基于吩嗪-1-羧酸的新型麦角甾醇生物合成抑制剂叔醇类衍生物的合成及杀菌活性研究

汤显军 鲁星亮 杨丹 张敏 熊永通 吴清来 李俊凯

引用本文: 汤显军, 鲁星亮, 杨丹, 张敏, 熊永通, 吴清来, 李俊凯. 基于吩嗪-1-羧酸的新型麦角甾醇生物合成抑制剂叔醇类衍生物的合成及杀菌活性研究[J]. 有机化学, 2020, 40(8): 2491-2501. doi: 10.6023/cjoc202004011 shu
Citation:  Tang Xianjun, Lu Xingliang, Yang Dan, Zhang Min, Xiong Yongtong, Wu Qinglai, Li Junkai. Synthesis and Fungicidal Activities of Novel Tertiary Alcohol Ergosterol Biosynthesis Inhibitors Based on Phenazine-1-carboxylic Acid[J]. Chinese Journal of Organic Chemistry, 2020, 40(8): 2491-2501. doi: 10.6023/cjoc202004011 shu

基于吩嗪-1-羧酸的新型麦角甾醇生物合成抑制剂叔醇类衍生物的合成及杀菌活性研究

    通讯作者: 吴清来, wql106@163.com; 李俊凯, junkaili@sina.com
  • 基金项目:

    国家重点研发计划(No.2018YFD200500)和国家自然科学基金(No.31672069)资助项目

摘要: 在前期以天然产物吩嗪-1-羧酸为先导化合物进行新农药的开发研究中发现,吩嗪-1-甲醇具有优异的杀菌活性.基于上述发现,通过将吩嗪-1-甲醇作为次级先导化合物,并结合麦角甾醇生物合成抑制剂氯苯嘧啶醇,设计并合成了16个吩嗪-1-芳基(5-嘧啶)甲醇衍生化合物.杀菌测试表明部分目标化合物对水稻纹枯病及辣椒疫病表现出中等抑制活性.有趣的是大多数目标化合物对抗吩嗪-1-羧酸特征性杀菌谱水稻纹枯病活性是显著下降的;而对于抗氯苯嘧啶醇特征性杀菌谱小麦白粉病(Erysiphe graminis)表现出中等以上的杀菌活性.这表明目标化合物的作用机制可能不同于吩嗪-1-羧酸,但相似于氯苯嘧啶醇.进一步的麦角甾醇生物合成抑制实验证明了目标化合物的作用方式与氯苯嘧啶醇相同.

English

  • 杀菌剂对于保护农作物安全及提高作物产量是不可或缺的[1-2].然而长期不限制地使用已商品化的杀菌剂不仅会导致植物病害真菌抗性的增加, 也会导致一系列生物环境问题, 如对有益生物的毒性、环境污染等[3-4].为了解决现有杀真菌剂的负面影响, 有必要开发具有新的作用机制和生物环境友好型高活性杀菌剂.

    天然产物时常可以作为开发具有上述特征农药的先导化合物[5-7].吩嗪-1-羧酸(Phenazine-1-carboxylic acid, PCA, 图 1)广泛存在许多微生物的次生代谢物中, 如链霉菌(Streptomyces)[8]、假单胞杆菌(Pseudomonads) 等[9].现有研究表明, 吩嗪-1-羧酸具有许多优良的生物学功能, 如杀真菌活性[10-12]、抗白血病[13]及抗肿瘤活性[14].同时, 也表现出许多优异的生物农药属性, 如对人和牲畜安全低毒[15]、自然环境中易降解及显著提高作物产量等[16-17].目前, 吩嗪-1-羧酸已经在中国注册登记为“申嗪霉素”, 用于防治水稻纹枯病[18-20].基于上述优点, 吩嗪-1-羧酸是一个理想的先导化合物用于新农药创制.我们团队一直立志于对吩嗪-1-羧酸进行结构改造以提高其农业利用价值[21-24], 例如将吩嗪-1-羧酸与氨基酸成功进行耦合以提高其在植物体内的传导性[25], 吩嗪-1-羧酸衍生化合物吩嗪-1-甲醇(EC50=41.82 mg/L, 图 1)表现出更好的抗禾谷镰刀菌(Fusarium graminearum)(吩嗪-1-羧酸: EC50=127.28 mg/L)活性[21].本文作为上述研究的继续, 吩嗪-1-甲醇被作为次级先导化合物以开发更高活性的吩嗪-1-羧酸衍生物.

    图 1

    图 1.  目标化合物的设计策略
    Figure 1.  Design strategy for target compounds

    农用杀真菌剂氯苯嘧啶醇(图 1)已经被成功开发出来, 其主要作用机理是抑制真菌体内麦角甾醇的合成, 从而杀死真菌[26].氯苯嘧啶醇主要被用来防治禾谷类白粉病、辣椒炭疽病等[27].氯苯嘧啶醇具有典型的芳基嘧啶三级醇骨架结构, 并占有较大的空间位阻效应.考虑到吩嗪-1-甲醇与氯苯嘧啶醇两者结构的相似性, 本文采用活性亚结构拼接法将取代苯环、嘧啶与吩嗪环进行结构拼接, 设计合成16个吩嗪-1-芳基(5-嘧啶基)甲醇衍生物(Scheme 1).测试了16个中间体及16目标化合物在体外对6种重要作物病原真菌的抑制活性, 同时也测试了16个目标化合物在体内抗小麦白粉病活性.更进一步选择部分目标化合物进行麦角甾醇生物合成抑制实验, 以验证目标化合物可能的作用模式.

    图式 1

    图式 1.  目标化合物6a~6p的合成路线
    Scheme 1.  Synthetic route of target compounds 6a~6p

    Reagents and conditions: (a) oxalyl chloride, CH2Cl2, DMF, reflux, 8 h; (b) substituted benzene, AlCl3, 0~60 ℃, 7 h; (c) n-butyllithium, THF, below -80 ℃, 1 h; (d) THF, below -80 ℃, 4 h

    在合成关键中间体3a~3p时, 采用傅克酰基化反应.在步骤(b)以相应的反应试剂取代苯作为反应溶剂时, 得到的产率比二氯甲烷作溶剂要高.如果反应温度高于60 ℃, 化合物3a~3p的产率会迅速下降.反应(c)和(d)为“一锅法”, 其中正丁基锂:5-溴嘧啶:3a~3p的物质的量比为1.2:1.2:1时, 得到的6a~6p产率最高.

    所有的中间体3a~3p和目标化合物6a~6p的结构表征均通过核磁共振氢谱、碳谱和高分辨质谱确定.在核磁共振氢谱中, 中间体3a~3p吩嗪环上氢质子化学位移值在δ 7.73~8.42;而相应化合物6a~6p吩嗪环上氢质子化学位移值在δ 6.98~8.38, 表明化合物6a~6p吩嗪环上氢质子可能受到嘧啶环的屏蔽效应, 使得其向高场移动.在核磁共振碳谱中, 中间体3a~3p的羰基碳化学位移值在δ 194.23~199.03;目标化合物6a~6p中与羟基直接相连的叔碳化学位移值在δ 80.65~82.58, 这与常见的叔醇碳化学位移值相比, 其处于相对的较低场, 可能由于受到邻近三个芳基的去屏蔽效应.在高分辨质谱中, 所有合成化合物均存在较强的[M+H]+峰.为了进一步确定目标化合物结构骨架, 选取化合物6f进行单晶衍射实验, 其单晶结构如图 2所示.

    图 2

    图 2.  目标化合物6f的X单晶衍射结构
    Figure 2.  X-ray single crystal structure of target compound 6f

    由于中间体化合物3a~3p对吩嗪-1-羧酸的羧基部分进行了较大改变, 故对化合物3a~3p也进行了部分杀菌活性测定(表 1), 结果表明化合物3a~3p几乎丢失了原有吩嗪-1-羧酸所具有的抗真菌活性.目标化合物6a~6p对6种常见作物病原菌的杀菌活性结果见表 2, 结果表明, 在50 mg/L的条件下, 与吩嗪-1-羧酸的杀菌活性相比较, 6a~6p的活性是降低的, 但整体上6a~6p的杀菌活性要远好于相应的3a~3p.此外在50 mg/L的条件下, 对水稻纹枯病的抑制活性中, 化合物6b(68.81%)、6f (76.45%)、6g (71.39%)、6i (69.70%)表现出中等的抑制活性.化合物6b (69.39%)、6h (79.95%)对辣椒疫病表现出与氯苯嘧啶醇(AC)(72.09%)相近的抑制活性.为了进一步确定目标化合物对水稻纹枯病及辣椒疫病的活性, 选取化合物6b6f6g进行EC50测试(表 3), 结果表明, 对于抗水稻纹枯病和辣椒疫病, 6b (EC50=21.62, 18.68 mg/L)、6f (EC50=22.04, 66.98 mg/L)和6g (EC50=14.48L, 17.52 mg/L)相对于吩嗪-1-羧酸(EC50=6.30, 7.11 mg/L)和氯苯嘧啶醇(EC50=6.85, 10.39 mg/L)均表现出活性下降.

