Cellular metabolism in lymphocytes of a novel thioether-phospholipid-AZT conjugate with anti-HIV-1 activity

We previously synthesized a thioetherphospholipid - AZT conjugate (3'-azido - 3'-deoxy-5'-(1-hexadecylthio-2-methoxypropyl)-phosphothymidine, CP-102) w ith potent anti-HIV-1 activity and significant reduction in cell cytotoxici ty compared to AZT alone. To study the cellular metabolism of the conjugate compound we synthesized a double-tritium-labeled thioetherphospholipid-AZT conjugate (3'-azido-3'-deoxy-5'-(1-[9, 10-H-3]-S-octadecylthio-2-O-methoxy propyl)-phosphothymidine-[methyl- [H-3]CP-102). The intracellular radioacti ve metabolic products of [H-3]CP-102 treated human lymphoblastoid CEM-SS ce lls were analyzed by HPLC and thin-layer chromatography. Results of this in vestigation provide evidence that a putative intracellular lipid cleavage e nzyme metabolizes [H-3]CP-102 to form a thioetherdiglyceride compound that migrates with an authentic 1-S-octadecyl-2-O-methyl-thioglycerol standard o n TLC. The thioetherdiglyceride metabolite did not react with the ninhydrin reagent indicating it did not contain a primary amine such as that found o n serine or ethanolamine containing phospholipids. Also, the product did no t contain a phosphatidic acid group based on migration characteristics in t he TLC plate. The other major hydrophilic metabolite was 3'-azido-3 '-deoxy thymidine-[methyl-H-3]-monophosphate (AZT- MP) with lesser amounts of AZT, AZT-DP and AZT-TP. In summary, the best interpretation of these data is tha t the thioetherphospholipid-AZT conjugate, [H-3]CP-102, is cleaved by a put ative intracellular lipid cleavage enzyme to release a thioetherdiglyceride compound and AZT-MP. The resulting AZT-MP was either dephosphorylated to A ZT or sequentially phosphorylated to AZT-DP and, ultimately, to AZT-TP, the known inhibitory metabolite against HIV-1 reverse transcriptase. Phospholi pid-nucleoside conjugates may provide a unique approach for developing anti -HIV-l prodrugs that do not have a strict requirement for a nucleoside kina se for initial activation of the prodrug to an antiviral form. (C) 2001 Els evier Science B.V. All rights reserved.