CN116574138B - 二溴酪氨酸-铱配合物及其制备方法与应用 - Google Patents
二溴酪氨酸-铱配合物及其制备方法与应用 Download PDFInfo
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Abstract
本发明属于有机物合成领域,具体涉及二溴酪氨酸-铱配合物及其制备方法与应用。本发明以酪氨酸为起始原料,合成了天然产物二溴酪氨酸衍生物,并证明此天然产物通过凋亡杀死宫颈癌细胞。进一步合成含有二溴酪氨酸的两个环金属铱配合物,Ir4和Ir5。Ir4和Ir5导致细胞内活性氧升高等一系列线粒体损伤,染色质聚集,以及凋亡相关蛋白caspase‑3的激活,能够诱导宫颈癌细胞发生凋亡。Ir4和Ir5也可以诱导细胞内谷胱甘肽水平降低、脂质过氧化物积累、谷胱甘肽过氧化物酶4下调等铁死亡的特征,且Ir4和Ir5均对宫颈癌3D细胞球有较好的毒性作用,其中Ir5对细胞球杀伤力最显著。最终Ir4,Ir5可通过凋亡和铁死亡双重方式诱导宫颈癌细胞死亡。
Description
技术领域
本发明属于有机物合成领域,具体涉及二溴酪氨酸-铱配合物及其制备方法与应用。
背景技术
抗癌药物触发细胞凋亡死亡是杀死癌细胞的主要方法之一。然而,由于癌细胞对凋亡具有获得性或内在抗性,诱导凋亡在肿瘤中的效果有限,许多肿瘤细胞表现出耐药性。非凋亡性死亡通常包括坏死、自噬、副凋亡、胀亡、铁死亡、焦亡等。铁死亡与癌症免疫治疗、转移、能量代谢和耐药性有关,是一种非凋亡细胞死亡,由于其能够克服传统凋亡介导的癌症治疗的一些局限性而受到越来越多的关注。铁死亡是由不受限制的脂质过氧化驱动的,伴随着GSH的消耗和ROS的产生,接下来抑制脂质修复酶谷胱甘肽过氧化物酶4(GPX4)的生物合成。GPX4利用辅助因子GSH,还原具有细胞毒性的脂质过氧化物,它的下调被认为是铁死亡的一个重要标志。通过化合物的结构修饰可获得新的细胞死亡模式或多个细胞死亡途径,这是一个有效的策略来克服耐药最终导致肿瘤细胞死亡。
海洋生物资源以其丰富的生物多样性和化合物多样性成为近期海洋药物研究开发的热潮。许多海洋天然产物具有显著的生物活性,例如,从海绵、海鞘、软珊瑚、海兔、苔藓虫等提取分离出来的酰胺类、萜类、大环内酯类、肽类等具有良好的抗肿瘤活性。海洋生物的多样性为人类提供了许多结构新颖、活性优良的先导化合物。国际上已投入应用的海洋药物经典的例子有头孢霉素、阿糖腺苷、阿糖胞苷等,这些也是较早开发成功的现代海洋药物,现已广泛用于临床。二溴酪氨酸是一种广泛存在于海洋生物海绵中的氨基酸,并且有抗癌活性。在非铂类药物中,铱配合物因其催化细胞内反应、定位于特定亚细胞器、作用机制不同于铂类药物而备受关注。已有文献报道,用6-氨基己酸将两活性物质连接,活性增强。将金属铱配合物与海洋天然产物连接,以期望可以发挥更好的抗癌活性,其研究对海洋药物抗肿瘤的发展具有重要的意义。
发明内容
本发明提供了一种二溴酪氨酸-铱配合物Ir4,结构式如图1所示。
本发明还提供了二溴酪氨酸-铱配合物Ir4的制备方法,包括以下步骤:
(1)将L-酪氨酸加入到甲醇中,冰浴下滴加氯化亚砜,加热回流过夜,反应完毕后旋蒸干溶液后乙酸乙酯洗涤,抽滤得到物质2;
(2)将物质2和N-溴代琥珀酰亚胺NBS溶解在二氯甲烷中,室温搅拌,减压下旋干,萃取,得到产物3;
(3)将产物3中加入甲醇和二碳酸二叔丁酯,加入三乙胺,室温下搅拌。除去溶剂,萃取,纯化得到产物4;
(4)将产物4、碘甲烷溶解在N,N-二甲基甲酰胺DMF中,加入碳酸钾搅拌,反应完成后旋干溶剂,萃取,粗产物纯化得到物质5;
