BACKGROUND OF THE INVENTION Infection by hepatitis C virus ("HCV") is a compelling human medical problem. HCV is recognized as the causative agent for most cases of non-A, non-B hepatitis, with an estimated human seroprevalence of 1% globally [Purcell, R. H.,"Hepatitis C virus: Historical perspective and current concepts"FEMS Microbiology Reviews 14, pp. 181-192 (1994); Van der Poel, C. L., "Hepatitis C Virus. Epidemiology, Transmission and Prevention in Hepatitis C Virus. Current Studies in Hematology and Blood Transfusion, H. W. Reesink, Ed., (Basel: Karger), pp. 137-163 (1994)]. Four million individuals may be infected in the United States alone [Alter, M. J. and Mast, E. E.,"The Epidemiology of Viral Hepatitis in the United States, Gastroenterol. Clin.

North Am. 23, pp. 437-455 (1994)].

Upon first exposure to HCV only about 20% of infected individuals develop acute clinical hepatitis while others appear to resolve the infection spontaneously. In most instances, however, the virus establishes a chronic infection that persists for decades

[Iwarson, S."The Natural Course of Chronic Hepatitis" FEMS Microbiology Reviews 14, pp. 201-204 (1994)]. This usually results in recurrent and progressively worsening liver inflammation, which often leads to more severe disease states such as cirrhosis and hepatocellular carcinoma [Kew, M. C., "Hepatitis C and Hepatocellular Carcinoma", FEMS Microbiology Reviews, 14, pp. 211-220 (1994); Saito, I., et al."Hepatitis C Virus Infection is Associated with the Development of Hepatocellular Carcinoma"Proc. Natl. Acad. Sci. USA 87, pp. 6547-6549 (1990)]. Unfortunately, there are no broadly effective treatments for the debilitating progression of chronic HCV.

The HCV genome encodes a polyprotein of 3010- 3033 amino acids [Choo, Q.-L., et al."Genetic Organization and Diversity of the Hepatitis C Virus", Proc. Natl. Acad. Sci. USA, 88, pp. 2451-2455 (1991); Kato, N. et al., Molecular Cloning of the Human Hepatitis C Virus Genome From Japanese Patients with Non-A, Non-B Hepatitis", Proc. Natl. Acad. Sci. USA, 87, pp. 9524-9528 (1990); Takamizawa, A. et al.,"Structure and Organization of the Hepatitis C Virus Genome Isolated From Human Carriers", J. Virol., 65, pp. 1105-1113 (1991)]. The HCV nonstructural (NS) proteins are presumed to provide the essential catalytic machinery for viral replication. The NS proteins are derived by proteolytic cleavage of the polyprotein [Bartenschlager, R. et al.,"Nonstructural Protein 3 of the Hepatitis C Virus Encodes a Serine-Type Proteinase Required for Cleavage at the NS3/4 and NS4/5 Junctions", J. Virol., 67, pp. 3835-3844 (1993); Grakoui, A. et al.

"Characterization of the Hepatitis C Virus-Encoded Serine

Proteinase: Determination of Proteinase-Dependent Polyprotein Cleavage Sites", J. Virol., 67, pp. 2832-2843 (1993); Grakoui, A. et al., Expression and Identification of Hepatitis C Virus Polyprotein Cleavage Products",.

Virol., 67, pp. 1385-1395 (1993); Tomei, L. et al.,"NS3 is a serine protease required for processing of hepatitis C virus polyprotein", J. Virol., 67, pp. 4017-4026 (1993)].

Proteolytic processing of the HCV polyprotein by virally-encoded proteases generates several nonstructural (NS) proteins with enzymatic activities essential for the replicative cycle of the virus [P.

Neddermann et al., Biol. Chem., 378, pp. 469-476 (1997)]. NS2 encodes a presumed metalloprotease, NS5B is a RNA-dependent RNA polymerase, and NS3 is a bifunctional enzyme with a serine protease localized to the N-terminal 181 residues of the protein and a RNA helicase in the C- terminal 465 amino acids. The NS3 protease performs an intramolecular cleavage at the NS3/NS4A junction to form a tight noncovalent NS3-NS4A complex necessary for efficient processing of the remaining polyprotein [C.

Failla et al., J. Viroi., 69, pp. 1769-i777 (1995); R.

Bartenschlager et al., J. Virol., 69, pp. 7519-7528 (1995); Y. Tanji et al., J. Virol., 69, pp. 1575-1581 (1995)]. To date, no evidence exists to suggest that the serine protease and helicase domains are separated by proteolytic processing of NS3 in vivo. This may reflect economical packaging of these enzymatic components, or could imply a functional interdependence between the two domains.

Numerous studies have demonstrated that the serine protease [J. L. Kim et al., Cell, 87, pp. 343-355

(1996); W. Markland et al., J. Gen. Virol., 78, pp. 39- 43 (1997).; C. Steinkuhler et al., J. Virol., 70, pp.

6694-6700 (1996) and RNA helicase domains [J. A. Suzich et al., J. Virol., 67, pp. 6152-6158 (1993); C. L. Tai et al., J. Virol., 70, pp. 8477-8484 (1996); L. Jin et al., Arch. Biochem. Biophys., 323, pp. 47-53 (1995); and F.

Preugschat et al., J. Biol. Chem., 271, pp. 24449-24457 (1996)] of NS3 can be expressed independently and isolated as catalytically active species. However, emerging evidence suggests that the NS3 protease and helicase domains may contact one another and modulate NS3 catalytic activities. Examples include apparent differences in pH optima of ATPase and RNA unwinding activities between a contiguous NS3 protein complexed with the NS4A cofactor [K. A. Morgenstern et al., J.

Virol., 71, pp. 3767-3775 (1997); Z. Hong et al., J.

Virol., 70, pp. 4261-4268 (1996)] and an isolated NS3 helicase domain [C. L. Tai et al., J. Virol., (1996), supra; L. Jin et al., Arch. Biochem. Biophys., (1995), supra; F. Preugschat et al., J. Biol. Chem., (1996), supra; and Y. Gwack et al., Biochem. Biophys. Res.

Commun., 225, pp. 654-659 (1996). Similarly, the ATPase activities of both proteins differ in their sensitivity to polynucleotide stimulation. Contiguous NS3 appears to have a lower apparent dissociation constant for poly (U) than does the helicase domain [J. A. Suzich et al., J.

Virol., (1993), supra; F. Preugschat et al., J. Biol.

Chem., (1996), supra; K. A. Morgenstern et al., J.

