Note: Descriptions are shown in the official language in which they were submitted.
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HETEROCYCLIC TRIPEPTIDES AS HEPATITIS C INHIBITORS
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, NSSA, and NSSB) 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-NSSA, NSSA-NSSB 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. NSSB 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
1o 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;
W O 00/59929; W O 00/31129; W O 01 /02424; W O 01 /07407; 1/110 01 /16357;
W O 01 /32691; W O 01 /40262; W O 01 /58929; W O 01 /64678; W O 01 /74768;
W O 01 /77113; W O 01 /81325; W O 02/08187; W O 02/08198; W O 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.
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). Furthermore, the
3o compounds are active in cell culture and have good pharmacokinetic profile
in vivo.
A further advantage of the present invention is that it provides compounds
that are
orally bioavailable in mammals.
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SUMMARY OF THE INVENTION
Included in the scope of the invention is a compound of formula (I):
Me0
0
o Ra
Rvo~N~N S
R'
O O H~ R
o (I)
wherein R' is hydroxy or NHSOZR'A wherein R'A is (C,_8)alkyl, (C3_,)cycloalkyl
or
{(C,_6)alkyl-(C3_~)cycloalkyl }, which are all optionally substituted from 1
to 3 times
with halo, cyano, nitro, O-(C,_6)alkyl, amido, amino or phenyl, or R'A is Cs
or C,o aryl
which is optionally substituted from 1 to 3 times with halo, cyano, nitro,
(C,_6)alkyl,
O-(C,_6)alkyl, amido, amino or phenyl; R2 is t-butyl, -CHZ-C(CH3)3, or -CH2-
1 o cyclopentyl; R3 is t-butyl or cyclohexyl and R4 is cyclobutyl,
cyclopentyl, or
cyclohexyl; 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 pharmaceutically 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 interferon (pegylated or not), or ribavirin, or one or more other anti-
HCV
2o 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
agent, administered together or separately.
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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
therapeutically
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
1o 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 centre of a compound of formula
I, the
designation is done in the context of the whole compound and not in the
context of
2o the substituent or asymmetric centre 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 8257, 249-264 (1970)).
As used herein the term "(1 R, 2S)-vinyl-ACCA" refers to a compound of
formula:
S
OH
HzN ,,,,R
O
3o namely, ( 1 R, 2S) 1-amino-2-ethenylcyclopropylcarboxylic acid.
The term "(C,_6)alkyl" as used herein, either alone or in combination with
another
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substituent, means acyclic, straight or branched chain alkyl substituents
containing
from 1 to 6 carbon atoms and includes, for example, methyl, ethyl, propyl,
butyl,
1-methylethyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl and hexyl.
Similarly, the term "(C,_S)alkyl" means acyclic, straight of branched chain
alkyl
containing 1 to 8 carbon atoms, e.g. octyl.
The term "(C3_,)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 "{(C,_6)alkyl-(C3_,)cycloalkyl}"as used herein means a cycloalkyl
radical
containing from 3 to 7 carbon atoms directly linked to an alkylene radical
containing
1 to 6 carbon atoms; for example, cyclopropylmethyl, cyclopentylethyl,
cyclohexylmethyl, cyclohexylethyl and cycloheptylpropyl. In the instance where
R°"
is a {(C,_6)alkyl-(C3_6)cycloalkyl}, this group is attached to the S02 group
via the (C,_
6)alkyl (i.e. the alkylene portion).
The term "C6 or C,o aryl" as used herein, either alone or in combination with
another
radical, means either an aromatic monocyclic group containing 6 carbon atoms
or
2o an aromatic bicyclic group containing 10 carbon atoms. For example, aryl
includes
phenyl, 1-naphthyl or 2-naphthyl.
The term "O-(C,_s)alkyl" as used herein, either alone or in combination with
another
radical, means the radical -O-(C,_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,
3o chloro, fluoro or iodo.
The term "pharmaceutically acceptable salt' means a salt of a compound of
formula
(I) 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,
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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.
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, which is hereby
incorporated by reference in its entirety.
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
1o biologically or otherwise undesirable, formed with inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric
acid,
phosphoric acid, and the like, and organic acids such as acetic acid,
trifluoroacetic
acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic
acid,
butyric acid, camphoric acid, camphorsulfonic acid, cinnamic acid, citric
acid,
digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid,
glycerophosphoric
acid, hemisulfic acid, hexanoic acid, formic acid, fumaric acid, 2-
hydroxyethanesulfonic acid (isethionic acid), lactic acid, hydroxymaleic acid,
malic
acid, malonic acid, mandelic acid, mesitylenesulfonic acid, methanesulfonic
acid,
naphthalenesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic
acid,
2o pamoic acid, pectinic acid, phenylacetic acid, 3-phenylpropionic 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,
3o 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).
