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三类基于香豆素母核衍生物的合成及其抗炎活性

赵秀娟, 杨恒俐, 吴金叶, 郑晓琦, 张耀苹, 林玉萍, 虎春艳

赵秀娟,杨恒俐,吴金叶,等. 三类基于香豆素母核衍生物的合成及其抗炎活性[J]. 中国药科大学学报,2025,56(1):40 − 48. DOI: 10.11665/j.issn.1000-5048.2024072101
引用本文: 赵秀娟,杨恒俐,吴金叶,等. 三类基于香豆素母核衍生物的合成及其抗炎活性[J]. 中国药科大学学报,2025,56(1):40 − 48. DOI: 10.11665/j.issn.1000-5048.2024072101
ZHAO Xiujuan, YANG Hengli, WU Jinye, et al. Synthesis and anti-inflammatory activity of three series of coumarin-based derivatives[J]. J China Pharm Univ, 2025, 56(1): 40 − 48. DOI: 10.11665/j.issn.1000-5048.2024072101
Citation: ZHAO Xiujuan, YANG Hengli, WU Jinye, et al. Synthesis and anti-inflammatory activity of three series of coumarin-based derivatives[J]. J China Pharm Univ, 2025, 56(1): 40 − 48. DOI: 10.11665/j.issn.1000-5048.2024072101

三类基于香豆素母核衍生物的合成及其抗炎活性

基金项目: 

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云南省科学技术厅-云南中医药大学应用基础研究联合专项资助项目(202301AZ070001-061)

详细信息
    通讯作者:

    虎春艳: Tel:13078754255 E-mail:hchuny@126.com

  • 中图分类号: R914;R965

Synthesis and anti-inflammatory activity of three series of coumarin-based derivatives

Funds: 

This study was supported by the Applied Basic Research Joint Special Funds of Yunnan Provincial Science and Technology Department and Yunnan University of Chinese Medicine(202301AZ070001-061)

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  • 摘要:

    以4-羟基香豆素为原料出发,合成得到3个系列共22个香豆素衍生物,其中8个衍生物未见文献报道,并采用小鼠巨噬细胞模型对其体外抗炎活性及作用机制进行初步研究。结果表明,大部分衍生物均可显著抑制促炎因子NO的生成,其中化合物2e2f2g2h2i2j4e4f的抗炎活性优于阳性对照药物地塞米松。进一步实验发现,化合物2h4f可显著抑制RAW264. 7巨噬细胞内促炎因子IL-6、TNF-α和IL-1β的生成,可作为先导化合物进行深入研究。

    Abstract:

    In this work, starting from 4-hydroxycoumarin, three series of 22 coumarin derivatives, among which 8 have not been reported in the literature, were synthesized and their in vitro anti-inflammatory activities and mechanisms of action were preliminarily investigated using mouse macrophage model. The results showed that most of the derivatives could significantly inhibit the production of pro-inflammatory factor NO, with compounds 2e, 2f, 2g, 2h, 2i, 2j, 4e, and 4f showing better anti-inflammatory activity than the positive control drug dexamethasone. Further experiments showed that compounds 2h and 4f significantly inhibited the production of pro-inflammatory factors IL-6, TNF-α and IL-1β in RAW264.7 macrophages, and could, therefore, be used as lead compounds for further studies.

  • 炎症是机体对导致组织损伤的多种有害刺激的生物反应[1],被认为是一种抵抗细胞损伤的防御机制,可以消除有害刺激,促进组织修复和愈合。然而,如果其过度发展和不受控制,可能会变成病理性的,例如严重的心肌炎会影响心脏功能,脑部炎症可压迫中枢神经系统,声带炎症可阻塞咽喉而引起窒息[2]。另外,由于近年来城镇化、老龄化进程的不断加快和人们生活方式的改变,炎症性疾病已越来越成为危害全球人类健康的重要因素[3]。目前,常用来治疗炎症的药物包括糖皮质激素抗炎药和非甾体类抗炎药等[4]。经研究发现,长期使用此类药物可能引起胃肠道反应,甚至导致免疫抑制和加重感染等严重的或不可逆的不良反应[56]。因此,研发安全有效的新型抗炎药物是研究热点之一。

    香豆素存在于许多天然产物中,也是药物化学中具有药理活性优势的母核片段,具有抗凝血、抗炎、抗氧化、抗菌及抗癌等作用[7]。文献报道,香豆素衍生物在抗炎药物研发中具有巨大的潜力[810],例如尿石素可抑制细胞因子IL-6、TNF-α和NO的生成[11],含杂环结构的衍生物也表现出良好的抗炎效果[12]。本文在前期研究的基础上[1315],以4-羟基香豆素为原料,分别经加成、缩合、取代和环化等反应,合成得到3个系列共22个香豆素衍生物(路线1),其结构均经1H NMR和13C NMR确证。其中,有8个化合物未见文献报道,另外14个已知化合物均未见抗炎活性相关报道。以地塞米松为阳性药,采用脂多糖诱导巨噬细胞(RAW264.7)炎症模型对香豆素衍生物的体外抗炎活性及作用机制进行初步研究,为炎症性疾病寻找潜在先导化合物。

