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 5 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 5 月

论文答辩日期：

2015 年 5 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。

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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 8 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 6 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 9 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.
1. The novel total synthesis of icaritin (1a), a naturally occurring with importance
bioactive 8-prenylflavonoid, was performed via a reaction sequence of 8 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.
2. 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.
3. 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).
4. 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).
5. 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.
6.

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 1 Introduction............................................................................................................1
1.1 Overview of flavonoids .................................................................................................1
1.1.1 The structure of flavonoids and related natural products....................................1
1.1.2 Pharmacological activities of flavonoids ............................................................3
1.2 Prenylated flavonoids ....................................................................................................4

1.3 Synthesis of flavonoids..................................................................................................6
1.4 Claisen rearrangement ...................................................................................................8
1.5 Baker-Venkatarama reaction .........................................................................................9
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 2 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 Detivatives

2-(4- methoxyphenyl)flavone. ...................................................................................22
2.2.9 Synthesis of 5-hydroxy-3,7-bis(methoxymethoxy)-2-(4-methoxy-..................21
phenyl)-8-3-methylbut-2-enyl)flavonol (10a) and 5-hydroxy-3,7-bis- .....................21
(methoxymethoxy)-2-(4-methoxyphenyl)-6-(1,1-dimethylallyl)flavonol. ................21

2.2.10 Synthesis of icaritin.........................................................................................23
2.3 Result and discussion ..................................................................................................23
2.4 Summary......................................................................................................................27
Chapter 3 The First Total Synthesis of Sophoflavescenol, Flavenochromane C and
Citrusinol .................................................................................................................................28
3.1 Introduction .................................................................................................................28
3.2 Experimental................................................................................................................29
3.2.1 General ..............................................................................................................29
3.2.2 Synthesis of 2-hydroxy-4,6-bis(methoxymethoxy)acetophenone ....................30
3.2.3 Synthesis of 2’-hydroxyl-4,4’,6’-trimethoxymethoxylchalcone.......................30
3.2.4 Synthesis of 5,4’,7-trimethoxymethylflavone...................................................31
3.2.5 Synthesis of 3-hydroxy-5,4’,7-trimethoxymethylflavone ................................31
3.2.6 Synthesis 3,5-hydroxyl-4’,7-dimethoxymethylflavone. ...................................32
3.2.7 Synthesis of 3,4’,7-tris-O-methoxymethylkaempferol .....................................32
3.2.8 Synthesis of 5-O-Prenyl-3,4’,7-tris-O-methoxymethylkaempferol. .................32
3.2.9 Synthesis of 5-hydroxy-8-Prenyl-3,4’,7-tris-O-methoxymethyl- .....................33
kaempferol .................................................................................................................33
3.2.10 Synthesis of 7,8-(2,2-dimethyl-2H-pyran)-5,4’-dihydroxyflavone ................33
3.2.11 Synthesis of sophoflavescenol ........................................................................34
3.2.12 Synthesis of 8-prenylkaempferol ....................................................................34
3.2.13 Synthesis of flavenochromane C ....................................................................35
3.2.14 Synthesis of 4'-​desmethyl-​β-​anhydroicaritin ..................................................35
3.2.15 Synthesis of citrusinol.....................................................................................35
3.3 Result and discussion ..................................................................................................36
3.4 Summary......................................................................................................................45
Chapter 4 Synthesis of Icaritin and β-Anhydroicaritin Mannich Base Derivetives and
Their Cytotoxic Activities on Hela cells ................................................................................46
4.1. Introduction ................................................................................................................46
4.2 Experimental................................................................................................................49
4. 2.1 General .............................................................................................................49

4.2.2 Synthesis of β-anhydroicaritin ..........................................................................49
IV


PhD Thesis

4.2.3 General experimental procedure for Mannich base derivatives........................49
4.3 Assay for cytotoxic activity.........................................................................................56
4.4 Results and discussion .................................................................................................56
4.5 Summary......................................................................................................................59
Chapter 5

