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

ABSTRACT 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 ( K i = 5.25 ± 0.73 nM), SP‐9 (5.09 ± 0.44 nM), and SP‐10 (5.74 ± 0.55 nM), significantly outperforming tacrine ( K i = 105.03 ± 8.85 nM) under identical conditions. For BChE, SP‐9 showed the strongest inhibition ( K i = 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.