23 卷周强等 233 [16] 关环即 Algar-Flynn-Oyamada(AFO) 反应以其原料及试剂易得操作简单而成为目前为止最为广泛采用的一种黄酮醇合成法. 因此本研究采用 AFO 法, 即以邻羟基苯乙酮和苯甲醛为原料一锅和分步进行合成, 进一步用牛津杯法检测合成化合物对枯草

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1 DOI: /SP.J 应用与环境生物学报 Chin J Appl Environ Biol 2017,23 ( 2 ) : 黄酮醇类化合物的合成与抗菌活性 * 周强 1, 2 王淳 1** 李云萍 1 蒲文臣 1 中国科学院成都生物研究所成都中国科学院大学北京摘要以邻羟基苯乙酮和苯甲醛为原料, 采用 Algar-Flynn-Oyamada(AFO) 反应, 通过一锅法和分步法合成化合物 ; 进一步用牛津杯法以甲氧西林为细菌阳性试验对照 DMSO 为阴性对照, 检测合成化合物对枯草芽孢杆菌酿酒酵母菌黑曲霉菌的抑菌能力. 共合成 20 种黄酮醇类化合物, 其中包括 1 个新化合物 ; 合成化合物对酿酒酵母菌和黑曲霉菌并无明显抑制效果 (d < 9 mm), 而对枯草芽孢杆菌具有不同程度的抑制作用, 其中含卤素取代的黄酮醇对枯草芽孢杆菌具有显著的抑制作用 (d > 15 mm). 构效关系研究表明, 黄酮醇类化合物能有效对枯草芽孢杆菌形成抑制作用, 且黄酮醇骨架上含有卤素取代 ( 化合物 ) 表现出较好的抑菌作用 ;5- 甲氧基取代黄酮醇 ( ) 类化合物尽管不是活性最好的, 但与相应的 5 位无取代的黄酮醇比较, 如 12 vs 2 13 vs 3 14 vs 1, 活性有所提高. 本研究获得了一些具有较好抗菌活性的黄酮醇化合物, 可为药物发现和合成提供结构依据. ( 图 2 表 2 参 19) 关键词黄酮醇 ; 合成 ; 抗菌活性 ; 枯草芽孢杆菌 ; 构效关系 CLC R914.5 Synthesis and antibacterial activity of flavonols* ZHOU Qiang 1, 2, WANG Chun 1**, LI Yunping 1 & PU Wenchen 1 1 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu , China 2 University of Chinese Academy of Sciences, Beijing , China Abstract Flavonol, also known as 3-hydroxylflavonone, is a kind of natural flavonoid compounds that have various important physiological and pharmacological activities. This study aimed to establish the structure-activity relationship of flavonols against antibacterial activity, in order to provide the basis for further drug design. In all, 20 flavonols were synthesized, and their antibacterial activities were identified. Twenty flavonols, including 1 new compound, were synthesized by using the one-pot method or three-step approach, based on the Algar Flynn Oyamada (AFO) reaction. Their anti-microbial activities against Bacillus subtilis, Saccharomycetes, and Aspergillus niger were subsequently evaluated using the Oxford cup method by using dimethyl sulfoxide as a negative control and methicillin as a positive control. Our results indicated that these flavonols possess satisfactory antibacterial activity to Bacillus subtilis, but have no obvious inhibitive effect against Saccharomycetes and Aspergillus niger. Structure-activity relationship indicated that halo-substitution at either A- or B-ring (compounds 7, 8, 17, 19, and 20) might strengthen the antibacterial activity. In addition, 5-methoxy-substituted flavonols (compounds 12, 13, and 14) exhibited improved antibacterial activities relative to the ones with non-substitution at the 5-position (12 vs. 2, 13 vs. 3, 14 vs. 1). Our results provide preliminary data for designing and discovering new leading compounds with antibacterial activities. Keywords flavonol; synthesis; antibacterial activity; Bacillus subtilis; structure-activity relationship 黄酮醇, 即 3- 羟基黄酮, 是一类独特的黄酮化合物, 广泛存在于植物界中, 数量众多, 结构复杂多样, 具有许多重要的生物活性和药理作用, 如抗肿瘤清除自由基解毒抗炎提高人体免疫力等生物活性 [1-3]. 