CN116854704A - 具有抗肝癌活性的瑞香烷二萜衍生物及其制备方法和用途 - Google Patents
具有抗肝癌活性的瑞香烷二萜衍生物及其制备方法和用途 Download PDFInfo
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Abstract
具有抗肝癌活性的瑞香烷二萜衍生物及其制备方法和用途,涉及植物医药技术领域,所述具有抗肝癌活性的瑞香烷二萜衍生物是指YHE噁二唑衍生物和YHD噁二唑衍生物,所述YHE噁二唑衍生物具有式(I)和式(II)所示的化学结构,所述YHD噁二唑衍生物具有式(III)和式(IV)所示的化学结构;与传统的提取分离方法相比,本发明对其提取分离工艺进行了优化采用浸膏碱水解的方法富集到了大量的瑞香烷型原酸酯类二萜,为提取分离过程提供了新思路,同时本发明还为本领域提供了新的以YHD、YHE为母核噁二唑衍生物,为制备抗原发性肝癌感染或治疗原发性肝癌的药物提供了先导化合物。
Description
技术领域
本发明涉及植物医药技术领域,涉及芫花中瑞香烷型二萜的制备方法和应用,尤其涉及药用植物芫花的花蕾中具有抗肿瘤活性的12-O-debenzoyl-yuanhuacine噁唑衍生物及12-hydroxydaphnetoxin噁唑衍生物的制备和应用。
背景技术
肝癌是世界上最常见的肿瘤之一,也是癌症相关死亡的主要原因,原发性肝癌(Hepatocellular carcinoma,HCC)是最常见的癌症形式,约占病例的90%。其病因包括病毒性肝炎(乙型和丙型)、酒精、肥胖、饮食致癌物等。目前的治疗方式,包括手术切除和肝移植,均被发现效果不佳。并且靶向治疗仅限于索拉非尼、乐伐替尼、瑞戈非尼、雷莫芦单抗和卡博替尼,这有利于延长患者的生存期,但具有耐药性(European Journal of MedicinalChemistry,2021,224:113690)。因此,迫切需要开发替代治疗策略。
1,3,4-噁二唑衍生物作为抗有丝分裂药物已成功用于治疗癌症。大多数抗有丝分裂药物以微管为靶点,微管是细胞骨架中负责有丝分裂纺锤体形成的动态元件,是细胞分裂过程中染色体分离所必需的。噁唑衍生物可以抑制微管蛋白聚合,阻止肿瘤细胞有丝分裂,从而起到抗肿瘤的活性(Bioorganic&Medicinal Chemistry Letters,2006,16:1191-1196)。
芫花(Daphne genkwa Sieb.et Zucc.)为瑞香科(Thymelaeaceae)瑞香属(DaphneLinn.)植物。芫花资源储量丰富,广泛分布于山东、河南、陕西以及长江流域各省区。芫花具有泻水逐饮、祛痰止咳和解毒杀虫等功效,民间主要运用该药进行引产、抗早孕以及治疗多种疑难病症。瑞香烷型原酸酯类二萜是芫花中的特征性成分,具有多样的生物活性,特别是在抗肿瘤方面的作用而备受关注(Bioorganic&Medicinal Chemistry Letters,2013,23:2500-2504)。芫花中主要代表化合物有芫花酯甲、芫花烯,但目前芫花酯甲、芫花烯含量有限,其衍生物类型少,缺少相应的抗肝癌的先导化合物,这限制了其在药物研发中的应用。12-O-debenzoyl-yuanhuacine(YHD)、12-hydroxydaphnetoxin(YHE)是芫花干燥花蕾中的芫花烯、芫花酯甲或相同骨架C12不同酰基取代化合物的水解产物,分子中存在5/7/6三环骨架,具有高度氧化性、多酯键以及多个手性中心,在C9、C13、C14位置的手性羟基之间形成特定的原酸酯结构。
YHD、YHE具有显著的生物活性,以该化合物为起始原料进行半合成修饰在医药工业上具有广阔的应用前景。通过调研文献,以YHD、YHE为母核,基于活性片段组合设计合成了一系列具有显著抑制肝细胞癌活性的YHD噁二唑衍生物和YHE噁二唑衍生物,它不同于目前已知的肝癌抑制剂。
发明内容
为解决上述技术问题,本发明以YHD、YHE为母核制备了一系列具有较高抗肝癌活性的噁二唑衍生物或其药学上可接受的盐,并进一步提供了包含所述衍生物或其药学上可接受的盐的药物组合物,以及所述衍生物或其药学上可接受的盐的制备方法。此外,还提供了所述衍生物或其药学上可接受的盐或者其药物组合物在制备抗原发性肝癌感染或治疗原发性肝癌的药物中的用途。
具体地,通过以下几个方面的技术方案实现了本发明:
本发明提供了一种具有抗肝癌活性的瑞香烷二萜衍生物物,其为YHE噁二唑衍生物和YHD噁二唑衍生物;其中,所述YHE噁二唑衍生物具有式(I)和式(II)所示的化学结构;所述YHD噁二唑衍生物具有式(III)和式(IV)所示的化学结构。
所述式(Ⅰ)和式(Ⅲ)中的R选自:
所述式(Ⅱ)和式(Ⅳ)中的R选自:
优选地,所述具有抗肝癌活性的YHD、YHE瑞香烷型原酸酯类二萜衍生物选自以下化合物或其在药学上可接受的盐:
本发明还提供所述的具有抗肝癌活性的YHD、YHE瑞香烷二萜衍生物的制备方法,包括如下步骤:
以a1-a12为起始原料经过步骤i得到中间体b1-b12,再经过步骤ii得到片段c1-c12,最后经过步骤iii拼合YHD、YHE,得到化合物YHD1-12,YHE1-10。
具体的,所述制备方法包括下述步骤:
(1)b1-b12的合成:将a1-a12溶于溶剂中加入1.5-3倍量HOBt,1.5-3倍量EDC搅拌0.5-4小时,滴加2-4倍量水合肼,室温反应,除去溶剂,萃取,洗涤,干燥,抽滤,纯化;
(2)c1-c12的合成:将b1-b12溶于溶剂中加入1-3倍量三甲氧基氯乙烷和3-5倍量醋酸,110-130℃反应,后处理方式同(1);
(3)YHD1-12的合成:将YHD和碱溶于溶剂中,室温下搅拌,加入相应的噁二唑片段,室温反应,除去溶剂,萃取,洗涤,干燥,抽滤,纯化;
(4)YHE1-10的合成:将YHE和碱溶于溶剂中,室温下搅拌,加入相应的噁二唑片段,室温反应,后处理方式同(3)。
上述制备方法,其中:
所述溶剂选自无水乙腈,N,N-二甲氨基甲酰胺,1,4-二氧六环;
所述碱选自无水氢氧化钠,无水氢氧化钾,无水碳酸铯,无水氢化钠。
一种药物组合物,包含所述具有抗肿瘤活性YHD、YHE噁唑衍生物或其在药学上可接受的盐以及药学上可接受的载体。
所述具有抗肿瘤活性的YHD、YHE噁唑衍生物或其在药学上可接受的盐以及所述药物组合物在制备抗原发性肝癌感染或治疗原发性肝癌的药物中的应用。
本发明的有益效果:
(1)与传统的提取分离方法相比,本发明对其提取分离工艺进行了优化采用浸膏碱水解的方法富集到了大量的瑞香烷型原酸酯类二萜,为提取分离过程提供了新思路。
(2)为本领域提供了新的以YHD、YHE为母核噁二唑衍生物,为制备抗原发性肝癌感染或治疗原发性肝癌的药物提供了先导化合物。
附图说明
图1化合物YHD1对Hep3B细胞的抑制活性;
图2化合物YHD1诱导肝癌细胞G0/G1周期阻滞;
图3化合物YHD1诱导肝癌细胞凋亡;
图4化合物YHD1诱导肝癌细胞线粒体损伤。
具体实施方式
下面参照具体的实施例对本发明做进一步说明。应当理解,此处所描述的具体实施例仅用于解释本发明,并不用于限定本发明的范围。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道购买得到的常规产品。
制备YHD、YHE的方法:
1、将10kg芫花干燥花蕾以95%乙醇冷浸提取3次,浓缩得到浸膏,以水溶解,用石油醚萃取3-5次,取石油醚层减压浓缩;
2、石油醚层以甲醇溶解,大孔吸附树脂HP20拌样,分别用80%、95%浓度的乙醇洗脱,收集95%乙醇馏分,减压浓缩得到馏分Fr.3;
3、馏分Fr.3用无水甲醇溶解,分批次加入氢氧化钡,体系pH保持为8-9,TLC监测反应,减压浓缩后以水溶解,石油醚萃取3-5次,得石油醚层;
4、石油醚层减压浓缩用甲醇溶解,硅胶拌样,以二氯甲烷甲醇体系洗脱,收集二氯甲烷甲醇比30∶1、20∶1和10∶1馏分,减压浓缩得到Fr.d-f;
5、使用RP-HPLC,以65%乙腈水溶液的条件制备得到活性母核YHD共800mg;以40%乙腈水的条件制备得到活性母核YHE共200mg。
YHD:浅黄色油状,1H NMR(600MHz,Chloroform-d)δ7.59(s,1H),6.64(dd,J=15.4,10.6Hz,1H),6.03(m,1H),5.84(m,1H),5.63(d,J=15.4Hz,1H),5.11(s,1H),5.10(s,1H),4.72(d,J=2.5Hz,1H),4.24(s,1H),3.90(m,2H),3.83(m,1H),3.76(d,J=12.7Hz,1H),3.74(d,J=2.4Hz,1H),3.53(s,1H),2.48(d,J=7.3Hz,1H),2.08(m,2H),1.87(s,3H),1.80(brs,3H),1.37(q,J=7.3Hz,2H),1.25–1.28(m,4H),1.20(d,J=7.3Hz,3H),0.88(t,J=7.0Hz,3H).13C NMR(150MHz,Chloroform-d)δ210.0,161.0,144.6,139.2,136.6,134.9,128.8,122.8,116.8,113.2,84.9,80.6,78.6,77.1,72.8,71.8,65.1,64.3,60.8,47.7,45.3,34.8,32.7,31.4,28.8,22.6,18.9,18.8,14.1,10.1.HR-ESI-MS:m/z calcd forC30H41O9[M+H]+545.2700found 545.2730.
