TIME-COURSE OF BENOXACOR METABOLISM AND IDENTIFICATION OF BENOXACOR METABOLITES ISOLATED FROM SUSPENSION-CULTURED ZEA-MAYS CELLS 1 H AFTER TREATMENT

Extracts of suspension-cultured Zea mays (cv. Black Mexican Sweet) cel ls treated with [C-14]benoxacor for 0.25-24 h were analyzed by HPLC an d TLC to investigate the metabolic fate of benoxacor. Thin layer chrom atography determined that benoxacor was rapidly metabolized to six det ectable metabolites within 0.5 h. Twelve metabolites were detected in extracts from cells treated for 24 h. Analysis of cell extracts by rev ersed phase HPLC determined that the glutathione conjugate [mono-(GSH) ] of benoxacor was present in all samples analyzed, based on cochromat ography with a mono(GSH) conjugate standard. The abundance of the mono (GSH) conjugate increased as treatment time increased. The presence of a di(GSH) conjugate was detected in extracts of cells treated for 0.5 h and reached a maximum level 2 h after treatment. Three predominant metabolites present in samples treated with benoxacor for 1 h were sub jected to structural analysis by H-1-NMR or mass spectrometry followin g purification by conventional HPLC methodologies. These structural an alyses determined that two of the metabolites were the catabolic formy lcarboxamide and carboxycarboxamide derivatives of benoxacor. A third metabolite was determined to be the mono(GSH) conjugate of benoxacor. This metabolite consisted of a single glutathione molecule linked via the cysteinyl sulfhydryl group to the N-dichloroacetyl alpha-carbon of benoxacor. Structures of the metabolites and postulated pathways of t heir biosynthesis in vivo are presented.