本論文主旨在研究設計與合成新穎組織蛋白酶S抑制劑，探討結構與活性的關係（structure-activity relationship，SAR），並進一步應用於抗癌症轉移之研究。研究方向分為二部分：第一部分係探討α-酮醯胺胜肽系列化合物之合成與抗癌轉移的研究；第二部分係探討醛基胜肽系列化合物之合成與抗癌轉移的研究。

首先我們利用Passerini multicomponent reaction(MCR)合成一系列α-酮醯胺胜肽化合物，建立一系列P3位置衍生物，進行芳香環取代基效應、脂環族骨架結構、提昇選擇性和氨基甲酸酯置換醯胺四個方向進行結構與活性關係的探討。

接著我們利用兩次醯胺偶合反應建構醛基胜肽系列化合物之主要片段方法，合成一系列組織蛋白酶S彈頭(warhead)位置衍生物，增加彈頭結構的多元性與新穎性，並進行結構與活性關係的探討。

總結本論文藉由建立α-酮醯胺胜肽系列和醛基胜肽系列兩系列衍生物之結構活性關係，尋找抑制組織蛋白酶S的最佳結構化合物，進行抗癌症轉移生理活性之研究。

This dissertation focuses on the design and synthesis of novel Cathepsin S inhibitors used as anticancer agents in metastasis research. For further detailed studies on structure-activity relationships (SARs), the research is subdivided into two parts: a) dipeptide synthesis of α-ketoamide series and it’s anticancer activity discussion; b) dipeptide synthesis of aldehyde series and it’s anticancer activity discussion.
Passerini multicomponent reaction is the key step for the synthesis of α-ketoamide. To construct structure-activity relationships, we focus on modifying analogues of P3 site series. Our strategy is divided into the following four parts: a) aromatic ring substitution effect; b) aliphatic ring size effect to compare it to the aromatic ring; c) enhancing selectivity for cathepsin S; and d) changing amide moiety to urea or thiourea.
The peptides of aldehyde series were constructed by double amide coupling to synthesize the main skeleton. We synthesized a series of warhead analogues to enhance the diversity and novelty and discovered the SARs of the aldehyde series.
We constructed the SARs for α-ketoamide peptide series and aldehyde peptides series in order to search the best cathepsin S inhibitor for further research on their role as anticancer agent in metastasis.

中文摘要 I
英文摘要 II
謝誌 III
目錄 V
表目錄 XII
圖目錄 XIII
縮寫對照表 XVI

壹、緒 論 1
1.1 前言 1
1.2 癌症轉移過程之簡介 2
1.3 以癌症轉移過程為標靶之藥物治療 3
1.3.1 生長因子標靶 4
1.3.2 抗附著試劑 5
1.3.3 基質金屬蛋白酶抑制劑 6
1.3.4 抗血管新生療法 6
1.3.5 抗癌症轉移訊號傳導治療 7
1.4 組織蛋白酶S之背景介紹 8
1.4.1 半胱胺酸組織蛋白酶之簡介 8
1.4.2 半胱胺酸組織蛋白酶S之介紹 12
1.4.3 半胱胺酸組織蛋白酶S與癌症之關係 13
1.5 組織蛋白酶S抑制劑之簡介 15
1.5.1 組織蛋白酶S抑制劑分類 16
1.5.2 組織蛋白酶S抑制劑之設計 16
1.5.3 組織蛋白酶S可逆共價性抑制劑 17
1.5.4 組織蛋白酶S可逆非共價性抑制劑 22
1.5.5 組織蛋白酶S不可逆共價性抑制劑 22
貳、研究構想 25
2.1 α-酮醯胺胜肽系列之合成設計 25
2.2 醛基胜肽系列之合成計 27
2.3 CTSS抑制劑開發 28
参、結果與討論 30
3.1 α-酮醯胺胜肽化合物之合成策略 30
3.2 α-酮醯胺胜肽合成與CTSS抑制活性之研究 31
