CN114456116A - 一种以萘酰亚胺为骨架的小分子抗癌剂及其制备方法和应用 - Google Patents
一种以萘酰亚胺为骨架的小分子抗癌剂及其制备方法和应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及生物医药技术领域,具体涉及种以萘酰亚胺为骨架的小分子抗癌剂及其制备方法和应用。
背景技术
肺癌是癌症相关死亡的最常见原因,是常见的恶性肿瘤,肺癌一半以上的患者(约55%)在诊断时就出现了转移性肺癌。所以,开发响应肿瘤微环境的小分子药物一直是研究热点,但是开发疗效好、特异性强的小分子抗癌药物仍面临很大挑战。
萘酰亚胺在1980年代初期首次被认为是嵌入细胞毒剂,从那时起,已经评估了许多衍生物的抗肿瘤活性,其中有米托萘胺和氨萘非特进入临床试验。已经证明,连接臂至少包含一个氨基和具有至少一个3-硝基的1,8-萘酰亚胺部分有利于增强萘酰亚胺系列中的细胞毒活性。大多数萘酰亚胺具有荧光特性,并表现出广泛的生物学特性,如抗癌、抗菌、抗病毒和镇痛作用。1,8-萘二甲酰亚胺还是具有高抗肿瘤活性的物质。但是这类物质对正常细胞和癌细胞的选择性差,所以在癌症治疗中,这类衍生物毒副作用大,无法区分正常细胞和癌细胞,在临床应用中具有很大的局限性。
氮芥是一种轻度有效的核DNA烷基化剂,可在体内形成活性亲电子基团形成活性缺电子中间体或其他化合物。这些活性中间体可以通过形成共价键与生物大分子中的一些富电子基团发生亲电反应,从而抑制相应的生物大分子的活性。由于氮芥剂与DNA鸟嘌呤上的N7氮原子结合选择性较差,对正常细胞具有毒性。因此近些年来的研究发现通过引入药物片段开发氮芥基杂交分子可以提高抗肿瘤作用,为开发出选择性强和毒性低的抗肿瘤分子提供了新的策略。
发明内容
发明目的:针对现有技术存在的问题,本发明提供了一种以萘酰亚胺为骨架的小分子抗癌剂,其是一种基于偶氮键连接的可激活小分子抗肿瘤药物,该小分子抗癌剂可以降低氨萘非特和氮芥分子自身对正常细胞的高抗肿瘤活性,提高对如顺铂耐药肺癌细胞的抑制作用,同时本发明的小分子在癌细胞内有荧光团的信号可以被检测到,有效提供诊断和治疗肿瘤一体化策略。
本发明还提供以萘酰亚胺为骨架的小分子抗癌剂的制备方法和应用。
技术方案:为了实现上述目的,本发明所述一种以萘酰亚胺为骨架的小分子抗癌剂,所述抗癌剂其结构如式I所示:
其中,所述小分子抗癌剂由氨萘非特和1,1-二甲基乙二胺合成获得。
本发明所述的以萘酰亚胺为骨架的小分子抗癌剂的制备方法,其特征在于,包括如下步骤:
(1)在室温下向苯胺和2-溴乙醇的混合物中添加碳酸钾加,再加入乙醇溶液;将所得混合物加热搅拌分出有机层,干燥,旋转蒸发浓缩得粗品,粗品纯化得到产物S1;
(2)将产物S1溶解在三氯氧磷中并回流,减压浓缩反应混合物并稀释,然后洗涤,干燥并减压浓缩;纯化得到产物S2;
(3)将氯化锡(II)加入浓盐酸中,所得溶液逐滴加入到3-硝基-1,8-萘二甲酸酐的搅拌悬浮液乙醇中,将所得悬浮液搅拌回流,然后冷却至室温;过滤收集沉淀产物,洗涤,所得固体干燥得到产物S3;
(4)将产物S3溶于乙醇中,加入1,1-二甲基乙二胺的乙醇溶液,然后加热回流,在室温下缓慢冷却溶液,过滤分离固体,洗涤,所得固体干燥得到产物S4;
(5)在惰性气体中,向S4的搅拌溶液中加入NaNO2水溶液,将溶液搅拌后加入TFA并将溶液再搅拌,随后将S2溶于乙腈加入上述反应液中,并将混合物搅拌并加入水,混合物萃取,合并有机层,干燥,除去溶剂;经色谱分离后得到小分子抗癌剂化合物CFJ001。
其中,步骤(1)中在室温下将苯胺和2-溴乙醇按摩尔比1:2-3加入三颈圆底烧瓶中,将碳酸钾加入反应瓶中,再加入乙醇溶液,将所得混合溶液在78-80℃下搅拌4-6小时。
其中,步骤(2)中将产物S1溶解在三氯氧磷中,控制反应温度在100-105℃回流3-4h,反应结束后,先用二氯甲烷稀释后再用饱和食盐水洗涤。
其中,步骤(4)中将产物S3溶解在乙醇中,加入1,1-二甲基乙二胺的乙醇溶液,然后在78-80℃下加热回流2.5-3小时。
其中,步骤(5)中在Ar气氛围在0℃下将产物S4溶于ACN和H2O的混合溶液加入两颈圆底烧瓶中,0℃搅拌后,将NaNO2的水溶液加入圆底烧瓶中,将溶液搅拌后,加入TFA并将溶液再搅拌,随后将S2溶于乙腈加入反应液中,并将混合物在0℃下搅拌之后加入水。
作为优选,所述小分子抗癌剂的制备方法,包括如下步骤:
(1)在室温下将苯胺和2-溴乙醇的混合物添加到三颈圆底烧瓶中,将30mmol碳酸钾加入反应瓶中,再加入50mL乙醇溶液。将所得混合物加热至80℃并在此温度下搅拌4小时。真空去除溶剂,用二氯甲烷萃取(3×50mL),分出有机层,无水硫酸钠干燥,旋转蒸发浓缩得粗品。粗品用柱层析色谱法纯化得到产物S1;
