WO2012103807A1 - 一种双功能抗血小板聚集药物及其用途 - Google Patents

一种双功能抗血小板聚集药物及其用途 Download PDF

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WO2012103807A1
WO2012103807A1 PCT/CN2012/070804 CN2012070804W WO2012103807A1 WO 2012103807 A1 WO2012103807 A1 WO 2012103807A1 CN 2012070804 W CN2012070804 W CN 2012070804W WO 2012103807 A1 WO2012103807 A1 WO 2012103807A1
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medicament
platelet
platelet aggregation
activity
clopidogrel
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丁忠仁
胡亮
张思
杜洪光
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复旦大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/167—Purine radicals with ribosyl as the saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to the field of pharmaceutical technology, and in particular to an antiplatelet agent and its use in the prevention and treatment of arterial thrombotic diseases.
  • antiplatelet drugs include cyclooxygenase inhibitors such as aspirin, thienopyridine ⁇ 2 ⁇ ⁇ 2 receptor antagonists, such as clopidogrel (prapidogrel prasugrel, platelet fibrinogen receptor) Body antagonists and citrate diesterase inhibitors.
  • cyclooxygenase inhibitors such as aspirin, thienopyridine ⁇ 2 ⁇ ⁇ 2 receptor antagonists, such as clopidogrel (prapidogrel prasugrel, platelet fibrinogen receptor) Body antagonists and citrate diesterase inhibitors.
  • clopidogrel prapidogrel prasugrel, platelet fibrinogen receptor
  • antiplatelet drugs with different mechanisms of action are used in combination: triple application, It is used to achieve the clinical effect of increasing the curative effect and reducing the side effects.
  • the prevention and treatment of cardiovascular and cerebrovascular diseases is very important. So far, there is no correlation between ⁇ 2 ⁇ 22 receptor antagonism and citrate diesterase inhibition. Functional drug reports.
  • the object of the present invention is to provide a bifunctional antiplatelet drug, and more particularly to a bifunctional antiplatelet aggregation drug and use thereof, in order to overcome the deficiencies of the prior art.
  • the antiplatelet agent of the present invention has both ⁇ 2 ⁇ 2 receptor antagonism and phosphodiesterase inhibition dual activity.
  • the bifunctional antiplatelet aggregation drug of the present invention (abbreviated as BF061) has the structure of the formula (I), and has the chemical formula: C 2 () H 25 N 5 0 4 S , molecular weight; 431.5 i
  • the compound BF061 of the formula (I) of the present invention was subjected to in vivo test, and the results showed that the compound BF061 of the formula (I) of the present invention has both P2Y 12 receptor antagonist and phosphodiesterase inhibitor dual activity; the BF061 of the present invention A variety of agonist-induced platelet aggregation has a good inhibitory effect; the experimental results of the arterial thrombosis model in mice indicate that the BF061 has a significant antithrombotic activity similar to clopidogrel, and the side effects of bleeding are not obvious.
  • BF061 The in vivo antithrombotic effect of BF061 was studied. The results showed that in the 10% _ ferric chloride induced mouse thrombus model, BF061 25mg/kg tail vein injection significantly prolonged the embolization time of mesenteric arterioles. The time required for the formation of a stable thrombus with a diameter of 30 uM after administration was 418. 7 soil 55 2 seconds, which was significantly longer than that of the control group, 116 4 ⁇ 10, 0, while the clopidogrel group was embolized. 702. 7 ⁇ 81. 4 (shown in Figure 1).
  • the bleeding tendency of BF061 after intravenous administration was studied. The results showed that the bleeding tendency of BF061 after intravenous administration was weaker than that of clopidogrel. Specifically, the amount of bleeding in the tail vein of the mouse was measured within 10 minutes after tail-cutting. The amount of bleeding in the control group was 76 ⁇ L 4 L, and the amount of oral administration of clopidogrel 30 mg/kg was ⁇ 8, 6 ⁇ 2, 5 ⁇ , BF061 25 mg. /kg tail static ' pulse injection dose of 10, 5 ⁇ 1. 2 ⁇ , (as shown in Table 5).
  • the experimental results confirmed that the compound of formula (I) of the present invention has the dual activity of ⁇ 2 ⁇ 22 receptor antagonism and acid diesterase inhibition; the experimental results provide an anti-platelet mechanism of the drug, which is different from the known anti-platelet drugs.
