THE USE OF 5'-PHOSPHATE DERIVATIVES OF NUCLEOSIDE ANALOGS AS INHIBITORS OF HIV-1 REPLICATION

A series of phosphate esters of nucleoside analogues was synthesized a nd tested for anti-HIV avtivity in cell culture. A number of these com pounds were potent inhibitors of HIV-replication with ED(50)less than or equal to 10 mM. The most potent compounds were phosphate esters of the most potent nucleosides, 3'-fluoro-3'-deoxythymidine (FLT) and 3'- azido-3'-deoxythymidine (AZT). In cell culture, the inhibition by one of these derivatives was shown to be reversed by thymidine. Also, the AZT analogue was less active against AZT-resistant virus. None of the compounds tested was directly inhibitory of HIV reverse transcriptase. The compounds were found to be labile; the rate of hydrolysis and the identity of the products varied depending on the substituents on the phosphorus atom. Activation of the most active analogue, FLT-5'-(metho xyglycinyl) S-(N-methylcarbamoylmethyl))thiophosphate (JCA-304), invol ved a pH-dependent hydrolysis, which increased with increasing pH. The hydrolysis was not dependent on HIV proteinase, the presence of MT4 c ells or enzymatic activity originating from the culture medium, The pr oduct of hydrolysis of JCA-304 was the free nucleoside (FLT); FLT-5'-m onophosphate was not detected. The corresponding acyclovir analogue wa s not active against thymidine-kinase-deficient herpes simplex virus i n Vero cells. Hydrolysis of FLT-5'-(S-(N-methylcarbamoylmethyl))thioph osphate (JCA-319) was observed In cell culture medium but not in buffe r at the same pH. The product was identified as FLT-5'monophosphate. N either of these compounds was seen as an intermediate in the hydrolysi s of JCA-304. The results suggest that the compunds are prodrugs activ ated by a non-enzymatic hydrolysis and a cytosolic phosphorylation yie lding potent inhibitors of HIV reverse transcriptase. The cellular sta bility of substituted 5'-phosphate derivatives cannot be predicted fro m their behaviour in buffer or from the hydrolysis of closely related analogues.