YHE:浅黄色油状,1H NMR(600MHz,Chloroform-d)δ7.71(m,2H),7.61(s,1H),7.38(m,3H),5.14(m,2H),4.87(d,J=2.4Hz,1H),4.27(s,1H),3.99(s,1H),3.94(m,1H),3.92(d,J=12.4Hz,1H),3.83(d,J=2.5Hz,1H),3.79(d,J=12.4Hz,1H),3.59(s,1H),2.57(q,J=7.3Hz,1H),1.92(s,3H),1.80(brs,3H),1.28(d,J=7.3Hz,3H).13C NMR(150MHz,Chloroform-d)δ209.9,161.0,145.0,136.9,135.7,129.7,128.2,126.2,117.7,113.1,85.7,81.3,79.0,77.1,72.4,72.2,65.2,64.6,60.7,47.8,44.9,35.1,19.1,19.0,10.1.HR-ESI-MS:m/z calcd for C27H30O9Na[M+Na]+521.1788found 521.1788.
实施例1:化合物YHD5的制备:
取10mL反应瓶,将YHD(9.8mg,0.02mmoL),氢化钠(2.15mg,0.09mmoL)置于其中,在冰水浴下加入1mL干燥DMF溶解,室温搅拌5分钟,加入相应噁二唑取代物(11.47mg,0.05mmoL),室温下反应2小时。TLC监测反应完成,加入1mL水淬灭反应,加入2mL乙酸乙酯萃取三次,饱和氯化钠溶液洗涤有机相,无水硫酸钠干燥后减压除去溶剂,用RP-HPLC纯化得到YHD5,结构鉴定数据如下:
YHD5:无色油状,1H NMR(600MHz,Chloroform-d)δ8.08(m,2H),7.51(s,1H),7.21(m,2H),6.65(dd,J=15.4,10.7Hz,1H),6.04(m,1H),5.85(m,1H),5.65(d,J=15.4Hz,1H),5.51(d,J=13.3Hz,1H),5.10(m,3H),4.70(d,J=2.6Hz,1H),4.18(s,1H),4.11(d,J=12.1Hz,1H),4.04(m,1H),3.90(s,1H),3.72(d,J=2.6Hz,1H),3.54(d,J=12.1Hz,1H),3.45(s,1H),2.57(q,J=7.3Hz,1H),2.09(q,J=7.2Hz,2H),1.87(s,2H),1.78(brs,3H),1.38(m,2H),1.29–1.25(m,4H),1.20(d,J=7.3Hz,3H),0.89(t,J=6.9Hz,3H).13C NMR(150MHz,Chloroform-d)δ207.5,164.7,164.3,163.5,158.9,145.0,139.3,136.5,135.0,129.6,129.5,128.8,122.9,120.0,116.9,116.7,116.6,113.0,85.2,80.7,78.8,78.7,77.1,74.1,66.2,65.9,64.7,62.4,47.2,44.7,34.6,32.8,31.4,28.9,22.6,19.0,18.9,14.2,10.2.HR-ESI-MS:m/z calcd for C39H45N2O10FNa[M+Na]+743.2956found743.2964.
实施例2:化合物YHD8的制备:
取10mL反应瓶,将YHD(12.4mg,0.02mmoL),氢化钠(2.7mg,0.11mmoL)置于其中,在冰水浴下加入1mL干燥DMF溶解,室温搅拌5分钟,加入相应噁二唑取代物(17.4mg,0.06mmoL),室温下反应2小时。TLC监测反应完成,加入1mL水淬灭反应,加入2mL乙酸乙酯萃取三次,饱和氯化钠溶液洗涤有机相,无水硫酸钠干燥后减压除去溶剂,用RP-HPLC纯化得到YHD8,结构鉴定数据如下:
YHD8:无色油状,1H NMR(600MHz,Chloroform-d)δ7.51(m,4H),7.39(m,2H),7.01(d,J=16.4Hz,1H),6.65(dd,J=15.5,10.7Hz,1H),6.04(dd,J=15.2,10.7Hz,1H),5.85(dt,J=14.7,7.0Hz,1H),5.65(d,J=15.4Hz,1H),5.47(d,J=13.2Hz,1H),5.09(m,3H),4.70(d,J=2.6Hz,1H),4.18(s,1H),4.12(d,J=12.1Hz,1H),4.05(m,1H),3.89(s,1H),3.71(d,J=2.6Hz,1H),3.53(d,J=12.1Hz,1H),3.45(s,1H),2.58(q,J=7.3Hz,1H),2.09(q,J=8.0,7.6Hz,2H),1.87(s,3H),1.80(brs,3H),1.38(m,2H),1.29-1.25(m,4H),1.21(d,J=7.3Hz,3H),0.88(t,J=7.0Hz,3H).13C NMR(150MHz,Chloroform-d)δ207.5,164.9,163.0,158.9,145.1,139.3,138.5,136.6,136.2,135.0,133.2,129.5×2,128.9×2,128.8,122.8,117.0,112.9,110.3,85.3,80.7,78.8,78.4,77.1,74.1,66.1,65.8,64.6,62.4,47.2,44.7,34.6,32.8,31.4,28.9,22.6,19.1,18.9,14.2,10.2.HR-ESI-MS:m/zcalcd for C41H47N2O10NaCl[M+Na]+785.2817,found 785.2810.