Sythesis of Kaempferide Mannich Base Derivatives and Their

Antiproliferative Activity on Hela Cells................................................................................60
5.1 Introdution ...................................................................................................................60
5.2 Experimental................................................................................................................61
5.2.1 General methods ...............................................................................................61
5.2.2 Synthesis of rhoifolin ........................................................................................61
5.2.3 Synthesis of acacetin.........................................................................................62
5.2.4 Synthesis of 2-(4-methoxyphenyl)-5,7-bis(benzyloxy)flavone ........................62
5.2.5 Synthesis of 3-hydroxy-2-(4-methoxyphenyl)-5,7-bis(benzyloxy)-.................63
flavone........................................................................................................................63
5.2.6 Synthesis of kaempferide ..................................................................................64
5.3 General experimental procedure for synthesis of Mannich base derivatives. .............63
5.4 Assay for antiproliferative activity ..............................................................................66
5.5 Results and discussion .................................................................................................67
5.6 Summary......................................................................................................................70
Chapter 6 Promoting Hydrolysis of Flavonoid Glycosides by Microwave Irradiation...71
6.1 Introduction .................................................................................................................71

6.2 Experimental................................................................................................................72
6.2.1 General experimental procedures .....................................................................72
6.2.2 Microwave assistance hydrolysis of hesperidin, naringin and rutin. ................72
6.3 Results and discussion .................................................................................................73
Conclusion ................................................................................................................................77
References .................................................................................................................................79
Publication ................................................................................................................................94
Acknowledgements ...................................................................................................................95
附录 A 合成化合物一览表......................................................................................................96
附录 B 化合物谱图 ..................................................................................................................99

List of Schemes
Scheme 1.1 Synthesis of icaritin .................................................................................................7

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Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Detivatives

Scheme 1.2 Reagents and conditions: Synthesis of icaritin and β-anhydroicaritin ....................7
Scheme 1.3 Synthesis of (+)-isoamijiol and (+)-dolasta-1(15),7,9-triesn-14-o1probes..8
Scheme 1.4 Synthesis of 5-epi-vibsanin in E .............................................................................9
Scheme 1.5 Synthesis of xanthohumol and soxanthohumol……………………………..9
Scheme 1.6 Baker-Venkatarama rearrangement synthetic flavonoids…………………10
Scheme 1.7 The mechanism DMDO in the synthesis of β-D-glucopyranoside and α-Dmannopyrano side. ....................................................................................................................11
Scheme 1.8 The mechanism of DMDO in the synthesis flavonols ..........................................11
Scheme 1.9 The mechanism of Mannich reaction ....................................................................13
Scheme 1.10 Synthesis of 8-aminomethylated derivatives of oroxylin....................................13
Scheme 2.1 The novel total synthetic route of icaritin..............................................................18
Scheme 2.2 Strategy for the regioselective syntheses of 6-(1,1-dimethylallyl)- and 8-(3,3dimethylally)-flavonoid…………………………………………………………..21

Scheme 3.1. The novel total synthetic routes of sophoflavescenol, flavenochromane C and
citrusinol…………………………………………………………………………….30
Scheme 3.2 The mechanism of DMDO in the synthesis flavonol……………………...38
Scheme 4.1 Synthesis of icaritin and β-anhydroicaritin Mannich base derivatives…49
Scheme 4.2 Active phenol ortho-hydrogen via the enol form followed by the Mannich
reaction……………………………………………………………………………………60
Scheme 4.3 The mechanism of the Mannich reaction of β-anhydroicaritin…………...61
Scheme 5.1 Synthesis of kaempferide Mannich base derivatives…………………… 64
Scheme 6.1. Synthesis routes of flavonoid aglycones from flavonoid glycosides by microwave
irradiation hydrolysis ................................................................................................................76

List of Figures

IV


PhD Thesis

Figure 1.1 Basic structure of flavonoids .....................................................................................2
Figure 1.2 Chemical structures of the most common flavonoid subclasses. ..............................3
Figure 1.3 Scanning electron micrograph of an apoptotic Hela cell…………………..14
Figure 1.4 Multiphoton fluorescence image of cultured Hela cells with a fluorescent protein
targeted to the Golgi apparatus (orange), microtubules (green) and

counterstained for

DNA (cyan). ………………………………………………….…… 14
Figure 1.5 Typical cell survival curve…………………………………........................15
Figure 1.6 Exemple of the plate arrangement and color development………………...15
Figure 1.7 Cell viability detection mechanism with CCK-8…………………………...15