Virol., (1997), supra; A. Kanai et al., FEBS Lett., 376, pp. 221-224 (1995)]. Aside from these differences, both proteins display nearly indistinguishable kinetic parameters for NTP hydrolysis when stimulated with

saturating poiynucleotide J. A. Suzich et al., J.

Virol., (1993), supra; K. A. Morgenstern et al., J.

Virol., (1997), supra], both dispiay 3'-5' directionality for translocation along a polynucleotide substrate, and the helicases of both proteins effectively unwind duplex RNA: RNA substrates [C. L. Tai et al., J.

Virol., (1996), supra; Z. Hong et al., J. Virol., (1996), supra.

In addition to HCV, all flavi-and pestiviruses sequenced to date contain conserved helicase sequence motifs in their homologous NS3 proteins, suggesting that this enzyme plays an important role in the HCV replicative cycle [R. H. Miller et al., Proc. Natl. Acad.

Sci. USA, 87, pp. 2057-2061 (1990)]. Consistent with this possibility, helicase encoding sequences have been identified in other viruses and helicases are suggested to catalyze the separation of double-stranded nucleic acid structures during transcription and genome replication [G. Kadare et al., J. Virol., 71, pp. 2583- 2590 (1997)]. Previous studies with poliovirus, a positive-stranded RNA virus of the Picornaviridae family, show that mutation of conserved sequence motifs in the 2C helicase inhibits virus replication and proliferation [C.

Mirzayan et al., Virology, 189, pp. 547-555 (1992)].

Similar mutational studies on the helicases encoded by herpes simplex virus type 1 and bovine papilloma virus also show that these enzymes are critical for virus replication [P. MacPherson et al., Virology, 204, pp.

403-408 (1994); R. Martinez et al., J. Virol., 66, pp.

6735-6746 (1992)]. Thus, the ability to inhibit helicase activity in HCV may provide an avenue for the therapeutic treatment of HCV infection.

SUMMARY OF THE INVENTION The present invention provides compounds which inhibit HCV NS3 helicase. These compounds are mono-, di- and tri-substituted pentacycles, wherein the pentacyclic ring may contain up to 3 heteroatoms and may be saturated, partially unsaturated or fully unsaturated.

The compounds of this invention may be formulated into pharmaceutically acceptable compositions useful for the treatment and the prevention of HCV infection. Such compositions and the methods of using them to treat or prevent HCV infection are also part of the present invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a compound by formula 1: 1, wherein: the ring containing Xl, X and X3 is saturated, partially unsaturated or fully unsaturated; each of L1 and L2 is independently selected from a single or double bond, (Cl-Clo)-straight or branched alkyl, (C2-C1o)-straight or branched alkenyl or (C2-CiD)- straight or branched alkynyl, wherein, in said alkyl, alkenyl or alkynyl chain, any-CH2-group is optionally replaced with-O-,-S-,-NH-,-C (0)-,-C (=S)-,-C (=NOH), -S (0)- or-S (0) 2- ; any =CH-or-CH= group is optionally replaced with =N-or-N=; and any hydrogen atom is optionally replaced by R R2, or R3 ;

each of W1 and w2 is independently selected from H, a monocyclic or bicyclic ring system, wherein in said ring system: i. each ring comprises 5 to 6 ring atoms independently selected from C, N, 0 or S; ii. no more than 4 ring atoms are selected from N, 0 or S; iii. any CH2 is optionally replaced with C (O); iv. any S is optionally replaced with S (0) or S(02); v. up to 3 hydrogen atom bound to said ring atoms are optionally and independently replaced with R', R2, or R3; and vi. said bicyclic ring system is optionally benzofused; each of X1, X2, and X3 are independently selected from C, CH, CH2, N, NH, 0, S, S (0) or S (02), wherein any hydrogen atom present in X2 or X3 is optionally and independently replaced with Rl, R2, or R3; Y is selected from-OH,-N (R4) 2,-SH,-S (02) R4, -S (0) R4,-N (R") C (=NR4) N (R4) 2,-C (=NR4) N (R4) 2,-C (0) OR4 -C (O) N (R4) 2,-C (0) R4,-C (H) RlORa,-C (Rl) 2-N (R4) 2, -C (R2-OR,-C(R')2-N(R)R\-N(R)R-OR; each R1 is independently selected from H, (C1- C6)-straight or branched alkyl, or (C2-C6)-straight or branched alkenyl, wherein up to 2 hydrogen atoms in said alkyl or alkenyl are optionally and independently replaced with R2 or R3; each R2 is a monocyclic or bicyclic ring system wherein in said ring system: i. each ring comprises 5 to 6 ring atoms independently selected from C, N, 0 or S;

ii. no more than 4 ring atoms are selected from N, 0 or S; iii. any CH2 is optionally replaced with C (O); iv. any S is optionally replaced with S (0) or S (0) 2; and v. up to 3 hydrogen atoms bound to said ring atoms are optionally and independently replaced with R3 or (CH2) nR3 ; wherein n is 1,2 or 3; each R³ is independently selected from-F,-Cl,-Br, -I,-CN,-N02,-CF3,-OCF3,-OR4,-OC (0) Rq,-OC (0) OR4, -C (0) R,-C (C) OR4,-C (0) N (R4) 2,-C (0) C (0) R4-C (O) C (O) OR -C (0) C (O) N (R4)2, -C(=NR4) N (R4)2, -SR4, -S(O)R4, -S(O)2R4, -N (R4)2, -N(R4)C(O)R4, -N (R4)C(O)OR4, -N(R4)C(O)N(R4)2, -N (R4) C (O) C (C) (R4)C(O)C(O)OR4, -N(R4)C(O)C(O)N(R4)2, -N(R4)C(=NR4)N(R4) 2,-N (R4) C(=NR4)N (R4) 2,-N (R4)(O)2R4, -N(R4)S(O) 2N (R') 2,-P (0) (OR) N (R4)2, -P(O)(OR4)2, -OR², -OC(O)R²,-OC (O)OR²,(O)OR², -C(O)R², -C(O)OR², -C (0) N (R4) (R),-C (0) C (0) R2,-C (O) C (O)OR2, -C (0) C (0) N (R4)(R²), -C(=NR4)N(R4)(R²), -SR², -S(O)R², S (O)2R², -N(R4) R²),-N (R4)C(O)R², -N(R4)C(O)OR², -N(R4)C(O)N(R4)(R²), -N (R4)C(O)C(O)R², -N(R4)C(O)C(O)OR2, -N (R4) C (O) C (C) N (R4)(R2), -N(R4)C(=NR4)N(R4)(R2), -N(R4)C(=NR4)N(R4)(R²), -N(R4)S(O)2R2, -N(R4)S(O)2N(R4)(R2), -P (0) (OR) N(R4)(R²), -P(O)(OR4)(OR²), or -P(O)(OR²)2; and each R4 is independently selected from H, (C1-C6)- straight or branched alkyl, or (C2-C6)-straight or branched alkenyl, wherein up to 3 hydrogen atoms in said alkyl or alkenyl are optionally and independently replaced with R2; or, when two R4 groups are bound to the same atom, said two R4 groups are taken together with the atom to which they are bound to form a monocyclic ring system wherein in said ring system:

