【作者】 蔡双莲；

【导师】 汪秋安；

【作者基本信息】 湖南大学 ， 有机化学， 2019， 博士

【摘要】 黄酮类化合物有抗炎、抗癌和心脑血管保护等多种多样的生物学活性与药理作用,在化学、生物学与药物学领域均受到广泛关注。自然界不同来源的黄酮其结构与含量差别大,难以提取和纯化,同时,各种疾病治疗也要求不断设计和开发新药物,因此,开展各种新型黄酮衍生物的合成与生物活性筛选,对进一步研发高效低毒性药物先导化合物具有重要的研究意义和潜在经济价值。鉴于Mannich碱与羧酸基团是许多天然产物和药物中的重要活性结构单位,并且直接影响药物的溶解性、体内吸收部位以及生物利用度,本文开展了系列黄酮Mannich碱和黄酮羧酸（酯）衍生物的合成及其生物活性的研究,其中一些化合物具有良好的抗癌和抗菌作用。主要研究内容简述如下:1.首先以来源丰富且廉价的天然资源二氢黄酮柚皮苷为原料,经酸水解得到柚皮素,再经I₂/吡啶氧化脱氢制得芹菜素,然后在苄基保护黄酮羟基的条件下,利用DMDO氧化获得苄基保护黄酮醇化合物,最后,用Pd/C/H₂去苄基保护制备山奈酚等黄酮类天然产物。2.以芹菜素、木犀草素、二氢杨梅素和几种黄酮醇等黄酮化合物作为反应底物,与甲醛、二级胺（六氢吡啶、吡咯烷和吗啡啉等）在酸催化下发生Mannich反应,得到18种新的黄酮Mannich碱化合物。然后,将这些Mannich碱酸化成盐,制备得到13种黄酮Mannich碱盐酸盐。再分别以Mannich碱盐酸盐化合物为底物,与一级胺类化合物（氨基硫脲,4-甲基氨基硫脲,乙醇胺,异丙醇胺,乙二胺,丙二胺等）发生胺交换反应,获得22种新型黄酮一级胺Mannich碱的化合物。3.研究考察了Mannich反应和胺交换反应的合成条件和结果,通过实验数据与量子化学理论计算相结合,深入探讨发生胺基交换过程中的S_N2反应机理,从而揭示了氢键在反应中所起的关键作用。为了仔细考察氢键的复杂作用,采用DFT方法,利用Gaussian09程序对一系列胺分子和Mannich碱及其反应过程进行计算与模拟,在分子几何构型优化基础上,求得分子轨道、频率、键能与键级、自然键轨道与电荷分布等数据,并选择部分代表性体系,采用内禀反应坐标（intrinsic reaction coordinate,IRC）分析过渡态反应路径,观察其过渡态的结构与运动变化,比较氢键参数及其在反应过程中的变化特点,计算反应的活化能,以评估反应难易和程度。理论计算的结果充分说明和解释了实验现象,从而揭示胺基交换反应中氢键所起的作用,以及氢键对参与交换的胺分子的选择规律。4.采用MTT比色法和牛津杯法对所合成的黄酮Mannich碱衍生物和黄酮类化合物分别进行了抗癌细胞活性和抗菌活性测试,通过比较和分析,探讨Mannich碱的取代与活性的一些规律。抗癌测试结果表明,（1）取代基类型和位置对黄酮衍生物抗癌活性有明显的影响,如Mannich碱中N,N-二异丙基衍生物的活性都强于N,N-二甲基;（2）末端-CH₃有利于抗癌活性的提高;（3）含有多个末端-NH₂或-OH等极性基团则不利于抗癌活性;（4）黄酮类底物中的C环C-3原子上有羟基对抗HeLa、HCC1954、MCF-7等癌细胞增殖活性有帮助,但不利于抗SK-OV-3癌细胞增殖活性,而C-2和C-3之间的双键有利于抗癌细胞增殖作用;（5）黄酮B环羟基数目与抗癌活性并无明显正相关,但是在3’与4’位同时含有-OH的分子往往抗癌活性比较好。在抗菌活性方面,考察了化合物的抗大肠杆菌、抗铜绿假单胞菌、抗金黄色葡萄球菌和抗白色念珠菌活性,结果表明,（1）分子结构上有（甲）氨基硫脲、二丙基、吡咯烷基、哌啶（氢化吡啶）基的黄酮Mannich碱具有较好的抗菌活性,而有二甲基、吗啉基的黄酮Mannich碱相比黄酮类底物其活性并无改善;（2）多数情况下,酚羟基数目的增多也有利于提高抗菌活性。5.通过Williamson醚合成法制备了系列黄酮羧酸及其甲酯衍生物,并对这些化合物分别进行了体外抗肿瘤活性与抗菌活性的测试（所选菌株和癌细胞株与上述相同）。结果表明,黄酮羧酸酯衍生物的抗癌细胞增殖活性比相应黄酮类底物更高,而黄酮羧酸衍生物在多数情况下能提高黄酮类底物的抗菌活性。更多还原

