CN111848657B - 靶向酪氨酸激酶识别的可逆性荧光化合物及其制备方法和应用 - Google Patents
靶向酪氨酸激酶识别的可逆性荧光化合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供靶向酪氨酸激酶识别的可逆性荧光化合物及其制备方法和应用。具体地,本发明提供一类具有荧光淬灭现象的新结构化合物,该化合物由c‑Met抑制剂与BODIPY染料通过脂肪链连接组成。本发明还提供上述化合物的制备方法及其在制备治疗和/或诊断肿瘤的药物中的应用,所述药物通过对肿瘤细胞中c‑Met蛋白激酶活性可视化实现对肿瘤的治疗和/或诊断作用。
Description
技术领域
本发明涉及一类靶向酪氨酸激酶c-MET识别的可逆性荧光化合物及其制备方法和用途。
背景技术
癌症是威胁人类健康的重要杀手,肿瘤个体化治疗体系的核心和成功的关键在于寻找并确定个体肿瘤所依赖的驱动型癌基因,通过开发针对药物敏感预测标志物的特异性探针,可遴选出携带驱动基因表型的敏感病人进行靶向治疗。这一基于特定分子分型的个体化治疗模式是肿瘤靶向治疗的主流,在癌症诊疗中具有巨大价值。
在抗肿瘤药物靶标研究中,蛋白激酶高达75%;绝大多数已上市的小分子肿瘤靶向抑制剂均为酪氨酸蛋白激酶抑制剂(Tyrosine Kinase Inhibitor,TKI)。c-Met也是受体型酪氨酸激酶(receptor tyrosine kinase,RTK)家族的一员,其天然配体为肝细胞生长因子(HGF,hepatocyte growth factor),共同形成HGF/c-Met信号通路。[Cancer lett.,2012,319(1):109-117.]c-Met信号通路不但在胚胎正常发育、伤口愈合、肝脏再生中具有重要作用,而且在病理状态下,c-Met的过度表达或过度激活具有诱导肿瘤细胞增殖、转移及肿瘤血管生成的作用。在许多实体瘤(肺癌、恶性胶质瘤、卵巢癌、胃癌、结直肠癌、肾癌等)中,该通路多通过MET基因的扩增,引起c-Met蛋白的高表达和激酶活性的增加。[Nat.Rev.Mol.Cell Biol.,2003,4:915-925.]大量的临床数据提示c-Met的表达量与预后、转移性和耐药性紧密相关,因此针对c-Met靶点的肿瘤分子靶向诊断和治疗具有重要意义。
目前已上市和临床在研的代表性c-Met抑制剂有如下所示:
近年来,以c-Met为靶点的小分子靶向药物研究广泛,发展迅速。多个选择性c-Met激酶抑制剂已在临床测试的不同阶段,比如tivantinib(ARQ 197)[Mol CancerTher.2010,9(6):1544-53.],Tepotinib、AMG337、JNJ-38877618和SGX523[Mol CancerTher,2009,8(12),3181-3190.]等。此外,非选择性c-Met激酶抑制剂如crizotinib与cabozantinib已被FDA批准,分别适用于ALK重组的非小细胞肺癌和转移性甲状腺髓样癌。但不加筛选的患者在接受c-Met激酶抑制剂的靶向治疗中总体有效率偏低。[GeneChromosone Canc.,2008,47(12):1025-1037.]“肿瘤异质性”(Tumor Heterogeneity)现象在MET基因的突变和扩增上得到了充分体现。在胃癌病患中,MET基因的扩增率为16-30%;而在遗传性肾乳头状细胞癌病患中,MET基因点突变的发生率为100%。如何针对敏感预测标志物c-Met筛选出适合c-Met激酶抑制剂治疗的病患人群,实现病患的精确分子分型,是靶向治疗获取成功的关键。
目前检测c-Met在活检或手术样本中异常活化状况的手段包括:(1)通过免疫组化或Western blotting的方法检查c-Met的表达水平;(2)利用荧光原位杂交方法检测MET基因的拷贝数;(3)DNA测序来探测MET基因的点突变状况。但是这些方法检测费用高、耗时长、操作复杂,在肿瘤病患的分子分型中无法广泛开展。因此,亟需开发简单、便捷、准确检测c-Met表达量的方法用于肿瘤的初步筛查。
小分子化合物的荧光成像具有成本低、选择性好、灵敏度高、响应速度快、操作简单便捷等优点,在药物高通量筛选、细胞成像、靶标定位及疾病诊断等方面已有大量的应用。
