CA2538205C - 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

NUCLE051nES WITH ANTI HEPATITIS B VIRUS ACT1YITY
Background of the Inrentiou This invention is in the area of methods for the treatment of hepatitis B
virus (also referred to as "HBV") that includes admuustermg an effective amount of one or more of the active compounds disclosed herein, or a pharmaceutically acceptable derivative or prodrug of one of these 1o 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. Aftex a two to six month incubation period in which the host is unaware of the infection, HBV infection can lead to xute hepatitis and liver damage, that causes abdominal pain, jaundice, and elevated blood levels of certain Z 0 enrymes. HBV can cause fitlminant 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 infections can lead to chronic persistent hepatitis. Patients infected with chronic persistent HBV are most common in developing countries. By mid-1991, there were approumatety 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 WO 96!40164 PCT/U596/10026 hepatocellular carcinoma, a primary liver cancer. In western industrialized countries, high risk groups for IiBV 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 s accounts for why HBV infection is common among patients with AIDS or HIV-associated infections. However, HBV is more contagious than H1V.
Daily treatments with a-interferon, a genetically engineered prntein, has shown promise. A human serum-derived vaccine has also been developed to immunize patients against hIBV. Vaccines have been 1o 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 ezpensive_ Further, each batch of vaccine prepared from different serum must be tested in chimpanzees to ensure safety. In 15 addition, the vaccine does not help the patienES already infected with the virus.
European Patent Application Na. 92304530.6 discloses that a group of 1,2-ozathiolane nucleosides are useful in the treatment of hepatitis B
infections. It has been reported that the 2-hydrnzymethyl-5-(cytosin-1-y1)-20 1,3-oxarhiolarte has anti-hepatitis B activity. Doong, et al., Proc. of Natl.
Acad, Sci. USA, 88, 8495-8499 (1991); Chang, et al., J. of Biologic Chem., VoI 267(20), 13938-13942. The and-hepatitis B activity of the (-) and (+)-enantiomers of 2-hydrozymethyl-5-(5-fluorocytQSin-I-yl)-1;3-oxathiolane has been published by Furman, et al., in Antimicrobial Agents 2 s and Chemotheraov, Dec. 1992, pages 268fr2692.
PCT/US92/03144 (International Publication No. WO 92/18517) filed by Yale University discloses a number of 9-L-nucleosides for the treatment of both I38V and HIV. Other drugs ezlored for the treatment of HBV .
include adenosine arabinoside, thymosin, acyclovir, phosphonoformate, WO 96140164 PCTNS9611002b zldavudinc, (+y-cyanidanol, quinacrine, and 2'-fluoroarabinosyl-5-iodan~dl.
M essential step in the mode of anion of purine and pyrimidine nucleosides against viral dexases, and In particular, HBV and HIV, is their r~abolic acdvation by cellular and viral bltases, to yield the mono-, dl-, and ttiphosphate derivatives. The biologically active spacies of many auclaoaides is the triphaspahte form, Which inhibits DNA polymerase or reNerae transcriptase, or causes chain termination. The nucleo~de derivatives that have beat developed far the treabaent of HBV and HIV to 1 o dace have been presatted for administration to the host in nnghosphorylated form, notwithstanding the fact that the nucleoside must be phosphorylatied is the cell prior bo exhibiting its antiviral effort, because the triphasphate form has typically either been dephospborylated prior to r~a~ching the cell or is poorly absorbed by the xll. NucleoudGS in generat cross cell 15 membrarxs very inefficiently and era gatdslly not very not very potent ju ~. Attempts at modifytt~g nucleotides to increase the absorption and pararcy of nucloolides have been described by R. Tones and N.
l3iscawtberger, MtivJrrr! Reseal, Z9 (199 1-1'~, 2 0 In light of the fact that hepatitis B virus has readied ~d~iCC levels worldwide, and has aevae and often tragic effocfs on the infocted patient, there remains a strong aeod to provide nevi effective pharmaceutical agents to treat humans infocted with the virus that have loW toxidty W the host.
Therefore; it Is another object of the present invention to provide a z 5 method and composition for the treatment of human patients or other hosts infected with HBV.
_3-Wp 9fil4U164 PCT/US95/10026 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 Rt NHz N/\ N N/ / F
\ ~ 1N
N O~N
NaN N O' \ N
HO HO HO
p O O
wherein:
R' is hydrogen, fluoxo, bromo, chloro, iodo, methyl or ethyl; and R2 is OH, Cl, NHs, or H; or a pharmaceutically acceptable salt of the compound, optionally in a pharmaceutically acceptable carrier or diluent.
In an alternative embodiment, the $-L-enantiomer of a compound of the formula:
Rs NO
O
wherein Rs is adenine, xanthine, hypozanthine, 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:

Y~
l3ase AO
YZ
O
wherein B is a purine or pyrimidine base;
Yl, Y2, Y', and Y' are independently H, OH, N3, NR'R2, N02, NOR', -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 Y', Y~, 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 Y°
are in the arabino (aythro} configuration, and Y' and Y' are in the threo (ribose) configuration. R is H, monophosphate, diphosphate, triphosphate, alkyl, aryl or a phosphate derivative, as described in more detail below. R', R2, and R3 are independently alley! (and in particular lower alkyl), aryl, arallryl, atkaryl, 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 (l.c., 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 in vivo, or that has activity ~ in itself. Nonlimiting examples are the 5' and 1V~-pyrinudine or N6-purine acylated or alkylated derivatives of the active compound.
-S-WO 96/40164 PCTIUS96Il0U2b In a preferred embodiment of the invention, the nucleoside is provided as the monophosphate, diphosphate or triphosphate in a fonnuiation that protects the compound from dephosphorylation. Formulations include tiposomes, 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 prndrug), for example an ester, that stabilizes the phosphate in vivo. In an alternative embodiment of this invention, a stabilized phosphate derivative, as described further 1o below, of FTC, 13CH-189, or 3TC is provided for the treatment of hepatits.
The disclosed nucleosides, or their pharmaceutically acceptable prodxugs or salts or pharmaceutically acceptable formulations containing these compounds are useful in the prevention and treatment of H13V
infections and other related conditions such as anti-HBV antibody positive and H$V-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 P~PhY~tically to prevent or retard the progression of clinical ikltless 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 of anti-HBV agents that can be used in alternation ar coubination therapy include but are not limited to the Z-hydroxymethyl-5-{5-fluorocytosin-1-yl)-1,3-oxathiolane {"FTC", see WO 92/14743), its physiologically acceptable derivative, or physiologically acceptable salt; the 2-hydroxymethyl-5- -(cytosin-1-yl)-1,3-oaathiotane (including the racemic BCH-189 form, or -b-WO 96I40Ib4 PCT/L1S96/10026 3TC (BCH-189 enriched with the (-)-enxntiomer)) its physiologit~llly acceptable derivative, or physiologically acceptable salt; 2'-fluoro-5-ethyl-arabinosyluracil (FEAL>); carbovir, or interferon.
