臺灣博碩士論文加值系統

由天然藥物Distamycin及Netropsin研究指出，此類化合物對於DNA小凹槽上的A-T鹼基對有專一性且極強之鍵結能力，並具有抗腫瘤和抗生素的潛力性，由於此類藥物具高毒性，因此無法作為臨床應用藥物。因此在過去幾十年間，科學家主要透過修飾不同芳香環使化合物能辨識更多鹼基對且能與C-G鹼基對作用之外，希望能降低對於細胞的毒性及副作用，然而對於改變醯胺架橋的研究少之又少。
本篇研究中，主要以烯烴、亞胺或烷基來修飾醯胺架橋並分為三個方向：(一)嘗試利用威悌反應(Wittig reaction)及麥克莫里反應(McMurry Reaction)合成烯類架橋的1H-吡咯及噻吩衍生物以提高與DNA小凹槽的結合力並比較吡咯與噻吩之間差別；(二)將E-1,2-二(2-噻吩基)乙烯先以甲醯化反應後，再進行縮合得到亞胺(imine)架橋化合物45及46來比對烯類架橋與亞胺架橋之差異；(三)以苯甲醚或2,5-雙(吡咯基甲基)吡咯為連結劑，將兩段Distamycin衍生物連結，形成螺旋型化合物16a及16b。
最後將所合成化合物與小牛胸腺DNA進行DNA之熔點實驗與溴化乙錠螢光滴定實驗等性質測試來探討設計藥物之潛力。

Research has showed that natural product; such as Netropsin and Distamycin have specific and extremely strong bonding ability with A-T base pair within the DNA minor groove. These compounds are useful as potential antitumor agents and antibiotics, but the major drawback is the high toxicity that hampered their clinical application. As such, scientists have concentrated on designing of more specific DNA base pairs recognitions, including C-G base pair, with the hope of reducing toxicity and improving drug efficacy. However, most research have concentrated on variation of the pyrrole ring with other heterocycles or aromatics while retaining the amide linkage. Till today, there has been little research on the derivatization of the amide linkage.
This thesis focuses on the derivatization of amide linkage using alkene, imine or alkane, and can be divided into three directions. First, we attempted to use wittig and McMurry reaction to synthesize alkene linked 1H-pyrrole or thiophene ring to improve binding according to theoretical calculation of Vinylexin. Secondly, we further replaced alkene linkage with imine using the E-1,2-bis(2-thionyl)ethene to carry out formylation reaction and then condensation with amine to give compound 45 and 46 for comparing alkenyl with imine linking bridge. Thirdly, helical compound 16 a,b and 17 were prepared by connecting two fragment of Distamycin derivatives. Using the phenyl ether and 2,5-bis(2-pyrrolylmethyl)pyrrole as linker. Finally, all compounds prepared were studied for their binding to minor groove by DNA melting point experiments and Ethidium bromide fluorescent titration experiments with calf thymus DNA, or splenic sequenced DNA.
Keywords:Netropsin, Distamycin, pyrrole, thiohene, alkene, imine, DNA minor groove.

