WO2003068244A1 - Methods of inhibiting orthopoxvirus replication with nucleoside compounds - Google Patents

Methods of inhibiting orthopoxvirus replication with nucleoside compounds Download PDF

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Publication number
WO2003068244A1
WO2003068244A1 PCT/US2003/003703 US0303703W WO03068244A1 WO 2003068244 A1 WO2003068244 A1 WO 2003068244A1 US 0303703 W US0303703 W US 0303703W WO 03068244 A1 WO03068244 A1 WO 03068244A1
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Prior art keywords
methyl
amino
ribofuranosyl
pyrimidine
pyrrolo
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PCT/US2003/003703
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French (fr)
Inventor
David B. Olsen
Robert L. Lafemina
Anne B. Eldrup
Sanjib Bera
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Merck & Co., Inc.
Isis Pharmaceuticals, Inc.
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Priority to EP03707772A priority Critical patent/EP1476169B1/en
Priority to CA2474563A priority patent/CA2474563C/en
Priority to US10/504,445 priority patent/US7323453B2/en
Priority to JP2003567425A priority patent/JP2005527499A/en
Priority to AU2003209045A priority patent/AU2003209045B2/en
Publication of WO2003068244A1 publication Critical patent/WO2003068244A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/655—Azo (—N=N—), diazo (=N2), azoxy (>N—O—N< or N(=O)—N<), azido (—N3) or diazoamino (—N=N—N<) compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P31/20—Antivirals for DNA 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 methods of inhibiting orthopoxvirus replication and methods for treating orthopoxvirus infections with certain nucleoside compounds and derivatives thereof.
  • the compounds are particularly useful for inhibiting the replication of vaccinia, variola, cowpox, and monkeypox virus and for the treatment of vaccinia, variola, cowpox, and monkeypox virus infections.
  • Another aspect of the present invention provides for the use of the nucleoside compounds and derivatives thereof and their pharmaceutical compositions for the manufacture of a medicament for the inhibition of orthopoxvirus replication and/or for the treatment of orthopoxvirus infection.
  • Yet a further aspect of the present invention provides for the nucleoside compounds and derivatives thereof and their pharmaceutical compositions for use as a medicament for the inhibition of orthopoxvirus replication and/or for the treatment of orthopoxvirus infection.
  • Orthopoxvirus is a genus of the Poxviridae family of complex DNA viruses that replicate in the cytoplasm of vertebrate and invertebrate cells.
  • the Poxviridae family is characterized by having a large complex virion containing enzymes that synthesize mRNA, a genome composed of a single linear double- stranded DNA molecule of 130-300 kilobase pairs with a hairpin loop at each end, and a cytoplasmic site of replication.
  • Members of the orthopoxvirus genus include cowpox, monkeypox, vaccinia, and variola virus [for a description of the Poxviridae family, reference is made to B.
  • Variola virus is the agent responsible for smallpox infections. Smallpox infections were effectively eradicated subsequent to the introduction of prophylactic vaccinations with cowpox and vaccinia virus. However, most of the human populations worldwide are no longer immune to smallpox as a result of the discontinuation of routine vacination in the early 1980' s.
  • Cidofovir is the generic name for (S)-l-[3-hydroxy-2-(phosphonylmethoxy)- propyl]cytosine [(S)-HPMPC] which is currently the leading agent for the treatment of orthopoxvirus infections in humans. It is potent against vaccinia and cowpox virus infection in mice when administered subcutaneously or intraperitoneally.
  • cidofovir' s therapeutic utility is limited by safety concerns as well as lack of oral bioavailablity [see D.F.
  • Ribavirin has also been found to inhibit vaccinia virus and other orthopoxvirus replication in cell culture (see J.H. Huffman et al., "In vitro effect of 1- ⁇ -D-ribofuranosyl-l,2,4-triazole-3-carboxarnide on DNA and RNA viruses," Antimicrobial Agents and Chemotherapy, 3: 235-241 (1973) and D.F. Smee et al., "Potential of the IMP dehydrogenase inhibitors for antiviral therapies of poxvirus infections," Antiviral Res., 37: A89 (1998)].
  • Ribavirin was also reported to suppress vaccinia virus-induced lesions in a mouse model and to effectively treat vaccinia keratitis in rabbits.
  • ribavirin causes anemia during prolonged administration and at high doses has certain immunosuppressive properties limiting its clinical usefulness against orthopoxvirus.
  • anti-orthopoxvirus agents particularly as a result of the threat of either variola (smallpox) or monkeypox viruses in biowarfare or bioterrorism.
  • variola smallpox
  • monkeypox viruses in biowarfare or bioterrorism.
  • anti-orthopoxvirus agents should be effective when administered orally and be safe and well-tolerated by the host.
  • nucleoside compounds of the present invention and certain derivatives thereof are potent inhibitors of orthopoxvirus replication and in particular of vaccinia, variola, cowpox, and monkeypox virus replication.
  • the instant nucleoside compounds and derivatives thereof are useful to treat orthopoxvirus infection and in particular vaccinia, variola, cowpox, and monkeypox virus infection.
  • nucleoside compounds and certain derivatives thereof which are useful as inhibitors of the replication of orthopoxvirus and in particular as inhibitors of the replication of vaccinia, variola, cowpox, and monkeypox virus. It is another object of the present invention to provide nucleoside compounds and certain derivatives thereof which are useful in the treatment of orthopoxvirus infection and in particular in the treatment of vaccinia, variola, cowpox, and monkeypox virus infection. It is another object of the present invention to provide pharmaceutical compositions comprising the nucleoside compounds of the present invention in association with a pharmaceutically acceptable carrier.
  • compositions comprising the nucleoside compounds and derivatives of the present invention for use as inhibitors of orthopoxvirus replication and in particular as inhibitors of vaccinia, variola, cowpox, and monkeypox virus replication.
  • nucleoside compounds and certain derivatives thereof and their pharmaceutical compositions for use as a medicament for the inhibition of orthopoxvirus replication and/or the treatment of orthopoxvirus infection and in particular for the inhibition of vaccinia, variola, cowpox, and monkeypox virus replication and/or the treatment of vaccinia, variola, cowpox, and monkeypox virus infection.
  • the present invention provides a method for inhibiting orthopoxvirus replication and/or a method for treating orthopoxvirus infection in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of a compound of structural formula I:
  • A is N or C-R9;
  • Rl is C2-4 alkenyl, C2-4 alkynyl, or Ci_4 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino, Ci_4 alkoxy, C ⁇ _4 alkylthio, or one to three fluorine atoms;
  • R2 is amino, fluorine, hydroxy, CI_IQ alkylcarbonyloxy, mercapto, or Ci-4 alkoxy;
  • R3 and R4 are each independently selected from the group consisting of hydrogen, cyano, azido, halogen, hydroxy, C ⁇ _i6 alkylcarbonyloxy, C2-I8 alkenylcarbonyloxy, Cl-lO alkyloxycarbonyloxy, C3-6 cycloalkylcarbonyloxy,
  • R5 is hydrogen, C ⁇ _i6 alkylcarbonyl, C2-I8 alkenylcarbonyl, Cl-lO alkyloxycarbonyl, C3-6 cycloalkylcarbonyl, C3-6 cycloalkyloxycarbonyl, P3O9H4, P2O6H3, or P(O)Rl3Rl4;
  • 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
  • compositions containing the compounds alone or in combination with other agents active against orthopoxvirus and in particular against vaccinia, variola, cowpox, and monkeypox virus as well as methods for the inhibition of orthopoxvirus replication and for the treatment of orthopoxvirus infection with such compositions.
  • the present invention provides a method for inhibiting orthopoxvirus replication and/or a method for treating orthopoxvirus infection in a mammal in need thereof comprising administering to the mammal a therapeutically effective amount of a compound of structural formula I:
  • A is N or C-R9;
  • Rl is C2-4 alkenyl, C2-4 alkynyl, or C ⁇ _4 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino, C ⁇ _4 alkoxy, Ci_4 alkylthio, or one to three fluorine atoms;
  • R2 is amino, fluorine, hydroxy, C ⁇ _ ⁇ o alkylcarbonyloxy, mercapto, or Ci-4 alkoxy; R and R are each independently selected from the group consisting of hydrogen, cyano, azido, halogen, hydroxy, C ⁇ _i6 alkylcarbonyloxy, C2-18 alkenylcarbonyloxy, Cl-10 alkyloxycarbonyloxy, C3-6 cycloalkylcarbonyloxy,
  • R5 is hydrogen, C ⁇ _i6 alkylcarbonyl, C2-18 alkenylcarbonyl, Cl-lO alkyloxycarbonyl, C3-6 cycloalkylcarbonyl, C3-6 cycloalkyloxycarbonyl, P3O9H4, P2O6H3, or P(O)Rl3Rl4 ;
  • R6 and R7 are each independently hydrogen, methyl, hydroxymethyl, or fluoromethyl
  • R8 is hydrogen, C ⁇ _4 alkyl, C2-4 alkynyl, halogen, cyano, carboxy, Ci-4 alkyloxycarbonyl, azido, amino, C1.4 alkylamino, di(C ⁇ _4 alkyl)amino, hydroxy, Ci-6 alkoxy, Ci-6 alkylthio, C ⁇ _6 alkylsulfonyl, or (Ci_4 alkyl) ⁇ -2 aminomethyl
  • A is N or C-R9;
  • Rl is Ci-3 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino, Ci-3 alkoxy, C ⁇ _3 alkylthio, or one to three fluorine atoms;
  • R2 is hydroxy, Ci-1 alkylcarbonyloxy, fluoro, or Ci-3 alkoxy;
  • R3 is hydrogen, halogen, hydroxy, C ⁇ _i6 alkylcarbonyloxy, amino, or C ⁇ _3 alkoxy;
  • R5 is hydrogen, Ci_i6 alkylcarbonyl, P3O9H4, P2O6H3, or PO3H2;
  • R8 is hydrogen, amino, or C _4 alkylamino;
  • R9 is hydrogen, cyano, methyl, halogen, or CONH2; and
  • RlO and RU are each independently hydrogen, halogen, hydroxy, amino, C ⁇ _4 alkylamino, di(C ⁇ _4 alkyl)amino, or C3.6 cycloalkylamino.
  • a second embodiment of the present invention is the method of inhibiting orthopoxvirus replication and/or treating orthopoxvirus infection with a compound of structural formula II wherein
  • Rl is methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, or aminomethyl
  • R2 is hydroxy, C ⁇ _i6 alkylcarbonyloxy, fluoro, or methoxy
  • R3 is hydrogen, fluoro, hydroxy, C ⁇ _i6 alkylcarbonyloxy, amino, or methoxy
  • R5 is hydrogen, Ci-16 alkylcarbonyl, or P3O9H4;
  • R8 is hydrogen or amino
  • R9 is hydrogen, cyano, methyl, halogen, or CONH2;
  • RlO and Rl 1 are each independently hydrogen, halogen, hydroxy, amino, C ⁇ _4 alkylamino, di(C ⁇ _4 alkyl)amino, or C3-6 cycloalkylamino.
  • Illustrative of the invention is a method for inhibiting orthopoxvirus replication and/or treating orthopox infection wherein the compound is selected from: 4-amino-7-(2-C-methyl- ⁇ -D-ribofuranosyl)-7H-pyrrolo[2,3-( ]pyrimidine, 4-methylamino-7-(2-C-methyl- ⁇ -D-ribofuranosyl)-7H-pyrrolo[2,3-(i]pyrimidine, 4-dimethylamino-7-(2-C-methyl- ⁇ -D-ribofuranosyl)-7H-pyrrolo[2,3- ]pyrimidine, 4-cyclopropylamino-7-(2-C-methyl- ⁇ -D-ribomranosyl)-7H-pyrrolo
  • FIG. 1 Further illustrative of the invention is a method for inhibiting orthopoxvirus replication and/or treating orthopox infection wherein the compound is selected from: 4-a ⁇ no-7-(2-C-methyl- ⁇ -D-ribofuranosyl)-7H-pyrrolo[2,3- ⁇ i]pyrimidine, 4-amino-7-(2-C-fluoromethyl- ⁇ -D-ribofuranosyl)-7H-py ⁇ olo[2,3- ⁇ pyrimidine,
  • the orthopoxvirus replication is selected from the group consisting of vaccinia virus replication, variola virus replication, cowpox virus replication, and monkeypox virus replication.
  • the orthopoxvirus replication is vaccinia virus replication or variola virus replication.
  • the orthopoxvirus infection is selected from the group consisting of vaccinia virus infection, variola virus infection, cowpox virus infection, and monkeypox virus infection.
  • the orthopoxvirus infection is vaccinia virus infection or variola virus infection.
  • Another aspect of the present invention provides the novel nucleoside derivative, 6-amino-2-fluoro-9-(2-C-methyl- ⁇ -D-ribofuranosyl)purine or a pharmaceutically acceptable salt thereof.
  • alkyl groups specified above are intended to include those alkyl groups of the designated length in either a straight or branched configuration.
  • 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).
  • alkoxy refers to straight or branched chain alkoxides of the number of carbon atoms specified (e.g., Ci_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.].
  • alkylsulfonyl refers to straight or branched chain alkylsulfones of the number of carbon atoms specified (e.g., C ⁇ _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., C ⁇ _4 alkyloxycarbonyl), or any number within this range [i.e., methyloxycarbonyl (MeOCO-), ethyloxycarbonyl, or butyloxycarbonyl].
  • alkyloxycarbonyloxy refers to straight or branched chain alkyl carbonates of the present invention of the number of carbon atoms specified (e.g., Ci-io alkyloxycarbonyloxy), or any number within this range [i.e., methyloxycarbonyl oxy (MeOCOO-), ethyloxycarbonyloxy, or butyloxycarbonyloxy].
  • alkylcarbonyloxy refers to straight or branched chain alkanoic acid derivatives of alcohols of the present invention of the number of carbon atoms specified (e.g., Ci-16 alkylcarbonyloxy), or any number within this range [i.e., methylcarbonyloxy (MeCOO-), ethylcarbonyloxy, or n-octylcarbonyloxy].
  • cycloalkylcarbonyloxy refers to cyclic alkanoic acid derivatives of alcohols of the present invention of the number of carbon atoms specified (e.g., C3-6 cycloalkylcarbonyloxy), or any number within this range [i.e., cyclopropylcarbonyloxy, cyclopentylcarbonyloxy, or cyclohexylcarbonyloxy].
  • alkenylcarbonyloxy refers to a straight or branched chain alkenoic acid derivatives of alcohols of the present invention having two to eighteen total carbon atoms and containing one to three double bonds in the alkene chain.
  • aryl includes both phenyl, naphthyl, and pyridyl. The aryl group is optionally substituted with one to three groups independently selected from Ci-4 alkyl, halogen, cyano, nitro, trifluoromethyl, C ⁇ _4 alkoxy, and Ci_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.
  • 5 '-triphosphate refers to a triphosphoric acid ester derivative of the 5'-hydroxyl group of a nucleoside compound of the present invention having the following general structural formula HI:
  • Rl-Rl 1 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 IV and V, respectively,
  • 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 carrier, 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 treating orthopoxvirus infections with a compound of the present invention in combination with one or more agents useful for treating orthopoxvirus infections.
  • agents active against orthopoxviruses include, but are not limited to, cidofovir, ribavirin, levovirin, and viramidine.
  • Levovirin is the L-enantiomer of ribavirin which has shown immunomodulatory activity similar to ribavirin.
  • Niramidine represents an analog of ribavirin disclosed in WO 01/60379 (assigned to IC ⁇ Pharmaceuticals).
  • the individual components of the combination can be administered separately at different times during the course of therapy or concurrently 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 interpreted accordingly. It will be understood that the scope of combinations of the compounds of this invention with other agents useful for treating orthopoxvirus infection includes in principle any combination with any pharmaceutical composition for treating orthopoxvirus infection.
  • the dose of each compound may be either the same as or different from the dose when the compound is used alone.
  • Ribavirin, levovirin, and viramidine may exert their anti-orthopoxvirus effects by modulating intracellular pools of guanine nucleotides via inhibition of the intracellular enzyme inosine monophosphate dehydrogenase (IMPDH).
  • IMPDH is the rate-limiting enzyme on the biosynthetic route in de novo guanine nucleotide biosynthesis.
  • Ribavirin is readily phosphorylated intracellularly and the monophosphate derivative is an inhibitor of IMPDH.
  • 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 IMPDH 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 IMPDH 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)].
  • pharmaceutically acceptable is meant that the carrier
  • compositions comprising the 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 orthopoxvirus replication comprising an effective amount of a compound of the present invention and a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions useful for treating orthopoxvirus infection are also encompassed by the present invention.
  • 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 orthopoxvirus.
  • Agents active against orthopoxvirus include, but are not limited to, cidofovir, ribavirin, levovirin, and viramidine.
  • Another aspect of the present invention provides for the use of the nucleoside compounds and derivatives thereof and their pharmaceutical compositions for the manufacture of a medicament for the inhibition of orthopoxvirus replication, in particular vaccinia virus, variola virus replication, cowpox virus replication, and monkeypox virus replication and/or the treatment of orthopoxvirus infection, in particular vaccinia virus, variola virus infection, cowpox virus infection, and monkeypox virus infection.
  • nucleoside compounds and derivatives thereof and their pharmaceutical compositions for use as a medicament for the inhibition of orthopoxvirus replication, in particular vaccinia, variola, cowpox, and monkeypox virus replication, and/or for the treatment of orthopoxvirus infection, in particular vaccinia, variola, cowpox, and monkeypox virus 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.
  • compositions include compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation), or nasal administration, although the most suitable route in any given case will depend on the nature and severity of the conditions being treated and on the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy. In practical use, 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 pharmaceutical 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 preferred 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 being preferred
  • 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, corn 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 pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists.
  • 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.
  • 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 nucleoside compounds having the ⁇ -D stereochemical configuration for the five-membered furanose ring as depicted in the structural formula below, that is, nucleoside compounds in which the substituents at C-l and C-4 of the five-membered furanose ring have the ⁇ -stereochemical configuration ("up" orientation as denoted by a bold line).
  • 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.
  • An example of keto-enol tautomers which are intended to be encompassed within the compounds of the present invention is 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, dihydrochloride, 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, ferrous, lithium, magnesium, manganic, mangamous, potassium, sodium, zinc, and the like. Particularly preferred 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- ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, 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, or acyl derivatives of alcohols, such as acetate or maleate, can be employed. Included are those esters and acyl groups known in the art for modifying the solubility or hydrolysis characteristics for use as sustained-release or prodrug formulations.
  • nucleoside compounds and derivatives thereof of the present invention can be prepared following synthetic methodologies well-established in the practice of nucleoside and nucleotide chemistry. Reference is made to the following text for a description of synthetic methods used in the preparation of the compounds of the present invention: "Chemistry of Nucleosides and Nucleotides," L.B.
  • a representative general method for the preparation of compounds of the present invention is outlined in Scheme 1 below.
  • This scheme illustrates the synthesis of compounds of the present invention of structural formula 1 7 wherein the furanose ring has the ⁇ -D-ribo configuration.
  • the starting material is a 3,5-bis-O- protected alkyl furanoside, such as methyl furanoside, of structural formula L ⁇ .
  • the C-2 hydroxyl group is then oxidized with a suitable oxidizing agent, such as a chromium trioxide or chromate reagent, Dess-Martin periodinane, or by Swem oxidation, to afford a C-2 ketone of structural formula 1 ⁇ 2.
  • 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 J 2 in a suitable organic solvent, such as tetrahydrofuran, diethyl ether, and the like, affords the C-2 tertiary alcohol of structural formula .
  • 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 MeLi, across the carbon
  • a good leaving group (such as CI, Br, and I) is next introduced at the C-l (anomeric) position of the furanose sugar derivative by treatment of the furanoside of formula L ⁇ with a hydrogen halide in a suitable organic solvent, such as hydrogen bromide in acetic acid, to afford the intermediate furanosyl halide 1 ⁇ 4.
  • a C-l sulfonate such methanesulfonate (MeSO 2 O-), trifluoromethane- sulfonate (CF 3 SO 2 O-), or p-toluenesulfonate (-OTs), may also serve as a useful leaving group in the subsequent reaction to generate the glycosidic (nucleosidic) linkage.
  • the nucleosidic linkage is constructed by treatment of the intermediate of structural formula 1 ⁇ 4 with the metal salt (such as lithium, sodium, or potassium) of an appropriately substituted lH-pyrrolo[2,3-d]pyrimidine 1-5, such as an appropriately substituted 4-halo-lH-pyrrolo[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, l-methyl-2- pyrrolidinone, or N,N-dimethylformamide (DMF).
  • the metal salt such as lithium, sodium, or potassium
  • an appropriately substituted lH-pyrrolo[2,3-d]pyrimidine 1-5
  • the displacement reaction can be catalyzed by using a phase-transfer catalyst, such as TDA-1 or triethylbenzyl- ammonium chloride, in a two-phase system (solid-liquid or liquid-liquid).
  • a phase-transfer catalyst such as TDA-1 or triethylbenzyl- ammonium chloride
  • TDA-1 or triethylbenzyl- ammonium chloride in a two-phase system (solid-liquid or liquid-liquid).
  • solid-liquid or liquid-liquid solid-liquid or liquid-liquid.
  • Optional introduction of an amino group at the 4-position of the pyrrolo[2,3-d]pyrimidine nucleus is effected by treatment of the 4-halo intermediate 1 ⁇ 6 with the appropriate amine, such as alcoholic ammonia or liquid ammonia, to generate a primary amine at the C-4 position (-N ⁇ 2 ), an alkylamine to generate a secondary amine (-NHR), or a dialkylamine to generate a tertiary amine (-NRR').
  • a 7H-pyrrolo[2,3-d]pyrimidin-4(3H)one compound may be derived by hydrolysis of 1 ⁇ 6 with aqueous base, such as aqueous sodium hydroxide.
  • nucleoside compounds of the present invention were prepared according to procedures detailed in the following examples. The examples are not intended to be limitations on the scope of the instant invention in any way, and they should not be so construed. Those skilled in the art of nucleoside and nucleotide synthesis will readily appreciate that known variations of the conditions and processes of the following preparative procedures can be used to prepare these and other compounds of the present invention. All temperatures are degrees Celsius unless otherwise noted.
  • Step A 3,5-Bis-O-(2,4-dichlorophenylmethyl)-l-6>-methyl- -D-ribofuranose
  • Step C 3,5-Bis-Q-(2,4-dichlorophenylmethyl -2-C-methyl-l- ⁇ -methyl- ⁇ -D- ribofuranose
  • a solution of MeMgBr in anhydrous Et 2 O (0.48 M, 300 mL) at -55 °C was added dropwise a solution of the compound from Step B (17.40 g, 36.2 mmol) in anhydrous Et O (125 mL).
  • the reaction mixture was allowed to warm to -30°C and stirred for 7 h at -30°C to -15°C, then poured into ice-cold water (500 mL) and the mixture vigorously stirred at room temperature for 0.5 h.
  • Step D 4-Chloro-7-r3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl- ⁇ -D- ribofuranosyl]-7H-py ⁇ olo[2,3-(f
  • ⁇ Br 5.7 M in acetic acid, 20 mL, 114 mmol
  • Step E 4-Chloro-7-(2-C-methyl- ⁇ -D-ribofuranosvn-7H-pyrrolo[2,3-
  • Step F 4-Amino-7-(2-C-methyl- ⁇ -D-ribofuranosyl -7H-pyrrolor2,3- ⁇ lpyrimidine
  • methanolic ammonia saturated at 0°C; 150 mL
  • the crude mixture was purified on a silica gel column with C ⁇ 2 Cl 2 /MeO ⁇ (9/1) as eluent to give the title compound as a colorless foam (0.8 g), which separated as an amorphous solid after treatment with MeCN.
  • the amorphous solid was recrystallized from methanol/acetonitrile; m.p. 222°C. lH NMR (DMSO- ): ⁇ 0.62 (s, 3H, CH 3 ), 3.57-3.67 (m, IH, H-5'), 3.75-3.97 (m,
  • Step A 3,5-Bis-0-(2,4-dichlorophenylmethyl)-2-C-ethyl-l- ⁇ 9-methyl- ⁇ -D- ribofuranose
  • EtMgBr 3.0 M, 16.6 mL
  • Et 2 O 100 mL
  • the reaction mixture was stirred at -78 °C for 15 min, allowed to warm to -15°C and stirred for another 2 h, and then poured into a stirred mixture of water (300 mL) and Et 2 O (600 mL).
  • Step B 4-Chloro-7-[3.5-bis-Q-(2,4-dichlorophenylmethyl)-2-C-ethyl- ⁇ -D- ribofuranosyll-7H-pyrrolo[2,3-(f1pyrimidine
  • Step C 4-Chloro-7-(2-C-ethyl- ⁇ -D-ribofuranosyn-7H-pyrrolo[2,3- flpyrimidine
  • dichloromethane 8 mL
  • boron trichloride 4.50 mL, 4.50 mmol
  • the mixture was stirred at -78°C for lh, then at -10°C for 3h.
  • reaction was quenched by addition of methanol/dichloromethane (1:1) (10 mL), stirred at -15°C for 30 min, and neutralized by addition of aqueous ammonium hydroxide. The mixture was evaporated under diminished pressure and the resulting oil purified on silica gel using methanol/dichloromethane (1:9) as eluent. Fractions containing the product were pooled and evaporated in vacuo to give the desired product (112 mg) as a colorless foam.
  • Step D 4-Amino-7-(2-C-ethyl- ⁇ -D-ribofuranosyl -7H-pyrrolo[2,3--
  • Step A 2-Amino-4-chloro-7-[3,5-bis-O-(2,4-dichlorophenvImethyl)-2-C- methyl- ⁇ -D-ribofuranosyl -7H-pyrrolo [2 ,3 - d ⁇ pyrimidine
  • HBr 5.7 M in acetic acid; 3 mL
  • Step B 2-Amino-4-chloro-7-(2-C-methyl- ⁇ -D-ribofuranosyl -7H-pyrrolor2,3- d ⁇ pyrimidine
  • Step C 2-Amino-7-(2-C-methyl- ⁇ -D-ribofuranosyl -7H-pyrrolo[2,3- ⁇ f1pyrimidin-4(3H)-one
  • S-Acyl-2-thioethyl (SATE) pronucleotides are discussed in C.R. Wagner, N.N. Iyer, and E.I. Mclntee, "Pronucleotides: Toward the In Nivo Delivery of Antiviral and Anticancer Nucleotides," Med. Res. Rev., 20: 1-35 (2000), which is incorporated by reference herein in its entirety.
  • SATE derivatives of nucleosides are also disclosed U.S. Patent Nos. 5,770,725; 5,849,905; and 6,020,482, the contents of each of which are incorporated by reference herein in their entirety.
  • 2-Mercaptoethanol (5 g, 64 mmol) was dissolved in CH 2 C1 2 (50 mL). To this solution was added triethylamine (7.67 mL, 57.6 mmol), and the reaction mixture was cooled in an ice bath to 0 °C. Acetic anhydride (4.54 mL, 48 mmol) was added dropwise in 10 min, and the reaction mixture was stirred for 1 h at 0 °C. The reaction mixture was then allowed to come to room temperature over a period of 2 h.
  • the reaction mixture was diluted with CH 2 C1 2 (50 mL), washed with water (75 mL), 5% aqueous ⁇ aHCO 3 (75 mL) and brine (75 mL).
  • the organic phase was dried over anhydrous Na 2 SO and concentrated in vacuo to give an oil.
  • the oil was then dissolved in anhydrous THF (40 mL) and anhydrous triethylamine (7.76 mL) was added.
  • activated molecular sieves (4 A) was kept at room temperature for 10 min.
  • the reaction mixture was cooled in an ice bath to 0°C and diisopropylphosphoramidous dichloride (6.47 g, 32.03 mmol) was added.
  • the reaction mixture was stirred at 0 °C for 2 h under inert atmosphere. Hexane (40 mL) was added to the reaction mixture and the precipitate formed was filtered. The filtrate was concentrated to one fourth of the volume, purified by loaded silica gel column chromatography and eluted with hexane containing 3 % triethylamine and incremental amount of ethyl acetate (0 to 7 %) to give the title compound as an oil (2.36 g).
  • nucleoside 5'-triphosphate Derivatives The nucleoside 5'-triphosphates of the present invention were prepared following general procedures described in Chem. Rev. 100: 2047 (2000).
  • the triphosphate derivatives were purified by anion exchange (AX) chromatography using a 30 x 100 mm Mono Q column (Pharmacia) with a buffer system of 50 mM Tris, pH 8. Elution gradients were typically from 40 mM NaCl to 0.8 M NaCl in two column volumes at 6.5 mL/min. Appropriate fractions from anion exchange chromatography were collected and desalted by reverse-phase (RP) chromatography using a Luna C18 250 x 21 mm column (Phenomenex) with a flow rate of 10 mL/min. Elution gradients were generally from 1% to 95% methanol in 14 min at a constant concentration of 5 mM triethylammonium acetate (TEAA).