  • Aspirin, clopidogrel, prasugrel, cilostazol and platelet fibrinogen receptor antagonist, the medicament of the present invention can be used as an antithrombotic drug for preventing and treating arterial thrombotic diseases such as coronary heart disease and stroke.
  • Figure 1 shows that ⁇ F061 shows an in vivo antithrombotic effect similar to clopidogrel in a ferric chloride-induced mouse thrombus model.
  • ferric chloride-induced thrombus was recorded for 0 minutes, 3-5 minutes, and vascular imaging data at 5. 5 minutes.
  • a small blood vessel thrombus appeared at 3.5 minutes, and a large-scale thrombus was observed at 5. 5 minutes.
  • the purpose of this example was to demonstrate the anti-phosphodiesterase activity of BF061.
  • the citrate diesterase was purified from human platelets, and the enzyme reaction was carried out in 200 ⁇ L of PBS buffer (137 mM NaCl, 2. 7 mM KCL 8. 8 mM Na 2 HP0 4 , 1.5 mM KH 2 P0 4 , 1 raM CaCl 2 , 1 mM MgCl 2 , 10 ⁇ M cAMP, pH 7. 4), the substrate is 25 ⁇ cAMP (Si gma), the system contains phosphodiesterase 10 ⁇ , The residual substrate concentration after the 30 minute enzyme reaction was measured to calculate the activity of the phosphodiesterase. The residual substrate concentration was determined by high pressure liquid chromatography. The column was a C18 column (Kromasi 1 4.
  • the binding force between the ligand ADP and the ⁇ 2 ⁇ 12 receptor was determined by atomic force microscopy (Asylum Research, Santa Barbara, CA), and the probe 'previously in ADP (500 ng/ ⁇ L, 20 L) The coating was overnight, and finally ADP was coupled to the probe, and the P2Y ]2 receptor was expressed in CH0-K1 cells.
  • the binding conditions of the probe to the cell surface receptor were measured at a pressure of 3 ⁇ /s, contact time. 0. 2 s , contact force 100 pN, cantilever correction coefficient about 20 pN/nm c
  • Table 2 After BF061 treatment, the binding force between the ligands ADP and ⁇ 2 ⁇ ⁇ 3 receptors is significantly weakened, indicating that BF061 is obvious. ⁇ 2 ⁇ 12 receptor antagonism.
  • the purpose of this example is to demonstrate that BF061 can inhibit ADP-induced human platelet aggregation.
  • Platelet preparation Platelets were obtained from healthy volunteers who signed informed consent. Volunteers did not take any antiplatelet drugs such as aspirin or clopidogrel within 20 days prior to blood collection.
  • the anticoagulant was ACD (85 mmo 1 3 - 1 sodium c itrate, 71, 38 ol L - 1 citric acid, and 27. 78 mmol L - 1 glucose). Centrifuge at 300 xg for 20 minutes to obtain a blood-rich small blood paddle. After taking the supernatant, centrifuge at 900 x g for 10 minutes to obtain platelets, and finally resuspend with Tyrode's buffer (138 mmol L - ⁇ NaCl, 2.
  • Platelet aggregation assay Platelet aggregation in platelet aggregation instrument (Model 400VS, Chrono-Log, Havers ton, PA), parameter setting: Stirring speed 900 rpm, temperature 37 ° C, before adding agonist ADP, platelet The solvent or drug was pretreated for 3 minutes, the platelet aggregation curve was monitored for 5 minutes, and the aggregation rate was defined as 100% in the solvent control group.
  • Example 4 Inhibitory effect of BF061 10 ⁇ 1 ⁇ 2 on other various agonist-induced human platelet aggregation
  • the purpose of this example was to demonstrate that BF061 can inhibit U46619, arachidonic acid and collagen-induced human platelet aggregation.
  • BF061 inhibits U46619, arachidonic acid and collagen-induced human platelet aggregation
  • Mouse (C57BL/6J) platelets were obtained in vitro, labeled with green fluorescence and injected into the normal mouse blood circulatory system through the tail vein.
  • the mouse mesenteric artery was isolated, treated with ferric chloride, and the blood vessel wall was induced to induce thrombus.
  • the thrombus formation process was observed under a fluorescence microscope (Le ica. Yang 00 ), and the thrombus formation image was recorded in real time using a confocal scanning microscope (Leica TCS SPE ). data.
