WO1998017281A1 - MONOPHOSPHATE PRODRUGS OF β-L-FD4C AND β-L-FddC AS POTENT ANTIVIRAL AGENTS - Google Patents

Abstract

The present invention relates to certain prodrug forms of the L-dideoxynucleoside analogs β-L-FD4C and β-L-FddC, especially β-L-Fd4C, which preferably contain S-acyl-2-thioethyl-bearing 5'monophosphate groups which exhibit excellent activity against hepatitis B virus (HBV) and human immunodeficiency virus (HIV). In particular, the compounds according to the present invention show potent inhibition of the replication of the virus in combination with very low toxicity to the host cells (i.e, animal or human tissue) and unexpectedly high therapeutic indices. The prodrug form of β-L-FD4C exhibits particularly effective inhibition of HBV in comparison to β-L-FD4C and markedly improved therapeutic index.

Description

Monophosphate Prodrugs of β-L-FD4C & β-L-FddC as Potent Antiviral Agents

FIELD OF THE INVENTION

The present invention relates to prodrug forms of dideoxy nucleoside analogs. These compounds exhibit significant activity against retroviruses, including human immunodeficiency virus (HIV), and hepatitis B virus (HBV). This invention also relates to pharmaceutical compositions containing these compounds and to methods of inhibiting the growth or replication of HIV and HBV as well as treating hepatitis B viral infections in animals and in particular, humans.

BACKGROUND OF THE INVENTION

Of the six approved anti-HIV drugs, five of them are nucleoside analogs including the first approved anti-AIDS drug AZT (3'-azido-2',3'-dideoxythymidine), two 2',3'-dideoxy nucleosides, ddl (2',3'-dideoxyinosine) and ddC (2',3'-dideoxycytidine) and one C2'-3' double bond bearing derivative, D T (2',3'-dideoxy-2',3'-didehydrothymidine) and the newly approved L-nucleoside, (2-hydroxymethyl-l ,3-oxathiolan-4-yl)cytosine (-3TC). The target of this viral inhibition appears to be the viral reverse transcriptase. Because hepatitis B virus (HBV) relies on reverse transcriptase-like activity for DNA replication, it was postulated that nucleoside analogs with activity against HIV might also have activity against HBV as well.

A large number of nucleoside analogs exhibiting potent antiviral activities are now in various stages of preclinical and clinical evaluation. These include β-L-FD4C (Lin, T-S.; Luo, M-Z.; Liu, M-C; Zhu, Y-L.; Gullen, E.; Dutschman, G.E.; Cheng, Y-C. J. Med. Chem. 1996, 39, 1757) and β-L-FddC (Lin, T-S.; Luo, M-Z.; Liu, M-C; Pai, S.B.; Dutschman, G.E.; Cheng, Y-C. J. Med. Chem. 1994, 37, 798), among others, such as 3TC, FTC.

Mechanistically, before a nucleoside analog can be incorporated into viral DNA, the cell must first phosphorylate the nucleoside analogs to their triphosphorylated form(s). (Jones. R.J.; Bischofberger, N. Antiviral Res. 1995, 27, 1). This phosphorylation process generally occurs in three successive steps. In many cases, among these three successive phosphorylation steps, the first phosphorylation step is rate limiting. Further conversion to the di- and triphosphorylated forms are catalyzed by less specific kinases.

The lack of antiviral activity of some nucleoside analogs appears to be due to the inefficiency of the kinase capable of carrying out the first phosphorylation step. Therefore, to by-pass the first highly selective and regulated phosphorylation step, the use of nucleotide prodrugs has been investigated intensively (Abstracts published in Antiviral Res. (suppl.) 1993, p. 45-142). In particular, design and synthesis of novel neutral monophosphate-bearing prodrugs (pronucleotides) of the nucleosides of interest is highly important because these neutral nucleotides can penetrate cell membranes much more readily than their corresponding 5'-monophosphate dianion counterparts. Once inside the cell, pronucleotides should decompose cleanly to generate the corresponding 5'-monophosphate dianion species. These nucleotides will then be further phosphorylated to their di- and triphosphates.

A newly reported ester linker for converting the 5'-monophosphate dianion species of a nucleoside analog to a neutral prodrug is S-acyl-2-thioethyl (SATE). Several bis(SATE) esters of AZT (3'-azido-2',3'-dideoxythymidine) [Lefebvre, I.; Perigaud, C; Pompon, A.; Aubertin, A-M.; Girardet, J-L.; Kirn, A.; Gosselin, G.; Imbach, J-L. J. Med. Chem. 1995, 38, 3941] and D4T (2',3'-dideoxy-2',3'-didehydrothymidine) [Girardet, J-L.; Perigaud, C; Aubertin, A-M.; Gosselin, G.; Kirn, A.; Imbach, J-L. Bioorg. Med. Chem. Lett. 1995, 5, 2981] showed marked anti-HIV activity in thymidine kinase-deficient (TK ) cell lines. In addition, the bis(SATE) phosphotriester derivative of ddA (2',3'-dideoxyadenosine) [Perigaud, C; Aubertin, A-M.; Benzaria, S.; Pelicano, H.; Girardet, J-L.; Maury, G.; Gosselin, G.; Kirn, A.; Imbach, J-L. Biochem. Pharmacol. 1994, 48, 11] displayed a potent inhibitory effect in various HIV- 1 -infected cell lines. The production of neutral, monophosphate-bearing prodrug forms of recently discovered, more potent nucleoside analogs (e.g. β-L-FD4C and β-L-FddC) is desired to further increase their therapeutic usefulness.

Other ester linkers known in the art for the conversion of a nucleoside analog mono- phosphate into a neutral form include modifications of SATE such as methyl(SATE), isopropyl(SATE), t-butyl(SATE), and phenyl(SATE), as well as other thioesters such as (pivaloyloxy)methyl and S-[(2-hydroxyethyl)sulfidyl]-2-thioethyl (Lefebvre, I., et al.), and cyclic phosphate moieties (Meier, C, Angew. Chem. Int. Ed. Engl 1996, 35, 70 and Meier, et al. Bioorg. Med. Chem. Lett.. 1997, 7, 99). Prodrug forms resulting from the use of these ester linkers should have decomposition schemes substantially similar to Figure 2.

OBJECTS OF THE INVENTION

It is an object of the invention to provide neutral, monophosphate-bearing prodrug forms of β-L-FD4C and β-L-FddC.

It is another object of the invention to provide a method of treating viral infections with neutral, monophosphate-bearing prodrug forms of β-L-FD4C and β-L-FddC.

It is a further object of the invention to provide pharmaceutical compositions which are effective as anti-HIV and anti-HBV agents.

It is yet another object of the invention to provide methods of treating HBV and/or HIV infections in humans which utilize significantly less prodrug form of β-L-FD4C or β-L- FddC on a molar basis than the free nucleoside analog β-L-FD4C or β-L-FddC to produce substantially the same therapeutic result.

These and/or other objects of the invention may be readily gleaned from the description of the invention, which follows.

SUMMARY OF THE INVENTION

The present invention relates to the surprising discovery that certain prodrug forms of the L-dideoxynucleoside analogs β-L-FD4C and β-L-FddC, especially β-L-Fd4C which contain S-acyl-2-thioethyl-bearing 5'monophosphate groups exhibit unexpected activity against Hepatitis B virus (HBV) and Human immunodeficiency virus (HIV). In particular, the compounds according to the present invention show potent inhibition of the replication of the virus in combination with very low toxicity to the host cells (i.e., animal or human tissue). The prodrug form of β-L-FD4C exhibits particularly effective inhibition of HBV in comparison to β-L-FD4C and a markedly improved therapeutic index. In certain aspects of the present invention, the prodrug form of β-L-FD4C or β-L-FddC can produce the same therapeutic prophylactic result as β-L-FD4C or β-L-FD4C using less than 1/2, 1/5 or even 1/8 the molar concentration of the free nucleoside form. This is an unexpected result.

Compounds according to the present invention exhibit primary utility as agents for inhibiting the growth or replication of HBV, HIV and other viruses, most preferably HBV and HIV. Certain of these agents also may be useful for inhibiting the growth or replication of other viruses or for treating other viral infections and/or related disease states. In addition, certain of these agents may be useful as intermediates for producing or synthesizing related chemical species, including other prodrug forms of L-nucleosides.

Compounds of the present invention find particular use in combating viral infections which afflict animals, and in particular, humans suffering from HBV or HIV viral infections. Compounds according to the present invention offer great potential as therapeutic agents against disease states for which there presently are few real therapeutic options. The compounds according to the present invention may be used alone or in combination with other agents or other therapeutic treatments.

Compounds according to the present invention are prodrug forms of β-L-FD4C and β- L-FddC which are appropriate for increasing the intracellular availability of 5'- monophosphate forms of the novel nucleoside analogs β-L-FD4C and β-L-FddC. In addition to providing higher concentrations of nucleoside within the cytosol compared to β-L-FD4C, the prodrug form of this agent also surprisingly exhibits reduced toxicity compared to β-L- FD4C

The present invention also relates to methods for inhibiting the growth or replication of viruses, especially, for example, HBV and/or HIV, comprising exposing the virus to an inhibitory effective amount or concentration of at least one of the disclosed prodrug L- nucleoside analogs. The molar concentration of the prodrug form which may be administered effectively for the inhibition of the virus is significantly less than the effective concentration of the free nucleoside form. The present invention may also be used for treating viral infections in animals and in humans.

