3,5-二溴水杨醛缩-4-氨基安替比林席夫碱Cu(域)配合物的合成及其抗癌活性

[收稿日期]2023-04-15　[修回日期]2023-07-14
[基金项目]安徽省高校自然科学研究重点项目(2022AH051442);国家级大学生创新创业训练计划项目(202210367008)
[作者单位]蚌埠医学院1.生命科学学院,2.公共基础学院,3.临床医学院,安徽蚌埠233030
[作者简介]陈司燮阳(2000-),男,学生.
[通信作者]胡　静,博士,讲师.E⁃mail:hj0310055@
[文章编号
]1000⁃2200(2023)08⁃1080⁃06㊃药　学㊃
3,5⁃二溴水杨醛缩⁃4⁃氨基安替比林席夫碱Cu(Ⅱ)
配合物的合成及其抗癌活性评价
陈司燮阳1,李　茹2,柴　俊2,张涤清3,殷华军3,陈莎莉3,胡　静2
[摘要]目的:设计并合成新的3,5⁃二溴水杨醛缩⁃4⁃氨基安替比林席夫碱Cu(Ⅱ)配合物([CuL 2]),并评价其抗癌活性㊂方法:利用3,5⁃二溴水杨醛与4⁃氨基安替比林发生缩合反应,合成新的3,5⁃二溴水杨醛缩⁃4⁃氨基安替比林席夫碱配体(HL)㊂将HL 与Cu(Ac)2㊃H 2O 通过溶剂热法合成了[CuL 2],并利用X 射线单晶衍射㊁多晶粉末衍射(PXRD)㊁红外光谱(FT⁃IR)和热重分析(TGA⁃DSC)等技术对[CuL 2]进行表征㊂MTT 法检测HL 及[CuL 2]对MDA⁃MB⁃231细胞㊁SMMC⁃7721细胞㊁CNE⁃2Z 细胞和A⁃549细胞的体外抗肿瘤活性㊂流式细胞术(FCM)分析[CuL 2]对MDA⁃MB⁃231细胞凋亡和周期的影响㊂结果:X 射
线单晶衍射分析显示,[CuL 2]为单核金属配合物,属单斜晶系,空间群为C 2/c ,具有微扭曲的四方形配位结构;PXRD 显示,[CuL 2]结晶度良好,晶体结构符合单晶测试的分析结果;配体和配合物的FT⁃IR 特征峰与分子结构一致;TGA⁃DSC 显示,[CuL 2]在270℃以下具有良好的热稳定性㊂MTT 实验结果显示,当HL 与Cu(Ⅱ)形成配合物时,[CuL 2]抗癌活性优于HL,其抑制MDA⁃MB⁃231细胞和SMMC⁃7721细胞活性较强,略低于顺铂㊂FCM 分析结果显示,[CuL 2]可诱导MDA⁃MB⁃231细胞凋亡(P <0.05);[CuL 2]诱导MDA⁃MB⁃231细胞阻滞在G 0/G 1期和S 期(P <0.05)㊂结论:合成得到[CuL 2],其可抑制MDA⁃
MB⁃231细胞和SMMC⁃7721细胞活性,诱导MDA⁃MB⁃231细胞凋亡和细胞周期阻滞㊂[关键词]3,5⁃二溴水杨醛缩⁃4⁃氨基安替比林;Cu(Ⅱ)配合物;晶体结构;抗癌活性
[中图法分类号]R 914.3 [文献标志码]A DOI :10.13898/ki.issn.1000⁃2200.2023.08.015
Synthesis of Cu (Ⅱ)complex based on 3,5⁃dibromosalicylaldehyde⁃4⁃aminoantipyrine
Schiff base and its anticancer activity evaluation
CHEN Si⁃xie⁃yang 1,LI Ru 2,CHAI Jun 2,ZHANG Di⁃qing 3,YIN Hua⁃jun 3,CHEN Sha⁃li 3,HU Jing 2
(1.School of Life Science ,2.School of Public Base ,3.School of Clinical Medicine ,
Bengbu Medical College ,Bengbu Anhui 233030,China )
[Abstract ]Objective :To design and synthesize a novel Cu(Ⅱ)complex based on 3,5⁃dibromosalicylaldehyde⁃4⁃aminoantipyrine Schiff base ([CuL 2]),and evaluate its anticancer activity.Methods :A novel Schiff base ligand,3,5⁃dibromosalicylalde⁃4⁃aminoantipyrine (HL),was synthesized by condensation reaction