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 degrees 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.