(5)在冷二氯甲烷,加入产物5和三氟乙酸室温反应。蒸发溶剂,调PH到7,纯化得到产物6;
(6)将1,10-菲罗啉-5,6-二酮和对羧基苯甲醛、醋酸铵放入冰醋酸中,加热,反应后将溶液用氨水中和,静置后抽滤得到物质7;
(7)将物质7,6-氨基己酸甲酯盐酸盐、2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐HATU和N,N-二异丙基乙胺DIEA加入到DMF中,室温搅拌24h,反应完成后旋干溶剂,水洗,粗产物经纯化得到物质8;
(8)将物质8在氢氧化钠的醇溶液中水解,加热回流,然后浓缩乙醇,调节PH,得到的沉淀抽滤即为物质9;
(9)将物质9,物质6,HATU和DIEA加入到DMF中,搅拌,反应完成后旋干溶剂,水洗,粗产物纯化得到物质11;
(10)将IrCl3·nH2O和bzq加入到乙二醇-乙醚和水的混合溶液中,加热回流,过滤即得Ir(ppy)2Cl2;
(11)将物质7,[Ir(bzq)2Cl]2混悬于CH2Cl2/CH3OH的溶液中,在氩气环境下加热回流,冷却至室温后,加入过量的KPF6,纯化,得到产物10;
(12)将物质10和物质6、2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐HATU和N,N-二异丙基乙胺DIEA加入到DMF中,搅拌,反应完成后旋干溶剂,水洗,粗产物纯化得到物质Ir4。
进一步的,所述步骤(1)中回流过夜具体为60℃;步骤(2)中所述萃取为乙酸乙酯和水萃取;步骤(3)中所述萃取为使用乙酸乙酯萃取,纯化为层析柱纯化;步骤(4)中所述萃取为乙酸乙酯萃取,所述纯化为过硅胶柱纯化。
进一步的,所述步骤(5)中冷二氯甲烷温度为-5~4℃,所述纯化为硅胶柱纯化;步骤(6)中所述加热为100~140℃加热6h;步骤(7)所述纯化为硅胶柱层析纯化;步骤(8)所述加热回流为100℃加热回流4h。
进一步的,所述步骤(9)中纯化为硅胶柱层析法纯化;步骤(10)IrCl3·nH2O和bzq摩尔比1:2,乙二醇-乙醚和水的混合溶液中乙二醇-乙醚和水的体积比为3:1;步骤(11)所述纯化为硅胶柱层析纯化;步骤(12)中纯化为硅胶柱层析纯化。
本发明还提供了一种二溴酪氨酸-铱配合物Ir5,结构式如图1所示。
本发明还提供了二溴酪氨酸-铱配合物Ir5的制备方法,将上述物质11和[Ir(bzq)2Cl]2混悬于CH2Cl2/CH3OH的溶液中,在氩气环境下加热回流4h,冷却至室温后,加入过量的KPF6,通过真空蒸发获得粗产物,经纯化后得到产物Ir5。
进一步的,所述CH2Cl2/CH3OH的溶液CH2Cl2与CH3OH体积比为2:1;所述纯化为硅胶柱层析纯化。
本发明还提供了二溴酪氨酸-铱配合物Ir4或Ir5在制备抗肿瘤药物中的应用。
本发明具有以下有益效果:
本专利以酪氨酸为起始原料,合成了天然产物二溴酪氨酸衍生物,并证明此天然产物通过凋亡杀死宫颈癌细胞。进一步合成含有二溴酪氨酸的两个环金属铱配合物,用6-氨基己酸将二溴酪氨酸衍生物和金属铱配合物连接起来合成Ir5,而Ir4直接缩合连接。两种配合物可以有效积累到溶酶体,并且在细胞内导致活性氧升高等一系列线粒体损伤,染色质聚集,以及凋亡相关蛋白caspase-3的激活,两种配合物以及二溴酪氨酸衍生物均能够诱导宫颈癌细胞发生凋亡。另外研究中发现,我们检测到Ir4,Ir5处理后的细胞GSH和铁死亡相关蛋白GPX4的下调,以及MDA含量升高,线粒体形态改变,触发宫颈癌细胞铁死亡,而二溴酪氨酸衍生物几乎不能诱导宫颈癌细胞发生铁死亡。相对于Ir4和二溴酪氨酸衍生物,Ir5对3D细胞球表现出较强的抗肿瘤活性。最终Ir4,Ir5可通过凋亡和铁死亡双重方式诱导宫颈癌细胞死亡。
