Stilbene-pyridazinone hybrids: design, synthesis and in vitro antiplatelet activity screening.

A series of stilbene analogues, in which a phenyl ring was replaced by the pyridazin-3(2H)-one nucleus, was designed and synthesized to be explored as platelet aggregation inhibitors. The proposed stilbene-pyridazinone hybrids were successfully obtained from simple starting materials and by Wittig's reaction. Most of the target compounds displayed improved in vitro activity in comparison with the standard drug, resveratrol, highlighting as the most potent the analogues 10d and 10e, with inhibition percentages of 94.15 % at 100 µM and 100 % at 50 µM, respectively. The pharmacokinetic and toxicity (ADME/T) properties of the novel hybrids were also estimated with the SwissADME and ProTox-II web servers.

Novel compounds of hybrid structure pyridazinone-coumarin as potent inhibitors of platelet aggregation.

Aim: The current limitations of antiplatelet therapy promote the search for new antithrombotic agents. Here we describe novel platelet aggregation inhibitors that combine pyridazinone and coumarin scaffolds in their structure. Results: The target compounds were synthesized in good yield from maleic anhydride, following a multistep strategy. The in vitro studies demonstrated significant antiplatelet activity in many of these compounds, with IC50 values in the low micromolar range, revealing that the activity was affected by the substitution pattern of the two selected cores. Additional studies point out their effect as inhibitors of glycoprotein (Gp) IIb/IIIa activation. Conclusion: This novel hybrid structure can be considered a good prototype for the development of potent platelet aggregation inhibitors.

2-Substituted 4-, 5-, and 6-[(1E)-3-oxo-3-phenylprop-1-en-1-yl]pyridazin-3(2H)-ones and 2-substituted 4,5-bis[(1E)-3-oxo-3-phenylprop-1-en-1-yl]pyridazin-3(2H)-ones as potent platelet aggregation inhibitors: design, synthesis, and SAR studies.

A set of regioisomeric 2-substituted pyridazin-3(2H)-ones containing a 3-oxo-3-phenylprop-1-en-1-yl fragment at either position 4, 5 or 6 and 2-substituted pyridazin-3(2H)-ones containing the same fragment both at positions 4 and 5 have been synthesized and evaluated as antiplatelet agents. The study allows the identification of a new highly potent platelet aggregation inhibitor (4c).

Design, synthesis, and structure-activity relationships of a novel series of 5-alkylidenepyridazin-3(2H)-ones with a non-cAMP-based antiplatelet activity.

5-alkylidenepyridazin-3-ones with four points of diversity (R2, R6, X, Y) have been synthesized and evaluated as platelet aggregation inhibitors. Several derivatives eliciting antiplatelet activity in the low micromolar range (e.g., 14e, 14k, 14p, 14v, IC50 congruent with 1 microM) were identified. Structure-activity relationships studies on these compounds revealed the key molecular determinants of this new family of antiplatelet agents: (a) two ester groups in the alkoxy moieties; (b) lipophilic substituents at the N2 position of the pyridazin-3-one. The preliminary results of a pharmacological study aimed at determining the mechanism of action of a set of representative compounds revealed that, unlike other pyridazinones, the documented antiplatelet effect is not a consequence of a PDE-III inhibitory activity.

Parallel regulation by olanzapine of the patterns of expression of 5-HT2A and D3 receptors in rat central nervous system and blood cells.

