(19)
(11)EP 2 943 496 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
06.09.2017 Bulletin 2017/36

(21)Application number: 14701637.2

(22)Date of filing:  08.01.2014
(51)Int. Cl.: 
C07D 491/04  (2006.01)
C07D 498/04  (2006.01)
A61K 31/34  (2006.01)
(86)International application number:
PCT/US2014/010579
(87)International publication number:
WO 2014/110066 (17.07.2014 Gazette  2014/29)

(54)

MACROCYCLIC BENZOFURAN AND AZABENZOFURAN COMPOUNDS FOR THE TREATMENT OF HEPATITIS C

MAKROCYCLISCHE BENZOFURAN- UND AZABENZOFURANVERBINDUNGEN ZUR BEHANDLUNG VON HEPATITIS C

COMPOSÉS BENZOFURANE ET AZABENZOFURANE MACROCYCLIQUES POUR LE TRAITEMENT DE L'HÉPATITE C


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 10.01.2013 US 201361750967 P

(43)Date of publication of application:
18.11.2015 Bulletin 2015/47

(73)Proprietor: Bristol-Myers Squibb Company
Princeton, NJ 08543 (US)

(72)Inventors:
  • WANG, Tao
    Wallingford, Connecticut 06492 (US)
  • ZHANG, Zhongxing
    Wallingford, Connecticut 06492 (US)
  • KADOW, John F.
    Wallingford, Connecticut 06492 (US)

(74)Representative: Vögeli-Lange, Regina 
Swords Laboratories, Ireland Swiss Branch, Steinhausen Hinterbergstrasse 16
6330 Cham
6330 Cham (CH)


(56)References cited: : 
WO-A1-2009/101022
WO-A1-2011/106929
WO-A1-2012/003164
WO-A2-2004/041201
WO-A1-2010/030592
WO-A1-2011/112769
WO-A1-2012/078545
  
      
    Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


    Description

    FIELD OF THE INVENTION



    [0001] The invention relates to novel compounds, including their salts, which have activity against hepatitis C virus (HCV) and which are useful in treating those infected with HCV. The invention also relates to compositions and methods of making and using these compounds.

    BACKGROUND OF THE INVENTION



    [0002] Hepatitis C virus (HCV) is a major human pathogen, infecting an estimated 170 million persons worldwide - roughly five times the number infected by human immunodeficiency virus type 1. A substantial fraction of these HCV infected individuals develop serious progressive liver disease, including cirrhosis and hepatocellular carcinoma (Lauer, G. M.; Walker, B. D. N. Engl. J. Med. 2001, 345, 41-52).

    [0003] HCV is a positive-stranded RNA virus. Based on a comparison of the deduced amino acid sequence and the extensive similarity in the 5'-untranslated region, HCV has been classified as a separate genus in the Flaviviridae family. All members of the Flaviviridae family have enveloped virions that contain a positive stranded RNA genome encoding all known virus-specific proteins via translation of a single, uninterrupted, open reading frame.

    [0004] Considerable heterogeneity is found within the nucleotide and encoded amino acid sequence throughout the HCV genome. At least six major genotypes have been characterized, and more than 50 subtypes have been described. The major genotypes of HCV differ in their distribution worldwide, and the clinical significance of the genetic heterogeneity of HCV remains elusive despite numerous studies of the possible effect of genotypes on pathogenesis and therapy.

    [0005] The single strand HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non-structural (NS) proteins. In the case of HCV, the generation of mature non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. The first one is believed to be a metalloprotease and cleaves at the NS2-NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 (also referred to as NS3 protease) and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components. The complex formation of the NS3 protein with NS4A seems necessary to the processing events, enhancing the proteolytic efficiency at all of the sites. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B (also referred to as HCV polymerase) is a RNA-dependent RNA polymerase that is involved in the replication of HCV. The HCV NS5B protein is described in "Structural Analysis of the Hepatitis C Virus RNA Polymerase in Complex with Ribonucleotides (Bressanelli; S. et al., Journal of Virology 2002, 3482-3492; and Defrancesco and Rice, Clinics in Liver Disease 2003, 7, 211-242.

    [0006] Currently, the most effective HCV therapy employs a combination of alpha-interferon and ribavirin, leading to sustained efficacy in 40% of patients (Poynard, T. et al. Lancet 1998, 352, 1426-1432). Recent clinical results demonstrate that pegylated alpha-interferon is superior to unmodified alpha-interferon as monotherapy (Zeuzem, S. et al. N. Engl. J. Med. 2000, 343, 1666-1672). However, even with experimental therapeutic regimens involving combinations of pegylated alpha-interferon and ribavirin, a substantial fraction of patients do not have a sustained reduction in viral load. Thus, there is a clear and important need to develop effective therapeutics for treatment of HCV infection.

    [0007] HCV-796, an HCV NS5B inhibitor, has shown an ability to reduce HCV RNA levels in patients. The viral RNA levels decreased transiently and then rebounded during dosing when treatment was with the compound as a single agent but levels dropped more robustly when combined with the standard of care which is a form of interferon and ribavirin. The development of this compound was suspended due to hepatic toxicity observed during extended dosing of the combination regimens. US patent 7,265,152 and the corresponding PCT patent application WO2004/041201 describe compounds of the HCV-796 class. Other compounds have been disclosed; see for example, WO2009/101022, as well as WO 2012/058125.

    [0008] What is therefore needed in the art are additional compounds which are novel and effective against hepatitis C. Additionally, these compounds should provide advantages for pharmaceutical uses, for example, with regard to one or more of their mechanism of action, binding, inhibition efficacy, target selectivity, solubility, safety profiles, or bioavailability. Also needed are new formulations and methods of treatment which utilize these compounds.

    SUMMARY OF THE INVENTION



    [0009] One aspect of the invention is a compound of formula I, including pharmaceutically acceptable salts thereof:

    wherein

    n, m are 1;

    R1 is methyl;

    R2 is phenyl that is substituted with 1-2 halo

    R3 is hydrogen;

    R5 is selected from hydrogen, halo, alkyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, alkoxy, hydroxyalkyloxy, alkoxyalkyloxy, COOR11 and CON(R12)(R13);

    R4 and R6 are hydrogen;

    R11 is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy;

    R12, R13 are each independently hydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy; or

    R12 and R13 can form a ring by joining two atoms, one from each of R12 and R13;

    R14 is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy;

    R7 is hydrogen;

    R21 and R22 are independently hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl, alkylsulfonyl, or alkylsulfonylalkyl;

    or N(R21)(R22) taken together is azetidinyl, pyrrolidinyl, piperidinyl, or piperazinyl, and is substituted with 0-2 substituents selected from alkyl, hydroxyalkyl, and hydroxy;

    X, Y are independently selected from CR31R32, CO, O, NR33, NCN, S, SO, and S(O)2;

    R31, R32 are each independently alkyl or cycloalkyl, further substituted with 0-3 substituents; or

    R31 and R32 can form a ring by joining two atoms, one from each of R31 and R32;

    R33 is hydrogen, alkyl, cycloalkyl, or S(O)2R11 with 0-3 substituents selected from halo, haloalkoxy, OR11, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, and NR14CONR12R13;

    Z is an alkylene or alkenylene chain containing 0-12 groups selected from O, NR41, S, S(O), S(O)2, C(O), C(O)O, C(O)NR41, C(S)NR41, OC(O)NR41, NR41C(O)NR42, and Ar2, provided that any O or S atom does not directly bond to another O or S atom, such that ring A is 11-24 membered; and further wherein the alkylene or alkenylene chain is substituted with 0-6 substituents selected from the group of alkyl, cycloalkyl, halo, alkoxy, haloalkyl, OH, OR11, haloalkoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, NR14CONR12R13, and Ar3;

    R41, R42 is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo, haloalkoxy, OR11, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, and NR14CONR12R13;

    Ar2 is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and is substituted with 0-3 substituents selected from cyano, halo, alkyl, cycloalkyl, haloalkyl, OH, OR11, haloalkoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, and NR14CONR12R13;

    Ar3 is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and is substituted with 0-3 substituents selected from cyano, halo, alkyl, cycloalkyl, haloalkyl, OH, OR11, haloalkoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, and NR14CONR12R13.



