WO2007059330A2 - Analogue du nucleoside de cubane - Google Patents

Analogue du nucleoside de cubane Download PDF

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Publication number
WO2007059330A2
WO2007059330A2 PCT/US2006/044835 US2006044835W WO2007059330A2 WO 2007059330 A2 WO2007059330 A2 WO 2007059330A2 US 2006044835 W US2006044835 W US 2006044835W WO 2007059330 A2 WO2007059330 A2 WO 2007059330A2
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alkyl
compound
salt
patient
cubane
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PCT/US2006/044835
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WO2007059330A3 (fr
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Miroslav Trampota
Randall B. Murphy
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Medkura Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals

Definitions

  • the present invention provides cubane nucleoside analogs useful as anti-viral and anti-cancer agents. Methods for preparing cubane nucleoside analogs .are also provided. Certain cubane nucleoside analogs disclosed herein are potent and/ or selective inhibitors of viral DNA synthesis.
  • the invention also provides pharmaceutical compositions containing 5 one or more cubane nucleoside analogs and one or more pharmaceutically acceptable carriers, excipients, or diluents. Such pharmaceutical compositions may contain a cubane nucleoside analog as the sole active agent or may contain a combination of a cubane nucleoside analog and one or more other pharmaceutically active agents.
  • the invention also provides methods for treating cancer and viral infections in mammals.
  • nucleosides have been used successfully for a number of years in the treatment of cancer, HIV/AIDS, Hepatitis B, Hepatitis C 5 Epstein-Barr virus and herpes simplex viruses.
  • a "nucleoside” is a molecule that includes a purine or pyrimidine base covalently bound to a ribose sugar.
  • a “nucleoside analog” is a synthetic molecule that i D resembles a naturally occurring nucleoside, but that lacks a bond site needed to link it to an adjacent nucleotide.
  • a nucleoside analog may include a purine or pyrimidine base covalently bound to a hydroxyl substituted alkoxy group rather that a ribose sugar.
  • Nucleoside analogs include AZT, acyclovir, combivir, abacavir, emtricitabine, ddl, ddC, d4T, L-FMAU (stavudine), and 3TC. Nucleoside analogs act via a variety of cellular mechanisms 0 to inhibit DNA synthesis, thereby preventing viral and malignant cell replication.
  • nucleoside analogs for the treatment of viral infections and cancer exists. Nucleoside analogs of new and distinct structural classes are particularly desirable. 5 Nucleoside analogs for the treatment of HIV
  • HIV-I Human immunodeficiency virus type-1
  • AIDS acquired immune deficiency syndrome
  • HIV-I fusion, transcription, or proteolysis targets one of only three steps in the HTV-I replication cycle.
  • RT inhibitors which inhibit synthesis of viral DNA
  • NRTIs nucleoside/nucleotide reverse transcriptase inhibitors
  • NRTIs non-nucleoside reverse transcriptase inhibitors
  • Nucleoside (analog) reverse transcriptase inhibitors are the most common class of compounds used in current anti-HTV therapy. Seven NRTIs, which are approved by the FDA, lack a 3 -hydroxyl group in the sugar moiety. Once the nucleoside analog is phosphorylated to the active form and incorporated into an elongating DNA strand by HIV-I reverse transcriptase (RT) DNA synthesis is terminated. Due to their mechanism of action, these NRTIs are often referred to as "chain terminators”. Retroviral reverse transcripatases, including the HIV-I RT, are exceptionally error prone. This high error rate has led to the development of distinct HIV-I strains, many of which are resistant to inhibition by existing NRTIs.
  • Hepatitis B (HBV) infection is a worldwide public health problem affecting 300 million individuals. Chronic HBV infection increases the risk of developing hepatocellular carcinoma about 150-fold. Like HIV-I, HBV requires an RNA-directed DNA polymerase to copy its viral genome. Likewise, nucleoside analogs may be used effectively to reduce Hepatitis B viral DNA synthesis. For example, lamivudine, a (-) enantiomer of 2'3'- dideoxy-3 '-thiacytidine, phosphorylated to the triphosphate, which competes with the other triphosphates for incorporation into viral DNA, causing chain termination.
  • Flaviviridae are a family of at least 66 viruses, of which almost half have been associated with human disease.
  • the most well-known Flaviviridae include Hepatitis C virus (HCV), Dengue fever virus, yellow fever virus, West Nile virus, and Japanese encephalitis.
  • Flaviviruses also cause diseases that have an economic impact in domestic and wild animals
  • HCV infection is the most common chronic blood-borne infection in the United States. There are about 36,000 new infections every year, of which 25-30% are symptomatic. It is estimated that 3.9 million (1.8%) Americans have been infected.
  • the mosquito-borne flavivirus, Dengue is estimated to cause 100 million cases of Dengue fever, 500,000 cases of Dengue hemorrhagic fever and 25,000 deaths each year with 2.5 billion people at risk worldwide.
  • West Nile virus (WNV) a mosquito-borne infective agent, is the causative agent of West Nile (WN) fever. A common complication of this infection is encephalitis. First reported in the Western Hemisphere in 1999, WNV is now endemic in the United States.
  • Ribavirin (1 '-D-ribofuranosyl- 1 ,2,4-triazole-3-carboxamide), a guanosine nucleoside analog, has been shown to be active in combination with interferon among patients with chronic HCV.
  • ribavirin Unlike many antiviral nucleoside analogs, ribavirin has a modified base instead of a modified sugar. Although its mechanism(s) of action are uncertain, it appears to affect the phosphorylation of guanosine into the trisphosphate nucleotide, resulting in a depletion of the intracellular GTP required for virus replication. Combination interferon/ ribavarin therapy provides moderate response rates in HCV infected patients with higher response rate for patients infected with selected HCV strains. Even using pegylated interferon, which is better tolerated than interferon itself, the combination with ribavirin is still poorly tolerated, leading to a large number of patients who discontinue treatment.
  • Herpes simplex viruses (HSV-I and HSV-2) produce a wide variety of illnesses, including mucocutaneous infections, infections of the central nervous system, and an occasional infection of visceral organs; many of these conditions may be life-threatening.
  • the eight known human herpesviruses (HHVs) are divided by genomic and biologic behavior into three groups: the alpha-herpesviruses (HSV-I, HSV-2, and varicella-zoster), the beta- herpesviruses (cytomegalovirus, HHV-6, HHV-7) and the gamma-herpesviruses (Epstein- Barr virus, Kaposi's sarcoma-associated herpes virus [KSHV], or HHV-8). Although all herpesviruses share common structural features, the biologic and epidemiologic features of each of the herpesviruses are distinct.
  • Herpes viruses have been successfully treated with nucleoside analogs.
  • acyclovir and its related compounds famciclovir and valacyclovir are used to treat mucocutaneous and visceral HSV infections.
  • Acyclovir is selectively phosphorylated by HSV-infected cells to acyclovir-monophosphate.
  • Cellular enzymes then phosphorylate acyclovir-monophosphate to acyclovir-triphosphate, a competitive inhibitor dGTP of viral DNA polymerase.
  • acyclovir-triphosphate is incorporated into the growing DN ' chain of the virus and causes chain termination.
  • Acyclovir has potent in vitro activity against both HSV-I and HSV-2.
  • Acyclovir-resistant strains of HSV are being identified with increasing frequency, especially in HIV-infected persons. Serious infections such as HSV encephalitis still have a high percentage of mortality despite aggressive treatment for both HIV patients and patients not infected with HIV. Thus a need exists for new classes of nucleoside analogs, effective against HSV replication.
  • Epstein Barr Virus is a pathogen associated with infectious diseases and malignancies in humans.
  • EBV Epstein Barr Virus
  • nucleoside analogs including acyclovir, gancyclovir, FIAU, and D-FMAU have shown activity against EBV replication in cell culture. The compounds are thought to act by incorporation of their 5 -monophosphate forms into viral DNA as a result of a preferential interaction of EBV DNA polymerase with their 5 -triphosphate metabj3h " tes.._RecenJly, L ⁇ activity against EBV and HBV.
  • L-FMAU may be phosphorylated stepwise to its 5 - triphosphate form, L-FMAU-TP, in human cell cultures. Additional nucleoside analogs of novel structural classes are needed to treat EBV infections.
