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WO2012047764A1 - Peptides antiviraux thérapeutiques - Google Patents

Peptides antiviraux thérapeutiques Download PDF

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Publication number
WO2012047764A1
WO2012047764A1 PCT/US2011/054382 US2011054382W WO2012047764A1 WO 2012047764 A1 WO2012047764 A1 WO 2012047764A1 US 2011054382 W US2011054382 W US 2011054382W WO 2012047764 A1 WO2012047764 A1 WO 2012047764A1
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Prior art keywords
compound
optionally substituted
alkoxy
alkyl
individual
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Inventor
Brad Buckman
Vladimir Serebryany
John B. Nicholas
Scott Seiwert
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Intermune Inc
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Intermune Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to compounds, processes for their synthesis, compositions and methods for the treatment of hepatitis C virus (HCV) infection.
  • HCV hepatitis C virus
  • HCV infection is the most common chronic blood borne infection in the United States. Although the numbers of new infections have declined, the burden of chronic infection is substantial, with Centers for Disease Control estimates of 3.9 million (1.8%) infected persons in the United States.
  • Chronic liver disease is the tenth leading cause of death among adults in the United States, and accounts for approximately 25,000 deaths annually, or approximately 1% of all deaths. Studies indicate that 40% of chronic liver disease is HCV-related, resulting in an estimated 8,000-10,000 deaths each year. HCV-associated end-stage liver disease is the most frequent indication for liver transplantation among adults.
  • Antiviral therapy of chronic hepatitis C has evolved rapidly over the last decade, with significant improvements seen in the efficacy of treatment. Nevertheless, even with combination therapy using pegylated IFN-0C plus ribavirin, 40% to 50% of patients fail therapy, i.e., are nonresponders or relapsers. These patients currently have no effective therapeutic alternative. In particular, patients who have advanced fibrosis or cirrhosis on liver biopsy are at significant risk of developing complications of advanced liver disease, including ascites, jaundice, variceal bleeding, encephalopathy, and progressive liver failure, as well as a markedly increased risk of hepatocellular carcinoma.
  • 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 of the virus.
  • ORF open reading frame
  • NS structural and non-structural
  • the generation of mature nonstructural proteins (NS2, NS3, NS4, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases.
  • the first viral protease cleaves at the NS2-NS3 junction of the polyprotein.
  • the second viral protease is serine protease contained within the N-terminal region of NS3 (herein referred to as "NS3 protease").
  • NS3 protease mediates all of the subsequent cleavage events at sites downstream relative to the position of NS3 in the polyprotein (i.e., sites located between the C-terminus of NS3 and the C-terminus of the polyprotein).
  • NS3 protease exhibits activity both in cis, at the NS3-NS4 cleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites.
  • the NS4A protein is believed 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 formation of the complex between NS3 and NS4A is necessary for NS3-mediated processing events and enhances proteolytic efficiency at all sites recognized by NS3.
  • the NS3 protease also exhibits nucleoside triphosphatase and RNA helicase activities.
  • NS5B is an RNA-dependent RNA polymerase involved in the replication of HCV RNA.
  • compounds that inhibit the action of NS5A in viral replication are potentially useful for the treatment of HCV.
  • a dashed line represents the presence or absence of a bond X is NR X or O (oxygen); R x is H (hydrogen) or Ci_ 6 hydrocarbyl;
  • R 1 is , wherein Z is
  • Z' is NR 7 , CHR 7 , S (sulfur), or O (oxygen)
  • Y is N (nitrogen) or CR 14 ;
  • -C(0)OR a and aryl optionally substituted with one or more substituents each independently selected from the group consisting of halo, amino, C 1-6 alkyl optionally substituted with up to 5 fluoro, C 2 -6 alkenyl, C 2 _ 6 alkynyl, -C(0)NR 2b R 2c , -NHC(0)NR 2b R 2c , -C(0)OR 2d , heteroaryl, C 1-6 alkoxy optionally substituted with up to 5 fluoro, C 1-6 alkoxy optionally substituted by Ci_ 6 alkoxy, and Ci_ 6 alkoxy optionally substituted by -NR 12 R 13 ;
  • R 2a is selected from the group consisting of H (hydrogen), C 1-6 alkyl, cycloalkyl, and heterocyclyl;
  • R 2b and R 2c are taken together with the nitrogen to which they are attached to form piperazinyl or morpholinyl, each optionally substituted with one or more substituents independently selected from the groups consisting of optionally substituted Ci_ 6 alkyl, C 2 _ 6 alkenyl, C 2 _ 6 alkynyl, optionally substituted C 1-6 alkoxy, -C(0)OR 2d , -C(0)R 2e , optionally substituted aryl, and optionally substituted heteroaryl; and R 2d and R 2e are each separately selected from the group consisting of -H (hydrogen), C 1-4 alkoxy, C 1-6 alkyl, C3-7 cycloalkyl, aryl, arylalkyl and heteroaryl;
  • R 3 is H (hydrogen), -S0 2 R 9 , or -SO 2 NR 10 R u ;
  • R 4 is H (hydrogen) or C 1-4 alkyl;
  • R 5 is optionally substituted thiazole or -C(0)NR 12 R 13 ;
  • R 6 is H (hydrogen) or Ci_ 4 alkyl;
  • R 7 is C M2 hydrocarbyl, optionally substituted arylalkyl, optionally substituted aryl, or optionally substituted heteroaryl;
  • R 9 is H (hydrogen) or C 1-12 hydrocarbyl; and
  • R 10 , R 11 , R 12 , R 13 , and R 14 are independently selected from H (hydrogen) or C 1-12 hydrocarbyl; provided that the compound is not selected from the group consisting of:
  • aryl optionally substituted with one or more substituents each independently selected from the group consisting of halo, amino, C 1-6 alkyl optionally substituted with up to 5 fluoro, C 2 -6 alkenyl, C 2 _ 6 alkynyl, - C(0)NR 2b R 2c , -NHC(0)NR 2b R 2c , -C(0)OR 2d , heteroaryl, and Ci_ 6 alkoxy optionally substituted with up to 5 fluoro or optionally substituted with C 1-6 alkoxy.
  • the compound is selected from the group consisting of:
  • Some embodiments provide a pharmaceutical composition
  • a pharmaceutical composition comprising: a) a compound disclosed herein; and b) a pharmaceutically acceptable carrier or excipient.
  • Some embodiments provide a method of inhibiting NS3/NS4 protease activity comprising contacting a NS3/NS4 protease with a compound or a composition herein.
  • the contacting is conducted in vivo, such as wherein a sustained viral response is achieved.
  • the contacting is conducted ex vivo.
  • Some embodiments provide a method of treating liver fibrosis in an individual, the method comprising administering to the individual an effective amount of a compound or composition herein.
  • Some embodiments provide a method of increasing liver function in an individual having a hepatitis C virus infection, the method comprising administering to the individual an effective amount of a compound a composition herein.
  • Some embodiments provide a method of treating hepatitis by modulating NS3/NS4 protease comprising contacting a NS3/NS4 protease with a compound disclosed herein.
  • Some embodiments provide a method of treating a hepatitis C virus infection in an individual, the method comprising administering to the individual an effective amount of a composition comprising a compound disclosed herein.
  • the methods herein further comprise identifying a subject suffering from a hepatitis C infection and administering the compound to the subject in an amount effective to treat the infection.
  • the methods herein further comprises administering to the individual an effective amount of a nucleoside analog, such as ribavirin, levovirin, viramidine, an L-nucleoside, or isatoribine.
  • a nucleoside analog such as ribavirin, levovirin, viramidine, an L-nucleoside, or isatoribine.
  • the methods herein further comprise administering to the individual an effective amount of a human immunodeficiency virus 1 protease inhibitor, such as ritonavir.
  • a human immunodeficiency virus 1 protease inhibitor such as ritonavir.
  • the methods herein further comprise administering to the individual an effective amount of an NS5B RNA-dependent RNA polymerase inhibitor.
  • the methods herein further comprise administering to the individual an effective amount of an NS5A inhibitor.
  • the methods herein further comprise administering to the individual an effective amount of interferon-gamma (IFN- ⁇ ), such as wherein the IFN- ⁇ is administered subcutaneously in an amount of from about 10 ⁇ g to about 300 ⁇ g.
