CN113546176B - 一种特异性用于抑制眼内血管新生药物合成方法 - Google Patents
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Abstract
本发明提供了一种特异性用于抑制眼内血管新生药物合成方法，包括以下步骤：(1)将1eq的阿帕替尼和1eq的大黄酸加入到反应瓶中，再加入100ml二氯甲烷溶解；(2)待上述反应体系降至常温，依次加入1.1eq的1‑(3‑二甲氨基丙基)‑3‑乙基碳二亚胺盐酸盐和1.1eq 1‑羟基苯并三唑，加料完毕后，反应体系在常温下搅拌10小时；(3)向反应体系中加入水猝灭反应，用50ml乙酸乙酯萃取，通过100ml的有机相用饱和NaCl洗涤，再经无水NaSO4干燥、过滤，脱除溶剂后得到产物。本发明的有益效果为：可以特异性靶向眼睛，抑制眼睛新生血管形成，达到预防或治疗眼睛血管生成相关疾病的效果。
Description
技术领域
本发明涉及生物医学技术领域，尤其涉及一种特异性用于抑制眼内血管新生药物合成方法。
背景技术
眼科类疾病的发病机理有多种，各种原因引起的新生血管的过度生成是其主要病理因素之一，如视网膜新生血管性眼病、角膜新生血管性眼病及虹膜新生血管性眼病等，抑制新生血管的过度生成是治疗这类疾病的关键。
传统治疗上述疾病主要是采取中医疗法和激光等物理疗法，但是疗效并不理想。阻断新生血管生长可能成为治疗此类疾病的新方法，因此血管生成抑制剂成为目前治疗新生血管异常疾病中极具重视的一类药物。尽管目前市面上这些血管抑制剂呈现出极大的前景，但其缺陷也十分明显：传统用于抑制血管生成的药物，如舒尼替尼、阿帕替尼等作用靶点不特异，对组织特异性血管的专一性和选择性不够高，效果有限，因此用药量较大，容易引起较大的副作用。因此，一个好的抗血管生成药物应该对组织特异性血管具有选择性，做到只需要低剂量的药物就能达到高效率的血管生成抑制效果。当前，亟待于开发新的用于特异性抑制眼睛血管生成的药物。
血管新生是一个受多种促血管及抑血管因子，包括VEGF/VEGFR、PDGF/PDGFR、FGF/FGFR等严密调控的过程，其中，VEGF/VEGFR2是其中最为重要的血管生成因子。阿帕替尼是一种选择性抑制VEGF2的小分子化合物，在肿瘤血管发生和细胞增殖中起作用。提供组织特异性的阿帕替尼或其类似物的给药方式有利于其更好的发挥药效，减少副作用。然而，目前市面上流通的替尼类药物，其对受药组织的特异性选择方面还有很大的缺陷。
荧光是包括蛋白质在内的各种生物有机分子的固有特性之一。目前已发现的携带有荧光团的小分子药物如阿霉素(doxorubicin)、柔红霉素(daunorubicin)、甲萘醌(menadione)、玫瑰树碱(ellipticine)和哈马洛(harmalol)等，使得这些药物在细胞培养中可以在亚细胞水平上可视化，从而指示功能部位。此外，一些强荧光化合物在体内具有阻止特异性分布，为合成组织特异性的改造药物带来可能。
发明内容
本发明的目的在于提供一种特异性用于抑制眼内血管新生药物合成方法，克服了现有阿帕替尼药物作用靶点不特异、用药量大等问题，本发明中使用的大黄酸(Rhein)是可以在眼睛中特异性分布的红色荧光化合物。
为了实现上述发明目的，本发明采用以下技术方案：一种特异性用于抑制眼内血管新生药物合成方法，包括以下步骤：
(1)将1eq的阿帕替尼和1eq的大黄酸加入到反应瓶中，加入二氯甲烷100ml溶解；
(2)待上述反应体系降至常温，依次加入1.1eq的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)和1.1eq1-羟基苯并三唑(HOBT),加料完毕后，反应体系在常温下搅拌10小时；
(3)向反应体系中加入水猝灭反应，然后用乙酸乙酯50ml萃取，有机相用饱和NaCl100ml洗涤，再经无水NaSO4干燥、过滤，脱除溶剂后得到产物。
