Design, synthesis, and biological activities of novel isobutyrophenone derivatives

This compounds also exhibited potential Mg-Fba enzyme inhibitory activity with IC50 data in the range of 28.91-69.82 µM.. Moreover, it has exhibited the highest type II fructose-1,6-bisp

Dissertation for Doctor Degree Northwest A＆F University in 2019

Design, Synthesis, and Biological Activities

of Novel Isobutyrophenone Derivatives

Research Field: Structure and Bioactivities

of Natural Products

Co-Supervisor: Assoc Prof Li Ding

Date of submission: 2019.05

曹 蔚 教授 博导 西北农林科技大学化学与药学院 委员

答辩日期：2019 年 5 月 30 日

本研究得到陕西省科技统筹重点项目-战略性新兴产业重大产 品（群）项目（编号：2015KTCQ02-14）与国家自然科学基金面上

项目（21572182）资助。

摘要

农业病虫害造成经济作物的减产和品质下降，导致农业经济面临很大威胁。过量使用合成农药不仅污染环境而且影响人类和动物的健康。由于结构多样性的天然产物及其类似物是新药先导化合物发现的重要来源。 课题组前期从高等真菌青柄多孔菌

（Polyporus picipes）中分离得到的 2,4-二羟基苯乙酮具有抗菌作用，在其抗植物病源

真菌构效关系研究的基础上，本文研究包括两部分：第一部分设计、合成异丁酰苯酚类化合物及其构效关系研究，首次证实了它们抗真菌活性的潜在靶标之一 II 型果糖-1,6-

关系及化合物与α-糖苷酶的结合机制，首次证明了异丁酰苯酚类化合物的降血糖作用。主要结果如下：

1、设计合成了 43 个全新的 4-甲基-异丁酰苯二酚的脂类和醚类的衍生物。采用生长速率法进行植物病源真菌抑制活性的筛选和评价。结果表明部分化合物对七种植物病

源真菌（番茄灰霉病菌（Botrytis cinerea）、番茄早疫病菌（Alternaria solani）、苹果炭 疽菌（Colletotrichum gloeosporiodes）、马铃薯干腐病菌(Fusarium solani）、小麦赤霉病 菌（F Graninearum）、苹果腐烂病菌（Cytospora sp.）和稻瘟病菌（Magnaporthe grisea）

与嘧菌酯相近。综上所述，结果表明，异丁酰苯酚衍生物 12b 可以进一步优化，并可作为新型杀菌剂先导化合物。

5、评价了 76 个异丁酰苯酚衍生物对α-葡萄糖苷酶抑制作用。构效关系研究显示苯环的 5 位甲基和 4 位羟基碘代烷基酯化对维持高抑制α-葡萄糖苷酶的活性起重要

与酶结合的机制表现出输水作用和氢键是它们结合的主要次级相互作用。肠外翻实验和小鼠体内的降糖实验表明化合物 12d 能作用于糖苷酶，从而达到降低血糖的效果。

关键词：1-（2,4-二羟基-5-甲基苯基）-2-甲基丙-1-酮，异丁酰间苯二酚，抗真菌，II 型果糖-1,6-二磷酸醛缩酶抑制活性，α-葡萄糖苷酶抑制活性

ABSTRACT

The agricultural diseases and insect pests caused to decrease the production and quality of cash crops, led to significantly threaten the agricultural economy The excessive use of synthetic pesticides not only resulted in the environment pollution but also affected to the human healthy and animals Natural products and their analogs are important sources to discovery of biologically active compounds and new drug lead compounds Acetophenone

isolated from the higher fungus Polyporus picipes exhibits antifungal and antibacterial

properties Our pervious studies, isobutyrophenone which is one of acetophenone derivative displayed have good antifungal activity, so, in this study focused on the discovery and structural optimization of potential targets related to isobutyrophenone The antifungal activities of all synthesis compounds were tested against seven phytopathogenic fungi The potential targets for their antifungal activity were confirmed as inhibitor type II fructose-1,6-bisphosphate aldolase for the first time Their α-glucosidase inhibitory activities were evaluated and the structure-antifungal relationships were also discussed The main

results are as follows:

1 A series 46 compounds of 1-(2,4-dihydroxy-5-methylphenyl)- 2-methylpropan

-1-one (3) derivatives were designed and synthesized The antifungal activity of

synthetic compounds were evaluated for against seven phytopathogenic fungi including

Botrytis cinerea, Alternaria solani, C.gloeosporiodes, Fusarium solani, F Graninearum,

Cytospora sp (Valsa mali） and Magnaporthe grisea Compounds 4a, 5a, 5c, 5d, 5e exhibited

as a good antifungal activities Antifungal properties of compound 5a exhibited strong with

relatively high selectivity to against B Cinerea, C Gloeosporiodes and , Fusarium solani

with EC50 values 4.12, 6.56 and 9.87 μg/mL, respectively, 5c has the best inhibitory activity

against Cytospora sp, EC50 is 1.42 μg/mL; 5d had the best inhibitory activity against F

Graninearum and Fusarium solani, with EC50 of 6.51 and 2.67 μg/mL, respectively, 5e also

had a good inhibitory activity against Fusarium graminearum with an EC50 of 2.49 μg/mL

