單偉光,施　航,占扎君

(浙江工業(yè)大學(xué) 藥學(xué)院，浙江 杭州 310014)

雷公藤紅素衍生物合成與活性測(cè)定

單偉光,施航,占扎君

(浙江工業(yè)大學(xué) 藥學(xué)院，浙江 杭州 310014)

摘要：雷公藤紅素是一種從中藥雷公藤中提取的一種具有很強(qiáng)抗癌活性的三萜化合物，為找到具有更好抗癌活性和成藥性的雷公藤紅素衍生物，在雷公藤紅素C-29羧基設(shè)計(jì)并合成10個(gè)新型的衍生物，并通過MMT法，測(cè)定了衍生物對(duì)A549和HepG2腫瘤細(xì)胞的抗癌活性，實(shí)驗(yàn)結(jié)果顯示10個(gè)衍生物都具有很好的抗癌活性，化合物C4和C8抗癌活性最強(qiáng)，并且具有良好的水溶性，是較好的體內(nèi)實(shí)驗(yàn)備選化合物.

關(guān)鍵詞：雷公藤紅素；抗癌；毒性；MTT法

Synthesis and anti-cancer evaluation of novel celastrol analogues

SHAN Weiguang, SHI Hang, ZHAN Zhajun

(College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 3100, China)

Abstract:Celastrol, a natural quinone methide triterpenoid originally isolated from root bark of the traditional Chinese herb “Thunder of God Vine”, showed remarkable anticancer activity. In order to find the celasrol analogues of better anticancer activity and druggability, Ten novel celastrol analogues were designed and synthesized. Their cytotoxicity were evaluated against 2 human cancer cell lines(A549 and HepG2) by MTT method. Ten celastrol analogues all showed better inhibitions than positive drug. Among all of the derivatives, C4 and C6 were the most suitable candidate compounds for in vivo experiment in the future because of their high activity and water solubility.

Key words:celastrol; anticancer; cytotoxicity; MTT method

雷公藤紅素(celastrol)是一種五環(huán)的木栓烷型三萜化合物，又名南蛇藤堿，是第一個(gè)從雷公藤根部提取出來的三萜類化合物[1-2].三萜類化合物雷公藤紅素具有獨(dú)特的化學(xué)結(jié)構(gòu)基團(tuán)，能夠和半胱氨酸殘基中的巰基發(fā)生邁克爾加成，生成共軛加成產(chǎn)物[3]，該化合物在體內(nèi)通過發(fā)生加成反應(yīng)，影響蛋白或酶的活性、調(diào)節(jié)多種細(xì)胞信號(hào)途徑等，進(jìn)而產(chǎn)生藥理作用.據(jù)報(bào)道，雷公藤紅素具有多種顯著的生物活性，例如，抗腫瘤、抗免疫與抗炎、抗病毒以及抗神經(jīng)衰退性疾病等[4-6].雷公藤紅素的抗腫瘤活性在很久之前已經(jīng)被研究證實(shí)，但是很長(zhǎng)一段時(shí)間機(jī)制不明確，直到2006年Yang等[7]首次實(shí)驗(yàn)證明雷公藤紅素可以誘導(dǎo)癌細(xì)胞凋亡，使得腫瘤細(xì)胞壞死，從而引發(fā)了雷公藤紅素抗癌機(jī)制的研究熱浪；之后相繼有研究表明雷公藤紅素對(duì)多種腫瘤細(xì)胞的治療都有很好的療效，例如前列腺癌細(xì)胞、神經(jīng)膠質(zhì)瘤細(xì)胞、口腔鱗狀癌細(xì)胞、黑素瘤、乳腺癌、白血病和肺癌等等[8-10].

國(guó)內(nèi)外開展了不少雷公藤紅素修飾物的研究[11-13]，主要是針對(duì)C-3羥基和C-29羧基成一些酯基和一些酰胺鍵，本次研究的創(chuàng)新處在于在C-29位的常規(guī)修飾基團(tuán)中加入一些鹵元素、苯環(huán)、六圓環(huán)及羥基，試圖改變整個(gè)分子的結(jié)構(gòu)以及電子云密度，從而產(chǎn)生不同的抗癌活性.

