Chemical and biological evaluation of hydrolysis products of cyclophosphamide

Article abstract of DOI:10.1021/jm00049a018

³¹P NMR spectroscopy was used to study the products of the decomposition of cyclophosphamide (1) in buffered solutions at pH's ranging between 1.2 and 8.6 at 20 °C and at pH 7.4 at 37 °C. At pH 1.2, 1 undergoes a rapid breakdown (t( 1/2 ) = 1.4 days) of the two P-N bonds, giving compounds 2 [HN(CH₂CH₂Cl)₂] and 3 [H₂N(CH₂)₃OP(O)(OH)₂] as hydrochlorides. No intermediates were detected. At pH's between 5.4 and 8.6, hydrolysis of 1 during 17 days leads to the sole and previously unknown nine-membered ring compound 13. 13 results from the intramolecular alkylation of 1 giving the bicyclic compound 7 followed by the exothermal hydrolytic breakdown of the P- N bond of its six-membered ring. At pH 2.2 and 3.4, the two hydrolytic pathways coexist since, beside compounds 2 and 3, the hydrochloride of compound 9 [Cl(CH₂)₂NH(CH₂)₂NH(CH₂)₃OP(O)(OH)₂] is formed, resulting from the acid-catalyzed breakdown of the P-N bond in the nine-membered ring compound 13. At pH 2.2, the presence of chloride ion affected neither the stability of 1 nor the contribution of the two competing hydrolytic pathways. 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 °C. Under physiological conditions (pH 7.4, 37 °C) after 6 days, 45% of 1 is hydrolyzed (t( 1/2 ) = 6.6 days), leading essentially (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 found 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 exhibit any direct cytotoxic efficacy on the colony- forming capacity of L1210 cells in vitro, and they had no antitumor activity in vivo against P388 leukemia.