This compounds also exhibited potential Mg-Fba enzyme inhibitory activity with IC50 data in

the range of 28.91-69.82 µM

2 The 3, 4 and 5-modified derivatives were synthesized with the isobutyrophenone skeleton The structure-activity relationship (SAR) study showed that the introduction of a

group with the chloroacetyl group can significantly improve the antifungal activity and broad

spectrum of the compound Among them, compound 12b were showed the best antifungal activity against 7 plant pathogenic fungi, with the EC50 ranged from 1.22 to 39.44 µg/mL

Moreover, it has exhibited the highest type II fructose-1,6-bisphosphate aldolase inhibitory activity with IC50 of 3.63 μM, which is higher nearly 7.9 times than compound 5e (IC50 =

28.91 μM) In vivo fruit tests showed that compound 12b has the practical potential to protect

fruits (or plants) from pathogens, and its protective ability is similar to that of azoxystrobin Cytotoxicity results of synthesized compounds on normal cells line NIH3T3 and 293T and silkworms shown that all compounds were non-toxicity and safe with living environment of

the organism Therefore, compounds 12b are well worth the consideration as a safe and

effective fungicidal product to control plant diseases

3 α-glucosidase inhibitory activity of 76 new isobutyrylphenol derivatives were evaluated Structure-activity relationship (SAR) studies have shown that the introduction of the bromine atom on the 3-phenyl, iodomethyl group at 4 position and the methyl group on the 5-phenyl ring were play an important role to increase the inhibitory of α-glucosidase

activity Among them, compound 12d exhibits the best α-Glucosidase inhibitory activity with

an IC50 of 0.57 μM The interaction mechanism of the inhibitors with α-glucosidase showed

that compound 12d could bind to other site of α-glucosidase enzyme with hydrogen bonds

interactions The everts intestinal sleeves in vitro and the sucrose loading test in vivo on the

rat α-glucosidase inhibition of the lead compound proved that they have significant postprandial hypoglycemic effects These compounds may serve as lead for the future research in order to identify a potent and safer therapy for the treatment of diabetes mellitus

KEY WORDS: 1-(2,4-dihydroxy-5-methylphenyl)-2-methylpropan-1-one, isobutyrophenone derivatives, antifungal, inhibitory activity against Class II Fba and alpha-glucosidase inhibitors

Contents

摘要 I ABSTRACT III

Chapter 1 Literature Review 1

1.1 Introduction 1

1.2 Natural products of Isobutylphenone 2

1.2.1 Identification and Bioactivity
of Callistemon 2

1.2.2 Identification and Bioactivity of Hypericum 5

1.2.3 Identification and Bioactivity of Pilidiostigma 8

1.2.4 Identification and Bioactivity of Corymbia
 9

1.3 Synthesis of Isobutylphenone Deivatives 14

1.3.1 Antifungal 14

1.3.2 Anti bacterial, antimalarial and anticancer 16

1.4 Research Objectives 18

Chapter 2 Synthesis Ether and Ester Devivatives of Isobutylphenone and Biological Activity 20

2.1 Introduction 20

2.2 Experimental 21

2.2.1 Chemistry 21

2.2.2 Synthesis of ether and ester derivatives of isobutylphenone 21

2.3 Bioactivity 38

2.3.1 Fungal Materials 38

2.3.2 Cultures 38

2.3.3 In Vitro Antifungal Bioassay 38

2.3.4 Enzyme Inhibition Activity 39

2.3.5 Homology modeling 40

2.3.6 Molecular docking 40

2.4 Results and discussion 41

2.4.1 Antifungal activity 41

2.4.2 Mg-Fba inhibitory activities 45

2.4.3 Molecular Modeling Studies 46

Chapter 3 Synthesis of Bromo Isobutylpheone Derivatives and Antifungal Activity 49

3.1 Introduction 49

3.2 Experimental 50

3.2.1 General procedure for the synthesis of intermediate compounds 50

3.2.2 General procedure to synthesize compound 8a-8f, 10a-10d, 12a-12m by conventional method 52

3.3 Antifungal Activity 62

3.3.1 Fungal Materials 62

3.3.2 Cultures 62

3.3.3 In Vitro Antifungal Bioassay 62

3.3.4 Effect of inhibition on Hyphal Morphology 62

3.3.5 In Vivo Testing on apple fruits Infected by C Gloeosporiodes 62

3.3.6 Enzyme inhibition activity assays 63

3.3.7 Cytotoxicity assay 63

3.3 8 Stability assay 64

3.4 Results and Discussion 65

3.4.1 Fungicidal Activities 65

3.4.2 In Vivo Testing on apple fruits Infected by C Gloeosporiodes 68

3.4.3 Effect of inhibition on Hyphal Morphology 69

3.4.4 Enzyme Activity 70

3.5 Cytotoxicity assay 74

3.5.1 Cytotoxicity on normal cells line 74

3.5.2 Silkworm toxicity study 74

3.6 Stability studies 75

3.7 Summary 77

Chapter 4 Alpha-Glucosidase Inhibiting Activity of Isobutyrophenone Derivatives 78