- Any method of alternation can 1x used that provides treatment to the patient. Nonlimiti;ng 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 ei~ective 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 io 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 HIY, 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'~eozythymidine (AZT~, 2',3'-dideozyinosine (DDn, 2',3'-dideoxycytidine {DDC), Z',3'-dideoxy-2',3'-didehydrothymidine (D4T), 2-hydrozymethyl-5-(5-fiuorocyWsin-1-yl)-1,3-oxathiolane (FTC, or Z-hydroxymethyl-5-(cytosin-1-yl)-1,3-ozathiolane (racemic BCH-189 or Z 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 Frgures to Figure 1 is an illustration of the chemical structures of B-L-2',3'-dideoxycytidine (B-L-FddC), B-D-2',3'-dideoxycytidine (B-D-ddC), B-L-2',3'-dideoxy-5-fluoroeytidine (B-L-ddC), (-)-B-L-2-hydroaymethyi-5-(5-fluorocytosin-1-y1r1,3,oxathiolane (( )-B-L-FTC), (+)-&D-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-dioxolane ((+)-&D-FDOG~, and B-L-2-amino-6-(R~-9-[(4-hydroaymethyl)-tetrahydrofiuan-1-yl]purine.
Figure 2 is an illustration of the numbering scheme used in the chemical nomenclature for nucleosides in this text.
2 0 Detailed lDescrlption of the Invention As used herein, the term "enantiomerically pure" refers to a nucleoside composition that includes at least approximately 9596, and preferably approximately 97%, 9$%, 99%, or 10096 of a single enantiomer of that nucleoside.
The term alkyl, as used herein, unless otherwise specified, refers to a saturated straight, branched, or cyclic, primary, secondary, or tertiary hydrocarbon of C~ to C,o, and specifically includes methyl, ethyl, gropyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, _8-neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl, ' 2,2-dimethylbutyl, arid 2,3-dimethylbutyl. The alkyl group can be optionally substituted with one or more moieties selected from the group consisting of hydroxyl, amino, alkylamino, arylamitto, alkoxy, aryloxy, vitro, cyano, sulfonic acid, sulfate, phosphoric acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known Lo those skilled in the art, for example, as taught in Greene, et al., "Protective Groups in Organic Synthesis," John Wiley and Sons, Second Edition, i99I. The term lower alkyl, as used herein, and unless otherwise 1o spocified, refers to a C, to C, 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, propionyl, valeryl, raproic, 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 z 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 Whey and Sons, Second F~ctition, 1991.
. The term purine or pyrimidine base includes, but is not limited to, adenine, 1V6-alkylpurines, N6-acylpurines (wherein acyl is C(O)(alkyl, aryl, ' alkylaryl, or arylalkyl), N6-bcnzylpurine, ~-halopurine, N6-vinylpurinc, N6-acetylenic purirte, 1Vb-acyl purine, N6-hydroxyalkyl purine, 1V~-thioalkyl _9-purine, Nz-alkyIpurines, Ni-alkyl-trthiopurines, thymine, cytosine, 6-a~apyrimidine, 2- and/or 4-metcaptopyrmidine, uracil, Cs_ ~YIPY~dines, Cs-benzylpyrimidines, C3-halopyrimidines, CS-vinylpyrimidine, Cs-acetylrnic pyrimidine, Cs-aryl pyrimidine, Cs-hydroxyalkyl purine, Cs-amidopyrimidine, Cs.cyanopyrimidine, Cs-nitropyrimidine, G"5-aminc~pYrimidine, N1 allrylpurines, Nz-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl, triazalopyridinyl, imidazolopyridinyl, pyrrolopyzimidinyl, pyrawlopyrimidinyl. Functional oxygen and nitrogen groups on the base can be protected as neo~ssar~r or l0 desired. Suitable protecting groups are well known to those slQlled in the art, and include trimethylsilyl, dimethylhexylsilyl, t-butyWimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as acetyl and propionyl, methylsulfonyi, and p-Loluyisulfonyl.
As usod herein, the term natural amino acid includes but is not limited 15 to alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, ~YP~1~Y1. oninyl, glycinyl, serinyl, rhreoninYl, cysteinyl, tyrosinyl, aspataginyi, giutaminyl, aspartoyl, glutaoyl, iysinyl, argininyl, and histidinyl.
The invention as disclosed herein is a method and composition for the z o treatment of HBV infection and oilier viruses replicating in a like manner, in humans or other host animals, that includes administering an effective amount of one or morn of the above-identified compounds, or a physiologi~lly acceptable derivative, or a physiologically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier. The z ~ compounds of this invention either possess anti-HBV activity, or are metabolized to a compound or compounds that exhibit anti-I3BV activity.

WO 46/40164 PC'F1U596/t002fi I. Structure and Preparation of Active Nucleosides The compounds used in the methods disclosed herein are enantiomers of 2',3'-didcoxycytidine, 2',3'-dideoxy-5-(halo or methylxytidine, 2-s hydmxymethyl-5-(5-fiuorocytosin-1-yl)-1,3-dioxolane, or 2-amino-b-(OH, Cl, NH2, or I~-9-[(4-hydtnxymethyl)-tettahydrofuran-1-yl]purine.
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 (CHZOR and the pyrimidine or purine base, respectively) can be zither 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 Cl' 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-z 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-D-enantiomeric form, because it has been discovered that the B-D-enantiomer of FDOC is surprisingly less toxic than the H-L-enantiomer of FDOC.
_11-' WO 96/40164 ' P(:TNS96Ii0026 The nucleosides disclosed herein can be administered as any derivative that upon administration to the rodpterct, is capable of providing ditscfty or indiroctly. the parrot active compound, or that exhibits activity in itself.
1n oae embodiment, dye hydrogen of the s~-QH gt~oup is by a C,-Cm alkyl, including Ci to Cs alkyl; aryl in which the noo-carbonyl moiety of tha ester group is seladed from straight, b:aa~hed. or cyclic Ci-Cm alkyl including Ci to Cs atkyl, phenyl, or benryl; a naturally occurring or rwiinaturally occurring amino acid; allooxyalkyl including mahoxymethyl;
to aralkyt including benzyl; aryloxyalkyl such as phartoxymethyl; aryl ineludirig phenyl optionally substituted with haloga~, C, b C4 alkyl or Cs to C, alkoxy; a dicarboxylie acid such as ~cinic acid; sulfonate eaters such as alkyl or aralkyl sulphonyl including metheneeulfonyl; or a mono, dl or triphosphate ester.
is Une or both hydmgens of the amino groups on the purine or pyxlmidiile base can be replaced by a Ci-Cm alkyl, inctud'mg C, to Cs alkyl; aryl in which the rwn-carbonyl moiety of the esta~ group is aeiected from straight, branched. or cyclic Ci-C~ alkyl, including C, to Cs alkyl, phenyl, or berizyl; sllwxyalkyl includiug methaxymethyl; aratkyl including 2 o benzyl; arybxyalkyl such at phenoxyrndhyl; aryl including phenyl optiartally aubstitutod with halogea, C, to C~ alkyl or C, to C, alkozy.
