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研究生:詹振勳
研究生(外文):Chen-Hsin Chan
論文名稱:銅縮氨基硫脲類複合物做為第二型拓樸異構酶抑制劑之合成與生物性評估
論文名稱(外文):Synthesis and Biological evaluation of Thiosemicarbazone-Copper complexes for Topoisomerase II inhibition
指導教授:陳傳霖陳傳霖引用關係
指導教授(外文):Chuan-Lin Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物醫學影像暨放射科學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:74
中文關鍵詞:缩氨基硫脲類化合物第二型拓樸異構酶
外文關鍵詞:ThiosemicarbazoneTopoisomerase II
相關次數:
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  • 下載下載:3
  • 收藏至我的研究室書目清單書目收藏:0
目的:缩氨基硫脲類化合物(Thiosemicarbazone,TSC)具有抗微生物、 抗病毒及抗癌等特性,其銅缩氨基硫脲錯合物藥物亦具有不錯的抑制腫瘤細胞能力。本研究發展一系列新銅缩氨基硫脲藥物,探討其抑制腫瘤第二型拓撲異構酶活性及此些藥物對腫瘤細胞的毒殺能力。
材料與方法:利用簡單一步驟加成反應,分別合成出四類缩氨基硫脲衍生物;2-乙醯比啶缩氨基硫脲(Pm4pT,Pm44mT)、2-雙比啶缩氨基硫脲(Dp4pT,Dp44mT)、2-甲醛比啶缩氨基硫脲(P4pT,P44mT)及2-甲醛喹林缩氨基硫脲(Q44mT)等七種缩氨基硫脲衍生物,再進行銅離子配位,可得到銅缩氨基硫脲錯合物(Cu[Pm4pT]Cl,Cu[Pm44mT]Cl, Cu[Dp4pT]Cl, Cu[Dp44mT]Cl,Cu[P4pT]Cl,Cu[P44mT]Cl及Cu[Q44mT]Cl) 等。由Topoisomerase IIα藥物篩檢測定可決定此類銅缩氨基硫脲錯合物對於第二型拓樸酶之抑制能力,再利用其銅缩氨基硫脲錯合物進行細胞毒殺試驗,以驗證腫瘤細胞之第二型拓撲異構酶表現與藥物抑制腫瘤能力之關係;並以最佳藥物進行腫瘤細胞型態分析與利用流式細胞儀分析其細胞週期。
結果:成功合成銅缩氨基硫脲錯合物,並且此些錯合物皆有不錯的抑制第二型拓樸酶活性之能力,其藥物抑制濃度在0.78~21.21μM之間;其中以Cu[Dp4pT]Cl有最佳之抑制效果(IC50為0.78±0.14μM);對於 

高表現的第二型拓樸酶的LL/2 細胞毒殺試驗,此類銅缩氨基硫脲錯合物其抑制濃度約在0.26~0.69μM之間,其中亦以Cu[Dp4pT]Cl的抑制效果最佳(GI50為0.26±0.02μM);而低表現第二型拓樸酶的MCF-7細胞其抑制濃度約在0.64~1.16μM之間。腫瘤細胞型態分析,在加入不同濃度之Cu[Dp4pT]Cl於培養之LL/2細胞 (第二型拓撲異構酶高表現)與MCF-7細胞(第二型拓撲異構酶低表現)後皆有明顯細胞凋亡之變化。細胞週期部份,可以明顯地看到經過Cu[Dp4pT]Cl處理過後的LL/2與MCF-7細胞在細胞週期S與G2/M期明顯地增加。
結論:成功合成出七個銅缩氨基硫脲衍生物,其中以Cu[Dp4pT]Cl對於第二型拓樸酶之抑制能力或及LL/2 細胞毒殺試驗中皆有最佳結果。在腫瘤細胞型態分析,Cu[Dp4pT]Cl也能夠有效造成腫瘤細胞凋亡狀態;細胞週期分析部份,Cu[Dp4pT]Cl能夠有效地抑制第二型拓樸異構酶高表現之S期與G2/M期。以上實驗結果得知此藥物可有效抑制第二型拓樸酶活性,未來可進行Cu-64放射性標幟作為腫瘤表現第二型拓撲異構酶的正子造影探針。

Objectives: Thiosemicarbazone compounds (TSC) have anti-microbial, anti-viral and anti-cancer properties, and their copper complexe drugs also have a better activity to inhibit tumor cell. In this study we synthesized several novel thiosemicarbazone-Cu complexes and investigate the in vitro biological activity for topoisomerase II inhibition.
