Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • 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

Definitions

• the present invention is concerned with carbocyclic nucleoside compounds and certain derivatives thereof, their synthesis, and their use as inhibitors of RNA-dependent RNA viral polymerase.
• the compounds of the present invention are inhibitors of RNA-dependent RNA viral replication and are useful for the treatment of RNA-dependent RNA viral infection. They are particularly useful as inhibitors of hepatitis C virus (HCV) NS5B polymerase, as inhibitors of HCV replication, and for the treatment of hepatitis C infection.
• HCV hepatitis C virus
• Hepatitis C virus (HCV) infection is a major health problem that leads to chronic liver disease, such as cirrhosis and hepatocellular carcinoma, in a substantial number of infected individuals, estimated to be 2-15% of the world's population.
• HCV Hepatitis C virus
• World Health Organization there are more than 200 million infected individuals worldwide, with at least 3 to 4 million people being infected each year. Once infected, about 20% of people clear the virus, but the rest harbor HCV the rest of their lives.
• Ten to twenty percent of chronically infected individuals eventually develop liver-destroying cinhosis or cancer.
• the viral disease is transmitted parenterally by contaminated blood and blood products, contaminated needles, or sexually and vertically from infected mothers or carrier mothers to their off-spring.
• Cunent treatments for HCV infection which are restricted to immunotherapy with recombinant interferon- ⁇ or pegylated interferon- ⁇ , alone or in combination with the nucleoside analog ribavirin, are effective in only about 50% of the infected population (J. Gillis, "Doctors Close in on Hepatitis C Suppression, Data Show," Washington Post, Thursday, April 18,
• RNA-dependent RNA polymerase RNA-dependent RNA polymerase
• the HCV virion is an enveloped positive-strand RNA virus with a single oligoribonucleotide genomic sequence of about 9600 bases which encodes a polyprotein of about 3,010 amino acids.
• the protein products of the HCV gene consist of the structural proteins C, El, and E2, and the non-structural proteins NS2, NS3, NS4A and NS4B, and NS5A and NS5B.
• the nonstructural (NS) proteins are believed to provide the catalytic machinery for viral replication.
• the NS3 protease releases NS5B, the RNA-dependent RNA polymerase from the polyprotein chain.
• HCV NS5B polymerase is required for the synthesis of a double-stranded RNA from a single-stranded viral RNA that serves as a template in the replication cycle of HCV.
• NS5B polymerase is therefore considered to be an essential component in the HCV replication complex [see K. Ishi, et al., "Expression of Hepatitis C Virus NS5B Protein: Characterization of Its RNA Polymerase Activity and RNA Binding,"
• carbocyclic nucleoside compounds of the present invention and certain derivatives thereof are potent inhibitors of RNA- dependent RNA viral replication and in particular HCV replication.
• the 5'- triphosphate derivatives of these carbocyclic nucleoside compounds are inhibitors of RNA-dependent RNA viral polymerase and in particular HCV NS5B polymerase.
• the instant carbocyclic nucleoside compounds and derivatives thereof are useful to treat RNA-dependent RNA viral infection and in particular HCV infection.
• compositions comprising the carbocyclic nucleoside compounds and derivatives thereof of the present invention for use as inhibitors of RNA-dependent RNA viral polymerase and in particular as inhibitors of HCV NS5B polymerase.
• compositions comprising the carbocyclic nucleoside compounds and derivatives thereof of the present invention for use as inhibitors of RNA-dependent RNA viral replication and in particular as inhibitors of HCV replication.
• Rl is C2-4 alkenyl, C2-4 alkynyl, or Ci_4 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino, C ⁇ _4 alkoxy, C ⁇ _4 alkylthio, or one to three fluorine atoms;
• R2 is hydrogen, fluorine, amino, hydroxy, mercapto, Ci_4 alkoxy, C ⁇ _8 alkylcarbonyloxy, or C ⁇ _4 alkyl;
• R3 and R4 are each independently selected from the group consisting of hydrogen, cyano, azido, halogen, hydroxy, mercapto, amino, Ci_4 alkoxy, Ci_8 alkylcarbonyloxy, C2-4 alkenyl, C2-4 alkynyl, and Ci-4 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino, C ⁇ _4 alkoxy, Ci-4 alkylthio, or one to three fluorine atoms;
• R5 is hydrogen, CMO alkylcarbonyl, P3O9H4, P2O6H3, or P(O)R13R14 ;
• R6 and R7 are each independently hydrogen, methyl, hydroxymethyl, or fluoromethyl;
• R8 is hydrogen, C1.4 alkyl, C2-4 alkynyl, halogen, cyano, carboxy, Ci_4 alkyloxycarbonyl, azido, amino, Ci-4 alkylamino, di(C ⁇ _4 alkyl)amino, hydroxy, Ci-6 alkoxy, Ci-6 alkylthio, C ⁇ -6 alkylsulfonyl, or (Ci-4 alkyl) ⁇ -2 aminomethyl;
• n 0, 1, or 2;
• Rll is hydrogen, hydroxy, halogen, C ⁇ _4 alkoxy, amino, Ci_4 alkylamino, di(C ⁇ _4 alkyl)amino, C3-6 cycloalkylamino, or di(C3_6 cycloalkylamino); each Rl2 is independently hydrogen or C . alkyl; Rl7, Rl8 5 and Rl9 are each independently hydrogen or C ⁇ . alkyl;
• Rl5 is hydrogen, C ⁇ _6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C ⁇ -4 alkylamino, CF3, or halogen;
• R20 i hydrogen, Ci-4 alkyl, or phenyl C ⁇ -2 alkyl; with the proviso that when B is
• X is CH2; Y is N; RlO is NH2; R 2 and R3 are ⁇ -OH; and R4, R5, R6, R7, R8, an d Rll are hydrogen, then R 1 is not ⁇ -methyl.
• the compounds of formula I are useful as inhibitors of RNA-dependent RNA viral polymerase and in particular of HCV NS5B polymerase. They are also inhibitors of RNA-dependent RNA viral replication and in particular of HCV replication and are useful for the treatment of RNA-dependent RNA viral infection and in particular for the treatment of HCV infection.
• compositions containing the compounds alone or in combination with other agents active against RNA-dependent RNA virus and in particular against HCV as well as methods for the inhibition of RNA-dependent RNA viral replication and for the treatment of RNA-dependent RNA viral infection.
• the present invention relates to compounds of structural formula I of the indicated stereochemical configuration:
• n 0, 1, or 2;
• B is
• R2 is hydrogen, fluorine, amino, hydroxy, mercapto, Ci_4 alkoxy, C ⁇ _8 alkylcarbonyloxy, or C 1-4 alkyl;
• R and R4 are each independently selected from the group consisting of hydrogen, cyano, azido, halogen, hydroxy, mercapto, amino, C ⁇ _4 alkoxy, C ⁇ _8 alkylcarbonyloxy, C2-4 alkenyl, C2-4 alkynyl, and C ⁇ _4 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino, C ⁇ -4 alkoxy, Ci-4 alkylthio, or one to three fluorine atoms;
• R5 is hydrogen, C ⁇ _ ⁇ o alkylcarbonyl, P3O9H4, P2O6H3, or P(O)R13R14 ;
• R6 and R7 are each independently hydrogen, methyl, hydroxymethyl, or fluoromethyl
• R8 is hydrogen, Ci-4 alkyl, C2-4 alkynyl, halogen, cyano, carboxy, Ci-4 alkyloxycarbonyl, azido, amino, Ci-4 alkylamino, di(Ci_4 alkyl)amino, hydroxy, Ci-6 alkoxy, C ⁇ _6 alkylthio, C ⁇ _6 alkylsulfonyl, or (Ci_4 alkyl) ⁇ -2 aminomethyl
• Rll is hydrogen, hydroxy, halogen, C ⁇ _4 alkoxy, amino, C ⁇ _4 alkylamino, di(C ⁇ _4 alkyl)amino, C3_6 cycloalkylamino, or di(C3_6 cycloalkylamino); each Rl2 is independently hydrogen or Ci_6 alkyl;
• Rl7, Rl8 5 and Rl9 are each independently hydrogen or C ⁇ -6 alkyl
• Rl5 is hydrogen, C ⁇ _6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C ⁇ _4 alkylamino, CF3, or halogen;
• R20 is hydrogen, C ⁇ _4 alkyl, or phenyl C ⁇ -2 alkyl; with the proviso that when B is
• X is CH2; Y is N; RlO is NH2; R 2 and R are ⁇ -OH; and R4, R5, R6, R7, R8, and Rll are hydrogen, then Rl is not ⁇ -methyl.
