Involvement of thromboxane A2 and tyrosine kinase in the synergistic interaction of platelet activating factor and calcium ionophore A23187 in human platelet aggregation

The present study was carried out to examine the mechanisms of the synergistic interaction of PAF and A23187 mediated platelet aggregation. We found that platelet aggregation mediated by subthreshold concentrations of PAF (5 nM) and A23187 (1 mM) was inhibited by PAF receptor blocker (WEB 2086, IC50 = 0.65 mM) and calcium channel blockers, diltiazem (IC50 = 13 mM) and verapamil (IC50 = 18 mM). Pretreatment of platelets with PAF and A23187 induced rise in intracellular calcium and this effect was also blocked by verapamil. While examining the role of the down stream signaling pathways, we found that platelet aggregation induced by the co-addition of PAF and A23187 was also inhibited by low concentrations of phospholipase C (PLC) inhibitor (U73122; IC50 = 10 mM), a cyclooxygenase inhibitor (indomethacin; IC50 = 0.2 mM) and inhibitor of TLCK, herbimycin A with IC50 value of 5 mM. The effect was also inhibited by a specific TXA2 receptor antagonist, SQ 29548 with very low IC50 value of 0.05 mM. However, the inhibitors of MAP kinase, PD98059 and protein kinase C, chelerythrine had no effect on PAF and A23187-induced platelet aggregation. These data suggest that the synergism between PAF and A23187 in platelet aggregation involves activation of thromboxane and tyrosine kinase pathways.     

Inhibitory effects of 2'-hydroxychalcones on rat lens aldose reductase and rat platelet aggregation

Inhibitory effects of synthetic 2'-hydroxychalcone derivatives on rat lens aldose reductase (RLAR) and on platelet aggregation were investigated for the prevention or the treatment of chronic diabetic complications. 5'-chloro-4,2'-dihydroxychalcone (8) and 5'-chloro-3,2'-dihydroxychalcone (27) exhibited a potent inhibitory effect on rat platelet aggregation induced by ADP (IC50=0.10 and 0.06 mg/ml, respectively) and collagen (IC50=44 and 16 microg/ml, respectively) but showed relatively weak inhibitory activities on RLAR.     

Inhibitory effects of acetylmelodorinol, chrysin and polycarpol from Mitrella kentii on prostaglandin E₂ and Thromboxane B₂ production and platelet activating factor receptor binding

Acetylmelodorinol, chrysin and polycarpol, together with benzoic acid, benzoquinone and stigmasterol were isolated from the leaves of Mitrella kentii (Bl.) Miq. The compounds were evaluated for their ability to inhibit prostaglandin E? (PGE?) and thromboxane B? (TXB?) production in human whole blood using a radioimmunoassay technique. Their inhibitory effect on platelet activating factor (PAF) receptor binding to rabbit platelet was determined using 3H-PAF as a ligand. Among the compounds tested, chrysin showed a strong dose-dependent inhibitory activity on PGE(2) production (IC?? value of 25.5 μM), which might be due to direct inhibition of cyclooxygenase-2 (COX-2) enzymatic activity. Polycarpol, acetylmelodorinol and stigmasterol exhibited significant and concentration-dependent inhibitory effects on TXB? production with IC?? values of 15.6, 19.1 and 19.4 μM, respectively, suggesting that they strongly inhibited COX-1 activity. Polycarpol and acetylmelodorinol showed strong dose-dependent inhibitory effects on PAF receptor binding with IC?? values of 24.3 and 24.5 μM, respectively.     

A novel class of platelet activating factor (PAF) antagonists. II. Modification of the 2-position of the glycerol backbone of PAF-sulfonamide isosteres.

