English

• 香豆素又称1, 2-苯并吡喃酮, 其结构中的苯环和吡喃酮环可形成一个较大的共轭体系, 这种结构特征使得香豆素具有很强的可修饰性, 能够引入多种功能性基团^[1], 使其在生物医药、功能材料和光电材料等领域广泛应用^[2].此外, 香豆素类化合物具有多种生物活性, 如抗炎^[3]、抗病毒^[4]、抗菌^[5]、抗氧化^[6]和抗肿瘤^[7~9]等.近十几年来, 人们对香豆素类化合物的研究不断深入, 发现许多香豆素类化合物主要通过调控细胞周期、诱导细胞凋亡或对多种酶和信号通路产生影响等途径抑制肿瘤细胞的增殖^{[10, 11]}.

  微管在细胞有丝分裂和生长发育过程中起着非常重要的作用, 因此微管蛋白是抗癌药物研究中的一个重要靶点.研究发现, 多种含3, 4, 5-三甲氧基苯基片段的化合物通过抑制微管蛋白聚合来抑制肿瘤细胞的增殖^[12].如2-氨基-4-(3, 4, 5-三甲氧基苯基)-5-芳基噻唑作为微管蛋白聚合抑制剂, 对多药耐药肿瘤细胞表现出有效的抑制活性^[13]; 3-(3, 4, 5-三甲氧基苯胺基)苯并呋喃作用于微管蛋白的秋水仙碱结合位点, 从而有效抑制肿瘤细胞的增殖^[14].

  酰胺类化合物具有抗菌^[15]、抗肿瘤^[16]和杀虫^[17]等生物活性, 广泛应用于药物、材料和工业溶剂等方面.酰胺结构是蛋白质的基本结构, 具有低毒和生物利用度高等优点, 因此在药物分子设计中常被用到^[18].如温倩雯等^[19]合成一系列4-(5H-嘧啶并[5, 4-b]吲哚-2-基-氨基)苯甲酰胺类化合物, 结果显示该类化合物对肿瘤细胞增殖有明显抑制作用; 张明千等^[20]合成了一系列1, 3, 4-噻二唑酰胺类化合物, 发现部分化合物对人前列腺癌细胞(PC-3)的抑制作用要优于阳性对照药达卡巴嗪.

  在前期工作中我们设计合成了一系列含3, 4, 5-三甲氧基苯基的三级酰胺类化合物, 发现含有4-甲基香豆素基团的化合物有较好的抗肿瘤活性.利用药物化学中的拼合原理, 将3, 4, 5-三甲氧基苯基和香豆素这两个药效基团通过酰胺片段连接(图 1), 设计合成了19个目标化合物, 并测试了该类化合物对人食管癌细胞(EC-109)、人胃癌细胞(MGC-803)和人前列腺癌细胞(PC-3)的抑制作用, 探索其构效关系, 为此类化合物的进一步研究提供参考.

  图 1

  

  图 1.  目标化合物4的分子设计示意图

  Figure 1.  Design strategy of the target compounds 4

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  1.   结果与讨论

  1.1   化合物的合成

  3, 4, 5-三甲氧基苯胺和取代基苄氯在N, N-二甲基甲酰胺和碳酸钾作用下发生亲核取代反应生成化合物2, 再与氯乙酰氯反应得到化合物3, 最后, 再和香豆素通过Williamson反应得到目标化合物4.在合成化合物2的过程中, 通常会有二取代副产物产生, 因此, 需要严格控制反应物之间的物质的量比和反应时间.参考文献[21]中的合成方法, 控制两种原料的物质的量比为1:1, 与参考文献不同的是没有对反应进行加热而是在室温下进行.实验过程中通过薄层色谱对反应进行监测, 当体系中出现二取代杂质点时停止反应, 此时单取代化合物2的收率最高.最后一步反应的溶剂选择也非常关键, 参考文献[22]用丙酮作溶剂, 发现完成反应需要12 h, 收率约为20%.为了提高收率缩短反应时间, 分别使用乙腈、乙醇和四氢呋喃等溶剂, 对比发现乙腈作溶剂时, 反应时间缩短到5 h, 大部分收率提高到60%以上.

  1.2   化合物的抗肿瘤活性

  选用5-氟尿嘧啶作阳性对照药, 用四甲基偶氮唑盐(MTT)法评价该类化合物对人食管癌细胞(EC-109)、人胃癌细胞(MGC-803)和人前列腺癌细胞(PC-3)三种肿瘤细胞的抑制作用, 其IC₅₀值见表 1.

  表 1

  

  表 1  化合物4a~4s的抗肿瘤活性[IC₅₀/(μmol•L^-1)]

  Table 1.  Anti-cancer activity [IC₅₀/(μmol•L^-1)] of the compounds 4a~4s

  下载: 导出CSV

  Compd.	PC-3	EC-109	MGC-803
  4a	5.62±1.27	16.24±1.53	18.90±1.28
  4b	14.24±1.55	38.73±0.42	43.88±1.27
  4c	31.51±2.94	10.96±1.24	＞80
  4d	45.58±2.02	43.24±2.18	＞80
  4e	32.91±1.81	44.94±3.06	14.65±1.82
  4f	＞80	＞80	＞80
  4g	76.28±2. 59	46.31±1.25	78.29±3.64
  4h	＞80	36.183±1.72	＞80
  4i	47.93±1.30	27.93±1.30	56.94±1.52
  4j	＞80	72.86±1.37	67.61±1.32
  4k	12.81±1.63	55.68±3.09	＞80
  4l	42.46±2.42	51.33±3.82	33.32±7.06
  4m	39.33±2.02	46.75±0.64	76.19±2.42
  4n	4.18±0.41	14.32±2.48	17.92±2.38
  4o	14.62±1.51	42.93±2.04	＞80
  4p	9.72±1.28	24.19±1.82	20.19±1.84
  4q	42.18±1.39	＞80	47.17±1.24
  4r	32.06±1.59	48.91±2.08	＞80
  4s	＞80	60.63±3.47	＞80
  5-Fu	9.79±0.17	20.42±1.83	21.21±3.61

  从表 1可以看出, 大部分化合物对肿瘤细胞具有一定的抑制作用, 值得注意的是, 化合物4a和4n对三种肿瘤细胞的抑制作用均优于阳性对照药5-氟尿嘧啶, 其中, 化合物4n对人前列腺癌细胞(PC-3)的抑制活性最强, 其IC₅₀值为4.18 μmol/L.化合物4c对人食管癌细胞(EC-109)具有选择性抑制作用, 其IC₅₀值为10.96 μmol/ L; 化合物4e对人胃癌细胞(MGC-803)具有选择性抑制作用, 其IC₅₀值为14.65 μmol/L.对比化合物4b~4j对人前列腺癌细胞(PC-3)的抑制活性发现, 取代基R¹的种类和位置对化合物的抗肿瘤活性有明显的影响. R¹为给电子取代基(4b和4c)时的抑制活性优于R¹为吸电子取代基(4d、4e和4f)时的抑制活性, 其抑制活性顺序为OCH₃＞CH₃＞Br＞F＞Cl; R¹位于对位(4b、4c、4d和4f)时的抗肿瘤活性普遍优于位于间位(4g、4h、4i和4j)时的抗肿瘤活性.比较含有不同香豆素基团的化合物4n~4s对三种肿瘤细胞的抑制作用发现, 当香豆素基团的4位上含有甲基(4n和4p)时, 化合物对人前列腺癌细胞(PC-3)和人胃癌细胞(MGC-803)均有较好抑制的作用.