(2)将产物S1溶解在20mL三氯氧磷中并回流3小时。减压浓缩反应混合物并用100mL二氯甲烷稀释,然后用饱和食盐水(3×50mL)洗涤,用无水硫酸镁干燥并减压浓缩;通过柱层析色谱法纯化得到产物S2;
(3)将氯化锡(II)加入3.5mL浓盐酸中,所得溶液逐滴加入到3-硝基-1,8-萘二甲酸酐的搅拌悬浮液乙醇中,将所得悬浮液搅拌回流2小时,然后冷却至室温。过滤收集沉淀产物,依次用水、乙醇和乙醚洗涤。所得红棕色固体在真空烘箱中干燥得到产物S3;
(4)将步骤3中所得的固体S3溶于乙醇中,加入1,1-二甲基乙二胺的乙醇溶液,然后加热回流约2.5h。在室温下缓慢冷却溶液。观察到沉淀物,然后通过真空过滤分离固体,用3mL乙醇洗涤两次。所得黄色固体在真空烘箱中干燥得到产物S4;
(5)在Ar气氛围下,在0℃下,向S4的20mL(ACN/H2O=1/1)的搅拌溶液中加入含75mgNaNO2的蒸馏水溶液,将溶液搅拌10分钟后加入115mgTFA并将溶液再搅拌30分钟,随后将S2溶于5mL乙腈加入上述反应液中,并将混合物在0℃下搅拌4小时之后,加入100mL水。混合物用二氯甲烷(3×50mL)萃取,合并有机层,用无水硫酸镁干燥,真空除去溶剂;经色谱分离后得到小分子抗癌剂化合物CFJ001。
其反应式如下所示:
本发明所述的小分子抗癌剂在制备抗肿瘤药物中的应用。
其中,所述肿瘤包括宫颈癌,肝癌,肺癌和非小细胞肺癌。
进一步地,包括宫颈癌,肝癌,人源肿瘤人非小细胞肺癌和顺铂耐药肺癌。
本发明所述的小分子抗癌剂在活细胞中对人源癌细胞系如宫颈癌(HeLa),肝癌(HepG2),肺癌耐药细胞株(A549R),非小细胞肺癌(A549)具有一定的抗癌活性,对正常细胞人胚肺成纤维细胞(HLF)的毒副作用明显小于氨萘非特。
本发明所述的小分子抗癌剂在制备对SDT的特异性响应以及对各种金属离子的选择性筛选的药物中的应用。
本发明所述小分子抗癌剂的设计理念就是为了降低对正常细胞的毒性,提高抗肿瘤活性。本发明中的细胞实验也证实了小分子通过诱导自噬依赖性的铁死亡发挥抗肿瘤作用。本发明所述的小分子抗癌剂体外对SDT的特异性响应以及对各种金属离子的选择性筛选的药物中的应用,主要用途是小分子抗癌剂在癌细胞内可以释放出荧光团氨萘非特,荧光信号可以帮助诊断肿瘤,同时对顺铂耐药肺癌细胞具有明显的抑制作用。对SDT的特异性响应是为了证明小分子的偶氮键在常氧条件下就可以断裂。对金属离子的选择性筛选是为了证明药物分子选择性好,排除体内金属离子的干扰。
本发明所述的小分子抗癌剂诱导细胞死亡的机制为在细胞器诱导活性氧ROS上升,线粒体膜电位下降,诱导细胞自噬,脂质过氧化以及铁死亡发挥抗肿瘤作用杀死癌细胞的。
本发明设计合成了一种以萘酰亚胺为基本骨架,ANF(氨萘非特)为荧光团的小分子抗癌剂。荧光团通过偶氮苯间隔物与DNA烷基化抗癌剂结合,偶氮键的引入可作为荧光的猝灭剂和苯胺芥的灭活剂。在肿瘤微环境中,药物分子被激活后释放出荧光团和活性氮芥分子。该分子可通过诱导细胞自噬使细胞发生脂质过氧化,随着细胞内亚铁离子含量的增加,自噬依赖性的铁死亡被诱导。同时DNA烷化剂可诱导核DNA损伤,从而引起细胞死亡。本发明研究表明该小分子抗癌剂具有潜在的抗癌活性,尤其针对肺癌耐药性细胞株展现出优于阳性对照药物的抗癌活性。
本发明中小分子的设计思路主要是通过偶氮键将两个小分子抗癌药连接起来,降低对正常细胞的毒性,发挥前药的作用。现有技术中所报道的偶氮键都是在厌氧条件下断开的,但是本发明设计的小分子偶氮键在常氧条件下就可以断裂,这也是本发明首次提出的。本发明化合物结构设计最大的优势就是降低了氨萘非特和氮芥分子自身对正常细胞的毒性,而小分子在细胞内释放的荧光团可以帮助进行诊断肿瘤。
有益效果:与现有技术相比,本发明具有如下优点:
1、本发明制备方法简单,易于操作,且产率高,能有效降低合成成本,小分子药物进入细胞后释放了荧光团和小分子抗癌剂,可以在细胞内反应发挥很好的抗肿瘤效果,并且对肺癌耐药株细胞具有很好的抑制作用。
2、本发明的小分子抗癌剂可以在常氧条件下发挥抗肿瘤作用,和之前报道的以萘酰亚胺为骨架的小分子相比,本发明对正常细胞的毒性明显小于对肺癌耐药株细胞的毒性。通过引入偶氮键,有效的降低了苯胺氮芥和氨萘非特对正常细胞的毒性。
3、本发明的小分子抗癌剂可以在癌细胞内出释放荧光团,通过追踪荧光团的信号,可以帮助诊断肿瘤,为开发肿瘤的诊断和治疗一体化的小分子药物提供了新的思路。
附图说明
图1为CFJ001,ANF,Afatinib对A549R细胞的细胞毒性曲线图。
