Note: Descriptions are shown in the official language in which they were submitted.
CA 02836827 2013-12-17
1
Alpha Amino Acid Ester-Drug Conjugates Hydrolysable by Carboxylesterases.
This invention relates to a general method of increasing or prolonging the
activity of a
compound which modulates the activity of an intracellular enzyme or receptor
by the
covalent conjugation of an alpha amino acid ester motif to the modulator. The
invention
also relates to modulators to which an alpha amino acid ester motif has been
covalently
conjugated, and to a method for the identification of such conjugates having
superior
properties relative to the parent non-conjugated modulator. The invention
further relates to
the use of modulators containing amino acid ester motifs that allow the
selective
accumulation of amino acid conjugates inside cells of the monocyte-macrophage
lineage.
Background to the invention
Many intracelular enzymes and receptors are targets for pharmaceutically
useful drugs
which modulate their activities by binding to their active sites. Examples
appear in Table 1
below. To reach the target enzymes and receptors, modulator compounds must of
course
cross the cell membrane from plasma/extracellular fluid. In general, charge
neutral
modulators cross the cell membrane more easily than charged species. A dynamic
equilibrium is then set up whereby the modulator equilibrates between plasma
and cell
interior. As a result d the equilibrium, the intracellular residence times and
concentrations
of many modulators of intracellular enzymes and receptors are often very low,
especially in
cases where the modulator is rapidly cleared from the plasma. The potencies of
the
modulators are therefore poor despite their high binding affinities for the
target enzyme or
receptor.
It would therefore be desirable if a method were available for increasing the
intracellular
concentration of a given modulator of an intracellular enzyme or receptor.
This would result
in increased potency, and by prolonging the residency of the modulator inside
the cell
would result in improved pharmacokinetic and phamiacodynamic properties. More
consistent exposure and reduced dosing frequencies would be achieved. A
further benefit
could be obtained if the drug could be targeted to the specific target cells
responsible for its
therapeutic action, reducing systemic exposure and hence side effects.
Brief description of the Invention
This invention provides such a method, and describes improved modulators
incorporating
the structural principles on which the method is based. It takes advantage of
the fact that
lipophilic (low polarity or charge neutral) molecules pass through the cell
membrane and
CA 02836827 2013-12-17
2
enter cells relatively easily, and hydrophilic (higher polarity, charged)
molecules do not.
Hence, if a lipophilic motif is attached to a given modulator, allowing the
modulator to enter
the cell, and if that motif is converted in the cell to one of higher
polarity, it is to be expected
that the modulator with the higher polarity motif attached would accumulate
within the cell.
Providing such a motif is attached to the modulator in a way which does not
alter its binding
mode with the target enzyme or receptor, the accumulation of modulator with
the higher
polarity motif attached is therefore expected to result in prolonged and/or
increased activity.
The present invention makes use of the fact that there are carboxylesterase
enzymes
within cells, which may be utilised to hydrolyse an alpha amino acid ester
motif attached to
a given modulator to the parent acid. Therefore, a modulator may be
administered as a
covalent conjugate with an alpha amino acid ester, in which form it readily
enters the cell
where it is hydrolysed efficiently by one or more intracellular
carboxylesterases, and the
resultant alpha amino acid-modulator conjugate accumulates within the cell,
increasing
overall potency and/or active residence time. It has also been found that by
modification of
the alpha amino acid motif or the way in which it is conjugated, modulators
can be targeted
to monocytes and macrophages. Herein, unless "monocyte" or "monocytes" is
specified,
the term macrophage or macrophages will be used to denote macrophages
(including
tumour associated macrophages) and/or monocytes.
Detailed description of the invention
Hence in one broad aspect the present invention provides a covalent conjugate
of an alpha
amino acid ester and a modulator of the activity of a target intracellular
enzyme or receptor,
wherein: the ester group of the conjugate is hydrolysable by one or more
intracellular
carboxylesterase enzymes to the corresponding acid; and the alpha amino acid
ester is
covalently attached to the modulator at a position remote from the binding
interface
between the modulator and the target enzyme or receptor, and/or is conjugated
to the
modulator such that the binding mode of the conjugated modulator and the said
corresponding acid to the target enzyme or receptor is the same as that of the
unconjugated modulator.
Looked at in another way, the invention provides a method of increasing or
prolonging the
intracellular potency and/or residence time of a modulator of the activity of
a target
intracellular enzyme or receptor comprising structural modification of the
modulator by
covalent attachment thereto of an alpha amino acid ester at a position remote
from the
binding interface between the modulator and the target enzyme or receptor,
and/or such
CA 02836827 2013-12-17
3
that the binding mode of the conjugated modulator and the said corresponding
acid to the
target enzyme or receptor is the same as that of the unconjugated modulator,
the ester
group of the conjugate being hydrolysable by one or more intracellular
carboxylesterase
enzymes to the corresponding acid.
As stated, the invention is concerned with modification of modulators of
intracellular
enzymes or receptors. Although the principle of the invention is of general
application, not
restricted by the chemical identity of the modulator or the identity of the
target enzyme or
receptor, it is strongly preferred that the modulator be one that exerts its
effect by reversible
binding to the target enzyme or receptor, as opposed to those whose effect is
due to
covalent binding to the target enzyme or receptor.
Since for practical utility the carboxylesterase-hydrolysed conjugate is
required to retain the
intracellular binding activity of the parent modulator with its target enzyme
or receptor,
attachment of the ester motif must take account of that requirement, which
will be fulfilled if
the alpha amino acid carboxylesterase ester motif is attached to the modulator
such that
the binding mode of the corresponding carboxylesterase hydrolysis product (ie
the
corresponding acid) to the target is essentially the same as the unconjugated
modulator. In
general this is achieved by covalent attachment of the carboxylesterase ester
motif to the
modulator at a position remote from the binding interface between the
modulator and the
target enzyme or receptor. In this way, the motif is arranged to extend into
solvent, rather
than potentially interfering with the binding mode,
In addition, the amino acid carboxylesterase motif obviously must be a
substrate for the
carboxylesterase if the former is to be hydrolysed by the latter within the
cell. Intracellular
carboxylesterases are rather promiscuous in general, in that their ability to
hydrolyse does
not depend on very strict structural requirements of the amino acid ester
substrate. Hence
most modes of covalent conjugation of the amino acid carboxylesterase motif to
a
modulator will allow hydrolysis. Attachment by a flexible linker chain will
usually be how this
is achieved.
It will be appreciated that any chemical modification of a drug may subtly
alter its binding
geometry, and the chemistry strategy for linkage of the carboxylesterase ester
motif may
introduce additional binding interactions with the target, or may substitute
for one or more
such interactions. Hence the requirement that the hydrolysed conjugate's
binding mode to
the target is the same as the unconjugated modulator is to be interpreted as
requiring that
CA 02836827 2013-12-17
4
there is no significant perturbation of the binding mode, in other words that
the binding
mode is essentially the same as that of the unconjugated modulator. When the
requirement
is met, the main binding characteristics of the parent modulator are retained,
and the
modified and unmodified modulators have an overall common set of binding
characteristics. The "same binding mode" and "remote attachment" viewpoints
are similar
because, as stated above, the usual way of achieving the "same binding mode"
requirement is to attach the carboxylesterase motif at a point in the
modulator molecule
which is remote from the binding interface between the inhibitor and the
target enzyme or
receptor. However, it should be noted that these requirements do not imply
that the
conjugate and/or its corresponding acid must have the same in vitro or in vivo
modulatory
potency as the parent modulator. In general, however, it is preferred that the
esterase-
hydrolysed carboxylic acid has a potency in an in vitro enzyme- or receptor-
binding assay
no less than one tenth of the potency of the parent modulator in that assay,
and that the
ester has a potency in a cellular activity assay at least as high as that of
the parent
modulator in the same assay.
Although traditional medicinal chemistry methods of mapping structure-activity
relationships
are perfectly capable of identifying an attachment strategy to meet the
foregoing "same
binding mode" and "remote attachment" requirements, modern techniques such as
NMR
and X-ray crystallography have advanced to the point where it is very common
for the
binding mode of a known modulator of an enzyme or receptor to be known, or
determinable. Such information is in the vast majority of cases in the public
domain, or can
be modelled using computer based modelling methods, such as ligand docking and
homology modelling, based on the known binding modes of structurally similar
modulators,
or the known structure of the active site of the target enzyme or receptor.
With knowledge
of the binding mode of the modulator obtained by these techniques, a suitable
location for
attachment of the carboxylesterase ester motif may be identified, usually (as
stated above)
at a point on the modulator which is remote from the binding interface between
the inhibitor
and the target enzyme or receptor.
Intracellular carboxylesterase enzymes capable of hydrolysing the ester group
of the
conjugated alpha amino acid to the corresponding acid include the three known
human
carboxylesterase ("hCE") enzyme isotypes hCE-1 (also known as CES-1), hCE-2
(also
known as CES-2) and hCE-3 (Drug Disc. Today 2005, 10, 313-325). Although these
are
considered to be the main enzymes other carboxylester enzymes such as
biphenylhydrolase (BPH) may also have a role in hydrolysing the conjugates.
CA 02836827 2013-12-17
The broken cell assay described below is a simple method of confirming that a
given
conjugate of modulator and alpha amino acid ester, or a given alpha amino acid
ester to be
assessed as a possible carboxylesterase ester motif, is hydrolysed as
required. These
enzymes can also be readily expressed using recombinant techniques, and the
recombinant enzymes may be used to determine or confirm that hydrolysis
occurs.
It is a feature of the invention that the desired conjugate retains the
covalently linked alpha
amino acid motif when hydrolysed by the carboxylesterase(s) within the cell,
since it is the
polar carboxyl group of that motif which prevents or reduces clearance of the
hydrolysed
conjugate from the cell, and thereby contributes to its accumulation within
the cell. Indeed,
the cellular potency of the modified modulator is predominantly due to the
accumulation of
the acid and its modulation of the activity of the target (although the
unhydrolysed ester
also exerts its activity on the target for so long as it remains
unhydrolysed). Since cells in
general contain several types of peptidase enzymes, it is preferable that the
conjugate, or
more especially the hydrolysed conjugate (the corresponding acid), is not a
substrate for
such peptidases. In particular, it is strongly preferred that the alpha amino
acid ester group
should not be the C-terminal element of a dipeptide motif in the conjugate.
However, apart
from that limitation on the mode of covalent attachment, the alpha amino acid
ester group
may be covalently attached to the modulator via its amino group or via its
alpha carbon. In
some cases the modulator will have a convenient point of attachment for the
carboxylesterase ester motif, and in other cases a synthetic strategy will
have to be
devised for its attachment.
It has been found that cells that only express the carboxylesterases hCE-2,
and/or hCE-3
and recombinant forms of these enzymes will only hydrolyse amino acid ester
conjugates
to their resultant acids if the nitrogen of the alpha amino acid group is
either unsubstituted
or is directly linked to a carbonyl group, whereas cells containing hCE-1, or
recombinant
hCE-1 can hydrolyse amino acid conjugates with a wide range of groups on the
nitrogen.
This selectivity requirement of hCE-2 and hCE-3 can be turned to advantage
where it is
required that the modulator should target enzymes or receptors in certain cell
types only. It
has been found that the relative amounts of these three carboxylesterase
enzymes vary
between cell types (see Figure 1 and database at
http:/symatlas.gnf.org/SymAtlas (note
that in this database hCE3/CES3 is referred to by the symbol FLJ21736)) If the
modulator
is intended to act only in cell types where hCE-1 is found, attachment of a
carboxylesterase
ester motif wherein the amino group is directly linked to a group other than
carbonyl results
CA 02836827 2013-12-17
6
in the hydrolysed modulator conjugate accumulating preferentially in cells
with effective
concentrations of hCE-1. Stated in another way, specific accumulation of the
acid derived
from the modulator conjugate in hCE-1 expressing cells can be achieved by
linking the
amino acid ester motif to the modulator in such a way that the nitrogen atom
of the amino
acid ester is not linked directly to a carbonyl, or is left unsubstituted.
Macrophages are known to play a key role in inflammatory disorders through the
release of
cytokines in particular TNFoc and IL-1 (van Roon et al Arthritis and
Rheumatism , 2003,
1229-1238). In rheumatoid arthritis they are major contributors to joint
inflammation and
joint destruction (ConeII in N.Eng J. Med. 2004, 350, 2591-2602). Macrophages
are also
involved in tumour growth and development (Naldini and Carraro in Curr Drug
Targets
Inflamm Allergy ,2005, 3-8). Hence agents that selectively target macrophage
cells could
be of value in the treatment of cancer, inflammation and autoimmune disease.
Targeting
specific cell types would be expected to lead to reduced side-effects. The
present invention
enables a method of targeting modulators to macrophages, which is based on the
above
observation that the way in which the carboxylesterase ester motif is linked
to the
modulator determines whether it is hydrolysed by specific carboxylesterases,
and hence
whether or not the resultant acid accumulates in different cell types.
Specifically it has been
found that macrophages contain the human carboxylesterase hCE-1 whereas other
cell
types do not. In the conjugates of the invention, when the nitrogen of the
ester motif is
substituted but not directly bonded to a carbonyl group moiety the ester will
only be
hydrolysed by hCE-1 and hence the esterase-hydrolysed modulator conjugates
will only
accumulate in macrophages.
There are of course many possible ester groups which may in principle be
present in the
carboxylesterase ester motif for attachment to the modulator. Likewise, there
are many
alpha amino acids, both natural and non-natural, differing in the side chain
on the alpha
carbon, which may be used as esters in the carboxylesterase ester motif. Some
alpha
amino acid esters are rapidly hydrolysed by one or more of the hCE-1,
-2 and ¨3 isotypes or cells containing these enzymes, while others are more
slowly
hydrolysed, or hydrolysed only to a very small extent. In general, if the
carboxylesterase
hydrolyses the free amino acid ester to the parent acid it will, subject to
the N-carbonyl
dependence of hCE-2 and hCE-3 discussed above, also hydrolyse the ester motif
when
covalently conjugated to the modulator. Hence, the broken cell assay and/or
the isolated
carboxylesterase assay described herein provide a straightforward, quick and
simple first
screen for esters which have the required hydrolysis profile. Ester motifs
selected in that
CA 02836827 2013-12-17
7
way may then be re-assayed in the same carboxylesterase assay when conjugated
to the
modulator via the chosen conjugation chemistry, to confirm that it is still a
carboxylesterase
substrate in that background. Suitable types of ester will be discussed below,
but at this
point it may be mentioned that it has been found that t-butyl esters of alpha
amino acids
are relatively poor substrates for hCE-1, -2 and ¨3, whereas cyclopentyl
esters are
effectively hydrolysed. Suitable alpha amino acids will also be discussed in
more detail
below, but at this point it may be mentioned that phenylalanine,
homophenylalanine,
phenylglycine and leucine are generally suitable, and esters of secondary
alcohols are
preferred.
As stated above, the alpha amino acid ester may be conjugated to the modulator
via the
amino group of the amino acid ester, or via the alpha carbon (for example
through its side
chain) of the amino acid ester. A linker radical may be present between the
carboxylesterase ester motif and the modulator. For example, the alpha amino
acid ester
may be conjugated to the modulator as a radical of formula (IA), (IB) or (IC):
wherein
Flz
R.K R
N¨ Y-L-X-(CH2),i¨
________________________________ L ¨Y1 ¨ (A1 k3)5 __
H R4¨N
(IA) H (IB)
Fli L ¨Y1¨ (Alk3)s +
HNr1;75
(IC)
R1 is an ester group which is hydrolysable by one or more intracellular
carboxylesterase
enzymes to a carboxylic acid group;
R2 is the side chain of a natural or non-natural alpha amino acid;
R4 is hydrogen; or optionally substituted C1-C6 alkyl, C3-C7cycloalkyl, aryl
or heteroaryl or -
(C=0)R3, -(C=0)0R3, or ¨(C=0)NR3wherein R3 is hydrogen or optionally
substituted (C1-
C6)alkyl;
CA 02836827 2013-12-17
8
B is a monocyclic heterocyclic ring of 5 or 6 ring atoms wherein R1 is linked
to a ring carbon
adjacent the ring nitrogen shown, and ring B is optionally fused to a second
carbocyclic or
heterocyclic ring of 5 or 6 ring atoms in which case the bond to L may be from
a ring atom
in said second ring
Y is a bond, ¨C(=0)-, -S(=0)2-, -C(=0)0-, -C(=0)NR3-, -C(=S)-NR3-, -C(=NH)NR3-
or -
S(=0)2NR3- wherein R3 is hydrogen or optionally substituted C1-C6 alkyl;
YI is a bond, -(C=0)-, -S(02)-, -C(=0)0-, -0C(=0)-, ¨(C=0)NR3-, -NR3(C=0)-,
-S(02)NR3-, -NR3S(02)-, or -NR3(C=0)NR5-, wherein R3 and R5 are independently
hydrogen or optionally substituted (C1-C6)alkyl,
L is a divalent radical of formula ¨(Alkl)m(Q)n(Alk2)p- wherein
m, n and p are independently 0 or 1,
a is (i) an optionally substituted divalent mono- or bicyclic carbocyclic or
heterocyclic radical having 5 - 13 ring members, or (ii), in the case where
both m
and p are 0, a divalent radical of formula ¨X2-Q1- or ¨01-X2- wherein X2 is
¨0-, -S- or NRA- wherein RA is hydrogen or optionally substituted C1-C3 alkyl,
and
Q' is an optionally substituted divalent mono- or bicyclic carbocyclic or
heterocyclic
radical having 5 - 13 ring members,
Alkl and Alk2 independently represent optionally substituted divalent C3-C7
cycloalkyl radicals, or optionally substituted straight or branched, C1-C6
alkylene,
C2-C6 alkenylene, or C2-C6 alkynylene radicals which may optionally contain or
terminate in an ether (-0-), thioether (-S-) or amino
(¨NRA-) link wherein RA is hydrogen or optionally substituted Cl-C3 alkyl;
X represents a bond, -C(=0)-; -S(=0)2-; ¨NR3C(=0)-, -C(=0)NR3-,¨NR3C(=0)NR5- ,
-NR3S(=0)2-, or -S(=0)2NR3- wherein R3 and R5 are independently hydrogen or
optionally
substituted C1-C6 alkyl;
z is 0 or 1;
s is 0 or 1; and
CA 02836827 2013-12-17
9
Alk3 represents an optionally substituted divalent C3-C7 cycloalkyl radical,
or optionally
substituted straight or branched, C1-C6 alkylene, C2-C6 alkenylene ,or C2-C6
alkynylene
radical which may optionally contain or terminate in an ether (-0-), thioether
(-S-) or amino
(¨NRA-) link wherein RA is hydrogen or optionally substituted C1-C3 alkyl;
The term "ester" or "esterified carboxyl group" means a group R90(C=0)- in
which R9 is the
group characterising the ester, notionally derived from the alcohol R9OH.
As used herein, the term "(Ca-Cb)alkyr wherein a and b are integers refers to
a straight or
branched chain alkyl radical having from a to b carbon atoms. Thus when a is 1
and b is 6,
for example, the term includes methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, sec-butyl,
t-butyl, n-pentyl and n-hexyl.
As used herein the term "divalent (Ca-Cb)alkylene radical" wherein a and b are
integers
refers to a saturated hydrocarbon chain having from a to b carbon atoms and
two
unsatisfied valences.
As used herein the term "(Ca-Cb)alkenyl" wherein a and b are integers refers
to a straight or
branched chain alkenyl moiety having from a to b carbon atoms having at least
one double
bond of either E or Z stereochemistry where applicable. The term includes, for
example,
vinyl, ally!, 1- and 2-butenyl and 2-methyl-2-propenyl.
As used herein the term "divalent (Ca-Cb)alkenylene radical" means a
hydrocarbon chain
having from a to b carbon atoms, at least one double bond, and two unsatisfied
valences.
As used herein the term "Ca-Cb alkynyr wherein a and b are integers refers to
straight
chain or branched chain hydrocarbon groups having from two to six carbon atoms
and
having in addition one triple bond. This term would include for example,
ethynyl, 1-
propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-
pentynyl, 2-
hexynyl, 3-hexynyl, 4-hexynyl and 5-hexryl.
As used herein the term "divalent (Ca-Cb)alkynylene radical" wherein a and b
are integers
refers to a divalent hydrocarbon chain having from 2 to 6 carbon atoms, and at
least one
triple bond.
CA 02836827 2013-12-17
As used herein the term "carbocyclic" refers to a mono-, bi- or tricyclic
radical having up to
16 ring atoms, all of which are carbon, and includes aryl and cycloalkyl.
As used herein the term "cycloalkyl" refers to a monocyclic saturated
carbocyclic radical
having from 3-8 carbon atoms and includes, for example, cyclopropyl,
cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
As used herein the unqualified term "aryl" refers to a mono-, bi- or tri-
cyclic carbocyclic
aromatic radical, and includes radicals having two monocyclic carbocyclic
aromatic rings
which are directly linked by a covalent bond. Illustrative of such radicals
are phenyl,
biphenyl and napthyl.
As used herein the unqualified term "heteroaryl" refers to a mono-, bi- or tri-
cyclic aromatic
radical containing one or more heteroatoms selected from S, N and 0, and
includes
radicals having two such monocyclic rings, or one such monocyclic ring and one
monocyclic aryl ring, which are directly linked by a covalent bond.
Illustrative of such
radicals are thienyl, benzthienyl, fury!, benzfuryl, pyrrolyl, imidazolyl,
benzimidazolyl,
thiazolyl, benzthiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyl, oxazolyl,
benzoxazolyl,
isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl, benztriazolyl,
thiadiazolyl, oxadiazolyl,
pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl and
indazolyl.
As used herein the unqualified term "heterocycly1" or "heterocyclic" includes
"heteroaryl" as
defined above, and in its non-aromatic meaning relates to a mono-, bi- or tri-
cyclic non-
aromatic radical containing one or more heteroatoms selected from S, N and 0,
and to
groups consisting of a monocyclic non-aromatic radical containing one or more
such
heteroatoms which is covalently linked to another such radical or to a
monocyclic
carbocyclic radical. Illustrative of such radicals are pyrrolyl, furanyl,
thienyl, piperidinyl,
imidazoly1, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl,
pyridinyl, pyrrolidinyl,
pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl, benzfuranyl,
pyranyl, isoxazolyl,
benzimidazolyl, methylenedioxyphenyl, ethylenedioxyphenyl, maleimido and
succinimido
groups.