4.1 Introduction 78

4.2 Experimental 80

4.2.1 α-Glucosidase inhibition assay 80

4.2.2 Kinetic studies 80

4.2.3 Fluorescence Measurement 81

4.2.4 Atomic Force Microscopy (AFM) Measurements 81

4.2.5 Molecular docking analysis 81

4.2.6 Sucrose-loading test 82

4.2.7 Inhibitory activity of target compounds against α-glucosidase on everted intestine sleeves 83

4.2.8 Statistical analysis 83

4.3 Results and discussion 83

4.3.1 α-Glucosidase inhibition assay 83

4.3.2 Kinetics of α-glucosidase inhibition 87

4.3.3 Fluorescence spectra 88

4.3.4 Thermodynamic parameters and nature of the binding forces 90

4.3.5 Synchronous Fluorescence 91

4.3.6 Atomic Force Microscopy Measurements 92

4.3.7 Molecular docking analysis 94

4.3.8 Reduction of postprandial blood glucose by target compounds 95

4.3.9 Inhibition of α-glucosidase activity on everted intestine sleeves 
 96

4.4 Summary 98

Chapter 5 Conclusion 100

5.1 Research results 100

5.2 The innovation of this thesis 101

Reference 108

致 谢 124

About the Author 125

Chapter 1 Literature Review

1.1 Introduction

Acetophenone and its derivatives are one of the commonly used chemical scaffolds, which plays an important role in medicinal chemistry and agrochemicals These naturally occurring compounds were found in many foods, such as apples, apricots, bananas, beef, cheese, and cauliflowers (Müller-Schwarze, & Houlihan, 1991) It is also widely presented in

the fungi as Lindgomyces madisonensis (G416) (Paguigan et al 2016), as well as in many

plants, such as Camellia sinensis (Dong et al 2012, Emami et al 2018) These of structurally diverse compounds have shown significant cytotoxicity (Kouloura et al 2012; Yang et al 2015), anti-imflammatory (Bali, Ohri, and Deb 2012; Sala et al 2001), antiproliferative (Miyake et al 2016), anti-tuberculosis (Huang et al., 2017), anti-cancer (Ryu et al, 2016), anti-bacterial ( Maree et al., 2014), antifungal (Shaikh et al., 2016) Moreover, they also good substrates for several organic transformations (Wakade et al., 2017)

In recent years, acetophenone and their derivatives have also been recognized as important intermediates for the synthesis of agrochemicals, pharmaceuticals and functional materials (Dayan et al., 2014, Morita et al., 2016) For example, haloacetophenones have been

synthesized as newly potent nematicides against Meloidogyne incognita (Tocco et al 2017)

Acetophenone benzoylhydrazones have been as antioxidant agents (Emami et al 2018) 1,2,3-Triazole tethered acetophenones have been demonstrated as a potent antifungal agents

against some human pathogenic fungal strains such as: Candida albicans, Fusarium

oxysporum, Aspergillus flavus, Aspergillus niger, and Cryptococcus neoformans (Shaikh et al

2016)

2,4-Dihydroxy-5-methylacetophenone were isolated from the higher fungus Polyporus

picipes (Polypo-raceae) (Simonsen et al 2004) In our previous studies, 2,4-dihydroxy-5-methylacetophenone and its derivatives exhibited antifungal activity against

some important plant fungal pathogens as: Cytospora sp., Glomerella cingulate, Pyricularia

oryzaecar, Botrytis cinerea and Alternaria solani (weishi et al., 2016) Due to the broad

activity of acetophenone and its derivatives, the potential of this compound as pharmaceuticals, agrochemicals still need to be investigated

1.2 Natural products of Isobutylphenone

1.2.1 Identification and Bioactivity
of Callistemon

Six alkylphloroglucinol derivatives (1–6) were isolated from dried powdered leaves

and stems of Callistemon citrinus (Curtis) All the compounds were evaluated on sEH

inhibitory effect The compounds (5 and 6, as a mixture) exhibited the highest sEH inhibitory

activity with an IC50 values of 0.7 µM (Khanh et al 2016)

Myrtucommulone D (7) were isolated from the twigs and leaves of Callistemon

salignus which showed significant antibacterial activity against Staphylococcus aureus and

three drug resistant S aureus strains with MIC values of 1.953 and 0.975 µg/mL,

respectively Isomyrtucommulone B (8) displayed remarkable antibacterial activity

against Escherichia coli with an MIC value of 0.122 µg/mL Cytotoxic assay revealed that

isomyrtucommulone B (8) was the most active against HCT116 with an IC50 value of 2.09 ±

0.10μM (Qin et al 2017)

The extract of leaves of Callistemon lanceolatus comprised six acylphloroglucinols

derivative (9-15) All of the compounds showed very strong antibacterial activity (Rattanaburi

et al 2013).

Viminalins A-O (16-30) were isolated from the fruits of Callistemon viminalis The

cytotoxic activities of compounds 16-30 were evaluated and compound 21 displayed

moderate cytotoxic activity against HepG-2 (IC50 9.2 µM), MCF-7 (IC50 17.0 µM ) and U2-

OS (IC50 27.4 µM) cells (Wu et al 2017).

