Tris-azaaromatic quaternary ammonium salts: Novel templates as antagonists at nicotinic receptors mediating nicotine-evoked dopamine release

A series of tris-azaaromatic quaternary ammonium salts has been synthesized and evaluated for their ability to inhibit neuronal nicotinic acetylcholine receptors (nAChRs) mediating nicotine-evoked [³H]dopamine release from superfused rat striatal slices and for inhibition of [³H]nicotine and [³H]methyllycaconitine binding to whole rat brain membranes. The 3-picolinium compound 1,3,5-tri-{5-[1-(3-picolinium)]-pent-1-ynyl}benzene tribromide (tPy3PiB), 3b, exhibited high potency and selectivity for nAChR subtypes mediating nicotine-evoked [³H]dopamine release with an IC₅₀ of 0.2 nM and I_max of 67%.     

Tetrakis-azaaromatic quaternary ammonium salts: novel subtype-selective antagonists at neuronal nicotinic receptors that mediate nicotine-evoked dopamine release

A series of tetrakis-azaaromatic quaternary ammonium salts was synthesized to identify compounds with higher affinity and selectivity as antagonists at neuronal nicotinic receptor subtypes (nAChR) that mediate nicotine-evoked DA release. A high hit rate was achieved in identifying potent analogs that inhibit these nAChRs. Three tetrakis analogs, 11j, 11f, and 11g, were identified as potent (IC(50)=3, 28 and 56nM, respectively) antagonists at these receptors. These compounds represent a novel structural class of nicotinic receptor antagonists.     

Development of subtype-selective ligands as antagonists at nicotinic receptors mediating nicotine-evoked dopamine release

N-n-Alkylation of nicotine converts it from an agonist into an antagonist at neuronal nicotinic acetylcholine receptor subtypes mediating nicotine-evoked dopamine release. Conformationally restricted analogues exhibit both high affinity and selectivity at this site, and are able to access the brain due to their ability to act as substrates for the blood-brain barrier choline transporter.     

Novel bis-2,2,6,6-tetramethylpiperidine (bis-TMP) and bis-mecamylamine antagonists at neuronal nicotinic receptors mediating nicotine-evoked dopamine release

By linking two or three mecamylamine or 2,2,6,6-tetramethylpiperidine (TMP) molecules together via a linear lipophilic bis-methylene linker or a specially designed conformationally restricted tris-linker, a series of bis- and tris-tertiary amine analogs has been synthesized and evaluated as potent antagonists at nAChRs mediating nicotine-evoked [³H]dopamine release from rat striatal slices. Compounds 7e, 14b and 16 demonstrated high potency in decreasing nicotine-evoked [³H]dopamine release (IC₅₀ = 2.2, 46, and 107 nM, respectively). The preliminary structure–activity data obtained with these new analogs suggest the importance of the length of the methylene linker in the bis-analog series. Such bis-tertiary amino analogs may provide a new strategy for the design of drugable ligands that have high inhibitory potency against nAChRs mediating nicotine-evoked dopamine release in striatum, which have been suggested to be target receptors of interest in the development of potential smoking cessation therapies.     

WIN-55,212-2 and SR-141716A alter nicotine-induced changes in locomotor activity, but do not alter nicotine-evoked [3H]dopamine release