i. each ring comprises 5 to 6 ring atoms independently selected from C, N, O or S; ii. no more than 4 ring atoms are selected from N, O or S; iii. any CH2 is optionally replaced with C (O) ; iv. any S is optionally replaced with S (0) or S (0) 2; and v. up to 3 hydrogen atoms bound to said ring atoms are optionally and independently replaced with C1-C6 straight or branched alkyl.

It should be understood that when L1 (or L2) is a single bond, the portion of the compound designated as W-L-X-has the structure W-X-gor W2-C-). When L1 (or L2) is a double bond, the portion of the compound designated as W1-Ll-Xl-has the structure W1=Xl- (or W2- C-).

It should also be understood that the terms "alkyl","alkenyl"and"alkynyl", when used to define L1 or L2 denote straight or branched chains which are bound to the rest of the molecules at two ends (i. e., one end is bound to Wu (or) and the other is bound to X1 (or C)). This means, with respect to the definitions of Ll and L2, the terms"alkyl","alkenyl"and"alkynyl"have a slightly different meaning than that which is known in the art. This minor difference, however, would be well understood by those in the art. For example, if L1 is a C3 straight alkyl chain, then the portion of the molecule containing L1 would be represented by W1-CH2-CH2-CH2-Xi.

In this example L1 is-CH2-CH2-CH2-, rather than the accepted definition of a C3 straight alkyl chain-- -CH2-CH2-CH3--which is chemically incompatible with the depicted structure of the molecule

It should also be noted that formula I is intended to encompass structures wherein L1 and/or L2 may be bound to the rest of the molecule at either end by a single or a double bond. Therefore, in the above example, when L1 is a C3 straight alkyl chain, then the portion of the molecule containing Li could also be represented by W1=CH-CH2-CH2-Xl-, W1=CH-CH2-CH=X1-, or W-CH2-CH2-CH=X-.

The above definitions include certain polymers and multimers which are not part of the present invention. Thus, whenever R3 replaces a hydrogen atom in Wl, W2, RI or R2, any R4 present in said hydrogen atom- replacing R3 group is not substituted with R2. This restriction eliminates those undesired polymers.

The invention includes all stereoisomers and racemic forms of the above compounds. The invention also includes pharmaceutically acceptable salts of the compounds. And the invention includes tautomers of the above compounds.

The term"monocyclic ring system", as used herein, includes saturated, partially unsaturated and fully unsaturated ring structures. The term"bicyclic ring system", as used herein, includes systems wherein each ring is independently saturated, partially unsaturated and fully unsaturated. Examples of monocyclic and bicyclic ring systems useful in W1, w2 and R2 include, but are not limited to, cyclopentane, cyclopentene, indane, indene, cyclohexane, cyclohexene, cyclohexadiene, benzene, tetrahydronaphthalene, decahydronaphthalene, naphthalene, pyridine, piperidine, pyridazine, pyrimidine, pyrazine, 1,2,3-triazine, 1,2,4- triazine, 1,3,5-triazine, 1,2,3,4-tetrazine, 1,2,4,5-

tetrazine, 1, 2,3,4-tetrahydroquinoline, quinoline, 1,2,3,4-tetrahydroisoquinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,5-naphthyridine, 1,6-naphthyridine, 1,7-naphthyridine, 1,8-naphthyridine, 2,6-naphthyridine, 2,7-naphthyridine, pteridine, acridine, phenazine, 1, 10-phenatroline, dibenzopyrans, 1- benzopyrans, phenothiazine, phenoxazine, thianthrene, dibenzo-p-dioxin, phenoxathiin, phenoxthionine, morpholine, Ihiomorpholine, tetrahydropyan, pyran, benzopyran,, 4-dioxane, 1,3-dioxane, dihyropyridine, dihydropyran, 1-pyrindine, quinuclidine, triazolopyridine, ß-carboline, indolizine, quinolizidine, tetrahydronaphtheridine, diazaphenanthrenes, thiopyran, tetrahydrothiopyran, benzodioxane, furan, benzofuran, tetrahydrofuran, pyrrole, indole, thiophene, benzothiopene, carbazole, pyrrolidine, pyrazole, isoxazole, isothiazole, imidazole, oxazole, thiazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4- oxadiazole, 1,3,4 oxadiazole, 1,2,5-oxadiazole, 1,2,3- thiadiazole, 1,2,4-thiadiazole, 1,3,4-thiadiazole, 1,2,5 thiadiazole, tetrazole, benzothiazole, benzoxazole, benzotriazoie, benzimidazole, benzopyrazole, benzisothiazole, benzisoxazole and purine.

Additional monocyclic and bicyclic structures falling within the above description may be found in A. R.

Katritzky, and C. W. Rees, eds."Comprehensive Heterocyclic Chemistry: Structure, Reactions, Synthesis and Use of Heterocyclic Compounds, Vol. 1-8,"Pergamon Press, NY (1984), the disclosure of which is herein incorporated by reference.

It should be understood that heterocycles referred may be attached to the rest of the compound by

any atom of the neterocycle which results in the creation of a stable structure.

The term"ring atom", as used herein, refers to a backbone atom that makes up the ring. Such ring atoms are selected from C, N, 0 or S and are bound to 2 or 3 other such ring atoms (3 in the case of certain ring atoms in a bicyclic ring system). The term"ring atom" does not include hydrogen.

According to one preferred embodiment, Xl is N, X2 is S, X3 is N and the pentacyclic ring which comprises X ;, X2 and X3 has the structure: According to another preferred embodiment, one of Ll or L2is a single bond and the other is a single bond or methylene.

According to another preferred embodiment, Y is -OH or N (R4) 2. Even more preferred is when Y is-NH (R4).

Most preferred is when Y is NH2.