Triketone−phloroglucinol−monoterpene hybrids (31 and 32) and (33) were isolated

from the plant Callistemon rigidus (Myrtaceae) Compounds 31−33 were tested on the

antibacterial effect against five Gram-positive and four Gram-negative strains Among them,

compound 31 exhibited moderate antibacterial activity against all Gram-positive bacteria with

MIC values ranging from 16 to 32 μg/mL (Cao et al 2016)

From the dichloromethane extract of the flowers of Helichrysum gymnocomum

(Asteraceae) two acylphloroglucinol derivatives, 34 and 35 were isolated Compound 35

showed potent activity against (6.3–45 µg/ml) for eight of the ten pathogens tested, including

Staphylococcus aureus (6.3 𝜇g/ml) and methicillin and gentamycin resistant strain of S

aureus (7.8 𝜇g/ml) (Drewes and van Vuuren 2008)

1.2.2 Identification and Bioactivity of Hypericum

Compounds from 36 to 42 were isolated from the dichloromethane extract of the

Greek endemic plant Hypericum jovis All six compounds demonstrated significant

antioxidant activity Two of them compounds 36 and 38 possessed activity at a cellular level

comparable to Trolox, protecting against ROS (Athanasas et al 2004)

(±)-Japonicols A−D (43a/43b−46a/46b), four pairs of new phloroglucinol-based

terpenoid enantiomers, were isolated from Hypericum japonicum Compounds 43a/43b,

44a/44b, and 45a/ 45b possess 2-oxabicyclo[3.3.1]nonane, pyrano[3,2-b]pyran, and

benzo[b]cyclopenta-[e]oxepine ring systems, respectively The effects of the phloroglucinols

on anti-Kaposi‟s sarcoma-associated herpesvirus were assessed, and 44a exhibited a moderate

inhibitory effect, with an EC50 value of 8.75 μM and a selectivity index of 16.06 (Hu et al 2016)

Three new acylphloroglucinol derivatives have been isolated from the hexane extract

of the aerial parts of Hypericum prolificum L prolificin A (47), prolifenone A (48), and

prolifenone B (49) Among them, prolificin A showed growth inhibition of human breast

(MCF-7), lung (NCI-H460), CNS (SF-268), stomach (AGS), and colon (HCT-116) tumor cell lines with IC50 values ranging from 23 to 36 μM (Henry et al 2006)

Eight new polyprenylated acylphloroglucinol derivatives, hypercohins B−J (50-57)

were isolated from the aerial parts of Hypericum cohaerens Hypercohins B−D (50−52)

exhibited moderate inhibitory activity (IC50 5.8−17.9 μM) against the tested tumor cell lines and moderate toxicities, while other isolates exhibited no inhibitory activities (Liu et al 2016)

Some of acylphloroglucinol derivatives, andinin A (58), uliginosin A (59), uliginosin B

(60), and isouliginosin B (61) were isolated from the underground plant parts of Hypericum

andinum Andinin A (58) displayed antidepressant-like activity in a mouse forced-swimming

test when administered orally at doses of 3, 10, and 30 mg/kg (Ccana-Ccapatinta et al 2014).

Some compounds (62-68) have been isolated from Hypericum papuanum and the

bicyclic compounds hyperguinones A and B (67/68, 69/69a) and hyperpapuanone (70/70a), respectively In which the compound (70/70a), showed the most potent antibacterial activity

against all of the three tested bacteria with MICs values against B cereus were 8 μg/mL, S

epidermidis 8 μg/mL, M luteus 16 μg/mL (Winkelmann et al 2001).

1.2.3 Identification and Bioactivity of Pilidiostigma

Dibenzofurans 71-76 were
isolated from the leaves of Pilidiostigma glabrum which

exhibited potential inhibited the synthesis of PGE2 in 3T3 cells and the synthesis of nitric oxide in RAW264 macrophages with IC50 values in the low micromolar range (Shou et al 2012)

Pilidiostigmin (77) a novel dimeric acylphloroglucinol derivative, was isolated from

the leaves of the Australian plant species Pilidiostigma glabrum (Myrtaceae) Pilidiostigmin

inhibited the synthesis of nitric oxide in LPS-stimulated RAW 264.7 macrophages was 21.0%, 37.1%, and 60.6% at concentrations of 0.29, 0.87, and 7.8 𝜇M, respectively (Shou et al 2013)

1.2.4 Identification and Bioactivity of Corymbia


Myrtucommulones A (78) and D (79) and myrtucommulones F-I (80-83) were isolated

from the seeds of the Queensland tree Corymbia scabrida Compounds myrtucommulones A,

D, and F-I also inhibited the specific binding of [3H]3-methylhistidylTRH to HEK cell membranes expressing recombinant rat TRH receptors-2 with IC50 values of 39, 11, 16, 24, 31,

and 16 μM, respectively (Carroll et al 2008)

The myrtle phenolic compounds semimyrtucommulone (84) and myrtucommulone A

(85) were isolated from Myrtus communis L showed potent protective effect in simplified

models of oxidative degradation of cholesterol and LDL, two processes that are well known to play a role in cardiovascular diseases These plant-derived phenolics were also qualified as interesting dietary antioxidants with potential antiatherogenicity (Rosa et al 2008)

Eight phloroglucinol derivatives (86-95) were isolated from the leaves of Myrtus

communis (Myrtaceae) Among them, racemic 94 exhibited significant antimicrobial activities

against Staphylococcus aureus and Bacillus subtilis Comparable antimicrobial activities were

also found for the enantiomers (+)-94 and (-)-94 obtained from 94, suggesting that the

stereochemistry was not essential for its antimicrobial activity (Tanaka et al 2018)

The dimeric nonprenylated acylphloroglucinol semimyrtucommulone (98), phloroglucinol myrtucommulone A (96) and 97 were obtained from the leaves of myrtle

(Myrtus communis) Myrtucommulone A showed significant antibacterial activity against

