CN110078688B - 半日花烷型二萜衍生物及其药物组合物与应用 - Google Patents
半日花烷型二萜衍生物及其药物组合物与应用 Download PDFInfo
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Abstract
本发明公开了半日花烷型二萜衍生物及其药物组合物与应用,属于药物技术领域。本发明在分离得到半日花烷型二萜类衍生物的基础上,证实了结构式为Ⅰ~Ⅻ中的任意一种半日花烷型二萜衍生物可作为核因子‑κB(NF‑κB)信号通路抑制剂,能够抑制NF‑κB信号通路并治疗NF‑κB介导的各类疾病。
Description
技术领域
本发明涉及半日花烷型二萜衍生物及其药物组合物与应用,属于药物技术领域。
背景技术
炎症反应是临床常见的一种病理过程,也是免疫系统对有害刺激的首要响应措施,其症状常表现为组织红、肿、发、热、痛等。一般情况下,炎症是有益的:炎症引起的渗出和组织增生可稀释有害刺激并限制其扩散;渗出液中的白细胞可消灭致炎因子和清除坏死组织。相反地,炎症也具有潜在的危害性,这表现为:炎症引起的重要器官的变性和坏死会严重影响其正常功能,过度的炎症反应可能会造成休克或多器官功能损伤。
诱发炎症的刺激有很多,当机体受到如病原微生物感染时,病原体相关分子模式(PAMPs)将被识别,识别得到的信号经过Toll样受体(TLR)、CD14受体及其他细胞因子受体的信号传导,进而引发一系列的防御性反应,促使体内细胞因子(Cytokines)、趋化因子(Chemokines)和生长因子(Growth Factors)的释放,并使淋巴细胞迁移至受刺激部位,最终引发炎症。细菌脂多糖(LPS)就是一种PAMPs,它是革兰氏阴性菌细胞壁中的一种成分,可诱导胞内多条炎症信号通路的活化,导致大量炎症介质的释放。在其刺激下,TLR4信号通路被激活,并导致MyD88依赖性和MyD88非依赖性的信号传导途径激活,进而活化核因子-κB(Nuclear Factor-κB,NF-κB)通路。而NF-κB通路又调控着下游诸多促炎细胞因子如肿瘤坏死因子(TNF-α)、白细胞介素-1β(IL-1β)、NO的表达,在炎症反应过程中起着重要作用。因此,可通过LPS诱导的巨噬细胞炎症反应作为体外研究细胞炎症机制的模型,这已成为研究细胞炎症反应的常见方式。
NF-κB是炎症过程中一个经典而重要的转录因子,通过与启动子和增强子中称为κB元件的离散DNA序列结合来诱导或抑制基因表达。在静息状态时,NF-κB与其抑制剂IκB(Inhibitors ofNF-κB)结合,保留在细胞质中。哺乳动物细胞中主要存在三个IκB,即IκBα、IκBβ和IκBε,NF-κB的激活通常涉及IκB激酶(IκB kinaseI,IKK)复合物对IκB的降解。在经典NF-κB激活途径中,外界刺激信号跨膜传递到细胞内并激活IKK,活化的IKK将NF-κB/IκB复合体中的IκBα磷酸化,并诱发IκB泛素化,随后由蛋白酶将其降解,从而使NF-κB得以释放并进入细胞核内与各种靶基因结合。受NF-κB调控的基因有很多,其中最具代表性的是一氧化氮合酶(iNOS)的基因。当iNOS表达降低时,其催化的终产物一氧化氮(NO)水平就会降低,表明NF-κB信号通路受到抑制。
而目前核因子-κB(NF-κB)信号通路抑制剂的细胞毒性较大、活性不够强(如IC50值较高)。
发明内容
针对现有技术中前核因子-κB(NF-κB)信号通路抑制剂存在的毒性大、活性差的技术问题,提供了半日花烷型二萜衍生物及其药物组合物与应用,半日花烷型二萜衍生物能够抑制NF-κB信号通路并治疗NF-κB介导的各类疾病。本发明立足于对益母草的长期研究,从中发现毒性小、活性好的化合物,为NF-κB信号通路相关疾病的药物开发奠定工作基础。
半日花烷型二萜衍生物,结构式为I~XII中的任意一种,其中
结构式II~V的通式为结构式II中R1=αOAc,R2=Δ5(6),R3=oH,R4=OC2H5,结构式III中R1=O,R2=Δ5(6),R3=OH,R4=OC2H5,结构式IV中R1=H,H,R2=αH,R3=βOH,R4=CH3,结构式V中R1=αOAc,R2=αH,R3=βOH,R4=OC2H5;
结构式VI和VII的通式为结构式VI中R1=αOAc,R2=Δ5(6),R3=OH,R4=CH3,Δ8(9),R6=OCH3,结构式VII中;R1=O,R2=αH,R3=βOH,R4=CH3,Δ8(9)R6=OCH3
药物组合物:含有药用载体和/或赋形剂,以及权利要求1所述结构式为I~XII中的任意一种半日花烷型二萜衍生物。
所述结构式为I~XII中的任意一种半日花烷型二萜衍生物或药物组合物作为核因子-κB(NF-κB)信号通路抑制剂,可以用于治疗由NF-κB介导的疾病。
所述半日花烷型二萜衍生物的提取方法,具体步骤如下:
(1)将益母草叶和/或枝进行干燥、粉碎;