Nicotine, the main psychoactive ingredient in tobacco, plays a key role in the development of cigarette smoking addiction. The endocannabinoid system has been demonstrated to have an important role in the motivational and reinforcing effects of drugs. The present study used behavioral and neurochemical techniques to study the interaction of cannabinoid receptors and nicotine pharmacology. In a locomotor activity experiment in rats, the CB(1)/CB(2) cannabinoid receptor agonist WIN-55,212-2 (0.28-2.8 mg/kg) attenuated nicotine (0.4 mg/kg)-induced hyperactivity, but did not alter nicotine (1.0 mg/kg)-induced hypoactivity. In contrast, the selective CB(1) cannabinoid receptor antagonist SR-141716A (1.0 mg/kg) diminished nicotine-induced hypoactivity, but did not alter nicotine-induced hyperactivity. In a neurochemical experiment, rat striatal slices preloaded with [(3)H]dopamine were superfused with WIN-55,212-2 or SR-141716A. A high concentration (100 microM) of WIN-55,212-2 evoked [(3)H]overflow, but this effect was not blocked by the cannabinoid receptor antagonist AM-251. SR-141716A did not evoke [(3)H]overflow, and neither WIN-55,212-2 nor SR-141716A altered nicotine-evoked [(3)H]overflow. Overall, these results indicate a behavioral interaction between cannabinoid receptors and nicotine pharmacology. Likely, WIN-55,212-2 and SR-141716A block nicotine-induced changes in behavior through an indirect mechanism, such as alteration in endocannabinoid regulation of motor circuits, rather than directly through blockade of nicotinic acetylcholine receptors.     

Involvement of alpha7 nicotinic acetylcholine receptors in activation of tyrosine hydroxylase and dopamine beta-hydroxylase gene expression in PC12 cells

Nicotine treatment increases intracellular free Ca(2+) concentration [Ca(2+)](i), stimulates catecholamine release, and elevates gene expression for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). However, the type of nicotinic acetylcholine receptors (nAChRs) mediating these events is unclear. The nAChR receptor antagonists alpha-bungarotoxin (alphaBTX) and methyllycaconitine greatly reduced the nicotine-triggered initial transient rise in [Ca(2+)](i) and prevented the second prolonged elevation of [Ca(2+)](i), suggesting the involvement of alpha7 nAChRs. Two specific alpha7 nicotinic agonists, 3-(2,4-dimethoxybenzilidene)anabaseine (DMXB) and E, E-3-(cinnamylidene)anabaseine (3-CA), were found to elicit a small, delayed increase in [Ca(2+)](i) with kinetics and magnitude similar to the second elevation observed with nicotine. This increase was inhibited by the inositol trisphosphate receptor antagonist xestospongin C. Exposure to 3-CA or DMXB for 6 or 24 h elevated TH and DBH mRNA levels two- to fourfold over control levels. These agonists were more effective than nicotine alone in increasing TH and DBH gene expression and significantly elevated [Ca(2+)](i) for up to 6 h. The increase in [Ca(2+)](i) or the elevation in TH mRNA by 3-CA was completely inhibited by alphaBTX. This study, for the first time, implicates stimulation of alpha7 nAChRs in the activation of TH and DBH gene expression.     

Novel bis-, tris-, and tetrakis-tertiary amino analogs as antagonists at neuronal nicotinic receptors that mediate nicotine-evoked dopamine release

A series of tertiary amine analogs derived from lead azaaromatic quaternary ammonium salts has been designed and synthesized. The preliminary structure-activity relationships of these new analogs suggest that such tertiary amine analogs, which potently inhibit nicotine-evoked dopamine release from rat striatum, represent drug-like inhibitors of α6-containing nicotinic acetylcholine receptors. The bis-tertiary amine analog 7 exhibited an IC₅₀ of 0.95 nM, while the tris-tertiary amine analog 19 had an IC₅₀ of 0.35 nM at nAChRs mediating nicotine-evoked dopamine release.     

Stimulation of nicotinic acetylcholine receptors protects motor neurons

The present study demonstrated that administration of nicotine prevented glutamate-induced motor neuronal death in primary cultures of the rat spinal cord. The nicotine-induced neuroprotection was inhibited by either dihydro-beta-erythroidin (DHbetaE) or alpha-bungarotoxin (alphaBT), suggesting that it is mediated through both alpha4beta2 and alpha7 nicotinic acetylcholine receptors (nAChRs). Both alpha4beta2 and alpha7 nAChRs were identified on rat spinal motor neurons by immunohistochemical methods. We also demonstrated that galantamine, an acetylcholinesterase inhibitor with allosteric nAChR-potentiating ligand properties, prevented glutamate-induced motor neuronal death. These results suggest that stimulation of nAChR may be used as a treatment for ALS.     

