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 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 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.     

Inhibition of uridine phosphorylase by pyrimidine nucleoside analogs and consideration of substrate binding to the enzyme based on solution conformation as seen by NMR spectroscopy

Some 3'- and/or 5'-substituted pyrimidine nucleosides, as well as anhydropyrimidine nucleosides, which have no flexibility about the N-glycosidic bond were studied as inhibitors of thymidine phosphorylase and uridine phosphorylase. The conformation of some analogs was also investigated in order to obtain information on substrate binding to the enzyme. The above compounds, including the potential anti-(human immunodeficiency virus) agent, 3'-azido-2',3'-dideoxy-5-methyluridine were not substrates for either thymidine phosphorylase or uridine phosphorylase. (The only exception was arabinofuranosyl-5-ethyluracil, which proved to be a poor substrate for uridine phosphorylase). The phosphorolysis of thymidine by thymidine phosphorylase was slightly or not at all altered by these pyrimidine nucloside analogs. The lowest Ki was obtained in the case of 3'-azido-2',3'-dideoxy-5-methyluridine and the highest in the case of 2'-deoxylyxofuranosyl-5-ethyluracil, when studying the analogs with flexible structure as inhibitors of uridine phosphorylase. The Ki for 2,3'- and 2,5'-anhydro-2'-deoxy-5-ethyluridine was 5-6 orders of magnitude higher than that for 2,2'-anhydro-5-ethyluridine. Competitive inhibition was observed in all cases. For these three molecules computer-aided molecular modelling predicts the following glycosidic torsion angles chi (O4,-C1,-N1-C2): 109 degrees for 2,2'-anhydro-5-ethyluridine, and 78 degrees and 71 degrees for 2,3'- and 2,5'-anhydro-2'-deoxy-5-ethyluridine respectively. These values are corroborated by high-resolution 13C- and 1H-NMR studies. 2'-Deoxy-5-ethyluridine is predicted to have a syn conformation with chi = 46 degrees and delta E about 2.5 kJ/mol over the minimum energy (in anti position, chi = -147 degrees). 1H and 13C data including homonuclear Overhauser enhancements complete the information about the solution conformation. Considering the Ki values obtained, it is likely that substrates of uridine phosphorylase will bind to the enzyme in the same conformation as 2,2'-anhydro-5-ethyluridine. The greater than 30 degrees deviation from the N-glycosidic torsion angle of 2,2'-anhydro-5-ethyluridine results in much higher Ki values.     

Synthesis and antitumor activity of 7-O-[2,6-dideoxy-2-fluoro-5-C-(trifluoromethyl)-alpha-L-talopyranosyl]- daunomycinone and -adriamycinone.

7-O-[2,6-Dideoxy-2-fluoro-5-C-(trifluoromethyl)-alpha-L-talopyranosyl]- daunomycinone and -adriamycinone have been prepared by the coupling of 3,4-di-O-acetyl-2,6-dideoxy-2-fluoro-5-C-(trifluoromethyl)-alpha-L- talopyranosyl iodide with daunomycinone. The key steps in this synthesis are the regioselective fluorination of methyl alpha-D-lyxopyranoside to give the 4-deoxy-4-fluoro-beta-L-ribopyranoside and the C-trifluoromethylation of the aldehydo-L-ribose derivative to give the 1,1,1-trifluoro-5-monofluoro-L-altritol derivative. Antitumor activities of the synthetic products were compared with those for the 2'-deoxy-2'-fluoro and 2',6'-dideoxy-5'-C-trifluoromethyl analogs.     

Synthesis and antiviral activity of D- and L-2'-azido-2',3'-dideoxy-4'-thiopyrimidine and purine nucleosides

Novel D- and L-2'-azido-2',3'-dideoxy-4'-thionucleosides were synthesized starting from L- and D-xylose via D- and L-4-thioarabitol derivative as key intermediates and evaluated for antiviral activity, respectively. When the final nucleosides were tested against HIV-1, HSV-1, HSV-2, and HCMV, they were found to be only active against HCMV without cytotoxicity up to 100 micrograms/ml.     

Preparation of 2'-deoxyribonucleosides with an identically 2H/13C-labeled sugar residue.