The therapeutic aspect according to the present invention relates to methods for treating viral infections in animal or human patients, in particular, HBV or HIV infections in humans comprising administering anti-viral effective amounts of the compounds according to the present invention to inhibit the growth or replication of the viruses in the animal or human patient being treated.

Pharmaceutical compositions based upon these novel chemical compounds comprise the above-described compounds in a therapeutically effective amount for treating or preventing a viral infection, preferably a Hepatitis B viral or HIV infection. The pharmaceutical compositions according to the present invention optionally include a pharmaceutically acceptable additive, carrier or excipient.

Certain of the compounds, in pharmaceutical dosage form, may be used as prophylactic agents for inhibiting the growth or replication of the viral infection. These may be particularly appropriate as anti-HBV or anti-HIV agents. In certain pharmaceutical dosage forms, the pro-drug form of the compounds according to the present invention are preferred.

The compounds according to the present invention are produced by various synthetic chemical methods which are readily known to those of ordinary skill in the art.

More particularly, the present invention relates to prodrug forms for increasing the intracellular availability of 5'-monophosphate forms of the novel nucleoside analogs β-L- FD4C and β-L-FddC, preferably β-L-FD4C Use of these compounds to treat viral infections, including HIV and HBV infections, is also contemplated by the present invention. The present invention relates to the use of neutral monophosphate prodrugs (pronucleotides), in which the free phosphoric acid group is masked as a phosphate triester by a transient protecting group. These phosphate protecting groups can be cleaved either enzymatically or hydro lytically once inside of the cells, with concomitant release of the β-L-FD4C or β-L- FddC monophosphate dianion (11 and 12, respectively). Phosphate protecting groups, according to the present invention, include, but are not limited by, those depicted in Figure 7, with the SATE containing groups being preferable.

SATE-bearing 5'-monophosphate prodrugs of both β-L-FD4C and β-L-FddC, 3 and 4, respectively, were found to be more active than their corresponding parent nucleosides (1 and 2) in a standard HBV assay. Of particular significance is the fact that the pronucleotide of β- L-FD4C, 4, exhibited an EC₅₀ value of 2 nM, which was more than 8-fold lower than that of β-L-FD4C 1 (EC₅₀ = 17 nM). This inhibition was most dramatic when an infrequent dosing schedule was maintained. This difference in activity is an unexpected result. Additionally, pronucleotide 3 was capable of inhibiting HBV virus replication by 90%; whereas its parent β-L-FD4C (1) inhibited virus replication to a level of about 70% in the same assay. Most unexpectedly, when evaluated in the standard cytotoxicity assay in the CEM cell line, pronucleotide 3 exhibited an IC₅₀ value of 52 μM, which was 4 times less cytotoxic than that of β-L-FD4C 1 (IC_JO = 13.0 μM).

The combined effect of greater efficacy of the prodrug forms with concomitant lowered cytotoxicity, especially and most dramatically in the case of prodrug forms of β-L- FD4C leads to an unexpectedly large increase in the therapeutic index of the prodrug form compared to the parent nucleotides. Most unexpected is the decrease in the cytotoxicity of the prodrug form of the nucleoside analog, since the motivation for forming the prodrug form was, in large part, to increase the intracellular concentration of the nucleoside analog at any given dose of the analog. Without being limited by way of theory, it may be postulated that the prodrug, which forms the 5'-nucleotide monophosphate in the cytosol, has a more direct effect on the inhibition of the virus (perhaps by way of inhibition of one or more rate-limiting enzymatic steps) and less impact on the mammalian cell. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the nucleoside analogs β-L-FD4C and β-L-FddC, from which prodrug forms according to the present invention are prepared.

Figure 2 depicts the proposed decomposition steps of bis(S ATE)-bearing pronucleotides.

Figure 3 outlines the synthesis of bisthioester linker-bearing monophosphate prodrugs of β-L-FD4C and β-L-FddC.

Figure 4 outlines the synthesis of monophosphate β-L-FD4C prodrug 33 as set forth in the experimental section of the present specification..

Figure 5 outlines the synthesis of monophosphate β-L-FD4C prodrug 40 as set forth in the experimental section of the present specification..

Figure 6 presents the effects of β-L-FD4C and the bis(SATE)-bearing prodrug form of β-L-FD4C in standard HBV assays under different dosing regimens.

Figure 7 shows certain representative phosphate protecting groups according to the present invention.

Figure 8 outlines the chemical synthesis of the precursor 5'-O-silyl protected β- LFd4C (13) which is used to synthesize prodrug forms of β-LFd4C according to the present invention. DET AILED DESCRIPTION OF THE INVENTION

The following definitions shall be used to describe the present invention.

The term "patient" is used throughout the specification to describe an animal, including a mammal such as a human, to whom treatment with the compositions according to the present invention is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal.

The term "inhibitory effective concentration" or "inhibitory effective amount" is used throughout the specification to describe concentrations or amounts of prodrug nucleoside compounds according to the present invention which substantially or appreciably inhibit the growth or replication of susceptible organisms, especially viruses such as HBV or HIV.

The term "therapeutic effective concentration" or "therapeutically effective amount" is used throughout the specification to describe concentrations or amounts of compounds according to the present invention which are therapeutically effective in treating viral infections, especially including HBV and HIV infections in humans. The terms inhibitory effective concentration, inhibitory effective amount, therapeutic effective concentration and therapeutically effective amount are all interrelated terms which may be synonymous, depending upon the infection or disease state treated and the therapeutic result desired, including prophylactic uses of compounds according to the present invention.

In the present invention, the prodrug forms of β-L-FD4C or β-L-FddC are significantly more active than their corresponding free nucleoside forms by a factor of at least 2. According to this aspect of the present invention, it is an unexpected result that the prodrug form of β-L-FD4C or β-L-FddC may be administered to a human patient in an amount which is no greater than about 1/2 the molar concentration of the free nucleoside to obtain substantially the same therapeutic result in the treatment of HBV or HIV infections. In preferred embodiments according to the present invention, the prodrug form of β-L-FD4C or β-L-FddC, especially the prodrug form of β-L-FD4C. is administered to a human patient in an amount which is no greater than about 1/5 the molar concentration of the free nucleoside. In another preferred embodiment according to the present invention, the prodrug form of β-L- FD4C is administered in an amount which is no greater than about 1/8 the molar concentration of β-L-FD4C In the present invention, the administration of the bis-S-acyl-2- thioether monophosphate of β-L-FD4C is preferred, with the bis-S-acetyl-2-thioether monophosphate of β-L-FD4C being even more preferred.

The term "transient neutral masking group" or "masking group" is used throughout the specfication to describe a moiety which may be used to create a prodrug form of 5'- monophosphates of β-L-FD4C and β-L-FddC which will aid the delivery of the nucleoside analog into a cell. While not being limited by way of theory, it is believed that the neutral prodrug form is able to passively cross the cell membrane and once inside the cell, the masking group is removed by enzymatic or hydrolytic cleavage, producing the active, dianionic 5'-monophosphate form of the nucleoside analog.

The masking groups used in the present invention exhibit the following characteristics: 1) they exhibit a neutral or substantially neutral charge; 2) they neutralize the dianionic charge of the 5'-monophosphate of the nucleoside analalog by formation of a phosphate triester; and 3) they undergo enzymatic or hydrolytic cleavage in the cytosol, with the 5'-monophosphate form of nucleoside analog being produced. The masking groups for use in the present invention (represented by substituents on the phosphate of the nucleoside to form phosphotriesters) include thioesters as depicted in Figure 7, which may be represented by the following chemical structures:

-OCH₂CH₂SSCH₂CH₂OH or -OCH₂-O-C(O)-C(CH₃)₃,

where R is a linear or branch-chained C, to C₅ alkyl, phenyl or substituted phenyl and X is a substituent such as OMe, Me, Et, Pr, I-Pr, t-Bu, H, Cl, F, Br, or NO₂.

Other appropriate masking groups may be used to produce prodrug forms of the L-

nucleoside analogs L-FD4C and β-L-FddC, including, for example,

or

See, McGuigan, et al., Antiviral Chemistry & Chemotherapy.1996. 7, 184. In the present invention, the SATE-bearing masking groups are preferred.

Compounds according to the present invention may be prepared by synthetic methods known in the art. A general synthetic scheme employs a step-wise synthesis in which the free 5' hydroxyl group on the sugar moiety is first protected, followed by selective protection of the 4-amino group of the 5-F-cytosine base. Deprotection at the 5' hydroxyl position, followed by phosphorylation of the now-free 5' hydroxyl groups and deprotection of the 4- amino protecting group produces the appropriate 5' phosphate triester prodrug compound, which generally represents the prodrug nucleoside in final form.

