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

A series of fluorescent dibenzodiazepinone-type muscarinic acetylcholine M(2)receptor (M2R) ligands was synthesized using various fluorescent dyes (5-TAMRA,lambda(ex)/lambda(em)approximate to 547/576 nm; BODIPY 630/650,lambda(ex)/lambda(em)approximate to 625/640 nm; pyridinium dye Py-1,lambda(ex)/lambda(em)approximate to 611/665 nm and pyridinium dye Py-5,lambda(ex)/lambda(em)approximate to 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 M(1)and M(4)receptors and moderate to pronounced M2R selectivity compared to the M(3)and M(5)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.