According to yet another preferred embodiment, neither Wl ncr W-is hydrogen. Even more preferred is when at least one of W or w2 is phenyl, methylphenyl or chlorophenyl and the other is selected from phenyl, methylphenyl, chlorophenyl, ethylphenyl, isopropylphenyl, t-butylphenyl, methoxyphenyl, cyclohexylphenyl, bisphenyl, furyl, thiophenyl, benzothiophenyl, naphthyl, phenylmethylphenyl, 3-chloro-4-methylphenyl, 3-fluoro-4- methylphenyl, methoxycarboxyphenyl, fluorenyl, oxofluorenyl, oxobenzochromenyl, phenoxyphenyl, benzyloxyphenyl, indanyl, benzoylphenyl, 3,4- methylenedioxyphenyl, hydroxyphenyl, thiadiazolylphenyl.

Some of the more preferred inhibitors are depicted in Table 1, below. Cpd #I Structure iCpd # : Structure H3c ci- H3C, CI I/ SN SN N N? SNHz Br 1'29 i N N cri \ ber 3 i N'NH2 C H3. s NH 2 I 3 0 w N \//z ci \/ N s c-i N N ci ci sus w w ' / j CI- cri- w N\ N Hz I Nliz 4 N 32 I HsCO; Cpd #i Structure Cpd #i Structure ci cri CI i H3C +-S ci N 'N 33 I/ 2 1 5 33 NH2 N N Ils I i /1 CI +. S ci- cl NH ' N Z cri vs O O CL ; \ CL N ci 'N i N/NHz 1 N NH 2 35 +OS i 1 nu2 i i CH3 F CH3 F (ci +, S N nu2 36 ,--NHz, N ,/ Cpd #1 Structure Cpd #1 Structure ci ci- ci ,/N. _S N H 2 I//NHz 9 I N 37 N N 9 N 37 v i Cl \ cri ci cl-ci- NtrS I , I fV+, S IV I-12--NH2 IO f \ 3g 10 38 N H3Ci H3C/ CH3 cl-0, ci ,/NS N,.. S N HZ i,--. NH 2 N -, oxo 0,"' i ,' ; i I t I _ I 12 INH2 4p C N --N Hz //-NH N Cpd#I Structure, Cpd # Structure \, i CF, ci nus 13N) s C N nu 2 N N cri ci ; . S I I CI "nu2 w N\NHZ i N+. S N 14 N 42 MU N ci Ber- i/ Cri N ( i- ci H C Br i \ \ ci s 15 43 s N Nu ,, i' i H3C 's CI j ,. s , w N 16 N\ 2 I 44 Cpd! Structure Cpd Structure j S- H3 w 1S 46 XN i O nus 17 NI/>'-NH2 45/\ +. S"JH N N N H 0 3 F F H3C I CI O /. _S, e N P * N o D 4 18 46 /I H-ci 3C F i H C C H3 p F, \ J. L y'0 H3C I CI_ w \ 1 \f0 F JJL 19 NH2 47 N NH N N Q) cr- /1/ I /. F F N hic N HZ 20 48 I /Ni. SNHz I N H3C 1/ I Cpd #11 Structure Cpd #1 Structure F zoo w I i 0 F < CL +, S F 21 NH2 49 N--S lI-NH N N 1 w ci CH F I NH , CI F in 22 N-90 , N NI/NHz 1 N Cl N, S W s N i i NU ! CI 0 iw 23 i N 51/S I (NI/N Hz ;-O i \ N , \ I N N 24 52 s 1 w N CH3 24 i N 52 i i Cpd # Structure, Cpd #i Structure ci i N+. S NHZ ; _ N+. I NC Hs 25 ; 53 N - I I' Cl, I /ci w e "Q' !" ! 0 0 CX Cl S N < N Nl 26 54 i XNH2 S hic HIC \ cri , /+, S w N+ NINH2 \ N 27 N 55 I V OH 28 L-N 56 ! i N+, s , OH NH 28 N 56 i w I i

The compounds of this invention may be synthesized by methods well-known in the art. For example, compounds containing the preferred thiadiaolyl core and wherein Y is NHZ or NH (R") may be synthesized by the scheme depicted below.

Scheme 1 Other compounds of this invention may be prepared by analogous routes of synthesis. Examples of synthesis step used in preparing compounds of this invention may be found, for example, in K. Hagiwara et al., J. Pesticide Sci. 17, pp. 251-259 (1992).

The HCV NS3 helicase contains a binding site for ATP (which is cleaved by the ATPase activity of the helicase) and a separate binding site for double-stranded polynucleotides (which are unwound by the unwinding activity of the helicase). The energy generated by cleavage of ATP is required for the unwinding activity.

The compounds of this invention are designed to bind Hepatitis C helicase and therefore are expected to inhibit, either directly or indirectly, the unwinding activity of the helicase. Therefore, the compounds of this invention can be assayed for their ability to inhibit ATP cleavage or to inhibit unwinding activity.

Assays for each of the activities are known in the art [M. E. Pullman et al.,"Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation", J. Biol. Chem., 235, pp. 3322-28 (1980); C. H. Gross et al.,"Mutational Analysis of Vaccinia Virus Nucleoside Triphosphate Phosphohydrolase II, a DExH Box RNA Helicase", J. Virol., 69, pp. 4727-36 (1995) and are described in detail in the Example section.

According to another embodiment, the invention

provides a composition comprising a compound of this invention or a pharmaceutically acceptable salt thereof, as described above, and a pharmaceutically acceptable carrier.

If pharmaceutically acceptable salts of the compounds of this invention are utilized in these compositions, those salts are preferably derived from inorganic or organic acids and bases. Included among such acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl- propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate. Base salts include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as dicyclohexylamine salts, N-methyl-D- glucamine, and salts with amino acids such as arginine, lysine, and so forth.

Also, the basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides,

bromides and iodides, aralkyl halides, such as benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.

The compounds utilized in the compositions and methods of this invention may also be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e. g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.

Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

According to a preferred embodiment, the compositions of this invention are formulated for pharmaceutical administration to a mammal, preferably a human being.

Such pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral"as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously.

Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension.

These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as those described in Pharmacopeia Helvetica (Ph. Helv.) or similar alcohol.

The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch.

Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non- irritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation.

Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administraticn of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral cil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or

dispersing agents.

The amount of compound present in the above- described composition should be sufficient to cause a detectable decrease in ATPase activity and/or unwinding activity of the HCV NS3 helicase, as measured by any of the assays described in the examples.