Figure 2.1 1H NMR spectrum of 1a……………………………………………………..26
Figure 2.2 13C NMR spectrum of 1a……………………………………………………27
Figure 3.1 1H NMR spectrum of 2b…………………………………………………….42
Figure 3.2 13C NMR spectrum of 2b……………………………………………………42
Figure 3.3 1H NMR spectrum of 1b…………………………………………………….43
Figure 3.4 13C NMR spectrum of 1b…………………………………………………...43
Figure 3.5 1H NMR spectrum of 3b…………………………………………………….44
Figure 3.6 13C NMR spectrum of 3b……………………………………………………44
Figure 4.1 The dose-response curve for CCK-8 assay of compounds compounds 2c, 8c, 11c,
16c, 19c, 20c and cis-Platin on Hela cells proliferation…………………………..48
Figure 5.1 The dose-response curve for CCK-8 assay of compounds 1d, 2d, 9d and cis-Platin
on Hela cell proliferation……………………………………………………..69
Figure 6.1 Graph of speed optimization of time, temperature, capacity of the microwave
irradiation in the reaction to hydrolysis the glycosidic bond of hesperidin
…………………………………………………………………………………………….75
Figure 6.2 Graph of speed optimization of time, temperature, capacity of the microwave
irradiation in the reaction to hydrolysis the glycosidic bond of naringin...75
Figure 6.3 Graph of speed optimization of time, temperature, capacity of the microwave
irradiation in the reaction to hydrolysis the glycosidic bond of rutin…….76

List of Tables

IV


Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Detivatives

Table 1.1 Biological activities of some prenylflavonoids...........................................................5
Table 3.1 Comparison of the NMR spectroscopic data of natural and synthetic flavonoid
1b………………………………………………………………………………39

Table 3.2 Comparison of the NMR spectroscopic data of natural and synthetic flavonoid
2b………………………………………………………………………………40
Table 3.3 Comparison of the NMR spectroscopic data of natural and synthetic flavonoid
3b………………………………………………………………………………41
Table 4.1 Half-inhibitory concentration [IC50 (μM] of compounds 1a, 2c-20c on Hela
cells……………………………………………………………………………………….47
Table 5.1 Half-inhibitory concentration [IC50 (μM)] of compounds 1d-10d on Hela cell line 69
Table 6.1 The yields of flavonoid aglycone hesperetin (2e), naringnin (2f) and quercetin (2g)
from

corresponding

flavonoid

glycosides

by

microwave

irradiation

hydrolysis…………………………………………………………………………………74

List of Symbols and Abbreviations

IV


PhD Thesis

µL, mL, L, µM

Microliter, milliliter, liter, micromole

pH

Potential of Hydrogen

min

Minute

%

Percent

MS

Mas spectrometry

s, d, t, q, b, m

Singlet, doublet, triplet, quartet, broad, multiplet

IR

Infrared spectroscopy

C NMR

Carbon Nuclear Magnetic Resonance

H NMR

Proton Nuclear Magnetic Resonance

HRMS

High-resolution mass spectrometry

ESI-MS

Electrospray ionisation mass spectrometry

EI-MS

Electron ionized mass spectrometry

Hz

Hertz

mp

Melting points

δ

Shifts

ppm

Parts per million

DMSO

Dimethylsulfoxyd

CDCl3

Chloroform

IC50

Half maximal inhibitory concentration

Ar

Aryl

Bn

Benzyl

DQQ

2,3-Dichloro-5,6-dicyano-1,4-benzoquinone

Hela

Human cervical cancer

MCF-7

Human breast cancer

MDA-MB-453

Androgen-responsive human breast carcinoma cell line

HL-60

Human acute promyelonic leukemia

LLC

Lewis lung carcinoma

A549 cells

Adenocarcinomic human alveolar basal epithelial cells

IV


PhD Thesis

Chapter 1 Introduction
1.1 Overview of flavonoids

Flavonoids are naturally occurring polyphenolic metabolites distributed throughout the
plant kingdom and found in substantial amounts in fruits, vegetables, grains, nuts, seeds, tea,
and traditional medicinal herbs

[1-3].

Within individual plants, flavonoids occur in every part

but are usually concentrated in the leaves and flowers [4]. Flavonoids are edible plant pigments
responsible for much of the coloring in nature. They play an important role in plant
metabolism, for instance as growth regulators and protect against ultraviolet light, oxidation
and heat. Plant-eating insects are deterred by their bitter taste. However, their bright colours
also help attract certain other insects to facilitate pollination.
Flavonoids were discovered firstly by Albert Szent-Györgyi, one of the most important
chemists from the start of the twentieth century. He received the Nobel Prize in 1937 for his
discovery and description of vitamin C. Szent-Györgyi discovered the flavonoids while he
was working on the isolation of vitamin C [5].

