CN114957190B - 南牡蒿素及其药物组合物和其制备方法与应用 - Google Patents
南牡蒿素及其药物组合物和其制备方法与应用 Download PDFInfo
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- CN114957190B CN114957190B CN202210627416.0A CN202210627416A CN114957190B CN 114957190 B CN114957190 B CN 114957190B CN 202210627416 A CN202210627416 A CN 202210627416A CN 114957190 B CN114957190 B CN 114957190B
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Abstract
本发明提供南牡蒿素及其药物组合物和其制备方法与应用,属于药物技术领域。本发明的结构式(I)所示36个倍半萜二聚体,南牡蒿素(arteriopodins,1–36)对人肝癌细胞株HepG2、Huh7和SK‑Hep‑1具有细胞毒活性,能够与可药用载体或赋型剂组成药物组合物,可用于制备抗肝癌药物。
Description
技术领域:
本发明属于药物技术领域。具体地,涉及36个结构新颖的倍半萜二聚体,南牡蒿素(arteriopodins,1–36),及其制备方法和应用,以及以化合物1–36为有效成分的药物组合物在制备抗肝癌的药物中的应用。
背景技术:
肝癌是全球居第七的常见恶性肿瘤,是一个全球性健康问题,其发病隐匿、进展快、预后差,从而导致死亡率高居全球所有癌症死亡率第三位。以索拉非尼为代表的4个化学合成抗肝癌药物主要作用于受体络氨酸激酶,两个免疫治疗药物纳武单抗和帕姆单抗属PD-1抑制剂,雷莫芦单抗为抗血管生成抑制剂,取得了显著的临床疗效,但易产生耐药性和毒副反应,不能满足临床需求。因此迫切需要开发新型有效的抗肝癌药物。天然产物结构丰富、活性多样,是药物发现的重要源泉,特别是一些化合物在抗肿瘤方面显示了独特的优势。
蒿属(Artemisia)作为菊科(Asteraceae)植物中分布最广、种类最多的一个属之一,为一、二年生或多年生草本植物,全世界共380余种,广布于非洲地区和北半球,我国有186种(有82个特有种),44变种,遍布于全国各地。该属植物多具有清热解毒、抗菌消炎、祛风除湿、通经活络、活血和止血等功效。该属中的一些植物如黄花蒿 (A.annua)、艾(A.argyi)、茵陈(A.capillaris)、奇蒿(A.anomala)等是著名的传统中药,常用于治疗疟疾、肝炎、癌症、湿疹、腹泻、跌打损伤和风湿病等疾病。迄今为止,国内外学者已对260余种蒿属植物进行了较深入的化学成分研究,其结构类型涉及单萜、倍半萜及其二聚体、二萜、三萜、黄酮、甾醇等,其中一些化合物具有抗疟疾、抗病毒、抗肿瘤、抗出血、抗凝血、抗氧化和抗溃疡等生物活性。其中倍半萜内酯类是蒿属植物的主要特征成分,其结构类型复杂多样,具有广泛的生物活性。特别是它们的抗肿瘤活性,引起了许多药物化学家的广泛关注。
倍半萜二聚体(sesquiterpenoid dimers)是蒿属植物中一类重要的化学成分,因其新颖的结构以及独特的药理活性受到国内外学者的广泛关注。迄今为止,国内外学者从15种蒿属植物中共发现了101个倍半萜二聚体,结构类型主要涉及愈创木烷倍半萜二聚体(91个)、愈创木烷-桉烷倍半萜二聚体(6个)、愈创木烷-oplopane倍半萜二聚体 (1个)、愈创木烷-艾里莫芬烷倍半萜二聚体(1个)、艾里莫芬烷倍半萜二聚体(1个)、以及杜松烷倍半萜二聚体。在生物合成方面来说,这些倍半萜二聚体是由两个相同或不同的单体倍半萜通过Diels-Alder[4+2]环加成、[2+2]环加成、酯键连接而成。虽然在蒿属植物中发现了大量的倍半萜二聚体,但愈创木倍半萜二聚体是主要类型,同时发现从特定种中发现的倍半萜二聚体数量且结构类型单一。例如:国外的研究小组共得到了 24个愈创木倍半萜二聚体,其中4个来自中亚苦蒿(A.absinthium)、4个分离于大籽蒿 (A.sieversiana)、7个来自艾蒿(A.argyi)、5个来源于A.caruifolia、2个来源于阴地蒿(A.sylvatica)、1个来源于A.leucodes、1个来源于奇蒿(A.anomala)。国内学者从 11中蒿中共分离得到了69个二聚体,其中屠鹏飞教授团队对蒿属植物的研究最多,从中共得到了33个化合物,包括10个愈创木倍半萜二聚体来源于奇蒿(A.anomala)、7 个愈创木倍半萜二聚体分离于中亚苦蒿(A.absinthium)、6个愈创木、5个愈创木-桉烷和1个愈创木-oplopane二聚体来自一支蒿(A.rupestris)、从绿栉齿叶蒿(A.freyniana) 中得到了1个愈创木烷-艾里莫芬和1个艾里莫芬倍半萜二聚体、从艾蒿(A.argyi)分离得到了1个愈创木烷-桉烷倍半萜二聚体、1个愈创木倍半萜二聚体源自大籽蒿(A.sieversiana)。另外,孔令仪教授团队从艾蒿(A.argyi)分离得到了12个愈创木倍半萜二聚体;叶阳教授团队从A.lavandulifolia和A.heptapotamica两种蒿属植物共中分离得到了15个愈创木倍半萜二聚体;姚新生院士团队从黄花蒿(A.annua)中分离得到了1 个杜松烷倍半萜二聚体;Aisa教授团队从大籽蒿(A.sieversiana)中得到了3个愈创木倍半萜二聚体;香港大学Brown等人从多花蒿(A.myriantha)中得到了4个愈创木倍半萜二聚体;冯小章等人从蒌蒿(A.selengensis)中得到了1个愈创木倍半萜二聚体 artselenoide。
南牡蒿(A.eriopoda)是菊科蒿属的一种多年生草本植物,全国各地多有分布。在我国部分地区,南牡蒿可代替中国传统中草药青蒿入药,具有祛风除湿,解毒的功效,常用于治疗风湿关节痛,头痛,浮肿,毒蛇咬伤等。但是一直以来对南牡蒿的研究较少,从中主要分离得到了5个桉烷型倍半萜、2个聚炔、1个谷甾醇和3个脂肪酸。
迄今未见南牡蒿中倍半萜抗肝癌活性报道。迄今为止,现有技术中无南牡蒿素(arteriopodins,1–36)的报道,也没有其作为有效成分的药物组合物的报道,也没有其药物组合物在制备或治疗肝癌药物中的应用报道。
发明内容:
本发明的目的在于提供一类新的具有药用价值如式(I)所示的南牡蒿(arteriopodins,1–36)的制备方法、药物组合物及其应用。本发明前期研究发现,南牡蒿乙醇提取物对三株肝癌细胞HepG2、Huh7和SK-Hep-1具有细胞毒活性,当浓度为200μg/mL时其抑制率为59.4%、58.9%和53.5%。为了阐明南牡蒿的活性成分,本发明以抗肝癌活性为导向,从中共分离鉴定了36个结构新颖的倍半萜二聚体,南牡蒿素(arteriopodins, 1–36)。该类化合物对人肝癌细胞株HepG2、Huh7和SK-Hep-1具有明显的细胞毒活性,能够用于制备抗肝癌药物。
为了实现本发明的上述目的,本发明提供了如下的技术方案:
本发明提供了一系列倍半萜二聚体类化合物,南牡蒿(arteriopodins,1–36),结构如下式(I)所示:
本发明另外提供了制备式I所示的化合物1–36的方法,取干燥的南牡蒿地上部分,粉碎,用3倍量的90%乙醇提取两次,合并提取液减压浓缩后得到的粗浸膏分散于水中,用乙酸乙酯萃取得到乙酸乙酯萃取部分;随后,将乙酸乙酯萃取部分过硅胶柱层析,并用丙酮-石油醚体积比0:100、5:95、10:90、20:80、40:60和100:0梯度洗脱得到8个流分Frs.A-1~A-6;Fr.A-4过MCI gel CHP 20P柱层析,用甲醇-水50:50、70:30、90:10 和100:0洗脱得到了四个亚流分Frs.A-4a-A-4d;Fr.A-4c再使用硅胶柱层析,以丙酮- 石油醚5:95、10:90和20:80划分成五个流分Frs.A-4c-1-A-4c-5;Fr.A-4c-4首先经过 Sephadex LH-20用甲醇-氯仿50:50洗脱,再用Rp-C18柱层析,以甲醇-水50:50、60:40、 70:30和80:20处理,最后经半制备高效液相HPLC在安捷伦XDB-C18柱上以55:45的乙腈-水和80:20甲醇-水纯化得到化合物1、2和3;Fr.A-4c-5经过Rp-C18反相柱层析,甲醇-水50:50、60:40和70:30梯度洗脱得到了流分Frs.A-4c-5a-A-4c-5f;Fr.A-4c-5a 经半制备HPLC在安捷伦XDB-C18柱上以60:40的乙腈-水和82:18甲醇-水反复纯化得到了化合物11、12、13、28、29和36;化合物14、16、17和18是流分Fr.A-4c-5c经过反复的Sephadex LH-20柱层析,用甲醇-氯仿50:50洗脱处理之后,再使用半制备HPLC 在Agilent XDB-C18柱,用以50:50的乙腈-水和75:25洗脱甲醇-水纯化得到;Fr.A-4c-5e 经过Sephadex LH-20柱层析,用甲醇-氯仿50:50洗脱处理之后,再用半制备HPLC在安捷伦XDB-C18柱上以48:52的乙腈-水和78:22甲醇-水纯化得到化合物6和15。Fr.A-5 经MCI gel CHP 20P柱层析,甲醇-水50:50、70:30、90:10和100:0洗脱得到了四个流分Frs.A-5a-A-5d;Fr.A-5c进一步用反相Rp-C18柱层析,用甲醇-水50:50、60:40、70:30和100:0梯度洗脱得到四个亚流分Frs.A-5c-1-A-5c-4;Fr.A-5c-1经反复的Sephadex LH-20柱层析,用甲醇-氯仿50:50处理之后,再用半制备HPLC在Agilent XDB-C18柱,用以50:50的乙腈-水和73:27洗脱甲醇-水纯化得到了化合物4、5、7、8、9和10;Fr. A-5c-2经过硅胶柱层析,以丙酮-氯仿5:95和10:90洗脱得到了五个流分Frs.A-5c-2a- A-5c-2e;Fr.A-5c-2a经过反复的硅胶柱层析以乙酸乙酯-氯仿5:95和10:90洗脱,在过Sephadex LH-20柱层析,用甲醇-氯仿50:50处理之后,在使用半制备HPLC在Agilent XDB-C18柱用以47:53的乙腈-水和75:25洗脱甲醇-水分离纯化得到化合物22、23和 24;Fr.A-5c-2b经过Sephadex LH-20柱层析用甲醇-氯仿50:50处理后,再使用半制备高效液相在安捷伦XDB-C18柱以乙腈-水55:45和甲醇-水80:20为流动相纯化得到化合物27、30、31和33;Fr.A-5c-2c经过Sephadex LH-20柱层析用甲醇-氯仿50:50处理之后,再半制备HPLC在安捷伦XDB-C18柱以乙腈-水48:52和甲醇-水76:24为流动相分离纯化得到化合物19、20和25;Fr.A-5c-3经反复的硅胶柱层析以丙酮-氯仿10:90和 20:80洗脱之后,再过Sephadex LH-20柱层析用甲醇-氯仿50:50处理,最后使用半制备 HPLC在安捷伦XDB-C18柱以乙腈-水50:50和甲醇-水75:25为流动相纯化得到化合物 21、26、32、34和35。
本发明提供了式I所示的化合物1–36在制备抗肝癌药物中的应用,本发明对所述应用的方法没有特殊的限定,选用本领域熟知的方法即可。
本发明同时提供了一种药物组合物,所述药物组合物包括式(I)所示的化合物1-36 中的至少一种和药学上可接受的载体或赋型剂。
并此,还提供了所述的药物组合物在制备抗肝癌药物中的应用。并同时提供了所述的药物组合物的制备方法:采用上述制备化合物的方法制备得到本发明化合物1-36,然后加入可药用载体。
当所述化合物1-36中的至少一种用于制备抗肝癌药物时,本发明优选将所述化合物1-36直接使用,或以药物组合物的形式使用
本发明提供的药物组合物,包括上述化合物1-36中的至少一种和药学上可接受的载体或赋型剂。在本发明中,所述药学上可接受的载体或赋型剂优选为固体、半固体或液体稀释剂、填料以及药物制品辅剂。本发明对所述药学上可接受的载体或赋型剂没有特殊的限定,选用本领域熟知的、对人和动物无毒且惰性的药学上可接受的载体和/或赋型剂即可。
本发明对所述药物组合物的制备方法没有特殊的限定,直接将化合物1-36中的至少一种与药学上可接受的载体或赋型剂混合即可,本发明对所述混合的过程没有特殊的限定,选用本领域熟知的过程能够得到药物组合物即可。
本发明提供了上述技术方案所述药物组合物在制备抗肝癌药物中的应用,对所述应用的方法没有特殊的限定,选用本领域熟知的方法即可。
在本发明中,当所述药物组合物用于制备抗肝癌药物时,所述组合物在药物中的含量优选为0.1~99%;在所述药物组合物中,所述化合物1-36中的至少一种在药物组合物中的含量优选为0.5~90%。本发明的药物组合物优选以单位体重服用量的形式使用。在本发明中,所制备的药物优选可经注射(静注、肌注)和口服两种形式给药。
与现有技术相比,本发明具备如下的优益性:
1.本发明提供了一系列新的倍半萜二聚体类化合物,南牡蒿(arteriopodins,1–36)。
2.本发明提供了制备新化合物1–36的新的方法,该方法原料易得,易于操作,适于工业化生成。
3.本发明提供了新化合物1–36作为有效成分的药物组合物,为新的抗肝癌药物提供了具有较好药用作用的新的药物。
4.本发明的化合物1–36对三株肝癌细胞(HepG2、Huh7、SK-Hep-1)的细胞毒活性均具有较强活性,化合物13、16、31和33对HepG2细胞具有明显的细胞毒活性,其IC50分别为14.3、12.2、17.2和16.0μM,活性与阳性对照索拉非尼(IC50,11.0μM);化合物12、18、27、28、30和32对HepG2细胞具有适中的细胞毒活性,其IC50在20.0 至45.3μM之间。化合物31和33对Huh7细胞具有与阳性对照索拉非尼(IC50,12.3μM) 相当的细胞毒活性,其IC50分别为10.3和18.3μM;化合物13、28和30具有适中的细胞毒活性,其IC50在26.4至39.5μM之间。化合物31和33对SK-Hep-1细胞具有明显的细胞毒活性,其IC50分别为22.3和19.0μM,活性与索拉非尼(IC50,18.1μM)相当;化合物24、28、30和34具有适中的细胞毒活性,其IC50在25.4至42.6μM之间。以上结果表明南牡蒿中分离得到的化合物1-36可作为药物用于治疗肝癌相关的疾病。
附图说明:
图1为本发明化合物1-36的结构式;
图2为是化合物12、14、16、17、19、27、29的X-ray单晶结构示意图。
具体实施方式:
为了更好地理解本发明的实质,下面结合附图,用本发明的试验例和实施例来进一步说明本发明南牡蒿(arteriopodins,1–36)制备方法、结构鉴定、药理作用,以及本发明的制备方法及药物组成,但不以此试验例和实施例来限定本发明。
下面将结合本发明中的实施例,对本发明中的技术方案进行清楚、完整地描述。显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:
本发明倍半萜二聚体,南牡蒿素(arteriopodins,1–36)(化合物1-36)的制备:
取干燥的南牡蒿地上部分15.7kg,粉碎,用3倍量的90%乙醇提取两次,合并提取液减压浓缩后得到的粗浸膏分散于水中,用乙酸乙酯萃取得到乙酸乙酯萃取部分550 g;随后,将乙酸乙酯萃取部分过硅胶柱层析,并用丙酮-石油醚体积比0:100、5:95、10:90、20:80、40:60和100:0梯度洗脱得到8个流分Frs.A-1~A-6;Fr.A-4 80g过MCI gel CHP 20P柱层析,用甲醇-水50:50、70:30、90:10和100:0洗脱得到了四个亚流分 Frs.A-4a-A-4d;14gFr.A-4c再使用硅胶柱层析,以丙酮-石油醚5:95、10:90和20:80 划分成五个流分Frs.A-4c-1-A-4c-5;Fr.A-4c-4首先经过Sephadex LH-20用甲醇-氯仿 50:50洗脱,再用Rp-C18柱层析,以甲醇-水50:50、60:40、70:30和80:20处理,最后经半制备高效液相HPLC在安捷伦XDB-C18柱上以55:45的乙腈-水和80:20甲醇-水纯化得到化合物1(67mg)、2(25mg)和3(17mg);10g Fr.A-4c-5经过Rp-C18反相柱层析,甲醇-水50:50、60:40和70:30梯度洗脱得到了流分Frs.A-4c-5a-A-4c-5f;460 mg Fr.A-4c-5a经半制备HPLC在安捷伦XDB-C18柱上以60:40的乙腈-水和82:18甲醇- 水反复纯化得到了化合物11(4mg)、12(12mg)、13(2mg)、28(2mg)、29(2mg) 和36(5mg);化合物14(70mg)、16(6mg)、17(5mg)和18(70mg)是流分Fr. A-4c-5c(1g)经过反复的Sephadex LH-20柱层析,用甲醇-氯仿50:50洗脱处理之后,再使用半制备HPLC在Agilent XDB-C18柱,用以50:50的乙腈-水和75:25洗脱甲醇- 水纯化得到;Fr.A-4c-5e(2.3g)经过Sephadex LH-20柱层析,用甲醇-氯仿50:50洗脱处理之后,再用半制备HPLC在安捷伦XDB-C18柱上以48:52的乙腈-水和78:22甲醇- 水纯化得到化合物6(4mg)和15(19mg)。
123g Fr.A-5经MCI gel CHP 20P柱层析,甲醇-水50:50、70:30、90:10和100:0 洗脱得到了四个流分Frs.A-5a-A-5d;18g Fr.A-5c进一步用反相Rp-C18柱层析,用甲醇-水50:50、60:40、70:30和100:0梯度洗脱得到四个亚流分Frs.A-5c-1-A-5c-4;2g Fr. A-5c-1经反复的Sephadex LH-20柱层析,用甲醇-氯仿50:50处理之后,再用半制备HPLC 在AgilentXDB-C18柱,用以50:50的乙腈-水和73:27洗脱甲醇-水纯化得到了化合物4 (1mg)、5(1.3mg)、7(15mg)、8(2mg)、9(5mg)和10(10mg);4g Fr.A-5c-2 经过硅胶柱层析,以丙酮-氯仿5:95和10:90洗脱得到了五个流分Frs.A-5c-2a-A-5c-2e;1.5g Fr.A-5c-2a经过反复的硅胶柱层析以乙酸乙酯-氯仿5:95和10:90洗脱,在过 Sephadex LH-20柱层析,用甲醇-氯仿50:50处理之后,在使用半制备HPLC在Agilent XDB-C18柱用以47:53的乙腈-水和75:25洗脱甲醇-水分离纯化得到化合物22(5mg)、 23(3mg)和24(7mg);1.7g Fr.A-5c-2b经过Sephadex LH-20柱层析用甲醇-氯仿50:50 处理后,再使用半制备高效液相在安捷伦XDB-C18柱以乙腈-水55:45和甲醇-水80:20 为流动相纯化得到化合物27(21mg)、30(5mg)、31(31mg)和33(6mg);1g Fr. A-5c-2c经过Sephadex LH-20柱层析用甲醇-氯仿50:50处理之后,再用半制备HPLC在安捷伦XDB-C18柱以乙腈-水48:52和甲醇-水76:24为流动相分离纯化得到化合物19(1 mg)、20(15mg)和25(7mg);2.4g Fr.A-5c-3经反复的硅胶柱层析以丙酮-氯仿10:90 和20:80洗脱之后,再过Sephadex LH-20柱层析用甲醇-氯仿50:50处理,最后使用半制备HPLC在安捷伦XDB-C18柱以乙腈-水50:50和甲醇-水75:25为流动相纯化得到化合物21(18mg)、26(11mg)、32(22mg)、34(22mg)和35(26mg)。
化合物1-36的结构数据:
旋光由Autopol VI旋光仪(Rudolph Research Analytical,Hackettstown,USA)测定;红外光谱(IR)采用KBr压片法,由Bio-Rad FTS-135型红外光谱仪(Hercules,California, USA)测定;紫外光谱由UV-2401PC型紫外光谱仪(Shimadzu,Kyoto,Japan)测定; ECD谱由Applied Photophysics圆二色谱仪(Agilent,Santa Clara,United States)测定;核磁共振谱(1D和2D NMR)用Avance III-600型超导核磁共振仪(Bruker,Bremerhaven,Germany)测定,以氘代氯仿作为溶剂;高分辨质谱(HRESIMS)用岛津LCMS-IT-TOF 型质谱仪(Shimadzu,Kyoto,Japan)测定;薄层层析硅胶板HSGF254是烟台江友硅胶开发有限公司产品;柱层析硅胶(200~300目)为临沂市海祥化工有限公司生产;葡聚糖凝胶LH-20(Sephadex LH-20)购自GE Healthcare Bio-Sciences AB公司;高效液相色谱仪为岛津公司生产,控制器型号是CBM-20A,泵型号是LC-20AR,检测器型号为SPD-M20A,柱温箱型号为AT-350,使用的色谱柱型号为Agilent-Eclipse XDB-C18 (5μm,9.4×250mm);色谱纯乙腈购自迈瑞达公司;MCI gel CHP20P(75~150μm) 购自Mitsubishi ChemicalCorporation(Tokyo,Japan);显色剂为10%H2SO4-EtOH溶液。
南牡蒿A1(arteriopodin A1,1)
分子式:C30H44O3
分子量:452
性状:无色油状物;
HRESIMS m/z:453.3356[M+H]+(calcd.for C30H45O3,453.3363);
IR(KBr)vmax:3447,1730,1658,1645,1454,1443,1382,1233,1193,1124,1075,1024 cm-1;
ECD(c 0.21,MeOH)λmax(Δε):208(+2.33),235(–0.48),265(+0.26)nm;
1H NMR和13C NMR(DEPT)数据见表1和2。
南牡蒿A2(arteriopodin A2,2)
分子式:C30H44O3
分子量:452
性状:无色油状物;
HRESIMS m/z:453.3359[M+H]+(calcd.for C30H45O3,453.3363);
IR(KBr)vmax:3445,1730,1714,1645,1455,1397,1384,1194,1103,1068cm–1;
ECD(c 0.18,MeOH)λmax(Δε):196(+7.81),240(–0.65),271(+0.27)nm;
1H NMR和13C NMR(DEPT)数据见表1和2。
南牡蒿A3(arteriopodin A3,3)
分子式:C30H44O3
分子量:452
性状:无色油状物;
HRESIMS m/z:453.3357[M+H]+(calcd.for C30H45O3,453.3363);
IR(KBr)vmax:3440,1714,1640,1455,1444,1382,1204,1052cm–1;
