CN116375586A - 氮芥活性探针及其应用 - Google Patents
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Abstract
本发明提供了氮芥活性探针及其应用，探针具有下式所示结构的化合物。本发明进一步提供了该氮芥活性探针的合成方法以及其在制备氮芥标记物或抗肿瘤药物中的应用，同时提供了以该氮芥活性探针作为活性组分的氮芥标记物或抗肿瘤药物。本发明探针可作为生物正交标记探针，尤其是用于标记组织和细胞中的氮芥，用于研究氮芥抗肿瘤、免疫抑制等方面的毒理机制；该探针可用于探究氮芥的直接作用蛋白靶点，有助于深入了解氮芥的作用机制；活性探针本身具有一定的抗肿瘤、免疫抑制的作用，可作为免疫抑制和/或抗肿瘤药物。
Description
技术领域
本发明涉及医学生物检测及药物技术领域，涉及一种氮芥活性探针及其在氮芥细胞内标记及抗肿瘤中的应用。
背景技术
氮芥是一种有机化合物，化学式为C5H11Cl2N，是最早用于临床并取得突出疗效的抗肿瘤药物，为双氯乙胺类烷化剂的代表。氮芥抗肿瘤化疗药物的起源，可用于治疗恶性淋巴瘤、肺癌、头颈癌和霍奇金病等，临床上也将其用于治疗牛皮癣、狼疮性肾病。氮芥还是糜烂性毒剂的一种，可引起局部皮肤的起疱和溃烂。研究者们还基于氮芥的结构，开发了大量的氮芥类药物，例如环磷酰胺、司莫司汀等。
氮芥进入体内后，通过分子内成环作用，形成高度活泼的乙烯亚胺离子，在中性或弱碱条件下迅速与多种有机物质的亲核基团(如蛋白质的羧基、氨基、巯基、核酸的氨基和羟基、磷酸根)结合，进行烷基化作用。氮芥最重要的反应是与鸟嘌呤第7位氮共价结合，产生DNA的双链内的交叉联结或DNA的同链内不同碱基的交叉联结。G1期及M期细胞对氮芥的细胞毒作用最为敏感，由G1期进入S期延迟。
尽管已有研究表明氮芥在体内可与各种生物大分子发生结合，但其在体内具体的蛋白靶点及其机制尚不清楚。由于缺乏有效的氮芥标记物，难以实现氮芥在体内具体作用靶标的确定。
发明内容
为了明确氮芥在体内具体的蛋白靶点，本发明开发了一种能在细胞和组织内可视，并能够用于靶点蛋白鉴定的氮芥活性探针，其还具有较强的抗肿瘤活性的活性，可用于制备相应药物。
本发明第一方面，提供了一种氮芥活性探针，具有式I所示结构的化合物：
其中，n为0-5间的正整数。
优选的，n为1或2。
本发明的第二方面，提供了上述氮芥活性探针的制备方法，反应结构式如下：
合成流程如下：将二(2-氯乙基)胺与溴代炔加入至有机溶剂中，在碱性试剂存在条件下，25-160℃反应8-72h，得到所述氮芥活性探针。
优选的，有机溶剂选自二氯甲烷、乙腈、正丁醇、甲醇、二甲亚砜中的至少一种；碱性试剂选自碳酸钾、氢氧化钠、氢氧化钾、甲醇钠、碳酸铯中的至少一种。
本发明的第三方面，提供了上述氮芥活性探针的应用，具体是在制备氮芥标记物或抗肿瘤药物中的应用。
优选的，氮芥标记物为细胞内氮芥标记物，通过荧光胶分析、基于质谱的靶标蛋白定量及位点鉴定明确氮芥的直接作用蛋白质靶标。所述抗肿瘤药物为抗黑色素瘤的药物。
本发明的第四方面，提供了一种氮芥标记物，其功能组分为上述所述的氮芥活性探针，其作用于细胞内，通过追踪分析实现具体作用靶标鉴定。
本发明的第五方面，提供了一种抗肿瘤药物，包括活性组分以及药学上可接受的辅料，所述活性组分包括上述氮芥活性探针。
本发明的有益保障及效果如下：
(1)实验结果显示本发明的氮芥活性探针可直接作用于细胞内，通过细胞成像实现氮芥在细胞内的追踪。该探针可作为生物正交标记探针，尤其是用于标记组织或细胞中的氮芥，用于研究氮芥抗肿瘤、免疫抑制等方面的药理毒理机制，探究氮芥的直接作用蛋白靶点，有助于深入了解氮芥的作用机制。
(2)氮芥活性探针对包括HaCaT细胞在内的肿瘤细胞活力的抑制作用呈浓度依赖关系，说明该活性探针本身具有一定的抗肿瘤的作用，可作为抗肿瘤药物。
