Hybridization into a Bitopic Ligand Increased Muscarinic Receptor Activation for Isopilocarpine but Not for Pilocarpine Derivatives.

Pilocarpine ( 1 ), a secondary metabolite of several Pilocarpus species, is a therapeutically used partial agonist of muscarinic acetylcholine receptors (mAChRs). The available pharmacological data and structure-activity relationships do not provide comparable data for all five receptor subtypes. In this study, pilocarpine ( 1 ), its epimer isopilocarpine ( 2 ), racemic analogues pilosinine ( 3 ) and desmethyl pilosinine ( 4 ), and the respective hybrid ligands with a naphmethonium fragment ( 5-C6 to 8-C6 ) were synthesized and analyzed in mini-G nano-BRET assays at the five mAChRs. In line with earlier studies, pilocarpine was the most active compound among the orthosteric ligands 1 - 4 . Computational docking of pilocarpine and isopilocarpine to the active M 2 receptor suggests that the trans- configuration of isopilocarpine leads to a loss of the hydrogen bond from the lactone carbonyl to N 6.52 , explaining the lower activity of isopilocarpine. Hybrid formation of pilocarpine ( 1 ) and isopilocarpine ( 2 ) led to an inverted activity rank, with the trans -configured isopilocarpine hybrid ( 6-C6 ) being more active. The hydrogen bond of interest is formed by the isopilocarpine hybrid ( 6-C6 ) but not by the pilocarpine hybrid ( 5-C6 ). Hybridization thus leads to a modified binding mode of the orthosteric moiety, as the binding mode of the hybrid is dominated by the high-affinity allosteric moiety.