I、開發以Lovastatin為基礎之羥基醯胺作為選擇性組蛋白去乙醯酶抑制劑；II、黃酮類Hispidulin之全合成研究

I. Development of Lovastatin–based Hydoxamates as Selective Inhibitors of Histone Deacetylase; II. Study on the Total Synthesis of Naturally Occurring Flavonoid Hispidulin

黃志強(chih chyang,huang)

指導教授 ： 黃偉展

臺北醫學大學/藥學院/生藥學研究所/碩士(2012年)

https://doi.org/10.6831/TMU.2012.00163

摘要

摘要
本論文分為I、開發以Lovastatin為基礎之羥基醯胺作為選擇性組蛋白去乙醯酶抑制劑；II、黃酮類Hispidulin之全合成研究。
I、開發以 Lovastatin 為基礎之羥基醯胺作為選擇性組蛋白去乙醯酶抑制劑
組蛋白抑制劑為癌症標靶治療發展趨勢之一，現今已知之組蛋白去乙醯酶抑制劑化學結構上包含了三個藥理活性基團：具有疏水性辨認部位及鏈長及可與金屬鋅產生螯合之基團，最近研究顯示臨床上使用之降血酯藥物 lovastatin具有中度抑制組蛋白去乙醯酶活性，因此我們希望以lovastatin作為先導化合物進行結構修飾，以進一步提升其活性，我們合成了同時具有烷基取代及與鋅離子螯合基團一系列化合物。酵素活性測試發現，化合物對於主要以異構酶1, 2, 3型酵素為主之子宮頸癌細胞萃取組蛋白去乙醯酶 (HeLa nuclear HDACs, IC50>30 µM) 無顯著抑制，進一步測試組蛋白去乙醯異構酶8 (HDAC8) 之酵素活性，發現其中化合物3c與3e具有酵素抑制活性，IC50分別為8.57 與7.69 µM，然而有趣的是分子模擬實驗顯示化合物3c與3e之aliphatic hydroxamate並無法進入組蛋白去乙醯異構酶8活性pocket與鋅離子產生螯合作用，酵素動力學實驗呈現其為非競爭型抑制，此結果亦支持分子模擬之假設，細胞生長抑制活性實驗顯示化合物3c具有較佳之抑制前列腺癌PC-3細胞生長活性 (GI50=3.3 µM)，優於已知之組蛋白去乙醯異構酶第8型制劑PCI34051 (GI50=16.6 µM)，且對正常之人類臍帶血內皮細胞無明顯毒性，經由本研究我們發現新一類組蛋白去乙醯異構酶第8型抑制劑其作用於另外活性部位。

Ⅱ、黃酮類Hispidulin 之全合成研究
妥瑞氏症(Tourette syndrome)是一種遺傳性的神經運動疾病，患者的身體會出現不自主的、重複性的動作稱為抽筋 (tics) 好發於兒童之慢性神經精神異常 (neuropsychiatric disorder)，可能會因疲勞或壓力而惡化，至今仍無有效之治療方法，病患終其一生都要與此症狀共處，我們最近發表之病例報告指出苦藍盤(Clerodendrum inerme L. Gaertn) 具有減緩患者妥瑞氏症發作頻率之效果。進一步以生物活性導向分離技術 (bioassay-guided isolation) 分析苦藍盤活性成分，發現其主要活性成分為天然物黃酮類：粗毛豚草素 (Hispidulin)，為了更進一步進獲得較大數量以進行動物體內試驗，本研究將以全合成方式製備粗毛豚草素，我們以3,4,5-trimethoxyphenol為起始物，進行Friedel–Crafts醯基化反應，接下來進行Baker–Venkataraman重排反應及Aldol縮合反應得到chalcone結構，以oxidative cyclization反應得到flavone結構，最後選擇性去甲基可得到粗毛豚草素。

關鍵字

組蛋白去乙醯酶黃酮類

並列摘要

Abstract
I. Development of Lovastatin–based Hydoxamates as Selective Inhibitors of Histone Deacetylase
Histone deacetylase (HDAC) inhibitors have been validated as a potential agent for targeted chemotherapy. The pharmacophore of HDAC inhibitor is composed of three moieties: a zinc-chelating group, a hydrophobic linear or cyclic linker as a connection group and a hydrophobic cap for surface recognition. Lovastatin, clinically used for treatment of hypercholesterolemia, has recently been reported to show moderate HDAC inhibitory activity. To enhance its enzyme activity, in the present study, we attempted to carry out structural modifications using lovastatin as a lead compound, A series of lovastatin-based hydroxamates with alkyl substitution were synthesized and tested for inhibitory activity against HeLa nuclear HDACs, mostly contained HDAC1, -2, -3 as well as HDAC8. Most did not inhibit HeLa nuclear HDACs (, IC50>30 µM), whereas compounds 3c and 3e exhibited HDAC8–selective inhibition with the IC50 of 8.57 and 7.69 µM, respectively. Notably, compound 3c can not enter into the catalytic HDAC8 pocket with a zinc cofactor at the bottom based on the molecular docking analysis. The enzyme kinetic study demonstrated that compound 3c inhibited enzyme in non-competitive manner, further supporting the molecular modeling result. Furthermore, compound 3c showed growth inhibitory activity against human prostate cancer PC-3, cells (GI50= 3.3 µM) superior to PCI34051 GI50= 16.6 µM), a HDAC8 inhibitor, but with significant cytotoxicity towards normal human umbilical vein endothelial cells. In conclusion, we have discovered a novel HDAC8-selective inhibitor which is not related to the catalytic site.

II. Study on the Total Synthesis of Naturally Occurring Flavonoid Hispidulin
Tourette syndrome, a hereditary neuropsychiatric disorder, is characterized by the repetitive involuntary movements known as the cramps (or tics) in children. Moreover, the symptoms may become serious under fatigue or stress status. There are some therapies used for treating the disorder, but are limited. In our recent case report, we demonstrated that Clerodendrum inerme L. Gaertn can reduce the incidence for Tourette syndrome symptoms in patient. Using bioassay-guided fractionation and isolation, we achieved the main active compound elucidated as a flavonoid hispidulin. In order to obtain a large amount for further in-vivo assessment, we designed total synthetic methodology to prepare hispidulin. We exploited 3,4,5-trimethoxyphenol as a starting material to carry out several reaction steps including Friedel-Crafts acylation, Baker-Venkataraman rearrangement, Aldol condensation for chalcone structure, oxidative cyclization reaction to produce flavone . Finally, a selective demethylation will give the target compound.

並列關鍵字

lovastatin ； HDAC ； hispidulin

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