Abstract:
This invention is directed to compounds of formula (I):  
                         
 
wherein R 1 , R 2 , X, and n are as defined herein, including all crystalline forms and pharmaceutically acceptable salts thereof, with the provisos that when X is CH 2 , n is 1, and R 1  is —COOH, then R 2  cannot be  
                         
 
wherein A is CH 3 —, CH 3 CH 2 — or a haloalkyl of 1 to 2 carbon atoms, and B is a halogen; and 
 
when X is CH 2 , n is 2, and R 1  is —COOH, then R 2  cannot be  
                         
The invention is also directed to compositions containing compounds of the invention and methods of using the compounds to treat or prevent hepatitis C virus infections.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application claims the benefit of U.S. Provisional Application No. 60/771,904, filed Feb. 8, 2006 the disclosure of which is incorporated by reference herein. 
     
    
     FIELD OF THE INVENTION  
       [0002]     A series of sulfonamide compounds are effective pharmaceuticals for the treatment of hepatitis C infection.  
       BACKGROUND OF THE INVENTION  
       [0003]     Hepatitis C is a common infection that can lead to chronic hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma. Infection with the hepatitis C virus (HCV) leads to chronic hepatitis in at least 85% of cases, is the leading reason for liver transplantation, and is responsible for at least 10,000 deaths annually in the United States (Hepatology, 1997, 26 (Suppl. 1), 2S-10S).  
         [0004]     The hepatitis C virus is a member of the Flaviviridae family, and the genome of HCV is a single-stranded linear RNA of positive sense (Hepatology, 1997, 26 (Suppl. 1), 11S-14S). HCV displays extensive genetic heterogeneity; at least 6 genotypes and more than 50 subtypes have been identified.  
         [0005]     There is no effective vaccine to prevent HCV infection. The only therapy currently available is treatment with interferon-α (INF-α) or combination therapy of INF-α with the nucleoside analog ribavirin (Antiviral Chemistry and Chemotherapy, 1997, 8, 281-301). However, only about 40% of treated patients develop a sustained response, so there is a need for more effective anti-HCV therapeutic agents.  
         [0006]     The HCV genome contains a number of non-structural proteins: NS2, NS3, NS4A, NS4B, NS5A, and NS5B (J. General Virology, 2000, 81, 1631-1648). NS5B is an RNA-dependent RNA polymerase that is essential for viral replication. Therefore, the inhibition of NS5B is a suitable target for the development of therapeutic agents.  
         [0007]     U.S. Pat. No. 3,506,646 relates to compounds that are derivatives of 6-aminosulfonyl compounds, in particular 1,2,5-benzothiadiazepine 1,1-dioxides with fused heterocycles, the intermediates used to synthesize them, and their use as diuretic and hypotensive agents.  
         [0008]     WO 98/08815 relates to substituted cyclic amine metalloprotease inhibitors.  
         [0009]     Biorganic and medicinal chemistry, 1996, 837-850 describes 5H-pyrrolo[1,2-b][1,2,5]benzothiadiazepines (PBTDs) as a novel class of non-nucleoside reverse transcriptase inhibitors.  
         [0010]     WO 03/043985 describes sulfonamides as peroxisome proliferator-activated receptor agonists.  
         [0011]     WO 02/02554 describes sulfonyl-pyrrolidine derivatives useful for the treatment of neurological disorders.  
       SUMMARY OF THE INVENTION  
       [0012]     This invention relates to a series of sulfonamide derivatives, processes for their preparation, pharmaceutical compositions containing them, and their use in therapy. The compounds are believed to be useful in the treatment of hepatitis C by virtue of their ability to inhibit hepatitis C polymerase (NS5B).  
         [0013]     This invention is directed to compounds of formula (I):  
                         
 
         [0014]     wherein:  
         [0015]     R 1  is H, —COOH, —CO 2 R 4 , cyano, tetrazole, a straight chain alkyl of 1 to 6 carbon atoms optionally substituted with OH, amine or —COOH, an optionally substituted —C(O)-alkyl, or an optionally substituted —C(O)-aryl, wherein R 4  is an alkyl, aryl, cycloalkyl or heteroaryl, any of which may be optionally substituted;  
         [0016]     R 2  is an aryl or a heteroaryl group optionally substituted with one to five substituents selected from the group consisting of halogen, —NO 2 , —CN, —N 3 , —CHO, —CF 3 , —OCF 3 , —R 3 , —OR 3 , S(O) m R 3 , —NR 3 R 3 , NR 3 S(O) m R 3 , —NR 3 C(O)R 3 , —C(O)R 3 , —C(O)OR 3 , —C(O)NR 3 R 3 , —OC(O)R 3 , —OC(O)OR 3 , —OC(O)NR 3 R 3 , NR 3 C(O)R 3 , —NR 3 C(O)OR 3 , and —NR 3 C(O)NR 3 R 3 , wherein m is 0, 1, or 2;  
         [0017]     R 3  is H, an alkyl of 1-6 carbon atoms, a branched alkyl of 1-8 carbon atoms, a cycloalkyl of 3 to 6 carbon atoms, phenyl, a heteroaryl, an alkenyl of 2-6 carbon atoms, or an alkynyl of 2-6 carbon atoms;  
         [0018]     X is CH 2 , CHOR 3 , or S; and  
         [0019]     n is 1 or 2;  
         [0020]     and all crystalline forms and pharmaceutically acceptable salts thereof, with the provisos that when X is CH 2 , n is 1, and R 1  is —COOH, R 2  cannot be  
                         
 
         [0021]     wherein:  
         [0022]     A is CH 3 —, CH 3 CH 2 — or a haloalkyl of 1 to 2 carbon atoms; and  
         [0023]     B is a halogen; and  
         [0024]     when X is CH 2 , n is 2, and R 1  is —COOH, R 2  cannot be  
                         
 
         [0025]     The present invention is also directed to compounds of formula (II):  
                         
 
         [0026]     wherein:  
         [0027]     R 1  is H, —COOH, —CN, tetrazole, —C(O)R 4 , or a hydroxyalkyl of 1 to 4 carbon atoms, wherein R 4  is an alkyl of 1 to 4 carbon atoms or an optionally substituted phenyl;  
         [0028]     R 5  is H, OH or —OCH 3 ; and  
         [0029]     X 1 -X 5  are independently H, a halogen, OH, NH 2 , an alkyl of 1 to 4 carbon atoms, —NH—C(O)—R 3 , wherein R 3  is an alkyl of 1 to 4 carbon atoms, an aryl, a cycloalkyl of 3 to 6 carbon atoms, or a heteroaryl;  
         [0030]     and all crystalline forms or pharmaceutically acceptable salts thereof, with the provisos that when R 1  is —COOH, R 5  is H, X 3  is a halogen, and X 4  is —CH 3  or —CF 3 , then X 1  cannot be NH 2 ; and  
         [0031]     when R 1  is —COOH, R 5  is H, X 2  is —CH 3  or —CF 3 , and X 3  is a halogen, then X 5  cannot be NH 2    
         [0032]     Another aspect of the present invention are compounds of formula (III):  
                         
