樹豆葉二苯乙烯類成分的甲醚化和體外抗腫瘤活性

李恩念, 周娟, 譚海波, 沈小玲, 胡英杰*

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樹豆葉二苯乙烯類成分的甲醚化和體外抗腫瘤活性

李恩念1, 周娟1, 譚海波2, 沈小玲1, 胡英杰1*

(1. 廣州中醫藥大學熱帶醫學研究所中藥新藥發現實驗室, 廣州 510405; 2. 中國科學院華南植物園, 中國科學院植物資源保護與可持續利用重點實驗室, 廣州 510650)

為獲得具有抗腫瘤活性的甲醚化先導化合物，對樹豆()葉中的二苯乙烯類成分進行甲醚化，并測定其對人腫瘤細胞的細胞毒性。將木豆素C、樹豆酮酸A、Cajanotone和木豆素與碘甲烷-碳酸鉀反應制備-甲基產物，通過波譜數據分析產物結構分別鑒定為：2-異戊烯基-3,5-二甲氧基二苯乙烯 (1)、樹豆酮酸A甲醚(2)、5--methyl cajanotone (3)和3--甲基木豆素(4)，其中化合物3是新化合物。腫瘤細胞增殖抑制實驗(CCK-8法)結果表明，木豆素C對乳腺癌MDA-MB- 231、宮頸癌HeLa、肝癌HepG2、結腸癌SW480及3種非小細胞肺癌細胞(A549, NCI-H460和NCI-H1299)的半數抑制濃度IC50分別為14.4、16.1、19.6、17.4和25.7~29.6mol L–1；木豆素對宮頸癌和結腸癌之外的5種腫瘤細胞有弱抑制作用，IC50為44.9~78.3mol L–1；而對照的3,4′,5-三甲氧基二苯乙烯(三--甲基白藜蘆醇)對乳腺癌MDA-MB-231、宮頸癌HeLa、結腸癌SW480和肝癌HepG2細胞有較強抑制作用(IC50為3.0~14.5mol L–1)。樹豆葉二苯乙烯甲醚化衍生物1~4對7種腫瘤細胞系無明顯細胞毒活性。

樹豆；二苯乙烯；甲醚化；細胞毒活性；化學成分

癌癥是嚴重威脅人類生命和健康的重大疾病。據統計，2014年全國惡性腫瘤估計死亡病例229.6萬例，腫瘤死亡率0.17%，肺癌和乳腺癌分別位居男女性發病的第1位[1]。尋找新的抗腫瘤先導化合物具有重要意義。一些酚類物質在甲醚化后抗腫瘤活性有所提高，例如:黃酮類化合物白楊素甲醚化后生成5,7-二甲氧基黃酮，能夠降低急性淋巴細胞白血病細胞增殖水平[2]。二苯乙烯類植物多酚白藜蘆醇(3,5,4′-三羥基二苯乙烯)及其類似物的生物活性,可因結構中甲氧基及其取代位置的不同而變化。Weng等[3–4]報道白藜蘆醇及其甲氧基類似物如三甲氧基白藜蘆醇可抑制肝癌細胞的轉移和浸潤性質, 還對非小細胞肺癌A549細胞增殖有明顯抑制作用，而白藜蘆醇對A549細胞無效；白藜蘆醇和三甲氧基白藜蘆醇在25mol L–1工作濃度下對乳腺癌MDA-MB-231細胞的生長有明顯抑制作用[5]。

豆科植物樹豆[(Linn.)Mill.](又名木豆)葉有多方面的藥理作用, 文獻報道，12-羥基木豆菧酸具有良好的自由基清除活性和體外抗腫瘤活性[6]；木豆素體外作用于乳腺癌細胞可引起細胞周期停滯并誘導細胞凋亡[7]；木豆菧H具有體外誘導人骨髓間充質細胞分化為成骨細胞的作用，并對多種腫瘤細胞表現出中等強度抑制作用[8]；木豆素C對多種人源腫瘤細胞株的增殖表現出明顯抑制作用[9–10]；樹豆酮酸A經動物試驗，具有顯著降低糖尿病大鼠的血糖、血脂的作用[11]。樹豆二苯乙烯類成分的結構與白藜蘆醇相似，其甲氧基類似物有可能也具有抗腫瘤生物活性，所以我們對源自于樹豆葉的4個二苯乙烯類成分木豆素C、樹豆酮酸A、Cajanotone和木豆素進行甲醚化反應[12]，反應產物經硅膠柱層析純化和波譜分析確定結構。以白藜蘆醇及其甲氧基衍生物為對照，檢測相關化合物的體外抗腫瘤活性。本文報道這類甲醚化衍生物的制備、結構鑒定，以及體外抗腫瘤活性的測試結果。

