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WO2012047764A1 - Therapeutic antiviral peptides - Google Patents

Therapeutic antiviral peptides 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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WIPO (PCT)
Prior art keywords
compound
optionally substituted
alkoxy
alkyl
individual
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PCT/US2011/054382
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French (fr)
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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Publication of WO2012047764A1 publication Critical patent/WO2012047764A1/en
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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

The invention relates to compounds of Formula (I) (in which the variables are defined as herein) and compositions, including pharmaceutical compositions, thereof. The invention further provides treatment methods, including methods of treating a hepatitis C virus infection and methods of treating liver fibrosis through administering to an individual in need thereof an effective amount of a subject compound or composition.

Description

THERAPEUTIC ANTIVIRAL PEPTIDES
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application Nos. 61/389,627, filed October 4, 2010; and 61/473,471, filed April 8, 2011; the disclosures of which are incorporated herein by reference in their entirety.
BACKGROUND
Field
[0002] The present invention relates to compounds, processes for their synthesis, compositions and methods for the treatment of hepatitis C virus (HCV) infection.
Description of the Related Technology
[0003] 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.
[0004] 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. [0005] The high prevalence of chronic HCV infection has important public health implications for the future burden of chronic liver disease in the United States. Data derived from the National Health and Nutrition Examination Survey (NHANES ΙΠ) indicate that a large increase in the rate of new HCV infections occurred from the late 1960s to the early 1980s, particularly among persons between 20 to 40 years of age. It is estimated that the number of persons with long-standing HCV infection of 20 years or longer could more than quadruple from 1990 to 2015, from 750,000 to over 3 million. The proportional increase in persons infected for 30 or 40 years would be even greater. Since the risk of HCV-related chronic liver disease is related to the duration of infection, with the risk of cirrhosis progressively increasing for persons infected for longer than 20 years, this will result in a substantial increase in cirrhosis-related morbidity and mortality among patients infected between the years of 1965-1985.
[0006] 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. In the case of HCV, 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. Apparently, 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. In addition, compounds that inhibit the action of NS5A in viral replication are potentially useful for the treatment of HCV. SUMMARY
[0007] Some embodiments rovide a compound represented by Formula I:
Figure imgf000004_0001
or a pharmaceutically acceptable salt thereof, wherein a dashed line represents the presence or absence of a bond X is NRX or O (oxygen); Rx is H (hydrogen) or Ci_6 hydrocarbyl;
R1 is
Figure imgf000004_0002
, wherein Z is
NR6, CHR6, S (sulfur), or O (oxygen), and Z' is NR7, CHR7, S (sulfur), or O (oxygen); Y is N (nitrogen) or CR14;
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, C1-6 alkyl optionally substituted with up to 5 fluoro, C2-6 alkenyl, C2_6 alkynyl, -C(0)NR2bR2c, -NHC(0)NR2bR2c, -C(0)OR2d, heteroaryl, C1-6 alkoxy optionally substituted with up to 5 fluoro, C1-6 alkoxy optionally substituted by Ci_6 alkoxy, and Ci_6 alkoxy optionally substituted by -NR 12 R 13 ;
R2a is selected from the group consisting of H (hydrogen), C1-6 alkyl, cycloalkyl, and heterocyclyl; R2b and R2c 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, C2_6 alkenyl, C2_6 alkynyl, optionally substituted C1-6 alkoxy, -C(0)OR2d, -C(0)R2e, optionally substituted aryl, and optionally substituted heteroaryl; and R2d and R2e are each separately selected from the group consisting of -H (hydrogen), C1-4 alkoxy, C1-6 alkyl, C3-7 cycloalkyl, aryl, arylalkyl and heteroaryl;
R3 is H (hydrogen), -S02R9, or -SO2NR10Ru; R4 is H (hydrogen) or C1-4 alkyl; R5 is optionally substituted thiazole or -C(0)NR12R13; R6 is H (hydrogen) or Ci_4 alkyl; R7 is CM2 hydrocarbyl, optionally substituted arylalkyl, optionally substituted aryl, or optionally substituted heteroaryl; R9 is H (hydrogen) or C1-12 hydrocarbyl; and R10, R11, R12, R13, and R14 are independently selected from H (hydrogen) or C1-12 hydrocarbyl; provided that the compound is not selected from the group consisting of:
Figure imgf000005_0001
Figure imgf000006_0001
Figure imgf000007_0001
is selected from the group consisting of optionally substituted heteroaryl, -C(0)OR2a, and aryl optionally substituted with one or more substituents each independently selected from the group consisting of halo, amino, C1-6 alkyl optionally substituted with up to 5 fluoro, C2-6 alkenyl, C2_6 alkynyl, - C(0)NR2bR2c, -NHC(0)NR2bR2c, -C(0)OR2d, heteroaryl, and Ci_6 alkoxy optionally substituted with up to 5 fluoro or optionally substituted with C1-6 alkoxy.
[0008] In some aspects, the compound is selected from the group consisting of:
(Compound 4),
Figure imgf000008_0001
(Compound 7), mpound 8),
pound 101),
(Compound 102),
Figure imgf000009_0001
(Compound 103),
Figure imgf000009_0002
und 105), and
Figure imgf000010_0001
(Compound 106).
[0009] These definitions of are understood to apply to structures depicted herein for which any one of those variables are not expressly defined.
[0010] Some embodiments provide a pharmaceutical composition comprising: a) a compound disclosed herein; and b) a pharmaceutically acceptable carrier or excipient.
[0011] Some embodiments provide a method of inhibiting NS3/NS4 protease activity comprising contacting a NS3/NS4 protease with a compound or a composition herein. In some aspects, the contacting is conducted in vivo, such as wherein a sustained viral response is achieved. In some aspects, the contacting is conducted ex vivo.
[0012] 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.
[0013] 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.
[0014] Some embodiments provide a method of treating hepatitis by modulating NS3/NS4 protease comprising contacting a NS3/NS4 protease with a compound disclosed herein. [0015] 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. In some aspects, 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.
[0016] In some aspects, 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.
[0017] In some aspects, the methods herein further comprise administering to the individual an effective amount of a human immunodeficiency virus 1 protease inhibitor, such as ritonavir.
[0018] In some aspects, the methods herein further comprise administering to the individual an effective amount of an NS5B RNA-dependent RNA polymerase inhibitor.
[0019] In some aspects, the methods herein further comprise administering to the individual an effective amount of an NS5A inhibitor.
[0020] In some aspects, the methods herein further comprise administering to the individual an effective amount of interferon-gamma (IFN-γ), such as wherein the IFN-γ is administered subcutaneously in an amount of from about 10 μg to about 300 μg.
[0021] In some aspects, 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. In some aspects, the IFN-OC is INFERGEN consensus IFN-OC.
[0022] In some aspects, 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.
[0023] These and other embodiments are described in greater detail below.
DETAILED DESCRIPTION
Definitions
[0024] 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.
[0025] As used herein, the term "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, γ-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.
[0026] The term "sustained viral response" (SVR; also referred to as a "sustained response" or a "durable response"), as used herein, refers to the response of an individual to a treatment regimen for HCV infection, in terms of serum HCV titer. Generally, 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.
[0027] "Treatment failure patients" as used herein 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.
[0028] "Treat," "treating," "treatment," or another form thereof 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. [0029] 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. Likewise, when compounds contain a double bond, there exists the possibility of cis- and trans- type isomeric forms of the compounds. Both cis- and trans- isomers, both in pure form as well as mixtures of cis- and trans- isomers, are contemplated. Thus, reference herein to a compound includes all of the aforementioned isomeric forms unless the context clearly dictates otherwise.
[0030] 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. Thus, reference herein to a compound includes all of the aforementioned forms unless the context clearly dictates otherwise.
[0031] The term "pharmaceutically acceptable salt," as used herein, and particularly when referring to a pharmaceutically acceptable salt of a compound, including a compound of Formula I , refers to any pharmaceutically acceptable salts of a compound, and preferably refers to an acid addition salt of a compound. With respect to compounds that contain a basic nitrogen, the preferred examples of 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. With respect to compounds that contain an acidic functional group, 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.
[0032] Isotopes may be present in the compounds described. Each chemical element as represented in a compound structure may include any isotope of said element. For example, in a compound structure a hydrogen atom may be explicitely disclosed or understood to be present in the compound. At any position of the compound that a hydrogen atom may be present, the hydrogen atom can be any isotope of hydrogen, including but not limited to hydrogen- 1 (protium) and hydrogen-2 (deuterium). Thus, reference herein to a compound encompasses all potential isotopic forms unless the context clearly dictates otherwise.
[0033] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the embodiments. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the embodiments.
[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments belong. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the embodiments, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
[0035] It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods and reference to "a dose" includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth. [0036] The term "optionally substituted," as used herein refers to a moiety or structural feature which may be unsubstituted, or may have one or more substituents. Thus, for example, "optionally substituted aryl" may be unsubstituted aryl, or may be an aryl with one or more substituents. The term "substituent" as used herein refers to a moiety that replaces one or more hydrogen atoms of the parent group for which it is a substituent. In some embodiments, 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. In some embodiments, 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. Examples include Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl (e.g., tetrahydrofuryl), aryl, heteroaryl, halo (e.g., chloro, bromo, iodo and fluoro), cyano, hydroxy, Ci-C6 alkoxy, aryloxy, sulfhydryl (mercapto), Ci-C6 alkylthio, arylthio, mono- and di-(Ci-C6)alkyl amino, quaternary ammonium salts, amino(Ci-C6)alkoxy, hydroxy(Ci-C6)alkylamino, amino(Ci-C6)alkylthio, cyanoamino, nitro, carbamyl, keto (oxo), carbonyl, carboxy, glycolyl, glycyl, hydrazino, guanyl, sulfamyl, sulfonyl, sulfinyl, thiocarbonyl, thiocarboxy, and combinations thereof. The protecting groups that can form the protective derivatives of the above substituents are known to those of skill in the art and can be found in references such as Greene and Wuts Protective Groups in Organic Synthesis; John Wiley and Sons: New York, 1999.
[0037] In some aspects, 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. Thus, the substituent may alter the degree of inhibition of NS3/NS4 protease activity or other activities dislcclosed herein.
[0038] The term "aryl" as used herein refers to an aromatic ring or aromatic ring system such as phenyl, naphthyl, biphenyl, and the like.
[0039] The term "heteroaryl" as used herein 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. The term "fused bicyclic heteroaryl" as used herein 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
[0040] The term "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. In 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. Examples of heterocyclic groups include, but are not limited to, morpholinyl, tetrahydrofuranyl, dioxolanyl, pyrolidinyl, pyranyl, piperidyl, piperazyl, and the like.
[0041] The term "hydrocarbyl" as used herein refers to a hydrocarbon moiety. The term "alkyl" as used herein refers to a hydrocarbon moiety which has no double or triple bonds, such as methyl, ethyl, propyl, cyclopropyl, etc.
