CN112479998A - 一种二氢喹啉类荧光探针及其制备方法与应用 - Google Patents

一种二氢喹啉类荧光探针及其制备方法与应用 Download PDF

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CN112479998A
CN112479998A CN202011278753.0A CN202011278753A CN112479998A CN 112479998 A CN112479998 A CN 112479998A CN 202011278753 A CN202011278753 A CN 202011278753A CN 112479998 A CN112479998 A CN 112479998A
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dihydroquinoline
fluorescent probe
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邓涛
刘芳
吴亚兰
黄文怡
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Guangzhou University Of Chinese Medicine Guangzhou Institute Of Chinese Medicine
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Abstract

本发明属于生物探针技术领域，具体公开了一种二氢喹啉类荧光探针及其制备方法与应用。一种二氢喹啉类荧光探针，其结构式如下式I所示：所述R为具有取代基的芳基、吡啶基或杂环。本发明提供的一类二氢喹啉类产物可以凭借其荧光开启特性作为新型的化学探针，用以高灵敏度且高选择性的检测·OH。此外本发明提供的一种二氢喹啉类化合物的绿色制备方法，在NaOH作为碱的水相反应条件下，许多芳香族乙酰基化合物都能和3‑甲酰‑1‑甲基喹啉盐发生反应，特点是在高度亲电性的C‑4位点上形成二氢喹啉。同时，本发明更换21种化合物底物应用上述方法成功合成了一系列二氢喹啉类产物，产率为25％‑93％。

Description

一种二氢喹啉类荧光探针及其制备方法与应用
技术领域
本发明属于生物探针技术领域，特别涉及一种二氢喹啉类荧光探针及其制备方法与应用。
背景技术
荧光探针作为化学和生物学前沿研究的强大化学工具之一，可以靶向检测或成像细胞内的重要物质。氧化还原平衡和ROS信号传导是细胞内重要的生理活动，因此，近几十年来响应细胞内活性氧(ROS)的荧光探针的研发和应用逐渐成为相关研究领域的热点和难点。ROS荧光分析需要设计合理的荧光探针，探针主要基于氧化反应实现对目标物质的检测。在所有的ROS中，羟基自由基(·OH)是生物有机体中反应活性最强的自由基，具有最强的氧化潜力，由于·OH在生物环境中的寿命极短、反应可控性差，开发这类探针的难度较大。有机骨架与反应活性的关系可为高灵敏度、高选择性的荧光探针的设计提供依据。芳香加成是·OH参与的最典型的化学反应之一，常被用于设计·OH探针，例如弱荧光的香豆素及其衍生物通过芳香加成反应生成强荧光产物7-羟基香豆素，这种现象可以用于检测·OH。基于C-H位点的夺氢反应是·OH参与的另一个重要的氧化反应，这与·OH的强氧化特性密切相关。在此基础上，目前已有几种化学荧光探针被用于·OH的检测和成像。总而言之，优异的·OH探针需要具备较优的荧光骨架以及对·OH高度敏感且有选择性的反应位点。
在所有的有机骨架中，荧光喹啉类因其功能多样性而备受关注。以往的研究表明：喹啉及其衍生物可用于构建供体-π-受体(D-π-A)类型的荧光分子，且在喹啉的芳香环上引入不同电性的取代基可调节其光物理特性。众所周知，喹哪啶红便是一种重要的喹啉盐染料，可作为DNA显色指示剂和荧光探针。尽管喹啉类化合物用于构建荧光分子已经得到广泛关注，但其还原形式二氢喹啉类化合物的合成研究较少，其在生物探针领域的应用更为稀少。
发明内容
为了克服上述现有技术的缺点与不足，本发明的首要目的在于提供一种二氢喹啉类荧光探针。
本发明另一目的在于提供上述二氢喹啉类荧光探针的制备方法。
本发明再一目的在于提供上述二氢喹啉类荧光探针在检测·OH中的应用。
本发明的目的通过下述方案实现：
一种二氢喹啉类荧光探针，其结构式如下式I所示：
Figure BDA0002780030230000021
所述R为具有取代基的芳基、吡啶基或杂环。
优选地，所述二氢喹啉类荧光探针，其结构式为以下3a～3u所示：
Figure BDA0002780030230000022
Figure BDA0002780030230000031
一种制备上述二氢喹啉类荧光探针的制备方法，包括如下步骤：
在水/乙醇的溶剂体系下，以无机碱为催化剂，将乙酮类化合物作为模板底物与喹啉盐类化合物发生亲核加成反应，得到二氢喹啉类荧光探针。
所述水/乙醇的体积比为1：0.8～1.5，优选为1:1。
所述无机碱为Na2CO3、KOH、NaOH、K2CO3、KF、K3PO4和NaHCO3中的至少一种；所述无机碱相对于乙酮类化合物的当量比为1～8；优选为2。
所述喹啉盐类化合物通过以下方法制备得到：将乙腈与喹啉-3-甲醛混合后再加入碘甲烷，然后在保护气氛下进行加热反应，反应结束后得到产物。