  • AX anion exchange
  • RP reverse-phase
  • Mass spectra of the purified triphosphates were determined using on- line HPLC mass spectrometry on a Hewlett-Packard (Palo Alto, CA) MSD 1100.
  • a Phenomenex Luna (C18(2)), 150 x 2 mm, plus 30 x 2 mm guard column, 3- ⁇ m particle size was used for RP HPLC.
  • a 0 to 50% linear gradient (15 min) of acetonitrile in 20 mM TEAA (triethylammonium acetate) pH 7 was performed in series with mass spectral detection in the negative ionization mode. Nitrogen gas and a pneumatic nebulizer were used to generate the electrospray.
  • the mass range of 150- 900 was sampled. Molecular masses were determined using the HP Chemstation analysis package.
  • the purity of the purified triphosphates was determined by analytical RP and AX HPLC.
  • Triphosphates were eluted with a gradient of 0 to 0.4 M NaCl at constant concentration of 50 mM Tris, pH 8. The purity of the triphosphates was generally >80%.
  • nucleoside 5'-monophosphates of the present invention were prepared following the general procedures described in Tetrahedron Lett. 50: 5065 (1967). EXAMPLE 12
  • Step A 2-Amino-4-chloro-7-[3,5-bis-0-(2,4-dichlorophenylmethyl -2-C- methyl- ⁇ -D-ribofuranosyl]-5-methyl-7H-py ⁇ olor2,3- ⁇ i1pyrimidine
  • HBr 5.7 M in acetic acid; 3.3 mL
  • Step B 2-Amino-4-chloro-7-r3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C,2-0- dimethyl- ⁇ -D-ribofaranosyl]-5-methyl-7H-pyrrolor2,3- ⁇ i]pyrimidine
  • Na ⁇ 60% in mineral oil, 29 mg
  • Mel 48 ⁇ L
  • reaction mixture stirred at room temperature for 24 h.
  • the reaction was quenched with MeO ⁇ and the mixture evaporated.
  • Step C 2-Amino-7-r3.5-bis-O-(2,4-dichlorophenylmethyl)-2-C,2-C>-dimethyl- ⁇ -D-riboft ⁇ ranosyl]-5-methyl-7H-pyrrolo[2.3- ⁇ pyrimidine-4(3H -one
  • Step D 2-Amino-5-methyl-7-(2-C,2-0-dimethyl- ⁇ -D-ribofuranosyl -7H- pyrrolo[2,3- ⁇ lpyrimidin-4(3H)-one
  • MeO ⁇ 5 mL
  • Et 3 N 0.2 mL
  • 10% Pd/C 24 mg
  • Step A 4-Chloro-7-r3,5-bis-O-(2,4-dichloro ⁇ henylmethyl)-2-C-methyl- ⁇ -D- ribofuranosyl]-5-methyl-7H-pyrrolor2,3-(J1pyrirnidine
  • HBr 5.7 M in acetic acid; 2.2 mL
  • the reaction mixture was stirred at 0 °C for 1 h and then at room temperature for 2 h, concentrated in vacuo and co-evaporated with toluene (2 x 15 mL).
  • Step C 4-A ⁇ mno-5-methyl-7-(2-C-methyl- ⁇ -D-ribofuranosyl)-7H-pyrrolo[2,3-
  • Step A 3,5-Bis-O-(2,4-dichlorophenylmethyl)-2-C-vinyl-l-Q-methyl- ⁇ -D- ribofuranose Cerium chloride heptahydrate (50 g, 134.2 mmol) was finely crushed in a pre-heated mortar and transferred to a round-bottom flask equipped with a mechanical stirrer. The flask was heated under high vacuum overnight at 160°C. The vacuum was released under argon and the flask was cooled to room temperature. Anhydrous THF (300 mL) was cannulated into the flask. The resulting suspension was stirred at room temperature for 4 h and then cooled to -78 °C.
  • Step B 4-Chloro-7-[3,5-bis-( -(2,4-dichlorophenylmethyl)-2-C-vinyl- ⁇ -D- ribofuranosyl]-7H-pyrrolo[2,3- ⁇ flpyrimidine
  • ⁇ Br (30% solution in AcO ⁇ , 20 mL, 75.6 mmol
  • Step C 4-Amino-7-r3,5-bis-6>-(2,4-dichlorophenylmethyl)-2-C-vinyl- ⁇ -D- ribofuranosyl]-7H-pyrrolor2,3--f
  • the compound from Step B (80, mg) was dissolved in the minimum amount of 1,4-dioxane and placed in a stainless steel bomb. The bomb was cooled to -78°C and liquid ammonia was added. The bomb was sealed and heated at 90°C for 24 h. The ammonia was allowed to evaporate and the residue concentrated to a white solid which was used in the next step without further purification.
  • Step D 4-Amino-7-(2-C-vinyl- ⁇ -D-ribofuranosyl)-7H-pyrrolor2,3- c ⁇ pyrimidine
  • Step A 4-Chloro-7-r3,5-bis-0-(2,4-dichlorophenylmethyl -2-C- hydroxymethyl- ⁇ -D-ribofuranosyl]-7H-py ⁇ olo[2,3- ⁇ pyrimidine
  • Step B 300 mg, 0.48 mmol
  • N-methylmorpholine-N-oxide 300 mg, 2.56 mmol
  • osmium tetroxide 4% solution in water, 0.3 mL.
  • the precipitate was removed by filtration through a celite plug, diluted with water (3 x), and extracted with EtOAc.
  • Step B 4-Amino-7-[3,5-bis-0-(2,4-dichloiOphenylmethyl)-2-C- hydroxymethyl- ⁇ -D-ribofuranosyl1-7H-pyrrolo[2,3-rf1pyrimidine
  • Step A The compound from Step A (150 mg, 0.23 mmol) was dissolved in the minimum amount of 1,4-dioxane (10 mL) and placed in a stainless steel bomb. The bomb was cooled to -78 °C and liquid ammonia was added. The bomb was sealed and heated at 90°C for 24 h. The ammonia was allowed to evaporate and the residue concentrated to a white solid which was used in the next step without further purification.
  • Step C 4-Amino-7-(2-C-hydroxymethyl- ⁇ -D-ribofuranosyl)-7H-pyrrolo[2,3-
  • Step A 4-Chloro-7-r3,5-bis-O-(2,4-dichlorophenylmethyl -2-C-fluoromethyl- ⁇ -D-ribofuranosyn-7H-pyrrolo[2,3-i]pyrimidine
  • DMAP 4- dimethylaminopyridine
  • 62 ⁇ L, 0.45 mmol 4- dimethylaminopyridine
  • the solution was cooled in an ice bath and p-toluenesulfonyl chloride (30 mg, 0.15 mmol) was added.
  • Step B 4-Amino-7-r3,5-bis-O-(2.4-dichlorophenylmethyl)-2-C-fluoromethyl- ⁇ -D-ribofuranosyl]-7H-pyrrolor2,3-(f
  • the compound from Step A (18 mg, 0.03 mmol) was dissolved in the minimum amount of 1,4-dioxane and placed in a stainless steel bomb. The bomb was cooled to -78 °C and liquid ammonia was added. The bomb was sealed and heated at 90 °C for 24 h. The ammonia was allowed to evaporate and the residue concentrated to a white solid which was used in the next step without further purification.
  • Step C 4-Amino-7-(2-C-fluoromethyl- ⁇ -D-ribofuranosyl ' )-7H-pyrrolor2,3- . ⁇ pyrimidine
  • Step A 7-[2,5-Bis-0-(tgrt-butyldime ⁇ hylsilyl s >- ⁇ -D-ribofuranosyl]-7H- pyrrolo[2,3-(flpyrimidine and 7-[3,5-Bis-Q-(tert-butyldimethylsilyl - ⁇ - D-ribofuranosyl1-7H-pyrrolor2,3--t1 pyrimidine
  • tubercidin 5.0 g, 18.7 mmol
  • DMF 18.5 mL
  • Step B 7-[2,5-Bis-0-(tert-butyldimethylsilyl - ⁇ -D-ribofuranosyl ]-4-[di-(4- methoxyphenyPphenylmethyll an ⁇ ino-7H-pyrrolor2,3- ⁇ pyrimidine and 7-[3,5-bis-Q-(tert-butyldimethylsilyl - ⁇ -D-ribofuranosyll-4-rdi-(4- methoxyphenyl)phenylmethyl1amino-7H-pyrrolor2,3- ⁇ ilpyrimidine
  • 4'-dimethoxytrityl chloride 2.8 g, 8.2 mmol
  • Step C 7-[2,5-Bis-O-(tert-butyldimethylsilyI)-3-0-tosyl- ⁇ -D-ribofuranosyl)]-
  • Step D 4-[di-(4-methoxyphenyl)phenylmethyl]amino-7-F3-O-tosyl- ⁇ -D- ribofuranosyl-7H-pyrrolor2,3- ⁇ lpyrimidine
  • IM solution in THF tetrabutylammonium fluoride
  • the mixture was cooled and diluted with ether (50 mL).
  • the solution was washed with water (3 x 50 mL), dried over anhydrous Na 2 SO , and concentrated to an oil.
  • the residue was purified by passing through a pad of silica gel using hexane: ethyl acetate (1:1) as eluent; yield 780 mg.
  • Step E 4-Amino-7-(3-deoxy-2-C-methyl- ⁇ -D-ribofuranosyl -7H-pyrrolo[2,3- d ⁇ - pyrimidine and 4-amino-7-(3-deoxy-2-C-methyl- ⁇ -D- arabinofuranosyl)-7H-pyrrolo-[2,3- ⁇ i]pyrimidine
  • a solution of C ⁇ 3 MgI (3.0 M solution in ether, 3.0 mL) in anhydrous toluene (3.75 mL) was cooled in an ice bath. To this was added a solution of the compound from Step D (500 mg, 0.8 mmol) in anhydrous toluene (3.7 mL).
  • the acetic acid was removed by repeated co-evaporation with ethanol and toluene. The residue was partitioned between chloroform and water. The aqueous layer was washed with chloroform and concentrated. The evaporated residue was purified on silica gel using 5-10% MeOH in CH 2 C1 2 as the eluent to furnish the desired compounds as white solids.
  • Step A 5-Deoxy-l,2-( -isopropylidene-D-xylofuranose
  • 1,2-O-Isopropylidene-D-xylofuranose 38.4 g, 0.2 mol
  • 4- dimethylaminopyridine 5 g
  • triethylamine 55.7 mL, 0.4 mol
  • dichloromethane 300 mL
  • p-Toluenesulfonyl chloride 38.13 g, 0.2 mol
  • the reaction mixture was stirred at room temperature for 2 h.
  • the reaction mixture was then poured into saturated aqueous sodium bicarbonate (500 mL) and the two layers were separated.
  • Step B 3,5-Bis-O-(2,4-dichlorophenylmethyl)-l-Q-methyl-4-methyl- ⁇ -D- ribofuranose
  • Step C 3,5-Bis-Q-(2,4-dichlorophenylmethyl -2,4-di-C-methyl-l-O-methyl- ⁇ -
  • Step D 4-Chloro-7-[3,5-bis-0-(2,4-dichlorophenylmethyl)-2,4-di-C-methyl- ⁇ - D-ribofuranosvH-7H-pyrrolo[2,3 ⁇ f1pyrimidine
  • Step E 4-Amino-7-(2,4-C-dimethyl- ⁇ -D-ribofuranosyl)-7H-pyrrolo[2,3-
  • Step A 3-Deoxy-3-fluoro-l-0-methyl-5-0-toluoyl- ⁇ -D-ribofuranose
  • 1,2-O-Isopropylidene-D-xylofuranose 9.0 g, 50 mmol
  • -toluoyl chloride 7.0 mL, 50 mmol
  • pyridine 50 mL
  • Water (10 mL) was added and the mixture was concentrated under reduced pressure.
  • the residue was dissolved in toluene (500 mL) and the solution was washed with water (200 mL) and saturated aqueous sodium bicarbonate (200 mL). The two layers were separated and the organic layer was evaporated.
  • the residue was dissolved in methanol (100 mL) and HCl in dioxane (4 M, 10 mL) was added.
  • Step B 3-Deoxy-3-fluoro-2-C-methyl-l-O-methyl-5-0-toluoyl- ⁇ -D- ribofuranose
  • Step C 4-Amino-7-(3-deoxy-3-fluoro-2-C-methyl- ⁇ -D-ribofuranosyl ' )-7H- pyrrolo[2,3-(i1pyrimidine
  • Step A 4-chloro-7-[3,5-bis-6>-(2,4-dichlorophenylmethyl)-2-C.2-Q-dimethyl- ⁇ -D-ribofuranosyl]-7H-pyrrolo[2,3-(flpyrimidine
  • Step D 6-chloro-7-[3,5-bis-6>-(2,4-dichlorophenylmethyl)-2-C.2-Q-dimethyl- ⁇ -D-ribofuranosyl]-7H-pyrrolo[2,3-(flpyrimidine
  • Step B 4-amino-7-r3,5-bis-O-(2,4-dichlorophenylmethyl -2-C2-0-dimethyl- ⁇ -D-ribofuranosyl1-7H-pyrrolo[2,3- ⁇ f
  • methanolic ammonia saturated at 0°C
  • the mixture was heated in a stainless steel autoclave at 80°C overnight, then cooled and the content evaporated in vacuo.
  • the crude mixture was purified on silica gel using ethyl acetate/hexane as the eluent. Fractions containing the product were pooled and evaporated in vacuo to give the desired product (504 mg) as colorless foam.
  • Step C 4-amino-7-(2-C.2-O-dimethyl- ⁇ -D-ribofuranosylV7H-pyrrolo[2,3- d] pyrimidine
  • Step A 7-r2,5-Bis-6-(tert-butyldimethylsilyl - ⁇ -D-ribofuranosyl s )1-4-[(4- methoxyphenyl)diphenylmethyl]amino-7H-pyrrolo[2,3-J1pyrimidine and 7-[3,5-bis-0-(tert-butyldimethylsilyl - ⁇ -D-ribofuranosyl1-4-[(4- methoxyphenyl)diphenylmethyl1amino-7H-pyrrolo[2,3-6 ⁇ pyrimidine
  • monomethoxytrityl chloride (0.30 g, 0.98 mmol
  • Step B 7-[2,5-Bis-( -(tert-butyldimethylsilyl - ⁇ -D-ervt ⁇ ro-pentofuranos-3- ulosyl]-4-[(4-methoxyphenyl)diphenylmethynamino-7H-pyrrolo[2,3- lpyrimidine
  • chromium trioxide 9 mg, 0.91 mmol
  • C ⁇ 2 C1 2 4 0°C
  • pyridine 147 ⁇ L, 1.82 mmol
  • acetic anhydride 86 ⁇ L, 0.91 mmol
  • Step C 7-r2,5-Bis-O-(tert-butyldimethylsilyl)-3-C-methyl- ⁇ -D-ribofuranosyl -
  • Step D 4-Amino-7-(3-C-methyl- ⁇ -D-xylofuranosyl -7H-pyrrolo[2,3-
  • Step A 3,5-Bis-O-(2,4-dichlorobenzyl -l-0-methvI- ⁇ -D-ribofuranose (1-2)
  • Step B 3,5-Bis-Q-(2,4-dichlorobenzyl)-l-O-methyl- ⁇ -D-eryt/ ⁇ ro- pentofuranos-2-ulose (2-3)
  • Dess-Martin periodinane 50.0 g, 118 mmol
  • anhydrous CH 2 C1 350 mL
  • argon (Ar) a solution of the compound from Step A (36.2 g, 75 mmol) in anhydrous CH 2 C1 2 (200 mL) dropwise over 0.5 h.
  • the reaction mixture was stirred at 0°C for 0.5 h and then at room temperature for 3 days.
  • Step C 3,5-Bis-Q-(2,4-dichlorobenzyl)-2-C-methyl-l-Q-methyl- ⁇ -D- ribofuranose (2-4) To a solution of MeMgBr in anhydrous Et 2 O (0.48 M, 300 mL) at
  • Step D 4-Chloro-7-[3,5-bis-0-(2,4-dichlorobenzyl)-2-C-methyl- ⁇ -D- ribofuranosyn-7H-pyrrolo[2,3- ⁇ lpyrimi dine (2-5)
  • ⁇ Br 5.7 M in acetic acid, 20 mL, 114 mmol
  • Step E 4-Chloro-7-(2-C-methyl- ⁇ -D-ribofuranosvn-7H-pyrrolo[2,3-
  • Step F 4-Amino-7-(2-C-methyl- ⁇ -D-ribomranosv ⁇ V7H-pyrrolor2,3- lpyrimidine (2-7)
  • Step E To the compound from Step E (1.54 g, 5.1 mmol) was added methanolic ammonia (saturated at 0°C; 150 mL). The mixture was heated in a stainless steel autoclave at 85°C for 14 h, then cooled and evaporated in vacuo. The crude mixture was purified on a silica gel column with C ⁇ C ⁇ /MeO ⁇ (9/1) as eluent to give the title compound as a colorless foam (0.8 g), which separated as an amorphous solid after treatment with MeCN. The amorphous solid was recrystallized from methanol/acetonitrile; m.p. 222°C.
  • Step G 4-Amino-7-r5-0-(tert-butyldimethylsilyl -2-C-methyl- ⁇ -D- ribofuranosyll-7H-pyrrolor2,3- ⁇ flpyrimidine (2-8)
  • Step H 4-(p-Methoxyphenyldiphenylmethylamino)-7-[5-( -(tert- butyldimethylsilyl)-2-C-methyl- ⁇ -D-ribofuranosyl]-7H-pyrrolo[2,3- d ⁇ pyrimidine (2-9)
  • Step I 4-(p-Methoxyphenyldiphenylmethylamino)-7-[5-O-(tert- butyldimethylsilyl)-3-O-(l-oxo-octyl)-2-C-methyl- ⁇ -D-ribofuranosvn- 7H-pyrrolo[2,3-(flpyrimidine (2-10)
  • To a solution of the compound from Step ⁇ (400 mg, 0.6 mmol) and anhydrous DMAP (73 mg, 0.6 mmol) in anhydrous C ⁇ 2 ⁇ 2 (7 mL) was added slowly triethylamine (250 ⁇ L, 1.8 mmol).
  • Step J 4-Amino-7-r5-0-(tert-butyldimethylsilyl -3-0-(l-oxo-octyl)-2-C- methyl- ⁇ -D-ribofuranosyl]-7H-pyrrolo[2,3-( lpyrimidine (2-11)
  • Step K 4-Amino-7-r2-C-methyl-3-0-(l-oxo-octyl - ⁇ -D-ribofuranosyl]-7H- pyrrolo[2,3- ⁇ flpyrimidine (2-12)
  • Step A 4-(p-Methoxyphenyldiphenylmethylamino)-7-[3-0-(l-oxo-octyl)-2-C- methyl- ⁇ -D-ribofuranosyl]-7H-pyrrolo[2,3-(i]pyrimidine (3-1)
  • a solution of the compound from Step I of Example 1 (1-10) (300 mg,
  • Step B 4-(p-Methoxyphenyldiphenylmethylamino)-7-r3,5-di-0-(l-oxo-octyl)-
  • the reaction mixture was stirred at this temperature for 2 h, diluted with CH2CI2 (20 mL) and washed with saturated aqueous sodium bicarbonate solution (2 x 10 mL) followed by water (10 mL).
  • the crude product obtained after evaporation was purified by column chromatography on silica gel eluting with 10% acetone in CH 2 C1 2 .
  • the title compound was obtained as a colorless foam (13.5 mg).
  • Step C 4-Amino-7-[2-C-methyl-3,5-di-0-(l-oxo-octyl)- ⁇ -D-ribofuranosyl]- 7H-pyrrolo[2,3--flpyrimidine (3-3)
  • Step A 5-Bromo-4-chloro-7H-pyrrolo[2,3- ⁇ lpyrimidine (4-2) To a solution of 4-chloro-7H-pyrrolo[2,3-( ]pyrimidine (4-1) (1.53 g,
  • Step B 5-(Trimethylstannyl)-4-chloro-7H-pyrrolo[2,3- ⁇ flpyrimidine (4-3)
  • Step C 5-Fluoro-4-chloro-7H-pyrrolo[2,3- ⁇ i]pyrimidine (4-4) To a solution of the compound from Step B (1.97 g, 6.20 mmol) in
  • Step D 4-Amino-5-fluoro-7-(2-C-methyl- ⁇ -D-ribofuranosyl -7H-pyrrolor2.3- (ilpyrimidine (4-7)
  • Step A 2-Amino-6-chloro-9-(2,3,5-tri-O-benzoyl-2-C-methyl- ⁇ -D- ribofuranosvDpurine
  • Step B 2-Amino-6-chloro-9-(2-C-methyl- ⁇ -D-ribofuranosyl)purine
  • Step C 2,6-Diamino-9-(2-C-methyl- ⁇ -D-ribofuranosyl purine To the compound from Step B (100 mg) was added ammonium hydroxide (5 mL) and the resulting mixture was stirred at 80°C in a Parr bomb overnight. The mixture was cooled and evaporated in vacuo and adsorbed onto silica and purified on silica gel using methanol/dichloromethane (1:4) as the eluent. Fractions containing the product were combined and evaporated in vacuo to give the desired product as a colorless powder.
  • Step D 6-Amino-2-fluoro-9-(2-C-methyl- ⁇ -D-ribofuranosyl)purine
  • nucleoside derivatives of the present invention were also evaluated for cellular toxicity and antiviral specificity in the counterscreens described below.
  • Reaction buffer components 200 ⁇ g/mL bovine serum albumin 100 mM KCl 2 mM ⁇ -mercaptoethanol 10 mM MgCl 2
  • 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 KCl. 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 h 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.
  • % inhibition [l-(cpm in test reaction - cpm in blank)/(cpm in control reaction - cpm in blank)] x 100.
  • 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.
  • the ability of the purine nucleoside derivatives of the present invention to inhibit HTV infectivity and HTV spread was measured in the following assays.
  • HIV-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. d. Inhibition of HIV Spread
  • the 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).
  • Cells were plated at 15-20,000 cells/well in 100 ⁇ L of appropriate media and incubated 18 h at 37°C, 5% CO 2 .
  • 100 ⁇ L of compound diluted in complete media was added to the cells for a final of 1% DMSO concentration.
  • the plates were incubated at 37°C and 5% CO 2 for 24 h.
  • 40 ⁇ L of CellTiter 96 Aqueous One Solution Cell Proliferation Assay reagent (MTS) (Promega) was added to each well, and the plates were incubated at 37°C and 5% CO 2 for 1 h.
  • the plates were agitated to mix well and absorbance at 490 nm was read using a plate reader. Metabolically active cells reduce MTS to formazan.
  • Formazan absorbs at 490 nm.
  • the absorbance at 490 nm in the presence of compound was compared to absorbance in cells without any compound added.
  • 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 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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Abstract

The present invention provides methods of inhibiting orthopoxvirus replication and/or treating orthopoxvirus infection with certain nucleoside compounds and derivatives thereof. These compounds are particularly useful as inhibitors of vaccinia virus and variola virus replication and/or for the treatment of vaccinia virus and variola virus infection. The nucleoside compounds may be administered alone or in combination with other agents active against orthopoxvirus infection, in particular against vaccinia virus or variola virus infection. Another aspect of the present invention provides for the use of such nucleoside compounds in the manufacture of a medicament for the inhibition of orthopoxvirus replication and/or for the treatment of orthopoxvirus infection. Yet a further aspect of the present invention provides such nucleoside compounds for use as a medicament for the inhibition of orthopoxvirus replication and/or for the treatment of orthopoxvirus infection.

Description

TITLE OF THE INVENTION
METHODS OF INHTBITΓNG ORTHOPOXVIRUS REPLICATION WITH
NUCLEOSIDE COMPOUNDS
FIELD OF THE INVENTION
The present invention is concerned with methods of inhibiting orthopoxvirus replication and methods for treating orthopoxvirus infections with certain nucleoside compounds and derivatives thereof. The compounds are particularly useful for inhibiting the replication of vaccinia, variola, cowpox, and monkeypox virus and for the treatment of vaccinia, variola, cowpox, and monkeypox virus infections. Another aspect of the present invention provides for the use of the nucleoside compounds and derivatives thereof and their pharmaceutical compositions for the manufacture of a medicament for the inhibition of orthopoxvirus replication and/or for the treatment of orthopoxvirus infection. Yet a further aspect of the present invention provides for the nucleoside compounds and derivatives thereof and their pharmaceutical compositions for use as a medicament for the inhibition of orthopoxvirus replication and/or for the treatment of orthopoxvirus infection.
BACKGROUND OF THE INVENTION Orthopoxvirus is a genus of the Poxviridae family of complex DNA viruses that replicate in the cytoplasm of vertebrate and invertebrate cells. The Poxviridae family is characterized by having a large complex virion containing enzymes that synthesize mRNA, a genome composed of a single linear double- stranded DNA molecule of 130-300 kilobase pairs with a hairpin loop at each end, and a cytoplasmic site of replication. Members of the orthopoxvirus genus include cowpox, monkeypox, vaccinia, and variola virus [for a description of the Poxviridae family, reference is made to B. Moss, "Poxviridae: The Viruses and Their Replication," in Fields Virology. B.N. Fields, et al., Eds., 3rd ed., Ch. 83, pages 2637- 2671 (1996)]. Variola virus is the agent responsible for smallpox infections. Smallpox infections were effectively eradicated subsequent to the introduction of prophylactic vaccinations with cowpox and vaccinia virus. However, most of the human populations worldwide are no longer immune to smallpox as a result of the discontinuation of routine vacination in the early 1980' s.
There are very few compounds available as therapeutics against orthopoxvirus infections. Two drugs under investigation are cidofovir and ribavirin. Cidofovir is the generic name for (S)-l-[3-hydroxy-2-(phosphonylmethoxy)- propyl]cytosine [(S)-HPMPC] which is currently the leading agent for the treatment of orthopoxvirus infections in humans. It is potent against vaccinia and cowpox virus infection in mice when administered subcutaneously or intraperitoneally. However, cidofovir' s therapeutic utility is limited by safety concerns as well as lack of oral bioavailablity [see D.F. Smee et al., "Effects of cidofovir on the pathogenesis of a lethal vaccinia virus respiratory infection in mice," Antiviral Res., 52: 55-62 (2001) and references cited therein]. Inhibition of vaccinia virus is considered in the art to be predictive of inhibitory activity against other orthopo viruses, including variola; see E. De Clercq, "Vaccinia Virus Inhibitors as a Paradigm for the Chemotherapy of Poxvirus Infections," Clin. Microbiol. Rev.. 14: 382-397 (2001).
Ribavirin has also been found to inhibit vaccinia virus and other orthopoxvirus replication in cell culture (see J.H. Huffman et al., "In vitro effect of 1- β-D-ribofuranosyl-l,2,4-triazole-3-carboxarnide on DNA and RNA viruses," Antimicrobial Agents and Chemotherapy, 3: 235-241 (1973) and D.F. Smee et al., "Potential of the IMP dehydrogenase inhibitors for antiviral therapies of poxvirus infections," Antiviral Res., 37: A89 (1998)]. Ribavirin was also reported to suppress vaccinia virus-induced lesions in a mouse model and to effectively treat vaccinia keratitis in rabbits. However, ribavirin causes anemia during prolonged administration and at high doses has certain immunosuppressive properties limiting its clinical usefulness against orthopoxvirus.
Consequently, there exists a need for more effective anti-orthopoxvirus agents particularly as a result of the threat of either variola (smallpox) or monkeypox viruses in biowarfare or bioterrorism. Preferably such agents should be effective when administered orally and be safe and well-tolerated by the host.
It has now been found that nucleoside compounds of the present invention and certain derivatives thereof are potent inhibitors of orthopoxvirus replication and in particular of vaccinia, variola, cowpox, and monkeypox virus replication. The instant nucleoside compounds and derivatives thereof are useful to treat orthopoxvirus infection and in particular vaccinia, variola, cowpox, and monkeypox virus infection.
It is therefore an object of the present invention to provide nucleoside compounds and certain derivatives thereof which are useful as inhibitors of the replication of orthopoxvirus and in particular as inhibitors of the replication of vaccinia, variola, cowpox, and monkeypox virus. It is another object of the present invention to provide nucleoside compounds and certain derivatives thereof which are useful in the treatment of orthopoxvirus infection and in particular in the treatment of vaccinia, variola, cowpox, and monkeypox virus infection. It is another object of the present invention to provide pharmaceutical compositions comprising the nucleoside compounds of the present invention in association with a pharmaceutically acceptable carrier.