  • the experiment was divided into a solvent control intravenous administration group, a BF06 intravenous administration group, and a clopidogrel oral administration group.
  • mice Six to eight weeks old mice (BALB/C) were anesthetized with 10% chloral hydrate (400 mg kg- ; ), and the tail was cut off from the distal centimeter. The distal end of the tail was quickly immersed in 1 ml of 37 ⁇ . In physiological saline, for 10 minutes, the absorbance of physiological saline containing mouse blood was measured by a colorimetric method (Beckman, DU800) at a wavelength of 560 nm, and the standard curve was physiological saline containing mouse blood at different dilutions, according to the standard. The curve calculates the amount of bleeding.
  • the experiment was divided into a solvent control group, a clopidogrel oral administration group, and a BF061 intravenous administration group.
  • the administration time was the same as in Example 5.
  • the results are shown in Table 5. No obvious bleeding tendency was observed after intravenous administration of BF061.

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Abstract

本发明属于药物技术领域，涉及一种具有式（Ⅰ）结构的双功能抗血小板聚集药物及其在预防和治疗动脉血栓性疾病中的应用。本发明药物同时具有P2Y12受体拮抗和磷酸二酯酶抑制活性。该药物的体内和体外的抗血小板聚集活性实验结果，提供了该药物的抗血小板机制，该药物的结构式和抗血小板机制不同于已知的抗血小板药物阿司匹林、氯比格雷、普拉格雷、西洛他唑及血小板纤维蛋白原受体拮抗剂，本发明药物对于多种激动剂诱导的血小板聚集具有良好的抑制效果；小鼠体内动脉血栓模型实验结果表明该药物具有显著的与氯吡格雷相似的抗血栓活性，而出血副作用不明显。本发明药物可以作为抗血栓药物用于治疗冠心病、中风等动脉血栓性疾病，其中式（Ⅰ）如下：

Description

- 种双功能抗血小板聚集药物及其用途 技术领域 本发明属于药物技术领域，具体涉及一种抗血小板药物及其在预防和治 疗动脉血栓性疾病中的应用。
技术背景
随着生活水平的提高， 中风、 冠心病等动脉血栓性疾病己成为威胁我国 和发达国家人民健康的头号杀手。 研究显示， 血小板异常激活引起的血管 内血栓形成是动脉血栓性疾病冠心病和中风的病理基础， 故抑制血小板激活 的抗血小板药成为预防与治疗这类疾病的主要手段。 有关研究表明了抗血小 板药对冠心病和中风的疗效是肯定的， 但实践显示， 冠心病和中风的治疗效 果仍未尽如人意。 目前临床上广泛应用的抗血小板药包括有环氧化酶抑制 剂， 如阿司匹林， 噻吩吡啶类 Ρ2Υί2受体拮抗剂， 如氯吡格雷（c lopidogrel 普拉格雷 （prasugrel ) 、 血小板纤维蛋白原受体拮抗剂和辚酸二酯酶抑制 剂。 但上述药物或具有胃藤道毒性， 或具有出血倾向等副作用， 且在临床应 用中有耐受和抵抗现象， 故， 上述存在的缺陷局限了它们的应用。 更重要的 是， 这些药物都是通过单一的机制抑制血小板激活， 因此具有对抗单一的血 小板激动剂的局限性。 临床上多把不同作用机制的抗血小板药二联或:三联应 用， 用于达到增加疗效、 降低副作用的临床效果。 综上， 对心脑血管血栓性 疾病的防治显得十分重要。 迄今为止， 尚无有关同^具有 Ρ2Υί2受体拮抗和辚 酸二酯酶抑制双重功能的药物报道。
