Dopamine D4-like binding sites labeled by [3H]nemonapride include substantial serotonin 5-HT2A receptors in primate cerebral cortex

Dopamine D4-like binding sites are abundant in human cerebral cortex as detected by [3H]nemonapride. The extremely low density of D4 mRNA in human cerebral cortex is inconsistent with the high amount of D4-like binding sites. To investigate the nature of the D4-like receptors, [3H]nemonapride binding sites in the nonhuman primate cerebral cortex were characterized. Although [3H]nemonapride binding sites were D4-like, displaceable by clozapine but not raclopride, [3H]nemonapride binding was not displaced by selective D4 antagonists but was displaced by the selective 5-HT2A antagonist MDL100907. Using [3H]ketanserin as a 5-HT2A ligand, nemonapride showed high affinity for monkey (Ki = 10.4 nM) and cloned human (Ki = 9.4 nM) 5-HT2A receptors, while its affinity for rat receptors was lower (Ki = 140 nM). The present study demonstrates that cerebral cortical D4-like binding sites labeled by [3H]nemonapride in nonhuman primates consist of a very small portion of D4, but a substantial portion of 5-HT2A receptors. The unexpectedly high affinity of nemonapride for primate 5-HT2A receptor suggests reconsidering previous data from other studies using [3H]nemonapride, particularly those on D4-like receptors.     

Conformationally-flexible benzamide analogues as dopamine D3 and sigma 2 receptor ligands

A series of conformationally-flexible analogues was prepared and their affinities for D2-like dopamine (D2, D3 and D4) were determined using in vitro radioligand binding assays. The results of this structure-activity relationship study identified one compound, 15, that bound with high affinity (K(i) value=2nM) and moderate selectivity (30-fold) for D3 compared to D2 receptors. In addition, this series of compounds were also tested for affinity at sigma1 and sigma2 receptors. We evaluated the affinity of these dopaminergic compounds at sigma receptors because (a) several antipsychotic drugs, which are high affinity antagonists at dopamine D2-like receptors, also bind to sigma receptors and (b) sigma receptors are expressed ubiquitously and at high levels (picomoles per mg proteins). It was observed that a number of analogues displayed high affinity and excellent selectivity for sigma2 versus sigma1 receptors. Consequently, these novel compounds may be useful for characterizing the functional role of sigma2 receptors and for imaging the sigma2 receptor status of tumors in vivo with PET.     

Design, synthesis, and evaluation of hexahydrobenz[f]isoquinolines as a novel class of dopamine 3 receptor ligands

We previously identified hexahydrobenz[f]isoquinoline (4a) as a new class of dopamine 3 receptor (D(3)) ligand. Herein, we described the design, synthesis, and preliminary structure-activity relationships of new analogues of 4a as a novel class of D(3) ligands. Among these new analogues, compound 4 h is a potent D(3) ligand (K(i)=6.1 nM) and has a selectivity of 133-fold between D(3)- and D(2)-like receptors, and of 163-fold between D(3)- and D(1)-like receptors, respectively. Thus, compound 4 h represents a promising new lead compound for further design and optimization toward achieving highly potent and selective D(3) ligands.     

Design of novel hexahydropyrazinoquinolines as potent and selective dopamine D3 receptor ligands with improved solubility

We have recently reported hexahydropyrazinoquinolines as a new class of dopamine 3 (D(3)) receptor ligands with high-affinity to the D(3) receptor and excellent selectivity over the closely related D(1)-like and D(2)-like receptors. However, our previously reported most potent and selective D(3) ligands have poor aqueous solubility, which greatly hinders our in vivo studies aimed at evaluation of their therapeutic potential in animal models. In this study, we wish to report the design, synthesis, and evaluation of a series of new hexahydropyrazinoquinolines as D(3) ligands with improved solubility. Among them, compound 4g has a K(i) value of 9.7 nM for the D(3) receptor and displays a selectivity of >5000 and 466 times over the D(1)-like and D(2)-like receptors, respectively. Importantly, the hydrochloride salt form of compound 4g has a good aqueous solubility (>50 mg/mL) and represents a promising D(3) ligand for further in vivo evaluations of its therapeutic potential for the treatment of drug abuse, restless legs syndrome, schizophrenia, Parkinson's disease, and depression.     