• the compounds of formula I are useful as inhibitors of RNA-dependent RNA viral polymerase. They are also inhibitors of RNA-dependent RNA viral replication and are useful for the treatment of RNA-dependent RNA viral infection.
• B is
• Rl is C ⁇ _3 alkyl, wherein alkyl is unsubstituted or substituted with one to three fluorine atoms;
• R2 is hydroxy, fluoro, Ci_3 alkoxy, or C ⁇ _8 alkylcarbonyloxy
• R3 is hydrogen, halogen, hydroxy, amino, C ⁇ _3 alkoxy, or Ci_8 alkylcarbonyloxy
• R5 is hydrogen, Cl_8 alkylcarbonyl, P3O9H4, P2O6H3, or PO3H2
• R8 is hydrogen, amino, or Ci-4 alkylamino
• RlO and Rll are each independently hydrogen, halogen, hydroxy, amino, Ci-4 alkylamino, di(C ⁇ _4 alkyl)amino, or C3-.6 cycloalkylamino; with the proviso that when RlO is NH2, R and R3 are ⁇ -OH, and R5, R8, and RU are hydrogen, then Rl is not ⁇ -methyl.
• RlO is NH2
• R and R3 are ⁇ -OH
• R5, R8, and RU are hydrogen, then Rl is not ⁇ -methyl.
• Rl is methyl, fluoromethyl, difiuoromethyl, or trifluoromethyl
• R2 is hydroxy, fluoro, or methoxy
• R3 is hydrogen, fluoro, hydroxy, amino, or methoxy
• R5 is hydrogen or P3O9H4; R8 is hydrogen or amino; and
• RlO and Rll are each independently hydrogen, fluoro, hydroxy, or amino; with the proviso that when RlO is NH2, R2 and R3 are ⁇ -OH, and R5, R8, and Rll are hydrogen, then Rl is not ⁇ -methyl.
• Rl is C1-.3 alkyl, wherein alkyl is unsubstituted or substituted with one to three fluorine atoms;
• R2 is hydroxy, fluoro, C ⁇ _3 alkoxy, or C _8 alkylcarbonyloxy
• R3 is hydrogen, halogen, hydroxy, amino, Cl-3 alkoxy, or Ci_8 alkylcarbonyloxy
• R5 is hydrogen, Ci-8 alkylcarbonyl, P3O9H4, P2O6H3, or PO3H2
• R8 is hydrogen, amino, or Ci-4 alkylamino
• R9 is hydrogen, cyano, methyl, halogen, CONH2 or CSNH2;
• RlO and Rll are each independently hydrogen, halogen, hydroxy, amino, C1-.4 alkylamino, di(C ⁇ _4 alkyl)amino, or C3-6 cycloalkylamino.
• Rl is methyl, fluoromethyl, difluoromethyl, or trifluoromethyl
• R2 is hydroxy, fluoro, or methoxy
• R3 is hydrogen, fluoro, hydroxy, amino, or methoxy
• R5 is hydrogen or P3O9H4;
• R8 is hydrogen or amino
• R9 is hydrogen, cyano, methyl, halogen, CONH2 or CSNH2;
• RlO and RU are each independently hydrogen, fluoro, hydroxy, or amino.
• Rl is C ⁇ _3 alkyl, wherein alkyl is unsubstituted or substituted with one to three fluorine atoms; R is hydroxy, fluoro, C ⁇ _3 alkoxy, or C ⁇ _8 alkylcarbonyloxy;
• R3 is hydrogen, halogen, hydroxy, amino, C ⁇ _3 alkoxy, or C ⁇ _8 alkylcarbonyloxy
• R5 is hydrogen, C ⁇ _8 alkylcarbonyl, P3O9H4, P2O6H3, or PO3H2
• R8 is hydrogen, amino, or C ⁇ _4 alkylamino
• RlO and Rll are each independently hydrogen, halogen, hydroxy, amino, Ci-4 alkylamino, di(C _4 alkyl)amino, or C3-6 cycloalkylamino.
• Rl is methyl, fluoromethyl, difluoromethyl, or trifluoromethyl
• R2 is hydroxy, fluoro, or methoxy
• R3 is hydrogen, fluoro, hydroxy, amino, or methoxy
• R5 is hydrogen or P3O9H4;
• R8 is hydrogen or amino
• RlO and RU are each independently hydrogen, fluoro, hydroxy, or amino.
• Rl is Cl-3 alkyl, wherein alkyl is unsubstituted or substituted with one to three fluorine atoms;
• R2 is hydroxy, fluoro, Ci_3 alkoxy, or Ci-8 alkylcarbonyloxy
• R3 is hydrogen, halogen, hydroxy, amino, C ⁇ _3 alkoxy, or C ⁇ _8 alkylcarbonyloxy;
• R5 is hydrogen, Ci_8 alkylcarbonyl, P3O9H4, P2O6H3, or PO3H2;
• R8 is hydrogen, amino, or C ⁇ _4 alkylamino
• R9 is hydrogen, cyano, methyl, halogen, CONH2 or CSNH2;
• RlO and Rll are each independently hydrogen, halogen, hydroxy, amino, Ci-4 alkylamino, di(Ci-4 alkyl)amino, or C3..6 cycloalkylamino.
• Rl is methyl, fluoromethyl, difluoromethyl, or trifluoromethyl
• R2 is hydroxy, fluoro, or methoxy
• R is hydrogen, fluoro, hydroxy, amino, or methoxy
• R5 is hydrogen or P3O9H4;
• R8 is hydrogen or amino
• R9 is hydrogen, cyano, methyl, halogen, CONH2 or CSNH2;
• RlO and Rll are each independently hydrogen, fluoro, hydroxy, or amino.
• RNA-dependent RNA viral polymerase examples of compounds of the present invention of structural formula I which are useful as inhibitors of RNA-dependent RNA viral polymerase are the following:
• the carbocyclic nucleoside compounds of the present invention are useful as inhibitors of positive-sense single- stranded RNA-dependent RNA viral polymerase, inhibitors of positive-sense single- stranded RNA-dependent RNA viral replication, and/or for the treatment of positive- sense single-stranded RNA-dependent RNA viral infection.
• the positive-sense single-stranded RNA-dependent RNA virus is a Flaviviridae virus or a Picornaviridae virus.
• the Picornaviridae virus is a rhinovirus, a poliovirus, or a hepatitis A virus.
• the Flaviviridae virus is selected from the group consisting of hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, Japanese encephalitis virus, Banzi virus, and bovine viral diarrhea virus (BVDV).
• the Flaviviridae virus is hepatitis C virus.
