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.

Human immunodeficiency virus type 1 proteinase resistance to symmetric cyclic urea inhibitor analogs

“…Cell culture studies. The effects of test compounds on HIV-infected cell culture were analyzed as described previously (19). Briefly, 2 ϫ 10 4 MT4 cells/ on September 25, 2020 by guest http://aac.asm.org/ Downloaded from well were grown in microplates, and each well was inoculated with 10 to 20 50% tissue culture infective doses of virus.…”

Synthesis of Nucleoside Phosphate and Phosphonate Prodrugs

“…In the same study, 217 the use of a cyclic phosphorodiamidate reagent for the synthesis of a FLT o -(methynesulfonamino)phenyl methoxy glycine prodrug analog was also reported (Scheme 127 ). Cyclic phosphorochloridate 433 was reacted with glycine methyl ester in the presence of triethylamine to form phosphorodiamidate reagent 434 .…”

“…Another approach involving P(V) chemistry was developed by Nillroth et al for the synthesis of FLT-prodrugs. 217 FLT was first reacted with 2 equiv of o -chlorophenyl phosphorodichloridate and excess 1,2,4-triazole in the presence of triethylamine to form the nucleoside aryloxy triazolide phosphoramidate intermediate 431 . The subsequent addition of glycine methyl ester hydrochloride and triethylamine afforded the FLT-aryloxyphosphoramidate prodrug 432 in 80% yield (Scheme 126 ).…”

“…Amino acid nucleoside phosphoramidate monoesters can also be obtained by hydrolysis of phosphorothioamidate derivatives (Scheme 156 ). 216 , 246 2-Chloro-3-methyl-1,3,2-thiazaphospholidin-4-one 2-oxide was first reacted with glycine methyl ester hydrochloride in the presence of triethylamine to provide intermediate 506 . Subsequent reaction with FLT and hydrolysis with 10% Et 3 N in dichloromethane over silica gel at 40 °C afforded the amino acid nucleoside phosphoramidate monoesters 508 .…”

HIV-1 Protease Inhibitors Based on Acyclic Carbohydrates

“…The inhibitory effect of the synthesized compounds was determined with purified HIV-1 protease in a standardized assay. The results are presented as IC 50 values, i.e., the concentration of inhibitor resulting in 50% inhibition in this assay …”