(2)在室温条件下,采用体积分数为85%的乙醇水溶液对步骤(1)粉碎的益母草叶和/或枝进行提取3~4次,合并乙醇提取液,过滤,将滤液进行减压浓缩得到浸膏;
(3)将步骤(2)的浸膏混悬于水中,依次用石油醚、乙酸乙酯和正丁醇萃取得到石油醚萃取液、乙酸乙酯萃取液和正丁醇萃取液;
(4)将步骤(3)的乙酸乙酯萃取液减压浓缩,然后溶解于氯仿中并吸附于硅胶上,挥发溶剂并研碎,采用硅胶柱层析,采用氯仿/甲醇体系进行梯度洗脱,用薄层色谱法检视,分段收集得到A、B、C、D和E五部分;
(5)步骤(4)的D部分采用硅胶柱层析,采用氯仿/丙酮体系进行梯度洗脱,用薄层色谱法检视,分段收集得到D1、D2和D3三部分;
(6)步骤(5)的D1部分依次用LH-20凝胶色谱柱和C18色谱柱纯化得到结构式Ⅴ、结构式Ⅸ、结构式Ⅹ、结构式Ⅺ和结构式Ⅻ的半日花烷型二萜衍生物;
(7)步骤(5)的D3部分用提取型高效液相色谱层析分离,采用硅胶柱和体积比为9:2的乙酸乙酯/丙酮混合液洗脱得到结构式Ⅱ、结构式Ⅲ、结构式Ⅵ、结构式Ⅷ和结构式Ⅸ的半日花烷型二萜衍生物;
(8)步骤(4)的E部分用C18色谱柱层析,采用甲醇/水溶液进行梯度洗脱,用薄层色谱法检视,分段收集得到E1、E2、E3和E4四部分;
(9)步骤(8)的E1部分用硅胶柱层析,依次分别以氯仿与甲醇的体积比为1:0、50:1、30:1、20:1、10:1、5:1、1:1和0:1的氯仿/甲醇体系梯度洗脱得到结构式Ⅰ、结构式Ⅳ和结构式Ⅶ的半日花烷型二萜衍生物。
所述步骤(4)梯度洗脱用氯仿/丙酮混合液中的氯仿与丙酮的体积比依次为1:0、80:1、40:1、20:1、10:1、5:1、1:1、0:1的7个梯度。
优选的,步骤(4)梯度洗脱用氯仿/丙酮混合液中的氯仿与丙酮的体积比依次为40:1→10:1。
更优选的,步骤(4)梯度洗脱用氯仿/丙酮混合液中的氯仿与丙酮的体积比依次为30:1→10:1。
所述步骤(5)梯度洗脱用氯仿/丙酮混合液中的氯仿与丙酮的体积比依次为40:1、20:1、10:1、5:1、2:1、1:1、0:1的7个梯度。
优选的,步骤(5)梯度洗脱用氯仿/丙酮混合液中的氯仿与丙酮的体积比依次为20:1→5:1。
更优选的,步骤(5)梯度洗脱用氯仿/丙酮混合液中的氯仿与丙酮的体积比依次为10:1→5:1。
所述步骤(8)梯度洗脱用甲醇/水溶液中甲醇与水的体积比依次为3:10、4:10、5:10、6:10、7:10的5个梯度。
优选的,步骤(8)梯度洗脱用甲醇/水溶液中甲醇与水的体积比依次为4:10→7:10。
更优选的,步骤(8)梯度洗脱用甲醇/水溶液中甲醇与水的体积比依次为5:10→7:10。
所述结构式为Ⅰ~Ⅻ中的任意一种半日花烷型二萜衍生物,加入药用载体和/或赋形剂即可得到含结构式为Ⅰ~Ⅻ中的任意一种半日花烷型二萜衍生物的药物组合物;
结构式为Ⅰ~Ⅻ中的任意一种半日花烷型二萜衍生物可直接作为核因子-κB(NF-κB)信号通路抑制剂用于治疗由NF-κB介导的疾病,也可以以含结构式为Ⅰ~Ⅻ中的任意一种半日花烷型二萜衍生物的药物组合物的形式作为核因子-κB(NF-κB)信号通路抑制剂用于治疗由NF-κB介导的疾病;
药物组合物中所述结构式为Ⅰ~Ⅻ中的任意一种半日花烷型二萜衍生物的质量百分含量为0.1~99%;
优选的,药物组合物中所述结构式为Ⅰ~Ⅻ中的任意一种半日花烷型二萜衍生物的质量百分含量为0.5~90%;
药用载体和/或赋形剂对人体和动物均无毒,并且为惰性;
药用载体为固体稀释剂、半固体稀释剂、液体稀释剂、填料、药物制品辅剂的一种或多种;
药物组合物以单位体重服用量的形式使用;
药物可经注射(静注、肌注)和口服两种形式给药。
本发明的有益效果:
(1)本发明在分离得到半日花烷型二萜类衍生物的基础上,证实了该类半日花烷型二萜类衍生物能够抑制细胞NF-κB信号通路;
(2)本发明分离到的半日花烷型二萜类衍生物可以用于治疗NF-κB介导的各类炎症性疾病和免疫性疾病,包括但不限于脓毒症、类风湿性关节炎、痛风等;
(3)本发明分离到的半日花烷型二萜类衍生物可以用于信号通路研究中的阳性抑制剂,在具体研究过程中特异性阻断NF-κB信号通路,排除NF-κB信号通路的干扰,可作为工具化合物使用。
具体实施方式
下面结合具体实施方式对本发明作进一步详细说明,但本发明的保护范围并不限于所述内容。
实施例1:半日花烷型二萜衍生物的提取方法,具体步骤如下:
(1)将益母草枝进行干燥、粉碎;
(2)在室温条件下,采用体积分数为85%的乙醇水溶液对步骤(1)粉碎的益母草枝进行提取3次,每次提取24小时,合并乙醇提取液,过滤,将滤液进行减压浓缩得到浸膏;
(3)将步骤(2)的浸膏混悬于水中,依次用石油醚、乙酸乙酯和正丁醇萃取得到石油醚萃取液、乙酸乙酯萃取液和正丁醇萃取液;
