Synthesis, in silico and in vitro evaluation of N-phenylbenzimidazole derivatives targeting muscarinic acetylcholine receptors.

The development of efficient, orthosteric, and subtype-selective ligands for muscarinic acetylcholine receptors (mAChRs) remains a significant challenge, despite their critical role in numerous physiological and pathological processes. As part of ongoing efforts to optimize mAChR ligands based on an N-phenylbenzimidazole core, we synthesized and characterized a series of analogues featuring varied linkers and cyclic or non-cyclic amine substitutions. These compounds were evaluated both in silico and in vitro for binding pose, affinity, cytotoxicity, and functional activity. Several ligands demonstrated high binding affinity in the low nanomolar range and favorable properties predicting central nervous system penetration. Notably, even minor structural modifications led to significant shifts in receptor subtype selectivity, suggesting that kinetic parameters may play a more dominant role than static structural features. This highlights the need for broader compound libraries to support predictive modeling of subtype selectivity in mAChRs. All active compounds exhibited an antagonistic mode of action, which is particularly advantageous for the development of diagnostic imaging agents such as positron-emission tomography tracers.