近年来, 黄酮类化合物的抗菌活性日益受到重视 [4], 研究表明黄酮类化合物能够通过抑制细菌核酸合成 [5-6] [7-8] 影响细菌细胞膜的功效和抑制 [9] 细菌能量代谢等多种作用机制发挥抗菌抑菌作用, 黄酮醇类化合物作为黄酮化合物大家族中的一员, 其抗菌活性也受收稿日期 Received: 接受日期 Accepted: * 国家自然科学基金项目 ( ) 资助 Supported by the National Natural Science Foundation of China ( ) ** 通讯作者 Corresponding author ( wangchun@cib.ac.cn) 到研究人员的关注, 如 Mirzoeva 等研究发现蜂胶中槲皮素能增强细菌内膜的通透性, 引起膜电位的改变, 进而增强抑菌活性 [10]. 此外, 研究还发现高良姜素能诱导金黄色葡萄球菌细胞质膜损伤导致钾离子的渗出, 造成细胞的自我分解或细胞壁损伤和渗透溶解, 产生抑菌作用 [11]. 虽然目前有一些关于黄酮醇化合物抗菌活性的报道, 但仅局限于少数从天然产物中分离的黄酮醇类化合物, 直到目前还没有系统的关于黄酮醇类化合物抗菌作用的研究. 由于黄酮醇往往在植物界中与类似物共存, 从混合物中获取难度大, 化学合成是大量获取的途径. 黄酮醇的合成有多种方法, 如 Auwers 法 [12] Baker- [13-14] Venkataraman 法和黄酮过氧丙酮 (DMDO) 氧化法 [15], 但是这些方法效率低选择性及对底物适应性差. 查耳酮氧化

2 23 卷周强等 233 [16] 关环即 Algar-Flynn-Oyamada(AFO) 反应以其原料及试剂易得操作简单而成为目前为止最为广泛采用的一种黄酮醇合成法. 因此本研究采用 AFO 法, 即以邻羟基苯乙酮和苯甲醛为原料一锅和分步进行合成, 进一步用牛津杯法检测合成化合物对枯草芽孢杆菌酿酒酵母菌黑曲霉菌的抑菌能力, 并探讨化合物抗菌活性的构效关系, 旨在为发现具有抗菌活性的黄酮醇类先导化合物提供依据. 层旋蒸除去溶剂后经硅胶柱色谱纯化得到二氢黄酮醇中间体. 在 0 下将化合物二氢黄酮醇中间体 (0.2 mmol) 无水醋酸钾 (9 mmol) 溶于冰醋酸中 (7 ml), 将碘 (0.45 mmol) 的醋酸 (7 ml) 溶液, 逐滴加入到反应液中并加热至沸腾 (5 h), 冷却至室温后用冰水淬灭, 过量的碘用焦硫酸钠去除, 用乙酸乙酯萃取溶液, 合并有机相, 旋蒸除去溶剂后用硅胶柱色谱纯化得到终产物材料与方法 1.1 仪器与材料 NMR 谱由 Bruker Avance 600 和 Bruker Ascend 400 核磁共振仪记录 ( 1 H:600 MHz,400 MHz; 13 C:150 MHz,100 MHz),TMS 作为内标. ESI-MS 由 Bruker Daltonics Bio-TOF-Q III 质谱仪记录. 常压柱层析硅胶 H( 目 ) 和薄层层析硅胶 GF 254 购自青岛海洋化工厂. 细菌真菌培养所用主要试剂购自 Invitrogen 公司, 其余试剂均为国产分析纯试剂. 实验菌株为枯草芽孢杆菌酿酒酵母菌黑曲霉菌. 1.2 实验方法化合物的合成化合物 1-10 参照 Ozturk 等报道的方 [17] 法合成 ( 图 1), 该方法是在经典的合成黄酮醇的方法 AFO 反应法的基础上进行改进而来, 因为缩短了原有反应的反应步骤, 所以黄酮醇的总产率得到较大提升, 多数黄酮醇的产率在 50% 以上. 以化合物 1 为例, 向 25 ml 圆底烧瓶中加入邻羟基苯乙酮 (1.66 mmol) 苯甲醛 (1.66 mmol) 氢氧化钠 (5 mmol), 以甲醇 (10 ml) 为溶剂, 回流加热, 用薄层色谱检测至反应完全. 随后将反应体系冷却至室温, 加入氢氧化钠水溶液 (0.5 mol/l,10 ml) 和双氧水 (35%,0.684 ml), 搅拌 2-3 h 后倒进冰水中搅拌, 用乙酸乙酯萃取, 取有机层并旋蒸除去溶剂. 得到的粗产物经硅胶柱色谱纯化得到化合物 1. 图 1 化合物 1-10 的合成路线. R 1 R 2 R 3 R 4 R 5 分别为 H CH 3 OCH 3 Cl Br 或 N(CH 3 ) 2 等取代基. Fig. 1 The synthetic route for compounds 1 to 10. R 1, R 2, R 3, R 4, R 5 represent H, CH 3, OCH 3, Cl, Br or N(CH 3 ) 2, respectively. 化合物的合成, 采用一锅法未能得到黄酮醇, 因此 [18] 参照 Roy 等报道的方法合成 ( 图 2), 该方法步骤较多, 总产率不高, 多数黄酮醇的产率不超过 50%, 但是该方法能合成一些特殊结构的黄酮醇化合物, 如用其他合成方法很难得到的 5- 取代黄酮醇. 同时, 该方法对一些非 5- 取代黄酮醇也适用. 以化合物 16 为例, 向 50 ml 圆底烧瓶中加入 2- 羟基 5- 甲氧基苯乙酮 (2.4 mmol) 苯甲醛 (2 mmol) 氢氧化钠 10 mmol), 以甲醇 (20 ml) 为溶剂, 回流加热, 用薄层色谱检测至反应完全, 旋蒸除去溶剂后用硅胶柱色谱纯化得到化合物查耳酮中间体. 在 0 下将查耳酮中间体 (1 mmol) 溶解于甲醇 (45 ml) 中, 加入 10% 氢氧化钠水溶液 (15 ml) 30% 双氧水 (10 ml), 搅拌 1 h 后逐渐升至室温并继续搅拌 18 h. 