发明内容
本发明提供了一类对肿瘤细胞中c-Met蛋白激酶活性可视化的化合物,实现对肿瘤的治疗和/或诊断作用,为临床筛选c-Met抑制剂受益人群提供重要依据。
本发明以c-Met选择性小分子激酶抑制剂为“靶向元件”(targeting moiety),以生物兼容性好的BODIPY为荧光染料,开发针对敏感预测标志物c-Met的特异性探针。该类探针体积小、可高效结合靶点,并具有优异的成像特性,可在分子水平和细胞水平上获取c-Met的生物学信息,为临床筛选c-Met抑制剂受益人群提供重要依据。
本发明涉及c-Met抑制剂与BODIPY通过柔性烷基链连接的荧光染料,同时利用c-Met抑制剂中芳胺的光诱导电子转移(PET,Photoinduced-electron transfer)机制达到荧光off的目的,利用c-Met抑制剂的靶向性对肿瘤进行分子分型。同时本发明提供了这类荧光探针的合成方法,通过简便反应即可高产率的获得具有结构多样性和功能多样性的荧光探针分子。这是一类结构新颖的off-on探针,同时此类探针能够有效识别肿瘤细胞中表达的c-Met蛋白。
本发明的一个目的是提供一类具有荧光淬灭现象的新结构化合物,该化合物由c-Met抑制剂与BODIPY染料通过脂肪链连接组成,在靶蛋白识别前后具有荧光off-on的效应,可以高灵敏度的识别c-Met蛋白,其结构如通式Ⅰ所示。
本发明的另一个目的是提供上述化合物的制备方法及其在制备治疗和/或诊断肿瘤的药物中的应用,所述药物通过对肿瘤细胞中c-Met蛋白激酶活性的可视化,实现对肿瘤的治疗和/或诊断作用。
根据本发明的目的,本发明提供了一类如通式Ⅰ所示的BODIPY类化合物:
其中,
R为氢或C1-C6烷基;
“-”为负电荷,“+”为正电荷。
优选的,R为氢或乙基;
更优选地,所述通式Ⅰ所示的BODIPY类化合物选自如下具体化合物:
根据本发明的另一个目的,本发明提供了通式Ⅰ所示的BODIPY类化合物的制备方法:
包括以下步骤:
(a)化合物2与化合物1发生Friedel–Crafts反应,再向反应溶液中加入三氟化硼-乙醚复合物和碱,得到化合物3;
(b)化合物3与氧化剂发生氧化反应得到化合物4;
(c)化合物4与化合物5在还原剂的作用下发生还原胺化反应得到通式I的化合物;
上述步骤(a)中,化合物2首先与化合物1经过两次Friedel–Crafts反应,再向反应液中加入三氟化硼-乙醚复合物和碱。一般而言,该反应可在加热条件下进行,例如加热至50-70℃,使用的碱选自三乙胺或二异丙基乙胺。
上述步骤(b)中,化合物3经氧化剂氧化得到化合物4。一般而言,该反应在0℃到室温下进行,氧化剂选自Dess-Martin氧化剂或三氧化硫-吡啶复合物。
上述步骤(c)中,化合物4与化合物5经过还原剂的还原胺化得到化合物通式I。该反应在室温条件的弱酸或者中性条件下反应,所述弱酸选自乙酸,丙酸。所述的还原剂选自氰基硼氢化钠。
根据本发明的另一个目的,本发明提供了一种药物组合物,包含上述通式Ⅰ所示的BODIPY类化合物,以及药学上可接受的载体。
根据本发明的另一个目的,本发明提供了上述通式Ⅰ所示的BODIPY类化合物或所述药物组合物在制备c-Met蛋白染色制剂中的应用,具体的,提供了上述通式Ⅰ所示的BODIPY类化合物或所述药物组合物在制备荧光探针中的应用,所述荧光探针用于肿瘤细胞中c-Met蛋白染色成像。从而,本发明还提供了上述通式Ⅰ所示的BODIPY类化合物或所述药物组合物在制备治疗和/或诊断肿瘤的药物中的应用,所述药物通过对肿瘤细胞中c-Met蛋白激酶活性可视化实现对肿瘤的治疗和/或诊断作用。
一种使用上述通式Ⅰ所示的BODIPY类化合物对c-Met蛋白成像的操作可以为:将肿瘤细胞与1-1000nM通式Ⅰ所示的BODIPY类化合物共孵育5-30min。于荧光显微镜下观察肿瘤细胞的染色成像结果。
本发明的有益结果:
项目发现了一类新结构的BODIPY类化合物,将其与肿瘤细胞孵育后,可特异性地与c-Met蛋白结合,在不清洗的操作下即可在荧光显微镜下直接观察c-Met蛋白表达水平和亚细胞水平定位。这类荧光探针合成简单,具有简便性好、操作简单快速和实用性强的特点。
