The synthesis, hydrolysis, and antiviral evaluation of novel, lipophilic cycloSal-d4TMP derivatives 3a-h of the anti-HIV dideoxynucleoside 2',3'-dideoxy-2',3'-didehydrothymidine (d4T, 1) are reported. This pro-nucleotide concept has been designed to deliver d4TMP (2) by selective chemical hydrolysis. All compounds 3a-h were synthesized using phosphorus(III) chemistry in good yields and in somewhat lower yields using phosphorus(V) chemistry starting from substituted salicyl alcohols 6a-h. The phosphotriesters 3 were obtained without stereochemical preference with respect to the configuration at the phosphorus center as 1:1 diastereomeric mixtures. However, a few of the triesters 3 could be separated into the diastereomers by means of semipreparative HPLC. In a 1-octanol/phosphate buffer mixture, all compounds 3 exhibited 9-100-fold higher lipophilicity as judged from their Pa values as compared to d4T (1). Furthermore, in hydrolysis studies 3 decomposed under mild aqueous basic conditions releasing solely d4TMP (2) and the diols 6 following the designed tandem reaction sequence. A correlation of the electronic properties introduced by the substituents and the half-lives of triesters 3 was observed. Thus, by varying the substituent, the half-lives of 3 could be adjusted over a wide range of compounds still delivering d4TMP (2) selectively. Phosphotriesters 3 exhibited considerable activity against HIV-1 and HIV-2 in wild-type human T-lymphocyte (CEM/O) cells as well as mutant thymidine kinase-deficient (CEM/TK-) cells. Surprisingly, we observed a 3-80-fold difference in antiviral activity between the two diastereomers. Our data clearly prove that the cycloSal-d4TMPs deliver exclusively the nucleotide d4TMP not only under simulated hydrolysis conditions but also under cellular conditions and thus fulfill the thymidine kinase-bypass premise. Therefore, the cycloSal-nucleotide concept is the first reported pro-nucleotide system that delivers the dideoxynucleotide by a pH-driven, chemically activated, tandem reaction without the requirement of an enzymatic contribution.