1 材料和方法

1.1 儀器、試劑和耗材

DRX-400核磁共振儀、EQUINOX55型紅外波譜儀(德國Bruker公司)；TU-1901雙光束紫外可見分光光度計(北京普析通用儀器有限公司)；C506- Triple TOF 5600系統(美國AB SCIEX公司)；LC6000型高效制備液相色譜儀、LC3000型高效制備液相色譜儀(北京創新通恒有限公司)；UV D-170U、Chro- meleon色譜工作站高效液相色譜儀(美國Dionex公司)；SHIM-PACK色譜柱(日本SHIMADZU公司); 硅膠(200~300目)、硅膠TLC薄層預制板(青島海洋化工廠分廠)；Sephadex LH-20葡聚糖凝膠(美國GE Healthcare公司)；HPLC分析，使用Merck公司甲醇; 其他溶劑和試劑均為國產分析純產品。TLC顯色劑為6%香蘭素乙醇液-12%高氯酸溶液等體積混合液。

細胞培養基RPMI 1640 (德國Biological Industries公司，批號0012118)；胎牛血清(FBS)(澳大利亞Gibco公司，批號1704206)。

CO2細胞培養箱(上海力申科學儀器有限公司);垂直超凈工作臺(上海智城分析儀器制造有限公司);倒置光學顯微鏡(日本Olympus公司)；低速離心機(科大創新股份有限公司中佳分公司)；MK3型酶標儀(美國Thermo fisher公司)；Cell Counting Kit-8 (CCK-8)細胞增殖-毒性檢測試劑盒(日本同仁化學研究所)。

1.2 反應原料

木豆素C，樹豆酮酸A，Cajanotone和木豆素均從樹豆葉分離得到[8,10,13]。

1.3 細胞株

人乳腺癌MDA-MB-231細胞、人宮頸癌HeLa細胞、人肝癌HepG2細胞、人結腸癌SW480細胞、人非小細胞肺癌A549、NCI-H1299和NCI-H460均來自中國科學院上海生命科學研究院細胞資源中心。

1.4 樹豆菧類成分的甲醚化

木豆素C (500 mg, 1.7 mmol)溶于乙腈(5 mL),加入無水碳酸鉀(400 mg, 2.9 mmol)，于室溫(28℃~32℃)下攪拌20 min，緩慢滴入200L碘甲烷(460 mg, 3.2 mmol)，攪拌3 h (經TLC檢測反應至終點)，加水5 mL淬滅反應；加乙酸乙酯15 mL萃取反應液3次，合并乙酸乙酯溶液，減壓蒸干，得粗產物；粗產物經硅膠(200~300目)柱層析，以正己烷-乙酸乙酯(50∶1)洗脫，得產物1 (317 mg，圖1)，收率60.6%。

樹豆酮酸A (93 mg, 0.26 mmol)溶于乙腈(5 mL),加入無水碳酸鉀(120 mg, 0.87 mmol)，緩慢滴入20L碘甲烷(46 mg, 0.32 mmol)，后續處理過程與制備化合物1的相同，硅膠柱層析流動相為正己烷-乙酸乙酯(20∶1~10∶1)，得化合物2 (35.6 mg，圖1)，收率36.8%。

以Cajanotone (40 mg, 0.13 mmol)溶于乙腈(5 mL),加入無水碳酸鉀(60 mg, 0.44 mmol)，緩慢滴入10L碘甲烷(23 mg, 0.16 mmol)，后續處理過程與制備化合物1的相同，硅膠柱層析流動相為正己烷-乙酸乙酯(20∶1~10∶1)，得化合物3 (14.3 mg，圖1)，收率34.2%。

木豆素(236 mg, 0.70 mmol)溶于乙腈(5 mL)，加入無水碳酸鉀(300 mg, 2.17 mmol)，緩慢滴入80L碘甲烷(184 mg, 1.28 mmol)，后續處理過程與制備化合物1的相同，硅膠柱層析流動相為正己烷-乙酸乙酯(50∶1~20∶1)，得化合物4 (35 mg，圖1)，收率14.8%。