1.1.1 The structure of flavonoids and related natural products
The flavonoids are a very large and varied group of plant substances. However, they all
share the same basic chemical structure. The basic flavonoid structure is the flavan nucleus,
containing 15 carbon atoms arranged in three rings (C6-C3-C6), which are labeled as A, B and
C. Flavonoid are themselves divided into six subgroups: flavones, flavonols, flavanols,
flavanones, isoflavones, and anthocyanins, according to the oxidation state of the central C
ring as shown in Fig. 1.1. Their structural variation in each subgroup is partly due to the
degree and pattern of hydroxylation, methoxylation, prenylation, or glycosylation. Some of
the most common flavonoids include quercetin, a flavonol abundant in onion, broccoli, and
apple; catechin, a flavanol found in tea and several fruits; naringenin, the main flavanone in
grapefruit; cyanidin-glycoside, an anthocyanin abundant in berry fruits (black currant,
raspberry, blackberry, etc.); and daidzein, genistein and glycitein, the main isoflavones in

soybean [6]. Since a phenol group is always bound to one of the benzene rings, the flavonoids,
together with the phenolic acids and the non-flavonoid polyphenols, belong to the larger
group of polyphenols.
Six sub-classes can be distinguished, in which there are many bonds that are unique for
the individual substances. These substances differ from each other in the number of hydroxyl
groups they contain, how they are ordered in three dimensions and the extent to which these

-1-


Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Derivatives

groups are ‘taken’. This results in a large variety of flavonoids, which usually have a broad
range of different biochemical and physiological properties.

Fig. 1.1 Basic structure of flavonoids

Flavones and flavonols are the substrates for a range of modification reactions, including
glycosylation, methylation and acylation [7-9]. In plants, flavonoid aglycones occur in a variety
of structural forms. For convenience, the rings are labeled A, B, and C. The flavonoid
aglycons all consist of a benzene ring (A) condensed with a six-members rings (C) wich in the
2-position carries a phenyl ring (B) as substituent as shown in Fig. 1.2. The six-membered
ring condensed with the benzene ring is ether a γ-pyrone (favaonoids and flavonones) or its
dihydroderi vative (favaonols and flavonones) the position of the benzenoid substituent
divides the flavonoids (3-position). Flavonols differ from flavanones by a hydroxyl group in
the 3-position and a C2-C3 double bond. Authocyanidines are closely related to the
flavonoids. They differ from the latter in the C-ring, wich in authocyanidines is open, but their
biological properties are similar.
Flavonoids are often hydroxylated in positions 3, 5, 7, 3’, 4’ and 5’. Methyl ethers and
acectyl esters of the alconol groups are known to occur in nature when glycosides are formed,

the glycosidic linkage is normally located in positions 3 or 7 and the carbonhydrate can be Lrhamnose, D-glucose, glucorhamnose, galactose or arabinose [10-12]. Flavonoids can be further
divided into flavonols, flavones, flavanols, flavanones, anthocyanidins, and isoflavonoids
based on the saturation level and opening of the central pyran ring as shown in Fig. 1.2 [13-15].

-2-


PhD Thesis

O

O

OH

O

O

O
Flavonoid
(colourless)

O
Flavone
(yellow)

Isoflavonol
O

O

O

O

O
OH
O

Catechin
(colourless)

3'
2'
1
B 4'
8 O
7
2
1'
5'
6'
A
C 3
OH
6
5
4
O

Flavono-3-ol
(yellow)

OH
HO

OH

OH

Isoflavonol
(colourless)

IIsoflavonol

O

chalchone
5

O+

O
Authocyanidre
(red, blue, violet)

7

2'

3'

4'

5'
-

Quercetin

OH

OH

-

OH

OH

Kaempkerol

OH

OH

-

-

OH

-

Myricetin

OH

OH

-

OH

OH

OH

Morin

OH

OH

OH

-

OH

-

Fig. 1.2 Chemical structures of the most common flavonoids subclasses. The lower part of the figure shows
the generic structure of flavon-3-ols and some representative compounds where the hydroxyl groups of ring
B are shown.