It is another object of the present invention to provide pharmaceutical compositions comprising the nucleoside compounds and derivatives of the present invention for use as inhibitors of orthopoxvirus replication and in particular as inhibitors of vaccinia, variola, cowpox, and monkeypox virus replication.
It is another object of the present invention to provide pharmaceutical compositions comprising the nucleoside compounds and derivatives of the present invention for use in the treatment of orthopoxvirus infection and in particular in the treatment of vaccinia, variola, cowpox, and monkeypox virus infection.
It is another object of the present invention to provide pharmaceutical compositions comprising the nucleoside compounds and derivatives of the present invention in combination with other agents active against orthopoxvirus and in particular against vaccinia, variola, cowpox, and monkeypox virus. It is another object of the present invention to provide methods for the inhibition of orthopoxvirus replication and in particular for the inhibition of vaccinia, variola, cowpox, and monkeypox virus replication.
It is another object of the present invention to provide methods for the treatment of orthopoxvirus infection and in particular for the treatment of vaccinia, variola, cowpox, and monkeypox virus infection.
It is another object of the present invention to provide methods for the treatment of orthopoxvirus infection in combination with other agents active against orthopoxvirus and in particular for the treatment of vaccinia, variola, cowpox, and monkeypox virus infection in combination with other agents active against vaccinia, variola, cowpox, and monkeypox virus infection.
It is another object of the present invention to provide nucleoside compounds and certain derivatives thereof and their pharmaceutical compositions for use as a medicament for the inhibition of orthopoxvirus replication and/or the treatment of orthopoxvirus infection and in particular for the inhibition of vaccinia, variola, cowpox, and monkeypox virus replication and/or the treatment of vaccinia, variola, cowpox, and monkeypox virus infection.
It is another object of the present invention to provide for the use of the nucleoside compounds and certain derivatives thereof of the present invention and their pharmaceutical compositions for the manufacture of a medicament for the inhibition of orthopoxvirus replication and/or the treatment of orthopoxvirus infection and in particular for the inhibition of vaccinia, variola, cowpox, and monkeypox virus replication and/or the treatment of vaccinia, variola, cowpox, and monkeypox virus infection. These and other objects will become readily apparent from the detailed description which follows.
SUMMARY OF THE INVENTION
The present invention provides a method for inhibiting orthopoxvirus replication and/or a method for treating orthopoxvirus infection in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of a compound of structural formula I:
Figure imgf000005_0001
(I)
or a pharmaceutically acceptable salt thereof; wherein A is N or C-R9;
Rl is C2-4 alkenyl, C2-4 alkynyl, or Ci_4 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino, Ci_4 alkoxy, Cι_4 alkylthio, or one to three fluorine atoms; R2 is amino, fluorine, hydroxy, CI_IQ alkylcarbonyloxy, mercapto, or Ci-4 alkoxy; R3 and R4 are each independently selected from the group consisting of hydrogen, cyano, azido, halogen, hydroxy, Cι_i6 alkylcarbonyloxy, C2-I8 alkenylcarbonyloxy, Cl-lO alkyloxycarbonyloxy, C3-6 cycloalkylcarbonyloxy,
C3-6 cycloalkyloxycarbonyloxy, mercapto, amino, C1-.4 alkoxy, C2-4 alkenyl, C2-4 alkynyl, and Cχ-4 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino, Ci-4 alkoxy, Ci-4 alkylthio, or one to three fluorine atoms; R5 is hydrogen, Cι_i6 alkylcarbonyl, C2-I8 alkenylcarbonyl, Cl-lO alkyloxycarbonyl, C3-6 cycloalkylcarbonyl, C3-6 cycloalkyloxycarbonyl, P3O9H4, P2O6H3, or P(O)Rl3Rl4; 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, Cι_4 alkylamino, di(Cι_4 alkyl)amino, hydroxy, Cι_6 alkoxy, Ci_6 alkylthio, C\. alkylsulfonyl, or (C1-.4 alkyl)θ-2 aminomethyl; R9 is hydrogen, cyano, nitro, NHCONH2, CONR12R12, CSNR12R12, COOR12, C(=NH)NH2, hydroxy, Ci-3 alkoxy, amino, Cι_4 alkylamino, di(Ci-4 alkyl)amino, halogen, or Ci-3 alkyl, wherein alkyl is unsubstituted or substituted with one to three groups independently selected from halogen, amino, hydroxy, carboxy, and Ci-3 alkoxy;
RlO and RU are each independently hydrogen, hydroxy, mercapto, halogen, Ci-4 alkoxy, Cι_4 alkylthio, amino, Ci-4 alkylamino, di(Cι_4 alkyl)amino, C3-6 cycloalkylamino, di(C3_6 cycloalkyl)amino, phenyl-Ci-2 alkylamino, Ci-4 acylamino, Cι_8 alkylcarbonyloxy, or OCH(Cι_4 alkyl)O(C=O)Cι_4 alkyl; each Rl2 is independently hydrogen or Cι_6 alkyl; and Rl3 and Rl4 are each independently hydroxy, OCH2CH2SC(=O)Cι_4 alkyl, OCH2θ(C=O)OCi_4 alkyl, NHCHMeCO2Me, OCH(Ci-4 alkyl)O(C=O)Cι_4 alkyl,
Figure imgf000006_0001
Also encompassed within the present invention are pharmaceutical compositions containing the compounds alone or in combination with other agents active against orthopoxvirus and in particular against vaccinia, variola, cowpox, and monkeypox virus as well as methods for the inhibition of orthopoxvirus replication and for the treatment of orthopoxvirus infection with such compositions. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for inhibiting orthopoxvirus replication and/or a method for treating orthopoxvirus infection in a mammal in need thereof comprising administering to the mammal a therapeutically effective amount of a compound of structural formula I:
Figure imgf000007_0001
(I)
or a pharmaceutically acceptable salt thereof; wherein A is N or C-R9;
Rl is C2-4 alkenyl, C2-4 alkynyl, or Cι_4 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino, Cι_4 alkoxy, Ci_4 alkylthio, or one to three fluorine atoms;
R2 is amino, fluorine, hydroxy, Cι_ιo alkylcarbonyloxy, mercapto, or Ci-4 alkoxy; R and R are each independently selected from the group consisting of hydrogen, cyano, azido, halogen, hydroxy, Cι_i6 alkylcarbonyloxy, C2-18 alkenylcarbonyloxy, Cl-10 alkyloxycarbonyloxy, C3-6 cycloalkylcarbonyloxy,
C3-6 cycloalkyloxycarbonyloxy, mercapto, amino, Ci_4 alkoxy, C2-4 alkenyl, C2-4 alkynyl, and Ci-4 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino, Cι_4 alkoxy, Cι_4 alkylthio, or one to three fluorine atoms; R5 is hydrogen, Cι_i6 alkylcarbonyl, C2-18 alkenylcarbonyl, Cl-lO alkyloxycarbonyl, C3-6 cycloalkylcarbonyl, C3-6 cycloalkyloxycarbonyl, P3O9H4, P2O6H3, or P(O)Rl3Rl4;
R6 and R7 are each independently hydrogen, methyl, hydroxymethyl, or fluoromethyl; R8 is hydrogen, Cι_4 alkyl, C2-4 alkynyl, halogen, cyano, carboxy, Ci-4 alkyloxycarbonyl, azido, amino, C1.4 alkylamino, di(Cι_4 alkyl)amino, hydroxy, Ci-6 alkoxy, Ci-6 alkylthio, Cι_6 alkylsulfonyl, or (Ci_4 alkyl)θ-2 aminomethyl; R9 is hydrogen, cyano, nitro, NHCONH2, CONR12R12, CSNR12R12, COOR12, C(=NH)NH2, hydroxy, Ci-3 alkoxy, amino, Ci-4 alkylamino, di(Cι_4 alkyl)amino, halogen, or Ci-3 alkyl, wherein alkyl is unsubstituted or substituted with one to three groups independently selected from halogen, amino, hydroxy, carboxy, and Ci_3 alkoxy;
RlO and RU are each independently hydrogen, hydroxy, mercapto, halogen, Ci-4 alkoxy, Ci-4 alkylthio, amino, Ci-4 alkylamino, di(Ci_4 alkyl)amino, C3-6 cycloalkylamino, di(C3_6 cycloalkyl)amino, phenyl-Cι_2 alkylamino, C1.4 acylamino, Cι_8 alkylcarbonyloxy, or OCH(Cι_4 alkyl)O(C=O)Cι_4 alkyl; each Rl2 is independently hydrogen or Cι_6 alkyl; and
Rl3 and Rl4 are each independently hydroxy, OCH2CH2SC(=O)Ci_4 alkyl,
OCH2θ(C=O)OCι_4 alkyl, NHCHMeCO2Me, OCH(Cι_4 alkyl)O(C=O)Cι_4 alkyl,
ιCH3
Figure imgf000008_0002
Figure imgf000008_0001
In one embodiment of the present invention is the method of inhibiting orthopoxvirus replication and/or treating orthopoxvirus infection with a compound of structural formula II which is of the stereochemical configuration:
Figure imgf000008_0003
or a pharmaceutically acceptable salt thereof; wherein A is N or C-R9;
Rl is Ci-3 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino, Ci-3 alkoxy, Cι_3 alkylthio, or one to three fluorine atoms; R2 is hydroxy, Ci-1 alkylcarbonyloxy, fluoro, or Ci-3 alkoxy; R3 is hydrogen, halogen, hydroxy, Cι_i6 alkylcarbonyloxy, amino, or Cι_3 alkoxy; R5 is hydrogen, Ci_i6 alkylcarbonyl, P3O9H4, P2O6H3, or PO3H2; R8 is hydrogen, amino, or C _4 alkylamino; R9 is hydrogen, cyano, methyl, halogen, or CONH2; and
RlO and RU are each independently hydrogen, halogen, hydroxy, amino, Cι_4 alkylamino, di(Cι_4 alkyl)amino, or C3.6 cycloalkylamino.
In a second embodiment of the present invention is the method of inhibiting orthopoxvirus replication and/or treating orthopoxvirus infection with a compound of structural formula II wherein
Rl is methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, or aminomethyl;
R2 is hydroxy, Cι_i6 alkylcarbonyloxy, fluoro, or methoxy;
R3 is hydrogen, fluoro, hydroxy, Cι_i6 alkylcarbonyloxy, amino, or methoxy; R5 is hydrogen, Ci-16 alkylcarbonyl, or P3O9H4;
R8 is hydrogen or amino;
R9 is hydrogen, cyano, methyl, halogen, or CONH2; and
RlO and Rl 1 are each independently hydrogen, halogen, hydroxy, amino, Cι_4 alkylamino, di(Cι_4 alkyl)amino, or C3-6 cycloalkylamino. Illustrative of the invention is a method for inhibiting orthopoxvirus replication and/or treating orthopox infection wherein the compound is selected from: 4-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-( ]pyrimidine, 4-methylamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-(i]pyrimidine, 4-dimethylamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3- ]pyrimidine, 4-cyclopropylamino-7-(2-C-methyl-β-D-ribomranosyl)-7H-pyrrolo[2,3-^pyrimidine, 4-amino-7-(2-C-hydroxymethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-<f]pyrimidine, 4-amino-7-(2-C-fluoromethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-( lpyrimidine, 4-amino-5-methyl-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-- lpyrimidine, 4-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-( ]pyrimidine-5- carboxamide,
4-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-( |pyrimidine-5- carbonitrile,
4-amino-5-bromo-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-( ]pyrimidine, 4-amino-5-chloro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrtolo[2,3--flpyrimidine, 4-amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-rf]pyrimidine, 2,4-diamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-( ]pyrimidine, 2-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-(i]pyrimidine, 2-amino-4-cyclopropylarmno-7-(2-C-memyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3- (fjpyrimidine,
2-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-<i]pyrimidin-4(3H)-one, 7-(2-C-methyl-β-D-ribomranosyl)-7H-pyrtθlo[2,3-- ]pyrimidin-4(3H)-one, 4-amino-2-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrtθlo[2,3-^pyrimidine, 9-(2-C-methyl-β-D-ribofuranosyl)-2-ammo-6-hydroxypurine, 9-(2-C-methyl-β-D-ribofuranosyl)-2-amino-6-cyclopropylaminopurine, 9-(2-C-methyl-β-D-ribofuranosyl)-2,6-diaminopurine, 9-(2-C-methyl-β-D-ribofuranosyl)-2-amino-6-methylaminopurine, 6-amino-2-fluoro-9-(2-C-methyl-β-D-ribofuranosyl)purine, 2' -C-methyl-adenosine, 4-amino-7-[2-C-methyl-3-O-(l-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3- ( jpyrimidine, and
4-amino-7- [2- C-methyl-3 ,5 -di- 0-( 1 -oxo-octyl)- β-D-ribofuranosyl] -7H-pyrrolo [2,3- < ]pyrimidine; and the corresponding 5'-triphosphates; or a pharmaceutically acceptable salt thereof.
Further illustrative of the invention is a method for inhibiting orthopoxvirus replication and/or treating orthopox infection wherein the compound is selected from: 4-aπήno-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-<i]pyrimidine, 4-amino-7-(2-C-fluoromethyl-β-D-ribofuranosyl)-7H-pyιτolo[2,3-^pyrimidine,
4-amino-5-methyl-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-(i]pyrimidine, 4-amino-5-bromo-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyι olo[2,3-d]pyrimidine, 4-amino-5-chloro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine, 4-amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-<i]pyrimidine, 4-armno-2-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine, 6-amino-2-fluoro-9-(2-C-methyl-β-D-ribofuranosyl)purine, 2'-C-methyl-adenosine,
4-anaino-7-[2-C-methyl-3-O-(l-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3- rf]pyrimidine, and 4-amino-7-[2-C-methyl-3,5-di-0-(l-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-
<2]pyrimidine; and the corresponding 5'-triphosphates; or a pharmaceutically acceptable salt thereof. In a third embodiment of the methods of the present invention, the orthopoxvirus replication is selected from the group consisting of vaccinia virus replication, variola virus replication, cowpox virus replication, and monkeypox virus replication. In a class of this embodiment, the orthopoxvirus replication is vaccinia virus replication or variola virus replication. In a fourth embodiment of the methods of the present invention, the orthopoxvirus infection is selected from the group consisting of vaccinia virus infection, variola virus infection, cowpox virus infection, and monkeypox virus infection. In a class of this embodiment, the orthopoxvirus infection is vaccinia virus infection or variola virus infection. Another aspect of the present invention provides the novel nucleoside derivative, 6-amino-2-fluoro-9-(2-C-methyl-β-D-ribofuranosyl)purine or a pharmaceutically acceptable salt thereof.
Throughout the instant application, the following terms have the indicated meanings: The 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.
The term "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.).
The term "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.). The term "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).
The term "alkoxy" refers to straight or branched chain alkoxides of the number of carbon atoms specified (e.g., Ci_4 alkoxy), or any number within this range [i.e., methoxy (MeO-), ethoxy, isopropoxy, etc.]. The term "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.].
The term "alkylamino" refers to straight or branched alkylamines of the number of carbon atoms specified (e.g., Cι_4 alkylamino), or any number within this range [i.e., methylamino, ethylamino, isopropylamino, t-butylamino, etc.].
The term "alkylsulfonyl" refers to straight or branched chain alkylsulfones of the number of carbon atoms specified (e.g., Cι_6 alkylsulfonyl), or any number within this range [i.e., methylsulfonyl (MeSO2-), ethylsulfonyl, isopropylsulfonyl, etc.].
The term "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., Cι_4 alkyloxycarbonyl), or any number within this range [i.e., methyloxycarbonyl (MeOCO-), ethyloxycarbonyl, or butyloxycarbonyl]. The term "alkyloxycarbonyloxy" refers to straight or branched chain alkyl carbonates of the present invention of the number of carbon atoms specified (e.g., Ci-io alkyloxycarbonyloxy), or any number within this range [i.e., methyloxycarbonyl oxy (MeOCOO-), ethyloxycarbonyloxy, or butyloxycarbonyloxy]. The term "alkylcarbonyloxy" refers to straight or branched chain alkanoic acid derivatives of alcohols of the present invention of the number of carbon atoms specified (e.g., Ci-16 alkylcarbonyloxy), or any number within this range [i.e., methylcarbonyloxy (MeCOO-), ethylcarbonyloxy, or n-octylcarbonyloxy].
The term "cycloalkylcarbonyloxy" refers to cyclic alkanoic acid derivatives of alcohols of the present invention of the number of carbon atoms specified (e.g., C3-6 cycloalkylcarbonyloxy), or any number within this range [i.e., cyclopropylcarbonyloxy, cyclopentylcarbonyloxy, or cyclohexylcarbonyloxy].
The term "alkenylcarbonyloxy" refers to a straight or branched chain alkenoic acid derivatives of alcohols of the present invention having two to eighteen total carbon atoms and containing one to three double bonds in the alkene chain. The term "aryl" includes both phenyl, naphthyl, and pyridyl. The aryl group is optionally substituted with one to three groups independently selected from Ci-4 alkyl, halogen, cyano, nitro, trifluoromethyl, Cι_4 alkoxy, and Ci_4 alkylthio.
The term "halogen" is intended to include the halogen atoms fluorine, chlorine, bromine and iodine. The term "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.
The term "5 '-triphosphate" refers to a triphosphoric acid ester derivative of the 5'-hydroxyl group of a nucleoside compound of the present invention having the following general structural formula HI:
Figure imgf000013_0001
(III)
wherein Rl-Rl 1 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 IV and V, respectively,
Figure imgf000013_0002
(V) The term "5'-(S-acyl-2-thioethyl)phosphate" or "SATE" refers to a mono- or di -ester derivative of a 5'-monophosphate nucleoside derivative of the present invention of structural formulae VI and VII, respectively, as well as pharmaceutically acceptable salts of the mono-ester,
Figure imgf000014_0001
The term "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 carrier, 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. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.
The terms "administration of and "administering a" compound 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 treating orthopoxvirus infections with a compound of the present invention in combination with one or more agents useful for treating orthopoxvirus infections. Such agents active against orthopoxviruses include, but are not limited to, cidofovir, ribavirin, levovirin, and viramidine. Levovirin is the L-enantiomer of ribavirin which has shown immunomodulatory activity similar to ribavirin. Niramidine represents an analog of ribavirin disclosed in WO 01/60379 (assigned to ICΝ Pharmaceuticals). In accordance with this method of the present invention, the individual components of the combination can be administered separately at different times during the course of therapy or concurrently 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 interpreted accordingly. It will be understood that the scope of combinations of the compounds of this invention with other agents useful for treating orthopoxvirus infection includes in principle any combination with any pharmaceutical composition for treating orthopoxvirus infection. When a compound of the present invention or a pharmaceutically acceptable salt thereof is used in combination with a second therapeutic agent active against orthopoxvirus, the dose of each compound may be either the same as or different from the dose when the compound is used alone. Ribavirin, levovirin, and viramidine may exert their anti-orthopoxvirus effects by modulating intracellular pools of guanine nucleotides via inhibition of the intracellular enzyme inosine monophosphate dehydrogenase (IMPDH). IMPDH is the rate-limiting enzyme on the biosynthetic route in de novo guanine nucleotide biosynthesis. Ribavirin is readily phosphorylated intracellularly and the monophosphate derivative is an inhibitor of IMPDH. Thus, inhibition of IMPDH represents another useful target for the discovery of inhibitors of orthopoxvirus replication. Therefore, 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 IMPDH 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)]. By "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.
Also included within the present invention are pharmaceutical compositions comprising the nucleoside compounds and derivatives thereof of the present invention in association with a pharmaceutically acceptable carrier. Another example of the invention is 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.
Also included within the present invention are pharmaceutical compositions useful for inhibiting orthopoxvirus replication comprising an effective amount of a compound of the present invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions useful for treating orthopoxvirus infection are also encompassed by the present invention. Additionally, 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 orthopoxvirus. Agents active against orthopoxvirus include, but are not limited to, cidofovir, ribavirin, levovirin, and viramidine.
Another aspect of the present invention provides for the use of the nucleoside compounds and derivatives thereof and their pharmaceutical compositions for the manufacture of a medicament for the inhibition of orthopoxvirus replication, in particular vaccinia virus, variola virus replication, cowpox virus replication, and monkeypox virus replication and/or the treatment of orthopoxvirus infection, in particular vaccinia virus, variola virus infection, cowpox virus infection, and monkeypox virus infection. Yet a further aspect of the present invention provides for the nucleoside compounds and derivatives thereof and their pharmaceutical compositions for use as a medicament for the inhibition of orthopoxvirus replication, in particular vaccinia, variola, cowpox, and monkeypox virus replication, and/or for the treatment of orthopoxvirus infection, in particular vaccinia, variola, cowpox, and monkeypox virus infection.
The pharmaceutical 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 compositions include compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation), or nasal administration, although the most suitable route in any given case will depend on the nature and severity of the conditions being treated and on the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy. In practical use, 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). In preparing the compositions for oral dosage form, any of the usual pharmaceutical 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 preferred over the liquid preparations.
Because of their ease of administration, 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, corn 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. When 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.
Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, 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 pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, 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. For example, 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. Preferably compounds of structural formula I are administered orally. For oral administration to humans, 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. For oral administration, 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 nucleoside compounds having the β-D stereochemical configuration for the five-membered furanose ring as depicted in the structural formula below, that is, nucleoside compounds in which the substituents at C-l and C-4 of the five-membered furanose ring have the β-stereochemical configuration ("up" orientation as denoted by a bold line).
Figure imgf000019_0001
β-D-
Some of the compounds described herein contain olefinic double bonds, and unless specified otherwise, are meant to include both E and Z geometric isomers.
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. An example of keto-enol tautomers which are intended to be encompassed within the compounds of the present invention is illustrated below:
Figure imgf000020_0001
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.
Alternatively, 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 stereochemistry of the substituents at the C-2 and C-3 positions of the furanose ring of the compounds of the present invention of structural formula I is denoted by squiggly lines which signifies that substituents Rl, R2, R3 and R can have either the α (substituent "down") or β (substituent "up") configuration independently of one another. Notation of stereochemistry by a bold line as at C-l and C-4 of the furanose ring signifies that the substituent has the β-configuration (substituent "up").
Figure imgf000020_0002
(I)
The compounds of the present invention may be administered in the form of a pharmaceutically acceptable salt. The term "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, dihydrochloride, 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, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide and valerate. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof include, but are not limited to, salts derived from inorganic bases including aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, mangamous, potassium, sodium, zinc, and the like. Particularly preferred 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- ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.
Also, in the case of a carboxylic acid (-COOH) or alcohol group being present in the compounds of the present invention, pharmaceutically acceptable esters of carboxylic acid derivatives, such as methyl, ethyl, or pivaloyloxymethyl, or acyl derivatives of alcohols, such as acetate or maleate, can be employed. Included are those esters and acyl groups known in the art for modifying the solubility or hydrolysis characteristics for use as sustained-release or prodrug formulations. Preparation of the Nucleoside Compounds and Derivatives of the Invention
The nucleoside compounds and derivatives thereof of the present invention can be prepared following synthetic methodologies well-established in the practice of nucleoside and nucleotide chemistry. Reference is made to the following text for a description of synthetic methods used in the preparation of the compounds of the present invention: "Chemistry of Nucleosides and Nucleotides," L.B.
Townsend, ed., Vols. 1-3, Plenum Press, 1988, which is incorporated by reference herein in its entirety.
The synthesis of 9-(2'-C-methyl-β-D-ribofuranosyl)purines of structural formula VHI is described in U.S. Patent No. 3,480,613, the contents of which are incorporated herein in their entirety.
Figure imgf000022_0001
A representative general method for the preparation of compounds of the present invention is outlined in Scheme 1 below. This scheme illustrates the synthesis of compounds of the present invention of structural formula 1 7 wherein the furanose ring has the β-D-ribo configuration. The starting material is a 3,5-bis-O- protected alkyl furanoside, such as methyl furanoside, of structural formula LΛ. The C-2 hydroxyl group is then oxidized with a suitable oxidizing agent, such as a chromium trioxide or chromate reagent, Dess-Martin periodinane, or by Swem oxidation, to afford a C-2 ketone of structural formula 1^2. Addition of 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 MeLi, across the carbonyl double bond of J 2 in a suitable organic solvent, such as tetrahydrofuran, diethyl ether, and the like, affords the C-2 tertiary alcohol of structural formula . A good leaving group (such as CI, Br, and I) is next introduced at the C-l (anomeric) position of the furanose sugar derivative by treatment of the furanoside of formula Lθ with a hydrogen halide in a suitable organic solvent, such as hydrogen bromide in acetic acid, to afford the intermediate furanosyl halide 1^4. A C-l sulfonate, such methanesulfonate (MeSO2O-), trifluoromethane- sulfonate (CF3SO2O-), or p-toluenesulfonate (-OTs), may also serve as a useful leaving group in the subsequent reaction to generate the glycosidic (nucleosidic) linkage. The nucleosidic linkage is constructed by treatment of the intermediate of structural formula 1^4 with the metal salt (such as lithium, sodium, or potassium) of an appropriately substituted lH-pyrrolo[2,3-d]pyrimidine 1-5, such as an appropriately substituted 4-halo-lH-pyrrolo[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 NaΗMDS) in a suitable anhydrous organic solvent, such as acetonitrile, tetrahydrofuran, l-methyl-2- pyrrolidinone, or N,N-dimethylformamide (DMF). The displacement reaction can be catalyzed by using a phase-transfer catalyst, such as TDA-1 or triethylbenzyl- ammonium chloride, in a two-phase system (solid-liquid or liquid-liquid). The optional protecting groups in the protected nucleoside of structural formula 1^6 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," 3rd ed., lohn Wiley & Sons, 1999. Optional introduction of an amino group at the 4-position of the pyrrolo[2,3-d]pyrimidine nucleus is effected by treatment of the 4-halo intermediate 1^6 with the appropriate amine, such as alcoholic ammonia or liquid ammonia, to generate a primary amine at the C-4 position (-NΗ2), an alkylamine to generate a secondary amine (-NHR), or a dialkylamine to generate a tertiary amine (-NRR'). A 7H-pyrrolo[2,3-d]pyrimidin-4(3H)one compound may be derived by hydrolysis of 1^6 with aqueous base, such as aqueous sodium hydroxide. Alcoholysis (such as methanolysis) of 1^6 affords a C-4 alkoxide (-OR), whereas treatment with an alkyl mercaptide affords a C-4 alkylthio (-SR) derivative. Subsequent chemical manipulations well-known to practitioners of ordinary skill in the art of organic/medicinal chemistry may be required to attain the desired compounds of the present invention. Scheme 1
Figure imgf000024_0001
1 -1 1 -2
Pg = protecting group R = lower alkyl
Figure imgf000024_0002
The examples below provide citations to literature publications, which contain details for the preparation of final compounds or intermediates employed in the preparation of final compounds of the present invention. The nucleoside compounds of the present invention were prepared according to procedures detailed in the following examples. The examples are not intended to be limitations on the scope of the instant invention in any way, and they should not be so construed. Those skilled in the art of nucleoside and nucleotide synthesis will readily appreciate that known variations of the conditions and processes of the following preparative procedures can be used to prepare these and other compounds of the present invention. All temperatures are degrees Celsius unless otherwise noted.
EXAMPLE 1
4-Amino-7-(2-C-methyl-β-D-arabino ranosyl -7H-pyrtolo[2,3- ]pyrimidine
Figure imgf000025_0001
To chromium trioxide (1.57 g, 1.57 mmol) in dichloromethane (DCM) (10 mL) at 0°C was added acetic anhydride (145 mg, 1.41 mmol) and then pyridine (245 mg, 3.10 mmol). The mixture was stirred for 15 min, then a solution of 7-[3,5- ( -[l,l,3,3-tetrakis(l-methylethyl)-l,3-disiloxanediyl]-β-D-ribofuranosyl]-7H- pyrrolo[2,3-<i]pyrimidin-4-amine [for preparation, see J. Am. Chem. Soc. 105: 4059 (1983)] (508 mg, 1.00 mmol) in DCM (3 mL) was added. The resulting solution was stirred for 2 h and then poured into ethyl acetate (10 mL), and subsequently filtered through silica gel using ethyl acetate as the eluent. The combined filtrates were evaporated in vacuo, taken up in diethyl ether/TΗF (1:1) (20 mL), cooled to -78°C and methylmagnesium bromide (3M, in TΗF) (3.30 mL, 10 mmol) was added dropwise. The mixture was stirred at -78°C for 10 min, then allowed to come to room temperature (rt) and quenched by addition of saturated aqueous ammonium chloride (10 mL) and extracted with DCM (20 mL). The organic phase was evaporated in vacuo and the crude product purified on silica gel using 5% methanol in dichloromethane as eluent. Fractions containing the product were pooled and evaporated in vacuo. The resulting oil was taken up in TΗF (5 mL) and tetrabutylammonium fluoride (TBAF) on silica (1.1 mmol/g on silica) (156 mg) was added. The mixture was stirred at room temperature for 30 min, filtered, and evaporated in vacuo. The crude product was purified on silica gel using 10% methanol in dichloromethane as eluent. Fractions containing the product were pooled and evaporated in vacuo to give the desired compound (49 mg) as a colorless solid. IH NMR (DMSO-d6): δ 1.08 (s, 3H), 3.67 (m, 2H), 3.74 (m, IH), 3.83 (m, IH), 5.19
(m, IH), 5.23 (m, IH), 5.48 (m, IH), 6.08 (IH, s), 6.50 (m, IH), 6.93 (bs, 2H), 7.33 (m, IH), 8.02 (s, IH).
