CA2219132C - Nucleosides with anti-hepatitis b virus activity - Google Patents

Abstract

A method for the treatment of a host, and in particular, a human, infected with HBV is provided that includes administering an HBV-treatment amount of the stabilized nucleotide of a nucleoside which exhibits anti-hepatitis B activity.

Description

NUCLEOSIDES WITH ANTI-HEPATITIS B VIRUS ACTIVITY
Background of the Invention 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 1 o 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 cell regeneration associated with the infection.
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, jaundice, and elevated blood levels of certain 2 o 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 2 5 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 hepatocellular carcinoma, a primary liver cancer. In western industrialized countries, high risk gmups 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 ' 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 1 o 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 addition, the vaccine does not 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-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane has anti-hepatitis B activity. Doong, et al., Proc. of Natl.
Acad. Sci. USA, 88, 8495-8499 (1991); Chang, et al., ~ of Biological them., Vol 267(20), 13938-13942. The anti-hepatitis B activity of the (-) and (+)-enantiomers of 2-hydroxyrnethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane has been published by Furman, et al., in ~ntimicrobial Agents 2 5 end Chemotheranv, Dec. 1992, pages 2686-2692.
PCT/US92/03144 (International Publication No. WO 92/18517) filed .
by Yale University discloses a number of B-L-nucleosides for the treatment of both HBV and HIV. Other drugs exlored for the treatment of HBV .
include adenosine arabinoside, thymosin, acyclovir, phosphonoformate, zidovudine, (+)-cyanidanol, quinacrine, and 2'-fluamarabinosyl-S-iodourxit.
M essential step in the mode of action of purine and pyrimidine nucleosides against viral diseases, and in particular, HBV and HIV, is their s metabolic activation by cellular and viral ldnases, to yield the mono-, di-, and triphosphate derivatives. The biologically active species of many nucleosides is the triptlospahte form. which inhibits DNA potymerase or reverse transcriptase, or causes chain termina4on. The nucleoside derivatives that have been developed for the treatment of HBV and HIV to io date have been pt~ese~ntsd for admini~ to the host in unphosphorylated form, notwithstanding the fact that the nucleoside must be phosphorytated in the cell prior to exhibiting its antivirat effect, because the triphosphate form has typically either been deghosphorylated prior to reaching the cell or is poorly absorbed by the cell. Nucleotides in general cross sell 15 membranes very inefficicndy and are ge~c~erally nrot very not very potent ~
yes. Attempts at modifying nucleotides to incr~e t3he absorption and potency of nucleotides have been described by R.. Jones and N.
Bischofbergar, Anttv~irat Rtseax)t, 17 (1995) 1-17, 2 o In tight of the fact that hepatitis B virus has trached epidemic levels worldwide, and has severs and often tragic effects on the infectod patient, there remains a strong need w provide new effective phannaaeutical agents to treat humans infectod with the virus that have Mw toxicity to the halt.
Therefore, it is another object of the present invention to provide a 2 5 method and composition for the treatment of human patients or other hosts infectod with HBV.
_3_ Summary of the Invention A method for the treatment of a host, and in particular, a human, infected with HBV is provided that includes administering an HBV-treatment amount of a nucleoside of the formula:

NHZ
R
N~ N N / / F
I ~~ ~J ~N\ I
N O N O' HO HO HO~
O O '~~' wherein:
Ri is hydrogen, fluoro, bromo, chloro, iodo, methyl or ethyl; and R2 is OH, Cl, NH2, or H; or a pharmaceutically acceptable salt of the compound, optionally in a pharmaceutically acceptable carrier or diluent.
In an alternative embodiment, the B-L-enantiomer of a compound of the formula:
Rs HO
O
wherein RS is adenine, xanthine, hypoxanthine, or other purine, including , an alkylated or halogenated purine is administered to a host in an HBV-treatment amount as described more fully herein. ' In another alternative embodiment, the nucleoside is of the formula:

WO 96/40164 PCT/US96/10026~
RO
O
wherein B is a purine or pyrimidine base;
y1, Y2, y3, and Y4 are independently H, OH, N3, NR1R2, N02, l0 NOR3, -O-alkyl, -O-aryl, halo (including F, Cl, Br, or n, -CN, -C(O)NH2, SH, -S-alkyl, or -S-aryl, and wherein typically three of Yl, y2, Y3, and Y4 are either H or OH. The -OH substituent, when present, is typically a Y' or Y3 group. As illustrated in the structure, Y2 and Y~ are in the arabino (erythro) configuration, and Y1 and Y3 are in the threo (ribose) configuration. R is H, monophosphate, diphosphate, triphosphate, alkyl, acyl or a phosphate derivative, as described in more detail below. R1, R2, and R3 are independently alkyl (and in particular lower alkyl), aryl, aralkyl, alkaryl, acyl, or hydrogen.
In a preferred embodiment, the nucleoside is provided as the indicated 2 o enantiomer and substantially in the absence of its corresponding enantiomer (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 2 5 nucleosides. A prodrug, as used herein, refers to a pharmaceutically acceptable derivative of the specifically disclosed nucleoside, that is converted into the nucleoside on administration ' viv , or that has activity in itself. Nonlimiting examples are the 5' and N4-pyrimidine or N6-purine acylated or alkylated derivatives of the active compound.

In a preferred embodiment of the invention, the nucleoside is provided as the monophosphate, diphosphate or triphosphate in a formulation that .
protects the compound from dephosphorylation. Formulations include liposomes, lipospheres, microspheres or nanospheres (of which the latter ' three can be targeted to infected cells). In an alternative preferred embodiment, the nucleoside is provided as a monophosphate, diphosphate or triphosphate derivative (i.e., a nucleotide prodrug), for example an ester, that stabilizes the phosphate inin vivo. In an alternative embodiment of this invention, a stabilized phosphate derivative, as described further to below, of FTC, BCH-189, or 3TC is provided for the treatment of hepatits.
The disclosed nucleosides, or their pharmaceutically acceptable prodrugs or salts or pharmaceutically acceptable formulations containing these compounds are useful in the prevention and treatment of HBV
infections and other related conditions such as anti-HBV antibody positive and HBV-positive conditions, chronic liver inflammation caused by HBV, 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 2 o individuals who are anti-HBV antibody or HBV-antigen positive or who have been exposed to HBV.
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-HBV agents, to provide effective anti-HBV treatment. Examples 2 5 of anti-HBV agents that can be used in alternation or combination therapy include but are not limited to the 2-hydrozymethyl-5-(5-fluorocytosin-1-yl)-1,3-ozathiolane ("FTC", see WO 92/14743), its physiologically acceptable derivative, or physiologically acceptable salt; the 2-hydrozymethyl-5-(cytosin-1-yl)-1,3-ozathiolane (including the racemic BCH-189 form, or _6..

