AU2003201382B8 - Nucleosides with anti-hepatitis B virus activity - Google Patents

Description

AUSTRALIA Patents Act 1990 Centre National de-la Recherche Scientifique, Emory University and The UAB Research Foundation IP AUSTRALIA RECEIVED 2 0 MAR 2003 COMPLETE SPECIFICATION CANBERRA - FEP STANDARD PATENT Invention Title: Nucleosides with anti-hepatitis B virus activity The following statement is a full description of this invention, including the best method of performing it known to us: 141479370 NUCLEOSIDES WITH ANTI-BEPATTIS B VIRUS ACTIViTY Background of the Invention 5 This invention is in the area of methods for the treatment of hepatitis B virus (also referred to as "HBV") that includes administering an effective amount of one or more of the active compounds disclosed herein, or a pharmaceutically acceptable derivative or prodrug of one of these 10 compounds. HBV is second only to tobacco as a cause of human cancer. The mechanism by which HBV induces cancer is unknown, although it is postulated that it may directly trigger tumor development, or indirectly trigger tumor development through chronic inflammation, cirrhosis, and i5 cell regendration associated with the infection. I t Hepatitis B virus has reached epidemic levels worldwide. After a two to six month incubation period in which the host is unaware of the infection, HBV infection can lead to acute hepatitis and liver damage, that causes abdominal pain, jaundi, and elevated blood levels of certain 20 enzymes. HBV can cause fulminant hepatitis, a rapidly progressive, often fatal form of the disease in which massive sections of the liver are destroyed. Patients typically recover'from acute viral hepatitis. In some patients, however, high levels of viral antigen persist in the blood for an extended, or indefinite, period, causing a chronic infection. Chronic 25 infections can lead to chronic persistent hepatitis. Patients infected with chronic persistent HBV are most common in developing countries. By mid-1991, there were approximately 225 million chronic carriers of HBV in Asia alone, and worldwide: almost 300 million carriers. Chronic persistent hepatitis can cause fatigue, cirrhosis of the liver, and 1A hepatocellular carcinoma, a primary liver cancer. In western industrialized countries, high risk groups for HBV infection include those in contact with HBV carriers or their blood samples. The epidemiology of HBV is in fact very similar to that of acquired immunodeficiency syndrome, which 5 accounts for why HBV infection is common among patients with AIDS or HIV-associated infections. However, HBV is more contagious than HIV. Daily treatments with a-interferon, a genetically engineered protein, has shown promise. A human serum-derived vaccine has also. been developed to immunize patients against HBV. Vaccines have been 10 produced through genetic engineering. While the vaccine has been found effective, production of the vaccine is troublesome because the supply of human serum from chronic carriers is limited, and the purification procedure is long and expensive. Further, each batch of vaccine prepared from different serum must be tested in chimpanzees to ensure safety. In 15 addition, the vaccine doesnot help the patients already infected. with the virus. European Patent Application No. 92304530.6 discloses that a group of 1,2-oxathiolane nucleosides are useful in the treatment of hepatitis B infections. It has been reported that the 2-hydroxymetbyl-5-(cytosin-1-yl) 20 1,3-oxathiolane has anti-hepatitis B activity. Doong, et al., Proc. of Nat. Aca. Sci. USA. 88, 8495-8499 (1991); Chang, et al., L of ioloiL Chm., Vol 267(20), 13938-13942. The anti-hepatitis B activity of the (-) and (+)-enantiomers of 2-hydroxymethyl-5-(5-fluorocytosin-l-yl)-l,3 oxathiolanc has been published by Furman, at al., in AntimicroiaLAgents 25 and Chemothepy, Dec. 1992, pages 2686-2692. PCT/US92/O3144 (Interqational Publication No. WO 92/18517) filed by Yale University discloses a number of B-L-nucleosides for the treatment of both HBV and HUV. Other drugs exlored for the treatment of HBV include adenosine arabinoside, thymosin, acyclovir, phosphonoformate, zidovudine. (+)-cyanidanol, quinacrine, and 2'-fluoroarabinosyl-5 iodourail. An essendal step in the mode of action of purine and pyrimidine nucleosides against viral diseases, and in particular, HBV and HIV, is their 5 metabolic activation by cellular and viral kinases, to yield the mono-, di-, and triphosphate derivatives. The biologically active species of many nucleosides is the triphospahte form, which inhibits DNA polymerase or reverse transcriptase, or causes chain termination. The nucleoside derivatives that have been developed for the treatment of HBV and HIV to 10 date have been presented for administration to the host in unphosphorylated form, notwithstanding the fact that the nucleoside must be phosphorylated in the cell prior to exhibiting its antiviral effect, because the triphosphate form has typically either been dephosphorylated prior to reaching-the cell or is poorly absorbed by the cell. Nucleotides in general cross cell 15 membranes very inefficiently and are generally not very not very potent in vig. Attempts at modifying nucleoddes to increase the absorption and potency of nucleotides have been described by R. Jones and N. Bischofberger, Antiviral Research, 27 (1995) 1-17, the contents of which are incorporated herein by reference. 20 In light of the fact that hepltitis B virus has reached epidemic levels worldwide, and has severe and often tugic effects on the infected patient, there remains a strong need to provide new effective pharmaceutical agents to treat humans infected with the virus that have low toxicity to the host. Therefore, it is another object of the present invention to provide a 25 method and composition fbr the treatinent of human patients or other hosts infected with HBV.

summary of the Inenton A method for the treatment of a host, and in particular, a human, infected with HBV is provided that includes administering an HBV 5 treatment amount of a nucleoside of the formula: R NH2NH 2 N H F 10 HN NY N X N HO HO HO wherein: R is hydrogen, fluoro, bromo, chlor, iodo, methyl or ethyl; and R 2 15 is OH Cl, NH 2 , or H; ora pharmaceutically acceptable salt of the compound, optionally in a pharmaceutically acceptable carrier or diluent. In an alternative embodiment, the B-Lenantiomer of a compound of the formula: 20 25 wherein R 5 is adenine, xanthine, hypoxanthine, or other purine, including an alkylated or halogenated purine is administered to kzhost in an REV tratment amount as described more fully herein. In another alternative embodiint, the nucleoside is of the formula: -4- L' ' Base ROO 0 wherein B is a purine or pyrimidine base; Y', Y, Y, and Y9 arc independently H, OH, N,, NR'R&, NO 1 , 1o NOR 3 , -O-alkyl, -o-aryl, halo (including F, Cl, Br, or 1), -CN, C(O)NH 2 , SH, -S-alkyl, or -5-aryl, and wherein typically three of Y,9, 9, and 9 are either H or OR. The -OH substituent, when present, is typically a Y' or Y group. As Illustrated in the structure, Y and 9 arn in the arabino (erythro) configuration, and Y' and Y are in the threo (ribose) .15 configuration. R is H, mohophosphate, diphosphate, triphosphate, alkyl, acyl, or a phosphate derivative, as described in more detail below. R, R 2 and e are independently alkyl (and in particular lower alkyl), aryl, anlkyl, alkaryl, acyl, or hydrogen. In a preferred embodiment, the nucleoside is provided as the indicated 20 enantiomer and substantially in the absence of its corresponding enantioner (i.e., in enantiomerically enriched form). In another embodiment, the invention includes a method for the treatment of humans infected with HBV that includes administering an HBV treatment amount of a prodrug of the specifically disclosed 25 nucleosides. A prodrug, as used herein,. refers to a pharmaceutically acceptable derivative of the specifically disclosed nucleoside, that is converted into the nucleosideen admintrton in vivo or that has activity in itielf. Nonlmiting examp r the 5' and N-pyrimidine or N'-purine acylated or alkylated derivatives of the active compound.

In a preferred embodiment of the invention, the nucleoside is provided as the mfonlophosphate, diphosphate or triphosphate in a formulation that protects the compound from dephosphorylation. Formulations include liposomes, lipospheres, microspheres or nanospheres (of which the latter 5 three can be targeted to infected cells). In an alternative preferred embodiment, the nucleoside is provided as a modophosphate, diphosphate or triphosphate derivative (i.e., a nucleotide prodrug), for example an ester, that stabilizes the phosphate in viv. In an alternative embodiment of this invention, a stabilized phosphate derivative, as described further 10 below, of FTC, BCH-189, or 3TC is provided for the treatment of hepatits. The disclosed nucleosides, or their pharmaceutically acceptable prodrags or salts or pharmaceutically acceptable formulations containing these compounds are useful in the prevention and treatment of HBV 15 infections and other related conditions such as anti-HBV antibody positive and HBV-positive conditions, chronic liver inflammation caused by BEV, cirrhosis, acute hepatitis, fulminant hepatitis, chronic persistent hepatitis, and fatigue. These compounds or formulations can also be used prophylactically to prevent or retard the progression of clinical illness in 20 individuals who are anti-HBV antibody or HBV-antigen positive or who have been exposed to BEV. In one embodiment of the invention, one or more of the active compounds is administered in alternation or combination with one or more other anti-HBEV agents, to provide effective anti-BEV treatment. Examples 25 of anti-HBV agents that can .be used in alternation or combination therapy include but are not limited to the 2-hydroxymethyl-5-(5-fluorocytosin-1-yl) 1,3-oxathiolane ("FTC", see Wp 92/14743),.its physioldgically acceptable derivative, or physiologically acceptable salt; th.2-hydroxymethyl-5 (cytosin-I-yl)-l,3-oxathiolane (including the racemic BCH-189 form, or 3TC (BCH-189 enriched with the (-)-enantiomer)) its physiologically acceptable derivative, or physiologically acceptable salt; 2'-fluoro-5-ethyl arabinosyluracil (FEAU); carbovir, or interferon. Any method of alternation can be used that provides treatment to the S patient. Nonimiting examples of alternation patterns include 1-6 weeks of administration of an effective amount of one agent followed by 1-6 weeks of administration of an effective amount of a second anti-HBV agent. The alternation schedule can include periods of no treatment, Combination therapy generally includes the simultaneous administration of an effective 10 ratio of dosages of two or more anti-HBV agents. In light of the fact that HBV is often found in patients who are also anti-HIV antibody or HIV-antigen positive or who have been exposed to HIV, the active anti-HBV compounds disclosed herein or their derivatives or prodrugs can be administered in the appropriate circumstance in is combination or alternation.,with and-HIV medications, including but not limited to 3'-azido-3-deoxythymidine (AZT). 2',3'-dideoxyinosine (DDI), 2',3'-dideoxycytidine (DDC), 2',3'-dideoxy-2',3'-didelydrothymidine (D4T), 2-hydroxymethyl-5-(5-floorocytosin-1-yl)-1,3-oxathiolane (FTC), or 2-hydroxymethyl-5-(cytosiq-1-yl)-1,3-oxathiolane (racemic BCH-189 or 20 BCH-189 enriched with the (-)-enantiomer, 3TC). Non-nucleoside RT inhibitors such as the Tibo class of compounds, nevirapine, or pyrimidinone can also be administered in combination with the claimed compounds. The active anti-HBV agents can also be administered in combination 25 with antibiotics, other antiviral compounds, antifungal agents, or other phaimaceutical agents administered for the treatment of secondary Infections. In one embodiment, the nucleoside is provided as a phosphate derivative that is stabilized to decrease or eliminate dephosphorylation prior -7- 0 to uptake into the infected cell. A number of stablized phosphate derivative groups in the 5'-position of the nucleoside are known and have been published in the literature. In one embodiment, the nucleoside is administered as a SATE derivative, as disclosed in iore detail below. Any 5 alternative stablized phosphate derivative can be placed in the 5'-position of the nucleoside that does not materially adversely affect the activity of the compound. Brief Description of the Figures 10 Figure 1 is an illustration of the chemical structures of 5-L-2',3' dideoxycytidine (B-L-FddC), f-D-2',3'-dideoxycytidine (8-D-ddC), B-L 2',3'-dideoxy-5-fluorocytidine (h-L-ddC), (-)-B-L-2-hydroxymethyl-5-(5 fluorocytosin-1-yl)-1,3-oxathiolane ((-)-5-L-FTC). (+)-h-D-2 15 hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-dioxolane ((+)-8-D-FDOC), and h-L-2-amino-6-(R5-9-[(4-hydroxymethyl)-tetahydrofuran-1-yl]purine. Figure 2 is an illustration of the numbering scheme'used in the chemical nomenclature for nucleosides in this text. 20 Detailed Description of the Invention As used herein, the term "enantiomerically pure" refers to a nucleoside comlposition that includes.at least approximately 95%, and preferably approximately 97%, 98%, 99%, or 100% of a single enantiomer of that 25 nucleoside. The term alkyl, as used herein, unless otherwise specified, refers to a saturated straight, branched, gr cyclic, primary. secondary, or tertiary hydrocarbon of C, to Cl 0 , and specifically includes methyl, ethyl, propyl, isopropyl. butyl, isobutyl, :-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmcthyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The alkyl group can be optionally substituted with one or more moieties selected from the group consisting of hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, 5 nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al., "Protective Groups in Organic Synthesis," John Wiley and Sons, Second Edition, 1991. The term lower alkyl, as used herein, and unless otherwise 10 specified, refers to a C, to C 4 ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, or t-butyl group. As used herein, the term acyl specifically includes but is not limited to acetyl, propionyl, butyryl, pentanoyl. 3-methylbutyryl, hydrogen succinate, 3-chlorobenzare, benzoyl, acetyl, pivaloyl, mesylate, 15 propionyl, valeryl, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, and oleic. The term aryl, as used herein, and unless otherwise.specified, refers to phenyl, biphenyl, or naphthyl, and preferably.phenyl. The aryl group can be optionally substituted with pne or mors moieties selected from the group 20 consisting of hydroxyl, amino, alkylamino, arylamino, alkoxy. aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonatc, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al., "Protective Groups in Organic Synthesis," John Wiley and Sons, Second Edition, 25 1991. The term purine or pyrimidine base includes, but is not limited to, adenine, N'-alkylpurines, N-acylpurinfs (wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl), N- izylpurine, N-halopurine. N'-vinylpurine, N'-acetylenic purine. N'-acyl purine, N6-hydroxyalkyl purine, Nd-thialkyl -9-.

purine, N-alkylpurines, N 2 -alkyl-6-thiopurines, thymine, cytosine. 6 azapyrimidine, 2- and/or 4-mercaptopyrmidine, urcil. C alkylpyrimidines, e-baizylpyrimidins, &-halopyrimidines,

&

vinylpyrimnidine, C-acetylenic pyrimidine, C-acyl pyrimidine, C 5 5 hydroxyalkyl purine, C -amidopyrinmdine, C -cyanopyrimidine, C nitropyrimidine, C-aminopyTimidine, Ni-alkylpurines, Nkalkyl-6 thiopurines, 5-azacytidinyl, 5-azanuracilyl, triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl Functional oxygen and nitrogen groups on the base can be protected as necessary or 10 desired. Suitable protecting groups are well kmown to those skilled in the an, and include trimethylsilyl, dimethylhexyLsilyl, t-butyldimethylsilyl, and :-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as acetyl and propionyl, methylsulfonyl, and p-toluylsulfonyl. As used herein, the term natural amino acid includes but is not limited 15 to alanyl, valinyl, leucinylisoiencinyl. proUnyl, phenylalaninyl, tryptophanyl, methloninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, gluaninyl, aspartoyl, glutaoyl. lysinyl, argininyl, and histidinyl. The invention as disclosed herein is a method and composition for the 20 treatment of HBV infection and other viruses replicating in a like manner, in humans or other host animals, that includes administering an effective amount of one or more of the above-identified compounds, or a physiologically acceptable derivative, or a physiologically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier. The 25 compounds of this invention either possess and-HBV activity, or are metabolized to a compound or-compounds that exhibit ant-HBV activity. -10-, . Structure and Preparation of Active Nucleosides Stereochemistr The compounds used in the methods disclosed herein are enantiomers of 2',3'-dideoxycytidine, 2',3'-dideoxy-5-(halo or methyl)cytidine, 2 5 hydroxymethyl-5-(5-fluorocytosin-1-yl)-1.3-dioxolane, or 2-amino-6(OH, Cl, NH 2 , or H)-9-[(4-hydroxymethyl)-tetrahydrofuran-1-yljpurine. Since the 1' and 4' carbons of the sugar or dioxolanyl moiety (referred to below generically as the sugar moiety) of the nucleosides are chiral, their nonhydrogen substituents (CH 2 OR and the pyrimidine or purine base, 10 respectively) can be either cis (on the same side) or trans (on opposite sides) with respect to the sugar ring system. The four optical isomers therefore are represented by the following configurations (when orienting the sugar moiety in a horizontal plane such that the "primary" oxygen (that between the CI' and C4'-atoms; e Figure 2) is in back): cis (with both 15 groups "up", which corresponds to the configuration of naturally occurring rtucleosides), cis (with both groups "down", which is a nonnaturally occurring configuration), trans (with the C2 substituenL"up" and the C5 substituent "down'), and trans (with the C2 substituent 'down' and the C5 substituent "up"). As indicated schematically in Figure 1, the "D 20 nucleosides" are cis nucleosides in a natural configuration and the "L nucleosides" are cis nucleosides in the nonnaturally occurring configuration. The nucleosides useful in the disclosed method to treat HBV infection are B-L-enantiomers, with the exception of PDOC, which is used in its B 25 D-enantiomeric form, because it has been discovered that the 5-D enaitiomer of FDOC is surprisingly less toxic than the 5-L-enantiomer of

