Reactive Metabolite Trapping Studies on Imidazo- and 2-Methylimidazo[2,1-b]thiazole-based Inverse Agonists of the Ghrelin Receptor

The current study examined the bioactivation potential of ghrelin receptor inverse agonists, 1-{2-[2-chloro-4-(2 H -1,2,3-triazol-2-yl)benzyl]-2,7-diazaspiro[3.5]nonan-7-yl}-2-(imidazo[2,1- b ]thiazol-6-yl)ethanone (1) and 1-{2-[2-chloro-4-(2 H -1,2,3-triazol-2-yl)benzyl]-2,7-diazaspiro[3.5]nonan-7-yl}-2-(2-methylimidazo[2,1- b ]thiazol-6-yl)ethanone (2), containing a fused imidazo[2,1- b ]thiazole motif in the core structure. Both compounds underwent oxidative metabolism in NADPH- and glutathione-supplemented human liver microsomes to yield glutathione conjugates, which was consistent with their bioactivation to reactive species. Mass spectral fragmentation and NMR analysis indicated that the site of attachment of the glutathionyl moiety in the thiol conjugates was on the thiazole ring within the bicycle. Two glutathione conjugates were discerned with the imidazo[2,1- b ]thiazole derivative 1. One adduct was derived from the Michael addition of glutathione to a putative S -oxide metabolite of 1, whereas, the second adduct was formed via the reaction of a second glutathione molecule with the initial glutathione- S -oxide adduct. In the case of the 2-methylimidazo[2,1- b ]thiazole analog 2, glutathione conjugation occurred via an oxidative desulfation mechanism, possibly involving thiazole ring epoxidation as the rate-limiting step. Additional insights into the mechanism were obtained via 18 O exchange and trapping studies with potassium cyanide. The mechanistic insights into the bioactivation pathways of 1 and 2 allowed the deployment of a rational chemical intervention strategy that involved replacement of the thiazole ring with a 1,2,4-thiadiazole group to yield 2-[2-chloro-4-(2 H -1,2,3-triazol-2-yl)benzyl]-2,7-diazaspiro[3.5]nonan-7-yl)-2-(2-methylimidazo[2,1- b ][1,3,4]thiadiazol-6-yl)ethanone (3). These structural changes not only abrogated the bioactivation liability but also retained the attractive pharmacological attributes of the prototype agents.