P2Y_(12)受体抑制剂的合成及其抗血小板聚集活性研究
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摘要
随着社会的进步,越来越多的生活方式类疾病正不断威胁着人类的健康。根据世界卫生组织(WHO)2008年的统计,全球近1/4的死亡病例源于心脑血管类疾病。因此,研究心脑血管类疾病的病理及治疗是富有挑战的课题。
研究显示动脉粥样硬化血栓是缺血性心脏病和血管阻塞性脑疾病形成的主要原因,血小板在动脉血栓形成进程中起关键性的作用。由于血小板细胞膜上有多种受体,它们分别可以与不同的血小板激动剂结合导致血小板激活。这些激动剂包括凝血酶、血栓素A2(TXA2)、胶原质和二磷酸腺苷(ADP)等。
P2Y12受体抑制剂是抗血小板研究领域的热点,由于其具有很强的激活信号放大作用。该放大作用不仅表现在对血小板聚集信号的放大,同时也放大随后的多步信号传导,包括血小板颗粒的释放和血小板促凝活性的增强。因此,这是一个较理想的靶标。早些时间上市的药物属于噻吩吡啶类,包括噻氯匹啶、氯吡格雷、普拉格雷。该类药物作用靶点明确、效果明显,是抗血栓领域主流药物。该类药物都属于前药,进入体内主要经过肝脏CYP2C19酶代谢成活性形态来发挥作用。这些活性药物相对不稳定并且与P2Y12受体形成不可逆结合。因此,被抑制的血小板在其7-10天的生命周期里完全失去活性,必须要等血液形成新的血小板且未被活性代谢物抑制才能恢复血小板功能。因此,开发出可逆的P2Y12受体抑制剂成为近年来的研究热点。
在可逆的P2Y12受体抑制剂研究中,人们期望当血浆药物水平下降时,药物可以从血小板P2Y12受体解离重新进入血浆,同时血小板功能恢复。依然处于临床试验阶段的坎格雷拉(Cangrelor)属于这类药物。但坎格雷拉只能静脉给药。于2011年7月上市的药物替格瑞洛(Ticagrelor, AZD6140, Brilinta)是第一个口服可逆P2Y12受体抑制剂。临床试验证明的替格瑞洛比氯吡格雷对ADP诱导的血小板聚集效果更好。更重要的证据来自于对已经预服用氯吡格雷的病人,替格瑞洛可以更进一步增强疗效。
本课题选择可逆P2Y12受体抑制剂为目标,从替格瑞洛分子结构出发,设计出全新的合成路线：从容易获得的手性氨基酸出发,经过区域选择Diels-Alder反应来性构建桥环分子4,再通过桥环自身的手性诱导,对其双键二羟基化引入手性二醇,再氢化开环获得另外两个绝对构型的手性中心,然后通过烷基化成功制备手性环戊烷胺片段S-1。
在合成手性环丙烷胺S-2片段时,利用樟脑磺酰胺的手性诱导作用,对肉桂酰胺衍生物15进行手性选择环丙烷化反应。再通过水解脱除手性辅基得环丙羧酸17。再通过Curtis重排顺利制得手性环丙烷胺S-2。
对巯基嘧啶S-3的合成,我们优化了文献报道的方法,通过容易制备的偶氮化合物21进行氯代,后在普通铂碳催化氢化条件下可以大量制备巯基嘧啶S-3。该方法简化了合成工艺,为大规模生产奠定了基础。
在成功合成了关键的片段后,先用手性环戊烷胺S-1片段对二氯巯基嘧啶S-3取代,将产物用亚硝酸异戊酯处理环合,制得含核心片段三唑并嘧啶结构的关键中间体24.再用设计的基团对24进行取代后脱除丙酮叉保护。至此,顺利完成新型P2Y12受体抑制剂的合成。
通过新颖的合成工艺建立起来的化学合成平台,为类似物的合成创造了条件。根据同源模建原理,利用牛视紫红质蛋白结晶,模拟出P2Y12受体的三维结构,建立了计算机模拟的受体-配体结合模型。对模型的深入研究,初步了解替格瑞洛分子与P2Y12受体之间的结合。识别出手性环丙烷胺片段及二氟苯基团对整个分子活性的贡献。此外,我们还完成化合物抗血小板的活性测试手段。至此,完成了一整套平台的建立。
从计算机模型预测的结果,我们设计、合成出一大批替格瑞洛的类似物。通过体外抗血小板聚集活性测试,发现化合物T-34和T-47活性优于对照药物替卡瑞洛,化合物T-17, T-19, T-25, T-37和T-49的活性与替格瑞洛相当。进一步的构效关系(SAR)的研究,得出初步结论,结果如下：
1.苯基烷胺类化合物系列,苄位氟取代分子(T-1)比阳性对照替格瑞洛(EC50=71.5uM)活性略弱。其他的开三元环分子则大幅度失去活性。
2.环化苯基烷胺类化合物系列,烷基芳基稠合的六元环分子、烷基烷基六元环分子及稠合五元环分子都大幅度失去活性。可能是分子构象由于环合的因素产生大幅度的改变而失去活性。
3.芳杂烷胺类化合物系列,吡啶类分子(T-34)比阳性对照替格瑞洛活性强。同时吡啶类分子T-37与替格瑞洛活性相当。这个结果有非常积极的意义。同时我们也发现有些五元杂环分子(T-16和T-17)及双环分子(T-12)都有较好的活性。
4.脂肪胺类化合物系列,直链脂肪胺T-47含六个碳原子的活性优于阳性对照替格瑞洛。另一个直链分子T-49和含三元环的分子T-25及含氟烯烃T-19也有非常好的活性。整个系列具有相当高的活性水平。可作为后期结构修饰的先导物进一步优化,为研发新型抗血板药物打下坚实的基础。
Along with the social development, more and more people are suffered from the diseases associated, with their lifestyle. Cardiovascular disease is the leading cause of deaths worldwide according to WHO's statistics. The causes of cardiovascular disease are diverse but atherosclerosis and/or hypertension are the most common. Additionally, aging also accounts for a number of physiological and morphological changes that alter cardiovascular function and subsequently increased risk of cardiovascular disease.
