Probing the Mechanism of Action and Decomposition of Amino Acid Phosphomonoester Amidates of Antiviral Nucleoside Prodrugs

Abstract
The decomposition pathways in peripheral blood mononuclear cells (PBMCs) and the in vitro anti-HIV-1 activity of the structurally similar 3‘-azido-3‘-deoxythymidine (AZT) phosphoramidates 1−6 and 3‘-fluoro-3‘-deoxythymidine (FLT) phosphoramidates 7−10 are reported. The AZT phosphoramidates exhibited no cytotoxicity toward CEM cells at concentrations as high as 100 μM, whereas the FLT phosphoramidates 9 and 10 had CC50 values of 95.6 and 35.1 μM, respectively. All 10 compounds exhibited no cytotoxicity toward PBMCs at concentrations as high as 100 μM and were effective at inhibiting viral replication. In particular, the AZT phosphomonoester amidate 4 displayed comparable antiviral acitivity to the parent nucleoside analog AZT. Mechanistic studies on the amino acid carbomethoxy ester phosphomonoester amidates revealed that their decomposition pathway differs from that of amino acid carbomethoxy ester aryl phosphodiester amidates of nucleotide prodrugs. AZT phosphomonoester amidates are internalized by lymphocytes to the same extent as AZT by a nonsaturable process. In lymphocytes, the amino acid carbomethoxy ester phosphomonoester amidates of AZT are not significantly metabolized to either AZT or the mono-, di-, or triphosphate of AZT. The amount of active anabolite, AZT-5‘-triphosphate, formed in PBMCs incubated with the AZT phosphomonoester amidates 3 and 4 was 2- and 3-fold less than that observed after treatment with AZT, respectively. In contrast, FLT phosphomonoester amidates are rapidly converted to FLT-5‘-monophosphate by a process that is antagonized by the corresponding AZT derivative 4. These results suggest that the metabolism of aromatic amino acid carbomethoxy ester phosphomonoester amidate nucleotide prodrugs by PBMCs does not require prior conversion to the corresponding carboxylic acid before proceeding to P−N bond cleavage.

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