Amino acid phosphoramidate monoesters of 3′-azido-3′-deoxythymidine

Relationship between antiviral potency and intracellular metabolism

S. L. Chang, G. W. Griesgraber, Peter Southern, Carston R Wagner

Research output: Contribution to journalArticle

62 Citations (Scopus)

Abstract

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
Volume44
Issue number2
DOIs
StatePublished - Jan 18 2001

Fingerprint

Zidovudine
Metabolism
Antiviral Agents
Amino Acids
Blood Cells
Blood
Phenylalanine
Amides
HIV-1
Esters
Bearings (structural)
Virus Integration
Derivatives
Virus Internalization
Carbamates
Viral DNA
Hydrolases
Cytotoxicity
Bioactivity
Cell Extracts

Cite this

@article{043dbbd1ed5c4dc2903559b5a3fd2a31,
title = "Amino acid phosphoramidate monoesters of 3′-azido-3′-deoxythymidine: Relationship between antiviral potency and intracellular metabolism",
abstract = "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.",
author = "Chang, {S. L.} and Griesgraber, {G. W.} and Peter Southern and Wagner, {Carston R}",
year = "2001",
month = "1",
day = "18",
doi = "10.1021/jm000260r",
language = "English (US)",
volume = "44",
pages = "223--231",
journal = "Journal of Medicinal Chemistry",
issn = "0022-2623",
publisher = "American Chemical Society",
number = "2",

}

TY - JOUR

T1 - Amino acid phosphoramidate monoesters of 3′-azido-3′-deoxythymidine

T2 - Relationship between antiviral potency and intracellular metabolism

AU - Chang, S. L.

AU - Griesgraber, G. W.

AU - Southern, Peter

AU - Wagner, Carston R

PY - 2001/1/18

Y1 - 2001/1/18

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0035905850&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035905850&partnerID=8YFLogxK

U2 - 10.1021/jm000260r

DO - 10.1021/jm000260r

M3 - Article

VL - 44

SP - 223

EP - 231

JO - Journal of Medicinal Chemistry

JF - Journal of Medicinal Chemistry

SN - 0022-2623

IS - 2

ER -