CN107474038B - 一类靶向雌激素受体荧光探针及其制备和使用方法 - Google Patents

一类靶向雌激素受体荧光探针及其制备和使用方法 Download PDF

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CN107474038B
CN107474038B CN201710580544.3A CN201710580544A CN107474038B CN 107474038 B CN107474038 B CN 107474038B CN 201710580544 A CN201710580544 A CN 201710580544A CN 107474038 B CN107474038 B CN 107474038B
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董春娥
杨录
胡志烨
刘现军
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Abstract

本发明属于生物、医药技术领域，公开了一类靶向雌激素受体荧光探针及其制备和使用方法。本发明的荧光探针对雌激素受体具有高亲和力且对雌激素受体有较强的激动或拮抗活性，有潜在的癌症治疗功效。共聚焦荧光成像测试结果表明该类荧光探针选择性对表达雌激素受体相关癌症细胞成像，并且对雌激素受体在细胞中表达进行定位，为相关癌症的早期诊断、治疗及治预后评价提供更为详细、精准的信息。考虑该类荧光探针同时具有诊断与治疗功效，且可用于活细胞检测，成本低、相关操作简便及快速，具有开发和应用价值。

Description

一类靶向雌激素受体荧光探针及其制备和使用方法
技术领域
本发明属于生物、医药技术领域，涉及一类具有癌症诊断和治疗功能的靶向雌激素受体荧光探针及其制备和使用方法。
背景技术
雌激素受体属于核受体，通过与雌激素配体，如雌二醇结合发挥激素作用。当雌激素信号通路发生异常时，常常会引起多种疾病，如子宫内膜癌、卵巢癌、骨质疏松、动脉硬化、阿尔茨海默氏病等，尤其目前70％以上的乳腺癌与雌激素受体过表达有关。
目前在临床上治疗乳腺癌主要是采用激素替代治疗法，如一线药物他莫昔芬、雷洛昔芬和拉索昔芬等主要是通过调节雌激素受体介导的信号通路来实现乳腺癌的治疗。但是这些药物常存在一定副作用，以他莫昔芬为例，长期使用会产生耐药性以及增加患子宫内膜癌的风险。通过研究雌激素受体在体内的表达以实现疾病的早期诊断和治疗对患者的病情控制有着重要的意义。
18F-FES(18氟-氟雌二醇)是一种基于正电子发射型计算机断层成像技术的雌激素受体放射性示踪剂，可以对雌激素受体在体内的表达进行定量。临床上使用18F-FES需另外考虑绝经期及相关的干预治疗影响肿瘤组织对其的吸收。雌激素受体表达不仅作为相关肿瘤诊断、预后及治疗过程中的重要指标，同时雌激素受体的表达程度与肿瘤的恶性程度相关。虽然18F-FES目前已能用于检测雌激素受体的表达，但作为一种放射性技术，往往存在副作用，同时不能实时动态的检测治疗过程中雌激素受体的表达情况，此外，其分辨率有待提高，还未能运用于手术导航，指导临床肿瘤切除。因此，靶向雌激素受体荧光探针正好弥补18F-FES以上不足，在可视化治疗、个性化治疗及手术导航等领域可提供更为精准的信息，用于患者病情监测和疗效评价。于此，构建了一类靶向雌激素受体荧光探针，实现对活细胞中雌激素受体表达进行定位，同时作为新颖的雌激素受体调节剂，这类探针可望运用于相关疾病的体内成像及相关领域。
发明内容
本发明所要解决的技术问题是提供一类具有癌症诊断和治疗功能的靶向雌激素受体荧光探针及其制备和使用方法。
本发明所提供的一类靶向雌激素受体荧光探针，具有以下通式所示的结构：
Figure BDA0001352160740000021
其中，
R1为H、OH、或B(OH)2；