3TC (BCH-189 enriched with the (-)-enantiomer)) its physiologically acceptable derivative, or physiologically acceptable salt; 2'-fluoro-5-ethyl-arabinosyluracil (FEAL>]; carbovir, or interferon.
- Any method of alternation can be used that provides treatment to the patient. Nonlimiting 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 to 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 combination or alternation with anti-HIV medications, including but not limited to 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (DDn, 2',3'-dideaxycytxdine (DDC), 2',3'-dideoxy-2',3'-didehydrothymidine (D4T), 2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC), or 2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (racemic BCH-189 or 2 o 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 2 5 with antibiotics, other antiviral compounds, antifungal agents, or other pharmaceutical 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 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 more detail below. Any 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 Figure 1 is an illustration of the chemical structures of B-L-2',3'-dideozycytidine (B-L-FddC), B-D-2',3'-dideozycytidine (B-D-ddC), B-L-2',3'-dideozy-5-fluorocytidine (B-L-ddC), (-)-B-L-2-hydrozymethyl-5-(5-fluorocytosin-1-yl)-1,3-ozathiolane ((-)-B-L-FTC), (+)-B-D-2-hydrozymethyl-5-(5-fluorocytosin-1-yl)-1,3-diozolane ((+)-B-D-FDOC), and B-L-2-amino-6-(R'~-9-[(4-hydrozymethyl)-tetrahydrofuran-1-yl]purine.
Figure 2 is an illustration of the numbering scheme used in the chemical nomenclature for nucleosides in this tent.
2 o Detailed Description of the Invention As used herein, the term "enantiomerically pure" refers to a nucleoside composition that includes at least approzimately 95 96 , and preferably approximately 97 ~ , 98 9& , 99 96 , or 100 ~'o of a single enantiomer of that 2 5 nucleoside.
The term alkyl, as used herein, unless otherwise specified, refers to a saturated straight, branched, or cyclic, primary, secondary, or tertiary ' hydrocarbon of C1 to Clo, and specifically includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, _g_ WO 96/40164 PCT/US96/1002Ei neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 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 vitro, cyano, sulfonic acid, sulfate, phosphoric 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 1o specified, refers to a C1 to C4 ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, or t-butyl group.
As used herein, the term aryl specifically includes but is not limited to acetyl, propionyl, butyryl, pentanoyl, 3-methylbutyryl, hydrogen succinate, 3-chlorobenzoate, 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 one or more moieties selected from the group 2 o consisting of hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, vitro, cyano, sulfonic acid, sulfate, phosphoric 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, 25 1991.
The term purine or pyrimidine base includes, but is not limited to, adenine, N6-alkylpurines, N6-acylpurines (wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl), N6-benzylpurine, N6-halopurine, N6-vinylpurine, N6-acetylenic purine, N6-acyl purine, N6-hydroxyalkyl purine, N6-thioalkyl purine, N2-alkylpurines, N2-alkyl-6-thiopurines, thymine, cytosine, 6-azapyrimidine, 2- and/or 4-mercaptopyrmidine, uracil, CS-alkylpyrimidines, CS-benzylpyrimidines, CS-halopyrimidines, CS-vinylpyrimidine, Cs-acetylenic pyrimidine, CS-acyl pyrimidine, CS-hydroxyalkyl purine, CS-amidopyrimidine, CS-cyanopyrimidine, CS-nitropyrimidine, CS-aminopyrimidine, N2-alkylpurines, N2-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl. Functional oxygen and nitrogen groups on the base can be protected as necessary or 1 o desired. Suitable protecting groups are well lrnown to those skilled in the art, and include trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as acetyl and propionyl, methylsulfonyl, and p-toluylsulfonyl.
As used herein, the term natural amino acid includes but is not limited to alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaoyl, lysinyl, argininyl, and histidinyl.
The invention as disclosed herein is a method and composition for the 2 o 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 2 5 compounds of this invention either possess anti-HBV activity, or are metabolized to a compound or compounds that exhibit anti-HBV activity.

I. Structure and Preparation of Active Nucleosides The compounds used in the methods disclosed herein are enantiomers ' of 2',3'-dideozycytidine, 2',3'-dideoxy-5-(halo or methyl)cytidine, 2-hydrozymethyl-5-(5-fluorocytosin-1-yl)-1,3-diozolane, or 2-amino-6-(OH, Cl, NH2, or H)-9-[(4-hydroxymethyl)-tetrahydrofuran-1-yl]purine.
Since the 1' and 4' carbons of the sugar or diozolanyl moiety (referred to below generically as the sugar moiety) of the nucleosides are chiral, their nonhydrogen substituents (CH20R and the pyrimidine or purine base, 1 o respectively) can be either cis (on the same side) or traps (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 C1' and C4'-atoms; see Figure 2) is in back): cis (with both groups "up", which corresponds to the configuration of naturally occurring nucleosides), cis (with both groups "down", which is a nonnaturally occurring configuration), traps (with the C2 substituent "up" and the CS
substituent "down"), and traps (with the C2 substituent "down" and the CS
substituent "up"). As indicated schematically in Figure 1, the "D-2 o 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 FDOC, which is used in its B-2 5 D-enantiomeric form, because it has been discovered that the B-D-enantiomer of FDOC is surprisingly less toacic than the B-L-enantiomer of FDOC.