Platelets aggregate plays very important roles in atherosclerosis. Studies show that platelets aggregate uses fibrinogen and von Willebrand factor (vWF) as a connecting agent. The most abundant platelet aggregation receptor is glycoprotein Ⅱb/Ⅲa (gpⅡb/Ⅲa) Activated platelets will adhere, to the collagen via glycoprotein (GP) Ⅰa. Aggregation and adhesion act together to form the platelet plug.
Platelet aggregation is stimulated by ADP, thromboxane, and a2receptor-activation. Human platelets have three types of P2receptors:P2X1, P2Y1and P2Y12. After thoroughly explorations, scientists have documented only P2Y12is clinically correlated available.
P2Y12belongs to the Gi class of a group of G protein-coupled (GPCR) purinergic receptors.P2Y12protein is found mainly but not exclusively on the surface of blood platelets, and is an important regulator in blood clotting. As this receptor is involved in platelet aggregation, and is a potential target for the treatment of thromboembolisms and other clotting disorders. Nowadays, pharmaceutical industry has developed two major drug classes:Thienopyridines series, containing Clopidogrel, Prasugrel and Ticlopidine. The other is nucleotide/nucleoside analogs, which contains Cangrelor, Elinogrel and Ticagrelor.
Ticagrelor was approved by the US Food and Drug Administration on July20,2011for treating of acute coronary syndrome. Like the thienopyridines prasugrel, clopidogrel and ticlopidine, ticagrelor blocks adenosine diphosphate (ADP) receptors of subtype P2Y12-In contrast to the other anti-platelet drugs, ticagrelor has a binding site different from ADP. This binding format makes it an allosteric antagonist, and the blockage is reversible. Moreover, the drug does not need hepatic activation, which might work better for patients with genetic variants regarding the enzyme CYP2C19.
In this dissertation, a series of me-too drugs derivated from Ticagrelor were designed, synthesized and assayed. We developed a new synthetic route to access the key subunit S-1. Started from chiral amino acid, the bridge ring4was constructed via regio-selective Diels-Alder reaction. Depending on the rigid structure of the bridge, two chiral centers were created via dihydroxylation from olefin. Then after remove the chiral auxiliary group and reduction to free hydroxyl group and amine, the key intermediate contains four desired chiral centers was successfully created. Then after alkylation and reduction,S-1subunit was synthesized. The route avoids the reported highly expensive starting material though it takes some steps longer.
After S-1was prepared in large scale, novel P2Y12inhibitors were fully assembeled using a novel route. Meanwhile, we also established a computer model for Ticagrelor and P2Y12, which is created with Homology Modeling from bovine rhodopsin crystal protein data. After investigate the binding model, the chiral cycpropal part along with the attaching difluorophenyl ring was identified for the further exploration area as the binding is not clear form the established model. The assay protocol was also created for testing very inhibitor to evaluate the anti-platelet aggregation.
Thus far, all of the working templates were created and we designed four major series of analogues based on the model and also with basic medicinal chemistry concepts. After the synthesis work and the bioassays, we got some promise results such as T-37and T-47are more potent than the control Ticagrelor. Moreover, some analogues like T-17, T-19, T-25, T-34, and T-49show very similar potency as Ticagrelor. Some useful SARs were also generated as follows.
1) For phenylalkylamine series, then benzyl position substituted with fluorine shows slightly less potency than Ticagrelor. The other ring-opened analogues lost most of potency. We decided inhale the further exploration this area.
2) For cyclized phenylalkylamine series, all of the analogues including alkyl-aromatic additional ring or alkyl-alkyl additional ring are lose potency no matter the ring size.
3) Introducing heterocycle containing Nitrogen is an interesting idea as we identified T-37, a pyridine ring containing compound, shows more potent than Ticagrelor. Also, another pyridine ring containing compound T-34has very similar potency as Ticagrelor. It's a very encouraging result as it shows that the molecular can be improved based on our strategies. Meanwhile, some five-membered ring (T-16and T-17) and bicyclic ring (T-12) show moderate potency.
4) In the alkyl amine series, we also find the six carbon straight link (T-47) has better potency than Ticagrelor. Besides, five carbon straight link (T-49), the analogue containing a cycpropal ring (T-25) and also fluoroolefin (T-19) show ideal potency. Actually, the whole series have better potency over the other series.
Based on these scattered data and SARs, our work paved a practicable methods for synthesis, evaluation compounds with the aim for treating platelets aggregate. Some interesting compounds were identified with good potency and their further profile is under investigating. It's also give chance for further modification the other part like cyclopental ring and also the propel sulfur parts in the future.