Unless otherwise specified in the context in which it occurs, the term
"substituted" as
applied to any moiety herein means substituted with up to four compatible
substituents,
each of which independently may be, for example, (C1-C6)alkyl, (C1-C6)alkoxy,
hydroxy,
hydroxy(CI-C6)alkyl, mercapto, mercapto(C1-C6)alkyl, (C1-C6)alkylthio, phenyl,
halo
CA 02836827 2013-12-17
11
(including fluoro, bromo and chloro), trifluoromethyl, trifluoromethoxy,
nitro, nitrile (-CN),
oxo, -0001-1, -COORA, -CORA, -SO2RA,
-CONH2, -SO2NH2, -CONHRA, -SO2NHRA, -CONRARB, -SO2NRARB, -NH2, -NHRA,
-NRARB, -000NH2, -OCONHRA , -OCONRARB, -NHCORA, -NHCOORA,
-NRBCOORA, -NHSO2ORA, -NRBS020H, -NRBSO2ORA,-NHCONH2, -NRACONH2,
-NHCONHRB,-NRACONHRB, -NHCONRARB, or -NRACONRARB wherein RA and RB are
independently a (C1-C6)alkyl, (C3-C6) cycloalkyl , phenyl or monocyclic
heteroaryl having 5
or 6 ring atoms. An "optional substituent" may be one of the foregoing
substituent groups.
The term "side chain of a natural or non-natural alpha-amino acid" refers to
the group 131 in
a natural or non-natural amino acid of formula NH2-CH(R1)-COOH.
Examples of side chains of natural alpha amino acids include those of alanine,
arginine,
asparagine, aspartic acid, cysteine, cystine, glutamic acid, histidine, 5-
hydroxylysine, 4-
hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine,
praline, serine,
threonine, tryptophan, tyrosine, valine, a-aminoadipic acid, a-amino-n-butyric
acid, 3,4-
dihydroxyphenylalanine, homoserine, a-methylserine, omithine, pipecolic acid,
and
thyroxine.
Natural alpha-amino acids which contain functional substituents, for example
amino,
carboxyl, hydroxy, mercapto, guanidyl, imidazolyl, or indolyl groups in their
characteristic
side chains include arginine, lysine, glutamic acid, aspartic acid,
tryptophan, histidine,
serine, threonine, tyrosine, and cysteine. When R2 in the compounds of the
invention is one
of those side chains, the functional substituent may optionally be protected.
The term "protected" when used in relation to a functional substituent in a
side chain of a
natural alpha-amino acid means a derivative of such a substituent which is
substantially
non-functional. For example, carboxyl groups may be esterdied (for example as
a C1-C6
alkyl ester), amino groups may be converted to amides (for example as a NHCOC1-
C6 alkyl
amide) or carbamates (for example as an NHC(.0)0CI-C6 alkyl or NHC(=0)0CH2Ph
carbamate), hydroxyl groups may be converted to ethers (for example an 0C1-C6
alkyl or a
0(C1-C6 alkyl)phenyl ether) or esters (for example a OC(=0)C1-C6 alkyl ester)
and thiol
groups may be converted to thioethers (for example a tert-butyl or benzyl
thioether) or
thioesters (for example a SC(=0)C1-C6 alkyl thioester).
CA 02836827 2013-12-17
12
Examples of side chains of non-natural alpha amino acids include those
referred to below
in the discussion of suitable R2 groups for use in compounds of the present
invention.
The ester arours R1
In addition to the requirement that the ester group must be hydrolysable by
one or more
intracellular enzymes, it may be preferable for some applications (for example
for systemic
administration of the conjugate) that it be resistant to hydrolysis by
carboxylester-
hydrolysing enzymes in the plasma, since this ensures the conjugated modulator
will
survive after systemic administration for long enough to penetrate cells as
the ester. It is a
simple matter to test any given conjugate to measure its plasma half life as
the ester, by
incubation in plasma. However, it has been found that esters notionally
derived from
secondary alcohols are more stable to plasma carboxylester-hydrolysing enzymes
than
those derived from primary alcohols. Furthermore, it has also been found that
although
esters notionally derived from tertiary alcohols are generally stable to
plasma
carboxylester-hydrolysing enzymes, they are often also relatively stable to
intracellular
carboxylesterases. Taking these findings into account, it is presently
preferred that Ri in
formulae (IA), (IB) and (IC) above, is an ester group of formula -(C=0)0R9
wherein Rg is (i)
R7FI5CH- wherein R7 is optionally substituted (CI-C3)alkyl-(Z1)a-(Ci-C3)alkyl-
or (C2-
C3)alkenyl-(Z1),-(Ci-C3)alkyl- wherein a is 0 or 1 and Z1 is -0-, -S-, or -NH-
, and Ra is
hydrogen or (C1-C3)alkyl- or R7 and Rg taken together with the carbon to which
they are
attached form an optionally substituted C3-C7 cycloalkyl ring or an optionally
substituted
heterocyclic ring of 5- or 6-ring atoms; or (ii) optionally substituted phenyl
or monocyclic
heterocyclic ring having 5 or 6 ring atoms. Within these classes, Rg may be,
for example,
methyl, ethyl, n- or iso-propyl, n- or sec-butyl, cyclohexyl, allyl, phenyl,
benzyl, 2-, 3- or 4-
pyridylmethyl, N-methylpiperidin-4-yl, tetrahydrofuran-3-y1 or methoxyethyl.
Currently
preferred is where Rg is cyclopentyl.
The amino acid side chain R.,
Subject to the requirement that the ester group RI be hydrolysable by
intracellular carboxylesterase enzymes, the selection of the side chain group
R2
, can determine the rate of hydrolysis. For example, when the carbon in R2
adjacent
to the alpha amino acid carbon does not contain a branch eg when R2 is ethyl,
isobutyl or benzyl the ester is more readily hydrolysed than when R2 is
branched eg
isopropyl or t-butyl.
Examples of amino acid side chains include
CA 02836827 2013-12-17
13
C1-C6 alkyl, phenyl, 2,- 3-, or 4-hydroxyphenyl, 2,- 3-, or 4-methoxyphenyl,
2,-
3-, or 4-pyridylmethyl, benzyl, phenylethyl, 2-, 3-, or 4-hydroxybenzyl, 2,- 3-
,
or 4-benzyloxybenzyl, 2,- 3-, or 4- C1-C6 alkoxybenzyl, and benzyloxy(C1-C6
alkyl)-
groups;
the characterising group of a natural a amino acid, in which any functional
group may be
protected;
groups -[Alk]R6 where Alk is a (C1-C6)alkyl or (C2-C6)alkenyl group optionally
interrupted by
one or more -0-, or -S- atoms or -N(R7)- groups [where R7 is a hydrogen atom
or a (C1-
C6)alkyl group], n is 0 or 1, and R6 is an optionally substituted cycloalkyl
or cycloalkenyl
group;
a benzyl group substituted in the phenyl ring by a group of formula -OCH2COR9
where 138 is
hydroxyl, amino, (C1-COalkoxy, phenyl(Cl-COalkoxy, (C1-C6)alkylamino, di((C1-
C6)alkyl)amino, phenyl(C1-C6)alkylamino, the residue of an amino acid or acid
halide, ester
or amide derivative thereof, said residue being linked via an amide bond, said
amino acid
being selected from glycine, a or 13 alanine, valine, leucine, isoleucine,
phenylalanine,
tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine,
glutamine, lysine,
histidine, arginine, glutamic acid, and aspartic acid;
a heterocyclic(C1-C6)alkyl group, either being unsubstituted or mono- or di-
substituted in
the heterocyclic ring with halo, nitro, carboxy, (C1-C6)alkm, cyano, (C1-
C6)alkanoyl,
trifluoromethyl (C1-C6)alkyl, hydroxy, formyl, amino, (C1-C6)alkylamino, di-
(C1-
C6)alkylamino, mercapto, (C1-C6)alkylthio, hydroxy(C1-C6)alkyl, mercapto(C1-
C6)alkyl or (C1-
C6)alkylphenylmethyl; and
a group -CRaRaRc in which:
each of Ra, Rb and Fic is independently hydrogen, (C1-C6)alkyl, (C2-
C6)alkenyl, (C2-
C6)alkynyl, phenyl(C1-C6)alkyl, (C3-C8)cycloalkyl; or
Ra is hydrogen and Ra and Rb are independently phenyl or heteroaryl such as
pyridyl; or
CA 02836827 2013-12-17
14
I:1c is hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, phenyl(CI-
C6)alkyl, or
(C3-Ce)cycloalkyl, and 13, and Rb together with the carbon atom to which they
are
attached form a 3 to 8 membered cycloalkyl or a 5-to 6-membered heterocyclic
ring; or
Fla, Rb and 13, together with the carbon atom to which they are attached form
a
tricyclic ring (for example adamantyl); or
IR, and Rb are each independently (C1-C6)alkyl, (C2-C6)alkenyl, (C2-
C6)alkynyl,
phenyl(C1-C6)alkyl, or a group as defined for Rc below other than hydrogen, or
Ra
and Rb together with the carbon atom to which they are attached form a
cycloalkyl
or heterocyclic ring, and Ft, is hydrogen, -OH, -SH, halogen, -CN, -CO2H, (C1-
C4)perfluoroalkyl, -CH2OH, -0O2(C1-C6)alkyl, -0(C1-C6)alkyl, -0(C2-C6)alkenyl,
-
S(C1-C6)alkyl, -SO(C1-C6)alkyl, -S02(C1-C6) alkyl, -S(C2-C6)alkenyl, -SO(C2-
C6)alkenyl, -S02(C2-C6)alkenyl or a group -O-W wherein 0 represents a bond or -
0-
-S-, -SO- or -SO2- and W represents a phenyl, phenylalkyl, (C3-C8)cycloalkyl,
(C3-
C6)cycloalkylalkyl, (C4-C6)cycloalkenyl, (C4-C6)cycloalkenylalkyl, heteroaryl
or
heteroarylalkyl group, which group W may optionally be substituted by one or
more
substituents independently selected from, hydroxyl, halogen, -CN, -CO2H, -
0O2(C1-
C6)alkyl, -CONH2, -CONH(CI-C6)alkyl, -CONH(C1-C6alky1)2, -CHO, -CH2OH, (Cr
C4)perfluoroalkyl, -0(C1-C6)alkyl, -S(C1-C6)alkyl, -SO(CI-C6)alkyl, -S02(C1-
C6)alkyl, -
NO2, -NH2, -NH(CI-C6)alkyl, -N((CI-C6)alky1)2, -NHCO(CI-C6)alkyl, (C1-
C6)alkyl, (C2-
C6)alkenyl, (C2-C6)alkynyl, (C3-C6)cycloalkyl, (C4-C6)cycloalkenyl, phenyl or
benzyl.
Examples of particular R2 groups include benzyl, phenyl, cyclohexylmethyl,
pyridin-3-
ylmethyl, tert-butoxymethyl, iso-butyl, sec-butyl, tert-butyl, 1-benzylthio-1-
methylethyl, 1-
methylthio-1-methylethyl, and 1-mercapto-1-methylethyl, phenylethyl. Presently
preferred
R2 groups include phenyl, benzyl, tert-butoxymethyl, phenylethyl and iso-
butyl.
The group R4
As mentioned above, if the modulator is intended to act only in cell types
where hCE-1 is
present, such as macrophages, the amino group of the carboxylesterase ester
motif should
be substituted such that it is directly linked to a group other than carbonyl.
In such cases R4
may be optionally substituted C1-C6 alkyl, C3-C7cycloalkyl, aryl or
heteroaryl, for example
methyl, ethyl, n- or iso-propyl, cyclopropyl, cyclopentyl, cyclohexyl, phenyl,
or pyridyl. In
cases where macrophage specificity is not required, R4 may be H, -(C=0)F13, -
(C=0)0R3,
CA 02836827 2013-12-17
or ¨(C=0)NR3 wherein R3 is hydrogen or optionally substituted (C1-C6)alkyl,
for example
methyl, ethyl, or n- or iso-propyl, and CH2CH2OH.
The ring or ring system B
Ring or ring system B may be one chosen from, for example, the following:
D i b:H. R1
N ..
410 NH 0 NH
H
R1 R1
¨0
1- - - -
D i R1 R1
N r, i 10 NH 0NH
H
The radical ¨Y-L-X-ECH,1,-
When the alpha amino acid ester is conjugated to the inhibitor as a radical of
formula (IA)
this radical (or bond) arises from the particular chemistry strategy chosen to
link the amino
acid ester motif R1CH(R2)NH- to the modulator. Clearly the chemistry strategy
for that
coupling may vary widely, and thus many combinations of the variables Y, L, X
and z are
possible.
It should also be noted that the benefits of the amino acid ester
carboxylesterase motif
described above (facile entry into the cell, carboxylesterase hydrolysis
within the cell, and
accumulation within the cell of active carboxylic acid hydrolysis product) are
best achieved
when the linkage between the amino acid ester motif and the modulator is not a
substrate
for peptidase activity within the cell, which might result in cleavage of the
amino acid from
the molecule. Of course, stability to intracellular peptidases is easily
tested by incubating
the compound with disrupted cell contents, and analysing for any such
cleavage.
With the foregoing general observations in mind, taking the variables making
up the radical
¨Y-L-X1CH21,- in turn:
z may be 0 or 1, so that a methylene radical linked to the modulator is
optional.
CA 02836827 2013-12-17
16
specific preferred examples of Y include a bond, ¨(C=0)-, -(C=0)NH-, and
-(C=0)0-. However, for hCE-1 specificity when the alpha amino acid ester is
conjugated to the inhibitor as a radical of formula (IA), Y should be a bond.
In the radical L, examples of Alkl and Alk2 radicals, when present, include
¨CH2-, ¨CH2CH2- ¨CH2CH2CH2-, ¨CH2CH2CH2CH2-, ¨CH=CH-,
¨CH=CHCH2-, ¨CH2CH=CH-, CH2CH=CHCH2-,¨CC-, ¨CECCH2-, CH2CEC-, and
CH2CECCH2. Additional examples of All(' and Alk2 include ¨CH2W-,
¨CH2CH2W- ¨CH2CH2WCH2-, -CH2CH2WCH(CH3)-, ¨CH2WCH2CH2-,
¨CH2WCH2CH2WCH2-, and ¨WCH2CH2- where W is ¨0-, -S-, -NH-,
¨N(CH3)-, or ¨CH2CH2N(CH2CH2OH)CH2-. Further examples of Alkl and Alk2
include divalent cyclopropyl, cyclopentyl and cyclohexyl radicals.
In L, when n is 0, the radical is a hydrocarbon chain (optionally substituted
and
perhaps having an ether, thioether or amino linkage). Presently it is
preferred that
there be no optional substituents in L. When both m and p are 0, L is a
divalent
mono- or bicyclic carbocyclic or heterocyclic radical with 5 - 13 ring atoms
(optionally substituted). When n is 1 and at least one of m and p is 1, L is a
divalent
radical including a hydrocarbon chain or chains and a mono- or bicyclic
carbocyclic
or heterocyclic radical with 5 - 13 ring atoms (optionally substituted). When
present,
Q may be, for example, a divalent phenyl, naphthyl, cyclopropyl, cyclopentyl,
or
cyclohexyl radical, or a mono-, or bi-cyclic heterocyclic radical having 5
to13 ring
members, such as piperidinyl, piperazinyl, indolyl, pyridyl, thienyl, or
pyrrolyl radical,
but 1,4-phenylene is presently preferred.
Specifically, in some embodiments of the invention, m and p may be 0 with n
being
1. In other embodiments, n and p may be 0 with m being 1. In further
embodiments,
m, n and p may be all 0. In still further embodiments m may be 0, n may be 1
with Q
being a monocyclic heterocyclic radical, and p may be 0 or 1. Alkl and A1k2,
when
present, may be selected from ¨CH2-,
¨CH2CH2-, and ¨CH2CH2CH2- and Q may be 1,4-phenylene.
Specific examples of the radical ¨Y-L-X[CH2]- include ¨C(=0)- and ¨C(=0)NH- as
well as
-(CH2)v-, -(CH2)O-, -C(=0)-(CF12)v-, -C(=0)-(CH2),0-, -C(.0)-NH-(CH2)w-, -
C(=0)-NH-
(CH2)w0-
CA 02836827 2013-12-17
17
4-0--(CH2),0 and+¨
wherein v is 1, 2, 3 or 4 and w is 1, 2 or 3, such as -CH2-, -CH20-, -C(=0)-
CH2-, - C(=0)-
CH20-, -C(=0)-NH-CH2-, and -C(=0)-NH-CH20-.
The radical ¨L-Y1-
When the alpha amino acid ester is conjugated to the inhibitor as a radical of
formula (IB)
this radical (or bond) arises from the particular chemistry strategy chosen to
link the alpha
carbon of the amino acid ester motif in formula (IB) or (IC) to the modulator.
(In the latter
case, the ¨L-Y1- radical is indirectly linked to the alpha carbon through the
intervening ring
atoms of ring system B.) Clearly the chemistry strategy for that coupling may
vary widely,
and thus many combinations of the variables L and Y1 are possible.
For example, L may be as discussed above in the context of the radical
¨Y-L-X-[CH2]2-. For example, in some embodiments m and n are 1 and p is 0; Q
is ¨0-;
and Alk1 is an optionally substituted, straight or branched, C1-C6 alkylene,
C2-C6 alkenylene
or C2-C6 alkynylene radical which may optionally contain or terminate in an
ether (-0-),
thioether (-S-) or amino (NR'-) link wherein RA is hydrogen or optionally
substituted C1-C4
alkyl. In other embodiments, m, n and p may each be 1, and in such cases Q may
be, for
example a 1,4 phenylene radical, or a cyclopentyl, cyclohexyl, piperidinyl or
piperazinyl
radical. In all embodiments, Y1 may be, for example, a bond, or ¨(C=0)-, -
(C=0)NH-, and -
(C=0)0-.
For compounds of the invention which are to be administered systemically,
esters with a
slow rate of carboxylesterase cleavage are preferred, since they are less
susceptible to
pre-systemic metabolism. Their ability to reach their target tissue intact is
therefore
increased, and the ester can be converted inside the cells of the target
tissue into the acid
product. However, for local administration, where the ester is either directly
applied to the
target tissue or directed there by, for example, inhalation, it will often be
desirable that the
ester has a rapid rate of esterase cleavage, to minimise systemic exposure and
consequent unwanted side effects. Where the esterase motif is linked to the
modulator via
its amino group, as in formula (IA) above, if the carbon adjacent to the alpha
carbon of the
alpha amino acid ester is monosubstituted, ie R2 is CH2R1 (Rz being the mono-
substituent)
then the esters tend to be cleaved more rapidly than if that carbon is di- or
tri-substituted,
as in the case where R2 is, for example, phenyl or cyclohexyl. Similarly,
where the esterase
CA 02836827 2013-12-17
18
motif is linked to the modulator via a carbon atom as in formulae (IB) and
(IC) above, if a
carbon atom to which the RINHCH(Ri)- or R1-(ring B)- esterase motifs are
attached is
unsubstituted, ie RINHCH(R1)- or R1-(ring B)- is attached to a methylene (-
CH2)- radical,
then the esters tend to be cleaved more rapidly than if that carbon is
substituted, or is part
of a ring system such as a phenyl or cyclohexyl ring.
Modulators of Intracellular Enzymes and Receptors
The principles of this invention can be applied to modulators of a wide range
of intracellular
targets which are implicated in a wide range of diseases. As discussed, the
binding modes
of known modulators to their targets are generally known soon after the
modulators
themselves become known. In addition, modem techniques such as X-ray
crystallography
and NMR are capable of revealing such binding topologies and geometries, as
are
traditional medicinal chemistry methods of characterising structure-activity
relationships.
With such knowledge, it is straightforward to identify where in the structure
of a given
modulator an carboxylesterase ester motif could be attached without disrupting
the binding
of the modulator to the enzyme or receptor by use of structural data. For
example, Table 1
lists some intracellular enzyme or receptor targets where there is published
crystal
structural data.