附图说明
图1为试验实施例1中荧光探针7在乙醇中的UV-Vis吸收光谱;
图2为试验实施例1中荧光探针7在乙醇中的荧光光谱;
图3为试验实施例1中荧光淬灭效率实验,荧光探针7与淬灭荧光染料3a在乙醇中的荧光光谱;
图4为试验实施例3中荧光探针7与不同浓度c-Met蛋白孵育后荧光曲线图;
图5为试验实施例4中荧光探针7在MKN45和MKN74中的荧光成像照片及不同浓度下荧光强度的柱状图。
具体实施方式
下面的实施例用于具体地说明本发明化合物的制备,以及其作为开关型荧光探针分子的应用,但本发明并不局限于这些实施例。
核磁共振氢谱(1HNMR)用BrukerAMX-400型、Gemini-300型或AMX–600型核磁共振仪记录,溶剂为氘代氯仿(CDCl3)或氘代二甲基亚砜(DMSO-d6)。化学位移δ的单位为ppm,耦合常数J的单位为Hz。所用微波为CEM-discovery微波反应器。所有反应溶剂均按照常规方法进行纯化。柱层析用硅胶(200-300目)为青岛海洋化工分厂生产。薄层层析使用GF254高效板,为烟台化工研究所生产。制备型薄层层析板由自己制备,固定相采用GF254(HG/T2354-92)硅胶和羧甲基纤维素钠(800-1200)制备,分别为青岛海洋化工有限公司和中国医药(集团)上海化学试剂公司生产。所有溶剂均为分析纯试剂,所用试剂均购自国药集团化学试剂有限公司。采用紫外荧光等方法显色。减压蒸除有机溶剂在旋转蒸发仪中进行。吸收光谱在Hitachi U-3010紫外-可见分光光度计测试,荧光光谱在Horiba FluoroMax 4荧光分光光度计测试,细胞荧光成像由Leica TCS SP8 STED测试。实验中所用激酶由本实验室利用昆虫杆状病毒表达系统表达纯化蛋白激酶区重组蛋白;多聚谷氨酸-酪氨酸肽段【Poly(Glu,Tyr)4:1】及钒酸钠购自Sigma公司;抗磷酸化单抗PY99购自Santa Cruz公司;辣根过氧化物酶标记羊抗鼠二抗购自Calbiochem公司;ATP及OPD购自上海生工;其余所用试剂均购自国药集团化学试剂有限公司。反应酶标板(#2592)购自Corning公司。实验读板用全波长型酶标仪为Molecular Device公司产品,型号:SpectraMax 190;实验用水为国药集团产蒸馏水。
制备实施例1
合成路线同中国专利201510418869.2(申请号)。圆底烧瓶中加入2,4-二甲基-吡咯(2.2eq.),溶于无水二氯甲烷,氮气保护,冰浴,用恒压低液漏斗滴入4-溴丁酰氯(1.0eq.),滴加完成后加热回流反应8h,旋干二氯甲烷,加入甲苯和二氯甲烷(甲苯与二氯甲烷体积比=19:1),冰浴,加入三乙胺(7.0eq.),用恒压低液漏斗滴入三氟化硼-乙醚络合物(7.0eq.),滴加完成后,去除冰浴,室温反应10-20min,50℃加热反应3h,反应液冷却,倒入冰水中淬灭,二氯甲烷萃取,硫酸钠干燥,旋干溶剂,过柱纯化,获得产品3a,红棕色固体,产率30%,1H NMR(400MHz,CDCl3)δ6.06(s,2H),3.78(t,J=5.9Hz,2H),3.05–2.96(m,2H),2.53(s,6H),2.41(s,6H),1.89(s,1H),1.85–1.78(m,2H).
制备实施例2
圆底烧瓶中加入2,4-二甲基-3-乙基-吡咯(2.2eq.),溶于无水二氯甲烷,氮气保护,冰浴,用恒压低液漏斗滴入4-溴丁酰氯(1.0eq.),滴加完成后加热回流反应8h,旋干二氯甲烷,加入甲苯和二氯甲烷(甲苯与二氯甲烷体积比=19:1),冰浴,加入三乙胺(7.0eq.),用恒压低液漏斗滴入三氟化硼-乙醚络合物(7.0eq.),滴加完成后,去除冰浴,室温反应10-20min,50℃加热反应3h,反应液冷却,倒入冰水中淬灭,二氯甲烷萃取,硫酸钠干燥,旋干溶剂,过柱纯化,获得产品3b,红棕色固体,产率30%,1HNMR(400MHz,CDCl3)δ4.38(s,1H),3.49(t,J=5.9Hz,2H),3.05–2.96(m,2H),2.43(t,J=6.5Hz,2H),2.23(q,J=6.8Hz,4H),2.21(s,6H),1.89(s,6H),1.35(t,J=6.8Hz,6H).