2o 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: antiviral 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-, (i-, S- 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-570310. 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:
- US Application No. 10/198,680, which corresponds to PCT/CA02/01127,
both filed 18 July 2002 (Boehringer Ingelheim),
- US Application No. 10/198,384, which corresponds to PCT/CA02/01128,
both filed 18 July 2002 (Boehringer Ingelheim),
- US Application No. 10/198,259, which corresponds to PCT/CA02/01129,
both filed 18 July 2002 (Boehringer Ingelheim),
- WO 02/100846 A1 and WO 02/100851 A2 (both Shire),
-' WO 01/85172 A1 and WO 02/098424 A1 (both GSK),
- WO 00/06529 and WO 02/06246 A1 (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
internal
ribosomeal entry site (IRES). A specific example of inhibitors of another
target in
1o 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~ (GIaxoSmithKline), 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
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2001-3 (ICN), Bam 205 (Novelos), XTL-001 (XTL), Imino-Sugars (Nonyl-DNJ)
(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 B~ (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-
~3-, 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.
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.
3o 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
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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
1o 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
Preferred are compounds of formula 1 as defined above wherein R' is hydroxy,
2o NHS02Me, NHS02-cyclopropyl, or NHS02Ph. More preferably, R' is NHS02Me or
hydroxy. Most preferably, R' is hydroxy.
Preferred are compounds of formula 1 as defined above wherein R2 is t-butyl or
CH2-C(CH3)3. More preferably, R2 is CH2-C(CH3)3.
Preferably R3 is t-butyl.
Preferably, compounds of formula 1 as defined above wherein R4 is cyclopentyl
or
cyclohexyl. More preferably R4 is cyclopentyl.
More preferably, a compound of formula 1 as defined above wherein R' is
hydroxy,
R2 is CH2-C(CH3)3, R3 is t-butyl and R4 is cyclopentyl.
More preferably, a compound of formula 1 wherein R' is hydroxy, R2 and R3 each
is
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t-butyl and R4 is cyclopentyl.
More preferably, a compound of formula 1 wherein R' is hydroxy, RZ is CH2-
C(CH3)3, R3 is cyclohexyl and R4 is cyclopentyl.
More preferably, a compound of formula 1 wherein R' is hydroxy, RZ is CH2-
C(CH3)3, and R3 and R4 each is cyclohexyl.
More preferably, a compound of formula 1 wherein R' is hydroxy, R2 is
~o cyclopentylmethyl, R3 is t-butyl, and R° is cyclobutyl.
More preferably, a compound of formula 1 wherein R' is hydroxy, R2 is CH2-
C(CH3)3, R3 is t-butyl and R4 is cyclobutyl.
More preferably, a compound of formula 1 wherein R' is NHS02Me, RZ is CH2-
C(CH3)3 R3 is t-butyl and R4 is cyclopentyl.
More preferably, a compound of formula 1 wherein R' is NHS02Ph, R2 is CH2-
C(CH3)3, R3 is t-butyl and R4 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), ~- (beta), b- (delta), ~y- (gamma), tau- or c~
(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
3o 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
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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
1 o 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.
2o 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.
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, 19t" Ed. Mack
Publishing Company, Easton, Penn., (1995).
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Dosage levels of between about 0.01 and about 100mg/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 I 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
3o 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
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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-, [3-, 8-, c~-, 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 I.
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
2o 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
(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
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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.
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 (b) 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; DIEA: diisopropylethylamine; DIPEA:
diisopropylethylamine; 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
2o 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 22°); tert-
butyl or t-
butyl: 1,1-dimethylethyl; Tbg: ten'-butyl glycine: tent leucine; TFA:
trifluoroacetic
acid; and THF: tetrahydrofuran.
Synthesis of compounds of formula (I):
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, incorporated herein by reference.
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Preparation of Thioureas
Preparation of Thiourea 2a
g O
H2N' _NHZ CI ~ II ~
H2N~N
H
(2a)
Thiourea (5.0 gm, 66mmol) was dissolved in toluene (50mL) and tert-butylacetyl
chloride (8.88 gm, 66mmol) was added. The mixture was heated at reflux for 14h
to
give a yellow solution. The mixture was concentrated to dryness, and the
residue
partitioned between EtOAc and sat. NaHC03. The yellow organic phase was dried
over MgS04, filtered and concentrated to give a yellow solid. The solid was
dissolved into a minimum amount of EtOAc and triturated with hexane to give 2a
as
a white solid (8.52 g; 75%). M.S. (electrospray): 173 (M-H)- 175 (M+H)+.
Reverse
Phase HPLC Homogeneity (0.06 % TFA ; CH3CN : H20) : 99 %.
Preparation of Thiourea 2b
H
CI H2N\ /NH2 H2N\ /N
O ~ + ~S ~ ~S O
(2b)
Using the procedure described above and using commercially available
cyclopentyl
acetyl chloride instead of tert-butylacetyl chloride yielded thiourea 2b.