(4)将步骤(3)的乙酸乙酯萃取液减压浓缩回收溶剂得到乙酸乙酯萃取部分,然后将乙酸乙酯萃取部分溶解于氯仿中并吸附于硅胶上,挥发溶剂氯仿后研碎,采用硅胶柱层析,采用氯仿/丙酮混合液进行梯度洗脱,用薄层色谱法检视,分段收集得到A、B、C、D和E五部分;其中梯度洗脱用氯仿/甲醇体系中的氯仿与甲醇的体积比依次为1:0、80:1、40:1、20:1、10:1、5:1、1:1、0:1;
(5)步骤(4)的D部分采用硅胶柱层析,采用氯仿/丙酮体系进行梯度洗脱,用薄层色谱法检视,分段收集得到D1、D2和D3三部分;其中梯度洗脱用氯仿/丙酮体系中的氯仿与丙酮的体积比依次为40:1、20:1、10:1、5:1、2:1、1:1、0:1;
(6)步骤(5)的D1部分依次用LH-20凝胶色谱柱和C18色谱柱纯化得到结构式Ⅴ、结构式Ⅸ、结构式Ⅹ、结构式Ⅺ和结构式Ⅻ的半日花烷型二萜衍生物;
(7)步骤(5)的D3部分用提取型高效液相色谱层析分离,采用硅胶柱和体积比为9:2的乙酸乙酯/丙酮混合液洗脱得到结构式Ⅱ、结构式Ⅲ、结构式Ⅵ、结构式Ⅷ和结构式Ⅸ的半日花烷型二萜衍生物;
(8)步骤(4)的E部分用C18色谱柱层析,采用甲醇/水溶液进行梯度洗脱,用薄层色谱法检视,分段收集得到E1、E2、E3和E4四部分;其中梯度洗脱用甲醇/水溶液中甲醇与水的体积比依次为3:10、4:10、5:10、6:10、7:10;
(9)步骤(8)的E1部分用硅胶柱层析,分别以氯仿与甲醇的体积比为1:0、50:1、30:1、20:1、10:1、5:1、1:1和0:1的氯仿/甲醇混合液梯度洗脱得到结构式Ⅰ、结构式Ⅳ和结构式Ⅶ的半日花烷型二萜衍生物;
本实施例的半日花烷型二萜衍生物,
结构式II~V的通式为结构式II中R1=αOAc,R2=Δ5(6),R3=OH,R4=OC2H5,结构式III中R1=O,R2=Δ5(6),R3=OH,R4=OC2H5,结构式IV中R1=H,H,R2=αH,R3=βOH,R4=CH3,结构式V中R1=αOAc,R2=αH,R3=βOH,R4=OC2H5;
结构式VI和VII的通式为结构式VI中R1=αOAc,R2=Δ5(6),R3=OH,R4=CH3,Δ8(9),R6=OCH3,结构式VII中R1=O,R2=αH,R3=βOH,R4=CH3,Δ8(9),R6=OCH3;
本实施例结构式X和结构式XI的半日花烷型二萜衍生物的13C NMR数据(δin ppm,Jin Hz)见表1,
本实施例结构式X和结构式XI的半日花烷型二萜衍生物的1H-NMR数据(600MHz)(δin ppm,Jin Hz)见表2,
表1结构式X和结构式XI的半日花烷型二萜衍生物的13C NMR数据(δin ppm,JinHz)
Measured at 150MHz;
表2结构式Ⅹ和结构式Ⅺ的半日花烷型二萜衍生物的1H-NMR数据(600MHz)(δinppm,Jin Hz)
b Measured at 600MHz;
化合物Ⅹ无色油状,通过高分辨HRESIMS(m/z 417.2245,[M+Na]+)可知其分子式为C22H34O6,不饱和度为6。13C和DEPT NMR谱图(表2)显示该分子共有22个碳信号:6个季羰(包括1个羰基:δC 213.3;1个酯基:δC 171.8;1个烯碳:δC 138.1;1个连氧季碳:δC 110.0)、4个次甲基(包括1个烯碳:δC 143.9;2个连氧次甲基:δC 103.6,δC 84.0)、6个亚甲基、6个甲基(包括2个甲氧基:δC 56.8,δC 49.2)。进一步分析化合物Ⅹ的HMBC谱,H-20与C-1,C-5,C-9有相关信号,H-5与C-1,C-4,C-6,C-10,C-19相关,可知C-5,C-6,C-9,C-10形成了一个五元呋喃环(即C-6-O-C-9),H-17与C-7,C-8相关,结合1H-1H COSY谱中H-2与H-1/H-3相关,H-5与H-6相关,H-8与H-17相关可推断得出化合物Ⅹ中A/B环的连接方式;与已知化合物1-((1S,3R,3aS,7aS)-3-(2-(furan-3-yl)ethyl)-3-methoxy-3a,7,7–trimethyloctahydroisobenzofuran-1-yl)propan-1-one的核磁数据比较,C-1到C-12比较相似,进一步确定化合物Ⅹ中A/B环的连接方式;
相对构型参考已知类似物
1-((1S,3R,3aS,7aS)-3-(2-(furan-3-yl)ethyl)-3-methoxy-3a,7,7–trimethyloctahydroisobenzofuran-1-yl)propan-1-one来确定,在化合物Ⅹ的ROESY谱中,H-20与H-6,H-11相关,H-19与H-6相关,H-1,8与H-5相关,同种植物同种类型化合物生源途径一致,表明化合物Ⅹ的相对构型为(5S),(6S),(9R),(10S),但是由于化合物量少不易结晶,且通过ROESY谱也无法确定C-15的相对构型,所以其绝对构型不能确定;
