Synthesis and activity of substituted carbamates as potentiators of the alpha4beta2 nicotinic acetylcholine receptor

The synthesis and structure-activity relationship of a series of carbamate potentiators of alpha4beta2 nAChR is reported herein. These compounds were highly selective for alpha4beta2 over other nAChR subtypes. In addition, compounds increased the response of alpha4beta2 nAChRs to acetylcholine, as measured with patch-clamp electrophysiology.     

Discovery, synthesis, and structure activity of a highly selective alpha7 nicotinic acetylcholine receptor antagonist

Nicotinic acetylcholine receptors (nAChRs) that contain an alpha7 subunit are widely distributed in neuronal and nonneuronal tissue. These receptors are implicated in the release of neurotransmitters such as glutamate and in functions ranging from thought processing to inflammation. Currently available ligands for alpha7 nAChRs have substantial affinity for one or more other nAChR subtypes, including those with an alpha1, alpha3, alpha6, and/or alpha9 subunit. An alpha-conotoxin gene was cloned from Conus arenatus. Predicted peptides were synthesized and found to potently block alpha3-, alpha6-, and alpha7-containing nAChRs. Structure-activity information regarding conotoxins from distantly related Conus species was employed to modify the C. arenatus derived toxin into a novel, highly selective alpha7 nAChR antagonist. This ligand, alpha-CtxArIB[V11L,V16D], has low nanomolar affinity for rat alpha7 homomers expressed in Xenopus laevis oocytes, and antagonism is slowly reversible. Kinetic analysis provided insight into the mechanism of antagonism. alpha-CtxArIB interacts with five ligand binding sites per alpha7 receptor, and occupation of a single site is sufficient to block function. The peptide was also shown to be highly selective in competition binding assays in rat brain membranes. alpha-CtxArIB[V11L,V16D] is the most selective ligand yet reported for alpha7 nAChRs.     

Discovery of substituted 4-aminoquinazolines as selective Toll-like receptor 4 ligands

The Toll-like receptors (TLRs) are critical components of the innate immune system that regulate immune recognition in part through NF-κB activation. A human cell-based high throughput screen (HTS) revealed substituted 4-aminoquinazolines to be small molecular weight activators of NF-κB. The most potent hit compound predominantly stimulated through the human TLR4/MD2 complex, and had less activity with the mouse TLR4/MD2. There was no activity with other TLRs and the TLR4 activation was MD-2 dependent and CD14 independent. Synthetic modifications of the quinazoline scaffold at the 2 and 4 positions revealed trends in structure–activity relationships with respect to TLR dependent production of the NF-κB associated cytokine IL-8 in human peripheral blood mononuclear cells, as well as IL-6 in mouse antigen presenting cells. Furthermore, the hit compound in this series also activated the interferon signaling pathway resulting in type I interferon production. Substitution at the O-phenyl moiety with groups such as bromine, chlorine and methyl resulted in enhanced immunological activity. Computational studies indicated that the 4-aminoquinazoline compounds bind primarily to human MD-2 in the TLR4/MD-2 complex. These small molecules, which preferentially stimulate human rather than mouse innate immune cells, may be useful as adjuvants or immunotherapeutic agents.     

5-Azidoepibatidine: an exceptionally potent photoaffinity ligand for neuronal alpha 4 beta 2 and alpha 7 nicotinic acetylcholine receptors

Racemic 5-azidoepibatidine [(+/-)-1] was synthesized via 5-aminoepibatidine as a candidate photoaffinity ligand with exceptionally high affinity at the mammalian neuronal nicotinic receptors (K(i) values of 0.027 nM for alpha 4 beta 2 and 9.7 nM for alpha 7) and excellent photoreactivity.     

Biaryl piperidines as potent and selective delta opioid receptor ligands

The design and synthesis of novel opiates are reported. Based on the message-address principle a novel class of 4,4- and 3,3-biaryl piperidines was designed and synthesized. Biological evaluation confirmed that these compounds exhibit high affinity and selectivity for the delta opioid receptor. Key structure–activity relationships that influence affinity, selectivity, functional activity and clearance are reported.     