发明内容
本发明的目的是为克服现有技术的缺陷， 提供一种具有双功能抗血小板 药物， 具体涉及一种双功能抗血小板聚集药物及其用途。 本发明的抗血小板 药物同时具有 Ρ2Υί2受体拮抗和磷酸二酯酶抑制双重活性。
具体而言， 本发明的双功能抗血小板聚集药物 （简称 BF061 ) , 其具有 式 （ I ) 的结构， 其化学式为： C2()H25N504S , 分子量为； 431.5 i
Figure imgf000004_0001
本发明的式 （ I ) 化合物 BF061进行了体夕卜体内试验， 结果表明， 本发 明的式（ I )化合物 BF061同时具有 P2Y12受体拮抗和磷酸二酯酶抑制双重活 性；本发明的 BF061对于多种激动剂诱导的血小板聚集具有良好的抑制效果； 小鼠体内动脉血栓模型实验结果表明该 BF061具有显著的与氯吡格雷相似的 抗血栓活性， 而出血副作用不明显。
(1) 对 BF061 的抗磷酸二酯酶活性进行研究， 结果表明： BF06 ΙθμΜ 对血小板磷酸二酯酶活性的抑制率为 6, 7 士 1, 2%, 3()μΜ 对磷酸二酯酶活 性的抑制率为 24.9 士 0.8%, ΙΟθμΜ对磷酸二酯酶活性的抑制率为 53.3 ± 2.0%, 明显高于 3 -异丁基 4 甲基黄嘌呤（ΙΒΜΧ, 非特异性磷酸二酯酶抑制 剂） 100 μΜ对磷酸二酯酶活性的抑制率 33.3 土 3.3%。 与之相比， 坎格雷 洛 （Cangrelor, 新一代 P2Y12受体拮抗剂） 100 nM对辚酸二酯酶活性无抑 制效果 （如表 1所示） 。
(2) 对 BF061的 Ρ2Υί2受体拮抗作用进行研究， 结果表明： BF06130 μΜ 时将 P2Yi2受体与其配体 ADP之间的结合力由 76, 7 士 1, 3 pN降至 51 .2 士 L 4 pN, 与坎格雷洛 ΙΟΟηΜ时的拮抗作用相似， 坎格雷洛 100 nM:时将 P2Yi2 受体与其配体 ADP之间的结合力由 74 9 + 0, 7 pN降至 44.6 士 0 9 pN 与之相比, MRS2179 (P2Y1受体拮抗剂） 在 300 ηΜ时则对 P2Y12受体与其配 体 ADP之间的结合力无明显影响，其结合力仅由 82, 6 士 L 5pN变为 84.1 + 1.4 pN (如表 2所示） 。
(3) 对 BF061对 ADP诱导的人血小板聚集的抑制效果进行研究， 结果 表明： BF061浓度依赖性抑制 ADP lOuM诱导的人血小板聚集， 其 IC50为 3.2 士丄.5 μΜ。 （如表 3所示）。
(4) 对 BF061 1()μ 对其他多种激动剂诱导的人血小板聚集的抑制效果 进行研究， 结果表明： 画 时 BF061 可完全抑制 [J46619 ΐ μΜ,花生四烯酸 0. 5mM诱导的血小板聚集，对胶原 2 !4诱导的血小板聚集的抑制率为 90 % (如 表 4所示） 。
( 5 ) 对 BF061 的体内抗血栓效果进行研究， 结果表明： 在 10 % _三氯化 铁诱导的小鼠血栓模型中， BF061 25mg/kg 尾静脉注射给药显著延长肠系膜 小动脉的栓塞时间， 给药后形成直径 30 uM 稳定血栓所需时间栓塞^间为 418. 7 土 55 2秒， 较对照组栓塞 ^间 116 4 士 10, 0明显延长， 而氯比格 雷给药组栓塞 ^间为 702. 7 士 81. 4 (如图 1所示） 。
( 6 ) 对 BF061 静脉给药后的出血倾向进行研究， 结果表明： 与氯比格 雷口服有较强的出血倾向相比， BF061 静脉给药后的出血倾向较弱。 具体为 剪尾后测量小鼠尾静脉 10分钟内出血量， 对照组出血量为 7 6 士 L 4 L, 氯比格雷 30mg/kg口服给药出血量为 ί8, 6 ± 2, 5 μΐ, BF061 25mg/kg尾静' 脉注射给药出血量为 10, 5 士 1. 2 μΐ, (如表 5所示） 。