1.5 結構鑒定

化合物1 白色粉末，ESI-MS: 308.9 [M + H]+。1H NMR (400 MHz, CDCl3):7.55 (2H, d,= 7.6 Hz, H-2′, H-6′), 7.42 (2H, m, H-3′, H-5′), 7.41 (1H, m, H-4′), 7.30 (2H, d,= 12 Hz, H-7, H-8), 6.79 (1H, d,= 2.4 Hz, H-4), 6.46 (1H, d,= 2 Hz, H-6), 5.16 (1H, t,= 6.4 Hz, H-2′′), 3.95 (3H, s, 3-OCH3), 3.88 (3H, s, 5-OCH3), 3.48 (2H, d,= 9.2 Hz, H-1′′), 1.84 (3H, s, H-4′′), 1.71 (3H, s, H-5′′);13C NMR (100 MHz, CDCl3):158.5 (C, C-3), 158.3 (C, C-5), 137.5 (C, C-1, C-1′), 130.9 (CH, C-3′′), 130.3 (CH, C-7), 128.6 (CH, C-3′, C-5′), 127.6 (CH, C-4′′), 126.8 (CH, C-8), 126.5 (CH, C-2′, C-6′), 123.4 (CH, C-2′′), 121.1 (C, C-2), 101.4 (CH, C-4), 98.1 (CH, C-6), 55.6 (CH3, 3-OCH3), 55.3 (CH3, 5- OCH3), 25.9 (CH3, C-4′′), 24.4 (CH2, C-1′′), 17.9 (CH3, C-5′′)。以上數據與5--methyl-longistilin C一致[14]。

化合物2 白色粉末，ESI-MS: (neg.) 367.1 [M - H]–。1H NMR (400 MHz, CDCl3):11.59 (1H, s, H-COOH), 8.06 (2H, s,= 7.6 Hz, H-2′, H-6′), 7.61 (1H, t,= 7.2 Hz, H-4′), 7.52 (2H, t,= 7.6 Hz, H-3′, H-5′), 6.46 (1H, s, H-3), 4.95 (1H, t,= 7.2 Hz, H-7), 3.85 (3H, s, 4-OCH3), 3.48 (3H, s, 2-OCH3), 3.27 (2H, d,= 6 Hz, H-1′′), 1.61 (3H, s, H-4′′), 1.59 (3H, s, H-5′′);13C NMR (100 MHz, CDCl3):197.1 (C, C-8), 171.2 (C, 1-COOH), 163.6 (C, C-4), 162.5 (C, C-2), 137.0 (C, C-1′), 136.7 (C, C-6), 133.0 (C, C-4′), 131.6 (C, C-3′′), 128.7 (CH, C-3′, C-5′), 123.1 (C, C-5), 122.7 (CH, C-2′′), 105.2 (C, C-1), 98.6 (CH, C-3), 55.7 (CH3, 4-OCH3), 51.9 (CH3, 2-OCH3), 41.6 (CH2, C-7), 25.6 (CH3, C-5′′), 24.9 (CH2, C-1′′), 17.8 (CH3, C-4′′)。以上數據與5--methyl-cajanonic acid A的一致[15]。

化合物3 白色粉末，ESI-MS/: (pos.) 347.0 [M + Na]+。1H NMR (400 MHz, CDCl3):8.01 (2H, d,= 7.6 Hz, H-2′, H-6′), 7.57 (1H, t,= 7.2 Hz, H-4′), 7.46 (2H, t,= 7.2 Hz, H-3′, H-5′), 6.41 (1H, d,= 2 Hz, H-6), 5.00 (1H, t,= 6.4 Hz, H-2′′), 4.26 (2H, s, H-7), 3.78 (3H, s, 3-OCH3), 3.74 (3H, s, 5- OCH3), 3.26 (2H, d,= 6.4 Hz, H-1′′), 1.59 (3H, s, H- 4′′), 1.51 (3H, s, H-5′′);13C NMR (100 MHz, CDCl3):197.8 (C, C-8), 158.4 (C, C-3, C-5), 136.7 (C, C-1′), 134.9 (C, C-1), 133.1 (CH, C-4′), 131.2 (C, C-3′′), 128.6 (CH, C-2′, C-6′), 123.2 (CH, C-2′′), 121.1 (C, C-2), 106.6 (CH, C-4), 97.5 (CH, C-6), 55.6 (CH3, 3- OCH3), 55.2 (CH3, 5-OCH3), 43.2 (CH2, C-7), 25.6 (CH3, C-5′′), 24.8 (CH2, C-1′′), 17.8 (CH3, C-4′′)。因此，化合物3的結構確定為5--methyl-cajanotone (表1，圖2)[16]。