1合成研究

1.1實(shí)驗(yàn)材料與儀器

實(shí)驗(yàn)材料：雷公藤紅素(課題組自提分離)；鹵代烷試劑(AR，Aladdin Chemistry Co.Ltd.)；碳酸氫鈉(CR，天津永大化學(xué)試劑有限公司)；無水硫酸鈉(CR，上海四赫維化工有限公司)；硅膠(青島海洋化工廠)；溶劑均為國(guó)產(chǎn)分析純.

實(shí)驗(yàn)儀器：磁力攪拌器(杭州大衛(wèi)科教儀器)；真空干燥箱(上海精宏實(shí)驗(yàn)設(shè)備有限公司)；真空油泵(臺(tái)州博奧真空設(shè)備有限公司)；恒溫鼓風(fēng)干燥箱(上海精宏實(shí)驗(yàn)設(shè)備有限公司)；數(shù)控超聲波清洗器(昆山市禾創(chuàng)超聲儀器有限公司)；旋轉(zhuǎn)蒸發(fā)儀(BUCHI)；循環(huán)水式多用真空泵(杭州大衛(wèi)科教儀器)；紫外燈(上海顧村電光儀器廠)；電子天平(max 220 g，d=0.1 mg，德國(guó)塞多利斯)；冰箱(青島海爾股份有限公司).

1.2衍生物的合成

如圖1所示，取紅素(50 mg, 0.11 mmol)溶于N,N-二甲基甲酰胺(DMF，4 mL)中，加入碳酸氫鈉少量，再加入相應(yīng)的二鹵代烷、鹵代環(huán)烷烴(0.3 mmol)，室溫下攪拌數(shù)小時(shí).TLC法檢測(cè)反應(yīng)程度，觀察雷公藤紅素點(diǎn)消失后，反應(yīng)終止.加入去離子水(15 mL)，乙酸乙酯萃取3 次，合并有機(jī)層.有機(jī)層再用飽和食鹽水洗3次，無水硫酸鈉干燥，旋轉(zhuǎn)蒸發(fā)儀濃縮得棕色油狀物.粗產(chǎn)物經(jīng)快速柱層析(油醚：丙酮)法分離純化，產(chǎn)物經(jīng)減壓蒸除溶劑、真空干燥得深棕色固體C1～C10.所得產(chǎn)物經(jīng)氫譜、碳譜和質(zhì)譜等方法確定其結(jié)構(gòu).雷公藤紅素衍生物的合成路線為

1.3衍生物的圖譜歸屬

C1：1H-NMR(CDCl3,500 MHz):0.55(3H,s,CH3),1.12(3H,s,CH3),1.21(3H,s,CH3),1.25(3H,t,J=7.0 Hz,H-2′),1.27(3H,s,CH3),1.45(3H,s,CH3),2.25(3H,s,CH3),3.98(2H,m,H-1′),6.35(1H,d,J=7.0 Hz7.0,H-7),6.55(1H,s,H-1),7.03(1H,d,J=7.0 Hz,H-6).13C-NMR(CDCl3,125 MHz):10.2,14.0,18.4,21.6,28.7,29.3,29.8,30.6,30.7,31.6,32.8,33.6,34.8,36.4,38.2,39.5,40.2,43.0,44.3,45.1,60.3,117.3,118.2,119.5,127.5,134.3,146.1,164.9,170.4,178.2,178.4.ESI-MS:m/z479.3[M+H]+.

C2:1H-NMR(CDCl3,500 MHz):0.55(3H,s,CH3),1.09(3H,s,CH3),1.21(3H,s,CH3),1.27(3H,s,CH3),1.45(3H,s,CH3),2.19(3H,s,CH3),3.45(2H,t,J=6.5 Hz,H-2′),4.10(1H,m,H-1′a),4.27(1H,m,H-1′b),6.31(1H,d,J=7.0 Hz,H-7),6.49(1H,s,H-1),7.09(1H,d,J=7.0 Hz,H-6).13C-NMR(CDCl3,125 MHz):10.2,18.4,21.5,28.4,28.8,29.6,29.8,30.6,30.7,31.5,32.7,33.5,34.6,36.3,38.1,39.4,40.4,42.8,44.2,44.9,63.9,117.0,118.0,119.5,127.3,134.0,146.1,164.6,169.8,177.7,178.2.ESI-MS:m/z559.3[M+H]+.