of 3,5⁃dibromosalicylalde and 4⁃aminoantipyrine.The [CuL 2]was synthesized by solvothermal method with HL and Cu(Ac)2㊃H 2O.The [CuL 2]was characterized by X⁃ray single crystal diffraction,polycrystaline powder X⁃ray diffraction (PXRD),Fourier transform infrared spectroscopy (FT⁃IR)and thermogravimetric analysis (TGA⁃DSC).The in vitro anticancer activity of HL and [CuL 2]on MDA⁃MB⁃231,SMMC⁃7721,CNE⁃2Z and A⁃549cells was
determined by MTT assay.The effect of [CuL 2]on apoptosis and cell cycle of MDA⁃MB⁃231cells was analyzed by flow cytometry (FCM).Results :X⁃ray single crystal diffraction analysis showed that [CuL 2]was a mononuclear metal complex,belonging to the monoclinic crystal system,with space group of C 2/c and 4⁃coordination micro⁃torsion square structure;PXRD showed that the crystallinity of [CuL 2]was good,and the crystal structure accorded with the results of single crystal test;the FT⁃IR characteristic peaks were consistent with the molecular structures of ligand and complex;TGA⁃DSC analysis showed that [CuL 2]had good thermal stability below 270℃.The MTT assay results showed that when HL forms a complex with Cu (Ⅱ),[CuL 2]had better anticancer activity than
HL,and its inhibitory activity on MDA⁃MB⁃231cells and SMMC⁃7721cells was stronger,which was slightly lower than that of cisplatin.The FCM analysis results showed that [CuL 2]could induce apoptosis in MDA⁃MB⁃231cells (P <0.05),and induce cell cycle arrest in G 0/G 1and S phases in MDA⁃MB⁃231cells (P <0.05).Conclusions :The [CuL 2]is synthesized,which can inhibit the activity
of MDA⁃MB⁃231and SMMC⁃7721cells,and induce cell cycle
arrest and apoptosis in MDA⁃MB⁃231cells.[Key words ]3,5⁃dibromosalicylalde⁃4⁃aminoantipyrine;Cu (Ⅱ)complex;crystal structure;anticancer
activity
临床上作为抗癌化疗药物的金属配合物种类有
限,其中Pt(Ⅱ)和Ru(Ⅲ)配合物研究最多,但此类
药物耐药和不良反应是最大的障碍,因此,探索出具
有良好的抗肿瘤新型金属配合物迫在眉睫㊂其中含
席夫碱的过渡金属配合物具有催化㊁抗氧化㊁抗菌㊁
