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

Abstract

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 therapeutic benefits, different series of 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides structurally related to diazoxide were developed. 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 purpose, two selected promising compounds [7-chloro-3-isopropylamino-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 phenobarbitalinduced 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 process of the selected PCO drugs. These results greatly help the optimization of the lead compounds.

Hydroxylated Analogues of ATP-Sensitive Potassium Channel Openers Belonging to the Group of 6- and/or 7-Substituted 3-Isopropylamino-4H-1,2,4-benzothiadiazine 1,1-Dioxides: Toward an Improvement in Sulfonylurea Receptor 1 Selectivity and Metabolism Stability

“…The use of this type of microsome is a well-established method both for producing expected in vivo phase I metabolites and for determining the metabolic weakness of drugs. The residue of biotransformation of compound 13a was analyzed using the slightly modified described liquid chromatography (LC) conditions . As shown in Figure , the parent product 13a was eluted at a retention time of approximately 13 min.…”

“…The in vitro biological test system selected to metabolize the parent compounds was the phenobarbital (PB)-induced male rat liver microsome system . The parent compounds were dissolved in methanol and added directly to the incubation medium, yielding a final substrate concentration of 200 μM and a final percentage in methanol of <1%.…”

“…Thus, potassium channel openers (PCOs) can interfere with several physiological processes, such as the release of insulin from pancreatic β-cells and contractile activity in smooth muscle cells. , For several years, selective activation of pancreatic K ATP channels has been known to be of therapeutic value for the treatment of critical metabolic disorders such as diabetes, obesity, and hyperinsulinemia. , Taking into account these potential therapeutic benefits, we have developed a series of new compounds belonging to 3-isopropylamino-4 H -1,2,4-benzothiadiazine 1,1-dioxides over the past decade. Among these drugs, compounds 1–6 (Figure ) have been described as potent and selective pancreatic PCOs. − Structure–activity relationships, assessed from previous studies, indicated that the presence of a small branched alkylamino chain at position 3 (i.e., an isopropylamino chain) as well as the presence of one or two halogen atoms (preferably Cl or F) at position(s) 6 and/or 7 and/or the presence of a small electron-donating group (i.e., OMe) at position 7 were favorable to both in vitro activity and selectivity for pancreatic tissue. − However, a recent in vitro metabolic study demonstrated the rapid biotransformation of compound 1a , by hydroxylation, to compound 8a (X = H, and Y = Cl) or, by dealkylation of the alkylamino side chain, to compound 7 (Figure ) …”

Metabolomics as a Challenging Approach for Medicinal Chemistry and Personalized Medicine

“…Nuclear magnetic resonance (NMR) and mass spectrometry (MS) are the principal techniques used for molecular identification and characterization . Historically, gas chromatography (GC) coupled with MS was the first method used to explore the metabolome, but proton NMR ( 1 H NMR) rapidly emerged as another powerful method and has long been a widely employed platform for nontargeted metabolomics. ,, Indeed, NMR spectroscopy, which was and remains an important analytical tool in the pharmaceutical industry − for the structural determination of drugs, fragment-based discovery, protein–ligand explorations, high-throughput ligand affinity screening, and ADME studies, , also provides many features required for metabolomic analysis . It is considered a universal, nondestructive detection method that is both highly reproducible and robust.…”

Nuclear magnetic resonance: a key metabolomics platform in the drug discovery process

“…Indeed, NMR is routinely used in the main steps leading to drug development such as high-resolution structural determination of drugs [4,5], fragment-based lead discovery [6,7], protein-ligand studies, high-throughput ligand affinity screening [7,8] and absorption, distribution, metabolism and excretion (ADME) studies [9]. NMR is also used in the lead optimization process to study ligand-receptor interactions [10].…”