Reaction of homopiperazine with endogenous formaldehyde. A carbon hydrogen addition metabolite/product identified in rat urine and blood.

Drug reactivity and bioactivation are of major concern to the development of potential drug candidates in the pharmaceutical industry (Evans et al., 2004, Baillie 2006). Identifying potentially problematic compounds as soon as possible in the discovery process is of great importance, so often early in vitro screening is employed 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 1 carbon and 1 hydrogen atom), which were detected in urine and blood. The identification of these +13 Da products, their origin and mechanism of formation are described in detail through analyses of a representative homopiperazine compound (AZX) by Liquid Chromatography (LC)-UV-Mass Spectrometry (MS), 1H Nuclear Magnetic Resonance and chemical tests.