WO1998017281A1 - PROMEDICAMENTS A BASE DE MONOPHOSPHATES DE β-L-FD4C ET DE β-L-FddC UTILISES COMME PUISSANTS AGENTS ANTIVIRAUX - Google Patents

PROMEDICAMENTS A BASE DE MONOPHOSPHATES DE β-L-FD4C ET DE β-L-FddC UTILISES COMME PUISSANTS AGENTS ANTIVIRAUX Download PDF

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WO1998017281A1
WO1998017281A1 PCT/US1997/018860 US9718860W WO9817281A1 WO 1998017281 A1 WO1998017281 A1 WO 1998017281A1 US 9718860 W US9718860 W US 9718860W WO 9817281 A1 WO9817281 A1 WO 9817281A1
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fd4c
mmol
hbv
present
prodrug
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PCT/US1997/018860
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English (en)
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Xiuyan Li
Shu-Hui Chen
Ellen Carmichael
Terrence W. Doyle
Yung-Chi Cheng
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Vion Pharmaceuticals, Inc.
Yale University
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Priority to AU49886/97A priority Critical patent/AU4988697A/en
Publication of WO1998017281A1 publication Critical patent/WO1998017281A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids

Definitions

  • 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.
  • retroviruses including human immunodeficiency virus (HIV), and hepatitis B virus (HBV).
  • HBV human immunodeficiency virus
  • HBV hepatitis B virus
  • 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).
  • AZT 3'-azido-2',3'-dideoxythymidine
  • ddl 2',3'-dideoxyinosine
  • dC 2',3'-dideoxycytidine
  • D T (2',3'-dideoxy-2',3'-didehydrothy
  • HBV hepatitis B virus
  • nucleoside analogs exhibiting potent antiviral activities are now in various stages of preclinical and clinical evaluation. These include ⁇ -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 ⁇ -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.
  • 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.
  • nucleotide prodrugs have been investigated intensively (Abstracts published in Antiviral Res. (suppl.) 1993, p. 45-142).
  • 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.
  • 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).
  • SATE S-acyl-2-thioethyl
  • AZT 3'-azido-2',3'-dideoxythymidine
  • 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. ⁇ -L-FD4C and ⁇ -L-FddC is desired to further increase their therapeutic usefulness.
  • 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.
  • the present invention relates to the surprising discovery that certain prodrug forms of the L-dideoxynucleoside analogs ⁇ -L-FD4C and ⁇ -L-FddC, especially ⁇ -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).
  • HBV Hepatitis B virus
  • HCV Human immunodeficiency virus
  • 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 ⁇ -L-FD4C exhibits particularly effective inhibition of HBV in comparison to ⁇ -L-FD4C and a markedly improved therapeutic index.
  • the prodrug form of ⁇ -L-FD4C or ⁇ -L-FddC can produce the same therapeutic prophylactic result as ⁇ -L-FD4C or ⁇ -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.
  • prodrug forms of ⁇ -L-FD4C and ⁇ - L-FddC which are appropriate for increasing the intracellular availability of 5'- monophosphate forms of the novel nucleoside analogs ⁇ -L-FD4C and ⁇ -L-FddC.
  • the prodrug form of this agent also surprisingly exhibits reduced toxicity compared to ⁇ -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.
  • 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.
  • 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.
  • 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.
  • the present invention relates to prodrug forms for increasing the intracellular availability of 5'-monophosphate forms of the novel nucleoside analogs ⁇ -L- FD4C and ⁇ -L-FddC, preferably ⁇ -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.
  • Phosphate protecting groups can be cleaved either enzymatically or hydro lytically once inside of the cells, with concomitant release of the ⁇ -L-FD4C or ⁇ -L- FddC monophosphate dianion (11 and 12, respectively).
  • Phosphate protecting groups 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 ⁇ -L-FD4C and ⁇ -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.
  • 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.
  • Figure 1 shows the nucleoside analogs ⁇ -L-FD4C and ⁇ -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 ⁇ -L-FD4C and ⁇ -L-FddC.
  • Figure 4 outlines the synthesis of monophosphate ⁇ -L-FD4C prodrug 33 as set forth in the experimental section of the present specification..
  • Figure 5 outlines the synthesis of monophosphate ⁇ -L-FD4C prodrug 40 as set forth in the experimental section of the present specification..
  • Figure 6 presents the effects of ⁇ -L-FD4C and the bis(SATE)-bearing prodrug form of ⁇ -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 ⁇ - LFd4C (13) which is used to synthesize prodrug forms of ⁇ -LFd4C according to the present invention.
  • 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.
  • a mammal such as a human
  • the term patient refers to that specific animal.
  • 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.
  • 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.
  • 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.
  • the prodrug forms of ⁇ -L-FD4C or ⁇ -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 ⁇ -L-FD4C or ⁇ -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.
  • the prodrug form of ⁇ -L- FD4C is administered in an amount which is no greater than about 1/5 the molar concentration of ⁇ -L-FD4C.
  • the administration of the bis-S-acyl-2- thioether monophosphate of ⁇ -L-FD4C is preferred, with the bis-S-acetyl-2-thioether monophosphate of ⁇ -L-FD4C being even more preferred.
  • 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 ⁇ -L-FD4C and ⁇ -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:
  • R is a linear or branch-chained C, to C 5 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 NO 2 .
  • nucleoside analogs L-FD4C and ⁇ -L-FddC including, for example,
  • 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.
  • an exemplary synthetic route employed for the preparation of preferred pronucleotides 3 & 4 from the corresponding L-nucleoside analogs is outlined in Figure 3.
  • N-Troc protection of two known intermediates in the synthesis of parent nucleosides ⁇ -L-FD4C and ⁇ -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.
  • prodrug nucleoside 33 proceeds through amidine blocked ⁇ -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.
  • synthesis of the di- benzylsubtitutedphosphotriester nucleoside analog 40 proceeds by reacting amidine blocked ⁇ -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.
  • 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.
  • 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.
  • 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.
  • one or more of the compounds according to the present invention is formulated preferably in admixture with a pharmaceutically acceptable carrier.
  • 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.
  • enteric coated formulations such as tablets, capsules or the like
  • Intravenous and intramuscular formulations are preferably administered in sterile saline.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an oral route of administration is preferred and in particular, enteric forms of tablets, capsules and the like are especially preferred.
  • any of the usual pharmaceutical media may be used.
  • suitable carriers and additives including water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents and the like may be used.
  • 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.
  • the tablets or capsules may be enteric-coated or sustained release by standard techniques.
  • 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.
  • the carrier will usually comprise sterile water or aqueous sodium chloride solution, though other ingredients, including those which aid dispersion, may also be included.
  • 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.
  • 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.
  • 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 may be employed advantageously prophylactically to prevent infection or to prevent the occurrence of clinical symptoms associated with the viral infection.
  • 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.
  • the antiviral compound utilized should be as low in toxicity and preferably non-toxic to the patient.
  • 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.
  • 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.
  • 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.
  • 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.
  • an inhibitory effective amount of the bis thioacetate prodrug of ⁇ -L-Fd4C is administered to a patient suffering from an HBV infection to alleviate the symptoms of such infection.
  • an inhibitory effective amount of the prodrug form ⁇ -L-Fd4C, more preferably, the bis-S-acyl-2-thioether monophosphate of ⁇ -LFd4C is administered to a patient suffering from an HIV infection and/or AIDS to alleviate the symptoms of such infection.
  • 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 ⁇ -L nucleoside analog.
  • Lithium bis(trimethysilyl) amide in THF (1M, 32.2 mL, 32.20 mmol) was added to 6 ml of THF under N 2 , 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
  • TMSC1 5.0 mL, 64.46 mmol
  • 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 NaHCO 3 solution and 300 mL ether, and then extracted twice with NaHCO 3 and once with NaCl solution. The aqueous layers were re-extracted with 100 mL of ether, and the organic layers were combined, dried over MgSO 4 , filtered, and the solvent was removed in vacuo.
  • 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 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.
  • 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.
  • 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 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 10 4 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 32 P 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, ⁇ -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 ⁇ -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.
  • 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.
  • CEM Human T-cell Lymphatic Leukemia Cell
  • the change in the therapeutic index of the ⁇ -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.

