Synthesis,Characterization, and Cytotoxicity of Some New 5-Aminopyrazole and Pyrazolo[1,5-a]pyrimidine Derivatives

5-Amino-N-aryl-3-[(4-methoxyphenyl)amino]-1H-pyrazole-4-carboxamides 4a–c were synthesized by the reaction of N-(aryl)-2-cyano-3-[(4-methoxyphenyl)amino]-3-(methylthio)acrylamides 3a–c with hydrazine hydrate in ethanol. The reaction of 5-amino-N-aryl-1H-pyrazoles 4a–c with acetylacetone 5 or 2-(4-methoxybenzylidene)malononitrile 8 yielded the pyrazolo[1,5-a]pyrimidine derivatives 7a–c and 10a–c, respectively. The structures of the synthesized compounds were established based on elemental analysis and spectral data (IR, MS, ¹H-NMR, and ¹³C-NMR). Representative examples of the new synthesized products were screened for their in vitro cytotoxic activity against Ehrlich Ascites Carcinoma (EAC) cells.     

Synthesis,Anti-Inflammatory,and Ulcerogenicity Studies of Novel Substituted and Fused Pyrazolo[3,4-d]pyrimidin-4-ones

In the present investigation, some new pyrazolo[3,4-d]pyrimidin-4(5H)-one derivatives (7–12) as well as fused pyrazolo[3′,4′:4,5]pyrimido[1,2-b]pyridazin-4(1H)-one (14–16) and 7,8,9,10-tetrahydropyrazolo[3′,4′:4,5]pyrimido[1,2-b]-cinnolin-4(1H)-one (17) ring systems were synthesized using the starting compound 5-amino-6-methyl-1-phenyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (5). The structures of the newly synthesized compounds were elucidated by IR, ¹H NMR, ¹³C NMR, mass spectroscopy, and elemental analysis. The theoretical calculation of their lipophilicity as C log P was performed. The anti-inflammatory activity of all newly synthesized compounds was evaluated using the carrageenan-induced paw edema test in rats using indomethacin as the reference drug. Ulcer indices for the most active compounds were calculated. Seven compounds (10b, 11a–f) showed consistently good anti-inflammatory activity. In particular, 5-{[4-(4-bromophenyl)-3-(4-chlorophenyl)-1,3-thiazol-2(3H)-ylidene]amino}-6-methyl-1-phenyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (11e) and its 3,4-bis(4-chlorophenyl) analog (11f) were found to be the most effective among the other derivatives, showing activity comparable to that of indomethacin with minimal ulcerogenic effects. Correlation of the biological data of the active compounds with their theoretically calculated C log P values revealed that lipophilicity influences the biological response.     

Synthesis of [11C]Am80 via Novel Pd(0)-Mediated Rapid [11C]Carbonylation Using Arylboronate and [11C]Carbon Monoxide

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Synthesis and Antimicrobial Activity of 6-Thioxo-6,7-dihydro-2H-[1,2,4]triazino[2,3-c]-quinazolin-2-one Derivatives

Potassium 8-R¹-9-R²-10-R³-3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-thiolates 2.1–2.26 were synthesized via cyclocondensation of 6-R-3-(3-R¹-4-R²-5-R³-aminophenyl)-1,2,4-triazin-5-ones 1.1–1.26 with carbon disulfide, potassium hydroxide, and ethanol or with potassium O-ethyl dithiocarbonate in 2-propanol. The corresponding thiones 3.1–3.26 were obtained by treatment of 2.1–2.26 with hydrochloric acid. It was found that the nature of the substituents in positions 3, 4, and 5 of the corresponding 6-R-3-(3-R¹-4-R²-5-R³-aminophenyl)-1,2,4-triazin-5-ones were affected on the terms of the reaction. The structures of compounds were proven by a complex of physicochemical methods (¹H, ¹³C NMR, LC–MS, and EI-MS). The results of the antibacterial and antifungal activity assay allowed the determination of the high sensitivity of Staphylococcus aureus ATCC 25923 (MIC 6.25–100 μg/mL, MBC 12.5–200 μg/mL) to the synthesized compounds.     

