Chemical stability and fate of the cytostatic drug ifosfamide and its N-dechloroethylated metabolites in acidic aqueous solutions

P-31 NMR spectroscopy was used to study the products of the decomposition o f the antitumor drug ifosfamide (IF, 1d) and its N-dechloroethylated metabo lites, namely, 2,3-didechloroethylIF (1a) and 2- (1b) and 3-dechloroethylIF (1c), in buffered solutions at acidic pH. The First stage of acid hydrolysi s of these four oxazaphosphorines is a P-N bond cleavage of the six-membere d ring leading to the phosphoramidic acid monoesters(2a-d) of type R'HN(CH2 )(3)OP(O)(OH)-NHR, with R and/or R' = H or (CH2)(2)Cl. The electron-withdra wing chloroethyl group at the endocyclic and/or exocyclic nitrogens counter acts the endocyclic P-N bond hydrolysis. This effect is even more marked wh en the N-chloroethyl group is in the exocyclic position since the order of stability is 1d > 1c > 1b > 1a. In the second stage of hydrolysis, the rema ining P-N bond is cleaved together with an intramolecular attack at the pho sphorus atom by the non-P-linked nitrogen of the compounds 2a-d. This leads to the formation of a 2-hydroxyoxazaphosphorine ring with R = H (3a coming from compounds 2a,c) or (CH2)(2)Cl (3b coming from compounds 2b,d) and to the release of ammonia or chloroethylamine. The third step is the P-N ring opening of the oxazaphosphorines 3a,b leading to the phosphoric acid monoes ters, H2N(CH2)(3)OP(O)(OH)(2) (4a) and Cl(CH2)(2)HN(CH2)(3)OP(O)(OH)(2) (4b -1), respectively. For the latter compound, the chloroethyl group is partia lly (at pH 5.5) or totally (at pH 7.0) cyclized into aziridine (4b-2), whic h is then progressively hydrolyzed into an N-hydroxyethyl group (4b-3). Com pounds 3a,b are transient intermediates, which in strongly acidic medium ar e not observed with P-31 NMR. In this case, cleavage of the P-N bond of the type 2 phosphoramidic acid monoesters leads directly to the type 4 phospho ric acid monoesters. The phosphate anion, derived from P-O bond cleavage of these latter compounds,is only observed at low levels after a long period of hydrolysis. Compounds la-e and some of their hydrolytic degradation prod ucts (4b-1, 4b-2, diphosphoric diester [Cl(CH2)(2)NH(CH2)(3)OP(O)(OH)](2)O (5), and chloroethylamine) did not exhibit, as expected, any antitumor effi cacy in vivo against P388 leukemia. P-31 NMR determination of the N-dechlor oethylated metabolites of IF or its structural isomer, cyclophosphamide (CP ), and their degradation compounds could provide an indirect and accurate e stimation of chloroacetaldehyde amounts formed from CP or IF.