3.2.1 CTSS抑制活性測試 33
3.2.2 芳香環取代基效應探討 34
3.2.3 脂環族骨架結構探討 35
3.2.4 提昇選擇性探討 37
3.2.5 胺基甲酸酯置換醯胺探討
3.3 醛基胜肽化合物之合成策略 43
3.4 醛基胜肽化合物之合成研究 45
3.4.1 引入Michael acceptors之合成 46
3.4.2 引入α-酮醯胺基團之合成 48
3.4.3 引入氮-胜肽基團之合成 50
3.4.4 引入腙基團之合成 51
3.4.5 引入氮-氰基團之合成 52
3.4.5.1 氮-氰基團化合物之單肽衍生物合成 54
3.4.5.2 氮-氰基團化合物之P3位置衍生物合成 55
3.4.5.3 氮-氰基團化合物之氮上取代衍生物合成 56
3.5 醛基胜肽化合物之抑制CTSS活性 58
3.5.1 氮-氰基團衍生物系列之抑制CTSS活性 59
3.5.2 癌症轉移抑制活性測試 60
3.5.2.1 纖維粘連蛋白和鈣黏著素降解試驗 61
3.5.2.2 抗癌細胞侵入與抗癌細胞遷移試驗 62
3.5.2.3 皮膚癌細胞株A2058毒性測試 63
肆、總 結 66
伍、實驗部份 68
5.1 一般實驗方法 68
5.2 第一部分：α-酮醯胺胜肽化合物之實驗方法及光譜資料 71
5.2.1 化合物48的合成 71
5.2.2 化合物45的合成 72
5.2.3 化合物44的合成 5.2.4 化合物43的合成 73
5.2.5 化合物50a+50a’的合成 74
5.2.6 化合物50b+50b’的合成 75
5.2.7 化合物50c+50c’的合成 77
5.2.8 化合物50d+50d’的合成 79
5.2.9 化合物50e+50e’的合成 80
5.2.10 化合物50f+50f’的合成 82
5.2.11 化合物50g+50g’的合成 83
5.2.12 化合物50h+50h’的合成 85
5.2.13 化合物50i+50i’的合成 86
5.2.14 化合物50j+50j’的合成 87
5.2.15 化合物50k+50k’的合成 89
5.2.16 化合物50l+50l’的合成 90
5.2.17 化合物50m+50m’的合成 92
5.3 第二部分：醛基胜肽化合物之實驗方法及光譜資料94
5.3.1 化合物59的合成 94
5.3.2 化合物58的合成 95
5.3.3 化合物57的合成 96
5.3.4 化合物65的合成 96
5.3.5 化合物42的合成 97
5.3.6 化合物62a的合成 98
5.3.7 化合物62b的合成 99
5.3.8 化合物68的合成 100
5.3.9 化合物69的合成 101
5.3.10 化合物70的合成 101
5.3.11 化合物71的合成 102
5.3.12 化合物66的合成 102
5.3.13 化合物63a的合成 104
5.3.14 化合物63b的合成 104
5.3.15 化合物56的合成 106
5.3.16 化合物55a的合成 107
5.3.17 化合物55b的合成 108
5.3.18 化合物54a的合成 109
5.3.19 化合物78的合成 111
5.3.20 化合物54b的合成 112
5.3.21 化合物79的合成 113
5.3.22 化合物54c的合成 114
5.3.23 化合物80的合成 115
5.3.24 化合物81的合成 116
5.3.25 化合物54d的合成 117
5.3.26 化合物82a的合成 118
5.3.27 化合物83a的合成 119
5.3.28 化合物84a的合成 120
5.3.29 化合物54e的合成 121
5.3.30 化合物82b的合成 122
5.3.31 化合物83b的合成 123
5.3.32 化合物84b的合成 124
5.3.33 化合物54f的合成 125
5.3.34 化合物86a的合成 126
5.3.35 化合物54g的合成 128
5.3.36 化合物86b的合成 129
5.3.37 化合物54h的合成 130
5.3.38 化合物86c的合成 131
5.3.39 化合物54i的合成 132
陸、參考資料 134
附錄一、化合物編號對照表 142
附錄二、核磁共振光譜資料 144
附錄三、論文口試投影片 197

1. 行政院衛生署統計處網頁
2. Hua, S. C.; Lu, C. H.; Chang, T. C. The mechanism of cancer metastasis and advances in potential treatment for anaplastic cancer. J. Inter. Med. Taiwan. 2008, 19, 472-480.
3.. (a) Thiery, J. P. Epithelial-mesenchymal transitions in tumour
progression. Nat. Rev. Cancer 2. 2002, 442-454. (b)Raymond, W.
S.; Aidan, F.; Ramaswamy, S. Genome-wide views of cancer
metastasis. Drug Discovery Today:Disease Mechanisms. 2005, 2,
165-169.
