Title of Invention	"A COMPOUND OF THE 3`-O-VALINYL ESTER OF 2`-DEOXY-[BETA]-L-CYTIDINE"
Abstract	The present invention relates to compounds, compositions and methods for the treatment of a host infected with a hepatitis B virus. Specifically, compounds and compositions of 3"-esters of 2"-deoxy-$g(b)-L-nucleosides are disclosed, which can be administered either alone or in combination with other anti-hepatitis B agents. Compounds and compositions of 3",5"-esters of 2"-deoxy-ß-L-nucleosides are disclosed, which can be administered either alone or in combination with other anti-hepatitis B agents, are also disclosed.

Title of Invention

"A COMPOUND OF THE 3`-O-VALINYL ESTER OF 2`-DEOXY-[BETA]-L-CYTIDINE"

Abstract

The present invention relates to compounds, compositions and methods for the treatment of a host infected with a hepatitis B virus. Specifically, compounds and compositions of 3"-esters of 2"-deoxy-$g(b)-L-nucleosides are disclosed, which can be administered either alone or in combination with other anti-hepatitis B agents. Compounds and compositions of 3",5"-esters of 2"-deoxy-ß-L-nucleosides are disclosed, which can be administered either alone or in combination with other anti-hepatitis B agents, are also disclosed.