用 2 mol/l 盐酸水溶液淬灭, 并用二氯甲烷萃取, 合并有机图 2 化合物的合成路线. R 1 R 2 R 3 R 4 R 5 R 6 分别为 H CH 3 OCH 3 Cl Br F 等取代基. Fig. 2 The synthetic route for compounds 11 to 20. R 1, R 2, R 3, R 4, R 5, R 6 represent H, CH 3, OCH 3, Cl, Br or F, respectively 化合物的结构鉴定 3-Hydroxy-2-phenyl-4H-chromen- 4-one (1): yellow solid; MP: H NMR (400 MHz, DMSO-d 6 ) δ 8.23 (d, J = 7.6 Hz, 2H), 8.13 (d, J = 7.8 Hz, 1H), (m, 2H), 7.58 (t, J = 7.8 Hz, 2H), (m, 2H). 13 C NMR (100 MHz, DMSO-d 6 ) δ , , , , , , , , , , , , HR-ESIMS: [M-H] - (calc. for C 15 H 9 O 3, ). 2-(3,4-Dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one (2): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ 8.25 (dd, J = 8.0, 1.5 Hz, 1H), 7.91 (dd, J = 8.6, 2.0 Hz, 1H), 7.86 (d, J = 2.0 Hz, 1H), 7.70 (ddd, J = 8.6, 7.1, 1.6 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.03 (d, J = 8.6 Hz, 1H), 4.00 (s, 3H), 3.97 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , , , , 56.05, HR-ESIMS: [M-H] - (calc. for C 17 H 13 O 5, ). 3-Hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one (3): yellow solid; MP: H NMR (400 MHz, DMSO-d 6 ) δ 9.46 (s, 1H), (m, 2H), 8.12 (dd, J = 8.0, 1.3 Hz, 1H), (m, 2H), 7.47 (ddd, J = 8.0, 6.7, 1.4 Hz, 1H), (m, 2H), 3.86 (s, 3H). 13 C NMR (100 MHz, DMSO-d 6 ) δ , , , , , , , , , , , , , HR-ESIMS: [M-H] - (calc. for C 16 H 11 O 4, ). 3-Hydroxy-2-(3-methoxyphenyl)-4H-chromen-4-one (4): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ 8.28 (dd, J = 8.0, 1.6 Hz, 1H), (m, 2H), 7.74 (ddd, J = 8.5, 7.1, 1.5 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), (m, 2H), 7.06 (dd, J = 8.3, 2.2 Hz, 2H), 3.93 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , , , , HR- Chin J Appl Environ Biol 应用与环境生物学报

3 234 黄酮醇类化合物的合成与抗菌活性 2 期 ESIMS: [M-H] - (calc. for C 16 H 11 O 4, ). 3-Hydroxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (5): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ 8.26 (dd, J = 8.0, 1.5 Hz, 1H), 7.72 (ddd, J = 8.6, 7.1, 1.6 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.55 (s, 2H), (m, 1H), 7.04 (s, 1H), 3.98 (s, 6H), 3.94 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , , 61.02, HR-ESIMS: [M-H] - (calc. for C 18 H 15 O 6, ). 2-(Benzo[d][1,3]dioxol-5-yl)-3-hydroxy-4H-chromen-4-one (6): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ 8.24 (dd, J = 8.0, 1.5 Hz, 1H), 7.87 (dd, J = 8.4, 1.8 Hz, 1H), 7.78 (d, J = 1.7 Hz, 1H), 7.70 (ddd, J = 8.6, 7.1, 1.7 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), (m, 1H), (m, 2H), 6.07 (s, 2H). 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , , , , , 77.34, 77.02, HR-ESIMS: [M-H] - (calc. for C 16 H 9 O 5, ). 