Background and PurposeThe aim of this study was to develop potent and long‐acting antagonists of muscarinic ACh receptors. The 4‐hexyloxy and 4‐butyloxy derivatives of 1‐[2‐(4‐oxidobenzoyloxy)ethyl]‐1,2,3,6‐tetrahydropyridin‐1‐ium were synthesized and tested for biological activity. Antagonists with long‐residence time at receptors are therapeutic targets for the treatment of several neurological and psychiatric human diseases. Their long‐acting effects allow for reduced daily doses and adverse effects.Experimental ApproachThe binding and antagonism of functional responses to the agonist carbachol mediated by 4‐hexyloxy compounds were investigated in CHO cells expressing individual subtypes of muscarinic receptors and compared with 4‐butyloxy analogues.Key ResultsThe 4‐hexyloxy derivatives were found to bind muscarinic receptors with micromolar affinity and antagonized the functional response to carbachol with a potency ranging from 30 nM at M1to 4 μM at M3receptors. Under washing conditions to reverse antagonism, the half‐life of their antagonistic action ranged from 1.7 h at M2to 5 h at M5receptors.Conclusions and ImplicationsThe 4‐hexyloxy derivatives were found to be potent long‐acting M1‐preferring antagonists. In view of current literature, M1‐selective antagonists may have therapeutic potential for striatal cholinergic dystonia, delaying epileptic seizure after organophosphate intoxication or relieving depression. These compounds may also serve as a tool for research into cognitive deficits.