CN111848722B - 一种雷公藤红素衍生物及其制备方法与应用 - Google Patents
一种雷公藤红素衍生物及其制备方法与应用 Download PDFInfo
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Abstract
本发明属于医药领域,公开了一种雷公藤红素衍生物及其制备方法与应用。所述雷公藤红素衍生物,在雷公藤红素的29位羧酸上通过酰胺键的形成与二肽进行偶联,所述二肽的羧基与叔丁基相连。所述雷公藤红素衍生物可以降低雷公藤红素的细胞毒性、提高稳定性,并增强对肿瘤细胞的选择性,从而提高其作为抗癌成分的实际应用价值。
Description
技术领域
本发明属于医药领域,特别涉及一种雷公藤红素衍生物及其制备方法与应用。
背景技术
雷公藤红素(celastrol)主要来源于中药雷公藤(Tripterygium wilfordii HookF)的根部,其重要药物组成部分包含有醌甲基三萜类化合物,该天然化合物成分具有多种药理活性,包括抗炎、抗氧化、心肌保护、神经元变性、抗肥胖、抑制癌细胞生长、诱导细胞凋亡、抑制肿瘤细胞的侵袭和转移等。近期研究表明,雷公藤红素通过多种分子靶标展示其药理作用,例如SERCA、Hsp90-Cdc37、NF-κB/IKKβ、VEGFR1、VEGFR2及PI3K/Akt信号通路等。最近研究展示雷公藤红素在EGFR(表皮生长因子受体)突变体中诱导了实质性的细胞毒性作用并调动了胞质钙。因此,雷公藤红素成为了非小细胞肺癌(NSCLC)的新候选药物。
基于我们前期的研究发现,雷公藤红素能通过抑制SERCA和P-gp来刺激钙离子介导的自噬、ATP耗竭以及诱导抗药性癌细胞的附属敏感性。
肌浆网-内质网(sarco-endoplasmic reticulum,SR)钙离子转运ATP酶(SERCA)是唯一将钙离子从细胞质转运到SR的泵。SERCA抑制剂可诱导细胞质和线粒体中钙离子的积累与及SR的耗竭,从而触发可导致癌细胞死亡的多种信号通路。雷公藤红素可通过激活钙离子/钙调素依赖性蛋白激酶(Ca2+/calmodulin-dependent kinase kinase-β-AMP-activated protein kinase-mTOR)的途径继而抑制SERCA,从而诱导在抗凋亡的癌症和RASF/RAFLS中的细胞死亡,并改善AIA(佐剂性关节炎)大鼠的关节炎病情。
多重抗药性(multidrug-resistance,MDR)是一种癌细胞的现象,其特征是对许多抗癌药物具有交叉的抗药性。由多重抗药性基因(简称为MDR1或ABCB1)编码的人类P-糖蛋白(P-gp)是一种ABC转运蛋白(ATP-binding cassette membrane transporter),并具有药物外排泵的作用。P-gp在肿瘤细胞中的过度表达降低了细胞内药物的浓度,从而降低了多种抗癌药物的细胞毒性。由于这些MDR转运体在临床肿瘤学中带出的重要性,因此对有效的P-gp抑制剂研发已经进行了深入研究。迄今为止,P-gp抑制剂已开发了三代。维拉帕米(verapamil,VPM)是P-gp抑制剂的原型,并可增强加许多抗癌药物在细胞内的积累,如肺癌细胞A549和肝癌细胞HepG2中阿霉素(doxorubicin)的积累。伐司扑达(Valspodar,PSC833)为环孢菌素D(cycl osporin D)衍生物的P-gp抑制剂,能增加在白血病细胞中柔红霉素(daunorubicin)的摄取。
尽管雷公藤红素展示了良好的治疗效能,但它亦具备一些不良的药理特性(如高毒性、稳定性差等),使得其进一步的应用面临阻碍。