PC'1'/US96/10026 The nuclerosides disclosed herein can be administered as any derivative ' that upon administration to the recipient, is capable of providing directly or indinxtly, the parent active compound, or that exhibits activity in itself. In 5 one embodiment, the hydrogen of the 5'-(JIi group is replaced by a C,-CZo alkyl, including C, to Cs alkyl; aryl in which the non-carbonyl maicty of the ester group is selected from straight, bran~claed, or cyclic C,-C~ alkyl including C, w Cs alkyl, phenyl, ar bertzyl; a naturally occurring or nonnaturally occurring amino acid; atkaxyalkyl including methoxymethyl;
1o aralkyi including benzyl; aryloxyalkyl such as phenaxymethyl; aryl including phenyl optionally substituted with halogen, C, to C, alkyl or C, to C, alkoxy; a dicarboxylic acid such as succinic acid; sulfonate esters such ax alkyl or aralkyl sulphonyl including methancsulfonyl; or a mono, di or triphosphate ester.
15 One or both hydrogens of the amine groups an the purine or PYnmidine base can be replaced by a C,-C~ alkyl, including C, to Cs alkyl; aryl in which the non-caurbcmyl moiety of the ester group is selected from straight, branched, or cyclic C,-C~ alkyl, including C, to Cs alkyl, phenyl, or benxyl; alkaxyalkyl n,rc1ading mc~thoaymcthyl; aralkyl including 2 o henry); aryloxyalkyl such as phenaxymethyl; aryl including pixnyl optionally substituted) with halogen, C, to C~ alkyl or C, to C, alkoxy.
The active nucleoside can also be provided as a ~'-ether lipid, as disclosed in the following references, r Kucera, L.S.,, N. Iyer, E. Leake, A, Raben, Modest E.J., D.
2 5 L. W. , and C. Piantadoai. 1990. hlovel membrane-interactive ether lipid analogs that inhibit infectious HIY«1 production and induce defective virus formation. AIDS Res Hum Retroviruses. 6:491-5171; Piantadosi, C'., J..
Marasco C.J., S.L. Morris-Nfatschke, K.L. Meyer, F. Gumus, J.R.
Surles, K.S. Ishaq, L.S. Kucera., N. Iyer, C.A. Wallen, S. Piantadosi, and -1~-E.J. 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 human immunodeficiency virus type 1 replication in CEM and HT4-6C
cells by 3'-deo~ythymidine diphosphate dimyristoylglycerol, a lipid prodrug of 3,-deoxythymidine. Antimicrob Agents Chemother.
36:2025.2029; Hostetler, K.Y., L.M. Stuhmiller, H.B. 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. 265:6112.7.
Nucleotide Prodru~
Any of the nucleosides described herein, or any other nucleoside that has anti-hepatitis B activity, can be administered as a nucleotide prodrug to increase the activity, bioavalability, stability or otherwise alter the properties of the nucleoside. A number of nucleotide prodrug ligands are lrnown. 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 2 o than the parent phosphate, and which does not materially adversely affect the anti-hepatits B activity of the nucleoside. Phosphonates are included as phosphate derivatives. In general, alkylation, acylation or other lipophilic modification of the mono, di or triphosphoate of the nucleoside will increase the stability of the nucleotide. Examples of substituent groups that 2 5 can replace one or more hydrogens on the the phosphate moiety are alkyl, aryl, steroids, carbohydrates, including sugars, 1,2-diacylglycerol and alcohols. Many are described in R. Jones and N. Bischoft~erger, Antiviral Research, 27 (1995) 1-17. Any of these can be used in combination with the disclosed nucleosides to achieve a desired effect. Nonlimiting examples of nucleotide prodrugs are described in the following references.
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anti-HIV activity of some haloalky phosphoramidate derivatives of 3'-azido-3'deoxythylmidine (AZT); potent activity of the trichloroethyl -methoxyalaninyl compound. Antiviral Res. 15, 255-263; McGuigan, C., WO 96/40164 PCT/US96/10026.
Pathirana, R.N., Mahmood, N., Devine, K.G. and Hay, A.J. (1992) Aryl phosphate derivatives of AZT retain activity against HIV 1 in cell lines which are resistant to the action of AZT. Antiviral Res. 17, 311-321;
McGuigan, C., Pathirana, R.N., Choi, S.M., Kinchington, D. and 5 O'Connor, T.J. (1993a) Phosphoramidate derivatives of AZT as inhibitors of HIV; studies on the carbonyl terminus. Antiviral Chem. Chemother. 4, 97-101; McGuigan, C., Pathirana, R.N., Balzarini, J. and De Clercq, E.
(1993b) Intracellular delivery of bioactive AZT nucleotides by aryl phosphate derivatives ~of AZT. J. Med. Chem. 36, 1048-1052.
1 o Alley hydrogen phosphonate derivatives of the anti-HIV agent AZT
may be less toxnc than the parent nucleoside analogue. Antiviral Chem.
Chemother. 5, 271-277; Meyer, R. B., Jr., Shuman, D.A. and Robins, R.K. (1973) Synthesis of purine nucleoside 3',5'-cyclic phosphoramidates.
Tetrahedron Lett. 269-272; Nagyvary, J. Gohil, R.N., Kirchner, C.R. and 15 Stevens, J.D. (1973) Studies on neutral esters of cyclic AMP, Biochem.
Biophys. Res. Commrrrc. 55, 1072-1077; Namane, A. Gouyette, C., Fillion, 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 Leser, 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',5'monophosphates. 'HNMR and a-ray crystallographic study of the diasteromers of thymidine phenyl cyclic 3',5'-monophosphate. J. Am.
25 Chem. Soc. 109, 4058-4064; Nerbonne, J.M., Richard, S., Nargeot, J.
and Lester, H.A. (1984) New photoactivatable cyclic nucleotides produce intracellular jumps in cyclic AMP and cyclic GMP concentrations. Nature 301, 74-76; Neumann, J.M., Herv~, M., Debouzy, J.C., Guerra, F.L, Gouyette, C., Dupraz, B. and Huynh-Dinh, T. (1989) Synthesis and transmembrane transport studies by NMR of a glucosyl phospholipid of thymidine. J. Am. Chem. Soc. 111, 4270-4277; Ohno, R., Tatsumi, N., Hirano, M., Imai, K. Mizoguchi, H., Nakamura, T., Kosaka, M., Takatuski, K. , Yamaya, T. , Toyama, K. , Yoshida, T. , Masaoka, T. , Hashimoto, S., Ohshima, T., Kimura, L, Yamada, K. and Kimura, J.
(1991) Treatment of myelodysplastic syndromes with orally administered 1-~i-D-rabinofuranosylcytosine -5'stearylphosphate. Oncology 48, 451-455.
Palomino, E., Kessle, D. and Horwitz, J.P. (1989) A dihydropyridine 1 o carrier system for sustained delivery of 2' , 3' dideoaynucleosides to the brain. J. Med. Chem. 32, 622-625; Perkins, R.M., Barney, S., Wittrock, R. , Clark, P. H. , Levin, R. Lambert, D. M. , Petteway, S. R. , Serafinowska, H.T., Bailey, S.M., Jackson, S., Harnden, M.R. Ashton, R., Sutton, D., Harvey, J.J. and Brown, A.G. (1993) Activity of BRL47923 and its oral prodrug, SB203657A against a rauscher murine leukemia virus infection in mice. Antiviral Res. 20 (Suppl. n. 84;
Piantadosi, C., Marasco, C.J., Jr., Moms-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 2 o novel ether lipid nucleoside conjugates for anti-HIV-1 activity. J. Med.
Chem. 34, 1408-1414; Pompon, A., Lefebvre, L, Imbach, J.L., Kahn, S.
and Farquhar, D. (1994) Decomposition pathways of the mono- and bis(pivaloyloaymethyl) esters of azidothymidine-5'-monophosphate in cell extract and in tissue culture medium; an application of the ' on-line ISRP-cleaning' HPLC technique. Antiviral Chem. Chemother. 5, 91-98;
Postemark, T. (1974) Cyclic AMP and cyclic GMP. Annu. Rev.
Pharmacol. 14, 23-33; Prisbe, 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 phosphonate derivatives of 9-[(1,3-dihydroxy-2-propoxy)methyl] guanine J. Med. Chem. 29, 671-675; Pucch, F., Gosselin, G., Lefebvre, L, Pompon, A., Aubertin, A.M. Dirn, A. and Imbach, J.L. (1993) Intracellular delivery of nucleoside monophosphate through a reductase-mediated activation process. Antiviral Res. 22, 155-174; Pugaeva, V.P., Klochkeva, S.L, Mashbits, F.D. and Eizengart, R.S. (1969).
Toxicological assessment and health standard ratings for ethylene sulfide in the industrial atmosphere. Gig. Trf. Prof. Zabol. 13, 47-48 CChem. Abstr. 72, 212); Robins, R.K. (1984) The potential of nucleotide 1o 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-~3-D-arabinofuranosylcytosine and its 1V~-acyl and 2.2'-anhydro-3'0-acyl derivatives as potential prodrugs. J. Med.
Chem. 25, 171-178; Ross, W. (1961) Increased sensitivity of the walker turnout towards aromatic nitrogen mustards carrying basic side chains following glucose pretreatment. Biochem. Pharm. 8, 235-240; Ryu, e. K. , 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 1-(3-D-arabinofuranosylcytosine 5'diphosphate[-], 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., Morozumi, M., Kodama, K., Machida, J., Kuninaka, A. and Yoshino, H. (1980) Synthetic nucleosides and nucleotides. XVI. Synthesis and biological evaluations of a series of 1-(3-D-arabinofuranosylcytosine 5'-alley or arylphosphates. Chem. Pharm.
_ Bull. 28, 2915-2923; Sastry, J.K., Nehete, P.N., Khan, S., Nowak, B.J., Plunkett, W., Arlinghaus, R.B. and Farquhar, D. (1992) Membrane-permeable dideoxyuridine 5'-monophosphate analogue inhibits human immunodeficiency virus infection. Mol. Pharmacol. 41, 441-445; Shaw, ' J.P., Jones, R.J. Arimilli, M.N., Louie, M.S., Lee, W.A. and Cundy, K. C. ( 1994) Oral bioavailability of PMEA from PMEA prodrugs in male Sprague-Dawley rats. 9th Annual 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, 1 o 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" .
~aration of the Active Co unds The nucleosides used in the disclosed method to treat HBV infections in a host organism can be prepared according to published methods. $-L-Nucleosides can be prepared from methods disclosed in, or standard 2 o modifications of methods disclosed in, for example, the following publications: Jeong, et al., J. of Med. Chem., ~ø, 182-195, 1993;
European Patent Application Publication No. 0 285 884; Gnu-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;
2 5 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, and J. C. Martin, Preparation of the geometric isomers of DDC, DDA, D4C and D4T as potential anti-HIV agents, Bioorg. Med. Chem. Lett. l:ti5-68 (1991); Fujimori, S., N. Iwanami, Y.
Hashimoto, and K. Shudo, A convenient and stereoselective synthesis of 2'-deoxy-B-L-ribonucleosides, Nucleosides & Nucleotides 11:341-349 (1992); G6nu-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; synthesis and biological evaluation, Antiviral Chem. Chemother. 2:83-92 (1991); Holy, A, Synthesis of 2'-deoxy-L-uridine, Tetrahedron Lett. 2:189-192 (1992); Holy, A., Nucleic acid components and their analogs. CLIII. Preparation of 2'-deoxy-L-1o 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'-deoaynucleoside from a sugar 2-aminooxazoline through a 2.2'-anhydronucleoside intermediate. In: Townsend LB, Tipson RS, ed.
Nucleic Acid Chem. New York: Wiley, 1992: 347-353. vol 1) (1992);
Okabe, M., R.-C. Sun, S. Tan, L. Todaro, and D. L. Coffen, Synthesis of the dideoxynucleosides ddC and CNT from glutamic acid, ribonolactone, and pyrimidine bases. J Org Chem. 53:4780-4786 (1988);
Robins, M. J., T. A. Khwja, and R. K. Robins. Purine nucleosides.
XXIX. Synthesis of 21-deoxy-L-adenosine and 21-decay-L-guanosine and 2o their alpha anomers. J Org Chem. 35:363-639 (1992); G6nu-Dellac, C., Gosselin G., Aubertin A-M, Obert G., Kirn A., and Imbach J-L, 3'-Substituted thymine a-L-nucleoside derivatives as potential antiviral agents;
synthesis and biological evaluation. Andviral Chem. Chemother. 2(2):83-92 (1991); GCnu-Dellac, C., Gosselin G., Imbach J-L; Synthesis of new 2 5 2'-deoxy-3'-substituted-a-L-threo-pentofuranonucleosides of thymine as a potential antiviral agents. Tet Lett 32(1):79-82 (1991); G6nu-Dellac, C., Gosselin G., Imbach J-L. Preparation of new acylated derivatives of L-arabino-furanose and 2-deoxy-1-erythro-pentofuranose as precursors for the synthesis of 1-pentofuranosyl nucleosides. 216:240-255 (1991); and G6nu-Dellac, C., Gosselin, G., Puech, F, et al. Systematic synthesis and antiviral evaluation of a-L-arabinofuranosyl and 2'-deoxy-a-L-erythro-pento-furanosyl nucleosides of the five naturally occurnng nuclei acid bases. 10(b):1345-1376 (1991).
2',3'-Did~xycytidine (DDC) is a known compound. The D-enantiomer of DDC
is currently being marketed by Hoffinan-LaRoche under the name Zalcitabine for use in the treatment ofpersons infected with HTV. See U.S. Patent Nos. 4,879,277 and 4,900,828.
Enantiomerically pure (3-D-dioxolane-nucleosides such as (i-D-FDOC can be prepared such as disclosed in detail in WO 92/010497. The process involves the intitial preparation of {2R,4R)- and (2R,45)-4-acetoxy-2-(protected-oxymethyl)-dioxolane from 1,6-anhydromannose, a sugar that contains all of the necessary stereochernistry for the entantiomerically pure final product, including the correct diastereomeric configuration about the 1 position of the sugar (that becomes the 4'-position in the later formed nucleoside). The (2R,4R)- and (2R,4S)-4-acetoxy-2-(protected-oxymethyl)-dioxolane is condensed with a desired heterocyclic base in the presence of SnCl4, other Lewis acid, or trimethylsilyl triflate in an organic solvent such as dichloroethane, acetonitrile, or methylene chloride, to provide the stereochemically pure dioxolane-nucleoside.
Enzymatic methods for the separation of D and L enantiomers of cis-nucleosides are disclosed in, for exarxaple, Nucleosides and Nucleotides, 12(2), 225-236 (1993);
European Patent Application Nos. 92304551.2 and 92304552.0 filed by Biochem Pharma, Inc.; and PC'F Publication Nos. WO 91/11186, WO 92/14729, and WO
92/14743 filed by Emory University.
Separation of the acylated and alkyiated racemic mixture of D and L
enantiomers of cis-nucleosides can be accomplished by high performance liquid chromatography with chiral stationary phases, as disclosed in PCT
Publication No. WO 92/ 14729.
Mono, di, and triphosphate derivative of the active nucleosides can be prepared as described according to published methods. The monophosphate can be prepared according to the procedure of Imai et al. , J. OrE, Chem., 34(6), 1547-1550 (June 1969). The diphosphate can be prepared according to the procedure of Davisson et al., J. Org. Chem., 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 io (1965).
General Procedures for the Prtyaration of Bic l~yl-2-thioetlavD
l~hos~hoester of ~-L-dideoxvnucleosides Ibis «A1'E~ (3-L ddx MPl I O O
O ~ R,~ ~OH II R~-~S~-O P--N(iP~)2 SH