  图式 1

  

  图式 1.  目标化合物4的合成路线

  Scheme  1.  Synthetic routes of the target compounds 4

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  2.   结论

  利用拼合原理, 设计并合成了19个未见文献报道的新型香豆素类衍生物.用MTT法对合成的化合物进行抗肿瘤活性筛选, 结果显示, 化合物4a和4n对三种肿瘤细胞的抑制作用均优于阳性对照药5-氟尿嘧啶.此外, 化合物4c对人食管癌细胞(EC-109)的抑制作用以及化合物4e和4p对人胃癌细胞(MGC-803)的抑制作用也都优于阳性对照药5-氟尿嘧啶.

  3.   实验部分

  3.1   仪器与试剂

  ZF7三用紫外分析仪(巩义市予华仪器有限责任有限公司); SHB-Ⅲ循环水式多用真空泵(郑州长城科工贸有限公司); CP214电子天平(奥豪斯仪器有限公司); RE5299旋转蒸发仪(上海亚荣生化仪器厂); Q-TOF Micro高分辨质谱测定仪(美国Waters公司); DPX-400FT超导核磁共振仪(美国Bruker公司).所用试剂均为市售分析纯, 使用前按常规方法处理.实验所用的肿瘤细胞:人食管癌细胞(EC-109)、人胃癌细胞(MGC-803)和人前列腺癌细胞(PC-3)由郑州大学基础医学院药理系提供.

  3.2   实验方法

  3.2.1   中间体2的合成

  将2.00 g (10.92 mmol)的3, 4, 5-三甲氧基苯胺溶于N, N-二甲基甲酰胺中, 加入取代基苄氯(10.92 mmol)和1.50 g (10.92 mmol)的碳酸钾, 室温搅拌, 通过薄层色谱(TLC)监测.反应结束后抽滤, 收集滤液, 加入水和乙酸乙酯萃取三次, 合并有机相, 无水硫酸镁干燥后旋干, 柱层析分离(洗脱剂:石油醚-乙酸乙酯, V:V=6:1), 得到目标化合物2a~2n.

  N-苄基-3, 4, 5-三甲氧基苯胺(2a):白色固体, 收率49%. m.p. 84~85 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.37 (d, J=7.0 Hz, 2H), 7.32 (t, J=7.5 Hz, 2H), 7.22 (t, J=7.2 Hz, 1H), 6.02 (t, J=6.0 Hz, 1H), 5.89 (s, 2H), 4.23 (d, J=6.0 Hz, 2H), 3.64 (s, 6H), 3.50 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆)δ: 153.3, 145.4, 140.4, 128.5, 128.2, 127.4, 126.6, 90.1, 60.1, 55.4, 46.9; HRMS calcd for C₁₆H₂₀NO₃ [M+H]⁺ 274.1443, found 274.1448.

  N-(4-甲氧基苄基)-3, 4, 5-三甲氧基苯胺(2b)^[22]:白色固体, 收率58%. m.p. 101~102 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.23 (d, J=8.6 Hz, 2H), 6.81 (d, J=8.7 Hz, 2H), 5.82 (s, 2H), 4.15 (s, 2H), 3.73 (s, 3H), 3.72 (s, 6H), 3.69 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 158.1, 153.3, 145.1, 131.9, 130.4, 128.7, 113.6, 90.4, 60.1, 55.4, 55.0, 46.5; HRMS calcd for C₁₇H₂₂NO₄ [M+H]⁺ 304.1549, found 304.1553.

  N-(4-甲基苄基)-3, 4, 5-三甲氧基苯胺(2c):白色固体, 收率54%. m.p. 78~79 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.25 (d, J=7.9 Hz, 2H), 7.12 (d, J=7.8 Hz, 2H), 5.96 (t, J=6.0 Hz, 1H), 5.88 (s, 2H), 4.17 (d, J=6.0 Hz, 2H), 3.64 (s, 6H), 3.50 (s, 3H), 2.27 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 153.3, 145.4, 137.3, 135.6, 128.8, 128.6, 127.3, 90.2, 60.1, 55.4, 46.7, 20.6; HRMS calcd for C₁₇H₂₂NO₃ [M+H]⁺ 288.1600, found 288.1602.

  N-(4-氟苄基)-3, 4, 5-三甲氧基苯胺(2d):白色固体, 收率46%. m.p. 115~116 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.40 (dd, J=8.6, 5.7 Hz, 2H), 7.14 (t, J=8.9 Hz, 2H), 6.08 (s, 1H), 5.88 (s, 2H), 4.22 (s, 2H), 3.64 (s, 6H), 3.50 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 162.3, 159.9, 158.7, 153.3, 147.0, 144.6, 98.7, 90.6, 60.1, 55.9, 55.4; HRMS calcd for C₁₆H₁₉FNO₃ [M+H]⁺ 292.1349, found 292.1354.

  N-(4-溴苄基)-3, 4, 5-三甲氧基苯胺(2e):灰色固体, 收率55%. m.p. 102~103 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.51 (d, J=8.4 Hz, 2H), 7.33 (d, J=8.4 Hz, 2H), 6.09 (t, J=6.1 Hz, 1H), 5.86 (s, 2H), 4.21 (d, J=6.1 Hz, 2H), 3.64 (s, 6H), 3.50 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 153.3, 145.1, 140.0, 131.1, 129.5, 128.7, 119.5, 90.2, 60.1, 55.5, 46.1; HRMS calcd for C₁₆H₁₈Br-NO₃Na [M+Na]⁺ 374.0368, found 374.0365.

  N-(4-氯苄基)-3, 4, 5-三甲氧基苯胺(2f):灰色固体, 收率53%. m.p. 94~95 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.48~7.27 (m, 4H), 6.08 (t, J=6.1 Hz, 1H), 5.86 (s, 2H), 4.23 (d, J=6.0 Hz, 2H), 3.64 (s, 6H), 3.50 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 153.3, 131.1, 130.1, 129.2, 128.9, 128.1, 98.8, 90.5, 60.1, 55.9, 55.5, 46.3; HRMS calcd for C₁₆H₁₈ClNO₃Na [M+Na]⁺ 330.0873, found 330.0876.

  N-(3-甲氧基苄基)-3, 4, 5-三甲氧基苯胺(2g):白色固体, 收率49%. m.p. 90~91 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.23 (t, J=8.0 Hz, 1H), 6.95 (d, J=6.5 Hz, 2H), 6.79 (dd, J=8.1, 1.8 Hz, 1H), 5.91 (s, 2H), 4.20 (s, 2H), 3.73 (s, 3H), 3.65 (s, 6H), 3.51 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 159.3, 153.3, 144.9, 141.8, 129.3, 119.7, 113.1, 112.0, 98.7, 90.6, 60.1, 55.4, 54.9, 47.1; HRMS calcd for C₁₇H₂₂NO₄ [M+H]⁺ 304.1549, found 304.1555.

  N-(3-甲基苄基)-3, 4, 5-三甲氧基苯胺(2h):黄色油状液体, 收率52%. ¹H NMR (400 MHz, DMSO-d₆) δ: 7.20 (dd, J=9.7, 4.8 Hz, 2H), 7.16 (d, J=7.4 Hz, 1H), 7.04 (d, J=7.1 Hz, 1H), 5.97 (t, J=5.9 Hz, 1H), 5.90 (d, J=8.2 Hz, 2H), 4.18 (d, J=5.8 Hz, 2H), 3.64 (s, 6H), 3.50 (s, 3H), 2.29 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 153.3, 145.3, 140.2, 137.2, 128.1, 128.0, 127.3, 124.5, 90.2, 60.1, 55.9, 55.4, 47.0, 21.0; HRMS calcd for C₁₇H₂₂NO₃ [M+H]⁺ 288.1600, found 288.1603.

