EP1966181A2 - Anthranilic acid derivatives useful in treating infection with hepatitis c virus - Google Patents

Anthranilic acid derivatives useful in treating infection with hepatitis c virus

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Publication number
EP1966181A2
EP1966181A2 EP06851777A EP06851777A EP1966181A2 EP 1966181 A2 EP1966181 A2 EP 1966181A2 EP 06851777 A EP06851777 A EP 06851777A EP 06851777 A EP06851777 A EP 06851777A EP 1966181 A2 EP1966181 A2 EP 1966181A2
Authority
EP
European Patent Office
Prior art keywords
acetyl
amino
benzoic acid
compound
alkyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06851777A
Other languages
German (de)
French (fr)
Inventor
Jonathan David Bloom
Shabana Insaf
Thomas Nittoli
Alan Gordon Sutherland
Kevin J. Curran
Karen Wheless
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wyeth LLC
Original Assignee
Wyeth LLC
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Filing date
Publication date
Application filed by Wyeth LLC filed Critical Wyeth LLC
Publication of EP1966181A2 publication Critical patent/EP1966181A2/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/62—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/66—Nitrogen atoms not forming part of a nitro radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C235/18—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the singly-bound oxygen atoms further bound to a carbon atom of a six-membered aromatic ring, e.g. phenoxyacetamides
    • C07C235/24—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the singly-bound oxygen atoms further bound to a carbon atom of a six-membered aromatic ring, e.g. phenoxyacetamides having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
    • C07C251/32—Oximes
    • C07C251/34—Oximes with oxygen atoms of oxyimino groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
    • C07C251/48—Oximes with oxygen atoms of oxyimino groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with the carbon atom of at least one of the oxyimino groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C317/00—Sulfones; Sulfoxides
    • C07C317/26—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C317/32—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C07C317/34—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having sulfone or sulfoxide groups and amino groups bound to carbon atoms of six-membered aromatic rings being part of the same non-condensed ring or of a condensed ring system containing that ring
    • C07C317/36—Sulfones; Sulfoxides having sulfone or sulfoxide groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having sulfone or sulfoxide groups and amino groups bound to carbon atoms of six-membered aromatic rings being part of the same non-condensed ring or of a condensed ring system containing that ring with the nitrogen atoms of the amino groups bound to hydrogen atoms or to carbon atoms

Definitions

  • This invention is directed to a series of anthranilic acid compounds that are useful in the treatment of Hepatitis C viral infections.
  • Hepatitis C is a common viral infection that can lead to chronic Hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma. Infection with the Hepatitis C virus (HCV) leads to chronic Hepatitis in at least 85% of cases, is the leading reason for liver transplantation, and is responsible for at least 10,000 deaths annually in the United States (Hepatology, 1997, 26 (Suppl. 1), 2S- 10S).
  • HCV Hepatitis C virus
  • the Hepatitis C virus is a member of the Flaviviridae family, and the genome of HCV is a single-stranded linear RNA of positive sense (Hepatology, 1997, 26 (Suppl. 1), 11S-14S).
  • HCV displays extensive genetic heterogeneity; at least 6 genotypes and more than 50 subtypes have been identified. [0004] There is no effective vaccine to prevent HCV infection. The only therapy currently available is treatment with interferon- ⁇ (INF- ⁇ ) or combination therapy of INF- ⁇ with the nucleoside analog ribavirin (Antiviral Chemistry and Chemotherapy, 1997, 8, 281-301). However, only about 40% of treated patients develop a sustained response, so there is a need for more effective anti-HCV therapeutic agents.
  • INF- ⁇ interferon- ⁇
  • ribavirin Antiviral Chemistry and Chemotherapy, 1997, 8, 281-301
  • the HCV genome contains a number of non-structural proteins: NS2, NS3, NS4A, NS4B, NS5A, and NS5B (J. General Virology, 2000, 81, 1631-1648).
  • NS5B is an RNA-dependent RNA polymerase which is essential for viral replication, and therefore, the inhibition of NS5B is a suitable target for the development of therapeutic agents.
  • R 1 , R 2 , R 3 , R 4 and R 5 are independently selected from the group consisting of hydrogen, halogen, ORn, CX3, a Ci-C 6 alkyl group, (CH 2 ) n CH 2 OH, (CH 2 ) n CO 2 Rj 2 . and (CH 2 ) n -5-tetrazolyl, where one but not more than one of Ri, R 2 , R 3 , R 4 or R 5 is selected from the group consisting of (CH 2 ) n CO 2 R) 2 .
  • Rn and Ri 2 are independently selected from the group consisting of hydrogen and a CpC 6 alkyl group;
  • X is a halogen;
  • n is 0 or 1;
  • R 6 , R 7 , R 8 , R9 and Rio are independently selected from the group consisting of hydrogen, halogen, ORi 3 , SR 14 , C(Y)3, a Ci-C 6 alkyl group and phenyl;
  • Y is halogen;
  • R 1 is selected from the group consisting Of -C(O)R 6 and wherein R 6 and R 7 are independently selected from H, Ci-C 12 alkyl, C 3 -Ci 2 cycloalkyl, Ci-Cn heterocycloalkyl, C 2 -C 9 heteroaryl or C 6 -Ci 2 aryl, any of which may be optionally substituted
  • G is selected from H or hydroxyl
  • Y is O, NH or S
  • X is selected from the group consisting of H, halo, Ci-C] 2 alkyl, C 3 -Ci 2 cycloalkyl, Ci-Ci 2 perfluoroalkyl, Ci-Ci 2 alkoxy, Ci-Ci 2 alkylthio, amino, Ci-C 6 alkylamino, C 2 -Ci 2 dialkylamino, and CN, and n is 0, 1, 2 or 3, or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof
  • R 1 is selected from -C(O)R 6 and , wherein R 6 and R 7 are independently selected from Ci-C 6 alkyl, C 3 -Ci 2 cycloalkyl, Ci-Cn heterocycloalkyl, C 2 -C 9 heteroaryl or C 6 -Ci 2 aryl, any of which may be optionally substituted
  • X is selected from the group consisting of H, halo, alkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, perfluoroalkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 6 carbon atoms, amino, alkylamino of 1 to 4 carbon atoms, dialkylamino of 2 to 8 carbon atoms, and CN
  • G is selected from H or hydroxyl, or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof.
  • R 1 is selected from -C(O)R 6 and , wherein R 6 and R 7 are independently selected from Ci-Ce alkyl, C 3 -Ci 2 cycloalkyl, Ci-Cn heterocycloalkyl, C 2 -C 9 heteroaryl or C 6 -Ci 2 aryl, any of which may be optionally substituted
  • X is selected from the group consisting of H, halo, alkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, perfluoroalkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 6 carbon atoms, amino, alkylamino 1 to 6 carbon atoms, dialkylamino of 2 to 12 carbon atoms, and CN
  • G is selected from H or hydroxyl, or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof.
  • the present invention also includes compositions containing compounds of formulas (I), (El) and (III), and methods of using these compounds and compositions thereof to treat or prevent Hepatitis C virus infection, or to inhibit replication of a Hepatitis C virus.
  • alkyl includes both straight and branched alkyl moieties, which can contain as many as 12 carbon atoms. Preferably, the alkyl moiety contains between 1 to 6 carbon atoms, though 1 to 4 carbon atoms is more preferable.
  • cycloalkyl refers to alicyclic hydrocarbon groups having 3 to 12 carbon atoms, but more preferably contain 3 to 6 carbon atoms, and includes but is not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, or adamantyl. These moieties may be further substituted.
  • heterocycloalkyl refers to a non-aromatic heterocyclic ring system (monocyclic or bicyclic) where the moieties contain 1 to 4 heteroatoms selected from the group consisting of S, N, and O, and include but is not limited to: Pyrrolidine, pyrroline, 1,3-dioxolane, imidazoline, imidazolidine, pyrazoline, pyrazolidine, pyran, piperidine, dioxane, morpholine, dithioxane, thiomorpholine, piperazine, azetidinyl, hexahydroazepinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothienyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazoly
  • a heterocycloalkyl moiety preferably contains 1-11 carbon atoms. These moieties may be further substituted.
  • aryl is defined as an aromatic hydrocarbon moiety and may be substituted or unsubstituted.
  • An aryl group preferably contains 6 to 12 carbon atoms and may be selected from, but not limited to, the group: phenyl, ⁇ -naphthyl, ⁇ -naphthyl, biphenyl, anthryl, tetrahydronaphthyl, phenanthryl, fluorenyl, indanyl, biphenylenyl, acenaphthenyl, acenaphthylenyl, or phenanthrenyl groups.
  • An aryl group may be optionally mono- , di-, tri- or tetra-substituted with substituents selected from, but not limited to, the group consisting of alkyl, acyl, alkoxycarbonyl, alkoxy, alkoxyalkyl, alkoxyalkoxy, arylalkyl, alkylaryl, aryl, heteroaryl, heteroarylalkyl, cyano, halogen, hydroxy, nitro, trifluoromethyl, trifluoromethoxy, trifluoropropyl, amino, alkylamino, dialkylamino, dialkylaminoalkyl, hydroxyalkyl, alkoxyalkyl, alkylthio, -SO 3 H, - SO 2 NH 2 , -SO 2 NHalkyl, -SO 2 N(alkyl) 2 , -CO 2 H, CO 2 NH 2 , CO 2 NHalkyl, and - CO 2 N(alkyl) 2
  • heteroaryl is defined as an aromatic heterocyclic ring system (monocyclic or bicyclic) where the heteroaryl moieties are five or six membered rings containing 1 to 4 heteroatoms selected from the group consisting of S, N, and O, and include but is not limited to: (1) furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, N- methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole, 1,2,4-triazole,
  • a heterocyclic group contains 2 to 9 carbon atoms.
  • These moieties may be further substituted from substituents selected from the group consisting of alkyl, acyl, alkoxycarbonyl, alkoxy, alkoxyalkyl, alkoxyalkoxy, arylalkyl, alkylaryl, aryl, heteroaryl, heteroarylalkyl, cyano, halogen, hydroxy, nitro, trifluoroniethyl, trifluoromethoxy, trifluoropropyl, amino, alkylamino, dialkylamino, dialkylaminoalkyl, hydroxyalkyl, alkoxyalkyl, alkylthio, -SO 3 H, -SO 2 NH 2 , -SO 2 NHalkyl, -SO 2 N(alkyl) 2 , -CO 2 H, CO 2 NH 2 , CO 2 NHalkyl, and -CO 2 N(alkyl) 2 .
  • heteroaryl moieties include: alkyl, alkoxy, halogen, amino, alkylamino, dialkylamino, trifluoromethyl, and trifluoromethoxy.
  • a heteroaryl moiety contains 1 to 9 carbon atoms.
  • alkoxy is defined as Ci-C ⁇ - alkyl-O-; the term “aryloxy” is defined as aryl-O-; the term “heteroaryloxy” is defined as heteroaryl-O-; wherein alkyl, aryl, and heteroaryl are as defined above.
  • arylalkyl is defined as aryl-Ci-C ⁇ - alkyl-; arylalkyl moieties include benzyl, 1 -phenylethyl, 2-phenylethyl, 3- phenylpropyl, 2-phenylpropyl and the like.
  • alkylaryl is defined as C I -C O - alkyl-aryl, such as a tolyl group.
  • alkylthio is defined as C I -C O - alkyl-S-.
  • thio and mercapto refer to a -SH group.
  • alkylthioalkyl and “alkyloxyalkyl,” denote an alkyl group as defined above that is further substituted with an alkoxy or alkylthio as defined above.
  • arylthio and heteroarylthio denote a thio group that is further substituted with an aryl or heteroaryl group as defined above.
  • alkylamino and dialkylamino refer to moieties with one or two alkyl groups wherein the alkyl chain is 1 to 6 carbons or 1 to 4 carbons and the groups may be the same or different.
  • aminoalkyl refer to an alkyl group, as defined herein, which is further substituted with an amino group.
  • alkylaminoalkyl and dialkylaminoalkyl refer to monoalkylamino and dialkylamino moieties with one or two alkyl groups (the same or different) bonded to the nitrogen atom which is attached to an alkyl group of 1 to 6 carbon atoms.
  • alkylsulfinyl is defined as a R 1 SO- radical, where R' is an alkyl radical of 1 to 6 carbon atoms.
  • Alkylsulfonyl is a
  • R 1 SO 2 - radical where R 1 is an alkyl radical of 1 to 6 carbon atoms.
  • haloalkyl is defined as an alkyl group, as defined herein, which has been substituted with at least one halogen atom. This moiety may also be fully substituted by halogen atoms, such as a trifluoromethyl group.
  • perfluoroalkyl is defined as an alkyl group, as defined herein, which has been fully substituted with fluorine atoms, and thus, is defined by the formula -C n F 2n+ i. Examples include -CF3 and -
  • hydroxyalkyl is defined as an alkyl group, as defined herein, which has been substituted with at least one hydroxy group.
  • haloalkoxy and haloalkylthio are defined as an alkoxy or alkylthio group, as defined herein, further substituted by at least one halo atom.
  • alkylthioalkoxy are defined as an alkoxy group further substituted by another alkoxy group, such as CH 3 -O-CH 2 CH 2 -O-, or an alkylthio group.
  • alkoxyalkylthio and “alkylthioalkylthio” are defined as an alkylthio group further substituted by another alkoxy group or an alkylthio group.
  • oxo is defined as a carbonyl group
  • substituted is used herein to refer to an atom radical, a functional group radical or a moiety radical that replaces a hydrogen radical on a molecule. Unless expressly stated otherwise, it should be assumed that any of the moieties described herein may be optionally substituted with one or more groups selected from: alkyl, alkenyl, alkynyl, halogen, haloalkyl, hydroxyalkyl, nitro, amino, hydroxy, cyano, aminoalkyl, alkylamino, dialkylamino, monoalkylaminoalkyl, dialkylaminoalkyl, alkoxy, haloalkoxy, alkoxyalkyl, alkoxyalkoxy, alkylthioalkoxy, alkoxyalkylthio, alkylthioalkylthio, oxo, alkylthio, -SH, haloalkylthio, aryl, aryloxy, arylthio, oxo, alkyl
  • substituted refers to where a hydrogen radical on a molecule has been replaced by another atom radical, a functional group radical or a moiety radical; these radicals being generally referred to as "substituents.”
  • the compounds of this invention may contain an asymmetric carbon atom and may thus give rise to stereoisomers, such as enantiomers and diastereomers.
  • the stereioisomers of the instant invention are named according to the Cahn- Ingold-Prelog System. While shown without respect to stereochemistry in formulas (I), (II) and (III), the present invention includes all the individual possible stereoisomers; as well as the racemic mixtures and other mixtures of R and S stereoisomers (scalemic mixtures which are mixtures of unequal amounts of enantiomers) and prodrugs, pharmaceutically acceptable salts and pharmaceutically active metabolites thereof. It should be noted that stereoisomers of the invention having the same relative configuration at a chiral center may nevertheless have different R and S designations depending on the substitution at the indicated chiral center.
  • R 6 and R 7 being independently selected from H, Ci-C 6 alkyl, C 3 - C a cycloalkyl, Ci -Cn heterocycloalkyl, C 2 -Cg heteroaryl or C 6 -Ci 2 aryl any of which may be optionally substituted
  • X is selected from the group consisting of H, halo, Ci-C 6 alkyl, C 3 -Ci 2 cycloalkyl, Ci-Cn heterocycloalkyl, Ci-C 6 perfluoroalkyl, CpC 6 alkoxy, Ci-Ce alkylthio, amino, Ci-C 6 alkylamino, C 2 -Ci 2 dialkylamino, and CN, and optional substituents are selected from the group consisting Of Ci-C 6 alkyl, halogen, Ci-C 6 haloalkyl, Ci-C 6 hydroxyalkyl, nitro, amino
  • Another embodiment of the compounds of formula (I) is wherein Y is O or NH and X is selected from the group consisting of H, halo, Ci -C 4 alkyl, C 1 -C 4 alkoxy, and Ci-C 4 haloalkyl.
