A rigidification strategy was applied to the preclinical candidate donecopride, an acetylcholinesterase inhibitor possessing 5-HT4R agonist activity. Inspired by promising bioactive benzisoxazole compounds, we have conducted a pharmacomodulation study to generate a novel series of multitarget directed ligands. The chemical synthesis of the ligand was optimized and compounds were evaluated in vitro against each target and in cellulo. Structure-activity relationship was supported by docking analysis in human acetylcholinesterase binding site. Among the synthesized compounds, we have identified a novel hybrid 32a (3-[2-[1-(cyclohexylmethyl)-4-piperidyl]ethyl]-4-methoxy-1,2-benzoxazole) able to display nanomolar acetylcholinesterase inhibitory effects and nanomolar Ki for 5-HT4R.

Highlights • Conception and synthesis of new MTDLs combining 5-HT4R agonist activity and antioxidant properties • All compounds showed good %inhibition at 10-6M on 5-HT4R, with 9b and 9g showing Ki < 15 nM on 5-HT4R as well as agonist profile. • In vitro and in cellulo antioxidant effects were evaluated • 9b is the best compromise regarding the 5-HT4R activity and antioxidant properties