CN114736192B - 一种含7-羟基香豆素结构基团的荧光化合物 - Google Patents
一种含7-羟基香豆素结构基团的荧光化合物 Download PDFInfo
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Abstract
本发明公开了一种含7‑羟基香豆素结构基团的荧光化合物,其为7‑羟基香豆素报告基团通过2‑甲酰胺‑4,5‑二甲氧基苯甲酰胺连接2‑(6,7‑二甲氧基‑3,4‑二氢‑1H‑异喹啉‑2‑基)苯乙基结合基团形成的7‑羟基香豆素类荧光化合物,用于P‑糖蛋白特异性荧光探针。本发明开发了一种含7‑羟基香豆素结构基团的P‑糖蛋白示踪剂作为P‑糖蛋白特异性荧光探针,此荧光探针能够可靠地用于P‑糖蛋白的定位示踪,在体内具有特异性高、灵敏度高、安全性好的优点;对肿瘤、癫痫等疾病的P‑糖蛋白组织高表达区定位、细胞内P‑糖蛋白定位及其研究的可视性等问题提供了新的视角和选择。
Description
技术领域
本发明属于荧光化合物技术领域,具体涉及一种含7-羟基香豆素结构基团的荧光化合物。
背景技术
P-糖蛋白(P-glycoprotein,P-gp)是一种分子量170KD的跨膜糖蛋白,它具有能量依赖性″药泵″功能,使细胞内药物泵出细胞外,减低了细胞内的药物浓度,使细胞产生耐药性。P-gp的存在影响了大量治疗药物的吸收分布,包括抗癌药物、抗病毒药物、抗组胺药、抗癫痫药和镇痛药。
目前,对P-gp的成像主要通过正电子发射计算机断层扫描(positronemissiontomography,PET)技术。其中用于研究P-gp功能的PET示踪剂是用同位素标记的P-gp底物,如[11C]维拉帕米,[11C]洛培酰胺,[11C]秋水仙碱,[11C]卡维地洛,[64Cu]配合物,[68Ga]配合物和[99mTc]配合物等,由于其低基线脑吸收,它们仅被用于监测帕金森和阿尔兹海默病患者P-gp的下降,不能用于观察患者P-gp的增加。少量的同位素标记的P-gp抑制剂,如[11C]elacridar、[11C]laniquidar和[11C]tarquidar也被研究用于P-gp表达成像,但其实际效果仍待进一步考察。
此外,PET技术检查费用相对较高,并且受检者安全保障、同期使用药物对PET的影响等对实际操作与结果可信性带来困难。
发明内容
针对现有技术中的不足与难题,本发明旨在提供一种含7-羟基香豆素结构基团的荧光化合物,开发可视性P-糖蛋白示踪剂,对疾病预测、病灶组织定位、细胞内P-糖蛋白定位和表达量检测、P-糖蛋白结构及功能的进一步验证等具有重要意义。本发明通过在2-(6,7-二甲氧基-3,4-二氢-1H-异喹啉-2-基)苯乙基结合基团上添加7-羟基香豆素荧光基团,设计了P-糖蛋白特异性荧光探针。采用计算机辅助药物设计、荧光量子产率测定、细胞毒性实验、细胞和组织成像评价等方法,证实了化合物出色的P-糖蛋白共定位能力和安全性。
本发明通过以下技术方案予以实现:
一种含7-羟基香豆素结构基团的荧光化合物,其为7-羟基香豆素荧光基团通过2-甲酰胺-4,5-二甲氧基苯甲酰胺连接2-(6,7-二甲氧基-3,4-二氢-1H-异喹啉-2-基)苯乙基形成的7-羟基香豆素荧光化合物,其结构式如下式(I)所示:
所述7-羟基香豆素荧光化合物用于P-糖蛋白特异性荧光探针中。
与现有技术相比,本发明有益效果包括:
1、本发明开发了一种含7-羟基香豆素结构基团的荧光化合物用于P-糖蛋白特异性荧光探针,这些荧光探针能够可靠地用于P-糖蛋白的定位示踪,在体内具有特异性高、灵敏度高、安全性好的优点;对肿瘤、癫痫等疾病的P-糖蛋白组织高表达区定位、细胞内P-糖蛋白定位及其研究的可视性等问题提供了新的视角和选择。
2、本发明采用P-糖蛋白特异性示踪剂荧光显像,与目前使用的PET示踪剂相比,对人体无放射性伤害,安全性高,仅需荧光成像仪辅助操作,且结果分析简便直观。
