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| 題名: | 合成以蛇床子與肉桂酸為基礎之羥基醯胺作為組蛋白去乙醯酶抑制劑並促進神經母細胞瘤分化 |
| 作者: | 陽蘋 |
| 貢獻者: | 生藥學研究所 |
| 日期: | 2010 |
| 上傳時間: | 2010-10-21 09:09:33 (UTC+8) |
| 摘要: | 組蛋白去乙醯酶抑制劑為目前標靶治療的研究之發展趨勢，分析組蛋白去乙醯酶抑制劑的化學結構分為三個部分分別為：疏水性的基團、疏水性的鏈長、與鋅螯合產生活性的基團，本論文主要合成(1)以蛇床子為基礎之氮-羥基直鏈醯胺(2)芳香環取代之羥基肉桂酸醯胺，以作為有效及選擇性的組蛋白去乙醯酶抑制劑(HDACi)，並具有促進神經細胞瘤分化作用。
我們以氫化後的蛇床子素為模板合成不同碳鏈長度之氮-羥基直鏈醯胺6a~6k，並測試其抑制子宮頸癌細胞之組蛋白去乙醯酶(HeLa cell nuclear extract HDAC)之活性。其中化合物6c、6d、6g、6k (IC50=24.6, 28.9, 22.2, 23.8 nM)比suberoylanilide hydroxamic acid (SAHA, IC50=41.7 nM)效果更強，進一步測試組蛋白去乙醯酶亞型(HDAC-1, -4, -6, -8)活性，在第一、四、六型組蛋白去乙醯酶 (HDAC-1, -4, -6)抑制活性中，6c、6d、6g、6k均顯示與SAHA相當的活性，而在第八型組蛋白去乙醯酶 (HDAC8)的酵素抑制活性中6c、6d、6g、6k顯示比SAHA較強之活性，尤其6c為SAHA 12倍。近來有研究指出第八型組蛋白去乙醯酶與神經母細胞瘤分化(neuroblastoma cell differentiation)有關，因此我們將化合物6c、6d、6g、6k進行神經母細胞瘤SH-SY5Y 細胞活性實驗，發現6c比SAHA具有較佳之促進神經母細胞瘤分化的效果，進而將6c與SAHA分別與第八型組蛋白去乙醯酶進行分子模擬分析(molecular modeling analysis)，顯示6c與SAHA共同擁有之苯環都是作用酵素疏水區的口袋表面，但不同的是，與SAHA相較，由於6c苯環上較SAHA多了側鏈dimemthylpropane及 N-hydroxypropamide，故可與HDAC8產生更多的疏水作用 (hydrophobic interaction)，推測因此6c較SAHA具有更好的抑制活性。
以氮-羥基肉桂酸醯胺為模板，在鄰位與對位分別接上不同取代基之苯甲基，共合成十四個化合物11a~11e、17a~17f、22、27、32進行第一，四，六，八型組蛋白去乙醯酶(HDAC1, -4, -6, -8)抑制活性測定。在第八型(HDAC8)抑制活性，化合物11a、11c、11d、22、32較SAHA效果強，第一，六型(HDAC1, -6)均比SAHA弱，在第四型(HDAC4)則與SAHA類似，較不具抑制性，此結果顯示化合物11a、11c、11d、22、32為具有選擇性之第八型組蛋白去乙醯酶抑制劑(HDAC 8 inhibitor)， 而在神經母細胞瘤SH-SY5Y 細胞活性實驗發現，22與32均較SAHA具明顯促進神經母細胞瘤分化的效果。
綜合以上的活性試驗的結果顯示，以蛇床子為基礎之氮-羥基直鏈醯胺系列化合物，其對組蛋白去乙醯酶亞型(HDAC-1, -4, -6, -8)抑制活性均較SAHA強或與SAHA相當，此類化合物屬於為pan-histone deacetylase (HDAC) inhibitors。而芳香環取代之羥基肉桂酸醯胺系列化合物，雖對組蛋白去乙醯酶亞型(HDAC-1, -6)抑制活性遠比SAHA來得小，但對HDAC-8有很好的抑制活性，具有成為治療神經母細胞瘤藥物之潛力。
Histone deacetylase (HDAC) inhibitors had been used as potential agents for targeted cancer chemotherapy. From previous publications, HDAC inhibitors were know to consist of three major parts: a hydrophobic cap for surface recognition, a zinc-chelating group and a hydrophobic linker between the two functional groups. We focus on the synthesis of osthole- and cinnamate-based hydroxamates as histone deacetylase inhibitors and test their neuronal differentiation activities on neuroblastoma cells (SH-SY5Y).