Synthesis and Biological Activity of New Thiopyrano[2,3-d]thiazoles Containing a Naphthoquinone Moiety

Novel 11-substituted 3,11-dihydro-2H-benzo[6,7]thiochromeno[2,3-d][1,3]-thiazole-2,5,10-triones 4a–i were synthesized in 75–90% yields via the hetero-Diels-Alder reaction of 5-arylidene-4-thioxo-2-thiazolidinones with 1,4-naphthoquinone. The synthesized compounds were evaluated for their antineoplastic and antimycobacterial activities. A moderate selectivity against melanoma cancer cells (GI₅₀ (UACC-257-melanoma) = 0.22 μM) was demonstrated for 4i, whereas derivatives 4a, 4c, 4g, and 4h showed promising antimycobacterial activity at a low toxicity level.     

Substituted 2-[(2-Oxo-2H-[1,2,4]triazino [2,3-c]quinazolin-6-yl)thio]acetamides with Thiazole and Thiadiazole Fragments: Synthesis,Physicochemical Properties,Cytotoxicity, and Anticancer Activity

The series of novel N-R-2-[(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]acetamides with thiazole and thiadiazole fragments in a molecule were obtained by alkylation of potassium salts 1.1–1.4 by N-hetaryl-2-chloroacetamides and by aminolysis of activated acids 2.1–2.4 with N,N’-carbonyldiimidazole (CDI). The structures of compounds were determined by IR, ¹H NMR, MS, and EI-MS analysis. The results of cytotoxicity evaluated by the bioluminescence inhibition of bacterium Photobacterium leiognathi, Sh1 showed that the compounds have considerable cytotoxicity. The synthesized compounds were tested for anticancer activity in NCI against 60 cell lines. Among the highly active compounds 3.1, 3.2, and 6.5, 2-[(3-methyl-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]-N-(1,3-thiazol-2-yl)acetamide (3.1) was found to be the most active anticancer agent against the cell lines of colon cancer (GI₅₀ at 0.41–0.69 μM), melanoma (GI₅₀ 0.48–13.50 μM), and ovarian cancer (GI₅₀ 0.25–5.01 μM). The structure-activity relationship (SAR-analysis) was discussed.     

Synthesis,Structural Elucidation,and In Vitro Antitumor Activities of Some Pyrazolopyrimidines and Schiff Bases Derived from 5-Amino-3-(arylamino)-1H-pyrazole-4-carboxamides

The reaction of 5-amino-3-(arylamino)-1H-pyrazole-4-carboxamides 1a,b with acetylacetone 2 and arylidenemalononitriles 5a–c yielded the pyrazolo[1,5-a]-pyrimidine derivatives 4a,b and 7a–f respectively. On the other hand, Schiff bases 9a,b and 12a–j were obtained upon treatment of carboxamides 1a,b with isatin 8 and some selected aldehydes 11a–e. The newly synthesized compounds were characterized by analytical and spectroscopic data. Representative examples of the synthesized products 4a,b, 7e, 7f, 9b, 12b–f, 12h, and 12j were screened for their in vitro antitumor activities against different human cancer cell lines and the structure-activity relationship (SAR) was discussed.     

The plasma–occupancy relationship of the novel GABAA receptor benzodiazepine site ligand, α5IA,is similar in rats and primates

Background and purpose:

α5IA (3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine) is a triazolophthalazine with subnanomolar affinity for α1-, α2-, α3- and α5-containing GABA_A receptors. Here we have evaluated the relationship between plasma α5IA concentrations and benzodiazepine binding site occupancy in rodents and primates (rhesus monkey).

Experimental approach:

In awake rats, occupancy was measured at various times after oral dosing with α5IA (0.03–30 mg·kg⁻¹) using an in vivo {[³H]flumazenil (8-fluoro 5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester)} binding assay. In anaesthetized rhesus monkeys, occupancy was measured using {[¹²³I]iomazenil (ethyl 5,6-dihydro-7-iodo-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester)} γ-scintigraphy and a bolus/infusion paradigm. In both rat and rhesus monkey, the plasma drug concentration corresponding to 50% occupancy (EC₅₀) was calculated.