制备实施例3
合成路线同中国专利201510418869.2(申请号)。圆底烧瓶中加入实施例1得到的产品(1.0eq.),溶于无水二氯甲烷和无水二甲亚砜(DCM与DMSO体积比=4:1),氮气保护,冰浴,加入无水三乙胺(5.0eq.),加入三氧化硫-吡啶复合物(3.0eq.),撤出冰浴,室温反应30-60min,加水淬灭反应,二氯甲烷萃取,硫酸钠干燥,旋干溶剂,过柱纯化,获得产品4a,红棕色固体,产率37%,1H NMR(400MHz,CDCl3)δ9.91(s,1H),6.08(s,2H),3.33–3.24(m,2H),2.85–2.77(m,2H),2.54(s,6H),2.37(s,6H).
制备实施例4
圆底烧瓶中加入实施例2得到的产品(1.0eq.),溶于无水二氯甲烷和无水二甲亚砜(DCM与DMSO体积比=4:1),氮气保护,冰浴,加入无水三乙胺(5.0eq.),加入三氧化硫-吡啶复合物(3.0eq.),撤出冰浴,室温反应30-60min,加水淬灭反应,二氯甲烷萃取,硫酸钠干燥,旋干溶剂,过柱纯化,获得产品4b,红棕色固体,产率37%,1H NMR(400MHz,CDCl3)δ9.81(s,1H),3.23–3.14(m,2H),2.67–2.57(m,2H),2.54(s,6H),2.43(q,J=6.8Hz,4H),2.25(s,6H),1.28(t,J=6.8Hz,6H)
制备实施例5
化合物a和b的合成参照专利,WO 2012015677。将化合物b(2.33g,4.74mmol)和二异丙基乙胺(1.5mL,8.61mmol)溶于DMF,在氩气鼓泡20分钟后加入加入三(二亚苄基丙酮)二钯(99mg,0.11mmol)和Xantphos(127mg,0.22mmol),然后加入化合a(1g,4.36mmol),将反应置于100℃油浴中反应1小时后冷却至室温,加入2M的氢氧化钠水溶液(100mL)。将析出的固体抽滤后水洗,真空干燥后将所得固体溶于4M盐酸二氧六环溶液(20mL),50℃下加热反应30分钟后压蒸干溶剂得到产物5-a。1H-NMR(500MHz,CDCl3)δ8.42(d,J=1.5Hz,1H),8.34–8.26(m,2H),7.99(d,J=1.5Hz,1H),7.92–7.84(m,2H),7.55–7.49(m,2H),7.23(d,J=1.5Hz,1H),3.98(s,3H),3.50(s,2H).
制备实施例6荧光探针1:
圆底烧瓶中加入4a(1eq.),化合物5-a(1.3eq.),溶于1,2-二氯乙烷:甲醇(体积比)=5:1,加入氰基硼氢化钠(1.3eq.),室温反应6-8h,旋干反应溶液,粗品过柱纯化得产品。产物为棕红色固体,产率38%。1H NMR(500MHz,CDCl3)δ8.94(d,J=7.5Hz,1H),8.54(d,J=1.5Hz,1H),8.21(d,J=7.5Hz,1H),7.97(d,J=1.5Hz,1H),7.82(d,J=7.5Hz,1H),7.45–7.37(m,2H),7.05(d,J=1.6Hz,1H),6.96(t,J=1.5Hz,1H),5.60(s,1H),5.00(d,J=1.0Hz,1H),3.94(s,3H),3.68–3.58(m,3H),2.16(s,3H),2.08(s,3H),1.97–1.87(m,5H),1.57(q,J=7.1Hz,2H),0.90(s,3H).
制备实施例7荧光探针2:
圆底烧瓶中加入4b(1eq.),化合物5-a(1.3eq.),溶于1,2-二氯乙烷:甲醇(体积比)=5:1,加入氰基硼氢化钠(1.3eq.),室温反应6-8h,旋干反应溶液,粗品过柱纯化得产品。产物为棕红色固体,产率43%。1H NMR(500MHz,CDCl3)δ8.94(d,J=7.5Hz,1H),8.54(d,J=1.5Hz,1H),8.21(d,J=7.5Hz,1H),7.97(d,J=1.5Hz,1H),7.45–7.37(m,2H),7.05(d,J=1.6Hz,1H),3.94(s,3H),3.68–3.58(m,3H),2.56(q,J=8.0Hz,2H),2.16(s,3H),2.08(s,3H),2.00(q,J=8.0Hz,2H),1.97–1.87(m,5H),1.56(p,J=7.1Hz,2H),1.20(t,J=8.0Hz,3H),1.03(t,J=8.0Hz,3H),0.90(s,3H).