Synthesis of intermediates 3
Preparation of carbamate 3a
0 0
+ H N OH ~ '"O"N OH
O O
O O H O
(3a)
Tetrahydrofuran (350mL) was added to a flask containing carbonic acid
cyclopentyl
ester 2,5-dioxo-pyrrolidin-1-yl ester (9.OOg; 39.6mmol) and tent leucine
(6.24g;
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 THF was evaporated and the aqueous residue
diluted
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with water (100mL) and the reaction 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 then acidified with 1 N HCI (ca. 70mL - final pH <2). The turbid
solution was extracted with EtOAc (200 + 150mL). The extract was dried (MgS04)
and evaporated to give compound 3a as a white solid (8.68 g).
Preparation of carbamates 3b, 3c, and 3d
Using the procedure described above and using appropriate combinations of tert-
butyl glycine, or cyclohexyl glycine and carbonic acid cyclobutyl,
cyclopentyl, or
cyclohexyl ester 2,5-dioxo-pyrrolidin-1-yl ester, the following carbamates
were
prepared:
o ~ 0 0
O~N OH O~N OH QO~N OH
I I I I I I
H O H O H O
3b 3c 3d
Preaaration of intermediate 5
Preparation of intermediate 5a:
I o H H ...__ I
O
(2a)
O N O N
N,,, OMe ~ ~N,,, OMe
O
H O OH O H
O
(5a)
(4)
a-Bromoketone 4 (3.61 g ; 5.71 mmol) was combined with thiourea 2a (1.09g ;
6.28mmol) in isopropanol (140mL) and the yellow solution was placed into a pre-
heated oil bath of 70°C for 1.5h. The solution was cooled to R.T. and
evaporated to
dryness. The residue was dissolved in EtOAc. The EtOAc solution was washed
with saturated NaHC03 (2x), water (2x) and brine (1 x), dried (MgS04), and
evaporated to give the product as an orange-brown foam. Flash column
chromatography in 7:3 hexane : EtOAc removed the less polar impurities and 6:4
hexane : EtOAc provided pure product as a light yellow solid (3.05g ; 76%).
M.S.(electrospray) : 706.3 (M-H)- 708.4 (M+H)+ . Reverse Phase HPLC
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Homogeneity (0.06 % TFA; CH3CN : H20) : 99 %.
Preparation of intermediate 5b:
Using the procedure described above and using thiourea 2b instead of thiourea
2a,
the corresponding intermediate 5b is obtained:
y ..\ _N
HZN~H
O
(2b)
O N ~\
Q O~N ~Me
H O
(5b)
(4)
Preparation of intermediate 5c:
Using the procedure described above and using commercially available thiourea
1o instead of thiourea 2a, the corresponding intermediate 5c is obtained:
HZN- _NHz
NHZ
O~N ~\
OMe ~Me
O O NN
O OH H O
(5c)
(4)
EXAMPLE 1
Synthesis of Compound 100
...,... N w w ~N
mcv
O ~O~N~OH I ~ ~ O
O H ~~O p
/~ O
OuN \ (3a) ~O~N~N \
IO' O N~Me H O N~Me
H O O H O
(5a) (6a)
Step 1: Preparation of intermediate 6a
Boc-Dipeptide 5a (3.05g ; 4.31 mmol) was dissolved in 4N HCI/dioxane (22mL).
After stirring at R.T. for 30 min., the HCI salt precipitated. MeOH (2mL) was
added
to dissolve the precipitate. After 2h, the reaction mixture was evaporated to
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WO 03/064416 PCT/CA03/00091
dryness. The resulting HCI salt was dissolved in DCM (22mL) and DIEA (3.OmL ;
17.24mmol); carbamate 3a (1.158 ; 4.47mmol) and HATU (1.72g ; 4.516mmol)
were added. The solution was stirred at R.T. for 6h. The mixture was then
diluted
with EtOAc and the solution washed with saturated NaHC03 (2x), water (2x) and
brine (1 x), dried (MgS04), filtered and evaporated to obtain compound 6a as a
yellow solid. Flash column chromatography eluting first with hexane : EtOAc
7:3
and then 6:4 afforded pure Me-ester 6a as a white foam (3.25g ; 90%). M.S.
(electrospray) :831.4 (M-H)- 833.4 (M+H)+ 855.4 (M+Na)+ . Reverse Phase
HPLC Homogeneity (0.06 % TFA ; CH3CN : HZO) : 98 %.
Step 2: H~rolysis of Ester
° H ~ H
Me0 ~ N\ ~ N~N Me0 ~ N~ i N~N
I
~O~N N O \ LIOH ~O~N N O
H O O N Me ' H O O N~ OH
H H '.
O O
sa Compound 100
Methyl ester 6a (3.24mg; 3.89mmol) was dissolved in THF (40mL) and MeOH
(20mL), and an aqueous solution of LiOH (1.63mg ; 38.9mmol in 25mL) was added.
