Novel non-peptide lead structures forBradykinin B2-receptor antagonists

Summary A series of new non-peptide Bradykinin (BK) B₂-receptor antagonists is reported. These new leads belong to a larger set including both commercially or otherwise available potential candidates found by proprietary database searches using 3D-pharmacophore models as query, and several bis-benzamidino compounds selected from our tryptase-like protease inhibitor library on the basis of topological considerations, derived from the same models. Some of these compounds show functional competitive antagonistic activity, inhibiting in vitro the BK-induced contraction of isolated guinea-pig ileum (GPI) and rat uterus with a pA₂ up to 5.3 and 7.0, respectively. They display also binding affinity (IC₅₀ up to 0.56 μM) to the BK B₂-receptor in radioligand binding assays on GPI membrane preparations and on human IMR-90 fetal lung fibroblast cells expressing this receptor subtype. Furthermore, the results with the commercially available compounds, in some cases developed as therapeutics, show that the used 3D-pharmacophore model allows to predict to some certainty possible side actions of potential drugs.     

Increased α1 receptor density in brown adipose tissue of cafeteria-fed rats

A possible general corollary between ₁-receptor density in brown adipose tissue and the degree of activation of the tissue was investigated. For this purpose, the effect of cafeteria feeding on ₁-adrenergic receptors in brown adipose tissue of seven-week-old female rats was studied by the use of the ₁-antagonist (³H)prazosin. In cafeteria-fed rats, the K_D of the ₁-receptor for (³H)prazosin was unchanged (about 0.35 nM), but the receptor density was doubled (up to 40 fmol per mg of membrane protein). This was also observed when the results were expressed per unit of a plasma-membrane marker (5-nucleotidase). It was concluded that an increased ₁-receptor density is seen not only in cold-acclimated rats, but also in other conditions where brown fat is activated, and a possible general physiological significance of ₁-adrenergic pathways in brown adipose tissue is discussed.     

Mediation of serotonin-induced hyperventilation via 5-HT3-receptor in European eel Anguilla anguilla

The effects of serotonin (5-hydroxytryptamine) on ventilation were investigated by continuous measurements of intrabuccal pressure in unrestrained eel. Intravenous administration of 5-hydroxytryptamine (30 g·kg^-1) caused a large increase in ventilatory frequency (+100%) and amplitude (+140%). The 5-hydroxytryptamine-induced hyperventilation was blocked by the 5-HT₃-receptor antagonists metoclopramide (1.0 mg·kg^-1) or MDL72222 (1.0 mg·kg^-1), and was insensitive to the 5-HT_1/2-receptor antagonist methysergide (3.0 mg·kg^-1) and to the 5-HT₄-receptor antagonist DAU 6285 CL (3.0 mg·kg^-1). The hyperventilatory response to 5-hydroxytryptamine could be mimicked by the 5-HT₃ receptor agonist 1-phenylbiguanide (300 g·kg^-1). These results strongly implicate the 5-HT₃-receptor as the mediator of the 5-hydroxytryptamine-induced hyperventilation in eel.Abbreviations a.u. arbitrary units - 5-HT 5-hydroxytryptamine - SEM standard error of mean - VA ventilatory amplitude - VF ventilatory frequency - RBI 1-phenylbiguanide     

Theoretical study of the effect of N-oxides on the performances of energetic compounds

In order to study the effects of N-oxide on structure and performance, six categories of energetic compounds were systemically investigated. The results indicated that the C–C bonds in the rings were shortened, and the C–N bonds close to the N?→?O bond were elongated when N atoms was oxidized to form N?→?O bonds. N?→?O bonds can increase the densities of most categories of compounds, and the increment will increase with the number of N?→?O bonds. As to their detonation performances, almost all categories of compounds had an increased trend, except for some NO₂-, NHNO₂- and ONO₂-substituted compounds. The contribution of 1,2,3,4-tetrazine and 1,2,4,5-tetrazine to performances was better than that of pyrazine and [1,2,5] oxadiazolo [3,4-b] pyrazine on the whole, and the groups, especially energetic groups, made a huge contribution to performance. When R was a NH₂ or ONO₂ group, all compounds had lower impact sensitivities, and thus represent candidates for novel energetic compounds. However, other than the sixth category of compounds, all compounds had higher impact sensitivities when R was a NO₂ or NHNO₂ group, and have little significance in application.