Table 1
Nam et al., J Exp Med 201, 441
CD45 Autoimmune disease
(2005)
Lck Zhu et al., Structure 7, 651 (1999) Inflammation
Jin et al., J Biol Chem 279, 42818
ZAP-70 Autoimmune disease
(2004)
Huai et al., Biochemistry 42,
PDE4 Inflammation
13220 (2003)
Scapin et al., Biochemistry 43,
PDE3 Asthma
6091 (2004)
IMPDH Intchak et al., Cell 85,921 (1996) Psoriasis
Wang et al., Structure 6, 1117
p38 MAPK Inflammation
(1998)
Kiefer et al., J Biol Chem 278,
COX2 Inflammation
45763, (2003)
Schumacher et al., J Mol Biol 298,
Adenosine Kinase Inflammation
875 (2000)
Chandra et al., Biochemistry B
PLA2 Psoriasis
10914 (2002)
Essen et al., Biochemistry 36,
PLC Rheumatoid arthritis
1704, (1997)
Leiros et al., J Mol Biol 339, 805
PLO Inflammation
(2004)
CA 02836827 2013-12-17
19
iNOS Rosenfeld et al., Biochemistry 41,
Inflammation
13915 (2002)
Rudberg et al., J Biol Chem 279,
Inflammation
LTA4 hydrolase 27376 (2004)
Okamato et al., Chem Pharm Bull
ICE Rheumatoid arthritis
47, 11(1999)
Bertrand et al., J Mol Biol 333, 393
GSK313 Rheumatoid arthritis
(2003)
PKC Xu et al., JBC 279, 50401 (2004) Inflammation
PARP Rut et al., PNAS (USA) 93, 7481 Proliferative
(1996) disorders
MetAP2 Sheppard et al Bioorg Med Chem
Rheumatoid arthritis
Lett 14, 865 (2004)
Corticosteroid receptor Bledsoe at al, Cell 110, 93 (2002) Inflammation
PI3K Walker et al., Mol Cell Bid 6, 909 Proliferative
(2000) disorders
Proliferative
Raf Wan et al., Cell 116, 855 (2004) disorders
Yang et al., Nat Struct Biol 9, 940 Proliferative
AKT/PKB (2002) disorders
HDAC Finnin et al., Nature 401, 188 Proliferative
(1999) disorders
c-Abl Nagar et al., Cancer Res 62, 4236 Proliferative
(2002) disorders
IGF-1R Munshi et al., Acta Crystallogr Proliferative
Sect D 59, 1725 (2003) disorders
Thymidylate Stout et al., Structure 6, 839 Proliferative
Synthetase (1998) disorders
Glycinamide Klein et al., J Mol Biol 249, 153 Proliferative
Ribonucleotide
(1995) disorders
Formyttransferase
Purine Nucleoside Koelner et al., J Mol Biol 280, 153 Proliferative
Phosphorylase (1998) disorders
Hernandez-Guzman et al., J Bid Proliferative
Estrone Sulphatase Chem 278, 22989(2003) disorders
Stamos at al., J Biol Chem 277, Proliferative
EG F-RTK 46265 (2002) disorders
Lamers et al., J Mol Biol 285, 713 Proliferative
Src kinase (1999) disorders
McTigue et al., Structure 7, 319 Proliferative
VEGFR2 (19999) disorders
Hough at al., J Mol Biol 287, 579 Proliferative
Superoxide Dismutase (1999) disorders
Ornithine Almrud et al., J Mol Biol 295, 7 Proliferative
Decarboxylase (2000) disorders
Classen et al., PNAS (USA) 100, Proliferative
Topoisomerase II
10629(2003) disorders
Staker at al., PNAS (USA), 99, Proliferative
Topoisomerase I
15387 (2002) disorders
Matias et al., J Bid Chem 275, Proliferative
Androgen Receptor 26164 (2000) disorders
JNK Heo at al., EMBO J 23, 2185 Proliferative
(2004) disorders
CA 02836827 2013-12-17
Curtin of at., Bioorg Med Chem Proliferative
Famesyl Transf erase Lett 13, 1367(2003) disorders
Davis et al., Science 291, 134 Proliferative
CDK (2001) disorders
Dihydrofolate Gargaro et al., J Mol Biol 277, 119 Proliferative
Reductase (19981 disorders
Griffith et al., Mol Cell 13, 169 Proliferative
Flt3 (2004) disorders
Stams at al., Protein Sci 7, 556 Proliferative
Carbonic Anhydrase (1998) disorders
Thymidine Norman at at., Structure 12, 75 Proliferative
Phosphorylase (2004) disorders
Dihydropyrimidine Dobritzsch of at., MC 277, 13155, Proliferative
Dehydrogenase (2002) disorders
Van den Eisen at al., EMBO J 20, Proliferative
Mannosidase a 3008(2001) disorders
Peptidyl-prolyl Ranganathan et at., Cell 89, 875 Proliferative
isomerase (Pi n1) (1997) disorders
Egea at al., EMBO J 19,2592 Proliferative
Retinoid X Receptor (2000) disorders
Jain et al., Nat Struct Biol 3, 375 Proliferative
fl-Glucuronidase (1996) disorders
Glutathione Oakley at at., J Mol Blot 291, 913 Proliferative
Transferase (1999) disorders
Jez at al., Chem Bid 10, 361 Proliferative
hsp90 (2003) disorders
IMPDH Intchak et al., CeN 85, 921 (1996) Proliferative
disorders
Chandra et al., Biochemistry 41, Proliferative
Phospholipase A2 10914 (2002) disorders
Essen of at., Biochemistry 36, Proliferative
Phospholipase C 1704, (1997) disorders
Leiros et at., J Mol Biol 339, 805 Proliferative
Phosphollpase D (2004) disorders
Sheppard at at Bioorg Med Chem Proliferative
MetAP2 Lett 14, 865(2004) disorders
PTP-113 Andersen et al., J Biol Chem 275, Proliferative
7101 (2000) disorders
Fence!!i at a1.,J.Med Chem 2006, Proliferative
Aurora Kinase 49, 7249-7251 disorders
Komander at at., Biochem J 375, Proliferative
PDK-1 255 (2003) disorders
Istvan and Delsenhofer Science
HIVIGCoA reductase Atheriosclerosis
292, 1160(20011
Oxidosqualene Lenhart at at., Chem Biol 9,639 Hypercholesteroiaem
cyclase (2002) la
Pyruvate Mattevi at al., Science 255, 1544 Cardiovascular
dehydrogenase
stimulator (1992) disease
Zhang et al., Nature 388, 247 Cardiovascular
Adenylate cyclase (1997) .. disease
PPARy agonist Ebdurp et al., J Med Chem 48, Diabetes
1306 (2003)
CA 02836827 2013-12-17
21
Alcohol Bahnson et al., PNAS USA 94,
Alcohol poisoning
dehydrogenase 12797 (1997)
Hormone sensitive Wei et al., Nat Struct Biol 6, 340 Insulin resistant
lipase (1999) diabetes
Mathews et al., Biochemistry 37, Epilepsy
Adenosine kinase
15607 (1998)
Urzhmsee al.,Structure 5, 601
Aldose reductase Diabetes
(1997)
Tocchini-Valentini et al., PNAS
Vitamin 03 receptor USA 98, 5491 (2001) Osteoporosis
Protein tyrosine Andersen et al., J Biol Chem 275, Diabetes
phosphatase 7101 (2000)
Louis et al., Biochemistry 37, 2105 HIV
HIV Protease
(1998)
Bressanelli et al., PNAS USA 96,
HCV Polymerase Hepatitis C
13034 (1999)
Neuraminidase Taylor et al., J Med Chem 41, 798 Influenza
(1998)
Das et al., J Mol Biol 264, 1085
Reverse Transcriptase HIV
(1996)
Khayat et al., Biochemistry 42,
CMV Protease CMV infection
885 (2003)
Champness et al., Proteins 32,
Thymidine Kinase Herpes infections
350 (1998)
Molteni et al., Acta Crystallogr
HIV Integrase HIV
Sect D 57, 536 (2001)
For the purpose of illustration, reference is made to known inhibitors of 5 of
the above
intracellular targets, whose binding mode to the target is known. These
examples illustrate
how such structural data can be used to determine the appropriate positions
for the
attachment of the carboxylesterase ester motif. Schematics of the active sites
are shown
together with representative inhibitors. In general, positions remote from the
binding
interface between modulator and target, and therefore pointing away from the
enzyme
binding interface into solvent are suitable places for attachment of the
carboxylesterase
ester motif and these are indicated in the diagrams.
CA 02836827 2013-12-17
22
HDAC
Head Hydrophobic
Group Linker
Metal Binding
Group
\ HOH
iiiki U.r7..1
Solvent
illri ____________________________
Esterase
Motif
Attachment
Aurora kinase
0 Specific aurora kinase
7-----
ICI II Ar binding site
XJI
\ X ......}
X
=
2:0
Solvent - 01 C' X
70 N,
.1-1 lAdenine binding site
Esterase
motif
attachment
H bond acceptor site
P13 Kinase
Solvent
Esterase
motif
attachment
Adenine H-bond acceptor site
binding site
CA 02836827 2013-12-17
23
P38 MAP Kinase
Adenine
Kinase binding pocket
selectivity
pocket
x--- _____________ \\õ-- __ ,- H-bond acceptor site
0.
/
N 40, Solvent
NH2
...... 1* 0
.......... -,"''
Esterase
motif
attachment
1KK kinase
Kinase
H selectivity
Pocket
N--.1
SO(
SI
NL: 1 SI Solvent
N N 0 _____
_________________________ \
: H
Adenine ¨ \
binding
site H bond ________________ Esterase
H bond donor motif
acceptor ___________________ attachment
A similar approach can also be used for the other examples identified in Table
1.
The method of the invention, for increasing cellular potency and/or
intracellular residence
time of a modulator of the activity of a target intracellular enzyme or
receptor, may involve
several steps:
Step 1: Identify a position or positions on one or a plurality of modulator
molecules
sharing the same binding mode for the target enzyme or receptor, remote from
the binding
interface between the modulators and the target enzyme or receptor.
CA 02836827 2013-12-17
24
Usually such positions are identified from the X-ray co-crystal structure (or
structure
derived by nmr) of the target enzyme or receptor with a known modulator (or a
close
structural analogue thereof) bound to the enzyme or receptor, by inspection of
the
structure. Alternatively the X-ray crystal structure of the target enzyme or
receptor with the
modulator docked into the active site of the enzyme or receptor is modelled by
computer
graphics methods, and the model is inspected The presumption is that
structural
modification of the modulator at positions remote from the binding interface
is unlikely to
interfere significantly with the binding of the modulator to the active site
of the enzyme or
receptor. Suitable positions will normally appear from the co-crystal
structure or docked
model to be orientated towards solvent.
Step 2: Covalently modify the modulator(s) by attachment of an alpha amino
acid
ester radical, or a range of different alpha amino acid ester radicals at one
or more of the
positions identified in Step 1.
Attachment of alpha amino acid ester radicals (ie the potential
carboxylesterase motifs)
may be via an existing covalent coupling functionality on the modulator(s), or
via a suitable
functionality specifically introduced for that purpose. The carboxylesterase
motifs may be
spaced from the main molecular bulk by a spacer or linker element, to position
the motif
deeper into solvent and thereby reduce still further any small effect of the
motif on the
binding mode of the modulator and/or to ensure that the motif is accessible to
the
carboxylesterase by reducing steric interference that may result from the main
molecular
bulk of the modulator.
Performance of Step 2 results in the preparation of one or, more usually, a
small library of
candidate modulators, each covalently modified relative to its parent
inhibitor by the
introduction of a variety of amino acid ester radicals, at one or more points
of attachment
identified in Step 1.
Step 3: Test the alpha amino acid-conjugated modulator(s) prepared in step
2 to
determine their activity against the target enzyme or receptor.
As is normal in medicinal chemistry, the carboxylesterase motif version(s) of
the parent
modulator(s), prepared as a result of performing Steps 1 and 2, are preferably
tested in
assays appropriate to determine whether the expected retention of modulator
activity has in
CA 02836827 2013-12-17
fact been retained, and to what degree and with what potency profile. In
accordance with
the underlying purpose of the invention, which is to cause the accumulation of
modulator
activity in cells, suitable assays will normally include assays in cell lines
to assess degree
of cellular activity, and potency profile, of the modified modulators. Other
assays which may
be employed in Step 3 include in vitro enzyme or receptor modulation assays to
determine
the intrinsic activity of the modified modulator and its putative
carboxylesterase hydrolysis
product; assays to determine the rate of conversion of the modified modulators
to the
corresponding carboxylic acid by carboxylesterases; and assays to determine
the rate and
or level of accumulation of the carboxylesterase hydrolysis product (the
carboxylic acid) in
cells. In such assays, both monocytic and non-monocytic cells, and/or a panel
of isolated
carboxylesterases, can be used in order to identify compounds that show cell
selectivity.
If necessary or desirable, step 3 may be repeated with a different set of
candidate alpha
amino acid ester-conjugated versions of the parent modulator.
Step 4: From data
acquired in Step 3, select one or more of the tested alpha amino
acid ester-conjugated versions of the parent modulator(s) which cause
modulation of
enzyme or receptor activity inside cells, are converted to and accumulate as
the
corresponding carboxylic acid inside cells, and which show increased or
prolonged cellular
potency.
The above described Steps 1-4 represent a general algorithm for the
implementation of the
principles of the present invention. The application of the algorithm is
illustrated in Example
A below, applied to a known inhibitor of the intracellular enzyme
dihydrofolate reductase
(DHFR).
Example A
Folic (pteroylglutamic) acid is a vitamin which is a key component in the
biosynthesis of
purine and pyrimidine nucleotides. Following absorption dietary folate is
reduced to
dihydrofolate and then further reduced to tetrahydrofolate by the enzyme
dihydrofolate
reductase (DHFR). Inhibition of DHFR leads to a reduction in nucleotide
biosynthesis
resulting in inhibition of DNA biosynthesis and reduced cell division. DHFR
inhibitors are
widely used in the treatment of cancer (Bertino J, J.Cin. Oncol. 11,5-14,
1993), cell
proliferative diseases such as rheumatoid arthritis (Cronstein N., Pharmacol.
Rev. 57, 163-
1723), psoriasis and transplant rejection. DHFR inhibitors have also found use
as
CA 02836827 2013-12-17
26
antiinfective (Salter A., Rev. Infect. Dis. 4,196-236, 1982) and antiparasitic
agents (Plowe
C. BMJ 328, 545-548, 2004).
Many types of DHFR inhibitor compounds have been suggested, and several such
compounds are used as anti-cancer, anti-inflammatory, anti-infective and anti-
parasitic
agents. A general templates for known DHFR inhibitors is shown below:
N NH2 4.R
Linker
H2NA.N
G = N or CH
1
Methotrexate (S)-2-(4-(((2,4-diaminopteridin-6-yl)methyl)methylamino)-
benzamido)pentanedioic acid is the most widely used DHFR inhibitor and
contains a
glutamate functionality which enables it to be actively transported into, and
retained inside,
cells. However cancer cells can become resistant to methotrexate by modifying
this active
transport mechanism. Furthermore non-mammalian cells lack the active transport
system
and methotrexate has limited utility as an anti-infective agent. Lipophilic
DHFR inhibitors
such as trimetrexate (2,4-diamino-5-methyl-64(3,4,5-
trimethoxyanilino)methyliquinazoline)
(2 G = CH) (GB patent 1345502) and analogues such as (2 G = N) (Gangjee et al
J.Med.Chem. 1993, 36, 3437-3443) which can be taken up by passive diffusion
have
therefore been developed both to circumvent cancer cell resistance and for use
as anti-
infective agents.
OMe
OMe
N H2
NN OMe
H2N N G
G = CH or N
2
However agents that passively diffuse into cells will also exit the cell
readily and are not
readily retained inside the cell. Thus a DHFR inhibitor modified in accordance
with the
present invention, that is lipophilic but whose activity accumulates inside
the cell could
CA 02836827 2013-12-17
27
have significant advantages. Furthermore both classes of DHFR inhibitors have
side
effects which limit the doses that can be used in the clinic. A DHFR inhibitor
whose activity
accumulates selectively in macrophages could have value as macrophages, via
the
production of cytokines, are known to play a key role in inflammatory
disorders and
evidence is increasing that they have a negative role in cancer.
Step 1 of the General Algorithm Described Above
The nmr structure of DHFR with trimetrexate (2 G = CH) docked in the active
site is
published (Polshakov, V.I. at al, Protein Sc!. 1999, 8,467-481) and it is
apparent that the
most appropriate position to append a carboxylesterase motif in accordance
with the
invention was on the phenyl ring as shown below. It was inferred that
attachment at that
point in known close structural analogues of trimetrexate, such as (2 G = N)
would also be
suitable. Fgure 2 shows (2 G = N) docked into DHFR showing that a suitable
point for
attachment is the 4 position of the aromatic ring since this points away from
the active site
of the enzyme.
Stet) 2 of the General Algorithm Described Above
Compounds in which the carboxylesterase motif is linked via its alpha amino
acid nitrogen
were prepared as shown in schemes I and II. Within this series compounds with
and
without a carbonyl were made to identify potential macrophage selective
compounds.
CA 02836827 2013-12-17
28
Scheme I
BocNH 0,
BocNHcOH ' c' ----4-
H2N-con
0 0
0 0 _____
ci
0
0
NO2
0 N õCO
, __________________________________________ 0 NO
0 H 0
H2N SI H 0 'ICi
02N
NH2
Raney/Ni
A.-......, ....- H2
N N-
H2N _
NH2 0
0 ticc'n
NN
H
H2N N N''''
i LiOH
0
NH, 401 vicoH
0
H
, ......,--
H2N N N
CA 02836827 2013-12-17
29
Scheme II
BocNH4µ0H ------.-
BooNHc0n-1.- H2Nc(:),
0
0 0
0 H1
1.1
NO2
is 1,140,..so
0
02N40 r40,0
H2N
NH2
N )x-,e,CN
Raney/Ni
AH2
.,- e..,='..,
H2N N N -
NH2 0
0
I*1-"L'"--'''= N
i 1 H
i., ..õ,
H2N N N
i LiOH
0 Hi,' fOH
NH2
0
isrl-ksYN
),IH
....% ,..:-.
H2N N N
Compounds were also made in which the esterase motif was linked to the
modulator via
the alpha amino acid side chain schemes III and IV. Within this series
compounds with
CA 02836827 2013-12-17
alkyl substitution on the nitrogen were also prepared to identify macrophage
selective
compounds (scheme IV).
Scheme III
Br HO . NO2
411 NO2
Cs2Co3
c)HBoc .()0
IrCINHBoc
0
0
1 H2
0 0.---0
H2N.-...,
0 .NH,
(i) Raney/NI
NH, 41 (ii) TFA oyLINHBoc
N LX-L-`=7N
H2N N N +
WH2
N)1CN
A
LiOH
H2N N N
V
0
._....OH
H2N
0
NH, .
reiN
A H
H2N N N
CA 02836827 2013-12-17
31
Scheme IV
O . NO2
(-,' oyCjNHBoc
TFA
0 0 0.--0
0¨ri--,. NO2
Na(0Ac)3BH
0 . _____
0 0
*
a 0
02N
NH2
0 0 --0 0-11
N CN
A it 0¨
N 0
-... ..-,.;-õ, .;-.. H H2N
_________________________________ . NH2
N N
0
= N"'INFI
A
H2N N N
H2N
o
OH
0- ri
= /
0
NH2
NI)N
A H
H2N N N
CA 02836827 2013-12-17
32
Steo 3 of the General Algorithm Described Above
The compounds including the trimetrexate analogue (2 G = N) were tested in the
DHFR
enzyme assay, the cell proliferation assay, using both monocytic and non-
monocytic cell
lines, and the broken cell assay in order to assess the cleavability of the
esters by
monocytic and non-monocytic cell lines. Details of all these assays are given
below.
Step 4 of the General Alaorithm Described Above
As shown in Table 2 compounds were identified whose acids have activity
against the
enzyme comparable to the trimetrexate analogue (2 G = N). It can also be seen
that by
altering the way in which the esterase motif is linked can lead to a compound
(6) that is
100 fold more potent in U937 cells and 15 fold more potent in the HCT116 than
the
unmodified analogue (2 G=N).
In addition modifications of either the linker or the substituent on the
esterase motif nitrogen
allowed identification of compounds 4 and 5 that showed selective cleavage in
monocytic
but not non-monocytic cell lines and which were significantly more anti-
proliferative in
monocytic cell lines then non-monocytic cell lines (see table 2).
CA 02836827 2013-12-17
33
U937 (Monocytic cell line) HCT116
(non-monocytic cell
line)
Compound IC50 nM IC50 nM Ratio Acid IC50 nM Ratio
Acid
enzyme cell IC50 Produce cell prolif-
IC50 produced
'(acid) profit- cell/ d eration Cell/
2n/m1
eration enzyme 2ng/m1 enzyme
(2 G=N) 10 ' 2200 220 NA 1700 170 NA
....-
rak, 0.
14.
i
HEN '11
3 2700 5100 1.9 80 7300 1.4 180
i n (11)
(61 .
4 1033 310 0.3 970 6900 6.6 40
7 (25)
NNAtX&TN N cc" '
4000 310 0.8 210 6700 1.7 2
(10)
N., 1 011t 1140
=9.11:5)11 ID
6 1700 23 0.013 110 110 0.04 150
(8)
Kor -I, 'II
Table 2
Notes
1 The figures in brackets refer to the enzyme 1C5Os for the acid resulting
from
cleavage of the esters
CA 02836827 2013-12-17
34
2 The amount of acid produced after incubation of the ester for 80 minutes in
the
broken cell carboxylesterase assay described below
Using similar strategies the concept has successfully been applied to a range
of
intracellular targets as outlined in the examples below.
By way of further illustration of principles of this invention the following
Examples are
presented. In the compound syntheses described below:
Commercially available reagents and solvents (HPLC grade) were used without
further
purification.
Microwave irradiation was carried out using a CEM Discover focused microwave
reactor.
Solvents were removed using a GeneVac Series I without heating or a Genevac
Series II
with VacRamp at 30 C.
Purification of compounds by flash chromatography column was performed using
silica gel,
particle size 40-63 pm (230-400 mesh) obtained from Silicycle. Purification of
compounds
by preparative HPLC was performed on Gilson systems using reverse phase
ThermoHypersil-Keystone Hyperprep HS C18 columns (12 gm, 100 X 21.2 mm),
gradient
20-100% B ( A= water/ 0.1% TFA, B= acetonitrile/ 0.1% TFA) over 9.5 min, flow
= 30
ml/min, injection solvent 2:1 DMSO:acetonitrile (1.6 ml), UV detection at 215
nm.
1H NMR spectra were recorded on a Bruker 400 MHz AV spectrometer in deuterated
solvents. Chemical shifts (8) are in parts per million. Thin-layer
chromatography (TLC)
analysis was performed with Kieselgel 60 F254 (Merck) plates and visualized
using UV light.
Analytical HPLCMS was performed on Agilent HP1100, Waters 600 or Waters 1525
LC
systems using reverse phase Hypersil BDS C18 columns (5 gm, 2.1 X 50 mm),
gradient 0-
95% B ( A= water/ 0.1% TFA, B= acetonitrile/ 0.1% TFA) over 2.10 min, flow =
1.0 ml/min.
