臺灣博碩士論文加值系統

C型肝炎病毒已在全球流行病學造成嚴重威脅。約有七至八成之感染者將發展為長期慢性肝炎，並有可能導致肝硬化甚或肝癌。現時治療C型肝炎以干擾素－alpha（interferon-alpha）為主之療法卻療效有限。因此研發創新且更有效之治療方法不但重要並且迫切。WSP-9376為本實驗室由DOCK軟體所完成之電腦虛擬篩選找出之C型肝炎病毒解旋酶抑制劑（IC50 = 20 uM）。之後在一篇專利中發現其並為C型肝炎病毒NS5B聚合酶之抑制劑（IC50 = 44.5 uM）。因此將之選為先導化合物（lead compound）。發展C型肝炎藥物時，需同時考慮藥效與選擇性，以減低毒性及副作用，NS5B核醣核酸聚合酶（RNA-dependent RNA polymerase, RdRp）因此為合適之標的。其不但為C型肝炎病毒複製所必須，且哺乳動物中並未有功能上相對應之蛋白質。小分子與NS5B之結合區域（binding sites）較常見者為五處，四處為異位結合區域（allosteric binding sites），另一為活性區域（active site）。大部分活性區抑制劑屬於受質導向之核苷類似物（nucleoside analogues）或產物導向之焦磷酸類似物（pyrophosphate-mimics）。反觀異位結合區抑制劑，結構多樣性高且變化豐富。藉由結構跳躍式藥物設計原理（scaffold hopping），我們設計了四個系列之化合物，唯一順利合成出之為3-芳香基亞甲基喹啉-2,4-雙酮類。化合物44b為這之中抑制NS5B聚合酶活性較好的（IC50 = 8.4 uM）。雖然優化之程度與先導化合物（WSP-9376）相較並不顯著，但我們成功地找出了以喹啉為基礎架構之NS5B抑制劑，而且其與WSP-9376之結構相去甚遠。此外，這一系列化合物會對NS5B聚合酶之受質造成影響，因此其與NS5B之結合區域很可能位於活性區附近；換句話說，其將有可能成為另一系列不同於已發表之活性區抑制劑！

Hepatitis C virus (HCV) has become a serious etiological agent worldwide. HCV sets up a persistent infection in 70-80% of cases, and can lead to cirrhosis or hepatoma. Currently available interferon-based treatments show limited efficacy. Therefore, to develop new and more effective therapies is a vital and urgent issue. WSP-9376 was discovered as an HCV helicase inhibitor (IC50 = 20 uM) in our laboratory through computational virtual screening using the DOCK program. Coincidentally, this compound was found as an HCV NS5B polymerase inhibitor (IC50 = 44.5 uM) as well, thus being selected as our lead. When it comes to both potency and selectivity toward HCV, NS5B RNA-dependent RNA polymerase (RdRp) can be a proper target. Since it is essential for HCV RNA polymerization and there is no functional counterpart in mammalian cells. Five binding sites of NS5B, four allosteric binding sites and the active site, are commonly reported. Most of the active site inhibitors belong to nucleoside analogues (substrate-like) or pyrophosphate-mimics (product-like). On the contrary, numerous classes of structurally diversified allosteric inhibitors have been developed. By employing scaffold hopping strategies, four series of compounds were designed whereas only one was fulfilled in synthesis, i.e. 3-arylmethylene-1H-quinoline-2,4-diones. Among them, compound 44b is the most potent NS5B inhibitor with an IC50 of 8.4 uM against NS5B. Albeit there is only slight improvement in the inhibitory activity of 44b compared with the lead, it is more important that we have identified this core structure for the development of new NS5B inhibitor, even if the structure differs a lot from that of WSP-9376. Moreover, the binding site of this series of compounds may be the active site, since compound 44b has been shown to have influence to some extent on the substrates of NS5B. In other words, this is probably another series of active site inhibitors distinguished from those published previously.

Contents
Referees’ Approval i
Acknowledgements ii
Abstract in Chinese iii
Abstract in English iv
I. Introduction 1
1.1 History 1
1.2 Hepatitis C Virus (HCV) 2
1.3 Nowadays 5
1.4 NS5B as Target for Drug Development 7
II. The Concept of Inhibitor Design 14
III. Results and Discussion 18
3.1 Approaches toward the Synthesis of Target 1 18
3.1.1 Method I 18
3.1.2 Method II 22
3.2 Approaches toward the Synthesis of Target 2 26
3.3 Approaches toward the Synthesis of Target 3 30
3.4 Synthesis of Target 4 34
3.5 NS5B Inhibition Assay 42
3.6 Computer Modeling 44
IV. Conclusion 48
V. Experimental Section 49
5.1 Chemistry 49
5.2 NS5B Inhibition Assay 57
5.3 Molecular docking studies 60
References 62
Appendixes 71

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