• Another aspect of the present invention is concerned with a method for inhibiting RNA-dependent RNA viral polymerase, a method for inhibiting RNA- dependent RNA viral replication, and/or a method for treating RNA-dependent RNA viral infection in a mammal in need thereof comprising administering to the mammal a therapeutically effective amount of a compound of structural formula I.
• the RNA- dependent RNA viral polymerase is a positive-sense single-stranded RNA-dependent RNA viral polymerase.
• the positive-sense single- stranded RNA-dependent RNA viral polymerase is a Flaviviridae viral polymerase or a Picornaviridae viral polymerase.
• the Picornaviridae viral polymerase is rhinovirus polymerase, poliovirus polymerase, or hepatitis A virus polymerase.
• the Flaviviridae viral polymerase is selected from the group consisting of hepatitis C virus polymerase, yellow fever virus polymerase, dengue virus polymerase, West Nile virus polymerase, Japanese encephalitis virus polymerase, Banzi virus polymerase, and bovine viral diantiea virus (BVDV) polymerase.
• the Flaviviridae viral polymerase is hepatitis C virus polymerase.
• the RNA-dependent RNA viral replication is a positive-sense single-stranded RNA- dependent RNA viral replication
• the positive-sense single-stranded RNA-dependent RNA viral replication is Flaviviridae viral replication or Picornaviridae viral replication.
• the Picornaviridae viral replication is rhinovirus replication, poliovirus replication, or hepatitis A virus replication.
• the Flaviviridae viral replication is selected from the group consisting of hepatitis C virus replication, yellow fever virus replication, dengue virus replication, West Nile virus replication, Japanese encephalitis virus replication, Banzi virus replication, and bovine viral diarehea virus replication.
• the Flaviviridae viral replication is hepatitis C virus replication.
• the RNA- dependent RNA viral infection is a positive-sense single-stranded RNA-dependent viral infection.
• the positive-sense single-stranded RNA-dependent RNA viral infection is Flaviviridae viral infection or Picornaviridae viral infection.
• the Picornaviridae viral infection is rhinovirus infection, poliovirus infection, or hepatitis A virus infection.
• the Flaviviridae viral infection is selected from the group consisting of hepatitis C virus infection, yellow fever virus infection, dengue virus infection, West Nile virus infection, Japanese encephalitis virus infection, Banzi virus infection, and bovine viral dianhea virus infection.
• the Flaviviridae viral infection is hepatitis C virus infection.
• alkyl groups specified above are intended to include those alkyl groups of the designated length in either a straight or branched configuration.
• exemplary of such alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl, isohexyl, and the like.
• alkenyl shall mean straight or branched chain alkenes of two to six total carbon atoms, or any number within this range (e.g., ethenyl, propenyl, butenyl, pentenyl, etc.).
• alkynyl shall mean straight or branched chain alkynes of two to six total carbon atoms, or any number within this range (e.g., ethynyl, propynyl, butynyl, pentynyl, etc.).
• cycloalkyl shall mean cyclic rings of alkanes of three to eight total carbon atoms, or any number within this range (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl).
• cycloheteroalkyl is intended to include non-aromatic heterocycles containing one or two heteroatoms selected from nitrogen, oxygen and sulfur.
• 4-6-membered cycloheteroalkyl include azetidinyl, pynolidinyl, piperidinyl, mo ⁇ holinyl, thiamo ⁇ holinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, piperazinyl, and the like.
• alkoxy refers to straight or branched chain alkoxides of the number of carbon atoms specified (e.g., C ⁇ _4 alkoxy), or any number within this range [i.e., methoxy (MeO-), ethoxy, isopropoxy, etc.].
• alkylthio refers to straight or branched chain alkylsulfides of the number of carbon atoms specified (e.g., Ci-4 alkylthio), or any number within this range [i.e., methylthio (MeS-), ethylthio, isopropylthio, etc.].
• alkylamino refers to straight or branched alkylamines of the number of carbon atoms specified (e.g., C ⁇ _4 alkylamino), or any number within this range [i.e., methylamino, ethylamino, isopropylamino, t-butylamino, etc.].
• cycloalkylamino refers to saturated aminohydrocarbons containing one ring of the number of carbon atoms specified (e.g., C3-6 cycloalkylamino), or any number within this range [i.e., cyclopropylamino, cyclobutylamino, cyclopentylamino, and cyclohexylamino].
• alkylsulfonyl refers to straight or branched chain alkylsulfones of the number of carbon atoms specified (e.g., Ci_6 alkylsulfonyl), or any number within this range [i.e., methylsulfonyl (MeSO2-), ethylsulfonyl, isopropylsulfonyl, etc.].
• alkyloxycarbonyl refers to straight or branched chain esters of a carboxylic acid derivative of the present invention of the number of carbon atoms specified (e.g., Ci-4 alkyloxycarbonyl), or any number within this range [i.e., methyloxycarbonyl (MeOCO-), ethyloxycarbonyl, or butyloxycarbonyl].
• aryl includes both phenyl, naphthyl, and pyridyl.
• the phenyl, naphthyl, or pyridyl group wherever it occurs in the compounds of the present invention is optionally substituted with one to three groups independently selected from Ci-4 alkyl, halogen, cyano, nitro, trifluoromethyl, C ⁇ _4 alkoxy, and C ⁇ _4 alkylthio.
• halogen is intended to include the halogen atoms fluorine, chlorine, bromine and iodine.
• substituted shall be deemed to include multiple degrees of substitution by a named substituent. Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally.
• amino acyl residue refers to an ⁇ -, ⁇ -, or ⁇ -amino acyl group of structural formula
• n 0, 1, or 2 and Rl7, Rl8 ; R19 ; and R20 are as defined hereinabove.
• R20 i not hydrogen, the amino acyl residue contains an asymmetric center and is intended to include the individual R- and S-enantioners as well as RS-racemic mixtures.
• 5'-triphosphate refers to a triphosphoric acid ester derivative of the 5 '-hydroxyl group of a carbocyclic nucleoside compound of the present invention having the following general structural formula:
• B and R1-RH are as defined above.
• the compounds of the present invention are also intended to include pharmaceutically acceptable salts of the triphosphate ester as well as pharmaceutically acceptable salts of 5'-monophosphate and 5'-diphosphate ester derivatives of the structural formulae A and B, respectively,
• 5'-(S-acyl-2-thioethyl)phosphate or "SATE” refers to a mono- or di-ester derivative of a 5'-monophosphate carbocyclic nucleoside derivative of the present invention of structural formulae C and D, respectively, as well as pharmaceutically acceptable salts of the mono-ester,
• composition as in “pharmaceutical composition,” is intended to encompass a product comprising the active ingredient(s) and the inert ingredient(s) that make up the can ⁇ er, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.
• pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.
• administering a should be understood to mean providing a compound of the invention or a prodrug of a compound of the invention to the individual in need.
• Another aspect of the present invention is concerned with a method of inhibiting HCN ⁇ S5B polymerase, inhibiting HCV replication, or treating HCV infection with a compound of the present invention in combination with one or more agents useful for treating HCV infection.
• agents active against HCV include, but are not limited to, ribavirin, levovirin, viramidine, thymosin alpha- 1, interferon- ⁇ , interferon- ⁇ , pegylated interferon- ⁇ (peginterferon- ⁇ ), a combination of interferon- ⁇ and ribavirin, a combination of peginterferon- ⁇ and ribavirin, a combination of interferon- ⁇ and levovirin, and a combination of peginterferon- ⁇ and levovirin.
• Interferon- ⁇ includes, but is not limited to, recombinant interferon- ⁇ 2a (such as Roferon interferon available from Hoffmann-LaRoche, Nutley, NJ), pegylated interferon- ⁇ 2a (PegasysTM), interferon- ⁇ 2b (such as Intron-A interferon available from Schering Co ⁇ ., Kenilworth, NJ), pegylated interferon- ⁇ 2b (PeglntronTM), a recombinant consensus interferon (such as interferon alphacon-1), and a purified interferon- ⁇ product.
