Novel Xanomeline-Containing Bitopic Ligands of Muscarinic Acetylcholine Receptors: Design, Synthesis and FRET Investigation.

In the last few years, fluorescence resonance energy transfer (FRET) receptor sensors have contributed to the understanding of GPCR ligand binding and functional activation. FRET sensors based on muscarinic acetylcholine receptors (mAChRs) have been employed to study dual-steric ligands, allowing for the detection of different kinetics and distinguishing between partial, full, and super agonism. Herein, we report the synthesis of the two series of bitopic ligands, and and their pharmacological investigation at the M, M, M, and M FRET-based receptor sensors. The hybrids were prepared by merging the pharmacophoric moieties of the M/M-preferring orthosteric agonist Xanomeline and the M-selective positive allosteric modulator 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) . The two pharmacophores were connected through alkylene chains of different lengths (C3, C5, C7, and C9). Analyzing the FRET responses, the tertiary amine compounds , , and evidenced a selective activation of M mAChRs, while the methyl tetrahydropyridinium salts , , and showed a degree of selectivity for M and M mAChRs. Moreover, whereas hybrids showed an almost linear response at the M subtype, hybrids evidenced a bell-shaped activation response. This different activation pattern suggests that the positive charge anchoring the compound to the orthosteric site ensues a degree of receptor activation depending on the linker length, which induces a graded conformational interference with the binding pocket closure. These bitopic derivatives represent novel pharmacological tools for a better understanding of ligand-receptor interactions at a molecular level.