CN103483243B - 一种磺酸酯吡啶盐生物显影材料及其制备方法 - Google Patents

一种磺酸酯吡啶盐生物显影材料及其制备方法 Download PDF

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CN103483243B
CN103483243B CN201310422583.2A CN201310422583A CN103483243B CN 103483243 B CN103483243 B CN 103483243B CN 201310422583 A CN201310422583 A CN 201310422583A CN 103483243 B CN103483243 B CN 103483243B
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sulphonate
pyridinium salt
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CN103483243A (zh
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田玉鹏
李丹丹
孙贤顺
邵南起
吴杰颖
李胜利
周虹屏
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Guangdong Branch Road Heat Source Technology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
    • C07D213/38—Radicals substituted by singly-bound nitrogen atoms having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
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    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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    • A61K49/006—Biological staining of tissues in vivo, e.g. methylene blue or toluidine blue O administered in the buccal area to detect epithelial cancer cells, dyes used for delineating tissues during surgery
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo
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    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom

Abstract

本发明公开了一种磺酸酯吡啶盐生物显影材料及其制备方法，其中磺酸酯吡啶盐生物显影材料的结构式为：

Description

一种磺酸酯吡啶盐生物显影材料及其制备方法
一、技术领域
本发明涉及一种生物显影材料及其制备方法，具体地说是一种磺酸酯吡啶盐生物显影材料及其制备方法，具有人体宫颈癌细胞显影功能。
二、背景技术
继生物光学显微镜、普适性生物荧光显微镜、共聚焦荧光显微镜之后，双光子荧光显微镜已经成为新一代的细胞和组织的微观成像工具。与传统的单光子荧光共聚焦显微镜相比，双光子荧光显微镜在成像细胞和组织方面显示出了许多显著的优势：红外激发、生物样品穿透深、自发荧光低、生物样品的光损伤小、明显延长活细胞和组织的观测时间等。特别是双光子荧光显微镜在对活性生物样品的观测上体现出独特的优势，这使得生物科学家们得以在活的细胞和组织中观察和研究生命过程。荧光显微探测技术与荧光探针是相互依存互相促进的，新的荧光技术需要新的探针，新的探针将使探测技术更加完善。双光子荧光探针的研究是随着双光子荧光显微镜的出现而逐渐发展起来的，但其研究工作的相对滞后则限制了它们在双光子荧光显微镜上的广泛应用。因此，发展具有大吸收截面、高荧光量子产率的双光子荧光探针具有非常重要的科学意义和应用价值。
近年来，设计合成双光子荧光生物显影材料备受关注。众所周知，具有双光子活性的化合物，大多是具有大共轭体系的有机物，水溶性较差，不利于在生物体内的应用。因此，设计合成具有良好生物相容性的双光子荧光材料，是制备用于生物体双光子荧光探针的关键。
发明人对本申请的主题进行了如下的文献检索：
1、www.google.com网检索结果：（2013/9/2）
2、中国期刊网检索结果：
检索方式一：
篇名-磺酸酯吡啶盐生物显影材料无相关文献。
篇名-水溶性吡啶盐双光子荧光材料无相关文献。
检索方式二：
全文-磺酸酯吡啶盐生物显影材料无相关文献。
全文-水溶性吡啶盐双光子荧光材料无相关文献。
三、发明内容
本发明旨在提供一种磺酸酯吡啶盐生物显影材料及其制备方法，所要解决的技术问题是通过分子设计合成具有双光子活性的化合物，同时使其具有较好的水溶性和低毒性，以利于在生物体内的应用。
本发明以具有较好的双光子活性的吡啶盐为母体，引入磺酸酯提高化合物的水溶性，并在端基修饰柔性醚氧链基团，进一步增加水溶性和脂溶性，简洁高效地制备了具有π共轭体系的吡啶盐类衍生物（目标产物TM）。本发明目标产物TM在960nm左右具有较大的双光子吸收截面，能够完全溶于水安全地用于活体细胞显微成像，使之在生命科学研究领域具有明显的应用前景。