  • Cancer is the second leading cause of death in the United States, with over 500,000 cancer deaths annually in the US from this disease.
  • 5-fluorouracil has been used clinically in the treatment of malignant tumors, including, for example, carcinomas, sarcomas, skin cancer, cancer of the digestive organs, and breast cancer.
  • Derivatives of 5-fluorouracil with anti-cancer activity have been described in U.S. Pat. No.4,336,381, which is hereby incorporated by reference for its teachings in regard to 5-fluorouracil derivatives.
  • 5-fluorouracil causes serious adverse reactions, including death.
  • Cytosine arabinoside (also referred to as Cytarabin, araC, and Cytosar) is a nucleoside analog of deoxycytidine that was first synthesized in 1950 and introduced into clinical medicine in 1963. It is used to treat acute myeloid leukemia and is also active against acute lymphocytic leukemia, and to a lesser extent, is useful in chronic myelocytic leukemia and non-Hodgkin's lymphoma. The primary action of araC is inhibition of nuclear DNA synthesis.
  • 5-Azacytidine is a cytidine analog that is primarily used in the treatment of acute myelocytic leukemia and myelodysplastic syndrome.
  • 2-Fluoroadenosine- 5'-phosphate (Fludara, also referred to as FaraA) is one of the most active agents in the treatment of chronic lymphocytic leukemia.
  • the compound acts by inhibiting DNA synthesis.
  • 2-Chlorodeoxyadenosine is useful in the treatment of low grade B-cell neoplasms such as chronic lymphocytic leukemia, non-Hodgkin's lymphoma, and hairy-cell leukemia.
  • cancer cell lines often develop resistance to nucleoside analogs after extended treatment. Cellular resistance frequently is produced through pathways that pump the particular nucleoside analog out of the cell efficiently.
  • nucle.o.side.analogs whose_cancers-hav.e-already_dey.eloped_ resistance to treatment with existing nucleoside analogs.
  • nucleoside analogs and compositions containing such compounds for the treatment of human patients or other hosts infected with HIV, Hepatitis B, Hepatitis C, Herpes family viruses, Epstein Barr virus, and cancer. Because many viruses and cancers have developed resistance to currently used therapeutic agents, nucleoside analogs having novel structures are particularly desirable.
  • the present invention fulfills this need and provides further related advantages, which are described herein.
  • the invention provides compounds of Formula I useful for treatment and prevention of viral infections and disorders of cell proliferation, including cancer. Such compounds are referred to as cubane nucleoside analogs. Thus the invention provides compounds of Formula I
  • Ri, R 2 , Rs, Rj, R5, R 5 , and R 7 are independently chosen from: i) hydrogen, halogen, cyano, azido, -COOH, hydroxyl, Cj-C 2 haloalkyl, and d- C 2 haloalkoxy, and ii) Ci-C6alkyl, C 2 -C ⁇ alkenyl, C 2 -C 6 alkynyl, Ci-C ⁇ alkoxy, mono- and di-(Ci-
  • B is an optionally substituted purine or pyrimidine base.
  • compositions comprising a compound or salt of Formula I are also provided herein.
  • the invention also comprising packaged pharmaceutical compositions comprising a compound or salt of Formula I in a container together with instructions for using the composition to treat a patient having a viral infection or susceptible to a viral infection.
  • Other packaged pharmaceutical compositions provided herein include a compound or salt of Formula I in a container together with instructions for using the composition to treat a patient having cancer.
  • Methods of treating a patient having a viral infection or a patient susceptible to a viral infection comprising administering to the patient a therapeutically effective amount of a compound or salt of Formula I are included herein.
  • Methods of treating or prevent cancer in a patient comprising administering to the patient a therapeutically effective amount of a compound or salt of Formula I are further included herein.
  • Compounds of Formula I may be administered alone, with another compound of Formula I or in combination with another active agent. Methods of combination administration are included herein.
  • the invention further provides a method of inhibiting DNA replication in vivo comprising administering to a patient infected with a virus or having cancer a concentration of a compound or salt of Formula I sufficient to inhibit DNA replicon replication in vitro. Methods of preparing compounds and salts of Formula I are also provided.
  • compounds of Formula I may contain one or more asymmetric elements such as stereogenic centers, stereogenic axes and the like, e.g. asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms.
  • asymmetric elements such as stereogenic centers, stereogenic axes and the like, e.g. asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms.
  • These compounds can be, for example, racemates or optically active forms.
  • compounds having asymmetric centers it should be understood that all of the optical isomers and mixtures thereof are encompassed.
  • compounds with carbon-carbon double bonds may occur in Z- and E-forms, with all isomeric forms of the compounds being included in the present invention.
  • the single enantiomers i.e., optically active forms can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or by resolution of the racemates. Resolution of the racemates can also be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral HPLC column.
  • the invention is not limited to any one of the specific tautomers, but rather includes all tautomeric forms.
  • the present invention is intended to include all isotopes of atoms occurring in the present compounds.
  • Isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium and isotopes of carbon include ' 1 C, 13 C, and 14 C.
  • substituted means that any one or more hydrogens on the designated atom or group is replaced with a selection from the indicated group, provided that the designated atom's normal valence is not exceeded.
  • 2 hydrogens on the atom are replaced.
  • Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds or useful synthetic intermediates.
  • a stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive isolation from a reaction mixture, and subsequent formulation into an effective therapeutic agent.
  • substituents are named into the core structure. For example, it is to be understood that when (cycloalkyl)alkyl is listed as a possible substituent the point of attachment of this substituent to the core structure is in the alkyl portion.
  • Suitable groups that may be present on a "substituted" position include, but are not limited to, e.g., halogen; cyano; hydroxyl; azido; alkanoyl (such as a C 2 -C O alkanoyl group such as acyl or the like); carboxamido; alkyl groups (including cycloalkyl groups, having 1 to about 8 carbon atoms, or 1 to about 6 carbon atoms); alkenyl and alkynyl groups (including groups having one or more unsaturated linkages and from 2 to about 8, or 2 to about 6 carbon atoms); alkoxy groups having one or more oxygen linkages and from 1 to about 8, or from 1 to about 6 carbon atoms; aryloxy such as phenoxy; alkyl
  • alkyl is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups, having the specified number of carbon atoms.
  • C 1 - Ce alkyl as used herein includes alkyl groups having from 1 to about 6 carbon atoms.
  • Co-C n alkyl is used herein in conjunction with another group, for example,
  • alkyl (aryl)Co-C 2 alkyl, the indicated group, in this case aryl, is either directly bound by a single covalent bond (CQ), or attached by an alkyl chain having the specified number of carbon atoms, in this case from 1 to about 2 carbon atoms.
  • alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and sec-pentyl.
  • Alkenyl indicates a hydrocarbon chain of either a straight or branched configuration having one or more carbon-carbon double bond bonds, which may occur at any stable point along the chain.
  • alkenyl groups include ethenyl and propenyi.
  • alkenyl groups include ethenyl and propenyi.
  • Alkynyl indicates a hydrocarbon chain of either a straight or branched configuration having one or more triple carbon-carbon bonds that may occur in any stable point along the chain, such as ethynyl and propynyl.
  • Alkoxy represents an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n- propoxy, i- propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy,
  • Alkanoyl indicates an alkyl group as defined above, attached through a keto (-
  • Alkanoyl groups have the indicated number of carbon atoms, with the carbon of the keto group being included in the numbered carbon atoms. For example a
  • the terms "mono- or di-alkylamino" or “mono- and di-alkylamino” indicate secondary or tertiary alkyl amino groups, wherein the alkyl groups are as defined above and have the indicated number of carbon atoms. The point of attachment of the alkylamino group is on the nitrogen. Examples of mono- and di-alkylamino groups include ethylamino, dimethylamino, and methyl-propyl-amino. A mono- or di-(C 3 -
  • C 7 cycloalkylamino)Co-C 2 alkylamino group is an alkyl amino substituent in which a first alkyl group is chosen from C 3 -C 7 alkyl and an second alkyl group is chosen from Co-C 2 alkyl, wherein Co indicates the absence of a second alkyl group, i.e. a mono-C 3 -C 7 alkylamino. The point of attachment to the core structure is on the second, Co-C 2 alkyl group.