4. (a) Robert C. Bast Jr.; Donald W. Kufe.; Raphael E. Pollock.; Ralph R. Weichselbaum.; James F. Holland.; Emil Frei III.; Ted S. Gansler. Cancer Medicine, 6th Edition. (b) Laura, K. S.; Donna, P. S.; Elaina, M.; Chen, H.; Tsai, J.; Chu, L.; Lesley, T.; Michael, L.; Shelly, M.; Linda, G.; Jolanta, S. J.; Tang, C.; Axel, U.; Michael, E. B.; Evan, H.; Gerald, M. K.; Peter, H.; T. J. Powell. Inhibition of platelet-derived growth factor-mediated signal transduction and tumor growth by N-[4-(trifluoromethyl)-phenyl] 5-methylisoxazole -4-carboxamide. Clinical Cancer Research. 1997, 3, 1167-1177. (c) Brian, P. E.; David, A. C. The role of αv integrins during angiogenesis: insights into potential mechanisms of action and clinical development. J. Clin. Invest. 1999, 103, 1227-1230. (d) Hu, J.; Philippe, E.; Sang, Q. A.; Ghislain, O. Matrix metalloproteinase inhibitors as therapy for inflammatory and vascular diseases. Nature 2007, 6, 480-498. (e) Benjamin, G.; Asher, M.; Reuven, O.; Giannoula, K. Successful antiangiogenic therapy for neuroblastoma with thalidomide. J. Clin. Oncol. 2007, 25, 5321-5324. (f) Tetsu, A.; Junko, I.; Suguru, N.; Hiroshi, O.; Shun-ichi, W.; Noriki, I.; M asabumi S.; Yasuo, F. Genistein, a Specific Inhibitor of Tyrosine-specific Protein Kinases. J. Biol. Chem. 1987, 262, 5592-5595. (g) Mark, S. S.; Irena, S.; Frank, H.; Ivan, D. H.; Nir, O.; Alexander, L.; Terrence, R. B. Non-amine based analogues of lavendustin A as protein-tyrosine kinase inhibitors. J. Med. Chem. 1993, 36, 3010-3014.
5. (a)Jedeszko, C.; Sloane, B. F. Cysteine cathepsins in human cancer. Biol. Chem. 2004, 385, 1017-27. (b) Vito, T.; Kos, J.; Boris, T. Cysteine cathepsins (protease)--on the main stage of cancer? Cancer Cell. 2004, 5, 409-410.
6. Kirschke, H.; Schmidt, I.; Wiederanders, B. Cathepsin S The
cysteine proteinase from bovine lymphoid tissue is distinct from
cathepsin L (EC 3.4.22.15). Biochem. J. 1986, 240, 455-459.
7. Dieter, B.; Jadwiga, K. Thiol-dependent cathepsins: pathophysio- logical implications and recent advances in inhibitor design. Curr. Pharm. Des. 2002, 8, 1639-1658.
8. Olga, V.; Thomas, R.; Christoph, P.; Dusan, T.; Vito, T.; Boris, T. Emerging roles of cysteine cathepsins and their potential as drug targets. Curr. Pharm. Des. 2007, 13, 387-403.
9. Chang, W. S. W.; Wu, H. R.; Yeh, C. T.; Wu, C. W.; Chang, J. Y. Lysosomal cysteine protease cathepsins as a potential target for anti-cancer therapy. J. Cancer. Mol. 2007, 3, 5-14.
10. Vincent, L.; Sukanthini, T. Cathepsin S inhibitors. Expert. Opin. Ther. Patent. 2004, 14, 301-311.
11. http://en.wikipedia.org/wiki/Cysteine_protease.
12. Toshiyuki, N.; Nobuhiko, K. Involvement of cathepsins in the invasion, metastasis and proliferation of cancer cells. J. Med. Invest. 2005, 52, 1-9.
13. (a) Bing, W.; Jiusong, S.; Min, Y.; Anders, G.; Harold, A. C.; Raghu, K.; Shi, G. P. Cathepsin S controls angiogenesis and tumor growth via matrix-derived angiogenic factors. J. Biol. Chem. 2006, 281, 6020-6029. (b) Vasilena, G.; Zeng. W.; David, K.; Thomas, R.; Christoph, P.; Douglas, H.; Johanna A, J. Distinct roles for cysteine cathepsin genes in multistage tumorigenesis. Genes Dev. 2006, 5, 543-556.
14. Poole, A. R.; Tiltman, K. J.; Recklies, A. D.; Stoker, T. A. Differences in secretion of the proteinase cathepsin B at the edges of human breast carcinomas and fibroadenomas. Nature. 1978, 273, 545- 547.