Full Text	3'-PRODRUGS OF 2'-DEOXY-β-L-NUCLEOSIDES Field of the Invention The present invention relates to 3'-prodrugs of 2'-deoxy-β-L-nucleosides for the treatment of hepatitis B virus This application claims priority to U S provisional application no 60/212,100, filed on June 15, 2000 Background of the Invention Hepatitis B virus ("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 chrome 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 h\er damage, that causes abdominal pain, jaundice, and elevated blood levels of certain 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 m the blood for an extended, or indefinite, period, causing a chrome infection Chrome infections can lead to chrome persistent hepatitis Patients infected with chrome persistent HBV are most common m developmg countries Chrome persistent hepatitis can cause fatigue, cirrhosis of the liver and hepatocellular carcinoma, a primary liver cancer In western industrialized countries, high risk groups for HBV infection mclude those m contact with HBV earners or then 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 wi+h AIDS or HIV-associated infections However, HBV is more contagious than HIV Daily treatments with α-interferon, a genetically engineered protein, have shown promise A human serum-derived vaccine has also been developed to immunize patients against HBV Vaccmes have been produced through genetic engineering While the vaccme has been found effective, production of the vaccine is troublesome because the supply of human serum from chrome earners is limited, and the purification procedure is long and expensive Further, each batch of vaccme prepared from different serum must be tested m chimpanzees to ensure safety In addition, the vaccme does not help the patients already infected with the virus An essential step m the mode of action of purine and pynnudme nucleosides against viral diseases, and m particular, HBV and HIV, is their metabolic activation by cellular and viral kinases, to yield the mono-, di- and triphosphate derivatives The biologically active species of many nucleosides is the triphosphate form, which inhibits DNA polymerase or reverse transcriptase, or causes chain termination A number of synthetic nucleosides have been identified which exhibit activity against HBV The (-)-enantiomer of BCH-189 (2\3'-dideoxy-3'-thiacytidine), known as 3TC, claimed m U S Pacent 5,539,116 to Liotta, et al, is currently m clinical trials for tne treatment of hepatitis B See also EPA 0 494 119 Al filed by BioChem Pharma, Inc jS-2-Hydroxymethyl-5-(5-fluorocytosm-l-yI)-l,3-oxathiolane ("FTC"), claimed m U S Patent Nos 5,814,639 and 5,914,331 to Liotta et al, exhibits activity against HBV See Furman et al, "The Anti-Hepattis B Virus Activities, Cytotoxicities, and Anabolic Profiles of the (-) and (+) Enanuomers of cis-5-Fluoro-l-{2-(Hydroxymethyl)-l,3-oxatmolane-5-vl}-Cytosine" Antimicrobial Agents and Chemotherapy December 1992, page 2686-2692, and Cheng, et al, Journal of Biological Chermstrv Volume 267(20), 13938-13942 (1992) U S Patent Nos 5,565,438, 5,567,688 and 5,587,362 (Chu, et al) disclose the use of 2'-fluoro-5-methyl-β-L-arabmofuranolylundme (L-FMAU) for the treatment of hepatitis B and Epstein Barr viru> Penciclovir (?CV, 2-amino-1,9-dihydro-9- {4-hydroxy-3-(hydroxymethyl)butyl} -6H-purm-6-one) has established activity against hepatitis B See US Patent Nos 5,075,445 and 5 684 1:3 Adefovir (9-{2-(phosphonomethoxy)ethyl} adenine, also referred to as PMEA or {{2-(6-amino-9H-punn-9-yl)ethoxy}methylphosphomc acid), also has established activity against hepatitis B See, for example, U S Patent Nos 5,641,763 and 5,142,051 Yale University and The University of Georgia Research Foundation, Inc disclose the use of L-FDDC (5-fluoro-3'-thia-2',3'-dideoxycytidine) for the treatment of hepatitis B virus in WO 92/18517 Other drugs explored for the treatment of HBV mclude adenosine arabinoside, thymosin, acyclovir, phosphonoformate, zidovudine, (+)-cyanidanol, quinaenne, and 2'-fluoroarabinosyl-5 -lodouracil U S Patent Nos 5,444,063 and 5,684,010 to Emory University disclose the use of enantiomencally pure /3-D-l,3-dioxolane purine nucleosides to treat hepatitis B WO 96/40164 filed by Emory University, UAB Research Foundation, and the Centre National de la Recherche Scientifique (CNRS) discloses a number of (S-L-2',3'-dideoxynucleosides for the treatment of hepatitis B WO 95/07287 also filed by Emory University, UAB Research Foundation, and the Centre National de la Recherche Scientifique (CNRS) discloses 2' or 3' deoxy and 2',3'-dideoxy-ß-L-pentofuranosyl nucleosides for the treatment of HIV infection W096/13512 filed by Genencor International, Inc , and Lipitek, Inc, discloses the preparation of L-nbofuranosyl nucleosides as antitumor agents and virucides W095/32984 discloses lipid esters of nucleoside monophosphates as lrnmuno-suppresive drugs DE 4224737 discloses cytosine nucleosides and then- pharmaceutical uses Tsai et al, in Biochem Pharmacol 1994, 48(7), 1477-81, disclose the effect of the anti-HIV agent 2'-j5-D-F-2' 3'-dideoxynucleoside analogs on the cellular content of mitochondrial DNA and lactate production Galvez, J Chem Inf Comput Sci 1994, 55(5), 1198-203, describes molecular computation of/3-D-r azido-2',3'-dideoxy-5-fluorocytidine Mahmoudian, Pharm Research 1991, 5(1), 43-6, discloses quantitative structure-activity relationship analyses of HIV agents such as β-D-3'-azido-2',3'-dideoxy-5-fluorocytidine US Patent No 5,703,058 discloses (5-carboximido or 5-fluoro)-(2',3'-unsaturated or 3'-modified) pynmidrne nucleosides for the treatment of HIV or HBV Lm et al, discloses the synthesis and antiviral activity of various 3'-azido analogues of/3-D-nucleosides m J Med Chem 31(2), 336-340 (1988) WO 00/3998 filed by Novino Pharmaceuticals, Ltd discloses methods of preparing substituted 6-benzyl-4-oxopynmidines, and the use of such pynmidines for the treatment of HIV Novino Pharmaceuticals, Ltd was also first to disclose 21-deoxy-β-T-_ erythropentofuranonucleosides, and then use in the treatment of HBV m WO 00/09531 A method for the treatment of hepatitis B infection m humans and other host animals is disclosed that mcludes administering an effective amount of a biologically active 2'-deoxy-i3-L-erythro-pentofuranonucleoside (alternatively referred to as β-L-dN or a ß-L-2'-dN) or a Dharmaceuticalry acceptable salt or prodrug thereof, including β-L-deoxynbomynndine (β-L-dT), β-L-deoxynbocytidme (β-L-dC), β-L-deoxynboundine (β-L-dU), β-L-deoxynbo-"guanosme"(β-L-:dg β-L-deoxyriboadenosine (β-L-dA) and β-L-deoxynboinosirie (β-Lal); administered either alone or m combination, optionally in a pharmaceuticalry"~acceptabie earner 5' and N4 (cytidme) or N6 (adenosine) acylated or alkylated denvatives of the active compound, or the 5'-phospholipid or 5'-ether lipids were also disclosed Vanous prodrugs of antivirals have been attempted Most notably, U S Patent No 4,957,924 to Beauchamp discloses vanous therapeutic esters of acyclovir In light of the fact that hepatitis B virus has reached epidemic levels worldwide, and has severe and often tragic effects on the infected patent, there remains a strong need to provide new effective pharmaceutical agents to treat humans infected with the virus that have low toxicity to the host Therefore, it is an object of the present invention to provide compounds, compositions and methods for the treatment of human patents or other hosts infected with HBV Summary of the Invention 3'-Prodrugs of 2'-deoxy-ß-L-nucleosides, or their pharmaceutically acceptable salts or pharmaceutically acceptable formulations containing these compounds are useful in the prevention and treatment of hepatitis B infections and other related conditions such as anu-HBV antibody positive and HBV-positive conditions, chrome liver inflammation caused by HBV, cirrhosis, acute hepatitis, fulminant hepatitis, chrome persistent hepatitis, and fatigue These compounds or formulations can also be used prophylactically to prevent or retard the progression of cluneal illness in individuals who are anti-HBV antibody or HBV-antigen positive or who have been exposed to HBV A method for the treatment of a hepatitis B viral infection m a host, including a human, is also disclosed that includes administering an effective amount of a 3'-prodrug of a biologically active 2'-deoxy-β-L-nucleoside or a pharmaceutically acceptable salt thereof, administered either alone or in combination or alternation with another anti-hepatitis B virus agent, optionally in a pharmaceutically acceptable earner The term 2'-deoxy, as used in this specification, refers to a nucleoside that has no substituent m the 2'-position The term 3'-prodrug, as used herein, refers to a 2'-deoxy-ß-L-nucleoside that has a biologically cleavable moiety at the 3'-position, including, but not limited to acyl, and in one embodiment, an L-armno acid In one embodiment, the 2'-deoxy-β-L-nucleoside 3'-prodrug includes biologically cleavable moieties at the 3' and/or 5' positions Preferred moieties are ammo acid esters including valyl, and alkyl esters including acetyl Therefore, this invention specifically mcludes 3'-L-amino acid ester and 3',5'-L-diamino acid ester of 2'~β-L-deoxy nucleosides with any desired purine or pynrmdme base, wherein the parent drug has an EC50 of less than 15 rmcromolar, and preferably less than 10 micromolar in 2 2 15 cells, 3'-(alkyl or aryl ester)- or 3',5'-L-di(alkyl or aryl ester)-2'-β-L-deoxy nucleosides with any desired purine or pynmidine base, wherein the parent drug has an EC50 of less than 10 or 15 rmcromolar m 2 2 15 cells, and prodrugs of 3',5'-diesters of 2'-deoxy-β-L-nucleosides wherein (1) the 3' ester is an ammo acid ester and the 5' -ester is an alkyl or aryl ester, (11) both esters are ammo acid esters, (111) both esters are independently alkyl or aryl esters, and (IV) the 3' ester is independently an alkyl or aryl ester and the 5'-ester is an ammo acid ester, wherein the parent drug has an EC50 of less than 10 or 15 micromolar in 2 2 15 cells Examples of prodrugs falling within the invention are 3'-L-vahne ester of 2'-deoxy-β-L-cytidine, 3'-L-valine ester of 2'-deoxy-β-L-thymine, 3'-L-valine ester of 2'-deoxy-β-L-adenosine, 3'-L-valine ester of 2'-deoxy-β-L-guanosine, 3'-L-vahne ester of 2'-deoxy-β-L-5-fluoro-cytidine, 3'-L-vahne ester of 2'-deoxy-β-L-undine, 3'-acetyl ester of 2'-deoxy-β-L-cytidine, 3'-acetyl ester of 2'-deoxy-β-L-thymine, 3'-acetyl ester of 2'-deoxy-β-L-adenosine, 3'-acetyl ester of 2'-deoxy-β-L-guanosine, 3'-acetyl ester of 2'-deoxy-β-L-5-fluoro-cytidine, and 3'-esters of 2'-deoxy-β-L-(cytidine, 5-fluorocytidine, guanosine, undine, adenosine, or thymine) wherein (1) the 3' ester is an ammo acid, ester, or (n) the 3' ester is an alkyl or aryl ester Additional examples of prodrugs falling within the invention are 3',5'-L-drvaline ester of 2'-deoxy-β-I^cytidine (dival-L-dC), 3',5'-L-divalme ester of 2'-deoxy-β-L-thymine, 3',5'-L-divahne ester of 2'-deoxy-β-L-adenosine, 3',5'-L-divaline ester of 2'-deoxy-β-L-guanosine, 3',5'-L-divahne ester of 2'-deoxy-β-L-5-fluoro-cytidine, 3',5'-L-divalme ester of 2'-deoxy-β-L-undine 3',5'-diacetyl ester of 2'-deoxy-β-L-cytidme, 3',5'-diacetyl ester of 2'-deoxy-β-L-thymine, 3',5'-diacetyl ester of 2'-deoxy-β-L-adenosrne, 3',5'-diacetyl ester of 2'-deoxy-β-L-guanosine, 3',5'-diacetyl ester of 2'-deoxy-β-L-5-fluoro-cytidme, and 3',5'-diesters of 2'-deoxy-β-L-(cytidine, 5-fluorocytidme, guanosme, undme, adenosme, or thymine) wherein (l) the 3' ester is an ammo acid ester and the 5'-ester is an alkyl or aryl ester, (u) both esters are ammo acid esters, (in) both esters are independently alkyl or aryl esters, or (rv) the 3' ester is an alkyl or aryl ester and the 5'-ester is an ammo acid ester (Formula Removed) or its pharmaceutically acceptable salt thereof, wherein In a second embodiment the invention provides the P~L nucleoside 3'-prodrug defined by formula (I) R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative, R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or diphosphate, or a phosphate derivative, X is O, S, SO2 or CH2, and BASE is a purine or pyrrolidine base that may optionally be substituted In a preferred embodiment, X is O In one embodiment, R1 and/or R2 are an ammo acid residue In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10Ru), wherein R8 is the side chain of an ammo acid and wherein, as in proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and Rn are independently hydrogen, acyl (mcludmg an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl) In another embodiment of the present invention, the β-L nucleoside 3'-prodrug is a β-L-2'-deoxypurine of the formula (Formula Removed) or its pharmaceutically acceptable salt thereof, wherein R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-subshtuted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative, R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, Y is OR3, NR3R4 or SR3, and X1 and X2 are mdependently selected from the group consisting of H, straight chamed, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, halogen, OR5, NR5R6 or SR5, and R3, R4, R5 and R6 are mdependently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), dialkylammoalkylene (in particular, dimethylammomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative In one embodiment, the amino acid residue is of the formula C(O)C(R8)(R9)(NR10Ru), wherem R8 is the side chain of an ammo acid and wherem, as in proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocychc moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and R11 are independently hydrogen, acyl (mcludmg an acyl derivative attached to R8) or alkyl (mcludmg but not limited to methyl, ethyl, propyl, and cyclopropyl) In a particular embodiment, the β-L nucleoside 3'-prodrug is a β-L-2'-deoxyadenosine of the formula (Formula Removed) or its pharmaceutically acceptable salt thereof, wherein R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative, R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substtuted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, and R3 and R4 are mdependently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), dialkylarmnoalkylene (in particular, dimethylammomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative In a preferred embodiment, R1 is H In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10Rn)5 wherein R8 is the side chain of an ammo acid and wherem, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and Ru are mdependently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl) In another preferred embodiment, R2 is an amino acid residue, and in particular L-vahnyl In one embodiment, R3 is hydrogen, and R4 is dimethylaminomethylene In another embodiment, R3 is hydrogen, and R is acetyl In another embodiment, R3 is hydrogen, and R4 is L-vahnyl In another particular embodiment, the β-L nucleoside 3'-prodrug is β-L-2'-deoxyguanostne of the formula (Formula Removed) or its pharmaceutically acceptable salt thereof, wherem R1 is hydrogen, straight chamed, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, arallcylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, R2 is selected from the group consisting of straight chamed, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, and R5 and R6 are independently H, straight chamed, branched or cyclic alkyl (εspecially cyclopropyl), dialkylammoalkylene (in particular, dunethylammomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-subsututed aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative In a preferred embodiment, R1 is H In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10R"), wherem (Formula Removed) R is the side chain of an amino acid and wherein, as in proline, R8 can optionally be attached to R to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and R11 are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethvl, propyl, and cyclopropyl) In another preferred embodiment, R2 is an ammo acid residue, and m particular L-vahnyl In one embodiment, R5 is hydrogen, and R6 is dimethylammomethylene In another embodiment, R5 is hydrogen, and R6 is acetyl In another embodiment, R5 is hydrogen, and R6 is L-vahnyl In another particular embodiment the β-L nucleoside 3'-prodrug is β-L-2'-deoxyinosine or pharmaceutically acceptable salt or prodrug thereof of the formula (Formula Removed) or its pharmaceutically acceptable salt thereof, wherem R1 is hydrogen, straight chained, branched or cychc alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, and R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO aryloxyalkyl, CO-substtuted aryl, alkylsulfonyl, arylsulfonvl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative In a preferred embodiment, R1 is H In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10R1,)) wherein R8 is the side chain of an ammo acid and wherem, as in prolme, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and Rn are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl) In another preferred embodiment, R2 is an ammo acid residue, and in particular L-valinyl In another embodiment of the present invention, the β-L nucleoside 3'-prodrug is /?-L-2'-deoxypynmidme of the formula (Formula Removed) or its pharmaceutically acceptable salt thereof, wherem R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-ar\loxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, R2 is selected from the group consistmg of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonvl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, Y is OR3, NR3R4 or SR', X1 is selected from the group consisting of H, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, halogen, OR5, NR*R6 or SR5, and R3,R4,R5andR6 are independently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), dialkylamrnoalkylene (in particular, drmethylarnrnomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10R!1), wherein R8 is the side chain of an ammo acid and wherein, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocychc moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and Ru are mdependently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl) In one particular embodiment, the 0-L nucleoside 3'-prodrug is ß-L-2'-deoxycytidme of the formula (Formula Removed) or its pharmaceutically acceptable salt thereof, wherein R1 is hydrogen, straight chamed, branched or cychc alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-an loxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, R2 is selected from the group consisting of straight chamed, branched or cyclic alkyi CO-alkyl, CO-aryl CO alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative, X1 is selected from the group consisting of H, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, halogen, OR5, NR5R6 or SR5, and R3, R4, R5 and R6 are rndependently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), diaUcylammoalkylene (m particular, dimethylaminomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative In one embodiment, X1 is hydrogen In another embodiment, X1 is a halogen, namely fluorine, chlorine, bromine or iodine In a preferred embodiment, R1 is H In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10R11), wherein R8 is the side chain of an ammo acid and wherein, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and R11 are independently hydrogen, acyl (rncluding an acyl derivative attached to R8) or alkvl (including but not limited to methyl, ethyl, propyl, and cyclopropyl) In another preferred embodiment, R2 is an ammo acid residue, and in particular L-vahnyl In one embodiment, R3 is hydrogen, and R4 is dimethylammomethylene In another embodiment, R3 is hydrogen, and R4 is acetyl In another embodiment, R3 is hydrogen, and R4 is L-valmyl In another embodiment, the β-L-nucleoside 3'-prodrug is β-L-2'-deoxyundine of the formula (Formula Removed) or its pharmaceutically acceptable salt thereof, wherein R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative, and R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono di, or triphosphate, or a phosphate derivative In a preferred embodiment, R1 is H In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10R'l), wherein R8 is the side chain of an ammo acid and wherein, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and R11 are independently hydrogen, acyl (mcludmg an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl and cyclopropyl) In another preferred embodiment, R2 is an amino acid residue, and in particular L-vaknyl In another embodiment, the β-L-nucleoside 3'-prodrug is ß-L-thymidrne of the formula (Formula Removed) or its pharmaceutically acceptable salt thereof, wherein R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyL ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, and R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative In a preferred embodiment, R1 is H In one embodiment, the ammo acid residue is of the formula CCO^CR^CR^CNR10^1), wherein R8 is the side chain of an ammo acid and wherein, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and Rn are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl) In another preferred embodiment, R2 is an ammo acid residue, and in particular L-vahnyl the invention also provides combinations of at least two of the herein described prodrugs The invention further provides at least one of the described 3'-prodrugs in combination or alternation with a second nucleoside that exhibits activity against hepatitis B, including but not limited to a parent drug of any of the prodrugs defined herein, 1 e 2'deoxy-β-L-nucleosides, including 2'-deoxy- β-L-cytidine, 2'-deoxy- β-Lthymine,2'-deoxy- β-L-adenosine,2'-deoxy- β-L-guanine,2'-deoxy- β—L-5-fluorocytidine Alternatively, the 3'-produrgs can be administered in combination or alternation with other anti-hepatitis B virus agent such as (-)-cis-2',3'-dideoxy-3'-thiacytidine, cis-2'3'-dideoxy-3'-thia-5-fluorocytidine, L-FMAU, adefovir, famciclovir, and entecivir, or any other compound that exhibits an EC50 of less than 10 or 15 micromolar in 2 2 15 cells, or their prodrugs or pharmaceutically acceptable salts The invention further includes administering the prodrug in combination or alternation with an immune modulator or other pharmaceutically active modifier of viral replication, including a biological material such as protein, peptide, oligonucleotide, or gamma globulin, including but not limited to interferon,interleukin or an antisense oligonucleotides to genes which express or regulate hepatitis B replication The efficacy of the parents of the anti-HBV compound can be measured according to the concentration of compound necessary to reduce the replication rate of the virus in vitro, according to methods set forth more particularly herein, by 50% (1 e the compound's EC 50 ) In preferred embodiments the parent of the prodrug compound exhibits an EC50 of less than 15 or preferably, less than 10 micromolar in vitro, when tested in 2 2 15 cells transferred with the hepatitis virion In accordance with the present invention it relates to a A compound of the 3'-0-valmyl ester of 2'-deoxy-[beta]-L-cytidine (Formula Removed) or a pharmaceutically acceptable salt thereof BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Figure 1 a and lb are non-limiting illustrating examples according to the present invention of the synthesis of 3'- and 5'-vaniyl esters of 2'-deoxy-β-L-cytidme (β-L-dC) from 2'-deoxy-β-L-cytidine, respectively Figure 2 is a non-limiting illustrative example according to the present invention of the synthesis of N4-acetyl-2'-deoxy-β-L-cytidine from 2'-deoxy-β-L-cytidine Figure 3 is a non-limiting illustrative example according to the present invention of the synthesis of N-[(dmiethylamino)methylene]-2'-deoxy-β-L-cytidrne from 2'-deoxy-β-L-cytidine Figure 4 is a non-limiting illustrative example according to the present invention of the synthesis of 3',5'-di-0-acetyl-2'-deoxy-β-L-cytidine from 2'-deoxy-β-L-cytidine Figure 5 is a non-hmiting illustrative example accordmg to the present invention of the synthesis of 3',5'-di-0-vahnyl ester of 2'-deoxy-β-L-cytiduie from 2'-deoxy-β-L- cytidine Figure 6 is a non-hmiting illustrative example according to the present invention of the synthesis of N-(Boc-valmyl) ester of 2'-deoxy-β-L-cyhdine from 2'-deoxy-β-L-cytidme Figure 7 is a non-limiting illustrative example according to the present invention of the synthesis of 3',5' N-tn-(L-valmyl)-2'-deoxy-β-L-cytidme from 3'55' N-tn-(Boc-L-valmyl)-2' -deoxy-β-L-cyudine Figure 8 is a hne graph depicting a standard calibration technique useful for the determination of solubility of various nucleosides Figure 8a is the calibration curve determined for nature β-D-deoxynbocytosme Figure 8b is the calibration curve determined for the 3',5'-divalinyl ester of β-L-deoxynbocytosine Figure 9a is a non-hmiting example of a HPLC profile used to assess the stability of the 3\5'-divahnyl ester of β-L-deoxynbocytosme at a pH of 7 42 The HPLC profile mdicates the presence of the 3',5'-divalmyl ester of β-L-deoxynbocytosine along with 3 active metabolites, the 3'-vahnyl ester of β-L-deoxynbocytosme, the 5'-vahnyl ester of β-L-deoxynbocytosine and L-dC Figure 9b is a hne graph depicting the relative concentrations of the 3\5'-divahnyl ester of β-L-deoxynbocytosme and its metabolites over time Similarly, Figure 10a and 11a are non-lrmitmg examples of HPLC profiles used to assess the stability of the 3',5'-divahnyl ester of β-L-deoxynbocytosme at a pH of 7 20 and 4 51, respectively At these pH's, the HPLC profile mdicates the presence of the 3',5'-divahnyl ester of β-L-deoxynbocytosme along with 3 active metabolites, the 3'-vahnyl ester of B-L-deoxynbocytosine, the 5'-valinyl ester of β-L-deoxynbocytosme and L-dC Figure 10b and lib are line graphs depicting the relative concentrations of the 3\5'-divahnyl ester of β-L-deoxynbocytosine and its metabolites over time Figure 12 is a non-limiting example of a HPLC profile used to assess the stability of the 3',5'-divahnyl ester of β-L-deoxynbocytosine at a pH of 1 23 At this pH, the HPLC profile only indicates the presence of the 3',5'-divahnyl ester of β-L-deoxynbocytosine without any decomposition into any of its 3 active metabolites Figure 13 is a line graph depicting the in vitro metabolism of 3',5'-divahnyl ester of β-L-deoxynbocytosine m human plasma Figure 14 is a line graph depictmg the intracellular metabolism of β-L-deoxynbocytosme (L-dC) in HepG2 cells Figure 15 is a line graph depictmg the intracellular accumulation of L-dC m primary human hepatocytes Figure 16 is a bar graph depicting the antiviral dose response of L-dC upon treatment of a chronic hepatitis B virus infection for 28 days m the woodchuck model of chronic Hepatitis B virus infection Figure 17 is a line graph depictmg the antiviral activity of L-dC in the woodchuck model of chrome hepatitis B virus infection Figure 18 are lme graphs indicating the body weights of individual woodchucks treated for 28 days with L-dC (0 01-10 mg/kg/day) orally Figure 19 are lme graphs indicating the body weights of individual woodchucks treated for 12 Weeks with L-dC (1 mg/kg/day) orally Detailed Description of the Invention The invention as disclosed herein is a compound, a method and composition for the treatment of hepatitis B virus in humans and other host animals The method mcludes the administration of an effective HBV treatment amount of a 3'-prodrug of a ß-L-nucleoside as desenbed herein or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable earner The compound of this invention either possesses antiviral (1 e, anh-HBV) activity, or is metabolized to a compound that exhibits such activity In summary, the present invention includes the following features (a) β-L-2'-deoxy-nucleoside 3'-prodrugs, as described herein, and pharmaceutically acceptable salts, esters and compositions thereof, (b) ß-L-2'-deoxy-nucleoside 3'-prodrugs as described herein, and pharmaceutically acceptable salts, esters and compositions thereof for use m the treatment or prophylaxis of a hepatitis B infection, especially in individuals diagnosed as having a hepatitis B infection or bemg at nsk of becoming infected by hepatitis B, (c) use of these β-L-2'-deoxy-nucleoside 3'-prodrugs, and pharmaceutically acceptable salts, esters and compositions thereof in the manufacture of a medicament for treatment of a hepatitis B infection, (d) pharmaceutical formulations comprising the j3-L-2'-deoxy-nucleoside 3'-prodrugs or pharmaceutically acceptable salts thereof together with a pharmaceutically acceptable earner or diluent, (e) β-L-2'-deoxy-nucleoside 3'-prodrugs, or their pharmaceutically acceptable salts, esters and compositions as described herein substantially in the absence of the opposite enanuomers of the described nucleoside, or substantially isolated from other chemical entities, (f) processes for the preparation of ß-L-2'-deoxy-nucleoside 3'-prodrugs, as described in more detail below, (g) processes for the preparation of ß-L-2'-deoxy-nucleoside 3'-prodrugs substantially in the absence of enantiomers of the described nucleoside, or substantially isolated from other chemical entities, (h) the treatment of a host infected with hepatitis B that includes the administration of an effective amount of a 3'-prodrug of a ß-L-2'-deoxy-nucleoside, its pharmaceutically acceptable salt, ester or composition with a second anti-hepatius B agent, (l) the treatment of a host infected with hepatitis B that includes the administration of an effective amount of a 3'-prodrug of a β-L-2'-deoxy-nucleoside, its pharmaceutically acceptable salt, ester or composition with the parent of a different β-L--2'-deoxynucleoside, (j) the treatment of a host infected with hepatitis B that includes the administration of an effective amount of a 3'-prodrug of a β-L--2'-deoxy-cytidine, its pharmaceutically acceptable salt or ester with the parent of a second anti-hepatitis B agent, (k) the treatment of a host infected with hepatitis B that mcludes the administration of an effective amount of the 3',5'-divalyl or diacetyl ester of ß-L-2'-deoxy-cytidine, or its pharmaceutically acceptable salt or ester thereof, with a second anti-hepatitis B agent, and (1) the treatment of a host infected with hepatitis B that mcludes the administration of an effective amount of the 3',5'-divalyl or diacetyl ester of ß-L-2'-deoxy-cytidine, or its pharmaceutically acceptable salt or ester thereof, with β-L-2'-deoxy-thymidine, or its pharmaceutically acceptable salt A particularly preferred combination is the 3',5'-prodrug ofß-L-dC (also referred to as L-dC) with the parent ß-L-dT (also referred to as L-dT), and in particular, the 3',5'-divalyl or 3',5'-diacetyl ester of ß-L-dC in combination with ß-L-dT The oral bioavailability of L-dC as the neutral base and the HC1 salt is low in rodents and non-human primates It has been discovered that there is significant competition of L-dC with other nucleosides or nucleoside analogs for absorption, or transport, from the gastrointestinal tract and competition of other nucleosides or nucleoside analogs for the absorption with L-dC In order to improve oral bioavailability and reduce the potential for drug-drug interaction, a pharmacokinetic screen in monkeys was established This screen identified 3'-prodrugs of L-dC that had higher oral bioavailability than the parent molecule and a reduced effect on the bioavailability of other nucleosides or nucleoside analogs used in combination Examples of such nucleosides or nucleoside analogs used m combination with the prodrugs of L-dC are L-dT, L-dA, lamivudme or FTC It was discovered using this approach, that the 3',5'-divaline ester of L-dC had higher oral bioavailability than the parent L-dC and a reduced interaction with other nucleosides or nucleoside analogs when used in combination as compared to L-dC Pharmacokinetic studies also showed that the 3',5'-divahne ester of L-dC was converted to the parent L-dC through de-estenfication in the gastrointestinal mucosa, blood or liver The 3',5'-divahne ester of L-dC is apparently actively transported from the gastrointestinal lumen after oral delivery into the bloodstream by an ammo acid transporter function in the mucosa of the gastrointestinal tract This accounts for the increase in oral bioavailability compared to the parent L-dC that would be transported primarily by a nucleoside transporter function It would also explain the reduced competition for uptake of the 3',5'-divahne ester of L-dC with other nucleosides or nucleoside analogs that are transported by the nucleoside transporter function and not the ammo acid transporter function As partial de-estenfication of the divahne ester of L-dC occurs prior to complete absorption, the monovahne ester contmues to be absorbed using the ammo acid transporter function Therefore, the desired outcome of better absorption, or bioavailability, and reduced competition with other nucleosides or nucleoside analogs for uptake into the bloodstream is maintained I Compounds Defined by this Invention In a first embodiment, the 2'-deoxy-ß-L-nucleoside 3'-prodrug includes biologically cleavable moieties at both the 3' and 5' positions Preferred moieties are L-amino acid esters such as L-valyl, and alkyl esters such as acetyl This invention specifically mcludes 3',5'-L-amino acid-β-L-2'-deoxy nucleosides with any desired purine or pynmidme base, wherein the parent drug has an EC50 of less than 15 micromolar, and preferably less than 10 micromolar, in 2 2 15 cells, 3',5'-(alkyl or aryl)-β-L-2'-deoxy nucleosides with any desired purine or pynmidme base, wherein the parent drug has an EC50 of less than 15, and preferably less than 10 micromolar in 2 2 15 cells, and prodrugs of 3',5'-diesters of 2'-deoxy-β-L-nucleosides wherem (1) the 3' ester is an ammo acid ester and the 5'-ester is an alkyl or aryl ester, (11) both esters are ammo acid esters, (m) both esters are independently alkyl or aryl esters, and (IV) the 3' ester is independently an alkyl or aryl ester and the 5'-ester is an ammo acid ester, wherem the parent drug has an EC50 on dosmg of less than 15 micromolar in 2 2 15 cells, Examples of 3'-prodrugs falling within the invention are 3,5'-L-vahne ester of 2'-deoxy-ß-L-cytidine, 3',5'-L-vahne ester of 2'-deoxy-β-L-thymine, 3',5'-L-valme ester of 2'-deoxy-β-L-adenosine, 3',5'-L-vakne ester of 2'-deoxy-β-L-guanosine, 3',5'-L-vahne ester of 2'-deoxy-β-L-5-fluoro-cytidine, 3',5'-L-vaIine ester of 2'-deoxy-β-L-undine, 3',5'-acetyl ester of 2'-deoxy-β-L-cytidine, 3',5'-acetyl ester of 2'-deoxy-β-L-thymine, 3',5'-acetyl ester of 2'-deoxy-β-L-adenosine, 3',5'-acetyl ester of 2'-deoxy-β-L-guanosrne, 3',5'-acetyl ester of 2'-deoxy-β-L-5-fluoro-cytidine, and 3',5'-diesters of 2'-deoxy-β-L-(cytidine, 5-fluorocytidine, guanosme, undine, adenosine, or thymine) wherein (1) the 3' ester is an amino acid ester and the 5'-ester is an alkyl or aryl ester, (n) both esters are ammo acid esters, (m) both esters are mdependently alkyl or aryl esters or (IV) the 3' ester is an alkyl or aryl ester and the 5'-ester is an amino acid ester In one embodiment, the invention provides the β-L nucleoside 3'-prodrug defined by formula (I) (Formula Removed) or its pharmaceutically acceptable salt thereof, wherein R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, R2 is selected from the group consistmg of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, X is O, S, SO2 or CH2) and BASE is a purine or pvnmidine base that may optionally be substituted In a preferred embodiment, X is O In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10Rn), wherein R is the side chain of an amino acid and wherein, as in proline, R can optionally be attached to R1 to form a ring structure, or alternatively, Rs is an alkyl, aryl, heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and Ru are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl) In a first subembodiment R2 is C(O)-alkyl (including lower alkyl) or aryl, and BASE is adenine, protected adenine, cytosme, protected cytosine or thymine In a second subembodiment R2 is C(O)-lower alkyl and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine In a third subembodiment R2 is C(O)-methyl and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine In a fourth subembodiment R2 is C(O)C(R8)(H)(NR10Rn), and BASE is adenine protected adenine, cytosme, protected cytosine or thymine In a fifth subembodiment R2 is C(O)C(R8)(H)(NR10Rn), R8 is isopropyl, at least one of R10 and Ru is hydrogen, and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine In a sixth subembodiment R2 is C(O)C(R8)(H)(NR10Ru), R8 is an amino acid side chain, and BASE is adenine, protected adenine, cytosme, protected cytosme, or thymine In a seventh subembodiment R2 is C(O)C(R8)(H)(NR,0Rn), R8 is a nonpolar amino acid side chain and BASE is adenine, protected adenine, cytosme, protected cytosme or thymine Nonhmiting examples of subembodiments can be defined by formula (T) m which (1) R2 is C(O)-methyl and BASE is adenine (2) R2 is C(O)-methyl and BASE is protected adenine (3) R2 is C(0>methyl and BASE is cytosme (4) R2 is C(O)-methyl and BASE is protected cytosme (5) R2 is C(O)-methyl and BASE is thymine (6) R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is adenine (7) R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected adenine (8) R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is cytosme (9) R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected cytosine (10) R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is thymine Li a eighth subembodiment X is O, R2 is C(O)-alkyl (including lower alkyl) or aryl, and BASE is adenine, protected adenine, cytosine, protected cytosine, or thymine In a ninth subembodiment X is O, R2 is C(O)-lower alkyl and BASE is adenine, protected adenine, cytosme, protected cytosme or thymine In a tenth subembodiment X is O, R2 is C(O)-methyl and BASE is adenine, protected adenine, cytosine, protected cytosme or thymine In an eleventh subembodiment X is 0, R2 is C(O)C(R8)(H)(NR10Rn), and BASE is adenine, protected adenine, cytosme, protected cytosme or thymine In a twelfth subembodiment X is O, R2 is C(O)C(R8)(H)(NR10Ru), R8 is isopropyl, at least one of R10 and Ru is hydrogen, and BASE is adenine, protected adenine, cytosme, protected cytosme or thymine In a thirteenth subembodiment X is 0, R2 is C(O)C(R8)(H)(NR10Ru), R8 is an ammo acid side chain, and BASE is adenine, protected adenine, cytosme, protected cytosme, or thymine In a fourteenth subembodiment X is O, R2 is C(O)C(R8)(H)(NR10Rn), R8 is a nonpolar ammo acid side chain, at least one of R5 and R6 is hydrogen and B is adenine, protected adenine, cytosme, protected cytosme or thymine Nonkmiting examples of subembodiments can be defined by formula (I) m which (1) X is O, R2 is C(O)-methyl and BASE is adenine (2) X is O, R2 is C(O)-methyl and BASE is protected adenine (3) X is O, R2 is C(O)-methyl and BASE is cytosme (4) X is O, R2 is C(O)-methyl and BASE is protected cytosme (5) X is 0, R2 is C(O)-methyl and BASE is thymine (6) X is 0, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is adenine (7) X is O, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected adenine (8) X is 0, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is cytosine (9) X is O, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected cytosine (10) X is O, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is thymine In a fifteenth subembodiment X is 0, R1 is hydrogen, R2 is C(O)-alkyl (mcludmg lower alkyl) or aryl, and BASE is adenine, protected adenine, cytosine, protected cytosine, or thymine In a sixteenth subembodiment X is O, R1 is hydrogen, R2 is C(O)-lower alkyl and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine In a seventeenth subembodiment X is O, R1 is hydrogen, R2 is C(O)-methyl and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine In a eighteenth subembodiment X is O, R1 is hydrogen, R2 is C(O)C(R8)(H)(NR10Ru), and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine In a nineteenth subembodiment X is O, R1 is hydrogen, R2 is C(O)C(R8)(H)(NR10Ru), R8 is isopropyl, at least one of R10 and Ru is hydrogen, and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine In a twentieth subembodiment X is O, R1 is hydrogen, R2 is (XCOCCR)CHXNR^R11), R8 is an ammo acid side chain, and BASE is adenine, protected adenine, cytosine, protected cytosine, or thymine In a twentx first subembodiment X is O, R1 is hydrogen, R2 is C(O)C(R8)(H)(NR10R ), R8 is a nonpolar ammo acid side chain, at least one of R5 and R6 is hydrogen and B is adenine, protected adenine, cytosine, protected cytosine or thymine Nonhmiting examples of subembodiments can be defined by formula (I) in which (1) X is O, R1 is Inarogen, R2 is C(O)-methyl and BASE is adenine (2) X is O, R1 is Indrogen, R2 is C(O)-methyl and BASE is protected adenine (3) X is 0, R1 is hydrogen, R2 is C(O)-methyl and BASE is cytosine (4) X is 0, R1 is hydrogen, R2 is C(O)-methyl and BASE is protected cytosine (5) X is 0, R1 is hydrogen, R2 is C(O)-methyl and BASE is thymine (6) X is O, R1 is hydrogen, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is adenine (7) X is O, R1 is hydrogen, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected adenine (8) X is 0, R1 is hydrogen, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is cytosine (9) X is O, R1 is hydrogen, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected cytosine (10) X is O, Rl is hydrogen, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is thymine In a twenty-second subembodiment X is 0, R1 and R2 are independently C(O)-alkyl (mcludmg lower alkyl) or aryl, and BASE is ademne, protected adenine, cytosine, protected cytosine, or thymine In a twenty-third subembodiment X is O, R1 and R2 are independently C(O)-lower alkyl and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine In a twenty-fourth subembodiment X is O, R1 and R2 are independently C(O)-methyl and BASE is ademne, protected ademne, cytosine, protected cytosine or thymine In a twenty-fifth subembodiment X is O, R1 and R2 are independently C(O)C(R8)(H)(NR10Ru), and BASE is ademne, protected ademne, cytosine, protected cytosine or thymine In a twent\ -sixth subembodiment X is O, R1 and R2 are independently C(O)C(R8)(H)(NR,0Rn), R8 is isopropyl, at least one of R10 and R11 is hydrogen, and BASE is ademne, protected ademne, cytosine, protected cytosine or thymine In a twentv-seventh subembodiment X is O, R1 and R2 are independently C(O)C(R8)(H)(NRI0RH), R8 is an amino acid side chain, and BASE is ademne, protected ademne, cytosine, protected cytosine, or thymine In a twenty-eighth subembodrment X is O, R1 and R2 are independently C(O)C(R8)(H)(NR,0Ru), R8 is a nonpolar ammo acid side chain, at least one of R5 and R6 is hydrogen and B is adenine, protected adenine, cytosine, protected cytosme or thymine Nonhmiting examples of subembodiments can be defined by formula (I) in which (1) X is O, R1 and R2 are independently C(O)-methyl and BASE is adenine (2) X is O, R1 and R2 are independently C(O)-methyl and BASE is protected adenine (3) X is O, R1 and R2 are independently C(O)-methyl and BASE is cytosme (4) X is O, R1 and R2 are independently C(O)-methyl and BASE is protected cytosme (5) X is O, R1 and R2 are independently C(O)-methyl and BASE is thymine (6) X is O, R1 and R2 are independently C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is adenine (7) X is O, R1 and R2 are mdependently C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected adenine (8) X is O, R1 and R2 are mdependently C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is cytosine (9) X is O, R1 and R2 are mdependently C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected cytosine (10) X is O, R1 and R2 are mdependently C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is thymine In another embodiment of the present invention, the 0-L nucleoside 3'-prodrug is a ß-L-2'-deoxypunne of the formula (Formula Removed) or its pharmaceutically acceptable salt thereof, wherein R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyL CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or tnphosphate, or a phosphate derivative, R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or tnphosphate, or a phosphate derivative, Y is OR3, NR3R4 or SR3, and X1 and X2 are independently selected from the group consisting of H, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, halogen, OR5, NR^6 or SR5, and R3, R4, R5 and R6 are mdependently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), dialkylammoalkylene (in particular, dimethylammomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or tnphosphate, or a phosphate denvauve In one embodiment, the amino acid residue is of the formula C(O)C(R8)(R9)(NRI0R11), wherein R8 is the side chain of an amino acid and wherein, as in prolme, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and R11 are mdependently hydrogen, acyl (including an acyl denvative attached to R8) or alkyl (mcludmg but not limited to methyl, ethyl, propyl, and cyclopropyl) In a particular embodiment, the β-L nucleoside 3'-prodrug is a ß-L-2'-deoxyadenosme of the formula (Formula Removed) or its pharmaceutically acceptable salt thereof, wherein R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative, R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative, and R3 and R4 are independently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), dialkylaminoalkylene (m particular, dimethylammomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative In a preferred embodiment, R1 is H In one embodiment, the amino acid residue is of the formula C^CCR^O^CNR10^1), wherein R8 is the side chain of an ammo acid and wherem, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and R11 are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl) In another preferred embodiment, R2 is an ammo acid residue, and in particular L-vahnyl In one embodiment, R3 is hydrogen, and R is dimethylammomethylene In another embodiment, R3 is hydrogen, and R4 is acetyl In another embodiment, R3 is hydrogen, and R4 is L-vahnyl In another particular embodiment, the β-L nucleoside 3'-prodrug is ß-L-2'-deoxyguanosme of the formula (Formula Removed) or its pharmaceutically acceptable salt thereof, wherein R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, and R5 and R6 are independently H, straight chamed, branched or cyclic alkyl (εspecially cyclopropyl), dialkylaminoalkylene (in particular, dnnethylammomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative In a preferred embodiment, R1 is H In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10Rn), wherein R8 is the side chain of an ammo acid and wherein, as in proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryL heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and R11 are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl) In another preferred embodiment, R2 is an ammo acid residue, and m particular L-vahnyl In one embodiment, R5 is hydrogen, and R is dimemylaminomethylene In another embodiment, R5 is hydrogen, and R6 is acetyl In another embodiment, R5 is hydrogen, and R6 is L-vahnyl In another particular embodiment, the β-L nucleoside 3'-prodrug is /8-I^2'-deoxyinosine or pharmaceutically acceptable salt or prodrug thereof of the formula (Formula Removed) or its pharmaceuticalh acceptable salt thereof, wherein R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-anloxyalkyl, CO-subsututed aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl amino acid residue, mono, di, or triphosphate, or a phosphate derivative, and R2 is selected from the group consisting of straight chained, branched or cychc alkyl, CO-alkyl, CO-anl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative In a preferred embodiment, R1 is H In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10Rn), wherein R8 is the side chain of an ammo acid and wherein, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and Rn are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl) In another preferred embodiment, R2 is an ammo acid residue, and in particular L-valmyl In another embodiment of the present invention, the \|3-L nucleoside 3'-prodrug is /3-L-2'-deoxypynrnidine of the formula (Formula Removed) or its pharmaceuticalh acceptable salt thereof, wherein R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-ars 'oxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, R2 is selected from the group consisting of straight chained, branched or cychc alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, Y is OR3, NR3R4 or SR , X is selected from the group consisting of H, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, halogen, OR5, NR5R6 or SR5, and R3, R4, R5 and R6 are independently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), dialkylarmnoalkylene (in particular, dimethylamrnomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10Rn), wherein R is the side chain of an amino acid and wherein, as in proline, R can optionally be attached to R10 to form a rmg structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocychc moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and R11 are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl) In one particular embodiment, the β-L nucleoside 3'-prodrug is jS-L-2'-deoxycytidine of the formula (Formula Removed) or its pharmaceuticallv acceptable salt thereof, wherein R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-arvloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkyisulfonyl, amino acid residue, mono, di, or tnphosphate, or a phosphate derivative, and R3 and R4 are independently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), dialkylaminoalkylene (ni particular, dimethylaminomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkyisulfonyl, amino acid residue, mono, di, or tnphosphate, or a phosphate derivative In a preferred embodiment, R1 is H In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10R11), wherein R8 is the side chain of an ammo acid and wherein, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and Ru are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl) In another preferred embodiment, R2 is an ammo acid residue, and in particular L-vahnyl In one embodiment, R3 is hydrogen, and R4 is dimethylammomethylene In another embodiment, R3 is hydrogen, and R4 is acetyl In another embodiment, R3 is hydrogen, and R4 is L-vahnyl In another embodiment, the β-L-nucleoside 3'-prodrug is j3-L-2'-deoxyundme of the formula (Formula Removed) or its pharmaceutically acceptable salt thereof, wherein R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative, and R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative In a preferred embodiment, R1 is H In one embodiment, the amino acid residue is of the formula C(O)C(R8)(R9)(NR10Rn), wherem R8 is the side chain of an amino acid and wherem, as in proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and R11 are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl) In another preferred embodiment, R2 is an amino acid residue, and in particular L-vahnyl In another embodiment, the β-L-nucleoside 3'-prodrug is β-L-thymidine of the formula (Formula Removed) or its phannaceutically acceptable salt thereof, wherein R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyI, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, and R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative In a preferred embodiment, R1 is H In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10Ru), wherein R is the side chain of an ammo acid and wherem, as in proline, R can optionally be attached to R10 to form a nng structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety, R9 is hydrogen, alkyl (including lower alkyl) or aryl, and R10 and Rn are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl) In another preferred embodiment, R2 is an ammo acid residue, and in particular L-vahnyl II Definitions and Use of Terms The term alkvl, as used herein, unless otherwise specified, refers to a saturated straight, branched, or cyclic, primary, secondary, or ternary hydrocarbon of C1 to C10 and specifically includes methyl, tnfluoromethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, /-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dunethylbutyl The term includes both substituted and unsubstituted alkyl groups Moieties with which the alkyl group can be substituted are selected from the group consisting of hydroxyl, amino, alkylamino, arylarnino, alkoxy, aryloxy, nitro, cyano, sulfomc acid, sulfate, phosphomc acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught m Greene, et al, Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, hereby incorporated by reference The term lower alkyl, as used herein, and unless otherwise specified, refers to a Q to C4 saturated straight, branched, or if appropriate, a cyclic (for example, cyclopropyl) alkyl group, including both substituted and unsubstituted forms Unless otherwise specifically stated m this application, when alkyl is a suitable moiety, lower alkyl is preferred Similarly, when alkyl or lower alkyl is a suitable moiety, unsubstituted alkyl or lower alkyl is preferred The term "protected" as used herein and unless otherwise defined refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent its further reaction or for other purposes A wide variety of oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis Non-limiting examples of protecting groups are taught in Greene, et al, Protective Groups m Organic Synthesis, John Wiley and Sons, Second Edition, 1991 The term aryl, as used herein, and unless otherwise specified, refers to phenyl, biphenyl, or naphth>l and preferably phenyl The term mcludes both substituted and unsubstituted moieties The aryl group can be substituted with one or more moieties selected from the group consisting of hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano sulfomc acid, sulfate, phosphomc 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 purine or pynmidme base mcludes, but is not limited to, adenine, N6-alkylpunnes, N^-acylpunnes (wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl), N6- benzylpunne, N6halopurine, N6-vinylpunne, N6-acetylenic punne, punne, N^hydroxyalkyl punne, N6-thioalkyl punne, N2-alkylpunnes, N2-alkyl-6-thiopunnes, thymine, cytosine, 5-fluorocytosine, 5-methylcytosnie, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4-mercaptopyrmidme, uracil, 5-halouracil, including 5-fluorouracil, C^-alkylpynmidines, C5-benzylpynmidines, C5-halopynmidines, C5-vinylpynmidine, C5-acetylemc pynrmdine, C5-acyl pynmidine, C5-hydroxyalkyl punne, C5-amidopynmidnie, C5-cyanopynmidine, C5-nitropynmidine, C5-aminopynmidine, N2-alkylpunnes, N2-alkyl-6-thiopunnes, 5-azacytidinyl, 5-azauracilyl, tnazolopyndinyl, lmidazolopyndinyl, pyrrolopyrimidinyl, and pyrazolopynmidinyl Punne bases include, but are not limited to, guanine, adenine, hypoxanthine, 2,6-diaminopunne, and 6-chloropunne Functional oxygen and mtrogen groups on the base can be protected as necessary or desired Suitable protecting groups are well known to those skilled m the art, and mclude rnmethylsilyl, dimethylhexylsilyl, r-butyldimethylsilyl, and /-butyldiphenylsilyl, tntyl, alkyl groups, and acyl groups such as acetyl and propionyl, methanesulfonyl, and β-toluenesulfonyl The term acyl refers to a carboxyhc acid ester m which the non-carbonyl moiety of the ester group is selected from straight, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkyl including benzyl, aryloxyalkyl such as phenoxymethyl aryl includmg phenyl optionally substituted with halogen, C1 to C4 alkyl or C1 to C4 alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl including methanesulfonyl, the mono, di or Diphosphate ester, tntyl or monomethoxytntyl, substituted benzyl, tnalkylsilyl (ε g dimethyl-t-butylsilyl) or diphenylmethylsilyl Aryl groups m the esters optimally compnse a phenyl group The term "lower acyl" refers to an acyl group in which the non-carbon\ 1 moiety is lower alkyl The term amino acid includes naturally occurring and synthetic α, ß  or δ ammo acids, and mcludes but is not limited to, ammo acids found in proteins, 1 e glycine, alanine, valine, leucme, isoleucme, methionine, phenylalanine, tryptophan, prolme, serine threonine, cysteine, tyosme, asparagine, glutamme, aspartate, glutamate, lysine, argimne and hishdme In a orefened embodiment, the ammo acid is in the L-configuration Alternatively, the amiro acid can be a denvauve of alanyl, vahnyl, leucmyl, isoleuccinyl, prohnyl, phemlalanun1, tryptophanyl, methiomnyl, glycmyl, sermyl, threomnyl, cystemyl, tyrosmyl, asparaginv glutammyl, aspartoyl, glutaroyl, lysmyl, argimnyl, hisudinyl, β-alanyl, β-valm\l, β-kucinyl, β-isoleuccinyl, β-prohnyl, β-phenylalamnyl, β-tryptophanyl, β-methioninyl, β-glycmyl, β-sennyl, β-threoninyl, β-cysteinyl, β-tyrosmyl, β-asparaginyl, β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl or β-histidinyl The term heteroaryl or heteroaromatic, as used herein, refers to an aromatic moiety that includes at least one sulfur, oxygen, nitrogen or phosphorus in the aromatic ring The term heterocyclic refers to a nonaromatic cyclic group wherein there is at least one heteroatom, such as oxygen, sulfur, nitrogen or phosphorus m the ring Nonhmiting examples of heteroanl and heterocyclic groups include furyl, furanyl, pyndyl, pynmidyl, thienyl, isothiazolyl, lmidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoqumolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl, benzumdazolyl, purmyl, carbazolyl, oxazolyl, thiazolyl, isothiazolyl, 1,2,4-thiadiazolyl, isooxazolyl, pyrrolyl qumazohnyl, cinnohnyl, phthalazmyl, xanthmyl, hypoxanthinyl, thiophene, furan, p>Trole, isopyrrole, pyrazole, imidazole, 1,2,3-tnazole, 1,2,4-tnazole, oxazole, isoxazole, thiazole, isothiazole, pynmidme or pyndazine, and ptendmyl, azindrnes, thiazole, isothiazole, 1,2,3-oxadiazole, thiazme, pyndine, pyrazme, piperazine, pyrrohdme, oxaziranes, phenazme, phenothiazine, morphohnyl, pyrazolyl, pyndazmyl, pyrazinyl, qumoxalmvl, xanthmyl, hypoxanthinyl, ptendmyl, 5-azacytidinyl, 5-azauracilyl, tnazolopyndinyl, lmidazolopyndmyl, pyrrolopynmidmyl, pyrazolopynmidmyl, adenine, N6-alkylpunnes, N6-benzyIpunne, N6-halopunne, N6-vmypunne, N6-acetylemc purine, N6-acyl punne,N6-hydroxyalkyl punne, N6-thioalkyl purine, thymine, cytosine, 6-azapynmidine, 2-mercaptopyrmidine, uracil, N5-alkylpynmidmes, NS-benzylpynmidines, N5-halopynmidmes, N5-vmylpynmidme, N5-acetylemc pynmidme, N5-acyl pynmidme, N5-hydroxyalkyl punne, and N6-thioalkyl punne, and isoxazolyl The heteroaromatic and heterocyclic moieties can be optionally substituted as descnbed above for aryl, including substituted with one or more substituent selected from halogen, haloalkyl, alkyl, alkoxy, hydroxy, carboxyl cenvauves, amido, ammo, alkylammo, dialkylarmno The heteroaromatic can be partially or totally hydrogenated as desired As a nonlimitmg example, dihydropyndme can be used m place of pyndine Functional oxygen and nitrogen groups on the heteroaryl gro jp can be protected as necessary or desired Suitable protecting groups are well known to those skilled m the art, and mclude tnmethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, tntyl or substituted tntyl, alkyl groups, acyl groups such as acet\ and propionyl, methanesulfonyl, and β-toluenelsulfonyl As used herein, the term "substantially free of enantiomer" or "substantially m the absence of enantiomer" refers to a nucleoside composition that mcludes at least 95% to 98 % by weight, and even more preferably 99% to 100% by weight, of the designated enantiomer of that nucleoside In a preferred embodiment, m the methods and compounds of this invention, the compounds are substantially free of enantiomers Similarly, the term "isolated" refers to a nucleoside composition that mcludes at least 85% or 90% by weight, preferably 95% to 98% by weight, and even more preferably 99% to 100% by weight, of the nucleoside, the remainder comprising other chemical species or enantiomers The term "independently" is used herein to indicate that the variable that is independently applied vanes independently from application to application Thus, m a compound such as R"XYR", wherem R" is "independently carbon or nitrogen," both R" can be carbon, both R" can be nitrogen, or one R" can be carbon and the other R" nitrogen The term host, as used herein, refers to a unicellular or multicellular organism in which the virus can replicate, including cell lines and animals, and preferably a human Alternatively, the host can be carrying a part of the hepatitis B viral genome, whose replication or function can be altered by the compounds of the present invention The term host specifically refers to infected cells, cells transfected with all or part of the HBV genome and animals, in particular, primates (including chimpanzees) and humans In most animal applications of the present invention, the host is a human patient Veterinary applications, in certain indications, however, are clearly anticipated by the present invention (such as chimpanzees) The terms "pharmaceutically acceptable salts" and "pharmaceutcally acceptable complexes" are used throughout the specification to describe any pharmaceutically acceptable form of a nucleoside compound, which, upon administration to a patient, provides the nucleoside compound and exhibit minimal, if any, undesired toxicologjcal effects Pharmaceutically acceptable salts mclude those derived from pharmaceutically acceptable inorganic or organic bases and acids Nonhmitrng 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 acid, tannic acid, palmoic acid, algmic acid, polyglutamic acid, naphthalenesulforuc acids, naphthalenedisulfomc acids, and polygalacturonic acid, (b) base addition salts formed with cations such as those derived from alkali metals, those derived from alkaline earth metals, sodium, potassium, zinc, calcium, bismuth, banum, magnesium, aluminum, copper, cobalt, mckel, cadmium, sodium, potassium, and the like, or with an organic cation formed from N,N-dibenzylethylene-diamine, ammonium, or ethylenediamme, or (c) combinations of (a) and (b), e g, a zinc tannate salt or the like Pharmaceutically acceptable prodrugs refer to a compound that is metabolized, for example hydrolyzed or oxidized, m the host to form the compound of the present invention Typical examples of prodrugs include compounds that have biologically labile protecting groups on a functional moiety of the active compound Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, dephosphorylated to produce the active compound The compounds of this invention possess antiviral activity against HBV, or are metabolized to a compound that exhibits such activity III Nucleotide Salt or Prodrug Formulations In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compound as a pharmaceutically acceptable salt may be appropriate Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids, which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate and α-glycerophosphate Suitable inorganic salts may also be formed, including, sulfate, nitrate, bicarbonate and carbonate salts Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made Any of the nucleosides described herem can be administered as a nucleotide prodrug to increase the actmry, bioavailability, stability or otherwise alter the properties of the nucleoside A number of nucleotide prodrug hgands are known In general, alkylahon, acylahon or other lipophilic modification of the mono, di or triphosphate of the nucleoside will increase the stability of the nucleotide Examples of substituent groups that can replace one or more hydrogens on the phosphate moiety are alkyl, aryl, steroids, carbohydrates, including sugars, 1,2-diacylglycerol and alcohols Many are described m R Jones and N Bischofberger, Antiviral Research, 27 (1995) 1-17 Any of these can be used in combination with the disclosed nucleosides to achieve a desired effect The active β-L-3'-prodrug nucleoside can also be provided as a 5'-phosphoether lipid or a 5'-ether lipid, as disclosed m the following references, which are incorporated by reference herein Kucera, L S , N Iyer, E Leake, A Raben, Modest E K, D L W , and C Piantadosi 1990 "Novel membrane-interactive ether lipid analogs that inhibit infectious HIV-1 production and induce defective virus formation " AIDS Res Hum Retro Viruses 6 491-501, Piantadosi, C , J Marasco C J, S L Morns-Natschke, K L Meyer, F Gumus, J R Surles, K S Ishaq, L S Kucera, N Iyer, C A Wallen, S Piantadosi, and E J Modest 1991 "Synthesis and evaluation of novel ether hpid nucleoside conjugates for anh-HIV activity " J Med Chem 34 1408 1414, Hosteller, K Y , D D Richman, D A Carson, L M Stuhmiller, GM T \an Wyk, and H van den Bosch 1992 "Greatly enhanced inhibition of human immunodeficiency virus type 1 replication in CEM and HT4-6C cells by 3'-deoxythymidine diphosphate dimynstoylglycerol, a lipid prodrug of 3,-deoxythymidine " Antimicrob Agents Chemother 36 2025 2029, Hosetler, KY, LM Stuhmiller, HB Lenting, H van den Bosch, and D D Richman, 1990 "Synthesis and antiretrovrral activity of phosphohpid analogs of azidothymidine and other antiviral nucleosides " / Biol Chem 265 61127 Nonhmiting examples of U S patents that disclose suitable lipophilic subsbtuents that can be covalentlv incorporated mto the nucleoside, preferably at the 5'-OH position of the nucleoside or lipophilic preparations, include US Patent Nos 5,149,794 (Sep 22, 1992, Yatvin et al), 5,194,654 (Mar 16, 1993, Hostetler et al, 5,223,263 (June 29, 1993, Hostetler et al), 5,256,641 (Oct 26,1993, Yatvin et al), 5,411,947 (May 2,1995, Hostetler et al), 5,463,092 (Oct 31, 1995, Hostetler et al), 5,543,389 (Aug 6, 1996, Yatvin et al), 5,543,390 (Aug 6, 1996, Yatvin et al), 5,543,391 (Aug 6, 1996, Yatvin et al), and 5,554,728 (Sep 10, 1996, Basava et al), all of which are incorporated herein by reference Foreign patent applications that disclose lipophilic substituents that can be attached to the nucleosides of the present invention, or lipophilic preparations, include WO 89/02733, W0 90/00555, W0 91/16920, W0 91/18914, W0 93/00910, W0 94/26273, W0 96/15132, EP 0 350 287, EP 939170M 4, and W0 91/19721 The 3'-prodrug can be administered as any derivative that upon administration to the recipient, is capable of providing directly or indirectly, the 3'-prodrug of the parent compound or that exhibits activity itself Nonlimiting examples are the pharmaceutically acceptable salts (alternatively referred to as "physiologically acceptable salts"), and the N4 pynmidine or N2 and/or N-punne alkylated (m particular with drmethylarmnomethylene) or acylated (in particular with acetyl or annnoacetyl) derivatives of the active compound In one nonlimiting embodiment, the acyl group is a carboxyhc acid ester in which the non-carbonyl moiety of the ester group is selected from straight, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkyl including benzyl, aryloxyalkyl such as phenoxymethyl, aryl including phenyl optionally substituted with halogen, C1 to C4 alkyl or C1 to C4 alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl including methanesulfonyl, phosphate, including but not limited to mono, di or triphosphate ester, tntyl or monomethoxytntyl, substituted benzyl, tnalkylsilyl (ε g , dimethyl-5-butylsilyl) or diphenylmethylsilyl Aryl groups m the esters optionally comprise a phenyl group Modifications of the 3'-prodrug or parent compound, and especially at the N4 pvnrmdmyl, or N2 and/or N6 purine positions, can affect the bioavailability and rate of metabolism of the active species, thus providing control over the delivery of the active species Further, the modifications can affect that antiviral activity of the compound, in some cases increasing the activity over the parent compound This can easily be assessed by preparing the derivative and testing its antiviral activity according to the methods described herein, or other method known to those skilled m the art IV Stereochemistry Since the 1' and 4' carbons of the sugar (referred to herein genencally as the sugar moiety) of the nucleosides are crural, their nonhydrogen substituents (CH2OR and the pynmidine or purine base, respectively) can be either cis (on the same side) or trans (on opposite sides) with respect to the sugar ring system The four optical isomers therefore are represented by the following configurations (when orienting the sugar moiety in a horizontal plane such that the "primary" oxygen (that between the C1' and C4'-atoms is m back) "ß" or "cis" (with both groups "up", which corresponds to the configuration of naturally occurring nucleosides, 1 e, the D configuration), "ß" or cis (with both groups "down", which is a nonnaturally occurring configuration, 1 e , the L configuration), "a "or "trans" (with the C2 substituent "up" and the C5 substituent "down"), and "a " or trans (with the C2 substituent "down" and the C5 substituent "up") The nucleosides of the present invention are of the β-L-configuration In a preferred embodiment, the 2'-deoxy-ß-L-nucleoside is administered substantially in the form of a smgle isomer, 1 e, at least approximately 95% m the designated stereoconfiguration V Combination and Alternation Therapy In combination therapy, effective dosages of two or more agents are administered together, whereas during alternation therapy an effective dosage of each agent is administered serially The dosages will depend on absorption, inactivation and excretion rates of the drug as well as other factors known to those of skill m the art It is to be noted that dosage values will also vary with the seventy of the condition to be alleviated It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions For example, m any of the embodiments described herem, if the 3'-prodrug of the ß-I,-2'-deoxynucleoside of the present invention is administered m combination or alternation with a second nucleoside or nonnucleoside reverse transcriptase inhibitor that is phosphorylated to an active form, in one embodiment, a second compound is one that can be phosphorylated by an enzyme that is different from that which phosphorylates the selected β-L-2'-nucleoside of the present invention in vivo Examples of kinase enzymes are thymidine kinase, cytosme kinase, guanosine kinase, adenosme kinase, deoxycytidine kinase, 5'-nucleotidase and deoxy-guanosine kinase Thus, in one embodiment the invention provides a combination of two or more nucleoside prodrugs of the present invention, preferably nucleosides that are phosphorylated by distinct enzymes, or that act through distinct biological pathways In another embodiment the invention provides at least one prodrug in combination or alternation with a nucleoside that exhibits activity against hepatitis B, including but not limited to a parent drug of any of the prodrugs defined herem, I e 2'-deoxy-β-L-nucleosides, including 2'-deoxy-β-L-cytidrne, 2'-deoxy-β-L-thymine, 2'-deoxy-ß-L-adenosine, 2'-deoxy-β-L-guamne, 2'-deoxy-β-L-5-fluoiocytidme, 2',3,-dideoxy-3'-thiacyhdine, 2',3'-dideoxy-3'- thia-5-fluorocytidine Alternatively, the compounds of the present invention can also be administered in combination or alternation with any other known anti-hepatits B virus agent, such as entecivir, cis-2-hydroxymethyl-5-(5-fluorocytosin-l-yl)-l,3-oxathiolane, preferably substantially in the form of the (-)-optical isomer ("FTC", see WO 92/14743), the (-)-enantiomer of cis-2-hydroxymethyl-5-(cytosm-l-yl)-l,3-oxathiolane (3TC), β-D-l,3-dioxolane purine nucleosides as described m US Patent Nos 5,444,063 and 5,684,020, ß-D-dioxolane nucleosides such as β-D-dioxolanyl-guanine (DXG), β-D-dioxolanyl-2,6-diaminopunne (DAPD), and β-D-dioxolanyl-6-chloropunne (ACP), L-FDDC (5-fluoro-3'-thia-2',3'-dideoxycytidine), L-enantiomers of 3' fluoro-modifled /9-2'-deoxynbonucleoside 5'-tnphosphates, carbovir, mterferon, penciclovir and famciclovir, L-FMAU, famciclovir, penciclovir, BMS-200475, bis pom PMEA (adefovir, dipivoxil), lobucavir, ganciclovir, or nbavann, or any other compound that exhibits an EC50 of less than 15 micromolar m 2 2 15 cells, or their prodrugs or pharmaceuncally acceptable salts Combination and alternation therapy can also be undertaken to combat drug resistance It has been recognized that drug-resistant variants of viruses can emerge after prolonged treatment with an antiviral agent Drug resistance most typically occurs by mutation of a gene that encodes for an enzyme used in viral replication The efficacy of a drug against hepatitis B infection can be prolonged, augmented, or restored by administering the compound in combination or alternation with a second, and perhaps third, antiviral compound that induces a different mutation from that caused by the principle drug Alternatively, the pharmacokinetics, biodistnbution or other parameter of the drug can be altered by such combination or alternation therapy In general, combination therapy is typically preferred over alternation therapy because it induces multiple simultaneous stresses on the virus In another embodiment, the prodrug is administered in combination or alternation with an immune modulator or other pharmaceuncally active modifer of viral replication, including a biological material such as a protein, peptide, oligonucleotide, or gamma globulin, including but not limited to mterfereon, mterleukm, or an anusense oligonucleotides to genes which express or regulate hepatitis B replication Any method of alternation can be used that provides treatment to the patient Nonhmiting examples of alternation patterns mclude 1-6 weeks of administration of an effective amount of one agent followed by 1-6 weeks of admmistration of an effective amount of a second anti-HBV agent The alternation schedule can include periods of no treatment Combination therapy generally mcludes the simultaneous administration of an effective 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-HrV 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 m the appropriate circumstance m combination or alternation with anh-HIV medications The second antiviral agent for the treatment of HIV, m one embodiment, can be a reverse transcriptase inhibitor (a "RTF), which can be either a synthetic nucleoside (a "NRTT') or a non-nucleoside compound (a "NNRTT') In an alternative embodiment, in the case of HIV, the second (or third) antiviral agent can be a protease inhibitor In other embodiments, the second (or third) compound can be a pyrophosphate analog, or a fusion binding inhibitor A list compiling resistance data collected in vitro and in vivo for a number of antiviral compounds is found in Schinazi, et al, Mutations m retroviral genes associated with drug resistance, International Antiviral News Volume 1(4), International Medical Press 1996 The active anti-HBV agents can also be administered m combination with antibiotics, other antiviral compounds, antifungal agents or other pharmaceutical agents administered for the treatment of secondary infections VI Pharmaceutical Compositions Humans suffering from any of the disorders described herein, including hepatitis B, can be treated by administering to the patient an effective amount of a 3'-prodrug of a ß-L-2'-deoxy-nucleoside of the present invention, or a pharmaceutically acceptable salt thereof, in the presence of a pharmaceutically acceptable earner or diluent The active materials can be administered by any appropriate route, for example, orally, parenterally, intravenously, rntradermally, subcutaneously, or topically, m liquid or solid form The active compound is included in the pharmaceutically acceptable earner or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount of compound to mhibit viral replication in vivo without causing senous toxic effects m the patient treated By "inhibitory amount" is meant an amount of active ingredient sufficient to exert an inhibitory effect as measured by, for example, an assay such as the ones described herein A preferred dose of the compound for all of the abovementioned conditions will be in the range from about 1 to 50 mg/kg, preferably 1 to 20 mg/kg, of body weight per day, more generally 0 1 to about 100 mg per kilogram body weight of the recipient per day The effective dosage range of the pharmaceutically acceptable prodrug can be calculated based on the weight of the parent nucleoside to be dehvered If the prodrug exhibits activity in itself, the effective dosage can be estimated as above using the weight of the prodrug, or by other means known to those skilled in the art The compound is conveniently administered m 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 plasma concentrations of the active compound of from about 0 2 to 70 µM, preferably about 1 0 to 10 µM This may be achieved, for example, by the intravenous injection of a 0 1 to 5% solution of the active ingredient, optionallv in salme, or administered as a bolus of the active ingredient The concentration of active compound m 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 seventy of the condition to be alleviated It is to be further understdod 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 mtended to limit the scope or practice of the claimed composition The acnve ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of tune A preferred mode of administration of the active compound is oral Oral compositions will generally include an inert diluent or an edible earner They may be enclosed in gelatin capsules or compressed mto tablets For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used m 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, Pnmogel, or com starch, a lubricant such as magnesium stearate or Sterotes, a glidant such as colloidal silicon dioxide, a sweetening agent such as sucrose or saccharin, or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring When the dosage unit form is a capsule, it can contain, m addition to material of the above type, a liquid earner such as a fatty oil In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents The compound can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors The compound or a pharmaceutically acceptable derivative or salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, anhinflammatones, protease inhibitors, or other nucleoside or nonnucleoside antiviral agents Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methyl parabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as 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 m ampoules, disposable svnnges or multiple dose vials made of glass or plastic If admmisterea intravenously, preferred carriers are physiological saline or phosphate buffered salme (PBS) In a preferred embodiment, the active compounds are prepared with earners that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems Biodegradable, (Formula Removed) Alternatively, the 3'-derivative is derived from an aminoacyl moiety The key starting material for this process is also an appropriately substituted β-L nucleoside The ß-L nucleoside can be purchased or can be prepared by any known means including standard coupling reactions with an L-sugar moiety These aminoacyl derivatives can be made by first selectively protecting the 5'-hydroxyl with a suitable oxygen protecting group, such as an acyl or silyl protecting group, and optionally protecting any free amine m the heterocyclic or heteroaromatic base Subsequently, the free 3'-hydroxyl can be coupled to an N-protected a or ß ammo acid Subsequently, the β-L-nucleoside is coupled to the aminoacyl using standard coupling reagents that promote the coupling Some non-lrmitmg examples of coupling reagents are Mitsunobu-type reagents (ε g dialkyl azodicarboxylates such as dnsopropyl azodicarboxylate and diethyl azodicarboxylate) with tnphenyl phosphine or various types of carbodiimides The coupling reaction can be earned out at any temperature that achieves the desired results, 1 e, that is suitable for the reaction to proceed at an acceptable rate without promoting decomposition or excessive side products Am reaction solvent can be selected that can achieve the necessary temperature and that can solubihze the reaction components Non-hmiting examples are any aprotic solvent including, but not limiting to, alkyl or halo-alkyl solvents such as hexane, cyclohexane, dichloromethane or dichloroethane, toluene, acetone, ethyl acetate, dithianes, THF, dioxane, acetonitnle, diethyl ether, pyridine, dirnethylformarnide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide, or any combination thereof Scheme 1 is a non-limiting example of the preparation of a β-L-3'-aminoacyl-nucleoside denved from L-deoxynbonucleoside (Scheme Removed) B Method for the preparation of ß-L-5'-denvatives of β-L-nucleosides β-L-5'-derivatives of a β-L-nucleoside can be made by any means known in the art, particularly by known methods to protect primary alcohols with acyl moieties, 1 e via an anhydride or with the aid of a coupling agent As a non-limiting example, the β-L-5'-denvatives can be prepared according to the following reaction sequence (Sequence Removed) In a preferred embodiment, the 5'-derivative is denved from an aminoacyl moiety The key starting material for this process is an appropriately substituted β-L-nucleoside The β-L-nucleoside can be purchased or can be prepared by any known means including standard coupling reactions with an L-sugar moiety, such as deoxynbose The aminoacyl derivatives can be made by selectively coupling an amino acid to a β-L-nucleoside, preferably without any additional protection of the nucleoside The coupling reaction can be achieved using appropriate coupling reagents that promote the coupling Some non-lrmitmg examples of coupling reagents are Mitsunobu-type reagents (e g dialkyl azodicarboxylates such as dnsopropyl azodicarboxylate and diethyl azodicarboxylate) with tnphenyl phosphine or various types of carbodiimides The coupling reaction can be earned out at any temperature that achieves the desired results, 1 e, that is suitable for the reaction to proceed at an acceptable rate without promoting decomposition or excessive side products Any reaction solvent can be selected that can achieve the necessary temperature and that can solubihze the reaction components Non-hmiting examples are any aprotic solvent including, but not limiting to, alkyl or halo-alkyl solvents such as hexane, cyclohexane, dichloromethane or dichloroethane, toluene, acetone, ethyl acetate, dithianes, THF, dioxane, acetomtnle, diethyl ether, pyridine, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide, or any combination thereof Scheme 2 is a non-lrmitmg example of the preparation of a β-L-5'-aminoacyl-nucleoside derived from L-deoxynbonucleoside (Scheme Removed) C Method for the preparation of β-L-3' 5'-bis-0-denvatives of β-L-nucleosides β-L-3',5'-bis-0-denvatives of a β-L-nucleoside can be made by any means known in the art, particularly by known methods to protect primary and secondary alcohols with acyl moieties, 1 e via an anhydride or with the aid of a coupling agent As a non-limiting example, the 3',5'-bis-0-derivatives can be prepared according to the following reaction sequence (Sequence Removed) In a preferred embodiment, the 3',5'-bis-ßderivative is derived from an aminoacyl moiety The key starting material for this process is also an appropriately substituted ß-L-nucleoside The 3',5'-bis-0-derivatives of the β-L-nucleosides can be purchased or can be prepared by any known means including standard coupling reactions with an L-sugar moiety, such as deoxvnbose Subsequently, the free 3'- and 5'-hydroxyl can be coupled to a N-protected a or ß ammo acid The coupling reaction can be achieved using appropriate coupling reagents that promote the coupling Some non-hrmtmg examples of coupling reagents are Mitsunobu-type reagents (ε g dialkyl azodicarboxylates such as dusopropyl azodicarboxylate and diethyl azodicarboxylate) with tnphenyl phosphme or various types of carbodirmides The coupling reaction can be earned out at any temperature that achieves the desired results, 1 e, that is suitable for the reaction to proceed at an acceptable rate without promoting decomposition or excessive side products Any reaction solvent can be selected that can achieve the necessary temperature and that can solubilize the reaction components Non-hrmtmg examples are any aprotic solvent including, but not limiting to, alkyl or halo-alkyl solvents such as hexane, cyclohexane, dichloromethane or dichloroethane, toluene, acetone, ethyl acetate, dithianes, THF, dioxane, acetonitnle, diethyl ether, pyridine, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide, or any combination thereof Scheme 3 is a non-hrmting example of the preparation of a β-L-3',5'-di-aminoacyl-nucleoside denved from L-deoxynbonucleoside Scheme 3 (Scheme Removed) D Optional method for the extension of the aminoacvl moiety The title compounds can be made by reacting the 3' and 5'-hydroxyl with a suitable derivative, such as an acyl, and in particular an aminoacyl group If the nucleoside is denvatized with an aminoacyl moiety, it may be desirable to further couple the free amine to a N-protected a or ß ammo acid The coupling reaction can be achieved using appropriate coupling reagents that promote the couplmg Some non-limiting examples of coupling reagents are Mitsunobu-type reagents (e g dialkyl azodicarboxylates such as dnsopropyl azodicarboxylate and diethyl azodicarboxylate) with tnphenyl phosphine or various types of carbodiirnides The couplmg reaction can be earned out at any temperature that achieves the desired results, l e, that is suitable for the reaction to proceed at an acceptable rate without promoting decomposition or excessive side products Any reaction solvent can be selected that can achieve the necessary temperature and that can solubihze the reaction components Non-limiting examples are any aprotic solvent including, but not limiting to, alkyl or halo-alkyl solvents such as hexane, cyclohexane, dichloromethane or dichloroethane, toluene, acetone, ethyl acetate, dithianes, THF, dioxane, acetomtnle, diethyl ether, pyndine, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide, or any combination thereof E Optional method for denvatization of the heteroaromatic or heterocyclic base The title compounds can be made by optionally protecting any free amnio in the heterocyclic or heteroaromatic base, for example N4-cytosine, N6-adenrne or N2-guanme For example, the amine can be protected by an acyl moiety or a dialkylaminomethylene moiety by the following general protocol (Scheme Removed) The protection can be earned out at any temperature that achieves the desired results, 1 e, that is suitable for the reaction to proceed at an acceptable rate without promoting decomposition or excessive side products Any reaction solvent can be selected that can achieve the necessary temperature and that can solubihze the reaction components Non-hmiting examples are any aprotic solvent including, but not limiting to, alkyl or halo-alkyl solvents such as hexane, cyclohexane, dichloromethane or dichloroethane, toluene, acetone, ethyl acetate, dithianes, THF, dioxane, acetomtnle, diethyl ether, pyridine, dimethylformarmde (DMF), dimethylsulfoxide (DMSO), dimethylacetamide, or any combmation thereof Subsequently, the free 3'-hydroxyl can be coupled to a N-protected a or ß ammo acid The coupling reaction can be achieved using appropriate coupling reagents that promote the coupling Some non-hmiting examples of couplmg reagents are Mitsunobu-type reagents (ε g dialkyl azodicarboxylates such as dusopropyl azodicarboxylate and diethyl azodicarboxylate) with tnphenyl phosphine or various types of carbodnmides The couplmg reaction can be earned out at any temperature that achieves the desired results, 1 e, that is suitable for the reaction to proceed at an acceptable rate without promoting decomposition or excessive side products Any reaction solvent can be selected that can achieve the necessary temperature and that can solubilize the reaction components Non-limiting examples are any aprotic solvent including, but not limiting to, alkyl or halo-alkyl solvents such as hexane, cyclohexane, dichloromethane or dichloroethane, toluene, acetone, ethyl acetate, dithianes, THF, dioxane, acetonitnle, diethyl ether, pyridine, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide, or any combination thereof In an alternate embodiment, the N4- or N^-acyl derivative is derived from an aminoacyl moiety, and can be prepared accordmg to the following reaction sequence, by optionally protecting the free hydroxyls, followed by a condensation reaction with the appropriately protected ammo ester, and the removal of the hydroxyl protecting groups, if necessary (Scheme Removed) Examples Example 1 N-mMTr-2'-deoxy-β-L-cytidine (1, Figure 1) β-L-dC (1 g, 4 40 mmol) was taken up m dry pyridine (44 ml) After transient protection with tnmethylsilyl group (TMSC1, 3 34 ml, 26 4 mmol) followed by addition of mMTrCl (3 38 mg, 11 mmol) and 4-dimethylaminopyndine (DMAP, 540 mg, 4 40 mmol) the reaction mixture was stirred for 3 days at room temperature {A Nyilas, C Glemarec, J Chattopadhyaya, Tetrahedron Lett 1990, 46. 2149-2164} After sodium bicarbonate extraction the organic layer was washed with water, evaporated and taken up m dioxane (40 mL) Aqueous ammonia (8 5 ml) was added dropwise and the reaction mixture was stirred overnight After evaporation of all volatile materials, the solid residue was purified on silica gel column {eluent stepwise gradient of MeOH (0-10%) in CH2CI2}, giving the desired compound 1 (1 02 g, 46 5%) as a foam 1H NMR (DMSO-d6) δ ppm 8 39 (br s, 1H, N#, D20 exchangeable), 7 70 (d, 1H, H-6, J= 7 3 Hz), 7 4-6 8 (m, 14H, (C6H5)(C6H4)OCH ), 6 23 (d, 1H, H-5, J = 7 3 Hz), 6 02 (t, 1H, H-l', J = 6 5 Hz), 5 16 (d, 1H, 0#-3\ J = 3 8 Hz, D20 exchangeable), 4 9 (br s, 1H, OH-5\ D20 exchangeable), 4 1 (m, 1H, H-3'), 3 7 (m, 4H, H-4', OCH,), 3 5 (m, 2H, H-S\ H-5"), 2 1-1 8 (2m, 2H, H-V, H-2"), FABO, (GT) m/e 498 (M-H), 382 (B), 226 (M-mMTr), FAB>0 (GT) δ00 (M+H)+, 273 (mMTr), UV (EtOH 95) λmax = 279 ran,λmax= 250 nm Example 2 S'-L-N-(tert-butoxycarbonyI) valine ester of 4NmMTr-2'-deoxy-β-L-cytidme (2, Figure 1) To a solution of compound 1 0- g, 2 00 mmol) in dry DMF (34 ml) were added successively 4-dunethylammopyndine (DMAP, 37 mg, 0 3 mmol), N-(tert-butoxy-carbonyl)-L-vahne (Boc-Val-OH, 587 mg, 2 7 mmol), and NN -dicyclohexylcarbodiumde (DCC, 660 mg, 3 2 mmol) {L M Beauchamp, G F Orr, P De Miranda, T Burnette, T A Krenitsky, Antiviral Chem Chemother 1992, 3, 157-164 } The solution was stirred at room temperature After 40h, the reaction mixture was recharged with additional DMAP (37 mg, 0 3 mmol), Boc-Val-OH (587 mg, 2 7 mmol) and DCC (660 mg, 3 2 mmol) and stirred at room temperature for 40h The mixture was filtered, the DMF was removed from the filtrate under reduced pressure, and the residue was chromatographed on a silica gel column {eluent stepwise gradient of MeOH (0-10%) m CH2CI2} to afford the desired compound 2 (515 mg, 37%) as a foam 1H NMR (DMSO-d6) δ ppm 8 44 (br s, 1H, Nff, D20 exchangeable), 7 7-6 8 (m, 15H,, H-6 and (C6H5)2C(C6H4)OCH3), 6 26 (d, 1H, H-5, J = 73 Hz), 606 (t, 1H, H-1', J = 6 6 Hz), 5 7 (bs, 1H, OH-3\ D2O exchangeable), 42-40 (m, 3H, H-V, H-4' and CH), 3 8-3 9 (m, 2H, H-5\ H-5"), 3 7 (s, 3H,, OCH3), 2 0-1 9 (m, 3H, H-T, H-2", CH), 1 36 (s, 9H, (CH3)3C), 0 86 (m, 6H, (CH3)2CH), FAB0 (GT) 384 (B+2H)+, 273 (mMTrf, 57 (CH3)3C)+,UV (EtOH 95) λmax = 279 nm, λmax = 249 nm Example 3 5'-L-valine ester of 2'-deoxy-ß-L-cytidine hydrochloride (3, Figure 1) Compound 2 (500 mg, 0 715 mmol) was dissolved in a 20% solution of tnfluoroacetic acid in CH2CI2 (25 ml) and tnisopropylsilane (1 47 ml, 71 5 mmol) was added The reaction mixture was stirred at room temperature for 1 h and the valine ester was precipitated m Et20 as the tnfluoroacetate salt After several coevaporations with water, the precipitate was taken up m water (2 ml), treated with a saturated solution of HC1 m dioxane (20 ml) and evaporated under reduced pressure This treatment was repeated 3 times and the desired compound 3 was finally precipitated m ether (207 mg, 73%) as the hydrochloride salt 1H NMR (DMSO-d6) δ ppm 9 7 (br s, 1H, 1/2NH2, D20 exchangeable), 8 6 (br s, 4H, 1/2NH, NH3, D20 exchangeable), 7 98 (d, 1H, , H-6 J = 7 8 Hz), 617 (d, 1H, H-5, J = 7 8 Hz), 6 11 (pt, 1H, H-\'), 5 5 (bs, OCH3), 2 3-2 1 (m, 3H, H-T, H-2 CH) 0 94 (dd, 6H, (CH3)2CH, J = 3 7 and 6 6 Hz), FABO, (GT) m/e 361 (M+Cl), 325 (M-H), 116 (Val-H), 110 (B), 216 (BocVal-H), FAB>0 (GT) δ53 (2M+H)+, 327 (M+H)+, 112 (B+2H)+, )+, {α}D20 - 28 57 (c = 0 49 in DMSO), UV (EtOH 95) λmax = 272 ran (ε 8700), \™ = 255 nm (s 7600), HPLC it = 8 37nun (gradient from 0 to 50% CH3N in 20 mM tnethyl ammonium acetate buffer programmed over a 30 mm period with a flow rate of 1 ml/mm) Example 4 ^V'-Acetyl-2'-deoxy-ß-L-cytidme (4, Figure 2) To a suspension of the nucleoside, ß-L-dC (415 mg, 1 83 mmol) in NN-dmiethylformamide (9 2 ml) was added acetic anhydride (207 µl 2 20 mmol) and the mixture was stirred at room temperature for 24h [V Bhat, B G Ugarkar, V A Sayeed, K Grimm, N Kosora, P A Domenico, E Stacker, Nucleosides & Nucleotides, 1989, 8 (2), 179-183] After removal of the DMF under reduced pressure, the resulting residue was purified by silica gel column chromatography [eluant 15% MeOH in CH2CI2] to afford the desired compound (310 mg, 63%) which was crystallized from ethanol, rap 128-170°C, 1H NMR (DMSO-4) δ ppm 10 86 (s, 1H, NH, D2O exchangeable), 8 31 (d, 1H,, H-6, J = 7 5 Hz), 7 18 (d, 1H, H-S, J = 7 5 Hz), 6 09 (t, 1H, HA ', J = 6 3 Hz), 5 25 (d, 1H, OH-3', D20 exchangeable, J = 4 2 Hz), 5 03 (t, 1H, OH-5', D20 exchangeable, J = 5 0 Hz), 4 1-4 2 (m, 1H, H-3'), 3 8 (m, 1H, H~4\ 3 4-3 6 (m, 2H, 2H, H-5\ H-5"), 2 2-2 3 (m, 1H, H-2), 2 08 (s, 3H, CH3), 2 0-1 9 (m 1H, H-Z"), FAB0 (GT) δ08 (3M+H)+, 539 (2M+H)+. 362 (M+G+H)+» 270 (M+H)+. 154 (B+2Hf> 117 (S)+, UV (H20) λmax = 297 nm (ε 8300), λmax = 270 nm (ε 3500), U - 245 nm (ε 14400), λmax= 226 nm (ε 5800), [α]D20 - 81 31 (c = 1 07 in DMSO) Example 5 N4- [(DI methylamin o)methylene]-2 '-deoxy-ß-L-cytidme (5, Figure 3) The title compound was prepared according to a published procedure developed for the preparation of the corresponding D-enantiomer [S G Kerr, and TI Kalman, J Phaim Sci 1994, 83, 582-5S6] A solution of L-dC (500 mg, 2 20 mmol) in DMF (4 8 ml) was treated with dimeth\l!ormamide dimethylacetal (2 8 ml, 21 08 mmol), and stirred at room temperature overmgn The solution was evaporated under reduced pressure, and coevaporated with ethanol Crystallization from ethanol/ether yielded the title compound (501 2 mg, 81%) as light yellow crystals mp 174-176°C (lit 188-190°C for the D-enantiomer), 1H NMR (DMSO-d6) δ ppm 8 60 (s, 1H, N=CH), 8 00 (d, 1H, H-6), 6 15 (t, J= 6 6 Hz, 1H, H-l'), 5 96 (d, J = 7 2 Hz, 1H, H-5), 5 22 (d, J = 4 2 Hz, 1H, OH-31), 5 01 (t, J = 5 2 Hz, 1H, Ott-50 (GT) δ47 (3M -H)+, 565 (2M+H)+, 283 (M+H), FAB Example 6 3',5'-Di-0-acetyl-2'-deoxy-β-L-cytidine (6, Figure 4) The title compound has been synthesized in one step starting from the L-dC and following a procedure developed by Bremer et al [R G Bremer, W Rose, J A Dunn, J E Mae Diarmid and J Bardos, J Med Chem 1990, 33, 2596-2603] for the preparation of the D-enanuomer A solution of L-dC (765 mg, 3 37 mmol) and acetyl chloride (960 (A, 13 48 mmol) m glacial acetic acid (4 8 ml) was stirred at room temperature for 10 mm, then dry chloroform (3 5 ml) was added and the starring was continued for 24h The solution was evaporated under reduced pressure and coevaporated with ethanol Crystallization from ethanol yielded 78% of the desired compound, mp 192 193°C (lit 187-189°C for the D-enantiomer [Bremer et al J Med Chem 1990, 33, 2596-2603]), 1H NMR (DMSO-d6) δ ppm 9 8 and 8 7 (2 br s, 6 08 (t, 1H, H-\\ J = 6 7 Hz), 5 2 (m, 1H, H-3'), 4 3-4 1 (m, 3H, H-4', H-5', H-5"), 2,4-2,5 (m, 2H, H-2', H-2"), 2 06 and 2 03 (2 s, 6H, 2 CH3), FAB (CH3COO), FAB>0 (GT) 623 (2M+H)+. 312 (M+H)+» 201 (S)+, 112 (B+2H)+> 43 (CH3CO)+, [α]D20 36 27 (c = 1 02 in DMSO), UV (MeOH) max = 277 nm (ε 9900), min= 246 nm (ε 5000) Example 7 3',5'-L-V-(t-Butoxycarbonyl)valine diester of 2'-deoxy-β-L-cytidine (9, Figure 5) A solution o: N-[(dimethylamino)methylene]-2'-deoxy-ß-I-cytidme (7, 500 mg, 1 77 mmol) in DMT (35 ml) was treated with Boc-Val-OH (1 31 g, 6 03 mmol), DMAP (86 5 mg, 0 71 mmol), l-β-dimethylaminopropyl)-3-ethylcarbodimude hydrochlonde (EDC) (1 36 g, 7 09 mmol), and stirred at room temperature for 40 hours Additional quantities of Boc-Val-OH (655 mg, 3 01 mmol), DMAP (43 2 mg, 0 35 mmol), EDC (680 mg, 3 55 mmol) were added, and the solution was stirred for an additional 20 hours After evaporation under reduced pressure, the residue was taken up m CH2CI2, and extracted several times with water The organic layer was washed with brine (100 ml), dried (Na2SO4), and evaporated under reduced pressure to give 8 as a crude material, which was used for the next step without further purification The residue was taken up in dioxane (18 ml), treated with aq 26% NH4OH, and stirred at room temperature for 1 hour The solution was evaporated under reduced pressure, and the residue was purified by chromatography on silica gel using a stepwise gradient of MeOH (0-5%) m CH2CI2, to give the title compound (698 7 mg, 58% from 9) 1H NMR (DMSO-d6) δ ppm 7 58 (d, 1H, H-6), 7 29-7 18 (m, 4H, NH-Boc and NH2), 6 20 (t, J= 6 6 Hz, 1H, H-l'), 5 75 (d, J = 7 3 Hz, 1H, H-5), 5 20 (br s, 1H, H-3'), 4 29 (m, 2H, H-5' and H-5"), 4 14 (br s, 1H, H-4'), 3 86 (m, 2H, CH-NH-Boc), 2 31-2 21 (m, 2H, H-2' and H-2"), 2 13-1 98 (m, 2H, CH(iPr)), 1 38 and 1 36 (2s, 18H, tBu), 0 88 and 0 85 (2 d, J = 6 8 Hz, 12H, CH(CH3)2), l3C NMR (DMSO-d6) δ ppm 172 67 and 172 46, 166 41, 156 64 and 155 70, 141 39, 95 43, 85 78, 82 03, 79 14, 75 57, 64 90, 60 37 and 6011, 37 40, 30 33, 29 00, 19 83-19 12, FAB>0 (GT) 626 (M+H)+> 112 (B+2H)+, 255 (M-Boc)', FABO, (GT) m/z 1249 (2M-H), 624 (M-H) Example 8 3,5'-L-Valine ester of 2'-deoxy-β-L-cytidine hydrochloride (10, Figure 5) A solution of 9 (675 mg, 1 08 mmol) m dioxane (30 ml) was treated with a solution of 26% HC1 in dioxane (30 ml), and stirred at room temperature for 1 hr 55 The resulting white suspension was evaporated under reduced pressure The white solid residue was taken up m the minimal amount of MeOH and precipitated in ether to give the title compound 10 as a white sohd mp 187°C (decomp ), 1H NMR (DMSO-d6) δ ppm 9 79 (br s, 1H, 1/2NH2), S 72 (br s, 7H, 1/2NH2 and NH3+), 8 04 (d, 1H, H-6), 6 21 (d, J = 7 8 Hz, 1H, H-5), 6 16 (t, J = 6 9 Hz, 1H, H-l'), 5 39 (m, 1H, H-3'), 4 50-4 40 (m, 3H, H-4', H-5' and H-5"), 3 90 (2 br d, 2d, CH-NH), 2 63-2 50 (2m, 2H, H-2' and H-2"), 2 21 (m, 2H, CH(iPr)), 1 02-0 94 (r 12H, CH(CH3) 2) I3C NMR (DMSO-efe) δ ppm 169 50 and 168 94, 161 02, 148 50, 145 26, 95 18, 87 19, 82 15, 76 14, 65 77 and 65 59, 58 12 and 58 07, 37 00, 30 16,19 26-18 51, FAB>0 (GT) 426 (M+H)+, 112 (B+2H)+, FAB Example 9 N-Boc-Valmyl ester of 2'-deoxy-ß-L-cytidine (13, Figure 6) A mixture of L-dC (1 80 g, 7 92 mmol) and tnethylamine (8 8 ml, 63 14 mmol) in anhydrous THF (80 ml) was treated with chlorotnmethylsilane (6 ml, 47 28 mmol) and stirred at room temperature overnight The reaction was quenched by addition of an aqueous saturated solution of NH4CI (26 ml) and water (10 mL) The aqueous layer was extracted three rimes with EtOAc The organic layers were combined, washed with bnne, dried (Na2SO4) and evaporated under reduced pressure to give a crude light yellow foam-oil containing 11, which was used for the next step without further purification This residue was taken up in CH2CI2 (104 ml), treated with iV-(tert-butoxycarbonyl)-L-vahne (Boc-Val-OH, 1 72 g, 7 90 mmol), benzotnazol-l-yloxy-tns(dunethylammo) phosphonium hexafluorophosphate (BOP, 4 20 g, 9 50 mmol), tnethylannne (2 2 ml, 15 78 mmol), and stirred at room temperature for 2 days The solution was diluted with EtOAc and extracted twice with sat NaHC03 The organic layer was dried (Na2SO4) and evaporated under reduced pressure to give 12 as a crude material, which was used for the next step without further purification This residue was taken up in dioxane (80 ml), treated with aq 26% NH4OH solution, and stirred at room temperature for 6h45 The solution was evaporated under reduced pressure, coevaporated with absolute EtOH, and the residue was purified by chromatography on silica gel, using a stepwise gradient of MeOH (5-10%) in CH2CI2, to give the title compound 13 as a foam (1 64 g, 48 5% overall yield) 1H NMR (DMSO-d6) δ ppm 10 88 (s, 1H, NH-4), 8 40 (d, 1H, H-6), 7 26 (d, J = 7 4 Hz, 1H, H-5), 7 06 (d, J= 8 2 Hz, 1H, CH-NH-Boc), 6 15 (t, J= 6 3 Hz, 1H, H-l1), 5 32 (d, J = 4 2 Hz, 1H, OH-3'), 5 09 (t, J= 5 2 Hz, 1H, OH-5') 4 27 (m, 1H, H-3'), 4 06 (pt, J = 7 5 Hz, 1H, CH-NH-Boc), 3 91 (m, 1H, H-4') 3 63 (m 2H, H-5' and H-5"), 235 (m, 1H, H-2"), 2 06 (m, 2H, H-2' and CH(CH3)2), 1 43 (s, m, tBu), 0 92 (pt, J = 6 6 Hz, 6H, CH(CTh)?), 13C NMR (DMSO-rf6) δ ppm 174 41, 162 94 15647, 155 24, 146 10, 96 06, 88 79, 87 10, 79 09, 70 75, 6178, 61 55, 41 74, 30 63, 29 02, 19 91 and 19 10, FAB>0 (GT) δ53 (2M+H)+, 427 (M+H)+ 311 (B+2H)+, 255 (M-Boc)+, FAB Example 10 3',5'-N-Tnvalyl-2'-deoxycytidine (14, Figure 7) The starting material, 3',5'-N-tri(Boc-valyl)-2'-deoxycyhdine was dissolved in CH2CI2, but there was some insoluble material so the sample was filtered through Perhta This resulted in an increase in the volume of the CH2CI2 used The HCl/dioxane reagent was then added with stirring Within a few seconds some bubbling could be observed m the solution and then the mixture became cloudy The mixture was stirred at room temperature for about 1 hr During this time the precipitate became more crystalline The mixture was quickly filtered, the filtercake was washed with CH2CI2, and then it was dried on the pump to give, 0 16g (69%) of cream-white crystals The reagents and conditions are more explicitly described below in Table 1 (Table Removed) Example 11 HPLC 4ssay Method for DiBocValyI-2'-dC and DiBocVaIyl-2'-dU A 10mg/mL sample was made by dissolving the desired compound in absolute ethanol The solution was then diluted with a solution that contained 50% MeOH and 50% KH2PO4 (0 015M, pH=3 30-3 50) until a concentration of 0 16mg/mL was obtained (Note all solvents used were degasified before use ) 20µL of the solution was then immediately mjected into an HPLC column from WATERS (NOVAPAK C18 - 4pm - 3,9 X 150mm) The flow rate was set at lmL/min with a column temperature of 35°C To detect the compounds, the wavelength detection was set at 275nm for Di-Boc 2'dC, 260nm for Di-Boc2'dU and 204 for impurities after 15 minutes The column was run with KH2P04 (0 015M, pH=3 30 - 3 50, adjusted with H3PO4 10% v/v) in Pump A and HPLC grade acetonitnle in Pump B The gradient pattern is indicated in Table 2 Table 2 (Table Removed) VIII Anti-HBV Activity of the Active Compounds Human DNA polymerases and mitochondrial function were not affected by L-dC in vitro L-dC was non-cytotoxic to human peripheral blood mononuclear cells (PBMCs), bone marrow progenitor cells and numerous cell lmes of human and other non-human mammalian origin The antiviral activity and safety of L-dC was investigated in two studies using the woodchuck model of chrome hepatitis B infection In the initial study, woodchucks chronically infected with WHV (>10n genome equivalents/ML serum) were treated with a liquid formulation of L-dC by the oral route once a day for 28 days Control animals received lamivudine or the liquid formulation without drug In the L-dC treated groups, viral load decreased in a dose-dependent manner At the highest dose tested (10 mg/kg/day), viral load decreased by as much as 6 logs from baseline by quantitative polymerase chain reaction (PCR) assay Post-treatment virus rebound was detected by Week 2 All animals gamed weight and there was no drug-related toxicity observed during the four-week treatment phase or eight-week post-treatment follow-up period The in vitw 50% effective concentration (EC50) for reduction in extracellular viral deoxyribonucleic acid (DNA) by L-dC was 0 24 µM against HBV and 0 87 µM_ against DHBV In addition L-dC reduced intracellular HBV DNA rephcative intermediates (RI) with an EC50 of 0 51 µM The 90% effective concentration (EC90) of L-dC against HBV replication was 1 07 µM Structure activity relationships (SAR) show that replacement of the hydroxyl group in the 3' position (3'-OH) broadened the antiviral activity from hepadnaviruses to other viruses including human immunodeficiency virus (HIV) and certain herpes viruses Substitution in the base decreased antiviral potency and selectivity The second study using the woodchuck model of chronic hepatitis B virus infection tested the antiviral effect and safety of L-dC in combination with a second investigational nucleoside [ß-L-2'-deoxythymidme (L-dT)] Included m this study was a treatment group in which L-dC was used as a single agent (1 mg/kg/day) There was no drug-related toxicity observed for L-dC alone or in combination with L-dT during the 12-week treatment phase or 12-week post-treatment follow-up period There were no changes in body weight relative to control animals or serum chemistry and hematologic parameters End-of-treatment liver biopsies showed no histomorphological evidence of fatty changes (microvesicular steatosis) The combination of L-dC (1 mg/kg/day) plus L-dT (1 mg/kg/day) was synergistic and reduced viral load by up to 8 logs from baseline Antiviral nucleosides and nucleoside analogs exert their antiviral effect as intracellular triphosphate derivatives at the level of the viral polymerase during virus replication Like natural nucleosides (D-deoxycytidine and D-thymidrne) and antiviral nucleoside analogs (ε g, lamivudine and zidovudine), L-dC was activated rntracellularly by phosphorylation In human hepatocytes, deoxycytidrne kinase (dCK) was responsible for the dose-dependent initial conversion of L-dC to a 5'-monophosphate (MP) derivative L-dC-MP was then converted to a 5'-diphosphate (DP) form, which was subsequently converted to the predominant intracellular 5'-triphosphate (TP) metabolite The L-dC-TP level reached 72 4 joM in HepG2 cells exposed to 10 µM L-dC (90 1 µM in primary human hepatocytes) at 24 hours and had an intracellular half-life of 15 5 hours In endogenous polymerase assays, L-dC-TP inhibited the vinon-associated DNA polymerase of WHV with a 50% inhibitory concentration (IC50) of 1 82 µM The detailed mechanism of inhibition of HBV DNA polymerase by L-dC is under investigation Exposure of HepG2 cells or human hepacytes in pnman culture to L-dC also produced a second TP derivative, 0-L-2'-deoxyundrne 5'-tnphosphate (L-dU-TP) The L-dU-TP level reached 18 2 \|iM in HepG2 cells exposed to 10 µM L-dC (43 5 µM in primary human hepatocytes) at 24 hours In endogenous polymerase assays, L-dU-TP inhibited vmon-associated DNA polymerases of WHV with an IC50 of 5 26 uM In primary human hepatocyte cultures and in a human hepatoma cell line (HepG2), the major metabolite of L-dC was L-dC-TP Exposure of these cells to L-dC also ted to the formation of L-dU-TP In vitro pharmacological assays showed that L-dC-TP inhibited hepadnaviral DNA synthesis with an IC50 of 1 82 mM, against vinon-associated DNA polymerase L-dU-TP inhibited hepadnaviral DNA synthesis with an IC50 of 5 26 µM L-dC-TP and L-dU-TP did not inhibit human DNA polymerases α, ß and  up to concentrations of 100 µM, the highest concentration tested The ability of the active compounds to inhibit the growth of virus in 2 2 15 cell cultures (HepG2 cells transformed with hepatitis virion) can be evaluated as described in detail below A summary and description of the assay for antiviral effects in this culture system and the analysis of HBV DNA has been described (Korba and Milman, 1991, Antiviral Res , 15 217) The antiviral evaluations are performed on two separate passages of cells All wells, m all plates, are 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 5-fold (for HBV virion DNA) or 3 0-fold (for HBV DNA replication intermediates) from the average levels for these HBV DNA forms in untreated cells are considered to be statistically significant (P Typical values for extracellular HBV virion DNA m untreated cells range from 50 to 150 pg/ml culture medium (average of approximately 76 pg/ml) Intracellular HBV DNA rephcation intermediates m untreated cells range from 50 to 100 µg/pg cell DNA (average approximately 74 pg/ftg 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 (Korba and Milman, 1991, Antiviral Res, 15 217) The maimer in which the hybridization analyses are performed results in an equivalence of approximately 1 0 pg of intracellular HBV DNA to 2-3 genomic copies per cell and 1 0 pg/ml of extracellular HBV DNA to 3 x 105 viral particles/mL Examples Example 12 Solubility Study The solubility of natural deoxynbocytosme (D-dC), the 3'-vahnyl ester of L-dC and the 3',5'-divalmyl estei of L-dC in water was compared The solubility of L-dC was assessed first by analyzing the HPLC data (i e area under the curve) by successive injections of various well-known concentrations of ß-L-dC, as shown m Table 3 The HPLC was run on a Nova-Pack CI 8 column (3 9 x 150 mm) on a gradient of 0 to 25% of CH3CN in 20 mM tnethylammonium acetate buffer (TEAAc) programmed over a fifteen minute period with a flow rate of 1 mL per minute The concentration of the solution versus the area under the cun e produced a linear relationship with y=4150049477 x -4334 46845 (Figure 8a) Table 3 (Table Removed) From this, a saturated solution was prepared with natural deoxynbocytosme (D-dC), 3 samples were taken and injected mto the HPLC The concentration of this saturated solution was determined to be 1 07, 1 08 and 0 96 mol/L, therefore, the saturated solution had an average saturated concentration of 1 03 mol/L or 272 g/L The results are tabulated in Table 4 Table 4 (Table Removed) Similarly, the solubility of 3'-valmyl ester hydrochloride of β-L-dC m water was evaluated The calibration curve was determined by successive injections of various concentrations of the 3'-Valrnyl ester hydrochloride of β-L-dC into the HPLC and measuring the area under the curve, as shown in Table 5 Again, the HPLC was run on a Nova-Pack CI 8 column (3 9 x 150 mm) on a gradient of 0 to 25% of CH3CN m 20 mM tnethylammonium acetate buffer (TEAAc) programmed over a fifteen minute period with a flow rate of 1 mL per minute The concentration of the solution versus the area under the curve produced a linear relationship with y=3176423963 x -33051 63 Table 5 (Table Removed) From this, a saturated solution was attempted for 3'-valinyl ester hydrochloride of ß-L-dC, however, one was not obtained Therefore, the maximum quantity of 3 '-vahnyl ester hydrochloride of β-L-dC readily available in the laboratory was dissolved in water 3 samples were collected, and were determined from the area under the curve from the HPLC, to have an average concentration of 1 013, 0 996 and 1 059 mol/L The results are tabulated m Table 6 (Table Removed) Table 6 (Table Removed) All three resul s fell within the predicted range calculated from the calibration curve, indicating complete solubility of the compound at those high concentrations, indicating that a saturated solution o" this sample is greater than the average of the three samples, l e greater than 1 023 mo L or 408g/L The solubility of 3,5'-divalrnyl ester hydrochloride of β-L-dC in water was evaluated The call-ration curve was determined by successive injections of various concentrations of the 3',5'-divaknyl ester hydrochloride of β-L-dC into the HPLC and measuring the area under the curve, as shown in Table 7 The HPLC was run on a Nova-Pack CI 8 column (3 9 x 150 mm) on a gradient of 0 to 25% of CH3CN in 20 mM tnethylammomum acetate buffer (TEAAc) programmed over a fifteen minute period with a flow rate of 1 mL per minute The concentration of the solution versus the area under the curve produced a linear relationship with y=3176423963 x -33051 63 (Figure 8b) Table 7 (Table Removed) From this, a saturated solution was attempted for 3\5'-divalinyl ester hydrochloride of β-L-dC, however, one was not obtamed Therefore, the maximum quantity of 3',5'-divahnyl ester hydrochloride of β-L-dC readily available in the laboratory was dissolved in water 3 samples were collected, and were determined from the area under the curve from the HPLC, to have an average concentration of 2 8, 2 4 and 2 4 mol/L The results are tabulated in Table 8 (Table Removed) All three results fell within the predicted range calculated from the calibration curve, indicating complete solubility of the compound at those high concentrations, indicating that a saturated solution of this sample is greater than the average of the three samples, l e more than 2 5 mol/L or 133" g/L Similar solubility studies were done on 5'-valmyl ester hydrochloride of β-L-dC (more than 5 1 moll or 1664 g/L) and 3'5'-diacetyl ester hydrochloride of β-L-dC (3 3 mol/L or 1148 g/L) The cumulative results are tabulated in Table 9 (Table Removed) Example 13 Log P Study - Phosphate Buffer Approximately 1 5 mg of D-dC was dissolved in 2 2 mL of 0 02 M phosphate buffer solution (A, 100 mL, pH 7 2), made from a mixture of monobasic potassium phosphate solution (28 5 mL) and dibasic potassium phosphate solution (71 5 mL), saturated with octanol-1 (B) To 1 mL of this solution, 1 mL of octanol-1 (B) saturated with 0 02 M phosphate buffer solution (A) was added The resultant mixture was shaken and centnfuged, three samples from each phase was collected and analyzed by HPLC, as shown in Table 10 The HPLC was run on a Nova-Pack C18 column (39x150 mm) on a gradient of 0 to 25% of CH3CN m 20 mM tnethylammomum acetate buffer (TEAAc) programmed over a fifteen minute period with a flow rate of 1 mL per mmute It was found that the log P of D-dC is -1 41, therefore, D-dC prefers water to octanol Table 10 (Table Removed) Similarly, approximately 1 5 mg of L-dC-3'-valrne ester hydrochloride was dissolved in 2 5 mL of 0 02 M phosphate buffer solution (A, 100 mL, pH 7 2), made from a mixture of monobasic potassium phosphate solution (28 D mL) and dibasic potassium phosphate solution (71 5 mL) The solution was then saturated with octanol-1 (B) To 1 mL of this solution, 1 mL of octanol-1 (B) saturated with 0 02 M phosphate buffer solution (A) was added The resultant mixture was shaken and centnfuged, three samples from each phase was collected and analyzed by HPLC, as shown in Table 11 The HPLC was run on a Nova-Pack CI 8 column (3 9 x 150 mm) on a gradient of 0 to 25% of CH3CN in 20 mM tnethylammomum acetate buffer (TEAAc) programmed over a fifteen minute period with a flow rate of 1 mL per minute Table 11 (Table Removed) It was found that the log P of L-dC-3'-valine ester hydrochloride is -1 53, therefore, L-dC- 3'-valine ester prefers water to octanol to a greater degree than D-dC Log P values were calculated for L-dC-5'-valine ester hydrochloride and L-dC-3',5'-divalme ester hvdrochlonde The results are tabulated m Table 12 However, it should be noted that the log P value for L-dC-3',5'-divahne ester hydrochloride is probably lower than the one measured (-0 86) Significant conversion of the divahne ester into the 3'- or 5'-monovahnyl ester or even L-dC was observed during the experiment 50% of conversion of L-dC-3',5'-divaline ester hydrochloride was detected m the aqueous phase and 14% m the organic phase This conversion is due to the instability of the esters in the phosphate buffer at a pH of 7 (see examples 15 and 16) (Table Removed) Table 12 (Table Removed) Example 14 Log P' Study - MdhQ Water In order to avoid the conversion of the divahne ester into the mono esters and L-dC, an alternate log P study was performed using MilhQ water (A') instead of the phosphate buffer (pH of 6 5 instead of 7 2) It is important to note that only the hydrochloride form of the divaknyl ester can be considered m water Approximately 1 5 mg of L-dC-3',5'-divahnyl ester hydrochloride was dissolved in 2 2 mL of MilhQ water (A', pH 6 5) saturated with octanol-1 (B) To 1 mL of this solution, 1 mL of octanol-1 (B) saturated with MilhQ water (A') was added The resultant mixture was shaken and centnfuged, three samples from each phase was collected and analyzed by HPLC, as shown in Table 13 The HPLC was run on a Nova-Pack CI8 column (3 9 x 150 mm) on a gradient of 0 to 25% of CH3CN in 20 mM tnethylammonium acetate buffer (TEAAc) programmed over a fifteen minute period with a flow rate of 1 mL per minute It was found that the log P' of the 3',5'-divahne under these conditions was -2 72, indicating the strong effect of the counter ions rn the phosphate buffer No conversion of the divahne to the monoesters or L-dC was observed m either the aqueous or organic phases Table 13 (Table Removed) Similarly, approximately 15 mg of L-dC-5'-valmyl ester hydrochloride was dissolved in 2 2 mL of MilhQ water (A', pH 6 5) saturated with octanol-1 (B) To 1 mL of this solution, 1 mL of octanol-1 (B) saturated with MilhQ water (A') was added The resultant mixture was shaken and centnfuged three samples from each phase was collected and analyzed by HPLC, as shown in Table 14 The HPLC was run on a Nova-Pack CI8 column (3 9 x 150 mm) on a gradient of 0 to 25% of CH3CN m 20 mM tnethylammonium acetate buffer (TEAAc) programmed over a fifteen minute period with a flow rate of 1 mL per minute It was found that the log P of the 5'-valine under these conditions was -2 75, again a value lower than found in the log P study using the phosphate buffer Table 14 (Table Removed) Under these conditions, the log P' values for L-dC-5'-valinyl ester hydrochloride and L-dC-3',5'-divalmvl ester hydrochloride are very similar (Table 15) Table 15 (Table Removed) Example 15 Stabilitv Study at pH 7 4 The rate of decomposition of each metabolite of L-dC-3' -valine ester hydrochloride was calculated The half-life of L-dC-3'-valine ester hydrochloride at pH of 7 40 was determined to be 7 hours m a 0 2M Tns-HCl solution at 37 °C In these conditions, L-dC-3'-valine ester hydrochloride is simply transformed to L-dC No cytosine was detected, thus, there was no detectable glycoside bond breakage Similarly, the rate of decomposition of each metabolite of L-dC-3',5'-divahne ester hydrochloride was calculated The half-life of L-dC-3',5'-divahne ester hydrochloride at pH of 7 42 was determined to be 2 4 hours in a 0 2M Tns-HCl solution at 37 CC In these conditions,L-dC-3',5'-divaline ester hydrochloride is partially hydrolyzed into the 3'- and 5'-vahnyl-L-dC, which are later transformed into L-dC No cytosine was detected, thus, there was no detectable glycoside bond breakage (Scheme 4, Figures 9a and 9b) (Scheme Removed) 4 Example 16 Stability Study at pH 7 20 The half-life of I-dC-3',5'-divaline ester hydrochloride at pH of 7 20 was determined to be 2 2 hours in a 20 mM phosphate buffer In these conditions,L-dC-3',5'-divahne ester hydroch-onde is partially hydrolyzed into the 3'- and 5'-vahnyl-L-dC, which are later transformed into L-dC No cytosine was detected, thus, there was no detectable glycoside bond breakage (Scheme 5, Figures 10a and 10b) Scheme 5 (Scheme Removed) Example 17 Stability Study at pH 4 5 The half-life of L-dC-3'-valine ester hydrochloride at pH of 4 5 was determined to be 8 6 days in a 20 mM acetate buffer Again, L-dC-3'-valine ester hydrochloride is simply transformed to L-dC No cytosine was detected, thus, there was no detectable glycoside bond breakage Similarly, the half-life of L-dC-3',5'-divalme ester hydrochloride at pH of 4 51 was determined to be 44 hours m a 20 mM acetate buffer In these conditions,L-dC-3',5'-divahne ester hydrochloride is partially hydrolyzed into the 3'- and 5'-vahnyl-L-dC, which are later transformed into L-dC No cytosine was detected, thus, there was no detectable glycoside bond breakage (Figures 11a and lib) Example 18 Stability Study at pH 1 2 The half-life of L-dC-3'-valine ester hydrochloride at pH of 1 2 was determined to be greater than 48 hours m a 135 mM KC1-HC1 buffer solution No cytosine was detected, thus, there was no detectable glycoside bond breakage Similarly, stability studies were done on L-dC-5'-valine ester hydrochloride This compound is fully stable at a pH of 1 2, with no other metabolites or decomposition products detected for up to 23 hours No glycosidic bond breakage was detected up to 2 days in solution The 3',5'-diacetyl ester of L-dC was found to have a half life at a pH of 1 2 of 11 2 hours Under these conditions the compound was partially hydrolyzed into the 3'- or 5'-denvatives, which were later transformed into L-dC No glycosidic bond breakage was detected up to 2 days m solution The 3',5'-divahnyl ester of L-dC was found to be fully stable at a pH of 1 23 since no other compounds were detected up to 48 hours m these conditions No glycosidic bond breakage was detected up to 2 days m solution (Figurel2) Alternatively, when the N4 position of L-dC is masked with dimethylammo-methylene or acetyl, the half-kfe of the compound at a pH of 1 2 is only 26 minutes or 50 minutes, respectively Example 19 Single Dose Bioavailability of L-dC in the Cynomologus Monkey The pharmacokinetics of L-dC following IV and oral admrxustration of L-dC to cynomologus monkeys was determined In this study, 10 mg/kg tritium ([3H]) radiolabeled L-dC was administered to three cynomologus monkeys as a single IV dose Following a six week washout period, the same three monkeys received an identical oral dose of L-dC Blood samples for pharmacokinetic analysis were collected pre-dose and at 0 25, 0 5, 1, 2, 3, 6, 8 and 24 hours after dosing Unne samples for pharmacokinetic analyses were collected via pan catch pre-dose and over the following intervals post-dose 0-2, 2-4, 4-8, and 8-12 hours, and then over 12-hour intervals thereafter through 336 hours post-dose The drug was detected and the concentration determined using a reverse-phase high-performance liquid chromatography technique The blood and unne drug level data were analyzed by a non-modeling mathematical method and AUC's derived by the linear trapezoidal rule Intravenous administration of L-dC The mean Cmax of L-dC after IV administration was 95 7 µM and occurred at the earliest sampling time (15 minutes post-dose) for all animals I-dC plasma concentrations decreased over time following the IV bolus with a mean t½ of 1 59 hours The total clearance (CL) and renal clearance (CLR) of L-dC following IV administration averaged 0 53 L/h/kg and 0 46 L/h/kg, respectively The mean apparent volume of distribution (Vd) of 1 22 L/kg indicated that L-dC had a significant extravascular tissue distribution Urinary excretion was rapid, with 71% of the administered dose recovered within 2 hours L-dC accounted for the majority (94%) of the dose recovered in the unne The renal clearance (0 46 L/h/kg) accounted for 87% of total L-dC clearance and suggested that renal excretion was the major route of elimination L-dU was detected m the plasma and unne, indicating that metabolic elimination of L-dC also occurred following IV administration Low levels of L-dU were detected m plasma at the limit of detection (lower limit of detection (LLOD) = 0 1 µM) Renal excretion of L-dU was 4 0% of the total dose recovered m unne With the exception of L-dU, no other metabolites were detected in the plasma or unne Oral administration of L-dC The Cmax was 3 38 µM and occurred at a Tmax of 2 33 hours The plasma concentration of L-dC declined m a biphasic manner with a mean terminal t½ of 2 95 hours and was below detection limits by 24 hours in all monkeys L-dC was absorbed from the gastrointestinal tract with a mean oral bioavailability (F) of 16 4% L-dU was detected m the plasma and unne, which suggested that metabolic elimination of L-dC occurred following oral admmistration Low levels of L-dU were detected in plasma at the LLOD With the exception of L-dU, no other metabolites were detected in the plasma or unne Approximately 8 5% of the administered oral dose was recovered in the unne within 12 hours After 72 hours 15 5% ± 8% was recovered L-dC accounted for the majonty (-69%) of drug excreted in the unne Renal excretion of L-dU was 29% of the total recovered dose Feces were not collected Table 16 presents a summary of pharmacokinetic results for IV and oral administration of L-dC m cynomologus monkeys Table 16 Pharmacokinetic Analysis after Intravenous and Oral Administration of L-dC (10 mg/kg) in the Cynomologus Monkey (Table Removed) Mean value (±SD) Example 20 Single-Dose Bioavailability of L-dC in the Rhesus Monkey The pharmacokinetics of L-dC following oral administration m the rhesus monkey was determined In this study, 10 mg/kg [3H] radiolabeled L-dC was administered to three rhesus monkeys as a single oral dose Blood samples for pharmacokinetic analysis were collected pre-dose and at 0 25, 0 5, 1, 2, 3, 6, 8 and 24 hours after dosing Urine samples for pharmacokinetic analyses were collected via pan catch pre-dose and over the following intervals post-dose 0-2, 2-4, 4-8 and 8-12 hours, and then at 12-hour intervals thereafter through 336 hours post-dose The drug was detected and concentration determined using a reverse-phase HPLC technique The blood and unne drug level data were analyzed by a non-modeling mathematical method and AUCs derived by the linear trapezoidal rule The average AUC025-8 and Cmax values were 12 2 mgMh and 3 23 mgM, respectively The Cmax occurred at a Tmax of 0 83 hours The mean t½ was 3 34 hours and the L-dC plasma concentration was below detection levels by 24 hours in all monkeys The mean renal clearance of L-dC was 0 273 L/h/kg No metabolites were observed in the plasma of monkeys receivmg L-dC Approximately 8 5% of the administered oral dose (oral bioavailability of L-dC -16%) was recovered in the unne within 8 hours After 48 hours 15% was recovered L-dC accounted for the majority (-77%) of drug excreted m the unne Renal excretion of L- dU was 23% of the total recovered dose With the exception of L-dU, no other metabolites v, ere detected The AUC and Cmax for L-dC after oral administration to rhesus monkeys were similar to that observed in cynomologus monkeys Example 21 Single-Dose Bioavailability of L-dC in the Rat The pharmacokinetics and bioavailability of L-dC m rats was determined In this study, 10 mg/kg [3H] radiolabeled L-dC was administered to three female Sprague-Dawley rats as a single IV dose A second group of three animals received an identical oral dose of L-dC Blood samples for pharmacokinetic analyses were collected at 0 17, 0 33, 0 5,1, 2, 3, 4, 6, 8 and 24 hours after dosing Urine was also collected at 8 and 24 hours after dosmg The drug was detected and the concentration determined m plasma and urine using a reverse-phase HPLC technique The data were analyzed by a non-modeling mathematical method and the AUCs derived by the linear trapezoidal rule Intravenous administration of L-dC The average AUC0 25- g value was 30 1 mM h The Cmax of L-dC was 911 mgM and occurred at the earliest sampling tune (10 minutes post-dose) for all animals L-dC plasma concentrations declmed m a brphasic manner following the TV bolus with a mean t½ of 1 21 hours The CL of L-dC averaged 1 44 L/h/kg The mean Vd of 2 53 L/kg indicated that L-dC had a significant extravascular tissue distribution. No metabolites were observed m the plasma of rats receiving L-dC L-dC accounted for the majority of radioactivity recovered in the urine L-dU was detected m the urine, which suggested that metabohc elimination of L-dC occurred following IV administration Oral administration of L-dC The average AUC0.25- 8 value was 4 77 mM h The mean Cmax was 1 50 mgM and occurred at a Tmax of 1 0 hour The plasma concentration of L-dC declined with a t½ of 2 52 hours L-dC had limited uptake from the gastrointestinal tract with a mean oral bioavailability (F) of 15 4% No metabohtes were observed m the plasma of rats following oral administration of L-dC L-dC accounted for the majority of radioactivity recovered in the urine L-dU was detected m the plasma and urine, which suggested that metabolic elimination of L-dC occurs following oral administration Table 17 presents a summary of pharmacokinetic results for both IV and oral L-dC Table 17 Pharmacokinetic Analysis after Intravenous and Oral Administration of L-dC (10 mg/kg) in the Rat (Table Removed) Example 22 Single-Dose Bioavailability of L-dC m the Woodchuck The pharmacokinetics and bioavailability of L-dC m woodchucks was determined In this study, 10 mg/kg [3H] radiolabeled L-dC was administered to three woodchucks as a single IV dose Blood samples for pharmacokinetic analyses were collected at 2, 5,15, and 30 minutes and 1 0, 1 5, 2 0, 3 0, 4 0, and 24 hours post-dose After a seven-day washout period, the same animals received 10 mg/kg L-dC as a single oral dose Blood samples for pharmacokinetic analvses were collected at 15 and 30 minutes and 10, 1 5, 2 0, 3 0, 4 0, 8 0, and 24 hours post-dose Unne was collected over the 24-hour post-dose period Plasma drug levels, CL, t½ and F were determined Drug levels were determined using an HPLC method with in-line radioactivity detection and scintillation counting Intravenous admimstration of L-dC The mean Cmax of L-dC was 112 µM. and occurred at the earliest sampling time (2 minutes post-dose) for all animals L-dC plasma concentrations decknedin a biphasic manner following the IV bolus with a mean t½ of 2 85 hours The CL of L-dC averaged 0 39 L/h/kg The mean Vd was 1 17 L/kg L-dC accounted for the majority of radioactivity recovered in the urine L-dU was detected in the plasma and urine, indicating that metabolic elimination of L-dC occurred following IV administration The levels of L-dU detected intermittently m plasma were at or below the limit of assay quantitation with a mean Cmax of 0 75 µM Oral administration of L-dC The Cmax was 1 37 µM and occurred at a Tmax of 3 hours L-dC plasma concentrations decreased with a mean t½ of 5 22 hours L-dC was absorbed from the gastrointestinal tract with an oral bioavailability ranging from 5 60 to 16 9% with an average of 9 57% L-dC accounted for the majority of radioactivity recovered m the unne L-dU was detected m the plasma and urine, indicating that metabolic elimination of L-dC occurred following oral administration L-dU m the plasma was near the limit of quantitation with a mean Cmax of 0 19 µM Table 18 presents a summary of pharmacokinetic results for both IV and oral L-dC Table 18 Pharmacokinetic Analysis of L-dC (10 mg/kg) after Intravenous and Oral Administration in the Woodchuck (Table Removed) Example 23 Bioavailability of the Prodrugs of L-dC The bioavailability of L-dC, the 5'-monoester of L-dC, the divahne ester of L-dC, and the diacetyl ester of L-dC was evaluated m cynamologous monkeys, with and without L-dT When the divaline ester of L-dC was orally administered to monkeys, approximately 73% of the dose was absorbed Of the absorbed divahne ester of L-dC, more than 99% was rapidly converted to L-dC to give a high concentration of L-dC m the plasma and no detectable divahne ester of L-dC A low plasma concentration of the monovahne ester of L-dC was detected early after oral administration of divaline ester of L-dC A low plasma concentration of ß-L-2'-deoxyundrne (L-dU) was detected intermittently No other metabolites were detected. The results are provided in Table 19 As indicated, the combination of the 3',5'-divalyl ester of L-dC with L-dT provided the largest bioavailability ofL-dC Table 19 (Table Removed) Example 24 Single Dose Bioavailability of Dival-L-dC in Cynomologus Monkey Three make non-naive cynomologus monkeys (macaca fasciculans) received 10 mg/kg of dival-L-dC intravenously with a tracer amount of tritium ([3H] ) labeled drub (250 uCi) dissolved in sterile 9 0% saline Following a 6 week washout period, the same three animals received an identical oral dose of dival-L-dC Blood samples were collected m hepanmzed tubes at pre-dose (-18 hours) and 0 25, 0 50, 1, 2, 3, 4, 6, 8, and 24 hours after dosing Urine was also collected from 0-2, 2-4, 4-8, 8-12 and then at 12-hour intervals until 336 hours post-dose The drug was quantitated in plasma and urine with a liquid chromatography-mass spectrometry (LC-MS) technique After administration of dival-L-dC, the plasma concentration time course of L-dC was analyzed by a non-modeling mathematical method and the area under the time-concentration curves (AUC) derived by the linear trapezoidal rule The bioavailability (F) of L-dC following TV and PO administration of dival-L-dC was calculated from the L-dC AUCs, where F = AUCpo/AUCiv x doseiv/dosepo Intravenously administered dival-L-dC was converted rapidly to L-dC following intravenous administration Dival-L-dC was detected m the plasma at 15 minutes (1 39 uM) and at 30 minutes (0 36 µM, 1 of 3 animals) [lower limit of quantitation (LLOQ) = 0 23 µM or 100 ng/mL] Dival-L-dC was not detected in the plasma after 30 minutes post-dosing The partially de-estenfied form of dival-L-dC, ß-L-2'-deoxycytidine-5'-valine ester, was detected in plasma at 15 minutes (3 23 µM) and decreased in concentration to 0 08 µM by 2 hours (LLOQ = 0 031 µM or 10 ng/mL) L-dC represented the majority of drug present in the plasma following intravenous administration The average AUCo 25→8 value for L-dC was 19 8 µM h The mean peak plasma concentration (Cmax) of L-dC was 24 6 µM (LLOQ = 0 088 µM or 20 ng/mL) and occurred at the earliest sampling time (15 minutes post-dose) in all animals The plasma concentration of L-dC declined m a biphasic manner with a mean t½ of 1 73 hours The total body clearance (CL) and apparent volume of distribution (Vd) of L-dC averaged 1 01 L/h/kg and 2 46 17kg, respectively, indicating that L-dC had significant extravascular tissue distribution The binding of dival-L-dC and L-dC to human plasma protems ex vivo was 13 3% ± 2 6% and 19 7% ± 5 9%, respectively The impact of human plasma protein binding on dival-L-dC and L-dC free-drug levels was minimal, suggesting that drug interactions involving binding site displacement are not anticipated Urinary excretion was rapid with 58 ± 3% of the administered dose of dival-L-dC excreted within 2 hours following intravenous administration L-dC accounted for the majority (-93%) of drug excreted in the urine L-dU was also detected in the plasm? and urme This suggested that metabolic elimination of L-dC also occurs following administration of dival-L-dC Low levels of L-dU were detected in plasma at intermittent time points in two of three animals at concentrations ranging from 0 22 µM to 0 88 µM (LLOQ = 0 22 jiM or 50 ng/mL) There were no detectable levels of L-dU at any time point m the third monkey Renal excretion of L-dU and the partially de-esterfied form of dival-L-dC, j6-L-2'-deoxycvtidine-5'-valine ester was minor, accounting for approximately 2 5% and 3 7% of the total recovered dose, respectively Dival-L-dC was detected m the urme of one of three animals at 2 hours following IV administration, which accounted for approximately 0 15% of the recovered dose Because of the intermittent low concentrations of both the monovaline esters and L-dU in the plasma and urine, it was not feasible to perform pharmacokinetic analysis of these metabolites The appearance of the monovaline ester of dival-L-dC was not unexpected as it represents and intermediate in the conversion of dival-L-dC to L-dC In addition, in vitro cellular metabolism studies m monkey, rat and human primary hepatocytes and m extracts of HepG2 cells demonstrated that L-dC was not directly deamrnated to L-dU but that L-dC monophosphate (-MP) is converted to L-dU-MP, which is either activated to L-dU disphosphate (-DP), and triphosphate (-TP), or metabolized to L-dU, which is then detected m the extracellular compartment (plasma) L-dU was non-cytotoxic (CC50 > 200 uM) and L-dU-TP had an IC50 in vitro against hepatitis B virus deoxyribonucleic acid (DNA) polymerase of 5 26 µM (see Microbiology and Virology, Section 10) Orally admimstered dival-L-dC also was converted rapidly to L-dC following oral administration and was not detectable in plasma samples at any time point (LLOQ of dival-L-dC in solution = 0 23 µM or 100 ng/mL) The partially de-estenfied metabolite of dival-L-dC, j3-L-2'-deoxycytidine-5'-valine ester, was detected in plasma at 30 mmutes and 1 hour at concentrations ranging from 0 034 /? to 0 107 /3 (LLOQ of monoester in solution = 0 031 \|iM or 10 ng/mL) Dival-L-dC was not detected m the plasma L-dC represented the majority (>99% at Cmax) of the plasma drug levels following oral administration of dival-L-dC The average AUCo 25- 8 value for L-dC was 14 0 µM h The Cmax of L-dC was 8 26 µM (LLOQ of L-dC m solution = 0 088 µM or 20 ng/mL) and occurred at 0 67 hours following administration of dival-L-dC The plasma concentration of L-dC declined m a biphasic manner with a mean t½ of 2 28 hours The mean oral bioavailability of L-dC following administration of dival-L-dC was 72 7% ± 22% L-dU was also detected m the plasma indicating the metabolic elimination of L-dC occurs following oral administration of dival-L-dC Low levels of L-dU were detectable in the plasma from 30 minutes to 4 hours m two of three animals of concentrations ranging from 0 24 µM to 0 66 µM (LLOQ of L-dU in solution = 0 22 µM or 50 ng/mL) and m one animal only at 8 hours at a concentration of 0 39 µM After oral administration, dival-L-dC was rapidly absorbed from the gastrointestinal tract and converted to L-dC by first-pass mtestmal and/or hepatic metabolism Neither dival-L-dC nor L-dC metabolism was associated with liver microsomal enzymes Following administration of high dose levels of dival-L-dC, the monovahne ester of L-dC as transiently detected prior to conversion to L-dC No dival-L-dC was detected after oral administration Intermittent low plasma levels of L-dU were detected at, or below, the lower limit of assay quantitation L-dU was formed by dearmnation of L-dC following cellular uptake of l-dC Approximately31 ± 8% of the administered oral dose was recovered in the urine within 4 hours After 72 hours 39 ± 8% was recovered L-dC accounted for the majority (~95%) of drug excreted in the urine Renal excretion of L-dU and the partially de-estenfied form of dival-L-dC, β-L--2'-deoxycytidine-5'-valine ester was minor, accounting for approximately 2 5% and 0 2% of the total recovered dose, respectively No dival-L-dC was detected in the urine Table 20 represents a summary of pharmacokinetic results for L-dC following both IV and oral dosmg of dival-L-dC Table 20 Pharmacokinetic Analysis after Intravenous and Oral Administration of Dival-L-dC (10 mg/kg) m Cynomologus Monkeys Pharmacokinetic Parameter2 (Table Removed) Table 21 presents a schematic of metabolite formation form dival-L-dC, the monovahne derivative of L-dC, L-dC and L-dU following IV and oral administration of dival-L-dC The Cmax of each metabolite is also noted Table 21 Metabolite Formation for IV and PO Administration of Dival-L-dC Intravenous (10 mg/kg Dival-L-dQ (Table Removed) Example 25 Oral Bioavailability of L-dC via Dival-L-dC in Cynomologus Monkey Three male non-naive cynomologus monkeys (macaca fasciculans) received 10 mg/kg of dival-L-dC orally with a tracer amount of [3H]-labeled drug (250 µCi) dissolved m sterile 0 9% saline Blood samples were collected in hepannized tubes at pre-dose (~18 hours) and 0 25, 0 50,1, 2, 3, 4, 6, 8 and 24 hours after dosmg Urine was collected from 0-2, 2-4, 4-8, 8-12 and then at 12-hour intervals until 336 hours post-dose The drug was quantitated in plasma and in urme using HPLC analysis After administration of dival-L-dC the plasma concentration time course of L-dC was analyzed by a non-modeling mathematical method and the area under the time-concentration curves (AUC) derived by the linear trapezoidal rule Dival-L-dC was rapidly absorbed and converted to L-dC following oral administration. Radiochromatographic high pressure liquid chromatography (HPLC) analysis of plasma samples confirmed that the majority of the recovered radioactivity was L-dC Dival-L-dC was detected in only one animal at 15 minutes post-dose at a concentration of 0 35 µM The partially de-estenfied form of dival-L-dC, ß -L-2'-deoxycytidine-5 '-valine ester, was not detected m the plasma or urine Approximately 26% of the administered oral dose was recovered m the urme within 8 hours After 72 hours 31 % was recovered L-dC accounted for the majonty (~89%) of drug excreted m the urme Renal excretion of L-dU was minor, accounting for approximately 10% of the recovered dose No dival-L-dC or its partially de-estenfied form, and no other metabolites were detected in the urme The overall pharmacokinetic profile was comparable to that determined m the pharmacokinetic stud\ as demonstrated by similar Cmax to AUC ratios Low levels of L-dU were detected in the plasma in two of three animals with an average Cmax of 0 33 µM No L-dU was detected in the plasma of the third animal The level of L-dU was at or below the limit of quantitation rrecluding pharmacokinetic analysis Example 26 In vitro metabolism of Dival-L-dC Studies were conducted to determine the stability and protein binding of dival-L-dC and its de-estenfied metabolites m human plasma. Dival-L-dC was mcubated in human plasma at 37°C and samples analyzed at various tune points up to 24 hours (Figure 13) No dival-L-dC was detectable at 24 horns with complete conversion to L-dC Two additional metabolites (β-L-2'-deoxycytidme-5'-vahne ester and β-L-2'-deoxycytidine-valine ester) were also noted The transient nature of these metabolites indicated that they are intermediates in the conversion of dival-L-dC to L-dC The in vitro half-life of dival-L-dC in human plasma at 37°C was determined to be approximately 39 mm The impact of human plasma protein binding on free levels of dival-L-dC and L-dC was also investigated using an ultrafiltration method Plasma protem binding of dival-L-dC was 13 3% i 2 6% The binding of L-dC to plasma proteins was 19 7% ±5 9% This study shows that the impact of human plasma protem binding on dival-L-dC and L-dC is minimal and suggests that drug interactions involving binding site displacement are not anticipated Example 27 Metabolic Activation and Intracellular Profile of L-dC The cellular metabolism of L-dC was examined using HepG2 cells and human primary hepatocytes High pressure liquid chromatography (HPLC) analysis demonstrated that L-dC was extensively phosphorylated m hepatocytes The predominant metabolite m HepG2 cells exposed to 10 µM L-dC for 24 hours was L-dC-TP which reached 72 4 ± 1 8 µM (see Table 23) In primary human hepatocytes, the L-dC-TP concentration at 24 hours was 90 1 ± 37 uM, similar to the level of phosphorylation in HepG2 cells Exposure of hepatocytes to L-dC led to activation of a second 5'-triphosphate derivative, L-dU-TP In HepG2 cells exposed to 10 µM L-dC, the L-dU-TP level reached 18 2 µM (43 5 pM in primary human hepatocytes) at 24 hours In primary rat and monkey hepatocytes the extent of phosphorylation of L-dC was slightly lower Table 23 Activation of L-dC(10 µM) in Hepatocytes Metabolite (10 /iM) (Table Removed) (a) Cells were incubated for 24 hours with [3H]-L-dC, specific activity HepG2 assay = 0 5 Ci/mmol, human, monkey and rat hepatocyte assay = 1 0 Ci/mmol In addition to the phosphorylated derivatives of L-dC and L-dU, formation of a [ß-L-2'-deoxyhponucleotide metabolite was noted In HepG2 cells and primary hepatocyte cultures exposed to 10 µM L-dC for 24 hours, [3-L-2'-deoxycytidme-5l-diphosphochohne (L-dC-Dβ-chohne) was detected at a concentration of 25 6 µM (range 25 6 - 25 7 µM) and 12 3 µM (range 8 82 -15 8 µM), respectively The metabolic profile obtained after a 24-hour exposure of HepG2 cells to 10 µM [3H]-L-dC is shown in Figure 14 The apparent intracellular half-life of the L-dC-TP was 15 5 ± 0 34 hours, which correlated with prolonged antiviral activity following drug withdrawal in the virus rebound experiments The phosphorylation pattern detected in primary human hepatocytes was qualitatively and quantitatively similar to that obtained using HepG2 cells (Figure 15) Example 28 Cellular Kinases Associated with Metabolic Activation D-Deoxycytidine (dCyd) is a natural substrate of cytosohc dCyd kinase (dCK) and mitochondrial thymidine kinase (TK2) for conversion to dCyd-5'-monophosphatc (dCMP) Cytosohc thymidine kinase (TK1) and TK2 utilize D-thymidine (Thd) as a natural substrate for conversion to Thd 5'-monophosphate (TMP) The cellular kinase involved m the initial phosphorylation of L-dC was identified m competition studies using L-dC and the natural endogenous Thd and oCyd Intracellular phosphorylation of L-dC was decreased m a dose-dependent fashion b\ dCyd but not by Thd Thus, dCyd acted as an inhibitor of L-dC phosphorylation The change in intracellular phosphorylation of L-dC was similar when HcpG2 cells were exposed to both Thd and dCyd or dCyd alone The inhibition of L-dC phosphorylation by only the natural deoxypynmidine, dCyd, suggested that dCK was involved in I-dC phosphorylation The role of these pynmiduie nucleoside kinase activities in the phosphorylation of L-dC was further investigated in kinase deficient cell lines There was a significant decrease in the mount of phosphorylated metabolites of L-dC m dCK deficient cells However, no significant difference was observed m L-dC phosphorylation m TK1 deficient cells These data were consistent with the competition studies described above and indicated that dCK plays a critical role m the phosphorylation of L-dC to L-dC-MP Usmg cytosohc extracts of HepG2 cells as an enzyme source, steady state kinetics for L-dC, Thd, and dCyd phosphorylation were similar as indicated by the apparent Michaehs-Menten constant (Km) and maximum initial velocity (Vmax) values (L-dC Km of 5 75 mM and Vmax, of 1 12 mmol/min/mg protein, Thd Km of 4 06 mM and Vmax of 1 26 nmol/min/mg protein, dCyd Km of 4 85 mM and Vmax of 2 15 nmol/min/mg protein) In addition, the efficiency of I^dC, Thd, and dCyd phosphorylation were similar as defined by their corresponding Vmax/Km in values (0 19, 0 31, and 0 44, respectively) In addition, the extent of intracellular phosphorylation of L-dC was compared to that of the natural endogenous substrates, Thd and dCyd in woodchuck liver extracts This was done to support antiviral testing in the woodchuck model of chrome hepatitis B virus infection Phosphorylation of L-dC was similar to that of the endogenous substrates Furthermore, the level of phosphorylation of L-dC was comparable to that of L-dC and that of the endogenous substrates m human hver extracts Example 29 Antiviral Activity Against Hepadnavirus of L-dC The antiviral activity of L-dC against human hepatitis B virus was measured by the reduction in extracellular HBV DNA and rephcative intermediates compared to untreated control cells in the HBV-expressmg hepatoma cell line 2 2 15 (see Table 24) Confirmatory testing of the antiviral activity of L-dC using a panel of ribonucleic acid (RNA) and DNA viruses was performed bv the NIH Antiviral Research and Antimicrobial Chemistry Program L-dC did not inhibit replication of any virus other than hepadnaviruses (HBV, DHBV) L-dC had potent antiviral activity agamst HBV replication in vitro, reducing extracellular HBV DNA production with an EC50 of 0 24 µM (EC90 1 06 iiM) L-dC also reduced intracellular HBV DNA replicative intermediates (RI) with an EC50 of 0 5 µM Furthermore, L-dC produced a dose-dependent inhibition of duck hepatitis B virus (DHBV) DNA synthesis in primary duck hepatocyte (PDH) cultures with an EC50 of 0 87 µM Table 24 In vitro Antiviral Activity, Selectivity and Cytotoxicity of L-dC (Table Removed) a. PDH, pnman. duck hepatocytes, PBMC, peripheral blood mononuclear cells, HFF, human foreskin fibroblast, Daudi, Burkitt's B-cell lymphoma, MDCK, canine kidney epitheual cells, CV-I, African green monkey kidney fibroblast cells, KB, human nasopharyngeal carcinoma, MA-i 04, Rhesus monkey kidney epithelial cells b EC50 = 50% effective concentration c CC50 =50% cytotoxic concentration d nd = not determined e Result presented in µg/mL rather than µM No cytotoxicity was detected at the maximum concentrations of L-dC tested m any of the cell lmes or primary cell types used to support replication of the various DNA and RNA viruses No toxicity was seen m human PBMCs, HFF, or other cell types of mammalian origin Example 30 Antiviral Activity of L-dC in Woodchucks - 28 Days Woodchucks chronically infected with WHV are widely accepted as a model of HBV infection and ha\e proven to be useful m the evaluation of anti-HBV agents It has been proven to be a positive predictor of antiviral activity of therapies for chrome HBV infection and has served as a sensitive system for evaluation of the safety of nucleosides and their analogs L-dC was given orally to woodchucks once daily at 0 01 to 10 mg/kg/day for 28 days The serum levels of WHV DNA during 28 days of drug treatment and 56 days of post-treatment follow-up were determined by DNA dot-blot hybridization (detection limit of approximately 107 genome equivalents (geq)/mL serum) and by quantitative PCR (detection limit of 300 geq/mL serum)(l) WHV DNA replication was significantly inhibited within the first few days of treatment and was maintained throughout the treatment phase Once a day oral delivery of L-dC produced a strong antiviral effect, which was dose-dependent as determined using the DNA dot-blot hybridization assay (Figure 16) Figure 17 presents the antiviral activity of L-dC for individual animals treated with 10 mg/kg/day for 28 days m the woodchuck model of chrome hepatitis B infection Notably, in the L-dC 10 mg/kg/day treatment group, by day 14 to 28, viral load had dropped by 2-6 logs from baseline as measured by quantitative PCR assay Following drag withdrawal, viral reboind reached near pre-treatment levels between Weeks 1 and 2 In the lamivucine treated group (10 mg/kg/day, orally), the HBV viral load decreased by approximately 0 5 log to 1 0 log (geq/mL, data not shown) which is consistent with previous studies using similar concentrations of lamivudine, which is a cytidine nucleoside analog (30) xample 31 Viral Rebound in L-dC Treated Cells Viral rebound in L-dC treated 2 2 15 cells occurred after drug withdrawal HBV replication returned to 50% of pretreatment levels by day 18 post-treatment The kinetics of viral rebound after L-dC treatment suggested that a significant antiviral effect continued after drug withdrawal, which was consistent with the intracellular half-hfe of L-dC-TP (15 5 hours in HepG2 cells) Example 32 Antiviral Activity Against Drug-Resistant HBV of L-dC In controlled cluneal studies of lamivudine (100 mg once daily), administered to HBV-mfected patients, the prevalence of YMDD-mutant HBV was 14 to 32% after one year of treatment and as much as 58% after two to three years of treatment (18-20) Mutant virus was associated with evidence of diminished treatment response relative to lamivudine-treated patients without YMDD mutations Genotypic analysis of viral isolates obtained from patients who showed evidence of renewed HBV replication while receiving lamivudine suggests that a reduction in HBV sensitivity to lamivudine is associated with mutations resulting m a methionine to valine or isoleucine substitution m the YMDD motif of the catalytic domain of HBV polymerase (position 552) and a leucine to methionine substitution at position 528 HBV recombinants containing the YMDD mutation are lamivudine-resistant and slightly less replication-competent than wild-type HBV in vitro (21) The triphosphate derivative of L-dC will be tested against wild type and mutant HBV DNA polymerase to compare IC50 values In addition, antiviral testing of L-dC against lamivudine-resistant HBV isolates and recombinant viruses with mutations at positions 552 and 528 will be performed In addition, selection of L-dC drug-resistant HBV mutants in vivo during chrome treatment of WHV-infected woodchucks is also being considered The relevance of selection of drug-resistant mutants in the woodchuck m vivo model is uncertain because the spectrum of lamivudine-resistant mutants in the woodchuck does not match that identified in HBV-mfected patients (20-22) A subset of this long-term study (12 to 24 months) could provide information relevant to treatment-related elimination of HBV covalently closed circular (ccc) DNA from infected hepatocytes At the present time, it is not possible to use the DHBV in vitro model to select drug-resistant mutations because the primary duck hepatocytes used in this model cannot be sustained in cell culture for the extended periods required to select drug-resistant virus Example 33 Combination Antiviral Activity and Cytotoxicity of L-dT + L-dC The anti-HBV activity and cytotoxicity of a combination of L-dT and L-dC at near equimolar ratios were tested in 2 2 15 cells and found to by synergistic at ratios of 1 1, 1 3, and 3 1 (see Table 25) Table 25 Combination Antiviral Activity of L-dT + L-dC in HBV Infected 2 215 Cells (Table Removed) a CC50 = drug concentration at which a 50% inhibition of neutral red dye uptake (as compared to untreated cultures) was observed b EC90 = drug concentration at which a 10-fold reduction of HBV virion DNA levels (as compared to untreated cultures) was observed c EC90 values are used for calculation of the Selectivity Index (SI) since reductions of HBV DNA levels that are less than three-fold are generally not statistically significant in this assay system d Analysis of the effectiveness of the drug combination treatments by the CalcuSyn combination evaluation program (Biosoft, Inc) Example 34 Human Bone Marrow Progenitor Cells Toxicity Assay for L-dC The myelosuppressive effects of certain nucleoside analogs have highlighted the need to test for potential effects on the growth of human bone marrow progenitor cells in clonogenic assays In particular, anemia and neutropenia are the most common drug-related clinical toxicities associated with the ann-HIV drug zidovudine (ZDV) This toxicity has been modeled m an in vitro assay that employs bone marrow cells obtained from healthy volunteers (Sommadossi J-P, Carlisle R. 'Toxicity of 3'-azido-3'-deoxythynudine and 9-(l,3-dihydroxy-2-propoxymethyl)guanme for normal human hematopoietic progenitor cells m vitro" Antmncrob Agents Chemother 1987, 31(3), 452-454) ZDV has been shown to directly inhibit human granylocyte-marcrophage colony-forming (CFU-GM) and erythroid burst-forming (BFU-E) activity at clinically relevant concentrations of 1-2 µM Using human bone marrow clonogenic assays with ZDV as a positive control and larmvudme as a negative control, L-dC had an IC50 m CFU-GM and BFU-E of >10 µM (see Table 26) Table 26 Bone Marrow Toxicity of L-dC in Granulocyte Macrophage Progenitor and Erythrocyte Precursor Cells (Table Removed) a Values represent the results of three independent experiments performed rn triplicate Example 35 Mitochondrial Toxicity Assay for L-dC Antiviral nucleoside analogs approved for HIV therapy such as ZDV, stavudine (d4T), didanosine (ddl), and zalcitabine (ddC) have also been associated with clinically limiting delayed toxicities such as penpheral neuropathy, myopathy, and pancreatitis (8-11) These clinical adverse events have been attributed to inhibition of mitochondrial function due to reduction in mitochondrial DNA (mtDNA) content and nucleoside analog incorporation into mtDNA In addition, a particular nucleoside analog, fialundme (FIAU) caused hepatic failure, pancreatitis, neuropathy, myopathy and lactic acidosis due to direct mitochondrial toxicity Drug-associated increases m lactic acid production can be considered a marker of impaired mitochondrial function or oxidative phosphorylation To assess the potential of L-dC to produce mitochondrial toxicity, several in vitro studies were conducted using the human heparoma cell line HepG2 These studies mcluded analysis of lactic acid production, mtDNA content and determination of changes m morphology (εg, loss of cnstae, matrix dissolution and swelling, and lipid droplet formation) of mitochondrial ultrastructure The effects of L-dC on mitochondna are presented m Table 27 No differences were observed in lactic acid levels produced m cells chronically treated with L-dC and m untreated cells Lactic acid production m the ZDV and FIAU treated cells increased by 100% compared to vehicle control Exposure of HepG2 cells for 14 days to L-dC at concentrations up to 10 µM had no effect on mitochondna! DNA content compared to an 87% reduction in the ddC-treated cells Following 14 days of exposure to 10 µM L-dC, the ultrastructure of HepG2 cells, and m particular mitochondria, were examined by transmission electron microscopy No discernible changes m cell architecture or mitochondrial morphology were detected The size and organization of mitochondrial cnstae were normal ZDV-treated cells showed typical swollen mitochondna with loss of cnstae Mitochondna] morphology also was abnormal in the ddC- and FIAU-treated cells Table 27 Effect of L-dC on Hepatocyte Proliferation, Mitochondrial Function, and Morphology in HepG2 Cells (Table Removed) CC50 after 14 days of treatment Example 36 Human DNA Polymerases α, ß and  Toxicity Assay for L-dC Nucleosides and nucleoside analogs are usually metabolized within cells to their TP derivatives Cellular DNA polymerases are routinely responsible for normal nuclear and mitochondrial DNA synthesis and repair Because the TP metabolites are potential substrates for DNA polymerases, studies were undertaken to determine if L-dC-TP inhibited human DNA polymerases The nucleoside analog 3'-ammo-3'-deoxythymidme (AMT) TP inhibited human DNA polymerase a by 30% at a concentration of 10 µM Human DNA polymerases ß and were inhibited by ddC-TP by 50% (5 µM) and 35% (2 5 µM), respectively L-dC-TP and L-dU-TP were not inhibitory to human DNA polymerases α, ß and  up to concentrations of 100 jµM (Table 28) These results suggest that the TP of L-dC and L-dU have a low affinity for these nuclear and mitochondrial human DNA polymerases, which is consistent with the favorable safety profile of L-dC observed in vitro and m vivo Table 28 Effect of L-dC-TP on Hepatitis Virus DNA Polymerase and Human DNA Polymerases α, ß and  (Table Removed) a Each set of data represents the arithmetic mean value and, where presented, the standard deviation of three independent experiments b WHV DNA polymerase c 3'-Amino-3'-deoxvthyrmdme TP inhibited pol α30% at 10 µM, ddC-TP inhibited pol ß 50% at 5 mM and pol 7 35% at 3 5 µM d Human DNA polymerase data for lamivudrne-TP and L-EMAU-TP from Chang, et al (13), and Yao, et al (14), respectively Example 37 Toxicity Assay in Rats for Dival-L-dC The toxicity associated with a single oral dose of dival-L-dC in rats was determined A total of 40 animals (Sprague-Dawley rats, six to eight weeks of age) were studied, ten animals each (five males and five females) were randomized to receive a single oral dose of dival-L-dC at one of three doses selected from the dose-range finding portion of the study (500, 1000, or 2000 mg/kg) or control article Animals were observed for 15 days Cage side observations for monbundity and mortality were documented twice daily Clinical observations and body weight were documented once daily on Days 1, 8, 14, and 15 Also on Day 15 blood samples for hematology and serum chemistries were collected After completion of Day 15 evaluations, all animals were euthanized and subjected to a comprehensive gross necropsy, which included macroscopic examination of the external body surface, all orifices, and the cranial, thoracic, and abdominal cavities and then-contents Body and selected organ weight and organ-to-body and organ-to-brain weight ratios also were documented No overt signs of toxicity were observed during the study, and no treatment-related effects on body weight, organ weight, or clinical pathology parameters were seen No treatment-related abnormalities were noted in hematology or serum chemistry profiles Furthermore, there were no treatment-related macroscopic lesions observed at necropsy Based on the results of this study, the NOAEL for dival-L-dC following a single oral dose in the rat was 2000 n g kg Example 38 Toxicity Assay in Monkeys for Dival-L-dC The potential toxicity of five escalating doses of dival-L-dC in cynomologus monkeys was determined Four animals (two males and two females) each received a total of five oral dival-L-dC doses, one at each dose level (20,100, 500,1000, and 2000 mg/kg), on Days 1,4, 7,10, and 14, respectively Cage side observations for monbundity and mortality were documented twice daily Clinical observations were documented daily Blood samples for hematology and serum chemistries were collected and body weight measured before treatment on Days 1, 4, 7, 10, and 14, and before necropsy on Day 17 After completion of Day 17 evaluations, all animals were euthanized and a complete necropsy performed, including macroscopic examination and comprehensive tissue collection No treatment-related clinical abnormalities were observed Following the initial dose on Day 1, each animal demonstrated a loss m body weight of approximately 0 6 kg From Day 4 through the remainder of the study all animals maintained body weight The following observations were noted m the individual hematology profiles At Day 17, erythrocyte counts (RBG, hemoglobm (HGB), and hematocrit (HCT) were lower (by approximately 15% to 27%, cumulatively in all four animals when compared to values obtained on Day 1 Exclusive of Animal No 1001 (Male), at each timepomt changes m these parameters were The following observations were noted m the individual serum chemistry profiles Day 17 blood urea nitrogen (BUN) values were decreased (by ~43%, cumulatively) in all four monkeys when compared to Day 1 values These cumulative changes result from interim variations of -39% to +46% These changes were consistent in all monkeys on study, however, the toxicological relevance is uncertain Based on the results of this study, the NOAEL for dival-L-dC following a single oral dose by gavage in the monkey was 2000 mg/kg Example 39 28 Day Toxicity Assay m Woodchucks for L-dC The woodchuck model of chrome hepatitis B infection has been valuable for the preclinical toxicological evaluation of nucleoside analogs This model identified the delayed severe hepatocellular toxicity induced by FIAU m humans not seen m preclinical evaluation m rodents or primates The FIAU-mduced toxicity observed m woodchucks, including significant weight loss, wasting, and hepatocellular damage seen on liver biopsy, was identified beginning six to eight weeks from commencement of treatment and was similar to that observed m the FIAU-treated HBV-infected patents The antiviral activity and safety of L-dC as well as post-treatment viral rebound m woodchuck hepatitis virus (WHV) infected woodchucks was determined Male and female woodchucks were infected as neonates by subcutaneous inoculation of diluted serum of WHV earners and were all chrome earners of WHV Animals (16 to 18 months of age) were randomized to comparable groups on the basis of body weight, g-glutamyl transferase (GGT) levels, sex, and serum WHV DNA concentration (>10n genome equivalents/mL serum) measured by quantitative dot blot analysis Three animals each received L-dC at doses of 0 01, 01, 10, or 10 0 mg/kg/day orally for 28 days In addition, three animals received lamivudrne at 10 mg/kg/day orally for 28 days Four animals received vehicle control according to the same schedule All animals were monitored for rebound of WHV for an additional 56 days post-treatment Blood samples for WHV DNA levels were obtained on Days -7, 0, 1, 3, 7, 14, 21, and 28, and WHV DNA levels were also obtained post-treatment on Days 1, 3, 7, 14, 28, and 56 WHV DNA levels were detected by a polymerase chain reaction (PCR) technique Body weights were obtained concurrently and drug dosage was adjusted accordingly If clinical evidence of toxicity was observed, clinical biochemical and hematological tests were to be performed Post-mortem examination, including histologic evaluation of tissues, was to be performed on one animal that died during the study No toxicity was observed during the four-week treatment period or eight-week post-treatment follow-up period Furthermore, there was no weight loss in any L-dC treatment group compared to control animals (Figure 18) All animals gamed weight in a fashion similar to control animals during the 84-day protocol period One ammal (#98051) m the 0 1 mg/kg/d group died on the eighth day after treatment ended Postmortem examination revealed a large hepatic carcinoma (8x5x2 cm) in the left lateral lobe of the liver and death was attributed to the hepatic malignancy Hepatocellular neoplasms are seen m this model as early as nine months of age and have been a cause of death as early as 15 months of age Death m this ammal was attributed to hepatocellular carcinoma, which is an expected part of the natural history of WHV infection, and was not considered related to L-dC treatment smce there was no indication that drug toxicity was a factor in the death of the ammal Example 40 Twelve-Week Toxicity Assay in Woodchucks for L-dC The antiviral activity and safety of L-dC in woodchucks was determined In this study, four animals each received L-dC 1 0 mg/kg/day or vehicle control orally for 12 weeks Four additional annuals received L-dC along with another nucleoside analog, L-dT The animals were randomized into comparable groups, stratified by sex, weight, and pretreatment serum WHV DNA and GGT levels WHV DNA and body weight were measured on Days 0, 1, 3, 7, 14, 21, 28, 42, 56, and 84 as well as on post-treatment Days 7, 14, 21, 28, 42, 56, 70, and 84 WHV DNA levels were determined by quantitative PCR Appropriate samples for hematology, serum chemistries, WHV serology, and liver biopsy were collected pretreatment and on Day 84 Plasma drag levels were determined from samples collected 2 5 hours post-dose on Days 0, 14 and 84 L-dC (1 mg/kg/day, orally) was well tolerated and showed no drug-related toxicity through 12 weeks of treatment or during 12 weeks of follow-up WHV viremia in chronically infected woodchucks treated for 12 weeks with L-dC (1 mg/kg/d, orally) decreased by 0 5 to 1 logl0 by the end of 12 weeks of treatment, similar to the response in the 28 day study at this dose This study mcluded additional groups treated with L-dT 1 mg/kg/day, and L-dC (1 mg/kg/day) plus L-dT (1 mg/kg/day) administered in combination This combination of L-dC and L-dT reduced viral load to the limit of detection, similar to that seen during treatment with L-dC or L-dT at 10 mg/kg/day in the 28 day study There as no difference in weight between the animals in the groups treated with L-dC and the control group (see Figure 19) One animal in the control group died at Week 8, necropsy revealed the cause of death to be aortic degeneration and rupture Although unusual, spontaneous rupture of the ascending aorta has been observed historically in both uninfected and WHV-infected woodchucks The weight of all animals decreased slightly during the 24-week study period. Previous experience has determined that this slight decrease in weight was due to the approach of a hibernation cycle (B Tennant, DVM, Marmotech, Inc) Serum chemistries and hematology from all animals were in the normal range before and after 12 weeks of treatment Liver tissue histomorphology as evaluated by microscopy was normal for all groups There was no evidence of fatty change (microvesicular steatosis) Example 41 Repeated-Dose Toxicokinetics of Dival-L-dC in the Cynomologus Monkeys The potential toxicity and pharmacokinetics of dival-L-dC after oral administration for 25 days to cynomologus monkeys was determined Eight animals (four males and four females) were randomized to receive dival-L-dC via gavage at one of three doses (500, 1000, or 2000 mg/kg) or vehicle control once daily for 25 days (total N = 32) Cage side observations for monbundity and mortality were documented twice daily, and clinical observations documented once daily Body weights were documented before treatment on Days 1,8, 15 and 25 and before necropsy on Day 26 Food consumption was documented daily and reported for weekly intervals as a daily average Physical and ophthalmologic examinations and urinalysis were performed before treatment and at necropsy After completion of Day 26 evaluations, all animals were euthanized and subjected to a comprehensive gross necropsy, which included macroscopic examination of the external body surface, all orifices, and the cranial, thoracic, and abdominal cavities and then-contents Body and selected organ weight and organ-to-body and organ-to-bram weight ratios also were documented Tissue obtamed by comprehensive gross necropsy was evaluated histomorphologically by a board-certified veterinary pathologist A Body Weights All animals either maintained or gamed body weight during the course of the study, except for Animal Nos 2002 (500 mg/kg group), and 4001 and 4003 (2000 mg/kg group), which demonstrated a weight loss of 0 1 kg on Day 25 (compared to Day 1) The statistically significant differences between the males m the control group and the males m the dival-L-dC treated groups are not considered toxicologically relevant as the pre-study mean body weight for control group animals was greater than the mean body weights for the treatment groups by 0 13-0 25 kg B Food Consumption During the course of the study, all animals maintained adequate food consumption with expected variability The mean biscuit consumption was less than control males for the 500 mg/kg group males on Days 8/9,15/16, and 16/17, 1000 mg/kg group males on Days 24/25, and 2000 mg/kg group makes on Days 8/9, 15/16, 16/17, 20/21 and 23/24 The only difference noted m the females was a decrease m food consumption m the 2000 mg/kg group females on Day 7/8 1 hese differences are not considered toxicologically relevant C Clinical Pathology Hematology On Day 1 prior to the initiation of treatment, there were no differences between the control and treatment groups for any hematological parameter On Day 26, a number of statistically significant differences were noted m the erythrocyte indices, mcludmg a decreased red blood cell count (RBC_ (all treated females), decreased hemoglobin (HGB) (all treated males), and decreased hematocrit (HCT) (all treatment groups, both sexes) The males also demonstrated a reduced RBC, but the differences were not statistically significant Hemoglobm concentration was also lower in the treated females, but was not statistically significant Relative to Day 1 the RBC, HGB and HCT were decreased on Day 26 in the control and dival-LdC treated males and females However, the relative decreases observed for the control animals were less than those noted for the dival-L-dC treated animals These results are indicative of a clinically relevant nonhemolytic anemia, however, any dose response phenomenon was minimal, and histopathologic evaluation suggests that the bone marrow remained responsive Therefore, any progressive or permanent effects are considered unlikely In the white blood cell count, there were decreased absolute polymorphonuclear leukocytes (APLY) (500 MG/KG and 1000 mg/kg group females and 2000 mg/kg group males and females), decreased percent polymorphonuclear leukocytes (PLY) (1000 mg/kg and 2000 mg/kg group females), and increased percent lymphocytes (LYM) (2000 mg/kg group males and 1000 mg/kg and 2000 mg/kg group females) Serum Chemistry The mean alkaline phosphatase (ALK) levels for all treated males were significantly less the make control group mean ALK on Day 26 The mean globulin (GLOB) and calcium (CAL) levels were also elevated m the 2000 mg/kg group males on Day 26 These changes were not considered to be clinically relevant The mean potassium (K) values were greater m the 1000 mg/kg and 2000 mg/kg group males than the control group and could be related to the observed non-hemolyhc anemia present rn those treatment groups There were no changes in any serum chemistry parameter in the females on Day 26 Urinalysis The mean urinary pH was slightly decreased in the 2000 mg/kg group males and the 1000 mg/kg and 2000 mg/kg group females, but the differences were not statistically significant Noteworthy and consistent with acidification of the unne was a lack of crystals in the unne from the high dose males and females D Organ Weights Statistically significant decreases m organ weights were noted for the lungs (absolute) of the 1000 mg/kg and 2000 mg/kg group males and the relative thymus (thymus brain) of the 2000 mg/kg group males However, these differences were not considered toxicologically relevant E Pathology Macroscopic There were no macroscopic findings that were interpreted as related to the administration of the dival-L-dC All macroscopic findings were typical of those commonly present as incidental findings in non-human primates Microscopic Thymic atrophy was the only microscopic finding that was interpreted as a treatment-related finding The incidence and seventy of thymic atrophy was increased m the 1000 mg/kg and 2000 mg/kg group males and females, but was not affected m the 500 mg/kg group animals However, the clinical significance of the thymic atrophy was interpreted as equivocal The dose-response relationship was weak, not all 1000 mg/kg and 2000 mg/kg group males were affected and thymic atrophy typically occurs as primates age Other microscopic findings present m this study were commonly minor inflammatory or degenerative changes of the usual type and incidence observed in primates of this age Toxicokinetics Blood samples for hematology and serum chemistries were collected pretreatment Day 1 and before necropsy on Day 26 Blood samples were collected for pharmacokinetic analvsis on Day 25 from each animal at each of the following times after dosing 0 5, 1, 2, 4, 6, 8, 12, and 24 hours Plasma was prepared from blood and analyzed for concentrations of dival-L-dC and three metabolites L-dC, L-dU and the partially de-estenfied form of dival-L-dC, ß-L-2'-deoxycybdine-5'-valine ester Only L-dC and ß-L-2'-deoxycyhdine-5'-valine ester wee quantifiable The mean plasma concentration-time data for the 1000 and 2000 mg/kg group were subjected to noncompartmental pharmacokinetic analysis using WinNonlin 1 5 (Model 200) Analysis of the 500 mg/kg mg/kg group is in progress Plasma concentrations of ß-L-2'-deoxycytidme-5'-vahne ester on Day 25 reached maximal values (Cmax) at 1 hour (median Tmax) post oral administration of dival-L-dC, compared to a median Tmax of 2-4 hours for L-dC However, ß-L-2'-deoxycytidme-5,-valme ester Cmax values were approximately 2 orders of magnitude lower than for L-dC After reaching Cmax, concentrations of L-dC declmed in an apparent bi-exponenual manner for each group Estimated terminal phase mean half-lives were approximately 4-5 hours for males and females in both dosage groups These half-life estimates should be viewed as minimal values, however, because most individual estimates were based on data from 6 to 12 hours post-dose, at which time the terminal phases may not have been completely characterized Mean β-L-2'-deoxycyudine-5'-valine ester concentrations also declined after reaching CMAX, but the terminal phases were not adequately defined to allow estimation of half-lives Mean Cmax values for L-dC and β-L-2'-deoxycytidine-5'-vahne ester were similar for males and females within each dosage group, except for 1000 mg/kg group makes, which were lower bv half the concentration values of the 2000 mg/kg group males Therefore, CmaX appeared to increase with dosage only for the 1000 mg/kg group males Comparison of L-dD AUClast between males and females showed trends similar to those noted for Cmax with the males sin the 1000 mg/kg group having values lower by approximately half the AUClast values of the 2000 mg/kg group males Comparison of ß-L-2'-deoxycytidme-5'-vahne ester AUC]ast between males and females showed an absence of sex related differences and AUCast appeared to mcrease m a directly proportional manner to increases in dosages The data suggest that following oral administration of dival-L-dC is a rapid conversion to the de-estenfied form of dival-L-dC, ß-L-2,-deoxycytidme-5'-valme ester, and then L-dC but overall exposure is 100 fold higher for L-dC than for ß-L-2'-deoxycytidme-5'-vahne ester Overall exposure to metabolite β-L-2'-deoxycytidme-5,-valme ester appears to mcrease in an approximately linear manner with increases in dosages A summary of toxicokmebc results is presented in Table 29 Table 29 Pharmacokinetic Analysis of Repeated-Dose Dival-L-dC 1000 mg/kg and 2000 mg/kg Administered Orally m the Monkey Pharmacokinetic Parameter1 (Table Removed) m 1 Mean values (+ SD) at Day 25 2 n = 4 for all parameters for both L-dC and β-L-2'-deoxycytidine-5'-vahne ester except for 2000 mg/kg group females, for which n = 3 and for L-dC AUC and t^, 1000 mg/kg group females, where n = 2 due to inadequate characterization of the terminal phase 3 Median (rather than mean) values are presented for Tmax, and T^ NA Not Applicable ID Insufficient Data to define terminal phase for all animals Example 42 Repeated-Dose Toxicokinetics of Dival-L-dC m the Rat The potential toxicity and pharmacokinetics of dival-L-dC after oral administration for 28 days to rats was determined Twenty animals each (10 males and 10 females) were randomized to receive dival-L-dC via gavage at one of three doses (500, 1000, or 2000 mg/kg) or vehicle control once daily for 28 days Cage side observations for monbundity and mortality were documented twice daily Clinical observations were documented once daih Body weights were documented before dosing on Days 1, 8, 15, 22, and 28 and before necropsy on Day 29 Food consumption was documented weekly Blood samples for hematology and serum chemistries also were collected before necropsy on Day 29 After completion of Day 29 evaluations, all animals were euthanized and subjected to a comprehensive gross necropsy, which mcluded macroscopic examination of the external body surlace, all orifices, and the cranial, thoracic, and abdominal cavities and their contents Bod\ and selected organ weight and organ-to-body and organ-to-brain weight ratios also were documented Tissue obtained by comprehensive gross necropsy was evaluated histomorphologically by a board-certified veterinary pathologist A Body Weights The mean body weight values for the 2000 mg/kg group males on Days 22 and 28 were significantly lower than the mean value for the male control group The mean body weight value for the 2000 mg/kg group females on Day 28 was also significantly lower than the mean value for the control group females B Food Consumption Food consumption was reduced in the 2000 mg/kg group males throughout the duration of the study Also, the food consumption of the 1000 mg/kg group males during the third week of the study was significantly less than the control group males The food consumption was significantly reduced in the 1000 mg/kg and 2000 mg/kg females during the second, third, and fourth weeks of the study C Clinical Pathology Hematology On Day 29, a number of statistically significant differences were noted in the erythrocyte indices The red blood cell count (RBC) was significantly reduced m both males and females at all three dose levels (500, 1000 and 2000 mg/kg) The hemoglobin concentration (HGB) was decreased m the 2000 mg/kg group males, the 1000 mg/kg group females and the 2000 mg/kg group females A decrease in hematocrit (HCT) was noted m the 1000 mg/kg and 2000 mg/kg group males and females The mean cell volume (MCV) was significantly increased in the 500, 1000 and 2000 mg/kg group males and m the 500 and 1000 mg/kg group females The mean cell hemoglobin (MCH) was significantly mcreased in the 500, 1000 and 2000 mg/kg group males and females The mean cell hemoglobin concentration (MCHC) was increased m the 1000 mg/kg females The nucleated red blood cell count (NRC, absolute and relative) was decreased in the 1000 mg/kg and 2000 mg-'kg males and mcreased m the 2000 mg/kg females These changes indicate a treatment-related mild responsive anemia. The white blood cell count (WBC) was decreased m the 2000 mg/kg males There was a reduction in the monocytes (MNO, absolute and percentage) m the 2000 mg/kg group males Platelets (PLT) were increased in the 2000 mg/kg males However, these changes were quantitatively small and the toxicological relevance is uncertain Serum Chemistry Mean globulin (GLOB) levels were decreased m the 2000 mg/kg group males and the 1000 mg/kg group females on Day 29 The albumin/globulin ratios were increased m the 1000 and 2000 mg/kg group males and the 1000 mg/kg group females The alkaline phosphatase (ALK) levels were elevated in the 500 mg/kg group females The cholesterol (CHOL) levels were mcreased m the 1000 mg/kg females These minor changes did not form dose-response-related patterns or trends to suggest that these values were toxicologically relevant D Organ Weights Significant decreases in absolute organ weights were noted for the lungs (2000 mg/kg group males and females) and thymus (2000 mg/kg group males, 1000 mg/kg group females and 2000 mg/kg group females) Also significant was the decrease m the mean absolute organ weight for the prostate and seminal vesicles in the 2000 mg/kg group males The mean absolute heart weights were decreased m the 1000 mg/kg and 2000 mg/kg group females The salivary glands mean weight was decreased in 2000 mg/kg group females The mean spleen weight was mcreased m the 2000 mg/kg group females The relative (to body) organ weight changes included an mcreased brain weight in the 2000 mg/kg group males and females An increase m the mean tests weight of the 1000 mg/kg and 2000 mg/kg group males was also noted The relative thymus weight was reduced m the 2000 mg/kg group males and the 1000 mg/kg and 2000 mg/kg group females The mean relative spleen weight was increased m the 2000 mg/kg group females Also, the relative (to brain weight) organ weight changes mcluded a decreased relative lung weight in the 2000 mg/kg group males The relative thymus weights were decreased in the 1000 mg/kg and 2000 mg/kg group males and females The relative prostate and seminal \esicle mean weights were also decreased in the 2000 mg/kg group males The mean relanve heart weight was reduced m the 2000 mg/kg group females as was the mean relati\ e weight of the salivary glands The relative spleen weight was increased in 2000 mgkg group females The decreases in organ weights (thymus, lung, heart, salivary glands, prostate, seminal vesicles, and brain) were interpreted as secondary to the generalized body weight loss presented in the 1000 mg/kg and 2000 mg/kg group animals Thymic atrophy, which was observed microscopically in the 1000 mg/kg and 2000 mg/kg group animals, was consistent with the decreased thymus weights observed Other tissues with decreased weights did not have microscopic correlates The increased spleen weights were interpreted as a consequence of erythropoietic activity observed microscopically E Pathology Microscopic The incidences of thymic atrophy and lymphoid necrosis were increased m the 1000 mg/kg and 2000 mg/kg group animals, but were not affected m the 500 mg/kg group animals However, the clinical significance of thymic atrophy and lymphoid necrosis was interpreted as equivocal because the dose-response relationship was weak Also, these thymic changes are often present as non-specific changes m animals stressed by a variety of factors, and significant body weight reductions were observed m the 1000 mg/kg and 2000 mg/kg group animals m this study Erythropoiesis in spleen was mcreased in the 1000 mg/kg and 2000 mg/kg group males and females sufficiently to distinguish them from the controls, but the spleens from the 500 mg/kg group animals were similar to controls Hematopoiesis in liver was mcreased m the 2000 mg/kg group males and females sufficiently to distinguish them from the controls, but the livers from 500 mg/kg and 1000 mg/kg group animals were similar to those m controls Hyperplasia m sternal bone marrow was observed in the 2000 mg/kg group males and females Erythropoiesis m spleen, mcreased hematopoiesis m liver and hyperplasia m bone marrow were all interpreted as expected and appropriate responses to the mild anemia observed as a part of the hematology results These results confirm the responsive nature of the anemia during contmued treatment There were several other microscopic changes present m this study These were most commonly manor inflammatory or degenerative changes of the usual type and mcidence observed in rodent gavage studies Toxicokinetics An additional 54 animals (27 males and 27 females) had samples collected for pharmacokinetic analyses on Days i and 28 On both days, samples were collected at each of six timepoints (alternating two animals per timepoint) 0 5, 1, 2, 4, 8 and 24 hours post-dosing Plasma was prepared from blood and analyzed for concentrations of dival-L-dC and three metabolites L-dC, L-dU and the partially de-estenfied form of dival-L-dC, /3-L-2,-deoxycytidme-5'-vahne ester Only L-dC and j3-L-2'-deoxycytidme-5l-vahne ester were qualifiable The mean plasma concentration-time data for the 1000 and 2000 mg/kg group were subjected to noncompartmental pharmacokinetic analysis using WmNonhn 1 5 (Model 200) Analysis of the 500 mg/kg group is in progress Mean plasma concentrations of the metabolite, L-dC, reached maximal values (Cmax) at 2 hours post-dose (Tmax) for the 1000 m/kg dose group and at 1-4 hours post-dose for the 2000 mg/kg dose group Mean Cmax values for males and females were comparable within each of the 1000 mg/kg and 2000 mg/Kg- osage groups and were similar on Day 28 versus Day 1 in both groups Cmax increased via dose m most cases but the extent cf the increase was variable After reaching Cmax, cone .ntrations of L-dC declined in an apparent bi-exponenhal manner for each group Es x ii-ued terminal phase half-lives for th 1000 mg/kg dose group (9-17 hours) tended to be longe for the 2000 mg/kg dose group (& S hours), but the half-life estimates should be interpreted with caution The estimation or he half-lives required using only three data pomts am the data tended to be variable Also one of three data points used was at 4 hours, at which time the terminal phase may not have been established Tiast for L-dC concentration. vocurred at 24 hours for all data sets AUClast was comparable for males and females with. wahic group, and did not appear to bt substantially different on Day 28 versus Day 1 Although Cmax for L-dC did not appear to mcrease with increased dosage of dival-L-dC in a c manner, as noted above, AUCast of L-dC mcreased with dival-L-dC m a relation that appeared to be approximatel' proportional to dose Plasma mean concentrations of ß-L-2'-deoxycytidine-5'-valine rster reached naxnnal values (Cmax) at 1 to 2 hours dose (Tmax) Mean Cmax values Tor males and females were similar within each dosage group witn a trend toward higher values for remales Cmax values ^vere approximated 4% to 50% higher for females on each Day 1 and Day 28 except for females m the 2000 mg/kg group, where j8-L 2-deo cycytidine-51-alme ester Cmax values were approxb by 164% higher than males on Day 28 When comparing values within each gender, - values on Day 28 wee similar to Day 1 except for females in the 2000 mg/kg group for which Cmax values were 130% higher on Day 28 than Day 1 Cmax increased with dosage in each case, but by a factor that was generally less than linearly proportional to dose The apparent terminal elimination phase of ß-L-2'-deoxycyudine-5 '-valine ester was not well characterized and therefore half-lives were not reported Tlast for 0-L-2'-deoxycytidine-5'-vahne ester concentrations occurred at 4-8 hours for the 1000 mg/kg dose group and at 8-24 hours for the 2000 mg/kg dose group As was noted for Cmax, the AUClast was 25% to 50% higher for females than males AUCast was consistently slightly higher on Day 28 vs Day 1 for both males and females (30% to 62%) AUC\|ast increased with dosage in a relationship that appeared to be approximately linearly proportional to dose These data suggest that both L-dC and ß-L-2'-deoxycytidine-5'-vahne ester reached systemic circulation relatively rapidly Overall exposure as measured by Cmax was 10 to 40 fold greater for L-dC than ß-L-2'-deoxycytidme-5'-valine ester and 35 to 80 fold greater as measured by AUClast Exposure appeared to mcrease proportionally to dosage within the dosage range of 1000-2000 mg/kg/day Overall exposure on Day 29 of L-dC was comparable to that observed on Day 1 while ß-L-2l-deoxycytidine-5l-vahne ester exposure was generally greater on Day 28, suggesting that accumulation of ß-L-2'-deoxycytidine-5'-valme ester may occur during repeated dosing A summary of toxicokmetic results is presented m Table 30 Table 30 Pharmacokinetic Analysis of Single- and Repeated-Dose Dival-L-dC 1000 mg/kg and 2000 mg/kg Administered Orally in the Rat Pharmacokinetic Parameter (Table Removed) NA = Not Applicable, a terminal phase was not adequately characterized ID = Insufficient Data to define terminal phase for all animals Example 43 S Typhimurutm and E Colt Plate Incorporation Mutation Assay (Genotoxicity) Dival-L-dC when administered orally to animals is rapidly converted to L-dC to give high plasma concentrations of L-dC and no detectable dival-L-dC Therefore, the mutagenicity studies conducted in vitro were performed using L-dC This study was conducted in accordance with FDA GLP regulations L-dC was tested for its potential to cause mutation at the hisudine operon of Salmonella typhimunum strains TA98, TA100, TA1535, and TA1537 and at the tryptophan opeton of Escherichia coh strain WP2uvrA L-dC at concentrations of 50, 100, 500, 1000, and 5000 mg/plate plus positive and negative controls were tested Test strains were exposed to L-dC or control m the absence of exogenous activation and in the presence of induced rat liver S-9 extract plus eofactors After incubation for approximately 68 hours, L-dC and controls were evaluated for the number of revertants per plate and integrity of the background microcolony lawn Both negative and positive controls fulfilled the requirements of the test The results of both definitive and confirmatory assays indicated that L-dC did not induce any significant increase rn the number of revertant colonies for any of the test strains in the presence or absence of induced rat liver S-9 extract Based on the study findings, it was concluded that there was no evidence of mutagenicity in the S typhimunum or E coh plate incorporation mutation assay with L-dC concentrations up to 5000 mg/plate Example 44 Chromosomal Aberration Assay Dival-L-dC when administered orally to animals is rapidly converted to L-dC to give high plasma concentrations of L-dC and no detectable dival-L-dC Therefore, the mutagenicity studies conducted in vitro were performed using L-dC This study was conducted in accordance with FDA GLP regulations L-dC was tested for its potential to induce chromosomal aberrations m cultured CHO cells In the definitive assay, L-dC at concentrations of 100 500, 1000, and 5000 mg/mL and positive and negative controls were tested with and without metabolic activation After continuous treatment for 18 hours, toxicity was determined by the reduction in relative cell growth (RCG) and relative mitotic mdex (RMI) Based on the RCG and RMI results, chromosomal aberrations were scored from the three highest concentrations (500, 1000, and 5000 mg/mL) One hundred metaphases were scored from each of the duplicate cultures at each concentration (mcludmg positive and negative controls) A confirmatory assay was performed without activation only with L-dC concentrations of 1 0,10, 100, 500,1000, and 5000 mg/mL After continuous treatment for 18 hours the reduction m RCG and RMI were determined Based on RCG and RMI results, chromosomal aberrations were scored from the three highest concentrations (500, 1000, and 5000 mg/mL) One hundred metaphases were scored from each of the duplicate cultures at each concentration level (mcludmg positive and negative controls) Results from the definitive and confirmatory assays indicated that L-dC did not mduce a statistically significant increase (defined as a β-value £0 05 determined by the Chi-square test) m the percentage of cells with aberrations at any of the concentrations tested, both with and without metabolic activation, compared to solvent controls Based on the study findings, it wa> concluded that there was no evidence of chromosomal aberrations in the CHO assay after exposure to L-dC at concentrations up to 5000 mg/mL, and L-dC is not considered to be a cLstogenic agent Example 45 Mouse Micronucleus Assay Dival-L-dC when administered orally to animals is rapidly converted to L-dC to give high plasma concentrations of L-dC and no detectable dival-I-dC Therefore, the mutagenicity studies conducted in vitro were performed using I-dC This study was conducted in accordance with FDA GLP regulations Assuming an oral bioavailability of 10-20% m the rodent (see Pharmacology and Toxicology, Section 8 17 3) exposure to L-dC (2000 mg/kg dose) would reach or exceed 400 mg/kg This level of exposure would exceed the expected human exposure by 20 to 50-fold L-dC was tested for its potential to induce micronucleated polychromatic erythrocytes (MPCE) in the bone marrow cells of male and female mice L-dC at concentrations of 500, 1000, and 2000 mg/kg and positive and negative controls were tested Study drag was administered by oral garage as a single dose Two harvests approximately 24 and 48 hours after L-dC or negative control admimstration were performed, and a single harvest approximately 24 hours after positive control administration was performed Five male and five female mice per dose group per harvest time were used The percentage of polychromatic erythrocytes (PCE) and MPCE frequency were determined for each traiepoint The results of the study indicate that there was no statistically significant increase (defined as a β-value £0 025 determined by a one-tailed Student's t-test) m the number of MPCE at any timepoint in any L-dC dose group compared to negative control A reduction of more than 20% versus the vehicle control in the percentage of PCE, as an indication of toxicity, was observed at each test article dose level at the 24 hour harvest tune in both sexes (-30 5% to -43 1% for the males and -26 1% to -32 2% for the females) The reduction also mdicates an appropriate exposure of test article to the target tissue However, this reduction of more than 20% was not observed at any test article dose level at the 48 hour harvest tune m eidier sex This study mdicates that, under the conditions of the test and according to the criteria set for evaluating the test results, L-dC was negative m the micronucleus assay to male or female animals at doses up to 2000 mg/kg Example 46 Integrated Summary of Toxicologic Findings Conventional cell-based assays were used to assess the cytotoxicity of L-dC and any cellular metabolites L-dC was non-cytotoxic (50% cytotoxic concentration, CC50, >2000 µM) to the human hepatoma cell line 2 2 15, which is routinely used to determine the anti-I-EBV activity of potential antiviral agents L-dC was not cytotoxic to human human peripheral blood mononuclear cells (PBMCs, CC50 >100 µM) and to human bone marrow progenitor cells (50% inhibitory concentration, IC50, > 10 µM in granulocyte-macrophage colony forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) assays) (Table Removed) a PBMC, peripheral blood mononuclear cells, HFF, human foreskin fibroblast, Daudi, Burkitt's B-cell lymphoma, MDCK, canine kidney epithelial cells, CV-1, African green monkey kidney fibroblast cells, MA-104, rhesus monkey kidney epithelial cells b CC30 = 50% cytotoxic concentration, '>' indicates that no CC30 was reached at the highest drug concentration tested c NIH, Antiviral Research and Antimicrobial Chemistry Program d R Schinazi, Emory University, Veterans Affairs Medical Center e Result presented m µg/mL rather than µM In addition, L-dC was not cytotoxic to numerous other cell lines of human and other mammalian ongin No discernible changes in the function, morphology, or DNA content of mitochondria were noted and there was no lactic acid accumulation m L JC treated hepatocytes (IC50 > 10 µM) The triphosphate form of L-dC was not inhibitory to the human DNA. polymerases α, ß and , up to concentrations of 100 µM In acute single dose (including 500, 1000, and 2000 mg/kg single oral dose) toxicology studies in rats and in monkeys (dose escalation over days 1, 4, 7,10 and 14 up to 2000 mg/kg) there were no overt signs of toxicity nor were there any dival-L-dC related effects on body weight, food consumption, or clinical pathology parameters (hematology and serum chemistry) In addition, there were no macroscopic lesions observed at necropsy, nor were there any microscopic findings on histomorphological analysis attributable to dival-L-dC Based on the results of these studies, the no observed adverse effect level (NOAEL) for dival-L-dC, following a single dose by oral gavage in the Sprague-Dawley rat and cynomologus monkey was 2000 mg/kg In the subchronic (25 day) toxicology study in monkeys, the NOAEL was less than 500 mg/kg for dival-L-dC Thymic atrophy was the only microscopic finding that was possibly related to dnal-L-dC, but the clinical significance was interpreted as equivocal A mild non-hemolytic anemia (decreased red blood cell count, decreased hemoglobin, and hematocrit) and decrease in the absolute and percent polymorphonuclear leukocyte counts of no apparent consequence were noted at the 500 mg/kg dose level Other than the hematological changes there were no other toxicities identified m any dose group In the subchronic (28 day) toxicology study m rats, the NOAEL was less than 500 mg/kg for dival-L-dC Oral administration of the dival-L-dC for 28 days to the rat at a dose of 2000 mg/kg resulted m treatment related changes that included a mild macrocytic anemia, reduced thymus weight, increased spleen weight (females only), reduced body weight, and hematopoiesis m the spleen, liver and sternal bone marrow Oral admmistration of dival-L-dC for 28 days to the rat at a dose of 1000 rog/kg resulted in treatment related changes that mcluded a mild macrocytic anemia, thymic atrophy (females only) and hematopoiesis m the spleen The histomorphological changes seen in the liver, spleen and bone marrow reflect a hematological response to the mild anemia. Oral admmistration of the dival-L-dC for 2S days to the rat at a dose of 500 mg/kg resulted m a mild macrocytic anemia Other than the hematological changes and hematopoietic responses noted there were no other toxicities identified m any dose group In normal healthy woodchucks or woodchucks chronically infected with hepatitis B virus (εfficacy model for treatment of HBV infection), no toxicity was observed during acute (10 mg/kg single dose IV and PO) and subchronic (28 days at 10 mg/kg/day orally and 12 weeks at 1 mg/kg/day orally) studies of animals receiving L-dC There was no weight loss in the L-dC treatment groups compared to control animals, clinical pathology parameters (hematology and serum chemistry) were in the normal range and liver biopsies taken at end of treatment m the 12-week study showed no evidence of fatty change (microvesicular steatosis) L-dC was not mutagenic in the S typhimunum or E coh plate incorporation mutagenicity assay at concentrations up to 5000 µg/plate There was no evidence of chromosomal aberrations m the Chinese hamster ovary (CHO) assay after exposure to L-dC at concentrations up to 5000 µg/mL (or 22 0 mM) In the mouse nucronucleus assay, L-dC was not clastogemc to male or female animals at doses up to 2000 mg/kg Mild anemia noted m the monkey was not associated with any clinical correlates even at the highest dose (2000 mg/kg) and m the rat at 500 mg/kg In addition the reticulocyte counts were unchanged Although there was no formal reversibility component m these studies it is apparent that a hematological rebound can occur as indicated by the extramedullary hematopoiesis seen in the spleen and liver at the higher doses m the rat Table 32 Interspecies Comparison of Doses by Weight and Body Surface Area (Table Removed) Similar hematological changes at comparable or lower doses were observed in preclinical toxicity studies of lamivudine (Epivrr-HBVT™), and valacyclovir (Valtrex™) Both of these appnn ed drugs are members of the same well-charactenzed class (nucleoside or nucleoside analog) as dival-L-dC The choice of lamivudine for comparison is based on the fact that it is a cvtosine derivative as is dival-L-dC and on its approval for the treatment of chronic hepatitis B infection The choice of valacyclovir for comparison is based on the fact that it is a valine ester prodrug of the nucleoside acyclovir This invention has been described with reference to its preferred embodiments Variations and modifications of the invention, will be obvious to those skilled in the art from the foregoing detailed description of the invention It is mtended that all of these variations and modifications be included within the scope of this invention WE CLAIM: 1: A compound of the 3'-0-valinyl ester of 2'-deoxy-[beta]-L-cytidine (Formula Removed) or a pharmaceutically acceptable salt thereof. 2: A pharmaceutical composition comprising a 3'-0-valinyl ester of 2'-deoxy-[beta]-L-cytidine as claimed in claim l the formula (Formula Removed) or a pharmaceutically acceptable salt thereof, in combination with compound such as herein described. 3. A compound substantially as herein described with reference to foregoing description, examples, tables and accompanying drawings.