研究显示动脉粥样硬化血栓是缺血性心脏病和血管阻塞性脑疾病形成的主要原因,血小板在动脉血栓形成进程中起关键性的作用。由于血小板细胞膜上有多种受体,它们分别可以与不同的血小板激动剂结合导致血小板激活。这些激动剂包括凝血酶、血栓素A2(TXA2)、胶原质和二磷酸腺苷(ADP)等。
P2Y12受体抑制剂是抗血小板研究领域的热点,由于其具有很强的激活信号放大作用。该放大作用不仅表现在对血小板聚集信号的放大,同时也放大随后的多步信号传导,包括血小板颗粒的释放和血小板促凝活性的增强。因此,这是一个较理想的靶标。早些时间上市的药物属于噻吩吡啶类,包括噻氯匹啶、氯吡格雷、普拉格雷。该类药物作用靶点明确、效果明显,是抗血栓领域主流药物。该类药物都属于前药,进入体内主要经过肝脏CYP2C19酶代谢成活性形态来发挥作用。这些活性药物相对不稳定并且与P2Y12受体形成不可逆结合。因此,被抑制的血小板在其7-10天的生命周期里完全失去活性,必须要等血液形成新的血小板且未被活性代谢物抑制才能恢复血小板功能。因此,开发出可逆的P2Y12受体抑制剂成为近年来的研究热点。
在可逆的P2Y12受体抑制剂研究中,人们期望当血浆药物水平下降时,药物可以从血小板P2Y12受体解离重新进入血浆,同时血小板功能恢复。依然处于临床试验阶段的坎格雷拉(Cangrelor)属于这类药物。但坎格雷拉只能静脉给药。于2011年7月上市的药物替格瑞洛(Ticagrelor, AZD6140, Brilinta)是第一个口服可逆P2Y12受体抑制剂。临床试验证明的替格瑞洛比氯吡格雷对ADP诱导的血小板聚集效果更好。更重要的证据来自于对已经预服用氯吡格雷的病人,替格瑞洛可以更进一步增强疗效。
本课题选择可逆P2Y12受体抑制剂为目标,从替格瑞洛分子结构出发,设计出全新的合成路线：从容易获得的手性氨基酸出发,经过区域选择Diels-Alder反应来性构建桥环分子4,再通过桥环自身的手性诱导,对其双键二羟基化引入手性二醇,再氢化开环获得另外两个绝对构型的手性中心,然后通过烷基化成功制备手性环戊烷胺片段S-1。
在合成手性环丙烷胺S-2片段时,利用樟脑磺酰胺的手性诱导作用,对肉桂酰胺衍生物15进行手性选择环丙烷化反应。再通过水解脱除手性辅基得环丙羧酸17。再通过Curtis重排顺利制得手性环丙烷胺S-2。
对巯基嘧啶S-3的合成,我们优化了文献报道的方法,通过容易制备的偶氮化合物21进行氯代,后在普通铂碳催化氢化条件下可以大量制备巯基嘧啶S-3。该方法简化了合成工艺,为大规模生产奠定了基础。
在成功合成了关键的片段后,先用手性环戊烷胺S-1片段对二氯巯基嘧啶S-3取代,将产物用亚硝酸异戊酯处理环合,制得含核心片段三唑并嘧啶结构的关键中间体24.再用设计的基团对24进行取代后脱除丙酮叉保护。至此,顺利完成新型P2Y12受体抑制剂的合成。
通过新颖的合成工艺建立起来的化学合成平台,为类似物的合成创造了条件。根据同源模建原理,利用牛视紫红质蛋白结晶,模拟出P2Y12受体的三维结构,建立了计算机模拟的受体-配体结合模型。对模型的深入研究,初步了解替格瑞洛分子与P2Y12受体之间的结合。识别出手性环丙烷胺片段及二氟苯基团对整个分子活性的贡献。此外,我们还完成化合物抗血小板的活性测试手段。至此,完成了一整套平台的建立。
从计算机模型预测的结果,我们设计、合成出一大批替格瑞洛的类似物。通过体外抗血小板聚集活性测试,发现化合物T-34和T-47活性优于对照药物替卡瑞洛,化合物T-17, T-19, T-25, T-37和T-49的活性与替格瑞洛相当。进一步的构效关系(SAR)的研究,得出初步结论,结果如下：
1.苯基烷胺类化合物系列,苄位氟取代分子(T-1)比阳性对照替格瑞洛(EC50=71.5uM)活性略弱。其他的开三元环分子则大幅度失去活性。
2.环化苯基烷胺类化合物系列,烷基芳基稠合的六元环分子、烷基烷基六元环分子及稠合五元环分子都大幅度失去活性。可能是分子构象由于环合的因素产生大幅度的改变而失去活性。
3.芳杂烷胺类化合物系列,吡啶类分子(T-34)比阳性对照替格瑞洛活性强。同时吡啶类分子T-37与替格瑞洛活性相当。这个结果有非常积极的意义。同时我们也发现有些五元杂环分子(T-16和T-17)及双环分子(T-12)都有较好的活性。
4.脂肪胺类化合物系列,直链脂肪胺T-47含六个碳原子的活性优于阳性对照替格瑞洛。另一个直链分子T-49和含三元环的分子T-25及含氟烯烃T-19也有非常好的活性。整个系列具有相当高的活性水平。可作为后期结构修饰的先导物进一步优化,为研发新型抗血板药物打下坚实的基础。
Along with the social development, more and more people are suffered from the diseases associated, with their lifestyle. Cardiovascular disease is the leading cause of deaths worldwide according to WHO's statistics. The causes of cardiovascular disease are diverse but atherosclerosis and/or hypertension are the most common. Additionally, aging also accounts for a number of physiological and morphological changes that alter cardiovascular function and subsequently increased risk of cardiovascular disease.
Platelets aggregate plays very important roles in atherosclerosis. Studies show that platelets aggregate uses fibrinogen and von Willebrand factor (vWF) as a connecting agent. The most abundant platelet aggregation receptor is glycoprotein Ⅱb/Ⅲa (gpⅡb/Ⅲa) Activated platelets will adhere, to the collagen via glycoprotein (GP) Ⅰa. Aggregation and adhesion act together to form the platelet plug.
Platelet aggregation is stimulated by ADP, thromboxane, and a2receptor-activation. Human platelets have three types of P2receptors:P2X1, P2Y1and P2Y12. After thoroughly explorations, scientists have documented only P2Y12is clinically correlated available.
P2Y12belongs to the Gi class of a group of G protein-coupled (GPCR) purinergic receptors.P2Y12protein is found mainly but not exclusively on the surface of blood platelets, and is an important regulator in blood clotting. As this receptor is involved in platelet aggregation, and is a potential target for the treatment of thromboembolisms and other clotting disorders. Nowadays, pharmaceutical industry has developed two major drug classes:Thienopyridines series, containing Clopidogrel, Prasugrel and Ticlopidine. The other is nucleotide/nucleoside analogs, which contains Cangrelor, Elinogrel and Ticagrelor.
Ticagrelor was approved by the US Food and Drug Administration on July20,2011for treating of acute coronary syndrome. Like the thienopyridines prasugrel, clopidogrel and ticlopidine, ticagrelor blocks adenosine diphosphate (ADP) receptors of subtype P2Y12-In contrast to the other anti-platelet drugs, ticagrelor has a binding site different from ADP. This binding format makes it an allosteric antagonist, and the blockage is reversible. Moreover, the drug does not need hepatic activation, which might work better for patients with genetic variants regarding the enzyme CYP2C19.