图2为CFJ001本身和荧光团氨萘非特的吸收和发射光谱以及对不同金属离子的选择性的光谱图;其中(a)是化合物CFJ001(10μM)和氨萘非特(10μM)在PBS中的的紫外吸收谱图;(b)是化合物CFJ001(10μM)和氨萘非特(10μM)在PBS中的的荧光发射谱图;(c)是化合物CFJ001(10μM)在PBS缓冲液(10mM,1%DMSO,pH 7.4)中的响应SDT(1mM)的荧光光谱图;(d)是(c)600nm处响应SDT(1mM)的荧光强度随时间的变化;(e)是化合物CFJ001(10μM)对各种金属离子的响应,记录600nm处的荧光强度变化;(f)在不同激发波长下用SDT(1mM)处理的CFJ001(10μM)的相应发射光谱;
图3为CFJ001在细胞内被激活的LC-MS图;
图4为CFJ001在细胞内的共定位图;
图5为CFJ001诱导细胞自噬的免疫荧光共聚焦成像图和TEM图;其中A是细胞自噬免疫荧光成像图;B是A中荧光强度定量分析柱状图;C是透射电镜TEM图;
图6为CFJ001诱导细胞产生活性氧流式图;
图7为CFJ001诱导线粒体膜电位下降共聚焦成像图和流式图;其中A是小分子抗癌剂CFJ001作用于A549R细胞的线粒体膜电位变化共聚焦成像图;B是A中荧光强度定量分析柱状图;C是小分子抗癌剂CFJ001作用于A549R细胞的线粒体膜电位变化流式数据图;
图8为CFJ001用商用探针BODIPY-581/591C11和FeRhoNox-1分别检测细胞发生脂质过氧化和铁死亡的共聚焦成像图;其中A是A549R细胞与小分子CFJ001孵育后,然后用特定的亚铁探针FerroOrange染色的共聚焦成像图;B是A中图像的相对荧光强度定量分析柱状图;C是在CFJ001处理后,通过ICP-MS测量细胞的总铁含量柱状分析图;D是A549R细胞与小分子CFJ001孵育后,然后用BODIPY-581/591C11染色,用于分析细胞脂质过氧化水平的共聚焦成像图;
图9为CFJ001与各种蛋白作用的蛋白印迹图;其中A、B、C、D、E分别是不同浓度的CFJ001对A549R细胞处理后LC3II/LC3I、p62和p53、γH2AX、GPX4和NCOA4蛋白表达影响的蛋白印迹图;
图10为实施例1制得的S1的1H NMR谱图;
图11为实施例1制得的S2的1H NMR谱图;
图12为实施例1制得的S4的1H NMR谱图;
图13为实施例1制得的CFJ001的1H NMR谱图;
图14为实施例1制得的CFJ001的13C NMR谱图;
图15为实施例1制得的CFJ001的质谱图(HR-MS)。
具体实施方式
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的内容仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可从商业途径获得。
实施例1
在室温下将14.82mmol苯胺和29.64mmol 2-溴乙醇的混合物添加到三颈圆底烧瓶中。将30mmol碳酸钾加入反应瓶中,再加入50mL无水乙醇溶液。将所得混合物加热至80℃并在此温度下搅拌4小时。真空去除溶剂,用二氯甲烷萃取(3×50mL),分出有机层,无水硫酸钠干燥,旋转蒸发浓缩得粗品。粗品再用柱层析(二氯甲烷/甲醇=98/2)纯化得到产物S1,产率为60%。Analytical TLC(100%DCM),Rf=0.3;1H NMR(400MHz,DMSO-d6)δ7.17–7.09(m,2H),6.67(dd,J=8.1,1.3Hz,2H),6.58–6.52(m,1H),4.78(t,J=5.4Hz,2H),3.54(q,J=6.1Hz,4H),3.45–3.37(m,4H).
将20mmol产物S1溶解在20mL三氯氧磷中并在100℃下回流3小时。减压浓缩反应混合物并用100mL二氯甲烷稀释,然后用饱和食盐水(3×50mL)洗涤,用无水硫酸镁干燥并减压浓缩。通过柱色谱(二氯甲烷/甲醇=99/1)纯化得到产物S2,产率为65%。AnalyticalTLC(100%DCM),Rf=0.6;1H NMR(400MHz,CDCl3)δ7.35–7.26(m,2H),6.82(dd,J=7.8,6.7Hz,1H),6.74–6.72(m,2H),3.83–3.60(m,8H).
将4.1mmol 3-硝基-1,8-萘二甲酸酐溶于10mL无水乙醇中,所得溶液加入50mL圆底烧瓶中,将21.2mmol氯化锡(II)溶于3.5mL37%的浓盐酸缓慢滴加到圆底烧瓶中,将所得悬浮液搅拌并在80℃下回流2小时,然后冷却至室温。过滤收集沉淀产物,依次用水、乙醇和乙醚洗涤。所得红棕色固体在真空烘箱中干燥得到产物S3,产率为90%。Analytical TLC(10%MeOH in DCM),Rf=0.6;1H NMR(400MHz,DMSO-d6)δ8.17–8.08(m,2H),7.98(d,J=2.3Hz,1H),7.67(dd,J=8.3,7.3Hz,1H),7.40(d,J=2.3Hz,1H).