附图说明
图1是氮芥活性探针I-1的核磁共振氢谱；
图2是氮芥活性探针I-1的核磁共振碳谱；
图3是氮芥活性探针I-2的核磁共振氢谱；
图4是氮芥活性探针I-2的核磁共振碳谱；
图5是氮芥活性探针I-1在细胞中的荧光成像结果；
图6是氮芥活性探针I-1基于荧光胶分析的直接标记结果；
图7是不同浓度氮芥活性探针I-1对HaCaT细胞活力的抑制作用。
具体实施方式
现结合实施例和附图，对本发明作详细描述，但本发明的实施不仅限于此。
本发明所用试剂和原料均市售可得或可按文献方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按体积计算。
本文中提供的所有数值范围旨在清楚地包括落在范围端点之间的所有数值及它们之间的数值范围。可对本发明提到的特征或实施例提到的特征进行组合。本说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以任何可提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。
如本文所用，“含有”、“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”；“主要由……构成”、“基本上由……构成”和“由……构成”属于“含有”、“具有”或“包括”的下位概念。
本文中的数值范围包括其端点以及该数值范围内的各具体数值点和子范围。例如，1～3包括端点1和3、其中的具体整数数值点2和非整数数值点(例如但不限于：1.2、1.5、1.8、2.1、2.3、2.4、2.8等，以及其子范围(例如但不限于：1～2、2～3、1～1.2、1.5～1.8等)。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本申请中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1：活性探针I-1的合成
将0.1mmol二(2-氯乙基)胺与0.1mmol溴代丙炔加入至二氯甲烷中，加入0.05mmol碳酸钾在20℃反应20h，将反应液旋干，柱层析(二氯甲烷)分离得到油状物I-1，产率为60.9％。
该氮芥活性探针I-1的核磁共振氢谱以及核磁共振碳谱分别如图1和图2所示。
实施例2：活性探针I-2的合成
将0.1mmol二(2-氯乙基)胺与0.1mmol溴代丁炔加入至乙腈中，加入0.05mmol碳酸钾在80℃反应8h，将反应液旋干，柱层析(二氯甲烷)分离得到油状物I-2，产率为50.2％。
该氮芥活性探针I-2的核磁共振氢谱以及核磁共振碳谱分别如图3和图4所示。
实施例3：氮芥活性探针I-1作为氮芥标记探针在细胞成像中的应用
将HaCaT细胞铺于培养皿，生长至80％分组，分别为空白组、低浓度探针组、中浓度探针组和高浓度探针组。空白组加入0.1％DMSO，低浓度探针组加入10μM的I-1，中浓度探针组加入50μM的I-1，高浓度探针组加入100μM的I-1，孵育时间2h。
将100μM叠氮罗丹明、100μM三[(1-苄基-1H-1,2,3-三唑-4-基)甲基]胺、1mM硫酸铜预混合后，加入细胞中孵育，反应完成后，加入PBS洗涤3次。通过DAPI进行细胞核染色。采用荧光显微镜拍照，结果见图5，对氮芥的行踪能够实现有效追踪。
实施例4：荧光胶分析的直接标记实验
取-80℃保存的皮肤组织置于冰上融化，加入lysis buffer，研磨，超声破碎；4℃，台式离心机，20000g，30min；取上层清液，蛋白质浓度定量，稀释至一定浓度待用。将HNP-1按如下表1配制：
表1 HNP-1及其封闭液配置
