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

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 o

分 类 号 密 级

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

学 科 专 业Organic chemistry

研 究 方 向Organic synthesis of products

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

Flavonoids and Flavonoid Mannich Base Derivatives

By NGUYEN VAN SONM.S (Vinh University of Education, Vietnam) 2008

A dissertation submitted in partial satisfaction of the

Requirements for the degree ofDoctor of Science

inOrganic Chemistry

In the Graduate School

ofHunan University

SupervisorsProfessor WANG QIU ANApril 25, 2015

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。

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。

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

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,

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。

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。

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

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 trihydroxyacetophenone and substituted benzaldehydes with overall yields 23%, 17% and 16%, respectively The key step of the synthetic route is regioselective microwave assistance

2,4,6-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

2d-10d 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

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

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

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

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

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-D-mannopyrano 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-(3,3-dimethylally)-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

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

Table 1.1 Biological activities of some prenylflavonoids 5Table 3.1 Comparison of the NMR spectroscopic data of natural and synthetic flavonoid

from corresponding flavonoid glycosides by microwave irradiation hydrolysis………74

List of Symbols and Abbreviations

µL, mL, L, µM Microliter, milliliter, liter, micromole

MDA-MB-453 Androgen-responsive human breast carcinoma cell line

A549 cells Adenocarcinomic human alveolar basal epithelial cells

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

2

-groups are ‘taken’ This results in a large variety of flavonoids, which usually have a broad

range of different biochemical and physiological properties

5 6

A

B C

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

L-rhamnose, D-glucose, glucoL-rhamnose, 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]

Catechin (colourless)

(yellow)

Flavonoid (colourless)

chalchone

Flavono-3-ol (yellow) Authocyanidre(red, blue, violet)

1 2 3 4 1'

3' 4' 5' 6' 5

6

2' O OH

O + OH

O OH

Quercetin Kaempkerol Myricetin Morin

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 [16-20], anti-allergic [21],cytotoxicity to HeLa cells [22], anti-microbial (antibacterial) [23], antifungal [24], antiviral [25], anti-diarrheal activities [26] 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

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]

Flavonoid compounds of isopentenyl ether compounds can effectively promote into the

biological cells membranes more easily absorbed by the organism Examples include:

bacterial, cancer, lowering blood pressure, a wide range of physiological activity,

anti-inflammatory, anti-HIV, etc as shown in Table 1.1.

Table 1.1 Biological activities of some prenylflavonoids

Active molecules and source Biological and/or pharmacological

Xanthoangelol and 4-hydroxy derricin from

Angelica keinskei Koidrumi

Antibacterial activity [against (+) pathogenic bacteria]

Gram-Antiulcer

[46]

Macarangin from M denticulata

6,8-diprenyleriodictiol, dorsmanin C and

Citotoxicity (against three human

tumour cell lines) [50]Prenyflavonoids from leaves of Macaranga

conifera

Cyclooxygenase-1 (COX-1) and

Known prenylated flavonoids from stem

bark of Artocarpus kemando Xanthoangelol

DNA strand scission activity and

[53]

2’,4,4’-trihydroxychalcone, 6- and 8-prenyl

eriodictiol from liquorice

Induction activity of DNA damage

(rec-assay)Isoliquiritigenin

alba

Herpes simplex type 1 (HSV-1)

Kuraridin and kurarinon from root bark of

Synthetic prenylated flavonoids Electronic transfer inhibition in

mitochondrial inner membrane [58]

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 alkali-

catalyzed 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-dride), leading to 2-hydroxyflavanones and flavones [60] The synthesis of chalcones and anthocyanidins has been described in detail by Dhar [62] Biotransformation of flavonoids in the gut can release these cinnamic acid (phenolic acids) derivatives [63] 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 Flynn-Oyamada reaction and europium-promoted pre-nylation etc., from anhydrous

Algar-phloroglucin with 4.2% overall yield was obtained as shown in Figure 1.3 [64]

MOMO

OMe

O

OMOM O

OH

MOMO

OMe O

OMOM O

HO

OMe

HCl

Scheme 1.1 Synthesis of icaritin

Icaritin and β-anhydroicaritin have also been synthesized by using icariin as shown in

Scheme 1.2 [65]

O O O OH

O

OCH 3

Rha Glu

O O O OH

HO

OCH 3

Rha

O OH O OH

HO

OCH 3

O OH O OH

O

OCH 3

O O O OH

O

OCH 3

Rha

O O O OH

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 ever-increasing 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

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 syntheses of natural products have been appeared earlier in a book [68] and in review articles

[69] 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.

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

vibsane-diterpenes, 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

O O

14h 5-epi-vibsanin E

Scheme 1.4 Synthesis of 5-epi-vibsanin E

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.

20h

OH

O O

H+ or heat

OH-xanthohumol iso 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 Venkatarama 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.