In this dissertation, a series of me-too drugs derivated from Ticagrelor were designed, synthesized and assayed. We developed a new synthetic route to access the key subunit S-1. Started from chiral amino acid, the bridge ring4was constructed via regio-selective Diels-Alder reaction. Depending on the rigid structure of the bridge, two chiral centers were created via dihydroxylation from olefin. Then after remove the chiral auxiliary group and reduction to free hydroxyl group and amine, the key intermediate contains four desired chiral centers was successfully created. Then after alkylation and reduction,S-1subunit was synthesized. The route avoids the reported highly expensive starting material though it takes some steps longer.
After S-1was prepared in large scale, novel P2Y12inhibitors were fully assembeled using a novel route. Meanwhile, we also established a computer model for Ticagrelor and P2Y12, which is created with Homology Modeling from bovine rhodopsin crystal protein data. After investigate the binding model, the chiral cycpropal part along with the attaching difluorophenyl ring was identified for the further exploration area as the binding is not clear form the established model. The assay protocol was also created for testing very inhibitor to evaluate the anti-platelet aggregation.
Thus far, all of the working templates were created and we designed four major series of analogues based on the model and also with basic medicinal chemistry concepts. After the synthesis work and the bioassays, we got some promise results such as T-37and T-47are more potent than the control Ticagrelor. Moreover, some analogues like T-17, T-19, T-25, T-34, and T-49show very similar potency as Ticagrelor. Some useful SARs were also generated as follows.
1) For phenylalkylamine series, then benzyl position substituted with fluorine shows slightly less potency than Ticagrelor. The other ring-opened analogues lost most of potency. We decided inhale the further exploration this area.
2) For cyclized phenylalkylamine series, all of the analogues including alkyl-aromatic additional ring or alkyl-alkyl additional ring are lose potency no matter the ring size.
3) Introducing heterocycle containing Nitrogen is an interesting idea as we identified T-37, a pyridine ring containing compound, shows more potent than Ticagrelor. Also, another pyridine ring containing compound T-34has very similar potency as Ticagrelor. It's a very encouraging result as it shows that the molecular can be improved based on our strategies. Meanwhile, some five-membered ring (T-16and T-17) and bicyclic ring (T-12) show moderate potency.
4) In the alkyl amine series, we also find the six carbon straight link (T-47) has better potency than Ticagrelor. Besides, five carbon straight link (T-49), the analogue containing a cycpropal ring (T-25) and also fluoroolefin (T-19) show ideal potency. Actually, the whole series have better potency over the other series.
Based on these scattered data and SARs, our work paved a practicable methods for synthesis, evaluation compounds with the aim for treating platelets aggregate. Some interesting compounds were identified with good potency and their further profile is under investigating. It's also give chance for further modification the other part like cyclopental ring and also the propel sulfur parts in the future.
引文
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8. Kroll, M., J. Hellums, et al. "Platelets and shear stress." Blood (1996),88(5),1525-1541.
9. Bahou WF, Nierman WC, Durkin AS, Potter CL, Demetrick DJ. "Chromosomal assignment of the human thrombin receptor gene:localization to region q13 of chromosome 5". Blood (1993), 82 (5),1532-7.
10. Samuel Chackalamannil; Wang, Yuguang; Greenlee, William J.; Hu, Zhiyong; Xia, Yan; Ahn, Ho-Sam; Boykow, George; Hsieh, Yunsheng et al. "Discovery of a Novel, Orally Active Himbacine-Based Thrombin Receptor Antagonist (SCH 530348) with Potent Antiplatelet Activity". Journal of Medicinal Chemistry (2008),51 (11),3061-4.
11. Serebruany VL, Kogushi M, Dastros-Pitei D, Flather M, Bhatt DL. "The in-vitro effects of E5555, a protease-activated receptor (PAR)-1 antagonist, on platelet biomarkers in healthy volunteers and patients with coronary artery disease", Thromb Haemost. (2009),102(1),111-9.
12. Marcum JA, McKenney JB. et al. "Anticoagulantly active heparin-like molecules from mast cell-deficient mice". Am. J. Physiol. (1986),250, H879-888.
13. Chuang YJ, Swanson R. et al. "Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin". J. Biol. Chem. (2001), 276(18),14961-14971.
14. Bjork I, Lindahl U. "Mechanism of the anticoagulant action of heparin". Mol. Cell. Biochem. (1982),48(3),161-182.
15.李端,殷明,药理学,第六版,人民卫生出版社,275-277.
16. Lever R. and Page C.P. "Novel drug opportunities for heparin". Nat. Rev. Drug Discov. (2002), 1 (2),140-148.
17. Coombe D.R. and Kett W.C. "Heparan sulfate-protein interactions:therapeutic potential through structure-function insights". Cell. Mol. Life Sci. (2005),62 (4),410-424.
18. Ahmed I, Majeed A, Powell R "Heparin induced thrombocytopenia:diagnosis and management update". Postgrad Med J(2007),83 (983),575-82.
19. Haruyama H. and Wuthrich K. "Conformation of recombinant desulfatohirudin in aqueous solution determined by nuclear magnetic resonance". Biochemistry (1989),28 (10), 4301-4312.
20. Rydell TJ, Tulinsky A. et al. "Refined structure of the Hirudin-Thrombin complex". J. Mol. Biol. (1991),221 (2),583-601.
21. Holbrook AM, Pereira JA, Labiris R, McDonald H, Douketis JD, Crowther M, Wells PS "Systematic overview of warfarin and its drug and food interactions". Arch. Intern. Med. (2005),165(10),1095-106.
22. Whitlon DS, Sadowski JA, Suttie JW "Mechanism of coumarin action:significance of vitamin K epoxide reductase inhibition". Biochemistry (1978),17 (8),1371-7.