  • a more specific embodiment of the compounds of formula (I) is wherein n is 0, 1, or 2, and G is H. Another specific embodiment is wherein R and R (when present) are Cj-C 4 alkyl, wherein methyl is preferable, Y is NH. Another more specific embodiment is wherein n is 2 and X is halo, preferably Cl or F. Especially more preferable is wherein one X is Cl and the other X is F.
  • a embodiment of the compounds of formula (IT) is wherein R 6 and R 7 are independently selected from H, Ci-C 6 alkyl, C 3 -Ci 2 cycloalkyl, or C 6 -Ci 2 aryl, any of which may be optionally substituted, though it is more preferred that R 6 or R 7 be Ci-C 4 alkyl.
  • Another embodiment of the compounds of formula (II) is wherein G is H.
  • Another embodiment of the compound of formula (H) is wherein X is selected from the group H, halo, C]-C 4 alkyl, Ci -C 4 perfluoroalkyl and Ci-C 4 alkoxy, with X being halo the most preferred.
  • a more specific embodiment is where in n is 0, 1 , or 2, and the most specific embodiments are where the compound of formula (II) is either - ⁇ [N-(2-acetyl-5-chloro-4-fluorophenyl)glycyl]amino ⁇ benzoic acid or 2-(2-([5- chloro-4-fluoro-2-( 1 -methoxyimino-ethyl)-phenylamino] acetylamino ⁇ benzoic acid.
  • R 6 and R 7 are independently selected from H, Ci-C 6 alkyl, C 3 -C 12 cycloalkyl, or C 6 -C ⁇ aryl, any of which may be optionally substituted, though it is more preferred that R 6 or R 7 be Ci-C 4 alkyl.
  • a more specific embodiment of the compounds of formula (HI) is wherein G is H.
  • Another embodiment of the compound of formula (III) is wherein X is selected from the group H, halo, Cj-C 4 alkyl, Ci-C 4 perfluoroalkyl and Ci-C 4 alkoxy, with X being halo the most preferred.
  • a more specific embodiment is where in n is 1, 2, or 3 and at least one X is halo, though it more preferable that n be 1 or 2.
  • Preferred compounds of this invention are: 2- ⁇ [N-(6-acetyl-l ,3-benzodioxol-5-yl)glycyl]amino ⁇ benzoic acid; 2-( ⁇ [N-[2-acetyl-4-chloro-5-(methylthio)phenyl]glycyl ⁇ amino) benzoic acid;
  • inventive compounds are acids and a desired salt may be prepared by any suitable method known to the art, including treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary, or tertiary); an alkali metal or alkaline earth metal hydroxide; or the like.
  • an inorganic or organic base such as an amine (primary, secondary, or tertiary); an alkali metal or alkaline earth metal hydroxide; or the like.
  • suitable salts include organic salts derived from amino acids such as glycine and arginine; ammonia; primary, secondary, and tertiary amines; and cyclic amines, such as piperidine, morpholine, and piperazine; as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.
  • an inventive compound is a base
  • a desired salt may be prepared by any suitable method known in the art, including treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid, such as glucuronic acid or galacturonic acid, alpha-hydroxy acid, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.
  • an inorganic acid such as hydrochloric acid,
  • a prodrug is intended to mean a compound that is converted under physiological conditions or by solvolysis or metabolically to a specified compound that is pharmaceutically active.
  • a prodrug containing such a moiety may be prepared according to conventional procedures by treatment of a compound of this invention containing, for example, an amido, carboxylic acid, or hydroxyl moiety with a suitable reagent.
  • a “pharmaceutically active metabolite” is intended to mean a pharmacologically active compound produced through metabolism in the body of a specified compound.
  • Prodrugs and active metabolites of compounds of this invention of the above-described Formulas may be determined using techniques known in the art, for example, through metabolic studies. See, e.g., “Design of Prodrugs,” (Bundgaard, ed.), 1985, Elsevier Publishers B. V., Amsterdam, The Netherlands.
  • a “pharmaceutically acceptable salt” is intended to mean a salt that retains the biological effectiveness of the free acids and bases of a specified compound and that is not biologically or otherwise undesirable.
  • Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-l,4-dioates, hexyne-l,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates,
  • the present invention also provides for a method of inhibiting the Hepatitis C RNA-dependent RNA polymerase NS5B.
  • the method comprises contacting a cell with an amount of a compound of formulas (I), (II) or (III) effective to decrease or prevent NS5B function.
  • the cell may be a mammalian cell and more specifically a human cell.
  • the cell may also be a bacterial cell such as for example E coli.
  • the cell may include but is not limited to, a neuronal cell, an endothelial cell, a glial cell, a microglial cell, a smooth muscle cell, a somatic cell, a bone marrow cell, a liver cell, an intestinal cell, a germ cell, a myocyte, a mononuclear phagocyte, an endothelial cell, a tumor cell, a lymphocyte cell, a mesangial cell, a retinal epithelial cell, a retinal vascular cell, a ganglion cell or a stem cell.
  • the cell may be a normal cell, an activated cell, a neoplastic cell, a diseased cell, or an infected cell.
  • the present invention further provides a compound of the invention for use as an active therapeutic substance for treating or preventing Hepatitis C infection.
  • Compounds of formulas (I), (II) and (III) are of particular use for the treatment or prevention of infection with Hepatitis C virus.
  • a preferred embodiment of the method of the present invention involves treating or preventing a Hepatitis C viral infection in a mammal comprising providing the mammal with an effective amount of at least one compound of formula (I), (II), or (HI).
  • This embodiment can further comprise providing the mammal with at least one biologically active agent, which can be selected from the group consisting of interferon, a pegylated interferon, ribavirin, protease inhibitors, polymerase inhibitors, small interfering RNA compounds, anti-sense compounds, nucleotide analogs, nucleoside analogs, immunoglobulins, immunomodulators, hepatoprotectants, anti-inflammatory agents, antibiotics, antivirals, and anti- infective compounds.
  • This list only provides examples and is not meant to be exhaustive.
  • the method of the present invention involves inhibiting replication of a Hepatitis C virus comprising contacting the Hepatitis C virus with an effective amount of at least one compound of formula (I), (II), or (III).
  • This embodiment can further comprise providing the mammal with at least one biologically active agent, which can be selected from the group consisting of interferon, a pegylated interferon, ribavirin, protease inhibitors, polymerase inhibitors, small interfering RNA compounds, anti-sense compounds, nucleotide analogs, nucleoside analogs, immunoglobulins, immunomodulators, hepatoprotectants, anti-inflammatory agents, antibiotics, antivirals, and anti- infective compounds.
  • at least one biologically active agent which can be selected from the group consisting of interferon, a pegylated interferon, ribavirin, protease inhibitors, polymerase inhibitors, small interfering RNA compounds, anti-sense compounds, nucleot
  • the compounds, or the prodrugs, the pharmaceutically active metabolites or the pharmaceutically acceptable salts thereof, and the pharmaceutical compositions of the present invention are preferably provided orally or subcutaneously.
  • the compounds may be provided by intralesional, intraperitoneal, intramuscular or intravenous injection; infusion; liposome-mediated delivery; topical, nasal, anal, vaginal, sublingual, uretheral, transdermal, intrathecal, ocular or otic delivery.
  • a compound of the invention is in the form of a unit dose. Suitable unit dose forms include tablets, capsules and powders in sachets or vials.
  • Such unit dose forms may contain from 0.1 to 100 mg of a compound of the invention and preferably from 2 to 50 mg. Still further preferred unit dosage forms contain 5 to 25 mg of a compound of the present invention.
  • the compounds of the present invention can be administered orally at a dose range of about 0.01 to 100 mg/kg or preferably at a dose range of 0.1 to 10 mg/kg. Such compounds may be administered from 1 to 6 times a day, more usually from 1 to 4 times a day.
  • the effective amount will be known to one of skill in the art; it will also be dependent upon the form of the compound. One of skill in the art could routinely perform empirical activity tests to determine the bioactivity of the compound in bioassays and thus determine what dosage to administer.
  • the compounds or the prodrugs, the pharmaceutically active metabolites or the pharmaceutically acceptable salts thereof, and the pharmaceutical compositions of the invention may be formulated with conventional excipients, such as a filler, a disintegrating agent, a binder, a lubricant, a flavoring agent, a color additive, or a carrier.
  • the carrier may be for example a diluent, an aerosol, a topical carrier, an aqueous solution, a nonaqueous solution or a solid carrier.
  • the carrier may be a polymer or a toothpaste.
  • a carrier in this invention encompasses any of the standard pharmaceutically accepted carriers, such as phosphate buffered saline solution, acetate buffered saline solution, water, emulsions such as an oil/water emulsion or a triglyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules.
  • standard pharmaceutically accepted carriers such as phosphate buffered saline solution, acetate buffered saline solution, water, emulsions such as an oil/water emulsion or a triglyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules.
  • such compounds When provided orally or topically, such compounds would be provided to a subject by delivery in different carriers.
  • such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, talc, vegetable fats or oils, gums, or glycols.
  • the specific carrier would need to be selected based upon the desired method of delivery, for example, phosphate buffered saline (PBS) could be used for intravenous or systemic delivery and vegetable fats, creams, salves, ointments or gels may be used for topical delivery.
  • PBS phosphate buffered saline
  • the compounds, or the prodrugs, the pharmaceutically active metabolites or the pharmaceutically acceptable salts thereof, and the pharmaceutical compositions of the present invention may be delivered together with suitable diluents, preservatives, solubilizers, emulsif ⁇ ers, adjuvants and/or carriers useful in treatment or prevention of Hepatitis C viral infection.
  • compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (for example, Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumins or gelatin to prevent absorption to surfaces, detergents (for example, TWEEN 20, TWEEN 80, PLURONIC F68, bile acid salts), solubilizing agents (for example, glycerol, polyethylene glycerol), anti-oxidants (for example ascorbic acid, sodium metabisulfate), preservatives (for example, thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (for example, lactose, mannitol), covalent attachment of polymers such as polyethylene glycol, complexation with metal ions, or incorporation of the compound into or onto particulate preparations of hydrogels or liposomes, micro-emulsions, micelles, unilamellar or multilamellar
  • compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of the compound or composition.
  • the choice of compositions will depend on the physical and chemical properties of the compound capable of treating or preventing a Hepatitis C viral infection.
  • the compound, or the prodrugs, the pharmaceutically active metabolites or the pharmaceutically acceptable salts thereof, and the pharmaceutical compositions of the present invention may be delivered locally via a capsule that allows a sustained release of the compound over a period of time.
  • Controlled or sustained release compositions include formulation in lipophilic depots (for example, fatty acids, waxes, oils).
  • the present invention further provides controlled-release therapeutic dosage forms for the pharmaceutical composition in which the composition is incorporated into a delivery system.
  • the dosage form controls release of the pharmaceutical composition in such a manner that an effective concentration of the composition in the blood can be maintained over an extended period of time, but also the release of the composition should be such that the concentration in the blood remains relatively constant over the extended period of time to improve therapeutic results and/or minimize side effects. Additionally, a controlled release system would affect minimal peak to trough fluctuations in blood plasma levels of the pharmaceutical composition.
  • the present invention further provides a method of treating Hepatitis C infection in humans, which comprises administering to the infected individual an effective amount of a compound or a pharmaceutical composition of the invention.
  • the present invention is also directed to a pharmaceutical composition that contains a compound of formula (I):
  • R 1 is selected from the group consisting of -C(O)R 6 and wherein R 6 and R 7 are independently selected from H, alkyl, cycloalkyl, heterocycloalkyl, heteroaryl or aryl, any of which maybe optionally substituted
  • G is selected from H or hydroxyl
  • Y is O
  • X is selected from the group consisting of H, halo, alkyl, cycloalkyl, perfluoroalkyl, alkoxy, alkylthio, amino, alkylamino, dialkylamino, and CN
  • n is 0, 1, 2 or 3, or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof, and a pharmaceutically acceptable carrier.
  • the present invention is also directed to a pharmaceutical composition that contains a compound of formula (II):
  • R 1 is selected from -C(O)R 6 and , wherein R 6 and
  • R 7 are independently selected from Ci-C 6 alkyl, C3-Ci 2 cycloalkyl, Ci-Cn heterocycloalkyl, C 2 -C 9 heteroaryl or C 6 -Ci 2 aryl, any of which may be optionally substituted
  • X is selected from the group consisting of H, halo, alkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, perfluoroalkyl, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 6 carbon atoms, amino, alkylamino of 1 to 4 carbon atoms, dialkylamino of 2 to 8 carbon atoms, and CN
  • G is selected from H or hydroxyl, or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof, and a pharmaceutically acceptable carrier.
  • the present invention is also directed to a pharmaceutical composition that contains a compound of formula (HI):
  • R 1 is selected from -C(O)R 6 and ' , wherein R 6 and R 7 are independently selected from Ci-C 6 alkyl, C 3 -Ci 2 cycloalkyl, Ci-Cn heterocycloalkyl, C 2 -Cg heteroaryl or C 6 -Ci 2 aryl, any of which may be optionally substituted
  • X is selected from the group consisting of H, halo, alkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, perfluoroalkyl, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 6 carbon atoms, amino, alkylamino of 1 to 6 carbon atoms, dialkylamino of 2 to 12 carbon atoms, and CN
  • G is selected from H or hydroxyl, or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof, and a pharmaceutically acceptable carrier.
  • the present invention provides a method of treating or preventing a Hepatitis C viral infection in a mammal comprising providing the mammal with an effective amount of at least one pharmaceutical composition, wherein the at least one pharmaceutical composition includes a compound of formula (I), (II), or (III). [0060] The method of the present invention further comprises providing the mammal with an effective amount of at least one biologically active agent.
  • the at least one biologically active agent is provided prior to the at least one pharmaceutical composition, concurrently with the at least one pharmaceutical composition or after the at least one pharmaceutical composition, wherein the biologically active agent is selected from the group consisting of interferon, a pegylated interferon, ribavirin, protease inhibitors, polymerase inhibitors, small interfering RNA compounds, anti- sense compounds, nucleotide analogs, nucleoside analogs, immunoglobulins, immunomodulators, hepatoprotectants, anti-inflammatory agents, antibiotics, antivirals, and anti-infective compounds.
  • the biologically active agent is selected from the group consisting of interferon, a pegylated interferon, ribavirin, protease inhibitors, polymerase inhibitors, small interfering RNA compounds, anti- sense compounds, nucleotide analogs, nucleoside analogs, immunoglobulins, immunomodulators, hepatoprotectants, anti-inflammatory agents, antibiotics, anti
  • the compounds of the present invention, or the prodrugs, the pharmaceutically active metabolites or the pharmaceutically acceptable salts thereof, or the isomers thereof are also useful in treating and preventing viral infections, in particular hepatitis C infection, and diseases in living hosts when used in combination with each other (i.e. pharmaceutical compositions comprising the compounds, or the prodrugs or pharmaceutically acceptable salts thereof, are administered concurrently with each or sequentially, in either order).
  • the combination of compounds provided herein may further be provided to a subject in respective pharmaceutical compositions, concurrently with or sequentially to other biologically active agents, including but not limited to the group consisting of interferon, a pegylated interferon, ribavirin, protease inhibitors, polymerase inhibitors, small interfering RNA compounds, anti-sense compounds, nucleotide analogs, nucleoside analogs, immunoglobulins, immunomodulators, hepatoprotectants, anti-inflammatory agents, antibiotics, antivirals, and anti- infective compounds.