实施例3:化合物YHD9的制备:
取10mL反应瓶,将YHD(9.8mg,0.02mmoL),氢化钠(2.15mg,0.09mmoL)置于其中,在冰水浴下加入1mL干燥DMF溶解,室温搅拌5分钟,加入相应噁二唑取代物(9.85mg,0.05mmoL),室温下反应2小时。TLC监测反应完成,加入1mL水淬灭反应,加入2mL乙酸乙酯萃取三次,饱和氯化钠溶液洗涤有机相,无水硫酸钠干燥后减压除去溶剂,用RP-HPLC纯化得到YHD9,结构鉴定数据如下:
YHD9:无色油状,1H NMR(600MHz,Chloroform-d)δ7.51(s,1H),7.48(d,J=16.3Hz,
1H),7.07(d,J=1.7Hz,1H),7.02(dd,J=8.1,1.7Hz,1H),6.85(m,2H),6.65(dd,J=15.4,10.6Hz,1H),6.03(m,3H),5.85(m,1H),5.65(d,J=15.4Hz,1H),5.48(d,J=13.3Hz,1H),5.08(m,3H),4.70(d,J=2.6Hz,1H),4.17(s,1H),4.08(d,J=12.1Hz,1H),3.90(s,1H),3.72(d,J=2.6Hz,1H),3.54(d,J=12.1Hz,1H),3.45(s,1H),2.57(q,J=7.3Hz,1H),2.09(q,J=6.8Hz,2H),1.87(s,3H),1.79(brs,3H),1.38(m,2H),1.29-1.25(m,4H),1.20(d,J=7.3Hz,3H),0.88(t,J=7.0Hz,3H).13C NMR(150MHz,Chloroform-d)δ207.5,165.3,162.8,158.9,149.6,148.7,145.0,140.1,139.5,139.3,136.5,135.0,129.2,128.8,124.0,122.9,116.9,113.0,108.8,107.8,106.2,101.8,85.2,80.7,78.8,78.7,77.1,74.1,66.2,65.9,64.6,62.4,47.2,32.8,31.4,28.9,22.6,19.0,18.9,14.2,10.2.HR-ESI-MS:m/z calcd for C42H48N2O12Na[M+Na]+795.3105,found 795.3098.
实施例4:化合物YHD10的制备:
取10mL反应瓶,将YHD(14.0mg,0.02mmoL),氢化钠(3.5mg,0.14mmoL)置于其中,在冰水浴下加入1mL干燥DMF溶解,室温搅拌5分钟,加入相应噁二唑取代物(27mg,0.08mmoL),室温下反应2小时。TLC监测反应完成,加入1mL水淬灭反应,加入2mL乙酸乙酯萃取三次,饱和氯化钠溶液洗涤有机相,无水硫酸钠干燥后减压除去溶剂,用RP-HPLC纯化得到YHD10,结构鉴定数据如下:
YHD10:无色油状,1H NMR(600MHz,Chloroform-d)δ7.57(m,3H),7.51(s,1H),7.42(m,3H)7.04(d,J=16.5Hz,1H),6.65(dd,J=15.4,10.7Hz,1H),6.04(m,1H),5.84(m,1H),5.65(d,J=15.4Hz,1H),5.49(d,J=13.3Hz,1H),5.09(m,3H),4.70(d,J=2.6Hz,1H),4.17(s,1H),4.09(d,J=12.1Hz,1H),4.03(m,1H),3.91(s,1H),3.73(d,J=2.6Hz,1H),3.55(d,J=12.1Hz,1H),3.45(s,1H),2.57(q,J=7.3Hz,1H),2.09(q,J=8.0,7.6Hz,3H),1.87(s,3H),1.79(brs,3H),1.38(m,2H),1.29–1.25(m,4H),1.20(d,J=7.3Hz,3H),0.89(t,J=6.8Hz,3H).13C NMR(150MHz,Chloroform-d)δ207.5,165.1,163.0,158.9,144.9,139.9,139.3,136.5,135.0,134.7,130.3×2,129.2,128.8,127.8×2,122.9,116.9,113.0,109.7,85.2,80.6,78.7,78.7,77.1,74.1,66.2,65.9,64.6,62.4,47.2,44.8,34.6,32.8,31.4,28.9,22.6,19.0,18.9,14.2,10.2.HR-ESI-MS:m/z calcd for C41H48N2O10Na[M+Na]+751.3207,found 751.3212.
