Some studies of the active intermediates formed in the microsomal metabolism of cyclophosphamide and isophosphamide

Abstract

Evidence is presented in support of the following metabolic pathways, in the liver, of the antitumour agent cyclophosphamide 2-[bis(2-chloroethyl)amino]-tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide. The drug is first converted, presumably by the mixed-function oxidases, into 4-hydroxycyclophosphamide which may then break down by elimination of acrolein from its tautomeric form, aldophosphamide, to yield phosphoramide mustard [ N, N-bis(2-chloroethyl)phosphorodiamidic acid], a known cytotoxic agent. In competition with this process is the enzymic conversion of 4-hydroxycyclophosphamide (by dehydrogenation) and aldophosphamide (by oxidation) into the known in vivo metabolites of cyclophosphamide, 4-ketocyclophosphamide and carboxyphosphamide, respectively, each of which has low cytotoxicity. 4-Hydroxycyclophosphamide, which was too unstable to allow identification directly by conventional procedures, was trapped by reaction with ethanol. The resulting two, apparently isomeric, ethyl derivatives, (1) were amenable to mass spectrometry, (2) yielded acrolein 2,4-dinitrophenylhydrazone on treatment with acidic 2,4-dinitrophenylhydrazine, (3) were hydrolysed in water (pH 4.3), each isomer apparently regenerating 4-hydroxycyclophosphamide, (4) were highly toxic to Walker tumour cells in culture. Phosphoramide mustard was also isolated after in vitro metabolism of cyclophosphamide. On the basis of a bioassay involving Walker tumour cells in whole animals it appeared that, of the known metabolites of cyclophosphamide, only phosphoramide mustard possesses the cytoxicity and biological half-life appropriate to the active antitumour metabolite. Four other metabolites of low cytotoxicity were isolated and identified, namely, 4-ketocyclophosphamide, carboxyphosphamide, 2-(2-chloroethylamino)tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide, and 3-hydroxypropyl- N, N-bis(2-chloroethyl)phosphorodiamidate. The significance of metabolic detoxification processes in relation to the selective cytotoxicity of cyclophosphamide towards tumour cells in vivo is discussed. The metabolic activation of isophosphamide appears to follow a pathway similar to that of cyclophosphamide.