R1

R2Base

Scheme 1.6 Baker-Venkatarama rearrangement synthetic flavonoid

The O-benzoate (24h) of 2-hydroxy-4-methoxypropiophenone (23h, R1=OMe, R2=OMe)

was smoothly rearranged by potassium hydroxide in pyridine to 1,3-diketone (25h, R1=OMe,

R2=OMe), which cyclised to 7-methoxy-4’-methoxyflavone (26h, R1=OMe, R2=OMe) when warmed with acetic-hydrochloric acids These substances have usually been prepared by the Allan-Robinson [73] synthesis (e.g., 23h-26h) and the present work confirmed the view that

the Bake-Venkataraman rearrangement is probably involved in this method

1.6 DMDO in organic synthesis

The 1990s relied on the discovery of the easy access to these oxiranes via epoxidation of glycals by dimethyldioxirane (DMDO) [74] An example of a methodologically simpler procedure employing Oxone/acetone system in biphasic conditions (CH2Cl2– aqueous NaHCO3, to keep the pH of the reaction medium ≥8) was reported for the epoxidation of allenes [75]

O AcO

AcO

OAc Oxone, acetone

aq NaHCO3CH2Cl2

0 oC to rt, 6.5 h 87%

O AcO

AcO

OAc

O AcO

O AcO

AcO

OAc

O AcO

7:1

31h

Schemes 1.7 The mechanism of DMDO in the synthesis of β-D-glucopyranoside and

α-D-mannopyranoside

Halcomb and Danishefsky reported on the reaction of tri-O-acetyl glucal 27h with a

DMDO [76] solution to give a mixture of products whose composition was not further

investigated In our hands, the in situ epoxidation of 27h (3.00 g, 11.02 mmol) gave a mixture

28h of epoxides 28h and 29h in an 87% overall yield as shown in Scheme 1.7 To prove the

stereochemical outcome of the epoxidation of 27h, the mixture of 28h and 29h was subjected

to methanolysis to give, after chromatography, a mixture of known methyl glycosides 30h

(β-D-gluco) and 31h (α-D-manno) in a 95% total yield and a 7:1 ratio[77]

p-TsOH

OH

Scheme 1.8 The mechanism of DMDO in the synthesis flavonols

The DMDO method was used in synthesis of flavonoids compounds The reaction operation the solution in dichloromethane and acetone, with sodium carbonate and sodium bicarbonate buffer was added, the mixture was strongly stirred at 0 oC, then an aqueous solution of oxone® (2KHSO5·KHSO4·K2SO4) was slowly added dropwise, and the system was maintained at pH 9 After the completion of the reaction, extracted by dichloromethane,

the combined organic phase was then added p-toluenesulfonic acid and stirred for several

minutes, it will find that the reaction system turns from brown to blue-green solution with a

strong fluorescence, and epoxy compounds was catalyzed ring opening to form the flavonoids as shown in Scheme 1.8.

1.7 Mannich reaction

The Mannich reaction, also referred to as amine methylation reaction, is an important

organic reaction developed progressively since early 20th century and was named after

Germany chemist Carl Ulvich Franz Mannich (1877-1947) Mannich found in 1917 that the reaction of amine hydrochlorides, formaldehyde and C-H acid compounds, in particular ketones, can produce ketone bases Moreover, substances of characters similar to alkaloids can be produced by appropriate selection of reaction components Thereafter, a number of research reports were delivered by Mannich bases Specifically, reactions with aliphatic ketones, aromatic ketones and alicyclic ketones as the acid component were intensively investigated, thereby establishing the basis of Mannich reaction Mannich bases and derivatives thereof were firstly used as medicaments As time goes on, the products of Mannich reaction are widely spread in various fields of consumer goods production, for example, they can be used for the synthesis of sedative, acesodyne, fungicide, oedema inhibitor, antineoplastic, hepatic protectant, anticoagulant and the like in terms of medicament, and they also found use in terms of explosive, propellant, polymeric flocculant, corrosion inhibitor, vulcanizing accelerator, phytocide, dispersant, antioxidant, active dye, food flavorant, metal chelator The classical conditions of the Mannich reaction for the hydroxyl compounds are based on the ratio of substrate, amine and formaldehyde in alcohol with prolonged heating [78]

The Mannich reaction was used to convert a primary or secondary amine and two

carbonyl compound (one non-enolizable and one enolizable) to a β-amino carbonyl

compound, also known as a Mannich base [79,80], using an acid or base catalyst In the acid catalyzed mechanism both carbonyl compounds get protonated at the oxygen The enolizable

carbonyl compound, which has an α-hydrogen, then gets deprotonated to form an enol

intermediate The non-enolizable carbonyl compound reacts with the amine to form an

iminium ion as shown in Scheme 1.9 The enol intermediate then attacks the iminium ion

which after deprotonation provides the final Mannich base product

H

O

R1N H

R2

H H

O N H

R 1

R2

O H H -H2O

H H

O

R3

R3H

H H

enolized carbonyl compound

Scheme 1.9 The mechanism of the Mannich reaction

The classical conditions of the Mannich reaction for the hydroxyl compounds are based

on the substrate, amine and formaldehyde ratio in alcohol with prolonged heating In our case,

oroxylin A (32h), formaldehyde and primary or secondary amines in 1:1:1 ratio, respectively,

were refluxed and stirred in isopropanol for 1–2 h to afford the C-aminome-thylated

derivatives (33h) [81] as shown in Scheme 1.10.