将1.4mmol产物S3固体溶于6.5mL乙醇中,向溶液中加入1.47mmol 1,1-二甲基乙二胺(N,N'-二甲基乙二胺)的1mL乙醇溶液,然后在80℃下加热回流2.5h。在室温下缓慢冷却溶液。观察到沉淀物,然后通过真空过滤分离固体,用3mL乙醇洗涤两次。所得黄色固体在真空烘箱中干燥得到产物S4(氨萘非特)。产率为71%。Analytical TLC(10%MeOH inDCM),Rf=0.4;1H NMR(400MHz,DMSO-d6)δ8.10–8.01(m,2H),7.97(d,J=2.3Hz,1H),7.61(dd,J=8.2,7.3Hz,1H),7.29(d,J=2.2Hz,1H),6.01(s,2H),4.16(t,J=6.8Hz,2H),2.60(t,J=6.6Hz,2H),2.28(s,6H).
在Ar气氛围,0℃下将1mmol产物S4溶于20mL(ACN/H2O=1/1)溶液加入两颈圆底烧瓶中,0℃搅拌10分钟后,将75mg NaNO2溶于5mL蒸馏水所得的溶液加入圆底烧瓶中,将溶液搅拌10分钟后,加入115mg TFA(三氟乙酸)并将溶液再搅拌30分钟。随后将S2溶于5ml乙腈加入反应液中,并将混合物在0℃下搅拌4小时之后加入100mL水。混合物用二氯甲烷(3×50mL)萃取,合并有机层,用无水硫酸镁干燥,真空除去溶剂。经色谱分离(二氯甲烷/甲醇=90/10)后得到化合物CFJ001,产率为54%。Analytical TLC(20%MeOH in DCM),Rf=0.4;1HNMR(400MHz,CDCl3)δ9.14(d,J=1.9Hz,1H),8.70(d,J=1.9Hz,1H),8.63–8.58(m,1H),8.38(d,J=8.1Hz,1H),8.04–7.98(m,2H),7.82(t,J=7.8Hz,1H),6.86–6.81(m,2H),4.65(t,J=5.8Hz,2H),3.90(t,J=7.0Hz,4H),3.75(t,J=6.9Hz,4H),3.53(t,J=5.9Hz,2H),3.02(s,6H).13C NMR(101MHz,CDCl3)δ164.56,150.98,144.46,135.40,125.93,124.22,123.14,122.20,111.68,43.37,40.31.ESI-MS:C16H16N5O2[M+H+],Calculated:512.15,Found:512.16.
上述目标产物1H和13C NMR数据采用Bruker Avance II 400MHz光谱(美国)进行收集。电喷雾电离质谱(ESI-MS)数据通过使用Mass Lynx系统的Thermo LCQ-FLEET质谱仪(美国)测定。核磁数据通过MestReNova软件分析。
图10为实施例1制得的S1的1H NMR谱图,图11为实施例1制得的S2的1H NMR谱图,图12为实施例1制得的S4的1H NMR谱图,图13为实施例1制得的CFJ001的1H NMR谱图,图14为实施例1制得的CFJ001的13C NMR谱图,图15为实施例1制得的CFJ001的质谱图(HR-MS),证明化合物CFJ001合成成功。
实施例2
本发明实施例1制备的小分子抗癌剂用于肿瘤细胞治疗方面的应用。
方法:MTT比色法,测定对人源癌细胞系如宫颈癌(HeLa),肝癌(HepG2),肺癌耐药细胞株(A549R),非小细胞肺癌(A549)等在体外的抗癌活性和正常细胞人胚肺成纤维细胞(HLF)的毒副作用。HeLa,HepG2,A549和HLF细胞在具有10%胎牛血清和1%青霉素-链霉素溶液的DMEM培养基中37℃、5%CO2细胞培养箱中培养。A549R细胞在具有10%胎牛血清和1%青霉素-链霉素溶液的1640培养基中,37℃,5%CO2细胞培养箱中培养;具体将上述细胞以5000个细胞/孔的初始密度接种到96孔细胞培养板中,在培养24小时后移除培养基,分别设三组平行对比试验,五个浓度(40μM、20μM、10μM、5μM、2.5μM)的待测样品,继续37℃孵育48h。然后在每个孔中加入15μL,5mg/mL的MTT,于37度培养箱继续孵育4h后再加入150μLDMSO,震荡至紫色结晶完全溶解,并在酶标仪(型号为Tecan Infinite M1000 Pro)上读取490nm处的吸光度,同时以等浓度的顺铂为阳性对照,不加化合物的为空白对照;同时测定以厌氧培养箱中培养的活性。实施例1制得小分子抗癌剂的抗癌活性如表1和表2所示。
表1为常氧下有机化合物CFJ001和顺铂(CDDP)的IC50(μM)值
表2为厌氧下有机化合物CFJ001和顺铂(CDDP)的IC50(μM)值
结果表明,小分子抗癌剂CFJ001在常氧和厌氧条件下均表现出很强的抗增殖活性,IC50值在5-11μM的范围内。与顺铂相比,CFJ001显示出更高的抗癌活性,尤其是顺铂耐药细胞A549R。与小分子对照药Afatinib(百灵威)和氨萘非特(实施例1产物S4)相比,CFJ001对耐药癌细胞A549R也具有明显的抑制作用(如图1所示),并且常氧条件下对正常细胞HLF的毒性和顺铂比明显低了很多。这些结果表明CFJ001对耐药癌细胞具有潜在的抗增殖活性。
