CN112574240B - Egfr特异识别的荧光化合物,其制备方法和用途 - Google Patents
Egfr特异识别的荧光化合物,其制备方法和用途 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及一类靶向酪氨酸激酶EGFR突变识别的荧光化合物,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,在癌症诊疗中具有应用价值。
以肺癌为例,临床上按组织学类型将其分为非小细胞肺癌和小细胞肺癌。其中,非小细胞肺癌的发病率约为85%,通过存在于血液、体液及组织中的生物标志物进行分子分型,可以用来诊断、监测疾病的发生发展和反应治疗效果,指导靶向药物疗效监控。肺癌中以EGFR、ALK和KRAS等的突变和重排病人比例最高。比如,在近15%的白人肺癌患者和40%的亚裔肺癌患者中检测到EGFR突变,故靶向EGFR突变的小分子抑制剂(EGFR-TKIs)成为药物研发的发展方向,也是临床治疗的重要选择。第一代EGFR-TKIs为吉非替尼、厄洛替尼和埃克替尼;第二代的EGFR不可逆抑制剂以阿法替尼、达科米替尼和来那替尼为代表。第三代EGFR-TKI奥斯替尼(AZD9291)已成为一线推荐药物。相较于前两代的EGFR-TKI,第三代抑制剂具有更低的皮肤毒性以及对EGFRT790M更高的选择性。AZD9291对于EGFRT790M/L858R的激酶具有高度的选择性。因此,对癌症病人精准的分子分型并指导靶向药物的用药选择,使患者从治疗方案中获益,避免过度治疗和降低患者经济负担,减少医疗资源的浪费,这具有重要的临床应用意义。
目前临床上常用荧光原位杂交、逆转录PCR、第二代测序技术和免疫组织化学法等技术进行检测和诊断相关基因的异常,对患者进行分子分型。但这些技术耗时长、成本高、操作复杂,这使开发实时可视化分子分型的检测技术成为需求。基于荧光成像的可视化技术可实现样品的快速检测、进一步降低成本、并提高灵敏度。同时,可视化分子探针来源于靶标的小分子抑制剂,可以更精确的定位临床用药的可行性和相关耐药,更精确的指导临床用药和监控。
发明内容
本发明的目的是提供一类具有荧光淬灭现象的新结构化合物、其制备方法及其在诊断EGFR突变的肿瘤细胞中的应用。
一方面,本发明提供一类由以下通式I表示的化合物:
其中,R、R1和R2各自独立地选自H或C1-C6直链或支链烷基,
在具体实施方式中,所述EGFR抑制剂可选自阿法替尼、来那替尼以及AZD9291,优选地,为AZD9291。
在一个具体实施方式中,上述化合物选自以下化合物:
另一方面,本发明提供了上述化合物的制备方法,其包括以下步骤:
在室温下,弱酸或中性条件下,使式1的化合物与EGFR抑制剂衍生物2在还原剂的存在下反应得到通式I的化合物,
在具体实施方式中,所述弱酸选自乙酸或丙酸,所述还原剂可以为氰基硼氢化钠。
在以上方法步骤中的化合物1可以通过本领域已知的方法制备,例如可以按照中国专利申请201510418869.2公开的路线并适当选取R1和R2对应位置的取代基来制备。
根据本发明的方法,EGFR突变抑制剂与BODIPY通过柔性烷基链连接形成了本发明具有EGFR结合特异性的荧光化合物。
另一方面,本发明提供了上述化合物在制备用于诊断或检测EGFR突变的肿瘤细胞的试剂中的用途。
在具体实施方式中,所述EGFR突变包括EGFRT790M/L858R突变。
本发明的另一个目的是上述通式所示化合物在EGFR突变的肿瘤细胞检测中的应用:将肿瘤细胞与1-1000nM通式Ⅰ所示的化合物(探针)共孵育5min后,使用荧光显微镜、酶标仪或者流式分析仪等荧光检测仪器,检测关-开型探针在肿瘤细胞中荧光强度,以反映其与EGFR蛋白(野生型与T790M突变型)激酶的特异性结合。
本发明的效果
本发明开发了一类化合物,其是能够对EGFR突变特异识别的荧光探针,其与肿瘤细胞孵育后,可特异性地与EGFR突变蛋白结合,在不清洗的操作下即可在荧光显微镜下直接观察EGFR突变蛋白表达水平和亚细胞水平定位,可应用于肿瘤细胞的分子分型的特异性检测,能够对肿瘤病人进行有效的诊断和疗效监控。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:根据本申请实施例5制备的荧光探针1在乙醇中的吸收光谱。
图2:根据本申请实施例5制备的荧光探针1在乙醇中的荧光发射光谱(激发波长:497nm)。
图3:根据本申请实施例5制备的荧光探针1和实施例1的化合物3a在PBS中的荧光发射光谱(激发波长:497nm)。