FDOC.

rPmdnwl Pormuladnons nudosles dilosed herein can be administered as any denvative that Upon administraion to the recipient, is capable of providing directly or indirectly, the parent active compound, or that exhibits activity in itself. in 5 one embodiment, the hydrogen of the 5'-OH group is replaced by a C-C2o alkyl, including C, to Cs alkyl; acyl in which the non-carbonyl moiety of the ester group is selected from straight, branched, or cyclic C-Cm alkyl including C, to C 5 alkyl, phenyl, or benzyl; a naturally occurring or nonnaturally occurring amino acid; alkoxyalkyl including methoxymethyl; 10 aralkyl including benzyl; aryloxyalkyl such as phenoxymethyl; aryl including phenyl optionally substituted with halogen, C 1 to C 4 alkyl or C to C 4 alkoxy; a dicarboxylic acid such as succinic acid; sulfonate esters such as alkyl or aralkyl sulphonyl including mehanaulfoyl; or a mono, di or triphosphate ester. 15 One or both hydrogens. of the amino groups on the purine or pyrimidine base can be replaced by a C-C2, alkyl, including C, to Cs alkyl; acyl in which the non-carbonyl moiety of the ester group is selected from straight. branched, or cyclic C-C2 alkyl, including C, to C5 alkyl, phenyl, or benzyl; alkoxyilkyl including methoxymethyl; aralkyl including 20 benzyl; aryloxyalkyl such as phenoxymethyl; aryl including phenyl optionally substituted with halogen, C, to C 4 alkyl or C, to C 4 .alkoxy. The active nucleoside can also be provided as a 5'-ether lipid, as disclosed in the following references, which are incorporated by reference herein: Kucera, LS., N. lyer, E. ILake, A. Raben, Modest E.L, D. 25 L.W., and C. Piantadosi, 1990. Novel membrane-interactive ether lipid analogs that inhibit infectious HIV-1 production and induce defective virs formation. AIDS Res Hum Retrovirusds. 6:491-501; Piantadosi, C., I. Marasco CJ., S.L. Morris-Narschke, K.L. Meyer, F. Gumus, L R Surles, K.S. Ishaq, L.S. Kucera, N. Iyer, C.A. Wallen, S. Piantadosi, and -12- EJ. Modest. 1991. Synthesis and evaluation of novel ether lipid nucleoside conjugates for anti-HIV activity. J Med Chem. 34:1408.1414; Hostetler, K.Y., D.D. Richman, D.A. Carson, L.M. Stuhmiller, G.M. T. van Wijk, and H. van den Bosch. 1992. Greatly enhanced inhibition of 5 human immunodeficiency virus type 1 replication in CEM and HT4-6C cells by 3'-deoxythymidine diphosphate dimyristoylglycerol, a lipid prodrug of 3,-dwoxythymidinc. Andmicob ANents Chemother. 36:2025.2029; Hostetler, K.Y., L.M. Stubmiller, H.B. Lenting, H. van den Bosch, and D.D. Richman, 1990. Synthesis and antiretroviral activity 10 -of phospholipid analogs of azidothymidine and other antiviral nucleosides. J. Biol Chem. 265:6112.7. Nucleotide Prodrugs Any of the nucleosides described herein, or any other nucleosidethat 15 has anti-hepatitis B activity., can be administered as a nucleotide prodrug to increase the activity, biavalability, stability or otherwise alter the properties of the nucleoside. A number of nucleotide prodrug ligands are known. A nucleotide prodrug, as described herein, refers to a nucleoside that has a phosphate derivative on the 5'-position that is more stable in vivo 20 than the parent phosphate, and which does not materially adversely affect the anti-hepatits B activity of the nucleoside: Phosphonates arm included as phosphate derivatives. In general. alkylation, acylation or other Upophilic modification of the mono, di or triphosphoate of the nucleoside will increase the stability of the nucleodde. Examples of substituent groups that 25 can replace one or more hydrogens o'n the the phosphate moiety are alkyl, aryl, steroids, carbohydrates, including sugars, 1,2-diacylglycerol and alcohols. Many are described in R. Jones and N. Bischofberger, AntIral Research, 27 (1995) 1-.17. Any, of these can be used in combination with -13the disclosed nucleosides to achieve a desired effect. Nonlimiting examples of nuclotide prodrugs are described in the following references. Ho, D.H.W. (1973) Distribution of Kinase and deaminase of 1p-D arabinofuranosylcytosine in tissues of man and muse. Cancer Res. 33, 5 2816-2820; Holy, A. (1993) Isopolar phosphorous-modified nucleotide analogues. In: De Clercq (Ed.), Advances in Antiviral Drug Design, Vol. I, JAI Press, pp. 179-231; Hong, C.L, Nechacv, A., and West, C.R. (1979a) Synthesis and antitumor activity of 10-D-arabinofuranosylcytosine conjugates of cortisol and cortisone. Biochem. Biophys. Rs. Common. 88, 10 1223-1229; Hong, C.L, Nechaev, A., Kirisits, A.J. Buchheit, D.J. and West, C.R. (1980) Nucleoside conjugates as potential antitumor agents. 3. Synthesis and antitumor activity of 1-(j-D-arabinofuranosyl)cytosine conjugates of corticosteriods and selected lipophilic alcohols. J. Med. hem. 28, 171-177; Hostetler, K.Y., Stuhmiller, L.M., Lenting, H.D.M. 15 van den Bosch, H-. and Richman, D.D. (1990) Synthesis and andretrioviral activity of phospholipid analogs of azidothymidine and other antiviral nucleosides. 1. Bio. Cram. 265, 6112-6117; Hostetler, K.Y., Carson, D.A. and Richman, D.D. (1991); Phosphatidylazidothymidine: mechanism of 20 andretroviral action in CEM cells. J. Bio. Omem. 266, 11714-11717; Hosteler, K.Y., Korba, B. Sridhar, C., Gardener, M. (1994a) Antiviral activity of phosphatidyl-dideoxycytidine in hepatitis B-infected cells and enhanced hepatic uptake in mice. Antvral Res. 24, 59-67; Hostetler, K.Y., Richman, D.D., Sridhar, C.N. Feigner, P.L. Feigner, 1., 25 Ricci, J., Gardener, M.F. Selleseth,'D.W. and Ellis, M.N. (1994b) Phosphatidylazidothymidine and phosphatidyl-ddC: Assessment of uptake in mouse lymphoid tissues and antiviralfactivitics in human immunodeficiency virus-infect4 vells and in rauscher leukemia virus infected mice. Anrlmicrobal Agents Chemoher. 38, 2792-2797; Hunston, -14- R.N., Jones, A.A. McGuigan, C., Walker, R.T.. Baizarini, J., and De Clercq, E. (1984) Synthesis and biological properties of some cyclic phosphotriesters derived from 2 '-deoxy-5-fluorouridine. 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(1990a) Synthesis and evaluation of some novel phosphoramidate derivatives of 3'-azido-3 deoxythymidine (AZT) as and-IIV compounds. Antiviral Chem. Cemother. 1, 107-113; Mcuigan, C., O'Connor, T.J., Nicholls, S.R. 20 Nickson, C. and Kinchington, D. (1990b) Synthesis and anti-IV activity of some novel substituted dialky phosphate derivatives of AZT and ddCyd. Aniviral an. Chemother. 1, 355-360; McGuigan, C., Nicholls, S.,R. O'Connor, TJ., and Kinchington, D. (1990c) Synthesis of some novel dialkyl phosphate derivative of 3-hodified nucleosides as potential and 25 AIDS drugs. Antiviral Chn. Chemother. 1, 25-33; McGuigan, C., Devine, K.G., O'Connor, T.1., and Kinchington, D.(1991) Synthesis and ant-HIV activity of some halalky phosplieramidate derivatives of 3' azido-3'deoxythylmidine (AZT); potent activity of the trichloroethyl methozyalaninyl compound. Andviral Res. 15, 255-263; McGuign, C., -16- Pathirana, R.N., Mahmood, N., Devine, K.G. and Hay. A.J. (1992) Aryl phosphate derivatives of AZT retain activity against HIVI in cell lines which are resistant to the action of AZT. Antvrl Res. 17, 311-321; McGuigan, C., Pathirna, R.N., Choi, S.M., Kinchington, D. and 5 O'Connor, T.J. (1993a) Phosphoramidate derivatives of AZT as inhibitors of WV; studies on the carboxyl terminus. AnfiWral Gen. Chemother. 4, 97-101; McGuigan, C., Pathirana, R.N., Balzarini, Y. and De Clercq, E. (1993b) Intracellular delivery of bioactive AZT nucleotides by aryl phosphate derivatives of AZT. . Med. Cem. 36, 1048-1052. 10 Alky hydrogen phosphonate derivatives of the anti-HIV agent AZT may be less toxic than the parent nucleoside analogue. Aniviral Cem. Chemother. 5. 271-277; Meyer, R. B., Jr., Shuman, D.A. and Robins, R.K. (1973) Synthesis 6f purine nucleoside 3'5'-cyclic phospho'ramidates. Termhedmn Left. 269-272; Nagyvary, y. Gohil, R.N., Kirchner, C.R. and 15 Stevens, J.D. (1973) Studies on neutral esters of cyclic AMP, Biochem. Blophy. Res. Common. 55, 1072-1077; Namane, A. Gouyette, C., Billion, M.P., Fillion, G. and Huynh-Dinh, T. (1992) Improved brain delivery of AZT using a glycosyl phosphotriester prodrug. J. Med. Chem. 35, 3039-3044; Nargeot, J. Nerbonne, J.M. Engels, J. and Lesr, H.A. 20 (1983) Natl. Acad. Sci. U.S.A. 80, 2395-2399; Nelson, K.A., Bentrude, W.G., Stser, W.N. and Hutchinson, J.P. (1987) The question of chair twist equilibria for the phosphate rings of nucleoside cyclic 3''monophosphates. 'HNMR and x-ray crystallographic study of the diasteromers of thymidine phenyl cyclic 3',5'-monophosphate. 