The synthesis of C-Mel, a cephalosporin carbamate derivative of the clinically used alkylating agent melphalan, is described. C-Mel was designed as an anticancer nitrogen mustard prodrug that releases melphalan upon tumor-specific activation by targeted beta-lactamase (bL). The Km and kcat values for bL hydrolysis of C-Mel were 218 microM and 980 s(-1), respectively. In vitro cytotoxicity assays with 3677 human melanoma cells demonstrated that C-Mel was 40-fold less toxic than melphalan and was activated in an immunologically specific manner by L49-sFv-bL, a recombinant fusion protein that binds to the melanotransferrin antigen on melanomas and on some carcinomas. L49-sFv-bL in combination with C-Mel led to regressions and cures of established subcutaneous 3677 tumors in nude mice. The effects were significantly greater than those of melphalan, which did not result in any long-term regressions in this tumor model. The therapeutic effects were comparable to those obtained in mice treated with the previously described L49-sFv-bL/7-(4-carboxybutanamido)-cephalosporin mustard (CCM) combination. However, C-Mel may be more attractive than CCM for clinical development since the released drug is clinically approved.