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.