
LRMS 469 (MH+), 491 (MNa+), 959 (M~Na+).
Anal. Found: C, 56.20; H, 5.57; N, 5.88. Calc for C"H~~CI~N~OS: C, 56.29; H,
5.58: N, 5.97.
Preparation 76:
O-t-Suty1-N [(1,4-dichloro-7-isoquinofinyl)carbonyl]-DL-serine t-butyl ester
c1 c1
tBuO~
CI I / ~ N t8uD=C H I ~ i N
0 CI O CI
A solution of 1,4-dichloro-7-isoquinolinecarbonyl chloride (247 mg, 0.95 mmol)
in CHZCIz
(10 mL) was added to a solution of O-t-butyl-DL-serine t-butyl ester
hydrochloride (288 mg,
1.14 mmol) and NEt3 (330 p,L, 2.37 mmol) in CH~CIz (10 mL) and the mixture was
stirred at
room temperature for 3 h. The mixture was diluted with CH2Cl2 (30 mL), washed
with HCl
( 1 M), saturated aqueous NazCO3, saturated brine, dried (Na~S04) and
evaporated in vacuo.
The residue was crystallised from hexane to give O-t-butyl-N [(1,4-dichloro-7-
isoquinolinyl)carbonyl]-DL-serine t-butyl ester (378 mg, 0.86 mmol) as a white
solid.
mp 116-117 °C.
' H (CDC l3, 300 MHz) 8 1.1 (9H, m), 1.5 (9H, m), 3 .7 ( 1 H, dd), 3.9 ( 1 H,
dd), 4.8-4.9 ( 1 H, m),
7.15 (1H, d), 8.25-8.35 (2H, m), 8.4 (1H, s), 8.75 (1H, s) ppm.
LRMS 441 (MH+), 881 (M~H+), 903 (M2Na+).
Anal. Found: C, 57.15; H, 5.94; N, 6.27. Calc for C~,H26Cl2NzOd: C, 57.15; H,
5.94; N, 6.35.
Preparation 77:
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N-[( 1,4-Dichforo-7-isoquinolinyl)carbonyl]-DL-a-cyclopentylglycine t-butyl
ester
c1 c1
w w ~ w
CI I ~ ~ N tBuO.C N
0 CI ~ H 0 CI
A solution of 1,4-dichloro-7-isoquinolinecarbonyl chloride (148 mg, 0.57 mmol)
in CHZCh (6
mL) was added to a solution of DL-a-cyclopentylglycine t-butyl ester
hydrochloride {134 mg,
0.57 mmol) and NEt3 (200 pL, 1.44 mmol) in CH,Ch (5 mL) and the mixture was
stirred at
room temperature overnight. The reaction mixture was diluted with CHZCh (25
mL), washed
with dilute HCI (0.5 M), saturated aqueous Na~C03, brine, dried (Na~S04) and
evaporated in
vaczro. The residue was crystallised from i-Pr~O-hexane to give N [(1,4-
dichloro-7-
isoquinolinyl)carbonyl]-DL-a-cyclopentylglycine t-butyl ester (198 mg, 0.47
mmol) as a
white solid.
I H (CDC13, 300 MHz) 8 1.4-1.9 ( 17H, m), 2.3-2.5 ( I H, m), 4.8 ( 1 H, dd),
6.85 ( 1 H, d), 8.2-8.3
1 S (2H, m), 8.4 ( 1 H, s), 8.7 ( 1 H, s) ppm.
LRMS 423 (MH+), 440 {MNH.,+), 445 (MNa+), 845 (MZH+), 867 (MsNa+)
Anal. Found: C, 59.56; H, 5.72; N, 6.57. Calc for CZIH~4CIZNZO3: C, 59.58; H,
5.72; N, 6.62.
Preparation 78:
N Benzyl-N [(1,4-dichloro-7-isoquinolinyl)carbonyl]glycine t-butyl ester
c1 c1
y
CI ~ ~ N tBuO=C~N ~ N
0 CI ~ 0 CI
Oxalyl chloride (95 p.1, I .09 mmol) and then DMF (2 drops) were added to a
stirred
suspension of 1,4-dichloro-7-isoquinolinecarboxylic acid (130 mg, 0.54 mmol)
in CHZCh (10
mL), and the mixture was stirred for 30 min. to give a clear solution of the
corresponding acid
chloride. The solvents were evaporated in vacuo and the residue redissolved in
CH~Ch (10
mL). N Benzylglycine t-butyl ester hydrochloride (152 mg, 0.59 mmol) and NEt3
(200 uL,
1.44 mmol) were added and the mixture stirred at room temperature overnight.
The solvents
were evaporated in vacuo, and the residue was partioned between EtzO and
dilute HCI (1 M).
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The organic phase was washed with dilute HCl ( 1 M), aqueous Na~C03 ( 10 %, 20
mL),
saturated brine, dried (Na~SOa), and evaporated in vacuo. The residue was
extracted with hot
hexane, and the organic solution was decanted from the insoluble material. The
organic
solution was evaporated ire vcrcaro and the residue purified by column
chromatography upon
silica gel using hexane-EtOAc (80:20) as eluant to give N benzyl-N [( 1,4-
dichloro-7-
isoquinolinyl)carbonyl]glycine t-butyl ester (130 mg, 0.29 mmol) as an oil.
~ H (CDC13, 400 MHz) shows a l :2 mixture of rotamers 8 1.4 ( 1l3 of 9H, s),
1.5 (2/3 of 9H, s),
3.75 ( 1/3 of 2H, s), 4.1 (2/3 of 2H, s), 4.6 (2/3 of 2H, s), 4.85 ( 1/3 of
2H, s), 7.2-7.45 (5H, m),
7.9-8.05 ( 1 H, m), 8.2-8.5 (3 H, m) ppm.
LRMS 445 (MH+), 467 (MNa'), 889 (MPH''), 91 1 (M~Na+).
Preparation 79:
7-(Chloromethyl)-1,4-dichloro-isoquinoline
ci c,
El0=C I ~ NH ~ HO I a NH
0 0
LiBHa (530 mg, 24.3 mmol) was added portionwise to a stirred solution of ethyl
4-chloro-1-
oxo-1,2-dihydro-7-isoquinolinecarboxylate (3.06 g, 12.2 mmol) in THF (100 mL)
and the
mixture was stirred at room temperature for 1 h. The heterogeneous mixture was
quenched
with dilute HC( (2 M), and extracted with CH~Ch (2x100 mLj and EtOAc (5x100
mL). The
remaining solid was taken up in hot EtOH, and allowed to cool to yield a white
fluffy solid.
This solid was combined with the combined organic extracts, evaporated in
vacuo and
crystallised with EtOH to give 4-chloro-7-(hydroxymethyl)-1(2I~-isoquinolone
(2.19 g,
10.49 mmol) as a white solid.
mp 266-268 °C.
~H (DMSO-db, 300 MHz) s 4.6 (2H, d), 5.4 (1H, t), 7.4 f 1H, s), 7.7-7.8 (2H,
m), 8.2 (1H, s)
ppm.
LRMS 210 (Ml-I~), 419 (M~H+).
Anal. Found: C, 57.11; H, 3.81; N, 6.54. Calc for C,oH8C1N02: C, 57.29; H,
3.85; N, 6.68.
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c1 c1
I ~ ~1
HO / NH CI I / , N
0 CI
A solution of4-chloro-7-(hydroxymethyl)-1(21-isoquinolone (1.00 g, 4.77 mmol)
in POCI;
was stirred at 50 °C for 19 h. The reaction mixture was cooled in an
ice-bath, quenched by
the dropwise addition of dilute HCI (1 M) (reaction temperature < 30°C)
and then partioned
between water and EtOAc. The aqueous phase was reextracted with EtOAc and the
combined
organic extracts were dried (Na~SOa) and evaporated irz vacuo. The residue was
purified by
column chromatography upon silica gel using hexane-EtOAc (80:20) as eluant to
give 7-
(chloromethyl)-1,4-dichloroisoquinoline (870 mg, 3.53 mmol).
mp 139-141°C.
I H (CDC13, 400 M Hz) 8 4.8 (2 H, s), 7.9 ( 1 H, d), 8.1 ( 1 H, d), 8.3-8.4
(2H, m) ppm.
LRMS 241 [CI IH9ChON~H+; product of Me0 (from MeOH) substitution of C1]
Preparation 80:
N [(1,4-Dichloro-7-isoquinolinyl)methyl]-N methyl-DL-phenylglycine t-butyl
ester
c1 I ~ c1
/
CI I / i N tBu02C N I / i N
CI Me CI
7-(Chloromethyl)-1,4-dichloroisoquinoline (230 mg, 0.93 mmol) was added to a
solution of
N methyl-DL-phenylglycine t-butyl ester (248 mg, 0.96 mmol) and NEt3 (187 pL,
1.34
mmol) in CHzCl2 (5 mL), and the mixture heated at reflux for 15 h. [TLC
indicated
incomplete reaction]. The solvent was evaporated in vacuo, THF (30 mL) and
NEt3 (100 pL,
0.72 mmol) were added, and the mixture heated at reflux for 24 h. Although the
reaction was
still incomplete, the solvent was evaporated in vacuo, and the residue
purified by column
chromatography upon silica gel using hexane-EtzO (98:2) as eluant to give N [(
1,4-dichloro-
7-isoquinolinyl)methyl]-N methyl-DL-phenylglycine t-butyl ester (120 mg, 0.28
mmol) as a
colourless oil.
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The corresponding dihydrochloride salt vas prepared as follows: a solution of
the amine in
hexane was stirred with a solution of HCI in Et~O (0.5 M). The resulting white
precipitate was
collected by filtration and dried.
mp 120-122 °C.
IH (CDC13, 400 MHz) b 1.5 (9H, s), 2.25 (3H, s), 3.8 (1H, d), 3.9 (IH, d), 4.3
(1H, s), 7.3-7.4
(3 H, m), 7.45-7.~ (2 H, m), 7.95 ( 1 H, d), 8.15 ( 1 H, d), 8.2 ( 1 H, s),
8.3 ( 1 H, s) ppm.
l0 LRMS 432 (MH+).
Anal. Found: C, 56.62; H, 5.58; N, x.63. Calc for Ca3Hz.~CI~N~O,~HCI~H~O: C,
56.86; H,
5.60; N, 5.77.
f 5 Preparation 81:
N Benzyl-N [(1,4-dichloro-7-isoquinolinyl)methyl]glycine t-butyl ester
ci i
y ~1 y ~1
CI ~ N 1Bu02C~N
CI CI
20 7-(Chloromethyl)-1,4-dichloroisoquinoline (378 mg, 1.53 mmol) was added to
a stirred
solution of N benzyl glycine t-butyl ester (340 mg, 1.53 mmol) and NEt3 (256
p.L, 1.84
mmol) in THF (20 mL) and the mixture heated at reflux for 18 h. The solvent
was evaporated
in vacuo and the residue was purified by column chromatography upon silica gel
using
hexane-EtOAc (95:5 to 90:10) as eluant to give N benzyl-N.[(1,4-dichloro-7-
25 isoquinolinyl)methyl]glycine t-butyl ester (245 mg, 0.57 mmol).
The corresponding dihydrochloride salt was prepared as follows: a solution of
the amine in
Et20 was stirred with a solution of HCI in dioxane (0.5 M). The resulting
white precipitate
was collected by filtration and dried.
mp 140-143 °C.
~H (CDCl3, 400 MHz) 8 1.4 (9H, s), 3.3 (2H, s), 4.6 (2H, s), 4.8 (2H, s), 7.4-
7.45 (3H, m),
7.75-7.8 (2H, m), 8.3 5 ( 1 H, d), 8.4 ( 1 H, s), 8.45 ( 1 H, s), 8.8 ( 1 H,
d) ppm.
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LRMS 433 (MH+).
Anal. Found: C, 58.91; H, 5.38; N, 5.90. Calc for Cz3HZdCl~N~O~~HCI: C, 59.05;
H, 5.39; N,
5.99.
Preparation 82:
Na-[(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-NE-tent-butyloxycarbonyl-L-lysine
tart-butyl
ester
NHBoc
NHBoc
CI CI
\ \
O
II / ~N
tBuOZC ~ NH2, CISOz ~ ~ N tBuOZC ~ ~ O CI
CI
A solution of 1,4-dichloro-7-isoquinolinylsulphonyl chloride (250 mg, 0.84
mmol), NE-tert-
butyloxycarbonyl-L-lysine tent-butyl ester hydrochloride (286 mg, 0.84 mmol)
and
triethylamine (235 p.1, 1.69 mmol) in CHZCh (25 ml) was stirred at 23°C
for 3h. The reaction
mixture was washed witl3 water (2 x 20 ml), dried (MgS04) and concentrated in
vacuo to a
residue which upon trituration with hexane and then i-Pr~O gave Na [( 1,4-
dichloro-7-
isoquinolinyl)sulphonyl]-NE-tart-butyloxycarbonyl-L-lysine tart-butyl ester as
a white
powder (270 mg, 0.48 mmol)
'H (CDC13, 300 MHz) 8 1.1 (9H, s), 1.35-1.5 (13H, m), 1.6-1.85 (2H, m), 3.0-
3.2 (2H, m),
3.8-3.95 (1H, m), 4.45-4.6 (1H, br m), 5.35 (1H, d), 8.2 (1H, dd), 8.35 (1H,
d), 8.45 (1H, s),
8.8 (1H, d) ppm.
LRMS 562 (MH+), 584 (MNa~).
Anal. Found: C, 51.04; H, 5.96; N, 7.42. Calc for CZ~H33CIZN3O6S: C, 51.24; H,
5.91; N, 7.47.
Preparation 83:
Na [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-Ns-tart-butyloxycarbonyl-D-lysine
tart-butyl
ester
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NHBoc
NHBoc
CI
CI
\ ~ ~ O
CISO I / ~ N tBuO~C~~J-S / i N
tBuOZC NHZ z ~' ~I O CI
A solution of 1,4-dichloro-7-isoquinolinylsulphonyl chloride (250 mg, 0.84
mmol), NE-tert-
butyloxycarbonyl-D-lysine tent-butyl ester hydrochloride (286 mg, 0:84 mmol)
and
triethyiamine (23~ ~1, 1.69 mmof) in CH~CI, (25 ml) was stirred at 23°C
for 18 h. The
reaction mixture was concentrated in vacuo and the residue purified by colunm
chromatography upon silica gel using hexane-EtOAc (70:30) as eluant.
Crystallisation from i-
Pr~O gave Ncr-[( 1,4-dichloro-7-isoquinolinyl)sulphonylJ-N8-t-butyloxycarbonyl-
D-lysine
tart-butyl ester (285 mg, 0.51 mmol).
~H (CDC13, 400 MHz) 8 1.15 (9H, s), 1.2-I.SS (13H, m), 1.55-1.8 (2H, m), 3.05-
3.15 (2H, m),
3.85-3.9(lH,m),4.5-4.6(lH,m),5.4(IH,brd),8.2(lH,d),8.35(lH,d),8.45(lH,s),8.8
(1H, s) ppm.
LRMS 584 (MNa+).
Anal. Found: C, 51.18; H, 5.89; N, 7.33. Calc for C~qHg3CIZN3O6S: C, 51.24; H,
5.91; N, 7.47.
Preparation 84:
N [(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-L-glutamine tart-butyl ester
CONHZ CI C, ONHz CI
\ \
\ ~ 0
i N tBuO2C~N-S / ~ N
tBu02C ~ NHa CISOZ v ~ H 0 CI
CI
A solution of 1,4-dichloro-7-isoquinolinyl sulphonylchloride (250 mg, 0.84
mmol), L-
glutamine tart-butyl ester hydrochloride (201 mg, 0.84 mmol) and triethylamine
(235 p,1, 1.69
mmol) in CHZCl2 (25 ml) was stirred at 23°C for 18 h. The reaction
mixture was washed with
water (2 x 20 ml) and the solvent removed in vacuo to give N [(1,4-dichloro-7-
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isoquinolinyl)sulphonyl]-L-glutamine tert-butyl ester (309 mg, 0.67 mmol). An
analytical
sample was obtained following crystallisation from EtOAc.
1H (CDC13, 300 MHz) 8 1.05-1.15 (9H, s), 1.8-1.95 (1H, m), 2.1-2.25 (1H, m),
2.35-2.55 (2H,
m), 3.9-4.0 ( t H, m), 5.4-5.6 ( 1 H, br s), 5.6-5.8 ( I H, br s), 5.85 ( 1 H,
d), 8.2 ( 1 H, d), 8.3 5 ( 1 H,
d), 8.5 ( 1 H, s), 8.8 ( I H, s) ppm.
LRMS 462 (MH+), 479 (MNH,~~)
Anal. Found: C, 46.66; H, 4.54; N, 8.96. Calc for C,sH~,ChN305S: C, 46.75; H,
4.58; N, 9.09.
Preparation 85:
N-[(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-cyclopentylamin
o\s o ~
\S\CI ~ \H~
CI
CI /
N CI
N c1
1,4-Dichloro-7-isoquinolinylsulphonyl chloride (250 mg, 0.84 mmol) was added
to a solution
of cyclopentylamine (100 p.1, 1.0 mmol) and triethylamine (170 p1, I.22 mmol)
in CHZC12 (15
ml), and the reaction stirred at room temperature for 18 h. The solution was
diluted with
CHzCh, washed with 2M hydrochloric acid, saturated aqueous Na~C03 solution and
then
brine. This solution was dried (MgS04), and evaporated in vacuo, to give N-
[(1,4-dichloro-7-
isoquinolinyl)sulphonyl]-cyclopentylamine (250 mg, 0.72 mmol) as a white
crystalline solid.