UV spectra were recorded at 215 nm using a Gilson G1315A Diode Array Detector,
G1214A single wavelength UV detector, Waters 2487 dual wavelength UV detector,
Waters 2488 dual wavelength UV detector, or Waters 2996 diode array UV
detector. Mass
spectra were obtained over the range m/z 150 to 850 at a sampling rate of 2
scans per
second or 1 scan per 1.2 seconds using Micromass LCT with Z-spray interface or
Micromass LCT with Z-spray or MUX interface. Data were integrated and reported
using
OpenLynx and OpenLynx Browser software
The following abbreviations have been used:
Me0H = Me0H
CA 02836827 2013-12-17
Et0H = Et0H
Et0Ac = Et0Ac
Boc = tert-butoxycarbonyl
DCM = DCM
DMF = dimethylformamide
DMSO = dimethyl sulfoxide
TFA = trifluoroacetic acid
THF = tetrahydrofuran
Na2CO3 = sodium carbonate
HCI = hydrochloric acid
DIPEA = diisopropylethylamine
NaH = sodium hydride
NaOH = sodium hydroxide
NaHCO3= sodium hydrogen carbonate
Pd/C = palladium on carbon
TBME = tert-butyl methyl ether
N2 = nitrogen
PyBop = benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate
Na2SO4 = sodium sulphate
Et3N = triethylamine
NH3 = ammonia
TMSCI = trimethylchlorosilane
NH4CI = ammonium chloride
L1AIH4 = lithium aluminium hydride
PyBrOP = Bromo-tris-pyrrolidino phosphoniumhexafluorophosphate
MgSas = MgSO4
"BuLi = n-butyllithium
CO2 = carbon dioxide
EDCI = N-(3-DimethylaminopropyI)-Af-ethylcarbodiimide hydrochloride
Et20 = diethyl ether
LiOH = lithium hydroxide
HOBt = 1-hydroxybenzotriazole
ELS = Evaporative Light Scattering
TLC = thin layer chromatography
ml = millilitre
g = gram(s)
CA 02836827 2013-12-17
36
mg = milligram(s)
mol = moles
mmol = millimole(s)
LCMS = high performance liquid chromatography/mass spectrometry
NMR = nuclear magnetic resonance
r. t. --.- room temperature
min = minute(s)
h = hour(s)
CA 02836827 2013-12-17
37
INTERMEDIATES
The following building blocks were used for the synthesis of the modified
modulators:
NHBocHr
0 µ-.0 NHBocr ys>
(S)-2-tert-Butoxycarbonylamino-4- (S)-4-Bromo-2-tert-butoxycarbonylamino-
hydroxy-butyric acid cyclopentyl ester butyric acid cyclopentyl ester
11101
H2Nt2 )::),
H N
2 0 I-1)
(S)-2-Amino-4-methyl-pentanoic acid (S)-Amino-phenyl-acetic acid
cyclopentyl ester cyclopentyl ester
HOO NHe=-ir,Ø,..e=
NHBoc 0 I
(S)-2-tert-Butoxycarbonylamino- (S)-2-Benzyloxycarbonylamino-4-
pentanedioic acid 1-cyclopentyl ester bromo-butyric acid tert-butyl ester
HOO
NHBoc
(S)-2-tert-Butoxycarbonylamino-
pentanedioic acid 1-tert-butyl ester
CA 02836827 2013-12-17
38
Synthesis of (S)-2-tert-Butoxycaitoonylamino-4-hydroxy-butyric acid
cyclopentyl ester and
(S)-2-telt-Butoxvcarbonvlamino-4-hydroxy-butvric acid 1-tert-butyl ester
OH
, Si*
0 \ \
BoC20. EtaN. DCM
TBDMSCI.
H2N40H DEW, MeCN
H2N.eirOH Stage 2 BocNH4OH
0 Slap 1
0 0
yclo! D)entanEDCIol MAP 3
DCM
Br ti53S,
pp ?H
OH
DCM
BocNH
0 LI
Maga 5 BoeNH r0
0 Acom THF / H20
Stags 4 0 \
BocNH 40 0
0
Stage 1 ¨ Synthesis of (S)-2-Amino-4-(tert-butyl-dimethyl-silanyloxy)-butyric
acid
0
Hp140H
To a suspension of L-homoserine (1g, 8.4mmol) in acetonitrile (10m1) at 0 C
was added
1,8-diazabicyclo[5.4.01undec-7-ene (1.32m1, 8.8mmol, 1.05eq). Tert-butyl-
dimethyl-sily1
chloride (1.33g, 8.8mmol, 1.05eq) was then added portionwise over 5 min and
the reaction
mixture allowed to warm to r. t. and stirred for 16 h. A white precipitate had
formed which
was filtered off and washed with acetonitrile before drying under vacuum. The
title
compound was isolated as a white solid (1.8g, 92%). 1H NMR (500 MHz, DMSO), &
7.5
(1H, bs), 3.7 (1H, m), 3.35 (4H, bm), 1.95 (1H, m), 1.70 (1H, m), 0.9 (9H, s),
0.1 (6H, s).
CA 02836827 2013-12-17
39
Stage 2 ¨ Synthesis of (S)-2-tert-Butoxycarbonylamino-4-(tert-butyl-dimethyl-
silanyloxy)-
butyric acid
BccHN40H
0
A suspension of Stage 1 product (1.8g, 7.7mmol) in DCM (100m1) at 0 C was
treated with
triethylamine (2.15m1, 15.4mmol, 2eq) and di-tert-butyl dicarbonate (1.77g,
8.1mmol,
1.05eq). The reaction mixture was stirred at r. t. for 16 h for complete
reaction. The DCM
was removed under reduced pressure and the mixture was treated with Et0Ac /
brine. The
Et0Ac layer was dried over MgSat and evaporated under reduced pressure. The
crude
product was taken forward without further purification (2.53g, 99%). 'H NMR
(500 MHz,
CDCI3), 8: 7.5(1H, bs), 5.85 (1H, d, J = 6.5Hz), 4.3(1H, m), 3.75 (2H, m),
1.95 (2H, m),
1.40 (9H, s), 0.85 (9H, s), 0.1 (6H, s).
Stage 3 ¨ Synthesis of (S)-2-tert-Butoxycarbonylamino-4-(tert-butyl-dimethyl-
silanyloxy)-
butyric acid cyclopentyl ester
,I9l*
0 \
BocHN400
0
To a solution of Stage 2 product (2.53g, 7.6mmol) in DCM (50m1) at 0 C was
added
cyclopentanol (1.39m1, 15.3m1, 2eq), EDCI (1.61g, 8.4mmol, 1.1eq) and DMAP
(0.093g,
0.76mmol, 0.1eq). The reaction mixture was stirred for 16 hat r. t. before
evaporation
under reduced pressure. The crude residue was dissolved in Et0Ac (100m1) and
washed
with 1M HCI, 1M Na2CO3 and brine. The organic layer was then dried over MgSO4
and
evaporated under reduced pressure. The product was purified by column
chromatography
using Et0Ac / heptane (1:4) to yield the title compound (2.24g, 73%). LCMS
purity 100%,
CA 02836827 2013-12-17
m/z 402.5 [M++H], 1H NMR (250 MHz, CDCI3), 8: 5.2 (1H, d, J = 6.3Hz), 5.15
(1H,m), 4.2
(1H, m), 3.6 (2H, m), 2.0 (1H, m), 1.95-1.55 (9H, bm), 1.4 (9H,$), 0.85
(9H,$), 0.1 (6H,$).
Stage 4¨ Synthesis of (S)-2-tert-Butoxycarbonylamino-4-hydroxy-butyric acid
cyclopentyl
ester
H
BocHNON
0 I,/
Stage 3 product (1.57g, 3.9mmol) was dissolved in acetic acid:THF:water
(3:1:1, 100m1).
The reaction mixture was stirred at 30 C for 16 h for complete reaction. Et0Ac
(200m1) was
added and washed with 1M Na2CO3, 1M HCI and brine. The Et0Ac extracts were
dried
over MgSO4 and evaporated under reduced pressure to give the product as a
clear oil
which crystallised on standing (1.0g, 95%). LCMS purity 100%, m/z 310.3
[M++Na], 1Fi
NMR (250 MHz, CDCI3), 6:5.4 (1H, d, J = 6.5Hz), 5.2 (1H, m), 4.4 (1H, m), 3.65
(2H, m),
2.15 (1H, m), 1.9-1.55 (9H, bm), 1.45 (9H, s).
Stage 5 ¨ Synthesis of (S)-4-Bromo-2-tert-butoxycarbonylamino ¨butyric add
cyclopentyl
ester
Br
BocHN40\
0 1-----/
To a slurry of N-bromo succinimide (1.869, 10.4mmol) in DCM (16.2m1) was added
a
solution of triphenyl phosphine (2.56g, 9.74mmol) in DCM (7.2m1). The solution
was stirred
for a further 5 min after addition. Pyridine (338p1, 4.18mmol) was added,
followed by a
solution of Stage 4 product (1.00g, 3.48mmol) in DCM (8.8m1). The solution was
stirred for
18 h, concentrated under reduced pressure and the residual solvent azeotroped
with
toluene (3 x 16m1). The residue was triturated with diethyl ether (10m1) and
ethyl acetate:
heptane (1:9, 2 x 10m1). The combined ether and heptane solutions was
concentrated onto
silica and purified by column chromatography eluting with Et0Ac / heptane (1:9
to 2:8) to
provide the title compound (1.02g, 84%). 1H NMR (300 MHz, CDCI3), 6: 5.30-5.05
(2H, m),
CA 02836827 2013-12-17
41
4.45-4.30(1H, m), 3.45 (2H, t, J = 7.3 Hz), 2.50-2.30 (1H, m), 2.25- 2.10 (1H,
m), 1.95- 1.60
(8H, br m), 1.47 (9H, s).
Synthesis of (S)-2-Amino-4-methyl-oentanoic acid cyclooentyl ester
s.So-
0 0
\\,
S,
H2NfOH CYdoPentanol 10 0 H3Nico,0
Cyclohexane
0 0
Stage 1
NaHCO3 Stage 2
DCM
H2N
Stage 1 ¨ Synthesis of (S)-2-Amino-4-methyl-pentanoic acid cyclopentyl ester
toluene-4-
sulfonic acid
(1101
Ce(Ci H3N+ C)--
To a suspension of (S)-leucine (15g, 0.11mol) in cyclohexane (400m1) was added
cyclopentanol (103.78m1, 1.14mmol) and p-toluene sulfonic acid (23.93g,
0.13mol). The
suspension was heated at ref lux to effect' ulphate' . After ref luxing the
solution for 16 h, it
was cooled to give a white suspension. Heptane (500m1) was added to the
mixture and the
suspension was filtered to give the product as a white solid (35g, 85%). 1H
NMR (300 MHz,
Me0D), 1.01 (6H, t, J = 5.8Hz), 1.54-2.03 (11H, m), 2.39 (3H, s), 3.96 (1H, t,
J = 6.5Hz),
5.26-5.36 (1H, m), 7.25 (2H, d, J = 7.9Hz), 7.72 (2H, d, J = 8.3Hz).
Stage 2 ¨ Synthesis of (S)-2-Amino-4-methyl-pentanoic acid cyclopentyl ester
CA 02836827 2013-12-17
42
H2N----0
0
A solution of Stage 1 product (2.57g, 0.013mol) in DCM (5m1) was washed with
sat. aq.
NaHCO3 solution (2 x 3m1). The combined aq. Layers were back extracted with
DCM (3 x
4m1). The combined organic layers were dried (MgSO4), and the solvent removed
in vacuo
to give the title compound as a colourless oil (1.10g, 80%). 1H NMR (300 MHz,
CDCI3), 8:
0.90 (6H, t, J = 6.4Hz), 1.23-1.94(11H, m), 3.38 (1H, dd, J = 8.4, 5.9Hz),
5.11-5.22 (1H,
m).
Synthesis of (S)-Amino-nhenyl-acetic acid cyclooentyl ester
1101
0
H2N
il)
0
(S)-Amino-phenyl-acetic acid cyclopentyl ester was prepared from (S)-Amino-
phenyl-acetic
acid following the same procedure used for the synthesis of (S)-2-Amino-4-
methyl-
pentanoic acid cyclopentyl ester
Synthesis of (S)-2-tert-Butoxycarbonylamino-oentanedioic acid 1-cyclooentyl
ester
CA 02836827 2013-12-17
43
1----M-1 ____________________________________________ X1).
0 0 NH OH EDCI, DMAP
- 0 01"-'''N ifl:Finc0
Stage 1
H2 stage 2
EIOH
H01110):)
NHBoc
Stage 1 ¨ Synthesis of (S)-2-tert-Butoxycarbonylamino-pentanedioic acid 5-
benzyl ester 1-
cyclopentyl ester
[10 ailICYL)
NHBoc
To a stirred solution of (S)-2-tert-butoxycarbonylamino-pentanedioic acid 5-
benzyl ester
(5g, 14.8mmol) in a mixture of DCM (50m1) and DMF (30m1) at 0 C was added
cyclopentanol (2.7m1, 29.6mmol), EDC1 (4.25g, 22.2mmol) and DMAP (0.18g,
1.48mmol).
Stirring was continued at r. t. overnight, after which time LCMS showed
completion of
reaction. DCM was removed under reduced pressure. The reaction mixture was
diluted
with Et0Ac (200m1), washed with water (100m1), 1M aq HCI (50m1) followed by
sat aq
NaHCO3 (50m1). The Et0Ac layer was dried (Na2SO4), filtered and concentrated
in vacuo
to give a viscous oil which solidified on standing overnight. Trituration with
Et20 (2 x 10m1)
afforded the title compound as a white solid (43.78g, 80%). LCMS purity 94%.
Stage 2 ¨ Synthesis of (S)-2-tert-Butoxycarbonylamino-pentanedioic acid 1-
cyclopentyl
ester
H00"1:1).
NHBoc
CA 02836827 2013-12-17
44
A mixture of Stage 1 product (1.3g, 3.20mmol), and 10% Pd/ C (0.5g) in Et0H
(150m1) was
stirred under H2 (balloon) at r. I. for 4 h, after which time LCMS showed
completion of
reaction. The reaction mixture was filtered through a pad of celite, washed
with Et0H
(20m1) and concentrated in vacuo to give a white solid. To remove residual
Et0H the solid
was dissolved in toluene / THE mixture (5/1) (20m1) and concentrated in vacuo
to yield the
title compound (0.8g, 79%). 11-1 NMR (400 MHz, MOOD), 6:1.35 (9H, s), 1.60-
2.10 (10H,
m), 4.05(1 H, m), 5.20 (1H, m).
Synthesis of (S)-2-tert-Butozycatbonylamino-Dentanedioic acid 1-tett-butyl
ester
HOL-40j<
NHBoc
(S)-2-tert-Butoxycarbonylamino-pentanedioic acid 1-tert-butyl ester was
prepared from (S)-
2-tert-butoxycarbonylamino-pentanedioic acid 5-benzyl ester following the same
procedure
used for the synthesis of (S)-2-tert-Butoxycarbonylamino-pentanedioic acid 1-
cyclopentyl
ester
Synthesis of (S)-2-Benzyloxycarbonylamino-4-bromo-b4ric acid tent-butyl ester
0 0
Dibenzyldicarbonate, NaOH 4 ,i.,
40)(.0H ____________________________
Dioxane, water 1. 0 .........
NH2 0 NHZ 0
Stage 1
1) EtOCOC1, NEt3, THF
Stage 2
2) NaBH4, THF/water
.40 0
0.1Lisõ,..-13r NBS, PPh3, DCM, Pyridine
NHZ Stage 3 NHZ
Stage 1 ¨ Synthesis of (S)-2-Benzyloxycarbonylamino-succinic acid 1-tert-butyl
ester
CA 02836827 2013-12-17
0
NHZ 0
(S)-2-Amino-succinic acid 1-tert-butyl ester (0.9g, 4.75mmol) and sodium
hydroxide (0.28g,
7.13mmol, 1.5eq) were dissolved in 25% water in dioxane (50m1). The solution
was stirred
at 5 C and dibenzyldicarbonate (2g, 4.13mmol, 1.5eq) in dioxane (10m1) was
added slowly.
The mixture was stirred at 0 C for 1 h and then at r. t. overnight. Water
(10mi) was added
and the mixture was extracted with Et0Ac (2 x 20m1). The organic phase was
back
extracted with a sat. aq. Solution of sodium bicarbonate (2 x 10m1). The
combined aq.
Layers were acidified to pH 1 with 1M HG!, and extracted with Et0Ac (3 x
10m1). The
combined organic fractions were dried over MgSO4 and concentrated under
reduced
pressure. The product was purified by column chromatography (35% Et0Ac in
heptane) to
afford the title compound as a colorless oil (0.76g, 50%). m/z 346 [M+23]+, 1H
NMR (300
MHz, CDCI3), 8: 7.39-7.32 (5H, m), 5.72 (1H, d, J = 8.1Hz), 5.13 (2H, s), 4.58-
4.50 (1H, m),
3.10-2.99(1H, m), 2.94-2.83 91H, m), 1.45 (9H, s).
Stage 2 ¨ Synthesis of (S)-2-Benzyloxycarbonylamino-4-hydroxy-butyric acid
tert-butyl
ester
0
NHZ
To a solution of Stage 1 product (0.69, 1.87mmol) in anhydrous THF (20m1) at -
20 C was
slowly added triethylamine (0.032m1, 2.24mmol, 1.2eq) and ethyl chloroformate
(0.021m1,
2.24mmol, 1.2eq). The mixture was stirred at -20 C for 2 h. The solid formed
was filtered
off and washed with THF (2 x 10m1). The filtrate was added dropwise to a
solution of
sodium borohydride (0.2g, 5.61mmol, 3eq) at 0 C and stirred at r. t. for 4 h.
The solvent
was removed under reduced pressure, the residue was diluted with water (10m1)
acidified
to pH 5 with 1M HCI and extracted with Et0Ac. The organic fractions were
combined
washed with 10% aq. Sodium hydroxide, water and brine, dried on MgSO4 and
concentrated under reduced pressure to give the title compound as clear oil
(0.3g, 51%).
m/z 332 [M+23].
Stage 3 ¨ Synthesis of (S)-2-Benzyloxycarbonylamino-4-bromo-butyric acid tert-
butyl ester
CA 02836827 2013-12-17
46
0
õ4Ø1(1õõ,,Br
NHZ
To a solution of N-bromosuccinimide (0.52g, 2.91mmol, 3eq) in DCM (10m1) was
slowly
added a solution of triphenylphosphine (0.71g, 2.72mmol, 2.8eq) in DCM (10m1).
The
mixture was stirred at r. t. for 5 min. Pyridine (0.094m1, 1 .16mmol, 1.2eq)
and a solution of
Stage 2 product (0.3g, 0.97mmol, leg) in DCM (20m1) were added dropwise and
the
mixture stirred at r. t. overnight.
The solvent was removed under reduced pressure, the residue was azeotroped
with
toluene (2 x 15m1) and triturated with diethyl ether (2 x 25m1) and 10% Et0Ac
in heptanes.
The solutions from the trituration were combined and evaporated to dryness.
The crude
product was purified by column chromatography (15% Et0Ac in heptanes) to give
the title
compound as a clear oil (0.16g, 44%). miz 395 [M+23] 4, 1H NMR (300 MHz,
CDC13), 5:
7.39-7.30(5H, m), 5.40(1H, d, J = 6.8Hz), 5.12 (2H, s), 4.38 (1H, q, J =
7.7Hz), 3.47-3.38
(2H, m), 5.49-2.33 (1H, m), 2.28-2.13 (1H, m), 1.48 (9H, s).
EXAMPLE 1
This example describes the modification of the known HDAC (Histone
Deacetylase)
inhibitor Suberoylanilide hydroxamic acid (compound 7) herein referred to as
"SAHA", by
the attachment of amino acid ester motifs at points remote from the binding
interface with
the target, where no disruption of its binding mode occurs.
Compound 7: Suberoylanilide hydroxamic acid (SAHA)
0
HO, IN1
N
H
0 0
SAHA was purchased from BioCat GmbH, Heidelberg, Germany.
CA 02836827 2013-12-17
47
Standard wash procedure for resin chemistry
Resin was washed in the following sequence: DMF, Me0H, DMF, Me0H, DCM, Me0H,
DCM, Me0H x 2, TBME x 2.
Resin test cleavage
A small amount of functionalised hydroxylamine 2-chlorotrityl resin (ca 0.3ml
of reaction
mixture, ca 10mg resin) was treated with 2% TFA/DCM (0.5m1) for 10min at r.
t.. The resin
was filtered and the filtrate was concentrated by blowing with a stream of N2
gas. LCMS of
the residue was obtained.
Preparation of Suberic acid Derivatised Hydroxvlamine 2-Chlorotrityl Resin
Stage 1 ¨ Immobilisation to 2-chlorotrity1-0-NH2 resin
41,0 H OMe
0
To a round bottomed flask charged with 2-chlorotrity1-0-NH2 resin (6g, loading
1.14mmoVg,
6.84mmol) and DCM (60m1) was added DIPEA (5.30g, 41.0mmol, 6eq). Methyl 8-
chloro-8-
oxooctanoate (4.2g, 20.5mmol, 3eq) was slowly added to the reaction mixture
with orbital
shaking and the reaction mixture shaken for 48 h. The resin was filtered and
washed using
the standard washing procedure. The resin was dried under vacuum. LCMS purity
was
determined by ELS detection, 100%, m/z 204 [M++Hr.
Stage 2¨ Saponification
0-Ny,--jOH
0
To a round bottomed flask charged with Stage 1 resin (4g, loading 1.14mmoVg,
4.56mmol)
was added THE (16m1) and Me0H (16m1). To the reaction was added a solution of
NaOH
(0.91g, 22.8mmol, 5eq) in water (16m1). The reaction mixture was shaken for 48
h. The
resin was filtered and washed with water x 2, Me0H x 2, followed by the
standard wash
procedure. The resin was dried under vacuum. LCMS purity was determined by ELS
detection, 100% m/z 190 [M++Hr.
CA 02836827 2013-12-17
48
Preparation of SAHA derivatives
Compounds based on SAHA were prepared by the methods outlined below.
Compounds (8), (9) and (10) were prepared by the methodology described in the
following
scheme:
02N 401
Br _______________________________________ Boc,O, K,CO, 10
ICAO,. DMF 02N is
N 0,R THF __________ op all N = ,F1
Woe i H Swipe 2
0 boc 0
A n cYcloPon1Y1
WV
1-butyl
ElOac Stags 3
Illrolgon .
W/0,t4jr/3 0,R IN1301., DIPEA, DO* H2N is 0,
N A
H I
0 boc 0
Sninin 4 b0C 0
TFA/DCM Saps 5
I
''''....\.õ....s.N.13õ..,.............,......1017116
Na0H, meom (kw " ' Et all()
THF
I. IP
H H
HO witsõ,....¨õõ...,-,,........--,r,N
HN 0, 0, N OH
R
s H 101
0 10 Nbeci 0
. 0
R = Cyclopentyl Compound (9)
R = (-Butyl Compound (10)
TFNDCM Stage 7
IS
OH
= 11
H 0 0
Compound (9)
Stages 1 to 5 are exemplified for R = cyclopentyl
Stage 1 ¨ Synthesis of (S)-(3-Nitro-benzylamino)-phenyl-acetic acid
cyclopentyl ester
CA 02836827 2013-12-17
49
1101
02N $
0 T--)
3-Nitrobenzyl bromide (46mmol) was dissolved in DMF (180m1) and potassium
carbonate
(92mmol) added, followed by the (S)-phenylglycine ester (10.6g, 46mmol). The
reaction
was stirred for 17h at r. t. before evaporating to dryness. The residue was re-
dissolved in
Et0Ac (150m1) and washed with water (3 x 80m1), dried (Na2SO4) filtered and
concentrated
to dryness. After purification by flash column chromatography (30% Et0Ac /
hexane) the
ester of was obtained and used directly in Stage 2.
