【作者】 周美云；

【导师】 郑文杰； 陈填烽；

【作者基本信息】 暨南大学 ， 生物化学与分子生物学， 2015， 博士

【摘要】 有机含硒化合物具有活性高、毒性低、生物体相容性好等优点,因而受到科技工作者的广泛关注。本论文通过比较富硒大蒜中主要有机含硒、含硫化合物的抗氧化活性,推断富硒大蒜可能具有比普通大蒜更强的生物活性;通过二硒醚化合物结构与生物活性的构效关系研究,阐明了极化率是影响该类化合物生物活性的重要因素;通过优化合成方法,获得了三个系列的有机含硒化合物,新化合物超过20个,其中V-7、VI-8两个化合物具有显著的抗肿瘤活性,并揭示了可能的机制。本研究有助于加强对天然有机硒化合物生物活性的认识,同时亦为高选择性诱导肿瘤细胞凋亡诊疗试剂的开发提供新的选择。1)研究并比较了大蒜新素、大蒜素、硒代大蒜新素对DPPH自由基的清除能力,发现抗氧化能力:大蒜素>硒代大蒜新素>大蒜新素。采用密度泛函B3LYP方法在6-311+G(d,p)基组下,计算了硒代大蒜新素、大蒜新素的离解焓、电离势以及电子亲合势。发现硒代大蒜新素的C-H离解焓、电离势均小于大蒜新素,而电子亲和势则略大于后者,说明无论从抽氢或是电子转移机理考量,硒代大蒜新素清除自由基的能力均要强于大蒜新素。采用B3LYP/6-311+G(d,P)基组计算了硒代大蒜素、大蒜素反应物、过渡态和产物的能量,得到大蒜素的分解能垒为67.0 k J/mol,而硒代大蒜素为63.0 k J/mol,说明硒代大蒜素比大蒜素更易分解,以此解释了硒代大蒜素难以分离得到的原因。2)合成了6个二硒醚类化合物,其中2个为未见文献报道的新化合物。通过MTT法初步研究了它们对人肝癌细胞Hep G2、人宫颈癌细胞He La及人正常肝细胞L02的增殖抑制作用,得出化合物III-2对肿瘤细胞的抑制活性最好,尤其是对He La细胞的IC50值为2.9μg/m L,表现出较好的应用潜力。采用B3LYP/6-311G*(d)基组,计算6个化合物主要原子的电荷、静电势、前线轨道、偶极矩、极化率等参数,明确了分子中的硒原子对于化合物性质所呈现出的核心作用;通过相关性分析,得出该类化合物的极化率与抗肿瘤活性成线性相关,并得到其线性方程,为进一步优化该类分子结构提供了理论依据。3)针对目前苯并硒唑类衍生物合成过程繁琐、催化剂难以回收利用等问题,本文制备了经济实用的炭负载纳米铜催化剂,采用一锅法合成了16个苯并硒唑类化合物,其中6个为新化合物。以吗啡啉与邻溴异硒氰酸酯为模板,系统地探讨了反应温度、时间、溶剂、碱等对反应的影响,优化了反应条件。考察了活性炭负载纳米铜催化剂的制备条件,获得了最佳的制备工艺。采用SEM、XRD表征了催化剂的结构和组成,发现真正的活性成分是Cu+离子,为该类催化剂的制备与优化提供了理论依据。4)设计、合成了7个硒唑吡啶类衍生物,其中6个化合物为首次合成。采用MTT法筛选了它们对人肝癌细胞Hep G2、人乳腺癌细胞MCF-7及人正常肝细胞L02的增殖抑制作用,其中化合物V-7对MCF-7细胞增殖的抑制能力最强,并对人正常肝细胞L02表现出低毒性,说明V-7具有良好的选择性。流式细胞术结果显示,化合物V-7可以引起MCF-7细胞中Sub-G1峰细胞百分数呈剂量效应增加,而S期细胞百分数无明显变化,说明化合物V-7是通过诱导细胞凋亡来抑制MCF-7细胞增殖的。DHE荧光强度检测肿瘤细胞内ROS含量的变化,表明化合物V-7能够作为一种自由基清除剂来清除细胞内的ROS而诱导细胞凋亡。化合物V-7具有进一步优化,发展成为治疗肿瘤药剂的潜力。5)设计、合成了一类结构新颖的亚甲基异硒脲衍生物(化合物VI-1-VI-8)。利用MTT法筛选了它们对5种细胞(人宫颈癌细胞He La、Caski、Si Ha,恶性黑色素毒瘤细胞A375及人正常肝细胞L02)的增殖抑制作用,得出化合物VI-8对肿瘤细胞的抑制活性最好,尤其是对He La细胞的IC50值为2.4μM。化合物VI-8的亲油性最好,并且具有最好细胞的吸收效率,且靶向目标为溶酶体。应用胞吞抑制剂确定了化合物VI-8是受Dynasore介导的脂筏胞吞作用进入He La细胞的。深入研究证实化合物VI-8通过促使线粒体断裂而导致肿瘤细胞凋亡,经westernbolting实验证实VI-8诱导肿瘤细胞凋亡是通过磷酸化通路来完成的。该研究为亚甲基异硒脲衍生物的合成、高选择性地抗肿瘤的诊疗试剂的开发提供了理论基础和应用选择。更多还原