    表 1

    表 1  中间体3a~3p在50 mg/L条件下对4种植物病原真菌的抑制活性a
    Table 1.  Inhibition activity of the intermediate compounds 3a~3p against four pathogenic fungi at a concentration of 50 mg/L
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    Compd. Thanatephorus cucumeris Fusarium graminearum Alternaria alternata Colletotrichum gloeosporioides
    3a 15.15±0.52 <10.00 21.20±0.01 20.28±0.00
    3b 12.45±0.29 NA 22.03±0.36 10.61±0.24
    3c <10.00 NA <10.00 13.29±0.02
    3d 21.69±0.52 NA <10.00 NA
    3e 20.17±0.50 <10.00 11.24±0.02 10.96±0.75
    3f <10.00 NA <10.00 <10.00
    3g 11.92±0.20 <10.00 12.76±0.43 <10.00
    3h <10.00 NA <10.00 NA
    3i 45.36±0.14 26.81±0.22 32.45±0.00 34.65±0.40
    3j 10.65±0.51 NA <10.00 <10.00
    3k 18.62±0.33 NA <10.00 NA
    3l 14.02±0.01 NA <10.00 NA
    3m <10.00 NA 12.85±0.12 14.79±0.52
    3n 11.56±0.21 NA 13.82±0.38 16.94±0.20
    3o 17.62±0.16 NA <10.00 NA
    3p <10.00 NA <10.00 <10.00
    PCA 80.54±0.24 40.76±0.62 81.11±0.40 79.05±0.78
    a Values are the mean±SD of three replicates. PCA (phenazine-1-carboxylic acid) served as the positive control. NA: not active under this concentration.

    表 2

    表 2  目标化合物6a~6p在50 mg/L的条件下对6种植物病原真菌的抑制活性a
    Table 2.  Inhibition activity of the target compounds 6a~6p against six pathogenic fungi at a concentration of 50 mg/L
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    Compd. T.c. F.g. C.g. A.a. F.o. P.c.
    6a 24.55±0.20 14.38±0.16 19.19±0.42 21.47±0.73 10.58±0.19 40.53±0.02
    6b 68.81±0.24 53.04±0.40 44.36±0.26 57.06±0.15 46.10±0.31 69.39±0.17
    6c 34.37±0.12 NA <10.00 36.14±1.17 NA 48.02±0.06
    6d 13.94±0.43 NA <10.00 15.57±0.32 12.03±0.14 23.70±0.10
    6e NA 10.61±0.33 24.64±0.44 15.23±0.23 11.04±0.44 12.60±0.41
    6f 76.45±0.13 20.93±0.48 60.03±0.25 23.49±0.42 31.63±0.51 59.92±0.17
    6g 71.39±0.49 45.79±0.29 49.71±0.29 30.34±1.02 50.91±0.38 68.78±0.51
    6h 32.15±0.23 48.74±0.14 34.30±0.24 21.92±0.10 44.55±0.39 79.95±0.01
    6i 69.70±0.09 45.78±0.39 54.39±0.46 23.93±0.01 22.48±0.18 50.12±0.73
    6j 46.73±0.00 17.14±0.05 42.02±0.27 18.19±0.34 14.44±0.27 59.34±0.46
    6k 17.18±0.62 52.75±0.10 32.99±0.70 22.50±0.64 <10.00 15.85±0.68
    6l 28.45±0.02 12.53±0.05 12.45±0.20 20.25±0.05 15.66±0.57 10.65±0.01
    6m 16.35±0.50 14.54±0.27 25.89±0.35 19.87±0.25 17.34±0.22 21.99±0.00
    6n 23.83±0.24 <10.00 14.49±0.41 11.94±0.04 11.63±0.10 11.67±0.21
    6o 17.27±0.00 21.69±0.18 49.74±0.02 14.39±0.28 24.41±0.34 39.79±0.24
    6p NA 11.92±0.00 11.91±0.01 15.25±0.30 12.71±0.42 <10.00
    AC 98.81±0.26 74.60±0.33 74.36±0.51 96.00±1.13 47.55±0.90 72.09±0.23
    PCA 75.04±0.28 43.21±0.52 83.78±0.83 81.11±0.72 72.66±0.22 98.92±1.18
    a Values are the mean±SD of three replicates. PCA (phenazine-1-carboxylic acid) and AC (fenarimol) served as the positive control. NA: not active under this concentration. T.c.: Thanatephorus cucumeris, F.g.: Fusarium graminearum, C.g.: Colletotrichum gloeosporioides, A.a.: Alternaria alternata, F.o.: Fusarium oxysporum, P.c.: Phytophthora capsici.

    表 3

    表 3  目标化合物6b6f6g对水稻纹枯病和辣椒疫病的EC50 (mg/L)a
    Table 3.  EC50 (mg/L) of the target compounds 6b, 6f and 6g against Thanatephorus cucumeris and Phytophthora capsici
    下载: 导出CSV
    Comp. Thanatephorus cucumeris Phytophthora capsici
    Regression Eq.b Rc EC50/(mg•L1) (95%CL)d Regression Eq.b Rc EC50/(mg•L1)(95%CL)d
    6b y=-1.61+1.15x 0.9750 21.62 (10.95~40.09) y=-2.13+1.69x 0.9437 18.68 (11.64~27.07)
    6f y=-1.70+1.24x 0.9891 22.04 (12.41~34.84) y=-4.43+2.51x 0.9573 66.98 (47.61~100.52)
    6g y=-1.86+1.62x 0.9620 14.48 (12.18~17.13) y=-2.23+1.86x 0.9812 17.52 (11.80~26.15)
    AC y=-0.90+1.08x 0.9942 6.85 (5.37~8.60) y=-1.12+1.07x 0.9749 10.39 (4.74~18.20)
    PCA y=-0.91+1.14x 0.9903 6.30 (4.98~7.82) y=-1.17+1.38x 0.9785 7.11 (5.87~8.55)
    a PCA and AC served as the positive control. b Regression Eq. means the toxicity regression equation; c Correlation coefficient; d CL means the confidence interval.

    简单的构效关系表明(50 mg/L), 在苯环对位存在氯原子或氟原子取代时, 目标化合物对水稻纹枯病和辣椒疫病的抑制活性会显著增加.当苯环上出现给电子基团(如OCH3), 则目标化合物对所测病原真菌抑制活性大大减弱.同时当苯环为对位取代时, 随着取代基的空间位阻的增加, 化合物对水稻纹枯病、梨黑斑病及辣椒疫病的抑制率会显著下降, 例如: 6b (CH3)>6c (CH2- CH3)>6d (CH(CH3)2)>6e (C(CH3)3).

    为了测定化合物6a~6p结构中嘧啶醇亚结构片段的抗真菌活性, 选取氯苯嘧啶醇特征性杀菌谱小麦白粉病进行盆栽测试.目标化合物6a~6p的抗小麦白粉病活性测试结果见表 4.结果表明, 部分化合物表现出明显的抗小麦白粉病活性, 其中6g6i6j最具有潜力(图 3).在50 mg/L浓度下, 化合物6f6g6i6k对小麦白粉病的平均防效分别为79.50%、85.52%、79.48%、78.45%.有趣的是, 在此浓度下吩嗪-1-羧酸对小麦白粉病的平均仿效仅仅只有20.12%.有趣的是, 通过表 2中目标化合物对吩嗪-1-羧酸特征性杀菌谱水稻纹枯病抑制活性数据分析, 可以看到除化合物6f6g6i表现出中等抑制活性外, 绝大多数目标化合物都表现出较低的抑制活性; 然而多数目标化合物对抗小麦白粉病却表现出中等以上的活性.这表明从吩嗪-1-甲醇的一级醇结构转换成目标化合物6a~6p的三级醇结构, 使得化合物的杀真菌谱也发生了明显变化.为了进一步阐明这种变化, 选取6f6g6i6j进行了麦角甾醇生物合成抑制抑制实验.实验结果(图 4)表明, 化合物6f6g6i6j对植物病原真菌体内麦角甾醇合成的抑制率分别为44.04%、64.40%、40.61%、27.03%;而吩嗪-1-羧酸的抑制率仅仅为2.84%.结果阐明目标化合物分子结构中保留了嘧啶醇亚结构抗真菌活性, 而丢失了吩嗪-1-羧酸亚结构生物活性.