Stage 2 ¨ Synthesis of (S)-[tert-Butoxycarbonyl-(3-nitro-benzy1)-amino]-phenyl-
acetic acid
cyclopentyl ester
1110
02N 0
bac 0
The Stage 1 product (40.9mmol) was dissolved in THF (250m1) before addition of
potassium carbonate (61.4mmol) and water (150m1). Di-tert-butyl-dicarbonate
(163mmol)
was added and the reaction mixture heated to 50 C for 18 h. DCM was added the
resultant
mixture washed consecutively with 0.1 M HCI (150m1), sat. aq. NaHCO3 and water
(150
m1). The DCM layer was dried (Na2SO4), filtered and concentrated to dryness.
After
purification by flash column chromatography (5% Et0Ac / hexane) the title
ulphate was
isolated and used directly in Stage 3.
Stage 3¨ Synthesis of (S)1(3-Amino-benzy1)-tert-butoxycarbonyl-amino]-phenyl-
acetic acid
cyclopentyl ester
CA 02836827 2013-12-17
H2
b
N 0
l oc 0 0-0
The Stage 2 product (11.5mmol) was dissolved in Et0Ac (150m1) before addition
of Pd/C
(10% wet) catalyst (0.8g) and hydrogenated under balloon pressure at r. t. for
18 h. The
reaction mixture was filtered through a pad of celite and evaporated to
dryness to give a
solid.
Stage 4¨ Resin coupling
0
411L,.0 ,)rN 0
0 401 bloc 0
Hydroxylamine 2-chlorotrityl resin derivatized with suberic acid (1.0 g,
loading 0.83mmol/g)
was swollen in DMF (15m1) and PyBOP (1.36g, 2.61mmol) added, followed by DIPEA
(1.5m1, 8.7mmol). Stage 3 product (2.61mmol) was dissolved in DCM (15m1) and
added to
the reaction mixture. The reaction was shaken for 24 h at r. t.. The resin was
filtered and
washed using the standard wash procedure. The resin was dried under vacuum.
Stage 5 ¨ Synthesis of (S)43-(7-Hydroxycarbamoyl-heptanoylamino)-benzylamino]-
phenyl-
acetic acid cyclopentyl ester compound (8)
110
0
HON 0
N
0 0
The Stage 4 product (loading 0.83mmol) was gently shaken in 2% TFA/DCM (10m1)
for
20min. The resin was filtered. The filtrate was evaporated under reduced
pressure at r. t..
CA 02836827 2013-12-17
51
The resin was re-treated with 2% TFA/DCM (10m1) and was filtered after 20min.
The
combined fiftrates were evaporated to dryness under reduced pressure at r. t.
to give an
oily residue. The residue was allowed to stand in 20% TFA/DCM for 40 min.
After
evaporation to dryness, also under reduced pressure at r. t., the crude
product was purified
by preparative HPLC.
Analytical data for Compound 8
LCMS purity 100%, m/z 496 [M++Hr, 1F1NMR (400MHz, Me0D), 8: 1.30-1.70 (1611,
m),
2.00 (2H, t), 2.30 (2H, t), 4.05 (2H, dd), 5.00 (1H, m), 5.15 (1H, m), 7.05
(1H, m), 7.30 (21-1,
m), 7.40 (5H, m), 7.75 (1H, m).
Analytical data for Compound (10)
LCMS purity 97%, m/z 484 [M++Hr, 1H NMR (400 MHz, Me0D), 8: 1.30 (13H, m),
1.45-
1.65 (4H, m), 1.93-2.05 (2H, m), 2.20-2.40 (2H, m), 3.99 (2H, q), 4.65-4.95
(1H, m) 7.05
(1H, d), 7.25-7.33(2 H, m), 7.35-7.50 (5H, m), 7.75 (1H, s).
Stage 6 ¨ Saponification
0
N OH
401 0 boc 0
The Stage 5 product where R = Et (1.4g, loading 0.83mmol) was suspended in THF
(8.6m1)
and Me0H (8.6m1) and 1.4M sodium hydroxide solution (5.98mmol) was added. The
mixture was shaken for 24 h and the resin was filtered and washed with water x
2, Me0H x
2, followed by the standard wash procedure. The resin was dried under vacuum.
Stage 7 ¨ Synthesis of (S)43-(7-Hydroxycarbamoyl-heptanoylamino)-benzylaminol-
phenyl-
acetic acid (9)
CA 02836827 2013-12-17
52
#
0
HO, I-N1 1 OH
N
H 0 1
0 0
Stage 6 product (1.44g, loading 0.83mmol) was then gently shaken in 2% TFA/DCM
(10m1)
for 20 min. The resin was filtered and the filtrate evaporated under reduced
pressure at r. t..
The resin was re-treated with 2% TFA/DCM (10m1) and was filtered after 20 min.
The
combined filtrates were evaporated to dryness under reduced pressure at r. t.
to give an
oily residue. The residue was allowed to stand in 20% TFA/DCM for 40 min.
After
evaporation to dryness, under reduced pressure at r. t., the crude product was
purified by
preparative HPLC to yield compound (9). LCMS purity 100%, m/z 428 [M+4-H], 'H
NMR
(400MHz, Me0D), 8: 1.20-1.35 (4H, m), 1.50-1.65 (4H, m), 2.00 (2H, m), 2.30
(2H, m), 4.00
(2H, dd), 4.90 (1H, m), 7.05 (1H, m), 7.25-7.50 (7H, m), 7.70 (1H, m).
Compound (24) was prepared following the same methodology described for the
synthesis
of compound (8).
({(R)44-7-Hydroxycarbamoyl-heptanoylamino)-phenyl]-phenyl-methyl)-amino)acetic
acid
cyclopentyl ester (24)
401
0
ID
H
HO.ily-,.--LN NI1110 0
H
0
LCMS purity 95%, m/z 496 [M++H], 'H NMR (400 MHz, DMSO), 6:1.30-1.50 (6H, m),
1.50-
1.70 (8H, m), 1.80 (2H, m), 2.10 (2H, t), 2.45 (2H, t), 4.1 (2H, dd), 5.25
(1H, m), 5.35 (1H,
m), 7.45 (2H, d), 7.60 (5H, m), 7.80 (2H, d), 10.00-10.10 (2H, br s), 10.50
(1H, s).
CA 02836827 2013-12-17
53
EXAMPLE 2
This example describes the modification of the known Aurora Kinase A ("Aurora
A")
inhibitor N-(4-(7-methoxy-6-methoxy-quinoline-4-yloxy)-phenyl}-benzamide
(compound
(11)) by the attachment of an amino acid ester motif at a point where no
disruption of its
binding mode occurs.
Compound (11): N-(4-(7-methoxy-6-methoxy-quinoline-4-yloxy)-phenyl)-benzamide
0 N
0 I
/
? 0
=0
N
1110
H
Compound (11) was prepared as described in US Patent No. 6,143,764
Compounds based on compound (11) were prepared by the methods outlined below.
Compounds (12) and (13) were prepared by the method described in the following
scheme:
CA 02836827 2013-12-17
54
HO 0 CI
HO
Et3N NH
0
+
NH, 0 0 ___________
Stage 1 ...-
0 0
0 0 N
140 C
el \
I. 0 0 N Stage 2
neat ...
I 0
0 HO a I 0 0
1 CI1114-11F NH
NH
0*
0 0
cyclohexene/ethanol
Pd/C Stage 3
0
O
N Ph3P, DIAD, DCM HO 0 N
,
I -.4 _________
OH ,
I
NHBoc Stage 4 rj .-=
o o
I
0 I o 0
o
NH BocKIH)-.1r0,,,.\
0 Li NH
0 1101 0 0
.TFA/DCM
Stage ...'s-, 0LI)
Stage 6 LION, THF/water O() 0 N
I
NH, 0= ..,
I 0 opi
Compound (12) NH
AOOH OH 0
0 N
rõ.
, -- dk"0 0 N
, --
I I
NHBoc ..-- NH2 ..---
0 o
I I o
o
NH TFA/DCM
____________________________________ ....
I.1 NH
S
Stage 7
0 110 Compound (13) 0 0
Stage 1 ¨ Synthesis of N-(4-Hydroxy-phenyl)-benzamide
CA 02836827 2013-12-17
HO 0NH
0
To a solution of 4-aminophenol (4.27g, 39.1mmol) in DMF (50m1) at 0 C under an
atmosphere of argon was added triethylamine (7.44m1, 53.4mmol, 1.5eq). The
reaction was
stirred for 10 min before slow addition of benzoyl chloride (5g, 35.6mmol, 1
eq) over a
period of 5 min. The reaction mixture was allowed to warm to r. t. and stirred
over 18 h. The
DMF was removed under reduced pressure and the mixture was treated with Et0Ac
/
water. Precipitation of a white solid resulted, this was filtered off and
dried under reduced
pressure to give the title compound (8.0g, 96%). 1H NMR (270 MHz, DMSO), 8:
10.0 (1H,
s), 9.35 (1H, s), 7.9 (2H, d, J = 7.2Hz), 7.5 (5H, m), 6.75 (2H, d, J =
7.4Hz).
Stage 2 ¨ Synthesis of N44-(7-Benzyloxy-6-methoxy-quinolin-4-yloxy)-pheny1]-
benzamide
001 0
0
=
0
NH
0 11101
To a round bottomed flask charged with 4-chloro-6-methoxy-7-benzyloxyquinoline
[see
Org. Synth. Col. Vol. 3, 272 (1955) and US006143764A (Kirin Beer Kabushiki
Kaisha) for
methods of synthesis] (1.09g, 3.6mmol) was added Stage 1 product (2.33g, 10.9
mmol, 3
eq). Reaction was heated to 140 C for 3 h. After cooling to r. t., water was
added to the
reaction mixture and the mixture extracted 3 times with Et0Ac. The combined
Et0Ac layer
was washed with 5% aq. NaOH, brine and dried over MgSO4. The solvent was
removed
under reduced pressure and purified by column chromatography eluting with
Et0Ac /
heptane (2:1) to obtain the title compound (0.56g, 32%). m/z 477 [M++H].
Stage 3 ¨ Synthesis of N44-(7-Hydroxy-6-methoxy-quinolin-4-yloxy)-pheny1]-
benzamide
CA 02836827 2013-12-17
56
HO 0 N
,
I
0
I 0 .NH
0 40
A mixture of Stage 2 product (0.56g, 1.17mmol) and 10% Pd/C (0.08g) in 10%
cyclohexene
/ Et0H (80m1) was heated under ref lux for 3 h. The Pd/C catalyst was filtered
through a pad
of celite, washing twice with Me0H. The filtrate was concentrated under
reduced pressure
to yield the title compound as a yellow solid (0.34g, 75%). m/z 387 (M++H].
Stage 4 ¨ Synthesis of (S)-444-(4-Benzoylamino-phenoxy)-6-methoxy-quinolin-7-
yloxy]-2-
tert-butoxycarbonylamino-butyric acid cyclopentyl ester
0')
Of.""s0 õI ti.
i
NHBoc
0
I 0 0N
0 01
To a solution of Stage 3 product (0.2g, 0.52mmol) in anhydrous DCM (30m1) at 0
C was
added (S)-2-tert-Butoxycarbonylamino-4-hydroxy-butyric acid cyclopentyl ester
(0.223g,
0.78mmol, 1.5eq) in 5m1 of DCM. Ph3P (0.557g, 2.1mmol, 4.1eq) and DIAD
(0.412m1,
2.1mmol, 4.1eq) were then added and the reaction mixture allowed to warm to r.
t. and
stirred for 16 h. The crude reaction mixture was evaporated under reduced
pressure and
purified by column chromatography to give the title compound (0.135g, 46%).
m/z 656.3
[M++H].
CA 02836827 2013-12-17
57
Stage 5¨ Synthesis of ((S)-2-amino-4-[4-(4-benzoylamino-phenoxy)-6-methoxy-
quinolin-7-
yloxy]-butyric acid cyclopentyl ester) (12)
oL)
,
NH2 0
1 0 'NH
0 0
To a solution of Stage 4 product (5.8mg, 0.009mmol) in DCM (1m1) was added TEA
(1mI).
The reaction mixture was allowed to stir for 16 h before evaporation under
reduced
pressure, azeotroping with toluene to remove the traces of TFA. Compound (12)
was
isolated as an off-white solid (4.7mg). LCMS purity 95%, m/z 556.2 [M1-+H], 1H
NMR (270
MHz, DMSO), 5:10.4 (1H, s), 8.8 (1H, d, J = 6.5Hz), 8.55 (2H,bs), 8.01 (4H,
m), 7.65 (4H,
m), 7.35 (1H, d, J = 7.6Hz), 6.75 (1H, d, J = 6.5Hz), 5.25 (1H, m), 4.35 (3H,
m), 4.0 (3H, s),
2.4 (2H, m), 1.85-1.4 (8H, bm).
Stage 6 ¨ Synthesis of (S)-4-[4-(4-Benzoylamino-phenoxy)-6-methoxy-quinolin-7-
yloxy]-2-
tert-butoxycarbonylamino-butyric acid
OH
(iji. ,,,,, 0 N
NHBoc I /
? 0, 0
vi 0
To a solution of Stage 4 product (17mg, 0.02mmol) in THF (1m1) was added 2M
NaOH
(0.026m1, 0.046mmol, 2eq). After 16 h, the reaction was incomplete so an
additional 2
equivalents of NaOH was added. Stirring was completed after 6 h and the THF
was
CA 02836827 2013-12-17
58
removed under reduced pressure. The aq. Layer was diluted with 3m1 of water
and
acidified to pH 6 with 1M HCI. The title compound was extracted into Et0Ac,
dried over
M9SO4 and isolated as a white solid. This was used directly in Stage 7 without
further
purification.
Stage 7¨ Synthesis of (S)-444-(4-benzoylamino-phenoxy)-6-methoxy-quinolin-7-
yloxy]-2-
tert-butylcarbonylamino-butyric acid (13)
OH
0....).õ.".........õ,0 0 ......
I N
NH, 0=/
I 0
= 0
N
*
To a solution of Stage 6 product (6.5mg, 0.011mmol) in DCM (1m1) was added TFA
(1mI).
The reaction was allowed to stir for 6 h and then evaporated under reduced
pressure to
give the title compound (13) as an off-white solid (90%). LCMS purity 100%,
miz 488.2
[M++H], 11-1NMR (300 MHz, Me0D), 6: 8.75 (1H, d, J = 7.8Hz), 8.00 (4H, m),
7.65 (4H, m),
7.4 (1H, d, J = 7.6Hz), 6.95 (1H, d, J = 8.0Hz), 4.6 (2H, m), 4.3 (1H, m), 4.2
(3H, s), 2.6
(2H, m).
Compound (14) was prepared by the method described in the following scheme:
CA 02836827 2013-12-17
59
HO
HO, I 0
+NH
NH2 0 * ------0-stage 1
0 =
0 o aihi N,
Oil 0 0 Nõ
140 C
DMF Stage 2 ...--
I ________________________________ - 0 971
I
01
NHI to-'".5.-NH
A o to 0 =
cyclohexene/ethanol
PcUC Stage 3
C:r.=
0r,0 0 N, ... K2CO3, DMF HO
0 =
0 N,
I I
NHZ Stage 4 ar 0
I
0
0 =
NH Dal 1(
I NH
0
0 = 0 0
Pd(OH)2, H2
Et0Ac Stage 5
0j(
0,I t'i
NH2 r)
T o .NH
Compound (14) 0 =
Stage 1, 2 and 3 are the same as described above for the synthesis of compound
(12).
Stage 4¨ Synthesis of (S)-444-(4-13enzoylamino-phenoxy)-6-methoxy-quinolin-7-
yloxy]-2-
benzyloxycarbonylamino-butyric acid tert-butyl ester
CA 02836827 2013-12-17
Oj<
, .
NHZ
0
I 0 0NH
0 0
The Stage 3 product (0.15g, 0.39mmol), (S)-2-Benzyloxycarbonylamino-4-bromo-
butyric
acid tert-butyl ester (0.16g, 0.43mmol, 1.1eq) and K2CO3 (0.11g, 0.78mmol,
2eq) were
dissolved in anhydrous DMF (10m1) under an atmosphere of nitrogen. The
reaction was
stirred at 35 C overnight before the DMF was removed under reduced pressure.
The
residue was dissolved in DCM and washed with water followed by brine. The
organic layer
was dried over MgSO4 and evaporated under reduced pressure. Column
chromatography
(eluting with 1% MeOH/ DCM) afforded the title compound (0.16g, 60%). miz 678
[M+H]
+,1H NMR (300 MHz, CDCI3), 5: 8.49(1H, d, J = 5.3Hz), 7.98 (1H, s), 7.92 (2H,
dd, J = 8.2,
1.4Hz), 7.80-7.72 (2H, m), 7.63-7.48 (4H, m), 7.43-7.29 (4H, m), 7.24-7.17
(2H, m), 6.64
(1H, d, J = 8.9Hz), 6.49 (1H, d, J = 5.3Hz), 5.15 (2H, s), 4.66-4.57 (1H, m),
4.43-4.34 (1H,
m), 3.85 (3H, s), 2.55-2.33 (2H, m), 1.41 (9H, m).
Stage 5- Synthesis of -(S)-2-Amino-4-[4-(4-benzoylamino-phenoxy)-6-methoxy-
quinolin-
7-yloxy]-butyric acid tert-butyl ester (14)
0
or,,0%=,,.0 0 N
, .
I
NH, 0
I 0 op
NH
0*
CA 02836827 2013-12-17
61
The Stage 4 product (0.045g, 0.066mmol), was dissolved in anhydrous Et0Ac
(5m1) and
Pd(OH)2/C was added under an atmosphere of nitrogen. The reaction was degassed
and
stirred under an atmosphere of hydrogen at r. t. overnight. The catalyst was
filtered off
through a pad of celite and the solvent removed under reduced pressure.
Compound (14)
was purified by preparative HPLC. m/z 544 [M+Hr,'H NMR (300 MHz, CD30D), .6:
8.67
(1H, d, J = 6.8Hz), 7.98 (41-I,d, J = 8.7Hz), 7.90 (1H,$), 7.68-7.51 (41-1,
m), 7.42-7.36 (2H,
m), 6.97 (11-I, d, J = 6.6Hz), 4.52 (2H, t, J = 5.7Hz), 4.28 (1H, t, J =
6.5Hz), 4.13 (3H, s),
2.69-2.45 (2H,m ), 1.53 (9H, s).
Compound (25) was prepared by the method described in the following scheme:
0),..r..............0 Ail N,
a AcOH. NaCNBH4 0'()
NHz
o' cr NH 0
I 0 at Me0H I 0 ah
VI
1114-1111" NH Stags 1 NH
Compound (12) 0' . Compound (25) 0 io
Stage 1 ¨ Synthesis of (S)-444-(4-Benzoylamino-phenoxy)-6-methoxy-quinolin-7-
yloxy]-2-
cyclohexylamino-butyric acid cyclopentyl ester (25)
H
cfN.:0 la N.
0 0 0 ilr
a 0
* 0
Vi 0
To compound (12) (37mg, 0.066mmol) in anhydrous Me0H (1m1) was added 100p1 of
a
1M solution of cyclohexanone in Me0H and 1 drop of acetic acid. The reaction
mixture
was stirred at r. t. for 3 h. Sodium cyanoborohydride (10.3mg, 0.165mmol) was
then added
and the reaction was left stirring 4 h at r. t., prior to concentration under
vacuum.
Purification by preparative HPLC afforded the title compound (25) as a di-TFA
salt. rn/z 638
[M+H]. 111 NMR (300 MHz, CD30D) 6; 8.72 (1H, Cl, J = 6.8 Hz), 8.02-7.98 (4H,
m), 7.93
(1H, s), 7.67 (1H, s), 7.66-7.53(3H, m), 7.42 (2H, m), 6.99 (1H, d, J = 6.8
Hz), 5.38 (1H,
CA 02836827 2013-12-17
62
m), 4.49 (3H, m), 4.14 (3H, s), 3.27 (11H, m), 2.66 (2H, m), 2.20 (2H, m),
12.05-1.46 (16H,
m).
EXAMPLE 3
This example describes the modification of the known P38 kinase inhibitor 6-
Amino-5-(2,4-
difluoro-benzoy1)-1-(2,6-difluoro-pheny1)-1H-pyridin-2-one (compound 3258) by
the
attachment of an amino acid ester motif at a point where no disruption of its
binding mode
occurs.
Compound (15): 6-Amino-5-(2,4-difluoro-benzoy1)-1-(2,6-difluoro-pheny1)-1H-
pyridin-2-one
F
0 NH2 I.
* '' N
F F 0F
Compound (15) was prepared as described in W003/076405.
Compounds based on compound (15) were prepared by the methods outlined below.
Compounds (16) and (17) were prepared by the method described in the following
scheme:
CA 02836827 2013-12-17
F
63
F c2H
= NH2 ili .................rit f.) K2CO2. DMF
F (:).õ.....s.ricri)
ar = NH2 4
NHBoc
NHBoc SUS* I
'lir"- F 0 Si N F
F F 0
TFA / DCM Stage 2
1
0
0 NH: 4. NH2 (:).'"'YILOII Na0H, M o NH
OH
, 141 0
. .4----
N NH Stags 3 SO ' N ,
,...