~H (CDCl3, 300MHz) 8 1.4 (2H, m), 1.5-1.7 (4H, m), 1.85 (2H, m), 3.75 (1H, m),
4.6 (1H, d),
8.25 ( 1 H, d), 8.4 ( 1 H, d), 8.5 ( 1 H, s), 8.95 ( 1 H, s) ppm.
LRMS 346 (MH+)
Anal. Found: C, 48.68; H, 4.02; N, 7.97. Calc. for C,4H,4ChN~OzS: C, 48.71; H,
4.09; N,
8.11%.
Preparation 86:
1,4-Dichloro-7-(1-pyrrolidinylsulphonyl)isoquinoline
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o ~o
o\ ~o ~Ss
CI \ N
CI / CI ~ /
NCI
NCI
Pyrrolidine (96 mg, 1.35 mmol) was added to a solution of 1,4-dichloro-7-
isoquinolinylsulphonyl chloride (20 mg, 0.67 mmol) in CH~CI~ (5 ml), and the
reaction stirred
at room temperature for 72 h. The mixture was concentrated irr vacaro, and the
residual solid
triturated with water, filtered and dried. The crude product was purified by
column
chromatography upon silica gel using EtOAc-hexane (50:50) as eluant, and
recrystallised
from i-Pr,O, to give 1,4-dicliloro-7-(I-pyrrolidinylsulphonyl)isoquinoline (67
mg, 0.20
mmol) as a white solid,
~ H (CDC13, 300MHz) 8 I .8 (4H, m), 3.35 (4H, m), 8.25 ( I H, d), 8.4 ( I H,
d), 8.5 ( I H, s), 8.85
( 1 H, s) ppm.
LRMS : 331, 333 (MH*)
Anal. Found: C, 47.23; H, 3.60; N, 8.32. Calc. for C,3H,ZNZCIzO~S: C, 47.14;
H, 3.65; N,
8.46%.
Preparation 87:
tert-Butyl (2R)-1-[(1,4-dichloro-7-isoquinolinyl)sulphonyl]-2-
piperidinecarboxylate
o~~ ~o
G s \ SAN
HN HN Iri a
_ CI /
0~ 0 0
OH 0 N CI
Concentrated HzS04 (2.0 ml) was added to an ice-cold solution of 2-(R)-
piperidine carboxylic
acid (415 mg, 3.21 mmol) in dioxan (10 ml). Condensed isobutyiene (40 ml) was
carefully
added, and the reaction stirred at room temperature in a sealed vessel for 21
h. The reaction
mixture was poured into an ice-cooled solution of EtzO (100 ml) and 5N NaOH
(20 ml), the
mixture allowed to warm to room temperature with stirring, and then diluted
with water. The
phases were separated, the organic layer washed with 1N NaOH, then
concentrated in vacuo,
to half the volume, and extracted with 2N HC1. The combined acidic extracts
were basified
using 1N NaOH, and extracted with CHZC1~, the combined organic solutions dried
(MgS04)
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and evaporated in vaczro to afford tert-butyl 2(R)-piperidine carboxylate (210
mg, 1.14 mmol)
as an oil.
~ H (CDC13, 300MHz) 8 l .4-l .6 ( 1 I H, m), 1.75 (3H, m), 1.9 ( 1 H, m), 2.65
( I H, m), 3.1 ( 1 H,
m), 3.2 ( 1 H, m) ppm.
LRMS 186 (MH+).
1,4-Dichloro-7-isoquinolinylsulphonyl chloride (245 mg, 0.83 mmol) was added
to a solution
of tert-butyl 2(R)-piperidine carboxylate (153 mg, 0.83 mmol) and
triethylamine (170 p,1,
1.22 mmol) in CH~CI~ (IS ml), and the reaction stirred at room temperature for
18 h. The
solution was diluted with CH~CI~, washed with 2M hydrochloric acid, saturated
Na,C03
solution and then brine, dried (MgSO.~), and evaporated in vacuo. The residual
oil was
purified by column chromatography upon silica gel using an elution gradient of
pentane-
EtOAc (100:0 to 90:10), to give tent-butyl (2R)-I-[(1,4-dichloro-7-
isoquinolinyl)sulphonyl]-
2-piperidinecarboxylate, (290 mg, 0.65 mmol) as a colourless film.
~H (CDCl3, 400MHz) & 1.3 (9H, s), 1.55 (2H, m), 1.7-1.85 (3H, m), 2.2 (1H, m),
3.3 (1H, dd),
3 .9 ( 1 H, dd), 4.75 ( 1 H, d), 8. t 5 ( 1 H, d), 8.3 S ( 1 H, dd), 8.45 ( 1
H, s), 8.8 ( 1 H, s) ppm.
LRMS 462, 464 (MNHd+)
Anal. Found: C, 50.99; H, 4.95; N, 6.10. Calc. For C~gH2zCIzNZO4S; C, 51.24;
H, 4.98; N,
6.29%.
Preparation 88:
Methyl 4-{ [( 1,4-dichloro-7-isoquinolinyl)sulphonyl]amino}-1-methyl-4-
piperidinecarboxylate
°s
N CI ~ i
I N CI
H N- -CO H
a x HZN COzMe
A solution of 4-amino-1-methyl-4-piperidinecarboxylic acid (4.0 g, 15.6 mmol)
in methanolic
HCI (100 ml) was stirred under reflux for 20 h. The cooled mixture was
concentrated in vacuo
and azeotroped with CHZC12 to give an oil. This was dissolved in ice-cold
NaZC03 solution
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and extracted with CHZCh (2 x). The combined organic extracts were dried
(MgSO.,) and
evaporated iu vacuo to afford 4-amino-1-methyl-4-piperidinecarboxylate ( 1.6g,
9.3 mmol) as
an oil.
'H (CDCI;, 400MHz) 8 1.4-1.65 (4H, m), 2.1-2.25 (2H, m), 2.35 (3H, s), 2.4-
2.55 (4H, m),
3.75 (3 H, s) ppm.
LRMS 173 (MH+)
1,4-Dichloro-7-isoquinolinylsulphonyl chloride (I.0 g, 3.37 mmol) was added to
a solution of
methyl 4-amino-I-methyl-4-piperidinecarboxylate (700 mg, 4.0 mmol) and
triethylamine
(700 p,1, 1.0 mmol) in CH~CI~ (60 ml), and the. reaction stirred at room
temperature for 18 h.
The mixture was concentrated in vacaro, and the residue purified by column
chromatography
upon silica gel using an elution gradient of CH,Ch-MeOH-0.880 NH3 (97:3:0.3 to
95:5:0.5)
IS to give methyl 4-{[(1,4-dichloro-7-isoquinolinyl)sulphonyl]amino}-l-methyl-
4-
piperidinecarboxylate (700 mg, 1.62 mmol) as a white solid.
~H (CDC13, 400MHz) ~ 2.05 (2H, m), 2.25 (6H, m), 2.4 (2H, m), 2.55 (2H, m),
3.5 (3H, s),
8.25 ( 1 H, d), 8.4 ( I H, d), 8.5 ( 1 H, s), 8.85 ( I H, s) ppm.
LRMS 432, 434 (MH~)
Preparation 89:
N-[(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N-(methyl)cycloleucine ethyl ester
o~ i, o~ y
N ~ N
H CO,Et I CO=Et
CI / CI /
N CI N Ct
IC~C03 (238 mg, 1.73 mmol) was added to a solution of N-[(1,4-dichloro-7-
isoquinolinyl)sulphonyl]-cycloleucine ethyl ester (300 mg, 0.72 mmol) in DMF
(5 ml), and
the mixture stirred at room temperature for 40 min. Methyl iodide (47 p.1,
0.76 mmol) was
added and the reaction stirred for a further 30 min. at room temperature. The
mixture was
poured into water, extracted with EtOAc, and the combined organic extracts
washed with
water, then brine, dried (NazS04) and evaporated in vacuo. The residual yellow
solid was
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purified by column chromatography upon silica gel using EtOAc-hexane (20:80)
as eluant to
give N-[(1,4-dichloro-7-isoquinolinyl)sulphonyl]-N-(methyl)cycloleucine ethyl
ester (204
mg, 0.47 mmol) as a white solid.
~H (CDCI3, 400MHz) 8 1.25 (3H, t), 1.75 (4H, m), 2.1 (2H, m), 2.4 (2H, m) 3.05
(3H, s), 4.2
(2H,q),8.25(lH,d),8.35(lH,d),8.5(lH,s),8.9(lH,s)ppm.
LRMS 431, 433 (MH+)
Anal. Found: C, 50.12; H, 4.66; N, 6.43. Calc. for C,BHzoCI~N~OaS: C, 50.12;
H, 4.67; N,
6.49%.
Preparation 90:
4-Bromo-1-chloro-7-isoquinolinesulphonyl chloride
l5
0
°\ ~o
a s~
6r ~ / B ~ / ~ ~ a
-- ~ -~ a
o i o ~
i' \o I i
H H N CI
H
A suspension of isoquinolinol (10 g, 68.9 mmol) in MeCN (250 ml) at
50°C, was treated with
N bromosuccinimide (12.6 g, 70.8 mmol) whereupon almost complete solution
occurred
before a thick white precipitate was formed. After heating under reflux for 3
h, the reaction
mixture was cooled in ice and the solid filtered, washed with MeCN, and dried
to afford 4-
bromo-1-(2I~-isoquinolone (7.6 g, 34.0 mmol).
'H (DMSO-d6, 300MHz) 8 7.55 (1H, s), 7.6 (1H, m), 7.75 (1H, d), 7.85 (1H, m),
8.2 (1H, d),
11.55 (1H, br s) ppm.
LRMS 223, 225 (MH+).
4-Bromo-1-(2F~-isoquinolone (7.5 g, 33.0 mmol) was added portionwise to
chlorosulphonic
acid (23 ml, 346 mmol) and the resultant solution heated to 100°C for 2
%z days. After
cooling, the reaction mixture was poured carefully onto ice to give a white
solid which was
filtered, washed with water, MeCN, and Et~O and air-dried to give a cream
solid. 4-Bromo-1-
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oxo-1,2-dihydro-7-isoquinolinesulphonyl chloride 013.5 g) was immediately used
without
further drying.
mp >300°C.
I H (DMSO-d~, MHz) 8 7.45 ( I H, s), 7.7 ( 1 H, d), 8.0 ( 1 H, d), 8.45 ( 1 H,
s), 1 1.55 ( 1 H, br s)
PP~n.
To a stirred solution of 4-bromo-I-oYO-1,2-dihydro-7-isoquinolinesulphonyl
chloride 013.5
g) in acetonitrile (200 ml) was added portionwise POC13 (10 ml, 110 mmol). The
resultant
heterogeneous mixture was heated under reflux for 24 h, allowed to cool, and
the supernatant
decanted from the brown oily residues and concentrated to a solid. Extraction
of the solid
into EtOAc _gave, after solvent removal, a sticky solid which was triturated
with Et,O to
afford the title compound (3.83 g, 1 I .0 mmol) as a white solid.
IS
mp 120.5-121°C.
I H (DMSO-d~, 300MHz) 8 8.2 (2H, m), 8.5 ( I H, s), 8.6 ( 1 H, s) ppm.
Anal. Found: C, 31.21; H, 1.27; N, 4.08. Calc for C9H4BrCI2NO~S~0.25H20:
C, 31.29; H, 1.31; N, 4.05.
Preparation 91:
N [(4-Bromo-I-chloro-7-isoquinolinyl)sulphonyl]-D-proline tart-butyl ester
0
~ \S~ci / ~
N 1
Br / //
--~ Br
CO,Bu
N- -CI
N CI
4-Bromo-1-chloro-7-isoquinolinesulphonyl chloride (400 mg, 1.17 mmol) in
CHzC(z (20 ml)
was treated with (D)-proline tart-butyl ester hydrochloride (250 mg, 1.20
mmol) and
triethylamine (410 p.l, 2.94 mmol) and stirred at room temperature for 2 h.
The reaction was
diluted with CH~Ch, washed consecutively with water, 10% aqueous citric acid
and brine, and
then dried (MgSOd) and concentrated in vacuo to give an off white solid.
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This was purified by column chromatography upon silica gel eluting with EtOAc -
he;cane
(16:84) to give N [(4-bromo-1-chloro-7-isoquinolinyl)sulphonyl]-D-proline tent-
butyl ester
(350 mg, 0.74 mmol) as a white solid.
J I7lp 128.5-129.5°C.
~H (CDC13, 300MHz) b 1.1 (9H, s), 1.85-2.0 (3H, m), 2.2 (1H, m), 3.5 (2H, m),
4.4 (IH, dd),
8.3 (2 H, m), 8.6 ( 1 H, s), 8.9 ( 1 H, s) ppm.
LRMS 475, 477 (MH+):
Anal. Found: C, 45.41; H, 4.21; N, 5.83. Calc for Ci$H,oBrCIN,04S: C, 45.44;
H, 4.24; N,
5.89.
I ~ Preparation 92:
N-{[(4-Bromo-I-chloro-7-isoquinolinyl)sulphonyl]-N-[2-
(dimethylamino)ethyl]cycloleucine
ethyl ester hydrochloride
0 0~ so ~ 0 0
O~S,~ \ SAN CO,Et \ \S~N~O Et
CI z
Br / ~ Br ~ /
Br /
/ NCI / NMez
N CI N CI
Triethylamine (1.02 ml, 7.33 mmol) was added to a solution of 4-bromo-1-
chloroisoquinolinylsulphonyl chloride (1.0 g, 2.93 mmol) in CH~C12 (25 ml) and
the reaction
stirred at room temperature for 2 h. The reaction was washed consecutively
with 1N HC1,
Na~C03 solution, and brine, then dried (Na2S04) and evaporated in vacuo. The
residual oil
was crystallised from CH~C12-i-PrzO to give N-{[(4-bromo-1-chloro-7-
isoquinolinyl)sulphonyl]cycloleucine ethyl ester (380 mg, 0.82 mmol) as a
solid.
'H (CDCl3, 300MHz) 8 1.2 (3H, t), 1.6-1.8 (4H, m), 2.0 (2H, m), 2.15 (2H, m),
4.05 (2H, q),
8.25 (1H, d), 8.35 (1H, d), 8.6 (1H, s), 8.9 (1H, s) ppm.
LRMS 484 (MNa+)
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K~C03 (157 mg, 1.14 mmol) was added to a solution of N-{[(4-bromo-1-chloro-7-
isoquinolinyl)sulphonyl]cycloleucine ethyl ester (300 mg, 0.65 mmol) in DMF (5
ml), and the
solution stirred for 5 min. N,N-dimethylaminoethyl chloride hydrochloride (112
mg, 0.78
mmol) was added and the reaction stirred at room temperature for 36 h. The
reaction mixture
was partitioned between water and EtOAc, the layers separated, and the aqueous
phase
extracted with EtOAc. The combined organic solutions were washed with brine,
dried
(Na~SOa) and evaporated in vaczro. The residue was purified by column
chromatography upon
silica gel using CH,Ch-MeOH-0.880 NH3 (95:5:0.5) as eluant, to give a gum.
This was
dissolved in an Et~O-EtOAc solution, ethereal HCI added and the mixture
evaporated in
vaczro. The resulting solid was triturated with water, filtered and dried to
give N-{[(4-bromo-
1-chloro-7-isoquinolinyl)sulphonyl]-N-[2-(dimethylamino)ethyl]cycloleucine
ethyl ester
hydrochloride (90 mg, 0.16 mmol) as a solid.
1H (CDC13, 300MHz) 8 1.3 (3H, t), 1.65 (2H, m), 1.8 (2H, m), 2.15 (2H, m), 2.4
(2H, m), 2.9
(6H, m), 3.6 (2 H, m), 4.0 (2 H, m), 4.2 (2 H, q), 8.2 ( 1 H, d), 8.4 ( 1 H,
d), 8.65 ( 1 H, s), 8.80. ( I H,
s) ppm.
L,RMS 534 (MH+)
Anal Found: C, 44.17; H, 4.97; N, 7.24. Calc. for CZ,Hz~BrC1N30dS~HCI: C,
44.30; H, 4.96;
N, 7.38%.
Preparation 93:
Ethyl 3-{[(1,4-dichloro-7-isoquinolinyl)sulphonyl]amino}-2,2-
dimethylpropanoate
hydrochloride
0
o~ ~o o~s~ ~ /co2ec
~S ~ N
SCI / IH
CI /
CI /
/ N' -CI
N CI
The title compound was obtained as a white solid (86%) from 1,4-
dichlorosulphonyl chloride
. and ethyl 3-amino-2,2-dimethylpropanoate hydrochloride, following a similar
procedure to
that described in preparation 90.
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I H (CDC13, 300MHz) 8 1.25 (9H, m), 3.0 (2H, d), 4.1 (2H, q), 5.4 ( 1 H, t),
8.2 ( 1 H, d), 8.4
(l H,d),8.5(lH,s),8.9(IH,s)ppm.
LRMS 404, 406 (MH+)
Anal. found : C, 47.39; H, 4.44: N, 6.73. Calc. for Ct6HIgCI~N~OaS: C, 47.42;
H, 4.48; N,
6.91 %.
Preparation 94:
N-[(1,4-Dichloro-7-isoquinolinyl)sulphonyl]-N-[2-(tetrahydro-2Hpyran-2-
yloxy)ethyl]cyc.loleucine ethyl ester
0
oysr ~ ow io
COZEt \ SAN COZEt
CI
c1
NCI ~ OTHP
N CI
K~C03 (238 mg, 1.73 mmol) was added to a solution ofN-[(1,4-dichloro-7-
isoquinolinyl)sulphonyl]cycloleucine ethyl ester (600 mg, 1.44 mmol) in DMF
(10 ml), and
the suspension stirred at room temperature for 30 min. A solution of 2-(2-
bromoethoxy)tetrahydro-2H-pyran (J.C.S. 1948; 4187) (316 mg, 1.44 mmol) in DMF
(4 ml)
was added, followed by sodium iodide (10 mg), and the reaction stirred at
70°C for 23 h. The
cooled mixture was poured into water, and extracted with EtOAc. The combined
organic
extracts were washed with brine, dried (MgSO~), and evaporated in vacuo. The
residual
yellow oil was purified by column chromatography upon silica gel using hexane-
EtzO (75:25)
as eluant, azeotroped with CH~Ch and dried under vacuum to afford N-[(1,4-
dichloro-7-
isoquinolinyl)sulphonyl]-N-[2-(tetrahydro-2H pyran-2-yloxy)ethyl]cycloleucine
ethyl ester
(341 mg, 0.63 mmol) as a solid.
