Antagonistic properties of 4-(hexyloxy)benzoate derivatives and their N -methyl ammonium salts at muscarinic acetylcholine receptors

Muscarinic acetylcholine receptors (mAChRs) regulate numerous physiological functions and represent established therapeutic targets for various central and peripheral disorders. This study investigates structural analogues of the long-acting muscarinic antagonist KH-5, focusing on the impact of modifications to the nitrogen-containing heterocycle on receptor binding, affinity, and duration of functional antagonism. A series of 4-(hexyloxy)benzoate derivatives and their N-methylated quaternary ammonium salts were synthesised, characterised, and tested for binding affinity at all five muscarinic receptor subtypes (M ₁ –M ₅ ). Molecular docking and dynamics simulations revealed that stable hydrogen bonding to N ^6.52 and salt bridge formation with D3.32 were critical for ligand-receptor interactions. Experimental data indicated that compounds containing an azabicyclo[2.2.2]octan-1-ium group (e.g., 10a, 11a ) exhibited the highest affinity and potency (up to 250-fold compared to lead compounds). However, these compounds had shorter functional antagonism half-lives compared to lead compounds, likely due to reduced molecular flexibility. Conversely, analogues retaining moderate flexibility (e.g., 7g ) showed a balance between potency and sustained action. Structure- activity analysis demonstrated that charged nitrogen and the presence of a 4-hexyloxy substituent were critical for binding and prolonged activity. Shortening the alkoxy chain markedly decreased affinity and abolished long-term antagonism. Docking energies alone were insufficient to distinguish binders from non-binders; however, molecular dynamics-based metrics such as stable hydrogen bonding to N ^6.52 better predicted affinity. These findings emphasise the need for optimal spatial arrangement and charge distribution for high-affinity, long-residence muscarinic antagonists and provide a framework for future design of therapeutic agents targeting mAChRs.