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Graphical abstract To study the effects of N-oxide on the structure and performance of energetic compounds, and to propose theoretical guidance for the design of novel compounds, the six categories (94 species) listed in the figure were investigated systemically by density functional theory methods and some empirical formulae

     

Differences in H-2 recombinant mice in the β-naphthoflavone inducibility of the mixed function monooxygenase,P1-450

The influence of the major histocompatibility complex (H-2 in mouse) on induction of cytochrome P-450-dependent monooxygenase (P₁-450) by the prototype polyaromatic hydrocarbon (PAH), -naphthoflavone, was investigated in C57BL/10 Sn (B10) recombinant congenic mice. The cytosolic Ah-receptor level, as measured by specific binding with [³H]-2, 3, 7, 8-tetrachlorodibenzo-p-dioxin, was significantly lower in B10.A and B10.A (5R) than in either B10, B10.BR, or B10.A(2R), suggesting that the D region of H-2 influences Ah-receptor levels. The responsiveness to -naphthoflavone, as determined by increased catalytic activity toward benzo(a)pyrene and 7-ethoxycoumarin, was considerably lower in 1310, B10.A, and B10.A(5R) than in B10.BR and somewhat lower than in B10.A(2R) or B10.A(4R) mice. The lower PAH responsiveness in B10.A and B10.A(5R) correlated with their lower Ah-receptor levels while that in B10 appeared to reflect a K-A region influence on PAH responsiveness that was not due to changed Ah-receptor levels. Thus, we conclude that more than one H-2 locus may influence PAH responsiveness, and by different mechanisms.     

Response of the rat heart to catecholamines and thyroid hormones

Catecholamines and thyroid hormones have a similar influence on heart function and metabolism, but this may occur in a differential manner and to a different extent In this study, the effects of norepinephrine (NE) and of triiodothyronine (T₃) were studied in regard to the function of the left (LV) and right ventricle (RV) and to the oxidative pentose phosphate pathway (PPP). NE was applied in rats as continuous i. v. infusion (0.2 mg/kg/h) for three days. T₃ was given as daily s.c. injections (0.2 mg/kg) for the same period of time. LV, and RV function was measured in the closed-chest trapanal-anesthetized animals using special Millar ultraminature catheter pressure transducers. NE induced an increase in heart rate, in mean arterial pressure, and in total peripheral resistance (TPR). The cardiac RNA/DNA and the left ventricular weight/body weight ratios were increased by about 40%. These effects were prevented by simultaneous -and -receptor blockade with prazosin and metoprolol, respectively, but not by verapamil which abolished the hemodynamic effects. RVSP was significantly elevated by NE in a dose-dependent manner. The functional effects of T₃ on the LV were not as pronounced as those induced by NE. Heart rate and LV dp/dt_max were increased by T₃ and this increase was prevented by concomitant -receptor blockade with, metoprolol. In contrast to NE, T₃ induced an increase in cardiac output and a concominant decrease in TPR. The RNA/DNA ratio was elevated and cardiac hypertrophy had developed after treatment for three days with T₃. These changes were not affected by -receptor blockade with metoprolol. RVSP was increased by T₃ to a lesser extent than with NE. In metabolic terms in turned out that only NE, but not T₃ had a stimulating effect on the cardiac PPP. NE increased the mRNA and activity of glucose-6-phosphate dehydrogenase (G-6-PD), the first and regulating enzyme of this pathway. However, there was no effect of T₃ on G-6-PD activity nor on 6-phosphogluconate dehydrogenase activity, one of the following enzymes in the pathway within the first 5 days of T₃ treatment. These results demonstrate that the functional effects of T₃ were not as pronounced as or even different from those of NE, and that T₃ lacked a stimulating effect on the cardiac PPP.     

