臺灣博碩士論文加值系統

本研究團隊在過去的研究中發現一個 β-nitrostyrene 類化合物，3,4-methylenedioxy-β-nitrostyrene（MNS）對於血小板凝集反應具有強效的抑制效果。此外， MNS 之苯甲醯基（benzoyl）取代衍生物 4-O-benzoyl-3- methoxyl-β-nitrostyrene（BMNS）對於由 thrombin 誘發血小板凝集的抑制能力約為 MNS 的 8 倍，且為非選擇性酪氨酸激酶抑制劑 genistein 與 tyrphostin A47 的 100 倍。另經由進一步研究指出 β-nitrostyrene 類化合物為一群具有抗血小板能力的新型酪氨酸激酶抑制劑，可以經由阻斷 GP IIb/IIIa 的活化而達到抑制血小板凝集的效果。綜上所述可以看出 β-nitrostyrene 類化和物具有開發為抗血小板藥物的潛力。因此為了進一步尋找更強效的抗血小板凝集的藥物，本研究團隊擬合成一系列新型的 β-nitrostyrene 衍生物，並進一步的探討其化學結構與活性的關係（structure-activity relationships, SAR）。在本研究中，共合成 47 個化合物，並測其抗血小板凝血活性。分析其活性結果，並歸納其 SAR 如下：
1. 所有化合物或市售之 β-nitrostyrene 類化合物皆具有抑制由thrombin 與 collagen 兩者所誘導之血小板凝集活性。
2. 對於 Series A 化合物而言，修飾其 B 環上取代基（R）之位置與基團時，可改變其活性；其中para-位置取代時，活性普遍較佳。
3. 對於 Series B 化合物而言：
a. 當 A 環部份改以其他芳香環取代（如CYT-RX17與CYT-RX18）時，其活性下降。
b. 當 A 環部份 para 位置改以 hydroxyl group 時，R3 取代基由 OCH3 變更為 OCH2CH3 時，其活性下降。
c. 當 A 環部份 para 位置改以 hydroxyl group 以其 isoteres（如 Cl 與 CH3）或其他基團（如 NO2 與 OCH3）時，其活性皆上升。其中以 NO2 與 Cl 取代為最佳，此數據顯示 A 環部份 para 位置取代拉電子基團或鹵素原子，可增加 β-nitrostyrene 類化合物之活性。
d. 當將 β-nitrostyrene 修飾為 β-methyl-β-nitrostyrenes（即R4由H取代改變為CH3時；如CYT-RX19與CYT-RX20，以及CYT-RX21與CYT-RX22），其活性下降。

In previous studies, 3,4-methylenedioxy-β-nitrostyrene (MNS) and 4-O-benzoyl-3-methoxyl-β-nitrostyrene (BMNS) exhibited inhibitory effector platelet aggregation via suppression of tyrosine kinases (Src and Syk). Furthermore, BMNS possessed 8 times greater potency than MNS, and 100 times greater potency than non-selective tyrosine kinases inhibitors (genistein and tyrphostin A47) in inhibiting thrombin-induced pletlet aggregation. Accordingly, β-nitrostyrenes derivatives were be as potential anti-platelet aggregation agent. In order to research and develop potent antiplatelet β-nitrostyrenes, we designed and synthesized a series of β-nitrostyrenes derivatives and evaluate their anti-platelet activities. On the basis of their bioactivities, the proposed structure-activity relationships(SARs) were as following.
1. All of the synthesized or commercial β-nitrostyrene derivatives inhibited platelet aggregation induced by thrombin or collagen.
2. For the compounds of Series A, the substitutes on the B ring influence the bioactivity. Of these, the derivatives with the para- substitutes are more potency.
3. For the compounds of Series B,
a. When the benzene ring (A ring) was replaced by thiophenyl or furanyl ring, (ex CYT-RX17 and CYT-RX18), the activity was decreased.
b. While the part of A ring in para-position is changed to hydroxyl group , the activity decreases.
c. While the para-hydroxyl group in the A ring was modified by its isoteres (e.g. Cl or CH3), or other groups (e.g. NO2 or OCH3), the activity all increases.
d. The bioactivities of β-methyl-β-nitrostyrenes (CYT-RX20 and CYT-RX22) were less then β-nitrostyrenes (CYT-RX19 and CYT-RX21).

