摘要
心脑血管疾病是一类由于心脏或血管病变导致的循环系统功能紊乱的疾病的统称,是世界范围内尤其是亚洲地区的首要死亡原因,对人类的生命健康构成了严重威胁。在中国,每年死于心脑血管疾病约有300万人,为死亡人口总数的41%左右。因此对心脑血管疾病的预防和治疗是药物研发领域的迫切任务。
由于心脑血管系统的复杂性,其致病因素颇为繁杂,而血栓形成在心脑血管疾病的发病机制中起到关键作用；与血栓栓塞密切相关的血小板聚集和氧化应激也是心脑血管疾病发病的两种始动因素。迄今为止,临床上用于预防和治疗血栓的抗凝血剂主要有两种类型：肝素类及维生素K拮抗剂,然而这些药物存在给药不方便、起效慢、治疗窗窄、复杂的药物相互作用等诸多局限性。因此,研究和开发高效低毒、高选择性的新型抗凝血药物就成为心脑血管药物研发的热点。
本论文的研究内容分为两大部分：第一部分为新型凝血因子Xa (FXa)抑制剂的设计、合成与抗凝血及FXa抑制活性的研究；第二部分为新型川芎酸酚酯类衍生物的设计、合成与抗血小板聚集及抗内皮细胞氧化损伤生物活性评价。
1.新型FXa抑制剂的设计、合成与抗凝血活性和FXa抑制活性的研究
FXa位于人体内、外两条凝血途径的交汇点,抑制FXa能够同时切断两条凝血途径,因此FXa已经成为抗凝血药物研发的一个有吸引力的靶点。目前已经有三种FXa抑制剂：利伐沙班(rivaroxaban)、阿哌沙班(apixaban)和依度沙班(edoxaban)在美国、欧洲和日本上市。以利伐沙班为活性先导化合物,通过分析“利伐沙班-FXa复合体”的晶体结构,发现FXa中存在S1、S4两个结合口袋,两口袋之间存在一定的夹角；利伐沙班分子也可分为三部分：P1区、P4区和两者之间的linker。FXa的S1口袋为较小的疏水口袋,容纳利伐沙班的P1区5-氯噻吩；S4口袋为大的疏水口袋,与利伐沙班的P4区苯环形成π-π堆积作用；两口袋之间的Gly219与利伐沙班的linker形成两个氢键。根据先导化合物与FXa的结合模式,对先导化合物的P4区、linker进行结构改造,通过变换linker的角度和P4区的体积、疏水性等,设计了双酰基哌嗪类FXa抑制剂和氨基苄胺类FXa抑制剂,以期得到活性改善的新型FXa抑制剂。为验证设计思想的合理性,用计算机辅助药物设计软件对所设计的化合物进行了对接分析和虚拟筛选,分析结果表明目标分子的结构修饰具有理论上的合理性。
对设计的化合物进行了定向合成,通过成环反应、重氮化反应、酰化反应等实验步骤,共合成了两个系列40个FXa抑制剂。所合成的双酰基哌嗪类FXa抑制剂和氨基苄胺类FXa抑制剂的结构经光谱确证。
对所合成的FXa抑制剂进行了抗凝血试验。以利伐沙班作为对照药,用STAGO凝血酶原时间试剂盒,与凝血酶原时间测定仪中测定化合物对贫血小板血浆的凝血酶原时间的影响,以“致凝血时间加倍浓度”(PTCT2, clotting time doubling concentration for prothrombin time)评价化合物抗凝血活性。其中部分化合物表现出良好的抗凝血活性,化合物H10, H13, H14, H17, PTCT2值分别为1.3,3.1,2.7,1.0μM,其中化合物H17抗凝血活性优于先导化合物利伐沙班(PTCT2=1.3μM)。
与此同时,对化合物的FXa抑制活性进行了测定。FXa可以催化发色底物S-2222生成对硝基苯胺,在405nm下有紫外吸收峰。以利伐沙班作为对照药,通过酶标仪测定FXa抑制剂对405nm下吸光度的影响,得到化合物对FXa的抑制活性IC50值。活性结果显示部分化合物显示出良好活性。其中化合物H17对FXa的抑制活性(IC50=1.9μM)略高于阳性对照药利伐沙班(IC50=3.3μM)。
总之,通过合理药物设计和虚拟筛选设计了两个系列的FXa抑制剂,并通过定向合成得到40个化合物,通过活性测定发现了活性先导化合物,为进一步研究开发奠定了基础。
2.新型川芎酸酚酯类衍生物的设计、合成与抗血小板和抗内皮细胞氧化损伤生物活性评价
川芎嗪是川芎的主要活性物质之一。药理学研究证明,川芎嗪表现出多种心脑血管药理活性,如清除自由基、抗血小板聚集与血栓形成、保护内皮细胞等。然而,川芎嗪药理活性不强、体内代谢快、生物利用度低,因此对其进行结构改造,改善其药效学和药代动力学性质,以研发高效抗心脑血管疾病药物有重要的意义。本课题组对川芎嗪进行了长期、大量的结构改造工作,对川芎嗪衍生物的结构改造和构效关系有深入的认识。
川芎嗪的代谢产物3,5,6三甲基吡嗪甲酸的药理活性研究表明其具有抗凝血活性和降低血浆低密度脂蛋白水平等作用。川芎所含的另一种活性成分肉桂酸类化合物具有较好的抗氧化、清除自由基的作用。肉桂酸衍生物奥扎格雷(ozagrel)是选择性的血栓烷A2合成酶抑制剂,具有抗血小板聚集活性。依据药物化学中的拼合原理,将3,5,6三甲基吡嗪甲酸的结构和肉桂酸的结构拼合,设计了一系列新型川芎酸酚酯类衍生物。
对所合成川芎酸酚酯类衍生物进行了体外抗血小板聚集活性测定保护过氧化损伤的血管内皮细胞的活性试验。以奥扎格雷和氯吡格雷作为阳性对照药,用微量反应板酶标仪比浊法测定所合成的化合物对血小板聚集率的影响。活性实验结果表明：川芎酸酚酯类衍生物F'1, F2, F3, F5, F'7和F'9表现出良好的活性(IC50分别为24.4,26.4,9.6,41.8,28.2和37.5μM),均超过了阳性对照药奥扎格雷(IC50=144.1μM),尤其是化合物F3活性最为显著,为阳性对照药奥扎格雷活性的15倍以上,接近氯毗格雷活性(IC50=7.6pM)。
以MTT比色法对所合成的川芎酸酚酯类衍生物进行了保护血管内皮细胞过氧化损伤活性试验,以药物硫辛酸和常用抗氧化剂丁基羟基茴香醚(BHA)为阳性对照。活性试验数据显示：化合物F2,F5,F'5,F'9,F10,F'10表现出了较好的内皮细胞保护作用(EC50分别为24.0,8.8,29.9,21.4,2.2和1.7μM)。尤其是化合物F'10,活性远高于阳性对照药硫辛酸和BHA(EC50分别为68.0和111.4μM)。
综上所述,依据拼合原理设计了一系列川芎酸酚酯类衍生物(20个),并对合成的川芎嗪衍生物进行了抗血小板聚集活性和血管内皮细胞保护活性的测定,发现其中一些化合物具有良好的活性,具有进一步研究的价值。
There are two main parts in this thesis:the first part is the design, synthesis and anti-coagulation activity research of novel factor Xa inhibitors; the second part is the design, synthesis and anti-platelet activity and anti-oxidation activity research of novel ligustrazine derivatives.