2 0 O Base O p Y~ Y3 R,~S~O P-.O 2Y4 Bis (SATE) ~i-L-ddoMP
_ 3 o y1, y2, y3, and Y~ are independently H, OH, N3, NR1R2, NO2, NOR3, -O-alkyl, -O-aryl, halo (including F, Cl, Br, or I), -CN, -C(O)NH2, SH, -S-alkyl, or -S-aryl, and wherein typically three of Y1, Y2, Y3, and Y4 are either H or OH. The -OH substituent, when present, is typically a Y~ or Y3 group.
As illustrated in the structure, YZ and Y4 are in the arabino (erythro) configuration, and Y' and Y3 are in the threo (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). (i) ICH2CH20H, DBU/C6HSCH3; (ii) C12PN(iPr)2, NEt3/THF; (iii) ~i-L-dideoxynucleoside, 1H--tetrazole/THF, then 1o C1C6H4C03H/CH2C121H-Tetrazole (0.21g, 3.0 mmol) was added to a stirred solution of /3-L-dideoxynucleoside (1.0 mmol) and the appropriate phosphoramidite ~ (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 dichloromethane (2.5 mL) was added; the mixture was then allowed to warm to room temperature over 1 h. Sodium sulfite ( 10 ~6 solution, 1.3 mL) was added to the mixture to destroy the excess 3-chloroperoxybenzoic acid, after which the organic layer was separated and the aqueous layer washed with dichloromethane (2 x 10 mL). The combined organic layers 2 o 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) ~3-L-ddxrri~.