In a continuing effort to obtain more potent platelet activating factor (PAF) antagonists, we tried to synthesize a series of PAF-sulfonamide isosteres in which the substituent at the 2-position was modified to an acetoxy equivalent other than the methoxy group. These modifications produced highly active PAF antagonists. Compound 3-[2-(5-methyl-2H-tetrazol-2-yl)-3-(octadecylcarbamoyloxy) propylaminosulfonyl]propylquinolinium iodide (52) showed the most potent activity in the in vitro inhibitory effect on PAF-induced platelet aggregation in rabbit platelet-rich plasma (IC50 = 125 nM) and also in the in vivo protective effect on PAF-induced lethality in mice, with prolonged duration of action. Optically active enantiomers of this compound were synthesized and the (S)-(-)-isomer (IC50 = 87 nM) was found to be three times more potent that the (R)-(+)-isomer (IC50 = 289 nM), clearly exemplifying the enantioselectivity in the PAF-antagonist action of this novel compound.     

A novel class of platelet activating factor (PAF) antagonists. I. Synthesis and structure-activity studies on PAF-sulfonamide isosteres.

New platelet activating factor (PAF) antagonists, 3 were synthesized by replacing the charged phosphate and trimethylammonium moieties with sulfonamide and heterocyclic quaternary ammonium functionalities, respectively (PAF-sulfonamide isosteres). Darmstoff phosphatidic acid analogues of this class (Darmstoff-sulfonamide isosteres), 6 were also synthesized. The activity of these compounds as PAF antagonists was evaluated from their in vitro inhibitory effect on PAF-induced platelet aggregation in rabbit platelet-rich plasma. Among the compounds tested, some of the 2-methoxypropane derivatives with an octadecylcarbamoyloxy or octadecylcarbamoylthio side chain at the 1-position and a propylsulfonamide function bearing a terminal polar substituent such as a quaternary quinolinium or substituted quinolinium group at the 3-position were found to be the most potent (IC50 = 0.3-0.6 microM).     

Lactone and cyclic ether analogues of platelet-activating factor. Synthesis and biological activities

Six-membered lactone and tetrahydropyran analogues of platelet-activating factor (PAF), 4-11, and related antagonistic derivatives 41-46 were synthesized. None of the delta-lactones 4-7 showed PAF-like activities, while the corresponding cyclic ethers 8, 9 and 11 were slightly active. Some of the cyclic antagonists showed more potent inhibitory activities than the open chain antagonist CV-3988 against platelet aggregation (rabbit platelet-rich plasma, IC50) and hypotension (rat, ID50) induced by C16-PAF: e.g. dl-3-[6-[O-(trans-3-heptadecylcarbamoyloxytetrahydropyran-2- yl)methyl]phosphonoxy]hexylthiazolium (inner salt) (41d) (IC50 5.5 x 10(-7) M, ID50 0.046 mg/kg, i.v.); dl-3-[5-[O-(cis-3-heptadecylcarbamoylthiotetrahydropyran-2- yl)methyl]phosphonoxy]pentylthiazolium (inner salt) (43c) (IC50 5.7 x 10(-7) M, ID50 0.076 mg/kg, i.v.).     

((吡啶-3-基)甲氧基)芳酸衍生物的合成及抗血小板聚集活性

以具有活血化瘀作用的中药有效成分阿魏酸为先导物,按生物电子等排原理,设计合成了6个((吡啶-3-基)甲氧基)芳酸衍生物,其结构经IR,1H NMR,13C NMR及MS确证.体外药效筛选结果显示,部分((吡啶-3-基)甲氧基)芳酸衍生物对二磷酸腺苷(ADP)诱导的血小板聚集具有较好的抑制活性,其中化合物1a的抑制作用明...     