附图说明
图1为Ir4和Ir5的结构图;
图2为Ir4的质谱;
图3为Ir5的质谱;
图4为Ir4的核磁图;
图5为Ir5的核磁图;
图6为在乙腈和缓冲液中Ir4的紫外和荧光图谱;
图7为在乙腈和缓冲液中Ir5的紫外和荧光图谱;
图8为Ir4、Ir5和物质6的溶血性实验;
图9为随时间变化Ir4、Ir5和物质6进入细胞含量变化;
图10为Ir4的亚细胞器定位;
图11为Ir5的亚细胞器定位;
图12为Ir4、Ir5和物质6的ROS染色;
图13为Ir4、Ir5和物质6的DHE染色;
图14为Ir4、Ir5和物质6的JC-1染色实验;
图15为Ir4、Ir5和物质6的Hoechst 33342染色;
图16为GSH的消耗;
图17为GSH和GSSG变化柱状图;
图18为GSH和NAC对细胞存活率影响;
图19为MDA检测;
图20为不同死亡抑制剂对细胞的影响;
图21为Western bolt实验;
图22为透射电镜图;
图23为药物对MCTSs的影响。
具体实施方式
实施例1
将L-酪氨酸(MW=181,1.81g)加入到甲醇中,冰浴下滴加氯化亚砜1ml,60度回流过夜,反应完毕后旋蒸干溶液后乙酸乙酯洗涤,抽滤得到物质2,产率98%。将物质2(MW=195,1.95g)和N-溴代琥珀酰亚胺NBS(MW=178,3.56g)溶解在二氯甲烷中,室温搅拌24h,减压下旋干,乙酸乙酯和水萃取,得到产物3,产率96%;将产物3(MW=353,2.82g)中加入40mL甲醇和二碳酸二叔丁酯(MW=218,2.18g),加入三乙胺1.5mL,室温下搅拌24h。除去溶剂,乙酸乙酯萃取,层析柱纯化得到产物4,产率93%。产物4(MW=451,3.6g)、碘甲烷(MW=142,63μL)溶解在N,N-二甲基甲酰胺(DMF)中,加入碳酸钾(MW=138,2.2g)室温搅拌24h,反应完成后旋干溶剂,用乙酸乙酯萃取,粗产物过硅胶柱得到物质5,产率80%;在冷却到0度的二氯甲烷30mL中,加入产物5(MW=465,2.3g)和三氟乙酸(5mL)室温反应12h。蒸发溶剂,调PH到7,硅胶柱纯化得到产物6,产率95%。
将1,10-菲罗啉-5,6-二酮(MW=210,1g)和对羧基苯甲醛(MW=150,0.75g)、醋酸铵(MW=77,6g)放入到100mL冰醋酸中,130度加热6h,反应后将溶液用氨水中和,静置后抽滤得到物质7,产率89%。
将物质7(MW=340,0.68g),6-氨基己酸甲酯盐酸盐(MW=181,0.36g)、HATU(MW=380,0.76g)和DIEA(MW=129,1mL)加入到20mL DMF中,室温搅拌24h,反应完成后旋干溶剂,水洗,粗产物过硅胶柱得到物质8,产率81%。将物质8(MW=467,0.93g)在氢氧化钠(MW=40,0.32g)的醇溶液中水解,100度加热回流4h,然后浓缩乙醇,用1M HCl调节PH,得到的沉淀抽滤即为物质9,产率80%。将物质9(MW=453,0.91g),物质6(MW=365,0.73g),2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(HATU,MW=380,0.76g)和DIEA(MW=129,1mL)加入到20mL DMF中,室温搅拌24h,反应完成后旋干溶剂,水洗,粗产物过硅胶柱得到物质11,产率83%。
