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,
12-Cn and
13-Cn, and their pharmacological investigation at the M
1, M
2, M
4, and M
5 FRET-based receptor sensors. The hybrids were prepared by merging the pharmacophoric moieties of the M
1/M
4-preferring orthosteric agonist Xanomeline
10 and the M
1-selective positive allosteric modulator 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone)
11. The two pharmacophores were connected through alkylene chains of different lengths (C3, C5, C7, and C9). Analyzing the FRET responses, the tertiary amine compounds
12-C5,
12-C7, and
12-C9 evidenced a selective activation of M
1 mAChRs, while the methyl tetrahydropyridinium salts
13-C5,
13-C7, and
13-C9 showed a degree of selectivity for M
1 and M
4 mAChRs. Moreover, whereas hybrids
12-Cn showed an almost linear response at the M
1 subtype, hybrids
13-Cn evidenced a bell-shaped activation response. This different activation pattern suggests that the positive charge anchoring the compound
13-Cn 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.
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