1. Am. 25 Chem. Soc. 109, 4058-4064; Nerbonne, J.M., Richard, S., Nargeot, J. and Ister, H.A. (1984) New photoactivatable cyclic nucleotides produce intracellular jumps in cyclic AMP and dyclic GMP concentations. Nature 301, 74-76; Neumann, J.M., fervd, M., Debouzy, J.C., Guerra, F.., Gouyette, C., Dupraz, B. and Huynh-Dihh, T. (1989) Synthesis and -17transmembrane transport studies by NMR of a glucosyl phospholipid of thymidine. J. Am. Chem. Soc. I11, 4270-4277; Ohno, R., Tatsumi, N., Hirano, M., Imai, K. Mizoguchi, H., Nakamura, T., Kosaka, M., Takatuski, X., Yamaya, T., Toyama, K., Yoshida, T., Masaoka, T., 5 Hashimoto, S., Ohshima, T., Kimura, L, Yamada, K. and Kimura, J. (1991) Treatment of myelodysplastic syndromes with orally administered 1-p-D-rabinofuranosylcytosine -5'stearylphosphate. Oncology 48, 451 455. Palomino, E., Kessle, D. and Horwitz, J.P. (1989) A dihydropyridine 10 carrier system for sustained delivery of 2',3'dideoxynucleosides to the brain. J. Med. Chem. 32, 622-625; Perkins, R.M., Barney, S., Wittrock, R., Clark, P.H., Lavin, R. Lambert, D.M., Petteway, S.R. Sezafinowska, H.T., Bailey, S.M., Jackson, S., Harnden, M.R. Ashton, R., Sutton, D., Harvey, J.J. and Brown, A.G. (1993) Activity of 15 BRL47923 and its oral prodrug, SB203657A against a rauscher marine leukemia virus infection in mice. Antiviral Res. 20 (Suppl. I). 84; Piantadosi, C., Marasco, C.J., Jr., Morris-Natschke, S:L., Meyer, K.L., Gumus, F., Surles, J.R., Ishaq, K.S., Kucera, L.S. Iyer, N., Wallen, C.A., Piantadosi, S. and Modest, E.J. (1991) Synthesis and evaluation of 20 novel ether lipid nucleoside conjugates for anti.-IV-I activity. 1. Med. Chem. 34, 1408-1414; Pompon, A., Lefebvre, L, Imbach, J.L;, Kahn, S. and Farqubar, D. (1994) Decomposition pathways of the mono- and bis(pivaloyloxymethyl) esters of azidothymidine-5'-monophosphate in cell extract and in tissue culture mediurn; an application of the 'on-line ISRP 25 cleaning' HPLC technique.. Antiviral Chem. Chemother. 5, 91-98; Postmark, T. (1974) Cyclic AMP and cyclic GMP. Annu. Rev. Pharmacol. 14, 23-33; Prisb% E.J., Martin, J.C.M., McGee, D.P.C., Barker, M.F., Smee, D.F. Duke, A.E., Matthews, T.R. and Verheyden, J.P.J. (1986) Synthesis and antiherpes virus activity of phosphate an -18-, phosphonate derivatives of 9 -[(1,3-dihydroxy-2-propxy)mcthyl] guanine. . Med. Chem. 29, 671-675; Pucch, F., Gosselin, G., Lefebvre, L, Pompon, A., Aubertin, A.M. Dim, A. and Imbach, J.L. (1993) Intracellular delivery of nucleoside monophosphate through a reductae 5 mediated activation process. Antiviral Res. 22, 155-174; Pugaeva, V.P., Klochkeva, S.I, Mashbits, F.D. and EizengartR.S..(1969). Toxicological assessment and health standard ratings for ethylene sulfide in the industrial atmosphere. Gig. Trf. Prof. Zabol. 13, 47-48 (Chem. Abstr. 72, 212); Robins, R.K. (1984) The potential of nucleotide 10 analogs as inhibitors of retroviruses and tumors. Pharm, Res. 11-18; Rosowsky, A., Kim. S.H., Ross and J. Wick, M.M. (1982) Lipophilic 5' (alkylphosphate) esters of 1-0-D-arabinofiranosylcytosine and its M-acyl and 2.2'-anhydro-3'0-acyl derivatives as potential prodrugs. 3. Med. Chem. 25, 171-178; Ross, W. (1961) Increased sensitivity of the walker 15 turnout towards aromatic nitrogen mustards carrying basic side chains following glucose pretreatment. Blochem. Pharm. 8, 235-240; Ryu, ec, Ross, R.J. Matsushita, T., MacCoss, M., Hong, C.I. and West, C.R. (1982). Phospholipid-nucleoside conjugates. 3. Synthesis and preliminary biological evaluation of l-0-D-arabinoftranosylcytosine 5'diphosphatef-], 20 2-diacylglycerols. J. Med. Chem. 25, 1322-1329; Saffhill, R. and Hume, W.J. (1986) The degradation of 5-iododeoxyurindine and 5 bromoeoxyuridine by serum from different sources and its consequences for the use of these compounds for incorporation into DNA. Chem. Biol. Interact. 57, 347-355; Saneyoshi, M., Morozuml, M., Kodama, K., 25 Machida, J., Kuninaka, A. and Yoshino, H. (1980) Synthetic nucleosides and nucleotides. XVI Synthesis and biological evaluations of a series of 1-0-D-arabinofuranosylcytosine 5'-aky-orarylphosphates. Chem. Pharm. Bull. 28, 2915-2923; Sastry, J. ., Nehete, P.N., Khan, S., Nowak, B.J., Plunkett, W., Arlinghaus, R.B. and Farquhar, D. (1992) Membrane -19permesble dideoxyuridine 5'-monophosphate analogue inhibits human immunodeficiency virus infection. Mol. Pharmacol. 41, 441-445; Shaw, J.P., Jones, R.J. Arimilli, M.N., Loue, M.S., Lee, W.A. and Cundy, K.C. (1994) Oral bioavailability of PMEA from PMEA prodrugs in male S Sprague-Dawley rats. 9th AnnWal AAPS Meeting. San Diego, CA (Abstract). Shuto, S., Ueda, S., Imamura, S., Fukukawa. K. Matsuda, A. and Ueda. T. (1987) A facile one-step synthesis of 5'phosphatidylnucleosides by an enzymatic two-phase reaction. Tetrahedron Lett. 28, 199-202; Shuto, S.; Itoh, H., Ueda, S., Imamura, 1q S., Kukukawa, K., Tsujino, M., Matsuda, A. and Ueda, T. (1988) A facile enzymatic synthesis of 5'-(3-sn-phosphatidyl)nucleosides and their antileukemic activities. Chem. Pharm. Bull. 36, 209-217. A preferred phosphate prodrug group is the S-acyl-2-thioethyl group, also referred to as "SATE". Praation of the Active Comnomds The nucleosides used in the disclosed method to treat HBV infections in a host organism can be prepared according to published methods. 8-L Nucleosides can be preparedifrom methods disclosed in, or standard 20 modifications of methods disclosed in, for example, the following publications: Jeong, et al., J. of Med, Chem., 26, 182-195, 1993; European Patent Application Publication No. 0 285 884; Gdnu-Dellac, C., G. Gosselin, A.-M. Aubertin, G. Obert, A. Kirn, and J.-L. Imbach, 3 Substituted thymine a-L-nucleoside derivatives as potential antiviral agents; 25 synthesis and biological evaluation, Antiviral Chem. Chemother. 2:83-92 (1991); Johansson, K. N. G., B. G. Lindborg, and R. Noreen, European Patent Application 352 248; Mansuri .M'. M., V. Farina, J. E. Starrett, D. A. Benigni, V. Brankovan, agn J. C. Martin, Preparation of the geometric isomers of DDC, DDA, D4C and D4T as potential anti-HV agents, Binnru. Mad. Chem. -1t 1:65-68 (1991); Fujimori, S., N. Iwanami, Y. Hashimoto, and K. Shudo, A convenient and stereoselective synthesis of 2'-deoxy-B-L-ribonucleosides, Nuclensides & Nucleotides 1:341-349 (1992); Gdnu-Dellac, C., G. Gosselin, A.-M. Aubertin, G. Obert, A. 5 Kim, and J.-L. Imbach, -3-Substituted thynine a-L-nucleoside derivatives as potendal antiviral agents; synthesis and biological evaluation, Antivial Chem. Chemother. 2:83-92 (1991); Holy, A, Synthesis of 2'-deoxy-L uridine, Tetahedron . 2:189-192 (1992); Holy, A., Nucleic acid components and their analogs. CLI, Preparadon of 2'-deoxy-L 10 ribonucleosides of the pyrimidine series. Collect Czech Chem Commun. 37:4072-4087 (1992); Holy, A, 2'-deoxy-L-uridine: Total synthesis of a uracil 2'-deoxynucleoside from a sugar 2-aminooxazoline through a 2.2' anhydronucleoside interediate. In: Townsend LB, Tipson RS, ed. Nucleic Acid Chem. New York: Wiley, 1992: 347-353. vol 1) (1992); 15 Okabe, M., R.-C. Sun, S,. Tan, L. Todaro, and D. L. Coffee, Synthesis of the dideoxynucleosides ddC and CNT from glutamic acid, ribonolactone, and pyrimidine bases. T Qrg Chem. 53:4780-4786 (1988); Robins, M. J., T. A. Khwja, and R. K. Robins. Purine nucleosides. XXI. Synthesis of 21-deozy-L-adenosine and 21-deoxy-L-guanosine and 20 their alpha anomers. I rM Chem. 35:363-639 (1992); GEnu-Dellac, C., Gosselin G., Aubertin A-M, Obert G., Kirn A., and Imbach J-L, 3' Substituted thymine cz-L-nucleoside derivatives as potential antiviral agents; synthesis and biological evaluation. Antiviral Chcm. Chemother. 2(2):83 92 (1991); Genu-Dellac, C., Goselin G., Imbach J-L; Synthesis of new 25 2'-deoxy-3'-substituted-a-L-threo-entofranonucleosides of thymine as a potential antiviral agents. Tet La 32(1):79-82 (1991); Gdnu-Dellac, C., Gosselin G., Imbach J-L. Preparatio4 of new acylatd derivatives of L arabino-furanose and 2-deoxy-1-eythro-pentofuranose as precursors for the synthesis of 1-pentofuranosyl nucleosides. 216:240-255 (1991); and Genu- Dellac, C., Goselin G., Puech F, et a1. Systematic synthesis and antiviral evaluation of a-L-arabinofuranosyl and 2'-deoxy-aeL-crythro-pento furanosyl nucleosides of the five natural occurring nuclei acid bases. 10(b):1345-1376 (1991). 5 2',3'-Dideoxycytidine (DDC) is a known compound. The D enantiomer of DDC is currently being marketed by Hoffnan-LaRoche under the name Zalcitabine for use in the treatment of persons infected with HIV. See U.S. Patent Nos. 4,879,277 and 4,900,828. Enantiomrically pure 8-D-dioxolane-nucleosides such as B-D-FDOC 10 can be prepared as disclosed in detail in PCT/US91/09124. The process involves the initial preparation of (2R,4R)- and (2R,4S)-4-acetoxy-2 (protected-oxymethyl)-dioxolane from 1,6-anhydromannose, a sugar that contains all of the necessary stereochemistry for the enantiomerically pure final product, including the correct diastereomeric configuration about the 15 1 position of the sugar (tht becomes the 4-position in the later.formed nucleoside). The (2R,4R)- and (2R,4S)-4-acetoxy-2-(protected-4 oxymethyl)-dioxolane is condensed with a desired heterocyclic base in the presence of SnC! 4 , other Lewis acid, or trimethylsilyl triflate in an organic solvent such as dichloroethane, acetonitrile, or methylene chloride, to 20 provide the stereochemically pure dioxolane-nucleoside. Enzymatic methods for the separation of D and L enantiomers of cis nucleosides are disclosed in, for example, Nucleosides and Nucleotides, 12(2), 225-236 (1993); European Patent Application Nos. 92304551.2 and 92304552.0 filed by Biochem Pharma, Inc.; and PCr Publication Nos. 25 WO 91/11186, WO 92/14729, and WO 92/14743 filed by Emory University. Separation of the acylated or alkylated racemic mixture of D and L enantiomers of cis-nucleoside* can be accomplished by high performance -22-.