因而药物化合家希望通过合成或半合成的方法,对雷公藤红素进行结构上的修饰,以期获得具新活性的雷公藤红素衍生物,来降低其毒性。目前,雷公藤红素的2及3位上的结构修饰已被报导对许多癌细胞株具有良好的细胞毒性,但仍需进一步开发研究,以寻求性能更好的结构修饰方法。
因此,希望提供一种可改善雷公藤红素原有的不良药理特性(对正常细胞毒性大)的雷公藤红素衍生物。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此,本发明提出一种雷公藤红素衍生物,可以降低雷公藤红素的细胞毒性和提高稳定性,并增强对肿瘤细胞的选择性,从而提高其作为抗癌成分的实际应用价值。
一种雷公藤红素衍生物,在雷公藤红素的29位羧酸上通过酰胺键的形成与二肽进行偶联,所述二肽的羧基与叔丁基相连。
申请人发现,雷公藤红素通过在29位羧酸上与二肽偶联可制备低细胞毒性的无活性前体药物,而该前体药物需由癌细胞中的特异性蛋白酶激活,才会具有较强的细胞毒性,因此药物的安全性得到提高。由于羧酸易于被转化,较为适合作为修饰位点,因此将雷公藤红素29位上的羧基转化成酰胺键可提高在体内外的稳定性,不易被酶水解,并可作为氢键供体提高该药物成分的生物活性。通过叔丁基对羧基进行保护可提高化合物的稳定性,并有助于调节药物亲脂性,还可增强对SERCA的抑制效果。
优选的,所述二肽选自天冬氨酸-谷氨酸、谷氨酸-天冬氨酸、天冬氨酸-天冬氨酸或谷氨酸-谷氨酸。与上述二肽底物相连的雷公藤红素衍生物可被癌细胞中的酶水解,并产生抗肿瘤效应,因此可作为良好的前体药物。
更优选的,所述二肽的氨基酸之间以α键或γ键连接。
优选的,所述雷公藤红素衍生物的结构式如式(I)或式(II)所示:
上述雷公藤红素衍生物的制备方法,包括以下步骤:在1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐、1-羟基苯并三唑和三乙胺的条件下,雷公藤红素与所述二肽经缩合反应生成所述雷公藤红素衍生物。
优选的,反应所用溶剂为二氯甲烷和/或四氢呋喃。
上述雷公藤红素衍生物在制备抗肿瘤药物中的应用。
一种抗肿瘤药物,包括上述的雷公藤红素衍生物,还包括药学上可接受的辅料。
优选的,所述药学上可接受的辅料为溶剂、润湿剂、乳化剂、增稠剂、赋形剂、悬浮剂、崩解剂、填充剂、润滑剂或稀释剂中的至少一种。
相对于现有技术,本发明的有益效果如下:
(1)相比于雷公藤红素,本发明所述雷公藤红素衍生物对正常细胞的毒性有所降低,同时对一些肿瘤细胞的选择性得到提高;
(2)本发明所述雷公藤红素衍生物保留了对SERCA和P-gp双重靶向的抗肿瘤活性,具备良好的抗肿瘤效应。
附图说明
图1表示伐司扑达、雷公藤红素(化合物1)、化合物3和化合物6在人类P-糖蛋白上的对接姿势;
图2表示毒胡萝卜素、雷公藤红素(化合物1)、化合物3和化合物6在SERCA上的对接姿势;
图3表示采用雷公藤红素(Celastrol)、化合物3和化合物6处理后HeLa细胞中Ca2+的动态变化;
图4表示在毒胡萝卜素或化合物C存在下,雷公藤红素(Celastrol)、化合物3和化合物6处理后HeLa细胞中Ca2+的动态变化;
图5表示使用维拉帕米、雷公藤红素(化合物1)、化合物3和化合物6后,A549细胞中罗丹明123的积累量;
图6表示雷公藤红素(化合物1)、化合物3和化合物6对LO2、HepG2和HepAD38诱导细胞凋亡的诱导活性。
具体实施方式
为了让本领域技术人员更加清楚明白本发明所述技术方案,现列举以下实施例进行说明。需要指出的是,以下实施例对本发明要求的保护范围不构成限制作用。