  • IFN- ⁇ interferon-gamma
  • the methods herein further comprise administering to the individual an effective amount of interferon-alpha (IFN-Oc), such as monoPEG-ylated consensus IFN-OC administered at a dosing interval of every 8 days to every 14 days, for example once every 7 days.
  • IFN-Oc interferon-alpha
  • the IFN-OC is INFERGEN consensus IFN-OC.
  • the methods herein further comprise administering an effective amount of an agent selected from 3'-azidothymidine, 2',3'-dideoxyinosine, 2',3'- dideoxycytidine, 2-,3-didehydro-2',3'-dideoxythymidine, combivir, abacavir, adefovir dipoxil, cidofovir, and an inosine monophosphate dehydrogenase inhibitor.
  • an agent selected from 3'-azidothymidine, 2',3'-dideoxyinosine, 2',3'- dideoxycytidine, 2-,3-didehydro-2',3'-dideoxythymidine, combivir, abacavir, adefovir dipoxil, cidofovir, and an inosine monophosphate dehydrogenase inhibitor.
  • the terms "individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, murines, primates, including simians and humans, mammalian farm animals, mammalian sport animals, and mammalian pets.
  • liver function refers to a normal function of the liver, including, but not limited to, a synthetic function, including, but not limited to, synthesis of proteins such as serum proteins (e.g., albumin, clotting factors, alkaline phosphatase, aminotransferases (e.g., alanine transaminase, aspartate transaminase), 5'- nucleosidase, ⁇ -glutaminyltranspeptidase, etc.), synthesis of bilirubin, synthesis of cholesterol, and synthesis of bile acids; a liver metabolic function, including, but not limited to, carbohydrate metabolism, amino acid and ammonia metabolism, hormone metabolism, and lipid metabolism; detoxification of exogenous drugs; a hemodynamic function, including splanchnic and portal hemodynamics; and the like.
  • serum proteins e.g., albumin, clotting factors, alkaline phosphatase, aminotransferases (e.g., alanine transa
  • sustained viral response refers to the response of an individual to a treatment regimen for HCV infection, in terms of serum HCV titer.
  • a sustained viral response refers to no detectable HCV RNA (e.g., less than about 500, less than about 200, or less than about 100 genome copies per milliliter serum) found in the patient's serum for a period of at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, or at least about six months following cessation of treatment.
  • Treatment failure patients generally refers to HCV- infected patients who failed to respond to previous therapy for HCV (referred to as “non- responders") or who initially responded to previous therapy, but in whom the therapeutic response was not maintained (referred to as “relapsers").
  • the previous therapy generally can include treatment with IFN-OC monotherapy or IFN-0C combination therapy, where the combination therapy may include administration of IFN-OC and an antiviral agent such as ribavirin.
  • Treating refers to the use of a compound, composition, therapeutically active agent, or drug in the diagnosis, cure, mitigation, treatment, or prevention of disease or other undesirable condition in a mammal; or the use of a compound, composition, therapeutically active agent, or drug in a manner intended to affect the structure or any function of the body of a mammal.
  • Asymmetric carbon atoms may be present in the compounds described. All such stereoisomers, both in a pure form or as a mixture of isomers, are intended to be included in the scope of a recited compound. In certain cases, compounds can exist in tautomeric forms. All tautomeric forms are intended to be included in the scope.
  • Alternate forms including alternate solid forms, are included in the embodiments.
  • Alternate solid forms such as polymorphs, solvates, hydrates, and the like, are alternate forms of a chemical entity that involve at least one of: differences in solid packing arrangements, non-covalent interactions with another compound such as water or a solvent.
  • Salts involve at least one ionic interaction between an ionic form of a chemical entity of interest and a counter-ion bearing an opposite charge.
  • Salts of compounds can be prepared by methods known to those skilled in the art. For example, salts of compounds can be prepared by reacting the appropriate base or acid with a stoichiometric equivalent of the compound.
  • a prodrug is a compound that undergoes biotransformation (chemical conversion) to a parent compound (such as a compound described herein) in the body of an animal.
  • a parent compound such as a compound described herein
  • pharmaceutically acceptable salt refers to any pharmaceutically acceptable salts of a compound, and preferably refers to an acid addition salt of a compound.
  • pharmaceutically acceptable salts are acid addition salts of pharmaceutically acceptable inorganic or organic acids, for example, hydrohalic, sulfuric, phosphoric acid or aliphatic or aromatic carboxylic or sulfonic acid.
  • Examples of pharmaceutically acceptable inorganic or organic acids as a component of an addition salt include but are not limited to, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid acetic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbi acid c, nicotinic acid, methanesulfonic acid, p-toluensulfonic acid or naphthalenesulfonic acid acid.
  • the preferred examples of pharmaceutically acceptable salts include, but are not limited to, alkali metal salts (sodium or potassium), alkaline earth metal salts (calcium or magnesium), or ammonium salts derived from ammonia or from pharmaceutically acceptable organic amines, for example C1-C7 alkylamine, cyclohexylamine, triethanolamine, ethylenediamine or tris-(hydroxymethyl)-aminomethane.
  • Isotopes may be present in the compounds described. Each chemical element as represented in a compound structure may include any isotope of said element.
  • a hydrogen atom may be explicitely disclosed or understood to be present in the compound.
  • the hydrogen atom can be any isotope of hydrogen, including but not limited to hydrogen- 1 (protium) and hydrogen-2 (deuterium).
  • reference herein to a compound encompasses all potential isotopic forms unless the context clearly dictates otherwise.
  • substituted refers to a moiety that replaces one or more hydrogen atoms of the parent group for which it is a substituent.
  • a substituent consists of from 0-10 carbon atoms, from 0-26 hydrogen atoms, from 0-5 oxygen atoms, from 0-5 nitrogen atoms, from 0-5 sulfur atoms, from 0-7 fluorine atoms, from 0-3 chlorine atoms, from 0-3 bromine atoms, and/or from 0-3 iodine atoms.
  • a substituent may comprise at least one carbon atom or one heteroatom selected from N, O, S, P, F, CI, Br, and I, and may comprise 0-12 carbon atom, 0- 6 carbon atoms, or 0-3 carbon atoms, and 0-12 heteroatoms, 0-6 heteroatoms, 0-3 heteroatoms, or 1 heteroatom.
  • Ci-C 6 alkyl examples include Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 6 cycloalkyl, C 3 -C 6 heterocycloalkyl (e.g., tetrahydrofuryl), aryl, heteroaryl, halo (e.g., chloro, bromo, iodo and fluoro), cyano, hydroxy, Ci-C 6 alkoxy, aryloxy, sulfhydryl (mercapto), Ci-C 6 alkylthio, arylthio, mono- and di-(Ci-C 6 )alkyl amino, quaternary ammonium salts, amino(Ci-C 6 )alkoxy, hydroxy(Ci-C 6 )alkylamino, amino(Ci-C 6 )alkylthio, cyanoamino, nitro, carbamyl, keto (oxo
  • a compound of formula (I) that is "optionally substituted” leaves the ability of the compound of formula (I) to inhibit NS3/NS4 protease activity qualitatively intact and/or other activities disclosed herein intact.
  • the substituent may alter the degree of inhibition of NS3/NS4 protease activity or other activities dislcclosed herein.
  • aryl refers to an aromatic ring or aromatic ring system such as phenyl, naphthyl, biphenyl, and the like.
  • heteroaryl refers to an aromatic ring or aromatic ring system having one or more oxygen atom, nitrogen atom, sulfur atom, or a combination thereof, which are part of the ring or ring system.
  • examples include thienyl, furyl, pyridinyl, quinolinyl, thiazolyl, benzooxazolyl, benzothiazolyl, benzoimidazolyl, benzothiazolyl, benzothienyl, benzofuryl, pyridinyl, imidazolyl, thiazolyl, oxazolyl, and the like.
  • fused bicyclic heteroaryl refers to heteroaryl having a ring system of two rings, wherein two adjacent ring atoms are shared by both rings of the system.
  • Preferred monocyclic ring systems are of 4, 5, 6, 7, or 8 members.
  • Six membered monocyclic rings preferably contain from one to three heteroatoms wherein each heteroatom is individually selected from oxygen, sulfur, and nitrogen.