【Abstract】 Flavonoids exhibit a variety of biological activities and pharmacological effects,such as anticancer,antioxidant,anti-inflammatory,antibacterial and cardiocerebrovascular protection.They have attracted extensive attention in the fields of chemistry,pharmacology and medicine.There are great differences in the structure and content of flavonoids from different sources in nature,so it is difficult to extract and purify them.On the other hand,it is also necessary to design and develop new drugs for the treatment of various diseases.Therefore,the synthesis and biological activity screening for various new flavonoid derivatives are of great significance and potential economic value for the further research and development of high-efficiency and low-toxicity drug lead compounds.In view of the fact that Mannich bases and carboxylic groups are important active units of many natural products and medicinal plants,and that they directly affect the solubility,absorption sites and bioavailability of drugs.In this paper,the synthesis and biological activity of a series of flavonoids Mannich base and flavonoid carboxylic acid（ester）derivatives have been studied.Many of the compounds have good anticancer and antibacterial effects.The main research contents are summarized as follows:1.Naringenin was obtained at first by acid hydrolysis of naringin and then reduced to apigenin by dehydrogenation using I₂/pyridine.After protecting the hydroxyl groups on flavonoids with benzyl group,DMDO was employed to oxidize the products to 5,7,4’-tribenzyl flavonol compounds.Finally,under the catalysis of Pd/C/H₂,the benzyl flavonoids were converted to kaempferol through debenzylation process.2.Eighteen secondary amine Mannich base derivatives of flavonoids were obtained by the acid-catalyzed Mannich reaction of flavonoid substrates,such as apigenin,kaempferol,quercetin,myricetin,luteolin and dihydromyricetin,with formaldehyde and some secondary amines（dimethylamine,diethylamine,dipropylamine,pyrrolidine,morpholine,N-methylpiperazine,hexahydropyridine,etc.）.These Mannich bases were then acidified to form salts,resulting in thirteen flavonoid Mannich bases.After that,twenty-two new Mannich base derivatives of flavonoids based on primary amine were obtained by using Mannich base hydrochloride derivatives 5,18,20,35,47 as substrates to undergo amine exchange reaction with primary amines such as thiosemicarbazone,4-methyl thiosemicarbazone,ethanolamine,isopropanolamine,ethylenediamine,and propylenediamine,etc.3.The optimum conditions were found and established for Mannich reaction and amine exchange reaction.The S_N2 reaction mechanism of amine exchange reaction was well understood in detail based both on the experimental data and on the quantum chemistry theory.The experimental data and phenomena indicate that the amine-exchange reaction is in line with the S_N2 reaction characteristics,and it is found that hydrogen bonding plays a key role in the reaction.In order to investigate the complex effects of hydrogen bonds on the reaction,the density functional theory（DFT）method was used to calculate and simulate a series of amine molecules,Mannich bases and their reaction processes by using the Gaussian09 program.After optimizing the molecular geometry,the frequency,molecular orbital,natural bond orbital and charge distribution,bond energy and bond order are calculated.The structure and reaction path of transition states for some selected representative systems were analyzed by using intrinsic reaction coordinates（IRC）.The hydrogen bond parameters and their variation characteristics in the reaction process were also investigated.Moreover,the activation energies were calculated to evaluate the difficulty and extent of the reaction.The results of these theoretical calculations well explain and support the experimental phenomena,thus revealing the crucial role of hydrogen bonds in the exchange reaction of amino groups and the preference patterns of hydrogen bonds on the amine molecules involved in the exchange.4.The antitumor and antibacterial activities of the synthesized flavonoid Mannich base derivatives and flavonoid substrates were tested by MTT colorimetry and Oxford cup method in vitro.Some patterns and rules of substitution and activity of Mannich base are discussed by means of comparison and analysis.（1）In terms of anti-tumor properties,the type and position of substituents on flavonoid molecules have some effects on their activity intensity.For example,the anti-cancer activity of N-diisopropyl derivatives in Mannich base is stronger than that of N-dimethyl derivatives.（2）The presence of-CH₃ at the end of the substituent is beneficial to the improvement of anticancer activity.（3）But the presence of polar groups such as terminal-NH₂ or-OH is not conducive to the improvement of anticancer activity.（4）In flavonoid substrates,hydroxyl on C3 atom of C ring is helpful for the anti-HeLa activity,the anti-HCC1954 activity and the anti-MCF-7 activity,but not for the anti-SK-OV-3 activity.The double bond between C2 and C3 was also found to be beneficial to the anticancer activity.（5）No positive correlation was found between the number of hydroxyl groups in B ring of flavonoids and their anticancer activity.In general,however,molecules containing 3’-OH and 4’-OH tend to have good anticancer activity.When it comes to antibacterial properties（against Escherichia coli,Pseudomonas aeruginosa,Staphylococcus aureus,and Candida albicans）,（1）Flavonoid Mannich bases with amino residues such as（Methyl-）thiosemicarbazide,dipropyl,pyrrolidine and hydropyridine in molecular structures tend to have good antibacterial activity,while the antibacterial activities of those flavonoid derivatives with dimethyl and morpholine groups are not improved compared with their corresponding flavonoid substrates.（2）In most cases,the increase in the number of phenolic hydroxyl groups is also beneficial to the improvement of antibacterial activity,but the situation of myricetin and quercetin is not exactly consistent with this.5.A series of flavonoid carboxylic acid derivatives and their methyl ester derivatives were prepared by Williamson ether synthesis,and their antitumor and antibacterial activities were tested in vitro（the strains and cancer cell lines selected are the same as those mentioned above）.The results showed that the anticancer activity of flavonoid carboxylic ester derivatives was higher than that of corresponding flavonoid substrates.In terms of antibacterial activity,the carboxyl group can improve the antibacterial activity of the compounds in most cases.更多还原