Synthesis, conversion and biological activity of 5-substituted 2'-deoxyuridines modified by carbohydrates]

5-Benzyloxymethyl(Bom)-2'-deoxyuridine and its alpha-anomer were used as the key compounds for syntheses of thymidine analogues or 3'-derivatives. Anomeric 5-Bom-2'-deoxyuridines were synthesized from 5-Bom-uracil and 2-deoxy-3,5-di-O-p-toluyl-alpha-D-ribo-furanosyl chloride by means of the silyl method. 5-Bom-2'-deoxyuridine was transformed successively to 3',5'-di-O-mesyl derivative, 2,3'-anhydro-1-(2-deoxy-5-O-p-toluyl-beta-D-xylofuranosyl)-5-Bom-uracil and 3'-azido-2',3'-dideoxy-5-Bom-uridine. Treatment of the last with SnCl4 in methylene dichloride--methanol led to 3'-azido-2',3'-dideoxy-5-methoxymethyluridine. Under the same conditions the 5-methoxymethyl derivative was obtained from 3',5'-di-O-p-toluyl-5-Bom-2'-deoxyuridine. Interaction of 1-(2-deoxy-alpha-D-ribofuranosyl)-4-Bom-uracil with SnCl4 in methylene dichloride as well as the hydrogen transfer hydrogenolysis in the presence of cyclohexene and Pd(OH)2/C in ethanol led to 1-(2-deoxy-alpha-D-ribofuranosyl)-5-hydroxymethyluracil. Only 3'-azido-2',3'-dideoxy-5-Bom-uridine showed a cytotoxic activity against CaOv cells in vitro: in 10(-5)-10(-4) M concentrations it inhibits the thymidine incorporation into DNA by 78.8-95.1%. Elucidation of antitumor activity in vivo showed that this nucleoside inhibits growth of solid tumours, Ca755 and LLC, by 79 and 79-83%, respectively, but has no therapeutic effect against lympholeukemia P388.     

2'',3''-Dideoxy-3'' aminonucleoside 5''-triphosphates are the terminators of DNA synthesis catalyzed by DNA polymerases.

It is shown that 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates with adenine, guanine, cytosine and thymine bases are effective inhibitors of DNA polymerase I, calf thymus DNA polymerase alpha and rat liver DNA polymerase beta. The effect of the above-mentioned compounds is markedly higher than corresponding action of the well-known DNA synthesis inhibitors arabinonucleoside 5'-triphosphates and 2',3'-dideoxynucleoside 5'-triphosphates. 2',3'-dideoxy-3'-aminonucleoside 5'-monophosphate residues incorporate into the 3'-terminus of the primer and terminate the DNA chain elongation. The possibility of using 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates as terminators for DNA sequencing by the polymerization method is demonstrated.     

Synthesis and studies of 3'-C-trifluoromethyl nucleoside analogues bearing adenine or cytosine as the base

3'-Deoxy-3'-C-CF3, 2',3'-dideoxy-3'-C-CF3 and 2',3'-unsaturated-3'-C-CF3 nucleoside derivatives of adenosine and cytidine have been synthesized. All these derivatives were prepared by glycosylation of adenine and uracil with a suitable peracylated 3-trifluoromethyl sugar precursor. The resulting protected nucleosides were subject to appropriate chemical modifications to afford the target nucleoside derivatives. Additionally, the chemical stability in acidic and neutral media of the 2',3'-dideoxy-3'-C-CF3 and 2',3'-unsaturated-3'-C-CF3 nucleoside derivatives of adenosine was compared to that of their parent nucleosides 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxy-2',3'-didehydroadenosine (d(4)A). Our results confirm that addition of a trifluoromethyl group at C-3' on such nucleoside derivatives appears to confer increased chemical stability toward acid-catalyzed cleavage of the glycosidic bond comparatively to their parent counterparts. When evaluated for their antiviral activity in cell culture experiments, two compounds, namely, 2',3'-dideoxy-3'-C-CF3-adenosine and 2',3'-dideoxy-2',3'-didehydro-3'-C-CF3-cytidine exhibited moderate anti-HBV activity with EC50 values of 10 and 5 microM, respectively.     

