Note: Descriptions are shown in the official language in which they were submitted.
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TRIPEPTIDES HAVING A HYDROXYPROLINE ETHER OF A SUBSTITUTED QUINOLINE FOR THE
INHIBITION OF NS3 (HEPATITIS C)
FIELD OF THE INVENTION
The present invention relates to compounds, processes for their synthesis,
compositions and methods for the treatment of hepatitis C virus (HCV)
infection. In
particular, the present invention provides novel peptide analogs,
pharmaceutical
compositions containing such analogs and methods for using these analogs in
the
treatment of HCV infection.
BACKGROUND OF THE INVENTION
Hepatitis C virus (HCV) is the major etiological agent of post-transfusion and
community-acquired non-A non-B hepatitis worldwide. It is estimated that over
200
million people worldwide are infected by the virus. A high percentage of
carriers
become chronically infected and many progress to chronic liver disease, so-
called
chronic hepatitis C. This group is in turn at high risk for serious liver
disease such
as liver cirrhosis, hepatocellular carcinoma and terminal liver disease
leading to
death.
The mechanism by which HCV establishes viral persistence and causes a high
rate
of chronic liver disease has not been thoroughly elucidated. It is not known
how
HCV interacts with and evades the host immune system. In addition, the roles
of
cellular and humoral immune responses in protection against HCV infection and
disease have yet to be established. Immunoglobulins have been reported for
prophylaxis of transfusion-associated viral hepatitis, however, the Center for
Disease Control does not presently recommend immunoglobulins treatment for
this
purpose. The lack of an effective protective immune response is hampering the
development of a vaccine or adequate post-exposure prophylaxis measures, so in
the near-term, hopes are firmly pinned on antiviral interventions.
Various clinical studies have been conducted with the goal of identifying
pharmaceutical agents capable of effectively treating HCV infection in
patients
afflicted with chronic hepatitis C. These studies have involved the use of
interferon-
alpha, alone and in combination with other antiviral agents. Such studies have
shown that a substantial number of the participants do not respond to these
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therapies, and of those that do respond favorably, a large proportion were
found to
relapse after termination of treatment.
Until recently, interferon (IFN) was the only available therapy of proven
benefit
approved in the clinic for patients with chronic hepatitis C. However the
sustained
response rate is low, and interferon treatment also induces severe side-
effects (i.e.
retinopathy, thyroiditis, acute pancreatitis, depression) that diminish the
quality of life
of treated patients. Recently, interferon in combination with ribavirin has
been
approved for patients non-responsive to IFN alone. However, the side effects
caused by IFN are not alleviated with this combination therapy. Pegylated
forms of
interferons such as PEG-Intron and Pegasys can apparently partially address
these deleterious side-effects but antiviral drugs still remain the avenue of
choice for
oral treatment of HCV.
Therefore, a need exists for the development of effective antiviral agents for
treatment of HCV infection that overcome the limitations of existing
pharmaceutical
therapies.
HCV is an enveloped positive strand RNA virus in the Flaviviridae family. The
single
strand HCV RNA genome is approximately 9500 nucleotides in length and has a
single open reading frame (ORF) encoding a single large polyprotein of about
3000
amino acids. In infected cells, this polyprotein is cleaved at multiple sites
by cellular
and viral proteases to produce the structural and non-structural (NS)
proteins. In the
case of HCV, the generation of mature nonstructural proteins (NS2, NS3, NS4A,
NS4B, NS5A, and NS5B) is effected by two viral proteases. The first one, as
yet
poorly characterized, cleaves at the NS2-NS3 junction (henceforth referred to
as
NS2/3 protease); the second one is a serine protease contained within the N-
terminal region of NS3 (NS3 protease) and mediates all the subsequent
cleavages
downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans,
for the
remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites. The NS4A protein
appears to serve multiple functions, acting as a cofactor for the NS3 protease
and
possibly assisting in the membrane localization of NS3 and other viral
replicase
components. The complex formation of the NS3 protease with NS4A seems
necessary to the processing events, enhancing the proteolytic efficiency at
all of the
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sites. The NS3 protein also exhibits nucleoside triphosphatase and RNA
helicase
activities. NS5B is a RNA-dependent RNA polymerase that is involved in the
replication of HCV.
A general strategy for the development of antiviral agents is to inactivate
virally
encoded enzymes that are essential for the replication of the virus.
The following is a list of patent applications published in the last few years
that
disclose HCV NS3 protease inhibitor peptide analogs that are structurally
different
from the compounds of the present invention:
GB 2,337,262; JP10298151; JP 11126861; JP 11292840; JP 2001-103993;
US 6,159,938; US 6,187,905; WO 97/43310; WO 98/17679; WO 98/22496;
WO 98/46597; WO 98/46630; WO 99/38888; WO 99/50230; WO 99/64442;
WO 99/07733; WO 99/07734; WO 00/09543; WO 00/09558; WO 00/20400;
WO 00/59929; WO 00/31129; WO 01/02424; WO 01/07407; WO 01/16357;
WO 01/32691; WO 01/40262; WO 01/58929; WO 01/64678; WO 01/74768;
WO 01/77113; WO 01/81325; WO 02/08187; WO 02/08198; WO 02/08244;
WO 02/08251; WO 02/08256; WO 02/18369; WO 02/60926 and WO 02/79234.
One advantage of the present invention is that it provides tripeptide
compounds that
are inhibitory to the NS3 protease, an enzyme essential for the replication of
the
hepatitis C virus. Furthermore, the compounds are able to inhibit HCV RNA
replication in the replicon cell model.
A further advantage of one aspect of the present invention resides in the fact
that
the compounds specifically inhibit the NS3 protease and do not show
significant
inhibitory activity against other serine proteases such as human leukocyte
elastase
(HLE), porcine pancreatic elastase (PPE), or bovine pancreatic chymotrypsin,
or
cysteine proteases such as human liver cathepsin B (Cat B).
SUMMARY OF THE INVENTION
Included in the scope of the invention is a compound of formula (1):
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N-R2
N
MeO N S
o R3 0
R\OANN
1
H R
O O H R O
(1)
wherein R' is hydroxy or NHSOQR1A wherein R'A is (C1_8)alkyl, (C3-7)cycloalkyl
or
{(C1_6)alkyl-(C3.7)cycloalkyl}, which are all optionally substituted from 1 to
3 times
with halo, cyano, nitro, O-(C1-6)alkyl, amido, amino or phenyl, or R'" is C6
or C10 aryl
which is optionally substituted from 1 to 3 times with halo, cyano, nitro,
(C1_6)alkyl,
O-(C1-6)alkyl, amido, amino or phenyl; R2 is (C4_6)cycloalkyl; R3 is t-btuyl
or (C5-6)
cycloalkyl and R4 is (C4_6)cycloalkyl; or a pharmaceutically acceptable salt
thereof.
Included within the scope of this invention is a pharmaceutical composition
comprising an anti-hepatitis C virally effective amount of a compound of
formula I, or
a therapeutically acceptable salt thereof, in admixture with a
pharmaceutically
acceptable carrier medium or auxiliary agent.
According to one embodiment, the pharmaceutical composition of this invention
further comprises interferon (pegylated or not), or ribavirin, or one or more
other
anti-HCV agents, or any combination of the above.
Another important aspect of the invention involves a method of treating a
hepatitis C
viral infection in a mammal by administering to the mammal an anti-hepatitis C
virally effective amount of a compound of formula I, a pharmaceutically
acceptable
salt thereof, or a composition as described above, alone or in combination
with one
ore more of: interferon (pegylated or not), or ribavirin, or one or more other
anti-HCV
agents, administered together or separately.
Another important aspect of the invention involves a method of preventing a
hepatitis C viral infection in a mammal by administering to the mammal an anti-
hepatitis C virally effective amount of a compound of formula I, a
pharmaceutically
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acceptable salt thereof, or a composition as described above, alone or in
combination with one ore more of: interferon (pegylated or not), or ribavirin,
or one
or more other anti-HCV agent, all of which administered together or
separately.
Also within the scope of this invention is the use of a compound of formula I,
as
described herein, for the manufacture of a medicament for the treatment or
prevention of hepatitis C viral infection.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Definitions
As used herein, the following definitions apply unless otherwise noted:
With reference to the instances where (R) or (S) is used to designate the
absolute
configuration of a substituent or asymmetric center of a compound of formula
1, the
designation is done in the context of the whole compound and not in the
context of
the substituent or asymmetric center alone.
The designation "P1, P2, and P3" as used herein refer to the position of the
amino
acid residues starting from the C-terminus end of the peptide analogs and
extending
towards the N-terminus (i.e. P1 refers to position 1 from the C-terminus, P2:
second
position from the C-terminus, etc.) (see Berger A. & Schechter I.,
Transactions of the
Royal Society London series B257, 249-264 (1970)).
As used herein the term "vinyl-ACCA" refers to a compound of formula:
S
H N"R OH
z
0
namely, (1R, 2S) 1-amino-2-ethenylcyclopropylcarboxylic acid.
The term "(C1.g)alkyl" as used herein, either alone or in combination with
another
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substituent, means acyclic straight or branched alkyl substituents containing
for 1 to
8 carbon atoms and includes, for example, methyl, ethyl, 2-methylhexyl, 1,1-
dimethylhexyl (or t-butyl) and octyl.
The term "(C3_7)cycloalkyl" as used herein, either alone or in combination
with
another substituent, means a cycloalkyl substituent containing from 3 to 7
carbon
atoms and includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and
cycloheptyl.
The term "((C1_6)alkyl-(C3.6)cycloalkyl}" as used herein means a cycloalkyl
radical
containing from 3 to 6 carbon atoms directly linked to an alkylene radical
containing
1 to 6 carbon atoms; for example, cyclopropylmethyl, cyclopentylethyl,
cyclohexylmethyl, and cyclohexylethyl. In the instance where R1A is a
{(C1_6)alkyl-
(C3_6)cycloalkyl), this group is attached to the SO2 group via the (C1_6)alkyl
(i.e. the
alkylene portion).
The term "C6 or C10 aryl" as used herein, either alone or in combination with
another
radical, means either an aromatic monocyclic group containing 6 carbon atoms
or
an aromatic bicyclic group containing 10 carbon atoms. For example, aryl
includes
phenyl, 1-naphthyl or 2-naphthyl.
The term "O-(C1_6) alkyl" as used herein, either alone or in combination with
another
radical, means the radical -O-(C1_6)alkyl wherein alkyl is as defined above
containing
up to six carbon atoms, and includes methoxy, ethoxy, propoxy, 1 -
methylethoxy,
butoxy and 1,1-dimethylethoxy. The latter radical- is known commonly as tert-
butoxy.
The term "halo" as used herein means a halogen substituent selected from
bromo,
chloro, fluoro or iodo.
The term "pharmaceutically acceptable salt" means a salt of a compound of
formula
(1) which is, within the scope of sound medical judgment, suitable for use in
contact
with the tissues of humans and lower animals without undue toxicity,
irritation,
allergic response, and the like, commensurate with a reasonable benefit/risk
ratio,
generally water or oil-soluble or dispersible, and effective for their
intended use.
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The term includes pharmaceutically-acceptable acid addition salts and
pharmaceutically-acceptable base addition salts. Lists of suitable salts are
found in,
e.g., S.M. Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19.