In more specific embodiments of synthesizing compounds according to the present invention, an exemplary synthetic route employed for the preparation of preferred pronucleotides 3 & 4 from the corresponding L-nucleoside analogs is outlined in Figure 3. In the present invention, N-Troc protection of two known intermediates (in the synthesis of parent nucleosides β-L-FD4C and β-L-FddC), 13 & 14, provided the desired products 15 and 16, respectively. These intermediates were then desilylated to provide the corresponding 5'- carbinols 17 and 18 in modest yield. Reaction of 17 & 18 with the appropriate phosphoramidite 19, in the presence of 1-H tetrazole, followed by in situ oxidation with MCPBA, afforded the desired 5'-monophosphates 20 and 21, and thereafter, the final products 3 and 4, upon deprotection of the N-Troc protecting group.

In another exemplary synthetic route, as set forth in figure 4, synthesis of prodrug nucleoside 33 proceeds through amidine blocked β-L-FD4C 30 by reaction with the 2-hydroxybenzylalcohol phosphorous monochloride to produce an intermediate which is further reacted in the presence of meta-chloroperbenzoic acid to produce the corresponding N-blocked phosphotriester nucleoside followed by deprotection of the N-amidine group ustilizing ammonia in methanol to produce the mono-benzylphosphotriester nucleoside analog 33.

In another synthetic route, as set forth in figure 5, synthesis of the di- benzylsubtitutedphosphotriester nucleoside analog 40 proceeds by reacting amidine blocked β-L-FD4C 30 with tris(pyrrolidino) phosphine 34 in the presence of tetrazole and methylene chloride to produce intermediate nucleoside analog 35, which is subsequently reacted with the pivaloylbenzyl alcohol intermediate 39 in the presence of TMS-imidazole and tetrazole to produce a phosphorous intermediate which is subsequently oxidized to an amidine protected di-benzylsubstituted phosphtriester intermediate which is deprotected in ammonia/methanol to produce the di-benzylsubstituted phosphotriester nucleoside 40.

During chemical synthesis of the various compositions according to the present invention, one of ordinary skill in the art will be able to practice the present invention without undue experimentation. In particular, one of ordinary skill in the art will recognize the various steps that should be performed to introduce a particular substituent at the desired position of the base or a substituent at the desired position on the sugar moiety. In addition, chemical steps which are taken to "protect" functional groups such as hydroxyl or amino groups, among others, as well as "de-protect" these same functional groups, will be recognized as appropriate within the circumstances of the syntheses.

The therapeutic aspect according to the present invention relates to methods for treating retroviral infections in animal or human patients, in particular, HBV or HIV infections in humans comprising administering anti- viral effective amounts of the compounds according to the present invention to inhibit the growth or replication of the viruses in the animal or human patient being treated.

Pharmaceutical compositions based upon these novel chemical compounds comprise the above-described compounds in a therapeutically effective amount for treating a viral, preferably a Hepatitis B viral or HIV infection, optionally in combination with a pharmaceutically acceptable additive, carrier or excipient. One of ordinary skill in the art will recognize that a therapeutically effective amount will vary with the infection or condition to be treated, its severity, the treatment regimen to be employed, the pharmacokinetics of the agent used, as well as the patient (animal or human) treated.

In the pharmaceutical composition aspect according to the present invention, one or more of the compounds according to the present invention is formulated preferably in admixture with a pharmaceutically acceptable carrier. In general, it is preferable to administer the pharmaceutical composition in orally-administrable form, more particuarly as enteric coated formulations such as tablets, capsules or the like, but certain formulations may be administered via a parenteral, intravenous, intramuscular, transdermal, buccal, subcutaneous, suppository or other route. Intravenous and intramuscular formulations are preferably administered in sterile saline. Of course, one of ordinary skill in the art may modify the pharmaceutical compositions within the teachings of the specification to provide numerous formulations for a particular route of administration without rendering the compositions of the present invention unstable or compromising their therapeutic activity.

The amount of compound included within therapeutically active formulations according to the present invention is an effective amount for treating the infection or condition, in its most preferred embodiment, an HBV infection or an HIV infection. In general, a therapeutically effective amount of the present compound in dosage form usually ranges from slightly less than about 0.5 mg./kg. to about 50 mg./kg. of the patient or considerably more, depending upon the compound used, the condition or infection treated and the route of administration. In the case of HBV infections, the compound is preferably administered in amounts ranging from about 0.5 mg/kg to about 50 mg kg, more preferably about 1 mg/kg to about 20 mg/kg. In the case of the use of the prodrug form of β-L-Fd4C as an anti-HIV agent, the compound is preferably administered in an amount ranging from about 0.5 mg/kg to about 50 mg/kg, more preferably about 1 mg/kg to about 20 mg/kg depending upon the pharmacokinetics of the agent in the patient. This dosage range generally produces effective blood level concentrations of active compound ranging from about 0.04 to about 100 micrograms/cc of blood in the patient.

Administration of the active compound may range from continuous (intravenous drip) to several oral administrations per day (for example, Q.I.D.) and may include oral, topical, parenteral, intramuscular, intravenous, sub-cutaneous, transdermal (which may include a penetration enhancement agent), buccal and suppository administration, among other routes of administration.

To prepare the pharmaceutical compositions according to the present invention, a therapeutically effective amount of one or more of the compounds according to the present invention is preferably intimately admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dose. A carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral. In the present invention, an oral route of administration is preferred and in particular, enteric forms of tablets, capsules and the like are especially preferred. In preparing pharmaceutical compositions in oral dosage form, any of the usual pharmaceutical media may be used. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives including water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents and the like may be used. For solid oral preparations such as powders, tablets, capsules, and for solid preparations such as suppositories, suitable carriers and additives including starches, sugar carriers, such as dextrose, mannitol, lactose and related carriers, diluents, granulating agents, lubricants, binders, disintegrating agents and the like may be used. If desired, the tablets or capsules may be enteric-coated or sustained release by standard techniques. In the case of enteric coated forms, the active prodrug compound is generally formulated with an acid-stable coating in order to avoid dissolution of the tablet in the stomach and to promote dissolution in the duodenum, jejunum or ileum.

For parenteral formulations, the carrier will usually comprise sterile water or aqueous sodium chloride solution, though other ingredients, including those which aid dispersion, may also be included. Of course, where sterile water is to be used and maintained as sterile, the compositions and carriers must also be sterilized. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.

In particularly preferred embodiments according to the present invention, the pharmaceutical compositions are used to treat retroviral infections of mammals and in particular humans. The compounds are used to treat HBV infections, including chronic HBV infection, and numerous retroviral infections, especially HIV, including AIDS. Generally, to treat HBV or HIV infections, the compositions preferably will be administered in oral dosage form in amounts ranging from about 250 micrograms up to about 500 mg or more up to four times a day. The present compounds are preferably administered orally in enteric dosage form, but may be administered parenterally, topically or in suppository form.

The compounds according to the present invention, especially the prodrug forms of β-LFd4C, because of their unexpectedly low toxicity to host cells and unexpectedly high therapeutic index, may be employed advantageously prophylactically to prevent infection or to prevent the occurrence of clinical symptoms associated with the viral infection. Thus, the present invention also encompasses methods for the therapeutic or prophylactic treatment of viral infections, and in particular HBV or HIV infections. This prophylactic method comprises administering to a patient in need of such treatment an amount of a one or more of the prodrug compounds according to the present invention effective for alleviating, and/or preventing the viral infection. In the prophylactic treatment according to the present invention, it is preferred that the antiviral compound utilized should be as low in toxicity and preferably non-toxic to the patient. Consequently, the preferred compound for use in the prophylactic aspects according to the present invention is β-LFd4C having a bis-S-acetyl-2- thioethyl substituent as the phosphate transient protecting group (AA of Figure 7, R= Me). It is particularly preferred in this aspect of the present invention that the compound which is used should be maximally effective against the virus and should exhibit a minimum of toxicity to the patient (thus, have a high therapeutic index). In the case of the prodrug form of β-L-Fd4C, this compound may be administered within the same dosage range for therapeutic treatment (i.e., about 250 micrograms up to about 500 mg from one to four times per day for an oral dosage form) as a prophylactic agent to prevent the rapid proliferation of HIV or alternatively, to prolong the onset of AIDS in a patient.

In addition, compounds according to the present invention may be administered alone or in combination with other agents, especially including other compounds of the present invention. Certain compounds according to the present invention may be effective for enhancing the biological activity of certain agents according to the present invention by reducing the metabolism or inactivation of other compounds and as such, are co-administered for this intended effect. In the case of the prodrug compounds of β- LFd4C or β-L-FddC, more preferably, β-L-Fd4C, this compound may be effectively combined with any one or more of the standard anti-HIV agents which are presently utilized including AZT, ddC, ddl, d4T and other L-nucleoside compounds such as 3TC, (-)FTC, among others, and non-nucleoside agents such as protease inhibitors.

