WO2004096197A2

WO2004096197A2 - 5-aza-7-deazapurine nucleosides for treating flaviviridae

Info

Publication number: WO2004096197A2
Application number: PCT/IB2004/001740
Authority: WO
Inventors: Paolo La Colla; Gilles Gosselin; Frank Seela; David Dukhan; Frédéric Leroy
Original assignee: Universita Degli Studi Di Cagliari; Centre National De La Recherche Scientifique; Universitat Osnabruck Laboratorium Fur Organic And Biorganic Chemie
Priority date: 2003-05-02
Filing date: 2004-05-03
Publication date: 2004-11-11
Also published as: WO2004096197A8; WO2004096197A3

Abstract

This invention is directed to a method for treating a host, especially a human, infected with hepatitis C, flavivirus and/or pestivirus, comprising administering to that host an effective amount of an anti-hepacivirus, anti-flavivirus or anti-pestivirus biologically active acyclic ester or pentofuranonucleoside that has a 5-aza-7-deazapurine nucleoside base. Also claimed are pharmaceutical compositions of the present invention that may be administered alone or in combination and/or alternation with another antiviral agent, and a use of these nucleoside analogues in the manufacture of a medicament.

Description

5-AZA-7-DEAZAPURINE NUC EOSIDES FOR TREATING FLΛVIVIRIDΛE

CROSS-REFERENCE FOR THE INVENTION

This application claims priority to U.S. Provisional No. 60/467,465, filed May 2, 2003.

FIELD OF THE INVENTION

The present invention is in the area of pharmaceutical chemistry and, in particular, is a 5-aza-7-deazapurine nucleoside and derivatives thereof, their synthesis and their use as εaxύ-Flaviviridae agents in the treatment of hosts infected with Flaviviridae.

BACKGROUND OF THE INVENTION

The family of Flaviviridae viruses include pestiviruses, flaviviruses and hepatitis C virus. The pestivirus genus includes bovine viral diarrhea virus (VDV), classical swine fever virus (CSFV, also known as hog cholera virus), and Border disease virus (DN) of sheep (Moennig et al., Adv. Vir. Res. 1992, 7:53-98). Pestiviras infections of domesticated livestock (i.e., cattle, pigs, and sheep) cause significant economic losses worldwide. VDN causes mucosal disease in cattle and is of significant economic importance to the livestock industry (Meyers, G. and Thiel, H-J., Adv. In Viral Res., 1996, ¥7:53-118; Moennig et al, Adv. Vir. Res. 1992, 41:53-98).

Human pestiviruses have not been as extensively characterized as animal pestiviruses. However, serological surveys indicate considerable pestivirus exposure in humans. Pestivirus infections in man have been implicated in several diseases including congenital rain injury, infantile gastroenteritis, and chronic diarrhea in human immunodeficiency viras (HIN) positive patients (M. Giangaspero et al., Arch. Virol. Suppl, 1993, 7:53-62; M. Giangaspero βt al, Int. J. Std. Aids, 1993, 4(5) .300-302).

The flavivirus genus includes more than 68 members that are separated into groups on the basis of serological relatedness (Calisher et al., J. Gen. Virol, 1993, 70:37-

43). Clinical symptoms vary and include fever, encephalitis and hemorrhagic fever {Fields Virology, Ed.: Fields, B.Ν., Knipe, D.M., and Howley, P.M.; Lippincott-Raven Publishers, Philadelphia, PA; 1996; Chapter 31, pp. 931-59). Flavivirases of global concern that are associated with human disease include the dengue hemorrhagic fever viras (DHF or DENN), yellow fever viras (YFV), West Nile virus (WNN), shock syndrome and Japanese encephalitis viras (S.B. Halstead, Rev. Infect. Dis., 1984, 6:251- 64; S.B. Halstead, Science, 1988, 239:476-81; T.P. Monath, New Engl. J. Med., 1988, 379:641-3).

Hepatitis C viras (HCV) is the leading cause of chronic liver disease worldwide (Ν. Boyer et al., J. Hepatol. 2000, 32:98-112). HCV causes a slow-growing viral infection and is the major cause of cirrhosis and hepatocellular carcinoma (DiBesceglie, A.M. and B.R. Bacon, Scientific American, 1999, Oc/.:80-85; Ν. Boyer et al., J. Hepatol. 2000, 32:98-112). An estimated 170 million people are infected with HCV worldwide (Ν. Boyer et al., J. Hepatol. 2000, 32:98-112). Cirrhosis caused by chronic HCV infection accounts for 8-12,000 deaths per year in the United States, and HCV infection is the leading indication for liver transplant.

HCV is known to cause at least 80% of post-transfusion hepatitis and a substantial proportion of sporadic acute hepatitis. Preliminary evidence implicates HCV in many cases of "idiopathic" chronic hepatitis, "cryptogenic" cirrhosis, and probably hepatocellular carcinoma unrelated to other hepatitis viruses. A small proportion of healthy persons appear to be chronic HCV carriers, but this varies geographically and epidemiologically. The numbers may substantially exceed those for HBV although this information is still preliminary, and it is still unclear how many of these people have subclinical chronic liver disease {The Merck Manual, 1992, 16^th Ed., Chpt. 69, p. 901). HCV is classified as a member of the Flaviviridae family of viruses that includes the genera flavivirases, pestiviruses and hapacivirases, which include hepatitis C viruses (CM. Rice, "Flaviviridae: The viruses and their replication," Fields Virology, B.N. Fields, D.M. Knipe and P.M. Howley, Editors; 1996, Lippincott-Raven Publishers, Philadelphia, PA; Chpt. 30, pp. 931-59). HCV is an enveloped virus containing a positive-sense single-stranded RNA genome of approximately 9.4 k. The viral genome consists of a 5 '-untranslated region (UTR), a long open reading frame (ORF) encoding a polyprotein precursor of approximately 3011 amino acids, and a short 3' -UTR. The 5'- UTR is the most highly conserved part of the HCV genome and is important for the initiation and control of polyprotein translation. Translation of the HCV genome is initiated by a cap-independent mechanism known as internal ribosome entry. This mechanism involves the binding of ribosomes to an RNA sequence known as the internal ribosome entry site (IRES). An RNA pseudoknot structure has recently been determined to be an essential structural element of the HCV IRES. Viral structural proteins include a nucleocapsid core protein (C) and two envelope glycoproteins, El and E2. HCV also encodes two proteinases, a zinc-dependent metalloproteinase encoded by the NS2-NS3 region, and a serine proteinase encoded in the NS3 region. These proteinases are required for cleavage of specific regions of the precursor polyprotein into mature peptides. The carboxyl half of nonstractural protein 5, NS5, contains the RNA- dependent RNA polymerase. The function(s) of the remaining non-structural proteins, NS4A, NS4, and NS5A (the amino terminal half of non-structural protein 5) are the subjects of ongoing studies. The non-structural protein NS4A appears to be a serine protease (Hsu et al., Nat. Biotechnol, April 23, 2003; [retrieved on April 23, 2003]; retrieved from Entrez PubMed, Internet URL: http://www.ncbi.nlm.nih.gov/EntrezΛ, while studies on NS4 suggest its involvement in translational inhibition and consequent degradation of host cellular proteins (Forese et al., Virus Res., Dec. 2002, 90(1-2) .T19- 31). The non-structural protein NS5A has been shown to inhibit p53 activity on a p21 promoter region via its ability to bind to a specific DNA sequence, thereby blocking p53 activity (Gong et al., Zonghua Gan Zang Bing Za Zhi, March 2003, 11(3): 162-5). Both NS3 and NS5A have been shown to be involved with host cellular signaling transduction pathways (Giannini et al., Cell Death Diff, Jan. 2003, 10 Suppl. 7.S27-28). Examples of antiviral agents that have been identified as active against the

Flaviviridae family of viruses include: (1) Interferon

Interferons (IFNs) are compounds that have been commercially available for the treatment of chronic hepatitis for nearly a decade. IFNs are glycoproteins produced by immune cells in response to viral infection. IFNs inhibit viral replication of many viruses, including HCV, and when used as the sole treatment for hepatitis C infection,

IFN suppresses serum HCV-RNA to undetectable levels. Additionally, IFN normalizes serum amino transferase levels. Unfortunately, the effects of IFN are temporary and a sustained response occurs in only 8%-9% of patients chronically infected with HCV (Gary L. Davis. Gastroenterology 118:S104-S114, 2000). A number of patents disclose HCV treatments using interferon-based therapies.

For example, U.S. Patent No. 5,980,884 to Blatt et al. discloses methods for retreatment of patients afflicted with HCV using consensus interferon. U.S. Patent No. 5,942,223 to Bazer et al. discloses an anti-HCV therapy using ovine or bovine interferon-tau. U.S. Patent No. 5,928,636 to Alber et al. discloses the combination therapy of interleukin-12 and interferon alpha for the treatment of infectious diseases including HCV. U.S. Patent

No. 5,908,621 to Glue et al. discloses the use of polyethylene glycol modified interferon for the treatment of HCV. U.S. Patent No. 5,849,696 to Chretien et al. discloses the use of thymosins, alone or in combination with interferon, for treating HCV. U.S. Patent No. 5,830,455 to Valtuena et al. discloses a combination HCV therapy employing interferon and a free radical scavenger. U.S. Patent No. 5,738,845 to Imakawa discloses the use of human interferon tau proteins for treating HCV. Other interferon-based treatments for HCV are disclosed in U.S. Patent No. 5,676,942 to Testa et al., U.S. Patent No. 5,372,808 to Blatt et al., and U.S. Patent No. 5,849,696.

(2) Ribavirin (Battaglia, A.M. et al., Ann. Pharmacother, 2000,. 34, 487-494);

Berenguer, M. et al. Antivir. Ther., 1998, 3 (Suppl. 3), 125-136).

Ribavirin (l-β-D-ribofuranosyl-l-l,2,4-triazole-3-carboxamide) is a synthetic, non-interferon-inducing, broad spectrum antiviral nucleoside analog. It is sold under the trade names Virazole™ (The Merck Index, 11th edition, Editor: Budavari, S., Merck & Co., Inc., Rahway, NJ, pi 304, 1989); Rebetol (Sobering Plough) and Co-Pegasus

(Roche). United States Patent No. 3,798,209 and RE29,835 (ICN Pharmaceuticals) disclose and claim ribavirin. Ribavirin is structurally similar to guanosine, and has in vitro activity against several DNA and RNA viruses including Flaviviridae (Gary L. Davis. Gastroenterology 118:S104-S114, 2000). U.S. Patent No 4,211,771 (to ICN Pharmaceuticals) discloses the use of ribavirin as an antiviral agent. Ribavirin reduces serum amino transferase levels to normal in 40% of patients, but it does not lower serum levels of HCV-RNA (Gary L. Davis. Gastroenterology 118:S104-S114, 2000). Thus, ribavirin alone is not effective in reducing viral RNA levels. Additionally, ribavirin has significant toxicity and is known to induce anemia.