    [0010] The invention also relates to pharmaceutical compositions comprising a compound of formula 1, including a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

    [0011] In addition, the invention provides one or more compounds according to the invention for the treatement of treating hepatitis C infection comprising administering a therapeutically effective amount of a compound of formula I to a patient.

    [0012] Also provided as part of the invention are one or more methods for making the compounds of formula I.

    [0013] The present invention is directed to these, as well as other important ends, hereinafter described.

    DETAILED DESCRIPTION OF THE EMBODIMENTS



    [0014] Unless otherwise specifically set forth elsewhere in the application, the following terms may be used herein and shall have the following meanings: "Hydrogen" or "H" refers to hydrogen, including its isotopes, such as deuterium. "Halo" means fluoro, chloro, bromo, or iodo. "Alkyl" means a straight or branched alkyl group composed of 1 to 6 carbons. "Alkenyl" means a straight or branched alkyl group composed of 2 to 6 carbons with at least one double bond. "Cycloalkyl" means a monocyclic ring system composed of 3 to 7 carbons. "Hydroxyalkyl," "alkoxy" and other terms with a substituted alkyl moiety include straight and branched isomers composed of 1 to 6 carbon atoms for the alkyl moiety. "Halo" includes all halogenated isomers from monohalo substituted to perhalo substituted in substituents defined with halo, for example, "Haloalkyl" and "haloalkoxy", "halophenyl", "halophenoxy." "Aryl" means a monocyclic or bicyclic aromatic hydrocarbon groups having 6 to 12 carbon atoms, or a bicyclic fused ring system wherein one or both of the rings is a phenyl group. Bicyclic fused ring systems consist of a phenyl group fused to a four- to six-membered aromatic or non-aromatic carbocyclic ring. Representative examples of aryl groups include, but are not limited to, indanyl, indenyl, naphthyl, phenyl, and tetrahydronaphthyl. "Heteroaryl" means a 5 to 7 membered monocyclic or 8 to 11 membered bicyclic aromatic ring system with 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Parenthetic and multiparenthetic terms are intended to clarify bonding relationships to those skilled in the art. For example, a term such as ((R)alkyl) means an alkyl substituent further substituted with the substituent R. Substituents which are illustrated by chemical drawing to bond at variable positions on a multiple ring system (for example a bicyclic ring system) are intended to bond to the ring where they are drawn to append.

    [0015] The invention includes all pharmaceutically acceptable salt forms of the compounds. Pharmaceutically acceptable salts are those in which the counter ions do not contribute significantly to the physiological activity or toxicity of the compounds and as such function as pharmacological equivalents. These salts can be made according to common organic techniques employing commercially available reagents. Some anionic salt forms include acetate, acistrate, besylate, bromide, camsylate, chloride, citrate, fumarate, glucouronate, hydrobromide, hydrochloride, hydroiodide, iodide, lactate, maleate, mesylate, nitrate, pamoate, phosphate, succinate, sulfate, tartrate, tosylate, and xinofoate. Some cationic salt forms include ammonium, aluminum, benzathine, bismuth, calcium, choline, diethylamine, diethanolamine, lithium, magnesium, meglumine, 4-phenylcyclohexylamine, piperazine, potassium, sodium, tromethamine, and zinc.

    [0016] Some of the compounds of the invention possess asymmetric carbon atoms. The invention includes all stereoisomeric forms, including enantiomers and diastereomers as well as mixtures of stereoisomers such as racemates. Some stereoisomers can be made using methods known in the art. Stereoisomeric mixtures of the compounds and related intermediates can be separated into individual isomers according to methods commonly known in the art. The use of wedges or hashes in the depictions of molecular structures in the following schemes and tables is intended only to indicate relative stereochemistry, and should not be interpreted as implying absolute stereochemical assignments.

    [0017] The invention is intended to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include deuterium and tritium. Isotopes of carbon include 13C and 14C. Isotopically-labeled compounds of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed. Such compounds may have a variety of potential uses, for example as standards and reagents in determining biological activity. In the case of stable isotopes, such compounds may have the potential to favorably modify biological, pharmacological, or pharmacokinetic properties.

    [0018] As set forth above, the invention is directed to one or more compounds of formula I, including pharmaceutically acceptable salts thereof:

    wherein

    n, m are 1;

    R1 is methyl;

    R2 is phenyl that is substituted with 1-2 halo

    R3 is hydrogen, halo, or alkyl;

    R5 is selected from hydrogen, halo, alkyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, alkoxy, hydroxyalkyloxy, alkoxyalkyloxy, COOR11 and CON(R12)(R13);

    R4 and R6 are hydrogen;

    R11 is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy;

    R12, R13 are each independently hydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy; or

    R12 and R13 can form a ring by joining two atoms, one from each of R12 and R13;

    R14 is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy;

    R7 is hydrogen;

    R21 and R22 are independently hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl, alkylsulfonyl, or alkylsulfonylalkyl;

    or N(R21)(R22) taken together is azetidinyl, pyrrolidinyl, piperidinyl, or piperazinyl, and is substituted with 0-2 substituents selected from alkyl, hydroxyalkyl, and hydroxy;

    X, Y are independently selected from CR31R32, CO, O, NR33, NCN, S, SO, and S(O)2;

    R31, R32 are each independently alkyl or cycloalkyl, further substituted with 0-3 substituents; or

    R31 and R32 can form a ring by joining two atoms, one from each of R31 and R32;

    R33 is hydrogen, alkyl, cycloalkyl, or S(O)2R11 with 0-3 substituents selected from halo, haloalkoxy, OR11, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, and NR14CONR12R13;

    Z is an alkylene or alkenylene chain containing 0-12 groups selected from the group consisting of O, NR41, S, S(O), S(O)2, C(O), C(O)O, C(O)NR41, C(S)NR41, O-C(O)NR41, NR41C(O)NR42, and Ar2, provided that any O or S atom does not directly bond to another O or S atom, such that ring A is 11-24 membered; and further wherein the alkylene or alkenylene chain is substituted with 0-6 substituents selected from alkyl, cycloalkyl, halo, alkoxy, haloalkyl, OH, OR11, haloalkoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, NR14CONR12R13, and Ar3;

    R41, R42 is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo, haloalkoxy, OR11, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, and NR14CONR12R13;

    Ar2 is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and is substituted with 0-3 substituents selected from cyano, halo, alkyl, cycloalkyl, haloalkyl, OH, OR11, haloalkoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, and NR14CONR12R13;

    Ar3 is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and is substituted with 0-3 substituents selected from cyano, halo, alkyl, cycloalkyl, haloalkyl, OH, OR11, haloalkoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, and NR14CONR12R13.



    [0019] It is also preferred that R2 is phenyl substituted with fluoro or F.

    [0020] In certain embodiments it is also preferred that R5 is CONR12R13.

    [0021] It is also preferred that X and Y are each NR33.

    [0022] Further preferred is the embodiment wherein R33 is hydrogen or is S(O)2CH3.

    [0023] It is also preferred that A be equal to or greater than 12 members. In other embodiments, it is preferred that A be equal to or greater than 15 members. In other embodiments, it is preferred that A be equal to or greater than 17 members. In yet other embodiments, it is preferred that A be equal to or greater than 19 members. By way of non-limiting example, a compound of the invention wherein ring A has 15 members could be represented as follows (with ring numbers included for illustration):



    [0024] Also preferred are compounds of formula I, including pharmaceutically acceptable salts thereof, which are selected from the group of:













    and


    Pharmaceutical Compositions and Methods of Treatment



    [0025] The compounds according to the various embodiments herein set forth demonstrate activity against HCV NS5B, and can be useful in treating HCV and HCV infection. Therefore, another aspect of the invention is a composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

    [0026] Another aspect of the invention is a composition further comprising an additional compound having anti-HCV activity.

    [0027] Another aspect of the invention is a composition where the compound having anti-HCV activity is an interferon or a ribavirin. Another aspect of the invention is wherein the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, interferon lambda, and lymphoblastoid interferon tau.

    [0028] Another aspect of the invention is a composition where the compound having anti-HCV activity is a cyclosporin. Another aspect of the invention is where the cyclosporin is cyclosporin A.