15. Pedro, L. F.; Xavier, F.; Alfons, N.; Eva, F.; Nerea, P.; Bonnie, F. S.; Shi, G. P.; Harold, A. C.; Elias, C.; Antonio, C. Expression of cathepsins B and S in the progression of prostate carcinoma. Int. J. Cancer. 2001, 95, 51-55.
16. Kos, J.; Sekirnik, A.; Cimerman, N.; Kayser, K.; Stremmer, A.; Fiehn, W.; Werle, B. Cathepsin S in tumours, regional lymph nodes and sera of patients with lung cancer: relation to prognosis. Br. J. Cancer. 2001, 85, 1193-1200.
17. Flannery, T.; Gibson, D.; Mirakhur, M.; McQuaid, S.; Greenan, C.; Trimble, A.; Walker, B.; McCormick, D.; Johnston, P. G. The clinical significance of cathepsin S expression in human astrocytomas. Am. J. Pathol. 2003, 163, 175-182.
18. John, O. L.; Sheila, Z. Advanced in cathepsin S inhibitor design. Current Opinion in Drug Discovery & Development 2006, 9, 471-482.
19. Reik, L.; Klaus, S.; Elke, D.; Michael, G. Interaction of papain-like cysteine protease with dipeptide-derived nitriles. J. Med. Chem. 2005, 48, 7688-7707.
20. Kazutoshi, S.; Hidefumi, S.; Toru, O. Paecilopeptin, a new cathepsin S inhibitor Produced by Paecilomyces carneus. Biosci. Biotechnol. Biochem. 2002, 66, 2444-2448.
21. Cywin, C. L.; Firestone, R. A.; McNeil, D. W.; Grygon, C. A.; Crane, K. M.; White, D. M.; Kinkade, P. R.; Hopkins, J. L.; Davidson, W.; Labadia, M. E.; Wildeson, J.; Morelock, M. M.; Peterson, J. D.; Raymond, E. L.; Brown, M. L.; Spero, D. M. The design of potent hydrazones and disulfides as cathepsin S inhibitors. Bioorg. Med. Chem. 2003, 11, 733-740.
22. Inagaki, H.; Tsuruoka, H.; Hornsby, M.; Lesley, S. A.; Spraggon, Glen.; Ellman, J. A. Characterization and optimization of selective, nonpeptidic inhibitors of cathepsin S with an unprecedented binding mode. J. Med. Chem. 2007, 50, 2693-2699.
23. Zhou, N. E.; Guo, D.; Thomas, G.; Reddy, A. V. N.; Kaleta, J.; Purisima, E.; Menard, R.; Micetich, R. G.; Singh, R. 3-Acylamino -azetidin-2-one as a novel class of cysteine proteases inhibitors. Bioorg. Med. Chem. Lett. 2003, 139–141.
24. Marquis, R. W.; Ru, Y.; LoCastro, S. M.; Zeng, J.; Yamashita, D. S.; Oh, H.; Erhard, K. F.; Davis, L. D.; Tomaszek, T. A.; Tew, D.; Salyers, K.; Proksch, J.; Ward, K.; Smith, B.; Levy, M.; Cummings, M. D.; Haltiwanger, R. C.; Trescher, G.; Wang, B.; Hemling, M. E.; Quinn, C. J.; Cheng, H-Y.; Lin, F.; Smith, W. W.; Janson, C. A.; Baoguang, Z.; McQueney, M. S.; D'Alessio, K.; Lee, C. P.; Marzulli, A.; Dodds, R. A.; Blake, S.; James, I. E.; Gress, C. J.; Bradley, B. R.; Lark, M. W.; Gowen, M.; Veber, D. F. Azepanone- based inhibitors of human and rat cathepsin K. J. Med. Chem. 2001, 44, 1380-1395.
25. Edwards, P. D.; Wolanin, D. J.; Andisik, D. W.; Davis, M. W. Peptidyl alpha-ketoheterocyclic inhibitors of human neutrophil
elastase 2 effect of varying the heterocyclic ring on in vitro potency. J. Med. Chem. 1995, 38, 76-85.
26. Walker, B.; Lynas, J. F.; Meighan, M. A.; Bromme, D. Evaluation of dipeptide α-keto-β-aldehyde as new inhibitors of cathepsin S. Biochem. Biophys. Res. Commun. 2000, 275, 401-405.
27. Kissei Yakuhin Kogyo KK : JP1311037 (2001).
28. Donkor, I, O.; Assefa, H.; Liu, J. Structure basis for the potent calpain inhibitory activity of peptidyl α-ketoacids. J. Med. Chem. 2008, 51, 4346–4350.