O

O OH

H3CO

HO

O

O OH

H3CO HO R

R = Methyl benzyl amino (C8H10N)

Scheme 1.10 Synthesis of 8-aminomethylated derivatives of oroxylin

1.8 Hela cell line

Hela cell line is a cell type in an immortal cell line used in scientific research It is the oldest and most commonly used human cell line[82,83] George Gey was able to isolate one specific cell, multiply it, and start a cell line Gey named the sample Hela, after the initial

letters of Henrietta Lacks' name As the first human cells grown in a lab that were "immortal" (they do not die after a few cell divisions), they could be used for conducting many experiments This represented an enormous boon to medical and biological research [84]

Figure 1.3 Scanning electron micrograph of an apoptotic HeLa cell Zeiss Merlin HR-SEM

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) Nikon RTS2000MP custom laser scanning microscope anatomical terminology

Hela cells were also the first human cells to be successfully cloned in 1955 by Theodore Puck and Philip I Marcus at the University of Colorado, Denver [85] Demand for the HeLa cells quickly grew Since they were put into mass production, Henrietta's cells have been used

by scientists around the globe for "research into cancer, AIDS, the effects of radiation and toxic substances, gene mapping, and countless other scientific pursuits".Hela cells have been used to test human sensitivity to tape, glue, cosmetics, and many other products There are almost 11,000 patents involving Hela cells [84]

Hela cells have been used in the study of the expression of the papillomavirus E2 and apoptosis [86] Hela cells have also been used to study canine distemper virus' ability to induce apoptosis in cancer cell lines [87] This virus' ability to induce apoptosis could play an important role in developing treatments for tumor cells resistant to radiation and chemotherapy Hela cells have also been used in a number of cancer studies including those involving sex steroid hormones such as Estradiol, estrogen, and estrogen receptors along with estrogen like compound such as Quercetin and its cancer reducing properties [88] There have

also been studies on Hela cells, the effects of flavonoids and antioxidants with estradiol on cancer cell proliferation Hela cells were used to investigate the phytochemical compounds and the fundamental mechanism of the anticancer activity of the ethanolic extract of mango peel (EEMP) EEMP was found to contain various phenolic compounds and to activate death through apoptosis of human cervical malignant Hela cells which suggests EEMP may help to prevent cervical cancer as well as other types of cancers [89] Hela cells have been used in research involving fullerenes to induce apoptosis as a part of Photodynamic therapy as well as

in in vitro cancer research using cell lines [90].Further Hela cells have also been used to define cancer markers in RNA, and have been used to establish an RNAi based identification system and interference of specific cancer cells [91]

1.9 Assay for antiproliferative activity

The standard CCK-8 assay is a sensitive nonradioactive colorimetric assay for determining the number of viable cells in cell proliferation and cytotoxicity assays The solution is added directly to the cells and no pre-mixing of components is required CCK-8 uses Dojindo's tetrazolium salt, WST-8[2-(2-methoxy-4-nitrophenyl)-3- (4-nitrophenyl)-5-(2,4-disul-fophenyl)-2H-tetrazolium, monosodium salt], which produces a water-soluble formazan upon reduction in the presence of an electron carrier The amount of yellow formazan produced in cells is directly proportional to the number of living cells Briefly, at the end of the culture period, 10 μL of the CCK-8 solution was added to each well of the culture plate After 2-4 h incubation, absorbance at 450 nm was measured with a universal microplate spectrophotometer (Biotek Instruments, Gene Company Limited, Winooski, VT) The cytotoxic effect was indicated as a percentage of surviving cells (ratio of surviving cells after treatment and without treatment) using the following formula:

Cell viability = (absorption of sample - absorption of background)/ (absorption of control - absorption of background) × 100% [92]

Figure 1.5 Typical cell survival curve Figure 1.6 Exemple of the plate arrangement and

color development

With the WST-8 is reduced to an orange-colored formazan through electron mediator, Methoxy PMS by NADH and NADPH activity which are generated by cellular activities as

1-indicated in the Fig 1.7 The amount of WST-8 formazen is dependent on the activity of

cellular dehydrogenase, so WST-8/1-Methoxy PMS system can be used to determine the number of living cells and cell viability

Fig 1.7 Cell viability detection mechanism with CCK-8

Chapter 2 Total Synthesis of Icaritin via Microwave

Assistance Claisen Rearrangement

2.1 Introduction

Icaritin (1a), the aglycone of icariin, is natural prenylated flavonoid isolated from