The yellow reaction mixture was stirred for 5.5h and then concentrated to
provide an
off-white suspension. The suspension was dissolved in EtOAc and brine,
prepared
with deionized water. The pH of the resulting solution was adjusted to 6 by
the
addition of 1 N HCI. The layers were separated and the aqueous layer further
extracted with EtOAc (2x) . The combined EtOAc extracts were washed with
2o deionized water (2x), deionized water prepared brine (1x), dried (MgS04),
filtered
and evaporated to obtain compound 100 as a pale yellow-white solid (3.02g; 95%
yield).
Conversion to Na Salt:
The neutral compound 100 (1.22g; 1.49mmol) was dissolved in MeOH (30mL) and 1
equivalent 0.01 N NaOH (14.85mL) was added - no product precipitation. The
clear
yellow solution was concentrated, diluted with deionized water, frozen and
lyophilized to obtain the product (Na salt ) as a yellow-white amorphous solid
(1.24g; 99 % yield )
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Na Salt : MW : 840.98 C42H53N609SNa
M.S.(electrospray) : 817.3 (M-H)- 819.4 (M+H)+ 841.4 (M+Na)+ . Reverse Phase
HPLC Homogeneity (0.06 % TFA ; CH3CN : H20) : 98 %.
'H NMR (400 MHz,DMSO-d6): ca, 6:1 mixture of rotamers ; S 8.11-7.65 (m, 4H) ,
7.33 (bs, 1 H) , 7.18-6.97 (m, 2H), 6.36-6.08 (m, 1 H), 5.55-5.33 (m, 1 H),
4.98 (d, J
= 18.0 Hz, 1 H), 4.85 (bs, 1 H), 4.80 (d, J = 10.4 Hz, 1 H), 4.50-4.41 (m, 1
H), 4.22-
4.02 (m, 2H), 3.92 (s, 3H), 2.72-2.45 (m, 1 H), 2.50 ( under DMSO, s, 2H),
2.40-
2.26 (m, 1 H), 1.89-1.43 (m, 9H), 1.37-1.30 (m, 1 H), 1.30-1.12 (m, 1 H), 1.03
& 0.90
(2x s, 9H), 0.98 & 0.94 (2x s, 9H).
EXAMPLE 2
Synthesis of Compound 101
s
Me0 \ N\ I /~NHz
N
~CI
~O
~ O
~O~N N \
,; OMe
O N
O H
O O
(6b) (6c)
By following the same procedure as described in the first step of example 1,
and
using Boc-dipeptide 5c instead of 5a, compound 6b was obtained.
Compound 6b (70mg, 0.095mmol) was dissolved in 2mL of DCM and successively
treated with DIEA (0.045mL, 0.26mmol) and pivaloyl chloride (0.015mL,
0.12mmol).
After stirring for 1 h at 40°- an additional pivaloyl chloride
(0.015mL, 0.12mmol) was
2o added and stirring was continued for an additional 2h. After the solution
was
concentrated, the residue was dissolved in EtOAc. The solution was washed with
a
saturated solution of NaHC03 and brine, dried (MgS04) and concentrated to
afford
82mg of crude compound 6c which was used without purification.
Methyl ester derivative 6c was hydrolyzed as in step 2 of example 1 and
purified by
preparative HPLC using a YMC Combi-Prep. ODS-AQ column, 50 x 20mm. ID, S -
5micron, 120 A, and a linear gradient program from 2 to 100% AcCN /water
(0.06%
TFA).
Fractions were analyzed by analytical HPLC, and the pure fractions were
combined,
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WO 03/064416 PCT/CA03/00091
concentrated, frozen and lyophilized to yield compound 101 as the
trifluoroacetate
salt:
S H
~ ~~N~
Me0 ~ N N
/ / O
O
O
-O- _N N \
H OH
O N ,;