  • Five-membered rings preferably have one or two heteroatoms wherein each heteroatom is individually selected from oxygen, sulfur, and nitrogen. Examples include, but are not limited to, quinolinyl, benzooxazolyl, benzothiazolyl, benzoimidazolyl, benzothiazolyl, benzothienyl, benzofuryl, and the like
  • heterocyclic or “heterocyclyl” or “heterocycloalkyl” used herein refers to cyclic non-aromatic ring system radical having at least one ring in which one or more ring atoms are not carbon, namely heteroatom.
  • the heteroatoms are independently selected from oxygen, sulfur, and nitrogen.
  • fused ring systems the one or more heteroatoms may be present in only one of the rings and each ring in the fused system is non- aromatic.
  • Preferred monocyclic ring systems are of 4, 5, 6, 7, or 8 members.
  • Six membered monocyclic rings preferably contain from one to three heteroatoms wherein each heteroatom is individually selected from oxygen, sulfur, and nitrogen.
  • Five-membered rings preferably have one or two heteroatoms wherein each heteroatom is individually selected from oxygen, sulfur, and nitrogen.
  • heterocyclic groups include, but are not limited to, morpholinyl, tetrahydrofuranyl, dioxolanyl, pyrolidinyl, pyranyl, piperidyl, piperazyl, and the like.
  • hydrocarbyl refers to a hydrocarbon moiety.
  • alkyl refers to a hydrocarbon moiety which has no double or triple bonds, such as methyl, ethyl, propyl, cyclopropyl, etc.
  • alkyl, alkenyl and alkynyl include straight- and branched-chain and cyclic monovalent substituents. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and the like.
  • the alkyl, alkenyl and alkynyl substituents contain Ci-Cio (alkyl) or C 2 -Cio (alkenyl or alkynyl).
  • they contain Ci-C 6 (alkyl), C 2 -C 6 (alkenyl or alkynyl), or C3-C6 cycloalkyl.
  • alkyl refers to a radical of a fully saturated hydrocarbon, including, but not limited to, methyl, ethyl, n-propyl, isopropyl (or i-propyl), n-
  • fully saturated hydrocarbons defined by the following general formula's: the general formula for linear or branched fully saturated hydrocarbons not containing a cyclic structure is C n H2n+2; the general formula for a fully saturated hydrocarbon containing one ring is C n H2 n ; the general formula for a fully saturated hydrocarbon containing two rings is C n H 2(n _i ) ; the general formula for a saturated hydrocarbon containing three rings is C n H 2 ( n -2).
  • alkyl such as propyl, butyl, etc.
  • alkenyl used herein refers to a monovalent straight or branched chain radical of from two to twenty carbon atoms containing a carbon double bond including, but not limited to, 1-propenyl, 2-propenyl, 2-methyl-l-propenyl, 1-butenyl, 2-butenyl, and the like.
  • alkynyl used herein refers to a monovalent straight or branched chain radical of from two to twenty carbon atoms containing a carbon triple bond including, but not limited to, 1-propynyl, 1-butynyl, 2-butynyl, and the like.
  • arylalkyl refers to one or more aryl groups appended to an alkyl radical.
  • arylalkyl groups include, but are not limited to, benzyl, phenethyl, phenpropyl, phenbutyl, and the like.
  • alkoxy used herein refers to straight or branched chain alkyl radical covalently bonded to the parent molecule through an— O— linkage.
  • alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, n-butoxy, sec -butoxy, t-butoxy and the like.
  • aryloxy refers to an aryl radical covalently bonded to the parent molecule through an— O— linkage.
  • alkylthio refers to straight or branched chain alkyl radical covalently bonded to the parent molecule through an— S— linkage.
  • arylthio refers to an aryl radical covalently bonded to the parent molecule through an— S— linkage.
  • alkylamino refers to nitrogen radical with one or more alkyl groups attached thereto.
  • monoalkylamino refers to nitrogen radical with one alkyl group attached thereto and dialkylamino refers to nitrogen radical with two alkyl groups attached thereto.
  • R refers to a substituent selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl and heterocycle.
  • halo refers to fluoro, chloro, bromo, or iodo.
  • cyanoamino used herein refers to nitrogen radical with nitrile group attached thereto.
  • sulfamyl used herein refers to -S0 2 NH 2 .
  • thiocarboxy used herein refers to CSOH.
  • a radical indicates species with a single, unpaired electron such that the species containing the radical can be covalently bonded to another species.
  • a radical is not necessarily a free radical. Rather, a radical indicates a specific portion of a larger molecule.
  • the term "radical” can be used interchangeably with the term "group.”
  • a substituted group is derived from the unsubstituted parent structure in which there has been an exchange of one or more hydrogen atoms for another atom or group.
  • variable groups of the compound of Formula I are discussed below in more detail:
  • R can be selected from the group consisting of optionally substituted heteroaryl, -C(0)OR a , and aryl optionally substituted with one or more substituents each independently selected from the group consisting of halo, amino, Ci_ 6 alkyl optionally substituted with up to 5 fluoro, C 2 -6 alkenyl, C 2 _ 6 alkynyl, -C(0)NR 2b R 2c , -NHC(0)NR 2b R 2c , -C(0)OR 2d , heteroaryl, Ci_ 6 alkoxy optionally substituted with up to 5 fluoro, Ci-6 alkoxy optionally substituted by C 1-6 alkoxy, and C 1-6 alkoxy optionally substituted by
  • R 2 may be selected from the group consisting of optionally substituted heteroaryl, -C(0)OR 2a , and aryl optionally substituted with one or more substituents each independently selected from the group consisting of halo, amino, C 1-6
  • R 2a can be selected from the group consisting of H (hydrogen), Ci_ 6 alkyl, t-butyl, cycloalkyl, and heterocyclyl;
  • R 2b and R 2c can be taken together with the nitrogen to which they are attached to form piperazinyl or morpholinyl, each optionally substituted with one or more substituents independently selected from the groups consisting of optionally substituted C 1-6 alkyl, C 2 _ 6 alkenyl, C 2 _ 6 alkynyl, optionally substituted Ci_ 6 alkoxy, -C(0)OR 2d , -C(0)R 2e , optionally substituted aryl, and optionally substituted heteroaryl; and R 2d and R 2e can each be separately selected from the group consisting of -H (hydrogen), Ci_ alkoxy, Ci_ 6 alk l, C 3 _ 7 cycloalkyl,
  • R may be , or -C(0)OiBu, wherein m is an integer between 1 and 6, and n is an integer between 0-5.
  • R 2 is -C(0)OR 2a or aryl optionally substituted with one or more substituents each independently selected from the group consisting of C 2 -6 alkenyl, C 2 _ 6 alkynyl, -C(0)NR 2b R 2c , -C(0)OR 2d , heteroaryl, and Ci_ 6 alkoxy optionally substituted by C 1-6 alkoxy.
  • R is phenyl substituted with 1 to 3 substituents independently selected from the group consisting of: heteroaryl, -C(0)NR 2b R 2c , or Ci-6 alkoxy optionally substituted by C 1-6 alkoxy; wherein in some embodiments, R 2b and R 2c are taken together with the nitrogen to which they are attached to form piperazinyl, wherein piperazinyl is optionally substituted with C 1-6 alkyl or -C(0)OR 2d ; wherein R 2d is
  • R 4 may be H (hydrogen) or C 1-4 alkyl, such as a C 3-
  • R may be iBu
  • R 1 may b wherein Z may be
  • R 1 may be wherein Z' may be NR 7 ,
  • Y may be N (nitrogen) or CR 14 .
  • Y may be N (nitrogen) and Z may be NR 6 .
  • Y may be N (nitrogen) and Z' may be NR 7 .
  • R 1 may be
  • R may be linked to the remainder of the molecule through any available position on the 6-membered ring.
  • R may be C 1-12 hydrocarbyl, optionally substituted arylalkyl, optionally substituted aryl, or optionally substituted heteroaryl, such as an optionally substituted 5 or 6 membered aryl or heteroaryl. In some aspects, R is optionally
  • R may be an unsubstituted aryl or heteroaryl. In some aspects, R may be an unsubstituted aryl, such as unsubstituted phenyl.
  • R 5 may be optionally substituted thiazole or -C(0)NR 12 R 13 .
  • R 5 is optionally substituted thiazole, wherein the thiazole is unsubstituted or substituted with an alkyl, alkenyl or alkynyl such as a CrC 4 alkyl or C 2 -C4 alkenyl or alkynyl group, such as C3 or C 4 alkyl or alkenyl.
  • R 5 may be optionally substituted thiazole or -C(0)NR 12 R 13 .