制备实施例8
将化合物a(0.2g,0.456mmol)、对氨基苯硼酸频哪醇酯(0.150g,0.684mmol)和碳酸铯(0.297g,0.913mmol)溶于二氧六环(2mL)和水(0.4mL),氩气鼓泡15分钟后加入四三苯基磷钯(0.056g,0.0456mmol)。微波100℃条件下反应30分钟后用乙酸乙酯和水萃取反应。合并有机相并用饱和氯化钠溶液洗3次后用无水硫酸钠干燥后减压蒸发除去溶剂,柱层析得到化合物5-c。
制备实施例9荧光探针3:
圆底烧瓶中加入4a(1eq.),化合物5-c(1.3eq.),溶于1,2-二氯乙烷:甲醇(体积比)=5:1,加入氰基硼氢化钠(1.3eq.),室温反应6-8h,旋干反应溶液,粗品过柱纯化得产品。产物为棕红色固体,产率62%。1H NMR(500MHz,CDCl3)δ8.94(d,J=7.5Hz,1H),8.70(d,J=1.5Hz,1H),8.21(d,J=7.5Hz,1H),8.16–8.08(m,2H),7.97(d,J=1.5Hz,1H),7.62(dd,J=7.5,1.5Hz,1H),7.52(t,J=1.6Hz,1H),7.31–7.25(m,2H),7.05(d,J=1.6Hz,1H),6.80–6.74(m,2H),5.60(s,1H),5.00(d,J=1.0Hz,1H),3.94(s,3H),3.60(s,1H),3.39(t,J=7.1Hz,2H),2.16(s,3H),2.08(s,3H),1.97–1.87(m,5H),1.57(q,J=7.1Hz,2H),0.90(s,3H).
制备实施例10荧光探针4:
圆底烧瓶中加入4b(1eq.),化合物5-c(1.3eq.),溶于1,2-二氯乙烷:甲醇(体积比)=5:1,加入氰基硼氢化钠(1.3eq.),室温反应6-8h,旋干反应溶液,粗品过柱纯化得产品。产物为棕红色固体,产率68%。1H NMR(500MHz,CDCl3)δ8.94(d,J=7.5Hz,1H),8.70(d,J=1.5Hz,1H),8.21(d,J=7.5Hz,1H),8.16–8.08(m,2H),7.97(d,J=1.5Hz,1H),7.62(dd,J=7.5,1.5Hz,1H),7.52(t,J=1.6Hz,1H),7.31–7.25(m,2H),7.05(d,J=1.6Hz,1H),6.80–6.74(m,2H),3.94(s,3H),3.60(s,1H),3.39(t,J=7.1Hz,2H),2.56(q,J=8.0Hz,2H),2.16(s,3H),2.08(s,3H),2.00(q,J=7.9Hz,2H),1.97–1.87(m,5H),1.57(q,J=7.1Hz,2H),1.20(t,J=8.0Hz,3H),1.03(t,J=8.0Hz,3H),0.90(s,3H).
制备实施例11
将化合物a和二异丙基乙胺溶于DMF,在氩气鼓泡20分钟后加入加入三(二亚苄基丙酮)二钯和Xantphos,然后加入化合物胺甲酸叔丁酯,将反应置于100℃油浴中反应1小时后冷却至室温,加入2M的氢氧化钠水溶液(100mL)。将析出的固体抽滤后水洗,真空干燥后将所得固体溶于4M盐酸二氧六环溶液(20mL),50℃下加热反应30分钟后压蒸干溶剂得到产物5-e。1H NMR(500MHz,CDCl3)δ8.70(d,J=1.5Hz,1H),7.93(d,J=7.5Hz,1H),7.84(d,J=11.0Hz,1H),7.74(d,J=1.5Hz,1H),7.63(t,J=1.5Hz,1H),7.57(d,J=1.6Hz,1H),7.43(dd,J=7.5,1.4Hz,1H),7.11(d,J=11.0Hz,1H),7.05(t,J=1.5Hz,1H),3.97(s,3H),3.50(s,2H),3.29(t,J=7.7Hz,2H),2.79(t,J=7.7Hz,2H).
制备实施例12荧光探针5:
圆底烧瓶中加入4a(1eq.),化合物5-e(1.3eq.),溶于1,2-二氯乙烷:甲醇(体积比)=5:1,加入氰基硼氢化钠(1.3eq.),室温反应6-8h,旋干反应溶液,粗品过柱纯化得产品。产物为棕红色固体,产率35%。1H NMR(500MHz,CDCl3)δ8.60(d,J=1.5Hz,1H),7.96(d,J=1.6Hz,1H),7.84–7.77(m,2H),7.62(t,J=1.6Hz,1H),7.32(dd,J=7.5,1.4Hz,1H),7.11(d,J=10.8Hz,1H),6.96(t,J=1.5Hz,1H),5.60(s,1H),5.40(d,J=11.0Hz,1H),5.00(d,J=1.0Hz,1H),3.94(s,3H),3.68–3.58(m,3H),3.29(t,J=7.1Hz,2H),2.79(t,J=7.1Hz,2H),2.16(s,3H),2.08(s,3H),1.97–1.87(m,5H),1.57(q,J=7.1Hz,2H),0.90(s,3H).