ECD(c 0.27,MeOH)λmax(Δε):203(+2.75),235(–0.91),270(+0.43)nm;
1H NMR和13C NMR(DEPT)数据见表1和2。
南牡蒿B1(arteriopodin B1,4)
分子式:C30H44O7
分子量:516
性状:无色油状物;
HRESIMS m/z:517.3157[M+H]+(calcd.for C30H45O7,517.3160);
IR(KBr)vmax:3443,1758,1707,1626,1456,1384,1168,1155cm–1;
ECD(c 0.20,MeOH)λmax(Δε):243(–0.45),283(+0.58)nm;
UV(MeOH)λmax(logε):247(2.82)nm;
1H NMR和13C NMR(DEPT)数据见表1和2。
南牡蒿B2(arteriopodin B2,5)
分子式:C30H44O7
分子量:516
性状:无色油状物;
HRESIMS m/z:517.3158[M+H]+(calcd.for C30H45O7,517.3160);
IR(KBr)vmax:3444,1760,1706,1627,1457,1445,1384,1295,1248,1159,1026cm–1;
ECD(c 0.26,MeOH)λmax(Δε):260(–1.97),296(+0.56)nm;
UV(MeOH)λmax(logε):259(3.10)nm;
1H NMR和13C NMR(DEPT)数据见表1和2。
南牡蒿C1(arteriopodin C1,6)
分子式:C29H40O4;
分子量:452;
性状:白色粉末;
HRESIMS m/z:453.3001[M+H]+(calcd.for C29H41O4,453.2999);
IR(KBr)vmax:1750,1707,1682,1632,1449,1384,1257,1143,1070,1011cm–1;
ECD(c 0.01,MeOH)λmax(Δε):212(+4.21),240(–2.11),262(+0.12),290(–1.14)nm;
UV(MeOH)λmax(logε):219(3.14)nm;
1H NMR和13C NMR(DEPT)数据见表1和3。
南牡蒿C2(arteriopodin C2,7)
分子式:C29H40O7;
分子量:500;
性状:白色粉末;
HRESIMS m/z:501.2853[M+H]+(calcd.for C29H41O7,501.2847);
IR(KBr)vmax:3429,1749,1713,1618,1572,1450,1378,1242,1144,1091cm–1;
ECD(c 0.20,MeOH)λmax(Δε):213(+2.57),241(–2.88),286(+0.874)nm;
UV(MeOH)λmax(logε):221(3.11)nm;
1H NMR和13C NMR(DEPT)数据见表1和3。
南牡蒿C3(arteriopodin C3,8)
分子式:C29H39NO5;
分子量:481;
性状:白色粉末;
HRESIMS m/z:482.2895[M+H]+(calcd.for C29H40NO5,482.2901)
IR(KBr)νmax:3440,1746,1711,1632,1454,1384,1165,1104cm–1;
ECD(c 0.22,MeOH)λmax(Δε):207(+0.38),227(+1.83),247(+0.38)nm;
UV(MeOH)λmax(logε):215(2.94)nm;
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1H NMR和13C NMR(DEPT)数据见表1和3。
南牡蒿C4(arteriopodin C4,9)
分子式:C29H39NO5;
分子量:481;
性状:白色粉末;
HRESIMS m/z:482.2882[M+H]+(calcd.for C29H40NO5,482.2901);
IR(KBr)vmax:3443,1745,1706,1632,1454,1384,1260,1165,1049cm–1;
ECD(c 0.18,MeOH)λmax(Δε):221(+3.69),245(–0.01),264(+0.71)nm;
UV(MeOH)λmax(logε):215(3.08)nm:
1H NMR和13C NMR(DEPT)数据见表1和3。
南牡蒿D(arteriopodin D,10)
分子式:C30H40O7;
分子量:512;
性状:白色粉末;
HRESIMS m/z:513.2844[M+H]+(calcd.for C30H41O7,513.2847);
IR(KBr)νmax:3428,1751,1712,1620,1562,1451,1382,1274,1169,1096cm–1;
ECD(c 0.20,MeOH)λmax(Δε):216(+1.32),269(–1.58)nm;
UV(MeOH)λmax(logε):220(2.98)nm;
1H NMR和13C NMR(DEPT)数据见表1和3。
南牡蒿E(arteriopodin E,11)
分子式:C30H42O6;
分子量:498;
性状:白色粉末;
HRESIMS m/z:521.2865[M+Na]+(calcd.for C30H42O6Na,521.2874);
IR(KBr)vmax:3445,1762,1711,1633,1454,1384,1253,1133,1076cm–1;
ECD(c 0.19,MeOH)λmax(Δε):236(–0.14),268(+0.23),309(–0.58)nm;
UV(MeOH)λmax(logε):214(2.84)nm;
1H NMR和13C NMR(DEPT)数据见表1和4。
南牡蒿F1(arteriopodin F1,12)
分子式:C30H40O8
分子量:528
性状:白色单斜晶体(MeOH–H2O);
HRESIMS m/z:527.2650[M-H]-(calcd.for C30H39O8,527.2650)
IR(KBr)vmax:3432,1767,1708,1666,1632,1455,1382,1280,1264,1166,1098cm–1;
ECD(c 0.23,MeOH)λmax(Δε):217(–6.00),256(+0.93)nm;
UV(MeOH)λmax(logε):218(2.80)nm;
1H NMR和13C NMR(DEPT)数据见表1和4。
化合物12的X-单晶衍射结构
化合物12的晶体数据:Crystallographic data for compound 12:C30H40O8·CH4O,M= 560.66,α=90°,β=98.2520(10)°,γ= 90°,/>T=100.(2)K,space group P1211,Z=2,μ(Cu Kα)=0.786mm-1, 22677measured reflections,5521independent reflections(Rint=0.0501).The final R1 values were 0.0387(I>2σ(I)).The final wR(F2)values were 0.1065(I>2σ(I)).The final R1 values were 0.0388(all data).The final wR(F2)values were0.1068(all data).The goodness of fit on F2 was 1.060.Flack parameter=0.02(4).CCDC 2175134.