The active nucleoside can also be provided :s a S'-ether fipid, as disclosed in the following references,:
iCuoera, L.S., N. iyer, B. Leaks, A. Rabea, Modest E.1., D.
z5 L.W., and C. Piantadosi. 1990. Novel mernbrati~intersdive ether lipid analogs that inhibit infectious HIV-1 production and induce defective virus formation. A1DS Res Hum Retrovituses. 6:491-501; Piantadosi, C., I.
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Surles, K.S. Ishaq, L.S. Kucera, N. Iyer, C.A. Wallen, S. 1?iaetadosi, and WO 96/40164 PCT/US961t0026 B.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. ltichman, D.A. Carson, L.M. Stuhtniller, G,M. T.
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Any of the nucleosides described herein, or any other nucleoside that i5 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 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 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 Iipophilic modification of the mono, di or triphosphoate of the nucleoside will increase the stability of the nucleotide. Examples of substituent groups that 2 s cart 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. Bischofberger, Antiviral Research, 27 (1995) I-I'7. Any of these can be used in combination with the disclosed nucleosides to achieve a desired effect. Nonlimiting examples of nucleotide pmdrugs are described in the following references. .
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Chemother. I, 107-113; McGuigan, C., O'Connor, T.J., Nieholls, S.R.
2o Nickson, C. and Kinchington, D. (1990b) Synthesis and anti-HIV activity of Borne novel substituted dialky phosphate derivatives of AZT and ddCyd.
Antiviml Deem. Chernother. l, 355-360; McGuigan, C., Nicholls, S.R., O'Connor, T.J., and Kinchington, D. (1990c) Synthesis of some novel diatkyl phosphate derivative of 3'-modified nucleosides as potential anti-AIDS drugs. Aruiviral C~rrr. Ctother. 1, 25-33; McGuigan, C., Devine, K.G., O'Connor, T.J., and Kinchington, D.(199I) Synthesis and anti-HIV activity of some haloalky phosphoramidate derivatives of 3'-azido-3'deoxythylmidine (AZT); potent activity of the trichloroethyl methoxyalaninyl compound. Mtivtral Res. 15, 255-263; McGuigan, C., 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. .lruivirnl Res. 17, 311-321;
McGuigan, C., Fathirana, R.N., Choi, S.M., Kinchington, D. and O'Cannor, T.J. (1993x) Phosphoialnidate derivatives of AZT as inhibitors of HIV; studies on the carbonyl terminus. Antiviral Chem. C7temother. 4, 97-101; McGuigan, C., Parhirana, R.N., B~alzarini, J. and De Clercq, E.
(1993b) Intracellular delivery of bioactive AZT nucleotides by aryl phosphate derivatives of AZT. J. Med. t:7sem. 3as, 1048-1052.
io Alky hydrogen phosphonate derivatives of the anti-HIV agent AZT
may be less tonic than the parent nucleoside analogue. Antiviral tfiem.
C~entothtr. 5, 271-217; Meyer, R. H., Jr., Shaman, D.A. and Robins, R.K. (1973) Synthesis of purine nucleoside 3',5'-cyclic phosphoramidates.
Tetrahedron Left. 2b9-272; Nagyvary, J. Gohil, R.N., Kirchner, C.R, and Stevens, J.D. (1973) Studies on neutral esters of cyclic AMP, Biochem.
Biophys. Res t.onsmun. 55, 1072-10'17; Namane, A. Gouyette, C., ' Fillion, M.1'., Fillion, G. and Huynh-Dinh, T. (199Z) Improved brain delivery of AZT using a glycosyl phosphotriester prodrug. J. Med. Chem.
35, 3039-3044; Nargeot, !. Nezbonne, 1.M. Bngels, J. and I,eser, H.A.
no (1983) Natl. Acad. Sci. U.S.A. 80, 2395-2399; Nelson, K.A., Hentrude, W.G., Stser, W.N. and Hutchinson, J.P. (I987) The question of chair-twist equilibria for the phosphate rings of nucleoside cyclic 3',3'monopl~sphatec. ~I~TMR and a-ray crystallographic study of the diasteromers of thymidine pheayi cyclic 3',5'-monophosphate. 1. Am.
z5 Chem. Soc. 109, 4058-4064; Nerbonne, J.M., Richard, S., Nargeot, J.
and Lester, H.A. (1984) New photoactivatable cyclic nueieotides produce intracellular jumps in cyclic AMP and cyclic GMP concentrations. Nature 341, 74-76; Neumarut, J.M., Herv~, M., Debouzy, J.C., Guerta, F.L, Gouyetie, C., Dupraz, B. and Huynh-Dinh, T. (1989) Synthesis and WO 96!40164 PCT/IJ596l10028 transmembrane transport studies by NMR of a glucosyl phospholipid of thymidine. J. Am. Chem. Sec. 111, 4270-4277; Ohno, R., Tatsumi, N., Hirano, M., imai, K. Mixoguchi, H., Nakamura, T., Kosaka, M., Takatuski, K., Ysmaya, T., Toyama, K., Yoshida. T., Masaoka, T., Hashimoto, S., Ohshima, T., IClmura, L, Yamada, K. and Kimura, J.
(I99I) Treatment of myelodysplastic syndromes with orally administered 1-~i-D-rabinofuranosylcytosine -5'stearylphosphate. Oncology 48, 451-455.
Palomino, E., Kcssie, D. and Horwitz, J.P. {1989) A dihydropyridine l0 carrier system for sustained delivery of 2',3'dideaaynucleosides to the brain. J. Med. Chem. 32, 622-625; Perkins, R.M., Barney, S., Wittroek, R., Clark, P.H., Levin, R. Lambert, D.M., PeEteway, S.R., Serafinowsl~, H.T., Banley, S.M., Jackson, S., Hamden, M.R. Ashton, R., Sutton, D., Harvey, J.J. and Brown, A.G. (1993) Activity of >3RI~47923 and its oral prodrug, SB20365yA against a rauscher routine leukemia virus infection in mice. Antiviral Res. 20 (Suppl. n. 84;
Piantadosi, C., Marasco, C.J., Ir., Morns-Natschke, S_L., Meyer, K.L., Gumus, F., Surles, J.R., Ishaq, K.S., Kuceia, L.S. Iyer, N., Wallen, C.A., Piantadosi, S. snd 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., Isfebvre, L, lmba~ch, J.L., Kahn, S.
and Farquhar, D. (1994) Decomposition pathways of the mono- and bis(pivaloyloaymetllyl) esters of azidothymidine-5'-monophosphate in cell extract and in tissue culture medium; an application of the ' on-line 1SRP-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., Smce, D.F. Duke, A.E., Matthews, T.R. and Verheyden, 1.P.J. (1986) Synthesis and antiherpes virus activity of phosphate an WO 96/4016A PC'flI3S96It0026 phosphonate derivatives of 9-[(I,3-dihydroxy-2-propoxy)methyl] guanine.
1. Med. Chem. 29, 671-675; Pucch, F., G~selin, G., lxfebvre, L, Pompon, A., Aubertin, A.M. Dirn, A. and Imbach, J.L. (1993) Intracellulaur 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 Eiaengart, R.S. (I969).