目錄
指導教授推薦書
口試委員會審定書
授權書 iii
誌謝 iv
中文摘要 v
英文摘要 vii
目錄 ix
圖目錄 xi
表目錄 xii
第一章 緒論 1
1.1 研究背景 1
1.2 研究背景與動機 8
1.3 研究目標與設計 11
第二章 實驗材料及方法 13
2.1 實驗器材及儀器 13
2.2 試藥 14
2.3 合成方法 18
2.4 合成生物活性實驗方法 22
第三章 結果與討論 24
3.1 合成部份及其產率（表3.1.1） 24
3.2 活性部分 24
第四章 結論 38
對於 Series A 化合物而言（圖4.1）： 38
參考文獻 42
附錄光譜性質及物理性質 47

圖目錄
圖 1.1.1：血小板之結構說明圖 2
圖 1.3.1：CYT-RX11~ CYT-RX16 ＆ CYT-RX19~ CYT-RX22 12
圖 1.3.2：CYT-RX17~ CYT-RX18取代基之結構說明圖 12
圖 1.3.3：單取代、雙取代、三取代及多取代基之結構說明圖 12
圖 2.3.1：CYT-RX1~ CYT-RX10 ＆ CYT-RX23~ CYT-RX43 19
圖 2.3.2：CYT-RX19 & CYT-RX21化學合成流程圖 20
圖 2.3.3：CYT-RX20 & CYT-RX22化學合成流程圖 20
圖 2.3.4：CYT-RX44 & CYT-RX45化學合成流程圖 21
圖 2.3.5：CYT-RX46 & CYT-RX47化學合成流程圖 21
圖 3.1.1：苯環上含氮取代基化學合成流程圖 31
圖 4.1：SERIES A化合物取代基最佳活性整理圖 41
圖 4.2：SERIES B化合物取代基最佳活性整理圖 41

表目錄
表 3.1.1：各化學合成產物之產率圖表 30
表 3.2.1：化合物CYT-RX1~CYT-RX10活性分析一覽表 32
表 3.2.2：化合物CYT-RX11~ CYT-RX16活性分析一覽表 33
表 3.2.3：化合物CYT-RX16~ CYT-RX22活性分析一覽表 33
表 3.2.4：化合物CYT-RX23~ CYT-RX34活性分析一覽表 34

行政院衛生署(2007年).台灣96年衛生統計動向.衛生署統計資訊網.取自http://health99.doh.gov.tw/media/pubic/pdf/21593.pdf
(1962) Platelet aggregation: Part II Some results from a new method of study. J Clin Pathol, 15(5): 452-455.
(2002) Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. Bmj, 324(7329): 71-86.
Asahi, M, Yanagi, S, Ohta, S, Inazu, T, Sakai, K, Takeuchi, F, Taniguchi, T, Yamamura, H (1992) Thrombin-induced human platelet aggregation is inhibited by protein-tyrosine kinase inhibitors, ST638 and genistein. FEBS Lett, 309(1): 10-14.
Behan, MW, Storey, RF (2004) Antiplatelet therapy in cardiovascular disease. Postgrad Med J, 80(941): 155-164.
Bennett, JS, Vilaire, G (1979) Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest, 64(5): 1393-1401.
Bhatt, DL, Fox, KA, Hacke, W, Berger, PB, Black, HR, Boden, WE, Cacoub, P, Cohen, EA, Creager, MA, Easton, JD, Flather, MD, Haffner, SM, Hamm, CW, Hankey, GJ, Johnston, SC, Mak, KH, Mas, JL, Montalescot, G, Pearson, TA, Steg, PG, Steinhubl, SR, Weber, MA, Brennan, DM, Fabry-Ribaudo, L, Booth, J, Topol, EJ (2006) Clopidogrel and aspirin versus aspirin alone for the prevention of atherothrombotic events. N Engl J Med, 354(16): 1706-1717.
Carter, KC, Finnon, YS, Daeid, NN, Robson, DC, Waddell, R (2002) The effect of nitrostyrene on cell proliferation and macrophage immune responses. Immunopharmacol Immunotoxicol, 24(2): 187-197.
Cattaneo, M (2004) Aspirin and clopidogrel: efficacy, safety, and the issue of drug resistance. Arterioscler Thromb Vasc Biol, 24(11): 1980-1987.
Colman, PD, White, GH (1990) Complexities in the management of arterial compromise due to thoracic outlet syndrome. Aust N Z J Surg, 60(12): 1000-1002.