Cardiovascular disease (CVDs) is a class of diseases caused by dysfunction of circulatory system, which seriously threaten people's health and life. CVDs is the leading cause of death world wide, especially in Asian regions. In China, nearly3million people die of CVDs annually, taking up about41%of the total death toll. Therefore it is an urgent task for researchers to develop novel and efficient anti-CVDs agents.
Thrombosis plays a critical role in the pathogenesis of some cardiovascular diseases such as acute coronary syndromes. Until relatively recently, pharmacologic prophylaxis and treatment of venous thromboembolism were based on two types of anticoagulants, heparins (unfractionated heparin, low-molecular-weight heparins and fondaparinux) and vitamin K antagonists (e.g. warfarin). Although these agents considerably reduce thromboembolic events, they have several limitations, such as inconvenient for long-term use, slow onset of action, a narrow therapeutic window and their multiple drug and food interactions. To overcome these shortcomings, the research and development of new anticoagulants with high selectivity, better anticoagulative activity and low toxicity is needed.
Part1.Design, synthesis and anti-coagulation activity research of novel factor Xa inhibitors
FXa is a trypsin-like serine protease that sits at the junction of both the extrinsic and the intrinsic pathways. The FXa is therefore an attractive and specific druggable target for new anticoagulant agents.
Up to now, three oral direct FXa inhibitors, rivaroxaban, apixaban and edoxaban, have been approved for anti-thrombosis therapy for their high efficiency and high selectivity. However, in addition to a great success in the clinical application, rivaroxaban and other drugs have been found to have some side effects, such as causing anemia or bleeding. Therefore, research and development for new FXa inhibitors with higher efficacy and lowertoxicity is an important direction of the current anti-thrombotic drug development.
There have been numerous studies on FXa's structure and its interaction with FXa inhibitors. Based on these studies, we designed two series of new FXa inhibitors by changing P4area and linker region of leading compounds. In order to verify the reasonableness of the design, we carried out the docking analysis of the designed compounds, using compouter-aided drug design software sybyl7.3. The docking results showed that the design of the target compounds is theoretically rational. The synthesized compounds were tested for their in vitro anti-coagulation activities and FXa inhibitory activities. Results showed that some of the compounds exhibited good activities. Among which, H17displayed higher FXa inhibitory activity (IC50=1.9μM) and better anti-coagulant activity (PTCT2=1.0μM) than that of the positive control drug rivaroxaban (IC50=3.3μM, PTCT2=1.3μM).
In summary, we designed and synthesize two series of FXa inhibitors with potent anticoagulation activities and FXa inhibitory activity, which are worth further investigation and development.
Part2. Design, synthesis and biological evaluation of novel Ligustrazine derivatives Ligustrazine is an active component of Chinese traditional medicine Chuanxiong. Currently ligutrazine is an important drug for the treatment of ischemic cardiovascular disease in China. It is reported that ligustrazine exihibited various cardiovascular activities, such as free radical-scavenging, endothelium-protection and antiplatelet. However, ligustrazine was found dissatisfactory to be applicated in clinical practice because of its moderate activity, rapid metabolism and short half life time. Thus, it is meaningful to develop new generation of the ligustrazine drugs for treatment of CVDs from molecular modification.
During our structure modification studies of ligustrazine, we found that some ligustrazine metabolites possessed improved cardiovascular activities than ligustrazine. The main metabolite of ligustrazine,3,5,6-trimethylpyrazine-2-carboxylic acid, displayed moderate activity on lowering the level of serum cholesterol and low density lipoprotein in vivo, and potent anticoagulation activity. Cinnamic acid and some of its derivatives have been reported to display anti-oxidative property. It is believed that cinnamic acids are potential scavengers of free radicals (such as O2·) and other oxidative species. Moreover, ozagrel, a derivative of cinnamic acid, is reported as a highly selective thromboxane (TXA2) synthase inhibitor, which has the ability to preclude the TXA2induced platelet aggregation.
Inspired by the structural characteristics and the drug-like properties of cinnamic acid and in continuation of our work, we conjugated the Ligustrazinyloxy group with cinnamic acid group to obtain a novel series of trimethylpyrazine-2-carbonyloxy-cinnamic acids, in the hope of getting novel ligustrazine derivatives with high anti-oxidative activity or anti-platelet aggregation activity.
This series of ligstrazine derivatives were evaluated for their anti-platelet aggregation activity and endothelial cells-protection activity.