Patterns of protein expression can be used to identify biomarkers of disease, prognosis or treatment response. Peripheral 5-HT2A and D3 receptors have been proposed as protein markers in schizophrenia. We investigated the possible parallel regulation of these candidate biomarkers in central nervous system (CNS) and peripheral blood cells by a comparative study of the effects of antipsychotic treatment on the expression of the receptors in both systems in rats. Acute (24 and 48 h) and subchronic (16 days) treatment of rats with olanzapine induced a significant decrease in 5-HT2A receptor density both in frontal cortex (Bmax=76.2%, 83.0% and 46.0% of control after 24 h, 48 h and 16 days of treatment, respectively; P<0.01) and blood platelets (Bmax approximately 55% of control at all times measured; P<0.01), without any changes in receptor affinity. Furthermore, olanzapine induced redistribution in 5-HT2A-like immunoreactivity and time-dependent remodelling of synaptic circuits involved in the activity of pyramidal and GABAergic neurons in frontoparietal motor cortex of treated rats, as assessed by immunohistochemical studies. D3 receptor mRNA levels increased significantly by 52.5% (P<0.01) and 21.1% (P<0.05) in nucleus accumbens, and by 53.4% (P<0.05) and 91.7% (P<0.01) in lymphocytes, after acute (24 h and 48 h) treatment with olanzapine, returning to levels similar to control after subchronic treatment (16 days). In conclusion, we observed in rats after olanzapine treatment: (1) parallelism in the regulation of 5-HT2A receptors in frontal cortex and in blood platelets; (2) parallelism in the regulation of D3 mRNA levels in nucleus accumbens and lymphocytes. These results endorse the interest in future studies aimed at validating these receptors as candidate biomarkers in schizophrenia.

(-)-Trans-epsilon-viniferin, a polyphenol present in wines, is an inhibitor of noradrenaline and 5-hydroxytryptamine uptake and of monoamine oxidase activity.

(-)-Trans-epsilon-viniferin (epsilon-viniferin, 5-200 microM), a dimer of resveratrol, concentration-dependently inhibited the uptake of [3H]noradrenaline and [3H]5-HT by synaptosomes from rat brain (being slightly but significantly more selective against [3H]noradrenaline) and the uptake of [3H]5-HT by human platelets. On the other hand, epsilon-viniferin (5-200 microM) concentration-dependently inhibited the enzymatic activity of commercial (human recombinant) monoamine oxidase (MAO) isoform (MAO-A and MAO-B) activity, being slightly but significantly more selective against MAO-B than against MAO-A. Taking into account that the principal groups of drugs used to treat major depression are noradrenaline/5-HT uptake or MAO inhibitors, under the assumption that epsilon-viniferin exhibits a similar behaviour in humans in vivo, our results suggest that this natural polyphenol may be of value as a structural template for the design and development of new antidepressant drugs with two important biochemical activities combined in the same chemical structure: noradrenaline/5-HT uptake and MAO inhibitory activity.

Beta-hexosaminidase isoenzyme profiles in serum, plasma, platelets and mononuclear, polymorphonuclear and unfractionated total leukocytes.

OBJECTIVES: The relative proportion in percentage of the isoenzyme A of beta-hexosaminidase (Hex) is the single discriminatory function most frequently used for the biochemical screening of heterozygote Tay-Sachs disease carriers. It has been suggested that the assay of the Hex isoenzymes in homogeneous cell preparations is preferable to that in mixed total leukocytes which present greater interindividual variation. The major aim of our study was the evaluation of this hypothesis. DESIGN AND METHODS: Total Hex and its Hex A and Hex B isoenzymes were determined in different samples of serum and plasma (n = 81) as well as in lysates of platelets (n = 75), and mononuclear (n = 81), polymorphonuclear (n = 81) and mixed total leukocytes (n = 33). RESULTS: The interindividual variations found for % Hex A in the different biological samples were: plasma (CV = 23.4%), platelets (CV = 10.2%), mononuclear (CV = 5.7%), polymorphonuclear (CV = 5.3%) and total leukocytes (CV = 7.1%). Although the relative proportion of Hex A was significantly greater in polymorphonuclear than in mononuclear leukocytes (P < 0.001), a statistical significance was not attained for the correlation between the relative proportions of blood polymorphonuclear cells and Hex A in mixed total leukocytes (r = 0.220). CONCLUSIONS: The use of total leukocyte lysates does not appear to introduce a significant increase for the interindividual variation of the Hex A isoenzyme relative proportion in relation to the use of homogeneous cell preparations.

New platelet aggregation inhibitors based on pyridazinone moiety.