The term "pharmaceutically-acceptable acid addition salt" means those salts
which
retain the biological effectiveness and properties of the free bases and which
are not
biologically or otherwise undesirable, formed with inorganic acids such as
hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic
acid,
nitric acid, phosphoric acid, and the like, and organic acids such as acetic
acid,
trichloroacetic acid, trifluoroacetic acid, adipic acid, alginic acid,
ascorbic acid,
aspartic acid, benzenesulfonic acid, benzoic acid, 2-acetoxybenzoic acid,
butyric
acid, camphoric acid, camphorsulfonic acid, cinnamic acid, citric acid,
digluconic
acid, ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphoric
acid,
hemisulfic acid, heptanoic acid, hexanoic acid, formic acid, fumaric acid, 2-
hydroxyethanesulfonic acid (isethionic acid), lactic acid, maleic acid,
hydroxymaleic
acid, malic acid, malonic acid, mandelic acid, mesitylenesulfonic acid,
methanesulfonic acid, naphthalenesulfonic acid, nicotinic acid, 2-
naphthalenesulfonic acid, oxalic acid, pamoic acid, pectinic acid,
phenylacetic acid,
3-phenylpropionic acid, picric acid, pivalic acid, propionic acid, pyruvic
acid, salicylic
acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, p-
toluenesulfonic acid,
undecanoic acid, and the like.
The term "pharmaceutically-acceptable base addition salt" means those salts
which
retain the biological effectiveness and properties of the free acids and which
are not
biologically or otherwise undesirable, formed with inorganic bases such as
ammonia
or hydroxide, carbonate, or bicarbonate of ammonium or a metal cation such as
sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese,
aluminum, and the like. Particularly preferred are the ammonium, potassium,
sodium, calcium, and magnesium salts. Salts derived from pharmaceutically-
acceptable organic nontoxic bases include salts of primary, secondary, and
tertiary
amines, quaternary amine compounds, substituted amines including naturally
occurring substituted amines, cyclic amines and basic ion-exchange resins,
such as
methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine,
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triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine,
diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol,
dicyclohexylamine,
lysine, arginine, histidine, caffeine, hydrabamine, choline, betaine,
ethylenediamine,
glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-
ethylpiperidine, tetramethylammonium compounds, tetraethylammonium
compounds, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-
methylmorpholine,
dicyclohexylamine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine,
N,N'-dibenzylethylenediamine, polyamine resins, and the like. Particularly
preferred
organic nontoxic bases are isopropylamine, diethylamine, ethanolamine,
trimethylamine, dicyclohexylamine, choline, and caffeine.
The term "antiviral agent" as used herein means an agent (compound or
biological)
that is effective to inhibit the formation and/or replication of a virus in a
mammal.
This includes agents that interfere with either host or viral mechanisms
necessary
for the formation and/or replication of a virus in a mammal. Antiviral agents
include,
for example, ribavirin, amantadine, VX-497 (merimepodib, Vertex
Pharmaceuticals),
VX-498 (Vertex Pharmaceuticals), Levovirin, Viramidine, Ceplene (maxamine),
XTL-
001 and XTL-002 (XTL Biopharmaceuticals).
The term other anti-HCV agent as used herein means those agents that are
effective for diminishing or preventing the progression of hepatitis C related
symptoms of disease. Such agents can be selected from: anti-viral agents,
immunomodulatory agents, inhibitors of HCV NS3 protease, inhibitors of HCV
polymerase or inhibitors of another target in the HCV life cycle.
The term "immunomodulatory agent" as used herein means those agents
(compounds or biologicals) that are effective to enhance or potentiate the
immune
system response in a mammal. Immunomodulatory agents include, for example,
class I interferons (such as a-, P- and omega interferons, tau-interferons,
consensus interferons and asialo-interferons), class II interferons (such as y-
interferons) and pegylated interferons.
The term "inhibitor of HCV NS3 protease" as used herein means an agent
(compound or biological) that is effective to inhibit the function of HCV NS3
protease
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in a mammal. Inhibitors of HCV NS3 protease include, for example, those
compounds described in WO 99/07733, WO 99/07734, WO 00/09558, WO
00/09543, WO 00/59929 or WO 02/060926, the Boehringer Ingelheim clinical
candidate identified as BILN 2061 and the Vertex/Eli Lilly pre-development
candidate
identified as VX-950 or LY-57031 0. Particularly, compounds # 2, 3, 5, 6, 8,
10, 11,
18, 19, 29, 30, 31, 32, 33, 37, 38, 55, 59, 71, 91, 103, 104, 105, 112, 113,
114, 115,
116, 120, 122, 123, 124, 125, 126 and 127 disclosed in the table of pages 224-
226
in WO 02/060926, can be used in combination with the compounds of the present
invention .
The term "inhibitor of HCV polymerase" as used herein means an agent (compound
or biological) that is effective to inhibit the function of an HCV polymerase
in a
mammal. This includes, for example, inhibitors of HCV NS5B polymerase.
Inhibitors
of HCV polymerase include non-nucleosides, for example, those compounds
described in:
WO 2003/010140, (Boehringer Ingelheim),
WO 2003/010141, (Boehringer Ingelheim),
- WO 2003/007945, (Boehringer Ingelheim),
WO 02/100846 Al and WO 02/100851 A2 (both Shire),
WO 01/85172 Al and WO 02/098424 Al (both GSK),
WO 00/06529 and WO 02/06246 Al (both Merck),
- WO 01/47883 and WO 03/000254 (both Japan Tobacco) and
EP 1 256 628 A2 (Agouron).
Furthermore other inhibitors of HCV polymerase also include nucleoside
analogs,
for example, those compounds described in:
WO 01/90121 A2 (Idenix),
- WO 02/069903 A2 (Biocryst Pharmaceuticals Inc.), and
WO 02/057287 A2 and WO 02/057425 A2 (both Merck/Isis).
Specific examples of inhibitors of an HCV polymerase, include JTK-002, JTK-003
and JTK-109 (Japan Tobacco).
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The term "inhibitor of another target in the HCV life cycle" as used herein
means an
agent (compound or biological) that is effective to inhibit the formation
and/or
replication of HCV in a mammal other than by inhibiting the function of the
HCV NS3
protease. This includes agents that interfere with either host or HCV viral
mechanisms necessary for the formation and/or replication of HCV in a mammal.
Inhibitors of another target in the HCV life cycle include, for example,
agents that
inhibit a target selected from a helicase, an HCV NS2/3 protease and an
inhibitor of
internal ribosomal entry site (IRES). A specific example of inhibitors of
another
target in the HCV life cycle include ISIS-14803 (ISIS Pharmaceuticals).
The term "HIV inhibitor" as used herein means an agents (compound or
biological)
that is effective to inhibit the formation and/or replication of HIV in a
mammal. This
includes agents that interfere with either host or viral mechanisms necessary
for the
formation and/or replication of HIV in a mammal. HIV inhibitors include, for
example, nucleosidic inhibitors, non-nucleosidic inhibitors, protease
inhibitors, fusion
inhibitors and integrase inhibitors.
The term "HAV inhibitor" as used herein means an agent (compound or
biological)
that is effective to inhibit the formation and/or replication of HAV in a
mammal. This
includes agents that interfere with either host or viral mechanisms necessary
for the
formation and/or replication of HAV in a mammal. HAV inhibitors include
Hepatitis A
vaccines, for example, Havrix (GlaxoSmithKline), VAQTA (Merck) and Avaxim
(Aventis Pasteur).
The term "HBV inhibitor" as used herein means an agent (compound or
biological)
that is effective to inhibit the formation and/or replication of HBV in a
mammal. This
includes agents that interfere with either host or viral mechanisms necessary
for the
formation and/or replication of HBV in a mammal. HBV inhibitors include, for
example, agents that inhibit HBV viral DNA polymerase or HBV vaccines.
Specific
examples of HBV inhibitors include Lamivudine (Epivir-HBV ), Adefovir
Dipivoxil,
Entecavir, FTC (Coviracil ), DAPD (DXG), L-FMAU (Clevudine ), AM365 (Amrad),
Ldt (Telbivudine), monoval-LdC (Valtorcitabine), ACH-126,443 (L-Fd4C)
(Achillion),
MCC478 (Eli Lilly), Racivir (RCV), Fluoro-L and D nucleosides, Robustaflavone,
ICN
2001-3 (ICN), Bam 205 (Novelos), XTL-001 (XTL), Imino-Sugars (Nonyl-DNJ)
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(Synergy), HepBzyme; and immunomodulator products such as: interferon alpha
2b, HE2000 (Hollis-Eden), Theradigm (Epimmune), EHT899 (Enzo Biochem),
Thymosin alpha-1 (Zadaxin ), HBV DNA vaccine (PowderJect), HBV DNA vaccine
(Jefferon Center), HBV antigen (OraGen), BayHep Be (Bayer), Nabi-HB (Nabi)
and
Anti-hepatitis B (Cangene); and HBV vaccine products such as the following:
Engerix B, Recombivax HB, GenHevac B, Hepacare, Bio-Hep B, TwinRix, Comvax,
Hexavac.
The term "class I interferon" as used herein means an interferon selected from
a
group of interferons that all bind to receptor type I. This includes both
naturally and
synthetically produced class I interferons. Examples of class I interferons
include a-
, R-, omega interferons, tau-interferons, consensus interferons, asialo-
interferons.
The term "class II interferon" as used herein means an interferon selected
from a
group of interferons that all bind to receptor type II. Examples of class II
interferons
include y-interferons.
The pharmaceutical compositions of the invention may contain one or more
additional active agents selected, for example, from antiviral agents,
immunomodulatory agents, other inhibitors of HCV NS3 protease, inhibitors of
HCV
polymerase, inhibitors of another target in the HCV life cycle, HIV
inhibitors, HAV
inhibitors and HBV inhibitors. Examples of such agents are provided in the
Definitions section above.
Specific preferred examples of some of these agents are listed below:
^ antiviral agents: ribavirin and amantadine;
^ immunomodulatory agents: class I interferons, class II interferons and
pegylated
interferons;
^ inhibitor of another target in the HCV life cycle that inhibits a target
selected
from: NS3 helicase, HCV NS2/3 protease or internal ribosome entry site (IRES);
^ HIV inhibitors: nucleosidic inhibitors, non-nucleosidic inhibitors, protease
inhibitors, fusion inhibitors and integrase inhibitors; or
^ HBV inhibitors: agents that inhibit HBV viral DNA polymerase or is an HBV
vaccine.
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As discussed above, combination therapy is contemplated wherein a compound of
formula (1), or a pharmaceutically acceptable salt thereof, is co-administered
with at
least one additional agent selected from: an antiviral agent, an
immunomodulatory
agent, another inhibitor of HCV NS3 protease, an inhibitor of HCV polymerase,
an
inhibitor of another target in the HCV life cycle, an HIV inhibitor, an HAV
inhibitor
and an HBV inhibitor. Examples of such agents are provided in the Definitions
section above. These additional agents may be combined with the compounds of
this invention to create a single pharmaceutical dosage form. Alternatively
these
additional agents may be separately administered to the patient as part of a
multiple
dosage form, for example, using a kit. Such additional agents may be
administered
to the patient prior to, concurrently with, or following the administration of
wherein a
compound of formula (1), or a pharmaceutically acceptable salt thereof.
As used herein, the term "treatment" means the administration of a compound or
composition according to the present invention to alleviate or eliminate
symptoms of
the hepatitis C disease and/or to reduce viral load in a patient.
As used herein, the term "prevention" means the administration of a compound
or
composition according to the present invention post-exposure of the individual
to the
virus but before the appearance of symptoms of the disease, and/or prior to
the
detection of the virus in the blood.
Preferred embodiments
Preferably, compounds of formula 1 as defined above wherein R1 is hydroxy,
NHSO2Me, NHSO2cyclopropyl or NHSO2Ph. More preferably, R' is NHSO2-
cyclopropyl or NHSO2Ph. Alternatively, most preferably, R' is hydroxy.
Preferably, compounds of formula 1 as defined above wherein R2 is cyclopentyl
or
cyclohexyl. Most preferably, R2 is cyclopentyl.