2-(4-Chlorophenyl)-3-hydroxy-4H-chromen-4-one (7): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ (m, 3H), 7.72 (ddd, J = 8.7, 7.1, 1.7 Hz, 1H), 7.59 (d, J = 8.5 Hz, 1H), (m, 2H), 7.43 (ddd, J = 8.1, 7.1, 1.0 Hz, 1H), 7.07 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , HR-ESIMS: [M-H] - (calc. for C 15 H 8 35 ClO 3, ), [M-H] - (calc. for C 15 H 8 37 ClO 3, ). 2-(4-Bromophenyl)-3-hydroxy-4H-chromen-4-one (8): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ 8.25 (dd, J = 8.0, 1.4 Hz, 1H), 8.14 (d, J = 8.7 Hz, 2H), (m, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.59 (d, J = 8.5 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 7.09 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , HR-ESIMS: [M-H] - (calc. for C 15 H 8 79 BrO 3, ), [M-H] - (calc. for C 15 H 8 81 BrO 3, ). 3-Hydroxy-2-(p-tolyl)-4H-chromen-4-one (9): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ 8.25 (dd, J = 8.0, 1.5 Hz, 1H), 8.16 (d, J = 8.3 Hz, 2H), 7.70 (ddd, J = 8.6, 7.1, 1.6 Hz, 1H), 7.59 (d, J = 8.3 Hz, 1H), (m, 1H), 7.35 (d, J = 8.2 Hz, 2H), 6.99 (s, 1H), 2.44 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , , HR-ESIMS: [M-H] - (calc. for C 16 H 11 O 3, ). 2-(4-(Dimethylamino)phenyl)-3-hydroxy-4H-chromen-4- one (10): red solid; MP: H NMR (400 MHz, CDCl 3 ) δ (m, 3H), (m, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 6.81 (d, J = 9.1 Hz, 3H), 3.07 (s, 6H). 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , , HR-ESIMS: [M-H] - (calc. for C 17 H 14 NO 3, ). 3-hydroxy-5-methoxy-2-(p-tolyl)-4H-chromen-4-one (11): yellow solid; MP: H NMR (400 MHz, CDCl3) δ 8.01 (s, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.58 (t, J = 8.4 Hz, 1H), 7.36 (s, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.17 (d, J = 9.2 Hz, 1H), 6.78 (d, J = 8.2 Hz, 1H), 4.03 (s, 3H), 2.37 (s, 3H), 2.34 (s, 3H).13C NMR (150 MHz, CDCl3) δ , , , , , , , , , , , , , , , 56.45, 19.99, HR-ESIMS: ; [M+Na] + (calc. for C 18 H 16 NaO 4, ). 2-(3,4-dimethoxyphenyl)-3-hydroxy-5-methoxy-4Hchromen-4-one (12): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ (m, 2H), 7.60 (t, J = 8.4 Hz, 1H), 7.43 (s, 1H), 7.18 (d, J = 8.5 Hz, 1H), 7.04 (d, J = 9.1 Hz, 1H), 6.81 (d, J = 8.2 Hz, 1H), 4.06 (s, 3H), 4.01 (s, 3H), 3.99 (s, 3H) 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , , 77.36, 77.04, 76.73, 56.48, HR-ESIMS: ; [M+Na] + (calc. for C 18 H 16 NaO 6, ). 