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An efficient procedure for the synthesis of 3,5-disubstituted pyrazoles has been achieved through cyclocondensation reaction of 1,2-allenic ketones and hydrazines under extremely mild conditions. As an application, this methodology provides a highly efficient route to 5-(5-methyl-pyrazol-3-yl)-2′-deoxycytidine.

An improved strategy for the synthesis of protected 5-hydroxymethyl-2′-deoxycytidine phosphoramidite (XI), useful for site-specific incorporation into oligonucleotides, is described.

The 2-deoxy-D-arabino-hexopyranosyl nucleoside analogues of thymidine, deoxyuridine and deoxycytidine have been prepared by sugar-base condensation and a 3,4-unsaturated bond was introduced via the bisdithiocarbonates. Hydrogenolysis in the presence of Pd on carbon afforded the 2,3,4-trideoxy-D-glycero-hexopyranosyl analogues.

Cytosine nucleosides substituted at C-5 with differently substituted aryls and heteroaryls have been prepared via a palladium catalyzed reaction utilizing 5-chloromercuri or acetoxymercuri nucleosides and haloarenes or heteroarenes.

Reaction of 5-methylcytosine nucleosides and nucleotide with Na2S2O8 resulted in an oxidation of the 5-methyl group, while treatment of them and cytosine nucleosides with KHSO5 gave the corresponding N3-oxides.

2′-Deoxycytidine is prepared from 2-deoxy-α-D-ribofuranosyl chloride via novel stereoselective glycosylation conditions.

We report a four-step synthesis of 2'-deoxy-2-deuteroadenosine from 2'-deoxyadenosine in 38% overall yield. The more accessible 2'-deoxy-8-deuteroadenosine was also prepared and incorporated into DNA by automated solid phase synthesis (80% deuterium) using N6-benzoyl-2'-deoxy-8-deuteroadenosine-3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) in combination with acetyl-protected deoxycytidine and phenoxyacetyl-protected purine phosphoramidites.

An efficient process to synthesize 5′-O-dimethoxytrityl-N4-benzoyl-5-methyl-2 ′-deoxycytidine in high yield and quality is described. Final benzoylation was improved by developing a method to selectively hydrolyze benzoyl ester impurities. This inexpensive approach was scaled up to multi-kilogram quantities for routine use in oligonucleotide therapeutics.

[Chemical reaction: See text] Depurination is an important degradation pathway for antisense phosphorothioate oligonucleotides under conditions of thermal stress. We present evidence showing that depurinated oligonucleotides react with cytosine-containing sequences giving products containing a 6-(2-deoxy-beta-D-erythro-pentofuranosyl)-3-(2-oxopropyl)imidazo[1,2-c]pyrimidin-5(6H)-one residue. Further, we demonstrate that the same product is formed upon treatment of 2'-deoxycytidine with 4-oxo-2-pentenal, the latter being an expected byproduct of serial elimination reactions at apurinic sites. In addition to being important for synthetic oligonucleotides, apurinic site formation in cellular DNA is a common occurrence. Because repair of these sites can result in the production of 4-oxo-2-pentenal, it is interesting to speculate whether 6-(2-deoxy-beta-D-erythro-pentofuranosyl)-3-(2-oxopropyl)imidazo[1,2-c]pyrimidin-5(6H)-one residues can form in vivo.

Transformation of 2′-deoxyuridine and uridine analogs to protected 2′-deoxycytidine and cytidine analogs has been investigated by two different methods. First, traditional triazolation protocol and second p-nitrophenoxylation method. Our studies conclude that the triazolation method is better and suitable for commercial scale--up.

We report methodology which enables direct phosphorylation of 3′-deoxycytidine exclusively either at the 5′-hydroxyl or the 2′-hydroxyl. Protection of the base is not required. Standard phosphoramidochloridates in combination with pyridine and tert-butyl magnesium chloride is employed, in which the ratio of nucleoside to Grignard reagent is crucial. These findings, which appear to be general for 3′-deoxycytidines, are not applicable to 3′-deoxyuridine or 3′-deoxyguanosine.

Although 2′-deoxy-β- d -5-azacytidine (Decitabine) and β- d -5-azacytidine display potent antileukemic properties, their therapeutic use is hampered by their sensitivity to nucleophiles and to deamination catalysed by cytidine deaminase. As shown earlier [Shafiee M., Griffon J.-F., Gosselin G., Cambi A., Vincenzetti S., Vita A., Erikson S., Imbach J.-L., Maury G., Biochem. Pharmacol. 56 (1998) 1237–1242], β- l -enantiomers of cytidine derivatives are resistant to cytidine deaminase. We thus synthesized several 5-azacytosine β- l -nucleoside analogues to evaluate their enzymatic and biological properties. 2′-Deoxy-β- l -5-azacytidine ( l -Decitabine), β- l -5-azacytidine, 1-(β- l -xylo-furanosyl)5-azacytosine, and 1-(2-deoxy-β- l -threo-pentofuranosyl)5-azacytosine were stereospecifically prepared starting from l -ribose and l -xylose. d - and l -enantiomers of 2′-deoxy-β-5-azacytidine were weak substrates of human recombinant deoxycytidine kinase (dCK) compared to β- d -deoxycytidine, whereas both enantiomers of β-5-azacytidine or the l -xylo-analogues were not substrates of the enzyme. As expected, none of the presently reported derivatives of β- l -5-azacytidine was a substrate of human recombinant cytidine deaminase (CDA). The prepared compounds were tested for their activity against HIV and HBV and they did not show any significant activity or cytotoxicity. In the case of l -Decitabine, this suggests that the enantioselectivities of concerned enzymes other than dCK and CDA might not be favourable.

5′-Azido-3′-carbomethoxymethyl-4-N-benzoyl-2′,3′,5′-trideoxy-cytidine 17 and 5′-O-t-butyldimethylsilyl-3′-carboxymethyl-4-N-benzoyl-2′,3′-dideocytidine 22 were efficiently synthesized from 2′-deoxyuridine via a new method which transformed the uracil heterocycle to 4-N-benzoylcytosine (four steps, 60 % overall yield). The amide-linked deoxycytidine-thymidine dimer analog was synthesized.

Various 3'-azido analogues of pyrimidine deoxyribonucleosides have been synthesized and tested against human immunodeficiency virus (HIV-1, HTLV-III/LAV) in human peripheral blood mononuclear cells. Among these compounds, the 3'-azido analogues of thymidine (2), 3-(3-oxo-1-propenyl)thymidine (21), 2'-deoxyuridine (1), 2'-deoxy-5-bromouridine(5), 2'-deoxy-5-fluorocytidine (19), 2'-deoxy-5-iodouridine (6), 2'-deoxycytidine (18), 2'-deoxy-5-fluorouridine (4), 2'-deoxy-5-thiocyoanatouridine (16), 2'-deoxy-5-methylcytidine (20), 2'-deoxy-5-aminouridine (7), and 2'-deoxy-5-hydroxyuridine (10) were found to have significant antiviral activity, with EC50 values of 0.002, 0.01, 0.2, 1.0, 1.0, 1.1, 1.2, 4.8, 5.1, 5.1, 6.2, and 10 microM, respectively. The structure-activity relationships are discussed.