C3:1H-NMR(CDCl3,500 MHz):0.55(3H,s,CH3),1.09(3H,s,CH3),1.21(3H,s,CH3),1.27(3H,s,CH3),1.45(3H,s,CH3),2.19(3H,s,CH3),3.64(2H,t,J=6.5 Hz,H-2′),4.08(1H,m,H-1′a),4.23(1H,m,H-1′b),6.34(1H,d,J=7.0 Hz,H-7),6.53(1H,s,H-1),7.01(1H,d,J=7.0 Hz,H-6).13C-NMR(CDCl3,125 MHz):10.2,18.6,21.6,28.6,29.6,29.7,29.8,30.8,31.5,32.7,33.6,34.7,36.3,38.2,39.5,40.5,41.6,42.9,44.3,45.0,64.2,117.1,118.1,119.5,127.4,134.0,146.1,164.7,169.8,177.9,178.4.ESI-MS:m/z513.3[M+H]+.

C4:1H-NMR(CDCl3,500 MHz):0.55(3H,s,CH3),1.12(3H,s,CH3),1.21(3H,s,CH3),1.26(3H,s,CH3),1.44(3H,s,CH3),2.20(3H,s,CH3),3.78(2H,m,H-2′),4.01(1H,m,H-1′a),4.09(1H,m,H-1′b),6.35(1H,d,J=7.0 Hz,H-7),6.53(1H,s,H-1),7.02(1H,d,J=7.0 Hz,H-6).13C-NMR(CDCl3,125 MHz):10.2,18.5,21.6,28.7,29.3,29.8,30.6,30.7,31.6,32.8,33.6,34.8,36.4,38.2,39.5,40.2,43.0,44.3,45.1,60.9,66.1,117.2,118.2,119.6,127.5,134.1,146.1,164.8,170.0,178.4,178.5.ESI-MS:m/z485.3[M+H]+.

C5:1H-NMR(CDCl3,500 MHz):0.55(3H,s,CH3),0.97(3H,m,H-3′),1.12(3H,s,CH3),1.19(3H,s,CH3),1.27(3H,s,CH3),1.45(3H,s,CH3),2.24(3H,s,CH3),3.82(1H,m,H-1′a),3.93(1H,m,H-1′b),6.35(1H,d,J=7.0 Hz,H-7),6.54(1H,s,H-1),7.02(1H,d,J=7.0 Hz,H-6).13C-NMR(CDCl3,125 MHz):10.2,14.0,18.4,21.6,21.7,28.7,29.3,29.7,30.6,30.7,31.6,32.8,33.5,34.8,36.4,38.2,39.4,40.4,43.0,44.3,45.1,66.3,117.3,118.2,119.5,127.4,134.0,146.0,164.7,170.0,178.3,178.4.ESI-MS:m/z493.1[M+H]+.

C6:1H-NMR(CDCl3,500 MHz):0.54(3H,s,CH3),1.09(3H,s,CH3),1.21(3H,s,CH3),1.27(3H,s,CH3),1.45(3H,s,CH3),2.19(3H,s,CH3),3.44(2H,t,J=6.5 Hz H-3′),3.98(1H,m,H-1′a),4.10(1H,m,H-1′b),6.33(1H,d,J=7.0 Hz,H-7),6.52(1H,s,H-1),7.00(1H,d,J=7.0 Hz,H-6).13C-NMR(CDCl3,125 MHz):10.2,18.6,21.6,28.6,29.2,29.3,29.6,29.8,30.6,30.7,31.5,32.7,33.5,34.6,36.3,38.1,39.4,40.4,42.8,44.3,44.9,62.0,117.0,118.1,119.5,127.4,134.0,146.0,164.6,169.7,178.3,178.2.ESI-MS:m/z573.3[M+H]+.