Anti-platelet aggregation assay:The inhibition of platelet aggregation of the compounds was tested using microplate-reader-nephelometry. The positive control drugs are ozagrel and clopidogrel. Among all the synthesized compounds, F'1, F2, F3, F5, F'7and F'9were the most potent platelet aggregation inhibitors with IC50values24.4,26.4,9.6,41.8,28.2and37.5μM, respectively, much more active than that of ozagrel (IC50=144.1μM), but slightly lower than that of clopidogrel (IC50=7.6μM). Protecting vascular endothelial cells against hyperoxic acute injury assay:These ligustrazine derivatives have been tested for protecting vascular endothelial cells against hyperoxic acute injury. The viability of injured endothelial cells is assessed by methylthiazolyltetrazolium (MTT) assay. Among all the ligustrazine derivatives, F2, F5, F'5, F'9, F10, F'10displayed remarkable protection activity (EC50=24.0,8.8,29.9,21.4,2.2,1.7μM, respectively). Particularly, the compound F'10presented almost40times higher potency than lipoic acid.
In summary, a series of novel ligustrazine derivatives were designed, synthesized and biologically evaluated for their inhibitory activities against the platelet aggregation, and their protective effect on the damaged vacular endothelial cells. The results showed that some compounds exhibited high activities in one or both of the assays, demonstrating the potential values in further development of cardiovascular agents.
引文
[1]Ohira, T; Iso, H., Cardiovascular disease epidemiology in Asia:an overview. Circ J,2013,77, (7), 1646-1652.
[2]Finegold, J.A.; Asaria, P.; Francis, D.P., Mortality from ischaemic heart disease by country, region, and age:statistics from World Health Organisation and United Nations. Int J Cardiol,2013,168, (2), 934-945.
[3]Jani, B.; Rajkumar, C., Ageing and vascular ageing. Postgrad Med J,2006,82, (968),357-362.
[4]Jousilahti, P.; Vartiainen, E.; Tuomilehto, J.; Puska, P., Sex, age, cardiovascular risk factors, and coronary heart disease:a prospective follow-up study of 14 786 middle-aged men and women in Finland. Circulation,1999,99, (9),1165-1172.
[5]Barrett-Connor, E.; Khaw, K., Family history of heart attack as an independent predictor of death due to cardiovascular disease. Circulation,1984,69, (6),1065-1069.
[6]Carretero, O.A.; Oparil, S., Essential hypertension part I:definition and etiology. Circulation, 2000,101, (3),329-335.
[7]Lewington, S.; Clarke, R.; Qizilbash, N.; Peto, R.; Collins, R., Age-specific relevance of usual blood pressure to vascular mortality:a meta-analysis of individual data for one million adults in 61 prospective studies. Lancet,2002,360, (9349),1903-1913.
[8]Singer, D.R.; Kite, A., Management of hypertension in peripheral arterial disease:does the choice of drugs matter? Eur J Vase Endovasc Surg,2008,35, (6),701-708.
[9]Law, M.; Wald, N.; Morris, J., Lowering blood pressure to prevent myocardial infarction and stroke:a new preventive strategy. Health Technol Assess,2003,7, (31),1-94.
[10]Berg, K.; Dahlen, G.; Christophersen, B.; Cook, T.; Kjekshus, J.; Pedersen, T., Lp(a) lipoprotein level predicts survival and major coronary events in the Scandinavian Simvastatin Survival Study. Clin
Genet,1997,52, (5),254-261. [11] Reiner, Z.; Catapano, A.L.; De Backer, G.; Graham, I.; Taskinen, M.-R.; Wiklund, O.; Agewall, S.; Alegria, E.; Chapman, M.J.; Durrington, P., ESC/EAS Guidelines for the management of dyslipidaemias The Task Force for the management of dyslipidaemias of the European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS). Eur Heart J,2011,32, (14), 1769-1818.
[12]Collaboration, E.R.F., Diabetes mellitus, fasting blood glucose concentration, and risk of vascular disease:a collaborative meta-analysis of 102 prospective studies. The Lancet,2010,375, (9733), 2215-2222.
[13]Campisi, R.; Czernin, J.; Schoder, H.; Sayre, J.W.; Marengo, F.D.; Phelps, M.E.; Schelbert, H.R., Effects of long-term smoking on myocardial blood flow, coronary vasomotion, and vasodilator capacity. Circulation,1998,98, (2),119-125.
[14]Kim, J.W.; Park, C.G; Hong, S.J.; Park, S.M.; Rha, S.W.; Seo, H.S.; Oh, D.J.; Rho, Y.M., Acute and chronic effects of cigarette smoking on arterial stiffness. Blood Press,2005,14, (2),80-85.
[15]Howard, G.; Wagenknecht, L.E.; Burke, G.L.; Diez-Roux, A.; Evans, G.W.; McGovern, P.; Nieto, F.J.; Tell, GS.; investigators, A., Cigarette smoking and progression of atherosclerosis:The Atherosclerosis Risk in Communities (ARIC) Study. Jama,1998,279, (2),119-124.
[16]Attvall, S.; Fowelin, J.; Lager, I.; Schenck, H.; Smith, U., Smoking induces insulin resistance—a potential link with the insulin resistance syndrome. J Intern Med,1993,233, (4),327-332.
[17]Facchini, F.S.; Hollenbeck, C.B.; Jeppesen, J.; Ida Chen, Y.-D.; Reaven, G., Insulin resistance and cigarette smoking. The Lancet,1992,339, (8802),1128-1130.
[18]Nogueira, J.B., Air pollution and cardiovascular disease. Revista portuguesa de cardiologia: orgao oficial da Sociedade Portuguesa de Cardiologia= Portuguese journal of cardiology:an official journal of the Portuguese Society of Cardiology,2009,28, (6),715-733.
[19]Sun, Q.; Hong, X.; Wold, L.E., Cardiovascular effects of ambient particulate air pollution exposure. Circulation,2010,121, (25),2755-2765.