抗肿瘤等多种生物活性[1-4],近年来得到了广泛的研究,是开发具有抗癌活性化合物的研究热点㊂
水杨醛类席夫碱是研究较多的一类化合物,其
分子结构中的亚胺或亚胺基团起着关键作用,使得
席夫碱配体表现出很强的配位活性㊂吡唑啉酮类衍
生物在极性羰基的两侧具有N⁃苯基和⁃CH基团,因
此具有较大的偶极矩和较强的碱性,也是潜在的优
良配体㊂水杨醛衍生物可与吡唑啉酮类衍生物化合
生成多种席夫碱,具有良好的抗肿瘤㊁抗菌㊁清除自
由基等生物活性[5],可以应用于生物㊁药理㊁临床等方面㊂
研究[6]表明席夫碱衍生物的金属基配合物往往比配体具有更多的生物活性㊂特别是Cu(Ⅱ)㊁Ni (Ⅱ)㊁Fe(Ⅱ/Ⅲ)和Zn(Ⅱ)几种过渡金属基于其稳定性和低毒性,被认为是形成配合物的理想金属离子[3,7]㊂其中,过渡金属中的铜元素是人体不可缺少的微量元素,为体内多种酶系的重要成分㊂许多铜配合物对癌细胞具有细胞毒性,而铜基癌症化疗药物是金属配合物疗法的一种研究新方向,主要机制被认为是通过产生活性氧或复合物插入模式与DNA结合等其他机制诱导细胞死亡[8-10],这些研究让Cu(Ⅱ)配合物作为抗癌药物成为可能㊂本研究利用3,5⁃二溴水杨醛与4⁃氨基安替比林发生缩合反应,合成新的3,5⁃二溴水杨醛缩⁃4⁃氨基安替比林席夫碱配体(HL),并将配体与Cu(Ac)2㊃H2O通过溶剂热法合成配合物[CuL2],利用X射线单晶衍射㊁红外光谱㊁PXRD粉末衍射和热重分析等手段对[CuL2]进行了表征,通过MTT法和FCM法研究[CuL2]抗肿瘤细胞活性㊁诱导细胞凋亡及对细胞周期的影响㊂现作报道㊂
1　材料与方法
1.1　仪器与试剂　X射线单晶衍射仪(Bruker Apex Smart ApexⅡ型);红外光谱仪(Nicolet Nexus 470型);多晶粉末衍射仪(Bruker D8型);同步热分析仪(STA449⁃F5);Beckman Coulter流式细胞仪(美国Beckman公司)㊂4⁃氨基安替比林(分析纯, 98%)和3,5⁃二溴水杨醛(分析纯,98%)(上海阿拉丁生化科技股份有限公司);一水合醋酸铜(分析纯,99%)(百灵威科技有限公司);三乙胺(分析纯,99.0%)㊁二甲基亚砜(DMSO,分析纯,99.7%)和乙醇(分析纯,99.9%)(国药控股股份有限公司);胎牛血清(FBS)㊁RPMI⁃1640培养基和DMEM培养基(美国Invitrogen公司);青霉素/链霉素(西格玛试剂);MTT试剂(上海百时生物科技有限公司);胰蛋白酶(美国Gibco公司);Annexin V⁃FITC/PI双染细胞凋亡检测试剂盒㊁细胞周期试剂盒(上海碧云天生物技术有限公司);人MDA⁃MB⁃231细胞㊁A⁃549细胞㊁SMMC⁃7721细胞(美国ATCC公司);人CNE⁃2Z细胞(南京凯基生物科技发展有限公司)㊂1.2　配体HL的合成　称取1.0g4⁃氨基安替比林于三颈烧瓶中,加20mL无水乙醇,不断搅拌至完全溶解㊂再称取1.38g3,5⁃二溴水杨醛完全溶解于20mL无水乙醇,逐滴加入溶有4⁃氨基安替比林的三颈烧瓶中,升温至60°C,回流搅拌加热5h㊂反应完成后冷却至室温,将所得到的黄色产物进行减压抽滤,用少量无水乙醇洗涤2次,常温下干燥,收集3,5⁃二溴水杨醛缩⁃4⁃氨基安替比林(C18H15 O2N3Br2)黄色粉末HL,产率为89%,合成路线见图1㊂
1.3　配合物[CuL2]的合成　精确称取0.02g新合成的HL,置于10mL聚四氟乙烯内衬中,用8mL无水乙醇溶解,再准确称取0.004g醋酸铜和0.004g 无水K2CO3加入内衬中,装入高压釜中密封㊂放入65℃烘箱反应3d后,常温下冷却至室温,弃去黄色上清液,得墨绿色晶体[CuL2],洗涤㊁干燥,计算产率为51.2%㊂合成路线见图
1㊂
1.4　[CuL2]结构测试方法　采用X射线衍射法确定[CuL2]的单晶结构㊂显微镜下挑选出外观尺寸合适的晶体,于173K条件下,经过石墨单色器化的Mo⁃Ka射线(λ=0.71073Å),在单晶衍射仪上获取X⁃射线衍射数据,并进行结构的解析㊂晶体学数据已存入剑桥晶体数据中心,CCDC编号为
2257607㊂
