CN112062797B - 一种二聚体前药及其制备方法和应用 - Google Patents
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Abstract
Description
技术领域
本发明涉及一种二聚体前药及其制备方法和应用,属于前药载体技术领域。
背景技术
肿瘤治疗主要包括手术治疗、化疗、光动力疗法、放疗、免疫治疗等方法,其中化疗是最为主要的临床治疗方式。然而,由于化疗药物的毒性往往是没有选择性的,在杀伤肿瘤细胞的同时,也会对正常组织造成极大的损害,所以传统的化疗往往会带来严重的毒副作用。因此,研究人员越来越关注前药的研究。
前药,也称前体药物、药物前体、前驱药物等,是指药物经过化学结构修饰后得到的在体外无活性或活性较小、在体内经酶或非酶的转化释放出活性药物而发挥药效的化合物。前药不仅可以降低化疗过程中的毒副作用,而且可以减少药物的使用剂量。
前药活化过程的研究对于前药而言,具有重要意义。荧光分子通过可响应键与药物键合,而响应键的断裂会伴随荧光打开或者荧光通道转变,根据荧光信号的变化来检测前药的活化过程,这是目前检测前药活化过程的主要手段。但是,目前大多数可以实现自监测的前药都是使用不具备治疗作用的荧光分子结合传统化疗药物,仅仅通过一个通道荧光信号观测药物活化过程,而且其中大多数不是近红外的荧光,难以在动物水平观测前药的活化。
发明内容
本发明的目的在于提供一种二聚体前药及其制备方法和应用。
本发明所采取的技术方案是:
一种二聚体前药,构式为:
上述二聚体前药的制备方法,包括以下步骤:
优选的,上述二聚体前药的制备方法,包括以下步骤:
优选的,步骤1)所述2,3,3-三甲基吲哚、碘乙烷的摩尔比为1:(4~6)。
优选的,步骤2)所述环己酮、三氯氧磷的摩尔比为1:(2~3)。
本发明的有益效果是:本发明的二聚体前药具有双重荧光效应,可以通过ICT(光诱导电子转移)和FRET(荧光共振能量转移)效应猝灭CyNH2和DOX的荧光,很好地监测药物活化过程,并降低毒性,且利用CyNH2和DOX的协同效应还可以增强对肿瘤的治疗效果。
具体来说:
1)本发明的二聚体前药中的荧光分子CyNH2无需光照,作用于细胞,产生ROS,杀伤细胞,具有很好的治疗效果;
2)本发明的二聚体前药可以同时利用ICT和FRET效应,淬灭前药中两个通道的荧光,响应后两个通道荧光能够打开,可实现自监测药物活化过程;
3)本发明的二聚体前药可以利用荧光分子CyNH2的线粒体靶向特性,使得二聚体前药作用于线粒体,通过损伤线粒体DNA进行杀伤;
4)本发明的二聚体前药中的荧光分子CyNH2和化疗药物DOX的协同效应,可以增强对肿瘤的治疗。
附图说明
图1为CyNH-SS-DOX的合成路线图。
图2为CyNH-SS-DOX的核磁共振氢谱图。
图3为CyNH-SS-DOX响应前后的吸收光谱图。
图4为CyNH-SS-DOX对各种物质响应的荧光光谱图。
图5为CyNH-SS-DOX在相同时间、不同浓度DTT孵育的两个通道荧光光谱图。
图6为CyNH-SS-DOX在相同浓度DTT、不同时间孵育的两个通道荧光光谱图。
图7为4T1细胞系下游离药物的MTT法测试结果图。
图8为231细胞系下游离药物的MTT法测试结果图。
图9为4T1细胞系下CyNH-SS-DOX和CyNH-CC-DOX的MTT法测试结果。
图10为小鼠活体成像结果。
图11为不同时间点肿瘤切片的两个通道荧光成像结果。
图12为体内治疗小鼠肿瘤体积变化曲线。
图13为体内治疗小鼠肿瘤重量结果。
图14为体内治疗小鼠肿瘤大小实物图。