Preferably, R3 is t-butyl or cyclohexyl. Most preferably,R3 is t-butyl.
Preferably, compounds of formula 1 as defined above wherein R4 is cyclobutyl
or
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cyclopentyl. Most preferably, R4 is cyclopentyl.
More preferably, a compound of formula 1 as defined above wherein R1 is
hydroxy,
R2 and R4 each is cyclopentyl and R3 is t-butyl.
More preferably, a compound of formula 1 wherein R' is hydroxy, R2 is
cyclobutyl,
R3 is t-butyl and R4 is cyclopentyl.
More preferably, a compound of formula 1 wherein R1 is hydroxy, R2 is
cyclohexyl,
R3 is t-butyl and R4 is cyclopentyl.
More preferably R1 is NHSO2Ph, R2 and R4 each is cyclopentyl and R3 t-butyl.
More preferably, a compound of formula 1 wherein R1 is hydroxy, R2 is
cyclopentyl,
R3 is t-butyl and R4 is cyclobutyl.
More preferably, a compound of formula 1 wherein R1 is hydroxy, R2 is
cyclopentyl,
R3 is t-butyl and R4 is cyclohexyl.
More preferably, a compound of formula 1 wherein R1 is hydroxy, R2 and R4 each
is
cyclopentyl and R3 is cyclohexyl.
More preferably, a compound of formula 1 wherein R1 is hydroxy, R2, R3 and R4
each is cyclopentyl.
According to an alternate embodiment, the pharmaceutical composition of this
invention may additionally comprise another anti-HCV agent. Examples of anti-
HCV
agents include, a- (alpha), (3- (beta), S- (delta), y- (gamma) or co- (omega)
interferon,
ribavirin and amantadine.
According to another alternate embodiment, the pharmaceutical composition of
this
invention may additionally comprise another inhibitor of HCV NS3 protease.
According to another alternate embodiment, the pharmaceutical composition of
this
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invention may additionally comprise an inhibitor of HCV polymerase.
According to yet another alternate embodiment, the pharmaceutical composition
of
this invention may additionally comprise an inhibitor of other targets in the
HCV life
cycle, including but not limited to, helicase, NS2/3 protease or internal
ribosome
entry site (IRES).
The pharmaceutical composition of this invention may be administered orally,
parenterally or via an implanted reservoir. Oral administration or
administration by
injection is preferred. The pharmaceutical composition of this invention may
contain
any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or
vehicles. In some cases, the pH of the formulation may be adjusted with
pharmaceutically acceptable acids, bases or buffers to enhance the stability
of the
formulated compound or its delivery form. The term parenteral as used herein
includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-
articular,
intrasynovial, intrasternal, intrathecal, and intralesional injection or
infusion
techniques.
The pharmaceutical composition may be in the form of a sterile injectable
preparation, for example, as a sterile injectable aqueous or oleaginous
suspension.
This suspension may be formulated according to techniques known in the art
using
suitable dispersing or wetting agents (such as, for example Tween 80) and
suspending agents.
The pharmaceutical composition of this invention may be orally administered in
any
orally acceptable dosage form including, but not limited to, capsules,
tablets, and
aqueous suspensions and solutions. In the case of tablets for oral use,
carriers
which are commonly used include lactose and corn starch. Lubricating agents,
such
as magnesium stearate, are also typically added. For oral administration in a
capsule form, useful diluents include lactose and dried corn starch. When
aqueous
suspensions are administered orally, the active ingredient is combined with
emulsifying and suspending agents. If desired, certain sweetening and/or
flavoring
and/or coloring agents may be added..
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Other suitable vehicles or carriers for the above noted formulations and
compositions can be found in standard pharmaceutical texts, e.g. in
"Remington's
Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19th Ed. Mack
Publishing Company, Easton, Penn., (1995).
Dosage levels of between about 0.01 and about 1 00mg/kg body weight per day,
preferably between about 0.1 and about 50mg/kg body weight per day of the
protease inhibitor compound described herein are useful in a monotherapy for
the
prevention and treatment of HCV mediated disease. Typically, the
pharmaceutical
composition of this invention will be administered from about 1 to about 5
times per
day or alternatively, as a continuous infusion. Such administration can be
used as a
chronic or acute therapy. The amount of active ingredient that may be combined
with the carrier materials to produce a single dosage form will vary depending
upon
the host treated and the particular mode of administration. A typical
preparation will
contain from about 5% to about 95% active compound (w/w). Preferably, such
preparations contain from about 20% to about 80% active compound.
As the skilled artisan will appreciate, lower or higher doses than those
recited above
may be required. Specific dosage and treatment regimens for any particular
patient
will depend upon a variety of factors, including the activity of the specific
compound
employed, the age, body weight, general health status, sex, diet, time of
administration, rate of excretion, drug combination, the severity and course
of the
infection, the patient's disposition to the infection and the judgment of the
treating
physician. Generally, treatment is initiated with small dosages substantially
less
than the optimum dose of the peptide. Thereafter, the dosage is increased by
small
increments until the optimum effect under the circumstances is reached. In
general,
the compound is most desirably administered at a concentration level that will
generally afford antivirally effective results without causing any harmful or
deleterious side effects.
When the composition of this invention comprise a combination of a compound of
formula 1 and one or more additional therapeutic or prophylactic agent, both
the
compound and the additional agent should be present at dosage levels of
between
about 10 to 100%, and more preferably between about 10 and 80% of the dosage
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normally administered in a monotherapy regimen.
When these compounds or their pharmaceutically acceptable salts are formulated
together with a pharmaceutically acceptable carrier, the resulting composition
may
be administered in vivo to mammals, such as man, to inhibit HCV NS3 protease
or
to treat or prevent HCV virus infection. Such treatment may also be achieved
using
a compound of this invention in combination with agents which include, but are
not
limited to: a-, P-, S-, co-, tau, or y-interferon, ribavirin, amantadine;
other inhibitors of
HCV NS3 protease; inhibitors of HCV polymerase, inhibitors of other targets in
the
HCV life cycle, which include but not limited to, helicase, NS2/3 protease, or
internal
ribosome entry site (IRES); or combinations thereof. The additional agents may
be
combined with compounds of this invention to create a single dosage form.
Alternatively these additional agents may be separately administered to a
mammal
as part of a multiple dosage form.
Accordingly, another embodiment of this invention provides a method of
inhibiting
HCV NS3 protease activity in a mammal by administering a compound of the
formula 1.
In a preferred embodiment, this method is useful in decreasing the NS3
protease
activity of the hepatitis C virus infecting a mammal.
If the pharmaceutical composition comprises only a compound of this invention
as
the active component, such method may additionally comprise the step of
administering to said mammal an agent selected from an immunomodulatory agent,
an antiviral agent, a HCV NS3 protease inhibitor, an inhibitor of HCV
polymerase or
an inhibitor of other targets in the HCV life cycle such as helicase, NS2/3
protease
or IRES. Such additional agent may be administered to the mammal prior to,
concurrently with, or following the administration of the composition of this
invention.
A compound of formula 1 set forth herein may also be used as a laboratory
reagent.
A compound of this invention may also be used to treat or prevent viral
contamination of materials and therefore reduce the risk of viral infection of
laboratory or medical personnel or patients who come in contact with such
materials
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CA 02474156 2009-11-13
(e.g. blood, tissue, surgical instruments and garments, laboratory instruments
and
garments, and blood collection apparatuses and materials).
A compound of formula 1 set forth herein may also be used as a research
reagent.
A compound of formula 1 may also be used as positive control to validate
surrogate
cell-based assays or in vitro or in vivo viral replication assays.
Further details of the invention are illustrated in the following examples
which are
understood to be non-limiting with respect to the appended claims.
Methodology
In general, the compounds of formula 1, and intermediates therefore, are
prepared
by known methods using reaction conditions which are known to be suitable for
the
reactants. Several such methods are disclosed in WO 00/09543, WO 00/09558 and
US Pat. No. 6,323,180.
Compounds of formula I wherein R' is NHSO2R'" as defined herein are prepared
by
coupling the corresponding acid of formula I (i.e. R' is hydroxy) with an
appropriate
sulfonamide of formula R'" SO2NH2 in the presence of a coupling agent under
standard conditions. Although several commonly used coupling agents can be
employed, TBTU and HATU have been found to be practical. The sulfonamides are
available commercially or can be prepared by known methods.
The following schemes illustrate two convenient processes using known methods
for
preparing the compounds of formula 1 when R' is OR
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Scheme 1:
O
O
0 2 N - ~- O
02N O
RXJLHOH+ HCIH O HOMe R, XxNN N OMe
O H O O H O
X = O, NH 3 6a O
4or5
O MeO
MeO ( )/ N\ OMe N O
OH O
0 RAN OH 3 = OMe
R\XN 11 Me 9 RX O N N OMe
O H O H O
O H 0
7a 10a
Meo
MeO
N 0 ~ N H z
NS N-R
3 Sr N R2 0
O R s=< 3
R~XJLH N ~Me NH2 _-~ R~XAN
O H N N OH
O H O O H 0
13a Compounds of formula I
when R1 is OH
Scheme 2:
O 0 N-Z
MeO OMe Me0 N Br N--~ S
Me0
N-R2
O O S=<
+ NH2 O
BocN \ BocN 8
OMe OMe BocN \
0 N O H Me
O H O O H
O
15 18 H 2 21 H
//N-R N-R2
N=1 N(
MeO N\ s Meo S
0 R3
R\XJIN kyOH
H O 0 O
0 R3 OH-o R3
X = O, NH _ Rl~ 0A, N N RtOAN N \
4 or 5 H 0 0 N OMe H O O N
H H
0 0
Compounds of formula I
when R1 is OH
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EXAMPLES
Temperatures are given in degrees Celsius. Solution percentages express a
weight
to volume relationship, and solution ratios express a volume to volume
relationship,
unless stated otherwise. Nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker 400 MHz spectrometer; the chemical shifts (8) are
reported in
parts per million. Flash chromatography was carried out on silica gel (Si02)
according to Still's flash chromatography technique (W.C. Still et al., J.
Org. Chem.,
(1978), 43, 2923).
Abbreviations used in the examples include:
DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM: dichloromethane; DIAD:
diisopropylazodicarboxylate; DIEA: diisopropylethylamine; DIPEA:
diisopropylethyl
amine; DMF: N, N-dimethylformamide; DMAP: 4-(dimethylamino) pyridine; EtOAc:
ethyl acetate; HATU: [O-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate]; HPLC: high performance liquid chromatography; MS: mass
spectrometry (MALDI-TOF: Matrix Assisted Laser Disorption Ionization-Time of
Flight, FAB: Fast Atom Bombardment); Me: methyl; MeOH: methanol; Ph: phenyl;
R.T.: room temperature (18 to 222); tert-butyl or t-butyl: 1,1-dimethylethyl;
Tbg: tert-
butyl glycine: tert-leucine; TBTU: 2-(1 H-benzotriazole-1 -yl)-1,1,3,3-
tetramethyl
uronium tetrafluoroborate; TFA: trifluoroacetic acid; and THF:
tetrahydrofuran.
Synthesis of compounds of formula (I):
The dipeptide intermediate 15 (Scheme 2) and 2-carbomethoxy-4-hydroxy-7-
methoxyquinoline 9 (Scheme 1) were synthesized according to the methods
described in WO 00/09543.
