title: Synthesis and biological activity of aromatic amino acid phosphoramidates of 5-fluoro-2'-deoxyuridine and 1-beta-arabinofuranosylcytosine: evidence of phosphoramidase activity.
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-fluoro-2'-deoxy-5'- uridyl N-(1-carbomethoxy-2-indolylethyl)phosphoramidate (5b), 1-beta-arabinofuranosylcytosine 5'-N-(1-carbomethoxy-2-phenylethyl) phosphoramidate (8a), and 1-beta-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 microM, while the FUdR conjugates 5a,b exhibited IC50 values within a range of 0.30-0.40 microM. 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.
authors.affiliation: Department of Medicinal Chemistry, University of Minnesota, Minneapolis 55455, USA.
authors.forename: T W
authors.initials: TW
authors.lastname: Abraham
authors.forename: T I
authors.initials: TI
authors.lastname: Kalman
authors.forename: E J
authors.initials: EJ
authors.lastname: McIntee
authors.forename: C R
authors.initials: CR
authors.lastname: Wagner
mesh_terms: D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003561:Cytarabine / Q000737:chemistry*; D005467:Floxuridine / Q000737:chemistry*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D009943:Organophosphorus Compounds / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured
chemical_list: D009943:Organophosphorus Compounds; D005467:Floxuridine; D003561:Cytarabine; D013940:Thymidylate Synthase; D010744:Phosphoric Monoester Hydrolases