实验结果证实， 本发明的式 （ I ) 化合物 BF061 同^具有 Ρ2Υί2受体拮 抗和 酸二酯酶抑制双重活性； 实验结果提供了该药物的抗血小板机制， 其不 同于已知的抗血小板药物阿司匹林、 氯比格雷、 普拉格雷、西洛他唑及血小板纤 维蛋白原受体拮抗剂， 本发明药物可以作为抗血栓药物用于预防和治疗冠心病、 中风等动脉血栓性疾病》
附图说明
图 1是 β F061在 氯化铁诱导的小鼠血栓模型中显示与氯比格雷类似的 体内抗栓效果，
其中显示了， 记录 氯化铁诱导血栓 0分钟， 3 5分钟， 5. 5分钟时血管 影像资料， 对照组 3. 5分钟时可见血管小血栓出现， 5. 5分钟时可见明显大范 围血栓将血管阻塞， BF061和氯比格雷给药组 3. 5分钟和 5. 5分钟均未见小血 栓或大范围血栓形成。
具体实施方式
下面结合具体实施倒， 进一步阐述本发明。 应理解， 这些实施例仅用于 说明本发明而不用于限制本发明的范圈》 下列实施例中未注明具体条件的实 验方法， 通常按照常规条件如 Alan D. Michel son等人， 在血小板第二版（New York: Elsevier Press, 2007)中所述的条件， 或按照制造厂商所建议的条 件。 除非另外说明， 文中所使 ]¾试剂来自国药试剂有限公司，百分比和份数 按重量计算。
除非另行定义，所有专业与科学用语与本领域熟练人员所熟悉的意义相 同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中„ 文中所述的较佳实施方法与 ^料仅作示范之用。
实施例 1. BF061的 ίΦ制磷酸二酯酶活性
本实施例的目的是为了证明 BF061的抗磷酸二酯酶活性。
在本实施例中， 辚酸二酯酶纯化自人血小板， 酶反应在 200 μ L PBS缓 冲液 ( 137 mM NaCl, 2. 7 mM KCL 8. 8 mM Na2HP04, 1. 5 mM KH2P04, 1 raM CaCl2, 1 mM MgCl2, 10 μ M cAMP, pH 7. 4)中完成， 底物为 25 μΜ的 cAMP ( si gma公 司） ， 体系中含有磷酸二酯酶 10 Ά , 通过测量 30分钟酶反应后的残余底物 浓度^算磷酸二酯酶的活性。 残余底物浓度用高压液相色谱测定， 色谱柱为 C18柱 (Kromasi 1 4. 6 X 150 mm, Eka-chemi cals, Bohns, Sweden) 0 结果 如表 1所示， 显示 BF061有明显的磷酸二酯酶活性抑制效果。 表] h BF061抑制磷酸二酯酶活性
Figure imgf000006_0001
实施例 2. BF061 的 P2Y1S受体拮抗作用
本实施例的目的是证明 BF061_具有 Ρ2Υί3受体拮抗作用。
在本实施例中， 配体 ADP和 Ρ2Υ12受体之间的结合力通过原子力显微镜 (Asylum Research, Santa Barbara, CA )测定，探针'预先在 ADP ( 500 ng/ μ L, 20 L)中包被过夜， 最终将 ADP偶联在探针上， P2Y]2受体表达于 CH0- K1细 胞， 探针与细胞表面受体的结合力的测定条件：压力速度 3 μ ηι/s , 接触时间 0. 2 s , 接触力 100 pN, 悬臂矫正系数约 20 pN/nm c 结果如表 2所示， BF061 处理后， 配体 ADP和 Ρ2Υί3受体之间的结合力明显减弱， 显示 BF061具有明显 的 Ρ2Υ12受体拮抗作用。
表 2 : BF061拮抗 Ρ2Υ12受体与其配体结合
Figure imgf000007_0001
实施例 3. BF061对 ADP诱导的人血小板聚集的 ίΦ制效果
本实施例的目的是证明 BF061可以抑制 ADP诱导的人血小板聚集
血小板的制备： 血小板来自于签订知情同意书的健康志愿者， 志愿者在 取血前 20 天内未服用阿司匹林， 氯比格雷等任何抗血小板药物。 抗凝剂采 用 ACD (85 mmo 1 3— 1 sodium c itrate, 71, 38 ol L— 1 citric acid, and 27。 78 mmol L— 1 glucose) 。 300 x g 离心 20分钟得到富血小扳血桨, 取上清后 900 X g进一歩离心 10分钟得到血小板，最终用 Tyrode ' s buffer重悬 （138 mmol L—丄 NaCl, 2. 7 mmol L— 1 KC1, 2 mmol L— 1 MgCl2, 0. 42 mmol Na¾P04， 5 mmol L— 1 glucose, 10 mmo 1 L— 1 HEPES, 0, 2% bov ine serum albumin, and 0. 02 unit mL;1 apyrase, pH 7. 4) , 得到洗过的血小板， 血小板数目调整为 2. 5 X 108 个 血小板 /