作为本发明提供的一种特异性用于抑制眼内血管新生药物合成方法进一步优化方案，所述步骤(2)是通过加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和1-羟基苯并三唑作为缩合剂完成的。
作为本发明提供的一种特异性用于抑制眼内血管新生药物合成方法进一步优化方案，所述步骤(1)中阿帕替尼的用量为6.217g，9.717mmol。
作为本发明提供的一种特异性用于抑制眼内血管新生药物合成方法进一步优化方案，所述步骤(1)中大黄酸的用量为2.784g,9.717mmol。
作为本发明提供的一种特异性用于抑制眼内血管新生药物合成方法进一步优化方案，所述步骤(2)中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的用量为2.06g,10.69mmol。
作为本发明提供的一种特异性用于抑制眼内血管新生药物合成方法进一步优化方案，所述步骤(2)中1-羟基苯并三唑的用量为1.46g,10.69mmol。
作为本发明提供的一种特异性用于抑制眼内血管新生药物合成方法进一步优化方案，所述步骤(3)中用乙酸乙酯萃取的次数为五次。
与现有技术相比，本发明的有益效果为：
(1)、本发明的改造药物是由阿帕替尼和大黄酸进行缩合反应生成，其中，阿帕替尼可以通过靶向抑制血管生成因子VEGF/VEGFR2，有效抑制血管的新生，从而达到抗血管生成相关疾病的功能；大黄酸(Rhein)是一种具有红色荧光特性的小分子化合物，在眼睛中有特异性分布，可以用于辅助组织特异性靶向药物的合成。
(2)、本发明的改造药物可以特异性靶向眼睛，抑制眼睛新生血管形成，从而达到预防或治疗眼睛血管生成相关疾病的效果。
(3)、本发明药物可以特异性靶向眼睛，通过实验证实将本发明药物稀释成浓度为15μM的工作液，对三天的斑马鱼幼鱼进行浸泡处理，在第六天通过共聚焦进行观察，该药物可以特异性亲和斑马鱼眼睛，在抑制眼睛血管形成方面，通过活体成像和共聚焦观察可明显看到15μM浓度下对视网膜血管生成有明显的抑制作用，抑制率为75％以上。
附图说明
图1为本发明要去的合成过程示意图。
图2为本发明药物在斑马鱼眼内的组织特异性亲和情况的效果图。
图3为本发明实药物对斑马鱼视网膜血管生成影响表型图。
图4为本发明药物对斑马鱼视网膜血管过度新生的挽救表型图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。当然，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
实施例1
参见图1至图4，本发明提供其技术方案为，本发明药物对眼睛的特异性靶向作用，采用斑马鱼活体成像技术和共聚焦显微观察检测本发明药物在斑马鱼幼鱼不同组织的亲和情况。
收集野生型斑马鱼胚胎，至少从三个繁殖组中收集胚胎，将其混合加入E3培养液放入28.5℃恒温培养箱中培养待用，期间按时去除死胚胎和更换培养液。
斑马鱼的胚胎体积小、发育快、产胚胎量多、胚胎透明，通过结合96孔板进行处理能够实现高通量实验，可平行对不同处理时间点、处理时间段、药物浓度等参数进行优化。具体方法：在96孔板的每个小孔中放5条特定发育时期的斑马鱼胚胎(2dpf,3dpf和4dpf)，完全弃去孔中E3培养液后加200μL不同浓度的本发明药物工作液(5μM,10μM,15μM,20μM和30μM)，放置于28℃培养箱中培养。
在所处理胚胎发育前七天用体式荧光显微镜与激光共聚焦显微镜来观察幼鱼体内药物组织亲和情况。具体操作如下：按下表(2)配置斑马鱼胚胎固定液，用胚胎固定液固定胚胎后在荧光显微镜下观察所发明药物在斑马鱼幼鱼体内的组织特异性亲和情况。
实验结果如图2所示，本发明药物可以特异性的靶向斑马鱼胚胎眼睛部位。
实施例2