• Amgen's recombinant consensus interferon has the brand name Infergen®.
• Levovirin is the L-enantiomer of ribavirin which has shown immunomodulatory activity similar to ribavirin.
• Viramidine represents an analog of ribavirin disclosed in WO 01/60379 (assigned to ICN Pharmaceuticals).
• the individual components of the combination can be administered separately at different times during the course of therapy or concunently in divided or single combination forms.
• the instant invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment, and the term “administering" is to be inte ⁇ reted accordingly. It will be understood that the scope of combinations of the compounds of this invention with other agents useful for treating HCV infection includes in principle any combination with any pharmaceutical composition for treating HCV infection.
• the dose of each compound may be either the same as or different from the dose when the compound is used alone.
• the compounds of the present invention may also be administered in combination with an agent that is an inhibitor of HCV NS3 serine protease.
• HCV NS3 serine protease is an essential viral enzyme and has been described to be an excellent target for inhibition of HCV replication.
• HCV NS3 protease inhibitors Both substrate and non-substrate based inhibitors of HCV NS3 protease inhibitors are disclosed in WO 98/22496, WO 98/46630, WO 99/07733, WO 99/07734, WO 99/38888, WO 99/50230, WO 99/64442, WO 00/09543, WO 00/59929, and GB- 2337262.
• HCV NS3 protease as a target for the development of inhibitors of HCV replication and for the treatment of HCV infection is discussed in B.W. Dymock, "Emerging therapies for hepatitis C virus infection," Emerging Drugs, 6: 13-42 (2001).
• Ribavirin, levovirin, and viramidine may exert their anti-HCV effects by modulating intracellular pools of guanine nucleotides via inhibition of the intracellular enzyme inosine monophosphate dehydrogenase (IMPDH).
• IMPDH inosine monophosphate dehydrogenase
• Ribavirin is readily phosphorylated intracellularly and the monophosphate derivative is an inhibitor of IMPDH.
• inhibition of IMPDH represents another useful target for the discovery of inhibitors of HCV replication.
• the compounds of the present invention may also be administered in combination with an inhibitor of IMPDH, such as VX-497, which is disclosed in WO 97/41211 and WO 01/00622 (assigned to Vertex); another LMPDH inhibitor, such as that disclosed in WO 00/25780 (assigned to Bristol-Myers Squibb); or mycophenolate mofetil [see A.C. Allison and E.M. Eugui, Agents Action, 44 (Suppl.): 165 (1993)].
• an inhibitor of IMPDH such as VX-497, which is disclosed in WO 97/41211 and WO 01/00622 (assigned to Vertex)
• another LMPDH inhibitor such as that disclosed in WO 00/25780 (assigned to Bristol-Myers Squibb)
• mycophenolate mofetil see A.C. Allison and E.M. Eugui, Agents Action, 44 (Suppl.): 165 (1993)].
• the compounds of the present invention may also be administered in combination with the antiviral agent amantadine (1-aminoadamantane) [for a comprehensive description of this agent, see J. Kirschbaum, Anal. Profiles Drug Subs. 12: 1-36 (1983)].
• the compounds of the present invention may also be combined for the treatment of HCV infection with antiviral 2'-C-branched ribonucleosides disclosed in R. E. Hany-O'kuru, et al., J. Org. Chem.. 62: 1754-1759 (1997); M. S. Wolfe, et al., Tetrahedron Lett.. 36: 7611-7614 (1995); U.S. Patent No. 3,480,613 (Nov. 25, 1969); International Publication Number WO 01/90121 (29 November 2001); International Publication Number WO 01/92282 (6 December 2001); and International Publication Number WO 02/32920 (25 April 2002); the contents of each of which are inco ⁇ orated by reference in their entirety.
• Such 2'-C-branched ribonucleosides include, but are not limited to, 2'-C-methyl-cytidine, 2'-C-methyl-uridine, 2'-C- methyl-adenosine, 2'-C-methyl-guanosine, and 9-(2-C-methyl- ⁇ -D-ribofura ⁇ osyl)- 2,6-diaminopurine.
• pharmaceutically acceptable is meant that the carrier, diluent, or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• compositions comprising the carbocyclic nucleoside compounds and derivatives thereof of the present invention in association with a pharmaceutically acceptable carrier.
• a pharmaceutical composition made by combining any of the compounds described above and a pharmaceutically acceptable carrier.
• Another illustration of the invention is a process for making a pharmaceutical composition comprising combining any of the compounds described above and a pharmaceutically acceptable carrier.
• compositions useful for inhibiting RNA-dependent RNA viral polymerase in particular HCV NS5B polymerase comprising an effective amount of a compound of the present invention and a pharmaceutically acceptable carrier.
• Pharmaceutical compositions useful for treating RNA-dependent RNA viral infection in particular HCV infection are also encompassed by the present invention as well as a method of inhibiting RNA-dependent RNA viral polymerase in particular HCV NS5B polymerase and a method of treating RNA-dependent viral replication and in particular HCV replication.
• the present invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention in combination with a therapeutically effective amount of another agent active against RNA-dependent RNA virus and in particular against HCV.
• Agents active against HCV include, but are not limited to, ribavirin, levovirin, viramidine, thymosin alpha-1, an inhibitor of HCV NS3 serine protease, interferon- ⁇ , pegylated interferon- ⁇ (peginterferon- ⁇ ), a combination of interferon- ⁇ and ribavirin, a combination of peginterferon- ⁇ and ribavirin, a combination of interferon- ⁇ and levovirin, and a combination of peginterferon- ⁇ and levovirin.
• hiterferon- ⁇ includes, but is not limited to, recombinant interferon- ⁇ 2a (such as Roferon interferon available from Hoffmann-LaRoche, Nutley, NJ), interferon- ⁇ 2b (such as Intron-A interferon available from Schering Co ⁇ ., Kenilworth, NJ), a consensus interferon, and a purified interferon- ⁇ product.
• interferon- ⁇ 2a such as Roferon interferon available from Hoffmann-LaRoche, Nutley, NJ
• interferon- ⁇ 2b such as Intron-A interferon available from Schering Co ⁇ ., Kenilworth, NJ
• a consensus interferon such as Intron-A interferon available from Schering Co ⁇ ., Kenilworth, NJ
• a purified interferon- ⁇ product for a discussion of ribavirin and its activity against HCV, see J.O. Saunders and S.A. Raybuck, "Inosine Monophosphate Dehydrogenase: Consideration of
• Another aspect of the present invention provides for the use of the carbocyclic nucleoside compounds and derivatives thereof and their pharmaceutical compositions for the manufacture of a medicament for the inhibition of RNA- dependent RNA viral replication, in particular HCV replication, and/or the treatment of RNA-dependent RNA viral infection, in particular HCV infection.
• Yet a further aspect of the present invention provides for the carbocyclic nucleoside compounds and derivatives thereof and their pharmaceutical compositions for use as a medicament for the inhibition of RNA-dependent RNA viral replication, in particular HCV replication, and/or for the treatment of RNA-dependent RNA viral infection, in particular HCV infection.
• compositions of the present invention comprise a compound of structural formula I as an active ingredient or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.
• the compounds of structural formula I can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
• the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous).
• any of the usual phannaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations being prefened over the liquid preparations.
• oral liquid preparations such as, for example, suspensions, elixirs and solutions
• carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations
• tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques. Such compositions and preparations should contain at least 0.1 percent of active compound. The percentage of active compound in these compositions may, of course, be varied and may conveniently be between about 2 percent to about 60 percent of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that an effective dosage will be obtained.
