Regiospecific Introduction of Halogens on the 2-Aminobiphenyl Subunit Leading to Highly Potent and Selective M3 Muscarinic Acetylcholine Receptor Antagonists and Weak Inverse Agonists

Abstract

Muscarinic M 3 receptor antagonists and inverse agonists displaying high affinity and subtype selectivity over the antitarget M 2 are valuable pharmacological tools and may enable improved treatment of chronic obstructive pulmonary disease (COPD), asthma, or urinary incontinence. On the basis of known M 3 antagonists comprising a piperidine or quinuclidine unit attached to a biphenyl carbamate, 5-fluoro substitution was responsible for M 3 subtype selectivity over M 2 , while 3′-chloro substitution substantially increased affinity through a σ-hole interaction. Resultantly, two piperidinyl-and two quinuclidiniumsubstituted biphenyl carbamates OFH243 (13n), OFH244 (13m), OFH3911 (14n), and OFH3912 (14m) were discovered, which display two-digit picomolar affinities with K i values from 0.069 to 0.084 nM, as well as high selectivity over the M 2 subtype (46-to 68-fold). While weak inverse agonistic properties were determined for the biphenyl carbamates 13m and 13n, neutral antagonism was observed for 14m and 14n and tiotropium under identical assay conditions.

Synthesis, Characterization, and Application of Muscarinergic M3 Receptor Ligands Linked to Fluorescent Dyes

“…For attachment of the linker and the fluorescent dye, we chose the previously developed biphenylcarbamate 3b as a starting point as this compound shows high binding affinity (K i (M 3 ) = 0.069 nM) to the muscarinergic M 3 receptor subtype (Scheme 1). 12 The rationale for connecting other binding motifs or functional units via the piperidine nitrogen of 3b was derived from the structure of the bivalent ligand batefenterol (Figure 2). 24 Since the structure of batefenterol contains a 4-piperidinyl moiety directly attached to the oxygen atom of an N-(2biphenyl)-carbamate and not via a methylene unit such as in 3b, this led us to consider compound 3a as the second orthosteric binding motif for the present study.…”

“…Before turning to flow cytometric saturation binding experiments, the functional properties of the two most promising conjugates OFH611 (12) and OFH5503 (16) were investigated (Figure 3). As summarized in Figure 3, both OFH5503 (16) and OFH611 (12) behave as antagonists in G-protein-mediated signaling (determined via IP-One assay) and β-arrestin recruitment (determined via PathHunter assay). The antagonistic properties were further proven by the effects of conjugates 12 and 16 on the functional activity of the agonist carbachol (brown and yellow curves).…”

“…Table 5. Parameters Characterizing the Association and Dissociation of OFH611 (12) and OFH5503 (16) to or from the hM 3 R Dissociation constant determined by flow cytometric saturation binding at CHO-hM 3 R cells; mean values ± SEM from three independent experiments performed in triplicate. b Kinetically derived dissociation constant (± propagated error) calculated from k on and k off .…”

“…Radioligand binding studies with all subtypes of muscarinic receptors (M 1 , M 2 M 3 , M 4 , and M 5 ) were performed as described previously. , In brief, competition binding experiments were done using membranes of HEK293T cells transiently transfected with the cDNA of the human M 1 , M 2 M 3 , M 4 , and M 5 receptors (cDNA Resource Center, Bloomsberg, PA), respectively. Radioligand displacement assays were performed in binding buffer (25 mM HEPES, 5 mM MgCl 2 , 1 mM EDTA, 0.006% BSA at a pH of 7.4) with [ 3 H] N -methylscopolamine (specific activity = 82 Ci/mmol, Novandi, Södertälje, Sweden) at final concentrations of 0.2–0.4 nM.…”

“…Our general interest in this research field results from the recent development of high-affinity and subtype-selective antagonists for the M 3 receptor. 12 To make such compounds useful besides their role as potential drug candidates, they can be further elaborated into pharmacological tools. In this overall context, ligands conjugated to fluorescent dyes have become of particular interest.…”