  N-(3-氟苄基)-3, 4, 5-三甲氧基苯胺(2i):黄色固体, 收率60%. m.p. 75~76 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.36 (td, J=7.9, 6.3 Hz, 1H), 7.27~7.11 (m, 2H), 7.04 (td, J=8.4, 2.3 Hz, 1H), 6.10 (t, J=6.2 Hz, 1H), 5.88 (s, 2H), 4.26 (d, J=6.1 Hz, 2H), 3.64 (s, 6H), 3.50 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 159.7, 153.3, 153.2, 145.0, 141.4, 137.0, 135.7, 129.7, 129.6, 129.4, 128.8, 128.6, 127.4, 98.6, 90.4, 60.1, 55.4, 46.9; HRMS calcd for C₁₆H₁₈FNO₃Na [M+Na]⁺ 292.1349, found 292.1353.

  N-(3-氯苄基)-3, 4, 5-三甲氧基苯胺(2j):黄色油状液体, 收率43%. ¹H NMR (400 MHz, DMSO-d₆) δ: 7.43 (s, 1H), 7.35 (dd, J=3.4, 2.0 Hz, 2H), 7.31~7.26 (m, 1H), 6.12 (s, 1H), 5.88 (s, 2H), 4.26 (s, 2H), 3.65 (s, 6H), 3.51 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 153.3, 133.0, 130.1, 127.1, 126.6, 126.1, 98.9, 90.4, 60.1, 55.9, 55.4, 46.3; HRMS calcd for C₁₆H₁₉ClNO₃ [M+H]⁺ 330.0873, found 330.0874.

  N-((1H-苯并[d]咪唑-2-基)甲基)-3, 4, 5-三甲氧基苯胺(2k):黄色固体, 收率51%. m.p. 50~52 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.29 (s, 1H), 7.50 (dd, J=5.9, 3.2 Hz, 2H), 7.13 (dd, J=6.0, 3.2 Hz, 2H), 6.10 (s, 1H), 5.99 (s, 2H), 4.46 (s, 2H), 3.65 (s, 6H), 3.50 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 153.6, 153.3, 145.0, 129.0, 121.4, 90.4, 60.1, 55.5, 42.1; HRMS calcd for C₁₇H₂₀N₃O₃ [M+H]⁺ 314.1505, found 314.1508.

  N-(苯并[d]噻唑-2-基甲基)-3, 4, 5-三甲氧基苯胺(2l):黄色油状液体, 收率53%. ¹H NMR (400 MHz, DMSO-d₆) δ: 8.03 (d, J=7.9 Hz, 1H), 7.95 (d, J=8.1 Hz, 1H), 7.49 (t, J=7.6 Hz, 1H), 7.40 (t, J=7.2 Hz, 1H), 6.52 (s, 1H), 5.98 (s, 2H), 4.70 (d, J=5.2 Hz, 2H), 3.65 (s, 6H), 3.52 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 174.6, 153.4, 153.0, 144.5, 134.6, 129.2, 126.0, 124.8, 122.3, 122.2, 90.4, 60.1, 55.5, 45.9; HRMS calcd for C₁₇H₁₉N₂-O₃S [M+H]⁺ 331.1116, found 331.1124.

  N-(吡啶-2-基甲基)-3, 4, 5-三甲氧基苯胺(2m):黄色油状液体, 收率57%. ¹H NMR (400 MHz, DMSO-d₆) δ: 8.59~8.44 (m, 1H), 7.74 (td, J=7.7, 1.8 Hz, 1H), 7.38 (d, J=7.8 Hz, 1H), 7.25 (dd, J=6.9, 5.4 Hz, 1H), 6.13 (t, J=6.0 Hz, 1H), 5.90 (s, 2H), 4.33 (d, J=6.0 Hz, 2H), 3.64 (s, 6H), 3.50 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 159.8, 153.4, 148.7, 145.1, 136.6, 128.7, 122.0, 121.3, 90.2, 60.1, 55.4, 48.9; HRMS calcd for C₁₅H₁₉N₂O₃ [M+H]⁺ 275.1396, found 275.1398.

  N-((5-氯苯并[b]噻吩-3-基)甲基)-3, 4, 5-三甲氧基苯胺(2n):白色固体, 收率44%. m.p. 140~141 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.06 (d, J=1.9 Hz, 1H), 8.02 (d, J=8.6 Hz, 1H), 7.77 (s, 1H), 7.41 (dd, J=8.6, 2.0 Hz, 1H), 6.05 (t, J=5.8 Hz, 1H), 5.99 (s, 2H), 4.47 (d, J=5.8 Hz, 2H), 3.68 (s, 6H), 3.51 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 153.4, 145.2, 139.5, 138.5, 134.3, 129.3, 128.7, 126.3, 124.5, 124.4, 121.8, 90.2, 60.1, 55.5, 41.3; HRMS calcd for C₁₈H₁₉ClNO₃S [M+H]⁺ 364.0774, found 364.0779.

  3.2.2   中间体3的合成

  将0.50 g (2.09 mmol)的化合物2a~2n加入100 mL的圆底烧瓶中, 用N, N-二甲基甲酰胺溶解, 缓慢滴加氯乙酰氯(2.51 mmol), 室温搅拌, TLC检测反应完全后, 加入水和乙酸乙酯萃取三次, 合并有机相, 无水硫酸镁干燥, 旋干得化合物3a~3n.

  2-氯-N-苄基-N-(3, 4, 5-三甲氧基苯基)乙酰胺(3a):白色固体, 收率73%. m.p. 137~138 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.22 (s, 1H), 7.21 (d, J=1.3 Hz, 2H), 7.18~7.13 (m, 2H), 6.09 (s, 2H), 4.79 (s, 2H), 3.83 (s, 2H), 3.77 (s, 3H), 3.62 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.2, 152.6, 137.1, 135.9, 135.2, 128.3, 127.5, 126.8, 104.5, 60.0, 55.1, 52.5, 40.9; HRMS calcd for C₁₈H₂₁ClNO₄ [M+H]⁺ 350.1159, found 350.1162.

  2-氯-N-(4-甲氧基苄基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(3b):白色固体, 收率75%. m.p. 136~137 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.08 (d, J=8.7 Hz, 2H), 6.74 (d, J=8.7 Hz, 2H), 6.10 (s, 2H), 4.72 (s, 2H), 3.81 (s, 2H), 3.78 (s, 3H), 3.71 (s, 3H), 3.64 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.1, 158.3, 152.6, 137.2, 135.2, 129.7, 128.1, 112.8, 104.6, 60.0, 55.2, 54.3, 52.0, 40.9; HRMS calcd for C₁₉H₂₃ClNO₅ [M+H]⁺ 380.1265, found 380.1270.

  2-氯-N-(4-甲基苄基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(3c):白色固体, 收率68%. m.p. 143~144 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.05 (d, J=8.3 Hz, 2H), 7.02 (d, J=8.3 Hz, 2H), 6.10 (s, 2H), 4.74 (s, 2H), 3.82 (s, 2H), 3.78 (s, 3H), 3.64 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.1, 152.6, 137.2, 136.5, 135.3, 132.9, 128.2, 128.1, 104.6, 59.9, 55.2, 52.3, 40.9, 20.1; HRMS calcd for C₁₉H₂₃ClNO₄ [M+H]⁺ 364.1316, found 364.1318.

  2-氯-N-(4-氟苄基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(3d):白色固体, 收率78%. m.p. 119~121 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.22 (dd, J=8.6, 5.4 Hz, 2H), 6.98 (t, J=8.7 Hz, 2H), 6.17 (s, 2H), 4.82 (s, 2H), 3.89 (s, 2H), 3.85 (s, 3H), 3.72 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.2, 162.6, 160.2, 152.7, 137.2, 135.0, 131.8, 131.7, 130.1, 130.0, 114.4, 114.2, 104.4, 60.0, 55.2, 51.8, 40.8; HRMS calcd for C₁₈H₂₀ClFNO₄ [M+H]⁺ 368.1065, found 368.1072.

