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 (<b>1</b>) 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, <b>3</b>) connected by an aliphatic linker of variable length, were prepared. The novel heterobivalent hybrids <b>4a</b>–<b>f</b> along with the intermediate alcohols <b>5a</b>–<b>f</b> 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 <b>4d</b> exhibited high efficacy for β-arrestin2 engagement in M₁ mAChR and alcohol <b>5c</b> behaved much like <b>3</b> 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