Abstract

Drug reactivity and bioactivation are of major concern to the development of potential drug candidates in the pharmaceutical industry (Chem Res Toxicol 17:3–16, 2004; Chem Res Toxicol 19:889–893, 2006). Identifying potentially problematic compounds as soon as possible in the discovery process is of great importance, so often early in vitro screening is used to speed up attrition. Identification of reactive moieties is relatively straightforward with appropriate in vitro trapping experiments; however, on occasion unexpected reactive intermediates can be found later during more detailed in vivo studies. Here, we present one such example involving a series of compounds from an early drug discovery campaign. These compounds were found to react with endogenous formaldehyde from a rat in vivo study, resulting in the formation of novel +13-Da bridged homopiperazine products (equivalent to the addition of one carbon and one hydrogen atom), which were detected in urine and blood. The identification of these +13-Da products and their origin and mechanism of formation are described in detail through analyses of a representative homopiperazine compound [N-(3-(3-fluorophenyl)-1,2,4-thiadiazol-5-yl)-4-(4-isopropyl-1,4-diaze-pane-2-carbonyl)piperazine-1-carboxamide (AZX)] by liquid chromatography-UV-mass spectrometry, ¹H NMR, and chemical tests.