Combination of Interferon and Ribavirin

Schering-Plough sells ribavirin as Rebetol® capsules (200 mg) for administration to patients with HCV. The U.S. FDA has approved Rebetol capsules to treat chronic HCV infection in combination with Schering's alpha interferon-2b products Intron® A and PEG-Intron™. Rebetol capsules are not approved for monotherapy (i.e., administration independent of Intron® A or PEG-Intron), although Intron A and PEG-

Intron are approved for monotherapy (i.e., administration without ribavirin). Hoffman La Roche is selling ribavirin under the name Co-Pegasus in Europe and the United States, also for use in combination with interferon for the treatment of HCV. Other alpha interferon products include Roferon-A (Hoffmann-La Roche), Infergen® (Intermune, formerly Amgen's product), and Wellferon® (Wellcome Foundation) are currently FDA- approved for HCV monotherapy. Interferon products currently in development for HCV include: Roferon-A (interferon alfa-2a) by Roche, PEGASYS (pegylated interferon alfa- 2a) by Roche, INFERGEN (interferon alfacon-1) by InterMune, OMNIFERON (natural interferon) by Viragen, ALBUFERON by Human Genome Sciences, REBIF (interferon beta-la) by Ares-Serono, Omega Interferon by BioMedicine, Oral Interferon Alpha by

Amarillo Biosciences, and Interferon gamma-lb by InterMune.

The combination of IFN and ribavirin for the treatment of HCV infection has been reported to be effective in the treatment of IFN naϊve patients (for example,

Battaglia, A.M. et al., Ann. Pharmacother. 34:487-494, 2000). Combination treatment is effective both before hepatitis develops and when histological disease is present (for example, Berenguer, M. et al. Antivir. Ther. 3(Suppl. 3): 125-136, 1998). Currently, the most effective therapy for HCV is combination therapy of pegylated interferon with ribavirin (2002 NIH Consensus Development Conference on the Management of Hepatitis C). However, the side effects of combination therapy can be significant and include hemolysis, flu-like symptoms, anemia, and fatigue (Gary L. Davis. Gastroenterology 118:S104-S114, 2000).

(3) Substrate-based NS3 protease inhibitors (for example, Attwood et al, Antiviral peptide derivatives, PCT WO 98/22496, 1998; Attwood et al, Antiviral Chemistry and Chemotherapy 1999, 10, 259-273; Attwood et al, Preparation and use of amino acid derivatives as anti-viral agents, German Patent Pub. DE 19914474; Tung et al.

Inhibitors of serine proteases, particularly hepatitis C virus NS3 protease, PCT WO 98/17679), including alphaketoamides and hydrazinoureas, and inhibitors that terminate in an electrophile such as a boronic acid or phosphonate (for example, Llinas-Brunet et al, Hepatitis C inhibitor peptide analogues, PCT WO 99/07734). (4) Non-substrate-based inhibitors such as 2,4,6-trihydroxy-3-nitro-benzamide derivatives (for example, Sudo K. et al, Biochemical and Biophysical Research Communications, 1997, 238, 643-647; Sudo K. et al. Antiviral Chemisttγ and Chemotherapy, 1998, 9, 186), including RD3-4082 and RD3-4078, the former substituted on the amide with a 14 carbon chain and the latter processing a para- phenoxyphenyl group;

(5) Thiazolidine derivatives which show relevant inhibition in a reverse-phase HPLC assay with an NS3/4A fusion protein and NS5A/5B substrate (for example, Sudo K. et al, Antiviral Research, 1996, 32, 9-18), especially compound RD-1-6250, possessing a fused cinnamoyl moiety substituted with a long alkyl chain, RD4 6205 and RD4 6193; (6) Thiazolidines and benzanilides for example, as identified in Kakiuchi N. et al. J.

EBS Letters 421, 217-220; Takeshita N. et al. Analytical Biochemistry, 1997, 247, 242- 246;

(7) A phenan-threnequinone possessing activity against protease in a SDS-PAGE and autoradiography assay isolated from the fermentation culture broth of Streptomyces sp., Sch 68631 (for example, Chu M. et al, Tetrahedron Letters, 1996, 37, 7229-7232), and

Sch 351633, isolated from the fungus Penicillium griscofuluum, which demonstrates activity in a scintillation proximity assay (for example, Chu M. et al, Bioorganic and Medicinal Chemistry Letters 9, 1949-1952);

(8) Selective NS3 inhibitors based on the macromolecule elgin c, isolated from leech (for example, Qasim MA. et al, Biochemistry, 1997, 36, 1598-1607); (9) Helicase inhibitors (for example, Diana G.D. et al, Compounds, compositions and methods for treatment of hepatitis C, U.S. Pat. No. 5,633,358; Diana G.D. et al, Piperidine derivatives, pharmaceutical compositions thereof and their use in the treatment of hepatitis C, PCT WO 97/36554);

(10) Polymerase inhibitors such as nucleotide analogues, gliotoxin (for example, Ferrari R. et al. Journal of Virology, 1999, 73, 1649-1654), and the natural product ceralenin (for example, Lohmann V. et al, Virology, 1998, 249, 108-118);

(11) Antisense phosphorothioate oligodeoxynucleotides (S-ODN) complementary to sequence stretches in the 5' non-coding region (NCR) of the virus (for example, Alt M. et al, Hepatology, 1995, 22, 707-717), or nucleotides 326-348 comprising the 3' end of the NCR and nucleotides 371-388 located in the core coding region of the HCV RNA

(for example, Alt M. et al, Archives of Virology, 1997, 142, 589-599; Galderisi U. et al, Journal of Cellular Physiology, 1999, 757, 251-257).

(12) Inhibitors of IRES-dependent translation (for example, Ikeda N et al, Agent for the prevention and treatment of hepatitis C, Japanese Patent Pub. JP-08268890; Kai Y. et al. Prevention and treatment of viral diseases, Japanese Patent Pub . JP- 10101591).

(13) Nuclease-resistant ribozymes (for example, Maccjak, D. J. et al, Hepatology 1999, 30, abstract 995).

(14) Nucleoside analogs have also been developed for the treatment of Flaviviridae infections. Idenix Pharmaceuticals, Ltd. discloses branched nucleosides, and their use in the treatment of HCV and flavivirases and pestiviruses in US Patent Publication No. 2003/0050229 Al and US Patent Publication No. 2003/0060400 Al, which correspond to International Publication Nos. WO 01/90121 and WO 01/92282. A method for the treatment of hepatitis C infection (and flavivirases and pestiviruses) in humans and other host animals is disclosed in the Idenix publications that includes administering an effective amount of a biologically active 1', 2', 3' or 4'-branched β-D or β-L nucleosides or a pharmaceutically acceptable salt or prodrag thereof, administered either alone or in combination, optionally in a pharmaceutically acceptable carrier. See also U.S. Patent Publication Nos. 2004/0006002 and 2004/0006007 as well as WO 03/026589 and WO 03/026675. Idenix Pharmaceuticals, Ltd. also discloses in US Patent Publication No.

2004/0077587 pharmaceutically acceptable branched nucleoside prodrags, and their use in the treatment of HCV and flavivirases and pestivirases in prodrags. See also PCT Publication Nos. WO 04/002422, WO 04/002999, and WO 04/003000.

Emory University and the University of Georgia Research Foundation, Inc. (UGARF) discloses the use of 2'-fluoronucleosides for the treatment of HCV in US

Patent No. 6,348,587. See also International Patent Publication WO 99/43691.

BioChem Pharma Inc. (now Shire Biochem, Inc.) discloses the use of various 1,3-dioxolane nucleosides for the treatment of a Flaviviridae infection in International Publication No. WO 01/32153 (PCT/CAOO/01316; filed November 3, 2000). BioChem Pharma Inc. (now Shire Biochem, Inc.) also discloses various other 2'- halo, 2'-hydroxy and 2'-alkoxy nucleosides for the treatment of a Flaviviridae infection in International Publication No. WO 01/60315 (PCT/CA01/00197; filed February 19, 2001).

ICN Pharmaceuticals, Inc. discloses various nucleoside analogs that are useful in modulating immune response in US Patent Nos. 6,495,677 and 6,573,248. See also WO

98/16184, WO 01/68663, and WO 02/03997.

US Patent Publication Nos. 2003/083307 Al and US 2003/008841 Al, and the corresponding International Patent Publication Nos. WO 02/18404 (PCT/EP01/09633; published August 21, 2001); WO 02/100415 and WO 02/094289, filed by F. Hoffmann- La Roche AG discloses various nucleoside analogs for the treatment of HCV RNA replication.

Pharmasset Limited discloses various nucleosides and antimetabolites for the treatment of a variety of viruses, including Flaviviridae, and in particular HCV, in WO 02/32920, WO 01/79246, WO 02/48165, WO 03/068162, WO 03/068164 and 2004/013298. Merck & Co., Inc. and Isis Pharmaceuticals disclose in US Patent Publication No. 2002/0147160 and the corresponding International Patent Publication Nos. WO 02/057425 (PCT/US02/01531; filed January 18, 2002) and WO 02/057287 (PCT/US02/03086; filed January 18, 2002) various nucleosides, and in particular several pyrrolopyrimidine nucleosides, for the treatment of virases whose replication is dependent upon RNA-dependent RNA polymerase, including Flaviviridae, and in particular HCV. See also WO 2004/003138, WO 2004/007512, and WO 2004/009020.

US Patent Publication No. 2003/028013 Al as well as International Patent Publication Nos. WO 03/051899, WO 03/061576, WO 03/062255 WO 03/062256, WO 03/062257, and WO 03/061385, filed by Ribapharm, also are directed to the use of certain nucleoside analogs to treat hepatitis C viras.

(15) Other miscellaneous compounds including 1-amino-alkylcyclohexanes (for example, U.S. Patent No. 6,034,134 to Gold et al), alkyl lipids (for example, U.S. Pat. No. 5,922,757 to Chojkier et al), vitamin E and other antioxidants (for example, U.S. Pat. No. 5,922,757 to Chojkier et al), squalene, amantadine, bile acids (for example,

U.S. Pat. No. 5,846,964 to Ozeki et al), N-(phosphonoacetyl)-L-aspartic acid (for example, U.S. Pat. No. 5,830,905 to Diana et al), benzenedicarboxamides (for example, U.S. Pat. No. 5,633,388 to Diana et al), polyadenylic acid derivatives (for example, U.S. Pat. No. 5,496,546 to Wang et al), 2',3'-dideoxyinosine (for example, U.S. Pat. No. 5,026,687 to Yarchoan et al), and benzimidazoles (for example, U.S. Pat. No. 5,891,874 to Colacino et α/.).

