V. Gilard et al., CHEMICAL AND BIOLOGICAL EVALUATION OF HYDROLYSIS PRODUCTS OF CYCLOPHOSPHAMIDE, Journal of medicinal chemistry, 37(23), 1994, pp. 3986-3993
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.