[20]Donnan, GA.; Fisher, M.; Macleod, M.; Davis, S.M., Stroke. Lancet,2008,371, (9624), 1612-1623.
[21]Sims, N.R.; Muyderman, H., Mitochondria, oxidative metabolism and cell death in stroke. Biochim Biophys Acta,2010,1802, (1),80-91.
[22]Fuster, V.; Stein, B.; Ambrose, J.A.; Badimon, L.; Badimon, J.J.; Chesebro, J.H., Atherosclerotic plaque rupture and thrombosis. Evolving concepts. Circulation,1990,82, (3 Suppl),1147-59.
[23]Libby, P.; Theroux, P., Pathophysiology of coronary artery disease. Circulation,2005,111, (25), 3481-3488.
[24]Libby, P., Inflammation in atherosclerosis. Nature,2002,420, (6917),868-874.
[25]Demer, L.L., Vascular calcification and osteoporosis:inflammatory responses to oxidized lipids. Int J Epidemiol,2002,31, (4),737-741.
[26]Geng, Y.J.; Libby, P., Progression of atheroma:a struggle between death and procreation. Arterioscler Thromb Vasc Biol,2002,22, (9),1370-1380.
[27]Bogdanov, V.Y.; Balasubramanian, V.; Hathcock, J.; Vele, O.; Lieb, M.; Nemerson, Y, Alternatively spliced human tissue factor:a circulating, soluble, thrombogenic protein. Nat Med,2003, 9, (4),458-462.
[28]Seidl, S.E.; Potashkin, J.A., The promise of neuroprotective agents in Parkinson's disease. Front Neurol,2011,2,68.
[29]Varga-Szabo, D.; Braun, A.; Nieswandt, B., Calcium signaling in platelets. J Thromb Haemost, 2009,7,(7),1057-1066.
[30]Varga-Szabo, D.; Pleines, I.; Nieswandt, B., Cell adhesion mechanisms in platelets. Arterioscler Thromb Vase Biol,2008,28, (3),403-412.
[31]Murugappan, S.; Shankar, H.; Kunapuli, S.P., Platelet receptors for adenine nucleotides and thromboxane A2. Semin Thromb Hemost,2004,30, (4),411-418.
[32]Gryglewski, R.J., Prostacyclin among prostanoids. Pharmacol Rep,2008,60, (1),3-11.
[33]Niiya, K.; Hodson, E.; Bader, R.; Byers-Ward, V.; Koziol, J.A.; Plow, E.F.; Ruggeri, Z.M., Increased surface expression of the membrane glycoprotein Ⅱb/Ⅲa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation. Blood,1987,70, (2), 475-483.
[34]Ma, Y.Q.; Qin, J.; Plow, E.F., Platelet integrin alpha(IIb)beta(3):activation mechanisms. J Thromb Haemost,2007,5, (7),1345-1352.
[35]Scarborough, R.M.; Kleiman, N.S.; Phillips, D.R., Platelet Glycoprotein Ilb/IIIa Antagonists What Are the Relevant Issues Concerning Their Pharmacology and Clinical Use? Circulation,1999,100, (4), 437-444.
[36]Vickers, J.D., Binding of polymerizing fibrin to integrin alpha(IIb)beta(3) on chymotrypsin-treated rabbit platelets decreases phosphatidylinositol 4,5-bisphosphate and increases cytoskeletal actin. Platelets,1999,10, (4),228-237.
[37]Calvete, J.J., On the structure and function of platelet integrin alpha lIb beta 3, the fibrinogen receptor. Proc Soc Exp Biol Med,1995,208, (4),346-360.
[38]FitzGerald, G.A.; Reilly, I.A.; Pedersen, A.K., The biochemical pharmacology of thromboxane synthase inhibition in man. Circulation,1985,72, (6),1194-1201.
[39]Ching, S.; Ingram, D.; Hahnel, R.; Beilby, J.; Rossi, E., Serum levels of micronutrients, antioxidants and total antioxidant status predict risk of breast cancer in a case control study. J Nutr, 2002,132, (2),303-306.
[40]Wang, Y.; Chun, O.K.; Song, W.O., Plasma and Dietary Antioxidant Status as Cardiovascular Disease Risk Factors:A Review of Human Studies. Nutrients,2013,5, (8),2969-3004.
[41]Ames, B.N.; Shigenaga, M.K.; Hagen, T.M., Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci U S A,1993,90, (17),7915-7922.
[42]Madamanchi, N.R.; Runge, M.S., Mitochondrial dysfunction in atherosclerosis. Circ Res,2007, 100, (4),460-473.
[43]Cai, H.; Harrison, D.G, Endothelial dysfunction in cardiovascular diseases:the role of oxidant stress. Circ Res,2000,87, (10),840-844.
[44]Ceconi, C.; Boraso, A.; Cargnoni, A.; Ferrari, R., Oxidative stress in cardiovascular disease:myth or fact? Arch Biochem Biophys,2003,420, (2),217-221.
[45]Kumar, A.; Kar, S.; Fay, W.P., Thrombosis, physical activity, and acute coronary syndromes. Journal of Applied Physiology,2011,111, (2),599-605.
[46]A1 Dieri, R.; de Laat, B.; Hemker, H.C., Thrombin generation:What have we learned? Blood reviews,2012,26, (5),197-203.
[47]Mitrophanov, A.Y.; Reifman, J., Kinetic modeling sheds light on the mode of action of recombinant factor VIIa on thrombin generation. Thrombosis research,2011,128, (4),381-390.
[48]Bauer, K., Recent progress in anticoagulant therapy:oral direct inhibitors of thrombin and factor Xa. Journal of Thrombosis and Haemostasis,2011,9, (s1),12-19.
[49]Krupiczojc, M.A.; Scotton, C.J.; Chambers, R.C., Coagulation signalling following tissue injury: focus on the role of factor Xa. The international journal of biochemistry & cell biology,2008,40, (6), 1228-1237.
