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

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 approximately 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.

Interactions of conformationally biased north and south 2'-fluoro-2', 3'-dideoxynucleoside 5'-triphosphates with the active site of HIV-1 reverse transcriptase.

Molecular dynamics simulations of a ternary complex of HIV-1 reverse transcriptase (RT), double-stranded DNA, and bound dideoxynucleoside-5'-triphosphate (RT-DNA-ddNTP), utilizing the ddNTPs ddATP, betaFddATP, and alphaFddATP, explain the experimentally observed order of potency of these 5'-triphosphates as inhibitors of RT: ddATP > betaFddATP > alphaFddATP. On the basis of RT's known preference to bind the incoming dNTP (or ddNTP) with a north conformation at the polymerase site, alphaFddATP, which in solution prefers almost exclusively a north conformation, was predicted to be the most potent inhibitor. However, Tyr115, which appears to function as a steric gate to preclude the binding of ribonucleoside 5'-triphosphates, prevents the effective binding of alphaFddATP in its preferred north conformation. The south-biased betaFddATP, while able to bind to RT without hindrance by Tyr115, has to pay a high energy penalty to be flipped to the active north conformation at the polymerase site. Finally, the more flexible and less conformationally biased ddATP is able to switch to a north conformation at the RT site with a smaller energy penalty than betaFddATP. These results highlight the opposite conformational preferences of HIV-1 RT for alphaFddATP and betaFddATP and help establish conformational guidelines for optimal binding at the polymerase site of this enzyme.

Intracellular metabolism of CycloSaligenyl 3'-azido-2', 3'-dideoxythymidine monophosphate, a prodrug of 3'-azido-2', 3'-dideoxythymidine (zidovudine).

The administration of CycloSaligenyl 3'-azido-2',3'-dideoxythymidine monophosphate (CycloSal-AZTMP) to CEM cells resulted in a concentration- and time-dependent conversion to the 5'-monophosphate (AZTMP), 5'-diphosphate (AZTDP), and 5'-triphosphate (AZTTP) derivatives. High ratios of AZTMP/AZTTP were found in the CEM cell cultures treated with CycloSal-AZTMP. The intracellular T(1/2) of AZTTP in CEM cell cultures treated with either AZT and CycloSal-AZTMP was approximately 3 h. A variety of human T- and B-lymphocyte cell lines efficiently converted the prodrug to the AZT metabolites, whereas peripheral blood lymphocytes and primary monocyte/macrophages showed at least 10-fold lower metabolic conversion of the prodrug. CycloSal-AZTMP failed to generate marked levels of AZT metabolites in thymidine kinase-deficient CEM/TK(-) cells, an observation that is in agreement with the substantial loss of antiviral activity of CycloSal-AZTMP in CEM/TK(-) cells. The inability of CycloSal-AZTMP to generate AZTMP in CEM/TK(-) cells is presumably due to a relatively high hydrolysis rate of AZTMP to the parent nucleoside AZT, combined with the inability of CEM/TK(-) cells to phosphorylate AZT to AZTMP through the cytosolic salvage enzyme thymidine kinase.

CycloSal-2'-ara(ribo)-fluoro-2',3'-dideoxyadenosine monophosphates--an effort to solve the structure-activity relationship of 2'-fluoro-ddA.

Novel lipophilic cycloSal-triesters 3 and 4 from the ara- and ribo-configurated 2'-fluorinated ddAs 1 and 2, respectively, were prepared. The title compounds 3 and 4 delivered the corresponding monophosphates and thus, increasing the bioactivity or convert a formerly inactive compound into a RT inhibitor.

cycloSal-Pronucleotides of 2',3'-dideoxyadenosine and 2', 3'-dideoxy-2',3'-didehydroadenosine: synthesis and antiviral evaluation of a highly efficient nucleotide delivery system.

The synthesis, hydrolysis, and antiviral evaluation of novel, lipophilic cycloSal-ddAMP (9a-d) and cycloSal-d4AMP (10a-d) derivatives of the antiviral purine dideoxynucleoside analogues 2', 3'-dideoxyadenosine (ddA) (2) and 2',3'-dideoxy-2', 3'-didehydroadenosine (d4A) (3) are reported. These potential pronucleotides release ddAMP (7) or d4AMP (8) selectively by a controlled, chemically induced tandem reaction. All new compounds 9 and 10a-d were synthesized in good yields using our previously reported phosphorus(III) method starting from substituted salicyl alcohols 14a-h. The phosphotriesters 9 and 10 were obtained with a stereochemical preference of 2:1 with respect to the configuration at the phosphorus center. In an 1-octanol/water mixture phosphotriesters 9 and 10 exhibited 7-43-fold higher lipophilicity than the parent nucleosides ddA (2) and d4A (3) as judged by their log P values. In hydrolysis studies, 9 and 10 decomposed under mild aqueous basic conditions releasing solely ddAMP (7) and d4AMP (8), as well as the diols 14. Further hydrolysis studies under acidic conditions showed a marked increase in stability with respect to the acid-catalyzed cleavage of the glycosyl bond. Phosphotriesters 9 and 10 exhibited antiviral potencies against wild-type HIV-1 and HIV-2 strains in human T-lymphocyte (CEM/O) cells that were, respectively, 100- and 600-fold higher than those of ddA (2) and d4A (3). Furthermore, all triesters 9 and 10 were markedly more active than the corresponding ddI compounds 11 and 12, which supports the concept of the delivery of the adenine nucleotides. Studies with adenosine deaminase (ADA) and adenosine monophosphate deaminase (AMPDA) showed that the triesters were not substrates for enzymatic deamination. The studies reported herein demonstrate conclusively that the cycloSal triesters deliver exclusively the nucleotides ddAMP and d4AMP, not only under chemical-simulated hydrolysis but also under intracellular conditions fulfilling the adenosine deaminase bypass premise.

cycloSal-Pronucleotides of 2'-fluoro-ara- and 2'-fluoro-ribo-2',3'- dideoxyadenosine as a strategy to bypass a metabolic blockade.

Novel, lipophilic cycloSal triesters 4a-c and 5a-c were synthesized, respectively, from the ara- and ribo-configurated 2'-fluorinated-2', 3'-dideoxyadenosines 2 and 3. The cycloSal phosphotriesters were used as tools to study the effects of the two different sugar pucker conformations induced by two opposite configurations of the fluorine substituent at C2' of the dideoxyribose moiety. F-ara-ddA (2) is known to be an active anti-HIV agent, whereas the ribo-analogue 3 is inactive. Hydrolysis studies with the triester precursors 4a-c and 5a-c showed selective formation of the monophosphates of 2 and 3. The lipophilicity of the triester prodrugs was considerably increased by the cycloSal mask with respect to ddA (1), F-ara-ddA (2), and F-ribo-ddA (3). Phosphotriesters 4 and 5 proved to be completely resistant to ADA and AMPDA deamination. In parallel experiments, ribo-nucleoside 3 showed a 50-fold faster deamination rate relative to the ara-analogue 2. Against HIV in CEM cells, the phosphotriesters 4 proved to be 10-fold more potent than the parent nucleoside 2. Furthermore, the prodrugs 4 were active against MSV-induced transformation of C3H/3T3 fibroblasts, while 2 was inactive. More interestingly, the ribo-configurated phosphotriesters 5, prepared from the inactive F-ribo-ddA (3), showed a level of anti-HIV activity that was even higher than that of F-ara-ddA (2). Our findings clearly prove that the application of the cycloSal-pronucleotide concept to F-ribo-ddA (3) overcomes a metabolic blockade in the formation of the corresponding monophosphate.