Treating hepatitis C viral infections with thiosemicarbazone compounds

Abstract

Thiosemicarbazone compounds of formula: 1

Description

FIELD OF THE INVENTION

[0002] The present invention is directed to the use of thiosemicarbazone compounds to treat infection by the hepatitis C virus, treat or delay the onset of hepatitis C, and inhibit replication of the hepatitis C virus.

BACKGROUND OF THE INVENTION

[0003] The hepatitis C virus (HCV) is the major causative agent of parenterally-transmitted and sporadic non-A, non-B hepatitis. It is believed that about 3 percent of the world's population and 2 percent of the U.S. population have been infected with this agent at some time. Exposure to HCV can result in an overt acute disease, but in most cases the virus establishes a chronic infection that causes liver inflammation and slowly progresses into liver failure and cirrhosis, as described for example in Iwarson, FEMS Microbiol. Rev. 1994, 14: 201-204. Epidemiological surveys have also indicated an important role of HCV in the pathogenesis of hepatocellular carcinoma, as described for example in Kew, FEMS Microbiol. Rev. 1994, 14: 211-220. No vaccine or established therapy currently exists, although partial success has been achieved in a minority of cases by treatment with recombinant interferon-alpha, either alone or in combination with ribavirin. There is accordingly a need for the development of alternative anti-HCV therapies.

[0004] The following references provide technical background for the present invention:

[0005] D. J. Bauer, Brit. Med. Bull. 1985, 41: 309-314 discloses that certain para-substituted benzaldehyde thiosemicarbazones (e.g., p-aminobenzaldehyde thiosemicarbazone and p-methoxybenzaldehyde thiosemicarbazone) are effective against the vaccinia virus (i.e., the cowpox virus), and also discloses that the 1-methyl isatin beta-thiosemicarbazone (M-IBT) and 1-ethyl isatin beta-thiosemicarbazone (E-IBT) are active against the vaccinia virus and the smallpox virus. D. J. Bauer, Ann. N.Y. Acad. Sci., 1965, 130: 110-117 contains a similar disclosure.

[0006] U.S. Pat. No. 4,927,843 (Teitz, 1990) discloses that certain isatin thiosemicarbazone derivatives are useful against viruses of the Retroviridae family. The patent specifically discloses 1-methylisatin-β-4′:4′-diethylthiosemicarbazone (M-IBDET), 1-allylisatin-β-4′:4′-dimethylthiosemicarbazone (A-IBDMT), and 1-allylisatin-β-4′:4′-diethylthiosemicarbazone (A-IBDET). Teitz et al., Antiviral Res. 1994, 24: 305-314, discloses inhibition of HIV by M-IBDET and by 1-allylisatin-β-4′:4′-diallylthiosemicarbazone (A-IBDAT).

[0007] Ronen et al., Antimicrob. Agents and Chemotherapy 1987, 31: 1798-1802 analyzes the mode of inhibition of Moloney leukemia virus production by M-IBDET. Teitz et al., Chemotherapy 1994, 40: 195-200, discloses that A-IBDAT is also an effective inhibitor of the Money leukemia virus.

[0008] Peloquin et al., Phamacotherapy 1996, 16: 735-741, presents a pharmacokinetic evaluation of thiacetazone, which is a known antimycobacterial agent useful for treating tuberculosis.

[0009] Finch et al., Biochemical Pharmacology 2000, 59: 983-991 discloses that 3-aminopyridine-2-carboxaldehyde thiosemicarbazone is a human ribonucleotide reductase inhibitor useful for treating cancer.

[0010] U.S. Pat. No. 5,098,462 (Anderson et al., 1992) and U.S. Pat. No. 5,098,466 (Anderson et al., 1992) each disclose certain (aryl- and aza-aryl-aldehyde and ketone)-4-(aryl- or aza-aryl-)thiosemicarbarzones which are said to be useful for the control of weeds.

[0011] U.S. Pat. No. 5,281,597 (McCall et al; 1994) discloses certain heterocyclic and aromatic thiosemicarbazones which are said to be useful for the treatment of filariasis.

[0012] U.S. Pat. No. 5,344,842 (Missbach, 1994) discloses certain (4-oxo-thiazolidin-2-ylidene)-thiosemicarbazones which are said to be useful for the treatment of diseases of the rheumatoid type. Similarly, U.S. Pat. No. 5,641,776 (Missbach, 1997) discloses certain (4-oxo-[1,3]thiazinan-2-ylidene)-thiosemicarbazones which are said to be useful for the treatment of diseases of the rheumatoid type.

[0013] U.S. Pat. No. 5,376,685 (Stanek et al., 1994) discloses certain amidine and amide substituted aryl and aza-aryl-hydrazones which are said to be useful as SAMDC inhibitors, wherein SAMDC is an enzyme that plays an important role in polyamine synthesis occurring in the cells of mammals.

[0014] U.S. Pat. No. 5,942,527 (Kadaba et al., 1999) discloses certain thiosemicarbazones derived from certain pyridyl ketones which are said to be useful for the treatment of stroke and other neurological disorders.

SUMMARY OF TEE INVENTION

[0015] The present invention is directed to the use of certain thiosemicarbazone compounds for the treatment of infection by the hepatitis C virus, the treatment of hepatitis C, the delay in the onset of hepatitis C, prevention of hepatitis C, and the inhibition of the hepatitis C virus. More particularly, the present invention is a method for treating infection by the hepatitis C virus, treating hepatitis C or a related condition, delaying the onset of hepatitis C or a related condition, preventing hepatitis C or a related condition, or inhibiting replication of the hepatitis C virus, which comprises administering to a subject in need thereof an effective amount of a compound of Formula (I): 3

[0016] or a pharmaceutically acceptable salt thereof;

[0017] wherein Q is selected from the group consisting of: 4

[0018] wherein

[0019] X is S or O;

[0020] R1 is —H or —C1-4 alkyl; and

[0021] R2 is:

[0022] (1) —C1-6 alkyl,

[0023] (2) —O—C1-6 alkyl,

[0024] (3) —C3-8 cycloalkyl,

[0025] (4) —O—C3-8 cycloalkyl,

[0026] (5) —Si(Ra)3, in which one Ra group is phenyl optionally substituted with from 1 to 3 substituents each of which is independently —C1-6 alkyl or —O—C1-6 alkyl; and the other Ra groups are independently methyl, ethyl, methoxy, ethoxy, or phenyl optionally substituted with from 1 to 3 substituents each of which is independently —C1-6 alkyl or —O—C1-6 alkyl,

[0027] (6) —Cl or —Br,

[0028] (7) phenyl, optionally substituted with from 1 to 3 substituents each of which is independently —C1-6 alkyl, —O—C1-6 alkyl, or —C3-8 cycloalkyl, or

[0029] (8) —(C1-6 alkyl)-phenyl, in which the phenyl is optionally substituted with from 1 to 3 substituents each of which is independently —C1-6 alkyl, —O—C1-6 alkyl, or —C3-8 cycloalkyl; 5

[0030] wherein

[0031] each R3 is independently a —C1-6 alkyl group;

[0032] m is an integer from 2 to 9; and

[0033] t is zero, 1, 2, 3, or 4;

[0034] (C): 6

[0035] wherein

[0036] A is absent or —C(RbRc)—;

[0037] B is —C(RdRe)— or —NRf—;

[0038] each R4 is independently:

[0039] (1) —C1-6 alkyl,

[0040] (2) —O—C1-6 alkyl,

[0041] (3) —C3-8 cycloalkyl,

[0042] (4) —O—C3-8 cycloalkyl,

[0043] (5) —Cl or —Br,

[0044] (6) phenyl, optionally substituted with from 1 to 3 substituents each of which is independently —C1-6 alkyl, —O—C1-6 alkyl, or —C3-8 cycloalkyl, or

[0045] (7) —(C1-6 alkyl)-phenyl, in which the phenyl is optionally substituted with from 1 to 3 substituents each of which is independently —C1-6 alkyl, —O—C1-6 alkyl, or —C3-8 cycloalkyl;

[0046] R5 and R6 are each independently —H or —C1-4 alkyl; or R5 and R6 taken together form oxo;

[0047] u is an integer equal to zero, 1, or 2;

[0048] Rb and Rc are each independently —H or —C1-4 alkyl;

[0049] Rd and Re are each independently —H or —C1-4 alkyl; and

[0050] Rf is —H or —C1-4 alkyl; 7

[0051] wherein each R7 and each R8 is independently:

[0052] (1) —C1-6 alkyl,

[0053] (2) —O—C1-6 alkyl,

[0054] (3) —C3-8 cycloalkyl,

[0055] (4) —O—C3-8 cycloalkyl,

[0056] (5) —Cl or —Br,

[0057] (6) phenyl, optionally substituted with from 1 to 3 substituents each of which is independently —C1-6 alkyl, —O—C1-6 alkyl, or —C3-8 cycloalkyl, or

[0058] (7) —(C1-6 alkyl)-phenyl, in which the phenyl is optionally substituted with from 1 to 3 substituents each of which is independently —C1-6 alkyl, —O—C1-6 alkyl, or —C3-8 cycloalkyl; and

[0059] v and w are each integers independently equal to zero, 1, 2 or 3; 8

[0060] wherein R9 is:

[0061] (1) —C1-6 alkyl,

[0062] (2) —C3-8 cycloalkyl,

[0063] (3) phenyl, optionally substituted with from 1 to 3 substituents each of which is independently —C1-6 alkyl, —O—C1-6 alkyl, or —C3-8 cycloalkyl, or

[0064] (4) —(C1-6 alkyl)-phenyl, in which the phenyl is optionally substituted with from 1 to 3 substituents each of which is independently —C1-6 alkyl, —O—C1-6 alkyl, or —C3-8 cycloalkyl;

[0065] R10 is —H or —C1-4 alkyl; and

[0066] R11 is —H or —C1-4 alkyl; and 9

[0067] wherein each R12 and each R13 is independently:

[0068] (1) —C1-6 alkyl,

[0069] (2) —O—C1-6 alkyl,

[0070] (3) —C3-8 cycloalkyl,

[0071] (4) —O—C3-8 cycloalkyl,

[0072] (5) —Cl or —Br,

[0073] (6) phenyl, optionally substituted with from 1 to 3 substituents each of which is independently —C1-6 alkyl, —O—C1-6 alkyl, or —C3-8 cycloalkyl, or

[0074] (7) —(C1-6 alkyl)-phenyl, in which the phenyl is optionally substituted with from 1 to 3 substituents each of which is independently —C1-6 alkyl, —O—C1-6 alkyl, or —C3-8 cycloalkyl; and

[0075] x and y are each integers independently equal to zero, 1, 2 or 3.

[0076] Embodiments, aspects and features of the present invention are either further described in or will be apparent from the ensuing description, examples, and appended claims.

DETAILED DESCRIPTION OF THE INVENTION

[0077] A first embodiment of the present invention is a method for treating infection by the hepatitis C virus, treating hepatitis C or a related condition, delaying the onset of hepatitis C or a related condition, preventing hepatitis C or a related condition, or inhibiting replication of the hepatitis C virus, which comprises administering to a subject in need thereof an effective amount of a compound of Formula (II): 10

[0078] or a pharmaceutically acceptable salt thereof;

[0079] wherein X is S or O;

[0080] R1 is —H, methyl, or ethyl; and

[0081] R2 is:

[0082] (1) methyl,

[0083] (2) ethyl,

[0084] (3) methoxy,

[0085] (4) ethoxy,

[0086] (5) —C5-6 cycloalkyl,

[0087] (6) —Si(Ra)3, in which one Ra group is phenyl and the other Ra groups are independently methyl, ethyl, or phenyl,

[0088] (7) —Cl,

[0089] (8) —Br,

[0090] (9) phenyl, or

[0091] (10) —CH2-phenyl.

[0092] An aspect of the first embodiment is a method as set forth above wherein in the compound of Formula (II) or a pharmaceutically acceptable salt thereof:

[0093] X is S or O;

[0094] R1 is —H, methyl, or ethyl; and

[0095] R2 is:

[0096] (1) methyl,

[0097] (2) ethyl,

[0098] (3) methoxy,

[0099] (4) ethoxy, 11

[0100] (6) —Cl, or

[0101] (7) —Br.