EXAMPLE 2
4-Amino-7-(2-C-methyl-β-D-ribofuranosyl -7H-pyrrolo[2,3-(f|pyrimidine
Figure imgf000026_0001
Step A: 3,5-Bis-O-(2,4-dichlorophenylmethyl)-l-6>-methyl- -D-ribofuranose
A mixture of 2-0-acetyl-3,5-bis-0-(2,4-dichlorophenylmethyl)-l-O- methyl-α-D-ribofuranose [for preparation, see: Helv. Chim. Acta 78: 486 (1995)] (52.4 g, 0.10 mol) in methanolic K2CO3 (500 mL, saturated at room temperature) was stirred at room temperature for 45 min and then concentrated under reduced pressure. The oily residue was suspended in CH2C12 (500 mL), washed with water (300 mL + 5 x 200 mL) and brine (200 mL), dried (Na2SO ), filtered, and concentrated to give the title compound (49.0 g) as colorless oil, which was used without further purification in Step B below. lH NMR (DMSO-d6 ): δ 3.28 (s, 3H, OCH3), 3.53 (d, 2H, JSΛ = 4.5 Hz, H-5a, H-5b), 3.72 (dd, IH, J3,4 = 3.6 Hz, J3,2 = 6.6 Hz, H-3), 3.99 (ddd, IH, J2,ι = 4.5 Hz, /2,OH-2 = 9.6 Hz, H-2), 4.07 (m, IH, H-4), 4.50 (s, 2H, CH2Ph), 4.52, 4.60 (2d, 2Η, Jgem = 13.6 Hz, CH2Ph), 4.54 (d, 1Η, OΗ-2), 4.75 (d, IH, H-l), 7.32-7.45, 7.52-7.57 (2m, 10H, 2Ph). 13C NMR (DMSO-d6): δ 55.40, 69.05, 69.74, 71.29, 72.02, 78.41, 81.45, 103.44, 127.83, 127.95, 129.05, 129.28, 131.27, 131.30, 133.22, 133.26, 133.55, 133.67, 135.45, 135.92. Step B: 3,5-Bis-O-(2,4-dichlorophenylmethyl)-l-0-methyl- -D-eryt/tro- pentofuranos-2-ulose
To an ice-cold suspension of Dess-Martin periodinane (50.0 g, 118 mmol) in anhydrous CH2C12 (350 mL) under argon (Ar) was added a solution of the compound from Step A (36.2 g, 75 mmol) in anhydrous CH2C1 (200 mL) dropwise over 0.5 h. The reaction mixture was stirred at 0°C for 0.5 h and then at room temperature for 3 days. The mixture was diluted with anhydrous Et2O (600 mL) and poured into an ice-cold mixture of Na2S2O3.5H2O (180 g) in saturated aqueous NaHCO3 (1400 mL). The layers were separated, and the organic layer was washed with saturated aqueous NaHCO3 (600 mL), water (800 mL) and brine (600 mL), dried (MgSO ), filtered and evaporated to give the title compound (34.2 g) as a colorless oil, which was used without further purification in Step C below. IH NMR (CDC13): δ 3.50 (s, 3H, OCH3), 3.79 (dd, IH, J5a,5b = 113 Hz, JSΛΛ = 3.5 Hz, H-5a), 3.94 (dd, IH, J5 ,4 = 2.3 Hz, H-5b), 4.20 (dd, IH, J3Λ = 1.3 Hz, I3Λ = 8.4 Hz, H-3), 4.37 (ddd, IH, H-4), 4.58, 4.69 (2d, 2H, Jgem = 13.0 Hz, CH2Ph), 4.87 (d, 1Η, Η-l), 4.78, 5.03 (2d, 2H, Jgem = 12.5 Hz, CH2Ph), 7.19-7.26, 7.31-7.42 (2m, 10Η, 2Ph).
13C NMR (DMSO-d6): δ 55.72, 69.41, 69.81, 69.98, 77.49, 78.00, 98.54, 127.99, 128.06, 129.33, 129.38, 131.36, 131.72, 133.61, 133.63, 133.85, 133.97, 134.72, 135.32, 208.21.
Step C: 3,5-Bis-Q-(2,4-dichlorophenylmethyl -2-C-methyl-l-< -methyl-α-D- ribofuranose To a solution of MeMgBr in anhydrous Et2O (0.48 M, 300 mL) at -55 °C was added dropwise a solution of the compound from Step B (17.40 g, 36.2 mmol) in anhydrous Et O (125 mL). The reaction mixture was allowed to warm to -30°C and stirred for 7 h at -30°C to -15°C, then poured into ice-cold water (500 mL) and the mixture vigorously stirred at room temperature for 0.5 h. The mixture was filtered through a Celite pad (10 x 5 cm) which was thoroughly washed with Et2O. The organic layer was dried (MgSO ), filtered and concentrated. The residue was dissolved in hexanes (-30 mL), applied onto a silica gel column (10 x 7 cm, prepacked in hexanes) and eluted with hexanes and hexanes/EtOAc (9/1) to give the title compound (16.7 g) as a colorless syrup. IH NMR (CDCI3): δ 1.36 (d, 3H, JMe,OH = 0.9 Hz, 2C-Me), 3.33 (q, IH, OH), 3.41 (d, IH, J3ι4 = 3.3 Hz), 3.46 (s, 3H, OCH3), 3.66 (d, 2H, JSA = 3.7 Hz, H-5a, H-5b), 4.18 (apparent q, IH, H-4), 4.52 (s, IH, H-l), 4.60 (s, 2H, CH2Ph), 4.63, 4.81 (2d, 2Η, Jgem = 13.2 Hz, CH2Ph), 7.19-7.26, 7.34-7.43 (2m, 10Η, 2Ph). 1 C NMR (CDCI3): δ 24.88, 55.45, 69.95, 70.24, 70.88, 77.06, 82.18, 83.01, 107.63,
127.32, 129.36, 130.01, 130.32, 133.68, 133.78, 134.13, 134.18, 134.45, 134.58.
Step D: 4-Chloro-7-r3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-β-D- ribofuranosyl]-7H-pyιτolo[2,3-(f|pyrimidine To a solution of the compound from Step C (9.42 g, 19 mmol) in anhydrous dichloromethane (285 mL) at 0°C was added ΗBr (5.7 M in acetic acid, 20 mL, 114 mmol) dropwise. The resulting solution was stirred at 0°C for 1 h and then at room temperature for 3 h, evaporated in vacuo and co-evaporated with anhydrous toluene (3 x 40 mL). The oily residue was dissolved in anhydrous acetonitrile (50 mL) and added to a solution of the sodium salt of 4-chloro-lH-pyrrolo[2,3- <f]pyrimidine in acetonitrile [generated in situ from 4-chloro-lH-pyrrolo[2,3- <i]pyrimidine [for preparation, see J. Chem. Soc, 131 (I960)] (8.76 g, 57 mmol) in anhydrous acetonitrile (1000 mL), and NaΗ (60% in mineral oil, 2.28 g, 57 mmol), after 4 h of vigorous stirring at room temperature]. The combined mixture was stirred at room temperature for 24 h, and then evaporated to dryness. The residue was suspended in water (250 mL) and extracted with EtOAc (2 x 500 mL). The combined extracts were washed with brine (300 mL, dried over Na2SO4, filtered and evaporated. The crude product was purified on a silica gel column (10 cm x 10 cm) using ethyl acetate/hexane (1:3 and 1:2) as the eluent. Fractions containing the product were combined and evaporated in vacuo to give the desired product (5.05 g) as a colorless foam. lΗ NMR (CDCI3): δ 0.93 (s, 3Η, CH3), 3.09 (s, IH, OH), 3.78 (dd, IH, /5.,5- = 10.9 Hz, JyΛ = 2.5 Hz, H-5'), 3.99 (dd, IH, 75",4 = 2.2 Hz, H-5"), 4.23-4.34 (m, 2H, H-3\ H-4'), 4.63, 4.70 (2d, 2H, Jgem = 12.7 Hz, CH2Ph), 4.71, 4.80 (2d, 2Η, /gem = 12.1 Hz,CH2Ph), 6.54 (d, 1Η, , J5ι6 = 3.8 Ηz, Η-5), 7.23-7.44 (m, 10H, 2Ph).
13C NMR (CDCI3): δ 21.31, 69.10, 70.41, 70.77, 79.56, 80.41, 81.05, 91.11, 100.57, 118.21, 127.04, 127.46, 127.57, 129.73, 129.77, 130.57, 130.99, 133.51, 133.99,
134.33, 134.38, 134.74, 135.21, 151.07, 151.15 152.47. Step E: 4-Chloro-7-(2-C-methyl-β-D-ribofuranosvn-7H-pyrrolo[2,3-
<Jlpyrimidine
To a solution of the compound from Step D (5.42 g, 8.8 mmol) in dichloromethane (175 mL) at -78°C was added boron trichloride (1M in dichloromethane, 88 mL, 88 mmol) dropwise. The mixture was stirred at -78°C for
2.5 h, then at -30°C to -20°C for 3 h. The reaction was quenched by addition of methanol/dichloromethane (1:1) (90 mL) and the resulting mixture stirred at -15°C for 30 min, then neutralized with aqueous ammonia at 0°C and stirred at room temperature for 15 min. The solid was filtered and washed with CΗ2Cl2/MeOΗ (1/1, 250 mL). The combined filtrate was evaporated, and the residue was purified by flash chromatography over silica gel using CH2C12 and CH2Cl2:MeOH (99:1, 98:2, 95:5 and 90:10) gradient as the eluent to furnish desired compound (1.73 g) as a colorless foam, which turned into an amorphous solid after treatment with MeCN. iH NMR (OMSO-d6): δ 0.64 (s, 3H, CH3), 3.61-3.71 (m, IH, H-5'), 3.79-3.88 (m, IH, H-5"), 3.89-4.01 (m, 2H, H-3', H-4'), 5.15-5.23 (m, 3H, 2'-OH, 3'-OH, 5'-OH), 6.24 (s, IH, H-l'), 6.72 (d, IH, J5,6 = 3.8 Hz, H-5), 8.13 (d, IH, H-6), 8.65 (s, IH, H-
2).
13C NMR (DMSO-d6): δ 20.20, 59.95, 72.29, 79.37, 83.16, 91.53, 100.17, 117.63,
128.86, 151.13, 151.19, 151.45.
Step F: 4-Amino-7-(2-C-methyl-β-D-ribofuranosyl -7H-pyrrolor2,3- άlpyrimidine To the compound from Step E (1.54 g, 5.1 mmol) was added methanolic ammonia (saturated at 0°C; 150 mL). The mixture was heated in a stainless steel autoclave at 85°C for 14 h, then cooled and evaporated in vacuo. The crude mixture was purified on a silica gel column with CΗ2Cl2/MeOΗ (9/1) as eluent to give the title compound as a colorless foam (0.8 g), which separated as an amorphous solid after treatment with MeCN. The amorphous solid was recrystallized from methanol/acetonitrile; m.p. 222°C. lH NMR (DMSO- ): δ 0.62 (s, 3H, CH3), 3.57-3.67 (m, IH, H-5'), 3.75-3.97 (m,
3H, H-5", H-4', H-3'), 5.00 (s, IH, 2'-OH), 5.04 (d, IH, 73>OH,3' = 6.8 Hz, 3'-OH),
5.06 (t, IH, J5'0H,5',5" = 5.1 Hz, 5' -OH), 6.11 (s, IH, H-l'), 6.54 (d, IH, J5j6 = 3.6 Hz, H-5), 6.97 (br s, 2H, NH2), 7.44 (d, IH, H-6), 8.02 (s, IH, H-2).
13C NMR (DMSO-dό): δ 20.26, 60.42, 72.72, 79.30, 82.75, 91.20, 100.13, 103.08, 121.96, 150.37, 152.33, 158.15. LC-MS: Found: 279.10 (M-H+); calc. for C12H16N4O4+H+: 279.11.
EXAMPLE 3
4-Amino-7-(2-C-ethyl-β-D-ribofuranosyl')-7H-pyrrolor2,3-<flpyrimidine
Figure imgf000030_0001
Step A: 3,5-Bis-0-(2,4-dichlorophenylmethyl)-2-C-ethyl-l-<9-methyl-α-D- ribofuranose To diethyl ether (300 mL) at -78°C was slowly added EtMgBr (3.0 M, 16.6 mL) and then dropwise the compound from Step B of Example 2 (4.80 g, 10.0 mmol) in anhydrous Et2O (100 mL). The reaction mixture was stirred at -78 °C for 15 min, allowed to warm to -15°C and stirred for another 2 h, and then poured into a stirred mixture of water (300 mL) and Et2O (600 mL). The organic phase was separated, dried (MgSO4), and evaporated in vacuo. The crude product was purified on silica gel using ethyl acetate/hexane (1:2) as eluent. Fractions containing the product were pooled and evaporated in vacuo to give the desired product (3.87 g) as a colorless oil.
Step B: 4-Chloro-7-[3.5-bis-Q-(2,4-dichlorophenylmethyl)-2-C-ethyl-β-D- ribofuranosyll-7H-pyrrolo[2,3-(f1pyrimidine
To a solution of the compound from Step A (1.02 mg, 2.0 mmol) in dichloromethane (40 mL) was added ΗBr (5.7 M in acetic acid) (1.75 mL, 10.0 mmol) dropwise at 0°C. The resulting solution was stirred at room temperature for 2 h, evaporated in vacuo and co-evaporated twice from toluene (10 mL). The oily residue was dissolved in acetonitrile (10 mL) and added to a vigorously stirred mixture of 4- chloro-lH-pyιτolo[2,3-d]pyrimidine (307 mg, 2.0 mmol), potassium hydroxide (337 mg, 6.0 mmol) and tris[2-(2-methoxyethoxy)ethyl] amine (130 mg, 0.4 mmol) in acetonitrile (10 mL). The resulting mixture was stirred at room temperature overnight, and then poured into a stirred mixture of saturated ammonium chloride (100 mL) and ethyl acetate (100 mL). The organic layer was separated, washed with brine (100 mL), dried over MgSO , filtered and evaporated in vacuo. The crude product was purified on silica gel using ethyl acetate/hexane (1:2) as eluent to give the desired product (307 mg) as a colorless foam.
Step C: 4-Chloro-7-(2-C-ethyl-β-D-ribofuranosyn-7H-pyrrolo[2,3- flpyrimidine To a solution of the compound from Step B (307 mg, 0.45 mmol) in dichloromethane (8 mL) was added boron trichloride (IM in dichloromethane) (4.50 mL, 4.50 mmol) at -78°C. The mixture was stirred at -78°C for lh, then at -10°C for 3h. The reaction was quenched by addition of methanol/dichloromethane (1:1) (10 mL), stirred at -15°C for 30 min, and neutralized by addition of aqueous ammonium hydroxide. The mixture was evaporated under diminished pressure and the resulting oil purified on silica gel using methanol/dichloromethane (1:9) as eluent. Fractions containing the product were pooled and evaporated in vacuo to give the desired product (112 mg) as a colorless foam.
Step D: 4-Amino-7-(2-C-ethyl-β-D-ribofuranosyl -7H-pyrrolo[2,3--
Figure imgf000031_0001
To the compound from Step C (50 mg, 0.16 mmol) was added saturated ammonia in methanol (4 mL). The mixture was stirred at 75°C for 72 h in a closed container, cooled and evaporated in vacuo. The crude mixture was purified on silica gel using methanol/dichloromethane (1:9) as eluent. Fractions containing the product were pooled and evaporated in vacuo to give the desired product (29 mg) as a colorless powder. lΗNMR (200 MHz, OMSO-d6): δ 0.52 (t, 3H), 1.02 (m, 2H), 4.01-3.24 (m, 6H), 5.06
(m, IH), 6.01 (s, IH), 6.51 (d, IH), 6.95 (s br, 2H), 6.70 (d, IH), 7.99 (s, IH). LC-MS: Found: 295.2 (M+H+); calc. for C13H18N4O4+H+: 295.14. EXAMPLE 4
2-Amino-7-(2-C-methyl-β-D-ribofuranosyl -7H-pyrrolor2,3-^pyrimidin-4(3H)-one
Figure imgf000032_0001
Step A: 2-Amino-4-chloro-7-[3,5-bis-O-(2,4-dichlorophenvImethyl)-2-C- methyl- β-D-ribofuranosyl -7H-pyrrolo [2 ,3 - dλ pyrimidine To an ice-cold solution of the product from Step C of Example 2 (1.27 g, 2.57 mmol) in CΗ2C12 (30 mL) was added HBr (5.7 M in acetic acid; 3 mL) dropwise. The reaction mixture was stirred at room temperature for 2 h, concentrated under diminished pressure and coevaporated with toluene (2 x 15 mL). The resulting oil was dissolved in acetonitrile (MeCN) (15 mL) and added dropwise into a well- stirred mixture of 2-amino-4-chloro-7H-pyrrolo[2,3-(i]pyrimidine [for preparation, see Ηeterocvcles 35: 825 (1993)] (433 mg, 2.57 mmol), KOΗ (85%, powdered) (0.51 g, 7.7 mmol), tris-[2-(2-methoxyethoxy)ethyl] amine (165 μL, 0.51 mmol) in acetonitrile (30 mL). The resulting mixture was stirred at room temperature for lh, filtered and evaporated. The residue was purified on a silica gel column using hexanes/EtOAc, 5/1, 3/1 and 2/1, as eluent to give the title compound as a colorless foam (0.65 g).
Step B: 2-Amino-4-chloro-7-(2-C-methyl-β-D-ribofuranosyl -7H-pyrrolor2,3- d\ pyrimidine
To a solution of the product from Step A (630 mg, 1.0 mmol) in CΗ2C12 (20 mL) at -78°C was added boron trichloride (IM in CH2C12 ) (10 mL, 10 mmol). The mixture was stirred at -78°C for 2 h, then at -20°C for 2.5 h. The reaction was quenched with CH2Cl2/MeOH (1 : 1) (10 mL), stirred at -20°C for 0.5 h, and neutralized at 0°C with aqueous ammonia. The solid was filtered, washed with CH2Cl2/MeOH (1:1) and the combined filtrate evaporated in vacuo. The residue was purified on a silica gel column with CH2Cl2/MeOH, 50/1 and 20/1, as eluent to give the title compound as a colorless foam (250 mg).
Step C: 2-Amino-7-(2-C-methyl-β-D-ribofuranosyl -7H-pyrrolo[2,3- <f1pyrimidin-4(3H)-one
A mixture of the product from Step B (90 mg, 0.3 mmol) in aqueous NaOΗ (2N, 9 mL) was heated at reflux temperature for 5 h, then neutralized at 0°C with 2 N aqueous ΗC1 and evaporated to dryness. Purification on a silica gel column with CΗ2Cl /MeOΗ, 5/1, as eluent afforded the title compound as a white solid (70 mg).
IH NMR (200 MHz, CD3OD): δ 0.86 (s, 3H), 3.79 (m IH), 3.90-4.05 (m, 3H), 6.06 (s, IH), 6.42 (d, / = 3.7 Hz, IH), 7.05 (d, /= 3.7 Hz, IH).
EXAMPLE 5
2-Amino-4-cyclopropylamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3- lpyrimidine
Figure imgf000033_0001
A solution of 2-amino-4-chloro-7-(2-C-methyl-β-D-ribofuranosyl)-7H- pyrrolo [2,3 -d]pyrimi dine (Example 4, Step B) (21 mg, 0.07 mmol) in cyclopropylamine (0.5 mL) was heated at 70°C for two days, then evaporated to an oily residue and purified on a silica gel column with CΗ2Cl2/MeOΗ, 20/1, as eluent to give the title compound as a white solid (17 mg).
IH NMR (200 MHz, CD3CN): δ 0.61 (m, 2H), 0.81 (m, 2H), 0.85 (s, 3H), 2.83 (m, IH), 3.74-3.86 (m, IH), 3.93-4.03 (m, 2H), 4.11 (d, J= 8.9 Hz, IH), 6.02 (s, IH),
6.49 (d, J= 3.7 Hz, IH), 7.00 (d, /= 3.7 Hz, IH). EXAMPLE 6
4-Anιino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-(i1pyrimidine-5- carbonitrile
Figure imgf000034_0001
This compound was prepared following procedures described by Y. Murai et al. in Ηeterocycles 33: 391-404 (1992).
EXAMPLE 7
4-Amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolor2,3-^lpyrimidine-5- carboxamide
Figure imgf000034_0002
This compound was prepared following procedures described by Y. Murai et al. in Ηeterocycles 33: 391-404 (1992).
EXAMPLE 8
General process to SATE prodrug moiety
S-Acyl-2-thioethyl (SATE) pronucleotides are discussed in C.R. Wagner, N.N. Iyer, and E.I. Mclntee, "Pronucleotides: Toward the In Nivo Delivery of Antiviral and Anticancer Nucleotides," Med. Res. Rev., 20: 1-35 (2000), which is incorporated by reference herein in its entirety. SATE derivatives of nucleosides are also disclosed U.S. Patent Nos. 5,770,725; 5,849,905; and 6,020,482, the contents of each of which are incorporated by reference herein in their entirety.
Bis(S-acetyl-2-thioethyl)-N,N-diisopropylphosphoramidite
2-Mercaptoethanol (5 g, 64 mmol) was dissolved in CH2C12 (50 mL). To this solution was added triethylamine (7.67 mL, 57.6 mmol), and the reaction mixture was cooled in an ice bath to 0 °C. Acetic anhydride (4.54 mL, 48 mmol) was added dropwise in 10 min, and the reaction mixture was stirred for 1 h at 0 °C. The reaction mixture was then allowed to come to room temperature over a period of 2 h. The reaction mixture was diluted with CH2C12 (50 mL), washed with water (75 mL), 5% aqueous ΝaHCO3 (75 mL) and brine (75 mL). The organic phase was dried over anhydrous Na2SO and concentrated in vacuo to give an oil. The oil was then dissolved in anhydrous THF (40 mL) and anhydrous triethylamine (7.76 mL) was added. To this mixture was added activated molecular sieves (4 A) and was kept at room temperature for 10 min. The reaction mixture was cooled in an ice bath to 0°C and diisopropylphosphoramidous dichloride (6.47 g, 32.03 mmol) was added. The reaction mixture was stirred at 0 °C for 2 h under inert atmosphere. Hexane (40 mL) was added to the reaction mixture and the precipitate formed was filtered. The filtrate was concentrated to one fourth of the volume, purified by loaded silica gel column chromatography and eluted with hexane containing 3 % triethylamine and incremental amount of ethyl acetate (0 to 7 %) to give the title compound as an oil (2.36 g). iH NMR (CDCI3): δ 1.17 (s, 6H), 1.21 (s, 6H), 2.36 (s, 6H), 3.14 (t, J= 6.44 Hz), 3.51-3.84 (m, 6H); 13C NMR (CDCI3): δ 24.47, 24.61, 30.48, 42.85, 43.1, 61.88, 62.23, 195.26; 13P NMR (CDC13): δ 146.96.
EXAMPLE 9
5 '-Triphosphate Derivatives The nucleoside 5'-triphosphates of the present invention were prepared following general procedures described in Chem. Rev. 100: 2047 (2000).
EXAMPLE 10
Purification and Purity Analysis of 5 '-Triphosphate Derivatives The triphosphate derivatives were purified by anion exchange (AX) chromatography using a 30 x 100 mm Mono Q column (Pharmacia) with a buffer system of 50 mM Tris, pH 8. Elution gradients were typically from 40 mM NaCl to 0.8 M NaCl in two column volumes at 6.5 mL/min. Appropriate fractions from anion exchange chromatography were collected and desalted by reverse-phase (RP) chromatography using a Luna C18 250 x 21 mm column (Phenomenex) with a flow rate of 10 mL/min. Elution gradients were generally from 1% to 95% methanol in 14 min at a constant concentration of 5 mM triethylammonium acetate (TEAA).
Mass spectra of the purified triphosphates were determined using on- line HPLC mass spectrometry on a Hewlett-Packard (Palo Alto, CA) MSD 1100. A Phenomenex Luna (C18(2)), 150 x 2 mm, plus 30 x 2 mm guard column, 3-μm particle size was used for RP HPLC. A 0 to 50% linear gradient (15 min) of acetonitrile in 20 mM TEAA (triethylammonium acetate) pH 7 was performed in series with mass spectral detection in the negative ionization mode. Nitrogen gas and a pneumatic nebulizer were used to generate the electrospray. The mass range of 150- 900 was sampled. Molecular masses were determined using the HP Chemstation analysis package.
The purity of the purified triphosphates was determined by analytical RP and AX HPLC. RP HPLC with a Phenomonex Luna or lupiter column (250 x 4.6 mm), 5-μm particle size was typically run with a 2-70% acetonitrile gradient in 15 min in 100 mM TEAA, pH 7. AX HPLC was performed on a 1.6 x 5 mm Mono Q column (Pharmacia). Triphosphates were eluted with a gradient of 0 to 0.4 M NaCl at constant concentration of 50 mM Tris, pH 8. The purity of the triphosphates was generally >80%.
EXAMPLE 11 5 '-Monophosphate Derivatives
The nucleoside 5'-monophosphates of the present invention were prepared following the general procedures described in Tetrahedron Lett. 50: 5065 (1967). EXAMPLE 12
Mass Spectral Characterization of 5 '-Triphosphate Derivatives
Mass spectra of 5 '-triphosphates of the compounds of the present invention were determined as described in Example 10. Listed in the following table are the calculated and experimental masses for representative 5 '-triphosphates prepared according to the procedures of Example 9. The example numbers correspond to the parent compound of the 5 '-triphosphate.
Figure imgf000037_0002
EXAMPLE 13
r4-Amino-7-(2-C-methyl-β-D-ribomranosyl -7H-pyrrolo[2,3- ]-pyrimidine]-5' monophosphate
Figure imgf000037_0001
To the compound from Step F of Example 2 (14 mg, 0.05 mmol) (dried by coevaporation with pyridine and several times with toluene) was added trimethyl phosphate (0.5 mL). The mixture was stirred overnight in a sealed container. It was then cooled to 0°C and phosphorous oxychloride (0.0070 mL, 0.075 mmol) was added via a syringe. The mixture was stirred for 3 h at 0°C, then the reaction was quenched by addition of tetraethylammonium bicarbonate (TEAB) (IM) (0.5 mL) and water (5 mL). The reaction mixture was purified and analyzed according to the procedure described in Example 10. Electron spray mass spectrum (ES-MS): Found: 359.2 (M-FT), calc. for C12H17N4O7P- 1^: 359.1.
EXAMPLE 14
[4-Armno-7-(2-C-methyl-β-D-ribofuranosyl -7H-pyrrolo[213--f|-pyrimidine]-5'- diphosphate
Figure imgf000038_0001
To the compound from Step F of Example 2 (56 mg, 0.20 mmol) (dried by coevaporation with pyridine and several times with toluene) was added trimethyl phosphate (stored over sieves) (1.0 mL). The mixture was stirred overnight in a sealed container. It was then cooled to 0°C and phosphorous oxychloride (0.023 mL, 0.25 mmol) was added via a syringe. The mixture was stirred for 2 h at 0°C, then tributylamine (0.238 mL, 1.00 mmol) and tributylammonium phosphate (generated from phosphoric acid and tributylamine in pyridine, followed by repeated azeotropic evaporation with pyridine and acetonitrile) (1.0 mmol in 3.30 mL acetonitrile) was added. The mixture was stirred for an additional 30 min at 0°C, the sealed vial was then opened and the reaction quenched by addition of TEAB (IM) (1.0 mL) and water (5 mL). The reaction mixture was purified and analyzed according to the procedure described in Example 10. ES-MS: Found: 439.0 (M-Ef), calc. for d2H18N4O10P2- FT: 439.04. EXAMPLE 15
[4-Amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrtθlor2,3-^-pyrimidine]-5' triphosphate
Figure imgf000039_0001
To the compound from Step F of Example 2 (20 mg, 0.07 mmol) (dried by coevaporation with pyridine and several times with toluene) was added trimethyl phosphate (stored over sieves) (0.4 mL). The mixture was stirred overnight in a sealed container. It was then cooled to 0°C and phosphorous oxychloride (0.0070 mL, 0.075 mmol) was added via syringe. The mixture was stirred for 3 h at 0°C, then tributylamine (0.083 mL, 0.35 mmol), tributylammonium pyrophosphate (127 mg, 0.35 mmol) and acetonitrile (stored over sieves) (0.25 mL) were added. The mixture was stirred for an additional 30 min at 0°C, the sealed vial was then opened and the reaction quenched by addition of TEAB (IM) (0.5 mL) and water (5 mL). The reaction mixture was purified and analyzed according to the procedure described in
Example 10.
