臺灣博碩士論文加值系統

摘要
實體腫瘤常因血液供應不足導致細胞缺氧，形成 hypoxic cell，而造成對化療及放射療法產生抗性，致使治療實體腫瘤時，療效不佳。生物還原劑是新一類抗癌藥，選擇性作用於hypoxic cells。目前，生物還原劑依結構可分為四大類；其中以 aromatic N-oxide 類的tirapazamine ( TPZ ) 對 hypoxic cells 的細胞毒性選擇性最高，副作用最小，顯示極具抗癌新藥發展潛力。

本實驗室以TPZ 作為先導化合物，進行結構修飾，合成一系列 di-N-oxide 化合物，以篩選高效、高選擇、低毒性的抗癌藥物。標的產物以離體癌細胞毒性試驗(MTS assay)評估抗癌活性，另文獻報告苯三氮(benzotriazine)類化合物具抗炎活性；因而將本系列TPZ類似物以鹿角菜誘導浮腫試驗，評估其抗炎活性。

初步結果顯示一系列tirapazamine類似物含氧狀態下之MTS 抗癌篩選上，並未呈現明顯的抗癌效果，但化合物9a之抗癌活性較先導化合物TPZ 佳。鹿角菜誘導浮腫之抗發炎試驗中，化合物4b、4c、6、7f、9a抗發炎效果較ibuprofen佳；其中又以9a最優，其抗發炎比leflunomide佳。

ABSTRACT

Solid tumor causes cells with deficiency of oxygen to form hypoxic cells because of the insufficient supply of the blood. Cancer patients resistant to chemotherapy and radiotherapy show a poor curative effect. The bioreductive agents are novel anticancer drugs which selectively act on hypoxic cells. At present, the bioreductive anticancer agents can be divided into four main classes in accordance with the structure. Among them, tirapazamine (TPZ) of aromatic N-oxides is highest in the cytotoxic selectivity of cell to hypoxic cell, whereas the side effect is minimum. The bioreductive agents, therefore, possess developmental potentiality of novel anticancer agents.

Regarding TPZ as the lead anticancer compound in this laboratory, we aimed at the synthesis of a series of di-N-oxides to develop a new class of bioreductive anticancer agents with higher selectivity and lower toxicity. The synthetic compounds were examined on the cytotoxic activity by MTS assay, and anti-inflammatory activity by a carrageenan-induced edema model.

All the compounds displayed insignificant anticancer effect under normal oxygen conditions, but 9a showed better cytotoxic effect than TPZ. In carrageenan-induced edema model, 4b, 4c, 6, 7f, and 9a revealed better anti-inflammatory activity than ibuprofen. Among them 9a showed most potent, even superior to leflunomide.

目錄
頁次
正文目錄..................................................................................................I
附圖目錄..................................................................................................III
中文摘要……………………………………………………………......V
英文摘要………………………………………………………………..VI

第一章、緒論…………………………………………………………...1
第一節、前言………………………………………………………1
壹、癌症的簡介………………………………………..…….1
貳、癌症的治療………………………….…………………..6
第二節、研究目的…………………………………….…………..15
壹、缺氧細胞(hypoxic cell)的簡介………………………...15
貳、克服hypoxic cell 的策略……………..………………17
參、Tirapazamine (TPZ)的簡介...………………………....20
第三節、研究方法……………………………………….………..23
壹、藥物設計原理…………………………………………...23
貳、合成反應概要……………………………………………26
第二章、材料與方法……………………………………………………32
第一節、儀器與材料……………………………………………...32
壹、重要儀器………………………………………………....32
貳、試藥來源………………………………………………....33
第二節、合成實驗………………………………………………...35
第三節、生物活性試驗……………………………………………46
壹、生物活性試驗原理………………………………………46
貳、生物活性試驗方法……………………………………..47


第三章、結果……………………………………………………………50
第一節、化學合成…………………………………………………52
第二節、生物活性試驗(抑制腫瘤細胞活性試驗)……………….80
第四章、討論……………………………………………………………85
第一節、化學合成…………………………………………………85
第二節、生物活性試驗……………………………………………90
第五章、結論……………………………………………………………91
第六章、參考文獻………………………………………………………92

























表目錄
頁次
Table 1. The cell cytotoxicity (％) of TPZ analogs ………….....….80
Table 2. Effect of pre-treatment with tirapzamine analogs 4a~4h
on inflammation..............................................................81
Table 3. Effect of pre-treatment with tirapzamine analogs 7a~7h
on inflammation..............................................................82
Table 4. Effect of pre-treatment with tirapzamine analogs 11, 13, 14 on inflammation.........................................................83
Table 5. Effect of pre-treatment with tirapzamine analogs 6, 9a,
10a, 16 on inflammation.................................................84