23. Freedman MD "Oral anticoagulants:pharmacodynamics, clinical indications and adverse effects". J Clin Pharmacol (1992),32 (3),196-209.
24. Holford NH. "Clinical pharmacokinetics and pharmacodynamics of warfarin. Understanding the dose-effect relationship". Clin Pharmacokinet (1986),11,483-504.
25. Horton JD, Bushwick BM "Warfarin therapy:evolving strategies in anticoagulation". Am Fam Physician (1999),59 (3),635-46.
26. Sneader, W. "The discovery of aspirin:A reappraisal". BMJ (Clinical research ed.) (2000),321 (7276),1591-4.
27. John Robert Vane "Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs". Nature-New Biology (1971),231 (25),232-5.
28. Vane JR, Botting RM "The mechanism of action of aspirin". Thromb Res (2003),110 (5-6), 255-8.
29. Kimura Y, Tani T, Kanbe T, Watanabe K, Arzneimittel-Forschung (1985),35(7A),1144-9.
30. Halkes PH, van Gijn J, Kappelle LJ, Koudstaal PJ, Algra A "Aspirin plus dipyridamole versus aspirin alone after cerebral ischaemia of arterial origin (ESPRIT), randomised controlled trial". The Lancet (2006),367 (9523),1665-73.
31. Loo MH, Egan D, Vaughan ED, Marion D, Felsen D, Weisman S "The effect of the thromboxane A2 synthesis inhibitor OKY-046 on renal function in rabbits following release of unilateral ureteral obstruction". J. Urol. (1987),137 (3),571-6.
32. Hennerici, M. G.; Bots, M. L.; Ford, I.; Laurent, S.; Touboul, P. J. "Rationale, design and population baseline characteristics of the PERFORM Vascular Project:an ancillary study of the Prevention of cerebrovascular and cardiovascular Events of ischemic origin with teRutroban in patients with a history oF ischemic strOke or tRansient ischeMic attack (PERFORM) trial". Cardiovascular Drugs and Therapy (2010),24 (2),175.
33. Sorbera, LA, Serradell, N, Bolos, J, Bayes, M "Terutroban sodium". Drugs of the Future (2006), 31 (10),867-873.
34. Michael Gent, J. Donald Easton, VladimirC. Hachinski, Edouard Panak, Jane Siourella, JohnA. Blakely, DavidJ. Ellis, JohnW. Harbison, RobinS. Roberts, AlexanderG.G. Turpie, "the Canadian American Ticlopidine Study (CATS) in thromboembolic stroke", The Lancet (1989), 333,1215-1220
35. CAPRIE Steering Committee "A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE)". The Lancet (1996),16 (9038),1329-39.
36. Pereillo JM, Maftouh M, Andrieu A, Uzabiaga MF, Fedeli O, Savi P, Pascal M, Herbert JM, Maffrand JP, Picard C "Structure and stereochemistry of the active metabolite of clopidogrel". Drug Metab. Dispos. (2002),30 (11),1288-95.
37. Stockl, KM; Le L; Zakharyan A; et al. "Risk of rehospitalization for patients using clopidogrel with a proton pump inhibitor". Arch Intern Med (2010),170 (8),704-10.
38. Baker WL, White CM. Role of Prasugrel, a Novel P2Y12 Receptor Antagonist, in the Management of Acute Coronary Syndromes. American Journal of Cardiovascular Drugs (2009),9 (4),213-229.
39. Michelle O'Donoghue,; Elliott M. Antman,; Eugene Braunwald, et al. "The Efficacy and Safety of Prasugrel With and Without a Glycoprotein Ⅱb/Ⅲa Inhibitor in Patients With Acute Coronary Syndromes Undergoing Percutaneous", J Am Coll Cardiol. (2009),54(8),678-685.
40. Wiviott SD, Braunwald E, McCabe CH et al. "Prasugrel versus clopidogrel in patients with acute coronary syndromes". NEngl J Med (2007),357 (20),2001-15.
41. Bhatt DL et al. "Effect of Platelet Inhibition with. Cangrelor during PCI on Ischemic Events" New England Journal of Medicine (2013), (March 10,2013, published initially online).
42. van Giezen JJ, Humphries RG. Semin Thromb Hemost (2005),31,195-204.
43. Christopher P Cannon, Robert A Harrington,Stefan James, et al. "Comparison of ticagrelor with clopidogrel in patients with a planned invasive strategy for acute coronary syndromes (PLATO), a randomised double-blind study". The Lancet (2010),375,283-293.
44. Christopher P. Cannon et al., "Safety, Tolerability, and Initial Efficacy of AZD6140, the First Reversible Oral Adenosine Diphosphate Receptor Antagonist, Compared With Clopidogrel, in Patients With Non-ST-Segment Elevation Acute Coronary Syndrome". J Am Coll Cardiol (2007),50,1844-1851.
45. Dorsam RT, Kunapuli SP "Central role of the P2Y12 receptor in platelet activation". J. Clin. Invest. (2004),113 (3),340-5.
46. Murugappa S, Kunapuli SP "The role of ADP receptors in platelet function". Front. Biosci. (2006),11 (1),1977-86.
47. Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, TMurden P, Nurden A, Julius D, Conley PB "Identification of the platelet ADP receptor targeted by antithrombotic drugs". Nature (2001),409 (6817),202-7.
48. Hubbard R "The molecular weight of rhodopsin and nature of the rhodopsin-digitonin complex". J Gen Physiol (1954),37,381-399.
49. Overington JP, Al-Lazikani B, Hopkins AL "How many drug targets are there?". Nat Rev Drug Discov (2006),5(12),993-6.
50. Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, Chae PS, Pardon E, Calinski D, Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH, Steyaert J, Skiniotis G, Weis WI, Sunahara RK, Kobilka BK "Crystal structure of the human beta2 adrenergic receptor-Gs protein complex". Nature (2011),477,549-555.
51. Wettschureck N, Offermanns S "Mammalian G proteins and their cell type specific functions". Physiol. Rev. (2005),85 (4),1159-204.