本发明药物对斑马鱼视网膜血管生成的影响
采用斑马鱼活体成像技术和共聚焦显微观察检测本发明药物对斑马鱼视网膜血管生成的影响。
收集转基因斑马鱼Tg(fli1ep:EGFP-CAAX)的胚胎，至少从三个繁殖组中收集胚胎，将其混合加入E3培养液放入28.5℃恒温培养箱中培养待用，期间按时去除死胚胎和更换培养液。
根据上述实施例中不同处理时间、处理时段以及处理浓度实验，我们最终选择以下处理方案：培养至第三天，在96孔板的每个小孔中放入5条斑马鱼幼鱼，将孔中残留E3培养液吸净后加入200μL 15μM的本发明药物工作液，放置于28℃培养箱中浸泡处理三天。
在斑马鱼胚胎发育第六天通过体式荧光显微镜与激光共聚焦显微镜观察幼鱼视网膜血管发育表型。
对斑马鱼幼鱼视网膜血管发育表型进行数据统计。在GraphPad Prism8软件上进行差异分析，P<0.05代表显著性差异。
实验结果见图3所示，与正常对照组相比，在本发明药物处理下，新生血管的数量显著减少。
实施例3
本发明药物对斑马鱼视网膜血管过度新生的治疗效果
采用斑马鱼活体成像技术和共聚焦显微观察检测本发明药物对斑马鱼视网膜血管过度新生的影响。
收集转基因斑马鱼Tg(fli1ep:EGFP-CAAX)的胚胎，至少从三个繁殖组中收集胚胎，将其混合加入E3培养液放入28.5℃恒温培养箱中培养待用，期间按时去除死胚胎和更换培养液。
收集2dpf的斑马鱼胚胎给予含有300mM葡萄糖的胚胎培养液(E3)处理，诱导斑马鱼胚胎血管过度新生模型。
培养至3dpf，在96孔板的每个小孔中放入5条上述高糖处理过的斑马鱼幼鱼，将孔中残留E3培养液吸净后加入200μL 15μM的本发明药物工作液，放置于28℃培养箱中继续浸泡处理三天。
于斑马鱼胚胎发育第六天通过体式荧光显微镜与激光共聚焦显微镜观察幼鱼视网膜血管发育表型。
对斑马鱼幼鱼视网膜血管发育表型进行数据统计。在GraphPad Prism8软件上进行差异分析，P<0.05代表显著性差异。
实验结果见图4所示，与病理模型相比，本发明药物处理下，斑马鱼胚胎血管过度新生表型明显被挽救。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，任何在此基础上，对其中配方和工艺的局部变动，都应在本发明的保护范围之内。
Claims (2)
1.一种特异性用于抑制眼内血管新生药物合成方法，其特征在于，包括以下步骤：
(1)将1eq的阿帕替尼和1eq的大黄酸加入到反应瓶中，再加入100ml二氯甲烷溶解；
所述步骤(1)中阿帕替尼的用量为6.217g，9.717mmol；
所述步骤(1)中大黄酸的用量为2.784g,9.717mmol；
(2)待上述反应体系降至常温，依次加入1.1eq的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和1.1eq 1-羟基苯并三唑，加料完毕后，反应体系在常温下搅拌10小时；
所述步骤(2)是通过加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和1-羟基苯并三唑作为缩合剂完成的；
所述步骤(2)中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的用量为2.06g,10.69mmol；
所述步骤(2)中1-羟基苯并三唑的用量为1.46g,10.69mmol；
(3)向反应体系中加入水猝灭反应，用50ml乙酸乙酯萃取，通过100ml的有机相用饱和NaCl洗涤，再经无水NaSO4干燥、过滤，脱除溶剂后得到产物。
2.根据权利要求1所述的特异性用于抑制眼内血管新生药物合成方法，其特征在于，所述步骤(3)中用乙酸乙酯萃取的次数为五次。
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