29. Liu, H.; Tully, D. C.; Epple, R.; Bursulaya, B.; Li, J.; Harris, J. L.; Williams, J. A.; Russo, R.; Tumanut, C.; Roberts, M. J.; Alper, P. B.; He, Y.; Karanewsky, D. S. Design and synthesis of arylaminoethyl amides as noncovalent inhibitors of cathepsin S. Part 1. Bioorg. Med. Chem. Lett. 2005, 15, 4979-4984.
30. Gustin, D. J.; Sehon, C. A.; Wei, J.; Cai, H.; Meduna, S. P.; Khatuya, H.; Sun, S.; Gu, Y.; Jiang, W.; Thurmond, R. L.; Karlesson, L.; Edwards, J. P. Discovery and SAR studies of a novel series of noncovalent cathepsin S inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 1687-1691.
31. Yamashita, DS.; Dodds, RA. Cathepsin K and the design of inhibitors of cathepsin K. Curr. Pharm. Des. 2000, 6, 1-24.
32. Lescop, C.; Herzner, H.; Siendt, H.; Bolliger, R.; Hennebohle, M.; Weyermann. P.; Briguet, A.; Courdier-Fruh, I.; Erb, M.; Foster, M.; Meier, T.; Magyar, J. P.; Sprecher, A. Novel cell-penetrating α-keto-amide calpain inhibitors as potential treatment for muscular dystrophy. Bioorg. Med. Chem. Lett. 2005, 15, 5176-5181.
33 Adams, J.; Behnke, M.; Chen, S.; Cruickshank, A. A.; Dick, L. R.; Grenier, L.; Klunder, J. M.; Ma, Y. T.; Plamondon, L.; Stein, R. L. Potent and selective inhibitors of the proteasome : dipeptidyl boronic acids. Bioorg. Med. Chem. Lett. 1998, 8, 333–338.
34. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Delivery Rev. 2001, 46, 3–26.
35. 清華大學生命科學院呂平江教授實驗室提供。
36. Ekici, O. D.; Gotz, M. G.; James, K. E.; Li, Z. Z.; Rukamp, B. J.; Asgian, J. L.; Caffrey, C. R.; Hansell, E.; Dvorak, J.; McKerrow, J. H.; Potempa, J.; Travis, J.; Mikolajczyk, J.; Salvesen, G. S.; Power, J. C. Aza-peptide Michael acceptors : a new class of inhibitors specifics for caspases and other clan CD cysteine proteases. J. Med. Chem. 2004, 47, 1899-1892.
37. Kokotos, G.; Six, D. A.; Loukas, V.; Smith, T.; Constantinou, V.; Hadjipavlou, D.; Kotsovolou, S.; Chiou, A.; Beltzner, C. C.; Dennis, E. A. Inhibition of group IVA cytosolic phospholipase A2 by novel 2-oxoamides in vitro, in cells, and in vivo. J. Med. Chem. 2004, 47, 3615-3628.
38. Xing, R.; Hanzlik, R. P. Azapeptide as inhibitor and active site titrants for cysteine proteinases. J. Med. Chem. 1998, 41, 1344-1351.
39. Verhelst, S. H. L.; Witte, M. D.; Arastu-Kapur, S.; Fonovic, M.; Bogyo, M. Novel aza peptide inhibitors and active-sites probes of papain-family cysteinse protease. ChemBioChem. 2006, 7, 943-950.
40. Loser, R.; Frizler, M.; Schilling, K.; Gutschow, M. Azadipeptide nitriles : highly potent and proteolytically stable inhibitors of papain-like cysteine protease. Angew. Chem. Int, Ed. 2008, 47, 4331-4334.
41. Ward, Y. D.; Thomson, D. S.; Frye, L. L.; Cywin, C. L.; Morwick, T.; Emmauel, M. J.; Zindell, R.; McNeil, D.; Bekkali, Y.; Girardot, M.; Hrapchak, M.; DeTuri, M.; Grane, K.; White, D.; Pav, S.; Wang, Y.; Hao, M, H.; Grygon, C. A.; Labadia, M. E.; Freeman, D. M.; Davidson, W.; Hopkins, J. L.; Brown, M. L.; Spero, D. M. Design and synthesis of dipeptide nitriles as reversible and potent cathepsin S inhibitors. J. Med. Chem. 2002, 45, 5471-5482.
42. 陳若君，清華大學化學系未發表之研究成果。
43. 蔡明宗，清華大學化學系未發表之研究成果。