Isolation, purification, and characterization of two new chemical decomposition products of methylazoxyprocarbazine

Abstract

We have previously reported that the antineoplastic agent, procarbazine, in aqueous solutions was chemically oxidized to its azoxy metabolites (methylazoxy and benzylazoxy). To determine if there was additional metabolism of the most active metabolite, methylazoxyprocarbazine, it was incubated in the presence and absence of CCRF-CEM human leukemia cells. Incubations were extracted, and potential metabolites were detected by HPLC with UV detection and by combined HPLC and thermospray mass spectrometric analysis. The major metabolite identified by HPLC with UV detection of the extracts was N-isopropyl-p-formylbenzamide; this was identified by comparison of its retention time with that of a synthesized standard. This identification was further corroborated by HPLC/thermospray mass spectrometry (LC/MS). Analysis of the extracts by LC/MS also showed the presence of a closely eluting peak that had a protonated molecular ion at m/z 207. This new metabolite was identified as N-isopropyl-(benzene-1,4-bis-carboxamide) by 1H NMR and gas chromatography/ion trap mass spectrometry. This metabolite is postulated to arise from breakage of the N-N bond in the hydrazine portion of the molecule. Reconstructed ion (m/z 236) current profiles from the analysis of the cell extracts indicated that there was only a trace amount of methylazoxyprocarbazine left after a 72-hr incubation. Interestingly, a peak with the same molecular weight as the parent compound (methylazoxyprocarbazine) was observed in the cellular incubations and also in extracts of control incubations in which methylazoxyprocarbazine was incubated in medium without cells. This unknown was silylated and identified as a hydroxyazo compound by an ion trap mass spectrometer operated under both single and multiple-stage mass analysis. Formation of this decomposition product appears to involve a novel intramolecular rearrangement of methylazoxyprocarbazine in solution. This pathway may be responsible for the formation of the ultimate cytotoxic species by chemical decomposition of procarbazine.