SYNTHESIS AND IN-VITRO ACTIVITY OF LONG-CHAIN XYCARBONYL)PHOSPHINYL]-3'-AZIDO-3'-DEOXYTHYMIDINES AGAINST WILD-TYPE AND AZT-RESISTANT AND FOSCARNET-RESISTANT STRAINS OF HIV-1

Lipophilic esters of '-azido-3'-deoxy-5'-O-(carboxyphosphinyl)thymidin e (PFA-AZT) were synthesized and tested for antiretroviral activity in CD4(+) HT4-6C cells infected with either wild-type HIV-1(LAI), a PFA- resistant strain encoding a single-point mutation in reverse transcrip tase (E89K), or an AZT-resistant clinical isolate (A018-post). Arbuzov condensation of 1-octadecyl, 1-eicosanyl, and 1-docosanyl chloroforma te with trimethyl phosphite yielded the corresponding dimethyl long-ch ain alkyl triesters of PFA. Selective removal of one methyl group from the triesters with sodium iodide yielded monosodium salts, whereas tr eatment with bromotrimethylsilane cleaved both methyl groups while lea ving the long-chain alkyl group intact. Neutralization of the resultin g [(alkyloxy)carbonyl]phosphonic acids with 2 equiv of sodium methoxid e afforded disodium salts of the phosphonic acid moiety. Similar chemi stry was used to obtain the mono- and disodium salts of the cholestero l ester of PFA. Reaction of the triesters with phosphorous pentachlori de, followed by coupling with AZT and O-demethylation with sodium iodi de, afforded -O-[[(1-octadecyloxy)carbonyl]phosphinyl]thymidine (9a), -O-[[(1-eicosanyloxy)carbonyl]phosphinyl]thymidine (9b), -O-[[(1-docos anyloxy)carbonyl]phosphinyl]thymidine (9c), and 3'-azido-3'-deoxy-5'-O -[[(3 eta-cholest-5-enyloxy)carbonyl]phosphiny]thymidine (9d). Concent rations of 9a-d found to inhibit replication of wild-type HTV-1(LAI) b y 50% (EC50 values) as measured in a plaque reduction assay were in th e 0.1-0.3 mu M range as compared with 0.013 mu M for AZT and 133 mu M for PFA. The concentration at which toxicity was observed in 50% of th e host cells (TC50 values) as measured by a visual grading scale of ce llular morphology was 10 mu M for 9a and 9d, 32 mu M for 9b, and 320 m u M for 9c. Thus, the TC50/EC50 ratio or selectivity index (SI) was 10 0 for 9a, 230 for 9b, and 1000 for 9c but only 33 for 9d, suggesting t hat the straight-chained fatty alcohol esters were more therapeuticall y selective. Similar TC50 and SI values were obtained for rapidly divi ding 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 mu M, whereas the EC50 Of PFB was > 1000 mu M and that of AZT was 0.009 mu M; thus, E89K was highly resistant to PFA but not cross-resistant to either AZT or the lipophilic PFA-AZT conjugates. In viral replication assays against the A018C-post isolate, the mean EC50 values of 9a-c were 0.30, 0.53, and 0.77 mu M as compared with 2.9 mu M for AZT and 65 mu M for PFA; thus , the virus recovered from a patient pretreated with AZT was not cross -resistant to either PFA or 9a-c. A notable feature of these results w as that, in addition to being >1000-fold more potent than PFA against the PFA-resistant mutant, the lipophilic PFA-AZT conjugates were more potent than PFA, as well as AZT, against AZT-resistant HIV-1.