The use of deuterated analogs in qualitative and quantitative investigations of the metabolism of cyclophosphamide (NSC-26271).
Ring-deuterated analogs of cyclophosphamide (CP) (4-d2, 5-d, 4,6-d4, and 4,5,6-d6 derivatives) have been used to study the influence of deuterium substitution on the rates of metabolic pathways involving oxidation at C-4, and on the rate of elimination of acrolein from aldophosphamide. The magnitude of the deuterium isotope effect (kH/kD) associated with appropriate C-deuteration has been related to antitumor activity against the ADJ/PC6 murine plasma cell tumor. Isotope effects of 2.2 and 1.8 respectively, for the formation of 4-ketocyclophosphamide (4-keto-CP) and carboxyphosphamide, caused little or no change in antitumor activity (4-d2 and 4,6-d4 analogs compared with CP), but an isotope effect of about 5.3 for the beta-elimination pathway, consequent on 5,5-dideuteration, was paralleled by a marked drop in potency (7-13-fold increase in ED90) of 5,5-dideuterated analogs compared with that of CP. Analogs tetradeuterated in the bis(2-chloroethyl)amino function were used to quantitate CP and 4-keto-CP in human plasma and urine using stable-isotope dilution and direct-insertion electron impact mass spectrometry. The negative optical rotation of CP recovered from human urine after administration of the racemlc drug gave evidence for stereoselectivity in the metabolism.