Myrtucommulones J, K, and L (99−101) and myrtucommulone A (102) were extracted

of the leaves of Myrtus communis All the compounds were evaluated the antibacterial

potential against Staphylococcus aureus Compound 99/99a, however, showed a certain

specificity, being 35-fold more active against S aureus with respect to normal eukaryotic cells with a MIC of 0.38 μM (Cottiglia et al 2012)

Phytochemical investigation on Myrtus communis Linn afforded

myrtucommuacetalone (103), myrtucommulone M (104), and compound 105 In which, compound 103 also exhibited significant antiproliferative activity (IC50 < 0.5 μg/mL) against

T-cell proliferation Myricetin (105) exerted a significant inhibition (IC50 = 1.6 μg/mL) on

zymosan-stimulated whole blood phagocytes ROS production (Choudhary et al 2013)

The polyphenol-enriched fraction of an ethanolic hops extract (Humulus lupulus) was

separated to provide four acylphloroglucinol-glucopyranosides (106-109) The aglycon 110

exhibited inhibition of COX-1 activity with an IC50 of 3.8 μM The inhibitory potential of the

glucosides was 106 > 107> 108 slowing that increasing the length of the acyl side chain, led

to decreased potency (Bohr et al 2005)

Psorothatins A−C (111−113) were isolated from 
 the native American plant

Psorothamnus fremontii Psorothatin C (113) showed highest activity against

methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium,

with IC50 values in the range 1.4−8.8 μg/mL (Yu et al 2015)

2016, Adams et al survefed the healing properties of 14 phloroglucionl derived

compounds (114-120) from the species Pilidiostigma glabrum, Myoporum montanum,

Geijera parviflora, and Rhodomyrtus psidioides for assessing their ability to induce or

suppress Collagen I and Collagen III expression in human skin fibroblasts in culture

7-geranyloxycoumarin (114) was able to significantly increase Collagen I (23.7%, p b 0.0002)

expression in comparison to control Significant suppression of Collagen III was observed for the compounds flindersine (11.1%, p b 0.02), and (N-acetoxymethyl) flindersine (27%, p b 0.00005) These finding implicated that these compounds could potentially alter the expression of different collagens in the skin, enabling the development of new wound healing therapies and new approaches for treating various skin diseases as well as photo (sun) damaged, and aged skin (Adams et al 2016)

1.3 Synthesis of Isobutylphenone Deivatives

1.3.1 Antifungal

Acylphloroglucinol derivatives reseability natural product were synthesized to identify

antifungal activity against Cryptococcus spp Compound (122) showed the most promising

with antifungal activity (MICs, 1.5−2.1 μg/mL) and low cytotoxicity against the mammalian

Vero and LLC-PK1 cell lines (IC50 values >50 μg/mL) (Yu et al 2016)

A series of dipeptides with benzothiazole and amide-imidazole scaffolds were designed and synthesized to find new regents for drug-resistant fungal infections Several

compounds, such as 125a-125e showed better antifungal activity than FLC with MIC

values in the range of 0.125 - 2 𝜇g/mL, particularly for Candida albicans, C neoformans, and Candida tropicalis (Zhao et al 2016)

The antifungal compounds were synthesized from p-hydroxybenzaldehyde (126a–

126f) and p- hydroxybenzoic acid (131a–131f) The antifungal activity were evaluated against

standard fungal strains, such as Trichophyton rubrum CCT 5507 URM 1666, Trichophyton

mentagrophytes ATCC 11481, and Candida albicans ATCC 10231 Minimum inhibitory

concentrations and minimum fungicidal concentrations were determined according to Clinical and Laboratory Standards Institute protocols M38-A2, M27-A3, and M27-S4 The amine

series 127b–127e, especially 127c and 127e, were effective against filamentous fungi and

yeast (MIC from 7.8 to 31.2 µg/mL) (Caneschi et al 2017)

1.3.2 Anti bacterial, antimalarial and anticancer

Fifteen natural-product-like acylphloroglucinol congeners were synthesized, which

possessed different acyl side chains Compound 138j was identified as a promising lead for

the generation of new anti-MRSA drug development

It was discovered by optimization of the side chain length in light of the potency, the breadth of the antibacterial spectrum, the rate of bactericidal action, as well as the membrane

strain (JCSC 2172), and its MIC was 3-4 ford of magnitude lower than that of vancomycin (Tan et al 2017)

Furohyperforin (139) showed a similar potency as the natural product (1.7 µM),

suggesting a specific role for the oxygen-sensitive enolized β-dicarbonyl This was further confirmed by the observation that the introduction of an oxygen or an halogen at the α-carbon

of the β-dicarbonyl (compounds 140 and 141) substantially maintained or even increased the

activity (2.0 𝜇M for 140; 0.6 𝜇M for 141) (Verotta et al 2007)

Myrtucommulones are a class of pharmacologically active natural products which offer various alternatives to the treatment of pain, inflammation and cancer 2017, Charpentier

and Jauch have successfully synthesized MCA (142) via developing an efficient, eco-friendly

and cheap multicomponent reaction This protocol was interesting method for the synthesis of Myrtucommulones for medicinal studies and related compounds (Charpentier and Jauch 2017)

A series of symmetric polyoxygenated dibenzofurans (143-151) with 2-methylbutyryl

moieties at C-4 and C-6 were obtained from phloroglucinol through a sequence of including monoacylation, iodination, Suzuki-Miyaura coupling, oxidative dimerization and cyclization Some of the compounds were active against Gram-positive bacteria, including multi resistant

Staphylococcus aureus Compound 148 with propyl chains at C-2 and C-8 exhibited the best

antibacterial activity with MIC values of 0.56, 2.23 and 4.64 µg/mLagainst NRS 402 S

aureus (VISA), and ATCC29212 (E.faecalts) and ATCC25923 S aureus (MSSA),

respectively (Oramas-Royo et al 2017).