Search for antimetabolites among 5-trimethylsilyluracil nucleosides and their nonglycoside analogs]

The reaction of 2'-deoxy-5-trimethylsilyl(Tms)uridine with methanesulfonyl chloride led to the corresponding 3',5'-di-O-mesyl derivative, which was treated with lithium toluylate in DMF to give 2,3'-anhydro-1-(2-deoxy-5-O-p-toluyl-beta-D-xylofurano- syl)-5-Tms-uracil. Under these conditions 1-(2,3-dideoxy-5-O-p-toluyl-alpha-D- glycero-pent-2-enofuranosyl)-5-Tms-uracil was obtained from 1-(2-deoxy-alpha-D-ribofuranosyl)-5-Tms-uracil. Interaction of 2,3'-anhydronucleoside with LiN3 in DMF and successive deacylation with MeONa-MeOH gave 3'-azido-2',3'-dideoxy-5-Tms-uridine. Hydrogenation of this compound with 10% Pd/C in ethanol gave 3'-azido-2',3'-dideoxy-5-Tms-uridine. From 2,4,5-tris-Tms-uracil and 2,3-didehydrofurane in 1,2-dichloroethane in the presence of SnCl4 1-(2-tetrahydrofuranyl)-5-Tms-uracil was prepared. In a similar way 1-[(1,3-dioxy-2-propoxy)methyl]-5-Tms-uracil was synthesized by condensation of silylated uracil with 1,3-dibenzyloxy-2-acetoxymethylglycerol followed by the hydrogen transfer hydrogenolysis with cyclohexene--20% Pd(OH)2/C. None of the compounds exhibits cytotoxic activity against CaOv in vitro. The acycloderivative in concentration of 250 micrograms/ml has no effect on the HSV-1 and vaccinia virus replication in vitro. 3'-Azidonucleoside in dose of 100-750 mg/kg as well as 1-(2-tetrahydrofuranyl)-5-Tms-uracil in dose of 160-800 mg/kg were devoid of antitumour activity against P388 in vivo.     

Synthesis and biological evaluation of 2',3'-didehydro-2',3'-dideoxy-9-deazaguanosine, a monophosphate prodrug and two analogues, 2',3'-dideoxy-9-deazaguanosine and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine

2',3'-Didehydro-2',3'-dideoxy-9-deazaguanosine (1), its monophosphate prodrug (2), and two analogues, 2',3'-dideoxy-9-deazaguanosine (3) and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine (4), have been synthesized from benzoylated 9-deazaguanosine (5). Basic hydrolysis of 5, selective protection of the 2-amino and 5'-hydroxy functions with isobutyryl and silyl groups, respectively, followed by reaction with thiocarbonyldiimidazole gave the cyclic thiocarbonate, which, upon reaction with triethyl phosphite, followed by deprotection, afforded 1. Treatment of 1 with phenyl methoxyalaninylphosphochloridate and N-methylimidazole gave 2. Catalytic hydrogenation of 1 gave 3. Hydrodediazoniation of 1 with tert-butyl nitrite and tris(trimethylsilyl)silane gave 4. Compounds 1-4 were found to be inactive against the human immunodeficiency virus and exhibited minimal to no cytotoxic activity against the L1210 leukemia, CCRF-CEM lymphoblastic leukemia, and B16F10 melanoma in vitro.     

Synthesis and antiviral evaluation of unnatural beta-L-enantiomers of 3'-fluoro- and 3'-azido-2',3'-dideoxyguanosine derivatives

3'-fluoro-2',3'-dideoxy- (3) and 3'-azido-2',3'-dideoxy- (4) beta-L-ribofuranonucleoside derivatives of guanine have been synthesized and their antiviral properties examined. All these derivatives were regioselectively and stereospecifically prepared by glycosylation of 2-N-acetyl-6-O-(diphenylcarbamoyl)guanine 5 with a suitable peracylated L-xylo-furanose sugar 6, followed by appropriate chemical modifications. The prepared compounds were tested for their activity against HIV and HBV viruses, but they did not show significant activity.     

2',3'-didehydro-2',3'-dideoxy-5-chlorocytidine is a selective anti-retrovirus agent

2',3'-Didehydro-2',3'-dideoxy-5-chlorocytidine (D4CC) is, in contrast with 2',3'-dideoxy-5-chlorocytidine (ddClCyd) and 2',3'-didehydro-2',3'-dideoxy-5-chlorouridine (D4CU), a potent and selective inhibitor of the replication of human immunodeficiency virus (HIV) types 1 and 2, simian immunodeficiency virus (SIV) and simian AIDS related virus (SRV). D4CC is a poor inhibitor of the phosphorylation of [5-3H]2'-deoxycytidine (dCyd) by partially purified MT-4 cell dCyd kinase (Ki: 612 microM). The findings that (i) D4CC has little, if any, affinity for MT-4 cell Cyd/dCyd deaminase, (ii) D4CU is not antivirally active and (iii) the antiretroviral action of D4CC can be reversed by dCyd, but not dThd, indicate that D4CC is antivirally active as its Cyd metabolite (D4CC 5'-triphosphate) and does not need to be deaminated (to the corresponding Urd metabolite) to exert its antiretroviral action.     