Full Text

3'-PRODRUGS OF 2'-DEOXY-β-L-NUCLEOSIDES
Field of the Invention
The present invention relates to 3'-prodrugs of 2'-deoxy-β-L-nucleosides for the treatment of hepatitis B virus
This application claims priority to U S provisional application no 60/212,100, filed on June 15, 2000
Background of the Invention
Hepatitis B virus ("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 chrome 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 h\er damage, that causes abdominal pain, jaundice, and elevated blood levels of certain 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 m the blood for an extended, or indefinite, period, causing a chrome infection Chrome infections can lead to chrome persistent hepatitis Patients infected with chrome persistent HBV are most common m developmg countries Chrome persistent hepatitis can cause fatigue, cirrhosis of the liver and hepatocellular carcinoma, a primary liver cancer In western industrialized countries, high risk groups for HBV infection mclude those m contact with HBV earners or then 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 wi+h AIDS or HIV-associated infections However, HBV is more contagious than HIV
Daily treatments with α-interferon, a genetically engineered protein, have shown promise A human serum-derived vaccine has also been developed to immunize patients against HBV Vaccmes have been produced through genetic engineering While the vaccme has been found effective, production of the vaccine is troublesome because the supply of human serum from chrome earners is limited, and the purification procedure is long and expensive Further, each batch of vaccme prepared from different serum must be tested m chimpanzees to ensure safety In addition, the vaccme does not help the patients already infected with the virus
An essential step m the mode of action of purine and pynnudme nucleosides against viral diseases, and m particular, HBV and HIV, is their metabolic activation by cellular and viral kinases, to yield the mono-, di- and triphosphate derivatives The biologically active species of many nucleosides is the triphosphate form, which inhibits DNA polymerase or reverse transcriptase, or causes chain termination
A number of synthetic nucleosides have been identified which exhibit activity against HBV The (-)-enantiomer of BCH-189 (2\3'-dideoxy-3'-thiacytidine), known as 3TC, claimed m U S Pacent 5,539,116 to Liotta, et al, is currently m clinical trials for tne treatment of hepatitis B See also EPA 0 494 119 Al filed by BioChem Pharma, Inc
jS-2-Hydroxymethyl-5-(5-fluorocytosm-l-yI)-l,3-oxathiolane ("FTC"), claimed m U S Patent Nos 5,814,639 and 5,914,331 to Liotta et al, exhibits activity against HBV See Furman et al, "The Anti-Hepattis B Virus Activities, Cytotoxicities, and Anabolic Profiles of the (-) and (+) Enanuomers of cis-5-Fluoro-l-{2-(Hydroxymethyl)-l,3-oxatmolane-5-vl}-Cytosine" Antimicrobial Agents and Chemotherapy December 1992, page 2686-2692, and Cheng, et al, Journal of Biological Chermstrv Volume 267(20), 13938-13942 (1992)
U S Patent Nos 5,565,438, 5,567,688 and 5,587,362 (Chu, et al) disclose the use of 2'-fluoro-5-methyl-β-L-arabmofuranolylundme (L-FMAU) for the treatment of hepatitis B and Epstein Barr viru>
Penciclovir (?CV, 2-amino-1,9-dihydro-9- {4-hydroxy-3-(hydroxymethyl)butyl} -6H-purm-6-one) has established activity against hepatitis B See US Patent Nos
5,075,445 and 5 684 1:3
Adefovir (9-{2-(phosphonomethoxy)ethyl} adenine, also referred to as PMEA or {{2-(6-amino-9H-punn-9-yl)ethoxy}methylphosphomc acid), also has established activity against hepatitis B See, for example, U S Patent Nos 5,641,763 and 5,142,051
Yale University and The University of Georgia Research Foundation, Inc disclose the use of L-FDDC (5-fluoro-3'-thia-2',3'-dideoxycytidine) for the treatment of hepatitis B virus in WO 92/18517
Other drugs explored for the treatment of HBV mclude adenosine arabinoside, thymosin, acyclovir, phosphonoformate, zidovudine, (+)-cyanidanol, quinaenne, and 2'-fluoroarabinosyl-5 -lodouracil
U S Patent Nos 5,444,063 and 5,684,010 to Emory University disclose the use of enantiomencally pure /3-D-l,3-dioxolane purine nucleosides to treat hepatitis B
WO 96/40164 filed by Emory University, UAB Research Foundation, and the Centre National de la Recherche Scientifique (CNRS) discloses a number of (S-L-2',3'-dideoxynucleosides for the treatment of hepatitis B
WO 95/07287 also filed by Emory University, UAB Research Foundation, and the Centre National de la Recherche Scientifique (CNRS) discloses 2' or 3' deoxy and 2',3'-dideoxy-ß-L-pentofuranosyl nucleosides for the treatment of HIV infection
W096/13512 filed by Genencor International, Inc , and Lipitek, Inc, discloses the preparation of L-nbofuranosyl nucleosides as antitumor agents and virucides
W095/32984 discloses lipid esters of nucleoside monophosphates as lrnmuno-suppresive drugs
DE 4224737 discloses cytosine nucleosides and then- pharmaceutical uses
Tsai et al, in Biochem Pharmacol 1994, 48(7), 1477-81, disclose the effect of the anti-HIV agent 2'-j5-D-F-2' 3'-dideoxynucleoside analogs on the cellular content of mitochondrial DNA and lactate production
Galvez, J Chem Inf Comput Sci 1994, 55(5), 1198-203, describes molecular computation of/3-D-r azido-2',3'-dideoxy-5-fluorocytidine
Mahmoudian, Pharm Research 1991, 5(1), 43-6, discloses quantitative structure-activity relationship analyses of HIV agents such as β-D-3'-azido-2',3'-dideoxy-5-fluorocytidine
US Patent No 5,703,058 discloses (5-carboximido or 5-fluoro)-(2',3'-unsaturated or 3'-modified) pynmidrne nucleosides for the treatment of HIV or HBV
Lm et al, discloses the synthesis and antiviral activity of various 3'-azido analogues of/3-D-nucleosides m J Med Chem 31(2), 336-340 (1988)
WO 00/3998 filed by Novino Pharmaceuticals, Ltd discloses methods of preparing substituted 6-benzyl-4-oxopynmidines, and the use of such pynmidines for the treatment of HIV
Novino Pharmaceuticals, Ltd was also first to disclose 21-deoxy-β-T-_ erythropentofuranonucleosides, and then use in the treatment of HBV m WO 00/09531 A method for the treatment of hepatitis B infection m humans and other host animals is disclosed that mcludes administering an effective amount of a biologically active 2'-deoxy-i3-L-erythro-pentofuranonucleoside (alternatively referred to as β-L-dN or a ß-L-2'-dN) or a Dharmaceuticalry acceptable salt or prodrug thereof, including β-L-deoxynbomynndine (β-L-dT), β-L-deoxynbocytidme (β-L-dC), β-L-deoxynboundine (β-L-dU), β-L-deoxynbo-"guanosme"(β-L-:dg β-L-deoxyriboadenosine (β-L-dA) and β-L-deoxynboinosirie (β-Lal); administered either alone or m combination, optionally in a pharmaceuticalry"~acceptabie earner 5' and N4 (cytidme) or N6 (adenosine) acylated or alkylated denvatives of the active compound, or the 5'-phospholipid or 5'-ether lipids were also disclosed
Vanous prodrugs of antivirals have been attempted Most notably, U S Patent No 4,957,924 to Beauchamp discloses vanous therapeutic esters of acyclovir
In light of the fact that hepatitis B virus has reached epidemic levels worldwide, and has severe and often tragic effects on the infected patent, there remains a strong need to provide new effective pharmaceutical agents to treat humans infected with the virus that have low toxicity to the host
Therefore, it is an object of the present invention to provide compounds, compositions and methods for the treatment of human patents or other hosts infected with HBV
Summary of the Invention
3'-Prodrugs of 2'-deoxy-ß-L-nucleosides, or their pharmaceutically acceptable salts or pharmaceutically acceptable formulations containing these compounds are useful in the prevention and treatment of hepatitis B infections and other related conditions such as anu-HBV antibody positive and HBV-positive conditions, chrome liver inflammation caused by HBV, cirrhosis, acute hepatitis, fulminant hepatitis, chrome persistent hepatitis, and fatigue These compounds or formulations can also be used prophylactically to prevent or retard the progression of cluneal illness in individuals who are anti-HBV antibody or HBV-antigen positive or who have been exposed to HBV
A method for the treatment of a hepatitis B viral infection m a host, including a human, is also disclosed that includes administering an effective amount of a 3'-prodrug of a biologically active 2'-deoxy-β-L-nucleoside or a pharmaceutically acceptable salt thereof, administered either alone or in combination or alternation with another anti-hepatitis B virus agent, optionally in a pharmaceutically acceptable earner The term 2'-deoxy, as used in this specification, refers to a nucleoside that has no substituent m the 2'-position The term 3'-prodrug, as used herein, refers to a 2'-deoxy-ß-L-nucleoside that has a biologically cleavable moiety at the 3'-position, including, but not limited to acyl, and in one embodiment, an L-armno acid
In one embodiment, the 2'-deoxy-β-L-nucleoside 3'-prodrug includes biologically cleavable moieties at the 3' and/or 5' positions Preferred moieties are ammo acid esters including valyl, and alkyl esters including acetyl Therefore, this invention specifically mcludes 3'-L-amino acid ester and 3',5'-L-diamino acid ester of 2'~β-L-deoxy nucleosides with any desired purine or pynrmdme base, wherein the parent drug has an EC50 of less than 15 rmcromolar, and preferably less than 10 micromolar in 2 2 15 cells, 3'-(alkyl or aryl ester)- or 3',5'-L-di(alkyl or aryl ester)-2'-β-L-deoxy nucleosides with any desired purine or pynmidine base, wherein the parent drug has an EC50 of less than 10 or 15 rmcromolar m 2 2 15 cells, and prodrugs of 3',5'-diesters of 2'-deoxy-β-L-nucleosides wherein (1) the 3' ester is an ammo acid ester and the 5' -ester is an alkyl or aryl ester, (11) both esters are ammo acid esters, (111) both esters are independently alkyl or aryl esters, and (IV) the 3' ester is independently an alkyl or aryl ester and the 5'-ester is an ammo acid ester, wherein the parent drug has an EC50 of less than 10 or 15 micromolar in 2 2 15 cells
Examples of prodrugs falling within the invention are 3'-L-vahne ester of 2'-deoxy-β-L-cytidine, 3'-L-valine ester of 2'-deoxy-β-L-thymine, 3'-L-valine ester of 2'-deoxy-β-L-adenosine, 3'-L-valine ester of 2'-deoxy-β-L-guanosine, 3'-L-vahne ester of 2'-deoxy-β-L-5-fluoro-cytidine, 3'-L-vahne ester of 2'-deoxy-β-L-undine, 3'-acetyl ester of 2'-deoxy-β-L-cytidine, 3'-acetyl ester of 2'-deoxy-β-L-thymine, 3'-acetyl ester of 2'-deoxy-β-L-adenosine, 3'-acetyl ester of 2'-deoxy-β-L-guanosine, 3'-acetyl ester of 2'-deoxy-β-L-5-fluoro-cytidine, and 3'-esters of 2'-deoxy-β-L-(cytidine, 5-fluorocytidine, guanosine, undine, adenosine, or thymine) wherein (1) the 3' ester is an ammo acid, ester, or (n) the 3' ester is an alkyl or aryl ester
Additional examples of prodrugs falling within the invention are 3',5'-L-drvaline ester of 2'-deoxy-β-I^cytidine (dival-L-dC), 3',5'-L-divalme ester of 2'-deoxy-β-L-thymine, 3',5'-L-divahne ester of 2'-deoxy-β-L-adenosine, 3',5'-L-divaline ester of 2'-deoxy-β-L-guanosine, 3',5'-L-divahne ester of 2'-deoxy-β-L-5-fluoro-cytidine, 3',5'-L-divalme ester of 2'-deoxy-β-L-undine 3',5'-diacetyl ester of 2'-deoxy-β-L-cytidme, 3',5'-diacetyl ester of 2'-deoxy-β-L-thymine, 3',5'-diacetyl ester of 2'-deoxy-β-L-adenosrne, 3',5'-diacetyl ester of 2'-deoxy-β-L-guanosine, 3',5'-diacetyl ester of 2'-deoxy-β-L-5-fluoro-cytidme, and 3',5'-diesters of 2'-deoxy-β-L-(cytidine, 5-fluorocytidme, guanosme, undme, adenosme, or thymine) wherein (l) the 3' ester is an ammo acid ester and the 5'-ester is an alkyl or aryl ester, (u) both esters are ammo acid esters, (in) both esters are independently alkyl or aryl esters, or (rv) the 3' ester is an alkyl or aryl ester and the 5'-ester is an ammo acid ester
(Formula Removed)
or its pharmaceutically acceptable salt thereof, wherein
In a second embodiment the invention provides the P~L nucleoside 3'-prodrug defined by formula (I)
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative,
R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or diphosphate, or a phosphate derivative,
X is O, S, SO2 or CH2, and
BASE is a purine or pyrrolidine base that may optionally be substituted
In a preferred embodiment, X is O
In one embodiment, R1 and/or R2 are an ammo acid residue
In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10Ru), wherein
R8 is the side chain of an ammo acid and wherein, as in proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and Rn are independently hydrogen, acyl (mcludmg an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl)
In another embodiment of the present invention, the β-L nucleoside 3'-prodrug is a β-L-2'-deoxypurine of the formula (Formula Removed)
or its pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-subshtuted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative,
R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative,
Y is OR3, NR3R4 or SR3, and
X1 and X2 are mdependently selected from the group consisting of H, straight chamed, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, halogen, OR5, NR5R6 or SR5, and
R3, R4, R5 and R6 are mdependently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), dialkylammoalkylene (in particular, dimethylammomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative
In one embodiment, the amino acid residue is of the formula C(O)C(R8)(R9)(NR10Ru), wherem
R8 is the side chain of an ammo acid and wherem, as in proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocychc moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and R11 are independently hydrogen, acyl (mcludmg an acyl derivative attached to R8) or alkyl (mcludmg but not limited to methyl, ethyl, propyl, and cyclopropyl)
In a particular embodiment, the β-L nucleoside 3'-prodrug is a β-L-2'-deoxyadenosine of the formula
(Formula Removed)
or its pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative,
R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substtuted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, and
R3 and R4 are mdependently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), dialkylarmnoalkylene (in particular, dimethylammomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative
In a preferred embodiment, R1 is H
In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10Rn)5 wherein
R8 is the side chain of an ammo acid and wherem, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and Ru are mdependently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl)
In another preferred embodiment, R2 is an amino acid residue, and in particular L-vahnyl
In one embodiment, R3 is hydrogen, and R4 is dimethylaminomethylene In another embodiment, R3 is hydrogen, and R is acetyl In another embodiment, R3 is hydrogen, and R4 is L-vahnyl
In another particular embodiment, the β-L nucleoside 3'-prodrug is β-L-2'-deoxyguanostne of the formula
(Formula Removed)
or its pharmaceutically acceptable salt thereof, wherem
R1 is hydrogen, straight chamed, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, arallcylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative,
R2 is selected from the group consisting of straight chamed, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, and
R5 and R6 are independently H, straight chamed, branched or cyclic alkyl (εspecially cyclopropyl), dialkylammoalkylene (in particular, dunethylammomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-subsututed aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate
derivative
In a preferred embodiment, R1 is H
In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10R"), wherem
(Formula Removed)
R is the side chain of an amino acid and wherein, as in proline, R8 can optionally be attached to R to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and R11 are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethvl, propyl, and cyclopropyl)
In another preferred embodiment, R2 is an ammo acid residue, and m particular L-vahnyl
In one embodiment, R5 is hydrogen, and R6 is dimethylammomethylene
In another embodiment, R5 is hydrogen, and R6 is acetyl
In another embodiment, R5 is hydrogen, and R6 is L-vahnyl
In another particular embodiment the β-L nucleoside 3'-prodrug is β-L-2'-deoxyinosine or pharmaceutically acceptable salt or prodrug thereof of the formula
(Formula Removed)
or its pharmaceutically acceptable salt thereof, wherem
R1 is hydrogen, straight chained, branched or cychc alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, and
R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO aryloxyalkyl, CO-substtuted aryl, alkylsulfonyl, arylsulfonvl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative
In a preferred embodiment, R1 is H
In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10R1,)) wherein
R8 is the side chain of an ammo acid and wherem, as in prolme, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and Rn are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl)
In another preferred embodiment, R2 is an ammo acid residue, and in particular L-valinyl
In another embodiment of the present invention, the β-L nucleoside 3'-prodrug is /?-L-2'-deoxypynmidme of the formula
(Formula Removed)
or its pharmaceutically acceptable salt thereof, wherem
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-ar\loxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative,
R2 is selected from the group consistmg of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonvl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative,
Y is OR3, NR3R4 or SR',
X1 is selected from the group consisting of H, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, halogen, OR5, NR*R6 or SR5, and
R3,R4,R5andR6 are independently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), dialkylamrnoalkylene (in particular, drmethylarnrnomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative
In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10R!1), wherein
R8 is the side chain of an ammo acid and wherein, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocychc moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and Ru are mdependently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl)
In one particular embodiment, the 0-L nucleoside 3'-prodrug is ß-L-2'-deoxycytidme of the formula
(Formula Removed)
or its pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, straight chamed, branched or cychc alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-an loxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl ammo acid residue, mono, di, or triphosphate, or a phosphate derivative,
R2 is selected from the group consisting of straight chamed, branched or cyclic alkyi CO-alkyl, CO-aryl CO alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative,
X1 is selected from the group consisting of H, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, halogen, OR5, NR5R6 or SR5, and
R3, R4, R5 and R6 are rndependently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), diaUcylammoalkylene (m particular, dimethylaminomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative
In one embodiment, X1 is hydrogen
In another embodiment, X1 is a halogen, namely fluorine, chlorine, bromine or iodine
In a preferred embodiment, R1 is H
In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10R11), wherein
R8 is the side chain of an ammo acid and wherein, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and R11 are independently hydrogen, acyl (rncluding an acyl derivative attached to R8) or alkvl (including but not limited to methyl, ethyl, propyl, and cyclopropyl)
In another preferred embodiment, R2 is an ammo acid residue, and in particular L-vahnyl
In one embodiment, R3 is hydrogen, and R4 is dimethylammomethylene
In another embodiment, R3 is hydrogen, and R4 is acetyl
In another embodiment, R3 is hydrogen, and R4 is L-valmyl
In another embodiment, the β-L-nucleoside 3'-prodrug is β-L-2'-deoxyundine of the formula
(Formula Removed)
or its pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative, and
R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono di, or triphosphate, or a phosphate derivative
In a preferred embodiment, R1 is H
In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10R'l), wherein
R8 is the side chain of an ammo acid and wherein, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and R11 are independently hydrogen, acyl (mcludmg an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl and cyclopropyl)
In another preferred embodiment, R2 is an amino acid residue, and in particular L-vaknyl
In another embodiment, the β-L-nucleoside 3'-prodrug is ß-L-thymidrne of the formula
(Formula Removed)
or its pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyL ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, and
R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative
In a preferred embodiment, R1 is H
In one embodiment, the ammo acid residue is of the formula CCO^CR^CR^CNR10^1), wherein
R8 is the side chain of an ammo acid and wherein, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and Rn are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl)
In another preferred embodiment, R2 is an ammo acid residue, and in particular L-vahnyl
the invention also provides combinations of at least two of the herein described prodrugs
The invention further provides at least one of the described 3'-prodrugs in combination or alternation with a second nucleoside that exhibits activity against hepatitis B, including but not limited to a parent drug of any of the prodrugs defined herein, 1 e 2'deoxy-β-L-nucleosides, including 2'-deoxy- β-L-cytidine, 2'-deoxy- β-Lthymine,2'-deoxy- β-L-adenosine,2'-deoxy- β-L-guanine,2'-deoxy- β—L-5-fluorocytidine Alternatively, the 3'-produrgs can be administered in combination or alternation with other anti-hepatitis B virus agent such as (-)-cis-2',3'-dideoxy-3'-thiacytidine, cis-2'3'-dideoxy-3'-thia-5-fluorocytidine, L-FMAU, adefovir, famciclovir, and entecivir, or any other compound that exhibits an EC50 of less than 10 or 15 micromolar in 2 2 15 cells, or their prodrugs or pharmaceutically acceptable salts
The invention further includes administering the prodrug in combination or alternation with an immune modulator or other pharmaceutically active modifier of viral replication, including a biological material such as protein, peptide, oligonucleotide, or gamma globulin, including but not limited to interferon,interleukin or an antisense oligonucleotides to genes which express or regulate hepatitis B replication
The efficacy of the parents of the anti-HBV compound can be measured according to the concentration of compound necessary to reduce the replication rate of the virus in vitro, according to methods set forth more particularly herein, by 50% (1 e the compound's EC 50 ) In preferred embodiments the parent of the prodrug compound exhibits an EC50 of less than 15 or preferably, less than 10 micromolar in vitro, when tested in 2 2 15 cells transferred with the hepatitis virion

In accordance with the present invention it relates to a A compound of the 3'-0-valmyl ester of 2'-deoxy-[beta]-L-cytidine
(Formula Removed)
or a pharmaceutically acceptable salt thereof
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure 1 a and lb are non-limiting illustrating examples according to the present invention of the synthesis of 3'- and 5'-vaniyl esters of 2'-deoxy-β-L-cytidme (β-L-dC) from 2'-deoxy-β-L-cytidine, respectively
Figure 2 is a non-limiting illustrative example according to the present invention of the synthesis of N4-acetyl-2'-deoxy-β-L-cytidine from 2'-deoxy-β-L-cytidine
Figure 3 is a non-limiting illustrative example according to the present invention of the synthesis of N-[(dmiethylamino)methylene]-2'-deoxy-β-L-cytidrne from 2'-deoxy-β-L-cytidine
Figure 4 is a non-limiting illustrative example according to the present invention of the synthesis of 3',5'-di-0-acetyl-2'-deoxy-β-L-cytidine from 2'-deoxy-β-L-cytidine
Figure 5 is a non-hmiting illustrative example accordmg to the present invention of the synthesis of 3',5'-di-0-vahnyl ester of 2'-deoxy-β-L-cytiduie from 2'-deoxy-β-L-
cytidine
Figure 6 is a non-hmiting illustrative example according to the present invention of the synthesis of N-(Boc-valmyl) ester of 2'-deoxy-β-L-cyhdine from 2'-deoxy-β-L-cytidme
Figure 7 is a non-limiting illustrative example according to the present invention of the synthesis of 3',5' N-tn-(L-valmyl)-2'-deoxy-β-L-cytidme from 3'55' N-tn-(Boc-L-valmyl)-2' -deoxy-β-L-cyudine
Figure 8 is a hne graph depicting a standard calibration technique useful for the determination of solubility of various nucleosides Figure 8a is the calibration curve determined for nature β-D-deoxynbocytosme Figure 8b is the calibration curve determined for the 3',5'-divalinyl ester of β-L-deoxynbocytosine
Figure 9a is a non-hmiting example of a HPLC profile used to assess the stability of the 3\5'-divahnyl ester of β-L-deoxynbocytosme at a pH of 7 42 The HPLC profile mdicates the presence of the 3',5'-divalmyl ester of β-L-deoxynbocytosine along with 3 active metabolites, the 3'-vahnyl ester of β-L-deoxynbocytosme, the 5'-vahnyl ester of β-L-deoxynbocytosine and L-dC Figure 9b is a hne graph depicting the relative concentrations of the 3\5'-divahnyl ester of β-L-deoxynbocytosme and its metabolites over time
Similarly, Figure 10a and 11a are non-lrmitmg examples of HPLC profiles used to assess the stability of the 3',5'-divahnyl ester of β-L-deoxynbocytosme at a pH of 7 20 and 4 51, respectively At these pH's, the HPLC profile mdicates the presence of the 3',5'-divahnyl ester of β-L-deoxynbocytosme along with 3 active metabolites, the 3'-vahnyl ester of B-L-deoxynbocytosine, the 5'-valinyl ester of β-L-deoxynbocytosme and L-dC
Figure 10b and lib are line graphs depicting the relative concentrations of the 3\5'-divahnyl ester of β-L-deoxynbocytosine and its metabolites over time
Figure 12 is a non-limiting example of a HPLC profile used to assess the stability of the 3',5'-divahnyl ester of β-L-deoxynbocytosine at a pH of 1 23 At this pH, the HPLC profile only indicates the presence of the 3',5'-divahnyl ester of β-L-deoxynbocytosine without any decomposition into any of its 3 active metabolites
Figure 13 is a line graph depicting the in vitro metabolism of 3',5'-divahnyl ester of β-L-deoxynbocytosine m human plasma
Figure 14 is a line graph depictmg the intracellular metabolism of β-L-deoxynbocytosme (L-dC) in HepG2 cells
Figure 15 is a line graph depictmg the intracellular accumulation of L-dC m primary human hepatocytes
Figure 16 is a bar graph depicting the antiviral dose response of L-dC upon treatment of a chronic hepatitis B virus infection for 28 days m the woodchuck model of chronic Hepatitis B virus infection
Figure 17 is a line graph depictmg the antiviral activity of L-dC in the woodchuck model of chrome hepatitis B virus infection
Figure 18 are lme graphs indicating the body weights of individual woodchucks treated for 28 days with L-dC (0 01-10 mg/kg/day) orally
Figure 19 are lme graphs indicating the body weights of individual woodchucks treated for 12 Weeks with L-dC (1 mg/kg/day) orally
Detailed Description of the Invention
The invention as disclosed herein is a compound, a method and composition for the treatment of hepatitis B virus in humans and other host animals The method mcludes the administration of an effective HBV treatment amount of a 3'-prodrug of a ß-L-nucleoside as desenbed herein or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable earner The compound of this invention either possesses
antiviral (1 e, anh-HBV) activity, or is metabolized to a compound that exhibits such activity
In summary, the present invention includes the following features
(a) β-L-2'-deoxy-nucleoside 3'-prodrugs, as described herein, and pharmaceutically acceptable salts, esters and compositions thereof,
(b) ß-L-2'-deoxy-nucleoside 3'-prodrugs as described herein, and pharmaceutically acceptable salts, esters and compositions thereof for use m the treatment or prophylaxis of a hepatitis B infection, especially in individuals diagnosed as having a hepatitis B infection or bemg at nsk of becoming infected by hepatitis B,
(c) use of these β-L-2'-deoxy-nucleoside 3'-prodrugs, and pharmaceutically acceptable salts, esters and compositions thereof in the manufacture of a medicament for treatment of a hepatitis B infection,
(d) pharmaceutical formulations comprising the j3-L-2'-deoxy-nucleoside 3'-prodrugs or pharmaceutically acceptable salts thereof together with a pharmaceutically acceptable earner or diluent,
(e) β-L-2'-deoxy-nucleoside 3'-prodrugs, or their pharmaceutically acceptable salts, esters and compositions as described herein substantially in the absence of the opposite enanuomers of the described nucleoside, or substantially isolated from other chemical entities,
(f) processes for the preparation of ß-L-2'-deoxy-nucleoside 3'-prodrugs, as described in more detail below,
(g) processes for the preparation of ß-L-2'-deoxy-nucleoside 3'-prodrugs substantially in the absence of enantiomers of the described nucleoside, or substantially isolated from other chemical entities,
(h) the treatment of a host infected with hepatitis B that includes the administration of an effective amount of a 3'-prodrug of a ß-L-2'-deoxy-nucleoside, its pharmaceutically acceptable salt, ester or composition with a second anti-hepatius B agent,
(l) the treatment of a host infected with hepatitis B that includes the administration of an effective amount of a 3'-prodrug of a β-L-2'-deoxy-nucleoside, its
pharmaceutically acceptable salt, ester or composition with the parent of a different β-L--2'-deoxynucleoside,
(j) the treatment of a host infected with hepatitis B that includes the administration of an effective amount of a 3'-prodrug of a β-L--2'-deoxy-cytidine, its pharmaceutically acceptable salt or ester with the parent of a second anti-hepatitis B agent,
(k) the treatment of a host infected with hepatitis B that mcludes the administration of an effective amount of the 3',5'-divalyl or diacetyl ester of ß-L-2'-deoxy-cytidine, or its pharmaceutically acceptable salt or ester thereof, with a second anti-hepatitis B agent, and
(1) the treatment of a host infected with hepatitis B that mcludes the administration of an effective amount of the 3',5'-divalyl or diacetyl ester of ß-L-2'-deoxy-cytidine, or its pharmaceutically acceptable salt or ester thereof, with β-L-2'-deoxy-thymidine, or its pharmaceutically acceptable salt
A particularly preferred combination is the 3',5'-prodrug ofß-L-dC (also referred to as L-dC) with the parent ß-L-dT (also referred to as L-dT), and in particular, the 3',5'-divalyl or 3',5'-diacetyl ester of ß-L-dC in combination with ß-L-dT The oral bioavailability of L-dC as the neutral base and the HC1 salt is low in rodents and non-human primates It has been discovered that there is significant competition of L-dC with other nucleosides or nucleoside analogs for absorption, or transport, from the gastrointestinal tract and competition of other nucleosides or nucleoside analogs for the absorption with L-dC In order to improve oral bioavailability and reduce the potential for drug-drug interaction, a pharmacokinetic screen in monkeys was established This screen identified 3'-prodrugs of L-dC that had higher oral bioavailability than the parent molecule and a reduced effect on the bioavailability of other nucleosides or nucleoside analogs used in combination Examples of such nucleosides or nucleoside analogs used m combination with the prodrugs of L-dC are L-dT, L-dA, lamivudme or FTC
It was discovered using this approach, that the 3',5'-divaline ester of L-dC had higher oral bioavailability than the parent L-dC and a reduced interaction with other nucleosides or nucleoside analogs when used in combination as compared to L-dC Pharmacokinetic
studies also showed that the 3',5'-divahne ester of L-dC was converted to the parent L-dC through de-estenfication in the gastrointestinal mucosa, blood or liver
The 3',5'-divahne ester of L-dC is apparently actively transported from the gastrointestinal lumen after oral delivery into the bloodstream by an ammo acid transporter function in the mucosa of the gastrointestinal tract This accounts for the increase in oral bioavailability compared to the parent L-dC that would be transported primarily by a nucleoside transporter function It would also explain the reduced competition for uptake of the 3',5'-divahne ester of L-dC with other nucleosides or nucleoside analogs that are transported by the nucleoside transporter function and not the ammo acid transporter function As partial de-estenfication of the divahne ester of L-dC occurs prior to complete absorption, the monovahne ester contmues to be absorbed using the ammo acid transporter function Therefore, the desired outcome of better absorption, or bioavailability, and reduced competition with other nucleosides or nucleoside analogs for uptake into the bloodstream is maintained
I Compounds Defined by this Invention
In a first embodiment, the 2'-deoxy-ß-L-nucleoside 3'-prodrug includes biologically cleavable moieties at both the 3' and 5' positions Preferred moieties are L-amino acid esters such as L-valyl, and alkyl esters such as acetyl This invention specifically mcludes 3',5'-L-amino acid-β-L-2'-deoxy nucleosides with any desired purine or pynmidme base, wherein the parent drug has an EC50 of less than 15 micromolar, and preferably less than 10 micromolar, in 2 2 15 cells, 3',5'-(alkyl or aryl)-β-L-2'-deoxy nucleosides with any desired purine or pynmidme base, wherein the parent drug has an EC50 of less than 15, and preferably less than 10 micromolar in 2 2 15 cells, and prodrugs of 3',5'-diesters of 2'-deoxy-β-L-nucleosides wherem (1) the 3' ester is an ammo acid ester and the 5'-ester is an alkyl or aryl ester, (11) both esters are ammo acid esters, (m) both esters are independently alkyl or aryl esters, and (IV) the 3' ester is independently an alkyl or aryl ester and the 5'-ester is an ammo acid ester, wherem the parent drug has an EC50 on dosmg of less than 15 micromolar in 2 2 15 cells,
Examples of 3'-prodrugs falling within the invention are 3,5'-L-vahne ester of 2'-deoxy-ß-L-cytidine, 3',5'-L-vahne ester of 2'-deoxy-β-L-thymine, 3',5'-L-valme ester of
2'-deoxy-β-L-adenosine, 3',5'-L-vakne ester of 2'-deoxy-β-L-guanosine, 3',5'-L-vahne ester of 2'-deoxy-β-L-5-fluoro-cytidine, 3',5'-L-vaIine ester of 2'-deoxy-β-L-undine, 3',5'-acetyl ester of 2'-deoxy-β-L-cytidine, 3',5'-acetyl ester of 2'-deoxy-β-L-thymine, 3',5'-acetyl ester of 2'-deoxy-β-L-adenosine, 3',5'-acetyl ester of 2'-deoxy-β-L-guanosrne, 3',5'-acetyl ester of 2'-deoxy-β-L-5-fluoro-cytidine, and 3',5'-diesters of 2'-deoxy-β-L-(cytidine, 5-fluorocytidine, guanosme, undine, adenosine, or thymine) wherein (1) the 3' ester is an amino acid ester and the 5'-ester is an alkyl or aryl ester, (n) both esters are ammo acid esters, (m) both esters are mdependently alkyl or aryl esters or (IV) the 3' ester is an alkyl or aryl ester and the 5'-ester is an amino acid ester
In one embodiment, the invention provides the β-L nucleoside 3'-prodrug defined by formula (I)
(Formula Removed)
or its pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative,
R2 is selected from the group consistmg of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative,
X is O, S, SO2 or CH2) and
BASE is a purine or pvnmidine base that may optionally be substituted
In a preferred embodiment, X is O
In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10Rn), wherein
R is the side chain of an amino acid and wherein, as in proline, R can optionally be attached to R1 to form a ring structure, or alternatively, Rs is an alkyl, aryl, heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and Ru are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl)
In a first subembodiment R2 is C(O)-alkyl (including lower alkyl) or aryl, and BASE is adenine, protected adenine, cytosme, protected cytosine or thymine
In a second subembodiment R2 is C(O)-lower alkyl and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine
In a third subembodiment R2 is C(O)-methyl and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine
In a fourth subembodiment R2 is C(O)C(R8)(H)(NR10Rn), and BASE is adenine protected adenine, cytosme, protected cytosine or thymine
In a fifth subembodiment R2 is C(O)C(R8)(H)(NR10Rn), R8 is isopropyl, at least one of R10 and Ru is hydrogen, and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine
In a sixth subembodiment R2 is C(O)C(R8)(H)(NR10Ru), R8 is an amino acid side chain, and BASE is adenine, protected adenine, cytosme, protected cytosme, or thymine
In a seventh subembodiment R2 is C(O)C(R8)(H)(NR,0Rn), R8 is a nonpolar amino acid side chain and BASE is adenine, protected adenine, cytosme, protected cytosme or thymine
Nonhmiting examples of subembodiments can be defined by formula (T) m which
(1) R2 is C(O)-methyl and BASE is adenine
(2) R2 is C(O)-methyl and BASE is protected adenine
(3) R2 is C(0>methyl and BASE is cytosme
(4) R2 is C(O)-methyl and BASE is protected cytosme
(5) R2 is C(O)-methyl and BASE is thymine
(6) R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is adenine
(7) R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected adenine
(8) R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is cytosme
(9) R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected cytosine
(10) R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is thymine
Li a eighth subembodiment X is O, R2 is C(O)-alkyl (including lower alkyl) or aryl, and BASE is adenine, protected adenine, cytosine, protected cytosine, or thymine
In a ninth subembodiment X is O, R2 is C(O)-lower alkyl and BASE is adenine, protected adenine, cytosme, protected cytosme or thymine
In a tenth subembodiment X is O, R2 is C(O)-methyl and BASE is adenine, protected adenine, cytosine, protected cytosme or thymine
In an eleventh subembodiment X is 0, R2 is C(O)C(R8)(H)(NR10Rn), and BASE is adenine, protected adenine, cytosme, protected cytosme or thymine
In a twelfth subembodiment X is O, R2 is C(O)C(R8)(H)(NR10Ru), R8 is isopropyl, at least one of R10 and Ru is hydrogen, and BASE is adenine, protected adenine, cytosme, protected cytosme or thymine
In a thirteenth subembodiment X is 0, R2 is C(O)C(R8)(H)(NR10Ru), R8 is an ammo acid side chain, and BASE is adenine, protected adenine, cytosme, protected cytosme, or thymine
In a fourteenth subembodiment X is O, R2 is C(O)C(R8)(H)(NR10Rn), R8 is a nonpolar ammo acid side chain, at least one of R5 and R6 is hydrogen and B is adenine, protected adenine, cytosme, protected cytosme or thymine
Nonkmiting examples of subembodiments can be defined by formula (I) m which
(1) X is O, R2 is C(O)-methyl and BASE is adenine
(2) X is O, R2 is C(O)-methyl and BASE is protected adenine
(3) X is O, R2 is C(O)-methyl and BASE is cytosme
(4) X is O, R2 is C(O)-methyl and BASE is protected cytosme
(5) X is 0, R2 is C(O)-methyl and BASE is thymine
(6) X is 0, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is adenine
(7) X is O, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected adenine
(8) X is 0, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is cytosine
(9) X is O, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected cytosine
(10) X is O, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is thymine
In a fifteenth subembodiment X is 0, R1 is hydrogen, R2 is C(O)-alkyl (mcludmg lower alkyl) or aryl, and BASE is adenine, protected adenine, cytosine, protected cytosine, or thymine
In a sixteenth subembodiment X is O, R1 is hydrogen, R2 is C(O)-lower alkyl and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine
In a seventeenth subembodiment X is O, R1 is hydrogen, R2 is C(O)-methyl and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine
In a eighteenth subembodiment X is O, R1 is hydrogen, R2 is C(O)C(R8)(H)(NR10Ru), and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine
In a nineteenth subembodiment X is O, R1 is hydrogen, R2 is C(O)C(R8)(H)(NR10Ru), R8 is isopropyl, at least one of R10 and Ru is hydrogen, and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine
In a twentieth subembodiment X is O, R1 is hydrogen, R2 is (XCOCCR)CHXNR^R11), R8 is an ammo acid side chain, and BASE is adenine, protected adenine, cytosine, protected cytosine, or thymine
In a twentx first subembodiment X is O, R1 is hydrogen, R2 is C(O)C(R8)(H)(NR10R ), R8 is a nonpolar ammo acid side chain, at least one of R5 and R6 is hydrogen and B is adenine, protected adenine, cytosine, protected cytosine or thymine
Nonhmiting examples of subembodiments can be defined by formula (I) in which
(1) X is O, R1 is Inarogen, R2 is C(O)-methyl and BASE is adenine
(2) X is O, R1 is Indrogen, R2 is C(O)-methyl and BASE is protected adenine
(3) X is 0, R1 is hydrogen, R2 is C(O)-methyl and BASE is cytosine
(4) X is 0, R1 is hydrogen, R2 is C(O)-methyl and BASE is protected cytosine
(5) X is 0, R1 is hydrogen, R2 is C(O)-methyl and BASE is thymine
(6) X is O, R1 is hydrogen, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is adenine
(7) X is O, R1 is hydrogen, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected adenine
(8) X is 0, R1 is hydrogen, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is cytosine
(9) X is O, R1 is hydrogen, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected cytosine
(10) X is O, Rl is hydrogen, R2 is C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is thymine
In a twenty-second subembodiment X is 0, R1 and R2 are independently C(O)-alkyl (mcludmg lower alkyl) or aryl, and BASE is ademne, protected adenine, cytosine, protected cytosine, or thymine
In a twenty-third subembodiment X is O, R1 and R2 are independently C(O)-lower alkyl and BASE is adenine, protected adenine, cytosine, protected cytosine or thymine
In a twenty-fourth subembodiment X is O, R1 and R2 are independently C(O)-methyl and BASE is ademne, protected ademne, cytosine, protected cytosine or thymine
In a twenty-fifth subembodiment X is O, R1 and R2 are independently C(O)C(R8)(H)(NR10Ru), and BASE is ademne, protected ademne, cytosine, protected cytosine or thymine
In a twent\ -sixth subembodiment X is O, R1 and R2 are independently C(O)C(R8)(H)(NR,0Rn), R8 is isopropyl, at least one of R10 and R11 is hydrogen, and BASE is ademne, protected ademne, cytosine, protected cytosine or thymine
In a twentv-seventh subembodiment X is O, R1 and R2 are independently C(O)C(R8)(H)(NRI0RH), R8 is an amino acid side chain, and BASE is ademne, protected ademne, cytosine, protected cytosine, or thymine
In a twenty-eighth subembodrment X is O, R1 and R2 are independently C(O)C(R8)(H)(NR,0Ru), R8 is a nonpolar ammo acid side chain, at least one of R5 and R6 is hydrogen and B is adenine, protected adenine, cytosine, protected cytosme or thymine
Nonhmiting examples of subembodiments can be defined by formula (I) in which
(1) X is O, R1 and R2 are independently C(O)-methyl and BASE is adenine
(2) X is O, R1 and R2 are independently C(O)-methyl and BASE is protected adenine
(3) X is O, R1 and R2 are independently C(O)-methyl and BASE is cytosme
(4) X is O, R1 and R2 are independently C(O)-methyl and BASE is protected cytosme
(5) X is O, R1 and R2 are independently C(O)-methyl and BASE is thymine
(6) X is O, R1 and R2 are independently C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is adenine
(7) X is O, R1 and R2 are mdependently C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected adenine
(8) X is O, R1 and R2 are mdependently C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is cytosine
(9) X is O, R1 and R2 are mdependently C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is protected cytosine
(10) X is O, R1 and R2 are mdependently C(O)C(R8)(H)(NH2), R8 is isopropyl and BASE is thymine
In another embodiment of the present invention, the 0-L nucleoside 3'-prodrug is a ß-L-2'-deoxypunne of the formula (Formula Removed)
or its pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyL CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or tnphosphate, or a phosphate derivative,
R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or tnphosphate, or a phosphate derivative,
Y is OR3, NR3R4 or SR3, and
X1 and X2 are independently selected from the group consisting of H, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, halogen, OR5, NR^6 or SR5, and
R3, R4, R5 and R6 are mdependently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), dialkylammoalkylene (in particular, dimethylammomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or tnphosphate, or a phosphate denvauve
In one embodiment, the amino acid residue is of the formula C(O)C(R8)(R9)(NRI0R11), wherein
R8 is the side chain of an amino acid and wherein, as in prolme, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and R11 are mdependently hydrogen, acyl (including an acyl denvative attached to R8) or alkyl (mcludmg but not limited to methyl, ethyl, propyl, and cyclopropyl)
In a particular embodiment, the β-L nucleoside 3'-prodrug is a ß-L-2'-deoxyadenosme of the formula
(Formula Removed)
or its pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative,
R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative, and
R3 and R4 are independently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), dialkylaminoalkylene (m particular, dimethylammomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative
In a preferred embodiment, R1 is H
In one embodiment, the amino acid residue is of the formula C^CCR^O^CNR10^1), wherein
R8 is the side chain of an ammo acid and wherem, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and R11 are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl)
In another preferred embodiment, R2 is an ammo acid residue, and in particular L-vahnyl
In one embodiment, R3 is hydrogen, and R is dimethylammomethylene In another embodiment, R3 is hydrogen, and R4 is acetyl In another embodiment, R3 is hydrogen, and R4 is L-vahnyl
In another particular embodiment, the β-L nucleoside 3'-prodrug is ß-L-2'-deoxyguanosme of the formula
(Formula Removed)
or its pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative,
R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, and
R5 and R6 are independently H, straight chamed, branched or cyclic alkyl (εspecially cyclopropyl), dialkylaminoalkylene (in particular, dnnethylammomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative
In a preferred embodiment, R1 is H
In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10Rn), wherein