(Ir2(bzq)4Cl2)的合成,将IrCl3·nH2O(1.192g,2mmol)和bzq(0.72g,4mmol)以摩尔比1:2加入到乙二醇-乙醚和水的混合溶液(3:1,v/v)中,加热回流24h,过滤即得Ir(bzq)2Cl2。
将物质7(MW=340,0.68g),[Ir(bzq)2Cl]2(MW=1168,0.117g)混悬于CH2Cl2/CH3OH(2:1,v/v)的溶液中,在氩气环境下加热回流4h。冷却至室温后,加入过量的KPF6。硅胶柱层析纯化,得到产物10,产率75%。
将物质10(MW=1034,0.1034g)和物质6(MW=365,0.0365g)、2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐HATU(MW=380,0.038g)和N,N-二异丙基乙胺(DIEA)(MW=129,0.05mL)加入到20mLDMF中,室温搅拌24h,反应完成后旋干溶剂,水洗,粗产物过硅胶柱得到物质Ir4,产率71%。
将物质11(MW=800,0.160g)和[Ir(bzq)2Cl]2(MW=1168,0.117g)混悬于CH2Cl2/CH3OH(2:1,v/v)的溶液中,在氩气环境下加热回流4h。冷却至室温后,加入过量的KPF6。通过真空蒸发获得粗产物。硅胶柱层析纯化,得到产物Ir5,产率70%。
实施例2生物活性实验
(1)对实施例中的化合物Ir4和Ir5的细胞毒性进行试验,试验方法如下:
细胞毒性实验采用MTT法测定:取处于对数生长期的Siha细胞以5×103/孔均匀接种于96孔板,培养12h。待细胞贴壁,分别以浓度梯度的方式加入Ir4、Ir5、物质6和物质10(DMSO处理组作为对照组,不接种细胞组作为空白组)。孵育完成后,加入MTT于96孔板中于37℃孵育4h,随后小心吸出培养液,室温下加入150μL/孔DMSO溶解甲鐕,振荡摇匀后,采用酶标仪于570nm波长处测定OD值,并计算细胞存活率。
表1.Ir4和Ir5的细胞毒性试验结果
两种铱配合物抗肿瘤效果均强于物质10,Ir5抗肿瘤活性优于Ir4,且Ir5细胞毒性将近是物质6的9倍,Ir5的抗肿瘤活性可能归因于其独特的结构。
(2)生物安全性
新鲜红细胞经1000rpm离心10min分离,预冷的PBS洗涤3次,稀释至终浓度(5%,v/v)。将不同浓度的Ir4和Ir5分别添加到450μL的红细胞中,并孵育5h以150rpm的速度轻轻摇动。孵育结束时,将溶液以1000×g离心15分钟,并使用酶标仪在540nm处测定溶血活性。能够完全裂解红细胞的Triton X-100(PBS中的0.1%)用作阳性对照,而PBS用作阴性对照。红细胞的溶血率由以下等式计算:溶血率(%)=(样品组-阴性对照)/(阳性对照-阴性对照)×100%。
图8中显示,15μM和30μM的Ir4和Ir5所测得溶血率均小于5%,但浓度为60μM时两配合物溶血率均略大于5%,100μM和200μM的物质6溶血率均小于5%,说明铱配合物在所用浓度范围相对安全。
(3)药物完全进入细胞所需时间
取对数生长的Siha细胞接种于6孔板中,细胞贴壁后,分别在1、3、6、12、24h加入Ir4或Ir5(浓度为15μM),收集细胞,PBS洗涤三次,流式检测。
流式结果显示(图9),12h前进入细胞的药物逐渐增加,表明在12h以前配合物进入细胞呈时间依赖性增加;而24h进入细胞的药物与12h基本相当,这表明大约12h左右Ir4和Ir5已经完全进入细胞。
(4)亚细胞器共定位
