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.

Mitigating Heterocycle Metabolism in Drug Discovery

“…52 The high intrinsic clearance in both HLM and MLM of their thiophene analogue 73 was mitigated by replacing the ring with either a thiazole (74) or isothiazole (75), and the metabolic stability was improved when the furan on analogue 76 was replaced with an oxazole (77). The more metabolic stable analogues were all more polar than the analogue 73.…”

“…For example, in their anti-diabetes program on Gprotein-coupled receptor 119 (GPR119) agonists, Kalgutkar et al observed that the pyrimidine compound 137 underwent metabolic activation and GSH conjugation to give two major metabolites 138 and 139 (Figure 38). 75 The authors proposed that the GSH adducts formed through a nucleophilic aromatic substitution reaction onto the pyrimidine. The negatively charged Meisenheimer complex that forms in the proposed mechanism would be stabilized by the 5-cyano group on the pyrimidine ring.…”

“…GPR119 agonists containing 5-substituted pyrimidines: propensity for GSH adduct formation. 75,76 Journal of Medicinal Chemistry conjugation. The 5-methyl analogue 144 showed a significant increase in GSH adduct formation on the pyrazin-2(1H)-one ring over the 5-chloro analogue, while the 5-cyano analogue 145 had negligible metabolism at the pyrazin-2(1H)-one ring.…”

“…However, in some instances, an electron deficient ring was more prone to nucleophilic attack by GSH as demonstrated by Kalgutkar et al in their work with the GPR119 agonist containing a 4-cyanopyrimidine. 75 Also, Linton et al found that the electron deficient imidazopyrimidine, while relatively stable to CYPs, was susceptible to nucleophilic oxygen attack mediated by AO. 84 Heterocycles are a broad class of compounds, covering a diverse set of ring systems with various types of heteroatoms.…”

Practical approaches to resolving reactive metabolite liabilities in early discovery

“…Replacement of the bridging nitrogen atom of an atypical antipsychotic clozapine with an oxygen atom in loxapine or with sulfur in quetiapine ( Figure 2) prevents bioactivation of the dibenzoxazepine moiety and results in a drug with improved safety profile (Uetrecht et al, 1997). Furthermore, examples where resolution of RM liability (while maintaining primary pharmacology and disposition attributes) occurred through rational medicinal chemistry design has been considered to be a success story have also been presented (Bavetsias et al, 2010;Cox et al, 2010;Crawford et al, 2012;Finlay et al, 2012;Hartz et al, 2009;Kalgutkar et al, 2010Kalgutkar et al, , 2011Kalgutkar et al, , 2013Sarabu et al, 2012;Subramanian et al, 2010;Zhang et al, 2008). For instance, fluorofelbamate was specifically designed to eliminate the RM liability of felbamate on the basis of its bioactivation mechanism ( Figure 3) (Roecklein et al, 2007).…”