Competition, Selectivity and Efficacy of Analogs of A-84543 for Nicotinic Acetylcholine Receptors with Repositioning of Pyridine Nitrogen

Abstract

Nicotinic acetylcholine receptors (nAChRs) play a crucial role in a number of clinically relevant mental and neurological pathways, as well as autonomic and immune functions. The development of subtype-selective ligands for nAChRs therefore is potentially useful for targeted therapeutic management of conditions where nAChRs are involved. We tested if selectivity for a particular nAChR subtype can be achieved through small structural modifications of a lead compound containing the nicotinic pharmacophore by changing the distance between the electronegative elements. For this purpose, analogs of A-84543 were designed, synthesized and characterized as potentially new nAChR subtype-selective ligands. Compounds were tested for their binding properties in rat cerebral cortical tissue homogenates, and subtype-selectivity was determined using stably transfected HEK cells expressing different nAChR subtypes. All compounds synthesized were found to competitively displace [(3)H]-epibatidine ([(3)H]EB) from the nAChR binding site. Of all the analogues, H-11MNH showed highest affinity for nAChRs compared to a ~ fivefold to tenfold lower affinity of A-84543. All other compounds had affinities >10,000 nM. Both A-84543 and H-11MNH have highest affinity for α2β2 and α4β2 nAChRs and show moderate affinity for β4- and α7-containing receptors. H-11MNH was found to be a full agonist with high potency at α3β4, while A-84543 is a partial agonist with low potency. Based on their unique pharmacological binding properties we suggest that A-84543 and its desmethylpyrrolidine analog can be useful as pharmacological ligands for studying nAChRs if selective pharmacological and/or genetic tools are used to mask the function of other receptors subtypes.

Keywords: A-85380; Cerebral cortex; Epibatidine; H-11MNH; Nicotine; Saturation.

Fig. 1. A-84543 meets the criteria of a nicotinic receptor pharmacophore

The typical nicotinic structure at left, was modified as seen at right in compound H-11MNH. This new molecule fits the pharmacophore model. The pharmacophore usually consists of a protonated nitrogen and a hydrogen bond donor. The optimum distance between the protonated nitrogen, which acts as a hydrogen bond acceptor and the hydrogen bond donor, is estimated to be 5.5 Å based on measurements obtained from one of the highest affinity nicotinic compounds, epibatidine. a–b, is the distance between the electron acceptor and electron donor; C, center of the electronegative element.

Fig. 2. Schematic representation of the synthesis of A-84543 and its analogs

Synthesis of compounds H-11MNH, H-11ONH, H-11ONM and H-11PNH was initiated by combining N-Boc-L-prolinol with appropriate hydroxypyridines using the Mitsunobu reaction. The N-Boc protected purified products were then deprotected to give the hydrogen or methyl analogue. A = N-Boc-L-prolinol; B, H, I = 3, 2, and 4-hydroxy pyridines respectively.

Fig. 3. Representative saturation binding curve for [³H]EB in membrane homogenates of rat brain cortex

Plot shows representative curves for three independent binding experiments. Total binding was done in the presence of increasing concentrations (0.1pM–3nM) of [³H]EB. Non-specific binding was determined in parallel preparations under identical conditions in the presence of 300μM (−)-nicotine. Specific binding was then calculated as the difference between total and non-specific binding. Each point represents the average of triplicate determinations for each concentration. Data was analyzed by nonlinear least square regression with GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA). The Mean B_max value was 84 fmol/mg protein and the K_D = 35pM for 10 mg tissue.

Fig. 4. Competition curves of nicotinic ligands for binding sites labeled with 150pM [³H]EB

Competition assays were performed in adult rat brain cortex homogenates labeled with 150pM [³H]EB. Binding of A-84543 (◆), H-11ONM (*), (●) H-11MNH, (+) H-11ONH and H-11PNH (X), were compared to that of A-85380 (■), a high affinity nAChR agonist, DHβE (▲) a high affinity nAChR antagonist, and Nicotine (▼). The data were analyzed by nonlinear least square regression method using GraphPad Prism 5 software (GraphPad Software Inc. San Diego, CA). IC₅₀ and K_i values are presented in Table II. The data shown is representative of n = 3–6 independent experiments using triplicates.