实施例5:化合物YHD12的制备:
取10mL反应瓶,将YHD(14.9mg,0.02mmoL),氢化钠(3.26mg,0.14mmoL)置于其中,在冰水浴下加入1mL干燥DMF溶解,室温搅拌5分钟,加入相应噁二唑取代物(21.26mg,0.08mmoL),室温下反应2小时。TLC监测反应完成,加入1mL水淬灭反应,加入2mL乙酸乙酯萃取三次,饱和氯化钠溶液洗涤有机相,无水硫酸钠干燥后减压除去溶剂,用RP-HPLC纯化得到YHD12,结构鉴定数据如下:
YHD12:黄色油状,1H NMR(600MHz,Chloroform-d)δ7.47(m,4H),6.78(d,J=16.3Hz,
1H),6.71(d,J=8.4Hz,2H),6.65(m,1H),6.04(dd,J=15.4,10.9Hz,1H),5.84(m,1H),5.64(dd,J=15.5,3.9Hz,1H),5.49(d,J=13.3Hz,1H),5.10(m,3H),5.02(d,J=13.5Hz,1H),4.69(d,J=2.6Hz,1H),4.14(s,1H),4.01(m,2H),3.92(s,1H),3.75(d,J=2.6Hz,1H),3.58(d,J=12.2Hz,1H),3.45(s,1H),3.03(s,6H),2.56(m,1H),2.09(q,J=6.9Hz,3H),1.86(s,3H),1.78(brs,3H),1.38(m,3H),1.29–1.25(m,4H),1.20(d,J=7.3Hz,3H),0.88(t,J=6.9Hz,3H).13C NMR(150MHz,Chloroform-d)δ207.5,166.0,162.3,158.9,144.8,140.2,139.2,136.5,134.9,132.0,129.3×2,128.9×2,123.0,116.9,113.1,112.2,104.3,85.2,80.6,79.0,78.8,77.1,74.2,66.4,66.0,64.6,62.3,47.2,44.8,40.4×2,34.6,32.8,31.4,28.9,22.6,19.0,18.8,14.2,10.2.HR-ESI-MS:m/z calcd forC43H54N3O10[M+Na]+772.3809,found 772.3806.
实施例6:化合物YHE1的制备:
取10mL反应瓶,将YHE(19mg,0.03mmoL),氢化钠(4.55mg,0.19mmoL)置于其中,在冰水浴下加入1mL干燥DMF溶解,室温搅拌5分钟,加入相应噁二唑取代物(11mg,0.06mmoL),室温下反应2小时。TLC监测反应完成,加入1mL水淬灭反应,加入2mL乙酸乙酯萃取三次,饱和氯化钠溶液洗涤有机相,无水硫酸钠干燥后减压除去溶剂,用RP-HPLC纯化得到YHE1,结构鉴定数据如下:
YHE1:无色油状,1H NMR(600MHz,Chloroform-d)δ7.71(m,2H),7.53(s,1H),7.49(d,J=16.3Hz,1H),7.38(m,3H),7.07(d,J=1.7Hz,1H),7.03(dd,J=8.0,1.7Hz,1H),6.86(d,J=10.3Hz,1H),6.84(d,J=1.9Hz,1H),6.03(s,2H),5.49(d,J=10.2Hz,1H),5.13(m,2H),5.08(d,J=10.2Hz,1H),4.84(d,J=2.4Hz,1H),4.20(s,1H),4.16(m,1H),4.11(d,J=12.1Hz,1H)3.97(s,1H),3.81(d,J=2.5Hz,1H),3.55(d,J=12.1Hz,1H),3.51(s,1H),2.66(q,J=7.3Hz,1H),1.92(s,3H),1.80(brs,3H),1.28(d,J=7.2Hz,3H).13C NMR(150MHz,Chloroform-d)δ207.5,165.3,162.8,158.9,149.6,148.7,145.0,139.6,136.6,135.8,129.7,129.2,128.2×2,126.2×3,124.0,117.8,113.1,108.8,106.2,101.8,85.7,81.0,79.2,78.7,77.1,74.1,66.2,65.9,64.6,62.4,47.2,44.8,34.7,19.1,19.0,10.2.HR-ESI-MS:m/z calcd for C39H38N2O12Na[M+Na]+749.2322,found 749.2322.