优选地，所述乙腈：喹啉-3-甲醛：碘甲烷的摩尔体积比为3～6mL：6～10mmol：35～45mmol；更优选为5mL：8mmol：40mmol。所述加热反应为在40～60℃反应12～30h。更优选为在50℃反应24h。
所述乙酮类化合物与喹啉盐类化合物的摩尔比为1：0.8～1.5；优选为1：1.2。
所述亲核加成反应的时间为5～20h；反应温度为25-60℃。
所述二氢喹啉类荧光探针在检测·OH中的应用。
所述二氢喹啉类荧光探针在制备检测·OH的比率式荧光探针和近红外荧光探针中的应用。
本发明相对于现有技术，具有如下的优点及有益效果：
本发明提供的一类二氢喹啉类化合物可以凭借其荧光开启特性作为新型的化学探针，用以高灵敏度且高选择性的检测·OH。此外本发明提供的一种二氢喹啉类化合物的绿色制备方法，在NaOH作为碱的水相反应条件下，许多芳香族乙酰基化合物都能和3-甲酰-1-甲基喹啉盐发生反应，特点是在高度亲电性的C-4位点上形成二氢喹啉。同时，本发明更换21种化合物底物应用上述方法成功合成了一系列二氢喹啉类产物，产率为25％-93％。
附图说明
图1为实施例3所得3c和·OH之间氧化反应的可能机制示意图。
图2为实施例1至7中手持紫外光下合成探针与·OH反应时的荧光图片(A)，化合物3a-3g与·OH反应后的归一化荧光光谱，最大荧光强度归一化为1(B)，荧光产物3a-pr在激发态下HOMO(左)和LUMO(右)的Kohn-Sham等值面(0.05au)(C)。
图3中A)为DIC图像；B)为在加入H2O2前10μM 3c和100μM EDTA-Fe2+孵育后Hela细胞的绿色荧光通道成像；C)为DIC图像；D)为Hela细胞经过10μM 3c和100μM EDTA-Fe2+/1.0mM H2O2共同孵育20min后的绿色荧光通道成像，标尺为20μm。
图4为化合物3c对活细胞内·OH的荧光成像；其中A)为细胞内DNA的DAPI染色；B)为3c孵育后绿色通道下的细胞成像；C)为商品化的溶酶体红色荧光探针孵育后红色通道下的细胞成像；D)为A)、B)和C)的合并图；E)为DIC和所有通道的合并图；F)为Image J对共定位实验的定量分析，标尺为20μm。
具体实施方式
下面结合实施例和附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例中所用试剂如无特殊说明均可从市场常规购得。
化学品从正规商业渠道购买，其他试剂属于AR级，不需要经进一步纯化使用，除非另外说明。LysoTracker Red DND-99和黄嘌呤氧化酶分别从Feiyubio(中国)和Solarbio(中国)购买。在整个实验过程中，使用Milli-Q Plus纯水系统获取去离子水。必要时采用高效液相色谱技术(Agilent HPLC 1260,USA)，使用反相C18柱(250x 4.6mm)进行分离。读板则采用Varioskan LUX酶标仪，联合SkanIt 4.1处理软件(Thermo Fisher)。紫外-可见吸收光谱由TU-1900分光光度计(Persee,Beijing)采集。ESI-MS由Agilent 6420液质联用仪获取，高分辨质谱(HRMS)由Maxis Impact HD Mass Spectrometer(Bruker)收集。AVANCE IIIHD 400MHz digital NMR spectrometer(Switzerland)记录1H和13C核磁谱图，数据报告包含内容如下：化学位移，多重性(s＝单重峰，d＝双重峰，m＝多重峰)，耦合常数(J值)以及位移积分。细胞成像由Leica共聚焦显微镜(TCS-SPE-II)记录分析。
实施例中制备目标产物二氢喹啉类荧光探针的反应式如下所示：
Figure BDA0002780030230000051
所述3-甲酰基-1-甲基喹啉盐通过以下方法制备得到：
Figure BDA0002780030230000052
在装有5mL乙腈(ACN)和8mmol喹啉-3-甲醛的圆底烧瓶中加入40mmol碘甲烷。然后在氮气保护下将混合物加热至50℃，反应液搅拌24小时。反应结束后，将混合物冷却至室温，大量黄色固体析出，抽滤得到目标产物，产率为92％。
实施例1
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的2-萘乙酮作为模板底物与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3a，产率高达90％(黄色固体)。1H NMR(400MHz,CDCl3)δ9.10(s,1H),8.42(s,1H),7.92–7.86(m,2H),7.77–7.74(m,2H),7.49–7.43(m,2H),7.09–7.04(m,2H),6.94–6.90(m,1H),6.88(s,1H),6.76(d,J＝8.0Hz,1H),4.69–4.66(m,1H),3.35–3.21(m,5H).13C NMR(100MHz,CDCl3)δ198.52,187.56,151.84,138.23,135.48,134.35,132.57,130.44,129.78,129.77,128.39,128.30,127.68,127.62,126.64,126.50,124.46,124.02,114.84,113.09,47.31,39.45,32.20.ESI[M+H]+m/z calcd.for C23H20NO2 342.14,found 342.15[M+H]+.