WO 96/40164 PCT/US96/t0026 ~_ (3-L-2',3'-Dideoxyadenosin-5'-yl bis (2-pivaloylthioethyl) phosphate [Bis {SATE) Q-L-ddAM~.

(CIi3)3C-C~ ~C4~2-O PN(iPr)2 (3-L-ddA. 1 H-tecrazoleJ'THF
then C1C6H4C0;H/CH2CI2 then silica gel column chromatography (CH3~3C~C~ iCH2-~ O-O
S-CHZ
Bis (SATE)(3-I,_ddAMp Following the above general procedure, pure Bis(SATE)(3-L-ddAMP_ was obtained as a colorless oil in 72 q6 yield after silica gel column chromatography [eluent: stepwise gradient of methanol (0-3R6) in dichloromethane]; ' NMR (DMSO - d6) b ppm: 8.26 and 8.13 (2s, 2H
each, H-2 and H-8), 7.20 (br s, 2H, NHS, 6.24 (t, 1H, H-1'; J=6.0 Hz), 4.35 - 4.25 (m, 1H, H-4'), 4.25-4.00 (m, 2H, H-5', 5"), 3.96 (m, 4H, 2 SCH2CH20), 3.04 (t, 4H, 2 SCH2CH20 ; J = 6.3 Hz), 2.5 - 2.4 (m, 2H, H-2',2") 2.2-2.0 (m, 2H, H-3',3"), 1.15 [s, 18H, 2 (CH3)3CJ; 3i~~NMR
(DMSO-d6) 8 ppm = -0.76 (s) ; LTV (EtOH) , ~, ~ = 259 nm (E 15400);

mass spectrum (performed in: glycerol, thioglycerol, 1:1, u/u), FAB > O
604 (M+H)+, 136 (BHP+.
('~neral ~he~ne for the steros~~ecifC vnthes'>ic of 3'-substituted ~i-Ir ' dideoxvnucleosides off RO
O
Compound 8 (see Fig. 1/2 of the French patent See Appendiz 4 RO-~~~~
ov ox O RO Base 2 0 RO ease OH
O
RO-~~~~se r Ro ox a r Y «erythrOo»
RO ~ cont7guration Y
HO-~~~~~
O
«thneo»
4 0 configuration O U
V = aryl (CH3-C, C6H3-C) X = Leaving gmup [CH3 S02, CH3 C6H4 S02, CF3 SOx.1 Y~ Y' = F~ N3~ NRiRa [RuR2 = H~ ~yl~ aryi]~
NOZ, NOR [R = H, alkyl, acyl], O-alkyl, O-aryl, etc.
~~= l.-(3-Azido-2-3-dideoxy-Q-L-erythro-pentofuranosyl) thymine [/3-L-AZTj H~ CH3 N
HO
O N P(C H

