Studies on the synthesis and bioactivity of natural prenylated flaconoid and flavonoid mannich base derivatives

学校代号 10532 分类号 学 密 号 LB2012012 级 PhD Thesis Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Derivatives 学位申请人姓名 NGUYEN VAN SON (阮文山) 培 养 单 位College of Chemistry and Chemical Engineering 导师姓名及职称 Professor Wang Qiu An 学 科 专 业Organic chemistry 研 究 方 向Organic synthesis of products 论文提交日期 2015 year month 学校代号：10532 学 号：LB2012012 密 级： 湖南大学博士学位论文 异戊烯基黄酮类和黄酮 Mannich 碱衍生 物的合成与生物活性研究 学位申请人姓名： NGUYEN VAN SON (阮文山) 导师姓名及职称： 汪秋安教授 培 养 单 位： 化学化工学院 专 业 名 称： 有机化学 论文提交日期: 2015 年 月 论文答辩日期： 2015 年 月 28 日 答辩委员会主席： 安德烈教授 　　　　　 Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Derivatives By NGUYEN VAN SON M.S (Vinh University of Education, Vietnam) 2008 A dissertation submitted in partial satisfaction of the Requirements for the degree of Doctor of Science in Organic Chemistry In the Graduate School of Hunan University Supervisors Professor WANG QIU AN April 25, 2015 湖 南 大 学 学位论文原创性声明 本人郑重声明：所呈交的论文是本人在导师的指导下独立进行研究所取得的研究 成果。除了文中特别加以标注引用的内容外，本论文不包含任何其他个人或集体已经 发表或撰写的成果作品。对本文的研究做出重要贡献的个人和集体，均已在文中以明 确方式标明。本人完全意识到本声明的法律后果由本人承担。 作者签名： 日期： 年 月 日 学位论文版权使用授权书 本学位论文作者完全了解学校有关保留、使用学位论文的规定，同意学校保留并 向国家有关部门或机构送交论文的复印件和电子版，允许论文被查阅和借阅。本人授 权湖南大学可以将本学位论文的全部或部分内容编入有关数据库进行检索，可以采用 影印、缩印或扫描等复制手段保存和汇编本学位论文。 本学位论文属于 1、保密□，在 年解密后适用本授权书。 2、不保密□。 （请在以上相应方框内打“√”） 作者签名： 日期： 年 月 日 导师签名： 日期： 年 月 日 I PhD Thesis 摘 要 黄酮类是一类广泛分布于植物界的酚类次级代谢产物，是天然产物的重要组成部 分。这类化合物具有多种生物活性和有效的医疗应用，如抗癌和抗肿瘤活性、抗炎和 抗病毒活性、抗菌和抗真菌活性、抗心血管疾病、酶抑制活性、抗自由基和抗氧化活 性。 异戊烯黄酮是一类独特的天然黄酮类化合物，其特征是在黄酮骨架上存在着异戊烯 基侧链。C-异戊烯化的黄酮可以增强其对 p-糖蛋白的亲和力和对细胞膜的通透性，可 以显著提高黄酮类化合物的生物活性。具有显著抗癌活性的天然异戊烯基黄酮类，可 以作为日益增长的保健食品的先导化合物和人类疾病治疗新的药物来源。然而，黄酮 类和异戊烯基类黄酮在自然界植物中的含量低且来源有限，这些因素严重影响其生物 活性价值的开发和利用。因此，黄酮类和异戊烯基类黄酮的化学合成将解决其实用性 难题。另一方面，黄酮类化合物在药物研发中存在着溶解性差、生物利用度低等缺点, 限制了它们的应用。Mannich 反应是合成 β-胺基酮和邻胺基酚类等含氮有机化合物的 有效方法, 被广泛应用于天然产物和有机药物分子的合成。 含氮的 Mannich 碱结构单 元是一类重要的药理活性基团, 它可以有效提高化合物的生物活性、生物利用度和水 溶性。因此进行黄酮 Mannich 碱衍生物的合成与生物活性研究具有重要意义。本论文 围绕异戊烯基黄酮类和黄酮 Mannich 碱衍生物的合成与生物活性进行了系列研究。 1、淫羊藿素（1a）的全合成。本论文以 2,4,6-三羟基苯乙酮和 4-羟基苯甲酸为原料， 通过 Baker-Venkatarama 反应、选择性苄基或甲氧基甲基保护、二甲基过氧丙酮 （DMDO）氧化、O-异戊烯基化反应、微波促进的 Claisen 重排和脱保护基等 步反应， 以 23%的总产率合成了具有重要意义的生物活性物质 8-异戊烯基类黄酮淫羊藿素。对 该合成的关键反应步骤微波促进的 Claisen 重排进行了探讨。 2、首次全合成了 Sophoflavescenol (1b) 、 Flavenochromane C（2b）和 Citrusinol (3b) 等三种有良好药理活性如细胞毒性、抗癌和治疗性功能勃起障碍的天然异戊烯基 黄酮类或异戊烯基侧链成环的黄酮类化合物。全合成是以 2,4,6-三羟基苯乙酮和取代苯 甲醛为初始原料，分别通过甲氧甲基保护、羟醛缩合、环合反应、DMDO 氧化、O-异 戊烯化、微波促进的 Claisen 重排、脱保护基、异戊烯基环合作用和 DDQ 脱氢等反应 步骤。1b、2b 和 3b 的总产率分别为 23%，17%和 16%。其中最为关键的步骤是从 5O-异戊烯基黄酮通过微波促进的 Claisen 重排得到 8-异戊烯基黄酮类。 3、异戊烯基黄酮类淫羊藿素（1a）在微波条件下和甲酸反应以 89％的收率得到另 一种天然产物 β-去水淫羊藿黄素（2c）。以 1a 和 2c 为底物，分别与甲醛、各种仲胺 在酸性醇溶液中进行 Mannich 反应，合成得到 18 个 位胺甲基化的 Mannich 碱衍生物 3c-11c 和 12c-20c。对这些化合物采用标准 CCK-8 法对宫颈癌 Hela 细胞系的细胞毒性 II Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Detivatives 潜力进行了测试，以抗癌药物顺铂为阳性对照，结果表明绝大部分化合物对 Hela 细胞 表现出中等强度的细胞毒性。 4、山奈素（3,5,7-三羟基-4'-甲氧基黄酮, 1d)是对许多人肿瘤细胞系有抗癌活性的黄 酮类天然产物，我们以来源丰富且廉价的柚皮苷为原料首次通过半合成得到山奈素。 以山奈素与各种仲胺和甲醛进行 Mannich 反应，得到 种山奈素 Mannich 碱衍生物 2d10d。胺甲基化的位置，优先发生在黄酮环上的 C-6 和 C-8 位置。所有合成化合物以标 准 CCK-8 法测试其对宫颈癌 Hela 细胞系的细胞毒活性，结果表明，所有的目标化合物 表现出对 Hela 细胞的中度到良好的细胞毒性（IC50 值为 12.48-70.52 μmol•L-1），化合 物 1d，2d，5d-9d 及 10d 的细胞毒性效果分别为优于或等于阳性对照药物顺铂。 5、通过使用微波加热方法水解黄酮苷类化合物橙皮苷（1a），柚皮苷（1b）和芦丁 （1c）中的糖基，分别得到相应的黄酮苷元橙皮素（2e），柚皮素（2f）和槲皮素 （2g）。研究了微波加热水解过程中的影响因素，如微波的功率，反应温度和照射时 间的反应产率的影响，优化了反应条件。黄酮苷元的产率为 90-95%。研究结果表明微 波可以大大加快黄酮苷的水解速率，缩短反应时间，并提高了黄酮苷元的产率。优化 的反应条件是：微波功率 500-600 W，照射时间 30-45 分钟，反应温度为 80-90 摄氏度。 微波协助的方法具有高效省时、低碳环保、产品纯度和产率更高的优点。 