Epimedium Genus [93,94] It has been shown to exhibit an interesting spectrum of

pharmacological effects, such as antiosteoporosis activity [95] and estrogen regulation [96].Recently, icaritin was recognized as a novel anticancer agent by remove strongly inhibiting growth of breast cancer MDA-MB-453 and MCF-7 cells at the concentrations of 2-3 μM [96] Icaritin also “induces cell death in activated hepatic stellate cells through mitochondrial activated apoptosis and ameliorates the development of liver fibrosis in rats” [98] Additionally, icaritin and its 3,7-bis-2-hydroxyethyl derivative can act as good phosphodiasterase-5 (PDE-5) inhibitor, which were claimed to be useful for improving the sexual performance and treatment of sexual problems [99]

On the other hand, natural resources of icaritin (1a) is limited due to the low contents in

Epimedium plants, which negatively influenced their further bioactivity evaluation Therefore,

chemical synthesis of 1a will be a very important alternative approach for addressing the

problem of its availability So far, 1a has been synthesized via an eight-step strategy including

Houben-Hoesch acylation, Algar-Flynn-Oyamade reaction and Europium-promoted prenylation

etc., starting from anhydrous phloroglucin but only 4.2% overall yield was obtained [100] This method is considered time consuming, low efficient and inappropriate for larger scale synthesis

of 1a.

As the continuation of our study on the chemistry and biology of flavonoids [101-103], we herein

report the novel total synthesis of 1a through 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 with a reaction sequence of 8 steps in overall yields of 23% The key step of the synthetic route

was para-Claisen rearrangement under microwave assistance.

O OH

OH

OBn

O

O OBn

O

O OH

O

O OH O

O O

O

O OH

OMOM OMe

O

O OH

HO

OH OMe

10a

6a 7a

8a

1a

5a 4a

OH OMe OMe

OMOM OMe

h a

MOMO

OH

O

BnO BnO

e

f g

j b

1 2 3 4

4a 5 6 7

1'

2' 3'4' 5' 6' 8a

8

k

O

O OMOM

OMe

O

O OMOM

OMe MOMO

Reagents and conditions: (a) BrBn, K2CO3, acetone, reflux, 12 h, 85%; (b) (Me)2SO4, NaOH then SOCl2,

CH2Cl2, reflux, 86%; (c) K2CO3, acetone, reflux, 24 h, 70%; (d) DMDO, 0 oC, 24 h, r.t then

p-toluensulfonic acid, 2 h, r.t, 75%; (e) 5% Pd/C, 24 h, r.t, 76%; (f) K2CO3, acetone, MOMCl, 6 h, r.t, 78%; (g) K2CO3, acetone, 3,3-dimethylallylbromide, 50 oC, reflux, 12 h, 89%; (h) N,N-Dietyllaniline, 190 o C, 36

h, reflux, 83% of 9a; (h-j) N,N-Dietyllaniline, microwave assistance, 190 oC, reflux, 30 min, 85% of 10a;

by the EI method The chemical shifts (δ) were measured by ppm, and coupling constant (J)

was calculated in hertz (Hz) While melting points were determined by an XRC-1 apparatus

and were uncorrected Microwave irradiation was performed with XH-MC-1 microwave reactor (Beijing Xinghu Science and Technology Development Co., China) Column chromatography was carried out on silica gel 200–300 mesh (Qingdao Ocean Chemical Products of China) Commercially available AR or chemical pure reagents, and anhydrous solvent removed water and redistilled were employed.

2.2.2 Synthesis of 2-hydroxy-4,6-bis(benzyloxy)acetophenone (2a).

The solution of 2,4,6-trihydroxyacetophenone (5 g, 29.76 mmol) and anhydrous K2CO3

(15 g, 108.6 mmol) in 120 mL dry acetone was refluxed at 65 oC for 1 h Then BnBr (7.5 mL, 63.05 mmol) was added dropwise After stirring for 16 h, the organic phase was separated The solvent was removed, and the residue was purified by column chromatography on silica

gel (petroleum ether/EtOAc, v/v, 30:1) to give 2a (8.8 g, 85%) as white solids, m.p 95-96 oC;

1H NMR (400 MHz, CDCl3): δ 14.04 (d, J = 2.0 Hz, 1H, 2-OH), 7.39–7.31 (m, 10H, ArH), 6.16 (s, 1H, 5-H), 6.07 (d, J = 10.5 Hz, 1H, 3-H), 5.05 (t, J = 6.0 Hz, 4H, 2OCH2), 2.54 (s, 3H, Me); 13C NMR (100 MHz, CDCl3): δ 203.2, 167.6, 165.1, 162.0, 135.9, 135.6, 128.8,

128.7, 128.5, 128.0, 127.7, 106.4, 94.8, 92.4, 71.1, 70.3, 33.7; EIMS: m/z 348 [M]+

.