3-hydroxy-5-methoxy-2-(4-methoxyphenyl)-4H-chromen- 4-one(13): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ 8.23 (d, J = 8.8 Hz, 2H), 7.59 (t, J = 8.3 Hz, 1H), 7.37 (s, 1H), 7.16 (d, J = 8.5 Hz, 1H), 7.06 (d, J = 8.8 Hz, 2H), 6.80 (d, J = 8.1 Hz, 1H), 4.05 (s, 3H), 3.91 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , , 56.46, HR- ESIMS: [M+Na] + (calc. for C 17 H 14 NaO 5, ). 3-hydroxy-5-methoxy-2-phenyl-4H-chromen-4-one(14): yellow solid; MP: H NMR (400 MHz, Acetone) δ (m, 2H), 8.11 (s, 1H), 7.71 (t, J = 8.4 Hz, 1H), 7.59 (t, J = 7.5 Hz, 2H), 7.51 (t, J = 7.3 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 6.98 (d, J = 8.1 Hz, 1H), 3.98 (s, 3H). 13 C NMR (100 MHz, Acetone) δ , , , , , , , , , , , , , HR-ESIMS: ; [M+Na] + (calc. for C 16 H 12 NaO 4, ). 3-Hydroxy-2-(naphthalen-1-yl)-4H-chromen-4-one (15): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ 8.35 (dd, J = 8.1, 1.5 Hz, 1H), 8.04 (d, J = 8.3 Hz, 1H), 7.96 (dd, J = 6.6, 2.7 Hz, 1H), (m, 2H), 7.73 (ddd, J = 8.6, 7.2, 1.6 Hz, 1H), (m, 1H), 7.55 (pd, J = 6.8, 1.7 Hz, 3H), 7.48 (t, J = 7.6 Hz, 1H), 6.59 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , , , , , , , HR-ESIMS: [M-H] - (calc. for C 19 H 11 O 3, ). 3-Hydroxy-6-methoxy-2-phenyl-4H-chromen-4-one (16): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ 8.25 (d, J = 7.4 Hz, 2H), (m, 5H), 7.31 (dd, J = 9.2, 3.1 Hz, 1H), 7.05 (s, 1H), 3.92 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , , HR-ESIMS: 应用与环境生物学报 Chin J Appl Environ Biol

4 23 卷周强等 235 [M-H] - (calc. for C 16 H 11 O 4, ). 6-Chloro-3-hydroxy-2-phenyl-4H-chromen-4-one (17): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ (m, 3H), 7.65 (dd, J = 9.0, 2.6 Hz, 1H), (m, 4H), 7.00 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , HR-ESIMS: [M-H] - (calc. for C 15 H 35 8 ClO 3, ), [M-H] - (calc. for C 15 H 37 8 ClO 3, ). 3-Hydroxy-7-methoxy-2-phenyl-4H-chromen-4-one (18): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ (m, 2H), 8.14 (d, J = 8.9 Hz, 1H), (m, 3H), (m, 3H), 3.94 (s, 3H). 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , 99.86, 77.34, 77.02, 76.70, HR- ESIMS: [M-H] - (calc. for C 16 H 11 O 4, ). 6-Bromo-3-hydroxy-2-phenyl-4H-chromen-4-one (19): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ 8.38 (d, J = 2.4 Hz, 1H), (m, 2H), 7.77 (dd, J = 9.0, 2.4 Hz, 1H), (m, 4H), 7.01 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) δ , , , , , , , , , , , , HR-ESIMS: [M- H] - (calc. for C 15 H 79 8 BrO 3, ), [M-H] - (calc. for C 15 H 81 8 Br O 3, ). 6-Fluoro-3-hydroxy-2-phenyl-4H-chromen-4-one (20): yellow solid; MP: H NMR (400 MHz, CDCl 3 ) δ 8.24 (d, J = 7.9 Hz, 2H), 7.88 (dd, J = 8.0, 3.0 Hz, 1H), (m, 5H), 7.01 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) δ (d, J = 2.7 Hz), (d, J = Hz), , , , , , , , (d, J = 25.9 Hz), (d, J = 8.1 Hz), (d, J = 8.3 Hz), (d, J = 23.7 Hz). HR-ESIMS: [M-H] - (calc. for C 15 H 8 FO 3, ) 牛津杯法检测抗菌活性化合物的抗菌活性采用牛津杯法 [19]. 