Key results:

In rats, α5IA occupancy was dose- and time-dependent with maximum occupancy occurring within the first 2 h. However, rat plasma EC₅₀ was time-independent, ranging from 42 to 67 ng·mL⁻¹ over a 24 h time course with the average being 52 ng·mL⁻¹ (i.e. occupancy decreased as plasma drug concentrations fell). In rhesus monkeys, the EC₅₀ for α5IA displacing steady-state [¹²³I]iomazenil binding was 57 ng·mL⁻¹.

Conclusions and implications:

Rat plasma EC₅₀ values did not vary as a function of time indicating that α5IA dissociates readily for the GABA_A receptor in vivo. These data also suggest that despite the different assays used (terminal assays of [³H]flumazenil in vivo binding in rats and [¹²³I]iomazenil γ-scintigraphy in anaesthetized rhesus monkeys), these techniques produced similar plasma α5IA EC₅₀ values (52 and 57 ng·mL⁻¹ respectively) and that the plasma–occupancy relationship for α5IA translates across these two species.     

Synthesis,characterization, and antimicrobial evaluation of carbostyril derivatives of 1H-pyrazole

A new series of 12 derivatives of 4-pyrazolyl-N-arylquinoline-2,5-dione (4a–l) were synthesized by one pot base catalyzed cyclocondensation reaction of 1-aryl-5-chloro-3-methyl-1H-pyrazole-4-carbaldehyde (1a–c), Meldrum’s acid (2) and 3-arylamino-5,5-disubstitutedcyclohex-2-enone (3a–d). All the compounds were characterized by elemental analysis, FT-IR, ¹H NMR and ¹³C NMR spectral data and were screened, against six bacterial pathogens, namely Bacillus subtilis, Clostridium tetani, Streptococcus pneumoniae, Salmonella typhi, Vibrio cholerae, Escherichia coli and antifungal activity, against two fungal pathogens Aspergillus fumigatus and Candida albicans, using broth microdilution MIC (minimum inhibitory concentration) method. Some of the compounds were found to be equipotent or more potent than commercial drugs, against most of the employed strains, as evident from the screening data.     

Protective effects of compound FLZ,a novel synthetic analogue of squamosamide,on β-amyloid-induced rat brain mitochondrial dysfunction in vitro

Aim:

The aim of the present study was to assess the effects of N-[2-(4-hydroxyphenyl)ethyl]-2-(2,5-dimethoxyphenyl)-3-(3-methoxy-4-hydroxyphenyl) acrylamide (compound FLZ), a novel synthetic analogue of squamosamide, on the dysfunction of rat brain mitochondria induced by Aβ_25–35 in vitro.

Methods:

Isolated rat brain mitochondria were incubated with aged Aβ_25–35 for 30 min in the presence and absence of FLZ (1–100 μmol/L). The activities of key mitochondrial enzymes, the production of hydrogen peroxide (H₂O₂) and superoxide anion (O₂^·-), and the levels of glutathione (GSH) in mitochondria were examined. Mitochondrial swelling and the release of cytochrome c from mitochondria were assessed by biochemical and Western blot methods, respectively.

Results:

Incubation of mitochondria with aged Aβ_25–35 inhibited the activities of α-ketoglutarate dehydrogenase (α-KGDH), pyruvate dehydrogenase (PDH) and respiratory chain complex IV. It also resulted in increased H₂O₂ and O₂^·- production, and decreased the GSH level in mitochondria. Furthermore, it induced mitochondrial swelling and cytochrome c release from the mitochondria. The addition of FLZ (100 μmol/L) prior to treatment with Aβ_25–35 significantly prevented these toxic effects of Aβ_25–35 on the mitochondria.

Conclusion:

FLZ has a protective effect against Aβ_25–35-induced mitochondrial dysfunction in vitro.     

