CN111606839A - 诊疗联用分子先导化合物合成方法及在线粒体成像中应用 - Google Patents
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Abstract
诊疗联用分子先导化合物合成方法及在线粒体成像中应用，该先导化合物由吲哚花菁母核和苯丁酸氮芥构成，在同一分子结构中集线粒体靶向、荧光成像诊断、光动力治疗与化疗于一体。本发明还公开了在细胞线粒体成像的应用。本发明中的先导化合物结构简单、分子量小，具有确定的化学结构，易于制备、纯化和进一步修饰。通过细胞成像实验证明，借助近红外荧光成像可发现先导化合物结构中的正电性和线粒体负电性作用可以显著靶向于细胞的线粒体。因此，本发明涉及的线粒体靶向诊疗联用单分子多功能先导化合物在细胞器成像、肿瘤治疗方面具有良好的应用前景。
Description
技术领域
本发明涉及一种诊疗联用分子先导化合物合成方法及在线粒体成像中应用，属于生物医药领域。
背景技术
在细胞和分子水平对细胞表面受体、信号通路及细胞代谢的改变以及基因的异常突变等相关疾病进行实时、原味的筛查和诊断，一直是分子影像技术努力的方向，也是实现精准医疗有限收到之一，并在科学研究和临床应用中得到了快速的发展。当前，主要的分子影像诊断技术有光学成像、核素成像、磁共振成像及计算机断层成像等。其中，光学分子成像具有低能量、无辐射及对信号检测灵敏度高，且具有实时监测标记的活体生物体内细胞活动和基因行为，正成为分子成像中研究热点[Chen C,Tian R,Zeng Y,et al.Bioconjugate Chemistry,2020,31:276-292]。光学成像中的荧光成像技术能够在三维尺度上，动态标记特定组织及病理状态下目标分子在细胞内的时空分布，实现在活细胞、组织中“靶向性”示踪，能够将活体中的这些目标分子，进行细胞及组织/器官层面上的可视化检测，追踪其在活体生物内的生理过程，对这些生物分子及其与机体构效关系进行实时和原味呈现。同时，荧光成像技术还具有非侵入性、安全性及高时空分辨率的特点，使其在肿瘤诊断、药物分布和代谢以及内源性生物分子检测等诸多领域被广泛用，并成为当前生物医学领域的一个重要工具[6-12]。
在光学影像技术中，所用荧光染料及其本身固有的吸收和荧光波长成为该诊断技术的关键，主要是由于波长与组织穿透性之间的关系会影像其在生物体内的诊断情况。相较于其它波长的光，近红外光具有组织穿透性强，受内源性分子自发荧光干扰显著减小的特点，因此在近红外光谱区域的荧光光敏剂具有良好的组织渗透性和低背景的综合优点，容易透过生物组织用于活体荧光成像[Kobayashi H, Ogawa M,Alford R,et al.ChemicalReviews,2010,110: 2620-2640]。其中，花菁类染料在生物成像研究中发现其具有背景干扰小、灵敏度高、组织穿透力强以及适用于组织荧光等优点，已成为近红外荧光染料的热点母核结构。同时，花菁类染料的生物相容性和对生物样品的低毒性，使其适用于生物体进行活体实时和原味近红外荧光成像诊断。此外，近红外吲哚花菁染料不仅拥有优良的近红外荧光成像诊断性能，还显示出光依赖性的细胞毒性，可作为光敏剂具有光动力治疗的性能[Yang Q,Jin H,Gao Y,et al.ACS Applied Materials&Interfaces,2019,11:15417-15425]。
发明内容
本发明所要解决的是针对现有临床对未来个性化医疗需求靶向药物、诊疗联用药物，以克服目前恶性肿瘤诊治领域出现的成像与治疗模式单一、诊治效果差、单一疗法对肿瘤的不敏感性等缺点，实现针对恶性肿瘤早期的近红外荧光成像诊断以及原位光动力治疗与化疗协同治疗和治疗过程疗效评估的技术问题。
