Synthesis of a Series of Structurally Diverse MB327 Derivatives and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor

Abstract

A novel series of 30 symmetric bispyridinium and related N-heteroaromatic bisquaternary salts with a propane-1,3-diyl linker was synthesized and characterized for their binding affinity at the MB327 binding site of nicotinic acetylcholine receptor (nAChR) from Torpedo californica. Compounds targeting this binding site are of particular interest for research into new antidotes against organophosphate poisoning, as therapeutically active 4-tert-butyl-substituted bispyridinium salt MB327 was previously identified as a nAChR re-sensitizer. Efficient access to the target compounds was provided by newly developed methods enabling N-alkylation of sterically hindered or electronically deactivated heterocycles exhibiting a wide variety of functional groups. Determination of binding affinities toward the MB327 binding site at the nAChR, using a recently developed mass spectrometry (MS)-based Binding Assay, revealed that several compounds reached affinities similar to that of MB327 (pK =4.73±0.03). Notably, the newly prepared lipophilic 4-tert-butyl-3-phenyl-substituted bispyridinium salt PTM0022 (3 h) was found to have significantly higher binding affinity, with a pK value of 5.16±0.07, thus representing considerable progress toward the development of more potent nAChR re-sensitizers.

Synthesis of a Series of Non‐Symmetric Bispyridinium and Related Compounds and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor

“…All target compounds synthesized in the context of this study were catalogued with a certain PTM number (Pharmacy and Toxicology Munich). Binding affinities toward the nAChR were determined in MS Binding Assays using [ 2 H 6 ]MB327 as marker and nAChR‐enriched membranes prepared from Torpedo californica electroplaque tissue as target (Table ) . In the following, binding data for compounds are given and discussed with respect to the substitution pattern using bispyridinium salt 1 a as reference compound.…”

“…Notably, the increased binding affinity of dimethylamino‐substituted compounds is in accordance with recently published structure–affinity relationships found for symmetric bispyridinium compounds. The dimethylamino group was identified as potential substitute for the lipophilic 4‐ tert ‐butyl substituent, as it enhances binding affinity as well . Although 3‐methoxy‐substituted 1 k (PTM0040, p K i =4.35±0.08, Table , entry 12) showed a lower binding affinity than unsubstituted 1 a (PTM0029, p K i =4.52±0.06, Table , entry 2), the p K i value for the bifunctional compound 1 l (PTM0038, Table , entry 13), with an additional 4‐ tert ‐butyl group next to the 3‐methoxy moiety, increased significantly (determined by a two‐sided t‐test, α =0.05) about 0.6 log units ( 1 l , PTM0038: p K i =4.97±0.02) relative to the p K i value of compound 1 a (PTM0029).…”

“…MS Binding Assays were performed with [ 2 H 6 ]MB327 as marker and nAChR‐enriched membranes, prepared from Torpedo californica electroplaque tissue, as previously described …”

“…We reported on the synthesis of 30 novel symmetric bispyridinium and related salts structurally analogous to MB327 and their binding affinities toward the MB327 binding site. The most affine compound, lipophilic 3‐phenyl‐4‐ tert ‐butyl‐substituted bispyridinium salt PTM0022 (Figure ), showed a p K i value significantly higher than that of MB327 . Furthermore, the study provided vital information on the influence of various functional groups and structural motives on binding affinity.…”

“…First attempts to create nAChR re‐sensitizers with increased potency were previously published by us . We reported on the synthesis of 30 novel symmetric bispyridinium and related salts structurally analogous to MB327 and their binding affinities toward the MB327 binding site.…”