  • alkylthio indicates an alkyl group as defined above attached through a sulfur linkage, i.e. a group of the formula alkyl-S-. Examples include ethylthio and pentylthio.
  • aminoalkyl indicates an alkyl group as defined above substituted with at least one amino substituent.
  • hydroxyalkyl indicates an alkyl group as defined above, substituted with at least one hydroxyl substituent. In certain instances the alkyl group of the aminoalkyl or hydroxyalkyl group may be further substituted.
  • aryl indicates aromatic groups containing only carbon in the aromatic ring or rings. Typical aryl groups contain 1 to 3 separate, fused, or pendant rinjr and from 6 to about 18 ring atoms, without heteroatoms as ring members. When indicated, such aryl groups may be further substituted with carbon or non-carbon atoms or groups. Such substitution may include fusion to a 5 to 7-membered saturated cyclic group that optionally contains 1 or 2 heteroatoms independently chosen from N, O, and S, to form, for example, a 3,4-methylenedioxy-phenyl group.
  • Aryl groups include, for example, phenyl, naphthyl, including 1- naphthyl and 2-naphthyl, and bi-phenyl.
  • aryl and alkyl are as defined above, and the point of attachment is on the alkyl group. This term encompasses, but is not limited to, benzyl, phenylethyl, and piperonyl.
  • (aryl)alkoxy and (aryl)alkylthio aryl, alkylthio, and alkoxy are as defined above and the point of attachment is through the oxygen atom of the alkoxy group or the sulfur group of the alkylthio.
  • alkoxy is a Coalkoxy the aryl is attached through an oxygen bridge; if the alkylthio is a Coalkylthio the aryl is attached through the sulfur.
  • Cycloalkyl indicates saturated hydrocarbon ring groups, having the specified number of carbon atoms, usually from 3 to about 8 ring carbon atoms, or from 3 to about 7 carbon atoms.
  • Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl as well as bridged or caged saturated ring groups such as norborane or adamantane, and cubane.
  • (cycloalkyl)alkyl and “(cycloalkyl)alkoxy” the terms cycloalkyl, alkyl, carbohydryl, and alkoxy are as defined above, and the point of attachment is on the alkyl, carbohydryl, or alkoxy group respectively. These terms include examples such as cyclopropylmethyl, cyclohexylmethyl, cyclohexylpropenyl, and cyclopentylethyoxy.
  • (cycloalkyl)alkylamino indicates an amino group substituted with at least one (cycloalkyl)alkyl or cycloalkyl (when the alkyl is a Co alkyl).
  • the amino group may be a secondary, in which case the other nitrogen atom valence is occupied by a hydrogen atom or a tertiary amino wherein containing an additional alkyl or (cycloalkyl)alkyl substituent.
  • Haloalkyl indicates both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, substituted with 1 or more halogen atoms, generally up to the maximum allowable number of halogen atoms.
  • Example- of haloalkyl include, but are not limited to, trifluoromethyl, difiuoromethyl, 2-fluoroethyl, and penta-fluoroethyl.
  • Haloalkoxy indicates a haloalkyl group as defined above attached through an oxygen bridge.
  • haloalkoxy include, but are not limited to, trifluoromethoxy, difluoromethoxy, 2-fluoroethyoxy, and pentafluoroethoxy
  • Halo or “halogen” as used herein includes fiuoro, chloro, bromo, and iodo.
  • “Pharmaceutical compositions” are compositions comprising at least one active agent, such as a compound or salt of Formula I, and at least one other substance, such as a carrier, excipient, or diluent. Pharmaceutical compositions meet the U.S. FDA's GMP (good manufacturing practice) standards for human or non-human drugs.
  • Salts of the compounds of the present invention include inorganic and organic acid and base addition salts.
  • the salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with . stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two.
  • the appropriate base such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like
  • salts of the present compounds further include solvates of the compounds and of the compound salts.
  • “Pharmaceutically acceptable salts” includes derivatives of the disclosed compounds wherein the parent compound is modified by making non-toxic acid or base salts thereof, and further refers to pharmaceutically acceptable solvates of such compounds and such salts.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • the pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH 2 ) n -COOH where n is 0-4, and the like.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phospho
  • prodrugs includes any compounds that become compounds of Formula I when administered to a mammalian subject, e.g., upon metabolic processing of the prodrug.
  • prodrugs include, but are not limited to, acetate, formate and benzoate and like derivatives of functional groups (such as alcohol or amine groups) in the compounds of Formula I.
  • an effective amount means an amount effective, when administered to a human or non-human patient, to provide any therapeutic benefit such as an amelioration of symptoms, e.g., an amount effective to decrease the symptoms of a viral infection, a preferably an amount sufficient to reduce the symptoms of an HIV, HBV, other viral infection or cancer. In certain circumstances a patient suffering from a viral infection or cancer may not present symptoms.
  • a therapeutically effective amount of a compound is also an amount sufficient to provide a positive effect on any indicia of disease, e.g. an amount sufficient to prevent a significant increase or significantly reduce the detectable level of virus or viral antibodies in the patient's blood, serum, or tissues.
  • a therapeutically effective amount may be an amount sufficient to reduce abnormal cell proliferation or an amount sufficient to reduce the mitotic figure index.
  • a significant increase or reduction in the detectable level of virus or viral antibodies is any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student's T-test, where p ⁇ 0.05.
  • the compounds described herein may also be used prophylactically to prevent cancer or viral infection in a patient susceptible to viral infection or cancer. When used prophylactically and effective amount is an amount sufficient to produce a statistically significant reduction in the risk of developing the condition being treated prophylactically.
  • nucleoside is a molecule that includes a purine or pyrimidine base covalently bound to a ribose sugar.
  • a “nucleoside analog” is synthetic molecule that resembles naturally occurring nucleoside, but that lacks a bond site needed to link it to an adjacent nucleotide.
  • a nucleoside analog may include a purine or pyrimidine base covalently bound to a hydroxyl substituted alkoxy group rather that a ribose sugar.
  • Nucleoside analogs include AZT, acyclovir, combivir, abacavir, emtricitabine, ddl, ddC, d4T, L-FMAU (stavudine),and 3TC. Nucleotide analogs, which include a phosphate group, are not included within the definition of nucleoside analogs.
  • a "patient” is any human or non-human animal in need of medical treatment.
  • Medical treatment can include treatment of an existing condition, such as a disease or disorder, or prophylactic or preventative treatment.
  • the patient is a human patient.
  • the invention also includes compounds of Formula I in which one or more of the following conditions are met.
  • the invention includes compounds and salts of the Formula I in which
  • B is a purine base of the formula
  • the invention includes compounds and salts of Formula I in which B is a purine or pyrimidine base of the formula:
  • the invention includes compounds and salts of Formula I in which B is a purine or pyrimidine base of the formula
  • the invention also provides compounds and salts of Formula I in which B is a pyrimidine base of the formula:
  • X is O or S.
  • Rio is amino, hydroxyl, nitro, halogen, d-C 4 alkyl, C 2 -C 4 alkenyl, Ci-C 4 alkoxy, C 2 - C4alkanoyl, hydroxyCi-C 4 alkyl, aminoCi-C 4 alkyl, mono-and di-CrC 8 alkylamino, (C 3 - C 7 cycloalkyl)Co-C 2 alkyl 5 (C3-C 7 cycloalkenyl)C 0 -C 2 alkyl, Ci-C 4 alkylthio, haloCi-C 4 alkenyl, haloC i -C 4 alkyl, or haloC i -C ⁇ alkoxy .
  • Ru is halogen, hydroxy, amino, Ci-C ⁇ alkyl, or Ci-C ⁇ alkoxy.
  • Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethylhexylsilyl, t- butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as acetyl and propionyl, methanesulfonyl, and p- toluenesulfonyl.
  • the invention includes compounds and salts of Formula I in which one or more of the following conditions is met for the Ri to R 7 variables.
  • Ri to R 7 are Ci-C2alkyl substituted with hydroxyl.