以下实施例中所用的原料、试剂或装置如无特殊说明,均可从常规商业途径得到,或者可以通过现有已知方法得到。
雷公藤红素从成都三叶生物科技有限公司(中国成都)购买。硅胶FCP200-300目用作柱层析。所有化学品均购自毕得医药科技有限公司(中国上海)和百灵威科技有限公司(中国香港)。在25℃下,用Bruker AV-600核磁共振波谱仪在CDCl3中收集1H-NMR和13C-NMR谱。所有化学位移都以百万分之一的标准δ标记法报告,使用CDCl3残余质子讯号的峰值作为内标。用安捷伦6230(LC-ESI-MS/MS)以电喷雾电离(ESI)进行质谱分析。采用数位显微熔点测定仪进行了熔点分析。所有化合物都经1H-NMR、13C-NMR、IR、LC-ESI-MS和HPLC确认纯度均在98.0%以上。
实施例1
将雷公藤红素(化合物1)与二肽通过雷公藤红素29位羧酸上以酰胺键的形式进行偶联,得到以下一系列雷公藤红素衍生物(化合物2-7):
实施例2
对实施例1的雷公藤红素(化合物1)与雷公藤红素衍生物(化合物2-7)采用MTT法进行细胞毒性的测试,所用细胞为人正常肝细胞LO2和肺上皮细胞BEAS-2B,测试结果如表1所示:
表1
由表1可知,与化合物1相比,化合物2-7对正常细胞的毒性得到明显的下降,证实本发明对雷公藤红素的结构修饰有效提高了其性能。
实施例3
通过测试雷公藤红素衍生物的选择性指数(正常细胞的IC50/癌细胞的IC50),得到的结果为:与天然的雷公藤红素(化合物1)相比,实施例1中的一系列雷公藤红素衍生物对肿瘤细胞的选择性得到提高。其中化合物3对肝癌细胞株HepAD38的选择特异性,相比于正常细胞系株LO2和CCD19Lu,分别高出了30倍以上和10倍以上;其中相比于正常细胞系株CCD19Lu,化合物6对肝癌细胞株HepAD38和肝癌细胞株HepG2选择性得到提高,均提高了12倍以上;具体测试数据如表2所示:
表2
实施例4
实施例1中化合物3的具体制备方法为:
将N-Fmoc-L-天冬氨酸-β-叔丁酯(500mg)溶解在20mL四氢呋喃:二氯甲烷(3:2)中,并混合用10mL二氯甲烷溶解的L-天冬氨酸二叔丁基酯盐酸盐(340mg)。加入1-羟基苯并三唑(245mg)后,将烧瓶冷却至0℃,然后滴加N,N'-二异丙基碳二亚胺(0.28mL)。反应1小时后,自然升温至室温,并搅拌过夜。反应完毕,过滤所得混合物以除去沉淀的尿素。滤液用1M盐酸洗涤3次,10%碳酸氢钠溶夜洗涤3次,盐水洗涤1次,无水硫酸钠干燥,过滤浓缩至适当量,产物在乙酸乙酯中重结晶,得到中间产物;
将该中间产物用20%哌啶溶液(5mL)溶解,并反应30分钟,哌啶通过与甲醇共蒸发3次除去,可去除中间产物上的保护基Fmoc,制得二肽化合物3';
将雷公藤红素(30mg)溶解在二氯甲烷中,并加入1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(32mg),1-羟基苯并三唑(21mg)及二肽化合物3'(50mg),在0℃搅拌反应30分钟。加入50μL三乙胺,并继续在室温下反应过夜。反应完毕,混合物用水洗涤2次,无水硫酸钠干燥,过滤浓缩至适当量,经柱层析(二氯甲烷:甲醇=100:1)分离纯化,得到化合物3,产率为38%。
实施例5
实施例1中化合物6的具体制备方法为:
将N-Fmoc-L-谷氨酸-1-叔丁酯(400mg)溶解在20mL四氢呋喃:二氯甲烷(3:2)中,并混合用10mL二氯甲烷溶解的L-谷氨酸二叔丁酯盐酸盐(406mg)。加入1-羟基苯并三唑(190mg)后,将烧瓶冷却至0℃,然后滴加N,N'-二异丙基碳二亚胺(0.22mL)。反应1小时后,自然升温至室温,并搅拌过夜。反应完毕,过滤所得混合物以除去沉淀的尿素。滤液用1M盐酸洗涤3次,10%碳酸氢钠溶夜洗涤3次,盐水洗涤1次,无水硫酸钠干燥,过滤浓缩至适当量,产物在乙酸乙酯中重结晶,得到中间产物;
将该中间产物用20%哌啶溶液(5mL)溶解,并反应30分钟,哌啶通过与甲醇共蒸发3次除去,可去除中间产物上的保护基Fmoc,制得二肽化合物6';
将雷公藤红素(30mg)溶解在四氢呋喃:二氯甲烷(3:2)中,并加入1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(32mg),1-羟基苯并三唑(21mg)及二肽化合物6'(79mg),在0℃搅拌反应30分钟。加入50μL三乙胺,并继续在室温反应过夜。反应完毕,混合物用水洗涤2次,无水硫酸钠干燥,过滤浓缩至适当量,经柱层析(二氯甲烷:甲醇=100:1)分离纯化,得到化合物6,产率为36%。
实施例6
雷公藤红素衍生物的药物靶点预测
使用Autodock 4.2Lamarckian算法和Autodock工具1.5.7rc1进行对接研究。基于小鼠P-gp结构(PDB程式码:4M1M)透过MODELLER 9.11软件建立了人P-gp的同源性模型。对于P-gp定义的对接,涵盖药物结合结构域。对于SERCA定义的对接,选择Ca2+结合位点,将伐司扑达(Valspodar)和毒胡萝卜素(thapsigargin)分別作为P-gp和SERCA对接计算的对照药物。对接参数设置为每个周期运行250次,进行2500000次能量评估,并进行三次独立计算。对接姿态的视觉化是通過VMD(伊利诺伊大学香槟分校贝克曼研究所的视觉分子动力学、理论和计算生物物理小組)完成的。对结合位点的必需氨基酸进行了测定,并与毒胡萝卜素(thapsigargin)、伐司扑达(Valspodar)和雷公藤红素(化合物1)进行了比较,并比较雷公藤红素衍生物(化合物3和化合物6)与SERCA和P-gp模型的分子间相互作用。
如图1和图2所示,其中化合物3(-9.7kcal/mol对P-gp,-6.3kcal/mol对SERCA),化合物6(-9.7kcal/mol对P-gp及-7.5kcal/mol对SERCA),与常规抑制剂(毒胡萝卜素、伐司扑达)的最稳定的结构有显著不同的位点。
通过比较毒胡萝卜素(thapsigargin)、雷公藤红素(化合物1)和雷公藤红素衍生物(化合物3和化合物6)对接至ATP酶(SERCA)的最低结合能(LBE,kcal/mol),结果如表3所示:
表3
通过比较伐司扑达(valspodar)、维拉帕米(verapamil)、雷公藤红素(化合物1)和雷公藤红素衍生物(化合物3和化合物6)对接至人类P-糖蛋白(P-gp)的最低结合能(LBE,kcal/mol),结果如表4所示:
表4
通过比较毒胡萝卜素(thapsigargin)、雷公藤红素(化合物1)和雷公藤红素衍生物(化合物3和化合物6)对接至钙离子转运ATP酶(SERCA)的预测抑制常数pKi[μM]值,结果如表5所示:
表5
pKi-1 | pKi-2 | pKi-3 | 平均pKi | 标准差 | |
化合物1 | 3.83 | 3.98 | 4.11 | 3.973 | 0.140 |
化合物3 | 18.6 | 27.65 | 18.66 | 21.637 | 5.207 |
化合物6 | 5.53 | 2.39 | 2.3 | 3.406 | 1.839 |