• the active compounds can also be administered intranasally as, for example, liquid drops or spray.
• the tablets, pills, capsules, and the like may also contain a binder such as gum tragacanth, acacia, co starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin.
• a dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.
• tablets may be coated with shellac, sugar or both.
• a syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry or orange flavor.
• Compounds of structural formula I may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant such as hydroxy-propylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the phannaceutical fonns suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
• Any suitable route of administration may be employed for providing a mammal, especially a human with an effective dosage of a compound of the present invention.
• oral, rectal, topical, parenteral, ocular, pulmonary, nasal, and the like may be employed.
• Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
• compounds of structural formula I are administered orally.
• the dosage range is 0.01 to 1000 mg/kg body weight in divided doses. In one embodiment the dosage range is 0.1 to 100 mg/kg body weight in divided doses. In another embodiment the dosage range is 0.5 to 20 mg/kg body weight in divided doses.
• the compositions are preferably provided in the form of tablets or capsules containing 1.0 to 1000 milligrams of the active ingredient, particularly, 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and 1000 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
• the effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Such dosage may be ascertained readily by a person skilled in the art. This dosage regimen may be adjusted to provide the optimal therapeutic response.
• the compounds of the present invention contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers.
• the present invention is meant to comprehend carbocyclic nucleoside compounds having the stereochemical configuration for the five-membered carbocycle depicted in the structural formula below, that is, carbocyclic nucleoside compounds in which the substituents at the positions denoted as 1 and 4 in the formula below have a cis relative configuration.
• keto-enol tautomers Some of the compounds described herein may exist as tautomers such as keto-enol tautomers.
• the individual tautomers as well as mixtures thereof are encompassed with compounds of structural formula I.
• Example of keto-enol tautomers which are intended to be encompassed within the compounds of the present invention are illustrated below:
• Compounds of structural formula I may be separated into their individual diastereoisomers by, for example, fractional crystallization from a suitable solvent, for example methanol or ethyl acetate or a mixture thereof, or via chiral chromatography using an optically active stationary phase.
• a suitable solvent for example methanol or ethyl acetate or a mixture thereof
• any stereoisomer of a compound of the structural formula I may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known configuration.
• the compounds of the present invention may be administered in the form of a pharmaceutically acceptable salt.
• pharmaceutically acceptable salt refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts of basic compounds encompassed within the term “pharmaceutically acceptable salt” refer to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid.
• Representative salts of basic compounds of the present invention include, but are not limited to, the following: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochlori.de, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt
• suitable pharmaceutically acceptable salts thereof include, but are not limited to, salts derived from inorganic bases including aluminum, ammonium, calcium, copper, ferric, fenous, lithium, magnesium, manganic, mangamous, potassium, sodium, zinc, and the like. Particularly prefened are the ammonium, calcium, magnesium, potassium, and sodium salts.
• Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, cyclic amines, and basic ion-exchange resins, such as arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N- ethylmo ⁇ holine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, mo ⁇ holine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.
• basic ion-exchange resins such as arginine, betaine, caffeine
• esters of carboxylic acid derivatives such as methyl, ethyl, or pivaloyloxymethyl
• acyl derivatives of alcohols such as acetate, octanoate, or maleate
• esters and acyl groups known in the art for modifying the solubility or hydrolysis characteristics for use as sustained-release or prodrug formulations.
• the compounds of the present invention can be prepared following modifications of procedures described by Bindu Madhavan et al. in J. Org. Chem., 51: 1287-1293 (1986) and J. Med. Chem., 31: 1798-1804 (1988) as well as synthetic methodologies well-established in the practice of nucleoside and nucleotide chemistry, as described in "Chemistry of Nucleosides and Nucleotides," L.B. Townsend, ed., Vols. 1-3, Plenum Press, 1988, which is inco ⁇ orated by reference herein in its entirety.
• Reaction Schemes A-B illustrate the methods employed in the synthesis of the compounds of the present invention of structural formula I. All substituents are as defined above unless indicated otherwise.
• the starting material is the known oxirane of structural formula A-1, whose synthesis has been described in J. Med. Chem., 31: 1798-1804 (1988).
• the carbocyclic "nucleosidic" linkage is constructed by opening of the oxirane in A-1 with the metal salt (such as lithium, sodium, or potassium) of an appropriately substituted purine or 7-deaza-purine A-6, such as an appropriately substituted 4-halo-lH- pynOlo[2,3-d]pyrimidine, which can be generated in situ by treatment with an alkali hydride (such as sodium hydride), an alkali hydroxide (such as potassium hydroxide), an alkali carbonate (such as potassium carbonate), or an alkali hexamethyldisilazide (such as Na ⁇ MDS) in a suitable anhydrous organic solvent, such as acetonitrile, tetrahydrofuran,
• a suitable anhydrous organic solvent such as aceton
• the ring-opening reaction can be catalyzed by using a phase-transfer catalyst, such as TDA-1 or triethylbenzylammonium chloride, in a two-phase system (solid-liquid or liquid-liquid).
• a phase-transfer catalyst such as TDA-1 or triethylbenzylammonium chloride
• the cyclopentanol hydroxyl group in A-2 is then oxidized with a suitable oxidizing agent, such as a chromium trioxide or chromate reagent, Dess-Martin periodinane, or by Swern oxidation, to afford a cyclopentanone of structural formula A-3.
• Grignard reagent such as an alkyl, alkenyl, or alkynyl magnesium halide (for example, MeMgBr, EtMgBr, vinylMgBr, allylMgBr, and ethynylMgBr) or an alkyl, alkenyl, or alkynyl lithium, such as MeLi, across the carbonyl double bond of A-3 in a suitable organic solvent, such as tetrahydrofuran, diethyl ether, and the like, affords the tertiary cyclopentanol of structural formula A-4.
• a Grignard reagent such as an alkyl, alkenyl, or alkynyl magnesium halide (for example, MeMgBr, EtMgBr, vinylMgBr, allylMgBr, and ethynylMgBr) or an alkyl, alkenyl, or alkynyl lithium, such as MeL
• the optional protecting groups in the protected carbocyclic nucleoside of structural formula A-4 are then cleaved following established deprotection methodologies, such as those described in T.W. Greene and P.G.M. Wuts, "Protective Groups in Organic Synthesis,” 3 rd ed., John Wiley & Sons, 1999.
• the appropriate amine such as alcoholic ammonia or liquid ammonia
• a representative general method for the preparation of compounds of the present invention wherein X is CH2 is outlined in Scheme B below.
• This Scheme illustrates the synthesis of compounds of the present invention of structural formula B-7.
• a useful starting material is the aminocyclopentanetriol of structural formula B-2, which is prepared from commercially available (lR)-(-)-2-azabicyclo[2.2.1]hept- 5-en-3-one (B-l) in a similar fashion as that described in J. Org. Chem., 46: 3268 (1981) for the preparation of the coreesponding racemic form.
• Elaboration of the amino functionality in B-2 into a substituted purine or 7-deaza-purine is carried out by methods analogous to those described in J. Med.
• the 1,3-diol in the derived intermediate B-3 is protected in the form of its (1,1,3,3-tetraisopropyldisiloxanylidene) (TEPDS) derivative B-4.
• TPDS (1,1,3,3-tetraisopropyldisiloxanylidene)
• the cyclopentanol hydroxyl group in B-4 is then oxidized with a suitable oxidizing agent, such as a chromium trioxide or chromate reagent, Dess-Martin periodinane, or by Swern oxidation, to afford a cyclopentanone of structural formula B-5.