Thymidine with the stereoselectively 2H/13C-Labeled sugar moiety, (2'R)(5'S)-[1',2',3',4',5'-(13)C5;2',5'-(2)H2]-thymidine, was synthesized from uniformly 13C-labeled glucose, via the selectively deuterated ribose derivative prepared by the stereo-controlled deuteride transfer reactions. The labeled sugar moiety of the thymidine was then transferred to 2'-deoxyadenosine, 2'-deoxyguanosine, and 2'-deoxyuridine, by the enzymatic transglycosylation reactions by purine and pyrimidine nucleoside phosphorylases, in good yields. Labeled 2'-deoxyuridine was chemically converted to 2'-deoxycytidine. Consequently, all of the 2'-deoxynucleosides prepared by this method has the identically labeled sugar moiety. By using DNA oligomers containing the identically labeled sugar residue for NMR studies, any possible complexity in NMR data analyses expected to be observed for DNA oligomers containing variously labeled nucleosides can be minimized.     

A comparison of the conformations adopted by some 5-bromovinyl-2''-deoxyuridines and a correlation with their antiviral properties: an X-ray study.

Crystal structures of (Z)-5-(2-bromovinyl)-2'-deoxyuridine, 3',5'-di-O-acetyl-(E)-5-(2-bromovinyl)-2'-deoxyuridine and 3',5'-di-O-p-chlorobenzoyl-5-(2-dibromovinyl)-2'-deoxyuridine are compared with each other and with that of the most potent antiviral agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (E-BVDU) reported earlier. A comparison of the conformation of 3',5'-di-O-acetyl-pyrimidine nucleoside structures in which intermolecular hydrogen bond network formation is minimized, with those of their parent compounds has shown that the greatest change in rotation about the glycosyl bond and in the sugar ring pucker is exhibited by E-BVDU. Upon acylation this molecule changes from C2'-endo/C3'-exo conformation to C3'-endo/C4'-exo conformation. The relevance of these structures upon the biological activity of the nucleosides and in particular to their ability to be a substrate for thymidine kinase is discussed.     

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.     

Synthesis of 2',3'-dideoxy-3'-C-(hydroxymethyl)-4'-thiopentofuranosyl nucleosides as potential antiviral agent.

1-O-Acetyl-2,3-dideoxy-3-C-(hydroxymethyl)-4-thiofuranose derivative was synthesized from (S,S)-1,4-bis(benzyloxy)-2,3-epoxybutane derived from (+)-diethyl L-tartrate and the enantiomerically pure (E)-5-(2-bromovinyl)-1-[2',3'-dideoxy-3'-C-(hydroxymethyl)-beta-D-4'- thiopentofuranosyl]uracil 4 was obtained via coupling of silylated uracil followed by palladium-mediated coupling of methyl acrylate.     

Anti-HIV drugs: comparative toxicities in murine fetal liver and bone marrow erythroid progenitor cells

The toxicity of anti-HIV drugs, 3'-azido-3'-deoxythymidine (zidovudine, AZT), 2',3'-dideoxycytidine (DDC), 2',3'-dideoxy-2',3'-didehydrothymidine (d4T) and ribavirin was studied in vitro in murine fetal liver cells (FLC) and in bone marrow cells. These studies indicate that d4T is the least toxic drug and ribavirin is the most toxic agent in both models. However, the murine FLC system was found to be a more sensitive model for the assessment of toxicity of anti-HIV agents towards erythroid progenitor cells as indicated by the IC50 values.     

An alternative synthetic method for 4'-C-ethynylstavudine by means of nucleophilic substitution of 4'-benzoyloxythymine nucleoside

For the synthesis of 2',3' -didehydro-3' -deoxy-4' -C-ethynylthymidine (8: 4' -Ed4T), a recently reported promising anti-HIV agent, a new approach was developed. Since treatment of 1-(2,5-dideoxy-beta-l-glycero-pent-4-enofuranosyl)thymine with Pb(OBz)4 allowed the introduction of a 4'-benzoyloxy leaving group, nucleophilic substitution at the 4' -position became feasible for the first time. Thus, reaction between the 4'-benzoyloxy derivative (11) and Me3SiC identical with CAl(Et)Cl as a nucleophile led to the isolation of the desired 4'-"down"-ethynyl derivative (15) stereoselectively in 62% yield.     