Psorothatin C (152) was synthesized from phlorogluciol, which is a known compound

displaying activity against methicillin-resistant Staphylococcus aureus and

vancomycin-resistant Enterococcus faecium, with IC50 values of 1.4 μg/mL against MRSA

33591 and 2.4 μg/mL against S aureus ATCC 29213 (Fobofou et al 2016)

1.4 Research Objectives

Although great success has been achieved over the past decades in studying the isolated, synthesis and evaluated bioactivity of acetophenone and its derivatives by different research groups, synthesizing novel of acetophenone derivatives remain important for the discovery and development of new agrochemicals and pharmaceuticals

In previously work, we had been synthesized of lipophilic acetophenone derivatives with different acyl groups Biological showed that the test compounds with an isobutyryl group exhibited the highest antifungal activity As a continuation of this work:

1 Synthesis 3, 4 and 5-modified derivatives with the isobutyrophenone skeleton and ether and ester derivatives of isobutyrophenone were synthesized with different substituents

2 The evaluation of antifungal activity of those compounds against seven pathogenic

fungi: Botrytis cinerea, Alternaria solani, C.gloeosporiodes, Fusarium solani, F

Graninearum, Cytospora sp（Valsa mali）and Magnaporthe grisea by using a mycelia growth

inhibition method Testing on fruits infected by C Gloeosporiodes in vivo and the effect of

inhibition on hyphal morphology were evaluated

3 Evaluation of the potential enzymatic activities against Mg-Fba and the identification of the binding-modes of the most active compounds The enzymatic activity against Class II Fba was also were further analyzed carefully by kinetic mechanism and molecular docking algorithms

4 Evaluation of their ability to inhibit α-glucosidase of all the synthesis compounds The relationship between the structure and the biological activity of all test compounds were also discussed

Chapter 2 Synthesis Ether and Ester Devivatives of

Isobutylphenone and Biological Activity

2.1 Introduction

Natural products play an important role in discovery and development of new drugs (L Cantrell, Franck E, and Stephen O, 2012) The presence of isobutylphenone in many plants not only have composited of structurally diverse compounds but also the extracts of that compounds has showed significant cytotoxicity (Kouloura et al 2012; Yang et al 2015), anti-imflammatory (Bali et al 2012; Sala et al 2001) , antiproliferative (Miyake et al 2016) , anti-tuberculosis (Huang et al 2017) , anti-cancer (Ryu et al 2016), anti-bacterial (Maree et al 2014), antifungal (Shaikh et al 2016), or as good substrats for several organic transformations (Wakade et al 2017)

Agricultural crops are facing tremendous losses due to pests, diseases, and weed damages which result in direct economic losses including reduction in grain yield and quality Despite the availability of effective synthetic fungicides, new fungicides with novel chemical structures which have higher potency and broader spectrum of activity against resistant fungal strains are still needed

The rice blast fungus, which is caused by the filamentous ascomycete fungus

Magnaporthe oryzae (M grisea) This plant pathogens has been reported in more than 85

countries (Kato 2001), mainly concentrated in Africa, Latin America and Asia (Law et al 2017) In the rice producing countries, yield loss due to rice blast ranges from approximately 10–30% annually and this rate may be reached to 50% during disease epidemics (Ashkani et

al 2015; Skamnioti and Gurr 2009).In addition, the scab fungus can produce mycotoxins called polyketides, which contaminate grain-based foods, causing a serious food safety and economic problem in the United States (Cleveland et al 2003) Class II fructose 1,6-bisphosphate aldolase (Fba) is an enzyme critical for bacterial, fungal, and protozoan glycolysis/gluconeogenesis Importantly, humans lack this type of aldolase, having instead a class I FBA that is structurally and mechanistically distinct from class II Fbas As such, it has been suggested that Class II FBA could be a viable drug target and a lot of attentions are focused on the drug design of the enzyme

In previously work, we have reported the design and synthesis the libraries of

acetophenone from the cultures of the higher fungus Polyporus picipes, which exhibited

potential antifungal agent against several phytopathogens (Ma et al 2013; Nandinsuren et al 2016; Shi et al 2016).In this some of lipophilic acetophenone derivatives, which attached isobutyryl group at 2 position 2,4-dihydroxytoluene exhibited the highest antifungal activity (Shi et al 2016) In addition, the compound containing isobutyryl group increase biological

activity of the synthesis compounds has also been demonstrated by Yu et al (Yu et al 2016)

As a continuation, in this study we proceeded to synthesize the ether and ester derivatives of isobutyrophenone The potential of antifungal, enzyme inhibition activity of target compounds were evaluated The relationship between structure and activity were also discussed