2.2.3 Synthesis of 4-methoxybenzoyl chloride (3a).

The solution of 4-hydroxybenzoic acid (10 g, 74.07 mmol) in 100 mL 20% NaOH (aq) was strongly stirred at 40 oC and then (CH3)2SO4 (17 mL, 63.05 mmol) was added dropwise The mixture was stirred for 4 h, cooled to room temperature, acidated and solution was filtered, the obtained residue was washed with H2O and dried The obtained white solid was poured into 30 mL of dichloromethane and refluxed Then thionyl chloride (SOCl2) (8.8 mL,

121 mmol) was put into thereaction system The reaction mixture was stirred under reflux for

4 h The solvent was removed under reduced pressure obtained 3a (10.6 g, 86%) as white

solid, m.p 20-22 oC; 1H NMR (400 MHz, CDCl3): δ 8.0 (d, J = 8.9 Hz, 2H, 2-H and 6-H), 6.88 (d, J = 8.9 Hz, 2H, 3-H and 5-H), 3.86 (s, 3H, OMe); EIMS: m/z 170 [M]+

.

2.2.4 Synthesis of 5,7-bis(benzyloxy)-2-(4-methoxyphenyl)flavone (4a).

To a solution of compound 2a (5 g, 29.24 mmol) and anhydrous K2CO3 (15 g, 0.1 mol) in

70 mL of dry acetone was stirred at room temperature for 30 min, then 4-methoxybenzoyl

choloride (3a) (7.5 mL, 63.07 mmol) was added dropwise The temperature was increased up

to 65 oC, after refluxed for 24 h, the organic phase was separated The solvent was removed, and the residue was purified by column chromatography on silica gel (petroleum

ether/EtOAc, v/v, 7:1) to afford 4a (5.42 g, 70%) as yellow powder, m.p 150-152 oC; 1H NMR (400 MHz, CDCl3): δ 7.83 (d, J = 8.6 Hz, 2H, 2’-H and 4’-H), 7.62 (d, J = 8.6 Hz, 2H,

3’-H, 5’-H ), 7.02-7.44 (m, 10H, ArH), 6.65 (d, J = 8.6 Hz, 2H, 6-H), 6.59 (d, J = 8.6 Hz, 1H, 3-H), 6.50 (s, 1H, 8-H), 5.24 (d, J = 8.5 Hz, 2H, 5-OCH2), 5.12 (s, 7-OCH2), 3.88 (s, 3H, 4’-OMe); 13C NMR (100 MHz, CDCl3): δ 177.4, 163.6, 162.8, 162.0, 160.7, 159.7, 136.4, 135.8,

The solution of compound 4a (1.5 g, 2.15 mmol) in 70 mL solvent (acetone and CH2Cl2,

v/v, 3:4) and (8 g Na2CO3 and 3.5 g NaHCO3 in 70 mL of water), the mixture was strongly stirred at 0 oC, then 13 g Oxone in 200 mL of water was slowly added dropwise in for 5 hours The solution was adjusted to pH 9 After stirring for 24 h, the organic phase was separated The aqueous phase was extracted with dichloromethane(30 mL  3) The organic phase was combined and washed with NaCl (aq) and Na2S2O3 (aq) three times, dried over anhydrous sodium sulfate The solvent was removed under reduced pressure, the residue was

added 5 mg p-toluensulfonic acid in dry acetone (20 mL) The solution was stirred at room

temperature for 2 h The crude solid was recrystallized from CH3OH to afford 5a (1.16 g,

75%) as yellow powder, m.p 254-256 oC; 1H NMR (400 MHz, CDCl3): δ 8.18 (d, J = 9.0 Hz, 2H, 2’-H and 6’-H), 7.61 (d, J = 7.3 Hz, 2H, ArH), 7.45–7.35 (m, 8H, ArH), 7.04 (d, J = 9.0

Hz, 2H, 3’-H and 5’-H), 6.66 (d, J = 2 1 Hz, 1H, 8-H), 6.50 (d, J = 2.1 Hz, 1H, 6-H), 5.31 (s,

1H, 3-OH), 5.25 (s, 2H, 5-OCH2), 5.16-5.12 (m, 2H, 7-OCH2), 3.89 (s, 3H, 4’-OMe); 13C NMR (100 MHz, CDCl3): δ 171.8, 166.4, 160.6, 159.2, 158.5, 142.5, 137.6, 136.4, 135.7,

130.9, 128.9, 128.5, 127.8, 126.9, 126.7, 122.2, 114.0, 106.7, 97.5, 95.3, 71.3, 70.4, 58.0; ESIMS: m/z 480 [M]+

.