本实施例通过和顺铂进行活性对照,偶氮键的引入降低了小分子对正常细胞的毒性,提高了对肺癌耐药细胞的抗肿瘤活性,并且与用临床用药Afatinib和ANF作为对照,发现小分子CFJ001对A549R的抗增殖作用更好。
实施例3
研究CFJ001的光谱特性和选择性以及体外对SDT的响应。
方法:化合物CFJ001及荧光团(氨萘非特)在37℃下的紫外吸收光谱使用Lambda365紫外-可见分光光度计检测。将化合物CFJ001及荧光团用DMSO溶解并制备为10mM的浓储液,用PBS(10mM)(pH=7.4)将化合物和荧光团的浓储液分别稀释成10μM(含有1%DMSO)的样品溶液,在测试时,石英比色皿与光源路径为1cm,吸收波长范围设为200-800nm,化合物及荧光团在37℃下的荧光发射光谱通过FS5荧光分光光度计检测。用PBS(10mM)将化合物及荧光团的浓储液分别稀释成10μM(含有1%DMSO)的样品溶液,在415nm激发光波长下使用FS5荧光分光光度计记录下435-800nm范围内的发射光谱。
通过荧光光谱仪测试CFJ001(10μM)在PBS缓冲液(10mM,1%DMSO,pH 7.4)中对SDT响应的激发和发射光谱。测试CFJ001(10μM)和1mM SDT(连二亚硫酸钠,国药)孵育40min后的荧光光谱。各种无机盐用PBS(10mM,1%DMSO,pH=7.4)稀释获得实验所需的浓度,然后与CFJ001(10μM)孵育40min,荧光发射光谱通过FS5荧光分光光度计检测。激发波长:λex=415nm。通过荧光光谱仪测试CFJ001(10μM)对连二亚硫酸钠(SDT)、各种无机盐FeCl3(1mM),CuCl2(1mM),CaCl2(1mM),FeCl2(1mM),KCl(1mM),MgCl2(1mM),MnCl2(1mM),ZnCl2(1mM),NaCl(1mM),Cys(100μM),Hcy(100μM),GSH(1mM),NADPH(100μM)的选择性。CFJ001和氨萘非特(ANF)的吸收和发射光谱如图2所示,在415nm激发时,ANF在600nm附近显示荧光带,而CFJ001由于偶氮部分荧光猝灭而未显示荧光。在本实施例中为了证明荧光团的假定释放,首先研究了CFJ001在体外对SDT的响应,由于偶氮键淬灭了探针的荧光,几乎没有荧光信号在600nm处检测到。添加1mM SDT后,在600nm处检测到信号发射。CFJ001的荧光强度逐渐增加并增强约85倍,直到在40分钟时达到饱和点。CFJ001检测到的荧光强度显示出时间依赖性增加,证实荧光团的释放确实通过与SDT反应得到促进。为了测试CFJ001的选择性,将其与SDT、各种细胞还原剂(GSH、Cys、Hcy、NADPH)和无机盐(FeCl3、CuCl2、CaCl2、FeCl2、KCl、MgCl2、MnCl2、NaCl、ZnCl2)一起孵育。结果显示只有用SDT处理才能大大增强其荧光。而对于其他潜在干扰物,在相同条件下观察到非常低的荧光强度。结果表明CFJ001不会受到这些潜在干扰物的影响。另外,当对反应后的溶液使用不同的激发波长时,发现只有415nm激发才能观察到比较强的荧光信号,说明不同的激发波长对荧光团的荧光强度有一定的影响,该结果排除了对后续共焦成像实验的某些干扰。本实施例排除了后面共聚焦实验所用的一些染料的激发和发射不会干扰荧光团的信号,说明本发明的偶氮键有效的猝灭了荧光团的荧光,但是在还原条件下,偶氮键可以被连二亚硫酸钠特异性还原重新释放出荧光团。
实施例4
研究CFJ001在细胞内的释放。
方法:将A549R细胞(1x106个)接种到10cm培养皿中,在细胞培养箱中培养12小时。然后将CFJ001(5μM)添加到细胞培养皿中并孵育48小时。吸出培养基并用PBS缓冲液洗涤细胞3次。将细胞在含有1mM PMSF的50μL RIPA裂解缓冲液中裂解30min,每5min震荡一次。通过在4℃下以13400rpm离心20分钟去除细胞碎片。收集含有蛋白质的液体上清液。加入0.5mL甲醇沉降,用液相色谱质谱连用仪LC-MS分析药物在细胞内的释放。
本实施例研究化合物CFJ001在A549R细胞内的是否被激活,如图3所示,在药物处理48h的细胞裂解液中加入甲醇沉降后,LC-MS显示小分子CFJ001在细胞内被部分激活,还有一部分以小分子自身的形式存在于细胞中。本实施例证明了药物分子在细胞内可以被激活释放出荧光团活性药。
实施例5
研究CFJ001的亚细胞定位。
方法:将A549R细胞(5x104个)接种到35mm共聚焦培养皿(JET BIOFIL,加拿大)中进行共聚焦显微镜共定位研究。过夜培养后,将细胞与化合物CFJ001(5μM)在37℃下孵育48h。随后,将培养基吸出,PBS洗两次后,分别向孔中加入含有Mito-Tracker Green(0.25μM),Lyso-Tracker Green(2μM),ER-Tracker Green(1μM),Hoechst 33342:1X用培养基稀释好的染料,并在37℃下染色30分钟。去除染色培养基并用PBS洗涤细胞3次,并立即使用共聚焦显微镜在共聚焦显微镜(A1,尼康,日本)下使用100倍油浸物镜观察。药物和染料的激发和发射波长分别为CFJ001:λex/λem=405/570-620nm;Hoechst33342:λex/λem=405/425-475nm;LTG:λex/λem=488/500-550nm;MTG:λex/λem=488/500-550nm;ETG:λex/λem=488/500-550nm。