[0102] Exemplary of the compounds employed in the method of the present invention as set forth in the first embodiment is a compound selected from the group consisting of: 1213

[0103] and pharmaceutically acceptable salts thereof.

[0104] A second embodiment of the present invention is a method for treating infection by the hepatitis C virus, treating hepatitis C or a related condition, delaying the onset of hepatitis C or a related condition, preventing hepatitis C or a related condition, or inhibiting replication of the hepatitis C virus, which comprises administering to a subject in need thereof an effective amount of a compound of Formula (III): 14

[0105] wherein m is an integer from 5 to 9;

[0106] or a pharmaceutically acceptable salt thereof.

[0107] Exemplary of the compounds employed in the method of the present invention as set forth in the second embodiment is 15

[0108] or a pharmaceutically acceptable salt thereof.

[0109] A third embodiment of the present invention is a method for treating infection by the hepatitis C virus, treating hepatitis C or a related condition, delaying the onset of hepatitis C or a related condition, preventing hepatitis C or a related condition, or inhibiting replication of the hepatitis C virus, which comprises administering to a subject in need thereof an effective amount of a compound of Formula (IV): 16

[0110] or a pharmaceutically acceptable salt thereof;

[0111] wherein:

[0112] A is absent or —CH2—;

[0113] B is —CH2— or —NRf—, with the proviso that when B is —NRf—, A is absent;

[0114] each R4 is independently:

[0115] (1) —C1-4 alkyl,

[0116] (2) —O—C1-4 alkyl, or

[0117] (3) —Cl or —Br,

[0118] R5 and R6 are both —H; or R5 and R6 taken together form oxo;

[0119] u is zero, 1, or 2; and

[0120] Rf is —H or methyl.

[0121] Exemplary of the compounds employed in the method of the present invention as set forth in the third embodiment is a compound selected from the group consisting of: 17

[0122] and pharmaceutically acceptable salts thereof.

[0123] A fourth embodiment of the present invention is a method for treating infection by the hepatitis C virus, treating hepatitis C or a related condition, delaying the onset of hepatitis C or a related condition, preventing hepatitis C or a related condition, or inhibiting replication of the hepatitis C virus, which comprises administering to a subject in need thereof an effective amount of a compound of Formula (V): 18

[0124] or a pharmaceutically acceptable salt thereof;

[0125] wherein each R7 and each R8 is independently:

[0126] (1) —C1-4 alkyl,

[0127] (2) —O—C1-4 alkyl, or

[0128] (3) —Cl or —Br; and

[0129] v and w are each integers independently equal to zero, 1, or 2.

[0130] Exemplary of the compounds employed in the method of the present invention as set forth in the fourth embodiment is 19

[0131] or a pharmaceutically acceptable salt thereof.

[0132] A fifth embodiment of the present invention is a method for treating infection by the hepatitis C virus, treating hepatitis C or a related condition, delaying the onset of hepatitis C or a related condition, preventing hepatitis C or a related condition, or inhibiting replication of the hepatitis C virus, which comprises administering to a subject in need thereof an effective amount of a compound of Formula (VI): 20

[0133] or a pharmaceutically acceptable salt thereof;

[0134] wherein R9 is —C6-8 cycloalkyl;

[0135] R10 is —H or methyl; and

[0136] R11 is —H or methyl.

[0137] Exemplary of the compounds employed in the method of the present invention as set forth in the fifth embodiment is 21

[0138] or a pharmaceutically acceptable salt thereof.

[0139] A sixth embodiment of the present invention is a method for treating infection by the hepatitis C virus, treating hepatitis C or a related condition, delaying the onset of hepatitis C or a related condition, preventing hepatitis C or a related condition, or inhibiting replication of the hepatitis C virus, which comprises administering to a subject in need thereof an effective amount of a compound of Formula (VII): 22

[0140] or a pharmaceutically acceptable salt thereof;

[0141] wherein R12 and R13 are each independently:

[0142] (1) —H,

[0143] (2) —C1-6 alkyl,

[0144] (3) —O—C1-6 alkyl,

[0145] (4) —C5-6 cycloalkyl,

[0146] (5) —O—C5-6 cycloalkyl,

[0147] (6) —Cl or —Br,

[0148] (7) phenyl, optionally substituted with from 1 to 3 substituents each of which is independently —C1-6 alkyl or —O—C1-6 alkyl, or

[0149] (8) —(C1-6 alkyl)-phenyl, in which the phenyl is optionally substituted with from 1 to 3 substituents each of which is independently —C1-6 alkyl or —O—C1-6 alkyl.

[0150] Exemplary of the compounds employed in the method of the present invention as set forth in the sixth embodiment is 23

[0151] or a pharmaceutically acceptable salt thereof.

[0152] The present invention also includes a compound of Formula (I) as defined and described above for use in (a) treating infection by the hepatitis C virus, (b) treating hepatitis C or a related condition, (c) delaying the onset of hepatitis C or a related condition, (d) preventing hepatitis C or a related condition, or (e) inhibiting replication of the hepatitis C virus. The present invention further includes use of a compound of Formula (I) as defined and described above as a medicament for (a) treating infection by the hepatitis C virus, (b) treating hepatitis C or a related condition, (c) delaying the onset of hepatitis C or a related condition, (d) preventing hepatitis C or a related condition, or (e) inhibiting replication of the hepatitis C virus. The present invention also includes use of a compound of Formula (I) as defined and described above in the preparation of a medicament for (a) treating infection by the hepatitis C virus, (b) treating hepatitis C or a related condition, (c) delaying the onset of hepatitis C or a related condition, (d) preventing hepatitis C or a related condition, or (e) inhibiting replication of the hepatitis C virus.

[0153] As used herein, the term “C1-6 alkyl” refers to a linear or branched chain alkyl group having from 1 to 6 carbon atoms, and is selected from the hexyl alkyl and pentyl alkyl isomers, n-, iso-, sec- and t-butyl, n- and isopropyl, ethyl and methyl. “C1-4 alkyl” has an analogous definition.

[0154] The term “C3-8 cycloalkyl” refers to a cyclic ring selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl. “C5-6 cycloalkyl” has an analogous definition.

[0155] The term “substituted” includes mono- and poly-substitution by a named substituent to the extent such single and multiple substitution is chemically allowed and results in a chemically stable compound.