  • other biologically active agents including but not limited to the group consisting of interferon, a pegylated interferon, ribavirin, protease inhibitors, polymerase inhibitors, small interfering RNA compounds, anti-sense compounds, nucleotide analogs, nucleoside analogs, immunoglobulins, immunomodulators, hepatoprotectants, anti-inflammatory agents, antibiotics, antivirals, and
  • the present invention further provides combination therapy with one or more anthranilic acid derivatives, i.e., at least two pharmaceutical compositions, each comprising a different compound, or prodrug or a pharmaceutically acceptable salt thereof, of the present invention, are provided to a subject in need thereof either concurrently with each other or sequentially, and such therapy may further comprise providing concurrently or sequentially other medicinal agents or potentiators, such as acyclovir, famicyclovir, valgancyclovir and related compounds, ribavirin and related compounds, amantadine and related compounds, various interferons such as, for example, interferon-alpha, interferon- beta, interferon-gamma and the like, as well as alternative forms of interferons such as pegylated interferons.
  • anthranilic acid derivatives i.e., at least two pharmaceutical compositions, each comprising a different compound, or prodrug or a pharmaceutically acceptable salt thereof, of the present invention, are provided to a subject
  • combinations of, for example ribavirin and interferon may be administered as an additional combination for a multiple combination therapy with at least one of the compounds of the present invention.
  • the combination therapy with any of the above-described biologically active agents may also be sequential, that is the treatment with a first pharmaceutical composition comprising a compound, or prodrug or a pharmaceutically acceptable salt thereof, of the invention followed by treatment with a second pharmaceutical composition comprising a second compound of the invention, wherein the second compound is different than the first compound; alternatively, treatment may be with both two or more pharmaceutical compositions, wherein each pharmaceutical composition comprises a different compound, or prodrug or a pharmaceutically acceptable salt thereof, of the invention, at the same time.
  • the sequential therapy can be within a reasonable time after the completion of the first therapy with the pharmaceutical composition.
  • Treatment with the respective pharmaceutical compositions, each comprising a different compound, or prodrug or a pharmaceutically acceptable salt thereof, of the present invention, at the same time may be provided in the same daily dose or in separate doses.
  • Combination therapy may also be provided wherein a pharmaceutical composition comprising at least one compound, or prodrug or a pharmaceutically acceptable salt thereof, of the present invention is administered in a composition further comprising at least one biologically active agent, i.e. in a single dose.
  • the dosages for both concurrent and sequential combination therapy will depend on absorption, distribution, metabolism and excretion rates of the components of the pharmaceutical composition as well as other factors known to one of skill in the art. Dosage values of the pharmaceutical composition will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules may be adjusted over time according to the individual's need and the professional judgment of the person administering or supervising the administration of the pharmaceutical compositions.
  • the compounds of the invention may be used for the treatment of HCV in humans in combination therapy mode with other inhibitors of the HCV polymerase.
  • the compounds, or the prodrugs, the pharmaceutically active metabolites or the pharmaceutically acceptable salts thereof, of the present invention may be used for the treatment of HCV in humans in combination therapy mode with other inhibitors of the HCV life cycle such as, for example, inhibitors of HCV cell attachment or virus entry, HCV translation, HCV RNA transcription or replication, HCV maturation, assembly or virus release, or inhibitors of HCV enzyme activities such as the HCV nucleotidyl transferase, helicase, protease or polymerase.
  • combination therapies of the pharmaceutical compositions include any chemically compatible combination of a compound, or prodrug or a pharmaceutically acceptable salt thereof, of this inventive group with other compounds, or the prodrugs or pharmaceutically acceptable salts thereof, of the inventive group or other compounds outside of the inventive group, as long as the combination does not eliminate the anti- viral activity of the compound of this inventive group or the anti-viral activity of the pharmaceutical composition itself.
  • interferon-alpha as used herein means the family of highly homologous species-specific proteins that inhibit viral replication and cellular proliferation and modulate immune response.
  • Typical suitable interferon-alphas include, but are not limited to, recombinant interferon alpha-2b such as INTRON- A INTERFERON available from Schering Corporation, Kenilworth, NJ, recombinant interferon alpha-2a such as Roferon interferon available from Hofman-La Roche, Nutley, NJ, a recombinant interferon alpha-2C, such as BEROFOR ALPHA 2 INTERFERON available from Boehringer Ingelheim Pharmaceutical, Inc., Ridgef ⁇ eld, Conn., interferon alpha-nl, a purified blend of natural alpha interferons such as SUMIFERON available from Sumitomo, Japan or as Wellferon interferon alpha-nl (INS) available from Glaxo-Wellcome Ltd., London, Great Britain, or a consensus alpha interferon such as those described in U.S.
  • recombinant interferon alpha-2b such as INTRON- A INTERFERON available from
  • Patent Nos. 4,897,471 and 4,695,623 (the contents of which are hereby incoprpoated by reference in their entireties, specifically examples 7, 8 or 9 thereof) and the specific product available from Amgen, Inc., Newbury Park, Calif., or interferon alpha-n3 a mixture of natural interferons made by Interferon Sciences and available from the Purdue Frederick Co., Norwalk, Conn., under the ALFERON trademark.
  • the use of interferon alpha-2a or alpha 2b is preferred. Since interferon alpha 2b, among all interferons, has the broadest approval throughout the world for treating chronic hepatitis C infection, it is most preferred. The manufacture of interferon alpha 2b is described in U.S. Pat. No.
  • pegylated interferon as used herein means polyethylene glycol modified conjugates of interferon, preferably interferon alpha-2a and alpha-2b.
  • the preferred polyethylene-glycol-interferon alpha-2b conjugate is PEG.sub.l2000-interferon alpha 2b.
  • PEG.sub.l2000-IFN alpha as used herein means conjugates such as are prepared according to the methods of International Application No. WO 95/13090 and containing urethane linkages between the interferon alpha-2a or alpha-2b amino groups and polyethylene glycol having an average molecular weight of 12000.
  • R 1 is , by the methods described below in schemes 3-6, or by other methods know by skilled practitioners of the art.
  • phenylamine derivatives of this invention are prepared by Method 1, as described in Scheme 1. Accordingly, a methyl anthranilate derivative is acylated with a reagent of formula L ⁇ CH 2 C(O)L 2 to give compound 2, wherein L 1 is a leaving group such as halo, O-mesyl, O-tolyl or O-triflate, and L 2 is a leaving groups such as halo.
  • the reagent is bromoacetyl bromide.
  • the ortho acylation of compound 3 is accomplished according to the method of Sugasawa, (Sugasawa, Tsuotomu, Toyoda, Tatsuo, Adachi, Makoto; Sasakura, Kazuyuki. Aminohaloborane in Organic Synthesis. 1. Specific Ortho Substitution Reaction of Anilines. J. Am. Chem. Soc. (1978), 100(15), 4842-52) by treating the compound with a Lewis acid, or a combination of Lewis acids, such as boron tribomide, aluminum trichloride, and the corresponding nitrile, followed by acid hydrolysis to give the aminoketone 4.
  • a Lewis acid or a combination of Lewis acids, such as boron tribomide, aluminum trichloride, and the corresponding nitrile, followed by acid hydrolysis to give the aminoketone 4.
  • phenoxy and thiophenyl derivates of this invention are prepared by Method 2, as illustrated in Scheme 2.
  • ortho-acylphenol or ortho- acylthiophenol derivative 7 is prepared by reacting the corresponding acid chloride and a Lewis acid with a substituted phenol or thiophenol 6.
  • This methodology is described in Mewshaw, et al (Mewshaw, R.E.; Marquis, K.L.; Shi, X.; McGaughey, G.; Stack, G.; et al.; Tetrahedron; EN; 54; 25; 1998; 7081-7108).
  • Ester 8 is then converted to acid 9 by basic hydrolysis.
  • Compound 9 is then coupled to an anthranilic acid derivative using standard peptide coupling methods to yield final product 10.
  • An example of a standard coupling method is converting compound 9 into the corresponding acid halide which can then reacted the anthranilic acid derivative in a non-reactive solvent.
  • This conversion can be effected with reagents like oxalyl chloride, thionyl chloride, phosphorus trichloride, phosphorus pentachloride, Ph 3 PBr 2 , Ph 3 P/CBrCl 3 , Ph 3 P/CCl 4 and the like.
  • reagents like oxalyl chloride, thionyl chloride, phosphorus trichloride, phosphorus pentachloride, Ph 3 PBr 2 , Ph 3 P/CBrCl 3 , Ph 3 P/CCl 4 and the like.
  • reagents like oxalyl chloride, thionyl chloride, phosphorus trichloride, phosphorus pentachloride, Ph 3 PBr 2 , Ph 3 P/CBrCl 3 , Ph 3 P/CCl 4 and the like.
  • One skilled in the art would be aware of other similar reagents that could be used to form an acid halide from
  • Scheme 3 illustrates a general method of preparing oxime derivatives 13 starting from the corresponding aminoketone 4.
  • the aminoketone 4 is reacted with hydroxylamine, or a derivative thereof, such as an alkoxyamine (i.e. CH 3 ONH 2 ), H 2 NOSO 3 H or HON(SO 3 Na), in the presence of an acid or a base to obtain oxime 11.
  • an acid is preferable, such as HCl.
  • the oxime is then reacted with benzyl ester 2 under basic conditions, as described previously herein, to form oxime ester 12, which is then saponified to yield the final product 13.
  • Scheme 4 shows alternative methods of yielding final product 13 by introducing the oxime functionality at different points of the synthesis described in Scheme 1.
  • ester 5 can be first reacted with hydroxylamine, or a derivative thereof, as described herein, to form the oxime, which can then be saponified to obtain the final compound 13.
  • acid compound 1 (which was the final product shown in Scheme 1), can be reacted with hydroxylamine, or a derivative thereof, as described herein, to form the oxime final product.
  • Scheme 5 shows a method of preparing the corresponding oximes of the phenol or thiophenol derivatives of the present invention.
  • the 2-acylphenol or 2- acylthiophenol 7 is reacted with hydroxylamine, or a derivative thereof, such as an alkoxyamine (i.e. CH 3 ONH 2 ), H 2 NOSO 3 H or HON(SO 3 Na), in the presence of an acid or a base, and then is reacted with the appropriate acid halide in the presence of a Lewis acid, as described previously herein, to obtain oxime 14.
  • an acid is preferable, such as HCl.
  • the oxime is then hydrolyzed under basic conditions, as described previously herein, to form oxime acid 15, which is then coupled to a anthranilic acid derivative using standard peptide coupling methods, as described herein, to yield the final product 16.
  • Scheme 6 illustrates a method of preparing the oxime 16 from the corresponding acid 10 (which was the final product in Scheme 2) by reacting it with hydroxylamine, or a derivative thereof, such as an alkoxyamine, H 2 NOSO 3 H or HON (SO 3 NA), in the presence of an acid or base.
  • hydroxylamine or a derivative thereof, such as an alkoxyamine, H 2 NOSO 3 H or HON (SO 3 NA)
  • an acid such HCl
  • Scheme 7 shows the specific synthesis of 2- ⁇ [N-(2-acetyl-5-chloro-4- fluorophenyl)glycl] amino ⁇ benzoic acid using the general methodology described in Scheme 1.
  • NS5B from the BK strain (genotype Ib) is expressed in E. coli as a protein in which the 21 C-terminal amino acids are replaced with a short linker and a hexahistidine tag (GSHHHHHH; SEQ ID NO: 1).
  • the purified protein is mixed with radioactive nucleotides and allowed to replicate a heteropolymeric RNA substrate, primed by an endogenous short hairpin, resulting in an approximately
  • the radioactive product is captured on a filter and quantitated after removal of the unincorporated nucleotides.
  • CTP 10 mM cytidine 5 '-triphosphate
  • Bovine Serum Albumin 10 mg/ml NEB (10OX at 10 mg/ml) #007-BSA
  • A-[33P]-GTP (NEN-easytides NEG/606H 3000 Ci/mmol, 370 MBq/ml, 10 mCi/ml)
  • EDTA ethylenediaminetetraacetic acid
  • pH 8 GIC-BRL #15575-020
  • Dibasic sodium phosphate (7-hydrate) Na 2 HPO 4 .7H 2 O; Baker#3824-07
  • Phosphoric acid Baker, #0262.02
  • RNA Spin down a tube of RNA (5 ⁇ g/tube stored in 75% ethanol and 0.3 M sodium acetate) in a microcentrifuge for 20 minutes at 4 0 C. One tube is enough for 1 - 1.5 plates. Remove as much ethanol from the tube as possible by inverting the tube. Be gentle, pellet RNA may not adhere to the tube. Vacuum dry the RNA. Resuspend the RNA by adding 1 ml of DEPC water, close the cap of the tube tightly. To dissolve RNA, incubate RNA solution on ice for -60 minutes and gently vortex. Spin briefly to ensure all RNA solution is down to the bottom of the tube before opening cap. Gently transfer RNA solution into a 5 ml or larger tube. Add another 3 ml of DEPC water (total 4 ml of volume). [0087] Add the following volumes of reagents
  • step (8) three more times.
  • step (8) Remove polypropylene bottom. Spot dry filter at the bottom with paper towel. Air dry filter plate on a bench for 1 hour. Add 40 ⁇ l Super Mix scintillant. Seal top of the plate with a tape. Place plate into a Packard carrier or micro- beta carrier.
  • Percent inhibition is calculated after background subtraction as a percent reduction of activity relative to the positive control (average value of the plate excluding the negative controls). For the primary screen hits were chosen as showing >75 % inhibition.
  • Clone A cells (licensed from Apath, LLC) are derived from Huh-7 cells (human hepatoma cell line) and constitutively express of the HCV replication proteins with concomitant amplification the HCV replicon (Ib) genome.
  • Cells are maintained and passaged in DMEM/ 10% FCS/ 1 mg/ml G418 (Geneticin from Gibco #1181 1-023; other media components as described below in "elisa media”). Care should be taken to maintain cell monolayers at a subconfluent state by 1 :3 or 1 :4 passages every 3-4 days.
  • the replicon is extremely sensitive to the cellular metabolism/proliferation state and replicon copy number will rapidly decline in confluent monolayers (resting cells). Under ideal conditions each cell has, on average, 1000 copies of the HCV replicon genome.
  • DMEM Dulbecco's Modified Eagle Media
  • FCS Fetal Calf Serum
  • NS 5a monoclonal antibody (Virostat #1873)
  • the process for each plate involves:
  • Read plates with optical density (O.D.) 450 filter Read plates with optical density (O.D.) 450 filter.

Abstract

The present invention is directed to compounds of formula (I): wherein R1 is selected from the group consisting of -C(O)R and (a), wherein R6 and R7 are independently selected from H, alkyl, cycloalkyl, heterocycloalkyl, heteroaryl or aryl, any of which may be optionally substituted, G is selected from H or hydroxyl, Y is O, NH or S, X is selected from the group consisting of H, halo, alkyl, cycloalkyl, perfluoroalkyl, alkoxy, alkylthio, amino, alkylamino, dialkylamino, and CN, and n is 0, 1, 2 or 3, or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof. This invention is also directed to compositions containing compounds of the invention and methods of using both the inventive compounds and compositions thereof to treat or prevent Hepatitis C virus infection, or to inhibit replication of a Hepatitis C virus.

Description

ANTHRANILIC ACID DERIVATIVES USEFUL IN TREATING INFECTION WITH HEPATITIS C VIRUS
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] This invention is directed to a series of anthranilic acid compounds that are useful in the treatment of Hepatitis C viral infections.