Material and Methods: Thiosemicarbazone-copper complexes were prepared via a multi-step synthesis (total yield > 70%). The crystal structures of three complexes Cu[Q44mT]Cl, Cu[Dp4pT]Cl and Cu[Dp44mT]Cl have been determined by crystal X-ray diffraction technique .The biological character of these complexes were evaluated by in vitro Topoisomerase IIα inhibition assays and antiproliferative activity in LL/2(mouse Lewis lung carcinoma) and MCF-7(human breast adenocarcinoma) cells which expressed different level of Topo-IIα .DNA fragmentation were detected byHoechst33342 staining after 24hr treated with Cu[Dp4pT]Cl (0.25 μM). In DNA distribution , LL/2 cells were analyzed by flow cytometry after 24hr treated Cu[Dp4pT]Cl (0.5 μM).
Results: A series of thiosemicarbazone-Cu complexes were synthesized successfully. In agarose gel TopoII α inhibition assay clearly show the 10 μM Cu[TSC]Cl complexes possessed inhibition activity for Topoisomerase IIα, but the TSC ligands are not suitable. Among, the Cu[Dp4pT]Cl complexe was showed higher Topoisomerase IIα inhibition activity (IC50 0.78±0.14 μM) then others (over 3 μM). The antiproliferation study, the Cu[Dp4pT]Cl was also revealed the higher inhibition activity for high Topoisomerase IIα expressing lung cancer cell (LL2) than low Topoisomerase IIα expressing cells(MCF-7). DNA fragmentation (DAPI staining) study was observed from LL/2 and MCF-7 cancer cells treated with different drug concentration of Cu[Dp4pT]Cl . DNA distribution profiles obtained the percentage of cells in S and G2/M phase had increased significantly.
Conclusions: We have synthesized a serious thiosemicarbazone-Cu complexes successfully. The Cu(TSC)Cl complexes are more effective topoisomerase IIα inhibitor than the free thiosemicarbazone ligands. Among of these complexes, the Cu[Dp4pT]Cl possessed the high anti-topoisomerase II activity and antiproliferation activity of high Topo-IIα expression cell (LL2). Futher the Cu[Dp4pT]Cl might be applied to develop 64Cu-based positron emission tomography probe for noninvasive imaging of Topo-Iiα expresion in vivo.

目錄
中文摘要................................................................................................... I
英文摘要................................................................................................. III
目錄.......................................................................................................... V
圖目錄.................................................................................................... VII
表目錄..................................................................................................... IX
前言........................................................................................................... 1
研究目的................................................................................................. 14
材料與方法
1. 材料
1-1縮氨基硫脲類化合物及其銅錯合物之標準品合成材料...... 15
1-2第二型拓樸異構酶抑制試驗所需材料................................... 15
1-3細胞實驗所需材料................................................................... 15
1-4儀器........................................................................................... 16
2. 方法
2-1合成N-phenyl-Thiosemicarbazone類化合物.......................... 17
2-2合成N,N-dimethyl -Thiosemicarbazone類化合物.................. 19
2-3合成N-phenyl-Cu(II) -Thiosemicarbazone錯合物與 N,N-dimethyl-Cu(II) -Thiosemicarbazone錯合物.................. 21
2-4細胞培養液配置....................................................................... 23
2-5 細胞培養.................................................................................. 24
2-6第二型拓樸異構酶抑制試驗................................................... 24
2-7腫瘤細胞毒殺試驗................................................................... 25
2-8 腫瘤細胞型態分析.................................................................. 26
2-9 細胞週期分析.......................................................................... 26
結果與討論............................................................................................. 28
1. Thiosemicarbazone合成與特性................................................ 28