liquid chromatography with chiral stationary phases, as discosed in PCr Publication No. WO 92/14729. Mono, di, and triphosphate derivative of rt active nucleosides can be prepared as described according to published methods. The 5 monophosphate can be prepared according to the procedure of Imai et al., J. Or&. Chem., 34(6), 1547-1550 (June 1969), The diphosphate can be prepared according to the procedure of Davisson et al., J. Qr. m.. 52(9), 1794-1801 (1987). The triphosphate can be prepared according to the procedure of Hoard et al., J. Am. Chem. Soc., 87(8), 1785-1788 10 (1965). General Procedures for thePreparation of BIs (S-acvi-2-thioetby Phosphoester of B-LdIdeoxvucleosides ffis (SATE 8-L ddxMl 15 0 SH' F 2P 20 Base 20 0 25 Bis (SATE) A-L-ddxMP 30 Y', , and Y 4 are ind gently H, OH, N, N'Rt NO 1 , NOR, 0-alkyl, -23- -0-aryl, halo (mcluding F, Cl, Br, or 1). -CN. -C(O)NH 2 , SH, -S-aikyl, or -S-aryl, and wherein typically three of Y', y2 y,, and y' are either H or OH. The -OH substituent, when present, is typically a Y' or Y group. As illustrated in the structure, Y2 and Y are in the arabino (erythro) 5 configuration, and Y' and Y are in the thro (ribose) configuration. The base is a purine or pyrimidine. Alternatively, the psuedo-sugar moiety is a 1,3-oxathiolane (as in FTC and BCH-189 or 3TC or is a 1,3-dioxolane derivative). (1) ICH 2