为了解决上述技术问题，本发明采用如下技术方案：
一种诊疗联用分子先导化合物合成方法，包括以下步骤：(i)在避光条件下，取IR780(0.334g，0.5mmol)溶于10mL除水的DMF中，在氮气保护下搅拌加入溶解有哌嗪(0.173g，2mmol)的除水DMF 溶液，加热至80℃反应4h；反应溶液逐渐由绿色变为蓝色，用TLC 对反应完成度进行监测，待反应完成后，将反应体系冷却至室温，减压浓缩得到粗产品；采用硅胶柱层析进行纯化，用甲醇：二氯甲烷(v:v)＝1:30，得到红色固体中间体(产率75％)；见以下反应路线：
(ii)在避光条件下，取30mL除水二氯甲烷，搅拌加入中间体(5mg， 6.6μmol)和苯丁酸氮芥(2mg，6.6μmol)，接着加入BOP试剂 (3mg，6.6μmol)，然后加入三乙胺数滴，在氮气保护下室温搅拌反应1-2h；用TLC对反应完成度进行监测，待反应完成后，将反应体系冷却至室温，减压浓缩得到粗产品；采用硅胶柱层析进行纯化，用甲醇：二氯甲烷(v:v)＝0:100-1:20，得到红色固体目标产物见以下反应路线：
该先导化合物为有机小分子，在同一分子结构中具有线粒体靶向、近红外荧光成像诊断、近红外光动力治疗协同化学治疗过个功能，其分子结构由吲哚花菁母核和苯丁酸氮芥构成。其分子结构中的吲哚花菁母核正电性为靶向于线粒体基团；吲哚花菁母核为光动力基团；苯丁酸氮芥为化学治疗基团，其功能结构式如式I所示，
所述先导化合物用于在线粒体成像中。
采用上述技术方案的有益效果是：
本发明所述的单分子多功能诊疗联用先导化合物能够通过荧光成像,对肿瘤进行近红外定位且可以通过活体荧光成像监测本发明所述先到化合物的靶向肿瘤富集,并于肿瘤病灶引导精准的光动力产生大量ROS,达到光动力治疗和化学治疗协同治疗, 有效改善了疗效。
附图说明
图1为本发明所述单分子多功能诊疗联用先导化合物的1H NMR。
图2为本发明所述单分子多功能诊疗联用先导化合物的13C NMR。
图3为本发明所述单分子多功能诊疗联用先导化合物的高分辨质谱图。
图4为本发明所述单分子多功能诊疗联用先导化合物的紫外-可见吸收光谱图。
图5为本发明所述单分子多功能诊疗联用先导化合物的荧光激发谱和发射谱图。
图6为本发明所述单分子多功能诊疗联用先导化合物的线粒体靶向成像。HepG2细胞与Mito-Tracker green和先导化合物共染色。(a) 先导化合物通道；(b)Mito-Trackergreen通道；(c)图像(a) 和图像(b)的叠加图像；(d)对应的差分干涉对比图像；(e) Mito-Tracker green和先导化合物的强度相关图(皮尔森系数0.95 和重叠系数0.94)；(f)线性ROI 1中的Mito-Tracker green和先导化合物的强度曲线。
具体实施方式
下面将结合具体实施例详细描述本发明的内容。应当注意的是，下面所述是对本发明的解释而不是限定。
1、本发明所述诊疗联用分子先导化合物合成方法；
包括以下步骤：(i)在避光条件下，取IR780(0.334g，0.5mmol) 溶于10mL除水的DMF中，在氮气保护下搅拌加入溶解有哌嗪(0.173 g，2mmol)的除水DMF溶液，加热至80℃反应4h；反应溶液逐渐由绿色变为蓝色，用TLC对反应完成度进行监测，待反应完成后，将反应体系冷却至室温，减压浓缩得到粗产品；采用硅胶柱层析进行纯化，用甲醇：二氯甲烷(v:v)＝1:30，得到红色固体中间体(产率75％)；见以下反应路线：
