Discovery, Optimization, and Biological Characterization of 2,3,6-Trisubstituted Pyridine-Containing M₄ Positive Allosteric Modulators

Herein we describe the discovery and optimization of a new series of 2,3-disubstituted and 2,3,6-trisubstituted muscarinic acetylcholine receptor 4 (M₄ ) positive allosteric modulators (PAMs). Iterative libraries enabled rapid exploration of one-dimensional structure-activity relationships (SAR) and identification of potency-enhancing heterocycle and N-alkyl pyrazole substituents. Further optimization led to identification of the potent, receptor-subtype-selective, brain-penetrant tool compound 24 (7-[3-[1-[(1-fluorocyclopentyl)methyl]pyrazol-4-yl]-6-methyl-2-pyridyl]-3-methoxycinnoline). It is efficacious in preclinical assays that are predictive of antipsychotic effects, producing dose-dependent reversal of amphetamine-induced hyperlocomotion in rats and mice, but not in M₄ knockout mice. Cholinergic-related adverse effects observed in rats treated with 24 at unbound plasma concentrations more than 3-fold higher than an efficacious dose in the hyperlocomotion assay were fewer and less severe than those observed in rats treated with the nonselective M₄ agonist xanomeline, suggesting a receptor-subtype-selective PAM has the potential for an improved safety profile.