Time Course of Benoxacor Metabolism and Identification of Benoxacor Metabolites Isolated from Suspension-Cultured <i>Zea mays</i> Cells 1 h after Treatment

Abstract

Extracts of suspension-cultured Zea mays (cv. Black Mexican Sweet) cells treated with [14C]benoxacor for 0.25−24 h were analyzed by HPLC and TLC to investigate the metabolic fate of benoxacor. Thin layer chromatography determined that benoxacor was rapidly metabolized to six detectable metabolites within 0.5 h. Twelve metabolites were detected in extracts from cells treated for 24 h. Analysis of cell extracts by reversed phase HPLC determined that the glutathione conjugate [mono(GSH)] of benoxacor was present in all samples analyzed, based on cochromatography 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 subjected to structural analysis by 1H-NMR or mass spectrometry following purification by conventional HPLC methodologies. These structural analyses determined that two of the metabolites were the catabolic formylcarboxamide 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 α-carbon of benoxacor. Structures of the metabolites and postulated pathways of their biosynthesis in vivo are presented. Keywords: Benoxacor; herbicide safener; CGA-154281; glutathione S-transferase; BMS cells; xenobiotic metabolism; xenobiotic detoxification; maize; dehalogenase/dechlorinase enzymes; glutathione conjugation; dichloroacetamide; substituted benzoxazine; herbicide safener metabolism