Novel Mechanism for Dehalogenation and Glutathione Conjugation of Dihalogenated Anilines in Human Liver Microsomes: Evidence for <i>ipso</i> Glutathione Addition

The objective of the present study was to investigate the influence of halogen position on the formation of reactive metabolites from dihalogenated anilines. Herein we report on a proposed mechanism for dehalogenation and glutathione (GSH) conjugation of a series of <i>ortho</i>-, <i>meta-,</i> and <i>para</i>-dihalogenated anilines observed in human liver microsomes. Of particular interest were conjugates formed in which one of the halogens on the aniline was replaced by GSH. We present evidence that a (4-iminocyclohexa-2,5-dienylidene)halogenium reactive intermediate (QX) was formed after oxidation, followed by <i>ipso</i> addition of GSH at the imine moiety. The <i>ipso</i> GSH thiol attacks at the <i>ortho-</i>carbon and eventually leads to a loss of a halogen and GSH replacement. The initial step of GSH addition at the <i>ipso</i> position is also supported by density functional theory, which suggests that the <i>ipso</i> carbon of the chloro, bromo, and iodo (but not fluoro) containing 2-fluoro-4-haloanilines is the most positive carbon and that these molecules have the favorable highest occupied molecular orbital of the aniline and the lowest unoccupied orbital from GSH. The <i>para</i>-substituted halogen (chloro, bromo, or iodo but not fluoro) played a pivotal role in the formation of the QX, which required a delocalization of the positive charge on the <i>para</i>-halogen after oxidation. This mechanism was supported by structure–metabolism relationship analysis of a series of dihalogenated and monohalogenated aniline analogues.