毒胡萝卜素 | 318.92 | 462.75 | 168.75 | 316.807 | 147.011 |
通过比较伐司扑达(valspodar)、雷公藤红素(化合物1)和雷公藤红素衍生物(化合物3和化合物6)对接至人类P-糖蛋白(P-gp)的预测抑制常数pKi[μM]值,结果如表6所示:
表6
如表3所示,化合物3和化合物6与SERCA的相互作用均比毒胡萝卜素强;如表4所示,化合物3和化合物6与P-gp的最低结合能与伐司扑达相近,但比维拉帕米要较低。如表5和表6所示,与SERCA相比,化合物3和化合物6对人类P-糖蛋白具有更高的亲和力和抑制能力。
在进一步探究雷公藤红素、雷公藤红素衍生物(化合物3和化合物6)对P-gp和SERCA的分子对接情况时发现,化合物3在其最稳定结构的情况下,连接至三萜20位的N-H肽基团,与Gln946(氢键)的相互作用与伐司扑达相似;而化合物6的A环有助于与Phe343产生疏水相互作用。
实施例7
雷公藤红素衍生物对SERCA的抑制及钙激活作用
使用FLIPR钙6检测试剂盒(Molecular Devices)测定细胞内胞浆Ca2+动态。将HeLa细胞种在黑壁/透明底的96孔板中,密度为每孔10000个细胞,并在处理前培养过夜。第二天,用钙6试剂在37℃和5%CO2下处理HeLa细胞2小时。然后,将指定浓度的雷公藤红素(celastrol)、雷公藤红素衍生物(化合物3和化合物6)和毒胡萝卜素添加到孔中,并立即在FLIPR-Tetra高通量实时荧光检测分析系统(Molecular Devices)上进行数据获取。在室温下,以1秒的读取间隔进行三次独立的实验。
前期我们通过对类风湿性关节炎患者的滑膜成纤维细胞和多重抗药性肿瘤细胞进行研究发现,雷公藤红素在类风湿关节炎(RA)和多重抗药性癌症中的良好疗效,并且表明其作用机理通过与SERCA相关的细胞内钙离子流动,从而诱导细胞的自噬性死亡。因此,我们希望探明雷公藤红素衍生物,即化合物3和化合物6是否会在癌细胞中产生类似的钙离子的流动作用。
如图3所示,FLIPR钙6的分析结果表明:与雷公藤红素的作用效果相同的是,对HeLa细胞单次使用雷公藤红素衍生物(化合物3、化合物6)均会引起钙流动的变化。而图4则显示:将HeLa细胞与毒胡萝卜素预先培养以耗尽SERCA依赖性的钙离子的储存,会有效抑制雷公藤红素衍生物3和6诱导的钙离子流动。同时,阴性对照的预处理,化合物C(Dorsomorphin,一种AMPK抑制剂,且与SERCA无已知相互作用)不会影响这些化合物诱导钙离子的流动变化。
因此,化合物3和化合物6可通过特异性靶向SERCA来调节在癌细胞HeLa中的钙离子流动,以实现抗肿瘤的作用。
实施例8
雷公藤红素衍生物对P-gp的抑制作用
本发明对P-gp活性的测定是通过在A549细胞系中,测定罗丹明(Rho)123染料在细胞内的积累情况从而得出。A549细胞在37℃下分別与对照组(Ctrl)、10μM维拉帕米(verapamil,即VPM)、0.5μM雷公藤红素、0.5μM化合物3或0.5μM化合物6孵育4小时,并设置一组未染色组(unstained)。随后,将5μg/mL罗丹明123添加到每个孔中,并将孔再孵育1小时。在磷酸盐缓冲液中进行洗涤,将细胞置于蒸馏水中,细胞内罗丹明123的含量通过CellQuest荧光分光光度法进行定量测定。使用Prism软件对至少三次独立实验的实验数据进行平均值±SEM统计标示。