南牡蒿F2(arteriopodin F2,13)
分子式:C30H40O7
分子量:512
性状:白色粉末
HRESIMS m/z:511.2699[M+H]+(calcd.for C30H39O7,511.2690)
IR(KBr)vmax:3441,1768,1713,1648,1456,1378,1264,1164,1082cm–1;
ECD(c 0.16,MeOH)λmax(Δε):215(–5.48),243(–4.48),270(+0.60),316(–0.86)nm;
UV(MeOH)λmax(logε):239(2.96)nm;
1H NMR和13C NMR(DEPT)数据见表1和4。
南牡蒿F3(arteriopodin F3,14)
分子式:C30H42O7
分子量:514
性状:白色斜方晶体(MeOH–H2O)
HRESIMS m/z:515.2999[M+H]+(calcd.for C30H43O7,515.3003);
IR(KBr)vmax:3433,1790,1712,1628,1456,1382,1362,1250,1223,1180,1105cm–1;
ECD(c 0.24,MeOH)λmax(Δε):215(+0.83),234(–1.77),54(+0.01),278(–0.54)nm;
UV(MeOH)λmax(logε):218(2.80)nm;
1H NMR和13C NMR(DEPT)数据见表4和5。
化合物14的X-单晶衍射结构
化合物14的晶体数据:C30H42O7,M=514.63, α=90°,β=90°,γ=90°,/>T=100.(2)K,space groupP212121,Z=4,μ(Cu Kα)=0.717mm-1,28964measured reflections,5317independentreflections(Rint=0.0481).The final R1 values were 0.0290(I>2σ(I)).The finalwR(F2) values were 0.0716(I>2σ(I)).The final R1 values were 0.0296(all data).The final wR(F2) values were 0.0721(all data).The goodness of fit on F2 was1.046.Flack parameter=0.02(4). CCDC 2175136./>
南牡蒿F4(arteriopodin F4,15)
分子式:C30H42O7
分子量:514
性状:白色粉末
HRESIMS m/z:515.2999[M+H]+(calcd.for C30H43O7,515.3003);
IR(KBr)vmax:3522,3435,1789,1711,1628,1455,1384,1250,1166,1105cm–1;
ECD(c 0.23,MeOH)λmax(Δε):205(–8.55),230(–7.84)nm;
UV(MeOH)λmax(logε):220(3.59)nm;
1H NMR和13C NMR(DEPT)数据见表4和5。
南牡蒿F5(arteriopodin F5,16)
分子式:C30H44O5
分子量:484
性状:白色斜方晶体(MeOH–H2O);
HRESIMS m/z:483.3145[M-H]-(calcd.for C30H43O5,482.3116);
IR(KBr)vmax:3443,1782,1713,1659,1634,1455,1376,1245,1111,1063cm–1;
ECD(c 0.22,MeOH)λmax(Δε):237(–7.47),270(+0.43),314(–0.69)nm;
UV(MeOH)λmax(logε):241(2.81)nm;
1H NMR和13C NMR(DEPT)数据见表5和6。
化合物16的X-单晶衍射结构
化合物16的晶体数据:C30H44O5·CH4O,M=516.69, α=90°,β=90°,γ=90°,/>T=100.(2)K,spacegroup P212121,Z=4,μ(Cu Kα)=0.657mm-1,27658measured reflections,5547independent reflections(Rint=0.0416).The final R1 values were 0.0297(I>2σ(I)).The final wR(F2)values were 0.0751(I>2σ(I)).The final R1 values were0.0305(all data).The final wR(F2)values were 0.0760(all data).The goodness offit on F2 was 1.052.Flack parameter= 0.04(4).CCDC 2175135.
南牡蒿F6(arteriopodin F6,17)
分子式:
分子量:
性状:
HRESIMS m/z:
IR(KBr)vmax:
ECD(c 0.21,MeOH)λmax(Δε):
1H NMR和13C NMR(DEPT)数据见表5和6。
化合物17的X-单晶衍射结构
化合物17的晶体数据:C30H38O7,M=510.60, α=90°,β=93.5300(10)°,γ=90°,/>T=100.(2)K,spacegroup P1211,Z=2,μ(Cu Kα)=0.729mm-1,30680measured reflections,5145independentreflections(Rint=0.0413).The final R1 values were 0.0271(I>2σ(I)).The finalwR(F2) values were 0.0686(I>2σ(I)).The final R1 values were 0.0272(all data).The final wR(F2) values were 0.0687(all data).The goodness of fit on F2 was1.061.Flack parameter=0.04(4). CCDC 2175138.
南牡蒿F7(arteriopodin F7,18)
分子式:C29H38O7
分子量:498
性状:白色粉末
HRESIMS m/z:499.2701[M+H]+(calcd.for C29H39O7,499.2690)
IR(KBr)vmax:3501,1803,1765,1710,1667,1660,1455,1384,1268,1027cm–1;
ECD(c 0.21,MeOH)λmax(Δε):235(–2.89),2.88(–0.76)nm;
UV(MeOH)λmax(logε):240(2.83)nm;
1H NMR和13C NMR(DEPT)数据见表5和6。
南牡蒿F8(arteriopodin F8,19)
分子式:C28H38O6
分子量:468
性状:白色单斜晶体(MeOH–H2O);
HRESIMS m/z:469.2591[M+H]+(calcd.for C28H37O6,469.2585);
IR(KBr)vmax:3411,1788,1713,1611,1456,1444,1384,1240,1167,1089cm–1;
ECD(c 0.21,MeOH)λmax(Δε):218(+2.61),235(–4.30),308(+0.76)nm;
UV(MeOH)λmax(logε):224(2.84)nm;
1H NMR和13C NMR(DEPT)数据见表5和6。
化合物19的X-单晶衍射结构
化合物19的晶体数据:C28H36O6,M=468.57, α=90°,β=97.2540(10)°,γ=90°,/>T=100.(2)K,spacegroup P1211,Z=2,μ(Cu Kα)=0.702mm-1,19324measured reflections,4701independentreflections(Rint=0.0801).The final R1 values were 0.0389(I>2σ(I)).The finalwR(F2) values were 0.1003(I>2σ(I)).The final R1 values were 0.0438(all data).The final wR(F2) values were 0.1031(all data).The goodness of fit on F2 was1.042.Flack parameter= 0.19(10).CCDC 2175137.