A New Highly Deuterated [18F]AV-45, [18F]D15FSP,for Imaging β-Amyloid Plaques in the Brain

[¹⁸F]AV-45 (florbetapir f18, Amyvid) is an FDA-approved PET imaging agent targeting Aβ plaques in the brain for diagnosis of Alzheimer’s disease (AD). Its metabolites led to a high background in the brain and large bone uptake of [¹⁸F]F^–, produced from dealkylation of the PEG chain. To slow down the in vivo metabolism, we report the design, synthesis, and evaluation of a highly deuterated derivative, [¹⁸F]D15FSP, and compared it with N-methyl-deuterated [¹⁸F]D3FSP and nondeuterated [¹⁸F]AV-45. D15FSP displayed excellent binding affinity (K_i = 7.52 nM) to Aβ aggregates. In vitro autoradiography of [¹⁸F]D15FSP, [¹⁸F]D3FSP, and [¹⁸F]AV-45 showed excellent binding to Aβ plaques in human AD brain sections. Biodistribution studies displayed lower bone uptake at 120 min for [¹⁸F]D15FSP compared to that for [¹⁸F]D3FSP and [¹⁸F]AV-45 (1.44 vs 4.23 and 4.03%ID/g, respectively). As the highly deuterated [¹⁸F]D15FSP displayed excellent Aβ binding affinity, high initial brain penetration, and lower bone retention, it might be suitable for PET imaging in detecting Aβ plaques.     

Blockade of Tau Hyperphosphorylation and Aβ1–42 Generation by the Aminotetrahydrofuran Derivative ANAVEX2-73, a Mixed Muscarinic and σ1 Receptor Agonist,in a Nontransgenic Mouse Model of Alzheimer's Disease

The main objective of the present study was to establish whether the mixed σ₁/muscarinic ligand ANAVEX2-73, shown to be neuroprotective in Alzheimer''s disease (AD) models in vivo and currently in clinical phase I/IIa, could have the ability to reduce the appearance of hyperphosphorylated Tau and amyloid-β_1–42 (Aβ_1–42) in the Aβ_25–35 mouse model of AD. We therefore first confirmed that Aβ_25–35 injection induced hyperphosphorylation of Tau protein, by showing that it rapidly decreased Akt activity and activated glycogen synthase kinase-3β (GSK-3β) in the mouse hippocampus. Second, we showed that the kinase activation, and resulting Tau alteration, directly contributed to the amyloid toxicity, as co-administration of the selective GSK-3β inhibitor 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxidiazole blocked both Tau phosphorylation and Aβ_25–35-induced memory impairments. Third, we analyzed the ANAVEX2-73 effect on Tau phosphorylation and activation of the related kinase pathways (Akt and GSK-3β). And fourth, we also addressed the impact of the drug on Aβ_25–35-induced Aβ_1–42 seeding and observed that the compound significantly blocked the increase in Aβ_1–42 and C99 levels in the hippocampus, suggesting that it may alleviate amyloid load in AD models. The comparison with PRE-084, a selective and reference σ₁ receptor agonist, and xanomeline, a muscarinic ligand presenting similar profile as ANAVEX2-73 on M1 and M2 subtypes, confirmed that both muscarinic and σ₁ targets are involved in the ANAVEX2-73 effects. The drug, acting synergistically on both targets, but with moderate affinity, presents a promising pharmacological profile.     

Search for Compounds with Hypoglycemic Activity in the Series of 1-[2-(1H-Tetrazol-5-yl)-R1-phenyl]-3-R2-phenyl(ethyl)ureas and R1-Tetrazolo[1,5-c]quinazolin-5(6H)-ones

Methods of 1-[2-(1H-tetrazol-5-yl)-R¹-phenyl]-3-R²-phenyl(ethyl)ureas and R¹-tetrazolo[1,5-c]quinazolin-5(6H)-ones synthesis were designed. IR, LC-MS, ¹H NMR, and elemental analysis data evaluated the structure and purity of the obtained compounds. Different products, depending on the reaction conditions, were distinguished and discussed. The preliminary hypoglycemic activity of 36 synthesized compounds was revealed. Docking studies to 11β-hydroxysteroid dehydrogenase 1, γ-peroxisome proliferator-activated receptor, and dipeptidyl peptidase-4 were conducted. Eight of these substances were further tested on glucocorticoid-induced insulin resistance models, namely glucose tolerance, oral rapid insulin, and adrenalin tests. One of the most active compounds turned out to be tetrazolo[1,5-c]quinazolin-5(6H)-one 3.1, exceeding the reference drugs Metformin (50 and 200 mg/kg) and Gliclazide (50 mg/kg).     