圖目錄
頁次
圖一、細胞週期…………………………………………………………..3
圖二、Alkylating agents作用機轉……………………………………..8
圖三、Alkylating agents對DNA可能的作用…………………...........9
圖四、正常組織血管與癌症組織血管圖……....................................15
圖五、癌細胞剖面圖........................…..……………...........……….15圖六、放射線在有氧及無氧狀態下的選擇力...................................16
圖七、生物還原抗癌藥之共通機轉………………............................19
圖八、生物還原抗癌藥機轉圖例……………………………………..19
圖九、TPZ的HCR實驗….............................................................20
圖十、TPZ作用機轉……………………………………………………21
圖十一、TPZ對DNA雙股影響之假說機圖......................................21
圖十二、TPZ於放療增敏的作用機制.............................................22
圖十三、化合物1之1H-NMR＆13C圖譜……………………………94
圖十四、化合物2之1H-NMR圖譜…＆13C圖譜.........……...…….95
圖十五、化合物3之1H-NMR圖譜＆13C圖譜…......……..……….96
圖十六、化合物4a之1H-NMR＆13C圖譜……………………………97
圖十七、化合物4b之1H-NMR＆13C圖譜…………………………...98
圖十八、化合物4c之1H-NMR＆13C圖譜…………………………...99
圖十九、化合物4d之1H-NMR. ＆13C圖譜………………………100
圖二十、化合物4e之1H-NMR................…………….…………….101
圖二十一、化合物4f之1H-NMR...............………………………….102
圖二十二、化合物4g之1H-NMR＆13C圖譜………………………103
圖二十三、化合物4h之1H-NMR＆13C圖譜..…………………….104
圖二十四、化合物5之1H-NMR圖譜＆13C圖譜………………….105
圖二十五、化合物6之1H-NMR＆13C圖譜……………………….106
圖二十六、化合物7a之1H-NMR圖譜＆13C圖譜…………………107
圖二十七、化合物7b之1H-NMR圖譜＆13C圖譜….......…….….108
圖二十八、化合物7c之1H-NMR＆13C圖譜………………………109
圖二十九、化合物7d之1H-NMR＆13C圖譜………………………..110
圖三十、化合物7e之1H-NMR＆13C圖譜………………..……….111
圖三十一、化合物7f之1H-NMR＆13C圖譜………………………112
圖三十二、化合物7g之1H-NMR＆13C圖譜………………………..113
圖三十三、化合物7h之1H-NMR＆13C圖譜……………………...114
圖三十四、化合物9a之1H-NMR................……………………....115
圖三十五、化合物10a之1H-NMR................……………………..116
圖三十六、化合物11之1H-NMR＆13C圖譜………………………117
圖三十七、化合物12之1H-NMR圖譜…......……………………..118
圖三十八、化合物13之1H-NMR＆13C圖譜……………………...119
圖三十九、化合物14之1H-NMR＆13C圖譜……………………...120
圖四十、化合物15之1H-NMR＆13C圖譜……....………………...121
圖四十一、化合物16之1H-NMR＆13C圖譜……………………...122

