Isatin hemiaminal derivatives as dual-acting therapeutic agents against diarrhea: M3-AChR modulation, antibacterial activity, and in vivo gastrointestinal protection

Diarrhea remains a prevalent gastrointestinal disorder, frequently driven by bacterial infections and disrupted gut motility. To develop more effective therapeutics, a new series of isatin-based hemiaminal ethers 3a-k was synthesized and evaluated through integrated in silico, in vitro, and in vivo studies. Preliminary molecular docking against the muscarinic acetylcholine receptor (M3-AChR), a key regulator of gut motility, identified compounds 3h and 3k as the most active candidates, exhibiting binding affinities of -9.6 and -9.5 kcal/mol. These values were significantly higher than the parent isatin (-6.1 kcal/mol) and the native ligand BS46 (-8.4 kcal/mol), and were comparable to the reference standards, atropine (-8.4 kcal/mol) and loperamide (-9.8 kcal/mol), indicating strong M3-AChR inhibitory potential. Additionally, these derivatives exhibited significant inhibitory activity against diarrhea-causing pathogens, including E. coli, S. typhimurium, S. flexneri, and S. aureus, with IC₅₀ values 3h (1.66-4.82 μM) and 3k (1.4-5.3 μM). Based on its promising in silico and in vitro performance, the most active menthyl-isatin conjugate 3h was evaluated in vivo using the castor oil-induced diarrhea model in Swiss-Webster mice. The compound exhibited a dose-dependent protective effect, significantly delaying the onset diarrhea, reducing fecal mass by > 70 %, and decreasing defecation frequency. Histopathological analysis further validated these findings results, revealing preserved mucosal morphology, improved villus structure, and reduced necrotic cellular damage by 80 %. Pharmacokinetic and safety profiling indicated that 3h possess favorable drug-likeness, high intestinal absorption, and low blood-brain barrier penetration. Collectively, these results highlight dual therapeutic relevance of 3h in both motility and infection-driven diarrheal pathways.