Discovery of N-sulfonyl-7-azaindoline derivatives as potent,orally available and selective M4 muscarinic acetylcholine receptor agonists

We designed and synthesized novel N-sulfonyl-7-azaindoline derivatives as selective M₄ muscarinic acetylcholine receptor agonists. Modification of the N-carbethoxy piperidine moiety of compound 2, an M₄ muscarinic acetylcholine receptor (mAChR)-preferring agonist, led to compound 1, a selective M₄ mAChR agonist. Compound 1 showed a highly selective M₄ mAChR agonistic activity with weak hERG inhibition in vitro. A pharmacokinetic study of compound 1 in vivo revealed good bioavailability and brain penetration in rats. Compound 1 reversed methamphetamine-induced locomotor hyperactivity in rats (1–10 mg/kg, po).     

Activity of cytisine and its brominated isosteres on recombinant human alpha7, alpha4beta2 and alpha4beta4 nicotinic acetylcholine receptors

Effects of cytisine (cy), 3-bromocytisine (3-Br-cy), 5-bromocytisine (5-Br-cy) and 3,5-dibromocytisine (3,5-diBr-cy) on human (h) alpha7-, alpha4beta2- and alpha4beta4 nicotinic acetylcholine (nACh) receptors, expressed in Xenopus oocytes and cell lines, have been investigated. Cy and its bromo-isosteres fully inhibited binding of both [alpha-(125)I]bungarotoxin ([alpha-(125)I]BgTx) to halpha7- and [(3)H]cy to halpha4beta2- or halpha4beta4-nACh receptors. 3-Br-cy was the most potent inhibitor of both [alpha-(125)I]BgTx and [(3)H]cy binding. Cy was less potent than 3-Br-cy, but 5-Br-cy and 3,5-diBr-cy were the least potent inhibitors. Cy and 3-Br-cy were potent full agonists at halpha7-nACh receptors but behaved as partial agonists at halpha4beta2- and halpha4beta4-nACh receptors. 5-Br-cy and 3,5-diBr-cy had low potency and were partial agonists at halpha7- and halpha4beta4-nACh receptors, but they elicited no responses on halpha4beta2-nACh receptors. Cy and 3-Br-cy produced dual dose-response curves (DRC) at both halpha4beta2- and halpha4beta4-nACh receptors, but ACh produced dual DRC only at halpha4beta2-nACh receptors. Low concentrations of cy, 3-Br-cy and 5-Br-cy enhanced ACh responses of oocytes expressing halpha4beta2-nACh receptors, but at high concentrations they inhibited the responses. In contrast, 3,5-diBr-cy only inhibited, in a competitive manner, ACh responses of halpha4beta2-nACh receptors. It is concluded that bromination of the pyridone ring of cy produces marked changes in effects of cy that are manifest as nACh receptor subtype-specific differences in binding affinities and in functional potencies and efficacies.     

Alpha-conotoxin OmIA is a potent ligand for the acetylcholine-binding protein as well as alpha3beta2 and alpha7 nicotinic acetylcholine receptors

The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the alpha-subunit of nicotinic acetylcholine receptors and in particular the homomeric alpha7 nicotinic receptor. We report the isolation and characterization of an alpha-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the alpha7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the alpha3beta2 nAChR indicating that alpha-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of alpha3beta2 nAChRs. alpha-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17-amino-acid, two-disulfide bridge peptide. The ligand is the first alpha-conotoxin with higher affinity for the closely related receptor subtypes, alpha3beta2 versus alpha6beta2, and selectively blocks these two subtypes when compared with alpha2beta2, alpha4beta2, and alpha1beta1deltaepsilon nAChRs.     

Benzoxazole piperidines as selective and potent somatostatin receptor subtype 5 antagonists

SAR studies of a recently described SST5R selective benzoxazole piperidine lead series are described with particular focus on the substitution pattern on the benzyl and benzoxazole side-chains. Introduction of a second meta substituent at the benzyl unit significantly lowers residual hH1 activity and insertion of substituents onto the benzoxazole periphery entirely removes remaining h5-HT_2B activity. Compounds with single digit nM activity, functional antagonism and favorable physicochemical properties endowed with a good pharmacokinetic profile in rats are described which should become valuable tools for exploring the pharmacological role of the SST5 receptor in vivo.     