Synthesis and serotonin 2 (5-HT2) receptor antagonist activity of 5-aminoalkyl-substituted pyrrolo[3,2-c]azepines and related compounds

A series of 5-aminoalkylpyrrolo[3,2-c]azepine derivatives was synthesized and their serotonin 2 (5-HT2) receptor antagonist and antiplatelet aggregation activities were evaluated. 5-HT2 receptor antagonist activity was largely determined by the nature of the substituent at the 8-position as well as the aminoalkyl group at the 5-position of the pyrrolo[3,2-c]azepine ring. Compound 18a, 5-[3-[4-(4-fluorophenyl)piperazin-1-yl]propyl]-8-hydroxy-1-methyl- 1,4,5,6,7,8-hexahydropyrrolo[3,2-c]azepin-4-one, was recognized as having potent 5-HT2 receptor antagonist activity with weak alpha1 adrenoceptor blocking activity and no significant D2 receptor binding affinity, while the corresponding isomeric pyrrolo[3,4-c]azepine derivative (22) displayed only weak 5-HT2 receptor antagonist activity. After racemic 18a was resolved directly via diastereomeric salt formation, each enantiomer was evaluated precisely. The 5-HT2 receptor antagonist activity of 18a was found to reside primarily in (-)-18a (which was about 14-fold more potent than (+)-18a in isolated guinea pig arteries). Consequently, (S)-(-)-18a (SUN C5174) displayed the overall best profile with potent 5-HT2 receptor antagonist activity (pA2=8.98+/-0.06) and high selectivity versus other receptors. SUN C5174 showed a marked inhibitory effect on the platelet aggregation induced by serotonin in combination with collagen and adenosine diphosphate (ADP) in canine or human platelet-rich plasma (IC50=6.5 to 16 nM). Moreover, this compound significantly inhibited the mortality rate in mouse acute pulmonary thromboembolytic death induced by collagen and serotonin at oral doses of 0.3 mg/kg or higher. SUN C5174 is currently undergoing clinical evaluation.     

Evaluation of affinity interaction between small molecules and platelets by open tubular affinity capillary electrochromatography

In this paper, an open tubular affinity capillary electrochromatography (OT‐ACEC) was developed by physical adsorption of rabbit platelets on the inner surface of capillary. The interactions between small molecules include adenosine diphosphate (ADP) (positive control), protocatechuic acid (negative control) and seven natural products (salvianolic acid B, salvianic acid A sodium, hydroxysafflor yellow A, ferulic acid, chlorogenic acid, sinapic acid, caffeic acid) and platelets were evaluated by their retention factors and binding constants obtained based on peak‐shift assay. Then, the activities of anti‐platelet aggregation induced by thrombin (THR), ADP and arachidonic acid (AA) for those small molecules (except ADP) were evaluated by turbidimetric method. The results indicate that: (i) ADP, a platelet aggregation inducer, had strong interaction with platelet, while protocatechuic acid that had no inhibition on platelet aggregation behaved no specific interaction; (ii) there was a positive correlation between the anti‐platelet aggregation activities of small molecules and their interactions with platelet, generally those compounds with higher binding constants with platelet exhibited higher activities. Therefore, the OT‐ACEC method developed in the present study can be a potential method to evaluate affinity interactions between small molecules and platelets, so as to predict the biological activities such as anti‐platelet aggregation for the small molecules.     

吡嗪类化合物的合成及其抗血小板凝集活性测试

为提高川芎嗪的抗血小板凝集活性,分别以不同的二胺、2,3-丁二酮和硫辛酸为起始原料,采用溴代、水解、环化、氧化、氢化、取代等反应,通过四条路线合成了7个川芎嗪衍生物,其结构经1H NMR、13C NMR及ESI-MS确证。采用Born比浊法初步测试了化合物的体外抗血小板凝集活性,结果显示,化合物1(IC50=0. 26mmol/L)、2(IC50=0. 27mmol/L)和7(IC50=0. 21mmol/L)对由二磷酸腺苷(ADP)诱导的血小板凝集具有一定的抑制活性,优于先导化合物川芎嗪(IC50=0. 49mmol/L)。因此,在不改变川芎嗪药效团的前提下对其进行不同程度的环化,能明显提高川芎嗪的抗血小板凝集活性,此研究为后期化合物的结构修饰提供了一定的参考价值。     

Rapulasides A and B: two novel intermolecular rearranged biiridoid glucosides from the roots of Heracleum rapula

Two novel intermolecular rearranged biiridoid glucosides, rapulasides A and B (1 and 2), have been isolated from the roots of Heracleum rapula. Their structures were identified by extensive spectral analysis especially different NMR techniques. NOESY experiment, with the help of Dreiding molecular model, was used to elucidate their relative stereochemistry. Both compounds were tested for their inhibitory effects on rabbit platelet aggregation induced by PAF, ADP, or AA, respectively. Only trends of inhibition were observed for them.     

