APPLICATION OF BIOCATALYSIS TO DRUG METABOLISM: PREPARATION OF MAMMALIAN METABOLITES OF A BIARYL-BIS-SULFONAMIDE AMPA ( -AMINO-3- HYDROXY-5-METHYLISOXAZOLE-4-PROPIONIC ACID) RECEPTOR POTENTIATOR USING Actinoplanes missouriensis

LY451395 (2-propanesulfonamide, N-[(2R)-2-[4 -[2-[methylsulfonyl)amino]ethyl][1,1 -biphenyl]-4-yl]propyl]-) is a potent and highly selective potentiator of the -amino-3-hydroxy-5-methylisoxazole4-propionic acid (AMPA) receptors. It is a biaryl-bis-sulfonamide and is known to be highly metabolized in preclinical species. In those metabolism studies, the metabolite structures were proposed exclusively by the analysis of mass spectrometric data. Although mass spectrometry is clearly a technique of choice for rapid identification of drug metabolites, occasionally, nuclear magnetic resonance spectroscopy is required to unambiguously assign and characterize, particularly, the regioand stereochemistry of metabolic changes. Nuclear magnetic resonance spectroscopy, in general, is less sensitive than other detection methods and demands several micrograms of material for the analysis. To support full structure characterization of metabolites by NMR, in this study we demonstrated the application of a microbial-based surrogate biocatalytic system to produce sufficient amounts of the mammalian metabolites of LY451395. The results revealed that incubation of LY451395 with Actinoplanes missouriensis NRRL B3342 generated several metabolites that were previously detected in the in vivo metabolism studies of the preclinical species. Subsequent large-scale bioconversion resulted in the isolation of seven mammalian metabolites in milligram quantities for structural characterization by nuclear magnetic resonance spectroscopy. Furthermore, a selected group of metabolites generated from the microbial conversion served as analytical standards to monitor and quantify drug metabolites during clinical investigations. Glutamate is the major excitatory transmitter in the brain. Several recent breakthroughs in the molecular biology and pharmacology of the -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-subtype of glutamate receptors have led to the discovery of selective and potent AMPA receptor potentiators. These molecules enhance synaptic transmission and play important roles in plasticity and cognitive processes (Parsons et al., 2002). At Eli Lilly and Company, we have developed several novel series of biarylsulfonamides, which are potent potentiators of AMPA receptors in vitro (Gates et al., 2001; Miu et al., 2001). They have displayed functional central nervous system activity after systemic administration (Baumbarger et al., 2001a,b; Vandergriff et al., 2001; O’Neill et al., 2005) and are shown to be active in rodent models of cognition (Quirk and Nisenbaum, 2002; O’Neill et al., 2004) and depression (Li et al., 2001, 2003; Skolnick et al., 2001). LY451395 (2-propanesulfonamide, N-[(2R)-2-[4 -[2-[methylsulfonyl)amino]ethyl][1,1 -biphenyl]-4-yl]propyl]-), a biaryl-bis-sulfonamide, is a selective and potent potentiator of AMPA receptors. In vivo metabolism of LY451395 in mouse, rat, dog, and human has generated several phase I oxidative metabolites (D. Coutant, W. Annes, T. Gillespie, T. Rash, W. Knebel, B. Peterson, and M. Goldberg; and W. Annes, T. Gillespie, B. Peterson, and D. Coutant, manuscripts in preparation), which required unambiguous structural characterization by spectroscopic means, including NMR. In addition, metabolite reference standards were required to monitor and quantify the metabolites during clinical investigations. Even though mammalian liver microsomes and/or hepatocytes can be used to generate drug metabolites, in our case, multiple incubations from different mammalian sources are required to cover the entire range of metabolites. Furthermore, the limited capacity of these systems requires a larger incubation volume to generate several milligrams of the metabolites. The use of microbial biocatalysis to produce mammalian metabolites of drugs has long been well recognized (Azerad, 1999; Lacroix et al., 1999; Preisig et al., 2003). Microbes are attractive biocatalytic systems because of their chemo-, regio-, and stereoselectivity, impressive catalytic efficiency, and capability of accepting a wide range of Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.106.009522. ABBREVIATIONS: AMPA, -amino-3-hydroxy-5-methylisoxazole-4-propionic acid; HPLC, high-performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; MS, n-stage mass spectrometry; WET, water eliminated through transverse gradients; DQFCOSY, double-quantum filtered correlation spectroscopy; ROESY, rotating frame nuclear Overhauser effect spectroscopy; HSQC, heteronuclear single-quantum coherence; HMBC, heteronuclear multiple bond correlation; CIGAR-HMBC, constant time inverse-detected gradient accordion rescaled long-range heteronuclear multiple bond correlation. 0090-9556/06/3406-925–931$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 6 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 9522/3112952 DMD 34:925–931, 2006 Printed in U.S.A. 925 at A PE T Jornals on N ovem er 6, 2017 dm d.aspurnals.org D ow nladed from