[0042] As used herein, the term "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. Typically, the alkyl, alkenyl and alkynyl substituents contain Ci-Cio (alkyl) or C2-Cio (alkenyl or alkynyl). Preferably they contain Ci-C6 (alkyl), C2-C6 (alkenyl or alkynyl), or C3-C6 cycloalkyl.
[0043] The term "alkyl" as used herein refers to a radical of a fully saturated hydrocarbon, including, but not limited to, methyl, ethyl, n-propyl, isopropyl (or i-propyl), n-
Figure imgf000017_0001
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 CnH2n+2; the general formula for a fully saturated hydrocarbon containing one ring is CnH2n; the general formula for a fully saturated hydrocarbon containing two rings is CnH2(n_i); the general formula for a saturated hydrocarbon containing three rings is CnH2(n-2). When a more specific term for alkyl (such as propyl, butyl, etc.) is used without specifying linear or branched, the term is to be interpreted to include linear and branched alkyl.
[0044] The term "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.
[0045] The term "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.
[0046] The term "arylalkyl" used herein refers to one or more aryl groups appended to an alkyl radical. Examples of arylalkyl groups include, but are not limited to, benzyl, phenethyl, phenpropyl, phenbutyl, and the like. [0047] The term "alkoxy" used herein refers to straight or branched chain alkyl radical covalently bonded to the parent molecule through an— O— linkage. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, n-butoxy, sec -butoxy, t-butoxy and the like. When a more specific term for alkoxy (such as propoxy, butaoxy, etc.) is used without specifying linear or branched, the term is to be interpreted to include linear and branched alkoxy.
[0048] The term "aryloxy" used herein refers to an aryl radical covalently bonded to the parent molecule through an— O— linkage.
[0049] The term "alkylthio" used herein refers to straight or branched chain alkyl radical covalently bonded to the parent molecule through an— S— linkage.
[0050] The term "arylthio" used herein refers to an aryl radical covalently bonded to the parent molecule through an— S— linkage.
[0051] The term "alkylamino" used herein refers to nitrogen radical with one or more alkyl groups attached thereto. Thus, monoalkylamino refers to nitrogen radical with one alkyl group attached thereto and dialkylamino refers to nitrogen radical with two alkyl groups attached thereto.
[0052] The substituent "R" appearing by itself refers to a substituent selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl and heterocycle.
[0053] The term "halo" used herein refers to fluoro, chloro, bromo, or iodo.
[0054] The term "cyanoamino" used herein refers to nitrogen radical with nitrile group attached thereto.
[0055] The term "carbamyl" used herein refers to RNHCOO-.
[0056] The term "keto" and "carbonyl" used herein refers to C=0.
[0057] The term "carboxy" used herein refers to -COOH.
[0058] The term "sulfamyl" used herein refers to -S02NH2.
[0059] The term "sulfonyl" used herein refers to -S02-.
[0060] The term "sulfinyl" used herein refers to -SO-.
[0061] The term "thiocarbonyl" used herein refers to C=S.
[0062] The term "thiocarboxy" used herein refers to CSOH.
[0063] As used herein, a radical indicates species with a single, unpaired electron such that the species containing the radical can be covalently bonded to another species. Hence, in this context, 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."
[0064] As used herein, 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.
[0065] As employed herein, the following terms have their accepted meaning in the chemical literature.
Ac Acetyl
aq. aqueous
BINAP 2,2'-bis(diphenylphosphino)- 1 , 1 '-binaphthyl
Boc tert-Butoxycarbonyl
Bu Butyl
°C Temperature in degrees Centigrade
CDI Ι,Γ-Carbonyldiimidazole
dL deciliter
DBU l,8-Diazabicyclo[5.4.0]undec-7-ene
DCM Dichloromethane
DIAD Diisopropyl azodicarboxylate
DIEA N,N-Diisopropylethylamine
DMF N,N-Dimethylformamide
DMSO Dimethyl sulfoxide
EA or EtOAc Ethyl acetate
Et Ethyl
eq. equivalents
g grams
HATU N,N,N',N'-Tetramethyl-0-(7-azabenzotriazol- 1 -yl)uronium hexafluorophosphate
HRMS High resolution mass spectrometry
hrs hours
HPLC High-performance liquid chromatography (or high-pressure liquid chromatography)
LC-MS Liquid chromatography - Mass spectrometry M Molar
Me Methyl
MeOH Methanol
Min Minutes
mg Milligrams
MHz Megahertz
mL Milliliters
mM Millimolar
mmol Millimoles
mol Moles
nM Nanomolar
NMP N-Methyl-2-pyrrolidone
NMR Nuclear magnetic resonance
PE Petroleum ether
Ph Phenyl
ppm parts-per-million
prep Preparative
t Tert
t-Bu or tBu tert-Butyl
Tert tertiary
TEA Triethyl amine
THF Tetrahydrofuran
TLC Thin layer chromatography
tR Retention time
UV Ultraviolet
\L Microliters
μΜ Micromolar
Compounds
[0066] Unless otherwise indicated, if a term is used to describe more than one structural feature of the compounds disclosed herein, it should be assumed that the term has the same meaning for all of those features. Similarly, a subgroup of that term applies to every structural feature described by that term.
[0067] The variable groups of the compound of Formula I are discussed below in more detail:
Figure imgf000021_0001
(I) or a pharmaceutically acceptable salt thereof.
[0068] In some embodiments, 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, C2-6 alkenyl, C2_6 alkynyl, -C(0)NR2bR2c, -NHC(0)NR2bR2c, -C(0)OR2d, heteroaryl, Ci_6 alkoxy optionally substituted with up to 5 fluoro, Ci-6 alkoxy optionally substituted by C1-6 alkoxy, and C1-6 alkoxy optionally substituted by
Figure imgf000021_0002
In some embodiments, R 2 may be selected from the group consisting of optionally substituted heteroaryl, -C(0)OR2a, and aryl optionally substituted with one or more substituents each independently selected from the group consisting of halo, amino, C1-6 alkyl optionally substituted with up to 5 fluoro, C1-6 alkoxy optionally substituted with up to 5 fluoro or optionally substituted by Ci_6 alkoxy, C2_6 alkenyl, C2_6 alkynyl, - C(0)NR2bR2c, -NHC(0)NR2bR2c, -C(0)OR2d, and heteroaryl. In some embodiments, R2a can be selected from the group consisting of H (hydrogen), Ci_6 alkyl, t-butyl, cycloalkyl, and heterocyclyl; R2b and R2c 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 C1-6 alkyl, C2_6 alkenyl, C2_6 alkynyl, optionally substituted Ci_6 alkoxy, -C(0)OR2d, -C(0)R2e, optionally substituted aryl, and optionally substituted heteroaryl; and R2d and R2e can each be separately selected from the group consisting of -H (hydrogen), Ci_ alkoxy, Ci_6 alk l, C3_7 cycloalkyl,
Figure imgf000021_0003
aryl, arylalkyl and heteroaryl. For example, R may be
Figure imgf000022_0001
, or -C(0)OiBu, wherein m is an integer between 1 and 6, and n is an integer between 0-5.
[0069] In some embodiments, R2 is -C(0)OR2a or aryl optionally substituted with one or more substituents each independently selected from the group consisting of C2-6 alkenyl, C2_6 alkynyl, -C(0)NR2bR2c, -C(0)OR2d, heteroaryl, and Ci_6 alkoxy optionally substituted by C1-6 alkoxy. In some embodiments, R is phenyl substituted with 1 to 3 substituents independently selected from the group consisting of: heteroaryl, -C(0)NR2bR2c, or Ci-6 alkoxy optionally substituted by C1-6 alkoxy; wherein in some embodiments, R2b and R2c are taken together with the nitrogen to which they are attached to form piperazinyl, wherein piperazinyl is optionally substituted with C1-6 alkyl or -C(0)OR2d; wherein R2d is
Ci_6 alkyl
[[00070] In some embodiments, R4 may be H (hydrogen) or C1-4 alkyl, such as a C3-
4 branched alkyl. In some aspects, R may be iBu
[0071] In some embodiments, R1 may b wherein Z may be
NR6, CHR6, S (sulfur), or O (oxygen), R1 may be
Figure imgf000022_0002
wherein Z' may be NR7,
CHR 7 , S (sulfur), or O (oxygen), or R 1 may be
Figure imgf000022_0003
. In some embodiments, Y may be N (nitrogen) or CR14. In some aspects, Y may be N (nitrogen) and Z may be NR6 . Further Y may be N (nitrogen) and Z' may be NR7. For example, R1 may be
Figure imgf000022_0004
[0072] In embodiments where R is
Figure imgf000023_0001
may be linked to the remainder of the molecule through any available position on the 6-membered ring. For
Figure imgf000023_0002
[0074] In some embodiments, R may be C1-12 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
7 7
substituted aryl. R may be an unsubstituted aryl or heteroaryl. In some aspects, R may be an unsubstituted aryl, such as unsubstituted phenyl.
[0075] In some embodiments, R5 may be optionally substituted thiazole or -C(0)NR12R13. In some aspects, R5 is optionally substituted thiazole, wherein the thiazole is unsubstituted or substituted with an alkyl, alkenyl or alkynyl such as a CrC4 alkyl or C2-C4 alkenyl or alkynyl group, such as C3 or C4 alkyl or alkenyl. For example, R5 may
Figure imgf000023_0003
[0076] In some embodiments, R , R , and R may be independently selected from H (hydrogen) or C1-12 hydrocarbyl. C1-12 hydrocarbyl may be alkyl, alkenyl or alkynyl, for instance C1-C4 alkyl or C2-C4 alkeneyl or alkynyl group, such as C3 or C4 alkyl or alkenyl.
1^2 13 · 12
In some embodiments, one of either R or R" may be H (hydrogen). One of either R or
R 13
may be alkenyl, for instance where the point of unsaturation is between the terminal
carbon and the adjacent carbon thereto. For instance, R5 may be
Figure imgf000023_0004
[0077] The dashed line represents the presence or absence of a bond. Thus, the cylclopropyl is substituted with an ethyl or etheylene group depending on whether there is an absence of an additional bond forming a single bond or a presence of an additional bond forming a double bond. [0078] In some embodiments, X may be NRX or O (oxygen). In some embodiments, Rx may be H (hydrogen) or Ci_6 hydrocarbyl, such as Ci_4 alkyl, for example methyl. In some embodiments, Rx may be H (hydrogen). In some aspects, X is NH or X is O (oxygen).
3 9
[0079] In some embodiments, R may be H (hydrogen), -S02R , or - SO2NR10Ru In some aspects, R3 may be H (hydrogen). In some aspects, R3 may be -S02R9 wherein R9 may be H (hydrogen) or C1-12 hydrocarbyl. When R9 is C1-12 hydrocarbyl, C1-12 hydrocarbyl may be C1-12 alkyl, or C2_i2 alkenyl or alkynyl, such as Q-Q alkyl or C2-C8 alkenyl or alkynyl group, or C4-C8 alkyl or alkenyl. The alkyl, alkenyl or alkynyl such as C4- C8 alkyl or alkenyl may contain a cycloalkyl group such as cyclopropyl. Alternatively or in addition to a cyclopropyl group, R9 may contain alkenyl, for instance where the point of etween the terminal carbon and the adjacent carbon thereto. In some as ects,
enyl)cyclopropyl,
[0080] In some embodiments, R3 may be -SO2NR10Rn wherein R10 and R11 are independently selected from H (hydrogen) or C1-12 hydrocarbyl. In some embodiments, one of either R10 or R11 may be H (hydrogen). In some embodiments, R10 and R11 are each independently C1-6 alkyl. In some embodiment R10 and R11 are each methyl. In some embodiments, X may be NRX, and R3 may be -S02R9.
