Biosynthesis and Identification of an N-oxide/N-glucuronide Metabolite and First Synthesis of an N-O-glucuronide Metabolite of Lu AA21004

This article describes the biosynthesis and identification of a new class of metabolites, a piperazine N -oxide/ N -glucuronide metabolite 4-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-1-β-d-glucuronic acid-piperazine 1-oxide ( 4 ). The metabolite was found in urine and plasma from humans and animals dosed with 1-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazine hydrobromide (Lu AA21004, 1 ), as a novel multimodal antidepressant under development for treatment of depression. Human liver microsomes in combination with uridine 5′-diphosphoglucuronic acid were used as an in vitro system to generate enough material of 4 to perform one- and two-dimensional 1H and 13C NMR experiments for structure elucidation. Based on rotating frame Overhauser enhancement spectroscopy NMR experiments, the distance correlation between a piperazine proton and the anomeric proton of the glucuronic acid moiety is of a magnitude similar to that of the H-3′ and H-5′ protons and can only be explained by proximity in space and the postulated structure ( 4 ). The structural analog, the N-O -glucuronic acid conjugate 6-{4-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazin-1-yloxy}-1-β-d-glucuronic acid ( 3 ) was also observed in biological samples from humans and animals and the first organic synthesis and structural identification of this metabolite is also reported. Treatment of the glucuronide metabolites 3 and 4 with β-glucuronidase gave mainly the expected hydrolysis product, the hydroxyl amine 4-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazin-1-ol ( 2 ).