实施例7:化合物YHE2的制备:
取10mL反应瓶,将YHE(13.7mg,0.02mmoL),氢化钠(3.28mg,0.13mmoL)置于其中,在冰水浴下加入1mL干燥DMF溶解,室温搅拌5分钟,加入相应噁二唑取代物(17.53mg,0.08mmoL),室温下反应2小时。TLC监测反应完成,加入1mL水淬灭反应,加入2mL乙酸乙酯萃取三次,饱和氯化钠溶液洗涤有机相,无水硫酸钠干燥后减压除去溶剂,用RP-HPLC纯化得到YHE2,结构鉴定数据如下:
YHE2:无色油状,1H NMR(600MHz,Chloroform-d)δ8.08(m,2H),7.71(m,2H),7.53(s,1H),7.38(m,3H),7.21(m,2H),5.52(d,J=13.3Hz,1H),5.13(m,3H),4.84(d,J=2.6Hz,1H),4.21(s,1H),4.14(m,2H),3.97(s,1H),3.81(d,J=2.6Hz,1H),3.55(d,J=12.1Hz,1H),3.52(s,1H),2.65(q,J=7.3Hz,1H),1.91(s,3H),1.78(brs,3H),1.26(d,J=7.3Hz,3H).13C NMR(150MHz,Chloroform-d)δ207.4,165.8,164.1,158.8,144.8,136.5,135.6,129.6,129.4,129.4,128.1×2,126.1×2,119.9,119.9,117.6,116.6,116.4,113.0,85.6,80.9,79.0,78.6,77.1,74.0,66.1,65.8,64.6,62.3,47.1,44.6,34.6,18.9,18.8,10.1.HR-ESI-MS:m/z calcd for C36H35N2O10NaF[M+Na]+697.2173,found 697.2177.
实施例8:化合物YHE-6的制备:
取10mL反应瓶,将YHE(18.4mg,0.02mmoL),氢化钠(4.4mg,0.18mmoL)置于其中,在冰水浴下加入1mL干燥DMF溶解,室温搅拌5分钟,加入相应噁二唑取代物(29.4mg,0.11mmoL),室温下反应2小时。TLC监测反应完成,加入1mL水淬灭反应,加入2mL乙酸乙酯萃取三次,饱和氯化钠溶液洗涤有机相,无水硫酸钠干燥后减压除去溶剂,用RP-HPLC纯化得到YHE6,结构鉴定数据如下:
YHE6:无色油状;
1H NMR(600MHz,Chloroform-d)δ8.28(m,2H),7.72(m,3H),7.63(d,J=16.5Hz,1H),7.54(s,1H),7.38(m,3H),7.19(d,J=16.5Hz,1H),5.49(d,J=13.3Hz,1H),5.14(m,3H),4.84(d,J=2.6Hz,1H),4.21(s,1H),4.15(m,2H),3.97(s,1H),3.80(d,J=2.6Hz,1H),3.52(d,J=12.8Hz,2H),2.66(q,J=7.3Hz,1H),1.92(s,3H),1.80(brs,3H),1.26(d,J=7.3Hz,3H).
13C NMR(150MHz,Chloroform-d)δ207.5,164.2,163.5,159.0,148.5,144.9,140.8,137.0,136.6,135.7,129.7,128.3×2,128.2×2,126.2×2,124.5×2,117.8,113.9,113.1,85.7,81.0,79.2,78.5,77.1,74.1,66.0,65.9,64.7,62.5,47.2,44.7,34.7,19.1,19.0,10.2.
HR-ESI-MS:m/z calcd for C38H37N3O12Na[M+Na]+750.2275,found 750.2268.
实施例9:化合物YHE9的制备:
取10mL反应瓶,将YHE(14mg,0.02mmoL),氢化钠(3.07mg,0.12mmoL)置于其中,在冰水浴下加入1mL干燥DMF溶解,室温搅拌5分钟,加入相应噁二唑取代物(17mg,0.07mmoL),室温下反应2小时。TLC监测反应完成,加入1mL水淬灭反应,加入2mL乙酸乙酯萃取三次,饱和氯化钠溶液洗涤有机相,无水硫酸钠干燥后减压除去溶剂,用RP-HPLC纯化得到YHE9,结构鉴定数据如下:
YHE9:无色油状;
1H NMR(600MHz,Chloroform-d)δ7.71(m,2H),7.57(m,3H),7.53(s,1H),7.40(m,6H),7.04(d,J=16.5Hz,1H),5.51(d,J=13.3Hz,1H),5.13(s,2H),5.10(d,J=13.3Hz,1H),4.84(d,J=2.6Hz,1H),4.20(s,1H),4.15(m,1H),4.11(d,J=12.1Hz,1H),3.98(s,1H),3.82(d,J=2.6Hz,1H),3.56(d,J=12.1Hz,1H),3.52(s,1H),2.66(q,J=7.3Hz,1H),1.90(s,3H),1.79(brs,3H),1.26(d,J=7.4Hz,3H).
13C NMR(150MHz,Chloroform-d)δ207.5,165.1,163.0,158.9,144.9,139.9,136.6,135.8,134.7,130.3,129.7,129.2×2,128.2×2,127.8×2,126.2×2,117.8,113.2,109.7,85.7,81.0,79.2,78.7,77.1,74.2,66.2,66.0,64.7,62.5,47.2,44.8,34.7,19.1,19.0,10.2.
HR-ESI-MS:m/z calcd for C38H38N2O10Na[M+Na]+705.2424,found 705.2421.
