臺灣博碩士論文加值系統

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

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