实验方法为 : 实验细菌转接在配置的培养基 ( 蛋白胨 20 g/l, 牛肉膏 10 g/l,nacl 5 g/l, 琼脂 17 g/l) 上, 真菌 ( 土豆 200 g/l, 葡萄糖 20 g/l, 琼脂 17 g/l). 然后于牛津杯孔 ( 直径为 8 mm) 中加 200 μl 的不同浓度的样品 ( 浓度梯度为 μg/ml), 每个以甲氧西林为细菌阳性试验对照,DMSO 为阴性对照. 加样后分别放置于 31 ( 细菌 ) 培养 24 h,28 ( 真菌 ) 培养 3 d, 测定抑菌圈, 每个样品重复 3 次. 2 结果与讨论我们以不同官能团取代的邻羟基苯乙酮和苯甲醛为原料, 通过一锅法和分步法合成了 20 种黄酮醇类化合物, 产率在 10%-78% 之间 ( 表 1), 目前为止所有目标化合物均未有抗表 1 黄酮醇化合物的制备 Table 1 The preparation of flavonols 化合物结构产率化合物结构产率 Compound Structure Yield (r/%) Compound Structure Yield (r/%) * Chin J Appl Environ Biol 应用与环境生物学报

5 236 黄酮醇类化合物的合成与抗菌活性 2 期续表 1 Table 1 (Continued) 化合物结构产率化合物结构产率 Compound Structure Yield (r/%) Compound Structure Yield (r/%) R 1 R 2 R 3 R 4 R 5 R 6 分别代表 H CH 3 OCH 3 Cl Br F 等不同取代基. *: 新化合物. R 1, R 2, R 3, R 4, R 5, R 6 represent different substituents such as H, CH 3, OCH 3, Cl, Br, F, respectively *: New compound. 菌性研究的相关报道, 所有化合物结构经 1 H NMR 13 C NMR 和 HR-ESIMS 波谱鉴定确认. 将这些化合物用牛津杯法检测其抗菌活性, 结果发现对酿酒酵母菌和黑曲霉菌并无明显抑制效果 (d < 9 mm), 而对枯草芽孢杆菌的抗菌效果明显. 如表 2 所示,20 种黄酮醇在大于 5 μg/ml 浓度下对枯草芽孢杆菌具抑制效果, 抑制作用随着浓度变大而增强, 大部分化合物在浓度为 40 μg/ml 时达到最大. 少部分化合物如 1 6 由于浓度过高, 影响其溶解性和扩散性而导致其抑菌效果不再增强. 在 20 种黄酮醇类化合物中, 抑菌效果明显 (d > 15 mm), 分析这其中几种化合物的结构发现都是具有卤素取代的黄酮醇. 根据抑菌作用的强弱和取代基团的关系分析可发现 : 无论在黄酮醇 A 环还是 B 环上具有卤素取代的黄酮醇的抑菌效果都有很大提高 ( 化合物 );B 环对位单取代基 ( ),3 例外, 有利于提高抑菌活性. 甲氧基的位置与多寡对化合物的抑菌活性影响不大. 值得一提的是, 我们合成了 4 个 5- 甲氧基取代黄酮醇 ( ), 这类化合物尽管不是活性最好的, 但与相应的 5 位无取代的黄酮醇比较, 如 12 vs 2 13 vs 3 14 vs 1, 活性有所提高. 该类化合物的合成方法及更多 5- 取代黄酮醇的抗菌活性的系统研究本课题组正在进行中. 表 2 黄酮醇化合物的抗菌活性 Table 2 Antibacterial activity of flavonols 抑菌圈直径 ( 枯草芽孢杆菌 ) (d/mm) 化合物 Inhibitory zone diameter (Bacillus subtilis) Compound 40 μg/ml 20 μg/ml 10 μg/ml 5 μg/ml 2.5 μg/ml < < < < < < < Methicillin* 12.2 *: 阳性对照. *: positive control. 应用与环境生物学报 Chin J Appl Environ Biol

6 23 卷周强等结论以邻羟基苯乙酮和苯甲醛为原料, 采用一锅法和分步法经 AFO 反应合成了 20 种黄酮醇类化合物, 进一步对它们进行了抗菌活性研究, 发现黄酮醇化合物对细菌具有选择性抑制的效果, 而对真菌无效. 通过构效关系分析发现具有卤素取代黄酮醇具有较好的抑菌活性. 此外,5- 取代黄酮醇较 5- 位无取代的黄酮醇也表现出增强的抗菌活性. 本研究结果可为天然黄酮醇类化合物的药效结构分析先导药物分子的设计与合成提供重要的依据. 参考文献 [References] 1 Schlörke O, Zeeck A. Orsellides A-E: an example for 6-deoxyhexose derivatives produced by Fungi [J]. Eur J Org Chem, 2006 (4): Shaaban KA, Shepherd MD, Ahmed TA, Nybo SE, Leggas M, Rohr J. Pyramidamycins A-D and 3-hydroxyquinoline-2-carboxamide; cytotoxic benzamides from Streptomyces sp. DGC1 [J]. J Antibiot, 2012, 65 (12): Amado NG, Fonseca B, Cerqueira DM, Neto VM, Abreu JG. Flavonoids: potential Wnt/beta-catenin signaling modulators in