Glial α1-receptors probably inhibit the high-affinity uptake of noradrenaline into astrocytes in the rat brain in vivo

The effect of ₂-receptor blockage on the extraneuronal turnover of noradrenaline (NA) has been studied in the intact rat brain. Tropolone and yohimbine, along with reserpine or desmethylimipramine, were given 30 min after intracerebroventricular injection of [7-³H]NA, i.e. after the tracer had been stored or inactivated. Tropolone given alone did not change the fractions of³H-activity recovered as [³H]NA from hypothalamus, septum, striatum and pons-medulla, but in the presence of yohimbine improved the [³H]NA recovery in all areas except pons-medulla. The maximum effect was seen in the hypothalamus of reserpine-treated rats. Since the ₂-autoreceptors were blocked, the increased [³H]NA recovery does not reflect a down-regulated neuronal NA turnover. Instead it seems to show that a fraction greater than normal of neuronally released NA had been taken up into astrocytes and remained unmetabolized if catechol-O-methyltransferase was inactive. It is assumed that yohimbine enabled the protective tropolone effect by blocking astrocytic ₂-receptors that otherwise, either by itself or by antagonizing -receptor-induced hyperpolarization or cAMP formation, had impaired parameters that stimulate the high-affinity NA Uptake₁ of astrocytes (e.g. membrane potential, Na⁺, K⁺-ATPase) or control the gap junction permeability in the glial syncytium.     

s-Block metallabenzene: aromaticity and hydrogen adsorption

Second group metal dimers can replace the carbon atom in benzene to form metallabenzene (C₅H₆M₂) compounds. These complexes possess some aromatic character and promising hydrogen adsorption properties. In this study, we investigated the aromatic character of these compounds using aromaticity indices and molecular orbital analysis. To determine the nature of interactions between hydrogen and the metallic center, variation-perturbational decomposition of interaction energy was applied together with ETS-NOCV analysis. The results obtained suggest that the aromatic character comes from three π orbitals located mainly on the C₅H₅ ^? fragment. The high hydrogen adsorption energy (up to 6.5 kcal mol^?1) results from two types of interaction. In C₅H₆Be₂, adsorption is controlled by interactions between the empty metal orbital and the σ orbital of the hydrogen molecule (Kubas interaction) together with corresponding back-donation interactions. Other C₅H₆M₂ compounds adsorb H₂ due to Kubas interactions enhanced by H₂–π interactions.

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Graphical Abstract First π orbital in C₅H₆Be₂

     

GABA(A) Receptor α Subunits Differentially Contribute to Diazepam Tolerance after Chronic Treatment

Background

Within the GABA_A-receptor field, two important questions are what molecular mechanisms underlie benzodiazepine tolerance, and whether tolerance can be ascribed to certain GABA_A-receptor subtypes.

Methods

We investigated tolerance to acute anxiolytic, hypothermic and sedative effects of diazepam in mice exposed for 28-days to non-selective/selective GABA_A-receptor positive allosteric modulators: diazepam (non-selective), bretazenil (partial non-selective), zolpidem (α₁ selective) and TPA023 (α_2/3 selective). In-vivo binding studies with [³H]flumazenil confirmed compounds occupied CNS GABA_A receptors.

Results

Chronic diazepam treatment resulted in tolerance to diazepam''s acute anxiolytic, hypothermic and sedative effects. In mice treated chronically with bretazenil, tolerance to diazepam''s anxiolytic and hypothermic, but not sedative, effects was seen. Chronic zolpidem treatment resulted in tolerance to diazepam''s hypothermic effect, but partial anxiolytic tolerance and no sedative tolerance. Chronic TPA023 treatment did not result in tolerance to diazepam''s hypothermic, anxiolytic or sedative effects.