According to another embodiment, the compositions of this invention may further comprise an anti-viral agent effective against hepatitis C virus infection. Such agents include, but are not limited to, inhibitors of HCV NS3 protease, such as those described in WO 98/17679; inhibitors of HCV polymerase; IMPDH inhibitors, such as mycophenolic acid, mycophenolate mofetil, and those described in any of United States Patents 5,380,879; 5,441,953; 5,493,030; 5,633,279; 5,444,072; 5,536,747; 5,538,969; 5,554,612; and 5,807,876 or in PCT publications WO 95/22535; WO 95/22538; WO 95/22536; WO 95/22537; WO 95/22534; and WO 97/40028 (the disclosures of each of the above-cited patents and publications are herein incorporated by reference); interferons, such as alpha-interferon; ribavirin; and ZSX (California Institut of Molecular Medicine, Inc.).

The amount of the anti-viral agent present in the compositions of this invention should be between 10- 100% of the amount of that agent normally used in a monotherapy for anti-viral activity. Such amounts are known in the art and/or described in the patent applications referred to above.

It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body

weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of active ingredients will also depend upon the particular compound and anti-viral agent, if present, in the composition.

According to another embodiment, the invention provides a method of detecting HCV helicase activity in a biological sample suspected of containing a polypeptide having HCV NS3 helicase activity comprising the step of contacting said biological sample with a compound of this invention. The term"biological sample", as used herein includes cell cultures or extracts thereof; biopsied material obtained from a mammal or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof. The term"biological sample" also includes living organisms, in which case"contacting a compound of this invention with a biological sample suspected of containing HCV NS3 helicase"is synonymous with the term"administrating said compound to the living organism." The term"polypeptide having HCV helicase activity", as used herein, means any polypeptide which demonstrates activity in at least, but not limited to, the unwinding assay described in Example 57. Preferably, that term means a polypeptide encoded by a naturally occurring or an experimentally produced strain of hepatitis C virus or a polypeptide having a consensus amino acid sequence derived from polypeptides having the aforementioned activities encoded by two or more strains of hepatitis C virus.

The term"detecting HCV helicase activity", as used herein includes inhibiting the helicase activity, if present, in said sample; determining the presence or absence of HCV helicase activity in said sample; quantitating the HCV helicase activity in said sample; and, in the case of a living organism, treating and/or reducing the severity of an HCV viral infection. Each of these is clearly useful in diagnostic and therapeutic applications relating to HCV.

In a preferred embodiment, the invention provides a method of treating an HCV viral infection in a mammal comprising the step of administering to said mammal a pharmaceutically acceptable composition described above. In this embodiment, if the patient is also administered an anti-viral agent, it may be delivered together with the compound of this invention in a single dosage form, or, as a separate dosage form.

When administered as a separate dosage form, the anti- viral agent may be administered prior to, at the same time as, or following administration of a pharmaceutically acceptable composition comprising a compound of this invention.

In order that this invention be more fully understood, the following examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.

Example 1 Synthesis of Compound 1 5-Amino-3-phenyl-2-p-tolyl- 1,2,4 thiadiazol-2-ium; chloride Step 1 To a solution of 1.51 g (14.2 mmol) of p- toluidine in 20 mL of CH2C12 was added dropwise 1.60 mL (2.0 g, 14.2 mmol) of benzoyl chloride. The white suspension was treated with 20 mL of saturated aqueous NaHCO3. The resulting biphasic mixture was stirred for 3 hours at room temperature. The layers were separated and the organic layer was washed with 20 mL of 0.5 N aqueous HC1 and 20 mL of saturated aqueous NaCl and dried over Na2SO4. The clear solution was concentrated to give 2.50 g (83.1 % yield) N-p-tolyl-benzamide as a white solid which was used without further purification in Step 2.

Step 2 A 25 mL round bottom flask equipped with a stirbar and reflux condenser was charged with 1.15 g (5.45 mmol) of N-p-tolyl-benzamide and treated with 2.0 mL (3.26 g, 27.4 mmol) of thionyl chloride. The reaction mixture was heated to reflux for 1 hr and the excess thionyl chloride was removed by concentration in vacuo to give N-p-tolyl-benzimidoyl chloride which was used without further purification in Step 3.

Step 3 The crude imino chloride was dissolved in 10 mL of acetonitrile and treated with 600 mg (5.88 mmol) of 1- amino-2-thio-3,4,5-triazoie at room temperature. The resulting suspension was allowed to stir for 12 hr and filtered. The crude product was recrystallized from methanol to yield 1.00 g (60.3 % yield) of the title compound as a white crystalline solid: 1H NMR (500 MHz, DMSO-d6) 8 10.4 (1 H, s, NH2), 10.2 (1 H, s, NH2), 7.7- 7.3 (9 H, m, Ar), 2.5 (3 H, s, Me) ppm; MS 268.1 (M+H).

Example 2 Synthesis of Compound 2 5-Methylaminc-3-phenyl-2-phenyl- 1,2,4 thiadiazol-2-ium; bromide Steps 1 and 2 were carried out as described above.

Step 3 The crude imino chloride (prepared from 2.0 of benzanilide by similar method described in Example 1, Step 3) was dissolved in 10 mL of acetone and treated with 2.0 g (24.7 mmol) of sodium thiocyanate. The resulting orange suspension was stirred at room temperature for 8 hrs and filtered. The filtrate, containing the crude isothiocyanate, was concentrated to an orange crystalline solid and dissolved in 10 mL of

acetonitrile. The reaction mixture was then treated with 3.0 mL (38.7 mmol) of 40% solution of aqueous methylamine. The resulting yellow solution was allowed to stir at rcom temperature for 8 hrs and then decanted to remove a small amount of black precipitate. The yellow solution was then concentrated to a solid, which was azeotroped from acetonitrile (2x 10 mL) and methanol (lx 10 mL) and then dissolved in 20 mL of carbon tetrachloride. To the stirring solution was then added 1 mL (3.10 g, 19.4 mmol) of bromine. The resulting red suspension was allowed to stir for 8 h and then concentrated to a thick red oil. The crude product was triturated with methanol followed by recrystallization from methanol, to yield 570 mg (1.64 mmol, 16.2 %) of a light yellow solid: 1H NMR (500 MHz, CDC13) 10.3 (1 H, s, NHCH3), 7.6-7.2 (10 H, m, Ar), 3.4 (3 H, d, j = 5.0 Hz, NHCH3) ppm; LC/MS: 268 (M+H).

Compounds 3 through 55 were prepared as described in Example 1 and 2.

Example 3 Compound 3 MS (M+l) = 254 1H NMR (500 MHz; DMSO-d6) 5 = 10.5 (1 H, s, NH2), 10.1 (1 H, s, NH2), 7.. 7-7.5 (10 H, m, Ar) ppm.