5'-O-ester prodrugs of potent and selective anti-HIV agent--2',3'-dideoxy-3'-fluoro-2-thiothymidine (S2FLT): synthesis and anti-HIV activity

Novel synthesis of 2',3'-dideoxy-3'-fluoro-2-thiothymidine (SFLT) based on transformation of appropriately protected 1-beta-D-threo-ribofuranosylthymine is presented. The synthesis and evaluation of SFLT 5'-O-ester prodrugs enzymatic hydrolysis, as well as their anti-HIV activity, is also described.     

Synthesis and antiviral evaluation of 3'-C-trifluoromethyl nucleoside derivatives bearing adenine as the base

3'-deoxy-3'-C-trifluoromethyl- (3), 2',3'-dideoxy-3'-C-trifluoromethyl- (5) and 2',3'-dideoxy-2',3'-didehydro-3'-C-trifluoromethyladenosine (6) derivatives have been synthesized and their antiviral properties examined. All these derivatives were stereospecifically prepared by glycosylation of adenine with a trifluoromethyl sugar precursor (1), followed by appropriate chemical modifications. The prepared compounds were tested for their activity against HIV, but they did not show an antiviral effect.     

Synthesis of 2',3'-dideoxy-3'-fluoro-L-ribonucleosides as potential antiviral agents from D-sorbitol

2',3'-Dideoxy-3'-fluoro-L-ribonucleosides were synthesized as potential antiviral agents. The key intermediate, methyl 5-O-benzoyl-2,3-dideoxy-3-fluoro-L-ribofuranoside, which was prepared from D-sorbitol, was condensed with pyrimidine and purine bases to obtain the respective nucleosides. Among them, the cytosine analogue 2',3'-dideoxy-3'-fluoro-alpha-L-cytidine showed a moderate anti-HBV activity.     

Synthesis and biological evaluation of L- and D-configurations of 2',3'-dideoxy-4'-C-methyl-3'-oxacytidine analogues

Novel L- and D-configuration 2',3'-dideoxy-4'-C-methyl-3'-oxacytidine and their 5-fluoro analogues have been synthesized from 1-benzyloxy-2-propanone and L-ascorbic acid in eight steps and evaluated for biological activity.     

Synthesis of 3'-deoxy-3'-C-methyl nucleoside derivatives

2',3'-Dideoxy-3'-C-methyl nucleosides bearing the five naturally occurring nucleic acid bases were synthesized. Additionally, the 3'-deoxy-3'-C-methyl nucleoside analogues bearing 5-aminoimidazole-4-carboxamide as well as 1,2,4-triazole-3-carboxamide moieties were prepared. The synthesis of the corresponding 2',3'-dideoxy-3'-C-methyl triazole derivative was also accomplished. The dideoxynucleoside derivatives were prepared by radical deoxygenation from their 3'-deoxy-3'-C-methyl parent ribonucleosides. When evaluated for their antiviral activity in cell culture experiments, none of these compounds showed any significant antiviral activity.     

Synthesis and cytotoxic properties of anomeric 5-substituted 3'-azido-2',3'-dideoxyuridines]

Glycosylation of trimethylsilyl derivatives of 5-benzyloxymethyl- and 5-hydroxymethyluracil with 3-azido-2,3-dideoxy-5-O-benzoyl-D-ribofuranosyl chloride (prepared from ethyl 3-azido-2,3-dideoxy-5-O-benzoyl-D-ribofuranoside) and subsequent deacylation gave in both cases a mixture of anomeric 3'-azido-2',3'-dideoxy-5-benzyloxymethyl-or 5-hydroxymethyluridines. The anomers were separated by preparative TLC and their structures were studied by UV, IR and 1H-NMR spectroscopy. It is shown that 1-(3-azido-2,3-dideoxy-alpha-D-ribofuranosyl)-5-benzyloxymethyluracil has cytotoxic activity in vitro: in 10(-5)-10(-4) M concentrations it inhibits the thymidine incorporation into DNA of CaOv cells on 78.6-95.2%.     

Synthesis of 5-deoxy-5-fluoro and 5-deoxy-5,5-difluoro derivatives of kanamycin B and its analogs. Study on structure-toxicity relationships.

5-Deoxy-5-fluoro- (1), 5.3'-dideoxy-5-fluoro- (2), and 5,3',4'-trideoxy-5-fluoro-kanamycin B (3) have been prepared by treatment of 5-epihydroxyl precursors (prepared by the Mitsunobu reaction) with DAST as the key step. 5,3'-Dideoxy-5,5-difluoro- (26) and 5,3',4'-trideoxy-5,5-difluoro-kanamycin B (27) were also prepared by treatment of the corresponding 5-oxo derivatives with DAST. These 5-deoxy-5-fluoro and 5-deoxy-5,5-difluoro derivatives showed markedly decreased toxicity as compared with the parent compounds.     

