CHEMICAL AND BIOLOGICAL EVALUATION OF HYDROLYSIS PRODUCTS OF CYCLOPHOSPHAMIDE

P-31 NMR spectroscopy was used to study the products of the decomposit ion of cyclophosphamide (1) in buffered solutions at pH's ranging betw een 1.2 and 8.6 at 20 degrees C and at pH 7.4 at 37 degrees C. At pH 1 .2, 1 undergoes a rapid breakdown (t(1/2) = 1.4 days) of the two P-N b onds, giving compounds 2 [HN(CH2CH2Cl)(2)] and 3 [H2N(CH2)(3)OP(O)(OH) (2)] as hydrochlorides. No intermediates were detected. At pH's betwee n 5.4 and 8.6, hydrolysis of 1 during 17 days leads to the sole and pr eviously unknown nine-membered ring compound 13. 13 results from the i ntramolecular alkylation of 1 giving the bicyclic compound 7 followed by the exothermal hydrolytic breakdown of the P-N bond of its six-memb ered ring. At pH 2.2 and 3.4, the two hydrolytic pathways coexist sinc e, beside compounds 2 and 3, the hydrochloride of compound 9 [Cl(CH2)( 2)NH(CH2)(2)NH(CH2)(3)OP(O)(OH)(2)] is formed, resulting from the acid -catalyzed breakdown of the P-N bond in the nine-membered ring compoun d 13. At pH 2.2, the presence of chloride ion affected neither the sta bility of 1 nor the contribution of the two competing hydrolytic pathw ays. At pH's ranging from 3.4 to 8.6, there is little degradation of 1 since more than 95% of initial 1 was still present after 7 days at 20 degrees C. Under physiological conditions (pH 7.4, 37 degrees C) afte r 6 days, 45% of 1 is hydrolyzed (t(1/2) = 6.6 days), leading essentia lly (30% of initial 1) to the nine-membered ring compound 13. The rate of hydrolysis of 13 and the nature of its hydrolysis products were fo und to depend on pH over the range 0-8.6. After a single ip injection to mice, compounds 3, 9, and 13 were less toxic than 1. They did not e xhibit any direct cytotoxic efficacy on the colony-forming capacity of L1210 cells in vitro, and they had no antitumor activity in vivo agai nst P388 leukemia.