Mono- and Bis-Ureido Sulfapyridines as Nanomolar Dual Cholinesterase Inhibitors: Enzyme Kinetics, Molecular Docking, and ADME/Tox Insights

A series of mono- and bis-ureido sulfapyridine derivatives (SP-1–SP-10) were synthesized and evaluated as dual cholinesterase inhibitors targeting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). All compounds exhibited nanomolar inhibition. The most potent AChE inhibitors were SP-8 (Ki = 5.25 ± 0.73 nM), SP-9 (5.09 ± 0.44 nM), and SP-10 (5.74 ± 0.55 nM), significantly outperforming tacrine (Ki = 105.03 ± 8.85 nM) under identical conditions. For BChE, SP-9 showed the strongest inhibition (Ki = 26.38 ± 2.07 nM) compared with tacrine (172.03 ± 32.42 nM). Kinetic analyses revealed competitive inhibition toward AChE for all derivatives, whereas BChE inhibition displayed both competitive and noncompetitive patterns depending on structural features. Glide XP docking using AChE (PDB: 7XN1) and BChE (PDB: 4BDS), complemented by MM-GBSA binding free energy calculations, supported favorable ligand accommodation within the catalytic pockets and highlighted the importance of aromatic and hydrophobic interactions. In silico ADME/Tox analysis revealed a potency–drug-likeness trade-off: mono-ureido derivatives complied with Lipinski's rule of five and showed high predicted oral absorption, whereas bis-ureido derivatives, despite superior enzymatic potency, exhibited multiple drug-likeness violations. These findings identify the ureido–sulfapyridine scaffold as a potent enzymatic dual cholinesterase inhibitory framework, warranting further structural optimization to improve pharmacokinetic properties. © 2026 Wiley-VCH GmbH.