6 5~3 mMTrO DEAD
O DPPA O
~TfiF
$ HEN
O' \N.
~--~, i m'rviTrG--~ / ..
AcOH/HZO
O
HEN iCH3 O N
HO-~~1 O
(3-L-AZT
4096 yield from $, A mixture of diethyl azodicarboxylate (0.46 mL; 2.9 mmol) and Biphenyl phosphorazidate (0.62 ml; 2.9 mmol) in THF (2.9 ml) was added dropwise over 30 min. to a solution of 1-(2-deoxy-5-O-monomethoaytrityl-~i-L-threo-pentofuranosyl) thymine $ [0.5 g, 0.97 mmol] and , triphenylphosphine (0.76g, 2.9 mmol) in THF 11.6 ml) at 0°C. The mixture was stirred 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 1 o 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 j3-L-AZT (105 mg, 409b, crystallized from ethyl acetate). The physicochemical data of (3-L-AZT were in accordance with literature data [J. Wengel, J-Lau, E. B.
Ledersen, C.N. Nielsen, J. Org. Chem. 5~ (11), 3591-3594 (1991)].

~'~neral Scheme for the Stereos~~ec~c Synthesis of 2' subct~t~.tP
_ uSl~~i~
RO-~~~Base HO
O
Compound j2 [see Fig. 1/2 of the French parent]
See Appendix 4 OV
Base RO-~~~ Base RO XO
O
O

Ro ~ 1 r O RO 8ase O
1 ~X
Ro o r HO~~eaae \/O
«th~o»
RO-~~~~ conti oration Y
O
HO-~~Base Y
O
«erythro»
configuration O O
V = aryl [CH3-C C6H5-C]
X = Leaving group [CH3 SO2, CH3 C6H4S02,H, CF3 SOZ]
Y. y' = F~ N3~ NRtR2 [RuR2 = H~ ~yi~ ~Yl]~
3 o N02, NOR [R = H, alkyl, acyl], O-alkyl, O-aryl, etc.

~;$A~ = 1-(2-Fluoro-2,3-dideoxy-(3-L-threo-pentofuranosyl)-5-fluorocytosine [2'-F-(3-L (3-L-FddC]

HEN F H3C0 ~ ~ CHZ O F
'N
CHiC6H~CH=C1 O
Bz0-1~~ DBU/CH;C:~I
HO Bz0 HO
969 yield O O
~ DAST.
CH_Ch, CsHsY
O
HwN F F
,~ ( (NH,)iCe(N03)s O N ~--BZp y 6096 yield 7'~ yield O
Lawesson's reagent/CHZC12 re0ux IOdC
BZO
2'-F-~-L-Fddc 2 5 6496 yield Hitherto unlrnown 2'-F-(3-L-FddC was synthesized in five steps from 1-(5-O-benzoyl-3-deoxy-(3-L-erythro-pentofuranosyl)-5-fluorouraci1 ~7 with an overall yield of 289. m.p. 209-210°C (crystallized from absolute ethanol); IJV (>rt OH) 7v.m,x 276 m, (~, 9000), 7v,m;n 226 (e, 4000); 19F-NMR (DMSO-d6) 8 ppm : -179.7 (m, F2,) , -167.2 (dd, F5; JF.6 = 7.3 Hz, JF.1. = l.SHz); 1H-NMR (DMSO-d6) Sppm : 8.30 (d , 1H, H-6; J6,F = 7.3 Hz), 7.8-7.5 (br s, 2H, NHS, 5.80 (d, 1H, H-1' J1.,F = 17.4 Hz), 5.34 (t, 1H, OH-5'; J = 4.8 Hz), 5.10 (dd, 1H, H-2'; J2.,F = 51.2 Hz; J2.,3. _ 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 spectra (performed in: glycerol-thioglycerol, 1:1 u/u), FAB > 0:248 (M+H)+, 130 (BHP+; FAB < 0:246 (M-H)- ; [acji°n = -16.5~ (-c 0.85, DMSO). Anal. Calc. for CgHI1N303F2 : C, 43.73; H, l0 9.49; N. 17.00; F. 15.37 . Found: C, 43.56; H, 4.78; N, 16.75; F, 14.96.