第六章、參考文獻

1.http://www.uhealthy.com/cancer-chinese/cancer-news/news-031.htm
2.Brown, J. M. Tumor microenvironment and the response to anticancer
therapy. Cancer Biol. Ther. 2000, 1( 5 ), 453 – 458.
3.Brown, J. M. Exploiting the hypoxic cancer cell : Mechanism and therapeutic strategies. Mol. Med. Today. 2000, 6, 157–162.
4.Workman, P. ; Stratford, I. J. Bioreductive drugs into the next millennium .
Anti–Cancer Drug Design. 1998,13, 519–528.
5.Brown, J. M. ; Wang, L. H. Tirapazamine : Laboratory data relevant to clinical activity. Anti–Cancer Drug Design. 1998, 13, 529–539.
6.Fuchs, T. ; Chowdhury, G. ; Barnes, C. L. et al. 3-Amino- 1, 2 , 4 – benzotriazine 4-oxide : Characterization of a new metabolite arising from bioreductive processing of the antitumor agent 3-amino -1, 2,4 - benzotriazine 1, 4-dioxide (Tirapazamine) J. Org. Chem. 2001, 66, 107–114.
7.Bump, E. A. ; Foye, W. O. Radiosensitizer and Radioprotective Agent.In Wolff M. E. editor, Burger，s Medicinal Chemistry and Drug Discovery. Vol. 4: Therapeutic Agents. Wiley – interscience. N Y, 1997, p5 – 69 .
8.Kelson, A. B.; McNamara, J. P.; Pandey, A. et al. 1,2,4-benzotriazine 1,4-dioxides. An important class of hyopoxic cytoxin with antitumor activity. Anti-Cancer Drug Design 1998, 13, 575-592.
9.Hay, M. P.; Gamage, S. A.; Brown, J. M.; et al. Structure-activity relationships of 1,2,4-benzotriazine 1,4-dioxides as hypoxia selective analogues of tirapazamine. J. Med. Chem. 2003, 46, 168-182.
10.Hay, M. P.; Gamage, S. A.; Brown, J. M.; et al. DNA-targeted 1,2,4-benzotriazine 1,4-dioxides : Potent analogues of the hypoxia- selective cytotoxin tirapazamine. J. Med. Chem. 2004, 47, 475-488.
11.Nagasawa, H.; Yamashita, M.; Mikamo, N. et al. Design, synthesis and biological activites of antiangiogenic hypoxic cytotoxin, trazine-N-oxide derivatives. Comparative Biochem. Phys. 2002, 132, 33-40.
12.Brown, J. M. The hypoxic cell : A target for selective cancer therapy.
Cancer Res. 1999, 59, 5863 – 5870.
13.www.healthheal.com/asiapolbax/polbax.pdf
14.http://med.mc.ntu.edu.tw/~onc/Lecture/cancer1.html
15.蘇純閨：化學療法。藥理學。初版。合記圖書出版社，台北市。頁數：615-629。
16.http://hk.geocities.com/regenso/d4.html
17.http://www.kfsyscc.org/patients/faq/cat20_sub02.html
18.www.cgmh.org.tw/intr/intr5/c6700/teaching/ cancer/子宮頸癌/淺談放療.htm
19.www.fcf.org.tw/me-005a.htm
20.Denny, W. A. The role of hypoxia-activated prodrug in cancer therapy. Lancet Oncology 2000, 1(9), 25-29.
21.http: //www.24drs.com/consumer/disease/O2/index.asp
22.http: //www.vghtpe.gov.tw/~ped/newpage120.htm
23.Naylor, M. A.; Thomson, P. Recent advances in bioreductive drug targeting. Mini Review in Med. Chem. 2001, 1, 17-29.
24.Gatzemeier, U.; Pawel, J.; Boyer, M. et al. Tirapazamine plus cisplatin vs. cisplatin in advanced non-small-cell lung cancer : a report of the international CATAPULTⅠstudy group. J. Clin. Oncol. 2000, 18, 1351-1359.
25.Denny, W. A. Prodrug strategies in cancer therapy. Eur. J. Med. Chem. 2001, 36, 577-595.
26.Cerecetto, H.; Gonzalez, M. N-oxide as hypoxia selective cytotoxins. Mini Reviews in Med. Chem. 2001, 1, 219-231.
27.Zarranz, B.; Jaso, A.; Aldana, I.; Monge, A. Synthesis and antimycobacterial activity of new quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives. Bioog. Med. Chem. 2003, 11, 2149-2156.
28.Harold, E. K.; Mary, A. K.; Inn, H. et al. Neutron radiation enhances cisplatin cytotoxicity independently of apoptosis in human head and neck carcinoma cells. Clin. Cancer Res. 2000, 6, 4142-4147.
29. Winter C. A., Risley E. A., Nuss C. W. Carrageenin-Induced Edema in Hind Paw of the Rat as an Assay for Antiinflammatory Drugs. Proc. Soc. Exp. Biol. Med. 1962; 111: 544-7.
30.Gentilli M., Mazoit J.X., Bouaziz H., Fletcher D., Casper R.F., Benhamou D., Savouret J.F. Resveratrol decreases hyperalgesia induced by carrageenan in the rat hind paw. Life. Sci. 2001 Feb 2; 68(11): 1317-21.
31.Amann R., Schuligoi R. Inhibition of carrageenan-induced edema by indomethacin or sodium salicylate does not prevent the increase of nerve growth factor in the rat hind paw. Neurosci. Lett. 2000 Jan 14; 278(3): 173- 6.
32. Regan, BA. Hanifin, JW. Anti-inflammatory and analgesic profile of amidines of
3-amino-1,2,4-benzotriazine and 3-amino-1,2,4-benzotriazine-1-oxide. J Pharm Sci. 1980, 7, 789-793.
33. Antonio Monge, Francisco J. Hypoxia-selective agents derived from 2-quinoxalinecarbonotrile 1,4-di-N-oxide. J. Med. Chem. 1995, 38, 4488-4494