实施例10:化合物YHE10制备:
取10mL反应瓶,将YHE(13.6mg,0.02mmoL),氢化钠(3.25mg,0.13mmoL)置于其中,在冰水浴下加入1mL干燥DMF溶解,室温搅拌5分钟,加入相应噁二唑取代物(21.58mg,0.08mmoL),室温下反应2小时。TLC监测反应完成,加入1mL水淬灭反应,加入2mL乙酸乙酯萃取三次,饱和氯化钠溶液洗涤有机相,无水硫酸钠干燥后减压除去溶剂,用RP-HPLC纯化得到YHE10,结构鉴定数据如下:
YHE10:亮黄色油状;
1H NMR(600MHz,Chloroform-d)δ7.71(m,2H),7.52(s,1H),7.49(d,J=16.2Hz,1H),7.45(m,2H),7.38(m,3H),6.79(d,J=16.2Hz,1H),6.72(d,J=8.3Hz,2H),5.50(d,J=13.4Hz,1H),5.13(m,2H),5.04(d,J=13.4Hz,1H),4.84(d,J=2.6Hz,1H),4.17(s,1H),4.14(m,J=2.7Hz,1H),4.05(d,J=12.1Hz,1H),3.99(s,1H),3.83(d,J=2.6Hz,1H),3.59(d,J=12.1Hz,1H),3.52(s,1H),3.03(s,6H),,2.65(q,J=7.4Hz,1H),1.91(s,3H),1.78(brs,3H),1.26(d,J=7.3Hz,3H).
13C NMR(150MHz,Chloroform-d)δ207.5,166.0,162.4,158.9,151.6,144.8,140.2,136.6,135.8,129.6,129.3×2,128.2×3,126.2×3,117.7,113.2,112.2,104.3,85.7,81.0,79.2,79.0,77.1,74.2,66.4,66.0,64.7,62.4,47.2,44.9,40.4×2,34.7,19.1,19.0,10.2.
HR-ESI-MS:m/z calcd for C40H43N3O10Na[M+Na]+748.2846,found 748.2845.
实施例11:抗肝癌活性筛选
为了证明本发明所述的YHD、YHE噁二唑衍生物具有潜在治疗肝癌的效果,对制备的化合物进行了肝癌药效学筛选。具体的抗肝癌化合物筛选方法及结果,如下所示:
(1)体外活性初筛
利用MTT法进行化合物初筛,以索拉非尼(sorafenib)作为阳性对照。选择生长状态良好的肝癌细胞或正常肝细胞,胰蛋白酶消化,用细胞完全培养基分散成单细胞悬液,进行细胞计数,调整细胞浓度5×104个/mL,轻轻吹打细胞使其均匀分散,均匀铺于96孔板中(100μL/孔),CO2培养箱中培养过夜。第二天弃掉培养基,分别加入指定浓度的药物,并设置空白组以扣除背景的影响,每组设3个复孔。药物于CO2培养箱中作用72小时后,取出96孔板,以提前配制好的MTT溶液,20μL/孔,于培养箱中继续孵育4小时后。随后弃掉MTT溶液,每孔加入150μL DMSO溶液溶解甲瓒,室温振荡5分钟使结晶充分溶解。酶标仪调至OD 490nm处,检测各孔的吸光度值,实验重复3次。细胞生长抑制率按照如下公式计算:抑制率(%)=(A对照-A加药)/(A对照-A空白)×100%。计算不同浓度下的抑制率,并绘制量-效曲线。计算生长抑制率50%时的浓度,即为IC50值。结果表明大部分化合物具有相当程度抑制肝癌细胞增殖的活性,如表1所示。YHD、YHE、YHD1以及sora的生物选择指数,如表2所示。
表1 YHDs与YHEs的抗肝癌细胞增殖活性
a N.T.代表未检测.