 
         [0033]     wherein:  
         [0034]     R 1  is H, —COOH, —CN, tetrazole, —C(O)R 4 , or a hydroxyalkyl of 1 to 4 carbon atoms, wherein R 4  is an alkyl of 1 to 4 carbon atoms or an optionally substituted phenyl; and  
         [0035]     X 1 -X 5  are independently H, a halogen, OH, NH 2 , an alkyl of 1 to 4 carbon atoms, —NH—C(O)R 3 , wherein R 3  is an alkyl of 1 to 4 carbon atoms, an aryl, a cycloalkyl of 3 to 6 carbon atoms or a heteroaryl;  
         [0036]     and all crystalline forms and pharmaceutically acceptable salt thereof, with the provisos that when R 1  is —COOH, X 3  is a halogen, and X 4  is —CH 3  or —CH 2 CH 3 , then X 1  cannot be NH 2 ; and  
         [0037]     when R 1  is —COOH, X 2  is —CH 3  or —CH 2 CH 3 , and X 3  is a halogen, then X 5  cannot be NH 2    
         [0038]     The present invention is also directed to compounds of formula (IV):  
                         
 
         [0039]     wherein:  
         [0040]     R 1  is H, —COOH, —CN, tetrazole, —C(O)R 4 , or a hydroxyalkyl of 1 to 4 carbon atoms, wherein R 4  is an alkyl of 1 to 4 carbon atoms or an optionally substituted phenyl; and  
         [0041]     X 1  to X 5  are independently H, a halogen, OH, NH 2 , an alkyl of 1 to 4 carbon atoms, —NH—C(O)R 3 , wherein R 3  is an alkyl of 1 to 4 carbon atoms, an aryl, a cycloalkyl of 3 to 6 carbon atoms, or a heteroaryl;  
         [0042]     and all crystalline forms and pharmaceutically acceptable salts thereof.  
         [0043]     The present invention is also directed to pharmaceutical compositions comprising a compound of the present invention and a pharmaceutically acceptable carrier.  
         [0044]     The present invention also includes methods of treating or preventing a hepatitis C infection in humans, comprising administering an effective amount of a compound of formula (Ia):  
                         