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Abstract

L'invention concerne certaines formes promédicamenteuses d'analogues du L-didéoxynucléoside, le β-L-FD4C et le β-L-FddC, notamment le β-L-Fd4C, qui de préférence contiennent des groupes 5'monophosphates portant un S-acyl-2-thioéthyle, et qui présentent une excellente activité contre le virus de l'hépatite B (HBV) et le virus de l'immunodéficience humaine (VIH). En particulier, les composés de l'invention présentent un puissante inhibition de la réplication du virus, associée à une très faible toxicité pour les cellules hôtes (c'est-à-dire les tissus animaux ou humains), et des index thérapeutiques étonnamment élevés. La forme promédicamenteuse du β-L-FD4C présente une inhibition particulièrement efficace de l'HBV, par comparaison avec le β-L-FD4C, et un index thérapeutique nettement augmenté.
PCT/US1997/018860 1996-10-24 1997-10-23 PROMEDICAMENTS A BASE DE MONOPHOSPHATES DE β-L-FD4C ET DE β-L-FddC UTILISES COMME PUISSANTS AGENTS ANTIVIRAUX WO1998017281A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU49886/97A AU4988697A (en) 1996-10-24 1997-10-23 Monophosphate prodrugs of beta-l-fd4c and beta-l-fddc as potent antiviral agents

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US73615696A 1996-10-24 1996-10-24
US08/736,156 1996-10-24

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WO1998017281A1 true WO1998017281A1 (fr) 1998-04-30

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AU (1) AU4988697A (fr)
WO (1) WO1998017281A1 (fr)