(16) Other compounds currently in clinical development for treatment of hepatitis c viras include, for example: Interleukin-10 by Schering-Plough, IP-501 by Intemeuron, Merimebodib VX-497 by Vertex, AMANTADINE (Symmetrel) by Endo Labs Solvay, HEPTAZYME by RPI, IDN-6556 by Idun Pharma., XTL-002 by XTL., HCV/MF59 by

Chiron, CIVACIR by NABI, LEVOVIRIN by ICN, VIRAMIDINE by ICN, ZADAXIN (thymosin alfa-1) by Sci Clone, CEPLENE (histamine dihydrochloride) by Maxim, VX 950 / LY 570310 by Vertex Eli Lilly, ISIS 14803 by Isis Pharmaceutical/Elan, IDN-6556 by Idun Pharmaceuticals, Inc. and JTK 003 by AKROS Pharma. It has been recognized that drag-resistant variants of virases can emerge after prolonged treatment with an antiviral agent. Drug resistance most typically occurs by mutation of a gene that encodes for an enzyme used in viral replication, and, for example, in the case of HIV, reverse transcriptase, protease, or DNA polymerase. It has been demonstrated that the efficacy of a drag against viral infection can be prolonged, augmented, or restored by administering the compound in combination or alternation with a second, and perhaps third, antiviral compound that induces a different mutation from that caused by the principle drug. Alternatively, the pharmacokinetics, biodistribution, or other parameter of the drug can be altered by such combination or alternation therapy. In general, combination therapy is typically preferred over alternation therapy because it induces multiple simultaneous pressures on the viras. One cannot predict, however, what mutations will be induced in the viral genome by a given drug, whether the mutation is permanent or transient, or how an infected cell with a mutated viral sequence will respond to therapy with other agents in combination or alternation. This is exacerbated by the fact that there is a paucity of data on the kinetics of drag resistance in long-term cell cultures treated with modern antiviral agents.

In view of the severity of diseases associated with pestivirases, flavivirases, and hepatitis C viras, and their pervasiveness in animals and humans, it is an object of the present invention to provide a compound, method and composition for the treatment of a host infected with any member of the family Flaviviridae, including hepatitis C viras.

Thus, it is another object of the present invention to provide a method and pharmaceutically-acceptable composition for the prophylaxis and treatment of a host, and particularly a human, infected with any member of the family Flaviviridae.

It is still another object of the invention to provide nucleoside derivative compounds having optionally substituted 2-azapurine base members and congeners thereof, or a physiologically acceptable salt, ester or prodrag thereof, for the manufacture of a medicament to be used in the prophylaxis or treatment of a host infected with a pestivirus, flavivirus or hepatitis C viras. SUMMARY OF THE INVENTION

Methods and compositions for the treatment of pestiviras, flavivirus and hepatitis C viras infections are described that include administering an effective amount of a beta- D or beta-L-nucleoside of Formula (I), or an ester of Formula (II), or a pharmaceutically acceptable salt or prodrug thereof.

In a first principal embodiment, a compound of Formula (I), or a pharmaceutically acceptable salt or prodrug thereof, is provided:

(I) wherein: R¹ is OH; phosphate or phosphonate (including mono-, di-, or triphosphate or a stabilized phosphate prodrug); acyl (including lower acyl); O-acyl; H; alkyl; O-alkyl (including lower alkyl); sulfonate ester including alkyl or arylalkyl sulfonyl including methanesulfonyl and benzyl, wherein the phenyl group is optionally substituted with one or more substituents as described in the definition of an aiyl given herein; optionally substituted arylsulfonyl; lipid, including a phospholipid; amino acid; carbohydrate; peptide; cholesterol; any of which may be O-linked to the furanyl ring; or another pharmaceutically acceptable leaving group that, in vivo, provides a compound wherein R¹ is independently OH or O-phosphate;

Each R² and R³ independently is H or OH; and

Base is:

wherein:

Z is H, OH, SH, NH₂, halo, CF₃, C_M alkyl, C alkylamino, di(Cι. alkyl)amino, C_3-6 cycloalkylamino, or C_1- alkoxy, or

Y is O, S, orNR⁴; and

R⁴ is independently is hydrogen, optionally substituted or unsubstituted lower alkyl, lower haloalkyl, optionally substituted or unsubstituted lower alkenyl, lower haloalkenyl, optionally substituted or unsubstituted aryl, arylalkyl such as unsubstituted or substituted phenyl or benzyl, or an optionally substituted or unsubstituted acyl; and all tautomeric, enantiomeric and diastereomeric forms thereof.

In a second principal embodiment, an ester of Formula II, or a pharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:

Base is as defined above for formula (i); and all tautomeric forms thereof; and in alternative embodiment, is any purine or pyrimidine base. The active compounds of the present invention can be administered in combination, alternation or sequential steps with another anti-HCV agent. In combination therapy, effective dosages of two or more agents are administered together, whereas in alternation or sequential-step therapy, an effective dosage of each agent is administered serially or sequentially. The dosages given will depend on absorption, inactivation and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. In preferred embodiments, an anti-HCV (anti- pestiviras or anti-flaviviras) compound that exhibits an EC₅₀ of 10-15 μM, or preferably less than 1-5 μM, is desirable. HCV is a member of the family, Flaviviridae; however, HCV now has been placed in a new monotypic genus, hepacivirus. Therefore, in one embodiment, the flavivirus or pestivirus is not HCV.

In particular, the present invention provides the following: a) a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of Formula (II) , or a pharmaceutically acceptable salt or prodrug thereof; b) a pharmaceutical composition comprising a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of Formula (II), or a pharmaceutically acceptable salt or prodrug thereof, together with a pharmaceutically acceptable carrier, excipient or diluent; c) a pharmaceutical composition comprising a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of Formula (II), or a pharmaceutically acceptable salt or prodrug thereof, with one or more other effective antiviral agents, optionally with a pharmaceutically acceptable carrier or diluent; d) a pharmaceutical composition for the treatment or prophylaxis of a pestivirus, flavivirus or HCV infection in a host, especially a host diagnosed as having or being at risk for such infection, comprising a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of Formula (II), or a pharmaceutically acceptable salt or prodrug thereof, together with a pharmaceutically acceptable carrier or diluent; e) a pharmaceutical formulation comprising the beta-D- or beta-L-nucleoside compound of Formula (I), an ester of Formula (II), or a pharmaceutically acceptable salt or prodrug thereof, together with a pharmaceutically acceptable carrier, excipient or diluent; f) a method for the treatment of a pestivirus, flaviviras or HCV infection in a host comprising a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of Formula (II), or a pharmaceutically acceptable salt or prodrug thereof, optionally with a pharmaceutically acceptable carrier, excipient or diluent; g) a method for the treatment of a pestivirus, flavivirus or HCV infection in a host comprising a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of Formula (II), or a pharmaceutically acceptable salt or prodrug thereof, with one or more other effective antiviral agents, optionally with a pharmaceutically acceptable carrier, excipient or diluent; h) a method for the treatment of a pestivirus, flaviviras or HCV infection in a host comprising a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of

Formula (II), or a pharmaceutically acceptable salt or prodrug thereof, with one or more other effective antiviral agents, optionally with a pharmaceutically acceptable carrier, excipient or diluent; i) a method for the treatment of a pestivirus, flaviviras or HCV infection in a host comprising a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of

Formula (II), or a pharmaceutically acceptable salt or prodrug thereof, with one or more other effective antiviral agents, optionally with a pharmaceutically acceptable carrier, excipient or diluent; j) a method for the treatment of a pestivirus, flaviviras or HCV infection in a host comprising a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of

Formula (II), or a pharmaceutically acceptable salt or prodrug thereof, with one or more other effective antiviral agents, optionally with a pharmaceutically acceptable carrier, excipient or diluent; k) use of a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of Formula (II), or a pharmaceutically acceptable salt or prodrug thereof, optionally with a pharmaceutically acceptable carrier or diluent, for the treatment of a pestivirus, flavivirus or HCV infection in a host; 1) use of a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of Formula (II), or a pharmaceutically acceptable salt or prodrug thereof, with one or more other effective antiviral agents, optionally with a pharmaceutically acceptable carrier or diluent, for the treatment of a pestiviras, flaviviras and/or HCV infection in a host; m) use of a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of Formula

(II), or a pharmaceutically acceptable salt or prodrug thereof, optionally with a pharmaceutically acceptable carrier or diluent, in the manufacture of a medicament for the treatment of a pestivirus, flavivirus and/or HCV infection in a host; n) use of a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of Formula (II), or pharmaceutically acceptable salt or prodrug thereof, with one or more other effective antiviral agents and optionally with a pharmaceutically acceptable carrier, excipient or diluent, in the manufacture of a medicament for the treatment of a pestiviras, flaviviras and/or HCV infection in a host; o) a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of Formula (II), or a pharmaceutically acceptable salt or prodrug thereof, substantially in the absence of enantiomers of the described nucleoside, or substantially isolated from other chemical entities; p) a process for the preparation of a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of Formula (II), or a its pharmaceutically acceptable salt or prodrug thereof, as provided in more detail below; and q) a process for the preparation of a beta-D- or beta-L-nucleoside compound of Formula (I), an ester of Formula (II), or a pharmaceutically acceptable salt or prodrug thereof, substantially in the absence of enantiomers of the described nucleoside or substantially isolated from other chemical entities.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound, method and composition for the treatment of a pestivirus, flaviviras and/or hepatitis C in humans or other host animals that includes administering an effective anti-pestiviras, anti-flaviviras or anti-HCV treatment amount of a beta-D- or beta-L-nucleoside of Formula (I) or an ester of Formula (II), as described herein, or a pharmaceutically acceptable salt or prodrug thereof, optionally in a pharmaceutically acceptable carrier. The compounds of this invention either possess antiviral activity, or are metabolized to a compound that exhibits such activity.

Flavivirases included within the scope of this invention are discussed generally in Fields Virology, Editors: Fields, .N, Rnipe, D.M. and Howley, P.M.; Lippincott-Raven Pulishers, Philadelphia, PA; Chapter 31 (1996). Specific flavivirases include, without limitation: Absettarov; Alfuy; Apoi; Aroa; Bagaza; Banzi; Bououi; Bussuquara;

Cacipacore; Carey Island; Dakar bat; Dengue virases 1, 2, 3 and 4; Edge Hill; Entebbe bat; Gadgets Gully; Hanzalova; Hypr; Ilheus; Israel turkey meningoencephalitis; Japanese encephalitis; Jugra; Jutiapa; Kadam; Karshi; Kedougou; Kokoera; Koutango; Kumlinge; Kunjin; Kyasanur Forest disease; Langat; Louping ill; Meaban; Modoc; Montana myotis leukoencephalitis; Murray valley encephalitis; Naranjal; Negishi;

Ntaya; Omsk hemorrhagic fever; Phnom-Penh bat; Powassan; Rio Bravo; Rocio; Royal Farm; Russian spring-summer encephalitis; Saboya; St. Louis encephalitis; Sal Vieja; San Perlita; Saumarez Reef; Sepik; Sokuluk; Spondweni; Stratford; Temusu; Tyuleniy; Uganda S, Usutu, Wesselsbron; West Nile; Yaounde; Yellow fever; and Zika. Pestivirases included within the scope of this invention are also discussed generally in Fields Virology (Id). Specific pestiviruses include, without limitation: bovine viral diarrhea virus ("VDV"); classical swine fever viras ("CSFV") also lαiown as hog cholera viras); and border disease viras ("DV").