    [0029] Another aspect of the invention is a composition where the compound having anti-HCV activity is selected from the group consisting of interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, interfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine 5'-monophospate dehydrogenase inhibitor, amantadine, and rimantadine.

    [0030] Another aspect of the invention is a composition where the compound having anti-HCV activity is effective to inhibit the function of a target selected from HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH, and a nucleoside analog for the treatment of an HCV infection.

    [0031] Another aspect of the invention is a composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, an interferon and ribavirin.

    [0032] Another aspect of the invention is a compound according to the invention for use in inhibiting the function of the HCV replicon comprising contacting the HCV replicon with a compound of formula I or a pharmaceutically acceptable salt thereof.

    [0033] Another aspect of the invention is a compound according to the invention for use in inhibiting the function of the HCV NS5B protein comprising contacting the HCV NS5B protein with a compound of formula I or a pharmaceutically acceptable salt thereof.

    [0034] Another aspect of the invention is a compound according to the invention for use in treating an HCV infection in a patient comprising administering to the patient a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. In another embodiment the compound is effective to inhibit the function of the HCV replicon. In another embodiment the compound is effective to inhibit the function of the HCV NS5B protein.

    [0035] Another aspect of the invention is a compound according to the invention for use in treating an HCV infection in a patient comprising administering to the patient a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in conjunction with (prior to, after, or concurrently) another compound having anti-HCV activity.

    [0036] Another aspect of the invention is a compound according to the invention wherein the other compound having anti-HCV activity is an interferon or a ribavirin.

    [0037] Another aspect of the invention is a compound according to the invention where the interferon is selected from interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, interferon lambda, and lymphoblastoid interferon tau.

    [0038] Another aspect of the invention is a compound according to the invention where the other compound having anti-HCV activity is a cyclosporin.

    [0039] Another aspect of the invention is a compound according to the invention where the cyclosporin is cyclosporin A.

    [0040] Another aspect of the invention is a compound according to the invention where the other compound having anti-HCV activity is selected from interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, interfering RNA, anti-sense RNA, Imiqimod, ribavirin, an inosine 5'-monophospate dehydrogenase inhibitor, amantadine, and rimantadine.

    [0041] Another aspect of the invention is a compound according to the invention wherein the other compound having anti-HCV activity is effective to inhibit the function of a target selected from the group consisting of HCV metalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH, and a nucleoside analog for the treatment of an HCV infection.

    [0042] Another aspect of the invention is a compound according to the invention wherein the other compound having anti-HCV activity is effective to inhibit the function of target in the HCV life cycle other than the HCV NS5B protein.

    [0043] "Therapeutically effective" means the amount of agent required to provide a meaningful patient benefit as understood by practitioners in the field of hepatitis and HCV infection.

    [0044] "Patient" means a person infected with the HCV virus and suitable for therapy as understood by practitioners in the field of hepatitis and HCV infection.

    [0045] "Treatment," "therapy," "regimen," "HCV infection," and related terms are used as understood by practitioners in the field of hepatitis and HCV infection.

    [0046] The compounds of this invention are generally given as pharmaceutical compositions comprised of a therapeutically effective amount of a compound or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier and may contain conventional excipients. Pharmaceutically acceptable carriers are those conventionally known carriers having acceptable safety profiles. Compositions encompass all common solid and liquid forms including for example capsules, tablets, lozenges, and powders as well as liquid suspensions, syrups, elixers, and solutions. Compositions are made using common formulation techniques, and conventional excipients (such as binding and wetting agents) and vehicles (such as water and alcohols) are generally used for compositions. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, 17th edition, 1985.

    [0047] Solid compositions are normally formulated in dosage units and compositions providing from about 1 to 1000 mg of the active ingredient per dose are preferred. Some examples of dosages are 1 mg, 10 mg, 100 mg, 250 mg, 500 mg, and 1000 mg. Generally, other agents will be present in a unit range similar to agents of that class used clinically. Typically, this is 0.25-1000 mg/unit.

    [0048] Liquid compositions are usually in dosage unit ranges. Generally, the liquid composition will be in a unit dosage range of 1-100 mg/mL. Some examples of dosages are 1 mg/mL, 10 mg/mL, 25 mg/mL, 50 mg/mL, and 100 mg/mL. Generally, other agents will be present in a unit range similar to agents of that class used clinically. Typically, this is 1-100 mg/mL.

    [0049] The invention encompasses all conventional modes of administration; oral and parenteral methods are preferred. Generally, the dosing regimen will be similar to other agents used clinically. Typically, the daily dose will be 1-100 mg/kg body weight daily. Generally, more compound is required orally and less parenterally. The specific dosing regimen, however, will be determined by a physician using sound medical judgment.

    [0050] The invention also encompasses methods where the compound is given in combination therapy. That is, the compound can be used in conjunction with, but separately from, other agents useful in treating hepatitis and HCV infection. In these combination methods, the compound will generally be given in a daily dose of 1-100 mg/kg body weight daily in conjunction with other agents. The other agents generally will be given in the amounts used therapeutically. The specific dosing regimen, however, will be determined by a physician using sound medical judgment.