'H (CDC13, 400MHz) 6 1.3 (3H, t), 1.55 (4H, m), 1.65-1.8 (6H, m), 2.15 (2H,
m), 2.4 (2H,
m),3.5(lH,m),3.7(3H,m),3.8(IH,m),3.95(IH,m),4.2(2H,q),4.55(lH,m),8.35(2H,
s), 8.45 ( 1 H, s), 8.9 ( I H, s) ppm.
LRMS 545 (MH+), 562 (MNH4+)
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Anal. Found: C, 52.31; H, x.58; N, 4.84. Calc. for C24H3oC1aN~O6S~0.3H,0:
C, 52.33; H, 5.60; N, 5.09%.
Preparation 9~:
N-[(1,4-dichloro-7-isoquinolinyl)methylJcycloleucine methyl ester
N COZMe
CI / -~ CI
N' -CI
N CI
7-Chloromethyl-1,4-dichloro-isoquinoline (400 mg, 1.62 mmol) was added to a
suspension of
cycloleucine methyl ester (255 mg, 1.78 mmol), K~C03 (500 mg, 3.62 mmol) and
sodium
iodide (15 mg) and the resultant mixture heated to 75°C for 2'/ h.
After cooling, the reaction
mixture was poured into water and extracted with CH~CI~ (2 x 60 ml). The
organic extracts
were washed with water, brine, dried (Na~SO~) and concentrated in vacuo to
give an oil. This
was purified by column chromatography upon silica gel eluting with hexane -
EtOAc (85
15) to give N-[(1,4-dichloro-7-isoquinolinyl)methyl]cycloleucine methyl ester
(414 mg, 1.17
mmol) as a yellow oil.
A sample of this oil was treated with ethereal HCI, and the mixture evaporated
to give the
hydrochloride salt of the title compound as a white solid. '
1H (CDCI~, 300MHz) b 1.4-1.8 (5H, m), 2.0 (3H, m), 3.75 (3H, s), 4.15 (2H, s),
8.25 (3H, m),
8.5 ( 1 H, s), 10.5 (2H, br s) ppm.
Anal. found: C, 52.53; H, 4.99; N, 6.84. Calc. for C,~H,8C1zN20~~HCI: C,
52.39; H, 4.91; N,
7.19%.
Preparation 96:
(1-Aminocyclopentyl)(4-methyl-1-piperazinyl)methanone dihydrochloride
i /
\ I O O N N
~O~N OH _~O~N NJ Fi N N
H O ' \ H O 0
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I-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.49 g, 13.0
mmol) was
added portionwise to a cooled (4°C) solution of hydroxybenzotriazole
hydrate ( 1.49 g, I I.0
mmol) and 1-[(tort-butoxycarbony!)amino]cyclopentanecarboxylic acid (2.29 g,
10.0 mmol)
in DMF (IS 1111), and the mixture stirred for 30 min. N-Methylpiperazine (1.10
g, 1 I.0 mmol)
was added, the reaction stirred for 30 min. allowed to warm to room
temperature and stirring
continued for a further 17 h. The reaction mixture was evaporated in vacuo ,
and the residual
yellow oil partitioned between saturated Na~C03 solution and EtOAc. The layers
were
separated, the aqueous phase extracted with EtOAc, and the combined organic
solutions dried
(MgSOa) and concentrated in vaczro. The residual solid was pre-adsorbed onto
silica gel and
purified by column chromatography upon silica gel using an elution gradient of
CH~C1~-MeOH-0.880 NHS (97.5:2.5:0.25 to 90:10: I) and triturated with Et~O to
afford tert-
butyl 1-[(4-methyl-1-piperaziny!)carbonyl]cyclopentylcarbamate (2.31 g, 7.4
mmol) as a
crystalline solid.
mp 171-175°C
~H (CDC13, 300MHz) 8 1.4 (9H, s), 1.7 (6H, m), 2.25 (3H, s), 2.4 (6H, m), 3.65
(4H, m), 4.7
( 1 H, br s).
LRMS 312 (MH+)
A suspension of tart-butyl 1-[(4-methyl-1-
piperaziny!)carbonyl]cyclopentylcarbamate (2.2 g,
7.06 mmol) in EtOAc (120 ml) at 4°C was saturated with HCl gas, and the
reaction then
stirred for 4 h. The mixture was azeotroped with EtOAc, then dry Et20, and
dried under
vacuum to afford (1-aminocyclopentyl)(4-methyl-1-piperazinyl)methanone
dihydrochloride
(2.1 g) as a white solid.
mp 267-270°C (Decomp)
Anal. Found: C, 43.29; H, 7.99; N, 13.84. Calc. for C"H~,N30~2HC1~H~O: C,
43.71; H, 8.34;
N, 13.90%.
LRMS 212 (MH+)
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PCS9482 Compounds
As indicated above, suitable inhibitor compounds (agents) for use in the
present
invention are disclosed in GB patent application No. 9908410.5 (incorporated
herein
by reference) and in US patent application No. 09/546410 (incorporated herein
by
reference) and European patent application No. 00302778.6 (incorporated herein
by
reference) and in Japanese patent application No. 2000-104725 (incorporated
herein
by reference). It is to be understood that if the following teachings refer to
further
statements of inventions and preferred aspects then those statements and
preferred
aspects have to be read in conjunction with the aforementioned statements and
preferred aspects - viz pharmaceutical compositions either comprising an iUPA
and/or an iMMP and a growth factor (as well as the. uses thereof) or
comprising an
iUPA and an iMMP and an optional,growth factor (as well as the uses thereof).
~5
The PCS9482 compounds are pyridine derivatives useful as urokinase inhibitors,
and in
particular to 2-diaminomethyleneaminopyridine derivatives, alternatively named
as 2-
pyridylguanidine derivatives, useful as urokinase inhibitors.
20 The PCS9482 compounds are of the general formula (I) :-
Rz
R, Ra
CI)
1V R~
or a pharmaceutically acceptable salt thereof, or solvate of either entity,
25 wherein
R~ is H, halogen, CN, C~_6 alkyl optionally substituted by one or moi~e
halogen, or C1_6 alkoxy
optionally substituted by one or more halogen,
30 RZ and R3 are each independently H, halogen, Ci_6 alkyl optionally
substituted by one or more
halogen or C,_6 alkoxy, aryl, (C"alkylene)COZH, (C"alkylene)COz(C,_6 alkyl),
(C~
alkylene)CONRSR6, CH=CHR~, CH=CHCO~H, CH=CHCONRSR6, CH=CHSOZNRSR6,
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C CR', 0(C"; alkylene)OH, O(C"; alkylene)ORB, ORg, O(Cm alkylene)CONRSR6,
CH~ORs or
CH~NRSR6,
R~ is N=C(NH~)~ or NHC(=NH)NH~,
RS and RG are each independently H, Ci_~ alkyl optionally substituted by OH or
CO~H, het(C,_
~, alkylene) or aryl(C,_~, alkylene), or can be taken together with the
nitrogen to which they are
attached, to form a 4- to 7-membered saturated ring optionally containing an
additional
hetero-moiety selected from O, S or NR9,
and which ring is optionally benzo-fused,
and which optionally benzo-fused ring is optionally substituted by up to three
substituents
independently selected from OH, halogen, CO~H, CO~(C,_~ alkyl) and C,_r,
alkyl,
R' is C~_~ alkyl, aryl or het;
R~ is C,_6 alkyl, aryl, het, aryl(CHCO~H) or aryl(C,_~, alkylene);
R~ is H, C,.6 alkyl, or CO(C,_~ alkyl);
wherein "aryl", including the aryl moiety of the aryl(Ci_6 alkylene) group,
means phenyl
optionally substituted by up to three substituents independently selected from
halogen, C,_6
alkyl, (C"alkylene)CO~H, (C"alkylene)CO~(C,_6 alkyl), (C"alkylene)CN, C,_6
alkoxy, CN,
(C~ alkylene)CONRSR6, CH=CHCO~H, CH=CHCONRSR6, CH=CHSOzNR5R6, O(C";
alkylene)OH, CH~NRSR6, and O(C"; alkylene)CONRSR6;
"het" means an optionally benzo-fused 5- or 6-membered saturated or
unsaturated heterocycle
linked by any available atom in the heterocyclic or benzo-ring (if present),
which heterocyclic
group is selected from dioxolyl, furyl, thienyl, pyrrolyl, oxazolyl,
thiazolyl, isoxazolyl,
isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, thiadiazolyl, triazolyl,
tetrazolyl, pyridyl,
pyrimidinyl, pyridazinyl, pyrazinyl, and pyranyl,
and which optionally benzo-fused heterocycle is optionally substituted by up
to three
substituents independently selected from halogen, C,~ alkyl, (C"alkylene)CO~H,
(C,;
alkylene)CO~(C,_6 alkyl), (C"alkylene)CN, (C"alkylene)CONRSR6, CH=CHCOZH,
CH=CHCONRSR6, CH=CHSO~NRSR6, 0(Cm alkylene)OH, CHZNRSR6, and 0(Cm
alkylene)CONRSR6;
n is 0,1 or 2;
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m is t or 2;
and wherein the "C-alkylene" linking groups in the definitions above are
optionally
substituted by one or more Ci_~ alkyl;
with the proviso that R~, R' and R' are not all H;
hereinafter referred to as "substances of the invention".
''Alkyl" groups and the alkyl moiety of ''alkoxy" groups can be straight-
chain, branched or
cyclic where the number of carbon atoms allows.
"Halogen" means F, CI, Br or I.
The two definitions given for the R'' moiety are of course tautomeric. The
skilled man will
realise that in certain circumstances one tautomer will prevail, and in other
circumstances a
mixture of tautomers will be present.
Preferably Rt is H, CN, halogen or methyl optionally substituted by one or
more halogen.
More preferably R~ is H, CN, CI, Br or methyl.
Most preferably R' is C1 or Br.
Preferably R' is H, halogen, Cl_6 alkyl optionally substituted by one or more
halogen, aryl,
CH~ORB, (C"alkylene)CONRSR6, COzH or CHzNR5R6.
More preferably RZ is H, C1, methyl, phenyl, CONHCHZPh, CHzOPh, CHZNCH3Bn, or
pyrrolidinomethyl.
Most preferably RZ is H.
Preferably R3 is H, Cl, Br, CF3, aryl, (C"alkylene)COzH, (C~ alkylene)COZ(C,_6
alkyl), (C~
alkylene)CONRSR6, CH=CHR', CH=CHCOzH, CH=CHCONRSR6, CH=CHSOzNR5R6,
C=CR', O(Cm-alkylene)OH, O(Cm alkylene)ORB, ORB, O(Cm alkylene)CONRSR6,
CHZORB,
or CHZNRSR6.
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More preferably R3 is CH=CHCO,H, (2-carboxypyrrolidino)SO~CH=CH,
(cyanophenyl)CH=CH, or (carboxyphenyl)CH=CH.
Yet more preferably R3 is CH=CHCO,H, (2-carboxypyrrolidino)SO,CH=CH, (3-
cyanophenyl)CH=CH, or (3-carboxyphenyl)CH=CH.
Most preferably R3 is (2-carboxypyrrolidino)SO~CH=CH, (3-cyanophenyl)CH=CH, or
(3-
carboxyphenyl)CH=CH.
t0
l5
A preferable group of substances of the invention are those'wherein R~ is H,
CN, CI, Br or
methyl; R' is H,, CI, methyl, phenyl, CONHCHaPh, CH~OPh, CH~NCH3Bn, or
pyrrolidinomethyl; and R3 is CH=CHCO~H, (2-carboxypyrrolidino)SO~CH=CH, (3-
cyanophenyl)CH=CH, or (3-carboxyphenyl)CH=CH.
A yet more preferable group of substances of the invention are those in which
R~ is CI or Br;
R'' is H; and R3 is (2-carboxypyrrolidino)SO~CH=CH, (3-cyanophenyl)CH=CH, or
(3-
carboxyphenyl)CH=CH.
20 A further preferred group of substances of the invention are those
mentioned below in the
Examples and the salts and solvates thereof.
In the Synthetic Methods below, unless otherwise specified, the substituents
are as defined above
with reference to the compounds of formula (I) above. .
Where desired or necessary the compound of formula (I) is converted into a
pharmaceutically
acceptable salt thereof. A pharmaceutically acceptable salt of a compound of
formula (I) may be
conveniently be prepared by mixing together solutions of a compound of formula
(I) and the
desired acid or base, as appropriate. The salt may be precipitated from
solution and collected by
filtration, or may be collected by other means such as by evaporation of the
solvent.
Snthetic Methods
Method 1
Compounds of formula (I) can be obtained from the corresponding 2-
aminopyridine
derivative (II)
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by reaction with cyanamide (NHZCN) or a reagent which acts as a "NHC*=NH"
synthon such
as carboxamidine derivatives, e.g. 1H pyrazole-1-carboxamidine (M. S.
Bernatowicz, Y. Wu,
G. R. Matsueda, J. Org. Chef»., 1992, 57, 2497), the 3,5-dimethylpyrazole
analogue thereof
(M.A.Brimble et al, J.Che»r.Soc.Perkin Trans.I (1990)311), simple O-
alkylthiouronium salts
or S-alkylisothiouronium salts such as O-methylisothiourea (F.EI-Fehail et al,
J.NIed.Chem.( 1986), 29, 984), S-methylisothiouronium sulphate (S.Botros et
al,
J.Med.Chem..(1986)29,874; P. S. Chauhan et al, Ind J. Chenz, 1993, 32B, 858)
or S-
ethylisothiouronium bromide (M.L.Pedersen et al, J.Org.Chem.(1993) 58, 6966).
Alternatively aminoiminomethanesulphinic acid, or aminoiminomethanesulphonic
acid may
be used (A.E.Miller et al, Svnthc~sis (1986) 777; K.Kim et al, Tet.Lett.(1988)
29,3183).
Rz
R~
(II)
Hz
Other methods for this transformation are known to those skilled in the art
(see for example,
"Comprehensive Organic Functional Group Transformations", 1995, Pergamon
Press, Vol 6
p639, T. L. Gilchrist (Ed.); Patai's "Chemistry of Functional Groups", Vol. 2.
"The
Chemistry of Amidines and Imidates", 1991, 488).
2-Aminopyridines (II) may be prepared by standard published methods (see for
example,
"The Chemistry of Heterocyclic Compounds" Vol. 38 Pt. 2 John Wiley & Sons, Ed.
F. G.
Kathawala, G. M. Coppolq, H. F. Schuster) including, for example, by
rearrangement from
the corresponding carboxy-derivative (Hoffmann, Curtius, Lossen, Schmidt-type
rearrangements) and subsequent deprotection.
Alternatively, 2-aminopyridines may be prepared by direct displacement of a
ring hydrogen
using the Chichibabin reaction (A. F. Pozharskii et. al. Russian Chem.
Reviews, 1978, 47,
1042. C. K. McGill et. al. Advances in Heterocyclic Chemistry 1988, Vol. 44,
1)
2-Aminopyridines (II) may alternatively be prepared from the corresponding 2-
halopyridines
by direct displacement of a leaving group such as C1 or Br with a nitrogen
nucleophile such as
azide (followed by reduction), or by ammonia, or through Pd-catalysis with a
suitable amine
(such as benzylamine) followed by deprotection using standard conditions well-
known in the
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WO 01/49309 PCT/IB00/01935
art. Examples of such chemistry is outlined in "The Chemistry of Heterocyclic
Compounds"
Vol. 14, Pts. 2 and 3 John W iley & Sons, in particular Pt. 2, ( 1961 ), Pt. 3
( 1962), Pt. 2 -
supplement (1974) and Pt. 3 - supplement (1974).
2-Halopyridines may be prepared by methods well known in the literature. For
example, by
treatment of 2-hydroxypyridines (2-pyrimidinones) with halogenating agents
such as SOCI,
(Y. S. Lo. Et. Al. Syn. Conam., 1988, 19, 5~3), POC13 (M. A. Waiters, Syn.
Comm., 1992, 22.
2829), or POBr3 (G. J. Quallich, J. Org. Chenz, 1992, 57, 761). Alternatively,
2-
alkoxypyridines may be transformed to the corresponding 2-aminopyridines under
Vilsmeir-
Haack conditions such as POC13 + DMF (L-L Lai et. Al. J. Chem. Res. (S), 1996,
194). The
corresponding N-oxide may be treated with suitable halogenating reactions to
directly
produce 2- .halopyridines - e.g. POC13/PC15 (M. A. Waiters, Tetrahedron Lett.,
199, 42,
7575). Direct halogenation of the 2-position is possible iri the presence of
certain ring
substituents (M. Tiecco et. al. Tetrahedron, 1986, 42, 1475, K. J. Edgar, J.
Org Chenr.,
l5 1990, 55, 5287).
