Cyclosaligenyl-2 ',3 '-didehydro-2 ',3 '-dideoxythymidine monophosphate: Efficient intracellular delivery of d4TMP

Cyclosaligenyl-2',3'-didehydro-2',3'-dideoxythymidine-5'-monophosphate (cyc loSal-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 antivira l 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 re lease of d4TMP in a variety of cell lines, including both wild-type CEM and thymidine kinase-deficient CEM/TK- cells. Under similar experimental condi tions, exposure of CEM/TK- cells to d4T failed to result in significant d4T TP levels. The intracellular conversion of cycloSal- d4TMP proved to be bot h time and dose dependent. The half-life of d4TTP generated intracellularly from d4T- or cycloSal-d4TMP-treated CEM cells was similar to 3.5 h, and th e intracellular ratios of d4TTP/d4TMP in cells exposed to cycloSal-d4TMP gr adually increased from 1 to 3.4 upon prolonged incubation. Radiolabeled cyc loSal- d4TMP could be separated as its two R-p and S-p diastereomers on hig h-performance liquid chromatography. The R-p diastereomer of cycloSal- d4TM P was 3- to 7-fold more efficient in releasing d4TMP and generating d4TTP t han the S-p cycloSal- d4TMP diastereomer. This correlated well with the 5-f old more pronounced antiviral activity of the R-p diastereomer versus the S -p diastereomer. d4TMP is a poor substrate for the cytosolic 5'(3')-deoxyri bonucleotidase (V-max/K-m for d4TMP: 0.08 of V-max/K-m for dTMP) and is onl y slowly hydrolyzed to d4T. This contributes to the efficient conversion of the prodrug of d4TTP.