[0081] In some aspects of a compound of Formula I, unless otherwise specified, groups indicated as "optionally substituted" can be optionally substituted with one or more group(s) individually and independently selected from Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, aryl, heteroaryl, halo, cyano, hydroxy, Cp C6 alkoxy, aryloxy, sulfhydryl, Ci-C6 alkylthio, arylthio, mono- and di-(Ci-C6)alkyl amino, quaternary ammonium salts, amino(Ci-C6)alkoxy, hydroxy(Ci-C6)alkylamino, amino(Cr C6)alkylthio, cyanoamino, nitro, carbamyl, oxo, carbonyl, carboxy, sulfamyl, sulfonyl, sulfinyl, thiocarbonyl, and thiocarboxy.
[0082] In some aspects, the compound is selected from the group consisting of:
Figure imgf000025_0001
-24-
Figure imgf000026_0001
(Compound 7),
Figure imgf000026_0002
(Compound 102),
Figure imgf000026_0003
(Compound 103),
Figure imgf000027_0001
(Compound 106).
[0083] In some aspects, the compound is not selected from:
Figure imgf000028_0001
-27-
Figure imgf000029_0001
-28-
Figure imgf000030_0001
[0084] The present embodiments provide for a method of inhibiting NS3/NS4 protease activity comprising contacting a NS3/NS4 protease with a compound disclosed herein.
[0085] 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.
[0086] The present embodiments provide a method of treating liver fibrosis in an individual, the method comprising administering to the individual a compound disclosed herein.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] The present embodiments provide a compound or a composition disclosed herein for use in treating HCV infection in an individual.
[0092] The present embodiments provide a compound or a composition disclosed herein for use in treating liver fibrosis in an individual.
[0093] 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
[0094] The present embodiments further provide compositions, including pharmaceutical compositions, comprising compounds disclosed herein. [0095] 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. Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3 ed. Amer. Pharmaceutical Assoc.
[0096] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, 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.
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] In some embodiments, 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. 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.
[0102] In many embodiments, a subject compound may inhibit enzymatic activity of an HCV NS3 protease with an IC50 of less than about 50 μΜ, e.g., a subject compound inhibits an HCV NS3 protease with an IC50 of less than about 40 μΜ, less than about 25 μΜ, less than about 10 μΜ, less than about 1 μΜ, 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.
[0103] In many embodiments, 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.
[0104] In many embodiments, a subject compound may inhibit HCV viral replication. For example, 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.
Treating a hepatitis virus infection
[0105] The methods and compositions described herein may be generally useful in treatment of an of HCV infection.
[0106] 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. [0107] In general, 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.
[0108] 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.
[0109] Some embodiments involve administering an effective amount of a compound disclosed herein optionally in combination with an effective amount of one or more additional antiviral agents. In some embodiments, 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. In some embodiments, 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.
[0110] In some embodiments, 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.
[0111] In many embodiments, 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. [0112] 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.
[0113] As one non-limiting example, levels of serum alanine aminotransferase (ALT) are measured, using standard assays. In general, an ALT level of less than about 45 international units is considered normal. In some embodiments, 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.
[0114] 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.
[0115] In many embodiments, an effective amount of a compound disclosed herein and an additional antiviral agent may be a synergistic amount. As used herein, 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.
Fibrosis
[0116] Some embodiments provide methods for treating liver fibrosis (including forms of liver fibrosis resulting from, or associated with, HCV infection), 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.
[0117] Whether treatment with a compound disclosed herein, and optionally one or more additional antiviral agents, is effective in reducing liver fibrosis may be determined by any of a number of well-established techniques for measuring liver fibrosis and liver function. 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. Based on analysis of the liver biopsy, a score may be assigned. A number of standardized scoring systems exist which provide a quantitative assessment of the degree and severity of fibrosis. These include the METAVIR, Knodell, Scheuer, Ludwig, and Ishak scoring systems.
[0118] 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). The definitions of each stage in the METAVIR system are 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.
[0119] 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; ΙΠ. Portal inflammation; and Γ . Fibrosis. In the Knodell staging system, 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. Knodell (1981) Hepatol. 1:431. [0120] In the Scheuer scoring system scores are as follows: score: 0, no fibrosis; score: 1, enlarged, fibrotic portal tracts; score: 2, periportal or portal-portal septa, but intact architecture; score: 3, fibrosis with architectural distortion, but no obvious cirrhosis; score: 4, probable or definite cirrhosis. Scheuer (1991) J. Hepatol. 13:372.
[0121] 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.
[0122] 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.
[0123] In some embodiments, 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. In particular embodiments, 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.
[0124] 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. [0125] 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. Those skilled in the art can readily measure such indices of liver function, using standard assay methods, many of which are commercially available, and are used routinely in clinical settings.
[0126] 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 ΠΙ peptide, 7S domain of type IV collagen, C-terminal procollagen I peptide, and laminin. Additional biochemical markers of liver fibrosis include α-2-macroglobulin, haptoglobin, gamma globulin, apolipoprotein A, and gamma glutamyl transpeptidase.
[0127] 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. Those skilled in the art can readily measure such serum markers of liver fibrosis, using standard assay methods, many of which are commercially available, and are used routinely in clinical settings. 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.
[0128] As used herein, 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. [0129] 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.
[0130] Whether treatment with a compound disclosed herein, and optionally one or more additional antiviral agents, is effective in reducing the incidence of a disorder associated with cirrhosis of the liver can readily be determined by those skilled in the art.
[0131] Reduction in liver fibrosis increases liver function. Thus, the embodiments provide methods for increasing liver function, generally involving administering a therapeutically effective amount of a compound disclosed herein, and optionally one or more additional antiviral agents. 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, γ-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.
[0132] Whether a liver function is increased is readily ascertainable by those skilled in the art, using well-established tests of liver function. Thus, synthesis of 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.
[0133] 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.
[0134] 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. For example, 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.
Dosages, Formulations, and Routes of Administration
[0135] In the subject methods, the active agent(s) (e.g., compounds as described herein, and optionally one or more additional antiviral agents) may be administered to the host using any convenient means capable of resulting in the desired therapeutic effect. Thus, 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.
Other antiviral or antifibrotic agents
[0136] As discussed above, 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).
[0137] In some embodiments, the method further may include administration of one or more interferon receptor agonist(s).
[0138] In other embodiments, the method may further include administration of pirfenidone or a pirfenidone analog.
[0139] Additional antiviral agents that are suitable for use in combination therapy 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.
[0140] In some embodiments, 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. Of course, 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.
[0141] In some embodiments, 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.
[0142] In some embodiments, 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.
[0143] In some embodiments, an additional antiviral agent may be administered during the entire course of NS3 inhibitor compound treatment. In other embodiments, 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.
Methods of Treatment
Monotherapies
[0144] The compounds described herein may be used in acute or chronic therapy for HCV disease. In many embodiments, 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.
[0146] In connection with the above-described methods for the treatment of HCV disease in a patient, 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. In some embodiments, 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.
[0147] 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.
[0148] Those of skill will readily appreciate that 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.
[0149] In many embodiments, multiple doses of NS3 inhibitor compound are administered. For example, 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.
Combination therapies with a TNF-cc antagonist and an interferon
[0150] 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.
Subjects Suitable for Treatment
[0151] In certain embodiments, 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.
[0152] 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).
[0153] 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). Such individuals may also, but need not, have elevated serum ALT levels.
[0154] 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). 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). [0155] In particular embodiments of interest, individuals have an HCV titer of at least about 105, at least about 5 x 105, or at least about 106, or at least about 2 x 106, 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.
[0156] Also of interest are 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. In particular embodiments of interest, 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. In other embodiments, 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. In still other embodiments, 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).
EXAMPLES
Preparation of NS3 Inhibitors Methodology
[0157] The 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. EXAMPLE I - PREPARATION OF COMPOUND 3
Figure imgf000046_0001
Compound 3
EXAMPLE I-A BUILDING BLOCKS PREPARATION
REACTION SCHEME:
Figure imgf000046_0002
STAGE IF:
Figure imgf000046_0003
[0158] trans-N-Boc-4-hydroxy-proline (500 mg, 2.16 mmol, 1.0 eq.) and N,N- dimethylformamide (10 mL) were charged in a 25 mL round bottom flask. HATU (729 mg, 2.59 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 (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.
• 1H NMR (500 MHz, CDC13) δ ppm 9.80 (br. s., 1 H) 7.23 (br. s., 1 H) 5.75 (quin, 1 H) 5.31 (d, 7=17.09 Hz, 1 H) 5.17 (d, 7=10.38 Hz, 1 H) 4.54 (br. s., 1 H) 4.28 (t, 7=7.48 Hz, 1 H) 3.55 - 3.61 (m, 1 H) 3.47 - 3.53 (m, 1 H) 2.28 - 2.37 (m, 1 H) 2.07 - 2.15 (m, 2 H) 1.97 (dd, 7=7.93, 5.80 Hz, 1 H) 1.83 (br. s., 1 H) 1.62 - 1.74 (m, 2 H) 1.49 - 1.51 (m, 12 H) 1.38 (dd, 7=7.71, 4.35 Hz, 1 H) 0.81 - 0.91 (m, 2 H).
• LC-MS: purity 100% (UV), tR 1.73 min m/z [M+Na]+ 480.25 (MET/CR/1278).
STAGE 2F:
Figure imgf000047_0001
[0159] Stage If intermediate (741 mg, 1.62 mmol, 1.0 eq.) and dioxane (4 mL) were charged into a 25 mL round bottom flask. 4M hydrogen chloride in dioxane (4 mL) was added dropwise and the reaction mixture stirred at 50°C for 1 hour. The solvent was removed in vacuo to give 560 mg (88% yield, hydrochloride salt) of the title compound as a white solid. LC-MS: purity 95% (UV), tR 1.05 min m/z [M+H]+ 358.40 (MET/CR/1278).
STAGE 3F:
Figure imgf000048_0001
[0160] N-(3-trifluoromethyl-phenyl)-te/t- leucine (178 mg, 0.65 mmol, 1.0 eq.) and 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.
