Phenylalanyl-aminocyclophosphamides as model prodrugs for proteolytic activation: Synthesis, stability, and stereochemical requirements for enzymatic cleavage

4-Aminocyclophosphamide (4-NH2-CPA, 7) was proposed as a prodrug moiety of phosphoramide mustard. Four diastereomers of phenylalanine-conjugates of 4-NH2-CPA were synthesized and their stereochemistry was assigned based on chromatographic and spectroscopic data. All diastereomers were stable in phosphate buffer but only the cis-(4R)-isomer of 15 was efficiently cleaved by α-chymotrypsin with a half-life of 20 min, which is much shorter than the 8.9 h to >12 h half-lives found for the other diastereomers. LC–MS analysis of the proteolytic products of cis-(4R)-15 indicated that 4-NH2-CPA was released upon proteolysis and further disintegrated to phosphoramide mustard. These results suggest the feasibility of using peptide-conjugated cis-(4R)-4-NH2-CPA as potential prodrugs for proteolytic activation in tumor tissues.

Graphical abstractA series of novel phenylalanine-conjugated 4-aminocyclophosphamide isomers were synthesized and evaluated as potential prodrugs for proteolytic activation.Figure optionsDownload full-size imageDownload as PowerPoint slide