制备实施例13荧光探针6:
圆底烧瓶中加入4b(1eq.),化合物5-e(1.3eq.),溶于1,2-二氯乙烷:甲醇(体积比)=5:1,加入氰基硼氢化钠(1.3eq.),室温反应6-8h,旋干反应溶液,粗品过柱纯化得产品。产物为棕红色固体,产率46%。1H NMR(500MHz,CDCl3)δ8.60(d,J=1.5Hz,1H),7.96(d,J=1.6Hz,1H),7.84–7.77(m,2H),7.32(dd,J=7.5,1.4Hz,1H),7.11(d,J=10.8Hz,1H),5.40(d,J=11.0Hz,1H),3.94(s,3H),3.68–3.58(m,3H),3.29(t,J=7.1Hz,2H),2.79(t,J=7.1Hz,2H),2.56(q,J=8.0Hz,2H),2.16(s,3H),2.08(s,3H),2.00(q,J=8.0Hz,2H),1.97–1.87(m,5H),1.56(p,J=7.1Hz,2H),1.20(t,J=8.0Hz,3H),1.03(t,J=8.0Hz,3H),0.90(s,3H).
制备实施例14
将化合物3-溴喹啉化合物(0.5g,1.26mmol),Pd(PPh3)4(0.072g,0.063mmol),频哪醇硼酸酯(0.54g,1.64mmol)和碳酸铯(0.82g,2.52mmol)溶于1,4-二乙烷中(5mL)和水(1mL)。用氮气鼓泡15分钟后,在微波条件下在120℃下反应30分钟。然后用乙酸乙酯萃取,合并有机相后用饱和食盐水洗三次。用无水硫酸钠干燥后柱层析得到化合物3-Boc保护苯胺喹啉化合物。将3-Boc保护苯胺喹啉化合物溶于二氯甲烷(2mL)和甲醇(2mL),加入4M的HCl的二氧六环溶液(5mL)。将反应液在室温下搅拌4小时至反应完全,减压蒸发除去溶解得到3-苯胺喹啉化合物5-g。1H NMR(400MHz,DMSO)δ9.11(d,J=2.3Hz,1H),8.33(d,J=2.2Hz,1H),8.14(s,1H),7.91(d,J=8.6Hz,1H),7.81(d,J=0.6Hz,1H),7.77(s,1H),7.74(d,J=9.6Hz,1H),7.57(dd,J=8.6,3.3Hz,1H),6.97(d,J=9.6Hz,1H),6.72(d,J=8.6Hz,1H),5.42(s,1H),4.43(t,J=7.3Hz,1H),3.85(s,2H),3.27(t,J=7.2Hz,1H).
制备实施例15荧光探针7:
圆底烧瓶中加入4a(1eq.),化合物5-g(1.3eq.),溶于1,2-二氯乙烷:甲醇(体积比)=5:1,加入氰基硼氢化钠(1.3eq.),室温反应6-8h,旋干反应溶液,粗品过柱纯化得产品。产物为棕红色固体,产率46%。1H NMR(500MHz,CDCl3)δ9.09(d,J=2.2Hz,1H),8.13(s,1H),8.05(d,J=8.6Hz,1H),7.75(s,1H),7.69(s,1H),7.61–7.56(m,2H),7.53(d,J=8.5Hz,2H),7.36(d,J=9.6Hz,1H),6.93(d,J=9.6Hz,1H),6.72(d,J=8.5Hz,2H),6.03(s,2H),4.55–4.48(m,2H),3.90(s,3H),3.40–3.30(m,4H),3.04–2.97(m,2H),2.52(s,6H),2.35(s,6H),1.97–1.88(m,2H).
制备实施例16荧光探针8:
圆底烧瓶中加入4b(1eq.),化合物5-g(1.3eq.),溶于1,2-二氯乙烷:甲醇(体积比)=5:1,加入氰基硼氢化钠(1.3eq.),室温反应6-8h,旋干反应溶液,粗品过柱纯化得产品。产物为棕红色固体,产率23%。1H NMR(500MHz,CDCl3)δ8.76(d,J=1.5Hz,1H),8.16–8.07(m,2H),7.96(d,J=1.6Hz,1H),7.81–7.73(m,2H),7.52(dd,J=7.5,1.4Hz,1H),7.31–7.25(m,2H),7.11(d,J=10.8Hz,1H),6.80–6.74(m,2H),5.40(d,J=11.0Hz,1H),3.94(s,3H),3.60(s,1H),3.39(t,J=7.1Hz,2H),3.29(t,J=7.1Hz,2H),2.79(t,J=7.1Hz,2H),2.56(q,J=8.0Hz,2H),2.16(s,3H),2.08(s,3H),2.00(q,J=7.9Hz,2H),1.97–1.87(m,5H),1.56(p,J=7.1Hz,2H),1.20(t,J=8.0Hz,3H),1.03(t,J=8.0Hz,3H),0.90(s,3H).