Coma-Canella, I, Velasc, A (2007) Variability in individual responsiveness to aspirin: clinical implications and treatment. Cardiovasc Hematol Disord Drug Targets, 7(4): 274-287.
Dore, JC, Viel, C (1975) [Research in antitumoral chemotherapy. X. Cytotoxic and antitumoral activity of beta-nitrostyrenes and of composed nitrovinyl derivatives]. Farmaco Sci, 30(2): 81-109.
Furman, MI, Grigoryev, D, Bray, PF, Dise, KR, Goldschmidt-Clermont, PJ (1994) Platelet tyrosine kinases and fibrinogen receptor activation. Circ Res, 75(1): 172-180.
Gorelick, PB, Weisman, SM (2005) Risk of hemorrhagic stroke with aspirin use: an update. Stroke, 36(8): 1801-1807.
Hamilton, JR (2009) Protease-activated receptors as targets for antiplatelet therapy. Blood Rev, 23(2): 61-65.
Hankey, GJ, Eikelboom, JW (2003) Antiplatelet drugs. Med J Aust, 178(11): 568-574.
Hsieh, PW, Hwang, TL, Wu, CC, Chiang, SZ, Wu, CI, Wu, YC (2007) The evaluation and structure-activity relationships of 2-benzoylaminobenzoic esters and their analogues as anti-inflammatory and anti-platelet aggregation agents. Bioorg Med Chem Lett, 17(6): 1812-1817.
Hynes, MS (1992) Pro: whole blood transfusions are useful in patients undergoing cardiac surgery. J Cardiothorac Vasc Anesth, 6(6): 756-760.
Jackson, SP, Schoenwaelder, SM, Yuan, Y, Salem, HH, Cooray, P (1996) Non-receptor protein tyrosine kinases and phosphatases in human platelets. Thromb Haemost, 76(5): 640-650.
Kaap, S, Quentin, I, Tamiru, D, Shaheen, M, Eger, K, Steinfelder, HJ (2003) Structure activity analysis of the pro-apoptotic, antitumor effect of nitrostyrene adducts and related compounds. Biochem Pharmacol, 65(4): 603-610.
Kawamura, M, Imura, Y, Moriya, N, Kita, S, Fukushi, H, Sugihara, H, Nishikawa, K, Terashita, Z (1996) Antithrombotic effects of TAK-029, a novel GPIIb/IIIa antagonist, in guinea pigs: comparative studies with ticlopidine, clopidogrel, aspirin, prostaglandin E1 and argatroban. J Pharmacol Exp Ther, 277(1): 502-510.
Khaw, KT (1996) Epidemiology of stroke. J Neurol Neurosurg Psychiatry, 61(4): 333-338.
Kim, JH, Kim, JH, Lee, GE, Lee, JE, Chung, IK (2003) Potent inhibition of human telomerase by nitrostyrene derivatives. Mol Pharmacol, 63(5): 1117-1124.
Lee, HS (2006) Antiplatelet property of Curcuma longa L. rhizome-derived ar-turmerone. Bioresour Technol, 97(12): 1372-1376.
Lincoff, AM (2001) Anticoagulant and antiplatelet drugs. Catheter Cardiovasc Interv, 54(4): 514-520.
Luttrell, LM, Daaka, Y, Lefkowitz, RJ (1999) Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Curr Opin Cell Biol, 11(2): 177-183.
Mackman, N, Tilley, RE, Key, NS (2007) Role of the extrinsic pathway of blood coagulation in hemostasis and thrombosis. Arterioscler Thromb Vasc Biol, 27(8): 1687-1693.
Maguire, PB, Wynne, KJ, Harney, DF, O'Donoghue, NM, Stephens, G, Fitzgerald, DJ (2002) Identification of the phosphotyrosine proteome from thrombin activated platelets. Proteomics, 2(6): 642-648.
Maurice, DH, Haslam, RJ (1990) Molecular basis of the synergistic inhibition of platelet function by nitrovasodilators and activators of adenylate cyclase: inhibition of cyclic AMP breakdown by cyclic GMP. Mol Pharmacol, 37(5): 671-681.
Mc, GJ, Brian, PW, Hemming, HG (1948) The fungistatic activity of ethylenic and acetylenic compounds; the effect of the affinity of the substituents for electrons upon the biological activity of ethylenic compounds. Ann Appl Biol, 35(1): 25-36.