[0156] The symbol “” in front of an open bond in the structural formula of a group marks the point of attachment of the group to the rest of the molecule.

[0157] When A is “absent” in Formula (IV), it is understood that B and the ring carbon adjacent to A are directly connected by a single bond. In other words, when A is absent, the compound of Formula (IV) is represented as follows: 24

[0158] The term “therapeutically effective amount” (or alternatively and more simply “effective amount”) as used herein means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease being treated and/or the prevention or delay in onset or recurrence of a pathology.

[0159] The expression “pharmaceutically acceptable” means that the salt, carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0160] The term “subject” (or alternatively “patient”) as used herein refers to a human or other animal, typically a mammal, who is susceptible to HCV infection and who is the object of treatment, observation or experiment.

[0161] The term “administration” and variants thereof (e.g., “administering” a compound) in reference to the present invention mean providing a compound of Formula (I) or a pharmaceutical composition comprising Compound I to the subject or individual in need of treatment. When Compound I is provided in combination with one or more other active agents useful for treating HCV infection or hepatitis C, “administration” and its variants are each understood to include concurrent and time-separated (e.g., alternating) provision of Compound I and other agents.

[0162] A “related condition” is a condition which is or can be caused, directly or indirectly, by the hepatitis C virus or with which HCV is associated.

[0163] Compounds of Formula (I) may be administered in the form of pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to all acceptable salts of the compounds of Formula (I) (in the form of water- or oil-soluble or dispersible products) and includes the conventional non-toxic salts formed from inorganic and organic acids or the quaternary ammonium salts formed by reaction with, e.g., alkyl halides. The salt (e.g., hydrochloride salt) can be used as a dosage form for modifying the solubility or hydrolysis characteristics of the compound or can be used in sustained release or pro-drug formulations.

[0164] In the methods and uses of the present invention, the compound of Formula (I) may be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and vehicles. Accordingly, the present invention includes the methods of treating infection by the hepatitis C virus, treating hepatitis C or a related condition, delaying the onset of hepatitis C or a related condition, preventing hepatitis C or a related condition, or inhibiting replication of the hepatitis C virus, as heretofore described in which Compound I is administered as a pharmaceutical composition comprising Compound I and a pharmaceutically acceptable carrier, adjuvant or vehicle.

[0165] These pharmaceutical compositions may be in the form of orally-administrable suspensions or tablets, nasal sprays, sterile injectible preparations, for example, as sterile injectible aqueous or oleagenous suspensions or suppositories.

[0166] When administered orally as a suspension, these compositions are prepared according to techniques known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents known in the art. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.

[0167] When administered by nasal aerosol or inhalation, these 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 solubilizing or dispersing agents known in the art.

[0168] The injectible solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

[0169] When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters of polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.

[0170] The compounds of this invention can be administered orally to humans in a dosage range of 0.01 to 1000 mg/kg body weight per day in a single dose or in divided doses. One preferred dosage range is 0.1 to 200 mg/kg body weight per day orally in a single dose or in divided doses. Another preferred dosage range is 0.5 to 100 mg/kg body weight per day orally in single or divided doses. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

[0171] The compounds of Formula (I) employed in the present invention in which Q is the group defined in A, B, C, D and E can be prepared by reacting the corresponding carbonyl compound with thiosemicarbazide in the presence of a suitable solvent (e.g., an alcohol such as methanol or ethanol) and with or without an acid catalyst (e.g., aqueous HCl or aqueous acetic acid) at room temperature or with moderate heating to give the thiosemicarbazone. Illustrative is the preparation of heteroaryl thiosemicarbazones of Formula (II) by reaction of the corresponding ketone or aldehyde VIII with thiosemicarbazide IX: 25

[0172] Thiosemicarbazide IX is widely available commercially (see, e.g., suppliers listed in Chem Sources—USA, 2000 edition, Chemical Sources International, Inc., Clemson, S.C.) or can be prepared from hydrazine sulfate and ammonium thiocyanate in the presence of sufficient potassium carbonate to half neutralize the hydrazine sulfate. Many of the starting ketones and aldehydes for preparing thiosemicarbazones I are also commercially available, and the others can be prepared by the person of ordinary skill in the art without undue experimentation by means known in the art. For example, compounds 1 and 2 can be prepared from the commercially available 5-bromothiophene-2-carboxaldehyde or 5-bromo-2-furaldehyde by suitable protection of the aldehyde functionality and subsequent metallation, silylation and deprotection using procedures known in the art. Compounds 3-9 can be prepared from commercially available ketones. Compound 10 can be prepared from the ketone prepared by aminating 4-methylpyrimidine with the alkali metal amide of cyclooctylamine, followed by Friedel-Crafts acylation with an acetyl halide. Alternatively, the ketone required for the preparation of Compound 10 can be obtained through modification of procedures described in Indian J. Chem. Sect. B 1977, 15B: 1129-1132.

[0173] Compounds of Formula (I) in which Q is the group defined by F can be prepared by methods known in the art. Compound 11, for example, can be prepared as described in Ismail, Pak. J. Sci. Ind. Res. 1995, 38: 67-70 or El-Deen et al., Arch. Pharmacal Res. 1994, 17: 294-297.

[0174] Abbreviations used in the instant specification, particularly in the Examples, include:

[0175] DMEM=Dulbecco's modified minimum essential medium (also referred known as Dulbecco's modified eagle medium)

[0176] DMSO=dimethylsulfoxide

[0177] FCS=fetal calf serum

[0178] MS=mass spectrometry

[0179] NMR=nuclear magnetic resonance

[0180] PBS=phosphate buffered saline

[0181] SDS=sodium dodecyl sulfate

[0182] SRB=sulphorhodamine B

[0183] TCA=trichloroacetic acid

[0184] TMB=3,3′,5,5′-tetramethylbenzidine

[0185] The following examples serve only to illustrate the invention and its practice. The examples are not to be construed as limitations on the scope or spirit of the invention.