    表 4

    表 4  目标化合物6a~6p在50 mg/L浓度下抗小麦白粉病活性a
    Table 4.  Fungicidal activity of the target compounds 6a~6p against wheat powdery mildew under greenhouse conditions at 50 mg/L
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    Compd. Disease index/% Control efficiencyb/%
    6a 23.42 63.44±3.45 cd
    6b 27.14 57.42±3.25 cde
    6c 37.65 47.27±3.11 g
    6d 35.23 43.50±3.04 g
    6e 45.28 29.17±4.10 h
    6f 14.89 79.50±4.03 b
    6g 9.54 85.52±5.20 b
    6h 34.74 52.17±5.92 ef
    6i 14.63 79.48±1.32 b
    6j 22.21 65.11±3.06 c
    6k 15.37 78.45±4.32 b
    6l 27.01 57.90±0.29 cde
    6m 49.59 22.04±3.06 hi
    6n 33.96 53.76±5.00 ef
    6o 30.57 55.84±4.69 de
    6p 44.91 29.44±3.45 h
    AC 2.78 96.00±3.64 a
    PCA 55.92 20.12±6.35 i
    a Values are the mean±SD of three replicates, PCA and AC served as the positive Control. b Control efficiency values marked by the same letter within 18 treatments were not significantly different according to the waller-duncan test at P=0.05.

    图 3

    图 3.  化合物6g6i6j在50 mg/L浓度下抗小麦白粉病活性
    Figure 3.  Fungicidal activity of compounds 6g, 6i and 6j against wheat powdery mildew under greenhouse conditions at 50 mg/L

    CK: the negative control

    图 4

    图 4.  化合物6f6g6i6j对麦角甾醇生物合成的抑制活性
    Figure 4.  Inhibitory activities of compounds 6f, 6g, 6i and 6j on ergosterol biosynthesis

    PCA and AC were served as the positive control. For test compounds, the statistical significance of differences in the mean values at P=0.5 was analyzed by the Waller-Duncan test (SPSS v.21 for windows). Histograms with letters a, b, c, d, e and f demonstrate significant difference between all test compounds

    以吩嗪-1-羧酸衍生化合物吩嗪-1-甲醇为级次先导化合物, 利用活性亚结构拼接原理, 将氯苯嘧啶醇活性结构与吩嗪-1-甲醇结合, 设计、合成了系列新型吩嗪-1-芳基(5-嘧啶基)甲醇.生物活性测试结果表明, 多数目标化合物在50 mg/L下对抗吩嗪-1-羧酸特征杀菌谱水稻纹枯病表现出活性下降, 但对抗氯苯嘧啶醇特征杀菌谱小麦白粉病表现出较好的抑制活性.通过麦角甾醇生物合成抑制实验进一步阐明了目标化合物的可能作用方式.

    除四氢呋喃(THF)通过重蒸除水制备外, 其他试剂均为市售分析纯. XT-4A熔点测定仪; Bruker AVANCE Ⅲ HD型400 MHz核磁共振仪(CDCl3为溶剂, TMS为内标); Bruker APEX Ⅳ型傅里叶变换质谱仪(ESI源); Bruker AXS D8 QUEST X射线单晶衍射仪; Agilent 7890 A型气质联用仪.

    3.2.1   吩嗪-1-甲酰氯(2)的合成

    向单口反应瓶中加入2.5 g (11.2 mmol)吩嗪-1-羧酸, 30 mL二氯甲烷, 1~2滴N, N-二甲基甲酰胺(DMF), 缓慢加入3.0 g草酰氯, 加热回流反应至固体完全消失, 继续回流反应2~3 h, 与旋转蒸发仪上脱去溶剂, 加入少量二氯甲烷溶解再脱去溶剂, 用于下步反应.

    3.2.2   吩嗪-1-芳基甲酮(3a~3p)的合成

    以化合物3a为例:向上述装有吩嗪-1-甲酰氯的单口瓶中加入30 mL无水苯溶液, 缓慢加入8.4 g (22.4 mmoL)无水三氯化铝, 室温反应7 h, 薄层色谱(TLC)跟踪.反应完毕, 在搅拌下将反应液倒入装有5%稀盐酸和碎冰的烧杯中, 淬灭1 h, 分出有机层, 水层用二氯甲烷萃取3次, 合并有机相, 用饱和碳酸钠水溶液洗涤, 再水洗至中性, 分出有机层, 干燥, 减压蒸去溶剂, 用二氯甲烷重结晶, 得到淡黄色固体, 收率73%. m.p. 115.4~117.7 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.40 (dd, J=7.2, 2.8 Hz, 1H), 8.24 (d, J=8.2 Hz, 1H), 8.07~8.02 (m, 1H), 7.96~7.80 (m, 4H), 7.91 (d, J=2.4 Hz), 7.79~7.74 (m, 1H), 7.59 (t, J=7.2 Hz, 1H), 7.43 (t, J=7.2 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ: 196.7, 143.6, 143.4, 142.7, 141.7, 139.7, 137.9, 135.4, 133.4, 131.7, 131.5, 131.2, 130.7, 130.4, 130.3, 130.3, 130.2, 129.5, 128.4; HR- ESI-MS calcd for C19H13N2O [M+H]+ 285.1022, found 285.1017.

    其他该类化合物合成方法类似.

    吩嗪-1-(4-甲基苯基)甲酮(3b):淡黄色固体, 收率82%. m.p. 154.5~155.9 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.41 (dd, J=8.4, 1.6 Hz, 1H), 8.26 (dd, J=8.8, 0.8 Hz, 1H), 8.10 (dd, J=8.8, 0.8 Hz, 1H), 7.96~7.76 (m, 4H), 7.79 (d, J=8.0 Hz, 2H), 7.25 (d, J=8.0 Hz, 2H), 2.44 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 196.3, 144.4, 143.6, 143.5, 142.8, 141.7, 140.0, 135.4, 131.5, 131.1, 130.6, 130.4, 130.3, 129.5, 129.4, 129.2, 129.1, 21.8; HR-ESI-MS calcd for C20H15N2O [M+H]+ 299.1179, found 299.1174.

    吩嗪-1-(4-乙基苯基)甲酮(3c):淡黄色固体, 收率88%. m.p. 149.3~151.5 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.41 (dd, J8.4, 1.6 Hz, 1H), 8.26 (dd, J=8.8, 0.8 Hz, 1H), 8.10 (dd, J=8.8, 0.8 Hz, 1H), 7.97~7.76 (m, 4H), 7.82 (d, J=8.4 Hz, 2H), 7.28 (d, J=8.0 Hz, 2H), 2.74 (q, J=7.2 Hz, 2H), 1.28 (t, J=7.6 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ: 196.3, 150.5, 143.6, 143.5, 142.8, 141.7, 140.0, 135.6, 131.5, 131.1, 130.6, 130.5, 130.3, 129.5, 129.4, 129.1, 128.0, 29.0, 15.1; HR-ESI-MS calcd for C21H17N2O [M+H]+ 313.1335, found 313.1331.

    吩嗪-1-(4-异丙基苯基)甲酮(3d):淡黄色固体, 收率71%. m.p. 126.4~129.0 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.40 (dd, J=8.4, 1.6 Hz), 8.26 (dd, J=8.8, 0.8 Hz, 1H), 8.11 (dd, J=8.8, 0.8 Hz, 1H), 7.97~7.76 (m, 4H), 7.84 (d, J=8.4 Hz, 2H), 7.31 (d, J=8.4 Hz, 2H), 2.99 (m, J=6.8 Hz, 1H), 1.29 (d, J=6.8 Hz, 6H); 13C NMR (101 MHz, CDCl3) δ: 196.3, 155.1, 143.6, 143.5, 142.8, 141.7, 140.0, 135.7, 131.4, 131.1, 130.6, 130.3, 129.5, 129.4, 129.1, 126.6, 34.3, 23.7; HR-ESI-MS calcd for C22H19N2O [M+H]+ 327.1492, found 327.1487.