Synthesis of dipeptide 1
OH OH
+ OMe 3 O ;'0 OyN OH HZNO N
O Me
III
O O O --{
P2 P1 P2-P1 O
1
A mixture of Boc-hydroxyproline (50.0g, 216mmol), vinyl-ACCA methyl ester
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(42.25g, 238mmo1, 1.1 equiv.), TBTU (76.36g, 238mmol, 1.1 equiv.) and DI PEA
(113mL, 649mmo1, 3equiv.) in DMF (800mL) was stirred at R.T. under a nitrogen
atmosphere. After 3.5h, the solvent was evaporated and the residue extracted
with
EtOAc. The extract was washed with hydrochloric acid (10%), saturated sodium
bicarbonate and brine. The organic phase was then dried over magnesium
sulfate,
filtered and evaporated to afford an oil. After drying overnight under high
vacuum,
dipeptide 1 was obtained as a yellow foam (72.0 g, 94%, purity >95% by HPLC).
Preparation of dipeptide 3
0 0
OH
O -I-/NO, O / NOZ
C~A OMe HCI.H
O O H xaAOMe OMe 11 O H, O H
1 2 3
Dipeptide 1 (72.0g, 203mmol), triphenylphosphine (63.94g, 243.8mmol,
1.2equiv.)
and 4-nitrobenzoic acid (41.08g, 245.8mmol, 1.2equiv) were dissolved in dry
THE
(1.4L) The stirred solution was cooled to 0 C under a nitrogen atmosphere.
Diethyl
azodicarboxylate (38.4mL, 244mmol, 1.2equiv.) was then added dropwise over 45
min and the reaction allowed to warm to R.T. After 4h, the solvent was
evaporated.
The residue was divided into four portions. Each of these was purified by
chromatography over fine silica gel (10-40 m mesh, column diameter 12cm,
column
length 16cm) using a gradient of 2 :1 hexane/EtOAc to 1:1 hexane/EtOAc to pure
EtOAc. In this manner, the Boc-dipeptide ester 2 was obtained as an amorphous
white solid after evaporation of the solvents and drying of the residues under
high
vacuum at 70 C for 1 h (108.1 g, quantitative-105%). A solution of 4N hydrogen
chloride in dioxane was added to the Boc-dipeptide ester 2 (108g, 243mmol)
resulting in a colorless solution. The solution was stirred at R.T. for 1 h.
The solvent
was evaporated and the residue placed under high vacuum for 3h affording the
hydrochloride salt of compound 3 as an amorphous solid. The solid was used as
such.
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Synthesis of carbamates 4
Preparation of carbamate 4a:
H N OMe \/O~CI i~ON OMe ~O~N OMe~N OH
z -i I I I
O H O H 0 H O
4a
At 0 C, was added to a solution of tent-butyl glycine methyl ester (590mg,
3.25mmol)
in THE (8mL). vinylchloroformate (0.55mL, 6.47mmol) and triethylamine (1.15mL,
8.25mmol). The temperature was allowed to rise to R.T. The solution was
stirred
for 16h. The solution was concentrated and the residue disolved in EtOAc. The
EtOAc solution was washed with a 10% aqueous solution of citric acid (2x), a
saturated aqueous solution of NaHCO3 (2x) and brine, dried and concentrated to
yield the corresponding vinyl carbamate (608mg) as a colorless oil. The oil
was
dissolved in DCM (4mL), cooled at 0 C, and diiodomethane (0.15mL, 1.86mmol)
and diethylzinc (95 L, 0.93mmol) were added. A white solid appeared at first
but
dissolved with time (- 1 h). The suspension/solution was stirred at R.T. for
5h. A
saturated solution of ammonium chloride was added and the solution extracted
with
EtOAc (2x). The organic extract was dried (MgSO4) and concentrated. The
residue
was purified by flash column chromatography. Elution with hexane:EtOAc 95:5
gave
the corresponding methyl ester as a colorless oil (96mg, 90% yield).
A solution the methyl ester (93mg; 0.41 mmol) in THE (5mL), MeOH (1 mL) and an
aqueous solution of LiOH (45mg ; 1.81 mmol) in water (2mL) was stirred for 4h.
The
solution is dilute with water and extracted with EtOAc (2x). The aqueous
solution
was acidified by the addition of 1 N HCI, and the acidic solution was
extracted with
EtOAc (2x). The combined organic extracts were dried (MgSO4), filtered and
evaporated to obtain the desired carbamate 4a as a white solid (53mg; 60%
yield)
Preparation of carbamate 4b
II0 0
.N + H N OH --~ ON OH
1 Y
O O z
O O H O
4b
THE (350mL) was added to a flask containing carbonic acid cyclopentyl ester
2,5-
dioxo-pyrrolidin-1-yl ester (9.00g; 39.6mmol) and tent butyl glycine (6.24g;
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47.5mmol) resulting in a suspension. Distilled water (100mL) was added with
vigorous stirring. A small amount of solid remained undissolved. Triethylamine
(16.6mL; 119mmol) was then added resulting in a homogenous solution which was
stirred at R.T. After 2.5h, the THE was evaporated and the aqueous residue
diluted
with water (100mL). The reaction was rendered basic by the addition of 1 N
NaOH
(25mL - final pH >10). The solution was washed with EtOAc (2 x 200mL) and the
aqueous phase acidified with 1 N HCI (ca. 70mL - final pH <2). The turbid
solution
was extracted with EtOAc (200 + 150mL). The extract was dried (MgSO4) and
evaporated to give carbamate 4b as a white solid (8.68g).
Preparation of other carbamates
Using the procedure described above and using appropriate combinations of tern
butyl glycine, cyclopentyl glycine, or cyclohexyl glycine and carbonic acid
cyclobutyl,
cyclopentyl, or cyclohexyl ester 2,5-dioxo-pyrrolidin-1 -yl ester, carbamates
of the
following formulas were prepared:
R.O1N OH ROAN OH ROAN OH
R H O R H O R H O
a a
Preparation of Ureas 5
al
OH
N OBn OAN OBn --a 0, 1 OBn QNAN Y
H H H H H
CIH O O O O
5
A solution of tert-butyl glycine benzyl ester hydrochloride salt (2.55g;
9.89mmol) in
THE (20mL) and pyridine (2.OmL; 24.73mmol) was cooled to 00 C. Phenyl
chloroformate (1.30mL; 10.19mmol) was added dropwise to the cooled solution.
The
resulting suspension was stirred for 5min at 00 C, then at R.T. for 1.5h. The
reaction
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mixture was diluted with EtOAc, washed with 10% citric acid (2x) water (2x)
saturated NaHCO3 (2x), water (2x) and brine (1x), dried (MgSO4), filtered and
evaporated to gain the crude compound as a nearly colorless oil (3.73g ;
>100%;
assume 9.89mmol). The crude product (1.01 g; 2.97mmol) was dissolved in DMSO
(6.5mL) and cyclopentylamine was added dropwise. The reaction mixture was
stirred at R.T. for 45min. The reaction mixture was diluted with EtOAc. The
organic
phase was washed with washed with 10% citric acid (2x) water (2x) saturated
NaHCO3 (2x), water (2x) and brine (1x), dried (MgSO4), filtered and evaporated
to
give the crude cyclopentyl urea -Tbg-OBn product as a nearly colorless oil.
The
crude material was purified by flash column chromatography with silica using
hexane:EtOAc 9:1 to remove the less polar impurities and 7:3 to elute the
purified
product as a thick colourless oil (936mg; 95%). The ester benzyl ester product
(936mg; 2.82mmol) was deprotected under a hydrogen filled balloon at R.T. in
absolute ethanol (15mL) solution by stirring the solution with 10% Pd/C
(93.6mg) for
5.5h. The reaction mixture was filtered through a 0.45micron filter and
evaporated to
dryness to provide urea 5 as a white solid (668.8mg ; 98%)
'H NMR (400 MHz,DMSO-d6): 812.39 (s, 1H), 6.09 (d, J = 7.4 Hz, 1 H) , 5.93 (d,
J
= 9.4 Hz, 1 H), 3.90 (d, J = 9.4 Hz, 1 H), 3.87-3.77 (m, 1 H), 1.84-1.72 (m,
2H),
1.63-1.42 (m, 4H), 1.30-1.19 (m, 2H), 0.89 (s, 9H).
M.S.(electrospray) : 241.0 (M-H)- 243.0 (M+H)+ . Reverse Phase HPLC
Homogeneity (0.06% TFA; CH3CN : H20) : 99%.
Synthesis of tipeptide 6
o
/ \ O2N
O2N O
aOxON OH O
\ Q 1111
N N cMe
H O HCI H N Me O N
4b O H O H O O H O
3 6
Carbamate 4b (6.15g, 22.5mmol) and TBTU (7.72g, 24.7mmol) were suspended in
DCM and the suspension was stirred rapidly. DIPEA (3.92mL, 22.5mmol) was
added at R.T. and after 10 min, the reaction was nearly homogeneous. A
solution of
dipeptide 3 (10.39g, 23.6mmol) in anhydrous DCM (100mL) containing DIPEA
(4.11 mL, 23.62mmol) was then poured into the reaction. The resulting yellow
solution was allowed to stir for 14h. The solvent was then evaporated yielding
a
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yellow syrup which was extracted with EtOAc (300 + 150mL) and washed with
0.05N HCI (2 x 200mL), saturated Na2CO3 (300mL) and brine (150mL). The
combined extracts were dried over MgSO4 and evaporated to yield the tripeptide
6
as a pale yellow foam (15.68g, quantitiative).
Synthesis of tripeptide 7:
O2N O
O OH
O a O \
OxN N OMe OxN N OM'
O N O N
H I I
O O O H
O
6 7
The tripeptide 6 (15.68g) was dissolved in THE (200mL) and water (30mL) was
added. The resulting solution was cooled to 0 C and a solution of lithium
hydroxide
monohydrate (1.18g, 28.12mmol) was added over 3 min with vigorous stirring.
After
3h at 0 C, the excess base was neutralized with 1 N HCI (final pH ca. 6) and
the THE
evaporated, resulting in an aqueous suspension (yellow gum). The mixture was
extracted with EtOAc (2 x 200mL) and washed with saturated NaHCO3 (2 x 300mL).
The combined extracts were dried over MgSO4 and evaporated to yield a pale
yellow foam. Flash chromatography of the foam over silica gel using EtOAc as
eluent afforded 7 as a white amorphous solid (9.77g, 91 %).
Preparation of Thioureas 8
Synthesis of thiourea 8a:
N----O -------- a. _)II H2N A H H B H 2 N 'J~ WO
H
8a
To a solution of tert-butyl isothiocyanate (5.OmL; 39.4mmoL) in DCM (200mL)
was
added cyclopentylamine (4.67mL; 47.3mmoL) followed by DIEA and the reaction
mixture was stirred at R.T. for 2h. The mixture was diluted with EtOAc, washed
with
a 10% aqueous solution of citric acid (2x), saturated NaHCO3 (2x), H2O (2x)
and
brine (1x). The organic layer was dried over anhydrous MgSO4, filtered and
evaporated to yield N-tert-butyl - N'-cyclopentyl thiourea as a white solid
(3.70g;
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WO 03/064456 PCT/CA03/00090
47% yield). The N-tert-butyl-N'-cyclopentyl thiourea (3.70g) was dissolved in
concentrated HCI (46mL). The dark yellow solution was heated at a gentle
reflux.
After 40min the reaction mixture was allowed to cool to R.T. and thereafter
cooled in
ice and rendered basic to pH 9.5 with solid and a saturated aqueous solution
of
NaHCO3. The product was extracted into EtOAc (3x). The combined EtOAc extracts
were washed with H2O (2x) and brine (1x). The organic layer was dried (MgSO4),
filtered and concentrated to yield a beige solid (2.46g crude). Trituration of
the crude
material in hexane/ EtOAc 95 / 5 provided, after filtration, the N-
cyclopentythiourea
8a as a white solid (2.38; 90% yield).