  2-氯-N-(4-溴苄基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(3e):白色固体, 收率73%. m.p. 169~172 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.51 (d, J=8.3 Hz, 2H), 7.20 (d, J=8.3 Hz, 2H), 6.56 (s, 2H), 4.83 (s, 2H), 4.19 (s, 2H), 3.67 (d, J=17.6 Hz, 9H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 165.7, 153.0, 137.1, 136.9, 136.5, 136.0, 131.2, 130.4, 120.4, 105.8, 60.0, 56.0, 52.1; HRMS calcd for C₁₈H₂₀Br- ClNO₄ [M+H]⁺ 428.0264, found 428.0269.

  2-氯-N-(4-氯苄基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(3f):白色固体, 收率79%. m.p. 166~167 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.27 (d, J=2.8 Hz, 2H), 7.19 (d, J=8.4 Hz, 2H), 6.18 (s, 2H), 4.82 (s, 2H), 3.90 (s, 2H), 3.86 (s, 3H), 3.73 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 166.4, 153.8, 138.3, 136.1, 135.4, 133.8, 130.7, 128.7, 105.4, 61.0, 56.2, 53.0, 41.8; HRMS calcd for C₁₈H₂₀- Cl₂NO₄ [M+H]⁺ 384.0769, found 384.0773.

  2-氯-N-(3-甲氧基苄基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(3g):白色固体, 收率75%. m.p. 143~144 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.13 (t, J=8.1 Hz, 1H), 6.77~6.70 (m, 3H), 6.13 (s, 2H), 4.76 (s, 2H), 3.84 (s, 2H), 3.78 (s, 3H), 3.69 (s, 3H), 3.64 (s, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 161.0, 154.5, 148.4, 133.1, 133.0, 131.0, 124.2, 116.3, 109.3, 108.2, 100.3, 55.7, 50.9, 50.0, 48.3, 36.6; HRMS calcd for C₁₉H₂₂ClNO₅Na [M+Na]⁺ 402.1084, found 402.1085.

  2-氯-N-(3-甲基苄基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(3h):白色固体, 收率72%. m.p. 93~95 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.10 (t, J=7.5 Hz, 1H), 7.04~6.97 (m, 2H), 6.93 (d, J=7.5 Hz, 1H), 6.11 (s, 2H), 4.75 (s, 2H), 3.84 (s, 2H), 3.78 (s, 3H), 3.63 (s, 6H), 2.23 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.4, 152.6, 137.1, 135.7, 135.2, 129.0, 127.5, 127.3, 125.3, 104.5, 59.9, 55.1, 52.6, 40.9, 39.8, 20.3; HRMS calcd for C₁₉H₂₃ClNO₄ [M+H]⁺ 364.1316, found 364.1321.

  2-氯-N-(3-氟苄基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(3i):白色固体, 收率77%. m.p. 110~112 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.22~7.17 (m, 1H), 6.96 (s, 1H), 6.92 (dd, J=9.1, 1.1 Hz, 2H), 6.13 (s, 2H), 4.77(s, 2H), 3.84 (s, 2H), 3.78 (s, 3H), 3.66 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.4, 163.0, 160.5, 152.7, 138.4, 138.3, 137.3, 135.1, 129.03, 129.0, 123.8, 123.8, 115.1, 114.9, 113.8, 113.6, 104.3, 60.0, 55.2, 52.1, 40.8; HRMS calcd for C₁₈H₂₀ClFNO₄ [M+H]⁺ 368.1065, found 368.1066.

  2-氯-N-(3-氯苄基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(3j):白色固体, 收率70%. m.p. 85~86 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.19 (d, J=1.6 Hz, 1H), 7.17 (s, 1H), 7.08 (d, J=6.8 Hz, 1H), 6.13 (s, 2H), 4.75 (s, 2H), 3.84 (s, 2H), 3.79 (s, 3H), 3.67 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.5, 152.8, 137.8, 137.3, 135.0, 133.2, 128.8, 128.3, 127.0, 126.4, 104.4, 60.0, 55.2, 52.1, 40.7; HRMS calcd for C₁₈H₁₉Cl₂NO₄Na [M+Na]⁺ 406.0589, found 406.0593.

  2-氯-N-((1H-苯并[d]咪唑-2-基)甲基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(3k):黄色固体, 收率76%. m.p. 134~135 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.57 (dd, J=6.0, 3.2 Hz, 2H), 7.23 (dd, J=6.0, 3.1 Hz, 2H), 6.90 (s, 2H), 5.12 (s, 2H), 4.26 (s, 2H), 3.70 (s, 6H), 3.64 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 168.6, 165.8, 162.3, 153.0, 150.3, 137.2, 136.1, 121.9, 105.9, 59.9, 55.9, 47.4, 43.0, 41.5, 35.8, 21.0; HRMS calcd for C₁₉H₂₀ClN₃O₄Na [M+Na]⁺ 412.1040, found 412.1053.

  2-氯-N-(苯并[d]噻唑-2-基甲基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(3l):黄色固体, 收率70%. m.p. 147~148 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.90 (d, J=8.1 Hz, 1H), 7.82 (d, J=7.9 Hz, 1H), 7.41 (dd, J=11.2, 4.1 Hz, 1H), 7.34 (dd, J=11.1, 4.1 Hz, 1H), 6.43 (s, 2H), 5.22 (s, 2H), 3.92 (s, 2H), 3.77 (s, 3H), 3.68 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.8, 165.6, 152.9, 151.3, 137.5, 135.1, 134.6, 125.3, 124.5, 121.9, 120.8, 104.1, 59.9, 55.2, 50.9, 40.3; HRMS calcd for C₁₉H₂₀ClN₃O₄ [M+H]⁺ 407.0832, found 407.0837.

  2-氯-N-(吡啶-2-基甲基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(3m):黄色固体, 收率71%. m.p. 121~122 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.46 (d, J=4.3 Hz, 1H), 7.63 (td, J=7.7, 1.6 Hz, 1H), 7.36 (d, J=7.8 Hz, 1H), 7.15 (dd, J=6.8, 5.2 Hz, 1H), 6.37 (s, 2H), 4.96 (s, 2H), 3.90 (s, 2H), 3.77 (s, 3H), 3.69 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.8, 155.0, 152.8, 147.1, 137.2, 136.9, 135.7, 122.5, 121.9, 104.3, 59.9, 55.2, 53.9, 40.7; HRMS calcd for C₁₇H₂₀ClN₂O₄ [M+H]⁺ 351.1112, found 351.1108.

  2-氯-N-((5-氯苯并[b]噻吩-3-基)甲基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(3n):白色固体, 收率67%. m.p. 154~156 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.67 (d, J=8.6 Hz, 1H), 7.59 (d, J=1.8 Hz, 1H), 7.25 (d, J=1.9 Hz, 1H), 7.23 (d, J=3.6 Hz, 1H), 6.04 (s, 2H), 5.00 (s, 2H), 3.82 (s, 2H), 3.77 (s, 3H), 3.56 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.2, 152.7, 138.3, 137.5, 137.2, 134.7, 130.0, 129.8, 128.1, 124.0, 122.7, 121.1, 104.6, 60.0, 55.2, 45.4, 40.8; HRMS calcd for C₂₀H₂₀Cl₂NO₄S [M+H]⁺ 440.0490, found 440.0496.

  3.2.3   目标化合物4的合成

  在50 mL圆底烧瓶中加入0.20 g (0.48 mmol)的化合物3, 不同取代香豆素(0.48 mmol), 碳酸钾(0.96 mmol)和乙腈加热回流5 h.反应结束后, 冷却至室温, 抽滤得滤液, 柱层析分离(洗脱剂:石油醚-乙酸乙酯, V:V=4:1), 得到目标产物4a~4s.