    吩嗪-1-(4-叔丁基苯基)甲酮(3e):淡黄色固体, 收率88%. m.p. 167.9~169.0 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.41 (dd, J=8.8, 1.6 Hz, 1H), 8.26 (dd, J=8.8, 0.8 Hz, 1H), 8.12 (dd, J=8.8, 0.8 Hz, 1H), 7.96~7.77 (m, 4H), 7.84 (d, J=8.4 Hz, 2H), 7.47 (d, J=8.4 Hz, 2H), 1.36 (s, 9H); 13C NMR (101 MHz, CDCl3) δ: 196.3, 157.3, 143.6, 143.5, 142.8, 141.7, 140.0, 135.2, 131.4, 131.1, 130.6, 130.4, 130.3, 129.5, 129.4, 129.1, 125.4, 35.2, 31.1; HR-ESI-MS calcd for C23H21N2O [M+H]+ 341.1648, found 341.1644.

    吩嗪-1-(4-氟苯基)甲酮(3f):淡黄色固体, 收率81%. m.p. 172.8~173.9 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.41 (dd, J=7.2, 2.8 Hz, 1H), 8.25 (dd, J=8.8, 0.8 Hz, 1H), 8.04 (dd, J=8.8, 0.8 Hz, 1H), 7.96~7.76 (m, 4H), 7.92 (d, J=2.4 Hz, 2H), 7.10 (t, J=8.8 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ: 195.2, 167.2, 164.7, 143.6, 143.4, 142.7, 141.5, 139.4, 132.9, 132.8 (d, JCF=10.1 Hz), 132.8, 131.9, 131.2, 130.8, 130.2, 129.5, 129.4, 115.7; HR-ESI- MS calcd for C19H12FN2O [M+H]+ 303.0928, found 303.0924.

    吩嗪-1-(4-氯苯基)甲酮(3g):淡黄色固体, 收率66%. m.p. 202.4~205.0 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.41 (dd, J=6.8, 3.6 Hz, 1H), 8.24 (dd, J=8.8, 0.8 Hz, 1H), 8.02 (dd, J=8.8, 0.8 Hz, 1H), 7.93 (dd, J=8.4, 5.2 Hz, 2H), 7.88~7.75 (m, 4H), 7.40 (dd, J=8.8, 4.8 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ: 195.2, 167.2, 164.7, 143.6, 143.4, 142.7, 141.5, 139.4, 134.5, 132.8, 131.9, 131.2, 130.8, 130.2, 129.5, 129.4, 115.7, 115.5; HR-ESI- MS calcd for C19H12ClN2O [M+H]+ 319.0633, found 319.0630.

    吩嗪-1-(4-溴苯基)甲酮(3h):淡黄色固体, 收率77%. m.p. 210.2~213.5 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.44 (dd, J=6.4, 3.6 Hz, 1H), 8.27 (dd, J=8.8, 0.8 Hz, 1H), 8.05 (dd, J=8.8, 0.8 Hz, 1H), 7.96~7.93 (m, 2H), 7.91~7.84 (m, 1H), 7.81 (d, J=8.4 Hz, 2H), δ 7.80 (dd, J=7.2, 1.2 Hz, 1H), 7.42 (d, J=8.8 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ: 195.6, 143.7, 143.4, 142.7, 141.5, 139.8, 139.2, 136.5, 132.1, 131.5, 131.2, 130.8, 130.2, 129.7, 129.6, 129.4, 128.7; HR-ESI-MS calcd for C19H12BrN2O [M+H]+ 363.0128, found 363.0125.

    吩嗪-1-(2, 3-二甲基苯基)甲酮(3i):淡黄色固体, 收率90%. m.p. 139.4~141.8 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.40 (dd, J=8.4, 1.2 Hz, 1H), 8.26 (dd, J=8.8, 0.8 Hz, 1H), 8.11 (dd, J=8.8, 0.8 Hz, 1H), 7.95~7.83 (m, 2H), 7.87 (dd, J=6.8, 1.6 Hz, 1H), 7.82~7.73 (m, 2H), 7.56 (dd, J=8.0, 1.6 Hz, 1H), 7.19 (d, J=8.0 Hz, 1H), 2.35 (s, 3H), 2.30 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 196.5, 143.6, 143.5, 143.2, 142.8, 141.8, 140.1, 136.9, 135.8, 131.3, 131.1, 130.5, 130.4, 129.7, 129.5, 129.4, 129.0, 128.4, 20.1, 19.7; HR-ESI-MS calcd for C21H17N2O [M+H]+ 313.1335, found 313.1331.

    吩嗪-1-(2, 4-二甲基苯基)甲酮(3j):淡黄色固体, 收率79%. m.p. 153.2~155.0 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.36 (dd, J=6.0, 4.0 Hz, 1H), 8.22 (dd, J=8.8, 0.8 Hz, 1H), 8.06 (dd, J8.8, 0.8 Hz, 1H), 7.93~7.73 (m, 4H), 7.23 (d, J=8.0 Hz, 1H), 7.17 (s, 1H), 6.88 (d, J=8.0 Hz, 1H), 2.72 (s, 3H), 2.35 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 199.0, 143.5, 143.4, 142.8, 141.7, 140.8, 138.6, 136.1, 134.8, 132.4, 132.0, 131.8, 131.6, 131.1, 130.6, 130.4, 129.9, 129.5, 129.4, 29.7, 20.8; HR-ESI-MS calcd for C21H17N2O [M+H]+ 313.1335, found 313.1329.

    吩嗪-1-(2, 5-二甲基苯基)甲酮(3k):淡黄色固体, 收率69%. m.p. 111.4~113.0 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.40 (dd, J=7.6, 2.4 Hz, 1H), 8.25 (dd, J=8.8, 0.8 Hz, 1H), 8.08 (dd, J=8.8, 0.8 Hz, 1H), 7.96~7.77 (m, 4H), 7.23 (q, J=7.8 Hz, 2H), 7.17 (s, 1H), 2.65 (s, 3H), 2.19 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 199.0, 143.5, 143.4, 142.8, 141.7, 140.8, 138.6, 136.1, 134.8, 132.4, 132.0, 131.8, 131.6, 131.1, 130.6, 130.4, 129.9, 129.5, 129.4, 29.7, 20.9; HR-ESI-MS calcd for C21H17N2O [M+H]+ 313.1335, found 313.1330.

    吩嗪-1-(4-苯氧基苯基)甲酮(3l):淡黄色固体, 收率97%. m.p. 151.7~153.2 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.41 (dd, J=8.0, 2, 0 Hz, 1H), 8.26 (dd, J=8.8, 0.8 Hz, 1H), 8.13 (dd, J=8.8, 0.8 Hz, 1H), 7.98~7.79 (m, 4H), 7.87 (d, J=8.8 Hz, 2H), 7.43 (t, J=8.0 Hz, 2H), 7.21 (t, J=7.2 Hz, 1H), 7.11 (d, J=7.2 Hz, 2H), 6.99 (d, J=8.8 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ: 195.2, 162.4, 155.3, 143.6, 143.5, 142.8, 141.66, 139.9, 132.6, 132.5, 131.5, 131.1, 130.7, 130.3, 130.1, 129.5, 129.4, 129.2, 124.7, 120.3, 118.9, 117.1, 100.0; HR-ESI-MS calcd for C25H17N2O2 [M+H]+ 377.1285, found 377.1278.

    吩嗪-1-(4-甲氧基苯基)甲酮(3m):淡黄色固体, 收率93%. m.p. 167.8~169.0 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.38 (dd, J=8.8, 1.6 Hz, 1H), 8.24 (dd, J=8.8, 0.8 Hz, 1H), 8.09 (dd, J=8.8, 0.8 Hz, 1H), 7.97~7.74 (m, 4H), 7.85 (d, J=8.8 Hz, 2H), 6.91 (d, J=8.8 Hz, 2H), 3.87 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 195.2, 163.9, 143.6, 143.5, 142.8, 141.7, 140.1, 132.7, 131.3, 131.1, 131.0, 130.6, 130.3, 129.5, 129.5, 129.1, 113.7, 55.5; HR-ESI-MS calcd for C20H15N2O2 [M+ H]+ 315.1128, found 315.1121.