为了评估CFJ001在癌细胞中的激活过程,通过荧光成像研究了A549R细胞中CFJ001的激活过程。与CFJ001孵育后,A549R癌细胞显示出明显的红色荧光。这证明了荧光团的释放。为了研究CFJ001在细胞内的分布,将各种细胞器特异性染料和CFJ001与A549R细胞共同孵育。此外,基于染料和CFJ001的像素强度,通过使用Image J软件计算Pearson相关系数(PCC)。在溶酶体中观察到较高的共定位系数,表明CFJ001主要定位于溶酶体(皮尔逊相关系数,0.63),部分定位于线粒体(皮尔逊相关系数,0.11)。这些结果表明细胞内的红色荧光是由于细胞中荧光团ANF的产生。在CFJ001孵育48小时后,在细胞核中观察到一个明显的共定位荧光信号(图4),这表明激活的CFJ001在48小时内从溶酶体逃逸到细胞核。本实施例证明在常氧条件下,小分子抗癌剂CFJ001在癌细胞内就可以被激活释放出荧光团,这是和现有技术(Chem.Commun.,2018,54,7983--7986)报道不一样的,现有技术中偶氮键一般都是在厌氧条件下断开。而本发明设计的小分子偶氮键在常氧条件下就可以断裂,与顺铂相比,发现本发明的小分子对正常细胞的毒性小,但是对于顺铂耐药细胞A549R,发现小分子的活性优于顺铂、临床用药Afatinib和氨萘非特;也降低了氨萘非特和氮芥分子自身对正常细胞的毒性,而小分子在细胞内释放的荧光团可以帮助进行诊断肿瘤。
实施例6
化合物CFJ001可诱导细胞产生自噬小体。
方法:将A549R细胞(5x104个)接种到共聚焦培养皿中,并在细胞培养箱中培养12小时。然后将不同终浓度的CFJ001(5μM)、(10μM)添加到细胞中并孵育24小时。用PBS缓冲液洗涤细胞两次。处理后,用4%多聚甲醛(在PBS中)固定细胞15分钟,并在0.2%Triton X-100(在PBS中)中渗透15分钟。用PBS洗涤步骤后,将细胞与1.5%BSA(在PBS中)在室温下孵育30分钟,然后在4℃下与一抗孵育过夜。用PBS洗涤细胞,然后在室温下用FITC标记的山羊抗兔IgG孵育1小时。最后,用PBS洗涤细胞3次,用DAPI核染料染色10分钟。最后,用PBS缓冲液洗涤细胞两次,并立即使用荧光共聚焦显微镜分析样品。对于MAPLC3B的检测,一抗是兔多克隆抗LC3B抗体(abcam,UK)并以1/1000稀释度使用。二抗是AlexaFluor FITC标记的山羊抗IgG兔(Beyotime,中国),并以1/500稀释度使用。DAPI(λex=405nm,λem=500-550nm);FITC(λex=488nm,λem=500-550nm)。
将A549R细胞((5x106个)在10cm培养皿中培养,分别用5μM CFJ001和1%DMSO处理48小时,每个样品用1.5mL微量离心管收集。然后将细胞用冷PBS洗涤两次,并在4℃下用冷固定溶液(磷酸盐缓冲液中的2.5%戊二醛)固定过夜。之后,将细胞样品脱水、固定、包埋、切片,最后用透射电子显微镜(日立,日本)观察。
考虑到药物定位于溶酶体,与自噬的调节密切相关。为了验证CFJ001诱导细胞自噬,对A549R细胞进行了免疫荧光染色。LC3免疫反应性使用FITC偶联抗体进行可视化,如图5所示,随着孵育时间的延长,CFJ001处理的细胞中的荧光信号明显强于对照组。为了更加直观观察细胞形态的变化,通过透射电镜(TEM)直接检测了CFJ001对A549R细胞的影响。在CFJ001处理后的TEM图像中清晰可见自噬空泡的形成。并且还发现线粒体的形态受损。这说明药物在细胞内活化后可以诱导细胞自噬。通过细胞自噬过程会导致活性氧产生,线粒体膜电位下降,诱导细胞死亡。
实施例7
化合物CFJ001可促进活性氧(ROS)生成。
方法:将A549R细胞接种(3×105个)在6孔细胞板中,在37℃下孵育18小时。与CFJ001(5μM、10μM、20μM、30μM)进一步孵育4小时后,收集细胞并用PBS洗涤两次。用PBS将细胞重新悬浮并在37℃下暴露于荧光探针2',7'-二氯荧光素二乙酸酯(DCFH-DA,10μM)(KGT010-1,凯基生物)30分钟。然后洗涤细胞,用PBS重新悬浮并通过BD FACSverse流式细胞仪分析样品,并用FlowJo 7.6软件分析数据。FL1通道:λex=488nm、λem=500-560nm。