  • Ri is hydroxymethyl and R 3 , R 4 , Re, and R 7 are hydrogen, e.g. the invention includes compounds such as
  • Ri is hydroxymethyl and R 2 , R3, R 4 , Re 9 and R 7 are all hydrogen
  • Ri is hydroxymethyl and R 3 , R 4 , R5, Re, and R 7 are all hydrogen
  • the invention provides cubane nucleoside analogs such as:
  • Ri is hydroxymethyl and R 2 , R 3 , R 4 , Rs 5 R ⁇ , and R 7 are all hydrogen, e.g., the invention includes cubane nucleoside analogs such as
  • Ri and R 2 are hydroxymethyl; and R 3 , R ⁇ , and R 7 are hydrogen, e.g, the invention includes cubane nucleoside analogs such as
  • R 1 is hydroxymethyl; R 2 is hydroxyl; and R 3 , R 4 , R 5 , R 6 , and R 7 are hydrogen, ⁇ .g, the invention includes cubane nucleoside analogs such as
  • R 1 , R 2 , and Rs are hydroxymethyl; and R 3 and Re are hydrogen, e.g, the invention includes cubane nucleoside analogs such as
  • Cubane nucleoside analogs include all pharmaceutically acceptable forms of compounds of Formula I, such a salts, hydrates, solvates, crystal forms, polymorphs, chelates, non-covalent complexes, esters, clathrates, prodrugs, and mixtures of such compounds.
  • Pharmaceutically acceptable salts are a preferred pharmaceutically acceptable form.
  • NUCLEOSIDE ANALOG PRODRUG FORMULATIONS Cubane nucleoside analogs described herein can be administrated as a nucleoside analog prodrug to increase the activity, bioavailability, stability or otherwise alter the properties of the nucleoside analog.
  • a number of nucleoside analog prodrug ligands are known.
  • alkylation, acylation or other lipophilic modification of the mono-, di- or triphosphate of the nucleoside analog increases the stability of the nucleotide.
  • substituent groups that can replace one or more hydrogens on the phosphate moiety are alkyl, aryl, steroids, carbohydrates, including sugars, 1,2-diacylglycerol and alcohols.
  • the active nucleoside analog can also be provided as a S'-phosphoether lipid or a 5'- ether lipid, as disclosed.
  • the cubane nucleoside analogs disclosed herein may also be modified by the addition of a lipophilic substituent covalently bound to the nucleoside analog.
  • anti-microbial compositions including anti-bacterial compositions, comprising a compound or salt thereof of Formula I, together with a carrier, diluent, or excipient.
  • the invention provides pharmaceutical compositions comprising a compound or salt of salt thereof of Formula I, together with a pharmaceutically acceptable carrier, diluent, or excipient.
  • the pharmaceutical composition may be formulated as any pharmaceutically useful form, e.g., as an aerosol, a cream, a gel, a pill, a capsule, a tablet, a syrup, a transdermal patch, or an ophthalmic solution.
  • Compounds and salts of Formula I can be administered as the neat chemical, but are preferably administered as a pharmaceutical composition or formulation. Accordingly, the invention provides pharmaceutical formulations comprising a compound or pharmaceutically acceptable salt of Formula I, together with one or more pharmaceutically acceptable carrier, excipient, adjuvant, diluent, or other ingredient.
  • Compounds of general Formula I may be administered orally, topically, parenterally, by inhalation or spray, sublingually, transdermally, via buccal administration, rectally, as an ophthalmic solution, or by other means, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, excipients, adjuvants, and vehicles.
  • compositions of the invention may contain a pharmaceutically acceptable carrier, one or more compatible solid or liquid filler diluents or encapsulating substances, which are suitable for administration to a patient.
  • Carriers must be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the patient being treated.
  • the carrier can be inert or it can possess pharmaceutical benefits of its own.
  • the amount of carrier employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound.
  • Exemplary pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, and corn oil; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the TWEENs; wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphat
  • pharmaceutically acceptable carriers for systemic administration include sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions, emulsifiers, isotonic saline, and pyrogen-free water.
  • Preferred carriers for parenteral administration include propylene glycol, ethyl oleate, pyrrolidone, ethanol, and sesame oil.
  • Optional active agents may be included in a pharmaceutical composition, which do not substantially interfere with the activity of the compound of the present invention.
  • Effective concentrations of one or more of the compounds of the invention including pharmaceutically acceptable salts, esters or other derivatives thereof are mixed with a suitable pharmaceutical carrier, excipients, adjuvant, or vehicle.
  • a suitable pharmaceutical carrier such as dimethylsulfoxide (DMSO)
  • DMSO dimethylsulfoxide
  • surfactants such as TWEEN
  • dissolution in aqueous sodium bicarbonate such as sodium bicarbonate.
  • Derivatives of the compounds, such as salts of the compounds or prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.
  • the resulting mixture may be a solution, suspension, emulsion or the like.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility t the compound in the chosen carrier or vehicle.
  • the effective concentration sufficient for ameliorating the symptoms of the disease, disorder or condition treated may be empirically determined.
  • the pharmaceutical compositions containing compounds of general Formula I may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents, such as sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide pharmaceutically elegant and palatable preparations.
  • Oral formulations contain between 0.1 and 99% (weight %) of a compound of the invention and usually at least about 5% of a compound of the present invention. Some embodiments contain from about 25% to about 50% or from 5% to 75 % of a compound of invention.
  • Compounds of the invention can be incorporated into oral liquid preparations such as aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, for example. Moreover, formulations containing these compounds can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can contain conventional additives, such as suspending agents (e.g., sorbitol syrup, methyl cellulose, glucose/sugar, syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats), emulsifying agents (e.g., lecithin, sorbitan monsoleate, or acacia), non-aqueous vehicles, which can include edible oils (e.g., almond oil, fractionated coconut oil, silyl este ⁇ s 3 propylene glycol and ethyl alcohol), and preservatives (e.g., methyl or propyl p-hydroxybenzoate and sorbic acid).
  • suspending agents e.g., sorbitol syrup, methyl cellulose, glucose/sugar, syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats
  • emulsifying agents e.g
  • Orally administered compositions also include liquid solutions, emulsions, suspensions, powders, granules, elixirs, tinctures, syrups, and the like.
  • the pharmaceutically acceptable carriers suitable for preparation of such compositions are well known in the art.
  • Oral formulations may contain preservatives, flavoring agents, sweetening agents, such as sucrose or saccharin, taste-masking agents, and coloring agents.
  • Typical components of carriers for syrups, elixirs, emulsions and suspensions included ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent.
  • typical suspending agents include methylcellulose, sodium carboxymethyl cellulose, AVICEL RC-591, tragacanth and sodium alginate;
  • typical wetting agents include lecithin and polysorbate 80; and
  • typical preservatives include methyl paraben and sodium benzoate.
  • Aqueous suspensions contain the active material(s) in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents; may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esterr derived from fatty acids and a hexitol such as polyoxyethylene sorbitol substitute, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan substitute.
  • Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations.
  • These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • compositions of the invention may also be in the form of oil-in- water emulsions.
  • the oily phase may be a vegetable oil, for example olive oil or peanut oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally- occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monoleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monoleate.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, ka
  • Tablets typically comprise conventional pharmaceutically compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrants such as starch, alginic acid and croscarmelose; lubricants such as magnesium stearate, stearic acid and talc. Glidants such as silicon dioxide can be used to improve flow characteristics of the powder mixture. Coloring agents, such as the FD&C dyes, can be added for appearance. Sweeteners and flavoring agents, such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets. Capsules (including time release and sustained release formulations) typically comprise one or more solid diluents disclosed above. The selection of carrier components often depends on secondary considerations like taste, cost, and shelf stability.
  • compositions may also be coated by conventional methods, typically with pH or time-dependent coatings, such that the subject compound is released in the gastrointestinal tract in the vicinity of the desired topical application, or at various times to extend the desired action.
  • dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methylcellulose phthalate, ethy cellulose, Eudragit coatings, waxes and shellac.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water or an oil medium for example peanut oil, liquid paraffin or olive oil.
  • compositions may be in the form of a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.
  • the sterile injectable preparation may also be sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3- butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are useful in the preparation of injectables.
  • Compounds of Formula I may be administered parenterally in a sterile medium.
  • Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrathecal injection or infusion techniques.
  • the drug depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
  • adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
  • the carrier comprises at least about 90% by weight of the total composition.
  • Compounds of Formula I may also be administered in the form of suppositories for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non- irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non- irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials are cocoa butter and polyethylene glycols.