Insect nicotinic acetylcholine receptor: conserved neonicotinoid specificity of [(3)H]imidacloprid binding site

The insect nicotinic acetylcholine receptor (nAChR) is a major target for insecticide action. The rapidly expanding use of neonicotinoid insecticides of varied structures makes it increasingly important to define similarities and differences in their action, particularly for the first-generation chloropyridinyl compounds versus the second-generation chlorothiazolyl derivatives. We have shown with Musca domestica that a convenient and relevant determination of the neonicotinoid insecticide target is a binding site assay with [(3)H]imidacloprid ([(3)H]IMI). This study uses membranes from the aphids MYZUS: persicae and Aphis craccivora and from heads of the flies DROSOPHILA: melanogaster and Musca domestica to characterize the [(3)H]IMI binding sites relative to their number and possible species variation in structure-activity relationships. With emphasis on commercial neonicotinoids, six potent chloropyridinyl compounds are compared with the corresponding six chlorothiazolyl analogues (syntheses are given for chemicals prepared differently than previously described). The preference for chloropyridinyl versus chlorothiazolyl is not dependent on the insect species examined but instead on other structural features of the molecule. The chlorothiazolyl substituent generally confers higher potency in the clothianidin and desmethylthiamethoxam series and the chloropyridinyl moiety in the imidacloprid, thiacloprid, acetamiprid, and nitenpyram series. Two chlorothiazolyl compounds compete directly with the chloropyridinyl [(3)H]IMI for the same binding sites in MYZUS: and DROSOPHILA: membranes. This study shows conserved neonicotinoid specificity of the [(3)H]IMI binding site in each of the four insect species examined.     

Selectivity of coronaridine congeners at nicotinic acetylcholine receptors and inhibitory activity on mouse medial habenula

The inhibitory activity of coronaridine congeners on human (h) α4β2 and α7 nicotinic acetylcholine receptors (AChRs) is determined by Ca²⁺ influx assays, whereas their effects on neurons in the ventral inferior (VI) aspect of the mouse medial habenula (MHb) are determined by patch-clamp recordings. The Ca²⁺ influx results clearly establish that coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to hα4β2 and hα7 subtypes, and with the following potency sequence, for hα4β2: (±)-18-methoxycoronaridine [(±)-18-MC] > (+)-catharanthine > (±)-18-methylaminocoronaridine [(±)-18-MAC] ∼ (±)-18-hydroxycoronaridine [(±)-18-HC]; and for hα7: (+)-catharanthine > (±)-18-MC > (±)-18-HC > (±)-18-MAC. Interestingly, the inhibitory potency of (+)-catharanthine (27 ± 4 μM) and (±)-18-MC (28 ± 6 μM) on MHb (VI) neurons was lower than that observed on hα3β4 AChRs, suggesting that these compounds inhibit a variety of endogenous α3β4* AChRs. In addition, the interaction of bupropion with (−)-ibogaine sites on hα3β4 AChRs is tested by [³H]ibogaine competition binding experiments. The results indicate that bupropion binds to ibogaine sites at desensitized hα3β4 AChRs with 2-fold higher affinity than at resting receptors, suggesting that these compounds share the same binding sites. In conclusion, coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to other AChRs, by interacting with the bupropion (luminal) site. Coronaridine congeners also inhibit α3β4*AChRs expressed in MHb (VI) neurons, supporting the notion that these receptors are important endogenous targets for their anti-addictive activities.     

Asymmetric total synthesis of tetrahydroprotoberberine derivatives and evaluation of their binding affinities at dopamine receptors

Cocaine addiction remains a serious challenge for clinical and medical research because there is no effective pharmacological treatment. l-THP, a natural product isolated from Corydalis yanhusuo W.T. Wang, is one of the most frequently used traditional herbs to treat drug addiction in China. Our laboratory first reported that its demethylated metabolites l-ICP, l-CD, and l-CP had high affinity at dopamine D1, D2, and D5 receptors. Here we report the chemical synthesis of these metabolites and other derivatives and their binding affinities at dopamine receptors. The synthesis of these bioactive metabolites will allow further in vivo study of their potential in treating cocaine addiction.     