取Siha细胞接种于Nest共聚焦皿中,细胞贴壁后,加入Ir4和Ir5(浓度为15μM),继续孵育12h。去除培养液,加入Mito-Tracker Red(MTR)、Lyso-Tracker Red(LTR)和ER-Tracker Red(ERTR)工作液,37℃孵育30min。去除工作液,加入37℃新鲜的细胞培养液,共聚焦激光显微镜下观察。
Ir4(图10)和Ir5(图11)的共聚焦荧光图像与线粒体荧光染料(MTR)和内质网荧光染料(ERTR)的相关系数均比较低,而与溶酶体荧光染料(LTR)的相关系数较高,分别为0.79和0.81,表明配合物被细胞摄取后在溶酶体中积累。
(5)ROS的产生
取对数生长的Siha细胞接种于Nest共聚焦皿中,细胞贴壁后,加入Ir4和Ir5(浓度为15μM),物质6(浓度为100μM),继续孵育12h,用稀释后的DCFH-DA或DHE工作液染色30min,去除工作液,加入37℃新鲜的细胞培养液,共聚焦激光显微镜下观察。
DCFH-DA本身没有荧光,可以自由穿过细胞膜,进入细胞内后,可以被细胞内的酯酶水解生成DCFH。而DCFH不能通透细胞膜,从而使探针很容易被装载到细胞内。细胞内的活性氧可以氧化无荧光的DCFH生成有荧光的DCF。如图12所示DCF荧光大量聚集,说明活性氧的产生。为了进一步探究活性氧的种类,用DHE检测细胞内的超氧阴离子生成情况(图13),三种化合物均能够在Siha细胞中引起·O2-的产生。
(6)线粒体膜电位的检测
取对数生长的Siha细胞接种于Nest共聚焦皿中,细胞贴壁后,加入Ir4和Ir5(浓度为15μM)。去除培养液,加入JC-1工作液,37℃孵育15-20min。去除工作液,加入37℃新鲜的细胞培养液,共聚焦激光显微镜下观察。
在正常细胞中,线粒体膜电位较高,JC-1以多聚体(aggregates)的形式聚集在线粒体内,呈现红色荧光;而在线粒体功能受损的细胞中,线粒体膜电位较低,JC-1以单体(monomer)的形式分散在线粒体内,呈现绿色荧光。如图14所示红色荧光明显下降,而绿色荧光显著增加,表明三种药物能使线粒体膜电位下降,这与大多数细胞通过凋亡途径死亡一致。
(7)Hoechst 33342染色实验
取对数生长的Siha细胞接种于Nest共聚焦皿中,细胞贴壁后,加入Ir4和Ir5(浓度为15μM)。去除培养液,加入Hoechst 33342工作液,避光孵育15min。去除工作液,PBS洗涤三次,共聚焦激光显微镜下观察。
图15采用Hoechst 33342染色技术,用共聚焦检测,以确定Ir4和Ir5是否通过凋亡发挥抗癌作用。经Ir4、Ir5和物质6处理后,Siha细胞表现出明显的凋亡形态,包括核收缩和染色质凝结。
(8)GSH的消耗能力
GSH和Ir4和Ir5(浓度为15μM)、物质6(浓度为100μM)混合物反应4小时。然后,加入DTNB检测残留的GSH,在412nm处,DTNB检测GSH量测得紫外曲线。
GSH标志着细胞氧化还原稳态发生变化的一个重要指标,图16中将Ir4、Ir5和物质6和GSH共同孵育,发现三种物质均能引起GSH的降低,其中Ir4、Ir5相对物质6更加显著。
(9)细胞内GSH的检测
将6孔板的Siha细胞与Ir4、Ir5(浓度为15μM)和物质6(浓度为100μM)孵育12小时。GSH检测试剂盒检测GSH和GSSG含量。
图17结果显示氧化型谷胱甘肽(GSSG)与还原性谷胱甘肽(GSH)之间的比值显著增加,表明GSH被耗尽,谷胱甘肽的平衡被破坏。GSH是GPX4清除脂质活性氧的必需辅助因子,GSH的减少会导致GPX4活性降低,从而导致细胞抗氧化能力的降低,脂质活性氧堆积,表明可能会发生细胞的氧化性死亡,即铁死亡。