    [0051] Some examples of compounds suitable for compositions and methods are listed in Table 1.
    Table 1
    Brand NamePhysiological ClassType of Inhibitor or TargetSource Company
    NIM811   Cyclophilin Inhibitor Novartis
    Zadaxin   Immuno-modulator Sciclone
    Suvus   Methylene blue Bioenvision
    Actilon (CPG10101)   TLR9 agonist Coley
    Batabulin (T67) Anticancer β-tubulin inhibitor Tularik Inc., South San Francisco, CA
    ISIS 14803 Antiviral antisense ISIS Pharmaceuticals Inc, Carlsbad, CA/Elan Phamaceuticals Inc., New York, NY
    Summetrel Antiviral antiviral Endo Pharmaceuticals Holdings Inc., Chadds Ford, PA
    GS-9132 (ACH-806) Antiviral HCV Inhibitor Achillion / Gilead
    Pyrazolopyrimidine compounds and salts From WO-2005047288 26 May 2005 Antiviral HCV Inhibitors Arrow Therapeutics Ltd.
    Levovirin Antiviral IMPDH inhibitor Ribapharm Inc., Costa Mesa, CA
    Merimepodib (VX-497) Antiviral IMPDH inhibitor Vertex Pharmaceuticals Inc., Cambridge, MA
    XTL-6865 (XTL-002) Antiviral monoclonal antibody XTL Biopharmaceuticals Ltd., Rehovot, Israel
    Telaprevir (VX-950, LY-570310) Antiviral NS3 serine protease inhibitor Vertex Pharmaceuticals Inc., Cambridge, MA/ Eli Lilly and Co. Inc., Indianapolis, IN
    HCV-796 Antiviral NS5B Replicase Inhibitor Wyeth / Viropharma
    NM-283 Antiviral NS5B Replicase Inhibitor Idenix / Novartis
    GL-59728 Antiviral NS5B Replicase Inhibitor Gene Labs / Novartis
    GL-60667 Antiviral NS5B Replicase Inhibitor Gene Labs / Novartis
    2'C MeA Antiviral NS5B Replicase Inhibitor Gilead
    PSI 6130 Antiviral NS5B Replicase Inhibitor Roche
    R1626 Antiviral NS5B Replicase Inhibitor Roche
    2'C Methyl adenosine Antiviral NS5B Replicase Inhibitor Merck
    JTK-003 Antiviral RdRp inhibitor Japan Tobacco Inc., Tokyo, Japan
    Levovirin Antiviral ribavirin ICN Pharmaceuticals, Costa Mesa, CA
    Ribavirin Antiviral ribavirin Schering-Plough Corporation, Kenilworth, NJ
    Viramidine Antiviral Ribavirin Prodrug Ribapharm Inc., Costa Mesa, CA
    Heptazyme Antiviral ribozyme Ribozyme Pharmaceuticals Inc., Boulder, CO
    BILN-2061 Antiviral serine protease inhibitor Boehringer Ingelheim Pharma KG, Ingelheim, Germany
    SCH 503034 Antiviral serine protease inhibitor Schering Plough
    Zadazim Immune modulator Immune modulator SciClone Pharmaceuticals Inc., San Mateo, CA
    Ceplene Immunomodulator immune modulator Maxim Pharmaceuticals Inc., San Diego, CA
    CellCept Immunosuppressant HCV IgG immunosuppressant F. Hoffmann-La Roche LTD, Basel, Switzerland
    Civacir Immunosuppressant HCV IgG immunosuppressant Nabi Biopharmaceuticals Inc., Boca Raton, FL
    Albuferon - α Interferon albumin IFN-α2b Human Genome Sciences Inc., Rockville, MD
    Infergen A Interferon IFN alfacon-1 InterMune Pharmaceuticals Inc., Brisbane, CA
    Omega IFN Interferon IFN-ω Intarcia Therapeutics
    IFN-β and EMZ701 Interferon IFN-β and EMZ701 Transition Therapeutics Inc., Ontario, Canada
    Rebif Interferon IFN-β1a Serono, Geneva, Switzerland
    Roferon A Interferon IFN-α2a F. Hoffmann-La Roche LTD, Basel, Switzerland
    Intron A Interferon IFN-α2b Schering-Plough Corporation, Kenilworth, NJ
    Intron A and Zadaxin Interferon IFN-α2b/α1-thymosin RegeneRx Biopharma. Inc., Bethesda, MD/ SciClone Pharmaceuticals Inc, San Mateo, CA
    Rebetron Interferon IFN-α2b/ribavirin Schering-Plough Corporation, Kenilworth, NJ
    Actimmune Interferon INF-γ InterMune Inc., Brisbane, CA
    Interferon-β Interferon Interferon-β-1a Serono
    Multiferon Interferon Long lasting IFN Viragen/ Valentis
    Wellferon Interferon Lympho-blastoid IFN-αn1 GlaxoSmithKline plc, Uxbridge, UK
    Omniferon Interferon natural IFN-α Viragen Inc., Plantation, FL
    Pegasys Interferon PEGylated IFN-α2a F. Hoffmann-La Roche LTD, Basel, Switzerland
    Pegasys and Ceplene Interferon PEGylated IFN-α2a/ immune modulator Maxim Pharmaceuticals Inc., San Diego, CA
    Pegasys and Ribavirin Interferon PEGylated IFN-α2a/ribavirin F. Hoffmann-La Roche LTD, Basel, Switzerland
    PEG-Intron Interferon PEGylated IFN-α2b Schering-Plough Corporation, Kenilworth, NJ
    PEG-Intron / Ribavirin Interferon PEGylated IFN-α2b/ribavirin Schering-Plough Corporation, Kenilworth, NJ
    IP-501 Liver protection antifibrotic Indevus Pharmaceuticals Inc., Lexington, MA
    IDN-6556 Liver protection caspase inhibitor Idun Pharmaceuticals Inc., San Diego, CA
    ITMN-191 (R-7227) Antiviral serine protease inhibitor InterMune Pharmaceuticals Inc., Brisbane, CA
    GL-59728 Antiviral NS5B Replicase Inhibitor Genelabs
    ANA-971 Antiviral TLR-7 agonist Anadys
    Boceprevir Antiviral serine protease inhibitor Schering Plough
    TMS-435 Antiviral serine protease inhibitor Tibotec BVBA, Mechelen, Belgium
    BI-201335 Antiviral serine protease inhibitor Boehringer Ingelheim Pharma KG, Ingelheim, Germany
    MK-7009 Antiviral serine protease inhibitor Merck
    PF-00868554 Antiviral replicase inhibitor Pfizer
    ANA598 Antiviral Non-Nucleoside NS5B Polymerase Inhibitor Anadys Pharmaceuticals, Inc., San Diego, CA, USA
    IDX375 Antiviral Non-Nucleoside Replicase Inhibitor Idenix Pharmaceuticals, Cambridge, MA, USA
    BILB 1941 Antiviral NS5B Polymerase Inhibitor Boehringer Ingelheim Canada Ltd R&D, Laval, QC, Canada
    PSI-7851 Antiviral Nucleoside Polymerase Inhibitor Pharmasset, Princeton, NJ, USA
    PSI-7977 Antiviral Nucleotide NS5B Polymerase Inhibitor Pharmasset, Princeton, NJ, USA
    VCH-759 Antiviral NS5B Polymerase Inhibitor ViroChem Pharma
    VCH-916 Antiviral NS5B Polymerase Inhibitor ViroChem Pharma
    GS-9190 Antiviral NS5B Polymerase Inhibitor Gilead
    Peg-interferon lambda Antiviral Interferon ZymoGenetics/Brist ol-Myers Squibb

    Synthesis Methods



    [0052] The compounds may be made by methods known in the art, including those described below. Some reagents and intermediates are known in the art. Other reagents and intermediates can be made by methods known in the art using commercially available materials. The variables (e.g. numbered "R" substituents) used to describe the synthesis of the compounds are intended only to illustrate how to make and are not to be confused with variables used in the claims or in other sections of the specification. Abbreviations used within the schemes generally follow conventions used in the art.

    [0053] Abbreviations used in the schemes generally follow conventions used in the art. Chemical abbreviations used in the specification and examples are defined as follows: "NaHMDS" for sodium bis(trimethylsilyl)amide; "DMF" for N,N-dimethylformamide; "MeOH" for methanol; "NBS" for N-bromosuccinimide; "Ar" for aryl; "TFA" for trifluoroacetic acid; "LAH" for lithium aluminum hydride; "DMSO" for dimethylsulfoxide; "h" for hours; "rt" for room temperature or retention time (context will dictate); "min" for minutes; "EtOAc" for ethyl acetate; "THF" for tetrahydrofuran; "EDTA" for ethylenediaminetetraacetic acid; "Et2O" for diethyl ether; "DMAP" for 4-dimethylaminopyridine; "DCE" for 1,2-dichloroethane; "ACN" for acetonitrile; "DME" for 1,2-dimethoxyethane; "HOBt" for 1-hydroxybenzotriazole hydrate; "DIEA" for diisopropylethylamine.

    [0054] For the section of compounds in the 0000 series all Liquid Chromatography (LC) data were recorded on a Shimadzu LC-10AS or LC-20AS liquid chromotograph using a SPD-10AV or SPD-20A UV-Vis detector and Mass Spectrometry (MS) data were determined with a Micromass Platform for LC in electrospray mode.

    [0055] HPLC Method (i.e., compound isolation). Compounds purified by preparative HPLC were diluted in methanol (1.2 mL) and purified using a Shimadzu LC-8A or LC-10A or Dionex APS-3000 or Waters Acquity automated preparative HPLC system.

    Examples:


    Preparation of Intermediates, Compound 3 and Compound 4:



    [0056] 



    [0057] Step 1: To a mixture of 2-(4-fluorophenyl)-3-(methylcarbamoyl)-6-nitrobenzofuran-5-yl trifluoromethanesulfonate (400 mg), (3-(tert-butylcarbamoyl)-5-nitrophenyl)boronic acid (345 mg)and Cs2CO3 (846 mg) in dioxane (8 mL) and water (1.5 mL) was added tetrakis(triphenylphosphine)palladium(O) (150 mg). The mixture was flushed with nitrogen and then heated at 99°C for 16 hours. The mixture was diluted with water and extracted with EtOAc (2 x 100mL). The organic layers were combined, washed with brine, dried over MgSO4 and concentrated. The residue was purified by silica gel column (hexanes/EtOAc = 3:1) to give 5-(3-(tert-butylcarbamoyl)-5-nitrophenyl)-2-(4-fluorophenyl)-N-methyl-6-nitrobenzofuran-3-carboxamide 2 (350 mg).
    Compound 2
    MS (2M+H)+ Calcd. 1069.3
    MS (2M+H)+ Observ. 1069.2
    Retention Time 1.88 minutes
    LC Condition
    Solvent A 5 % ACN: 95% Water : 10mM Ammonium Actetate
    Solvent B 95 % ACN: 5% Water : 10mM Ammonium Actetate
    Start % B 0
    Final % B 100
    Gradient Time 2 min
    Flow Rate 1 mL/min
    Wavelength 220
    Solvent Pair ACN: Water: Ammonium Actetate
    Column Phenomenex LUNA C18, 30x2, 3u