L-Xylose ~CH~ZCO, HZSO,, CuSO,, /~ 'then NH,OH
HCUHZO
then NaHC03lHi0 O /
OBZ
B20 O _~sCOCI Oy0 OH a / pyridine HO O ---~ CH3~ Bz0 Ac0 OH 1 CsHsN-CHCI3 (CH3C0)20, OAc <S)C(Im)z H2S0, O
/ (CHZCI)Z
O '' O / glycosidic (CH3Si)3SiH, AIBN O' O ~ndeasadon Bz0 O
/ Tolueae 0 OBz O-C-im B O Bz0 I) CH3COOH 8596 2) (CH3COlzU.
8z0 -Pyridine ~Z~Z . H~
O OAC / P~dine - CH3COOH I
O O'~ ~ Ac0 OB ,~z 14 Bz0 ease ~y~ Ho caodeasation o I) CsHsOC(SX5 Bz0 Base 2) Bu3SnH, AIBN
Ac0 / diwcane OBz O Bz0 Base 2 0 y~
O
CH30Na / CH30H =~ HO Base ~3 / ~~H z NH3 / CH30H
O
HO Base _.,"j" 1Q OH
HO '~ ~ ~ _ ~.Lr) HO Base O
~ ~ tR=TaDPS7 0 RCI / pyridine or Z
NH3 / CH30H (R _- gz or Ac) RCl / pyridine RO Base HO I) C6HsOC(Sxl OH 1 O DMAP / CH3CNR0 ~) ~~(Sxl RO Basz ,1Z 2) Bu3SaH. AIBN O _ 3 DAP / CH CN
/ Tolueae Q 2) Bu~,SnH, A18N O
3 0 /dioxane 8 Scheme I: Bases = purines or pyrimidines, eveatuellement conveoablenient protegees; R
Beazoyl (Bz) Ac~tyl (Ac), monomethoxytrityl (mMTr) or tertiarybutyldipheaylsilyl (TBDPS)) II. Anti-HBV Activity of Nucleosides The ability of the active compounds to inhibit HBV can be measured by various experimental techniques. The assay used herein to evaluate the 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-HBV activity are provided below for B-L-2',3'-dideoxycytidine (B-L-FddC), B-L-2',3'-dideoxy-5-1o fluorocytidine (B-L-ddC), and (+)-B-D-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-dioxolane ((+)-B-D-FDOC). The toxicity and anti-HBV activity of (-)-B-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 evaluated similarly.
The samples of B-L-ddC and B-L-5-FddC used in the anti-HBV assays were characteriaxd as follows.
2'.3'-Dideox~r-B-L 'dine B-L-DDC]. m.p. = 220-220°C; UV
(EtOH 95) max 273 nm, ,min 252 nm; NMR-'H (DMSO-d6) Sppm =

7.89 (d. 1H. H-6; J = 7.4 Hz). 7.15-6.95 (d large, 2H, NHS, 5.91 (dd.
1H, H-1'; J = 3.0 et 6.5 Hz), 5.66 (d, 1H, H-5; J = 7.4 Hz), 4.99 [t.
1H, OH-5'; J - 5.2 Hz]. 4.05-3.95 (m, 1H, H-4'), 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 2 5 (m. 1H, H-5"; after D20 exchange: dd, 3.50 ppm, J = 4,1 et 12.0 Hz), 2.30-2.15 (m. 1H, H-2'), 1.9-1.65 (m. 3H, H-2", 3' et 3"); [a]DZ°-103.6 (c 0.8 MeOH); mass spectrum (performed in: glycerol-thioglycerol, 50 . 50. v/v); FAB ~ 0 423 [2M+H]+, 304 [M+glycerol+H]+. 212 [M+H]+, 112 [BHP]+, 101 [s]+; FAB < O 210 (M-H]-. Anal. Calc. for ~13N3~ (M = 211.21); C 51.18; H 6.20; N 19.89 found; C 51.34; H
6.25; N 20.12.
2'.3'-Dideoxyr-B-L-5-fluorocyrtidine ra_T _S_Fmru~~ m.p, = 158-160°C; UV (EtOH 95) ~,max 281 nm (E, 8100) et 237 nm (s, 8500); min .
260 nm (E, 5700) et 225 nm (s, 7800); NMR - 1H (DMSO-d6) 8ppm 8.28 (d. 1H, H-6; J - 7.4 Hz), 7.7-7.4 (d large, 2H, NHS, 5.83 (dd poorly resolved, 1H, H-1'), 5.16(t. 1H, OH-5'; J = 5.1 Hz), 4.05-3.95 (m, 1H, H-4'), 3.8-3.70 [m,1H, H 5'; after D20 exchange: dd, 3.71 ppm. J = 2.7 et ~.3 Hz], 3.60-3.50 [m. 1H, H-5"; after D20 exchange: dd, 3.52 nnm;
1o J = 3.3 et 12.3 Hz], 2.35-Z.15 (m, 1H, H-2'). 1.95-1.75 (m, 3H, H-2", 3' et 3"): [a]DZO-80.0 (-c 1.0, DMSO); Mass spectrum [performed in: 3-nitrobenzyl alcohol] FAB > 0 230 [M+H]+ et 101 [s]+; FAB < O 228 [M-II]-. Anal. Calculated for C9H,2N3F03(M = 229.21); C 47.16; II 5.28; N
18.33, F 8.29, Found. C 16.90; H 5.28; N 18.07; F 8.17.
The antiviral evaluations were performed on two separate passages of cells, two cultures per passage (4 cultures total). 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
2 o 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 < 0.05] (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 2 5 cell basis in these 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 extracellular HBV virion DNA in untreated cells range from 50 to 150 pg/ml culture medium (average of approximately 76 _34_ WO 96/40164 PCT/US96/10026~
pg/ml). Intracellular HBV DNA replication intermediates in untreated cells range from 50 to 100 pg/ug cell DNA (average approximately 74 pg/ug cell DNA). In general, depressions in the levels of intracellular HBV DNA due to treatment with antiviral compounds are less pronounced, and occur more slowly, than depressions in the levels of HBV virion 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/ml culture medium to 3 x 105 1 o 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, 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 -20°C so that each individual aliquot would be subjected to a single freeze-thaw cycle. The daily test aliquots were thawed, suspenders 2 o into culture medium at room temperature and immediately added to the cell cultures. The compounds were tested at 0.01 to 10 ~cM for antiviral activity. The compounds were tested for toxicity at concentrations from 1 to 300 ~cM. The results are provided in Table 1.

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s~rrutE sa~r (~u,E 26) Example 2 Toxicity Of Compounds The ability of the active compounds to inhibit the growth of virus in 2.2.15 cell cultures (HepG2 cells transformed with hepatitis virion) was evaluated. As illustrated in Table 1, no significant toxicity (greater than 509b 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 ~M, however, all three compounds exhibited less toucity at this concentration than B-D-ddC. It appears that the ICso of B-L-ddC and B-L-FddC is approximately twice that of B-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 triplicate cultures. Uptake of neutral red dye was used to determine the relative level of toxicity. The absorbance of internalized dye at 510 nM
(Aslo) was used for the quantitative analysis. Values are presented as a percentage of the average As,o values (t standard deviations) in 9 separate cultures of untreated cells maintained on the same 96-well plate as the test 2 o compounds. The percentage of dye uptake in the 9 control cultures on plate 40 was 100 t 3. At 150-190 ~,M 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).