【Abstract】 Organoselenium compounds have been paid extensive attention by scientists owing to high activity, low toxicity, and good biocompatibility. In this paper, we investigated the antioxidant activity of major Se-containing- and S-containing compounds in selenium-enriched garlic and inferred that selenium enriched garlic may have the stronger biological activity than ordinary one; based on the structure-activity relationship of diselenide compounds, we clarified that the polarization was an important factor to affect the biological activity of these bioactivities; in addition, we obtained a series of organoselenium including more than 20 new compounds by optimizing synthesis method, and found that two compounds had significant antitumor activity, and revealed the possible mechanism of antitumor. Our study contributed to the understanding of natural organic selenium compounds with biological activities, and provided a new choice for the development of anticancer therapeutics agent with high selectivity. 1) We investigated the DPPH free radical scavenging ability of allitridin, allicin and Se-Allitridin, and found that antioxidant capacity of allicin was the strongest, Se-allitridin was the second, allitridin was the weakest. It found that Se-allitridin has less C-H dissociation enthalpy and ionization potential, but slightly larger electron affinity than allitridin by calculating C-H dissociation enthalpies and ionization potential and electron affinity of the above-mentioned three compounds at the 6-311+G(D, P) level of theory in the gas phase, reflecting Se-allitridin with the stronger ability to scaveng free radicals than allitridin from either extracting hydrogen or electron transfer mechanism considerations. Decomposition barriers of allicin and Se-allicin was determined as 67.0, 63.0 k J/mol respectively by calculating the energy differences between their optimizing configuration and the corresponding transition states, which explained why Se-allicin was difficult to obtain. 2) Six diselenide compounds were synthesized, in which two compounds have not been reported in literature. A preliminary study on the inhibitory effect of six diselenide compounds on Hep G2, Hela and L02 cell was carried out by MTT method. These compounds were able to inhibit the growth of tumor cells in different degrees, in which the inhibitory activity of compound III-2 on Hela cells is the best with the IC50 value at 2.9 g/m L, exhibiting good potential for application. The key effect of selenium atoms in molecules on the bioactivity of their compounds was clarified by calculating atom charge, electrostatic potential, frontier orbital, dipole moments, and polarizability of these compounds at the B3LYP/6-311G*(d) level of theory in the gas phase. Through the correlation analysis, it found that polarization of the compounds with antitumor activity presented the linear correlation, providing a theoretical basis for the further optimization of the molecular structure. 3) The post processing in selenium azole derivatives synthesis is cumbersome and catalyst recycling after reaction was difficult until now. Aiming at these questions, copper catalyst supported by carbon was prepared and used to one pot synthesis of 16 benzene selenium azole compounds including 6 new compounds. With morpholine and adjacent bromine different selenium cyanate ester as reaction template, the paper systematically discussed the influences of reaction temperature, reaction time, reaction solvent, base on the reaction, and obtained the optimized condition. Preparation conditions of activated copper catalyst supported by carbonwere investigated to obtain the best preparation process of catalyst. SEM and XRD were employed to characterize the structure and composition of catalyst, identifying that Cu+ ion was the active component of the catalyst and providing theory basis for catalyst preparation and optimization. 4) Seven selenazolopyridine derivatives were designed and synthesized, in which six compounds were synthesized for the first time. Their in vitro anticancer activities were tested and the result showed that 2-(phenylamino) selenazolo [5,4-b] pyridine(PSe D, compound V-7) can highly inhibit human breast carcinoma MCF-7 cells, has low toxicity to an normal cell line L02. Compound V-7 can result in the increase of the proportion of apoptotic cells expressed a dose-dependent reflected by the Sub-G1 cell population, but no significant change of the S phase population, which referred that V-7 compound induce the apoptosis of MCF-7 cells. Further investigation on the intracellular mechanisms demonstrated that PSe D had a capability to scavenge intracellular reactive oxygen species(ROS) with the result of cell apoptosis. More potential drug for the treatment of tumor will be discovered through the further optimization of selenazolopyridine derivatives. 5) A novel series of methylene carbamimidoselenoate derivatives have been designed, synthesize and well characterized. Firstly the synthesized compounds(VI-1-VI-8) were screened against a panel of five cell lines to evaluate their in vitro anticancer activity by MTT assay. Compound VI-8 was distinguished from other compounds, which had a highest growth inhibitory effect on cancer cells, especially on human cervical cancer He La cells. Compound VI-8 was monitored to enter the cancer cells by targeting lysosome with high cellular uptake potency by fluorescence imaging technique. It confirmed that dynasore-mediated lipid raft endocytosis was mainly involved in the internalization of VI-8 in He La cells. Further investigation on intracellular mechanisms revealed that compound VI-8 was able to cause apoptosis in cancer cells by inducing mitochondrial dysfunction, with an elevated expression of phosphorylated p53. On the basis of these results, we suggests this novel series of organoselenium compounds synthesized may be candidates for further research as a kind of chemotherapeutic agents for cancer therapy.更多还原