2. X光晶體繞射分析.................................................................... 29
3. 第二型拓樸異構酶抑制試驗................................................... 32
4. 細胞毒殺試驗........................................................................... 38
5. 腫瘤細胞型態分析................................................................... 39
6. 細胞週期分析........................................................................... 42
結論......................................................................................................... 45
參考文獻................................................................................................. 46
附錄......................................................................................................... 52

圖目錄
圖一、縮氨基硫脲類化合物結構式......................................................... 1
圖二、縮氨基硫脲類化合物與其金屬錯合物抑制機制圖.................... 2
圖三、抑制核糖核苷酸還原酶之thiosemicarbazone化合物結構式...... 3
圖四、抑制多重抗藥性之Isatin-β-thiosemicarbazones結構式............... 4
圖五、Shao等人合成之化合物結構式..................................................... 6
圖六、Di-2-pyridyl thiosemicarbazone化學結構..................................... 7
圖七、臨床上治療惡性腫瘤常用的化療藥物結構..................................9
圖八、TSC-24之化學結構式...................................................................10
圖九、α-Heterocyclic-N4-Substituted Thiosemicarbazone結構式與其金
屬錯合物..................................................................................... 11
圖十、Wei等人合成一系列縮氨基硫脲類化合物之結構式................12
圖十一、縮氨基硫脲類化合物與其銅錯合物合成圖............................28
圖十二、X光晶體繞射之結構圖..............................................................32
圖十三、TSC與Cu(TSC)Cl藥物抑制酵素活性之DNA電泳圖.........34
圖十四、Cu[Dp4pT]Cl、Cu[Pm4pT]Cl與Cu[P4pT]Cl抑制第二型拓樸
異構酶之DNA電泳圖............................................................35
圖十五、Cu[Dp44mT]Cl、Cu[Pm44mT]Cl與Cu[P44mT]Cl抑制第二
型拓樸異構酶之DNA電泳圖................................................36
圖十六、Cu[Q44mT]Cl抑制第二型拓樸異構酶之DNA電泳
圖.............................................................................................37
圖十七、腫瘤細胞型態分析...................................................................41.
圖十八、LL/2細胞周期分析圖................................................................43
圖十九、MCF-7細胞周期分析圖...........................................................44














表目錄
表一、抑制核糖核苷酸還原酶活性與毒殺腫瘤細胞能力之比較....... 3
表二、不同MDR1表現量之細胞毒殺能力之比較................................5
表三、縮氨基硫脲類化合物與其銅錯合物抑制第二型拓樸異構酶活性與細胞毒殺試驗.........................................................................12
表四、Cu[Dp4pT]Cl、Cu[Dp44mT]Cl與Cu[Q44mT]Cl晶體結構...31
表五、Cu[Dp4e4mT]Cl、Cu[Dp44mT]Cl與Cu[Q44mT]Cl之鍵長.....32
表六、Cu[Dp4e4mT]Cl、Cu[Dp44mT]Cl與Cu[Q44mT]Cl之鍵角.......32
表七、第二型拓樸異構酶抑制試驗數據................................................37
表八、細胞毒殺試驗數據........................................................................39
表九、LL/2細胞之細胞周期分佈............................................................43
表十、MCF-7細胞之細胞周期分佈........................................................44




1. Yu. Y ,Kalinowski D. S, Kovacevic Z, Siafakas A. R, Jansson P. J, Stefani C, Lovejoy D. B, Sharpe P. C, Bernhardt P. V, Richardson D. R. Thiosemicarbazones from the old to new: iron chelators that are more than just ribonucleotide reductase inhibitors. J. Med. Chem. 2009; 52,5271–5294.
2. West D. X, Liberta A. E. Thiosemicarbazone complexes of copper(II): structural and biological studies. Coord. Chem. Rev. 1993; 123, 49–71.
3. Beraldo H, Gambino D. The wide pharmacological versatility of semicarbazones, thiosemicarbazones and their metal complexes. Mini- Rev. Med. Chem. 2004; 4, 31–39.
4. Kalinowski D. S, Quach P, Richardson D. R. Thiosemicarbazones: the new wave in cancer treatment. Future Med. Chem. 2009; 1, 1143–1151.
5. Tisato F, Marzano C, Porchia M, Pellei M, Santini C. Copper in diseases and treatments, and copper-based anticancer strategies. Med. Res. Rev. 2010; 30 , 708–749.
6. Brockman RW, Sidwell RW, Arnett G, Shaddix S. Heterocyclic
thiosemicarbazones: correlation between structure, inhibition of ribonucleotide reductase, and inhibition of DNA viruses. Proc Soc
Exp Biol Med 1970; 133, 609-614.