1H NMR (500 MHz, CDC13) δ ppm 9.90 (br. s., 1 H) 7.27 (d, 7=18.01 Hz, 1 H) 7.21 - 7.25 (m, 1 H) 6.95 (d, 7=7.32 Hz, 1 H) 6.76 - 6.79 (m, 1 H) 5.67 (ddd, 7=17.20, 10.11, 8.85 Hz, 1 H) 5.27 (d, 7=17.09 Hz, 1 H) 5.16 (d, 7=10.38 Hz, 1 H) 4.58 - 4.65 (m, 2 H) 4.38 (t, 7=8.09 Hz, 1 H) 3.95 (d, 7=7.63 Hz, 1 H) 3.67 - 3.83 (m, 3 H) 2.18 - 2.28 (m, 1 H) 2.08 (q, 7=9.00 Hz, 1 H) 1.97 - 2.04 (m, 1 H) 1.88 - 1.93 (m, 1 H) 1.69 - 1.75 (m, 1 H) 1.62 (d, 7=4.43 Hz, 1 H) 1.50 (s, 3 H) 1.39 (dd, 7=9.46, 5.95 Hz, 1 H) 1.09 (s, 9 H) 0.82 - 0.91 (m, 2 H)).
LC-MS: purity 98% (UV), tR 2.22 min m/z [M+H]+ 615.55 (MET/CR/1278). EXAMPLE I-B - FINAL STEP IN PREPARATION OF COMPOUND 3
REACTION SCHEME
Figure imgf000049_0001
Compound 3
[0161] The P2-hydroxy starting material, l-isopropyl-2-chloro-4-(4,5-diisopropyl- thiazol-2-yl)-benzimidazole and anhydrous dimethylsulfoxide were charged into a reaction vessel. Potassium te/t-butoxide was added in a single portion and the reaction mixture stirred at ambient temperature. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic extracts were washed with water, dried over magnesium sulfate, filtered and the solvent removed in vacuo.
• 68 mg (44% yield) as an off-white solid after preparative HPLC.
• 1H NMR (500 MHz, CDC13) δ ppm 9.80 (br. s., 1 H) 8.19 (dd, J=7.40, 1.30 Hz, 1 H) 7.36 (br. s., 1 H) 7.27 (d, J=7.78 Hz, 1 H) 7.22 - 7.26 (m, 2 H) 7.03 (br. s., 1 H) 6.77 (br. s., 1 H) 6.69 (d, J=6.10 Hz, 1 H) 6.41 - 6.48 (m, 2 H)
5.69 - 5.73 (m, 1 H) 5.64 - 5.69 (m, 1 H) 5.29 (d, J=17.09 Hz, 1 H) 5.18 (d, J=10.38 Hz, 1 H) 4.53 - 4.66 (m, 3 H) 4.35 (spt, J=6.99 Hz, 1 H) 4.10 (dd, J=12.36, 3.51 Hz, 1 H) 3.99 (d, J=10.22 Hz, 1 H) 3.24 (spt, J=6.87 Hz, 1 H)
2.70 - 2.80 (m, 1 H) 2.65 (dd, J=14.27, 7.86 Hz, 1 H) 2.08 (q, J=8.70 Hz, 1 H) 1.97 (dd, J=7.78, 6.26 Hz, 1 H) 1.71 - 1.78 (m, 1 H) 1.64 - 1.68 (m, 1 H) 1.53 (s, 3 H) 1.41 (dd, J=6.87, 1.68 Hz, 6 H) 1.37 - 1.41 (m, 1 H) 1.29 (d, J=6.87 Hz, 3 H) 1.22 (d, J=6.87 Hz, 2 H) 1.09 (s, 9 H) 0.82 - 0.93 (m, 2 H).
• LC-MS: purity 98% (UV), tR 5.16 min m/z [M+H]+ 898.20 (MET/CR/1426.
• HRMS: Found: 898.3602, calculated for C44H54F3N706S2 (M+H)+: 898.3607. EXAMPLE II - PREPARATION OF COMPOUND 4
Figure imgf000050_0001
Compound 4
EXAMPLE II-A BUILDING BLOCKS PREPARATION
Figure imgf000050_0002
Compound 4
2-CHLORO-BENZIMIDAZOLE BUILDING BLOCKS PREPARATION
REACTION SCHEME:
Figure imgf000051_0001
STAGE 1A: PREPARATION OF ETHYL l-ISOPROPYL-2-OXO- BENZIMIDAZOLE-4-CARBOXYLATE
Figure imgf000051_0002
[0162] l-isopropyl-2-oxo-4-bromo-benzimidazole (32.6 g, 0.123 mmol, 1 eq.), triethylamine (58 g, 0.564 mol, 4.5 eq.) and ethanol (800 mL) were charged into a 2L pressure vessel. Bis(triphenylphosphine)palladium dichloride (4.4 g, 6.26 mmol, 5 mol%) was added and the reaction mixture heated at 100°C under 10 bar of carbon monoxide for 15 hours. The reaction mixture was left to cool down to room temperature and the pressure released. Checking the reaction extent by LCMS showed a 25% conversion ratio. The reaction mixture was filtered to remove a small amount of black solid. Fresh catalyst (5 mol%) and triethylamine (50 mL) were added and the reaction mixture was heated at 100°C under 8.5 bars of carbon monoxide for another 15 hours. LCMS analysis of an aliquot showed the conversion has reached 74%. The reaction mixture was filtered to remove extra black solid. Fresh catalyst (5 mol%) was added and the reaction mixture was heated at 100°C under 8.5 bars of carbon monoxide for a further 15 hours. LCMS analysis of an aliquot showed the reaction to be complete. The solvent was removed in vacuo and the residue diluted with ethyl acetate (300 mL). The organic phase was washed with 1M hydrochloric acid (300 mL), water (300 mL) and brine (300 mL), dried over magnesium sulfate, filtered and the solvent removed in vacuo. The residue was purified by flash column chromatography using a ethyl acetate / heptane gradient. After combining the relevant fractions, the solvent was removed in vacuo to give 25.6 g (78% yield) of the title compound as a free flowing pale yellow solid.
• 1H NMR (500 MHz, CDC13) δ ppm 9.03 (br. s., 1 H) 7.65 (dd, 7=8.09, 0.92 Hz, 1 H) 7.29 (d, 7=8.39 Hz, 1 H) 7.09 (t, 7=8.01 Hz, 1 H) 4.74 (spt, 7=7.02 Hz, 1 H) 4.44 (q, 7=7.07 Hz, 2 H) 1.55 (d, 7=7.02 Hz, 6 H) 1.43 (t, 7=7.17 Hz, 3 H).
• LC-MS: purity 90% (UV), tR 1.86 min m/z [M+H]+ 248.95 (MET/CR/1278).
STAGE 2A: PREPARATION OF 1-ISOPROPYL-2-OXO-BENZIMIDAZOLE-4-
CARBOXYLIC ACID
Figure imgf000052_0001
[0163] 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 aqueous phase was acidified to pH = 2 with hydrochloric acid and then extracted twice with an isopropanol / chloroform mixture (3:1, 100 mL). 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.
• 1H NMR (500 MHz, CDC13) δ ppm 13.09 (br. s., 1 H) 10.56 (br. s., 1 H) 7.48 (d, J=7.93 Hz, 1 H) 7.48 (d, J=7.93 Hz, 1 H) 7.06 (t, J=7.93 Hz, 1 H) 4.60 (spt, J=6.97 Hz, 1 H) 1.44 (d, J=7.02 Hz, 6 H).
• LC-MS: purity 95% (UV), tR 1.54 min m/z [M+H]+ 220.95 (MET/CR/1278). STAGE 3A: PREPARATION OF 1-ISOPROPYL-2-OXO-BENZIMIDAZOLE-4-
CARBOXYLIC ACID AMIDE
Figure imgf000053_0001
[0164] l-Isopropyl-2-oxo-benzimidazole-4-carboxylic acid (2.855 g, 12.96 mmol, 1.0 eq.) was charged into a 100 mL round bottom flask and the flask placed on top of an ice bath. Thionyl chloride (28 mL) was added portion wise and the reaction mixture stirred at ambient temperature for 15 hours. Thionyl chloride was removed in vacuo and the residue diluted with dry dioxane (20 mL). Ammonia in dioxane (0.5 M, 39 mL, 19.44 mmol, 1.5 eq) was added dropwise over 10 min. The reaction mixture was then stirred at ambient temperature for 15 hours. The solvent was removed in vacuo and the residue triturated with water (20 mL) and dioxane (5 mL) leading to the precipitation of a solid which was collected by filtration. After further drying under high vacuum for 4 hours 3.68 g (94%) of the title compound was isolated as a beige solid.
• 1H NMR (500 MHz, CDC13) δ ppm 9.66 (br. s., 1 H) 7.26 (d, 7=7.78 Hz, 1 H) 7.18 (d, 7=7.78 Hz, 1 H) 7.07 (m, 7=7.78, 7.78 Hz, 1 H) 6.00 (br. s., 2 H) 4.75 (spt, 7=6.99 Hz, 1 H) 1.55 (d, 7=7.02 Hz, 6 H).
• LC-MS: purity 92% (UV), tR 1.38 min m/z [M+H]+ 219.90 (MET/CR/1278).
STAGE 4A: PREPARATION OF 1-ISOPROPYL-2-OXO-BENZIMIDAZOLE-4-
CARBOTHIOIC ACID AMIDE
Figure imgf000053_0002
[0165] l-Isopropyl-2-oxo-benzimidazole-4-carboxylic acid amide (1.67 g, 8.0 mmol, 1.0 eq.) and dioxane (18 mL) were charged into a pressure tube. Lawesson's reagent (2.46 g, 6.0 mmol, 0.8 eq.) was added and the reaction mixture heated at 85°C for 50 minutes. The reaction mixture was left to cool down to ambient temperature and the solvent removed in vacuo. The residue was purified by flash column chromatography using a ethyl acetate / heptanes gradient to give 1.35 g (51% corrected yield) of the desired product as yellow oil which was used in the next step without further purification. • 1H NMR (500 MHz, CDC13) δ ppm 9.66 (br. s., 1 H) 7.26 (d, J=7.78 Hz, 1 H) 7.18 (d, J=7.78 Hz, 1 H) 7.07 (m, J=7.78, 7.78 Hz, 1 H) 6.00 (br. s., 2 H) 4.75 (spt, J=6.99 Hz, 1 H) 1.55 (d, J=7.02 Hz, 6 H).
• LC-MS: purity 68% (UV), tR 1.52 min m/z [M+H]+ 235.95 (MET/CR/1278).
STAGE 5A: PREPARATION OF l-ISOPROPYL-2-OXO-4-(4-CYCLOPROPYL-
THIAZOL-2-YL)-BENZIMIDAZOLE
Figure imgf000054_0001
[0166] l-Isopropyl-2-oxo-benzimidazole-4-carbothioic acid amide (200 mg, 0.85 mmol, 1.0 eq.) and N,N-dimethylformamide (2 mL) were charged into a 10 mL vial. 1- Cyclopropyl-2-bromoethanone (138 mg, 0.85 mmol, 1.0 eq.) was added and the reaction mixture stirred at ambient temperature for 15 hours. Saturated aqueous sodium hydrogencarbonate (2 mL) was added and the reaction mixture extracted with ethyl acetate (3 x 2 mL). The organic extracts were combined, washed with water (2 x 2mL), dried over magnesium sulfate, filter and the solvent removed in vacuo to give 125 mg (45% yield) of the desired product as a yellow oil which was used in the next step without any further purification.