O H
O
Compound 101
'H NMR (400 MHz,DMSO-ds): ca, 85:15 mixture of rotamers, major isomer
description; 8 8.56 (s, 1 H), 8.40-8.22 (m, 1 H), 8.17 (d, J = 8.9 Hz, 1 H),
7.67 (bs, 1 H),
7.52 (bs, 1 H), 7.24-7.15 (m, 1 H), 7.03 (d, J = 8.3 Hz, 1 H), 5.78-5.67 (m, 1
H), 5.61-
5.53 (m, 1 H), 5.19 (dd, J = 17.2, 1.6 Hz, 1 H), 5.09-5.03 (m, 1 H), 4.63-4.55
(m, 1 H),
4.49-4.39 (m, 2H), 4.11-3.92 (m, 2H), 3.95 (s, 3H), 2.62-2.53 (m, 1 H), 2.33-
2.25 (m,
1 H), 2.06-1.98 (m, 1 H), 1.72-1.25 (m, 10H), 1.30 (s, 9H), 0.97 (s, 9H).
1o M.S.(electrospray) : 803.3 (M-H)- 805.3 (M+H)+ . Reverse Phase HPLC
Homogeneity (0.06 % TFA; CH3CN : H20) : 97
EXAMPLE 3
Preparation of compound 102
By following the procedure described in example 1 and using carbamate 3d
instead
of 3a and using preparative HPLC to purify the final compound as described in
example 2, compound 102 was obtained as the trifluoroacetate salt:
S H
I i~-N
Me0 ~ ~ N
O
O
~O~N N ~\
H ~H
O ~I1N
O H
O
Compound 102
'H NMR (400 MHz,DMSO-ds): ca, 90:10 mixture of rotamers, major isomer
description; 812.37 (s, 1 H), 8.54 (s, 1 H), 8.40-8.06 (m, 2H), 7.67-7.40 (m,
2H), 7.26
(d, J = 8.0 Hz, 1 H), 7.26-7.13 (m, 1 H), 5.77-5.65 (m, 1 H), 5.62-5.49 (m, 1
H), 5.23-
5.16 (m, 1 H), 5.10-5.03 (m, 1 H), 4.57-4.37 (m, 3H), 4.03-3.88 (m, 2H), 3.94
(s, 3H),
2.64-2.54 (m, 1 H), 2.41 (s, 2H), 2.37-2.22 (m, 1 H), 2.04-1.95 (m, 1 H), 1.79-
1.21 (m,
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16H), 1.17-0.85 (m, 5H), 1.04 (s, 9H).
M.S.(electrospray) : 843.5 (M-H)- 845.4 (M+H)+ . Reverse Phase HPLC
Homogeneity (0.06 % TFA; CH3CN : H20) : 99
EXAMPLE 4
Preparation of compound 103
S H
I ,>--N
Me0 \ N~ N
/ / O
O
~O~N N ~\
H O ~H
O ~N
H O
Compound103
By following the procedure described in example 1 but using carbamate 3c
instead
of 3a and using preparative HPLC to purify the final compound as described in
example 2, compound 103 was obtained as the trifluoroacetate salt:
1H NMR (400 MHz,DMSO-ds): ca, 85:15 mixture of rotamers, maior isomer
description; 512.35 (s, 1 H), 8.52 (m, 1 H), 8.43-8.06 (m, 2H), 7.67-7.41 (m,
2H), 7.26
(d, J = 7.8 Hz, 1 H), 7.24-7.11 (m, 1 H), 5.77-5.65 (m, 1 H), 5.62-5.48 (m, 1
H), 5.23-
5.15 (m, 1 H), 5.10-5.03 (m, 1 H), 4.52-4.38 (m, 2H), 4.15-4.05 (m, 1 H), 4.03-
3.89 (m,
2H), 3.94 (s, 3H), 2.63-2.52 (m, 1 H), 2.41 (s, 2H), 2.35-2.23 (m, 1 H), 2.05-
1.96 (m,
1 H), 1.81-1.41 (m, 11 H), 1.38-0.86 (m, 12H), 1.04 (s, 9H).
M.S.(electrospray) : 857.5 (M-H)- 859.4 (M+H)+ . Reverse Phase HPLC
Homogeneity (0.06 % TFA; CH3CN : H20) : 99
EXAMPLE 5
Preparation of compound 104
S H
I ~~N
Me0 ~ ~ N
I / / O
O
O
~O~N N ~\
H ~H
O ~N
O H
O
Compound 104
By following the procedure described in example 1 but using carbamate 3b
instead
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WO 03/064416 PCT/CA03/00091
of 3a and Boc-dipeptide 5b instead of 5a and using preparative HPLC to purify
the
final compound as described in example 2, compound 104 was obtained as the
trifluoroacetate salt:
'H NMR (400 MHz,DMSO-ds): ca, 3:1 mixture of rotamers ; 88.02 (s, 1 H) , 7.90
(s,
1 H) , 7.84 (d, J = 7.OHz, 1 H) , 7.70 (s, 1 H) , 7.33 (d, J = 2.2 Hz, 1 H),
7.14 (dd, J =
2.5, 8.2 Hz, 1 H), 7.09 (dd, J = 2.2, 9.2 Hz, 1 H), 6.29-6.08 (m, 1 H), 5.54-
5.32 (m,
1 H), 4.99 (d, J = 15.9Hz, 1 H), 4.80 (d, J = 10.0 Hz, 1 H), 4.76-4.64 (m, 1
H), 4.46
(dd, J = 6.7 , 13.9 Hz, 1 H), 4.19-4.08 (m, 3H), 3.92 (s, 3H), 2.70-2.61 (m,
2H),
2.37-2.09 (m, 4H), 2.03-1.82 (m, 1 H), 1.85 (bs, 2H), 1.77-1.44 (m, 8H), 1.35-
1.29
(m, 1 H), 1.29-1.15 (m, 4H), 0.98 & 0.90 (2x s, 9H).
