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 M<inf>1</inf>/M<inf>4</inf> muscarinic acetylcholine receptor (mAChR) orthosteric agonist xanomeline (1) and the putative M<inf>1</inf> 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 M<inf>1</inf> mAChR and alcohol 5c behaved much like 3 at M<inf>1</inf> mAChR and showed full antagonism in both G<inf>i</inf> activation and β-arrestin2 engagement at M<inf>4</inf> mAChR. Moreover, docking simulations on the M<inf>1</inf> mAChR model were performed to elucidate how the binding mode of the proposed compounds is influenced by the linker length.