  • R 5 is optionally substituted thiazole, wherein the thiazole is unsubstituted or substituted with an alkyl, alkenyl or alkynyl such as a CrC 4 alkyl or C 2 -C4 alkenyl or alkynyl group, such as C3 or C 4 alkyl or alkenyl.
  • R 5 may be optionally substituted thiazole or -C(0)NR 12 R
  • R , R , and R may be independently selected from H (hydrogen) or C 1-12 hydrocarbyl.
  • C 1-12 hydrocarbyl may be alkyl, alkenyl or alkynyl, for instance C 1 -C4 alkyl or C 2 -C 4 alkeneyl or alkynyl group, such as C 3 or C 4 alkyl or alkenyl.
  • one of either R or R" may be H (hydrogen).
  • One of either R or R" may be H (hydrogen).
  • alkenyl for instance where the point of unsaturation is between the terminal
  • R 5 may be
  • X may be NR X or O (oxygen).
  • R x may be H (hydrogen) or Ci_ 6 hydrocarbyl, such as Ci_ 4 alkyl, for example methyl.
  • R x may be H (hydrogen).
  • X is NH or X is O (oxygen).
  • R may be H (hydrogen), -S0 2 R , or - SO 2 NR 10 R u
  • R 3 may be H (hydrogen).
  • R 3 may be -S0 2 R 9 wherein R 9 may be H (hydrogen) or C 1-12 hydrocarbyl.
  • R 9 is C 1-12 hydrocarbyl
  • C 1-12 hydrocarbyl may be C 1-12 alkyl, or C 2 _i 2 alkenyl or alkynyl, such as Q-Q alkyl or C 2 -C 8 alkenyl or alkynyl group, or C 4 -C 8 alkyl or alkenyl.
  • alkyl, alkenyl or alkynyl such as C 4 - C 8 alkyl or alkenyl may contain a cycloalkyl group such as cyclopropyl.
  • R 9 may contain alkenyl, for instance where the point of etween the terminal carbon and the adjacent carbon thereto. In some as ects,
  • R 3 may be -SO 2 NR 10 R n wherein R 10 and R 11 are independently selected from H (hydrogen) or C 1-12 hydrocarbyl. In some embodiments, one of either R 10 or R 11 may be H (hydrogen). In some embodiments, R 10 and R 11 are each independently C 1-6 alkyl. In some embodiment R 10 and R 11 are each methyl. In some embodiments, X may be NR X , and R 3 may be -S0 2 R 9 .
  • groups indicated as “optionally substituted” can be optionally substituted with one or more group(s) individually and independently selected from Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 6 cycloalkyl, C 3 -C 6 heterocycloalkyl, aryl, heteroaryl, halo, cyano, hydroxy, Cp C 6 alkoxy, aryloxy, sulfhydryl, Ci-C 6 alkylthio, arylthio, mono- and di-(Ci-C 6 )alkyl amino, quaternary ammonium salts, amino(Ci-C 6 )alkoxy, hydroxy(Ci-C 6 )alkylamino, amino(Cr C 6 )alkylthio, cyanoamino, nitro, carbamyl, oxo, carbonyl
  • the compound is selected from the group consisting of:
  • the compound is not selected from:
  • the present embodiments provide for a method of inhibiting NS3/NS4 protease activity comprising contacting a NS3/NS4 protease with a compound disclosed herein.
  • the present embodiments provide for a method of treating hepatitis by modulating NS3/NS4 protease comprising contacting a NS3/NS4 protease with a compound disclosed herein.
  • the present embodiments provide a method of treating liver fibrosis in an individual, the method comprising administering to the individual a compound disclosed herein.
  • the present embodiments provide a method of increasing liver function in an individual having a hepatitis C virus infection, the method comprising administering to the individual a compound disclosed herein.
  • the present embodiments provide use of the compound or a composition disclosed herein for the manufacture of a medicament for treating HCV infection in an individual.
  • the present embodiments provide use of the compound or a composition disclosed herein for the manufacture of a medicament for treating liver fibrosis in an individual.
  • the present embodiments provide use of the compound or a composition disclosed herein for the manufacture of a medicament for increasing liver function in an individual having a hepatitis C virus infection.
  • the present embodiments provide a compound or a composition disclosed herein for use in treating HCV infection in an individual.
  • the present embodiments provide a compound or a composition disclosed herein for use in treating liver fibrosis in an individual.
  • the present embodiments provide a compound or a composition disclosed herein for use in increasing liver function in an individual having a hepatitis C virus infection.
  • compositions comprising compounds disclosed herein.
  • a subject pharmaceutical composition comprises a subject compound; and a pharmaceutically acceptable excipient.
  • a wide variety of pharmaceutically acceptable excipients is known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C.
  • compositions such as vehicles, adjuvants, carriers or diluents
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • Preferred embodiments provide for a method of inhibiting NS3/NS4 protease activity comprising contacting a NS3/NS4 protease with an effective amount of a composition comprising a compound disclosed herein.
  • Preferred embodiments provide a method of treating a hepatitis C virus infection in an individual, the method comprising administering to the individual an effective amount of a composition comprising a compound disclosed herein.
  • Preferred embodiments provide a method of treating liver fibrosis in an individual, the method comprising administering to the individual an effective amount of a composition comprising a compound disclosed herein.
  • Preferred embodiments provide a method of increasing liver function in an individual having a hepatitis C virus infection, the method comprising administering to the individual an effective amount of a composition comprising a compound disclosed herein.
  • a subject compound may inhibit the enzymatic activity of a hepatitis virus C (HCV) NS3 protease. Whether a subject compound inhibits HCV NS3 protease can be readily determined using any known method. Typical methods may involve a determination of whether an HCV polyprotein or other polypeptide comprising an NS3 recognition site is cleaved by NS3 in the presence of the agent.
  • HCV hepatitis virus C
  • a subject compound inhibits NS3 enzymatic activity by a detectable amount, such as at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, compared to the enzymatic activity of NS3 in the absence of the compound.
  • a subject compound may inhibit enzymatic activity of an HCV NS3 protease with an IC 50 of less than about 50 ⁇ , e.g., a subject compound inhibits an HCV NS3 protease with an IC 50 of less than about 40 ⁇ , less than about 25 ⁇ , less than about 10 ⁇ , less than about 1 ⁇ , less than about 100 nM, less than about 80 nM, less than about 60 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, or less than about 1 nM, or less.
  • a subject compound may inhibit the enzymatic activity of a hepatitis virus C (HCV) NS3 helicase. Whether a subject compound inhibits HCV NS3 helicase can be readily determined using any known method. In many embodiments, a subject compound inhibits NS3 enzymatic activity by a detectable amount, such as at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, compared to the enzymatic activity of NS3 in the absence of the compound.
  • HCV hepatitis virus C
  • a subject compound may inhibit HCV viral replication.
  • a subject compound may inhibit HCV viral replication by a detectable amount, such as at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, compared to HCV viral replication in the absence of the compound.
  • Whether a subject compound inhibits HCV viral replication can be determined using methods known in the art, including an in vitro viral replication assay.
  • compositions described herein may be generally useful in treatment of an of HCV infection.
  • Whether a subject method is effective in treating an HCV infection may be determined by a reduction in viral load, a reduction in time to seroconversion (virus undetectable in patient serum), an increase in the rate of sustained viral response to therapy, a reduction of morbidity or mortality in clinical outcomes, or other indicator of disease response.
  • an effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents is an amount that is effective to produce a detectable effect described herein, such as to reduce viral load or achieve a sustained viral response to therapy.
  • Whether a subject method is effective in treating an HCV infection may be determined by directly or indirectly observing or measuring any effect or parameter which may be associated with the effective treatment of HCV infection such as, but not limited to, measuring viral load, or by measuring a parameter associated with HCV infection, including, but not limited to, liver fibrosis, elevations in serum transaminase levels, and necroinflammatory activity in the liver. Indicators of liver fibrosis are discussed in detail below.
  • an effective amount of a compound disclosed herein optionally in combination with an effective amount of one or more additional antiviral agents.
  • an effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents may be an amount that is effective to reduce viral titers to undetectable levels, e.g., to about 1000 to about 5000, to about 500 to about 1000, or to about 100 to about 500 genome copies/mL serum.
  • an effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents may be an amount that is effective to reduce viral load to lower than 100 genome copies/mL serum.