摘要 i
Abstract ii
圖目錄 vii
表目錄 xi
流程目錄 xii
第１章 緒論 1
1.1 去氧核醣核酸 1
1.2 生物中DNA所扮演的角色 3
1.3 作用於DNA之抗癌藥物 4
1.4 凹槽鍵結試劑(DNA binding reagents) 7
1.4.1 大小凹槽之選擇性 7
1.4.2 作用原理 8
1.4.3 對於A-T或C-G鹼基對之選擇性 9
1.5 設計作用於C-G鹼基對之藥物 11
1.6 辨識A-T/ T-A或C-G/ G-C之鹼基對 14
1.7 修飾小凹槽試劑之架橋 22
1.7.1 置換成烷基架橋 23
1.7.2 置換成烯類架橋 24
1.7.3 置換成重氮架橋 25
第２章 結果與討論 29
2.1 烯類架橋化合物 29
2.1.1 研究動機 29
2.1.2 Vinylexin衍生物(15)之合成策略及製備 30
2.1.3 路徑I (Path I) 30
2.1.4 路徑II (Path II) 34
2.1.5 以噻吩為主體的Vinylexin衍生物之製備 40
2.2 具醯胺架橋化合物 41
2.2.1 研究動機 41
2.2.2 含亞胺架橋化合物之製備 42
2.3 螺旋型化合物 44
2.3.1 研究動機 44
2.3.2 製備以二苯醚(diphenyl ether)為連結劑之螺旋型化合物45及46 46
2.3.3 製備以2,5-雙(吡咯基甲基)吡咯為連結劑之螺旋型化合物47 49
2.4 性質測試 54
2.4.1 DNA熔點實驗 54
第３章 結論 57
第４章 實驗部分 58
4.1 實驗儀器、藥品及試藥 58
4.2 合成與鑑定： 59
4.2.1 (1H-pyrrole-2,5-diyl)dimethanol (17) 60
4.2.2 1H-pyrrole-2,5-carbaldehyde (18) 60
4.2.3 Pyrrole-2-carboxaldehyde (20) 61
4.2.4 Ethyl 2-cyano-3-(1H-pyrrol-2-yl)acrylate (21) 61
4.2.5 Ethyl-α-cyano-5-formyl-2-pyrroleacrylate (22) 62
4.2.6 1-Tosyl-1H-pyrrole-2-carbaldehyde (23) 62
4.2.7 (1-Tosyl-1H-pyrrol-2-yl)methanol (24) 63
4.2.8 2-(Bromomethyl)-1-tosyl-1H-pyrrole (25) 63
4.2.9 2-[(Bromotriphenylphosphoranyl)methyl]-1-tosylpyrrole (26) 64
4.2.10 2,5-Bis (piperidinomethyl)pyrrole (29) 64
4.2.11 (2,5-Pyrroldiyldimethyl)-bis(triphenylphosphoniumiodine (30) 65
4.2.12 E-1,2-di-2-thienylethylene (32) 65
4.2.13 E-1-(2-Formyl-5-thienyl)-2-(2-thienyl)ethane (33) 66
4.2.14 2,2,2-Trichloro-1-(1-methyl-pyrrol-2-yl)ethenone (35) 66
4.2.15 2,2,2-Trichloro-1-(1-methyl-4-nitro-pyrrol-2-yl)ethenone (36) .67
4.2.16 N-(3-(dimethylamino)propyl)-1-methyl-4-nitro-pyrrole-2-carboxamide (38) 67
4.2.17 4-amino-N-(3-(dimethylamino)propyl)-1-methyl-pyrrole-2-carboxamide (39) 68
4.2.18 N-(3-(dimethylamino)propyl)-1-methyl-4-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-1-methyl-pyrrole-2-carboxamide (40) 68
4.2.19 N-(3-(dimethylamino)propyl)-1-methyl-4-(4-amino-1-methyl-pyrrole-2-carboxamido)-1-methyl-pyrrole-2-carboxamide (41) 69
4.2.20 E-2-[(N-dimethylpropyl)-2-carboxamido-1-methylpyrroyl-4-imino]thienyl-E-5-(2-thienyl)ethene (42) 69
4.2.21 E-2-(N-dimethylpropyl-2-carboxamido-1-methylpyrroyl-4-carboxamido)-N-(1-methylpyrroyl-4-imino)thienyl-E-5-(2-thienyl)ethene (43) 70
4.2.22 4,4’-oxydi(phenyl-azo-2-1H-pyrrole) (45) 70
4.2.23 4,4’-oxy-di(phenyl-azo-2-methylpyrrole) (46) 71
4.2.24 2,2,2-Trichloro-1-(1H-pyrrol-2-yl)ethenone (50) 71
4.2.25 N-(3-(dimethylamino)propyl)-1H-pyrrole-2-carboxamide (51) .72
4.2.26 N-(3-(dimethylamino)propyl)-1-methylpyrrole-2-carboxamide (52) 72
4.2.27 2,5-Bis(2-pyrrolylmethyl)pyrrole (tripyrrane) (55) 73
第５章 參考資料 74
第６章 附錄 81
6.1 核磁共振光譜圖 81