Discovery of a Novel Class of Bicyclo[3.1.0]hexanylpiperazines as Noncompetitive Neuropeptide Y Y1 Antagonists

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Stimulation of locus coeruleus neurons by non-I1/I2-type imidazoline receptors: an in vivo and in vitro electrophysiological study

1. Imidazoline binding sites have been reported to be present in the locus coeruleus (LC). To investigate the role of these sites in the control of LC neuron activity, we studied the effect of imidazolines using in vivo and in vitro single-unit extracellular recording techniques.
2. In anaesthetized rats, local (27 pmoles) and systemic (1 mg kg⁻¹, i.v.) administrations of 2-(2-benzofuranyl)-2-imidazoline (2-BFI), a selective I-imidazoline receptor ligand, increased the firing rate of LC cells (maximal increase: 22±5%, P<0.001 and 16±7%, P<0.001 respectively). Chronic pretreatment with the irreversible monoamine oxidase inhibitor clorgyline (3 mg kg⁻¹, i.p., every 12 h for 14 days) abolished this effect.
3. In rat midpontine brain slices containing the LC, bath application (1 mM) of the imidazolines 2-BFI, 2-(4,5-dihydroimidaz-2-yl)-quinoline (BU224), idazoxan, efaroxan, phentolamine and (2-2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline (RX821002) reversibly stimulated LC cells. The maximal effect was ∼90% except for RX821002 and efaroxan which induced smaller maximal effects (∼58% and ∼35% respectively). Simultaneous application of idazoxan and 2BFI did not lead to additive effects.
4. Bath application of the α₂-adrenoceptor antagonists, yohimbine (1–10 μM) and N-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (10 μM), failed to modify LC activity. The irreversible blockade of α₂-adrenoceptors with EEDQ (10 μM) did not alter the effect of idazoxan or that of efaroxan. Previous application of clorgyline (10 μM) did not modify the excitatory effect of 2-BFI or efaroxan.
5. Changes in the pH of the bathing solution (6.84–7.84) did not influence the effect caused by idazoxan. Bath application of 2-BFI (1 mM) reversed the inhibition induced by diazoxide (300 μM), an ATP-sensitive K⁺ channel opener, whereas application of glibenclamide (3 μM), an ATP-sensitive K⁺ channel blocker, partially blocked the effect of 2-BFI.
6. This study shows that imidazoline compounds stimulate the firing rate of LC neurons. This effect is not mediated by α₂-adrenoceptors nor by I₁ or I₂-imidazoline receptors but involves a different subtype of imidazoline receptor. Our results indicate that this receptor is located extracellularly and modulates ATP-sensitive K⁺ channels.

     

Characterization of calcitonin gene-related peptide(CGRP) receptors in intramural coronary arteries from male and female Sprague Dawley rats