Related Background Art
[0002] Hepatitis C is a common viral infection that can lead to chronic Hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma. Infection with the Hepatitis C virus (HCV) leads to chronic Hepatitis in at least 85% of cases, is the leading reason for liver transplantation, and is responsible for at least 10,000 deaths annually in the United States (Hepatology, 1997, 26 (Suppl. 1), 2S- 10S). [0003] The Hepatitis C virus is a member of the Flaviviridae family, and the genome of HCV is a single-stranded linear RNA of positive sense (Hepatology, 1997, 26 (Suppl. 1), 11S-14S). HCV displays extensive genetic heterogeneity; at least 6 genotypes and more than 50 subtypes have been identified. [0004] There is no effective vaccine to prevent HCV infection. The only therapy currently available is treatment with interferon-α (INF-α) or combination therapy of INF-α with the nucleoside analog ribavirin (Antiviral Chemistry and Chemotherapy, 1997, 8, 281-301). However, only about 40% of treated patients develop a sustained response, so there is a need for more effective anti-HCV therapeutic agents.
[0005] The HCV genome contains a number of non-structural proteins: NS2, NS3, NS4A, NS4B, NS5A, and NS5B (J. General Virology, 2000, 81, 1631-1648). NS5B is an RNA-dependent RNA polymerase which is essential for viral replication, and therefore, the inhibition of NS5B is a suitable target for the development of therapeutic agents.
[0006] In US patent 5,741,926, aniline derivatives, including anthranilic acids, of the below formula, are claimed and the compounds are stated to have antihyperglycemic activity,
where R1, R2, R3, R4 and R5 are independently selected from the group consisting of hydrogen, halogen, ORn, CX3, a Ci-C6 alkyl group, (CH2)nCH2OH, (CH2)nCO2Rj2. and (CH2)n-5-tetrazolyl, where one but not more than one of Ri, R2, R3, R4 or R5 is selected from the group consisting of (CH2)nCO2R)2. and (CH2)n-5- tetrazolyl; Rn and Ri2 are independently selected from the group consisting of hydrogen and a CpC6 alkyl group; X is a halogen; n is 0 or 1; R6, R7, R8, R9 and Rio are independently selected from the group consisting of hydrogen, halogen, ORi3, SR14, C(Y)3, a Ci-C6 alkyl group and phenyl; Y is halogen; A is C=O; and B is selected from the group consisting of NH, oxygen and sulfur. This reference does not teach or suggest that these compounds may be used to treat or prevent Hepatitis C viral infections. BRIEF SUMMARY OF THE INVENTION
[0007] The present invention is directed to compounds of formula (I):
(I) '
wherein R1 is selected from the group consisting Of -C(O)R6 and wherein R6 and R7 are independently selected from H, Ci-C12 alkyl, C3-Ci2 cycloalkyl, Ci-Cn heterocycloalkyl, C2-C9 heteroaryl or C6-Ci2 aryl, any of which may be optionally substituted, G is selected from H or hydroxyl, Y is O, NH or S, X is selected from the group consisting of H, halo, Ci-C]2 alkyl, C3-Ci2 cycloalkyl, Ci-Ci2 perfluoroalkyl, Ci-Ci2 alkoxy, Ci-Ci2 alkylthio, amino, Ci-C6 alkylamino, C2-Ci2 dialkylamino, and CN, and n is 0, 1, 2 or 3, or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof. [0008] This invention also includes the compounds of formula (II):
(II) '
wherein R1 is selected from -C(O)R6 and , wherein R6 and R7 are independently selected from Ci-C6 alkyl, C3-Ci2 cycloalkyl, Ci-Cn heterocycloalkyl, C2-C9 heteroaryl or C6-Ci2 aryl, any of which may be optionally substituted, X is selected from the group consisting of H, halo, alkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, perfluoroalkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 6 carbon atoms, amino, alkylamino of 1 to 4 carbon atoms, dialkylamino of 2 to 8 carbon atoms, and CN, and G is selected from H or hydroxyl, or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof. [0009] The present invention is also directed to the compounds of formula (IE):
(HI) 7
wherein R1 is selected from -C(O)R6 and , wherein R6 and R7 are independently selected from Ci-Ce alkyl, C3-Ci2 cycloalkyl, Ci-Cn heterocycloalkyl, C2-C9 heteroaryl or C6-Ci2 aryl, any of which may be optionally substituted, X is selected from the group consisting of H, halo, alkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, perfluoroalkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 6 carbon atoms, amino, alkylamino 1 to 6 carbon atoms, dialkylamino of 2 to 12 carbon atoms, and CN, and G is selected from H or hydroxyl, or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof.
[0010] The present invention also includes compositions containing compounds of formulas (I), (El) and (III), and methods of using these compounds and compositions thereof to treat or prevent Hepatitis C virus infection, or to inhibit replication of a Hepatitis C virus.
DETAILED DESCRIPTION OF THE INVENTION
[0011] For purposes of this invention the term "alkyl" includes both straight and branched alkyl moieties, which can contain as many as 12 carbon atoms. Preferably, the alkyl moiety contains between 1 to 6 carbon atoms, though 1 to 4 carbon atoms is more preferable. The term "cycloalkyl" refers to alicyclic hydrocarbon groups having 3 to 12 carbon atoms, but more preferably contain 3 to 6 carbon atoms, and includes but is not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, or adamantyl. These moieties may be further substituted.
[0012] For the purposes of this invention the term "heterocycloalkyl" refers to a non-aromatic heterocyclic ring system (monocyclic or bicyclic) where the moieties contain 1 to 4 heteroatoms selected from the group consisting of S, N, and O, and include but is not limited to: Pyrrolidine, pyrroline, 1,3-dioxolane, imidazoline, imidazolidine, pyrazoline, pyrazolidine, pyran, piperidine, dioxane, morpholine, dithioxane, thiomorpholine, piperazine, azetidinyl, hexahydroazepinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothienyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dihydro-1,4- dioxanyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl and indoline. A heterocycloalkyl moiety preferably contains 1-11 carbon atoms. These moieties may be further substituted. [0013] For purposes of this invention the term "aryl" is defined as an aromatic hydrocarbon moiety and may be substituted or unsubstituted. An aryl group preferably contains 6 to 12 carbon atoms and may be selected from, but not limited to, the group: phenyl, α-naphthyl, β-naphthyl, biphenyl, anthryl, tetrahydronaphthyl, phenanthryl, fluorenyl, indanyl, biphenylenyl, acenaphthenyl, acenaphthylenyl, or phenanthrenyl groups. An aryl group may be optionally mono- , di-, tri- or tetra-substituted with substituents selected from, but not limited to, the group consisting of alkyl, acyl, alkoxycarbonyl, alkoxy, alkoxyalkyl, alkoxyalkoxy, arylalkyl, alkylaryl, aryl, heteroaryl, heteroarylalkyl, cyano, halogen, hydroxy, nitro, trifluoromethyl, trifluoromethoxy, trifluoropropyl, amino, alkylamino, dialkylamino, dialkylaminoalkyl, hydroxyalkyl, alkoxyalkyl, alkylthio, -SO3H, - SO2NH2, -SO2NHalkyl, -SO2N(alkyl)2 , -CO2H, CO2NH2, CO2NHalkyl, and - CO2N(alkyl)2. Preferred substituents for aryl moieties include: alkyl, alkoxy, halogen, amino, alkylamino, dialkylamino, trifluoromethyl, and trifluoromethoxy. [0014] For purposes of this invention the term "heteroaryl" is defined as an aromatic heterocyclic ring system (monocyclic or bicyclic) where the heteroaryl moieties are five or six membered rings containing 1 to 4 heteroatoms selected from the group consisting of S, N, and O, and include but is not limited to: (1) furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, N- methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole, 1,2,4-triazole, 1- methyl-l,2,4-triazole, 1 H-tetrazole, 1 -methyltetrazole, benzoxazole, benzothiazole, benzofuran, benzisoxazole, benzimidazole, N-methylbenzimidazole, azabenzimidazole, indazole, quinazoline, quinoline, pyrrolidinyl; (2) a bicyclic aromatic heterocycle where a phenyl, pyridine, pyrimidine or pyridizine ring is: (i) fused to a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (ii) fused to a 5 or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (iii) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with either one oxygen or one sulfur atom; or (iv) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one heteroatom selected from O, N or S. Preferably a heterocyclic group contains 2 to 9 carbon atoms. These moieties may be further substituted from substituents selected from the group consisting of alkyl, acyl, alkoxycarbonyl, alkoxy, alkoxyalkyl, alkoxyalkoxy, arylalkyl, alkylaryl, aryl, heteroaryl, heteroarylalkyl, cyano, halogen, hydroxy, nitro, trifluoroniethyl, trifluoromethoxy, trifluoropropyl, amino, alkylamino, dialkylamino, dialkylaminoalkyl, hydroxyalkyl, alkoxyalkyl, alkylthio, -SO3H, -SO2NH2, -SO2NHalkyl, -SO2N(alkyl)2 , -CO2H, CO2NH2, CO2NHalkyl, and -CO2N(alkyl)2. Preferred substituents for heteroaryl moieties include: alkyl, alkoxy, halogen, amino, alkylamino, dialkylamino, trifluoromethyl, and trifluoromethoxy. Preferably a heteroaryl moiety contains 1 to 9 carbon atoms.
[0015] For the purposes of this invention the term "alkoxy" is defined as Ci-Cβ- alkyl-O-; the term "aryloxy" is defined as aryl-O-; the term "heteroaryloxy" is defined as heteroaryl-O-; wherein alkyl, aryl, and heteroaryl are as defined above. [0016] For purposes of this invention the term "arylalkyl" is defined as aryl-Ci-Cβ- alkyl-; arylalkyl moieties include benzyl, 1 -phenylethyl, 2-phenylethyl, 3- phenylpropyl, 2-phenylpropyl and the like.
[0017] For purposes of this invention the term "alkylaryl" is defined as CI-CO- alkyl-aryl, such as a tolyl group.
[0018] For purposes of this invention the term "alkylthio" is defined as CI-CO- alkyl-S-. For the purpose of this invention the terms "thio" and mercapto" refer to a -SH group.
[0019] For purposes of this invention "alkylthioalkyl," and "alkyloxyalkyl," denote an alkyl group as defined above that is further substituted with an alkoxy or alkylthio as defined above.
[0020] For purposes of this invention "arylthio" and "heteroarylthio," denote a thio group that is further substituted with an aryl or heteroaryl group as defined above. [0021] The terms "alkylamino" and "dialkylamino" refer to moieties with one or two alkyl groups wherein the alkyl chain is 1 to 6 carbons or 1 to 4 carbons and the groups may be the same or different. The term "aminoalkyl" refer to an alkyl group, as defined herein, which is further substituted with an amino group. The terms "monoalkylaminoalkyl" and "dialkylaminoalkyl" refer to monoalkylamino and dialkylamino moieties with one or two alkyl groups (the same or different) bonded to the nitrogen atom which is attached to an alkyl group of 1 to 6 carbon atoms. [0022] "Acyl" is a radical of the formula -(C=O)-alkyl or -(C=O)-perfluoroalkyl wherein the alkyl radical or perfluoroalkyl radical is 1 to 6 carbon atoms; preferred examples include, but are not limited to, acetyl, propionyl, butyryl, and trifluoroacetyl.
[0023] For purposes of this invention the term "alkylsulfinyl" is defined as a R1SO- radical, where R' is an alkyl radical of 1 to 6 carbon atoms. Alkylsulfonyl is a
R1SO2- radical, where R1 is an alkyl radical of 1 to 6 carbon atoms.
[0024] For purposes of this invention the term "haloalkyl" is defined as an alkyl group, as defined herein, which has been substituted with at least one halogen atom. This moiety may also be fully substituted by halogen atoms, such as a trifluoromethyl group.
[0025] For purposes of this invention the term "perfluoroalkyl" is defined as an alkyl group, as defined herein, which has been fully substituted with fluorine atoms, and thus, is defined by the formula -CnF2n+i. Examples include -CF3 and -
CF2CF3.
[0026] For purposes of this invention the term "hydroxyalkyl" is defined as an alkyl group, as defined herein, which has been substituted with at least one hydroxy group.
[0027] For purposes of this invention the terms "haloalkoxy" and "haloalkylthio" are defined as an alkoxy or alkylthio group, as defined herein, further substituted by at least one halo atom.
[0028] For purposes of this invention the terms "alkoxyalkoxy" and
"alkylthioalkoxy" are defined as an alkoxy group further substituted by another alkoxy group, such as CH3-O-CH2CH2-O-, or an alkylthio group. For purposes of this invention the terms "alkoxyalkylthio" and "alkylthioalkylthio" are defined as an alkylthio group further substituted by another alkoxy group or an alkylthio group.
[0029] For purposes of this invention the term "oxo" is defined as a carbonyl group
(i.e. C=O).
[0030] The term "substituent" is used herein to refer to an atom radical, a functional group radical or a moiety radical that replaces a hydrogen radical on a molecule. Unless expressly stated otherwise, it should be assumed that any of the moieties described herein may be optionally substituted with one or more groups selected from: alkyl, alkenyl, alkynyl, halogen, haloalkyl, hydroxyalkyl, nitro, amino, hydroxy, cyano, aminoalkyl, alkylamino, dialkylamino, monoalkylaminoalkyl, dialkylaminoalkyl, alkoxy, haloalkoxy, alkoxyalkyl, alkoxyalkoxy, alkylthioalkoxy, alkoxyalkylthio, alkylthioalkylthio, oxo, alkylthio, -SH, haloalkylthio, aryl, aryloxy, arylthio, heteroaryl, heteroaryloxy, heteroarylthio, acyl, -CO2-alkyl, -SO3H, -SO2NH2, -SO2NH-alkyl, -SO2NH-(alkyl)2, -CO2H, - CO2NH2, -CO2NH-alkyl and -CO2N-(alkyl)2.
[0031] For the purposes of this invention the term "substituted" refers to where a hydrogen radical on a molecule has been replaced by another atom radical, a functional group radical or a moiety radical; these radicals being generally referred to as "substituents."
[0032] The compounds of this invention may contain an asymmetric carbon atom and may thus give rise to stereoisomers, such as enantiomers and diastereomers. The stereioisomers of the instant invention are named according to the Cahn- Ingold-Prelog System. While shown without respect to stereochemistry in formulas (I), (II) and (III), the present invention includes all the individual possible stereoisomers; as well as the racemic mixtures and other mixtures of R and S stereoisomers (scalemic mixtures which are mixtures of unequal amounts of enantiomers) and prodrugs, pharmaceutically acceptable salts and pharmaceutically active metabolites thereof. It should be noted that stereoisomers of the invention having the same relative configuration at a chiral center may nevertheless have different R and S designations depending on the substitution at the indicated chiral center.
[0033] An embodiment of the present invention is where the compound of formula (I) is defined by R6 and R7 being independently selected from H, Ci-C6 alkyl, C3- C a cycloalkyl, Ci -Cn heterocycloalkyl, C2-Cg heteroaryl or C6-Ci2 aryl any of which may be optionally substituted, X is selected from the group consisting of H, halo, Ci-C6 alkyl, C3-Ci2 cycloalkyl, Ci-Cn heterocycloalkyl, Ci-C6 perfluoroalkyl, CpC6 alkoxy, Ci-Ce alkylthio, amino, Ci-C6 alkylamino, C2-Ci2 dialkylamino, and CN, and optional substituents are selected from the group consisting Of Ci-C6 alkyl, halogen, Ci-C6 haloalkyl, Ci-C6 hydroxyalkyl, nitro, amino, hydroxy, cyano, Ci-C6 alkylamino, C2-Ci2 dialkylamino, Ci-C6 alkoxy, C]-C6 haloalkoxy, C2-Ci2 alkoxyalkyl, C2-Cn alkoxyalkoxy, oxo, Ci-C6 alkylthio, mercapto, Ci-C6 haloalkylthio, C6-Ci2 aryl, C6-Ci2 aryloxy, C6-Ci2 arylthio, C2-Cg heteroaryl, C2-Cg heteroaryloxy, C2-C9 heteroarylthio, acyl, -CO2-Ci-C6 alkyl, -SO3H, - SO2NH2, -SO2NH-Ci-C6 alkyl, -SO2NH-(Ci-C6 alkyl)2, -CO2H, -CO2NH2, - CO2NH- Ci-C6 alkyl and -CO2N-(C1-C6 alkyl)2.
