本文選題：金屬卟啉　切入點：微波輔助合成　出處：《廣東工業(yè)大學(xué)》2014年博士論文

【摘要】：卟啉及金屬卟啉類化合物是自然界中分布較為廣泛的一類化合物,由于其獨特的結(jié)構(gòu)和性能、特別是與腫瘤細胞有特殊的親和力而在腫瘤細胞內(nèi)有聚集作用,在臨床醫(yī)學(xué)領(lǐng)域具有作為抗腫瘤藥物及相關(guān)診斷試劑潛在的應(yīng)用前景。G-四鏈體DNA是富含鳥嘌呤堿基重復(fù)序列的DNA,在金屬離子存在條件下4個鳥嘌呤堿基之間通過環(huán)狀氫鍵的互聯(lián)作用循環(huán)排列連接而形成的DNA二級結(jié)構(gòu)。端粒及部分可形成四鏈體的原癌基因序列在細胞生長、增殖、凋亡及腫瘤細胞形成過程中都具有重要的作用,從而成為當(dāng)下研究最熱門的小分子抗腫瘤藥物靶點之一。目前已有大量關(guān)于卟啉類化合物與各種四鏈體相互作用的報道,也得到了很多有意義的結(jié)論。然而,目前研究的四鏈體主要集中在端粒四鏈體,對于也可形成四鏈體構(gòu)型的原癌基因序列卻鮮有報道�；诖�,本文分別用常規(guī)及微波輔助合成方法制備了一系列卟啉及金屬卟啉化合物,通過體外活性篩選研究了目標(biāo)化合物對不同腫瘤細胞株及正常細胞株生長的抑制作用,進一步考查了幾種金屬卟啉和原癌基因c-myc四鏈體的分子識別機制。具體獲得了以下幾個方面的結(jié)果： 1.5,10,15,20-四對甲氧基苯基卟啉銅(Ⅱ)的合成及其與c-myc G4DNA的分子識別 第一步采用對甲氧基苯甲醛和吡咯為原料,以丙酸為溶劑反應(yīng)制得了5,10,15,20-四對甲氧基苯基卟啉(TMOPP);第二步以TMOPP和乙酸銅為原料,在DMF中反應(yīng),粗產(chǎn)物過硅膠柱分離純化得到5,10,15,20-四對甲氧基苯基卟啉銅(II)(CuTMOPP)。中間產(chǎn)物TMOPP及目標(biāo)產(chǎn)物CuTMOPP經(jīng)ESI-MS質(zhì)譜、’HNMR、13C NMR、紫外-可見光譜、熒光光譜及紅外光譜等方法進行了表征。常規(guī)與微波輔助合成方法制得的TOMPP的產(chǎn)率分別為21.6%和22.7%,CuTMOPP的產(chǎn)率分別為63.1%和64.1%。微波輔助合成目標(biāo)化合物的產(chǎn)率略有提高、差別并不明顯,但微波輔助合成技術(shù)大大縮短了反應(yīng)時間,特別是第二步中常規(guī)方法要反應(yīng)5h,而微波輔助合成只需要20min,明顯提高了反應(yīng)效率。 進一步采用紫外-可見光譜、圓二色光譜、FRET熔點實驗及PCR-Stop擴增實驗等方法研究了該化合物和c-myc G4DNA相互作用的機制。結(jié)果發(fā)現(xiàn)目標(biāo)化合物溶液加入c-myc G4DNA以后,紫外-可見光譜當(dāng)中出現(xiàn)明顯的減色和紅移,說明CuTMOPP可以結(jié)合c-myc G4DNA;從c-myc G4DNA溶液中加入目標(biāo)化合物前后的圓二色光譜來看,加入目標(biāo)化合物后,CD信號的強度稍微減弱,表明CuTMOPP對c-myc G4DNA的構(gòu)型有細微但可以觀察到的影響,說明該化合物很可能以靜電結(jié)合的方式與c-myc G4DNA相互作用；從PCR-Stop擴增實驗結(jié)果來看,目標(biāo)化合物可以進一步抑制c-myc相關(guān)酶的復(fù)制。 2.5-[4-(4-溴代丁氧基)苯基]-10,15,20-三對甲氧基苯基卟啉銅(Ⅱ)的合成及其與c-myc G4DNA的分子識別 第一步采用對羥基苯甲醛、對甲氧基苯甲醛及吡咯作為原料,以丙酸為溶劑反應(yīng),制得5-對羥基苯基-10,15,20-三對甲氧基苯基卟啉(p-HTMOPP);第二步以p-HTMOPP和1,4-二溴丁烷為原料,以DMF作為溶劑進行反應(yīng),制得5-[4-(4-溴代丁氧基)苯基]-10,15,20-三對甲氧基苯基卟啉(p-BrTMOPP);第三步以p-BrTMOPP和乙酸銅為原料,以DMF為溶劑制得目標(biāo)產(chǎn)物5-[4-(4-溴代丁氧基)苯基]-10,15,20-三對甲氧基苯基卟啉銅(Ⅱ)(p-CuBrTMOPP),各步粗產(chǎn)物均過柱純化。采用1H NMR、13C NMR、ESI-MS質(zhì)譜、紫外-可見光譜,熒光光譜及紅外光譜等方法對中間產(chǎn)物及目標(biāo)化合物進行了表征。采用常規(guī)方法合成p-HTMOPP、p-BrTMOPP和p-CuBrTMOPP所得到的產(chǎn)率分別為5.1%、84.7%和77.9%；采用微波輔助合成法所得的產(chǎn)率分別為5.73%、89.2%和66.7%。兩種合成方法比較,產(chǎn)率差別不大,但微波輔助合成明顯簡化了反應(yīng)過程,縮短了反應(yīng)時間,提高了反應(yīng)效率。 采用MTT法對目標(biāo)化合物p-CuBrTMOPP對不同腫瘤細胞株的生長抑制作用進行了研究。通過IC50值可以看出,以順鉑作為陽性對照,該化合物對于乳腺癌細胞株(MCF-7)有很強的抑制作用,而對于其他幾個腫瘤細胞株的抑制作用不明顯。 采用紫外-可見光譜、熒光光譜、圓二色光譜、熱變性熔點實驗及PCR-Stop擴增實驗等研究了目標(biāo)化合物p-CuBrTMOPP和c-myc G4DNA的分子識別機制。從紫外-可見光譜中加入四鏈體后化合物吸收峰出現(xiàn)的減色、紅移,熒光光譜中出現(xiàn)的熒光強度減弱,說明p-CuBrTMOPP與c-myc G4DNA之間發(fā)生了相互結(jié)合；加入目標(biāo)化合物前后,c-myc G4DNA溶液的CD光譜中,CD信號幾乎沒有變化,表明目標(biāo)化合物對c-myc G4DNA的構(gòu)型沒有明顯的影響。熱變性熔點實驗發(fā)現(xiàn)目標(biāo)化合物在一定程度上可以穩(wěn)定c-myc G4DNA的四鏈體構(gòu)型,不過效果不是很明顯,這也與CD光譜實驗結(jié)論是一致的。結(jié)合幾種實驗結(jié)果說明該化合物可能以靜電結(jié)合或溝槽結(jié)合方式和c-myc G4DNA發(fā)生相互作用,最終導(dǎo)致拓撲異構(gòu)酶的復(fù)制受到抑制。 3.5-[4-(4-溴代丁氧基)苯基]-10,15,20-三對甲氧基苯基卟啉鋅(Ⅱ)的合成及其與c-myc G4DNA的分子識別 5-[4-(4-溴代丁氧基)苯基]-10,15,20-三對甲氧基苯基卟啉鋅(p-ZnBrTMOPP)的合成和p-CuBrTMOPP相比,除了第三步中乙酸銅改為乙酸鋅以外其他條件完全相同。對p-ZnBrTMOPP進行表征,結(jié)果和預(yù)期相符。合成目標(biāo)化合物采用常規(guī)方法和微波輔助方法對產(chǎn)率影響較小。 采用MTT法對目標(biāo)化合物p-ZnBrTMOPP對不同腫瘤細胞株的抑制作用進行研究,從IC50值可以看出,以順鉑作為陽性對照,該化合物對所篩選的幾個腫瘤細胞株的抑制作用并不明顯,而對其他腫瘤細胞株是否有抑制作用,還有待于進一步篩選。 在對目標(biāo)化合物p-ZnBrTMOPP和c-myc G4DNA的相互作用機制研究發(fā)現(xiàn),此類化合物也可以結(jié)合并且穩(wěn)定c-myc的四鏈體構(gòu)型,但是對其構(gòu)型影響不是很明顯,很可能是通過外部堿基堆積發(fā)生作用的,功能上來看也可以抑制相關(guān)DNA的復(fù)制,具體的生物機制影響還在研究中。