New series of pyridazinone derivatives (4, 5 and 6) were synthesized in good yields following a synthetic strategy based on singlet oxygen oxidation of alkyl furans, in which a suitable ß(α)-substituted γ-hydroxybutenolide (10 or 11) or a bicyclic lactone (12 or 13) was the key intermediate. The synthesized compounds were tested in vitro as antiplatelet agents and some of them (compounds 4b, 4d and 5b) exhibited potent inhibitory effects on collagen-induced platelet aggregation with IC50 values in the low µM range. Studies performed with the most active compound of these series (4b) demonstrated its lack of activity as inhibitor of platelet aggregation induced by other agonists as thrombin, ionomycin or U-46619 suggesting a selective action on the biochemical mechanisms triggered by collagen in the platelets.

Antioxidant activity and inhibitory effects of hydralazine on inducible NOS/COX-2 gene and protein expression in rat peritoneal macrophages.

This study investigated the effects of the peripheral vasodilator hydralazine on in vitro generation of reactive species of oxygen (ROS), nitrogen (RNS) and prostaglandin (PG) biosynthesis in elicited murine peritoneal macrophages, and on the gene expression and protein synthesis of two key enzymes in the inflammatory process, inducible NO(*) synthase (NOS-2) and inducible cyclooxygenase 2 (COX-2). Hydralazine at 0.1-10 mM inhibited both extracellular and intracellular ROS production by inflammatory macrophages, by a ROS-scavenging mechanism probably affecting superoxide radical (O(2)(*-))-generation by xanthine oxidase (XO) and nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase. Hydralazine at 0.1-10 mM significantly reduced NO(*) generation, and this effect was attributable to an inhibition of NOS-2 gene expression and protein synthesis. At 1-10 mM, hydralazine also effectively blocked COX-2 gene expression which perfectly correlated with a reduction of protein levels and PGE(2) synthesis. These data suggest that hydralazine, at the concentrations tested, show antioxidant properties and strongly attenuates the macrophage activation.

Discovery and preliminary SAR of 5-arylidene-2,2-dimethyl-1,3-dioxane- 4,6-diones as platelet aggregation inhibitors.

We herein document the discovery of 5-arylidene-2,2-dimethyl-1,3-dioxane-4,6-diones as a novel family of platelet aggregation inhibitors. The preliminary optimization study enabled us to establish the most salient features of the structure-activity relationships in this series as well as to identify novel derivatives that are upto 60 times more potent than the hit structure 1 and slightly superior to the reference drug Milrinone.

Differences in U root-to-shoot translocation between plant species explained by U distribution in roots.

Accumulation and distribution of uranium in roots and shoots of four plants species differing in their cation exchange capacity of roots (CECR) was investigated. After exposure in hydroponics for seven days to 100 micromol U L(-1), distribution of uranium in roots was investigated through chemical extraction of roots. Higher U concentrations were measured in roots of dicots which showed a higher CECR than monocot species. Chemical extractions indicated that uranium is mostly located in the apoplasm of roots of monocots but that it is predominantly located in the symplasm of roots of dicots. Translocation of U to shoot was not significantly affected by the CECR or distribution of U between symplasm and apoplasm. Distribution of uranium in roots was investigated through chemical extraction of roots for all species. Additionally, longitudinal and radial distribution of U in roots of maize and Indian mustard, respectively showing the lowest and the highest translocation, was studied following X-ray fluorescence (XRF) analysis of specific root sections. Chemical analysis and XRF analysis of roots of maize and Indian mustard clearly indicated a higher longitudinal and radial transport of uranium in roots of Indian mustard than in roots of maize, where uranium mostly accumulated in root tips. These results showed that even if CECR could partly explain U accumulation in roots, other mechanisms like radial and longitudinal transport are implied in the translocation of U to the shoot.

Inhibitory effects of cis- and trans-resveratrol on noradrenaline and 5-hydroxytryptamine uptake and on monoamine oxidase activity.

