微波辅助金属卟啉化合物的合成及其与c-myc G4 DNA的分子识别

发布时间：2018-04-10 00:21

本文选题：金属卟啉　切入点：微波辅助合成　出处：《广东工业大学》2014年博士论文

【摘要】：卟啉及金属卟啉类化合物是自然界中分布较为广泛的一类化合物,由于其独特的结构和性能、特别是与肿瘤细胞有特殊的亲和力而在肿瘤细胞内有聚集作用,在临床医学领域具有作为抗肿瘤药物及相关诊断试剂潜在的应用前景。G-四链体DNA是富含鸟嘌呤碱基重复序列的DNA,在金属离子存在条件下4个鸟嘌呤碱基之间通过环状氢键的互联作用循环排列连接而形成的DNA二级结构。端粒及部分可形成四链体的原癌基因序列在细胞生长、增殖、凋亡及肿瘤细胞形成过程中都具有重要的作用,从而成为当下研究最热门的小分子抗肿瘤药物靶点之一。目前已有大量关于卟啉类化合物与各种四链体相互作用的报道,也得到了很多有意义的结论。然而,目前研究的四链体主要集中在端粒四链体,对于也可形成四链体构型的原癌基因序列却鲜有报道。基于此,本文分别用常规及微波辅助合成方法制备了一系列卟啉及金属卟啉化合物,通过体外活性筛选研究了目标化合物对不同肿瘤细胞株及正常细胞株生长的抑制作用,进一步考查了几种金属卟啉和原癌基因c-myc四链体的分子识别机制。具体获得了以下几个方面的结果： 1.5,10,15,20-四对甲氧基苯基卟啉铜(Ⅱ)的合成及其与c-myc G4DNA的分子识别第一步采用对甲氧基苯甲醛和吡咯为原料,以丙酸为溶剂反应制得了5,10,15,20-四对甲氧基苯基卟啉(TMOPP);第二步以TMOPP和乙酸铜为原料,在DMF中反应,粗产物过硅胶柱分离纯化得到5,10,15,20-四对甲氧基苯基卟啉铜(II)(CuTMOPP)。中间产物TMOPP及目标产物CuTMOPP经ESI-MS质谱、’HNMR、13C NMR、紫外-可见光谱、荧光光谱及红外光谱等方法进行了表征。常规与微波辅助合成方法制得的TOMPP的产率分别为21.6%和22.7%,CuTMOPP的产率分别为63.1%和64.1%。微波辅助合成目标化合物的产率略有提高、差别并不明显,但微波辅助合成技术大大缩短了反应时间,特别是第二步中常规方法要反应5h,而微波辅助合成只需要20min,明显提高了反应效率。进一步采用紫外-可见光谱、圆二色光谱、FRET熔点实验及PCR-Stop扩增实验等方法研究了该化合物和c-myc G4DNA相互作用的机制。结果发现目标化合物溶液加入c-myc G4DNA以后,紫外-可见光谱当中出现明显的减色和红移,说明CuTMOPP可以结合c-myc G4DNA;从c-myc G4DNA溶液中加入目标化合物前后的圆二色光谱来看,加入目标化合物后,CD信号的强度稍微减弱,表明CuTMOPP对c-myc G4DNA的构型有细微但可以观察到的影响,说明该化合物很可能以静电结合的方式与c-myc G4DNA相互作用；从PCR-Stop扩增实验结果来看,目标化合物可以进一步抑制c-myc相关酶的复制。 2.5-[4-(4-溴代丁氧基)苯基]-10,15,20-三对甲氧基苯基卟啉铜(Ⅱ)的合成及其与c-myc G4DNA的分子识别第一步采用对羟基苯甲醛、对甲氧基苯甲醛及吡咯作为原料,以丙酸为溶剂反应,制得5-对羟基苯基-10,15,20-三对甲氧基苯基卟啉(p-HTMOPP);第二步以p-HTMOPP和1,4-二溴丁烷为原料,以DMF作为溶剂进行反应,制得5-[4-(4-溴代丁氧基)苯基]-10,15,20-三对甲氧基苯基卟啉(p-BrTMOPP);第三步以p-BrTMOPP和乙酸铜为原料,以DMF为溶剂制得目标产物5-[4-(4-溴代丁氧基)苯基]-10,15,20-三对甲氧基苯基卟啉铜(Ⅱ)(p-CuBrTMOPP),各步粗产物均过柱纯化。采用1H NMR、13C NMR、ESI-MS质谱、紫外-可见光谱,荧光光谱及红外光谱等方法对中间产物及目标化合物进行了表征。采用常规方法合成p-HTMOPP、p-BrTMOPP和p-CuBrTMOPP所得到的产率分别为5.1%、84.7%和77.9%；采用微波辅助合成法所得的产率分别为5.73%、89.2%和66.7%。两种合成方法比较,产率差别不大,但微波辅助合成明显简化了反应过程,缩短了反应时间,提高了反应效率。采用MTT法对目标化合物p-CuBrTMOPP对不同肿瘤细胞株的生长抑制作用进行了研究。通过IC50值可以看出,以顺铂作为阳性对照,该化合物对于乳腺癌细胞株(MCF-7)有很强的抑制作用,而对于其他几个肿瘤细胞株的抑制作用不明显。采用紫外-可见光谱、荧光光谱、圆二色光谱、热变性熔点实验及PCR-Stop扩增实验等研究了目标化合物p-CuBrTMOPP和c-myc G4DNA的分子识别机制。从紫外-可见光谱中加入四链体后化合物吸收峰出现的减色、红移,荧光光谱中出现的荧光强度减弱,说明p-CuBrTMOPP与c-myc G4DNA之间发生了相互结合；加入目标化合物前后,c-myc G4DNA溶液的CD光谱中,CD信号几乎没有变化,表明目标化合物对c-myc G4DNA的构型没有明显的影响。热变性熔点实验发现目标化合物在一定程度上可以稳定c-myc G4DNA的四链体构型,不过效果不是很明显,这也与CD光谱实验结论是一致的。结合几种实验结果说明该化合物可能以静电结合或沟槽结合方式和c-myc G4DNA发生相互作用,最终导致拓扑异构酶的复制受到抑制。 3.5-[4-(4-溴代丁氧基)苯基]-10,15,20-三对甲氧基苯基卟啉锌(Ⅱ)的合成及其与c-myc G4DNA的分子识别 5-[4-(4-溴代丁氧基)苯基]-10,15,20-三对甲氧基苯基卟啉锌(p-ZnBrTMOPP)的合成和p-CuBrTMOPP相比,除了第三步中乙酸铜改为乙酸锌以外其他条件完全相同。对p-ZnBrTMOPP进行表征,结果和预期相符。合成目标化合物采用常规方法和微波辅助方法对产率影响较小。采用MTT法对目标化合物p-ZnBrTMOPP对不同肿瘤细胞株的抑制作用进行研究,从IC50值可以看出,以顺铂作为阳性对照,该化合物对所筛选的几个肿瘤细胞株的抑制作用并不明显,而对其他肿瘤细胞株是否有抑制作用,还有待于进一步筛选。在对目标化合物p-ZnBrTMOPP和c-myc G4DNA的相互作用机制研究发现,此类化合物也可以结合并且稳定c-myc的四链体构型,但是对其构型影响不是很明显,很可能是通过外部碱基堆积发生作用的,功能上来看也可以抑制相关DNA的复制,具体的生物机制影响还在研究中。
[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.

【学位授予单位】：广东工业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：O627;R914.5

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