附图说明
图1为本发明荧光化合物的设计图。
图2为本发明荧光化合物S1-M02合成路线图。
图3为本发明荧光化合物S1-M02紫外吸收曲线图。
图4为本发明荧光化合物S1-M02的荧光光谱图。
图5为本发明荧光化合物S1-M02细胞存活曲线图。
图6为本发明荧光化合物S1-M02在LLC-PK1和LLC-PK1-MDR1细胞中的染色图。
图7为本发明荧光化合物S1-M02与P-糖蛋白共定位成像。
图8为本发明荧光化合物S1-M02肝脏切片染色。
具体实施方式
下面结合附图,对本发明作进一步地说明。
实施例1荧光化合物的设计与合成
如图1所示,2-(6,7-二甲氧基-3,4-二氢-1H-异喹啉-2-基)苯乙基为结合基团对P-gp进行定位,7-羟基香豆素作为荧光基团显像,通过2-甲酰胺-4,5-二甲氧基苯甲酰胺连接两者合成可进行P-gp特异性定位成像的7-羟基香豆素荧光化合物S1-M02。
为确定设计的小分子是否能与P-gp结合及其在实际应用中的可能性,我们采用加拿大化学计算集团公司开发的化学计算及分子模拟软件MOE进行了理化性质和分子对接计算。
结果显示,S1-M02具有良好的预测结合能力及适宜的理化性质,具体如表1所示,化合物S1-M02对接评分为-15.0457kcal/mol,表现出计算出较高的p-糖蛋白结合能力,且具有较好的理化性质,其log P(o/w)为4.86,分子量为679.73,适合动物和人体应用。
表1 S1-M02的理化性质及对接得分
Compounds | lopP(o/w) | Mol.weight | S(kcal/mol) |
S1-M02 | 4.86 | 679.73 | -15.0457 |
7-羟基香豆素荧光化合物S1-M02以对硝基苯乙基溴、盐酸--1,2,3,4-四氢-6,7-二甲氧基异喹啉、4,5-二甲氧基-2-硝基-苯甲酸、2,4-二羟基苯甲醛、、2,2-二甲基-1,3-二恶烷-4,6-二酮等为原料,经多步有机反应生成。具体合成路线如图2所示。
实施例2荧光化合物S1-M02的相关光学性质测定
化学合成S1-M02后,进行了荧光性质测定。
(一)紫外吸光度的测量
标准物质香豆素-153(Coumarin-153,C-153)溶于无水乙醇中,化合物S1-M02,溶于DMSO中,样品浓度均为30μM。检测吸光度用日本SHIMADZU公司生产的紫外-可见光分光光度计UV-2450检测,结果如图3所示。
(二)荧光光谱的测量
首先用上述测量紫外吸光度的最大吸收波长作为固定的发射波长,扫描出激发曲线,得到最大激发波长,再用最大激发波长作为固定激发波长,扫描出发射曲线,得到最大发射波长。化合物的激发曲线和发射曲线用日本HITACHI公司生产的荧光光谱仪F-7000 FLSpectrophotometer检测。调整EX Slit:10.0nm or 5.0nm,EM Slit:10.0nm or 5.0nm,PMTVoltage:400V,上样量200μl。结果如图4所示
(三)绝对量子产率的测量
化合物的绝对量子产率用日本HORIBA公司生产的稳态-瞬态荧光光谱仪FluoroMax-4-TCSPC检测,选择合适的激发波长范围和发射波长范围拟合量子产率曲线,得到绝对量子产率,由绝对量子产率可以计算出化合物相对于C-153的量子产率。其中对照品C-153用无水乙醇校正,荧光化合物用DMSO校正,结果如表2所示。
表2 S1-M02的量子产率
实施例3荧光化合物的细胞毒性检测
通过噻唑蓝(MTT)实验进行了化合物细胞毒性检测。
1、取对数生长期细胞,离心收集后,用完全培养基重悬,制成单细胞悬液,调整细胞浓度至1.5×10^4个/ml,96孔板每孔加入200ul细胞悬液。
2、实验组分别于铺板后24h分别加入化合物,浓度梯度为100nM,200nM,1μM,2μM,10μM。37℃,5%CO2条件下继续培养2-3天。