1H NMR (400 MHz,DMSO-d6): 57.58 (bs, 1 H), 7.19 (bs, 1 H), 6.76 (bs, 1 H),
4.34
(bs, 1 H) , 1.92-1.79 (m,2H), 1.66-1.55 (m, 2H), 1.55-1.30 (m,4H). MS; es+
144.9(M + H)+, es 142.8 (M - H)
Preparation of thiourea 8b
Using the procedure describe above and using commercially available
cyclobutylamine instead of cyclopentylamine yielded thiourea 8b:
N----O
"0 , S S
H2N
A H H B H2N H
8b
Preparation of thiourea 8c
Using the procedure describe above and using commercially available
cyclohexylamine instead of cyclopentylamine yielded thiourea 8c.
-),N - -O
;~~ S N"-'O 8c
H2N A H H B H2N H
Preparation of thiourea 8d
GI 0 N S N N H NAN"L
H2N I/ H H 2
KSCN H
8d
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To a solution of the KSCN (4.60g; 47.33mmoL) in acetone (35mL), at 0 C, was
added dropwise benzoylchloride (5.OmL; 43.03mmoL) . The milky solution was
stirred in an ice bath for 1.5h, then, cyclopropylamine (3.2mL; 46.OmmoL) was
added dropwise. The reaction mixture was stirred for 1.5h at 0 C, then, more
cyclopropylamine (0.50mL, 7.22mmoL) was added and the reaction mixture stirred
at R.T. for an additional 30min. The reaction mixture was poured into ice/H20
(300mL), stirred for 5min. and the light yellow solid was filtered , washed
several
times with H2O and dried to provide N-benzyloxy-N'-cyclopropyl thiourea (
6.62g).
This thiourea was suspended in a solution of 2N NaOH (50mL) and heated at
reflux
for 15min. The solution was cooled to R.T., saturated with solid NaCl and
extracted
with EtOAc (3x). The combined EtOAc extracts were washed with H2O (2x) and
brine (1x), dried (MgSO4), filtered and evaporated to give the crude product
as an
off-white solid. The solid was triturated in hexane/EtOAc 5/5 to provide the N-
cyclopropyl thiourea 8d as a white crystalline solid (2.5g ; 50% yield).
'H NMR (400 MHz,DMSO-d6): 57.92 (bs, 1 H), 7.61 (bs, 1 H), 7.13 (bs, 1 H),
2.39
(bs, 1H) , 0.67-0.63 (m, 2H), 0.51-0.44 (m, 2H).
MS; es+ 116.9 (M + H)+, es : 114.8 (M - H)-
EXAMPLE 11
Preparation of Compound 100
Step 1: Synthesis of tripeptide 10:
MeO N
OMe
OH
g i0 N_ OMe O
O/ N N +
H O OMe N
O N . OH ON \
7 H O 9 H OMe
O YN
H O
To tripeptide 1 (1.0g; 2.09mmol) dissolved in THE (35mL), hydroxyquinoline 9
(729mg; 3.13mmol) and triphenylphosphine (1.1g ; 4.2mmol) were added. The
25 yellow suspension was cooled in an ice bath and DIAD (821 L , 4.2mmol) was
added dropwise. The solution was stirred at ice bath temperature for 30 min,
and at
R.T. for 16h. The solution was evaporated to dryness and the residue was
dissolved
in EtOAc, washed with a saturated sodium bicarbonate solution (2x), water (2x)
and
brine (lx), dried (MgS04), filtered and evaporated to obtain a yellow oil
which
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WO 03/064456 PCT/CA03/00090
precipitated on standing. The crude solid was suspended in DCM and the
insoluble
material was filtered off. The solution was concentrated and the residue
purified by
flash chromatography in Hexane : EtOAc; 5 : 5 to remove all less polar
impurities
and in CHCI3:EtOAc; 80:20 till all the Ph3P=O has eluted. The desired compound
was eluted with CHC13:EtOAc; 65:35 as a white solid (1 g; 70% yield).
M.S.(electrospray) : 693.3 (M-H)- 695.4 (M+H)+ 717.4 (M+Na)+ .
Reverse Phase HPLC Homogeneity (0.06% TFA ; CH3CN : H20) : 99%.
Step 2 : Selective monohydrolysis of ester 10:
0 0
MeO I \ N\ OH Me0 O,Na
O 0
c/~I fOI 0
0 N 0 O N N
0Me HMe
O H O O N..
11 0
Tripeptide 10 (1g; 1.44mmol) was dissolved in THE (lOmL) and MeOH (5mL), water
(5mL) and a 1 N NaOH aqueous solution (1.5mL) were added and the solution
stirred at R.T. for 2h. The mixture was evaporated to dryness and then co-
evaporating with MeOH:toluene (1:1; 4x), toluene (2x) and diethyl ether (2x)
to
obtain the product (water-free) as a white flaky solid (1.04g; 100% yield)
M.S.(electrospray) : 679.3 (M-H)- 681.3 (M+H)+ 703.3 (M+Na)+.
Reverse Phase HPLC Homogeneity (0.06% TFA ; CH3CN : H20) : 95%.
Step 3 : Synthesis of diazoketone 12:
0
Me0 N- O_Na O
MeO N\ NZ
O
0 O
OH N
N
O OMe O H Me
O ?N~
O 0 0 No4,o
11 12 0
Sodium salt 11 (assume 1.44mmol) was dissolved in THE (1 6mL), triethylamine
(301 L; 2.16mmol) was added and the solution cooled to 0 C .
Isobutylchloroformate (280 L; 2.16mmol) was added dropwise and the white
suspension was stirred at 0 C for 75min, followed by the addition of a
solution of
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diazomethane (0.67M in diethyl ether; 13mL; 8.64mmol). The reaction mixture is
stirred 1 h at 0 C, 45min at R.T. and evaporated to provide a thick
suspension. This
suspension was dissolved in EtOAc and water. The organic solution was washed
with saturated NaHCO3 (2x), water (2x) and brine (1 x), dried (MgSO4),
filtered and
evaporated to give the diazoketone product as an ivory solid (crude material
used
for next step; assume 1.44mmol).
M.S.(electrospray) : 703.3 (M-H)- 705.3 (M+H)+ . Reverse Phase HPLC
Homogeneity (0.06% TFA ; CH3CN : H20) : 91 %-
Step 4 : Synthesis of bromoketone 13:
0 0
Me0 \ N\ NZ Me0 N\ Br
O O
0 O
p' N N ON N
H O O HMe H O O HMe
12 0 13 0
At 0 C, to a solution of diazoketone 12 (1.44mmol) in THE (24mL) was added
dropwise an HBr solution (1.OmL) and the mixture was stirred for 1 h. The
solution
was diluted with EtOAc, washed with a saturated NaHCO3 solution(2x), water
(2x)
and brine (1x), dry (MgSO4), filtered and evaporated to give the desired
bromoketone as an ivory-beige solid (1.1 g; assume 1.44mmol).
M.S.(electrospray) : 757.3 (M) 759.3 (M+2)
Step 5 : Synthesis thiazolyl tripeptide 14:
0 s
Me0 Br MOO N~ I N'
O H2 N N 0
H 8a
ON N Y ON N ?N~q
H O
OMO~..) H O e
O H O H
13 0 14
a-Bromoketone 13 (0.40mmol) and N-cyclopentylthiourea (68.5mg; 0.48mmol) were
dissolved in isopropanol (15mL) and the yellow solution was heated at 70 C for
75min. The solution was allowed to cool to R.T., and evaporated to dryness.
The
residue was dissolved in EtOAc. The solution was washed with saturated NaHCO3
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(2x), water (2x) and brine (1x), dried (MgSO4), filtered and concentrated to
give the
product as an orange-brown foam. Flash column chromatography in hexane:EtOAc
7:3 removed less polar impurities and 6 : 4 retrieved the desired compound as
a
light yellow foam (218mg; 69%).
M.S.(electrospray) : 801.4 (M-H)- 803.4 (M+H)+ 825 (M+Na)+ .
Reverse Phase HPLC Homogeneity (0.06% TFA ; CH3CN : H20) : 99%.
Step 6: Hydrolysis of ester 14:
H S H
MeO N\ I N N MeO N\ N~N
Iõ b lõ b
O O '~Y o o
O N N ~\~% O 0 N ~\~
H O O NH 11 H O O NH 1L OH
O O
14
Compound 100
A solution of methyl ester 14 (145mg; 0.181 mmol) in THE (3mL), MeOH (1.5mL)
and
an aqueous solution of LiOH (75.8mg; 1.81 mmol) in water (1.5mL) was stirred
for
18h. The organic solution was concentrated to provide an off-white suspension
which was diluted with EtOAc and brine to obtain a total solution. The pH was
adjusted to 6 by the addition of 1 N HCI and the organic layer was extracted
further
with EtOAc (2x). The combined organic extracts were washed with water (2x),
brine
(1x), dried (MgSO4), filtered and evaporated to give the desired compound as a
yellow solid (138.2mg; 97% yield).
Conversion to Na Salt:
Compound 100 (138.2mg; 0.175mmol) was dissolved in MeOH (30mL) and 1
equivalent 0.01 N NaOH (17.5mL) was added. The clear yellow solution was
concentrated to remove MeOH and diluted with water, frozen and lyophililzed to
obtain the product (Na salt) as a yellow amorphous solid (139mg; theoretical
yield :
142mg; MW Na salt : 810.95)
M.S.(electrospray) : 787.2 (M-H)- 789.3 (M+H)+ 811.3 (M+Na)+ . Reverse
Phase HPLC Homogeneity (0.06% TFA ; CH3CN : H20) : 98%.
'H NMR (400 MHz,DMSO-d6): ca, 5:1 mixture of rotamers ;50 8.14 (bs, 1 H) ,
8.02
(d, J = 9.2 Hz, 1 H) , 7.89 (d, J = 6.7 Hz, 1 H), 7.49-7.36 (m, 2H), 7.27 (bs,
1 H),
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7.06-6.96 (m, 2H), 6.10-5.90 (m, 1 H), 5.33 (s, 1 H), 5.01 (d, J = 16.8 Hz, 1
H), 4.84
(d, J = 10.6 Hz, 1 H), 4.79-4.65 (m, 1 H), 4.47-4.40 (m, 1 H), 4.30 (d, J =
11.5 Hz,
1 H), ), 4.15 (d, J = 8.8 Hz, 1 H), 4.00-3.85 (m, 2H), 3.90 (s, 3H), 2.37-2.26
(m, 1 H),
2.15-1.91 (m, 2H), 1.80-1.23 (m, 18H), 0.96 & 0.86 (2x s, 9H).
EXAMPLE 2
Preparation of Compound 101
Using the same procedure described in example 1 and using N-cyclobutyl
thiourea
8b instead of using N-cyclopentyl thiourea 8a in step 5 and resulted in
compound
101 as theTFA salt:
S H
/>-N
MeO N N b
O
N
ON
H OH
O N..
H
O
Compound 101
1H NMR (400 MHz,DMSO-d6): ca, 90:10 mixture of rotamers, major isomer
description; 508.59 (s, 1 H), 8.45-8.39 (m, 1 H), 8.25 (bs, 1 H), 8.20 (d, J =
9.2 Hz,
1 H), 7.84 (bs, 1 H), 7.74 (s, 1 H), 7.32-7.26 (m, 1 H), 7.01 (d, J = 8.3 Hz,
1 H), 5.78-
5.66 (m, 2H), 5.20 (dd, J = 17.0, 1.6 Hz, 1 H), 5.09-5.04 (m, 1 H), 4.53-4.36
(m, 4H),
4.05-3.92 (m, 2H), 3.97 (s, 3H), 2.63-2.55 (m, 1 H), 2.44-2.29 (m, 3H), 2.07-
1.95 (m,
3H), 1.79-1.23 (m, 12H), 0.96 (s, 9H).