2.2 Experimental

2.2.1 Chemistry

Commercial solvents and reagents were of reagent grade (Aladdin Chemistry Co Ltd Shanghai, China), or were purified by standard methods before use Thin-layer chromatography (TLC) was performed on silica gel 60 F254 (Qingdao Marine Chemical Ltd., P

R China) Column chromatography (CC) was performed over silica gel (200 – 300 mesh, Qingdao Marine Chemical Ltd.) The melting points of the products were determined on an X-4 apparatus (Beijing Tech Instrument Co., Beijing, P R China) and are uncorrected MS spectra were taken on a BrukerDaltonics esquire 6000 (ESI-ION TRAP) Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance spectrometer (Unity plus 400 or

500 MHz) (Bruker Biospin, Rheinstetten, Germany) with tetramethysilane (TMS) as internal standard Chemical shift values (were given in parts per million (ppm)), the coupling constants (J) are expressed in hertz (Hz)

2.2.2 Synthesis of ether and ester derivatives of isobutylphenone

2.2.2.1 General procedure for the synthesis of intermediate compounds 2 and 3

Synthesis of 2,4-dihydroxytoluene (2) (Xie et al 2004)

A mixture of 2,4-dihydroxybenzaldehyde (11.04 g, 80 mmol) and sodium cyanoborohydride (6.03 g, 96 mmol ) in 240 mL of THF was added methyl orange ( 2-3

drops) as an indicator, giving the solution a yellow color; a solution of 4 N HCl (60 mL) was

slowly added to the reaction system, keeping the solution orange

Scheme 2-1 Synthesis of ether and ester derivatives of isobutylphenone

The reaction mixture was stirred for 16 h at room temperature Water was added, and the mixture was extracted with ethyl acetate three times Ethyl acetate layer was washed with brine solution and finally dries over MgSO4 Purification by CC on silica gel eluting with petroleum ether (PE) - ethyl acetate (EA): (10: 1) gave as a white solid (2) (8.23 g); yield:

82.8%; m.p 103.2–104.7 °C; 1H NMR (400MHz, CD3OD) δ 6.84 (d, J = 8.1 Hz, 1H, ArH), 6.28 (d, J = 2.4 Hz, 1H, ArH), 6.20 (dd, J = 8.1, 2.4 Hz, 1H, ArH), 2.06 (s, 3H, CH3) 13C NMR (100 MHz, CD3OD) δ 157.2, 157.1, 131.9, 116.5, 107.3, 103.2, 15.4 HRMS (ESI), m/z

calcd for C7H8O2[ M+ H]+ 125.0597; found 125.0598

Synthesis of compound 1-(2,4-dihydroxy-5-methylphenyl)-2-methylpropan-1-one (3)

To a solution of 2,4-dihydroxytoluene 2 (1.24 g, 10 mmol) in BF3.Et2O (12mL) was added isobutyric anhydride (3.31mL, 20 mmol) The reaction mixture was heated to 90 oC and stirred for 16 h After cooling, water (100mL) was added and mixture was neutralized with saturated aqueous NaHCO3 and diluted with ethyl acetate (EA) (200 mL) After filtration, the solution was extracted with EA (3 x 30 mL) The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo Purification by CC on silica gel eluting with

PE–EA (15: 1 5: 1) gave 3 as a yellow solid (1.45 g)

2.2.2.2 General Procedure To Synthesized Compounds 4a-e by Conventional Method

To a solution of compound 3 (194.23 mg, 1 mmol) and anhydrous potassium

carbonate (279.2 mg, 2 mmol) in dry acetone (3 mL), different alkylhalides (1.2 mmol) was added The mixture was refluxed in oil bath at 58 oC for 3h After cooling and filtration, the solution was concentrated under vacuum and then purified by CC on silica gel eluting with

PE : EA (20 : 1) to obtain the title compounds 4a–e

1-(2,4-Dihydroxy-5-methylphenyl)-2-methylpropan-1-one (3) Yellow solid; yield

1-(4-ethoxy-2-hydroxy-5-methylphenyl)-2-methylpropan-1-one (4b)

Yellow oily liquid; yield 64.0%; 1H NMR (400 MHz, CDCl3) δ 12.97 (s, 1H, OH),

7.48 (s, 1H, ArH), 6.37 (s, 1H, ArH), 4.06 (q, J = 7.0 Hz, 2H, CH2), 3.52 (dt, J = 13.7, 6.8 Hz,

1H, CH), 2.14 (s, 3H, CH3), 1.44 (t, J = 7.0 Hz, 3H, CH3), 1.22 (d, 6H, 2CH3) 13C NMR (101 MHz, CDCl3) δ 208.9, 164.7, 163.8, 130.9, 118.2, 111.4, 99.9, 64.1, 34.6, 19.6, 15.9, 14.7

ESI – MS m/z: [M+H]+ 223.18

1-(4-(allyloxy)-2-hydroxy-5-methylphenyl)-2-methylpropan-1-one (4c)

Light yellow solid; yield 76.5%; m.p 49.0–51.0 oC 1H NMR (400 MHz, CDCl3) δ 12.98 (s, 1H, OH), 7.52 (s, 1H, ArH), 6.40 (s, 1H, ArH), 6.26–5.88 (m, 1H, -CH=CH2 ), 5.43 (m, 1H, CH2=CH), 5.31 (m, 1H, CH2=CH), 4.59 (dt, J = 5.1, 1.5 Hz, 2H, CH2), 3.54 (dt, J =