    吩嗪-1-(3-氟-4-甲氧基苯基)甲酮(3n):淡黄色固体, 收率72%. m.p. 185.6~187.0 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.42 (dd, J=8.4, 1.6 Hz, 1H), 8.26 (dd, J=8.8, 0.8 Hz, 1H), 8.09 (dd, J=8.8, 0.8 Hz, 1H), 7.99~7.77 (m, 4H), 7.70 (dd, J=11.6, 2.0 Hz, 1H), 7.60~7.58 (m, 1H), 6.95 (t, J=8.4 Hz, 1H), 3.96 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 194.5, 153.2, 152.3, 152.2, 150.7, 143.6, 143.5, 142.7, 141.6, 139.4, 137.4, 135.1, 131.7, 131.2, 130.8, 130.3, 129.5 (d, JCF=15.3 Hz), 129.3, 128.0, 128.0, 117.5, 117.3, 113.7, 112.2, 56.3; HR-ESI-MS calcd for C20H14F- N2O2 [M+H]+ 333.1034, found 333.1026.

    吩嗪-1-(3-氯-4-甲氧基苯基)甲酮(3o):淡黄色固体, 收率90%. m.p. 187.2~189.9 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.40 (dd, J=8.4, 1.6 Hz, 1H), 8.24 (dd, J=8.8, 0.8 Hz, 1H), 8.07 (dd, J=8.8, 0.8 Hz, 1H), 7.96~7.76 (m, 4H), 7.94 (s, 1H), 7.72 (d, J2.0 Hz, 1H), 6.92 (d, J=8.8 Hz, 1H) 3.96 (s, 1H); 13C NMR (101 MHz, CDCl3) δ: 194.4, 159.1, 143.7, 143.5, 142.7, 141.6, 139.4, 132.2, 131.8, 131.60, 131.2, 131.0, 130.8, 130.3, 129.5, 129.4, 129.3, 127.8, 122.9, 112.1, 111.1, 56.4; HR-ESI-MS calcd for C20H14ClN2O2 [M+H]+ 349.0738, found 349.0733.

    吩嗪-1-(3-溴-4甲氧基苯基)甲酮(3p):淡黄色固体, 收率42%. m.p. 185.9~187.6 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.42 (dd, J=8.4, 1.2 Hz, 1H), 8.27 (d, J=8.8 Hz, 1H), 8.15 (d, J=2.0 Hz, 1H), 8.10 (d, J=8.8 Hz, 1H), 8.00~7.73 (m, 4H), 7.83~7.72 (m, 1H), 6.91 (d, J=8.8 Hz, 1H), 3.98 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 194.2, 159.9, 143.6, 143.5, 142.7, 141.6, 139.3, 135.3, 132.0, 131.8, 131.2, 130.8, 130.3, 129.5, 129.4, 129.3, 111.9, 111.0, 56.5; HR-ESI-MS calcd for C20H14BrN2O2 [M+H]+ 393.0233, found 393.0230.

    3.2.3   5-嘧啶锂(5)的合成

    称取0.59 g (3.75 mmol) 5-溴嘧啶于50 mL圆底烧瓶中, 加入30 mL无水四氢呋喃, 氮气置换并保护密封条件下, 降温至-78 ℃以下.量取1.8 mL的正丁基锂(2.5 mol/L的正己烷溶液), 缓慢注入烧瓶中反应40 min并保持温度-78 ℃以下, 得到淡黄色反应液.直接用于下步反应.

    3.2.4   吩嗪-1-芳基(5-嘧啶基)甲醇(6a~6p)的合成

    以吩嗪-1-苯基(5-嘧啶基)甲醇(6a)的合成为例, 称取0.7 g (2.5 mmoL)吩嗪-1-(苯基)甲酮(3a), 用无水四氢呋喃20 mL溶解, 缓慢注入上述所得反应液中, 滴加完毕后温度自然上升至室温, 反应4 h, TLC监测.反应完毕, 向反应液中滴入10%氯化铵水溶液, 淬灭10 min后加入15 mL二氯甲烷萃取, 干燥, 蒸去溶剂得粗品, 柱层析纯化(石油醚/乙酸乙酯, V:V=4:1)得黄色纯品, 收率31%. m.p. 175.2~177.9 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.12 (s, 1H), 8.68 (s, 2H), 8.33 (s, 1H), 8.25 (td, J=8.8, 0.8 Hz, 2H), 8.02 (d, J=0.8 Hz, 1H), 7.91~7.79 (m, 2H), 7.72 (dd, J=8.8, 7.2 Hz, 1H), 7.20 (q, J=8.4 Hz, 4H), 7.14 (dd, J=7.2, 1.2 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ: 157.6, 156.4, 144.4, 144.4, 143.3, 141.6, 141.6, 141.4, 140.5, 140.2, 131.5, 131.2, 130.6, 129.6, 129.4, 129.0, 128.4, 128.0, 127.7, 81.0; HR-ESI-MS calcd for C23H17N4O [M+H]+ 365.1397, found 365.1389.

    其他目标化合物的合成方法与之类似.

    吩嗪-1-(4-甲基苯基)(5-嘧啶基)甲醇(6b):黄色固体, 收率52%. m.p. 155.7~158.9 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.14 (s, 1H), 8.71 (s, 2H), 8.35 (s, 1H), 8.27 (td, J=8.8, 0.8 Hz, 2H), 8.03 (dd, J=8.4, 1.2 Hz, 1H), 7.93~7.81 (m, 2H), 7.74 (dd, J=8.8, 7.2 Hz, 1H), 7.22 (q, J=8.4 Hz, 4H), 7.16 (dd, J=7.2, 1.2 Hz, 1H), 2.39 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 157.5, 156.3, 144.4, 143.2, 141.6, 141.5, 141.4, 140.4, 140.4, 137.8, 131.4, 131.2, 131.2, 130.5, 129.5, 129.4, 129.1, 129.0, 127.7, 80.9, 21.1; HR-ESI-MS calcd for C24H19N4O [M+H]+ 379.1553, found 379.1547.

    吩嗪-1-(4-乙基苯基)(5-嘧啶基)甲醇(6c):黄色固体, 收率49%. m.p. 159.7~163.1 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.14 (s, 1H), 8.71 (s, 2H), 8.37 (s, 1H), 8.30~8.23 (m, 2H), 8.02 (dd, J=8.4, 1.2 Hz, 1H), 7.90~7.80 (m, 2H), 7.74 (dd, J=8.8, 7.2 Hz, 1H), 7.24 (q, J=8.4 Hz, 4H), 7.17 (dd, J=7.2, 1.2 Hz, 1H), 2.68 (q, J=7.6 Hz, 2H), 1.26 (t, J=7.6 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ: 157.5, 156.3, 144.4, 144.0, 143.2, 141.6, 141.6, 141.5, 140.4, 131.4, 131.3, 131.2, 130.5, 129.5, 129.4, 129.0, 127.9, 127.7, 80.9, 28.4, 15.4; HR-ESI-MS calcd for C25H21N4O [M+H]+ 393.1710, found 393.1703.

    吩嗪-1-(4-异丙基苯基)(5-嘧啶基)甲醇(6d):黄色固体, 收率22%. m.p. 154.1~158.3 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.13 (s, 1H), 8.71 (s, 2H), 8.38 (s, 1H), 8.27 (dd, J=8.8, 1.2 Hz, 1H), 8.24 (dd, J=8.8, 0.8 Hz, 1H), 8.02 (dd, J=8.4, 1.2 Hz, 1H), 7.89~7.79 (m, 2H), 7.74 (dd, J=8.8, 7.2 Hz, 1H), 7.30~7.22 (m, 4H), 7.17 (dd, J=7.2, 1.2 Hz, 1H), 2.94 (dt, J=13.4, 6.8 Hz, 1H), 1.28 (d, J=0.8 Hz, 6H); 13C NMR (101 MHz, CDCl3) δ: 157.4, 156.3, 148.7, 144.4, 143.2, 141.6, 141.6, 141.5, 140.5, 140.4, 131.4, 131.3, 131.2, 130.5, 129.5, 129.4, 129.0, 127.7, 126.4, 81.0, 33.7, 23.9, 23.9; HR-ESI-MS calcd for C26H23N4O [M+H]+ 407.1866, found 407.1861.