6、本论文共合成异戊烯基黄酮类以及黄酮 Mannich 碱衍生物 55 个，其中有 26 个是 未见文献报道的新化合物，所合成的化合物结构已经核磁共振氢谱 (1H NMR), 核磁共 振碳谱 (13C NMR), 质谱 (MS) 或 (HRMS), 红外光谱 (IR) 等波谱方法进行了结构表征。 关键词：异戊烯基黄酮类；全合成； Mannich 碱；微波协助 Clasien 重排；生物 活性。 III Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Detivatives Abstract Flavonoids are phenolic secondary metabolites which are widely distributed throughout the plant kingdom They have been isolated from various plant, and are a class of importance natural products These compounds have a variety biological activities and potent medical applications, such as anti-tumor and anti-cancer activity, antibacterial and antiviral activity, anti-cardiovascular disease, enzyme inhibitory activity, anti-free radical and antioxidant activity, etc Prenylated flavonoids are a unique class of naturally occurring flavonoids characterised by the presence of a prenylated side chain on the flavonoid skeleton C-prenylation of flavonoids can enhance binding affinity toward p-glycoprotein and increase ability to permeate cell membranes, which can significantly improve the biological activity of flavonoids Thus, prenylated flavonoids show promise as lead compounds for the development of nutraceuticals in plants and as new pharmacological agents for the treatment of human diseases On the other hand, natural resources of flavonoids and prenylflavonoid are limited due to the low contents in the plants kingdom They were negatively influenced their further bioactivity evaluation Therefore, chemical synthesis of flavonoids and prenylflavonoid will be a very important alternative approach for addressing the problem of its availability Mannich reaction is an effective method for the synthesis of β-amino ketones and phenols such as O-amino nitrogen compounds, it is widely used in the synthesis of natural products and organic drug molecules Mannich base structure containing amine moiety is an important class of pharmacological active groups, which can effectively improve the biological activity, bioavailability, and water-soluble of compounds Therefore, the synthesis of bioactive flavonoids Mannich base derivatives has great significance In this thesis, the synthesis and bioactivity of prenylated flavonoids natural products and flavonoid Mannich base derivatives have been studied The novel total synthesis of icaritin (1a), a naturally occurring with importance bioactive 8-prenylflavonoid, was performed via a reaction sequence of steps including Baker-Venkataraman reaction, chemoselective benzyl or methoxymethyl protection, dimethyldioxirane (DMDO) oxidation, O-prenylation, Claisen rearrangement and deprotection, starting from 2,4,6-trihydroxyacetophenone and 4-hydroxybenzoic acid in overall yields of 23% The key step was Claisen rearrangement under microwave irradiation The first total synthesis of Sophoflavescenol (1b), Flavenochromane C (2b) and Citrusinol (3b), three naturally occurring prenylated or prenyl-cyclizen flavonoids have IV PhD Thesis importance activities such as cytotoxicity against some cancer cell lines and treatment for erectile dysfunction, were achieved through methoxymethyl protection, aldol condensation, cyclization, DMDO oxidation, O-prenylation, microwave assistance Claisen rearrangement, deprotection, cyclization of prenyl group and DDQ dehehydrogenation, starting from 2,4,6trihydroxyacetophenone and substituted benzaldehydes with overall yields 23%, 17% and 16%, respectively The key step of the synthetic route is regioselective microwave assistance Claisen