• a suitable oxidizing agent such as a chromium trioxide or chromate reagent, Dess-Martin periodinane, or by Swern oxidation, to afford a cyclopentanone of structural formula B-5.
• Grignard reagent such as an alkyl, alkenyl, or alkynyl magnesium halide (for example, MeMgBr, EtMgBr, vinylMgBr, allylMgBr, and ethynylMgBr) or an alkyl, alkenyl, or alkynyl lithium, such as MeLi, across the carbonyl double bond of B-5 in a suitable organic solvent, such as tetrahydrofuran, diethyl ether, and the like, affords the tertiary cyclopentanol of structural formula B ⁇ .
• a Grignard reagent such as an alkyl, alkenyl, or alkynyl magnesium halide (for example, MeMgBr, EtMgBr, vinylMgBr, allylMgBr, and ethynylMgBr) or an alkyl, alkenyl, or alkynyl lithium, such as MeL
• TEPDS protecting group in the protected carbocyclic nucleoside of structural formula B-6 is then cleaved following established deprotection methodologies, such as by treatment with tetrabutylammonium fluoride in THF or triethylamine dihydrogen fluoride in THF.
• the appropriate amine such as alcoholic ammonia or liquid ammonia
• Step A (1 ⁇ ,2 ⁇ ,3 ⁇ V2-( ' Benzyloxymetl yl)-cyclopent-4-ene-l ,3-diol (1-31
• the mixture was filtered through a fritted funnel pre-cooled to -20 °C into a pre-cooled round-bottom flask.
• the excess benzyl chloromethyl ether and the solvent were removed by evaporation under diminished pressure at -10 °C.
• the residue was dissolved in pre-cooled (-20 °C) methanol (100 mL).
• the resulting solution was added to a solution of Rose Bengal (316 mg), sodium acetate (732 mg), and thiourea (13.26 g) in 500 mL of methanol which had been pre-saturated with oxygen and cooled to -10 °C.
• the reaction vessel was illuminated with two 100-watt flood lamps and stined at -10 °C for 24 h with continuous bubbling of oxygen.
• Step B (l ⁇ ,2 ⁇ ,3 ⁇ ,4 ⁇ ,5 ⁇ -3-(Benzyloxymethyl)-6-oxabicyclo[3.1.01hexane-2,4- diol (1-4)
• Compound L3 from Step A (2.0 g, 9.1 mmol) was dissolved in 50 mL of dichloromethane and cooled to 0 °C. To this was added met ⁇ -chloroperoxybenzoic acid (MCPBA) (77%, 3.5 g, 15.64 mmol) in portions. The resulting solution was stined at room temperature for 2 d at which point the product and met -chlorobenzoic acid precipitated out.
• MCPBA met ⁇ -chloroperoxybenzoic acid
• Step C ( ⁇ V(l ⁇ .2 ⁇ ,3 ⁇ .4 ⁇ ,5 ⁇ -(3-(BenzyloxymethylV4-(p- anisyldiphenylmethoxy)-6-oxabicyclor3.1.OIhexan-2-ol (1-5)
• L4 260 mg, 1.1 mmol
• p-anisylchloro- diphenylmethane 460 mg, 1.49 mmol
• Step D ( ⁇ -(l ⁇ ,3 ⁇ ,4 ⁇ ,5 ⁇ )-3-(Benzyloxymethyl)-4-(p-anisyldiphenylmethoxy - 6-oxa-bicyclo[3.1.0]hexan-2-one (1-6)
• Methylphosphonic acid (7 mg, 0.05 mmol) was added to a solution of 1-5 (260 mg, 0.52 mmol) and 1,3-dicyclohexylcarbodiimide (420 mg, 2.03 mmol) in methylsulfoxide (2.5 mL) cooled to 0 °C. After the mixture had stined at room temperature for 16 h, a solution of oxalic acid (335 mg in 3.35 mL of water) was added and stirring was continued for an additional 2 h. The mixture was filtered and the filtrate diluted with ethyl acetate (30 mL). The resulting solution was extracted three times with brine (10 mL).
• Step E ( ⁇ -(l ⁇ ,2 ⁇ ,3 ⁇ ,5 ⁇ ' )-3-(Benzyloxymethyl -2-(p-anisyldiphenylmethoxy - 4-methylene-6-oxa-bicvclo[3.1.0]hexane (1-7)
• n-butyllithium (0.375 mL of a 1.6 M solution in hexanes, 0.6 mmol). The solution was allowed to come to room temperature, stined for 20 min, and then re-cooled to -78 °C. To this mixture was added a solution of L6 (135 mg, 0.27 mmol) in 1.5 mL THF. The resulting solution was allowed to come to room temperature and stined overnight.
• reaction mixture was diluted with water (30 mL) and extracted three times with diethyl ether (60 mL). The combined ether extracts were dried over anhydrous sodium sulfate and evaporated under diminished pressure. The residue was purified by flash chromatography on silica gel using 5:1 hexanes/ethyl acetate as eluant to give title compound 1 7 (130 mg), whose proton and carbon-13 ⁇ MR spectral data matched those given in Bindu Madhavan, GN. et al., J. Med. Chem. 1988, 31, 1798- 1804.
• Step F ( ⁇ V(l ⁇ .2 ⁇ .4 ⁇ .5 ⁇ -2-(2-Amino-4-chloro-7H-pynolor2,3-JJpyrimidin-7- yl)-4-(benzyloxymethyl)-5-(p-anisyldiphenylmethoxy)-3-methylene- cvclopentanol (1-8)
• Step G ( ⁇ -(2 ⁇ ,4 ⁇ ,5 ⁇ )-2-(2-Amino-4-chloro-7H-pynolor2,3- 1pyrimidin-7- yl)-4-(benzyloxymethyl)-5-(p-anisyldiphenylmethoxy)-3- methylenecyclopentanone (1-9)
• Compound L8 (1 eq) is oxidized by dissolving it in anhydrous dichloromethane and adding the solution to an ice-cold suspension of Dess Martin periodinane (4 eq) in anhydrous dichloromethane under argon.
• Step I ( ⁇ )-(l ⁇ OH,2 ⁇ ,3 ⁇ ,5 ⁇ -5-(2-Amino-4-chloro-7H-pynolor2,3- 1pyrimidin-7-yl)-3-(benzyloxymethyl)-l-methyl-4- methylenecyclopentane- 1 ,2-diol
• This compound is prepared by dissolving compound 1-10 in 80% acetic acid and stirring overnight. The solvent is removed by evaporation under diminished pressure and the residue coevaporated twice with toluene. The residue is purified by chromatography on silica gel to furnish the title compound.
• Step J ( ⁇ -(l ⁇ O ⁇ .2 ⁇ .3 ⁇ ,5 ⁇ V5-(2-Amino-4-chloro-7H-pynolor2,3-
• This compound is prepared by treating a solution of the compound from Step I in anhydrous dichloromethane with boron trichloride at -70 °C for several h. The reaction is quenched with ammonia in methanol and the solvents are removed by evaporation under diminished pressure. Purification of the residue on a silica gel column affords the desired product 1-11.
• Step K ( ⁇ V2-Amino-7-[(l ⁇ .2 ⁇ O ⁇ .3 ⁇ ,4 ⁇ V2.3-dihvdroxy-4-hydroxymethyl-2- methyl-5-methylene-cyclopentyl]-3,7-dihydro-4H-pynolo[2,3-
• Step B ( ⁇ )-(2 ⁇ .3 ⁇ .5 ⁇ )-3-(Benzyloxymethyl)-5-(4-chloro-7H-pynolor2,3-
• Step C ( ⁇ )-(l ⁇ O ⁇ .2 ⁇ .3 ⁇ .5 ⁇ )-3-(Benzyloxymethyl)-5-(4-chloro-7H- pynolo[2,3-(f
• Step B The product from Step B is processed according to the procedure detailed in Step ⁇ of Example 1 to give the title compound.