EXAMPLE 1

RHEPLISA Assay—A Cell-Based Assay for Measuring HCV Replication

[0186] A cell-based assay for measuring the HCV replication (RHEPLISA) was developed in a 96-well microplate format. The assay utilizes Huh-7 cellular clone (Huh7_HBI10A) containing a HCV-replicon and is based on the detection of the viral NS3 protein by ELISA. About 5000 Huh7_HBI10A cells (or Huh-7 cells as negative control), resuspended in 100 μL of complete DMEM were seeded into each well of a 96 well microtiter plate (Falcon sterile by Becton Dickinson). After 4 hours, 50 μL of an appropriate dissolution medium (e.g., DMEM) containing the compounds to test or DMSO (3%) were added, and incubated (37° C., 5% CO2) for 4 days. The medium was removed by inversion and the cells were fixed by addition of 200 μL/well of ice-cold isopropanol 100%. After an incubation of 20 minutes at 4° C. the plate was washed with Washing Buffer 1 (=PBS 1×) and 300 μL of Buffer A (=PBS 1×, Triton ×100 0.1%, SDS 0.02%, 5% non-fat dry milk) was added in each well. The plates were incubated for 30 minutes at room temperature, and the blocking solution was removed by inversion. 100 μL of 1:2000 primary antibody (primary antibody=anti-NS3 mouse polyclonal 10E5/24) in Buffer A were added, and the plate was incubated for 120 minutes at room temperature. The plate was washed with solution Washing Buffer 2 (=PBS 1×, Triton ×100 0.1%, SDS 0.02%), followed by addition of 100 μL of 1:2000 secondary antibody AP-conjugated (anti-mouse IgG (Fc SPECIFIC) adsorbed with human IgG and rat serum proteins (SIGMA) or 1:4000 secondary antibody peroxidase-conjugated (anti-mouse IgG (Fc SPECIFIC)) adsorbed with bovine, horse and human serum proteins (SIGMA) in Buffer A. The plate was incubated for 120 minutes at room temperature and washed with Washing Buffer 2.

[0187] When secondary antibody AP-conjugated was used, 100 μL of AP substrate (SIGMA 104 Phosphatase substrate tablets; i.e., one tablet in 5 ml of AP buffer: diethanolamine solution 10%, pH 9.6-9.8.) were added to each well, and the plate was read in a conventional ELISA plate reader set at 405-620 nm.

[0188] When peroxidase-conjugated secondary was used, 100 μL of TMB substrate (SIGMA) were added to each well, and the plate was incubated for 20′ in the dark. 50 μL of 0.5M H2SO4 were then added, and the plate was read in a conventional ELISA plate reader set at 450-620 nm.

[0189] The specific compounds disclosed above (i.e., compounds 1 to 11) have all exhibited IC50 values of less than about 50 μM in the RHEPLISA assay.

EXAMPLE 2

Cytoxicity Assay (SRB)

[0190] About 5000 Huh7—HBI10A cells, resuspended in 100 μL of complete DMEM were seeded into each well of a 96 well microtiter plate (Falcon sterile by Becton Dickinson) and incubated (37° C., 5% CO2) for 24 hours. The medium was removed and the cells were fixed by incubation with 50 μL of 50% TCA for 1 hour at 4° C., 200 μL of ice-cold isopropanol 100% for 20 minutes at 4° C., or 100 μL of 10% paraformaldehyde for 20 minutes at room temperature. The plate was washed with water when either TCA or paraformaldehyde was used to fix the cells and with PBS1× when isopropanol was used to fix the cells. All washings were run 3 times. After drying the plate, 200 μL of SRB 1× solution (prepared fresh from a SRB solution 10×:4% sulforhodamine B (Sigma) in 10% acetic acid) was added to each well and incubated for 30 minutes at room temperature. The SRB 1× solution was removed by inversion and the plate was washed in 1% acetic acid 3 times. To each well was added 200 μL of 10 mM Tris pH 10.5, the plate was shaken till the color of the solution in the wells was uniform, and the plate was read in a conventional ELISA plate reader set at 570 nm.

[0191] The specific compounds disclosed above have all exhibited cytotoxicity in the foregoing assay of less than about 20 percent using a 2 μM dose of the compound. Each of the compounds exhibited anti-HCV activity in the RHEPLISA assay at concentrations well below a concentration that would be associated with significant cytotoxicity in the instant assay.

[0192] The Huh-7 cell line is well known and is described in, for example, H. Nakabayashi et al., Cancer Research 1982, 42: 3858-3863 and in EP 1,043,399.

[0193] HCV replication systems can be obtained using techniques such as those described in Lohmann et al., Science 1999, 285: 110-113 (hereinafter referred to as “Lohman et al. 1999”). The development of this system was based on an experimental strategy that allowed selection of cells capable of supporting HCV replication. Selection can be achieved by using bicistronic RNA replicons expressing a selectable marker, the neomycin phosphotransferase. Transfection of these replicons in the human hepatoma cell line Huh-7, followed by cultivation in the presence of neomycin sulfate (G418), permits the isolation of clones that support HCV replication.

[0194] Plasmids pHCVNeol17.wt was assembled by several subcloning steps and contains the cDNA coding for an HCV bicistronic replicon identical to replicon I377neo/NS3-3′/wt described by Lohmann et al. 1999, under the control of a T7 promoter.

[0195] The nucleic acid sequence for the pHCVNeol7.wt coding strand (SEQ. ID. No.: 1) is as follows:

gccagccccc gattgggggc gacactccac catagatcac tcccctgtga ggaactactg 60
tcttcacgca gaaagcgtct agccatggcg ttagtatgag tgtcgtgcag cctccaggac 120
cccccctccc gggagagcca tagtggtctg cggaaccggt gagtacaccg gaattgccag 180
gacgaccggg tcctttcttg gatcaacccg ctcaatgcct ggagatttgg gcgtgccccc 240
gcgagactgc tagccgagta gtgttgggtc gcgaaaggcc ttgtggtact gcctgatagg 300
gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac catgagcacg aatcctaaac 360
ctcaaagaaa aaccaaaggg cgcgccatga ttgaacaaga tggattgcac gcaggttctc 420
cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct 480
ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 540
acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca 600
cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc 660
tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga 720
aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc 780
cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc 840
ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg 900
ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct 960
gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc 1020
tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc 1080
ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc 1140
agcgcatcgc cttctatcgc cttcttgacg agttcttctg agtttaaaca gaccacaacg 1200
gtttccctct agcgggatca attccgcccc tctccctccc ccccccctaa cgttactggc 1260
cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg 1320
ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct 1380
aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca 1440
gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg 1500
aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct 1560
gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa 1620
tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt 1680
atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa 1740
aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgataatacc 1800
atggcgccta ttacggccta ctcccaacag acgcgaggcc tacttggctg catcatcact 1860
agcctcacag gccgggacag gaaccaggtc gagggggagg tccaagtggt ctccaccgca 1920
acacaatctt tcctggcgac ctgcgtcaat ggcgtgtgtt ggactgtcta tcatggtgcc 1980
ggctcaaaga cccttgccgg cccaaagggc ccaatcaccc aaatgtacac caatgtggac 2040
caggacctcg tcggctggca agcgcccccc ggggcgcgtt ccttgacacc atgcacctgc 2100
ggcagctcgg acctttactt ggtcacgagg catgccgatg tcattccggt gcgccggcgg 2160
ggcgacagca gggggagcct actctccccc aggcccgtct cctacttgaa gggctcttcg 2220
ggcggtccac tgctctgccc ctcggggcac gctgtgggca tctttcgggc tgccgtgtgc 2280
acccgagggg ttgcgaaggc ggtggacttt gtacccgtcg agtctatgga aaccactatg 2340
cggtccccgg tottcacgga caactcgtcc cctccggccg taccgcagac attccaggtg 2400
gcccatctac acgcccctac tggtagcggc aagagcacta aggtgccggc tgcgtatgca 2460
gcccaagggt ataaggtgct tgtcctgaac ccgtccgtcg ccgccaccct aggtttcggg 2520
gcgtatatgt ctaaggcaca tggtatcgac cctaacatca gaaccggggt aaggaccatc 2580
accacgggtg cccccatcac gtactccacc tatggcaagt ttcttgccga cggtggttgc 2640
tctgggggcg cctatgacat cataatatgt gatgagtgcc actcaactga ctcgaccact 2700
atcctgggca tcggcacagt cctggaccaa gcggagacgg ctggagcgcg actcgtcgtg 2760
ctcgccaccg ctacgcctcc gggatcggtc accgtgccac atccaaacat cgaggaggtg 2820
gctctgtcca gcactggaga aatccccttt tatggcaaag ccatccccat cgagaccatc 2880
aaggggggga ggcacctcat tttctgccat tccaagaaga aatgtgatga gctcgccgcg 2940
aagctgtccg gcctcggact caatgctgta gcatattacc ggggccttga tgtatccgtc 3000
ataccaacta gcggagacgt cattgtcgta gcaacggacg ctctaatgac gggctttacc 3060
ggcgatttcg actcagtgat cgactgcaat acatgtgtca cccagacagt cgacttcagc 3120
ctggacccga ccttcaccat tgagacgacg accgtgccac aagacgcggt gtcacgctcg 3180
cagcggcgag gcaggactgg taggggcagg atgggcattt acaggtttgt gactccagga 3240
gaacggccct cgggcatgtt cgattcctcg gttctgtgcg agtgctatga cgcgggctgt 3300
gcttggtacg agctcacgcc cgccgagacc tcagttaggt tgcgggctta cctaaacaca 3360
ccagggttgc ccgtctgcca ggaccatctg gagttctggg agagcgtctt tacaggcctc 3420
acccacatag acgcccattt cttgtcccag actaagcagg caggagacaa cttcccctac 3480
ctggtagcat accaggctac ggtgtgcgcc agggctcagg ctccacctcc atcgtgggac 3540
caaatgtgga agtgtctcat acggctaaag cctacgctgc acgggccaac gcccctgctg 3600
tataggctgg gagccgttca aaacgaggtt actaccacac accccataac caaatacatc 3660
atggcatgca tgtcggctga cctggaggtc gtcacgagca cctgggtgct ggtaggcgga 3720
gtcctagcag ctctggccgc gtattgcctg acaacaggca gcgtggtcat tgtgggcagg 3780
atcatcttgt ccggaaagcc ggccatcatt cccgacaggg aagtccttta ccgggagttc 3840
gatgagatgg aagagtgcgc ctcacacctc ccttacatcg aacagggaat gcagctcgcc 3900
gaacaattca aacagaaggc aatcgggttg ctgcaaacag ccaccaagca agcggaggct 3960
gctgctcccg tggtggaatc caagtggcgg accctcgaag ccttctgggc gaagcatatg 4020
tggaatttca tcagcgggat acaatattta gcaggcttgt ccactctgcc tggcaacccc 4080
gcgatagcat cactgatggc attcacagcc tctatcacca gcccgctcac cacccaacat 4140
accctcctgt ttaacatcct ggggggatgg gtggccgccc aacttgctcc tcccagcgct 4200
gcttctgctt tcgtaggcgc cggcatcgct ggagcggctg ttggcagcat aggccttggg 4260
aaggtgcttg tggatatttt ggcaggttat ggagcagggg tggcaggcgc gctcgtggcc 4320
tttaaggtca tgagcggcga gatgccctcc accgaggacc tggttaacct actccctgct 4380
atcctctccc ctggcgccct agtcgtcggg gtcgtgtgcg cagcgatact gcgtcggcac 4440
gtgggcccag gggagggggc tgtgcagtgg atgaaccggc tgatagcgtt cgcttcgcgg 4500
ggtaaccacg tctcccccac gcactatgtg cctgagagcg acgctgcagc acgtgtcact 4560
cagatcctct ctagtcttac catcactcag ctgctgaaga ggcttcacca gtggatcaac 4620
gaggactgct ccacgccatg ctccggctcg tggctaagag atgtttggga ttggatatgc 4680
acggtgttga ctgatttcaa gacctggctc cagtccaagc tcctgccgcg attgccggga 4740
gtccccttct tctcatgtca acgtgggtac aagggagtct ggcggggcga cggcatcatg 4800
caaaccacct gcccatgtgg agcacagatc accggacatg tgaaaaacgg ttccatgagg 4860
atcgtggggc ctaggacctg tagtaacacg tggcatggaa cattccccat taacgcgtac 4920
accacgggcc cctgcacgcc ctccccggcg ccaaattatt ctagggcgct gtggcgggtg 4980
gctgctgagg agtacgtgga ggttacgcgg gtgggggatt tccactacgt gacgggcatg 5040
accactgaca acgtaaagtg cccgtgtcag gttccggccc ccgaattctt cacagaagtg 5100
gatggggtgc ggttgcacag gtacgctcca gcgtgcaaac ccctcctacg ggaggaggtc 5160
acattcctgg tcgggctcaa tcaatacctg gttgggteac agctcccatg cgagcccgaa 5220
ccggacgtag cagtgctcac ttccatgctc accgacccct cccacattac ggcggagacg 5280