1. In this study we characterized the CGRP-receptor subtype by Schild-plot analysis using the C-terminal fragment, human-αCGRP(8–37), a putative competitive CGRP₁-receptor selective antagonist. In addition, the effect of rat-αCGRP was compared with that of homologous peptides rat-βCGRP, rat-amylin, rat-adrenomedullin and [Cys(Acm)^2,7]-human-αCGRP, a putative selective CGRP₂-receptor agonist, in the left coronary arteries of 3 months old male and female Sprague Dawley rats.
2. Isolated rings from the distal, intramural part of the left anterior descending (LAD) coronary artery in both groups of rats were mounted on a double wire-myograph. The arteries were then stretched to their optimal lumen diameter for active tension development and precontracted with 10⁻⁵ M prostaglandin F_2α (PGF_2α), after which agonists were added to the organ bath in a cumulative manner.
3. Rat-αCGRP induced endothelium-independent relaxations in male and female Sprague-Dawley rats. Rat-βCGRP concentration-response relations (10⁻¹¹–10⁻⁷ M) were similar to those of rat-αCGRP in either sex. The maximal relaxations induced by rat-amylin and rat-adrenomedullin, both at 10⁻⁶ M, were significantly (P<0.05) lower than those induced by rat-α- and rat-βCGRP. In contrast, the selective CGRP₂-receptor agonist [Cys(Acm)^2,7]-human-αCGRP failed to induce significant relaxations at the highest concentration used (10⁻⁷ M) in the coronary arteries of male and female rats.
4. The C-terminal fragment, human-αCGRP(8–37) blocked concentration-dependently (10⁻⁷–10⁻⁶ M) the rat-αCGRP-induced relaxation in 10⁻⁵ M PGF_2α-precontracted coronary arteries. The slopes of the regression lines of the Schild-plots for both male and female rats were not significantly (P>0.05) different from unity and the pA₂ values for human-αCGRP(8–37) were 6.93 and 6.98 in arteries from male and female rats, respectively. There was no significant (P>0.05) difference in estimated pK_B values for human-αCGRP(8–37) between male (6.99±0.10, n=13) and female (6.95±0.08, n=13) rats.
5. The concentration-response relationships for rat-α- and rat-βCGRP were similar in male and female Sprague Dawley rats. The predominant CGRP receptor subtype in small intramural coronary arteries appeared to belong to the CGRP₁-receptor subtype in both sexes.

     

AT-1001: A High Affinity and Selective α3β4 Nicotinic Acetylcholine Receptor Antagonist Blocks Nicotine Self-Administration in Rats

Genomic and pharmacologic data have suggested the involvement of the α3β4 subtype of nicotinic acetylcholine receptors (nAChRs) in drug seeking to nicotine and other drugs of abuse. In order to better examine this receptor subtype, we have identified and characterized the first high affinity and selective α3β4 nAChR antagonist, AT-1001, both in vitro and in vivo. This is the first reported compound with a Ki below 10 nM at α3β4 nAChR and >90-fold selectivity over the other major subtypes, the α4β2 and α7 nAChR. AT-1001 competes with epibatidine, allowing for [³H]epibatidine binding to be used for structure-activity studies, however, both receptor binding and ligand-induced Ca²⁺ flux are not strictly competitive because increasing ligand concentration produces an apparent decrease in receptor number and maximal Ca²⁺ fluorescence. AT-1001 also potently and reversibly blocks epibatidine-induced inward currents in HEK cells transfected with α3β4 nAChR. Importantly, AT-1001 potently and dose-dependently blocks nicotine self-administration in rats, without affecting food responding. When tested in a nucleus accumbens (NAcs) synaptosomal preparation, AT-1001 inhibits nicotine-induced [³H]dopamine release poorly and at significantly higher concentrations compared with mecamylamine and conotoxin MII. These results suggest that its inhibition of nicotine self-administration in rats is not directly due to a decrease in dopamine release from the NAc, and most likely involves an indirect pathway requiring α3β4 nAChR. In conclusion, our studies provide further evidence for the involvement of α3β4 nAChR in nicotine self-administration. These findings suggest the utility of this receptor as a target for smoking cessation medications, and highlight the potential of AT-1001 and congeners as clinically useful compounds.     

Prostanoid receptors of the EP3 subtype mediate inhibition of evoked [3H]acetylcholine release from isolated human bronchi