[0034] Another embodiment of the compounds of formula (I) is wherein Y is O or NH and X is selected from the group consisting of H, halo, Ci -C4 alkyl, C1-C4 alkoxy, and Ci-C4 haloalkyl.
[0035] A more specific embodiment of the compounds of formula (I) is wherein n is 0, 1, or 2, and G is H. Another specific embodiment is wherein R and R (when present) are Cj-C4 alkyl, wherein methyl is preferable, Y is NH. Another more specific embodiment is wherein n is 2 and X is halo, preferably Cl or F. Especially more preferable is wherein one X is Cl and the other X is F. [0036] A embodiment of the compounds of formula (IT) is wherein R6 and R7 are independently selected from H, Ci-C6 alkyl, C3-Ci2 cycloalkyl, or C6-Ci2 aryl, any of which may be optionally substituted, though it is more preferred that R6 or R7 be Ci-C4 alkyl.
[0037] Another embodiment of the compounds of formula (II) is wherein G is H. Another embodiment of the compound of formula (H) is wherein X is selected from the group H, halo, C]-C4 alkyl, Ci -C4 perfluoroalkyl and Ci-C4 alkoxy, with X being halo the most preferred. A more specific embodiment is where in n is 0, 1 , or 2, and the most specific embodiments are where the compound of formula (II) is either -{[N-(2-acetyl-5-chloro-4-fluorophenyl)glycyl]amino}benzoic acid or 2-(2-([5- chloro-4-fluoro-2-( 1 -methoxyimino-ethyl)-phenylamino] acetylamino } benzoic acid. [0038] An embodiment of the compounds of formula (HI) is wherein R6 and R7 are independently selected from H, Ci-C6 alkyl, C3-C12 cycloalkyl, or C6-C^ aryl, any of which may be optionally substituted, though it is more preferred that R6 or R7 be Ci-C4 alkyl.
[0039] A more specific embodiment of the compounds of formula (HI) is wherein G is H. Another embodiment of the compound of formula (III) is wherein X is selected from the group H, halo, Cj-C4 alkyl, Ci-C4 perfluoroalkyl and Ci-C4 alkoxy, with X being halo the most preferred. A more specific embodiment is where in n is 1, 2, or 3 and at least one X is halo, though it more preferable that n be 1 or 2. 040] Preferred compounds of this invention are: 2-{[N-(6-acetyl-l ,3-benzodioxol-5-yl)glycyl]amino}benzoic acid; 2-({[N-[2-acetyl-4-chloro-5-(methylthio)phenyl]glycyl}amino) benzoic acid;
2-({[N-[2-acetyl-4-chloro-5-(dimetheylamino)phenyl]glycyl} amino)benzoic acid;
2-({[N-[2-acetyl-4-chloro-5-(methylamino)phenyl]glycyl}amino) benzoic acid;
2-{[N-(2-acetyl-5-chloro-4-fluorophenyl-N-methylglycyl]amino} benzoic acid; 2-({[N-[2-acetyl-4-chloro-5-(methylsulfinyl)phenyl]glycyl}amino) benzoic acid; 2- { [N-(2-acetylphenyl)glycyl]amino}benzoic acid;
2- {[N-(5-chloro-4-methyl-2-propionylphenyl)glycyl]amino} benzoic acid; 2-{[N-(2-acetyl-4,5-dimethylphenyl)glycyl]amino}benzoic acid; 2-{[N-(2-acetyl-4-bromo-5-choloro phenyl)glycyl]amino}benzoic acid;
2-{[N-(5-chloro-4-fluoro-2-propionylphenyl)glycyl]amino}benzoic acid; 2- {[N-(2-acetyl-4,5-difluorophenyl)glycyl]amino}benzoic acid;
2-{[N-(2-acetyl-4-chloro-5-ethoxyphenyl)glycyl]amino}benzoic acid; 2-{[N-(2-acetyl-4-chloro-5-fluorophenyl)glycyl]amino}benzoic acid; 2-{[N-(2-acetyl-4,5-dichlorophenyl)glycyl]amino}benzoic acid; 2- { [N-(2-acetyl-4-chloro-5-methoxyphenyl)glycyl]amino}benzoic acid; 2- { [N-(2-acetyl-4-fluoro-5-methylphenyl)glycyl]amino}benzoic acid; 2-{[N-(2-acetyl-5-chloro-4-fluorophenyl)glycyl]amino}benzoic acid; 2- { [N-(4-chloro-2-propionylphenyl)glycyl]amino}benzoic acid; 2- { [N-(2-acetyl-4-chlorophenyl)glycyl]amino}benzoic acid;
2-(2-([5-chloro-4-fluoro-2-(l-methoxyimino-ethyl)-phenylamino]-acetylamino}- benzoic acid; 2-({[4,5-dimethyl-2-(trifluoroacetyl)phenoxy]acetyl}amino} benzoic acid;
2- {[(2-acetyl-4,5-dimethoxyphenoxy)acetyl]amino}benzoic acid;
2-[2-(2-acetyl-5-methylphenoxy)acetylamino]-benzoic acid;
2- {[(2-acetyl-4-ethylphenoxy)acetyl]amino}benzoic acid;
2- { [(2-acetyl-4-methylphenoxy)acetyl]amino}benzoic acid;
2- { [(2-acetyl-5-fluorophenoxy)acetyl]amino}benzoic acid;
2- { [(2-acetyl-5-methoxyphenoxy)acetyl]amino}benzoic acid;
2- {[(2-acetyl-5-chlorophenoxy)acetyl]amino}benzoic acid;
2- {[(2-benzoyl4-methylphenoxy)acetyl]amino}benzoic acid;
2- { [(2-acetyl-4, 5 -dimethylphenoxy)acetyl] amino } -5 -hydroxybenzoic acid;
2- {[(2-acetyl-4,5-dimethylphenoxy)acetyl]amino}benzoic acid; 2-{[(2-isobutyryl-4,5-dimethylphenoxy)acetyl]amino}benzoic acid; 2- {[(2-acetyl-5-ethylphenoxy)acetyl]amino}benzoic acid; 2- {[(4,5-dimethyl-2-pentanoyphenoxy)acetyl]amino}benzoic acid; 2- {[(2-butyryl-4,5-dimethylphenoxy)acetyl]amino}benzoic acid; 2- {[(4,5-dimethyl-2-propionyphenoxy)acetyl]amino}benzoic acid;
2-{[(2-acetyl-4-chloro-5-fluorophenoxy)acetyl]amino}benzoic acid; 2- { [(2-acetyl-5-chloro-4-fluorophenoxy)acetyl]amino}benzoic acid;
2- {[(2-acetyl-4-fluoro-5-methylphenoxy)acetyl]amino}benzoic acid;
2- {[(2-acetyl-4,5-difluorophenoxy)acetyl]amino}benzoic acid;
2- {[(4-chloro-2-propionylphenoxy)acetyl]amino}benzoic acid
2-{[(2-acetyl-4-chloro-5-methylphenoxy)acetyl]amino}benzoic acid
2-{[(2-acetyl-4-fluorophenoxy)acetyl]amino}benzoic acid;
2- { [(2-acetyl-4-chlorophenoxy)acetyl]amino}benzoic acid;
2-{[N-(2-acetyl-4-bromophenyl)glycyl]amino}benzoic acid; and
2- { [(2-acetyl-4-ethyl-3-hydroxphenoxy)acetyl]amino}benzoic acid. [0041] The inventive compounds are acids and a desired salt may be prepared by any suitable method known to the art, including treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary, or tertiary); an alkali metal or alkaline earth metal hydroxide; or the like. Illustrative examples of suitable salts include organic salts derived from amino acids such as glycine and arginine; ammonia; primary, secondary, and tertiary amines; and cyclic amines, such as piperidine, morpholine, and piperazine; as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.
[0042] If an inventive compound is a base, a desired salt may be prepared by any suitable method known in the art, including treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid, such as glucuronic acid or galacturonic acid, alpha-hydroxy acid, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.
[0043] A "prodrug" is intended to mean a compound that is converted under physiological conditions or by solvolysis or metabolically to a specified compound that is pharmaceutically active. A prodrug may be a derivative of one of the compounds of this invention that contains a moiety, such as for example -CO2R, - PO(OR)2 or -C=NR, that may be cleaved under physiological conditions or by solvolysis. Any suitable R substituent may be used that provides a pharmaceutically acceptable solvolysis or cleavage product. A prodrug containing such a moiety may be prepared according to conventional procedures by treatment of a compound of this invention containing, for example, an amido, carboxylic acid, or hydroxyl moiety with a suitable reagent. A "pharmaceutically active metabolite" is intended to mean a pharmacologically active compound produced through metabolism in the body of a specified compound. Prodrugs and active metabolites of compounds of this invention of the above-described Formulas may be determined using techniques known in the art, for example, through metabolic studies. See, e.g., "Design of Prodrugs," (Bundgaard, ed.), 1985, Elsevier Publishers B. V., Amsterdam, The Netherlands. A "pharmaceutically acceptable salt" is intended to mean a salt that retains the biological effectiveness of the free acids and bases of a specified compound and that is not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-l,4-dioates, hexyne-l,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycollates, tartrates, methane-sulfonates (mesylates), propanesulfonates, naphthalene- 1 -sulfonates, naphthalene-2-sulfonates, and mandelates. In the case of compounds or salts that are solids it is understood by those skilled in the art that the inventive compounds or salts may exist in different crystal forms, all of which are intended to be within the scope of the present invention and specified formulas. [0044] In one embodiment, the present invention also provides for a method of inhibiting the Hepatitis C RNA-dependent RNA polymerase NS5B. The method comprises contacting a cell with an amount of a compound of formulas (I), (II) or (III) effective to decrease or prevent NS5B function. The cell may be a mammalian cell and more specifically a human cell. The cell may also be a bacterial cell such as for example E coli. The cell may include but is not limited to, a neuronal cell, an endothelial cell, a glial cell, a microglial cell, a smooth muscle cell, a somatic cell, a bone marrow cell, a liver cell, an intestinal cell, a germ cell, a myocyte, a mononuclear phagocyte, an endothelial cell, a tumor cell, a lymphocyte cell, a mesangial cell, a retinal epithelial cell, a retinal vascular cell, a ganglion cell or a stem cell. The cell may be a normal cell, an activated cell, a neoplastic cell, a diseased cell, or an infected cell.
[0045] The present invention further provides a compound of the invention for use as an active therapeutic substance for treating or preventing Hepatitis C infection. Compounds of formulas (I), (II) and (III) are of particular use for the treatment or prevention of infection with Hepatitis C virus.
[0046] A preferred embodiment of the method of the present invention involves treating or preventing a Hepatitis C viral infection in a mammal comprising providing the mammal with an effective amount of at least one compound of formula (I), (II), or (HI). This embodiment can further comprise providing the mammal with at least one biologically active agent, which can be selected from the group consisting of interferon, a pegylated interferon, ribavirin, protease inhibitors, polymerase inhibitors, small interfering RNA compounds, anti-sense compounds, nucleotide analogs, nucleoside analogs, immunoglobulins, immunomodulators, hepatoprotectants, anti-inflammatory agents, antibiotics, antivirals, and anti- infective compounds. This list only provides examples and is not meant to be exhaustive.
[0047] In another embodiment of the method of the present invention involves inhibiting replication of a Hepatitis C virus comprising contacting the Hepatitis C virus with an effective amount of at least one compound of formula (I), (II), or (III). This embodiment can further comprise providing the mammal with at least one biologically active agent, which can be selected from the group consisting of interferon, a pegylated interferon, ribavirin, protease inhibitors, polymerase inhibitors, small interfering RNA compounds, anti-sense compounds, nucleotide analogs, nucleoside analogs, immunoglobulins, immunomodulators, hepatoprotectants, anti-inflammatory agents, antibiotics, antivirals, and anti- infective compounds. This list only provides examples and is not meant to exhaustive.
[0048] The compounds, or the prodrugs, the pharmaceutically active metabolites or the pharmaceutically acceptable salts thereof, and the pharmaceutical compositions of the present invention are preferably provided orally or subcutaneously. The compounds may be provided by intralesional, intraperitoneal, intramuscular or intravenous injection; infusion; liposome-mediated delivery; topical, nasal, anal, vaginal, sublingual, uretheral, transdermal, intrathecal, ocular or otic delivery. In order to obtain consistency in providing the compound of this invention it is preferred that a compound of the invention is in the form of a unit dose. Suitable unit dose forms include tablets, capsules and powders in sachets or vials. Such unit dose forms may contain from 0.1 to 100 mg of a compound of the invention and preferably from 2 to 50 mg. Still further preferred unit dosage forms contain 5 to 25 mg of a compound of the present invention. The compounds of the present invention can be administered orally at a dose range of about 0.01 to 100 mg/kg or preferably at a dose range of 0.1 to 10 mg/kg. Such compounds may be administered from 1 to 6 times a day, more usually from 1 to 4 times a day. The effective amount will be known to one of skill in the art; it will also be dependent upon the form of the compound. One of skill in the art could routinely perform empirical activity tests to determine the bioactivity of the compound in bioassays and thus determine what dosage to administer.
[0049] The compounds or the prodrugs, the pharmaceutically active metabolites or the pharmaceutically acceptable salts thereof, and the pharmaceutical compositions of the invention may be formulated with conventional excipients, such as a filler, a disintegrating agent, a binder, a lubricant, a flavoring agent, a color additive, or a carrier. The carrier may be for example a diluent, an aerosol, a topical carrier, an aqueous solution, a nonaqueous solution or a solid carrier. The carrier may be a polymer or a toothpaste. A carrier in this invention encompasses any of the standard pharmaceutically accepted carriers, such as phosphate buffered saline solution, acetate buffered saline solution, water, emulsions such as an oil/water emulsion or a triglyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules.
[0050] When provided orally or topically, such compounds would be provided to a subject by delivery in different carriers. Typically, such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, talc, vegetable fats or oils, gums, or glycols. The specific carrier would need to be selected based upon the desired method of delivery, for example, phosphate buffered saline (PBS) could be used for intravenous or systemic delivery and vegetable fats, creams, salves, ointments or gels may be used for topical delivery. [0051] The compounds, or the prodrugs, the pharmaceutically active metabolites or the pharmaceutically acceptable salts thereof, and the pharmaceutical compositions of the present invention may be delivered together with suitable diluents, preservatives, solubilizers, emulsifϊers, adjuvants and/or carriers useful in treatment or prevention of Hepatitis C viral infection. Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (for example, Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumins or gelatin to prevent absorption to surfaces, detergents (for example, TWEEN 20, TWEEN 80, PLURONIC F68, bile acid salts), solubilizing agents (for example, glycerol, polyethylene glycerol), anti-oxidants (for example ascorbic acid, sodium metabisulfate), preservatives (for example, thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (for example, lactose, mannitol), covalent attachment of polymers such as polyethylene glycol, complexation with metal ions, or incorporation of the compound into or onto particulate preparations of hydrogels or liposomes, micro-emulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of the compound or composition. The choice of compositions will depend on the physical and chemical properties of the compound capable of treating or preventing a Hepatitis C viral infection. [0052] The compound, or the prodrugs, the pharmaceutically active metabolites or the pharmaceutically acceptable salts thereof, and the pharmaceutical compositions of the present invention may be delivered locally via a capsule that allows a sustained release of the compound over a period of time. Controlled or sustained release compositions include formulation in lipophilic depots (for example, fatty acids, waxes, oils).
[0053] The present invention further provides controlled-release therapeutic dosage forms for the pharmaceutical composition in which the composition is incorporated into a delivery system. The dosage form controls release of the pharmaceutical composition in such a manner that an effective concentration of the composition in the blood can be maintained over an extended period of time, but also the release of the composition should be such that the concentration in the blood remains relatively constant over the extended period of time to improve therapeutic results and/or minimize side effects. Additionally, a controlled release system would affect minimal peak to trough fluctuations in blood plasma levels of the pharmaceutical composition.