Synthesis of 2-(5-bromo-2,3-dimethoxyphenyl)-5-(aminomethyl)-1H-pyrrole analogues and their binding affinities for dopamine D2, D3, and D4 receptors

A series of 2-(5-bromo-2,3-dimethoxyphenyl)-5-(aminomethyl)-1H-pyrrole analogues was prepared and their affinity for dopamine D(2), D(3), and D(4) receptors was measured using in vitro binding assays. The results of receptor binding studies indicated that the incorporation of a pyrrole moiety between the phenyl ring and the basic nitrogen resulted in a significant increase in the selectivity for dopamine D(3) receptors. The most selective compound in this series is 2-(5-bromo-2,3-dimethoxyphenyl)-5-(2-(3-pyridal)piperidinyl)methyl-1H-pyrrole (6p), which has a D(3) receptor affinity of 4.3 nM, a 20-fold selectivity for D(3) versus D(2) receptors, and a 300-fold selectivity for D(3) versus D(4) receptors. This compound is predicted to be a useful ligand for studying the functional role of dopamine D(3) receptors in vivo.     

Discovery of a muscarinic M3 receptor antagonist with high selectivity for M3 over M2 receptors among 2-[(1S,3S)-3-sulfonylaminocyclopentyl]phenylacetamide derivatives

In the course of developing a metabolically stable M3 receptor antagonist from the prototype antagonist, J-104129 (1), introduction of certain substituents into the cyclopentane ring of 1 was found to be effective not only in improving metabolic stability but also in greatly enhancing the subtype selectivity. Among the cyclopentane analogues, sulfonamide derivatives (10f) and (10g) displayed 160- and 310-fold selectivity for M3 over M2 receptors, and both were significantly more selective than the prototype antagonist (120-fold). Subsequent derivatization of the sulfonamide series led to the highly selective M3 receptor antagonists (10h, 10i and 10j) with >490-fold selectivity for M3 over M2 receptors. Among them, p-nitrophenylsulfonamide (J-107320, 10h) exhibited 1100-fold selectivity for M3 receptors (Ki = 2.5 nM) over M2 receptors (Ki = 2800 nM) in the human muscarinic receptor binding assay using [3H]-NMS as a radio ligand.     

Benzimidazole derivatives. Part 1: Synthesis and structure-activity relationships of new benzimidazole-4-carboxamides and carboxylates as potent and selective 5-HT4 receptor antagonists

New benzimidazole-4-carboxamides 1-16 and -carboxylates 17-26 were synthesized and evaluated for binding affinity at serotonergic 5-HT4 and 5-HT3 receptors in the CNS. Most of the synthesized compounds exhibited moderate-to-very high affinity (in many cases subnanomolar) for the 5-HT4 binding site and no significant affinity for the 5-HT3 receptor. SAR observations and structural analyses (molecular modeling, INSIGHT II) indicated that the presence of a voluminous substituent in the basic nitrogen atom of the amino moiety and a distance of ca. 8.0 A from this nitrogen to the aromatic ring are of great importance for high affinity and selectivity for 5-HT4 receptors. These results confirm our recently proposed model for recognition by the 5-HT4 binding site. Amides 12-15 and esters 24 and 25 bound at central 5-HT4 sites with very high affinity (Ki = 0.11-2.9 nM) and excellent selectivity over serotonin 5-HT3, 5-HT2A, and 5-HT1A receptors (Ki > 1000-10,000 nM). Analogues 12 (Ki(5-HT4) = 0.32 nM), 13 (Ki(5-HT4) = 0.11 nM), 14 (Ki(5-HT4) = 0.29 nM) and 15 (Ki(5-HT4) = 0.54 nM) were pharmacologically characterized as selective 5-HT4 antagonists in the isolated guinea pig ileum (pA2 = 7.6, 7.9, 8.2 and 7.9, respectively), with a potency comparable to the 5-HT4 receptor antagonist RS 39604 (pA2 = 8.2). The benzimidazole-4-carboxylic acid derivatives described in this paper represent a novel class of potent and selective 5-HT4 receptor antagonists. In particular, compounds 12-15 could be interesting pharmacological tools for the understanding of the role of 5-HT4 receptors.     

Design and synthesis of new benzimidazole-arylpiperazine derivatives acting as mixed 5-HT1A/5-HT3 ligands

A series of new benzimidazole-arylpiperazine derivatives III were designed, synthesized and evaluated for binding affinity at serotoninergic 5-HT(1A) and 5-HT(3) receptors. Compound IIIc was identified as a novel mixed 5-HT(1A)/5-HT(3) ligand with high affinity for both serotonin receptors and excellent selectivity over alpha(1)-adrenergic and dopamine D(2) receptors. This compound was characterized as a partial agonist at 5-HT(1A)Rs and a 5-HT(3)R antagonist, and was effective in preventing the cognitive deficits induced by muscarinic receptor blockade in a passive avoidance learning test.     