Toxicological assessment and health standard ratings for ethylene sulfide in the industrial atmosphere. Gig. Trf. Pm~ ?abol. 13, 47-48 CChem. Abstr. 72, 212); Robins, R.K. (1984) The potential of nucleotide 1 o analogs as inhibitors of retroviruses and tumors. Pharm. Res. ! 1-18;
Rosowsky, A., Kim. 5.H., Ross and J. Wick, M.M. (1982) Lipophilic 5'-(alkylphosphate) esters of 1-p-D-arabinofuranosylcytosine and its 1V~-aryl and 2.2'-anhydro-3'0-aryl 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, 23S-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-~-D-arabinofuranosyleytosine S'diphasphate[-j, Z~iaeylglycemls. J. Med. Chem. 25, 1322-1329; Saffhill, R. and Hume, W.J. (1986) The degradation of 5-iododeoxyurindine and S-bromoeoxyuridine by serum from different sources and its consequences for the use of these compounds for incorporation into DNA. Chem. Bial.
Interact. 57, 347-355; Saneyoshi, M., Morozumi, M., Kodama, K., Machida, J., Kuninaka, A. and Yoshino, H. (I980) Synthetic nucleosides and nucleotides. XVI. Synthesis and biologics! evaluations of a series of 1-p-D-arabinofuranosyleytosine 5'-alley or arylphosphatrs. 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-WO 96/40164 PCTlUS96J10026 permeable dideoxyuridine 5'-monophosphate analogue inhibits human ilnmunodeficieacy virus infection. Mol. 1'harnlacol. 41, 441-445; Shaw, ' J.P., Jones, R.J. Arirnilli, M.N., I:ouie, M.S., Lee, W.A. and Cundy, K.C. (I994) Oral bioavaiIability of PMF.A from PMEA prodrugs in male Sprague-Dawley rats. 9th Annual AAPS Moelirtg. San Diego, CA
(Abstract). Shuto, S., Ueda, S., Imarnura, S., Fukukawa, K. Matsuda, A.
and Ueda, T. (19$'~ A facile ono-step synthesis of 5'phosphatidylnucleosides by an enrymatic two-phase reaction.
Tetrahedron Left. 28, 199-202; Shuto, S., Itch, H., Ueda, S., Imamura, 1o S., Knkukawa, K., Tsu,~ielo, 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 prefenced phosphate prodrug group is the S-aryl-2-thioethyi group, also referred to as "SATE".
hreu~ration of the Active Compounds The nucleosides used in the disclosed method to treat HBV infections in a host organism can be prepared a~ccoidittg to published methods. &L-Nucloosides can be prepared from methods disclosed in, or standard modifications of methods disclosed in, for example, the following publications: Jeang, et a1.,1. of Med. Chem., ~, 182-195, 1993;
European Patent Application Publication No. 0 285 884; Gbnu-DeIlac, C., G. Gosselin, A.-M. Aubertin, G. Obert, A. Kirn, and 1.-L. Imbach, 3-Substituted thymine a-L-n~xleoside derivatives as potential antiviral agents;
z 5 synthesis and biological evaluation, ,pi,~~tiviral Chem. Chemother. 2:83-(I991); 3ohansson, K. N. G., B. G. Lindbor~g, and R. Noreen, European Patent Application 352 248; Mansuri, M. M., V. Farina, J. E. 5tarrett, D.
A. Berzigni, V. Brankovan, and J. C. Martin, Preparation of the geometric isomers of DDC, DDA, D4C and D4T as potential anti-HIV agents, WO 96!40164 PCTIU596ft0026 1:65-b8 (1991); Fujimori, S., N_ Iwanami, Y, Hashimoto, and K. Shudo, A convenient and stereoselective synthesis of 2'-deoxy-B-~L-ribonucieosides, Nucleosides do ucleotides 11:341-349 . (1992); GErtu-Dellac, C., G. Gosselin, A.-M. Aubertin, G. Obett, A.
Kirn, and J.-L. Imbach, 3-Substiwted thymine ac-L-nucleoside derivatives as potential antivirai agents; synthesis and biological evaluation, Antiviral Chem. Chemothe~ 2:83-92 (1991); Holy, A, Synthesis of 2'-deoxy-L-uridine, ~~. 2:189-192 (1992); Holy, A., Nucleic acid components and their analogs. CLI)Z. Preparation of 2'-deoxy-L-1o riba~nucleosides of the pyrimidine series. Coll~C,~h Chem Commun.
3y:4072-4087 (1992); Holy, A, 2'~eoxy-L-uridine: Total synthesis of a uracil 2'-deoxynucleoside from a sugar 2-aminooxazoline through a 2.2'-anhydronucleoside intermediate. In: Townsend LB, Tipsan RS, od.
Nucleic Acid Chem. New York: Whey, 1992: 347-353. vol 1) (1992);
Okabe, M., R.-C. Sun, S. Tan, L. Todaro, and D. L. Coffee, Synthesis of the dideoxynucleosides ddC and CNT fmm glutamic acid, ribonolactone, and pyrinudine bases. J~~ 53:4780-4786 (1988):
Robins, M. J., T. A. Khwja, and R. K. Robins. Purine nucleosides.
XXpC. Synthesis of 21-deoxy-L-adenosirve and 21-deoxy-L-guanosine and 2 o their alpha anomers. ~,QC~ Chem. 33:363-b39 (1992); Gnu-Dellac, C., Gosselin G., Aubertin A-M, Obert G., Kirn A., and Imbach J-L, 3'-SubstiWted thymine a-L-nucleoside derivatives as potential antiviral agents;
synthesis and biological evaluation. A.ntivi_raf Chem hemQther. 2(2):83-92 (1991); GEnu-Dellac, C., Gosselin G., Imbach J-L; Synthesis of new ~ s 2'-deoxy-3'-substituted-a-L-threo-pentofuranonucleosides of thymine as a potential antiviral agents. Tel Lett 32(1):79-82 (1991); Gnu-Dellac, C., Gosselin G., Imbach 1-L. Preparation of new acylatod derivatives of L-arabino-furanose and 2-deaxy-1-erythro-penwfvranose as precursors for the synthesis of 1-pentofiuanosyl nucleosides. 216:240-255 (1991); and G~nu-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 occurring nuclei acid bases. 10(b):1345-1376 (1991).
2',3'-Dideoxycytidine (DDC) is a known compound. The D-enantiomer of DDC
is currently being marketed by Hoffman-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.
Enantiomerically pure ~i-D-dioxolane-nucleosides such as ~3-D-FDOC can be prepared such as disclosed in detail in WO 92/010497. The process involves the intitiaI
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 entantiomerically pure final product, including the correct diastereorneric 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 example, Nucleosides and Nucleotides, 12(2), 225-236 (1993);
European Patent Application Nos. 92304551.2 and 92304552.0 filed by Biochem Pharma, Inc.; and PCT Publication Nos. WO 91/11186, WO 92/14729, and WO
92/14743 filed by Emory University.
Separation of the acylated and alkylated racemic mixture of D and L
enantiomers of cis-nucleosides can be acconrzplished by high performance Liquid chromatography with chiral stationary phases, as disclosed in PCT
Publication No. WO 92114729.