M.S.(electrospray) : 773.4 (M-H)- 775.4 (M+H)+ . Reverse Phase HPLC
Homogeneity (0.06% TFA; CH3CN : H20) : 98%
EXAMPLE 3
Preparation of Compound 102
Using the same procedure described in example 1 and using N-cyclohexyl
thiourea
8c instead of using N-cyclopentyl thiourea 8a in step 5 and resulted in
compound
102 as the TFA salt:
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S H
I />-N
MeO N N b
O
N \
ON
H OH OH
.
O
Compound 102
1H NMR (400 MHz,DMSO-d6): ca, 90:10 mixture of rotamers, major isomer
description; 58.61 (s, 1 H), 8.26-8.17 (m, 2H), 8.13-8.04 (m, 1 H), 7.81 (bs,
1 H), 7.73
(bs, 1 H), 7.32-7.24 (m, 1 H), 7.07 (d, J = 8.2 Hz, 1 H), 5.78-5.65 (m, 2H),
5.23-5.15
(m, 1 H), 5.09-5.03 (m, 1 H), 4.51-4.43 (m, 3H), 4.05-3.77 (m, 3H), 3.97 (s,
3H), 2.64-
2.55 (m, 1 H), 2.38-2.27 (m, 1 H), 2.05-1.95 (m, 3H), 1.79-1.71 (m, 2H), 1.66-
1.19 (m,
16H), 0.96 (s, 9H).
M.S.(electrospray) : 801.4 (M-H)- 803.4 (M+H)+ . Reverse Phase HPLC
Homogeneity (0.06% TFA; CH3CN : H20) : 99%
EXAMPLE 4
Preparation of compound 103
S}-N s>-N
\
Meo N I N b Meo N_ I N b
O
IOI O
0 N TY N \ /~ 0O,1u\N N
H OH I II H N \
O O N HZN,S \ O O Nis`
H O // O H O O O
O
Compound 100 Compound 103
Compound 100 (30mg, 0.038mmol) was combined with HATU (17mg, 0.045mmol)
and dissolved in anhydrous DMF (4mL). The solution was stirred at R.T. before
DIPEA (26 L, 0.15mmol) was added dropwise over ca. 1min. The mixture was
stirred for 60min. at R.T. and analyzed by analytical HPLC for the formation
of the
activated ester. Following this, a solution of benzenesulfonamide (23mg,
0.15mmol),
DMAP (17mg, 0.14mmol) and DBU (22 L, 0.15mmol) was added in DMF (1 mL).
The reaction mixture was stirred 24h at R.T. before being poured into EtOAc
(5OmL)
and washed with sat. NaHCO3, and sat. brine solutions. The organic phase was
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dried over MgSO4i filtered and concentrated. The residue was reconstituted in
DMSO and purified by preparative HPLC. Lyophilization gave the final product
(17
mg, 48%) as a pale yellow amorphous solid.
1H-NMR (400MHz, DMSO-d6), 510.89 (s, 1 H), 8.84 (s, 1H), 8.20 (d, J= 8.8 Hz,
1H),
8.18-8.08 (m, 2H), 7.89 (d, J = 7.6 Hz, 2H), 7.70 (dd, J = 7.6, 7.6 Hz, 2H),
7.58 (dd,
J = 7.7, 7.7 Hz, 2H), 7.28 (bs, 1 H), 7.11 (d, J = 6.6 Hz, 1 H), 5.75 (bs, 1
H), 5.37-5.25
(m, 1 H), 5.12 (d, J = 17 Hz, 1 H), 4.92 (d, J = 12 Hz, 1 H), 4.58-4.42 (m,
3H), 4.24
(bs, 1 H), 4.05 (d, J = 7.8 Hz, 1 H), 3.97 (s, 3H), 3.93 (d, J = 7.8 Hz, 1 H),
2.69-2.61
(m, 1 H), 2.35-2.21 (m, 1 H), 2.13-1.98 (m, 3H), 1.78-1.68 (m, 4H), 1.68-1.51
(m, 8H),
1.50-1.41 (m, 4H), 1.29-1.22 (m, 1 H), 0.99 (s, 9H).
MS (electrospray): 928.5 (M + H)+, and 926.5 (M - H)-.
RP-HPLC: Rt = 7.3 minutes (homogeneity = 99%).
Using the synthetic sequence shown in Scheme 2 the following compounds were
prepared:
EXAMPLE 5
Step 1: synthesis of dipeptide 16:
O 0
MeO N` OMe MeO N\ O~Na
O O
-~YO O ~O 0 O N
OMe OMe
O O
15 16
Dipeptide 15 (4.0g; 7.02mmol) was dissolved in THE (20mL) and MeOH (10mL),
water (1 OmL) and a 1 N NaOH aqueous solution (1.05 equivalents; 7.4mL) was
added. The solution stirred at R.T. for 2.75h. The mixture was evaporated to
dryness. The residue was diluted with water, frozen and lyophilized to provide
sodium salt 16 as a white amorphous solid (4.28g).
M.S.(electrospray) : 554.2 (M-H)- 556.3 (M+H)+ 578.2 (M+Na)+ .
Reverse Phase HPLC Homogeneity (0.06% TFA ; CH3CN : H20) : 96%.
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Step 2: synthesis of dipeptide diazoketone 17:
o O
Meo I N\ ONa Meo i N\ iNZ
O O
1O N N ~
O e
H
O .Me O He
N NI
O O
16 17
Sodium salt 16 (assume 7.02mmol) was dissolved in THE (78mL); triethylamine
(1.37mL; 9.83mmol) was added and the solution cooled to 0 C .
Isobutylchloroformate (1.28mL; 9.83mmol) was added dropwise and the white
suspension was stirred at 0 C for 2h, , followed by the addition of a solution
of
diazomethane (0.67M in diethyl ether; 63mL; 42.13mmol). The reaction mixture
was
stirred 1 h at 0 C, 1.25h at R.T. and evaporated to provide a thick
suspension. This
suspension was dissolved in EtOAc and water. The organic solution was washed
with saturated NaHCO3 (2x), water (2x) and brine (1x), dried (MgSO4), filtered
and
evaporated to give the diazoketone 17 as an beige solid (crude material used
for
next step; assume 7.02mmol).
M.S.(electrospray) : 578.2 (M-H)- 580.3 (M+H)+ . Reverse Phase HPLC
Homogeneity (0.06% TFA ; CH3CN : H20) : 90%-
Step 3: synthesis of dipeptide bromoketone 18:
o O
Meo NZ MeO N\ Br
O O 31 O N~ O 0 N~ Me
N
O 11 Me II
O O
17 18
At 0 C, to a solution of diazoketone (assume 1.44mmol) in THE (116mL) was
added
dropwise a 48% aqueous HBr solution (5.1 mL) and the mixture was stirred for
2h.
The solution was diluted with EtOAc, washed with a saturated NaHCO3
solution(2x),
water (2x) and brine (1x), dry (MgSO4), filtered and evaporated to give the
desired
bromoketone as a beige solid (4.25g; 6.72mmol).
M.S.(electrospray) : 632 (M) 634.2 (M+2)
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Step 4: synthesis of dipeptide 19:
O ~H
Me0 N\ Br MeO N N
O H 2 N N
H 8a
1OY N Y O N
OMe OH OMe
O H,. 0 O N
O H O
18 19
a-Bromoketone 18 (512mg; 0.81 mmol) and N-cyclopentylthiourea (128.4mg;
0.89mmol) were dissolved in isopropanol (20mL). The resulting yellow solution
was
heated at 70 C for 1.5h. The solution was allowed to cool to R.T., and
evaporated
to dryness. The residue was diluted with EtOAc. The EtOAc solution was washed
with saturated NaHCO3 (2x), water (2x) and brine (1x), dried (MgSO4), filtered
and
concentrated to give the product as an orange-brown foam. Flash column
chromatography in hexane:EtOAc 7:3 removed less polar impurities and 6 : 4
retrieved the desired compound as a light yellow solid (411.5mg; 75%).
M.S.(electrospray) : 676.3 (M-H)- 678.3 (M+H)+
Reverse Phase HPLC Homogeneity (0.06% TFA ; CH3CN : H20) : 99%-
Step 5: synthesis of tripeptide 20:
~
H H
MeO N~ I N N Meo Nzt N` i N N
lõ b lõ b
11 O 0, N" 'N OH O
H H O
O
1OuN ' \i\N~N N N qOMe
IoI HMe 5 H H o
O N O H19 20
The Boc-dipeptide (32.6g ; 0.048mmol) was dissolved in 4N HCI/dioxane (3mL)
and
stirred at R.T. After 2h, the reaction mixture was worked-up by evaporating to
dryness. The HCI salt thus obtained as an off-white solid. The HCI salt was
subjected to high vacuum for 30min. To a solution of the HCI salt in DCM (2mL)
and
DIEA (33 l_; 0.192mmol), was added urea 5 (13.96mg ; 0.058mmol), followed by
HATU coupling agent (21.90mg ; 0.058mmol). The reaction mixture was stirred at
R.T. for 3h. The reaction mixture was diluted with EtOAc, washed with
saturated
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NaHCO3 (2x), water (2x) and brine (lx), dried (MgSO4), filtered and evaporated
to
give the crude product as a thick yellow oil (assume 0.048mmol).
M.S. (electrospray) : 800.4 (M-H)- 802.4 (M+H)+ . Reverse Phase HPLC
Homogeneity (0.06% TFA ; CH3CN : H20) : 95%.
Step 6: Hydrolysis of tripeptide methyl ester 20:
S H S H
MeO ~-N Me0 \ N}-N
)*IN b b
0 0
II
'N'N
O N NN N \
H H YX OMe H H OH
N`
H O H O
A solution of methyl ester 20 (77.80mg; 0.097mmol) in THE (2mL), and MeOH
(1 mL), and an aqueous solution of LiOH (40.7mg; 0.97mmol) in water (1 mL),
was
10 stirred for 16h. The organic solution was concentrated to provide an off-
white
suspension. The crude material was purified by preparatory HPLC (YMC
Combiscreen ODS-AQ, 50 x20mm ID S-5micron,120A ; a.=220nm) using a linear
gradient and 0.06% TFA CH3CN / H2O . The pure fractions were combined,
concentrated and converted to the sodium salt.
Conversion to Na Salt:
The concentrated fractions were diluted with EtOAc and a few mis of brine,
(basified to pH -13 with 5N NaOH, then, neutralized to pH 5.5 - 6.0 with 1 N
HCI).
The product was extracted with EtOAc (3x), washed with water (2x) and brine
(lx),
dried (MgSO4), filtered and evaporated to dryness to provide the neutral
product as
a yellow solid (52.7mg ; 70%). The neutral product (49.4mg ; 0.0627mmo1) was
dissolved in MeOH (1 OmL) and 1 equivalent 0.01 N NaOH (6.27mL) was added.
The clear yellow solution was concentrated to remove MeOH and diluted with
water,
frozen and lyophililzed to give the product (Na salt ) as a yellow amorphous
solid
(50.8mg; theoretical yield : 50.8mg; MW Na salt : 809.75)
1H NMR (400 MHz,DMSO-d6): ca, 8:1 mixture of rotamers ;S 8.20 (bs, 1 H) , 7.96
(d, J = 8.8 Hz, 1 H) , 7.86 (d, J = 5.7 Hz, 1 H), 7.51-7.47 (m, 2H), 7.26 (s,
1 H), 6.97
(d, J = 8.6 Hz, 1 H), 6.19-6.02 (m, 1 H), 5.33 (bs, 1 H), 4.99 (d, J = 16.8
Hz, 1 H),
4.77 (d, J = 10.0 Hz, 1 H), 4.52-4.41 (m, 2H), 4.34 (d, J = 11.0 Hz, 1 H), ),
4.04-3.96
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(m, 2H), 3.89 (s, 3H), 3.79-3.68 (m, 1 H), 3.68-3.15 (under water peak, 2H),
2.45-
2.36 (m, 1 H), 2.05-1.92 (m, 2H), 1.82-1.35 (m, 16H), 1.35-1.12 (m, 2H), 0.91
&
0.84 (2x s, 9H).