F F 0F F F 0
Compound (17) Compound (16)
Stage 1 ¨ Synthesis of cyclopentyl (S)-4-(4-[6-Amino-5-(2,4-difluorobenzoy1)-2-
oxo-2H-
pyridin-1-y11-3,5-difluorophenoxy)-2-tert-butoxycarbonylaminobutyrate
io NH: 4110
* NHBoc
''' N
F
F F 0
To a stirred mixture of 6-amino-5-(2,4-difluorobenzoy1)-1-(2,6-difluoro-4-
hydroxy-pheny1)-
1H-pyridin-2-one [prepared by methods described in W003/076405] (100mg,
0.265mmo1)
and K2CO3 in DMF (1.5 ml) was added (L)-5-bromo-2-tert-
butoxycarbonylaminopentanoic
acid cyclopentyl ester (96mg, 0.265 mmol). The reaction mixture was stirred at
60 C for 2
h. The reaction mixture was diluted with Et0Ac (15m1) and washed with sat aq
NaHCO3
(3m1) and water (10ml). The Et0Ac layer was dried (Na2SO4), filtered and
concentrated to
dryness. Purification by flash chromatography (20% Et0Ac / heptane) yielded
the title
compound as a white solid (50mg, 29%). LCMS purity 100%, m/z 648 [M4-FH], 11-1
NMR
(400 MHz, Me0D), 8: 1.30 (9H, s), 1.40-1.65 (6H, m), 1.70-1.85 (2H, m), 1.95-
2.30 (2H, m),
4.00-4.10 (2H, m), 4.154.20 (1H, m), 5.05-5.10 (1H, m), 5.65 (1H, d), 6.70-
6.80 (2H, m),
6.95-7.05 (2H, m), 7.25-7.45 (2H, m).
Stage 2 ¨ Synthesis of cyclopentyl (S)-2-Amino-4-{4-[6-amino-5-(2,4-
difluorobenzoy1)-2-
oxo-2H-pyridin-1-y1]-3,5-difluorophenoxy}butanoate trifluoroacetate (16)
, ..
CA 02836827 2013-12-17
64
0
0 NH2
N NH,
0
A mixture of Stage 1 product (10mg) and 20% TFA / DCM (0.5m1) was allowed to
stand at
r. T. For 3 h. The reaction mixture was concentrated to dryness by blowing
under N2. The
residue was triturated with Et20 (0.3ml x 2) to give compound (16) as a white
solid (9.3mg,
91%). LCMS purity 100%, m/z 548 [M++Fl], 1H NMR (400 MHz, Me0D), 8: 1.55-1.80
(6H,
m), 1.85-2.00 (2H, m), 2.30-2.50 (2H, m), 4.15-4.30 (3H, m), 5.25-5.35 (1H,
m), 5.75 (1H,
d), 6.85-6.95 (2H, m), 7.05-7.15 (2H, m), 740-7.55 (2H, m).
Stage 3¨ Synthesis of (S)-2-Arnino-4-(446-amino-5-(2,4-difluorobenzoy1)-2-oxo-
2H-pyridin-
1-y11-3,5-difluorophenoxy)butanoic acid (17)
0
le ====I').
0 NH,LOH
N NH,
0
To a solution of compound (16) (20mg, 0.0317mmol) in a mixture of Me0H (0.3m1)
and
THE (0.3m1) was added 2M aq NaOH (0.3m1). The reaction mixture was allowed to
stand at
AT for 3h. Upon completion the reaction mixture was evaporated to dryness by
blowing
under a flow of N2, acidified to pH 1-2 by dropwise addition of 2N1 aq HCI.
The resulting
white solid formed was collected by filtration. Yield= 9mg, 48%., LCMS purity
97%, trilz
480 [M++H], 1H NMR (400 MHz, Me0D), 6: 2.35-2.55 (2H, m, CH2), 4.15-4.20 (1H,
m, CH), 4.25-4.35 (2H, m, CH2), 5.75 (1H, d, CH), 6.85-7.00 (2H, m, Ar), 7.05-
7.20
(2H, m, Ar), 7.40-7.55 (2H, m, Ar).
Compound (18) was prepared by the method described in the following scheme:
CA 02836827 2013-12-17
= 1.1112 0
a N
F 0 .1. NI.1
K CO DMF
arrAcrk= 2 3.
NHZ Stage 1 0 2
N F.
F
0
H2, Pd(OH)2 Stage 2
Et0Ac
o NH,4NH
'"ThAel<
N
FF
Compound (18)
Stage 1 ¨ Synthesis of (S)-4-{4-[6-Amino-5-(2,4-difluoro-benzoy1)-2-oxo-2H-
pyridin-1-y1]-
3,5-difluoro-phenoxy)-2-benzyloxycarbonylamino-butyric acid tert-butyl ester
0 NH2 "=7*Ny10
NHZ
N
0
To a solution of 6-Amino-5-(2,4-difluorobenzoyI)-1-(2,6-difluoro-4-
hydroxypheny1)-1H-
pyridin-2-one (100mg, 0.26mmol) and (S)-2-benzyloxycarbonylamino-4-bromo-
butyric acid
t-butyl ester (108mg, 0.29mmol) in acetone (2mL) was added sodium iodide
(79mg,
0.53mmol) and potassium carbonate (146mg, 1.06mmol). The reaction was heated
at
reflux for 12h, cooled and partitioned between water (20mL) and ethyl acetate
(20mL). The
aqueous layer was re-extracted with ethyl acetate (2x10mL) and the combined
organic
extracts washed with brine (20mL), dried (MgSO4) and concentrated under
reduced
pressure to give a yellow oil. This residue was subjected to column
chromatography [silica
gel, 40% ethyl acetate-heptane] to give the desired product (186mg, 79%) as a
colourless
solid, m/z 670 [M+H].
Stage 2 ¨ Synthesis of (S)-2-Amino-4-{4-[6-amino-5-(2,4-difluoro-benzoy1)-2-
oxo-2H-
pyridin-1-y1]-3,5-difluoro-phenoxy)-butyric acid tert-butyl ester
CA 02836827 2013-12-17
66
0
F
11
0 NH2 001 C)'0 (
, N NH2
(10 /
\ F
F F 0
(S)-4-{4-[6-Am ino-5-(2,4-difluoro-benzoy1)-2-oxo-2H-pyridi n-1 -y1]-3,5-
difluoro-phenoxy)-2-
benzyloxycarbonylamino-butyric acid tert-butyl ester
(140mg, 0.2mmol) was dissolved in ethyl acetate (15mL) containing 10%
palladium
hydroxide on carbon (20mg) and stirred under a hydrogen atmosphere (1atm) for
lh. The reaction mixture was purged with N2, and filtered through Celite
washing
with additional ethyl acetate. The filtrate was concentrated under reduced
pressure
to give a solid which was subjected to column chromatography [silica gel:
5%Me0H
in dichloromethane]. This gave the desired product (60mg, 54%) as a grey
solid:
LCMS purity 98%, m/z 536 [M+H], 1H NMR (300 MHz, CDCI3) 7.65-7.44 (1H, m),
7.39-7.29 (2H, m), 6.96-6.82 (2H, m), 6.66 (2H, br d, J=8.1 Hz), 5.82 (1H, d,
J=9.9
Hz), 4.20-4.07 (3H, m), 3.48 (1H, dd, J=4.8, 8.7 Hz), 2.22-2.15 (1H, m), 1.91-
1.84
(1H, m), 1.62 (2H, br s), 1.43 (9H, s).
EXAMPLE 4
This example describes the modification of the known DHFR inhibitor 5-Methyl-6-
((3,4,5-
trimethoxyphenylamino)methyl)pyrido[2,3-/pyrimidine-2,4-diamine (compound (2
G=N)) by
the attachment of an amino acid ester motif at a point where no disruption of
its binding
mode occurs.
Compound (2 G=N): 5-Methyl-6-((3,4,5-trimethoxyphenylamino)methyl)pyrido[2,3-
c]pyrimidine-2,4-diamine
OMe
el OMe
NH2
N N OMe
./. H
H2N N N
CA 02836827 2013-12-17
67
Compound (2 G=N) was prepared by a modification of the method described in
J.Med.Chem. 1993, 36, 3437-3443.
2,4-Diamino-5-methylpyrido[2,3-4pyrimidine-6-carbonitrile (0.10g, 0.5mmol),
3,4,5-
trimethoxyaniline (0.10g, 0.55mmol) and Raney nickel (0.7g, damp) in acetic
acid (20m1)
were stirred at r. t. under an atmosphere of hydrogen. After 2 h the reaction
mixture was
filtered through celite and the solvent evaporated under reduced pressure. The
crude
residue was purified by reverse phase HPLC to afford compound (2 G=N) as a
solid
(22mg, 16%). LCMS purity 94%, m/z 371.1 [M+H], 1H NMR (400 MHz, DMSO), 8: 8.5
(1H,
s), 7.0 (2H, bs), 6.2 (2H, bs), 6.0 (2H, s), 5.7 (1H, m), 4.2 (2H, d), 3.7
(6H, s), 3.5 (3H, s),
2.7 (3H, s).
Compounds based on compound (2 G=N) were prepared by the methods outlined
below.
Compounds (6) and (19) were prepared by the method described in the following
scheme:
CA 02836827 2013-12-17
68
rJ
DIPEA, DCM 0
02N =
+ H2. f
S19991
0 ID 02N * 0
Formic acid
Et3N, Et0H Stage 2
PcVC
1.12k ,.CN =
..===;:j Nf00
,
Raney Ni H2N N N
AcOH H 0
H2N
Stage 3
V
1-12N 0 0
LAOH, Et0H
H2N N N Stage 4
Compound (6)
=
N
0
NLVLVSN
..===
H2N N N
Compound (19)
Stage 1 ¨ Synthesis of (S)-cyclopentyl 4-methyl-2-(4-nitrobenzamido)pentanoate
0
1101
H II
0 NO
02N
CA 02836827 2013-12-17
69
4-Nitrobenzoyl chloride (0.60g, 3.9mmol) in DCM (2m1) was added dropwise over
10 min to
a solution of (S)-cyclopentyl 2-amino-4-methylpentanoate (0.70g, 3.5 mmol) and
diisopropylethylamine (0.94m1, 5.3mmol) in DCM (10m1) at -5 C under an
atmosphere of
nitrogen. On completion of the addition, the reaction mixture was allowed to
warm to r. t.
and stirred for a further 30 min. The reaction mixture was poured on to sat.
aq. NaHCO3
and the aqueous layer was extracted with DCM. The organic extracts were
combined,
washed with brine, dried over MgSO4 and evaporated under reduced pressure
afford the
title compound as an oily solid in quantitative yield. LCMS purity 92%, m/z
347.1 [M+H].
Stage 2 ¨ Synthesis of (S)-cyclopentyl 2-(4-aminobenzamido)-4-methylpentanoate
0
0
H2N
Triethylamine (1.09g, 10.8mmol) and formic acid (0.509, 10.8mmol) were
dissolved in
Et0H (10m1) and added to a solution of Stage 1 product (1.2g, 3.4mmol) in Et0H
(10m1).
10% Pd/C (approximately 10 mol%) was added and the mixture was heated to ref
lux. After
1 h the hot reaction mixture was filtered through celite and the residue was
washed with
Me0H. The filtrate and washings were combined and evaporated and the residue
was
partitioned between DCM and sat. aq. NaHCO3. The organic layer was washed with
brine,
dried over MgSO4 and evaporated under reduced pressure to furnish the title
compound as
a white solid (0.80g, 73%). LCMS purity 97%, m/z 319.2 [M+H], 1H NMR (400 MHz,
CDCI3), 8:7.6 (2H, dd), 6.6(2H, dd), 5.2 (1H, m), 6.4(1H, d) 4.7 (1H, m), 4.0
(2H, s), 1.9
(2H, m), 1.7 (5H, m), 1.6 (4H, m), 0.9 (6H, dd).
Stage 3¨ Synthesis of (S)-2-(4-[(2,4-Diamino-5-methyl-pyrido[2,3-d]pyrimidin-6-
ylmethyl)-
amino]-benzoylamino)-4-methyl-pentanoic acid cyclopentyl ester (6)
CA 02836827 2013-12-17
0
NLKN
N H2
HNC 0
0
H2N N N
2,4-Diamino-5-methylpyrido[2,3-4pyrimidine-6-carbonitrile (0.47g, 2.4mmol),
Stage 2
product (300mg, 0.94mmol) and Raney nickel (1g, damp) in acetic acid (40m1)
were stirred
at r. t. under an atmosphere of hydrogen. After 48 h the reaction mixture was
filtered
through celite and the solvent evaporated under reduced pressure. The material
was
loaded in Me0H onto an SCX column and eluted off with a 1% ammonia solution in
Me0H.
The crude product was then adsorbed onto silica and purified by column
chromatography
(10% Me0H/DCM) to afford compound (6) (60mg,13%). LCMS purity 95 %, m/z 506.1
[M+H], 1H NMR (400 MHz, DMSO), 5: 8.5 (1H, s), 8.2 (1H, d), 7.7 (2H, d), 7.0
(2H, bs), 6.7
(2H, d), 6.5 (1H, m), 6.2 (2H, bs), 5.1 (1H, m), 4.4 (1H, m), 4.3 (2H, d), 2.7
(3H, s), 1.7
(11H, m), 0.9 (6H, dd).
Stage 4 ¨ Synthesis of (S)-2-(4-((2,4-diamino-5-methylpyrido[2,3-djpyrimidin-6-
yl)methylamino)benzamido)-4-methylpentanoic acid (19)
o('OH
NH2
1401
NN
H2N N N
Stage 3 product (39 M) was suspended in Et0H (1.0m1). A solution of 1M lithium
hydroxide (1561.11) was added to the above and the suspension allowed to stir
for 48 h. The
Et0H was subsequently removed under reduced pressure, the residual diluted
with water
and taken down to pH 4 with dilute acetic acid. The solution was washed with
DCM,
evaporated and subjected to SCX purification to afford compound (19). LCMS
purity 92%,
m/z 438 [M+H]; 1H NMR (400 MHz, DMSO) 8: 8.5 (1H, s), 8.1 (1H, d), 7.7 (2H,
d), 7.2 (2H,
CA 02836827 2013-12-17
71
br s), 6.7 (2H, d), 6.5 (1H, t), 6.4 (2H, br s), 4.4 (1H, m), 4.3 (2H, d), 2.7
(3H, s), 1.8 ¨ 1.6
(2H, m), 1.6¨ 1.5 (1H, m), 0.9 (6H, dd).
Compounds (5) and the corresponding acid were prepared by the method described
in the
following scheme:
= AcOH, Na(0Ac)38H4
H2N.0 DCM
Stage 1
02N 0
0 02N
Raney Ni
H2NNH2.H20 Stage 2
Et0H
NH2
N.)rON
./.1ts F40,0
Raney Ni, H2N N N
H2 gas
H2N
AcOH
Stage 3
= _____________________________________________ tilc D
N N
UOH, Et0H
H2N N N Stage 4
Compound (5)
cOH
N
0
N '"=-= N
H
H2N N N
Stage 1 ¨ Synthesis of (S)-Cyclopentyl 4-methyl-2-(4-
nitrobenzylamino)pentanoate
CA 02836827 2013-12-17
72
410 N
H
0
02N
To a solution of (S)-cyclopentyl 2-amino-4-methylpentanoate (2.00g, 10.0mmol)
and 4-
nitrobenzaldehyde (3.04g, 20.0mmol) in DCM (40m1) was added glacial acetic
acid (2
drops). The solution was allowed to stir at r. t. for 1 h whereupon sodium
triacetoxyborohydride (6.40g, 30.2mmol) was added in a single a portion. After
stirring for
3 h, the solution was poured on to aq. 1M NCI, allowed to stir for 30 min,
neutralised with
aq. 1M NaOH and extracted with DCM. The combined organics were washed with
water
and brine, dried over MgSO4, and evaporated under reduced pressure. The crude
material
was purified by chromatography (5% Et0Ac / isohexane) to furnish the title
compound as
an oil (1.51g). This was used without further purification for the following
step. LCMS purity
71%, m/z 335.1 [M-H].
Stage 2 - Synthesis of (S)-2-(4-Amino-benzylamino)-4-methyl-pentanoic acid
cyclopentyl
ester
0 N
H 0
H2N
Stage 1 product (0.90g, 2.7mmol) was dissolved in Et0H (5m1) and added to a
suspension
of Raney nickel (- 0.5g) and hydrazine monohydrate (0.38m1, 8.1mmol) in Et0H
(5m1).
After heating under ref lux for 1 h the hot reaction mixture was filtered
through celite and the
residue was washed with Me0H. The filtrate and washings were combined and
evaporated
and the residue was partitioned between DCM and sat. aq. Sodium hydrogen
carbonate.
The organic layer was washed with brine, dried over MgSO4 and evaporated under
reduced pressure. The crude material was purified by chromatography (20% Et0Ac
/
isohexane) to furnish the title compound as an oil (0.50g, 61%). LCMS purity
99%, m/z
CA 02836827 2013-12-17
73
305.2 [M+H], 1F1 NMR (400 MHz, CDCI3), 8: 7.1 (2H, d), 6.6 (2H, d), 5.2 (1H,
m), 3.7(1H,
d), 3.5 (1H, d), 3.2 (1H, t), 1.9 (2H, m), 1.7 (5H, m), 1.6 (4H, m), 0.9 (6H,
dd).
Stage 3¨ Synthesis of (S)-244-[(2,4-Diamino-5-methyl-pyrido[2,3-d]pyrimidin-6-
ylmethyl)-
amino]-benzylamino}-4-methyl-pentanoic acid cyclopentyl ester (5)
N )3,
NH2
0
el H
H
,..;:-.....,. .-;.-
H2N N N
2,4-Diamino-5-methylpyrido[2,3-cf]pyrimidine-6-carbonitrile (0.16g, 0.83mmol),
Stage 2
product (100mg, 0.33mmol) and Raney nickel (1g, damp) in acetic acid (10m1)
were stirred
at r. t. under an atmosphere of hydrogen. After 5 h the reaction mixture was
filtered through
celite and the solvent evaporated under reduced pressure. The material was
loaded in
Me0H onto an SCX column and eluted with a 1% ammonia solution in Me0H. The
crude
product was then adsorbed onto silica and purified by column chromatography
(10%
Me0H/DCM) to afford the title compound (5) (30mg, 19%). LCMS purity 95 %, m/z
492.1
[M+Hr, 1H NMR (400 MHz, DMSO), 8: 8.5 (1H, s), 7.2 (2H, bs), 7.0 (2H, d), 6.6
(2H, d), 6.2
(2H, bs), 5.8 (1H, m), 5.1 (1H, m), 4.2 (2H, s), 3.6 (1H, m), 3.4(1H, m), 3.1
(1H, m), 2.7
(3H, s), 1.5 (11H, m), 0.8 (6H, dd).
Stage 4 ¨ Synthesis of (S)-2-{4-[(2,4-Diamino-5-methyl-pyrido[2,3-d]pyrimidin-
6-ylmethyl)-
aminoFbenzylamino)-4-methyl-pentanoic acid
,,..(.0H
NH2
* N
0
NN
H
--- ..-
H2N N N
CA 02836827 2013-12-17
74
Stage 3 product (391iM) was suspended in Et0H (1.0m1). A solution of 1M
lithium
hydroxide (156 1) was added to the above and the suspension allowed to stir
for 48 h. The
Et0H was subsequently removed under reduced pressure, the residual diluted
with water
and taken down to pH 4 with dilute acetic acid. The solution was washed with
DCM,
evaporated and subjected to SCX purification to afford the title compound LCMS
: 95%
purity at At 0.52 and 1.91 min, m/z (ES) 424 [M+Hr; 1H NMR (400 MHz, DMSO) 8:
8.5
(1H, s), 7.1 (2H, d), 7.0 (2H, br s), 6.6 (2H, d), 6.2 (2H, br s), 5.7 (1H,
t), 4.3 (2H, d),
3.6 (1H, m), 3.3 (2H, obscured by water), 2.7 (3H, s), 1.8 (1H, m), 1.3 (1H,
m), 1.2
(1H, m).
Compound (3) was prepared by the method described in the following scheme:
CA 02836827 2013-12-17
0 OH
+
NaH. THF. DMF * 0 ,.=riL ,ID
0
NHBocrr0,,,,N
Li
02N
0 Stage 1
02N NHBoc
Formic aV stage 2
Et0H
Nii.),,CN 0...Nri(e0
+
(101 NHBoc
1 H2N N N H2N
Raney Ni
AcOH Stage 3
0 -0
N
xi ot ,.....4
0
NHBoc
xrN I µ`==
,A, ...- .... H TFA / DCM
H2N N N
Stage 4
Ii2k * 00j-)
NH2
1 ---1 j ill
H2N N N
Compound (3)
Stage 1 ¨ Synthesis of (S)-Cyclopenty1-2-(tert-butoxycarbonylamino)-4-(4-
nitrophenoxy)butanoate
0
NHBoc
02N
To a solution of 4-nitrophenol (2.18g, 15.7mmol) in tetrahydrofuran (100m1) at
09-C under
nitrogen was added sodium hydride (0.63g, 15.7mmol). After warming to r. t.
and stirring for
10 min, a solution of (S)-cyclopenty1-4-bromo-2-(ten-
butoxycarbonylamino)butanoate (5.0g,
14.3mmol) in DMF (20m1) was added. The reaction was heated to 602C for 10 h,
after
CA 02836827 2013-12-17
76
which the reaction was cooled to r. t. and poured onto ether / sodium
carbonate. The
organic layer was collected and washed with 2M aq. Sodium carbonate solution,
1M HC1
and brine before being dried over MgSO4 and concentrated under reduced
pressure to
afford an oil which solidified upon standing to yield the title compound
(4.0g, 69%). LCMS
purity 97%, m/z 407.1 [M+H], 1H NMR (400 MHz, CDC13), 8: 8.2 (2H, d), 7.4 (1H,
d), 7.1
(2H, d), 5.1 (1H, m), 4.1 (3H, m), 2.1 (2H, m), 1.8 (2H, m), 1.6 (6H, m), 1.4
(9H, s).
Stage 2¨ Synthesis of (S)-Cyclopenty1-4-(4-aminophenoxy)-2-
(ter/butoxycarbonylamino)butanoate
01A0L)
NHBoc
H2N
Triethylamine (0.77m1, 5.2 mmol) and formic acid (0.19m1, 5.2 mmol) were
dissolved in
Et0H (4m1) and added to a solution of Stage 1 product (0.7g, 1.7mmol) in Et0H
(4m1). 10%
Pd/C (approximately 10 mol%) was added and the mixture was heated to ref lux.
After 2 h
the hot reaction mixture was filtered through celite and the residue was
washed with
Me0H. The filtrate and washings were combined and evaporated and the residue
was
partitioned between DCM and sat. aq. Sodium hydrogen carbonate. The organic
layer was
washed with brine, dried over MgSO4 and concentrated under reduced pressure.