This study investigated for the first time the potential effects of cis- and trans-resveratrol (c-RESV and t-RESV) on noradrenaline (NA) and 5-hydroxytryptamine (5-HT) uptake by synaptosomes from rat brain, on 5-HT uptake by human platelets, and on monoamine oxidase (MAO) isoform activity. Both c-RESV and t-RESV (5-200 microM) concentration-dependently inhibited the uptake of [3H]NA and [3H]5-HT by synaptosomes from rat brain and the uptake of [3H]5-HT by human platelets. In both experimental models, t-RESV was slightly more efficient than c-RESV. Furthermore, in synaptosomes from rat brain, the RESV isomers were less selective against [3H]5-HT uptake than the reference drug fluoxetine (0.1-30 microM). On the other hand, both c-RESV and t-RESV (5-200 microM) concentration-dependently inhibited the enzymatic activity of commercial (human recombinant) MAO isoform (MAO-A and MAO-B) activity, c-RESV being slightly less effective than t-RESV. In addition, both RESV isomers were slight but significantly more selective against MAO-A than against MAO-B. Since the principal groups of drugs used in the treatment of depressive disorders are NA/5-HT uptake or MAO inhibitors, under the assumption that the RESV isomers exhibit a similar behaviour in humans in vivo, our results suggest that these natural polyphenols may be of value as structural templates for the design and development of new antidepressant drugs with two important biochemical activities combined in the same chemical structure: NA/5-HT uptake and MAO inhibitory activity.

Differential regulation of platelet aggregation and aminophospholipid exposure by calpain.

Aggregation, exposure of procoagulant phospholipids and shedding of microparticles are platelet responses that depend on activating conditions. To determine how these different responses are interconnected, we simultaneously measured fibrinogen (Fg) binding and aminophospholipid exposure on activated platelets by means of flow cytometry. Low calcium ionophore (A23187) concentrations induced Fg binding but not annexin V binding. In contrast, high A23187 concentrations induced annexin V binding but not Fg binding. Collagen, both alone and in the presence of thrombin, induced both Fg and annexin V binding. Dual labelling was found on 38 +/- 9% of platelets stimulated by thrombin plus collagen. The regulatory role of calpain in these platelet functions was investigated. When calpain was partially inhibited by 2 microg/ml calpeptin, Fg binding still occurred but aminophospholipid exposure was limited. By contrast, complete inhibition of calpain by 100 microg/ml calpeptin or E64d decreased Fg binding but enhanced aminophospholipid exposure. In these latter conditions, cytosolic calcium-extruding systems were inhibited. The results suggest that (i) conditions that favour aminophospholipid exposure tend to decrease the aggregation process and (ii) calpain determines the switch to either aggregation or aminophospholipid exposure by controlling intracellular calcium.

Design, synthesis, and vasorelaxant and platelet antiaggregatory activities of coumarin-resveratrol hybrids.

We have synthesized the coumarin-resveratrol hybrid 4 and its dimethoxy derivative 3 by a very direct synthetic route involving a Pechmann procedure. Compound 4 has also been synthesized by an alternative route (Perkin), which also allowed the synthesis of compounds 9-13. In addition, we have evaluated the potential vasorelaxant activity of the new compounds in endothelium-containing rat aorta rings pre-contracted with noradrenaline, as well as the inhibitory effects on platelet aggregation induced by thrombin in washed human platelets. The compounds reported here relaxed vascular smooth muscle and inhibited platelet aggregation with a profile similar to that of trans-resveratrol (t-RESV) and, in some cases, showed activity higher than that of the natural compound. This is the case for compound 13, which has a vasorelaxant activity that is twice as high as that of t-resveratrol and a platelet antiaggregant activity that is six times higher. These results suggest that these novel compounds may have potential as structural templates for the design and subsequent development of new vasodilatory and platelet antiaggregatory drugs.

[Information on cardiovascular risk in hypertense patients monitored in primary care. Does it improve our efficacy?]. / Información sobre el riesgo cardiovascular a hipertensos seguidos en atención primaria. Mejora nuestra eficacia?