Method 2
Compounds of formula (I) can be obtained from the corresponding 2-
aminopyridine
derivative (II) as defined in Metliod 1 above, via reaction with a reagent
which acts as a
protected amidine(2+) synthon
(III):
.~ 2+ "
PNH~NHPi
such as a compound PNHC(=Z)NHP~, PN=CZ~NHP~ or PNHCZt=NP', where Z is a group
such as 0, or S and Z~ is a leaving group such as CI, Br, I, mesylate,
tosylate, alkyloxy, etc.,
and where P and P~ may be the same or different and are N-protecting groups
such as are
well-known in the art, such as t-butoxycarbonyl, benzyloxycarbonyl,
arylsulphonyl such as
toluenesulphonyl, vitro, etc.
Examples of reagents that act as synthons (III) include N, N'-protected-S-
alkylthiouronium
derivatives such as N, N'-bis(t-butoxycarbonyl)-S-Me-isothiourea, N, N'-
bis(benzyloxycarbonyl)-S-methylisothiourea, or sulphonic acid derivatives
ofthese (J. Org.
Chem. 1986, 51, 1882), or S-arylthiouronium derivatives such as N, N'-bis(t-
butoxycarbonyl)-S-(2,4-dinitrobenzene) (S. G. Lammin, B. L. Pedgrift, A. J.
Ratcliffe, Tet.
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Ll'tl. 1996, 37, 6815), or mono-protected analogues such as [(4-methoxy-2,3,6-
trimethylphenyl)sulphonyl]-carbamimidothioic acid methyl ester or the
corresponding
2,2,5,7,8-pentamethylchroman-6-sulphonyl analogue (D. R. Kent, W. L. Cody, A.
M.
Doherty, Tet. Lett., 1996, 37, 871 I), or S-methyl-N-nitroisothiourea
(L.Fishbein et al,
J.Arrr.Chenr.Soc. (1954) 76, 1877) or various substituted thioureas such as N,
N'- bis(t-
butoxycarbonyl)thiourea (C. Levallet, J. Lerpiniere, S. Y. Ko, Tet. 1997, 53,
5291) with or
without the presence of a promoter such as a Mukaiyama's reagent (Yong, Y.F.;
Kowalski,
J.A.; Lipton. N1.A. J. Org. Chenr., 1997, 62, 1540), or copper, mercury or
silver salts,
particularly with mercury (II) chloride. Suitably N-protected O-alkylisoureas
may also be
used such as 0-methyl-N-nitroisourea (N.Heyboer et al, Rec.Chinz.Trav.Pays-Bas
( 1962)81,69). Alternatively other guanylation agents known to those skilled
in the art such as
i-H-pyrazolc-1-[N,N'-bis(t-butoxycarbonyl)]carboxamidine, the corresponding
bis-Cbz
derivative (M. S. Bernatowicz, Y. Wu, G. R. Matsueda, Tet. Lett. 1993, 34,
3389) or mono-
Boc or mono-Cbz derivatives may be used (B. Drake.. Synthesis, 1994, 579, M.
S.
IS Bernatowicz.. Tet. Lett. 1993, 34, 3389). Similarly, 3,5-dimethyl-1-
nitroguanylpyrazole may
be used (T.Wakayima et al, Tet.Lett.( 1986)29,2143).
The reaction can conveniently be carried out using a suitable solvent such as
dichloromethane, N,N-dimethylformamide (DMF), methanol.
The reaction is also conveniently carried out by adding mercury (II) chloride
to a mixture of
the aminopyridine (II) and a thiourea derivative of type (III) in a suitable
base / solvent
mixture such as triethylamine l dichloromethane.
Rz
Rr
NHP'
(I~
The product of this reaction is the protected pyridinylguanidine (IV), which
can conveniently
be deprotected to give (I) or a salt thereof. For example, if the protecting
group P andlor P1 is
t-butoxycarbonyl, conveniently the deprotection is carried out using an acid
such as
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trifluoroacetic acid (TFA) or hydrochloric acid, in a suitable solvent such as
dichloromethane,
to give a trifluoroacetate (tritlate) salt of (I), either as the mono- or
ditriflate.
If P and/or P' is a hydrogenolysable group, such as benzyloxycarbonyl, the
deprotection could
be performed by hydrogenolysis.
Other protection l deprotection regimes include
nitro (K.Suzuki et al, Chc~nt.Pltctrnt.Btrll. (1985)33,1528, Nencioni et al,
.L~I~Ied.Chem.( 1991 )34,3373, B.T.Golding et al,
J.C.S.Chent.Conzm.(1994)2613;
l0 p-toluenesulphonyl (J.F.Callaghan et ai, Tetrahedron (1993) 49 3479;
mesitylsulphonyl (Shiori et al, Chent.Pltarnt.Bull.(1987)35,2698,
ibid.(1987)35,2561, ibid.,
(1989)37,3432, ibid., (1987)35,3880, ibid., (1987)35,1076;
2-adamantoyloxycarbonyl (Iuchi et al, ibid., ( 1987) 35, 4307; and
methylsulphonylethoxycarbonyl (Filippov et al, Syrt.Lett.(1994)922)
It will be apparent to those skilled in the art that other protection and
subsequent deprotection
regimes during synthesis of a compound of the invention may be achieved by
conventional
techniques, for example as described in ''Protective Groups in Organic
Synthesis" by T W
Greene and P G M Wuts, John Wiley and Sons Inc. (1991), and by P.J.Kocienski,
in "Protecting
Groups", Georg Thieme Verlag ( I 994).
Method 3
Compounds with the formula (I) can be obtained from compounds of formula (V):
Rz
R' R3
1V Z
where Z is a suitable leaving group such as CI, Br or OPh, by displacement of
the leaving
group by the free base of guanidine.
The free base of guanidine may conveniently be generated in situ from a
suitable salt, such as
the hydrochloride, carbonate, nitrate, or sulphate with a suitable base such
as sodium hydride,
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potassium hydride, or another alkali metal base, preferably in a dry non-
protic solvent such as
tetrahydrofuran (THF), DMSO, N,N-dimethylformamide (DMF), ethylene glycol
dimethyl
ether (DME), N,N-dimethyl acetamide (DMA), toluene or mixtures thereof.
Alternatively it
can be generated from a suitable salt using an alkoxide in an alcohol solvent
such as
potassium t-butoxide in t-butanol, or in a non-erotic solvent as above.
The thus formed free guanidine can be combined with the compound of formula
(V) and the
reaction to form compounds of formula (I) can be carried out at from room
temperature to
200°C, preferably from about 50°C to 150°C, preferably
for between 4 hours and 6 days.
Method 4
Compounds of the formula (f) when one.or more of R~-3 contains a hydroxy
group, may be
prepared from a suitably "protected" hydroxy derivative, i.e. a compound of
the formula (t)
l5 where one or more of R~-3 contains a corresponding "0P2", where Pz is a
suitable 0
protecting group such as O-benzyl. The benzyl group may be removed for example
by
catalytic hydrogenation using a palladium on charcoal catalyst in a suitable
solvent such as
ethanol at about 20°C and elevated pressure, optionally in the presence
of an excess of an acid
such as HCI or AcOH, or TEA, or by other known deprotection methods.
Suitable O-protecting groups and protection/deprotection can be found in the
texts by Greene
and Wuts, and Kocienski, supra.
Method 5
Compounds of the invention where Rz or R3 is or contains a carboxylic acid
group or
carbamoyl group can be made from the corresponding compound where the
subsfituent is or
contains a nitrile by full or partial hydrolysis. Compounds of the invention
where Rz or R3 is
or contains a carboxylic acid group can be made from the corresponding
compound where the
substituent is a carbamoyl moiety, by hydrolysis. The hydrolysis can be
carried out by
methods well-known in the art, for example those mentioned in "Advanced
Organic
Chemistry" by J.March, 3rd edition (Wiley-Interscience) chapter 6-5, and
references therein.
Conveniently the hydrolysis is carried out using concentrated hydrochloric
acid, at elevated
temperatures, and the product forms the hydrochloride salt.
Compounds of the formula (I) where one or more of R~, Rz or R3 is or contains
Cl or Br may
be dehalogenated to give the corresponding hydrido compounds of formula (I) by
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hydrogenolysis, suitably using a palladium on charcoal catalyst, in a suitable
solvent such as
ethanol at about 20°C and at elevated pressure.
Compounds of formula (I) in which one or more of Rz or R3 contains an amide
moiety may be
made via reaction of an optionally protected corresponding carboxy compound,
by coupling
with the amine of choice, e.g. via initial formation of the corresponding acid
halide or mixed
anhydride, and subsequent reaction with the amine, followed by deprotection if
appropriate.
Such transformations are well-known in the art.
Certain of the compounds of formula (1) which have an electrophilic group
attached to an
aromatic ring may be made by reaction of the corresponding hydrido compound
with an
electrophilic reagent.
For example sulphonylation of the aromatic ring using standard reagents and
methods. such
as fuming sulphuric acid, gives a corresponding sulphonic acid. This can then
be optionally
IS converted into the corresponding sulphonamide by methods known in the art,
for example by
firstly converting to the acid chloride followed by reaction with an amine.
Certain of the substances of the invention can be made via cross-coupling
techniques such as by
reaction of a compound containing a bromo-substituent attached to e.g. an
aromatic ring, with
e.g. a boronic acid derivative, an olefin or a tin derivative by methods well-
known in the art, for
example by the methods described in certain of the Preparations below.
Certain of the substances of the invention having an electrophilic substituent
can be made via
halogen/metal exchange followed be reaction with an electrophilic reagent. For
example a
bromo-substituent may react with a lithiating reagent such as n-butyllithium
and subsequently an
electrophilic reagent such as CO~, an aldehyde or ketone, to give respectively
an acid or an
alcohol
Substances of the invention are available by either the methods described
herein in the
Methods and Examples or suitable adaptation thereof using methods known in the
art. It is to
be understood that the synthetic transformation methods mentioned herein may
be carried out
in various different sequences in order that the desired compounds can be
efficiently
assembled. The skilled chemist will exercise his judgement and skill as to the
most efficient
sequence of reactions for synthesis of a given target compound.
EXAMPLES AND PREPARATIONS
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Melting points were determined using a Gallenkamp melting point apparatus and
are
uncorrected. Nuclear magnetic resonance data were obtained using a Varian
Unity 300 or
Varian Inova 400 spectrometer, and are quoted in parts per million from
tetramethylsilane.
Mass spectral data were obtained on a Finnigan Mat. TSQ 7000 or a Fisons
Instruments Trio
1000. The calculated and observed ions quoted refer to the isotopic
composition of lowest
mass. Reference to "ether" in this section should be read as diethyl ether,
unless specified
otherwise. "Ph" represents the phenyl group.
"Bn" represents the benzyl group. "Me" represents the methyl group. "TLC"
means thin layer
chromatography. "RT" means room temperature. "EtOAc" means ethyl acetate.
Other
abbreviations are standard and well-known in the art. Nomenclature has been
allocated using
the IUPAC NamePro software available from Advanced Chemical Development Inc.
Example 1: N'-(5-Met~l-2-~ ridin~)guanidine (I; R' = CH3Rz = R3 = H)
Trifluoroacetic acid (2 ml) was added with care to tert-butyl N [(tert-
butoxycarbonyl)amino][(5-methyl-2-pyridinyl)imino]methylcarbamate (111 mg,
0.32 mmol)
and the solution stirred at RT for 2 h, diluted with toluene and evaporated to
dryness. The
solid was azeotroped with methylene chloride, and recrystallised from methanol
to give the
trifluoroacetic acid salt of N'-(5-methyl-2-pyridinyl)guanidine as a cream-
coloured solid (32
mg, 0.1 mmol):
IH (8, d6-DMSO, 300 MHz); 2.2 (3H, s), 6.95 (1H, d), 7.7 (1H, d), 8.1 (1H, s),
8.35 (4H, br
s), 11.05 (1H, br s);
LRMS 151 (MH).
Other compounds of formula (I; R4 is N=C(NH2)2) prepared by the same method
are listed in
Table 1 below.
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
x
x
N
i
O
O
M
O
v 00
C/7
..; -...
, x .
~: .. N
yn 4~ ~ ..
~. M
w U' U
N
o_
z
U.U
~ ~ 0
x
~ N o0
W w ~ U o ri
U _-_
0
.l . ... .,
~ I.
i .~.._.___....
_...U _:_.. ..
'..
:'
. _ o , , ;; ! w:
_ I
I
j ~ i ,
x
~
_ ,.
_
I ~~.
i
I
U
V ' ~.I
'~~i
0
U .. . .
.
..
~
~ ~ ... . _ . . .
.
o a -- ~. .
x
W
335
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
e-:
~,
_
' ~
O w
, i
p ~
,.r
i s -~
x ~
. ..
~ 2 ..
.~
M .'~~ .
~
.._
Ii t
~
~
a
~ b o0
i
. . i i
i.
i
r
N ri
...,.L~i.-.,~~,
I )
~~ . '
I, I
~ : I',I
I '
I,~'I i
I
i ~,
;1 ,,I,
-:
._
f
yw:
. i V'
r _ i_.
i . _
.. i
C i_
I
i ...~'
i ~
,..
I
..,
..
M w _ _
tri- ,
..
_
U
336
CA 02395487 2002-06-26
WO
01/49309
PCT/IB00/01935
~ -s
,~
....~
. N
:: ~_x.~...~,,
x .
~
r
wr m
00
,-
i.M _ , .
~~.._ ....
'
. .' : .
,~,~
4-i I
.~ -
o .:
z
~n ~j U
. ~
i
!.
w
z U n
- .
..
; ~
, x,
- s l
-.
I
n
. i
i
_ ..-
;, .........
. .
. .. _
%, .
._.
O ..
_ ...
U ,
A,
337
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351
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Example 36: 3-((E)-2-t5-Chloro-2- ~(diaminomethylene)aminol-3-
R r~idinyl~ethenvl)benzoic acid
{I: R~ = C1; R'' = H; R' = E-CH=CH~3-C~H4-CO~H~
N'-5-chloro-3-[(E)-2-(3-cyanophenyl)ethenyl]-2-pyridinylguanidine (85 mg, 0.2
mmol) was heated to
retlux in conc. HC1 ( 1.5 ml) and acetic acid (0.5 ml) for 48 h. Solvent was
removed in vacuo and the
residue azeotropically dried with toluene to give a light brown solid which
was triturated with diethyl
ether to Qive 3-((E)-2-{5-chloro-2-[(diaminomethylene)amino]-3-
pyridinyl}ethenyl)benzoic acid as
an off white solid (65 mg, 0.2 mmol):
~ H (8, CF3C0~ D. 400 M Hz) 7.2 ( 1 H, d), 7.4 ( 1 H, d), 7.5 ( 1 H, t), 7.8 (
1 H, d), 8:1 ( 1 H, d), 8.3 ( 1 H, s),
8.45 ( 1 H, s), 8.55 ( 1 H, s);
LRMS 317, 319 (MH);
M. Pt. >275°C;
El. Anal. - Found: C, 49.36; H, 4.24; N, 15.51. Calcd for C,SH,3CINaO,.HCi +
2/3 water: C, 49:35; H,
4.23; N, 15.35.
Preparation l: t-Butyi (E)-3~2-amino-5-chloro-3-pyridinyl)-2-propenoate
A mixture of 3-bromo-5-chloro-2-pyridinamine (C. W. Murtiashaw, R.
Breitenbach, S. W. Goldstein,
a0 S. L. Pezzullo, J. Quallich, R. Sarges, J. Org. Chern., 1992, 57, 1930)
(8.56 g, 41.4 mmol), t-butyl
acrylate (12.m1, 82 mmol), tri-o-tolylphosphine (2.92 g, 9.6 mmol) and
palladium acetate (540 mg,
2.4 mmol) in triethylamine (130 ml) was heated in a sealed bomb to
150°C for 10 hours. The reaction
mixture was filtered, the residue washed with EtOAc and the combined filtrates
evaporated to a dark
brown oil. Purification by column chromatography upon silica gel using hexane-
EtOAc (7:3) as
?5 eluant and subsequent crystallisation from hexane at -78°C gave the
title compound as a bright yellow
solid (4.75 g, 18.6 mmol).
~H (8, CDC13, 300 MHz) 1.5 (9H, s), 4.7 (2H, br s), 6.3 ( 1 H, d), 7.45 ( 1 H,
d), 7.55 ( 1 H , s), 8.0 ( 1 H,
s);
LRMS 255, 257 (MH);
30 El. Anal. - Found: C, 56.55; H, 5.94; N, 10.91. Calcd for C,ZH,SC1N~02: C,
56.58; H, 5.94; N, 10.99.
Preparation 2: (E)-3 ~2-Amino-5-chloro-3-p ry idinylZ 2-propenoic acid
t-Butyl (E)-3-(2-amino-5-chloro-3-pyridinyl)-2-propenoate (2 g, 7.8 mmol), was
stirred in 3 ml of
35 trifluoroacetic acid at ambient temperature for 1 hour. The reaction
mixture was diluted with toluene,
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WO 01/49309 PCT/IB00/01935
evaporated to dryness, and the residue triturated with diethyl ether to yield
the title compound as
a pale yellow solid (1.89 g, 6.0 mmol).
~ H (8, d~-DMSO, 300 MHz) 5.0-7.5 (br s), 6.5 ( I H, d), 7.65 ( I H, d), 7.95
( 1 H ,s), 8.0 ( 1 H, s);
LRMS i 99, 201 (MH);
El. Anal. - Found: C, 38.41; H, 2.49; N, 8.87. Calcd for CBH~CIN,O~.CF;CO~H:
C, 38.42; H, 2.58; N,
8.96.
Preparation 3~ t-Butyl 3-(2-amino-5-chloro-3-pyridinyl)propanoate
To a solution of t-butyl (E~-3-(2-amino-5-chloro-3-pyridinyl)-2-propenoate
(500 mg, 2.0 mmol) in
ethanol ( 10 ml) at RT was added sodium borohydride (317 mg, 8.4 mmol)
portionwise and the
mixture stirred for 16 h. After the addition of water, the. ethanol removed in
vacuo and the mixture
extracted with diethyl ether. The ethereal extracts were dried over MgSO~,
evaporated to dryness and
purified by column chromatography upon silica gel using hexane- EtOAc (7:3) as
eluant to give t-
l5 butyl 3-(2-amino-5-chloro-3-pyridinyl)propanoate as a colourless oil (340
mg, l .3 mmol).