化合物Ⅺ无色油状;高分辨HRESIMS(m/z 403.2089,[M+Na]+)可知其分子式为C21H32O6。不饱和度为6,13C和DEPT NMR谱图(表2)显示该分子共有21个碳信号:6个季羰(包括1个羰基:δC 214.9;1个酯基:δC 172.0;1个烯碳:δC 138.8;1个连氧季碳:δC 108.4)、4个次甲基(包括1个烯碳:δC 143.6;2个连氧次甲基:δC 103.6,δC 84.1)、6个亚甲基、5个甲基(包括2个甲氧基:δC 56.7)。仔细对比化合物Ⅺ与Ⅹ的13C-NMR和1H-NMR数据(表1,表2)发现。化合物Ⅺ和Ⅹ非常相似,化合物Ⅺ只比Ⅹ少了一个甲氧基信号,其他部分几乎是一样的。通过碳谱数据对比,发现只有几个碳的数据差异大一点,他们分别是化合物Ⅺ的C-8(δC29.6),C-9(δC 108.4),C-11(δC 34.9)和化合物ⅩC-8(δC 31.4),C-9(δC 111.0),C-11(δC29.3),而且化合物Ⅺ的分子量正好比Ⅹ少了一个CH2,综合以上信息可以推断出化合物Ⅺ的C-9位的取代基由化合物Ⅹ的甲氧基变成了羟基,碳谱数据的差异是由于C-9位取代基的改变造成周围碳在空间上所处的化学环境变化从而导致化学位移变化;通过2D-NMR也可以确定化合物Ⅺ的平面结构;相对构型同样参考化合物Ⅹ与已知类似物1-((1S,3R,3aS,7aS)-3-(2-(furan-3-yl)ethyl)-3-methoxy-3a,7,7–trimethyloctahydroisobenzofuran-1-yl)propan-1-one来确定;在ROESY谱中,H-20与H-6,H-11相关,H-19与H-6相关,H-18与H-5相关,综合以上信息化合物Ⅺ的相对构型确定为为(5S),(6S),(9R),(10S),但是由于化合物Ⅺ量少不易结晶,且通过ROESY谱也无法确定C-15的相对构型,所以其绝对构型不能确定;
本实施例结构式Ⅻ的半日花烷型二萜衍生物的1H and 13C NMR数据(δin ppm,JinHz)见表3,
表3结构式Ⅻ的半日花烷型二萜衍生物的1H and 13C NMR数据(δin ppm,Jin Hz)
a Measured at 150MHz,b Measured at 600MHz
化合物Ⅻ无色油状;高分辨HRESIMS(m/z 387.2140,[M+Na]+)可知其分子式为C21H32O5,不饱和度为6,13C和DEPT NMR谱图(表3)显示该分子共有21个碳信号:6个季羰(包括1个羰基:δC 213.4;1个酯基:δC 174.7;1个烯碳:δC 133.4;1个连氧季碳:δC 111.1)、3个次甲基(包括1个烯碳:δC 146.8;1个连氧次甲基:δC 84.1)、7个亚甲基(包括1个连氧亚甲基:δC 71.2)、5个甲基(包括1个甲氧基:δC 49.2)。仔细对比化合物Ⅻ与Ⅹ的13C-NMR和1H-NMR数据发现。化合物Ⅻ和Ⅹ非常相似,最显著的区别是化合物Ⅻ的C-15位化学位移变成了(δC 71.2),此外与化合物Ⅹ对比,化合物Ⅻ缺少一个甲氧基信号,其他部分化学位移几乎是一模一样的。化合物Ⅻ结构与Ⅹ很相似,唯一的区别是由Ⅹ(C-15)位的甲氧基变成了Ⅻ的亚甲基结合C-15的化学位移和高分辨HRESIMS信息,这样就可以解释了。相对构型同样参考化合物Ⅹ与已知化合物1-((1S,3R,3aS,7aS)-3-(2-(furan-3-yl)ethyl)-3-methoxy-3a,7,7–trimethy loctahydroisobenzofuran-1-yl)propan-1-one来确定;在ROESY谱中,H-20与H-6,H-11,H-12相关,H-19与H-6相关,H-18与H-5相关,综合以上信息化合物Ⅻ的相对构型确定为为(5S),(6S),(9R),(10S),但是由于化合物Ⅻ量少不易结晶,所以其绝对构型不能确定;
本实施例结构式Ⅱ、结构式Ⅴ、结构式Ⅵ和结构式Ⅶ的半日花烷型二萜衍生物的13C NMR数据(δinppm,Jin Hz)见表4,
本实施例结构式Ⅱ和结构式Ⅴ的半日花烷型二萜衍生物的1H NMR(δin ppm,JinHz)见表5,
本实施例结构式Ⅵ和结构式Ⅶ的半日花烷型二萜衍生物的1H NMR(δin ppm,JinHz)见表6;
表4结构式Ⅱ、结构式Ⅴ、结构式Ⅵ和结构式Ⅶ的半日花烷型二萜衍生物的13C NMR数据(δin ppm,Jin Hz)
a Measured at 150MHz in CDCl3,b Measured at 150MHz inAcetone-d6;
表5结构式Ⅱ和结构式Ⅴ的半日花烷型二萜衍生物的1H NMR(δin ppm,JinHz)
a Measured at 600MHz;
表6结构式Ⅵ和结构式Ⅶ的半日花烷型二萜衍生物的1H NMR(δin ppm,Jin Hz)
a Measured at 600MHz.
