The use of 5′-Phosphate Derivatives of Nucleoside Analogues as Inhibitors of HIV-1 Replication

Abstract

A series of phosphate esters of nucleoside analogues was synthesized and tested for anti-HIV activity in cell culture. A number of these compounds were potent inhibitors of HIV-replication with ED50 ≥ 10 nM. 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′-(methoxyglycinyl S-(N-methylcarbamoylmethyl))thiophosphate (JCA-304), involved a pH-dependent hydrolysis, which increased with increasing pH. The hydrolysis was not dependent on HIV proteinase, the presence of MT4 cells or enzymatic activity originating from the culture medium. The product of hydrolysis of JCA-304 was the free nucleoside (FLT); FLT-5′-monophosphate was not detected. The corresponding acyclovir analogue was not active against thymidine-kinase-deficient herpes simplex virus in Vero cells. Hydrolysis of FLT-5′-(S-(N-methylcarbamoylmethyl))thiophosphate (JCA-319) was observed in cell culture medium but not in buffer at the same pH. The product was identified as FLT-5′-monophosphate. Neither of these compounds was seen as an intermediate in the hydrolysis of JCA-304. The results suggest that the compounds are prodrugs activated by a non-enzymatic hydrolysis and a cytosolic phosphorylation yielding potent inhibitors of HIV reverse transcriptase. The cellular stability of substituted 5′-phosphate derivatives cannot be predicted from their behaviour in buffer or from the hydrolysis of closely related analogues.