    [0058] Step 2: To a solution of 5-(3-(tert-butylcarbamoyl)-5-nitrophenyl)-2-(4-fluorophenyl)-N-methyl-6-nitrobenzofuran-3-carboxamide (350 mg) in MeOH (50 mL) was added palladium on carbon (69.7 mg). The mixture was stirred under hydrogen with a balloon for 16 hours. Pd/C was removed by filtration and the filtrate was concentrated to give 6-amino-5-(3-amino-5-(tert-butylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methylbenzofuran-3-carboxamide 3 (200 mg).
    Compound 3
    MS (M+H)+ Calcd. 475.2
    MS (M+H)+ Observ. 475.2
    Retention Time 0.80 min
    LC Condition
    Solvent A 90% Water -10% Methanol-0.1% TFA
    Solvent B 10% Water -90% Methanol-0.1% TFA
    Start % B 50
    Final % B 100
    Gradient Time 2 min
    Flow Rate 1 mL/min
    Wavelength 220
    Solvent Pair Water - Methanol- TFA
    Column PHENOMENEX-LUNA 2.0 x 30mm 3um


    [0059] Step 3: To a solution of 6-amino-5-(3-amino-5-(tert-butylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methylbenzofuran-3-carboxamide (100 mg) and pyridine (0.170 mL) in CH2Cl2 (10 mL) was added methanesulfonyl chloride (72.4 mg) slowly at 0 °C. The mixture was stirred at room temperature for 16 hours. The reaction was quenched with aqueous NaHCO3 and extracted with CH2Cl2. The organic layer was concentrated and the residue was purified by preparative HPLC to give 5-(3-(tert-butylcarbamoyl)-5-(methylsulfonamido)phenyl)-2-(4-fluorophenyl)-N-methyl-6-(methylsulfonamido)benzofuran-3-carboxamide 4 (92 mg).
    Compound 4
    MS (M+H)+ Calcd. 631.2
    MS (M+H)+ Observ. 631.3
    Retention Time 1.17 min
    LC Condition
    Solvent A 90% Water -10% Methanol-0.1% TFA
    Solvent B 10% Water -90% Methanol-0.1% TFA
    Start % B 50
    Final % B 100
    Gradient Time 2 min
    Flow Rate 1 mL/min
    Wavelength 220
    Solvent Pair Water - Methanol- TFA
    Column PHENOMENEX-LUNA 2.0 x 30mm 3um

    General Procedure for the Preparation of Compounds 1001 - 1006:



    [0060] 



    [0061] A mixture of 5-(3-(tert-butylcarbamoyl)-5-(methylsulfonamido)phenyl)-2-(4-fluorophenyl)-N-methyl-6-(methylsulfonamido)benzofuran-3-carboxamide 4 (10 mg, 1 eq.), 1,4-dibromide (1 eq.) and K2CO3 (8.77 mg, 4 eq.) in acetonitrile (5 mL) and DMF (2 mL) was heated at 80 °C for 16 hours. All the solvents were removed under vacuum. The residue purified by preparative HPLC.
    LC Condition
    Solvent A 90% Water -10% Methanol-0.1% TFA
    Solvent B 10% Water -90% Methanol-0.1% TFA
    Start % B 30
    Final % B 100
    Gradient Time 2 min
    Flow Rate 1 mL/min
    Wavelength 220
    Solvent Pair Water - Methanol- TFA
    Column PHENOMENEX-LUNA 2.0 x 30mm 3um
    Cmpd#StructureMS (M+H)+ Calcd.MS (M+H)+ Observ.Retention Time (min)
    1001

    685.2 685.4 1.60
    1002

    699.2 699.4 1.69
    1003

    713.2 713.4 1.83
    1004

    727.3 727.5 1.99
    1005

    741.3 741.5 2.05
    1006

    761.2 761.5 1.89

    General Procedure for the Preparation of Compounds 1051 - 1058:



    [0062] 



    [0063] To a solution of 6-amino-5-(3-amino-5-(tert-butylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methylbenzofuran-3-carboxamide 3 (10 mg, 1 eq.) in CH2Cl2 (10 mL) was added diacylchloride or dicarbonochloridate or diisothiocyanate (0.8 eq.) and DIPEA (0.022 mL, 6 eq.). The mixture was stirred at room temperature for 16 hours. All the solvents were removed under vacuum. The residue was dissolved in 1.5 mL of DMF and purified by preparative HPLC.
    LC Condition
    Solvent A 90% Water -10% Methanol-0.1% TFA
    Solvent B 10% Water -90% Methanol-0.1% TFA
    Start % B 30
    Final % B 100
    Gradient Time 2 min
    Flow Rate 1 mL/min
    Wavelength 220
    Solvent Pair Water - Methanol- TFA
    Column PHENOMENEX-LUNA 2.0 x 30mm 3um
    Cmpd#StructureMS (M+H)+ Calcd.MS (M+H)+ Observ.Retention Time (min)
    1051

    585.2 585.4 1.64
    1052

    641.3 641.5 2.22
    1053

    669.3 669.5 2.29
    1054

    589.2 589.3 1.65
    1055

    633.2 633.4 1.81
    1056

    677.3 677.4 1.82
    1057

    647.2 647.4 1.90
    1058

    703.3 703.5 2.26

    Preparation of Compound 2001:



    [0064] 



    [0065] Step 1: Cs2CO3 (705 mg) and tetrakis(triphenylphosphine)palladium(O) (62 mg) were added into a solution of 2-(4-fluorophenyl)-3-(methylcarbamoyl)-6-nitrobenzofuran-5-yl trifluoromethanesulfonate (500 mg) and 3-(6-methyl-4,8-dioxo-1,3,6,2-dioxazaborocan-2-yl)benzoic acid (360 mg) in dioxane (9 mL) and water (3 mL). The reaction was heated at 90°C for 4 hours. After being cooled to room temperature, the mixture was poured into ice water, which was adjusted to pH 3 by 2N aqueous HCl solution. The compound 101 formed as a brown solid and was isolated via filtration.
    Compound 101
    MS (M+H)+ Calcd. 435.1
    MS (M+H)+ Observ. 435.4
    Retention Time 1.37 minutes
    LC Condition
    Solvent A 5 % ACN: 95% Water : 10mM Ammonium Actetate
    Solvent B 95 % ACN: 5% Water : 10mM Ammonium Actetate
    Start % B 0
    Final % B 100
    Gradient Time 2 min
    Flow Rate 1 mL/min
    Wavelength 220
    Solvent Pair ACN: Water: Ammonium Actetate
    Column Phenomenex LUNA C18, 30x2, 3u


    [0066] Step 2: iPr2NEt (89 mg) and was added into a solution of 3-(2-(4-fluorophenyl)-3-(methylcarbamoyl)-6-nitrobenzofuran-5-yl)benzoic acid (150 mg), 2-amino-2-methylpropan-1-ol (37 mg) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (171 mg) in DMF (2 mL). The reaction was stirred at room temperature for 16 hours, before 20 mL of EtOAc was added. The organic layer was washed with water (2 x 10 mL), brine (5 mL) and dried over MgSO4. After filtration, removal of solvents under vacuum afforded a residue which was used as was.
    Compound 102
    MS (M+H)+ Calcd. 506.2
    MS (M+H)+ Observ. 506.6
    Retention Time 1.64 minutes
    LC Condition
    Solvent A 5 % ACN: 95% Water : 10mM Ammonium Actetate
    Solvent B 95 % ACN: 5% Water : 10mM Ammonium Actetate
    Start % B 0
    Final % B 100
    Gradient Time 2 min
    Flow Rate 1 mL/min
    Wavelength 220
    Solvent Pair ACN: Water: Ammonium Actetate
    Column Phenomenex LUNA C18, 30x2, 3u