(10)GSH和NAC对细胞存活率影响
取处于对数生长期的Siha细胞以5×103/孔均匀接种于96孔板,培养12h。待细胞贴壁,加入GSH和NAC预处理1h,换入新鲜的培养液,加入Ir4和Ir5(浓度为15μM)和物质6(浓度为100μM)孵育24h。孵育完成后,加入MTT于96孔板中于37℃孵育4h,随后小心吸出培养液,室温下加入150μL/孔DMSO溶解甲鐕,震荡摇匀后,采用酶标仪于570nm波长处测定OD值,并计算细胞存活率。
图18中当补充GSH时,细胞存活率增加,同时也证明了Ir4和Ir5通过消耗GSH来提高效果。此外,NAC作为合成GSH的底物,在Ir4和Ir5处理过程中表现出类似GSH的保护作用。
(11)细胞内MDA的检测
将6孔板的Siha细胞与Ir4、Ir5和物质6孵育12小时。MDA检测试剂盒检测MDA含量。
丙二醛(MDA)的量,过氧化脂质的重要末端代谢物,被定义作为脂质过氧化的指标。经Ir4和Ir5处理后的Siha细胞的丙二醛的含量增加了一倍,图19结果表明,Ir4和Ir5可以产生脂质过氧化来杀死癌细胞,这一结果说明铁死亡参与了Ir4和Ir5诱导的细胞死亡。
(12)抑制剂对药物作用的影响
细胞接种于96孔板,培养24小时,加入不同抑制剂(3-MA:1mM;Z-VAD-FMK:25μM;Necrostatin-1:60μM)1h后加入15μM Ir4、Ir5和100μM物质6,对照组只加入相应且相同浓度的Ir4、Ir5和物质6,作用24h后加入MTT孵育4h,随后小心吸出培养液,室温下加入150μL/孔DMSO溶解甲鐕,震荡摇匀后,采用酶标仪于570nm波长处测定OD值,并计算细胞存活率。
图20使用不同的细胞死亡抑制剂,包括Fer-1(铁死亡抑制剂)、Nec-1(坏死抑制素)、Z-VAD-FMK(泛半胱天蛋白酶抑制剂)、3-MA(自噬抑制剂)和4-PAB(内质网应激抑制剂)验证Ir4、Ir5和物质6诱导的细胞死亡模式。Z-VAD-FMK和Fer-1能够增加细胞活力,其他抑制剂对细胞活力的影响较小,表明了Ir4和Ir5通过凋亡和铁死亡诱导细胞死亡,而物质6几乎不参与细胞内铁死亡的发生。
(13)Western bolt蛋白免疫印迹实验
a.细胞的药物处理:取Siha细胞以1×106/孔均匀接种于10cm的细胞培养皿中,预培养12h,以使细胞贴壁。加入配合物r4、Ir5(浓度为15μM)和物质6(浓度为100μM),37℃孵育24h。胰酶消化收集细胞于1.5mL离心管中,4℃预冷的PBS洗涤2遍,接着放置于碎冰上,待提取蛋白。
b.细胞总蛋白的提取:往1.5mL离心管中加入适量胞浆裂解液(含RIPA,PMSF和磷酸化酶抑制剂),冰上裂解1h,结束后4℃,12000rpm,离心15min,吸取上清于新的EP管中,加热变性。
c.SDS-聚丙烯酰胺凝胶电泳:放置并固定电泳玻璃板,加入3mL分离胶(根据需要检测蛋白的分子量确定不同浓度的分离胶),室温放置30min。凝固后,加入配制好的5%浓缩胶并插入塑料梳子,室温放置30min。安装好垂直电泳槽,加入新配的1×电泳缓冲液。每孔加入50μg蛋白,以蛋白Marker作为分子量参照。接通电源,分别设置浓缩胶跑胶电压50V、30min和分离胶跑胶电压100V、90min。
d.转膜:电泳完后,小心取出分离胶,根据蛋白Marker的分子量小心切取包含目标蛋白的凝胶,根据胶面大小修剪PVDF膜(标记,甲醇活化3min)。按照阴极板-海绵-3张滤纸-凝胶-PVDF膜-3张滤纸-海绵-阳极板的顺序放好,赶气泡,转膜夹夹好。放入转膜槽中,安装好后插上电源,调节恒流220mA在冰浴条件下转膜约1~1.5h(根据蛋白分子量大小适当调整转膜时间)。
