Chemical and biological evaluation of hydrolysis products of cyclophosphamide.
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 degrees C and at pH 7.4 at 37 degrees C. At pH 1.2, 1 undergoes a rapid breakdown (t1/2 = 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 degrees C. Under physiological conditions (pH 7.4, 37 degrees C) after 6 days, 45% of 1 is hydrolyzed (t1/2 = 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.
IMRCP Laboratory, Université Paul Sabatier, Toulouse, France.
V
V
Gilard
R
R
Martino
M C
MC
Malet-Martino
B
B
Kutscher
A
A
Müller
U
U
Niemeyer
J
J
Pohl
E E
EE
Polymeropoulos
D000818:Animals; D003520:Cyclophosphamide / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007928:Lethal Dose 50; D007939:Leukemia L1210 / Q000473:pathology; D007941:Leukemia P388 / Q000473:pathology; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D051379:Mice; D012996:Solutions; D014407:Tumor Cells, Cultured; D014867:Water
D012996:Solutions; D014867:Water; D003520:Cyclophosphamide