The major objective of this research is to determine the chemical mechanism of cyclophosphamide activation and its role in the drug's oncotoxic specificity. Our primary focus will be on the mechanisms of conversion of 4-hydroxycyclophosphamide to the active cyctoxic agent phosphoramide mustard via aldophosphamide and to deactivated products generated by exchange reactions at the 4-position. The specific aims of these studies include an understanding of 1) the mechanism of substitution at C-4 of 4-hydroxycyclophosphamide and its importance with respect to cyclophosphamide deactivation; 2) the nature and significance of bifunctional catalysis, especially by phosphate, in the conversion of 4-hydroxycyclophosphamide to aldophosphamide; 3) the lifetime of the aldophosphamide intermediate and its possible role as a transport form of the drug, and 4) the synthesis of potentially longer-lived 4-hydroxycyclophosphamide analogs. Complete understanding of these chemical mechanisms will provide evidence to support or refute our hypothesis that intracellular phosphate is a major determinant in cyclophosphamide's specificity and should also provide a chemical rationale for the design and synthesis of cytotoxic agents with greater oncotoxic specificty.