1.1.2 Pharmacological activities of flavonoids
Flavonoids have been shown to have a wide range of biological and pharmacological
activities in in vitro studies. Examples include: anti-inflammatory
cytotoxicity to HeLa cells
anti-diarrheal activities

[22],

[26].

anti-microbial (antibacterial)

[23],

[16-20],

antifungal

anti-allergic
[24],

antiviral

[21],
[25],

Flavonoids have also been shown to inhibit topoisomerase

enzymes [27,28] and to induce DNA mutations in the mixed-lineage leukemia (MLL) gene in in
vitro studies

[29]

.

Flavonoids are regarded as safe and easily obtainable, making them ideal

candidates for cancer chemoprevention or associated agents in clinical treatment

[30-32].

Almost all artificial agents currently being used in cancer therapy are highly toxic and
produce severe damage to normal cells [33,34]. The ideal anticancer agent would exert minimal
adverse effects on normal tissues with maximal capacity to kill tumor cells and/or inhibit

-3-


Studies on the Synthesis and Bioactivity of Natural Prenylated Flavonoids and Flavonoid Mannich Base Derivatives

tumor growth [35,36]. The lack of substantial toxic effects for long-term therapies and inherent
biological activity of flavonoids make them ideal candidates for new therapeutics

[37,38].

Indeed, flavonoids have been shown to reveal cytotoxic activity toward various human cancer
cells with little or no effect on normal cells, and this fact has stimulated large interest in
developing of potential flavonoid-based chemotherapeutics for anticancer treatment [39,40].
Flavonoids increase the antioxidant enzyme activity, remove or reduce oxygen free
radicals and lipid peroxides and anti-aging effects achieved through

[41].

Studies have shown

that Epimedium flavonoids can improve the function of the neuroendocrine system of aged
rats with anti-aging effect. This is antioxidant capacity from its electronic capabilities for
hydrogen or for the phenolic hydroxyl groups. Numerous studies show that the hydroxy group
on the ring B is flavonoids exert anti-oxidation and removal of free radicals of the main active
site. The number of phenolic hydroxyl groups on the B ring of flavonoids was directly affects
their antioxidant activity [42]. Within a certain range, is proportional to the antioxidant activity
of flavonoids and phenolic hydroxyl number. This may be related to the number of flavonoids
phenolic hydroxyl hydrogen bond formation, the number of active radicals and the stability of
the free radicals and other factors [43].

1.2 Prenylated flavonoids
Prenylated flavonoids or prenylflavonoids are a sub-class of flavonoids. They are widely
distributed throughout the plant kingdom. The natural distribution and structural variation of
prenylated flavonoids have been reviewed by Barron and Ibrahim [44] in 1996. They are given
in the list of adaptogens in herbalism. Chemically they have a prenyl group attached to their
flavonoid backbone. It is usually assumed that the addition of hydrophobic prenyl groups
facilitate attachment to cell membranes. Prenylation may increase the potential activity of its
original flavonoid.
Within the flavonoid class of natural products the prenylated sub-class is quite rich in
structural variety and pharmacological activity. They are exists in many natural medicinal

plants. The presence, in different forms, of the isoprenoid chain can lead to impressive
changes in biological activity, mostly attributed to an increased affinity for biological
membranes and to an improved interaction with proteins. Molecules, such as xanthohumol
and sophoraflavanone G, while being very structurally simple, show numerous
pharmacological applications and are ideal candidates for SAR aimed to the discovery of new
drugs [45].

-4-


PhD Thesis

Flavonoid compounds of isopentenyl ether compounds can effectively promote into the
biological cells membranes more easily absorbed by the organism. Examples include: antibacterial, anti-cancer, lowering blood pressure, a wide range of physiological activity, antiinflammatory, anti-HIV, etc as shown in Table 1.1.
Table 1.1 Biological activities of some prenylflavonoids

Active molecules and source
Xanthoangelol and 4-hydroxy derricin from
Angelica keinskei Koidrumi
Macarangin from M. denticulata 6,8diprenyleriodictiol, dorsmanin C and

Biological and/or pharmacological
activity

lit

Antibacterial activity [against Gram(+) pathogenic bacteria]

[46]

