Balancing Rigidity and Flexibility: Optimised 4-(Hexyloxy)benzoate Antagonists with Enhanced Affinity and Tuneable Duration at Muscarinic Receptors

Muscarinic acetylcholine receptors (mAChRs) are key regulators of diverse physiological processes and longstanding therapeutic targets. Building on the long-acting antagonist KH-5, we synthesised and evaluated a series of 4-(hexyloxy)benzoate derivatives and their quaternary N-methylated analogues to explore how structural modifications influence receptor affinity and the duration of functional antagonism. Our structure-activity analysis revealed that introducing a rigid azabicyclo[2.2.2]octan-1-ium group boosted binding affinity (up to 250-fold compared to parental compounds) yet reduced the half-life of functional antagonism. In contrast, analogues with moderate flexibility maintained high potency while preserving longer receptor residence time. Computational docking and molecular dynamics (MD) simulations demonstrated that stable hydrogen bonding with residue N6.52 and salt-bridge formation with D3.32 were critical for sustained ligand binding to the receptor, with MD-derived metrics outperforming docking energies in predicting biological activity. Crucially, a positively charged nitrogen and a 4-hexyloxy substituent are essential features for high-affinity binding and prolonged antagonism. Shortening the alkyl chain resulted in a marked loss of affinity and abolished sustained activity. These findings underscore the need to balance molecular rigidity with conformational flexibility and charge distribution in the design of long-residence mAChR antagonists, offering a framework for further development of mAChR-targeted long-acting antagonists.