    吩嗪-1-(4-叔丁基苯基)(5-嘧啶基)甲醇(6e):黄色固体, 收率34%. m.p. 162.3~164.7 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.13 (s, 1H), 8.71 (s, 2H), 8.38 (s, 1H), 8.28 (td, J=8.8, 0.8 Hz, 2H), 8.04 (dd, J=8.4, 1.2 Hz, 1H), 7.92~7.81 (m, 2H), 7.76 (dd, J=8.8, 7.2 Hz, 1H), 7.41 (d, J=8.4 Hz, 2H), 7.27 (d, J=8.4 Hz, 2H), 7.18 (dd, J=6.8, 1.2 Hz, 1H), 1.35 (s, 9H); 13C NMR (101 MHz, CDCl3) δ: 157.5, 156.3, 150.9, 150.9, 144.4, 143.3, 141.7, 141.5, 141.2, 140.5, 140.5, 131.4, 131.3, 131.2, 130.5, 129.6, 129.4, 129.0, 127.5, 125.3, 80.9, 34.6, 31.3; HR- ESI-MS calcd for C27H25N4O [M+H]+ 421.2023, found 421.2018.

    吩嗪-1-(4-氟苯基)(5-嘧啶基)甲醇(6f):黄色固体, 收率23%. m.p. 182.4~184.8 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.16 (s, 1H), 8.70 (s, 2H), 8.39 (s, 1H), 8.30 (dd, J=8.8, 1.2 Hz, 2H), 8.27 (dd, J=8.8, 0.8 Hz, 1H), 8.02 (dd, J=8.4, 1.2 Hz, 1H), 7.93~7.82 (m, 2H), 7.75 (dd, J=8.8, 7.2 Hz, 1H), 7.34 (dd, J8.8, 5.2 Hz, 2H), 7.13 (dd, J=7.2, 1.2 Hz, 1H), 7.08 (t, J=8.8 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ: 163.6, 161.1, 157.7, 156.3, 144.4, 143.3, 141.5, 141.2, 140.5 (d, J=15.1 Hz), 140.0, 131.6, 131.3, 131.1, 130.8, 129.6, 129.5, 129.3, 128.9, 115.4, 115.2, 80.7; HR-ESI-MS calcd for C23H16FN4O [M+H]+ 383.1303, found 383.1299.

    吩嗪-1-(4-氯苯基)(5-嘧啶基)甲醇(6g):黄色固体, 收率57%. m.p. 172.3~174.9 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.17 (s, 1H), 8.70 (s, 2H), 8.38 (s, 1H), 8.31 (dd, J=8.8, 1.2 Hz, 1H), 8.27 (dd, J=8.8, 1.2 Hz, 1H), 8.02 (dd, J=8.4, 1.2 Hz, 1H), 7.95~7.81 (m, 2H), 7.76 (dd, J=8.8, 7.2 Hz, 1H), 7.37 (d, J=8.8 Hz, 2H), 7.31 (d, J=8.8 Hz, 2H), 7.13 (dd, J=7.2, 1.2 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ: 157.7, 156.3, 144.4, 143.4, 143.1, 141.44, 140.9, 140.4, 139.7, 134.0, 131.6, 131.3, 131.1, 130.8, 129.6, 129.3, 129.1, 128.9, 128.6, 80.7; HR-ESI-MS calcd for C23H16ClN4O [M+H]+ 399.1007, found 399.1004.

    吩嗪-1-(4-溴苯基)(5-嘧啶基)甲醇(6h):黄色固体, 收率42%. m.p. 176.9~180.5 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.17 (s, 1H), 8.70 (s, 2H), 8.38 (s, 1H), 8.31 (dd, J=8.8, 1.2 Hz, 1H), 8.27 (dd, J=8.8, 1.2 Hz, 1H), 8.02 (dd, J=8.4, 0.8 Hz, 1H), 7.95~7.82 (m, 2H), 7.76 (dd, J=8.8, 7.2 Hz, 1H), 7.52 (d, J=8.4 Hz, 2H), 7.25 (d, J=8.4 Hz, 2H), 7.14 (dd, J=7.2, 1.2 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ: 157.7, 156.3, 144.4, 143.4, 143.1, 141.4, 140.9, 140.4, 139.7, 134.0, 131.6, 131.3, 131.1, 130.8, 129.6, 129.3, 129.1, 128.9, 128.6, 80.7; HR-ESI-MS calcd for C23H16BrN4O [M+H]+ 443.0502, found 443.0499.

    吩嗪-1-(2, 3-二甲基苯基)(5-嘧啶基)甲醇(6i):黄色固体, 收率40%. m.p. 187.0~188.9 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.14 (s, 1H), 8.71 (s, 2H), 8.37 (s, 1H), 8.29~8.23 (m, 2H), 8.03 (dd, J=8.4, 1.2 Hz, 1H), 7.91~7.79 (m, 2H), 7.74 (dd, J=8.8, 7.2 Hz, 1H), 7.18 (dd, J=7.2, 1.2 Hz, 2H), 7.13 (d, J=7.2 Hz, 1H), 6.98 (dd, J=7.2, 1.2 Hz, 1H), 2.29 (s, 3H, CH3), 2.26 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 157.4, 156.3, 144.4, 143.2, 141.7, 141.7, 141.5, 140.6, 140.4, 136.7, 136.5, 131.4, 131.3, 131.2, 130.5, 129.5, 129.5, 129.4, 129.0, 128.9, 125.2, 80.9, 20.1, 19.4; HR-ESI-MS calcd for C25H21N4O [M+ H]+ 393.1710, found 393.1703.

    吩嗪-1-(2, 4-二甲基苯基)(5-嘧啶基)甲醇(6j):黄色固体, 收率39%. m.p. 151.0~153.1 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.14 (s, 1H), 8.74 (s, 1H), 8.57 (s, 2H), 8.31 (dd, J=8.8, 1.2 Hz, 1H), 8.27 (dd, J=8.4, 0.8 Hz, 1H), 8.06 (dd, J=8.4, 1.2 Hz, 1H), 7.94~7.81 (m, 2H), 7.76 (dd, J=8.8, 7.2 Hz, 1H), 7.23 (dd, J=7.2, 1.2 Hz, 1H), 7.15 (s, 1H), 6.84 (d, J=7.2 Hz, 1H), 6.41 (d, J=8.0 Hz, 1H), 2.35 (s, 3H), 2.30 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 157.5, 156.4, 144.5, 143.3, 141.7, 140.8, 140.5, 140.4, 139.3, 138.4, 138.1, 134.0, 131.5, 131.2, 130.6, 130.5, 129.6, 129.5, 129.0, 129.0, 125.7, 82.5, 22.4, 20.8; HR-ESI-MS calcd for C25H21N4O [M+H]+ 393.1710, found 393.1703.

    吩嗪-1-(2, 5-二甲基苯基)(5-嘧啶基)甲醇(6k):黄色固体, 收率46%. m.p. 154.0~157.5 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.14 (s, 1H), 8.74 (s, 1H), 8.61 (s, 2H), 8.31 (dd, J=8.8, 1.2 Hz, 1H), 8.28 (dd, J=8.4, 1.2 Hz, 1H), 8.06 (dd, J=8.4, 1.2 Hz, 1H), 7.93~7.83 (m, 2H), 7.77 (dd, J=8.8, 7.2 Hz, 1H), 7.24 (dd, J=7.2, 1.2 Hz, 1H), 7.21 (d, J7.8 Hz, 1H), 7.11 (dd, J=7.6, 0.8 Hz, 1H), 6.37 (d, J=0.8 Hz, 1H), 2.27 (s, 3H), 2.17 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 157.5, 156.5, 144.5, 143.3, 141.9, 141.7, 140.6, 140.5, 140.4, 135.4, 134.6, 133.1, 131.5, 131.2, 130.7, 130.5, 129.7, 129.6, 129.5, 129.0, 82.6, 22.1, 21.1; HR-ESI-MS calcd for C25H21N4O [M+ H]+ 393.1710, found 393.1703.

    吩嗪-1-(4-苯氧基苯基)(5-嘧啶基)甲醇(6l):黄色固体, 收率57%. m.p. 193.4~195.1 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.15 (s, 1H), 8.73 (s, 2H), 8.38 (s, 1H), 8.30 (dd, J=8.8, 1.2 Hz, 1H), 8.27 (dd, J=8.8, 1.2 Hz, 1H), 8.04 (dd, J=8.4, 1.2 Hz, 1H), 7.93~7.83 (m, 2H), 7.76 (dd, J=8.8, 7.2 Hz, 1H), 7.44~7.24 (m, 4H), 7.25~7.09 (m, 1H), 7.10~6.93 (m, 5H); 13C NMR (101 MHz, CDCl3) δ: 157.6, 157.2, 156.6, 156.3, 144.4, 143.3, 141.6, 141.4, 140.5, 140.2, 139.0, 131.5, 131.2, 131.2, 130.7, 129.8, 129.6, 129.3, 129.2, 129.0, 123.7, 119.3, 118.2, 80.8; HR-ESI-MS calcd for C29H21N4O2 [M+H]+ 457.1659, found 457.1650.