52. Xie, X. Q.; Chen, J. Z.; Billings, E. M., "3D structural model of the G-protein-coupled cannabinoid CB2 receptor". Proteins (2003),53, (2),307-319.
53. Orry, A. J.; Wallace, B. A., "Modeling and docking the endothelin G-protein-coupled receptor". Biophys J (2000),79 (6),3083-3094.
54. Huang, X.; Shen, J.; Cui, M.; Shen, L.; Luo, X.; Ling, K.; Pei, G; Jiang, H.; Chen, K., "Molecular dynamics simulations on SDF-1 alpha:binding with CXCR4 receptor. " Biophys J (2003),84(1),171-184.
55. Krzysztof Palczewski et al, "Crystal Structure of Rhodopsin:A G Protein-Coupled Receptor". Science (2000),739,289.
56. Gibas, C.; Jambeck, P., Developing Bioinformatics Computer Skills. O'Reilly & Associates, Inc, Sebastopol, (2001).
57. http://www.ebi.ac.uk/swissprot/
58. Altschul, S. F.; Madden, T. L.; Schaffer, A. A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D. J., "Gapped BLAST and PSI-BLAST:a new generation of protein database search programs". Nucleic Acids Res (1997),25 (17),3389-402.
59. Thompson, J. D.; Higgins, D. G.; Gibson, T. J., "Clustal-W-improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice". Nucleic Acids Res (1994),22(22),4673-4680.
60. Bockaert, J.; Pin, J. P., "Molecular tinkering of G protein-coupled receptors:an evolutionary success". Embo J(1999),18(7),1723-1729.
61. www.pdb.org
62. Sali, A.; Blundell, T. L., "Comparative protein modelling by satisfaction of spatial restraints". J Mol Biol (1993),234(3),779-815.
63. Laskowski, R. A.; Macarthur, M. W.; Moss, D. S.; Thornton, J. M., "Procheck-ap Program to Check the Stereochemical Quality of Protein Structures". J Appl Crystallogr (1993),26, 283-291.
64. Brian Springthorpe et al, "From ATP to AZD6140:The discovery of an orally active reversible P2Y12 receptor antagonist for the prevention of thrombosis". Bioorg. Med. Chem, Lett. (2007), 17,6013-6018.
65. Fjellstrom, Q. et al. WO 2009/093972.
66. Bonnert, R. et al, WO 98/28300.
67. Ingall, AH et al, EP0508687.
68. Allen R. Ritter and Marvin J. Miller, J. Org. Chem. (1994),59,4602-4611.
69. Brock T. Shireman and Marvin J. Miller, Tetrahedron Letters (2000),41,9537-9540.
70. Howard Ensign Simmons, Jr.; Smith, R.D. "4-Substituted-2,3,5-pyrrolidinetriones". J. Am. Chem. Soc. (1958),80,532.
71. Simmons, H.E.; Smith, R.D.. "A New Synthesis of Cyclopropanes". J. Am. Chem. Soc. (1959), 81,4256.
72. Howard E. Simmons, Ronald D. Smith "A new synthesis of cyclopropanes from olefins". J. Am. Chem. Soc. (1958),80 (19),5323-5324.
73. A. William Johnson, Robert B. LaCount. "The Chemistry of Ylids. VI. Dimethylsulfonium Fluorenylide-A Synthesis of Epoxides". J. Am. Chem. Soc. (1961),83 (2),417-423.
74. Corey, E.J.; Chaykovsky, M. "Dimethyloxosulfonium Methylide ((CH3)2SOCH2) and Dimethylsulfonium Methylide ((CH3)2SCH2), Formation and Application to Organic Synthesis". J. Am. Chem. Soc. (1965),87 (6),1353-1364.
75. Aggarwal, V. K.; Richardson, J. "The complexity of catalysis:origins of enantio-and diastereocontrol in sulfur ylide mediated epoxidation reactions". Chemical Communications (2003),2644.
76. Jose M. Fraile, Jose 1. Garcia, Victor Martinez-Merino, Jose A. Mayoral, and Luis Salvatella J. Am. Chem. Soc, (2001),123 (31),7616-7625.
77. Larsson, U. et al. WO 2005/095358.
2. Ross R. "The pathogenesis of atherosclerosis:a perspective for the 1990s". Nature (1993),362 (6423),801-9.
3. Ross R; Ross, Russell. "Atherosclerosis — An Inflammatory Disease". New England Journal of Medicine (1999),340 (2),115-26.
4. Erling Falk, Prediman K. Shah, Valentin Fuster, "Coronary Plaque Disruption". Circulation (1995),92,657-671.
5. Eric J. Topol, Jay S. Yadav, "Recognition of the Importance of Embolization in Atherosclerotic Vascular Disease". Circulation (2000),101,570-580.
6. Furie, Bruce; Furie, Barbara "Mechanisms of Thrombus Formation". The New England Journal of Medicine (2008),359,9.
7. Storey, Robert F., "Biology and Pharmacology of the Platelet P2Y12 Receptor". Curr Pharm Des. (2006),12,1255-1259.
8. Kroll, M., J. Hellums, et al. "Platelets and shear stress." Blood (1996),88(5),1525-1541.
9. Bahou WF, Nierman WC, Durkin AS, Potter CL, Demetrick DJ. "Chromosomal assignment of the human thrombin receptor gene:localization to region q13 of chromosome 5". Blood (1993), 82 (5),1532-7.
10. Samuel Chackalamannil; Wang, Yuguang; Greenlee, William J.; Hu, Zhiyong; Xia, Yan; Ahn, Ho-Sam; Boykow, George; Hsieh, Yunsheng et al. "Discovery of a Novel, Orally Active Himbacine-Based Thrombin Receptor Antagonist (SCH 530348) with Potent Antiplatelet Activity". Journal of Medicinal Chemistry (2008),51 (11),3061-4.
11. Serebruany VL, Kogushi M, Dastros-Pitei D, Flather M, Bhatt DL. "The in-vitro effects of E5555, a protease-activated receptor (PAR)-1 antagonist, on platelet biomarkers in healthy volunteers and patients with coronary artery disease", Thromb Haemost. (2009),102(1),111-9.