如图5所示,对照组中A549细胞的罗丹明123积累很差,仅有26.5%,从而反映了P-gp介导的荧光染料的外排。在已知的P-gp抑制剂维拉帕米(verapamil)存在下,10μM的药物浓度便能完全抑制P-gp的活性,使罗丹明123在A549细胞中的积累得到显着增加,达到82.8%的水平。在雷公藤红素的浓度(0.5μM)比维拉帕米低20倍的情况下,发现A549细胞中罗丹明123的累积为71.7%。而在相同浓度的情况下,化合物6的积累值为72.7%,处于与雷公藤红素相当的水平;而化合物3的积累值更高,达到84.7%。这些数据表明化合物3和化合物6可以有效抑制P-gp的活性,且化合物3表现出对P-gp活性的最强抑制作用。这些结果与用化合物3和化合物6进行P-gp的对接计算结果相吻合。
实施例9
雷公藤红素衍生物对细胞产生的凋亡作用
使用细胞凋亡检测试剂盒(Santa Cruz biotechnology)检测细胞凋亡相关的细胞生长和增殖抑制,所用细胞株包括LO2,HepG2和HepAD38。将细胞在6孔培养板中培养,37℃孵育24小时后,将指数生长的细胞放入浓度均为1μM的雷公藤红素(化合物1)、化合物3或化合物6中孵育24小时,并设置一组对照组(Ctrl);收集细胞,用PBS缓冲液洗涤两次,再用100μL 1×结合缓冲液悬浮,然后用1μL annexin V-FITC和10μL PI染色,在黑暗和室温下反应15分钟。使用FACSCalibur流式细胞仪和Cell Quest软件(Becton–Dickinson)计数凋亡细胞。
如图6所示,与对照组相比,经浓度为1μM的雷公藤红素处理后,annexin V-FITC与LO2细胞的结合的凋亡率显着增加,达到24.6%;而采用化合物3和化合物6处理的则较少,分别为12.3%和9.84%。当LO2细胞用雷公藤红素(化合物1)、化合物3和化合物6处理时,数据点会分散并转移到Q3面,这表明细胞已进入早期的细胞凋亡。实验结果表明,化合物3和化合物6诱导正常细胞的凋亡率比雷公藤红素(化合物1)要低,显示对正常细胞的毒性较小。此外,在对HepAD38癌细胞的毒性作用比较中,化合物3的凋亡率(21.4%)高于化合物1(13.9%)和化合物6(6.34%),表明化合物3对HepAD38癌细胞具有更高的选择性杀伤效果。
实施例10
一种抗肿瘤药物,包括实施例1中化合物2-7的至少一种,以及作为溶剂的去离子水。
Claims (7)
1.一种雷公藤红素衍生物,其特征在于,在雷公藤红素的29位羧酸上通过酰胺键的形成与二肽进行偶联,所述二肽的羧基与叔丁基相连;
所述二肽选自天冬氨酸-谷氨酸、谷氨酸-天冬氨酸、天冬氨酸-天冬氨酸或谷氨酸-谷氨酸;所述二肽的氨基酸之间以α键或γ键连接。
3.权利要求1-2中任一项所述雷公藤红素衍生物的制备方法,其特征在于,包括以下步骤:在1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐、1-羟基苯并三唑和三乙胺的条件下,雷公藤红素与所述二肽经缩合反应生成所述雷公藤红素衍生物。
4.根据权利要求3所述的制备方法,其特征在于,反应所用溶剂为二氯甲烷和/或四氢呋喃。
5.权利要求1-2中任一项所述雷公藤红素衍生物在制备抗肿瘤药物中的应用。
6.一种抗肿瘤药物,其特征在于,包括权利要求1-2中任一项所述的雷公藤红素衍生物,还包括药学上可接受的辅料。
7.根据权利要求6所述的抗肿瘤药物,其特征在于,所述药学上可接受的辅料为溶剂、润湿剂、乳化剂、增稠剂、赋形剂、悬浮剂、崩解剂、填充剂、润滑剂或稀释剂中的至少一种。
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