Fxxmpie 3 Anti-Hepatitis B Virus Activity The positive treatment control, B-D-2',3'-dideaxycytosine [B-D-ddC], induced significant depressions of HBV DNA replication at the concentration used. Previous studies have indicated that at 9-12 ~M of B-D-ddC, a 9096 depression of HBV 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 1 indicates that all three test compounds ((B-L-FddC), (B-L-ddC), and B-D-FDOC)), were potent inhibitors of HBV
replication, causing depression of HBV virion DNA and HBV RI to a degree comparable to, or greater than, that observed following treatment with B-D-ddC.
Example 4 The effect of selected B-L-derivatives against Hepatitis B virus replication in transfected Hep G-2 cells is described in Table 4.

WO 96/40164 PCT/US96/100Zb z ~
z N
a~
a M
~b N
O
4 chn '~ O
Y1 O ~ 00 V1 a p ~ v~
ri o n t~ ~~ s~
ro 8, -r ~ .- w ~~ .~
~ ra ~ o0 ~3 Example 5 The Comparative inhibitory effect of selected triphospahtes on woodchuck hepatitis virus DNA polymerase is set out in Table 5.
Table 2: Comparative inhibitory activities of L-nucleoside triphosphates on woochuck hepatitis virus DNA polymerase and human DNA polymerase ac and (3.
Inhibitor WHB DNA Pol DNA Pol a DNA Pol (3 ICsa (p.M) Ki (~.M) Ki (~M) (3-L-AZTPP 0.2 > 100 > 100 (3-L-ddATP 2.1 > 100 > 100 3-TC-TP 1.0 > 100 > 100 (3-L-SFDDCTP 2.0 > 100 > 100 1 o III. 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-HBV antibody positive and HBV-positive conditions, chronic liver inflammation caused by HBV, 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-antigen positive or who have been exposed to HBV.
2 0 Humans suffering from any of these conditions can be treated by administering to the patient an effective HBV-treatment amount of one or a mixture of the active compounds described herein or a pharmaceutically -acceptable derivative or salt thereof, optionally in a pharmaceutically acceptable carrier or diluent. The active materials can be administered by su~murs su~r c~u,E 2s~

any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, 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 1 to 60 mg/kg, preferably 1 to 20 mg/kg, of body weight per day, more generally 0.1 to 1 o about 100 mg per kilogram body weight of the recipient per day. The effective dosage range of the 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 15 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'-deoxythymidine (AZT), 2',3'-dideoxyinosine (DDn, 2',3'-dideoxycytidine (DDC), or 2',3'-dideoxy-2',3'-didehydrothymidine (D4T) for HIV indication.
2 o 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 should be administered to achieve peak 2 5 plasma concentrations of the active compound of from about 0.2 to 70 ~M, preferably about 1.0 to 10 pM. This may be achieved, for example, by the intravenous injection of a 0.1 to 5 q6 solution of the active ingredient, optionally in saline, or administered as a bolus of the active ingredient.

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 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 the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic 1 o acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, and polygalacturonic acid; (b) base addition salts formed with rations such as sodium, potassium, zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with an organic ration formed from N,N-dibenzylethylene-diamine, ammonium, or ethylenediamine; or (c) combinations of (a) and (b); e.g., a zinc tannate salt or the like.
Modifications of the active compound, specifically at the Nb or N4 and 5'-O positions, can affect the bioavailability and rate of metabolism of the 2 o 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 skill in the art. It is to be noted that 2 5 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 administration, the active 1 o 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 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 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 agent such as peppermint, methyl salicylate, or 2 0 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.
2 5 The active compound or pharmaceutically acceptable salt or derivative - thereof 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 active compound, or pharmaceutically acceptable derivative or salt 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, antiinflammatories, or other antivirals, including anti-I;iBV, anti-cytomegalovirus, or anti-HIV agents.
Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components:
1o 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 benryl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic 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 2 o 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 2 5 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 and Nova Pharmaceuticals, Inc.

L3poaomal suspensions (including liposotnea targeted to infected calls with monoclonal antibodies to viral antigens) are also preferred as pharmaceutically acceptable carriers. 'Thex may be prepared according to m~hods known to those skilled in flat art, for eaarmple, as described in U.S. Patalt No. 4,~z2,$I1.
For example, liposan~e frxmulatioeta may be prepared by dissolving a~mopria~te lipids) (such as a~amyl phosphatidyl ethanolamine, stearoyl phosphaxidyl cholin~e, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving bchir>d 1 o a thin film of driai lipid on the a~nrfa~oc of the co~atainer. M aqueous solution of the active compound c~ its mo~nophosphatc, diphasphate, and/or triphosphade derivatives arc than ink into the contxir~er. The container is then swirled by hind to free lipid rt~riaa fmm the sides of the container and w disperx lipid aggregates, y forming the liposomal suspension.
This invention has been described with reference to its preferred embodiments. Variations and modifications of the invention, will be obvious t4 thox skilled in the art from the foregoing detailed description of the invention. It is intendod that all of these variations and modifications 2 o be included within the scope of the appended claims.
-4~-

Claims (8)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR
PRIVILEGE IS CLAIMED ARE AS FOLLOWS:

1. Use of an effective amount of a bis(SATE).beta.-L-2',3'-dideoxyadenosine monophosphate to an infected patient, and wherein the bis{SATE) component of the nucleotide provides controlled delivery of the active species for treating a patient infected with hepatitis B.

2. Use of an effective amount of a bis(SATE).beta.-L-2',3'-dideoxyadenosine monophosphate to an infected patient, and wherein the bis(SATE) component of the nucleotide increases the activity of the nucleoside in vivo for treating a patient infected with hepatitis B.

3. A bis(SATE) monophosphate of the nucleoside .beta.-L-2',3'-dideoxyadenosine, wherein the bis(SATE) component of the nucleotide stabilizes the phosphate in vivo, and provides controlled delivery of the active species.

4. A pharmaceutical composition comprising an effective amount of a bis(SATE).beta.-L-2',3'-dideoxyadenosine monophosphate, wherein the bis(SATE) component of the nucleotide stabilizes the phosphate in vivo, and provides controlled delivery of the active species, in combination with a pharmaceutically acceptable carrier.

5. Use according to Claim 4, wherein the composition is suitable for oral delivery.

6. Use according to Claim 4, wherein the composition is in the form of a capsule.

7. Use according to Claim 4, wherein the composition is in the form of a tablet.

8. Use according to Claim 4, wherein the composition is suitable for parenteral administration.