12. Marcum JA, McKenney JB. et al. "Anticoagulantly active heparin-like molecules from mast cell-deficient mice". Am. J. Physiol. (1986),250, H879-888.
13. Chuang YJ, Swanson R. et al. "Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin". J. Biol. Chem. (2001), 276(18),14961-14971.
14. Bjork I, Lindahl U. "Mechanism of the anticoagulant action of heparin". Mol. Cell. Biochem. (1982),48(3),161-182.
15.李端,殷明,药理学,第六版,人民卫生出版社,275-277.
16. Lever R. and Page C.P. "Novel drug opportunities for heparin". Nat. Rev. Drug Discov. (2002), 1 (2),140-148.
17. Coombe D.R. and Kett W.C. "Heparan sulfate-protein interactions:therapeutic potential through structure-function insights". Cell. Mol. Life Sci. (2005),62 (4),410-424.
18. Ahmed I, Majeed A, Powell R "Heparin induced thrombocytopenia:diagnosis and management update". Postgrad Med J(2007),83 (983),575-82.
19. Haruyama H. and Wuthrich K. "Conformation of recombinant desulfatohirudin in aqueous solution determined by nuclear magnetic resonance". Biochemistry (1989),28 (10), 4301-4312.
20. Rydell TJ, Tulinsky A. et al. "Refined structure of the Hirudin-Thrombin complex". J. Mol. Biol. (1991),221 (2),583-601.
21. Holbrook AM, Pereira JA, Labiris R, McDonald H, Douketis JD, Crowther M, Wells PS "Systematic overview of warfarin and its drug and food interactions". Arch. Intern. Med. (2005),165(10),1095-106.
22. Whitlon DS, Sadowski JA, Suttie JW "Mechanism of coumarin action:significance of vitamin K epoxide reductase inhibition". Biochemistry (1978),17 (8),1371-7.
23. Freedman MD "Oral anticoagulants:pharmacodynamics, clinical indications and adverse effects". J Clin Pharmacol (1992),32 (3),196-209.
24. Holford NH. "Clinical pharmacokinetics and pharmacodynamics of warfarin. Understanding the dose-effect relationship". Clin Pharmacokinet (1986),11,483-504.
25. Horton JD, Bushwick BM "Warfarin therapy:evolving strategies in anticoagulation". Am Fam Physician (1999),59 (3),635-46.
26. Sneader, W. "The discovery of aspirin:A reappraisal". BMJ (Clinical research ed.) (2000),321 (7276),1591-4.
27. John Robert Vane "Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs". Nature-New Biology (1971),231 (25),232-5.
28. Vane JR, Botting RM "The mechanism of action of aspirin". Thromb Res (2003),110 (5-6), 255-8.
29. Kimura Y, Tani T, Kanbe T, Watanabe K, Arzneimittel-Forschung (1985),35(7A),1144-9.
30. Halkes PH, van Gijn J, Kappelle LJ, Koudstaal PJ, Algra A "Aspirin plus dipyridamole versus aspirin alone after cerebral ischaemia of arterial origin (ESPRIT), randomised controlled trial". The Lancet (2006),367 (9523),1665-73.
31. Loo MH, Egan D, Vaughan ED, Marion D, Felsen D, Weisman S "The effect of the thromboxane A2 synthesis inhibitor OKY-046 on renal function in rabbits following release of unilateral ureteral obstruction". J. Urol. (1987),137 (3),571-6.
32. Hennerici, M. G.; Bots, M. L.; Ford, I.; Laurent, S.; Touboul, P. J. "Rationale, design and population baseline characteristics of the PERFORM Vascular Project:an ancillary study of the Prevention of cerebrovascular and cardiovascular Events of ischemic origin with teRutroban in patients with a history oF ischemic strOke or tRansient ischeMic attack (PERFORM) trial". Cardiovascular Drugs and Therapy (2010),24 (2),175.
33. Sorbera, LA, Serradell, N, Bolos, J, Bayes, M "Terutroban sodium". Drugs of the Future (2006), 31 (10),867-873.
34. Michael Gent, J. Donald Easton, VladimirC. Hachinski, Edouard Panak, Jane Siourella, JohnA. Blakely, DavidJ. Ellis, JohnW. Harbison, RobinS. Roberts, AlexanderG.G. Turpie, "the Canadian American Ticlopidine Study (CATS) in thromboembolic stroke", The Lancet (1989), 333,1215-1220
35. CAPRIE Steering Committee "A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE)". The Lancet (1996),16 (9038),1329-39.
36. Pereillo JM, Maftouh M, Andrieu A, Uzabiaga MF, Fedeli O, Savi P, Pascal M, Herbert JM, Maffrand JP, Picard C "Structure and stereochemistry of the active metabolite of clopidogrel". Drug Metab. Dispos. (2002),30 (11),1288-95.
37. Stockl, KM; Le L; Zakharyan A; et al. "Risk of rehospitalization for patients using clopidogrel with a proton pump inhibitor". Arch Intern Med (2010),170 (8),704-10.
38. Baker WL, White CM. Role of Prasugrel, a Novel P2Y12 Receptor Antagonist, in the Management of Acute Coronary Syndromes. American Journal of Cardiovascular Drugs (2009),9 (4),213-229.
39. Michelle O'Donoghue,; Elliott M. Antman,; Eugene Braunwald, et al. "The Efficacy and Safety of Prasugrel With and Without a Glycoprotein Ⅱb/Ⅲa Inhibitor in Patients With Acute Coronary Syndromes Undergoing Percutaneous", J Am Coll Cardiol. (2009),54(8),678-685.
40. Wiviott SD, Braunwald E, McCabe CH et al. "Prasugrel versus clopidogrel in patients with acute coronary syndromes". NEngl J Med (2007),357 (20),2001-15.
41. Bhatt DL et al. "Effect of Platelet Inhibition with. Cangrelor during PCI on Ischemic Events" New England Journal of Medicine (2013), (March 10,2013, published initially online).