ES-MS: Found: 519.0 (M-Η+), calc. for C12H19N4O13P3- H+: 519.01.
EXAMPLE 16
7-(2-C-methyl-β-D-ribofaranosyl)-7H-pyrtθlo[2,3--f|pyrimidin-4(3H)-one
Figure imgf000040_0001
To the compound from Step E of Example 2 (59 mg, 0.18 mmol) was added aqueous sodium hydroxide (IM). The mixture was heated to reflux for lhr, cooled, neutralized with aqueous ΗC1 (2M) and evaporated in vacuo. The residue was purified on silica gel using dichloromethane/methanol (4:1) as eluent. Fractions containing the product were pooled and evaporated in vacuo to give the desired product (53 mg) as a colorless oil. lΗNMR (CD3CN): δ 0.70 (s, 3Η), 3.34-4.15 (overlapping m, 7H), 6.16 (s, IH), 6.57 (d, 3.6 Hz, IH), 7.37 (d, 3.6 Hz, IH), 8.83 (s, IH).
EXAMPLE 17
4-Amino-5-chloro-7-(2-C-memyl-β-D-ribofuι-anosyl)-7H-pyrrolo[2,3-<f|pyrimidine
Figure imgf000040_0002
To a pre-cooled solution (0°C) of the compound from Step F of Example 2 (140 mg, 0.50 mmol) in DMF (2.5 mL) was added N-chlorosuccinimide (0.075 g, 0.55 mmol) in DMF (0.5 mL) dropwise. The solution was stirred at room temperature for lh and the reaction quenched by addition of methanol (4 mL) and evaporated in vacuo. The crude product was purified on silica gel using methanol/dichloromethane (1:9) as eluent. Fractions containing the product were pooled and evaporated in vacuo to give the desired product (55 mg) as a colorless solid. iH NMR (CD3CN): δ 0.80 (s, 3H), 3.65-4.14 (overlapping m, 7H), 5.97 (s br, 2H),
6.17 (s, IH), 7.51 (s, IH), 8.16 (s, IH).
ES-MS: Found: 315.0 (M+H"), calc.for C12H15ClN4O + Ε : 315.09.
EXAMPLE 18
4-Armno-5-bromo-7-(2-C-methyl-β-D-ribo ranosyl -7H-pyrrolor2,3-^pyrimidine
Figure imgf000041_0001
To a pre-cooled solution (0°C) of the compound from Step F of Example 2 (28 mg, 0.10 mmol) in DMF (0.5 mL) was added N-bromosuccinimide (0.018 g, 0.10 mmol) in DMF (0.5 mL) dropwise. The solution was stirred at 0°C for 20 min, then at room temperature for 10 min. The reaction was quenched by addition of methanol (4 mL) and evaporated in vacuo. The crude product was purified on silica gel using methanol/dichloromethane (1:9) as eluent. Fractions containing the product were pooled and evaporated in vacuo to give the desired product (13.0 mg) as a colorless solid. iH ΝMR (CD3CΝ): δ 0.69 (s, 3H), 3.46-4.00 (overlapping m, 7H), 5.83 (s br, 2H), 6.06 (s, IH), 7.45 (s, IH), 8.05 (s, IH). ES-MS: Found: 359.1 (M+H+), calc.for C12H15BrN4O4 + HT: 359.04. EXAMPLE 19
2-Amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-6πpyrimidine
Figure imgf000042_0001
A mixture of 2-amino-4-chloro-7-(2-C-methyl-β-D-ribofuranosyl)-7H- pyrrolo[2,3-c pyrimidine (Example 4, Step B) (20 mg, 0.07 mmol) in EtOΗ (1.0 mL), pyridine (0.1 mL) and 10% Pd/C (6 mg) under Η2 (atmospheric pressure) was stirred overnight at room temperature. The mixture was filtered through a Celite pad which was thorougly washed with EtOH. The combined filtrate was evaporated and purified on a silica gel column with CH2Cl2/MeOH, 20/1 and 10/1, as eluent to give the title compound as a white solid (16 mg). iH NMR (200 MHz, CD3OD): δ 0.86 (s, 3H, 2'C-Me), 3.82 (dd, /5.4.= 3.6 Hz, J5.,5» = 12.7 Hz, IH, H-5'), 3.94-4.03 (m, 2H, H-5', H-4'), 4.10 (d, 73.4. = 8.8 Hz, IH, H-3'), 6.02 (s, IH, H-l'), 6.41 (d, J5>6 = 3.8 Hz, IH, H-5), 7.39 (d, IH, H-6), 8.43 (s, IH, H- 4). .ES MS: 281.4 (MET).
EXAMPLE 20
2-Amino-5-methyl-7-(2-C2-O-dimethyl-β-D-ribofuranosyl -7H-ρyrrolo[2,3- (i]pyrimidin-4(3H)-one
Figure imgf000042_0002
Step A: 2-Amino-4-chloro-7-[3,5-bis-0-(2,4-dichlorophenylmethyl -2-C- methyl-β-D-ribofuranosyl]-5-methyl-7H-pyιτolor2,3-<i1pyrimidine To an ice-cold solution of the product from Step C of Example 2 (1.57 g, 3.16 mmol) in CΗ2C12 (50 mL) was added HBr (5.7 M in acetic acid; 3.3 mL) dropwise. The reaction mixture was stirred at 0°C for 1 h and then at room temperature for 2 h, concentrated in vacuo and co-evaporated with toluene (2 x 20 mL). The resulting oil was dissolved in MeCN (20 mL) and added dropwise to a solution of the sodium salt of 2-amino-4-chloro-5-methyl-lH-pyrrolo[2,3- ]pyrimidine in acetonitrile [generated in situ from 2-amino-4-chloro-5-methyl-lH- pyrrolo[2,3-<i]pyrimidine [for preparation, see Liebigs Ann. Chem. 1984: 708-721] (1.13 g, 6.2 mmol) in anhydrous acetonitrile (150 mL), and NaΗ (60% in mineral oil, 248 mg, 6.2 mmol), after 2 h of vigorous stirring at room temperature]. The combined mixture was stirred at room temperature for 24 h and then evaporated to dryness. The residue was suspended in water (100 mL) and extracted with EtOAc (300 + 150 mL). The combined extracts were washed with brine (100 mL), dried over Na2SO4, filtered and evaporated. The crude product was purified on a silica gel column (5 x 7 cm) using ethyl acetate/hexane (0 to 30% EtOAc in 5% step gradient) as the eluent. Fractions containing the product were combined and evaporated in vacuo to give the desired product (0.96 g) as a colorless foam.
Step B: 2-Amino-4-chloro-7-r3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C,2-0- dimethyl-β-D-ribofaranosyl]-5-methyl-7H-pyrrolor2,3-ιi]pyrimidine To an ice-cold mixture of the product from Step A (475 mg, 0.7 mmol) in TΗF (7 mL) was added NaΗ (60% in mineral oil, 29 mg) and stirred at 0 °C for 0.5 h. Then Mel (48 μL) was added and reaction mixture stirred at room temperature for 24 h. The reaction was quenched with MeOΗ and the mixture evaporated. The crude product was purified on a silica gel column (5 x 3.5 cm) using hexane/ethyl acetate (9/1, 7/1, 5/1 and 3/1) as eluent. Fractions containing the product were combined and evaporated to give the desired compound (200 mg) as a colorless foam.
Step C: 2-Amino-7-r3.5-bis-O-(2,4-dichlorophenylmethyl)-2-C,2-C>-dimethyl- β-D-riboftιranosyl]-5-methyl-7H-pyrrolo[2.3-^pyrimidine-4(3H -one A mixture of the product from Step B (200 mg, 0.3 mmol) in 1,4- dioxane (15 mL) and aqueous NaOΗ (2N, 15 mL) in a pressure bottle was heated overnight at 135 °C. The mixture was then cooled to 0 °C, neutralized with 2N aqueous HCl and evaporated to dryness. The crude product was suspended in MeOH, filtered, and the solid thoroughly washed with MeOH. The combined filtrate was concentrated, and the residue purified on a silica gel column (5 x 5 cm) using CH2Cl2/MeOH (40/1, 30/1 and 20/1) as eluent to give the desired compound (150 mg) as a colorless foam.
Step D: 2-Amino-5-methyl-7-(2-C,2-0-dimethyl-β-D-ribofuranosyl -7H- pyrrolo[2,3-^lpyrimidin-4(3H)-one A mixture of the product from Step C (64 mg, 0.1 mmol) in MeOΗ (5 mL) and Et3N (0.2 mL) and 10% Pd/C (24 mg) was hydrogenated on a Parr hydrogenator at 50 psi at r.t. for 1.5 days, then filtered through a Celite pad which was thoroughly washed with MeOΗ. The combined filtrate was evaporated and the residue purified on a silica gel column (3 x 4 cm) with CΗ2Cl2/MeOΗ (30/1, 20/1) as eluent to yield 2-amino-5-methyl-7-(5-0-benzyl-2-C,2-0-dimethyl-β-D- ribofuranosyl)-7H-pyrrolo[2,3-rf]pyrimidin-4(3H)-one. The compound (37 mg) was further hydrogenated in EtOΗ (2 mL) with 10% Pd/C and under atmospheric pressure of hydrogen. After stirring 2 days at r.t., the reaction mixture was filtered through Celite, the filtrate evaporated and the crude product purified on a silica gel column (1 x 7 cm) with CΗ2Cl2/MeOΗ (30/1, 20/1 and 10/1) as eluent to yield the title compound (12 mg) after freeze-drying. iH NMR (200 MHz, CD3OD): δ 0.81 (s, 3H, 2'C-Me), 2.16 (d, 7H-6,C5-Me = 1-3 Hz, 3H, C5-Me), 3.41 (s, 3H, 2'-OMe), 3.67 (dd, 75.4.= 3.4 Hz, Js>#. = 12.6 Hz, IH, H-5'), 3.81-3.91 (m, 3H, H-5", H-4', H-3'), 6.10 (s, IH, H-l'), 6.66 (d, IH, H-6). ES MS: 323.3 (M-H)+.
EXAMPLE 21
4-Amino-5-methyl-7-(2-C-methyl-β-D-ribofuranosyl -7H-pyrrolo[2,3-(flpyrimidine
Figure imgf000045_0001
Step A: 4-Chloro-7-r3,5-bis-O-(2,4-dichloroρhenylmethyl)-2-C-methyl-β-D- ribofuranosyl]-5-methyl-7H-pyrrolor2,3-(J1pyrirnidine To an ice-cold solution of the product from Step C of Example 2 (1.06 g, 2.1 mmol) in CΗ2C12 (30 mL) was added HBr (5.7 M in acetic acid; 2.2 mL) dropwise. The reaction mixture was stirred at 0 °C for 1 h and then at room temperature for 2 h, concentrated in vacuo and co-evaporated with toluene (2 x 15 mL). The resulting oil was dissolved in MeCN (10 mL) and added dropwise into a solution of the sodium salt of 4-chloro-5-methyl-lH-pyrrolo[2,3--i]pyrimidine in acetonitrile [generated in situ from 4-chloro-5-methyl-lH-pyrrolo[2,3--f]pyrimidine [for preparation, see J. Med. Chem. 33: 1984 (1990)] (0.62 g, 3.7 mmol) in anhydrous acetonitrile (70 mL), and NaΗ (60% in mineral oil, 148 mg, 3.7 mmol), after 2 h of vigorous stirring at room temperature]. The combined mixture was stirred at room temperature for 24 h and then evaporated to dryness. The residue was suspended in water (100 mL) and extracted with EtOAc (250 + 100 mL). The combined extracts were washed with brine (50 mL), dried over Na2SO4, filtered and evaporated. The crude product was purified on a silica gel column (5 x 5 cm) using hexane/ethyl acetate (9/1, 5/1, 3/1) gradient as the eluent. Fractions containing the product were combined and evaporated in vacuo to give the desired product (0.87 g) as a colorless foam.
Ste B: 4-Chloro-5-methyl-7-(2-C-methyl-β-D-ribofuranosyl -7H-pyιτolor2.3-
(ilpyrimidine To a solution of the compound from Step A (0.87 g, 0.9 mmol) in dichloromethane (30 mL) at -78°C was added boron trichloride (IM in dichloromethane, 9.0 mL, 9.0 mmol) dropwise. The mixture was stirred at -78°C for 2.5 h, then at -30°C to -20°C for 3 h. The reaction was quenched by addition of methanol/dichloromethane (1:1) (9 mL) and the resulting mixture stirred at -15°C for 30 min, then neutralized with aqueous ammonia at 0°C and stirred at room temperature for 15 min. The solid was filtered and washed with CH2Cl2/MeOH (1/1, 50 mL). The combined filtrate was evaporated, and the residue was purified on a silica gel column (5 x 5 cm) using CH2C12 and CH2Cl2 MeOH (40/1 and 30/1) gradient as the eluent to furnish the desired compound (0.22 g) as a colorless foam.
Step C: 4-Aιmno-5-methyl-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-
(flpyrimidine
To the compound from Step B (0.2 g, 0.64 mmol) was added methanolic ammonia (saturated at 0°C; 40 mL). The mixture was heated in a stainless steel autoclave at 100°C for 14 h, then cooled and evaporated in vacuo. The crude mixture was purified on a silica gel column (5 x 5 cm) with CΗ2Cl /MeOΗ (50/1,
30/1, 20/1) gradient as eluent to give the title compound as a white solid (0.12 g).
IH NMR (OMSO-d6): δ 0.60 (s, 3H, 2'C-Me), 2.26 (s, 3H, 5C-Me), 3.52-3.61 (m, IH, H-5'), 3.70-3.88 (m, 3H, H-5", H-4', H-3'), 5.00 (s, IH, 2'-OH), 4.91-4.99 (m,
3H, 2'-OH, 3'-OH, 5'-OH), 6.04 (s, IH, H-l'), 6.48 (br s, 2H, NH2), 7.12 (s, IH, H-
6), 7.94 (s, IH, H-2). ES MS: 295.2 (MH1").
EXAMPLE 22
4-An no-7-(2-C-memyl-β-D-ribofuranosyl -7H-pyrrolor2,3-f|pyrimidine-5- carboxylic acid
Figure imgf000047_0001
The compound of Example 6 (0.035 g, 0.11 mmol) was dissolved in a mixture of aqueous ammonia (4 mL, 30 wt %) and saturated methanolic ammonia (2 mL), and a solution of H2O2 in water (2 mL, 35 wt %) was added. The reaction mixture was stirred at room temperature for 18 h. Solvent was removed under reduced pressure, and the residue obtained was purified by HPLC on a reverse phase column (Altech Altima C-18, lOx 299 mm, A = water, B = acetonitrile, 10 to 60 % B in 50 min, flow 2 mL/min) to yield the title compound (0.015 g, 41 %) as a white solid. iHNMR (CD3OD): δ 0.85 (s, 3H, Me), 3.61 (m, IH), 3.82 (m, IH) 3.99-4.86 (m, 2H), 6.26 (s, IH), 8.10 (s, 2H) 8.22(s, IH); 13C NMR (CD3OD): 20.13, 61.37, 73.79, 80.42, 84.01, 93.00, 102.66, 112.07, 130.07, 151.40, 152.74, 159.12, 169.30. HRMS (FAB) Calcd for C13H17N4O6 + 325.1148, found 325.1143.
EXAMPLE 23
4-Amino-7-(2-C-vinyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-<i1pyrimidine
Figure imgf000047_0002
Step A: 3,5-Bis-O-(2,4-dichlorophenylmethyl)-2-C-vinyl-l-Q-methyl-α-D- ribofuranose Cerium chloride heptahydrate (50 g, 134.2 mmol) was finely crushed in a pre-heated mortar and transferred to a round-bottom flask equipped with a mechanical stirrer. The flask was heated under high vacuum overnight at 160°C. The vacuum was released under argon and the flask was cooled to room temperature. Anhydrous THF (300 mL) was cannulated into the flask. The resulting suspension was stirred at room temperature for 4 h and then cooled to -78 °C. Ninylmagnesium bromide (IM in THF, 120 mL, 120 mmol) was added and stirring continued at -78 °C for 2 h. To this suspension was added a solution of 3,5-bis-O-(2,4- dichlorophenylmethyl)-l-( -methyl- -D-erythro-pentofuranose-2-ulose (14 g, 30 mmol) [from Example 2, Step B] in anhydrous THF (100 mL), dropwise with constant stirring. The reaction was stirred at -78 °C for 4 h. The reaction was quenched with saturated ammonium chloride solution and allowed to come to room temperature. The mixture was filtered through a celite pad and the residue washed with Et2O (2 x 500 mL). The organic layer was separated and the aqueous layer extracted with Et2O (2 x 200 mL). The combined organic layers were dried over anhydrous Νa2SO4 and concentrated to a viscous yellow oil. The oil was purified by flash chromatography (SiO , 10% EtOAc in hexanes). The title compound (6.7 g, 13.2 mmol) was obtained as a pale yellow oil.
Step B: 4-Chloro-7-[3,5-bis-( -(2,4-dichlorophenylmethyl)-2-C-vinyl-β-D- ribofuranosyl]-7H-pyrrolo[2,3-<flpyrimidine To a solution of the compound from Step A (6.4 g, 12.6 mmol) in anhydrous dichloromethane (150 mL) at -20 °C was added ΗBr (30% solution in AcOΗ, 20 mL, 75.6 mmol) dropwise. The resulting solution was stirred between -10°C and 0°C for 4 h, evaporated in vacuo and co-evaporated with anhydrous toluene (3 x 40 mL). The oily residue was dissolved in anhydrous acetonitrile (100 mL) and added to a solution of the sodium salt of 4-chloro-lH-pyrrolo[2,3- d]pyrimidine (5.8 g, 37.8 mmol) in acetonitrile (generated in situ as described in Example 2) at -20 °C. The resulting mixture was allowed to come to room temperature and stirred at room temperature for 24 h. The mixture was then evaporated to dryness, taken up in water and extracted with EtOAc (2 x 300 mL). The combined extracts were dried over Na2SO4, filtered and evaporated. The crude mixture was purified by flash chromatography (SiO2, 10% EtOAc in hexanes) and the title compound (1.75 g) isolated as a white foam. Step C: 4-Amino-7-r3,5-bis-6>-(2,4-dichlorophenylmethyl)-2-C-vinyl-β-D- ribofuranosyl]-7H-pyrrolor2,3--f|pyrimidine
The compound from Step B (80, mg) was dissolved in the minimum amount of 1,4-dioxane and placed in a stainless steel bomb. The bomb was cooled to -78°C and liquid ammonia was added. The bomb was sealed and heated at 90°C for 24 h. The ammonia was allowed to evaporate and the residue concentrated to a white solid which was used in the next step without further purification.
Step D: 4-Amino-7-(2-C-vinyl-β-D-ribofuranosyl)-7H-pyrrolor2,3- cπpyrimidine
To a solution of the compound from Step C (60 mg) in dichloromethane at -78 °C was added boron trichloride (IM in dichloromethane) dropwise. The mixture was stirred at -78 °C for 2.5 h, then at -30 °C to -20 °C for 3 h. The reaction was quenched by addition of methanol/dichloromethane (1:1) and the resulting mixture stirred at -15 °C for 0.5 h, then neutralized with aqueous ammonia at 0°C and stirred at room temperature for 15 min. The solid was filtered and washed with methanol/dichloromethane (1:1). The combined filtrate was evaporated and the residue purified by flash chromatography (SiO2, 10% methanol in EtOAc containing 0.1% triethylamine). The fractions containing the product were evaporated to give the title compound as a white solid (10 mg). lΗ NMR (DMSO-d6): δ 3.6 (m, 1Η, Η-5'), 3.8 (m, IH, H-5"), 3.9 (m d, 1-H, H-4'), 4.3 (t, IH, H-3'), 4.8-5.3(m, 6H, CH=CH2, 2'-OH, 3'-OH, 5'-OH) 6.12 (s, IH, H-l'), 6.59 (d, IH, H-5), 7.1 (br s, IH, NH2), 7.43 (d, IH, H-6), 8.01 (s, IH, H-2). ES-MS: Found: 291.1 (M-FT); calc. for C13H16N4O4 - H": 291.2.
EXAMPLE 24
4-Amino-7-(2-C-hvdroxymethyl-β-D-ribofuranosyl)-7H-pyrrolof2,3-^]pyrimidine
Figure imgf000050_0001
Step A: 4-Chloro-7-r3,5-bis-0-(2,4-dichlorophenylmethyl -2-C- hydroxymethyl-β-D-ribofuranosyl]-7H-pyιτolo[2,3-^pyrimidine To a solution of the compound from Example 23, Step B (300 mg, 0.48 mmol) in 1,4-dioxane (5 mL) were added N-methylmorpholine-N-oxide (300 mg, 2.56 mmol) and osmium tetroxide (4% solution in water, 0.3 mL). The mixture was stirred in the dark for 14 h. The precipitate was removed by filtration through a celite plug, diluted with water (3 x), and extracted with EtOAc. The EtOAc layer was dried over Νa2SO and concentrated in vacuo. The oily residue was taken up in dichloromethane (5 mL) and stirred over NaIO on silica gel (3 g, 10% NaIO4) for 12 h. The silica gel was removed by filtration and the residue was evaporated and taken up in absolute ethanol (5 mL). The solution was cooled in an ice bath and sodium borohydride (300 mg, 8 mmol) was added in small portions. The resulting mixture was stirred at room temperature for 4 h and then diluted with EtOAc. The organic layer was washed with water (2 x 20 mL), brine (20 mL) and dried over Na2SO4. The solvent was evaporated and the residue purified by flash chromatography (SiO , 2:1 hexanes EtOAc) to give the title compound (160 mg, 0.25 mmol) as white flakes.
Step B: 4-Amino-7-[3,5-bis-0-(2,4-dichloiOphenylmethyl)-2-C- hydroxymethyl-β-D-ribofuranosyl1-7H-pyrrolo[2,3-rf1pyrimidine
The compound from Step A (150 mg, 0.23 mmol) was dissolved in the minimum amount of 1,4-dioxane (10 mL) and placed in a stainless steel bomb. The bomb was cooled to -78 °C and liquid ammonia was added. The bomb was sealed and heated at 90°C for 24 h. The ammonia was allowed to evaporate and the residue concentrated to a white solid which was used in the next step without further purification. Step C: 4-Amino-7-(2-C-hydroxymethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-
(flpyrimidine
The compound from Step B (120 mg, 0.2 mmol) was dissolved in 1:1 methanol/dichloromethane, 10% Pd-C was added, and the suspension stirred under an Η2 atmosphere for 12 h. The catalyst was removed by filtration through a celite pad and washed with copious amounts of methanol. The combined filtrate was evaporated in vacuo and the residue was purified by flash chromatography (SiO2, 10% methanol in EtOAc containing 0.1% triethylamine) to give the title compound (50 mg) as a white powder. iH NMR (CD3OD): δ 3.12 (d, IH, CH2'), 3.33 (d, IH, CH2"), 3.82 (m, IH, H-5'), 3.99-4.1(m, 2H, H-4', H-5"), 4.3 (d, IH, H-3'), 6.2 (s, IH, H-l'), 6.58 (d, IH, H-5), 7.45 (d, IH, H-6), 8.05 (s, IH, H-2). LC-MS: Found: 297.2 (M+Ff1"); calc. for C12H16N4O5 + H+: 297.3.
EXAMPLE 25
4-Amino-7-(2-C-fluoromethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-<flpyrimidine
Figure imgf000052_0001
Step A: 4-Chloro-7-r3,5-bis-O-(2,4-dichlorophenylmethyl -2-C-fluoromethyl- β-D-ribofuranosyn-7H-pyrrolo[2,3-i]pyrimidine To a solution of the compound from Example 24, Step A (63 mg, 0.1 mmol) in anhydrous dichloromethane (5 mL) under argon, were added 4- dimethylaminopyridine (DMAP) (2 mg, 0.015 mmol) and triethylamine (62 μL, 0.45 mmol). The solution was cooled in an ice bath and p-toluenesulfonyl chloride (30 mg, 0.15 mmol) was added. The reaction was stirred at room temperature overnight, washed with NaΗCO3 (2 x 10 mL), water (10 mL), brine (10 mL), dried over Na2SO and concentrated to a pink solid in vacuo. The solid was dissolved in anhydrous THF (5 mL) and cooled in an icebath. Tetrabutylammonium fluoride (IM solution in THF, 1 mL, 1 mmol) was added and the mixture stirred at room temperature for 4 h. The solvent was removed in vacuo, the residue taken up in dichloromethane, and washed with NaHCO3 (2 x 10 mL), water (10 mL) and brine (10 mL). The dichloromethane layer was dried over anhydrous Na SO , concentrated in vacuo, and purified by flash chromatography (SiO2, 2:1 hexanes/EtOAc) to afford the title compound (20 mg) as a white solid.
Step B: 4-Amino-7-r3,5-bis-O-(2.4-dichlorophenylmethyl)-2-C-fluoromethyl- β-D-ribofuranosyl]-7H-pyrrolor2,3-(f|pyrimidine The compound from Step A (18 mg, 0.03 mmol) was dissolved in the minimum amount of 1,4-dioxane and placed in a stainless steel bomb. The bomb was cooled to -78 °C and liquid ammonia was added. The bomb was sealed and heated at 90 °C for 24 h. The ammonia was allowed to evaporate and the residue concentrated to a white solid which was used in the next step without further purification.
Step C: 4-Amino-7-(2-C-fluoromethyl-β-D-ribofuranosyl')-7H-pyrrolor2,3- .^pyrimidine
The compound from Step B (16 mg) was dissolved in 1: 1 methanol/dichloromethane, 10% Pd-C was added, and the suspension stirred under an
Η2 atmosphere for 12 h. The catalyst was removed by filtration through a celite pad and washed with copious amounts of methanol. The combined filtrate was evaporated in vacuo and the residue was purified by flash chromatography (SiO2, 10% methanol in EtOAc containing 0.1% triethylamine) to give the title compound (8 mg) as a white powder.
IH NMR (DMSO-d6): δ 3.6-3.7 (m, IH, H-5'), 3.8 - 4.3 (m, 5H, H-5", H-4', H-3',
CH2) 5.12 (t, IH, 5'-OH), 5.35 (d, IH, 3' -OH), 5.48 (s, IH, 2' -OH), 6.21 (s, IH, H- 1'), 6.52 (d, IH, H-5), 6.98 (br s, 2H, NH2), 7.44 (d, 1 H, H-6), 8.02 (s, IH, H-2).
19F NMR (DMSO-d6): δ -230.2 (t).
ES-MS: Found: 299.1 (M+HT), calc.for CizHisF t + H4: 299.27.
EXAMPLES 26 and 27
4-Amino-7-(3-deoxy-2-C-methyl-β-D-ribofuranosyl -7H-pyrrolo[2,3-< l pyrimidine and 4-amino-7-(3-deoxy-2-C-methyl-β-D-arabinofuranosyl)-7H-pyrrolo[2,3-rf] pyrimidine
Figure imgf000053_0001
Step A: 7-[2,5-Bis-0-(tgrt-butyldimeτhylsilyls>-β-D-ribofuranosyl]-7H- pyrrolo[2,3-(flpyrimidine and 7-[3,5-Bis-Q-(tert-butyldimethylsilyl -β- D-ribofuranosyl1-7H-pyrrolor2,3--t1 pyrimidine To a stirred solution of tubercidin (5.0 g, 18.7 mmol) in a mixture of pyridine (7.5 mL) and DMF (18.5 mL) was added silver nitrate (6.36 g, 38.8 mmol). This mixture was stirred at room temperature for 2 h. It was cooled in an ice bath and THF (37.4 mL) and tert-butyldimethylsilyl chloride (5.6 g, 37 mmol) was added and the mixture was stirred at room temperature for 2 h. The mixture was then filtered through a pad of celite and washed with THF. The filtrate and washings were diluted with ether containing a small amount of chloroform. The organic layer was washed successively with sodium bicarbonate and water (3 x 50 mL), dried over anhydrous sodium sulfate and concentrated. The pyridine was removed by coevaporation with toluene and the residue was purified by flash chromatography on silica gel using 5-7% MeOH in CH2C12 as the eluent; yield 3.0 g.