本发明磺酸酯吡啶盐生物显影材料的结构式为：
本发明磺酸酯吡啶盐生物显影材料的制备方法按以下步骤操作：
1）中间体M的合成
将4.10g(0.03mol)1,3-丙磺酸内酯和2.80g(0.03mol)γ-甲基吡啶溶于50mL丙酮中，加热回流反应4h，反应结束后冷却至室温，抽滤并用乙醚洗涤，真空干燥，得中间体M—甲基丙磺酸酯吡啶盐，为白色固体。
2）目标产物TM的合成
将3.25g（0.01mol）4-（双（2-（2-甲氧基乙氧基）乙基）氨基）苯甲醛和2.31g（0.01mol）中间体M溶解于50mL乙腈溶液中，然后加入催化剂六氢吡啶0.1mL，加热回流反应14h，反应结束后冷却至室温，旋蒸除去溶剂得到红色油状物，柱色谱分离（展开剂按体积比二氯甲烷:甲醇=2:1）后得到目标产物TM，为红色油状物。
本发明磺酸酯吡啶盐生物显影材料（简称目标产物TM）的合成路线如下：
与已有技术相比，本发明的有益效果体现在：
1、本发明目标产物TM与文献报道相比水溶性好，可以完全溶于水，并且在水中仍具有双光子活性（图1）。
2、本发明目标产物TM具有较低的毒性，20μMTM着色人体宫颈癌细胞(HepG2)在24h的孵化期间活性保持在95%左右（图2）。
3、本发明目标产物TM具有很好的细胞穿透性(图3)，且这种低能激发高能发射的光（960nm激发，600nm发射）具有对细胞损伤小的特点。
4、本发明目标产物TM稳定性好，所用的吡啶盐和磺酸酯等原料易得，成本低，合成简洁，产率高，是一种有实用价值的双光子荧光材料。
四、附图说明
图1是本发明目标产物TM水溶液的双光子荧光光谱，在960nm激光作为激发波长，得到浓度为10-3mol/LTM水溶液的双光子荧光光谱。
图2是MTT测试细胞毒性结果。通过MTT测试，使用人体宫颈癌细胞(HepG2)测定了TM的细胞毒性。结果表明活细胞被TM着色，且24h的孵化期、TM浓度范围为10μM-80μM，HepG2活性保持在90%以上，说明TM具有较低的细胞毒性。
图3是本发明目标产物TM单双光子荧光显影结果。其中(a)单光子作用图（470nm激发），(b)双光子作用图（960nm激发），(c)干扰对比图（DIC），(d)单双光子作用叠加图，(e)单双光子高共区域化散点图，（f）单双光子低共区域化散点图。由显影结果图3（f）可见，本发明目标产物TM可以显影人体宫颈癌细胞的溶酶体；由图3（e）和图3（f）单双光子高、低共区域化散点图的比较结果可知，双光子荧光显影具有更强的穿透性。
五、具体实施方式
1、中间体M的合成
称取4.10g(0.03mol)1,3-丙磺酸内酯和2.80g(0.03mol)γ-甲基吡啶，溶于50mL丙酮中，加热回流反应4h，反应结束后冷却至室温，抽滤并用乙醚洗涤，真空干燥，得中间体M—对甲基丙磺酸酯吡啶盐6.30g，为白色固体，产率91%。
lHNMR(DMSO-d6,400MHz),d(ppm):7.98(d,2H),7.05(d,2H),3.68(t,2H),2.83(t,2Hz),1.68(s,3H),1.25(m,2H).
2、目标产物TM的合成
将3.25g（0.01mol）4-（双（2-（2-甲氧基乙氧基）乙基）氨基）苯甲醛和2.31g（0.01mol）中间体M溶解于50mL乙腈溶液中，加入六氢吡啶0.1mL作为催化剂，加热至回流反应14h，反应结束后冷却至室温，旋蒸除去溶剂，柱色谱分离（展开剂按体积比为二氯甲烷:甲醇=2:1）后得到目标产物TM4.1g，为红色油状物，产率75%。
FT-IR(KBr,cm-1):2923(w),1641(w),1585(s),1524(m),1470(w),1399(w),1174(s),1120(m),1056(m),943(w),831(w),618(w),530(w).1H-NMR(400MHz,CD3COCD3):9.58(d,1H),8.94(d,2H),8.10(d,2H),7.63(d,2H),7.12(d,1H),6.85-6.83(q,2H),4.87-4.85(q,2H),3.59-3.58(t,4H),3.49-3.48(t,4H),3.29(s,6H),3.15-3.12(t,4H),1.89-1.86(q,4H),1.29(s,4H).13C-NMR(100MHz,DMSO-d6):153.8,149.8,143.6,141.7,130.3,122.4,117.1,115.5,111.5,71.3,69.7,67.8,50.1,40.1,27.4.MALDI-TOF:m/z,cal:522.24,found:523.37(M++H).Anal.Calc.forC26H38N2O7S:C,59.75;H,7.33;N,5.36.Found:C,59.78;H,7.35;N,5.33.

Claims (1)

1.一种磺酸酯吡啶盐生物显影材料，其特征在于其结构式为：
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CN106496102B (zh) * 2016-10-31 2019-02-05 安徽大学 一种线粒体双光子荧光粘度探针及其制备方法
CN107987014B (zh) * 2017-12-13 2021-02-09 安徽大学 一种吡啶磺酸内盐化合物及其制备方法和用途
CN112961103B (zh) * 2021-02-09 2021-07-30 广东海洋大学 一种含氮芥的三联吡啶配体及其制备方法和应用

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