    [0067] Step 3: To a solution of 2-(4-fluorophenyl)-5-(3-((1-hydroxy-2-methylpropan-2-yl)carbamoyl)phenyl)-N-methyl-6-nitrobenzofuran-3-carboxamide (108 mg) in ethyl ether (4 mL), was added palladium on carbon (1.137 mg). The mixture was stirred under hydrogen with a balloon for 16 hours. Pd/C was removed by filtration and the filtrate was concentrated to give 6-amino-2-(4-fluorophenyl)-5-(3-((1-hydroxy-2-methylpropan-2-yl)carbamoyl)phenyl)-N-methylbenzofuran-3-carboxamide 103 (95 mg).
    Compound 103
    MS (M+H)+ Calcd. 476.2
    MS (M+H)+ Observ. 476.5
    Retention Time 1.52 minutes
    LC Condition
    Solvent A 5 % ACN: 95% Water : 10mM Ammonium Actetate
    Solvent B 95 % ACN: 5% Water : 10mM Ammonium Actetate
    Start % B 0
    Final % B 100
    Gradient Time 2 min
    Flow Rate 1 mL/min
    Wavelength 220
    Solvent Pair ACN: Water: Ammonium Actetate
    Column Phenomenex LUNA C18, 30x2, 3u


    [0068] Step 4: NaH (3.71 mg) was added into the solution of 6-amino-2-(4-fluorophenyl)-5-(3-((1-hydroxy-2-methylpropan-2-yl)carbamoyl)phenyl)-N-methylbenzofuran-3-carboxamide (14.7 mg) and 3-chloro-2-(chloromethyl)prop-1-ene (3.86 mg) in DMF. The reaction was stirred at room temperature for 24 hours, before being quenched by MeOH. The compound 2001 was isolated by preparative HPLC.
    Compound 2001
    MS (M+H)+ Calcd. 528.2
    MS (M+H)+ Observ. 528.6
    Retention Time 1.77 minutes
    LC Condition
    Solvent A 5 % ACN: 95% Water : 10mM Ammonium Actetate
    Solvent B 95 % ACN: 5% Water : 10mM Ammonium Actetate
    Start % B 0
    Final % B 100
    Gradient Time 2 min
    Flow Rate 1 mL/min
    Wavelength 220
    Solvent Pair ACN: Water: Ammonium Actetate
    Column Phenomenex LUNA C18, 30x2, 3u

    Biological Methods



    [0069] The compound demonstrated activity against HCV NS5B as determined in the following HCV RdRp assays.

    [0070] HCV NS5B RdRp cloning, expression, and purification. The cDNA encoding the NS5B protein of HCV, genotype 1b, was cloned into the pET21a expression vector. The protein was expressed with an 18 amino acid C-terminal truncation to enhance the solubility. The E. coli competent cell line BL21(DE3) was used for expression of the protein. Cultures were grown at 37 °C for ∼ 4 hours until the cultures reached an optical density of 2.0 at 600 nm. The cultures were cooled to 20 °C and induced with 1 mM IPTG. Fresh ampicillin was added to a final concentration of 50 µg/mL and the cells were grown overnight at 20 °C.

    [0071] Cell pellets (3L) were lysed for purification to yield 15-24 mgs of purified NS5B. The lysis buffer consisted of 20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.5% triton X-100, 1 mM DTT, 1mM EDTA, 20% glycerol, 0.5 mg/ml lysozyme, 10 mM MgCl2, 15 ug/ml deoxyribonuclease I, and Complete TM protease inhibitor tablets (Roche). After addition of the lysis buffer, frozen cell pellets were resuspended using a tissue homogenizer. To reduce the viscosity of the sample, aliquots of the lysate were sonicated on ice using a microtip attached to a Branson sonicator. The sonicated lysate was centrifuged at 100,000 x g for 30 minutes at 4 °C and filtered through a 0.2 µm filter unit (Corning).

    [0072] The protein was purified using two sequential chromatography steps: Heparin sepharose CL-6B and polyU sepharose 4B. The chromatography buffers were identical to the lysis buffer but contained no lysozyme, deoxyribonuclease I, MgCl2 or protease inhibitor and the NaCl concentration of the buffer was adjusted according to the requirements for charging the protein onto the column. Each column was eluted with a NaCl gradient which varied in length from 5-50 column volumes depending on the column type. After the final chromatography step, the resulting purity of the enzyme is >90% based on SDS-PAGE analysis. The enzyme was aliquoted and stored at -80 °C.

    [0073] Standard HCV NS5B RdRp enzyme assay. HCV RdRp genotype 1b assays were run in a final volume of 60 µl in 96 well plates (Costar 3912). The assay buffer is composed of 20 mM Hepes, pH 7.5, 2.5 mM KCl, 2.5 mM MgCl2, 1 mM DTT, 1.6 U RNAse inhibitor (Promega N2515), 0.1 mg/ml BSA (Promega R3961), and 2 % glycerol. All compounds were serially diluted (3-fold) in DMSO and diluted further in water such that the final concentration of DMSO in the assay was 2%. HCV RdRp genotype 1b enzyme was used at a final concentration of 28 nM. A polyA template was used at 6 nM, and a biotinylated oligo-dT12 primer was used at 180 nM final concentration. Template was obtained commercially (Amersham 27-4110). Biotinylated primer was prepared by Sigma Genosys. 3H-UTP was used at 0.6 µCi (0.29 µM total UTP). Reactions were initiated by the addition of enzyme, incubated at 30 °C for 60 min, and stopped by adding 25 µL of 50 mM EDTA containing SPA beads (4 µg/µL, Amersham RPNQ 0007). Plates were read on a Packard Top Count NXT after > 1hr incubation at room temperature.

    [0074] Modified HCV NS5B RdRp enzyme assay. An on-bead solid phase homogeneous assay was also used to assess NS5B inhibitors (WangY-K, Rigat K, Roberts S, and Gao M (2006) Anal Biochem, 359: 106-111). The assay is a modification of the standard assay described above and was used in a 96-well or a 384-well format. The biotinylated oligo dT12 primer was captured on streptavidin-coupled beads (SPA beads (GE, RPNQ0007) or imaging beads (GE, RPNQ0261) by mixing primer and beads in buffer and incubating at room temperature for three hours. Unbound primer was removed after centrifugation. The primer-bound beads were resuspended in 3x reaction buffer (40 mM Hepes buffer, pH 7.5, 7.5 mM MgCl2, 7.5 mM KCl, dT primer coupled beads, poly A template, 3H-UTP, and RNAse inhibitor (Promega N2515). Compounds were serially diluted 1:3 in DMSO and aliquoted into assay plates. Equal volumes (20 µL for 96-well assay and 10 µL for 384-well assay) of water, 3X reaction mix, and enzyme in 20 mM Hepes buffer, pH 7.5, 0.1 mg/ml BSA were added to the diluted compound on the assay plate. Final concentration of components in 96-well assay: 0.36 nM template, 15 nM primer, 0.43 µM (1 µCi) 3H-UTP, 0.08 U/µL RNAse inhibitor, 7 nM NS5B enzyme, 0.033 mg mL BSA, and 2 µg/µL beads, 20 mM Hepes buffer, pH 7.5, 2.5 mM MgCl2, 2.5 mM KCl, 2% DMSO. Final concentration of components in 384-well assay: 0.2 nM template, 15 nM primer, 0.29 µM 3H-UTP (0.3 µCi), 0.08 U/µL RNAse inhibitor, 7 nM NS5B enzyme, 0.033 mg/mL BSA, and 0.33 µg/µL beads, 20 mM Hepes buffer, pH 7.5, 2.5 mM MgCl2, 2.5 mM KCl, 2% DMSO.

    [0075] Reactions were allowed to proceed for 4 hours at 30° C and terminated by the addition of 50 mM EDTA (10 µL). After incubating for at least 15 minutes, plates were read on a Packard NXT Topcount or Amersham LEADseeker multimodality imaging system.

    [0076] Cell lines. The cell lines used to evaluate compounds consist of a human hepatocyte derived cell line (Huh-7) that constitutively expresses a genotype 1a or 1b HCV replicon containing a Renilla luciferase reporter gene. These cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% FBS, 100 U/mL penicillin/streptomycin and 1.0 mg/mL G418.

    [0077] HCV replicon luciferase assay. To evaluate compound efficacy, HCV replicon cells were seeded in 96-well plates in DMEM containing 10% FBS at a cell density of 104/well. Following incubation at 37°C overnight, compounds serially diluted in DMSO were added to the cell plates. Alternatively, titrated compounds were transferred to sterile 384-well tissue-culture treated plates and the plates seeded with 50 µL of cells at a density of 2.4 x 103 cells/well in DMEM containing 4 % FCS (final DMSO concentration at 0.5 %). After 3 days incubation at 37°C, cells were analyzed for Renilla Luciferase activity using the EnduRen substrate (Promega cat #E6485) according to the manufacturer's directions. Briefly, the EnduRen substrate was diluted in DMEM and then added to the plates to a final concentration of 7.5 µM. The plates were incubated for at least 1 h at 37°C then read on a TopCount NXT Microplate Scintillation and Luminescence Counter (Packard) or Viewlux Imager (PerkinElmer) using a luminescence program. The 50% effective concentration (EC50) was calculated using the exponential form of the median effect equation where EC50 = 100- [(δFinh/δFcon) x100].