  N-苄基-2-((4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4a):白色固体, 收率59%. m.p. 138~140℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.42 (d, J=8.8 Hz, 1H), 7.22 (s, 1H), 7.21 (t, J=3.2 Hz, 2H), 7.16 (dd, J=6.6, 2.7 Hz, 2H), 6.83 (dd, J=8.8, 2.4 Hz, 1H), 6.59 (d, J=2.4 Hz, 1H), 6.15~6.08 (m, 2H), 6.07 (d, J=0.7 Hz, 1H), 4.80 (s, 2H), 4.46 (s, 2H), 3.78 (d, J=7.2 Hz, 3H), 3.64 (d, J=10.8 Hz, 6H), 2.32 (d, J=0.6 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.4, 160.1, 160.0, 154.0, 152.9, 151.5, 137.4, 135.9, 134.3, 128.4, 127.5, 126.9, 124.6, 113.1, 112.2, 111.2, 104.4, 100.2, 65.1, 59.9, 55.2, 52.3, 17.7; HRMS calcd for C₂₈H₂₈NO₇ [M+H]⁺ 490.1860, found 490.1873.

  N-(4-甲氧基苄基)-2-((4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4b):白色固体, 收率77%. m.p. 188~190 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.42 (d, J=8.8 Hz, 1H), 7.08 (d, J=8.6 Hz, 2H), 6.82 (dd, J=8.8, 2.5 Hz, 1H), 6.75 (d, J=8.6 Hz, 2H), 6.58 (d, J=2.4 Hz, 1H), 6.12 (s, 2H), 6.06 (d, J=0.9 Hz, 1H), 4.73 (s, 2H), 4.44 (s, 2H), 3.80 (s, 3H), 3.72 (s, 3H), 3.67 (s, 6H), 2.32 (d, J=0.9 Hz, 3H); ¹³C NMR (100MHz, CDCl₃) δ: 165.3, 160.1, 160.1, 158.3, 154.0, 152.8, 151.5, 137.3, 134.3, 129.7, 128.0, 124.6, 113.0, 112.8, 112. 2, 111.2, 104.4, 100.2, 65.1, 60.0, 55.3, 54.3, 51.7, 17.7; HRMS calcd for C₂₉H₃₀N₂O₇ [M+H]⁺ 520.1966, found 520.1963.

  N-(4-甲基苄基)-2-((4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4c):白色固体, 收率63%. m.p. 166~167℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.42 (d, J=8.8 Hz, 1H), 7.04 (t, J=5.6 Hz, 4H), 6.82 (dd, J=8.8, 2.5 Hz, 1H), 6.58 (d, J=2.4 Hz, 1H), 6.12 (s, 2H), 6.06 (d, J=1.0 Hz, 1H), 4.75 (s, 2H), 4.45 (s, 2H), 3.79 (s, 3H), 3.66 (s, 6H), 2.32 (d, J=0.9 Hz, 3H), 2.25 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 167.1, 132.9, 130.2, 129.5, 128.3, 96.39, 95.3, 94.8, 93.2, 92.3, 91.9, 91.9, 77.4, 77.1, 76.8, 55.2, 52.1, 44.8, 29.5, 27.1; HRMS calcd for C₂₉H₂₉NO₇Na [M+Na]⁺ 526.1836, found 526.1841.

  N-(4-氟苄基)-2-((4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4d):白色固体, 收率86%. m.p. 131~132 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.42 (d, J=8.8 Hz, 1H), 7.15 (dd, J=8.5, 5.4 Hz, 2H), 6.91 (t, J=8.6 Hz, 2H), 6.82 (dd, J=8.8, 2.4 Hz, 1H), 6.58 (d, J=2.4 Hz, 1H), 6.12 (s, 2H), 6.07 (d, J=0.9 Hz, 1H), 4.76 (s, 2H), 4.45 (s, 2H), 3.80 (s, 3H), 3.68 (s, 6H), 2.32 (d, J=0.9 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.5, 160.1, 159.9, 154.0, 152.9, 151.5, 137.7, 134.2, 131.8, 131.7, 130.2, 130.1, 124.7, 114.5, 114.3, 113.1, 112.1, 111.2, 104.3, 100.2, 65.0, 60.0, 55.3, 51.6, 17.6; HRMS calcd for C₂₈H₂₆FNO₇Na [M+Na]⁺ 530.1586, found 530.1592.

  N-(4-溴苄基)-2-((4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4e):白色固体, 收率65%. m.p. 181~182 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.49 (d, J=8.8 Hz, 1H), 7.43 (d, J=8.3 Hz, 2H), 7.13 (d, J=8.3 Hz, 2H), 6.89 (dd, J=8.8, 2.5 Hz, 1H), 6.65 (d, J=2.5 Hz, 1H), 6.20 (s, 2H), 6.14 (d, J=1.1 Hz, 1H), 4.81 (s, 2H), 4.53 (s, 2H), 3.87 (s, 3H), 3.76 (s, 6H), 2.39 (d, J=1.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.6, 160.1, 159.9, 153.9, 152.9, 151.5, 137.5, 134.8, 134.2, 130.6, 130.1, 124.7, 120.9, 113.1, 112.1, 111.2, 104.3, 100.2, 64.9, 60.0, 55.3, 51.8, 17.7; HRMS calcd for C₂₈H₂₆BrN-O₇Na [M+Na]⁺ 590.0785, found 590.0790.

  N-(4-氯苄基)-2-((4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4f):白色固体, 收率74%. m.p. 155~156 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.42 (d, J=8.8 Hz, 1H), 7.21 (d, J=4.2 Hz, 1H), 7.19 (s, 1H), 7.11 (d, J=8.4 Hz, 2H), 6.81 (dd, J=8.8, 2.3 Hz, 1H), 6.58 (d, J=2.3 Hz, 1H), 6.13 (s, 2H), 6.07 (d, J=0.8 Hz, 1H), 4.76 (s, 2H), 4.46 (s, 2H), 3.80 (s, 3H), 3.69 (s, 6H), 2.32 (d, J=0.7 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.6, 160.1, 159.9, 154.0, 153.0, 151.5, 137.5, 134.4, 134.2, 132.8, 129.8, 127.7, 124.7, 113.1, 112.1, 111.3, 104.3, 100.2, 65.0, 60.0, 55.3, 51.7, 17.7; HRMS calcd for C₂₈H₂₇ClNO₇ [M+H]⁺ 524.1471, found 524.1477.

  N-(3-甲氧基苄基)-2-((4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4g):白色固体, 收率82%. m.p. 131~132 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.42 (d, J=8.8 Hz, 1H), 7.13 (t, J=8.0 Hz, 1H), 6.82 (dd, J=8.8, 2.5 Hz, 1H), 6.74 (dd, J=12.8, 4.0 Hz, 3H), 6.59 (d, J=2.4 Hz, 1H), 6.15 (s, 2H), 6.06 (s, 1H), 4.76 (s, 2H), 4.47 (s, 2H), 3.79 (s, 3H), 3.67 (d, J=1.3 Hz, 9H), 2.32 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.4, 160.1, 160.0, 158.7, 154.0, 152.9, 151.5, 137.4, 134.4, 128.5, 124.6, 120.6, 113.8, 113.1, 112.4, 112.1, 111.2, 104.4, 100.2, 65.1, 60.0, 55.3, 54.3, 52.3, 17.6; HRMS calcd for C₂₉H₃₀NO₈ [M+H]⁺ 520.1966, found 520.1985.