rearrangement formed 8-prenylated flavonoids from 5-O-prenylflavonoids Preylated flavonoid icaritin (1a) upon treatment with formic acid under microwave assistance gave another natural product β-anhydroicaritin (2c) in good yield (89%) Based on Mannich reaction of 1a or 2c with various secondary amines and formaldehyde, two series eighteen new 6-aminomethylated flavonoids Mannich base derivatives 3c-11c and 12c-20c were synthesized Furthermore, their cytotoxic potential against cervical carcinoma Hela cell line were evaluated by the standard CCK-8 assay, the results showed that most of the target compounds exhibit moderate to potent cytotoxicity against Hela cells comparable with the positive control cis-Platin (DDP) Kaempferide (3,5,7-trihydroxy-4’-methoxyflavone, 1d), a naturally occurring flavonoid with potent anticancer activity in a number of human tumour cell lines, was first semisynthesized from naringin Based on Mannich reaction of kaempferide with various secondary amines and formaldehyde, nine novel kaempferide Mannich base derivatives 2d10d were synthesized The aminomethylation occurred preferentially in the position at C-6 and C-8 of the A-ring of kaempferide All the synthetic compounds were tested for antiproliferative activity against cervical carcinoma Hela cell line by the standard CCK-8 assay, the results showed that all target compounds exhibited moderate to potent cytotoxicity against Hela cells with IC50 values of 12.48-70.52 μmol/L, and compounds 1d, 2d, 5d, 6d, 7d, 8d, 9d and 10d were better than or equal to the activities of positive control cis-Platin (DDP) The efficient hydrolysis of flavonoid glycosides hesperidin (1e), naringin (1f) and rutin (1g) to corresponding flavonoid aglycone hesperetin (2e), naringnin (2f) and quercetin (2g) respectively by employing microwave irradiation method was studied The test was designed to investigate the influential factors of the hydrolysis process under a microwave irradiation such as power of microwave, reaction temperature and irradiation time The optimized parameters are: power 500-600 W, irradiation time 30-45 min, reaction temperature 80-90 oC The yields of flavonoid aglycone are 90-95% The results show that microwave assistance can greatly accelerate the hydrolysis rate of flavonoid glycosides, shorten the reaction time, increase the yield of flavonoid aglycone and product purities Fifty-five prenylated flavonoids and flavonoids Mannich base derivatives were IV Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Detivatives synthesized totally in this thesis, and twenty-six of them were new compounds The structures of all the synthesized compounds have been confirmed by IR, 1H NMR, 13C NMR and MS or HRMS techniques Keywords: Preylated Flavonoid; Total Synthesis; Microwave Irradiation; Claisen Rearrangement; Biological Activity IV PhD Thesis Contents 学位论文原创性声明与学位论文版权使用授权书 I 摘 要 II Abstract……………………………………………………………………………………………… IV List of Schemes……………………………………………………………………………X List of Figures……………………………………………………………………………XI List of Tables……………………………………………………………………………XII List of Symbols and Abbreviations………………………………… XIII Chapter Introduction 1.1 Overview of flavonoids 1.1.1 The structure of flavonoids and related natural products 1.1.2 Pharmacological activities of flavonoids 1.2 Prenylated flavonoids 1.3 Synthesis of flavonoids 1.4 Claisen rearrangement 1.5 Baker-Venkatarama reaction 1.6 DMDO in organic synthesis 10 1.7 Mannich reaction 12 1.8 Hela cell line 13 1.9 