CH

2 OH, DBU/CdHSCH; (ii) CI 2 PN(iPr), NFt/THF; (iii) A-L-dideoxynucleoside, 1H-tetrazole/THF. then 10 CICJL4CO 1 H/CH2Cl 2 1H-Tetrazole (0.21g. 3.0 mmol) was added to a stirred solution of j-L-dideoxynucleoside (1.0 mmol) and the appropriate phosphoramidite .C (1.2 mmol) in tetrahydrofuran (2mL) at room temperature. After 30 minutes, the reaction mixture was cooled to -40'C and a solution of 3-chloroperoxybenzoic acid (0.23 g, 1.3 mmol) in 15 dichloromethane (2.5 mL),was added; the mixture was then allowed to warm to room temperature over 1 h. Sodium sulfite (10% solution, 1.3 mL) was added to the mixture to destroy the excess 3-choroperoxybenzoic acid, after which die organic layer was separated and the aqueous layer washed with dichloromethane k2 x 10 mL). The combined organic layers 20 were washed with saturated aqueous sodium hydrogen carbonate (5 mL), then water (3 x 5 mL), dried over sodium sulfate, filtered and evaporated to dryness under reduced pressure. Column chromatography of the residue on silica gel afforded the title Bis(SATE) 0-L-ddxm2. -24- EXAMPn = -L-2',3'-Dideoxyadenosin-5'-yl bis (2-pivaloylthioethyl) phosphate [Bis (SATE) P-L-ddAME]. sC (c0-c e,-o PNQjPrh then CICdH4COiHCH2C 2 then silica -e column chromalography 10 N NH (cNac-c /cH-'o o ND . 20 15 Bis (SATE)0L-ddM 20 1M Following the above general procedure, pure Bis(SATE)-L-ddAMP was obtained as a colorless oil in 72% yield after silica gel column chromatography fluentt: stepwise gradient of methanol (0-3%) in dichloromethane]; ' NMR (DMSO,- d) 6 ppm: 8.26 and 8.13 (2s, 2H 25 each, H-2 and H-8), 7.20 (br s, 2H; NH2), 6.24 (t, 1H, H-1'; J=6.0 Hz), 4.35 - 4.25 (m, 1H, H-4'), 4.25-4.00 (in, 2H, H-5', 5"), 3.96 (m, 4H, 2

SCH

2

CH

2 O), 3.04 (t, 4H, 2 SCH'Ho ;.J = 6.3 Hz), 2.5-2.4 (m, 2H, H-2',2") 2.2-2.0 (m. 2H, H-5',3"), 1.15 [s, 18H, 2 (CH 3

)

1 C]; " 1 E'NMR (DMSO-d 6ppm= -076 (s) ;UV (EtOH) ,X 259 m (s 15400); -25mass spectrum (performed in: glycerol, thioglyccrol, 1:1, u/u), FAB>0 604 (M+H)*, 136 (BH*. General scheme for the stersneifc syntesl of 3%ubstituted 8-L:: 5 dideornucesides aH RO 0 Compound (se Fig. 1/2 t 10 the Proch pamnj Sae AppendiX 4 RO ox 15 20 1 a1. Ro- H 25 300 Y <<aryrhro>> 35 n <cihyo> 40 configunMU 0 V = acyl (CHyC, C 6 HS-C) -26- X group [CH, SO, CH CSI So, CF, So2] Y, Y' - F, N, NRR 2

[R

1 ,R2 = H, alkyl. aryi], NO, NOR [it = H, alkyl. acyl], 0-alkyl, O-aryl, etC. EXMPLE - 1-(3-Aido-2-34ideoxy-L-cythro-pentofuranosyl) 5 thynine [s-L-AZT H, N CH 1.0 N 10 Z...>N P(P{) H DEAD TH NN is - - -/ ACOH/Hz 00 CHH 20 0 13-L-AZT 40i Yield (mm a 25 30 -27- A mixture of diethyl azodicarboxylate (0.46 nil; 2.9 mmol) and diPhenyl phosphorzldatC (0.62 ml; 2.9 mmol) in THP (2.9 ml) was added dropwisc over 30 min. to a solution of 1.(2-dcoxy-5-O-monomethoxytrityl P-L-threo-pentofurInosyl) thymine A [0.5 g. 0.97 mmol] and 5 triphenylphosphinc (0.76g, 2.9 mmol) in THF 11.6 ml) at O'C. The mixture was stined for 3.5h at room temperature, and ethanol was added. After concentration to dryness in vacuo, the residue was dissolved in a mixture of acidic acid (240 ml) and water (60 ml) in order to remove the mMTr protecting group. The mixture was stirred for 5 hours at room 10 temperature and was diluted with toluene. The separated aqueous phase was concentrated to dryness in vacuo. The residue was purified over a silica gel column eluted with ethyl acetate to afford P-L-AZT (105 mg, 40%, crystallized from ethyl acetate). T1e physicochemical data of 0-L AZT were in accordance with literature data [J. Wengel, J-Lau, E.B. 15 Ledersen, C.N. Nielsen, L Org. Chem. j (11), 3591-3594 (1991)). -28- CeWMn ScheM for thn Stereneafc-Smb of 2'-su thted Mea dkleastnucleosdes A0 Compound U2 5 (s Rg. U of fhe Frtncb paneml sde Appendix 4 ov Ro- Base 10 goH ao I- 0 AX 15

-

o aihree~ 200 wecrydhron confgurtion 25 o 0 V = acyl (CH 3 -C C 6 HrC] X = Leaving group [CH, S02, CH 3

CJ

4 S0 2 ,H, CF 3 SO21 Y, Y' = F, N 3 , NRIR 2

[R

1

,R

2 = H, alkyl, aryl], 30 NO2, NOR [R = H, alkyl, acyl], 0-alky-, 0-aryl, etc. -29- EXAMPLE - 1-( 2 -Fluoro-2,3-dideoxy-p-L- petofuranosyl)-5 fluorocytosine (2'-F-P-L P-L-FddC] 0p 5r o N l oN Bzo DBU CH CN. 1 cO 0 '1 cDAST. 10 sCH 1 C1 H NL~cN 3 NN Ho, 15

CH

3

CNH

2 O 9 - 2 5y64%eyildd IMapcndCH 2

C

2 2 t2 20[F szo\>o aa 2'-F-0L-Fddc 25 64% yicld Hitherto unknown 2'-F-P-L-FddC was synthesized in five steps from 1-(5-0-benzoy13-deoxy--L ythw-pentofuranosyl)-5-fluOrouracil 12 with an ovell yield of 28%. m.p. 209-21rC (crystallized from absolute -30ethanol); UV (Et OH) ... 276 j(a, 9000). X. 226. (a, 4000); "F NMR (DMSO-d 4 ) 5 ppm : -179.7 (m, Fr) , -167.2 (dd, Fs; J,, = 7.3 Hz, JF.I. = 1.5Hz); IH-NMR (DMSO-d 6 ) 5ppm: 8.30 (d , IH, H-6; Js - 7.3 Hz), 7.8-7.5 (r s, 2H, NH 2 ), 5.80 (d, 1H, H-l' 1,,, = 17.4 Hz), 5.34 (t, 5 IH, OH-5'; J = 4.8 Hz), 5.10 (dd, IH, H-2'; 2g = 51.2 Hz; 12,r3' 3.4 Hz), 4.3 (m, 1H, H-4'), 3.8-3.6 (m, 2H, H-5',5"), 2.2-2.0 (m, 2H, H-3', H-3"); mass specta performedd in: glycerol-thioglycero, 1:1 u/u), FAB>O:248 (M+H*. 130 (BH2?; FAB<0:246 (M-H)~ ; (a 16.5 (-c 0.85, DMSO). Anal. Calc. for C,H 1

N

3 0 3

F

2 : C, 43.73; H, 10 9.49; N. 17.00; F. 15.37. Found: C, 43.56; H, 4.78; N, 16.75; F, 14.96. -31-.

L-Xylosc 'k 50 - 0,50,. Aba 14WOH s a/ C*HSCOCJ 0 7co osz IOH Rrs0 1 13 o 1) 5 : 7 -0 /TIW doin oCt su 14SO 0 2 7N NSn.2 H1 MzO- IzrO 0 OA Pr-ne- H> S4-"S I)~2 cBaccSnc1 AMN 300 20 he U:Ba ptn vpdiictww~eftcnehlfM mted -~~HOIYO Emcl(r cy A) 4 0 HOuiy(TCb0dIhY4 1 OFFBDPSI),O NHj35 -~ H32 if. Anti-HBV Activity of Nucleosidn The ability of the active compounds to inhibit HBV can be measured by various experimental techniques. The assay used herein to evaluate the 5 ability of the disclosed compounds to inhibit the replication of HBV is described in detail in Korba and Gerin, Antiviral Res. 19: 55-70 (1992). For purposes of illustration only, and without limiting the invention, the results of the evaluation of toxicity and anti-RBV activity are provided below for B-L-2',3'-didcoxycytidine (B-L-FddC), B-L-2',3*-dideoxy-5 10 fluorocytidine (2-L-ddC), and (+)-8-D-2-hydroxymethyl-5-(5 fluorocytosin-1-yl)-1,3-dioxolane ((+)-B-D-FDOC). The toxicity and anti HBV activity of (-)-8-L-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3 oxathiolane ((-)-B-L-FTC) and B-D-2',3'-dideoxycytidine (B-D-ddC) are included as controls. The other compounds disclosed herein can be 15 evaluated similarly. The samples of B-L-ddC and R-L-5-FddC used in the anti-HBV assays were characterized as follows. 2'.3'-Dideoxy-B--ctidine (8-L-DDC. m.p. = 220-220*C; UV 20 (EtOW 95) max 273 nm, Amin 252 nm; NMR-'H (DMSO-d) 5ppm 7.89 (d. IH. H-6; J = 7.4 Hz). 7.15-6.95 (d large, 2H, NH2), 5.91 (dd. 1H, H-1'; J - 3.0 et 6.5 Hz), 5.66 (d, 1, H-5; J = 7.4 Hz), 4.99 [t. IH, OH-5'; J - 5.2 Hz]. 4.05-3.95 (m, IH, H4'), 3.60-3.70 (m, 1H, H 5'; after D20 exchange: dd, 3.64:ppm, J = 3.6 et 12.0 Hz). 3.60-3.50 25 (m. 1H, H-5"; after D20 exchange: dd, 3.50 ppm, J = 4,1 et 12.0 Hz), 2.30-2.15 (m. IH, H-2', 1.9-1.65 (m. 3H, H-2", 3' et 3"); [aLD2-103.6 (c 0.8 MeOH); mass spectrum (performed in: glycerol-thioglycerol, 50: 50. v/v); FAB>0 423 (2M+H]*, 304 [M+glycerl+H]r. 212 [M+H]*, 112 [BH2]*, 101 [s]'; FAB<0 210 fM-HT. Anal. Cak.for -33-.