      1.  Synthesis of coumarin-based derivatives

    AS-400核磁共振谱仪(德国Bruker公司);SGW-X-4B显微熔点仪(上海仪电物理化学仪器有限公司);酶标仪(瑞士Tecan公司);GF254硅胶板、柱色谱硅胶(青岛海洋化工有限公司);试剂和溶剂均为市售分析级;DMEM培养基(武汉普诺赛生命科技有限公司)。小鼠单核巨噬细胞(RAW264.7)获自中国科学院(CAS)昆明细胞库。

    称取4-羟基香豆素162 mg(1 mmol)和芳香醛或杂环醛0.5 mmol置于25 mL圆底烧瓶中,依次加入无水乙醇 4 mL,哌啶0.1 mmol,室温搅拌下反应过夜。TLC检测反应完成后,加入二氯甲烷20 mL,并用水洗涤(3×10 mL),有机层经无水硫酸钠干燥浓缩,粗产物经柱色谱(二氯甲烷-甲醇, 50∶1)分离,分别得到化合物2a~2k,收率为80%~90%(表1)。

    Table  1.  Structure and inhibitory of coumarin-based derivatives (20 μmol/L) on RAW264.7
    Compd. R Yields /% Cell viability /% Compd. R Yields /% Cell viability/%
    2a 88.5 43.7 3a 78.8 49.9
    2b 86.7 42.4 3b 71.2 45.2
    2c 90.5 48.9 3c 49.8 40.0
    2d 85.8 45.8 3d 49.0 39.5
    2e 86.4 84.8 3e 59.8 43.4
    2f 87.9 51.8 4a 61.6 34.0
    2g 91.2 57.9 4b 64.3 34.2
    2h 87.4 97.5 4c 66.1 49.8
    2i 87.9 83.1 4d 76.9 49.8
    2j 85.6 63.0 4e 70.5 65.3
    2k 87.4 36.9 4f 84.1 98.5
    下载: 导出CSV 
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    称取4-羟基香豆素162 mg(1 mmol)、丙二腈66 mg(1 mmol)和芳香醛或杂环醛1 mmol于25 mL圆底烧瓶中,加入无水乙醇2 mL,哌啶17 mg(0.2 mmol)作为催化剂,加热回流反应1 h。TLC检测反应完成后,冷却至室温,抽滤,滤饼用无水乙醇洗涤(3×5 mL),分别得到化合物3a~3e,收率为40%~70%(表1)。

    称取4-羟基香豆素162 mg(1 mmol)、芳香醛0.5 mmol和乙酸铵385 mg(5 mmol)于25 mL圆底烧瓶中,加入水0.5 mL,85 ℃搅拌反应2 h。TLC检测反应完成后,冷却至室温,将反应物倒入冷水40~50 mL中搅拌析出固体,抽滤,滤饼经二氯甲烷洗涤(3×5 mL),分别得到化合物4a~4f,收率为60%~80%(表1)。

    化合物2a 白色固体,产率为88.5%,mp:173~175 ℃。1H NMR (400 MHz, CDCl3) δ: 8.09(2H, d, J = 8.0 Hz, Ar-H), 7.46(2H, t, J = 8.0 Hz, Ar-H), 7.25(2H, t, J = 8.0 Hz, Ar-H), 7.19(2H, d, J = 8.0 Hz, Ar-H), 7.03(1H, d, J = 4.0 Hz, Het-H), 6.84(1H, t, J = 4.0 Hz, Het-H), 6.81(1H, s, Het-H), 6.41(1H, s, CH); 13C NMR (100 MHz, CDCl3) δ: 171.20, 167.40, 152.81, 146.62, 131.37, 131.35, 126.51, 125.31, 125.28, 123.90, 123.87, 123.54, 122.97, 120.34, 115.79, 115.76, 104.19, 46.55, 45.44, 33.80, 22.85, 22.35, 9.07。

    化合物2b 白色固体,产率为86.7%,mp:163~165 ℃。1H NMR (400 MHz, CDCl3) δ: 8.45(1H, s, Het-H), 8.06(2H, d, J = 8.0 Hz, Ar-H), 7.59(1H, t, J = 8.0 Hz, Ar-H), 7.48(1H, s, Het-H), 7.45(2H, d, J = 8.0 Hz, Het-H), 7.25~7.23(4H, m, Ar-H), 7.04(1H, s, Ar-H), 6.43(1H, s, CH); 13C NMR (100 MHz, CDCl3) δ: 170.34, 167.33, 160.99, 153.02, 148.63, 136.42, 130.98, 130.95, 125.16, 125.13, 123.24, 121.88, 120.74, 120.51, 115.75, 115.72, 103.83, 52.57, 46.49, 39.98, 29.76, 22.82, 8.84, 7.68。