圖目錄
圖一、DNA雙股螺旋結構圖 2
圖二、DNA鹼基對之結構 2
圖三、DNA之作用機制 3
圖四、作用於DNA上之抗癌藥物分類 5
圖五、Tallimustine及Brostallicin之結構圖 6
圖六、天然藥物Distamycin及Netropsin之結構圖 7
圖七、裸露於DNA大小凹槽鹼基對示意圖 8
圖八、Netropsin與d (CGCGAATTCGCG)2之DNA作用示意圖 9
圖九、Distamycin A與d (CGCGAATTCGCG)2之作用示意圖 10
圖十、DE、P4E、P5E及P6E之結構圖 10
圖十一、Lexitropsin之設計概念圖 11
圖十二、Lexitropsin與d (GCGCGCAT)2之作用示意圖 11
圖十三、以吡啶修飾之Distamycin衍生物 12
圖十四、2-PyN與d (5’-CTTT-3’)作用示意圖 12
圖十五、2-PyN與d (5’-TGTCA-3’)作用示意圖 13
圖十六、2-ImN與d (5’-TTTC-3’)之結合示意圖 13
圖十七、Lexitropsin衍生物與G鹼基作用示意圖 14
圖十八、2-ImD與特定序列之DNA作用示意圖 15
圖十九、2-ImN-C4-P3與d(5’-TGTTA-3’)之作用示意圖 15
圖二十、頭對尾連結之髮夾型化合物示意圖 16
圖二十一、2-ImN-GABA-P3與5’-TGTTA-3’之結合示意圖 17
圖二十二、四種聚醯胺化合物之結構 18
圖二十三、四種聚醯胺化合物與特定序列DNA之結合強度示意圖 19
圖二十四、修飾N-甲基-3-羥基吡咯(Hp)之化合物 21
圖二十五、Isolexins與Vinylexins之結構圖 23
圖二十六、含烷基之化合物 23
圖二十七、具烯類架橋與對照組醯胺架橋之結構圖 24
圖二十八、具烯類架橋之凹槽試劑 25
圖二十九、具重氮架橋及對照組烯類架橋之化合物 26
圖三十、含重氮架橋之Distamycin衍生物 27
圖三十一、重氮化合物14作為DNA切割實驗式意圖 28
圖三十二、烯類架橋化合物之設計 30
圖三十三、Vinylexin衍生物之逆合成 30
圖三十四、Bhattacharya團隊設計之螺旋型化合物 45
圖三十五、化合物45及46之設計 45
圖三十六、化合物47之設計 46
圖三十七、DNA熔點之吸收與溫度關係圖 54
圖三十八、化合物42及43之結構圖 55
圖三十九、化合物42、43及Netropsin之Tm圖 56

表目錄
表一、不同連結劑與DNA之鍵結常數(M-1) 16
表二、修飾N-甲基-3-羥基吡咯(Hp)之化合物與特定序列DNA之平衡解離常數 20
表三、對於小凹槽鹼基對之辨識。 21
表四、具烯類架橋與醯胺架橋化合物之結合常數(× 105 M-1) 24
表五、化合物10至13與d(GCGATATATGCG)之DNA熔點實驗 26
表六、化合物17之氧化條件探討 32
表七、化合物17之溴化條件探討。 35
表八、化合物17修飾拉電子基之條件探討。 35
表九、合成化合物30之條件探討 37
表十、化合物30進行威悌反應條件探討 38
表十一、化合物29修飾拉電子基條件探討 39
表十二、2,5-雙(吡咯基甲基)吡咯衍生物之反應條件探討 50
表十三、DNA熔點實驗數據圖 56

流程目錄
流程一、實驗室之威悌反應方法 31
流程二、烯類架橋化合物15a之製備 31
流程三、化合物18之製備 32
流程四、2,5-雙醛基吡咯(19)之製備 33
流程五、威悌試劑(26)之製備 33
流程六、化合物28之製備 34
流程七、在酸性催化下，化合物17與親核試劑作用之反應機制。 35
流程八、Flitsch團隊合成雙烯類化合物之方法 36
流程九、化合物31進行取代反應而非威悌反應 36
流程十、延續Flitsch方法製備雙烯類化合物 36
流程十一、化合物30之製備 37
流程十二、威悌試劑30之合成 37
流程十三、推測威悌試劑(30)被分解的機制 39
流程十四、Nakayama團隊方法建構烯類架橋化合物16a 40
流程十五、化合物33之製備 40
流程十六、化合物16a之製備 41
流程十七、化合物45及46之逆合成 42
流程十八、化合物36之合成。 43
流程十九、化合物39之合成。 43
流程二十、化合物41之合成。 43
流程二十一、化合物42及43之合成 44
流程二十二、化合物45及48之合成。 47
流程二十三、化合物48及49之製備 47
流程二十四、前驅物51及52之製備 48
流程二十五、化合物53及54之製備 49
流程二十六、化合物56之合成 49
流程二十七、化合物60及61之合成策略 52
流程二十八、Rowland團隊之甲醯化條件 53
流程二十九、化合物55甲醯化反應之路徑示意圖 53

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