Milhazes, N, Calheiros, R, Marques, MP, Garrido, J, Cordeiro, MN, Rodrigues, C, Quinteira, S, Novais, C, Peixe, L, Borges, F (2006) Beta-nitrostyrene derivatives as potential antibacterial agents: a structure-property-activity relationship study. Bioorg Med Chem, 14(12): 4078-4088.
Milhazes, N, Martins, P, Uriarte, E, Garrido, J, Calheiros, R, Marques, MP, Borges, F (2007) Electrochemical and spectroscopic characterisation of amphetamine-like drugs: application to the screening of 3,4-methylenedioxymethamphetamine (MDMA) and its synthetic precursors. Anal Chim Acta, 596(2): 231-241.
Monroe, DM, Hoffman, M (2002) Coagulation factor interaction with platelets. Thromb Haemost, 88(2): 179.
Narasimhan, B, Belsare, D, Pharande, D, Mourya, V, Dhake, A (2004) Esters, amides and substituted derivatives of cinnamic acid: synthesis, antimicrobial activity and QSAR investigations. Eur J Med Chem, 39(10): 827-834.
Nash, GF, Turner, LF, Scully, MF, Kakkar, AK (2002) Platelets and cancer. Lancet Oncol, 3(7): 425-430.
Parikh, SA, Beckman, JA (2007) Contemporary use of clopidogrel in patients with coronary artery disease. Curr Cardiol Rep, 9(4): 257-263.
Patel, D, Moonis, M (2007) Clinical implications of aspirin resistance. Expert Rev Cardiovasc Ther, 5(5): 969-975.
Patton, GM (2004) Arterial thoracic outlet syndrome. Hand Clin, 20(1): 107-111, viii.
Putney, JW, Jr. (1990) Capacitative calcium entry revisited. Cell Calcium, 11(10): 611-624.
Raju, NC, Eikelboom, JW, Hirsh, J (2008) Platelet ADP-receptor antagonists for cardiovascular disease: past, present and future. Nat Clin Pract Cardiovasc Med, 5(12): 766-780.
Schlessinger, J, Ullrich, A (1992) Growth factor signaling by receptor tyrosine kinases. Neuron, 9(3): 383-391.
Schorlemmer, A, Matter, ML, Shohet, RV (2008) Cardioprotective signaling by endothelin. Trends Cardiovasc Med, 18(7): 233-239.
Shattil, SJ (1999) Signaling through platelet integrin alpha IIb beta 3: inside-out, outside-in, and sideways. Thromb Haemost, 82(2): 318-325.
Spinler, SA (2009) Safety and tolerability of antiplatelet therapies for the secondary prevention of atherothrombotic disease. Pharmacotherapy, 29(7): 812-821.
Taha, AS, McCloskey, C, Prasad, R, Bezlyak, V (2009) Famotidine for the prevention of peptic ulcers and oesophagitis in patients taking low-dose aspirin (FAMOUS): a phase III, randomised, double-blind, placebo-controlled trial. Lancet, 374(9684): 119-125.
Wagner, CL, Mascelli, MA, Neblock, DS, Weisman, HF, Coller, BS, Jordan, RE (1996) Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets. Blood, 88(3): 907-914.
Wang, WY, Hsieh, PW, Wu, YC, Wu, CC (2007) Synthesis and pharmacological evaluation of novel beta-nitrostyrene derivatives as tyrosine kinase inhibitors with potent antiplatelet activity. Biochem Pharmacol, 74(4): 601-611.
Wang, WY, Wu, YC, Wu, CC (2006) Prevention of platelet glycoprotein IIb/IIIa activation by 3,4-methylenedioxy-beta-nitrostyrene, a novel tyrosine kinase inhibitor. Mol Pharmacol, 70(4): 1380-1389.
Wiviott, SD (2008) Intensity of antiplatelet therapy in patients with acute coronary syndromes and percutaneous coronary intervention: the promise of prasugrel? Cardiol Clin, 26(4): 629-637.
Zhang, YH, Chung, KH, Ryu, CK, Ko, MH, Lee, MK, Yun, YP (2001) Antiplatelet effect of 2-chloro-3-(4-acetophenyl)-amino-1, 4-naph
thoquinone (NQ301): a possible mechanism through inhibition of intracellular Ca2+ mobilization. Biol Pharm Bull, 24(6): 618-622.