图4:根据本申请实施例5制备的荧光探针1与氧化性物种共孵育后的荧光变化(激发波长:497nm,荧光强度为506nm波长下的数据)。
图5:根据本申请实施例5制备的荧光探针1在野生型和突变型EGFR细胞中的“关-开型”荧光效应。A,左列:探针1在细胞中的分布(Ex=488nm);中间列:通过抗体识别的EGFR蛋白在细胞内的分布(Ex=552nm);右列:展示了细胞核、探针1和通过抗体识别的EGFR蛋白的融合信号图;比例尺,25μm。B,对A图右列的荧光强度用ImageJ软件定量结果;***,P<0.001。
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。吸收光谱用Hitachi U-3010紫外-可见分光光度计测试,荧光光谱用Horiba FluoroMax4荧光分光光度计测试,细胞荧光成像由Leica TCS SP8测试。EGFRWT和EGFRT790M/L858R激酶购自Eurofins公司;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。EGFR抗体(#4267)购自Cell Signaling Technology公司;Alexa Fluor555标记的抗兔二抗(A21428)和抗荧光淬灭的封片剂(P36970)购自Invitrogen公司。
制备实施例
实施例1化合物3a的合成
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).
实施例2化合物3b的合成
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%,1H NMR(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).
实施例3化合物4a的合成
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).
实施例4化合物4b的合成
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)
实施例5荧光探针1
5-2的合成:
微波管中加入原料5-1(3g,7.63mmol),溶于20ml DMA,依次加入甲基(2-(甲基氨基)乙基)氨基甲酸叔丁酯(1.72g,9.15mmol)和N,N-二异丙基乙胺(1.99g,22.8mmol),微波100℃反应4h。加入乙酸乙酯和水稀释,萃取,有机层无水硫酸钠干燥,旋干后柱层析分析,得浅棕色化合物5-2,收率77%。
5-3的合成:
化合物5-2溶于乙醇,依次加入饱和氯化铵溶液和铁粉,70℃加热反应3h。反应液中加入乙酸乙酯稀释,硅藻土过滤,滤液旋干,柱层析分离得到浅棕色化合物5-3,收率90%。
5-4的合成:
化合物5-3(1g,1.88mmol)溶于20mL无水四氢呋喃中,冰浴下依次滴加丙烯酰氯(187mg,2.07mmol),N,N-二异丙基乙胺(729mg,5.64mmol),滴加完后渐升至室温反应2小时。旋干溶剂,柱层析分离得白色固体化合物5-4 0.86g,产率78.1%。
5-5的合成:
化合物5-4(0.8g,1.37mmol)溶于10mL 1N HCl甲醇溶液,室温反应1小时,旋去溶剂后,得到5-5粗品0.64g,产率96.5%。
荧光探针1的合成:
化合物5-5(0.3g,0.62mmol)和实施例3制备的产物(282mg,0.93mmol)溶于5mL 1,2-二氯乙烷和5mL甲醇,滴加1滴乙酸,分2次加入氰基硼氢化钠(116.5mg,1.85mmol)室温反应2-3小时,旋干反应溶液,柱层析分离得到荧光探针1,棕红色固体55mg,产率11.5%。1HNMR(400MHz,CDCl3)δ9.41(s,1H),9.23(s,1H),8.63(s,1H),8.32(d,J=5.4Hz,1H),8.13(d,J=7.8Hz,1H),7.79(s,1H),7.39(d,J=7.8Hz,1H),7.33–7.21(m,2H),7.14(d,J=5.4Hz,1H),6.72(s,1H),6.46(dd,J=16.8,9.9Hz,1H),6.34(dd,J=16.9,1.6Hz,1H),5.94(s,2H),5.68(dd,J=9.9,1.7Hz,1H),3.89(s,3H),3.89(s,3H),3.14(m,2H),2.82(m,6H),2.63(s,3H),2.49(s,3H),2.46(s,6H),2.30(s,6H),1.85(m,2H).