血小板的聚集测定： 血小板的聚集在血小板聚集仪 （（Model 400VS, Chrono-Log, Havers ton , PA) , 参数设定： 搅拌速度 900 rpm , 温度 37。C , 在加入激动剂 ADP之前， 血小板用溶剂或药物预处理 3分钟， 血小板聚集曲 线监测时间为 5分钟， 聚集率定义以溶剂对照组为 100 %。
聚集抑制结果如表 3 所示， BF061 浓度依赖性抑制 10 μ M ADP ( Chrono log公司） 诱导的人血小板聚集， 其 IC50为 3. 2 士 i. 5 μ M。 表 3 : BF061浓度依赖性抑制 ADP诱导的人血小板聚集
Figure imgf000008_0001
实施例 4. BF061 10μ½对其他多种激动剂诱导的人血小板聚集的抑制效果 本实施例的目的是证明 BF061可以抑制 U46619 ,花生四烯酸和胶原诱导的人 血小板聚集。
血小板的制备和血小板的聚集测定方法同实施例 3。 结果如表 4所示， BF061 在 10 μ Μ 时明显抑制 U46619 ( Chrono- log 公司） ，花生四烯酸 ( Chrono- log公司） 和胶原 （ Chrono- log公司） 诱导的人血小板聚集。
表 4: BF061抑制 U46619、 花生四烯酸和胶原诱导的人血小板聚集
Figure imgf000008_0002
实施例 5. BF061的体内抗血栓效果
体外获取小鼠（C57BL/6J)血小板，标记绿色荧光后通过尾静脉注入正常 小鼠血液循环系统。 分离出小鼠肠系膜动脉， 用 氯化铁处理， 损伤血 管壁诱发血栓， 在荧光显微镜 ( Le ica. 陽 00 ) 下观察血栓形成过程， 使用 共聚焦扫描显微镜 （Leica TCS SPE ) 实时记录血栓形成影像资料。 实验分 为溶剂对照静脉给药组、 BF06 静脉给药组， 氯 格雷口服给药组。 BF061 给药 30分钟后 （氯吡格雷组为氯比格雷 30 mg/kg灌胃， 每天一次， 第二天 给药后 2小时）用 10 % :三氯化铁诱发血栓并录血栓形成影像资料。 结果如图 1 所示， BF061 静脉给药显著抑制≡氯化铁诱发的体内血栓形成过程， 抑制 效果类似与氯比格雷给药组。
实施例 6. BF061静脉给药后的出血倾向
6到 8周龄的小鼠（BALB/C)腹腔注射 1 0 % 水合氯醛 (400 mg kg— ;)麻醉, 尾部离远端 厘米处截断， 迅速将断尾远端浸入 1毫升 37 Ό的生理盐水中， 保持 10分钟， 采用比色法 （Beckman公司， DU800 ) 测定含小鼠血液的生理 盐水的吸光度， 波长为 560纳米， 标准曲线为不同稀释度含小鼠血液的生理 盐水， 根据标准曲线计算出血量。 实验分为溶剂对照组， 氯比格雷口服给药 组， BF061静脉给药组， 给药时间同实施例 5， 结果如表 5所示， BF061静脉 给药后未见明显的出血倾向。
表 5 : BF061副作用明显弱于氯比格雷
Figure imgf000009_0001
** ρ < 0. 0 i与对照组相比； < 0. 05与氯吡格雷组相比。

Claims

1- 一种双功能抗血小板聚集药物， 其特征在于， 其具有式 ( I ) 的化合 物结构，
其 , 分子量为： 43】.51，
Figure imgf000010_0001
( I )
2、 按权利要求 1所述的双功能抗血小板聚集药物， 其特征在于， 所述的 双功能是 Ρ2Υ;2受体拮抗和磷酸二酯酶抑制双重活性。
3、 权利要求 1或 2的双功能抗血小板聚集药物在制备抗血栓药物中的用 途。
4、 权利要求 1 或 2的双功能抗血小板聚集药物在制备预防和 /或治疗动 脉血栓性疾病药物中的用途。
5、 按权利要求 4的用途， 其特征在于， 所述的动詠血栓性疾病是冠心病或 中风。
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