Mono, di, and triphosphate derivative of the active nucleosides can be prcpared as described according to published methods. The monophosp6ate can be pttpa~d accordeng to the procedure of In~ai ct al., J. Ora. Chem., 34(6), 154? 1350 (June 1969). The diphosphate can be prepared according to the proceduze of Davisson et al., J. Orgasm., 52(9), 1794-1801 (1987). The triphosphate can be prepared according to the procedure of Hoard et a1.,1. Am. Chem. Soc., 87(8), 1785-1788 to (1965).
I O
S,~O ~ ~,~0 OH n R.~ ~O p,~g'~x --~SH '''CS-~ - 'S

2 o O Base ~ O ~t Yt Ya O p..._0 Y, 8is (SAT>~ p-L-ddoMP
an Y', Y2, Y', and Y4 are independently H, OH, Ns, NR'Ri, NOi, NOR3, -O-alkyl, _23_ -O-aryl, halo (including ~, Cl, Br, or n, -CN, -C{O)NHx, SH, -S-allryl, or -S-aryl, and wherein typically three of Y~, Y2, Y3, and Y4 are either H or OH. The -OH substituent, when present, is typically a Y' or Y' group.
As illustrated in the structure, Yx and Y4 are in the arabino (erythro) configuration, and Yt and Y3 are in the threo (ribose) configuration. The base is a purine or pyrimidine. AltGrnatirvely, the psuedo-sugar moiety is a 1,3-oaathiolane (as in FTC and BCH-184 or 3TC or is a 1,3-dioxolane derivative). (t) ICHZCH20H, DBU/C6HsCH~: (ii) CIxPN(iPr)x, NEt3lTHF; (iii) S-L-dideoxynucteoside, IH-tetrazole/THF, then Zo C1C~C03H/CH2C111H-Tetrazole (0.218, 3.0 mmol) was added to a stirred solution of p-I; dideoxynucleoside (1.0 mmol) and the appropriate phosphoramidite ~ (1.2 mmol) in tettahydrofuran (2mL) at room temperature. After 30 minutes, the reaction mixture was cooled to -40°C
and a soluti~ of 3-chiomperoxybenzaic acid {0.23 g, 1.3 mmol) in 1s dichlommethane (2.5 mL) was added; the mixture was then allowed to warm to room temperature over 1 h. Sodium sulfite (1096 solution, 1.3 mL) was added to the mixture to destiny the excess 3-chtoroperoxybenzoic acid, after which the organic layer was separated and the aqueous layer washed with dichloromethane {2 s 10 mL). The combined organic layers 20 were washed with saturated aqueous sodium hydrogen carbonato (5 mL), then water (3 a S mL), dried over sodium sulfate, filtered and evaporated to dryness under reduced pressure. Column chromatography of the residue on silica gel afforded the tine Bis(SA"Tfi) ~-L-ddxm~.

WO 96/4U164 PC'F/I3S96l10U26 = p-L-2',3'-Dideozyadenosin-5'-yI bis (2-pivaloylthioethyl) phosphate [Bis (SATE) (3-L-ddAMp].
~o (CH~3C-C~ ~CHZ_p PN(iPr)z S Cliz 2 ~-L-ddA. 1 H-teuazolelI'EiF
then CIC~HyC03HICf3~Ch then silica gcl column chromatography O
(Ct"ia)aC-C~ C
S-Cli2 $is (SATE)/3-L_ddAMp Following the above general procedure, pure l3is(SATE~~-L-ddAMP_ was obtained as a colorless oil in 72 % yield after silica gel column chromatogtaptty [eluent: stepwise gradient of methanol (0-3%) in dichloromethaale]; ' NMR (DMSO - d6) 8 ppm: 8.26 and 8. i3 (2s, 2H
each, H-2 and H-8), 7.20 (br s, 2H, NHS, 5.24 (t, 1H, H-1'; T=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 SCHzCHiO), 3.04 (t, 4H, 2 SCH1CH20 ; 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)3C]; 31~,NMR
(DMSO-db) 8 ppm = -0.76 (s) ; W {EtOH) , 7l ~ = 259 nm {e 15400);

mass spectrum (performed in: glycerol, thioglycerol, 1:1, ulu), FAB > O
604 {M+H)+, 136 (BHP+. .
~~~gueral s~~~,me for tie ~ros~xLt'N;~y~th~~,~~f 3'-cnbstit~ted B-L-OH
RO~~~
Y' 11~~YO
Compound 8 [see Fig. L/2 of tht French patent ~e Appendiz 4 Bass OX
Ro~Base ~/' .~/0 2 ~ ~ ease OH
O Ra Baae r RO-t 8iee V~-O~X
O
3 0 ~ ~o~aaae ~'' ~r «erythrOo»
conftgtuation RO
HO~~
~OIy <atueo»
4 0 contiguranon IE
v = any ~cH,-c, c6Hs-c) X = Leaving group [CH3 S4i, CH3 CaH, SOi, CF3 SOa]
Y. Y' = F. N3. W R: LRi.Rs = H. alkyl, aryl].
NOi, NOR [R = H, alkyl, aryl], O-alkyl, O-aryl, etc EX9~E = 1-(3-Arido-2-3-dideozy-~i-Irerythno-penwfuranosyl) thytnine [~i-L-AZTj '(C6FIs)3 D!~AD
PA p O
11N ~ CIi3 O~N
NO
a O
~-L-AZT
4096 yield from g A mixture of diethyl azodicarboxyiate (0.46 mL; 2.9 mmol) and diphenyl phosphorazidate (0.62 ml; 2.9 mmol) in 'TI-iF (2.9 ml) was added dropwise over 30 min. to a solution of 1-(2-decay-5-O-monomethoxytrityl-(i-L-three-pentofuranosyl) thymine $ [0.5 g, 0.97 mmol] and triphenylphosphine (0.76g, 2.9 rnmol) in THF l 1.6 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 1o temperature and was diluted with toluene. T'he sepazated aqueous phase was concentrated to dryness in vacuo. T'he residue was purified over a silica gel column eluted with ethyl acetate to afford ~-L-AZT' (105 mg, 40%, crystallized from ethyl acetate). The physicochemical data of p-L_ AZT were in accordance with literature data [J. Wengel, J-Lau, 1~.B.
Ledersen, C.N. Nielsen, J. Org. Chem. ~ (11), 3591-3594 (199I)].

~p~,~ral Scheme for the Stereas~fc Sv~~lhesis tt,~ ~~'_~,'b~tituted 9-1~.
RO~~
HO
L?
CO(IlpOtlnd j~
[see Fig. 1I2 of ~hc FrenCh patent' 5cx Appendix 4 OV
Beae RO RO
xo ~ OH
RO
r O R Hase O
1 ox RO
o r Ho 8°'s «thrto>s conti oration RO
'~yO
WO~~
Y
O
«erythtn»
configuration O O
v = acyl [CH3-C C6H5-C]
X .= Leaving group [CHs SO~,~ CHs Cs~~ H. ~3 Sue]
Y. 1" = F. Ns~ NRtRx fRt~Rz = H. ~Yl~ ~1'1]>
3 o NOs, NOR [R = H, alkyl, aryl], O-alkyl, O-aryl, etc.