Design and synthesis of 2-[4-[4-(m-(ethylsulfonamido)-phenyl) piperazin-1-yl]butyl]-1,3-dioxoperhydropyrrolo[1,2-c]imidazole (EF-7412) using neural networks. A selective derivative with mixed 5-HT1A/D2 antagonist properties.

A test series of 32 phenylpiperazines III with affinity for 5-HT1A and alpha1 receptors was subjected to QSAR analysis using artificial neural networks (ANNs), in order to get insight into the structural requirements that are responsible for 5-HT1A/alpha1 selectivity. Good models and predictive power were obtained for 5-HT1A and alpha1 receptors. A comparison of these models gives information for the design of the new ligand EF-7412 (5-HT1A:Ki(nM)= 27; alpha1: Ki(nM) > 1000). This derivative displayed affinity for dopamine D2 receptor (Ki = 22 nM) and is selective for all other receptor examined (5-HT2A, 5-HT3, 5-HT4 and Bz). EF-7412 acts an antagonist in vivo in pre- and postsynaptic 5-HT1A receptor sites and as an antagonist in dopamine D2 receptor.     

Selectivity of BAFF/BLyS and APRIL for binding to the TNF family receptors BAFFR/BR3 and BCMA

BAFF (B cell activating factor of the TNF family, also known as BlyS and TALL-1), a TNF family cytokine critical for the development and function of B cells, has been reported to bind to three receptors, BCMA (B cell maturation protein), TACI (transmembrane activator and CAML [calcium-modulator and cyclophilin ligand] interactor), and BAFFR (BAFF receptor), but with widely conflicting values for the affinity and selectivity of binding. BCMA and TACI additionally bind APRIL (a proliferation-inducing ligand), the TNF family ligand most homologous to BAFF. Using soluble, monomeric forms of the receptors, we demonstrate that BAFFR binds BAFF with K(D) approximately 16 nM, while BCMA binds with K(D) approximately 1.6 microM, indicating a approximately 100-fold selectivity for binding to BAFFR over BCMA. APRIL shows the opposite selectivity, binding to BCMA with K(D) approximately 16 nM while showing no detectable affinity for BAFFR (K(D) > 3 microM). The binding of BAFF or APRIL to these receptors is highly sensitive to assay-dependent avidity effects, likely explaining the widely ranging affinity values reported in the literature. Binding of BAFF to BCMA-Fc, a bivalent fusion protein consisting of the extracellular domain of BCMA fused to the hinge and CH1 and CH2 domains of human IgG1, in solution or coated onto an ELISA plate gave apparent binding affinities of approximately 0.63 and approximately 0.15 nM, respectively, compared to values of K(D(app)) 相似文献   

Design and synthesis of a piperazinylalkylisoxazole library for subtype selective dopamine receptor ligands

A piperazinylbutylisoxazole libary was designed, synthesized and screened for the binding affinities to dopamine D2, D3, and D4 receptors. Several ligands were identified to possess high binding affinity and selectivity for the D3 and D4 receptors over the D2 receptor. Compounds 6s and 6t showed K(i) values of 2.6 nM and 3.9 nM for the D3 receptor with 46- and 50-fold selectivity over the D2 receptor, respectively.     

Synthesis and binding affinities of methylvesamicol analogs for the acetylcholine transporter and sigma receptor

We synthesized methylvesamicol analogs 13-16 and investigated the binding characteristics of 2-[4-phenylpiperidino]cyclohexanol (vesamicol) and methylvesamicol analogs 13-16, with a methyl group introduced into the 4-phenylpiperidine moiety, to sigma receptors (sigma-1, sigma-2) and to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In competitive inhibition studies, (-)-o-methylvesamicol [(-)-OMV] (13) (Ki=6.7 nM), as well as (-)-vesamicol (Ki=4.4 nM), had a high affinity for VAChT. (+)-p-Methylvesamicol [(+)-PMV] (16) (Ki=3.0 nM), as well as SA4503 (Ki=4.4 nM), reported as a sigma-1 mapping agent for positron emission tomography (PET), had a high affinity for the sigma-1 receptor. The binding affinity of (+)-PMV (16) for the sigma-1 receptor (Ki=3.0 nM) was about 13 times higher than that for the sigma-2 (sigma-2) receptor (Ki=40.7 nM). (+)-PMV (16) (Ki=199 nM) had a much lower affinity for VAChT than SA4503 (Ki=50.2 nM) and haloperidol (Ki=41.4 nM). These results showed that the binding characteristics of (-)-OMV (13) to VAChT were similar to those of (-)-vesamicol and that (+)-PMV (16) bound to the sigma-1 receptor with high affinity. In conclusion, (-)-OMV (13) and (+)-PMV (16), which had a suitable structure, with a methyl group for labeling with 11C, may become not only a new VAChT ligand and a new type of sigma receptor ligand, respectively, but may also become a new target compound of VAChT and the sigma-1 receptor radioligand for PET, respectively.     