M.S.(electrospray) : 815.3 (M-H)- 817.3 (M+H)+ . Reverse Phase HPLC
Homogeneity (0.06 % TFA; CH3CN : H20) : 98 %.
EXAMPLE 6
Preparation of comaound 105
S H
I /~--N
Me0 ~ N N
O
/ /
O
N
O N
H OH
O N ,,.
O H
O
Compound 105
By following the procedure described in example 1 but using carbamate 3b
instead
of 3a and using preparative HPLC to purify the final compound as described in
example 2, compound 105 was obtained as the trifluoroacetate salt:
'H NMR (400 MHz, DMSO-ds): ca, 7:1 mixture of rotamers ; 88.58 (s, iH) , 8.19
(d,
J = 8.OHz, 1 H) , 7.76-7.50 (m, 2H), 7.35-7.20 (m, 1 H), 7.19 (d, J = 8.0 Hz,
1 H),
5.78-5.67 (m, 1 H), 5.65-5.50 (m, 1 H), 5.19 (d, J = 17.0 Hz, 1 H), 5.07 (d, J
= 10.2
Hz, 1 H), 4.51-4.42 (m, 2H), 4.42-4.31 (m, 1 H), 4.02 (d, J = 7.4 Hz, 1 H),
4.02-3.93
(m, 1 H), 3.97 (s, 3H), 2.63-2.52 (m, 1 H), 2.42 (s, 2H), 2.35-2.25 (m, 1 H),
2.07-1.95
(m, 3H), 1.90-1.76 (m, 1 H), 1.70-1.41 (m, 3H), 1.30-1.23 (m, 1 H), 1.04 (s,
9H), 0.96
& 0.87 (2xs, 9H),
M.S.(electrospray) : 803.4 (M-H)- 805.4 (M+H)+ . Reverse Phase HPLC
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Homogeneity (0.06 % TFA; CH3CN : H20) : 98 %.
EXAMPLE 7
0
/ N
~O~H~ H
O ~N~ /
O N~iSv
v H O O O
Compound 106
Compound 100 of example 1 (29.8mg, 0.036mmol) was combined with HATU (1.2
equiv., 19.7mg, 0.052mmol) and dissolved in anhydrous DMF (4mL). The solution
was stirred at R.T. and DIPEA (5 equiv., 31.4NL, 0.18mmol) was added dropwise
over ca. 1 min.. The mixture was stirred for 20min. at R.T. and analyzed by
1o analytical HPLC for the formation of the activated ester. A solution of
methanesulfonamide (5.8equiv., 19.7mg, 0.207mmol), DMAP (5.4equiv., 23.5mg,
0.193mmol) and DBU (4.8equiv., 25.8NL, 0.172mmol) was added in DMF (1 mL).
The reaction mixture was stirred 16h at R.T. before being concentrated in
vacuo.
The residue was reconstituted in DMSO and purified by preparative HPLC.
Lyophilization gave the final product (23mg, 71.3%) as an off-white amorphous
solid.
'H-NMR (400MHz, DMSO-d6), 812.35 (s, 1 H), 10.53 (s, 1 H), 8.87 (s, 1 H), 8.40-
8.20
(m, 1 H), 8.17 (d, J = 8.8 Hz, 1 H), 7.61 (bs, 1 H), 7.51 (bs, 1 H), 5.67-5.55
(m, 2H),
5.23-5.18 (m, 1 H), 5.10 (d, J= 12 Hz, 1 H), 4.68-4.57 (m, 1 H), 4.50 (bd, J=
12 Hz,
1 H), 4.46-4.37 (m, 1 H), 4.07 (d, J = 8.0 Hz, 1 H), 3.95 (s, 3H), 3.17 (s,
3H), 2.78-2.58
(m, 1 H), 2.42 (s, 2H), 2.29-2.19 (m, 1 H), 2.19-2.09 (m, 1 H), 1.71 (dd, J =
7.8, 7.6
Hz, 1 H), 1.67-1.55 (m, 4H), 1.55-1.37 (m, 4H), 1.04 (s, 9H), 0.98 (s, 9H),
0.97-0.87
(m, 2H).
MS (electrospray): 896.5 (M + H)+, and 894.5 (M - H)-.
RP-HPLC: Rt = 6.7 minutes (homogeneity = 100%).
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Preaaration of comaound 106
CA 02474031 2004-07-22
WO 03/064416 PCT/CA03/00091
EXAMPLE 8
Preparation of compound 107
N,; NHS \ I
ii ~~
O O O
Compound 107
Using the same procedure described in Example 7 and using benzenesulfonamide
instead of methylsulfonamide gave compound 107 as a pale yellow amorphous
solid
in 54% yield.