Example 4 Compound 4 MS (M+l) = 284 1H NMR (500 MHz; DMSO-d6) 5 = 10.3 (1 H, s, NH2), 10.0 (1 H, s, NH2), 7.7-7.6 (5 H, m, Ar), 7.5 (2 H, d, j = 8 Hz,

MeO-Ar), 7.0 (2 H, d, j = 8 Hz, MeO-Ar) 3.9 (3 H, s, MeO) ppm Example 5 Compound5 MS (M+l) = 288 1H NMR (500 MHz; DMSO-d6) 5 = 10.5 (1 H, s, NH2), 10.2 (1 H, s, NH2), 7.7-7.5 (9 H, m, Ar) ppm Example 6 Compound 6 MS (M+l) = 284 1H NMR (500 MHz; DMSO-d6) 5 = 10.4 (1 H, s, NH2), 10.1 (1 H, s, NH2), 7.7-7.1 (9 H, m, Ar), 3.9 (3 H, s, MeO) ppm Example 7 Compound 7 MS (M+l) = 268 1H NMR (500 MHz; DMSO-d6) 5 = 10.2 (1 H, s, NH2), 10. 1 (1 H, s, NH2), 7.8 (2 H, d, Ar), 7.7 (1 H, dd, Ar), 7.6 (2 H, dd, Ar), 7.4 (3 H, m, Ar), 7.3 (2 H, d, Ar), 5.3 (2 H, s, ArCH2) ppm Example 8 Compound 8 MS (M+l) = 284 1H NMR (500 MHz; DMSO-d6) 5 = 10.5 (1 H, s, NH2), 10.2 (1 H, s, NH2), 7.7-7.1 (9 H, m, Ar), 3.7 (3 H, s, MeO) ppm

Example 9 Compound 9 MS (M+l) = 284 1H NMR (500 MHz; DMSO-d6) 5 = 10.5 (1 H, s, NH2), 10.2 (1 H, s, NH2), 7.7-7.1 (9 H, m, Ar), 4.0 (3 H, s, MeO) ppm Example 10 Compound 10 MS (M+l) = 288 1H NMR (500 MHz; DMSO-d6) 6 = 10.5 (1 H, s, NH2), 10.1 (1 H, s, NH2), 7.9-7.5 (9 H, m, Ar) ppm Example 11 Compound 11 MS (M+l) = 268 1H NMR (500 MHz; DMSO-d6) 5 = 10.5 (1 H, s, NH2), 10.2 (1 H, s, NH2), (9 H, m, Ar), 2.6 (3 H, s, Me) ppm Example 12 Compound 12 MS (M+l) = 304 1H NMR (500 MHz; DMSO-d6) 5 = 10.6 (1 H, s, NH2), 10. 0 (1 H, s, NHz), 8.2 (1 H, d, Ar), 8.1 (1 H, dd, Ar), 7.9 (2 H, dd, Ar), 7.7 (2 H, dd, Ar), 7.6 (1 H, t, Ar), 7.45 (3 H, dd, Ar), 7.3 (2 H, t, Ar) ppm Example 13 Compound 13 MS (M+l) = 304 1H NMR (500 MHz; DMSO-d6) 5 = 10.5 (1 H, s, NH2), 10.0 (1 H, s, NH2), 8.3 (1 H, s, Ar), 8.1 (1 H, d, Ar), 8.0 (2 H,

dd, Ar), 7.7 (2 H, m, Ar), 7.55 (4 H, m, Ar), 7.4 (2 H, dd, Ar) ppm Example 14 Compound 14 MS (M+1) = 3C2 1H NMR (500 MHz; DMSO-d6) 5 = 10.6 (1 H, s, NH2), 10.2 (1 H, s, NH2),. 7-7.3 (8 H, m, Ar), 2.3 (3 H, s, Me) ppm Example 15 Compound 15 MS (M+l) = 302 1H NMR (500 MHz; DMSO-d6) 5 = 10.7 (1 H, s, NH2), 10.3 (1 H, s, NH2), 7.9-7.3 (8 H, m, Ar), 2.4 (3 H, s, Me) ppm Example 16 Compound 16 MS (M+l) = 268 1H NMR (500 MHz; DMSO-d6) 5 = 10.5 (1 H, s, NH2), 10.0 (1 H, s, NH2), 7.7-7.4 (9 H, m, Ar), 2.4 (3 H, s, Me) ppm Example 17 Compound 17 MS (M+l) = 282 1H NMR (500 MHz; DMSO-d6) 5 = 10.4 (1 H, s, NH2), 10. 1 (1 H, s, NH2), 7.55 (1 H, dd, Ar), 7.5 (2 H, d, Ar), 7.45 (2 H, d, Ar), 7.4 (2 H, dd, Ar), 7.35 (2 H, d, Ar), 2.7 (2 H, q, ArCH2CH3), 1.2 (3 H, t, CH2CH3) ppm

Example 18 Compound 18 MS (M+l) = 296 1H NMR (500 MHz; DMSO-d6) 5 = 10.4 (1 H, s, NH2), 10.1 (1 H, s, NH2), 7.55 (1 H, dd, Ar), 7.5 (2 H, d, Ar), 7.45 (2 H, d, Ar), 7.4 (2 H, dd, Ar), 7.35 (2 H, d, Ar), 3.0 (1 H, q, ArCH (C :-^.) 2), 1.2 (6 H, d, CH2 (CH3) 2) ppm Example 19 Compound 19 MS (M+1) = 310 1H NMR (500 MHz; DMSO-d6) 5 = 10.4 (1 H, s, NH2), 10. 1 (1 H, s, NH2), 7.55 (5 H, m, Ar), 7.45 (2 H, d, Ar), 7.4 (2 H, m, Ar), 1.3 (9 H, d, C (CH3) 3) ppm Example 20 Compound 20 MS (M+l) 268 1H NMR (500 MHz; DMSO-d6) 5 = 10.6 (1 H, s, NH2), 10.3 (1 H, s, NH2), 7.8-7.4 (9 H, m, Ar), 2.5 (3 H, s, Me) ppm Example 21 Compound 21 MS (M+l) = 318 1H NMR (500 MHz; DMSO-d6) 5 = 10.2 (1 H, s, NH2), 10. 1 (1 H, s, NH2), 8.0 (2 H, m, Ar), 7.9 (2 H, d, Ar), 7.75 (1 H, d, Ar), 7.7 (1 H, dd, Ar), 7.65 (2 H, d, Ar), 7.6 (1 H, d, Ar), 7.55 (3 H, m, Ar), 5.85 (2H, s, ArCH2N) ppm