化合物Ⅱ无色油状,高分辨HRESIMS(m/z 459.2353,[M+Na]+)可知其分子式为C24H36O7,不饱和度为7,13C和DEPT NMR谱图(表4)显示该分子有24个碳信号:9个季羰(包括1个羰基:δC 181.7;1个酯基:δC 171.1;4个烯碳:δC 166.2,δC 143.6,δC 138.4,δC 127.9;1个连氧季碳:δC 79.6)、2个次甲基(包括2个连氧次甲基:δC 104.2,δC 76.2)、7个亚甲基(1个连氧亚甲基:δC 63.2)、6个甲基。进一步分析化合物Ⅱ的HMBC谱,H-20与C-1,C-5,C-9,C-10有相关信号,H-3与C-4,C-5,C-23相关、H-24与C-23相关,H-17与C-7,C-8,C-9相关可推断化合物Ⅱ的A/B环结构片段。即C-3位被乙酰氧基取代,且两组双键分别位于C-5/C-6与C-8/C-9之间,C-7位的羰基由于共轭因素所以化学位移向高场偏移。再仔细分析该化合物的HMBC谱,H-11与C-12,C-9,C-8,C-10相关,H-15与C-13,C-14,C-16,C-21相关;结合1H-1HCOSY谱中H-14与H-15相关,H-11与H-12相关可得出化合物Ⅱ(C-9)位上的取代基即C环结构(五元呋喃环);化合物Ⅱ的平面结构基本上确定;
相对构型通过分子模型结合ROESY相关来确定:H-20与H-3/H-11(δH 2.50)有Roesy相关,H-19与H-3有Roesy相关,说明化合物Ⅱ的H-3、H-19与Me-20属于β取向,而C-3位的乙酰基属于α取向。至此,化合物Ⅱ可确定为3α-(acetyloxy)-15,16-epoxy-15-ethoxy-6,13-dihydroxylabd-5,8-dien-7-one);但是由于化合物Ⅱ量少不易结晶,且通过ROESY谱也无法确定C-13与C-15位取代基的相对构型,所以其绝对构型不能确定;
化合物Ⅴ无色油状;高分辨HRESIMS(m/z 461.2510,[M+Na]+)给出其分子式为C24H38O7,不饱和度为6。13C和DEPT NMR谱图(表4)显示该分子有24个碳信号:7个季羰(包括1个羰基:δC 199.3;1个酯基:δC 170.7;2个烯碳:δC169.8,δC 128.9;1个连氧季碳:δC 79.7)、4个次甲基(包括3个连氧次甲基:δC 104.2,δC 78.4,δC 71.1)、7个亚甲基(包括1个连氧亚甲基:δC 63.2)、6个甲基。仔细对比化合物Ⅴ与Ⅱ的碳谱数据,发现化合物Ⅴ的C-5(δC47.3)与C-6(δC71.1)为叔羰(其中一个含氧),而Ⅱ的C-5/C-6却是双键,其他部分只有少数几个C-5/C-6附近的化学位移有稍许的差异,这些差异是由附近的化学环境变化所引起的,结合高分辨给出的分子式,与化合物Ⅱ对比,可以确定化合物Ⅴ与Ⅱ的唯一差异在于C-5/C-6的双键被还原了,这个结构也可以通过HMBC谱证实;
相对构型通过Roesy谱和化合物Ⅱ来确定。在Roesy谱中H-20与H-3有Roesy相关,H-19与H-3有Roesy相关,说明H-3、H-19与Me-20属于β取向;H-5与H-18/H-6有Roesy相关,所以H-5,Me-18和H-6属于α取向。至此,化合物Ⅴ可确定为3α-acetyloxy-15,16-epoxy-15-ethoxy-6β,13-dihydroxylabd-8-en-7-one)。因为该化合物没有找到适宜做晶体衍射的单晶,且通过ROESY谱也无法确定C-13与C-15位取代基的相对构型,所以其绝对构型不能确定;
化合物Ⅵ无色油状。高分辨HRESIMS(m/z 441.1887,[M+Na]+)可得该化合物分子式为C23H30O7,不饱和度为6。13C和DEPT NMR谱图(表4)显示该分子有23个碳信号:10个季羰(包括1个羰基:δC 182.0;2个酯基:δC 171.7,δC 170.8;5个烯碳:δC 165.3,δC 144.4,δC138.8,δC 137.7,δC 128.8)、2个次甲基(包括1个烯碳:δC 144.5;2个连氧次甲基:δC103.7,δC 76.1)、4个亚甲基、6个甲基(包括1个甲氧基:δC 56.9)。仔细比较化合物Ⅵ与Ⅱ的碳谱数据(表6,表5),发现只有C-11到C-16的化学位移不同,其他部分化学位移的大小基本上差异是相同的,这个差异是由于氘代试剂不同的缘故。通过对比可知化合物Ⅵ具有和Ⅱ一样的A/B环结构。而C环结构通过二维谱图来确定。在HMBC谱中,H-15与C-13,C-16,C-21相关,H-14与C-13,C-15,C-12相关,H-11与C-8,C-9,C-10,C-12,C-13相关;结合1H-1H COSY谱中H-11与H-12相关,H-14与H-15相关可推断化合物Ⅵ的C环结构是一个五元不饱和内酯,且在C-15位上有一个甲氧基取代。至此化合物Ⅵ的平面结构已基本上确定了。通过对各种谱图的对比,化合物Ⅵ相对构型与Ⅱ相同。这个结论可以通过比旋光度对比可知,实验测得化合物Ⅵ与Ⅱ在浓度差不多的情况下具有同样符号的比旋光度,此外在ROESY谱中H-20与H-3有Roesy相关,H-19与H-3有Roesy相关,综合上述数据,将化合物Ⅵ定为3α-(acetyloxy)-15-methoxylabd-5,8,13-trien-13,16-olide-7-one。因为该化合物量少且无法结晶,通过ROESY谱也无法确定C-15位取代基的相对构型,所以其绝对构型不能确定;