 
         [0045]     wherein:  
         [0046]     R 1  is H, —COOH, —CO 2 R 4 , cyano, tetrazole, a straight chain alkyl of 1 to 6 carbon atoms optionally substituted with OH, amine, or —COOH, an optionally substituted —C(O)-alkyl, or an optionally substituted —(O)-aryl, wherein R 4  is an alkyl, aryl, cycloalkyl or a heteroaryl;  
         [0047]     R 2  is an aryl or a heteroaryl group optionally substituted with one to five substituents selected from the group consisting of halogen, —NO 2 , —CN, —N 3 , —CHO, —CF 3 , —OCF 3 , —R 3 , —OR 3 , —S(O) m R 3 , —NR 3 R 3 , —NR 3 S(O) m R 3 , —NR 3 C(O)R 3 , —C(O)R 3 , —C(O)OR 3 , —C(O)NR 3 R 3 , —OC(O)R 3 , —OC(O)OR 3 , —OC(O)NR 3 R 3 , NR 3 C(O)R 3 , —NR 3 C(O)OR 3 , and —NR 3 C(O)NR 3 R 3 , wherein m is 0, 1, or 2;  
         [0048]     R 3  is H, an alkyl of 1-6 carbon atoms, a branched alkyl of 1-8 carbon atoms, a cycloalkyl of 3 to 6 carbon atoms, phenyl, heteroaryl, an alkenyl of 2-6 carbon atoms, or an alkynyl of 2-6 carbon atoms;  
         [0049]     X is CH 2 , CHOR 3 , or S; and  
         [0050]     n is 1 or 2;  
         [0051]     and all crystalline forms and pharmaceutically acceptable salts thereof. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0052]     For purposes of this invention the term “alkyl” includes straight chain moieties with a length of up to 12 carbon atoms, but preferably 1 to 6 carbon atoms, and more preferably 1 to 4 carbons. The term “alkyl” also includes branched moieties of 3 to 12 carbon atoms, but preferably 1 to 8 carbon atoms. The term “alkenyl” refers to a radical aliphatic hydrocarbon containing one double bond and includes both straight and branched alkenyl moieties of 2 to 7 carbon atoms. Such alkenyl moieties may exist in the E or Z configurations; the compounds of this invention include both configurations. The term “alkynyl” includes both straight chain and branched moieties containing 2 to 7 carbon atoms having at least one triple bond. The term “cycloalkyl” refers to alicyclic hydrocarbon groups having 3 to 12 carbon atoms and includes but is not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, or adamantyl. Preferably cycloalkyl groups are 3 to 6 carbon atoms.  
         [0053]     For purposes of this invention the term “aryl” is defined as an aromatic hydrocarbon moiety having at least one aromatic ring, is mono-, bi- or tri-cyclic, and may be substituted or unsubstituted. An aryl group may be selected from but is not limited to: phenyl, α-naphthyl, β-naphthyl, biphenyl, anthryl, tetrahydronaphthyl, phenanthryl, fluorenyl, indanyl, biphenylenyl, acenaphthenyl, acenaphthylenyl, or phenanthrenyl. An aryl group may be optionally substituted with substituents selected from, but not limited to, the group consisting of alkyl, haloalkyl, acyl, alkoxycarbonyl, alkoxy, haloalkoxy, alkoxyalkyl, alkoxyalkoxy, cyano, halogen, hydroxy, nitro, trifluoromethyl, trifluoromethoxy, trifluoropropyl, amino, alkylamino, dialkylamino, dialkylaminoalkyl, hydroxyalkyl, alkoxyalkyl, alkylthio, mercapto, haloalkylthio, aryl, aryloxy, arylthio, heterocycloalkoxy, heterocycloalkylthio, —SO 3 H, —SO 2 NH 2 , —O 2 NHalkyl, —SO 2 N(alkyl) 2 , —CO 2 H, CO 2 NH 2 , CO 2 NHalkyl, and —CO 2 N(alkyl) 2 . Preferred substituents for aryl and heterocycloalkyl include: alkyl, halogen, amino, alkylamino, dialkylamino, trifluoromethyl, trifluoromethoxy, arylalkyl, and alkylaryl. Preferably an aryl group consists of 6 to 12 carbon atoms.  
         [0054]     For purposes of this invention, the term “heteroaryl” is defined as: (1) an aromatic heterocyclic ring system (monocyclic or bicyclic) where the heteroaryl moieties are selected from furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, N-methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole, 1,2,4-triazole, 1-methyl-1,2,4-triazole, 1H-tetrazole, 1-methyltetrazole, benzoxazole, benzothiazole, benzofuran, benzisoxazole, benzimidazole, N-methylbenzimidazole, azabenzimidazole, indazole, quinazoline, quinoline, and isoquinoline; and (2) a bicyclic aromatic heterocycle where a phenyl, pyridine, pyrimidine or pyridizine ring is: (a) fused to a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a 5 or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with either one oxygen or one sulfur atom; or (d) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one heteroatom selected from O, N or S. Preferably a heteroaryl group consists of 2 to 9 carbon atoms.  
         [0055]     For the purposes of this invention the term “hydroxyalkyl” is defined as an alkyl, as defined above, substituted with a hydroxyl group.  
         [0056]     The compounds of this invention may contain an asymmetric carbon atom and one or more asymmetric centers, and may thus give rise to optical isomers and diastereomers. While shown without respect to stereochemistry in formulas (I), (II), (III), and (IV), the present invention includes all optical isomers and diastereomers, racemic and resolved, enantiomerically pure R and S stereoisomers, and other mixtures of the R and S stereoisomers and pharmaceutically acceptable salts thereof.  
         [0057]     Pharmaceutically acceptable salts of the compounds of formulas (I), (II), (III), and (IV) with an acidic moiety can be formed from both organic and inorganic bases. For example, alkali metal salts such as sodium, lithium, and potassium, and N-tetraalkylammonium salts such as N-tetrabutylammonium salts. Similarly, when a compound of this invention contains a basic moiety, salts can be formed from organic and inorganic acids. For example, salts can be formed from acetic, propionic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, naphthalenesulfonic, benzenesulfonic, toluenesulfonic, camphorsulfonic, and similarly known acceptable acids.  
         [0058]     A preferred embodiment of the compounds of formula (I) is wherein X is CH 2  or S, especially where X is S and n is 1.  
         [0059]     Another preferred embodiment of the compounds of formula (I) is wherein X is CH 2 OR 3  and R 3  is H or CH 3 , especially where n is 1.  
         [0060]     Another preferred embodiment of the compounds of formula (I) is wherein R 1  is H, —COOH, —CN, tetrazole, —CH 2 OH, —C(O)—CH 3  or —C(O)-phenyl, but even more preferred is where R 1  is —COOH.  
         [0061]     Yet another preferred of the compounds of formula (I) is wherein R 2  is optionally substituted phenyl, especially where the phenyl ring is substituted by at least one substituent selected from OH, halogen, alkyl, amino, and —NR 3 C(O)R 3    
         [0062]     A preferred embodiment of the compounds of formula (II) is wherein R 5  is H, OH, or —OCH 3 .  
         [0063]     Another preferred embodiment of the compounds of formula (II) is wherein R 1  is —COOH.  
         [0064]     Yet another preferred embodiment of the compounds of formula (II) is wherein at least one of X 1 -X 5  is OH or —NH—C(O)—R 3 .  
         [0065]     A preferred embodiment of the compounds of formula (III) is wherein R 1  is —COOH.  
         [0066]     Another preferred embodiment of the compounds of formula (III) is wherein X 1 -X 5  are independently selected from H, NH 2 , OH, halogen and alkyl, especially where at least one of X 1 -X 5  is OH.  
         [0067]     A preferred embodiment of the compounds of formula (IV) is wherein R 1  is —COOH or H.  
         [0068]     Another preferred embodiment of the compounds of formula (IV) is wherein X 1 -X 5  are independently selected from H, halogen, NH 2 , alkyl and OH.  
         [0069]     Preferred compounds of the present invention include: 
    2,4-dichloro-6-{[(2S)-2-(2H-tetrazol-5-yl)pyrrolidin-1-yl]sulfonyl}phenol;     (2S)-1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]pyrrolidine-2-carbonitrile;     (4R)-4-hydroxy-1-[(3,4,5-trichloro-2-hydroxyphenyl)sulfonyl]-L-proline;     1-[(3,4,5-trichloro-2-hydroxyphenyl)sulfonyl]-L-proline;     1-[(3-bromo-5-chloro-2-hydroxyphenyl)sulfonyl]-L-proline;     (4R)-1-[(3,5-dibromo-2-hydroxyphenyl)sulfonyl]-4-hydroxy-L-proline;     (4R)-1-[(3-bromo-5-chloro-2-hydroxyphenyl)sulfonyl]-4-hydroxy-L-proline;     1-[(5-bromo-3-chloro-2-hydroxyphenyl)sulfonyl]-L-proline;     1-[(3,5-dibromo-2-hydroxyphenyl)sulfonyl]-L-proline;     (4R)-1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]-4-hydroxy-L-proline;     1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]-L-proline;     (4R)-1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]-4-methoxy-L-proline;     1-[(3,4,5-trichloro-2-hydroxyphenyl)sulfonyl]-D-proline;     1-[(3-chloro-5-fluoro-2-hydroxyphenyl)sulfonyl]-L-proline;     (4S)-4-hydroxy-1-[(3,4,5-trichloro-2-hydroxyphenyl)sulfonyl]-D-proline;     1-{(2S)-1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]pyrrolidin-2-yl}ethanone;     1-[(2-amino-5-chloro-4-methylphenyl)sulfonyl]-D-proline;     (4R)-1-[(3-chloro-5-fluoro-2-hydroxyphenyl)sulfonyl]-4-hydroxy-L-proline;     (4S)-1-[(2-amino-5-chloro-4-methylphenyl)sulfonyl]-4-hydroxy-D-proline;     1-[(2-amino-5-chloro-4-methylphenyl)sulfonyl]-L-proline; 2,4-dichloro-6-{[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]sulfonyl}phenol;     1-[(5-bromo-3-chloro-2-hydroxyphenyl)sulfonyl]-D-proline;     2,4-dichloro-6-(pyrrolidin-1-ylsulfonyl)phenol;     (4S)-1-[(2-amino-4-chloro-5-methylphenyl)sulfonyl]-4-hydroxy-D-proline;     (4R)-1-[(2-amino-5-chloro-4-methylphenyl)sulfonyl]-4-hydroxy-L-proline;     1-({5-chloro-4-methyl-2-[(2-thienylcarbonyl)amino]phenyl}sulfonyl)-L-proline; 1-[(3,5-dibromo-2-hydroxyphenyl)sulfonyl]-D-proline;     (4R)-1-[(2-amino-4-chloro-5-methylphenyl)sulfonyl]-4-hydroxy-L-proline;     1-{(2S)-1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]pyrrolidin-2-yl}propan-1-one;     {(2S)-1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]pyrrolidin-2-yl}(phenyl)methanone;     1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]-D-proline;     1-[(2,4,6-trichlorophenyl)sulfonyl]-L-proline;     1-({5-chloro-2-[(cyclopropylcarbonyl)amino]-4-methylphenyl}sulfonyl)-L-proline;     1-{[2-(benzoylamino)-5-chloro-4-methylphenyl]sulfonyl}-L-proline;     1-[(3-chloro-4-methylphenyl)sulfonyl]-L-proline;     1-[(2,4,5-trichlorophenyl)sulfonyl]-L-proline;     (2S)-1-[(3,4,5-trichloro-2-hydroxyphenyl)sulfonyl]piperidine-2-carboxylic acid;     (2R)-1-[(3,4,5-trichloro-2-hydroxyphenyl)sulfonyl]piperidine-2-carboxylic acid;     (2S)-1-[(3-bromo-5-chloro-2-hydroxyphenyl)sulfonyl]piperidine-2-carboxylic acid;     (2R)-1-[(3-bromo-5-chloro-2-hydroxyphenyl)sulfonyl]piperidine-2-carboxylic acid;     (2S)-1-[(3-chloro-5-fluoro-2-hydroxyphenyl)sulfonyl]piperidine-2-carboxylic acid;     (2R)-1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]piperidine-2-carboxylic acid;     (2S)-1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]piperidine-2-carboxylic acid;     (2R)-1-[(2-amino-5-chloro-4-methylphenyl)sulfonyl]piperidine-2-carboxylic acid;     (4R)-3-[(3,4,5-trichloro-2-hydroxyphenyl)sulfonyl]-1,3-thiazolidine-4-carboxylic acid;     (4S)-3-[(3,4,5-trichloro-2-hydroxyphenyl)sulfonyl]-1,3-thiazolidine-4-carboxylic acid;     (4R)-3-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]-1,3-thiazolidine-4-carboxylic acid;     (4R)-3-[(3,5-dibromo-2-hydroxyphenyl)sulfonyl]-1,3-thiazolidine-4-carboxylic acid;     (4R)-3-[(3-chloro-5-fluoro-2-hydroxyphenyl)sulfonyl]-1,3-thiazolidine-4-carboxylic acid;     (4S)-3-[(3-bromo-5-chloro-2-hydroxyphenyl)sulfonyl]-1,3-thiazolidine-4-carboxylic acid;     (4S)-3-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]-1,3-thiazolidine-4-carboxylic acid;     2,4-dichloro-6-(1,3-thiazolidin-3-ylsulfonyl)phenol;     (4S)-3-[(2-amino-5-chloro-4-methylphenyl)sulfonyl]-1,3-thiazolidine-4-carboxylic acid;     (4R)-3-[(2-amino-4-chloro-5-methylphenyl)sulfonyl]-1,3-thiazolidine-4-carboxylic acid;     (4S)-3-[(2-amino-4-chloro-5-methylphenyl)sulfonyl]-1,3-thiazolidine-4-carboxylic acid;     (4R)-3-[(3-bromo-5-chloro-2-hydroxyphenyl)sulfonyl]-1,3-thiazolidine-4-carboxylic acid;     (4S)-3-[(3-chloro-5-fluoro-2-hydroxyphenyl)sulfonyl]-1,3-thiazolidine-4-carboxylic acid; and     (4R)-3-[(2-amino-5-chloro-4-methylphenyl)sulfonyl]-1,3-thiazolidine-4-carboxylic acid.    
 
