Design synthesis and biological activities of novel isobutyrophenone derivatives

博 士 学 位 论 文 新型异丁酰苯衍生物的设计合成及其生 物活性研究 学 科 专 业 化学生物学 研 究 方 向 天然活性物质与构效关系 论 文 作 者 墙氏梅良 指 导 教 师 高锦明教授 联合指导教师 李定 副教授 论文提交时间 2019 年 5 月 Dissertation for Doctor Degree Northwest A＆F University in 2019 Design, Synthesis, and Biological Activities of Novel Isobutyrophenone Derivatives Major: Chemical Biology Research Field: Structure and Bioactivities of Natural Products Candidate: Tuong Thi Mai Luong Supervisor: Professor Gao Jinming Co-Supervisor: Assoc. Prof Li Ding Date of submission: 2019.05 分类号：TQ041 学校代码：10712 U D C： 547 研究生学号：2014070049 密 级：公开 西北农林科技大学博士学位论文 新型异丁酰苯衍生物的设计合成及其生 物活性研究 论文作者: 墙氏梅良 指导教师: 高锦明教授 指导小组： 答辩委员会： 张尊听 教授 博导 陕西师范大学化学化工学院 主席 段金友 教授 博导 西北农林科技大学化学与药学院 委员 谢卫青 教授 博导 西北农林科技大学化学与药学院 委员 党永军 教授 博导 复旦大学基础医学院 委员 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 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- 二磷酸醛缩酶（II 型 Fba）；第二部分测试了它们的α-糖苷酶的抑制活性，研究了构效 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） 表现出一定的广谱抗菌活性。其中，5a 对番茄灰霉病菌、苹果炭疽菌和马铃薯干腐病 菌具有较好的抑制活性，EC50 分别为 4.87 μg/mL，优于阳性对照药物多 菌灵；5c 对苹果腐烂病菌具有最好的抑制活性，EC50 为 1.42 μg/mL，较多菌灵的活性 提高了约 23 倍；5d 对小麦赤霉病菌和马铃薯干腐病菌具有最好的抑制活性，EC50 分别 为 6.67 μg/mL，5e 对小麦赤霉病菌也具有较好的抑制活性，EC50 为 2.49 μg/mL。 酶抑制实验显示化合物 3，5a，5b，5c，5d 和 5e 具有抑制 II 型果糖-1,6-二磷酸醛缩 酶酶活性，其 IC50 在 28.82 μM 之间。 2、进一步设计合成了 30 个新异丁酰苯酚的衍生物。构效关系研究表明，在苯环 3 位和 5 位引入溴原子，4-羟基与氯乙酰基酯化，可显著提高化合物的抗真菌活性和广 谱性。其中，化合物 12b 对 7 种植物病原真菌显示出最好的杀菌活性，EC50 在 1.44 μg/mL 之间. 同时，化合物 12b 还表现出具有最高的 II 型果糖-1,6-二磷酸醛缩酶抑 制活性，IC50 为 3.63 μM，较化合物 5e（IC50 = 28.91 μM）酶抑制活性提高了近 7.9 倍； 3、安全性评价表明以化合物 12b 为代表的异丁酰苯酚衍生物对两种正常细胞 NIH3T3 和 293T 没有毒性，环境危害实验评价对家蚕幼虫也表现出无毒的效果。离体果 实试验表明化合物 12b 具有保护果实(或植物)免受病原菌侵染的实际潜力，其防护能力 摘 要 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 作用。其中，化合物 12d 表现出最好的α-葡萄糖苷酶抑制活性，IC50 为 0.57 μM，12d 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 摘 要 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. Gloeosporiodes and , Fusarium solani with EC50 values 4.87 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 6.67 μg/mL, respectively, 5e also had a good inhibitory activity against Fusarium graminearum with an EC50 of 2. This compounds also exhibited potential Mg-Fba enzyme inhibitory activity with IC50 data in the range of 28.

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 bromo group at the 3 to 5 position of the benzene ring and the esterification of the 4-hydroxy 摘 要 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. 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.

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. 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. 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 .2 Natural products of Isobutylphenone.1 Identification and Bioactivity of Callistemon .2 Identification and Bioactivity of Hypericum .3 Identification and Bioactivity of Pilidiostigma .4 Identification and Bioactivity of Corymbia .3 Synthesis of Isobutylphenone Deivatives .2 Anti bacterial, antimalarial and anticancer. Synthesis Ether and Ester Devivatives of Isobutylphenone and Biological Activity .2 Synthesis of ether and ester derivatives of isobutylphenone.3 In Vitro Antifungal Bioassay .4 Enzyme Inhibition Activity.4 Results and discussion .2 Mg-Fba inhibitory activities.3 Molecular Modeling Studies.

Synthesis of Bromo Isobutylpheone Derivatives and Antifungal Activity .1 General procedure for the synthesis of intermediate compounds .2 General procedure to synthesize compound 8a-8f, 10a-10d, 12a-12m by conventional method.3 In Vitro Antifungal Bioassay.4 Effect of inhibition on Hyphal Morphology .5 In Vivo Testing on apple fruits Infected by C.6 Enzyme inhibition activity assays .4 Results and Discussion .2 In Vivo Testing on apple fruits Infected by C.3 Effect of inhibition on Hyphal Morphology .1 Cytotoxicity on normal cells line .2 Silkworm toxicity study. Alpha-Glucosidase Inhibiting Activity of Isobutyrophenone Derivatives .1 α-Glucosidase inhibition assay .4 Atomic Force Microscopy (AFM) Measurements .5 Molecular docking analysis. Sucrose-loading test .7 Inhibitory activity of target compounds against α-glucosidase on everted intestine sleeves .3 Results and discussion .1 α-Glucosidase inhibition assay .2 Kinetics of α-glucosidase inhibition .4 Thermodynamic parameters and nature of the binding forces .6 Atomic Force Microscopy Measurements .7 Molecular docking analysis .8 Reduction of postprandial blood glucose by target compounds .9 Inhibition of α-glucosidase activity on everted intestine sleeves .2 The innovation of this thesis. 124 About the Author.

Literature Review Chapter 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. 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. 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.

For example, haloacetophenones have been synthesized as newly potent nematicides against Meloidogyne incognita (Tocco et al. Acetophenone benzoylhydrazones have been as antioxidant agents (Emami et al. 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. 2,4-Dihydroxy-5-methylacetophenone were isolated from the higher fungus Polyporus picipes (Polypo-raceae) (Simonsen et al.

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. Due to the broad activity of acetophenone and its derivatives, the potential of this compound as pharmaceuticals, agrochemicals still need to be investigated.2 Natural products of Isobutylphenone 1.1 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.

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. Isomyrtucommulone B (8) displayed remarkable antibacterial activity against Escherichia coli with an MIC value of 0. Cytotoxic assay revealed that 2 Chapter 1.

Literature Review isomyrtucommulone B (8) was the most active against HCT116 with an IC50 value of 2.10μM (Qin et al. 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. 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 9.0 µM ) and U2- OS (IC50 27.4 µM) cells (Wu et al. 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.

Literature Review 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 4 Chapter 1. Literature Review Staphylococcus aureus (6.3 𝜇g/ml) and methicillin and gentamycin resistant strain of S.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. (±)-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[3.1]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.

Three new acylphloroglucinol derivatives have been isolated from the hexane extract of the aerial parts of Hypericum prolificum L. 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. Literature Review 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.