(ii)在避光条件下，取30mL除水二氯甲烷，搅拌加入中间体(5mg， 6.6μmol)和苯丁酸氮芥(2mg，6.6μmol)，接着加入BOP试剂 (3mg，6.6μmol)，然后加入三乙胺数滴，在氮气保护下室温搅拌反应1-2h；用TLC对反应完成度进行监测，待反应完成后，将反应体系冷却至室温，减压浓缩得到粗产品；采用硅胶柱层析进行纯化，用甲醇：二氯甲烷(v:v)＝0:100-1:20，得到红色固体目标产物见以下反应路线：
2、本发明所述诊疗联用分子先导化合物在经改造后最大吸收波长为775nm，最大激发波长为798nm，最大荧光发射波谱长为806nm，为近红外吸收区。
本发明所述的诊疗联用分子先导化合物是由吲哚花菁和苯丁酸氮芥，实现同一分子具有线粒体靶向、近红外荧光成像诊断、近红外光动力治疗和化学治疗协同的多个功能为一体。
将本发明所述的诊疗联用分子先导化合物用乙醇溶解后得配成5.0 μmol·L-1，在紫外-可见分光光度计测其吸光度值，在Vary Eclipse 荧光分光光度计上测量其荧光激发光谱和荧光发射光谱，结果见图1-1和1-2，其最大吸收波长为775nm，最大激发波长为798nm，最大荧光发射波谱长为806nm，为符合650～900nm近红外荧光染料的光学特征。
3、本发明所述诊疗联用分子先导化合物的线粒体靶向性。
本发明将HepG2细胞以5×104每孔接种于培养皿中，孵育24h，然后换成含有1μM单分子多功能诊疗联用先导化合物培养液，孵育 8h后换成含150nM线粒体靶向剂Mito-tracker green培养液，再次孵育半小时后用PBS清洗培养皿3次，加入1mL的PBS置于倒置荧光显微镜进行荧光成像拍摄，绿色荧光通道下拍摄先导化合物荧光，蓝色通道激发Mito-tracker green荧光，用Image J软件处理荧光图片。
本发明虽然已经给出了一些实施例，但是本领域的技术人员应当理解，在不脱离本发明精神的情况下，可以对本文的实施例进行改变。上述实施例只是示例性的，不应以本文的实施例作为本发明权利范围的限定。
Claims (3)
1.一种诊疗联用分子先导化合物合成方法，包括以下步骤：(i)在避光条件下，取IR780(0.334g，0.5mmol)溶于10mL除水的DMF中，在氮气保护下搅拌加入溶解有哌嗪(0.173g，2mmol)的除水DMF溶液，加热至80℃反应4h；反应溶液逐渐由绿色变为蓝色，用TLC对反应完成度进行监测，待反应完成后，将反应体系冷却至室温，减压浓缩得到粗产品；采用硅胶柱层析进行纯化，用甲醇：二氯甲烷(v:v)＝1:30，得到红色固体中间体(产率75％)；见以下反应路线：
(ii)在避光条件下，取30mL除水二氯甲烷，搅拌加入中间体(5mg，6.6μmol)和苯丁酸氮芥(2mg，6.6μmol)，接着加入BOP试剂(3mg，6.6μmol)，然后加入三乙胺数滴，在氮气保护下室温搅拌反应1-2h；用TLC对反应完成度进行监测，待反应完成后，将反应体系冷却至室温，减压浓缩得到粗产品；采用硅胶柱层析进行纯化，用甲醇：二氯甲烷(v:v)＝0:100-1:20，得到红色固体目标产物见以下反应路线：
2.根据权利要求1所述诊疗联用分子先导化合物合成方法，其特征在于：所述先导化合物为有机小分子，在同一分子结构中具有线粒体靶向、近红外荧光成像诊断、近红外光动力治疗协同化学治疗过个功能，其分子结构由吲哚花菁母核和苯丁酸氮芥构成。其分子结构中的吲哚花菁母核正电性为靶向于线粒体基团；吲哚花菁母核为光动力基团；苯丁酸氮芥为化学治疗基团，其功能结构式如式I所示，
3.根据权利要求1所述诊疗联用分子先导化合物合成方法，其特征在于：所述先导化合物用于在线粒体成像中。
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