'H NMR (DMSO-d6) 812.39 (s, 1 H), 10.89 (s, 1 H), 8.83 (s, 1 H), 8.40-8.22 (m,
1 H),
8.18 (d, J = 8.4 Hz, 1 H), 7.90 (s, 1 H), 7.88 (s, 1 H), 7.67-7.63 (m, 1 H),
7.63-7.54 (m,
3H), 7.54- 7.45 (m, 1 H), 7.30-7.15 (m, 1 H), 7.10 (d, J = 7.8 Hz, 1 H), 5.62-
5.51 (m,
1 H), 5.38-5.26 (m, 1 H), 5.16-5.08 (m, 1 H), 4.93 (d, J = 10.4 Hz, 1 H), 4.70-
4.58 (m,
1 H), 4.57-4.49 (m, 1 H), 4.48-4.39 (m, 1 H), 4.09 (d, J = 7.8 Hz, 1 H), 3.95
(s, 3H),
2.69-2.59 (m, 1 H), 2.41 (s, 2H), 2.28-2.16 (m, 1 H), 2.14-2.04 (m, 1 H), 1.72-
1.52 (m,
4H), 1.51-1.37 (m, 4H), 1.29-1.22 (m, 1 H), 1.03 (s, 9H), 1.00 (s, 9H), 0.99-
0.92 (m,
1 H).
MS (electrospray); 958.5 (M + H)+, and 956.5 (M - H)-.
RP-HPLC: Rt = 7.2 minutes (homogeneity = 95%).
EXAMPLE 9
NS3-NS4A protease assay
The enzymatic assay used to evaluate the present compound is described in WO
00/09543 and WO 00/59929.
EXAMPLE10
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
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CA 02474031 2004-07-22
WO 03/064416 PCT/CA03/00091
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. 200NL (10 000 cells) are distributed into each well of a
96-well
plate. The plate was then incubated at 37° with 5% COZ until the next
day.
Reagents and Materials:
Product ~~~~ Company Catalog Storage
#
DMEM Wisent Inc. 10013CV 4C
DMSO Sigma D-2650 RT
Dulbecco's PBS Gibco-BRL 14190-136 RT
Fetal Bovine Serum Bio-Whittaker 14-901 -20C/4C
F
Neomycin (G418) Gibco-BRL 10131-027 -20C/4C
Trypsin-EDTA Gibco-BRL 25300-054 -20C/4C
96-well plates Costar 3997 RT
PVDF 0.22Nm Filter Millipore SLGV025LS RT
Unit
Deep-Well Titer
Plate Beckman 267007 RT
Polypropylene
1o Preparation of Test Compound
lOpL 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 0.22NM Millipore Filter Unit. 900p1 was transfered into row
A of a
Polypropylene Deep-Well Titer Plate. Rows B to H, contain 400NL aliquots of
Assay
Medium (containing 0.5% DMSO), and are used to prepare serial dilutions (1/2)
by
transferring 400N1 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.
175NL 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% C02 for 72h.
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CA 02474031 2004-07-22
WO 03/064416 PCT/CA03/00091
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 (QiagenO, RNeasy
Handbook. 1999.). Briefly, assay medium was completely removed from cells and
100 NL of RLT buffer (QiagenO) containing 143 mM (3-mercaptoethanol was added
to each well of the 96-well cell-culture plate. The microplate was gently
shaken for
20 sec. 100 NL 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 QiagenO 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
2o 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 NL 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 NI RNase-free water. The microtubes with
total
3o cellular RNA are stored at -70°.
Quantification of Total Cellular RNA
RNA was quantified on the STORMO system (Molecular Dynamics~) using the
RiboGreen~ RNA Quantification Kit (Molecular Probes~). Briefly, the RiboGreen
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CA 02474031 2004-07-22
WO 03/064416 PCT/CA03/00091
reagent was diluted 200-fold in TE (1 OmM Tris-HCI pH =7.5, 1 mM EDTA).
Generally, 50NL of reagent was diluted in lOmL TE. A Standard Curve of
ribosomal
RNA was diluted in TE to 2Ng/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 100NL with TE.
Generally, column 1 of the 96-well plate was used for the standard curve and
the
other wells are used for the RNA samples to be quantified. 10NL of each RNA
sample that was to be quantified, was transferred to the corresponding well of
the
96-well plate and 90NL of TE was added. One volume (100NL) of diluted
RiboGreen
1o 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 ~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 DynamicsO). 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
determined from the standard curve and corrected for the 20X dilution.
Reagents and Materials:
_._-_.-..__.~.-_ --__-_._.____._..~_.___.__._._______.___--
_._..............._~.....__..._____.__.......-.._
_ ~. Company Catalog __.._-___
Product # Storage
DEPC Sigma D5758 4C
EDTA Sigma E5134 RT
Trizma-Base Sigma T8524 RT
Trizma-HCI Sigma T7149 RT
Collection Tube Strips Qiagen 19562 RT
.
Ribogreen RNA QuantitationMolecular 811490 -20C
Kit Probe
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.