  • Topical compositions of the present invention may be in any form including, for example, solutions, creams, ointments, gels, lotions, milks, cleansers, moisturizers, sprays, skin patches, and the like.
  • Such solutions may be formulated as 0.01% -10% (weight %) isotonic solutions, pH about 5-7, with appropriate salts.
  • Compounds of the invention may also be formulated for transdermal administration as a transdermal patch.
  • Topical compositions containing the active compound can be admixed with a variety of carrier materials well known in the art, such as, for example, water, alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, and the like.
  • carrier materials such as, for example, water, alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, and the like.
  • compositions suitable for use in topical carriers include, for example, emollients, solvents, humectants, thickeners and powders. Examples of each of these types of materials, which can be used singly or as mixtures of one or more materials, are as follows:
  • Emollients such as stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane-1 ,2-diol, butane-1 ,3-diol, mink oil, cetyl alcohol, iso-propyl isostearate, stearic acid, iso-butyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, dimethylpolysiloxane, di-n-butyl sebacate, iso-propyl myristate, iso-propyl palmitate, iso-propyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, arachis oil, castor oil, acety
  • the compounds of the invention may also be topically administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. Other formulations
  • compositions useful for attaining systemic delivery of the subject compounds include sublingual, buccal and nasal dosage forms.
  • Such compositions typically comprise one or more of soluble filler substances such as sucrose, sorbitol and mannitol, and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methylcellulose. Glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents disclosed above may also be included.
  • compositions for inhalation typically can be provided in the form of a solution, suspension or emulsion that can be administered as a dry powder or in the form of an aeroso 1 using a conventional propellant (e.g., dichlorodifluoromethane or trichlorofluoromethane). Additional components
  • compositions of the present invention may also optionally comprise an activity enhancer.
  • the activity enhancer can be chosen from a wide variety of molecules that function in different ways to enhance antimicrobial effects of compounds of the present invention.
  • Particular classes of activity enhancers include skin penetration enhancers and absorption enhancers.
  • compositions of the invention may also contain additional active agents can be chosen from a wide variety of molecules, which can function in different ways to enhance the antimicrobial or therapeutic effects of a compound of the present invention.
  • additional active agents can be chosen from a wide variety of molecules, which can function in different ways to enhance the antimicrobial or therapeutic effects of a compound of the present invention.
  • These optional other active agents, when present, are typically employed in the compositions of the invention at a level ranging from about 0.01% to about 15%. Some embodiments contain from about 0.1% to about 10% by weight of the composition. Other embodiments contain from about 0.5% to about 5% by weight of the composition.
  • the invention includes packaged pharmaceutical formulations.
  • packaged formulations include a pharmaceutical composition containing one or more compounds or salts of Formula I in a container and instructions for using the composition to treat an patient (typically a human patient) suffering from a viral infection or cancer or prevent a viral infection or cancer in an patient.
  • the instructions may also be instructions for using the composition cancer or a viral infection, wherein the viral infection is a Hepatitis B, Hepatitis C, HIV-I, HSV-I, HSV-2, or Epstein-Barr viral infection.
  • the compounds of the invention can be administered alone or as mixtures, and the compositions may further include additional drugs or excipients as appropriate for the indication.
  • the invention includes methods of preventing and treating viral infections and cancer, particularly HIV, HBV, HCV, HSV, and Epstein-Barr viral infections, by administering an effective amount of one or more compounds of Formula I to an patient at risk of contracting " viral infection or cancer or suffering from a viral infection or cancer.
  • Compounds of Formula I may also be administered to reduce abnormal cell proliferation, and in particular, cell hyperproliferation.
  • abnormal cell proliferation examples include, but are not limited to: benign tumors, including, but not limited to papilloma, adenoma, firoma, chondroma, osteoma, lipoma, hemangioma, lymphangioma, leiomyoma, rhabdomyoma, meningioma, neuroma, ganglioneuroma, nevus, pheochromocytoma, neurilemona, fibroadenoma, teratoma, hydatidiform mole, granuosa-theca, Brenner tumor, arrhenoblastoma, hilar cell tumor, sex cord mesenchyme, interstitial cell tumor, and thyoma as well as proliferation of smooth muscle cells in the course of development of plaques in vascular tissue; malignant tumors (cancer), including but not limited to carcinoma, including renal cell carcinoma, prostatic adenocarcinoma, bladder carcinoma
  • an effective amount of a compound of Formula I may be an amount sufficient to reduce the symptoms of the viral infection or cancer.
  • an effective amount of a compound of Formula I may be an amount sufficient to significantly reduce the amount of viral particles or antibodies against the detectable in a patient's tissues or bodily fluids.
  • An effective amount of a compound of Formula I may also be an amount sufficient to slow tumor growth, shrink tumor size, or reduce cell proliferation.
  • Methods of treatment also include inhibiting viral replication in vivo, in an patient at risk for a viral infection or suffering from such an infection, by administering a sufficient concentration of a compound of Formula I to inhibit viral replication in vitro.
  • a sufficient concentration of a compound administered to the patient is meant the concentration of the compound available in the patient's system to prevent or combat disease.
  • concentration by be ascertained experimentally, for example by assaying blood concentration of the compound, or theoretically, by calculating bioavailability.
  • the amount of a compound sufficient to inhibit viral infected cells in vitro may be determined with a conventional assay for viral replication or viral activity including the HIV reverse transcriptase assay of Example 6, the HBV replication assay of Example 7, and the HCV cell-based replicon assay of Example 8.
  • the invention also includes using compounds of Formula I in prophylactic therapies.
  • an effective amount of a compound of the invention is an amount sufficient to significantly decrease the treated patient's risk of contracting a viral infection or developing cancer.
  • Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day).
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient.
  • Frequency of dosage may also vary depending on the compound used and the particular disease treated. However, for treatment of most infectious disorders, a dosage regimen of 4 times daily or less is preferred and a dosage regimen of 1 or 2 times daily is particularly preferred.
  • the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
  • Compounds of the invention may be administered in combination with one or more other active agent, such as an additional anti-viral agent, immunomodulatory compound, antiinflammatory, anti-proliferative agent.
  • Pharmaceutical compositions described herein include single dosage forms contain a compound of Formula I and one or more other active agent, dosage forms containing more than one compound of Formula I and compositions formulated for separate administration of a compound of Formula I with another active agent. It has been recognized that drug-resistant variants of HTV and HBV can emerge after prolonged treatment with an antiviral agent. Drug resistance may occur by mutation of a gene that encodes for an enzyme used in viral replication, in the case of HIV, reverse transcriptase, protease, and DNA polymerase are known to mutate causing resistance.
  • a compound of Formula I maybe administered in combination, i.e., at the same time, as one or more other active agent. This is called combination therapy.
  • a compound of Formula I may also be administered serially with one or more additional active agent. Serial administration with a second active agent is known as alternation therapy. In general, combination therapy is typically preferred over alternation therapy because it induces multiple simultaneous stresses on the virus.
  • the second antiviral agent for the treatment of HTV can be a reverse transcriptase inhibitor (a "RTI"), which can be either a synthetic nucleoside (a "NRTI”) or a non-nucleoside compound (a "NNRTI”).
  • RTI reverse transcriptase inhibitor
  • NRTI synthetic nucleoside
  • NRTI non-nucleoside compound
  • the second (or third) antiviral agent can also be a protease inhibitor.
  • the second (or third) compound can be a pyrophosphate analog, or a fusion binding inhibitor.
  • a non-limiting list of compounds for combination or alternation therapy for the treatment of HBV include 3TC, FTC, L-FMAU, interferon, ⁇ -D-dioxolanyl- guanine (DXG N ⁇ D-dioxolanyl-2,6-diarninopurine (DAPD), and ⁇ -D- dioxolanyl-6-chloropurine (ACP), famciclovir, penciclovir, BMS-200475, bis pom PMEA (adefovir, dipivoxil); lobucavir, ganciclovir, and ribavirin.
