臺灣博碩士論文加值系統

摘要

Anthraquinones在自然界中廣泛分布，且具有許多的生物活性，例如:抗病毒、抗癌、抑制端粒酶….等，都有不錯的效果。流感病毒具有八個分離片段的核糖核蛋白(ribonucleoprotein, RNP)被包覆在脂蛋白膜裡，其膜上有兩種突出物一種是具有盒子形狀的蛋白，稱為neuraminidase (NA)，有九個主要的抗原型式，且具有酶的特性。另一種是三聚體的蛋白稱為haemagglutinin(HA)，具有13個主要的抗原型式。HA的功能是當病毒附著在細胞膜時，可以與各種細胞上特定的受體結合。流感病毒必須依賴宿主細胞提供能量並且提供複製及蛋白質合成時所需的生化物質，來產生下一帶。端粒位於染色體末端，可以保護染色體。在正常體細胞的染色體末端因為在複製時，端粒酶沒有表現，所以每當細胞分裂時會發生端粒縮短50~200個鹼基(造成末端複製問題)，而當它的長度變的危險短時這個侵蝕會引起細胞凋亡。另一方面，端粒酶在人類85~90%腫瘤細胞具有活性，且此酶可以讓端粒長度變長，藉由催化增加端粒的序列，維持端粒的長度所以使這些細胞變成不朽細胞。本研究係合成一系列1,2-diamidoanthraquinone衍生物進行生物活性評估，來探討此系列新穎化合物對人類反轉錄酶(hTERT)啟動子(promoter) 的活性，端粒序列複製法(TRAP)評估對端粒酶之活性，以及病毒活性之抑制。藥理活性主要藉由神經胺酸酶試驗(NA assay)及細胞毒性試驗(MTS assay)之結果對於抑制病毒活性進行數值交叉分析，及藉由分泌鹼性磷酸酶(SEAP assay)之基因活性表現和細胞毒性試驗(MTT assay)之結果對於端粒酶活性抑制進行數值交叉分析。在抑制病毒活性方面，此系列藥物在抑制病毒的同時，對細胞毒性也大，所以嚴格來說對抗病毒方面無作用。

Abstract

Anthraquinones are widely distributed in nature and are known to demonstrate various biological activities as antivirus, anti-cancer agents, telomerase inhibitors ¡¬.Wait, all have a good result. Influenza virus has eight separate segments of ribonucleoprotein (RNP). The envelope carries two types of protruding spikes. One is a box-shaped protein, called the neuraminidase (NA), of which there are nine major antigenic types, and which has enzymic properties. The other type of envelope spike is a trimeric protein called the haemagglutinin (HA) of which there are 13 major antigenic types. The haemagglutinin functions during attachment of the virus particle to the cell membrane, and can combine with specific receptors on a variety of cells. The influenza virus must rely on host cell which can offer the energy and the biochemical materials for replication and protein synthesis to produce next generation. Telomeres are the repeated sequences located at the ends of chromosomes in eukaryotes. Telomere has a key function in protecting chromosome ends from degradation, recombination, and end-to-end fusion. Since telomerase is not expressed in normal somatic cells, telomere is shortened by 50-200 bases whenever cell division happens (end-replication problem), and this erosion induce apoptosis when its length became critically short. On the other hand, telomerase is active in 85-90% of human tumor cells, and this enzyme combines and elongates telomeres by catalyzing the addition of telomere sequence, maintaining telomere length so that these cells are immortalized. By synthesizing a series of 1, 2-diamidoanthraquinone derivatives, we can study human telomerase reverse transcriptase (hTERT), telomeric repeat amplification protocol (TRAP) and the inhibition of virus activity. We use SEAP assay and MTT assay to monitor the hTERT expression; we can evaluate if compounds can inhibit the activity of telomerase by a mechanism consistent with interaction with G-quadruplex structure; and we use NA assay and MTS assay to examine the inhibition of virus activity and to precede the data cross-analysis. In inhibition of the virus activity, the results show that this series of compounds have lowers effects to inhibit the virus activity and have greater cytotoxicity in the same time. So this series of compounds have no effect in antivirus.