    化合物2c 白色固体,产率为90.5%。mp:179~181 ℃; 1H NMR (400 MHz, CDCl3) δ: 8.09(2H, d, J = 8.0 Hz, Ar-H), 7.47(2H, s, Ar-H), 7.26~7.20(6H, m, Ar-H), 6.81(2H, d, J = 8.0 Hz, Ar-H), 6.28(1H, s, CH), 3.84(4H, s, OCH2), 3.12(4H, s, NCH2); 13C NMR (100 MHz, CDCl3) δ: 170.54, 167.56, 152.90, 148.95, 132.35, 130.94, 130.91, 127.54, 127.51, 125.33, 125.30, 124.53, 124.50, 123.24, 123.21, 120.59, 115.73, 115.69, 115.63, 104.06, 67.10, 67.07, 53.52, 49.69, 49.66, 46.51, 46.48, 36.04, 8.79。

    化合物2d 黄色固体,产率为85.8%,mp:150~152 ℃。1H NMR (400 MHz, CDCl3) δ: 8.06(2H, d, J = 8.0 Hz, Ar-H), 7.82(1H, s, Het-H), 7.47(2H, t, J = 8.0 Hz, Ar-H), 7.41(2H, t, J = 8.0 Hz, Ar-H), 7.25~7.22(7H, m, Ar-H), 7.15(1H, s, Het-H), 6.46(1H, s, CH); 13C NMR (100 MHz, CDCl3) δ: 170.33, 167.15, 152.98, 141.16, 135.68, 134.77, 131.15, 129.90, 128.33, 128.30, 125.18, 125.15, 123.33, 123.30, 121.28, 121.25, 120.42, 118.50, 115.71, 103.59, 62.88, 52.73, 46.41, 36.52, 29.74, 22.77, 8.88, 7.73。

    化合物2e 黄色固体,产率为86.4%,mp:173~175 ℃。1H NMR (400 MHz, CDCl3) δ: 8.10(2H, d, J = 8.0 Hz, Ar-H), 7.48(2H, t, J = 8.0 Hz, Ar-H), 7.27~7.20(6H, m, Ar-H), 7.06 (2H, d, J = 4.0 Hz, Ar-H), 6.32 (1H, s, CH), 2.30 (1H, s, CH3); 13C NMR (100 MHz, CDCl3) δ: 170.57, 167.54, 152.95, 137.73, 134.64, 130.96, 130.93, 128.93, 128.90, 126.72, 126.70, 125.35, 125.33, 123.25, 123.23, 120.62, 115.65, 115.62, 103.99, 46.48, 46.15, 46.12, 36.47, 21.06, 8.82。

    化合物2f 白色固体,产率为87.9%,mp:163~165 ℃。1H NMR (400 MHz, DMSO-d6) δ: 7.82(2H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.47(2H, t, J = 8.0 Hz, Ar-H), 7.23~7.20(4H, m, Ar-H), 7.01(1H, d, J = 4.4 Hz, Ar-H), 6.22~6.20(2H, m, Ar-H), 6.10(1H, s, CH), 3.63(3H, s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ: 167.52, 164.62, 158.30, 156.18, 152.82, 130.84, 130.81, 129.91, 124.46, 124.43, 124.42, 123.17,121.83, 121.80, 120.77, 115.76, 115.73, 104.45, 103.18, 101.46, 55.18, 55.16, 46.06, 46.03, 32.69, 9.02。

    化合物2g 橙白色固体,产率为91.2%,mp:120~122 ℃。1H NMR (400 MHz, CDCl3) δ: 8.13(2H, s, Ar-H), 7.54~7.50(2H, m, Ar-H), 7.30~7.28(4H, m, Ar-H), 6.92~6.89(2H, m, Ar-H), 6.80(1H, d, J = 4.0 Hz, Ar-H), 6.37(1H, s, CH), 3.87(3H, s, OCH3), 3.74(3H, s, OCH3); 13C NMR (100 MHz, CDCl3) δ: 170.72, 167.58, 152.95, 148.73, 146.96, 133.50, 131.05, 131.02, 125.33, 125.30, 123.35, 123.32, 120.59, 118.98, 115.71, 115.68, 111.00, 118.80, 110.77, 104.05, 55.97, 55.83, 46.57, 46.55, 46.52, 36.42, 8.88。