• Step D ( ⁇ )-(l ⁇ O ⁇ .2 ⁇ ,3 ⁇ ,5 ⁇ )-3-(Benzyloxymethyl)-5-(4-chloro-7H- pynolor2,3- ⁇ i1pyrimidin-7-yl)-l-methyl-4-methylenecyclopentane-l,2- diol
• Step E ( ⁇ )-(l ⁇ O ⁇ .2 ⁇ ,3 ⁇ ,5 ⁇ )-5-(4-Chloro-7H-pynolor2,3-- 1pyrimidin-7-yl)-3-
• Step F ( ⁇ )-(l ⁇ OH,2 ⁇ .3 ⁇ .5 ⁇ )-5-(4-Amino-7H-pynolor2,3- ( 1pyrimidin-7-yl)-3-
• Step A ( ⁇ )- (l ⁇ .2 ⁇ ,4 ⁇ .5 ⁇ ) -2-(2-Amino-6-chloro-9H- ⁇ urin-9-yl)-4-
• Step B ( ⁇ )-(2 ⁇ ,4 ⁇ ,5 ⁇ )-2-(2-Amino-6-chloro-9H-purin-9-yl)-4-
• Compound 3 ⁇ 2 is obtained by taking the compound from Step A in anhydrous dichloromethane and adding the solution to an ice-cold suspension of Dess-Martin periodinane (4 eq) in anhydrous dichloromethane under argon. After stirring the solution at room temperature for 4 d, the mixture is diluted with ethyl acetate and poured into a solution of sodium thiosulfate in saturated sodium bicarbonate solution. The organic layer is separated and dried over anhydrous sodium sulfate. After evaporation, the residue is purified by flash chromatography on silica gel.
• Step C ( ⁇ )-(l ⁇ OH.2 ⁇ .4 ⁇ ,5 ⁇ )-2-(2-Amino-6-chloro-9H-purin-9-yl)-4-
• Step D ( ⁇ )-(l ⁇ OH.2 ⁇ .3 ⁇ ,5 ⁇ )-5-(2-Amino-6-chloro-9H-purin-9-yl)-3- (bcnzyl ⁇ xymethyl)- 1 -methyl-4-methylenecyclopentane- 1 ,2-diol
• Step E ( ⁇ )-(l ⁇ O ⁇ .2 ⁇ ,3 ⁇ ,5 ⁇ )-5-(2-Amino-6-cl ⁇ loro-9H-purin-9-yl)-3- (hvdroxymethyl)-l-methyl-4-methylenecyclopentane-l,2-diol (3-4)
• Step F ( ⁇ )- (l ⁇ ,2 ⁇ O ⁇ ,3 ⁇ ,4 ⁇ )-2-Amino-9-r2,3-dihvdroxy-4-(hvdroxymethyl)- 2-methyl-5-methylenecyclopentyn-l,9-dihydro-6H-purin-6-one (3-5)
• Step A (lR,4S,5R,6S)-5,6-Dihvdroxy-2-azabicyclo[2.2.nheptan-3-one (4-2)
• Step C ( 1R,2S ,3R,5R)-3-Amino-5-(hvdroxymethyl)cvclopentane- 1 ,2-diol hydrochloride (4-4)
• Step B (4,6-Dichloropyrimidine-5-yl)acetaldehyde (5-3) This compound was prepared by modification of the procedure described in J. Med. Chem.10: 665 (1967). A solution of 5 ⁇ 2 (3.0 g, 15.7 mmol) in dioxane (20 mL) was stined with 4-methylmo ⁇ holine-N-oxide (2.8 g, 24 mmol) and osmium tetroxide (4% solution in water, 6.2 mL) for one h.
• Step C 4,6-Dichloro-5-(2,2-diethoxyethyl)pyrimidine (5-4)
• Step A (lR,2S,3R,5R)-3- ⁇ f6-chloro-5-(2,2-diethoxyethyl)pyrimidin-4- yl1amino ⁇ -5-(hydroxymethyl)cyclopentane-l,2-diol (6-1)
• This compound was prepared from 54 and following the procedure described in J. Med. Chem., 27: 534 (1984).
• Step C (6aR,8R,9S,9aR)-8-(4-Chloro-7H-pynolor2,3- 1pyrimidin-7-yl)-
• Step D (6aR,8R,9aR)-8-(4-Chloro-7H-pynolor2,3- ⁇ i1pyrimidin-7-yl)-2,2,4,4- tetraisopropylhexahydrocvclopentarfl[l,3,5,2,41trioxadisilocin-9(6H)- one (6-4)
• Step F (lR,2R.3R,5R)-5-(4-Chloro-7H-pynolor2,3- 1 ⁇ yrirnidin-7-yl)-3-
• Step G (lR,2R,3R,5R)-5-f4-Amino-7H-pynolo[2,3- ⁇ 1pyrimidin-7-yl)-3-
• Examples 1, 2, and 3 can also prepared according to procedures depicted in Schemes 7, 8, and 9, respectively.
• This assay was used to measure the ability of the carbocyclic nucleoside derivatives of the present invention to inhibit the enzymatic activity of the RNA-dependent RNA polymerase (NS5B) of the hepatitis C virus (HCV) on a heteromeric RNA template.
• NS5B RNA-dependent RNA polymerase
• HCV hepatitis C virus
• Assay Buffer Conditions (50 ⁇ L -total/reaction) 20 mM Tris, pH 7.5 50 ⁇ M EDTA 5 mMDTT 2 mM MgCl 2 80 mM KCl
• RNAsin Promega, stock is 40 units/ ⁇ L
• t500 a 500-nt RNA made using T7 runoff transcription with a sequence from the NS2/3 region of the hepatitis C genome
• t500 a 500-nt RNA made using T7 runoff transcription with a sequence from the NS2/3 region of the hepatitis C genome
• purified hepatitis C NS5B form with 21 amino acids C-terminally truncated
• A,C,U,GTP Nucleoside triphosphate mix
• reaction buffer including enzyme and template t500.
• Carbocyclic nucleoside derivatives of the present invention were pipetted into the wells of a 96-well plate.
• the reaction was initiated by addition of the enzyme-template reaction solution and allowed to proceed at room temperature for 1-2 h.
• reaction was quenched by addition of 20 ⁇ L 0.5M EDTA, pH 8.0. Blank reactions in which the quench solution was added to the NTPs prior to the addition of the reaction buffer were included.
• %Inhibition [l-(cpm in test reaction - cpm in blank) / (cpm in control reaction - cpm in blank)] x 100.
• Representative compounds tested in the HCV NS5B polymerase assay exhibited ICso's less than 100 micromolar.
• the compounds of the present invention were also evaluated for their ability to affect the replication of Hepatitis C Virus RNA in cultured hepatoma (HuH- 7) cells containing a subgenomic HCV Replicon.
• the details of the assay are described below.
• This Replicon assay is a modification of that described in V. Lohmann, F. Ko er, J-O. Koch, U. Herian, L. Theilmann, and R. Bartenschlager, "Replication of a Sub-genomic Hepatitis C Virus RNAs in a Hepatoma Cell Line," Science 285:110 (1999).
• the assay was an in situ Ribonuclease protection, Scintillation Proximity based-plate assay (SPA). 10,000 - 40,000 cells were plated in 100-200 ⁇ L of media containing 0.8mg/mL G418 in 96-well cytostar plates (Amersham). Compounds were added to cells at various concentrations up to 100 ⁇ M in 1% DMSO at time 0 to 18 h and then cultured for 24-96 h.