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南牡蒿F9(arteriopodin F9,20)
分子式:C28H38O6
分子量:468
性状:白色粉末
HRESIMS m/z:469.2593[M+H]+(calcd.for C28H37O6,469.2585);
IR(KBr)vmax:3432,1789,1714,1681,1614,1459,1379,1241,1193,1109cm–1;
ECD(c 0.17,MeOH)λmax(Δε):217(+7.77),235(–9.08),310(+1.77)nm;
UV(MeOH)λmax(logε):224(3.09)nm;
1H NMR和13C NMR(DEPT)数据见表5和6。
南牡蒿F10(arteriopodin F10,21)
分子式:C30H40O7
分子量:512
性状:白色粉末
HRESIMS m/z:513.2856[M+H]+(calcd.for C30H41O7,513.2547);
IR(KBr)vmax:3436,1791,1711,1650,1632,1619,1597,1455,1384,1246,1183,1141, 1069cm–1;
ECD(c 0.23,MeOH)λmax(Δε):215(+1.31),236(–5.31),313(–1.31)nm;
UV(MeOH)λmax(logε):241(3.08)nm;
1H NMR和13C NMR(DEPT)数据见表5和6。
南牡蒿F11(arteriopodin F11,22)
分子式:C30H38O6
分子量:494
性状:白色粉末
HRESIMS m/z:495.2742[M+H]+(calcd.for C30H39O6,495.2741);
IR(KBr)vmax:3571,1756,1662,1591,1455,1442,1383,1278,1169cm–1;
ECD(c 0.19,MeOH)λmax(Δε):207(–1.50),226(+0.49),256(+0.33)nm;
UV(MeOH)λmax(logε):216(3.06)nm;
1H NMR和13C NMR(DEPT)数据见表5和7。
南牡蒿F12(arteriopodin F12,23)
分子式:C30H40O6
分子量:496
性状:白色粉末
HRESIMS m/z:497.2891[M+H]+(calcd.for C30H41O6,497.2898);
IR(KBr)vmax:3428,1728,1656,1631,1445,1378,1236,1218,1160,1044cm–1;
ECD(c 0.20,MeOH)λmax(Δε):208(–1.27),232(+0.72),250(–0.56),291(+0.76),323 (–0.66)nm;
UV(MeOH)λmax(logε):239(2.82)nm;
1H NMR和13C NMR(DEPT)数据见表5和7。
南牡蒿F13(arteriopodin F13,24)
分子式:C32H48O8
分子量:560
性状:白色粉末
HRESIMS m/z:583.3232[M+Na]+(calcd.for C32H48O8Na,583.3241);
IR(KBr)vmax:3445,1706,1633,1455,1384,1205,1048cm–1;
ECD(c 0.23,MeOH)λmax(Δε):202(–3.96),226(+2.38),299(+0.60)nm;
1H NMR和13C NMR(DEPT)数据见表5和7。
南牡蒿F14(arteriopodin F14,25)
分子式:C28H36O6
分子量:468
性状:白色粉末
HRESIMS m/z:469.2593[M+H]+(calcd.for C28H37O6,469.2585);
IR(KBr)vmax:3430,1747,1711,1627,1553,1444,1384,1136,1096,1082cm–1;
ECD(c 0.20,MeOH)λmax(Δε):203(–5.54),226(+0.75),242(–0.12),296(+1.66)nm;
UV(MeOH)λmax(logε):219(2.89)nm;
1H NMR和13C NMR(DEPT)数据见表5和7。
南牡蒿F15(arteriopodin F15,26)
分子式:C28H36O6
分子量:468
性状:白色粉末
HRESIMS m/z:491.2398[M+Na]+(calcd.for Na,491.2404);
IR(KBr)vmax:1748,1709,1626,1553,1454,1444,1383,1283,1185,1080cm–1;
ECD(c 0.19,MeOH)λmax(Δε):203(–10.14),226(+1.94),296(+2.90)nm;
UV(MeOH)λmax(logε):218(3.09)nm;
1H NMR和13C NMR(DEPT)数据见表5和7。
南牡蒿G1(arteriopodin G1,27)
分子式:C30H42O7
分子量:514
性状:白色三斜晶体(MeOH–H2O);
HRESIMS m/z:515.2997[M+H]+(calcd.for C30H43O7,515.3003);
IR(KBr)vmax:3331,1750,1705,1630,1590,1454,1384,1279,1180,1056cm–1;
ECD(c 0.22,MeOH)λmax(Δε):214(–5.92),287(–0.27)nm;
UV(MeOH)λmax(logε):219(2.80)nm;
1H NMR和13C NMR(DEPT)数据见表8和9。
化合物27的X-单晶衍射结构
化合物27的晶体数据:C30H42O7,M=514.63, α=89.6560(10)°,β=84.1740(10)°,γ=81.4640(10)°,/>T= 100.(2)K,space group P1,Z=2,μ(Cu Kα)=0.707mm-1,41783measured reflections,10366independent reflections(Rint=0.0497).The final R1 values were 0.0628(I>2σ(I)). The final wR(F2)values were 0.1697(I>2σ(I)).The final R1 values were0.0632(all data). The final wR(F2)values were 0.1704(all data).The goodnessof fit on F2 was 1.029.Flack parameter=0.20(7).CCDC 2175140./>
南牡蒿G2(arteriopodin G2,28)
分子式:C30H42O7
分子量:514
性状:白色粉末
HRESIMS m/z:515.3005[M+H]+(calcd.for C30H43O7,515.3003);
IR(KBr)vmax:3408,1752,1707,1630,1453,1382,1278,1160,1057cm–1;
ECD(c 0.18,MeOH)λmax(Δε):215(–4.80),276(+0.60)nm;
UV(MeOH)λmax(logε):219(2.84)nm;
1H NMR和13C NMR(DEPT)数据见表8和9。
南牡蒿G3(arteriopodin G3,29)
分子式:C30H44O7
分子量:516
性状:白色单斜晶体(MeOH–H2O)
HRESIMS m/z:539.2974[M+Na]+(calcd.for C30H44O7Na,539.2979);
IR(KBr)vmax:3442,1756,1703,1631,1455,1384,1249,1160,1056cm–1;
ECD(c 0.20,MeOH)λmax(Δε):199(–8.11),233(–0.48),280(–1.45)nm;
UV(MeOH)λmax(logε):220(2.94)nm;
1H NMR和13C NMR(DEPT)数据见表8和9。
化合物29的X-单晶衍射结构
化合物29的晶体数据:C30H44O7,M=516.65, α=90°,β=94.190(4)°,γ=90°,/>T=100.(2)K,spacegroup P1211,Z=2,μ(Cu Kα)=0.699mm-1,31887measured reflections,5211independentreflections(Rint=0.1979).The final R1 values were 0.0730(I>2σ(I)).The finalwR(F2) values were 0.1825(I>2σ(I)).The final R1 values were 0.1078(all data).The final wR(F2) values were 0.2074(all data).The goodness of fit on F2 was1.038.Flack parameter=0.4(2). CCDC 2175139.
