臺灣博碩士論文加值系統

組蛋白去乙醯酶抑制劑為現今抗癌標靶藥物的重要一員，隨著組蛋白去乙醯酶抑制劑的上市及臨床測試的相關資料累積，其使用範圍愈加廣闊，而許多問題也因而浮現，諸如臨床副作用、對固態腫瘤效果有限等。
本論文分為 I. 在脂鏈醯基羥胺嵌入芳香環吲哚開發新型組蛋白去乙醯酶抑制劑作為抗癌藥物； II. 鄰位芳香環氮-羥基肉桂酸醯胺結構優化作為選擇性第八型組蛋白去乙醯酶抑制劑，分別做探討。
I. 目前臨床使用的組蛋白去乙醯酶抑制劑Vorinostate (SAHA)及Romidepsin為治療皮膚T細胞淋巴瘤之血液腫瘤用藥，在臨床針對固態腫瘤時卻效果不彰，因此，我們希望發展出一系列新的組蛋白去乙醯酶抑制劑能有效對抗固態腫瘤；SAHA依其化學結構分為三部分：與鋅離子螯合之基團、疏水性長碳鏈及辨識部位，其中辨識部位依其結構不同能具有多種不同功能的變遷，我們以不同芳香環取代之吲哚當作新的辨識部位，合成化合物N-苯磺酰基吲哚4a-4d及三甲氧基苄基吲哚6，以期提升酵素抑制活性，進而增加抗癌活性；其中化合物4a、4b、4d及6在HeLa extract酵素測試中均顯示比SAHA有更佳的活性，而其中化合物4d在對人類非小細胞肺癌A549細胞的生長抑制活性(GI50 = 0.65 μM)，較SAHA(1.50 μM)為佳，另外在人類大腸癌HT29細胞中，造成組蛋白次單元體H3蛋白的乙醯化程度亦比SAHA高，值得對此系列化合物進一步研究。
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II. 組蛋白去乙醯酶為一個大家族，其中有不同亞型蛋白存在，研究顯示選擇性抑制劑與廣泛性抑制劑相較下，具有效減少副作用的產生之潛力，本研究以我們實驗室過去所發展之鄰位聯苯氮-羥基肉桂酸醯胺組蛋白去乙醯酶第八型抑制劑A22d為先導化合物，依據分子模擬之結果，設計在鄰位聯苯的第二苯環上做結構修飾，以增進對於酵素的抑制能力，我們合成了11a-11g其中化合物11d (IC50 = 29.3 nM)與文獻報導之組蛋白去乙醯酶第八型選擇性抑制劑PCI-34051 (IC50 = 27.1 nM)有相當之酵素抑制活性，並藉由分子嵌合模型佐證其在第八型組蛋白去乙醯酶的特徵性區域有交互作用，因而能造成高度選擇性。另外，我們延續實驗室過去所發展之能效對抗癌細胞的化合物A5e做結構修飾，得到七個新的化合物，其中化合物21和25 (IC50 = 292.5, 341.2 nM)具有中等的第八型組蛋白去乙醯酶抑制能力。