Opioid and melanocortin receptors: do they have overlapping pharmacophores?

We have identified compound 1 as a novel ligand for opioid and melanocortin (MC) receptors, which is derived from the overlapping of a well known structure for the delta opioid receptor, 2,6-dimethyltyrosine (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), and a small molecule for the MC receptor, Tic-DPhe(p-Cl)-piperidin-4-yl-N-phenyl-propionamide. Ligand 1 showed that there is an overlapping pharmacophore between opioid and MC receptors through the Tic residue. The ligand displayed high biological activities at the delta opioid receptor (Ki = 0.38 nM in binding assay, EC(50) = 0.48 nM in GTP-gamma-S binding assay, IC(50) = 74 nM in MVD) as an agonist instead of an antagonist and showed selective binding affinity (IC(50) = 2.3 muM) at the MC-3 receptor rather than at the MC-5 receptor. A study of the structure-activity relationships demonstrated that the residues in positions 2, 3, and the C-terminus act as a pharmacophore for the MC receptors, and the residues in positions 1 and 2 act as a pharmacophore for the opioid receptors. Thus, this structural construct can be used to prepare chimeric structures with adjacent or overlapping pharmacophores for opioid and MC receptors.     

Design, synthesis, and pharmacological evaluation of novel tetrahydroprotoberberine derivatives: selective inhibitors of dopamine D₁ receptor

A series of new tetrahydroprotoberberine (THPB) derivatives were designed, synthesized, and tested for their binding affinity towards dopamine (D(1) and D(2)) and serotonin (5-HT(1A) and 5-HT(2A)) receptors. Many of the THPB compounds exhibited high binding affinity and activity at the dopamine D(1) receptor, as well as high selectivity for the D(1) receptor over the D(2), 5-HT(1A), and 5-HT(2A) receptors. Among these, compound 19c exhibited a promising D(1) receptor binding affinity (K(i)=2.53nM) and remarkable selectivity versus D(2)R (inhibition=81.87%), 5-HT(1A)R (inhibition=61.70%), and 5-HT(2A)R (inhibition=24.96%). Compared with l-(S)-stepholidine (l-SPD) (D(1)K(i)=6.23nM, D(2)K(i)=56.17nM), compound 19c showed better binding affinity for the D(1) receptor (2.5-fold higher) and excellent D(2)/D(1) selectivity. Functional assays found compounds 18j, 18k, and 19c are pure D(1) receptor antagonists. These results indicate that removing the C10 hydroxy group and introducing a methoxy group at C11 of the pharmacophore of l-SPD can reverse the function of THPB compounds at the D(1) receptor. These results are in accord with molecular docking studies.     

Discovery of a novel CCR3 selective antagonist

In searching for a novel CCR3 receptor antagonist, we designed a library that included a variety of carboxamide derivatives based on the structure of our potent antagonists for human CCR1 and CCR3 receptors, and screened the new compounds for inhibitory activity against 125I-Eotaxin binding to human CCR3 receptors expressed in CHO cells. Among them, two 2-(benzothiazolethio)acetamide derivatives (1a and 2a) showed binding affinities with IC50 values of 750 and 1000 nM, respectively, for human CCR3 receptors. These compounds (1a and 2a) also possessed weak binding affinities for human CCR1 receptors. We selected la as a lead compound for derivatization to improve in vitro potency and selectivity for CCR3 over CCRI receptors. Derivatization of la by incorporating substituents into each benzene ring of the benzothiazole and piperidine side chain resulted in the discovery of a compound (1b) exhibiting 820-fold selectivity for CCR3 receptors (IC50 = 2.3 nM) over CCR1 receptors (IC50 = 1900 nM). This compound (1b) also showed potent functional antagonist activity for inhibiting Eotaxin (IC50 = 27 nM)- or RANTES (IC50 = 13 nM)-induced Ca2+ increases in eosinophils.     

