Synthesis and biological activity of aromatic amino acid phosphoramidates of 5-fluoro-2'-deoxyuridine and 1-β- arabinofuranosylcytosine: Evidence of phosphoramidase activity

Timothy W. Abraham, Thomas I. Kalman, Edward J. McIntee, Carston R. Wagner

Research output: Contribution to journalArticlepeer-review

84 Scopus citations

Abstract

The amino acid phosphoramidate diesters of FUdR (2) and Ara-C (6), 5- fluoro-2'-deoxy-5'-uridyl N-(1-carbomethoxy-2-phenylethyl)phosphoramidate (5a), 5-fiuoro-2'-deoxy-5'-uridyl N-(1-carbomethoxy-2- indolylethyl)phosphoramidate (5b), 1-β-arabinofuranosylcytosine 5'-N-(1- carbomethoxy-2-phenylethyl)phosphoramidate (8a), and 1-β- arabinofuranosylcytosine 5'-N-(1-carbomethoxy-2-indolylethyl)phosphoramidate (8b), were synthesized and tested for their antitumor activity against L1210 mouse lymphocytic leukemia cells and CCRF-CEM human T-cell lymphoblastic leukemia cells. Ara-C phosphoramidates 8a,b were found to be inactive at a concentration of 100 μM, while the FUdR conjugates 5a,b exhibited IC50 values within a range of 0.30-0.40 μM. Stability studies revealed that >99% of the phosphoramidates remained intact after incubation for >2 days in 20% calf or 20% human serum. Intracellular thymidylate synthase (TS) inhibition studies revealed that treatment of L1210 and CCRF-CEM cells with 5a or 5b resulted in significant inhibition of TS in intact and permeabilized cells, while treatment of L929 TK cells with these compounds did not result in inhibition of TS activity in intact cells. However, permeabilization of L929 TK- cells enhanced the activity of 5a,b toward intracellular TS by 900- and 1500-fold, respectively. In addition, incubation of cell-free extracts of CEM cells with radiolabeled 5b resulted in the rapid production of FUdR 5'- monophosphate and a lag in the generation of FUdR. Consequently, it is proposed that the metabolism of the phosphoramidate diesters of FUdR in proliferating tissue proceeds through two separate enzymatic steps involving P-N bond cleavage by an unknown phosphoramidase followed by P-O bond cleavage by phosphatases such as 5'-nucleotidase.

Original languageEnglish (US)
Pages (from-to)4569-4575
Number of pages7
JournalJournal of medicinal chemistry
Volume39
Issue number23
DOIs
StatePublished - 1996

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