In a particularly preferred pharmaceutical composition and method for treating HBV infections, an inhibitory effective amount of the bis thioacetate prodrug of β-L-Fd4C is administered to a patient suffering from an HBV infection to alleviate the symptoms of such infection. In a particularly preferred pharmaceutical composition and method for treating HIV infections, an inhibitory effective amount of the prodrug form β-L-Fd4C, more preferably, the bis-S-acyl-2-thioether monophosphate of β-LFd4C is administered to a patient suffering from an HIV infection and/or AIDS to alleviate the symptoms of such infection.

While not being limited by way of theory, it is believed that the compounds according to the present invention may induce their inhibitory effect on the growth or replication of HBV or HIV by primarily functioning as anti-metabolites of the reverse transcriptase enzyme of the virus after conversion to the 5'monophosphate form of the β-L nucleoside analog.

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

EXAMPLES

Synthesis of the preferred embodiment of this invention is detailed in Figures 3, 4 and 5 and detailed below in Example 1. References to the numbered compounds are the same as those found in the figures. Biological activity of the Bisthioaceate prodrug of β-LFd4C is set forth in Example 2, below.

Example 1 Materials and Methods

All reagents were purchased commercially except where indicated as otherwise. Solvents were distilled prior to use. Proton NMR spectra were recorded on a Varian EM390 or Bruker WM250 instrument and reported as ppm (delta) downfield from (CH₃)₄Si. Ultraviolet spectra were recorded on a Beckman 25 spectrophotometer. Analytical thin-layer chromatography (TLC) was performed using Merck EM Silica Gel 60 precoated sheets. Column chromatography employed Merck EM silica gel using standard organic solvents. Detailed reaction conditions and characterizations of each intermediate (including 'H NMR, ¹³C NMR and LRMS or HRMS and elemental analysis data for compounds are provided in this section.

Synthetic Procedure

Preparation of the prodrug form of β-L-F-d4C proceeds through synthesis of 5'-O- silyl ether protected β-L-Fd4C from D-glutamic acid. The synthesis of this derivative is essentially the same as presented in pending patent application serial number 08/663,674, filed June 14, 1996. After synthesizing the silyl ether protected L-nucleoside analog 13 or the amidine protected L-nucleoside analog 30, the synthesis follows the general schemes presented in Figures 3, 4 and 5 which are detailed below.

Preparation of Lactone Ester 23 From D-Glutamic Acid

To a suspension of D-glutamic acid (50.0 g) in water (70 mL) was added cone. HCI (70 mL) at 0°C, followed by slow addition of a solution of NaNO₂ (35.0 g) in water (75 mL) over a period of 2 hr. The reaction was then allowed to warm to r.t. and stirred for additional 15 hr. The solvent was evaporated in vacuo (below 50° C) to dryness. The reaction mixture was then stirred with EtOAc (150 mL). The insoluble fraction was filtered off and the solids were washed with EtOAc (2X50 mL). The combined filtrates were dried (Na^O . The solvent was evaporated in vacuo to afford 45 g (-100%) of the crude (R)-g-lactone (22) as a pale yellow syrup.

A solution of the above crude acid and the catalytic amount of p-TsOH (1.0 g) in EtOH (65 mL) and benzene (150 mL) was refluxed for 5 hr, and the solvent was distilled off under atmospheric pressure until the b.p. raised to 79°C The reaction mixture was cooled to r.t., and then diluted with benzene (500 mL). The resulting reaction mixture was washed with water, 10% Na^O_j solution, water and then dried with NajSO,,. The solvent was evaporated and the residue was distilled under high vacuum to provide 35.0 g (65% two-step yield) of the desired ester-lactone (23). Η NMR of 23 (CDC1₃, 300 MHz): δ 4.87 (m, IH), 4.14 (q, 2H), 2.55-2.23 (m, 4H), 1.22 (t, 3H). Preparation of Lactone Alcohol 24

To an ethanol solution (48 mL) of NaBH₄ (2.90 g) was added at 0°C an ethanol solution (72 mL) of the ester-lactone 23 (17.3 g, 109.5 mmol) over 10 min. The resulting reaction mixture was stirred at room temperature for 1 hr. The reaction was then quenched at 0^CC with 10% HCI until the pH of the soluton was 3. The solids were filtered off. The filtrates were cone, in vacuo, and the resulting residue was coevaporated with MeOH three times. The resulting residue was then purified with silica gel chromatography (10-20% EtOH/CH₂Cl₂) to afford 9.5 g (75%) of the desired alcohol 24 as a colorless liquid. *H NMR of 24 (CDClj, 300 MHz): δ 4.72 (m, IH), 3.99 (dd, J=3.1 Hz, J'=12.7 Hz, IH), 3.72 (dd, J=4.8 Hz, J'=12.6 Hz, IH), 2.76-2.18 (m, 4H).

Preparation of Silylether Lactone 25

To a dichloromethane solution (62 mL) of 24 (5.10 g, 43.97 mmol) was added imidazole (3.89 g, 57.16 mmol) and t-butylchlorodiphenylsilane (13.30 g, 12.58 mL) at 0°C The reaction was stirred at 0°C for 1 hr and then at r.t. for 2 hr. The reaction mixture was diluted with CH₂C1₂ (150 mL), and washed with water (3X40 mL) and brine (40 mL). The organic layer was dried and cone, in vacuo to yield a residue, which was chromatographed (20-30% EtOAc/Hexanes) to afford 15.2 g (97%) of the desired silylether 25 as a pale yellow syrup. 'H NMR of 25 (CDC1₃, 300 MHz): δ 7.70-7.39 (m, 10H), 4.61 (m, IH), 3.90 (d, J=3.3 Hz, J'=11.4 Hz, IH), 3.70 (dd, J=3.3 Hz, J'=11.4 Hz, IH), 2.70 (m, IH), 2.53 (m, lH), 2.26 (m, 2H), 1.08 (s, 9H).

Preparation of Phenylselenide Lactone 26

Lithium bis(trimethysilyl) amide in THF (1M, 32.2 mL, 32.20 mmol) was added to 6 ml of THF under N₂, and cooled to -78 °C Silylether lactone 25 (10.36 g, 29.30 mmol) dissolved in 20 ml THF was added slowly to the above solution in 45 min at -78 °C One hour after the addition, TMSC1 (5.0 mL, 64.46 mmol) was added dropwise over 5 min. This mixture was then stirred at -78 °C for 1 hr, and then warmed to room temperature and stirred for 2 hr. The reaction mixture was then cooled to -78 °C, and N-phenylseleno-phthalimide (11.0 g, 36.40 mmol) was added through a powder addition funnel over 1 hr. Stirring at -78 °C was continued for 3 h, followed by warming to room temperature for 30 min. The reaction mixture was poured into 150 ml of NaHCO₃ solution and 300 mL ether, and then extracted twice with NaHCO₃ and once with NaCl solution. The aqueous layers were re-extracted with 100 mL of ether, and the organic layers were combined, dried over MgSO₄, filtered, and the solvent was removed in vacuo. Purification of the crude material by column chromatography afforded 11.20 g of the trans-isomer 26 (yield 75 %). A minor quantity of the cis-isomer 200 mg (1%) was also obtained. 'H NMR of 26 (CDC1₃, 300 MHz): δ 7.75-7.32 (m, 15H), 4.40 (m, IH), 4.16 (dd, J=5.4 Hz, J'=9.2 Hz, IH), 3.90 (dd. J=2.9 Hz, J'=l 1.5 Hz, IH), 3.66 (dd, J=3.1 Hz, J'=11.5 Hz, IH), 2.75 (m, IH), 2.33 (m, IH), 1.10 (s, 9H). ¹³C NMR of 26 (CDC1₃, 75 MHz): δ 176.1, 135.8, 135.7, 135.6, 132.9, 132.5, 130.1, 129.5, 129.1, 128.0, 127.2, 78.8, 65.0, 37.3, 32.5, 27.0, 19.3.

Preparation of Lactol 27

A toluene solution (10 mL) of the phenylselenide lactone 26 (1.63 g, 3.20 mmol) was treated at -78°C with DIBAL-H (2.35 mL, 1.5 M). After 1 hr, an additional amount of DIBAL-H (0.32 mL) was added. After 30min, the reaction was quenched at -78 °C with a saturated solution of sodium potassium tartrate (40 mL). The reaction mixture was warmed to r.t. and extracted with EtOAc (2X50 mL). The combined organic layers was further washed with sodium potassium tartrate saturated solution until a clear solution was obtained. The organic layer thus obtained was dried and evaporated in vacuo. The residue was chromatographed (10-20% EtOAc/Hexanes) to provide 1.60 g (98%) of the desired lactol 27. 'H NMR of 27 (CDC1₃, 300 MHz): δ 7.80-7.26 (m, 15H), 5.64-5.44 (m, IH), 4.45 (m, IH), 4.10-3.52 (m, 4H), 2.79-2.00 (m, 2H), 1.13-1.06 (m, 9H).