gctaagcgta ggctggccag gggatctccc ccctccttgg ccagctcatc agctagccag 5340
ctgtctgcgc cttccttgaa ggcaacatgc actacccgtc atgactcccc ggacgctgac 5400
ctcatcgagg ccaacctcct gtggcggcag gagatgggcg ggaacatcac ccgcgtggag 5460
tcagaaaata aggtagtaat tttggactct ttcgagccgc tccaagcgga ggaggatgag 5520
agggaagtat ccgttccggc ggagatcctg cggaggtcca ggaaattccc tcgagcgatg 5580
cccatatggg cacgcccgga ttacaaccct ccactgttag agtcctggaa ggacccggac 5640
tacgtcccsc cagtggtaca cgggtgtcca ttgccgcctg ccaaggcccc tccgatacca 5700
cctccacgga ggaagaggac ggttgtcctg tcagaatcta ccgtgtcttc tgccttggcg 5760
gagctcgcca caaagacctt cggcagctcc gaatcgtcgg ccgtcgacag cggcacggca 5820
acggcctctc ctgaccagcc ctccgacgac ggcgacgcgg gatccgacgt tgagtcgtac 5880
tcctccatgc ccccccttga gggggagccg ggggatcccg atctcagcga cgggtcttgg 5940
tctaccgtaa gcgaggaggc tagtgaggac gtcgtctgct gctcgatgtc ctacacatgg 6000
acaggcgccc tgatcacgcc atgcgctgcg gaggaaacca agctgcccat caatgcactg 6060
agcaactctt tgctccgtca ccacaacttg gtctatgcta caacatctcg cagcgcaagc 6120
ctgcggcaga agaaggtcac ctttgacaga ctgcaggtcc tggacgacca ctaccgggac 6180
gtgctcaagg agatgaaggc gaaggcgtcc acagttaagg ctaaacttct atccgtggag 6240
gaagcctgta agctgacgcc cccacattcg gccagatcta aatttggcta tggggcaaag 6300
gacgtccgga acctatccag caaggccgtt aaccacatcc gctccgtgtg gaaggacttg 6360
ctggaagaca ctgagacacc aattgacacc accatcatgg caaaaaatga ggttttctgc 6420
gtccaaccag agaagggggg ccgcaagcca gctcgcctta tcgtattccc agatttgggg 6480
gttcgtgtgt gcgagaaaat ggccctttac gatgtggtct ccaccctccc tcaggccgtg 6540
atgggctctt catacggatt ccaatactct cctggacagc gggtcgagtt cctggtgaat 6600
gcctggaaag cgaagaaatg ccctatgggc ttegcatatg acacccgctg ttttgactca 6660
acggtcactg agaatgacat ccgtgttgag gagtcaatct accaatgttg tgacttggcc 6720
cccgaagcca gacaggccat aaggtcgctc acagagcggc tttacatcgg gggccccctg 6780
actaattcta aagggcagaa ctgcggctat cgccggtgcc gcgcgagcgg tgtactgacg 6840
accagctgcg gtaataccct cacatgttac ttgaaggccg ctgcggcctg tcgagctgcg 6900
aagctccagg actgcacgat gctcgtatgc ggagacgacc ttgtcgttat ctgtgaaagc 6960
gcggggaccc aagaggacga ggcgagccta cgggccttca cggaggctat gactagatac 7020
tctgcccccc ctggggaccc gcccaaacca gaatacgact tggagttgat aacatcatgc 7080
tcctccaatg tgtcagtcgc gcacgatgca tctggcaaaa gggtgtacta tctcacccgt 7140
gaccccacca ccccccttgc gcgggctgcg tgggagacag otagacacac tccagtcaat 7200
tcctggctag gcaacatcat catgtatgcg cccaccttgt gggcaaggat gatcctgatg 7260
actcatttct tctccatcct tctagctcag gaacaacttg aaaaagccct agattgtcag 7320
atctacgggg cctgttactc cattgagcca cttgacctac ctcagatcat tcaacgactc 7380
catggcctta gcgcattttc actccatagt tactctccag gtgagatcaa tagggtggct 7440
tcatgcctca ggaaacttgg ggtaccgccc ttgcgagtct ggagacatcg ggccagaagt 7500
gtccgcgcta ggctactgtc ccaggggggg agggctgcca cttgtggcaa gtacctcttc 7560
aactgggcag taaggaccaa gctcaaactc actccaatcc cggctgcgtc ccagttggat 7620
ttatccagct ggttcgttgc tggttacagc gggggagaca tatatcacag cctgtctcgt 7680
gcccgacccc gctggttcat gtggtgccta ctcctacttt ctgtaggggt aggcatctat 7740
ctactcccca accgatgaac ggggagctaa acactccagg ccaataggcc atcctgtttt 7800
tttttcctct ttttttcctt ttctttcctt tggtggctcc atcttagccc tagtcacggc 7920
tagctgtgaa aggtccgtga gccgcttgac tgcagagagt gctgatactg gcctctctgc 7980
agatcaagta cttctagaga attctagctt ggcgtaatca tggtcatagc tgtttcctgt 8040
gtgaaattgt tatcagctca caattccaca caacatacga gccggaagca taaagtgtaa 8100
agcctgggat gcctaatgag tgagctaact cacattagtt gcgttgcgct cactgcccgc 8160
tttccagtcg ggaaacctgt cgtgccagct ccattagtga atcgtccaac gcacggggag 8220
aggcggtttg cgtattgggc gcacttccgc ttcctcgctc actgactcgc tgcgctcgtt 8280
cgttcggctg cggcgagccg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 8340
atcaggggat aacgcaggaa agaccatgtg agcaaaaggc cagcaaaagg ccaggaaccg 8400
taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 8460
aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 8520
tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 8580
gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 8640
cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 8700
cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 8760
atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 8820
tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 8880
ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 8940
acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 9000
aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 9060
aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 9120
tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 9180
cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 9240
catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 9300
ccccagtgct gcaatgatac cgcgagaacc acgctcaccc gcaccagatt tatcagcaat 9360
aaaccagcca gccggaagtg cgctgcggag aagtggtcct gcaactttat ccgcctccat 9420
ccagtctatt agttgttgcc gggaagctag agtaagtagt tcgccagtca gcagtttgcg 9480
taacgtcgtt gccatagcaa caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 9540
attcagctcc ggctcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 9600
agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 9660
actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 9720
ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 9780
ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 9840
gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 9900
atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 9960
cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 10020
gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 10080
gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 10140
ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattaccat 10200
gacattaacc tataaaaata ggcgtatcac gaagcccttt cgtctagcgc gtttcggtga 10260
tgacggtgaa aacctctgac acttgcagct cccgcagacg gtcacagctt gtctgtaagc 10320
ggatgccggg agcaggcaag cccgtcaggg cgcgtcagtg ggtgttggcg ggtgtcgggg 10380
ctggcttaac tatgcggcat cagagcagat tgtactgaga gtacaccaga tgcggtgtga 10440
aataccgcac agatgcgtaa ggagaaaata ccgcatcagc ctccattcgc cattcagact 10500
ccgcaactgt tgggaagggc ggtcagtacg cgcttcttcg ctattacgcc aactggcgaa 10560
agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc aatcacgacg 10620
ttgtaaaacg acagccaatg aattgaagct tattaattct agactgaagc ttttaatacg 10680
actcactata