C,H 1N 3

O

3 (M - 211.21); C 51.18; H 6.20; N 19.89 found; C 51.34; H 6.25; N 20.12. 2 '-Dideoxy4L-fluoroc'tidie (h-M-FDDL m.p. = 158 160*C; UV (EtOH 95) %max 281 nm (s, 8100) ct 237 nm (s, 8500); min 5 260 nm (e, 5700) et 225 nm (a, 7800); NM - 'H (DMSO-d 6 ) 6ppm 8.28 (d. IM, H-6; J - 7.4 Hz), 7.7-7.4 (d large, 2H,,NH 2 ), 5.83 (dd poorly resolved, HI, H-l'), 5.16(t. IH, OH-5'; J = 5.1 Hz), 4.05-3.95 (m, IM, H-4'), 3.8-3.70 [m,1H, H 5'; after D20 exchange: dd, 3.71 ppm. J = 2.7 et 12.3 Hz], 3.60-3.50 (m. 1H, H-5"; after D0 exchange: dd, 3.52 gin; 10 I = 3.3 et 12.3 Hz]L 2.35-2.15 (m, 1H, H-2'). 1.95-1.75 (m, 3H, H-2", 3 et 3"): [ab)"-80.0 (-c 1.0, DMSO); Mass spectrum [performed in: 3 nitrobeuzyl alcohol] FAB>0 230 [M+H]+ et 101 [s]*; FAB< O 228 [M 1]'. Anal. Calculatedfor C,H 1 NFO,(M = 229.21); C 47.16; 115.28; N 18.33, F 8.29, Found. C 16.90; H 5.28; N 18.07; F 8.17. 15 The antiviral evaluations were performed on two separate passages of cells; two cultures per passage (4 cultures tol). All wells, in all plates, were seeded at the same density and at the same time. Due to the inherent variations in the levels of both intracellular and extracellular HBV DNA, only depressions greater than 3.0-fold (for HBV 20 virion DNA) or 2.5-fold (for HBV DNA replication intermediates) from the average levels for these HBV DNA forms in untreated cells are generally considered to be statistically significant [P<0,O5] (Korba and Gerin, Antiviral Res. 19; 55-70, 1992). The levels of integrated HBV DNA in each cellular DNA preparation (which remain constant on a per 25 cell basis in thes experiments) were used to calculate the levels of intracellular HBV DNA forms, thereby eliminating technical variations inherent in the blot hybridization assays. Typical values for extracc4ular HEV vision DNA in untreated cells range from 50 to 150 pg/mli culture medium (average of approximately 76 -34pg/ml). Intracellular HEV DNA replication intermediates in unteated cells range from 50 to 100 pg/ug cell DNA (avenge approximately 74 pg/ug cell DNA). In general, depressions in the levels of inrUcellular HBV DNA due to treatment with antiviral compounds are less pronounced, 5 and occur more slowly, than depressions in the levels of HBV vision DNA. For reference, the manner in which the hybridization analyses were performed for these experiments results in an equivalence of approximately 1.0 pg intracellular HBV DNA/ug cellular DNA to 2-3 genomic copies per cell and 1.0 pg of extracellular HBV DNA/mil culture medium to 3 x 10 30 viral particles/ml. Toxicity analyses were performed in order to assess whether any observed antiviral effects were due to a general effect on cell viability. The method used was based on the uptake of neutral red dye, a standard and widely used assay for cell viability in a variety of virus-host systems, 15 including HSV (herpes simplex virus) and HiV. The test compounds were used in the form of 40 mM stock solutions in DMSO (frozen on dry ice). Daily aliquots of the test samples were made and frozen at -2(*C so that each individual aliquot would be subjected to a single freeze-thaw cycle. The daily test aliquots were thawed, suspended 20 into culture medium at room temperature and immediately added to the cell cultures. The compounds were tested at 0.01 to 10 sM for antiviral activity. The compounds were tested for toxicity at concentrations from 1 to 300 yM. The results are provided in Table 1. -35- It c-i'0 .1dl rw go. - cocn 0 q t- 00 m 4 r' 4 Xe t-o~tSD ~ -c'ir6f Example 2 Toxicity Of Compounts The ability of the active compounds to inhibit the growth of virus in 2.2.15 cell cultures (HepG2 cells transformed with hepatitis vision) was evaluated. As illustrated in Table 1. no significant toxicity (greater than 5 50% depression of the dye uptake levels observed in untreated cells) was observed for any of the test compounds at the concentrations 100 pM. The compounds were moderately toxic at 300 pM, however, all three compounds exhibited less toxicity at this concentration than B-D-ddC. It appears that the IC, 0 of 8-L-ddC and B-L-FddC is approximately twice that 10 of 8-D-ddC. Toxicity analyses were performed in 96-well flat bottomed tissue culture plates. Cells for the toxicity analyses were cultured-and treated with test compounds with the same schedule as used for the antiviral evaluations. Each compound was tested at 4 concentrations, each in 15 triplicate cultures. Uptake of neutral red dye was used to determine the relative level of toxicity. The absorbance of internalized dye at 510 nM (Asio) was used for the quantitative analysis. Values are presented as a percentage of the average A5 10 values (± standard deviations) in 9 separate cultures of untreated cells maintained on the same 96-well plate as the test 20 compounds. The percentage of dye uptake in the 9 control cultures on plate 40 was 100 ± 3. At 15b-190 pM B-D-ddC, a 2-fold reduction in dye uptake (versus the levels observed in untreated cultures) is typically observed in these assays (Korba and Gerin, Antiviral Res. 19: 55-70, 1992). 25 -37- Example 3 Anti-Hepatitis B Virus Activity lie positive treatment control, B-D-2',3'-dideoxycywsine [h-D-dC], induced significant depressions of BV DNA replication at the concentration used. Previous studies have indicated that at 9-12 pM of 8 S D-ddC, a 90% depression of HEV RI (relative to average levels in untreated cells) is typically observed in this assay system (Korba and Gerin, Antiviral Res. 19: 55-70, 1992). This is consistent with the data presented in Table 1. The data presented in Table I indicates that all three test compounds 2.0 ((B-L-FddC), (B-L-ddC), and B-D-FDOC)), were potent Inhibitors of HBV replication, causing depression of HBV vision DNA and HBV RI to a degree comparable to, or greater than, that observed following treatment with S-D-ddC. Example 4 15 The effect of selected t-L-derivatives against Hepatitis B virus replication in transfected Hep G-2 cells is described in Table 4. C .4 -38ett II P. c Iin Um C' .2a a io C C2,C Example 5 The Comparative inhibitory effect of selected triphospahtes on woodchuck hepatitis virus DNA polymerase is set out in Table 5. 5 Table 2: Comparative Inhibitory activities of ,nucleoside triphosphates on woochuck hepatitis virs DNA polymerase and human DNA polymerase a and P. Inhibitor WHB DNA Pol DNA Pol a DNA Po ICso (pM) Ki ( M) Ki (piM) P-L-AZTPP 0.2 >100 >100 0-L-ddATP 2.1 >100 >100 3-TC-T 1.0 >100 >100 0-L-5FDDCTP 2. >100 ->100 10 IH. Preparation of Pharmaceutical Compositions The compounds disclosed herein and their pharmaceutically acceptable salts, prodrugs, and derivatives, are useful in the prevention and treatment of HBV infections and other related conditions such as anti-HEV antibody positive and RBV-positive conditions, chronic liver inflammation caused 15 by RBV, cirrhosis, acute hepatitis, fulminant hepatitis, chronic persistent hepatitis, and fatigue. These compounds or formulations can also be used prophylactically to prevent or retard the progression of clinical illness in individuals who are anti-HBV antibody or HBV-andgen positive or who have been exposed to HBV. 20 Humans suffering from any of these conditions can be treated by administering to the patient an effective HBV-tnatment amount of one or a mixture of the active compounds described herein or a pharmaceutically acceptable derivative or salthereof, optiohally in a pharmaceutically acceptable carrier or diluent. The active materials can be administered by -40any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutanmusly, or topically, in liquid or solid form. The active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a 5 therapeutically effective amount without causing serious toxic effects in the patient treated. A preferred dose of the active compound for all of the above mentioned conditions will be in the range from about I to 60 mg/kg, preferably 1 to 20 mg/kg, of body weight per day, more generally 0.1 to 10 about 100 mg per kilogram body weight of the recipient per day. The effective dosage range of die pharmaceutically acceptable derivatives can be calculated based on the weight of the parent nucleoside to be delivered. If the derivative exhibits activity in itself, the effective dosage can be estimated as above using the weight of the derivative, or by other means is known to those skilled in the art. In one embodiment, the active compound is administered as described in the product insert or Physician's Desk Reference for 3-azido-3'-deoxythymidiMe (A2T), 2',3dideoxyinosine (IDD), 2',3'-dideoxycytidine (DDC), or 2',3'-dideoxy-2',3' didehydrothymidine (D4T) for y indication. 20 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. A oral dosage of 50-1000 mg is usually convenient. Ideally the active ingredient sh'oild be administered to achieve p 25 plasma concentrations of the active compound of from about 0.2 to 70 pM, preferably about 1.0 to 10 pLM. This may be achieved, for example, by the intravenous injection of a 0. to 5% solution of the acive ingredient, optionally in saline, or adminiftered as-a bolt* of the active ingredient. -41- The active compound can be provided in the form of pharmaceutically acceptable salts. As used herein, the term pharmaceutically acceptable salts or complexes refers to salts or complexes of the nucleosides that retain the desired biological activity of the parent compound and exhibit minimal, if 5 any, undesired toxicological effects. Nonlimiting examples of such salts are (a) acid addition salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and die like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic 10 acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, and polygalacturonic acid; (b) base addition salts formed with cation; such as sodium, potassium, zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the 15 like, or with an organic cation formed from N,N dibenzylethylene-diamine, ammonium, or ethylenediamine; or (d) combinations of (a) and (b); e.g., a zinc tannate salt or the like. Modifications of the active compound, specifically at the N' or N' and 5'-O positions, can affect the bioavailability and rate of metabolism of the 20 active species, thus providing control over the delivery of the active species. The concentration of active compound in the drug composition will depend on absorption, inactivation, and excretion rates of the drug as well as other factors known to those of Sil in the art. It is to be noted dat 25 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 shod be adjusted over time according to the individual need and the professional judgment of the person administering -42or supervising the administration of the compositions, and that the concentration ranges set forth herein an exemplary only and are not intended to limit the scope Of practice of the claimed composition. The active ingredient may be administered at once, or may be divided into a 5 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 administration, the active 10 compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, Md/or adjuvant materials can be 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 i5 microcrystalline cellulose,'gum tragacanth 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 agedt such as peppermint, methyl salicylate, or 20 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. 25 -The active compound.or phamaceutically acceptable salt or derivative thereof can be administered as a component of an elixir, suspension. syrup, wafer, chewing gum or the lige. A syrup may contain, in addition to the -43-.