7. Shao J, Zhou B, Di Bilio AJ. A Ferrous-Triapine complex
mediates formation of reactive oxygen species that inactivate human
ribonucleotide reductase. Mol Cancer Ther 2006; 5, 586-92.
8. Hall IH, Lackey CB, Kistler TD. Cytotoxicity of copper and
cobalt complexes of furfural semicarbazone and thiosemicarbazone
derivatives in murine and human tumor cell lines. Pharmazie 2000;
55, 937-941
9. Yuan J,Lovejoy DB, Richardson DR. Novel di-2-pyridyl-derived
iron chelators with marked and selective antitumor activity: in vitro
and in vivo assessment. Blood 2004; 104,1450-1458.
10. Ludwig JA, Szakács G, Martin SE. Selective toxicity of
NSC73306 in MDR1-positive cells as a new strategy to circumvent
multidrug resistance in cancer. Cancer Res 2006; 66, 4808-4815.
11. Wu C, Shukla S, Calcagno AM, Hall MD,Gottesman MM, Ambudkar SV. Evidence for dual mode of action of thiosemicarbazone,NSC73306: a potent substrate of the multidrug resistancelinked ABCG2 transporter. Mol Cancer Ther 2007; 6,3287-3296.
12. Hiroshi T, Hirofumi A, Tatsuya Y, Kenji S, Seisuke F, Kuniko M,Yoshiki T,Yusuke N. A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature 2000 404, 42-49
13. Par Nordlund,Britt-Marie Sjoberg,Hans Eklund .Three-dimensional structure of the free radical protein of ribonucleotide reductase. Nature 1900;345, 593 - 598
14. Christian R. Kowol, Robert Trondl, Petra Heffeter, Vladimir B, Michael A. Jakupec, Alexander Roller,Markus Galanski Walter Berger, and Bernhard K. Keppler. Impact of Metal Coordination on Cytotoxicity of 3-Aminopyridine-2-carboxaldehyde Thiosemicarbazone (Triapine) and Novel Insights into Terminal Dimethylation. J. Med. Chem. 2009; 52, 5032–5043
15. Michael M, Gottesman Tito Fojo, Susan E. Bates. Multidrug resistance in cancer: role of ATP–dependent transporters. Nature Reviews Cancer 2002;28, 48-58
16. Chang-jie Chen, Janice E.Chi, Kazumitsu Ueda, Douglas P,Clark Ira Pastan, Michael M,Gottesman, Igor B Roninson. Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells.Cell 1986;47,381-389
17. Matthew D.Hall, Noeris K. Salam, Jennifer L, Hellawell,Henry M. Fales,Caroline B. Kensler. Synthesis, Activity, and Pharmacophore Development for Isatin-β-thiosemicarbazones with Selective Activity toward Multidrug-Resistant Cells. J. Med. Chem. 2009; 52, 3191–3204
18. Danuta S, Kalinowski,Yu Yu, Philip C. Sharpe,Mohammad Islam,Yi-Tyng Liao. Design, Synthesis, and Characterization of Novel Iron Chelators: Structure-Activity Relationships of the 2-Benzoylpyridine Thiosemicarbazone Series and Their 3-Nitrobenzoyl Analogues as Potent Antitumor Agents. J. Med. Chem. 2007; 50, 3716-3729.
19. Annahita Sallmyr, Jinshui Fan,Feyruz Virgilia Rassool. Genomic instability in myeloid malignancies: Increased reactive oxygen species (ROS), DNA double strand breaks (DSBs) and error-prone repair.cancer letter 2008;1,1-9
20. Jia Shao ,Zhong-Ying Ma , Ang Li , Ya-Hong Liu,Cheng-Zhi Xie , Zhao-Yan Qiang , Jing-Yuan Xu. Thiosemicarbazone Cu(II) and Zn(II) complexes as potential anticancer agents: Syntheses, crystal structure, DNA cleavage, cytotoxicity and apoptosis induction activity.J.Inor Bio.2014;136,13-23.