[50]Schulman, S., The role of ximelagatran in the treatment of venous thromboembolism. Pathophysiology of haemostasis and thrombosis,2005,34, (Suppl.1),18-24.
[51]Ansell, J.; Bergqvist, D., Current options in the prevention of thromboembolic disease. Drugs, 2004,64, (1),1-5.
[52]Kaku, S.; Uemura, T.; Saitoh, M.; Suzuki, K.-i.; Iwatsuki, Y.; Funatsu, T.; Kawasaki, T., Antithrombotic and anticoagulant effects of direct factor Xa inhibitor darexaban in rat and rabbit models of venous thrombosis. European journal of pharmacology,2013,699, (1),40-47.
[53]Morishima, Y.; Honda, Y.; Kamisato, C.; Shibano, T., Comparison of antithrombotic and hemorrhagic effects of edoxaban, a novel factor Xa inhibitor, with unfractionated heparin, dalteparin, lepirudin and warfarin in rats. Thrombosis research,2013,132, (2),234-239.
[54]van Montfoort, M.L.; Meijers, J.C., Anticoagulation beyond direct thrombin and factor Xa inhibitors:indications for targeting the intrinsic pathway? Thrombosis and haemostasis,2013,110, (2013),223-232.
[55]Bhunia, S.S.; Roy, K.K.; Saxena, A.K., Profiling the structural determinants for the selectivity of representative factor-Xa and thrombin inhibitors using combined ligand-based and structure-based approaches. Journal of chemical information and modeling,2011,51, (8),1966-1985.
[56]Mann, K.; Brummel, K.; Butenas, S., What is all that thrombin for? Journal of Thrombosis and Haemostasis,2003,1, (7),1504-1514.
[57]Toschi, V.; Lettino, M., Inhibitors of propagation of coagulation:factors V and X. British journal of clinical pharmacology,2011,72, (4),563-580.
[58]Cheng, J.W., Fondaparinux:a new antithrombotic agent. Clinical therapeutics,2002,24, (11), 1757-1769.
[59]Borensztajn, K.; Spek, C.A., Blood coagulation factor Xa as an emerging drug target. Expert opinion on therapeutic targets,2011,15, (3),341-349.
[60]Ansell, J., Factor Xa or thrombin:is factor Xa a better target? Journal of Thrombosis and Haemostasis,2007,5, (s1),60-64.
[61]Hermans, C.; Claeys, D., Review of the rebound phenomenon in new anticoagulant treatments. Current Medical Research and Opinion(?),2006,22, (3),471-481.
[62]Chattopadhyay, R.; Iacob, R.; Sen, S.; Majumder, R.; Tomer, K.B.; Lentz, B.R., Functional and structural characterization of factor Xa dimer in solution. Biophysical journal,2009,96, (3),974-986.
[63]de Candia, M.; Lopopolo, G.; Altomare, C., Novel factor Xa inhibitors:a patent review. Expert Opinion on Therapeutic Patents,2009,19, (11),1535-1580.
[64]Bode, W.; Mayr, I.; Baumann, U.; Huber, R.; Stone, S.R.; Hofsteenge, J., The refined 1.9 A crystal structure of human alpha-thrombin:interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. The EMBO Journal,1989,8, (11),3467.
[65]Katz, B.A.; Elrod, K.; Luong, C.; Rice, M.J.; Mackman, R.L.; Sprengeler, P.A.; Spencer, J.; Hataye, J.; Janc, J.; Link, J., A novel serine protease inhibition motif involving a multi-centered short hydrogen bonding network at the active site. Journal of molecular biology,2001,307, (5),1451-1486.
[66]Katz, B.A.; Elrod, K.; Verner, E.; Mackman, R.L.; Luong, C.; Shrader, W.D.; Sendzik, M.; Spencer, J.R.; Sprengeier, P.A.; Kolesnikov, A., Elaborate manifold of short hydrogen bond arrays mediating binding of active site-directed serine protease inhibitors. Journal of molecular biology,2003, 329,(1),93-120.
[67]Katz, B.A.; Spencer, J.R.; Elrod, K.; Luong, C.; Mackman, R.L.; Rice, M.; Sprengeier, P.A.; Allen, D.; Janc, J., Contribution of multicentered short hydrogen bond arrays to potency of active site-directed serine protease inhibitors. Journal of the American Chemical Society,2002,124, (39),11657-11668.
[68]Wang, J.-F.; Hao, P.; Li, Y.-X.; Dai, J.-L.; Li, X., Exploration of conformational transition in the aryl-binding site of human FXa using molecular dynamics simulations. Journal of molecular modeling, 2012,18, (6),2717-2725.
[69]Samama, M.M., Synthetic direct and indirect factor Xa inhibitors. Thrombosis research,2002, 106, (3), V267-V273.
[70]Lee, C.J.; Chandrasekaran, V.; Wu, S.; Duke, R.E.; Pedersen, L.G., Recent estimates of the structure of the factor VIIa (FVIIa)/tissue factor (TF) and factor Xa (FXa) ternary complex. Thrombosis research,2010,125, S7-S10.
[71]van Dieijen, G.; Tans, G.; Rosing, J.; Hemker, H.C., The role of phospholipid and factor VIIIa in the activation of bovine factor X. Journal of Biological Chemistry,1981,256, (7),3433-3442.
[72]Lopopolo, G.; Fiorella, F.; de Candia, M.; Nicolotti, O.; Mattel, S.; Carrupt, P.-A.; Altomare, C., Biarylmethoxy isonipecotanilides as potent and selective inhibitors of blood coagulation factor Xa. European Journal of Pharmaceutical Sciences,2011,42, (3),180-191.
[73]Quan, M.L.; Lam, P.Y.; Han, Q.; Pinto, D.J.; He, M.Y.; Li, R.; Ellis, C.D.; Clark, C.G; Teleha, C.A.; Sun, J.-H., Discovery of 1-(3'-Aminobenzisoxazol-5'-yl)-3-trifluoromethyl-N-[2-fluoro-4-[(2'-dimethylaminomethyl) imidazol-1-yl] phenyl]-1 H-pyrazole-5-carboxyamide Hydrochloride (Razaxaban), a Highly Potent, Selective, and Orally Bioavailable Factor Xa Inhibitor. Journal of medicinal chemistry,2005,48, (6), 1729-1744.
