PhD

dissertationProcarbazine is a 1,2-disubstituted hydrazine derivative that acts as a methylating agent in the treatment of Hodgkin's disease, leukemias, lymphomas, and brain tumors. The anticancer activity of this agent results from oxidation to azoprocarbazine and additional N-oxidation to a mixture of azoxy isomers. These isomers were synthesized and purified, and their cytotoxic effects against leukemia cell lines were evaluated. The methylazoxy isomer was shown to be the most cytotoxic metabolite. Results of these studies suggested that procarbazine may be further activated by the cells to the active metabolite, methylazoxyprocarbazine. Subsequent studies, however, revealed that the cells did not contain appreciable amounts of cytochrome P-450. Cells were incubated with procarbazine for 72 h, extracted, and analyzed by mass spectrometric techniques. Results of these studies showed that small amounts of the azoxy isomers were produced in cell incubations, but they were also present in control incubations in which procarbazine was incubated in media containing no cells. This represented new evidence of nonenzymatic activation of procarbazine. Results of in vitro cytotoxicity assays suggested that methylazoxyprocarbazine also may be activated in a time-dependent manner. Studies were carried out to determine what metabolites (if any) were present in cell incubations. There was very little methylazoxyprocarbazine left by 72 h, but there were significantly greater amounts of a more polar isomer of the same molecular weight. This metabolite was isolated by reversed-phase high pressure liquid chromatography (HPLC) and characterized by gas chromatography/mass spectrometry (GC/MS) analysis of its silyl derivative. It appeared that methylazoxyprocarbazine was capable of rearranging in solution to this more polar isomer. Thermospray LC/MS permitted the unambiguous structural assignment of an additional metabolite that eluted just before N-isopropyl-p-formylbenzamide, the major metabolite produced from methylazoxyprocarbazine. This additional metabolite had not been detected by previous HPLC studies. Reversed-phase HPLC was used to isolate and purify this metabolite. NMR characterization and GC/MS analysis of its silyl derivatives showed it to be a carboxamide. This new carboxamide was probably formed by breakage of the N-N bond of the rearranged isomer. Results of this research support the existence of previously undetermined, alternative pathways for the activation of procarbazine