The
residue was purified by column chromatography (gradient elution, 10-40% Et0Ac
in
hexane) to afford the title compound (0.3g, 46%). LCMS purity 93%, m/z 379.1
[M+H], 1F1
NMR (400 MHz, CDCI3), 8: 6.9 (2H, d), 6.8 (2H, d), 5.3 (2H, m), 4.4 (1H, m)
4.0 (2H, m),
2.3 (1H, m), 2.2 (1H, m), 1.9 (2H, m), 1.7 (4H, m), 1.6 (2H, m), 1.4 (9H, s).
Stage 3 ¨ Synthesis of (S)-cyclopenty1-2-(tert-butoxycarbonylamino)-4-(4-((2,4-
diamino-5-
methylpyrido[2,3-4pyrimidin-6-yl)methylamino)phenoxy)butanoate
0
OrA0,,0
NH2
NHBoc
N N
",
H2N N N H
CA 02836827 2013-12-17
77
2,4-Diamino-5-methylpyrido[2,3-c]pyrimidine-6-carbonitrile (0.50g, 2.5mmol),
Stage 2
product (0.38g, 1.0mmol) and Raney nickel (3g, damp) in acetic acid (40m1)
were stirred at
r. T. Under an atmosphere of hydrogen. After 16 h the reaction mixture was
filtered through
celite and the solvent evaporated under reduced pressure. The material was
loaded in
Me0H onto an SCX column and eluted with a 1% ammonia solution in Me0H. The
crude
product was then adsorbed onto silica and purified by column chromatography
(5%
Me0H/DCM) to afford the title compound (145mg, 26%). LCMS purity 95 A", m/z
566.2
[M+H], 1H NMR (400 MHz, DMSO), 8: 8.5 (11-1, s), 7.3 (2H, m), 7.0 (2H, m), 6.7
(2H, d), 6.6
(2H, d), 6.2 (2H, bs), 5.5 (1H, bs), 5.1 (1H, m), 4.1 (3H, m), 3.9 (2H, m),
2.6 (3H, s), 2.0
(1H, m), 1.8 (3H, m), 1.6 (6H, m), 1.4 (9H, s).
Stage 4¨ Synthesis of (S)-2-Amino-4-{4-[(2,4-diamino-5-methyl-pyrido[2,3-
d]pyrimidin-6-
ylmethyl)-amino]-phenoxy}-butyric acid cyclopentyl ester (3)
0
0,0jD
NH2
INC'L''''..N SI NH2
,7 ,.,.. H
H2N N N
To a solution of Stage 3 product (145mg, 0.26mmol) in DCM (3m1) was added
trifluoroacetic acid (3m1) and the reaction stirred for 30 min at r. t.. The
solvent was
evaporated under reduced pressure and the crude residue purified by loading in
Me0H
onto an SCX column and eluting with a 1% ammonia solution in Me0H to afford
compound (3) (39mg, 33%). LCMS purity 94 /0, rn/z 466.1 [M+H], 1H NMR (400
MHz,
DMSO), 6:8.5 (1H, s), 7.0 (2H, bs), 6.7 (2H, d), 6.6 (2H, d), 6.2 (2H, bs),
5.5 (1H, bs), 5.1
(1H, m), 4.1 (2H, s), 3.9 (2H, m), 3.4 (1H, m), 2.7 (3H, s), 2.0 (2H, m), 1.8
(3H, m), 1.6 (6H,
m).
Compound (4) was prepared by the method described in the following scheme:
CA 02836827 2013-12-17
78
0 OH
+
NaH, TI-IF, DMF 0 0 .1oCl)
____________________________________ 2
02N NHBoc4 I) Stage 1 NHBoc
02N
0
TFA / DCM Stage 2
I
Na(0Ac)3BH4
HN0
02N Me0H III NH3
02N
Stage 3
FEtsozif3acid
Stage 4
Et0H
0
NH2
0 0õ.y..0,0
HN0 + 1 CN
H2N H2N N N
Raney Ni
AcOH Stage 5
--ID
ts1Fijr, 411) o
HNy....,1
IN.71
I-I,N N N
Compound (4)
Stage 1 is the same as described for compound (3).
Stage 2 ¨ Synthesis of (S)-Cyclopentyl 2-amino-4-(4-nitrophenoxy)butanoate
CA 02836827 2013-12-17
79
= 00L)
02N NH2
To a solution of Stage 1 product (4.0g, 9.8mmol) in DCM (12m1) was added
trifluoroacetic
acid (12m1). After stirring at r. t. for 1 h the reaction was diluted with
DCM, cooled in ice and
neutralised by the addition of aq. Ammonia. The organic layer was collected
and washed
with water, aq. Sodium hydrogen carbonate and brine, then dried over MgSO4 and
concentrated under reduced pressure to afford the title compound as a yellow
oil (3.0g,
100%). LCMS purity 97%, rn/z 309.1 [M+H], 1H NMR (400 MHz, CDCI3), 8: 8.2 (2H,
d), 7.0
(2H, d), 5.2 (1H, m), 4.2 (2H, m), 3.6 (1H, dd), 1.7-1.5 (10H, m).
Stage 3¨ Synthesis of (S)-Cyclopentyl 2-(cyclohexylamino)-4-(4-
nitrophenoxy)butanoate
0
=
OrA,43,-.0
H
02N
To a flask containing Stage 2 product (1.0g, 3.3mmol) and cyclohexanone
(0.34m1,
3.3mmol) under nitrogen was added anhydrous Me0H (10m1). After stirring for 12
h at r. t.
sodium triacetoxyborohydride (2.07g, 9.75mmol) was added. After 4 h the
reaction was
poured slowly onto a mixture of DCM / aq. HCI (1M). After stirring for 10 min
the organic
layer was collected and washed with sodium hydrogen carbonate and brine, then
dried
over MgSO4 and concentrated under reduced pressure to afford the title
compound as a
yellow oily solid (1.21g, 95%). LCMS purity 92%, m/z 391.1 [M+H].
Stage 4¨ Synthesis of (S)-Cyclopentyl 4-(4-aminophenoxy)-2-
(cyclohexylamino)butanoate
0
* Orke-C>
H2N
CA 02836827 2013-12-17
Triethylamine (1.29m1, 9.3mmol) and formic acid (3480, 9.3mmol) were dissolved
in Et0H
(10m1) and added to a solution of Stage 3 product (1.2g, 3.1mmol) in Et0H
(10m1). 10%
Pd/C (approximately 10 mol%) was added and the mixture was heated to reflux.
After 30
min the hot reaction mixture was filtered through celite and the residue was
washed with
Me0H. The filtrate and washings were combined and evaporated and the residue
was
partitioned between DCM and sat. aq. NaHCO3. The organic layer was washed with
brine,
dried over MgSO4 and concentrated under reduced pressure to afford the title
compound
(1.01g, 92%). LCMS purity 94%, m/z 361.1 [M+H], 1FINMR (400 MHz, CDCI3), 8:
6.7 (2H,
d), 6.6 (2H, d), 5.2 (1H, m), 4.0 (1H, m), 3.9 (1H, m), 3.5 (1H, dd), 2.3 (1H,
m), 2.1 (1H, m),
1.9 (4H, m), 1.7 (7H, m), 1.6 (3H, m), 1.3-0.9 (5H, m).
Stage 5¨ Synthesis of (S)-2-Cyclohexylamino-4-(4-[(2,4-diamino-5-methyl-
pyrido[2,3-
dlpyrimidin-6-ylmethyl)-amino]-phenoxy)-butyric acid cyclopentyl ester (4)
is 0,,....d)(0L)
LI j....,
111 N
,,A, ...- r FIN. ...- H
H2N N N
2,4-Diamino-5-methylpyrido[2,3-djpyrimidine-6-carbonitrile (0.50g, 2.5mmol),
Stage 4
product (0.36g, 1.0mmol) and Raney nickel (3g, damp) in acetic acid (40m1)
were stirred at
r. t. under an atmosphere of hydrogen. After 48 h the reaction mixture was
filtered through
celite and the solvent evaporated under reduced pressure. The material was
loaded in
Me0H onto an SCX column and eluted with a 1% ammonia solution in Me0H. The
crude
product was then adsorbed onto silica and purified by column chromatography
(10%
Me0H/DCM) to afford the title compound (76 mg, 14%). LCMS purity 90 %, m/z
548.2
[M+Hr, 'H NMR (400 MHz, DMSO), 8: 8.5 (1H, s), 7.0 (2H, bs), 6.7 (2H, d), 6.6
(2H, d), 6.2
(2H, bs), 5.5 (1H, m), 5.1 (1H, m), 4.1 (2H, s), 3.9 (2H, m), 3.4 (1H, m), 2.7
(3H, s), 2.3 (1H,
m), 1.9 (1H, m), 1.8 (4H, m), 1.6 (11H, m), 1.1 (5H, m).
CA 02836827 2013-12-17
81
EXAMPLE 5
This example describes the modification of the known PI3 kinase inhibitor N-[5-
(4-Chloro-
3-methanesulfonyl-pheny1)-4-methyl-thiazol-2-y1]-acetamide (compound (20)) by
the
attachment of an amino acid ester motif at a point where no disruption of its
binding mode
occurs.
Compound (20): N-(5-(4-Chloro-3-methanesulfonyl-pheny1)-4-methyl-thiazol-2-y1]-
acetamide
0µt
I 1-- 7---
N
/110 S H
CI
a=r0
Compound (20) was prepared as described in W003072552
Compounds based on compound (20) were prepared by the methods outlined below.
Compounds (21) and (22) were prepared by the method described in the following
scheme:
CA 02836827 2013-12-17
82
i) Na2S03, NaHCO,
Chlorosulfonic acid water /dioxane
Si o 0 o
--==== CI -..--... CI
1101 0
CI Stage 1 ii) Mel, DMF 0¨ ¨=
a
Stage 2
Br2, dioxane Stage 3
1
.=
I \ NH2 HiCINH, EtCH
1/01 S
S.
Hol-40X1) +
a Stage 4
NHBoc
0= ¨0 070
EDCI, HOBt
Et3N, DMF w
QUA 5
I \ NJLI)LeC) ¨ I \ NL4OL)
0 s H TFA / DCM so S H
NHBoc -...
a Stage 6
a NH2
0= II OTO
Compound (21)
NaOH, THF, Me0H1 Stage 7
I t.---ti'l014
I P. ---.= NiL 0 f 1 TFA / DCM
is
a
NHBoc
Stage 8 5 s
NH2
a
0 =r0
70 Compound (22)
Stage 1 ¨ Synthesis of 2-Chloro-5-(2-oxo-propyI)-benzenesulfonyl chloride
CA 02836827 2013-12-17
83
o
CI
o=s=o
CI
4-Chlorophenyl acetone (4g, 0.023mol) was added dropwise to chlorosulfonic
acid (30m1,
0.45mo1) at -10 C under N2 with gentle stirring. The reaction mixture was
allowed to warm
to r. t. and stirring was continued for 18 h. The reaction mixture was
carefully quenched by
adding dropwise to crushed ice (500m1). The aq. Solution was extracted with
Et0Ac (3 x
250m1). Et0Ac layers combined, dried (Na2SO4), filtered and concentrated to
dryness in
vacuo to give the crude title compound (6.3g, 65%) which was used in the next
step without
further purification. LCMS purity 92%. 1H NMR (400 MHz, CDCI3), 8: 2.30 (3H,
s), 3.85 (2H,
s), 7.50 (1H, d), 7.65 (1H, d), 7.95 (1H, s).
Stage 2 ¨ Synthesis of 1-(4-Chloro-3-methanesulfonyl-phenyl)-propan-2-one
10I o
o=s=o
A mixture of Na2S03 (3.79g, 0.030mol) and NaHCO3 (2.53g, 0.030mol) in water
(90m1) was
stirred at 70 C. To this solution was added a solution of Stage 1 product
(4.65g, 0.015mol)
in dioxane (190m1). Stirring was continued at 70 C for 1 h. Upon cooling to r.
t. the reaction
mixture was concentrated to dryness in vacuo giving a brown solid. DMF (190m1)
was
added followed by Mel (1.88m1, 0.030mol). The reaction mixture was stirred at
40 C for 1 h.
After completion the reaction mixture was poured into water (90m1) and
extracted with
Et0Ac (500m1). The Et0Ac was dried (Na2SO4), filtered and concentrated in
vacuo to give
the title compound as a brown solid (2.49g, 67%) which was used in the next
step without
further purification. LCMS purity 81%, m/z 247 [M++H]; 1H NMR (400 MHz,
CDCI3), 8: 2.15
(3H, s), 3.20 (3H, s), 3.75 (2H, s), 7.35 (11, d), 7.45 (1H, d), 7.85 (1H, s).
Stage 3 ¨ Synthesis of 1-Bromo-1-(4-chloro-3-methanesulfonyl-phenyl)-propan-2-
one
CA 02836827 2013-12-17
84
Br
0 0
C1
0-1¨
¨S0¨
I
To a stirred solution of Stage 2 product (1.88g, 7.60mmol) in 1,4-dioxane
(120m1) bromine
(0.292m1, 5.72mmol) was added dropwise at r. t. giving a dark orange solution.
Stirring was
continued for 18 h. The reaction mixture was evaporated to dryness in vacuo
avoiding
heating above 30 C during evaporation. The residue was re-dissolved in Et0Ac
(100m1)
and washed with sat aq NaHCO3 (20m1) followed by water (20m1). The Et0Ac layer
was
dried (Na2504), filtered and concentrated in vacuo. Purification by flash
chromatography
(50% Et0Ac / heptane) gave the title compound as a yellow oil (2.0g, 80%).
LCMS purity
74%, m/z 325/327 [M++H];11-1 NMR (400 MHz, CDCI3), 6: 2.35 (3H, s), 3.25 (3H,
s), 5.35
(1H, s), 7.50 (1H, d), 7.65 (1H, dd), 8.05 (1H, s).
Stage 4¨ Synthesis of 5-(4-Chloro-3-methanesulfonyl-pheny1)-4-methyl-thiazol-2-
ylamine
, N
i ..-__NH2
0 S
CI
-- S ---
0--1 ¨0
A mixture of Stage 3 product (2g, 6.15mmol) and thiourea (468mg, 6.15mmol) in
Et0H
(65m1) was stirred at 70 C for 1.5 h. The reaction was then cooled to r. t.
and precipitation
occurred. The cream solid was collected by filtration to afford the title
compound (1.2g,
64%). LCMS purity 91%, m/z 303 [M++H],11-INMR (400 MHz, Me0D), 6: 2.35 (3H,
s), 3.35
(3H, s), 7.75-7.85 (2H, m), 8.15 (1H, s).
Stage 5 ¨ Synthesis of (S)-2-tert-Butoxycarbonylamino-445-(4-chloro-3-
methanesulfonyl-
pheny1)-4-methyl-thiazol-2-ylcarbamoyli-butyric acid cyclopentyl ester
CA 02836827 2013-12-17
=
0 0
S H
NHBoc
CI
To a stirred mixture of 2-tert-butoxycarbonylamino-pentanedioic acid 1-
cyclopentyl ester
(208mg, 0.66mmol), EDCI (190mg, 0.99mmol) and HOBt (107mg, 0.79mmol) in DMF
(1.5m1) was added dropwise a solution of Stage 4 product (200mg, 0.66mmol) in
DMF
(1.5m1) at r. t. Triethylamine (0.138m1,0.99mmol) was added and stirring was
continued for
18 h. The reaction mixture was diluted with water (10m1) and extracted with
Et0Ac (15m1).
The Et0Ac layer was washed with water (10m1), dried (Na2SO4), filtered and
concentrated
in vacuo. Purification by preparative TLC (70% Et0Ac/ heptane, Ri 0.5)
afforded the title
compound (160mg, 40%). LCMS purity 91%, m/z 600/602 [M++H), 11-1 NMR (400 MHz,
DMSO), 8: 1.45-1.55 (9H, s), 1.65-2.15 (10H, m), 2.45 (3H, s), 2.70 (2H, m),
3.45 (3H, s),
410-4.25 (1H, m), 5.25 (1H, m), 7.75-7.90 (2H, m), 8.25 (1H, s).
Stage 6 ¨ Synthesis of (S)-2-Amino-445-(4-chloro-3-methanesulfonyl-pheny1)-4-
methyl-
thiazol-2-ylcarbamoyll-butyric acid cyclopentyl ester (21)
I IN)3L-.10--0
S H
NH2
scr-1=--0
A solution of Stage 5 product (140mg, 0.233mmo1) in 20% TFA / DCM (2m1) was
allowed to
stand at r. t. for 3 h. After completion the reaction mixture was concentrated
in vacuo to
give compound (21) (143mg, 100%). LCMS purity 97%, m/z 500/502 [M++H], 'H NMR
(400
MHz, Me0D), 8: 1.35-1.85 (8H, m), 2.00-2.20 (2H, m), 2.25 (3H, s), 2.60 (2H,
m), 3.20 (3H,
s), 3.85-4.00 (1H, m), 5.10 (1H, m), 7.50-7.65 (2H, m), 7.95 (1H, s).
Stage 7¨ Synthesis of (S)-2-tert-Butoxycarbonylamino-445-(4-chloro-3-
methanesulfonyl-
pheny1)-4-methyl-thiazol-2-ylcarbamoy11-butyric acid
CA 02836827 2013-12-17
86
0 0
I rsNOH
0 S H
NHBoc
Cl
0-1---0
To a solution of Stage 5 product (20mg, 0.033mmol) in a mixture of THF (0.5m1)
and Me0H
(0.5m1) was added 2M aq. NaOH (0.5m1). The mixture was allowed to stand at r.
t. for 3 h.
Upon completion the reaction mixture was concentrated to near dryness and 1M
Ha
added dropwise until pH 1-2. The resultant precipitate was collected by
filtration under
slight pressure. The solid was washed with water (0.5m1) and thoroughly dried
in vacuo to
yield the title compound (12mg, 68%). LCMS purity 94%, m/z 532/534 [M++11, 1H
NMR
(400 MHz, CDC13), 8: 1.55-1.70 (9H, s), 2.15-2.55 (2H, m), 2.60 (3H, s), 275-
2.90 (2H, m),
3.55 (3H, s), 4.25-4.45 (1H, m), 7.85-8.00 (2H, m), 8.35 (1H, s).
Stage 8¨ Synthesis of (S)-2-Amino-445-(4-chloro-3-methanesulfonyl-pheny1)-4-
methyl-
thiazol-2-ylcarbamoyl]-butyric acid (22)
0 0
0 S H
NH2
Cl
0,0
A solution of Stage 7 product (12mg, 0.0225mmo1) in 20% TFA / DCM (0.3m1) was
allowed
to stand at r. t. for 3 h. After completion the reaction mixture was
concentrated in vacuo to
give the title compound (22) (12mg, 100%). LCMS purity 94%, m/z 432/434
[M4+H], 'H
NMR (400 MHz, Me0D), 8: 2.10-2.25 (2H, m), 2.30 (3H, s), 2.65-2.75 (2H, m),
3.25 (3H, s),
3.95-4.05 (1H, m), 7.60-7.80 (2H, m), 8.05 (1H, s).
Compound (23) was prepared by the method described in the following scheme:
CA 02836827 2013-12-17
87
- i) Na2S03, NaHCO3
Chlorosuttonic acid water / &mane
a
1.1 o ____,.... a SI o
4.-
40 0
CI Stage 1 01=0 ii) Mel. DMF 0=r0
a
Stage 2
Br2, dioxane stage 3
I
I rµ"---NH2 tipINH, Et0H
* S
HO"-U...y.t0"..j. + - _______
so
CI Stage 4 CI 0
NHBoc
OTO OT-0
EDCI, HOBt Stage 5
EN, DMF
40 s H HCI, Et20 is S H
NHBoc NH2
....................Ø
CI Stage 6 a
0-To o=ro
Compound (23)
Stages 1, 2, 3 and 4 are the same as described for the preparation of
compounds (21) and
(22)
Stage 5 ¨ Synthesis of (S)-2-Amino-4-[5-(4-chloro-3-methanesulfonyl-pheny1)-4-
methyl-
thiazol-2-ylcarbamoyl]-butyric acid tert-butyl ester
0 0
I 1 0 101 S H
NHBoc
CI
01---0
CA 02836827 2013-12-17
88
This compound was prepared from 2-tert-butoxycarbonylamino-pentanedioic acid 1-
tert-
butyl ester and 5-(4-chloro-3-methanesulfonyl-pheny1)-4-methyl-thiazol-2-
ylamine (Stage 4
product) following the procedure described for the synthesis of compound (21).
Stage 6 ¨ Synthesis of (S)-2-Amino-445-(4-chloro-3-methanesutfonyl-pheny1)-4-
methyl-
thiazol-2-ylcarbamoylpbutyric acid tert-butyl ester (23)
I r--NLyLO
0 S H
NH2
CI
0---'11 0
To a solution of Stage 5 product (50mg, 0.085mmol) in Et0Ac (0.25m1) was added
2M HCI
/ ether solution (0.25m1) at r. t. The reaction mixture was vigorously stirred
for 4 h. The
reaction was re-treated with a mixture of Et0Ac (0.25m1) and 2M HCI / ether
(0.25m1).
Stirring was continued for 1 h. The precipitate formed was collected by
filtration under
gravity, partitioned between Et0Ac (3m1) and sat. aq. NaHCO3 (0.5m1). The
Et0Ac layer
was washed with water (1m1), dried (Na2SO4), filtered and concentrated in
vacuo to give
compound (23) (6.5mg, 16%). LCMS purity 95%, m/z 488/490 [M++H], 'H NMR (400
MHz,
Me0D), 8: 1.35-1.40 (9H, s), 1.80-2.05 (2H, m), 2.30 (3H, s), 2.45-2.55 (2H,
m), 3.25 (3H,
s), 3.30-3.35 (1H, m), 7.60-7.70 (2H, m), 8.05 (1H, s).
Biological Assays
Histone deacetvlase inhibitory activity assay
The ability of compounds to inhibit histone deacetylase activities was
measured using the
commercially available HDAC fluorescent activity assay from Biomol. In brief,
the Fluor de
Lysrmsubstrate, a lysine with an epsilon-amino acetylation, is incubated with
the source of
histone deacetylase activity (HeLa cell nuclear extract) in the presence or
absence of
inhibitor. Deacetylation of the substrate sensitises the substrate to Fluor de
LysTM
developer, which generates a fluorophore. Thus, incubation of the substrate
with a source
CA 02836827 2013-12-17
89
of HDAC activity results in an increase in signal that is diminished in the
presence of an
HDAC inhibitor.