  • 3TC 3TC
  • FTC ⁇ ⁇ D-dioxolanyl- guanine
  • DAPD ⁇ D-dioxolanyl-2,6-diarninopurine
  • ACP ⁇ -D- dioxolanyl-6-chloropurine
  • a non-limiting list of antiviral agents that can be used in combination or alternation with the compounds disclosed herein for HIV therapy include cis-2-hydroxymethyl-5-(5- fluorocytosin-l-yl)-l,3-oxathiolane (FTC) ; the (-)-enantiomer of 2-hydroxymethyl-5- (cytosin-l-yl)-l,3-oxathiolane (3TC); carbovir, acyclovir, foscarnet, interferon, AZT, DDI, DDC, D4T, CS- (3'-azido-2',3'-dideoxy-uridine), and ⁇ -D-dioxolane nucleosides such as ⁇ - D-dioxolanyl-guanine (DXG), ⁇ -D-dioxolanyl-2,6- diaminopurine (DAPD), and ⁇ -D- dioxolanyl-6-chlor
  • Preferred protease inhibitors include crixivan (Merck), nelfinavir (Agouron), ritonavir (Abbott), saquinavir (Roche), DMP-266 (Sustiva) and DMP-450 (DuPont Merck).
  • a more comprehensive list of compounds that can be administered in combination o alternation with any of the disclosed nucleosides include (lS,4R)-4-[2-amino-6- cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-l- methanol succinate ("1592", a carbovir analog; GlaxoWellcome); 3TC: (-)- ⁇ -L-2' 5 3'-dideoxy-3'-thiacytidme (GlaxoWellcome); a- APA Rl 8893: a-nitro-anilino-phenylacetamide; A-77003; C2 symmetry-based protease inhibitor (Abbott); A-75925: C2 symmetry-based protease inhibitor (Abbott) ; AAP- BHAP: bisheteroarylpiperazine analog (Upjohn); ABT-538: C2 symmetry- based protease inhibitor (Abbott); AzddU'.S'-
  • VX-478 hydroxyethylsulphonamide protease inhibitor (Vertex); XM 323: cyclic urea protease inhibitor (Dupont Merck).
  • Non limiting examples of antiproliferative compounds that can be administered in combination a compound of Formula I include antifolates, 5-fluoropyrimidine (including 5- fluorouracil), a cytidine analogue such as ⁇ -L-l,3-dioxolanyl cytidine or ⁇ -L-l,3-dioxolanyl 5- fluorocytidine, antimetabolites (including purine antimetabolites, cytarabine, tudarabine, floxuridine, 6- mercaptopurine, methotrexate, and 6-thioguanine), hydroxyurea, mitotic inhibitors (including CPT-11, Etoposide (VP-21), taxol, and vinca alkaloids such as vincristine and vinblastine, an alkylating agent (including but not limited to busulfan, chlorambucil, cyclophosphamide, ifofamide, mechlorethamine, melphalan, and thi
  • 1,4-cubanedicarboxylic acid (2.20 g., 100 mmol)(I) is dissolved in 150 ml oxalyl chloride (Aldrich) containing 20 mg of t-butyl perbenzoate (Aldrich) as an initiator. This is photolyzed for a 12 hr. period with a 250 W low pressure hanovia lamp contained in a quartz immersion well. The temperature is maintained at ca. 5 0 C with the aid of a recirculating chiller. The reaction is quenched in cold methanol (IL). Solvent is removed using an R-14 ⁇ rotary evaporator (Buchi) keeping the bath temperature below 50 0 C.
  • IL cold methanol
  • the resultant oil is purified by column chromatography on silica gel using Hexane/EtOH (6:1) to yield 1.94 g (58% yield) of the pure tetraester (II)(one spot on TLC) material, see also Bashir-Hashemi, A., Photochemical Carboxylation of Cubanes, Angew Chem Int. Ed. (1993) 32: 612-613.
  • the monomethyl ester (1.37 g, 6.65 mmol) is dissolved in 15 ml of oxalyl chloride and warmed gently until it dissolves.
  • the oxalyl chloride is removed in the rotary evaporator with the bath temperature not allowed to remain over 40 0 C in order to avoid significant decomposition.
  • Residual halide is azeotroped away by adding 3 x 50 ml portions of 50:50 toluene/ethyl acetate and rotovaping away. The resultant residue is used immediately without further purification (the material is, however, stable at 4 C if kept dry)
  • 4-methoxycarbonylcubane-l -carbonyl chloride (11 mMol) is dissolved in 20 ml of toluene, and 1.6 g (11 mmol) of 1 -hydroxypyridine-2-thione sodium salt (Aldrich) is added. Then, 4-N,N-dimethylaminopyridine (71 mg, 0.58 mmol) is added, and the reaction flask is wrapped in aluminum foil to exclude light. The mixture is refluxed with stirring for 1 hour under nitrogen.
  • the residual brown oil is dissolved in 30 ml of toluene, and 30 ml of 15% aqueous NaOH is added. This mixture is refluxed overnight, cooled to room temperature, an separated. The organic layer is washed with two 30 ml portions of water. The combined aqueous layer is extracted with ether (two portions of each 30 ml), acidified to pH 2-3 while being kept cool, and extracted repeatedly with ethyl acetate (5 portions of 30 ml). The combined organic layer is washed with brine, dried over sodium sulfate and filtered twice through small pads of silica gel (230 mesh then 60-200 mesh), eluting with ethyl acetate.
  • HCl gas is bubbled through a suspension of 4.0 g (18.3 millimoles) (tert- butoxycarbonyl)aminocubane in 200 ml of wet methanol at -3O.degree. C. until the solution becomes homogeneous.
  • the methanol is removed in vacuo and 50 ml ether/acetone (4: 1 ) is added to the residue.
  • the solid (2.4 g, 85%) is collected by filtration and dried. The light ta solid is used without further purification.
  • 1 H NMR- 8.9 ppm (broad singlet, 3H) and 4.3-3.8 (multiplet, 7 H).
  • Cubane-1 ,4-dicarboxylic acid dimethyl esther(Pharmatech International Inc.) (3.10 g, 14.1 mmol) is dissolved in 15 ml of hot methanol in a 250 ml in a round-bottomed flask equipped with a condenser.
  • Solid reagent NaOH 0.560 g, 14.1 mmol
  • the reaction mixture is heated under refluxed for a 13-15 hr period.
  • the reaction mixture is then cooled to room temperature.
  • the reaction mixture is diluted with 120 ml of water and extracted 3 times with 50 ml portions of ether, which is discarded..
  • the aqueous portion is acidified to ca. pH 3 with 6N HCl and then extracted with chloroform (4 portions of ca. 50 ml each).
  • the combined chloroform extracts are dried and reduced by rotary evaporation to produce the diacid as an off-white powder in 70% yield.
  • EXAMPLE 10-P PREPARATION OF 1 -CYANOCUBANE-4-CARBOXYLIC ACID METHYL ESTER.
  • Phosphorous oxychloride (1 ml) is added to 2 mMol of 1 -carboxamido-cubane-4- carboxylic acid methyl ester in dry 1,2-dichloroethane. This mixture is heated to reflux for a 30-min period, washed with 5% sodium bicarbonate, brine, and water, dried with anhydrous magnesium sulfate, and solvent removed on the rotary evaporator. This gave 4.02 g. white solid. Column chromatography on silica using 80:20 toluene/methanol yielded pure materia (one spot on TLC). 1 H NMR :3.70 (singlet, 3H) and 4.32 (singlet, 6H)
  • 1,4-cubanedicarboxylic acid (2.20 g, 100 mMol) is dissolved in 150 ml oxalyl chloride (Aldrich) containing 20 ing of t-butyl perbenzoate (Aldrich) as an initiator. This is photolyzed for 12 hr with a 250 W low pressure hanovia lamp contained in a quartz immersion well. The temperature is maintained at ca. 5 0 C with the aid of a recirculating chiller. The reaction mixture is placed on a rotary evaporator and the oxalyl chloride removed, followed by successive portions of isopropyl acetate to azeotrope away any remaining oxalyl chloride.
  • the bath temperature is maintained below 50 0 C.
  • Cold (0 0 C) ethyl acetate (50 ml) cont ⁇ ing p-nitrobeniylTlel>l ⁇ mixture is allowed to remain overnight.
  • Solvent (1 L) is removed and the yellow oil is purified by column chromatography on silica gel using Hexane/EtOH ( 6:1) to yield 2.31 g (40% yield) of the pure (one spot on TLQmaterial.