    化合物2h 白色固体,产率为87.4%,mp:181~183 ℃。1H NMR (400 MHz, CDCl3) δ: 8.08(2H, d, J = 8.0 Hz, Ar-H), 7.48(2H, t, J = 8.0 Hz, Ar-H), 7.26(4H, d, J = 8.0 Hz, Ar-H), 7.21(4H, t, J = 8.0 Hz, Ar-H), 6.33(1H, s, CH); 13C NMR (100 MHz, CDCl3) δ: 170.56, 167.37, 152.94, 139.63, 131.14, 131.11, 130.99, 128.38, 128.35, 128.30, 128.21, 125.27, 125.24, 123.37, 123.33, 120.44, 115.69, 115.66, 103.57, 46.58, 46.55, 46.52, 36.39, 29.60, 8.81。

    化合物2i 白色固体,产率为87.9%,mp:141~143 ℃。1H NMR (400 MHz, CDCl3) δ: 8.07(2H, d, J = 8.0 Hz, Ar-H), 7.79~7.71(1H, m, Ar-H), 7.48(2H, t, J = 8.0 Hz, Ar-H), 7.34(2H, d, J =8.0 Hz, Ar-H), 7.25(3H, d, J = 8.0 Hz, Ar-H), 7.19(2H, d, J = 8.0 Hz, Ar-H), 6.30(1H, s, CH); 13C NMR (100 MHz, CDCl3) δ: 191.15, 170.62, 167.42, 152.93, 140.16, 132.52, 131.18, 131.15, 131.12, 128.80, 128.77, 125.28, 125.24, 123.39, 123.37, 120.40, 119.89, 119.19, 115.71, 115,69, 103.52, 46.55, 46.52, 36.45, 8.89。

    化合物2j 白色固体,产率为85.6%,mp:205~207 ℃。1H NMR (400 MHz, CDCl3) δ: 8.09(2H, d, J = 7.2 Hz, Ar-H), 7.48(2H, t, J = 8.0 Hz, Ar-H), 7.34(2H, t, J = 8.0 Hz, Ar-H), 7.25~7.22(2H, m, Ar-H), 6.56(2H, s, Ar-H), 6.33(1H, s, CH), 3.83(3H, s, OCH3), 3.68(6H, s, OCH3); 13C NMR (100 MHz, CDCl3) δ: 170.63, 167.44, 152.88, 136.99, 135.85, 131.05, 131.01, 125.26, 125.24, 123.37, 123.34, 120.49, 120.48, 115.68, 115,67, 104.26, 104.20, 103.78, 60.82, 56.15, 56.12, 52.55, 46.50,46.47, 36.90, 29.75, 8.82, 7.61。

    化合物2k 白色固体,产率为87.4%,mp:147~149 ℃。1H NMR (400 MHz, DMSO-d6) δ: 7.83(2H, d, J = 8.0 Hz, Ar-H), 7.49(2H, t, J = 8.0 Hz, Ar-H), 7.25(2H, d, J = 8.0 Hz, Ar-H), 7.22~7.20(1H, m, Ar-H), 7.11~7.06(2H, m, Ar-H), 6.98(1H, s, Ar-H), 6.57(1H, d, J = 8.0 Hz, Ar-H), 6.15(1H, s, CH); 13C NMR (100 MHz, DMSO-d6) δ: 167.73, 164.50, 155.02, 152.88, 132.50, 131.92, 131.22, 131.16, 129.11, 124.55, 124.52, 123.35, 120.57, 117.18, 115.88, 109.81, 103.64, 40.65, 40.44, 40.23, 40.02, 39.81, 39.60, 39.39, 33.53。

    化合物3a 白色固体,产率为78.8%,mp:172~174 ℃。1H NMR (400 MHz, CDCl3) δ: 7.90(1H, d, J = 8.0 Hz, Ar-H), 7.74(1H, t, J = 8.0 Hz, Ar-H), 7.54~7.49(2H, m, Ar-H), 7.41(1H, d, J = 8.0 Hz, Het-H), 7.05(1H, d, J = 3.2 Hz, Het-H), 6.97(1H, t, J = 4.0 Hz, Het-H), 4.84(1H, s, CH)。

    化合物3b 淡黄色固体,产率为71.2%,mp:175~177 ℃。1H NMR (400 MHz, CDCl3) δ: 7.90(1H, d, J = 8.0 Hz, Ar-H), 7.74(1H, t, J = 8.0 Hz, Ar-H), 7.53~7.46(2H, m, Ar-H), 6.15(1H, d, J = 3.2 Hz, Het-H), 5.99(1H, d, J = 2.0 Hz, Het-H), 4.56(1H, s, CH), 2.19(3H, s, CH3)。

    化合物3c 白色固体,产率为49.8%,mp:227~229 ℃; 1H NMR (400 MHz, DMSO-d6) δ: 7.93(1H, d, J = 8.0 Hz, Ar-H), 7.74(1H, t, J = 8.0 Hz, Ar-H), 7.54~7.47(2H, m, Ar-H), 7.40(2H, s, Ar-H), 7.18~7.13(2H, m, Ar-H), 4.43(1H, s, CH), 2.29(3H, s, CH3); 13C NMR (100 MHz, DMSO-d6) δ: 160.07, 158.46, 153.81, 152.65, 140.94, 136.82, 133.45, 129.61, 128.05, 125.22, 123.00, 119.79, 117.11, 113.51, 104.67, 58.63, 39.60, 39.39, 37.10, 21.15。