Step B: 7-[2,5-Bis-0-(tert-butyldimethylsilyl -β-D-ribofuranosyl ]-4-[di-(4- methoxyphenyPphenylmethyll anιino-7H-pyrrolor2,3-^pyrimidine and 7-[3,5-bis-Q-(tert-butyldimethylsilyl -β-D-ribofuranosyll-4-rdi-(4- methoxyphenyl)phenylmethyl1amino-7H-pyrrolor2,3-<ilpyrimidine To a solution of mixture of the compounds from Step A (3.0 g, 6.0 mmol) in anhydrous pyridine (30 mL) was added 4,4'-dimethoxytrityl chloride (2.8 g, 8.2 mmol) and the reaction mixture was stirred at room temperature overnight. The mixture was then triturated with aqueous pyridine and extracted with ether. The organic layer was washed with water, dried over anhydrous sodium sulfate and concentrated to a yellow foam (5.6 g). The residue was purified by flash chromatography over silica gel using 20-25% EtOAc in hexanes as the eluent. The appropriate fractions were collected and concentrated to furnish 2',5'-bis-O-(tert- butyldimethylsilyl)- and 3',5'-bis-O-(tert-butyldimethylsilyl) protected nucleosides as colorless foams (2.2 g and 1.0 g, respectively).
Step C: 7-[2,5-Bis-O-(tert-butyldimethylsilyI)-3-0-tosyl-β-D-ribofuranosyl)]-
4-[di-(4-methoxyρhenyl)phenylmethyl]amino-7H-pyrrolor2,3- -flpyrimidine
To an ice-cooled solution of 2',5'-bis-O-(tert-butyldimethylsilyl)- protected nucleoside from Step B (2.0 g, 2.5 mmol) in pyridine (22 mL) was added p- toluenesulfonyl chloride (1.9 g, 9.8 mmol). The reaction mixture was stirred at room temperature for four days. It was then triturated with aqueous pyridine (50%, 10 mL) and extracted with ether (3 x 50 mL) containing a small amount of CH2C1 (10 mL). The organic layer was washed with sodium bicarbonate and water (3 x 30 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated. Pyridine was removed by co-evaporation with toluene (3 x 25 mL). The residual oil was filtered through a pad of silica gel using hexane:ethyl acetate (70:30) as eluent; yield 1.4 g.
Step D: 4-[di-(4-methoxyphenyl)phenylmethyl]amino-7-F3-O-tosyl-β-D- ribofuranosyl-7H-pyrrolor2,3-^lpyrimidine A solution of the compound from Step C (1.0 g, 1.1 mmol) and THF (10 mL) was stirred with tetrabutylammonium fluoride (IM solution in THF, 2.5 mL) for 0.5h. The mixture was cooled and diluted with ether (50 mL). The solution was washed with water (3 x 50 mL), dried over anhydrous Na2SO , and concentrated to an oil. The residue was purified by passing through a pad of silica gel using hexane: ethyl acetate (1:1) as eluent; yield 780 mg.
Step E: 4-Amino-7-(3-deoxy-2-C-methyl-β-D-ribofuranosyl -7H-pyrrolo[2,3- d\- pyrimidine and 4-amino-7-(3-deoxy-2-C-methyl-β-D- arabinofuranosyl)-7H-pyrrolo-[2,3-<i]pyrimidine A solution of CΗ3MgI (3.0 M solution in ether, 3.0 mL) in anhydrous toluene (3.75 mL) was cooled in an ice bath. To this was added a solution of the compound from Step D (500 mg, 0.8 mmol) in anhydrous toluene (3.7 mL). The resulting mixture was stirred at room temperature for 3.5 h. It was cooled and treated with aqueous NH4CI solution and extracted with ether (50 mL containing 10 mL of CH2C12). The organic layer was separated and washed with brine (2 x 30 mL) and water (2 x 25 mL), dried over anhydrous Na2SO and concentrated to an oil which was purified by flash chromatography on silica gel using 4% MeOH in CH2C1 to furnish the 2-C-α-methyl compound (149 mg) and the 2-C-β-methyl compound (34 mg). These derivatives were separately treated with 80% acetic acid and the reaction mixture stirred at room temperature for 2.5 h. The acetic acid was removed by repeated co-evaporation with ethanol and toluene. The residue was partitioned between chloroform and water. The aqueous layer was washed with chloroform and concentrated. The evaporated residue was purified on silica gel using 5-10% MeOH in CH2C12 as the eluent to furnish the desired compounds as white solids. 4-Anuno-7-(3-deoxy-2-C-methyl-β-D-ribofuranosyl -7H-pyrrolo[2,3-(j]pyrimidine (9.0 mg): lΗ NMR (DMSO-d6): δ 0.74 (s, 3Η, CH3), 1.77 (dd, IH, H-3'), 2.08 (t, IH, H-3"), 3.59 (m, IH, H-5'), 3.73 (m, IH, H-5"), 4.15 (m, IH, H-4'), 5.02 (t, IH, OH-5'), 5.33 (s, IH, OH-2'), 6.00 (s, IH, H-l'), 6.54 (d, IH, H-7), 6.95 (br s, 2H, NH2), 7.47 (d, IH, H-8), 8.00 (s, IH, H-2); ES-MS: 263.1 [M-H].
4-Amino-7-(3-deoxy-2-C-methyl-β-D-arabino ranosyl)-7H-pyrrolo[2,3-d]pyrimidine (15 mg): lΗ NMR (DMSO-d6): δ 1.23 (s, 3Η, CH3), 2.08 (ddd, 2H, H-3'and 3"), 3.57 (m, 2H, H-5'and 5"), 4.06 (m, IH, H-4), 5.10 (s, IH, OH-2'), 5.24 (t, IH, OH-5'), 6.01 (s, IH, H-l'), 6.49 (d, IH, H-7),6.89 (br s, 2H, NH2), 7.35 (d, IH, H-8), 8.01 (s,lH,H-2). ES-MS: 265.2[M+H].
EXAMPLE 28
4-Amino-7-(2,4-C-dimethyl-β-D-ribofuranosyl -7H-pyrrolor2,3--f|pyrimidine
Figure imgf000056_0001
Step A: 5-Deoxy-l,2-( -isopropylidene-D-xylofuranose
1,2-O-Isopropylidene-D-xylofuranose (38.4 g, 0.2 mol), 4- dimethylaminopyridine (5 g), triethylamine (55.7 mL, 0.4 mol) were dissolved in dichloromethane (300 mL). p-Toluenesulfonyl chloride (38.13 g, 0.2 mol) was added and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then poured into saturated aqueous sodium bicarbonate (500 mL) and the two layers were separated. The organic layer was washed with aqueous citric acid solution (20%, 200 mL), dried (Na2SO ) and evaporated to give a solid (70.0 g). The solid was dissolved in dry TΗF (300 mL) and LiAlΗ]. (16.0 g, 0.42 mol) was added in portions over 30 min. The mixture was stirred at room temperature for 15. Ethyl acetate (100 mL) was added dropwise over 30 min and the mixture was filtered through a silica gel bed. The filtrate was concentrated and the resulting oil was chromatographed on silica gel (EtOAc/hexane 1/4) to afford the product as a solid (32.5 g).
Step B: 3,5-Bis-O-(2,4-dichlorophenylmethyl)-l-Q-methyl-4-methyl-α-D- ribofuranose
Chromium oxide (50 g, 0.5 mol), acetic anhydride (50 mL, 0.53 mol) and pyridine (100 mL, 1.24 mol) were added to dichloromethane (1 L) in an ice-water bath and the mixture was stirred for 15 min. 5-Deoxy-l,2-O-isopropylidene-D- xylofuranose (32 g, 0.18 mol) in dichloromethane (200 mL) was added, and the mixture was stirred at the same temperature for 30 min. The reaction solution was diluted with ethyl acetate (1 L) and filtered through a silica gel bed. The filtrate was concentrated to give a yellow oil. The oil was dissolved in 1,4-dioxane (1 L) and foimaldehyde (37%, 200 mL). The solution was cooled to 0°C and solid KOH (50 g) was added. The mixture was stirred at room temperature overnight and was then extracted with ethyl acetate (6 x 200 mL). After concentration, the residue was chromatographed on silica gel (EtOAc) to afford the product as an oil (1.5 g). The oil was dissolved in l-methyl-2-pyrrolidinone (20 mL) and 2,4-dichlorophenylmethyl chloride (4 g, 20.5 mmol) and NaH (60%, 0.8 g) were added. The mixture was stirred overnight and diluted with toluene (100 mL). The mixture was then washed with saturated aqueous sodium bicarbonate (3 x 50 mL), dried (Na2SO ) and evaporated. The residue was dissolved in methanol (50 mL) and HCl in dioxane (4 M, 2 mL) was added. The solution was stirred overnight and evaporated. The residue was chromatographed on silica gel (EtOAc/hexane: 1/4) to afford the desired product as an oil (2.01 g).
Step C: 3,5-Bis-Q-(2,4-dichlorophenylmethyl -2,4-di-C-methyl-l-O-methyl-α-
D-ribofuranose The product (2.0 g, 4.0 mmol) from Step B and Dess-Martin periodinane (2.0 g) in dichloromethane (30 mL) were stirred overnight at room temperature and then concentrated under reduced pressure. The residue was triturated with ether ether (50 mL) and filtered. The filtrate was washed with a solution of Na2S2O3.5H O (2.5 g) in saturated aqueous sodium bicarbonate solution (50 mL), dried (MgSO4), filtered and evaporated. The residue was dissolved in anhydrous Et2O (20 mL) and was added dropwise to a solution of MeMgBr in Et2O (3 M, 10 mL) at - 78 °C. The reaction mixture was allowed to warm to -30°C and stirred at -30°C to - 15°C for 5 h, then poured into saturated aqueous ammonium chloride (50 mL). The two layers were separated and the organic layer was dried (MgSO ), filtered and concentrated. The residue was chromatographed on silica gel (EtOAc/hexane: 1/9) to afford the title compound as a syrup (1.40 g).
Step D: 4-Chloro-7-[3,5-bis-0-(2,4-dichlorophenylmethyl)-2,4-di-C-methyl-β- D-ribofuranosvH-7H-pyrrolo[2,3~<f1pyrimidine
To the compound from Step C (0.70 g, 1.3 mmol) was added ΗBr (5.7 M in acetic acid, 2 mL). The resulting solution was stirred at room temperature for 1 h, evaporated in vacuo and co-evaporated with anhydrous toluene (3 x 10 mL). 4-Chloro-IH-pyrrolo[2,3-<f]pyrimidine (0.5 g, 3.3 mmol) and powdered KOΗ (85%, 150 mg, 2.3 mmol) were stirred in l-methyl-2-pyrrolidinone (5 mL) for 30 min and the mixture was co-evaporated with toluene (10 mL). The resulting solution was poured into the above bromo sugar residue and the mixture was stirred overnight. The mixture was diluted with toluene (50 mL), washed with water (3 x 50 mL) and concentrated under reduced pressure. The residue was chromatographed on silica gel eluting with EtOAc/ Hexane (15/85) to afford a solid (270 mg).
Step E: 4-Amino-7-(2,4-C-dimethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-
- lpyrimidine The compound from Step D (270 mg) was dissolved in dioxane (2 mL) and liquid ammonia (20 g) was added in a stainless steel autoclave. The mixture was heated at 100°C for 15, then cooled and evaporated. The residue was chromatographed on silica gel (EtOAc) to afford a solid (200 mg). The solid (150 mg) and Pd/C (10% 150 mg) in methanol (20 mL) were shaken under Η2 (30 psi) for 3 h, filtered and evaporated. The residue was chromatographed on silica gel (MeOH/CH2Cl2: 1/9) to afford the desired product as a solid (35 mg). iH NMR (OMSO-d6): δ 0.65 (s, 3H), 1.18 (s, 3H), 3.43 (m, 2H), 4.06 (d, IH, J 6.3 Hz), 4.87 (s, IH), 5.26 (br, IH), 5.08 (d, IH, 76.3 Hz), 5.25 (t, IH, J 3.0 Hz), 6.17 (s, IH), 6.54 (d, IH, J 3.5 Hz), 6.97 (s, br, 2H), 7.54 (d, IH, J 3.4 Hz), 8.02 (s, IH). 13C NMR (OMSO-d6): δ 18.19, 21.32, 65.38, 73.00, 79.33, 84.80, 90.66, 99.09,
102.41, 121.90, 149.58, 151.48, 157.38.
LC-MS: Found: 295.1 (M+H+); calculated for C13H18N4O4+H+: 295.1.
EXAMPLE 29
4-Amino-7-(3-deoxy-3-fluoro-2--C-methyl-β-D-ribofuranosyl -7H-pyrrolo[2,3- <f|pyrimidine
Figure imgf000059_0001
Step A: 3-Deoxy-3-fluoro-l-0-methyl-5-0-toluoyl-α-D-ribofuranose
1,2-O-Isopropylidene-D-xylofuranose (9.0 g, 50 mmol) and -toluoyl chloride (7.0 mL, 50 mmol) in pyridine (50 mL) were stirred for 30 min. Water (10 mL) was added and the mixture was concentrated under reduced pressure. The residue was dissolved in toluene (500 mL) and the solution was washed with water (200 mL) and saturated aqueous sodium bicarbonate (200 mL). The two layers were separated and the organic layer was evaporated. The residue was dissolved in methanol (100 mL) and HCl in dioxane (4 M, 10 mL) was added. The mixture was stirred at room temperature overnight and was then evaporated under reduced pressure. The resulting oil was chromatographed on silica gel (EtOAc/hexane: 1/1) to afford an oil (10.1 g). The oil was dissolved in dichloromethane (100 mL) and diethylaminosulfur trifluoride (DAST) (5.7 mL) was added. The mixture was stirred overnight and was then poured into saturated aqueous sodium bicarbonate solution (100 mL). The mixture was extracted with toluene (2 x 50 mL) and the combined organic layers were concentrated. The residue was chromatographed on silica gel (EtOAc/hexane: 15/85) to afford the title compound as an oil (1.50 g). Step B: 3-Deoxy-3-fluoro-2-C-methyl-l-O-methyl-5-0-toluoyl-α-D- ribofuranose
The product from Step A (1.0 g, 3.5 mmol) and Dess-Martin periodinane (2.5 g) in dichloromethane (20 mL) were stirred overnight at room temperature and was then concentrated under reduced pressure. The residue was triturated with diethyl ether (50 mL) and filtered. The filtrate was washed with a solution of Na2S2O3.5H2O (12.5 g) in saturated aqueous sodium bicarbonate (100 mL), dried (MgSO4), filtered and evaporated. The residue was dissolved in anhydrous THF (50 mL). TiCl (3 mL) and methyl magnesium bromide in ethyl ether (3 M, 10 mL) were added at -78°C and the mixture was stirred at -50 to -30°C for 2 h. The mixture was poured into saturated aqueous sodium bicarbonate solution (100 mL) and filtered through Celite. The filtrate was extracted with toluene (100 mL) and evaporated. The residue was chromatographed on silica gel (EtOAc/hexane: 15/85) to afford the title compound as an oil (150 mg).
Step C: 4-Amino-7-(3-deoxy-3-fluoro-2-C-methyl-β-D-ribofuranosyl')-7H- pyrrolo[2,3-(i1pyrimidine
The product from Step B (150 mg, 0.5 mmol) was dissolved in ΗBr (30%) in acetic acid (2 mL). After one hour, the mixture was evaporated under reduced pressure and co-evaporated with toluene (10 mL). 4-Chloro-IH-pyrrolo[2,3- - jpyrimidine (0.5 g, 3.3 mmol) and powdered KOΗ (85%, 150 mg, 2.3 mmol) were stirred in DMF (3 mL) for 30 min and the mixture was co-evaporated with toluene (2 mL). The resulting solution was poured into the above bromo sugar and the mixture was stirred overnight. The mixture was diluted with toluene (50 mL), washed with water (3 x 50 mL) and concentrated under reduced pressure. The residue was chromatographed on silica gel (EtOAc/hexane: 15/85) to afford an oil (60 mg). The oil was dissolved in dioxane (2 mL) and liquid ammonia (20 g) was added in a stainless steel autoclave. The mixture was heated at 85°C for 18 h, then cooled and evaporated. The residue was chromatographed on silica gel (methanol/dichloromethane: 1/9) to afford the title compound as a solid (29 mg). lΗ NMR (OMSO-d6): δ 0.81 (s, 3Η), 3.75 (m, 2H), 4.16 (m, IH), 5.09 (dd, IH, J 53.2, 7.8 Hz), 5.26 (br, IH), 5.77 (s, IH), 6.15 (d, IB, J 2.9 Hz), 6.59 (d, IH, J3.4 Hz), 7.02 (s br, 2H), 7.39 (d, IH, J 3.4 Hz), 8.06 (s, IH). 13C NMR (DMSO- 5): 19.40, 59.56, 77.24, 79.29, 90.15, 91.92, 99.88, 102.39,
121.17, 149.80, 151.77, 157.47.
19F NMR (OMSO-d6): δ 14.66 (m).
ES-MS: Found: 283.1 (M+H1"); calculated for
Figure imgf000061_0001
283.1.
EXAMPLE 30
4-Amino-7-(2-C,2-0-dimethyl-β-D-ribomranosyl -7H-pyrrolor2,3-f1pyrimidine
Figure imgf000061_0002
Step A: 4-chloro-7-[3,5-bis-6>-(2,4-dichlorophenylmethyl)-2-C.2-Q-dimethyl- β-D-ribofuranosyl]-7H-pyrrolo[2,3-(flpyrimidine To a pre-cooled (0°C) solution of the compound from Example 2, Step D (618 mg, 1.0 mmol) in TΗF (8 mL) was added methyl iodide (709 mg, 5.0 mmol) and NaΗ (60% in mineral oil) (44 mg, 1.1 mmol). The resulting mixture was stirred overnight at room temperature and then poured into a stirred mixture of saturated aqueous ammonium chloride (50 mL) and dichloromethane (50 mL). The organic layer was washed with water (50 mL), dried (MgSO4) and evaporated in vacuo. The resulting crude product was purified on silica gel using ethyl acetate/hexane as the eluent. Fractions containing the product were pooled and evaporated in vacuo to give the desired product (735 mg) as a colorless foam.
Step B: 4-amino-7-r3,5-bis-O-(2,4-dichlorophenylmethyl -2-C2-0-dimethyl- β-D-ribofuranosyl1-7H-pyrrolo[2,3-<f|pyrimidine To the compound from Step A (735 mg, 1.16 mmol) was added methanolic ammonia (saturated at 0°C) (20 mL). The mixture was heated in a stainless steel autoclave at 80°C overnight, then cooled and the content evaporated in vacuo. The crude mixture was purified on silica gel using ethyl acetate/hexane as the eluent. Fractions containing the product were pooled and evaporated in vacuo to give the desired product (504 mg) as colorless foam.
Step C: 4-amino-7-(2-C.2-O-dimethyl-β-D-ribofuranosylV7H-pyrrolo[2,3- d] pyrimidine
A mixture of the product from Step C (64 mg, 0.1 mmol), MeOΗ (5 mL), Et3N (0.2 mL) and 10% Pd/C (61 mg) was hydrogenated on a Parr hydrogenator at 50 psi at room temperature overnight. The mixture was filtered throught celite, evaporated in vacuo and filtered through a pad of silica gel using 2% methanol in dichloromethane as eluent. The desired product was collected and evaporated in vacuo. The compound was redissolved in methanol (10 mL) and 10% Pd/C (61 mg) was added. The mixture was hydrogenated on a Parr hydrogenator at 55 psi at room temperature for two weeks. The mixture was filtered through celite, evaporated in vacuo and purified on silica gel using 10% methanol in dichloromethane as eluent. Fractions containing the product were pooled and evaporated in vacuo to give the desired product (110 mg) as a colorless foam. lΗ NMR (DMSO-d6): δ 0.68 (s, 3Η,), 3.40 (s, 3H), 3.52-3.99 (overlapping m, 4H), 4.92 (d, IH), 5.07 (t, IH), 6.26 (s, IH), 6.55 (d, IH), 7.00s br, 2H), 7.46 (d, IH), 8.05 (s, IH). LC-MS: Found: 293.1 (M-H+); calc. for Ci2Hi6N4θ4-H+: 293.12.
EXAMPLE 31
4-Methylamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-< lpyrimidine
Figure imgf000062_0001
The compound from Step E of Example 2 (200 mg, 0.67 mmol) was added to methylamine (5 mL condensed in a small stainless steel autoclave) and warmed at 85°C for 48 h, then cooled and evaporated in vacuo. The crude mixture was purified on a silica gel with ethanol as the eluent to give the title compound which separated as an amorphous solid after treatment with MeCN. The amorphous solid was dissolved in water and lyophilized to give a colorless powder (144 mg). IH NMR (DMSO-dtf): δ 0.63 (s, 3H, CH3), 3.32 (s, 3H, N CH3), 3.58-3.67 (m, IH, H- 5'), 3.79-3.39 (m, 3H, H-5", H-4', H-3'), 5.03 (s, IH, 2'-OH), 5.04-5.11 (lH,3'-OH, IH, 5'-OH), 6.14 (s, IH, H-l'), 6.58 (d, IH, J5,6 = 3.6 Hz, H-5), 7.46 (d, IH, H-6), 7.70 (br s, IH, NH), 8.14 (s, IH, H-2). LC-MS: Found: 295.1 (M-H+); calc. for Ci3Hi8N4θ4+H+: 294.3.
EXAMPLE 32
4-Dimethylamino-7-(2-C-methyl-β-D-ribofuranosyl -7H-pyrrolor2,3-^pyrimidine
Figure imgf000063_0001
The compound from Step E of Example 2 (200 mg, 0.67 mmol) was added to dimethylamine (5 mL condensed in a small stainless steel autoclave) and warmed at 85 °C for 48 h, then cooled and evaporated in vacuo. The crude mixture was purified on a silica gel with ethanol as the eluent to give the title compound which separated as an amorphous solid after treatment with MeCN. The amorphous solid was dissolved in water and lyophilized to give a colorless powder (164 mg). IH NMR (OMSO~d6): δ 0.64 (s, 3H, CH3), 3.29 (s, 3H, N CH3), 3.32 (s, 3H, N CH3), 3.60-3.66 (m, IH, H-5'), 3.77-3.97 (m, 3H, H-5", H-4', H-3'), 5.04 (s, IH, 2'-OH), 5.06-5.11 (IH, 3'-OH, IH, 5'-OH), 6.21 (s, IH, H-l'), 6.69 (d, IH, J5,6 = 3.6 Hz, H- 5), 7.55 (d, IH, H-6), 8.13 (s, IH, H-2). LC-MS: Found: 309.3 (M-H+); calc. for Ci4H2θN4θ4+H+: 308.33.
EXAMPLE 33
4-Cyclopropylamino-7-(2-C-methyl-β-D-ribofuranosvD-7H-pyrrolor2,3-ι^1pyrimidine
Figure imgf000064_0001
The compound from Step E of Example 2 (200 mg, 0.67 mmol) was added to cyclopropylamine (5 mL condensed in a small stainless steel autoclave) and warmed at 85°C for 48 h, then cooled and evaporated in vacuo. The crude mixture was purified on a silica gel with ethanol as the eluent to give the title compound which separated as an amorphous solid after treatment with MeCN. The amorphous solid was dissolved in water and lyophilized to give a colorless powder (148 mg). iH NMR (DMSO-d6): δ 0.51- 0.58 (m, 2H), 0.64 (s, 3H, CH3), 0.74- 0.076 (m, 2H), 3.62-3.67 (m, IH, H-5'), 3.79-3.82 (m, 3H, H-5"), 3.92-3.96 (m, H-4', H-3'), 5.03 (s, IH, 2'-OH), 5.05-5.10 (IH, 3'-OH, IH, 5'-OH), 6.15 (s, IH, H-l'), 7.48 (d, IH, J5,6 = 3.6 Hz, H-5), 7.59 (d, IH, H-6), 8.13 (s, IH, H-2). LC-MS: Found: 321.1 (M-H+); calc. for C15H20N4O4+H+: 320.3.
EXAMPLE 34
4-Amino-7-(3-C-methyl-β-D-xylofuranosylN)-7H-pyrrolor2,3-(ilpyrimidine
Figure imgf000064_0002
Step A: 7-r2,5-Bis-6-(tert-butyldimethylsilyl -β-D-ribofuranosyls)1-4-[(4- methoxyphenyl)diphenylmethyl]amino-7H-pyrrolo[2,3-J1pyrimidine and 7-[3,5-bis-0-(tert-butyldimethylsilyl -β-D-ribofuranosyl1-4-[(4- methoxyphenyl)diphenylmethyl1amino-7H-pyrrolo[2,3-6πpyrimidine To a solution of mixture of the compounds from Step A of Examples 26 and 27 (0.32 g, 0.65 mmol) in anhydrous pyridine (6 mL) was added monomethoxytrityl chloride (0.30 g, 0.98 mmol) and the reaction mixture was stirred at room temperature overnight. The mixture was then concentrated and the residue was partitioned between CH2C12 (70 mL) and water (20 mL). The organic layer was washed with water and brine, dried (Na2SO ) and concentrated. The residue was purified on silica gel column using 5-13% EtOAc in hexanes as the eluent. The appropriate fractions were collected and concentrated to furnish 2',5'-bis-O-(tert- butyldimethylsilyl)- and 3',5'-bis-O-(tert-butyldimethylsilyl) protected nucleosides as colorless foams (343 mg and 84 mg, respectively).
Step B: 7-[2,5-Bis-( -(tert-butyldimethylsilyl -β-D-ervt ϊro-pentofuranos-3- ulosyl]-4-[(4-methoxyphenyl)diphenylmethynamino-7H-pyrrolo[2,3- lpyrimidine To a well-stirred suspension of chromium trioxide (91 mg, 0.91 mmol) in CΗ2C12 (4 mL) at 0°C were added pyridine (147 μL, 1.82 mmol) and then acetic anhydride (86 μL, 0.91 mmol). The mixture was stirred at room temperature for 0.5 h. Then the 2',5'-bis-O-(tert-butyldimethylsilyl) protected nucleoside from step A (343 mg 0.45 mmol) in CH2CI2 (2.5 mL) was added and the mixture stirred at room temperature 2 h. The mixture was then poured into ice-cold EtOAc (10 mL) and filtered through a short silica gel column using EtOAc as the eluent. The filtrate was evaporated and the residue purified on a silica gel column with hexanes and hexanes/EtOAc (7/1) as the eluent to give the title compound (180 mg).
Step C: 7-r2,5-Bis-O-(tert-butyldimethylsilyl)-3-C-methyl-β-D-ribofuranosyl -
4- [ (4-methoxyphenyl)diphenylmethyl1 amino-7H-pyrrolo [2,3- (f|pyrimidine and 7-[2,5-Bis-C>-(tert-butyldimethylsilyl)-3-C-methyl-β- D-xylofuranosyl -4- [(4-methoxyρhenyl)diphenylmethyl1 amino-7H- pyrrolo [2,3 -d\ pyrimidine To a mixture of MeMgBr (3.0 M solution in ether; 0.17 mL, 0.5 mmol) in anhydrous hexanes (1.5 mL) at room temperature was added dropwise a solution of the compound from Step B (78 mg, 0.1 mmol) in anhydrous hexanes (0.5 mL). After 2 h stirring at room temperature, the reaction mixture was poured into ice-cold water (10 mL) and diluted with EtOAc (20 mL), then filtered through Celite which was then thoroughly washed with EtOAc. The layers were separated and the organic layer was washed with brine, dried (Na2SO4) and concentrated. The residue was purified on a silica gel column using 8 to 25% EtOAc in hexanes as eluent to give the 3-C-methyl xylo- (60 mg) and the 3-C-methyl ribo-isomer (20 mg).
Step D: 4-Amino-7-(3-C-methyl-β-D-xylofuranosyl -7H-pyrrolo[2,3-
(flpyrimidine
To an ice-cold solution of 3-C-methyl-xylo isomer from Step C (60 mg, 0.08 mmol) in TΗF (2 mL) was added TBAF (1 M in TΗF; 0.32 mL, 0.32 mmol). The reaction mixture was stirred at room temperature for 5 h, then diluted with CΗ2C12 (50 mL), washed with water (3 x 15 mL), dried,and evaporated. The residue was dissolved in dioxane (0.3 mL) and 80% acetic acid (3 mL) was added. The reaction mixture was stirred at room temperature for 24 h and then evaporated. The residue was co-evaporated with dioxane, taken up in water (50 mL) and washed with CH2C12 (2 x 10 mL). The aqueous layer was concentrated and then freeze-dried. The residue was purified on silica gel column with CH2Cl2/MeOH (20/1 and 10/1) as the eluent to give the title compound as a white fluffy compound after freeze drying (10 mg).