WO 96140164 PCTlU596110026 E$~ ~ I-(2-Fluoro-2,3-dideoxy-(3-L-wren-pentofuranosyl)-5_ fluorocytosine [2'-F-~-L ~-L-FddC]

NON F H~CO CHZ_ F
o~N CH,C6~ N
DBUICH=CV
HO y~0 96~ yield DAST.
I O a CFIICh, CsH~V
O o ~F (NH,y~Ce(NO~)e H~O~~YN
15 F N CH3 ~ O~N
P
Bz0 8z0 6096 yitid ~7'b Yield O
O
f~wesson's ~xgendCHZCh roflux NHZ
20 ~ F
_CH~OHIf~(H3 ,~
10~C O~N
F
NO

2'-F-d-L-Fddc 2 5 6x96 yirld Hitherto unknown 2'-F-~i-L-FddC was synthesized in five steps from 1-(5-O-benzoyl-3-decay-~-I~-crydun-pentofuranosyl}-5-fluomutacil L7 with an overall yield of 2896, m.p. 209-210°C (crystallized from absolute WU 96140164 PC'1'1US96110026 ethanol); W (Et OH) l~,x 276 ~" (s, 9000), 7~ 226 (e, 4000); "F_ NMR (DMSO-d6) 8 ppm : -179.7 (m, F2,) , -167.2 (dd, Fs; JF.s = 7.3 Hz, JF.,. = l:SHz);'H-NMR (DMSO~ 800m : 8.30 (d , 1H, H-6: Ja.F = 7.3 . Hz), 7.8-7.5 (br s, 2H, NHS}, 5.80 (d, 1H, H-1' J,.,F = 17.4 Hz), 5.34 (t, 1H, OH-5'; J = 4.8 Hz), 5.10 (c~, 1H, H-2'; J=.,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-thioglycerpl, 1:1 v!u), FAB > 0:248 (M+H)*, 130 {BHP+; FAB < 0:246 (M-H)' ; [a]Z°a = -16.5-(-c 0.85, DMSO). Anal. Calc. far C9HmN30jF2 : C, 43.73; H, 1o 9.49; N. 17.00; F. 15.37 . Found: C, 43.56; H, 4.78; N, 16.75; F, 14.96.

G~Cylose «~iCO. HjSO,. CuSO,.
That NH,OH
HCIIH=O ~ NaHCO~hfiO
O ~~~,,/O
BxO~ ~H~OCI O-. O OBz 1 l p~~ HO-~ I ~~C~ Bz0 OH ~ 1 C3H~~I-CHCh (CH;CO),O. ~ OAc (S)COm)= ~ HZSO, O
l(CHiG~
glyoosidic (CH,S)),SiFi, AIHN O~ / O ooodmsation O
Bz0 / T~ ~z ZO O C lm ~ ~ Bzo--~8ase t) CH,cnpH ss~
HrSO, O
2) (CH300yj0, Bz0 ~~ ~Z~a-HxO
OAc lPYridioe-CH,COOHI
OAe -,.
L 14 Bz0 ease O
1) CaFf,OC(SxI
D~ / CH~CT~J
Bx0 Base 2) Hu3SnH, A~N
Ac0 ldioxaoe 08z O Bz0-~~Base CH,ONa/CH,OH ~ HO Base O
NH, ! Ctt,OH m NHr l CH,OH ~
,~ OH
H''OZ=mM'1'r) HO-~8ase O «
~ F (R=THDPSI) O
ltC1 l ~y.;a;ue « Z
NH, l CH,OH (R o Hz « Ac) RC1 I pyrid~oe RO Bax HO I) CatIspC(Sxi OH
O DMAP/CH,Ct~O ~ ~~(Sxl RO--~~Ba~e -rt 2) Bu,Saff- AIBN -l Talneoe Q 2) Hu~SoFI, AIBN O
3 d ! dioxane Schema I: Bases = pwines or pytimidines, eventuellemmt conveuablemeat pcotegeea; R
a Beazoyl (Bz) Acetyl (Ac), monomethoaytrityl (mMTr) or tertiarybutyldiphenylsilyl (-ranpsn WO 96140164 t'CTIUS96/10026 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 Germ, 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-Ir2',3'-dideozycytidine (B-L-lrddG7, B-L-2',3'-didemcy-S-1o fluorocytidine (B-L-ddC), and (+rB-D-2-hydrozymethyl-S-(5-fluorocytasin-1-yl)-1,3~iiozolane ((+}-$-D-FDOC). The toxicity and anti-HBV activity of (-)-&L-2-hydroxymethyl-5-(5-fluorocyWsin-1-yt)-1,3-oxathiohme ((-)-$-L-FTC) and B-D-2',3'-dideozycytidine (&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-HHV assays were characterized as follows.
2'.3~'-D'i~i~s~t-B-~v~j~e _f$-L'DDC1. m.p. = 220-220°C; W
2 o (EtOH 95) mix 273 nm, 7~min 252 nm; NMR-~H (DMSO-ds) 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, IH, H-5; J = 7.4 Hz), 4.99 [E.
1H, OH-5'; J - 5.2 Hz]. 4.05-3.95 (m, IH, H-4'), 3.64-3.70 (m, IH, H-5'; after DiO exchange: dd, 3.64 ppm, J = 3.6 et I2.0 Hz). 3.60-3.50 (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]p~°-103.6 (c 0.8 MeOH); mass spectrum (performed in: glytxrol-ttuoglycerol, 50 50. vlv); FAB>0 423 [2M+H]*, 304 (M+glyoerol+H]+. 212 [M+H]*, 112 [BHzJ*, 101 [s]+; FAB<O 210 [M-H]'. Anal. Calc. for WO 9b/40~ 6A PC'T/US9b/t002b C9H,3N303 (M = 211.21); C SI.18; H 6.20; N 19.89 found; C 51.34; H

6.25; N 20.12.
~ m.p. .- 1S8-160°C; UV (EtOH 95) kmax 281 am (e, 8100) et 237 nm (e, 8500); min 260 nm (e, 5700) et 225 nm (e, 7800); NMR - ~H (DMSO-d6) Sppm 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';1 = 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 Hzl, 3.60-3.50 [m. 1H, H-5"; after D20 exchange: dd, 3.52 ~r ;
1o J = 3.3 et 12.3 Hz], 2.35-2.15 (m, 1H, H-2'). 1.95-1.75 (m, 3H, H-2", 3' et 3"): jala~°-80.0 (~ 1.0, DMSO); Mass spectrum (performed in: 3-nitrobenzyl alcohol] FAB > 0 230 [M+H]+ et 101 [sl+; FAB C O 228 [M-IIl-. Anal. Calculated for C9H,zN~F'03(M = 229.2(); C 47.1b; 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 titan 3.0-fold (for HBV
Z o virion DNA) or 2.5-fold (for H13V DNA replication intermediates) from the average levels for these HBV DNA forms in unu~eated cells are generally considered to be statistically significant [P<0.051 (Korba and Germ, Antiviral Res. 19: 55-70, 1992). The leveEs of integrated HBV
DNA in each cellular DNA preparation (which remain constant on a per 2s cell basis in these experiments) were used to calculate the leveEs of intracellular HBV DNA forms, thereby eliminating technical variations inherent in the blot hybridization assays. .