A novel action of human apurinic/apyrimidinic endonuclease: excision of L-configuration deoxyribonucleoside analogs from the 3' termini of DNA

beta-l-Dioxolane-cytidine (l-OddC, BCH-4556, Troxacitabine) is a novel unnatural stereochemical nucleoside analog that is under phase II clinical study for cancer treatment. This nucleoside analog could be phosphorylated and subsequently incorporated into the 3' terminus of DNA. The cytotoxicity of l-OddC was correlated with the amount of l-OddCMP in DNA, which depends on the incorporation by DNA polymerases and the removal by exonucleases. Here we reported the purification and identification of the major enzyme that could preferentially remove l-OddCMP compared with dCMP from the 3' termini of DNA in human cells. Surprisingly, this enzyme was found to be apurinic/apyrimidinic endonuclease (APE1) (), a well characterized DNA base excision repair protein. APE1 preferred to remove l- over d-configuration nucleosides from 3' termini of DNA. The efficiency of removal of these deoxycytidine analogs were as follows: l-OddC > beta-l-2',3'-dideoxy-2', 3'-didehydro-5-fluorocytidine > beta-l-2',3'-dideoxycytidine > beta-l-2',3'-dideoxy-3'-thiocytidine > beta-d-2',3'-dideoxycytidine > beta-d-2',2'-difluorodeoxycytidine > beta-d-2'-deoxycytidine >/= beta-d-arabinofuranosylcytosine. This report is the first demonstration that an exonuclease can preferentially excise l-configuration nucleoside analogs. This discovery suggests that APE1 could be critical for the activity of l-OddC or other l-nucleoside analogs and may play additional important roles in cells that were not previously known.     

Synthesis of 3'-substituted-2',3'-deoxy-2'-methylidenepyrimidine nucleosides.

2'-deoxy-2'-methylideneuridine derivative 9 was converted into 2',3'-didehydro-2',3'-dideoxy-2'-phenyl-selenomethyl derivative 16, which was treated with NCS and tert-butyl carbamate to afford 3'-amino derivative 18 via a [2,3]-sigmatropic rearrangement. Treatment of 9 with DAST gave a mixture of 2',3'-didehydro-2', 3'-dideoxy-2'-fluoromethyl derivative 19 and 3'-"up"-fluoro-2'-methylidene derivative 20 in a ratio of 1.5 : 1. On the other hand, when 12 was treated with DAST, 19 and 3'-"down"-fluoro-2'-methylidene derivative 21 were obtained in a ratio of 1 : 1.6. These nucleosides were converted into the corresponding cytidine derivatives 4, 6, and 8, respectively. The reaction mechanisms as well as biological activity of these compounds will also be discussed.     

Enzymatic synthesis of β-D-2', 3'-unsaturated-5-fluorocytidine from β-D-2',3'-unsaturated thymidine

We have used crude preparations of N-deoxyribosyl transferases (NdRT-II) from Lactobacillus helveticus to catalyze the transfer of a glycosyl moiety from a donor nucleoside to an acceptor base. Optimal conditions for the transglycosylation reaction to make D-D4FC starting from D-D4T and 5-FC were determined after the analysis of several experimental parameters including reaction time, concentration of substrate, pH and the type of buffer. For the first time, a practical procedure for enzymatic synthesis of β-D-2',3'-unsaturated-5-fluorocytidine (β-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine, D-D4FC) from β-D-2',3'-unsaturated thymidine (D-D4T) has been established. This method will be useful in the manufacture of important nucleoside analogues for anti-viral therapy.     

Synthesis and antiviral evaluation of 2',3'-dideoxy-2'-fluoro-3'- C-hydroxymethyl-beta-D-arabinofuranosyl pyrimidine nucleosides

The synthesis and anti-HBV and anti-HIV activity of a number of 2',3'-dideoxy-2'-fluoro-3'-C-hydroxymethyl-beta-D-arabinofuranosyl pyrimidine nucleosides are reported.     

Synthesis of enantiomerically pure D-FDOC, an anti-HIV agent

The beta-D-enantiomer of FDOC (2',3'-dideoxy-5-fluoro-oxacytidine) exhibits potent anti-HIV-1 activity. It was obtained in optically pure form by employing a tandem kinetic resolution/chiral salt crystallization protocol. In addition, conditions were developed that allowed the unwanted butyrate ester of the L-enantiomer of FDOC to be racemized. This material could then be recycled in future resolutions.