Example 22 Compound 22 MS (M+l) = 260 1H NMR (500 MHz; DMSO-d6) 5 = 10.5 (1 H, s, NH2), 10.2 (1 H, s, NH2), 8. 1 (1 H, s, Ar), 7.9-7.7 (5 H, m, Ar), 6.9- 6.8 (2 H, m, Ar) ppm Example 23 Compound 23 MS (M+l) = 244 1H NMR (500 MHz; DMSO-d6) 5 = 10.5 (1 H, s, NH2), 10.0 (1 H, s, NH2), 8.2-8.1 (1 H, m, Ar), 8.0-7.8 (5 H, m, Ar), 7.3-7.2 (1 H, m, Ar), 7.2-7.1 (1 H, m, Ar) ppm Example 24 Compound 24 MS (M+l) = 310 1H NMR (500 MHz; DMSO-d6) 5 = 10.6 (1 H, s, NH2), 10. 0 (1 H, s, NH2), 8.2-7.6 (10 H, m, Ar) ppm Example 25 Compound25 MS (M+l) = 268 1H NMR (500 MHz; DMSO-d6) 5 = 10.6 (1 H, s, NH2), 10.2 (1 H, s, NH2), 7.8-7.4 (9 H, m, Ar) 2.4 (3 H, s, Me) ppm Example 26 Compound 26 MS (M+l) = 284 1H NMR (500 MHz; DMSO-d6) 5 = 10.6 (1 H, s, NH2), 10.2 (1 H, s, NH2), 7.7-7.1 (9 H, m, Ar) 3.8 (3 H, s, MeO) ppm

Example 27 Compound 27 MS (M+l) = 304 1H NMR (500 MHz; DMSO-d6) 5 = 10.5 (1 H, s, NH2), 10.2 (1 H, s, NH2), 8.1 (2 H, m, Ar), 8.0 (1 H, d, Ar), 7.7 (1 H, d, Ar), 7.6 (2 H, m, Ar), 7.5 (1 H, t, Ar), 7.45 (2 H, m, Ar), 7.3 (3 H, m, Ar) ppm Example 28 Compound 28 MS (M+l) = 304 1H NMR (500 MHz; DMSO-d6) 5 = 10.5 (1 H, s, NH2), 10.1 (1 H, s, NH2), 7.95 (3 H, m, Ar), 8.0 (2 H, dd, Ar), 7.7 (1 H, t, Ar), 7.6 (3 H, m, Ar), 7.5 (3 H, m, Ar), 7.4 (1 H, dd, Ar) ppm Example 29 Compound 29 MS (M+l) = 330 1H NMR (500 MHz; DMSO-d6) 5 = 10.7 (1 H, s, NH2), 10.3 (1 H, s, NH2), 8.1-7.5 (14 H, m, Ar) ppm Example 30 Compound 30 MS (M+l) = 342 1H NMR (500 MHz; DMSO-d6) 5 = 10.6 (1 H, s, NH2), 10.2 (1 H, s, NH2), 8.3-7.5 (12 H, m, Ar), 4.2 (2 H, s, CH2) ppm

Example 31 Compound 31 MS (M+1) = 336 This compound was produced by combinatorial chemistry techniques, and therefore, no NMR data is available. The same is true for all subsequent compounds for which NMR data is not reported.

Example 32 Compound 32 MS (M+l) = 344 Example 33 Compound 33 MS (M+l) = 302 Example 34 Compound 34 MS (M+l) = 330 Example 35 Compound 35 MS (M+l) = 312 Example 36 Compound 36 MS (M+l) = 286

Example 37 Compound 37 MS (M+1) = 346 Example 38 Compound 38 MS (M+l) = 312 Example 39 Compound 39 MS (M+l) = 298 Example 40 Compound 40 MS (M+l) = 318 1H NMR (500 MHz; DMSO-d6) 5 = 10.25 (s, 1 H), 10.0 (s, 1 H), 8.0-7.9 (m, 4 H), 7.8 (d, 2 H), 7.7 (dd, 1 H), 7.65 (dd, 2 H), 7.55 (dd, 2 H), 7.4 (d, 1 H), 5.5 (s, 2 H) ppm Example 41 Compound 41 MS (M+l) = 360 Example 42 Compound 42 MS (M+l) = 294 Example43 Compound 43 MS (M+l) = 344

1H NMR (500 MHz; DMSO-d6) 5 = 10.7 (1 H, s, NH), 7.6-7.2 (15 H, m, Ar), 5.0 (2H, d, J = 5 Hz, CH2Ph) ppm Example44 Compound 44 MS (M+l) = 358 1H NMR (500 MHz; DMSO-d6) 5 = 10.0 (1 H, s, NH), 7.6-7.2 (15 H, m, Ar), 4.0 (2H, m, CH2CH2Ph), 3.1 (2H, m, CHzCH2Ph) ppm Example 45 Compound 45 MS (M+l) = 330 Example 46 Compound 46 MS (M+l) = 330 Example 47 Compound 47 MS (M+l) = 358 Example 48 Compound 48 MS (M+1) = 372 Example 49 Compound 49 MS (M+1) = 356

Example 50 Compound 50 MS (M+l) = 270 Example51 Compound 51 MS (M+l) = 338 Example 52 Compound 52 MS (M+l) = 303 1H NMR (500 MHz; DMSO-d6) 5 = 10.2 (1 H, s, NH), 7.6-7.1 (9 H, m, Ar), 2.4 (3H, d, J = 5 Hz, CH3) ppm Example 53 Compound 53 MS (M+l) = 317 1H NMR (500 MHz; DMSO-d6) 5 = 10.3 (1 H, s, NH), 7.6-7.1 (9 H, m, Ar), 3.8 (2H, m, CH2CH3), 2.4 (3H, d, J = 5 Hz, CH2CH3) ppm Example 54 Compound 54 MS (M+l) = 379 1H NMR (500 MHz; DMSO-d6) 5 = 10.7 (1 H, s, NH), 7.6-7.1 (14 H, m, Ar), 5.0 (2H, d, J = 5 Hz, CH2Ph) ppm Example 55 Compound 55 MS (M+l) = 400

1H NMR (500 MHz; DMSO-d6) 5 = 10.7 (1 H, s, NH), 7.8-7.2 (15 H, m, Ar), 5.0 (2H, d, J = 5 Hz, CH2Ph) ppm Example 56 Synthesis of Compound 56 Compound 56 was synthesized by the scheme depicted below.

These procedures are described in detail in T.