化合物Ⅶ无色油状;高分辨HRESIMS(m/z 399.1775,[M+Na]+可得该化合物分子式为C21H28O6,不饱和度为8。13C和DEPT NMR谱图(表4))显示该分子有21个碳信号:8个季羰(包括2个羰基:δC 214.3,δC 197.4;1个酯基:δC 171.8;3个烯碳:δC 164.7,δC 137.8,δC129.5)、4个次甲基(包括1个烯碳:δC 144.4;2个连氧次甲基:δC 103.7,δC 72.0)、4个亚甲基、5个甲基(包括1个甲氧基:δC 56.9)。仔细比较化合物Ⅶ与Ⅵ的碳谱数据(表4)和氢谱数据(表6),发现C-11到C-16的化学位移几乎是一样的,因此可判断化合物Ⅶ具有和Ⅵ一样的C环(五元不饱和内酯)结构。其他部分化学位移也相差不大,最显著的差异是C-3位由(δC76.1)变成了(δC 214.3);C-5/C-6由(δC 138.8)/(δC 144.4)变成了(δC 53.1)/(δC 72.0)。通过与化合物Ⅵ的碳谱数据对比可知化合物Ⅶ的C-3位被氧化成了羰基,而C-5/C-6却被还原成了叔羰(其中一个含氧)。通过HMBC和1H-1H COSY谱也可以证实;至此化合物Ⅶ的平面结构已基本上确定了。化合物Ⅶ的相对构型通过ROESY谱来确定;在ROESY谱中Me-20与Me-19有Roesy相关,H-18与H-5,H-6互相之间都有Roesy相关,综合上述数据,将化合物Ⅶ定为6β-hydroxylabd-15-methoxy-8,13-dien-13,16-olide-3,7-dione。.因为该化合物量少且无法结晶,通过ROESY谱也无法确定C-15位甲氧基的相对构型,所以其绝对构型无法确定;
本实施例结构式Ⅲ的半日花烷型二萜衍生物的1H and 13C NMR数据(δin ppm,JinHz)见表7
表7结构式Ⅲ的半日花烷型二萜衍生物的1H and 13C NMR数据(δin ppm,Jin Hz)
a Measured at 150MHz,b Measured at 600MHz
化合物Ⅲ.无色针晶。高分辨HRESIMS(m/z 391.2130,[M-H]-)可得该化合物分子式为C22H32O6,不饱和度为7。13C和DEPT NMR谱图(表7)显示该分子有21个碳信号:8个季羰(包括2个羰基:δC 213.8,δC 182.0;4个烯碳:δC 164.7,δC 144.4,δC 137.8,δC 129.5)、3个次甲基(包括2个连氧次甲基:δC 103.7,δC 72.0)、4个亚甲基、5个甲基(包括1个甲氧基:δC56.9)。仔细比较化合物Ⅲ与化合物Ⅱ的碳谱数据(表7)和(表4),发现化合物Ⅲ与化合物Ⅱ的C-11到C-22化学位移几乎是一样的,而C-1到C-10的化学位移数据与HeterononeB相同。因此可判断化合物Ⅲ的A/B环结构与HeterononeB相同,而C环结构与Ⅱ相同,这个结论可通过该化合物的HMBC相关谱证实。在ROESY谱中,H-20与H-19相关,综合考虑相关类似物的构型分析,可得化合物Ⅲ的构型为5Z,8Z,10R。因为无法培养出适合做晶体衍射的单晶,所以该化合物绝对构型无法确定;将化合物Ⅲ定为15,16-epoxy-15-ethoxy-6,13-dihydroxylabd-5,8-dien-3,7-dione);
本实施例结构式Ⅰ的半日花烷型二萜衍生物的1H and 13C NMR数据(δin ppm,JinHz)见表8,
表8结构式Ⅰ的半日花烷型二萜衍生物的1H and 13C NMR数据(δin ppm,Jin Hz)
a Recorded at 100MHz.b Recorded at 400MHz
化合物Ⅰ白色无定形粉末,高分辨质谱HR-EI-MS(m/z 374.2091[M]+,calcd for374.2093),13C NMR谱和DEPT谱确定为C22H30O5,不饱和度为8。13C和DEPT NMR谱图(表8)显示该分子有22个碳信号:7个季羰(包括1个羰基:δC 199.1;1个酯基:δC 170.5;3个烯碳:δC169.2,δC 128.6,,δC 124.2;6个次甲基(包括3个烯碳:δC 143.0,δC 138.2,δC 110.4;2个连氧次甲基:δC 78.1,δC 70.8)、4个亚甲基、5个甲基。13C NMR和DEPT显示有22个碳信号。由上述数据可以推断化合物Ⅰ具有半日花烷型二萜的骨架,并与已知化合物6β-hydroxy-15,16-epoxylabda-8,13(16),14-trien-7-one相似,仔细比较两者不同之处发现:在化合物Ⅰ中,上述已知化合物中3位亚甲基(δC 43.4)被乙酰氧基(δC 21.2和170.5)氧化的次甲基(δC78.1)所取代,这一推断由以下两点得到证实:(1)化合物Ⅰ中3位H和C的化学位移明显向低场移动(δH 4.67和δC 78.1);(2)HMBC谱中H-3和H-22与C-21有明显的相关信号;