将A549R细胞接种(3×105个)在6孔细胞板中,在37℃下孵育18小时。用CFJ001(10μM)进一步孵育4h、12h、24h、48h后,收集细胞并用PBS洗涤两次。用PBS将细胞重新悬浮并在37℃下暴露于荧光探针2',7'-二氯荧光素二乙酸酯(DCFH-DA,10μM)30分钟。然后洗涤细胞,用PBS重新悬浮并通过BD FACSverse流式细胞仪分析样品,并用FlowJo 7.6软件分析数据。FL1通道:λex=488nm、λem=500-560nm。
鉴于CFJ001分布在A549R细胞的线粒体中,确定细胞凋亡是否由线粒体功能障碍引起。首先,利用2',7'-二氯荧光素(DCF)染色检测细胞内ROS水平的变化,如图6所示,随着孵育时间和浓度的增加,细胞内ROS的含量显着增加。定量结果表明,随着孵育浓度的增加,CFJ001诱导的ROS信号增加了22倍,随着孵育时间的增加,ROS增加了4倍。基于这些结果,可以推断在CFJ001引起的毒性氧化应激下,促进细胞产生活性氧ROS。
实施例8
化合物CFJ001可诱导线粒体膜电位下降。
方法:使用MMP Assay Kit JC-1(YEASEN)试剂盒测定MMP。A549R细胞以2×105个细胞/孔的密度接种于6孔细胞培养板中,在37℃、5%CO2的培养箱中培养12小时。然后,用不同浓度的CFJ001(0μM,5μM,10μM,20μM,30μM)处理细胞24小时。之后,将细胞用PBS洗涤两次,与10mg/mL JC-1染料在37℃下孵育30分钟。用1×Buffer洗涤后,收集细胞并重悬于500μL JC-1染色缓冲液中。然后,立即使用流式细胞仪分析样品。通道为FL1(λex=488nm;λem=510-550nm)和FL2(λex=525nm;λem=560-620nm)数据由FlowJo 7.6软件处理。
使用MMP Assay Kit JC-1(YEASEN)试剂盒测定MMP。将A549R细胞接种(5x104)到35mm共聚焦培养皿(JET BIOFIL,加拿大)中,在37℃、5%CO2的培养箱过夜培养后,将细胞用CFJ001(10μM)在37℃下处理24h。用JC-1工作溶液染色30分钟后,用1×Buffer缓冲液洗涤细胞3次,然后立即通过共聚焦显微镜(A1,尼康,日本)观察。绿色荧光通道:λex=488nm,λem=500-550nm。红色荧光通道:λex=561nm,λem=570-620nm。
在用CFJ001处理的细胞中,线粒体膜电位(MMP)(线粒体功能障碍的标志物)的丧失进一步证实了ROS对线粒体的损伤。MMP Assay Kit JC-1(YEASEN)用于测量A549R细胞的线粒体膜电位(MMP)。在健康细胞中,JC-1在线粒体中积累并形成发出红色荧光的复合物。相比之下,JC-1作为单体存在于细胞质中,并在凋亡细胞中发出绿色荧光。因此,A549R细胞用指定浓度的CFJ001(10μM)处理24小时,然后通过荧光显微镜进行分析。如图7所示,红色荧光减弱,绿色荧光增强,表明CFJ001诱导了A549R细胞中MMP的耗散。红色与绿色荧光强度的比率,从2.59(对照)降低到0.83。流式细胞仪还检测到由CFJ001引起的MMP信号降低了25%。总的来说,这些结果证实了CFJ001可诱导线粒体膜电位下降。
实施例9
化合物CFJ001诱导细胞脂质过氧化从而导致铁死亡。
方法:将A549R细胞以2×105个细胞/孔的密度接种到35mm共聚焦培养皿(JETBIOFIL,加拿大)中用于共聚焦显微镜检测,并在37℃下在5%CO2的环境中孵育12小时。分别与CFJ001(5μM)、ANF(5μM)和Afatinib(5μM)进一步孵育48小时后。用PBS洗涤细胞两次,然后加入5μM C11-BODIPY(Thermo Fisher,Cat#D3861)并孵育1小时。通过用PBS洗涤细胞两次来去除过量的C11-BODIPY。立即使用共聚焦显微镜分析样品。绿色荧光通道:λex=488nm,λem=500-550nm。红色荧光通道:λex=561nm,λem=570-620nm。
用脂质过氧化探针BODIPY-581/591C11(Thermo Fisher,Cat#D3861)来测定铁死亡,如图8所示细胞用药物处理48h后,和对照组相比,小分子抗癌剂CFJ001处理的细胞红色荧光逐渐减弱,绿色荧光逐渐增强,但是ANF和Afatinib处理的细胞没有明显的现象。这是因为C11-BODIPY的多不饱和丁二烯基部分的氧化导致荧光发射峰从~590nm转移到~510nm,与脂质ROS的生成成正比。结果证明了只有小分子抗癌剂CFJ001处理后的细胞发生了脂质过氧化。同时通过开启荧光探针FeRhoNox-1(MX4558-50UG,集奇生物)评估细胞内Fe2+水平以及通过ICP-MS评估加药前后细胞内的整体Fe含量,结果发现细胞内亚铁离子的水平升高,证明细胞发生了铁死亡。
实施例10
化合物CFJ001诱导细胞死亡机制研究。