Preparation of Sugar Acetate 28

To a dichloromethane solution (18 mL) of the lactol 27 (4.50 g, 8.80 mmol) was added triethylamine (1.59 mL, 11.44 mmol) and acetic anhydride (1.08 mL, 11.44 mmol) at 0°C A catalytic amount of DMAP was also added. The reaction was stirred at 0°C for 1 hr and then at r.t. for 12 hr. The reaction was quenched with a NaHCO₃ saturated solution (20 mL), and the resulting reaction mixture was extracted with CH₂C1₂ (2X100 mL). The combined organic layers were washed with brine, and dried and cone, in vacuo. The residue was purified through a short pack of silica gel column (using 15% ethyl acetate/hexanes as eluant) to provide 4.75 g (98%) of the corresponding sugar acetate derivative 28 as a thick oil.

Η NMR (300 MHz, CDC1₃): δ 7.74-7.20 (m, 15H), 6.44-6.22 (m, IH), 4.44-3.52 (m, 4H), 2.58-2.08 (m, 2H), 1.86 & 1.55 (s, IH), 1.02 & 0.92 (s, 9H). FAB mass calcd. for C₂₉H₃₅O₄SiSe (MH+): 554, found: 554.

Preparation of Phenylselenide Nucleoside 29

A mixture of 5-Fluorocytosine (1.11 g, 8.58 mmol) and ammonium sulfate (40 mg) in (TMS)₂NH (10 ml) was heated to reflux for 2 hr. A clear solution was resulted. The reaction mixture was cooled to r.t., and the solvent was removed in vacuo. The resulting white solids (bis-TMS-5-FC) were dried under high vacuum for 30 min.

To the bis-TMS-5-FC thus prepared was added a dichloroethane solution (30 mL) of sugar acetate derivative 28 (4.75 g, 8.58 mmol). To the above solution was then added at 0°C a dichloroethane solution (10 mL) of TMSOTf (1.99 mL, 10.30 mmol). The reaction mixture was stirred at 0°C for 30 min, and then at room temperature for 90 min. At this point, the reaction was quenched with NH₄C1 saturated solution (30 mL), and extracted with dichloroethane (250 mL). The organic layer was washed with brine, dried and cone, in vacuo. The resulting residue was chromatographed (60-80%) EtOAc/Hexanes, then 10% EtOH/CH₂Cl₂) to afford 5.40 g (100%) of the desired product phenylselenide nucleoside 29 as a white foam. Η NMR of 29 (300 MHz, CDC1₃): δ 8.00 (d, J=5.5 Hz, IH), 7.66-7.25 (m, 15H), 6.13 (dd, J=1.4 Hz, J'=4.9 Hz, IH), 4.32 (m, 11~ ), 4.11 (d, J=11.2 Hz, IH), 3.85 (dd, J=6.5 Hz, J'=l 1.6 Hz, IH), 3.69 (dd, J=2.3 Hz, J'=l 1.8 Hz, IH), 2.47 (m, IH), 2.06 (m, IH), 1.11 (s, 9H). ¹³C NMR of 29 (CDCl₃, 75 MHz): δ 156.2, 156.0, 151.9, 137.8, 135.8, 135.7, 135.5, 134.6, 132.6, 132.3, 130.2, 130.0, 129.4, 128.6, 128.1, 127.9, 126.9, 126.1, 125.7, 91.3, 80.5, 65.1, 44.9, 32.3, 27.1, 19.3. HRMS (FAB) calcd. for C₃₁H₃₅FN₃O₃SiSe (MH+): 624.1597, found: 524.1601

Preparation of Silylether Didehydro Nucleoside 13

To a THF solution of phenylselenide 29 (437 mg, 0.702 mmol) was added at 0°C 30% wt. hydrogen peroxide aqueous solution (0.22 mL, 7.02 mmol). The reaction was stirred at 0°C for 1 hr. At this point, pyridine (0.57 mL, 7.02 mmol) was added at 0°C The reaction was stirred at r.t. for 3 hr. The reaction mixture was then diluted with EtOAc (50 mL) and Et₂0 (10 mL), and then washed with NaHCO₃ saturated solution and brine. The organic layer was dried over Na^O^ and then evaporated in vacuo to afford a residue, which was purified with silica gel chromatography (5-10% EtOH/CH₂Cl₂) to provide 280 mg (86%) of the desired Silylether didehydro nucleoside 13. 'H NMR of 13 (CDC1₃, 300 MHz): δ 8.95 (bs, IH), 7.74-7.34 (m, 10H), 6.98 (d, J=1.5 Hz, IH), 6.10 (d, J=5.9 Hz, IH), 5.92 (d, =5.7 Hz, IH), 5.83 (bs, IH), 4.85 (s, IH), 3.95 (dd, J=3.1 Hz, J'=11.6 Hz, IH), 3.77 (dd, J=3.4 Hz, J'=11.7 Hz, IH), 1.05 (s, 9H). ¹³C NMR of 13 (CDC1₃, 75MHz): δ 158.6, 158.4, 154.4, 138.4, 135.7, 135.2, 133.1, 132.9, 132.7, 130.1, 130.0, 127.9, 127.7, 125.4, 125.0, 91.2, 87.2, 65.3, 27.0, 19.3. FAB mass calc. for C₂₅H₂₉FN₃0₃Si (MH^*): 467, found: 467 HRMS (FAB) calcd. for C₂₅H₂₉FN₃O₃Si (MH+): 466.1962, found: 466.1963.

Preparation of Silylether Dideoxy Nucleoside 14

To a degassed benzene solution (5 mL) of phenyl selenide nucleoside 29 (323 mg, 0.519 mmol) and catalytic amount of AIBN was added tributyltin hydride (0.279 mL, 1.038 mmol). The reaction mixture was heated to reflux for 1.5 hr. The reaction mixture was then cooled to room temperature, the solvent was removed in vacuo. The residue was purified by silica gel chromatography (5-10% EtOH/CH2C12) to afford 240 mg (100%) of the desired silylether didedeoxy nucleoside 14. Η NMR of 14 (CDC1₃, 300Mhz): δ 8.17 (d, J=4.2 Hz, IH), 7.71-7.28 (m, 10H), 6.40 (bs, IH), 5.98 (d, J=6.0 Hz, IH), 4.16-4.09 (m, 2H), 3.75-3.70 (m, IH), 2.60-1.78 (m, 4H). FAB mass calc. for C₉H,₃FN₃0₃ (MH⁺): 230, found:

230.

Cl Mass calcd. for C₂₅H₃₁FN₃O₃Si (MH+): 468, found: 468.

Preparation of 5'-TBDPS-N4-Troc protected derivative (15)

A dry dichloromethane solution (30 mL) of 13 (1.40 g, 3.01 mmol) prepared as above was treated at 0°C with anhydrous pyridine (365 uL, 4.52 mmol), followed by 2,2,2,- trichloroethyl chloroformate (622 uL, 4.52 mmol). The reaction was stirred at 0°C for 1 hr, and then diluted with CH₂C1₂ (100 mL). The reaction mixture was washed with a saturated solution of NaHCO3 and brine. The organic phase was then dried and cone, in vacuo. The residue was chromatographed (40% EtOAc/Hexanes) to afford 1.44 g (75%) of the desired N4-Troc protected derivative 15. Η NMR (300 Mhz, CDC1₃): δ 7.93 (d, J=5.3 Hz, IH), 7.64-7.35 (m, 1 IH), 6.92 (bs, IH), 6.22 (d, J=5.9 Hz, IH), 5.90 (d, J=5.6 Hz, IH), 4.90 (bs, IH), 4.82 (s, 2H), 4.00 (dd, J=2.7 Hz, J'=11.9 Hz, IH), 3.82 (dd, J=3.1 Hz, J'=11.8 Hz, IH), 1.05 (s, 9H).

Preparation of 5'-TBDPS-N4-Troc protected derivative (16)

A dichloromethane solution (30 mL) of 5'-TBDPS protected derivative 14 (910 mg, 1.947 mmol) was treated at 0°C with pyridine (236 uL, 2.92 mmol) and followed by 2,2,2- trichloroethyl chloroformate (402 uL, 2.92 mmol). After stirring 1 hr at 0°C, the reaction mixture was diluted with CH₂C1₂ (75 mL), and quenched with a saturated solution of NaHCO₃ (20 mL). The organic layer was washed with brine and dried and filtered. The filtrate was cone, in vacuo. The residue thus obtained was chromatographed (20-30% EtOAc/Hexanes) to afford 964 mg (77%) of the desired product 16 as a colorless oil. Η NMR (300 Mhz, CDC13): δ 8.24 (d, J=5.3 Hz, IH), 7.71-7.65 (m, 4H), 7.40-7.37 (m, 5H), 6.05-6.02 (m, IH), 4.85 (s, 2H), 4.20-4.09 (m, 2H), 3.72 (dd, J=2.8 Hz, J'=l 1.8 Hz. IH), 2.48-1.60 (m, 4H), 1.10 (s, 9H).