Pharmacological and immunological identification of native alpha7 nicotinic receptors: evidence for homomeric and heteromeric alpha7 receptors

Controversy surrounds the expression of alpha7 nicotinic acetylcholine receptors (nAChRs) in adrenal chromaffin cells. In these studies, alpha7 nAChRs expressed in bovine adrenal chromaffin cells are investigated. Using radiolabeled ligand binding techniques, [(125)I]alpha-bungarotoxin (alphaBGT) binding reaches equilibrium within 4 h and is saturable with a K(d) value of 4.2 nM. Using homologous competition experiments, the K(i) for binding of alphaBGT was 1.9 nM. These data are consistent with the expression of homomeric alpha7 nAChRs. Methyllycaconatine (MLA), which binds alpha7 nAChRs with high affinity, inhibits [(125)I]alphaBGT binding in a concentration-dependent manner with a K(i) of 30.6 nM; this value is approximately 10 fold higher than the reported affinity of MLA for alpha7 nAChRs. We also document the ability of bromoacetylcholine (brACh) to alkylate alpha7 nAChRs, as has been previous demonstrated for bovine adrenal alpha3beta4 nAChRs. When adrenal nAChRs are immunoprecipated with mAb319, an antibody which recognizes alpha7 nAChR protein, and then probed with mAb319 using Western blot analysis, a single band of approximately 53 kDa is identified. When adrenal nAChRs are immunoprecipated with mAb35, an antibody which recognizes alpha3 and alpha5 nAChR proteins, and then probed with mAb319 using Western blot analysis, a single band of approximately 53 kDa is identified. Together, these results support the expression of alpha7 nAChRs in bovine adrenal chromaffin cells. However, these data suggest that the subunit composition of some of these receptors may include heteromeric alpha7 nAChRs.     

Nicotine potentiates the neurogenic contractile response of rabbit bladder tissue via nicotinic acetylcholine receptors: nitric oxide and prostaglandins have no role in this process

Nicotine, a nicotinic acetylcholine receptors (nAChRs) agonist, has a role in modulation of the neurotransmitter release following nerve stimulation in both the central and peripheral nervous systems. The aim of this study was to determine whether electrical field stimulation (EFS)-evoked contractions are altered in rabbit bladder in the presence of nicotine and, if an alteration occurs, to investigate the effects of nitric oxide and prostaglandins on nicotine-induced alternation in isolated rabbit bladder. EFS-evoked contractile responses from rabbit bladder obtained were recorded with isometric force displacement transducers. Nicotine was added to preparations at various concentrations. The effects of hexamethonium, cadmium (Cd(2+)), indomethacin and N-nitro-L-arginine methyl ester (L-NAME) were tested on the EFS-evoked contractions in the presence of nicotine. Nicotine led to a dose-dependent increase in the amplitude of the EFS-evoked contractile responses. Cd(2+) and hexamethonium inhibited the nicotine-induced increase in EFS-evoked responses, whereas indomethacin and L-NAME had no effect. In conclusion, nicotine increased the EFS-evoked contractile responses possibly by facilitating release of neurotransmitters from nerve terminals by a mechanism dependent on the influx of Ca(2+) from voltage-gated Ca(2+) channels (VGCCs) via activation of nAChRs in isolated rabbit bladder. Nitric oxide and prostaglandins do not have a physiological role in the regulation of neurotransmitter release.     