方法:在蛋白质印迹研究中,A549R细胞用所需浓度的CFJ001处理,将A549R细胞分别以106个/皿的密度种在5个10cm的培养皿中,并在37℃下在5%CO2的环境中孵育24小时后,分别与CFJ001(1.25,2.5,5,10μM)和1%DMSO进一步孵育24小时后。每个样品用1.5mL微量离心管收集并用冰冷的PBS洗涤两次。将细胞在含有1mM PMSF的RIPA裂解缓冲液中裂解30min,每5分钟震荡一次。通过在4℃下以13000rpm离心20分钟去除细胞碎片后,收集含有蛋白质的液体上清液,并使用BCA蛋白质测定试剂盒(Beyotime)定量每个样品的总蛋白质含量。用PBS对样品进行稀释,加入SDS-PAGE上样缓冲液,并在95℃加热8min。蛋白按每孔40-50μg的浓度用12%SDS-PAGE凝胶进行电泳实验,并将目的蛋白转移到聚偏二氟乙烯(PVDF)膜上,设定恒定电流200mA,在冰浴下转膜1h。结束后,将PVDF膜在PBS缓冲液(10mM,5%脱脂奶粉,0.05%Tween-20)中封闭1h。按比例稀释的一抗溶液与PVDF膜在4℃下孵育过夜。结束后,将膜用PBST(10mM,0.05%Tween-20)洗涤4次,每次8min,用过氧化物酶偶联的相应二抗继续孵育1h后,将膜用PBST(10mM,0.05%Tween-20)洗涤4次,每次8min。将等体积配置好的ECL显影液覆盖在PVDF膜上,孵育两分钟后使用Power PacTM HC(新加坡)的BIORAD成像,通过Image J软件分析图像。
由于药物活化后产生的活性氮介分子和少量荧光团进入细胞核中,所以通过免疫印迹进行评估,如图9所示,在CFJ001处理的A549R细胞中观察到γH2AX表达明显增加。这进一步证实了CFJ001处理后的细胞的DNA双链受损。此外,还检测了肿瘤抑制因子p53对DNA损伤的反应,并观察到浓度依赖性的表达增加。综上所述,这些发现表明CFJ001被激活形成的氮介分子以及从溶酶体逃逸到细胞核的少量ANF在A549R细胞中进一步诱导DNA损伤。自噬可能会促进ROS的产生和随后的脂质过氧化物的积累。通过NCOA4介导的铁蛋白吞噬作用降解铁蛋白将释放铁以诱导铁死亡。所以通过蛋白印迹检测了NCOA4和GPX4这两种铁死亡调控基因的表达,NCOA4的表达上升增强了铁蛋白的吞噬,导致游离铁的增加,诱导铁死亡。这些结果证实了CFJ001可诱导细胞发生脂质过氧化和铁死亡。上述实施例有效证明了本发明的药物分子杀死癌细胞的作用机制,其优势就是药物分子可以诱导如肺癌耐药株细胞发生自噬依赖性的铁死亡。同时本发明的小分子在细胞内被激活后释放的荧光团氨萘非特可以帮助诊断肿瘤(实施例4),药物分子可以杀死细胞,提供诊断和治疗肿瘤一体化。
Claims (10)
2.根据权利要求1所述的小分子抗癌剂,其特征在于,所述小分子抗癌剂由氨萘非特和1,1-二甲基乙二胺合成获得。
3.一种权利要求1所述的以萘酰亚胺为骨架的小分子抗癌剂的制备方法,其特征在于,包括如下步骤:
(1)在室温下向苯胺和2-溴乙醇的混合物中添加碳酸钾,再加入乙醇溶液;将所得混合物加热搅拌分出有机层,干燥,旋转蒸发浓缩得粗品,粗品纯化得到产物S1;
(2)将产物S1溶解在三氯氧磷中并回流,减压浓缩反应混合物并稀释,然后洗涤,干燥并减压浓缩;纯化得到产物S2;
(3)将氯化锡(II)加入浓盐酸中,所得溶液逐滴加入到3-硝基-1,8-萘二甲酸酐的搅拌悬浮液乙醇中,将所得悬浮液搅拌回流,然后冷却至室温;过滤收集沉淀产物,洗涤,所得固体干燥得到产物S3;
(4)将产物S3溶于乙醇中,加入1,1-二甲基乙二胺的乙醇溶液,然后加热回流,在室温下缓慢冷却溶液,过滤分离固体,洗涤,所得固体干燥得到产物S4;
(5)在惰性气体中,向S4的搅拌溶液中加入NaNO2水溶液,将溶液搅拌后加入TFA并将溶液再搅拌,随后将S2溶于乙腈加入上述反应液中,并将混合物搅拌并加入水,混合物萃取,合并有机层,干燥,除去溶剂;经色谱分离后得到小分子抗癌剂化合物CFJ001。
4.根据权利要求3所述的制备方法,其特征在于,步骤(1)中在室温下将苯胺和2-溴乙醇按摩尔比1:2-3加入三颈圆底烧瓶中,将碳酸钾加入反应瓶中,再加入乙醇溶液,将所得混合溶液在78-80℃下搅拌4-6小时。
5.根据权利要求3所述的制备方法,其特征在于,步骤(2)中将产物S1溶解在三氯氧磷中,控制反应温度在100-105℃回流3-4h,反应结束后,先用二氯甲烷稀释后再用饱和食盐水洗涤。
6.根据权利要求3所述的制备方法,其特征在于,步骤(4)中将产物S3溶解在乙醇中,加入1,1-二甲基乙二胺的乙醇溶液,然后在78-80℃下加热回流2.5-3小时。
7.根据权利要求3所述的制备方法,其特征在于,步骤(5)中在Ar气氛围在0℃下将产物S4溶于ACN和H2O的混合溶液加入两颈圆底烧瓶中,0℃搅拌后,将NaNO2的水溶液加入圆底烧瓶中,将溶液搅拌后,加入TFA并将溶液再搅拌,随后将S2溶于乙腈加入反应液中,并将混合物在0℃下搅拌之后加入水。
8.一种权利要求1所述的小分子抗癌剂在制备抗肿瘤药物中的应用。
9.根据权利要求8所述的应用,其特征在于,所述肿瘤优选包括宫颈癌,肝癌,肺癌和非小细胞肺癌。
10.一种权利要求1所述的小分子抗癌剂在制备体外对SDT的特异性响应以及对各种金属离子的选择性筛选的药物中的应用。
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