Identification of the principal circulating metabolite of a synthetic 5,4 '-diaminoflavone (NSC 686288), an antitumor agent, in the rat

During the course of our study to develop analytical methodology for quanti tating the investigative antitumor agent 5-amino-2-(4-amino-3-fluorophenyl) -6,8-difluoro-7-methyl-4H-1-benzopyran-4-one (DAF; NSC 686288) in plasma, a significant concentration of a metabolite was observed in a post-dosed rat . The results of electron-ionization (EI) mass spectrometric analysis of th e metabolite suggested that N-acetylation had occurred, brit, interestingly , that only one of the compound's two primary amino groups had been transfo rmed. Comparing the mass spectra and gas chromatographic retention times of a mono-acetylated sample of DAF and that of the metabolite showed both to be the same. A retro-Diels-Alder (RDA) fragmentation of the B ring of DAF r esults in formation of two abundant product ions, each retaining one of the amino groups. The EI mass spectrum of mono-N-acetamido-d(3) DAF shows loss of ketene-d(2), leading to formation of an -NHD group. The ensuing RDA fra gmentation easily identifies which of the two product ions contains the deu terium, thereby allowing us to assign the site of N-acetylation as the amin o group on ring C (the 4' position) of DAF. (C) 2000 Elsevier Science B.V. All rights reserved.