Conclusions

Our data indicate that: (i) GABA_A-α₂/α₃ subtype selective drugs might not induce tolerance; (ii) in rodents quantitative and temporal variations in tolerance development occur dependent on the endpoint assessed, consistent with clinical experience with benzodiazepines (e.g., differential tolerance to antiepileptic and anxiolytic actions); (iii) tolerance to diazepam''s sedative actions needs concomitant activation of GABA_A-α₁/GABA_A-α₅ receptors. Regarding mechanism, in-situ hybridization studies indicated no gross changes in expression levels of GABA_A α₁, α₂ or α₅ subunit mRNA in hippocampus or cortex. Since selective chronic activation of either GABA_A α₂, or α₃ receptors does not engender tolerance development, subtype-selective GABA_A drugs might constitute a promising class of novel drugs.     

Lack of effect of chronic desipramine treatment on dopaminergic activity in the nucleus accumbens of the rat

The binding of [³H]SCH 23390 to dopamine (DA) D₁-receptors was measured in the nucleus accumbens of rats treated chronically with desipramine for 14 days. DA D₁ — and D₂-receptor binding using [³H]SCH 23390 and [³H]spiperone, respectively as ligands, was determined in rats treated for 28 days. NeitherB _max norK _d values were influenced by chronic desipramine treatment. In addition, chronic desipramine treatment (28 days) did not influence the dose dependent, quinpirole (10–1000 nM)-mediated inhibition of the electrically stimulated release of [³H]DA and [¹⁴C]ACh from nucleus accumbens slices or the dose dependent increase in [³H]DA release and decrease in [¹⁴C]ACh release in the presence of 1 and 10 M nomifensine. Therefore, our results suggest that the effect of chronic antidepressant treatment cannot be attributed to changes in either DA D₁₁-or D₂-receptor binding or DA D₂-receptor function in the nucleus accumbens.     

3D–QSAR studies of orvinol analogs as κ-opioid agonists

Orvinols are potent analgesics that target opioid receptors. However, their analgesic mechanism remains unclear and no significant preference for subtype opioid receptor has been achieved. In order to find new orvinols that target the κ-receptor, comparative 3D–QSAR studies were performed on 26 orvinol analogs using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The best predictions for the κ-receptor were obtained with the CoMFA standard model (q ²=0.686, r ²=0.947) and CoMSIA model combined steric, electrostatic, hydrophobic, and hydrogen bond donor/acceptor fields (q ²=0.678, r ²=0.914). The models built were further validated by a test set made up of seven compounds, leading to predictive r ² values of 0.672 for CoMFA and 0.593 for CoMSIA. The study could be helpful for designing and prepare new category κ-agonists from orvinols.      

Genetic evidence for α2-adrenergic receptor subtypes in rat brain,heart and adrenal gland

Summary A complementary DNA (cDNA) clone - cA₂-47 - corresponding to a new ₂-adrenergic receptor subtype has been isolated from a rat brain cDNA library and used as a hybridization probe to scrutinize the ₂-receptor poly(A⁺) RNAs in rat brain, heart and adrenal gland. Hybridization of the 5 half of the coding region of this cDNA at 37°C to rat brain poly(A+) RNA revealed a single band at 5.8 kb as the size of its corresponding mRNA. Under identical hybridization conditions, a human platelet ₂-receptor genomic probe failed to hybridize to any rat brain mRNAs.Under lower stringency conditions, hybridization of the full-length cDNA, cA₂-47, to selected rat tissue poly(A⁺) RNA showed the presence of four different sized mRNAs in brain and three in both heart and adrenal gland. Messages of 1.3 kb and 2.1 kb were common in all three tissues (although the band at 2.1 kb was slightly higher in the heart and adrenal gland). A 5.8 kb mRNA was unique to the brain and a slightly higher band at 6.0 kb was consistently present in heart and adrenal gland but was absent in the brain. A fourth message at 3.4 kb was found predominantly in the brain and was either absent or present at very low levels in the other tissues examined. Under the same conditions, a human platelet ₂-receptor probe hybridized to similar sized messages of 2.1 and 5.8 kb in rat brain and 2.2 and 6.0 kb in rat heart and adrenal gland. This probe, however, failed to detect the abundant 1.3 kb mRNA common to all tissues or the 3.4 kb message in rat brain. The extent of homology of these messages with cA₂-47 is not confined to limited regions of the cDNA since similar hybridization patterns were observed using either 5-noncoding or 5-coding regions of the probe.These results provide the first direct evidence of a surprisingly large range of mRNA sizes for members of the ₂-receptor family in brain, heart, and adrenal gland. The unique nature of certain members of the family in each of the tissues examined raises the curious possibility that these members might contribute to some of the individualized functions of the brain, cardiovasculature and adrenal gland.     