使用红外光谱仪(FT⁃IR)分析分子结构,KBr压片,在4000~500cm-1波数范围内扫描,获取样品HL和[CuL2]的光谱数据㊂利用多晶粉末衍射仪(PXRD)对[CuL2]结晶度和晶体结构进行判断,Cu 靶Kα射线,2θ=3°~50°测试,扫描速度为5°/min,实时收集数据㊂使用同步热分析仪研究晶体的热性能,扫描温度50~800℃,升温速率5K/min,操作在N2气中进行,获取样品的TGA⁃DSC曲线㊂1.5　细胞毒性实验　MTT法检测细胞毒性,以乳腺癌细胞株MDA⁃MB⁃231㊁肝癌细胞株SMMC⁃7721㊁鼻咽癌细胞株CNE⁃2Z和肺癌细胞株A⁃549为实验对象㊂A⁃549和CNE⁃2Z细胞在RPMI⁃1640培养基中培养,SMMC⁃7721和MDA⁃MB⁃231细胞在DMEM 培养基中培养㊂[CuL2]和HL先在DMSO中溶解,无菌水配制成5mmol/L的原液备用;将顺铂溶解在无菌水中,暗处配成5mmol/L浓度溶液㊂将[CuL2]原液和顺铂原液分别稀释为6.25㊁12.5㊁25㊁50μmol/L4个浓度,加入培养基,每孔100μL,培养箱5%CO2㊁37℃常规培养㊂孵育72h后,加入10μL(0.5mg/mL)MTT试剂,再将其置于培养箱4h,取出弃去培养液,在490nm波长下用酶标仪测量其吸光度(OD)值,用Graphpad Prism软件测定半数抑制浓度(IC50)㊂
1.6　细胞凋亡实验　用含有青霉素/链霉素和20%FBS的DMEM培养基对MDA⁃MB⁃231细胞传代培养㊂取对数生长期的细胞,在0㊁1㊁5㊁25μmol/ L药物中处理后,将细胞置于培养箱孵育24h后,弃去旧的培养基,用PBS洗涤2次㊂用不含EDTA 的胰蛋白酶处理细胞,再使用完全培养液终止消化㊂用冰冷的PBS洗涤2次,细胞浓度调整为1×105个/毫升,然后用Binding Buffer缓冲液悬浮细胞㊂在细胞悬液中加入5μL Annexin V⁃FITC,混合均匀后,再加入10μL PI混合,室温条件下避光孵育10min㊂1h内采用Beckman Coulter流式细胞仪检测㊂1.7　细胞周期实验　取对数生长期的细胞,用含0㊁1㊁5㊁25μmol/L药物的完全培养基孵育24h,然后弃去培养基㊂用PBS洗涤2次后,加入不含EDTA的胰蛋白酶,再用完全培养基终止消化㊂4℃离心,弃去上清液,收集并调整细胞浓度为1×106个/毫升,细胞用70%冷乙醇悬浮,固定密封,然后在4℃孵化过夜,离心后用PBS洗涤2次,加入提前配制的500μL PI/RNase A染色工作液,在冰浴中避光染色30min;采用Beckman Coulter流式细胞仪检测㊂
1.8　统计学方法　采用t检验㊁方差分析和q检验㊂
2　结果
2.1　配合物[CuL2]结构与表征　
2.1.1　晶体结构解析　单晶X射线衍射分析表明,[CuL2]为单核金属配合物,分子式为C36H28 O4N6CuBr4㊂配合物[CuL2]属于单斜晶系,C2/c空间群,其不对称单元结构中含1个Cu(Ⅱ)离子和2个去质子化配体㊂Cu(Ⅱ)离子分别与2个氧原子(分别来自两个配体的酚羟基)和2个氮原子(分别来自两个配体的C=N键的氮)配位,形成了四配位的略微扭曲的平面四方形[CuL2]结构(见图2)㊂单体的[CuL2]配合物分子以C⁃H π堆积以及氢键协同作用方式,形成三维空间分子结构(见图
3)㊂
2.1.2　FT⁃IR㊁PXRD和TGA⁃DSC分析　HL的FT⁃IR光谱图显示,在波数1591cm-1处有一强的特征吸收峰,指认为⁃CH=N⁃的伸缩振动[11-12];指纹区698cm-1和764cm-1处两峰表明有单取代苯的衍生物;967cm-1处有明显吸收峰,显示苯环上有1㊁2㊁3㊁5取代㊂在[CuL2]的FT⁃IR光谱图1627 cm-1处观察到一新的吸收峰,为亚胺基团和铜离子的电子效应;581cm-1和561cm-1处的两个峰,应归因于铜离子与O原子和N原子的配位㊂以上结果显示配体和配合物的FT⁃IR特征峰与分子结构一致(见图4)㊂PXRD检测出的[CuL2]晶体特征峰与结构模拟特征峰对应一致,表明其结晶度良好,晶体结构符合单晶测试的分析结果(见图5)㊂TGA⁃DSC 结果显示,[CuL2]在270℃以下具有良好的热稳定性,270℃后框架开始分解(见图6)㊂
2.2　细胞实验　
2.2.1　[CuL 2]对细胞活性的影响　当配体HL 与Cu(Ⅱ)形成配合物时,[CuL 2]抗肿瘤活性优于HL;
[CuL 2]对MDA⁃MB⁃231细胞和SMMC⁃7721细胞抗癌活性较强,略低于顺铂(见表1)㊂
2.2.2　[CuL 2]对MDA⁃MB⁃231细胞凋亡的影响　与Control 组相比,1㊁5㊁25μmol /L [CuL 2]可促进