總目錄
頁次
正文目錄 ------------------------------------------------------------------ Π

表目錄 ------------------------------------------------------------------ Ⅲ

圖目錄 ------------------------------------------------------------------ Ⅲ

附圖目錄 ------------------------------------------------------------------ IV

中文摘要 -------------------------------------------------------------------- VI

英文摘要 -------------------------------------------------------------------- VII





















正文目錄
頁次
第一章、緖論 --------------------------------------------------------------- 1
第一節、緖論 --------------------------------------------------------- 1
第二節、藥物設計原理 --------------------------------------------- 12
第三節、研究目的 -------------------------------------------------- 13

第二章、材料與方法 ------------------------------------------------------ 14
第一節、化學合成 --------------------------------------------------- 14
第二節、藥理活性實驗 --------------------------------------------- 17

第三章、實驗結果 --------------------------------------------------------- 30
第一節、化學合成結果 --------------------------------------------- 30
第二節、藥理試驗結果 --------------------------------------------- 51

第四章、討論與結論 ------------------------------------------------------ 61
第一節、化學合成 --------------------------------------------------- 61
第二節、藥理試驗 --------------------------------------------------- 62
第三節、結論 --------------------------------------------------------- 65

第五章、參考文獻 --------------------------------------------------------- 66

















表目錄
頁次
表1、化合物M1~M20一覽表 ----------------------------------------- 16
表2、化合物M1~M20之病毒(H1N1)抑制效果 --------------------- 51
表3、化合物M1~M20之BJ-hTERT-SEAP活化效果------------------ 52
表4、化合物M1~M20之BJ-CMV-SEAP活化效果 ----------------- 54
表5、化合物M1~M20之H1299-hTERT-SEAP抑制效果 ---------- 56
表6、化合物M1~M20之H1299-CMV-SEAP抑制效果 ------------ 58
表7、化合物M1~M20之抑制癌細胞生長IC50結果 ---------------- 60

圖目錄
頁次
圖1、人體端粒構造 --------------------------------------------------------- 1
圖2、Tetrahymena端粒延長機轉 ---------------------------------------- 2
圖3、G-tetrad的結構 ------------------------------------------------------- 3
圖4、human G-quadruplex的結構 -------------------------------------- 3
圖5、Oxytricha端粒酶上引子結構之影響模型 ----------------------- 4
圖6、hPOT 1-TTAGGGTTAG化合物的空間充填模型----------------- 4
圖7、hPOT 1調節端粒長度的模型 -------------------------------------- 5
圖8、具有穩定G-quadruplex的潛力化合物5﹑6﹑9﹑10﹑12﹑23﹑48及A9b ------------------------------------------------------------ 7
圖9、具活化作用之化合物19及42 ---------------------------------------- 7
圖10、具穩定G-quadruplex的2,7-diamidoanthraquinones衍生物----- 8
圖11、具抑制端粒酶活性的2,7-diamidoanthraquinones衍生物 ------ 8
圖12、流感病毒的結構 ------------------------------------------------------ 9
圖13、haemagglutinin的結構----------------------------------------------- 9
圖14、流感病毒複製過程 ------------------------------------------------- 10
圖15、Quinalizarin﹑Rhein﹑Alizarin化合物結構式 --------------- 11
圖16、Hypericin化合物結構式----------------------------------------- 11
圖17、ACID BLUE 40﹑ACID BLUE129﹑ALIZARIN VIOLET R和 REACTIVE BLUE 2化合物結構式---------------------------- 11
圖18、Chrysophanic acid化合物結構式---------------------------------- 12
圖19、Doxorubicine及Mitoxantrone的結構式------------------------- 12
圖20、1,2-Diamidoanthraquinone之藥物設計 ------------------------- 13
圖21、MDCK細胞膜式----------------------------------------------------- 17
圖22、MTS assay --------------------------------------------------------- 19
圖23、NA assay ------------------------------------------------------------ 20
圖24、hTERT-BJ1細胞模式 --------------------------------------------- 21
圖25、H1299細胞模式 --------------------------------------------------- 23
圖26、SEAP assay --------------------------------------------------------- 26
圖27、MTT assay ------------------------------------------------------------ 27
圖28、XTT assay ---------------------------------------------------------- 29
圖29、化合物M1、M8、M11、M12及M19之NA assay對照圖----- 62
圖30、化合物M1、M8、M11、M12及M19之 MTS assay對照圖-- 62
圖31、化合物M10、M15及M19之 PhTERT-SEAP與PCMV-SEAP圖- 63
圖32、化合物M1、M2、M18及對照組於C6 cells之濃度對照圖---- 64
圖33、化合物M1、M15、M19及對照組於Hep G2之濃度對照圖--- 64
圖34、化合物M15及M19結構式 -------------------------------------- 65