R8 is the side chain of an ammo acid and wherein, as in proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryL heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and R11 are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl)
In another preferred embodiment, R2 is an ammo acid residue, and m particular L-vahnyl
In one embodiment, R5 is hydrogen, and R is dimemylaminomethylene
In another embodiment, R5 is hydrogen, and R6 is acetyl
In another embodiment, R5 is hydrogen, and R6 is L-vahnyl
In another particular embodiment, the β-L nucleoside 3'-prodrug is /8-I^2'-deoxyinosine or pharmaceutically acceptable salt or prodrug thereof of the formula
(Formula Removed)
or its pharmaceuticalh acceptable salt thereof, wherein
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-anloxyalkyl, CO-subsututed aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl amino acid residue, mono, di, or triphosphate, or a phosphate derivative, and
R2 is selected from the group consisting of straight chained, branched or cychc alkyl, CO-alkyl, CO-anl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative
In a preferred embodiment, R1 is H
In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10Rn), wherein
R8 is the side chain of an ammo acid and wherein, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and Rn are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl)
In another preferred embodiment, R2 is an ammo acid residue, and in particular L-valmyl
In another embodiment of the present invention, the |3-L nucleoside 3'-prodrug is /3-L-2'-deoxypynrnidine of the formula
(Formula Removed)
or its pharmaceuticalh acceptable salt thereof, wherein
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-ars 'oxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative,
R2 is selected from the group consisting of straight chained, branched or cychc alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative,
Y is OR3, NR3R4 or SR ,
X is selected from the group consisting of H, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, halogen, OR5, NR5R6 or SR5, and
R3, R4, R5 and R6 are independently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), dialkylarmnoalkylene (in particular, dimethylamrnomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate
derivative
In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10Rn), wherein
R is the side chain of an amino acid and wherein, as in proline, R can optionally be attached to R10 to form a rmg structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocychc moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and R11 are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl)
In one particular embodiment, the β-L nucleoside 3'-prodrug is jS-L-2'-deoxycytidine of the formula
(Formula Removed)
or its pharmaceuticallv acceptable salt thereof, wherein
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-arvloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative,
R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl,
arylsulfonyl, aralkyisulfonyl, amino acid residue, mono, di, or tnphosphate, or a phosphate derivative, and
R3 and R4 are independently H, straight chained, branched or cyclic alkyl (εspecially cyclopropyl), dialkylaminoalkylene (ni particular, dimethylaminomethylene), CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkyisulfonyl, amino acid residue, mono, di, or tnphosphate, or a phosphate derivative
In a preferred embodiment, R1 is H
In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10R11), wherein
R8 is the side chain of an ammo acid and wherein, as m proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and Ru are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl)
In another preferred embodiment, R2 is an ammo acid residue, and in particular L-vahnyl
In one embodiment, R3 is hydrogen, and R4 is dimethylammomethylene
In another embodiment, R3 is hydrogen, and R4 is acetyl
In another embodiment, R3 is hydrogen, and R4 is L-vahnyl
In another embodiment, the β-L-nucleoside 3'-prodrug is j3-L-2'-deoxyundme of the formula
(Formula Removed)

or its pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative, and
R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative
In a preferred embodiment, R1 is H
In one embodiment, the amino acid residue is of the formula C(O)C(R8)(R9)(NR10Rn), wherem
R8 is the side chain of an amino acid and wherem, as in proline, R8 can optionally be attached to R10 to form a ring structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and R11 are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl)
In another preferred embodiment, R2 is an amino acid residue, and in particular L-vahnyl
In another embodiment, the β-L-nucleoside 3'-prodrug is β-L-thymidine of the formula
(Formula Removed)
or its phannaceutically acceptable salt thereof, wherein
R1 is hydrogen, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyI, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, ammo acid residue, mono, di, or triphosphate, or a phosphate derivative, and
R2 is selected from the group consisting of straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, mono, di, or triphosphate, or a phosphate derivative
In a preferred embodiment, R1 is H
In one embodiment, the ammo acid residue is of the formula C(O)C(R8)(R9)(NR10Ru), wherein
R is the side chain of an ammo acid and wherem, as in proline, R can optionally be attached to R10 to form a nng structure, or alternatively, R8 is an alkyl, aryl, heteroaryl or heterocyclic moiety,
R9 is hydrogen, alkyl (including lower alkyl) or aryl, and
R10 and Rn are independently hydrogen, acyl (including an acyl derivative attached to R8) or alkyl (including but not limited to methyl, ethyl, propyl, and cyclopropyl)
In another preferred embodiment, R2 is an ammo acid residue, and in particular L-vahnyl
II Definitions and Use of Terms
The term alkvl, as used herein, unless otherwise specified, refers to a saturated straight, branched, or cyclic, primary, secondary, or ternary hydrocarbon of C1 to C10 and
specifically includes methyl, tnfluoromethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, /-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dunethylbutyl The term includes both substituted and unsubstituted alkyl groups Moieties with which the alkyl group can be substituted are selected from the group consisting of hydroxyl, amino, alkylamino, arylarnino, alkoxy, aryloxy, nitro, cyano, sulfomc acid, sulfate, phosphomc acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught m Greene, et al, Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, hereby incorporated by reference
The term lower alkyl, as used herein, and unless otherwise specified, refers to a Q to C4 saturated straight, branched, or if appropriate, a cyclic (for example, cyclopropyl) alkyl group, including both substituted and unsubstituted forms Unless otherwise specifically stated m this application, when alkyl is a suitable moiety, lower alkyl is preferred Similarly, when alkyl or lower alkyl is a suitable moiety, unsubstituted alkyl or lower alkyl is preferred
The term "protected" as used herein and unless otherwise defined refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent its further reaction or for other purposes A wide variety of oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis Non-limiting examples of protecting groups are taught in Greene, et al, Protective Groups m Organic Synthesis, John Wiley and Sons, Second Edition, 1991
The term aryl, as used herein, and unless otherwise specified, refers to phenyl, biphenyl, or naphth>l and preferably phenyl The term mcludes both substituted and unsubstituted moieties The aryl group can be substituted with one or more moieties selected from the group consisting of hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano sulfomc acid, sulfate, phosphomc 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 purine or pynmidme base mcludes, but is not limited to, adenine, N6-alkylpunnes, N^-acylpunnes (wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl), N6-
benzylpunne, N6halopurine, N6-vinylpunne, N6-acetylenic punne, punne,
N^hydroxyalkyl punne, N6-thioalkyl punne, N2-alkylpunnes, N2-alkyl-6-thiopunnes, thymine, cytosine, 5-fluorocytosine, 5-methylcytosnie, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4-mercaptopyrmidme, uracil, 5-halouracil, including 5-fluorouracil, C^-alkylpynmidines, C5-benzylpynmidines, C5-halopynmidines, C5-vinylpynmidine, C5-acetylemc pynrmdine, C5-acyl pynmidine, C5-hydroxyalkyl punne, C5-amidopynmidnie, C5-cyanopynmidine, C5-nitropynmidine, C5-aminopynmidine, N2-alkylpunnes, N2-alkyl-6-thiopunnes, 5-azacytidinyl, 5-azauracilyl, tnazolopyndinyl, lmidazolopyndinyl, pyrrolopyrimidinyl, and pyrazolopynmidinyl Punne bases include, but are not limited to, guanine, adenine, hypoxanthine, 2,6-diaminopunne, and 6-chloropunne Functional oxygen and mtrogen groups on the base can be protected as necessary or desired Suitable protecting groups are well known to those skilled m the art, and mclude rnmethylsilyl, dimethylhexylsilyl, r-butyldimethylsilyl, and /-butyldiphenylsilyl, tntyl, alkyl groups, and acyl groups such as acetyl and propionyl, methanesulfonyl, and β-toluenesulfonyl
The term acyl refers to a carboxyhc acid ester m which the non-carbonyl moiety of the ester group is selected from straight, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkyl including benzyl, aryloxyalkyl such as phenoxymethyl aryl includmg phenyl optionally substituted with halogen, C1 to C4 alkyl or C1 to C4 alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl including methanesulfonyl, the mono, di or Diphosphate ester, tntyl or monomethoxytntyl, substituted benzyl, tnalkylsilyl (ε g dimethyl-t-butylsilyl) or diphenylmethylsilyl Aryl groups m the esters optimally compnse a phenyl group The term "lower acyl" refers to an acyl group in which the non-carbon\ 1 moiety is lower alkyl
The term amino acid includes naturally occurring and synthetic α, ß  or δ ammo acids, and mcludes but is not limited to, ammo acids found in proteins, 1 e glycine, alanine, valine, leucme, isoleucme, methionine, phenylalanine, tryptophan, prolme, serine threonine, cysteine, tyosme, asparagine, glutamme, aspartate, glutamate, lysine, argimne and hishdme In a orefened embodiment, the ammo acid is in the L-configuration Alternatively, the amiro acid can be a denvauve of alanyl, vahnyl, leucmyl, isoleuccinyl, prohnyl, phemlalanun1, tryptophanyl, methiomnyl, glycmyl, sermyl, threomnyl, cystemyl, tyrosmyl, asparaginv glutammyl, aspartoyl, glutaroyl, lysmyl, argimnyl, hisudinyl, β-alanyl, β-valm\l, β-kucinyl, β-isoleuccinyl, β-prohnyl, β-phenylalamnyl, β-tryptophanyl,
β-methioninyl, β-glycmyl, β-sennyl, β-threoninyl, β-cysteinyl, β-tyrosmyl, β-asparaginyl, β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl or β-histidinyl
The term heteroaryl or heteroaromatic, as used herein, refers to an aromatic moiety that includes at least one sulfur, oxygen, nitrogen or phosphorus in the aromatic ring The term heterocyclic refers to a nonaromatic cyclic group wherein there is at least one heteroatom, such as oxygen, sulfur, nitrogen or phosphorus m the ring Nonhmiting examples of heteroanl and heterocyclic groups include furyl, furanyl, pyndyl, pynmidyl, thienyl, isothiazolyl, lmidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoqumolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl, benzumdazolyl, purmyl, carbazolyl, oxazolyl, thiazolyl, isothiazolyl, 1,2,4-thiadiazolyl, isooxazolyl, pyrrolyl qumazohnyl, cinnohnyl, phthalazmyl, xanthmyl, hypoxanthinyl, thiophene, furan, p>Trole, isopyrrole, pyrazole, imidazole, 1,2,3-tnazole, 1,2,4-tnazole, oxazole, isoxazole, thiazole, isothiazole, pynmidme or pyndazine, and ptendmyl, azindrnes, thiazole, isothiazole, 1,2,3-oxadiazole, thiazme, pyndine, pyrazme, piperazine, pyrrohdme, oxaziranes, phenazme, phenothiazine, morphohnyl, pyrazolyl, pyndazmyl, pyrazinyl, qumoxalmvl, xanthmyl, hypoxanthinyl, ptendmyl, 5-azacytidinyl, 5-azauracilyl, tnazolopyndinyl, lmidazolopyndmyl, pyrrolopynmidmyl, pyrazolopynmidmyl, adenine, N6-alkylpunnes, N6-benzyIpunne, N6-halopunne, N6-vmypunne, N6-acetylemc purine, N6-acyl punne,N6-hydroxyalkyl punne, N6-thioalkyl purine, thymine, cytosine, 6-azapynmidine, 2-mercaptopyrmidine, uracil, N5-alkylpynmidmes, NS-benzylpynmidines, N5-halopynmidmes, N5-vmylpynmidme, N5-acetylemc pynmidme, N5-acyl pynmidme, N5-hydroxyalkyl punne, and N6-thioalkyl punne, and isoxazolyl The heteroaromatic and heterocyclic moieties can be optionally substituted as descnbed above for aryl, including substituted with one or more substituent selected from halogen, haloalkyl, alkyl, alkoxy, hydroxy, carboxyl cenvauves, amido, ammo, alkylammo, dialkylarmno The heteroaromatic can be partially or totally hydrogenated as desired As a nonlimitmg example, dihydropyndme can be used m place of pyndine Functional oxygen and nitrogen groups on the heteroaryl gro jp can be protected as necessary or desired Suitable protecting groups are well known to those skilled m the art, and mclude tnmethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, tntyl or substituted tntyl, alkyl groups, acyl groups such as acet\ and propionyl, methanesulfonyl, and β-toluenelsulfonyl
As used herein, the term "substantially free of enantiomer" or "substantially m the absence of enantiomer" refers to a nucleoside composition that mcludes at least 95% to 98
% by weight, and even more preferably 99% to 100% by weight, of the designated enantiomer of that nucleoside In a preferred embodiment, m the methods and compounds of this invention, the compounds are substantially free of enantiomers
Similarly, the term "isolated" refers to a nucleoside composition that mcludes at least 85% or 90% by weight, preferably 95% to 98% by weight, and even more preferably 99% to 100% by weight, of the nucleoside, the remainder comprising other chemical species or enantiomers
The term "independently" is used herein to indicate that the variable that is independently applied vanes independently from application to application Thus, m a compound such as R"XYR", wherem R" is "independently carbon or nitrogen," both R" can be carbon, both R" can be nitrogen, or one R" can be carbon and the other R" nitrogen
The term host, as used herein, refers to a unicellular or multicellular organism in which the virus can replicate, including cell lines and animals, and preferably a human Alternatively, the host can be carrying a part of the hepatitis B viral genome, whose replication or function can be altered by the compounds of the present invention The term host specifically refers to infected cells, cells transfected with all or part of the HBV genome and animals, in particular, primates (including chimpanzees) and humans In most animal applications of the present invention, the host is a human patient Veterinary applications, in certain indications, however, are clearly anticipated by the present invention (such as chimpanzees)
The terms "pharmaceutically acceptable salts" and "pharmaceutcally acceptable complexes" are used throughout the specification to describe any pharmaceutically acceptable form of a nucleoside compound, which, upon administration to a patient, provides the nucleoside compound and exhibit minimal, if any, undesired toxicologjcal effects Pharmaceutically acceptable salts mclude those derived from pharmaceutically acceptable inorganic or organic bases and acids Nonhmitrng 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 acid, tannic acid, palmoic acid, algmic acid, polyglutamic acid, naphthalenesulforuc acids, naphthalenedisulfomc acids, and polygalacturonic acid, (b) base addition salts formed with cations such as those derived from alkali metals, those derived
from alkaline earth metals, sodium, potassium, zinc, calcium, bismuth, banum, magnesium, aluminum, copper, cobalt, mckel, cadmium, sodium, potassium, and the like, or with an organic cation formed from N,N-dibenzylethylene-diamine, ammonium, or ethylenediamme, or (c) combinations of (a) and (b), e g, a zinc tannate salt or the like
Pharmaceutically acceptable prodrugs refer to a compound that is metabolized, for example hydrolyzed or oxidized, m the host to form the compound of the present invention Typical examples of prodrugs include compounds that have biologically labile protecting groups on a functional moiety of the active compound Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, dephosphorylated to produce the active compound The compounds of this invention possess antiviral activity against HBV, or are metabolized to a compound that exhibits such activity
III Nucleotide Salt or Prodrug Formulations
In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compound as a pharmaceutically acceptable salt may be appropriate Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids, which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate and α-glycerophosphate Suitable inorganic salts may also be formed, including, sulfate, nitrate, bicarbonate and carbonate salts
Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made
Any of the nucleosides described herem can be administered as a nucleotide prodrug to increase the actmry, bioavailability, stability or otherwise alter the properties of the nucleoside A number of nucleotide prodrug hgands are known In general, alkylahon, acylahon or other lipophilic modification of the mono, di or triphosphate of the nucleoside
will increase the stability of the nucleotide Examples of substituent groups that can replace one or more hydrogens on the phosphate moiety are alkyl, aryl, steroids, carbohydrates, including sugars, 1,2-diacylglycerol and alcohols Many are described m R Jones and N Bischofberger, Antiviral Research, 27 (1995) 1-17 Any of these can be used in combination with the disclosed nucleosides to achieve a desired effect
The active β-L-3'-prodrug nucleoside can also be provided as a 5'-phosphoether lipid or a 5'-ether lipid, as disclosed m the following references, which are incorporated by reference herein Kucera, L S , N Iyer, E Leake, A Raben, Modest E K, D L W , and C Piantadosi 1990 "Novel membrane-interactive ether lipid analogs that inhibit infectious HIV-1 production and induce defective virus formation " AIDS Res Hum Retro Viruses 6 491-501, Piantadosi, C , J Marasco C J, S L Morns-Natschke, K L Meyer, F Gumus, J R Surles, K S Ishaq, L S Kucera, N Iyer, C A Wallen, S Piantadosi, and E J Modest 1991 "Synthesis and evaluation of novel ether hpid nucleoside conjugates for anh-HIV activity " J Med Chem 34 1408 1414, Hosteller, K Y , D D Richman, D A Carson, L M Stuhmiller, GM T \an Wyk, and H van den Bosch 1992 "Greatly enhanced inhibition of human immunodeficiency virus type 1 replication in CEM and HT4-6C cells by 3'-deoxythymidine diphosphate dimynstoylglycerol, a lipid prodrug of 3,-deoxythymidine " Antimicrob Agents Chemother 36 2025 2029, Hosetler, KY, LM Stuhmiller, HB Lenting, H van den Bosch, and D D Richman, 1990 "Synthesis and antiretrovrral activity of phosphohpid analogs of azidothymidine and other antiviral nucleosides " / Biol Chem 265 61127
Nonhmiting examples of U S patents that disclose suitable lipophilic subsbtuents that can be covalentlv incorporated mto the nucleoside, preferably at the 5'-OH position of the nucleoside or lipophilic preparations, include US Patent Nos 5,149,794 (Sep 22, 1992, Yatvin et al), 5,194,654 (Mar 16, 1993, Hostetler et al, 5,223,263 (June 29, 1993, Hostetler et al), 5,256,641 (Oct 26,1993, Yatvin et al), 5,411,947 (May 2,1995, Hostetler et al), 5,463,092 (Oct 31, 1995, Hostetler et al), 5,543,389 (Aug 6, 1996, Yatvin et al), 5,543,390 (Aug 6, 1996, Yatvin et al), 5,543,391 (Aug 6, 1996, Yatvin et al), and 5,554,728 (Sep 10, 1996, Basava et al), all of which are incorporated herein by reference Foreign patent applications that disclose lipophilic substituents that can be attached to the nucleosides of the present invention, or lipophilic preparations, include WO 89/02733, W0 90/00555, W0 91/16920, W0 91/18914, W0 93/00910, W0 94/26273, W0 96/15132, EP 0 350 287, EP 939170M 4, and W0 91/19721
The 3'-prodrug can be administered as any derivative that upon administration to the recipient, is capable of providing directly or indirectly, the 3'-prodrug of the parent compound or that exhibits activity itself Nonlimiting examples are the pharmaceutically acceptable salts (alternatively referred to as "physiologically acceptable salts"), and the N4 pynmidine or N2 and/or N-punne alkylated (m particular with drmethylarmnomethylene) or acylated (in particular with acetyl or annnoacetyl) derivatives of the active compound In one nonlimiting embodiment, the acyl group is a carboxyhc acid ester in which the non-carbonyl moiety of the ester group is selected from straight, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkyl including benzyl, aryloxyalkyl such as phenoxymethyl, aryl including phenyl optionally substituted with halogen, C1 to C4 alkyl or C1 to C4 alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl including methanesulfonyl, phosphate, including but not limited to mono, di or triphosphate ester, tntyl or monomethoxytntyl, substituted benzyl, tnalkylsilyl (ε g , dimethyl-5-butylsilyl) or diphenylmethylsilyl Aryl groups m the esters optionally comprise a phenyl group
Modifications of the 3'-prodrug or parent compound, and especially at the N4 pvnrmdmyl, or N2 and/or N6 purine positions, can affect the bioavailability and rate of metabolism of the active species, thus providing control over the delivery of the active species Further, the modifications can affect that antiviral activity of the compound, in some cases increasing the activity over the parent compound This can easily be assessed by preparing the derivative and testing its antiviral activity according to the methods described herein, or other method known to those skilled m the art
IV Stereochemistry
Since the 1' and 4' carbons of the sugar (referred to herein genencally as the sugar moiety) of the nucleosides are crural, their nonhydrogen substituents (CH2OR and the pynmidine or purine base, respectively) can be either cis (on the same side) or trans (on opposite sides) with respect to the sugar ring system The four optical isomers therefore are represented by the following configurations (when orienting the sugar moiety in a horizontal plane such that the "primary" oxygen (that between the C1' and C4'-atoms is m back) "ß" or "cis" (with both groups "up", which corresponds to the configuration of naturally occurring nucleosides, 1 e, the D configuration), "ß" or cis (with both groups "down", which is a nonnaturally occurring configuration, 1 e , the L configuration), "a "or

"trans" (with the C2 substituent "up" and the C5 substituent "down"), and "a " or trans (with the C2 substituent "down" and the C5 substituent "up")
The nucleosides of the present invention are of the β-L-configuration In a preferred embodiment, the 2'-deoxy-ß-L-nucleoside is administered substantially in the form of a smgle isomer, 1 e, at least approximately 95% m the designated stereoconfiguration
V Combination and Alternation Therapy
In combination therapy, effective dosages of two or more agents are administered together, whereas during alternation therapy an effective dosage of each agent is administered serially The dosages will depend on absorption, inactivation and excretion rates of the drug as well as other factors known to those of skill m the art It is to be noted that dosage values will also vary with the seventy of the condition to be alleviated It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions
For example, m any of the embodiments described herem, if the 3'-prodrug of the ß-I,-2'-deoxynucleoside of the present invention is administered m combination or alternation with a second nucleoside or nonnucleoside reverse transcriptase inhibitor that is phosphorylated to an active form, in one embodiment, a second compound is one that can be phosphorylated by an enzyme that is different from that which phosphorylates the selected β-L-2'-nucleoside of the present invention in vivo Examples of kinase enzymes are thymidine kinase, cytosme kinase, guanosine kinase, adenosme kinase, deoxycytidine kinase, 5'-nucleotidase and deoxy-guanosine kinase
Thus, in one embodiment the invention provides a combination of two or more nucleoside prodrugs of the present invention, preferably nucleosides that are phosphorylated by distinct enzymes, or that act through distinct biological pathways In another embodiment the invention provides at least one prodrug in combination or alternation with a nucleoside that exhibits activity against hepatitis B, including but not limited to a parent drug of any of the prodrugs defined herem, I e 2'-deoxy-β-L-nucleosides, including 2'-deoxy-β-L-cytidrne, 2'-deoxy-β-L-thymine, 2'-deoxy-ß-L-adenosine, 2'-deoxy-β-L-guamne, 2'-deoxy-β-L-5-fluoiocytidme, 2',3,-dideoxy-3'-thiacyhdine, 2',3'-dideoxy-3'-
thia-5-fluorocytidine Alternatively, the compounds of the present invention can also be administered in combination or alternation with any other known anti-hepatits B virus agent, such as entecivir, cis-2-hydroxymethyl-5-(5-fluorocytosin-l-yl)-l,3-oxathiolane, preferably substantially in the form of the (-)-optical isomer ("FTC", see WO 92/14743), the (-)-enantiomer of cis-2-hydroxymethyl-5-(cytosm-l-yl)-l,3-oxathiolane (3TC), β-D-l,3-dioxolane purine nucleosides as described m US Patent Nos 5,444,063 and 5,684,020, ß-D-dioxolane nucleosides such as β-D-dioxolanyl-guanine (DXG), β-D-dioxolanyl-2,6-diaminopunne (DAPD), and β-D-dioxolanyl-6-chloropunne (ACP), L-FDDC (5-fluoro-3'-thia-2',3'-dideoxycytidine), L-enantiomers of 3' fluoro-modifled /9-2'-deoxynbonucleoside 5'-tnphosphates, carbovir, mterferon, penciclovir and famciclovir, L-FMAU, famciclovir, penciclovir, BMS-200475, bis pom PMEA (adefovir, dipivoxil), lobucavir, ganciclovir, or nbavann, or any other compound that exhibits an EC50 of less than 15 micromolar m 2 2 15 cells, or their prodrugs or pharmaceuncally acceptable salts
Combination and alternation therapy can also be undertaken to combat drug resistance It has been recognized that drug-resistant variants of viruses can emerge after prolonged treatment with an antiviral agent Drug resistance most typically occurs by mutation of a gene that encodes for an enzyme used in viral replication The efficacy of a drug against hepatitis B infection can be prolonged, augmented, or restored by administering the compound in combination or alternation with a second, and perhaps third, antiviral compound that induces a different mutation from that caused by the principle drug Alternatively, the pharmacokinetics, biodistnbution or other parameter of the drug can be altered by such combination or alternation therapy In general, combination therapy is typically preferred over alternation therapy because it induces multiple simultaneous stresses on the virus
In another embodiment, the prodrug is administered in combination or alternation with an immune modulator or other pharmaceuncally active modifer of viral replication, including a biological material such as a protein, peptide, oligonucleotide, or gamma globulin, including but not limited to mterfereon, mterleukm, or an anusense oligonucleotides to genes which express or regulate hepatitis B replication
Any method of alternation can be used that provides treatment to the patient Nonhmiting examples of alternation patterns mclude 1-6 weeks of administration of an effective amount of one agent followed by 1-6 weeks of admmistration of an effective
amount of a second anti-HBV agent The alternation schedule can include periods of no treatment Combination therapy generally mcludes the simultaneous administration of an effective 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-HrV 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 m the appropriate circumstance m combination or alternation with anh-HIV medications
The second antiviral agent for the treatment of HIV, m one embodiment, can be a reverse transcriptase inhibitor (a "RTF), which can be either a synthetic nucleoside (a "NRTT') or a non-nucleoside compound (a "NNRTT') In an alternative embodiment, in the case of HIV, the second (or third) antiviral agent can be a protease inhibitor In other embodiments, the second (or third) compound can be a pyrophosphate analog, or a fusion binding inhibitor A list compiling resistance data collected in vitro and in vivo for a number of antiviral compounds is found in Schinazi, et al, Mutations m retroviral genes associated with drug resistance, International Antiviral News Volume 1(4), International Medical Press 1996
The active anti-HBV agents can also be administered m combination with antibiotics, other antiviral compounds, antifungal agents or other pharmaceutical agents administered for the treatment of secondary infections
VI Pharmaceutical Compositions
Humans suffering from any of the disorders described herein, including hepatitis B, can be treated by administering to the patient an effective amount of a 3'-prodrug of a ß-L-2'-deoxy-nucleoside of the present invention, or a pharmaceutically acceptable salt thereof, in the presence of a pharmaceutically acceptable earner or diluent The active materials can be administered by any appropriate route, for example, orally, parenterally, intravenously, rntradermally, subcutaneously, or topically, m liquid or solid form
The active compound is included in the pharmaceutically acceptable earner or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount of compound to mhibit viral replication in vivo without causing senous toxic effects m the patient treated By "inhibitory amount" is meant an amount of active ingredient sufficient
to exert an inhibitory effect as measured by, for example, an assay such as the ones described herein
A preferred dose of the compound for all of the abovementioned conditions will be in the range from about 1 to 50 mg/kg, preferably 1 to 20 mg/kg, of body weight per day, more generally 0 1 to about 100 mg per kilogram body weight of the recipient per day The effective dosage range of the pharmaceutically acceptable prodrug can be calculated based on the weight of the parent nucleoside to be dehvered If the prodrug exhibits activity in itself, the effective dosage can be estimated as above using the weight of the prodrug, or by other means known to those skilled in the art
The compound is conveniently administered m 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 plasma concentrations of the active compound of from about 0 2 to 70 µM, preferably about 1 0 to 10 µM This may be achieved, for example, by the intravenous injection of a 0 1 to 5% solution of the active ingredient, optionallv in salme, or administered as a bolus of the active ingredient
The concentration of active compound m 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 seventy of the condition to be alleviated It is to be further understdod 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 mtended to limit the scope or practice of the claimed composition The acnve ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of tune
A preferred mode of administration of the active compound is oral Oral compositions will generally include an inert diluent or an edible earner They may be enclosed in gelatin capsules or compressed mto tablets For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used m 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, Pnmogel, or com starch, a lubricant such as magnesium stearate or Sterotes, a glidant such as colloidal silicon dioxide, a sweetening agent such as sucrose or saccharin, or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring When the dosage unit form is a capsule, it can contain, m addition to material of the above type, a liquid earner such as a fatty oil In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents
The compound can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors
The compound or a pharmaceutically acceptable derivative or salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, anhinflammatones, protease inhibitors, or other nucleoside or nonnucleoside antiviral agents Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methyl parabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as 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 m ampoules, disposable svnnges or multiple dose vials made of glass or plastic
If admmisterea intravenously, preferred carriers are physiological saline or phosphate buffered salme (PBS)
In a preferred embodiment, the active compounds are prepared with earners that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems Biodegradable,
(Formula Removed)
Alternatively, the 3'-derivative is derived from an aminoacyl moiety The key starting material for this process is also an appropriately substituted β-L nucleoside The ß-L nucleoside can be purchased or can be prepared by any known means including standard coupling reactions with an L-sugar moiety
These aminoacyl derivatives can be made by first selectively protecting the 5'-hydroxyl with a suitable oxygen protecting group, such as an acyl or silyl protecting group, and optionally protecting any free amine m the heterocyclic or heteroaromatic base Subsequently, the free 3'-hydroxyl can be coupled to an N-protected a or ß ammo acid
Subsequently, the β-L-nucleoside is coupled to the aminoacyl using standard coupling reagents that promote the coupling Some non-lrmitmg examples of coupling reagents are Mitsunobu-type reagents (ε g dialkyl azodicarboxylates such as dnsopropyl azodicarboxylate and diethyl azodicarboxylate) with tnphenyl phosphine or various types of carbodiimides
The coupling reaction can be earned out at any temperature that achieves the desired results, 1 e, that is suitable for the reaction to proceed at an acceptable rate without promoting decomposition or excessive side products
Am reaction solvent can be selected that can achieve the necessary temperature and that can solubihze the reaction components Non-hmiting examples are any aprotic solvent including, but not limiting to, alkyl or halo-alkyl solvents such as hexane, cyclohexane, dichloromethane or dichloroethane, toluene, acetone, ethyl acetate, dithianes, THF, dioxane,
acetonitnle, diethyl ether, pyridine, dirnethylformarnide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide, or any combination thereof
Scheme 1 is a non-limiting example of the preparation of a β-L-3'-aminoacyl-nucleoside denved from L-deoxynbonucleoside (Scheme Removed)
B Method for the preparation of ß-L-5'-denvatives of β-L-nucleosides
β-L-5'-derivatives of a β-L-nucleoside can be made by any means known in the art, particularly by known methods to protect primary alcohols with acyl moieties, 1 e via an anhydride or with the aid of a coupling agent As a non-limiting example, the β-L-5'-denvatives can be prepared according to the following reaction sequence
(Sequence Removed)
In a preferred embodiment, the 5'-derivative is denved from an aminoacyl moiety The key starting material for this process is an appropriately substituted β-L-nucleoside
The β-L-nucleoside can be purchased or can be prepared by any known means including standard coupling reactions with an L-sugar moiety, such as deoxynbose The aminoacyl derivatives can be made by selectively coupling an amino acid to a β-L-nucleoside, preferably without any additional protection of the nucleoside The coupling reaction can be achieved using appropriate coupling reagents that promote the coupling Some non-lrmitmg examples of coupling reagents are Mitsunobu-type reagents (e g dialkyl azodicarboxylates such as dnsopropyl azodicarboxylate and diethyl azodicarboxylate) with tnphenyl phosphine or various types of carbodiimides
The coupling reaction can be earned out at any temperature that achieves the desired results, 1 e, that is suitable for the reaction to proceed at an acceptable rate without promoting decomposition or excessive side products
Any reaction solvent can be selected that can achieve the necessary temperature and that can solubihze the reaction components Non-hmiting examples are any aprotic solvent including, but not limiting to, alkyl or halo-alkyl solvents such as hexane, cyclohexane, dichloromethane or dichloroethane, toluene, acetone, ethyl acetate, dithianes, THF, dioxane, acetomtnle, diethyl ether, pyridine, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide, or any combination thereof
Scheme 2 is a non-lrmitmg example of the preparation of a β-L-5'-aminoacyl-nucleoside derived from L-deoxynbonucleoside
(Scheme Removed)
C Method for the preparation of β-L-3' 5'-bis-0-denvatives of β-L-nucleosides
β-L-3',5'-bis-0-denvatives of a β-L-nucleoside can be made by any means known in the art, particularly by known methods to protect primary and secondary alcohols with acyl moieties, 1 e via an anhydride or with the aid of a coupling agent As a non-limiting
example, the 3',5'-bis-0-derivatives can be prepared according to the following reaction sequence
(Sequence Removed)
In a preferred embodiment, the 3',5'-bis-ßderivative is derived from an aminoacyl moiety The key starting material for this process is also an appropriately substituted ß-L-nucleoside The 3',5'-bis-0-derivatives of the β-L-nucleosides can be purchased or can be prepared by any known means including standard coupling reactions with an L-sugar moiety, such as deoxvnbose Subsequently, the free 3'- and 5'-hydroxyl can be coupled to a N-protected a or ß ammo acid The coupling reaction can be achieved using appropriate coupling reagents that promote the coupling Some non-hrmtmg examples of coupling reagents are Mitsunobu-type reagents (ε g dialkyl azodicarboxylates such as dusopropyl azodicarboxylate and diethyl azodicarboxylate) with tnphenyl phosphme or various types of carbodirmides
The coupling reaction can be earned out at any temperature that achieves the desired results, 1 e, that is suitable for the reaction to proceed at an acceptable rate without promoting decomposition or excessive side products
Any reaction solvent can be selected that can achieve the necessary temperature and that can solubilize the reaction components Non-hrmtmg examples are any aprotic solvent including, but not limiting to, alkyl or halo-alkyl solvents such as hexane, cyclohexane, dichloromethane or dichloroethane, toluene, acetone, ethyl acetate, dithianes, THF, dioxane,
acetonitnle, diethyl ether, pyridine, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide, or any combination thereof
Scheme 3 is a non-hrmting example of the preparation of a β-L-3',5'-di-aminoacyl-nucleoside denved from L-deoxynbonucleoside
Scheme 3
(Scheme Removed)
D Optional method for the extension of the aminoacvl moiety
The title compounds can be made by reacting the 3' and 5'-hydroxyl with a suitable derivative, such as an acyl, and in particular an aminoacyl group If the nucleoside is denvatized with an aminoacyl moiety, it may be desirable to further couple the free amine to a N-protected a or ß ammo acid The coupling reaction can be achieved using appropriate coupling reagents that promote the couplmg Some non-limiting examples of coupling reagents are Mitsunobu-type reagents (e g dialkyl azodicarboxylates such as dnsopropyl azodicarboxylate and diethyl azodicarboxylate) with tnphenyl phosphine or various types of carbodiirnides
The couplmg reaction can be earned out at any temperature that achieves the desired results, l e, that is suitable for the reaction to proceed at an acceptable rate without promoting decomposition or excessive side products
Any reaction solvent can be selected that can achieve the necessary temperature and that can solubihze the reaction components Non-limiting examples are any aprotic solvent including, but not limiting to, alkyl or halo-alkyl solvents such as hexane, cyclohexane, dichloromethane or dichloroethane, toluene, acetone, ethyl acetate, dithianes, THF, dioxane, acetomtnle, diethyl ether, pyndine, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide, or any combination thereof
E Optional method for denvatization of the heteroaromatic or heterocyclic base
The title compounds can be made by optionally protecting any free amnio in the heterocyclic or heteroaromatic base, for example N4-cytosine, N6-adenrne or N2-guanme For example, the amine can be protected by an acyl moiety or a dialkylaminomethylene moiety by the following general protocol
(Scheme Removed)
The protection can be earned out at any temperature that achieves the desired results, 1 e, that is suitable for the reaction to proceed at an acceptable rate without promoting decomposition or excessive side products
Any reaction solvent can be selected that can achieve the necessary temperature and that can solubihze the reaction components Non-hmiting examples are any aprotic solvent including, but not limiting to, alkyl or halo-alkyl solvents such as hexane, cyclohexane, dichloromethane or dichloroethane, toluene, acetone, ethyl acetate, dithianes, THF, dioxane, acetomtnle, diethyl ether, pyridine, dimethylformarmde (DMF), dimethylsulfoxide (DMSO), dimethylacetamide, or any combmation thereof
Subsequently, the free 3'-hydroxyl can be coupled to a N-protected a or ß ammo acid The coupling reaction can be achieved using appropriate coupling reagents that promote the coupling Some non-hmiting examples of couplmg reagents are Mitsunobu-type reagents (ε g dialkyl azodicarboxylates such as dusopropyl azodicarboxylate and diethyl azodicarboxylate) with tnphenyl phosphine or various types of carbodnmides
The couplmg reaction can be earned out at any temperature that achieves the desired results, 1 e, that is suitable for the reaction to proceed at an acceptable rate without promoting decomposition or excessive side products
Any reaction solvent can be selected that can achieve the necessary temperature and that can solubilize the reaction components Non-limiting examples are any aprotic solvent including, but not limiting to, alkyl or halo-alkyl solvents such as hexane, cyclohexane, dichloromethane or dichloroethane, toluene, acetone, ethyl acetate, dithianes, THF, dioxane,
acetonitnle, diethyl ether, pyridine, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide, or any combination thereof
In an alternate embodiment, the N4- or N^-acyl derivative is derived from an aminoacyl moiety, and can be prepared accordmg to the following reaction sequence, by optionally protecting the free hydroxyls, followed by a condensation reaction with the appropriately protected ammo ester, and the removal of the hydroxyl protecting groups, if necessary
(Scheme Removed)
Examples
Example 1 N-mMTr-2'-deoxy-β-L-cytidine (1, Figure 1)
β-L-dC (1 g, 4 40 mmol) was taken up m dry pyridine (44 ml) After transient protection with tnmethylsilyl group (TMSC1, 3 34 ml, 26 4 mmol) followed by addition of mMTrCl (3 38 mg, 11 mmol) and 4-dimethylaminopyndine (DMAP, 540 mg, 4 40 mmol) the reaction mixture was stirred for 3 days at room temperature {A Nyilas, C Glemarec, J Chattopadhyaya, Tetrahedron Lett 1990, 46. 2149-2164} After sodium bicarbonate extraction the organic layer was washed with water, evaporated and taken up m dioxane (40 mL) Aqueous ammonia (8 5 ml) was added dropwise and the reaction mixture was stirred overnight After evaporation of all volatile materials, the solid residue was purified on silica gel column {eluent stepwise gradient of MeOH (0-10%) in CH2CI2}, giving the desired compound 1 (1 02 g, 46 5%) as a foam 1H NMR (DMSO-d6) δ ppm 8 39 (br s, 1H, N#, D20 exchangeable), 7 70 (d, 1H, H-6, J= 7 3 Hz), 7 4-6 8 (m, 14H, (C6H5)(C6H4)OCH ), 6 23 (d, 1H, H-5, J = 7 3 Hz), 6 02 (t, 1H, H-l', J = 6 5 Hz), 5 16 (d, 1H, 0#-3\ J = 3 8 Hz, D20 exchangeable), 4 9 (br s, 1H, OH-5\ D20 exchangeable), 4 1 (m, 1H, H-3'), 3 7 (m, 4H, H-4', OCH,), 3 5 (m, 2H, H-S\ H-5"), 2 1-1 8 (2m, 2H, H-V, H-2"), FABO, (GT) m/e 498 (M-H), 382 (B), 226 (M-mMTr), FAB>0 (GT) δ00 (M+H)+, 273 (mMTr), UV (EtOH 95) λmax = 279 ran,λmax= 250 nm
Example 2 S'-L-N-(tert-butoxycarbonyI) valine ester of 4NmMTr-2'-deoxy-β-L-cytidme (2, Figure 1)
To a solution of compound 1 0- g, 2 00 mmol) in dry DMF (34 ml) were added successively 4-dunethylammopyndine (DMAP, 37 mg, 0 3 mmol), N-(tert-butoxy-carbonyl)-L-vahne (Boc-Val-OH, 587 mg, 2 7 mmol), and NN -dicyclohexylcarbodiumde (DCC, 660 mg, 3 2 mmol) {L M Beauchamp, G F Orr, P De Miranda, T Burnette, T A Krenitsky, Antiviral Chem Chemother 1992, 3, 157-164 } The solution was stirred at room temperature After 40h, the reaction mixture was recharged with additional DMAP (37 mg, 0 3 mmol), Boc-Val-OH (587 mg, 2 7 mmol) and DCC (660 mg, 3 2 mmol) and stirred at room temperature for 40h The mixture was filtered, the DMF was removed from the filtrate under reduced pressure, and the residue was chromatographed on a silica gel column {eluent stepwise gradient of MeOH (0-10%) m CH2CI2} to afford the desired compound 2 (515 mg, 37%) as a foam 1H NMR (DMSO-d6) δ ppm 8 44 (br s, 1H, Nff, D20 exchangeable), 7 7-6 8 (m, 15H,, H-6 and (C6H5)2C(C6H4)OCH3), 6 26 (d, 1H, H-5, J = 73 Hz), 606 (t, 1H, H-1', J = 6 6 Hz), 5 7 (bs, 1H, OH-3\ D2O exchangeable), 42-40 (m, 3H, H-V, H-4' and CH), 3 8-3 9 (m, 2H, H-5\ H-5"), 3 7 (s, 3H,, OCH3), 2 0-1 9 (m, 3H, H-T, H-2", CH), 1 36 (s, 9H, (CH3)3C), 0 86 (m, 6H, (CH3)2CH), FAB0 (GT) 384 (B+2H)+, 273 (mMTrf, 57 (CH3)3C)+,UV (EtOH 95) λmax = 279 nm, λmax = 249 nm
Example 3 5'-L-valine ester of 2'-deoxy-ß-L-cytidine hydrochloride (3, Figure 1)
Compound 2 (500 mg, 0 715 mmol) was dissolved in a 20% solution of tnfluoroacetic acid in CH2CI2 (25 ml) and tnisopropylsilane (1 47 ml, 71 5 mmol) was added The reaction mixture was stirred at room temperature for 1 h and the valine ester was precipitated m Et20 as the tnfluoroacetate salt After several coevaporations with water, the precipitate was taken up m water (2 ml), treated with a saturated solution of HC1 m dioxane (20 ml) and evaporated under reduced pressure This treatment was repeated 3 times and the desired compound 3 was finally precipitated m ether (207 mg, 73%) as the hydrochloride salt 1H NMR (DMSO-d6) δ ppm 9 7 (br s, 1H, 1/2NH2, D20 exchangeable), 8 6 (br s, 4H, 1/2NH, NH3, D20 exchangeable), 7 98 (d, 1H, , H-6 J = 7 8 Hz), 617 (d, 1H, H-5, J = 7 8 Hz), 6 11 (pt, 1H, H-\'), 5 5 (bs, OCH3), 2 3-2 1 (m, 3H, H-T, H-2 CH) 0 94 (dd, 6H, (CH3)2CH, J = 3 7 and 6 6 Hz), FABO, (GT) m/e 361 (M+Cl), 325 (M-H), 116 (Val-H), 110 (B), 216 (BocVal-H), FAB>0 (GT) δ53 (2M+H)+, 327 (M+H)+, 112 (B+2H)+, )+, {α}D20 - 28 57 (c = 0 49 in DMSO), UV (EtOH 95) λmax = 272 ran (ε 8700), \™ = 255 nm (s 7600), HPLC it = 8 37nun (gradient from 0 to 50% CH3N in 20 mM tnethyl ammonium acetate buffer programmed over a 30 mm period with a flow rate of 1 ml/mm)
Example 4 ^V'-Acetyl-2'-deoxy-ß-L-cytidme (4, Figure 2)
To a suspension of the nucleoside, ß-L-dC (415 mg, 1 83 mmol) in NN-dmiethylformamide (9 2 ml) was added acetic anhydride (207 µl 2 20 mmol) and the mixture was stirred at room temperature for 24h [V Bhat, B G Ugarkar, V A Sayeed, K Grimm, N Kosora, P A Domenico, E Stacker, Nucleosides & Nucleotides, 1989, 8 (2), 179-183] After removal of the DMF under reduced pressure, the resulting residue was purified by silica gel column chromatography [eluant 15% MeOH in CH2CI2] to afford the desired compound (310 mg, 63%) which was crystallized from ethanol, rap 128-170°C, 1H
NMR (DMSO-4) δ ppm 10 86 (s, 1H, NH, D2O exchangeable), 8 31 (d, 1H,, H-6, J = 7 5 Hz), 7 18 (d, 1H, H-S, J = 7 5 Hz), 6 09 (t, 1H, HA ', J = 6 3 Hz), 5 25 (d, 1H, OH-3', D20 exchangeable, J = 4 2 Hz), 5 03 (t, 1H, OH-5', D20 exchangeable, J = 5 0 Hz), 4 1-4 2 (m, 1H, H-3'), 3 8 (m, 1H, H~4\ 3 4-3 6 (m, 2H, 2H, H-5\ H-5"), 2 2-2 3 (m, 1H, H-2), 2 08 (s, 3H, CH3), 2 0-1 9 (m 1H, H-Z"), FAB0 (GT) δ08 (3M+H)+, 539 (2M+H)+. 362 (M+G+H)+» 270 (M+H)+. 154 (B+2Hf> 117 (S)+, UV (H20) λmax = 297 nm (ε 8300), λmax = 270 nm (ε 3500), U - 245 nm (ε 14400), λmax= 226 nm (ε 5800), [α]D20 - 81 31 (c = 1 07 in DMSO)
Example 5 N4- [(DI methylamin o)methylene]-2 '-deoxy-ß-L-cytidme (5, Figure 3)
The title compound was prepared according to a published procedure developed for the preparation of the corresponding D-enantiomer [S G Kerr, and TI Kalman, J Phaim Sci 1994, 83, 582-5S6] A solution of L-dC (500 mg, 2 20 mmol) in DMF (4 8 ml) was treated with dimeth\l!ormamide dimethylacetal (2 8 ml, 21 08 mmol), and stirred at room temperature overmgn The solution was evaporated under reduced pressure, and
coevaporated with ethanol Crystallization from ethanol/ether yielded the title compound (501 2 mg, 81%) as light yellow crystals mp 174-176°C (lit 188-190°C for the D-enantiomer), 1H NMR (DMSO-d6) δ ppm 8 60 (s, 1H, N=CH), 8 00 (d, 1H, H-6), 6 15 (t, J= 6 6 Hz, 1H, H-l'), 5 96 (d, J = 7 2 Hz, 1H, H-5), 5 22 (d, J = 4 2 Hz, 1H, OH-31), 5 01 (t, J = 5 2 Hz, 1H, Ott-50 (GT) δ47 (3M -H)+, 565 (2M+H)+, 283 (M+H), FAB Example 6 3',5'-Di-0-acetyl-2'-deoxy-β-L-cytidine (6, Figure 4)
The title compound has been synthesized in one step starting from the L-dC and following a procedure developed by Bremer et al [R G Bremer, W Rose, J A Dunn, J E Mae Diarmid and J Bardos, J Med Chem 1990, 33, 2596-2603] for the preparation of the D-enanuomer A solution of L-dC (765 mg, 3 37 mmol) and acetyl chloride (960 (A, 13 48 mmol) m glacial acetic acid (4 8 ml) was stirred at room temperature for 10 mm, then dry chloroform (3 5 ml) was added and the starring was continued for 24h The solution was evaporated under reduced pressure and coevaporated with ethanol Crystallization from ethanol yielded 78% of the desired compound, mp 192 193°C (lit 187-189°C for the D-enantiomer [Bremer et al J Med Chem 1990, 33, 2596-2603]), 1H NMR (DMSO-d6) δ ppm 9 8 and 8 7 (2 br s, 6 08 (t, 1H, H-\\ J = 6 7 Hz), 5 2 (m, 1H, H-3'), 4 3-4 1 (m, 3H, H-4', H-5', H-5"), 2,4-2,5 (m, 2H, H-2', H-2"), 2 06 and 2 03 (2 s, 6H, 2 CH3), FAB (CH3COO), FAB>0 (GT) 623 (2M+H)+. 312 (M+H)+» 201 (S)+, 112 (B+2H)+> 43 (CH3CO)+, [α]D20 36 27 (c = 1 02 in DMSO), UV (MeOH) max = 277 nm (ε 9900), min= 246 nm (ε 5000)
Example 7 3',5'-L-V-(t-Butoxycarbonyl)valine diester of 2'-deoxy-β-L-cytidine (9, Figure 5)
A solution o: N-[(dimethylamino)methylene]-2'-deoxy-ß-I-cytidme (7, 500 mg, 1 77 mmol) in DMT (35 ml) was treated with Boc-Val-OH (1 31 g, 6 03 mmol), DMAP
(86 5 mg, 0 71 mmol), l-β-dimethylaminopropyl)-3-ethylcarbodimude hydrochlonde (EDC) (1 36 g, 7 09 mmol), and stirred at room temperature for 40 hours Additional quantities of Boc-Val-OH (655 mg, 3 01 mmol), DMAP (43 2 mg, 0 35 mmol), EDC (680 mg, 3 55 mmol) were added, and the solution was stirred for an additional 20 hours After evaporation under reduced pressure, the residue was taken up m CH2CI2, and extracted several times with water The organic layer was washed with brine (100 ml), dried (Na2SO4), and evaporated under reduced pressure to give 8 as a crude material, which was used for the next step without further purification The residue was taken up in dioxane (18 ml), treated with aq 26% NH4OH, and stirred at room temperature for 1 hour The solution was evaporated under reduced pressure, and the residue was purified by chromatography on silica gel using a stepwise gradient of MeOH (0-5%) m CH2CI2, to give the title compound (698 7 mg, 58% from 9) 1H NMR (DMSO-d6) δ ppm 7 58 (d, 1H, H-6), 7 29-7 18 (m, 4H, NH-Boc and NH2), 6 20 (t, J= 6 6 Hz, 1H, H-l'), 5 75 (d, J = 7 3 Hz, 1H, H-5), 5 20 (br s, 1H, H-3'), 4 29 (m, 2H, H-5' and H-5"), 4 14 (br s, 1H, H-4'), 3 86 (m, 2H, CH-NH-Boc), 2 31-2 21 (m, 2H, H-2' and H-2"), 2 13-1 98 (m, 2H, CH(iPr)), 1 38 and 1 36 (2s, 18H, tBu), 0 88 and 0 85 (2 d, J = 6 8 Hz, 12H, CH(CH3)2), l3C NMR (DMSO-d6) δ ppm 172 67 and 172 46, 166 41, 156 64 and 155 70, 141 39, 95 43, 85 78, 82 03, 79 14, 75 57, 64 90,
60 37 and 6011, 37 40, 30 33, 29 00, 19 83-19 12, FAB>0 (GT) 626 (M+H)+> 112 (B+2H)+, 255 (M-Boc)', FABO, (GT) m/z 1249 (2M-H), 624 (M-H)
Example 8 3,5'-L-Valine ester of 2'-deoxy-β-L-cytidine hydrochloride (10, Figure 5)
A solution of 9 (675 mg, 1 08 mmol) m dioxane (30 ml) was treated with a solution of 26% HC1 in dioxane (30 ml), and stirred at room temperature for 1 hr 55 The resulting white suspension was evaporated under reduced pressure The white solid residue was taken up m the minimal amount of MeOH and precipitated in ether to give the title compound 10 as a white sohd mp 187°C (decomp ), 1H NMR (DMSO-d6) δ ppm 9 79 (br s, 1H, 1/2NH2), S 72 (br s, 7H, 1/2NH2 and NH3+), 8 04 (d, 1H, H-6), 6 21 (d, J = 7 8 Hz, 1H, H-5), 6 16 (t, J = 6 9 Hz, 1H, H-l'), 5 39 (m, 1H, H-3'), 4 50-4 40 (m, 3H, H-4', H-5' and H-5"), 3 90 (2 br d, 2d, CH-NH), 2 63-2 50 (2m, 2H, H-2' and H-2"), 2 21 (m, 2H, CH(iPr)), 1 02-0 94 (r 12H, CH(CH3) 2) I3C NMR (DMSO-efe) δ ppm 169 50 and 168 94, 161 02, 148 50, 145 26, 95 18, 87 19, 82 15, 76 14, 65 77 and 65 59, 58 12 and 58 07,
37 00, 30 16,19 26-18 51, FAB>0 (GT) 426 (M+H)+, 112 (B+2H)+, FAB Example 9 N-Boc-Valmyl ester of 2'-deoxy-ß-L-cytidine (13, Figure 6)
A mixture of L-dC (1 80 g, 7 92 mmol) and tnethylamine (8 8 ml, 63 14 mmol) in anhydrous THF (80 ml) was treated with chlorotnmethylsilane (6 ml, 47 28 mmol) and stirred at room temperature overnight The reaction was quenched by addition of an aqueous saturated solution of NH4CI (26 ml) and water (10 mL) The aqueous layer was extracted three rimes with EtOAc The organic layers were combined, washed with bnne, dried (Na2SO4) and evaporated under reduced pressure to give a crude light yellow foam-oil containing 11, which was used for the next step without further purification This residue was taken up in CH2CI2 (104 ml), treated with iV-(tert-butoxycarbonyl)-L-vahne (Boc-Val-OH, 1 72 g, 7 90 mmol), benzotnazol-l-yloxy-tns(dunethylammo) phosphonium hexafluorophosphate (BOP, 4 20 g, 9 50 mmol), tnethylannne (2 2 ml, 15 78 mmol), and stirred at room temperature for 2 days The solution was diluted with EtOAc and extracted twice with sat NaHC03 The organic layer was dried (Na2SO4) and evaporated under reduced pressure to give 12 as a crude material, which was used for the next step without further purification This residue was taken up in dioxane (80 ml), treated with aq 26% NH4OH solution, and stirred at room temperature for 6h45 The solution was evaporated under reduced pressure, coevaporated with absolute EtOH, and the residue was purified by chromatography on silica gel, using a stepwise gradient of MeOH (5-10%) in CH2CI2, to give the title compound 13 as a foam (1 64 g, 48 5% overall yield) 1H NMR (DMSO-d6) δ ppm 10 88 (s, 1H, NH-4), 8 40 (d, 1H, H-6), 7 26 (d, J = 7 4 Hz, 1H, H-5), 7 06 (d, J= 8 2 Hz, 1H, CH-NH-Boc), 6 15 (t, J= 6 3 Hz, 1H, H-l1), 5 32 (d, J = 4 2 Hz, 1H, OH-3'), 5 09 (t, J= 5 2 Hz, 1H, OH-5') 4 27 (m, 1H, H-3'), 4 06 (pt, J = 7 5 Hz, 1H, CH-NH-Boc), 3 91 (m, 1H, H-4') 3 63 (m 2H, H-5' and H-5"), 235 (m, 1H, H-2"), 2 06 (m, 2H, H-2' and CH(CH3)2), 1 43 (s, m, tBu), 0 92 (pt, J = 6 6 Hz, 6H, CH(CTh)?), 13C NMR (DMSO-rf6) δ ppm 174 41, 162 94 15647, 155 24, 146 10, 96 06, 88 79, 87 10, 79 09, 70 75, 6178, 61 55, 41 74, 30 63, 29 02, 19 91 and 19 10, FAB>0 (GT) δ53 (2M+H)+, 427 (M+H)+ 311 (B+2H)+, 255 (M-Boc)+, FAB
Example 10 3',5'-N-Tnvalyl-2'-deoxycytidine (14, Figure 7)
The starting material, 3',5'-N-tri(Boc-valyl)-2'-deoxycyhdine was dissolved in CH2CI2, but there was some insoluble material so the sample was filtered through Perhta This resulted in an increase in the volume of the CH2CI2 used The HCl/dioxane reagent was then added with stirring Within a few seconds some bubbling could be observed m the solution and then the mixture became cloudy The mixture was stirred at room temperature for about 1 hr During this time the precipitate became more crystalline The mixture was quickly filtered, the filtercake was washed with CH2CI2, and then it was dried on the pump to give, 0 16g (69%) of cream-white crystals The reagents and conditions are more explicitly described below in Table 1
(Table Removed)
Example 11 HPLC 4ssay Method for DiBocValyI-2'-dC and DiBocVaIyl-2'-dU
A 10mg/mL sample was made by dissolving the desired compound in absolute ethanol The solution was then diluted with a solution that contained 50% MeOH and 50% KH2PO4 (0 015M, pH=3 30-3 50) until a concentration of 0 16mg/mL was obtained (Note all solvents used were degasified before use ) 20µL of the solution was then immediately mjected into an HPLC column from WATERS (NOVAPAK C18 - 4pm - 3,9 X 150mm) The flow rate was set at lmL/min with a column temperature of 35°C To detect the compounds, the wavelength detection was set at 275nm for Di-Boc 2'dC, 260nm for Di-Boc2'dU and 204 for impurities after 15 minutes The column was run with KH2P04 (0 015M, pH=3 30 - 3 50, adjusted with H3PO4 10% v/v) in Pump A and HPLC grade acetonitnle in Pump B The gradient pattern is indicated in Table 2
Table 2 (Table Removed) VIII Anti-HBV Activity of the Active Compounds
Human DNA polymerases and mitochondrial function were not affected by L-dC in vitro L-dC was non-cytotoxic to human peripheral blood mononuclear cells (PBMCs), bone marrow progenitor cells and numerous cell lmes of human and other non-human mammalian origin
The antiviral activity and safety of L-dC was investigated in two studies using the woodchuck model of chrome hepatitis B infection In the initial study, woodchucks chronically infected with WHV (>10n genome equivalents/ML serum) were treated with a liquid formulation of L-dC by the oral route once a day for 28 days Control animals received lamivudine or the liquid formulation without drug In the L-dC treated groups, viral load decreased in a dose-dependent manner At the highest dose tested (10 mg/kg/day), viral load decreased by as much as 6 logs from baseline by quantitative polymerase chain reaction (PCR) assay Post-treatment virus rebound was detected by Week 2 All animals gamed weight and there was no drug-related toxicity observed during the four-week treatment phase or eight-week post-treatment follow-up period
The in vitw 50% effective concentration (EC50) for reduction in extracellular viral deoxyribonucleic acid (DNA) by L-dC was 0 24 µM against HBV and 0 87 µM_ against DHBV In addition L-dC reduced intracellular HBV DNA rephcative intermediates (RI) with an EC50 of 0 51 µM The 90% effective concentration (EC90) of L-dC against HBV replication was 1 07 µM Structure activity relationships (SAR) show that replacement of
the hydroxyl group in the 3' position (3'-OH) broadened the antiviral activity from hepadnaviruses to other viruses including human immunodeficiency virus (HIV) and certain herpes viruses Substitution in the base decreased antiviral potency and selectivity
The second study using the woodchuck model of chronic hepatitis B virus infection tested the antiviral effect and safety of L-dC in combination with a second investigational nucleoside [ß-L-2'-deoxythymidme (L-dT)] Included m this study was a treatment group in which L-dC was used as a single agent (1 mg/kg/day) There was no drug-related toxicity observed for L-dC alone or in combination with L-dT during the 12-week treatment phase or 12-week post-treatment follow-up period There were no changes in body weight relative to control animals or serum chemistry and hematologic parameters End-of-treatment liver biopsies showed no histomorphological evidence of fatty changes (microvesicular steatosis) The combination of L-dC (1 mg/kg/day) plus L-dT (1 mg/kg/day) was synergistic and reduced viral load by up to 8 logs from baseline
Antiviral nucleosides and nucleoside analogs exert their antiviral effect as intracellular triphosphate derivatives at the level of the viral polymerase during virus replication Like natural nucleosides (D-deoxycytidine and D-thymidrne) and antiviral nucleoside analogs (ε g, lamivudine and zidovudine), L-dC was activated rntracellularly by phosphorylation In human hepatocytes, deoxycytidrne kinase (dCK) was responsible for the dose-dependent initial conversion of L-dC to a 5'-monophosphate (MP) derivative L-dC-MP was then converted to a 5'-diphosphate (DP) form, which was subsequently converted to the predominant intracellular 5'-triphosphate (TP) metabolite The L-dC-TP level reached 72 4 joM in HepG2 cells exposed to 10 µM L-dC (90 1 µM in primary human hepatocytes) at 24 hours and had an intracellular half-life of 15 5 hours In endogenous polymerase assays, L-dC-TP inhibited the vinon-associated DNA polymerase of WHV with a 50% inhibitory concentration (IC50) of 1 82 µM The detailed mechanism of inhibition of HBV DNA polymerase by L-dC is under investigation Exposure of HepG2 cells or human hepacytes in pnman culture to L-dC also produced a second TP derivative, 0-L-2'-deoxyundrne 5'-tnphosphate (L-dU-TP) The L-dU-TP level reached 18 2 |iM in HepG2 cells exposed to 10 µM L-dC (43 5 µM in primary human hepatocytes) at 24 hours In endogenous polymerase assays, L-dU-TP inhibited vmon-associated DNA polymerases of WHV with an IC50 of 5 26 uM
In primary human hepatocyte cultures and in a human hepatoma cell line (HepG2), the major metabolite of L-dC was L-dC-TP Exposure of these cells to L-dC also ted to the formation of L-dU-TP In vitro pharmacological assays showed that L-dC-TP inhibited hepadnaviral DNA synthesis with an IC50 of 1 82 mM, against vinon-associated DNA polymerase L-dU-TP inhibited hepadnaviral DNA synthesis with an IC50 of 5 26 µM L-dC-TP and L-dU-TP did not inhibit human DNA polymerases α, ß and  up to concentrations of 100 µM, the highest concentration tested
The ability of the active compounds to inhibit the growth of virus in 2 2 15 cell cultures (HepG2 cells transformed with hepatitis virion) can be evaluated as described in detail below
A summary and description of the assay for antiviral effects in this culture system and the analysis of HBV DNA has been described (Korba and Milman, 1991, Antiviral Res , 15 217) The antiviral evaluations are performed on two separate passages of cells All wells, m all plates, are 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 5-fold (for HBV virion DNA) or 3 0-fold (for HBV DNA replication intermediates) from the average levels for these HBV DNA forms in untreated cells are considered to be statistically significant (P Typical values for extracellular HBV virion DNA m untreated cells range from 50 to 150 pg/ml culture medium (average of approximately 76 pg/ml) Intracellular HBV DNA rephcation intermediates m untreated cells range from 50 to 100 µg/pg cell DNA (average approximately 74 pg/ftg 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 (Korba and Milman, 1991, Antiviral Res, 15 217)
The maimer in which the hybridization analyses are performed results in an equivalence of approximately 1 0 pg of intracellular HBV DNA to 2-3 genomic copies per cell and 1 0 pg/ml of extracellular HBV DNA to 3 x 105 viral particles/mL
Examples
Example 12 Solubility Study
The solubility of natural deoxynbocytosme (D-dC), the 3'-vahnyl ester of L-dC and the 3',5'-divalmyl estei of L-dC in water was compared The solubility of L-dC was assessed first by analyzing the HPLC data (i e area under the curve) by successive injections of various well-known concentrations of ß-L-dC, as shown m Table 3 The HPLC was run on a Nova-Pack CI 8 column (3 9 x 150 mm) on a gradient of 0 to 25% of CH3CN in 20 mM tnethylammonium acetate buffer (TEAAc) programmed over a fifteen minute period with a flow rate of 1 mL per minute The concentration of the solution versus the area under the cun e produced a linear relationship with y=4150049477 x -4334 46845 (Figure 8a)
Table 3 (Table Removed)
From this, a saturated solution was prepared with natural deoxynbocytosme (D-dC), 3 samples were taken and injected mto the HPLC The concentration of this saturated solution was determined to be 1 07, 1 08 and 0 96 mol/L, therefore, the saturated solution had an average saturated concentration of 1 03 mol/L or 272 g/L The results are tabulated in Table 4
Table 4 (Table Removed)