[0054] The present invention further provides a method of treating Hepatitis C infection in humans, which comprises administering to the infected individual an effective amount of a compound or a pharmaceutical composition of the invention. [0055] The present invention is also directed to a pharmaceutical composition that contains a compound of formula (I):
(I) '
wherein R1 is selected from the group consisting of -C(O)R6 and wherein R6 and R7 are independently selected from H, alkyl, cycloalkyl, heterocycloalkyl, heteroaryl or aryl, any of which maybe optionally substituted, G is selected from H or hydroxyl, Y is O, NH or S, X is selected from the group consisting of H, halo, alkyl, cycloalkyl, perfluoroalkyl, alkoxy, alkylthio, amino, alkylamino, dialkylamino, and CN, and n is 0, 1, 2 or 3, or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof, and a pharmaceutically acceptable carrier.
[0056] The present invention is also directed to a pharmaceutical composition that contains a compound of formula (II):
(D) '
[0057] wherein R1 is selected from -C(O)R6 and , wherein R6 and
R7 are independently selected from Ci-C6 alkyl, C3-Ci2 cycloalkyl, Ci-Cn heterocycloalkyl, C2-C9 heteroaryl or C6-Ci2 aryl, any of which may be optionally substituted, X is selected from the group consisting of H, halo, alkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, perfluoroalkyl, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 6 carbon atoms, amino, alkylamino of 1 to 4 carbon atoms, dialkylamino of 2 to 8 carbon atoms, and CN, and G is selected from H or hydroxyl, or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof, and a pharmaceutically acceptable carrier. [0058] The present invention is also directed to a pharmaceutical composition that contains a compound of formula (HI):
(III) '
wherein R1 is selected from -C(O)R6 and ' , wherein R6 and R7 are independently selected from Ci-C6 alkyl, C3-Ci2 cycloalkyl, Ci-Cn heterocycloalkyl, C2-Cg heteroaryl or C6-Ci2 aryl, any of which may be optionally substituted, X is selected from the group consisting of H, halo, alkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, perfluoroalkyl, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 6 carbon atoms, amino, alkylamino of 1 to 6 carbon atoms, dialkylamino of 2 to 12 carbon atoms, and CN, and G is selected from H or hydroxyl, or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof, and a pharmaceutically acceptable carrier. [0059] The present invention provides a method of treating or preventing a Hepatitis C viral infection in a mammal comprising providing the mammal with an effective amount of at least one pharmaceutical composition, wherein the at least one pharmaceutical composition includes a compound of formula (I), (II), or (III). [0060] The method of the present invention further comprises providing the mammal with an effective amount of at least one biologically active agent. In an embodiment of the method of the present invention, the at least one biologically active agent is provided prior to the at least one pharmaceutical composition, concurrently with the at least one pharmaceutical composition or after the at least one pharmaceutical composition, wherein the biologically active agent is selected from the group consisting of interferon, a pegylated interferon, ribavirin, protease inhibitors, polymerase inhibitors, small interfering RNA compounds, anti- sense compounds, nucleotide analogs, nucleoside analogs, immunoglobulins, immunomodulators, hepatoprotectants, anti-inflammatory agents, antibiotics, antivirals, and anti-infective compounds.
[0061] The compounds of the present invention, or the prodrugs, the pharmaceutically active metabolites or the pharmaceutically acceptable salts thereof, or the isomers thereof are also useful in treating and preventing viral infections, in particular hepatitis C infection, and diseases in living hosts when used in combination with each other (i.e. pharmaceutical compositions comprising the compounds, or the prodrugs or pharmaceutically acceptable salts thereof, are administered concurrently with each or sequentially, in either order). The combination of compounds provided herein may further be provided to a subject in respective pharmaceutical compositions, concurrently with or sequentially to other biologically active agents, including but not limited to the group consisting of interferon, a pegylated interferon, ribavirin, protease inhibitors, polymerase inhibitors, small interfering RNA compounds, anti-sense compounds, nucleotide analogs, nucleoside analogs, immunoglobulins, immunomodulators, hepatoprotectants, anti-inflammatory agents, antibiotics, antivirals, and anti- infective compounds. The present invention further provides combination therapy with one or more anthranilic acid derivatives, i.e., at least two pharmaceutical compositions, each comprising a different compound, or prodrug or a pharmaceutically acceptable salt thereof, of the present invention, are provided to a subject in need thereof either concurrently with each other or sequentially, and such therapy may further comprise providing concurrently or sequentially other medicinal agents or potentiators, such as acyclovir, famicyclovir, valgancyclovir and related compounds, ribavirin and related compounds, amantadine and related compounds, various interferons such as, for example, interferon-alpha, interferon- beta, interferon-gamma and the like, as well as alternative forms of interferons such as pegylated interferons. Additionally, combinations of, for example ribavirin and interferon, may be administered as an additional combination for a multiple combination therapy with at least one of the compounds of the present invention. [0062] The combination therapy with any of the above-described biologically active agents may also be sequential, that is the treatment with a first pharmaceutical composition comprising a compound, or prodrug or a pharmaceutically acceptable salt thereof, of the invention followed by treatment with a second pharmaceutical composition comprising a second compound of the invention, wherein the second compound is different than the first compound; alternatively, treatment may be with both two or more pharmaceutical compositions, wherein each pharmaceutical composition comprises a different compound, or prodrug or a pharmaceutically acceptable salt thereof, of the invention, at the same time. The sequential therapy can be within a reasonable time after the completion of the first therapy with the pharmaceutical composition. Treatment with the respective pharmaceutical compositions, each comprising a different compound, or prodrug or a pharmaceutically acceptable salt thereof, of the present invention, at the same time may be provided in the same daily dose or in separate doses. Combination therapy may also be provided wherein a pharmaceutical composition comprising at least one compound, or prodrug or a pharmaceutically acceptable salt thereof, of the present invention is administered in a composition further comprising at least one biologically active agent, i.e. in a single dose. The dosages for both concurrent and sequential combination therapy (for combined pharmaceutical compositions comprising at least two compounds, or the prodrugs or a pharmaceutically acceptable salts thereof, of the invention or compositions comprising at least one compound, or prodrug or a pharmaceutically acceptable salt thereof, of the invention and at least one biologically active agent), will depend on absorption, distribution, metabolism and excretion rates of the components of the pharmaceutical composition as well as other factors known to one of skill in the art. Dosage values of the pharmaceutical composition will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules may be adjusted over time according to the individual's need and the professional judgment of the person administering or supervising the administration of the pharmaceutical compositions.
[0063] In a further embodiment, the compounds of the invention may be used for the treatment of HCV in humans in combination therapy mode with other inhibitors of the HCV polymerase.
[0064] In yet a further embodiment, the compounds, or the prodrugs, the pharmaceutically active metabolites or the pharmaceutically acceptable salts thereof, of the present invention may be used for the treatment of HCV in humans in combination therapy mode with other inhibitors of the HCV life cycle such as, for example, inhibitors of HCV cell attachment or virus entry, HCV translation, HCV RNA transcription or replication, HCV maturation, assembly or virus release, or inhibitors of HCV enzyme activities such as the HCV nucleotidyl transferase, helicase, protease or polymerase. [0065] It is intended that combination therapies of the pharmaceutical compositions include any chemically compatible combination of a compound, or prodrug or a pharmaceutically acceptable salt thereof, of this inventive group with other compounds, or the prodrugs or pharmaceutically acceptable salts thereof, of the inventive group or other compounds outside of the inventive group, as long as the combination does not eliminate the anti- viral activity of the compound of this inventive group or the anti-viral activity of the pharmaceutical composition itself. [0066] The term "interferon-alpha" as used herein means the family of highly homologous species-specific proteins that inhibit viral replication and cellular proliferation and modulate immune response. Typical suitable interferon-alphas include, but are not limited to, recombinant interferon alpha-2b such as INTRON- A INTERFERON available from Schering Corporation, Kenilworth, NJ, recombinant interferon alpha-2a such as Roferon interferon available from Hofman-La Roche, Nutley, NJ, a recombinant interferon alpha-2C, such as BEROFOR ALPHA 2 INTERFERON available from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefϊeld, Conn., interferon alpha-nl, a purified blend of natural alpha interferons such as SUMIFERON available from Sumitomo, Japan or as Wellferon interferon alpha-nl (INS) available from Glaxo-Wellcome Ltd., London, Great Britain, or a consensus alpha interferon such as those described in U.S. Patent Nos. 4,897,471 and 4,695,623 (the contents of which are hereby incoprpoated by reference in their entireties, specifically examples 7, 8 or 9 thereof) and the specific product available from Amgen, Inc., Newbury Park, Calif., or interferon alpha-n3 a mixture of natural interferons made by Interferon Sciences and available from the Purdue Frederick Co., Norwalk, Conn., under the ALFERON trademark. The use of interferon alpha-2a or alpha 2b is preferred. Since interferon alpha 2b, among all interferons, has the broadest approval throughout the world for treating chronic hepatitis C infection, it is most preferred. The manufacture of interferon alpha 2b is described in U.S. Pat. No. 4,503,901. [0067] The term "pegylated interferon" as used herein means polyethylene glycol modified conjugates of interferon, preferably interferon alpha-2a and alpha-2b. The preferred polyethylene-glycol-interferon alpha-2b conjugate is PEG.sub.l2000-interferon alpha 2b. The phrase "PEG.sub.l2000-IFN alpha" as used herein means conjugates such as are prepared according to the methods of International Application No. WO 95/13090 and containing urethane linkages between the interferon alpha-2a or alpha-2b amino groups and polyethylene glycol having an average molecular weight of 12000.
[0068] The following experimental details are set forth to aid in an understanding of the invention, and are not intended, and should not be construed, to limit in any way the invention set forth in the claims that follow thereafter.
[0069] The compounds of the present invention can be readily prepared according to the following reaction schemes or modification thereof.
[0070] Compounds of the present invention, wherein R1 is carbonyl containing moiety, can be synthesized as described in the schemes below (Scheme 1 and 2).
Subsequently, these compounds can be converted to the corresponding oximes, 7
where R1 is , by the methods described below in schemes 3-6, or by other methods know by skilled practitioners of the art.
Scheme 1 2
Scheme 2
10 [0071] The phenylamine derivatives of this invention are prepared by Method 1, as described in Scheme 1. Accordingly, a methyl anthranilate derivative is acylated with a reagent of formula L^CH2C(O)L2 to give compound 2, wherein L1 is a leaving group such as halo, O-mesyl, O-tolyl or O-triflate, and L2 is a leaving groups such as halo. Preferably the reagent is bromoacetyl bromide. The ortho acylation of compound 3 is accomplished according to the method of Sugasawa, (Sugasawa, Tsuotomu, Toyoda, Tatsuo, Adachi, Makoto; Sasakura, Kazuyuki. Aminohaloborane in Organic Synthesis. 1. Specific Ortho Substitution Reaction of Anilines. J. Am. Chem. Soc. (1978), 100(15), 4842-52) by treating the compound with a Lewis acid, or a combination of Lewis acids, such as boron tribomide, aluminum trichloride, and the corresponding nitrile, followed by acid hydrolysis to give the aminoketone 4. Reaction of methyl N-acetyl anthranilate 2 with aminoketone 4 in the presence of a base, preferably a tertiary amine base, such as N, N-diethylisopropylamine, triethylamine and pyridine, gives alkylation product 5. A skilled artisan would be knowledgeable of other bases that could be used to effect this alkylation. Hydrolysis of the ester under standard conditions affords final product 1. These compounds can be further derivatized to form the corresponding oximes (See Schemes 3 and 4).
[0072] The phenoxy and thiophenyl derivates of this invention are prepared by Method 2, as illustrated in Scheme 2. Accordingly, ortho-acylphenol or ortho- acylthiophenol derivative 7 is prepared by reacting the corresponding acid chloride and a Lewis acid with a substituted phenol or thiophenol 6. This methodology is described in Mewshaw, et al (Mewshaw, R.E.; Marquis, K.L.; Shi, X.; McGaughey, G.; Stack, G.; et al.; Tetrahedron; EN; 54; 25; 1998; 7081-7108). Alkylation with a reagent of formula ROC(O)L2, wherein R' is selected from alkyl or aryl and L2 is halo, affords ester 8. Preferably the reagent is ethyl iodoacetate. Ester 8 is then converted to acid 9 by basic hydrolysis. Compound 9 is then coupled to an anthranilic acid derivative using standard peptide coupling methods to yield final product 10. An example of a standard coupling method is converting compound 9 into the corresponding acid halide which can then reacted the anthranilic acid derivative in a non-reactive solvent. This conversion can be effected with reagents like oxalyl chloride, thionyl chloride, phosphorus trichloride, phosphorus pentachloride, Ph3PBr2, Ph3P/CBrCl3, Ph3P/CCl4 and the like. One skilled in the art would be aware of other similar reagents that could be used to form an acid halide from a carboxylic acid. These compounds can then be derivatized further to form the corresponding oximes, as shown in Schemes 5 and 6.
Scheme 3
Scheme 4
13 Scheme 5
15
16
Scheme 6
Scheme 7
Bromoacetyl bromide
[0073] Scheme 3 illustrates a general method of preparing oxime derivatives 13 starting from the corresponding aminoketone 4. The aminoketone 4 is reacted with hydroxylamine, or a derivative thereof, such as an alkoxyamine (i.e. CH3ONH2), H2NOSO3H or HON(SO3Na), in the presence of an acid or a base to obtain oxime 11. Use of an acid is preferable, such as HCl. The oxime is then reacted with benzyl ester 2 under basic conditions, as described previously herein, to form oxime ester 12, which is then saponified to yield the final product 13. [0074] Scheme 4 shows alternative methods of yielding final product 13 by introducing the oxime functionality at different points of the synthesis described in Scheme 1. Thus, ester 5 can be first reacted with hydroxylamine, or a derivative thereof, as described herein, to form the oxime, which can then be saponified to obtain the final compound 13. Alternatively, acid compound 1 (which was the final product shown in Scheme 1), can be reacted with hydroxylamine, or a derivative thereof, as described herein, to form the oxime final product. [0075] Scheme 5 shows a method of preparing the corresponding oximes of the phenol or thiophenol derivatives of the present invention. The 2-acylphenol or 2- acylthiophenol 7 is reacted with hydroxylamine, or a derivative thereof, such as an alkoxyamine (i.e. CH3ONH2), H2NOSO3H or HON(SO3Na), in the presence of an acid or a base, and then is reacted with the appropriate acid halide in the presence of a Lewis acid, as described previously herein, to obtain oxime 14. Use of an acid is preferable, such as HCl. The oxime is then hydrolyzed under basic conditions, as described previously herein, to form oxime acid 15, which is then coupled to a anthranilic acid derivative using standard peptide coupling methods, as described herein, to yield the final product 16.
[0076] Scheme 6 illustrates a method of preparing the oxime 16 from the corresponding acid 10 (which was the final product in Scheme 2) by reacting it with hydroxylamine, or a derivative thereof, such as an alkoxyamine, H2NOSO3H or HON (SO3NA), in the presence of an acid or base. Use of an acid, such HCl, is preferable.
[0077] Scheme 7 shows the specific synthesis of 2-{[N-(2-acetyl-5-chloro-4- fluorophenyl)glycl] amino} benzoic acid using the general methodology described in Scheme 1.
