We evaluated the pharmacological profiles of (2R)-N-[1-(6- aminopyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamide(compound A), which is a novel muscarinic receptor antagonist with M(2)-sparing antagonistic activity. Compound A inhibited [(3)H]NMS binding to cloned human muscarinic m1, m2, m3, m4, and m5 receptors expressed in Chinese hamster ovary cells with K(i) values (nM) of 1.5, 540, 2.8, 15, and 7.7, respectively. In isolated rat tissues, compound A inhibited carbachol-induced responses with 540-fold selectivity for trachea (K(B) = 1.2 nM) over atria (K(B) = 650 nM). In in vivo rat assays, compound A inhibited acetylcholine-induced bronchoconstriction and bradycardia with intravenous ED(50) values of 0.022 mg/kg and >/=10 mg/kg, respectively. Furthermore, in dogs, compound A (0.1-1 mg/kg p.o.) dose dependently shifted the methacholine concentration-respiratory resistance curves. In mice, compound A (10 mg/kg i.v.) did not inhibit oxotremorine-induced tremor. The brain/plasma ratio (K(p)) of compound A (3 mg/kg i.v.) was 0.13 in rats; this K(p) was less than that of scopolamine (1.7) and darifenacin (0.24). The inhibition of compound A (3 mg/kg i.v.) on ex vivo binding in rat cerebral cortex was almost similar to that of NMS. These findings demonstrate that compound A has high selectivity for M(3) receptors over M(2) receptors, displays a potent, oral M(3) antagonistic activity without inhibition of central muscarinic receptors because of low brain penetration. It is well known that central muscarinic antagonists may have diverse CNS effects, and M(2) receptors regulate cardiac pacing and act as autoreceptors in the lung and bladder. Thus, compound A may have fewer cardiac or CNS side effects than nonselective compounds.