在化合物Ⅰ的ROESY谱中,H-5α与H-18相关,为α取向,H-20与H-3和H-11相关,为β取向,从而确定化合物Ⅰ的相对构型;化合物Ⅰ的结构最终被确定为3α-acetoxy-6β-hydroxy-15,16-epoxylabda-8,13(16),14-trien-7-one,命名为leojaponin K;
本实施例结构式Ⅷ的半日花烷型二萜衍生物的1H and 13C NMR数据(δin ppm,JinHz)见表9,
表9结构式Ⅷ的半日花烷型二萜衍生物的1H and 13C NMR数据(δin ppm,Jin Hz)
a Recorded at 150MHz.b Recorded at 600MHz
化合物Ⅷ白色无定形粉末,高分辨质谱HR-EI-MS(m/z 305.2402[M+H]+,calcd.327.2425[M+Na]+)可得分子式为C20H32O2,不饱和度为5。13C NMR和DEPT谱图(表9)显示有20个碳信号:5个季羰(包括1个醛基:δC 206.3;2个烯碳:δC 139.9,δC 136.7)、4个次甲基(包括2个烯碳:δC 123.2,δC 118.8)、7个亚甲基(包括1个连氧季碳:δC 59.3)、4个甲基。由上述数据可以推断化合物Ⅷ具有半日花烷型二萜的骨架,并与已知化合物villenol(7)相似,仔细比较发现两者最主要的不同之处在于:villenol(7)中C-19位的羟甲基δC 64.7氧化成了化合物Ⅷ中的羰基δC 206.3,这一推断通过HMBC谱中H-3与C-5,C-19相关,H-5与C-4,C-19相关得到证实;
在化合物Ⅷ的ROESY谱中,H-5α与H-18相关,为α取向,H-20与H-19和H-11相关,为β取向,从而确定化合物Ⅷ的相对构型;因此,化合物Ⅷ的结构最终被确定为15-hydroxy-labda-7,13E-diene-4-al,命名为leojaponin L;
本实施例结构式Ⅳ和结构式Ⅸ的半日花烷型二萜衍生物的1H and 13C NMR数据(δin ppm,Jin Hz)见表10,
表10结构式Ⅳ和结构式Ⅸ的半日花烷型二萜衍生物的1H and 13C NMR数据(δinppm,Jin Hz)
化合物Ⅳ无色油状物质。通过高分辨质谱HR-EI-MS(m/z 366.2420[M]+,calcdfor 366.2406),可得分子式为C21H34O5,不饱和度为5。13C-NMR和DEPT谱图(表10)显示有21个碳信号:6个季碳(包括1个羰基:δC 199.3;2个烯碳:δC 170.2,δC 128.2;1个连氧季碳:δC79.5),3个次甲基(包括2个连氧次甲基:δC 105.4,δC71.0),7个亚甲基和5个甲基(包括1个甲氧基:δC 54.8);由上述数据可以推断化合物Ⅳ同样具有半日花烷型二萜的骨架,并与已知化合物(6β)-15,16-epoxy-15-ethoxy-6,13-dihydroxylabd-8-en-7-one相似,比较两者核磁谱图数据发现主要的不同之处在于(6β)-15,16-epoxy-15-ethoxy-6,13-dihydroxylabd-8-en-7-one中C-15位取代的乙氧基基团δC 63.0,15.2转化成了化合物Ⅳ中的甲氧基基团δC 54.8,通过HMBC谱中MeO与C-15相关,H-15与C-13,C-14,C-16相关证实上述推断;
化合物Ⅸ:无色油状物质。高分辨质谱HR-EI-MS(m/z 320.2344[M]+,calcd for320.2351),可得分子式为C20H32O3,不饱和度为5。13C-NMR和DEPT显示(表10)有20个碳信号:5个季碳(包括1个羧基:δC 184.9;2个烯碳:δC 136.6,δC 140.1),4个次甲基(2个烯碳:δC199.2,δC 123.2),7个亚甲基(包括1个连氧亚甲基:δC 59.4)和4个甲基。由上述数据可以推断化合物Ⅸ具有半日花烷型二萜的骨架,并与已知化合物villenol(7)相似,认真比较发现两者主要的不同之处在于villenol(7)中C-19位的羟甲基δC 64.7氧化成了化合物Ⅸ中的羰基δC 184.9,通过HMBC谱中H-3与C-5,C-19相关,H-5与C-4,C-18,C-19相关证实上述推断;
在ROESY谱中,H-15与H-16相关,可以推导出C-13和C-14间的双键是E构型。另外,H-18与H-5α相关,为α取向,H-20与H-11相关,为β取向,从而确定了化合物Ⅸ的相对构型,与已知化合物villenol(7)相同;由于化合物Ⅸ量少且不易结晶,其绝对构型未能确定。
实施例2:实施例1的结构式为Ⅰ~Ⅻ半日花烷型二萜衍生物的药理实验;
细胞培养:RAW264.7细胞(ATCC)在生长一段时间后逐渐汇合并将瓶底覆盖形成单层细胞膜,当贴壁细胞覆盖率达到70~80%时(约1~2天)进行传代;用PBS溶液(胎牛血清Gibco)洗细胞一次,加入1mL胰酶(Gibco),室温消化5min,加入10mL含10%胎牛血清的DMEM完全培养基,吹散细胞,放入细胞培养箱培养;
MTT细胞毒实验:在96孔板内,每孔加入4×104个对数生长期RAW264.7细胞,随后加入稀释好的化合物样品,在细胞培养箱内培养48h;每孔加入MTT(5mg/ml),37℃孵育4h,弃去上清,加入200μLDMSO溶解甲瓒沉淀,用酶标仪读取OD540/630,计算细胞存活率(%)(见表11);
