Isolation and identification of metabolites of porfiromycin formed in the presence of a rat liver preparation

The isolation and identification of the major metabolites of porfiromycin f ormed in the presence of a rat liver preparation under aerobic conditions w ere performed with high-performance liquid chromatography and electrospray ionization mass spectrometry. Porfiromycin was extensively metabolized by t he 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 eig ht metabolites was identified as mitosene analogs. Of these, three primary metabolites are 2-methylamino-7-aminomitosene, 1,a-cis and 1,2-trans-1-hydr oxy-2-methylamino-7-aminomitosene, which are consistent with those previous ly observed in hypoxia using purified rat liver NADPH-cytochrome c reductas e. Interestingly, 2-methylamino-7-aminomitosene is a reactive metabolite, w hich undergoes further activation at the C-10 position by the loss of carba mic acid and then links with the 7-amino group of the primary metabolites t o yield two dimeric adducts. In addition, three phosphate adducts, 10-decar bamoyl-2-methylamino-7-aminomitosene-10-phosphate, 1,a-cis and 1,2-trans-2- methylamino-7-aminomitosene-1-phosphate, were also identified in the incuba tion system. The configurations of the diastereoisomeric metabolites were d etermined with (HNMR)-H-1 and phosphatase digestion. On the basis of the me tabolite 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. (C) 2000 Wiley-Liss, Inc.