A 4‘-C-Ethynyl-2‘,3‘-Dideoxynucleoside Analogue Highlights the Role of the 3‘-OH in Anti-HIV Active 4‘-C-Ethynyl-2‘-deoxy Nucleosides

Abstract
4‘-C-Ethynyl-2‘-deoxynucleosides belong to a novel class of nucleoside analogues endowed with potent activity against a wide spectrum of HIV viruses, including a variety of resistant clones. Although favorable selectivity indices were reported for several of these analogues, some concern still exists regarding the 3‘-OH group and its role in cellular toxicity. To address this problem, we removed the 3‘-OH group from 4‘-C-ethynyl-2‘-deoxycytidine (1a). This compound was chosen because of its combined high potency and low selectivity index. The removal of the 3‘-OH was not straightforward; it required a different synthetic approach from the one used to synthesize the parent compound. Starting with glycidyl-4-methoxyphenyl ether, the target 4‘-C-ethynyl-2‘,3‘-dideoxycytidine analogue (rac -1h) was obtained after 13 steps. In a cellular assay, rac -1h was completely inactive (0.001−10 μM) against HIVLAI, demonstrating the critical importance of the 3‘-OH for antiviral activity. To determine whether the role of the 3‘-OH was essential for the phosphorylation of the compound by cellular kinases or for inhibition of DNA polymerization, we synthesized and tested the 5‘-triphosphate (rac -1h-TP) for its ability to inhibit HIV reverse transcriptase (RT). rac -1h-TP was slightly more potent than AZT-5‘-triphosphate against wild-type HIV RT, suggesting that the role of the 3‘-OH is crucial only for the activation of the drug by cellular kinases. The lipase-catalyzed resolution of rac -1h into ent -1h (β-d-dideoxyribo) and ent -14 (β-l-dideoxyribo) and the synthesis of the corresponding 5‘-triphosphates established the stereochemical assignment based on HIV RT's preference for the β-d-enantiomer, which was confirmed by assaying against the M184V variant, an RT mutant with a marked preference for incorporating nucleosides in the d-configuration.

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