  • an effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents may be an amount that is effective to achieve a 1.5-log, a 2-log, a 2.5-log, a 3-log, a 3.5-log, a 4-log, a 4.5-log, or a 5- log reduction in viral titer in the serum of the individual.
  • an effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents may be an amount that is effective to achieve a sustained viral response, e.g., non-detectable or substantially non- detectable HCV RNA (e.g., less than about 500, less than about 400, less than about 200, or less than about 100 genome copies per milliliter serum) is found in the patient's serum for a period of at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, or at least about six months following cessation of therapy.
  • a sustained viral response e.g., non-detectable or substantially non- detectable HCV RNA (e.g., less than about 500, less than about 400, less than about 200, or less than about 100 genome copies per milliliter serum) is found in the patient's serum for a period of at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, or
  • liver fibrosis As noted above, whether a subject method is effective in treating an HCV infection can be determined by measuring a parameter associated with HCV infection, such as liver fibrosis. Methods of determining the extent of liver fibrosis are discussed in detail below. In some embodiments, the level of a serum marker of liver fibrosis indicates the degree of liver fibrosis.
  • ALT serum alanine aminotransferase
  • an effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents is an amount effective to reduce ALT levels to less than about 45 IU/mL serum.
  • a therapeutically effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents may be an amount that is effective to reduce a serum level of a marker of liver fibrosis by a detectable amount, such as at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, or more, compared to the level of the marker in an untreated individual, or to a placebo-treated individual.
  • Methods of measuring serum markers include immunological-based methods, e.g., enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, and the like, using antibody specific for a given serum marker.
  • an effective amount of a compound disclosed herein and an additional antiviral agent may be a synergistic amount.
  • a "synergistic combination" or a “synergistic amount” of a compound disclosed herein and an additional antiviral agent is a combined dosage that is more effective in the therapeutic or prophylactic treatment of an HCV infection than the incremental improvement in treatment outcome that could be predicted or expected from a merely additive combination of (i) the therapeutic or prophylactic benefit of a compound disclosed herein when administered at that same dosage as a monotherapy and (ii) the therapeutic or prophylactic benefit of the additional antiviral agent when administered at the same dosage as a monotherapy.
  • liver fibrosis including forms of liver fibrosis resulting from, or associated with, HCV infection
  • methods for treating liver fibrosis generally involving administering a therapeutic amount of a compound disclosed herein, and optionally one or more additional antiviral agents.
  • Effective amounts of compounds disclosed herein, with and without one or more additional antiviral agents, as well as dosing regimens, are as discussed below.
  • liver fibrosis reduction may be determined by analyzing a liver biopsy sample.
  • An analysis of a liver biopsy comprises assessments of two major components: necroinflammation assessed by "grade” as a measure of the severity and ongoing disease activity, and the lesions of fibrosis and parenchymal or vascular remodeling as assessed by "stage” as being reflective of long-term disease progression. See, e.g., Brunt (2000) Hepatol. 31:241-246; and METAVIR (1994) Hepatology 20:15-20.
  • a score may be assigned.
  • the METAVIR scoring system is based on an analysis of various features of a liver biopsy, including fibrosis (portal fibrosis, centrilobular fibrosis, and cirrhosis); necrosis (piecemeal and lobular necrosis, acidophilic retraction, and ballooning degeneration); inflammation (portal tract inflammation, portal lymphoid aggregates, and distribution of portal inflammation); bile duct changes; and the Knodell index (scores of periportal necrosis, lobular necrosis, portal inflammation, fibrosis, and overall disease activity).
  • each stage in the METAVIR system is as follows: score: 0, no fibrosis; score: 1, stellate enlargement of portal tract but without septa formation; score: 2, enlargement of portal tract with rare septa formation; score: 3, numerous septa without cirrhosis; and score: 4, cirrhosis.
  • Knodell's scoring system also called the Hepatitis Activity Index, classifies specimens based on scores in four categories of histologic features: I. Periportal and/or bridging necrosis; II. Intralobular degeneration and focal necrosis; ⁇ . Portal inflammation; and ⁇ . Fibrosis.
  • scores are as follows: score: 0, no fibrosis; score: 1, mild fibrosis (fibrous portal expansion); score: 2, moderate fibrosis; score: 3, severe fibrosis (bridging fibrosis); and score: 4, cirrhosis. The higher the score, the more severe the liver tissue damage.
  • the Ishak scoring system is described in Ishak (1995) J. Hepatol. 22:696- 699. Stage 0, No fibrosis; Stage 1, Fibrous expansion of some portal areas, with or without short fibrous septa; stage 2, Fibrous expansion of most portal areas, with or without short fibrous septa; stage 3, Fibrous expansion of most portal areas with occasional portal to portal (P-P) bridging; stage 4, Fibrous expansion of portal areas with marked bridging (P-P) as well as portal-central (P-C); stage 5, Marked bridging (P-P and/or P-C) with occasional nodules (incomplete cirrhosis); stage 6, Cirrhosis, probable or definite.
  • the benefit of anti-fibrotic therapy can also be measured and assessed by using the Child-Pugh scoring system which comprises a multicomponent point system based upon abnormalities in serum bilirubin level, serum albumin level, prothrombin time, the presence and severity of ascites, and the presence and severity of encephalopathy. Based upon the presence and severity of abnormality of these parameters, patients may be placed in one of three categories of increasing severity of clinical disease: A, B, or C.
  • a therapeutically effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents may be an amount that effects a change of one unit or more in the fibrosis stage based on pre- and post-therapy liver biopsies.
  • a therapeutically effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents reduces liver fibrosis by at least one unit in the METAVIR, the Knodell, the Scheuer, the Ludwig, or the Ishak scoring system.
  • Secondary, or indirect, indices of liver function can also be used to evaluate the efficacy of treatment with a compound disclosed herein. Morphometric computerized semi- automated assessment of the quantitative degree of liver fibrosis based upon specific staining of collagen and/or serum markers of liver fibrosis can also be measured as an indication of the efficacy of a subject treatment method. Secondary indices of liver function include, but are not limited to, serum transaminase levels, prothrombin time, bilirubin, platelet count, portal pressure, albumin level, and assessment of the Child-Pugh score.
  • An effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents may be an amount that is effective to increase an index of liver function by a detectable amount, such as at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, or more, compared to the index of liver function in an untreated individual, or to a placebo-treated individual.
  • a detectable amount such as at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, or more, compared to the index of liver function in an untreated individual, or to
  • Serum markers of liver fibrosis can also be measured as an indication of the efficacy of a subject treatment method.
  • Serum markers of liver fibrosis include, but are not limited to, hyaluronate, N-terminal procollagen ⁇ peptide, 7S domain of type IV collagen, C-terminal procollagen I peptide, and laminin.
  • Additional biochemical markers of liver fibrosis include ⁇ -2-macroglobulin, haptoglobin, gamma globulin, apolipoprotein A, and gamma glutamyl transpeptidase.
  • a therapeutically effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents may be an amount that is effective to reduce a serum level of a marker of liver fibrosis by a detectable amount, such as at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, or more, compared to the level of the marker in an untreated individual, or to a placebo-treated individual.
  • a detectable amount such as at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, or more, compared to the level of
  • ELISA enzyme-linked immunosorbent assays
  • radioimmunoassays radioimmunoassays
  • a "complication associated with cirrhosis of the liver” refers to a disorder that is a sequellae of decompensated liver disease, i.e., or occurs subsequently to and as a result of development of liver fibrosis, and includes, but it not limited to, development of ascites, variceal bleeding, portal hypertension, jaundice, progressive liver insufficiency, encephalopathy, hepatocellular carcinoma, liver failure requiring liver transplantation, and liver-related mortality.
  • a therapeutically effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents may be an amount that is effective in reducing the incidence (e.g., the likelihood that an individual will develop) of a disorder associated with cirrhosis of the liver by a detectable amount, such as at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, or more, compared to an untreated individual, or to a placebo-treated individual.
  • a detectable amount such as at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about
  • liver function increases liver function.
  • Liver functions include, but are not limited to, synthesis of proteins such as serum proteins (e.g., albumin, clotting factors, alkaline phosphatase, aminotransferases (e.g., alanine transaminase, aspartate transaminase), 5 '-nucleosidase, ⁇ -glutaminyltranspeptidase, etc.), synthesis of bilirubin, synthesis of cholesterol, and synthesis of bile acids; a liver metabolic function, including, but not limited to, carbohydrate metabolism, amino acid and ammonia metabolism, hormone metabolism, and lipid metabolism; detoxification of exogenous drugs; a hemodynamic function, including splanchnic and portal hemodynamics; and the like.