川芎嗪衍生物的设计、合成及抗血小板凝集活性研究

分别以川芎嗪(TMP)和邻苯二胺为起始原料,经KMn O4氧化、酯缩合、环化、还原等步骤合成了7个新型的川芎嗪衍生物,其结构经1H NMR、13C NMR及HRMS确证。并采用Born比浊法初步测试了化合物的体外抗血小板凝集活性。结果表明,化合物3和7对二磷酸腺苷(ADP)诱导的血小板凝集抑制率IC50分别为0.23mmol/L和0.27mmol/L,优于母体化合物川芎嗪(0.42mmol/L)。     

Platelet activating factor (PAF) antagonists contained in medicinal plants: lignans and sesquiterpenes.

Hot aqueous extracts of medicinal plants were tested for their inhibitory effect on the binding of platelet activating factor (PAF) to rabbit platelets. The extracts of Forsythia suspensa VAHL. (Oleaceae), Arctium lappa L. (Compositae) and Centipeda minima (L.) A. BRAUN et ASCHERS (Compositae) showed significant activities. Since the main constituents of F. suspensa and A. lappa are lignans, 30 lignans were tested for their inhibitory effects on PAF binding to platelets and 9 lignans were found active. Four sesquiterpenes were isolated as active compounds from C. minima. In particular 6-O-angeloylplenolin and 6-O-senecioyplenolin are the most potent and specific PAF antagonists found in this study.     

Phenolic chemical composition of Petroselinum crispum extract and its effect on haemostasis

From the aqueous extract (Pc) of Petroselinum crispum (Mill) flat leaves specimens were isolated and identified the flavonoids apigenin (1), apigenin-7-O-glucoside or cosmosiin (2), apigenin-7-O-apiosyl-(1 --> 2)-O-glucoside or apiin (3) and the coumarin 2",3"-dihydroxyfuranocoumarin or oxypeucedanin hydrate (4). The inhibitory activity toward clotting formation and platelet aggregation was assessed for Pc flavonoids (1) and (2), and the coumarin (4). Pc showed no inhibition on clotting activity when compared with the control. On the other hand, a strong antiplatelet aggregation activity was observed for Pc (IC50 = 1.81 mg/mL), apigenin (IC50 = 0.036 mg/mL) and cosmosiin (IC50 = 0.18 mg/mL). In all cases ADP was used as inductor of platelet aggregation. Our results showed that Pc, apigenin and cosmosiin interfere on haemostasis inhibiting platelet aggregation. To the best of our knowledge this is the first report for the cosmosiin antiplatelet aggregation in vitro activity.     

Novel diphenylamine 2,4'-dicarboxamide based azoles as potential epidermal growth factor receptor inhibitors: synthesis and biological activity

Several hybrid molecules of diphenylamine-2,4'-dicarboxamide with various azolidinones and related heterocyclic rings have been synthesized and explored as epidermal growth factor receptor (EGFR) kinase inhibitors. Most of them displayed promising in vitro tyrosine kinase inhibition as well as potent cellular antiproliferative activity in the EGFR over-expressing breast cancer cell line (MCF-7). Compounds 12b and 13b that exhibited the highest inhibition in the kinase assay (89, 81% inhibition at 10 μM, respectively), showed potent antiproliferative effect against MCF-7 tumor cell line (IC(50) 1.04, 0.91 μM respectively). Molecular docking studies revealed that these compounds can bind to ATP binding site of the EGFR kinase domain and were involved in H-bonding with Met 793, in analogy to the known EGFR tyrosine kinase inhibitors. Moreover, compounds 15a-c possessed profound antitumor activity (IC(50) 0.59-0.73 μM) and significant EGFR-TK inhibition, making them of particular interest. In summary, the newly synthesized compounds provide promising new lead for the future design and development of anticancer agents of potential EGFR-TK inhibitory activity.     