Similarly, the solubility of 3'-valmyl ester hydrochloride of β-L-dC m water was evaluated The calibration curve was determined by successive injections of various concentrations of the 3'-Valrnyl ester hydrochloride of β-L-dC into the HPLC and measuring the area under the curve, as shown in Table 5 Again, the HPLC was run on a Nova-Pack CI 8 column (3 9 x 150 mm) on a gradient of 0 to 25% of CH3CN m 20 mM tnethylammonium acetate buffer (TEAAc) programmed over a fifteen minute period with a flow rate of 1 mL per minute The concentration of the solution versus the area under the curve produced a linear relationship with y=3176423963 x -33051 63
Table 5 (Table Removed)
From this, a saturated solution was attempted for 3'-valinyl ester hydrochloride of ß-L-dC, however, one was not obtained Therefore, the maximum quantity of 3 '-vahnyl ester hydrochloride of β-L-dC readily available in the laboratory was dissolved in water 3 samples were collected, and were determined from the area under the curve from the HPLC, to have an average concentration of 1 013, 0 996 and 1 059 mol/L The results are tabulated m Table 6 (Table Removed)
Table 6 (Table Removed)
All three resul s fell within the predicted range calculated from the calibration curve, indicating complete solubility of the compound at those high concentrations, indicating that a saturated solution o" this sample is greater than the average of the three samples, l e greater than 1 023 mo L or 408g/L
The solubility of 3,5'-divalrnyl ester hydrochloride of β-L-dC in water was evaluated The call-ration curve was determined by successive injections of various
concentrations of the 3',5'-divaknyl ester hydrochloride of β-L-dC into the HPLC and measuring the area under the curve, as shown in Table 7 The HPLC was run on a Nova-Pack CI 8 column (3 9 x 150 mm) on a gradient of 0 to 25% of CH3CN in 20 mM tnethylammomum acetate buffer (TEAAc) programmed over a fifteen minute period with a flow rate of 1 mL per minute The concentration of the solution versus the area under the curve produced a linear relationship with y=3176423963 x -33051 63 (Figure 8b)

Table 7
(Table Removed)
From this, a saturated solution was attempted for 3\5'-divalinyl ester hydrochloride of β-L-dC, however, one was not obtamed Therefore, the maximum quantity of 3',5'-divahnyl ester hydrochloride of β-L-dC readily available in the laboratory was dissolved in water 3 samples were collected, and were determined from the area under the curve from the HPLC, to have an average concentration of 2 8, 2 4 and 2 4 mol/L The results are tabulated in Table 8 (Table Removed)
All three results fell within the predicted range calculated from the calibration curve, indicating complete solubility of the compound at those high concentrations, indicating that a saturated solution of this sample is greater than the average of the three samples, l e more than 2 5 mol/L or 133" g/L
Similar solubility studies were done on 5'-valmyl ester hydrochloride of β-L-dC (more than 5 1 moll or 1664 g/L) and 3'5'-diacetyl ester hydrochloride of β-L-dC (3 3 mol/L or 1148 g/L) The cumulative results are tabulated in Table 9 (Table Removed)
Example 13 Log P Study - Phosphate Buffer
Approximately 1 5 mg of D-dC was dissolved in 2 2 mL of 0 02 M phosphate buffer solution (A, 100 mL, pH 7 2), made from a mixture of monobasic potassium phosphate solution (28 5 mL) and dibasic potassium phosphate solution (71 5 mL), saturated with octanol-1 (B) To 1 mL of this solution, 1 mL of octanol-1 (B) saturated with 0 02 M phosphate buffer solution (A) was added The resultant mixture was shaken and centnfuged, three samples from each phase was collected and analyzed by HPLC, as shown in Table 10 The HPLC was run on a Nova-Pack C18 column (39x150 mm) on a gradient of 0 to 25% of CH3CN m 20 mM tnethylammomum acetate buffer (TEAAc) programmed over a fifteen minute period with a flow rate of 1 mL per mmute It was found that the log P of D-dC is -1 41, therefore, D-dC prefers water to octanol
Table 10 (Table Removed)

Similarly, approximately 1 5 mg of L-dC-3'-valrne ester hydrochloride was dissolved in 2 5 mL of 0 02 M phosphate buffer solution (A, 100 mL, pH 7 2), made from a mixture of monobasic potassium phosphate solution (28 D mL) and dibasic potassium
phosphate solution (71 5 mL) The solution was then saturated with octanol-1 (B) To 1 mL of this solution, 1 mL of octanol-1 (B) saturated with 0 02 M phosphate buffer solution (A) was added The resultant mixture was shaken and centnfuged, three samples from each phase was collected and analyzed by HPLC, as shown in Table 11 The HPLC was run on a Nova-Pack CI 8 column (3 9 x 150 mm) on a gradient of 0 to 25% of CH3CN in 20 mM tnethylammomum acetate buffer (TEAAc) programmed over a fifteen minute period with a flow rate of 1 mL per minute
Table 11 (Table Removed)
It was found that the log P of L-dC-3'-valine ester hydrochloride is -1 53, therefore, L-dC-
3'-valine ester prefers water to octanol to a greater degree than D-dC
Log P values were calculated for L-dC-5'-valine ester hydrochloride and L-dC-3',5'-divalme ester hvdrochlonde The results are tabulated m Table 12 However, it should be noted that the log P value for L-dC-3',5'-divahne ester hydrochloride is probably lower than the one measured (-0 86) Significant conversion of the divahne ester into the 3'- or 5'-monovahnyl ester or even L-dC was observed during the experiment 50% of conversion of L-dC-3',5'-divaline ester hydrochloride was detected m the aqueous phase and 14% m the organic phase This conversion is due to the instability of the esters in the phosphate buffer at a pH of 7 (see examples 15 and 16) (Table Removed) Table 12 (Table Removed) Example 14 Log P' Study - MdhQ Water
In order to avoid the conversion of the divahne ester into the mono esters and L-dC, an alternate log P study was performed using MilhQ water (A') instead of the phosphate buffer (pH of 6 5 instead of 7 2) It is important to note that only the hydrochloride form of the divaknyl ester can be considered m water Approximately 1 5 mg of L-dC-3',5'-divahnyl ester hydrochloride was dissolved in 2 2 mL of MilhQ water (A', pH 6 5) saturated with octanol-1 (B) To 1 mL of this solution, 1 mL of octanol-1 (B) saturated with MilhQ water (A') was added The resultant mixture was shaken and centnfuged, three samples from each phase was collected and analyzed by HPLC, as shown in Table 13 The HPLC was run on a Nova-Pack CI8 column (3 9 x 150 mm) on a gradient of 0 to 25% of CH3CN in 20 mM tnethylammonium acetate buffer (TEAAc) programmed over a fifteen minute period with a flow rate of 1 mL per minute It was found that the log P' of the 3',5'-divahne under these conditions was -2 72, indicating the strong effect of the counter ions rn the phosphate buffer No conversion of the divahne to the monoesters or L-dC was observed m either the aqueous or organic phases
Table 13 (Table Removed)
Similarly, approximately 15 mg of L-dC-5'-valmyl ester hydrochloride was dissolved in 2 2 mL of MilhQ water (A', pH 6 5) saturated with octanol-1 (B) To 1 mL of this solution, 1 mL of octanol-1 (B) saturated with MilhQ water (A') was added The resultant mixture was shaken and centnfuged three samples from each phase was collected and analyzed by HPLC, as shown in Table 14 The HPLC was run on a Nova-Pack CI8 column (3 9 x 150 mm) on a gradient of 0 to 25% of CH3CN m 20 mM tnethylammonium
acetate buffer (TEAAc) programmed over a fifteen minute period with a flow rate of 1 mL per minute It was found that the log P of the 5'-valine under these conditions was -2 75, again a value lower than found in the log P study using the phosphate buffer
Table 14 (Table Removed)
Under these conditions, the log P' values for L-dC-5'-valinyl ester hydrochloride and L-dC-3',5'-divalmvl ester hydrochloride are very similar (Table 15)
Table 15 (Table Removed)
Example 15 Stabilitv Study at pH 7 4
The rate of decomposition of each metabolite of L-dC-3' -valine ester hydrochloride was calculated The half-life of L-dC-3'-valine ester hydrochloride at pH of 7 40 was determined to be 7 hours m a 0 2M Tns-HCl solution at 37 °C In these conditions, L-dC-3'-valine ester hydrochloride is simply transformed to L-dC No cytosine was detected, thus, there was no detectable glycoside bond breakage
Similarly, the rate of decomposition of each metabolite of L-dC-3',5'-divahne ester hydrochloride was calculated The half-life of L-dC-3',5'-divahne ester hydrochloride at
pH of 7 42 was determined to be 2 4 hours in a 0 2M Tns-HCl solution at 37 CC In these conditions,L-dC-3',5'-divaline ester hydrochloride is partially hydrolyzed into the 3'- and 5'-vahnyl-L-dC, which are later transformed into L-dC No cytosine was detected, thus, there was no detectable glycoside bond breakage (Scheme 4, Figures 9a and 9b)
(Scheme Removed) 4
Example 16 Stability Study at pH 7 20
The half-life of I-dC-3',5'-divaline ester hydrochloride at pH of 7 20 was determined to be 2 2 hours in a 20 mM phosphate buffer In these conditions,L-dC-3',5'-divahne ester hydroch-onde is partially hydrolyzed into the 3'- and 5'-vahnyl-L-dC, which are later transformed into L-dC No cytosine was detected, thus, there was no detectable glycoside bond breakage (Scheme 5, Figures 10a and 10b)
Scheme 5 (Scheme Removed)
Example 17 Stability Study at pH 4 5
The half-life of L-dC-3'-valine ester hydrochloride at pH of 4 5 was determined to be 8 6 days in a 20 mM acetate buffer Again, L-dC-3'-valine ester hydrochloride is simply transformed to L-dC No cytosine was detected, thus, there was no detectable glycoside bond breakage
Similarly, the half-life of L-dC-3',5'-divalme ester hydrochloride at pH of 4 51 was determined to be 44 hours m a 20 mM acetate buffer In these conditions,L-dC-3',5'-divahne ester hydrochloride is partially hydrolyzed into the 3'- and 5'-vahnyl-L-dC, which are later transformed into L-dC No cytosine was detected, thus, there was no detectable glycoside bond breakage (Figures 11a and lib)
Example 18 Stability Study at pH 1 2
The half-life of L-dC-3'-valine ester hydrochloride at pH of 1 2 was determined to be greater than 48 hours m a 135 mM KC1-HC1 buffer solution No cytosine was detected, thus, there was no detectable glycoside bond breakage
Similarly, stability studies were done on L-dC-5'-valine ester hydrochloride This compound is fully stable at a pH of 1 2, with no other metabolites or decomposition products detected for up to 23 hours No glycosidic bond breakage was detected up to 2 days in solution
The 3',5'-diacetyl ester of L-dC was found to have a half life at a pH of 1 2 of 11 2 hours Under these conditions the compound was partially hydrolyzed into the 3'- or 5'-denvatives, which were later transformed into L-dC No glycosidic bond breakage was detected up to 2 days m solution
The 3',5'-divahnyl ester of L-dC was found to be fully stable at a pH of 1 23 since no other compounds were detected up to 48 hours m these conditions No glycosidic bond breakage was detected up to 2 days m solution (Figurel2)
Alternatively, when the N4 position of L-dC is masked with dimethylammo-methylene or acetyl, the half-kfe of the compound at a pH of 1 2 is only 26 minutes or 50 minutes, respectively
Example 19 Single Dose Bioavailability of L-dC in the Cynomologus Monkey
The pharmacokinetics of L-dC following IV and oral admrxustration of L-dC to cynomologus monkeys was determined In this study, 10 mg/kg tritium ([3H]) radiolabeled L-dC was administered to three cynomologus monkeys as a single IV dose Following a six week washout period, the same three monkeys received an identical oral dose of L-dC Blood samples for pharmacokinetic analysis were collected pre-dose and at 0 25, 0 5, 1, 2, 3, 6, 8 and 24 hours after dosing Unne samples for pharmacokinetic analyses were collected via pan catch pre-dose and over the following intervals post-dose 0-2, 2-4, 4-8, and 8-12 hours, and then over 12-hour intervals thereafter through 336 hours post-dose The drug was detected and the concentration determined using a reverse-phase high-performance liquid chromatography technique The blood and unne drug level data were analyzed by a non-modeling mathematical method and AUC's derived by the linear trapezoidal rule
Intravenous administration of L-dC The mean Cmax of L-dC after IV administration was 95 7 µM and occurred at the earliest sampling time (15 minutes post-dose) for all animals I-dC plasma concentrations decreased over time following the IV bolus with a
mean t½ of 1 59 hours The total clearance (CL) and renal clearance (CLR) of L-dC following IV administration averaged 0 53 L/h/kg and 0 46 L/h/kg, respectively The mean apparent volume of distribution (Vd) of 1 22 L/kg indicated that L-dC had a significant extravascular tissue distribution
Urinary excretion was rapid, with 71% of the administered dose recovered within 2 hours L-dC accounted for the majority (94%) of the dose recovered in the unne The renal clearance (0 46 L/h/kg) accounted for 87% of total L-dC clearance and suggested that renal excretion was the major route of elimination
L-dU was detected m the plasma and unne, indicating that metabolic elimination of L-dC also occurred following IV administration Low levels of L-dU were detected m plasma at the limit of detection (lower limit of detection (LLOD) = 0 1 µM) Renal excretion of L-dU was 4 0% of the total dose recovered m unne With the exception of L-dU, no other metabolites were detected in the plasma or unne
Oral administration of L-dC The Cmax was 3 38 µM and occurred at a Tmax of 2 33 hours The plasma concentration of L-dC declined m a biphasic manner with a mean terminal t½ of 2 95 hours and was below detection limits by 24 hours in all monkeys L-dC was absorbed from the gastrointestinal tract with a mean oral bioavailability (F) of 16 4%
L-dU was detected m the plasma and unne, which suggested that metabolic elimination of L-dC occurred following oral admmistration Low levels of L-dU were detected in plasma at the LLOD With the exception of L-dU, no other metabolites were detected in the plasma or unne
Approximately 8 5% of the administered oral dose was recovered in the unne within 12 hours After 72 hours 15 5% ± 8% was recovered L-dC accounted for the majonty (-69%) of drug excreted in the unne Renal excretion of L-dU was 29% of the total recovered dose Feces were not collected
Table 16 presents a summary of pharmacokinetic results for IV and oral administration of L-dC m cynomologus monkeys
Table 16 Pharmacokinetic Analysis after Intravenous and Oral Administration of L-dC (10 mg/kg) in the Cynomologus Monkey

(Table Removed)
Mean value (±SD)
Example 20 Single-Dose Bioavailability of L-dC in the Rhesus Monkey
The pharmacokinetics of L-dC following oral administration m the rhesus monkey was determined In this study, 10 mg/kg [3H] radiolabeled L-dC was administered to three rhesus monkeys as a single oral dose Blood samples for pharmacokinetic analysis were collected pre-dose and at 0 25, 0 5, 1, 2, 3, 6, 8 and 24 hours after dosing Urine samples for pharmacokinetic analyses were collected via pan catch pre-dose and over the following intervals post-dose 0-2, 2-4, 4-8 and 8-12 hours, and then at 12-hour intervals thereafter through 336 hours post-dose The drug was detected and concentration determined using a reverse-phase HPLC technique The blood and unne drug level data were analyzed by a non-modeling mathematical method and AUCs derived by the linear trapezoidal rule
The average AUC025-8 and Cmax values were 12 2 mgMh and 3 23 mgM, respectively The Cmax occurred at a Tmax of 0 83 hours The mean t½ was 3 34 hours and the L-dC plasma concentration was below detection levels by 24 hours in all monkeys The mean renal clearance of L-dC was 0 273 L/h/kg No metabolites were observed in the plasma of monkeys receivmg L-dC
Approximately 8 5% of the administered oral dose (oral bioavailability of L-dC -16%) was recovered in the unne within 8 hours After 48 hours 15% was recovered L-dC accounted for the majority (-77%) of drug excreted m the unne Renal excretion of L-
dU was 23% of the total recovered dose With the exception of L-dU, no other metabolites v, ere detected
The AUC and Cmax for L-dC after oral administration to rhesus monkeys were similar to that observed in cynomologus monkeys
Example 21 Single-Dose Bioavailability of L-dC in the Rat
The pharmacokinetics and bioavailability of L-dC m rats was determined In this study, 10 mg/kg [3H] radiolabeled L-dC was administered to three female Sprague-Dawley rats as a single IV dose A second group of three animals received an identical oral dose of L-dC Blood samples for pharmacokinetic analyses were collected at 0 17, 0 33, 0 5,1, 2, 3, 4, 6, 8 and 24 hours after dosing Urine was also collected at 8 and 24 hours after dosmg The drug was detected and the concentration determined m plasma and urine using a reverse-phase HPLC technique The data were analyzed by a non-modeling mathematical method and the AUCs derived by the linear trapezoidal rule
Intravenous administration of L-dC The average AUC0 25- g value was 30 1 mM h The Cmax of L-dC was 911 mgM and occurred at the earliest sampling tune (10 minutes post-dose) for all animals L-dC plasma concentrations declmed m a brphasic manner following the TV bolus with a mean t½ of 1 21 hours The CL of L-dC averaged 1 44 L/h/kg The mean Vd of 2 53 L/kg indicated that L-dC had a significant extravascular tissue distribution. No metabolites were observed m the plasma of rats receiving L-dC
L-dC accounted for the majority of radioactivity recovered in the urine L-dU was detected m the urine, which suggested that metabohc elimination of L-dC occurred following IV administration
Oral administration of L-dC The average AUC0.25- 8 value was 4 77 mM h The mean Cmax was 1 50 mgM and occurred at a Tmax of 1 0 hour The plasma concentration of L-dC declined with a t½ of 2 52 hours L-dC had limited uptake from the gastrointestinal tract with a mean oral bioavailability (F) of 15 4% No metabohtes were observed m the plasma of rats following oral administration of L-dC
L-dC accounted for the majority of radioactivity recovered in the urine L-dU was detected m the plasma and urine, which suggested that metabolic elimination of L-dC occurs following oral administration
Table 17 presents a summary of pharmacokinetic results for both IV and oral L-dC
Table 17 Pharmacokinetic Analysis after Intravenous and Oral Administration of L-dC (10 mg/kg) in the Rat
(Table Removed)
Example 22 Single-Dose Bioavailability of L-dC m the Woodchuck
The pharmacokinetics and bioavailability of L-dC m woodchucks was determined In this study, 10 mg/kg [3H] radiolabeled L-dC was administered to three woodchucks as a single IV dose Blood samples for pharmacokinetic analyses were collected at 2, 5,15, and 30 minutes and 1 0, 1 5, 2 0, 3 0, 4 0, and 24 hours post-dose After a seven-day washout period, the same animals received 10 mg/kg L-dC as a single oral dose Blood samples for pharmacokinetic analvses were collected at 15 and 30 minutes and 10, 1 5, 2 0, 3 0, 4 0, 8 0, and 24 hours post-dose Unne was collected over the 24-hour post-dose period Plasma drug levels, CL, t½ and F were determined Drug levels were determined using an HPLC method with in-line radioactivity detection and scintillation counting
Intravenous admimstration of L-dC The mean Cmax of L-dC was 112 µM. and occurred at the earliest sampling time (2 minutes post-dose) for all animals L-dC plasma concentrations decknedin a biphasic manner following the IV bolus with a mean t½ of 2 85 hours The CL of L-dC averaged 0 39 L/h/kg The mean Vd was 1 17 L/kg L-dC
accounted for the majority of radioactivity recovered in the urine L-dU was detected in the plasma and urine, indicating that metabolic elimination of L-dC occurred following IV administration The levels of L-dU detected intermittently m plasma were at or below the limit of assay quantitation with a mean Cmax of 0 75 µM
Oral administration of L-dC The Cmax was 1 37 µM and occurred at a Tmax of 3 hours L-dC plasma concentrations decreased with a mean t½ of 5 22 hours L-dC was absorbed from the gastrointestinal tract with an oral bioavailability ranging from 5 60 to 16 9% with an average of 9 57% L-dC accounted for the majority of radioactivity recovered m the unne L-dU was detected m the plasma and urine, indicating that metabolic elimination of L-dC occurred following oral administration L-dU m the plasma was near the limit of quantitation with a mean Cmax of 0 19 µM
Table 18 presents a summary of pharmacokinetic results for both IV and oral L-dC
Table 18 Pharmacokinetic Analysis of L-dC (10 mg/kg) after Intravenous and Oral Administration in the Woodchuck (Table Removed)

