| Abstract
| - Lipophilic esters of3‘-azido-3‘-deoxy-5‘-O-(carboxyphosphinyl)thymidine(PFA-AZT) weresynthesized and tested for antiretroviral activity inCD4+ HT4-6C cells infected with eitherwild-type HIV-1LAI, a PFA-resistant strain encoding asingle-point mutation in reversetranscriptase (E89K), or an AZT-resistant clinical isolate (A018-post).Arbuzov condensationof 1-octadecyl, 1-eicosanyl, and 1-docosanyl chloroformate withtrimethyl phosphite yieldedthe corresponding dimethyl long-chain alkyl triesters of PFA.Selective removal of one methylgroup from the triesters with sodium iodide yielded monosodium salts,whereas treatment withbromotrimethylsilane cleaved both methyl groups while leaving thelong-chain alkyl groupintact. Neutralization of the resulting[(alkyloxy)carbonyl]phosphonic acids with 2 equiv ofsodium methoxide afforded disodium salts of the phosphonic acid moiety.Similar chemistrywas used to obtain the mono- and disodium salts of the cholesterolester of PFA. Reaction ofthe triesters with phosphorous pentachloride, followed by coupling withAZT and O-demethylation with sodium iodide, afforded3‘-azido-3‘-deoxy-5‘-O-[[(1-octadecyloxy)carbonyl]phosphinyl]thymidine (9a),3‘-azido-3‘-deoxy-5‘-O-[[(1-eicosanyloxy)carbonyl]phosphinyl]thymidine(9b),3‘-azido-3‘-deoxy-5‘-O-[[(1-docosanyloxy)carbonyl]phosphinyl]thymidine(9c), and 3‘-azido-3‘-deoxy-5‘-O-[[(3β-cholest-5-enyloxy)carbonyl]phosphinyl]thymidine(9d). Concentrations of9a−dfound to inhibit replication of wild-type HIV-1LAI by 50%(EC50 values) as measured in a plaquereduction assay were in the 0.1−0.3 μM range as compared with 0.013μM for AZT and 133μM for PFA. The concentration at which toxicity was observed in50% of the host cells (TC50values) as measured by a visual grading scale of cellular morphologywas 10 μM for 9a and9d, 32 μM for 9b, and 320 μM for9c. Thus, the TC50/EC50 ratioor selectivity index (SI) was100 for 9a, 230 for 9b, and 1000 for9c but only 33 for 9d, suggesting that thestraight-chainedfatty alcohol esters were more therapeutically selective. SimilarTC50 and SI values wereobtained for rapidly dividing CEM lymphoblasts as for HT4-6C cells.In assays against E89K,9a−c had mean EC50 values of 0.13,0.009, and 0.17 μM, whereas the EC50 of PFA was>1000μM and that of AZT was 0.009 μM; thus, E89K was highly resistant toPFA but not cross-resistant to either AZT or the lipophilic PFA-AZT conjugates. Inviral replication assays againstthe A018C-post isolate, the mean EC50 values of9a−c were 0.30, 0.53, and 0.77 μM ascomparedwith 2.9 μM for AZT and 65 μM for PFA; thus, the virus recoveredfrom a patient pretreatedwith AZT was not cross-resistant to either PFA or9a−c. A notable feature of theseresultswas that, in addition to being >1000-fold more potent than PFA againstthe PFA-resistantmutant, the lipophilic PFA-AZT conjugates were more potent than PFA, aswell as AZT, againstAZT-resistant HIV-1.
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