         [0126]     Compounds of the present invention inhibit the hepatitis C RNA-dependent RNA polymerase NS5B, and are therefore useful for the treatment of hepatitis C infection. The present invention accordingly provides a pharmaceutical composition that comprises a compound selected from formulas (I), (II), (III) and (IV) in combination or association with a pharmaceutically acceptable carrier. The compositions are preferably adapted for oral or subcutaneous administration. However, they may be adapted for other modes of administration. In order to obtain consistency of administration, it is preferred that a composition of the invention is in the form of a unit dose. Suitable unit dose forms include tablets, capsules and powders in sachets or vials. Such unit dose forms may contain from 0.1 to 100 mg of a compound of the invention, and preferably from 2 to 50 mg. Still further preferred unit dosage forms contain 5 to 25 mg of a compound of the present invention. The compounds of the present invention can be administered orally at a dose range of about 0.01 to 100 mg/kg, or preferably at a dose range of 0.1 to 10 mg/kg. Such compositions may be administered from 1 to 6 times a day, more usually from 1 to 4 times a day. The compositions of the invention may be formulated with conventional excipients, such as a filler, a disintegrating agent, a binder, a lubricant, a flavoring agent and the like.  
         [0127]     The present invention further provides a compound of the invention for use as an active therapeutic substance. Compounds of formulas (Ia), (II), (III), and (IV) are of particular use for the treatment of infection with hepatitis C virus.  
         [0128]     The present invention further provides a method of treating hepatitis C infection in humans, which comprises administering to the infected individual an effective amount of a compound of formulas (Ia), (II), (III), and (IV) or a pharmaceutical composition of the invention.  
       General Synthetic Schemes for Preparation of Compounds of the Present Invention  
       [0129]    
       
                 
         
             
             
         
       
       
          Reagents: (a) EDCI, HOBT, DIEA, DMF, RT, 6h; (b) 20% piperidine in DMF, RT, 20 min; (c) Pyridine, RT, ON; (d) 1:1 TFA-DCM, RT, 2h.  
       
     
         [0131]     Scheme 1 shows how compounds of formula (I) can be prepared on a solid support using a resin, for example Wang resin. The Fmoc protected amino acid of interest 2 was attached to the resin using coupling agents, for example EDCI, HOBT in the presence of a base, and DIEA in a polar solvent. DMF may be used as the polar solvent, but one skilled in the art would be aware of other appropriate solvents. After washing the excess reagents and solvent, the amino acid attached to the resin was deprotected using base in DMF. Appropriate bases include alkylamine bases, for example piperidine, but skilled artisans would be aware of other possible bases to use. The free amino acid was reacted with the sulfonyl chloride of interest 5 in a solvent such as pyridine. Depending on the nature of the groups on the sulfonyl chloride, a deprotection step was employed before cleaving the product from the resin using trifluoroacetic acid and DCM. One skilled in the art would of be aware of the possible protecting groups that can be used to protect various functional groups from the acidic cleavage conditions.  
                         