[0196] The different regions of pHCVNeol7.wt are as follows:

[0197] 1-341: HCV 5′ non-translated region, drives translation of the core-neo fusion protein;

[0198] 342-1181: core-neo fusion protein, selectable marker;

[0199] 1190-1800: Internal ribosome entry site of the encephalomyocarditis virus, drives translation of the HCV NS region;

[0200] 1801-7758: HCV polyprotein from non-structural protein 3 to non-structural protein 5B;

[0201] 1801-3696: Non-structural protein 3 (NS3), HCV NS3 protease/helicase;

[0202] 3697-3858: Non-structural protein 4A (NS4A), NS3 protease cofactor;

[0203] 3859-4641: Non-structural protein 4B (NS4B);

[0204] 4642-5982: Non-structural protein 5A (NS5A);

[0205] 5983-7755: Non-structural protein SB (NS5B); RNA-dependent RNA polymerase

[0206] 7759-7989: HCV 3′ non-translated region; and

[0207] 7990-10690: plasmid sequences comprising origin of replication, beta lactamase coding sequence, and T7 promoter.

[0208] Plasmid pHCVNeol7.wt was digested with the Scal endonuclease (New England Biolabs) and transcribed in vitro with the T7 Megascript kit (Ambion). Transcription mixtures were treated with DNase (0.2 U/mL) to completely remove template DNA, extracted and precipitated as described Lohmann et al. 1999, and resuspended with phosphate buffered saline.

[0209] RNA transfection using Huh-7 cells and selection of G418 resistant colonies was performed as described in Lohmann et al. 1999. Huh-7 cells were grown in DMEM (Gibco, BRL) supplemented with 10% FCS. The cells were passed twice a week 1 to 5, using 1× trypsin/EDTA (Gibco, BRL). Huh-7 cells were transfected with pHCVNeol7.wt and cultured in the presence of G418. Several G418 resistant colonies were isolated, expanded, and molecularly characterized. Analysis of nucleic acids by PCR/reverse transcription-PCR, Northern blot and metabolic labeling with 3H-uridine indicated that all clones contained replicon RNA but not replicon DNA, demonstrating that G418 resistance was due to replication of viral RNA genomes. Furthermore, western blot and immunoprecipitation experiments showed that these clones expressed all HCV proteins. Clones differed in terms of cell morphology and growth rate. Replicons RNA copy number (500-10000 molecules/cell) and viral protein expression also varied between different clones. Clone Huh7_HBI10A was chosen to develop the RHEPLISA assay because of its good growth rate and high average level of viral RNA.

[0210] While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, the practice of the invention encompasses all of the usual variations, adaptations and/or modifications that come within the scope of the following claims.

Claims

1. A method for treating infection by the hepatitis C virus, treating hepatitis C or a related condition, delaying the onset of hepatitis C or a related condition, preventing hepatitis C or a related condition, or inhibiting replication of the hepatitis C virus, which comprises administering to a subject in need thereof an effective amount of a compound of Formula (I):

2. The method according to claim 1, wherein the compound is a compound of Formula (II):

3. The method according to claim 2, wherein in the compound of Formula (II) or a pharmaceutically acceptable salt thereof: X is S or O; R1 is —H, methyl, or ethyl; and R2 is: (1) methyl, (2) ethyl, (3) methoxy, (4) ethoxy, 34(6) —Cl, or (7) —Br.

4. The method according to claim 3, wherein the compound is selected from the group consisting of:

5. The method according to claim 1, wherein the compound is a compound of Formula (III):

6. The method according to claim 5, wherein the compound is

7. The method according to claim 1, wherein the compound is a compound of Formula (IV):

8. The method according to claim 7, wherein the compound is selected from the group consisting of:

9. The method according to claim 1, wherein the compound is a compound of Formula (V):

10. The method according to claim 9, wherein the compound is

11. The method according to claim 1, wherein the compound is a compound of Formula (VI):

12. The method according to claim 11, wherein the compound is

13. The method according to claim 1, wherein the compound is a compound of Formula (VII):

14. The method according to claim 13, wherein the compound is

15. The method according to claim 1, which is a method for treating infection by the hepatitis C virus.

16. The method according to claim 1, which is a method for treating hepatitis C.

17. The method according to claim 1, which is a method for delaying the onset of hepatitis C.

18. The method according to claim 1, which is a method for preventing hepatitis C.

19. The method according to claim 1, which is a method for inhibiting replication of the hepatitis C virus.