Preparation of De-silylated N-troc protected derivative (17): A THF solution (37 mL) of 5'-silylated derivative 15 (1.44 g, 2.246 mmol) was treated with triethylamine-trihydrofluoride (1.47 mL, 8.99 mmol) at 0°C. The reaction was stirred at r.t. for 4 hr. At this point, second dose of reagent was added, and the reaction mixture was stirred at r.t. overnight. The solvent was then removed, and the residue was chromatographed (5-10% EtOH/CH2C12) to afford 690 mg (76%) of the 5'-desilylated derivative 17. Η NMR (300 Mhz, CDC13): δ 8.26 (d, J=6.2 Hz, IH), 6.95 (m, IH), 6.33- 6.30 (m, IH), 5.83 (d, J=6.0 Hz, IH), 4.94-4.92 (m, IH), 4.78 (s, 2H), 3.98 (dd, J=0.8 Hz, J'=11.7 Hz, IH), 3.86 (dd, J=2.0 Hz, J'=12.0 Hz, IH).

Preparation of Desilylated N4-Troc protected derivative (18):

A THF solution (13 mL) of 16 (957 mg, 0.672 mmol) was treated at 0°C with triethylamine trifhydrofluoride (438 uL, 2.688 mmol). The reaction mixture was stiired at r.t. overnight. At this point, second dose of the reagent was added, and the stirring was cont. for another 7 hr. The reaction was almost completed as indicated by TLC The solvent was then removed in vacuo, and the residue was chromatographed (5-10% EtOH/CH2C12) to afford 80 mg (8.3%) of the remaining starting material (16) along with 380 mg (63%) of the desired 5'- desilylated derivative 18 as a clear oil.

'H NMR (300 Mhz, CDC13 of 18): δ 8.72 (d, J=6.4 Hz, IH), 6.03 (d, J=6.4 Hz, IH), 4.82 (s, 2H), 4.25-4.12 (m, 2H), 3.84 (d, J=l 1.8 Hz, IH), 3.25 (bs, IH), 2.45-1.92 (m, 4H).

Preparation of Phosphorylating Agent (19):

A THF solution (40 mL) of 2-hydroxylethylthio acetate (3.17 g, 26.42 mmol) and triethylamine (8.09 mL, 58.12 mmol) was added dropwise (over 30 min) to a stirred solution of diisopropylphosphoramidous dichloride (2.66 g, 13.21 mmol) at -78°C The resulting mixture was stirred at -78 °C for 1 hr, and then at r.t. for 15 hr. The white solids formed during reaction was filtered. The filtrate was cone, in vacuo, the resulting residue was chromatographed (20% EtOAc/Hexanes) to afford 3.84 g (79%) of the desired phosphoramidite product 19 as a pale-yellow liquid. 'H NMR (300 Mhz, CDC13):_δ 3.74-3.12 (m, 3H), 3.09 (t, J=6.5 Hz, 2H), 2.31 (s, 3H), 1.14 (d, J=6.8 Hz, 6H).

Preparation of N4-Troc protected prodrug of β-L-FD4C (20):

To a THF solution (2 mL) of 5'-carbinol 17 (118 mg, 0.293 mmol) and phosphoramidite reagent 19 (130 mg, 0.352 mmol) was added 1-H tetrazole (61 mg, 0.879 mmol) at room temperature. The stirring was continued overnight at r.t. The reaction mixture was then treated with a dichlorometahne solution (2 mL) of MCPBA (93 mg, _70% pure, 0.381 mmol) at -40°C The stirring was continued at -40°C for 1 hr and then at room temperature for 2 hr. At this point, the reaction mixture was quenched with 10% Na2SO3 (10 mL), and extracted with EtOAc (3X20 mL). The combined organic layers were washed with brine and dried and filtered. The filtrate was cone, in vacuo, and the resulting residue was chromatographed (5-10% EtOH/CH2C12) to afford 173 mg (- 100%) of the desired product 20 as a clear oil. (This product was contaminated slightly with 3-chlorobenzoic acid as judged by 'H NMR).

Η NMR (300 Mhz, CDC13): δ 7.77 (d, J=5.9 Hz, IH), 6.99 (m, IH), 6.39 (m, IH), 5.97 (d, J=5.8 Hz. IH), 5.06 (bs, IH), 4.83 (s, 2H), 4.36-4.31 (m, 2H), 4.19-4.08 (m, 4H), 3.20-3.12 (m, 4H), 2.34 (s, 6H). LRMS (El) calcd. for C₂₀H₂₅C1₃FN₃O₁₀PS₂ (MH⁺): 686, found: 686.

Preparation of N4-Troc protected prodrug of β-L-FddC (21):

A THF solution (8 mL) of 5'-carbinol 18 (335 mg, 0.827 mmol) and 19 (396 mg, 1.075 mmol) was added 1-H tetrazole (174 mg, 2.481 mmol) at room temperature. After stirring at r.t. for 4 hr, the reaction mixture was cooled to -40 °C To this solution was then added a dichloromethane solution (5 mL) of MCPBA (172 mg, -70% pure, 1.075 mmol). The reaction was stirred at -40 °C for 1 hr, and then at r.t. overnight. The reaction mixture was diluted with EtOAc (75 mL) and washed with 10% Na2SO3 (15 mL). The aqueous layer was extracted with EtOAc (20 mL). The combined organic layers were washed with brine, and then dried (MgSO4). The organic layer was filtered and cone, in vacuo. The residue was chromatographed (60% EtOAc/Hexanes to 5% EtOH/CH2C12) to afford 575 mg (-100%) of the desired product 21 as a pale yellow oil.

Η NMR (300 Mhz, CDC13): δ 8.02 (d, J=6.2 Hz, IH), 5.98-5.95 (m, IH), 4.78 (s, 2H), 4.38- 4.06 (m, 7H), 3.14-3.08 (m, 4H), 2.60-1.91 (m, 10H).

Preparation of bisthioacetate prodrug of β-L-FD4C (3):

N4-Troc protected derivative 20 (188 mg, 0.274 mmol) was dissolved in methanol (9.1 mL). To this solution was added zinc dust (356 mg, 5.48 mmol). The resulting slurry was heated to reflux for 3 hr. The reaction was allowed to cool to room temperature, and the solvent was removed in vacuo. The residue was chromatographed (10-20% EtOH/CH2C12) to afford 100 mg (71 >) of the desired product 3 as a clear oil.

'H NMR (300 Mhz, CDC1₃): δ 7.66 (d, J=6.0 Hz, IH), 7.06 (bs, IH), 6.29-6.27 (m, IH), 6.03- 6.00 (m, IH), 5.04 (bs, IH), 4.31-4.28 (m, 2H), 4.17-4.09 (m, 4H), 3.19-3.14 (m, 4H), 2.37- 2.36 (m, 6H). LRMS (El) calcd. for C₁₇H₂₄FN₃PO₈S₂ (MH⁺): 512; found:

Preparation of bisthioacetate prodrug of β-L-FddC (4):

To a methanol solution (20 mL) of N-4 Troc protected derivative 21 (423 mg, 0.613 mol) was added zinc dust (796 mg, 12.26 mmol). The reaction mixture was heated to reflux for 3.5 hr. At this point, the solvent was removed in vacuo, and the resulting residue was chromatographed (5-10% EtOH/CH2C12) to afford 202 mg (64%) of the desired prodrug 4 as a clear oil. 'H NMR (300 Mhz, CDC13): δ 8.15 (bs, IH), 7.82 (d, J=6.5 Hz, IH), 5.97 (d, J=4.0 Hz, IH), 5.75 (bs, IH), 4.36-4.08 (m, 7H), 3.17-3.11 (m, 4H), 2.46-1.83 (m, 10H). LRMS calcd. for C17H26FN3PO8S2 (MH⁺): 514; found: 514.

Preparation of N-Protected β-L-FD4C (30).

To β-L-FD4C (5.0 g, 22 mmol) in 5 mL of ethanol was added at it N,N-dimethylformadehyde dimethyl acetate (4.48g, 35 mmol) and stirred for 2 hr. Solvent was then condensed to 1 mL, diethyl ether was added to precipitate the compound out as a white solid (4.05 g, 80%). 'H NMR of (I) (CDC1₃, 300 MHz): δ 8.82 (s, IH), 7.90 (d,

J=6 Hz, IH), 7.06 (br, IH), 6.28 (d, J=6 Hz, IH), 5.97 (d, J=6 Hz, IH), 4.98 (m, IH), 3.99

(dd, J=9.6, 2.7 Hz, IH), 3.84 (dd, J=9.6, 2.7 Hz, IH),

3.21 (s, 6H). FAB HRMS cald. for C₁₂H_I5N₄O₃F (MH⁺): 283.1206; found:

283.1211.

Preparation of 2-HydroxyBenzylPhosphorous Chloride Intermediate 32

To a solution of 2-hydroxybenzyl alcohol 31 (30g, 242mmol) and phosphorus trichloride (36.51 g, 261 mmol) in diethyl ether (300 mL) at -10°C was added dropwise with stirring a solution of Pyridne (40. lg, 507 mmol) in diethyl ether (60 mL) over 30 min. The mixture was then stirred at rt for 1 h and then stored at 10°C overnight. The mixture was filtered, and the solvent was removed. Hexane was added, and the mixture was filtered. The solvent was removed. The residue was distilled (1 mm Hg, 80-80 °C) giving the desired compound as a colorless liquid (66%). 'HNMR of (32) (CDC1₃, 300 MHz): δ 7.25-6.91 (m, 4H), 5.40 (m, IH), 4.97(m, IH). FAB HRMS cald. for C₇H₆O₂PCl (MH⁺): 187.9794; found: 187.9793.