1H NMR (500 MHz, CDC13) δ ppm 9.65 (br. s., 1 H) 7.38 (d, J=7.78 Hz, 1 H) 7.10 - 7.18 (m, 1 H) 7.00 - 7.10 (m, 1 H) 6.81 (s, 1 H) 4.70 - 4.82 (m, 1 H) 1.99 - 2.22 (m, 1 H) 1.56 (d, J=7.02 Hz, 6 H) 1.05 - 1.16 (m, 2 H) 0.89 - 1.04 (m, 2 H).
LC-MS: purity 92% (UV), tR 2.31 min m/z [M+H]+ 299.95 (MET/CR/1278).
STAGE 6A: PREPARATION OF l-ISOPROPYL-2-CHLORO-4-(4-CYCLOPROPYL-
THIAZOL-2-YL)- BENZIMIDAZOLE
Figure imgf000054_0002
[0167] l-Isopropyl-2-oxo-4-(4-cyclopropyl-thiazol-2-yl)-l,3-dihydro- benzoimidazole (400 mg, 1.0 mmol, 1.0 eq.) and phosphorous oxychloride (4 mL) were heated at 85°C for 15 hours. The reaction mixture was left to cool down to ambient temperature and the solvent removed in vacuo. Aqueous potassium carbonate (5 mL) was added to the residue then more potassium carbonate solution was added until pH=7. The aqueous phase was extracted with ethyl acetate (3 x 2 mL). The organic extracts were combined, washed with aqueous potassium carbonate (2 mL), dried over magnesium sulfate, filtered and the solvent removed in vacuo to give 191 mg (40% yield) of the desired product as a brown oil which was used in the next step without any further purification.
• 1H NMR (500 MHz, CDC13) δ ppm 8.23 (d, 7=7.78 Hz, 1 H) 7.52 (d, 7=8.09 Hz, 1 H) 7.34 (t, 7=8.01 Hz, 1 H) 6.98 (s, 1 H) 4.96 (spt, 7=6.99 Hz, 1 H) 2.13 - 2.24 (m, 1 H) 1.68 (d, 7=7.02 Hz, 6 H) 0.93 - 1.04 (m, 4 H).
• LC-MS: purity 87% (UV), tR 2.52 min m/z [M+H]+ 318.00 (MET/CR/1278).
EXAMPLE II-B FINAL STEP IN PREPARATION OF COMPOUND 4
Figure imgf000055_0001
Compound 4
REACTION SCHEME:
Figure imgf000056_0001
Compound 4
[0168] Compound 4 was made using the same procedure for making Compound 3 ple IB above.
• 36 mg (24% yield) as a white solid after preparative HPLC.
• 1H NMR (500 MHz, CDC13) δ ppm 9.70 - 9.87 (m, 1 H) 8.15 (d, J=7.48 Hz, 1 H) 7.29 (d, J=7.78 Hz, 1 H) 7.22 (t, J=7.93 Hz, 1 H) 7.11 (br. s., 1 H) 7.00 (s, 1 H) 5.89 (br. s., 1 H) 5.66 - 5.76 (m, 1 H) 5.28 (d, J=17.09 Hz, 1 H) 5.24 (d, J=9.61 Hz, 1 H) 5.17 (d, J=10.53 Hz, 1 H) 4.61 (spt, J=7.16 Hz, 1 H) 4.54 (t, J=7.86 Hz, 1 H) 4.37 (d, J=11.75 Hz, 1 H) 4.29 (d, J=9.46 Hz, 1 H) 4.17 (dd, J=11.83, 4.35 Hz, 1 H) 3.22 (spt, J=6.79 Hz, 1 H) 2.76 - 2.84 (m, 1 H) 2.67 - 2.76 (m, 1 H) 2.09 (q, J=8.80 Hz, 1 H) 1.96 (dd, J=7.17, 6.56 Hz, 1 H) 1.69 - 1.76 (m, 1 H) 1.65 - 1.69 (m, 1 H) 1.54 (dd, J=6.87, 3.36 Hz, 6 H) 1.51 (s, 3 H) 1.41 (s, 9 H) 1.39 (d, J=6.87 Hz, 6 H) 1.05 (s, 9 H) 0.98 - 1.04 (m, 1 H) 0.80 - 0.91 (m, 2 H).
• LC-MS: purity 100% (UV), tR 4.90 min m/z [M+H]+ 854.35 (MET/CR/1426).
• HRMS: Found: 854.3962, calculated for C42H59N7O8S2 (M+H)+: 854.3945. EXAMPLE III - PREPARATION OF COMPOUND 5
Figure imgf000057_0001
Compound 5
[0169] Compound 5 was made using the same procedure for making Compound 3 ple IB above.
• 32 mg (48% yield) as an off-white solid after preparative HPLC.
• 1H NMR (500 MHz, CDC13) δ ppm 8.19 (dd, J=7.40, 1.30 Hz, 1 H) 7.26 - 7.29 (m, 2 H) 7.26 (d, J=7.48 Hz, 1 H) 7.22 - 7.25 (m, 1 H) 7.09 (br. s., 1 H) 7.03 (s, 1 H) 6.90 (br. s., 1 H) 6.63 (d, J=7.63 Hz, 1 H) 6.45 (d, J=7.78 Hz, 1 H) 6.33 - 6.38 (m, 1 H) 5.76 - 5.85 (m, 1 H) 5.70 (br. s., 1 H) 5.27 (d, J=16.63 Hz, 1 H) 5.14 (d, J=9.92 Hz, 1 H) 4.62 (d, J=12.21 Hz, 1 H) 4.55 (t, J=8.01 Hz, 1 H) 4.32 (dt, J=13.58, 6.79 Hz, 1 H) 4.15 (dd, J=11.75, 3.36 Hz, 1 H) 4.04 (s, 1 H) 3.24 (spt, J=6.94 Hz, 1 H) 2.58 - 2.66 (m, 2 H) 2.07 - 2.15 (m, 1 H) 1.99 - 2.04 (m, 1 H) 1.46 - 1.51 (m, 1 H) 1.41 (dd, J=6.87, 1.83 Hz, 6 H) 1.28 (d, J=6.87 Hz, 3 H) 1.18 (d, J=6.87 Hz, 3 H) 1.08 (s, 9 H).
• LC-MS: purity 95% (UV), tR 4.85 min m/z [M+H]+ 781.95 (MET/CR/1426).
• HRMS: Found: 781.335, calculated for C4oH47F3N605S (M+H)+: 781.335. EXAMPLE IV - PREPARATION OF COMPOUND 6
Figure imgf000058_0001
Compound
[0170] Compound 6 was made using the same procedure for making Compound 3 ple IB above.
• 10 mg (49% yield) as a white solid after preparative HPLC.
• 1H NMR (500 MHz, CDC13) δ ppm 8.14 (d, J=7.63 Hz, 1 H) 7.29 (s, 1 H) 7.18 - 7.24 (m, 2 H) 6.97 (s, 1 H) 5.71 - 5.94 (m, 3 H) 5.25 (d, J=16.94 Hz, 1 H) 5.10 (d, J=10.38 Hz, 1 H) 4.50 - 4.64 (m, 2 H) 4.31 - 4.41 (m, 2 H) 4.01 - 4.18 (m, 2 H) 3.21 (spt, J=6.94 Hz, 1 H) 2.65 - 2.81 (m, 2 H) 2.06 - 2.16 (m, 1 H) 1.98 - 2.05 (m, 1 H) 1.50 - 1.55 (m, 6 H) 1.42 - 1.45 (m, 1 H) 1.41 (s, 9 H) 1.38 (d, J=6.87 Hz, 6 H) 1.04 (s, 9 H).
• LC-MS: purity 97% (UV), tR 4.48 min m/z [M+H]+ 737.70 (MET/CR/1426).
• HRMS: Found: 737.3686, calculated for QgHsiNeCvS (M+H)+: 737.3696.
EXAMPLE V PREPARATION OF COMPOUND 7
Figure imgf000059_0001
Compound 7
[0171] Compound 7 was made using the same procedure for making Compound 3 ple IB above.
• 57 mg (29% yield) as a white solid after preparative HPLC.
• 1H NMR (500 MHz, CDC13) δ ppm 9.74 (br. s., 1 H) 8.05 (s, 1 H) 7.76 (d, J=8.70 Hz, 1 H) 7.58 - 7.64 (m, 2 H) 7.51 (dd, J=7.55, 3.28 Hz, 3 H) 7.21 (br. s., 1 H) 7.00 (d, J=1.68 Hz, 1 H) 6.90 (d, J=9.61 Hz, 1 H) 5.69 (dt, J=17.70, 8.85 Hz, 1 H) 5.26 (d, J=16.78 Hz, 1 H) 5.15 (d, J=10.38 Hz, 1 H) 5.03 (br. s., 1 H) 4.53 (t, J=7.71 Hz, 1 H) 4.29 (d, J=9.77 Hz, 1 H) 4.22 (d, J=11.90 Hz, 1 H) 3.87 (dd, J=10.99, 3.20 Hz, 1 H) 2.39 - 2.52 (m, 2 H) 2.09 (q, J=8.29 Hz, 1 H) 1.94 (t, J=6.79 Hz, 1 H) 1.69 - 1.75 (m, 1 H) 1.50 (s, 3 H) 1.43 (d, J=5.49 Hz, 1 H) 1.38 (s, 9 H) 1.24 - 1.31 (m, 1 H) 1.05 (d, J=7.78 Hz, 1 H) 1.01 (s, 9 H) 0.80 - 0.90 (m, 2 H).
• LC-MS: purity 97% (UV), tR 3.74 min m/z [M+H]+ 764.05 (MET/CR/1426).
• HRMS: Found: 763.3480, calculated for QgHsoNeOsS (M+H)+: 763.3489.
EXAMPLE VI PREPARATION OF COMPOUND 8
Figure imgf000060_0001
Compound 8
[0172] Compound 8 was made using the same procedure for making Compound 3 mple IB above.
• 36 mg (18% yield) as a white solid after preparative HPLC.
1H NMR (500 MHz, CDC13) δ ppm 9.80 (br. s., 1 H) 8.06 (s, 1 H) 7.74 (d, J=8.85 Hz, 1 H) 7.57 - 7.62 (m, 2 H) 7.48 - 7.52 (m, 1 H) 7.45 (d, J=8.39 Hz, 2 H) 7.19 (br. s., 1 H) 7.06 (t, J=7.86 Hz, 1 H) 6.91 (d, J=2.14 Hz, 1 H) 6.86 (d, J=7.63 Hz, 1 H) 6.82 (br. s., 1 H) 6.80 (dd, J=8.85, 2.29 Hz, 1 H) 6.64 (d, J=8.24 Hz, 1 H) 5.67 (ddd, J=17.32, 9.92, 8.93 Hz, 1 H) 5.25 (d, J=17.24 Hz, 1 H) 5.15 (d, J=10.53 Hz, 1 H) 5.09 (br. s., 1 H) 4.60 (d, J=10.07 Hz, 1 H) 4.47 (t, J=8.09 Hz, 1 H) 4.06 (d, J=11.44 Hz, 1 H) 3.94 (d, J=9.92 Hz, 2 H) 2.37 - 2.49 (m, 2 H) 2.07 (q, J=8.65 Hz, 1 H) 1.93 (dd, J=8.01, 6.03 Hz, 1 H) 1.70 - 1.75 (m, 1 H) 1.49 (s, 3 H) 1.38 (dd, J=9.38, 5.87 Hz, 1 H) 1.11 - 1.14 (m, 1 H) 1.10 (s, 9 H) 0.80 - 0.89 (m, 2 H).