  • proteins such as serum proteins (e.g., albumin, clotting factors, alkaline phosphatase, aminotransferases (e.g., alanine transaminase, aspartate
  • liver function is increased is readily ascertainable by those skilled in the art, using well-established tests of liver function.
  • markers of liver function such as albumin, alkaline phosphatase, alanine transaminase, aspartate transaminase, bilirubin, and the like, can be assessed by measuring the level of these markers in the serum, using standard immunological and enzymatic assays.
  • Splanchnic circulation and portal hemodynamics can be measured by portal wedge pressure and/or resistance using standard methods.
  • Metabolic functions can be measured by measuring the level of ammonia in the serum.
  • Whether serum proteins normally secreted by the liver are in the normal range can be determined by measuring the levels of such proteins, using standard immunological and enzymatic assays. Those skilled in the art know the normal ranges for such serum proteins. The following are non-limiting examples.
  • the normal level of alanine transaminase is about 45 IU per milliliter of serum.
  • the normal range of aspartate transaminase is from about 5 to about 40 units per liter of serum.
  • Bilirubin is measured using standard assays. Normal bilirubin levels are usually less than about 1.2 mg/dL.
  • Serum albumin levels are measured using standard assays. Normal levels of serum albumin are in the range of from about 35 to about 55 g/L.
  • Prolongation of prothrombin time is measured using standard assays. Normal prothrombin time is less than about 4 seconds longer than control.
  • a therapeutically effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents may be an amount that is effective to increase liver function by a detectable amount, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more.
  • a therapeutically effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents is an amount effective to reduce an elevated level of a serum marker of liver function by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more, or to reduce the level of the serum marker of liver function to within a normal range.
  • a therapeutically effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents is also an amount effective to increase a reduced level of a serum marker of liver function by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more, or to increase the level of the serum marker of liver function to within a normal range.
  • the active agent(s) may be administered to the host using any convenient means capable of resulting in the desired therapeutic effect.
  • the agent may be incorporated into a variety of formulations for therapeutic administration. More particularly, the agents of the embodiments can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
  • a subject method may in some embodiments be carried out by administering a compound disclosed herein, and optionally one or more additional antiviral agent(s).
  • the method further may include administration of one or more interferon receptor agonist(s).
  • the method may further include administration of pirfenidone or a pirfenidone analog.
  • Additional antiviral agents may include, but are not limited to, nucleotide and nucleoside analogs.
  • Non-limiting examples include azidothymidine (AZT) (zidovudine), and analogs and derivatives thereof; 2',3'-dideoxyinosine (DDI) (didanosine), and analogs and derivatives thereof; 2',3'- dideoxycytidine (DDC) (dideoxycytidine), and analogs and derivatives thereof; 2',3'- didehydro-2',3'-dideoxythymidine (D4T) (stavudine), and analogs and derivatives thereof; combivir; abacavir; adefovir dipoxil; cidofovir; ribavirin; ribavirin analogs; and the like.
  • the method may further include administration of ribavirin.
  • Ribavirin, l- -D-ribofuranosyl-lH-l,2,4-triazole-3-carboxamide available from ICN Pharmaceuticals, Inc., Costa Mesa, Calif., is described in the Merck Index, compound No. 8199, Eleventh Edition. Its manufacture and formulation is described in U.S. Pat. No. 4,211,771. Some embodiments also involve use of derivatives of ribavirin (see, e.g., U.S. Pat. No. 6,277,830).
  • the ribavirin may be administered orally in capsule or tablet form, or in the same or different administration form and in the same or different route as the NS-3 inhibitor compound.
  • other types of administration of both medicaments as they become available are contemplated, such as by nasal spray, transdermally, intravenously, by suppository, by sustained release dosage form, etc. Any form of administration will work so long as the proper dosages are delivered without destroying the active ingredient.
  • the method may further includes administration of ritonavir.
  • Ritonavir 10-hydroxy-2-methyl-5-(l-methylethyl)-l-[2-(l-methylethyl)-4- thiazolyl]-3,6-dioxo-8,l l-bis(phenylmethyl)-2,4,7,12-tetraazatridecan-13-oic acid, 5- thiazolylmethyl ester [5S-(5R*, 8R*, 10R*, 11R*)], available from Abbott Laboratories, is an inhibitor of the protease of the human immunodeficiency virus and also of the cytochrome P450 3A and P450 2D6 liver enzymes frequently involved in hepatic metabolism of therapeutic molecules in man.
  • the method further includes administration of another protease inhibitor. In some embodiments, the method further includes administration of an NS5A inhibitor. In some embodiments, the method further includes administration of a helicase inhibitor. In some embodiments, the method further includes administration of a polymerase inhibitor.
  • an additional antiviral agent may be administered during the entire course of NS3 inhibitor compound treatment.
  • an additional antiviral agent may be administered for a period of time that is overlapping with that of the NS3 inhibitor compound treatment, e.g., the additional antiviral agent treatment can begin before the NS3 inhibitor compound treatment begins and end before the NS3 inhibitor compound treatment ends; the additional antiviral agent treatment can begin after the NS3 inhibitor compound treatment begins and end after the NS3 inhibitor compound treatment ends; the additional antiviral agent treatment can begin after the NS3 inhibitor compound treatment begins and end before the NS3 inhibitor compound treatment ends; or the additional antiviral agent treatment can begin before the NS3 inhibitor compound treatment begins and end after the NS3 inhibitor compound treatment ends.
  • the compounds described herein may be used in acute or chronic therapy for HCV disease.
  • the compounds as described herein may be administered for a period of about 1 day to about 7 days, or about 1 week to about 2 weeks, or about 2 weeks to about 3 weeks, or about 3 weeks to about 4 weeks, or about 1 month to about 2 months, or about 3 months to about 4 months, or about 4 months to about 6 months, or about 6 months to about 8 months, or about 8 months to about 12 months, or at least one year, and may be administered over longer periods of time.
  • the NS3 inhibitor compound can be administered 5 times per day, 4 times per day, tid, bid, qd, qod, biw, tiw, qw, qow, three times per month, or once monthly. In other embodiments, the NS3 inhibitor compound may be administered as a continuous infusion. [0145] In many embodiments, a compound described herein may be administered orally.
  • an NS3 inhibitor compound as described herein may be administered to the patient at a dosage from about 0.01 mg to about 100 mg/kg patient bodyweight per day, in 1 to 5 divided doses per day.
  • the NS3 inhibitor compound may be administered at a dosage of about 0.5 mg to about 75 mg/kg patient bodyweight per day, in 1 to 5 divided doses per day.
  • the amount of active ingredient that may be combined with carrier materials to produce a dosage form can vary depending on the host to be treated and the particular mode of administration.
  • a typical pharmaceutical preparation can contain from about 5% to about 95% active ingredient (w/w). In other embodiments, the pharmaceutical preparation can contain from about 20% to about 80% active ingredient.
  • dose levels can vary as a function of the specific NS3 inhibitor compound, the severity of the symptoms and the susceptibility of the subject to side effects.
  • Preferred dosages for a given NS3 inhibitor compound may be readily determinable by those of skill in the art by a variety of means.
  • a preferred means may be to measure the physiological potency of a given interferon receptor agonist.
  • an NS3 inhibitor compound may be administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid), over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more.
  • Some embodiments provide a method of treating an HCV infection in an individual having an HCV infection, the method comprising administering an effective amount of an NS3 inhibitor compound described herein, and effective amount of a TNF-oc antagonist, and an effective amount of one or more interferons.
  • the specific regimen of drug therapy used in treatment of the HCV patient is selected according to certain disease parameters exhibited by the patient, such as the initial viral load, genotype of the HCV infection in the patient, liver histology and/or stage of liver fibrosis in the patient.
  • Any of the above treatment regimens can be administered to individuals who have been diagnosed with an HCV infection. . Any of the above treatment regimens can be administered to individuals having advanced or severe stage liver fibrosis as measured by a Knodell score of 3 or 4 or no or early stage liver fibrosis as measured by a Knodell score of 0, 1, or 2. Any of the above treatment regimens can be administered to individuals who have failed previous treatment for HCV infection ("treatment failure patients," including non- responders and relapsers).