21. Susin SA,HK Lorenzo,N Zamzami, I Marzo, BE Snow. Molecular characterization of mitochondrial apoptosis-inducing factor.Nature,1999; 397, 441-446
22. Jun Yuan, David B,Lovejoy and Des R. Richardson. Novel di-2-pyridyl-derived iron chelators with marked and selective antitumor activity: in vitro and in vivo assessment. Blood 2004; 104, 1450-1458
23. Wang Chen,Jin Qi1,Yuemao Shen.Topoisomerase IIα rather than IIβ, is a promising target in development of anti-cancer drugs.Drug Discoveries & Therapeutics. 2012; 6,230-237.
24. John L, Nitiss.Targeting DNA topoisomerase II in cancer chemotherapy. Nature Reviews Cancer 2009;9, 338-350.
25. D.Andrew Burden, Neil Osheroffa. Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression,1998;1400,139-154.
26. K.R Hande .Etoposide: four decades of development of a topoisomerase II inhibitor. European Journal of Cancer,1998;34, 1514-1521.
27. He Huang,Qin Chen,Xin Ku,Linghua Meng, Liping Lin, Xiang Wang,Caihua Zhu. A Series of r-Heterocyclic Carboxaldehyde Thiosemicarbazones Inhibit Topoisomerase IIα
Catalytic Activity. J. Med. Chem. 2010; 53, 3048–3064.
28. Brian M. Zeglis, Vadim Divilov,Jason S. Lewis. Role of Metalation in the Topoisomerase IIα Inhibition and Antiproliferation Activity of a Series of α-Heterocyclic-N4-Substituted Thiosemicarbazones and Their Cu(II) Complexes. J. Med. Chem., 2011;54, 2391–2398.
29. Giuseppe Giaccone, Jannette van ,Ark-Otte,Giorgio Scagliotti,Giovanni Capranico,Paul van der Valka,Gonzalo Rubioa, Otilia Dalesio,Rafael Lopez,Franco Zunino,Jan Walboomers,Herbert M. Pinedo. Differential expression of DNA topoisomerases in non-small cell lung cancer and normal lung.Biochim Biophys Acta,1995;1264,337-346.
30. Lihui Wei1,Johnny Easmon, Ravneet K. Nagi1, Brian D. Muegge1, Laura A. Meyer1,Jason S. Lewis. 64Cu-Azabicyclo[3.2.2] Nonane Thiosemicarbazone Complexes: Radiopharmaceuticals for PET of Topoisomerase II Expression in Tumors.J.Nucl Med 2006 ;47,2034-2041.
31. A S Dobek,D L Klayman, E T Dickson Jr, J P Scovill , E C Tramont. Inhibition of clinically significant bacterial organisms in vitro by 2-acetylpyridine thiosemicarbazones. Antimicrob. Agents Chemother. 1980;18 ,127-136
32. DX West, JS Ives, J Krejci, MM Salberg, TL Zumbahlen. Copper(II) complexes of 2-benzoylpyridine 4N-substituted thiosemicarbazones. Polyhedron,1995;14,15-16.
33. David B. Lovejoy, Danae M. Sharp, Nicole Seebacher,Peyman
Obeidy, Thomas Prichard,Christian Stefani, Maram T. Basha, Philip C. Sharpe, Patric J. Jansson, Danuta S. Kalinowski,Paul V. Bernhardt, Des R. Richardson. Novel Second-Generation Di-2-Pyridylketone
Thiosemicarbazones Show Synergism with Standard
Chemotherapeutics and Demonstrate Potent Activity against Lung Cancer Xenografts after Oral and Intravenous Administration in Vivo. J.Med.Chem.2012;55, 7230-7244.
34. Rhee H. K, Park H. J, Lee S. K, Lee C.O, Choo H. Y. P.Synthesis, cytotoxicity, and DNA topoisomerase II inhibitory activity of
benzofuroquinolinediones. Bioorg. Med. Chem. 2007;15,1651–1658.
35. Kim J. S, Rhee H. K, Park H. J,Lee S. K, Lee C. O,Choo H. Y. P. Synthesis of 1-/2-substituted-[1,2,3]triazolo[4,5-g]phthalazine 4,9-diones and evaluation of their cytotoxicity and topoisomerase
II inhibition. Bioorg. Med. Chem. 2008; 16, 4545–4550.
36. Suzuki K,Yahara S, Maehata K,Uyeda M. Isoaurostatin, anovel topoisomerase inhibitor produced by Thermomonospora alba. J.Nat. Prod. 2001; 64, 204–207.

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