[74]Roehrig, S.; Straub, A.; Pohlmann, J.; Lampe, T.; Pernerstorfer, J.; Schlemmer, K.-H.; Reinemer, P.; Perzborn, E., Discovery of the novel antithrombotic agent 5-chloro-N-({(5 S)-2-oxo-3-[4-(3-oxomorpholin-4-yl) phenyl]-1,3-oxazolidin-5-yl} methyl) thiophene-2-carboxamide (BAY 59-7939):an oral, direct factor Xa inhibitor. Journal of medicinal chemistry,2005,48, (19), 5900-5908.
[75]Pinto, D.J.; Orwat, M.J.; Koch, S.; Rossi, K.A.; Alexander, R.S.; Smallwood, A.; Wong, P.C.; Rendina, A.R.; Luettgen, J.M.; Knabb, R.M., Discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl) phenyl)-4,5,6,7-tetrahydro-1 H-pyrazolo [3, 4-c] pyridine-3-carboxamide (Apixaban, BMS-562247), a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa. Journal of medicinal chemistry,2007,50, (22),5339-5356.
[76]Bauer, K.A., Fondaparinux:a new synthetic and selective inhibitor of Factor Xa. Best Practice & Research Clinical Haematology,2004,17, (1),89-104.
[77]Lobo, B.L., Emerging options for thromboprophylaxis after orthopedic surgery:A review of clinical data. Pharmacotherapy:The Journal of Human Pharmacology and Drug Therapy,2004,24, (7P2),66S-72S.
[78]Herbert, J.; Herault, J.; Bernat, A.; Van Amsterdam, R.; Lormeau, J.; Petitou, M.; Van Boeckel, C.; Hoffmann, P.; Meuleman, D., Biochemical and pharmacological properties of SANORG 34006, a potent and long-acting synthetic pentasaccharide. Blood,1998,91, (11),4197-4205.
[79]Herczeg, M.; Mezo, E.; Lazar, L.; Fekete, A.; Kover, K.E.; Antus, S.; Borbas, A., Novel syntheses of Idraparinux, the anticoagulant pentasaccharide with indirect selective factor Xa inhibitory activity. Tetrahedron,2013,69, (15),3149-3158.
[80]Chen, C.; Yu, B., Efficient synthesis of Idraparinux, the anticoagulant pentasaccharide. Bioorganic & medicinal chemistry letters,2009,19, (14),3875-3879.
[81]Buller, H.R.; Gallus, A.S.; Pillion, G.; Prins, M.H.; Raskob, G.E., Enoxaparin followed by once-weekly idrabiotaparinux versus enoxaparin plus warfarin for patients with acute symptomatic pulmonary embolism:a randomised, double-blind, double-dummy, non-inferiority trial. The Lancet, 2012,379, (9811),123-129.
[82]Eikelboom, J.W.; Weitz, J.I., Idrabiotaparinux treatment for venous thromboembolism. The Lancet, 2012,379, (9811),96-98.
[83]Weitz, J.I., Factor Xa and thrombin as targets for new oral anticoagulants. Thrombosis research, 2011,127, S5-S12.
[84]Yong, C.M.; Boyle, A.J., Factor Xa inhibitors in acute coronary syndromes and venous thromboembolism. Current vascular pharmacology,2010,8, (1),5-11.
[85]Walenga, J.M.; Prechel, M.; Jeske, W.P.; Hoppensteadt, D.; Maddineni, J.; Iqbal, O.; Messmore, H.L.; Bakhos, M., rivaroxaban-an oral, direct Factor Xa inhibitor-has potential for the management of patients with heparin-induced thrombocytopenia. British journal of haematology,2008,143, (1), 92-99.
[86]Salonen, L.M.; Holland, M.C.; Kaib, P.S.; Haap, W.; Benz, J.; Mary, J.L.; Kuster, O.; Schweizer, W.B.; Banner, D.W.; Diederich, F., Molecular recognition at the active site of factor Xa:cation-π interactions, stacking on planar peptide surfaces, and replacement of structural water. Chemistty-A European Journal,2012,18, (1),213-222.
[87]Samama, M.M., The mechanism of action of rivaroxaban-an oral, direct Factor Xa inhibitor-compared with other anticoagulants. Thrombosis research, 2011,127, (6),497-504.
[88]Misselwitz, F.; Berkowitz, S.D.; Perzborn, E., The discovery and development of rivaroxaban. Annals of the New York Academy of Sciences,2011,1222, (1),64-75.
[89]Perzborn, E.; Roehrig, S.; Straub, A.; Kubitza, D.; Misselwitz, F., The discovery and development of rivaroxaban, an oral, direct factor Xa inhibitor. Nature Reviews Drug Discovery,2010,10, (1), 61-75.
[90]Straub, A.; Roehrig, S.; Hillisch, A., Entering the Era of Non-Basic P1 Site Groups:Discovery of Xarelto(rivaroxaban). Current topics in medicinal chemistry,2010,10, (3),257-269.
[91]Thomas, T.F.; Ganetsky, V.; Spinler, S.A., rivaroxaban:an oral factor Xa inhibitor. Clinical therapeutics,2013,35, (1),4-27.
[92]Wong, P.C.; Pinto, D.J.; Zhang, D., Preclinical discovery of apixaban, a direct and orally bioavailable factor Xa inhibitor. Journal of thrombosis and thrombolysis,2011,31, (4),478-492.
