NMR Spectroscopic Studies of Intermediary Metabolites of Cyclophosphamide. 2. Direct Observation, Characterization, and Reactivity Studies of Iminocyclophosphamide and Related Species

Article abstract of DOI:10.1021/jm00385a019

4-Hydroxy-5,5-dimethylcyclophosphamide (6) was synthesized as a stable (to fragmentation) analogue of 4-hydroxycyclophosphamide (1).In anhydrous Me2SO-d₆ (<*>0.03 mol percent water), cis- and trans-6 were observed by multinuclear NMR spectroscopy to equlibrate with α,α-dimethylaldophosphamide (7) and 5,5-dimethyliminocyclophosphamide (8).Identification of 8 was based on 1H, 13C, and 31P chemical shifts, selective INEPT and two-dimensional NMR correlation experiments, and temperature-dependent equilibria data.The interconversion of cis-/trans-6 and -7 was also observed in lutidine buffer; 8 was not detected under the aqueous conditions.In Me2SO-d₆, hydroxy metabolite 1 underwent dehydration to give iminocyclophosphamide (5), as evidenced by chemical shift data and a selective INEPT experiment.Concentrations of cis-/trans-1, aldophosphamide (2), and 5 were found to be temperature-dependent with higher temperatures favoring 2 and 5 in a reversible manner, thus indicating that 1/2/5 were interconverting.The addition of small amount of water to Me2SO-d₆ solutions of imine 5 resulted in the immediate disappearance of its NMR signals.The role of imine 5 in the conversion of 1 to C-4 substituted analogues of 1 was elucidated for the formation of 4-cyanocyclophosphamide (3a) from 1 and sodium cyanate in lutidine buffer.