Preparation of Nucleoside Prodrug 33

To the protected β-L-FD4C 30 (150 mg, 0.397 mmol) in anhydrous THF (5 mL) at -40 °C was added diisopropylethylamine (257 mg, 2.0 mmol), followed by 32 (200 mg, 0.95 mmol). The reaction mixture was warmed to 0°C and stirred for 2 hr. The reaction mixture was then cooled to -40 °C, and mCPBA (363 mg, 2.1 mmol) in 2 mL of CH₂C1₂ was added and stirred for 4 hr. At the end of the reaction, 2.0 mL of 2M NH₃-CH₃OH (3.36 mmol) was added to remove the amidine protective group. The crude after removal of solvents was purified by column chromatography on silica gel with 5 % ethanol in CH₂C1₂ to provide the title compound (52.5 mg, 33 %) as white solids. Η NMR of (33) (CDC1₃, 300 MHz): δ 8.08 (br, IH), 7.98 (d, J=7.8 Hz, IH), 7.6-6.9 (m, 6H), 6.29 (m, IH), 6.03 (m, IH), 5.36-5.29 (m, 2H), 5.05 (m, IH), 4.49-4.42 (m, 2H). FAB HRMS cald. for C_I2H,₆N₃O₆PF (M+l): 396.0761; found: 396.0763. Preparation of Tris(pyrrolidino)phosphine 34

Phosphorus bichloride (25.2 g, 18.5 mmol) was placed in a 500 mL flask with 200 mL of anhydrous ether. The flask was fitted with a 125 mL addition funnel containing 86.75 g (60.5 mmol) of (trimethyl silyl) pyrrolidine. The flask was cooled to -10°C under Ar with magnetic stirring, after which (trimethylsilyl) pyrrolidine was added dropwise over the course of 1 hr. Stirring was maintained for an additional 1 hr, after which it was stopped to allow the small amount of precipitates to settle. The reaction mixture was filtered through a medium frit sintered glass funnel, and the salts were washed with an additional 50 mL of anhydrous ether. Ether and TMSCl were removed from the filtrate by rotary evaporation to give the crude phosphine. Distillation at reduced pressure afforded 40.0 g of the compound as a colorless liquid (86%). 'H NMR of (V) (CDC1₃, 300 MHz): δ 3.18 (m, 12H), 1.82 (m, 12H). El HRMS cald. for C₁₂H₂₄N₃P(M+16⁺): 257.1657; found: 257.1655.

Preparation of p-Hydroxybenzyl alcohol mono-TBDP silyl ether 37

To -hydroxybenzyl alcohol 36 (6.21 g, 50 mmol) CH₂C1₂ (100 mL) was added imidazole (4.42 g, 65 mmol) and stirred at -40°C TBDPC1 (15.12 g, 56 mmol) was then slowly added and the mixture was warmed to rt and stirred for 8 hr. The mixture was then diluted with CH₂C1₂ and washed with NaHCO₃ and brine. The crude was purified on column with 5%> diethyl ether in hexanes to provide 42 %> of the title compound 37 (35 %> of the double protected compound). Η NMR of (X) (CDC1₃, 300 MHz): δ7.70-7.39 (m, 1OH), 7.19 (d, J=9 Hz, 2H), 6.78 (d, J=9 Hz, 2H), 4.69 (s, 2H), 1.07 (s, 9H). FAB HRMS cald. for C₁₂H₂₅O₂Si 361.1624; found: 361.1629.

Preparation of p-Pivaloylbenzyl alcohol mono-TBDP silyl ether 38 and Desilylated Benzyl Alcohol 39

To 37 (880 mg, 2.58 mmol) in 20 mL anhydrous CH₂C1₂ was added at rt DMAP(630 mg, 5.16 mmol) and pivolyl chloride (622.4 mg, 5.16 mmol) sequentially. The reaction mixture was stirred at rt overnight. The mixture was then diluted with CH₂C1₂, and washed with brine and H₂O. The crude was purified by column chromatography with 5 %> ethyl ether in hexanes to afford 1.1 g of the title compound (100 %). 'H NMR of (XI) (CDC1₃, 300 MHz): δ 7.70-7.00 (m, 14H), 4.75 (s, 2H), 1.35 (s, 9H), 1.09 (s, 9H).

The above compound in THF (50 mL) was then treated with Et₃N-3HF (3.33 mL, 20.7 mmol) at 0°C for 30 min. then rt for 8 hr. Solvent was evaporated, the crude was purified by column chromatography with 20 %> ethyl ether in hexanes to afford 420 mg of the title compound 39 (78 %) as white solids. "H NMR of (XII) (CDC1₃, 300 MHz): δ 7.34 (d, J=8.4 Hz, 2H), 7.02 (d, J=8.4 Hz, 2H), 4.63 (s, 2H), 1.35 (s, 9H). El HRMS cald. for C₁₂H₁₆O₁₃(M⁺): 208.1099; found: 208.1095.

Preparation of Di(pyrrolidino)phosphine Nucleoside Compound 35 and Prodrug Nucleoside 40

To the protected β-L-FD4C 30 (160 mg, 0.567mmol) in anhydrous CH₂C1₂ (10 ml) was added 1.15 mL of 0.5 M tetrazole in CH₃CN (0.567 mmol), followed by tris(pyrrolidino)phosphine 34 (156mg, 0.624 mmol). After completion of the reaction (in 20 min.) TMS-imidazole (9.95 mg, 0.06 mmol) and 5 mL of 0.5 M tetrazole in CH₃CN were added, followed by the addition of 39 (310 mg, 1.49 mmol). After 20 min, the reaction mixture was cooled to -40°C, and mCPBA (489 mg) in 5 mL of CH₂C1₂ was added. The reaction was continuously stirred for 2hr. At this point, 1.68 mL of 2M NH₃-CH₃OH (3.36 mmol) was added to remove the amidine protective group. The crude product after removal of solvents was purified by column chromatography on silica gel with 10 % ethanol in CH₂C1₂ to provide the title compound 40 200 mg (51 %) as white solids. 'H NMR of 40 (CDC1₃, 300 MHz): δ 7.54 (d, J=6 Hz, IH), 7.35-7.02 (m, 9H), 6.18 (d, J=6 Hz, IH), 5.91 (d, J=6 Hz, IH), 5.03 (d, J=5.2 Hz, 4H), 4.14 (m, IH), 3.71 (m, 2H), 1.37 (s, 9H). FAB HRMS cald. for C₃₃H₃₉O₁₀PN₃F (MH⁺) 688.2433; found: 688.2435.

BIOLOGICAL ACTIVITY The human hepatoblastoma cell line HepG2 2.2.15 (2.2.15 cells) was used for evaluation of the compounds for inhibition of hepatitis B virus in vitro. The parent cell line, HepG2, was stably transfected with a plasmid containing replication competent DNA (Sells, M.A., Chen, M.-L., and G. Acs. Production of hepatitis B virus particles in HepG2 cells transfected with cloned hepatitis B virus DNA. Proc.Natl. Acad. Sci. USA 1987, 84, 1005- 1009). The resulting cell line, 2.2.15, contains integrated and episomal copies of HBV DNA and secretes infectious hepatitis B virions into the medium. The intracellular HBV DNA profile is identical to DNA isolated from livers of chronically infected patients (Sells, M.A., Zelent, A.Z., Shvartsman, M., and G. Acs. Replicative Intermediates of Hepatitis B Virus in HepG2 Cells that Produce Infectious Virions. J. Virol. 1988, 62, 2836-2844). Antiviral activity of various agents is determined by exposure of the cells to increasing concentrations of the agent and measuring the extracellular HBV DNA in the supernatant by dot blot hybridization technology. Intracellular viral replication intermediates can also be measured by Southern blot analysis.