13.7, 6.8 Hz, 1H, CH), 2.20 (s, 3H, CH3), 1.26 (d, 6H, 2CH3) 13C NMR (101 MHz, CDCl3) δ 208.7, 164.3, 162.9, 132.2, 130.8, 117.9, 117.6, 111.3, 99.9, 99.9, 68.6, 34.3, 19.2, 15.5 ESI–

MS m/z: [M+H]+ 251.18

2.2.2.3 General synthetic procedure for compounds 5a-i by conventional method

To a solution of different acid (0.75 mmol) in dichloromethane (2 mL) was added the

compound 3 (97.15 mg, 0.5 mmol) and 4–dimethylaminopyridine (DMAP) (6.1mg, 0.05

mmol) The solution was cooled to 0 oC and stirred for 15 min prior to addition of N,N –

dicyclohexylcarbodiimide (DCC) ( 123.79 mg, 0.6 mmol) After stirring for 4 h at room temperature The mixture was concentrated under vacuum and then purified by CC on silica

gel eluting with PE–EtOAC ( 20: 1-15: 1) to obtain corresponding compounds 5a–i

5-hydroxy-4-isobutyryl-2-methylphenyl acetate (5a)

White solid; yield: 63.8%; m.p 50.2–50.4 oC; 1H NMR (400 MHz, CDCl3) δ 12.49 (s,

1H, OH), 7.63 (s, 1H, ArH), 6.69 (s, 1H, ArH), 3.56 (dt, J =13.6, 6.8, 1H, CH), 2.33 (s, 3H,

9.3 ESI–MS m/z: [M-1]- 249.02

4-isobutyryl-6-methyl-1,3-phenylene dipropionate (5b2 )

Yellow oily liquid; yield 25.6%.1H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H, ArH), 6.90

(s, 1H, ArH), 3.31 (dt, J = 13.6, 6.8 Hz, 1H, CH-(CH3x2)), 2.60 (q, J =7.5 Hz, 4H, 2xCH2), 2.18 (s, 3H, CH3), 1.25 (t, J = 7.6 Hz, 6H, 2xCH3), 1.15 (d, J = 6.9 Hz, 6H, 2xCH3) 13C NMR (101 MHz, CDCl3) δ 204.8 (-C=O), 173.3 (-O=C-O-), 172.5 (-O=C-O-), 152.5 (ArC), 148.4 (ArC), 132.4 (ArC), 129.1 (ArC), 128.6 (ArC), 118.4 (ArC), 39.2 (-CH-(CH3x2)), 28.3 (2xCH2), 19.5 (CH3x2), 16.6 (H3C-Ar), 9.8 (CH3), 9.5 (CH3) ESI–MS m/z: [M+23]+ 329.24

5-hydroxy-4-isobutyryl-2-methylphenyl 2-iodoacetate (5e)

Yellow solid; yield 82.0%; m.p 72.2–74.5 oC 1H NMR (400 MHz, CDCl3) δ12.48 (s, 1H, OH), 7.64 (s, 1H, ArH), 6.71 (s, 1H, ArH), 3.92 (s, 2H, CH2), 3.56 (dt, J =13.6, 6.8 Hz,

1H, CH), 2.21 (s, 3H, CH3), 1.24 (d, J =6.8 Hz, 6H, 2CH3) 13C NMR (101 MHz, CDCl3) δ 210.0, 166.5, 162.7, 154.6, 132.3, 121.2, 116.9, 111.5, 35.2, 19.4, 15.8, -7.0 ESI–MS m/z: [2M+23]+ 747.07

4-isobutyryl-6-methyl-1,3-phenylene bis(2-methylpropanoate) (5f 2)

Yellow oil; yield 98.0%; 1H NMR (500 MHz, CDCl3) δ 7.52 (s, 1H), 6.85 (s, 1H),

3.43 – 3.18 (m, 1H), 3.00 – 2.66 (m, 2H), 2.19 (s, 3H), 1.33 (d, J = 7.0 Hz, 6H), 1.30(d, J = 7.0 Hz, 6H), 1.15 (d, J =6.9 Hz, 6H) 13C NMR (126 MHz, CDCl3) δ 204.3, 175.2, 174.6, 151.8, 147.7, 131.6, 129.1, 127.9, 117.7, 38.8, 34.4, 34.3, 19.1, 18.9, 18.8, 15.9 HRMS (ESI),

m/z calcd for C19H26O5Na+ [M+ Na]+ 357.1672; found 357.1666

5-hydroxy-4-isobutyryl-2-methylphenyl pivalate (5g1 )

Yellow oil; yield 64.7%; 1H NMR (500 MHz, CDCl3) δ 12.48 (s, 1H), 7.62 (s, 1H),

6.65 (s, 1H), 3.71–3.40 (m, 1H), 2.13 (s, 3H), 1.37 (s, 9H), 1.24 (d, J = 6.8 Hz, 6H) 13C NMR (126 MHz, CDCl3) δ 210.0, 176.0, 162.8, 155.8, 131.9, 121.2, 116.4, 111.9, 39.5, 35.2,

27.3, 19.5, 15.8 HRMS (ESI), m/z calcd for C16H22O4+ [M+ H]+ 279.1591; found 279.1590

4-isobutyryl-6-methyl-1,3-phenylene bis(2,2-dimethylpropanoate) (5g2)