 
         [0132]     Reagents: (g) Pyridine, THF, RT, 8 h; (h) aq. NaOH, EtOH, RT, 8 h.  
         [0133]     Alternately, the analogs can also be obtained by following Scheme 2. In this solution phase method, the amino acid ester of interest 8 was reacted with the sulfonyl chloride of choice 9 in pyridine, but other solvents may be used. The ester was hydrolyzed using a base like sodium hydroxide or lithium hydroxide to provide the required acid.  
         [0134]     The reagents used in the preparation of the compounds of this invention can be either commercially obtained or can be prepared by standard procedures described in the literature. In accordance with this invention, the compounds described are produced by the reaction schemes shown above.  
         [0135]     One skilled in the art would also understand how the methods of preparation detailed in Schemes 1 and 2 would also apply to compounds of formulas (II), (III) and (IV).  
       Specific Synthesis of Compounds of the Present Invention  
       [0136]     Examples 1, 2, 11 and 23 were synthesized in solution as shown in Scheme 2.  
       EXAMPLE 1  
     2,4-dichloro-6-{[(2S)-2-(2H-tetrazol-5-yl)pyrrolidin-1-yl]sulfonyl}phenol  
       [0137]     A mixture of (2S)-1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]pyrrolidine-2-carbonitrile (37 mg, 0.12 mmol), sodium azide (24 mg, 0.36 mmol), and triethylamine hydrochloride (25 mg, 0.18 mmol) in 1.5 mL of DMF was stirred at 120° C. for 6 hours. The reaction was then cooled to room temperature, acidified by adding 2 mL of 1N HCl, and concentrated. 2,4-dichloro-6-{[(2S)-2-(2H-tetrazol-5-yl)pyrrolidin-1-yl]sulfonyl}phenol (31 mg, 71% yield) was obtained after reverse phase chromatography.  1 H NMR (DMSO-d6) δ 7.89 (d, J=3.5 Hz, 1H), 7.60 (d, J=3.5 Hz, 1H), 5.46 (dd, 1H), 3.56 (m, 1H), 3.33 (m, 1H), 2.30 (m, 1H), 1.96 (m, 3H). HRMS: calcd for C 11 H 11 Cl 2 N 5 O 3 S, 364.00325; found (ESI-, [M−H]), 364.00349.  
       EXAMPLE 2  
     (2S)-1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]pyrrolidine-2-carbonitrile  
       [0138]     A mixture of (2S)-pyrrolidine-2-carbonitrile hydrochloride (34 mg, 0.2 mmol) and 3,5-dichloro-2-hydroxy-benzenesulfonyl chloride (57 mg, 0.22 mmol) in 2 mL of CH 2 Cl 2 /pyridine (1:1) was stirred at room temperature for 16 hours. Then the reaction mixture was concentrated and purified by reverse phase chromatography to give (2S)-1-(3,5-Dichloro-2-hydroxy-benzenesulfonyl)-pyrrolidine-2-carbonitrile (37 mg, 58% yield).  1 H NMR (DMSO-d6) δ 7.86 (d, J=2.8 Hz, 1H), 7.63 (d, J=2.8 Hz, 1H), 5.08 (br., 1H), 3.44(m, 1H), 3.18 (m, 1H), 2.54(m, 1H), 2.17 (m, 2H), 1.93 (m, 2H). HRMS: calcd for C 11 H 10 Cl 2 N 2 O 3 S, 320.9862; found (ESI-, [M−H]), 320.98639.  
         [0139]     Example 23 was prepared following the same procedure described for Example 2, except pyrrolidine was substituted for (2S)-pyrrolidine-2-carbonitrile hydrochloride.  
       EXAMPLE 11  
     1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]-L-proline  
       [0140]     Step 1. To a solution of L-proline methyl ester hydrochloride (8.24 g; 50.1 mmol) in pyridine (100 mL) was added 3,5 dichloro-2-hydroxybenzene sulfonyl chloride (15 g; 57.3 mmol) at 0° C. The reaction mixture was warmed to room temperature and stirred for additional 4 h. The mixture was concentrated and purified by flash chromatography on silica gel (25% ethyl acetate in hexane) to yield 1-(3,5-Dichloro-2-hydroxy-benzenesulfonyl)-pyrrolidine-2-carboxylic acid methyl ester (8.6 g; 49%).  
         [0141]     Step 2: The ester from step 1 was taken up in ethanol (175 mL) and 1 N sodium hydroxide was added and stirred overnight. The reaction mixture was then concentrated, diluted with water and extracted with ethyl acetate. The aqueous layer was acidified with 2N HCl to yield the desired compound as white solid (7.3 g; 89%). mp 107.4° C.; 1H NMR (CDCl3) δ 7.6 (d, 1H), 7.5 (d, 1H), 4.5 (dd, 1H), 3.4(m, 2H), 2.3 (m, 2H), 2.0 (m, 2H); MS (ESI) m/z 337.82; HRMS: calcd for C 11 H 11 Cl 2 NO5S, 337.96622; found (ESI-, [M−H]), 337.96619.  
         [0142]     Examples 3-10, 12-17, 18-19, 20-22, 24-58 were prepared in solid phase as described below for Example 20, using appropriately protected amino acids and sulfonyl chlorides, as shown in Scheme 1.  
       EXAMPLE 20  
     1-[(2-amino-5-chloro-4-methylphenyl)sulfonyl]-L-proline  
       [0143]     Step 1. 5-Chloro-2-(9H-fluoren-9-ylmethoxycarbonylamino)-4-methyl-benzenesulfonic acid. To a solution of 5-Chloro-2-amino-4-methyl-benzenesulfonic acid (3.3 g; 15.13 mmol) in water (40 mL) and sodium bicarbonate (2.8 g) was added a solution of 9-fluorenylmethoxycarbonyl chloride (4.3 g; 16.7 mmol) in dioxane (40 mL) at 0° C. The reaction mixture was stirred overnight and the volatiles were removed under reduced pressure. The aqueous layer was acidified with 2 N HCl, extracted with ethyl acetate, dried and concentrated. The residue was purified by flash column on silica gel (10% MeOH in EtOAc) to give a white solid (89%).  1 H NMR (DMSO-d6) δ 9.9 (s, 1H), 7.9 (d, J=8 Hz, 3H), 7.7 (d, J=8 Hz, 2H), 7.6 (s, 1H), 7.4 (m, 2H), 7.3 (m, 2H), 4.4 (d, J=7 Hz, 2H), 4.0 (t, J=7 Hz 1H), 2.3 (s, 3H).  
         [0144]     Step 2. (4-Chloro-2-chlorosulfonyl-5-methyl-phenyl)-carbamic acid 9H-fluoren-9-ylmethyl ester. Sulfonic acid (3.0 g; 6.7 mmol) from step 1 was dissolved in 3 mL of DMF, and 2.5 mL of thionyl chloride was added dropwise at room temperature. The resulting solution was stirred at room temperature for an additional 4 hours and then quenched with ice and water. The white solid precipitate was filtered and dried, and used without further purification.  
         [0145]     Step 3. Attachment of N-Fmoc-L-Proline to Wang Resin. Wang Resin (Ana Spec 100-200 mesh, 1% crosslinked; loading: 1.1 mmol/g; 5 g, 5.5 mmol) was swollen in anhydrous DMF (20 ml). A solution of N-Fmoc-L-Proline (7.4 g, 22 mmol), HOBT (3.37 g, 22 mmol), DMAP (268.8 mg, 2.2 mmol) and DIC (3.4 ml, 22 mmol) in anhydrous DMF (30 ml) was added to the resin. The mixture was shaken at room temperature on an orbital shaker overnight. The mixture was filtered and the resin washed with DMF (3×50 ml), MeOH (3×50 ml), CH 2 Cl 2  (3×50 ml), and dried.  
         [0146]     Step 4. Deprotection of Fmoc Group. The resin (5.5 mmol), prepared as described in step 1 above, was treated with a solution of 20% piperidine in DMF (2×50 ml, 10 min for the first time and 30 min for the second time) to remove the Fmoc protecting group from the resin. The mixture was filtered and the resin washed with DMF (3×50 ml), MeOH (3×50 ml), and CH 2 Cl 2  (3×50 ml).  
         [0147]     Step 5. Reaction with (4-Chloro-2-chlorosulfonyl-5-methyl-phenyl)-carbamic acid 9H-fluoren-9-ylmethyl ester. To the L-proline on Wang resin (5.5 mmol) was added a solution of (4-Chloro-2-chlorosulfonyl-5-methyl-phenyl)-carbamic acid 9H-fluoren-9-ylmethyl ester (5.1 g, 11 mmol) in 1:1 anhydrous CH 2 Cl 2  and pyridine (50 ml). After shaking at room temperature overnight, the mixture was filtered, washed with MeOH (3×50 ml) and CH 2 Cl 2  (5×50 ml).  
         [0148]     Step 6. Deprotection of Fmoc group. The resin (5.5 mmol) obtained from step 5 was reacted again with a solution of 20% piperidine in DMF (2×50 ml, 10 min for the first time and 30 min for the second time). The mixture was filtered and the resin washed with DMF (3×50 ml), MeOH (3×50 ml), CH 2 Cl 2  (3×50 ml), and dried.  
         [0149]     Step 7. Cleavage from resin. The above resin was treated with 1:1 TFA:CH 2 Cl 2  (50 ml) and was shaken at room temperature for 4 h. The mixture was filtered and the resin washed with CH 2 Cl 2  (3×10 ml). The combined CH 2 Cl 2  was concentrated and purified by HPLC.  1 H NMR (DMSO-d6) δ 7.4 (s, 1H), 6.8 (s, 1H), 6.3 (s, 2H), 4.3 (m, 1H), 3.2 (t, 2H), 2.2 (s, 3H), 2.1 (m, 1H), 1.9 (m, 1H), 1.65-1.8 (m, 2H). HRMS: calcd for C 12 H 15 ClN 2 O 4 S, 319.05139; found (ESI-FTMS, [M+H]), 319.05179.  
       EXAMPLES  
       [0150]     Examples of compounds of the present invention are listed in Table 1.  
                             TABLE 1                       Example           No.   Compound Name                                1   2,4-dichloro-6-{[(2S)-2-(2H-tetrazol-5-yl)pyrrolidin-           1-yl]sulfonyl}phenol       2   (2S)-1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]pyrrolidine-           2-carbonitrile       3   (4R)-4-hydroxy-1-[(3,4,5-trichloro-2-           hydroxyphenyl)sulfonyl]-L-proline       4   1-[(3,4,5-trichloro-2-hydroxyphenyl)sulfonyl]-L-proline       5   1-[(3-bromo-5-chloro-2-hydroxyphenyl)sulfonyl]-L-proline       6   (4R)-1-[(3,5-dibromo-2-hydroxyphenyl)sulfonyl]-4-hydroxy-           L-proline       7   (4R)-1-[(3-bromo-5-chloro-2-hydroxyphenyl)sulfonyl]-           4-hydroxy-L-proline       8   1-[(5-bromo-3-chloro-2-hydroxyphenyl)sulfonyl]-L-proline       9   1-[(3,5-dibromo-2-hydroxyphenyl)sulfonyl]-L-proline       10   (4R)-1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]-4-hydroxy-           L-proline       11   1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]-L-proline       12   (4R)-1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]-4-methoxy-           L-proline       13   1-[(3,4,5-trichloro-2-hydroxyphenyl)sulfonyl]-D-proline       14   1-[(3-chloro-5-fluoro-2-hydroxyphenyl)sulfonyl]-L-proline       15   (4S)-4-hydroxy-1-[(3,4,5-trichloro-2-           hydroxyphenyl)sulfonyl]-D-proline       16   1-{(2S)-1-[(3,5-dichloro-2-           hydroxyphenyl)sulfonyl]pyrrolidin-2-yl}ethanone       17   1-[(2-amino-5-chloro-4-methylphenyl)sulfonyl]-D-proline       18   (4R)-1-[(3-chloro-5-fluoro-2-hydroxyphenyl)sulfonyl]-4-           hydroxy-L-proline       19   (4S)-1-[(2-amino-5-chloro-4-methylphenyl)sulfonyl]-4-           hydroxy-D-proline       20   1-[(2-amino-5-chloro-4-methylphenyl)sulfonyl]-L-proline       21   2,4-dichloro-6-{[(2S)-2-(hydroxymethyl)pyrrolidin-1-           yl]sulfonyl}phenol       22   1-[(5-bromo-3-chloro-2-hydroxyphenyl)sulfonyl]-D-proline       23   2,4-dichloro-6-(pyrrolidin-1-ylsulfonyl)phenol       24   (4S)-1-[(2-amino-4-chloro-5-methylphenyl)sulfonyl]-4-           hydroxy-D-proline       25   (4R)1-[(2-amino-5-chloro-4-methylphenyl)sulfonyl]-4-           hydroxy-L-proline       26   1-({5-chloro-4-methyl-2-[(2-           thienylcarbonyl)amino]phenyl}sulfonyl)-L-proline       27   1-[(3,5-dibromo-2-hydroxyphenyl)sulfonyl]-D-proline       28   (4R)-1-[(2-amino-4-chloro-5-methylphenyl)sulfonyl]-4-           hydroxy-L-proline       29   1-{(2S)-1-[(3,5-dichloro-2-           hydroxyphenyl)sulfonyl]pyrrolidin-2-yl}propan-1-one       30   {(2S)-1-[(3,5-dichloro-2-           hydroxyphenyl)sulfonyl]pyrrolidin-2-yl}(phenyl)methanone       31   1-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]-D-proline       32   1-[(2,4,6-trichlorophenyl)sulfonyl]-L-proline       33   1-({5-chloro-2-[(cyclopropylcarbonyl)amino]-4-           methylphenyl}sulfonyl)-L-proline       34   1-{[2-(benzoylamino)-5-chloro-4-           methylphenyl]sulfonyl}-L-proline       35   1-[(3-chloro-4-methylphenyl)sulfonyl]-L-proline       36   1-[(2,4,5-trichlorophenyl)sulfonyl]-L-proline       37   (2S)-1-[(3,4,5-trichloro-2-           hydroxyphenyl)sulfonyl]piperidine-2-carboxylic acid       38   (2R)-1-[(3,4,5-trichloro-2-           hydroxyphenyl)sulfonyl]piperidine-2-carboxylic acid       39   (2S)-1-[(3-bromo-5-chloro-2-           hydroxyphenyl)sulfonyl]piperidine-2-carboxylic acid       40   (2R)-1-[(3-bromo-5-chloro-2-           hydroxyphenyl)sulfonyl]piperidine-2-carboxylic acid       41   (2S)-1-[(3-chloro-5-fluoro-2-           hydroxyphenyl)sulfonyl]piperidine-2-carboxylic acid       42   (2R)-1-[(3,5-dichloro-2-           hydroxyphenyl)sulfonyl]piperidine-2-carboxylic acid       43   (2S)-1-[(3,5-dichloro-2-           hydroxyphenyl)sulfonyl]piperidine-2-carboxylic acid       44   (2R)-1-[(2-amino-5-chloro-4-           methylphenyl)sulfonyl]piperidine-2-carboxylic acid       45   (4R)-3-[(3,4,5-trichloro-2-hydroxyphenyl)sulfonyl]-1,3-           thiazolidine-4-carboxylic acid       46   (4S)-3-[(3,4,5-trichloro-2-hydroxyphenyl)sulfonyl]-1,3-           thiazolidine-4-carboxylic acid       47   (4R)-3-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]-1,3-           thiazolidine-4-carboxylic acid       48   (4R)-3-[(3,5-dibromo-2-hydroxyphenyl)sulfonyl]-1,3-           thiazolidine-4-carboxylic acid       49   (4R)-3-[(3-chloro-5-fluoro-2-hydroxyphenyl)sulfonyl]-1,3-           thiazolidine-4-carboxylic acid       50   (4S)-3-[(3-bromo-5-chloro-2-hydroxyphenyl)sulfonyl]-1,3-           thiazolidine-4-carboxylic acid       51   (4S)-3-[(3,5-dichloro-2-hydroxyphenyl)sulfonyl]-1,3-           thiazolidine-4-carboxylic acid       52   2,4-dichloro-6-(1,3-thiazolidin-3-ylsulfonyl)phenol       53   (4S)-3-[(2-amino-5-chloro-4-methylphenyl)sulfonyl]-1,3-           thiazolidine-4-carboxylic acid       54   (4R)-3-[(2-amino-4-chloro-5-methylphenyl)sulfonyl]-1,3-           thiazolidine-4-carboxylic acid       55   (4S)-3-[(2-amino-4-chloro-5-methylphenyl)sulfonyl]-1,3-           thiazolidine-4-carboxylic acid       56   (4R)-3-[(3-bromo-5-chloro-2-hydroxyphenyl)sulfonyl]-1,3-           thiazolidine-4-carboxylic acid       57   (4S)-3-[(3-chloro-5-fluoro-2-hydroxyphenyl)sulfonyl]-1,3-           thiazolidine-4-carboxylic acid       58   (4R)-3-[(2-amino-5-chloro-4-methylphenyl)sulfonyl]-1,3-           thiazolidine-4-carboxylic acid                  
 
