Muscarinic acetylcholine receptors (mAChRs) comprise five distinct subtypes denoted M₁ to M₅. The antagonism of M₂ subtype could increase the release of acetylcholine from vesicles into the synaptic cleft and improve postsynaptic functions in the hippocampus via M₁ receptor activation, displaying therapeutic potentials for Alzheimer’s disease. However, drug development for M₂ antagonists is still challenged among different receptor subtypes. In this study, by optimizing a scaffold from virtual screening, we synthesized two focused libraries and generated up to 50 derivatives. By measuring potency and binding selectivity, we discovered a novel M₂ antagonist, ligand 47, featuring submicromolar IC₅₀, high M₂/M₄ selectivity (~30-fold) and suitable lipophilicity (cLogP = 4.55). Further study with these compounds also illustrates the structure–activity relationship of this novel scaffold. Our study could not only provide novel lead structure, which was easy to synthesize, but also offer valuable information for further development of selective M₂ ligands.

毒蕈碱型乙酰胆碱受体（mAChRs）包含五个不同的亚型，表示为M ₁至M ₅。M ₂亚型的拮抗作用可通过M ₁受体激活增加乙酰胆碱从囊泡向突触间隙的释放并改善海马突触后功能，从而显示出治疗阿尔茨海默氏病的潜力。但是，M ₂拮抗剂的药物开发仍在不同的受体亚型中受到挑战。在这项研究中，通过优化虚拟筛选的支架，我们合成了两个聚焦库并生成了多达50个衍生物。通过测量效能和结合选择性，我们发现了新型M ₂拮抗剂，配体47，具有亚微摩尔IC ₅₀，高M ₂ / M ₄选择性（〜30倍）和合适的亲脂性（cLogP = 4.55）。对这些化合物的进一步研究也说明了这种新型支架的结构-活性关系。我们的研究不仅可以提供易于合成的新颖的先导结构，而且还为进一步开发选择性M ₂配体提供了有价值的信息。