    吩嗪-1-(4-甲氧基苯基)(5-嘧啶基)甲醇(6m):黄色固体, 收率46%. m.p. 169.1~172.0 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.14 (s, 1H), 8.71 (s, 2H), 8.33 (s, 1H), 8.28 (td, J=8.8, 1.2 Hz, 2H), 8.03 (dd, J=8.4, 1.2 Hz, 1H), 7.92~7.81 (m, 2H), 7.75 (dd, J=8.8, 7.2 Hz, 1H), 7.27 (d, J=8.8 Hz, 2H), 7.17 (dd, J=7.2, 1.2 Hz, 1H), 6.92 (d, J=8.8 Hz, 2H), 3.84 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 159.2, 157.5, 156.3, 144.4, 143.3, 141.7, 141.6, 140.5, 136.6, 131.4, 131.2, 130.6, 129.6, 129.4, 129.0, 113.7, 80.8, 55.3; HR-ESI-MS calcd for C24H19N4O2 [M+H]+ 395.1503, found 395.1494.

    吩嗪-1-(3-氟-4-甲氧基苯基)(5-嘧啶基)甲醇(6n):黄色固体, 收率34%. m.p. 199.2~202.0 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.17 (s, 1H), 8.71 (s, 2H), 8.37 (s, 1H), 8.31 (dd, J=8.8, 1.2 Hz, 1H), 8.28 (dd, J=8.4, 0.8 Hz, 1H), 8.04 (dd, J=8.4, 1.2 Hz, 1H), 7.93~7.83 (m, 2H), 7.76 (dd, J=8.8, 7.2 Hz, 1H), 7.26~7.20 (m, 1H), 7.16 (dd, J=7.2, 1.2 Hz, 1H), 6.93 (dd, J=3.6, 1.2 Hz, 2H), 3.92 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 157.7, 156.3, 153.5, 151.0, 147.3 (d, J=15.1 Hz), 144.4, 143.3, 141.5, 141.0, 140.4, 139.8, 137.7, 131.6, 131.3, 131.1, 130.8, 129.6, 129.3, 129.0, 123.6, 115.9, 115.7, 112.7, 80.5, 56.3; HR-ESI-MS calcd for C24H18FN4O2 [M+H]+ 413.1408, found 413.1400.

    吩嗪-1-(3-氯-4-甲氧基苯基)(5-嘧啶基)甲醇(6o):黄色固体, 收率55%. m.p. 212.0~215.6 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.17 (s, 1H), 8.70 (s, 2H), 8.39 (s, 1H), 8.31 (dd, J=8.8, 1.2 Hz, 1H), 8.30~8.25 (m, 1H), 8.04 (dd, J=8.4, 1.2 Hz, 1H), 7.93~7.83 (m, 2H), 7.77 (dd, J=8.8, 7.2 Hz, 1H), 7.47 (d, J=2.4 Hz, 1H), 7.16 (dd, J=7.2, 1.2 Hz, 1H), 7.12 (dd, J=8.8, 2.4 Hz, 1H), 6.91 (d, J=8.8 Hz, 1H), 3.93 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 157.7, 156.3, 154.6, 144.4, 143.3, 141.5, 141.0, 140.4, 139.9, 137.8, 131.6, 131.3, 131.2, 130.8, 129.7, 129.6, 129.3, 128.9, 127.1, 122.8, 111.5, 80.4, 56.2; HR-ESI-MS calcd for C24H18ClN4O2 [M+H]+ 429.1113, found 429.1104.

    吩嗪-1-(3-溴-4-甲氧基苯基)(5-嘧啶基)甲醇(6p):黄色固体, 收率39%. m.p. 207.6~210.0 ℃; 1H NMR (400 MHz, CDCl3) δ: 9.17 (s, 1H), 8.71 (s, 2H), 8.37 (s, 1H), 8.31 (dd, J=8.8, 1.2 Hz, 1H), 8.29~8.25 (m, 1H), 8.06~8.00 (m, 1H), 7.94~7.82 (m, 2H), 7.76 (dd, J=8.8, 7.2 Hz, 1H), 7.26~7.20 (m, 1H), 7.16 (dd, J=7.2, 1.2 Hz, 1H), 6.93 (d, J=4.4 Hz, 2H), 3.92 (s, 3H); 13C NMR (101 MHz, CDCl3) δ: 157.7, 156.3, 153.5, 151.0, 144.4, 143.3, 141.5, 141.0, 140.4, 139.8, 137.7, 131.6, 131.3, 131.1, 130.8, 129.6, 129.3, 128.9, 123.6, 115.7, 112.8, 80.5, 56.3; HR-ESI-MS calcd for C24H18BrN4O2 [M+H]+ 473.0608, found 473.0598.

    3.3.1   离体杀菌活性测试

    采用离体平皿法[24]测定化合物3a~3p6a~6p在50 mg/L下对水稻纹枯病(Thanatephorus cucumeris)、小麦赤霉病(Fusarium graminearum)、香樟炭疽病(Colletotrichum gloeosporioides)、梨黑斑病(Alternaria alternate)、西瓜枯萎病(Fusarium oxysporum)、辣椒疫病(Phytophthora capsici)的杀菌活性.以商品化杀菌剂申嗪霉素和氯苯嘧啶醇原药作为对照药剂.每个处理重复3次实验.

    3.3.2   目标化合物抗小麦白粉病活性盆栽测试

    四叶期的小麦苗被喷洒浓度为50 mg/L的目标化合物[28-29], 培养箱培养24 h, 配制含有新鲜小麦白粉病孢子的悬浊液, 喷洒至小麦叶片进行接种, 接种完毕放入培养箱继续培养, 培养温度为20 ℃.观察病变的形成, 待空白组病变超过70%, 开始调查病情.每个处理重复三次实验.平均防效(%)=[(空白组病情指数—处理组病情指数)/空白组病情指数]×100%.

    麦角甾醇生物合成抑制实验采用文献描述的方法并做了部分改进[30].将带有新鲜水稻纹枯病菌菌丝的菌碟(直径为5 mm)放入150 mL马铃薯葡萄糖琼脂培养基(PDA)液体培养基中, 摇床培养3 d (150 r/min, 25 ℃).加入20 mg/L相应测试化合物, 继续培养48 h.培养完毕, 过滤得到菌丝, 烘干, 分别称取0.2 g干菌丝放入10 mL的甲醇和氯仿混合液(甲醇/氯仿, V:V=3:1)中进行萃取12 h, 依次加入蒸馏水、氯仿、0.5 mol/L的磷酸盐缓冲溶液(含2.0 mol/L氯化钾)各10 mL, 静置分层, 萃取氯仿层并旋蒸至干.加入10 mL的乙酸和乙醇混合液(V:V=4:1, 含1.4 mol/L氢氧化钾), 60 ℃下皂化60 min.正己烷萃取(10 mL), 转蒸脱去溶剂, 用适量乙醇溶解所得沉淀, 再在10 mL容量瓶中定容, 微孔滤膜(0.45 μm)过滤后, 待测.在设定的色谱条件下(气相色谱升温程序:初始温度70 ℃, 1 min; 以15 ℃/min升温至205 ℃; 以20 ℃/ min升温至270 ℃并保留23 min), 分别测定不同样品中的麦角甾醇浓度, 以计算测试化合物对菌丝中麦角甾醇含量的抑制率.每个处理重复三次实验.

    辅助材料(Supporting Information)中间体3a~3p及目标化合物6a~6p的核磁共振图谱和高分辨质谱, 麦角甾醇生物合成抑制实验气相色谱图谱.这些材料可以免费从本刊网站(http://sioc-journal.cn/)上下载.