表2YHD/YHE/YHD-1生物选择指数(SI)
a L-O2/HepG2或L-O2/Hep3B或L-O2/SMMC7721
(2)YHD1抑制肝癌细胞活力
为了进一步阐明化合物YHD1对肝癌细胞Hep3B的抑制作用,我们以YHC作为对照组,考察化合物YHD1在不同浓度下的Hep3B细胞的存活率,化合物YHD1在72小时作用下能浓度依赖性地抑制Hep3B细胞的活力,并且与相同作用浓度的YHC相比,化合物YHD1对Hep3B细胞的抑制活性更好,如图1所示。
(3)YHD1诱导肝癌细胞G0/G1周期阻滞
如图2所示,与对照组相比,Hep3B在不同浓度化合物YHD1处理后,细胞周期进程被显著地阻滞在G0/G1期,且呈浓度依赖性。
(4)YHD1诱导肝癌细胞凋亡
相差结果显示,与对照组相比,加药组中细胞数量呈浓度依赖性逐渐减少,皱缩变圆的细胞数量逐渐增多,漂浮细胞也逐渐增加。AO/EB染色结果显示,随着化合物YHD1浓度的增大,细胞密度减小,绿色荧光逐渐增强。Hoechst 33258染色结果显示,加药组中细胞出现了明显的胞质浓染以及核断裂的现象,并且这种凋亡现象随着给药浓度的加大而加深,证明了化合物YHD1可以诱导细胞发生浓度依赖性地凋亡(图3)。
(5)YHD1诱导肝癌细胞线粒体损伤
使用活性氧探针H2DCFH-DA检测化合物YHD1作用后的Hep3B的活性氧水平,结果显示,随药物浓度的增加,绿色荧光强度会逐渐增强,证明化合物YHD1会诱导活性氧的产生。MitoSOX活性氧指示剂可以选择性地将线粒体活性氧染色,实验结果表明,药物作用后,细胞线粒体活性氧呈浓度依赖性增高,证明化合物YHD1会诱导细胞线粒体产生活性氧。进而我们使用JC-1染色继续研究化合物YHD1对细胞线粒体功能的损伤情况。JC-1染色结果显示,Hep3B细胞中红色荧光随着加药浓度的增加逐渐减少,黄绿色荧光逐渐增加,这表明化合物YHD1对细胞线粒体造成损伤(图4)。
综上所述,通过应用上述药理学实验对肝癌细胞增殖进行抗肿瘤药物筛选,得到了化合物YHD1,发现其对肝癌Hep3B细胞系的抑制活性与阳性药Sora活性相当,具有进一步开发的价值,故本发明中的瑞香烷型原酸酯类二萜的醚衍生物具有良好的抗肝癌细胞增殖活性,具有进一步开发成为治疗肝癌药物的潜力。
Claims (10)
1.一种具有抗肝癌活性的瑞香烷二萜衍生物,其特征在于,所述衍生物具有式(I)、式(II)、式(III)或式(IV)所示的化学结构;
2.根据权利要求1所述的具有抗肝癌活性的瑞香烷二萜衍生物,其特征在于,式(Ⅰ)和式(Ⅲ)中R选自:
3.根据权利要求1所述的具有抗肝癌活性的瑞香烷二萜衍生物,其特征在于,式(Ⅱ)和式(Ⅳ)中R选自:
4.根据权利要求1所述的具有抗肝癌活性的瑞香烷二萜衍生物,其特征在于,所述衍生物为如下化合物:
5.一种权利要求4所述的具有抗肝癌活性的瑞香烷二萜衍生物的制备方法,其特征在于,包括如下步骤:
(1)b1-b12的合成:将a1-a12溶于溶剂中加入1.5-3倍量HOBt,1.5-3倍量EDC搅拌0.5-4小时,滴加2-4倍量水合肼,室温反应,除去溶剂,萃取,洗涤,干燥,抽滤,纯化;
(2)c1-c12的合成:将b1-b12溶于溶剂中加入1-3倍量三甲氧基氯乙烷和3-5倍量醋酸,110-130℃反应,后处理方式同步骤(1);
(3)YHD1-12的合成:将YHD和碱溶于溶剂中,室温下搅拌,加入相应的噁二唑片段,室温反应,除去溶剂,萃取,洗涤,干燥,抽滤,纯化;
(4)YHE1-10的合成:将YHE和碱溶于溶剂中,室温下搅拌,加入相应的噁二唑片段,室温反应,后处理方式同步骤(3);
所述YHD、YHE结构式为:
6.根据权利要求5所述的制备方法,其特征在于,所述YHD、YHE的制备方法如下:
(1)将芫花干燥花蕾以95%乙醇冷浸提取,浓缩得到浸膏,以水溶解,用石油醚萃取3-5次,取石油醚层减压浓缩;
(2)石油醚层以甲醇溶解,大孔吸附树脂HP20拌样,分别用80%、95%浓度的乙醇洗脱,收集95%乙醇馏分,减压浓缩得到馏分Fr.3;
(3)馏分Fr.3用无水甲醇溶解,分批次加入氢氧化钡,体系pH保持为8-9,TLC监测反应,减压浓缩后以水溶解,石油醚萃取3-5次,得石油醚层;
(4)石油醚层减压浓缩用甲醇溶解,硅胶拌样,以二氯甲烷甲醇体系洗脱,收集二氯甲烷甲醇比30∶1、20∶1和10∶1馏分,减压浓缩得到Fr.d-f;
(5)使用RP-HPLC,以65%乙腈水溶液的条件制备得YHD;以40%乙腈水的条件制备得到YHE。
7.一种药物组合物,其特征在于,包含权利要求1-4任一项所述的具有抗肝癌活性的瑞香烷二萜衍生物或其在药学上可接受的盐以及药学上可接受的载体。
8.权利要求1-4任一项所述的具有抗肝癌活性的瑞香烷二萜衍生物或其在药学上可接受的盐或权利要求7所述的药物组合物在制备抗肝癌药物中的应用。
9.权利要求1-4任一项所述的具有抗肝癌活性的瑞香烷二萜衍生物或其在药学上可接受的盐或权利要求7所述的药物组合物在制备治疗肝癌的药物中的应用。
10.根据权利要求9所述的应用,其特征在于,所述肝癌为原发性肝癌。
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