  N-(3-甲基苄基)-2-((4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4h):白色固体, 收率82%. m.p. 149~150 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.42 (d, J=8.8 Hz, 1H), 7.10 (t, J=7.5 Hz, 1H), 7.04~6.95 (m, 2H), 6.93 (d, J=7.4 Hz, 1H), 6.83 (dd, J=8.8, 2.5 Hz, 1H), 6.59 (d, J=2.4 Hz, 1H), 6.13 (s, 2H), 6.06 (d, J=1.0 Hz, 1H), 4.75 (s, 2H), 4.46 (s, 2H), 3.79 (s, 3H), 3.66 (s, 6H), 2.31 (d, J=0.9 Hz, 3H), 2.22 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.4, 160.1, 154.0, 152.8, 151.5, 137.4, 137.2, 135.8, 134.4, 129.0, 127.5, 127.4, 125.4, 124.63, 113.1, 112.1, 111.2, 104.4, 100.2, 65.1, 60.0, 55.2, 52.3, 20.3, 17.6; HRMS calcd for C₂₉H₂₉NO₇Na [M+Na]⁺ 526.1842, found 526.1843.

  N-(3-氟苄基)-2-((4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4i):白色固体, 收率62%. m.p. 143~144 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.42 (d, J=8.8 Hz, 1H), 7.19 (t, J=11.2 Hz, 1H), 6.95 (d, J=7.9 Hz, 1H), 6.93~ 6.88 (m, 2H), 6.82 (dd, J=8.8, 2.4 Hz, 1H), 6.58 (d, J=2.3 Hz, 1H), 6.15 (s, 2H), 6.06 (s, 1H), 4.78 (s, 2H), 4.47 (s, 2H), 3.80 (s, 3H), 3.68 (s, 6H), 2.32 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.6, 163.0, 160.5, 160.0, 159.9, 154.0, 153.0, 151.5, 138.4, 138.3, 137.5, 134.3, 129.1, 129.0, 124.7, 123.9, 123.9, 115.3, 115.0, 113.9, 113.7, 113.2, 112.1, 111.3, 104.3, 100.3, 65.0, 60.0, 55.3, 51.9, 17.6; HRMS calcd for C₂₈H₂₆FNO₇Na [M+Na]⁺ 530.1591, found 530.1592.

  N-(3-氯苄基)-2-((4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4j):白色固体, 收率64%. m.p. 145~146 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.42 (d, J=8.8 Hz, 1H), 7.22~7.12 (m, 3H), 7.08 (d, J=7.2 Hz, 1H), 6.81 (dd, J=8.8, 2.5 Hz, 1H), 6.58 (d, J=2.4 Hz, 1H), 6.15 (s, 2H), 6.06 (d, J=1.0 Hz, 1H), 4.76 (s, 2H), 4.47 (s, 2H), 3.80 (s, 3H), 3.69 (s, 6H), 2.31 (d, J=1.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.6, 160.0, 159.9, 154.0, 153.0, 151.5, 137.9, 137.6, 134.2, 133.3, 128.9, 128.3, 127.1, 126.5, 124.7, 113.1, 112.0, 111.3, 104.3, 100.3, 65.0, 60.0, 55.3, 51.8, 17.6; HRMS calcd for C₂₈H₂₆ClNO₇Na [M+Na]⁺ 546.1290, found 546.1295.

  N-((1H-苯并[d]咪唑-2-基)甲基)-2-((4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4k):白色固体, 收率60%. m.p. 146~147℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.36 (d, J=8.8 Hz, 1H), 7.18 (dd, J=5.8, 3.1 Hz, 2H), 6.69 (t, J=18.4 Hz, 1H), 6.61 (s, 1H), 6.34 (s, 2H), 6.06 (s, 1H), 4.98 (s, 2H), 4.54 (s, 2H), 3.79 (s, 3H), 3.70 (s, 6H), 2.29 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ: 167.7, 159.9, 159.7, 154.0, 153.3, 151.3, 148.7, 138.0, 134.5, 124.7, 121.9, 113.3, 111.6, 111.5, 103.8, 100.5, 64.9, 59.9, 55.4, 47.8, 17.6; HRMS calcd for C₂₉H₂₈N₃O₇ [M+H]⁺ 530.1922, found 530.1923.

  N-((苯并[d]噻唑-2-基)甲基)-2-((4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4l):白色固体, 收率62%. m.p. 175~176℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.94 (d, J=8.1 Hz, 1H), 7.82 (d, J=7.9 Hz, 1H), 7.40 (d, J=8.8 Hz, 2H), 7.33 (t, J=7.5 Hz, 1H), 6.82 (dd, J=8.8, 2.4 Hz, 1H), 6.66 (d, J=2.4 Hz, 1H), 6.50 (s, 2H), 6.06 (s, 1H), 5.21 (s, 2H), 4.58 (s, 2H), 3.79 (s, 3H), 3.71 (s, 6H), 2.31 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 166.1, 165.5, 160.0, 159.9, 154.0, 153.2, 151.4, 137.8, 134.6, 134.4, 125.3, 124.7, 124.5, 122.1, 120.8, 113.2, 112.1, 111.3, 104.1, 100.4, 65.0, 60.0, 59.4, 55.3, 50.8, 20.0, 17.6, 13.2; HRMS calcd for C₂₉H₂₇N₂O₇S [M+H]⁺ 547.1533, found 547.1538.

  N-(吡啶-2-基甲基)-2-((4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4m):白色固体, 收率55%. m.p. 142~143℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.54 (d, J=4.4 Hz, 1H), 7.60 (td, J=7.7, 1.5 Hz, 1H), 7.40 (d, J=8.8 Hz, 1H), 7.31 (d, J=7.8 Hz, 1H), 7.17~7.08 (m, 1H), 6.82 (dd, J=8.8, 2.4 Hz, 1H), 6.67 (d, J=2.3 Hz, 1H), 6.46 (s, 2H), 6.06 (s, 1H), 4.95 (s, 2H), 4.55 (s, 2H), 3.78 (s, 3H), 3.72 (s, 6H), 2.31 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 166.0, 160.1, 155.3, 154.0, 152.9, 151.5, 148.1, 137.4, 136.0, 135.2, 124.6, 122.1, 121.7, 113.0, 112.2, 111.1, 104.3, 100.4, 65.1, 59.9, 55.3, 54.1, 17.6; HRMS calcd for C₃₀H₂₇ClNO₇S [M+H]⁺ 491.1813, found 491.1817.

  N-((5-氯苯并[b]噻吩-3-基)甲基)-2-((4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4n):白色固体, 收率53%. m.p. 172~174 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.69 (d, J=8.6 Hz, 1H), 7.61 (d, J=1.9 Hz, 1H), 7.43 (d, J=8.8 Hz, 1H), 7.23 (dd, J=8.6, 1.9 Hz, 1H), 6.82 (dd, J=8.8, 2.5 Hz, 1H), 6.53 (d, J=2.5 Hz, 1H), 6.06 (s, 1H), 6.05 (s, 2H), 5.02 (s, 2H), 4.45 (s, 2H), 3.79 (s, 3H), 3.60 (s, 6H), 2.32 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.6, 160.0, 159.9, 154.0, 153.0, 151.4, 138.3, 137.4, 133.8, 130.1, 130.0, 128.3, 124.7, 124.1, 122.8, 121.1, 113.2, 111.9, 111.3, 104.6, 100.3, 65.1, 60.0, 55.3, 45.1, 17.6; HRMS calcd for C₃₀H₂₆ClNO₇SNa [M+Na]⁺ 602.1011, found 602.1016.