表1 Cajanotone與化合物3的核磁數據(溶劑為CDCl3)

化合物4 白色粉末，ESI-MS: (neg.) 351.0 [M - H]–。1H NMR (400 MHz, CDCl3):11.74 (1H, s, COO), 7.75 (1H, d,= 16 Hz, H-8), 7.53 (2H, t,= 7.6 Hz, H-2′, H-6′), 7.39 (2H, t,= 7.6 Hz, H-3′, H-5′), 6.81 (1H, d,= 16 Hz, H-7), 6.62 (1H, s, H-5), 5.23 (1H, t, H-2′′), 3.94 (3H, s, 4-OCH3), 3.93 (3H, s, 2-OCH3), 3.40 (2H, d,= 8 Hz, H-1′′), 1.81 (3H, s, H-4′′), 1.69 (3H, s, H-5′′);13C NMR (100 MHz, CDCl3):171.9 (C, 1-COOH), 161.4 (C, C-4), 161.3 (C, C-2), 140.3 (C, C- 6), 137.4 (C, C-1′), 131.9 (C, C- 3′′), 130.6 (CH, C-8), 128.7(CH, C-3′, C-5′), 127.7 (CH, C-7), 126.5 (CH, C-2', C-6'), 122.0 (CH, C-2′′), 116.6 (C, C-3), 104.4 (C, C-1), 102.8 (CH, C-5), 55.6 (CH3, 4-OCH3), 52.3 (CH3, 2-OCH3), 25.8 (CH3, C-4′′), 22.1 (CH2, C-1′′), 17.8 (CH3, C-5′′)。以上數據與5-- methyl-cajaninstilbene acid的一致[17]。

1.6 體外抗腫瘤活性研究

樣品對HeLa、SW480、HepG2、A549、NCI- H460、NCI-H1299、MDA-MB-231細胞的增殖抑制試驗(CCK-8法)：胰酶消化收集生長良好的細胞, 用新鮮培養液配成5×104個mL–1的單細胞懸液，按每孔100L接種于96孔板中，置于孵育箱中培養24 h使細胞貼壁。將樣品用細胞培養液稀釋至需要的百分濃度，按每孔100L替換原有的細胞培養液，每個濃度設3個復孔。試驗設置不含藥物的陰性對照，并對每個藥物作用濃度設置不含細胞的藥物空白對照。將細胞板置于孵育箱孵育72 h后取出, 每孔加入10L CCK-8，再置于孵育箱孵育1.5 h。取出細胞培養板，于450 nm檢測每孔吸收值A。樣品對腫瘤細胞增殖的抑制率(%)=100×(A陰性-A藥物)/ (A陰性-A空白)，并求得其對細胞增殖的半數抑制濃度IC50，即細胞增殖抑制率為50%時的作用濃度。