Data are expressed as a percentage of the control, measured in the absence of
inhibitor,
with background signal being subtracted from all samples, as follows:-
% activity = ((Sl ¨ B) / (S - B)) x 100
where S is the signal in the presence of substrate, enzyme and inhibitor, S
is the signal in
the presence of substrate, enzyme and the vehicle in which the inhibitor is
dissolved, and B
is the background signal measured in the absence of enzyme.
IC50 values were determined by non-linear regression analysis, after fitting
the results of
eight data points to the equation for sigmoidal dose response with variable
slope ( /0 activity
against log concentration of compound), using Graphpad Prism software.
Histone deacetylase activity from crude nuclear extract derived from HeLa
cells was used for
screening. The preparation, purchased from 4C (Seneffe, Belgium), was prepared
from
HeLa cells harvested whilst in exponential growth phase. The nuclear extract
was prepared
according to Dignam JD 1983 Nucl. Acid. Res. 11, 1475-1489, snap frozen in
liquid
nitrogen and stored at -80 C. The final buffer composition was 20 mM Hepes,
100 mM KCI,
0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF and 20 % (v/v) glycerol.
Aurora Kinase A Inhibitory Activity Assay
The ability of compounds to inhibit aurora kinase A activity was measured
using a
microplate assay. In brief, 96-well Flashplates (PerkinElmer Life Sciences)
were pre-
coated with myelin basic protein (MBP). MBP (100u1 of 100 mg/ml in PBS) was
incubated
at 372C for 1 h, followed by overnight incubation at 4QC. Plates were then
washed with PBS
and allowed to air dry.
To determine the activity of aurora kinase A, 40 ng enzyme (ProQuinase:
recombinant, full
length human aurora kinase A, N-terminally fused to GST and expressed by
baculovirus in
Sf21 insect cells) was incubated in assay buffer (50 mM Tris (pH7.5), 10 mM
NaCI, 2.5 mM
MgCl2, 1 mM DTT, 0.4 % DMSO), 10 pM ATP (Km of the enzyme) and 0.5 pCi Iv-P]-
ATP
and with varying concentrations of inhibitor. Wells lacking inhibitor were
used as vehicle
CA 02836827 2013-12-17
controls and wells containing no enzyme were used to measure the 'background'
signal.
Plates were incubated overnight at 30 C. After incubation the contents of the
wells were
removed and the plates washed three times with PBS containing 10 mM tetra
sodium
pyrophosphate prior to scintillation counting using a Wallac MicroBeta TriLux.
Dose response curves were generated from 10 concentrations (top final
concentration 10
pM, with 3-fold dilutions), using triplicate wells.
IC50 values were determined by non-linear regression analysis, after fitting
the data point
results to the equation for sigmoidal dose response with variable slope ( /0
activity against
log concentration of compound), using Xlfit software.
Dihydrofolate Reductase (DHFR) Inhibitory Activity Assay
The ability of compounds to inhibit DHFR activity was measured in an assay
based on the
ability of DHFR to catalyse the reversible NADPH-dependent reduction of
dihydrofolic acid
to tetrahydrofolic acid using a Sigma kit (Catalogue number CS0340). This uses
proprietary
assay buffer and recombinant human DHFR at 7.5 xl 04 Unit per reaction, NADPH
at 60
pM and dihydrofolic acid at 50 pM. The reaction was followed by monitoring the
decrease
in absorbance at 340 nm, for a 2 minute period, at room temperature, and the
enzyme
activity was calculated as the rate of decrease in absorbance. Enzyme
activity, in the
presence of inhibitor, was expressed as a percentage of inhibitor-free
activity and the
inhibitor IC50 was determined from a sigmoidal dose response curve using Xlf
it software
(To activity against log concentration of compound). Each sample was run in
triplicate and
each dose response curve was composed of 10 dilutions of the inhibitor.
P38 MAP Kinese a Inhibitory Activity Assay
The ability of compounds to inhibit p38 MAP kinase a (full length human enzyme
expressed
in E coli as an N-terminally GST-tagged protein) activity was measured in an
assay
performed by Upstate (Dundee UK). In a final reaction volume of 25 pl, p38 MAP
kinase a
(5-10 mU) was incubated with 25 mM Tris pH 7.5, 0.02 mM EGTA, 0.33 mg/ml
myelin
basic protein, 10 mM magnesium acetate, ATP 90 pM (Km 97 pM) and [y-3313]-ATP
(specific activity approx. 500 cpm/pmol). The reaction was initiated by the
addition of the
MgATP mix. After incubation for 40 minutes at room temperature, the reaction
was stopped
by the addition of 5 pl of a 3 % phosphoric acid solution. 10 Iii of the
reaction was then
CA 02836827 2013-12-17
91
spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM
phosphoric
acid and once in methanol, prior to drying and scintillation counting.
Dose response curves were generated from a % log dilution series of a stock
inhibitor
solution in DMSO. Nine dilutions steps were made from a top, final
concentration of 10 pM,
and a 'no compound' blank was included. Samples were run in duplicate. Data
from
scintillation counts were collected and subjected to free-fit analysis by
Graphpad Prism
software. From the curve generated, the concentration giving 50 % inhibition
was
determined.
PI 3-Kinase v Inhibition Assay
The measurement of PI 3-kinase y activity is dependent upon the specific and
high affinity
binding of the GRP1 pleckstrin homology (PH) domain to PIP3, the product of PI
3-kinase
activity. A complex is formed between europium-labelled anti-GST monoclonal
antibody, a
GST-tagged GRP1 PH domain, biotinylated PIP3 and streptavidin-
allophycocyanin(APC).
This complex generates a stable time-resolved fluorescence resonance energy
transfer
(FRET) signal, which is diminished by competition of PIP3, generated in the PI
3-kinase
assay, with the biotinylated PIP3.
The assay was performed at Upstate (Dundee, UK) as follows: in a final
reaction volume of
20 pl, PI 3-kinase y (recombinant N-terminally His6-tagged, full length human
enzyme,
expressed by baculovirus in Sf21 insect cells) was incubated in assay buffer
containing 10
pM phosphatidylinosito1-4,5-bisphosphate and 100 pM MgATP (Km of the enzyme
117
pM). The reaction was initiated by the addition of the MgATP mix. After
incubation for 30
minutes at room temperature, the reaction was stopped by the addition of 5 pl
of stop
solution containing EDTA and biotinylated phosphatidylinosito1-3,4,5-
trisphosphate. Finally,
pl of detection buffer was added, which contained europium-labelled anti-GST
monoclonal antibody, GST-tagged GRP1 PH domain and streptavidin-APC. The plate
was
then read in time-resolved fluorescence mode and the homogenous time-resolved
fluorescence (HTRF) signal was determined according to the formula HTRF =
10000 x
(Em665nm/Em620nm).
Duplicate data points were generated from a iii log dilution series of a stock
solution of
compound in DMSO. Nine dilutions steps were made from a top final
concentration of 10
pM, and a 'no compound' blank was included. HTRF ratio data were transformed
into %
CA 02836827 2013-12-17
92
activity of controls and analysed with a four parameter sigmoidal dose-
response (variable
slope) application. The concentration giving 50 % inhibition (IC50) was
determined.
Cell Proliferation inhibition Assay
Cancer cell lines (U937and HCT 116) growing in log phase were harvested and
seeded at
1000 ¨2000 cells/well (100 pl final volume) into 96-well tissue culture
plates. Following 24
h of growth cells were treated with compound. Plates were then re-incubated
for a further
72 ¨ 96 h before a WST-1 cell viability assay was conducted according to the
suppliers
(Roche Applied Science) instructions.
Data were expressed as a percentage inhibition of the control, measured in the
absence of
inhibitor, as follows:-
% inhibition = 100-((SI/S )x100)
where S' is the signal in the presence of inhibitor and S is the signal in
the presence of
DMSO.
Dose response curves were generated from 8 concentrations (top final
concentration 10
LIM, with 3-fold dilutions), using 6 replicates.
IC50 values were determined by non-linear regression analysis, after fitting
the results to
the equation for sigmoidal dose response with variable slope (% activity
against log
concentration of compound), using Graphpad Prism software.
LPS-stimulation of human whole blood
Whole blood was taken by venous puncture using heparinised vacutainers (Becton
Dickinson) and diluted in an equal volume of RPMI1640 tissue culture media.
100 pl was
plated in V-bottomed 96 well tissue culture plates. Inhibitor was added in 100
pl of
RPMI1640 media, and 2 h later the blood was stimulated with LPS (E coil strain
005:65,
Sigma) at a final concentration of 100 ng/m1 and incubated at 37 C in 5 '% CO2
for 6 h.
TNF-a levels were measured from cell-free supernatants by sandwich ELISA (R&D
Systems #OTA00B).
CA 02836827 2013-12-17
93
Broken Cell Carboxylesterase Assay
Preparation of cell extract
U937 or HCT 116 tumour cells (- le) were washed in 4 volumes of Dulbeccos PBS
(-
1litre) and pelleted at 525 g for 10 min at sec. This was repeated twice and
the final cell
pellet was resuspended in 35 ml of cold homogenising buffer (Trizma 10 mM,
NaCI 130
mM, CaCl2 0.5 mM pH 7.0 at 25 C). Homogenates were prepared by nitrogen
cavitation
(700 psi for 50 min at 4 C). The homogenate was kept on ice and supplemented
with a
cocktail of inhibitors at final concentrations of:
Leupeptin 1 gM
Aprotinin 0.1 gM
E648 gM
Pepstatin 1.5 gM
Bestatin 162 gM
Chymostatin 33 gM
After clarification of the cell homogenate by centrifugation at 525 g for 10
min, the resulting
supernatant was used as a source of esterase activity and was stored at -80 C
until
required.
Measurement of ester cleavage
Hydrolysis of esters to the corresponding carboxylic acids can be measured
using the cell
extract, prepared as above. To this effect cell extract (-30 gg / total assay
volume of 0.5
ml) was incubated at 37 C in a Tris- HCI 25 mM, 125 mM NaCI buffer, pH 7.5 at
25 C. At
zero time the ester (substrate) was then added at a final concentration of 2.5
gM and the
samples were incubated at 37 C for the appropriate time (usually 0 or 80 min).
Reactions
were stopped by the addition of 3 x volumes of acetonitrile. For zero time
samples the
acetonitrile was added prior to the ester compound. After centrifugation at
12000 g for 5
min, samples were analysed for the ester and its corresponding carboxylic acid
at room
temperature by LCMS (Sciex API 3000, HP1100 binary pump, CTC PAL).
Chromatography
was based on an AceCN (75*2.1mm) column and a mobile phase of 5-95%
acetonitrile in
water /0.1 % formic acid.
Quantification of hCE-1. hCE-2 and hCE-3 expression in monocytic and non-
monocytic cell lines
CA 02836827 2013-12-17
94
Gene-specific primers were used to PCR-amplify hCE-1, -2 and -3 from human
cDNA.
PCR products were cloned into a plasmid vector and sequence-verified. They
were then
serially diluted for use as standard curves in real-time PCR reactions. Total
RNA was
extracted from various human cell lines and cDNA prepared. To quantitate
absolute levels
of hCE's in the cell lines, gene expression levels were compared to the cloned
PCR
product standards in a real-time SYBR Green PCR assay. Figure 1 shows that hCE-
1 is
only expressed to a significant amount in a monocytic cell line.
Biological Results
The compounds referred to in Examples 1-5 above were investigated in the
enzyme
inhibition, cell proliferation and ester cleavage assays described above and
the results are
shown in Tables 3 and 4.
Potency
Table 3
HDAC Enzyme inhibition IC50 Cell proliferation IC50 nM Ratio
IC50
nM (HDAC - Hela cell (U937 cells) cell/enzyme
nuclear extract)
Unmodified Modulator 100 400 4
Compound (7)
(SAHA)
Modified Modulator 100 50 0.5
Compound (8)
(cyclopentyl ester)
Acid resulting from 70 Inactive NA
ester cleavage of
Modified Modulator
Compound (9)
Modified Modulator 130 1300 10
Compound (10)
(t-butyl ester)
The above results show that:
(I) the amino acid ester modified compounds (Compounds 8 and 10) and the
acid
(Compound 9) which would result from cleavage of the ester motif, have 1C5Os
in the
enzyme assay comparable to the value for the unmodified HDAC inhibitor (SAHA ¨
Compound 7) indicating that the alpha amino acid ester motif was attached to
SAHA at a
point which did not disrupt its binding mode.
CA 02836827 2013-12-17
(ii) even though the esters (Compounds 8 and 10) and acid (Compound 9) have
comparable activities to the unmodified inhibitor (SAHA ¨ Compound 7) there is
a
significant increase in the cellular potency of the esterase cleavable
cyclopentyl ester
(Compound 8) over the unmodified inhibitor (Compound 7) but a substantial
decrease in
cellular potency in the case of the esterase stable t-butyl ester (Compound
10), indicating
that the latter did not accumulate the acid in cells to generate increased
cellular potency.
(iii) the greater activity in the cell proliferation assay for Compound 8
over the
unmodified counterpart, Compound 7 (or the non-hydrolysable ester derivative,
Compound
10), indicates that the ester is hydrolysed to the parent acid Compound 9 in
the cell where
it accumulates and exerts a greater inhibitory effect.
Table 3 continued
Aurora kinase Enzyme inhibition IC50 Cell proliferation IC50 Ratio IC50
nM (Aurora kinase A) nM (U937 cells) cell/enzyme
Unmodified modulator 350 430 1.3
Compound(11)
Modified Modulator 2300 3.5 0.0015
Compound (12)
(cyclopentyl ester)
Acid resulting from 500 >5000 NA
ester cleavage of
Modified Modulator
Compound (13)
Modified Modulator 3000 75 0.025
Compound (14)
(t-butyl ester)
The above results show that:
(i) the alpha amino acid modified inhibitor, Compound 13, which would
result from
cleavage of the ester motif in Compound 12, has an IC50 value in the enzyme
assay
comparable to that of the unmodified aurora kinase inhibitor (Compound 11)
indicating that
it is possible to attach the alpha amino acid ester motif at a point which
does not disrupt the
binding to aurora kinase A.
(ii) even though the acid (Compound 13) has a comparable enzyme activity to
the
unmodified inhibitor (Compound 11) and the ester (Compound 12) is a weaker
inhibitor,
there is a significant increase in the cellular potency of Compound 12 over
the unmodified
inhibitor (Compound 11). The less readily cleaved t-butyl ester (Compound 14)
has a
comparable enzyme activity to the cleavable cyclopentyl ester (Compound 12)
but is some
20 fold less active in the cell assay
CA 02836827 2013-12-17
96
(iii) the greater activity in the cell proliferation assay of Compound 12
over both the
unmodified counterpart (Compound 11) and the less readily cleaved t-butyl
ester
(compound 14) indicates that the cyclopentyl ester is hydrolysed to the parent
acid in the
cell, where it accumulates, and exerts greater inhibitory effect.
Table 3 continued
P38 MAP kinase Enzyme inhibition Inhibition of TNFa production in Ratio
IC50
IC50 nM human whole blood WB/enzyme
(P38 MAP kinase) IC50 nM
Unmodified Modulator 50 300 6
Compound (15)
Modified Modulator 25 20 0.8
Compound
(16) (cyclopentyl ester)
Acid resulting from ester 30 not tested NA
cleavage of Modified
Modulator Compound
(17)
Modified Modulator 40 750 18
Compound
(18)
(t-butyl ester)
The above results show that:
(i) the alpha amino acid modified inhibitor Compound 16, and the acid
Compound 17
which would result from cleavage of the ester motif in Compound 16, have IC50
values in
the enzyme assay comparable to the value for the unmodified P38 MAP kinase
inhibitor
(Compound 15) indicating that it is possible to attach the alpha amino acid
ester motif at a
point which does not disrupt the binding to P38 MAP kinase.
(ii) the acid, Compound 17, has comparable activity against the enzyme to
the
unmodified inhibitor (Compound 15) and to the t-butyl ester (Compound 18).
However
there is a significant increase in the ability of the cyclopentyl ester
(Compound 16) to
inhibit TNF production inside monocytic cells present in whole blood compared
to the
unmodified inhibitor (Compound 15) and the less readily cleaved t-butyl ester
(Compound
18).
(iii) the greater activity in the whole blood assay for Compound 16 over
the unmodified
counterpart Compound 15 and the less readily cleaved t-butyl ester Compound 18
indicates that the cyclopentyl ester is hydrolysed to the parent acid in the
cell, where it
accumulates, and exerts a greater inhibitory effect.
CA 02836827 2013-12-17
97
Table 3 continued
DHFR Enzyme inhibition IC50 Cell proliferation IC50 nM Ratio
IC50
nM (DHFR) (U937 cells) cell/enzyme
Unmodified Modulator 10 2200 220
Compound (2 G=N)
Modified Modulator 1700 23 0.013
Compound (6)
(cyclopentyl ester)
Acid resulting from 10 not tested Not
applicable
ester cleavage of
Modified Modulator
Compound (19)
The above results show that:
(i) the alpha amino acid modified inhibitor, Compound 19, which would
result from
cleavage of the ester motif in Compound 6, has an IC50 value in the enzyme
assay
comparable to that of the unmodified DHFR inhibitor (Compound 2 (G=N))
indicating that it
is possible to attach the alpha amino acid ester motif at a point which does
not disrupt the
binding to DHFR.
(ii) even though the acid (Compound 19) has a comparable enzyme inhibitory
activity
to the unmodified inhibitor (Compound 2 (G=N)), the ester (Compound 6) is
significantly
more potent in inhibiting cell proliferation than the unmodified inhibitor
(Compound 2
(G=N)).
(iii) the greater activity in the cell proliferation assay of Compound 6
over the
unmodified counterpart (Compound 2 (G=N)) indicates that the cyclopentyl ester
is
hydrolysed to the parent acid in the cell, where it accumulates, and exerts
greater inhibitory
effect.
Table 3 continued
PI 3-Kinase Enzyme inhibition Inhibition of TNFa production in Ratio
IC50
IC50 nM human whole blood WB/enzyme
(P13-Kinase) IC50 nM
Unmodified Modulator 500 8500 17
Compound (20)
Modified Modulator 2700 400 0.15
Compound (21)
(cyclopentyl ester) ,
Acid resulting from 3600 Not tested not
applicable
ester cleavage of
Modified Modulator
(22)
CA 02836827 2013-12-17
98
Modified Modulator 7100 5200 0.75
Compound
(23)
(t-butyl ester)
The above results show that:
(i) the alpha amino acid ester modified inhibitor, Compound 21, and the
acid,
Compound 22, which would result from cleavage of the ester motif in Compound
21, have
IC50 values in the enzyme assay within a factor of 10 of the value for the
unmodified PI 3-
kinase inhibitor (Compound 20), indicating that it is possible to attach the
alpha amino acid
ester motif at a point which still retains reasonable binding to PI 3-kinase.
(ii) although the acid, Compound 22, has comparable activity to the
unmodified
inhibitor (Compound 20) and the ester (Compound 21), there is a significant
increase in
the potency of the ester to inhibit TNF production in monocytic cells present
in whole blood
compared to the unmodified inhibitor (Compound 20) and the less readily
cleaved t-butyl
ester (Compound 23).
(iii) the greater activity in the whole blood assay for Compound 21 over the
unmodified
counterpart Compound 20 and the less readily cleaved t-butyl ester Compound 23
indicates that the cyclopentyl ester is hydrolysed to the parent acid in the
cell, where it
accumulates, and exerts a greater inhibitory effect.
Selectivity
Table 4: Comparison of cell proliferation and ester cleavage for a monocytic
and a
non monocytic cell line.
HDAC U937 (Monocytic cell line) HCT116 (non-
monocytic cell line)
Compound Cell Acid Cell Acid
proliferation produced' proliferation produced'
IC50 nM ng/ml IC50 nM ng/ml
Unmodified 400 Not applicable 700 Not applicable
Modulator
Compound
(7)
Modified 60 110 2100 1
Modulator
Compound
(24)
1The amount of acid produced after incubation of the modified compound
(Compound 24)
for 80 min in the broken cell carboxylesterase assay described above.
CA 02836827 2013-12-17
99
The above results show:
i) that the unmodified compound (compound 7) shows no selectivity between a
monocytic and non-monocytic cell line whereas this can be achieved by
attaching an
appropriate ester motif, as in Compound 24.
ii) this selectivity correlates with the improved cleavage of the ester to
the acid by the
monocytic cell line.
iii) the improved cellular activity is only seen in the cell line where
acid is produced
indicating that this improvement in cellular potency is due to accumulation of
the acid.
Table 4 continued
Aurora U937 (Monocytic cell line) HCT116 (non-monocytic cell line)
Kinase A
Compound Cell Acid Cell Acid
proliferation produced' proliferation produced'
IC50 nM ng/ml IC50 nM ng/m1
Unmodified 430 NA 560 NA
Modulator
Compound
(10)
Modified 1900 50 6100 0
Modulator
Compound
(25)
'The amount of acid produced after incubation of the compound (25) for 80
minutes in the
broken cell carboxylesterase assay described above.
The above results show:
i) that the unmodified compound (compound (10) shows no selectivity between
a
monocytic and a non-monocytic cell line whereas this is achieved by attaching
an
appropriate ester motif, as in Compound 25.
ii) this selectivity correlates with the improved cleavage of the ester to
the acid by the
monocytic cell line.
iii) the improved cellular activity is only seen in the cell line where
acid is produced
indicating that this improvement in cellular potency is due to accumulation of
the acid
CA 02836827 2013-12-17
100
Table 4 continued
DHFR U937 (Monocytic cell line) HC7116 (non-monocytic cell
line,
Compound Cell Acid Cell Acid
proliferation produced' proliferation produced'
, 1050 nM ng/ml IC50 nM ngimi
Unmodified 2200 Not 1700 NA
Modulator applicable
Compound
(2 G=N)
Modified 310 210 6700 2
Modulator
Compound
(5)
I The amount of acid produced after incubation of compound (5) for 80 min in
the broken
cell carboxylesterase assay described above.
The above results show that:
(i) the unmodified compound (compound 2 G = N) shows no selectivity between
a
monocytic and non-monocytic cell lines whereas this can be achieved by
attaching an
appropriate ester motif as in compound 5.
(ii) this selectivity correlates with the improved cleavage of the ester to
the acid
by the monocytic cell line.
(iii) the improved cellular activity is only seen in the cell line where
acid is
produced indicating that this improvement in cellular potency is due to
accumulation of the
acid
