Differently fluorescence-labelled dibenzodiazepinone-type muscarinic acetylcholine receptor ligands with high M₂R affinity

A series of fluorescent dibenzodiazepinone-type muscarinic acetylcholine M₂ receptor (M₂R) ligands was synthesized using various fluorescent dyes (5-TAMRA, λ _ex/λ _em ≈ 547/576 nm; BODIPY 630/650, λ _ex/λ _em ≈ 625/640 nm; pyridinium dye Py-1, λ _ex/λ _em ≈ 611/665 nm and pyridinium dye Py-5, λ _ex/λ _em ≈ 465/732 nm). All fluorescent probes exhibited high M₂R affinity (pK _i (radioligand competition binding): 8.75-9.62, pK _d (flow cytometry): 8.36-9.19), a very low preference for the M₂R over the M₁ and M₄ receptors and moderate to pronounced M₂R selectivity compared to the M₃ and M₅ receptors. The presented fluorescent ligands are considered useful molecular tools for future studies using methods such as fluorescence anisotropy and BRET based MR binding assays.