[93]Pinto, D.J.; Orwat, M.J.; Wang, S.; Fevig, J.M.; Quan, M.L.; Amparo, E.; Cacciola, J.; Rossi, K.A.; Alexander, R.S.; Smallwood, A.M., Discovery of 1-[3-(Aminomethyl) phenyl]-N-[3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1 H-pyrazole-5-carboxamide (DPC423), a Highly Potent, Selective, and Orally Bioavailable Inhibitor of Blood Coagulation Factor Xa 1. Journal of medicinal chemistry,2001,44, (4),566-578.
[94]Zhang, P.; Bao, L.; Fan, J.; Jia, Z.J.; Sinha, U.; Wong, P.W.; Park, G.; Hutchaleelaha, A.; Scarborough, R.M.; Zhu, B.-Y., Anthranilamide-based< i> N,< i> N-dialkylbenzamidines as potent and orally bioavailable factor Xa inhibitors:P4 SAR. Bioorganic & medicinal chemistry letters, 2009,19, (8),2186-2189.
[95]Zhang, P.; Huang, W.; Wang, L.; Bao, L.; Jia, Z.J.; Bauer, S.M.; Goldman, E.A.; Probst, G.D.; Song, Y.; Su, T., Discovery of betrixaban (PRT054021),< i> N-(5-chloropyridin-2-yl)-2-(4-(< i> N,< i> N-dimethylcarbamimidoyl) benzamido)-5-methoxybenzamide, a highly potent, selective, and orally efficacious factor Xa inhibitor. Bioorganic & medicinal chemistry letters,2009,19, (8),2179-2185.
[96]Mathers, CD.; Boerma, T.; Ma Fat, D., Global and regional causes of death. Br Med Bull,2009, 92,7-32.
[97]Elahi, M.M.; Kong, Y.X.; Matata, B.M., Oxidative stress as a mediator of cardiovascular disease. Oxidative Medicine and Cellular Longevity,2009,2, (5),259-269.
[98]Munzel, T.; Gori, T.; Bruno, R.M.; Taddei, S., Is oxidative stress a therapeutic target in cardiovascular disease? Eur Heart J,2010,31, (22),2741-2748.
[99]Music in stroke rehabilitation. Lancet,2008,371, (9614),698.
[100]MULLER-DELP, J.M.; Gurovich, A.N.; Christou, D.D.; Leeuwenburgh, C., Redox balance in the aging microcirculation:new friends, new foes, and new clinical directions. Microcirculation, 2012,79,(1),19-28.
[101]Bae, Y.S.; Oh, H.; Rhee, S.G.; Yoo, YD., Regulation of reactive oxygen species generation in cell signaling. Mol Cells,2011,32, (6),491-509.
[102]Aliev, G; Li, Y.; H Palacios, H.; E Obrenovich, M., Oxidative Stress Induced Mitochondrial DNA Deletion as a Hallmark forthe Drug Development in the Context of the Cerebrovascular Diseases. Recent patents on cardiovascular drug discovery,2011,6, (3),222-241.
[103]Olmez, I.; Ozyurt, H., Reactive oxygen species and ischemic cerebrovascular disease, Neurochem Int,2012,60, (2),208-212.
[104]Park, J.G.; Oh, G.T., The role of peroxidases in the pathogenesis of atherosclerosis. BMB Rep, 2011,44, (8),497-505.
[105]Coller, B.S., Historical perspective and future directions in platelet research. J Thromb Haemost,2011,9 Suppl 1,374-395.
[106]Ueno, M.; Kodali, M.; Tello-Montoliu, A.; Angiolillo, D.J., Role of platelets and antiplatelet therapy in cardiovascular disease. JAtheroscler Thromb,2011,18, (6),431-442.
[107]Lowenberg, E.C.; Meijers, J.C.; Levi, M., Platelet-vessel wall interaction in health and disease. NethJMed,2010,68, (6),242-251.
[108]Brass, L.F.; Zhu, L.; Stalker, T.J., Novel therapeutic targets at the platelet vascular interface. Arterioscler Thromb Vasc Biol,2008,28, (3), s43-s50.
[109]张洁；王健；周其全,川芎嗪抗心肌缺血作用的研究进展.中国微循环,2003,7,(4),257-259.
[110]田禾；樊红亮,川芎嗪对猪离体冠状动脉的作用研究.中医研究,1997,10,(2),17-19.
[111]梁德；姚珍松,川芎嗪对家兔血管内皮细胞修复的影响.广州中医药大学学报,2001,18,(2),155-158.
[112]张志琳；包仕尧,川芎嗪对缺氧后血管内皮细胞功能保护作用的实验研究.江苏医药,2001,27,(5),346-348.
[113]林琳；郑永芳,mm—LDL激活ECV304 BKCa及丹参与川芎嗪的干预作用.中国医学科学院学报,2001,23,(4),386-389.
[114]赵丽霞；郭秀丽；张庆柱,川芎嗪抗缺血性心脑血管病的分子机制.齐鲁药事,2005,24,(5),298-300.
[115]王万铁；林丽娜；徐正祥,川芎嗪对心脏换瓣患者血小板聚集功能的影响.中国中西医结合杂志,2000,20,(1),13-14.
[116]王世军；史仁华；姬广臣；张连才；周恩平,葛根素,川芎嗪,丹参注射液对家兔软脑膜微循环的影响.微循环学杂志,1999,9,(4),19-21.
[117]陈开地；屈洪党,高粘滞血症患者血液流变学研究.中国微循环,1998,2,(2),108-109.
[118]张兆辉；赵保路,四甲吡嗪对活性氧自由基的清除作用.中国药理学报,1994,15,(3),229-231.
[119]夏腊梅；伍杰雄,川芎嗪对心肌细胞核钙转运功能异常的保护.中国临床药理学与治疗学2002,7,(2),124-126.
[120]史大卓；钟蓓,川芎嗪对缺氧缺糖培养心肌细胞蛋白质,RNA合成及一氧化氮合酶?….中国药学杂志,1998,33,(12),724-726.
[121]赵国胜；杨会杰,川芎嗪和丹参对大鼠心脏氧反常保护作用的比较.天津医药,1996,24,(5),289-292.