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  • 图 1  目标化合物的设计策略

    Figure 1  Design strategy for target compounds

    图式 1  目标化合物6a~6p的合成路线

    Scheme 1  Synthetic route of target compounds 6a~6p

    Reagents and conditions: (a) oxalyl chloride, CH2Cl2, DMF, reflux, 8 h; (b) substituted benzene, AlCl3, 0~60 ℃, 7 h; (c) n-butyllithium, THF, below -80 ℃, 1 h; (d) THF, below -80 ℃, 4 h

    图 2  目标化合物6f的X单晶衍射结构

    Figure 2  X-ray single crystal structure of target compound 6f

    图 3  化合物6g6i6j在50 mg/L浓度下抗小麦白粉病活性

    Figure 3  Fungicidal activity of compounds 6g, 6i and 6j against wheat powdery mildew under greenhouse conditions at 50 mg/L

    CK: the negative control

    图 4  化合物6f6g6i6j对麦角甾醇生物合成的抑制活性

    Figure 4  Inhibitory activities of compounds 6f, 6g, 6i and 6j on ergosterol biosynthesis

    PCA and AC were served as the positive control. For test compounds, the statistical significance of differences in the mean values at P=0.5 was analyzed by the Waller-Duncan test (SPSS v.21 for windows). Histograms with letters a, b, c, d, e and f demonstrate significant difference between all test compounds

    表 1  中间体3a~3p在50 mg/L条件下对4种植物病原真菌的抑制活性a

    Table 1.  Inhibition activity of the intermediate compounds 3a~3p against four pathogenic fungi at a concentration of 50 mg/L

    Compd. Thanatephorus cucumeris Fusarium graminearum Alternaria alternata Colletotrichum gloeosporioides
    3a 15.15±0.52 <10.00 21.20±0.01 20.28±0.00
    3b 12.45±0.29 NA 22.03±0.36 10.61±0.24
    3c <10.00 NA <10.00 13.29±0.02
    3d 21.69±0.52 NA <10.00 NA
    3e 20.17±0.50 <10.00 11.24±0.02 10.96±0.75
    3f <10.00 NA <10.00 <10.00
    3g 11.92±0.20 <10.00 12.76±0.43 <10.00
    3h <10.00 NA <10.00 NA
    3i 45.36±0.14 26.81±0.22 32.45±0.00 34.65±0.40
    3j 10.65±0.51 NA <10.00 <10.00
    3k 18.62±0.33 NA <10.00 NA
    3l 14.02±0.01 NA <10.00 NA
    3m <10.00 NA 12.85±0.12 14.79±0.52
    3n 11.56±0.21 NA 13.82±0.38 16.94±0.20
    3o 17.62±0.16 NA <10.00 NA
    3p <10.00 NA <10.00 <10.00
    PCA 80.54±0.24 40.76±0.62 81.11±0.40 79.05±0.78
    a Values are the mean±SD of three replicates. PCA (phenazine-1-carboxylic acid) served as the positive control. NA: not active under this concentration.
    下载: 导出CSV

    表 2  目标化合物6a~6p在50 mg/L的条件下对6种植物病原真菌的抑制活性a

    Table 2.  Inhibition activity of the target compounds 6a~6p against six pathogenic fungi at a concentration of 50 mg/L

    Compd. T.c. F.g. C.g. A.a. F.o. P.c.
    6a 24.55±0.20 14.38±0.16 19.19±0.42 21.47±0.73 10.58±0.19 40.53±0.02
    6b 68.81±0.24 53.04±0.40 44.36±0.26 57.06±0.15 46.10±0.31 69.39±0.17
    6c 34.37±0.12 NA <10.00 36.14±1.17 NA 48.02±0.06
    6d 13.94±0.43 NA <10.00 15.57±0.32 12.03±0.14 23.70±0.10
    6e NA 10.61±0.33 24.64±0.44 15.23±0.23 11.04±0.44 12.60±0.41
    6f 76.45±0.13 20.93±0.48 60.03±0.25 23.49±0.42 31.63±0.51 59.92±0.17
    6g 71.39±0.49 45.79±0.29 49.71±0.29 30.34±1.02 50.91±0.38 68.78±0.51
    6h 32.15±0.23 48.74±0.14 34.30±0.24 21.92±0.10 44.55±0.39 79.95±0.01
    6i 69.70±0.09 45.78±0.39 54.39±0.46 23.93±0.01 22.48±0.18 50.12±0.73
    6j 46.73±0.00 17.14±0.05 42.02±0.27 18.19±0.34 14.44±0.27 59.34±0.46
    6k 17.18±0.62 52.75±0.10 32.99±0.70 22.50±0.64 <10.00 15.85±0.68
    6l 28.45±0.02 12.53±0.05 12.45±0.20 20.25±0.05 15.66±0.57 10.65±0.01
    6m 16.35±0.50 14.54±0.27 25.89±0.35 19.87±0.25 17.34±0.22 21.99±0.00
    6n 23.83±0.24 <10.00 14.49±0.41 11.94±0.04 11.63±0.10 11.67±0.21
    6o 17.27±0.00 21.69±0.18 49.74±0.02 14.39±0.28 24.41±0.34 39.79±0.24
    6p NA 11.92±0.00 11.91±0.01 15.25±0.30 12.71±0.42 <10.00
    AC 98.81±0.26 74.60±0.33 74.36±0.51 96.00±1.13 47.55±0.90 72.09±0.23
    PCA 75.04±0.28 43.21±0.52 83.78±0.83 81.11±0.72 72.66±0.22 98.92±1.18
    a Values are the mean±SD of three replicates. PCA (phenazine-1-carboxylic acid) and AC (fenarimol) served as the positive control. NA: not active under this concentration. T.c.: Thanatephorus cucumeris, F.g.: Fusarium graminearum, C.g.: Colletotrichum gloeosporioides, A.a.: Alternaria alternata, F.o.: Fusarium oxysporum, P.c.: Phytophthora capsici.
    下载: 导出CSV

    表 3  目标化合物6b6f6g对水稻纹枯病和辣椒疫病的EC50 (mg/L)a

    Table 3.  EC50 (mg/L) of the target compounds 6b, 6f and 6g against Thanatephorus cucumeris and Phytophthora capsici

    Comp. Thanatephorus cucumeris Phytophthora capsici
    Regression Eq.b Rc EC50/(mg•L1) (95%CL)d Regression Eq.b Rc EC50/(mg•L1)(95%CL)d
    6b y=-1.61+1.15x 0.9750 21.62 (10.95~40.09) y=-2.13+1.69x 0.9437 18.68 (11.64~27.07)
    6f y=-1.70+1.24x 0.9891 22.04 (12.41~34.84) y=-4.43+2.51x 0.9573 66.98 (47.61~100.52)
    6g y=-1.86+1.62x 0.9620 14.48 (12.18~17.13) y=-2.23+1.86x 0.9812 17.52 (11.80~26.15)
    AC y=-0.90+1.08x 0.9942 6.85 (5.37~8.60) y=-1.12+1.07x 0.9749 10.39 (4.74~18.20)
    PCA y=-0.91+1.14x 0.9903 6.30 (4.98~7.82) y=-1.17+1.38x 0.9785 7.11 (5.87~8.55)
    a PCA and AC served as the positive control. b Regression Eq. means the toxicity regression equation; c Correlation coefficient; d CL means the confidence interval.
    下载: 导出CSV

    表 4  目标化合物6a~6p在50 mg/L浓度下抗小麦白粉病活性a

    Table 4.  Fungicidal activity of the target compounds 6a~6p against wheat powdery mildew under greenhouse conditions at 50 mg/L

    Compd. Disease index/% Control efficiencyb/%
    6a 23.42 63.44±3.45 cd
    6b 27.14 57.42±3.25 cde
    6c 37.65 47.27±3.11 g
    6d 35.23 43.50±3.04 g
    6e 45.28 29.17±4.10 h
    6f 14.89 79.50±4.03 b
    6g 9.54 85.52±5.20 b
    6h 34.74 52.17±5.92 ef
    6i 14.63 79.48±1.32 b
    6j 22.21 65.11±3.06 c
    6k 15.37 78.45±4.32 b
    6l 27.01 57.90±0.29 cde
    6m 49.59 22.04±3.06 hi
    6n 33.96 53.76±5.00 ef
    6o 30.57 55.84±4.69 de
    6p 44.91 29.44±3.45 h
    AC 2.78 96.00±3.64 a
    PCA 55.92 20.12±6.35 i
    a Values are the mean±SD of three replicates, PCA and AC served as the positive Control. b Control efficiency values marked by the same letter within 18 treatments were not significantly different according to the waller-duncan test at P=0.05.
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  • 发布日期:  2020-08-01
  • 收稿日期:  2020-04-07
  • 修回日期:  2020-04-28
  • 网络出版日期:  2020-05-11
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