Benzimidazole derivatives. Part 5: design and synthesis of new benzimidazole-arylpiperazine derivatives acting as mixed 5-HT1A/5-HT3 ligands

A series of new mixed benzimidazole-arylpiperazine derivatives were designed by incorporating in general structure III the pharmacophoric elements of 5-HT(1A) and 5-HT(3) receptors. Compounds 1-11 were synthesized and evaluated for binding affinity at both serotoninergic receptors, all of them exhibiting high 5-HT(3)R affinity (K(i)=10-62nM), and derivatives with an o-alkoxy group in the arylpiperazine ring showing nanomolar affinity for the 5-HT(1A)R (K(i)=18-150nM). Additionally, all the synthesized compounds were selective over alpha(1)-adrenergic and dopamine D(2) receptors (K(i)>1000-10,000nM). Compound 3 was selected for further pharmacological characterization due to its interesting binding profile as mixed 5-HT(1A)/5-HT(3) ligand with high affinity for both receptors (5-HT(1A): K(i)=18.0nM, 5-HT(3): K(i)=27.2nM). In vitro and in vivo findings suggest that this compound acts as a partial agonist at 5-HT(1A)Rs and as a 5-HT(3)R antagonist. This novel mixed 5-HT(1A)/5-HT(3) ligand was also effective in preventing the cognitive deficits induced by muscarinic receptor blockade in a passive avoidance learning test, suggesting a potential interest in the treatment of cognitive dysfunction.     

Preparation of an affinity chromatography matrix for the selective purification of the dopamine D2 receptor from bovine striatal membranes

A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.     

Synthesis, receptor binding and functional studies of mesoridazine stereoisomers

The four stereoisomers of mesoridazine were synthesized and evaluated in D2, 5-HT1A, 5-HT2A, 5-HT2C, D1, and D3 receptor binding and functional assays. Two isomers demonstrated potent D2 receptor binding (Ki < 3 nM) and functional antagonism (IC50 < or = 10 nM) activities. These two isomers also showed moderate affinity for the 5-HT2A and D3 receptors. A third isomer was devoid of significant D2 receptor binding, but did have moderate affinity for the 5-HT2A and D3 receptors. The fourth isomer demonstrated poor affinity for all the receptors tested. Most significantly, the stereochemistry of the sulfoxide moiety played a dominant role in the observed structure-activity relationship (SAR).     

The binding of SCH 39166 and SCH 23390 to 5-HT1C receptors in porcine choroid plexus

SCH 39166 is a novel benzonaphthazepine, which has been characterized as a potent and selective D1 antagonist. Recently, its D1 selective benzazepine predecessor, SCH 23390, has been shown to bind to 5-HT1C binding sites in the choroid plexus. Therefore, the present studies were undertaken to determine if SCH 39166 has any measurable affinity for 5-HT1C binding sites. Our results indicate that SCH 39166 exhibited poor affinity for the 5-HT1C receptor, with a Ki of 1327 nM. In contrast, SCH 23390 inhibited [3H]-mesulergine binding to 5-HT1C receptors with a Ki of 30 nM. The non-selective 5-HT antagonist, methysergide, inhibited binding with a Ki of 2.4 nM. Finally, studies with the stereoisomers of SCH 39166 and SCH 23390 demonstrated that stereoselectivity at the 5-HT1C site is significantly less than for the D1 site.     

Characterization of a new class of selective nonsteroidal progesterone receptor agonists

The identification of a new series of selective nonsteroidal progesterone receptor (PR) agonists is reported. Using a high-throughput screening assay based on the measurement of transactivation of a mouse mammary tumor virus promoter-driven luciferase reporter (MMTV-Luc) in human breast cancer T47D cells, a benzimidazole-2-thione analog was identified. Compound 1 showed an apparent EC50 of 53 nM and efficacy of 93% with respect to progesterone. It binds to PR with high affinity (Ki nM), but had no or very low affinity for other steroid hormone receptors. Structure-activity relationship studies of a series of benzimidazole-2-thione analogs revealed critical positions for high PR binding affinity and transactivation potency as well as receptor selectivity, as exemplified by 25. Compound 25 binds to human PR with high affinity (Ki nM) and had at least > 1000-fold selectivity for PR versus other steroid receptors. Molecular modeling studies suggested that these agonists overlap favorably with progesterone in the ligand-binding domain of PR. In T47D cells, compound 25 acted as a full agonist in the MMTV-Luc reporter assay, as well as in the induction of endogenous alkaline phosphatase activity with apparent EC50 values of 4 and 9 nM, respectively. In the immature rat model, compound 25 provided a significant suppression of estrogen-induced endometrium hypertrophy as measured by luminal epithelial height. In contrast, compound 25 was inactive in the luteinizing hormone release assay in young ovariectomized rats. These benzimidazole-2-thione analogs constitute a new series of nonsteroidal PR agonists with an excellent steroid receptor selectivity profile. The differential activities observed in the in vivo progestogenic assays in rat models suggest that these analogs can act as selective PR modulators.