Active Compounds, Physiologically Acceptable Salts and Prodrugs Thereof

Methods and compositions for the treatment of pestiviras, flavivirus and hepatitis

C viras infection are described that include administering an effective amount of a beta- D or beta-L-nucleoside of the Formulae (I), an ester of Formula (II), or a pharmaceutically acceptable salt or prodrug thereof. In a first principal embodiment, a compound of Formula (I), or a pharmaceutically acceptable salt or prodrug thereof, is provided:

wherein: R¹ is OH, phosphate or phosphonate (including mono-, di-, or triphosphate or a stabilized phosphate prodrug); acyl (including lower acyl); H; alkyl (including lower alkyl); sulfonate ester including alkyl or arylalkyl sulfonyl including methanesulfonyl and benzyl, wherein the phenyl group is optionally substituted with one or more substituents as described in the definition of an aryl given herein; optionally substituted arylsulfonyl; a lipid, including a phospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; any of which may be O-linked to the furanyl ring; or another pharmaceutically acceptable leaving group that when administered in vivo, provides a compound wherein R¹ is independently OH or O-phosphate; each R and R independently is H or OH; and Base is:

wherein:

Z is H, OH, SH, NH₂, halo, CF₃, C_1-4 alkyl, Cι_-4 alkylamino, di(Cι_-4 alkyl)amino, C_3-6 cycloalkylamino, or Cμ alkoxy Y is O, S, orNR⁴; and

wherein,

Base is as defined above for formula (i); and all tautomeric forms thereof.

In all embodiments, any optional substituents may be selected that do not adversely affect the properties of the molecule, and for example, may be selected from the group consisting of one or more halogen, amino, hydroxy, carboxy and alkoxy groups or atoms, among others. It is to be understood that all stereoisomeric and tautomeric forms of the compounds shown are included herein.

The active compounds of the present invention can be administered in combination, alternation or sequential steps with another anti-HCV agent. In combination therapy, effective dosages of two or more agents are administered together, whereas in alternation or sequential-step therapy, an effective dosage of each agent is administered serially or sequentially. The dosages given will depend on absorption, inactivation and excretion rates of the drug as well as other factors lαiown to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. In preferred embodiments, an anti-HCV (anti- pestivirus or anti-flaviviras) compound that exhibits an EC50 of 10-15 μM, or preferably less than 1-5 μM, is desirable.

The active compound can be administered as any salt or prodrug that upon administration to the recipient is capable of providing directly or indirectly the parent compound, or that exhibits activity itself. Nonlimiting examples are the pharmaceutically acceptable salts, which are alternatively referred to as "physiologically acceptable salts", and a compound that has been alkylated or acylated at the 5 '-position or on the purine or pyrimidine base, thereby forming a type of "phannaceutically acceptable prodrug". Further, the modifications can affect the biological activity of the compound, in some cases increasing the activity over the parent compound. This can easily be assessed by preparing the salt or prodrug and testing its antiviral activity according to the methods described herein, or other methods lαiown to those skilled in the art.

Definitions

The term "alkyl" as used herein, unless otherwise specified, includes a saturated straight, branched, or cyclic, primary, secondary, or tertiary hydrocarbon of typically Ci to do, and specifically includes methyl, trifluoromethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethybutyl, and 2,3- dimethylbutyl. The term includes both substituted and unsubstituted alkyl groups.

Moieties with which the alkyl group can be substituted with one or more substituents include but are not limited to halo, including Cl, F, Br and I so as to form, for eg., CF₃, 2- Br-ethyl, CH₂F, CH₂C1, CH₂CF₃, or CF₂CF₃; hydroxyl, for eg. CH₂OH; amino, for eg., CH₂NH₂, CH₂NHCH₃, or CH₂N(CH₃)₂; carboxylate; carboxamido; alkylamino; arylamino; alkoxy; aryloxy; nitro; azido, for eg., CH₂N₃; cyano, for eg., CH₂CN; thio; sulfonic acid; sulfate; phosphonic acid; phosphate; and phosphonate, either unprotected or protected as necessary, known to those skilled in the art, for eg., as taught in Greene et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition (1991), incorporated herein by reference. The term "lower alkyl" as used herein, and unless otherwise specified, includes a Ci to C₆ saturated straight, branched, or if appropriate, cyclic as in cyclopropyl, for eg., alkyl group, including both substituted and unsubstituted forms. Unless otherwise specifically stated in this application, when alkyl is a suitable moiety, lower alkyl is preferred. Similarly, when alkyl or lower alkyl is a suitable moiety, unsubstituted alkyl or lower alkyl is preferred.

The terms "alkylamino" and "arylamino" refer to an amino group that has one or two alkyl or aryl substituents, respectively.

The term "protected" as used herein and, unless otherwise defined, includes a group that is added to an oxygen, nitrogen or phosphorus atom to prevent its further reaction or for other purposes. Numerous oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis.

The term "aryl" as used herein and, unless otherwise specified, includes phenyl, biphenyl or naphthyl, and preferably phenyl. The term includes both substituted and unsubstituted moieties. The aryl group can be substituted with one or more moieties including but not limited to alkyl, hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, thio, alkylthio, carboxamido, carboxylate, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected or protected as necessary, as known to those skilled in the art, for eg., as taught in Greene et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition (1991), incorporated herein by reference.

The terms "alkaryl" and "akylaryl" refer to an alkyl group with an aryl sustituent. The terms "aralkyl" and "arylalkyl" refer to an aryl group with an alkyl substituent.

The term "halo" as used herein includes bromo, chloro, iodo and fluoro. The term purine or pyrimidine base includes, but is not limited to, adenine, N - alkylpurines, N⁶-acylpurines (wherein acyl is C(0)(alkyl, aryl, alkylaryl, or arylalkyl), N -benzylpurine, N -halopurine, N -vinylpurine, N -acetylenic purine, N -acyl purine, N⁶-hydroxyalkyl purine, N⁶-tlιioal__yl purine, N²-al__ylpurines, N²-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4-mercaptopyrmidine, uracil, 5-halouracil, including 5-fluoro- uracil, C⁵-alkylpyrimidines, C⁵-benzylpyrimidines, C⁵-halopyrimidines, C⁵-vinyl- pyrimidine, C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine, C⁵-hydroxyalkyl purine, C⁵- amidopyrimidine, C⁵-cyanopyrimidine, C⁵-nitiOpyrimidine, C⁵-aminopyrimidine, N²- alkylpurines, N -alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl, 5-aza-7-deazapurinyl, triazolopyridinyl, imidazolopyridinyl, pyrTolopyrimidinyl, and pyrazolopyrimidinyl. The term "acyl" includes a carboxylic acid ester in which the non-carbonyl moiety of the ester group is selected from straight, branched, or cyclic alkyl or lower alkyl; alkoxyalkyl including methoxymethyl; aralkyl including benzyl; aryloxyalkyl such as phenoxymethyl; aryl including phenyl optionally substituted with halogen, d-C₆ alkyl or Ci-Cβ alkoxy; sulfonate esters such as alkyl or aralkyl sulphonyl including methanesulfonyl; the mono-, di- or triphosphate ester; trityl or monomethoxytrityl; substituted benzyl; trialkylsilyl as, for eg., dimethyl-t-butylsilyl or diphenylmethylsilyl. Aryl groups in the esters optimally comprise a phenyl group. The term "lower acyl" includes an acyl group in which the non-carbonyl moiety is lower alkyl.

As used herein, the terms "substantially free of and "substantially in the absence of refer to a nucleoside composition that includes at least 85 or 90% by weight, preferably at least 95% or 98% by weight, and even more preferably at least 99% or 100%) by weight, of the designated enantiomer of that nucleoside. In a preferred embodiment, the compounds listed in the methods and compounds of this invention are substantially free of enantiomers other than for the one designated. Similarly, the term "isolated" refers to a nucleoside composition that includes at least 85% or 90%> by weight, preferably 95%> or98%> y weight, and even more preferably 99% or 100% by weight, of the nucleoside.

The term "host", as used herein, refers to a unicellular or multicellular organism in which the viras can replicate, including cell lines and animals, and preferably a human. Alternatively, the host can be carrying a part of the flaviviras or pestivirus genome, whose replication or function can be altered by the compounds of the present invention. The term host specifically refers to infected cells, cells transfected with all or part of the flaviviras or pestiviras genome and animals, in particular, primates (including chimpanzees) and humans. In most animal applications of the present invention, the host is a human patient. Veterinary applications, in certain indications, however, are clearly anticipated by the present invention such as in chimpanzees. The term "pharmaceutically acceptable salt or prodrug" is used throughout the specification to describe any pharmaceutically acceptable form (ester, phosphate ester, salt of an ester or a related group) of a nucleoside compound, which, upon administration to a patient, provides the nucleoside compound. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art. Phannaceutically acceptable prodrags refer to a compound that is metabolized, for example, hydrolyzed or oxidized, in the host to form the compound of the present invention. Typical examples of prodrags include compounds that have biologically labile protecting groups on a functional moiety of the active compound. Prodrags include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, dephosphorylated to produce the active compound. The compounds of this invention possess antiviral activity against flaviviras, pestiviras or HCV, or are metabolized to a compound that exhibits such activity.

Nucleotide Prodrug Formulations

Any of the nucleosides described herein can be administered as a nucleotide prodrug to increase the activity, bioavailability, stability or otherwise alter the properties of the nucleoside. A number of nucleotide prodrug ligands are known. In general, alkylation, acylation or other lipophilic modification of the mono-, di- or triphosphate of the nucleoside reduces polarity and allows passage into cells. Examples of 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. Many are described in R. Jones and N. Bischoferger, Antiviral Research, 1995, 27:1-17. Any of these can be used in combination with the disclosed nucleosides to achieve a desired effect.

In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compound as a pharmaceutically acceptable salt may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids, which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including, sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal

(for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

The active nucleoside can also be provided as a 5'-phosphoether lipid or a 5'- ether lipid, as disclosed in the following references, which are incorporated by reference herein: Kucera, L.S., N. Iyer, E. Leake, A. Raen, Modest E.K., D.L.W., and C. Piantadosi. 1990. "Novel membrane-interactive ether lipid analogs that inhibit infectious HIV-1 production and induce defective virus formation." AIDS Res. Hum.