e.封闭:转膜结束后,将PVDF膜蛋白膜朝上放入牛奶封闭液中,室温摇动封闭1.5~2h。
f.孵育抗体:TBST缓冲溶液室温摇动漂洗4×10min,一抗4℃孵育过夜(>12h)。TBST室温摇动漂洗4×10min,二抗室温孵育1.5~2h。
g.显影:二抗孵育完成后,TBST室温摇动漂洗4×10min。将PVDF膜与ECL发光液(等体积混合A液和B液)共孵育,并置于成像系统中进行曝光。
通过蛋白免疫印迹实验检测铱配合物作用后Siha蛋白(caspase-3,cleavedcaspase-3,GPX4)的表达水平。
Western blot对Ir4、Ir5和物质6孵育的癌细胞进行分析显示(图21),物质6对GPX4的表达基本无影响,而Ir4、Ir5抑制GPX4的表达,表示Ir4和Ir5诱导铁死亡的发生。Ir4、Ir5和物质6能使caspase-3减少,cleaved caspase-3增加,表明Ir4、Ir5和物质6诱导凋亡的发生。
(14)透射电镜拍摄
细胞接种于10cm皿中,培养24h长到约80%,加入15μM的Ir4、Ir5和100μM的物质6处理24h,收集细胞,800rpm离心6min,清洗1—2次得到细胞沉淀,缓慢滴加800μL戊二醛,4度保存,电镜检测。
透射电镜观察Ir4、Ir5和物质6理后细胞形态的改变(图22)。与对照组相比,Ir4和Ir5处理后的细胞表现出明显的典型铁死亡超微结构特征,包括线粒体嵴缩小、线粒体膜浓缩增加。
(15)配合物对多细胞肿瘤球体(MCTSs)的细胞毒性
Ir4,Ir5的细胞毒性对MCTSs的评估通过钙黄绿素AM/PI双染色。首先,如上所述构建MCTSs,并分别用化合物处理球体24小时。其次,处理过的球体用钙黄绿素AM(λex=488nm,λem=525±25nm)和PI(λex=535nm,λem=617±36nm)染色。最后,CLSM用于成像观察。
图23中未经药物处理的MCTSs中的Siha细胞球发出稳定的绿色荧光,表明细胞是存活的。物质6(100μM)处理的MCTSs整体显示出绿色荧光,可以观察到微弱的红光。相比之下,当MCTSs用Ir4和Ir5处理时,绿色荧光明显减弱,红色荧光逐渐增强,表明细胞已受损,其中Ir5处理组已经基本观察不到绿色荧光,Ir5的抗肿瘤效果强于Ir4和物质6。Ir4和Ir5在三维的MCTSs中仍能发挥良好的抗肿瘤能力。
Claims (5)
1.一种二溴酪氨酸-铱配合物Ir4,其特征在于,所述二溴酪氨酸-铱配合物Ir4结构式如式1所示
,
式1。
2.一种二溴酪氨酸-铱配合物Ir5,其特征在于,所述二溴酪氨酸-铱配合物Ir5结构式如式2所示
,
式2。
3.如权利要求2所述的二溴酪氨酸-铱配合物Ir5的制备方法,其特征在于,将物质11和[Ir(bzq)2Cl]2混悬于CH2Cl2/CH3OH的溶液中,在氩气环境下加热回流4 h,冷却至室温后,加入过量的KPF6,通过真空蒸发获得粗产物,经纯化后得到产物Ir5;物质11结构式如式11所示
,
式11。
4.根据权利要求3所述的制备方法,其特征在于,所述CH2Cl2/CH3OH的溶液CH2Cl2与CH3OH体积比为2:1;所述纯化为硅胶柱层析纯化。
5.如权利要求1所述二溴酪氨酸-铱配合物Ir4或如权利要求2所述二溴酪氨酸-铱配合物Ir5在制备抗肿瘤药物中的应用。
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