2.2.6 Synthesis of kaempferide (6a):

A solution of compound 5a (1000 mg, 2.08 mmol) and 650 mg 5% Pd/C in 15 mL

solvent (CH3OH/ EtOAc, v/v, 1:1) was stirring under H2 atmosphere (balloon) at room temperature After stirring for 24 h, the organic phase was separated The solvent was removed, and the residue was purified by column chromatography on silica gel (petroleum

ether/EtOAc, v/v, 2:1) to obtain 6a (446 mg, 76%) as yellow powder, m.p 224-226 oC (lit[104] 225-227 oC); 1H NMR (400 MHz, DMSO-d6): δ 12.47 (s, 1H, 5-OH), 10.90 (s, 1H, 7-OH),

9.53 (s, 1H, 3-OH), 8.14 (s, 2H, 2’-H and 6’-H), 7.11 (s, 2H, 3’-H and 5’-H), 6.47 (s, 1H,

8-H), 6.21 (s, 1H, 6-8-H), 3.84 (s, 3H, 4’-OMe); 13C NMR (100 MHz, DMSO-d6): δ 176.5 (C4),

164.5 (C7), 161.2 (C5), 160.9 (C4’), 156.7 (C8a), 146.7 (C2), 136.5 (C3), 129.8 (C2’ and

C6’), 123.7 (C1’), 114.5 (C3’ and C5’), 103.6 (C4a), 98.70 (C8), 93.9 (C6), 55.8 (4’-OMe); EIMS m/z 300 [M]+; HRMS (EI): m/z calcd for C16H12O6,300.0622 [M]+, found 300.0628.

2.2.7 Synthesis of

5-hydroxy-3,7-bis(methoxymethoxy)-2-(4-methoxy-phenyl)flavone (7a):

A solution of compound 6a (400 mg, 1.42 mmol) and dry K2CO3 (3.0 g, 21.72 mmol) in

20 mL dry acetone was stirred for 30 min at room temperature, then chloromethyl methoxy ether (0.5 mL, 3.69 mmol) was added dropwise The mixture was stirred for 6 h at room temperature, the organic phase was separated The solvent was removed in reduced pressure The residue was poured into 7 mL of ethanolic and 0.5 mL of HCl (3% HCl in EtOH) and stirred at room temperature for 30 min After diluting with H2O, the solution was extracted by

CH2Cl2, which was subsequently rinsed with H2O The combined extracts were then dried over anhydrous sodium sulfate, filtered, concentrated, the residue was purified by column

chromatography (petroleum ether/EtOAc, v/v, 7:1) to afford 7a (429 mg, 78%) as white

powder, m.p 158-159 oC; 1H NMR (400 MHz, CDCl3): δ 12.57 (s, 1H, 5-OH), 8.04 (d, J = 8.7 Hz, 2H, 2’-H and 6’-H), 7.01 (d, J = 8.7 Hz, 2H, 3’-H and 5’-H), 6.81 (s, 1H, 8-H), 6.73 (d, J = 1.8 Hz, 1H, 6-H), 5.35 (s, 2H, 7-MOM), 5.20 (s, 2H, 3-MOM), 3.89 (s, 3H, 4’-OMe),

3.56 (s, 3H, 7-MOM), 3.19 (s, 3H, 3-MOM); 13 C NMR (100 MHz, CDCl3): δ 177.6 (C4),

164.6 (C7), 161.2 (C5), 159.6 (C4’), 158.4 (C8a), 158.2 (C1), 137.4 (C3), 130.4 (C2’ and C6’), 123.2 (C1’), 113.7 (C3’ and C5’), 104.6 (C4a), 97.6 (C6), 96.8 (C8), 95.5 (7-MOM), 94.4 (3-MOM), 57.6 (4’-OMe), 56.6 (7-MOM), 55.4 (3-MOM); EIMS: m/z 388 [M]+

ether/EtOAc, v/v, 5:1) to afford 8a (211 mg, 89%) as white solid, m.p 76-77 oC; 1H NMR (400 MHz, CDCl3): δ 8.04 (d, J = 8.7 Hz, 2H, 2’-H and 6’-H), 7.01 (d, J = 8.7 Hz, 2H, 3’-H and 5’-H), 6.82 (d, J = 7.5 Hz, 1H, 8-H), 6.70 (d, J = 7.5 Hz, 1H, 6-H), 5.57 (d, J = 8.1 Hz, 1H, 2’’-H), 5.23 (s, 2H, 7-MOM ), 5.20 (s, 2H, 3-MOM), 4.69 (d, J = 6.5 Hz, 2H, 5-OCH2), 3.89 (s, 3H, 4’-OMe), 3.51 (s, 3H, 7-MOM), 3.19 (s, 3H, 3-MOM), 1.74 (s, 3H, 4’’-Me), 1.70 (s, 3H, 5’’-Me); 13C NMR (100 MHz, CDCl3): δ 173.8 (C4), 161.7 (C7), 161.2 (C5), 160.3

(C4’), 158.4 (C8a), 153.3 (C2), 137.5 (C3), 130.5 (C2’ and C6’), 123.1 (C1’), 119.8 (C2’’), 113.9 (C3’ and C5’), 109.9 (C4a), 99.9 (C8), 97.9 (C6), 95.6 (7-MOM), 94.5 (3-MOM), 66.9 (C1’’), 57.5 (4’-OMe), 56.5 (7-MOM), 55.3 (3-MOM), 25.3 (C5’’), 18.7 (C4’’); EIMS: m/z