实施例6荧光探针2
合成方法同实施例5中荧光探针1的制备方法,不同之处在于将实施例3的产物4a替换成实施例4的产物4b,即得荧光探针2,其为棕红色固体,产率15%。1H NMR(400MHz,CDCl3)δ9.85(s,1H),9.64(s,1H),9.08(s,1H),8.41(d,J=5.3Hz,1H),8.09(dd,J=6.3,2.4Hz,1H),7.76(s,1H),7.42(dd,J=6.5,2.4Hz,1H),7.34–7.26(m,2H),7.23(d,J=5.3Hz,1H),6.79(s,1H),6.43(s,1H),5.72–5.66(m,1H),3.99(s,3H),3.90(s,3H),3.02–2.90(m,4H),2.69(s,3H),2.63(d,J=8.3Hz,2H),2.48(m,8H),2.37(m,6H),2.31(s,6H),1.86(m,2H),1.02(t,J=7.5Hz,6H).
实验实施例
实验实施例1:代表性化合物的光谱性质测试
吸收光谱测试方法:将实施例5制备的荧光探针1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1的吸收光谱,结果如图1。
荧光发射光谱测试方法:将实施例5制备的荧光探针1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1的荧光光谱,结果如图2。
荧光淬灭效率实验:将实施例5制备的荧光探针1和实施例1的产物3a分别溶于DMSO配制成10mM母液,加入10mM pH=7.4的PBS溶液稀释至5μM,采用Horiba FluoroMax 4荧光光度计测试荧光探针1和实施例1的产物3a的荧光光谱,激发波长分别为均为494nm和496nm,结果如图3。
测试结果显示,荧光探针1在乙醇中的最大吸收波长为499nm,最大发射波长为506nm。荧光淬灭效果显示,在PBS中荧光探针1的荧光强度比未淬灭的实施例1中的产物3a的荧光弱220倍,具有显著的荧光淬灭特性。同时,在选择性和稳定性测试中发现,荧光探针1对过氧自由基、羟基自由基、超氧负离子、次氯酸根、过氧化氢、单线态氧、过氧化亚硝酰负离子和NO等均不具有明显的荧光响应(参见图4,对NO有约2倍荧光响应,但相对于220倍淬灭可忽略不计),表现出良好的稳定性和选择性。
实验实施例2:荧光探针1对野生型和突变型EGFR体外酪氨酸激酶活性的影响
实验方法:用酶联免疫吸附法(Enzyme-Linked Immunosorbent Assay,ELISA)检测荧光探针1对野生型EGFR(EGFRWT)和EGFRT790M/L858R激酶的抑制作用。主要步骤如下:酶反应底物Poly(Glu,Tyr)4:1稀释成2.5μg/孔,37℃反应过夜,包被酶标板。每孔加入用反应缓冲液(50mM HEPES pH 7.4,20mM MgCl2,0.1mM MnCl2,0.2mM Na3VO4,1mM DTT)稀释的ATP溶液(终浓度5μM),加入荧光探针1和阳性化合物AZD9291或溶剂对照,然后加入激酶启动反应,37℃摇床反应1h。T-PBS洗板三次,加入100μL抗体PY99(含BSA的T-PBS,1:500稀释)于37℃摇床反应0.5h。T-PBS洗板后,加入100μL辣根过氧化物酶标记的羊抗鼠的IgG(含BSA的T-PBS,1:2000稀释),37℃摇床反应0.5h。再次洗板后,加入含0.03%H2O2,2mg/mL的OPD(0.1mol/L)显色液100μL/孔,25℃避光反应1-10min。加入50μL/孔2M H2SO4终止反应,用可调波长式微孔板酶标仪(SpectraMax Plus384,Molecular Devices)读数,波长为490nm。IC50值由抑制曲线得到。