M.S.(electrospray) : 786.4 (M-H)- 788.3 (M+H)+ 810 (M+Na)+. Reverse Phase
HPLC Homogeneity (0.06% TFA ; CH3CN : H20) : 99%.
EXAMPLE 6
Compound 104:
Using the same procedure as described in example 6 and using the cyclobutyl
carbamate of tent-butyl glycine in step 5 instead of urea 5 and purifying the
crude
carboxylic acid after step 6 by preparative HPLC afforded the title compound
as a
TFA salt:
S H
/ --N
Me0 N N
N ~
O N
H OH
N.
O H
O
Compound 104
'H NMR (400 MHz,DMSO-d6): ca, 7:1 mixture of rotamers ;S 8.09 (bs, 1H) , 8.02
(d, J = 9.OHz, 1 H) , 7.88 (d, J = 6.5 Hz, 1 H), 7.45 (s, 1 H), 7.40 (s, 1 H),
7.28 (d, J =
2.3 Hz, 1 H), 7.16-7.08 (m, 1 H), 7.07-7.00 (m, 1 H), 6.10-5.95 (m, 1 H), 5.32
(bs,
1 H), 5.00 (d, J = 17.2 Hz, 1 H), 4.82 (d, J = 11.4 Hz, 1 H), 4.64-4.52 (m, 1
H), 4.48-
4.41 (m, 1 H), 4.29 (d, J = 11.7 Hz, 1 H), ), 4.12 (d, J = 8.6 Hz, 1 H), 3.97-
3.85 (m,
2H), 3.91 (s, 3H), 2.36-2.27 (m, 1 H), 2.18-2.04 (m, 2H), 2.03-1.81 (m, 6H),
1.77-
1.43 (m, 9H), 1.41-1.34 (m, 1H), 0.96 & 0.85 (2x s, 9H).
M.S.(electrospray) : 773.3 (M-H)- 775.4 (M+H)+ . Reverse Phase HPLC
Homogeneity (0.06% TFA; CH3CN : H20) : 99%.
EXAMPLE 7
Compound 105:
Using the same procedure as described in example 6 and using the cyclohexyl
carbamate of tent-butyl glycine in step 5 instead of urea 5 afforded the title
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compound as the sodium salt:
S H
/>-N
MeO N\ N b
a
N
o N Y
H off
0 N ,;
O H
O
Compound 105
1H NMR (400 MHz,DMSO-d6): ca, 5:1 mixture of rotamers; 8 8.29 (bs, 1 H) , 8.02
(d, J = 9.0Hz, 1 H) , 7.89 (d, J = 6.5 Hz, 1 H), 7.45 (s, 1 H), 7.40 (s, 1 H),
7.27 (d, J =
2.2 Hz, 1 H), 7.05 (d, J = 8.6 Hz, 1 H), 7.00 (dd, J = 2.0, 9.0 Hz, 1 H), 5.89
(bs, 1 H),
5.34 (s, 1 H), 5.08 (d, J = 17.0 Hz, 1 H), 4.91 (d, J = 9.4 Hz, 1 H), 4.43
(dd, J = 8.4,
16.8 Hz, 1 H), 4.36-4.24 (m, 1 H), 4.14 (d, J = 8.6 Hz, 1 H), ), 4.00-3.86 (m,
3H),
3.89 (s, 3H), 2.35-2.23 (m, 1 H), 2.04-1.91 (m, 5H), 1.79-1.41 (m, 1 OH), 1.39-
1.08
(m, 7H), 0.97 & 0.86 (2x s, 9H).
M.S.(electrospray) : 801.4 (M-H)- 803.4 (M+H)+ . Reverse Phase HPLC
Homogeneity (0.06% TFA; CH3CN : H20) : 98%.
EXAMPLE 8
Compound 106:
S H
Me0 \ N\ N
O
O N N
~
H OH
0
O
0
Using the same procedure as described in example 6 and using the cyclopentyl
carbamate of cyclohexyl glycine in step 5 instead of urea 5 afforded the title
compound as the sodium salt:
1H NMR (400 MHz,DMSO-d6): ca, 1 : 4 mixture of rotamers ;S 8.22 & 8.04 (2xs, 1
H)
, 8.00 (d, J = 9.2Hz, 1 H) , 7.91 (d, J = 6.5 Hz, 1 H), 7.46 (s, 1 H), 7.41
(s, 1 H), 7.27
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(d, J = 2.4 Hz, 1 H), 7.20 (d, J = 8.4 Hz, 1 H), 7.01 (dd, J = 2.4, 9.0 Hz, 1
H), 6.09-
5.98 (m, 1 H), 5.34 (s, 1 H), 4.98 (dd, J = 1.6, 17.4 Hz, 1 H), 4.80 (d, J =
11.9 Hz,
1 H), 4.73-4.67 (m, 1 H), 4.43-4.31 (m, 2H), 4.05-3.95 (m, 2H), 3.95-3.84 (m,
1 H),
3.90 (s, 3H), 2.38-2.29 (m, 1H), 2.03-1.92 (m, 2H), 1.83-1.21 (m, 24H), 1.21-
0.83
(m, 5H)
M.S.(electrospray) : 813.4 (M-H)- 815.4 (M+H)+ . Reverse Phase HPLC
Homogeneity (0.06% TFA; CH3CN : H20) : 99%.
EXAMPLE 9
Compound 107:
Using the same procedure as described in example 6 and using the cyclopentyl
carbamate of cyclopentyl glycine instead of urea 5 in step 5, afforded the
title
compound which was converted to its corresponding Na salt.
EXAMPLE10
NS3-NS4A protease assay
The enzymatic assay used to evaluate the present compound is described in
WO 00/09543 and WO 00/59929.
EXAMPLE11
Cell Based HCV RNA Replication Assay
Cell Culture
Huh7 cells that stably maintain a subgenomic HCV replicon were established as
previously described (Lohman et al., 1999. Science 285: 110-113) and
designated
as the S22.3 cell-line. S22.3 cells are maintained in Dulbecco's Modified
Earle
Medium (DMEM) supplemented with 10% FBS and 1 mg/mL neomycin (Standard
Medium). During the assay, DMEM medium supplemented with 10% FBS,
containing 0.5% DMSO and lacking neomycin was used (Assay Medium). 16 hours
prior to compound addition, S22.3 cells are trypsinized and diluted to 50 000
cells/ml
in Standard Medium. 200pL (10 000 cells) are distributed into each well of a
96-well
plate. The plate was then incubated at 370 with 5% CO2 until the next day.
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WO 03/064456 PCT/CA03/00090
Reagents and Materials:
Product Company Catalog # Storage
DMEM Wisent Inc. 1001 3CV 4 C
DMSO Sigma D-2650 RT
Dulbecco's PBS Gibco-BRL 14190-136 RT
Fetal Bovine Serum Bio-Whittaker 14-901 F -20 C/4 C
Neomycin (G418) Gibco-BRL 10131-027 -20 C/4 C
Trypsin-EDTA Gibco-BRL 25300-054 -20 C/4 C
96-well plates Costar 3997 RT
PVDF 0.22pm Filter Unit Millipore SLGV025LS RT
Deep-Well Titer Plate
Beckman 267007 RT
Polypropylene
Preparation of Test Compound
1 OpL of test compound (in 100% DMSO) was added to 2 ml of Assay Medium for a
final DMSO concentration of 0.5% and the solution was sonicated for 15 min and
filtered through a 0.22pM Millipore Filter Unit. 900pl was transfered into row
A of a
Polypropylene Deep-Well Titer Plate. Rows B to H, contain 400pL aliquots of
Assay
Medium (containing 0.5% DMSO), and are used to prepare serial dilutions (1/2)
by
transferring 400pl from row to row (no compound was included in row H).
Application of test compound to cells
Cell culture medium was aspirated from the 96-well plate containing the S22.3
cells.
175pL of assay medium with the appropriate dilution of test compound was
transferred from each well of the compound plate to the corresponding well of
the
cell culture plate (row H was used as the "No inhibition control"). The cell
culture
plate was incubated at 37 with 5% CO2 for 72h.
Extraction of Total Cellular RNA
Following the 72h incubation period, the total cellular RNA was extracted from
the
S22.3 cells of the 96-well plate using the RNeasy 96 kit (Qiagen(D, RNeasy
Handbook. 1999.). Briefly, assay medium was completely removed from cells and
100 pL of RLT buffer (Qiagen ) containing 143 mM P-mercaptoethanol was added
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CA 02474156 2004-07-22
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to each well of the 96-well cell-culture plate. The microplate was gently
shaken for
20 sec. 100 pL of 70% ethanol was then added to each microplate well, and
mixed
by pipetting. The lysate was removed and applied to the wells of a RNeasy 96
(Qiagen ) plate that was placed on top of a Qiagen Square-Well Block. The
RNeasy 96 plate was sealed with tape and the Square-Well Block with the RNeasy
96 plate was loaded into the holder and placed in a rotor bucket of a 4K15C
centrifuge. The sample was centrifuged at 6000 rpm (--5600 x g) for 4 min at
room
temperature. The tape was removed from the plate and 0.8 ml of Buffer RW1
(Qiagen RNeasy 96 kit) was added to each well of the RNeasy 96 plate. The
RNeasy 96 plate was sealed with a new piece of tape and centrifuged at 6000
rpm
for 4 min at room temperature. The RNeasy 96 plate was placed on top of
another
clean Square-Well Block, the tape removed and 0.8 ml of Buffer RPE (Qiagen
RNeasy 96 kit) was added to each well of the RNeasy 96 plate. The RNeasy 96
plate was sealed with a new piece of tape and centrifuged at 6000 rpm for 4
min at
room temperature. The tape was removed and another 0.8 ml of Buffer RPE
(Qiagen RNeasy 96 kit) was added to each well of the RNeasy 96 plate. The
RNeasy 96 plate was sealed with a new piece of tape and centrifuged at 6000
rpm
for 10 min at room temperature. Tape was removed, the RNeasy 96 plate was
placed on top of a rack containing 1.2-mL collection microtubes. The RNA was
eluted by adding 50 pL of RNase-free water to each well, sealing plate with a
new
piece of tape and incubated for 1 min at room temperature. The plate was then
centrifuged at 6000 rpm for 4 min at room temperature. The elution step was
repeated with a second volume of 50 pI RNase-free water. The microtubes with
total
cellular RNA are stored at -70 .
Quantification of Total Cellular RNA
RNA was quantified on the STORM system (Molecular Dynamics ) using the
RiboGreen RNA Quantification Kit (Molecular Probes ). Briefly, the RiboGreen
reagent was diluted 200-fold in TE (10mM Tris-HCI pH =7.5, 1 mM EDTA).
Generally, 50pL of reagent was diluted in 1 OmL TE. A Standard Curve of
ribosomal
RNA was diluted in TE to 2pg/mL and pre-determined amounts (100, 50, 40, 20,
10,
5, 2 and OpL) of the ribosomal RNA solution are then transferred in a new 96-
well
plate (COSTAR # 3997) and the volume was completed to 100pL with TE.