表11结构式为Ⅰ~Ⅻ半日花烷型二萜衍生物对RAW264.7的细胞毒性
从表11可知,结构式为Ⅰ~Ⅻ半日花烷型二萜衍生物对RAW264.7的细胞毒性很小,CC50均大于50μM;
LPS诱导RAW264.7生成一氧化氮(NO)实验:在96孔板内,每孔加入4×104个对数生长期RAW264.7细胞。加入稀释好的化合物,37℃孵育2h。瑞后,加入LPS(1μg/mL),37℃孵育24h。收集上清100μL,加入等体积Griess试剂,室温避光孵育5min,用酶标仪读取OD540/630,计算NO抑制率(见表12);
表12结构式为Ⅰ~Ⅻ半日花烷型二萜衍生物抑制LPS诱导RAW264.7生成NO
从表12可知,结构式为Ⅰ~Ⅻ半日花烷型二萜衍生物均能有效抑制由LPS诱导的RAW264.7生成NO,IC50在9.6-40.8μM之间,抗炎作用明显;
因此本发明结构式为Ⅰ~Ⅻ的半日花烷型二萜衍生物细胞毒性低,在体外均能有效抑制NF-κB信号通路标志性产物NO的生成,表明化合物的抗炎作用明显;
以结构式为Ⅰ~Ⅻ中的任意一种半日花烷型二萜衍生物为原料,加入药用载体和/或赋形剂即可得到含结构式为Ⅰ~Ⅻ中的任意一种半日花烷型二萜衍生物的药物组合物;
结构式为Ⅰ~Ⅻ中的任意一种半日花烷型二萜衍生物可直接作为核因子-κB(NF-κB)信号通路抑制剂用于治疗由NF-κB介导的疾病,也可以以含结构式为Ⅰ~Ⅻ中的任意一种半日花烷型二萜衍生物的药物组合物的形式作为核因子-κB(NF-κB)信号通路抑制剂用于治疗由NF-κB介导的疾病;
药物组合物中所述结构式为Ⅰ~Ⅻ中的任意一种半日花烷型二萜衍生物的质量百分含量为0.1~99%;
优选的,药物组合物中所述结构式为Ⅰ~Ⅻ中的任意一种半日花烷型二萜衍生物的质量百分含量为0.5~90%;
药用载体和/或赋形剂对人体和动物均无毒,并且为惰性;
药用载体为固体稀释剂、半固体稀释剂、液体稀释剂、填料、药物制品辅剂的一种或多种;
药物组合物以单位体重服用量的形式使用;
药物可经注射(静注、肌注)和口服两种形式给药。
以上,对本发明的实施方式进行了说明。但是,本发明不限定于上述实施方式。凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (6)
1.半日花烷型二萜衍生物,其特征在于,结构式为II、III、VI、VII、X、XI、XII中的任意一种,其中:
结构式VI和VII的通式为结构式VI中R1=aOAc,R2=Δ5(6),R3=OH,R4=CH3,R5=Δ8(9),R6=OCH3,结构式VII中R1=O,R2=αH,R3=βOH,R4=CH3,R5=Δ8(9),R6=OCH3;
2.药物组合物,其特征在于:含有药用载体和/或赋形剂,以及权利要求1所述结构式为II、III、VI、VII、X、XI、XII中的任意一种半日花烷型二萜衍生物。
3.权利要求1所述半日花烷型二萜衍生物的提取方法,其特征在于,具体步骤如下:
(1)将益母草叶和/或枝进行干燥、粉碎;
(2)在室温条件下,采用体积分数为85%的乙醇水溶液对步骤(1)粉碎的益母草叶和/或枝进行提取3~4次,合并乙醇提取液,过滤,将滤液进行减压浓缩得到浸膏;
(3)将步骤(2)的浸膏混悬于水中,依次用石油醚、乙酸乙酯和正丁醇萃取得到石油醚萃取液、乙酸乙酯萃取液和正丁醇萃取液;
(4)将步骤(3)的乙酸乙酯萃取液减压浓缩,然后溶解于氯仿中并吸附于硅胶上,挥发溶剂并研碎,采用硅胶柱层析,采用氯仿/甲醇体系进行梯度洗脱,用薄层色谱法检视,分段收集得到A、B、C、D和E五部分;
(5)步骤(4)的D部分采用硅胶柱层析,采用氯仿/丙酮体系进行梯度洗脱,用薄层色谱法检视,分段收集得到D1、D2和D3三部分;
(6)步骤(5)的D1部分依次用LH-20凝胶色谱柱和C18色谱柱纯化得到结构式Ⅹ、结构式Ⅺ和结构式Ⅻ的半日花烷型二萜衍生物;
(7)步骤(5)的D3部分用提取型高效液相色谱层析分离,采用硅胶柱和体积比为9:2的乙酸乙酯/丙酮混合液洗脱得到结构式Ⅱ、结构式Ⅲ和结构式Ⅵ的半日花烷型二萜衍生物;
(8)步骤(4)的E部分用C18色谱柱层析,采用甲醇/水溶液进行梯度洗脱,用薄层色谱法检视,分段收集得到E1、E2、E3和E4四部分;
(9)步骤(8)的E1部分用硅胶柱层析,分别以氯仿与甲醇的体积比为1:0、50:1、30:1、20:1、10:1、5:1、1:1和0:1的氯仿/甲醇混合液梯度洗脱得到结构式Ⅶ的半日花烷型二萜衍生物。
4.根据权利要求3所述半日花烷型二萜衍生物的提取方法,其特征在于:步骤(4)梯度洗脱用氯仿/甲醇混合液中的氯仿与甲醇的体积比依次为1:0、80:1、40:1、20:1、10:1、5:1、1:1、0:1。
5.根据权利要求3所述半日花烷型二萜衍生物的提取方法,其特征在于:步骤(5)梯度洗脱用氯仿/丙酮混合液中的氯仿与丙酮的体积比依次为40:1、20:1、10:1、5:1、2:1、1:1、0:1。
6.根据权利要求3所述半日花烷型二萜衍生物的提取方法,其特征在于:步骤(8)梯度洗脱用甲醇/水溶液中甲醇与水的体积比依次为3:10、4:10、5:10、6:10、7:10。
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