[Abstract]:Porphyrin and metalloporphyrin is a class of compounds widely distributed in nature, because of its unique structure and properties, especially with the tumor cells have a special affinity and accumulation in tumor cells, in the field of clinical medicine as antitumor drugs and potential diagnostic applications of.G- four chain DNA is rich in guanine repeat sequence of DNA, in the presence of metal ions between the 4 conditions of guanine interactions through hydrogen bond ring cycle arrangement formed by connecting the DNA two level structure. The original cancer gene sequence of telomere and part can form a chain of four body in cell growth, proliferation, apoptosis and tumor cells are formed an important role in the process, which has become one of the target of anti-tumor small molecules present study the most popular drugs. There are a large number of porphyrin compounds and various four The Interaction Chain reported, has also been a lot of meaningful conclusions. However, the four chain of current research mainly concentrated in the four chain for telomere, proto oncogene sequences can also form a chain of four body configurations are rarely reported. Based on this, this paper respectively by conventional and microwave assisted synthesis method for a a series of porphyrins and metalloporphyrins were synthesized, the in vitro study of inhibitory effect of target compounds on the growth of different tumor cell lines and normal cell lines, and further investigated the molecular recognition mechanism of several kinds of metal porphyrin and oncogene c-myc four chain specific body. Some results are obtained as follows:
Synthesis of 1.5,10,15,20- four pair of methoxy phenyl porphyrin copper (II) and molecular recognition of c-myc G4DNA
The first step in using p-methoxybenzaldehyde and pyrrole as raw materials, with propionic acid as solvent were prepared by the reaction of 5,10,15,20- four methoxy phenyl porphyrin (TMOPP); the second step is to TMOPP and copper acetate as raw material, reaction in DMF, the crude product with silica column purified 5,10,15,20- four methoxy phenyl porphyrin copper (II) (CuTMOPP). The intermediate product of TMOPP and target product CuTMOPP by ESI-MS mass spectrometry, 13C 'HNMR, NMR, UV Vis spectroscopy, fluorescence spectroscopy and infrared spectroscopy and other methods were investigated. The conventional and microwave assisted synthesis method prepared TOMPP production rates were 21.6% and 22.7%, the yield of CuTMOPP respectively 63.1% and 64.1%. microwave assisted synthesis of the target compounds yield increased slightly, the difference is not obvious, but the microwave assisted synthesis technology has greatly shortened the reaction time, especially the conventional method in the second step reaction to 5h, while the micro wave assisted synthesis of only 2 0min, significantly improved the efficiency of the reaction.
The UV Vis spectra, two circular dichroism, FRET melting experiments and PCR-Stop amplification experiment method to study the mechanism of the interaction between G4DNA and c-myc compounds. The results showed that the target compounds were added to c-myc G4DNA, UV Vis spectroscopy in obvious hypochromism and red shift, indicating that CuTMOPP can be combined with c-myc G4DNA from c-myc; G4DNA solution adding target compounds before and after round two color spectrum, with the target compound, CD signal intensity is slightly reduced, showed the effect of subtle but can be observed on the c-myc CuTMOPP G4DNA configuration, the compound is likely to combine with c-myc G4DNA electrostatic interaction; from PCR-Stop amplification experimental results show that the target compounds can further inhibit c-myc enzymes related to replication.
Synthesis of 2.5-[4- (4- brominated Ding Yangji) phenyl]-10,15,20- three pair of methoxy phenyl porphyrin copper (II) and its molecular identification with c-myc G4DNA
