RS67333 is a partial serotonin subtype 4 receptor (5-HT₄R) agonist that has been widely studied for its procognitive effect. More recently, it has been shown that its ability to promote the nonamyloidogenic cleavage of the precursor of the neurotoxic amyloid-β peptide leads to the secretion of the neurotrophic protein sAPPα. This effect has generated great interest in RS67333 as a potential treatment for Alzheimer’s disease (AD). We show herein that RS67333 is also a submicromolar acetylcholinesterase (AChE) inhibitor and therefore, could contribute, through this effect, to the restoration of the cholinergic neurotransmission that becomes altered in AD. We planned to pharmacomodulate RS67333 to enhance its AChE inhibitory activity to take advantage of this pleiotropic pharmacological profile in the design of a novel multitarget-directed ligand that is able to exert not only a symptomatic but also, a disease-modifying effect against AD. These efforts allowed us to select donecopride as a valuable dual (h)5-HT₄R partial agonist (K_i = 10.4 nM; 48.3% of control agonist response)/(h)AChEI (IC₅₀ = 16 nM) that further promotes sAPPα release (EC₅₀ = 11.3 nM). Donecopride, as a druggable lead, was assessed for its in vivo procognitive effects (0.1, 0.3, 1, and 3 mg/kg) with an improvement of memory performances observed at 0.3 and 1 mg/kg on the object recognition test. On the basis of these in vitro and in vivo activities, donecopride seems to be a promising drug candidate for AD treatment.

Background and Purpose: We recently identified donecopride as a pleiotropic compound able to inhibit AChE and to activate 5‐HT₄ receptors. Here, we have assessed the potential therapeutic effects of donecopride in treating Alzheimer's disease (AD).Experimental Approach: We used two in vivo animal models of AD, transgenic 5XFAD mice and mice exposed to soluble amyloid‐β peptides and, in vitro, primary cultures of rat hippocampal neurons. Pro‐cognitive and anti‐amnesic effects were evaluated with novel object recognition, Y‐maze, and Morris water maze tests. Amyloid load in mouse brain was measured ex vivo and effects of soluble amyloid‐β peptides on neuronal survival and neurite formation determined in vitro.Key Results: In vivo, chronic (3 months) administration of donecopride displayed potent anti‐amnesic properties in the two mouse models of AD, preserving learning capacities, including working and long‐term spatial memories. These behavioural effects were accompanied by decreased amyloid aggregation in the brain of 5XFAD mice and, in cultures of rat hippocampal neurons, reduced tau hyperphosphorylation. In vitro, donecopride increased survival in neuronal cultures exposed to soluble amyloid‐β peptides, improved the neurite network and provided neurotrophic benefits, expressed as the formation of new synapses.Conclusions and Implications: Donecopride acts like a Swiss army knife, exhibiting a range of sustainable symptomatic therapeutic effects and potential disease‐modifying effects in models of AD. Clinical trials with this promising drug candidate will soon be undertaken to confirm its therapeutic potential in humans.

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.