Synthesis and biological evaluation of Tc-99m-cyclopentadienyltricarbonyl-technetium-labeled A-85380: An imaging probe for single-photon emission computed tomography investigation of nicotinic acetylcholine receptors in the brain

We have designed (S)-(5-(azetidin-2-ylmethoxy)pyridine-3-yl)methyl cyclopentadienyltricarbonyl technetium carboxylate ([^99mTc]CPTT–A–E) with high affinity for nicotinic acetylcholine receptors (nAChRs) using (2(S)-azetidinylmethoxy)-pyridine (A-85380) as the lead compound to develop a Tc-99m-cyclopentadienyltricarbonyl-technetium (^99mTc)-labeled nAChR imaging probe. Because technetium does not contain a stable isotope, cyclopentadienyltricarbonyl rhenium (CPTR) was synthesized by coordinating rhenium, which is a homologous element having the same coordination structure as technetium. Further, the binding affinity to nAChR was evaluated. CPTR–A–E exhibited a high binding affinity to nAChR (K_i = 0.55 nM). Through the radiosynthesis of [^99mTc]CPTT–A–E, an objective compound could be obtained with a radiochemical yield of 33% and a radiochemical purity of greater than 97%. In vitro autoradiographic study of the brain exhibited that the local nAChR density strongly correlated with the amount of [^99mTc]CPTT–A–E that was accumulated in each region of interest. Further, the in vivo evaluation of biodistribution revealed a higher accumulation of [^99mTc]CPTT–A–E in the thalamus (characterized by the high nAChR density) when compared with that in the cerebellum (characterized by the low nAChR density). Although additional studies will be necessary to improve the uptake of [^99mTc]CPTT–A–E to the brain, [^99mTc]CPTT–A–E met the basic requirements for nAChR imaging.     

Neuronal nicotinic acetylcholine receptors from Drosophila: two different types of alpha subunits coassemble within the same receptor complex

Although neuronal nicotinic acetylcholine receptors from insects have been reconstituted in vitro more than a decade ago, our knowledge about the subunit composition of native receptors as well as their functional properties still remains limited. Immunohistochemical evidence has suggested that two alpha subunits, alpha-like subunit (ALS) and Drosophila alpha2 subunit (Dalpha2), are colocalized in the synaptic neuropil of the Drosophila CNS and therefore may be subunits of the same receptor complex. To gain further understanding of the composition of these nicotinic receptors, we have examined the possibility that a receptor may imbed more than one alpha subunit using immunoprecipitations and electrophysiological investigations. Immunoprecipitation experiments of fly head extracts revealed that ALS-specific antibodies coprecipitate Dalpha2, and vice versa, and thereby suggest that these two alpha subunits must be contained within the same receptor complex, a result that is supported by investigations of reconstituted receptors in Xenopus oocytes. Discrimination between binary (ALS/beta2 or Dalpha2/beta2) and ternary (ALS/Dalpha2/beta2) receptor complexes was made on the basis of their dose-response curve to acetylcholine as well as their sensitivity to alpha-bungarotoxin or dihydro-beta-erythroidine. These data demonstrate that the presence of the two alpha subunits within a single receptor complex confers new receptor properties that cannot be predicted from knowledge of the binary receptor's properties.     

Synthesis of quaternary ammonium salts of 16E-[4-(2-alkylaminoethoxy)-3-methoxybenzylidene]androstene derivatives as skeletal muscle relaxants

Synthesis of eighteen new quaternary ammonium salts of 16E-arylidene androstene derivatives as skeletal muscle relaxants is reported in the present study. The effects of possibly extended interonium distances on muscle relaxant activity are discussed. All the quaternary ammonium steroids produced reduction in the twitch responses, when screened for in vitro neuromuscular blocking activity using isolated chick biventer cervicis muscle preparation. However, the variable interonium distance, which is believed to range from 11 to 17 Å in these quaternary compounds and is associated with the built in flexibility of these structures about the single bonds on the moieties linked to ring D of the steroid skeleton, resulted in varied degrees of muscle relaxant activity. Some of the compounds also inhibited acetylcholinesterase activity in low concentrations so that they would not be directly suitable for use as muscle relaxants.     