  • Individuals who have been clinically diagnosed as infected with HCV are of particular interest in many embodiments.
  • Individuals who are infected with HCV are identified as having HCV RNA in their blood, and/or having anti-HCV antibody in their serum.
  • Such individuals include anti-HCV ELISA-positive individuals, and individuals with a positive recombinant immunoblot assay (RIBA).
  • RIBA positive recombinant immunoblot assay
  • Individuals who are clinically diagnosed as infected with HCV include naive individuals (e.g., individuals not previously treated for HCV, particularly those who have not previously received IFN-OC-based and/or ribavirin-based therapy) and individuals who have failed prior treatment for HCV ("treatment failure" patients).
  • naive individuals e.g., individuals not previously treated for HCV, particularly those who have not previously received IFN-OC-based and/or ribavirin-based therapy
  • treatment failure individuals who have failed prior treatment for HCV
  • Treatment failure patients include non-responders (i.e., individuals in whom the HCV titer was not significantly or sufficiently reduced by a previous treatment for HCV, e.g., a previous IFN-OC monotherapy, a previous IFN-OC and ribavirin combination therapy, or a previous pegylated IFN-OC and ribavirin combination therapy); and relapsers (i.e., individuals who were previously treated for HCV, e.g., who received a previous IFN-OC monotherapy, a previous IFN-OC and ribavirin combination therapy, or a previous pegylated IFN-OC and ribavirin combination therapy, whose HCV titer decreased, and subsequently increased).
  • non-responders i.e., individuals in whom the HCV titer was not significantly or sufficiently reduced by a previous treatment for HCV, e.g., a previous IFN-OC monotherapy, a previous IFN-OC and ribavirin combination therapy,
  • individuals have an HCV titer of at least about 10 5 , at least about 5 x 10 5 , or at least about 10 6 , or at least about 2 x 10 6 , genome copies of HCV per milliliter of serum.
  • the patient may be infected with any HCV genotype (genotype 1, including la and lb, 2, 3, 4, 6, etc. and subtypes (e.g., 2a, 2b, 3a, etc.)), particularly a difficult to treat genotype such as HCV genotype 1 and particular HCV subtypes and quasispecies.
  • HCV-positive individuals (as described above) who exhibit severe fibrosis or early cirrhosis (non-decompensated, Child' s-Pugh class A or less), or more advanced cirrhosis (decompensated, Child' s-Pugh class B or C) due to chronic HCV infection and who are viremic despite prior anti- viral treatment with IFN-OC-based therapies or who cannot tolerate IFN-OC-based therapies, or who have a contraindication to such therapies.
  • HCV-positive individuals with stage 3 or 4 liver fibrosis according to the METAVIR scoring system are suitable for treatment with the methods described herein.
  • individuals suitable for treatment with the methods of the embodiments are patients with decompensated cirrhosis with clinical manifestations, including patients with far-advanced liver cirrhosis, including those awaiting liver transplantation.
  • individuals suitable for treatment with the methods described herein include patients with milder degrees of fibrosis including those with early fibrosis (stages 1 and 2 in the METAVIR, Ludwig, and Scheuer scoring systems; or stages 1, 2, or 3 in the Ishak scoring system).
  • HCV protease inhibitors in the following sections can be prepared according to the procedures and schemes shown in each section.
  • the numberings in each of the following Preparation of NS3 Inhibitor sections are meant for that specific section only, and should not be construed or confused with the same numberings in other sections.
  • trans-N-Boc-4-hydroxy-proline 500 mg, 2.16 mmol, 1.0 eq.
  • N,N- dimethylformamide 10 mL
  • HATU 729 mg, 2.59 mmol, 1.2 eq.
  • Diisopropylethylamine (2.26 mL, 13.0 mmol, 2.0 eq.) was added as a single portion followed the amino sulfonamide building block (669 mg, 2.4 mmol, 1.1 eq.).
  • the reaction mixture was left to warm up to ambient temperature and stirring was continued for another 4 hours.
  • the reaction mixture was diluted with water (40 mL) and the pH adjusted to 3 with 1M aqueous hydrochloric acid.
  • the aqueous phase was further extracted with ethyl acetate (2 x 30 mL).
  • the organic extracts were combined, washed with water (5 x 15 mL), dried over sodium sulfate, filtered and the solvent removed in vacuo.
  • the residue was purified by flash column chromatography using a methanol/dichloromethane gradient to give 741 mg (75% yield) of the title compound as a white solid.
  • N-(3-trifluoromethyl-phenyl)-te/t- leucine 178 mg, 0.65 mmol, 1.0 eq.
  • N,N-dimethylformamide (6 mL) were charged in a 25 mL round bottom flask.
  • HATU (295 mg, 0.78 mmol, 1.2 eq.) was added as a single portion and the reaction mixture stirred at ambient temperature for 5 minutes then cooled to 0°C.
  • Diisopropylethylamine (0.65 mL, 388 mmol, 6.0 eq.) was added as a single portion followed by stage 2c intermediate (280 mg, 0.71 mmol, 1.1 eq.).
  • the reaction mixture was left to warm up to ambient temperature and stirring was continued for another 2 hours.
  • the reaction mixture was diluted with water (25 mL) and the pH adjusted to 3 with 1M aqueous hydrochloric acid.
  • the aqueous phase was further extracted with ethyl acetate (2 x 30 mL).
  • the organic extracts were combined, washed with water (5 x 15 mL), dried over sodium sulfate, filtered and the solvent removed in vacuo.
  • the residue was purified by flash column chromatography using a methanol/dichloromethane gradient to give 316 mg (72% yield) of the title compound as a beige foamy solid.
  • STAGE 1A PREPARATION OF ETHYL l-ISOPROPYL-2-OXO- BENZIMIDAZOLE-4-CARBOXYLATE
  • STAGE 2A PREPARATION OF 1-ISOPROPYL-2-OXO-BENZIMIDAZOLE-4-
  • Ethyl l-isopropyl-2-oxo-benzimidazole-4-carboxylate (25.6 g, 0.101 mmol, 1 eq.), water (125 mL) and tetrahydrofuran (250 mL) were charged into a 1L round bottom flask.
  • Sodium hydroxide (44.9 g, 1.01 mol, 10 eq.) was added portion wise over 5 minutes and the resulting reaction mixture heated at 70°C for 5 hours by which time no starting material could be detected by LCMS analysis.
  • the biphasic reaction mixture was left to cool down to room temperature and the phases were separated.
  • the organic phases were combined, dried over magnesium sulfate, filtered and the solvent removed in vacuo to give 22.4 g (99% yield) of the title compound as a pale pink solid.
  • STAGE 4A PREPARATION OF 1-ISOPROPYL-2-OXO-BENZIMIDAZOLE-4-
  • STAGE 5A PREPARATION OF l-ISOPROPYL-2-OXO-4-(4-CYCLOPROPYL-
  • STAGE 6A PREPARATION OF l-ISOPROPYL-2-CHLORO-4-(4-CYCLOPROPYL-
  • Recombinant E. coli or Baculovirus full-length NS3 was diluted to 3.33 ⁇ with assay buffer and the material was transferred to an eppendorf tube and placed in a water bath in a 4 °C refrigerator.
  • the appropriate amount of NS4A-2 diluted to 8.3mM in assay buffer was added to an equal the volume of NS3 above (conversion factor - 3.8 mg/272 ⁇ L assay buffer).
  • the material was transferred to an eppendorf tube and placed in water bath in a 4°C refrigerator.
  • NS3/NS4A-2 was removed and placed in a room temperature water bath for 10 minutes.
  • NS3/NS4A-2 was aliquoted at appropriate volumes and stored at -80 °C (E. coli NS3 run at 2 nM in assay, aliquot at 25 ⁇ L.
  • BV NS3 run at 3 nM in assay, aliquot at 30 ⁇ L).
  • Step a Sample compounds were dissolved to lOmM in DMSO then diluted to 2.5 mM (1:4) in DMSO. Typically, compounds were added to an assay plate at 2.5 mM concentration, yielding upon dilution a starting concentration of 50 ⁇ in the assay inhibition curve. Compounds were serial diluted in assay buffer to provide test solutions at lower concentrations.
  • Step 1 The E. coli. NS3/NS4A-2 was diluted to 4 nM NS3 (1:417.5 of 1.67 ⁇ stock - 18 uL 1.67 ⁇ stock + 7497 ⁇ . assay buffer).
