Abstracts Category : Other

Synthesis and pharmacological characterization of dibenzodiazepinone-type heterodimeric and fluorescently labeled muscarinic receptor ligands

by She Xueke

In humans, the family of muscarinic acetylcholine receptors (mAChR, MRs) comprises five subtypes (M1R-M5R), which are members of the class A GPCR superfamily and mediate the action of the neurotransmitter acetylcholine in the central and peripheral nervous system. For instance, the M2R, which binds to Gi/o heterotrimeric G-proteins, acts as a presynaptic autoreceptor in the brain and in the periphery. Accordingly, selective M2R antagonism in the CNS, resulting in enhanced cholinergic transmission, was suggested as an approach to increase cholinergic function in Alzheimer patients. MRs represent important drug targets, however, there is still a need for highly subtype selective MR ligands, as the development of selective agents has been challenging due to the high conservation of the acetylcholine (orthosteric) binding site. Due to the less conserved allosteric binding sites, the dualsteric ligand approach, i.e. the design of compounds, which simultaneously address the orthosteric and allosteric binding sites, is considered a promising strategy to develop MR ligands with improved subtype selectivity.This work was aiming at the synthesis and pharmacological characterization of dibenzodiazepinone-type heterodimeric MR ligands, which were prepared by linking different monomeric MR ligands (agonists, antagonists, orthosteric and allosteric ligands) through various linkers to a pharmacophoric moiety derived from the dibenzodiazepinone DIBA. The synthesis afforded heterodimeric ligands (DIBA-xanomeline, DIBA-TBPB, DIBA-77-LH-28-1, DIBA-propantheline and DIBA-4-DAMP) and DIBA-DIBA-type homodimeric ligands. Equilibrium competition binding studies with [3H]NMS at live CHO cells expressing the respective human MxR subtype (x = 1-5) revealed a M2R preference of all dimeric ligands with high M2R affinities (Ki values: 0.08-5.8 nM). These data demonstrated that the type of the linker (short vs. long, basic vs. non-basic, etc.) and the type of the second pharmacophoric group had only little impact on M2R binding. As non-DIBA-type monomeric and homodimeric reference compounds exhibited considerably lower M2R affinities than the synthesized DIBA-derived ligands, the high M2R affinity of the heterodimeric dibenzodiazepinone-type ligands is most likely conferred by the dibenzodiazepinone pharmacophore.Two tritium-labeled DIBA-derived heterodimeric ligands (DIBA-xanomeline- and DIBA-TBPB-type) were prepared and characterized by saturation and kinetic binding studies at the hM2R. Saturation binding experiments showed that these ligands address the orthosteric site of the M2R. The DIBA-TBPB-type dimeric radioligand ([3H]115) exhibited a very high M2R affinity (Kd value 0.13 nM). The investigation of the effect of allosteric MR ligands (gallamine, W84, LY2119620) on the equilibrium binding of [3H]115, and saturation binding studies with [3H]115 in the presence of the allosteric MR ligand W84 (Schild-like analysis) strongly suggested a competitive mechanism between [3H]115 and the investigated allosteric ligand.Advisors/Committee Members: Buschauer, Armin (advisor), Bernhardt, Gnther (advisor).