~H (b, CDC13, 300 MHz) 1.4 (9H, s), 2.5 (2H, t), 2.7 (2H, t), 4.6 (2H, br s),
7.2 (1H, d), 7.9 (1H, d);
LRMS 257, 259 (MH).
Preparation 4~ (E)-3-(~-Amino-5-chloro-3-pyridinyl -N methyl-2-propenamide
1-Hydroxybenzotriazole.H~O ( I 96 mg, I .4 mmol), methylamine.HCl ( 114 mg,
1.7 mmol), Hunig's,
base (1.58 ml, 9.1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.HC1
(555 mg, 2.8 mmol)
and (E7-3-(2-amino-5-chloro-3-pyridinyl)-2-propenoic acid.CF3CO2H (438 mg, 1.4
mmol) were
combined in DMF (5 ml) and stirred at RT for 16 h. The reaction mixture was
poured into water (50
ml), extracted with EtOAc (3 x 20 ml), and the combined organic extracts
washed with saturated
brine, dried over MgS04, and concentrated to a yellow solid. Trituration with
diethyl ether gave the
title compound (198 mg, 0.9 mmol).
1H (8, d6-DMSO, 300 MHz) 2.7 (3H, d), 6.25 (2H, br s), 6.45 (1H, d), 7.4 (1H,
d), 7.6 (1H, s), 7.87
(1H, br s), 7.9 (1H, s);
LRMS 212, 214 (MH);
M. Pt. 188-190°C;
El. Anal. - Found: 50.88; H, 4.81; N, 19.75. Calcd for C9H,oCIN30: C, 51.07;
H, 4.76; N, 19.86.
The following compounds of Preparations 5-9 were prepared similarly:
Preparation 5: 2-amino-N benz~isonicotinamide
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
The title compound was prepared from 2-aminoisonicotinic acid (L. W. Deady, O.
L. Korytsky, J. E.
Rowe, Aust. J. Chem., 1982, 35, 2025) and benzylamine:
' H (8, d~,-DMSO, 300 MHz) 4.4 (2 H, d), 6.05 (2 H, s), 6.8 ( 1 H, s), 6.8 ( 1
H, d), 7.2-7.4 (5 H, m), 8.0
( l H, d), 9.0 ( 1 H, br t);
LRMS 228 (MH); 455 (MPH).
Preparation 6' (E')-3- 2-Amino-5-chloro-3wridinyl)-N benzyl-2-propenamide
The title compound was prepared from (E~-3-(2-amino-5-chloro-3-pyridinyl)-2-
propenoic acid and
benzylamine as a yellow solid:
~H (~, db-DMSO, 300 MHz) 4.2 (2H, d), 6.25 (2H, brs), 6.6 (1H, d), 7.2-7.35
(5H, m), 7.45 (1H, d),
7.65 ( 1 H, s), 7.95 ( I H, s), 8.4 ( 1 H, br t);
LRMS 288, 290 (MH); 575, 577, 579 (MPH);
IS EI. Anal. - Found: C, 62.32; H, 4.93; N, 14.59. Calcd for C,SH,aC1N30: C,
62.61; H, 4.90; N, 14.60.
Preparation ~~-3-(2-Amino-5-chloro-3=pyridinyl)-I-(3-hydroxypiperidino)-2-
~ropen-1-one
The title compound was prepared from (E~-3-(2-amino-5-chloro-3-pyridinyl)-2-
propenoic acid and 3-
hydroxypiperidine as a white solid:
'H (b, d6-DMSO, 400 MHz) 1.25-1.55 (2H, m), 1.6-1.95 (2H, m), 2.6-3.15 (1H,
m), 3.2-4.3 (4H, m),
4.8-4.85 ( 1 H, m), 6.3 (2H, s), 7.1-7.2 ( 1 H, m), 7.5 ( 1 H, d), 7.9 ( 1 H,
s); 8.0 ( 1 H, d);
LRMS 282, 284 (MH); 563, 565, 567 (MZH).
Preparation 8' (~-3-(2-Amino-5-chloro-3-wridinylLN benzyl-N methyl-2-
propenamide
The title compound was prepared from (~-3-(2-amino-5-chloro-3-pyridinyl)-2-
propenoic acid and N
methyl benzylamine as a yellow solid following crystallisation from
diisopropyl ether:
'H (b, CDC13, 300 MHz) 3.1 (3H, s), 4.6-4.85 (4H, m), 6.8-6.85 (1H, m), 7.2-
7.45 (6H, m), 7.5-7.7
( 1 H, m), 7.95-8.05 ( 1 H, m);
LRMS 302, 304 (MH); 603 (MPH);
M. Pt. 106-109°C;
El. Anal. - Found: C, 63.33; H, 5.29; N, 13.67. Calcd for C,6H,6C1N30: C,
63.68; H,.5.34; N, 13.93.
Preparation 9' (~-3-(2-Amino-5-chloro-3-p ridinyll-1-morpholino-2-propen-1-one
354
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
The title compound was prepared from (~-3-(2- amino-S-chloro-3-pyridinyl)-2-
propenoic acid
and morphoiine as a yellow solid following crystallisation from isopropyl
alcohol and trituration with
diisopropyl ether:
' H (8, CDCI~, 400 MHz) 3.6-3.8 (8H, m), 4.8 (2H, br s), 6.8 ( 1 H, d), 7.SS (
I H, s), 7.6 ( l H, d), 8.0
S ( I H, s);
LRMS 268, 270 (MH).
Preparation IO' (E~-2-(~-Amino-S-chloro-3-pyridinyl)-N methyl-1-
ethenesulphonamide
A mixture of 3-bromo-S-chloro-2-pyridinamine (414 mg, 2 mmol), N-methyl ethene
sulphonamide
(266 mg, 2.2 mmol) and triethylamine (SSS p.1, 4 mmol), palladium acetate (18
mg, 0.08 mmol) and
tri-o-tolylphosphine (SO mg, 0.16 mmol) in DMF (0.S ml) in a TeflonT""
pressure vessel was
microwaved for 30 sec (full power), allowed to cool to RT and irradiated for a
further 30 sec. After
allowing to cool, the reaction mixture was diluted with water, extracted with
EtOAc and the organic
1S phase washed with saturated brine, dried over MgS04, and concentrated to a
brown semi-solid.
Purification by column chromatography on silica gel eluting with methylene
chloride - methanol
(9S:S), and then crystallisation from methanol gave the title compound (130
mg, 0.5 mmol):
'H (r5, db-DMSO, 400 MHz) 2.S (3H, s), 6.S (2H, br s), 6.95 (1H, br s), 7.1
(1H, d), 7.35 (1H, d), 7.95
( 1 H, s), 8.0 ( 1 H, s);
LRMS 248, 2S0 (MH);
M. Pt. 194-8°C;
El. Anal. - Found: C, 38.61; H, 4.04; N, 16.61. Calcd for C$H~oCINO~S: C,
38.79; H, 4.07; N, 16.97.
The following compounds of Preparations 11-1 S were prepared similarly:
2S
Preparation 11: S-Chloro-3-[(E1-2-phenylethenyl]-2-pyridinamine
The title compound was prepared from 3-bromo-S-chloro-2-pyridinamine and
stryene. Purification
by column chromatography on silica gel eluting with hexane - EtOAc (70:30)
gave an oil which
crystallised from hexane to give S-chloro-3-[(~-2-phenylethenyl]-2-
pyridinamine as a yellow solid:
'H (8, CDC13, 300 MHz) 4.S (2H, br s), 6.9 (1H, d), 7.0 (1H, d), 7.2-7.SS (5H,
m), 7.6 (1H, s), 8.0
(1H, s);
LRMS 231, 233 (MH);
El. Anal. - Found C, 67.33; H, 4.78; N, 12.00. Calcd for C,3H"ClN2: C, 67.68;
H, 4.81; N, 12.14.
3S
Preparation 12' S-Chloro-3-f(~-2-(4-methoxyphenyllethenyll-2-pyridinamine
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The title compound was prepared from 3-bromo-5-chloro-2-pyridinamine and 4-
methoxystryene.
Purification by column chromatography on silica gel eluting with hexane -
EtOAc (80:20) gave an oil
which crystallised from hexane to give a yellow solid:
~H (cS, CDC13, 300 MHz) 3.8 (3H, s), 4.5 (2H, br s), 6.75 (IH, d), 6.85-7.0
(3H, m), 7.4 (2H, d), 7.55
( l H, d), 7.95 ( l H, d);
LRMS 261, 263 (MH).
Preparation 13: ~-Chloro-3-[(E7-~2-pyridinyl)ethenyll-2-pYridinamine
The title compound .was prepared from 3-bromo-S-chloro-2-pyridinamine and 2-
vinylpyridine.
Purification by column chromatography on silica gel eluting with methylene
chloride.- methanol
(95:x) and repeated using hexane - EtOAc (70:30 to 50:50)-as eluant gave a
yellow solid:
~H (S, CDC13, 300 MHz) 4.7 (2H, br s), 7.05 (1 H, d), 7.2-7.35 (2H, m), 7.55
(1 H, d), 7.6-7.7 (2H, m),
IS 8.0(IH,d),8.6(lH,d);
LRMS 232, 234 (MH).
Preparation 14: 5-Chloro-3-[(E~-2-cyclohex lethenyll-2-pyridinamine
The title compound was prepared from 3-bromo-5-chloro-2-pyridinamine and
vinylcyclohexane.
Purification by column chromatography on silica gel eluting with hexane -
EtOAc (80:20) gave a pale
yellow oil. An analytical sample was prepared by crystallisation from hexane:
~H (8, CDC13, 300 MHz) 1.1-1.4 (5H, m), 1.5-1.8 (5H, m), 2.1-2.2 (1H, m), 4.5
(2H, br s), 6.0-6.2
(2H, m), 7.4 ( 1 H, d), 7.9 ( 1 H, d);
LRMS 237, 239 (MH);
EI. Anal. - Found: C, 65.85; H, 7.29; N, 11.84. Calcd for C,3H,~CIN~: C,
65.95; H, 7.24; N, 11.83.
Preparation 15: 3-[(~-2-(2-Amino-5-chloro-3-R rLidinyl)ethenyllbenzonitrile
The title compound was prepared from 3-bromo-5-chloro-2-pyridinamine and 3-
cyanostyrene.
Methylene chloride extracts of the crude reaction mixture were concentrated
and the desired product
purified by column chromatography on silica gel eluting with methylene
chloride - methanol (98:2) to
give a yellow solid:
~H (8, CDC13, 300 MHz) 6.4 (2H, br s), 7.2 (1H, d), 7.35 (1H, d), 7.55 (1H,
t), 7.7 (1H, d), 7.8-7.9
(3H, m), 8.15 (1H, s);
LRMS 256, 258 (MH);
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M. Pt. >275°C;
El. Anal. - Found: C, 65.49; H, 3.96; N, i6.2 l. Calcd for C,,,H,oCIN3: C,
6.76; H, 3.94; N, 1b.43.
Preparation 16' 3-[(E7-~-(1 3-Benzodioxol-5-yl)ethenv~-5-chloro-2-pyridinamine
S
A solution of 3-bromo-5-chloro-2-pyridinamine (318 mg, l.5 mmol), [(E~-2-(1,3-
benzodioxol-5-
yl)ethenyl](tributyl)stannane (250 mg, 1.7 mmol) (A. J. Bridges, A. Lee, C. E.
Schwatrz, M. J. Towle,
B. A. Littlefield, Biooy..Mec~ Chem., 1993, 1, 403), palladium acetate (19 mg,
0.08 mmol) and tri-o-
tolylphosphine (SO mg, 0.16 mmol) in DMF (0.5 ml) and triethylamine (0.5 ml)
in a teflon pressure
vessel was heated in a microwave (full power) for 20 sec, allowed to cool to
RT heated in a
microwave for a further 20 sec and then I min 20 see. After allowing to cool,
the reaction mixture
was poured into water (20 ml) and extracted with EtOAc (3 x 20 ml). The
combined extracts were
washed with water (2 x 20 ml), dried over MgSOd and concentrated.
Recrystallisation from EtOAc -
hexane gave the title compound as a brown solid (170 mg, 0.6 mmol):
~H (8, CDC13, 300 MHz) 4.55 (2H, br s), 6.0 (2H, s), 6.7 (1H, d), 6.8 (1H, d),
6.9-7:0 (2H, m), 7.05
( 1 H, s), 7.55 ( 1 H, s), 7.95 ( 1 H, s);
LRMS 275, 277 (MH).
Preparation 17: 5-Chloro-3-(2-phenylethynyl)-2wridinamine
A solution of 3-bromo-5-chloro-2-pyridinamine (414 mg, 2.0 mmol), phenyl
acetylene (225 mgr 2.2
mmol), copper (I) chloride (16 mg, O.16 mmol), triethylamine (555 p.1, 4.0
mmol) and
dichlorobis(triphenylphosphine)palladium (II) (32 mg, 0.05 mmol) in DMF (0.5
ml) in a teflon
pressure vessel was heated in a microwave (full power) for 30 sec, allowed to
cool to RT and reheated
for a further 30 sec. After cooling to RT, the reaction mixture was partioned
between water - EtOAc,
and the organic phase washed with sat. brine, dried over MgS04, and
concentrated. Purification by
column chromatography on silica gel eluting with methylene chloride-methanol
(99:1) and subsequent
crystallisation from hexane gave the title compound as a yellow solid (130 mg,
0.6 mmol):
~H (8, CDC13, 300 MHz) 5.0 (2H, br s), 7.3-7.4 (3H, m), 7.45-7.55(2H, m), 7.6
(1H, s), 8.0 (1H, br s);
LRMS 229, 231 (MH);*
M. Pt. 119-119°C;
EI. Anal. - Found: C, 66.53; H, 3.91; N, 12.00. Calcd for Ci3H9C1NZ + 1/3
water: C, 66.70; H, 4.13; N,
11.97.
Preparation 18: 5-Chloro-3-phenoxy-2-pyridinamine
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3-Bromo-~-chloro-2-pyridinamine (S20 mg, 2.5 mmoi), phenol (2.0 g, 2l .3
mmol). potassium
hydroxide (flakes, 85%, 600 mg, 9.1 mmol) and anhydrous copper (II) sulphate (
100 m~;, 0.6 mmol)
and dimethoxyethane (250 ~I) were heated together at 140°C for 2h,
allowed to cool and the mixture
poured into water (50 ml). EtOAc extracts (5 x 15 ml) were fltered through
eelite and extracted into
2N HC1 (4 x 10 ml). The combined aqueous extracts were basified with NaOH and
re-extracted into
EtOAc (3 x 20 ml), dried over MgSOa, and concentrated to a brown oil (230 mg).
Purification by
column chromatography on silica gel eluting with hexane - EtOAc (80:20) gave
the title compound as
a white solid (136 mg, 0.6 mmol). An analytical sample was prepared by
crystallisation from hexane:
~ H (S, CDC13, 400 M Hz) 4.7 (2 H, br s), 6.95 ( 1 H, s), 7.05 (2H, d), 7.2 (
1 H. t), 7.4 (2 H, dd), 7.8 ( 1 H,
t o s);
LRMS 22 i, 223 (MH);
El. Anal. - Found: C, 59.87; H, 4.1 l; N, 12.64. Calcd for Ci~H~CIN~O: C,
59.87; H, 4.1 l; N, 12.70.
Preparation 19' 3-(BenzyIoxX)-5-chloro-2-pyridinamine
1S
(This compound is known and synthesis by a different route is disclosed - J.
A. Bristol, I. Gross, R. G.
Lovey, Synthesis, 198 I , 971 ) .
The title compound was prepared from 3-bromo-5-chloro-2-pyridinamine and
benzyl alcohol using
the conditions of preparation 18:
20 1H (b, CDC13, 300 MHz) 4.65 (2H, br s), 5.0 (2H, s), 6.95 (1H, s), 7.3-7.45
(5H, m), 7.6 (IH, s);
LRMS 235, 237 (MH);
EI. Anal. - Found: C, 61.32; H, 4.70; N, 11.88. Calcd for C;ZH"CIN~O: C,
61.41; H, 4.72; N, 11.94.
Preparation 20: 2-[(2-Amino-5-chloro-3-pyridinXl oxy]-1-ethanol
The title compound was by the method of G. Mattern (Helv. Chimica Acta, 19.77,
60, 2062):
'H (8, CDC13, 300 MHz) 2.0 (1H, br s), 3.95-4.05 (2H, m), 4.I-4.2 (2H, m), 4.7
(2H, br s), 6.95 (1H,
s), 7.7 ( 1 H, br s);
LRMS I89, I9I (MH).
Preparation 2I' S-Chloro-3=(2-methoxyethox~_2-pyridinamine
The title compound was by the method of G. Mattern (Helv. Chimica Acta, 1977,
60, 2062):
'H (8, CDC13, 300 MHz) 3.4 (3H, s), 3.7-3.8 (2H, m), 4.1-4.2 (2H, m), 4.7 (2H,
br s), 6.95 (1H, s),
7.65 (1H, s);
LRMS 203, 20S (MH).
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Pr~aration 2w ~-f(~-Amino-5-chloro-3-~rridiny()oxy]-N benzylacetamide
The title compound was prepared by the method of P. Nedenskov, N. Clauson-
Kaas, J. Lei, H.-N.