Supramolecular structures and fluorescence properties of three transition-metal complexes

Three mononuclear transition-metal(II) compounds [Co(imidazole)₆]CO₃ · 5H₂O (1), [Co(nicotinate)₂(H₂O)₄] (2) and [Mn(isonicotinate)₂(H₂O)₄] (3) ware obtained under the hydrothermal conditions. It deserves to note that they all exhibit interesting supramolecular topologies: in a crystal host of 1 three crystallographically unique free H₂O molecules form a two-dimensional (2-D) water network based on the cyclic dodecamers with the occluded anions; both 2 and 3 possess 3-D network structures formed through the π-π stacking interactions of the pyridine rings and multi-types of O?Ow hydrogen-bonded interactions. All of these compounds show the strong fluorescence emissions with the peaks at 389, 650 and 390-420 nm (broad band) when excited at 220, 360 and 254 nm, respectively, which can be assigned to the charge transfer between the metal and the ligand.     

A series of POM-based hybrid materials with different copper/aminotriazole motifs

By controlling the reaction temperature, pH value of the system and the polyanion templates, three inorganic-organic hybrid materials based on Keggin polyoxometalate building blocks combined with Cu^I/II and 4-amino-1,2,4-triazole (4atrz) have been obtained by hydrothermal methods, namely, [Cu₃(μ₃-OH)(4atrz)₆][SiW₁₂O₄₀]·I·3H₂O (1), [Cu₄(4atrz)₆][SiW₁₂O₄₀] (2) and [Cu₆(4atrz)₆][PMo₁₂O₄₀]₂·H₂O (3). Crystal structure analysis reveals that the Cu^I/II/4atrz complexes in the three materials show tri-, tetra- and hexanuclear models, respectively. In compound 2, the copper clusters link the polyoxometalates into the chains by weak Cu-O bonds; while in the compounds 1 and 3, the copper clusters and the polyoxometalates stack by the ionic interactions. These compounds are further characterized by powder XRD, elemental analyses, FT-IR and thermogravimetric (TG) analyses. The electrochemical behavior of 3-CPE has been studied in the 1 M H₂SO₄ solution. The results exhibit that there are four pairs of redox waves attributable to the four consecutive two-electron processes of Mo(VI/V) couples and the redox process is surface-controlled.     

3D QSAR studies on peroxisome proliferator-activated receptor γ agonists using CoMFA and CoMSIA

The peroxisome proliferator-activated receptors (PPARs) have increasingly become attractive targets for developing novel anti-type 2 diabetic drugs. We employed comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) to study three-dimensional quantitative structure–activity relationship (3D QSAR) based on existing agonists of PPAR (including five thiazolidinediones and 74 tyrosine-based compounds). Predictive 3D QSAR models with conventional r² and cross-validated coefficient (q²) values up to 0.974 and 0.642 for CoMFA and 0.979 and 0.686 for COMSIA were established using the SYBYL package. These models were validated by a test set containing 18 compounds. The CoMFA and CoMSIA field distributions are in general agreement with the structural characteristics of the binding pockets of PPAR, which demonstrates that the 3D QSAR models built here are very useful in predicting activities of novel compounds for activating PPAR.      

