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The isolation and identification of the major metabolites of porfiromycin formed in the presence of a rat liver preparation under aerobic conditions were performed with high‐performance liquid chromatography and electrospray ionization mass spectrometry. Porfiromycin was extensively metabolized by the rat liver preparation in an aqueous 0.1 M potassium phosphate buffer (pH 7.4) containing an NADPH generating system at 37°C. A total of eight metabolites was identified as mitosene analogs. Of these, three primary metabolites are 2‐methylamino‐7‐aminomitosene, 1,2‐ cis and 1,2‐ trans ‐1‐hydroxy‐2‐methylamino‐7‐aminomitosene, which are consistent with those previously observed in hypoxia using purified rat liver NADPH‐cytochrome c reductase. Interestingly, 2‐methylamino‐7‐aminomitosene is a reactive metabolite, which undergoes further activation at the C‐10 position by the loss of carbamic acid and then links with the 7‐amino group of the primary metabolites to yield two dimeric adducts. In addition, three phosphate adducts, 10‐decarbamoyl‐2‐methylamino‐7‐aminomitosene‐10‐phosphate, 1,2‐ cis and 1,2‐ trans ‐2‐methylamino‐7‐aminomitosene‐1‐phosphate, were also identified in the incubation system. The configurations of the diastereoisomeric metabolites were determined with 1 HNMR and phosphatase digestion. On the basis of the metabolite profile, we propose in vitro metabolic pathways for porfiromycin. The findings provide direct evidence for understanding the reactive nature and hepatic metabolism of the drug currently in phase III clinical trials. © 2000 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89:191–198, 2000

Aspects of the degradations of mitomycin and porfiromycin were studied. The initial degradation processes of the compounds in an acidic medium were investigated. Influences of pH, buffers, and other additives such as halogenides and dioctyl sodium sulfosuccinate [sodium 1,4-bis(2-ethylhexyl)sulfosuccinate] were studied. The hydrogen ion catalyzes the degradation of both the uncharged and the protonated species. Anions also promote the degradation of the compounds in an acidic medium. Rate constants for all of the catalytic reactions could be determined. From the pH profiles, after correction for buffer influences, accurate pKa values for the aziridine nitrogens could be obtained. The protective influence of the dioctyl sulfosuccinate ion could be explained. From the data obtained a plausible mechanism for the initial acidic degradation reactions was developed.

The prototropic properties of mitomycin and porfiromycin were studied. pK0 values for two potentially basic groups and one acidic function could be established by titration. The kinetics of the tautom- erization preceding the prototropic reaction in an alkaline medium were investigated.