The first step in using the p-hydroxybenzene formaldehyde, p-methoxybenzaldehyde and pyrrole as raw materials, with propionic acid as solvent reaction to prepare 5- hydroxybenzoic -10,15,20- three methoxy phenyl porphyrin (p-HTMOPP); the second step is to p-HTMOPP and 1,4- dibromobutane with DMF as solvent for reaction, preparation of 5-[4- (4- bromo Ding Yangji) phenyl]-10,15,20- three methoxy phenyl porphyrin (p-BrTMOPP); the third step is to p-BrTMOPP and copper acetate as raw material, solvent to prepare the target product with 5-[4- DMF (4- bromide Ding Yangji) phenyl]-10,15,20- three methoxy phenyl porphyrin copper (II) (p-CuBrTMOPP), each step of rough the products were purified by 1H. NMR, 13C NMR, ESI-MS mass spectrometry, UV Vis spectroscopy, fluorescence spectroscopy and infrared spectroscopy and other methods of intermediates and target compounds were characterized by conventional methods. The synthesis of p-HTMOPP, p-BrTMOPP and p-CuBrTMOPP obtained yield The yields were 5.1%, 84.7% and 77.9% respectively. The yields obtained by microwave-assisted synthesis were 5.73%, 89.2% and 66.7%. two, respectively. The yields were not very different, but microwave-assisted synthesis significantly simplified the reaction process, shortened the reaction time and improved the reaction efficiency.
The target compounds p-CuBrTMOPP on different tumor cell growth inhibition was investigated by MTT method. As can be seen by IC50, with cisplatin as a positive control, the compound for breast cancer cell line (MCF-7) has a strong inhibitory effect, and the inhibitory effect of several other tumor cell lines is not obvious.
UV Vis spectra, fluorescence spectra, circular dichroism two, thermal denaturation melting experiment and PCR-Stop amplification experiment of target compounds p-CuBrTMOPP and G4DNA c-myc. The molecular recognition mechanism from UV Vis spectra with four peaks after the chain compounds of hypochromicity decreased fluorescence intensity appeared redshift, fluorescence spectrum the description of the interaction of p-CuBrTMOPP and c-myc G4DNA; after the addition of target compounds, the CD spectra of c-myc G4DNA solution, the CD signal is almost no change in configuration of the target compounds showed that c-myc G4DNA had no obvious effect. The thermal denaturation melting experiments found that four chain structure of target compounds can be stabilized by c-myc G4DNA in a certain degree but the effect is not obvious, this also with CD spectrum is consistent with the conclusion. The experimental results show that the combination of several compounds in electrostatic binding The interaction between the trench or the grooves and the c-myc G4DNA eventually leads to the inhibition of the replication of topoisomerase.
Synthesis of 3.5-[4- (4- brominated Ding Yangji) phenyl]-10,15,20- three pair of methoxy phenyl porphyrin zinc (II) and its molecular identification with c-myc G4DNA
5-[4- (4- bromo phenyl]-10,15,20- Ding Yangji) three methoxy phenylporphyrin zinc (p-ZnBrTMOPP) compared to the synthesis and p-CuBrTMOPP, in addition to the third step in copper acetate changed the conditions of other than zinc acetate identical. Characterization of p-ZnBrTMOPP, results and expectations. The target compound was synthesized by conventional method and microwave assisted method has little effect on the yield.
MTT method was used to study the inhibitory effect of compound p-ZnBrTMOPP on different tumor cell lines, can be seen from the IC50 value to cisplatin as a positive control, the inhibitory effects of compounds on several tumor cell lines were not obvious, but for other tumor cell lines have inhibitory effect, needs to be further screening.
Found in the study of the mechanism of interaction between p-ZnBrTMOPP and c-myc G4DNA of target compounds, such compounds can also be combined with the four chain structure and stability of c-myc, but the impact on the configuration is not very obvious, probably through the external base stacking effect, the point of the function can also inhibit DNA replication, also affected the biological research the specific mechanism.

