title: NMR spectroscopic studies of intermediary metabolites of cyclophosphamide. 2. Direct observation, characterization, and reactivity studies of iminocyclophosphamide and related species.
abstract: 4-Hydroxy-5,5-dimethylcyclophosphamide (6) was synthesized as a stable (to fragmentation) analogue of 4-hydroxycyclophosphamide (1). In anhydrous Me2SO-d6 (less than or equal to 0.03 mol % water), cis- and trans-6 were observed by multinuclear NMR spectroscopy to equilibrate with alpha, alpha-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 intercoverting. The addition of small amounts 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 cyanide in lutidine buffer.
mesh_terms: D003520:Cyclophosphamide / Q000031:analogs & derivatives* / Q000378:metabolism; D004121:Dimethyl Sulfoxide; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D013329:Structure-Activity Relationship
chemical_list: C035963:iminophosphamide; D003520:Cyclophosphamide; D004121:Dimethyl Sulfoxide