42. van Giezen JJ, Humphries RG. Semin Thromb Hemost (2005),31,195-204.
43. Christopher P Cannon, Robert A Harrington,Stefan James, et al. "Comparison of ticagrelor with clopidogrel in patients with a planned invasive strategy for acute coronary syndromes (PLATO), a randomised double-blind study". The Lancet (2010),375,283-293.
44. Christopher P. Cannon et al., "Safety, Tolerability, and Initial Efficacy of AZD6140, the First Reversible Oral Adenosine Diphosphate Receptor Antagonist, Compared With Clopidogrel, in Patients With Non-ST-Segment Elevation Acute Coronary Syndrome". J Am Coll Cardiol (2007),50,1844-1851.
45. Dorsam RT, Kunapuli SP "Central role of the P2Y12 receptor in platelet activation". J. Clin. Invest. (2004),113 (3),340-5.
46. Murugappa S, Kunapuli SP "The role of ADP receptors in platelet function". Front. Biosci. (2006),11 (1),1977-86.
47. Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, TMurden P, Nurden A, Julius D, Conley PB "Identification of the platelet ADP receptor targeted by antithrombotic drugs". Nature (2001),409 (6817),202-7.
48. Hubbard R "The molecular weight of rhodopsin and nature of the rhodopsin-digitonin complex". J Gen Physiol (1954),37,381-399.
49. Overington JP, Al-Lazikani B, Hopkins AL "How many drug targets are there?". Nat Rev Drug Discov (2006),5(12),993-6.
50. Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, Chae PS, Pardon E, Calinski D, Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH, Steyaert J, Skiniotis G, Weis WI, Sunahara RK, Kobilka BK "Crystal structure of the human beta2 adrenergic receptor-Gs protein complex". Nature (2011),477,549-555.
51. Wettschureck N, Offermanns S "Mammalian G proteins and their cell type specific functions". Physiol. Rev. (2005),85 (4),1159-204.
52. Xie, X. Q.; Chen, J. Z.; Billings, E. M., "3D structural model of the G-protein-coupled cannabinoid CB2 receptor". Proteins (2003),53, (2),307-319.
53. Orry, A. J.; Wallace, B. A., "Modeling and docking the endothelin G-protein-coupled receptor". Biophys J (2000),79 (6),3083-3094.
54. Huang, X.; Shen, J.; Cui, M.; Shen, L.; Luo, X.; Ling, K.; Pei, G; Jiang, H.; Chen, K., "Molecular dynamics simulations on SDF-1 alpha:binding with CXCR4 receptor. " Biophys J (2003),84(1),171-184.
55. Krzysztof Palczewski et al, "Crystal Structure of Rhodopsin:A G Protein-Coupled Receptor". Science (2000),739,289.
56. Gibas, C.; Jambeck, P., Developing Bioinformatics Computer Skills. O'Reilly & Associates, Inc, Sebastopol, (2001).
57. http://www.ebi.ac.uk/swissprot/
58. Altschul, S. F.; Madden, T. L.; Schaffer, A. A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D. J., "Gapped BLAST and PSI-BLAST:a new generation of protein database search programs". Nucleic Acids Res (1997),25 (17),3389-402.
59. Thompson, J. D.; Higgins, D. G.; Gibson, T. J., "Clustal-W-improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice". Nucleic Acids Res (1994),22(22),4673-4680.
60. Bockaert, J.; Pin, J. P., "Molecular tinkering of G protein-coupled receptors:an evolutionary success". Embo J(1999),18(7),1723-1729.
61. www.pdb.org
62. Sali, A.; Blundell, T. L., "Comparative protein modelling by satisfaction of spatial restraints". J Mol Biol (1993),234(3),779-815.
63. Laskowski, R. A.; Macarthur, M. W.; Moss, D. S.; Thornton, J. M., "Procheck-ap Program to Check the Stereochemical Quality of Protein Structures". J Appl Crystallogr (1993),26, 283-291.
64. Brian Springthorpe et al, "From ATP to AZD6140:The discovery of an orally active reversible P2Y12 receptor antagonist for the prevention of thrombosis". Bioorg. Med. Chem, Lett. (2007), 17,6013-6018.
65. Fjellstrom, Q. et al. WO 2009/093972.
66. Bonnert, R. et al, WO 98/28300.
67. Ingall, AH et al, EP0508687.
68. Allen R. Ritter and Marvin J. Miller, J. Org. Chem. (1994),59,4602-4611.
69. Brock T. Shireman and Marvin J. Miller, Tetrahedron Letters (2000),41,9537-9540.
70. Howard Ensign Simmons, Jr.; Smith, R.D. "4-Substituted-2,3,5-pyrrolidinetriones". J. Am. Chem. Soc. (1958),80,532.
71. Simmons, H.E.; Smith, R.D.. "A New Synthesis of Cyclopropanes". J. Am. Chem. Soc. (1959), 81,4256.
72. Howard E. Simmons, Ronald D. Smith "A new synthesis of cyclopropanes from olefins". J. Am. Chem. Soc. (1958),80 (19),5323-5324.
73. A. William Johnson, Robert B. LaCount. "The Chemistry of Ylids. VI. Dimethylsulfonium Fluorenylide-A Synthesis of Epoxides". J. Am. Chem. Soc. (1961),83 (2),417-423.
74. Corey, E.J.; Chaykovsky, M. "Dimethyloxosulfonium Methylide ((CH3)2SOCH2) and Dimethylsulfonium Methylide ((CH3)2SCH2), Formation and Application to Organic Synthesis". J. Am. Chem. Soc. (1965),87 (6),1353-1364.
75. Aggarwal, V. K.; Richardson, J. "The complexity of catalysis:origins of enantio-and diastereocontrol in sulfur ylide mediated epoxidation reactions". Chemical Communications (2003),2644.
76. Jose M. Fraile, Jose 1. Garcia, Victor Martinez-Merino, Jose A. Mayoral, and Luis Salvatella J. Am. Chem. Soc, (2001),123 (31),7616-7625.
77. Larsson, U. et al. WO 2005/095358.