The solution of 8a (190 mg, 0.41 mmol) in 8 mL of dry N,N-diethylaniline was stirred in

reflux under microwave-assistance (700 W) and nitrogen protection at 190 oC for 30 min Cooled to room temperature, the reaction was diluted with dilute hydrochloric acid (1N HCl), the mixture was extracted with ethyl acetate (20 mL  3), and dried over anhydrous sodium sulfate The organic phase was separated, the solvent was removed, and the residue was

purified by column chromatography on silica gel (petroleum ether/EtOAc, v/v, 7:1) to give

10a (161 mg, 85%) as pale yellow powder, m.p 179-180 oC; 1H NMR (400 MHz, CDCl3): δ 12.47 (s, 1H, 5-OH), 8.03 (d, J = 8.6 Hz, 2H, 2’-H and 6’-H), 6.95 (d, J = 8.6 Hz, 1H, 3’-H and 5’-H), 6.16 (s, 1H, 6-H), 5.25 (t, J = 6.7 Hz, 2H, 1H, 2’’-H), 5.19 (s, 2H, 7-MOM), 5.11 (s, 2H, 3-MOM), 3.83 (s, 3H, 4’-OMe), 3.42 (d, J = 2.4 Hz, 2H, 1’’-H), 3.31 (s, 3H, 7-MOM),

3.13 (s, 3H, 3-MOM), 1.79 (s, 3H, 4’’-Me), 1.70 (s, 3H, 5’’-Me); 13C NMR(100 MHz, CDCl3): δ 178.5 (C4), 161.5 (C7), 160.5 (C4’), 159.5 (C8a), 156.2 (C5), 153.9 ( C2), 135.3

(C3), 131.9 (C3’’), 130.5 (C2’ and C6’), 123.3 (C2’’), 122.5 (C1’), 113.9 (C3’ and C5’), 108.5 (C8), 105.7 (C4a), 100.1 (C6), 99.5 (7-MOM), 97.7 (3-MOM), 57.9 (4’-OMe), 56.3 (7-MOM), 55.4 (3-MOM), 31.7 (C1’’), 26.6 (C4’’), 16.3 (C5’’); EIMS: m/z 456 [M]+

.

The solution of 8a (190 mg, 0.41 mmol) in 8 mL of dry N,N-diethylaniline was stirred in

reflux and nitrogen protection at 190 oC for 36 h Cooled to room temperature, the reaction was diluted with dilute hydrochloric acid (1N HCl), the mixture was extracted with ethyl acetate (20 mL  3), and dried over anhydrous sodium sulfate The organic phase was separated, the solvent was removed, and the residue was purified by column chromatography

on silica gel (petroleum ether/EtOAc, v/v, 5:1) to give 9a (155 mg, yield: 83%) as pale yellow

powder, m.p 142-143 oC; 1H NMR (400 MHz, CDCl3): δ 12.42 (s, 1H, 5-OH), 7.84 (d, J = 8.7 Hz, 2H, 2’-H and 6’-H), 7.02 (d, J = 8.7 Hz, 2H, 3’-H and 5’-H), 6.35 (s, 1H, 8-H), 6.23 (dd, J = 9.3, 7.1 Hz, 1H, 2’’-H), 5.35 (s, 2H, 7-MOM), 5.28 (s, 2H, 3-MOM, 5.06 (dd, J = 5.4, 3.6 Hz, 1H, 3’’H), 5.01 (dd, J = 5.4, 3.3 Hz, 1H, 3’’-H), 3.89 (s, 3H, 4-OCH3), 3.57 (s, 3H, 7-OMOM), 3.47 (s, 3H, 3-OMOM), 1.65 (s, 3H, 4’’-Me), 1.59 (s, 3H, 5’’-Me); 13C NMR(100 MHz, CDCl3): δ 177.6 (C4), 162.6 (C7), 160.9 (C5), 159.8 (C4’), 158.5 (C2), 156.0 (C8a),

149.1 (C2’’), 136.3 (C3), 128.8 (C2’ and C6’), 123.0 (C1’), 114.4 (C6), 113.5 (C3’ and C5’), 111.9 (C3’’), 105.7 (C4a), 98.1 (7-MOM), 96.1 (3-MOM), 93.9 (C8), 55.9 (4’-OMe), 55.3 (7-MOM), 54.7 (3-MOM), 41.9 (C1’’), 29.1 (C4’’ and C5’’); EIMS: m/z 456 [M]+

.