IH NMR (CD3CN): δ 1.28 (s, 3H, CH3), 3.56 (br s, IH, OH), 3.78 (m, 3H, H-4', H- 5', H-5"), 4.10 (br s, IH, OH), 4.44 (d, IH, J v = 3.9 Hz, H-2'), 5.58 (d, IH, H-l'), 5.85 (br s, 2H, NH2), 6.15 (br s, IH, OH), 6.48 (d, IH, J5,6 = 3.7 Hz, H-5), 7.23 (d, IH, H-6), 8.11 (s, IH, H-2). ES-MS: 281 [MH]+.
EXAMPLE 35
4-Amino-7-(3-C-methyl-β-D-ribomranosyl -7H-pyrrolo[2,3-<i1pyrimidine
Figure imgf000066_0001
The ribo-isomer (20 mg) from Step C of Example 32 was deprotected using the procedure described in Step D of Example 32 to yield the title compound (4 mg).
IH NMR (CD3CN): δ 1.43 (s, 3H, CH3), 3.28 (br s, IH, OH), 3.58 (m, 2H, H-5', H- 5"), 3.99 ( m, IH, H-4'), 4.10 (br s, IH, OH), 4.62 (d, IH, JTγ = 8.1 Hz, H-2'), 5.69 (d, IH, H-l'), 5.88 (br s, 3H, OH, NH2), 6.45 (br s, IH, OH), 6.51 (d, IH, J5,6 = 3.7 Hz, H-5), 7.19 (d, IH, H-6), 8.12 (s, IH, H-2). ES-MS: 281 [MH]+.
EXAMPLE 36
2,4-Diamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolor2,3-fl1pyrimidine
Figure imgf000067_0001
A mixture of the product from Step B of Example 4 (24 mg) in aqueous ammonia (30%, 10 mL) was heated in a stainless steel autoclave at 100 °C overnight, then cooled and evaporated. The residue was purified on a silica gel column with CH2Cl2/MeOH (10/1 and 5/1) as the eluent to afford the title compound (15 mg).
IH NMR (DMSO-d6): δ 0.68 (s, 3H, CH3), 3.48-3.58 (m IH, H-5'), 3.68-3.73 (m, 2H, H-5", H-4'), 3.84 (m, IH, H-3'), 4.72 (s, IH, 2'-OH), 4.97-5.03 (m, 2H, 3'-OH, 5'- OH), 5.45 (br s, 2H, NH2), 6.00 (s, IH, H-l'), 6.28 (d, IH, J= 3.7 Hz, H-5), 6.44 (br s, 2H, NH2) 6.92 (d, IH I = 3.7 Hz, H-6). ES MS: 294.1 (M-H+). EXAMPLE 37
4-Amino-2-fluoro-7-(2-C-methyl-β-D-ribo ranosyl -7H-pyrtθlo[2,3-< lpyrimidine
Figure imgf000068_0001
To a solution of HF/pyridine (70%, 2 mL) diluted with pyridine (1 mL) at -30 °C is added the compound of Example 36 (60 mg, 0.2 mmol) in 0.5 mL pyridine followed by tert-butyl nitrite (36 μL, 0.3 mmol). Stirring is continued for 5 min at -25 °C. Then the solution is poured into ice-water (5 mL), neutralized with 2 N aqueous NaOH, and evaporated to dryness. The residue is purified on a silica gel column with CH2Cl2/MeOH (20/1 and 10/1) as the eluent to afford the title compound.
Scheme 2
Figure imgf000068_0002
(DCB = 2,4-dichlorobenzyl)
Figure imgf000068_0003
Figure imgf000069_0001
2-5 2-6
StepG StepH
Figure imgf000069_0003
Figure imgf000069_0002
2-7 z8
(TBS = tert-butyldimethylsilyl)
Figure imgf000069_0004
(MMTr = p-methoxyphenyldiphenylmethyl)
Figure imgf000070_0001
EXAMPLE 38
4-An no-7-[2-C-methyl-3-0-(l-oxo-octylVβ-D-ribofuranosyl]-7H-pyn.Olo[2,3- lpyrimidine (2-12)
Step A: 3,5-Bis-O-(2,4-dichlorobenzyl -l-0-methvI-α-D-ribofuranose (1-2)
A mixture of 2-0-acetyl-3,5-bis-0-(2,4-dichlorobenzyl)-l-0-methyl-α- D-ribofuranose (2-1) [for preparation, see: Ηelv. Chim. Acta 78: 486 (1995)] (52.4 g, 0.10 mol) in methanolic K2CO3 (500 mL, saturated at room temperature) was stirred at room temperature for 45 min. and then concentrated under reduced pressure. The oily residue was suspended in CΗ2C12 (500 mL), washed with water (300 mL + 5 x 200 mL) and brine (200 mL), dried (Na2SO4), filtered, and concentrated to give the title compound (49.0 g) as colorless oil, which was used without further purification in Step B below.
IH NMR (OMSO-d6 ): δ 3.28 (s, 3H, OCH3), 3.53 (d, 2H, J5A = 4.5 Hz, H-5a, H-5b), 3.72 (dd, IH, J3,4 = 3.6 Hz, J3,2 = 6.6 Hz, H-3), 3.99 (ddd, IH, J2ιl = 4.5 Hz, J2,0H-2 = 9.6 Hz, H-2), 4.07 (m, IH, H-4), 4.50 (s, 2H, CH2Ph), 4.52, 4.60 (2d, 2Η, Jgeτa = 13.6 Hz, CH2Ph), 4.54 (d, 1Η, OΗ-2), 4.75 (d, IH, H-l), 7.32-7.45, 7.52-7.57 (2m, 10H, 2Ph).
13C NMR (DMSO-dβ) δ 55.40, 69.05, 69.74, 71.29, 72.02, 78.41, 81.45, 103.44, 127.83, 127.95, 129.05, 129.28, 131.27, 131.30, 133.22, 133.26, 133.55, 133.67, 135.45, 135.92.
Step B: 3,5-Bis-Q-(2,4-dichlorobenzyl)-l-O-methyl-α-D-eryt/ιro- pentofuranos-2-ulose (2-3) To an ice-cold suspension of Dess-Martin periodinane (50.0 g, 118 mmol) in anhydrous CH2C1 (350 mL) under argon (Ar) was added a solution of the compound from Step A (36.2 g, 75 mmol) in anhydrous CH2C12 (200 mL) dropwise over 0.5 h. The reaction mixture was stirred at 0°C for 0.5 h and then at room temperature for 3 days. The mixture was diluted with anhydrous Et2O (600 mL) and poured into an ice-cold mixture of Na2S2O3.5H2O (180 g) in saturated aqueous NaHCO3 (1400 mL). The layers were separated, and the organic layer was washed with saturated aqueous NaHCO3 (600 mL), water (800 mL) and brine (600 mL), dried (MgSO4), filtered and evaporated to give the title compound (34.2 g) as a colorless oil, which was used without further purification in Step C below. iH NMR (CDC13): δ 3.50 (s, 3H, OCH3), 3.79 (dd, IH, J5a,5b = 11.3 Hz, J5aA = 3.5 Hz, H-5a), 3.94 (dd, IH, JSM = 2.3 Hz, H-5b), 4.20 (dd, IH, J3Λ = 1.3 Hz, J3,4 = 8.4 Hz, H-3), 4.37 (ddd, IH, H-4), 4.58, 4.69 (2d, 2H, Igsm = 13.0 Hz, CH2Ph), 4.87 (d, 1Η, Η-l), 4.78, 5.03 (2d, 2H, Jgem = 12.5 Hz, CH2Ph), 7.19-7.26, 7.31-7.42 (2m, 10Η, 2Ph). 13C NMR (OMSO-d6): δ 55.72, 69.41, 69.81, 69.98, 77.49, 78.00, 98.54, 127.99, 128.06, 129.33, 129.38, 131.36, 131.72, 133.61, 133.63, 133.85, 133.97, 134.72, 135.32, 208.21.
Step C: 3,5-Bis-Q-(2,4-dichlorobenzyl)-2-C-methyl-l-Q-methyl-α-D- ribofuranose (2-4) To a solution of MeMgBr in anhydrous Et2O (0.48 M, 300 mL) at
-55 °C was added dropwise a solution of the compound from Step B (17.40 g, 36.2 mmol) in anhydrous Et2O (125 mL). The reaction mixture was allowed to warm to -30°C and stirred for 7 h at -30°C to -15°C, then poured into ice-cold water (500 mL) and the mixture vigorously stirred at room temperature for 0.5 h. The mixture was filtered through a Celite pad (10 x 5 cm) which was thoroughly washed with Et2O. The organic layer was dried (MgSO4), filtered and concentrated. The residue was dissolved in hexanes (-30 mL), applied onto a silica gel column (10 x 7 cm, prepacked in hexanes) and eluted with hexanes and hexanes/EtOAc (9/1) to give the title compound (16.7 g) as a colorless syrup. lH NMR (CDC13): δ 1.36 (d, 3H, JMe,oH = 0.9 Hz, 2C-Me), 3.33 (q, IH, OH), 3.41 (d, IH, J3,4 = 3.3 Hz), 3.46 (s, 3H, OCH3), 3.66 (d, 2H, J5A = 3.7 Hz, H-5a, H-5b), 4.18 (apparent q, IH, H-4), 4.52 (s, IH, H-l), 4.60 (s, 2H, CH2Ph), 4.63, 4.81 (2d, 2Η, 7gem = 13.2 Hz, CH2Ph), 7.19-7.26, 7.34-7.43 (2m, 10Η, 2Ph).
13C NMR (CDCI3): δ 24.88, 55.45, 69.95, 70.24, 70.88, 77.06, 82.18, 83.01, 107.63, 127.32, 129.36, 130.01, 130.32, 133.68, 133.78, 134.13, 134.18, 134.45, 134.58.
Step D: 4-Chloro-7-[3,5-bis-0-(2,4-dichlorobenzyl)-2-C-methyl-β-D- ribofuranosyn-7H-pyrrolo[2,3-^lpyrimi dine (2-5) To a solution of the compound from Step C (9.42 g, 19 mmol) in anhydrous dichloromethane (285 mL) at 0°C was added ΗBr (5.7 M in acetic acid, 20 mL, 114 mmol) dropwise. The resulting solution was stirred at 0°C for 1 h and then at room temperature for 3h, evaporated in vacuo and co-evaporated with anhydrous toluene (3 x 40 mL). The oily residue was dissolved in anhydrous acetonitrile (50 mL) and added to a solution of sodium salt of 4-chloro-lH-pyrrolo[2,3-ii]pyrimidine [for preparation, see J. Chem. Soc, 131 (I960)] in acetonitrile [generated in situ from 4-chloro-lH-pyrrolo[2,3-<i]pyrimidine (8.76 g, 57 mmol) in anhydrous acetonitrile (1000 mL), and NaΗ (60%) in mineral oil, 2.28 g, 57 mmol), after 4 h of vigorous stirring at room temperature]. The combined mixture was stirred at room temperature for 24 h, and then evaporated to dryness. The residue was suspended in water (250 mL) and extracted with EtOAc (2 x 500 mL). The combined extracts were washed with brine (300 mL), dried over Na2SO4, filtered and evaporated. The crude product was purified on a silica gel column (10 cm x 10 cm) using ethyl acetate/hexane (1:3 and 1:2) as the eluent. Fractions containing the product were combined and evaporated in vacuo to give the desired product (5.05 g) as a colorless foam. 1H NMR (CDCI3): δ 0.93 (s, 3Η, CH3), 3.09 (s, IH, OH), 3.78 (dd, IH, J5',5" = 10.9 Hz, J5\4 = 2.5 Hz, H-5'), 3.99 (dd, IH, J5-,4 = 2.2 Hz, H-5"), 4.23-4.34 (m, 2H, H-3', H-4'), 4.63, 4.70 (2d, 2H, Jgeπι = 12.7 Hz, CH2Ph), 4.71, 4.80 (2d, 2Η, Λem = 12.1 Hz,CH2Ph), 6.54 (d, 1Η, , /5ι6 = 3.8 Ηz, Η-5), 7.23-7.44 (m, 10H, 2Ph). 13C NMR (CDCI3): δ 21.31, 69.10, 70.41, 70.77, 79.56, 80.41, 81.05, 91.11, 100.57, 118.21, 127.04, 127.46, 127.57, 129.73, 129.77, 130.57, 130.99, 133.51, 133.99, 134.33, 134.38, 134.74, 135.21, 151.07, 151.15 152.47.
Step E: 4-Chloro-7-(2-C-methyl-β-D-ribofuranosvn-7H-pyrrolo[2,3-
(Jlpyrimidine (2-6)
To a solution of the compound from Step D (5.42 g, 8.8 mmol) in dichloromethane (175 mL) at -78°C was added boron trichloride (IM in dichloromethane, 88 mL, 88 mmol) dropwise. The mixture was stirred at -78°C for 2.5 h, then at -30°C to -20°C for 3 h. The reaction was quenched by addition of methanol/dichloromethane (1:1) (90 mL) and the resulting mixture stirred at -15°C for 30 min., then neutralized with aqueous ammonia at 0°C and stirred at room temperature for 15 min. The solid was filtered and washed with CΗ2Cl2/MeOΗ (1/1, 250 mL). The combined filtrate was evaporated, and the residue was purified by flash chromatography over silica gel using CH2C12 and CH2Cl2:MeOH (99:1, 98:2, 95:5 and 90:10) gradient as the eluent to furnish desired compound (1.73 g) as a colorless foam, which turned into an amorphous solid after treatment with MeCN. iH NMR (DMSO-d6): δ 0.64 (s, 3H, CH3), 3.61-3.71 (m, IH, H-5'), 3.79-3.88 (m, IH, H-5"), 3.89-4.01 (m, 2H, H-3', H-4'), 5.15-5.23 (m, 3H, 2'-OH, 3'-OH, 5'-OH), 6.24 (s, IH, H-l'), 6.72 (d, IH, J5>6 = 3.8 Hz, H-5), 8.13 (d, IH, H-6), 8.65 (s, IH, H- 2).
13c NMR (ΩMSO-d6): δ 20.20, 59.95, 72.29, 79.37, 83.16, 91.53, 100.17, 117.63, 128.86, 151.13, 151.19, 151.45.
Step F: 4-Amino-7-(2-C-methyl-β-D-ribomranosvιV7H-pyrrolor2,3- lpyrimidine (2-7)
To the compound from Step E (1.54 g, 5.1 mmol) was added methanolic ammonia (saturated at 0°C; 150 mL). The mixture was heated in a stainless steel autoclave at 85°C for 14 h, then cooled and evaporated in vacuo. The crude mixture was purified on a silica gel column with C^C^/MeOΗ (9/1) as eluent to give the title compound as a colorless foam (0.8 g), which separated as an amorphous solid after treatment with MeCN. The amorphous solid was recrystallized from methanol/acetonitrile; m.p. 222°C. iH NMR (DMSO-d6): δ 0.62 (s, 3H, CH3), 3.57-3.67 (m, IH, H-5'), 3.75-3.97 (m, 3H, H-5", H-4', H-3'), 5.00 (s, IH, 2'-OH), 5.04 (d, IH, J3ΌH,3' = 6.8 Hz, 3'-OH), 5.06 (t, IH, J5OH,5-,s" = 5.1 Hz, 5' -OH), 6.11 (s, IH, H-l'), 6.54 (d, IH, J5,6 = 3.6 Hz, H-5), 6.97 (br s, 2H, NH2), 7.44 (d, IH, H-6), 8.02 (s, IH, H-2). 13C NMR (DMSO- 6): δ 20.26, 60.42, 72.72, 79.30, 82.75, 91.20, 100.13, 103.08, 121.96, 150.37, 152.33, 158.15. LC-MS: Found: 279.10 (M-H+); calc. for C12H16N4O4+H+: 279.11.
Step G: 4-Amino-7-r5-0-(tert-butyldimethylsilyl -2-C-methyl-β-D- ribofuranosyll-7H-pyrrolor2,3-<flpyrimidine (2-8)
To a solution of the compound from Step F (457 mg, 1.63 mmol) in anhydrous pyridine (3.5 mL) was added tert-butyldimethylsilyl chloride (370 mg, 2.45 mmol). The reaction mixture was stirred at room temperature for 24 h. The reaction mixture was then diluted with ethyl acetate (40 mL) which was washed with saturated aqueous sodium bicarbonate solution (20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and evaporated to an oil that was subjected to chromatography on silica gel eluting with 10% MeOΗ in CΗ2CI2. The appropriate fractions were collected, evaporated, and dried under high vacuum to furnish the title compound as a colorless foam (516 mg). iH NMR (DMSO-d6): δ 7.95 (s, IH), 7.35 (d, IH, J = 3.4Hz), 6.89 (bs, 2H, NH2),
6.44 (d, IH, I = 3.4Hz), 6.02 (s, IH), 5.01-4.98 (m, 2H), 3.92-3.70 (m, 3H), 3.40-3.25 (m, IH), 0.82 (s, 9H), 0.54 (s, 3H), 0.00 (s, 6H).
Step H: 4-(p-Methoxyphenyldiphenylmethylamino)-7-[5-( -(tert- butyldimethylsilyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3- d\ pyrimidine (2-9)
To a solution of the compound from Step G (394 mg, 1.0 mmol) in anhydrous pyridine (5 mL) was added p-methoxyphenylchlorodiphenylmethane (946 mg, 3.06 mmol) and 4-dimethylaminopyridine (DMAP) (123 mg, 1.0 mmol). The reaction mixture was stirred at room temperature for 20 h. It was then diluted with ethyl acetate (30 mL) and washed with saturated aqueous sodium bicarbonate solution (3 x 15 mL) followed by water (2 x 15mL). The organic layer was dried over anhydrous Na2SO4 and concentrated to an oil. The crude product was purified using column chromatography on silica gel eluting with 5% MeOΗ in CΗ2CI2. The appropriate fractions were collected and evaporated to give the title compound (540 mg). iH NMR (DMSO-_i6): δ 7.85 (s, IH), 7.65 (s, IH), 7.41 (d, IH, I = 3.8Hz), 7.25-7.03 (m, 12H), 6.78 (d, IH, I = 3.6 Hz), 6.69 (d, 2H, I = 9 Hz), 5.97 (s, IH), 5.00-4.94 (m, 2H), 3.85-3.62 (m, 4H), 3.59 (s, 3H), 0.83 (s, 9H), 0.55 (s, 3H), 0.003 (s, 6H).
Step I: 4-(p-Methoxyphenyldiphenylmethylamino)-7-[5-O-(tert- butyldimethylsilyl)-3-O-(l-oxo-octyl)-2-C-methyl-β-D-ribofuranosvn- 7H-pyrrolo[2,3-(flpyrimidine (2-10) To a solution of the compound from Step Η (400 mg, 0.6 mmol) and anhydrous DMAP (73 mg, 0.6 mmol) in anhydrous CΗ2θ2 (7 mL) was added slowly triethylamine (250 μL, 1.8 mmol). To the stirred solution was added octanoyl chloride (200 μL, 1.2 mmol) over 15 min. The reaction mixture was stirred for an additional 1.5 h. It was then diluted with methylene chloride (30 mL) and washed with saturated aqueous sodium bicarbonate solution (3 x 10 mL) and water (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was subjected to column chromatography on silica gel eluting with 5% MeOH in CH2C12 to afford the title compound as a light yellow foam (340 mg). iH NMR (OMSO-d6): δ 8.02 (s, IH), 7.75 (s, IH), 7.58 (d, IH, I - 3.6 Hz), 7.34-7.05 (m, 12H), 7.02 (d, IH, I = 3.6 Hz), 6.79 (d, 2H, I = 9.0 Hz), 6.01 (s, IH), 5.61 (s, IH), 5.34 (d, IH, I = 9.0 Hz), 4.19-4.14 (m, IH), 4.00-3.94 (m, IH), 3.67-3.62 (m, 4H), 3.48-3.40 (m, IH), 2.40-2.32 (m, 2H), 1.60-1.40 (m, 2H), 1.23 (bs, 8H), 0.91 (s, 9H), 0.84-0.80 (m, 3H), 0.67 (s, 3H), 0.07 (s, 6H).
Step J: 4-Amino-7-r5-0-(tert-butyldimethylsilyl -3-0-(l-oxo-octyl)-2-C- methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-( lpyrimidine (2-11) A solution of the compound from Step I (250 mg, 0.31 mmol) in 6:3:1 MeOΗ:acetic acid:H2O (10 mL) was stirred at 50°C for 12 h. The reaction mixture was then concentrated to dryness. The residue was diluted with ethyl acetate (30 mL) and washed with saturated aqueous sodium bicarbonate solution (3 15 mL) and water (2 x 10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The crude product (200 mg) was used without further purification in Step K below. Further purification of a small amount was accomplished by silica gel column chromatography using 5% MeOH in CH2CI2 as the eluent to give the title compound as a white foam . iH NMR (CDCI3): δ 8.29 (s,lH), 7.57 (d, IH, I = 3.8 Hz), 6.37 (d, IH, I = 3.8 HZ), 6.28 (s, IH), 5.33-5.28 (m, 3H), 4.29-4.23 (m, IH), 4.08-4.01 (m, IH), 3.86-3.79 (m,lH), 2.45-2.37 (m, 2H), 1.69-1.62 (m, 2H), 1.29-1.23 (m,8H), 0.97-0.84 (m, 12H), 0.11 (s, 6H).
Step K: 4-Amino-7-r2-C-methyl-3-0-(l-oxo-octyl -β-D-ribofuranosyl]-7H- pyrrolo[2,3-<flpyrimidine (2-12)
To a solution of the compound from Step I (230 mg, 0.44 mmol) in anhydrous TΗF (5mL), was added triethylamine (300 μL, 2.14 mmol) and triethylamine trihydrofluoride (750 μL, 4.5 mmol). The solution was stirred ovemight at room temperature. The reaction mixture was then diluted with ethyl acetate (30 mL) and washed with saturated aqueous sodium bicarbonate (3 x 10 mL) and water (10 mL). After drying the organic layer over anhydrous sodium sulfate and filtration, the solvent was evaporated. The resulting oil was purified on a silica gel column eluting with 1 : 1 acetone/CΗ2Cl2 followed by 10% MeOH in CH2CI2. The appropriate fractions were concentrated and lyophilized to afford the title compound as a colorless powder (90 mg).
IH NMR (CDC13): δ 8.30 (s, IH), 7.31 (d, IH, J = 3.8 Hz), 6.39 (d, IH, J = 3.8 Hz), 6.16 (s, IH), 5.44 (d, IH, I = 7.8 Hz), 5.23 (bs, 2H), 4.31-4.24 (m, IH), 4.14-4.06 (m, IH), 3.84-3.76 (m, IH), 2.48-2.40 (m, 2H), 1.80-1.50 (m, 3H), 1.34-1.23 (m, 7H), 0.95 (s, 3H), 0.88-0.55 (m, 3H).
Scheme 3
Figure imgf000076_0001
(MMTr = p-methoxyphenyldiphenylmethyl)
Figure imgf000077_0001
EXAMPLE 39
4-Amino-7-r2-C-methyl-3,5-di-O-(l-oxo-octyl -β-D-ribofuranosyl]-7H-pyrrolo[2,3- (flpyrimidine (3-3)
Step A: 4-(p-Methoxyphenyldiphenylmethylamino)-7-[3-0-(l-oxo-octyl)-2-C- methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-(i]pyrimidine (3-1) A solution of the compound from Step I of Example 1 (1-10) (300 mg,
0.37 mmol), anhydrous triethylamine (300 μL, 2.14 mmol) and triethylamine trihydrofluoride (750 μL, 4.5 mmol) in anhydrous TΗF (5 mL) was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium bicarbonate solution (3 x 20 mL) followed by water (2 x 15 mL). The organic layer was separated, dried over sodium sulfate, filtered, and evaporated. The crude product was purified on a silica gel column using 10-15% acetone in CΗ2CI2 as the eluent. The appropriate fractions were combined and evaporated to afford the title compound as a colorless foam (240 mg). lH NMR (DMSO-d6): δ 8.03 (s, IH), 7.79 (s, IH), 7.56 (d, IH, I = 3.8 Hz), 7.38-7.17 (m, 12H), 7.04 (d, IH, I = 3.8 Hz), 6.83 (d, 2H, I = 9.0 Hz), 6.13 (s, IH), 5.56 (s, IH), 5.31 (d, IH, I = 9 Hz), 5.21-5.16 (m, IH), 4.20-4.08 (m, IH), 3.38-3.70 (m, 4H), 3.65- 3.40 (m, 2H), 2.43-2.36 (m, 2H), 1.63-1.45 (m, 2H), 1.27 (bs, 8H), 0.91-0.84 (m, 3H), 0.74 (s, 3H).
Step B: 4-(p-Methoxyphenyldiphenylmethylamino)-7-r3,5-di-0-(l-oxo-octyl)-
2-C-methyl-β-D-ribofυranosyl]-7H-pyrrolor2,3-6πpyrimidine (3-2) A solution of the compound from Step B (18 mg, 0.026 mmol) and DMAP (3.5 mg, 0.028 mmol) in anhydrous CΗ2C12 (300 μL) was cooled in an ice bath for 10 minutes under an argon atmosphere. To this solution was added triethylamine (7.5 μL, 0.053 mmol) followed by octanoyl chloride (6.6 μL, 0.038 mmol). The reaction mixture was stirred at this temperature for 2 h, diluted with CH2CI2 (20 mL) and washed with saturated aqueous sodium bicarbonate solution (2 x 10 mL) followed by water (10 mL). The crude product obtained after evaporation was purified by column chromatography on silica gel eluting with 10% acetone in CH2C12. The title compound was obtained as a colorless foam (13.5 mg).
Step C: 4-Amino-7-[2-C-methyl-3,5-di-0-(l-oxo-octyl)-β-D-ribofuranosyl]- 7H-pyrrolo[2,3--flpyrimidine (3-3)
A solution of the compound from Step B (13 mg, 0.016 mmol) in 6:3:1 MeOΗ: acetic acid: Η O (500 μL) was stirred at 50°C for 15 h. The reaction mixture was then concentrated to dryness. The residue was diluted with ethyl acetate (15 mL) and washed with saturated aqueous sodium bicarbonate solution (3 x 5 mL) and water (2 x 5 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The crude product was purified by silica gel column chromatography eluting with 10% acetone in dichloromethane to afford the title compound as a white foam (6.0 mg). IH NMR (CDCI3): δ 8.29 (s, IH), 7.25 (d, IH, I = 3.4 Hz), 6.40 (d, IH, J = 4.0 Hz), 6.23 (s, IH), 5.22-5.39 (m, 3H), 4.60-4.39 (m, 4H), 2.47-2.35 (m, 4H), 1.82-1.60 (m, 4H), 1.27 (bs, 16 H), 0.87 (s, 3H), 0.873-0.80 (m, 6H). Scheme 4
Figure imgf000079_0001
4-6
Figure imgf000079_0002
EXAMPLE 40
4-Amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl -7H-pyrrolo[2,3-(flpyrimidine (4-7)
Figure imgf000080_0001
Step A: 5-Bromo-4-chloro-7H-pyrrolo[2,3-^lpyrimidine (4-2) To a solution of 4-chloro-7H-pyrrolo[2,3-( ]pyrimidine (4-1) (1.53 g,
10.0 mmol) in DMF (20 mL) was added N-bromosuccinimide (1.78 g, 10.0 mmol) in DMF (10 mL) dropwise at 0°C. The reaction mixture was stirred at 0°C for 30 min and then at room temperature for 1 h. Methanol (25 mL) was added, and the reaction mixture was stirred for an additional 1 h. The solvent was evaporated and the residue was crystallized from methanol to give the title compound as white solid.
Step B: 5-(Trimethylstannyl)-4-chloro-7H-pyrrolo[2,3-<flpyrimidine (4-3)
To a solution of the compound from Step A (0.92 g, 4 mmol) in TΗF (25 mL) was added n-BuLi (2.5 M solution in hexane, 3.48 mL) dropwise at -78°C. After the addition, the reaction mixture was stirred at -78°C for an additional 30 min. To this solution was added trimethyltin chloride (0.88 g, 4.4 mmol) in TΗF (8 mL) dropwise for a period of 10 min. The reaction mixture was brought to room temperature slowly and stirred at room temperature overnight. Saturated aqueous ammonium chloride (60 mL) was added and extracted with ethyl acetate (3 x 70 mL). The combined organic extracts were washed with brine, dried over Na2SO and evaporated to dryness. The residue was purified over silica gel to give the title compound as a colorless solid.
Step C: 5-Fluoro-4-chloro-7H-pyrrolo[2,3-<i]pyrimidine (4-4) To a solution of the compound from Step B (1.97 g, 6.20 mmol) in
CΗ3CN (60 mL) was added [l-(chloromethyl)-4-fluoro-l,4- diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate)] (SELECTFLUOR® fluorinating reagent) (2.40 g, 6.5 mmol) in one portion and the reaction mixture was stirred at room temperature for 7 h. The white precipitate was filtered off, and the filtrate was evaporated to dryness. The residue was purified over silica gel using ethyl acetate/hexane (3:7) as the eluent. Fractions containing the product were pooled and eveporated in vacuo to give the title compound as a colorless solid. 1H-NMR (MeOU-d4): δ 8.53 (s, IH), 7.37 (d, J = 2.8 Hz); 19F-NMR (DMSO-d6): δ -171.5.