Formulation of 3D pharmacophore models for bradykinin B2-receptor antagonists

In order to make a further contribution to the elucidation of the essential structural features for bradykinin (BK) antagonism, we extracted 3D pharmacophore models from a training set consisting of nine relatively rigid, small organic, non-peptide molecules, reported to be more or less active, competitive BK B₂-receptor antagonists. This was accomplished by means of the expert system Catalyst. The information contained in one of these models was then used to identify relevant structural features and perform consensus molecular dynamics simulations including, in addition to the non-peptide antagonist set, four prototypical linear and cyclic peptide antagonists, and BK itself. This combined approach allowed us to identify regions of the conformational space shared by this series of compounds, which includes highly flexible, and, hence, otherwise almost inaccessible for conventional conformational sampling techniques, peptide molecules. As a result, we obtained a relatively small number of extended pharmacophore models, whose average, together with the original Catalyst model, could be used to search proprietary 3D databases for potential candidates. The results of such a search are also reported.     

Hydrogen production by the hyperthermophilic bacterium <Emphasis Type="Italic">Thermotoga maritima</Emphasis> Part II: modeling and experimental approaches for hydrogen production

Background

Thermotoga maritima is a hyperthermophilic bacterium known to produce hydrogen from a large variety of substrates. The aim of the present study is to propose a mathematical model incorporating kinetics of growth, consumption of substrates, product formations, and inhibition by hydrogen in order to predict hydrogen production depending on defined culture conditions.

Results

Our mathematical model, incorporating data concerning growth, substrates, and products, was developed to predict hydrogen production from batch fermentations of the hyperthermophilic bacterium, T. maritima. It includes the inhibition by hydrogen and the liquid-to-gas mass transfer of H₂, CO₂, and H₂S. Most kinetic parameters of the model were obtained from batch experiments without any fitting. The mathematical model is adequate for glucose, yeast extract, and thiosulfate concentrations ranging from 2.5 to 20 mmol/L, 0.2–0.5 g/L, or 0.01–0.06 mmol/L, respectively, corresponding to one of these compounds being the growth-limiting factor of T. maritima. When glucose, yeast extract, and thiosulfate concentrations are all higher than these ranges, the model overestimates all the variables. In the window of the model validity, predictions of the model show that the combination of both variables (increase in limiting factor concentration and in inlet gas stream) leads up to a twofold increase of the maximum H₂-specific productivity with the lowest inhibition.

Conclusions

A mathematical model predicting H₂ production in T. maritima was successfully designed and confirmed in this study. However, it shows the limit of validity of such mathematical models. Their limit of applicability must take into account the range of validity in which the parameters were established.

     

Catecholamine receptor in the seawater eel intestine

Dose-response relationships of catecholamines on seawater eel intestinal ion transport were obtained; the potency order being (-)adrenalin>(-)noradrenalin=clonidine>(±)noradrenalin (racemic form of noradrenalin)>(-)phenylephrine>dopamine>(±)isoproterenol, indicating that ₂ are more potent than ₁- or -agonists. In addition, the effects of adrenalin were completely blocked by yohimbine (₂-antagonists) but not by prazosin (₁-antagonists) or propranolol (-antagonist). These results indicate the existence of an ₂-receptor in the seawater eel intestine. Adrenalin may activate the ₂-receptor physiologically, since adrenalin is the most potent stimulant and is the predominant catecholamine in American eel plasma (Hathaway and Epple 1989). Presumably ion and water absorption across the seawater eel intestine will be maintained by adrenalin. From the structure and the action of various agents used in the present study, structure-activity relationships of catecholamines are considered: hydroxyl groups on the benzene ring (catechol) seem to be essential for the ₂-action in the seawater eel intestine and the presence of OH and CH₃ on -carbon and amide, respectively, seems to potentiate the ₂-action.Abbreviations ACh acetylcholine - AD adrenalin - CONH 2-cyclooctyl-2-hydroxyethylamine; n-methyltransferase - DA dopamine - 5-HT serotonin - IBMX 3-isobutyl-1-methylxanthine - I _sc short-circuit current - MCh methacholine - NA noradrenalin - PD transepithelial potential difference - PNMT phenylethanolamine N-methyltransferase - R _t tissue resistance