title: Intrinsic electrophilicity of a 4-substituted-5-cyano-6-(2-methylpyridin-3-yloxy)pyrimidine derivative: structural characterization of glutathione conjugates in vitro.
abstract: Isopropyl 9-anti-[5-cyano-6-(2-methyl-pyridin-3-yloxy)-pyrimidin-4-yloxy]-3-oxa-7-aza-bicyclo[3.3.1]nonane-7-carboxylate (1) represents a prototypic compound from a lead chemical series of G protein-coupled receptor 119 agonists, intended for treatment of type 2 diabetes. When compound 1 was incubated with NADPH-supplemented human liver microsomes in the presence of glutathione, two thioether conjugates M4-1 and M5-1 were observed. Omission of NADPH from the microsomal incubations prevented the formation of M5-1 but not M4-1. The formation of M4-1 was also discerned in incubations of 1 and glutathione with human liver cytosol, partially purified glutathione transferase, and in phosphate buffer at pH 7.4. M4-1 was isolated, and its structure ascertained from LC-MS/MS and NMR analysis. The mass spectral and NMR data suggested that M4-1 was obtained from a nucleophilic displacement of the 6-(2-methylpyridin-3-yloxy) group in 1 by glutathione. In addition, mass spectral studies revealed that M5-1 was derived from an analogous displacement reaction on a monohydroxylated metabolite of 1; the regiochemistry of hydroxylation was established to be on the isopropyl group. Of great interest were the findings that replacement of the 5-cyano group in 1 with a 5-methyl group resulted in 2, which was practically inert toward reaction with glutathione. This observation suggests that the electron-withdrawing potential of the C5 cyano group serves to increase the electrophilicity of the C6 carbon (via stabilization of the transition state) and favors reaction with the nucleophilic thiol. The mechanistic insights gained from these studies should assist medicinal chemistry efforts toward the design of analogs that retain primary pharmacology but are latent toward reaction with biological nucleophiles, thus mitigating the potential for toxicological outcome due to adduction with glutathione or proteins.
authors.affiliation: Pharmacokinetics, Dynamics, and Metabolism Department, Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340, United States. amit.kalgutkar@pfizer.com
authors.forename: Amit S
authors.initials: AS
authors.lastname: Kalgutkar
authors.forename: Vincent
authors.initials: V
authors.lastname: Mascitti
authors.forename: Raman
authors.initials: R
authors.lastname: Sharma
authors.forename: Gregory W
authors.initials: GW
authors.lastname: Walker
authors.forename: Tim
authors.initials: T
authors.lastname: Ryder
authors.forename: Thomas S
authors.initials: TS
authors.lastname: McDonald
authors.forename: Yue
authors.initials: Y
authors.lastname: Chen
authors.forename: Cathy
authors.initials: C
authors.lastname: Preville
authors.forename: Arindrajit
authors.initials: A
authors.lastname: Basak
authors.forename: Kim F
authors.initials: KF
authors.lastname: McClure
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Kohrt
authors.forename: Ralph P
authors.initials: RP
authors.lastname: Robinson
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Munchhof
authors.forename: Peter
authors.initials: P
authors.lastname: Cornelius
mesh_terms: D000818:Animals; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000187:drug effects* / Q000378:metabolism*; D015394:Molecular Structure; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000819:agonists*
chemical_list: C519854:GPR119 protein, human; D011743:Pyrimidines; D043562:Receptors, G-Protein-Coupled; D005978:Glutathione