Extracellular domain nicotinic acetylcholine receptors formed by alpha4 and beta2 subunits

Models of the extracellular ligand-binding domain of nicotinic acetylcholine receptors (nAChRs), which are pentameric integral membrane proteins, are attractive for structural studies because they potentially are water-soluble and better candidates for x-ray crystallography and because their smaller size is more amenable for NMR spectroscopy. The complete N-terminal extracellular domain is a promising foundation for such models, based on previous studies of alpha7 and muscle-type subunits. Specific design requirements leading to high structural fidelity between extracellular domain nAChRs and full-length nAChRs, however, are not well understood. To study these requirements in heteromeric nAChRs, the extracellular domains of alpha4 and beta2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with alpha4beta2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain alpha4beta2 nAChRs formed from subunits with M1 were nearly identical to affinities of alpha4beta2 nAChRs when measured with [3H]epibatidine, cytisine, nicotine, and acetylcholine. Velocity sedimentation suggested that these extracellular domain nAChRs predominantly formed pentamers. The yield of these extracellular domain nAChRs was about half the yield of alpha4beta2 nAChRs. In contrast, [3H]epibatidine binding was not detected from the extracellular domain alpha4 and beta2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both alpha4 and beta2 play an important role for efficient expression of extracellular domain alpha4beta2 nAChRs that are high fidelity structural models of full-length alpha4beta2 nAChRs.     

Computational evidence for the ligand selectivity to the alpha4beta2 and alpha3beta4 nicotinic acetylcholine receptors

The homology models of the alpha4beta2 and alpha3beta4 nicotinic acetylcholine receptors (nAChRs) suggest that the two nAChR subtypes are different in their ligand-binding pockets due to the non-conserved residues in the beta-subunits. The docking of nicotine, epibatidine, A-84543, and the two analogs of A-84543 ligands 1 and 2 to the homology models of alpha4beta2 and alpha3beta4 is presented. It is found that the protonated amino groups of these ligands bind to the alpha-subunits, whereas the remaining parts of the ligands bind to the beta-subunits. The two non-conserved amino acids Lys77 and Phe117 in the beta2-subunit corresponding to Ile77 and Gln117 in the beta4-subunit are identified to be the key players determining the binding modes of the ligands. We demonstrate how the increase in the number of the atoms connecting the pyrrolidine and pyridine rings in A-84543, 1, and 2, and an introduction of the alkynyl substituent in the pyridine ring affect the binding and shift the selectivity of these ligands toward the beta2-containing receptors. Further improvement in affinity and selectivity in this and other series of the ligands may be achieved by designing molecules that would specifically target the non-conserved regions in nAChRs.     

Additional acetylcholine (ACh) binding site at alpha4/alpha4 interface of (alpha4beta2)2alpha4 nicotinic receptor influences agonist sensitivity

Nicotinic acetylcholine receptor (nAChR) α4 and β2 subunits assemble in two alternate stoichiometries to produce (α4β2)(2)α4 and (α4β2)(2)β2, which display different agonist sensitivities. Functionally relevant agonist binding sites are thought to be located at α4(+)/β2(-) subunit interfaces, but because these interfaces are present in both receptor isoforms, it is unlikely that they account for differences in agonist sensitivities. In contrast, incorporation of either α4 or β2 as auxiliary subunits produces isoform-specific α4(+)/α4(-) or β2(+)/β2(-) interfaces. Using fully concatenated (α4β2)(2)α4 nAChRs in conjunction with structural modeling, chimeric receptors, and functional mutagenesis, we have identified an additional site at the α4(+)/α4(-) interface that accounts for isoform-specific agonist sensitivity of the (α4β2)(2)α4 nAChR. The additional site resides in a region that also contains a potentiating Zn(2+) site but is engaged by agonists to contribute to receptor activation. By engineering α4 subunits to provide a free cysteine in loop C at the α4(+)α4(-) interface, we demonstrated that the acetylcholine responses of the mutated receptors are attenuated or enhanced, respectively, following treatment with the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate or aminoethyl methanethiosulfonate. The findings suggest that agonist occupation of the site at the α4(+)/(α4(-) interface leads to channel gating through a coupling mechanism involving loop C. Overall, we propose that the additional agonist site at the α4(+)/α4(-) interface, when occupied by agonist, contributes to receptor activation and that this additional contribution underlies the agonist sensitivity signature of (α4β2)(2)α4 nAChRs.     

Epibatidine analogues as selective ligands for the alpha(x)beta2-containing subtypes of nicotinic acetylcholine receptors

A series of epibatidine analogues was synthesized and characterized in vitro. These compounds are high affinity ligands for the nicotinic acetylcholine receptors (nAChR). They display binding selectivity for the alpha(x)beta2 subtypes of nAChRs over the alpha(x)beta4 subtypes, and especially for the alpha4beta2 and alpha2beta2 subtypes. Furthermore, most of these new nicotinic compounds display little, if any, agonist activities at alpha3beta4 nAChR. As a result they might become lead structures for the design and synthesis of highly selective ligands for nAChR subtypes containing the beta2 subunit.     