       Brief Description of Biological Test Procedure(s) and Text Summary of Results  
       [0151]     The ability of the compounds of the present invention to inhibit hepatitis C polymerase was established by the following experimental procedure:  
         [0152]     NS5B from the BK strain (1b subtype) is expressed in  E. coli  as a protein in which the 21 C-terminal amino acids are replaced with a short linker and a hexahistidine tag (GSHHHHHH). The purified protein is mixed with radioactive nucleotides and allowed to replicate a heteropolymeric RNA substrate, primed by an endogenous short hairpin, resulting in an approximately 760 nt product. The radioactive product is captured on a filter and quantitated after removal of the unincorporated nucleotides.  
         [0000]     Reagents:  
         [0000]     10 mM UTP (Promega # p116B)  
         [0000]     10 mM ATP (Promega # p113B)  
         [0000]     10 mM CTP (Promega # p114B)  
         [0000]     10 mM GTP (Promega # p115B)  
         [0000]     BSA 10 mg/ml NEB (100× at 10 mg/ml) #007-BSA  
         [0000]     RNaseIn (Promega #N251X) 40 U/μl  
         [0000]      33 P-GTP (NEN-easytides NEG/606H 3000 Ci/mmol, 370 MBq/ml, 10 mCi/ml)  
         [0000]     Falcon polypropylene 96-well plates (Becton Dickinson #351190)  
         [0000]     Millipore Multiscreen 96-well filtration plate #MADE NOB 50  
         [0000]     Optiphase Supermix (Wallac) formulated by Fisher  
         [0000]     Millipore Multiscreen liner for use in MicroBeta 1450-106 cassette (Wallac)  
         [0000]     PerkinElmer #1450-433  
         [0000]     1 M HEPES, pH 7.3  
         [0000]     Amersham Pharmacia Biotec (US16924-500 ml)  
         [0000]     1 M MgCl 2  (SIGMA #M1028)  
         [0000]     DTT (solid) (SIGMA #D9779)  
         [0000]     RNAse-free water (GIBCO-BRL #10977-023)  
         [0000]     Dimethyl sulfoxide (Aldrich #27685-5)  
         [0000]     Basilen Blue (Sigma, B5520)  
         [0000]     0.5M EDTA, pH 8 (GIBCO-BRL #15575-020)  
         [0000]     Dibasic sodium phosphate 7-hydrate (Na 2 HPO 4 .7H 2 O; Baker#3824-07)  
         [0000]     Phosphoric acid (Baker, #0262.02)  
         [0000]     Further Reagent Preparation:  
         [0153]     0.5 M Na Phosphate buffer. Per liter, weigh 134 g. Na 2 HPO 4 .7H 2 O; add water to 900 ml. Adjust pH to 7.0 with phosphoric acid. Top off with water to 1 L.  
         [0154]     Dilute nucleotides 1:1000 to 10 μM (GTP and CTP) or 1:100 to 100 μM (ATP and UTP) into RNAse-free water.  
         [0000]     Procedure:  
         [0000]     (1) Compounds 10 μl at 10 μg/ml in 15% DMSO  
         [0000]     When starting from 100 μg/ml compound stock in 1% DMSO:  
         [0000]     Dispense 5 μl 30% DMSO per well  
         [0000]     Dispense 5 μl compound (100 μg/ml) per well.  
         [0000]     When starting from 50 μg/ml compound stock in 15% DMSO:  
         [0000]     Add 10 μl compound per well.  
         [0155]     (2) Enzyme Mix:  
                                                                     Stock   Final Conc. (in 50 μl assay volume)           reactions   Per 20 μl mix (1 reaction)   Per 600                                DEPC H 2 0           17.06   μl   10236   μl       1 M HEPES,   20   mM   0.5   μl   300   μl       pH 7.5       1 M MgCl 2     5   mM   0.25   μl   150   μl       100 mM DTT   1   mM   0.5   μl   300   μl       100 μM UTP   0.5   μM   0.25   μl   150   μl       100 μM ATP   1   μM   0.5   μl   300   μl       10 μM CTP   0.08   μM   0.4   μl   240   μl       10 μM GTP   0.025   μM   0.125   μl   75   μl       BSA, 10 mg/ml   0.05   mg/ml   0.25   μl   150   μl       HCV RdRp   24   nM   0.16   μl   96   μl       NS5B d21BK       (500 ug/ml       or ˜7.5 uM)                                   Total: 20   μl   12   ml                  
 
