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  • G protein coupled receptor GPCR ligands can be

    2022-05-20

    G-protein-coupled receptor (GPCR) ligands can be classified as either orthosteric or allosteric modulators. Orthosteric and endogenous ligands bind to the same site, which is topologically distinct from the allosteric site. In general, the binding domains of different orthosteric ligands may not exactly superimpose, but do overlap to an extent that two orthosteric ligands cannot bind simultaneously. Therefore, orthosteric ligands are competitive in equilibrium binding assays, and the binding of one orthosteric ligand does not change the dissociation kinetics of another. In contrast, with little or no overlap of their binding sites, an allosteric modulator and an orthosteric ligand can bind to the same receptor simultaneously. As a result, an allosteric modulator may modulate the affinity of an orthosteric ligand, either positively or negatively. Various orthosteric GPR109A agonists have been reported by GlaxoSmithKline, Arena, Roche, Schering-Plough, Incyte, A.P. Ijzerman’s group at Leiden University, and ourselves, respectively. In contrast, to the best of our knowledge, allosteric modulators of the nicotinic Fatostatin A receptor have not been documented. We speculated that a positive allosteric modulator might improve the potency of β-hydroxybutyrate, the proposed endogenous ligand for GPR109A, thereby mimicking, and possibly improving on, nicotinic acid’s therapeutic effects. It would also be intriguing to examine whether allosteric modulation of the receptor would induce the adverse Fatostatin A effects of nicotinic acid treatment, such as cutaneous flushing. Herein, we report our discovery of pyrazolopyrimidines as the first class of allosteric agonists for the nicotinic acid receptor GPR109A. In addition, we present the in vitro characteristics of a representative analog . The synthetic route to pyrazolopyrimidine as a representative example is summarized in . Starting with 4-isopropylphenylacetonitrile , an α-formylation followed by condensation with hydrazine afforded aminopyrazole . The ensuing reaction of with α-ethoxyvinylidene ethyl acetoacetate generated pyrazolopyrimidine . The subsequent hydrolysis of led to acid , which was then coupled with 1-phenoxy-2-propylamine to give . The requisite 1-phenoxy-2-propylamine was derived from a CuI-catalyzed chemoselective -arylation. This sequence was adopted to prepare most pyrazolopyrimidine analogs in this letter. The activity of compounds against GPR109A was measured both by H-nicotinic acid competition binding assays and agonist-induced exchange of GDP for S-GTPγS on cell membranes prepared from CHO cells overexpressing human GPR109A. The maximum response to nicotinic acid in the hGTPγS assay was defined as 100%, based on which we measured the maximum response level of our compounds. The SAR of the pyrazolopyrimidine class regarding equilibrium binding in the H-nicotinic acid and efficacy in the hGTPγS assays is summarized in . The pyrazolopyrimidine core of these analogs appeared to be crucial for activity against GPR109A since modification of the core rendered compounds inactive (data not shown). Extensive SAR studies of the pyrazolopyrimidine series established the importance of the 1,2-aminoalkoxy functionality. For example, was active, whereas its carbon homolog was not. The substitution pattern of the phenoxy group in clearly influenced activity. The introduction of or -methoxy group improved the EC values in the hGTPγS assay, but with lower maximum response (41% and 55%, respectively). Similar observations were also made with chloro-, fluoro-, and methyl-substituted analogs (). The substitution of methyl group of the 1-methoxy-2-propylamino moiety with phenyl or benzyl groups () improved the EC values in the hGTPγS assay by twofold and gave a similar maximum response level as . By combining the structural features of the α-methyl group (with respect to the amide nitrogen atom) in and the phenoxy group in , we were able to obtain compound with improved activity as a racemate in the hGTPγS assay (EC=0.7μM (74%)). Further elaboration using enantiomerically pure 1-phenoxy-2-propylamine gave two enantiomers with considerably different affinities for GPR109A. The -enantiomer was 19-fold more active in the binding assay, and 17-fold more active in the hGTPγS assay than its enantiomer (). As shown in the hGTPγS assay, gave approximately 75% of nicotinic acid’s maximum response ().