[0078] The following are examples of the compounds of the present invention. These examples are not intended to encompass the entire scope of this invention and should not be viewed as a limitation of the scope of the invention. Table I
Example # Name Method of
Synthesis
1 2-{[(2-acetyl-4,5- 2 dimethylphenoxy)acetyl] amino } benzoic acid
2 2-{[(2-acetyl-4,5-dimethylphenoxy)acetyl]amino}- 2
5-hydroxybenzoic acid
3 2-{[(2-benzoyl-4- 2 methylphenoxy)acetyl] amino } benzoic acid
4 2-{[(2-acetyl-5- 2 chlorophenoxy)acetyl]amino}benzoic acid
5 2-{[(2-acetyl-4- 2 chlorophenoxy)acetyl]amino}benzoic acid
6 2-{[(4-chloro-2- 2 propionylphenoxy)acetyl] amino } benzoic acid
7 2-{[(2-acetyl-4- 2 fluorophenoxy)acetyl]amino}benzoic acid
8 2-{[(2-acetyl-5- 2 methoxyphenoxy)acetyl] amino } benzoic acid
9 2-{[(2-acetyl-5- 2 fluorophenoxy)acetyl] amino } benzoic acid
10 2-{[(2-acetyl-4-chloro-5- 2 methylphenoxy)acetyl]amino}benzoic acid
11 2-{[(2-acetyl-4-ethyl-3- 2 hydroxyphenoxy)acetyl] amino } benzoic acid
12 2-{[(2-acetyl-4- 2 ethylphenoxy)acetyl] amino } benzoic acid
13 2-{[N-(2-acetyl-4,5- • 1 dimethylphenyl)glycyl] amino } benzoic acid
14 2-{[(2-acetyl-4- 2 methylphenoxy)acetyl]amino}benzoic acid
15 2-{[(2-acetyl-4,5- 2 dimethoxyphenoxy)acetyl] amino } benzoic acid
16 2-[2-(2-acetyl-5-methyl-phenoxy)-acetylamino]- 2 benzoic acid
17 2-{[(4,5-dimethyl-2- 2 propionylphenoxy)acetyl] amino } benzoic acid
18 2-{[(2-butyryl-4,5- 2 dimethylphenoxy)acetyl] amino } benzoic acid
19 2-{[(4,5-dimethyl-2- 2 pentanoylphenoxy)acetyl] amino } benzoic acid
20 2-[2-(4,5-dimethyl-2-propionyl-phenylamino)- 1 acetylamino]-benzoic acid
21 2-{[(2-acetyl-4,5- 2 difluorophenoxy)acetyl] amino } benzoic acid Example # Name Method of
Synthesis
22 2-({[4,5-dimethyl-2- 2
(trifluoroacetyl)phenoxy]acetyl}amino)benzoic acid
23 2-{[(2-acetyl-4-fluoro-5- 2 methylphenoxy)acetyl] amino } benzoic acid
24 2-{[(2-acetyl-5- 2 ethylphenoxy)acetyl] amino } benzoic acid
25 2-{[N-(2-acetyl-4- 2 chlorophenyl)glycyl] amino } benzoic acid
26 2-{[N-(5-chloro-4-methyl-2- 1 propionylphenyl)glycyl] amino } benzoic acid
27 2-{[(2-isobutyryl-4,5- 2 dimethylphenoxy)acetyl] amino } benzoic acid
28 2-{[N-(2-acetyl-4-chloro-5- 1 methylphenyl)glycyl] amino } benzoic acid
29 2-{[N-(2-acetyl-4- 1 bromophenyl)glycyl]amino}benzoic acid
30 2-{[N-(2-acetyl-5-chloro-4- 1 methylphenyl)glycyl] amino } benzoic acid
31 2-{[N-(4-chloro-2- 1 propionylphenyl)glycyl] amino } benzoic acid
33 2-{[N-(2-acetyl-5-chloro-4- 1 fluorophenyl)glycyl] amino } benzoic acid
34 2-{[N-(2-acetyl-4-fluoro-5- 1 methylphenyl)glycyl] amino } benzoic acid
35 2-{[(2-acetyl-4-chloro-5- 1 fluorophenoxy)acetyl] amino } benzoic acid
36 2-{[(2-acetyl-5-chloro-4- 2 fluorophenoxy)acetyl] amino } benzoic acid
37 2-{[N-(2-acetyl-4-chloro-5- 2 fluorophenyl)glycyl] amino } benzoic acid
38 2-{[N-(2-acetyl-4-chloro-5- 1 methoxyphenyl)glycyl] amino } benzoic acid
39 2-{[N-(6-acetyl-l,3-benzodioxol-5- 1 yl)glycyl]amino}benzoic acid
40 2-{[N-(2-acetyl-4,5- 1 dichlorophenyl)glycyl] amino } benzoic acid
41 2-({N-[2-acetyl-4-chloro-5- 1
(methylthio)phenyl]glycyl} amino)benzoic acid
42 2-{[N-(5-chloro-4-fluoro-2- 1 propionylphenyl)glycyl] amino } benzoic acid
43 2-{[N-(2-acetyl-4-chloro-5- 1 ethoxyphenyl)glycyl] amino } benzoic acid
44 2-({N-[2-acetyl-4-chloro-5- 1
(dimethylamino)phenyl] glycyl } amino)benzoic acid
45 2-({N-[2-acetyl-4-chloro-5- 1 Example # Name Method of Synthesis
(methylamino)phenyl] glycyl } amino)benzoic acid
46 2-{[N-(2-acetyl-4-bromo-5- chlorophenyl)glycyl] amino } benzoic acid
47 2-{[N-(2-acetyl-4,5- difluorophenyl)glycyl] amino } benzoic acid
48 2-{[N-(2-acetylphenyl)glycyl]amino}benzoic acid 49 2-{[N-(2-acetyl-5-chloro-4-fluorophenyl)-N- methylglycyl] amino} benzoic acid
50 2-({N-[2-acetyl-4-chloro-5-
(methylsulfinyl)phenyl] glycyl } amino)benzoic acid
BIOLOGY
[0079] The ability of the compounds of the present invention to inhibit Hepatitis C
Polymerase was established by the following experimental procedure:
[0080] NS5B from the BK strain (genotype Ib) is expressed in E. coli as a protein in which the 21 C-terminal amino acids are replaced with a short linker and a hexahistidine tag (GSHHHHHH; SEQ ID NO: 1). The purified protein is mixed with radioactive nucleotides and allowed to replicate a heteropolymeric RNA substrate, primed by an endogenous short hairpin, resulting in an approximately
760 nt product. The radioactive product is captured on a filter and quantitated after removal of the unincorporated nucleotides.
Reagents:
10 mM uridine 5 '-triphosphate (UTP) (Promega # pi 16B)
10 mM adenine 5 '-triphosphate (ATP) (Promega # pi 13B)
10 mM cytidine 5 '-triphosphate (CTP) (Promega # pi 14B)
10 mM guanine 5 '-triphosphate (GTP) (Promega # pi 15B)
Bovine Serum Albumin (BSA) 10 mg/ml NEB (10OX at 10 mg/ml) #007-BSA
RNasein (Promega #N25 IX) 40 U/μl
A-[33P]-GTP (NEN-easytides NEG/606H 3000 Ci/mmol, 370 MBq/ml, 10 mCi/ml)
Falcon polypropylene 96 well plates (Becton Dickinson # 351190) Millipore Multiscreen assasy system-96 well-filtration plate #MADE NOB 50 Optiphase Supermix (Wallac) formulated by Fisher Millipore Multiscreen liner for use in microbeta 1450-106 casette [(Wallac) Perkin
Elmer #1450-433]
1 M (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) (HEPES), pH 7.3 Amersham Pharmacia Biotec (US 16924-500 ml) 1 M MgCl2 (SIGMA #M1028) Dithiothreitol (DTT) (solid) (SIGMA # D9779) RNase free water (GIBCO-BRL #10977-023) Dimethyl sulfoxide (Aldrich #27685-5) Basilen Blue (Sigma, B5520)
0.5M ethylenediaminetetraacetic acid (EDTA), pH 8 (GIBCO-BRL #15575-020) Dibasic sodium phosphate (7-hydrate) (Na2HPO4.7H2O; Baker#3824-07) Phosphoric acid (Baker, #0262.02)
[0081] Further reagent preparation:
- 0.5 M Na Phosphate buffer. Per liter, weigh 134 gr Na2HPO4.7H2O, add water to 900 ml. Adjust pH to 7.0 with phosphoric acid. Top off with water to 1 L.
- Dilute nucleotides 1 : 1000 to 10 μM (GTP and CTP) or 1 : 100 to 100 μM (ATP and UTP) into RNase free water.
[0082] Procedure:
(1) Compounds lOμl at 10 μg/ml in 15 % dimethylsulfoxide (DMSO)
[0083] When starting from 100 μg/ml compound stock in 1% DMSO: Dispense 5 μl 30 % DMSO per well Dispense 5 μl compound (100 μg/ml) per well.
[0084] When starting from 50 μg/ml compound stock in 15 % DMSO: Add 10 μl compound per well. (2) Enzyme Mix:
Total: 20 μl 12 ml
[0085] Add 20 μl enzyme mix into each well of the assay plate. Incubate compound and enzyme at room temperature for 15 minutes
(3) Template mix - prepare ahead
[0086] Spin down a tube of RNA (5μg/tube stored in 75% ethanol and 0.3 M sodium acetate) in a microcentrifuge for 20 minutes at 4 0C. One tube is enough for 1 - 1.5 plates. Remove as much ethanol from the tube as possible by inverting the tube. Be gentle, pellet RNA may not adhere to the tube. Vacuum dry the RNA. Resuspend the RNA by adding 1 ml of DEPC water, close the cap of the tube tightly. To dissolve RNA, incubate RNA solution on ice for -60 minutes and gently vortex. Spin briefly to ensure all RNA solution is down to the bottom of the tube before opening cap. Gently transfer RNA solution into a 5 ml or larger tube. Add another 3 ml of DEPC water (total 4 ml of volume). [0087] Add the following volumes of reagents
[0088] Add 20 μl template mix per reaction (i.e. 20 ng of pOF per reaction or ~3 nM)
(4) Incubate reaction at room temperature (22-25°C) for 2 hours.
(5) Stop reaction by adding 50 μl of 170 mM EDTA.
Final concentration of EDTA is 85 mM.
(6) Prewet filters of Millipore multiscreen assay plate by adding 200 μl of 0.5 M sodium phosphate buffer, pH 7.0 into each well. Let stand at room temperature for 2 - 3 minutes.
(7) Place the multiscreen filter plate onto a Millipore Manifold and turn on vacuum to allow buffer to flow through. Turn off vacuum. Transfer 80 μl of the reaction product into each well of the filter plate. Let stand for 2 - 3 minutes. Turn on vacuum to filter reaction product.
(8) Turn off vacuum. Add 200 μl of 0.5 M sodium phosphate buffer, pH 7.0 into each well to wash filter. Turn on vacuum.
Repeat step (8) three more times. (9) Remove polypropylene bottom. Spot dry filter at the bottom with paper towel. Air dry filter plate on a bench for 1 hour. Add 40 μl Super Mix scintillant. Seal top of the plate with a tape. Place plate into a Packard carrier or micro- beta carrier.
(10) Count plate using a Packard Topcount or micro-beta counter. Count (for example using Program 10) for 33P in Top count or 33P program in micro-beta.
[0089] Percent inhibition is calculated after background subtraction as a percent reduction of activity relative to the positive control (average value of the plate excluding the negative controls). For the primary screen hits were chosen as showing >75 % inhibition.
[0090] See, Ferrari et al. 1999. J. Virology 73: 1649-1654: "Characterization of soluble Hepatitis C virus RNA-dependent RNA polymerase expressed in E. coli and Takamizawa et al 1991" and J. Virology 65:1105-1113: "Structure and characterization of the Hepatitis C virus genome isolated from human carriers," both references are hereby incorporated by reference.
[0091] The compounds of the present invention inhibited Hepatitis C polymerase as summarized in Table II:
Table II Biological Data
Example [M+ 1]+ HCV PoI BK Replicon ELISA MTS
1 342 1.8 43.2 >147
2 358 0.23 136.3 >140
3 390 >6.4
4 348 0.53 21.9 >144
5 348 0.86 53.0 >144
6 332 1.5 111.3 >151
7 344 1.2 44.1 83.2
8 332 3.5 63.9 >151
9 362 0.14 22.1 115.6
10 358 >28 12.9 >140
11 341 0.21 31.9 >147
12 362 0.16 13.7 64.2 Example [M+l]+ HCV Pol BK Replicon ELISA MTS
13 328 2.8 48.1 >153
14 342 25.1 7.8 >147
15 328 1.9 70.8 >153
16 374 2.1
17 356 4.8 >141 >141
18 370 2.4 >136 >136
19 384 0.6 51.8 36.8
20 350 0.3 26.3 >143
21 355 0.9 18.5 87.5
22 396 1.8
23 346 0.4 14.4 >145
24 342 1.0 22.0 >147
25 347 0.23 11.7 >144
26 370 16.2 63.4 >136
27 375 0.8 5.8 44
28 361 0.1 3.1 >139
29 392 0.1 43.3 >128
30 361 3.4
31 365 0.017 2 >137
32 345 7.5
33 366 0.23 13.1 >137
34 366 0.11 11.5 131
35 377 >27
36 357 11.8
37 381 0.11 1.7 61.7
38 365 0.18 16.7 >137
39 393 0.29 12.1 40
40 390 4.1 72.6 >128
41 391 0.14 8.5 122.6
42 376 0.17 21.0 >133
43 358 0.199 13.9 >140
44 379 0.08 4.1 81.0
45 426 0.11 2.0 61.7
46 313 7.9
47 379 0.88
48 409
[0092] The ability of the compounds of the present invention to inhibit Hepatitis C virus replicon constitutively expressed in a human liver cell line was established by the following experimental procedure:
[0093] Clone A cells (licensed from Apath, LLC) are derived from Huh-7 cells (human hepatoma cell line) and constitutively express of the HCV replication proteins with concomitant amplification the HCV replicon (Ib) genome. Cells are maintained and passaged in DMEM/ 10% FCS/ 1 mg/ml G418 (Geneticin from Gibco #1181 1-023; other media components as described below in "elisa media"). Care should be taken to maintain cell monolayers at a subconfluent state by 1 :3 or 1 :4 passages every 3-4 days. The replicon is extremely sensitive to the cellular metabolism/proliferation state and replicon copy number will rapidly decline in confluent monolayers (resting cells). Under ideal conditions each cell has, on average, 1000 copies of the HCV replicon genome.
[0094] Reagents:
Elisa media:
Dulbecco's Modified Eagle Media (DMEM) (Gibco #12430-047)
2% Fetal Calf Serum (FCS) (HyClone #SH30070.03)
IX pen/strep (Gibco #15140-122)
IX Non-essential amino acids (NEAA) (Gibco #11140-050) no G418
Glutaraldehyde (Fisher #02957-4)
TWEEN-20, 10% (Roche #1332465)
TRITON X-100 (Sigma #T-8787)
Superblock in Phosphate Buffered Saline (PBS) (Pierce #37515)
NS 5a monoclonal antibody (Virostat #1873)
Goat antimouse-HRP monoclonal antibody (BioRad #172-1011)
3,3',5,5' tetramethylbenzidine (TMB) substrate (Sigma #T-0440)
[0095] Compound Dilution/Cell Plating:
Drug Plate Preparation (Mother Plate)
10 μl of compounds (in DMSO) are added to column 3 of the mother plate. 5 μl of
DMSO are added to the remaining columns. Mother plates are set aside until ready for serial dilution to be performed.
[0096] Control Drugs
Drug and Cell Addition:
The process for each plate involves:
Prepare cell plates (daughter plates) by adding 52μl of Elisa media to each well.
In Mother plates, serially transfer 50 μl/well from column 3 through column 12. Transfer 8 μl from mother plate to daughter plates (all 96 wells). Place daughter plates in incubator until cells are prepared.
Harvest Clone A cells and plate directly into daughter plates at 0.7x1 O^ cells/ml, lOO μl/well.
All plates are incubated at 37°C in 5% CO2 for 3 days.