Retro Viruses. 6:491-501; Piantadosi, C, J. Marasco C.J., S.L. Morris-Natschke, K.L. Meyer, F. Gumus, J.R. Surles, K.S. Ishaq, L.S. Kucera, N. Iyer, C.A. Wallen, S. Piantadosi, and E.J. Modest. 1991. "Synthesis and evaluation of novel ether lipid nucleoside conjugates for anti-HIN activity." J. Med. Chem. 34:1408.1414; Hosteller, K.Y., D.D. Richman, D.A. Carson, L.M. Stuhmiller, G.M. T. van Wijk, and H. van den

Bosch., 1992. "Greatly enhanced inhiition of human immunodeficiency virus type 1 replication in CEM and HT4-6C cells by 3'-deoxythymidine diphosphate dimyristoylglycerol, a lipid prodrug of 3-deoxythymidine." Antimicro. Agents Chemother. 36:2025.2029; Hosetler, K.Y., L.M. Stuhmiller, H.. Lenting, H. van den Bosch, and D.D. Richman, 1990. "Synthesis and antiretroviral activity of phospholipid analogs of azidothymidine and other antiviral nucleosides." J. Biol Chem. 265:61127.

Νonlimiting examples of U.S. patents that disclose suitable lipophilic substituents that can be covalently incorporated into the nucleoside, preferably at the 5' -OH position of the nucleoside or lipophilic preparations, include U.S. Patent Νos. 5,149,794 (Sep. 22, 1992, Yatvin et al); 5,194,654 (Mar. 16, 1993, Hosteller et al, 5,223,263 (June 29,

1993, Hostetler et al); 5,256,641 (Oct. 26, 1993, Yatvin et al); 5,411,947 (May 2, 1995, Hostetler et al); 5,463,092 (Oct. 31, 1995, Hostetler et al); 5,543,389 (Aug. 6, 1996, Yatvin et al); 5,543,390 (Aug. 6, 1996, Yatvin et al); 5,543,391 (Aug. 6, 1996, Yatvin et al); and 5,554,728 (Sep. 10, 1996; Basava et al), all of which are incorporated herein by reference. Foreign patent applications that disclose lipophilic substituents that can be attached to the nucleosides of the present invention, or lipophilic preparations, include WO 89/02733, W0 90/00555, W0 91/16920, W0 91/18914, W0 93/00910, W0 94/26273, W0 96/15132, EP 0 350 287, EP 93917054.4, and W0 91/19721.

Combination and Alternation Therapy

It has been recognized that drug-resistant variants of flavivirases, pestiviruses or HCN can emerge after prolonged treatment with an antiviral agent. Drag resistance most typically occurs by mutation of a gene that encodes for an enzyme used in viral replication. The efficacy of a drug against the viral infection can be prolonged, augmented, or restored by administering the compound in combination or alternation with a second, and perhaps third, antiviral compound that induces a different mutation from that caused by the principle drag. Alternatively, the pharmacokinetics, biodistriution or other parameter of the drug can be altered by such combination or alternation therapy. In general, combination therapy is typically preferred over alternation therapy because it induces multiple simultaneous stresses on the viras.

Any of the viral treatments described in the Background of the Invention can be used in combination or alternation with the compounds described in this specification. Νonlimiting examples include:

(1) Interferon

A number of patents disclose Flaviviridae, including HCV, treatments, using interferon-based therapies. For example, U.S. Patent No. 5,980,884 to Blatt et al. discloses methods for retreatment of patients afflicted with HCV using consensus interferon. U.S. Patent No. 5,942,223 to Bazer et al. discloses an anti-HCV therapy using ovine or bovine interferon-tau. U.S. Patent No. 5,928,636 to Alber et al. discloses the combination therapy of interleukin-12 and interferon alpha for the treatment of infectious diseases including HCV. U.S. Patent No. 5,849,696 to Chretien et al. discloses the use of thymosins, alone or in combination with interferon, for treating HCV. U.S. Patent No. 5,830,455 to Valtuena et al. discloses a combination HCN therapy employing interferon and a free radical scavenger. U.S. Patent No. 5,738,845 to Imakawa discloses the use of human interferon tau proteins for treating HCN. Other interferon-based treatments for HCV are disclosed in U.S. Patent No. 5,676,942 to Testa et al., U.S. Patent No. 5,372,808 to Blatt et al., and U.S. Patent No. 5,849,696. A number of patents also disclose pegylated forms of interferon, such as U.S. Patent Nos. 5,747,646, 5,792,834 and 5,834,594 to Hoffmann-La Roche Inc; PCT Publication No. WO 99/32139 and WO 99/32140 to Enzon; WO 95/13090 and US Patent Nos. 5,738,846 and 5,711,944 to Schering; and U.S. Patent No. 5,908,621 to Glue et al.. Interferon alpha-2a and interferon alpha-2b are currently approved as monotherapy for the treatment of HCV. For example, ROFERON®-A (Roche) is the recombinant form of interferon alpha-2a. Pegasys® (Roche) is the pegylated (i.e. polyethylene glycol modified) form of interferon alpha-2a. INTRON®A (Schering Corporation) is the recombinant form of Interferon alρha-2b, and PEG-INTRON® (Schering Corporation) is the pegylated form of interferon alpha-2b.

Other forms of interferon alpha, as well as interferon beta, gamma, tau and omega are currently in clinical development for the treatment of HCV. For example, INFERGEN (interferon alphacon-1) by InterMune, OMNIFERON (natural interferon) by Viragen, ALBUFERON by Human Genome Sciences, REBIF (interferon beta- la) by Ares-Serono, Omega Interferon by BioMedicine, Oral Interferon Alpha by Amarillo

Biosciences, and interferon gamma, interferon tau, and interferon gamma- lb by InterMune are in development.

(2) Ribavirin (for example, Battaglia, A.M. et al., Ann. Pharmacother, 2000,. 34, 487-494); Berenguer, M. et al. Antivir. Ther., 1998, 3 (Suppl. 3), 125-136).

Ribavirin (l-β-D-ribofuranosyl-l-l,2,4-triazole-3-carboxamide) is a synthetic, non- interferon-inducing, broad spectrum antiviral nucleoside analog. It is sold under the trade names VirazoleTM (The Merck Index, 11th edition, Editor: Budavari, S., Merck & Co., Inc., Rahway, NJ, pl304, 1989); Rebetol (Schering Plough) and Co-Pegasus (Roche). United States Patent No. 3,798,209 and RE29,835 (ICN Pharmaceuticals) disclose and claim ribavirin. Ribavirin is structurally similar to guanosine, and has in vitro activity against several DNA and RNA viruses including Flaviviridae (Gary L. Davis. Gastroenterology 118:S104-S114, 2000). For example, U.S. Patent No 4,211,771 (to ICN Pharmaceuticals) discloses the use of ribavirin as an antiviral agent. Ribavirin reduces serum amino transferase levels to normal in 40%> of patients, but it does not lower serum levels of HCV-RNA (for example, Gary L. Davis. Gastroenterology 118:S104-S114, 2000). Thus, ribavirin alone is not effective in reducing viral RNA levels. Additionally, ribavirin has significant toxicity and is lαiown to induce anemia.

Combination of Interferon and Ribavirin The current standard of care for chronic hepatitis C is combination therapy with an alpha interferon and ribavirin. The combination of interferon and ribavirin for the treatment of HCV infection has been reported to be effective in the treatment of interferon na^'ϊve patients (for example, Battaglia, A.M. et al., Ann. Pharmacother. 34:487-494, 2000), as well as for treatment of patients when histological disease is present (Berenguer, M. et al. Antivir. Ther. 3(Suppl. 3):125-136, 1998). Studies have show that more patients with hepatitis C respond to pegylated interferon-alpha/ribavirin combination therapy than to combination therapy with unpegylated interferon alpha. However, as with monotherapy, significant side effects develop during combination therapy, including hemolysis, flu-like symptoms, anemia, and fatigue, (for example, Gary L. Davis. Gastroenterology 118:S104-S114, 2000).

Combination therapy with PEG-INTRON® (peginterferon alpha-2b) and REBETOL® (Ribavirin, USP) Capsules is available from Schering Corporation. REBETOL® (Schering Corporation) has also been approved in combination with INTRON® A (Interferon alpha-2b, recombinant, Schering Corporation). Roche's Pegasys® (pegylated interferon alpha-2a) and COPEGUS® (ribavirin) are also approved for the treatment of HCV.

PCT Publication Nos. WO 99/59621, WO 00/37110, WO 01/81359, WO

02/32414 and WO 03/024461 by Schering Corporation disclose the use of pegylated interferon alpha and ribavirin combination therapy for the treatment of HCV. PCT Publication Nos. WO 99/15194, WO 99/64016, and WO 00/24355 by Hoffmann-La Roche Inc also disclose the use of pegylated interferon alpha and ribavirin combination therapy for the treatment of HCV.

(3) Substrate-based NS3 protease inhibitors (for example, Attwood et al., Antiviral peptide derivatives, PCT WO 98/22496, 1998; Attwood et al., Antiviral

Chemistry and Chemotherapy 1999, 10, 259-273; Attwood et al., Preparation and use of amino acid derivatives as anti-viral agents, German Patent Pub. DE 19914474; Tung et al. Inhibitors of serine proteases, particularly hepatitis C virus NS3 protease, PCT WO 98/17679), including alphaketoamides and hydrazinoureas, and inhibitors that terminate in an electrophile such as a boronic acid or phosphonate (Llinas-Branet et al, Hepatitis C inhibitor peptide analogues, PCT WO 99/07734).

(4) Non-substrate-based inhibitors, for example, 2,4,6-trihydroxy-3-nitro- benzamide derivatives (for example, Sudo K. et al., Biochemical and Biophysical Research Communications, 1997, 238, 643-647; Sudo K. et al. Antiviral Chemistry and Chemotherapy, 1998, 9, 186), including RD3-4082 and RD3-4078, the former substituted on the amide with a 14 carbon chain and the latter processing a para- phenoxyphenyl group;

(5) Thiazolidine derivatives which show relevant inhibition in a reverse- phase HPLC assay with an NS3/4A fusion protein and NS5A/5B substrate (for example Sudo K. et al., Antiviral Research, 1996, 32, 9-18), especially compound RD-1-6250, possessing a fused cinnamoyl moiety substituted with a long alkyl chain, RD4 6205 and RD4 6193;

(6) Thiazolidines and benzanilides (for example Kakiuchi N. et al. J. EBS Letters 421, 217-220; and Takeshita N. et al. Analytical Biochemistry, 1997, 247, 242- 246);

(7) A phenanthrenequinone possessing activity against protease in a SDS- PAGE and autoradiography assay isolated from the fermentation culture broth of Streptomyces sp., for example, Sch 68631 (for example, Chu M. et al., Tetrahedron Letters, 1996, 37, 7229-7232), and Sch 351633, isolated from the fungus Penicillium griscofuluum, which demonstrates activity in a scintillation proximity assay (for example, Chu M. et al., Bioorganic and Medicinal Chemistry Letters 9, 1949-1952); (8) Selective NS3 inhibitors, for example, those based on the macromolecule elgin c, isolated from leech (for example, Qasim M.A. et al., Biochemistry, 1997, 36, 1598-1607);

(9) Helicase inhibitors (for example Diana G.D. et al., Compounds, compositions and methods for tteatment of hepatitis C, U.S. Pat. No. 5,633,358; Diana

G.D. et al., Piperidine derivatives, pharmaceutical compositions thereof and their use in the treatment of hepatitis C, PCT WO 97/36554);

(10) Polymerase inhibitors for example nucleotide analogues, gliotoxin (for example, Ferrari R. et al. Journal of Virology, 1999, 73, 1649-1654), and the natural product ceralenin (for example, Lohmarm V. et al., Virology, 1998, 249, 108-118);

(11) Antisense phosphorothioate oligodeoxynucleotides (S-ODN) complementary to sequence stretches in the 5' non-coding region (NCR) of the viras (for example, Alt M. et al., Hepatology, 1995, 22, 707-717), or nucleotides 326-348 comprising the 3' end of the NCR and nucleotides 371-388 located in the core coding region of the HCV RNA (for example, Alt M. et al., Archives of Virology, 1997, 142,

589-599; Galderisi U. et al., Journal of Cellular Physiology, 1999, 181, 251-257).