    [0078] To assess cytotoxicity of compounds, Cell Titer-Blue (Promega) was added to the EnduRen-containing plates and incubated for at least 4 hrs at 37°C. The fluorescence signal from each well was read using a Cytoflour 400 (PE Biosystems) or Viewlux Imager.

    [0079] Compound EC50 data is expressed as A: < 100 nM; B = 100-1000 nM; C > 1000 nM). Representative data for compounds are reported in Table 2.
    Table 2
    Cmpd#StructureEC50 (uM) 1b
    1001

    0.1705 B
    1002

    B
    1003

    B
    1004

    0.0477 A
    1005

    B
    1006

    1.2100 C
    1051

    C
    1052

    0.7888 B
    1053

    B
    1054

    C
    1055

    C
    1056

    8.3380 C
    1057

    C
    1058

    C
    2001

     


    [0080] It will be evident to one skilled in the art that the present disclosure is not limited to the foregoing illustrative examples, and that it can be embodied in other specific forms without departing from the essential attributes thereof. It is therefore desired that the examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing examples, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.


    Claims

    1. A compound of formula I, including pharmaceutically acceptable salts thereof:

    wherein

    n, m are 1;

    R1 is methyl;

    R2 is phenyl that is substituted with 1-2 halo

    R3 is hydrogen;

    R5 is selected from hydrogen, halo, alkyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, alkoxy, hydroxyalkyloxy, alkoxyalkyloxy, COOR11 and CON(R12)(R13);

    R4 and R6 are hydrogen;

    R11 is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy;

    R12, R13 are each independently hydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy; or

    R12 and R13 can form a ring by joining two atoms, one from each of R12 and R13;

    R14 is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy;

    R7 is hydrogen;

    R21 and R22 are independently hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl, alkylsulfonyl, or alkylsulfonylalkyl;

    or N(R21)(R22) taken together is azetidinyl, pyrrolidinyl, piperidinyl, or piperazinyl, and is substituted with 0-2 substituents selected from alkyl, hydroxyalkyl, and hydroxy;

    X, Y are independently selected from CR31R32, CO, O, NR33, NCN, S, SO, and S(O)2;

    R31, R32 are each independently alkyl or cycloalkyl, further substituted with 0-3 substituents; or

    R31 and R32 can form a ring by joining two atoms, one from each of R31 and R32;

    R33 is hydrogen, alkyl, cycloalkyl, or S(O)2R11 with 0-3 substituents selected from halo, haloalkoxy, OR11, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, and NR14CONR12R13;

    Z is an alkylene or alkenylene chain containing 0-12 groups selected from the group consisting of O, NR41, S, S(O), S(O)2, C(O), C(O)O, C(O)NR41, C(S)NR41, O-C(O)NR41, NR41C(O)NR42, and Ar2, provided that any O or S atom does not directly bond to another O or S atom, such that ring A is 11-24 membered; and further wherein the alkylene or alkenylene chain is substituted with 0-6 substituents selected from alkyl, cycloalkyl, halo, alkoxy, haloalkyl, OH, OR11, haloalkoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, NR14CONR12R13, and Ar3;

    R41, R42 is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo, haloalkoxy, OR11, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, and NR14CONR12R13;

    Ar2 is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and is substituted with 0-3 substituents selected from cyano, halo, alkyl, cycloalkyl, haloalkyl, OH, OR11, haloalkoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, and NR14CONR12R13;

    Ar3 is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and is substituted with 0-3 substituents selected from cyano, halo, alkyl, cycloalkyl, haloalkyl, OH, OR11, haloalkoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, and NR14CONR12R13.


     
    2. A compound of claim 1 wherein halo is fluoro.
     
    3. A compound of claim 1 wherein X and Y are each NR33.
     
    4. A compound of claim 3 wherein R33 is hydrogen or S(O)2CH3.
     
    5. A compound of claim 1 wherein A is at least a 12-member ring.
     
    6. A compound of claim 5 wherein A is at least a 15-member ring.
     
    7. A compound of claim 6 wherein A is at least a 17-member ring.
     
    8. A compound of claim 7 wherein A is at least a 19-member ring.
     
    9. A compound according to claim 1, including pharmaceutically acceptable salts thereof, which is selected from the group of:













    and


     
    10. A composition comprising a compound according to anyone of claims 1 to 9 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
     
    11. A compound or pharmaceutically acceptable salt according to anyone of claims 1 to 9 or composition according to claim 10 for use as a medicament.
     
    12. A compound or pharmaceutically acceptable salt according to anyone of claims 1 to 9 or composition according to claim 10 for use in the treatment of hepatitis C infection.
     


    Ansprüche

    1. Verbindung der Formel I, einschließlich pharmazeutisch verträglicher Salze davon:

    worin

    n, m 1 sind;

    R1 Methyl ist;

    R2 Phenyl ist, das substituiert ist mit 1 bis 2 Halogenen;

    R3 Wasserstoff ist;

    R5 ausgewählt ist aus Wasserstoff, Halogen, Alkyl, Halogenalkyl, Hydroxyalkyl, Alkoxyalkyl, Alkoxy, Hydroxyalkyloxy, Alkoxyalkyloxy, COOR11 und CON(R12)(R13);

    R4 und R6 Wasserstoff sind;

    R11 Wasserstoff, Alkyl oder Cycloalkyl mit 0 bis 3 Substituenten ist, die ausgewählt sind aus Halogen, Hydroxyl, Alkoxy und Halogenalkoxy;

    R12, R13 jeweils unabhängig Wasserstoff, Alkyl oder Cycloalkyl mit 0 bis 3 Substituenten sind, ausgewählt aus Halogen, Hydroxyl, Alkoxy und Halogenalkoxy; oder

    R12 und R13 einen Ring bilden können, indem zwei Atome verbunden werden, von denen jeweils eines von R12 und R13 ist;

    R14 Wasserstoff, Alkyl oder Cycloalkyl mit 0 bis 3 Substituenten ist, die ausgewählt sind aus Halogen, Hydroxyl, Alkoxy und Halogenalkoxy;

    R7 Wasserstoff ist;

    R21 und R22 unabhängig Wasserstoff, Alkyl, Hydroxyalkyl, Alkoxyalkyl, Alkylsulfonyl oder Alkylsulfonylalkyl sind;

    oder N(R21)(R22) zusammengenommen Azetidinyl, Pyrrolidinyl, Piperidinyl oder Piperazinyl sind und substituiert sind mit 0 bis 2 Substituenten, die ausgewählt sind aus Alkyl, Hydroxyalkyl und Hydroxy;

    X, Y unabhängig ausgewählt sind aus CR31R32, CO, O, NR33, NCN, S, SO, und S(O)2;

    R31, R32 jeweils unabhängig Alkyl oder Cycloalkyl sind, weiter substituiert mit 0 bis 3 Substituenten; oder

    R31 und R32 einen Ring bilden können, indem zwei Atome verbunden werden, von denen jeweils eines von R31 und R32 ist;

    R33 Wasserstoff, Alkyl, Cycloalkyl oder S(O)2R11 mit 0 bis 3 Substituenten ist, die ausgewählt sind aus Halogen, Halogenalkoxy, OR11, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13 und NR14CONR12R13;

    Z eine Alkylen- oder eine Alkenylen-Kette ist, die 0 bis 12 Gruppen enthält, die ausgewählt sind aus O, NR41, S, S(O), S(O)2, C(O), C(O)O, C(O)NR41, C(S)NR41, O-C(O)NR41, NR41C(O)NR42 und Ar2 mit der Maßgabe, dass jedes O- oder S-Atom nicht direkt an ein anderes O- oder S-Atom gebunden ist, so dass Ring A 11-24-gliedrig ist; und ferner wobei die Alkylen- oder Alkenylen-Kette substituiert ist mit 0 bis 6 Substituenten, die ausgewählt sind aus Alkyl, Cycloalkyl, Halogen, Alkoxy, Halogenalkyl, OH, OR11, Halogenalkoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, NR14CONR12R13 und Ar3; ,

