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

Abstract We previously synthesized a thioetherphospholipid–AZT conjugate (3′-azido-3′-deoxy-5′-(1-hexadecylthio-2-methoxypropyl)-phosphothymidine, CP-102) with potent anti-HIV-1 activity and significant reduction in cell cytotoxicity 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- 3 H]- S -octadecylthio-2- O -methoxypropyl)-phosphothymidine-[methyl- 3 H], [ 3 H]CP-102). The intracellular radioactive metabolic products of [ 3 H]CP-102 treated human lymphoblastoid CEM-SS cells were analyzed by HPLC and thin-layer chromatography. Results of this investigation provide evidence that a putative intracellular lipid cleavage enzyme metabolizes [ 3 H]CP-102 to form a thioetherdiglyceride compound that migrates with an authentic 1- S -octadecyl-2- O -methyl-thioglycerol standard on TLC. The thioetherdiglyceride metabolite did not react with the ninhydrin reagent indicating it did not contain a primary amine such as that found on serine or ethanolamine containing phospholipids. Also, the product did not contain a phosphatidic acid group based on migration characteristics in the TLC plate. The other major hydrophilic metabolite was 3′-azido-3′-deoxythymidine-[methyl- 3 H]-monophosphate (AZT–MP) with lesser amounts of AZT, AZT–DP and AZT–TP. In summary, the best interpretation of these data is that the thioetherphospholipid–AZT conjugate, [ 3 H]CP-102, is cleaved by a putative intracellular lipid cleavage enzyme to release a thioetherdiglyceride compound and AZT–MP. The resulting AZT–MP was either dephosphorylated to AZT or sequentially phosphorylated to AZT–DP and, ultimately, to AZT–TP, the known inhibitory metabolite against HIV-1 reverse transcriptase. Phospholipid–nucleoside conjugates may provide a unique approach for developing anti-HIV-1 prodrugs that do not have a strict requirement for a nucleoside kinase for initial activation of the prodrug to an antiviral form.