active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavor. The active compound, or pharmaceutically acceptable derivative or salt thereof can also be mixed with other active materials that do not impair the 5 desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, antiinflammatories, br other andvirals, including anti-HBV, anti-cytmegalovirus, or anti-HIV agents. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: lo 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 ethylenediamineteraacetic acid; buffers such as acetates, citrates or is 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 intraveinously, preferred carriers are physiological 20 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. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl 25 acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled iathe art. the-materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. -44 Liposomal suspensions (including Uposomes 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 5 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 10 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 triphosphate derivatives are 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, thereby forming the liposomal '15 suspension. This invention has been described with reference to its preferred 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 20 be included within the scope of the appended claims. -45-.

Claims (3)

1. 46. The claims defining the invention are as follows: 1. A method of treating a patient infected with hepatitis B virus and HIV comprising administering to the patient a nucleotide prodrug of p-L-2',3'-dideoxyadenosine (P L-DDA) in combination with a second compound selected from: 5 (a) 3'-azido-3'-deoxythymidine (AZT); (b) 2',3'-dideoxyinosine (DDI); (c) 2',3'-dideoxy-2',3'-didehydrothymidine (D4T); (d) 2-hydroxymethyl 5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC); (e) a Tibo compound, nevirapine, or a pyrimidinone; or 10 (f) a physiologically acceptable salt thereof. 2. The method of claim 1, wherein the nucleotide prodrug of P-L-2',3' dideoxyadenosine (p-L-DDA) is administered in enantiomerically enriched form. 3. The method of claim 1, wherein the p-L-2',3'-dideoxyadenosine nucleotide is a monophosphate. 15 4. The method of claim 1, wherein the p-L-2',3'-dideoxyadenosine nucleotide is a diphosphate. 5. The method of claim 1, wherein the p-L-2',3'-dideoxyadenosine nucleotide is a triphosphate. 6. The method of claim 1, wherein the p-L-2',3'-dideoxyadenosine nucleotide prodrug 20 is an alkylated nucleotide. 7. The method of claim 1, wherein the p-L-2',3'-dideoxyadenosine nucleotide prodrug is an acylated nucleotide. 201226844_1
2. 47. 8. The method of claim 1, wherein the p-L-2',3'-dideoxyadenosine nucleotide prodrug is a lipophilic derivative of the nucleotide. 9. The method of claim 1, wherein the p-L-2',3'-dideoxyadenosine nucleotide prodrug is a derivative in which one or more hydrogens in the phosphate moiety of the 5 nucleotide is replaced by an alkyl. 10. The method of claim 1, wherein the p-L-2',3'-dideoxyadenosine nucleotide prodrug is a derivative in which one or more hydrogens in the phosphate moiety of the nucleotide is replaced by a steroid. 11. The method of claim 1, wherein the p-L-2',3'-dideoxyadenosine nucleotide prodrug 10 is a derivative in which one or more hydrogens in the phosphate moiety of the nucleotide is replaced by a carbohydrate. 12. The method of claim 1, wherein the p-L-2',3'-dideoxyadenosine nucleotide prodrug is a derivative in which one or more hydrogens in the phosphate moiety of the nucleotide is replaced by a sugar. 15 13. The method of claim 1, wherein the p-L-2',3'-dideoxyadenosine nucleotide prodrug is a derivative in which one or more hydrogens in the phosphate moiety of the nucleotide is replaced by a 1,2-diacylglycerol. 14. The method of claim 1, wherein the p-L-2',3'-dideoxyadenosine nucleotide prodrug is a derivative in which one or more hydrogens in the phosphate moiety of the 20 nucleotide is replaced by an alcohol. 15. The method of claim 1, wherein the prodrug component of the nucleotide increases the activity of the nucleoside in vivo. 16. The method of claim 1, wherein the second compound is 3'-azido-3'-deoxythymidine (AZT). 25 17. The method of claim 1, wherein the second compound is 2',3'-dideoxyinosine (DDI). 201226844 1
3. 48. 18. The method of claim 1, wherein the second compound is 2',3'-dideoxy-2',3' didehydrothymidine (D4T). 19. The method of claim 1, wherein the second compound is 2-hydroxymethyl-5-(5 fluorocytosin-1-yl)-1,3-oxathiolane (FTC). 5 20. The method of claim 1, wherein the second compound is a Tibo compound. 21. The method of claim 1, wherein the second compound is nevirapine. 22. The method of claim 1, wherein the second compound is a pyrimidinone. 23. The method of claim 1, wherein the second compound is a physiologically acceptable salt of a nucleotide prodrug of p-L-2',3'-dideoxyadenosine (p-L-DDA). 10 Dated: 16 August 2006 Centre National de la Recherche Scientifique, Emory University, The UAB Research 15 Foundation Patent Attorneys for the Applicants: BLAKE DAWSON WALDRON PATENT SERVICES 201226844_1