2.2.10 Synthesis of icaritin (1a).

The solution of 10a (120 mg, 0.26 mmol) in 15 mL CH3OH and hydrochloric acid (3 N, 2.5 mL) was stirred and refluxed for 2 hour under the nitrogen protection The solvent was removed under reduced pressure, the crude solid was recrystallized from CH3OHto give 1a

(92 mg, yield: 96%) as light yellow powder: m.p 231-232 oC (lit.[105]: 232-233 oC; 1H NMR

(400 MHz, DMSO-d6): δ 12.38 (s, 1H, 5-OH), 10.76 (s, 1H, 7-OH), 9.49 (s, 1H, 3-OH), 8.13 (d, J = 9.0 Hz, 2H, 2’-H and 6’-H), 7.13 (d, J = 9.1 Hz, 2H, 3’-H and 5’-H), 6.30 (s, 1H, 6- H), 5.18 (t, J = 6.8 Hz, 1H, 2’’-H), 3.85 (s, 3H, 4’-OMe), 3.44 (d, J = 6.7 Hz, 2H, 1’’-H),

1.75 (s, 3H, 4’’-Me), 1.63 (s, 3H, 5’’-Me); 13C NMR(100 MHz, DMSO-d6): δ 176.7 (C4),

161.7 (C7), 160.9 (C4’), 158.8 (C5), 153.9 (C8a), 146.6 (C2), 136.4 (C3), 131.5 (C3’’), 129.6 (C2’ and C6’), 124.1 (C1’), 122.9 (C2’’), 114.6 (C3’ and C5’), 106.1 (C4a), 103.5 (C8), 98.3 (C6), 55.9 (4’-OMe), 25.9 (C1’’), 21.7 (C4’’), 18.3 (C5’’); EIMS m/z 368 [M]+; HRMS (EI): m/z calcd for C21H20O6,368.1262 [M]+, found 368.1254

2.3 Result and discussion

The novel synthesis route of icaritin (1a) was shown in Scheme 2.1 The

2,4,6-trihydroxyacetophenone and 4-methoxybenzoyl chloride were prepared from anhydrous phloroglucin with Houben-Hoesch acylation[106]

, and 4-hydroxybenzoic acid via

O-methylation and acid chloration respectively Acylation of 2a with 3a gave rise to aryl ester

intermediate, which underwent the Baker-Venkataraman rearrangement and dehydrative

closure of the flavone C ring to afford 4a in good yield

Oxidation of 4a to the 3-hydroxyflavone 5a proved to be challenging According to our

previously published procedures [107]

, the oxidation of flavone 4a in one-step with

dimethyldioxirone (DMDO) generated in situ from oxone and acetone at low temperature,

followed by acid-induced rearrangement gave the hydroxyflavone 5a The oxidation reaction

was completed in excellent regioselective and high yield within a short time period, it was easily conducted on a large scale and product could be purified by recrystallization

Subsequently, Pd/C catalyzed hydrogenolysis of benzyl ether 5a gave kaempferide 6a, for

which the spectroscopic data was identical to that reported previously [108]

The selective O-methoxymethylation of 6a with chloromethylmethyl ether in dry acetone

gave compound 7a To our knowledge in flavonoid chemistry, the 5-hydroxyl group in

flavonoid easily form hydrogen bond with the adjacent carbonyl group, in fact,

O-methoxymethylation of the 5-hydroxyl group is unavailable -chemoselective 3-O- and

7-O-methoxymethyation of 6a was therefore readily achieved using two equivalents of MOMCl to

afford 7a Further O-prenylation at free 5-hydroxyl group of 7a gave 8a using

3,3-dimethylallylbromide as the electrophile reagent

The O-prenylated Claisen precursor 8a was subjected to double sigmatropic

rearrangement in N,N-diethylaniline under microwave heating leading to the corresponding

C-prenylated product 10a in 85% yield The rearrangement was assumed to proceed via the

intermediate 9a in domino Claisen rearrangement reaction [108]

The last step is careful deprotection of the MOM groups under mild acidic condition afforded the expected natural

product icaritin 1a

The Claisen rearrangement of the phenyl ether 8a as a key step in our, synthetic strategy

was investigated under microwave assistance and conventional heating respectively The effects on the regioselective and reaction rates by microwave assistance and conventional

heating were compared While conventional heating, the ortho-rearranged product 9a was

found to be preferred one, and was isolated in 83% yield in N,N-diethylaniline at 190 oC,

However, in the case of microwave assistance, the para-rearranged product 10a was selected

gained in 85% yield in the same system The two products showed difference of 1H NMR signals for the –C(CH3)2CH=CH2 in 9a at δ 5.01 and 5.06 (2H, 2dd) and –CH2-CH=C(CH3)2

in 10a at δ 3.42 (2H, d) to allow an unambiguous identification of two isomers The results

also demonstrated that microwave assistance could greatly accelerate the reaction rate of the Claisen rearrangement The reaction rates under microwave assistance (700 W) are 72 times higher than those acted by conventional heating at 190 oC Thus, we confirmed that the

microwave assistance procedure was suitable for synthesis of MOM protected icaritin 10a.