Coupling of Liquid Chromatography/Tandem Mass Spectrometry and Liquid Chromatography/Solid-Phase Extraction/NMR Techniques for the Structural Identification of Metabolites following In Vitro Biotransformation of SUR1-Selective ATP- Sensitive Potassium Channel Openers Florian Gillotin, Patrice Chiap, Michel Fre ´ de ´ rich, Jean-Claude Van Heugen, Pierre Francotte, Philippe Lebrun, Bernard Pirotte, and Pascal de Tullio Advanced Technology Corporation (A.T.C. s.a.), Academic Hospital of Lie ` ge (CHU), Lie ` ge, Belgium (F.G., P.C., J.-C.V.H.); Drug Research Center (CIRM), Laboratoire de Chimie Pharmaceutique et Laboratoire de Pharmacognosie, Universite ´ de Lie ` ge, Lie ` ge, Belgium (M.F., P.F., B.P., P.d.T.); and Laboratoire de Pharmacodynamie et de The ´ rapeutique, Universite ´ Libre de Bruxelles, Brussels, Belgium (P.L.) Received June 10, 2009; accepted October 27, 2009 ABSTRACT: SUR1-selective ATP-sensitive potassium channel openers (PCOs) have been shown to be of clinical value for the treatment of several metabolic disorders, including type I and type II diabetes, obesity, and hyperinsulinemia. Taking into account these promising thera- peutic benefits, different series of 3-alkylamino-4H-1,2,4-benzo- thiadiazine 1,1-dioxides structurally related to diazoxide were de- veloped. In view of the lead optimization process of the series, knowledge of absorption, distribution, metabolism, excretion, and toxicity parameters, and more particularly the metabolic fate of these compounds, is a fundamental requirement. For such a pur- pose, two selected promising compounds [7-chloro-3-isopro- pylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ 73) and 7-chloro-3-(3-pentylamino)-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ 157)] were incubated in the presence of phenobarbital- induced rat liver microsomes to produce expected mammal in vivo phase I metabolites. The resulting major metabolites were then analyzed by both mass spectrometry (MS) and NMR to completely elucidate their chemical structures. The two compounds were also further incubated in the presence of nontreated rats and human microsomes to compare the metabolic profiles. In the present study, the combined use of an exact mass liquid chromatography (LC)/tandem MS platform and an LC/solid-phase extraction/NMR system allowed the clarification of some unresolved structural assessments in the accurate chemical structure elucidation pro- cess of the selected PCO drugs. These results greatly help the optimization of the lead compounds. ATP-sensitive potassium channels (K ATP channels) found in a wide range of cell types are regulated by changes in intracellular ATP concentrations (Miki et al., 1999; Ballanyi, 2004; Ardehali and O’Rourke, 2005; Billman, 2008; Ko et al., 2008). These channels are formed by the combination of two different subunits, Kir6.x and SURx, the latter containing the regulatory sites for most channel modulators (Babenko et al., 1998). Several isoforms of Kir6.x (Kir6.1 and Kir6.2) and SURx (SUR1, SUR2A, SUR2B) have been reported (Inagaki et al., 1995; Hambrock et al., 1999). The combination of these different subunits led to specific K ATP channel subtypes. Acti- vation of K ATP channels creates an increase in the outflow of potas- sium ions through the cytoplasmic membrane and, consequently, an increase in membrane polarization. The physiological impact of this hyperpolarization depends on the tissue localization of the channel but generally results in a reduction of cell excitability. As a result, potassium channel openers (PCOs) can interfere with several impor- tant physiological processes such as insulin release from pancreatic B cells and contractile activity from smooth muscle cells (Lawson, 1996; Lebrun et al., 1997). Selective activation of pancreatic K ATP channels has been shown to be of therapeutic value for the treatment This work was supported by grants from the National Fund for Scientific Research (Belgium). P.d.T. and M.F. are Senior Research Associates for the National Fund for Scientific Research (Belgium). P.L. is Research Director for the National Fund for Scientific Research (Belgium). Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.109.028928. ABBREVIATIONS: K ATP channel, ATP-sensitive potassium channel; PCO, potassium channel opener; BPDZ 73, 7-chloro-3-isopropylamino-4H- 1,2,4-benzothiadiazine 1,1-dioxide; BPDZ 157, 7-chloro-3-(3-pentylamino)-4H-1,2,4-benzothiadiazine 1,1-dioxide; PB, phenobarbital; P450, cytochrome P450; LC, liquid chromatography; SPE, solid-phase extraction; MS/MS, tandem mass spectrometry; ACN, acetonitrile; COSY, correlation spectroscopy; BPDZ 44, 3-(1',2'-dimethylpropyl)amino-4H-pyrido[4,3-e][1,2,4]thiadiazine 1,1-dioxide; BPDZ 154, 6,7-dichloro-3- isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide; BPDZ 414, 6,7-difluoro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide; BPDZ 256, 6-chloro-3-cyclobutylamino-7-fluoro-4H-1,2,4-benzothiadiazine 1,1-dioxide. 0090-9556/10/3802-232–240$20.00 DRUG METABOLISM AND DISPOSITION Vol. 38, No. 2 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 28928/3549641 DMD 38:232–240, 2010 Printed in U.S.A. 232 at ASPET Journals on October 29, 2017 dmd.aspetjournals.org Downloaded from