         [0156]     Add 20 μl enzyme mix into each well of the assay plate. Incubate compound and enzyme at room temperature for 15 minutes.  
         [0000]     (3) Template Mix—Prepare Ahead.  
         [0157]     Spin down a tube of RNA (5 μg/tube stored in 75% ethanol and 0.3 M sodium acetate) in a microcentrifuge for 20 min. at 4° C. One tube is enough for 1 to 1½ plates. Remove as much ethanol from the tube as possible by inverting the tube. Be gentle, pellet RNA may not adhere to the tube. Vacuum dry the RNA. Resuspend the RNA by adding 1 ml of DEPC water, close the cap of the tube tightly. To dissolve RNA, incubate RNA solution on ice for ˜60 min. and gently vortex. Spin briefly to ensure all RNA solution is down to the bottom of the tube before opening cap. Gently transfer RNA solution into a 5 ml or larger tube. Add another 3 ml of DEPC water (total 4 ml of volume).  
         [0158]     Add the following volumes of reagents.  
                                                                             Final                   concentration           Per 600   Per 20       Stock   reactions   μl mix   (1 reaction)                                RNAse-free water           2.98   μl   1788   μl       Hepes, 1M   20   mM   0.5   μl   300   μl       Rnase Inhibitor   0.4   U/μl   0.5   μl   300   μl       (40 (1 reaction)/μl)         33 P-GTP 3000 Ci/mmol,   0.025   μM   0.0125   μl   7.5   μl       10 μCi/μl (3.3 μM)       POF   3   nM   16   μl   9600   μl                  
 