C7:1H-NMR(CDCl3,500 MHz):0.55(3H,s,CH3),1.09(3H,s,CH3),1.21(3H,s,CH3),1.27(3H,s,CH3),1.45(3H,s,CH3),2.19(3H,s,CH3),3.56(2H,t,J=6.5 Hz H-3′),3.95(1H,m,H-1′a),4.09(1H,m,H-1′b),6.31(1H,d,J=7.0 Hz,H-7),6.49(1H,s,H-1),7.01(1H,d,J=7.0 Hz,H-6).13C-NMR(CDCl3,125 MHz):10.1,18.6,21.5,28.5,29.6,29.7,29.8,30.4,30.7,31.5,32.6,33.4,34.7,36.3,38.1,39.3,40.5,41.0,42.8,44.2,44.9,61.0,117.1,118.1,119.5,127.3,133.9,146.o,164.6,169.8,177.9,178.2.ESI-MS:m/z527.3[M+H]+.

C8:1H-NMR(CDCl3,500 MHz):0.55(3H,s,CH3),1.10(3H,s,CH3),1.18(3H,s,CH3),1.27(3H,s,CH3),1.45(3H,s,CH3),2.21(3H,s,CH3),3.68(2H,m,H-3′),4.01(1H,m,H-1′a),4.13(1H,m,H-1′b),6.35(1H,d,J=7.0 Hz,H-7),6.54(1H,s,H-1),7.02(1H,d,J=7.0 Hz,H-6).13C-NMR(CDCl3,125 MHz):10.2,18.5,21.6,28.7,29.3,29.7,29.8,30.6,30.7,31.6,32.8,33.6,34.8,36.4,38.2,39.5,40.5,43.0,44.3,45.1,59.4,61.5,117.2,118.2,119.6,127.5,134.1,146.1,164.8,170.0,178.4,178.5.ESI-MS:m/z499.3[M+H]+.

C9:1H-NMR(CDCl3,500 MHz):0.50(3H,s,CH3),1.09(3H,s,CH3),1.20(3H,s,CH3),1.26(3H,s,CH3),1.43(3H,s,CH3),2.21(3H,s,CH3),4.94(1H,d,J=7.5 Hz,H-1′a),5.02(1H,d,J=7.5 Hz,H-1′b),6.33(1H,d,J=7.0 Hz,H-7),6.49(1H,s,H-1),7.01(1H,d,J=7.0 Hz,H-6),7.27-7.35(5H,m,H-Ar).13C-NMR(CDCl3,125 MHz):10.2,18.4,21.6,28.6,29.3,29.8,30.6,30.8,31.6,32.8,33.6,34.8,36.4,38.2,39.5,40.4,42.9,44.2,45.0,66.3,117.3,118.1,119.6,127.4,127.5,128.2,128.3,128,6 134,1 134.1,135,7,146.0,164.7,170.1,177.9,178.3.ESI-MS:m/z541.3[M+H]+.

C10:1H-NMR(CDCl3,500 MHz):0.53(3H,s,CH3),1.13(3H,s,CH3),1.21(3H,s,CH3),1.27(3H,s,CH3),1.46(3H,s,CH3),2.22(3H,s,CH3),3.64(H,m,H-1′a),3.80(H,m,H-1′b),6.36(1H,d,J=7.0 Hz,H-7),6.54(1H,s,H-1),7.03(1H,d,J=7.0 Hz,H-6).13C-NMR(CDCl3,125 MHz):10.2,18.4,21.6,25.7,25.8,28.7,29.7,29.7,29.8,29.9,30.6,30.8,31.6,32.8,33.5,34.9,36.5,38.2,39.5,40.5,43.0,44.4,45.1,69.7,117.0,118.1,119.5,127.5,134.0,146.1,164.7,169.9,178.3,178.4.ESI-MS:m/z547.3[M+H]+.

2衍生物抗腫瘤活性研究

2.1實(shí)驗(yàn)材料

實(shí)驗(yàn)細(xì)胞(人肺癌細(xì)胞株A549，人肝癌細(xì)胞株HepG2[14]；MEM培養(yǎng)基(南京凱基生物)；DMEM培養(yǎng)基(南京凱基生物)；RPMI-1640培養(yǎng)基(南京凱基生物)；胎牛血清(Hyclone)；胰酶(吉諾生物醫(yī)藥)；細(xì)胞凍存液(南京凱基生物)；PBS(吉諾生物醫(yī)藥)；MTT(Sigma)；DMSO(CR，上海凌峰化學(xué)有限公司).