实施例2
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的6-溴-2-萘乙酮与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3b，产率高达62％(黄色固体)。1H NMR(400MHz,d6-DMSO)δ9.10(s,1H),8.55(s,1H),8.27(d,J＝1.9Hz,1H),8.01(d,J＝8.0Hz,1H),7.94(s,2H),7.74–7.71(dm,1H),7.54(s,1H),7.20–7.14(m,2H),7.01–6.97(m,2H),4.58–4.55(m,1H),3.38(s,3H),3.30–3.22(m,2H).13C NMR(100MHz,d6-DMSO)δ198.3,187.3,153.0,138.6,136.4,134.9,132.2,131.2,130.5,130.4,130.1,129.7,128.0,127.8,126.7,125.3,124.4,122.5,114.1,114.1,47.8,39.2,31.5.ESI[M+H]+m/z calcd.for C23H19BrNO2 420.05,found 420.04[M+H]+.
实施例3
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的6-甲氧基-2-萘乙酮与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3c，产率高达58％(黄色固体)。1H NMR(400MHz,CDCl3)δ9.10(s,1H),8.35(d,J＝1.1Hz,1H),7.90–7.88(m,1H),7.76(d,J＝9.0Hz,1H),7.64(d,J＝8.7Hz,1H),7.10–7.03(m,4H),6.94–6.90(m,1H),6.88(s,1H),6.76(d,J＝8.1Hz,1H),4.68–4.65(m,1H),3.84(s,3H),3.32–3.20(m,5H).13C NMR(100MHz,CDCl3)δ198.2,187.6,159.7,151.8,138.2,137.2,132.5,131.3,130.3,129.8,127.9,127.6,127.0,126.6,124.8,124.4,119.5,114.9,113.1,105.7,55.4,47.2,39.4,32.2.ESI[M]+m/z calcd.for C24H21NO3 371.15,found371.15[M]+.
实施例4
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的6-噻吩基-2-萘乙酮与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3d，产率高达60％(黄色固体)。1H NMR(400MHz,CDCl3)δ9.19(s,1H),8.46(s,1H),8.02–7.93(m,3H),7.84–7.76(m,2H),7.47–7.46(m,1H),7.35–7.34(m,1H),7.17–7.11(m,3H),7.03–7.01(m,2H),6.96(s,1H),6.84(d,J＝8.1Hz,1H),4.77–4.74(m,1H),3.38–3.35(m,5H).13C NMR(100MHz,CDCl3)δ198.3,187.6,151.8,143.8,138.2,135.8,134.3,134.2,131.8,130.4,130.2,129.8,128.4,128.3,128.3,127.6,126.5,125.9,125.1,124.8,124.5,124.2,123.7,114.9,113.1,47.3,39.5,32.3.ESI[M]+m/z calcd.forC27H21NO2S 423.13,found 423.13[M]+.
实施例5
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的1-萘乙酮与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3e，产率高达53％(黄色油状物)。1H NMR(400MHz,CDCl3)δ9.03(s,1H),8.26–8.23(m,1H),7.82(d,J＝8.2Hz,1H),7.75–7.73(m,1H),7.65–7.63(m,1H),7.43–7.39(m,2H),7.33–7.29(m,1H),7.12–7.05(m,2H),6.95–6.91(m,1H),6.70(s,1H),6.65(d,J＝8.1Hz,1H),4.66–4.63(m,1H),3.40–3.34(m,1H),3.26–3.21(m,1H),3.01(s,3H).13C NMR(100MHz,CDCl3)δ203.1,187.5,151.7,138.0,135.9,133.8,132.4,130.1,129.9,128.4,128.3,127.6,126.3,126.2,125.8,124.6,124.4,114.6,113.1,50.2,39.2,32.2.ESI[M-H]-m/zcalcd.for C23H18NO2 340.14,found 340.13[M-H]-.