Eleven novel osthole-based N-hydroxamates, compound 6a~6k, were synthesized and screened for HDAC inhibitory activity by using HeLa nuclear extract. In this screening, compounds 6c, 6d, 6g and 6k showed similar activity (the IC50 was 24.6, 28.9, 22.2 and 23.8 nM, respectively) as suberoylanilide hydroxamic acid (SAHA, the IC50 was 41.7 nM), the potent HDAC inhibitor for the treatment of cutaneous T-cell lymphoma (CTCL) approved by FDA in 2006. After screening on different classes of HDAC enzymes, our compounds were active against both class I (HDAC-1, -8) and class II (HDAC-4, -6) indicated that all of them showed similar inhibitory activity against HDAC-1, -4 and -6, but were much more active against HDAC-8, especially the compound 6c with 12-fold active than SAHA. In the docking analysis, the branched side chains, the dimthylpropane and N-hydroxypropamide groups, of compound 6c made the hydrophobic interaction on HDAC 8 stronger than that of SAHA. Furthermore, the compound 6c also showed the best cellular activity on the promotion of neurite outgrowth and neuronal differentiation on SH-SY5Y neuroblastoma cells in these series of analogues.
Fourteen novel N-hydroxycinnamides, comuond 11a~11e, 17a~17f, 22, 27 and 32, substituted with ortho- or para- benzyl derivatives were synthesized and screened for their HDAC inhibitory activity (HDAC1, -4, -6, -8). Compounds 11a, 11c, 11d, 22 and 32 were highly selective against HDAC-8 and were 9-, 3-, 5-, 15- and 12-fold, respectively potent than that of SAHA. But in neuroblastoma differentiation experiment, only compounds 22 and 32 showed significant promoting effect, others lost their activity in the cell model system.
By these results, we found that osthole-based hydroxamates exhibited SAHA-like activity against HDAC-1, -4, -6 and -8. and were likely a wonderful skeleton for pan HDAC inhibitor design. On the other hand, the benzyl substituted N-hydroxycinnamides were selective toward HDAC 8 over other subtypes (HDAC-1, -4, -6). We think that this skeleton might be a good lead for the design of novel HDAC inhibitor with HDAC-8 selectivity. Although the preliminary data showed that some of the compounds were effective on the promotion of the neuroblastoma differentiation, but there still further estimations needed to be done to determinate their therapeutic potential on neuroblastoma. |
| 關聯: | 100頁 |
| 描述: | 目錄
中文摘要 i
Abstract iii
表目錄 viii
流程圖目錄 ix
圖目錄 x
附圖目錄 xi
一、緒論及研究目的 - 1 -
二、結果與討論 - 14 -
1. 化學合成 - 14 -
1.1以蛇床子為基礎之氮-羥基直鏈醯胺之合成 - 14 -
1.2 芳香環取代之羥基肉桂酸醯胺之合成 - 18 -
2. 化合物(6a~6k)對於抑制子宮頸癌細胞核萃取之組蛋白去乙醯酶(HeLa cell nuclear extract histone deacetylase)之活性 - 23 -
3. 化合物6c、6d、6g、6k、11a、11c、11d、22 、32 對組蛋白去乙醯酶1、4、6、8型(HDAC1，-4，-6，-8)酵素抑制活性 - 24 -
4. 以西方墨點法偵測化合物 6c、 6g、 6k、 22、 32影響 SH-SY5Y細胞株神經分化標記之表現: - 27 -
5. 化合物6c、32與SAHA對HDAC 8之分子模擬(molecular modeling)- 30 -
三、結論 - 32 -
四、實驗方法 - 33 -
1. 儀器與材料 - 33 -
(1). 一般儀器及方法 - 33 -
(2). 試劑及溶劑來源 - 34 -
2. 化學合成步驟及物理資料 - 37 -
3. 生物活性分析 - 88 -
(1). HDAC activity assay - 88 -
(2). Compound-Induced Neuronal Differentiation of Neuroblastoma Cells……………………………………………………………………...-89-
(3).Western Blotting - 90 -
(4). Molecular modeling - 91 -
五、參考文獻(References) - 93 -
六、附圖 - 95 -
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