Synthesis and Biological Evaluation of a Novel Series of Heterobivalent Muscarinic Ligands Based on Xanomeline and 1‑[3-(4-Butylpiperidin-1-yl)propyl]-1,2,3,4-tetrahydroquinolin-2-one (77-LH-28-1)

Novel bitopic hybrids, based on the M₁/M₄ muscarinic acetylcholine receptor (mAChR) orthosteric agonist xanomeline (1) and the putative M₁ mAChR allosteric agonist 1-[3-(4-butylpiperidin-1-yl)­propyl]-1,2,3,4-tetrahydroquinolin-2-one (77-LH-28-1, 3) connected by an aliphatic linker of variable length, were prepared. The novel heterobivalent hybrids 4a–f along with the intermediate alcohols 5a–f were pharmacologically evaluated in radioligand binding assays and some of them for their functional efficacies in bioluminescence resonance energy transfer (BRET)-based assays to give an insight into the structure–activity relationships of bivalent and linker-attached compounds in mAChRs. The hybrid 4d exhibited high efficacy for β-arrestin2 engagement in M₁ mAChR and alcohol 5c behaved much like 3 at M₁ mAChR and showed full antagonism in both G_i activation and β-arrestin2 engagement at M₄ mAChR. Moreover, docking simulations on the M₁ mAChR model were performed to elucidate how the binding mode of the proposed compounds is influenced by the linker length.