    化合物3d 白色固体,产率为49.0%,mp:249~251 ℃。1H NMR (400 MHz, DMSO-d6) δ: 8.47(1H, d, J = 2.4 Hz, Het-H), 7.93(1H, d, J = 8.0 Hz, Het-H), 7.77~7.72(2H, m, Ar-H), 7.52~7.46(4H, m, Ar-H), 7.41(2H, d, J = 8.0 Hz, Ar-H), 6.54(1H, t, J = 2.0 Hz, Het-H), 4.54(1H, s, CH); 13C NMR (100 MHz, DMSO-d6) δ: 160.11, 158.48, 154.01, 152.71, 141.81, 141.46, 139.25, 133.51, 129.38, 128.27, 125.23, 123.05, 119.71, 119.15, 117.13, 113.52, 108.30, 104.22, 58.23, 39.81, 39.39, 36.99。

    化合物3e 白色固体,产率为59.8%,mp:248~250 ℃。1H NMR (400 MHz, DMSO-d6) δ: 8.28(1H, s, Ar-H), 7.94(1H, d, J = 4.0 Hz, Ar-H), 7.76~7.73(2H, m, Ar-H), 7.59(2H, d, J = 4.0 Hz, Het-H), 7.54~7.43(4H, m, Ar-H), 7.14(1H, s, Het-H), 4.57(1H, s, CH); 13C NMR (100 MHz, DMSO-d6) δ: 160.12, 158.52, 154.09, 152.72, 142.73, 136.32, 136.09, 133.54, 129.97, 129.68, 125.24, 123.07, 121.31, 119.70, 118.79, 117.13, 113.52, 104.12, 58.13, 39.40, 39.60, 36.99。

    化合物4a 白色固体,产率为61.6%,mp:194~196 ℃。1H NMR (400 MHz, DMSO-d6) δ: 7.82(2H, d, J = 8.0 Hz, Ar-H), 7.47(2H, t, J = 8.0 Hz, Ar-H), 7.23~7.20(4H, m, Ar-H), 6.94(1H, s, Ar-H), 6.69(1H, d, J = 8.0 Hz, Ar-H), 6.47(1H, d, J = 8.0 Hz, Ar-H), 6.13(1H, s, CH), 2.10(3H, s, CH3); 13C NMR (100MHz, DMSO-d6) δ: 167.61, 164.66, 154.39, 153.26, 152.86, 130.88, 130.67, 130.11, 129.43, 129.31, 129.28, 126.73, 126.21, 126.04, 124.50, 124.43, 123.18, 120.80, 115.78, 115.65, 114.91, 114.87, 104.27, 103.78, 33.37, 21.24。

    化合物4b 白色固体,产率为64.3%,mp:212~214 ℃。1H NMR (400 MHz, DMSO-d6) δ: 7.91(2H, d, J = 8.0 Hz, Ar-H), 7.51(2H, t, J = 8.0 Hz, Ar-H), 7.37(1H, d, J = 2.4 Hz, Ar-H),7.26~7.23(4H, m, Ar-H), 7.16(1H, d, J = 2.8 Hz, Ar-H), 7.00~6.97 (1H, m, Ar-H), 6.28(1H, s, CH); 13C NMR (100 MHz, DMSO-d6) δ: 168.01, 164.55, 152.89, 151.22, 136.70, 135.40, 133.20, 131.88, 131.28, 123.33, 129.02, 128.83, 124.60, 124.41, 123.76, 123.47, 123.40, 120.45, 118.08, 116.88, 116.01, 115.96, 111.76, 103.45, 34.30。

    化合物4c 黄色固体,产率为66.1%,mp:244~246 ℃。1H NMR (400 MHz, DMSO-d6) δ: 7.94(1H, d, J = 8.0 Hz, Ar-H), 7.84(2H, d, J = 8.0 Hz, Ar-H), 7.51(2H, t, J = 8.0 Hz, Ar-H), 7.27~7.23(4H, m, Ar-H), 6.80(1H, d, J = 8.0 Hz, Ar-H), 6.22(1H, s, CH)。

    化合物4d 淡黄色固体,产率为76.9%,mp:281~283 ℃。1H NMR (400 MHz, DMSO-d6) δ: 7.83(2H, d, J = 8.0 Hz, Ar-H), 7.50(2H, t, J = 8.0 Hz, Ar-H), 7.26~7.21(4H, m, Ar-H), 6.41(2H, s, Ar-H), 6.21(1H, s, CH), 3.62(3H, s, OCH3), 3.55(6H, s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ: 168.29, 164.99, 152.95, 152.79, 152.74, 148.32, 148.10, 138.84, 135.93, 131.39, 131.36, 124.60, 123.44, 123.42, 123.38, 123.35, 123.26, 120.43, 120.41, 116.04, 115.95, 115.90, 105.15, 105.11, 103.96, 60.39, 56.23, 36.82。