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CA 02474031 2004-07-22
WO 03/064416 PCT/CA03/00091
37: 327-332). The system exploits the 5'-3' nucleolytic activity of AmpIiTaq
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
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 OD28o. Considering that 1 Ng of this RNA = 2.15 X 10" RNA copies, dilutions
are
made in order to have 108, 10', 106, 105, 104, 103 or 102 genomic RNA copies /
5NL.
Total cellular Huh-7 RNA was also incorporated with each dilution (50ng /
5NL). 5NL
of each reference standard (HCV Replicon + Huh-7 RNA) was combined with 45NL
of Reagent Mix, and used in the Real-Time RT-PCR reaction.
3o The Real-Time RT-PCR reaction was set-up for the experimental samples that
were
purified on RNeasy 96 -well plates by combining 5N1 of each total cellular RNA
sample with 45NL of Reagent Mix.
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CA 02474031 2004-07-22
WO 03/064416 PCT/CA03/00091
Reagents and Materials:
_.__...___..._._._._._.__._.__._......_......_..____.___.~_......_......_..__..
......_._....._..._....._._._____.....-__-___......_..__.__._..........
Product _......_..__._..__.._._Catalog Storage
COMPANY #
TaqMan EZ RT-PCR PE Applied BiosystemsN808-0236-20C
Kit
MicroAmp Optical PE Applied BiosystemsN801-0935RT
Caps
MicroAmp Optical
96- PE Applied BiosystemsN801-0560RT
Well Reaction Plate
Reagent Mix preparation:
Volume Volume for One
Component for Plate Final
!, one sample(NL) (91 samples conc.
+
(NL)
Dead Volume)
Rnase-free water 16.5 1617
5X TaqMan EZ buffer10 980 1 X
Mn(OAc)2 (25mM) 6 588 3mM
dATP (lOmM) 1.5 147 300NM
dCTP (lOmM) 1.5 . 147 300NM
dGTP (lOmM) 1.5 147 300NM
dUTP (20mM) 1.5 147 600NM
Forward Primer 1 98 200nM
(10NM)
Reverse Primer 1 98 200nM
(10NM)
PUTR probe (5NM) 2 196 200nM
rTth DNA polymerase
2 196 0.1 U/N
(2.5 U/NL) L
AmpErase UNG
0.5 49 0.01 U/N
(1 U/NL) L
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 (SE('~ ID NO. 2): 5' - TCC CGG GGC ACT CGC AAG
CAC CCT ATC AGG - 3'
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CA 02474031 2004-07-22
WO 03/064416 PCT/CA03/00091
Note: Those primers amplify a region of 256-nt present within the 5'
untranslated
region of HCV.
PUTR Probe Sequence (SEA ID NO. 3): 6FAM - TGG TCT GCG GAA CCG
GTG AGT ACA CC - AMR
No Template Controls (NTC): On each plate, 4 wells are used as "NTC". For
these
controls, 5N1 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
for 2 cycles
60°C 1 min
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 / Ng of total RNA [ge/Ng].
The RNA copy number [g.e./Ng] 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.lNg inh)l( g. e.lNg ctl)x 100J.
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CA 02474031 2004-07-22
WO 03/064416 PCT/CA03/00091
A non-linear curve fit with the Hill model was applied to the inhibition-
concentration
data, and the 50% effective concentration (ECso) was calculated by the use of
SAS
software (Statistical Software System; SAS Institute, Inc. Cart', 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
specifically, the compounds had ICso's below 0.1 NM in the NS3-NS4A protease
assay, and ECso's below 0.5 NM in the cell based HCV RNA replication assay.
EXAMPLE 11
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.
EXAMPLE 12
Pharmacokinetic properties ,
The present compounds also show good pharmacokinetic properties such as
detectable plasma levels in the rat at 1 hour and 2h after an oral dose of 4
or
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
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CA 02474031 2004-07-22
WO 03/064416 PCT/CA03/00091
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 (Tween-80). The dosing
volume was l0ml/kg via oral gavage.
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 NM 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 NM standard.
Results
When assayed in the preceding screen the compounds of examples 1 to 8 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
0.83 NM and 0.75 NM respectively.
This demonstration of in vivo oral absorption for the tripeptide compounds of
this
invention in noteworthy, in view of the poor oral absortion generally
attributed to this
class of peptides. The ready oral absorption renders the compounds useful for
treating of HCV infection.
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CA 02474031 2004-07-22
WO 03/064416 PCT/CA03/00091
SE(~UENCE LISTING
<110> BOEHRINGER INGELHEIM INTERNATIONAL GmbH
<120> HEPATITIS C INHIBITOR TRI-PEPTIDES
<130> 13/107
<150> 2,369,970
<151> 2002-02-01
<160> 3
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Forward primer
<400> 1
acgcagaaag cgtctagcca tggcgttagt 30
<210> 2
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Reverse primer
<400> 2
tcccggggca ctcgcaagca ccctatcagg 30
<210> 3
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> PUTR probe
<400> 3
tggtctgcgg aaccggtgag tacacc 26
-1/1-