实施例6
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的4-氟-1-萘乙酮与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3f，产率高达61％(黄色油状物)。1H NMR(400MHz,CDCl3)δ9.06(s,1H),8.43–8.41(m,1H),8.05–8.03(m,1H),7.74–7.71(m,1H),7.52–7.48(m,2H),7.11–7.07(m,2H),7.01–6.93(m,2H),6.74(s,1H),6.67(d,J＝8.1Hz,1H),4.65–4.63(m,1H),3.34–3.22(m,2H),3.05(s,3H).13C NMR(100MHz,CDCl3)δ201.6,187.5,162.2,159.6,151.6,138.0,132.3,132.2,131.9,131.9,129.9,129.8,129.8,128.8,127.7,126.7,126.6,126.2,126.1,126.1,124.6,124.0,123.9,120.7,120.6,114.6,113.1,108.2,108.0,50.0,39.2,32.5.ESI[M-H]-m/z calcd.for C23H17FNO2 358.13,found 357.13[M-H]-.
实施例7
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的4-甲氧基-1-萘乙酮与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3g，产率高达34％(黄色固体)。1H NMR(400MHz,CDCl3)δ9.05(s,1H),8.59(d,J＝8.4Hz,1H),8.24–8.22(m,1H),7.90(d,J＝8.2Hz,1H),7.52–7.41(m,2H),7.10–7.04(m,2H),6.95–6.93(m,1H),6.75(s,1H),6.71–6.69(m,2H),4.67–4.64(m,1H),3.98(s,3H),3.32–3.20(m,2H),3.08(s,3H).13C NMR(100MHz,CDCl3)δ201.2,187.5,158.8,151.6,138.0,131.9,131.7,129.9,128.4,127.7,127.6,126.3,126.0,125.7,125.6,124.5,122.0,114.8,113.1,102.2,55.8,49.6,39.2,32.9.ESI[M-H]-m/z calcd.for C24H20NO3370.15,found 370.15[M-H]-.
实施例8
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的1-羟基-2-萘乙酮与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3h，产率高达55％(黄色油状物)。1H NMR(400MHz,CDCl3)δ13.97(s,1H),9.10(s,1H),8.34(d,J＝8.3Hz,1H),7.71(d,J＝8.9Hz,1H),7.64(d,J＝8.1Hz,1H),7.53–7.49(m,1H),7.43–7.39(m,1H),7.16(d,J＝7.9Hz,1H),7.12–7.08(m,1H),7.00–6.98(m,1H),6.93–6.90(m,1H),6.88(s,1H),6.79(d,J＝8.1Hz,1H),4.68–4.65(m,1H),3.27–3.13(m,4H),3.11–3.05(m,1H).13C NMR(100MHz,CDCl3)δ204.5,187.5,162.7,151.7,138.2,137.3,130.0,129.8,127.8,127.5,125.8,125.8,125.2,125.1,124.6,124.4,118.4,114.6,113.4,113.2,47.3,39.4,32.8.ESI[M-H]-m/z calcd.for C23H18NO3356.14,found356.13[M-H]-.
实施例9
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的2-羟基-1-萘乙酮与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3i，产率高达90％(粉白色固体)。1H NMR(400MHz,d6-DMSO)δ10.22(s,1H),9.04(s,1H),7.78–7.74(m,2H),7.37–7.35(m,1H),7.33(s,1H),7.31–7.23(m,3H),7.20–7.16(m,1H),7.07–7.01(m,2H),6.88–6.86(m,1H),4.55–4.52(m,1H),3.45–3.39(m,1H),3.16–3.11(m,4H).13C NMR(100MHz,d6-DMSO)δ205.3,187.2,153.5,153.1,138.5,131.9,131.0,130.0,128.4,128.0,127.8,127.3,127.0,124.2,123.7,123.4,121.4,118.5,114.2,113.9,52.6,39.0,30.5.ESI[M-H]-m/z calcd.for C23H18NO3 356.14,found 356.13[M-H]-.