  • Tris (p-nitrobenzyl) ester is photolyzed for 4 hrs. using a high pressure mercury arc lamp (500 W: Hanovia) with a Pyrex filter at 5 0 C (maintained with a recirculated chiller) in a 0.5% (w/v) solution in heptane containing formic acid (obtained by saturating the heptane with 85% HCOOH in aqueous solution) and a catalytic amount (60 mg) of Pd(dba) 3 (Strem chemical).
  • the resultant solution is extracted 6X with 5% potassium bicarbonate to remove p-nitrobenzyl alcohol and other nitroaromatic products.
  • Tris(triethylsilyl)ester under conditions known in the art in 95% yield from the triethylsilyl chloride in pyridine.
  • .triethylsilyl ester is reduced by-Lithium Aluminum Hydride under- conditions — known in the art in refluxing THF (48 hr), followed by aqueous workup using potassium tartrate to avoid the presence of mineral acid, to provide hydroxymethyl-tris(triethylsilyl) ester Oil, m/e 441 (M+l) (64% yield).
  • Step B Cleavage of Benzyl Protecting group
  • EXAMPLE 21 1 -METHYLENE(N- 1 -5-C YCLOHEXYLURIDYL) ⁇ 5 O-TRIS(HYDROXYMETHYL)
  • Method B the preparation of the required (aminomethyl) cubane compound is described in the prior art example 12-P, supra.
  • the isocyanate is prepared from the acid chloride as used because it is unstable.
  • the acid chloride in toluene is added to a refluxing suspension of dry freshly pepared silver cyanate (from silver nitrate and potassium cyanate, filtering the ppt and drying) and then allowed to stand in the warm solution (with the heat turned off) for a 30 min-period.
  • 2.77 g (10 mMol) of the (aminomethyl) cubane derivative in the scheme above is dissolved in 20 ml of dry DMF, and is cooled to 4 C.
  • a solution of the isocyanate prepared from 11 mMoles (1.7 g) of the methacrolyl acid chloride as described above is added in one portion.
  • the solution is left for 48 hours at 4 0 C.
  • 50 ml of chloroform is added.
  • the resultant urea solution is worked up by washing with ice cold 0.1% citric acid (1 x 30 ml), 1% potassium bicarbonate (1 x 30 ml), brine (100 ml) and finally water.
  • the organic layer (predominantly chloroform with some DMF contained) is reduced at the rotary evaporator and remaining DMF is removed using the oil pump with a bath temperature of 50 0 C.
  • the resultant oil is added to a solution of 5 g of potassium bifluoride in 20 ml of 2N
  • General method C employs the fluorous version of the Mitsunobu reaction.
  • the advantage is that column chromatographic workup is generally not required. Flash chromatography is usually sufficient.
  • the reagents as developed by Curran and his colleagues are commercially available from Fluorous Technologies, Inc, Pittsburgh, PA. A detailed procedure for this reaction has been published in Application Note 20050511 from that Company. Briefly, 0.24 mMol of the 3-N-benzylated nucleobase (Uracil or Thymine) is dissolved in 2 ml of dry THF.
  • the invention additionally includes compounds of Formula A-B where A is a nucleoside base chosen from the bases shown in TABLE A covalently bound at the point of attachment indicated by a wavy line to B, a substituted cubane chosen from the cubanes shown in Table B.
  • A is a nucleoside base chosen from the bases shown in TABLE A covalently bound at the point of attachment indicated by a wavy line to B, a substituted cubane chosen from the cubanes shown in Table B.
  • EXAMPLE 28 INHIBITORY EFFECT OF COMPOUNDS AGAINST HIV- 1 REVERSE TRANSCRIPTASE Extension assays may be performed using a r(I) n (dC)i 2 .i 8 homopolymer template-primer (Pharmacia, Piscat ⁇ way, NJ.) and the HIV-I heterodimer p66/51 reverse transcriptase (RT, Biotechnology General, Rehovat, Israel).
  • the standard reaction mixture (100 ⁇ l) contains 100 mM Tris hydrochloride (pH 8.0), 50 mM KCl, 2 mM MgCl 2 , 0.05 units/ml r(I) n (dC)i 2 -ig, 5 mM DTT, 100 ⁇ g/ml Bovine Serum Albumin, and 1 ⁇ M 3 H-dCTP (23 Ci/mmol).
  • 3TCTP (0.001-50 ⁇ M) is used as a positive control.
  • Compounds are incubated 1 hr at 37 0 C. in the reaction mixture with 1 unit HTV-I RT.
  • the reaction is stopped with the addition of an equal volume of cold 10% TCA/0.05% sodium pyrophosphate and incubated 30 minutes at 4 0 C.
  • the precipitated nucleic acids are harvested onto fiberglass filter paper using a Packard manual harvester (Meriden, CT).
  • the radiolabel uptake in counts per minute (cpm) is determined using a Packard 9600 Direct Beta counter. Active compounds exhibit a statistically significant reduction in the number of counts (cpm). Certain active compounds exhibit a 2-fold decrease in the number of counts.
  • the ability of the active compounds to inhibit the growth of hepatitis virus in 2.2.15 cell cultures can be evaluated as follows.
  • the antiviral evaluations are optimally performed on two separate passages of cells. AU wells, in all plates, are seeded at the same density and at the same time.
  • the levels of integrated HBV DNA in each cellular DNA preparation are used to calculate the levels of intracellular HBV DNA forms, thereby ensuring that equal amounts of cellular DNA are compared between separate samples.
  • Typical values for extracellular HBV virion DNA in untreated cells range from 50 to 150 pg/ml culture medium.
  • Intracellular HBV DNA replication intermediates in untreated cells typically ranges from 50 to 100 ⁇ g/pg cell HBV DNA.
  • reductions in the levels of intracellular HB Vl DNA due to treatment with antiviral compounds are less pronounced, and occur more slowly, than reductions in the levels of HBV virion DNA.
  • Toxicity analyses are performed to assess whether any observed antiviral effects are due to a general effect on cell viability.
  • the method used herein is the measurement of the uptake of neutral red dye, a standard and widely used assay for cell viability in a variety of virus- host systems, including HSV and HTV.
  • Toxicity analyses are performed in 96-well flat bottomed tissue culture plates. Cells for the toxicity analyses are cultured and treated with test compounds with the same schedule as described for the antiviral evaluations below. Each compound are tested at 4 concentrations, each in triplicate cultures. Uptake of neutral red dye is used to determine the relative level of toxicity. The absorbance of internalized dye at 510 run is used for the quantitative analysis. Values are presented as a percentage of the average Abs 510 nm values in 9 separate cultures of untreated cells maintained on the same 96-well plate as the test compounds.
  • EXAMPLE 30 CELL-BASED ASSAY FOR HCV ANTI-VIRAL ACTIVITY Assays for determining anti-HCV activity have been previously disclosed. See for example U.S. patent application no. 2005/0222198, which is hereby incorporated by reference at pages 139-140 for its teaching regarding anti-HCV activity assays. Anti-HCV activity is determined as follows:
  • Huh-5-2 cells [a cell line with a persistent HCV replicon 13891uc-ubi-neo/NS3-3'/5.1; with firefly luciferase-ubiquitin- neomycin phosphotransferase fusion protein and EMCV-
  • IRES driven NS3-5B HCV polyprotein is cultured in RPMI medium (Gibco) supplemented with 10% fetal calf serum, 2 mM L-glutamine (Life Technologies), 1 *non- essential amino acids (Life Technologies); 100 IU/ml penicillin and 100 ug/ml streptomycin and 250 ug/ml G418 (Geneticin, Life Technologies).
  • Cells are seeded at a density of 7000 cells per well in 96 well View Plate® (Packard) in medium containing the same components as described above, except for G418. Cells are allowed to adhere and proliferate for 24 hr. At that time, culture medium is removed and serial dilutions of the test compounds are added in culture medium lacking G418.
  • Interferon alfa 2a 500 IU is used as a positive control. Plates are further incubated at 37° C. and 5% CO 2 for 72 hours. Replication of the HCV replicon in Huh-5 cells results in luciferase activity in the cells. Compounds active against HCV exhibit a statistically significant reduction in luciferase activity. Luciferase activity is measured by adding 50 ⁇ L of lxGlo-lysis buffer (Promega) for 15 minutes followed by 50 ⁇ L of the Steady-Glo Luciferase assay reagent (Promega). Luciferase activity is measured with a luminometer and the signal in each individual well is expressed as a percentage of the untreated cultures.