(12) Inhibitors of IRES-dependent translation (for example, Ikeda N et al., Agent for the prevention and tteatment of hepatitis C, Japanese Patent Pub. JP- 08268890; Kai Y. et al. Prevention and treatment of viral diseases, Japanese Patent Pub. JP-10101591).

(13) Nuclease-resistant ribozymes (for example Maccjak, D. J. et al., Hepatology 1999, 30, abstract 995).

(14) Nucleoside analogs have also been developed for the treatment of Flaviviridae infections. Examples include the following. Idenix Pharmaceuticals, Ltd. discloses branched nucleosides, and their use in the treatment of HCV and flavivirases and pestivirases in US Patent Publication No. 2003/0050229 Al and US Patent Publication No. 2003/0060400 Al, which correspond to International Publication Nos. WO 01/90121 and WO 01/92282. A method for the treatment of hepatitis C infection (and flavivirases and pestivirases) in humans and other host animals is disclosed in the Idenix publications that includes administering an effective amount of a biologically active 1', 2', 3' or 4'-branched β-D or β-L nucleosides or a pharmaceutically acceptable salt or prodrug thereof, administered either alone or in combination, optionally in a pharmaceutically acceptable carrier. See also U.S. Patent Publication Nos. 2004/0006002 and 2004/0006007 as well as WO 03/026589 and WO 03/026675. Idenix Pharmaceuticals, Ltd. also discloses in US Patent Publication No.

2004/0077587 pharmaceutically acceptable branched nucleoside prodrags, and their use in the treatment of HCV and flavivirases and pestiviruses in prodrags. See also PCT Publication Nos. WO 04/002422, WO 04/002999, and WO 04/003000.

Patent No. 6,348,587. See also International Patent Publication WO 99/43691.

98/16184, WO 01/68663, and WO 02/03997.

Pharmasset Limited discloses various nucleosides and antimetabolites for the treatment of a variety of viruses, including Flaviviridae, and in particular HCV, in WO 02/32920, WO 01/79246, WO 02/48165, WO 03/068162, WO 03/068164 and 2004/013298. Merck & Co., Inc. and Isis Pharmaceuticals disclose in US Patent Publication No. 2002/0147160 and the corresponding International Patent Publication Nos. WO 02/057425 (PCT/US02/01531; filed January 18, 2002) and WO 02/057287 (PCT/US02/03086; filed January 18, 2002) various nucleosides, and in particular several pyrrolopyrimidine nucleosides, for the tteatment of virases whose replication is dependent upon RNA-dependent RNA polymerase, including Flaviviridae, and in particular HCV. See also WO 2004/003138, WO 2004/007512, and WO 2004/009020.

(15) Miscellaneous compounds including, for example, 1-amino- alkylcyclohexanes (for example, U.S. Patent No. 6,034,134 to Gold et al.), alkyl lipids (for example, U.S. Pat. No. 5,922,757 to Chojkier et al.), vitamin E and other antioxidants (for example, U.S. Pat. No. 5,922,757 to Chojkier et al.), squalene, amantadine, bile acids (for example, U.S. Pat. No. 5,846,964 to Ozeki et al.), N- (phosphonoacetyl)-L-aspartic acid (for example, U.S. Pat. No. 5,830,905 to Diana et al.), benzenedicarboxamides (for example, U.S. Pat. No. 5,633,388 to Diana et al.), polyadenylic acid derivatives (for example, U.S. Pat. No. 5,496,546 to Wang et al.), 2',3'- dideoxyinosine (for example, U.S. Pat. No. 5,026,687 to Yarchoan et al.), and benzimidazoles for example, (U.S. Pat. No. 5,891,874 to Colacino et al.).

(16) Other compounds currently in clinical development for treatment of hepatitis c viras include, for example: Interleukin-10 by Schering-Plough, IP-501 by Interneuron, Merimebodib VX-497 by Vertex, AMANTADINE (Symmetrel) by Endo

Labs Solvay, HEPTAZYME by RPI, IDN-6556 by Idun Pharma., XTL-002 by XTL., HCV/MF59 by Chiron, CIVACIR by NABI, LEVOVIRIN by ICN, VIRAMIDINE by ICN, ZADAXIN (thymosin alfa-1) by Sci Clone, CEPLENE (histamine dihydrochloride) by Maxim, VX 950 / LY 570310 by Vertex/Eli Lilly, ISIS 14803 by Isis Pharmaceutical/Elan, IDN-6556 by Idun Pharmaceuticals, Inc. and JTK 003 by AKROS

Pharma. Pharmaceutical Compositions

Hosts, including humans, infected with pestiviras, flaviviras, HCV or another organism replicating through a RNA-dependent RNA viral polymerase, can be treated by administering to the patient an effective amount of the active compound or a pharmaceutically acceptable prodrug or salt thereof in the presence of a pharmaceutically acceptable carrier or diluent. The active materials can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form.

A preferred dose of the compound for pestiviras, flaviviras or HCV will be in the range from about 1 to 50 mg/kg, preferably 1 to 20 mg/kg, of body weight per day, more generally 0.1 to about 100 mg per kilogram body weight of the recipient per day. The effective dosage range of the pharmaceutically acceptable salts and prodrags can be calculated based on the weight of the parent nucleoside to be delivered. If the salt or prodrag exhibits activity in itself, the effective dosage can be estimated as above using the weight of the salt or prodrag, or by other means known to those skilled in the art.

The compound is conveniently administered in unit any suitable dosage form, including but not limited to one containing 7 to 3000 mg, preferably 70 to 1400 mg of active ingredient per unit dosage form. An oral dosage of 50-1000 mg is usually convenient. Ideally the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.2 to 70 μM, preferably about 1.0 to 10 μM. This may be achieved, for example, by the intravenous injection of a 0.1 to 5% solution of the active ingredient, optionally in saline, or administered as a bolus of the active ingredient. The concentration of active compound in the drug composition will depend on absorption, mactivation and excretion rates of the drug as well as other factors lαiown to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.

A preferred mode of administration of the active compound is oral. Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administtation, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can e included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as macrocrystalline cellulose, gum ttagacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents. The compound can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

The compound or a pharmaceutically acceptable prodrag or salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, anti- inflammatori.es, or other antivirals, including other nucleoside compounds. Solutions or suspensions used for parenteral, inttadermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetettaacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

If administered intravenously, preferred carriers are physiological saline or phosphate buffered saline (PBS).

In a preferred embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems, biodegradale, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) are also preferred as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811 (which is incorporated herein by reference in its entirety). For example, liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound or its monophosphate, diphosphate, and/or ttiphosphate derivatives is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, therbey forming the liposomal suspension.

Processes for the Preparation of Active Compounds

The nucleosides of the present invention can be synthesized by any means lαiown in the art. In particular, the synthesis of the present nucleosides can be achieved by either alkylating the appropriately modified sugar, followed by glycosylation or glycosylation followed by alkylation of the nucleoside, though preferably alkylating the appropriately modified sugar, followed by glycosylation. The following non-limiting embodiments illustrate some general and specific methodologies to obtain the nucleosides of the present invention.

General Synthesis of Ribofuranosyl-5-Aza-7-Deazapur_nes

The compounds of the present invention can be prepared by synthetic methods well known to those skilled in the art of nucleoside and nucleotide chemistry, such as taught by Townsend in Chemistry of Nucleosides and Nucleotides. Plenum Press, 1994. A representative general synthetic method is provided in Scheme 8. The starting material is a 3,5-is-O-protected beta-D-alkyl ribofuranoside, but it will be understood that any 2', 3', or 5 '-position may carry a protecting group to shield it from reacting. The 2'-C-OH then is oxidized with a suitable oxidizing agent in a compatible solvent at a suitable temperature to yield the 2'-keto-modified sugar. Possible oxidizing agents are Swern reagents, Jones' reagent (a mixture of chromic and sulfuric acids), Collins' reagent (dipyridine Cr(VI)oxide), Corey's reagent (pyridinium chlorochromate), pyridinium dichromate, acid dichromate, potassium permanganate, Mn0₂, rathenium tetroxide, phase transfer catalysts such as chromic acid or permanganate supported on a polymer, Cl₂-pyridine, H₂O₂-ammonium molydate, NarO₂-CAN, NaOCl in HOAc, copper chromate, copper oxide, Raney nickel, palladium acetate, Meerwin-Pondorf-

Verley reagent (aluminum t-butoxide with another ketone) and N-bromosuccinimide.

Next, addition of a Grignard reagent, such as, for example, an alkyl-, alkenyl- or alkynyl-magnesium halide like CH₃Mgr, CH₃CH₂Mgr, vinylMgr, allylMgr and ethynylMgr, or an alkyl-, alkenyl- or alkynyl-lithium, such as CH₃Li, in a suitable organic solvent, such as, for example, diethyl ether or THF, across the double bond of the 2'-carbonyl group provides a tertiary alcohol at this position. The addition of a hydrogen halide in a suitable solvent, such as, for example, Hr in HOAc, in the subsequent step provides a leaving group (LG) such as, for example, a chloro, bromo or iodo, at the C-l anomeric carbon of the sugar ring that later generates a nucleosidic linkage. Other suitable LGs include C-l sulfonates such as, for example, methanesulfonate, trifluoromethanesulfonate and/or p-toluenesulfonate .

The introduction in the next step of a metal salt (Li, Na or K) of an appropriately substituted 2-azapurine in a suitable organic solvent such as, for example, THF, acetonitrile of DMF, results in the formation of the desired nucleosidic linkage and addition of the desired 2-azapurine base. This displacement reaction may be catalyzed by a phase transfer catalyst like TDA-1 or triethylbenzylammonium chloride. The introduction of a "Z" substituent on the base formula (ioptionally may be performed subsequent to the initial addition of protecting groups. For example, the introduction of an amino group for "Z" is accomplished by the addition of an appropriate amine in an appropriate solvent to the 2'-C-halo intermediate just prior to the last step of removal of the protecting groups. Appropriate amines include alcoholic or liquid ammonia to generate a primary amine (-NH₂), an alkylamine to generate a secondary amine (-NHR), or a dialkylamine to generate a tertiary amine (-NRR'). Finally, the nucleoside can be deprotected by methods well known to those skilled in the art, as by Greene et al., Protective Groups in Organic Synthesis. John Wiley and Sons, Second Edition, 1991.