Example 23 Bioavailability of the Prodrugs of L-dC
The bioavailability of L-dC, the 5'-monoester of L-dC, the divahne ester of L-dC, and the diacetyl ester of L-dC was evaluated m cynamologous monkeys, with and without L-dT When the divaline ester of L-dC was orally administered to monkeys, approximately 73% of the dose was absorbed Of the absorbed divahne ester of L-dC, more than 99% was rapidly converted to L-dC to give a high concentration of L-dC m the plasma and no detectable divahne ester of L-dC A low plasma concentration of the monovahne ester of
L-dC was detected early after oral administration of divaline ester of L-dC A low plasma concentration of ß-L-2'-deoxyundrne (L-dU) was detected intermittently No other metabolites were detected. The results are provided in Table 19 As indicated, the combination of the 3',5'-divalyl ester of L-dC with L-dT provided the largest bioavailability ofL-dC
Table 19
(Table Removed)
Example 24 Single Dose Bioavailability of Dival-L-dC in Cynomologus Monkey
Three make non-naive cynomologus monkeys (macaca fasciculans) received 10 mg/kg of dival-L-dC intravenously with a tracer amount of tritium ([3H] ) labeled drub (250 uCi) dissolved in sterile 9 0% saline Following a 6 week washout period, the same three animals received an identical oral dose of dival-L-dC Blood samples were collected m hepanmzed tubes at pre-dose (-18 hours) and 0 25, 0 50, 1, 2, 3, 4, 6, 8, and 24 hours after dosing Urine was also collected from 0-2, 2-4, 4-8, 8-12 and then at 12-hour intervals until 336 hours post-dose The drug was quantitated in plasma and urine with a liquid chromatography-mass spectrometry (LC-MS) technique After administration of dival-L-dC, the plasma concentration time course of L-dC was analyzed by a non-modeling mathematical method and the area under the time-concentration curves (AUC) derived by the linear trapezoidal rule The bioavailability (F) of L-dC following TV and PO
administration of dival-L-dC was calculated from the L-dC AUCs, where F = AUCpo/AUCiv x doseiv/dosepo
Intravenously administered dival-L-dC was converted rapidly to L-dC following intravenous administration Dival-L-dC was detected m the plasma at 15 minutes (1 39 uM) and at 30 minutes (0 36 µM, 1 of 3 animals) [lower limit of quantitation (LLOQ) = 0 23 µM or 100 ng/mL] Dival-L-dC was not detected in the plasma after 30 minutes post-dosing The partially de-estenfied form of dival-L-dC, ß-L-2'-deoxycytidine-5'-valine ester, was detected in plasma at 15 minutes (3 23 µM) and decreased in concentration to 0 08 µM by 2 hours (LLOQ = 0 031 µM or 10 ng/mL) L-dC represented the majority of drug present in the plasma following intravenous administration The average AUCo 25→8 value for L-dC was 19 8 µM h The mean peak plasma concentration (Cmax) of L-dC was 24 6 µM (LLOQ = 0 088 µM or 20 ng/mL) and occurred at the earliest sampling time (15 minutes post-dose) in all animals The plasma concentration of L-dC declined m a biphasic manner with a mean t½ of 1 73 hours The total body clearance (CL) and apparent volume of distribution (Vd) of L-dC averaged 1 01 L/h/kg and 2 46 17kg, respectively, indicating that L-dC had significant extravascular tissue distribution The binding of dival-L-dC and L-dC to human plasma protems ex vivo was 13 3% ± 2 6% and 19 7% ± 5 9%, respectively The impact of human plasma protein binding on dival-L-dC and L-dC free-drug levels was minimal, suggesting that drug interactions involving binding site displacement are not anticipated
Urinary excretion was rapid with 58 ± 3% of the administered dose of dival-L-dC excreted within 2 hours following intravenous administration L-dC accounted for the majority (-93%) of drug excreted in the urine L-dU was also detected in the plasm? and urme This suggested that metabolic elimination of L-dC also occurs following administration of dival-L-dC Low levels of L-dU were detected in plasma at intermittent time points in two of three animals at concentrations ranging from 0 22 µM to 0 88 µM (LLOQ = 0 22 jiM or 50 ng/mL) There were no detectable levels of L-dU at any time point m the third monkey Renal excretion of L-dU and the partially de-esterfied form of dival-L-dC, j6-L-2'-deoxycvtidine-5'-valine ester was minor, accounting for approximately 2 5% and 3 7% of the total recovered dose, respectively Dival-L-dC was detected m the urme of one of three animals at 2 hours following IV administration, which accounted for approximately 0 15% of the recovered dose
Because of the intermittent low concentrations of both the monovaline esters and L-dU in the plasma and urine, it was not feasible to perform pharmacokinetic analysis of these metabolites The appearance of the monovaline ester of dival-L-dC was not unexpected as it represents and intermediate in the conversion of dival-L-dC to L-dC In addition, in vitro cellular metabolism studies m monkey, rat and human primary hepatocytes and m extracts of HepG2 cells demonstrated that L-dC was not directly deamrnated to L-dU but that L-dC monophosphate (-MP) is converted to L-dU-MP, which is either activated to L-dU disphosphate (-DP), and triphosphate (-TP), or metabolized to L-dU, which is then detected m the extracellular compartment (plasma) L-dU was non-cytotoxic (CC50 > 200 uM) and L-dU-TP had an IC50 in vitro against hepatitis B virus deoxyribonucleic acid (DNA) polymerase of 5 26 µM (see Microbiology and Virology, Section 10)
Orally admimstered dival-L-dC also was converted rapidly to L-dC following oral administration and was not detectable in plasma samples at any time point (LLOQ of dival-L-dC in solution = 0 23 µM or 100 ng/mL) The partially de-estenfied metabolite of dival-L-dC, j3-L-2'-deoxycytidine-5'-valine ester, was detected in plasma at 30 mmutes and 1 hour at concentrations ranging from 0 034 /? to 0 107 /3 (LLOQ of monoester in solution = 0 031 |iM or 10 ng/mL) Dival-L-dC was not detected m the plasma
L-dC represented the majority (>99% at Cmax) of the plasma drug levels following oral administration of dival-L-dC The average AUCo 25- 8 value for L-dC was 14 0 µM h The Cmax of L-dC was 8 26 µM (LLOQ of L-dC m solution = 0 088 µM or 20 ng/mL) and occurred at 0 67 hours following administration of dival-L-dC The plasma concentration of L-dC declined m a biphasic manner with a mean t½ of 2 28 hours The mean oral bioavailability of L-dC following administration of dival-L-dC was 72 7% ± 22%
L-dU was also detected m the plasma indicating the metabolic elimination of L-dC occurs following oral administration of dival-L-dC Low levels of L-dU were detectable in the plasma from 30 minutes to 4 hours m two of three animals of concentrations ranging from 0 24 µM to 0 66 µM (LLOQ of L-dU in solution = 0 22 µM or 50 ng/mL) and m one animal only at 8 hours at a concentration of 0 39 µM
After oral administration, dival-L-dC was rapidly absorbed from the gastrointestinal tract and converted to L-dC by first-pass mtestmal and/or hepatic metabolism Neither dival-L-dC nor L-dC metabolism was associated with liver microsomal enzymes Following administration of high dose levels of dival-L-dC, the monovahne ester of L-dC
as transiently detected prior to conversion to L-dC No dival-L-dC was detected after oral administration Intermittent low plasma levels of L-dU were detected at, or below, the lower limit of assay quantitation L-dU was formed by dearmnation of L-dC following cellular uptake of l-dC
Approximately31 ± 8% of the administered oral dose was recovered in the urine within 4 hours After 72 hours 39 ± 8% was recovered L-dC accounted for the majority (~95%) of drug excreted in the urine Renal excretion of L-dU and the partially de-estenfied form of dival-L-dC, β-L--2'-deoxycytidine-5'-valine ester was minor, accounting for approximately 2 5% and 0 2% of the total recovered dose, respectively No dival-L-dC was detected in the urine
Table 20 represents a summary of pharmacokinetic results for L-dC following both IV and oral dosmg of dival-L-dC
Table 20 Pharmacokinetic Analysis after Intravenous and Oral Administration of Dival-L-dC (10 mg/kg) m Cynomologus Monkeys
Pharmacokinetic Parameter2
(Table Removed)
Table 21 presents a schematic of metabolite formation form dival-L-dC, the monovahne derivative of L-dC, L-dC and L-dU following IV and oral administration of dival-L-dC The Cmax of each metabolite is also noted
Table 21 Metabolite Formation for IV and PO Administration of Dival-L-dC
Intravenous (10 mg/kg Dival-L-dQ
(Table Removed)
Example 25 Oral Bioavailability of L-dC via Dival-L-dC in Cynomologus Monkey
Three male non-naive cynomologus monkeys (macaca fasciculans) received 10 mg/kg of dival-L-dC orally with a tracer amount of [3H]-labeled drug (250 µCi) dissolved m sterile 0 9% saline Blood samples were collected in hepannized tubes at pre-dose (~18 hours) and 0 25, 0 50,1, 2, 3, 4, 6, 8 and 24 hours after dosmg Urine was collected from 0-2, 2-4, 4-8, 8-12 and then at 12-hour intervals until 336 hours post-dose The drug was quantitated in plasma and in urme using HPLC analysis After administration of dival-L-dC the plasma concentration time course of L-dC was analyzed by a non-modeling mathematical method and the area under the time-concentration curves (AUC) derived by the linear trapezoidal rule Dival-L-dC was rapidly absorbed and converted to L-dC following oral administration. Radiochromatographic high pressure liquid chromatography (HPLC) analysis of plasma samples confirmed that the majority of the recovered radioactivity was L-dC Dival-L-dC was detected in only one animal at 15 minutes post-dose at a concentration of 0 35 µM The partially de-estenfied form of dival-L-dC, ß -L-2'-deoxycytidine-5 '-valine ester, was not detected m the plasma or urine Approximately 26% of the administered oral dose was recovered m the urme within 8 hours After 72 hours 31 % was recovered L-dC accounted for the majonty (~89%) of drug excreted m the urme Renal excretion of L-dU was minor, accounting for approximately 10% of the recovered dose No dival-L-dC or its partially de-estenfied form, and no other metabolites were detected in the urme
The overall pharmacokinetic profile was comparable to that determined m the pharmacokinetic stud\ as demonstrated by similar Cmax to AUC ratios Low levels of L-dU were detected in the plasma in two of three animals with an average Cmax of 0 33 µM No L-dU was detected in the plasma of the third animal The level of L-dU was at or below the limit of quantitation rrecluding pharmacokinetic analysis
Example 26 In vitro metabolism of Dival-L-dC
Studies were conducted to determine the stability and protein binding of dival-L-dC and its de-estenfied metabolites m human plasma. Dival-L-dC was mcubated in human plasma at 37°C and samples analyzed at various tune points up to 24 hours (Figure 13) No dival-L-dC was detectable at 24 horns with complete conversion to L-dC Two additional metabolites (β-L-2'-deoxycytidme-5'-vahne ester and β-L-2'-deoxycytidine-valine ester) were also noted The transient nature of these metabolites indicated that they are intermediates in the conversion of dival-L-dC to L-dC The in vitro half-life of dival-L-dC in human plasma at 37°C was determined to be approximately 39 mm
The impact of human plasma protein binding on free levels of dival-L-dC and L-dC was also investigated using an ultrafiltration method Plasma protem binding of dival-L-dC was 13 3% i 2 6% The binding of L-dC to plasma proteins was 19 7% ±5 9% This study shows that the impact of human plasma protem binding on dival-L-dC and L-dC is minimal and suggests that drug interactions involving binding site displacement are not anticipated
Example 27 Metabolic Activation and Intracellular Profile of L-dC
The cellular metabolism of L-dC was examined using HepG2 cells and human primary hepatocytes High pressure liquid chromatography (HPLC) analysis demonstrated that L-dC was extensively phosphorylated m hepatocytes The predominant metabolite m HepG2 cells exposed to 10 µM L-dC for 24 hours was L-dC-TP which reached 72 4 ± 1 8 µM (see Table 23) In primary human hepatocytes, the L-dC-TP concentration at 24 hours was 90 1 ± 37 uM, similar to the level of phosphorylation in HepG2 cells Exposure of hepatocytes to L-dC led to activation of a second 5'-triphosphate derivative, L-dU-TP In HepG2 cells exposed to 10 µM L-dC, the L-dU-TP level reached 18 2 µM (43 5 pM in primary human hepatocytes) at 24 hours In primary rat and monkey hepatocytes the extent of phosphorylation of L-dC was slightly lower
Table 23 Activation of L-dC(10 µM) in Hepatocytes
Metabolite (10 /iM)
(Table Removed)
(a) Cells were incubated for 24 hours with [3H]-L-dC, specific activity HepG2 assay =
0 5 Ci/mmol, human, monkey and rat hepatocyte assay = 1 0 Ci/mmol
In addition to the phosphorylated derivatives of L-dC and L-dU, formation of a [ß-L-2'-deoxyhponucleotide metabolite was noted In HepG2 cells and primary hepatocyte cultures exposed to 10 µM L-dC for 24 hours, [3-L-2'-deoxycytidme-5l-diphosphochohne (L-dC-Dβ-chohne) was detected at a concentration of 25 6 µM (range 25 6 - 25 7 µM) and 12 3 µM (range 8 82 -15 8 µM), respectively
The metabolic profile obtained after a 24-hour exposure of HepG2 cells to 10 µM [3H]-L-dC is shown in Figure 14 The apparent intracellular half-life of the L-dC-TP was 15 5 ± 0 34 hours, which correlated with prolonged antiviral activity following drug withdrawal in the virus rebound experiments The phosphorylation pattern detected in primary human hepatocytes was qualitatively and quantitatively similar to that obtained using HepG2 cells (Figure 15)
Example 28 Cellular Kinases Associated with Metabolic Activation
D-Deoxycytidine (dCyd) is a natural substrate of cytosohc dCyd kinase (dCK) and mitochondrial thymidine kinase (TK2) for conversion to dCyd-5'-monophosphatc (dCMP) Cytosohc thymidine kinase (TK1) and TK2 utilize D-thymidine (Thd) as a natural substrate for conversion to Thd 5'-monophosphate (TMP) The cellular kinase involved m the initial phosphorylation of L-dC was identified m competition studies using L-dC and the natural endogenous Thd and oCyd Intracellular phosphorylation of L-dC was decreased m a dose-dependent fashion b\ dCyd but not by Thd Thus, dCyd acted as an inhibitor of L-dC phosphorylation The change in intracellular phosphorylation of L-dC was similar when HcpG2 cells were exposed to both Thd and dCyd or dCyd alone The inhibition of L-dC
phosphorylation by only the natural deoxypynmidine, dCyd, suggested that dCK was involved in I-dC phosphorylation
The role of these pynmiduie nucleoside kinase activities in the phosphorylation of L-dC was further investigated in kinase deficient cell lines There was a significant decrease in the mount of phosphorylated metabolites of L-dC m dCK deficient cells However, no significant difference was observed m L-dC phosphorylation m TK1 deficient cells These data were consistent with the competition studies described above and indicated that dCK plays a critical role m the phosphorylation of L-dC to L-dC-MP
Usmg cytosohc extracts of HepG2 cells as an enzyme source, steady state kinetics for L-dC, Thd, and dCyd phosphorylation were similar as indicated by the apparent Michaehs-Menten constant (Km) and maximum initial velocity (Vmax) values (L-dC Km of 5 75 mM and Vmax, of 1 12 mmol/min/mg protein, Thd Km of 4 06 mM and Vmax of 1 26 nmol/min/mg protein, dCyd Km of 4 85 mM and Vmax of 2 15 nmol/min/mg protein) In addition, the efficiency of I^dC, Thd, and dCyd phosphorylation were similar as defined by their corresponding Vmax/Km in values (0 19, 0 31, and 0 44, respectively)
In addition, the extent of intracellular phosphorylation of L-dC was compared to that of the natural endogenous substrates, Thd and dCyd in woodchuck liver extracts This was done to support antiviral testing in the woodchuck model of chrome hepatitis B virus infection Phosphorylation of L-dC was similar to that of the endogenous substrates Furthermore, the level of phosphorylation of L-dC was comparable to that of L-dC and that of the endogenous substrates m human hver extracts
Example 29 Antiviral Activity Against Hepadnavirus of L-dC
The antiviral activity of L-dC against human hepatitis B virus was measured by the reduction in extracellular HBV DNA and rephcative intermediates compared to untreated control cells in the HBV-expressmg hepatoma cell line 2 2 15 (see Table 24) Confirmatory testing of the antiviral activity of L-dC using a panel of ribonucleic acid (RNA) and DNA viruses was performed bv the NIH Antiviral Research and Antimicrobial Chemistry Program
L-dC did not inhibit replication of any virus other than hepadnaviruses (HBV, DHBV) L-dC had potent antiviral activity agamst HBV replication in vitro, reducing
extracellular HBV DNA production with an EC50 of 0 24 µM (EC90 1 06 iiM) L-dC also reduced intracellular HBV DNA replicative intermediates (RI) with an EC50 of 0 5 µM Furthermore, L-dC produced a dose-dependent inhibition of duck hepatitis B virus (DHBV) DNA synthesis in primary duck hepatocyte (PDH) cultures with an EC50 of 0 87 µM
Table 24 In vitro Antiviral Activity, Selectivity and Cytotoxicity of L-dC
(Table Removed)
a. PDH, pnman. duck hepatocytes, PBMC, peripheral blood mononuclear cells, HFF, human foreskin fibroblast, Daudi, Burkitt's B-cell lymphoma, MDCK, canine kidney epitheual cells, CV-I, African green monkey kidney fibroblast cells, KB, human nasopharyngeal carcinoma, MA-i 04, Rhesus monkey kidney epithelial cells
b EC50 = 50% effective concentration
c CC50 =50% cytotoxic concentration
d nd = not determined
e Result presented in µg/mL rather than µM
No cytotoxicity was detected at the maximum concentrations of L-dC tested m any of the cell lmes or primary cell types used to support replication of the various DNA and RNA viruses No toxicity was seen m human PBMCs, HFF, or other cell types of mammalian origin
Example 30 Antiviral Activity of L-dC in Woodchucks - 28 Days
Woodchucks chronically infected with WHV are widely accepted as a model of HBV infection and ha\e proven to be useful m the evaluation of anti-HBV agents It has been proven to be a positive predictor of antiviral activity of therapies for chrome HBV infection and has served as a sensitive system for evaluation of the safety of nucleosides and their analogs
L-dC was given orally to woodchucks once daily at 0 01 to 10 mg/kg/day for 28 days The serum levels of WHV DNA during 28 days of drug treatment and 56 days of post-treatment follow-up were determined by DNA dot-blot hybridization (detection limit of approximately 107 genome equivalents (geq)/mL serum) and by quantitative PCR (detection limit of 300 geq/mL serum)(l) WHV DNA replication was significantly inhibited within the first few days of treatment and was maintained throughout the treatment phase Once a day oral delivery of L-dC produced a strong antiviral effect, which was dose-dependent as determined using the DNA dot-blot hybridization assay (Figure 16)
Figure 17 presents the antiviral activity of L-dC for individual animals treated with 10 mg/kg/day for 28 days m the woodchuck model of chrome hepatitis B infection Notably, in the L-dC 10 mg/kg/day treatment group, by day 14 to 28, viral load had dropped by 2-6 logs from baseline as measured by quantitative PCR assay Following drag withdrawal, viral reboind reached near pre-treatment levels between Weeks 1 and 2
In the lamivucine treated group (10 mg/kg/day, orally), the HBV viral load decreased by approximately 0 5 log to 1 0 log (geq/mL, data not shown) which is consistent with previous studies using similar concentrations of lamivudine, which is a cytidine nucleoside analog (30)
xample 31 Viral Rebound in L-dC Treated Cells
Viral rebound in L-dC treated 2 2 15 cells occurred after drug withdrawal HBV replication returned to 50% of pretreatment levels by day 18 post-treatment The kinetics of viral rebound after L-dC treatment suggested that a significant antiviral effect continued after drug withdrawal, which was consistent with the intracellular half-hfe of L-dC-TP (15 5 hours in HepG2 cells)
Example 32 Antiviral Activity Against Drug-Resistant HBV of L-dC
In controlled cluneal studies of lamivudine (100 mg once daily), administered to HBV-mfected patients, the prevalence of YMDD-mutant HBV was 14 to 32% after one year of treatment and as much as 58% after two to three years of treatment (18-20) Mutant virus was associated with evidence of diminished treatment response relative to lamivudine-treated patients without YMDD mutations
Genotypic analysis of viral isolates obtained from patients who showed evidence of renewed HBV replication while receiving lamivudine suggests that a reduction in HBV sensitivity to lamivudine is associated with mutations resulting m a methionine to valine or isoleucine substitution m the YMDD motif of the catalytic domain of HBV polymerase (position 552) and a leucine to methionine substitution at position 528
HBV recombinants containing the YMDD mutation are lamivudine-resistant and slightly less replication-competent than wild-type HBV in vitro (21) The triphosphate derivative of L-dC will be tested against wild type and mutant HBV DNA polymerase to compare IC50 values In addition, antiviral testing of L-dC against lamivudine-resistant HBV isolates and recombinant viruses with mutations at positions 552 and 528 will be performed
In addition, selection of L-dC drug-resistant HBV mutants in vivo during chrome treatment of WHV-infected woodchucks is also being considered The relevance of selection of drug-resistant mutants in the woodchuck m vivo model is uncertain because the spectrum of lamivudine-resistant mutants in the woodchuck does not match that identified in HBV-mfected patients (20-22) A subset of this long-term study (12 to 24 months) could provide information relevant to treatment-related elimination of HBV covalently closed circular (ccc) DNA from infected hepatocytes At the present time, it is not possible to use
the DHBV in vitro model to select drug-resistant mutations because the primary duck hepatocytes used in this model cannot be sustained in cell culture for the extended periods required to select drug-resistant virus
Example 33 Combination Antiviral Activity and Cytotoxicity of L-dT + L-dC
The anti-HBV activity and cytotoxicity of a combination of L-dT and L-dC at near equimolar ratios were tested in 2 2 15 cells and found to by synergistic at ratios of 1 1, 1 3, and 3 1 (see Table 25)
Table 25 Combination Antiviral Activity of L-dT + L-dC in HBV Infected 2 215 Cells
(Table Removed)
a CC50 = drug concentration at which a 50% inhibition of neutral red dye uptake (as
compared to untreated cultures) was observed b EC90 = drug concentration at which a 10-fold reduction of HBV virion DNA levels (as
compared to untreated cultures) was observed c EC90 values are used for calculation of the Selectivity Index (SI) since reductions of HBV DNA levels that are less than three-fold are generally not statistically significant in this assay system d Analysis of the effectiveness of the drug combination treatments by the CalcuSyn combination evaluation program (Biosoft, Inc)
Example 34 Human Bone Marrow Progenitor Cells Toxicity Assay for L-dC
The myelosuppressive effects of certain nucleoside analogs have highlighted the need to test for potential effects on the growth of human bone marrow progenitor cells in clonogenic assays In particular, anemia and neutropenia are the most common drug-related clinical toxicities associated with the ann-HIV drug zidovudine (ZDV) This toxicity has been modeled m an in vitro assay that employs bone marrow cells obtained from healthy volunteers (Sommadossi J-P, Carlisle R. 'Toxicity of 3'-azido-3'-deoxythynudine and 9-(l,3-dihydroxy-2-propoxymethyl)guanme for normal human hematopoietic progenitor cells m vitro" Antmncrob Agents Chemother 1987, 31(3), 452-454) ZDV has been shown to directly inhibit human granylocyte-marcrophage colony-forming (CFU-GM) and erythroid burst-forming (BFU-E) activity at clinically relevant concentrations of 1-2 µM Using human bone marrow clonogenic assays with ZDV as a positive control and larmvudme as a negative control, L-dC had an IC50 m CFU-GM and BFU-E of >10 µM (see Table 26)
Table 26 Bone Marrow Toxicity of L-dC in Granulocyte Macrophage Progenitor and Erythrocyte Precursor Cells
(Table Removed)
a Values represent the results of three independent experiments performed rn
triplicate
Example 35 Mitochondrial Toxicity Assay for L-dC
Antiviral nucleoside analogs approved for HIV therapy such as ZDV, stavudine (d4T), didanosine (ddl), and zalcitabine (ddC) have also been associated with clinically limiting delayed toxicities such as penpheral neuropathy, myopathy, and pancreatitis (8-11) These clinical adverse events have been attributed to inhibition of mitochondrial function due to reduction in mitochondrial DNA (mtDNA) content and nucleoside analog incorporation into mtDNA In addition, a particular nucleoside analog, fialundme (FIAU) caused hepatic failure, pancreatitis, neuropathy, myopathy and lactic acidosis due to direct mitochondrial toxicity Drug-associated increases m lactic acid production can be considered a marker of impaired mitochondrial function or oxidative phosphorylation
To assess the potential of L-dC to produce mitochondrial toxicity, several in vitro studies were conducted using the human heparoma cell line HepG2 These studies mcluded analysis of lactic acid production, mtDNA content and determination of changes m morphology (εg, loss of cnstae, matrix dissolution and swelling, and lipid droplet formation) of mitochondrial ultrastructure The effects of L-dC on mitochondna are presented m Table 27
No differences were observed in lactic acid levels produced m cells chronically treated with L-dC and m untreated cells Lactic acid production m the ZDV and FIAU treated cells increased by 100% compared to vehicle control Exposure of HepG2 cells for 14 days to L-dC at concentrations up to 10 µM had no effect on mitochondna! DNA content compared to an 87% reduction in the ddC-treated cells Following 14 days of exposure to 10 µM L-dC, the ultrastructure of HepG2 cells, and m particular mitochondria, were examined by transmission electron microscopy No discernible changes m cell architecture or mitochondrial morphology were detected The size and organization of mitochondrial cnstae were normal ZDV-treated cells showed typical swollen mitochondna with loss of cnstae Mitochondna] morphology also was abnormal in the ddC- and FIAU-treated cells
Table 27 Effect of L-dC on Hepatocyte Proliferation, Mitochondrial Function, and Morphology in HepG2 Cells
(Table Removed)
CC50 after 14 days of treatment
Example 36 Human DNA Polymerases α, ß and  Toxicity Assay for L-dC
Nucleosides and nucleoside analogs are usually metabolized within cells to their TP derivatives Cellular DNA polymerases are routinely responsible for normal nuclear and mitochondrial DNA synthesis and repair Because the TP metabolites are potential substrates for DNA polymerases, studies were undertaken to determine if L-dC-TP inhibited human DNA polymerases
The nucleoside analog 3'-ammo-3'-deoxythymidme (AMT) TP inhibited human DNA polymerase a by 30% at a concentration of 10 µM Human DNA polymerases ß and were inhibited by ddC-TP by 50% (5 µM) and 35% (2 5 µM), respectively L-dC-TP and L-dU-TP were not inhibitory to human DNA polymerases α, ß and  up to concentrations of 100 jµM (Table 28) These results suggest that the TP of L-dC and L-dU have a low affinity for these nuclear and mitochondrial human DNA polymerases, which is consistent with the favorable safety profile of L-dC observed in vitro and m vivo
Table 28 Effect of L-dC-TP on Hepatitis Virus DNA Polymerase and Human DNA Polymerases α, ß and 
(Table Removed)
a Each set of data represents the arithmetic mean value and, where presented, the standard
deviation of three independent experiments b WHV DNA polymerase c 3'-Amino-3'-deoxvthyrmdme TP inhibited pol α30% at 10 µM, ddC-TP inhibited pol ß
50% at 5 mM and pol 7 35% at 3 5 µM d Human DNA polymerase data for lamivudrne-TP and L-EMAU-TP from Chang, et al (13), and Yao, et al (14), respectively
Example 37 Toxicity Assay in Rats for Dival-L-dC
The toxicity associated with a single oral dose of dival-L-dC in rats was determined A total of 40 animals (Sprague-Dawley rats, six to eight weeks of age) were studied, ten animals each (five males and five females) were randomized to receive a single oral dose of dival-L-dC at one of three doses selected from the dose-range finding portion of the study (500, 1000, or 2000 mg/kg) or control article Animals were observed for 15 days Cage side observations for monbundity and mortality were documented twice daily Clinical observations and body weight were documented once daily on Days 1, 8, 14, and 15 Also on Day 15 blood samples for hematology and serum chemistries were collected After completion of Day 15 evaluations, all animals were euthanized and subjected to a comprehensive gross necropsy, which included macroscopic examination of the external body surface, all orifices, and the cranial, thoracic, and abdominal cavities and then-contents Body and selected organ weight and organ-to-body and organ-to-brain weight ratios also were documented
No overt signs of toxicity were observed during the study, and no treatment-related effects on body weight, organ weight, or clinical pathology parameters were seen No treatment-related abnormalities were noted in hematology or serum chemistry profiles Furthermore, there were no treatment-related macroscopic lesions observed at necropsy Based on the results of this study, the NOAEL for dival-L-dC following a single oral dose in the rat was 2000 n g kg
Example 38 Toxicity Assay in Monkeys for Dival-L-dC
The potential toxicity of five escalating doses of dival-L-dC in cynomologus monkeys was determined Four animals (two males and two females) each received a total of five oral dival-L-dC doses, one at each dose level (20,100, 500,1000, and 2000 mg/kg), on Days 1,4, 7,10, and 14, respectively
Cage side observations for monbundity and mortality were documented twice daily Clinical observations were documented daily Blood samples for hematology and serum chemistries were collected and body weight measured before treatment on Days 1, 4, 7, 10, and 14, and before necropsy on Day 17 After completion of Day 17 evaluations, all animals were euthanized and a complete necropsy performed, including macroscopic examination and comprehensive tissue collection
No treatment-related clinical abnormalities were observed Following the initial dose on Day 1, each animal demonstrated a loss m body weight of approximately 0 6 kg From Day 4 through the remainder of the study all animals maintained body weight
The following observations were noted m the individual hematology profiles At Day 17, erythrocyte counts (RBG, hemoglobm (HGB), and hematocrit (HCT) were lower (by approximately 15% to 27%, cumulatively in all four animals when compared to values obtained on Day 1 Exclusive of Animal No 1001 (Male), at each timepomt changes m these parameters were The following observations were noted m the individual serum chemistry profiles Day 17 blood urea nitrogen (BUN) values were decreased (by ~43%, cumulatively) in all four monkeys when compared to Day 1 values These cumulative changes result from
interim variations of -39% to +46% These changes were consistent in all monkeys on study, however, the toxicological relevance is uncertain
Based on the results of this study, the NOAEL for dival-L-dC following a single oral dose by gavage in the monkey was 2000 mg/kg
Example 39 28 Day Toxicity Assay m Woodchucks for L-dC
The woodchuck model of chrome hepatitis B infection has been valuable for the preclinical toxicological evaluation of nucleoside analogs This model identified the delayed severe hepatocellular toxicity induced by FIAU m humans not seen m preclinical evaluation m rodents or primates The FIAU-mduced toxicity observed m woodchucks, including significant weight loss, wasting, and hepatocellular damage seen on liver biopsy, was identified beginning six to eight weeks from commencement of treatment and was similar to that observed m the FIAU-treated HBV-infected patents
The antiviral activity and safety of L-dC as well as post-treatment viral rebound m woodchuck hepatitis virus (WHV) infected woodchucks was determined Male and female woodchucks were infected as neonates by subcutaneous inoculation of diluted serum of WHV earners and were all chrome earners of WHV Animals (16 to 18 months of age) were randomized to comparable groups on the basis of body weight, g-glutamyl transferase (GGT) levels, sex, and serum WHV DNA concentration (>10n genome equivalents/mL serum) measured by quantitative dot blot analysis
Three animals each received L-dC at doses of 0 01, 01, 10, or 10 0 mg/kg/day orally for 28 days In addition, three animals received lamivudrne at 10 mg/kg/day orally for 28 days Four animals received vehicle control according to the same schedule All animals were monitored for rebound of WHV for an additional 56 days post-treatment Blood samples for WHV DNA levels were obtained on Days -7, 0, 1, 3, 7, 14, 21, and 28, and WHV DNA levels were also obtained post-treatment on Days 1, 3, 7, 14, 28, and 56 WHV DNA levels were detected by a polymerase chain reaction (PCR) technique Body weights were obtained concurrently and drug dosage was adjusted accordingly If clinical evidence of toxicity was observed, clinical biochemical and hematological tests were to be performed Post-mortem examination, including histologic evaluation of tissues, was to be performed on one animal that died during the study
No toxicity was observed during the four-week treatment period or eight-week post-treatment follow-up period Furthermore, there was no weight loss in any L-dC treatment group compared to control animals (Figure 18) All animals gamed weight in a fashion similar to control animals during the 84-day protocol period One ammal (#98051) m the 0 1 mg/kg/d group died on the eighth day after treatment ended Postmortem examination revealed a large hepatic carcinoma (8x5x2 cm) in the left lateral lobe of the liver and death was attributed to the hepatic malignancy Hepatocellular neoplasms are seen m this model as early as nine months of age and have been a cause of death as early as 15 months of age Death m this ammal was attributed to hepatocellular carcinoma, which is an expected part of the natural history of WHV infection, and was not considered related to L-dC treatment smce there was no indication that drug toxicity was a factor in the death of the ammal
Example 40 Twelve-Week Toxicity Assay in Woodchucks for L-dC
The antiviral activity and safety of L-dC in woodchucks was determined In this study, four animals each received L-dC 1 0 mg/kg/day or vehicle control orally for 12 weeks Four additional annuals received L-dC along with another nucleoside analog, L-dT The animals were randomized into comparable groups, stratified by sex, weight, and pretreatment serum WHV DNA and GGT levels
WHV DNA and body weight were measured on Days 0, 1, 3, 7, 14, 21, 28, 42, 56, and 84 as well as on post-treatment Days 7, 14, 21, 28, 42, 56, 70, and 84 WHV DNA levels were determined by quantitative PCR Appropriate samples for hematology, serum chemistries, WHV serology, and liver biopsy were collected pretreatment and on Day 84 Plasma drag levels were determined from samples collected 2 5 hours post-dose on Days 0, 14 and 84
L-dC (1 mg/kg/day, orally) was well tolerated and showed no drug-related toxicity through 12 weeks of treatment or during 12 weeks of follow-up WHV viremia in chronically infected woodchucks treated for 12 weeks with L-dC (1 mg/kg/d, orally) decreased by 0 5 to 1 logl0 by the end of 12 weeks of treatment, similar to the response in the 28 day study at this dose This study mcluded additional groups treated with L-dT 1 mg/kg/day, and L-dC (1 mg/kg/day) plus L-dT (1 mg/kg/day) administered in combination This combination of L-dC and L-dT reduced viral load to the limit of detection, similar to that seen during treatment with L-dC or L-dT at 10 mg/kg/day in the 28 day study There
as no difference in weight between the animals in the groups treated with L-dC and the control group (see Figure 19) One animal in the control group died at Week 8, necropsy revealed the cause of death to be aortic degeneration and rupture Although unusual, spontaneous rupture of the ascending aorta has been observed historically in both uninfected and WHV-infected woodchucks The weight of all animals decreased slightly during the 24-week study period. Previous experience has determined that this slight decrease in weight was due to the approach of a hibernation cycle (B Tennant, DVM, Marmotech, Inc) Serum chemistries and hematology from all animals were in the normal range before and after 12 weeks of treatment Liver tissue histomorphology as evaluated by microscopy was normal for all groups There was no evidence of fatty change (microvesicular steatosis)
Example 41 Repeated-Dose Toxicokinetics of Dival-L-dC in the Cynomologus Monkeys
The potential toxicity and pharmacokinetics of dival-L-dC after oral administration for 25 days to cynomologus monkeys was determined Eight animals (four males and four females) were randomized to receive dival-L-dC via gavage at one of three doses (500, 1000, or 2000 mg/kg) or vehicle control once daily for 25 days (total N = 32) Cage side observations for monbundity and mortality were documented twice daily, and clinical observations documented once daily Body weights were documented before treatment on Days 1,8, 15 and 25 and before necropsy on Day 26 Food consumption was documented daily and reported for weekly intervals as a daily average Physical and ophthalmologic examinations and urinalysis were performed before treatment and at necropsy After completion of Day 26 evaluations, all animals were euthanized and subjected to a comprehensive gross necropsy, which included macroscopic examination of the external body surface, all orifices, and the cranial, thoracic, and abdominal cavities and then-contents Body and selected organ weight and organ-to-body and organ-to-bram weight ratios also were documented Tissue obtamed by comprehensive gross necropsy was evaluated histomorphologically by a board-certified veterinary pathologist
A Body Weights
All animals either maintained or gamed body weight during the course of the study, except for Animal Nos 2002 (500 mg/kg group), and 4001 and 4003 (2000 mg/kg group), which demonstrated a weight loss of 0 1 kg on Day 25 (compared to Day 1) The statistically significant differences between the males m the control group and the males m the dival-L-dC treated groups are not considered toxicologically relevant as the pre-study mean body weight for control group animals was greater than the mean body weights for the treatment groups by 0 13-0 25 kg
B Food Consumption
During the course of the study, all animals maintained adequate food consumption with expected variability The mean biscuit consumption was less than control males for the 500 mg/kg group males on Days 8/9,15/16, and 16/17, 1000 mg/kg group males on Days 24/25, and 2000 mg/kg group makes on Days 8/9, 15/16, 16/17, 20/21 and 23/24 The only difference noted m the females was a decrease m food consumption m the 2000 mg/kg group females on Day 7/8 1 hese differences are not considered toxicologically relevant
C Clinical Pathology
Hematology On Day 1 prior to the initiation of treatment, there were no differences between the control and treatment groups for any hematological parameter On Day 26, a number of statistically significant differences were noted m the erythrocyte indices, mcludmg a decreased red blood cell count (RBC_ (all treated females), decreased hemoglobin (HGB) (all treated males), and decreased hematocrit (HCT) (all treatment groups, both sexes) The males also demonstrated a reduced RBC, but the differences were not statistically significant Hemoglobm concentration was also lower in the treated females, but was not statistically significant Relative to Day 1 the RBC, HGB and HCT were decreased on Day 26 in the control and dival-LdC treated males and females However, the relative decreases observed for the control animals were less than those noted for the dival-L-dC treated animals These results are indicative of a clinically relevant nonhemolytic anemia, however, any dose response phenomenon was minimal, and
histopathologic evaluation suggests that the bone marrow remained responsive Therefore, any progressive or permanent effects are considered unlikely
In the white blood cell count, there were decreased absolute polymorphonuclear leukocytes (APLY) (500 MG/KG and 1000 mg/kg group females and 2000 mg/kg group males and females), decreased percent polymorphonuclear leukocytes (PLY) (1000 mg/kg and 2000 mg/kg group females), and increased percent lymphocytes (LYM) (2000 mg/kg group males and 1000 mg/kg and 2000 mg/kg group females)
Serum Chemistry The mean alkaline phosphatase (ALK) levels for all treated males were significantly less the make control group mean ALK on Day 26 The mean globulin (GLOB) and calcium (CAL) levels were also elevated m the 2000 mg/kg group males on Day 26 These changes were not considered to be clinically relevant The mean potassium (K) values were greater m the 1000 mg/kg and 2000 mg/kg group males than the control group and could be related to the observed non-hemolyhc anemia present rn those treatment groups There were no changes in any serum chemistry parameter in the females on Day 26
Urinalysis The mean urinary pH was slightly decreased in the 2000 mg/kg group males and the 1000 mg/kg and 2000 mg/kg group females, but the differences were not statistically significant Noteworthy and consistent with acidification of the unne was a lack of crystals in the unne from the high dose males and females
D Organ Weights
Statistically significant decreases m organ weights were noted for the lungs (absolute) of the 1000 mg/kg and 2000 mg/kg group males and the relative thymus (thymus brain) of the 2000 mg/kg group males However, these differences were not considered toxicologically relevant
E Pathology
Macroscopic There were no macroscopic findings that were interpreted as related to the administration of the dival-L-dC All macroscopic findings were typical of those commonly present as incidental findings in non-human primates
Microscopic Thymic atrophy was the only microscopic finding that was interpreted as a treatment-related finding The incidence and seventy of thymic atrophy was increased m the 1000 mg/kg and 2000 mg/kg group males and females, but was not affected m the 500 mg/kg group animals However, the clinical significance of the thymic atrophy was interpreted as equivocal The dose-response relationship was weak, not all 1000 mg/kg and 2000 mg/kg group males were affected and thymic atrophy typically occurs as primates age Other microscopic findings present m this study were commonly minor inflammatory or degenerative changes of the usual type and incidence observed in primates of this age
Toxicokinetics Blood samples for hematology and serum chemistries were collected pretreatment Day 1 and before necropsy on Day 26 Blood samples were collected for pharmacokinetic analvsis on Day 25 from each animal at each of the following times after dosing 0 5, 1, 2, 4, 6, 8, 12, and 24 hours Plasma was prepared from blood and analyzed for concentrations of dival-L-dC and three metabolites L-dC, L-dU and the partially de-estenfied form of dival-L-dC, ß-L-2'-deoxycybdine-5'-valine ester Only L-dC and ß-L-2'-deoxycyhdine-5'-valine ester wee quantifiable The mean plasma concentration-time data for the 1000 and 2000 mg/kg group were subjected to noncompartmental pharmacokinetic analysis using WinNonlin 1 5 (Model 200) Analysis of the 500 mg/kg mg/kg group is in progress
Plasma concentrations of ß-L-2'-deoxycytidme-5'-vahne ester on Day 25 reached maximal values (Cmax) at 1 hour (median Tmax) post oral administration of dival-L-dC, compared to a median Tmax of 2-4 hours for L-dC However, ß-L-2'-deoxycytidme-5,-valme ester Cmax values were approximately 2 orders of magnitude lower than for L-dC After reaching Cmax, concentrations of L-dC declmed in an apparent bi-exponenual manner for each group Estimated terminal phase mean half-lives were approximately 4-5 hours for males and females in both dosage groups These half-life estimates should be viewed as
minimal values, however, because most individual estimates were based on data from 6 to 12 hours post-dose, at which time the terminal phases may not have been completely characterized Mean β-L-2'-deoxycyudine-5'-valine ester concentrations also declined after reaching CMAX, but the terminal phases were not adequately defined to allow estimation of half-lives Mean Cmax values for L-dC and β-L-2'-deoxycytidine-5'-vahne ester were similar for males and females within each dosage group, except for 1000 mg/kg group makes, which were lower bv half the concentration values of the 2000 mg/kg group males Therefore, CmaX appeared to increase with dosage only for the 1000 mg/kg group males
Comparison of L-dD AUClast between males and females showed trends similar to those noted for Cmax with the males sin the 1000 mg/kg group having values lower by approximately half the AUClast values of the 2000 mg/kg group males Comparison of ß-L-2'-deoxycytidme-5'-vahne ester AUC]ast between males and females showed an absence of sex related differences and AUCast appeared to mcrease m a directly proportional manner to increases in dosages
The data suggest that following oral administration of dival-L-dC is a rapid conversion to the de-estenfied form of dival-L-dC, ß-L-2,-deoxycytidme-5'-valme ester, and then L-dC but overall exposure is 100 fold higher for L-dC than for ß-L-2'-deoxycytidme-5'-vahne ester Overall exposure to metabolite β-L-2'-deoxycytidme-5,-valme ester appears to mcrease in an approximately linear manner with increases in dosages
A summary of toxicokmebc results is presented in Table 29
Table 29 Pharmacokinetic Analysis of Repeated-Dose Dival-L-dC 1000 mg/kg and 2000 mg/kg Administered Orally m the Monkey Pharmacokinetic Parameter1
(Table Removed)
m
1 Mean values (+ SD) at Day 25
2 n = 4 for all parameters for both L-dC and β-L-2'-deoxycytidine-5'-vahne ester except
for 2000 mg/kg group females, for which n = 3 and for L-dC AUC and t^, 1000 mg/kg
group females, where n = 2 due to inadequate characterization of the terminal phase
3 Median (rather than mean) values are presented for Tmax, and T^
NA Not Applicable
ID Insufficient Data to define terminal phase for all animals
Example 42 Repeated-Dose Toxicokinetics of Dival-L-dC m the Rat
The potential toxicity and pharmacokinetics of dival-L-dC after oral administration for 28 days to rats was determined Twenty animals each (10 males and 10 females) were randomized to receive dival-L-dC via gavage at one of three doses (500, 1000, or 2000 mg/kg) or vehicle control once daily for 28 days Cage side observations for monbundity and mortality were documented twice daily Clinical observations were documented once daih Body weights were documented before dosing on Days 1, 8, 15, 22, and 28 and before necropsy on Day 29 Food consumption was documented weekly Blood samples for hematology and serum chemistries also were collected before necropsy on Day 29 After completion of Day 29 evaluations, all animals were euthanized and subjected to a comprehensive gross necropsy, which mcluded macroscopic examination of the external body surlace, all orifices, and the cranial, thoracic, and abdominal cavities and their contents Bod\ and selected organ weight and organ-to-body and organ-to-brain weight ratios also were documented Tissue obtained by comprehensive gross necropsy was evaluated histomorphologically by a board-certified veterinary pathologist
A Body Weights
The mean body weight values for the 2000 mg/kg group males on Days 22 and 28 were significantly lower than the mean value for the male control group The mean body weight value for the 2000 mg/kg group females on Day 28 was also significantly lower than the mean value for the control group females
B Food Consumption
Food consumption was reduced in the 2000 mg/kg group males throughout the duration of the study Also, the food consumption of the 1000 mg/kg group males during the third week of the study was significantly less than the control group males The food consumption was significantly reduced in the 1000 mg/kg and 2000 mg/kg females during the second, third, and fourth weeks of the study
C Clinical Pathology
Hematology On Day 29, a number of statistically significant differences were noted in the erythrocyte indices The red blood cell count (RBC) was significantly reduced m both males and females at all three dose levels (500, 1000 and 2000 mg/kg) The hemoglobin concentration (HGB) was decreased m the 2000 mg/kg group males, the 1000 mg/kg group females and the 2000 mg/kg group females A decrease in hematocrit (HCT) was noted m the 1000 mg/kg and 2000 mg/kg group males and females The mean cell volume (MCV) was significantly increased in the 500, 1000 and 2000 mg/kg group males and m the 500 and 1000 mg/kg group females The mean cell hemoglobin (MCH) was significantly mcreased in the 500, 1000 and 2000 mg/kg group males and females The mean cell hemoglobin concentration (MCHC) was increased m the 1000 mg/kg females The nucleated red blood cell count (NRC, absolute and relative) was decreased in the 1000 mg/kg and 2000 mg-'kg males and mcreased m the 2000 mg/kg females These changes indicate a treatment-related mild responsive anemia.
The white blood cell count (WBC) was decreased m the 2000 mg/kg males There was a reduction in the monocytes (MNO, absolute and percentage) m the 2000 mg/kg group
males Platelets (PLT) were increased in the 2000 mg/kg males However, these changes were quantitatively small and the toxicological relevance is uncertain
Serum Chemistry Mean globulin (GLOB) levels were decreased m the 2000 mg/kg group males and the 1000 mg/kg group females on Day 29 The albumin/globulin ratios were increased m the 1000 and 2000 mg/kg group males and the 1000 mg/kg group females The alkaline phosphatase (ALK) levels were elevated in the 500 mg/kg group females The cholesterol (CHOL) levels were mcreased m the 1000 mg/kg females These minor changes did not form dose-response-related patterns or trends to suggest that these values were toxicologically relevant
D Organ Weights
Significant decreases in absolute organ weights were noted for the lungs (2000 mg/kg group males and females) and thymus (2000 mg/kg group males, 1000 mg/kg group females and 2000 mg/kg group females) Also significant was the decrease m the mean absolute organ weight for the prostate and seminal vesicles in the 2000 mg/kg group males The mean absolute heart weights were decreased m the 1000 mg/kg and 2000 mg/kg group females The salivary glands mean weight was decreased in 2000 mg/kg group females The mean spleen weight was mcreased m the 2000 mg/kg group females
The relative (to body) organ weight changes included an mcreased brain weight in the 2000 mg/kg group males and females An increase m the mean tests weight of the 1000 mg/kg and 2000 mg/kg group males was also noted The relative thymus weight was reduced m the 2000 mg/kg group males and the 1000 mg/kg and 2000 mg/kg group females The mean relative spleen weight was increased m the 2000 mg/kg group females
Also, the relative (to brain weight) organ weight changes mcluded a decreased relative lung weight in the 2000 mg/kg group males The relative thymus weights were decreased in the 1000 mg/kg and 2000 mg/kg group males and females The relative prostate and seminal \esicle mean weights were also decreased in the 2000 mg/kg group males The mean relanve heart weight was reduced m the 2000 mg/kg group females as was the mean relati\ e weight of the salivary glands The relative spleen weight was increased in 2000 mgkg group females
The decreases in organ weights (thymus, lung, heart, salivary glands, prostate, seminal vesicles, and brain) were interpreted as secondary to the generalized body weight loss presented in the 1000 mg/kg and 2000 mg/kg group animals Thymic atrophy, which was observed microscopically in the 1000 mg/kg and 2000 mg/kg group animals, was consistent with the decreased thymus weights observed Other tissues with decreased weights did not have microscopic correlates The increased spleen weights were interpreted as a consequence of erythropoietic activity observed microscopically
E Pathology
Microscopic The incidences of thymic atrophy and lymphoid necrosis were increased m the 1000 mg/kg and 2000 mg/kg group animals, but were not affected m the 500 mg/kg group animals However, the clinical significance of thymic atrophy and lymphoid necrosis was interpreted as equivocal because the dose-response relationship was weak Also, these thymic changes are often present as non-specific changes m animals stressed by a variety of factors, and significant body weight reductions were observed m the 1000 mg/kg and 2000 mg/kg group animals m this study
Erythropoiesis in spleen was mcreased in the 1000 mg/kg and 2000 mg/kg group males and females sufficiently to distinguish them from the controls, but the spleens from the 500 mg/kg group animals were similar to controls Hematopoiesis in liver was mcreased m the 2000 mg/kg group males and females sufficiently to distinguish them from the controls, but the livers from 500 mg/kg and 1000 mg/kg group animals were similar to those m controls Hyperplasia m sternal bone marrow was observed in the 2000 mg/kg group males and females Erythropoiesis m spleen, mcreased hematopoiesis m liver and hyperplasia m bone marrow were all interpreted as expected and appropriate responses to the mild anemia observed as a part of the hematology results These results confirm the responsive nature of the anemia during contmued treatment
There were several other microscopic changes present m this study These were most commonly manor inflammatory or degenerative changes of the usual type and mcidence observed in rodent gavage studies
Toxicokinetics An additional 54 animals (27 males and 27 females) had samples collected for pharmacokinetic analyses on Days i and 28 On both days, samples were collected at each of six timepoints (alternating two animals per timepoint) 0 5, 1, 2, 4, 8 and 24 hours post-dosing Plasma was prepared from blood and analyzed for concentrations of dival-L-dC and three metabolites L-dC, L-dU and the partially de-estenfied form of dival-L-dC, /3-L-2,-deoxycytidme-5'-vahne ester Only L-dC and j3-L-2'-deoxycytidme-5l-vahne ester were qualifiable The mean plasma concentration-time data for the 1000 and 2000 mg/kg group were subjected to noncompartmental pharmacokinetic analysis using WmNonhn 1 5 (Model 200) Analysis of the 500 mg/kg group is in progress
Mean plasma concentrations of the metabolite, L-dC, reached maximal values (Cmax) at 2 hours post-dose (Tmax) for the 1000 m/kg dose group and at 1-4 hours post-dose for the 2000 mg/kg dose group Mean Cmax values for males and females were comparable within each of the 1000 mg/kg and 2000 mg/Kg- osage groups and were similar on Day 28 versus Day 1 in both groups Cmax increased via dose m most cases but the extent cf the increase was variable After reaching Cmax, cone .ntrations of L-dC declined in an apparent bi-exponenhal manner for each group Es x ii-ued terminal phase half-lives for th 1000 mg/kg dose group (9-17 hours) tended to be longe for the 2000 mg/kg dose group (& S hours), but the half-life estimates should be interpreted with caution The estimation or he half-lives required using only three data pomts am the data tended to be variable Also one of three data points used was at 4 hours, at which time the terminal phase may not have been established Tiast for L-dC concentration. vocurred at 24 hours for all data sets AUClast was comparable for males and females with. wahic group, and did not appear to bt substantially different on Day 28 versus Day 1 Although Cmax for L-dC did not appear to mcrease with increased dosage of dival-L-dC in a c manner, as noted above, AUCast of L-dC mcreased with dival-L-dC m a relation that appeared to be approximatel' proportional to dose
Plasma mean concentrations of ß-L-2'-deoxycytidine-5'-valine rster reached naxnnal values (Cmax) at 1 to 2 hours dose (Tmax) Mean Cmax values Tor males and females were similar within each dosage group witn a trend toward higher values for remales Cmax values ^vere approximated 4% to 50% higher for females on each Day 1 and Day 28 except for females m the 2000 mg/kg group, where j8-L 2-deo cycytidine-51-alme ester Cmax values were approxb by 164% higher than males on Day 28 When comparing values within each gender, - values on Day 28 wee similar to Day 1 except
for females in the 2000 mg/kg group for which Cmax values were 130% higher on Day 28 than Day 1 Cmax increased with dosage in each case, but by a factor that was generally less than linearly proportional to dose
The apparent terminal elimination phase of ß-L-2'-deoxycyudine-5 '-valine ester was not well characterized and therefore half-lives were not reported Tlast for 0-L-2'-deoxycytidine-5'-vahne ester concentrations occurred at 4-8 hours for the 1000 mg/kg dose group and at 8-24 hours for the 2000 mg/kg dose group As was noted for Cmax, the AUClast was 25% to 50% higher for females than males AUCast was consistently slightly higher on Day 28 vs Day 1 for both males and females (30% to 62%) AUC|ast increased with dosage in a relationship that appeared to be approximately linearly proportional to dose
These data suggest that both L-dC and ß-L-2'-deoxycytidine-5'-vahne ester reached systemic circulation relatively rapidly Overall exposure as measured by Cmax was 10 to 40 fold greater for L-dC than ß-L-2'-deoxycytidme-5'-valine ester and 35 to 80 fold greater as measured by AUClast Exposure appeared to mcrease proportionally to dosage within the dosage range of 1000-2000 mg/kg/day Overall exposure on Day 29 of L-dC was comparable to that observed on Day 1 while ß-L-2l-deoxycytidine-5l-vahne ester exposure was generally greater on Day 28, suggesting that accumulation of ß-L-2'-deoxycytidine-5'-valme ester may occur during repeated dosing
A summary of toxicokmetic results is presented m Table 30
Table 30 Pharmacokinetic Analysis of Single- and Repeated-Dose Dival-L-dC 1000 mg/kg and 2000 mg/kg Administered Orally in the Rat
Pharmacokinetic Parameter
(Table Removed)
NA = Not Applicable, a terminal phase was not adequately characterized ID = Insufficient Data to define terminal phase for all animals
Example 43 S Typhimurutm and E Colt Plate Incorporation Mutation Assay (Genotoxicity)
Dival-L-dC when administered orally to animals is rapidly converted to L-dC to give high plasma concentrations of L-dC and no detectable dival-L-dC Therefore, the mutagenicity studies conducted in vitro were performed using L-dC This study was conducted in accordance with FDA GLP regulations L-dC was tested for its potential to cause mutation at the hisudine operon of Salmonella typhimunum strains TA98, TA100, TA1535, and TA1537 and at the tryptophan opeton of Escherichia coh strain WP2uvrA L-dC at concentrations of 50, 100, 500, 1000, and 5000 mg/plate plus positive and negative controls were tested Test strains were exposed to L-dC or control m the absence of exogenous activation and in the presence of induced rat liver S-9 extract plus eofactors After incubation for approximately 68 hours, L-dC and controls were evaluated for the number of revertants per plate and integrity of the background microcolony lawn
Both negative and positive controls fulfilled the requirements of the test The results of both definitive and confirmatory assays indicated that L-dC did not induce any significant increase rn the number of revertant colonies for any of the test strains in the presence or absence of induced rat liver S-9 extract Based on the study findings, it was

concluded that there was no evidence of mutagenicity in the S typhimunum or E coh plate incorporation mutation assay with L-dC concentrations up to 5000 mg/plate
Example 44 Chromosomal Aberration Assay
Dival-L-dC when administered orally to animals is rapidly converted to L-dC to give high plasma concentrations of L-dC and no detectable dival-L-dC Therefore, the mutagenicity studies conducted in vitro were performed using L-dC This study was conducted in accordance with FDA GLP regulations L-dC was tested for its potential to induce chromosomal aberrations m cultured CHO cells In the definitive assay, L-dC at concentrations of 100 500, 1000, and 5000 mg/mL and positive and negative controls were tested with and without metabolic activation After continuous treatment for 18 hours, toxicity was determined by the reduction in relative cell growth (RCG) and relative mitotic mdex (RMI) Based on the RCG and RMI results, chromosomal aberrations were scored from the three highest concentrations (500, 1000, and 5000 mg/mL) One hundred metaphases were scored from each of the duplicate cultures at each concentration (mcludmg positive and negative controls)
A confirmatory assay was performed without activation only with L-dC concentrations of 1 0,10, 100, 500,1000, and 5000 mg/mL After continuous treatment for 18 hours the reduction m RCG and RMI were determined Based on RCG and RMI results, chromosomal aberrations were scored from the three highest concentrations (500, 1000, and 5000 mg/mL) One hundred metaphases were scored from each of the duplicate cultures at each concentration level (mcludmg positive and negative controls)
Results from the definitive and confirmatory assays indicated that L-dC did not mduce a statistically significant increase (defined as a β-value £0 05 determined by the Chi-square test) m the percentage of cells with aberrations at any of the concentrations tested, both with and without metabolic activation, compared to solvent controls Based on the study findings, it wa> concluded that there was no evidence of chromosomal aberrations in the CHO assay after exposure to L-dC at concentrations up to 5000 mg/mL, and L-dC is not considered to be a cLstogenic agent
Example 45 Mouse Micronucleus Assay
Dival-L-dC when administered orally to animals is rapidly converted to L-dC to give high plasma concentrations of L-dC and no detectable dival-I-dC Therefore, the mutagenicity studies conducted in vitro were performed using I-dC This study was conducted in accordance with FDA GLP regulations Assuming an oral bioavailability of 10-20% m the rodent (see Pharmacology and Toxicology, Section 8 17 3) exposure to L-dC (2000 mg/kg dose) would reach or exceed 400 mg/kg This level of exposure would exceed the expected human exposure by 20 to 50-fold
L-dC was tested for its potential to induce micronucleated polychromatic erythrocytes (MPCE) in the bone marrow cells of male and female mice L-dC at concentrations of 500, 1000, and 2000 mg/kg and positive and negative controls were tested Study drag was administered by oral garage as a single dose Two harvests approximately 24 and 48 hours after L-dC or negative control admimstration were performed, and a single harvest approximately 24 hours after positive control administration was performed Five male and five female mice per dose group per harvest time were used The percentage of polychromatic erythrocytes (PCE) and MPCE frequency were determined for each traiepoint
The results of the study indicate that there was no statistically significant increase (defined as a β-value £0 025 determined by a one-tailed Student's t-test) m the number of MPCE at any timepoint in any L-dC dose group compared to negative control A reduction of more than 20% versus the vehicle control in the percentage of PCE, as an indication of toxicity, was observed at each test article dose level at the 24 hour harvest tune in both sexes (-30 5% to -43 1% for the males and -26 1% to -32 2% for the females) The reduction also mdicates an appropriate exposure of test article to the target tissue However, this reduction of more than 20% was not observed at any test article dose level at the 48 hour harvest tune m eidier sex
This study mdicates that, under the conditions of the test and according to the criteria set for evaluating the test results, L-dC was negative m the micronucleus assay to male or female animals at doses up to 2000 mg/kg
Example 46 Integrated Summary of Toxicologic Findings
Conventional cell-based assays were used to assess the cytotoxicity of L-dC and any cellular metabolites L-dC was non-cytotoxic (50% cytotoxic concentration, CC50, >2000 µM) to the human hepatoma cell line 2 2 15, which is routinely used to determine the anti-I-EBV activity of potential antiviral agents L-dC was not cytotoxic to human human peripheral blood mononuclear cells (PBMCs, CC50 >100 µM) and to human bone marrow progenitor cells (50% inhibitory concentration, IC50, > 10 µM in granulocyte-macrophage colony forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) assays)
(Table Removed)
a PBMC, peripheral blood mononuclear cells, HFF, human foreskin fibroblast, Daudi, Burkitt's B-cell lymphoma, MDCK, canine kidney epithelial cells, CV-1, African green monkey kidney fibroblast cells, MA-104, rhesus monkey kidney epithelial cells
b CC30 = 50% cytotoxic concentration, '>' indicates that no CC30 was reached at the highest drug concentration tested
c NIH, Antiviral Research and Antimicrobial Chemistry Program
d R Schinazi, Emory University, Veterans Affairs Medical Center
e Result presented m µg/mL rather than µM
In addition, L-dC was not cytotoxic to numerous other cell lines of human and other mammalian ongin No discernible changes in the function, morphology, or DNA content of mitochondria were noted and there was no lactic acid accumulation m L JC treated hepatocytes (IC50 > 10 µM) The triphosphate form of L-dC was not inhibitory to the human DNA. polymerases α, ß and , up to concentrations of 100 µM
In acute single dose (including 500, 1000, and 2000 mg/kg single oral dose) toxicology studies in rats and in monkeys (dose escalation over days 1, 4, 7,10 and 14 up to 2000 mg/kg) there were no overt signs of toxicity nor were there any dival-L-dC related effects on body weight, food consumption, or clinical pathology parameters (hematology and serum chemistry) In addition, there were no macroscopic lesions observed at necropsy, nor were there any microscopic findings on histomorphological analysis attributable to dival-L-dC Based on the results of these studies, the no observed adverse effect level (NOAEL) for dival-L-dC, following a single dose by oral gavage in the Sprague-Dawley rat and cynomologus monkey was 2000 mg/kg
In the subchronic (25 day) toxicology study in monkeys, the NOAEL was less than 500 mg/kg for dival-L-dC Thymic atrophy was the only microscopic finding that was possibly related to dnal-L-dC, but the clinical significance was interpreted as equivocal A mild non-hemolytic anemia (decreased red blood cell count, decreased hemoglobin, and hematocrit) and decrease in the absolute and percent polymorphonuclear leukocyte counts of no apparent consequence were noted at the 500 mg/kg dose level Other than the hematological changes there were no other toxicities identified m any dose group
In the subchronic (28 day) toxicology study m rats, the NOAEL was less than 500 mg/kg for dival-L-dC Oral administration of the dival-L-dC for 28 days to the rat at a dose of 2000 mg/kg resulted m treatment related changes that included a mild macrocytic anemia, reduced thymus weight, increased spleen weight (females only), reduced body weight, and hematopoiesis m the spleen, liver and sternal bone marrow Oral admmistration of dival-L-dC for 28 days to the rat at a dose of 1000 rog/kg resulted in treatment related changes that mcluded a mild macrocytic anemia, thymic atrophy (females only) and hematopoiesis m the spleen The histomorphological changes seen in the liver, spleen and bone marrow reflect a hematological response to the mild anemia. Oral admmistration of the dival-L-dC for 2S days to the rat at a dose of 500 mg/kg resulted m a mild macrocytic anemia Other than the hematological changes and hematopoietic responses noted there were no other toxicities identified m any dose group
In normal healthy woodchucks or woodchucks chronically infected with hepatitis B virus (εfficacy model for treatment of HBV infection), no toxicity was observed during acute (10 mg/kg single dose IV and PO) and subchronic (28 days at 10 mg/kg/day orally and 12 weeks at 1 mg/kg/day orally) studies of animals receiving L-dC There was no
weight loss in the L-dC treatment groups compared to control animals, clinical pathology parameters (hematology and serum chemistry) were in the normal range and liver biopsies taken at end of treatment m the 12-week study showed no evidence of fatty change (microvesicular steatosis)
L-dC was not mutagenic in the S typhimunum or E coh plate incorporation mutagenicity assay at concentrations up to 5000 µg/plate There was no evidence of chromosomal aberrations m the Chinese hamster ovary (CHO) assay after exposure to L-dC at concentrations up to 5000 µg/mL (or 22 0 mM) In the mouse nucronucleus assay, L-dC was not clastogemc to male or female animals at doses up to 2000 mg/kg
Mild anemia noted m the monkey was not associated with any clinical correlates even at the highest dose (2000 mg/kg) and m the rat at 500 mg/kg In addition the reticulocyte counts were unchanged Although there was no formal reversibility component m these studies it is apparent that a hematological rebound can occur as indicated by the extramedullary hematopoiesis seen in the spleen and liver at the higher doses m the rat
Table 32 Interspecies Comparison of Doses by Weight and Body Surface Area
(Table Removed)
Similar hematological changes at comparable or lower doses were observed in preclinical toxicity studies of lamivudine (Epivrr-HBVT™), and valacyclovir (Valtrex™) Both of these appnn ed drugs are members of the same well-charactenzed class (nucleoside or nucleoside analog) as dival-L-dC The choice of lamivudine for comparison is based on the fact that it is a cvtosine derivative as is dival-L-dC and on its approval for the treatment
of chronic hepatitis B infection The choice of valacyclovir for comparison is based on the fact that it is a valine ester prodrug of the nucleoside acyclovir
This invention has been described with reference to its preferred embodiments Variations and modifications of the invention, will be obvious to those skilled in the art from the foregoing detailed description of the invention It is mtended that all of these variations and modifications be included within the scope of this invention

WE CLAIM:
1: A compound of the 3'-0-valinyl ester of 2'-deoxy-[beta]-L-cytidine
(Formula Removed)
or a pharmaceutically acceptable salt thereof.
2: A pharmaceutical composition comprising a 3'-0-valinyl ester of 2'-deoxy-[beta]-L-cytidine as claimed in claim l the formula
(Formula Removed)
or a pharmaceutically acceptable salt thereof, in combination with compound such as herein described.
3. A compound substantially as herein described with reference to foregoing description, examples, tables and accompanying drawings.

Documents:

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Patent Number

245685

Indian Patent Application Number

IN/PCT/2002/01240/DEL

PG Journal Number

05/2011

Publication Date

04-Feb-2011

Grant Date

28-Jan-2011

Date of Filing

13-Dec-2002

Name of Patentee

IDENIX (CAYMAN) LIMITED

Applicant Address

WALKER SECRETARIES, WALKER HOUSE, GRAND CAYMAN, CAYMAN ISLAND.

Inventors:

#	Inventor's Name	Inventor's Address
1	MARTIN L. BRYANT	65 HICKORY LANE, CARLISLE, MA 01741, U.S.A.
2	GILLES GOSSELIN	82, RUE CALVIN, 400, AVENUE PAUL RIMBAUD, F-34080 MONTPELLIER, FRANCE.
3	JEAN-LOUIS IMBACH	1108, RUE LAS SORBES, IMPASSE DES LUQUES, F-34000 MONTPELLIER, FRANCE.

PCT International Classification Number

A61K 35/00

PCT International Application Number

PCT/US2001/19147

PCT International Filing date

2001-06-15

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/212,100	2000-06-15	U.S.A.