  N-((5-氯苯并[b]噻吩-3-基)甲基)-2-((2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4o):白色固体, 收率87%. m.p. 179~180 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.69 (d, J=8.6 Hz, 1H), 7.61 (d, J=1.7 Hz, 1H), 7.56 (d, J=9.5 Hz, 1H), 7.30 (d, J=8.5 Hz, 1H), 7.23 (dd, J=8.6, 1.8 Hz, 1H), 6.80 (d, J=6.7 Hz, 1H), 6.54 (s, 1H), 6.18 (d, J=9.5 Hz, 1H), 6.05 (s, 2H), 5.02 (s, 2H), 4.45 (s, 2H), 3.79 (s, 3H), 3.59 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 160.1, 159.9, 154.6, 153.0, 142.3, 138.2, 137.7, 137.3, 133.7, 130.0, 129.9, 128.3, 127.9, 124.1, 122.8, 121.0, 112.5, 112.3, 112.1, 104.5, 100.2, 65.1, 60.0, 55.3, 45.1; HRMS calcd for C₂₉H₂₄ClNO₇SNa [M+Na]⁺ 588.0854, found 588.0860.

  N-((5-氯苯并[b]噻吩-3-基)甲基)-2-((3-氯-4-甲基-2H-色烯-2-酮-7-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4p):白色固体, 收率36%. m.p. 188~189 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.69 (d, J=8.6 Hz, 1H), 7.60 (d, J=1.8 Hz, 1H), 7.45 (d, J=8.9 Hz, 1H), 7.22 (dd, J=8.6, 1.8 Hz, 1H), 7.18 (s, 1H), 6.86 (dd, J=8.9, 2.5 Hz, 1H), 6.53 (d, J=2.4 Hz, 1H), 6.04 (s, 2H), 5.01 (s, 2H), 4.45 (s, 2H), 3.79 (s, 3H), 3.60 (s, 6H), 2.47 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 165.4, 159.8, 156.1, 153.0, 151.8, 146.8, 138.2, 137.7, 137.4, 133.7, 130.0, 129.9, 128.3, 125.1, 124.1, 122.8, 121.0, 117.1, 112.8, 112.6, 104.5, 100.1, 65.0, 60.0, 55.3, 45.0, 15.2; HRMS calcd for C₃₀H₂₆Cl₂NO₇S [M+H]⁺ 614.0802, found 614.0815.

  N-((5-氯苯并[b]噻吩-3-基)甲基)-2-((2H-色烯-2-酮-4-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4q):白色固体, 收率68%. m.p. 223~224 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.80 (dd, J=7.9, 1.2 Hz, 1H), 7.68 (d, J=8.5 Hz, 1H), 7.59 (d, J=1.8 Hz, 1H), 7.54~7.42 (m, 1H), 7.26~7.24 (m, 1H), 7.23 (s, 2H), 7.20 (d, J=7.2 Hz, 1H), 6.05 (s, 2H), 5.39 (s, 1H), 5.03 (s, 2H), 4.55 (s, 2H), 3.79 (s, 3H), 3.56 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 163.9, 161.5, 153.1, 152.4, 138.3, 137.9, 137.3, 133.5, 131.6, 130.0, 129.8, 128.4, 124.1, 123.0, 122.8, 122.3, 121.0, 115.7, 114.3, 104.5, 90.1, 65.2, 60.0, 59.4, 55.2, 45.2; HRMS calcd for C₂₉H₂₅ClNO₇S [M+H]⁺ 566.1035, found 566.1026.

  N-((5-氯苯并[b]噻吩-3-基)甲基)-2-((4-甲基-2H-色烯-2-酮-6-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4r):白色固体, 收率73%. m.p. 169~170 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.69 (d, J=8.6 Hz, 1H), 7.63 (d, J=1.7 Hz, 1H), 7.24 (dd, J=8.6, 1.8 Hz, 1H), 7.16 (d, J=8.7 Hz, 1H), 6.92 (s, 1H), 6.90 (d, J=2.9 Hz, 1H), 6.20 (s, 1H), 6.01 (s, 2H), 5.02 (s, 2H), 4.44 (s, 2H), 3.78 (s, 3H), 3.56 (s, 6H), 2.19 (d, J=0.7 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 166.1, 159.8, 153.3, 152.9, 150.7, 147.3, 138.3, 137.7, 137.3, 134.0, 130.2, 130.0, 128.2, 124.2, 122.8, 121.1, 119.5, 117.5, 116.9, 114.6, 108.8, 104.6, 65.8, 60.0, 55.2, 45.1, 17.5; HRMS calcd for C₃₀H₂₇ClNO₇S [M+H]⁺ 580.1191, found 580.1097.

  N-((5-氯苯并[b]噻吩-3-基)甲基)-2-((7-甲基-2H-色烯-2-酮-4-基)氧基)-N-(3, 4, 5-三甲氧基苯基)乙酰胺(4s):白色固体, 收率59%. m.p. 219~220℃; ¹H NMR (400 MHz, CDCl₃) δ: 7.68 (dd, J=8.3, 5.2 Hz, 2H), 7.59 (d, J=1.7 Hz, 1H), 7.27~7.24 (m, 1H), 7.23 (s, 1H), 7.08~6.98 (m, 2H), 6.04 (s, 2H), 5.34 (d, J=16.6 Hz, 1H), 5.03 (s, 2H), 4.53 (s, 2H), 3.79 (s, 3H), 3.55 (s, 6H), 2.37 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 164.2, 164.0, 161.8, 153.1, 152.5, 142.9, 138.3, 137.8, 137.3, 133.5, 130.0, 129.8, 128.4, 124.2, 124.1, 122.8, 122.0, 121.0, 115.8, 111.8, 104.4, 89.2, 65.2, 60.0, 59.4, 55.2, 45.2, 20.7; HRMS calcd for C₃₀H₂₇ClNO₇S [M+H]⁺ 580.1191, found 580.1201.

  3.3   抗肿瘤活性测试

  测试所用的肿瘤细胞由郑州大学基础医学院药理系提供.所有化合物均用二甲基亚砜(DMSO)溶解, 配制成初始浓度为20 mmol/L的储备液, 放于4 ℃冰箱中保存, 使用前用培养基稀释成所需浓度, 使DMSO的终含量不超过0.1%.将肿瘤细胞培养于含5%CO₂的RPMI-1640培养基中, 加入适量的10%胎牛血清以及100 U/mL的青霉素和0.1 mg/mL的链霉素, 摇晃均匀, 配成细胞悬液.将细胞悬液按每孔100 μL于96孔板中培养24 h后, 弃去培养基, 然后加入不同浓度的药物, 每孔200 μL.培养48 h后, 取出放在超净台中操作, 每孔加入5 g/L的四甲基偶氮唑盐(MTT)溶液20 μL, 继续置于培养箱中培养4 h后, 取出至实验台, 移除96孔板中培养基, 每孔加入100 μL二甲基亚砜(DMSO)溶解的蓝紫色甲瓒颗粒, 置于摇床摇10 min后, 用酶标仪测定570 nm处的吸光度值A, 对所得数值进行处理, 分别计算出5-氟尿嘧啶和各化合物的平均值、标准差和存活率.通过存活率柱状图观察不同浓度药物作用的梯度情况, 用SPSS软件计算每个化合物的IC₅₀.

  辅助材料(Supporting Information)  化合物2a~2n、3a~3n和4a~4s的¹H NMR和¹³C NMR谱图.这些材料可以免费从本刊网站(http://sioc-journal.cn/)上下载.