Scheme 8

Pg = Protecting Group R = lower alkyl

The present invention is described by way of illustration in the following examples. It will be understood by one of ordinary skill in the art that these examples are in no way limiting and that variations of detail can be made without departing from the spirit and scope of the present invention.

EXAMPLES

Example 1 : Synthesis of β-D-2'-Deoxy-5-Aza-7-Deazaguanosine

The compound was synthesized according to the prior art process taught by Rosemeyer and Seela (Rosemeyer and Seela, J. Org. Chem., 1987, 52(23):5\36-43; F. Seela et al., Nucleic Acids Symposium Series, 1987, 18 (Symp. Chem. Nucleic Acid Compon., 7⁽.49-52.

Example 2: Synthesis of β-D-5-Aza-7-Deazaguanosine

The compound was synthesized according to the prior art process taught by

Robins and co-workers (Kim et al., J. Med. Chem., 1978, 27( :883-91; R.K. Robins and G.R. Revankar, U.S. Pat. 4,246,408). Example 3: Synthesis of 2-Amino-8-(methylpivalate)imidazo[l,2-«]-s-triazin-4- one

The compound was synthesized as follows: To a suspension of sodium hydride (60% in oil, 90 mg, 2.25 mmoles) in dry dimethylformamide (10 mL) was added 2-

Amino-imidazo[l,2-a]-s-triazin-4-one [for preparation see Journal of Medicinal Chemistry. 1978. Vol 21. No 9. 8831 (300 mg, 1.98 mmol) at 20°C and stirred for one hour. Chloromethyl pivalate was added and stirred at 20°C for 16 hours. The reaction mixture was evaporated to dryness. The residue was purified on silica gel using dichloromethane/methanol (98/2) as eluant to give the title compound (185 mg) as a white powder.

1H NMR (DMSO-dβ) δ ppm : 1.10 (s, 9H), 5.90 (s, 2H), 7.10 (br, 2H, NH₂), 7.30 (d, 1H, J=2.7 Hz), 7.35 (d, 1H, J= 2.7 Hz)

Mass spectrum : m/z (FAB>0) 266 (M+H)⁺, (FAB<0) 264 ( -H)^"

Example 4: Synthesis of 2-Ammo-8-(5-deoxy-β-D-ribofuranosyl)imidazo[l,2-a]-s- triazin-4-one

The following scheme illustrates the synthesis of 2-Amino-8-(5-deoxy-b-D- ribofuranosyl)imidazo[l,2-a]-s-triazin-4-one.

Step A: 2-Ammo-8-(2,3-O-isopropy_idene-β-D-ribofuranosyl)imidazo~[l,2-a]- triazin-4-one

To a solution of 2-Amino-8-(β-D-ribofuranosyl)imidazo[l,2-a]-s-triazin-4-one [for preparation see Journal of Medicinal Chemistry. 1978. Vol 21. No 9. 883] (4.35,

15.3 mmol) in acetone (43 mL) was added 2,2-dimethoxyproρane (43 L) and perchloric acid (10 drops). The mixture was stirred at 20°C for 6 hours. Then acetone (20 mL), 2,2-dimethoxypropane (20 L) and perchloric acid (10 drops) were added to the mixture and stirred 22 hours. This phase is repeated third once. The reaction mixture was neutralized by addition of pyridine and evaporated to dryness. The residue was purified on silica gel using dichloromethane/ethanol (95/5) as eluant to give the title compound (4.39 gr) as a yellow powder.

^!H NMR (DMSO-d₆) δ ppm: 1.34 (s, 3H, CH₃), 1.52 (s, 3H, CH₃), 3.60 (m, 2H), 4.25 (m, 1H), 4.95 (dd, 1H, J= 6.1 Hz, J= 2.6 Hz), 5.11 (dd, 1H, J= 6.1 Hz, J= 2.6 Hz), 5.98 (d, 1H, J= 2.6 Hz), 7.49 (br, 1H, NH), 7.70 (m, 2H), 8.52 (br, 1H, NH)

Step B: 2-Ammo-8-(5-deoxy~5-iodo-2,3-O-isopropylidene~β-D-ribofuranosyl)- imidazo- [l,2-a]-s-triazm-4~one

To a solution of methyltriphenoxyphosphonium iodide (12.3 g, 27.2 mmol) in dry dimethylformamide (60 mL) was added the compound from Step A (4.39 g, 13.5 mmol) and stirred at 20°C for 2 hours. The reaction mixture was poured into methanol and evaporated to dryness. The residue was treated with ethyl acetate. The organic layer was washed with aqueous sodium thiosulfate and water, dried over MgSO₄, filtered and evaporated to dryness. The crude product was purified on silica gel using dichloromethane/ethanol (96.5/3.5) as eluant to give the title compound (2.49 g) as a white powder.

1H NMR (DMSO-d₆) δ ppm: 1.32 (s, 3H, CH₃), 1.50 (s, 3H, CH₃), 3.39 (m, 2H), 4.30 (dt, 1H, J= 7.3 Hz, J= 3.1 Hz), 5.00 (dd, 1H, J= 6.4 Hz, J= 3.1 Hz), 5.34 (dd, 1H, J= 6.4 Hz, J= 2.1 Hz), 6.00 (d, 1H, J= 2.1 Hz), 7.10 (br, 2H, NH₂), 7.42 (d, 1H, J= 2.8 Hz), 7.47 (d, 1H, J= 2.8 Hz)

Step C: 2-Ammo-8-(5-deoxy-2,3-O-isopropy_idene-β-D-ribofuranosyl)- imidazo- [1 ,2-a] -s-triazin-4-one

A solution of the compound from Step B (1.02 g, 2.35 mmol), sodium acetate (418 mg, 5.09 mmol) in a mixture of ethanol (30 mL) and water (20 mL) was treated with 10%Pd/C (770 mg) and the mixture was hydrogenated at atmospheric pressure at 20 °C for 15 hours. The mixture was filtered through a Celite pad and evaporated to dryness. The crude product was purified on silica gel using dichloromethane/ethanol (95/5) as eluant to give the title compound (604 mg) as a yellow powder.

Step D: 2-Ammo-8-(5-deoxy-β-D-ribofuranosyl)imidazo[l,2-a]-s-triazin-4-one

A solution of the compound from Step C (540 mg, 1.76 mmol) in a mixture of trifluoroacetic acid (8 mL) and water (2 mL) was stirred at 20°C for 6 hours and then evaporated to dryness The crude product was purified on silica gel reverse-phase (C18) using water as eluant to give the title compound (288 mg) as a white powder.

^!H NMR (DMSO-d₆) δ ppm: 1.55 (d, 3H, J= 6.4 Hz, CH₃), 3.81 (m, 1H), 3.91 (m, 1H), 4.35 (m, 1H), 5.20 (d, 1H, J= 4.8 Hz, OH), 5.50 (d, 1H, J= 5.5 Hz, OH), 5.45 (d, 1H, J=

5.5 Hz), 6.92 (br, 2H, NH₂), 7.75 (d, 1H, J= 2.8 Hz), 7.82 (d, 1H, J= 2.8 Hz) ¹³C NMR (DMSO-d₆) δ ppm: 19.0, 72.8, 74.3, 79.94, 86.67, 108.53, 114.56, 150.0, 150.7, 165.3

Mass spectrum: m/z (FAB>0) 535 (2M+H)⁺, 268 (M+H)⁺, (FAB<0) 533 (2M-H)-, 266 (M-H)-

Example 5: Assessment of Biological Activity

Compounds can exhibit anti-flaviviras or pestiviras activity by inhibiting flaviviras or pestivirus polymerase, by inhibiting other enzymes needed in the replication cycle, or by other pathways.

The test compounds are dissolved in DMSO at an initial concentration, for example of 200 μM, and then serially diluted in culture medium.

Unless otherwise stated, baby hamster kidney (BHK-21) (ATCC CCL-10) and Bos Taurus (BT) (ATCC CRL 1390) cells are grown at 37°C in a humidified CO₂ (5%) atmosphere. BHK-21 cells are passaged in Eagle MEM additioned of 2 mM L- glutamine, 10% fetal bovine seram (FBS, Gibco) and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate and 0.1 mM non-essential amino acids. BT cells are passaged in

Dulbecco's modified Eagle's medium with 4 mM L-glutamine and 10% horse seram (HS, Gibco), adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose and 1.0 mM sodium pyravate. The vaccine strain 17D (YFV-17D) (Stamaril®, Pasteur Merieux) and Bovine Viral Diarrhea viras (BVDV) (ATCC VR-534) are used to infect BHK and BT cells, respectively, in 75 cm² bottles. After a 3 day incubation period at 37°C, extensive cytopathic effect can be observed. Cultures are freeze-thawed three times, cell debris are removed by centtifugation and the supernatant aliquoted and stored at -70°C. YFV-17D and BVDV are tittated in BHK-21 and BT cells, respectively, that were grown to confluency in 24-well plates.

Example 6: Phosphorylation Assay of Nucleoside to Active Triphosphate

To determine the cellular metabolism of the compounds, HepG2 cells are obtained from the American Type Culture Collection (Rockville, MD), and are grown in 225 cm² tissue culture flasks in minimal essential medium supplemented with non- essential amino acids, 1% penicillin-streptomycin. The medium is renewed every three days, and the cells are subcultured once a week. After detachment of the adherent monolayer with a 10 minute exposure to 30 mL of trypsin-EDTA and three consecutive washes with medium, confluent HepG2 cells are seeded at a density of 2.5 x 10⁶ cells per well in a 6-well plate and exposed to 10 μM of [³H] labeled active compound (500 dpm/pmol) for the specified time periods. The cells are maintained at 37°C under a 5% CO₂ atmosphere. At the selected time points, the cells are washed three times with ice- cold phosphate-buffered saline (PBS). Inttacellular active compound and its respective metabolites are extracted by incubating the cell pellet overnight at -20°C with 60% methanol followed by extraction with an additional 20 μL of cold methanol for one hour in an ice bath. The extracts are then combined, dried under gentle filtered air flow and stored at -20°C until HPLC analysis.