Efficiency of open channel blockers of neuronal nicotinic acetylcholine receptors as estimated from the structure/activity relationship

The participation of definite molecular fragments of bis-cationic ammonium compounds in their blocking effect upon neuronal nicotinic acetylcholine receptors (nAChRs) was deduced from the relationship between the blocking efficacy and three-dimensional molecular models of such compounds that have different fragments. The data on the structure and activity of 15 channel blockers were used for this purpose; predicted blocking effects of the substances were calculated. The correlation coefficients between the blocking activity of the compounds and their predicted efficacy were statistically significant (P > 0.95). The results suggest that HCNCCCCCNCH and HCCNCCCCCNCH fragments (atom chains) with the dimensions of 1.42 and 1.36 nm, respectively, provide the most positive contribution, while a HCNCCCCCNCH fragment with the dimension of 1.36 nm corresponds to highly negative contribution to the blocking activity of compounds. Using the data obtained, we identified the optimum compound structures. The mechanism of the blocking effect upon nAChRs is discussed. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 364–370, July–October, 2007.     

Affinities of bispyridinium non-oxime compounds to [H]epibatidine binding sites of Torpedo californica nicotinic acetylcholine receptors depend on linker length

The toxicity of organophosphorus nerve agents or pesticides arises from accumulation of acetylcholine and overstimulation of both muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs) due to inhibition of acetylcholinesterase (AChE). Standard treatment by administration of atropine and oximes, e.g., obidoxime or pralidoxime, focuses on antagonism of mAChRs and reactivation of AChE, whereas nicotinic malfunction is not directly treated. An alternative approach would be to use nAChR active substances to counteract the effects of accumulated acetylcholine. Promising in vitro and in vivo results were obtained with the bispyridinium compounds SAD-128 (1,1′-oxydimethylene bis(4-tert-butylpyridinium) dichloride) and MB327 (1,1′-(propane-1,3-diyl)bis(4-tert-butylpyridinium) di(iodide)), which were partly attributed to their interaction with nAChRs. In this study, a homologous series of unsubstituted and 4-tert-butyl-substituted bispyridinium compounds with different alkane linker lengths was investigated in competition binding experiments using [³H]epibatidine as a reporter ligand. Additionally, the effect of the well-characterised MB327 on the [³H]epibatidine equilibrium dissociation (K_D) constant in different buffers was determined. This study demonstrated that divalent cations increased the affinity of [³H]epibatidine. Since quaternary ammonium molecules are known to inhibit AChE, the obtained affinity constants of the tested bispyridinium compounds were compared with the inhibition of human AChE. In competition experiments, bispyridinium derivatives of longer linker length displaced [³H]epibatidine and inhibited AChE strongly. Bispyridinium compounds with short linkers, at most, have an allosteric interaction with the [³H]epibatidine binding sites and barely inhibited AChE. In dependence on alkane linker length, the bispyridinium compounds seemed to interact at different binding sites. However, the exact binding sites of the bispyridinium compounds responsible for the positive pharmacological effects have still not been identified, making predictive drug design difficult.     

Interaction of α-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7 nAChR-targeting α-conotoxin ImI, blocked α7 and muscle nAChRs without displacing α-bungarotoxin ( Ellison et al. 2003, 2004 ), suggesting binding at a different site. We synthesized α-conotoxin ImII, its ribbon isomer (ImII iso ), 'mutant' ImII(W10Y) and found similar potencies in blocking human α7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [¹²⁵I]-α-bungarotoxin from human α7 nAChRs in the cell line GH₄C₁ (IC₅₀ 17 and 23 μM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC₅₀ 2.0–9.0 μM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized α-bungarotoxin ( K _d and IC₅₀ 2.5–8.2 μM). On Torpedo nAChR, α-conotoxin [¹²⁵I]-ImII(W10Y) revealed specific binding ( K _d 1.5–6.1 μM) and could be displaced by α-conotoxin ImII, ImII iso and ImII(W10Y) with IC₅₀ 2.7, 2.2 and 3.1 μM, respectively. As α-cobratoxin and α-conotoxin ImI displaced [¹²⁵I]-ImII(W10Y) only at higher concentrations (IC₅₀≥ 90 μM), our results indicate that α-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.