         [0159]     Add 20 μl template mix per reaction (i.e. 20 ng of POF per reaction or ˜3 nM)  
         [0160]     (4) Incubate reaction at room temperature (22-25° C.) for 2 hours.  
         [0161]     (5) Stop reaction by adding 50 μl of 170 mM EDTA.  
         [0162]     Final concentration of EDTA is 85 mM.  
         [0163]     (6) Prewet filters of Millipore Multiscreen filter plate by adding 200 μl of 0.5 M sodium phosphate buffer, pH 7.0 into each well. Let stand at room temperature for 2-3 min.  
         [0164]     (7) Place the Multiscreen filter plate onto a Millipore Manifold and turn on vacuum to allow buffer to flow through. Turn off vacuum. Transfer 80 μl of the reaction product into each well of the filter plate. Let stand for 2-3 min. Turn on vacuum to filter reaction product.  
         [0165]     (8) Turn off vacuum. Add 200 μl of 0.5 M sodium phosphate buffer, pH 7.0 into each well to wash filter. Turn on vacuum.  
         [0166]     Repeat step (8) three more times.  
         [0167]     (9) Remove polypropylene bottom. Spot dry filter at the bottom with paper towel. Air dry filter plate on a bench for 1 hr. Add 40 μl Super Mix scintillant. Seal top of the plate with a tape. Place plate into a Packard carrier or MicroBeta carrier.  
         [0168]     (10) Count plate using a Packard Topcount or MicroBeta counter. Program 10 for  33 P in Top count or  33 P program in micro-beta.  
         [0000]     Analysis of Results  
         [0169]     Percent inhibition is calculated after background subtraction as a percent reduction of activity relative to the positive control (average value of the plate excluding the negative controls). For the primary screen hits were chosen as showing ≧75% inhibition.  
         [0170]     Table 2 shows the in vitro inhibitory activity for the compounds of the present invention towards HCV polymerase.  
                                         TABLE 2                                       HCV Polymerase           Example No.   Median IC50 (μM)                                        1   0.56           2   1.04           3   0.07           4   0.08           5   0.26           6   0.46           7   0.47           8   0.40           9   0.56           10   0.67           11   0.89           12   0.91           13   1.17           14   2.05           15   1.22           16   2.62           17   4.70           18   3.28           19   4.05           20   5.95           21   6.10           22   4.81           23   4.85           24   5.10           25   7.65           26   6.87           27   7.00           28   10.00           29   7.29           30   7.52           31   6.45           32   9.60           33   8.68           34   10.11           35   10.25           36   21.90           37   0.08           38   0.38           39   0.45           40   1.16           41   1.89           42   2.01           43   7.10           44   7.90           45   0.05           46   0.26           47   0.24           48   0.46           49   2.13           50   0.87           51   1.71           52   2.35           53   3.05           54   4.75           55   7.50           56   11.20           57   14.40           58   1.98                      
 
         [0171]