【學(xué)位授予單位】：廣東工業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：O627;R914.5

【參考文獻】

相關(guān)期刊論文 前10條

1 劉云,徐同寬,肖德寶,張軍,孫玉娥;四苯基卟啉的催化合成和微波合成研究[J];北京輕工業(yè)學(xué)院學(xué)報;1998年04期

2 黃興強;樊艷玲;吳天驕;吳臘梅;陳連清;;微波輻射下催化合成四(對硝基苯基)卟啉[J];江西師范大學(xué)學(xué)報(自然科學(xué)版);2009年06期

3 陳祖林;楊樺;廖海洋;潘英俊;肖衛(wèi)東;;ALA誘導(dǎo)結(jié)腸癌細胞內(nèi)PPIX產(chǎn)生和光動力學(xué)療法殺傷效應(yīng)研究[J];重慶醫(yī)學(xué);2009年05期

4 丁靜;孫舒婷;張諾;韓顏顏;陳欣;魏琴;;卟啉類顯色劑在重金屬離子分析中的研究及應(yīng)用[J];分析測試技術(shù)與儀器;2008年01期

5 張紅芬,潘景浩;卟啉及金屬卟啉的應(yīng)用[J];化學(xué)教育;2005年04期

6 ;Synthesis and anticancer activities of porphyrin induced anticancer drugs[J];Chinese Chemical Letters;2007年11期

7 劉杰,許東暉,梅文杰,蒲含林,黃錦汪,計亮年;水溶性卟啉及其系列金屬化合物的合成、抗癌活性及其作用機制研究[J];高等學(xué)�；瘜W(xué)學(xué)報;2001年09期

8 康敬萬,吳海霞,盧小泉,蘇碧泉,卓琳;水溶性鋅卟啉配合物的合成、表征及其與CTDNA的作用[J];高等學(xué)�；瘜W(xué)學(xué)報;2005年06期

9 師同順，孫浩然，曹錫章，陶建忠;四（對－硝基）苯基卟啉錳配合物的光譜電化學(xué)性質(zhì)[J];高等學(xué)�；瘜W(xué)學(xué)報;1994年07期

10 王淇;王成蹊;孫福強;賀麗敏;吳瓊;梁炳煥;邱永彬;梅文杰;;碳化硅管中微波輔助制備甲硝唑半抗原[J];廣東藥學(xué)院學(xué)報;2012年01期

，

本文編號：1728835