Typical values for extracelIular HHV virion DNA in untreated cells range from 50 to 150 pg/ml culture modium (average of approximately 76 WO 96/40164 PCTlUS96lI0o26 pg/ml). Intracellular HBV DNA replication intermediates in untreated cells range from 50 to 100 pg/ug cell DNA (average approximately 74 pglug cell DNA). In general, depressions in the levels of intracellular HBV DNA due to treatment with antiviral compounds are less pronounced, and occur mole slowly, than depressions in the levels of HHV virion DNA.
For reference, the manner in which the hybridization analyses were performed for these experiments results in an equivalence of approximately L0 pg intracellular HBV DNA/ug cellular DNA to 2-3 genomic copies per cell and 1.0 pg of extracellutar HHV DNA/ml culture medium to 3 x IOs 1o viral particles/mi.
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 simples 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, suspended 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 far toxicity at concentrations from 1 to 300 ACM. The results are provided in Table 1.

WO 96/401b4 PC'f/US96I10026 ....N
y1 0~0n o T
N

.C N

U

y W

V~ ~ ~ N
N

' ~ .~.--,..., .~ N W f N

$ ~ M oo.-.
d ~D O1M V1 .-i' ' N d ~ N
N

~!
O N ~O~ O

fV C O O O
CV

I I I
I

U -~ ~ ~ 000om o0 ~

N ", N
.~ 'a'd O
..v ..rt v v N , ' ~t N N_ 00 t~ O

~D etO

C O O O O

H ~ A ~ 1 1 I t~00N Oz .-n N

oo ~ nig M

w ~

r c 0 cmncEO o O O O C
N

m ,a N ..
' ~ Q o o L1 W

U

A

A n g o g w N 0 0 0 0 O ~ , 1 1 I 1 ~I M cnN o n p ~ ~~, "' -. o ~ Q ..
- 4 .

GTr W Q a ~ b m W ,Q

z A

. b U U .c A ~ A A

, ~ ..
c?' a ~

~yv 0 q V GrlC1~L

n a ca -3s-WO 96/40164 PCTIU596/t002fi Example 2 Toxicity De 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 s 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 itM, however, all three compounds exhibited less toxicity at this concentration than &D-ddC. It appears that the iC~ of &L-ddC and &IrFddC is approximately twice that of B-D-ddC.
Toxicity analyses were performed in 9Crwell flat bottomed tissue culture plates. Cells far the toxicity analyses were cultured and treated with test compounds with the same schedule as used for the antivirai 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 interna1ixed dye at 510 nM
(As~o) 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 compounds. The percentage of dye uptake in the 9 control cultures on plate d0 was 100 t 3. At 150-190 ~eM B-D-ddC, a 2-fold reduction in dys uptake (versus the levels observed in untreated cultures) is typically observod in these assays (Rorba and Gerin, Antiviral Res. 19: 55-70, 1992).

WO 96/40164 1'CT/U596/tOD26 Eacamtpk 3 Anti-Hepatitis B Vinrs Activity , The positive tr~nent control, &D-2',3'-dideoxycytosine [&D-ddC], induced significant depressions of HBV DNA replication at the concentration used. htevious studies have indicted that at 9-12 pM of B-D-ddC, a 90% depression of HBY RI (relative to average levels in untreated cells) is typically observed in this assay system (Korba and Germ, Antivira! 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 20 ((&L-FddC), (B-L.ddC), and B-D-FDQC)), wera potent inhibitors of HBV
replication, causing depression of HBY virion DNA and HSV RI to a degroc comparable to, or greater than, that observed following treatment with B-D-ddC.
Fxampfe 4 The effect of selected B-L-derivatives against Hepatitis B virus replication in transfectod fiep G-2 cells is described in Table 4.
_38-W0 9b/40164 PGTNS96110026 P~~ h x N

v' a z N ~

a U

N

~

U
W

,nO
~1G ~ 00h U ~

a o ~"a ~nr:

g o m. ~.~ z U ~ ~ ~ ~ ~ A
V
C~~1C~1f~N

w .o a WO 9b140t64 PC'TIU59b/Z002b Example 5 The Comparative inhibitory effect of selected triphospahtes on woodchuck hepatitis virus DNA polymexase is set out in Table 5.
Table 2: Comparative inhibitory activities of L..nucleoside triphosphates on woochuck hepatitis virus DNA polymerise and human DNA polymerise a and p.
Inhibitor WHB DNA Pol DNA Pol DNA Pol (i ICS (p,M) a Ki (pM) Ki (p.M) ~3-L-AZTPP 0.2 > 100 > 100 ~i-L-ddATP 2.1 > 100 > 100 3-TC-TP 1.0 > 100 > 100 [3-L-SFDDCTP2.0 > 100 > 100 l0 ).)(I. Preparation of Pharmaceutical Compositions The compounds disclosed herein and their pharmaceutically acceptable salts, prodrugs, and derivatives, are useful in the prevenEion 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 o 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 WO 96/40164 PCT/US96I1002b any appropriate route, for example, orally, parerltenxlly, 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 patitnt a therapeutically effective amount without causing serious toxic effects in the pati~t 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 mglkg, of body weight per day, more generally 0.1 to 1o about 100 mg per lalogram body weight of the recipient gcr day. The effective dosage range of the pharnlaceufically acceptable derivatives can be calculated based on the weight of the parent nucleoside to be delivered.
If the derivative exhibits activity in itself, the effxtive dosage can be estimated as above using the weight of the derivative, or by other means i5 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'~leoxythymidine (AZT), 2',3'-dideoxyinosine (DDn, 2',3'-dideoxycytidine (DISC), 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 NM. This may be achieved, for example, by the intravenous injection of a 0.1 to 596 solution of the active ingredient, optionally in saline, or administered as a bolus of the active ingredient.

WO 96/40164 PCT/US9611002b The active compound can be provided in the form of pharmaceutically acceptabie salts. As used herein, the term pharmaceutically acceptable salts or complexes refers to salts or complexes of the nucloosides 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, hydrabmmic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oaaHc acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic 1 o acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisuIfonic 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 1i6 or N' and 5'-O positions, can affect the bioavailability and late of metabolism of the Z 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 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 WO 9b/4U164 PCTNS96/10026 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.
t~ral compositions will generally include an inert diluent or an edible carrier. They may be eneiosed 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, andlor 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, iflrimogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a ~SUIe, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil.
1n 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 syrug may contain, in addition to the WO 96/90164 PC'f/U596/10026 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 s desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, antiinflammatories, or other antivirals, including anti-HBV, anti-cytomegalovirus, or anti-HIV agents.
Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following compon~ts:
to a sterile diIuent 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 bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or 15 phosphates and agents for the adjustment of tenacity 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 preferned embodiment, the active compounds are prepared with canters 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 use, 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.