Synthesis and antimetabolite properties of 5-substituted 2'-deoxyuridines. Anomeric 5-(2-aminohexafluoroprop-2-yl)- and 5-(2-trifluoroacetylaminohexafluoroprop-2-yl)-2'-deoxyuridine

Alkylation of 2,4-bis-O-(trimethylsilyl)uracil with hexafluoroacetone trifluoroacetylimine gave 5-(2-trifluoroacelylaminohexafluoroprop-2-yl)uracil, which was transformed by alkaline hydrolysis to 5-(2-aminohexafluoroprop-2-yl)uracil. The latter was glycosytated with 2-deoxy-3,5-di-O-p-toluoyl-alpha-D-ribofyranosyl chloride by means of various modifications of the silyl method leading to the predominant formation of beta-deoxynucleoside; after deacylation 1-(2-deoxy-beta-D-ribofuranosyl)-5-(2-aminohexafluoroprop-2-yl)ura cil was obtained. Interaction of silylated 5-(2-trifluoroacetylaminohexafluoroprop-2-yl)uracil with acylgalogenose gave anomeric O-substitutet deoxynucleosides, which were deblocked to give 5-(2-trifluoroacetylaminohexafluoroprop-2-yl)-2'-deoxyuridine and corresponding alpha-anomer. Alkaline hydrolysis of N-trifluoroacetyl group in both individual anomers produced 1-(2-deoxy-alpha-D-ribofuranosyl)-5-(2-aminohexafluoroprop-2-yl)ur acil and the abovementioned beta-anomer. Of all compounds synthesised only 1-(2-deoxy-beta-D-ribofuranosyl)-5-(2-aminohexafluoroprop-2-yl)ura cil has a moderate inhibitory effect on replication of vaccinia virus in vitro.     

Simultaneous determination of 3'-azido-2',3'-dideoxyuridine and novel prodrugs in rat plasma by liquid chromatography

3'-Azido-2',3'-dideoxyuridine (AZDU) is a nucleoside analog structurally similar to zidovudine (AZT) with proven activity against human immunodeficiency virus (HIV). The purpose of this study was to develop and validate a high-performance liquid chromatographic (HPLC) method to quantitatively determine AZDU and its novel prodrugs in rat plasma simultaneously. A reversed-phase gradient elution HPLC method was developed to quantitate AZDU and its prodrugs, N3-pivaloyloxymethyl-3'-azido-2',3'-dideoxyuridine (I), 5'-pivaloyloxymethyl-3'-azido-2',3'-dideoxyuridine (II), 5'-O-valinyl-3'-azido-2',3'-dideoxyuridine hydrochloride (III) and 5'-O-phenylalanyl-3'-azido-2',3'-dideoxyuridine hydrochloride (IV), in rat plasma. AZDU and its prodrugs were analyzed using an octadecyl silane column with a mobile phase consisting of 0.04 microM sodium acetate buffer, pH 5.0, and acetonitrile, running in a segmented gradient manner at a flow rate of 2 ml/min. Acetonitrile was increased from 10 to 50% during the first 8 min by 5% per min, followed by 10% per min until it reached 90% acetonitrile. 3'-Azido-2',3'-dideoxy-5-ethyluridine (CS-85) was used as an internal standard (25 microg/ml). Compounds were detected by UV absorption at 261 nm. Extraction recoveries for all compounds were greater than 80%. Retention times of AZDU, CS-85, prodrugs I, II, III and IV were 3.3, 5.2, 9.1, 8.8, 6.3 and 7.3 min, respectively. Calibration plots were linear over the range of 0.25-100 microg/ml for AZDU and prodrugs II, III, and IV and 0.5-100 microg/ml for prodrug I. The limit of quantitation was 0.25 microg/ml for prodrugs II, III and IV and 0.5 microg/ml for prodrug I. The intra- and inter-day variations were less than 10% and accuracies were greater than 90%. This method is rapid, sensitive and reproducible for the determination of AZDU and prodrugs in rat plasma.