实施例10
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的2-乙酰基菲与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3j，产率高达31％(黄色油状物)。1H NMR(400MHz,CDCl3)δ9.11(s,1H),8.61–8.58(m,2H),8.42(d,J＝1.8Hz,1H),8.11–8.18(m,1H),7.82–7.80(m,1H),7.73–7.67(m,2H),7.61–7.55(m,2H),7.10–7.06(m,2H),6.95–6.91(m,1H),6.88(s,1H),6.76(d,J＝8.1Hz,1H),4.71–4.68(m,1H),3.38–3.28(m,5H).13C NMR(100MHz,CDCl3)δ198.4,187.6,151.9,138.2,134.8,133.3,133.0,131.5,130.1,129.8,129.7,128.7,127.8,127.7,127.6,127.5,127.0,126.5,125.3,124.5,123.3,123.1,114.8,113.1,47.3,39.5,32.2.ESI[M-H]-m/zcalcd.for C27H20NO2 390.16,found 390.15[M-H]-.
实施例11
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的联苯乙酮与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3k，产率高达30％(黄色油状物)。1H NMR(400MHz,CDCl3)δ9.10(s,1H),7.96–7.93(m,2H),7.58–7.51(m,4H),7.40–7.35(m,2H),7.33–7.28(m,1H),7.13–7.05(m,2H),6.96–6.91(m,2H),6.81–6.79(m,1H),4.66–4.63(m,1H),3.32(s,3H),3.22–3.20(m,2H).13C NMR(100MHz,CDCl3)δ198.2,187.6,151.9,145.5,139.9,138.2,135.8,129.8,129.0,128.9,128.2,127.6,127.3,127.2,126.5,124.5,114.8,113.1,47.2,39.5,32.0.ESI[M-H]-m/zcalcd.for C25H20NO2 366.16,found 366.15[M-H]-.
实施例12
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的4-乙酰基吡啶与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3l，产率高达93％(黄色油状物)。1H NMR(400MHz,CDCl3)δ9.08(s,1H),8.68–8.66(m,2H),7.62–7.61(m,2H),7.14–7.10(m,1H),7.04–7.02(m,1H),6.97–6.93(m,2H),6.81(d,J＝8.2Hz,1H),4.61–4.58(m,1H),3.33(s,3H),3.23–3.12(m,2H).13C NMR(100MHz,CDCl3)δ198.0,187.5,151.9,150.8,142.8,138.2,129.6,127.8,126.0,124.6,121.3,114.2,113.3,47.3,39.5,31.8.ESI[M-H]-m/z calcd.for C18H15N2O2 291.12,found 291.11[M-H]-.
实施例13
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的3-乙酰基吡啶与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3m，产率高达85％(黄色油状物)。1H NMR(400MHz,CDCl3)δ9.08(s,1H),8.99–8.98(m,1H),8.65–8.63(m,1H),8.14–8.12(m,1H),7.31–7.27(m,1H),7.14–7.08(m,2H),6.98–6.93(m,2H),6.81(d,J＝8.1Hz,1H),4.64–4.61(m,1H),3.34(s,3H),3.27–3.13(m,2H).13C NMR(100MHz,CDCl3)δ197.5,187.5,153.2,151.8,149.7,138.2,135.7,132.3,129.6,127.8,126.2,124.6,123.5,114.4,113.2,47.4,39.5,31.7.ESI[M-H]-m/z calcd.forC18H15N2O2 291.12,found 291.11[M-H]-.
实施例14
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的2-乙酰基吡啶与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3n，产率高达91％(黄色油状物)。1H NMR(400MHz,CDCl3)δ9.06(s,1H),8.54–8.52(m,1H),7.91–7.89(m,1H),7.73–7.70(m,1H),7.34–7.32(m,2H),7.10–7.08(m,1H),6.98–6.94(m,1H),6.92(s,1H),6.82–6.80(m,1H),4.71–4.68(m,1H),3.70–3.65(m,1H),3.34(s,3H),3.20–3.15(m,1H).13C NMR(100MHz,CDCl3)δ199.5,187.4,153.7,151.7,148.7,138.3,136.7,130.0,127.3,126.8,124.4,121.9,115.1,112.8,47.1,39.4,30.9.ESI[M-H]-m/zcalcd.for C18H15N2O2 291.12,found 291.11[M-H]-.