S Heide, G. Olsen and G. Jansen (Acta ChemicaScandinavica, 1969, 23, 1791)
from 2-amino-5-chloro-
3-pyridinol (G. Mattern, Helv. ChimicaActa, 1977, 60, 2062) and N benzyl-a-
chloroacetamide. Sand
coloured solid:
~H (8, CDCI;, 400 MHz) 4.5-4.55 (2H, m), 4.6 (2H, s), 4.65 (2H, br s), 6.7
(1H, br s), 6.9 (1H, s), 7.2-
7.3 5 (5 H, m), 7.7 ( 1 H, s);
LRMS 292, 294 (MH);
EI. Anal. - Found: C, 56.92; H, 4.75; N, 13.93. Calcd for C,4H,aC1N30~ + 0.25
water: C, 56.76; H,
4.93; N, 14.18.
Preparation ~3~ Methvl 3-[(2-amino-5-chloro-3-pyridinyl)oxylmethylbenzoate
IS
The title compound was prepared using the method of Preparation 22 from 2-
amino-5-chloro-3-
pyridinol and methyl 3-(bromomethyl)benzoate to give a tan solid:
H (8, CDC13, 400 MHz) 3.9 (3 H, s), 4.7 (2H, br s), 5.1 (2H, s), 6.95 ( 1 H,
s), 7.5 ( 1 H, t), 7.6 ( t H, d),
7.65 (1H, s), 8.05 (1H, d), 8.1 (1H, s);
LRMS 293, 295 (MH); 585, 587 (MZH);
m. pt. 148-149.5°C;
EI. Anal. - Found: C, 57.08; H, 4.4I; N, 9.42. Calcd for Ci4Hi3CIN?O3: C,
57.44, H, 4.48; N, 9.57.
Preparation 24: 5-Chloro-3-(phenoxvmethyl)-2-pyridinamine
2S
Sodium hydride (80% in oil, 124 mg, 4.1 mmol) was added portionwise to a
solution of phenol (290
mg, 3.1 mmol) in anhydrous THF (15 ml). 5-Chloro-3-(chloromethyl)-2-
pyridinamine.HCl (R.
Herbert, D. G. Wibberley, J. Chem. Soc., 1969, 1504) (300 mg, 1.4 mmol) was
then added and the
reaction stirred at 50°C for 3 h. After removal of THF in vacuo, the
residue was partioned between
diethyl ether and 1N NaOH. The aqueous phase was removed, extracted with
diethyl ether and the
combined organics washed with saturated brine, dried over MgS04 and
concentrated to an oil which
upon triturating with hexane gave the title compound as a white solid (26S mg,
1.1 mmol):
~H (8, CDC13, 300 MHz) 4.85 (2H, br s), 4.9 (2H, s), 6.9-7.05 (3H, m), 7.25-
7.35 (2H, m), 7.4 (1H, s),
8.05 (1H, s);
LRMS 235, 237 (MH);
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Preparation 25: (2-Amino-3,5-dichloro-4- p ridinyllmethanol
To the hydrochloride salt of(2-amino-4-pyridinyl)methanol (J. M. Balkovec, M.
J. Szymoni~lca, J. V.
Heck, R. W. Ratcliffe; .I. Antibiotics, 1991, d4, I 172) (3.2 g, 20 mmol) in
conc HC1 (22 ml) at 75-
80°C was added, over 30 miss, hydrogen peroxide (1S% aq., 19.6 ml).
After stirring at 80°C for a
further 3 h, the reaction mixture was cooled in an ice bath and the resultant
yellow solid was removed
by filtration, triturated with diisopropylether and diethyl ether to give the
title compound as the
hydrochloride salt (3.3 g~ 14.3 mmol):
~H (8, db-DMSO, 300 MHz) 4.55 (2H, s), 8.0 (1 H, s);
LRMS 193, 195, 197 (MN);
M. Pt. 218 °C dec.;
EI. Anal. - Found: C, 31.36; H, 3.05; N, I 1.97. Calcd for C~,H6CI~N~O.HC1: C,
31.40; H, 3.07; N,
12.21.
IS Preparation 26: 3,5-Dichloro-4-(chloromethyl~pyridinamine
(2-Amino-3,5-dichloro-4-pyridinyl)methanoLHCI (2.2g, 9.6 mmol) was stirred in
thionyl chloride (5
ml) for 16 h at RT. The heterogeneous mixture was diluted with toluene, and
the white solid removed
by filtration, washed with diethyl ether and dried to give the title compound
as the hydrochloride salt
(2.27 g, 9.2 mmol):
~H (8, db-DMSO, 300 MHz) 4.75 (2H, s), 8.05 (1H, s);
LRMS 211, 213, 215, 217 (MH);
m. pt. 208-210°C;
El. Anal. - Found: C, 28.85; H, 2.48; N, 11.13. Calcd for C6HSC13NZ.HC1: C,
29.06; H, 2.44; N, 11.30.
Preparation 27: 3,5-Dichloro-4-(phenoxxmethyl)-2-p~ridinamine
Sodium phenoxide was prepared by the reaction of phenol (250 mg, 2.7 mmol) and
sodium hydride
(60% in oil, 106 mg, 2.7 mmol) in dry THF (IS ml) at RT. The solvent was
removed in vacuo and
replaced with dry DMF (10 ml), 3,5-dichloro-4-(chloromethyl)-2-pyridinamine
(300 mg, 1.2 mmol)
was added and the mixture heated to 60°C for 2.5 h. After cooling to
RT, the reaction mixture was
diluted with water (15 ml) and extracted with diethyl ether (4 x ISmI). The
combined ethereal
extracts were washed with water and saturated brine, dried over MgS04, then
concentrated to a solid.
This was crystallised from methylene chloride and hexane to give the title
compound as a white solid
(219 mg + 2"d crop of 45 mg, 1.0 mmol):
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'H (8, CDC13, 300 MHz) 5.0 (2H, br s), 5.2 (2H, s), 6.95-7.05 (3H, m), 7.25-
7.35 (2H, m), 8.05
( I H, s);
LRMS 269, 271, 273 (MH);
m. pt. 116-8°C;
EI. Anal. - Found: C, 53.10; H, 3.68; N, 10.33. Calcd for C,ZH,oCI~N~O + 0.1
water: C, 53.20; H,
3.79; N, 10.34.
Preparation 28: N~j(2-Amino-3,5-dichloro-4-p rL idinyl)methyl)-N benzyl-N
methvlamine
3,~-Dichloro-4-(chloromethyl)-2-pyridinamine.HCl (300 mg, 1.2 mmol) was
stirred in N
benzylmethylamine (3 ml) at RT for 48 h afterwhich the reaction mixture was
diluted with water to
give an oily precipitate. The supernatent liquor was removed, fresh water was
added and again the
aqueous layer removed. After trituration with hexane, the solid was dissolved
in methylene chloride,
dried over MgSO.~, and finally crystallised from methylene chloride - hexane
to give the title
compound as a fluffy white solid (190 mg, 0.6 mmol):
~H (c5, CDCIa, 400 MHz) 2.15 (3H, s), 3.6 (2H, s), 3.75 (2H, s), 4.85 (2H, br
s), 7.2-7.3 (5H, m), 7.9
( 1 H, s);
LRMS 296, 298, 300 (MH);
M. Pt. 124-6°C;
EI. Anal. - Found: C, 56.77; H, 5.10: N, 14.19. Calcd for C,4H,SChN3: C,
56.44; H, 5.04; N, 14.06.
Preparation 29' 3 5-Dichloro-4-(I-Ryrrolidinxlmethy~-2-Ryridinamine
The title compound was prepared using the method of preparation 28 using
pyrrolidine. White solid:
~H (8, CDC13, 400 MHz) 1.65-1.8 (4H, m), 2.6-2.7 (4H, m), 3.8 (2H, s), 4.9
(2H, br s), 7.95 (1H, s);
LRMS 246, 248, 250 (MH);
M. Pt. 98-101°C;
El. Anal. - Found: C, 48.77; H, 5.32; N, 16.98. Calcd for C,oH~3CI2N3: C,
48.79; H, 5.32; N, 17.07.
Preparation 30' t-butyl N [(t-butox~carbonyl~amino][~S-methyl-2-
pyridinYl)imino]methylcarbamate
To a solution of triethylamine (0.77 ml, 5.5 mmol) and 2-amino-5-picoline (200
mg, 1.8 mmol) in
methylene chloride (20 ml) at 0°C was added 1,3-bis(t-butoxycarbonyl)-2-
methyl-2-thiopseudourea
(0.59 g, 2.0 mmol) and mercury (II) chloride (0.55 g, 2.0 mmol). The reaction
mixture was stirred at
RT for 64 h, and the mercury residues filtered off for disposal. The filtrate
was chromatographed on
silica gel eluting with hexane - EtOAc (95:5 to 90:10) to give t-butyl N [(t-
butoxycarbonyl)amino][(5-
methyl-2-pyridinyl)imino]methyicarbamate compound (111 mg, 0.32 mmol):
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~H (8, CDC13, 300 MHz) 1.5 (18H, s), 2.3 (3H, s), 7.5 (1H, br d), 8.1 (1H, d),
8.2 (1H, br s), 10.8
( 1 H, br s), 1 l .5 ( 1 H, br s);
LRMS 351 (MH).
Other compounds of formula (IV; P and P~ are both CO~Bu') prepared by the same
method are listed
in Table 2 below.
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CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
~n
N
V1 ~ O L
~ n
V
\J
O ~O ....
Q ~ V1
~ m ,.~
t.
, ~n
Z U oho~
N
Fa'a
i. d
,~ M
r
ca
N C
w d
U
0
n.
U
a
n
J
c~
~ CC:,:r~ M
363
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WO 01/49309 PCT/IB00/01935
M
00 00 v~,Z ~ 2
-.c. --
-B .fl~ ,~
N o ~ ~
, Z x N ~ M
T ... ~ M Op
M Oy ~ ~
O O N m
O ? 00 O O I~ ~
M ~ ,]"~~~ ~j- ~ . z
-;
~; ~
U ~~ U
w x x 'r ~ r
.... r '. ~-- a ~ 0
Z
..
i
~
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.d .''x
Z ~ O
~ U z o
Cn N ~ x C' 0
00 L N
o ~c ~ U U ~o
~
0
~ ,
m x
x ~.
x x
M
m
364
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WO 01/49309 PCT/IB00/01935
m Z L ,; n
V WD v Z p '~ N I~ ~~,~
'/ '_'"
Z Z ~ ~ o tn .O ~ ~ t7
N tn ~ Z 2 x .-.
p N N b i~ ~' O 'r ~s ~ a ~ s..,
t!1~ ~_ .-. M 'C' v'1. O O~ ~ .O
O = " ,o O ~ O
'i"'rv ~ ~ j M ~ 00 .~..
~ N t ri w ~
~
r~ V7 L ~ V L ~ ~ v7 ~ ..~ .~ ~ n a
0 00 J. ~ ~ ~ Q 00 = ~ 0 00
r
~ ~r ~ . 'L7'r ~r ~ 'o ~ ~r 'r 'r ~
p
d' '~h'
p ~ ~ ~
O
t 1 i
1 t I
U U
~
U U r1 x x
Z
0
U
a,
L
U ~
!~'
M M M
365
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WO 01/49309 PCT/IB00/01935
N vp '-'
N ~ s.
1 n n i-;00
..-.N rs
N _ ~ 'fl_
N 'b
O ~ ~ x N
0 O yr n
O ~ O oo N
M ~ M ~ OO
n
Vf L ~ N ~1 /~ L
U T .o , -a -v .n
o_ox U 0~0~ x x
~ 0
a ~ r v ..rv ~.r'r
M
Z
d'n
v
T
_
T
x
b z V T m
0 N U ~ y l~
~ p M x N V1M
~ ~p ~ N O I~
rs"Tw o L U ~.,U vo
.-- .~ .-
w
0
00
L
Oa U
_ x x
0
N
U
U
II
x
U
U' W
M
366
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WO 01/49309 PCT/IB00/01935
".~~n ~; _ .a
~o ~ t wo
; N .-; '~..-:o '~ x
"O --~'O .-.
., ~, z x ...,
x ~..x ~ C'1W r
00 ~ ~. yrI\ ,-...O
O 'r O ~ 00
M ~ ~ r~v
M .~ O rJ ~ 't7
.. ~ 00 M ~ 'a 'T
.--: '~ N x x ~ 00
0 x -a ~n U
Z Z O o~0m (~ N
a
n
2 ~
,J ..
0
a~
cs d=
c~
ft d-
-o O -v O
o Z U ~ z N z
U x z U ~ ~i c~ x z U ~ N N
~ m
M ~ n, W O .,~ ~ pp W~1N M
1 rT.en, (~N ~ ~O 't~-
U .-.~. U
.,N, l~ ~ U .'o'~,~.-. c ~ vp~ ,j 'V,
c
co t~
0
U U
M x
x
0
U
p.
x
N
z
O O
U U
x
w
x x
U W U
c~ o
M '~'
367
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
_ ~ ~ _
i .~ c'?l~ V~ y n E N
N t~ t~ 00 v N zn _. _
N ~ ~ .-.'y'i ~ V1
d 'O .G N N ~ N O I~ 00
x ~ _ ~ ~ oo~ ~, _
O ~ W r ~ ; ~ ~; 'Q 'TJ
= ~,
~ v1 N .., O crx M
M C ~ ~ O -~ O r., ~ i r
l\ 00 M ~ W r wr
1 r~ ~ ~ ~ '~ ~ .D ~O
~ t~ ~ '~ "d ~ O ~O
~ ~ x x x N ~ o~o. Z
a ~O ve y s a o~
v
N
M N
tn v1
O d'
M N
b
U = Z U U ~ ~ U o,s
~ o v _ U ' v,.
W G _ b O M c
-- x W O N ~ ' O x O ~.
n U ~ y
; ~to U ~, x = w ~ ~ U ~ U '~'
w
N
i ~
U U
z x
0
..
U "T_ , o
U M
....:~
z o .
U
x .
V
x
x o
L
W U ' U -a
. W .~
.-r N
368
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
N
m M Z ~ ~' ~
i
~
N CV wr ~ ~ ~ n o=0,-,x O~
M ~ tn 00T N wr .-.N
a~ ~
p n' ~ (V ~ o p b ~ .~D
M M B
~ N ~ ~ v r~ = T..n
~ ~~ ~n _
CT
O pp a. fn ~ OO ~O v ~' N
0 0 ~ Z '~ o
L '. t~ o0
~
N
.-.
O
_
tn
'O
v U ~ Oi
~ z
O N U U N N
O T
M U M
~ ~ ~ -
t,im p c,.. U ,~ ~O
U U
~a
0
U O
~ :
c, ~ .
. o.
0
Q O
z ~ . z
U w U
o
w U U W U
369
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
M
V1 ~
!1 1 ~ ~" l .
1 00 ~" (n O ~
-.p ~ ~'~ O ~
.~ z _
N w .. 'C7Z N
z M z 2 ~:
N ~ _ N
CV ~ ~ ~ M t~ - y
~; " ~ ~. ~n
N n n 1
CT 'B "0 a N o0 ~
o ~ ~ z ~ ~ ~ ~ ~ o z
U ...,:. '. a U ~. t~ oo .:.
ci z
O M
h
'L7 0
v z ~ z ~ z
U U o 00
0 0
z U
~,
Cc,vO vc - U 'v z
~ ~-
U U
z z
0
z
z s
0
x
z
U
U
W U W
T w o
370
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
as ~ os o ~ -; cu
0 00
n ' ~ ~ o .'n ~?~ oo ..
.... ~
~' -v 'a ~- z ~ ~ oo '~
z -~ N '. ~ z -
w r1 x ~ T ~ ~ N
'o N
O o0 N o0 ~-. Op.-.
OMM I~ I~ 00 ~ n ~
L n
M VI i1 i1 r'v ~~UI c ,
U -o -n -n ~ ~ Z
U ~
D Zo ~ = ~ 2 ~ ~o ~ '
:r U .... '. r
o L
r. ,.
y, w
v
N
O
d.
d'
O
m V
'b
U -r Oi
~ z z
a U ~ ~ o
M U n
~ x o
.--m.. ~i N
rs.; vc ~ U U ~ .-,.
U U
0
U
a,
'. :r
x
U
U x -~
U ~ U .>,
W ~ W c.
371
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
.-: Z
,r~ .~ '~ o~ 'n ~ N ran~ v
-p ~ in I~ ~ 'b
.- o ~ ~ ~: z ~; z
M 0
~ o x ~ ~ M O
N ~ '~n ~
.."~..~ .- ~ ~ ~ a I~ Q~ O i.
O N ~ _ ~~ O 4 ~_
~ M v ~ <f'~ (s
U ~ ~O M ~ ,~'~"~ ran
"" ~ ~ '. ~. ~ U -a ....r
x ,~,.~ '
N N I~ N ~ ~ Z N O~
s 'r ~O ~ t~ 00 fl ~ a 'r I~ h 00
z
x
b o
U 'T
o z
U U c~
~ M O
O 00 o v, x U t~ t~
ps M '' ,
x
s,~n t~ - N :~ -
w U
U U
x x
0
U
n, X T
a~ ' x
~
V m o
x x v
c
U U
C
W W ~ c
o~ o
et N
372
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
~ .-:
, 'O "~ ,~Z ~ M ~
~ w 1
.-r:.. .:..~ v ~-w - .D
.r v 2 a ~, _ ~ c_1 .
N .
_ c~ "' v '_~ ~n T~d-
r1
O ~ -p ~ ~ r~ Z O M ~.;Z
'p W n 'O O
C' TJ .. v M Z
,n W n U a
00 0 -. m Caoo ~
t~ 000o U ... ri
Z
'.
O M
O
n
d'
'D
U V .n + U ~ U 2 U U
~-"z ~ V - ~, z O U
' ~ G z ~ O N o ~ U
0 Os N ~ x O ~ v1 Oyo0 . I~ 1 01
a, , ~ ~ + ~! a~ ~ o -: N .,
cc.~n ~; U .~Gl o ~nv; ti,vov; .-.. U -~ '
.T.