WQ 96140164 PCT/US96/100~b Liposontal suspensions rurcluding liposomes targeted to infected cells with monoclonal antibodies to viral antigens) are also preferred as pharma~ticxtlY acceptable carriers. Titex may be preparod according to m~Ods latawtt to those sldllod in the art, for exampk, as described in U.S. Patent No. ~t,522,$I I.
For example, liposome formulations may be by dissolving appropriate tipid(s) (such as straooyl phosphatidYl ahanolamine, st~earoyl phoephatidyl choliee, arachadoyl phosphatidyl c~oline, and chaksterotj in an inorganic solvatt that is then evaporat0d, kaving behind 1o a thin liLn of dried lipid on the surface of the container. M aqueous solution of the active compound or its motwphosphate, diphosphate. andlOr trlpltosphaec derivatives arc then introduced into the oantaina. 'fhe container is then swirled by hand to free Lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposo<nal is suspensio,t.
This invention has been described with reference to its preferred embodimatts. Variations and modifications of the invention, will be obvious to thox sfdlled in the art from the foregoing detailed description of the invention. It is intended that ail of these variations and modifications 2 o be included within the soopo of the appendod claims.

Claims (36)

1. Use of an effective amount of a bis(SATE)-.beta.-1-2',3'-dideoxyadenosine monophosphate, or a physiologically acceptable salt thereof, in combination with a second compound selected from: 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (DDI), 2',3'-dideoxy-2',3'-didehydrothymidine (D4T), 2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC), a non-nucleoside RT-inhibitor, and a physiologically acceptable salt thereof, in the treatment of a patient infected with hepatitis B virus and HIV.

2. The use of claim 1, wherein the bis(SATE)-.beta.-1-2',3'-dideoxyadenosine monophosphate is in enantiomerically enriched form.

3. The use of claim 1, wherein the bis(SATE) component of the nucleotide increases the activity of the bis(SATE)-.beta.-1-2',3'-dideoxyadenosine monophosphate, or a pharmaceutically acceptable salt thereof, in vivo.

4. The use of claim 1, wherein the second compound is 3'-azido-3'-deoxythymidine (AZT).

5. The use of claim 1, wherein the second compound is 2',3'-dideoxyinosine (DDI).

6. The use of claim 1, wherein the second compound is 2',3'-dideoxy-2',3'-didehydrothymidine (D4T).

7. The use of claim 1, wherein the second compound is 2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3 -oxathiolane (FTC).

8. The use of claim 1, wherein the second compound is a non-nucleoside RT-inhibitor.

9. The use of claim 1, wherein the compound is a physiologically acceptable salt of bis(SATE)-.beta.-1-2',3'-dideoxyadenosine monophosphate.

10. Use of an effective amount of a bis(SATE)-.beta.-L-2',3'-dideoxyadenosine monophosphate, or a physiologically acceptable salt thereof, in combination with a second compound selected from: 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (DDI), 2',3'-dideoxy-2',3'-didehydrothymidine (D4T), 2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC), a non-nucleoside RT-inhibitor, and a physiologically acceptable salt thereof, in the manufacture of a medicament for the treatment of a patient infected with hepatitis B virus and HIV.

11. The use of claim 10, wherein the bis(SATE)-.beta.-L-2',3'-dideoxyadenosine monophosphate is in enantiomerically enriched form.

12. The use of claim 10, wherein the bis(SATE) component of the nucleotide increases the activity of the bis(SATE)-.beta.-L-2',3'-dideoxyadenosine monophosphate, or a pharmaceutically acceptable salt thereof, in vivo.

13. The use of claim 10, wherein the second compound is 3'-azido-3'-deoxythymidine (AZT).

14. The use of claim 10, wherein the second compound is 2',3'-dideoxyinosine (DDI).

15. The use of claim 10, wherein the second compound is 2',3'-dideoxy-2',3'-didehydrothymidine (D4T).

16. The use of claim 10, wherein the second compound is 2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC).

17. The use of claim 10, wherein the second compound is a non-nucleoside RT-inhibitor.

18. The use of claim 10, wherein the compound is a physiologically acceptable salt of bis(SATE)-.beta.-L-2',3'-dideoxyadenosine monophosphate.

19. A combination for use in the treatment of a patient infected with hepatitis B virus and HIV, the combination comprising a bis(SATE)-p-L-2',3'-dideoxyadenosine monophosphate, or a physiologically acceptable salt thereof, with a second compound selected from: 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (DDI), 2',3'-dideoxy-2',3'-didehydrothymidine (D4T), 2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC), a non-nucleoside RT-inhibitor, and a physiologically acceptable salt thereof.

20. The combination of claim 19, wherein the bis(SATE)-.beta.-1-2',3'-dideoxyadenosine monophosphate is in an enantiomerically enriched form.

21. The combination of claim 19, wherein the bis(SATE) component of the nucleotide increases the activity of the bis(SATE)-.beta.-1-2',3'-dideoxyadenosine monophosphate in vivo.

22. The combination of claim 19, wherein the second compound is 3'-azido-3'-deoxythymidine (AZT).

23. The combination of claim 19, wherein the second compound is 2',3'-dideoxyinosine (DDI).

24. The combination of claim 19, wherein the second compound is 2',3'-dideoxy-2',3'-didehydrothymidine (D4T).

25. The combination of claim 19, wherein the second compound is 2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC).

26. The combination of claim 19, wherein the second compound is a non-nucleoside RT-inhibitor.

27. The combination of claim 19, wherein the compound is a physiologically acceptable salt of bis(SATE)-.beta.-1-2',3'-dideoxyadenosine monophosphate.

28. A pharmaceutical composition for use in the treatment of a patient infected with hepatitis B virus and HIV comprising a bis(SATE)-.beta.-1-2',3'-dideoxyadenosine monophosphate, or a physiologically acceptable salt thereof, with a second compound selected from: 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (DDI), 2',3'-dideoxy-2',3'-didehydrothymidine (D4T), 2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC), a non-nucleoside RT-inhibitor, and a physiologically acceptable salt thereof.

29. The pharmaceutical composition of claim 28, wherein the bis(SATE)-.beta.-L-2',3'-dideoxyadenosine monophosphate is in an enantiomerically enriched form.

30. The pharmaceutical composition of claim 28, wherein the bis(SATE) component of the nucleotide increases the activity of the bis(SATE)-.beta.-L-2',3'-dideoxyadenosine monophosphate in vivo.

31. The pharmaceutical composition of claim 28, wherein the second compound is 3'-azido-3'-deoxythymidine (AZT).

32. The pharmaceutical composition of claim 28, wherein the second compound is 2',3'-dideoxyinosine (DDI).

33. The pharmaceutical composition of claim 28, wherein the second compound is 2',3'-dideoxy-2',3'-didehydrothymidine (D4T).

34. The pharmaceutical composition of claim 28, wherein the second compound is hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC).

35. The pharmaceutical composition of claim 28, wherein the second compound is a non-nucleoside RT-inhibitor.

36. The pharmaceutical composition of claim 28, wherein the compound is a physiologically acceptable salt of bis(SATE)-.beta.-L-2',3'-dideoxyadenosine monophosphate.