附圖目錄
頁次
附圖1、化合物M1之1H-NMR, 13C-NMR圖譜 --------------------- 71
附圖2、化合物M1之IR, MS圖譜 ------------------------------------ 72
附圖3、化合物M2之1H-NMR, 13C-NMR圖譜 ---------------------- 73
附圖4、化合物M2之IR, MS圖譜 ------------------------------------ 74
附圖5、化合物M3之1H-NMR, 13C-NMR圖譜 ---------------------- 75
附圖6、化合物M3之IR, MS圖譜 ------------------------------------ 76
附圖7、化合物M4之1H-NMR, 13C-NMR圖譜 ---------------------- 77
附圖8、化合物M4之IR, MS圖譜 ------------------------------------ 78
附圖9、化合物M5之1H-NMR, 13C-NMR圖譜 ---------------------- 79
附圖10、化合物M5之IR, MS圖譜 ------------------------------------ 80
附圖11、化合物M6之1H-NMR, 13C-NMR圖譜 --------------------- 81
附圖12、化合物M6之IR, MS圖譜 ------------------------------------ 82
附圖13、化合物M7之1H-NMR, 13C-NMR圖譜 --------------------- 83
附圖14、化合物M7之IR, MS圖譜 ------------------------------------ 84
附圖15、化合物M8之1H-NMR, 13C-NMR圖譜 --------------------- 85
附圖16、化合物M8之IR, MS圖譜 ------------------------------------ 86
附圖17、化合物M9之1H-NMR, 13C-NMR圖譜 --------------------- 87
附圖18、化合物M9之IR, MS圖譜 ------------------------------------ 88
附圖19、化合物M10之1H-NMR, 13C-NMR圖譜 ------------------- 89
附圖20、化合物M10之IR, MS圖譜 ---------------------------------- 90
附圖21、化合物M11之1H-NMR, 13C-NMR圖譜 ------------------- 91
附圖22、化合物M11之IR, MS圖譜 ---------------------------------- 92
附圖23、化合物M12之1H-NMR, 13C-NMR圖譜 ------------------- 93
附圖24、化合物M12之IR, MS圖譜 ---------------------------------- 94
附圖25、化合物M13之1H-NMR, 13C-NMR圖譜 ------------------- 95
附圖26、化合物M13之IR, MS圖譜 ---------------------------------- 96
附圖27、化合物M14之1H-NMR, 13C-NMR圖譜 ------------------ 97
附圖28、化合物M14之IR, MS圖譜 --------------------------------- 98
附圖29、化合物M15之1H-NMR, 13C-NMR圖譜 ------------------ 99
附圖30、化合物M15之IR, MS圖譜 --------------------------------- 100
附圖31、化合物M16之1H-NMR, 13C-NMR圖譜 ----------------- 101
附圖32、化合物M16之IR, MS圖譜 --------------------------------- 102
附圖33、化合物M17之1H-NMR, 13C-NMR圖譜 ------------------ 103
附圖34、化合物M17之IR, MS圖譜 --------------------------------- 104
附圖35、化合物M18之1H-NMR, 13C-NMR圖譜 ------------------ 105
附圖36、化合物M18之IR, MS圖譜 ------------------------------[-- 106
附圖37、化合物M19之1H-NMR, 13C-NMR圖譜 ------------------ 107
附圖38、化合物M19之IR, MS圖譜 --------------------------------- 108
附圖39、化合物M20之1H-NMR, 13C-NMR圖譜 ------------------ 109
附圖40、化合物M20之IR, MS圖譜 -------------------------------- 110

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