Assay for antiproliferative activity 15 Chapter Total Synthesis of Icaritin via Microwave Assistance Claisen Rearrangement17 2.1 Introduction 17 2.2 Experimental 18 2.2.1 General 18 2.2.2 Synthesis of 2-hydroxy-4,6-bis(benzyloxy)acetophenone .19 2.2.3 Synthesis of 4-methoxybenzoyl chloride 19 2.2.4 Synthesis of 5,7-bis(benzyloxy)-2-(4-methoxyphenyl)flavone 19 2.2.5 Synthesis of 5,7-bis(benzyloxy)-3-hydroxy-2-(4-methoxyphenyl)- .20 flavone .20 2.2.6 Synthesis of kaempferide 20 2.2.7 Synthesis of 5-hydroxy-3,7-bis(methoxymethoxy)-2-(4-methoxy- 21 phenyl)flavone 21 2.2.8 Synthesis of 5-(3-methylbut-2-enyloxy)-3,7-bis(methoxymethoxy)- 21 IV Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Derivatives rwsh-141203-465 #5 RT: 0.99 AV: T: + c EI Full ms [ 49.50-500.50] NL: 1.13E6 382 100 327 95 90 85 80 75 70 65 84 Relative Abundance 60 55 135 50 45 40 35 85 30 25 383 326 20 56 325 218 328 57 324 15 10 77 70 69 50 152 107 92 98 100 121 136 148 164 179 191 205 150 200 367 465 314 219 220 242 252 269 297 298 281 299 250 300 329 353 351 350 384 422 385 412 450 m/z EIMS of compound 8c 1H 466 467 423 400 NMR spectrum of compound 9c - 116 - 500 PhD Thesis 13C NMR spectrum of compound 9c HRMS compound 9c - 117 - Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Derivatives 1H NMR spectrum of compound 10c 13C NMR spectrum of compound 10c - 118 - PhD Thesis EIMS of compound 10c 1H NMR spectrum of compound 11c - 119 - Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Derivatives 13C NMR of compound 11c HRMS of compound 11c 1H NMR spectrum of compound 12c - 120 - PhD Thesis 13C NMR spectrum of compound 12c rwsh-141111-425-3 #5 RT: 1.03 AV: NL: 8.52E5 T: + c EI Full ms [ 49.50-600.50] 327 100 95 90 85 80 382 75 70 65 Relative Abundance 60 55 50 45 40 35 324 135 380 30 25 328 20 15 383 58 77 152 10 70 100 107 245 164 179 133 148 150 314 207 354 410 281 298 208 100 367 200 242 353 246 269 250 300 350 384 388 400 425 426 450 500 550 m/z EIMS of 12c - 121 - 600 Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Derivatives 1H NMR spectrum of compound 13c 13C NMR spectrum of compound 13c - 122 - PhD Thesis 1H NMR spectrum of compound 14c 13C NMR spectrum of compound 14c - 123 - Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Derivatives 1H NMR spectrum of compound 15c 13C NMR spectrum of compound 15c - 124 - PhD Thesis 1H NMR o spectrum f compound 16c 13C NMR spectrum of compound 16c - 125 - Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Derivatives 1H NMR spectrum of compound 17c 13C NMR spectrum of compound 17c - 126 - PhD Thesis 1H NMR o spectrum f compound 18c 13C NMR spectrum of compound 18c - 127 - Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Derivatives HRMS of 18c 1H NMR spectrum of compound 19c - 128 - PhD Thesis 13C NMR spectrum of compound 19c HRMS of compound 19c - 129 - Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Derivatives 1H NMR spectrum of compound 20c 13C NMR spectrum of compound 20c - 130 - ... inhibition in mitochondrial inner membrane -5- [56] [57] [58] Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Derivatives 1.3 Synthesis of flavonoids... research -7- Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Derivatives Some of the variations of the aliphatic Claisen rearrangement offer stereoselective... shorten the reaction time, increase the yield of flavonoid aglycone and product purities Fifty-five prenylated flavonoids and flavonoids Mannich base derivatives were IV Studies on the Synthesis and