LC-MS: purity 100% (UV), tR 4.08 min m/z [M+H]+ 808.10 (MET/CR/1426). HRMS: Found: 807.3157, calculated for C4iH45F3N606S (M+H)+: 807.3152.
EXAMPLE VII-1 PREPARATIO OUND 101
Figure imgf000060_0002
toluene, 1 10°C
a 9b 9d
Figure imgf000061_0001
-60- [0173] Compound 9a (2.0 g, 15.267 mmol) was dissolved in a solution of HC1 (g) in MeOH (4 M, 50 mL). The mixture was heated to reflux overnight. The mixture was concentrated under reduced pressure to give crude compound 9b (2.7 g, yield 97%) as white solid. 1H NMR (300MHz, DMSO-d6): δ 8.57 (brs, 2H), 3.74 (s, 3H), 3.74-3.68 (m, 1H), 1.00 (s, 9H).
[0174] A flask was charged with compound 9b (543 mg, 3.75 mmol), compound 3 (455 mg, 2.5 mmol), BINAP (156 mg, 0.25mmol), Cs2C03 (1.793 g, 5.5 mmol) and toluene (15 mL), and then it was purged with nitrogen three times. Pd(OAc)2 (67.1 mg, 0.275 mmol) was added thereto. The reaction mixture was stirred at 110°C for 5 hrs. TLC (PE/EA =5/1) showed compound 9c was consumed. After being cooled to room temperature, the mixture was diluted with water (30 mL), extracted with ethyl acetate (20 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo. The residue was purified by prep-TLC (PE/EA =5/1) to provide compound 9d (205 mg, yield 33%) as yellow oil. 1H NMR (300MHz, CDC13): δ 1.34-121 (m, 1H), 7.05 (d, 7 =7.5 Hz, 1H), 6.93- 6.87 (m, 2H), 4.44 (d, 7 =10.2 Hz, 1H), 3.83 (d, 7 =10.2 Hz, 1H), 3.77 (s, 3H), 1.11 (s, 9H).
[0175] A flask was charged with compound 9d (230 mg, 0.934 mmol), TEA'HCl (383 mg, 2.8 mmol), NaN3 (182 mg, 2.8 mmol) and toluene (5 mL). The resulting mixture was stirred at 120°C overnight. TLC (PE/EA =1/5) showed compound 9d was consumed. After being cooled to room temperature, the mixture was quenched with water (20 mL), extracted with ethyl acetate (20 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo. The residue was purified by prep-TLC (DCM/MeOH =15/1) to afford compound 9e (80 mg, yield 30%) as white solid. 1H NMR (400MHz, DMSO-d6): δ 7.40 (s, 1H), 7.29-7.16 (m, 2H), 6.85 (d, 7 =8.0 Hz, 1H), 6.05 (d, 7 =10 Hz, 1H), 3.87 (d, 7 =10 Hz, 1H), 3.62 (s, 3H), 1.05 (s, 9H).
[0176] Compound 9e (290 mg, 1 mmol) was dissolved in THF (15 mL) and H20 (3 mL). To the solution was added LiOH monohydrate (444 mg, 10.6 mmol) in portions. The reaction mixture was stirred at room temperature for 2 days. LCMS showed compound 9e was consumed. The solvent was evaporated, and the aqueous layer was acidified to pH=3-4 with aq. HC1 (1 M), then extracted with ethyl acetate (20 mL x 3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo to give compound 9f (260 mg, yield 94%) as light red solid. MS (ESI) m / z (M+H)+ 275.9.
[0177] To a suspension of compound 9g (3.0 g, 12.99 mmol) in DMSO (60 mL) was added KOi-Bu (3.2 g, 28.6 mmol) at 0°C. The generated mixture was stirred for 15 min and then the compound 9h (4.74 g, 14.3 mmol) was added in one portion. The reaction was stirred at room temperature overnight. The reaction mixture was quenched with ice- water (30 mL), acidified to pH=3-4 with aq. citric acid (5%). The formed solid was collected by filtration and dried under vacuum to provide compound 9i (5.81g, yield 85%) as pale yellow solid. MS (ESI) m / z (M+H)+ 528.1.
[0178] To a solution of compound 9i (3.62 g, 6.86 mmol) in dry DCM (40 mL) was added compound 9j (2.62 g, 13.7 mmol), followed by adding HATU (3.91 g, 10.29 mmol) and DIEA (3.54 g, 27.44mmol). The reaction mixture was stirred at room temperature overnight. The solvent was evaporated, the resulting residue was diluted with EtOAc (50 mL), washed with aq. citric acid (5%) and saturated aq. NaHC03, followed by brine. The organic layer was dried over Na2S04, concentrated in vacuo to provide compound 9k (4.42 g, yield 97%) as yellow solid. MS (ESI) m / z (M+H)+ 665.3.
[0179] Compound 9k (2 g, 3.01 mmol) was dissolved in THF (30 mL) and H20 (6 mL). To the solution was added NaOH (1.20 g, 30.1 mmol) in portions. The reaction mixture was stirred at r.t for 2 days. LCMS showed compound 9k was consumed. The solvent was evaporated, and the aqueous layer was acidified to pH=3-4 with aq. HCl (1 M), then extracted with ethyl acetate (80 mL x 3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo to give compound 91 (1.89 g, yield 99%). MS (ESI) m / z (M+H)+ 637.1.
[0180] A mixture of compound 91 (200 mg, 0.314 mmol) and CDI (203 mg, 1.258 mmol) in DCM (10 mL) was refluxed for 3 hours under nitrogen atmosphere, then 1- methylcyclopropane-sulfonamide (339 mg, 2.517 mmol) and DBU (143 mg, 0.942 mmol) was added. The resulting mixture was stirred under reflux overnight. After the material was consumed, the reaction mixture was cooled to room temperature, diluted with water (30 mL) and extracted with EtOAc (50 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo. The residue was purified by prep-TLC (DCM/MeOH =15/1) to provide compound 9m (100 mg, yield 42%). MS (ESI) m / z (M+H)+ 754.0.
[0181] Compound 9m (90 mg, 0.119 mmol) was dissolved in DCM (5 mL) and TFA (0.3 mL). The reaction mixture was stirred at room temperature overnight. The mixture was concentrated to afford compound 9n, which was used in next step without further purification. [0182] A flask was charged with compound 6 (84 mg, 0.306 mmol), HATU (105 mg, 0.275 mmol), DIEA (80 mg, 0.6126 mmol) and dry DCM (5 mL), followed by a solution of compound 9n (100 mg, 0.157 mmol, neutralized by addition of DIEA) in dry DCM (5 mL). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with water, neutralized with aq. citric acid (5%), extracted with EtOAc (20 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo. The residue was purified by prep-HPLC to afford compound 101 (55 mg, yield 39%). MS (ESI) m l z (M+H)+ 911.4 -2 PREPARATION OF COMPOUND 102
Figure imgf000064_0001
102
[0183] A flask was charged with compound 10a (1.58 g, 7.86 mmol) and DCM (20 mL) to afford a mixture. Oxalyl chloride (2 g, 15.7 mmol) was added dropwise to the mixture at room temperature, followed by one drop of DMF. The resulting mixture was stirred at room temperature for 2 hrs. And then the mixture was concentrated to give compound 10b (1.7 g, yield 100%), which was used directly in the next step.
[0184] To a solution of compound 10c (1.46 g, 7.86 mmol) and TEA (1.19 g, 11.7 mmol) in DCM (20 mL) was added a solution of compound 10b ( 1.7 g, 7.86 mmol) in DCM dropwise at 0°C. The mixture was stirred at room temperature for 3 hrs. The mixture was poured into water and neutralized with aq. HC1 (1 N), extracted with EtOAc (50 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo to give compound lOd (2.9 g, yield 100%).
[0185] A flask was charged with compound lOd (2.9 g, 7.8mmol), TFA (3 mL) and DCM (10 mL) to afford a mixture. The mixture was stirred at room temperature for 4 hrs. The mixture was concentrated and the residue was diluted with water (30 mL) and basified with saturated aq. NaHC03 to pH=8, extracted with EtOAc (50 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo to give compound lOe (1.4 g, yield 66%).
[0186] A flask was charged with compound lOe (183 mg, 0.683 mmol), TEA (89 mg, 0.888 mmol) and DCM (4 mL). To the resulting mixture was added methyl chloroformate (85 mg, 0.888 mmol) at 0°C. The mixture was stirred for 2 hrs at room temperature. Then the mixture was was diluted with water (20 mL), extracted with EtOAc (20 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo to give compound lOf (200 mg, yield 90%).
[0187] A flask was charged with compound lOf (1.07 g, 3.28 mmol), compound 9b (570 mg, 3.936 mmol), BINAP (204 mg, 0.328 mmol), Cs2C03 (2.3 g, 7.2 mmol) and toluene (30 mL), and then it was purged with nitrogen three times. Pd(OAc)2 (103 mg, 0.459 mmol) was added thereto. The resulting mixture was stirred at 110°C for 24 hrs under N2 atmosphere. After being cooled to r.t., the mixture was diluted with water (30 mL), extracted with ethyl acetate (50 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo. The residue was purified by column chromatography on silica gel (PE/EA =3/1) to provide compound lOg (629 mg, yield 49%). MS (ESI) m / z (M+H)+ 392.2.
[0188] Compound lOg (500 mg, 1.3 mmol) was dissolved in MeOH (10 mL) and H20 (5 mL). To the solution was added NaOH (189 mg, 4.73 mmol) in portions. The reaction mixture was stirred at 40°C overnight. LCMS showed compound lOg was consumed. The solvent was evaporated, and the aqueous layer was acidified to pH=5-6 with aq. HC1 (1 M), then extracted with ethyl acetate (30 mL x 3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo to give compound lOh (380 mg, yield 78%). MS (ESI) m / z (M+H)+ 378.2.