• SPA Ribonuclease protection, Scintillation Proximity based-plate assay
• RNA viral genome RNA was fixed (20 min, 10% formalin), permeabilized (20 min, 0.25% Triton X-100/PBS) and hybridized (overnight, 50°C) with a single-stranded P RNA probe complementary to the (+) strand NS5B (or other genes) contained in the RNA viral genome.
• Cells were washed, treated with RNAse, washed, heated to 65 °C and counted in a Top-Count. Inhibition of replication was read as a decrease in counts per minute (cpm).
• Human HuH-7 hepatoma cells which were selected to contain a subgenomic replicon, carry a cytoplasmic RNA consisting of an HCV 5' non- translated region (NTR), a neomycin selectable marker, an EMCV IRES (internal ribosome entry site), and HCV non-structural proteins NS3 through NS5B, followed by the 3' NTR.
• NTR non- translated region
• EMCV IRES internal ribosome entry site
• HCV non-structural proteins NS3 through NS5B followed by the 3' NTR.
• Representative compounds tested in the replication assay exhibited EC 5 o's less than 100 micromolar.
• carbocyclic nucleoside derivatives of the present invention were also evaluated for cellular toxicity and anti-viral specificity in the counterscreens described below.
• Reaction buffer components 200 ⁇ g/mL bovine serum albumin lOO mM KCl 2 mM ⁇ -mercaptoethanol 10 mM MgCl 2 1.6 ⁇ M dA, dG, dC, dTTP - 33 P-dATP
• Terminate reaction by heating to 65 °C for 10 min; Load 50-100 ⁇ L aliquots onto Bio-spin 6 chromatography columns (Bio-Rad 732-
• the DNA template was diluted into an appropriate volume of 20 mM
• Tris-HCl, pH 7.5 and the enzyme was diluted into an appropriate volume of 20 mM Tris-HCl, containing 2 mM ⁇ -mercaptoethanol, and 100 mM KC1. Template and enzyme were pipetted into microcentrifuge tubes or a 96 well plate. Blank reactions excluding enzyme and control reactions excluding test compound were also prepared using enzyme dilution buffer and test compound solvent, respectively. The reaction was initiated with reaction buffer with components as listed above. The reaction was incubated for 1 hour at 37°C. The reaction was quenched by the addition of 20 ⁇ L 0.5M EDTA. 50 ⁇ L of the quenched reaction was spotted onto Whatman DE81 filter disks and air dried.
• the filter disks were repeatedly washed with 150 mL 0.3M ammonium formate, pH 8 until 1 mL of wash is ⁇ 100 cpm.
• the disks were washed twice with 150 mL absolute ethanol and once with 150 mL anhydrous ether, dried and counted in 5 mL scintillation fluid.
• the potential for inhibition of human DNA polymerase gamma was measured in reactions that included 0.5 ng/ ⁇ L enzyme; 10 ⁇ M dATP, dGTP, dCTP, and TTP; 2 ⁇ Ci/reaction [ ⁇ - 33 P]-dATP, and 0.4 ⁇ g/ ⁇ L activated fish sperm DNA (purchased from US Biochemical) in a buffer containing 20 mM Tris pH8, 2 mM ⁇ - mercaptoethanol, 50 mM KCl, 10 mM MgCl2, and 0.1 ⁇ g/ ⁇ L BSA. Reactions were allowed to proceed for 1 h at 37°C and were quenched by addition of 0.5 M EDTA to a final concentration of 142 mM.
• % inhibition [l-(cpm in test reaction - cpm in blank)/(cpm in control reaction - cpm in blank)] x 100.
• Assays were performed with a variant of HeLa Magi cells expressing both CXCR4 and CCR5 selected for low background ⁇ -galactosidase ( ⁇ -gal) expression.
• Cells were infected for 48 h, and ⁇ -gal production from the integrated HTV-1 LTR promoter was quantified with a chemiluminescent substrate (Galactolight Plus, Tropix, Bedford, MA).
• Inhibitors were titrated (in duplicate) in twofold serial dilutions starting at 100 ⁇ M; percent inhibition at each concentration was calculated in relation to the control infection.
• HTV human immunedeficiency virus
• the carbocyclic nucleoside derivatives of the present invention were also screened for cytotoxicity against cultured hepatoma (HuH-7) cells containing a subgenomic HCV Replicon in an MTS cell-based assay as described in the assay below.
• HuH-7 cell line is described in H. Nakabayashi, et al., Cancer Res., 42: 3858 (1982).
• Cell cultures were prepared in appropriate media at concentrations of approximately 1.5 x 10 5 cells/mL for suspension cultures in 3 day incubations and 5.0 x 10 4 cells/mL for adherent cultures in 3 day incubations. 99 ⁇ L of cell culture was transfened to wells ofa 96-well tissue culture treated plate, and 1 ⁇ L of 100-times final concentration of the test compound in DMSO was added. The plates were incubated at 37°C and 5% CO 2 for a specified period of time.
• Rhinovirus type 2 (RV-2), strain HGP, was used with KB cells and media (0.1% NaHCO 3 , no antibiotics) as stated in the Sidwell and Huffman reference.
• the virus obtained from the ATCC, was from a throat swab of an adult male with a mild acute febrile upper respiratory illness.
• Rhinovirus type 9 (RV-9), strain 211, and rhinovirus type 14 (RV-14), strain Tow, were also obtained from the American Type Culture Collection (ATCC) in Rockville, MD. RV-9 was from human throat washings and RV-14 was from a throat swab of a young adult with upper respiratory illness. Both of these viruses were used with HeLa Ohio-1 cells (Dr. Fred Hayden, Univ. of VA) which were human cervical epitheloid carcinoma cells. MEM (Eagle's minimum essential medium) with 5% Fetal Bovine serum (FBS) and 0.1% NaHCO 3 was used as the growth medium. Antiviral test medium for all three virus types was MEM with 5% FBS, 0.1% NaHCO3, 50 ⁇ g gentamicin/mL, and 10 mM MgCl2-
• Viruses Dengue virus type 2, New Guinea strain, was obtained from the Center for Disease Control. Two lines of African green monkey kidney cells were used to culture the virus (Vero) and to perform antiviral testing (MA-104). Both Yellow fever virus, 17D strain, prepared from infected mouse brain, and Banzi virus, H 336 strain, isolated from the serum of a febrile boy in South Africa, were obtained from ATCC. Vero cells were used with both of these viruses and for assay.
• Cells and Media
• MA-104 cells BioWhittaker, Inc., Walkersville, MD
• Vero cells ATCC
• Assay medium for dengue, yellow fever, and Banzi viruses was MEM, 2% FBS, 0.18% NaHC ⁇ 3 and 50 ⁇ g gentamicin/mL.
• Antiviral testing of the compounds of the present invention was performed according to the Sidwell and Huffman reference and similar to the above rhinovirus antiviral testing. Adequate cytopathic effect (CPE) readings were achieved after 5-6 days for each of these viruses.
• CPE cytopathic effect
• test medium was MEM, 1% FBS, 0.1% NaHCO3 and 50 ⁇ g gentamicin/mL.
• Antiviral testing of the compounds of the present invention was performed following the methods of Sidwell and Huffman which are similar to those used to assay for rhinovirus activity. Adequate cytopathic effect (CPE) readings were achieved after 5-6 days.
• CPE cytopathic effect
• EXAMPLE OF A PHARMACEUTICAL FORMULATION As a specific embodiment of an oral composition of a compound of the present invention, 50 mg of the compound of Example 1 or Example 2 is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size O hard gelatin capsule.

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