    R41, R42 Wasserstoff, Alkyl oder Cycloalkyl mit 0 bis 3 Substituenten sind, die ausgewählt sind aus Halogen, Halogenalkoxy, OR11, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13 und NR14CONR12R13;

    Ar2 Phenyl, 5-gliedriges Heteroaryl oder 6-gliedriges Heteroaryl ist und substituiert ist mit 0 bis 3 Substituenten, die ausgewählt sind aus Cyano, Halogen, Alkyl, Cycloalkyl, Halogenalkyl, OH, OR11, Halogenalkoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13 und NR14CONR12R13;

    Ar3 Phenyl, 5-gliedriges Heteroaryl oder 6-gliedriges Heteroaryl ist und substituiert ist mit 0 bis 3 Substituenten, die ausgewählt sind aus Cyano, Halogen, Alkyl, Cycloalkyl, Halogenalkyl, OH, OR11, Halogenalkoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13 und NR14CONR12R13.


     
    2. Verbindung nach Anspruch 1, worin Halogen Fluor ist.
     
    3. Verbindung nach Anspruch 1, worin X und Y jeweils NR33 sind.
     
    4. Verbindung nach Anspruch 3, worin R33 Wasserstoff ist oder S(O)2CH3.
     
    5. Verbindung nach Anspruch 1, worin A mindestens ein 12-gliedriger Ring ist.
     
    6. Verbindung nach Anspruch 5, worin A mindestens ein 15-gliedriger Ring ist.
     
    7. Verbindung nach Anspruch 6, worin A mindestens ein 17-gliedriger Ring ist.
     
    8. Verbindung nach Anspruch 7, worin A mindestens ein 19-gliedriger Ring ist.
     
    9. Verbindung nach Anspruch 1, einschließlich pharmazeutisch verträglicher Salze davon, die ausgewählt sind aus:













    und


     
    10. Zusammensetzung, umfassend eine Verbindung nach einem der Ansprüche 1 bis 9 oder ein pharmazeutisch verträgliches Salz davon und einen pharmazeutisch verträglichen Träger.
     
    11. Verbindung oder pharmazeutisch verträgliches Salz nach einem der Ansprüche 1 bis 9 oder Zusammensetzung nach Anspruch 10 zur Verwendung als ein Medikament.
     
    12. Verbindung oder pharmazeutisch verträgliches Salz nach einem der Ansprüche 1 bis 9 oder Zusammensetzung nach Anspruch 10 zur Verwendung in der Behandlung einer Infektion mit Hepatitis C.
     


    Revendications

    1. Composé de formule I, incluant les sels pharmaceutiquement acceptables de celui-ci:

    n, m sont 1 ;

    R1 est méthyle;

    R2 est phényle qui est substitué par 1-2 halogéno ;

    R3 est hydrogène;

    R5 est choisi parmi hydrogène, halogéno, alkyle, halogénoalkyle, hydroxyalkyle, alcoxyalkyle, alcoxy, hydroxyalkyloxy, alcoxyalkyloxy, COOR11 et CON(R12)(R13);

    R4 et R6 sont hydrogène;

    R11 est hydrogène, alkyle ou cycloalkyle avec 0-3 substituants choisis parmi halogéno, hydroxyle, alcoxy, et halogénoalcoxy;

    R12, R13 sont chacun indépendamment hydrogène, alkyle ou cycloalkyle avec 0-3 substituants choisis parmi halogéno, hydroxyle, alcoxy, et halogénoalcoxy; ou

    R12 et R13 peuvent former un cycle en joignant deux atomes, un de chacun parmi R12 et R13;

    R14 est hydrogène, alkyle ou cycloalkyle avec 0-3 substituants choisis parmi halogéno, hydroxyle, alcoxy, et halogénoalcoxy;

    R7 est hydrogène;

    R21 et R22 sont indépendamment hydrogène, alkyle, hydroxyalkyle, alcoxyalkyle, alkylsulfonyle, ou alkylsulfonylalkyle;

    ou N(R21)(R22) pris ensemble est azétidinyle, pyrrolidinyle, pipéridinyle, ou pipérazinyle, et est substitué par 0-2 substituants choisis parmi alkyle, hydroxyalkyle, et hydroxy;

    X, Y sont indépendamment choisis parmi CR31R32, CO, O, NR33, NCN, S, SO, et S(O)2;

    R31, R32 sont chacun indépendamment alkyle ou cycloalkyle, plus encore substitués par 0-3 substituants; ou

    R31 et R32 peuvent former un cycle en joignant deux atomes, un de chacun parmi R31 et R32;

    R33 est hydrogène, alkyle, cycloalkyle, ou S(O)2R11 avec 0-3 substituants choisis parmi halogéno, halogénoalcoxy, OR11, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, et NR14CONR12R13;

    Z est une chaîne alkylène ou alcénylène contenant 0-12 groupes choisis dans le groupe constitué par O, NR41, S, S(O), S(O)2, C(O), C(O)O, C(O)NR41, C(S)NR41, O-C(O)NR41, NR41C(O)NR42, et Ar2, étant entendu qu'aucun atome de O ou S ne se lie directement à un autre atome de O ou S, de sorte que le cycle A est à 11-24 chaînons; et de plus laquelle chaîne alkylène ou alcénylène est substituée par 0-6 substituants choisis parmi alkyle, cycloalkyle, halogéno, alcoxy, halogénoalkyle, OH, OR11, halogénoalcoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, NR14CONR12R13, et Ar3;

    R41, R42 sont hydrogène, alkyle ou cycloalkyle avec 0-3 substituants choisis parmi halogéno, halogénoalcoxy, OR11, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, et NR14CONR12R13;

    Ar2 est phényle, hétéroaryle à 5 chaînons ou hétéroaryle à 6 chaînons, et est substitué par 0-3 substituants choisis parmi cyano, halogéno, alkyle, cycloalkyle, halogénoalkyle, OH, OR11, halogénoalcoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, et NR14CONR12R13;

    Ar3 est phényle, hétéroaryle à 5 chaînons ou hétéroaryle à 6 chaînons, et est substitué par 0-3 substituants choisis parmi cyano, halogéno, alkyle, cycloalkyle, halogénoalkyle, OH, OR11, halogénoalcoxy, NH2, NR12R13, COOR11, CONR12R13, S(O)2R11, S(O)2NR12R13, et NR14CONR12R13.


     
    2. Composé selon la revendication 1, dans lequel halogéno est fluoro.
     
    3. Composé selon la revendication 1, dans lequel X et Y sont chacun NR33.
     
    4. Composé selon la revendication 3, dans lequel R33 est hydrogène ou S(O)2CH3.
     
    5. Composé selon la revendication 1, dans lequel A est au moins un cycle à 12 chaînons.
     
    6. Composé selon la revendication 5, dans lequel A est au moins un cycle à 15 chaînons.
     
    7. Composé selon la revendication 6, dans lequel A est au moins un cycle à 17 chaînons.
     
    8. Composé selon la revendication 7, dans lequel A est au moins un cycle à 19 chaînons.
     
    9. Composé selon la revendication 1, incluant les sels pharmaceutiquement acceptables de celui-ci, qui est choisi dans le groupe de:













    et


     
    10. Composition comprenant un composé selon l'une quelconque des revendications 1 à 9 ou un sel pharmaceutiquement acceptable de celui-ci et un véhicule pharmaceutiquement acceptable.
     
    11. Composé ou sel pharmaceutiquement acceptable selon l'une quelconque des revendications 1 à 9 ou composition selon la revendication 10, pour l'utilisation en tant que médicament.
     
    12. Composé ou sel pharmaceutiquement acceptable selon l'une quelconque des revendications 1 à 9 ou composition selon la revendication 10, pour l'utilisation dans le traitement d'une infection par hépatite C.
     




    REFERENCES CITED IN THE DESCRIPTION



    This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

    Patent documents cited in the description




    Non-patent literature cited in the description