结果分析:荧光探针1对EGFRWT和EGFRT790M/L858R的体外酪氨酸激酶活性抑制能力比阳性化合物AZD9291显著降低;而且荧光探针1对EGFRT790M/L858R的体外酪氨酸激酶活性抑制能力比对EGFRWT的略强,也提示了荧光探针1对EGFR驱动型突变(T790M)的选择性(表1)。
表1.荧光探针1与AZD9291对不同酪氨酸激酶的抑制IC50(nmol/L)
实验实施例3:荧光探针1在野生型和突变型EGFR细胞中的“关-开型”荧光效应
实验方法:将H1975、A549及PC9细胞接种于带有盖玻片的12孔细胞培养板中,待培养至细胞贴壁后,每孔加入50nmol/L的实施例5制备的荧光探针1孵育5min后,立即用新鲜PBS轻柔清洗细胞三次后,使用4%多聚甲醛固定细胞,15%BSA封闭液封闭细胞,4℃孵育EGFR抗体(1:50)过夜,在室温孵育荧光染料Alexa Fluor 555标记的抗兔二抗(1:200)2h,再用DAPI染料对细胞核染色后,最后用抗荧光淬灭的封片剂封片。细胞样品使用激光共聚焦显微镜采集图像。
结果分析:相较于A549细胞(EGFRWT)与PC9细胞(EGFRDel19),H1975细胞(EGFRT790M /L858R)荧光强度最大(图5),而且荧光信号聚集在细胞膜附近,并与EGFR抗体所染色区域重叠程度较高。这提示了荧光探针1可与EGFR的结合,改变构象解除了对荧光基团的位阻效应,产生荧光;而且相较于EGFRWT和EGFRDel19,探针1可高选择性地与EGFRT790M/L858R结合。这些数据提示了荧光探针1可用于基于EGFR激酶活性的分子分型,从而为指导EGFR靶向治疗的用药策略研究提供新的研究思路和有力工具。
Claims (6)
4.根据权利要求3所述的方法,其中,所述还原剂为氰基硼氢化钠。
5.如权利要求1或2所述的化合物在制备用于诊断或检测EGFR突变的肿瘤细胞的试剂中的用途。
6.根据权利要求5所述的用途,其中,所述EGFR突变包括EGFRT790M/L858R突变。
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---|
"Discovery of Nonpeptide,Reversible HER1/HER2 Dual-Targeting Small-Molecule Inhibitors as Near-Infrared Fluorescent Probes for Efficient Tumor Detection, Diagnostic Imaging, and Drug Screening";Shengnan Liu等;《Anal.Chem.》;20181221;第91卷;第1507-1515页 * |
Shengnan Liu等."Discovery of Nonpeptide,Reversible HER1/HER2 Dual-Targeting Small-Molecule Inhibitors as Near-Infrared Fluorescent Probes for Efficient Tumor Detection, Diagnostic Imaging, and Drug Screening".《Anal.Chem.》.2018,第91卷第1507-1515页. * |
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