MDA⁃MB⁃231细胞凋亡(P <0.05)(见图7㊁表2)㊂2.2.3[CuL 2]对MDA⁃MB⁃231细胞周期的影响　与Control 组相比,随着[CuL 2]浓度增加,G 0/G 1期细胞比例上升,S 期细胞比例下降,[CuL 2]诱导细胞阻滞在G 0/G 1期和S 期(P <0.05)(见图8㊁表3)㊂
表1　HL 及[CuL 2]的IC 50值(x ±s ;μmol /L )
药物MDA⁃MB⁃231细胞　SMMC⁃7721细胞CNE⁃2Z 细胞A⁃549细胞[CuL 2] 6.52±0.31
8.65±0.13
32.76±0.9132.76±0.91HL
91.61±2.3683.21±0.2477.36±0.6366.58±0.2顺铂 4.02±0.08 4.42±0.11 4.54±0.04
15.74±0.54
表2　[CuL 2]对MDA⁃MB⁃231细胞凋亡的影响(x ±s )
分组
n 凋亡率/%F
P
MS 组内
Control 组
3 4.17±0.74　
1μmol /L [CuL 2]组5μmol /L [CuL 2]组
337.14±0.38*
14.13±0.90*#
494.70<0.010.640
25μmol /L [CuL 2]组3
27.25±1.02*#▲
q 检验:与Control 组比较*P <0.05;与1μmol /L [CuL 2]组比较#P <
0.05;与5μmol /L [CuL 2]组比较▲P <0.05
表3　[CuL 2]对MDA⁃MB⁃231细胞周期影响(x ±s )
分组
n G 0/G 1期S 期G 2/M 期Control 组
347.29±1.34　32.75±1.46
19.96±0.501μmol/L [CuL 2]组351.26±1.49*
29.17±0.74*
19.57±1.55*
5μmol/L [CuL 2]组
359.41±1.68*#
24.84±1.18*#15.75±0.50*#
25μmol/L [CuL 2]组
3
63.24±2.53*#▲22.28±2.79*#▲14.48±1.04*#▲
F 48.5521.7522.50P <0.01
<0.01
<0.01
MS 组内
3.302
2.960
1.000
q 检验:与Control 组比较*P <0.05;与1μmol/L [CuL 2]组比较#P <0.05;与5μmol/L
[CuL 2]组比较▲P <0.053　讨论
有机化合物作为分子抗癌药物,一直被广泛应用于临床㊂而有机金属化合物比有机分子更容易合
成和修饰,有利于生成不良反应更小㊁活性范围更广的衍生物[13]㊂因此,开发高效㊁不良反应小㊁诱导细胞凋亡的金属化合物目前被广泛研究,例如用非铂
类金属化合物代替铂类化合物是改善不良反应的一种方法㊂相比之下,铜是一种重要的内源性金属,其配合物在癌细胞增殖过程中具有抗转移㊁抗血管生成活性㊁抑制DNA 合成㊁干扰线粒体等作用,铜(Ⅱ)配合物具有多方面的生物学特性和较高的生物相容性,是很有希望的抗癌药物[14]㊂
本研究以3,5⁃二溴水杨醛缩⁃4⁃氨基安替比林
席夫碱
HL 为原料,制备了[CuL 2]配合物,其单体为单核轻微扭曲的四方形配位结构,单体的[CuL 2]分子以C⁃H π堆积及氢键协同作用方式,形成三维
空间分子结构㊂[CuL 2]具有良好的结晶度,晶体结构符合单晶测试的分析结果,其在270℃以下具有良好的热稳定性
㊂
本研究还在不同的肿瘤细胞系中测试了[CuL 2]的体外抗肿瘤细胞活性,结果表明,席夫碱配体HL 对肿瘤细胞系影响微弱,[CuL 2]对MDA⁃MB⁃231和SMMC⁃7721细胞抑制作用优于HL,略低于顺铂㊂但是,铂类抗癌药物的临床应用受到其严重不良反应和对肿瘤治疗性耐药性的限制[15-16],在这一点上,铜配合物比顺铂更具有优势㊂细胞凋亡实验结果表明,[CuL 2]能明显诱导MDA⁃MB⁃231细胞早期凋亡㊂细胞周期实验结果表明,[CuL 2]可阻滞细胞在G 0/G 1期和S 期,因此,[CuL 2]作为一种新的具有抗癌活性的配合物,在药物化学中具有潜在的应用价值㊂
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及分布构建的列线图可以准确预测恶性风险㊂
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