3、吸除培养液,加5mg/ml MTT 10ul,再继续培养4小时,吸除MTT,加入DMSO100ul,于492nm波长下检测吸光度。同时设置调零孔(培养基、MTT、二甲基亚砜)。
4、结果如图5所示,S1-M02在100nM,200nM,1μM,2μM,10μM浓度下细胞存活率皆在75%以上,安全性较高。
实施例4荧光化合物S1-M02的应用
在具体实施中,荧光化合物S1-M02作为P-糖蛋白示踪剂。其在细胞与肝脏组织中定位P-糖蛋白效果良好。
(一)S1-M02在LLC-PK1和LLC-PK1-MDR1细胞内与P-糖蛋白的结合情况,如图6所示。
1、LLC-PK1-MDR1为转染人源MDR1(编码P-糖蛋白的基因)后的细胞。实验前两天分别将细胞(LLC-PK1细胞和LLC-PK1-MDR1细胞)种植于24孔板的爬片上;
2、给药前用PBS将细胞洗1遍,每孔加入150μl的化合物(2.5μM、5μM or10μM);
3、放入孵箱内培养1h后,用PBS将细胞洗2遍,取出爬片,将爬片覆盖在滴有抗荧光淬灭封片剂的载玻片上,指甲油固定,晾干。调节曝光时间,在Nikon荧光倒置显微镜ECFP滤光片下20倍镜成像。
4、结果显示,S1-M02在5μM浓度下对P-糖蛋白显示良好的特异性结合能力,其中LLC-PK1细胞在图中表示为LLC细胞,LLC-PK1-MDR1细胞在图中表示为LLC-MDR1细胞。
(二)S1-M02在LLC-PK1-MDR1-Apple细胞内与P-糖蛋白的共定位情况,如图7所示。
1、LLC-PK1-MDR1-Apple细胞为转染MDR1-Apple(Apple为红色荧光标记)后的细胞。实验前两天将LLC--PK1-MDR1-Apple细胞种植于24孔板的爬片上;
2、给药前用PBS将细胞洗1遍,每孔加入150μl的化合物(5μM);
3、放入孵箱内培养1h后,用PBS将细胞洗2遍,取出爬片,将爬片覆盖在滴有抗荧光淬灭封片剂的载玻片上,指甲油固定,在荧光倒置显微镜Ti(日本尼康)20倍物镜下观察切片P-糖蛋白红色荧光表达及各部位与化合物结合情况,使用的滤光片为TRITC和ECFP。
4、结果显示,S1-M02与P-糖蛋白荧光位置一致,S1-M02具有定位P-糖蛋白的能力。
(三)检测化合物在离体肝脏切片中与P-糖蛋白的结合情况,如图8所示。
1、将肝脏冠状面进行冰冻切片,切片厚度15μm,切片粘附于载玻片上;
2、用组化笔在组织周围画圈,实验组和对照组分别滴加化合物(5μM);
3、孵育30min后用TBS洗两遍,在荧光倒置显微镜Ti(日本尼康)20倍物镜下观察切片各部位与化合物结合情况,使用的滤光片为ECFP;
4、结果显示,S1-M02在人源P-糖蛋白高表达组(MDR1++组)中染色荧光明显多于对照组,表示S1-M02在肝脏中可特异性靶向P-糖蛋白。
以上所述仅表达了本发明的优选实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形、改进及替代,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (2)
1.一种含7-羟基香豆素结构基团的荧光化合物,其特征在于:所述荧光化合物为7-羟基香豆素荧光基团通过2-甲酰胺-4,5-二甲氧基苯甲酰胺连接2-(6,7-二甲氧基-3,4-二氢-1H-异喹啉-2-基)苯乙基结合基团形成的7-羟基香豆素荧光化合物,所述7-羟基香豆素荧光化合物的结构式如下式(1)所示:
,
式(Ⅰ)。
2.根据权利要求1所述的一种含7-羟基香豆素结构基团的荧光化合物,其特征在于:所述荧光化合物用于P-糖蛋白特异性荧光探针中,所述荧光探针用于P-糖蛋白的定位示踪。
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