Activation mechanisms of nucleoside phosphoramidate prodrugs

A series of 1-benzotriazolyl 5-fluorodeoxyuridyl phosphoramidate prodrugs containing a haloethylamine, piperidine or morpholine substituent were prepared previously in this laboratory and evaluated as inhibitors of the enzyme thymidylate synthase (TS). The growth inhibition observed by these prodrugs in L1210 mouse leukemia cells was consistent with a mechanism of action involving intracellular activation to the phosphoramidate anion followed by hydrolysis of the P-N bond to liberate FdUMP. The mechanism of activation of nucleoside phosphoramidate prodrugs was investigated during the course of this work. Tetrahydrofurfuryl phosphoramidates containing a haloethylamine, piperidine or morpholine substituent were synthesized and used as model compounds for nucleoside phosphoramidate prodrugs. Structure assignments for the products formed in the reactions of benzotriazolyl phosphoramidates were elucidated by comparison to authentic phosphate and phosphoramidates. Structure assignments for model thymidyl nucleoside phosphoramidate reaction products were made by analogy to the tetrahydrofurfuryl model system.;Information obtained in the elucidation of the complex product mixtures was sufficient to confirm the mechanism of activation of nucleoside phosphoramidate prodrugs. Cyclization of the haloethylamine, following hydrolysis of the benzotriazolyl group attached to phosphorus, is succeeded by nucleophilic attack at carbon and phosphorus of the resulting aziridinium ion intermediate to give the observed products. Nucleophilic attack by water at carbon and phosphorus occurs without selectivity, supporting a mechanism of action of haloethylamine nucleotide prodrugs that involves the intracellular release of FdUMP. Activation of the benzotriazolyl piperidyl phosphoramidates is followed by nucleophilic cleavage of the P-N bond; this reaction is subject to specific acid-catalysis and nucleophilic catalysis by HOBT. Thus, it was determined that the mechanism of action of the piperidyl nucleoside phosphoramidates is the intracellular release of the active nucleotide following P-N bond cleavage, presumably by the action of an endogenous phosphoramidase.