图15为主要器官的HE切片结果。
图16为体内治疗实验中各实验组小鼠体重变化曲线图。
具体实施方式
下面结合具体实施例对本发明做进一步的解释和说明。
实施例:
一种二聚体前药,其制备方法包括以下步骤(合成路线如图1所示):
1)将1.44g(9.0mmol)的2,3,3-三甲基吲哚和7.02g(45.0mmol)的碘乙烷分散在30mL的乙腈中,加热回流24h,再减压除去溶剂,用正己烷洗涤得到的粗产品3次,得到(白色固体);
2)在氮气气氛下,将3mL(40.95mmol)的二甲基甲酰胺和15mL的二氯甲烷在冰浴中混合,再将20mL的三氯氧磷的二氯甲烷溶液(含三氯氧磷2.63mL(17.25mmol))逐滴加入,并加入0.74g(7.5mmol)的环己酮,加热搅拌回流3h,再将反应混合物倒入冰中并静置12h,过滤,得到(淡黄色固体);
3)将1.89g(6.0mmol)的0.51g(3.0mmol)的和0.5g(6.0mmol)的乙酸钠分散在30mL的乙酸酐中,加热至130℃反应1h,再减压除去溶剂,用碳酸氢钠溶液洗涤得到的粗产品,用二氯甲烷萃取,减压除去溶剂,用硅胶快速层析法(二氯甲烷、甲醇体积比25:1)纯化粗产品,得到(绿色粉末);
4)将69.5mg(0.5mmol)的3-硝基苯酚和50.5mg(0.5mmol)的三乙胺分散在10mL的二甲基甲酰胺中,再加入10mL的的二甲基甲酰胺溶液(含256mg(0.4mmol)),常温搅拌12h,减压除去溶剂,再分散在二氯甲烷中,用碳酸氢钠溶液洗涤3次,用硫酸钠干燥,用硅胶快速层析法(二氯甲烷、甲醇体积比25:1)纯化粗产品,得到(金黄色粉末)。
5)将254mg(0.34mmol)的分散在10mL的甲醇中,加入1.3g(6.86mmol)的二氯化锡和1.4mL的浓盐酸(质量分数37%),加热回流12h,用饱和NaHCO3溶液中和反应混合物,用碳酸氢钠溶液洗涤,二氯甲烷萃取,减减压除去溶剂,用硅胶快速层析法(二氯甲烷、甲醇体积比20:1)纯化粗产品,得到(CyNH2,绿色粉末);
6)将100mg(0.191mmol)的和166μL的N,N-二异丙基乙胺混合,加入160mg(0.539mmol)的三光气和10mL的乙腈组成的混合物,再在冰浴中逐滴加入20mL的乙腈,加完后加热回流4h,再加入292mg(1.90mmol)的2-羟乙基二硫化物,室温搅拌12h,再用碳酸氢钠溶液洗涤反应液,用二氯甲烷提取,减压除去溶剂,用硅胶快速层析法(二氯甲烷、甲醇体积比20:1)纯化粗产品,得到(CyNH-SS-OH,蓝色固体)。
7)将73mg(0.104mmol)的CyNH-SS-OH和21mg(0.206mmol)的三乙胺分散在5mL的二氯甲烷中,冷却至0℃,再滴加2mL的对硝基氯甲酸苯酯的二氯甲烷溶液(含对硝基氯甲酸苯酯42mg(0.206mmol)),滴加完后室温搅拌6h,再用碳酸氢钠溶液洗涤反应液,用二氯甲烷提取,收集有机层,用无水Na2SO4干燥,浓缩干燥,得到(CyNH-SS-NPC)。
8)将75mg(0.086mmol)的CyNH-SS-NPC和21mg(0.206mmol)的三乙胺分散在2mL的二甲基甲酰胺中,再滴加5mL的盐酸阿霉素的二甲基甲酰胺溶液(含盐酸阿霉素61mg(0.105mmol)),滴加完后室温搅拌6h,反应液用二氯甲烷提取,收集有机层,用无水Na2SO4干燥,除去溶剂,用硅胶快速层析法(二氯甲烷、甲醇体积比10:1)纯化粗产品,得到(CyNH-SS-DOX),即二聚体前药。
对比例:
二聚体前药CyNH-CC-DOX的制备:
除了将实施例步骤6)中的2-羟乙基二硫化物替换成1,6-己二醇以外,其他与实施例中的二聚体前药CyNH-SS-DOX的制备过程完全一样。
性能测试:
1)CyNH-SS-DOX的核磁共振氢谱(1H NMR)如图2所示。
由图2可知:e为阿霉素甲氧基的质子氢,k为CyNH2荧光分子甲基的质子氢,f和g是二硫醇亚甲基的质子氢。
2)CyNH-SS-DOX的应用:
a)CyNH-SS-DOX的响应性:
在37℃的DMSO/PBS溶液(10/90,v/v,pH=7.4)中测试CyNH-SS-DOX的响应性,加入DTT(二硫苏糖醇)前后的吸收光谱如图3所示,CyNH-SS-DOX(10μM)对各种物质响应的荧光光谱如图4所示(左图为DOX通道,右图为CyNH2通道),CyNH-SS-DOX在相同时间、不同浓度DTT孵育的两个通道荧光光谱图如图5所示(左图为DOX通道,右图为CyNH2通道),CyNH-SS-DOX在相同浓度DTT、不同时间孵育的两个通道荧光光谱图如图6所示(左图为DOX通道,右图为CyNH2通道)。
由图3可知:CyNH-SS-DOX在加入DTT(二硫苏糖醇)后曲线由一个完整的峰型转变为两个独立的峰型,表现出二硫化物裂解反应。
由图4可知:对于非响应物质Met(蛋氨酸)、Glu(谷氨酰胺)、Lys(赖氨酸)、His(组氨酸)、Cys-Cys(胱氨酸),CyNH-SS-DOX(10μM)在DOX和CyNH2通道中都显示出非常微弱的荧光,而对于响应性物质DTT、Cys(半胱氨酸)、TCEP(三(2-羧乙基)膦)和GSH(谷胱甘肽),CyNH-SS-DOX(10μM)在DOX和CyNH2通道中都显示出很强的荧光。
由图5可知:相同孵育时间下,从0倍浓度组到1000倍浓度组,DOX通道荧光逐渐增强,CyNH2通道荧光也逐渐增强。
由图6可知:相同孵育浓度下,从0h组到24h组,DOX通道荧光逐渐增强,CyNH2通道荧光也逐渐增强。
b)体外细胞实验:
SNP和in SNP的制备:将PEG5k-PLA8K(聚乙二醇-聚乳酸)配成浓度10mg/mL的二甲基亚砜溶液,并将CyNH-SS-DOX配成浓度10mg/mL的二甲基亚砜溶液,取200μL的PEG5k-PLA8K溶液和50μL的CyNH-SS-DOX溶液混合,得到混合溶液,再取1mL的超纯水加入到10mL的烧瓶中,磁力搅拌下将混合溶液加入烧瓶中,再透析得到SNP(CyNH-SS-DOX的PEG5K-PLA8K颗粒),将CyNH-SS-DOX替换成CyNH-CC-DOX,参照同样的步骤,得到in SNP(CyNH-CC-DOX的PEG5K-PLA8K颗粒)。
进行游离DOX、游离CyNH2和DOX:CyNH2=1:1(DOX和CyNH2按照质量比1:1混合)的MTT细胞毒性实验,用4T1/231细胞株评价不同浓度的游离DOX、游离CyNH2和DOX:CyNH2=1:1的抗肿瘤作用,4T1和231细胞系下,游离药物的MTT结果分别如图7和图8所示,4T1细胞系下,CyNH-SS-DOX和CyNH-CC-DOX的MTT法测试结果如图9所示。
由图7所示:双药联用下,对于4T1肿瘤细胞有更好的抑制作用,通过计算,IC50下CI值为0.437,说明DOX和CyNH2之间具有协同效应(协同系数CI是衡量协同效应的黄金法则,如果CI=1,则为加法,如果<1,则为协同作用,如果大于1,则为拮抗性)。
由图8所示:双药联用下,对于231肿瘤细胞有更好的抑制作用,通过计算,IC50下CI值为0.137,说明DOX和CyNH2之间具有协同效应(协同系数CI是衡量协同效应的黄金法则,如果CI=1,则为加法,如果<1,则为协同作用,如果大于1,则为拮抗性)。