L. Gilchrist et al., J. Chem. Soc.-Perkin Trans. I 1981, pp. 3221-3224 and in E. J. Corey et al., J. Am.

Chem. Soc., 85, pp. 1788-1792 (1963), the disclosures of which are herein incorporated by reference.

MS (M+l) = 237 1H NMR (500 MHz; DMSO-d6) 5 = 7.4-7.2 (m, 10 H), 4.7 (m, 1 H), 3.35 (dd, 2 H), 2.95 (d, 2 H) ppm

Example 57 Assays A. Unwinding assay The standard 3'-tailed double-stranded RNA/DNA hybrid was prepared as described as follows. The long 98-nucleotide ("nt") RNA template was transcribed from a BsrBI-digested plasmid pSP65 (Promega, Madison, WI) in the presence of a-32P-GTP (New England Nuclear, Boston, MA). The short 34-nt DNA release strand corresponds to a SP6 RNA transcript from a BamHI-digested pSP64 (Promega).

Standard helicase reactions (20 1) were carried out as follows. HCV NS3 helicase (0.3 or 1 nM) was added to a mixture of 25 mM morpholinepropanesulfonic acid (MOPS)-NaOH (pH 6.5), 1 mM ATP, 0.5 mM MnCl2,2 mM dithiothreitol (DTT), 0.1 mg of bovine serum albumin (BSA) per ml, 4 units of RNasin (Promega), and 5 nM of 3'-tailed double-stranded RNA/DNA hybrid substrate.

Mixtures were incubated for 20 min at 37°C and stopped by the addition of 5 liters of 5X loading buffer [100 mM Tris-Cl (pH7.5), 20 mM EDTA, 50 % glycol, 0.5 % SDS, 0.1 % NP-40, 9.1 % bromophenol blue, and 0.1 % xylene cyanole). The reactions were then electrophoresed on 10% PAGE with 0.5x TBE and 0.1 % SDS. Gels were dried and exposed using Fuji 1500 phosphorimager (Fuji, Stamford, CT). Helicase activity was determined by radioactivity of the double-stranded substrate and single-stranded template.

B. Spectrophotometric Assay of ATPase Activity ATPase activity of HCV NS3 helicase in the presence or absence of inhibitor was monitored by

following the rate of ADP production using a coupled enzyme assay. In this assay, an amount of NADH equal to ADP is oxidized to NAD+ resulting in a decrease in absorbance at 340 nm. A reaction mix consisting of buffer, pH 7.0, coupling enzyme components, poly (rU), and HCV NS3 helicase was prepared and aliquoted into wells.

Various concentrations of inhibitor in DMSO were added to the wells and incubated for 15 minutes at 30°C.

Reactions were initiated with ATP. The final concentrations of assay components in 200 pi reaction are as follows: 44 mM MOPS, pH 7.0,8.8 mM NaCl, 2.2 mM MgCl2, 17.6% v/v glycerol, 2.5 mM PEP, 0.2 mM NADH, 12.5 pg/mL pyruvate kinase, 12.5 ug/mL lactate dehydrogenase, 1 uM poly (rU), 2 nM HCV NS3 helicase, 2% DMSO with varying concentrations of inhibitor, and 100 uM ATP.

Absorbance at 650 nm was used as a check for insolubility of inhibitors. The rate data was fitted to equation (1) to calculate the IC50 for the inhibitors. Y, B, and X are the observed rate, inverse of maximum rate in the absence of inhibitor, and inhibitor concentration, respectively.

Y=IC5o/(B*(X+IC5o))(1) C. HPLC Assay of ATPase Activity ATPase activity of HCV helicase in the presence or absence of inhibitor was also measured by quantifying the ADP produced from ATP using an HPLC method. A reaction mix consisting of buffer, pH 7.0, poly (rU), and HCV NS3 helicase was prepared and aliquoted into wells.

Various concentrations of inhibitor in DMSO were added to the wells and incubated for 15 minutes at 30°C.

Reactions were initiated with ATP. The final

concentrations of assay components in 200 ul reaction are as follows: 44 mM MOPS, pH 7.0,8.8 mM NaCl, 2.2 mM MgCl2, 17.6% v/v glycol, 1 uM poly (rU), 2 nM trHCV NS3 Helicase, 2% DMSO with varying concentrations of inhibitor, and 100 uM ATP.

After 15 minutes the reaction was quenched with 50 ul of 0.5 M EDTA. A Gilson UniPoint System Software (Gilson, Inc., Middletown, WI) controlled the autosampling of 20 uLs from each reaction well on a 96- well plate using Gilson components. The mobile phase was an isocratic solution of 0.15 M triethylamine, 6% methanol, and phosphoric acid to pH 5.5. ADP and ATP peaks were separated using the Phenomenex Columbus (Torrence, CA) 5 u, C18,100 Å, 100 X 4.60 mm reverse phase HPLC.

The results of these assays for compounds 1- 53 are depicted in Table 2, below: TABLE 2. Assays of HCV NS3 helicase inhibitors. Cmpd# Coupled NS3 HPLC ATPase Helicase Unwinding ATPase IC50 (NM) IC50 (pM) (%inhibition@ 30 NM) 1 B F F 2 G 3 F 4 F H 5 E G 6 C G 7 C H H 8 D 9 C G 10 A E 11 B E 12 A E 13 A E E 14 D 15 D 16 A E 17 A E 18 A E Cmpd# Coupled NS3 HPLC ATPase Helicase Unwinding ATPase IC50 (NM) IC50 (pM) (% inhibition@ 30 pM) 19 8 E 20 A F 21 A E 22 A E 23 E 24 E 25 F 26 A E 27 E 28 E 29 E 30 A E 31 A E 32 A E 33 C E 34 B F 35 A E 36 E 37 E 38 E 39 F 40 F 41 E 42 H 43 E 44 E 45 E 46 E 47 E 48 E 49 E 50 H 51 E 52 G 53 F 54 E 55 56 H

In the above table, values designated"A"represent >75% inhibition;"B"represents between 50 and 75% inhibition;"C"represents between 25 and 50% inhibition; and"D"represents less than 25% inhibition.

Values designated"E"represent an IC50 of less than 25 uM;"F"represents an IC50 of between 25 and 50 µM; "G"

represents an IC50 of between 50 and 75 uM; and"H" represents an IC50 of greater than 75 uM. Blank values reflect that a particular assay was not performed for that inhibitor.

While we have hereinbefore presented a number of embodiments of this invention, it is apparent that our basic construction can be altered to provide other embodiments which utilize the methods of this invention.

Therefore, it will be appreciated that the scope of this invention is to be defined by the claims appended hereto rather than the specific embodiments which have been presented hereinbefore by way of example.