3,5-Bicyclic aryl piperidines: a novel class of alpha4beta2 neuronal nicotinic receptor partial agonists for smoking cessation

3,5-Bicyclic aryl piperidines are a new class of high-affinity alpha4beta2 nicotinic receptor agents. We have sought nicotinic receptor partial agonists of the alpha4beta2 nicotinic acetylcholine receptor for smoking cessation, and a number of compounds fulfill potency, selectivity, and efficacy requirements in vitro. In vivo, selected agents demonstrate potent partial agonist efficacy on the mesolimbic dopamine system, a key measure of therapeutic potential for smoking cessation.     

Discovery and optimization of novel purines as potent and selective CB2 agonists

A focused screening strategy identified thienopyrimidine 1 as a hCB2 cannabinoid receptor agonist with moderate selectivity over the hCB1 receptor. This initial hit suffered from poor in vitro metabolic stability and high in vivo clearance. Structure-activity relationships describe the optimization and modification to a less lipophilic purine core. Examples from this novel series were found to be highly potent and fully efficacious agonists of the human CB2 receptor with excellent selectivity against CB1. Compound 10 possesses good biopharmaceutical properties, is highly water soluble and demonstrates robust oral activity in rodent models of joint pain.     

Discovery of substituted 2,4,4-triarylimidazoline derivatives as potent and selective neuropeptide Y Y5 receptor antagonists

Novel imidazoline derivatives were discovered to be potent neuropeptide Y Y5 receptor antagonists. High-throughput screening of Merck sample collections against the human Y5 receptor resulted in the identification of 2,4,4-triphenylimidazoline (1), which had an IC₅₀ of 54 nM. Subsequent optimization led to the identification of several potent derivatives.     

Novel substituted 4-aminomethylpiperidines as potent and selective human beta3-agonists. Part 2: arylethanolaminomethylpiperidines

The synthesis and SAR of a series of beta3 adrenoreceptor agonists based on a novel template derived from 4-aminomethylpiperidine coupled with a common pharmacophore, arylethylamine, is described. This combination led to the identification of human beta3 adrenoreceptor agonists with in vivo activity in a transgenic mouse model.     

Discovery and evaluation of potent, tyrosine-based alpha4beta1 integrin antagonists

Using disulphide cysteine-based inhibitors as lead structures, this communication describes our strategy for identifying more stable, potent antagonists of the alpha4beta1 integrin. These studies ultimately discovered potent, low molecular weight inhibitors based on D-thioproline-L-tyrosine.     

Discovery and evaluation of potent, cysteine-based alpha4beta1 integrin antagonists

Acyclic, disulphide derivatives of cysteine have been identified as moderately potent antagonists of alpha4beta1-mediated leukocyte cell adhesion to VCAM. This communication describes how they were discovered from a simple L-cystine derivative and using the structure-activity data of C*DThioPC* related cyclic peptides.     

Two neuronal nicotinic acetylcholine receptors, alpha4beta4 and alpha7, show differential agonist binding modes

Nicotinic acetylcholine receptors (nAChRs) are pentameric, neurotransmitter-gated ion channels responsible for rapid excitatory neurotransmission in the central and peripheral nervous systems, resulting in skeletal muscle tone and various cognitive effects in the brain. These complex proteins are activated by the endogenous neurotransmitter ACh as well as by nicotine and structurally related agonists. Activation and modulation of nAChRs has been implicated in the pathology of multiple neurological disorders, and as such, these proteins are established therapeutic targets. Here we use unnatural amino acid mutagenesis to examine the ligand binding mechanisms of two homologous neuronal nAChRs: the α4β4 and α7 receptors. Despite sequence identity among the residues that form the core of the agonist-binding site, we find that the α4β4 and α7 nAChRs employ different agonist-receptor binding interactions in this region. The α4β4 receptor utilizes a strong cation-π interaction to a conserved tryptophan (TrpB) of the receptor for both ACh and nicotine, and nicotine participates in a strong hydrogen bond with a backbone carbonyl contributed by TrpB. Interestingly, we find that the α7 receptor also employs a cation-π interaction for ligand recognition, but the site has moved to a different aromatic amino acid of the agonist-binding site depending on the agonist. ACh participates in a cation-π interaction with TyrA, whereas epibatidine participates in a cation-π interaction with TyrC2.