Histone deacetylase (HDAC) inhibitors are regarded as a promising therapeutic for treatment of cancer. However, current HDAC inhibitors have some unsatisfactory problems, including limited potency for solid tumors and dose-limiting side effects. This study includes： I. Aliphatic Hydroxamates Capped with Substituted Aryl Indole as Histone Deacetylase Inhibitors against Cancer Cells; II. Structure Optimization for Ortho-Aryl N-Hydroxycinnamides as HDAC8 Isoform-Selective Inhibitors I. Currently, SAHA and Romidepsin both were FDA-approved HDAC inhibitors to treat Cutaneous T cell lymphoma (CTCL). Depite their success in hematopoietic malignancy, HDAC inhibitors in clinical trials had still failed to heal solid tumorsm, thus, encouraging us to develop novel inhibitors against these tumors. The pharmacophore of SAHA is composed of three parts such as a zinc-binding group, a hydrophobic linker and a surface recognition cap. In this study, we used aryl indole as a surface recognition cap to develop a novel series of HDAC inhibitors. All compounds were screened for HeLa nucleus HDACs enzyme inhibition. Among them, compounds 4a, 4b, 4d and 6 exhibited potent inhibitory activity (IC50 = 209, 117, 104, 232 nM) comparable to SAHA (IC50 = 275 nM). Notably, compound 4d had the best cytotoxicity against non-small-cell lung carcinoma A549 cells (GI50 = 0.65 μM) compared to SAHA (GI50 = 1.50 μM). Western
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blot analysis related to intracellular HDAC inhibiton indicated compounds 4a, 4c, and 4d had significant effect on enhancing acetylation histone H3 and p21 in HT29 cells comparable to SAHA. Further molecular docking of 4d and SAHA interacted to histone deacetylase like protein (HDLP) demonstrated that the benzenesulfonyl of compound 4d may offer more interaction with the enzyme surface at HDAC than SAHA.Cutaneous T cell lymphoma (CTCL) II. HDAC family comprises eighteen isofirms. Studies had revealed that selective HDAC inhibitors may have less side effects than pan ones. We had delveloped a lead ortho-biphenyl N-hydroxycinnamide A22d with potent HDAC8 inhibition. Based on the molecular docking result for A22d with HDAC8, we extensively added different function groups to biphenyl moiety to enhance their enzyme inhibitory activity. Seven novelly synthesized compounds were screened for anti-HDAC 8 activity and compound 11d showed the best activity (IC50 = 29.3 nM) comparable to PCI-34051 (IC50 = 27.1 nM), a reportedly potent HDAC8 inhibitor. Molecular docking analysis suggested compound 11d may interact with HDAC 8 in a specific sub-pocket which allows to provide selectivity toward HDAC 8. Furthermore, based on the potent cytotoxicity by our previously developed HDAC 8 inhibitor A5e against tumor cells, we synthesized seven additional novel inhibitors. Of these, compounds 21 and 25 had moderate HDAC 8 inhibitory activity (IC50 = 292.5, 341.2 nM). Further cellular effects of HDAC 8 inhibitors in this study are being undertaken.

目錄 .................................................. I
表目錄 ............................................ V
圖目錄 ........................................... VI
流程圖目錄 .................................. VII
附圖目錄 .................................... VIII
中文摘要 .................................... XIII
Abstract ......................................... XV
第一章、緒論 ................................. 1
第二章、研究目的 ......................... 7
1. 在脂鏈醯基羥胺嵌入芳香環吲哚修開發新型組蛋白去乙醯酶抑制劑作為抗癌藥物 ............. 7
2. 鄰位芳香環氮-羥基肉桂酸醯胺結構優化作為選擇性第八型組蛋白去乙醯酶抑制劑 ............. 9
第三章、結果與討論 ................... 12
1. 在脂鏈醯羥胺嵌入芳香環吲哚開發新型組蛋白去乙醯酶抑制劑作為抗癌藥物 ................... 12
1.1 合成N-苯磺酰基吲哚(4a-d)之脂鏈醯基羥胺 ................. 12
1.2 合成N-三甲氧基苄基吲哚之長鏈醯基羥胺 .................. 14
1.3 化合物4a-4d與6對於抑制組蛋白去乙醯酶及非小細胞肺癌A549細胞生長抑制之活性 ............................................ 15
1.4 化合物4a-4d與6對於人類大腸上皮癌HT-29細胞之細胞毒性測試 ................... 16
1.5 化合物4d之分子模擬...................................................... 18
1.6 結論 ........................ 20
2. 鄰位芳香環氮-羥基肉桂酸醯胺結構優化作為選擇性第八型組蛋白去乙醯酶抑制劑........... 21
2.1 鄰位聯苯-氮-羥基肉桂酸醯胺11a-g之合成 .................. 21
2.2化合物11a-11g對HDAC 8酵素抑制活性試驗 ............. 23
2.3 化合物11d分子模擬 ........................................................ 24
2.4 三甲氧基苯胺氮-羥基肉桂酸醯胺15a-e、21和25之合成 ....................................... 26
2.5 化合物15a-e、21和25對HDAC 8酵素抑制活性測驗 ....................................... 30
2.6 結論........................ 31
第四章、實驗方法....................... 33
1. 儀器與材料.................... 33
1.1一般儀器及方法 ..... 33
1.2試劑來源 ................. 33
1.3 溶劑來源 ................ 34
1.4試劑製備 ................. 35
2. 化學合成步驟及物理資料 .......................................... 36
3. 酵素活性測定實驗步驟
3.1 HeLa nuclear HDAC inhibition assay................................ 66
3.2 The sulforhodamine B (SRB) growth inhibition assay ....... 66
第五章、參考資料....................... 68

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