Generally, column 1 of the 96-well plate was used for the standard curve and
the
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CA 02474156 2004-07-22
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other wells are used for the RNA samples to be quantified. 1 OpL of each RNA
sample that was to be quantified, was transferred to the corresponding well of
the
96-well plate and 90pL of TE was added. One volume (100pL) of diluted
RiboGreen
reagent was added to each well of the 96-well plate and incubated for 2 to 5
minutes
at room temperature, protected from light (a 10 p.L RNA sample in a 200 uL
final
volume generates a 20 X dilution). The fluorescence intensity of each well was
measured on the STORMO system (Molecular Dynamics ). A standard curve was
created on the basis of the known quantities of the ribosomal RNA and the
resulting
fluorescent intensities. The RNA concentration in the experimental samples was
1o determined from the standard curve and corrected for the 20X dilution.
Reagents and Materials:
__...._...._.._.-.-....... - ._..~... _ - __.-- ...... _.... _..... ~...- -_-
.... _ _~
Product Company Catalog # Storage
DEPC Sigma D5758 4 C
EDTA Sigma E5134 RT
Trizma-Base Sigma T8524 RT
Trizma-HCI Sigma T7149 RT
Collection Tube Strips Qiagen 19562 RT
Ribogreen RNA Quantitation Kit Molecular Probe 811490 -20 C
Rneasy 96 Kit Qiagen 74183 RT
Square-Well Blocks Qiagen 19573 RT
Real-Time RT-PCR
The Real-Time RT-PCR was performed on the ABI Prism 7700 Sequence Detection
System using the TaqMan EZ RT-PCR Kit from (Perkin-Elmer Applied
Biosystems ). RT-PCR was optimized for the quantification of the 5' IRES of
HCV
RNA by using the Taqman technology (Roche Molecular Diagnostics Systems)
similar to the technique previously described (Martell et al., 1999. J. Clin.
Microbiol.
37: 327-332). The system exploits the 5'-3' nucleolytic activity of AmpliTaq
DNA
polymerase. Briefly, the method utilizes a dual-labeled fluorogenic
hybridization
probe (PUTR Probe) that specifically anneals to the template between the PCR
primers (primers 8125 and 7028). The 5' end of the probe contains a
fluorescent
reporter (6-carboxyfluorescein [FAM]) and the 3' end contains a fluorescent
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quencher (6-carboxytetramethylrhodamine[TAMRA]). The FAM reporter's emission
spectrum was suppressed by the quencher on the intact hybridization probe.
Nuclease degradation of the hybridization probe releases the reporter,
resulting in an
increase in fluorescence emission. The ABI Prism 7700 sequence detector
measures the increase in fluorescence emission continuously during the PCR
amplification such that the amplified product was directly proportion to the
signal.
The amplification plot was analysed early in the reaction at a point that
represents
the logarithmic phase of product accumulation. A point representing a defined
detection threshold of the increase in the fluorescent signal associated with
the
exponential growth of the PCR product for the sequence detector was defined as
the
cycle threshold (CT). CT values are inversely proportional to the quantity of
input
HCV RNA; such that under identical PCR conditions, the larger the starting
concentration of HCV RNA, the lower the CT. A standard curve was created
automatically by the ABI Prism 7700 detection system by plotting the CT
against
each standard dilution of known HCV RNA concentration.
Reference samples for the standard curve are included on each RT-PCR plate.
HCV
Replicon RNA was synthesized (by T7 transcription) in vitro, purified and
quantified
by OD260. Considering that 1 pg of this RNA = 2.15 X 10" RNA copies, dilutions
are
made in order to have 108, 107, 106, 105, 104, 103 or 102 genomic RNA copies /
5pL.
Total cellular Huh-7 RNA was also incorporated with each dilution (50ng /
5pL). 5pL
of each reference standard (HCV Replicon + Huh-7 RNA) was combined with 45pL
of Reagent Mix, and used in the Real-Time RT-PCR reaction.
The Real-Time RT-PCR reaction was set-up for the experimental samples that
were
purified on RNeasy 96 -well plates by combining 5pl of each total cellular RNA
sample with 45pL of Reagent Mix.
Reagents and Materials:
Product COMPANY Catalog # Storage
TaqMan EZ RT-PCR Kit PE Applied Biosystems N808-0236 -20 C
MicroAmp Optical Caps PE Applied Biosystems N801-0935 RT
MicroAmp Optical 96-
PE Applied Biosystems N801-0560 RT
Well Reaction Plate
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Reagent Mix preparation:
Volume for Volume for One Plate
Final
Component one sample (NL) (91 samples +
conc.
(NL) Dead Volume)
Rnase-free water 16.5 1617
5X TagMan EZ buffer 10 980 1X
Mn(OAc)2 (25mM) 6 588 3mM
dATP (1OmM) 1.5 147 300pM
dCTP (10mM) 1.5 147 300pM
dGTP (10mM) 1.5 147 300pM
dUTP (20mM) 1.5 147 600pM
Forward Primer (10pM) 1 98 200nM
Reverse Primer (10pM) 1 98 200nM
PUTR probe (5pM) 2 196 200nM
rTth DNA polymerase
2 196 0.1 U/p L
(2.5 U/p L)
AmpErase UNG
0.5 49 0.01 U/pL
(1 U/pL)
Total Volume 45 4410
Forward Primer Sequence (SEQ ID. 1): 5' - ACG CAG AAA GCG TCT AGC CAT
GGC GTT AGT - 3'
Reverse Primer Sequence (SEQ ID NO. 2): 5'- TCC CGG GGC ACT CGC AAG
CAC CCT ATC AGG - 3'
Note: Those primers amplify a region of 256-nt present within the 5'
untranslated
lo region of HCV.
PUTR Probe Sequence (SEQ ID NO. 3): 6FAM I - TGG TCT GCG GAA CCG
GTG AGT ACA CC - AMR
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No Template Controls (NTC): On each plate, 4 wells are used as "NTC". For
these
controls, 5pl of water are added to the well in place of RNA.
Thermal Cycling Conditions:
50 C 2 min
60 C 30 min
95 C 5 min
95 C 15 sec
60 C 1 min for 2 cycles
90 C 15 sec
60 C 1 min for 40 cycles
Following the termination of the RT-PCR reaction the data analysis requires
setting
of threshold fluorescence signal for the PCR plate and a standard curve was
constructed by plotting the Ct value versus RNA copy number used in each
reference reaction. The Ct values obtained for the assay samples are used to
interpolate an RNA copy number based on the standard curve.
Finally, the RNA copy number was normalized (based on the RiboGreen RNA
quantification of the total RNA extracted from the cell culture well) and
expressed as
genome equivalents / pg of total RNA [ge/pg].
The RNA copy number [g.e./pg] from each well of the cell culture plate was a
measure of the amount of replicating HCV RNA in the presence of various
concentrations of inhibitor. The % inhibition was calculated with the
following
equation:
100 - ((g. e./Ng inh)/( g. e./Ng ctl)x 100].
A non-linear curve fit with the Hill model was applied to the inhibition-
concentration
data, and the 50% effective concentration (EC50) was calculated by the use of
SAS
software (Statistical Software System; SAS Institute, Inc. Cary, N.C.).
When the compounds of this invention were evaluated in the preceding enzymatic
and cell based assays, the compounds were found to be highly active. More
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CA 02474156 2009-11-13
specifically, the compounds had IC50's below 0.1 pM in the NS3-NS4A protease
assay, and EC50's below 0.5 pM in the cell based HCV RNA replication assay.
EXAMPLE 12
Specificity assays
The specificity assays used to evaluate the selectivity of this compound are
described in WO 00/09543.
When the compounds were evaluated in the specifity assays, the compounds of
formula 1 were found to be selective in that they do not show significant
inhibition in
the Human Leukocyte Elastase and Cathepsin B assays.
EXAMPLE13
Pharmacokinetic properties
The present compounds also show good pharmacokinetic properties such as
significant plasma levels in the rat at 1 hour and 2h after an oral dose of
5mg/kg.
More explicitly, the following assay, an in vivo oral absorption screen, was
used to
determine plasma levels of test compounds in a rat after oral administration:
Materials and Methods:
1. Method used to pool compounds ("cassette selection"):
The selection of compounds to be pooled into a "cassette" was based on their
structural similarity and physicochemical properties. A solid phase extraction
method applicable to all the selected compounds was established. Based on the
initial testing where each compound was spiked into rat plasma and run through
HPLC or HPLC/MS at a concentration of 0.5 NM, the retention time, ionic mass,
and
the possible separation among compounds by HPLC and/or HPLC/MS were used
as basis for pooling 3-4 compounds into one "cassette".
2. Oral vehicle and compound preparation:
Each "cassette" contains 3-4 compounds at 5 or 4mg/kg for each compound. The
cassettes were prepared as an oral suspension in 0.5% aqueous methylcellulose
and 0.3% of polyoxyethylene (20) sorbiton monooleate (TweenTM-80). The dosing
volume was 10ml/kg via oral gavage.
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3. Dosing and plasma sampling:
Male Sprague Dawley rats were fasted overnight in individual cages, with
access to
aqueous 10% dextrose. Two rats were dosed with each "cassette". Plasma
samples (-1 ml) were collected at 1 and 2h post-dosing from the 2 rats and
pooled
for extraction and analysis.
4. Compound extraction and analysis:
From each cassette, plasma samples at 1 and 2h, blank plasma, blank plasma
spiked with all the compounds at 0.5 pM of each, are extracted by the solid
phase
extraction method. Samples were analyzed by HPLC and HPLC/MS for comparison
purpose. Plasma concentrations are estimated based on the single concentration
of
0.5 pM standard.
When assayed in the preceding screen the compounds of examples 1 to 9 of this
invention were found to present in significant levels in the plasma at the 1
hour and
2 hour intervals following oral administration, averaging blood plasma levels
of 1.23
pM and 1.16 pM respectively. This demonstration of significant in vivo oral
absorption for the compounds of this invention is unexpected, in view of the
lower
oral absorption generally attributed to this class of peptides. The ready oral
absorption renders the compounds useful for treating of HCV infection.
The following table lists compounds representative of the invention. Further
in
keeping with the present disclosure, the compound all had IC50's below 0.1 pM
in
the NS3-NS4A protease assay, and EC50's below 0.5 pM in the cell based HCV
RNA replication assay.
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CA 02474156 2004-07-22
WO 03/064456 PCT/CA03/00090
TABLE 1
-
//N 2
N %(
MeO N S
3 O
R
O
R:O)LN N
H sill R1 0 ? N
H O (')
Cpd No. R R R R m/z
MH
789.3
100 OH tent- cl"
Butyl 101 OH tent- 775.4
Butyl Cl,
102 OH tart 803.4
Butyl CJI,
103 tert- 928.5
~s \ Butyl 01,
n
0
104 OH tent 775.4
Butyl
105 OH tert- 803.4
Butyl
106 OH ca" 815.4
107 OH 801.4
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CA 02474156 2004-07-22
Sequence listing.txt
SEQUENCE LISTING
<110> BOEHRINGER INGELHEIM INTERNATIONAL
<120> HEPATITIS C INHIBITOR TRI-PEPTIDES
<130> 13/106
<150> 2,370,396
<151> 2002-08-01
<160> 3
<170> FastSEQ for windows Version 4.0
<210> 1
<211> 30
<212> DNA
<213> Artifical Sequence
<220>
<223> Forward primier
<400> 1
acgcagaaag cgtctagcca tggcgttagt 30
<210> 2
<211> 30
<212> DNA
<213> Artifical Sequence
<220>
<223> Reverse Primer
<400> 2
tcccggggca ctcgcaagca ccctatcagg 30
<210> 3
<211> 26
<212> DNA
<213> Artifical sequence
<220>
<223> PUTR probe
<400> 3
tggtctgcgg aaccggtgag tacacc 26
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