Amino acid phosphoramidate monoesters of 3′-azido-3′-deoxythymidine: Relationship between antiviral potency and intracellular metabolism

S. L. Chang, G. W. Griesgraber, P. J. Southern, C. R. Wagner

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69 Scopus citations


A series of phosphoramidate monoesters of 3′-azido-3′-deoxythymidine (AZT) bearing aliphatic amino acid methyl esters (3a, 3c, 4a, 4c, 5-7) and methyl amides (3b, 3d, 4b, 4d) was prepared and evaluated for anti-HIV-1 activity in peripheral blood mononuclear cells (PBMCs). These compounds, which showed no cytotoxicity at concentrations of 100 μM, were effective at inhibiting HIV-1 replication at concentrations of 0.08-30 μM. Since the D-phenylalanine and D-tryptophan derivatives exhibited equivalent or enhanced antiviral activity compared to their L-counterparts, there appears to be no specific stereochemical requirement for the amino acid side chain. In addition, except for the D-phenylalanine derivatives, the methyl amides had greater antiviral activity than the corresponding methyl esters. On the basis of the observed antiviral activity of AZT phosphoramidate monoesters 3a and 4a in PBMCs and CEM cells, the mechanism of action of these two compounds was investigated. AZT-MP and substantial amounts of either phosphoramidate were detected in PBMCs and CEM cells treated with either 3a or 4a. Biological mechanistic studies demonstrated that 3a and 4a affect viral replication at a stage after virus entry and preceding viral DNA integration. Quantitation of the intracellular levels of AZT-TP in PBMCs and CEM cells treated with 3a and 4a in the presence and absence of exogenous thymidine correlated the intracellular levels of AZT-TP to the antiviral activity and suggested that AZT-TP was responsible for the activity observed. In addition, the reduced toxicity of 3a and 4a toward CEM cells relative to AZT correlated with reduced levels of total phosphorylated AZT and not AZT-TP. Stable carbamate analogues of 3a and 4a were prepared and shown to inhibit the production of AZT-MP from cell-free extracts of CEM cells, further suggesting that a phosphoramidate hydrolase may be responsible for intracellular P-N bond cleavage. Taken together, these results suggest that the biological activity and intracellular metabolism of nucleoside phosphoramidate monoesters are distinct from that of phosphoramidate diesters.

Original languageEnglish (US)
Pages (from-to)223-231
Number of pages9
JournalJournal of medicinal chemistry
Issue number2
StatePublished - Jan 18 2001


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