实施例15
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的3-乙酰基喹啉与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3o，产率高达92％(黄色固体)。1H NMR(400MHz,CDCl3)δ9.35(d,J＝2.2Hz,1H),9.20(s,1H),8.76(d,J＝2.0Hz,1H),8.13(d,J＝8.5Hz,1H),7.95–7.93(m,1H),7.84–7.80(m,1H),7.63–7.59(m,1H),7.20–7.15(m,2H),7.06–7.01(m,2H),6.86(d,J＝8.2Hz,1H),4.76–4.74(m,1H),3.39(s,4H),3.38(s,1H).13C NMR(100MHz,CDCl3)δ197.5,187.5,151.8,149.6,149.2,138.2,137.7,131.9,129.7,129.6,129.3,129.2,127.8,127.4,126.8,126.2,124.6,114.5,113.2,47.4,39.5,32.1.ESI[M-H]-m/z calcd.for C22H17N2O2 341.14,found 341.12[M-H]-.
实施例16
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的6-乙酰基喹啉与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3p，产率高达80％(黄色油状物)。1H NMR(400MHz,CDCl3)δ9.17(s,1H),8.96–8.95(m,1H),8.49(d,J＝1.9Hz,1H),8.26–8.19(m,2H),8.08(d,J＝8.9Hz,1H),7.44–7.41(m,1H),7.15–7.08(m,2H),7.00–6.98(m,2H),6.83–6.81(m,1H),4.74–4.71(m,1H),3.41–3.28(m,5H).13C NMR(100MHz,CDCl3)δ197.9,187.5,152.5,151.8,150.0,138.1,137.7,134.7,130.3,129.8,129.7,127.7,127.5,126.3,124.5,121.8,114.6,113.2,47.5,39.4,32.2.ESI[M-H]-m/z calcd.for C22H17N2O2 341.14,found 341.12[M-H]-.
实施例17
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的3-乙酰基苯并噻吩与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3q，产率高达85％(黄色固体)。1H NMR(400MHz,CDCl3)δ9.11(s,1H),7.93(s,1H),7.81–7.74(m,2H),7.37–7.28(m,2H),7.10–7.07(m,2H),6.98–6.96(m,1H),6.91(s,1H),6.78(d,J＝8.2Hz,1H),4.67–4.64(m,1H),3.29(s,3H),3.24–3.13(m,2H).13C NMR(100MHz,CDCl3)δ193.0,187.5,151.8,144.1,142.5,139.4,138.2,130.1,129.8,127.8,127.4,126.2,126.0,124.9,124.6,122.9,114.4,113.2,47.7,39.5,32.9.ESI[M-H]-m/zcalcd.for C21H176NO2S 346.09,found 346.09[M-H]-.
实施例18
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的1-甲基-3-乙酰基吲哚与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3r，产率高达30％(黄色固体)。1H NMR(400MHz,CDCl3)δ9.13(s,1H),8.33–8.31(m,1H),8.02(s,1H),7.27–7.20(m,3H),7.13–7.09(m,1H),6.98–6.90(m,3H),6.77(d,J＝8.0Hz,1H),4.61–4.59(m,1H),3.77(s,3H),3.26(s,3H),3.14–3.10(m,1H),2.78–2.72(m,1H).13C NMR(100MHz,CDCl3)δ193.6,187.8,151.7 138.0,137.4,137.1,130.1,127.6,126.5,124.5,123.2,122.7,122.4,116.3,115.2,113.0,109.5,49.4,39.4,34.1,33.6.ESI[M-H]-m/z calcd.for C22H19N2O2 343.15,found 343.15[M-H]-.
实施例19
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的N-甲基-2-乙酰基噻吩嗪与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3s，产率为37％(黄色油状物)。1H NMR(400MHz,CDCl3)δ9.10(s,1H),7.47–7.45(m,1H),7.36(d,J＝1.6Hz,1H),7.12–7.07(m,3H),7.04–7.02(m,1H),6.94–6.74(m,6H),4.60–4.58(m,1H),3.36(s,3H),3.32(s,3H),3.22–3.17(m,1H),3.06–3.00(m,1H).13CNMR(100MHz,CDCl3)δ197.9,187.5,151.7,145.9,145.3,138.1,136.2,130.3,129.8,127.9,127.7,127.1,126.8,126.2,124.5,123.1,122.7,122.2,114.8,114.4,113.3,113.1,47.2,39.5,35.6,32.5.ESI[M-H]-m/z calcd.for C26H21N2O2S 425.14,found425.13[M-H]-.