由图9可知:CyNH-SS-DOX对4T1肿瘤细胞具有明显更好的体外抗肿瘤活性,而CyNH-CC-DOX对4T1肿瘤细胞体外抗肿瘤活性更差。原因在于:CyNH-SS-DOX能通过二硫键的断裂充分释放DOX和CyNH2,对肿瘤杀伤效果更好,而CyNH-CC-DOX不能有效释放DOX和CyNH2,导致细胞毒性降低。
c)动物水平实验:
以4T1荷瘤小鼠为研究对象,静脉注射SNP和in SNP,采用活体成像系统观察小鼠体内药物分布,两组的测试结果如图10所示,在注射后36h时处死小鼠,通过采集肿瘤和主要器官进行活体外成像,对不同时间点肿瘤切片的两个通道荧光成像结果如图11所示。以4T1荷瘤小鼠为研究对象,采用4T1肿瘤模型研究其体内抗肿瘤作用,对荷瘤小鼠静脉注射PBS、DOX、CyNH2、in SNP和SNP,其中CyNH2等效剂量为2.5mg/kg,DOX等效剂量为5mg/kg,治疗期间对肿瘤体积进行测量记录,体内治疗小鼠肿瘤体积变化曲线结果如图12所示;治疗后牺牲小鼠取出肿瘤,对肿瘤重量进行测量记录,体内治疗小鼠肿瘤重量结果如图13所示;治疗后牺牲小鼠取出肿瘤进行拍照,体内治疗小鼠肿瘤大小实物图结果如图14所示;治疗小鼠牺牲,取出心、肝、脾、肺、肾、肿瘤,进行切片,并用HE(苏木精溶液,伊红溶液)染色,结果如图15所示;治疗期间对小鼠体重监测,得到的体内治疗实验中各实验组小鼠体重变化曲线如图16所示。
由图10可知:两个实验组中,SNP(1.0mg/kg)组,在静脉注射仅2h,该组近红外荧光就已清晰可见,而in SNP(1.0mg/kg)组的荧光则相对低得多,表明SNP通过血液循环迅速分布,其中二硫键因GSH浓度特别高而迅速断裂。
由图11可知:注射SNP的小鼠肿瘤组织中CyNH2的荧光强度明显高于in SNP,这可能是由于肿瘤细胞中GSH浓度过高,导致SNP通过二硫键释放出CyNH2,而in SNP完全不能释放CyNH2所致;静脉注射in SNP后36h,in SNP组DOX和CyNH2的荧光信号均较弱,而SNP组DOX和CyNH2的荧光信号在静脉注射SNP后12h增强,随着时间的延长DOX和CyNH2的荧光信号逐渐增强,这意味着SNP可以通过癌细胞摄取二硫键的断裂释放DOX和CyNH2,SNP的肿瘤特异性和在肿瘤中的特异性药物释放,使其成为一种高效、低毒的前药。
由图12可知:与PBS组和in SNP组相比,DOX、CyNH2或SNP治疗可以显著抑制4T1肿瘤生长,DOX、CyNH2和SNP对4T1肿瘤的抑制率分别为55.4%、52.9%和88.9%,实验结果表明实验组前药有更有效的治疗效果。
同时,体内治疗小鼠肿瘤重量结果图(图13)和体内治疗小鼠肿瘤大小实物图(图14)与IRT(肿瘤生长抑制率)数据一致。小鼠的主要器官的HE切片结果(图15)中,实验组SNP和PBS组主要切片细胞形态正常,表明对器官无明显影响。在整个实验过程中,所有小鼠的体重(图16)趋势是逐渐增加,表明治疗过程没有造成严重的系统性副作用。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
3.根据权利要求2所述的制备方法,其特征在于:步骤1)所述2,3,3-三甲基吲哚、碘乙烷的摩尔比为1:(4~6);步骤2)所述环己酮、三氯氧磷的摩尔比为1:(2~3)。
10.权利要求1所述的二聚体前药在制备抗癌药物中的应用。
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