Antiulcer
Antioxidant activity

[47]
[48]

dorsmanin F
Synthetic analogues of metabolites from

Aromatase inhibition

[49]

Broussonetia papyrifera
Artelastin and similar compounds from

Citotoxicity (against three human

Artocarpus

tumour cell lines)

Prenyflavonoids from leaves of Macaranga

Cyclooxygenase-1 (COX-1) and

conifera

COX-2 inhibition

Known prenylated flavonoids from stem
bark of Artocarpus kemando. Xanthoangelol
2’,4,4’-trihydroxychalcone, 6- and 8-prenyl
eriodictiol from liquorice.
5,7-Dihydroxy-6,8-diprenylflavonoids from

DNA strand scission activity and
chemopreventive

[54]

(rec-assay)
Isoliquiritigenin
HIV-inhibition

[55]

Herpes simplex type 1 (HSV-1)

alba

inhibition

Synthetic prenylated flavonoids

[52]

Induction activity of DNA damage

Leachianone G from root bark of Morus

Morus alba

[51]

[53]

Monotes africanus

Kuraridin and kurarinon from root bark of

[50]

Tyrosinase inhibition
Electronic transfer 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
The flavonoids are formed in plants and participate in the light-dependent phase of
photosynthesis during which they catalyze electron transport

[59].

They are synthesized from

the aromatic amino acids, pheny-lalamine and tyrosine, together with acetate units

[60].

Phenylalamine and tyrosine are converted to cinnamic acid and parahydroxy-cinnamic acid,
respectively, by the action of pheny-lalamine and tyrosine ammonia lyases [61]. Cinnamic acid
(or parahydroxycinnamic acid) condenses with acetate units to form the cinnamoyl structure
of the flavonoids (Fries rearrangement). A variety of phenolic acids, such as caffeic acid,
ferulic acid, and chlorogenic acid, are cinnamic acid derivatives. There is then alkalicatalyzed condensation of an ortho-hydroxyacetophenone with a benzaldehyde derivative
generating chalcones and flavonones as shown in Fig. 1.2, as well as a similar condensation
of an ortho- hydroxyacetophenone with a benzoic acid derivative (acid chloride or anhy[60].

dride), leading to 2-hydroxyflavanones and flavones
anthocyanidins has been described in detail by Dhar

[62].

The synthesis of chalcones and

Biotransformation of flavonoids in
[63].

the gut can release these cinnamic acid (phenolic acids) derivatives

Flavonoids are

complex and highly evolved molecules with intricate structural variation. In plants, they
generally occur as glycosylated and sulfated derivatives.
Icaritin is a native compound from Epimedium Genus, has many pharmacologi-cal and
biological activities, such as antiosteoporosis activity and estrogen regulation. The synthesis
of icaritin via an eight-step strategy including Houben-Hoesch acylation, one-pot AlgarFlynn-Oyamada reaction and europium-promoted pre-nylation etc., from anhydrous
phloroglucin with 4.2% overall yield was obtained as shown in Figure 1.3 [64].
OMe
HO

OH
HO
CH3CN, HCl (g)

MOMO

OH

ZnCl2
OH

OH

OH

MOMO

MOMCl

anisaldehyde

K2CO3

NaOH, H2O2

OMOM O

O

O

OH
OMOM O

HCl

OMe
OMe

OMe

MOMO
MOMO

MOMO

O

O

O

MOMCl

iPrBr
OMOM
O

OH
OMOM

K2CO3

OH

O

K2CO3

OH

O

O

OMe
MOMO

O

OMe

HCl

HO

O

Eu(fod)3, 80 oC
OMOM
OH

O

OH
OH

O

-6-


PhD Thesis
Scheme 1.1 Synthesis of icaritin

Icaritin and β-anhydroicaritin have also been synthesized by using icariin as shown in
Scheme 1.2 [65].
OCH3
HO

OCH3

O
O Rha
OH

a

d

O

HO

O
O Rha

O

OH

O

OCH3
Glu O

OCH3

O

O

c

O

O Rha
OH

OH

O

OH

O

b

b-anhydroicaritin

OCH3

OCH3
HO

O

O

d

O

HO
O

OH
OH

OH

O

OH

O

icaritin

Reagents and conditions: (a) cellulase, 37 °C, Na acetate pH = 5 buffered hydroalcoholic solution, 6 days;
(b) naringinase, 37 °C, Na acetate pH = 5 buffered hydroalcoholic solution, 11 days; (c) H2SO4, dioxane,
reflux, 24 h; (d) 2-bromoethanol, K2CO3, acetone, reflux, 8 h.
Scheme 1.2 Synthesis of icaritin and β-anhydroicaritin