取0.5μLDMSO或HNP-1加入50μL蛋白组溶液中，离心，最终溶液DMSO为1％，混合溶液在25℃孵育1h。在每份样品中按顺序加入TAMRA-azide、BTTAA/CuSO4、抗坏血酸钠进行Click反应，混合溶液在25℃震荡反应1h。反应结束后，每份样品中加入13.75μL loadingbuffer，煮样后冷却。取全部样品各40μL，10％SDS-PAGE，电泳。使用ChemiDoc MP检测荧光信号，考染检测蛋白质。
根据荧光胶实验结果可知，标记强度随着HNP-1浓度增大而增大，根据荧光条带与考马斯亮蓝条带对比，有一定的选择性，说明HNP-1可以特异性地标记蛋白质，其中在40-50、70kDa附近标记效果明显。黄色标注部分蛋白质CBB信号减弱，综合三次生物学重复结果，应该是探针浓度升高，导致蛋白质交联，分子量迁移造成。为了定量鉴定对HNP-1反应活性高的蛋白质，结合文献方法(Quantitative reactivity profiling predictsfunctional cysteines inproteomes.Nature.2010,468(7325):790-5)，后续选取1/10/100μM探针浓度进行质谱实验。Rhodamine和CBB的标记结果见图6。
实施例5：
采用含有0.01％EDTA的胰酶消化法传代培养HaCaT细胞，当细胞占瓶底有效面积80％时，胰酶消化后吹打成单细胞悬液，显微镜下细胞计数，稀释成每微升中含有100个细胞的悬液，以每孔100μl细胞悬液接种于96孔板中，置于37℃，5％CO2饱和湿度培养箱中培养，待细胞密度达80％时进行实验。
实验设置空白对照组、不同浓度氮芥活性探针I-1染毒组，每组3个复孔。配制不同浓度梯度的芥子气染毒液，染毒30min后更换新鲜培养基，细胞培养箱中培养24h后，每孔加入10μl CCK-8液，培养箱孵育1.5h后，酶标仪测定450nm下吸光度，计算细胞活力，其结果如图7所示。结构显示，氮芥活性探针I-1对细胞活力的抑制作用呈浓度依懒性，氮芥活性探针I-1的浓度越高，对细胞活力的抑制作用越强。
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可作出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (10)
1.一种氮芥活性探针，其特征在于，具有式I所示结构的化合物：
其中，n为0-5间的正整数。
2.根据权利要求1所述的氮芥活性探针，其特征在于：
其中，n为1或2。
3.权利要求1或2所述的氮芥活性探针的制备方法，其特征在于，包括如下步骤：
将二(2-氯乙基)胺与溴代炔加入至有机溶剂中，在碱性试剂存在条件下，25-160℃反应8-72h，分离后得到所述氮芥活性探针。
4.根据权利要求3所述的氮芥活性探针的制备方法，其特征在于：
其中，所述有机溶剂选自二氯甲烷、乙腈、正丁醇、甲醇、二甲亚砜中的至少一种；
所述碱性试剂选自碳酸钾、氢氧化钠、氢氧化钾、甲醇钠、碳酸铯中的至少一种。
5.根据权利要求3所述的氮芥活性探针的制备方法，其特征在于：
其中，二(2-氯乙基)胺与溴代炔的摩尔比为1:1，二(2-氯乙基)胺与碱性试剂之间的摩尔比为2:1，
产物分离方法为将反应液旋干，二氯甲烷柱层析分离得到油状物。
6.权利要求1或2所述的氮芥活性探针在制备氮芥标记物或抗肿瘤药物中的应用。
7.根据权利要求6所述的应用，其特征在于，所述氮芥标记物为细胞内氮芥标记物，所述抗肿瘤药物为抗黑色素瘤的药物。
8.根据权利要求7所述的应用，其特征在于，采用所述氮芥标记物确定氮芥的直接作用靶标。
9.一种氮芥标记物，其特征在于，其功能组分为权利要求1或2所述的氮芥活性探针。
10.一种抗肿瘤药物，其特征在于，包括活性组分以及药学上可接受的辅料，所述活性组分包括权利要求1或2所述的氮芥活性探针。
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