ABSTRACT
Some novel phosphoramidate derivatives of the nucleoside analogue stavudine have been prepared as membrane-soluble prodrugs of the bioactive free phosphate forms. Phenyl phosphates linked via nitrogen to methyl esterified amino acid analogues were studied, where the amino acid was an unnatural alpha-alkyl (or aryl) glycine or an alpha,alpha-dialkyl glycine. All compounds were characterized by a range of spectroscopic, spectrometric and analytical methods and were subjected to in vitro evaluation of their anti-human immunodeficiency virus efficacy. It is notable that certain unnatural amino acid derivatives could substitute for alanine with only a relatively small loss of activity and, moreover, that this activity did not fall-off with increasing alkyl chain length for the C2-C4 mono-alkyl series. These data are further probed by the application of our recently reported 31P-NMR-based carboxyl esterase assay, with informative results.
Subject(s)
Amides/chemistry , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Phosphoric Acids/chemistry , Stavudine/chemistry , Alanine , Amino Acid Substitution , Amino Acids/chemistry , Cell Line/drug effects , Cell Line/virology , HIV-1/drug effects , HIV-2/drug effects , Humans , Reverse Transcriptase Inhibitors/chemistry , Reverse Transcriptase Inhibitors/pharmacology , Stavudine/pharmacology , Structure-Activity RelationshipABSTRACT
The metabolism of different phosphoramidate prodrugs of d4T-MP, in which the phosphate group is linked to a phenyl group and the alkyl ester of an amino acid was studied in crude CEM cell extracts. Significant (80-100%) conversion to the amino acyl d4T-MP metabolite was obtained with derivatives containing L-alanine or methyl-L-aspartic acid. A lower degree of conversion was seen with derivatives containing L-phenylalanine, L-methionine, methyl-L-glutamic acid or L-leucine. Derivatives containing D-alanine, beta-alanine, glycine, L-valine or L-lactate showed no conversion to the amino acyl d4T-MP metabolite. Overall, there was a close correlation between the anti-HIV activity of these prodrugs and their conversion rate to the amino acyl d4T-MP metabolite. Our data suggest that the enzymes involved in the formation of the amino acyl d4T-MP metabolite have a rather stringent specificity for L-alanine as the amino acid moiety. In addition, these enzymes were found to be markedly species-dependent, their activities being highest in mouse serum, followed by guinea pig serum, but only minimal in human serum. Mouse serum therefore appears to be the medium of choice to isolate and identify the enzymes that are involved in the metabolism of these phosphoramidate prodrugs.
Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/drug effects , HIV-2/drug effects , Organophosphorus Compounds/pharmacology , Prodrugs/pharmacology , Stavudine/analogs & derivatives , Amino Acids , Animals , Anti-HIV Agents/chemistry , Anti-HIV Agents/metabolism , Cell Line , Cell Survival/drug effects , Dideoxynucleotides , Humans , Mice , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/metabolism , Prodrugs/chemistry , Prodrugs/metabolism , Stavudine/chemistry , Stavudine/metabolism , Stavudine/pharmacology , Structure-Activity Relationship , Thymidine Kinase/deficiencyABSTRACT
Phosphoramidate derivatives of the nucleoside analogue, 2',3'-dideoxy-2',3'-didehydro thymidine (d4T) have been prepared as potential membrane-soluble pro-drugs of the big-active free phosphate forms. In particular phenyl phosphates, linked via nitrogen to methyl-esterified amino acids, were studied. All compounds were fully characterised by a range of methods (high-field multinuclear NMR, mass spectrometry and high performance liquid chromatography (HPLC)) and were subjected to in vitro evaluation of their anti-HIV efficacy. The nature of the amino acid appeared to be extremely important for the eventual antiviral action. Of the amino acids studied, L-alanine was the most efficacious, whilst L-proline and glycine were particularly poor. However, an unnatural amino acid moiety, dimethylglycine, could substitute for alanine with little or no loss of activity.