南牡蒿G4(arteriopodin G4,30)
分子式:C30H42O6
分子量:498
性状:白色粉末
HRESIMS m/z:499.3047[M+H]+(calcd.for C30H43O6,499.3054);
IR(KBr)vmax:3440,1770,1706,1631,1455,1384,1250,1160,1056cm–1;
ECD(c 0.20,MeOH)λmax(Δε):221(–2.14),269(+0.36)nm;
UV(MeOH)λmax(logε):240(2.80)nm;
1H NMR和13C NMR(DEPT)数据见表8和9。
南牡蒿G5(arteriopodin G5,31)
分子式:C30H40O6
分子量:496
性状:白色粉末
HRESIMS m/z:497.2903[M+H]+(calcd.for C30H41O6,497.2898);
IR(KBr)vmax:3441,1764,1707,1650,1631,1455,1384,1276,1160,1056cm–1;
ECD(c 0.20,MeOH)λmax(Δε):215(–33.4),260(+3.25)nm;
UV(MeOH)λmax(logε):216(3.47),251(3.12)nm;
1H NMR和13C NMR(DEPT)数据见表8和9。
南牡蒿G6(arteriopodin G6,32)
分子式:C30H42O7
分子量:514
性状:白色粉末
HRESIMS m/z:515.2998[M+H]+(calcd.for C30H43O7,515.3003);
IR(KBr)vmax:3447,1761,1710,1633,1454,1383,1269,1161,1039cm–1;
ECD(c 0.21,MeOH)λmax(Δε):214(–9.84),260(+1.24)nm;
UV(MeOH)λmax(logε):220(2.85)nm;
1H NMR和13C NMR(DEPT)数据见表8和10。
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南牡蒿G7(arteriopodin G7,33)
分子式:C30H40O6
分子量:496
性状:白色粉末
HRESIMS m/z:497.2904[M+H]+(calcd.for C30H41O6,497.2898);
IR(KBr)vmax:3441,1763,1706,1632,1454,1384,1275,1159,1056cm–1;
ECD(c 0.21,MeOH)λmax(Δε):216(–7.47),269(+1.14)nm;
UV(MeOH)λmax(logε):220(2.98)nm;
1H NMR和13C NMR(DEPT)数据见表8和10。
南牡蒿G8(arteriopodin G8,34)
分子式:C30H42O8
分子量:528
性状:白色粉末
HRESIMS m/z:529.2805[M-H]-(calcd.for C30H41O8,529.2807);
IR(KBr)vmax:3429,1710,1624,1453,1278,1158,1106cm–1;
ECD(c 0.23,MeOH)λmax(Δε):214(–7.38)nm;
UV(MeOH)λmax(logε):220(2.94)nm;
1H NMR和13C NMR(DEPT)数据见表8和10。
南牡蒿H(arteriopodin H,35)
分子式:C30H42O5
分子量:484
性状:白色粉末
HRESIMS m/z:483.3108[M-H]-(calcd.for C30H41O5,483.3105);
IR(KBr)vmax:3440,1716,1632,1454,1384,1260,1169,1152,1105cm–1;
ECD(c 0.23,MeOH)λmax(Δε):205(–0.84),234(+1.16)nm;
UV(MeOH)λmax(logε):220(2.69)nm;
1H NMR和13C NMR(DEPT)数据见表8和10。
南牡蒿I(arteriopodin I,36)
分子式:C25H36O4
分子量:400
性状:白色粉末
HRESIMS m/z:423.2502[M+Na]+(calcd.for C25H36O4Na,423.2506);
IR(KBr)vmax:3440,1716,1632,1454,1384,1260,1169,1152,1105cm–1;
ECD(c 0.24,MeOH)λmax(Δε):215(–0.14),242(+0.44)nm;
UV(MeOH)λmax(logε):220(2.35)nm;
1H NMR和13C NMR(DEPT)数据见表8和10。
表21H NMR(δin ppm,J in Hz)data for compounds 1–5 in CDCl3 a.
aCompounds 1-3 were recorded in 600MHz;compounds 4 and 5 wererecorded in 800MHz. 表31H NMR(600MHz,δin ppm,J in Hz)data for compounds 6–10in CDCl3.
表41H NMR(600MHz,δin ppm,J in Hz)data for compounds 11–15.
aRecorded in CDCl3;brecorded in CD3OD.
1表61H NMR data(600MHz,J in Hz,δin ppm)of compounds 16–21.
2aRecorded in CDCl3;brecorded in CD3OD.
表71H NMR data(600MHz,J in Hz,δin ppm)of compounds 22–26.
aRecorded in CDCl3;brecorded in CD3OD.
表91H NMR(600MHz,δin ppm,J in Hz)data for compounds 27–31.
aRecorded in CDCl3;brecorded in CD3OD.
表101H NMR(600MHz,δin ppm,J in Hz)data for compounds 32–36.
aRecorded in CDCl3;brecorded in CD3OD.
实施例2:
化合物1-36对三株肝癌细胞株的细胞毒活性。
1.材料和方法
1.1材料
HepG2细胞株由中国科学院昆明植物研究所活性筛选中心赠予,SK-Hep-1和Huh7细胞株购自上海纪宁生物科技有限公司;培养基(Dulbecco's Modified Eagle Medium,DMEM)购自Thermo Fisher Scientific(苏州,中国);血清(fetal bovine serum,FBS)购自Life Technologies(NY,USA);RPMI-1640购自ThermoFisher Biochemical Products(北京,中国)。
1.2仪器
Flex Station 3台式多功能酶标仪(Bio-RAD 680,美国);分析天平(AG135,Metler Toledo,中国);恒温箱(DHP-9082,上海)。
1.3实验过程
1).取对数期生长的肝癌细胞,弃去旧培养基,用PBS清洗两遍,弃去PBS;
2).用0.25%的胰蛋白酶消化细胞,在显微镜下观察到细胞轮廓加深并有变圆趋势时,迅速吸去胰蛋白酶;
3)用含10%FBS的DMEM完全培养基终止消化并重悬细胞,取10μL细胞悬液,用细胞计数仪计数,并用培养基调整细胞浓度至1×104/mL,接种在96孔板上,每孔加入 100μL细胞悬液,在37℃,5%CO2的培养箱中孵育24h,使细胞贴壁;
4).吸去培养基,将稀释好的样品加入板中,每孔加入100μL,每个浓度设置3个复孔,培养箱中继续孵育48h;
5).吸去培养基,加入配好的MTT溶液(1mg/mL),每孔加入100μL,培养箱中孵育4h;