[0189] A flask was charged with compound 9n (47 mg, 0.072 mmol), compound lOh (35 mg, 0.093 mmol), HATU (54 mg, 0.144 mmol), DIEA (37mg, 0.288mmol) and dry DCM (5 mL). The reaction mixture was stirred at r.t. overnight. The mixture was diluted with water, neutralized with aq. HCl (1 M), extracted with EtOAc (20 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo. The residue was purified by prep-HPLC to afford compound 102 (10 mg, yield 13%). MS (ESI) m / z (M+H)+ 1013.2. -3 PREPARATION OF COMPOUND 103
Figure imgf000066_0001
[0190] A flask was charged with compound 11a (820 mg, 2.22 mmol), compound 9b (386 mg, 2.67 mmol), BINAP (138 mg, 0.222 mmol), Cs2C03 (1.6 g, 4.9 mmol) and toluene (30 mL), and then it was purged with nitrogen three times. Pd(OAc)2 (70 mg, 0.311 mmol) was added thereto. The reaction mixture was stirred at 110°C for 24 hrs under N2 atmosphere. After being cooled to r.t., the mixture was diluted with water (30 mL), extracted with ethyl acetate (50 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo. The residue was purified by column chromatography on silica gel (PE/EA =3/1) to provide compound lib (300 mg, yield 31%). MS (ESI) m / z (M+H)+ 434.2.
[0191] Compound lib (360 mg, 0.831 mmol) was dissolved in MeOH (10 mL) and H20 (5 mL). To the solution was added NaOH (130 mg, 3.32 mmol) in portions. The reaction mixture was stirred at 40°C overnight. LCMS showed compound lib was consumed. The solvent was evaporated, and the aqueous layer was acidified to pH=5-6 with aq. HC1 (1 M), then extracted with ethyl acetate (30 mL x 3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo to give compound 11c (300 mg, yield 86%). MS (ESI) m / z (M+H)+ 420.2.
[0192] A flask was charged with compound 9n (165 mg, 0.253 mmol), compound 11c (105 mg, 0.253 mmol), HATU (190 mg, 0.506 mmol), DIEA (129 mg, 1 mmol) and dry DCM (5 mL). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with water, neutralized with aq. HC1 (1 M), extracted with EtOAc (30 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo. The residue was purified by prep-HPLC to afford compound lid (45 mg, yield 17%). MS (ESI) m / z (M+H)+ 1055.5.
[0193] A flask was charged with compound lid (45 mg, 0.0427 mmol) and a solution of HC1 (g) in EtOAc (4 M, 3 mL). The mixture was stirred at room temperature for 2 hrs. The mixture was concentrated to give compound 103 as HC1 salt (30 mg, yield 70%). MS (ESI) m / z (M+H)+ 955.4. -4 PREPARATION OF COMPOUND 104
Figure imgf000067_0001
Figure imgf000068_0001
[0194] To a solution of compound 12a (2 g, 20 mmol) and TEA (3.64 g, 36 mmol) in DCM (100 mL) was added a solution of compound 10b (5.72 g, 26 mmol) in DCM dropwise at 0°C. The mixture was stirred at room temperature for 3 hrs. The mixture was poured into water and neutralized with aq. HCl (1 N), extracted with EtOAc (80 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo to give compound 12b (5.97 g, yield 80%).
[0195] A flask was charged with compound 12b (600 mg, 2.12 mmol), compound 9b (400 mg, 2.75 mmol), BINAP (130 mg, 0.212 mmol), Cs2C03 (1.52 g, 4.66 mmol) and toluene (20 mL), and then it was purged with nitrogen three times. Pd(OAc)2 (60 mg, 0.297 mmol) was added thereto. The reaction mixture was stirred at 110°C for 24 hrs under N2 atmosphere. After being cooled to r.t., the mixture was diluted with water (30 mL), extracted with ethyl acetate (50 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo. The residue was purified by column chromatography on silica gel (PE/EA =3/1) to provide compound 12c (230 mg, yield 31%). MS (ESI) m / z (M+H)+ 348.2.
[0196] Compound 12c (280 mg, 0.8 mmol) was dissolved in MeOH (10 mL) and H20 (5 mL). To the solution was added NaOH (130 mg, 3.32 mmol) in portions. The reaction mixture was stirred at 40°C overnight. The solvent was evaporated, and the aqueous layer was acidified to pH=5-6 with aq. HCl (1 M), then extracted with ethyl acetate (30 mL x 3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo to give compound 12d (100 mg, yield 38%). MS (ESI) m / z (M+H)+ 334.2. [0197] A flask was charged with compound 9n (160 mg, 0.245 mmol), compound 12d (81 mg, 0.245 mmol), HATU (186 mg, 0.49 mmol), DIEA (126 mg, 0.98 mmol) and dry DCM (5 mL). The reaction mixture was stirred at r.t. overnight. The mixture was diluted with water, neutralized with aq. HCl (1 M), extracted with EtOAc (30 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo. The residue was purified by prep-HPLC to afford compound 104 (40 mg, yield 16%). MS (ESI) m / z (M+H)+ 969.4. -5 PREPARATION OF COMPOUND 105
Figure imgf000069_0001
9n 105
[0198] A flask was charged with compound 13a (2.2 g, 10 mmol), compound 13b (1.9 g, 20 mmol), K2C03 (5.52 g, 40 mmol), Nal (1.5 g, 10 mmol) and DMF (15 mL). The reaction mixture was stirred at 100°C for 17 hrs under N2 protection. After cooling, the mixture was diluted with water, extracted with EtOAc (80 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo. The residue compound 13c was used directly in the next step.
[0199] A flask was charged with compound 13c (1.11 g, 4 mmol), compound 9b (0.87 g, 6 mmol), BINAP (500 mg, 0.8 mmol), Cs2C03 (5.2g, 15mmol) and toluene (15 mL), and then it was purged with nitrogen three times. Pd(OAc)2 (267 mg, 1.2 mmol) was added thereto. The reaction mixture was stirred at 110°C for 18 hrs under N2 atmosphere. After being cooled to r.t., the mixture was diluted with water (30 mL), extracted with ethyl acetate (50 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo. The residue was purified by column chromatography on silica gel (PE/EA =5/1) to provide compound 13d (110 mg, yield 10%). MS (ESI) m / z (M+H)+ 295.9.
[0200] Compound 13d (110 mg, 0.373 mmol) was dissolved in MeOH (4 mL) and H20 (1 mL). To the solution was added NaOH (450 mg, 1.11 mmol) in portions. The reaction mixture was stirred at 40°C overnight. The solvent was evaporated and diluted with water (20 mL), the aqueous layer was acidified to pH=5-6 with aq. HCl (1 M), then extracted with ethyl acetate (30 mL x 3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo to give compound 13e (50 mg, yield 48%). MS (ESI) m / z (M+H)+ 282.0.
[0201] A flask was charged with compound 9n (65 mg, 0.1 mmol), compound 13e (30 mg, 0.1 mmol), HATU (76 mg, 0.2 mmol), DIEA (50 mg, 0.4 mmol) and dry DCM (5 mL). The reaction mixture was stirred at r.t. overnight. The mixture was diluted with water, neutralized with aq. HCl (1 M), extracted with EtOAc (30 mLx3). The combined organic layer was washed with brine, dried over Na2S04, concentrated in vacuo. The residue was purified by prep-HPLC to afford compound 105 (11 mg, yield 12%). MS (ESI) m / z (M+H)+ 917.5.
EXAMPLE VII-6 PREPARATION OF COMPOUND 106
Scheme VII-6
Figure imgf000071_0001
Scheme II-6A
Figure imgf000071_0002
13a 14a
[0202] To a solution of 3-iodophenol (13a, 2 g, 9.09 mmol) in dry THF (50 mL) was successively added 2-(dimethylamino) ethanol (0.97 g, 10.9 mmol), and PPh3 (3.6 g, 13.63 mmol). The mixture was stirred at room temperature for 10 min, then cooled to 0 °C. DIAD (2.75 g, 13.63 mmol) was added dropwise to the cooled mixture at such a speed that the internal temperature was maintained below 5°C. Upon completion of the addition, the mixture was allowed to warm to room temperature and stirred overnight. The mixture was treated with aq. HC1 (1 M) to adjust the pH to 2. The aqueous layer was extracted with EtOAc (50 mL) and organic phase was discarded. The aqueous layer was treated with aq. NaOH (1 M) to adjust the pH to 11. The aqueous layer was extracted EtOAc (50 mLx3). The combined organic phase was washed with brine, dried over anhydrous Na2S04, and concentrated to give crude compound 14a (2.2 g, yield 83%) as a light yellow oil. 1H NMR (400MHz, CDC13): δ 7.28-7.27 (m, 2H), 6.98 (t, / = 8 Hz, 1H), 6.88 (m, 1H), 4.02 (t, / = 5.6 Hz, 2H), 2.70 (t, / = 5.6 Hz, 2H), 2.32 (s, 6H). Scheme VII-6B
Figure imgf000072_0001
[0203] A mixture of compound 14a (0.5 g, 1.718 mmol ), L-tert- Leucine (14b, 0.23 g, 1.718 mmol), CuBr«Me2S (71 mg, 0.3436 mmol), K2C03 (0.355 g, 2.577 mmol), and NMP (5 mL) was bubbled with nitrogen for 5 min, and then heated at 180°C for 1 hour by microwave. The mixture was filtered and the filtrate was acidified with aq. HCl (1 M) to pH= 4. The mixture was purified by prep-HPLC to give compound 14c (250 mg, yield 50%) as a light yellow solid. 1H NMR (400MHz, CDC13): δ 7.06-7.00 (m, 1H), 6.46-6.29 (m, 1H), 6.23-6.16 (m, 2H), 4.20-4.10 (m, 1H), 3.79 (m, 2H), 3.33 (m, 2H), 2.84 (s, 6H), 1.10 (s, 9H).
-6B
Figure imgf000072_0002
[0204] A flask was charged with compound 9n (222 mg, 0.34 mmol), DCM (8 mL), compound 14c (100 mg, 0.34 mmol), HATU (258 mg, 0.68 mmol) and DIEA (175 mg, 1.36 mmol). The mixture was stirred overnight at 35°C. Subsequently, the mixture was concentrated to dryness, diluted with water (20 mL) and the resulting aqueous mixture was acidified with aq. HCl (1 N) to pH=6. The mixture was extracted with EtOAc (30 mLx3), the combined organic layer was washed with brine, dried over Na2S04, and concentrated in vacuo to give a residue. The residue was purified by prep-HPLC to afford compound 106 (15 mg, yield 5%). MS (ESI) m / z (M+H)+ 930.5. HPLC Methods:
Figure imgf000073_0001
Figure imgf000073_0002
Figure imgf000073_0003
Column Symmetry Shield RP8
2.1 x 50mm, 3.5μπι column
40°C
Mobile phase A = Formic acid (aq) 0.1%
B = Formic acid (acetonitrile)
0.1%
Flow rate 0,6 mL/min
Injection 3μΙ.
volume
Detector 215nm (nominal)
Gradient Time (min) % Organic
0 5
5.0 100
7.00 100
7.10 5
Figure imgf000074_0001
EXAMPLE A: NS3-NS4 PROTEASE ASSAY
NS3 complex formation with NS4A-2.
[0205] Recombinant E. coli or Baculovirus full-length NS3 was diluted to 3.33μΜ 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. [0206] After equilibration to 4 °C, equal volumes of NS3 and NS4A-2 solutions were combined in an eppendorf tube, mixed gently with a manual pipettor, and incubated for 15 minutes in the 4 °C water bath. Final concentrations in the mixture are 1.67 μΜ NS3, 4.15 mM NS4A-2 (2485-fold molar excess NS4A-2).
