Vol. 58, Issue 5, 928-935, November 2000

Cyclosaligenyl-2',3'-didehydro-2',3'-dideoxythymidine Monophosphate: Efficient Intracellular Delivery of d4TMP

Jan Balzarini, Stefano Aquaro, Tina Knispel, Chiara Rampazzo, Vera Bianchi, Carlo-Federico Perno, Erik De Clercq, and Chris Meier

Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium (J.B., E.D.C.); Department of Experimental Medicine, University of Rome "Tor Vergata," Rome, Italy (S.A., C.-F.P.); Institut für Organische Chemie, Universität Hamburg, Hamburg, Germany (T.K., C.M.); Department of Biology, University of Padova, Padova, Italy (C.R., V.B.); and Istituto di Ricovero e Cura a Carattere Scientifico Lazzaro Spallanzani, Rome, Italy (C.-F.P.)

Cyclosaligenyl-2',3'-didehydro-2',3'-dideoxythymidine-5'-monophosphate (cycloSal-d4TMP) is a potent and selective inhibitor of human immunodeficiency virus replication in cell culture and differs from other nucleotide prodrug approaches in that it is designed to selectively deliver the nucleotide 5'-monophosphate by a controlled, chemically induced hydrolysis. Its antiviral efficacy in cell culture is at least as good as, if not superior to, that of d4T. CycloSal-d4TMP was found to lead to the efficient intracellular release of d4TMP in a variety of cell lines, including both wild-type CEM and thymidine kinase-deficient CEM/TK cells. Under similar experimental conditions, exposure of CEM/TK cells to d4T failed to result in significant d4TTP levels. The intracellular conversion of cycloSal-d4TMP proved to be both time and dose dependent. The half-life of d4TTP generated intracellularly from d4T- or cycloSal-d4TMP-treated CEM cells was ~3.5 h, and the intracellular ratios of d4TTP/d4TMP in cells exposed to cycloSal-d4TMP gradually increased from 1 to 3.4 upon prolonged incubation. Radiolabeled cycloSal-d4TMP could be separated as its two R_p and S_p diastereomers on high-performance liquid chromatography. The R_p diastereomer of cycloSal-d4TMP was 3- to 7-fold more efficient in releasing d4TMP and generating d4TTP than the S_p cycloSal-d4TMP diastereomer. This correlated well with the 5-fold more pronounced antiviral activity of the R_p diastereomer versus the S_p diastereomer. d4TMP is a poor substrate for the cytosolic 5'(3')-deoxyribonucleotidase (V_max/K_m for d4TMP: 0.08 of V_max/K_m for dTMP) and is only slowly hydrolyzed to d4T. This contributes to the efficient conversion of the prodrug of d4TTP.