Example 7: Bioavailability Assay in Cynomolgus Monkeys

Within 1 week prior to the study initiation, the cynomolgus monkey is surgically implanted with a chronic venous catheter and subcutaneous venous access port (VAP) to facilitate blood collection and underwent a physical examination including hematology and serum chemistry evaluations and the body weight was recorded. Each monkey (six total) receives approximately 250 μCi of H activity with each dose of active compound at a dose level of 10 mg/kg at a dose concentration of 5 mg/mL, either via an intravenous bolus (3 monkeys, IV), or via oral gavage (3 monkeys, PO). Each dosing syringe is weighed before dosing to gravimetrically determine the quantity of formulation administered. Urine samples are collected via pan catch at the designated intervals (approximately 18-0 hours pre-dose, 0-4, 4-8 and 8-12 hours post-dosage) and processed. Blood samples are collected as well (pre-dose, 0.25, 0.5, 1, 2, 3, 6, 8, 12 and 24 hours post-dosage) via the chronic venous catheter and VAP or from a peripheral vessel if the chronic venous catheter procedure should not be possible. The blood and urine samples are analyzed for the maximum concentration (C_max), time when the maximum concentration is achieved (T_max), area under the curve (AUC), half life of the dosage concentration (T_/.), clearance (CL), steady state volume and distribution (V_ss) and bioavailability (F).

Example 8: Bone Marrow Toxicity Assay

Human bone marrow cells are collected from nonnal healthy volunteers and the mononuclear population are separated by Ficoll-Hypaque gradient centrifugation as described previously by Sommadossi J-P, Carlisle R. "Toxicity of 3'-azido-3'- deoxythymidine and 9-(l,3-dihydroxy-2-propoxymethyl)guanine for normal human hematopoietic progenitor cells in vitro" Antimicrobial Agents and Chemotherapy 1987; 31:452-454; and Sommadossi J-P, Schinazi RF, Chu CK, Xie M-Y. "Comparison of cytotoxicity of the (-)- and (+)-enantiomer of 2',3'-dideoxy-3'-thiacytidine in normal human bone marrow progenitor cells" Biochemical Pharmacology 1992; 44:1921-1925. The culture assays for CFU-GM and BFU-E are performed using a bilayer soft agar or methylcellulose method. Drags are diluted in tissue culture medium and filtered. After 14 to 18 days at 37°C in a humidified atmosphere of 5% C0₂ in air, colonies of greater than 50 cells are counted using an inverted microscope. The results are presented as the percent inhibition of colony formation in the presence of drag compared to solvent control cultures.

Example 9: Mitochondria Toxicity Assay

HeρG2 cells are cultured in 12-well plates as described above and exposed to various concentrations of drags as taught by Pan-Zhou X-R, Cui L, Zhou X-J,

Sommadossi J-P, Darley-Usmer VM. "Differential effects of antiretroviral nucleoside analogs on mitochondrial function in HepG2 cells" Antimicrob Agents Chemother 2000; 44:496-503. Lactic acid levels in the culture medium after 4 day drug exposure are measured using a Boeliringer lactic acid assay kit. Lactic acid levels are normalized by cell number as measured by hemocytometer count. Example 10: Cytotoxicity Assay

Cells are seeded at a rate of between 5 x 10³ and 5 x 10⁴/well into 96-well plates in growth medium overnight at 37°C in a humidified C0₂ (5%) atmosphere. New growth medium containing serial dilutions of the drags is then added. After incubation for 4 days, cultures are fixed in 50% TCA and stained with sulforhodamineB. The optical density was read at 550 nm. The cytotoxic concentration was expressed as the concentration required to reduce the cell number by 50% (CC5₀).

Example 11: Cell Protection Assay (CPA)

The assay is performed essentially as described by Baginski, S. G.; Pevear, D. C; Seipel, M.; Sun, S. C. C; Benetatos, C. A; Chundura, S. K.; Rice, C. M. and M. S.

Collett "Mechanism of action of a pestiviras antiviral compound" PNAS USA 2000, 97(14), 7981-7986. MDBK cells (ATCC) are seeded onto 96-well culture plates (4,000 cells per well) 24 hours before use. After infection with BVDV (strain NADL, ATCC) at a multiplicity of infection (MOI) of 0.02 plaque forming units (PFU) per cell, serial dilutions of test compounds are added to both infected and uninfected cells in a final concentration of 0.5% DMSO in growth medium. Each dilution is tested in quadruplicate. Cell densities and viras inocula are adjusted to ensure continuous cell growth throughout the experiment and to achieve more than 90% virus-induced cell destruction in the untreated controls after four days post-infection. After four days, plates are fixed with 50%> TCA and stained with sulforhodamine B. The optical density of the wells is read in a microplate reader at 550 nm. The 50% effective concentration (EC50) values are defined as the compound concentration that acliieved 50%> reduction of cytopathic effect of the viras.

Example 12: Plaque Reduction Assay

For each compound the effective concentration is determined in duplicate 24-well plates by plaque reduction assays. Cell monolayers are infected with 100 PFU/well of virus. Then, serial dilutions of test compounds in MEM supplemented with 2% inactivated seram and 0.75% of methyl cellulose are added to the monolayers. Cultures are further incubated at 37°C for 3 days, then fixed with 50%> ethanol and 0.8% Crystal Violet, washed and air-dried. Then plaques are counted to determine the concentration to obtain 90% viras suppression.

Example 13: Yield Reduction Assay

For each compound the concenttation to obtain a 6-log reduction in viral load is determined in duplicate 24-well plates by yield reduction assays. The assay is performed as described by Baginski, S. G.; Pevear, D. C; Seipel, M.; Sun, S. C. C; Benetatos, C. A.; Chundura, S. K.; Rice, C. M. and M. S. Collett "Mechanism of action of a pestiviras antiviral compound" PNAS USA 2000, 97(14), 7981-7986, with minor modifications. Briefly, MDBK cells are seeded onto 24-well plates (2 x 105 cells per well) 24 hours before infection with BVDV (NADL strain) at a multiplicity of infection (MOI) of 0.1 PFU per cell. Serial dilutions of test compounds are added to cells in a final concentration of 0.5% DMSO in growth medium. Each dilution is tested in triplicate. After three days, cell cultures (cell monolayers and supernatants) are lysed by three freeze-thaw cycles, and viras yield is quantified by plaque assay. Briefly, MDBK cells are seeded onto 6-well plates (5 x 105 cells per well) 24 h before use. Cells are inoculated with 0.2 mL of test lysates for 1 hour, washed and overlaid with 0.5% agarose in growth medium. After 3 days, cell monolayers are fixed with 3.5% formaldehyde and stained with 1% crystal violet (w/v in 50% ethanol) to visualize plaques. The plaques are counted to determine the concentration to obtain a 6-log reduction in viral load.

Representative data is provided in Table 1.

Table 1

This invention has been described with reference to its prefened embodiments. Variations and modifications of the invention will be obvious to those skilled in the art from the foregoing detailed description of the invention. It is intended that all of these variations and modifications be included within the scope of this invention.

Claims

WE CLAIM:

1. A method of treating a host infected with a flaviviras or pestiviras, comprising administering an effective amount of a nucleoside of Formula (I):

or its tautomer, enantiomer, diastereomer, phanuaceutically acceptable salt, and/or prodrag, wherein:

R is OH, phosphate or phosphonate (including mono-, di-, or friphosphate or a stabilized phosphate prodrag); acyl (including lower acyl); H; alkyl (including lower alkyl); sulfonate ester including alkyl or arylalkyl sulfonyl including methanesulfonyl and benzyl, wherein the phenyl group is optionally substituted with one or more substituents as described in the definition of an aryl given herein; optionally substituted arylsulfonyl; a lipid, including a phospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; any of which may be O-linked; or another pharmaceutically acceptable leaving group that when administered in vivo, provides a compound wherein R¹ is independently OH or O-phosphate;

Each R² and R³ independently is H or OH; and

Base is:

wherein:

Z is. H, OH, SH, NH₂, halo, CF₃, CM alkyl, Ci-4 alkylamino, di(d_-4 alkyl)amino, C₃.₆ cycloalkylamino, or d- alkoxy;

Y is O, S, orNR⁴; and

R⁴ is independently is hydrogen, optionally substituted or unsubstituted lower alkyl, lower haloalkyl, optionally substituted or unsubstituted lower alkenyl, lower haloalkenyl, optionally substituted or unsubstituted aryl, arylalkyl, unsubstituted or substituted phenyl or benzyl, or an optionally substituted or unsubstituted acyl.

2. A method of treating a host infected with a flaviviras or pestiviras, comprising administering an effective amount of a ester of Formula (II):

or its tautomer, pharmaceutically acceptable salt, and/or prodrug, wherein: Base is:

wherein:

Z is H, OH, SH, NH₂, halo, CF₃, C alkyl, C alkylamino, di(Cι-4 alkyl)amino, C_3-6 cycloalkylamino, or d- alkoxy;

Y is O, S, or R⁴; and

3. The method of claim 1 wherein the host is a mammal.

4. The method of claim 3 wherein the mammal is a human.

5. The method of claim 2 wherein the host is a mammal.

6. The method of claim 5 wherein the mammal is a human.

7. The method of any one of claims 1, 3 or 4 wherein the compound of Formula (I) has the structure:

or a pharmaceutically acceptable salt or prodrag thereof.

The method of any one of claims 1, 3 or 4 wherein the compound of Formula (I) has the structure:

or a pharmaceutically acceptable salt or prodrag thereof.

9. The method of any one of claims 1, 3, or 4 wherein the compound of Formula (I) has the structure:

or a pharmaceutically acceptable salt or prodrag thereof.

10. The method of any one of claims 2, 5, or 6 wherein the compound of Formula (II) has the structure:

or a pharmaceutically acceptable salt or prodrag thereof.

11. A pharmaceutical composition comprising a compound of Formula (I), claim 1, or a pharmaceutically acceptable salt or prodrag thereof, optionally in combination with a pharmaceutically acceptable earner, diluent or excipient.

12. A pharmaceutical composition comprising a compound of Formula (II), claim 2, or pharmaceutically acceptable salt or prodrag thereof, optionally in combination with a pharmaceutically acceptable carrier, diluent or excipient.

13. A pharmaceutical composition comprising a compound of Formula (I), claim 1, having the structure:

or a pharmaceutically acceptable salt or prodrag thereof, optionally in combination with a pharmaceutically acceptable carrier, diluent or excipient.

14. A pharmaceutical composition comprising a compound of Formula (I), claim 1, having the structure:

15. A pharmaceutical composition comprising a compound of Formula (I), claim 1 , having the structure:

16. The pharmaceutical composition of any one of claims 11, 12, 13, 14, or 15, wherein the pharmaceutical composition is taken alone or optionally in combination and/or alternation with at least one other antiviral agent, optionally in combination with a pharmaceutically acceptable carrier, diluent or excipient.

17. Use of a compound of Formula (I), claim 1, for the manufacture of a medicament.

Use of a compound of Formula (II), claim 2, for the manufacture of a medicament.

19. A compound of the Formula (I) having the structure:

or a pharmaceutically acceptable salt or prodrug thereof.

20. A compound of the Formula (I) having the structure:

or a pharmaceutically acceptable salt or prodrag thereof.

21. A compound of the Formula (I) having the structure:

or a phannaceutically acceptable salt or prodrag thereof.

22. A compound of the Formula (II) having the structure:

or a pharmaceutically acceptable salt or prodrag thereof.