OBJECTIVES: To analyse how information to patients on their cardiovascular risk affects the latter's subsequent evolution and to see whether there are differences in the evolution of risk as a function of its being high, moderate, or low. DESIGN: Intervention study of patients who attended our scheduled hypertension clinics. SETTING: Health centre on the periphery of Murcia, Spain. PARTICIPANTS: Three hundred patients (139 men and 161 women) from 40 to 75 years old, with essential hypertension, treated or otherwise, controlled or otherwise, in the hypertension programme. They were divided into 3 groups of 100 patients: low, moderate, and high risk. INTERVENTIONS: There were 2 attendances of each patient: a) initially, at which cardiovascular risk was calculated; half of each risk group were informed of their cardiovascular risk; b) finally, a year later, at which the cardiovascular risk of all the patients was calculated. MAIN MEASUREMENTS: Calculation of cardiovascular risk on the Framingham scale. RESULTS: No significant differences were appreciated in low and moderate cardiovascular risk groups. In the high-risk group, the informed patients fell from an initial 23.6+/-2.5% to 20.1+/-2.6% after a year (P<.01); and in the non-informed group, from an initial 23.9+/-2.8% to a final 22.1+/-2.7% (P<.05). The difference in reduction of risk between informed and non-informed patients was significant (P<.05). CONCLUSIONS: Informing our hypertense patients about their cardiovascular risk is linked to a reduction in this risk when it is high.

Pyridazines part 41: synthesis, antiplatelet activity and SAR of 2,4,6-substituted 5-(3-oxo-3-phenylprop-1-en-1-yl)- or 5-(3-phenylprop-2-enoyl)pyridazin-3(2H)-ones.

As part of the optimization process of the lead compound I a focussed library of diversely substituted pyridazin-3(2H)-ones containing a 3-oxo-3-phenylprop-1-en-1-yl or 3-phenylprop-2-enoyl fragment at position 5 has been obtained and evaluated as antiplatelet agents. The structural modification at positions 2, 6 and 4 of the heterocyclic moiety allowed us to obtain preliminary information on the structure-activity relationship in this family.

Pyridazines. Part 28: 5-alkylidene-6-phenyl-3(2H)-pyridazinones, a new family of platelet aggregation inhibitors.

The synthesis and anti-platelet activity of several 5-alkylidene-6-phenyl-3(2H)-pyridazinones are described. The most active compounds are those that contain oxygenated functions (COOR, COMe) on the alkylidene fragment (6a, 6b and 6c).

Pyridazines. Part 36: Synthesis and antiplatelet activity of 5-substituted-6-phenyl-3(2H)-pyridazinones.

A convenient and efficient palladium-catalysed retro-ene-assisted method has been developed to prepare a series of 5-substituted-6-phenyl-3(2H)-pyridazinones as potential antiplatelet drugs. The most active compounds were those that contain a 3-phenyl-3-oxo-propenyl fragment or a phenylthio group at position 5 of the heterocyclic ring.

In vitro effects of resveratrol on the viability and infectivity of the microsporidian Encephalitozoon cuniculi.

Microsporidians of the genus Encephalitozoon are an important cause of disease in immunocompromised patients, and there are currently no completely effective treatments. The present study investigated the viability and infectivity of spores of Encephalitozoon cuniculi that had been exposed to resveratrol (RESV), a natural phytoalexin found in grapes and red wine. RESV at 50 microM showed significant sporicidal activity, and at 10 to 50 microM it reduced the capacity of the spores to infect dog kidney epithelial cells of the MDCK line. At 10 microM RESV also significantly inhibited intracellular development of the parasite, without affecting host cell viability. These results suggest that RESV may be useful in the treatment of Encephalitozoon infections.

Pyridazines. Part XXIX: synthesis and platelet aggregation inhibition activity of 5-substituted-6-phenyl-3(2H)-pyridazinones. Novel aspects of their biological actions.

A series of 6-phenyl-3(2H)-pyridazinones with a diverse range of substituents in the 5-position have been prepared and evaluated in the search for new antiplatelet agents. A significant dependence of the substituent on the inhibitory effect has been observed. The pharmacological study of these compounds confirms that modification of the chemical group at position 5 of the 6-phenyl-3(2H)-pyridazinone system influences both variations in the antiplatelet activity and the mechanism of action.