Step D: 4-Amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl -7H-pyrrolor2.3- (ilpyrimidine (4-7)
To a solution of the compound from Step C (0.075 g, 0.44 mmol), 2,3,5-tri-O-benzoyl-2-C-methyl-D-ribofuranose [Ηarry-O'kuru, Rogers E.; Smith, lennifer M.; Wolfe, Michael S, "A Short, Flexible Route toward 2'-C-Branched Ribonucleosides," J. Org. Chem., 62: 1754-1759 (1997)] (4-5) (0.25 g, 0.53 mmol) and triphenylphosphine (0.23 g, 0.88 mmol) in TΗF (15 mL) was added diethyl azodicarboxylate (DEAD) (0.14 mL, 0.88 mmol). The reaction mixture was stirred at room temperature overnight. The solution was directly adsorbed onto silica gel and purified over silica gel using ethyl acetate/hexane 1:9 as the eluent Appropriate fractions were dissolved in dioxane (3 mL) and liquid ammonia (4 mL) and the mixture was heated in a steel bomb at 85°C for 24 h. The solvent was evaporated and the residue was purified over silica gel using methanol/dichloromethane (1:9) as the eluent. Fractions containing the desired compound were pooled and evaporated in vacuo to give the title compound. lΗ-NMR (MeOH-d4): δ 8.07 (s, 1Η), 7.41 (d, J = 2.2 Ηz, 1Η), 6.25 (d, J = 1.8 Ηz), 4.09-3.95 (m, 3Η), 3.82 (dd, I = 2.7, 12.5 Hz, IH);
19F-NMR (MeOB.-d4): δ -170.4; mass spectrum: 321 (M+Na)+.
EXAMPLE 41
6-Amino-2-fluoro-9-(2-C-methyl-β-D-ribofuranosyl)purine
Figure imgf000082_0001
Step A: 2-Amino-6-chloro-9-(2,3,5-tri-O-benzoyl-2-C-methyl-β-D- ribofuranosvDpurine
To a pre-cooled solution of l,2,3,5-tetra-O-benzoyl-2-C-methyl-D- ribofuranose [Harry-O'kuru, Rogers E.; Smith, lennifer M.; Wolfe, Michael S, "A Short, Flexible Route toward 2'-C-Branched Ribonucleosides," J. Org. Chem.,62: 1754-1759 (1997)] (1.74 g, 3.00 mmol) in acetonitrile (15 mL) was added 2-amino-6- chloropurine (0.56 g, 3.30 mmol), then diazabicyclo[5.4.0]undec-7-ene (DBU) (1.37 g, 9.00 mmol), and then dropwise trimethylsilylmethyl trifluoromethanesulfonate
(TMS trifate) (2.67 g, 12.00 mmol). The resulting mixture was heated to 65°C for 4 h, then cooled and partitioned between saturated aqueous sodium bicarbonate (200 mL) and dichloromethane (200 mL). The organic phase was dried over magnesium sulfate, filtered and evaporated in vacuo. The resulting crude product was used directly in step B.
Step B: 2-Amino-6-chloro-9-(2-C-methyl-β-D-ribofuranosyl)purine
To the crude compound from Step A (2.54 g) in THF (18 mL) was added aqueous 2N Li OH (6 mL). The resulting mixture was stirred at room temperature for 3 h, the THF evaporated in vacuo and the resulting aqueous phase neutralized by addition of aqueous 2N hydrochloric acid. The mixture was adsorbed onto silica gel by evaporation in vacuo and purified on silica gel using methanol/dichloromethane (1:4) as the eluent. Fractions containing the product were combined and evaporated in vacuo to give the desired product as a colorless powder.
Step C: 2,6-Diamino-9-(2-C-methyl-β-D-ribofuranosyl purine To the compound from Step B (100 mg) was added ammonium hydroxide (5 mL) and the resulting mixture was stirred at 80°C in a Parr bomb overnight. The mixture was cooled and evaporated in vacuo and adsorbed onto silica and purified on silica gel using methanol/dichloromethane (1:4) as the eluent. Fractions containing the product were combined and evaporated in vacuo to give the desired product as a colorless powder.
Step D: 6-Amino-2-fluoro-9-(2-C-methyl-β-D-ribofuranosyl)purine
To a mixture of HF/pyridine (70%) (1 mL) in pyridine (1 mL) at -30°C was added the compound from Step C (29.6 mg, 0.10 mmol) in pyridine (0.5 mL), followed by addition of tert-butyl nitrite (0.018 mL, 0.15 mmol). The mixture was stirred for 5 minutes and then poured into ice water (5 mL), neutralized with 2N
NaOH and evaporated in vacuo. The crude product was purified on silica gel using methanol/dichloromethane (1:9 through 1:4) as the eluent. Fractions containing the desired product were pooled and evaporated in vacuo to give the desired compound as a colorless oil. iH-NMR (acetonitrile-^): δ 8.23 (s, IH), 5.93 (s, IH), (t, J = 8.4 Hz, IH), 4.00 (m,
2H), 3.81 (m, IH), 3.70 (s, IH), 3.60 (m, IH), 0.90 (s, 3H).
19F-NMR (MeCm-d4): -80;
Mass spectrum: 298.3 (M-H)+ and 597.1 (2M-H)+.
BIOLOGICAL ASSAYS
The assays employed to measure activity against vaccinia virus are described below:
a. Determination of In Vitro Antiviral Activity of Compounds Against Vaccinia Virus (Cvtopathic Effect Inhibition Assay):
Assay conditions are described in the article by Sidwell and Huffman, "Use of disposable microtissue culture plates for antiviral and interferon induction studies," Appl. Microbiol. 22: 797-801 (1971). Vaccinia virus, Lederle strain from the ATCC, was used with Vero cells and media (9% fetal bovine serum, 0.1% NaHCO3, no antibiotics) as stated in the article. Antiviral test medium was MEM with 2% FBS and 0.18% NaHCO3. Four and seven point titrations were used to assay compound inhibition. Final compound concentrations for 4-point titrations were 100, 10, 1, and 0.1 μM. Seven-point titrations were performed by preparing one-half log serial dilutions from maximum compound concentrations of either 100 μM or 320 μM. Virus was added to the assay plate approximately 5 min after the test compound. Assay plates were incubated with humidified air and 5% CO2 at 37°C. Cytotoxicity was monitored in the control cells microscopically for morphologic changes. Regression analysis of the virus CPE data and the toxicity control data gave the ED50 (50% effective dose) and CC50 (50% cytotoxic concentration). The selectivity index (SI) was calculated by the formula: SI = CC50 ÷ ED50. (S)-l-[3-hydroxy-2-(phosphonylmethoxy)-propyl]cytosine (cidofovir) was used as a positive control for anti-vaccinia virus testing.
Representative compounds tested in the anti-vaccinia virus assay exhibited ECso's less than 100 micromolar.
b. Determination of In Vitro Antiviral Activity of Compounds Against Vaccinia
Virus (Neutral Red Uptake Assay)
After performing the CPE inhibition assay above, an additional cytopathic detection method was used. McManus described the detection method in
"Microtiter Assay for Interferon: Microspectrophotometric Quantitation of Cytopathic Effect," Appl. Environ. Microbiol, 31 : 35-38 (1976). A Model EL309 microplate reader (Bio-Tek Instruments Inc.) was used at 540 nm to directly read the assay plate. ED50 and CD50 were calculated as above.
The nucleoside derivatives of the present invention were also evaluated for cellular toxicity and antiviral specificity in the counterscreens described below.
COUNTERSCREENS:
The ability of the nucleoside derivatives of the present invention to inhibit human DNA polymerases was measured in the following assays. a. Inhibition of Human DNA Polymerases alpha and beta:
Reaction Conditions: 50 μL reaction volume
Reaction buffer components:
Figure imgf000085_0001
200 μg/mL bovine serum albumin 100 mM KCl 2 mM β-mercaptoethanol 10 mM MgCl2
1.6 μM dATP, dGTP, dCTP, dTTP α-33P-dATP
Enzyme and template:
0.05 mg/mL gapped fish sperm DNA template 0.01 U/μL DNA polymerase cc or β
Preparation of gapped fish sperm DNA template: Add 5 μL IM MgCl2 to 500 μL activated fish sperm DNA (USB 70076);
Warm to 37°C and add 30 μL of 65 U/μL of exonuclease (GibcoBRL 18013-011);
Incubate 5 min at 37°C;
Terminate reaction by heating to 65 °C for 10 min;
Load 50-100 μL aliquots onto Bio-spin 6 chromatography columns (Bio-Rad 732- 6002) equilibrated with 20 mM Tris-HCl, pH 7.5 ;
Elute by centrifugation at l,000Xg for 4 min;
Pool eluate and measure absorbance at 260 nm to determine concentration.
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 β-mercaptoethanol, and 100 mM KCl. 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 h at 37°C. The reaction was quenched by the addition of 20 μL 0.5M EDTA. 50 μL 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 percentage of inhibition was calculated according to the following equation: % inhibition = [l-(cpm in test reaction - cpm in blank)/(cpm in control reaction - cpm in blank)] x 100.
b. Inhibition of Human DNA Polvmerase gamma :
The potential for inhibition of human DNA polymerase gamma was measured in reactions that included 0.5 ng/ μL enzyme; 10 μM dATP, dGTP, dCTP, and TTP; 2 μCi/reaction [α-33P]-dATP, and 0.4 μg/μL activated fish sperm DNA (purchased from US Biochemical) in a buffer containing 20 mM Tris pH8, 2 mM β- mercaptoethanol, 50 mM KCl, 10 mM MgCl2, and 0.1 μg/μL 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. Product formation was quantified by anion exchange filter binding and scintillation counting. Compounds were tested at up to 50 μM. The percentage of inhibition was calculated according to the following equation: % inhibition = [l-(cpm in test reaction - cpm in blank)/(cpm in control reaction - cpm in blank)] x 100.
The ability of the purine nucleoside derivatives of the present invention to inhibit HTV infectivity and HTV spread was measured in the following assays.
c. HTV Infectivity Assay
Assays were performed with a variant of HeLa Magi cells expressing both CXCR4 and CCR5 selected for low background β-galactosidase (β-gal) expression. Cells were infected for 48 h, and β-gal production from the integrated
HIV-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 μM; percent inhibition at each concentration was calculated in relation to the control infection. d. Inhibition of HIV Spread
The ability of the compounds of the present invention to inhibit the spread of the human immunedeficiency virus (HIV) was measured by the method described in U.S. Patent No. 5,413,999 (May 9, 1995), and I.P.Vacca, et al, Proc. Natl Acad. Sci., 91: 4096-4100 (1994), which are incorporated by reference herein in their entirety.
The 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. The HuH- 7 cell line is described in H. Nakabayashi, et al, Cancer Res., 42: 3858 (1982).
e. Cytotoxicity assay:
Cells were plated at 15-20,000 cells/well in 100 μL of appropriate media and incubated 18 h at 37°C, 5% CO2. 100 μL of compound diluted in complete media was added to the cells for a final of 1% DMSO concentration. The plates were incubated at 37°C and 5% CO2 for 24 h. After the incubation period, 40 μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay reagent (MTS) (Promega) was added to each well, and the plates were incubated at 37°C and 5% CO2 for 1 h. The plates were agitated to mix well and absorbance at 490 nm was read using a plate reader. Metabolically active cells reduce MTS to formazan. Formazan absorbs at 490 nm. The absorbance at 490 nm in the presence of compound was compared to absorbance in cells without any compound added. Reference: Cory, A. H. et al., "Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture," Cancer Commun. 3: 207 (1991).
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 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.
While the invention has been described and illustrated in reference to specific embodiments thereof, those skilled in the art will appreciate that various changes, modifications, and substitutions can be made therein without departing from the spirit and scope of the invention. For example, effective dosages other than the preferred doses as set forth hereinabove may be applicable as a consequence of variations in the responsiveness of the human being treated for severity of the HCN infection. Likewise, the pharmacologic response observed may vary according to and depending upon the particular active compound selected or whether there are present pharmaceutical carriers, as well as the type of formulation and mode of administration employed, and such expected variations or differences in the results are contemplated in accordance with the objects and practices of the present invention. It is intended therefore that the invention be limited only by the scope of the claims which follow and that such claims be interpreted as broadly as is reasonable.

Claims

WHAT IS CLAIMED IS:
1. A method of inhibiting orthopoxvirus replication comprising administering to a mammal in need of such treatment a therapeutically effective amount of a compound of structural formula I:
Figure imgf000089_0001
(0 or a pharmaceutically acceptable salt thereof; wherein A is N or C-R9; Rl is C2-4 alkenyl, C2-4 alkynyl, or Cι_4 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino, Ci_4 alkoxy, Ci-4 alkylthio, or one to three fluorine atoms;
R2 is amino, fluorine, hydroxy, Ci_io alkylcarbonyloxy, mercapto, or Cι_4 alkoxy;
R3 and R4 are each independently selected from the group consisting of hydrogen, cyano, azido, halogen, hydroxy, Cι_i6 alkylcarbonyloxy, C2-18 alkenylcarbonyloxy,
Cι_ιo alkyloxycarbonyloxy, C3-6 cycloalkylcarbonyloxy,
C3-6 cycloalkyloxycarbonyloxy, mercapto, amino, Cι_4 alkoxy, C2-4 alkenyl, C2-4 alkynyl, and C1.4 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino, Cι_4 alkoxy, Ci-4 alkylthio, or one to three fluorine atoms; R is hydrogen, Cι_i6 alkylcarbonyl, C2-I8 alkenylcarbonyl,
Ci-io alkyloxycarbonyl, C3-6 cycloalkylcarbonyl, C3-6 cycloalkyloxycarbonyl,
P3O9H4, P2O6H3, or P(O)R13R14;
R6 and R7 are each independently hydrogen, methyl, hydroxymethyl, or fluoromethyl; R8 is hydrogen, Cι_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, Ci_6 alkylthio, Cι_6 alkylsulfonyl, or (Cι_4 alkyl)θ-2 aminomethyl; R9 is hydrogen, cyano, nitro, NHCONH2, CONR12R12, CSNR12R12; COOR12, C(=NH)NH2, hydroxy, C1.3 alkoxy, amino, Cι_4 alkylamino, di(Ci-4 alkyl)amino, halogen, or Ci_3 alkyl, wherein alkyl is unsubstituted or substituted with one to three groups independently selected from halogen, amino, hydroxy, carboxy, and Ci_3 alkoxy;
RlO and RU are each independently hydrogen, hydroxy, mercapto, halogen, Cι_4 alkoxy, Cι_4 alkylthio, amino, Ci-4 alkylamino, di(Ci-4 alkyl)amino, C3_6 cycloalkylamino, di(C3_6 cycloalkyl)amino, phenyl-Ci-2 alkylamino, Ci-4 acylamino, Ci-8 alkylcarbonyloxy, or OCH(Cι_4 alkyl)O(C=O)Cι_4 alkyl; each Rl2 is independently hydrogen or Cι_6 alkyl; and
Rl3 and Rl4 are each independently hydroxy, OCH2CH2SC(=O)Cι_4 alkyl,
OCH2θ(C=O)OCι_4 alkyl, NHCHMeCO2Me, OCH(Ci-4 alkyl)O(C=O)Cι_4 alkyl,
Figure imgf000090_0001
2. A method of treating orthopoxvirus infection in a mammal in need thereof comprising administering a therapeutically effective amount of a compound of Claim 1.
3. The method of Claim 1 wherein the compound is of structural formula II:
Figure imgf000090_0002
(II)
or a pharmaceutically acceptable salt thereof; wherein A is N or C-R9; Rl is Ci-3 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino,
Cι_3 alkoxy, Ci-3 alkylthio, or one to three fluorine atoms;
R2 is hydroxy, Cι_i alkylcarbonyloxy, fluoro, or Cι_3 alkoxy;
R3 is hydrogen, halogen, hydroxy, Cι_i6 alkylcarbonyloxy, amino, or C1-.3 alkoxy;
R5 is hydrogen, Cι_i6 alkylcarbonyl, P3O9H4, P2O6H3, or PO3H2;
R8 is hydrogen, amino, or Cι_4 alkylamino;
R9 is hydrogen, cyano, methyl, halogen, or CONH2; and
RlO and Rl 1 are each independently hydrogen, halogen, hydroxy, amino, Ci-4 alkylamino, di(Cι_4 alkyl)amino, or C3-.6 cycloalkylamino.
4. The method of Claim 3 wherein
Rl is methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, or aminomethyl;
R2 is hydroxy, Ci_i6 alkylcarbonyloxy, fluoro, or methoxy; R3 is hydrogen, fluoro, hydroxy, Cι_i6 alkylcarbonyloxy, amino, or methoxy; R5 is hydrogen, Cι_i6 alkylcarbonyl, or P3O9H4;
R8 is hydrogen or amino;
R9 is hydrogen, cyano, methyl, halogen, or CONH2; and
RlO and RU are each independently hydrogen, halogen, hydroxy, amino, Ci-4 alkylamino, di(Ci-4 alkyl)amino, or C3-6 cycloalkylamino.
5. The method of Claim 4 wherein the compound is selected from the group consisting of: 4-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine, 4-methylamino-7-(2-C-methyl-β-D-ribo ranosyl)-7H-pyrrolo[2,3-( ]pyrimidine, 4-dimethylaι no-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine, 4-cyclopropylamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-(f|pyrimidine, 4-amino-7-(2-C-hydroxymethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-^pyrimidine, 4-amino-7-(2-C-fluoromethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-(f]pyrimidine, 4-amino-5-methyl-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-( lpyrimidine, 4-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyιτolo[2,3-^pyrimidine-5- carboxamide,
4-an ino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyιτolo[2,3-( Jpyrimidine-5- carbonitrile, 4-amino-5-bromo-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrtθlo[2,3-^pyrimidine, 4-amino-5-chloro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrtθlo[2,3-^pyrimidine, 4-aιmno-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-<i]pyrimidine, 2,4-diamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-cT]pyrimidine, 2-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
2-amino-4-cyclopropylamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3- d]pyrimidine,
2-amino-7-(2-C-methyl-β-D-ribomranosyl)-7H-pyrrolo[2,3-- ]pyrimidin-4(3H)-one, 7-(2-C-methyl-β-D-ribomranosyl)-7H-pyrrolo[2,3--^pyrimidin-4(3H)-one, 4-amino-2-fluoro-7-(2-C-methyl-β-D-riboforanosyl)-7H-pyιτolo[2,3-d]pyrimidine, 9-(2-C-methyl-β-D-ribofuranosyl)-2-amino-6-hydroxypurine, 9-(2-C-methyl-β-D-ribofuranosyl)-2-ammo-6-cyclopropylaminopurine, 9-(2-C-methyl-β-D-ribofuranosyl)-2,6-diaminopurine, 9-(2-C-methyl-β-D-ribofuranosyl)-2-amino-6-methylaminopurine, 6-amino-2-fluoro-9-(2-C-methyl-β-D-ribofuranosyl)purine, 2' -C-methyl-adenosine,
4-amino-7-[2-C-methyl-3-O-(l-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3- d]pyrimidine, and 4-amino-7-[2-C-methyl-3,5-di-O-(l-oxo-oc1yl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3- (i]pyrimidine; and the corresponding 5 '-triphosphates; or a pharmaceutically acceptable salt thereof.
6. The method of Claim 5 wherein the compound is selected from the group consisting of:
4-amάno-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-<i]pyrimidine, 4-amino-7-(2-C-fluoromethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-<i]pyrimidine, 4-amino-5-methyl-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyιτolo[2,3-rf]pyrimidine, 4-amino-5-bromo-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3--f]pyrimidine, 4-armno-5-chloro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-_ ]pyrimidine, 4-amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-ύ pyrimidine, 4-amino-2-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3- ]pyrimidine, 6-amino-2-fluoro-9-(2-C-methyl-β-D-ribofuranosyl)purine, 2' -C-methyl-adenosine, 4-anιino-7-[2-C-methyl-3-O-(l-oxo-oc1yl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3- <i]pyrimidine, and
4-amino-7-[2-C-methyl-3,5-di-O-(l-oxo-oc1yl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3- d]pyrimidine; and the corresponding 5 '-triphosphates; or a pharmaceutically acceptable salt thereof.
7. The method of Claim 6 wherein the compound is 4-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-<i]pyrimidine; or a pharmaceutically acceptable salt thereof.
8. The method of Claim 6 wherein the compound is 4-amino-7-[2-C-methyl-3-O-(l-oxo-oc1yl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3- <f|pyrimidine; or a pharmaceutically acceptable salt thereof.
9. The method of Claim 6 wherein the compound is 4-amino-7-(2-C-fluoromethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-(i]pyrimidine; or a pharmaceutically acceptable salt thereof.
10. The method of Claim 6 wherein the compound is 4-amino-7-[2-C-methyl-3,5-di-0-(l-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3- <i]pyrimidine; or a pharmaceutically acceptable salt thereof.
11. The method of Claim 6 wherein the compound is 4-amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyιτolo[2,3--i]pyrirrιidine; or a pharmaceutically acceptable salt thereof.
12. The method of Claim 2 wherein the compound is of structural formula II:
Figure imgf000094_0001
(ll) or a pharmaceutically acceptable salt thereof; wherein
A is N or C-R9;
Rl is Ci-3 alkyl, wherein alkyl is unsubstituted or substituted with hydroxy, amino,
Ci-3 alkoxy, Cι_3 alkylthio, or one to three fluorine atoms;
R2 is hydroxy, Cι_i alkylcarbonyloxy, fluoro, or Cι_3 alkoxy;
R3 is hydrogen, halogen, hydroxy, Ci-16 alkylcarbonyloxy, amino, or Ci-3 alkoxy;
R5 is hydrogen, Cι_i6 alkylcarbonyl, P3O9H4, P2O6H3, or PO3H2;
R8 is hydrogen, amino, or Ci-4 alkylamino;
R9 is hydrogen, cyano, methyl, halogen, or CONH2; and
RlO and RU are each independently hydrogen, halogen, hydroxy, amino, Ci-4 alkylamino, di(Cι_4 alkyl)amino, or C3-6 cycloalkylamino.
13. The method of Claim 12 wherein Rl is methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, or aminomethyl;
R2 is hydroxy, Cι_i6 alkylcarbonyloxy, fluoro, or methoxy; R3 is hydrogen, fluoro, hydroxy, Cι_i6 alkylcarbonyloxy, amino, or methoxy; R5 is hydrogen, Cι_i6 alkylcarbonyl, or P3O9H4; R8 is hydrogen or amino;
R9 is hydrogen, cyano, methyl, halogen, or CONH2; and
RlO and RU are each independently hydrogen, halogen, hydroxy, amino, Ci-4 alkylamino, di(Ci-4 alkyl)amino, or C3-6 cycloalkylamino.
14. The method of Claim 13 wherein the compound is selected from the group consisting of: 4-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3- ]pyrimidine, 4-methylamino-7-(2-C-methyl-β-D-ribomranosyl)-7H-pyιτolo[2,3-(i]pyrimidine, 4-dimethylamino-7-(2-C-methyl-β-D-ribofmanosyl)-7H-pyrtθlo[2,3--^pyrirrιidine, 4-cyclopiOpylamino-7-(2-C-methyl-β-D-ribo ranosyl)-7H-pyrrolo[2,3-<i]pyrimidine, 4-amino-7-(2-C-hydroxymethyl-β-D-ribofuranosyl)-7H-pyrtθlo[2,3--^pyrimidine, 4-amino-7-(2-C-fluoromethyl-β-D-ribofuranosyl)-7H-pyrtθlo[2,3-^pyrimidine, 4-amino-5-methyl-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrtθlo[2,3-^pyrimidine, 4-amdno-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pynOlo[2,3--/]pyrimidine-5- carboxamide, 4-an ino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pynOlo[2,3-<i]pyrimidine-5- carbonitrile,
4-amino-5-bromo-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrtOlo[2,3-d]pyrimidine, 4-amino-5-chloro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyπOlo[2,3-(i]pyrimidine, 4-amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-<i]pyrimidine, 2,4-diamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3--i]pyrimidine, 2-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
2-amino-4-cyclopropylamino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3- d pyrimidine,
2-an no-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-(i]pyrimidin-4(3H)-one, 7-(2-C-methyl-β-D-ribo ranosyl)-7H-pyrrolo[2,3-<f]pyrirnidin-4(3H)-one, 4-amino-2-fluoro-7-(2-C-methyl-β-D-ribomranosyl)-7H-pyrrolo[2,3-d]pyrimidine, 9-(2-C-methyl-β-D-ribofuranosyl)-2-amino-6-hydroxypurine, 9-(2-C-methyl-β-D-ribofuranosyl)-2-amino-6-cyclopropylaminopurine, 9-(2-C-methyl-β-D-ribofuranosyl)-2,6-diaminopurine, 9-(2-C-methyl-β-D-ribofuranosyl)-2-amino-6-methylaminopurine, 6-amino-2-fluoro-9-(2-C-methyl-β-D-ribofuranosyl)purine, 2' -C-methyl-adenosine,
4-amino-7-[2-C-methyl-3-O-(l-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3- -i]pyrimidine, and 4-amino-7-[2-C-methyl-3,5-di-O-(l-oxo-octyl)-β-D-ribofuranosyl]-7H-ρyrrolo[2,3- d pyrimidine; and the corresponding 5 '-triphosphates; or a pharmaceutically acceptable salt thereof.
15. The method of Claim 14 wherein the compound is selected from the group consisting of: 4-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-(i]pyrimidine, 4-amino-7-(2-C-fluoiOmethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-(i]pyrimidine, 4-amino-5-methyl-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-<f]pyrimidine, 4-amino-5-bromo-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrtθlo[2,3-^pyrimidine, 4-amino-5-chloro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-(i]pyrimidine, 4-amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-< ]pyrimidine, 4-amino-2-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-<i]pyrimidine, 6-amino-2-fluoro-9-(2-C-methyl-β-D-ribofuranosyl)purine, 2' -C-methyl-adenosine, 4-amino-7-[2-C-methyl-3-( -(l-oxo-octyl)-β-D-ribofuraιιosyl]-7H-pyrrolo[2,3- <i]pyrimidine, and
4-amino-7-[2-C-methyl-3,5-di-O-(l-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-
Figure imgf000096_0001
and the corresponding 5 '-triphosphates; or a pharmaceutically acceptable salt thereof.
16. The method of Claim 15 wherein the compound is 4-amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3- ]pyrimidine; or a pharmaceutically acceptable salt thereof.
17. The method of Claim 15 wherein the compound is 4-amino-7-[2-C-methyl-3-O-(l-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3- J]pyrimidine; or a pharmaceutically acceptable salt thereof.
18. The method of Claim 15 wherein the compound is 4-amino-7-(2-C-fluoromethyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-Jlpyrimidine; or a pharmaceutically acceptable salt thereof.
19. The method of Claim 15 wherein the compound is
4-amino-7-[2-C-methyl-3,5-di-O-(l-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3- flOpyrimidine; or a pharmaceutically acceptable salt thereof.
20. The method of Claim 15 wherein the compound is 4-amino-5-fluoro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; or a pharmaceutically acceptable salt thereof.
21. The method of Claim 1 wherein said orthopoxvirus replication is vaccinia virus or variola virus replication.
22. The method of Claim 2 wherein said orthopoxvirus infection is vaccinia virus or variola virus infection.
23. The method of Claim 22 in combination with a therapeutically effective amount of another agent active against orthopoxvirus.
24. The method of Claim 23 wherein said agent active against orthopoxvirus is cidofovir, ribavirin, levovirin, or viramidine.
25. Use of a compound of Claim 1 for the inhibition of orthopoxvirus replication in a mammal.
26. Use of a compound of Claim 1 for the treatment of orthopoxvirus infection in a mammal.
27. The use of Claim 26 wherein said orthopoxvirus infection is vaccinia virus infection or variola virus infection.
28. Use of a compound of Claim 1 in the manufacture of a medicament for the inhibition of orthopoxvirus replication in a mammal.
29. Use of a compound of Claim 1 in the manufacture of a ' medicament for the treatment of orthopoxvirus infection in a mammal.
30. The use of Claim 29 wherein said orthopoxvirus infection is vaccinia virus or variola virus infection.
31. The method of Claim 8 wherein the orthopoxvirus replication is vaccinia virus or variola virus replication.
32. The method of Claim 17 wherein the orthopoxvirus replication is vaccinia virus or variola virus replication.
33. A compound which is 6-amino-2-fluoro-9-(2-C-methyl-β-D- ribofuranosyl)purine or a pharmaceutically acceptable salt thereof.
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