6).吸去MTT溶液,加入DMSO,每孔加入100μL,培养箱中孵育10min;
7).使用酶标仪在490nm波长下测量吸光度值,通过公式抑制率=(阴性-实验组)/(阴性-空白组)×100%计算细胞抑制率,并用统计软件GraphPad prism 5计算IC50,实验重复3次。
表11化合物1–36的抗肝癌细胞毒活性
2.结果
对所有分离得到的进行了抗肝癌细胞毒活性评价,实验结果如表11所示:化合物13、16、31和33对HepG2细胞具有明显的细胞毒活性,其IC50分别为14.3、12.2、17.2 和16.0μmol/L,活性与阳性对照索拉非尼(IC50,11.0μmol/L);化合物12、18、27、 28、30和32对HepG2细胞具有适中的细胞毒活性,其IC50在20.0至45.3μmol/L之间;化合物1、3、6、19、21-24、29和34-36也具有一定的细胞毒活性,其IC50在52.1– 94.2μmol/L之间;然而,其它化合物对HepG2细胞的活性较弱,其IC50高于100μmol/L。
化合物31和33对Huh7细胞具有与阳性对照索拉非尼(IC50,12.3μmol/L)相当的细胞毒活性,其IC50分别为10.3和18.3μmol/L;化合物13、28和30具有适中的细胞毒活性,其IC50在26.4至39.5μmol/L之间;化合物1、12、16、27、32和34对Huh7 细胞也具有一定的细胞毒活性,其IC50在53.1–97.5μmol/L之间;其它化合物的活性较弱,其IC50高于100μmol/L。
化合物31和33对SK-Hep-1细胞具有明显的细胞毒活性,其IC50分别为22.3和19.0μmol/L,活性与索拉非尼(IC50,18.1μmol/L)相当;化合物24、28、30和4具有适中的细胞毒活性,其IC50在25.4至42.6μmol/L之间;化合物6、12、13、16、18、27、32、35和36对SK-Hep-1细胞表现出适中的细胞毒活性,其IC50在50.3至88.0μmol/L 之间,其它化合物活性较弱。
综上所示,化合物31和33对三株肝癌细胞HepG2、Huh7和SK-Hep-1的细胞毒活性最好,其IC50分别为17.2、10.3、22.3μmol/L和16.0、18.3、19.0μmol/L,活性与索拉非尼(IC50:11.0,12.3,and 18.1μmol/L)相当。
3.结论
实验结果显示,本发明的化合物1–36对三株肝癌细胞(HepG2、Huh7、SK-Hep-1) 的细胞毒活性均具有一定的细胞毒活性,化合物13、16、31和33对HepG2细胞具有明显的细胞毒活性,其IC50分别为14.3、12.2、17.2和16.0μM,活性与阳性对照索拉非尼(IC50,11.0μM);化合物12、18、27、28、30和32对HepG2细胞具有适中的细胞毒活性,其IC50在20.0至45.3μM之间。化合物31和33对Huh7细胞具有与阳性对照索拉非尼(IC50,12.3μM)相当的细胞毒活性,其IC50分别为10.3和18.3μM;化合物13、28和30具有适中的细胞毒活性,其IC50在26.4至39.5μM之间。化合物31和 33对SK-Hep-1细胞具有明显的细胞毒活性,其IC50分别为22.3和19.0μM,活性与索拉非尼(IC50,18.1μM)相当;化合物24、28、30和34具有适中的细胞毒活性,其IC50在25.4至42.6μM之间。以上结果表明南牡蒿中分离得到的化合物1-36可作为药物用于治疗肝癌相关的疾病。
制剂实施例
在以下制剂实施例中,选择常规试剂,并按照现有常规方法进行制剂制备,本应用例仅体现本发明所述化合物1-36中的至少一种能够制备成不同的制剂,对具体试剂和操作不作具体限定:
1.将本发明化合物1-36中的至少一种,用少量的DMSO溶解后,按常规加注射用水,精滤,灌封灭菌制成注射液,所述注射液的浓度为0.5~5mg/mL。
2.将本发明化合物1-36中的至少一种,用少量的DMSO溶解后,将其溶于无菌注射用水中,搅拌使溶解,用无菌抽滤漏斗过滤,再无菌精滤,分装于安瓿中,低温冷冻干燥后无菌熔封,得粉针剂。
3.将本发明化合物1-36中的至少一种,按其与赋形剂重量比为9:1的比例加入赋形剂,制成粉剂。
4.将本发明化合物1-36中的至少一种,按其与赋形剂重量比为5:1的比例加入赋形剂,制粒压片。
5.将本发明化合物1-36中的至少一种,按常规口服液制法制成口服液。
6.将本发明化合物1-36中的至少一种,按其与赋形剂重量比为5:1的比例加入赋形剂,制成胶囊。
7.将本发明化合物1-36中的至少一种,按其与赋形剂重量比为5:1的比例加入赋形剂,制成颗粒剂。
由以上实施例可知,本发明提供了一种南牡蒿中化合物及其制备方法和应用,药物组合物及其应用。本发明提供的南牡蒿素,主要包括36个结构新颖的倍半萜二聚体,这些化合物对肝癌细胞具有不同程度的细胞毒活性,能够与可药用载体或赋型剂组成药物组合物,能够用于制备抗肝癌药物。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (6)
1.如下结构式所示的南牡蒿素化合物27、28、30-34,
2.权利要求1所述的结构式所示的南牡蒿素化合物27、28、30-34的制备方法:取干燥的南牡蒿地上部分,粉碎,用3倍量的90%乙醇提取两次,合并提取液减压浓缩后得到的粗浸膏分散于水中,用乙酸乙酯萃取得到乙酸乙酯萃取部分;随后,将乙酸乙酯萃取部分过硅胶柱层析,并用丙酮-石油醚体积比0:100、5:95、10:90、20:80、40:60和100:0梯度洗脱得到6个流分Frs.A-1~A-6;Fr.A-4过MCI gel CHP 20P柱层析,用甲醇-水50:50、70:30、90:10和100:0洗脱得到了四个亚流分Frs.A-4a-A-4d;Fr.A-4c再使用硅胶柱层析,以丙酮-石油醚5:95、10:90和20:80划分成五个流分Frs.A-4c-1-A-4c-5;Fr.A-4c-5经过Rp-C18反相柱层析,甲醇-水50:50、60:40和70:30梯度洗脱得到了流分Frs.A-4c-5a-A-4c-5f;Fr.A-4c-5a经半制备HPLC在安捷伦XDB-C18柱上以60:40的乙腈-水和82:18甲醇-水反复纯化得到了化合物28;
Fr.A-5经MCI gel CHP 20P柱层析,甲醇-水50:50、70:30、90:10和100:0洗脱得到了四个流分Frs.A-5a-A-5d;Fr.A-5c进一步用反相Rp-C18柱层析,用甲醇-水50:50、60:40、70:30和100:0梯度洗脱得到四个亚流分Frs.A-5c-1-A-5c-4;Fr.A-5c-2经过硅胶柱层析,以丙酮-氯仿5:95和10:90洗脱得到了五个流分Frs.A-5c-2a-A-5c-2e;Fr.A-5c-2b经过Sephadex LH-20柱层析用甲醇-氯仿50:50处理后,再使用半制备高效液相在安捷伦XDB-C18柱以乙腈-水55:45和甲醇-水80:20为流动相纯化得到化合物27、30、31和33;Fr.A-5c-3经反复的硅胶柱层析以丙酮-氯仿10:90和20:80洗脱之后,再过Sephadex LH-20柱层析用甲醇-氯仿50:50处理,最后使用半制备HPLC在安捷伦XDB-C18柱以乙腈-水50:50和甲醇-水75:25为流动相纯化得到化合物32、34。
3.权利要求1所述的结构式所示的南牡蒿素化合物27、28、30-34在制备抗肝癌药物中的应用。
4.包括权利要求1所述的结构式所示的南牡蒿素化合物27、28、30-34的至少一种和药学上可接受的载体或赋型剂的药物组合物。
5.权利要求4所述的药物组合物在制备抗肝癌药物中的应用。
6.权利要求4所述的药物组合物的制备方法,其特征在于该方法包括下述步骤:先按权利要求2所述的方法,制备得到化合物27、28、30-34,然后取化合物27、28、30-34中的其中一种或其任意组合,加入可药用载体。
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