  • EXAMPLE 31 ASSAY FOR ANTI-TUMOR ACTIVITY CEM cells are human lymphoma cells (a T- lymphoblastoid cell line that can be obtained from ATCC, Rockville, Md.). The toxicity of a compound to CEM cells or other tumor cell line provides useful information regarding the activity of the compound against tumors. The toxicity is measured as IC 50 , the concentration of test compound that inhibits the growth of 50% of the tumor cells in the culture. Test compound solutions, are plated in triplicate in 50 ⁇ l growth medium at 2 times the final concentration and allowed to equilibrate at 37 0 C in a 5% CO 2 incubator. Cycloheximide may be used as a positive control.
  • Log phase cells are added in 50 ⁇ l growth medium to a final concentration of 2.5xlO 3 cells/well and incubated for 4 days at 37 0 C under a 5% CO 2 air atmosphere.
  • Control wells include media alone (blank) and cells plus media without drug.
  • 15 ⁇ l of Cell Titer 96 kit assay dye solution (Promega, Madison, Wis.) is added to each well and the plates are incubated 8 hr at 37 0 C in a 5% CO 2 incubator.
  • Promega Cell Titer 96 kit assay stop solution is added to each well and incubated 4-8 hr in the incubator.
  • Certain desirable cubane nucleoside analogs exhibit ICso values of from 100 nM to 20 mM in this assay against CEM or other tumor cell lines.
  • EXAMPLE 32 ASSAYS FOR NON-SPECIFIC CELL TOXICITY Example 32a. Measurement ofmtDNA.
  • Example 31b Assay for the analysis of intracellular metabolites.
  • CEM cells or cytoplasmic deoxycytidine kinase-deficient CEM cells are incubated with test compounds for 24 h. Untreated cells are used as a negative control. Cells are harvested by centrifugation, washed twice with ice-cold phosphate-buffered saline, and extracted with 60% methanol on ice for 15 min. The methanol-soluble fraction is evaporated to dryness, resuspended in water, and analyzed by ion-exchange HPLC using a Whatman Partisil-SAX column at a flow rate of 1 ml/min.
  • Intracellular metabolites are identified by a combination of authentic cold standards and enzyme digestion of methanol-soluble extracts. Methanol-soluble extracts are digested by alkaline phosphatase. Radioactivity is measured in tandem with a 150TR Radiomatic Flow Scintillation Analyzer.
  • Example 32c Assay for the activity of deoxycytidine kinase.
  • Mitochondrial deoxypyrimidine nucleoside kinase is purified by affinity chromatography w a thymidine analog ligand by using chronic lymphocytic leukemia cells harvested from patients byleukophoresis.
  • Enzyme (0.01 U of deoxycytidine kinase or 0.0006U of mitochondrial deoxypyrimidine nucleoside kinase; a unit is defined as the conversion of 1 nmol of substrate per min) is incubated with the kinase mixture containing 140 mM Tris (pH 7.5), 1.7 mM dithiothreitol, 8 mM NaF, and 2 mM ATP-MgCl 2 for 2 h at 37°C. Reaction mixtures are applied to DE-81 discs (Whatman), washed three times in ImM ammonium formate followed by one wash in ethanol, and dried. Radioactivity is elutedfrom the discs with a solution of 0.2 N HCl and 2 M NaCl, followed by scintillation counting.
  • Example 32d Chain termination assays.
  • Triphosphates of test compounds are analyzed for their chain elongation activities by using M13mpl9 phage DNA hybridized with a 5'- 32 P-22-mer oligonucleotide primer.
  • Incorporation of one template complementary nucleotide into the 3' terminus of the primer is carried out in an 8- ⁇ l mixture containing 1 U of HIV-RT or pol Y(I Unit is defined as the incorporation of 1 nmol of dTTP into activated DNA in 1 h), 50 mM Tris, 60 mMKCl, 1 mM dithiothreitol, 10 mM MgCl 2 , 0.05 ⁇ M 22-mer oligonucleotide (S'-GTAAAACGACGGCCAGTGAATT-S') annealed to M13mpl9 phage DNA (3 l -CATTTTGCTGCCGGTCACTTAAGCTCGA-5 1 ), and 0.5 ⁇ M nucleo
  • the effectiveness of various compounds is determined in a CEM cell assay.
  • the HIV inhibition assay method of acutely infected cells is an automated tetrazolium based colorimetric assay (see Pauwels, R. et al., "Rapid and automated tetrazolium-based calorimetric assay for the detection of anti-HTV compounds," J. Virol. Methods, (1988) 20: 309-321 and Weislow, O. S. et al., "New soluble-formazan assay for HIV-I cytopathic effects: application to high-flux screening of synthetic and natural products for AIDS- antiviral activity," J. Natl. Cancer Inst. (1989) 81: 577-586).
  • T-cell lines, CEM-SS, and MT- 2, and viruses HIV-I RF and HIV-I NL4-3 (pNL4-3) are obtained from the National Institutes of Health (AIDS Research and Reference Reagent Program, Bethesda, Md.)- Assays are performed in 96-well tissue culture plates.
  • CEM cells, a CD4 + cell line is grown in RPMI- 1640 medium (Gibco) supplemented with a 10% fetal calf serum and then treated with polybrene (2 ⁇ g/ml). An 80 microliter volume of medium containing 1 x 10 4 cells is dispensed into each well of the tissue culture plate.
  • test compound dissolved in tissue culture medium (or medium without test compound as a control) to achieve the desired final concentration and the cells were incubated at 37 0 C for a 1-hour period.
  • TCIDso the dose of virus that infects 50% of cells in tissue culture
  • test compound was determined by adding medium without virus to several wells containing test compound. Typically, the final concentrations of test compounds were 0.1, 1, 10, 100 and 500 ⁇ g /ml. Either azidothymidine (AZT) or dideoxyinosine (ddl) was included as a positive drug control in each tissue culture plate. Test compounds were dissolved in DMSO and diluted into tissui culture medium so that the final DMSO concentration did not exceed 1.5% in any case. DMSO was added to all control wells at an appropriate concentration. Following the addition of virus, cells were incubated at 37 C. in a humidified, 5% CO 2 atmosphere for a 7-day period. Test compounds could be added on days 0, 2 and 5 if desired.
  • ZTT azidothymidine
  • ddl dideoxyinosine
  • the 50% cytotoxicity concentration (CC50) was calculated as the concentration of compound that decreased the percentage of formazan produced in uninfected, compound- treated cells to 50% of that produced in uninfected, compound-free cells.
  • the therapeutic index was calculated by dividing the cytotoxicity (CC 50 ) by the antiviral activity (EC 5 0).

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Abstract

La présente invention concerne des analogues du nucléoside de cubane utiles en tant qu’agents anti-viral et anti-cancéreux. Elle concerne également des procédés de préparation desdits analogues. L’invention concerne, en outre, des compositions pharmaceutiques contenant un ou plusieurs analogues nucléosides de cubane et un ou plusieurs véhicules, excipients ou diluants pharmaceutiquement acceptables. L’invention concerne également des procédés destinés à traiter des infections cancéreuses et virales chez des mammifères. Les nucléosides de cubane décrits ici sont des composés répondant à la formule générale (I) dans laquelle B représente une base nucléoside de purine éventuellement substituée ou de pyrimidines et R1, R2, R3, R4, R5, R6, et R7 sont définis ici.
PCT/US2006/044835 2005-11-17 2006-11-17 Analogue du nucleoside de cubane WO2007059330A2 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5959100A (en) * 1996-03-27 1999-09-28 Nexstar Pharmaceuticals, Inc. Pyrimidine nucleosides as therapeutic and diagnostic agents
US6649600B1 (en) * 1999-11-12 2003-11-18 Biogen, Inc. Adenosine receptor antagonists and methods of making and using the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5959100A (en) * 1996-03-27 1999-09-28 Nexstar Pharmaceuticals, Inc. Pyrimidine nucleosides as therapeutic and diagnostic agents
US6649600B1 (en) * 1999-11-12 2003-11-18 Biogen, Inc. Adenosine receptor antagonists and methods of making and using the same

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