~-
u,
r,
U_ U
Z Z
z
U
U
II~ a,
:
, UI
W U
N
373
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
s. M
n ~ (' z
m E O Z ~n N .
~
~ 00 V ~ ~ N z n
N r~ V? ~ a
Z M ~_ ~
~ ~ v
O A
O _ .T.,~..~. ~ ~1 ~ O L
N -~ O N -fl
a ' ,.~
= L ., s V1 l1
~ d: = N z o0
U Z v; s, U
00 M J~ n ~ ~ ~ ~ ~t r;
v r ,..'
v l~ a C yr v.. .. (~ ~ ._.
s~ n
z .z
M
0
U 'T
U 2 ~ Z oN,,Z
U U ~ M
~ ~ L
0 N ~1
00 ~D z y ~
v~~wc - U '~,z
U U
2
,;:
_r
N
r z
c. U
O O
..
374
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
I~ v~f ~ M V'1
N ~ N ran,i.
r I~ x ~ N
N ,.~" .T.Z '~ ~ w
VI V'1 ~ M ~ 00
M
O ~O .L ~ L O Vl ~ .-.r
p ~. ~ O ~
~. M ..rn M M - r~
v~ ~ ~ ~n
N V'1L -~,N "' t.
U ,..,.; ~ - .- U ",_;
D o0 ~: Z O D o0 a~ N
'-
~r ~r C 'r ~r 'o M ~ i
n
Z
a
M
M
V
V1
M ~'
'a O -n O
U -r l~ z U z O~.-.rr
U Z z_ o U ~ z z
'T'~ U (~ N . Z U N t~~,
. 2~ ~ ~ ~ ~ U . 2 0 0
W .~ N _~ U ~t a~ 'r't
e ~ U '.~~ M- c~ N w U +.oU vo
v, ='
vo
~
0
M
N
U U
,:.. .~,
O O
N N
U U
N N
U U
O O
~n ~n
375
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
1 Ly In fill
1
C c vG op ~ V, ~n
.... w -
"' y
M ~ V r wr
c W n _ ~~ N fy
L - .L O ~n = ~ t~ 00 .
.D ~ M I
v ~ 'J _ n
O ~O
V'1 ~ O ~ r1 .:.,
Op ~ .... O ' ~
M ~ "' M M ~ .r~~a
T I ~ v n I~
U ~ ~ v; .r ~ 2 ,_
' c ~ " c-~ U :~ . -
--T. t~ n 00 ~ ~ M ~ rs
U
T. Z
M M
M M
N
'-' z N z ~ ~ ~ ~ z
~j--~~ '~ - ~ ~
~. U U va v U U
_w N " O O ~ o n 1 0
Q1 h O Z ~ ~O j O M Z V1
s ~ L ~ U . o 00~ N U d;
~ v, = U ',o~ .M-ti.W, ~n .. U
O M
00 i
N
00
M
U U
T
N
w
O O
I
U M
N N
z
z U
U
O O U
m ~n
376
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
w
'~. o Z ~ :.~
c,
C N T ....
O M ~ "' .T,::
a ~ ~ W O N
. C
V1 l~ VI ~ M
U ~ ~ -a ~ ~ U T
Z ~ a ~ p
U ~ 0
r ~ c~ ~ U :.
M
~s
~1' d'.T~.
d'a.i
o -~
z
U = UU U U x z U ~ v
N , -i U N N
x = ~ 00~ -~ O
O Y U ~O ; o r- ..'~~~ cy 0 O
0 '~ '' ~ ~ -. O ~f'~ N -r ..~ ~ N
t1.v, ~O -~ U '.:,.T..- rt.~t W . U O W vw n
0
M
0
V U
Z ~U
..
O
l
n
U U
m
377
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
T ~
W O N ,-,
t/1 L
.D .r ~ V1
M .~
z N y t~
r ""; V z y
~r .... _ a
M_ N ~
O V1 00 L O V1
M N ~ '~ M 00 tin
r~n " ~ "' ~ Z N L
U '_-' ~ U ?..: o0
N M %
00 ,..~.~ ~ . ..
.-.
_T t!1
a
M
N ~'
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z, ~ z
U z U U c~
M z ~ ,~
L
c-..,~ V~,'..Mr~ U O Z ' .
~o
0
U U
z ,=
U O a,
U U
N
378
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
~. M
~O N ., ~ i
v1 M ~ v~1 CI'.1.. ~ N
y r
.. ~ 'i L O
N ~ N ~ O ~ _'
N =
y p~ ~O S~ N ~
O x .. ~ D
M N O
M 0 ~..~Z O~ L
M c~ oo ~
~ ~ ~ r W s r%o
v~ ~n ~2iv, U c
Ll = " ' ~ ~ C~ ..
L 0
s~ oo - ~ ': ~
N
C~
p" ' O
~ G1
tn d
T
-zs
U .o ~ ~ U ~ V ~ O
-r~z U ~ U, o . U
~ o w ~ + U
~ c_oc "~ U o ~ rn '~. o
W o ~ U O .W. f~ W e ~ U O
M ~'
U U
= ~'
U ~~ ~ U~ U N z U
U U
379
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
-.
U
0oro
G~
vi
y
O
U
y
U
v U
C
C :3
J
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L
0
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y v a\
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cv-7
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6
C
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L
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tC.'O
'a
-~3 ~ ~ ~' -~o~.
U U ~'C7 ~C E-.L~
c~C.~ U 'L7N 4~,
'r~ 'r 'r ~ a
380
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
.c
a o 0
a~
u, O L
> s ~c O
z D _O U tn
O L N O
B O O O ""'CO O
O (~O N O ~ ~ C
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a~ .am c a .a o
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'. N
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~ O 'O (f3~
w ~ ~ O wN.,~ L (B
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~ d ~ ~ v0-~ O
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C cQ o
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381
<IMG>
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<IMG>
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
Preparation ~4;
methyl 4-[4-(4-{3-(2-[(tert-butoxycarbonyl)amino]ethoxy)phenyl}-3-
methylphenyl)-
piperidin-I-ylsulphonyl]-tetrahydro-2H-pyran-4-carboxylate;
methyl 4-[4-(4-{3-(2-aminoethoxy)phenyl}-3-methylphenyl)-piperidin-I-
ylsulphonyl]-
tetrahydro-2H-pyran-4-carboxylate;
Preparation 61;
methyl 4-[4-(4-{3-(2-oxoethoxy)phenyl}-3-methylphenyl)-piperidin-i-
ylsulphonylJ-
tetrahydro-2H-pyran-4-carboxylate; and
methyl 4-[4-(4-{3-(2,2-diethoxyethoxy)phenyl}-3-methylphenyl)-piperidin-1-
ylsulphonyl]-
tetrahydro-2H-pyran-4-carboxylate,
4-[4-(4-{ 6-[2-hydroxyethoxy] pyrid in-2-yl }-3-methylphenyl)piperidin-I -
ylsulphonyl]tetrahydro-2H-pyran-4-carboxylic acid;
methyl 4-{ [4-(4-{6-[2-hydroxyethoxy]pyridin-2-yl}-3-methylphenyl)piperidin-1-
yl]sulphonyl } tetrahydro-2 H-pyran-4-carboxylate;
methyl4-[4-(4-{6-[2-benzyloxy]ethoxypyridin-2-yl}-3-methylphenyl)-1,2,3,6
tetrahydropyridin-1-ylsulphonyl]tetrahydro-2H-pyran-4-carboxyfate; and
methyl 4-[4-(4-bromo-3-methylphenyl)-1,2,3,6-tetrahydropyridin-I-
ylsulphonyf]tetrahydro-
2H-pyran-4-carboxylate,
N-Hydroxy 4-{[4-(4-{6-[2-hydroxyethoxy]pyridin-2-yl}-3-methylphenyi)piperidin-
1-
yl]sulphonyl}tetrahydro-2H-pyran-4-carboxamide,
N-Hydroxy 4-{[4-(4-{6-[2-hydroxyethoxy]pyridin-2-yl}-3-inethylphenyl)piperidin-
I-
yl]sulphonyl}-piperidine-4-carboxamide,
N-Hydroxy 4-{[4-(-4-{6-[2-aminoethoxy]pyridin-2-yl}-3-methylphenyl)piperidin-I-
yl]sulphonyl}tetrahydro-2H-pyran-4-carboxamide,
and pharmaceutically acceptable salts thereof, and solvates thereof.
Moreover, persons skilled in the art will be aware of variations of, and
alternatives to, those
processes described herein, including in the Examples and Preparations
sections, which allow
the compounds deftned by formula (I) to be obtained, such as carrying out
certain bond-
forming or functional group interconversion reactions in different sequences.
Examples of the preparation of a number of intermediates and final compounds
are outlined
in the following synthetic schemes, where the abbreviations used are standard
and well-
known to the person skilled in the art. Routine variation of these routes can
give all the
required compounds of the invention.
Route I (Pyridkl alcohols)
399
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
i I\
ii
Br N Br Br N O~OR'°
R"'
Bu Sn~O OR,°
3
R"'
Br r Br
\ iii \ \
\ ~ ---
HN~ MeO~S
IIO
Br i I Br
i
\ ~ ~ --~ --~ \ - v--.~.
HN~ ~S~
Br ~ Br
\ 1 vi
v
Me0
z ~S~
O
i = NaH (1.1 equiv), HOCHZCHRI 1'0R10 (1 equiv) in toluene, reflux for 2 to 5
hours
ii = n-BuLi (1.1 equiv), Bu3SnCl (1.1 equiv), THF, -70°C to room
temperature.
Or, Pd(PPh3)4 (0.01 to 0.05 equiv), [SnMe3]z (1.1 equiv), dioxan, reflux for 2
to 5 hrs.
iii = BSA (0.5 equiv), MeCOzCHzSOzCI (1.2 equiv), THF, rt for 18 hours.
iv = MeSOzCI (1.2 equiv), Et3N (1.4 equiv), CHZCIz, rt, for an hour.
v = Et3SiH (3 equiv), CF3S03H (0.1 equiv), TFA:CHZCIz (1:1), rt, for 1-24 hrs.
vi = NaH (2 equiv), MeZC03 (4 equiv), toluene, reflux for 2 hours.
400
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
R10-alcohol protecting group- e.g. benzy! or dioxalane (for diols)
RI I'-H or a protected alcohol
Br ~. Br
~;,, ~
vii ~Z1 R2 viii _
Me0 S~ ''' -~ Me ~S~
2 II0
O
O OR'0 / ~ ~ O OH
ix ~ ~ ~ ~ R 1
n
Mec7 1 S~ ''' Me0 -1 R2
1~ a ~sd'i
0 0
vii = (VB), (1.3 equiv), K~C03 (3 equiv), DMSO, rt, 18-24 hours,
or KOtBu (2.5 equiv), (VA) or (VB) {excess), in THF, rt for 72 hours.
viii = Stille coupling-Pd(PPh3)4 (0.05 equiv), stannane (I.5 equiv), toluene,
reflex for 4 to 20
hours.
OR PdCh(PPh3)~ (0.05 equiv), stannane (I.1 equiv), THF, reflex for 17 hours.
ix = NH4+ HC03- (excess) Pd(OH)~/C, AcOH, MeOH, reflex for 20 hours,
OR IO% Pd/C, in MeOH or EtOH, 3.3 atmospheres, room temperature, for 6 to 17
hours,-
both methods also deprotect any benzyl group. (2N HCI, dioxan (3:1), rt, 75
mins at rt-
deprotects the dioxalane)
OR Pd(OH)2/C, NH4+ HC03' (excess), in MeOH:dioxan (2.5: I), 60°C for 2
hours.
R1 1 = H or deprotected alcohol
Similarly
when R1R2 when taken together, are a piperidine group:
401
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
Br b~ ~. Br
~i
Me0~ ~ -.~ Me0 S~I
II SOa z
O O
i
bn ~ ~N~O~OR~o H ~ ~N' O OH
N ~-..: ' ax N
w R> >' ~- w ~ R11
Me0 S~l Me0 S~J
z z
O O
R12 ~ ~N~O~OH
N w ~ R11
xi
Me0 S~l
0
x = NaH (3 equiv), tetra-nBuNH.~Br ( t equiv), BnN(CHzCH2Cl)z (0.95 equiv),
NMP, 60°C
for 6 hours.
xi = When R12 is Me, formaldehyde (4 equiv), Na(OAc)3BH (2 equiv), CHZCIz, 20
hrs at rt.
When R12 is Boc, (Boc)z0 (1.05 equiv), Et3N (1.l equiv), CHzCIz, rt for an
hour.
Route 2 (Phenyl alcohols)
xii _
Br I ~ OtBu B OH I ~ OtBu
~z
I
i Br ~ i ~ OtBu
xiii
Me0 O S~ ~MeO O SdJz
402
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
I
OtBu
I
vii R1 R2
~ MeO~s~l
O
Or
Br ~ OtBu
I ~ I
R7 R2 ~ x~ Me0_R1 R2
Me0 S~I ~S~~
a O
O
i r
I
OH ~ i v~0~
xiv R1 R2 xv R1 R2 ~ Ro
MeO~s~l ~ MeO~SdJ
p z
xii = nBuLi (1.1 equiv), B[OCH(CH3)~J3 (1.5 equiv), THF, -70°C to rt.
xiii = Suzuki coupling- arylboronic acid ( 1.2 to 1.5 equiv), CsF (2 to 2.6
equiv), P(o-tol)3 (0.1
equiv), Pd~(dba)z (0.005 equiv), DME, reflux for 6 to 50 hours.
xiv = Et3SiH (3 equiv), TFA:CH~CIz (1:l), rt for 2 to 24 hours.
xv = RlS glycidol (1 equiv), Et3N (catalytic), MeOH, reflux for 20 hours.
OR, Mitsunobu reaction -DEAD (1.5 equiv), PPh3 (1.5 equiv), HOCH(R11')CH~OR13'
(1.5
equiv) in THF, rt for 3 hours.
R11' is H or optionally protected alcohol
and R13' is optionally protected alcohol
For preparation 50 to S I, requires Bn deprotection using the conditions
described in ix.
Alternative route
403
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
O~ O~ O
O xxiv R1 R2 ~O - xvi R1 R2
---' Me0 ~- Me0 S~V
S~~ S~ z
2 2 O
i ~ R15
xxv ' O ~ ~ \ R~5' xiv R1 R2
R1 R2 ~ ~eO~S~J
MeO~S~J O
~cxiv = i- NaH (2.2 equiv), MezCO~ (5 equiv), toluene, MeOH (catalytic),
90°C, overnight.
ii- O(CH~CH~Br)~ ( 1.3 equiv), NMP, 90°C, 20 hrs.
xYV = Grignard reagant ( 1.1 equiv), THF, -78°C to rt over approx hr.
R15'-optionally protected alcohol, in prep 48 this is a t-butyl ether.
R15-OH, for prep 48.
Route 3 (Phe~l aminoalcohols)
8r . ~ ~ O~CH(OEt)z
R1 R2 ~ I ~ xiii _ ~ I r
MeO~S~IzJ Me0 R~S~1
fOl II
O
O~CH(OEt)z
s
ix R1 R2
MeO~S~lz
IIO
404
CA 02395487 2002-06-26
WO 01/49309 PCT/IB00/01935
O~CH(OEt)z
Br
i
a
R1 R2 I ~ ~ xii~ Me0 R1 S~1
Me0 S~l~ z
z
O
~ O'~.o
r ~ O~NRIaR,s
xvi R1 R2 ~ xvii
MeO~S~ R1 R2~
MeO~S
O
When R15 is a protecting group, eg. benzyl, deprotection, followed by
protection using an
alternative group eg Boc, can be used as shown below:
w O~NHR~4 , w ~ O~NRy4Ri
R2 \ xix T
R1 ~'J R1 R2,~
MeO~S~~ Me0~5
O O
xvi = IN HCl (1 to 2.3 equiv), acetone:dioxan (1:1), 70°C for 2 to 6
hours.
xvii = Reductive amination-amine (5.5 equiv), Na(OAc)3BH (3 to 4 equiv),
CH~C1~, rt,
overnight.
xviii = Pd(OH)2/C, MeOH, 50 psi, rt, 18 hrs.
xix = When RI6 is Boc,
(Boc)z0 (1 toll equiv), Et3N (optional, I equiv), DMAP (optional, cat), CH~Ch,
rt, 3 hrs.
Route 4 (aminoalkyl phenyls)
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8r
w ~ n
xiii R1 R2 ~ O
R1 R2
Me0 5~.1~ ~MeO~S~IJ
z O
O
~ W NR~4R~s
xvii R1 R2
MeO~S~ ,
IOl
Route 5 Heterocycles)
N ' ~ ii N '
N . R14 ~ ~ ~ ~N R14
Sr / SnMe3
O~ O O
O xx ~ ~ xxi Me0 ~r J 0
HN~ S~z ~ ~S~
O
O ~ OS02CF3
xvi xxii
Me0 S~lr~ --~- Me0 ~
2 ~S~
O O
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N N O
R14
ix
xxiii
~/IeO~S~
fIO
N N O
R14
MeO~s~l
IlO
xx= iso-PrSO~CI (1 equiv), Et3N (1.1 equiv), CHzCh, 3hours at rt.
xxi = n-BuLi ( 1.1 equiv), MeOCOCI ( l .2 equiv), THF -78° to rt.
xxii = 2,6-di-t-Bu-4-Me pyridine (2.5 equiv), (CF3S0~)~O (2.5 equiv), CH~Ch,
4°C to rt, 5
days.
xxiii = Pd~(dba)3 (0.02 equiv), vinyl triflate ( 1.1 equiv), Ph3As (0.21
equiv), CuI (0.1 equiv) in
NMP, 75°C for 5 hrs.