实施例20
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的N,N-二苯氨基苯基噻吩乙酮与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3t，产率为30％(黄色油状物)。1H NMR(400MHz,CDCl3)δ9.12(s,1H),7.64(d,J＝4.0Hz,1H),7.41(d,J＝8.7Hz,2H),7.23–7.19(m,4H),7.11–7.04(m,7H),7.01–6.96(m,5H),6.91(s,1H),6.81(d,J＝8.1Hz,1H),4.63–4.60(m,1H),3.33(s,3H),3.13–3.02(m,2H).13C NMR(100MHz,CDCl3)δ191.1,187.5,152.6,151.8,148.7,147.1,142.2,138.2,134.1,129.8,129.5,127.7,127.1,126.8,126.2,125.0,124.5,123.7,123.0,122.7,114.6,113.1,47.3,39.5,32.9.ESI[M]+m/z calcd.for C35H28N2O2S 540.19,found540.18[M]+.
实施例21
在水/乙醇(1:1)的溶剂体系下，以2eq NaOH为催化剂，以0.2mmol的(E)-4-(4’-二甲氨基苯基)-2-羰基-3-丁烯与0.24mmol的3-甲酰基-1-甲基喹啉盐在室温下搅拌反应5h，进行亲核加成反应得到目标产物3u，产率为25％(绿色固体)。1H NMR(400MHz,CDCl3)δ9.10(s,1H),7.43(d,J＝16.1Hz,1H),7.33–7.31(m,2H),7.14–7.09(m,2H),6.99–6.95(m,1H),6.89(s,1H),6.79(d,J＝8.2Hz,1H),6.58–6.56(m,2H),6.37(d,J＝16.0Hz,1H),4.56–4.53(m,1H),3.30(s,3H),2.94(s,6H),2.94–2.83(m,2H).13C NMR(100MHz,CDCl3)δ198.5,187.5,151.9,151.8,143.9,138.3,130.2,129.8,127.5,126.7,124.4,122.2,121.7,115.0,113.0,111.8,49.0,40.1,39.4,32.2.ESI[M-H]-m/z calcd.for C23H23N2O2359.18,found 359.18[M-H]-.
实施例22
本实施例与实施例1的不同之处在于反应条件不同，具体反应条件如表1所示：
表1制备化合物3a时的不同反应条件
Figure BDA0002780030230000131
a：分离产率，b：反应12h，c：反应20h.
实施例23二氢喹啉类化合物对·OH的检测能力
H2O2经过UV光解能产生·OH，这一反应通常被用来测试有机化合物与·OH的反应情况。测试时将合成的二氢喹啉类化合物荧光探针(20μM)分别与5mM H2O2加入H2O/ACN 1:9混合溶液中，然后在210nm功率5W的紫外光下照射。用96孔板测量时，每孔加入200μL反应混合物室温反应2.5小时，然后通过酶标仪记录荧光和吸收光谱。每组测试设置三个重复。
实验结果显示：反应溶液颜色和荧光变化直观有效证明了所有被测试的二氢喹啉类化合物都能与·OH发生反应。夺氢反应是羟基自由基介导的一种重要的氧化途径，可能是此类二氢喹啉类化合物的主要氧化机制。以实施例1制备的化合物3a为例，夺氢反应先后发生在41位以及邻近44位碳原子上，从而导致C41和C44之间形成双键，最终的荧光产物3a-pr(3a-pr为3a的氧化产物)由中间体通过双键重排形成。通过密度泛函理论(DFT)计算了3a分子中所有原子的Hirshfeld电荷以及亲电性，计算结果表明：相对于C44，C41具有更高的亲电性能，显然更易受到·OH的亲电进攻从而发生第一次夺氢反应。另外，HRMS和NMR对3a-pr的化学结构进行了表征，该结构含有烯醇基团。乙酰基的结构对相应氧化产物的荧光性质有很大的影响，结果显示：除了含有酚类基团的萘乙酮(如3h-pr和3i-pr)以外，大部分萘乙酮产物具有较高的量子产率。3h-pr(φ＝0.0049)和3i-pr(φ＝0.0031)的荧光猝灭很大可能是游离酚羟基的光诱导电子转移效应(PET)所致。相似的PET效应同样存在于吡啶类(3l-pr to 3n-pr)和喹啉类化合物(3o-pr和3p-pr)中。