  • the BV NS3/NS4A-2 was diluted to 6nM NS3 (1:278.3 of 1.67 ⁇ stock - 24 ⁇ . 1.67 ⁇ stock + 6655 ⁇ . assay buffer).
  • Step. 2 Using the manual multichannel pipettor, and being careful not to introduce bubbles into the plate, 50 assay buffer was added to wells AO 1 -H01 of a black Costar 96-well polypropylene storage plate.
  • Step 3 Using the manual multichannel pipettor, and being careful not to introduce bubbles into the plate, 50 of diluted NS3/NS4A-2 from step 1 was added to wells A02-H12 of the plate in step 2.
  • Step 4 Using the manual multichannel pipettor, and being careful not to introduce bubbles into the plate, 25 of the wells in drug dilution plate in step a was transferred to corresponding wells in assay plate in step 3. The tips on the multichannel pipettor were changed for each row of compounds transferred.
  • Step 5 Using the manual multichannel pipettor, and being careful not to introduce bubbles into the plate, the contents of the wells from the assay plate in step 4 were mixed by by aspirating and dispensing 35 of the 75 in each well five times. The tips on multichannel pipettor were changed for each row of wells mixed. [0214] Step 6. The plate was covered with a polystyrene plate lid, and the plate from step 5 containing NS3 protease and sample compounds was pre-incubated 10 minutes at room temperature.
  • the RETS1 substrate was diluted in a 15mL polypropylene centrifuge tube.
  • the RETS1 substrate was diluted to 8 ⁇ (1:80.75 of 646 ⁇ stock - 65 ⁇ . 646 ⁇ stock + 5184 ⁇ . assay buffer).
  • step 6 After the plate in step 6 finished pre-incubating, and using the manual multichannel, 25 of substrate was added to all wells on the plate. The contents of the wells of the plate were quickly mixed, as in step 5, mixing 65 of the 100 in the wells.
  • the plate was read in kinetic mode on the Molecular Devices SpectraMax Gemini XS plate reader. Reader settings: Read time: 30 minutes, Interval: 36 seconds, Reads: 51, Excitation ⁇ : 335nm, Emission ⁇ : 495nm, cutoff: 475nm, Automix: off, Calibrate: once, PMT: high, Reads/well: 6, Vmax pts: 21 or 28/51 depending on length of linearity of reaction
  • IC 50 S are determined using a four parameter curve fit equation, and converted to Ki's using the following Km's:
  • Neomycin phosphotransferase II (NPTII) in the HCV Sub-Genomic Replicon, GS4.3
  • HCV sub-genomic replicon (I377/NS3-3', accession No. AJ242652), stably maintained in HuH-7 hepatoma cells, was created by Lohmann et al. Science 285: 110- 113 (1999).
  • GS4.3 cells were maintained at 37 °C, 5%C0 2 , in DMEM (Gibco 11965- 092) supplemented with L-glutamine 200mM (100X) (Gibco25030-081), non-essential amino acids (NEAA)(Biowhittaker 13-114E), heat-inactivated (HI) Fetal Bovine Serum(FBS)(Hyclone SH3007.03) and 750 ⁇ g/mL geneticin (G418)(Gibco 10131-035). Cells were sub-divided 1:3 or 4 every 2-3 days.
  • GS4.3 cells 24 hrs prior to the assay, GS4.3 cells were collected, counted, and plated in 96-well plates (Costar 3585) at 7500 cells/well in 100 standard maintenance medium (above) and incubated in the conditions above. To initiate the assay, culture medium was removed, cells were washed once with PBS (Gibco 10010-023) and 90 Assay Medium (DMEM, L-glutamine, NEAA, 10% HI FBS, no G418) was added.
  • PBS Gibco 10010-023
  • Assay Medium DMEM, L-glutamine, NEAA, 10% HI FBS, no G418, was added.
  • Inhibitors were made as a 10X stock in Assay Medium, (3-fold dilutions from 10 ⁇ to 56 pM final concentration, final DMSO concentration 1%), 10 were added to duplicate wells, plates were rocked to mix, and incubated as above for 72 hrs.
  • NPTII Elisa kit was obtained from AGDIA, Inc. (Compound direct ELISA test system for Neomycin Phosphotransferase II, PSP 73000/4800). Manufacturer's instructions were followed, with some modifications. 10X PEB-1 lysis buffer was made up to include 500 ⁇ PMSF (Sigma P7626, 50 mM stock in isopropanol). After 72 hrs incubation, cells were washed once with PBS and 150 PEB-1 with PMSF was added per well. Plates were agitated vigorously for 15 minutes, room temperature, and then frozen at -70 °C. Plates were thawed, lysates were mixed thoroughly, and 100 were applied to an NPTII Elisa plate.
  • PMSF Sigma P7626, 50 mM stock in isopropanol
  • Lysate from DMSO-treated control cells was pooled, serially diluted with PEB-1 with PMSF, and applied to duplicate wells of the ELISA plate, in a range of initial lysate amount of 150 ⁇ -2.5 ⁇ L.
  • 100 buffer alone was applied in duplicate as a blank. Plates were sealed and gently agitated at room temperature for 2hrs. Following capture incubation, the plates were washed 5X 300 ⁇ with PBS-T (0.5% Tween-20, PBS-T was supplied in the ELISA kit).
  • a IX dilution of enzyme conjugate diluent MRS-2 (5X) was made in PBS-T, into which 1:100 dilutions of enzyme conjugates A and B were added, as per instructions. Plates were resealed, and incubated with agitation, covered, room temperature, for 2 hrs. The washing was then repeated and 100 of room temperature TMB substrate was added. After approximately 30 minutes incubation (room temperature, agitation, covered), the reaction was stopped with 50 3M sulfuric acid. Plates were read at 450nm on a Molecular Devices Versamax plate reader.
  • A indicates an EC 50 or IC 50 > 100 nM

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Abstract

L'invention concerne des composés de la formule (I) (dans laquelle les variables sont définies telles que par les présentes) et des compositions, comprenant des compositions pharmaceutiques, de ceux-ci. L'invention concerne en outre des méthodes de traitement, comprenant des méthodes de traitement d'une infection par le virus de l'hépatite C et des méthodes de traitement d'une fibrose du foie par l'administration à un individu qui en a besoin d'une quantité efficace d'un composé ou d'une composition selon la présente invention.
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US8957203B2 (en) 2011-05-05 2015-02-17 Bristol-Myers Squibb Company Hepatitis C virus inhibitors
US9527885B2 (en) 2011-05-05 2016-12-27 Bristol-Myers Squibb Company Hepatitis C virus inhibitors
US9630959B2 (en) 2011-06-22 2017-04-25 Purdue Pharma L.P. TRPV1 antagonists including dihydroxy substituent and uses thereof
US9273043B2 (en) 2011-06-22 2016-03-01 Purdue Pharma L.P. TRPV1 antagonists including dihydroxy substituent and uses thereof
US10450308B2 (en) 2011-06-22 2019-10-22 Purdue Pharma L.P. TRPV1 antagonists including dihydroxy substituent and uses thereof
US9296782B2 (en) 2012-07-03 2016-03-29 Gilead Sciences, Inc. Inhibitors of hepatitis C virus
US10603318B2 (en) 2012-07-03 2020-03-31 Gilead Pharmasset Llc Inhibitors of hepatitis C virus
US10335409B2 (en) 2012-07-03 2019-07-02 Gilead Pharmasset Llc Inhibitors of hepatitis C virus
US9499550B2 (en) 2012-10-19 2016-11-22 Bristol-Myers Squibb Company Hepatitis C virus inhibitors
US9334279B2 (en) 2012-11-02 2016-05-10 Bristol-Myers Squibb Company Hepatitis C virus inhibitors
US9643999B2 (en) 2012-11-02 2017-05-09 Bristol-Myers Squibb Company Hepatitis C virus inhibitors
US9598433B2 (en) 2012-11-02 2017-03-21 Bristol-Myers Squibb Company Hepatitis C virus inhibitors
US9409943B2 (en) 2012-11-05 2016-08-09 Bristol-Myers Squibb Company Hepatitis C virus inhibitors
US9580463B2 (en) 2013-03-07 2017-02-28 Bristol-Myers Squibb Company Hepatitis C virus inhibitors
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