[122]雷万龙；刘勇；袁群芳,川芎嗪对脑缺血保护作用的实验研究.中华神经科杂志,2000,33,(2),100-100.
[123]苗里宁,川芎嗪对老龄鼠再灌注肾损伤中一氧化氮的作用研究.中国老年学杂志,2000,20,(4),242-243.
[124]程先超；刘新泳；徐文方,川芎嗪心脑血管药理学研究进展.中国医院药学杂志,2005,25,(7),661-663.
[125]陆一敏；万福生；唐三保,川芎嗪对儿茶酚胺诱导大鼠心肌损伤时Bcl-2和Fas蛋白表达的影响.中国临床药理学与治疗学,2004,9,(2),197-200.
[126]徐浩,川芎嗪心血管药理与钙拮抗作用.中国中西医结合杂志,2003,23,(5),376-377.
[127]汤碧娥；陈晓明；张宁,川芎嗪对麻醉家兔缺血再灌注心肌的保护作用.金华职业技术学院学报,2007,7,(4),63-65.
[128]张兆辉；余绍祖,川芎嗪对局灶性缺血多形核白细胞功能的影响.浙江中西医结合杂志,2002,12,(3),154-156.
[129]王春霞；包仕尧；刘春风；张志琳,尼莫通和川芎嗪对脑缺血-再灌注时c-fos和bcl-2蛋白表达的影响.中国危重病急救医学,1999,11,(10),609-612.
[130]李建生；赵君玫；郭盛典；李建国；张卫红；程龙,川芎嗪和参麦注射液对老龄大鼠脑缺血再灌注多器官损伤保护作用.第四次全国中西医结合中青年学术研讨会论文集,2002.
[131]王万铁；陈寿权,川芎嗪注射液对脑缺血一再灌注损伤家兔血清白细胞介素—8的影响.中国中西医结合急救杂志,2000,7,(6),359-361.
[132]田晔；窦从让,川芎嗪对原代神经细胞缺血性再灌注后核因子一KB表达的影响及意义.中华临床杂志,2002,2,(11),16-17.
[133]Liao, F., Herbs of activating blood circulation to remove blood stasis. Clin Hemorheol Microcirc,2000,23, (2-4),127-131.
[134]Liao, F.; Jiao, L., Ligustrazine, allicin and shear-induced platelet aggregation. Clin Hemorheol Microcirc,2000,22, (2),167-168.
[135]Feng, L.; Ke, N.; Cheng, F.; Guo, Y.; Li, S.; Li, Q.; Li, Y., The protective mechanism of ligustrazine against renal ischemia/reperfusion injury. J Surg Res,2011,166, (2),298-305.
[136]Wu, H.J.; Hao, J.; Wang, S.Q.; Jin, B.L.; Chen, X.B., Protective effects of ligustrazine on TNF-alpha-induced endothelial dysfunction. Eur J Pharmacol,2012,674, (2-3),365-369.
[137]Gao, C.; Peng, H.; Wang, S.; Zhang, X., Effects of Ligustrazine on pancreatic and renal damage after scald injury. Burns,2012,38, (1),102-107.
[138]Gao, C.; Liu, Y; Ma, L.; Zhang, X.; Wang, S., Effects of Ligustrazine on pulmonary damage in rats following scald injury. Burns,2012,38, (5),743-750.
[139]叶云鹏；王世英,川芎嗪的代谢转化和结构改造研究.中国协和医科大学&,1993.
[140]陈学敏；曹永孝,2—羟甲基—3,5,6—三甲基吡嗪对心血管系统的药理作用.中国医药工业杂志,1998,29,(6),258-260.
[141]边晓丽；陈学敏；刘艳霞；潘清,川芎嗪及其衍生物对羟自由基的清除作用.中国医院药学杂志,2004,23,(11),678-679.
[142]刘新泳；葛蔚颖；徐丽君；张君仁；王海燕；潘淑菊,川芎嗪体内代谢产物的合成.山
东医科大学学报,1997,35,(1),80-80.
[143]刘建建.川芎嗪衍生物的设计,合成及其生物活性研究.山东大学,2006.
[144]Li, Z.; Yu, F.; Cui, L.; Chen, W.; Wang, S.; Zhan, P.; Shen, Y.; Liu, X., Ligustrazine derivatives. Part 8:design, synthesis, and preliminary biological evaluation of novel Ligustrazinyl amides as cardiovascular agents. Med Chem,2014,10,(1),81-89.
[145]李国宁.川芎嗪查尔酮类衍生物的设计,合成及其生物活性研究.山东大学,2012.
[146]张爽.川芎嗪代谢产物及其衍生物的设计,合成与生物活性研究.山东大学,2012.
[147]夏承建；王松；朱鹤孙,川芎嗪及其衍生物抗凝血活性的研究.中草药,2004,35,(8),911-913.
[148]Singh, P.K.; Singh, R.; Singh, S., Cinnamic acid induced changes in reactive oxygen species scavenging enzymes and protein profile in maize (Zea mays L.) plants grown under salt stress. Physiol Mol Biol Plants,2013,19, (1),53-59.
[149]Song, F.; Li, H.; Sun, J.; Wang, S., Protective effects of cinnamic acid and cinnamic aldehyde on isoproterenol-induced acute myocardial ischemia in rats. J Ethnopharmacol,2013,150, (1),125-130.
[150]Arii, K.; Igarashi, H.; Arii, T.; Katayama, Y., The effect of ozagrel sodium on photochemical thrombosis in rat:therapeutic window and combined therapy with heparin sodium. Life Sci,2002,71, (25),2983-2994.
[151]Park, S.I.; Jang, D.K.; Han, Y.M.; Sunwoo, Y.Y.; Park, M.S.; Chung, Y.A.; Maeng, L.S.; Im, R.; Kim, M.W.; Jeun, S.S.; Jang, K.S., Effect of combination therapy with sodium ozagrel and panax ginseng on transient cerebral ischemia model in rats. J Biomed Biotechnol,2010,2010,893401.