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JP2003508539A (ja) * 1999-09-08 2003-03-04 メタバシス・セラピューティクス・インコーポレイテッド 肝臓に特異的なドラッグデリバリーのためのプロドラッグ
WO2004043402A2 (fr) * 2002-11-12 2004-05-27 Pharmasset, Inc. Nucleosides modifies utilises comme agents antiviraux
EP1569652A2 (fr) * 2001-12-14 2005-09-07 Pharmasset Ltd. Nucleosides n sp 4 /sp -acylcytosines pour le traitement d'infections virales
EP1997829A1 (fr) 2001-12-21 2008-12-03 Human Genome Sciences, Inc. Protéines de fusion d'albumine
US7722886B2 (en) 2003-05-20 2010-05-25 Wyeth Compositions and methods for treatment of severe acute respiratory syndrome (SARS)
EP2206720A1 (fr) 2000-04-12 2010-07-14 Human Genome Sciences, Inc. Protéines de fusion d'albumine
US8076476B2 (en) 2007-11-15 2011-12-13 Avi Biopharma, Inc. Synthesis of morpholino oligomers using doubly protected guanine morpholino subunits
EP2431054A2 (fr) 2000-06-15 2012-03-21 Human Genome Sciences, Inc. Facteur delta de nécrose de tumeur humaine et epsilon
US8299206B2 (en) 2007-11-15 2012-10-30 Avi Biopharma, Inc. Method of synthesis of morpholino oligomers
US8623416B2 (en) 2009-11-25 2014-01-07 Michael Zasloff Formulations comprising aminosterols
US8691787B2 (en) 2005-12-13 2014-04-08 Spring Bank Pharmaceuticals, Inc. Nucleotide and oligonucleotide prodrugs
US8729058B2 (en) 2009-10-27 2014-05-20 Michael Zasloff Methods and compositions for treating and preventing viral infections
EP3037544A1 (fr) 2005-10-13 2016-06-29 Human Genome Sciences, Inc. Procedes et compositions destinees au traitement de patients atteints de lupus erythematosus systémique positifs pour des auto-anticorps
WO2018113710A1 (fr) * 2016-12-23 2018-06-28 江苏奥赛康药业股份有限公司 PROCÉDÉ DE PRÉPARATION D'UN COMPOSÉ β-NUCLÉOSIDE

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J. MED. CHEM., 1995, Vol. 38, LEFEBVRE et al., "Mononucleoside Phosphotriester with S-Acyl-2-Thioethyl Bioreversible Phoshate-Protecting Groups: Intracellular Delivery of 3'-Azido-2',3'-Dideoxythymidine 5'-Monophosphate", pages 3941-3950. *

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JP4979866B2 (ja) * 1999-09-08 2012-07-18 リガンド・ファーマシューティカルズ・インコーポレイテッド 肝臓に特異的なドラッグデリバリーのためのプロドラッグ
EP2295456A1 (fr) 2000-04-12 2011-03-16 Human Genome Sciences, Inc. Protéines de fusion d'albumine
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EP1569652A4 (fr) * 2001-12-14 2008-07-02 Pharmasset Inc Nucleosides n sp 4 /sp -acylcytosines pour le traitement d'infections virales
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WO2004043402A3 (fr) * 2002-11-12 2004-08-05 Pharmasset Ltd Nucleosides modifies utilises comme agents antiviraux
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US7722886B2 (en) 2003-05-20 2010-05-25 Wyeth Compositions and methods for treatment of severe acute respiratory syndrome (SARS)
US7892563B2 (en) 2003-05-20 2011-02-22 Wyeth Holdings Corporation Methods for treatment of severe acute respiratory syndrome (SARS)
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US10047114B2 (en) 2005-12-13 2018-08-14 Spring Bank Pharmaceuticals, Inc. Nucleotide and oligonucleotide prodrugs
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US8299206B2 (en) 2007-11-15 2012-10-30 Avi Biopharma, Inc. Method of synthesis of morpholino oligomers
US8076476B2 (en) 2007-11-15 2011-12-13 Avi Biopharma, Inc. Synthesis of morpholino oligomers using doubly protected guanine morpholino subunits
US9867835B2 (en) 2009-10-27 2018-01-16 Enterin Laboratories, Inc. Methods and compositions for treating and preventing viral infections
US8729058B2 (en) 2009-10-27 2014-05-20 Michael Zasloff Methods and compositions for treating and preventing viral infections
US10478444B2 (en) 2009-10-27 2019-11-19 Enterin, Inc. Methods and compositions for treating and preventing viral infections
US11419879B2 (en) 2009-10-27 2022-08-23 Enterin, Inc. Methods for treating and preventing viral infections
US8623416B2 (en) 2009-11-25 2014-01-07 Michael Zasloff Formulations comprising aminosterols
WO2018113710A1 (fr) * 2016-12-23 2018-06-28 江苏奥赛康药业股份有限公司 PROCÉDÉ DE PRÉPARATION D'UN COMPOSÉ β-NUCLÉOSIDE
US10752652B2 (en) 2016-12-23 2020-08-25 Jiangsu Aosaikang Pharmaceutical Co., Ltd. Method for preparing a ß-nucleoside compound

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