[0097] Elisa Assay:
Remove media from 96-well plates (cells should be ca 80% confluent) by flicking into sink.
Add 130 μl/well IX PBS + 0.05% glutaraldehyde.
Incubate 370C for 1 hour.
Remove by flicking into sink.
Wash 3X with 300 μl/well PBS, shaking 5 min each wash. Remove by flicking into sink.
Add 130 μl/well PBS + 0.05% TWEEN-20 + 0.1% TRITON X-100.
Incubate 370C for 10 minutes.
Remove by flicking into sink.
Add 300 μl/well Superblock in PBS.
Incubate 370C for 1 hour.
Remove by flicking into sink.
Wash 3x with 300 μl/well PBS, shaking 5 minutes each wash. Remove by flicking into sink.
During last wash, make a 1 :100 dilution of NS5a Monoclonal-antibody (Mab) in
Superblock + 0.02% TWEEN-20.
After last wash, add 50 μl/well diluted Mab.
Incubate 37°C for 1 hour.
Remove by flicking into sink.
Wash 3X with 300 μl/well PBS + 0.02% TWEEN-20, shaking 5 minutes each wash.
Remove by flicking into sink.
During last wash, make a 1:500 dilution of goat antimouse-HRP Mab in
Superblock + 0.02% TWEEN-20.
After last wash, add 50 μl/well diluted Mab. Incubate 370C for 1 hour.
Remove by flicking into sink.
Wash 5X with 300 μl/well PBS + 0.02% TWEEN-20, shaking 5 minutes each wash. Remove by flicking into sink.
Wash 3X with 300 μl/well PBS, shaking 5 minutes each wash. Remove by flicking into sink.
After last wash, add 130 μl/well room temperature TMB substrate.
Incubate until blue color develops.
Add 130 μl/well IN HCl to stop reaction (color turns from blue to yellow).
Read plates with optical density (O.D.) 450 filter.
ANALYSIS OF RESULTS: IC50 (μM); IC50 (μg/ml); % Inhibition
REFERENCE COMPOUNDS: Interferon-a2; 4-30 U/ml IC50

Claims

WHAT IS CLAIMED:
1. A compound of formula (I) :
(I)
wherein: '
R is selected from the group consisting of -C(O)R6 and wherein R6 and R7 are independently selected from H, C1-C12 alkyl, C3-Ci2 cycloalkyl, C]-Cn heterocycloalkyl, C2-C9 heteroaryl or C6-Ci2 aryl, any of which may be optionally substituted;
G is selected from H or hydroxyl;
Y is O, NH or S;
X is selected from the group consisting of H, halo, Ci -C 12 alkyl, C3-C12 cycloalkyl, C1-C12 perfluoroalkyl, Cj-Ci2 alkoxy, Q-Ci2 alkylthio, amino, Ci-C6 alkylamino, C2-Ci2 dialkylamino, and CN; and n is 0, 1, 2 or 3; or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof.
2. The compound of claim 1, wherein:
R6 and R7are independently selected from H, Ci-C6 alkyl, C3-C12 cycloalkyl, Ci-Cn heterocycloalkyl, C2-C9 heteroaryl or C6-Ci2 aryl, any of which may be optionally substituted; X is selected from the group consisting of H, halo, Ci-C6 alkyl, C3-C12 cycloalkyl, Ci-C11 heterocycloalkyl, C]-C6 perfluoroalkyl, Ci-C6 alkoxy, Ci-C6 alkylthio, amino, Ci-C6 alkylamino, C2-Cn dialkylamino, and CN; and optional substituents are selected from the group consisting Of Ci-C6 alkyl, halogen, Ci-C6 haloalkyl, CpC6 hydroxyalkyl, nitro, amino, hydroxy, cyano, Ci-C6 alkylamino, C2-Ci2 dialkylamino, Ci-C6 alkoxy, Ci-C6 haloalkoxy, C2-C12 alkoxyalkyl, C2-C12 alkoxyalkoxy, oxo, Ci-C6 alkylthio, mercapto, Ci-C6 haloalkylthio, Q-C12 aryl, C6-Ci2 aryloxy, C6-Cn arylthio, C2-C9 heteroaryl, C2-C9 heteroaryloxy, C2-C9 heteroarylthio, C2-C7 acyl, -CO2- Ci -C6 alkyl, -SO3H, - SO2NH2, -SO2NH-Ci-C6 alkyl, -SO2NH-(C1-C6 alkyl)2, -CO2H, -CO2NH2, - CO2NH-Ci-C6 alkyl and -CO2N-(Ci-C6 alkyl)2.
3. The compound of claim 2, wherein:
Y is O or NH; and
X is selected from the group consisting of H, halo, CpC4 alkyl, C1-C4 alkoxy, and C1-C4 haloalkyl.
4. The compound of claim 3, wherein n is O, 1 or 2 and G is H.
5. The compound of claim 4, wherein: R6 is Ci-C4 alkyl;
R7 is Ci -C4 alkyl, when present; and
Y is NH.
6. The compound of claim 5, wherein n is 2 and X is halo.
7. The compound of claim 6, wherein R6 is CH3 and, when present, R7 is CH3.
8. The compound of claim 7, wherein X is selected from Cl and F.
9. The compound of claim 8, wherein one X is Cl and the other X is F.
10. A compound of formula (II):
(H)
wherein:
R1 is selected from -C(O)R6 and , wherein R6 and R7 are independently selected from Ci-C6 alkyl, C3-C12 cycloalkyl, Ci-Cn heterocycloalkyl, C2-Cg heteroaryl or C6-Ci2 aryl, any of which may be optionally substituted;
X is selected from the group consisting of H, halo, alkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, perfluoroalkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 4 carbon atoms, amino, alkylamino of 1 to 4 carbon atoms, dialkylamino of 2 to 8 carbon atoms, and CN; and
G is selected from H or hydroxyl; or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof.
11. The compound of claim 10, wherein R6 and R7 are independently selected from Ci-C6 alkyl, C3-Ci2 cycloalkyl, or C6-Ci2 aryl, any of which may be optionally substituted.
12. The compound of claim 11 , wherein G is H.
13. The compound of claim 12, wherein the compound is: 2-{[N-6-acetyl-l,3-benzodioxol-5-yl)glycyl]amino}benzoic acid; 2-( { [N-[2-acetyl-4-chloro-5-(methylthio)phenyl] glycyl } amino) benzoic acid; 2-({[N-[2-acetyl-4-chloro-5-(dimetheylamino)phenyl]glycyl} amino)benzoic acid; 2-({[N-[2-acetyl-4-chloro-5-(methylamino)phenyl]glycyl}amino) benzoic acid; 2-{[N-(2-acetyl-5-chloro-4-(fluorophenyl)-N-methylglycyl]amino} benzoic acid; and 2-( { [N-[2-acetyl4-chloro-5-(methylsulfinyl)phenyl] glycyl } amino) benzoic acid.
14. The compound of claim 12, wherein X is selected from the group H, halo, Ci -C4 alkyl, C1-C4 perfluoroalkyl and Ci-C4 alkoxy.
15. The compound of claim 14, wherein R6 and R7 are C1-C4 alkyl.
16. The compounds of claim 15, wherein the compound is:
2-{[N-(2-acetyl-4-chloro-5-ethoxyphenyl)glycyl]amino}benzoic acid;
2-{[N-(2-acetyl-4-chloro-5-methoxyphenyl)glycyl]amino}benzoic acid; 2-{[N-(2-acetyl-4-fluoro-5-methylphenyl)glycyl]amino}benzoic acid; 2- {[N-(2-acetylphenyl)glycyl]amino}benzoic acid; 2- { [N-(5-chloro-4-methyl-2-propionylphenyl)glycyl]amino} benzoic acid; 2-[2-(4,5-dimethyl-2-propionyl-phenylamino)-acetylamino]-benoic acid; 2- {[N-(2-acetyl-5-chloro-4-methylphenyl)glycyl]amino}benzoic acid; and 2-{[N-(2-acetyl-4,5-dimethylphenyl)glycyl]amino}benzoic acid.
17. The compound of claim 15, wherein X is halo, if present.
18. The compound of claim 17, wherein n is 0, 1, or 2.
19. The compound of claim 18, wherein the compound is:
2- {[N-(2-acetyl-4-bromo-5-choloro phenyl)glycyl]amino}benzoic acid; 2-{[N-(5-chloro-4-fluoro-2-propionylphenyl)glycyl]amino}benzoic acid; 2- {[N-(2-acetyl-4,5-difluorophenyl)glycyl]amino}benzoic acid; 2-{[N-(2-acetyl-4-chloro-5-fluorophenyl)glycyl]amino}benzoic acid; 2- {[N-(2-acetyl-4,5-dichlorophenyl)glycyl]amino}benzoic acid; 2- { [N-(2-acetyl-5-chloro-4-fluorophenyl)glycyl]amino}benzoic acid; 2- {[N-(4-chloro-2-propionylphenyl)glycyl]amino}benzoic acid; 2-{[N-(2-acetyl-4-chloro-5-methylphenyl)glycyl]amino}benzoic acid; 2-{[N-(2-acetyl-4-chlorophenyl)glycyl]amino}benzoic acid; and 2- {[N-(2-acetyl-4-bromophenyl)glycyl]amino}benzoic acid.
20. The compound of claim 15, wherein the compound is 2-{[N-(2-acetyl-5- chloro-4-fluorophenyl)glycyl] amino } benzoic acid.
21. The compound of claim 15, wherein the compound is 2-(2-([5-chloro-4- fluoro-2-( 1 -methoxyimino-ethyl)-phenylamino]acetylamino}benzoic acid.
22. A compound of formula (III):
(m)
wherein: ?
R1 is selected from -C(O)R6 and , wherein R6 and R7 are independently selected from Ci-C6 alkyl, C3-Ci2 cycloalkyl, Ci-Cn heterocycloalkyl, C2-C9 heteroaryl or C6-Ci2 aryl, any of which may be optionally substituted;
X is selected from the group consisting of H, halo, alkyl of 1 to 4 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, C perfluoroalkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, alkylthio of 1 to 6 carbon atoms, amino, alkylaminol to 6 carbon atoms, dialkylamino of 2 to 12 carbon atoms, and CN; and
G is selected from H or hydroxyl; or a prodrug, a pharmaceutically acceptable salt, or a pharmaceutically active metabolite thereof.
23. The compound of claim 22, wherein R6 and R7 are independently selected from Ci-C6 alkyl, C3-Ci2 cycloalkyl and C6-C12 aryl, any of which may be optionally substituted.
24. The compound of claim 23, wherein G is H.
25. The compound of claim 24, wherein the compound is 2- {[(2-acetyl-5- methylphenoxy)acetyl] amino} benzoic acid.
26. The compound of claim 24, wherein X is selected from the group consisting of H, halo, Ci -C4 alkyl, Ci-C4 perfluoroalkyl and Ci-C4 alkoxy.
27. The compound of claim 26, wherein R6 and R7 are Ci-C4 alkyl.
28. The compounds of claim 27, wherein the compound is:
2- {[(2-acetyl-5-ethylphenoxy)acetyl]amino}benzoic acid;
2- { [(4,5-dimethyl-2-pentanoyphenoxy)acetyl]amino}benzoic acid;
2- { [(2-butyryl-4,5-dimethylphenoxy)acetyl]amino}benzoic acid;
2- {[(4,5-dimethyl-2-propionyphenoxy)acetyl]amino}benzoic acid;
2-({[4,5-dimethyl-2-(trifluoroacetyl)phenoxy]acetyl}amino} benzoic acid;
2- {[(2-acetyl-4,5-dimethoxyphenoxy)acetyl]amino}benzoic acid;
2- {[(2-acetyl-5-methylphenoxy)acetyl]amino}benzoic acid;
2- {[(2-acetyl-4-ethylphenoxy)acetyl] amino} benzoic acid;
2-{[(2-acetyl-4-methylphenoxy)acetyl]amino}benzoic acid;
2- {[(2-acetyl-5-methoxyphenoxy)acetyl]amino}benzoic acid;
2- {[(2-benzoyl4-methylphenoxy)acetyl]amino}benzoic acid;
2- {[(2-acetyl-4,5-dimethylphenoxy)acetyl]amino} -5-hydroxybenzoic acid;
2-{[(2-acetyl-4,5-dimethylphenoxy)acetyl]amino}benzoic acid; and
2- {[(2-isobutyryl-4,5-dimethylphenoxy)acetyl]amino}benzoic acid.
29. The compound of claim 27, wherein n is 1, 2 or 3 and at least one X is halo.
30. The compound of claim 29, wherein n is 1 or 2.
31. The compound of claim 30, wherein the compound is:
2- {[(2-acetyl-4-chloro-5-fluorophenoxy)acetyl]amino}benzoic acid; 2- { [(2-acetyl-5-chloro-4-fluorophenoxy)acetyl]amino}benzoic acid; 2- {[(2-acetyl-4-fluoro-5-methylphenoxy)acetyl]amino}benzoic acid; 2- {[(2-acetyl-4,5-difluorophenoxy)acetyl]amino}benzoic acid; 2- {[(4-chloro-2-propionylphenoxy)acetyl]amino}benzoic acid; , 2- { [(2-acetyl-4-chloro-5-methylphenoxy)acetyl]amino}benzoic acid; 2- {[(2-acetyl-5-fluorophenoxy)acetyl] amino} benzoic acid; 2- { [(2-acetyl-5-chlorophenoxy)acetyl]amino}benzoic acid; 2-{[(2-acetyl-4-fluorophenoxy)acetyl]amino}benzoic acid; and 2- {[(2-acetyl-4-chlorophenoxy)acetyl]amino}benzoic acid.
32. A method of inhibiting replication of a Hepatitis C virus comprising contacting a Hepatitis C virus with an effective amount of at least one compound from one of claims 1, 10 or 22.
33. The method of claim 32, further comprising contacting the Hepatitis C virus with at least one biologically active agent selected from the group consisting of interferon, a pegylated interferon, ribavirin, protease inhibitors, polymerase inhibitors, small interfering RNA compounds, anti-sense compounds, nucleotide analogs, nucleoside analogs, immunoglobulins, immunomodulators, hepatoprotectants, anti-inflammatory agents, antibiotics, antivirals, and anti- infective compounds.
34. A method of treating or preventing a Hepatitis C viral infection in a mammal comprising providing the mammal with an effective amount of at least one compound from one of claims 1, 10 or 22.
35. The method of claim 34, further comprising providing the mammal with at least one biologically active agent selected from the group consisting of interferon, a pegylated interferon, ribavirin, protease inhibitors, polymerase inhibitors, small interfering RNA compounds, anti-sense compounds, nucleotide analogs, nucleoside analogs, immunoglobulins, immunomodulators, hepatoprotectants, antiinflammatory agents, antibiotics, antivirals, and anti-infective compounds.
36. A method of inhibiting the Hepatitis C RNA-dependent RNA polymerase NS5B, comprising contacting a cell with an effective amount of a compound of claim 1 to decrease or prevent NS5B function.
37. A pharmaceutical composition, comprising a compound from one of claims 1 , 10 or 22, and a pharmaceutically acceptable carrier.
38. A method of treating or preventing a Hepatitis C viral infection in a mammal, comprising providing the mammal with an effective amount of at least one pharmaceutical composition of claim 37.
39. The method of claim 38, further comprising providing the mammal with an effective amount of at least one biologically active agent selected from the group consisting of interferon, a pegylated interferon, ribavirin, protease inhibitors, polymerase inhibitors, small interfering RNA compounds, anti-sense compounds, nucleotide analogs, nucleoside analogs, immunoglobulins, immunomodulators, hepatoprotectants, anti-inflammatory agents, antibiotics, antivirals, and anti- infective compounds.
EP06851777A 2005-12-01 2006-12-01 Anthranilic acid derivatives useful in treating infection with hepatitis c virus Withdrawn EP1966181A2 (en)

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