Platelet aggregation inhibitors from Populus sieboldii Miquel.

The water extract of Populus sieboldii Miquel (Salicaceae) inhibited arachidonic acid-induced platelet aggregation. Pyrocatechol and salicyl alcohol were isolated as active constituents. Pyrocatechol showed an inhibitory effect on platelet aggregation induced by arachidonic acid with IC100 value of 4 microM, which was 25 times more potent than aspirin.     

The first examples of ilexgenin A hybrids as a new class of multi-potent, anti-platelet agents

Seventeen novel ilexgenin A hybrids（lA-aspirin） and（IA-NO）,as donor hybrids（IA-NO will release NO in vivo and function as NO donor）,were designed and synthesized in order to develop new multi-targeting agents for the treatment of platelet disorders.Their in vitro activities against ADP,AA and thrombin were evaluated.As a result,IA hybrids achieved substantial increases in the three tested pathways compared with IA.Encouragingly,the most potent hybrid compounds 6d and 14d displayed about 8-fold higher potency than aspirin,and 3-fold higher potency than the simultaneous administration of aspirin and IA in inhibiting ADP-induced aggregation with IC₅₀ values of 0.15 mmol/L and 0.14 mmol/L,respectively. The results suggest these IA hybrids are good candidates for multi-target therapies,and especially,may be considered as promising ADP agonists.     

Inhibitory effect of hexahydrocurcumin on human platelet aggregation

The effects of hexahydrocurcumin on adenosine diphosphate (ADP)-induced human platelet aggregation were studied. Treatment of human platelet-rich plasma with hexahydrocurcumin resulted in an inhibitory effect on platelet aggregation, suggesting the potential of this compound as an anti-atherosclerogenic agent in humans.     

Synthesis,Antiproliferative, and Antiplatelet Activities of Oxime‐Containing 3,4‐Dihydroquinolin‐2(1H)‐One Derivatives

Some oxime‐containing 3,4‐dihydroquinolin‐2(1H)‐one derivatives were synthesized and evaluated for their antiplatelet and antiproliferative activities. These compounds were synthesized via alkylation of hydroxyl precursors followed by the reaction with NH₂OH. The preliminary assays indicated that (Z)‐7‐[2‐(4‐fluorophenyl)‐2‐(hydroxyimino)ethoxy]‐3,4‐dihydroquinolin‐2(1H)‐one (13c) is the most active against U46619 induced platelet aggregation with an IC₅₀ value of 3.51 μM. For the inhibition of AA‐induced aggregation, (E)‐6‐[2‐(hydroxyimino)propoxy]‐3,4‐dihydroquinolin‐2(1H)‐one (15 ) is the most potent with an IC₅₀ value of 1.85 μM. These oxime‐containing 3,4‐dihydroquinolin‐2(1H)‐one derivatives were inactive against thrombin induced platelet aggregation with an IC₅₀ value of greater than 26.78 μM. For the antiproliferative activity, most of these oxime‐containing 3,4‐dihydroquinolin‐2(1H)‐one derivatives were inactive while (Z)‐7‐[2‐(hydroxyimino)‐2‐(naphthalen‐2‐yl)ethoxy]‐3,4‐dihydroquinolin‐2(1H)‐one (13a) exhibited only marginal activities with GI₅₀ value of 7.63, 7.34 and 6.36 μM against the growth of NPC‐TW01, NCI‐H661, and Jurkat respectively.     

Biphenyls, a new class of compound that inhibits platelet-activating factors

Honokiol (1), a constituent of Magnoliae cortex, and related synthetic biphenyls inhibited the platelet aggregation induced by a few aggregating agents. In particular, compounds 3, 5, and 10 had an inhibitory effect on platelet activating factor (PAF), which provides a new class of PAF inhibitor.