  
  
• 1. [1]

     张韶瑜, 孟林, 高文远, 宋乃宁, 贾伟, 段宏泉, 中国中药杂志, 2005, 30, 410. doi: 10.3321/j.issn:1001-5302.2005.06.002Zhang, S.-Y.; Meng, L.; Gao, W.-Y.; Song, N.-Y.; Jia, W.; Duan, H.-Q. China J. Chin. Mater. Med. 2005, 30, 410(in Chinese). doi: 10.3321/j.issn:1001-5302.2005.06.002

  2. [2]

     杨国玉, 王彩霞, 黄立挺, 徐翠莲, 合成化学, 2011, 9, 337. doi: 10.3969/j.issn.1005-1511.2011.03.014Yang, G.-Y.; Wang, C.-X.; Huang, L.-T.; Xu, C.-L. Chin. J. Synth. Chem. 2011, 9, 337(in Chinese). doi: 10.3969/j.issn.1005-1511.2011.03.014

  3. [3]

     Fylaktakidou, K. C.; Hadjipavlou-Litina, D. J.; Litinas, K. E.; Nicolaides, D. N. Curr. Pharm. Des. 2004, 10, 3813. doi: 10.2174/1381612043382710

  4. [4]

     Putri, D. E. K.; Pranowo, H. D.; Haryadi, W. Mater. Sci. Forum 2019, 948, 101. doi: 10.4028/www.scientific.net/MSF.948.101

  5. [5]

     Shi, Y.; Zhou, C.-H. Biomed. Chem. Lett. 2011, 21, 956. doi: 10.1016/j.bmcl.2010.12.059

  6. [6]

     Atmaca, M.; Bilgin, H. M.; Obay, B. D.; Diken, H.; Kelle, M.; Kale, E. J. Physiol. Biochem. 2011, 67, 569. doi: 10.1007/s13105-011-0103-5

  7. [7]

     Zhao, H.; Neamati, N.; Pommier, Y.; Burke, T. R. J. Cheminf. 2010, 29, 1002. doi: 10.1002/chin.199816131

  8. [8]

     Weber, U. S.; Steffen, B.; Siegers, C. P. Res. Commun. Mol. Pathol. Pharmacol. 1998, 2, 193. doi: 10.1002/(SICI)1097-0134(19980201)30:2<193::AID-PROT9>3.0.CO;2-O

  9. [9]

     Abdizadeh, T.; Kalani, M. R.; Abnous, K.; Tayarani, Z.; Khash-yarmanesh, B. Z.; Abdizadeh, R.; Ghodsi, R.; Hadizadeh, F. Eur. J. Med. Chem. 2017, 132, 42. doi: 10.1016/j.ejmech.2017.03.024

  10. [10]

      Yang, E. B.; Zhao, Y. N.; Zhang, K.; Mack, P. Biochem. Biophys. Res. Commun. 1999, 260, 682. doi: 10.1006/bbrc.1999.0958

  11. [11]

      Kostova, I. Curr. Med. Chem. 2005, 5, 29. doi: 10.2174/156801105774574694

  12. [12]

      Prota, A. E.; Bargsten, K.; Zurwerra, D.; Field, J. J.; Diaz, J. F.; Altmann, K. H. Science 2013, 339, 587. doi: 10.1126/science.1230582

  13. [13]

      Romagnoli, R.; Baraldi, P. G.; Brancale, A.; Ricci, A.; Hamel, E.; Bortolozzi, R. J. Med. Chem. 2011, 54, 5144. doi: 10.1021/jm200392p

  14. [14]

      Romeo, S. O.; Andrea, B.; Ernest, H. J. Med. Chem. 2015, 58, 3209. doi: 10.1021/acs.jmedchem.5b00155

  15. [15]

      Anthony, N. G.; Breen, D.; Clarke, J.; Donoghue, G.; Waigh, R. D. J. Med. Chem. 2007, 50, 6116. doi: 10.1021/jm070831g

  16. [16]

      Kumar, N.; Kumar, S.; Abbat, S.; Nikhil, K.; Pruthi, V. Med. Chem. Res. 2016, 25, 1175. doi: 10.1007/s00044-016-1562-6

  17. [17]

      Eckelbarger, J. D.; Parker, M. H.; Yap, M. C.; Buysse, A. M.; Babcock, J.M.; Hunter, R. Pest. Manage. Sci. 2016, 73, 761. doi: 10.1002/ps.4359

  18. [18]

      郑玉国, 郭晴晴, 余忠林, 付如凯, 黄勇, 王永欢, 邓钊, 吴用, 精细化工中间体, 2015, 45, 1. http://www.cnki.com.cn/Article/CJFDTotal-HNHG201503002.htmZheng, Y.-G.; Guo, J.-J.; Yu, Z.-L.; Fu, R.-K.; Huang, Y.; Wang, Y.-H.; Deng, Z.; Wu, Y. Fine Chem. Intermed. 2015, 45, 1(in Chinese). http://www.cnki.com.cn/Article/CJFDTotal-HNHG201503002.htm

  19. [19]

      温借雯, 黎勇, 苏正颖, 万丽, 化学通报, 2019, 82, 350.Wen, J.-W.; Li, Y.; Su, Z.-Y.; Wan, L. Chemistry 2019, 82, 350(in Chinese).

  20. [20]

      张明千, 刘斌凯, 雷强, 龙跃, 有机化学, 2019, 39, 1064. doi: 10.6023/cjoc201809003Zhang, M.-Q.; Liu, B.-K.; Lei, Q.; Long, Y. Chin. J. Org. Chem. 2019, 39, 1064(in Chinese). doi: 10.6023/cjoc201809003

  21. [21]

      Fu, D.-J.; Song J.; Hou Y.-H.; Zhao R.-H.; Li J.-H.; Mao R.-W.; Zhang Y.-B.; Liu H.-M. Eur. J. Med. Chem. 2017, 138, 1076. doi: 10.1016/j.ejmech.2017.07.011

  22. [22]

      Fu, D.-J.; Yang, J.-J.; Li, P.; Hou, Y.-H.; Huang, S.-N, Pham, V.; Song, L. K.; Zi, X. L.; Xue, W.-L. Eur. J. Med. Chem. 2018, 157, 50. doi: 10.1016/j.ejmech.2018.07.060

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  图 1  目标化合物4的分子设计示意图

  Figure 1  Design strategy of the target compounds 4

  下载: 全尺寸图片 幻灯片
  

  图式 1  目标化合物4的合成路线

  Scheme  1  Synthetic routes of the target compounds 4

  下载: 全尺寸图片 幻灯片

  表 1  化合物4a~4s的抗肿瘤活性[IC₅₀/(μmol•L^-1)]

  Table 1.  Anti-cancer activity [IC₅₀/(μmol•L^-1)] of the compounds 4a~4s

  Compd.	PC-3	EC-109	MGC-803
  4a	5.62±1.27	16.24±1.53	18.90±1.28
  4b	14.24±1.55	38.73±0.42	43.88±1.27
  4c	31.51±2.94	10.96±1.24	＞80
  4d	45.58±2.02	43.24±2.18	＞80
  4e	32.91±1.81	44.94±3.06	14.65±1.82
  4f	＞80	＞80	＞80
  4g	76.28±2. 59	46.31±1.25	78.29±3.64
  4h	＞80	36.183±1.72	＞80
  4i	47.93±1.30	27.93±1.30	56.94±1.52
  4j	＞80	72.86±1.37	67.61±1.32
  4k	12.81±1.63	55.68±3.09	＞80
  4l	42.46±2.42	51.33±3.82	33.32±7.06
  4m	39.33±2.02	46.75±0.64	76.19±2.42
  4n	4.18±0.41	14.32±2.48	17.92±2.38
  4o	14.62±1.51	42.93±2.04	＞80
  4p	9.72±1.28	24.19±1.82	20.19±1.84
  4q	42.18±1.39	＞80	47.17±1.24
  4r	32.06±1.59	48.91±2.08	＞80
  4s	＞80	60.63±3.47	＞80
  5-Fu	9.79±0.17	20.42±1.83	21.21±3.61

  下载: 导出CSV
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