1. The release of neuronal [³H]acetylcholine (ACh) from isolated human bronchi after labelling with [³H]choline was measured to investigate the effects of prostanoids.
2. A first period of electrical field stimulation (S₁) caused a [³H]ACh release of 320±70 and 200±40 Becquerel (Bq) g⁻¹ in epithelium-denuded and epithelium-containing bronchi respectively (P>0.05). Subsequent periods of electrical stimulation (S_n, n=2, 3, and 4) released less [³H]ACh, i.e. decreasing S_n/S₁ values were obtained (0.76±0.09, 0.68±0.07 and 0.40±0.04, respectively).
3. Cumulative concentrations (1–1000 nM) of EP-receptor agonists like prostaglandin E₂, nocloprost, and sulprostone (EP₁ and EP₃ selective) inhibited evoked [³H]ACh release in a concentration dependent manner with IC₅₀ values between 4–14 nM and maximal inhibition of about 70%.
4. The inhibition of evoked [³H]ACh release by prostaglandin E₂, nocloprost and sulprostone was not affected by the DP-, EP₁- and EP₂-receptor antagonist AH6809 at a concentration of 3 μM, i.e. a 3–30 times greater concentration than its affinity (pA₂ values) at the respective receptors.
5. Circaprost (IP-receptor agonist; 1–100 nM), iloprost (IP- and EP₁-receptor agonist; 10-1000 nM) and U-46619 (TP-receptor agonist; 100–1000 nM) did not significantly affect [³H]ACh release.
6. Blockade of cyclooxygenase by 3 μM indomethacin did not significantly modulate evoked [³H]ACh release in epithelium-containing and epithelium-denuded bronchi. Likewise, the combined cyclo- and lipoxygenase inhibitor BW-755C (20 μM) did not affect evoked [³H]ACh release.
7. In conclusion, applied prostanoids appear to inhibit [³H]ACh release in epithelium-denuded human bronchi under the present in vitro conditions, most likely via prejunctional prostanoid receptors of the EP₃ subtype.

     

Effects of [3H]-BIDN,a novel bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates

1. The radiolabelled bicyclic dinitrile, [³H]-3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([³H]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of γ-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC₅₀, 35±3 nM) and dieldrin (IC₅₀, 93±7 nM), displaced [³H]-BIDN from rootworm membranes. When tested at 100 μM, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane(EBOB), 4-t-butyl-2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane-1-thione (TBPS), 1-phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [³H]-BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands.
2. Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and α and β endosulphan displace bound [³H]-BIDN from rootworm membranes by a competitive mechanism.
3. Rat brain membranes were also shown to possess a population of saturable, specific [³H]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [³H]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm.
4. Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABA_B receptors were unaffected.
5. Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl⁻ channels to which a number of insecticidally-active molecules bind.

     

Imaging Nicotine- and Amphetamine-Induced Dopamine Release in Rhesus Monkeys with [11C]PHNO vs [11C]raclopride PET

The radiotracer [¹¹C]PHNO may have advantages over other dopamine (DA) D₂/D₃ receptor ligands because, as an agonist, it measures high-affinity, functionally active D₂/D₃ receptors, whereas the traditionally used radiotracer [¹¹C]raclopride measures both high- and low-affinity receptors. Our aim was to take advantage of the strength of [¹¹C]PHNO for measuring the small DA signal induced by nicotine, which has been difficult to measure in preclinical and clinical neuroimaging studies. Nicotine- and amphetamine-induced DA release in non-human primates was measured with [¹¹C]PHNO and [¹¹C]raclopride positron emission tomography (PET) imaging. Seven adult rhesus monkeys were imaged on a FOCUS 220 PET scanner after injection of a bolus of [¹¹C]PHNO or [¹¹C]raclopride in three conditions: baseline; preinjection of nicotine (0.1 mg/kg bolus+0.08 mg/kg infusion over 30 min); preinjection of amphetamine (0.4 mg/kg, 5 min before radiotracer injection). DA release was measured as change in binding potential (BP_ND). Nicotine significantly decreased BP_ND in the caudate (7±8%), the nucleus accumbens (10±7%), and in the globus pallidus (13±15%) measured with [¹¹C]PHNO, but did not significantly decrease BP_ND in the putamen or the substantia nigra or in any region when measured with [¹¹C]raclopride. Amphetamine significantly reduced BP_ND in all regions with both radiotracers. In the striatum, larger amphetamine-induced changes were detected with [¹¹C]PHNO compared with [¹¹C]raclopride (52–64% vs 33–35%, respectively). We confirmed that [¹¹C]PHNO is more sensitive than [¹¹C]raclopride to nicotine- and amphetamine-induced DA release. [¹¹C]PHNO PET may be more sensitive to measuring tobacco smoking-induced DA release in human tobacco smokers.