1.4 Claisen rearrangement
Since its discovery in 1912 by Ludwig Claisen

[66]

the Claisen rearrangement has

stimulated the interest of several generations of organic chemists and its importance is everincreasing owing to its ability to form carbonecarbon and carboneheteroatom bonds. From
1960 the aliphatic Claisen rearrangement gained momentum with the discovery of its several
variations

[67].

Claisen rearrangement has a very important role in organic synthesis. You can

get a lot of widely used pharmaceutical intermediates. C-isopentenyl fragment prenyl group
by the Claisen rearrangement of the resulting dehydrated products icaritin other natural
structures play an important medicinal value. Microwave heating is a new, effective
shortening of the reaction time, increase the yield of the reaction, organic chemical synthesis
has become an effective means, and is widely used in organic synthesis. Microwave-assisted
heating promoted Claisen rearrangement has become an active 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 CeC
bond formation, which is extremely important in the synthesis of useful highly functionalized
compounds and complex natural products. Some applications of the rearrangement in the
[68]

syntheses of natural products have been appeared earlier in a book
[69]

and in review articles

on Claisen rearrangement in genera, covering only a portion of the relevant literature

published until 2004. The Claisen rearrangement as is one of the key steps from aldehyde 7h
as starting material (readily available from (R)-(γ)-limonene). The Claisen precursor 8h,
obtained from 7h was stereospecifically rearranged under thermal condition to 9h in 90%
yields, which was then converted to the natural products 10h and 11h in several steps as
shown in Scheme 1.3.
HO
OH

10h
O

H

200 oC, reflux

(+)-isoamijiol

H

HO

90%

O

9h

8h

7h

11h
(+)-dolasta-1(15), 7, 9-trien-14-ol

Scheme 1.3 Synthesis of (+)-isoamijiol and (+)-dolasta-1(15),7,9-triesn-14-o1

The (-)-5-epi-vibsanin E (14h), a functionalized natural product of the type vibsanediterpenes, isolated from Viburnum awabuki (Caplifoliaceae)

[70]

,

was synthesized by

Williams et al [71] by employing an aliphatic Claisen rearrangement as a key step. The Claisen
precursor allyl vinyl ether derivative 12 was heated at 185 oC under microwave irradiation
(MWI) to afford syn and anti-isomeric products 13i (41%) and 13i (11%), respectively. These
rearranged products 13i afforded the natural product 14h in few steps as shown in Scheme
1.4.
O

O

O

O

O
O

MWI, 185 oC

MOMO

O

syn 41%
anti 11% MOMO
12h

O

O
13i syn
13i. anti

14h
5-epi-vibsanin E

Scheme 1.4 Synthesis of 5-epi-vibsanin E

-8-


PhD Thesis

The O-prenylated Claisen precursor 18h, obtained from the starting material 17h, was
subjected to rearrangement at 200 oC in N,N-diethylaniline to afford 29h in 64% yield. The
rearrangement proceeds through the intermediate 19h by Claisene Cope rearrangement. The
rearranged product 20h was transformed to the natural product xanthohumol 21h in several

steps. The natural compound 21h furnished another natural product isoxanthohumol 22h upon
acid or base treatment as shown in Scheme 1.5.

HO

OH

MOMO

OMOM

MOMO

OMOM

N,N- diethylaniline
reflux, 200 oC, 64%

OH

O

O

OH

O
18h

17h

19h

OH
HO

O

HO

O

OH

OH

MOMO

OMOM

H+ or OHO

OH

heat

O

O

22h

O

O

20h

21h

iso xanthohumol

xanthohumol

Schemes 1.5 Synthesis of xanthohumol and isoxanthohumol

1.5 Baker-Venkatarama reaction
Conventional synthetic flavonoids mainly used Baker-Venkatarama method, which is
based on an aryl chloride and O-hydroxyacetophenone as raw material, then BakerVenkatarama rearrangement synthesized a series of β-propanediol, and then close the ring by
acidification to give flavonoids (1952, Ollis et al) [72] as shown in Scheme 1.6.

-9-