4 '-Ethynyl-2'-deoxynucleosides (4'-EdN) represent a new class of nucleoside analogues endowed with potent activity against a wide spectrum of HIV viruses, including a variety of resistant clones (Kodama et al. Antimicrob. Agents Chemother. 2001, 45, 1539). Although favorable selectivity indices (ca. 400-2600) were reported for a select group of analogues, some concern still exists regarding the 3'-OH group and its role in cellular toxicity. To study this problem, we elected to modify the structure of 4'-ethynyl-2'-deoxycytidine (4'-EdCyt), a compound that was identified amongst the most potent ones having a selectivity index in the lowest range of the spectrum. The removal of the 3'-OH group was not a trivial task and required a totally different synthetic approach to the one used for the 4'-EdN series. Starting with glycidyl 4-methoxyphenyl ether, 13 steps later the target cytidine analogue [(?)-4'-EddCyt) was obtained. The structure of the compound was confirmed by high resolution NMR spectroscopy and X-ray crystallography. The compound was completely inactive in vitro (0.001 ?M -10 ?M) against HIV-1-LAI, which demonstrated the critical importance of the 3'-OH group for antiviral activity. In order to determine whether the critical role of the 3'-OH is essential for the kinase(s) activation step(s) or for the inhibition of DNA polymerization, the 5'-triphoshate of (?)-4'-EddCyt was synthesized and tested for inhibition of reverse transcriptase (RT) in vitro. The 5'-triphosphate proved to be quite potent indeed suggesting that the role of the 3'-OH is only critical during activation by cellular kinases. Currently, our efforts are being directed to the resolution of both D- and L-enantiomers in order to select a candidate for a pro-drug approach that would by-pass the kinase activation step. Additional studies on a novel series of conformationally constrained, 2'-deoxy and dideoxy bicyclo[3.1.0]hexane analogues continues to determine the role of sugar conformation in RT inhibition and chain elongation after DNA incorporation.