The cell culture assay was performed essentially as described by Korba and Milman (Korba, B.E. and G. Milman. A cell culture assay for compounds which inhibit hepatitis B virus replication. Antiviral Research 1991, 15, 217-228) with few modifications. The 2.2.15 cell line was maintained in RPMI1640 supplemented with 5% fetal bovine serum, 2 mM glutamine and antibiotics. Prior to initiating the HBV assay, cells were seeded into 24-well tissue culture plates at a density of approximately 5 x 10⁴ cells per well. Since HBV replication does not take place until the cells reach confluency, cells were grown to confluency (approximately 7 days) prior to addition of the drug. All testing was done in quadruplicate. Dosing was performed every 2 days for a total of 4 doses. Thus, on days 1, 3, 5 and 7 after confluence, medium was removed and replaced with fresh medium containing drug. The first dose consisted of a drug concentration of 40 nM and 2 fold serial dilutions were performed down to 2.5 nM. A "no drug" control was included. On day 9 after the initial dose, medium was collected and assayed for presence of HBV DNA by slot blot analysis. Blots containing DNA from the medium were hybridized to ³²P labeled HBV DNA and quantitated on a Packard Instantlmager. Results are plotted as a percentage of the radioactivity in the "no drug" control. The parent drug, β-L-Fd4C inhibited viral DNA synthesis 50 % at a concentration of 7 nM. Two batches of parent drug were tested. One was dissolved in neutral phosphate buffered saline and the other batch was dissolved in DMSO. The lines on the graph were essentially superimposable. The β-L-Fd4C prodrug (dissolved in DMSO) inhibited HBV DNA synthesis 50% at a concentration of 4 nM. More notably, the parent drug did not reach 90% inhibition with the doses tested; however the prodrug reached 90 % inhibition at a concentration of 19 nM. See Figure 6.

In order to address the question of whether the parent drug maintains activity with less frequent dosing, a modified protocol was employed. Instead of dosing every 2 days for 4 doses, dosing was performed every 3 days for a total of 3 doses. Medium was collected on day 9 and medium was assayed for the presence of HBV DNA as above. Results were plotted as a percentage of the "no drug" control. See Figure 6.

Using this modified dosing protocol yielded a greater disparity between the parent and prodrug. The parent β-L-Fd4C reached 50% inhibition at a concentration of 17 nM. Even at 40 nM, inhibition did not go much below 50% in these experiments. The β-L-FD4C prodrug did not lose potency using this dosing regime. The 50% inhibition level was at 2 nM.

The measurement of cytotoxicity value of the nucleoside 1 and its monophosphate nucleotide 3 was performed in five cell lines. The results obtained from this study are listed in Table 1. The cytotoxicities of 1 and 3 in CEM cell line were found to be 13 uM and 52 uM, respectively. Both of 1 and 3 were not cytotoxic in the rest of the cell lines tested.

Table 1: In Vitro Cytotoxicity Evaluation of Analogs 1 & 3:

COMPOUNDS ID_so (μM)**

CEM 2.2.15 C26 B 6 M109 β-L-FD4C (7) 13 >70 >70 >70 >70

L-FD4CMP Prod. (9) 52 >70 >70 >70 >70

CEM: Human T-cell Lymphatic Leukemia Cell

2.2.15: H. Hepatoma Cell C26: Murine Colon Cancer B16: Murine Melanoma M109: Murine Lung Carcinoma

The change in the therapeutic index of the β-L-FD4C caused by the formation of a prodrug according to the present invention is unexpectedly large, as shown by the 8-fold decrease in the EC50 coupled with a 4-fold decrease in cytotoxicity (CEM cell line). The decrease in cytotoxicity is particularly anti-intuitive, since the formation of the neutral prodrug is known in the art to increase the ability of the prodrug to cross the cell membrane, thereby raising the intracellular concentration of the nucleoside analog at any given applied dose.

This demonstrated high selectivity indicates the usefulness of the prodrugs in treating viral infections, especially HBV and HIV infections.

It will be understood by those skilled in the art that the foregoing description and examples are illustrative of practicing the present invention, but are in no way limiting. Variations of the detail presented herein may be made without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

CLAIMSWe claim:

1. A compound according to the structure:

Where Y is

R¹ is selected from a C, to C₅ straight- or branch-chained alkyl group or a phenyl or substituted phenyl group;

R² is -CH₂ CH₂-S-S-CH₂ CH₂OH, -CH₂ O-C(S)C-(CH₃)₃ or -CH₂ O-C(O)-C(CH₃)₃ ; and

X is OMe, Me, Et, H, F, Cl, Br, I or NO₂ .

2. The compound of claim 1 according to the structure:

or

ojR¹

where R¹ is selected from a C, to C₅ straight- or branch-chained alkyl group or a phenyl or substituted phenyl group.

3. The compound according to claim 1 wherein R¹ is a C, to C₅ straight- or branch- chained alkyl group.

4. The compound according to claim 1 wherein R¹ is a phenyl or substituted phenyl group.

5. The compound according to claim 1 where X is OMe, methyl, ethyl, H, Cl or Br.

6. The compound according to claim 2 where R¹ is methyl, ethyl or t-butyl.

7. The compound according to claim 6 where R¹ is methyl.

8. The compound according to claim 1 where R² is

-CH₂ CH₂-S-S-CH₂CH₂OH, -CH₂ O-C(S)C-(CH₃)₃ or -CH₂ O-C(O)-C(CH₃)₃.

9. The compound according to claim 8 where R² is -CH₂ CH₂-S-S-CH₂ CH₂OH.

10. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to the structure:

where Y is

R¹ is selected from a C, to C₅ straight- or branch-chained alkyl group or a phenyl or substituted phenyl group;

R² is -CH₂ CH₂-S-S-CH₂ CH₂OH, -CH₂O-C(S)-C(CH₃)₃ or -CH₂O-C(O)-C(CH₃)₃; and

X is OMe, Me, Et, H, F, Cl, Br, I or NO₂ in combination with a pharmaceutically acceptable excipient, additive or carrier.

11. The composition according to claim 10 wherein said compound has the structure:

OJR¹

or

where R¹ is selected from a C, to C₅ straight- or branch-chained alkyl group or a phenyl or substituted phenyl group.

12. The composition according to claim 11 wherein R¹ is a C, to C₅ straight- or branch-chained alkyl group.

13. The composition according to claim 11 wherein R¹ is a phenyl or substituted phenyl group.

14. The composition according to claim 11 where X is OMe, methyl, ethyl, H, Cl or

Br.

15. The composition according to claim 13 where R¹ is methyl, ethyl or t-butyl.

16. The composition according to claim 16 where R¹ is methyl.

17. The composition according to claim 1 1 where R² is -CH₂ CH₂-S-S-CH₂ CH₂OH, -CH₂ O-C(S)C-(CH₃)₃ or -CH₂ O-C(O)-C(CH₃)₃

18. The composition according to claim 17 where R² is -CH₂ CH₂-S-S-CH₂ CH₂OH.

19. A method for treating a patient for an HBV infection, comprising administering to said patient an anti-HBV effective amount of a compound according to the structure:

Where Y is

R¹ is selected from a C, to C₅ straight- or branch-chained alkyl group or a phenyl or substituted phenyl group;

R² is -CH₂ CH₂-S-S-CH₂ CH₂OH, -CH₂ O-C(S)C-(CH₃)₃ or -CH₂ O-C(O)-C(CH₃)₃; and

X is OMe, Me, Et, H, F, Cl, Br, I or NO₂ .

20. The method according to claim 19 according to the structure:

or

jR¹

where R¹ is selected from a C, to C₅ straight- or branch-chained alkyl group or a phenyl or substituted phenyl group.

21. The method according to claim 19 wherein R¹ is a C, to C₅ straight- or branch- chained alkyl group.

22. The method according to claim 19 wherein R' is a phenyl or substituted phenyl group.

23. The method according to claim 19 where X is OMe, methyl, ethyl, H, Cl or Br.

24. The method according to claim 20 where R¹ is methyl, ethyl or t-butyl.

25. The method according to claim 24 where R¹ is methyl.

26. The method according to claim 19 where R² is

-CH₂ CH₂-S-S-CH₂ CH₂OH, -CH₂ O-C(S)C-(CH₃)₃ or -CH₂ O-C(O)-C(CH₃)₃.

27. The method according to claim 26 where R² is -CH₂ CH₂-S-S-CH₂ CH₂OH.

28. A method for treating a patient for an HIV infection, comprising administering to said patient an anti-HBV effective amount of a compound according to the structure:

where Y is

R¹ is selected from a C, to C₅ straight- or branch-chained alkyl group or a phenyl or substituted phenyl group;

R² is -CH₂ CH₂-S-S-CH₂ CH₂OH, -CH₂O-C(S)-C(CH₃)₃ or -CH₂O-C(O)-C(CH₃)₃ ; and X is OMe, Me, Et, H, F, Cl, Br, I or NO₂ .

29. The method according to claim 28 wherein said compound has the structure:

ojR¹

or

where R¹ is selected from a C, to C₅ straight- or branch-chained alkyl group or a phenyl or substituted phenyl group.

30. The method according to claim 28 wherein R¹ is a C, to C₅ straight- or branch- chained alkyl group.

31. The method according to claim 28 wherein R¹ is a phenyl or substituted phenyl group.

32. The method according to claim 28 where X is OMe, methyl, ethyl, H, Cl or Br.

33-. The method according to claim 29 where R¹ is methyl, ethyl or t-butyl.

34. The method according to claim 33 where R¹ is methyl.

35. The method according to claim 28 where R² is

-CH₂ CH₂-S-S-CH₂ CH₂OH, -CH₂ O-C(S)C-(CH₃)₃ or -CH₂ O-C(O)-C(CH₃)₃.

36. The method according to claim 35 where R² is -CH₂ CH₂-S-S-CH₂ CH₂OH.