在研究含萘乙酮的二氢喹啉类化合物3a-3g时，我们发现给电子效应可以有效影响氧化产物(3a-pr to 3g-pr)的光物理性质，简单的说，给电子基团导致明显的荧光红移，吸电基团则致使氧化产物荧光蓝移(图2A和图2B)。例如，与荧光峰值在514nm处的3a-pr相比，具有电子供体基团甲氧基的3c-pr的荧光峰值红移至556nm。如果在同样取代位置上更换成具有弱吸电性的溴原子，3b-pr的荧光峰值却轻微蓝移至512nm处，3e-pr,3f-pr和3g-pr的荧光光谱也表现出相同的变化趋势。DFT的计算结果还表明：与第一激发态S1相对应的前沿轨道位于3a-pr的不同区域，其中HOMO位于萘酮基团，而LUMO位于喹啉基团(图2C)。
此外，该反应还提供了构建比率式荧光探针的可能性，方法是用荧光染料取代非荧光乙酰基底物，比如携带荧光官能团N-甲基吩噻嗪的化合物3s。3s具有明亮荧光，波长在557nm处达到峰值，相应的氧化产物的荧光峰值在波长594nm处。另一方面，这种反应也可用于构造具有较大斯托克位移的近红外荧光探针，例如在绿光激发下，化合物3u的氧化产物发射近红外荧光(荧光峰值在波长722nm处)。
实施例24对细胞内·OH的成像实验
人类宫颈癌细胞Hela细胞被用作模式细胞进行成像研究，细胞内·OH分别由芬顿反应以及佛波酯刺激产生。细胞接种在35mm培养皿内，孵育条件为5％CO2，温度37℃，含10％FBS的DMEM。利用芬顿反应产生·OH的做法是往Hela细胞与EDTA-Fe2+的共孵育体系中加入H2O2。用PMA刺激产生ROS的产生，在加入探针前用1μg/mL佛波酯(PMA)孵育2h，随后用新鲜DMEM代替含药培养基，使用共聚焦荧光显微镜观察细胞，而另一组只加入探针孵育作为对照。在共定位实验中，加入10μM实施例3制备的3c和10μM商品化溶酶体红色荧光探针共同孵育细胞，使用莱卡共聚焦荧光显微镜(TCS-SPE-II)采集图像。3c和DCFH-DA的荧光通道为EX 488|EM 528/20，溶酶体红色荧光探针通道为EX 535|EM>600nm。
如图3所示，经过上述处理后细胞内产生明显绿色荧光，而在加入H2O2之前，细胞在3c和EDTA-Fe2+共同孵育下只表现出微弱的绿色荧光背景。化合物3c在亚细胞结构溶酶体的定位效果通过与商品化的溶酶体红色荧光探针共培育实现，如图4所示，随后运用软件image J对共定位实验进行定量分析，皮尔逊相关系数(PCC)为0.87，表明3c在溶酶体内定位良好。在进一步研究中，化合物3c与商品化ROS探针2’,7’-二氯荧光素二乙酸酯(DCFH-DA)进行比较，结果表明3c和DCFH-DA都能通过荧光开启方式对佛波酯-12-肉豆蔻酸-13-乙酸酯PMA诱导的细胞内ROS爆发现象产生信号响应。值得注意的是DCFH-DA在整个细胞中表现出荧光，而3c则表现出细胞器靶向性，主要定位在溶酶体中。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

1.一种二氢喹啉类荧光探针，其特征在于其结构式如下所示：
Figure FDA0002780030220000011
所述R为具有取代基的芳基、吡啶基或杂环。
2.根据权利要求1所述的二氢喹啉类荧光探针，其特征在于，其结构式为以下3a～3u所示：
Figure FDA0002780030220000012
3.一种制备权利要求1或2所述二氢喹啉类荧光探针的制备方法，其特征在于包括如下步骤：
在水/乙醇的溶剂体系下，以无机碱为催化剂，将乙酮类化合物作为模板底物与喹啉盐类化合物发生亲核加成反应，得到二氢喹啉类荧光探针。
4.根据权利要求3所述的制备方法，其特征在于：所述乙酮类化合物与喹啉盐类化合物的摩尔比为1：0.8～1.5。
5.根据权利要求3所述的制备方法，其特征在于：所述乙酮类化合物与喹啉盐类化合物的摩尔比为1:1.2。
6.根据权利要求3所述的制备方法，其特征在于：所述水/乙醇的体积比为1：0.8～1.5；所述无机碱为Na2CO3、KOH、NaOH、K2CO3、KF、K3PO4和NaHCO3中的至少一种。
7.根据权利要求3～6任一项所述的制备方法，其特征在于：所述无机碱相对于乙酮类化合物的当量比为1～8。
8.根据权利要求3所述的制备方法，其特征在于：所述亲核加成反应的时间为5～20h；反应温度为25-60℃。
9.根据权利要求1或2所述的二氢喹啉类荧光探针在检测·OH中的应用。
10.根据权利要求1或2所述的二氢喹啉类荧光探针在制备检测·OH的比率式荧光探针和近红外荧光探针中的应用。
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