IS Thiazoles
Br / N
viii
MeO~S~V~ MeO~S~J~
fOI II a
0
Route 6 (C~pentanediols)
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Br , Br
w
XXV I XXV II
Me0 S~I~ ~ Me0 S~IZ --
O
O
HO OH / I Br
Br
xxviii ~ I viii or xiii
Me0 S~1 J . Me0
o
y
~I
Me0 S~ ~ . v
O
xxvi =NaH (1.1 equiv), tetra-nBuNH4Br {1 equiv), CICHZCHCHCHzCI (I.1 equiv),
NMP, r.t
for 3 hours, then NaH ( 1.1 equiv), 2 days.
xxvii =NMO (1.1 equiv), OsOd (3 mol%), dioxan/water, r.t. 18 hours
OR
(a) AgOAc (2.3 equiv), AcOH, r.t for 18 hours (b) 1N NaOH, dixoan/water
xxviii = 2,2-Dimethoxypropane (2 equiv), TsOH (0.1 equiv), DMF, 50°C
for 4.5 hours.
EXAMPLES AND PREPARATIONS
Room temperature (rt) means 20 to 25°C. Flash chromatography refers to
column
chromatography on silica gel (Kieselgel 60, 230-400 mesh). Melting points are
uncorrected.
1H Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker
AC300, a
Varian Unity Inova-300 or a Varian Unity Inova-400 spectrometer and were in
all cases
consistent with the proposed structures. Characteristic chemical shifts are
given in parts-per-
million downfield from tetramethylsilane using conventional abbreviations for
designation of
major peaks: e.g. s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad. Mass
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WO 01/49309 PCT/IB00/01935
spectra were recorded using a Finnigan Mat. TSQ 7000 or a Fisons Intruments
Trio 1000
mass spectrometer. LRMS means low resolution mass spectrum and the calculated
and
observed ions quoted refer to the isotopic composition of lowest mass. Hexane
refers to a
mixture of hexanes (hplc grade) b.p. 65-70°C. Ether refers to diethyl
ether. Acetic acid refers
to glacial acetic acid. I-Hydroxy-7-aza-1H-1,2,3-benzotriazole (HOAt), N-
[(dimetiylamino)-
1H-1,3,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethaninium
hexafluorophosphate
N-oxide (1-(ATU) and 7-azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium
hexatluorophosphate (PyAOP) were purchased from PerSeptive Biosystems U.IC.
Ltd. "Me"
is methyl, ''Bu" is butyl, "Bn" is benzyl. Other abbreviations and terms are
used in
conjunction with standard chemical practice.
Example 1
N-Hydroxy 2-[(4-{4-[6-(2-hydroxyethoxy)pyridin-2-yl]-3-methylphenyl}piperidin-
1-
yl)sulphonyl]-2-methylpropanamide
Me
~N~O~OH
i
O N
HOHN - xs02 J
~e Me
N,N-Dimethylformamide (IOmI) was added to a solution of the acid from
preparation 70
(430mg, 0.93mmol) in pyridine (5m1), followed by chlorotrimethylsilane
(130p.1, 1.03mmol)
and the solution stirred for I '/z hours. I-(3-Dimethylaminopropyi)-3-
ethylcarbodiimide
hydrochloride (215mg, 1.1lmmol) and 1-hydroxybenzotriazole hydrate (130mg,
0.93mmo1)
were added, and the reaction stirred for a further 2 hours. Hydroxylamine
hydrochloride
(195mg, 2.8mmo1) was then added, and the reaction stirred at room temperature
overnight.
The reaction mixture was acidified to pH 1 using 2N hydrochloric acid, stirred
for an hour,
and then the pH re-adjusted to pH 4. Water (SOmI) was added, the resulting
precipitate
filtered, washed with water and dried under vacuum. This solid was purified by
column
chromatography on silica gel using dichloromethane:methanol:0.88 ammonia
(90:10:1) as
eluant to afford the title compound as a white solid, (220mg, 49%).
mp 137-140°C
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WO 01/49309 PCT/IB00/01935
~H nmr (DMSO-d6, 300MHz) 8: I.50 (s, 6H), 1.6I (m, 2H), I.80 (m, 2H}, 2.36 (s,
3H), 2.68
(m, 1 H), 3.05 (m, 2H), 3.72 (m, 4H), 4.25 (t, 2H), 4.79 (t, 1H), 6.76 (d, 1
H), 7.05 (d, 1 H),
7. I 7 (m, 2H), 7.35 (d, I H}, 7.76 (dd, 1 H), 9.00 (s, 1 H}, I0.55 (s, I H).
Example 2
N-Hydroxy 2-{[4-(4-{6-[2-(methoxy)ethoxy]pyridin-2-yl}-3-
methylplzenyl)piperidin-1-
yl]sulphonyl}-2-methylpropanamide
Me ~
O~~OMe
i
O N
HOHN~S02
Me Me
O-(7-Azabenzotriazol-1-yl)-N,N,N'N'-tetramethyluronium hexafluorophosphate
(425 mg,
0.95mmo1) and N-ethyldiisopropylamine ( 150p.1, 0.70mmol) were added to a
solution of the
acid from preparation 71 (300mg, 0.63mmol) in N,N-dimethylformamide (lOml),
and the
solution stirred at room temperature for 30 minutes. Hydroxylamine
hydrochloride (158mg,
l.9mmol) and additional N-ethyldiisopropylamine (4101, l.9mmol) were added,
and the
reacton stirred at room temperature overnight. The reaction mixture was
diluted with water
(20m1), and pH 7 buffer solution (20m1), and then extracted with ethyl acetate
(3x30m1). The
combined organic extracts were washed with brine (3x), water (2x), then dried
(MgS04),
filtered and evaporated in vacuo. The residue was triturated with di-isopropyl
ether to afford
the title compound as an off white solid, (220mg, 71%).
mp 134-138°C
'H nmr (DMSO-db, 300MHz) 8: 1.48 (s, 6H), 1.61 (m 2H), 1.80 (m, 2H), 2.36 (s,
3H), 2.66
(m, 1H), 3.05 (m, 2H), 3.28 (s, 3H), 3.62 (t, 2H), 3,78 (m, 2H), 4.38 (t, 2H),
6.78 (d, 1H), 7.06
(d, 1H), 7.16 (m, 2H), 7.35 (d, 1H), 7.76 (m, 1H).
Anal. Found: C, 59.65; H, 7.12; N, 7.69. C?4H33N306S,O.21-PrZO requires C,
59.59; H, 7.04;
N, 8.04%,
Example 3
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N-Hydroxy 4-{j4-(4-{6-[2-hydroxyethoxy]pyridin-2-yl}-3-methylphenyl)piperidin-
1-
yl]sulphonyl}tetrahydro-2H-pyran-4-carboxamide
Me
~N~O~''OH
i
D N
HOHN S~2
o~
Chlorotrimethylsilane (2.1 ml, 16.46mmol) was added to a solution of the acid
from
preparation 72 (7.55g, 14.96mmol) in N,N-dimethylformamide (150m1), and
pyridine
(150m1), and the solution stirred at room temperature under a nitrogen
atmosphere for 1 hour.
I-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.448, 17.95mmol)
and 1-
i0 hydroxy-7-azabenzotriazole (2.04g, 14.96mmol) were added, and stirring was
continued for a
further 45 minutes. Hydroxyiamine hydrochloride (3.12g, 44.8mmol) was then
added and the
reaction stirred at room temperature for 72 hours. The reaction mixture was
acidified to pH 2
using hydrochloric acid, stirred for 30 minutes, and the pH then re-adjusted
to pH 4 using 1N
sodium hydroxide solution. The mixture was extracted with ethyl acetate (3x),
the combined
organic extracts washed with brine, dried (MgS04), filtered and evaporated in
vacuo. The
residue was purified by column chromatography on silica gel using ethyl
acetate as eluant,
and recrystallised from methanol/ethyi acetate to afford the title compound as
a white solid,
(3.75g, 48%).
mp 193-194°C
~H nmr (DMSO-d6, 400MHz) 8: 1.61 (m, 2H), 1.79 (m, 2H), 1.92 (m, 2H), 2.36 (m
SH), 2.62
(m, 1H), 3.0I (m, 2H), 3.19 (m, ZH), 3.70 (m, 4H), 3.82 (m, 2H), 4.25 (t, 2H),
4.75 (br, t, 1H),
6.70 (d, 1 H), 7.01 (d, 1 H), 7.12 (m, 2H), 7.30 (d, 1 H), 7.62 (dd, 1 H),
9.10 (s, 1 H), 10.94 (s,
1 H).
LRMS : m/z 520 (M+1)+
Anal. Found: C, 57.73; H, 6.39; N, 7.99. Cz5H33N307S requires.C, 57.79; H,
6.40; N, 8.09°/a.
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Alternative route: Hydrogen chloride gas was bubbled through a solution of the
tert-butyl
ether from preparation 133 (3.0g, 5.22mmol) in anhydrous trifluoroacetic acid
(30m1) and
dichloromethane (30m1) for 10 minutes, then stirred at room temperature
overnight. Nitrogen
gas was bubbled through the reaction mixture for 1 hour and then SN NaOH
solution until the
solution was pH6. The resulting precipitate was cooled to 0°C, filtered
and washed with cold
water. The resulting solid was dissolved in hot ethyl acetate (500m1) and the
organic layer
was washed with water (3x250m1) and brine (250m1) and then dried (Na,SO.,),
filtered and
concentrated in vacuo. On cooling to 0°C overnight a solid formed and
was filtered, washed
with cold ethyl acetate and dried. The title compound was obtained as a beige
solid (1.6g,
60%).
Exayle 4
N-Hydroxy 4-{[4-(4-{6-[(2S)-2,3-dihydroxy-1-propoxy]pyridin-2-yl}-3-
methylphenyl)piperidin-1-yl]sulphonyl}tetrahydro-2H-pyran-4-carboxamide
l5
Me
~ 'N~O OH
~ , ~OH
0 N
Ho.N sot J
H
0
Chlorotrimethylsilane (168p.1, 1.32mmol) was added to a solution of the acid
from preparation
73 (318mg, 0.60mmol) in dichloromethane (6m1), and pyridine (2m1), and the
solution stirred
at room temperature under a nitrogen atmosphere for 1 hour. 1-(3-
Dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (138mg, 0.72mmo1) and 1-hydroxy-7-
azabenzotriazole
(90mg, 0.66mmoi) were added, and stirring was continued for a further hour.
Hydroxylamine
hydrochloride (124mg, 1.80mmol) was added and the reaction stirred at room
temperature for
2 hours. The reaction mixture was evaporated in vacuo, the residue dissolved
in methanol, the
solution acidified to pH 1 using hydrochloric acid (2M), then stirred for 10
minutes. The
solution was diluted with water, the pH adjusted to 6, and the resulting
precipitate filtered and
dried. The solid was purified by column chromatography on silica gel using
dichloromethane:methanol (90:10) as eluant, and recrystallised from
methanol/di-isopropyl
ether to give the title compound as a white solid, (200mg, 60%).
~H nmr (DMSO-db, 400MHz) b: 1.61 (m, 2H), 1.79 (m, 2H), 1.92 (m, 2H), 2.36 (m,
SH), 2.63
(m, 1H), 3.03 (m, 2H), 3.08-3.31 (m, 3H), 3.40 (m, 2H), 3.68-3.89 (m, 4H),
4.15 (m, 1H),
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WO 01/49309 PCT/IB00/01935
4.25 (m, 1 H), 4.56 (br, s, 1 H), 4.80 (br, s, I H), 6.75 (d, 1 H), 7.04 (d, 1
H), 7.14 (m, 2 H), 7.34
(d, 1 H), 7.75 (m, I H), 9. I 4 (s, 1 H), I 0.96 (s, I H).
LRMS : m/z 550 (M+1 )~
Anal. Found: C, 50.70; H, 6.00; N, 6.93. CZ6HasN30sS;0.6Hz0 requires C, 50.97;
H, 6.21; N,
6.86%.
Example 5
t0 N-Hydroxy 4-{[4-(4-{6-[(2R)-2,3-dihydroxy-I-propoxy]pyridin-2-yf}-3-
methylphenyl)piperidin-1-yl]sulphonyl}tetrahydro-2H-pyran-4-carboxamide
Me
O ,OH
~OH
NJ
Ho.N sot
H
0
IS The title compound was prepared from the acid from preparation 74,
following the procedure
described in example 4. The crude product was purified by crystallisation from
ethyl acetate
to give an off white solid ( I80mg, 58%).
mp 125-130°C
~H nmr (DMSO-d6, 400MHz) 8: 1.60 (m, 2H), 1.78 (m, 2H), 1.90 (m, 2H), 2.36 (m,
SH), 2.64
(m, 1H), 3.02 (m, ZH), 3.20 (m, 2H), 3.40 (m, 2H), 3.72 (m, 2H), 3.78 (m, 1H),
3.83 (m, 2H),
4.14 (m, 1H), 4.24 (m, 1H), 4.55 (dd, 1H), 4.80 (d, 1H), 6.75 (d, 1H), 7.03
(d, 1H), 7.15 (m,
2H), 7.32 (d IH), 7.75 (m, 1H), 9.14 (s, 1H), 10.95 (s, 1H).
LRMS : m/z 572 (M+23)+
Anal. Found: C, 55.32; H, 6.57; N, 7.28. Cz6H3sN30sS;HzO requires C, 55.02; H,
6.57; N,
7.40%.
Example 6
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N-Hydroxy 4-{[4-(4-{6-[2-hydroxyethoxy]pyridin-2-yl}-3-methylphenyl)piperidin-
1-
yl]sulphonyl}-piperidine-4-carboxamide dihydrochloride
Me
~N~O.~''OH
i
o NJ
I-~oHN S°2
2HCI
N
H
Hydrogen chloride gas was bubbled through an ice-cold solution of the
hydroxamic acid from
preparation 87 ( l3Smg, 0.22mmo1) in methanol (20m1), and the solution was
stirred at room
temperature. The reaction mixture was evaporated in vacuo, and the residue
azeotroped with
methanol. The solid was recrystallised from methanol/ether to afford the title
compound as a
white solid, (88mg, 64%).
~H nmr (DMSO-db, 400MHz) 8: 1.63 (m, 2H), 1.80 (m, 2H), 2.07 (m, 2H), 2.35 (s,
3H), 2.56-
2.72 (m, SH), 2.08 (m, 2H), 2.38 (m, 2H), 3.72 (m, 4H), 4.24 (t, 2H), 4.44-
4.67 (br, s, 2H),
6.76 (d, 1 H), 7.04 (d, 1 H), 7. I 7 (m, 2H), 7.34 (d, 1 H), 7.75 (m, 1 H),
8.97 (m, I H), 9.18 (m,
is 1H).
LRMS : m/z 519 (M+1)+
Exam~ie 7
N-Hydroxy 4- f [4-(4-~6=[2-hydroxyethoxy]pyridin-2-yl}-3-
methylphenyi)piperidin-I-
yl]sulphonyl}-I-methyl-piperidine-4-carboxamide
Me i
~N~O~OH
i
° NJ
HOHN SOZ
NJ
Me
The title compound was prepared from the acid from preparation 7S and
hydroxylamine
hydrochloride following a similar procedure to that described in example 1.
The reaction
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CA 02395487 2002-06-26
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mixture was acidified to pH 2 using hydrochloric acid, this mixture stirred
for 45 minutes,
then basified to pH 8 using sodium hydroxide solution (2N). This solution was
extracted with
ethyl acetate (3x), the combined organic extracts washed with water, then
brine, dried
(Na,SOa), filtered and evaporated in vacuo. The residue was dried at
60°C, under vacuum to
afford the title compound (39mg, 8%).
~H nmr (DMSO-d~, 400MHz) 8: 1.60 (m, 2H), 1.78 (m, 4H), 1.86 (m, 2H), 2.8 (s,
3H), 2.35
(s, 3H), 2.40 (m, 2H), 2.59-2.75 (m, 3H), 3.01 (m, 2H), 3.68 (m, 4H), 4.25 (t,
2H), 4.75 (t,
1 H), 6.75 (d, 1 H), 7.03 (d, 1 H), 7. I 5 (m, 2H), 7.32 (d, 1 H), 7.74 (m, 1
H), 9.06 (br, s, 1 H),
10.88 (br, s, 1 H).
LRMS : m/z 533 (M+1)+
Anal. Found: C, 57.91; H, 6.82; N, 10.24. C~~H36NdO6S;O.3HoO requires C,
58.04; H, 6.86; N,
10.41%.
Example 8
N-Hydroxy 2-[4-(4-{3-[(2S)-2,3-dihydroxy-1-propoxy]phenyl}-3-methylphenyl)-
piperidin-1-
ylsulphonyl]-2-methylpropanamide
Ma
OH
~OH
O
HOHN ~SOZ
Me Me
The title compound was prepared from the acid from preparation 77, following a
similar
procedure to that described in example 3. The crude product was recrystallised
from
methanol/di-isopropyl ether, to give the desired product (75mg, 24%) as a
white solid. The
mother liquors were evaporated in vacuo, and purified by column chromatography
on silica
gel using an elution gradient of dichloromethane:methanol (98:2 to 95:5) to
give an additional
(38mg, 12%) of the desired product.
mp 152-154°C
~H nmr (DMSO-d6, 400MHz) 8: 1.44 (s, 6H), 1.60 (m, 2H), 1.78 (m, 2H), 2.18 (s,
3H), 2.61
(m, 1H), 3.02 (m, 2H), 3.39 (m, 2H), 3.7I (m, 3H), 3.82 (m, 1H), 3.98 (m, 1H),
4.56 (m, 1H);
415
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
~~ TTENANT LES PAGES 1 A 415
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