[0207] After 15 minutes at 4 °C, the NS3/NS4A-2 eppendorf tube 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).
EXAMPLE B: NS3 INHIBITION ASSAY
[0208] 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 μΜ in the assay inhibition curve. Compounds were serial diluted in assay buffer to provide test solutions at lower concentrations.
[0209] Step 1. The E. coli. NS3/NS4A-2 was diluted to 4 nM NS3 (1:417.5 of 1.67μΜ stock - 18 uL 1.67 μΜ stock + 7497 μΐ. assay buffer). The BV NS3/NS4A-2 was diluted to 6nM NS3 (1:278.3 of 1.67 μΜ stock - 24 μΐ. 1.67 μΜ stock + 6655 μΐ. assay buffer).
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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.
[0215] While the plate from step 6 is pre-incubating, the RETS1 substrate was diluted in a 15mL polypropylene centrifuge tube. The RETS1 substrate was diluted to 8μΜ (1:80.75 of 646 μΜ stock - 65 μΐ. 646 μΜ stock + 5184 μΐ. assay buffer).
[0216] 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.
[0217] 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 λ: 335nm, Emission λ: 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
[0218] IC50S are determined using a four parameter curve fit equation, and converted to Ki's using the following Km's:
• Full-length E. coli NS3 - 2.03 μΜ
• Full-length BV NS3 - 1.74 μΜ
• where Ki = IC50/(l+[S]/Km))
Quantitation by ELISA of the selectable marker protein, Neomycin phosphotransferase II (NPTII) in the HCV Sub-Genomic Replicon, GS4.3
[0219] The 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). The replicon-containing cell culture, designated GS4.3, was obtained from Dr. Christoph Seeger of the Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania.
[0220] GS4.3 cells were maintained at 37 °C, 5%C02, 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 μg/mL geneticin (G418)(Gibco 10131-035). Cells were sub-divided 1:3 or 4 every 2-3 days. [0221] 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. Inhibitors were made as a 10X stock in Assay Medium, (3-fold dilutions from 10 μΜ 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.
[0222] An 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 μΜ 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. A standard curve was made. 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 μΕ-2.5 ^L. In addition, 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 300μΕ with PBS-T (0.5% Tween-20, PBS-T was supplied in the ELISA kit). For detection, 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.
[0223] Inhibitor effect was expressed as a percentage of DMSO-treated control signal, and inhibition curves were calculated using a 4-parameter equation: y=A+((B- A)/(l+((C/x)AD))), where C is half-maximal activity or EC50. EXAMPLES OF ACTIVITY:
Table 1 below provides examples of active compounds.
TABLE 1
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
A indicates an EC50 or IC50 > 100 nM
B indicates an EC50 or IC50 between 10 and 100 nM
C indicates an EC50 or IC50 of <10 nM Conclusion
[0225] Potent small molecule inhibitors of the HCV NS3 protease have been developed.
[0226] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

WHAT IS CLAIMED IS:
1. A compound represented by a formula (I):
Figure imgf000082_0001
or a pharmaceutically acceptable salt thereof,
wherein a dashed line represents the presence or absence of a bond;
X is NRX or O (oxygen); wherein Rx is H hydrogen) or Ci_6 hydrocarbyl;
Figure imgf000082_0002
wherein Z is NR6, CHR6, S (sulfur), or O (oxygen);
Z' is NR7, CHR7, S (sulfur), or O (oxygen);
Y is N (nitrogen) or CR14;
R is selected from the group consisting of optionally substituted heteroaryl, -C(0)OR2a, 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, C2-6 alkenyl, C2_6 alkynyl, -C(0)NR2bR2c, -NHC(0)NR2bR2c, -C(0)OR2d, heteroaryl, Ci_6 alkoxy optionally substituted with up to 5 fluoro, Ci-6 alkoxy optionally substituted by Ci_6 alkoxy, and Ci_6 alkoxy optionally substituted by -NR12R13;
R2a is selected from the group consisting of H (hydrogen), Ci_6 alkyl, cycloalkyl, and heterocyclyl;
R2b and R2c 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, C2_6 alkenyl, C2_6 alkynyl, optionally substituted C1-6 alkoxy, -C(0)OR2d, -C(0)R2e, optionally substituted aryl, and optionally substituted heteroaryl; R and R e are each separately selected from the group consisting of -H (hydrogen), Ci_4 alkoxy, Ci_6 alkyl, C3_7 cycloalkyl, aryl, arylalkyl and heteroaryl;
R3 is -H (hydrogen), -S02R9, or -SOaNR^R11;
R4 is -H (hydrogen) or C1-4 alkyl;
R5 is optionally substituted thiazole or -C(0)NR12R13;
R6 is -H (hydrogen) or C1-4 alkyl;
R' is C i_i2 hydrocarbyl, optionally substituted arylalkyl, optionally substituted aryl, or optionally substituted heteroaryl;
R9 is H (hydrogen) or C1-12 hydrocarbyl; and
R10 R11, R12, R13, and R14 are independently selected from H (hydrogen) or
C1-12 hydrocarbyl;
provided that the compound is not selected from the group consisting of:
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
-84-
Figure imgf000086_0001
2. The compound of claim 1, wherein X is NH.
3. The compound of claim 1, wherein X is O (oxygen).
4. The compound of any one of claims 1-3, wherein R is
Figure imgf000086_0002
5. The compound of claim 4, wherein R is an optionally substituted thiazole.
The compound of claim 5, wherein R5
7. The compound of claim 5, wherein R5 is
Figure imgf000086_0003
8. The compound of claim 4, wherein R5 is -C(0)NR12R13 .
9. The compound of claim 8, wherein R is
Figure imgf000086_0004
10. The compound of claim 4, wherein R6 is isopropyl
11. The compound of any one of claims 1-3, wherein R1 is
Figure imgf000087_0001
12. The compound of claim 11, wherein R is an optionally substituted aryl.
13. The compound of claim 12, wherein R is phenyl.
14. The compound of claims 1-13, wherein R2 is -C(0)OR2a or 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, Ci_6 alkoxy optionally substituted with up to 5 fluoro or optionally substituted by Ci_6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, -C(0)NR2bR2c, -NHC(0)NR2bR2c, -C(0)OR2d, and heteroaryl.
15. The compound of claim 14, wherein R2 is -C(0)OR2a.
16. The compound of claim 15, wherein R is -C(0)OiBu.
17. The compound of claim 14, wherein R is aryl optionally substituted with one or more substituents each independently selected from the group consisting of halo, Ci_6 alkyl optionally substituted with up to 5 fluoro, and Ci_6 alkoxy optionally substituted with up to 5 fluoro.
1 The compound of any one of claims 1-3, wherein R1 is
Figure imgf000087_0002
19. The compound of claim 18, wherein R2 is -C(0)OR2a or aryl optionally substituted with one or more substituents each independently selected from the group consisting of C2_6 alkenyl, C2_6 alkynyl, -C(0)NR2bR2c, -C(0)OR2d, heteroaryl, and Ci_6 alkoxy optionally substituted by Ci_6 alkoxy.
20. The compound of claim 18, wherein R is phenyl substituted with 1 to 3 substituents independently selected from the group consisting of: heteroaryl, -C(0)NR2bR2c, or Ci-6 alkoxy optionally substituted by Ci_6 alkoxy; wherein R2b and R2c are taken together with the nitrogen to which they are attached to form piperazinyl, wherein piperazinyl is optionally substituted with Ci_6 alkyl or -C(0)OR2d; wherein R2d is Ci_6 alkyl.
21. The compound of any one of claims 1-20, wherein X is NRX, and R3 is -
S02R9.
22. The compound of claim 21, wherein R9 is
Figure imgf000088_0001
(1-methylcyclopropyl) or
(cyclopropyl).
23. The compound of claim 21, wherein R9 is
Figure imgf000088_0002
(but-3- enyl)cyclopropyl .
24. The compound of claim 21, wherein R9 is
Figure imgf000088_0003
enyl)cyclopropyl .
25. The compound of any one of claims 1-20, wherein X is NRX, and R3 is -SO2NR10Rn wherein R10 and R11 are each independently Ci_6 alkyl.
26. The compound of any one of claims 1-20, wherein X is O (oxygen), and R is H (hydrogen).
27. The compound of any one of claims 1-26, wherein R4 is tert-butyl.
28. The compound of claim 1, wherein R is selected from the group consisting of optionally substituted heteroaryl, -C(0)OR2a, 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, Ci_6 alkoxy optionally substituted with up to 5 fluoro or optionally substituted by Ci_6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, -C(0)NR2bR2c, -NHC(0)NR2bR2c, -C(0)OR2d, and heteroaryl
29. The compound of any one of claims 1-28, wherein unless otherwise specified, groups indicated as "optionally substituted" are optionally substituted with one or more group(s) individually and independently selected from Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, aryl, heteroaryl, halo, cyano, hydroxy, Ci- C6 alkoxy, aryloxy, sulfhydryl, Ci-C6 alkylthio, arylthio, mono- and di-(Ci-C6)alkyl amino, quaternary ammonium salts, amino(Ci-C6)alkoxy, hydroxy(Ci-C6)alkylamino, amino(Ci- C6)alkylthio, cyanoamino, nitro, carbamyl, oxo, carbonyl, carboxy, sulfamyl, sulfonyl, sulfinyl, thiocarbonyl, and thiocarboxy.
30. The compound of claim 1 selected from:
Figure imgf000089_0001
-88-
Figure imgf000090_0001
-89-
Figure imgf000091_0001
-90-
Figure imgf000092_0001
(Compound 106).
31. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of any one of the preceding claims.
32. Use of the compound of any one of Claims 1-30 or a composition of Claim 31 for the manufacture of a medicament for treating HCV infection in an individual.
33. Use of the compound of any one of Claims 1-30 or a composition of Claim 31 for the manufacture of a medicament for treating liver fibrosis in an individual.
34. Use of the compound of any one of Claims 1-30 or a composition of Claim 31 for the manufacture of a medicament for increasing liver function in an individual having a hepatitis C virus infection.
35. A compound of any one of Claims 1-30 or a composition of Claim 31 for use in treating HCV infection in an individual.
36. A compound of any one of Claims 1-30 or a composition of Claim 31 for use in treating liver fibrosis in an individual.
37. A compound of any one of Claims 1-30 or a composition of Claim 31 for use in increasing liver function in an individual having a hepatitis C virus infection.
38. A method of treating HCV infection in an individual, the method comprising administering to the individual an effective amount of a compound of any one of Claims 1-30 or a composition of Claim 31.
39. The method of claim 38, further comprising identifying a subject suffering from a hepatitis C infection.
40. A method of treating liver fibrosis in an individual, the method comprising administering to the individual an effective amount of a compound of any one of claims 1-30 or a composition of claim 31.
41. The method of claim 40, further comprising identifying a subject suffering from a hepatitis C infection.
42. 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 of any one of claims 1-30 or a composition of claim 31.
43. The method of claim 42, further comprising identifying a subject suffering from a hepatitis C infection.
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