2.2實(shí)驗(yàn)儀器

超凈臺(tái)(SW-CJ-1F，上海博迅實(shí)業(yè)醫(yī)療設(shè)備廠)；CO2培養(yǎng)箱(Thermo scientific)；倒置相差顯微鏡(Olympus, CKX41)；酶標(biāo)儀(TECAN Sunrise)；高壓蒸汽滅菌鍋(上海博迅實(shí)業(yè)醫(yī)療設(shè)備廠)；96孔培養(yǎng)板(Corning Incorporated)；高速離心機(jī)(LG10-2.4A).

2.3實(shí)驗(yàn)操作

采用MTT法[15-16]，在37 ℃，體積比為5%的CO2培養(yǎng)箱內(nèi)，取對(duì)數(shù)生長(zhǎng)期的各細(xì)胞(3×104個(gè)/mL)，接種于96孔培養(yǎng)板(180 μL/孔)中貼壁生長(zhǎng).24 h后，加入不同濃度藥物及陽性對(duì)照物，以含細(xì)胞培養(yǎng)基為空白對(duì)照孔，每個(gè)濃度設(shè)5個(gè)平行孔.置于37 ℃，體積比為5% CO2培養(yǎng)箱中培養(yǎng).第48 h時(shí)加入5 mg/mL MTT溶液(20 μL/孔)，混勻后，繼續(xù)孵育4 h.第52 h時(shí)，停止培養(yǎng)，小心倒去96孔板內(nèi)培養(yǎng)基，加入DMSO(150 uL/孔)，振蕩溶解，10 min內(nèi)酶標(biāo)儀于570 nm波長(zhǎng)處測(cè)定OD值.最后根據(jù)IC50計(jì)算公式和軟件計(jì)算出各化合物IC50值.

2.4IC50實(shí)驗(yàn)結(jié)果和討論

選取雷公藤紅素以及10個(gè)衍生物，以卡鉑作為陽性藥，測(cè)出IC50值，如表1所示.合成所得的化合物C1～C10活性都要高于陽性對(duì)照藥卡鉑.C9和C10這兩個(gè)衍生物，芳烴衍生物C9活性要好于環(huán)烷烴衍生物C10.在直鏈烴衍生物中，羥基衍生物C4和C8活性最強(qiáng).

表1　雷公藤紅素衍生物的IC50

3結(jié)論

本實(shí)驗(yàn)對(duì)具有較強(qiáng)抗癌活性的木栓烷型三萜化合物雷公藤紅素C-29-羧基進(jìn)行修飾，得到了10個(gè)新型的衍生物，并對(duì)衍生物進(jìn)行抗癌活性測(cè)定，采用人肺癌細(xì)胞株A549和人肝癌細(xì)胞株HepG2，運(yùn)用MTT法算出了IC50.衍生物C1～C4以及C6～C8都表現(xiàn)出了比雷公藤紅素強(qiáng)的抗癌活性，引入了鹵素、苯環(huán)、羥基都明顯提升了化合物抗癌活性，而引入六圓環(huán)化合物活性有明顯下降.衍生物中化合物C4和C8抗癌活性最強(qiáng)，且C4和C8是衍生物中唯一兩個(gè)極性大于紅素的衍生物，可以作為未來體內(nèi)實(shí)驗(yàn)的候選藥物.

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(責(zé)任編輯：陳石平)

文章編號(hào)：1006-4303(2015)06-0607-04

中圖分類號(hào)：O626.4

文獻(xiàn)標(biāo)志碼：A

作者簡(jiǎn)介：?jiǎn)蝹ス?1961—)，男，浙江寧波人，教授，博士生導(dǎo)師，主要從事天然產(chǎn)物化學(xué)以及藥品質(zhì)量控制標(biāo)準(zhǔn)的研究，E-mail:swg@zjut.edu.cn.

基金項(xiàng)目：國(guó)家自然科學(xué)基金資助項(xiàng)目(20702049)

收稿日期：2015-04-15