2 結果和討論

我們利用樹豆天然二苯乙烯類成分木豆素C、樹豆酮酸A、Cajanotone和木豆素作為原料，以碘甲烷為甲醚化反應試劑，進行甲醚化反應，得到了2-異戊烯基-3,5-二甲氧基二苯乙烯 (1)、樹豆酮酸A--甲醚(2)、5--methyl cajanotone (3)和3--甲基木豆素 (4)。其中化合物3是新化合物。Zhang等報道,Cajanotone和木豆素對肺癌A549細胞的IC50分別為5.9和4.4mol L–1，對肝癌HepG2細胞的IC50為12.2~17.9mol L–1；木豆素C對這幾種腫瘤細胞呈顯著細胞毒活性(IC50為3.5~6.0mol L–1)[16]; 木豆素C的甲氧基衍生物對肝癌HepG2細胞的IC50為3.7mol L–1[14]。三甲氧基白藜蘆醇對MDA-MB- 231、HeLa、SW480和HepG2細胞具有較強細胞毒作用，IC50分別為3.0、9.2、14.5和13.0mol L–1。三甲氧基白藜蘆醇的體外抗腫瘤活性最強，4′-甲氧基白藜蘆醇次之，白藜蘆醇較弱。從表2可見，樹豆葉二苯乙烯類成分中，木豆素C對7種腫瘤細胞株均呈顯著的細胞增殖抑制作用(IC50為14.4~ 29.6mol L–1)，木豆素對除HeLa和SW480外的其余5個細胞株均呈較弱的細胞毒活性(IC50為44.9~ 78.3mol L–1)，但甲醚化產物1~4對參試7種腫瘤細胞株均無明顯細胞毒作用(IC50>100mol L–1)。這4種甲醚衍生物的甲氧基數目和取代位置與三甲氧基白藜蘆醇有較大差別。有報道，3,4,5,4′-四甲氧基二苯乙烯在體外對人宮頸癌HeLa細胞、人前列腺癌LNCaP細胞、人結腸腺癌HT-29細胞、人肝癌HepG2細胞的抑制作用顯著增強[18]。由此推測，甲氧基數目和取代位置對樹豆葉二苯乙烯類成分的抗腫瘤活性有顯著影響。我們的試驗為樹豆葉二苯乙烯類成分通過結構修飾改變體外抗腫瘤活性提供了參考依據。

表2 化合物1~4對人腫瘤細胞增殖的影響(IC50, μmol L–1)

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Methoxylation of the Stilbenes Originated from Leaves ofand Their Anti-tumor Activities

LI En-nian1, ZHOU Juan1, TAN Hai-bo2, SHEN Xiao-ling1, HU Ying-jie1*

(1. Laboratory of Herbal Drug Discovery, Tropical Medicine Institute, Guangzhou University of Chinese Medicine,Guangzhou 510405, China; 2. Key Laboratory of Plant Resources Conservation and Sustainable Utilization, Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China)

The aim was to obtain lead compounds with anticancer activity by-methylation of stilbene pheolic deriatives extracted from leaves of. Four derivatives, such as longistylin C, cajanonic acid A, cajanotone and cajaninstilbene acid, were treated with iodomethane and potassium carbohydrate under mild condition. The products were purified through silica gel column chromatography and their structures were identified by spectroscopic analysis as 2-isoprenyl-3,5-dimethoxy-stilbene (1),-methyl-cajanonic acid A (2), 5--methyl cajanotone (3) and 3--methyl cajaninstilbene acid (4), respectively. Among them, compound 3 was a new compound. Effects of 1–4 and their reactants, together with resveratrol, 4′--methyl resveratrol and 3,4′,5--trimethyl resveratrol using as positive controls, on the proliferation of the cell lines of human breast cancer (MDA-MB-231), cervical cancer (HeLa), liver cancer (HepG2), colon cancer (SW480), and non-small cell lung cancer (A549, NCI-H460 and NCI-H1299) were determined by CCK-8 assay. The results showed that longistilin C distinctly inhibited the proliferation of all the seven cell lines, with the half inhibitory concentrations (IC50) to MDA-MB-231, HeLa, HepG2, and SW480 at 14.4, 16.1, 19.6, and 17.4mol L–1(strong suppresion), with IC50values of 25.7–29.6mol L–1to other three cell lines of non-small cell lung cancer; cajaninstilbene acid had a weak inhibition to HepG2, A549, NCI-H460, NCI-H1299 and MDA-MB-231 cells with IC50as 44.9–78.3mol L–1; tri--methylated resveratrol showed significantly cytotoxity against MDA-MB-231, HeLa, SW480 and HepG2 cells with IC50of 16.1-17.4mol L–1;-methylated stilbenes 1-4 did not exhibited obvious cytotoxicity in the cell lines aboved.

; Stilbene; Methoxylation; Cytotoxicity; Chemical constituent

10.11926/jtsb.3938

2018-05-03

2018-06-15

廣東省教育廳重大項目(2014KZDXM026)；廣東省科技計劃項目(2016A020217015)資助

This work was supported by the Key Projects in Department of Education of Guangdong (Grant No. 2014KZDXM026), and the Plan Project for Science and technology in Guangdong (Grant No. 2016A020217015).

李恩念，男，碩士研究生，研究方向為中藥新藥研發。E-mail: ennian_li@163.com

E-mail: yingjiehu@gzucm.edu.cn