制备实施例17荧光探针9:
圆底烧瓶中加入4a(1eq.),化合物5-h(1.3eq.),溶于1,2-二氯乙烷:甲醇(体积比)=5:1,加入氰基硼氢化钠(1.3eq.),室温反应6-8h,旋干反应溶液,粗品过柱纯化得产品。产物为棕红色固体,产率38%。1H NMR(500MHz,CDCl3)δ9.00(s,1H),8.59(d,J=1.5Hz,1H),8.20(s,1H),8.06(dd,J=7.6,5.7Hz,1H),7.77(d,J=7.5Hz,1H),7.67–7.58(m,2H),7.42(dd,J=7.5,2.0Hz,1H),7.32(dd,J=7.5,1.4Hz,1H),6.97(t,J=1.5Hz,1H),6.86(s,1H),5.60(s,1H),5.00(d,J=1.0Hz,1H),3.82(s,2H),3.68–3.58(m,3H),2.76(s,3H),2.16(s,3H),2.08(s,3H),1.97–1.87(m,5H),1.57(q,J=7.1Hz,2H),0.90(s,3H).
制备实施例18荧光探针10:
圆底烧瓶中加入4a(1eq.),化合物5-i(1.3eq.),溶于1,2-二氯乙烷:甲醇(体积比)=5:1,加入氰基硼氢化钠(1.3eq.),室温反应6-8h,旋干反应溶液,粗品过柱纯化得产品。产物为棕红色固体,产率36%。1H NMR(500MHz,CDCl3)δ8.94(d,J=7.5Hz,1H),8.66(d,J=5.0Hz,2H),8.59(d,J=1.5Hz,1H),8.21(d,J=7.5Hz,1H),7.80(dd,J=25.1,6.3Hz,3H),7.60(t,J=1.5Hz,1H),7.32(dd,J=7.5,1.4Hz,1H),6.97(t,J=1.6Hz,1H),5.60(s,1H),5.00(d,J=1.0Hz,1H),3.68–3.58(m,3H),2.16(s,3H),2.08(s,3H),1.97–1.87(m,5H),1.57(q,J=7.1Hz,2H),0.90(s,3H).
(二)荧光及生物活性检测实施例
试验实施例1:代表性化合物的光谱性质测试
吸收光谱测试方法:荧光探针7溶于DMSO配制成10mM母液,加入乙醇稀释至5μM,采用Hitachi U-3010紫外-可见分光光度计测试荧光探针7的吸收光谱。
荧光发射光谱测试方法:荧光探针7溶于DMSO配制成10mM母液,加入乙醇稀释至5μM,采用Horiba FluoroMax 4荧光光度计测试荧光探针7的荧光光谱,激发波长为497nm,激发和发射的光栅均为5nm。
荧光淬灭效率实验:荧光探针7(制备实施例15得到)和荧光染料3a(制备实施例1得到)分别溶于DMSO配制成10mM母液,加入乙醇稀释至5μM,采用Horiba FluoroMax 4荧光光度计测试荧光探针7和3a的荧光光谱,激发波长均为497nm,激发和发射的光栅分别为0.5nm和1nm。
测试结果如图1-3所示,荧光探针7在乙醇中的最大吸收波长为497nm,最大发射波长为505nm。其荧光显著弱于荧光染料3a,表明探针的荧光被有效淬灭。
试验实施例2:代表性化合物的c-Met激酶活性
试验方法:(1)酶反应底物Poly(Glu,Tyr)4:1用无钾离子的PBS(10mM磷酸钠缓冲液,150mM NaCl,pH7.2-7.4)稀释成20μg/mL,125μL/孔包被酶标板,置37℃反应12-16小时。弃去孔中液体。洗板,用T-PBS(含0.1%Tween-20的无钾离子的PBS,200μL/孔)洗板三次,每次5分钟。于37℃烘箱中干燥酶标板1-2小时。(2)每孔加入以反应缓冲液(50mM HEPES pH7.4,50mM MgCl2,0.5mM MnCl2,0.2mM Na3VO4,1mM DTT)稀释的ATP溶液49μL,每孔中加入1μL待测试化合物,再加入50μL以反应缓冲液稀释的c-Met激酶域重组蛋白启动反应,每次实验需设无ATP对照孔两孔。置37℃摇床(100rpm)反应1小时。弃去孔中液体,T-PBS洗板三次。(3)加入抗体PY99稀释液(抗体用含BSA 5mg/mL的T-PBS 1:500稀释),100μL/孔,37℃摇床反应0.5小时。弃去孔中液体,T-PBS洗板三次。(4)加入辣根过氧化物酶标记的羊抗鼠二抗稀释液(抗体用含BSA 5mg/ml的T-PBS 1:2000稀释),100μL/孔,37℃摇床反应0.5小时。弃去孔中液体,T-PBS洗板三次。(5)加入2mg/ml的OPD显色液100μL/孔【用含有0.03%H2O2的0.1M柠檬酸-柠檬酸钠缓冲液(pH=5.4)稀释】,25℃避光反应1-10分钟。(6)加入2MH2SO450μL/孔中止反应,用可调波长式微孔板酶标仪VERSAmax读数,波长为490nm。(7)结果分析:
IC50值采用酶标仪随机附带软件以四参数法回归求得。
表1代表性实施例的激酶水平活性
荧光探针 | IC<sub>50</sub>(nM) |
1(制备实施例6) | 10-100 |
2(制备实施例7) | 1-10 |
3(制备实施例9) | 1-10 |
4(制备实施例10) | 100-1000 |
5(制备实施例12) | 10-100 |
6(制备实施例13) | 1-10 |
7(制备实施例15) | 10-100 |
8(制备实施例16) | 1-10 |
9(制备实施例17) | 1-10 |
10(制备实施例18) | 100-1000 |
测试结果显示,系列荧光探针均具有显著的c-Met抑制活性,特别是荧光探针2、3、6、8、9,激酶水平活性IC50在1-10nM,荧光探针1、5、7的激酶水平IC50在10-100nM。
试验实施例3:荧光探针7与c-Met蛋白共孵育的荧光光谱测试
实验方法:(1)荧光探针7加入DMSO配制成10-4M储液,涡旋均匀后超声10min,得到荧光探针7储液,待用。(2)c-Met蛋白溶于反应缓冲液(50mM HEPES pH 7.4,50mM MgCl2,0.5mM MnCl2,0.2mM Na3VO4,1mM DTT),加入荧光探针7储液(体系中DMSO浓度为1%),移液管搅拌混匀,室温孵育15min后测试荧光,激发波长480nm。
测试结果如图4所示,荧光探针7与不同浓度c-Met蛋白共孵育后,荧光逐渐增强,荧光强度具有良好的蛋白浓度依赖性。
试验实施例4:荧光探针7在c-Met不同表达水平细胞株MKN45和MKN74中的荧光成像
实验方法:(1)MKN45和MKN74均是胃癌细胞株,其中MKN45高表达c-Met,MKN74低表达c-Met,于玻璃底6孔板中用相应的培养基中培养,成像前倾掉培养基;(2)荧光探针7用溶剂DMSO,配置成2.5mM、5.0mM和10mM储备液;(3)PBS中加入荧光探针7储备液(体系中DMSO浓度为1%),混匀后加入到6孔板,孵育30min;(4)孵育完成后使用Leica TCS SP8STED成像,激发光波长为488nm,发射光检测波长范围为500-560nm。
结果如图5所示,c-Met高表达的MKN45细胞中具有显著的荧光,而在低表达的MKN74细胞株荧光较弱。统计显示,不同浓度如25nM、50nM和100nM的溶液中,MKN45细胞的荧光强度均显著高于MKN74的荧光强度。
Claims (9)
2.如权利要求1所述的一类如通式Ⅰ所示的BODIPY类化合物,其特征在于:
R为氢或乙基。
5.如权利要求4所述的一类如通式Ⅰ所示的BODIPY类化合物的制备方法,其特征在于:
步骤(a)中,所述Friedel–Crafts反应在50-70℃下进行,所述碱选自三乙胺或二异丙基乙胺;
步骤(b)中,所述氧化反应在0℃到室温下进行,所述氧化剂选自Dess-Martin氧化剂或三氧化硫-吡啶复合物;
步骤(c)中,所述还原胺化反应在室温条件的弱酸或者中性条件下反应,所述弱酸选自乙酸,丙酸;所述的还原剂选自氰基硼氢化钠。
6.一种药物组合物,包含权利要求1-3中任一项所述的一类如通式Ⅰ所示的BODIPY类化合物,以及药学上可接受的载体。
7.权利要求1-3中任一项所述的一类如通式Ⅰ所示的BODIPY类化合物或权利要求6所述的药物组合物在制备c-Met蛋白染色制剂中的应用。
8.权利要求1-3中任一项所述的一类如通式Ⅰ所示的BODIPY类化合物或权利要求6所述的药物组合物在制备荧光探针中的应用,所述荧光探针用于肿瘤细胞中c-Met蛋白染色成像。
9.权利要求1-3中任一项所述的一类如通式Ⅰ所示的BODIPY类化合物或权利要求6所述的药物组合物在制备治疗和/或诊断肿瘤的药物中的应用。
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