cancer [J]. Life Sci, 2011, 89: Betts JW, Sharili AS, Phee LM, Wareham DW. In vitro activity of epigallocatechin gallate and quercetin alone and in combination versus clinical isolates of methicillin-resistant Staphylococcus aureus [J]. J Nat Prod, 2015, 78 (8): Mori A, Nishino C, Enoki N, Tawata S. Antibacterial activity and mode of action of plant flavonoids against Proteus vulgaris and Staphylococcus aureus [J]. Phytochemistry, 1987, 26 (8): Plaper A, Golob M, Hafner I, Oblak M, Šolmajer T, Jerala R. Characterization of quercetin binding site on DNA gyrase [J]. Biochem Bioph Res Co, 2003, 306 (2): Sakagami Y, Mimura M, Kajimura K, Yokoyama H, Iinuma M, Tanaka T, Ohyama M. Anti-MRSA activity of sophoraflavanone G and synergism with other antibacterial agents [J]. Lett Appl Microbiol, 1998, 27 (2): Ikigai H, Nakae T, Hara Y, Shimamura T. Bactericidal catechins damage the lipid bilayer [J]. Biochim Biophys Acta, 1993, 1147 (1): Haraguchi H, Tanimoto K, Tamura Y, Mizutani K, Kinoshita T. Mode of antibacterial action of retrochalcones from Glycyrrhiza inflata [J]. Phytochemistry, 1998, 48 (1): Mirzoeva, OK, Grishanin, RN, Calder, PC. Antimicrobial action of propolis and some of its components: the effects on growth, membrane potential and motility of bacteria [J]. Microbiol Res, 1997, 152 (3): Cushnie TPT, Lamb AJ. Detection of galangin-induced cytoplasmic membrane damage in Staphylococcus aureus by measuring potassium loss [J]. J Ethnopharmacol, 2005, 101 (1-3): Bird CW, Cookson RC. The Mechanism of the von Auwers rearrangement of derivatives of 4-Methyl-4-polyhalomethyl-1-methylenecyclohexa-2,5-diene [J]. J Org Chem, 1959, 24 (3): Baker W. Molecular rearrangement of some o-acyloxyacetophenones and the mechanism of the production of 3-acylchromones [J]. J Chem Soc, 1933, 17 (21): Mahal HS, Venkataraman K. Synthetical experiments in the chromone group. Part XIV. The action of sodamide on 1-acyloxy-2- acetonaphthones [J]. J Chem Soc, 1934: Adam W, Golsch D, Hadjiarapoglou L, Patonay T. Epoxidation of flavones by dimethyldioxirane [J]. J Org Chem, 1991, 56 (26): Algar J, Flynn JP. A new method for the synthesis of flavonols [J]. Proc Roy Irish Acad, 1934, 42: Gunduz S, Goren AC, Ozturk T. Facile syntheses of 3-hydroxyflavones [J]. Org Lett, 2012, 14 (6): Kraus G, Roy S. Direct synthesis of chrysosplenol D [J]. J Nat Prod, 2008, 71: Carlson HJ, Bissell HD, Mueller MG. Antimala rial and antibacterial substances separated from higher plants [J]. J Bacteriol, 1946, 52 (2): Chin J Appl Environ Biol 应用与环境生物学报

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