    化合物4e 白色固体,产率为70.5%,mp:300~302 ℃。1H NMR (400 MHz, DMSO-d6) δ: 7.82(2H, d, J = 8.0 Hz, Ar-H), 7.50(2H, t, J = 8.0 Hz, Ar-H), 7.27~7.21(4H, m, Ar-H), 6.27(1H, s, Ar-H), 6.24(2H, s, Ar-H), 6.19(1H, s, CH), 3.60(6H, s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ: 168.25, 165.02, 160.55, 152.96, 152.91, 145.55, 131.47, 131.39, 124.67, 124.60, 123.38, 123,34, 123.19, 120.38, 120.33, 115.94, 115.91, 106.03, 106.01, 104.86, 104.79, 103.75, 96.47, 96.35, 55.30, 36.76。

    化合物4f 白色固体,产率为84.1%,mp:256~258 ℃。1H NMR (400 MHz, DMSO-d6) δ: 7.83(2H, d, J = 8.0 Hz, Ar-H), 7.51(2H, t, J = 8.0 Hz, Ar-H), 7.26(2H, d, J = 8.4 Hz, Ar-H), 7.23~7.20(4H, m, Ar-H), 7.11(2H, d, J = 8.4 Hz, Ar-H), 6.24(1H, s, CH); 13C NMR (100 MHz, DMSO-d6) δ: 168.26, 164.95,153.22, 153.01, 141.97, 131.68, 131.50, 131.46, 129.85, 129.23, 129.15, 129.04, 128.92, 128.56, 128.13, 128.10, 124.62, 123.41, 120.38, 120.32, 116.07, 115.97, 115.77, 103.60, 36.26。

    将巨噬细胞(RAW264.7)在5% CO2,37 ℃条件下用10%胎牛血清和1%青霉素-链霉素的DMEM培养。以每孔5×105个接种于96孔细胞培养板,将配制好的含20 μmol/L化合物的细胞培养基加入96孔板中孵育24 h。最后采用MTT法检测吸收度,计算细胞存活率。

    将巨噬细胞(RAW264.7)以5×105的细胞密度接种于96孔板并置于培养箱中24 h。取配制好的5、10、20 μmol/L目标化合物添加到每个孔里,2 h后加入LPS(1 μg/mL)继续培养24 h,每个浓度梯度重复3次。最后取细胞培养上清液,使用Griess Reagent法检测NO水平,并计算IC50

    将巨噬细胞(RAW264.7)以5×105的细胞密度接种于96孔板并置于培养箱中24 h。取化合物2h4f稀释至5、10、20 μmol/L添加到每个孔里,2 h后加入LPS(1 μg/mL)继续培养24 h。最后取细胞培养液上清液,按ELISA试剂盒说明方法检测IL-6、TNF-α和IL-1β水平。

    本文从香豆素出发,合成得到了3个系列共22个目标化合物(路线1)。首先,4-羟基香豆素和芳香醛或杂环醛采用哌啶催化,无水乙醇作溶剂,经Knoevenagel- Michael缩合反应[16],室温下搅拌得到11个双香豆素类衍生物(2a~2k)。具有反应条件温和、溶剂可回收和产率高等优点。其次,4-羟基香豆素、丙二腈和芳香醛或杂环醛在哌啶催化下,采用三组分“一锅法”[17]制备了5个四氢色烯类衍生物(3a~3e)。该反应一步即可获得目标产物,操作简便,不需要柱色谱纯化,且收率良好。最后, 4-羟基香豆素、芳香醛为原料,水作为溶剂,乙酸铵为催化剂加热反应,采用经典的Hantzsch反应[18],得到6个二氢吡啶类衍生物(4a~4f)。改进后的方案具有反应时间短、后处理简单方便等特点,为香豆素衍生物的合成提供了一条绿色合成途径。

    采用MTT法测定22个目标化合物的细胞毒性。结果显示(表1),双香豆素类衍生物大多无明显的毒性(2a~2k),而四氢色烯类衍生物(3a~3e)和二氢吡啶类衍生物(4e4f除外)对巨噬细胞RAW264.7显示出较大的毒性。在这3个系列化合物中,有8个化合物(2e2f2g2h2i2j4e4f)在20 μmol/L时细胞存活率均大于50%,具有较好的安全性,对这些化合物进行NO含量测定来进一步评估其体外抗炎活性。

    一氧化氮(NO)过量产生与炎症密切相关,抑制NO产生是治疗多种炎症性疾病的重要途径[19]。另外,地塞米松(Dex)为糖皮质激素抗炎药,可通过抑制免疫系统发挥抗炎作用,临床常用于抗感染、抗病毒等[2021],在抗炎活性评估中,常选择地塞米松为阳性对照药。本文中,利用LPS诱导RAW264.7细胞构建炎症模型,并检测8个化合物NO的生成(图1)。结果显示,当给药浓度为20 μmol/L时,化合物对NO的抑制作用均优于阳性药地塞米松。从这些衍生物的结构类型上看,双香豆素类衍生物和二氢吡啶类衍生物对NO的抑制活性均较强(IC50<13 μmol/L),并没有明显的差异。但从衍生物的结构上来看(表2),取代基对化合物的抗炎活性有一定的影响。当苯环上存在吸电子基团(EWD),特别是卤素原子(X),如Cl,Br时,双香豆素类衍生物和二氢吡啶类衍生物对NO的抑制活性要明显优于其他取代基,且氯原子的活性要高于溴原子,如化合物2h( IC50 = 6.76 μmol/L)、2i(IC50 = 7.92 μmol/L)和4f(IC50 = 5.49 μmol/L)。当苯环上存在给电子基团(EDG),如CH3,OCH3时,双香豆素类衍生物和二氢吡啶类衍生物的抗炎活性稍微减弱,如化合物2e(IC50 = 11.32 μmol/L)、2f(IC50 = 8.22 μmol/L)、2g(IC50 = 9.42 μmol/L)、2j(IC50 = 9.09 μmol/L)和4e(IC50 = 12.16 μmol/L)。此外,随着苯环上供电子基团(如OCH3)的增多,衍生物对NO的抑制作用也在逐渐减弱,且邻位取代活性优于间位取代。综上所述,本研究可以得出一个初步的构效关系:卤素原子(X) >吸电子基团(EWD) > 给电子基团(EDG)。特别是化合物2h4f在具有较优抗炎活性的同时还具有低毒性的特点(细胞活力大于 90%),进一步研究其对促炎因子IL-6、TNF-α和IL-1β的影响,以初步明确该类化合物的抗炎作用机制。

    Figure  1.  Inhibitory effect of compounds on LPS-induced NO expression in RAW264.7 cells ($\bar{x}\pm s$, n= 3)
    CON:Control;LPS: Lipopolysaccharide; DEX:Dexamethasone ### P<0.001 vs control group;*** P<0.001 vs LPS group
    Table  2.  In vitro anti-inflammatory activity of coumarin derivatives
    Compd.NO generation
    (IC50, μmol/L)
    Compd.NO generation
    (IC50, μmol/L)
    2e11.32 ± 1.022i7.92 ± 0.32
    2f8.22 ± 0.742j9.09 ± 0.44
    2g9.42 ± 1.524e12.16 ± 0.43
    2h6.76 ± 0.224f5.49 ± 0.44
    下载: 导出CSV 
    | 显示表格

    研究表明,当发生炎症时,促炎因子IL-6、TNF-α和IL-1β的表达水平会升高[22]。细胞毒性和抗炎活性筛选结果,化合物2h4f对RAW264.7细胞毒性较小,而对NO的抑制较为显著。因此,进一步测定化合物2h4f对LPS刺激RAW264.7细胞的IL-6、TNF-α和IL-1β表达水平的影响(图2)。研究发现:化合物2h4f均可抑制LPS诱导的RAW264.7细胞的IL-6的表达,且化合物4f抑制效果更为显著(图2-A);化合物2h4f均可显著抑制炎症因子TNF-α的表达,都呈剂量依赖性,且化合物4f对TNF-α的抑制作用趋近于正常组(图2-B);化合物2h4f均能显著抑制炎症因子IL-1β的表达,且高剂量(20 μmol/L)时对IL-1β的抑制作用趋近于正常组(图2-C)。研究结果显示两个化合物对促炎因子都具有一定的抑制作用,且呈现出剂量依赖性,说明化合物2h4f可能是通过抑制促炎因子IL-6、TNF-α和IL-1β的表达来发挥抗炎作用。

    Figure  2.  Inhibition of compounds 2h and 4f on LPS-induced pro-inflammatory factors IL-6 (A), TNF-α (B) and IL-1β (C) in RAW264.7 cells detected by ELISA ($\bar{x}\pm s $, n= 3)
    RAW264.7 cells were treated with compounds 2h and 4f (5, 10, and 20 μmol/L) for 2 h, and then with LPS for 24 h ### P<0.001 vs control group; * P<0.05,** P<0.01, *** P<0.001 vs LPS group

    天然产物对化学、生物学和药物发现都有着深远的影响,其巨大的结构多样性一直为药物化学家寻找具有药理活性的新分子实体提供重要的灵感来源[23]。香豆素核存在于多种植物中,在合成和药物化学领域是非常具有价值的支架[24]。研究表明,天然和合成香豆素衍生物的抗炎及其药理潜力可用于对抗动物体内的各种疼痛和炎症疾病[25]。其中,双香豆素衍生物具有抗肿瘤、抗炎、抗菌、抗HIV、抗结核和抗精神病等多种生物活性[26]。目前已有双香豆素在临床上作为抗凝血药物使用,如华法林,表明这类化合物相对安全。四氢色烯衍生物是合成化学中的重要中间体和一类具有多种生物学活性的药用支架,并且四氢色烯衍生物在医药领域有着广泛应用,如四氢吡喃并色烯γ-分泌酶抑制剂被用于治疗神经变性疾病。此外,二氢吡啶是一类重要的含氮杂环化合物,经研究发现,其母核结构能够与多种受体结合,诱导产生不同生物活性[27],尤其是在医药领域的应用较为普遍[2829]。综上所述,双香豆素衍生物、四氢色烯衍生物和二氢吡啶衍生物均相对安全,且被广泛应用于医药领域,但抗炎活性报道较少。所以高效、绿色、便捷地构建不同结构的香豆素衍生物并评估其抗炎活性具有很大的研究价值。

    因此,本文设计合成得到3个系列共22个香豆素衍生物。体外抗炎活性研究发现,化合物2e2f2g2h2i2j4e4f对NO的抑制作用优于阳性药物地塞米松。此外,化合物2h4f能显著抑制促炎因子IL-6、TNF-α和IL-1β的表达,尤其是化合物4f对促炎因子的抑制作用更为显著,可作为治疗炎症性疾病的潜在先导化合物。

    “广谱抗耐药性抗病毒药物研究”专栏征稿启事
    1. 抗病毒药物耐药性机制2. 抗耐药性抗病毒药物研究3. 广谱抗病毒药物研究4. 病毒感染的共性宿主靶标5. 抗病毒药物研究新策略与新技术6. 计算化学和人工智能在抗病毒药物研究的应用结构生物学与抗病毒药物新靶标发现与确证 1. 此次征稿类型为综述和研究论文,稿件内容应属未在国内外公开发表过的研究成果,论文格式参考《中国药科大学学报》投稿须知,并请附通信作者近照及个人介绍。2. 征文需要按照编辑部“三审制”进行评审,经过同行专家评审后决定最终是否录用。3. 所有稿件需通过“《中国药科大学学报》在线投稿系统” (https://manuscripts.cpu.edu.cn/zgykdx)提交,并备注“广谱抗耐药性抗病毒药物研究”专栏。4. 此次征文以《中国药科大学学报》正刊形式刊出,收稿截止日期为2025年3月31日。

  • 1.   Synthesis of coumarin-based derivatives

    Figure  1.   Inhibitory effect of compounds on LPS-induced NO expression in RAW264.7 cells ($\bar{x}\pm s$, n= 3)

    CON:Control;LPS: Lipopolysaccharide; DEX:Dexamethasone ### P<0.001 vs control group;*** P<0.001 vs LPS group

    Figure  2.   Inhibition of compounds 2h and 4f on LPS-induced pro-inflammatory factors IL-6 (A), TNF-α (B) and IL-1β (C) in RAW264.7 cells detected by ELISA ($\bar{x}\pm s $, n= 3)

    RAW264.7 cells were treated with compounds 2h and 4f (5, 10, and 20 μmol/L) for 2 h, and then with LPS for 24 h ### P<0.001 vs control group; * P<0.05,** P<0.01, *** P<0.001 vs LPS group

    Table  1   Structure and inhibitory of coumarin-based derivatives (20 μmol/L) on RAW264.7

    Compd. R Yields /% Cell viability /% Compd. R Yields /% Cell viability/%
    2a 88.5 43.7 3a 78.8 49.9
    2b 86.7 42.4 3b 71.2 45.2
    2c 90.5 48.9 3c 49.8 40.0
    2d 85.8 45.8 3d 49.0 39.5
    2e 86.4 84.8 3e 59.8 43.4
    2f 87.9 51.8 4a 61.6 34.0
    2g 91.2 57.9 4b 64.3 34.2
    2h 87.4 97.5 4c 66.1 49.8
    2i 87.9 83.1 4d 76.9 49.8
    2j 85.6 63.0 4e 70.5 65.3
    2k 87.4 36.9 4f 84.1 98.5
    下载: 导出CSV

    Table  2   In vitro anti-inflammatory activity of coumarin derivatives

    Compd.NO generation
    (IC50, μmol/L)
    Compd.NO generation
    (IC50, μmol/L)
    2e11.32 ± 1.022i7.92 ± 0.32
    2f8.22 ± 0.742j9.09 ± 0.44
    2g9.42 ± 1.524e12.16 ± 0.43
    2h6.76 ± 0.224f5.49 ± 0.44
    下载: 导出CSV
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出版历程
  • 收稿日期:  2024-07-20
  • 修回日期:  2024-08-26
  • 录用日期:  2024-09-08
  • 刊出日期:  2025-02-24

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