R2为H、2-Me、2-Et、2-F、2-Cl，2-CF3、2-CF2H、2-Br、2-OCF3、3-Me、3-Et、3-F、3-Cl，3-CF3、3-CF2H、3-Br、3-OCF3、2,5-Me、2,5-Et、2,5-F、2,5-Cl，2,5-CF3、2,5-CF2H、2,5-Br或2,5-OCF3；
R3为H、Me、Et、Br、Cl或CN；
R4为H、CN、COOH或
Figure BDA0001352160740000022
R5为H、CN、COOH、COOEt、COOMe、COO(CH2CH2O)nCH2CH2OMe、COO(CH2CH2O)nCH2CH2OEt或COO(CH2CH2O)nCH2CH2OH；
X为O、S或Se；
结构式中n取值为1～5。
通过细胞成像和体外乳腺癌活性实验，发现上述靶向雌激素受体荧光探针兼具诊断和治疗功能。
作为本发明进一步改进的技术方案，所述靶向雌激素受体荧光探针为以下化合物：
2-(4-(2,5-双(2-氟-4-羟基苯基)噻吩-3)苯亚甲基)丙二腈(7a)；或
3-(4-(2,5-双(2-氟-4-羟基苯基)噻吩-3-基)苯基)-2-氰基丙烯酸乙酯(7b)；或
2-(2-(2-(2-甲氧基乙氧基)乙氧基)乙基)-3-(4-(2,5-双(2-氟-4-羟基苯基)噻吩-3-基)苯基)-2-氰基丙烯酸(7c)；或
2-(4-(2,5-双(2-氟-4-羟基苯基)硒吩-3-基)苯亚甲基)丙二腈(8a)；或
3-(4-(2,5-双(2-氟-4-羟基苯基)硒吩-3-基)苯基)-2-氰基丙烯酸乙酯(8b)；或
2-(2-(2-(2-甲氧基)乙氧基)乙氧基)乙基)-3-(4-(2,5-双(2-氟-4-羟苯基)硒吩-3-基)苯基)-2-氰基丙烯酸(8c)；或
2-((2,5-双(2-氟-4-羟基苯基)噻吩-3-基)亚甲基)丙二腈(11a)；或
3-(2,5-双(2-氟-4-羟基苯基)噻吩-3-基)-2-氰基丙烯酸乙酯(11b)；或
2-(2-(2-(2-甲氧基)乙氧基)乙氧基)乙基(E)-3-(2,5-双(2-氟-4-羟基苯基)噻吩-3-基)-2-氰基丙烯酸酯(11c)；或
2-(4-(2-(2,5-双(2-氟-4-羟基苯基)噻吩-3-基)乙烯基)-3-氰基-5,5-二甲基-2(5H)-亚甲基)丙二腈(11d)；或
2-((3-(3-氟-4-羟基苯基)-7-羟基萘-1-基)亚甲基)丙二腈(14a)；或
2-氰基-3-(3-(3-氟-4-羟基苯基)-7-羟基萘-1-基)丙烯酸(14b)；或
2-氰基-3-(3-(3-氟-4-羟基苯基)-7-羟基萘-1-基)丙烯酸乙酯(14c)；或
2-(2-(2-(2-甲氧基)乙氧基)乙氧基)乙基-2-氰基-3-(3-(3-氟-4-羟基苯基)-7-羟基萘-1-基)丙烯酸乙酯(14d)。
所述靶向雌激素受体荧光探针是一类电子给体-受体结构，能够以特异性地高亲和力与雌激素受体结合，有效的激动或拮抗雌激素受体，影响雌激素信号通路，遏制相关肿瘤细胞的生长，用于癌症的治疗。
雌激素受体主要分布于细胞核和细胞浆中，此外在细胞膜上也有少量分布。共聚焦荧光成像结果表明靶向雌激素受体荧光探针与细胞作用后，在细胞核与细胞质中均有分布，定性表明了靶向雌激素受体荧光探针分子能够渗透细胞膜进入细胞与雌激素受体结合。
本发明还提供了一类靶向雌激素受体荧光探针的制备方法，包括以下步骤：
(a)在以1,4-二氧六环和甲苯为混合溶剂的有机溶剂体系中，以磷酸三钾和四丁基溴化
铵为助催化剂促进Suzuki反应；
(b)以二氯甲烷为溶剂、三溴化硼为脱甲基试剂完成脱甲基反应；
(c)以哌啶为催化剂完成Knoevenagel反应。
具体合成路线如下：
Figure BDA0001352160740000041
本发明还提供了一类靶向雌激素受体荧光探针的使用方法，用于制备诊断肿瘤药物和治疗肿瘤药物。
作为本发明进一步改进的技术方案，所述诊断肿瘤药物和治疗肿瘤药物还包括一种或多种药学上可接受的载体或赋形剂。
作为本发明所述的靶向雌激素受体荧光探针在乳腺癌诊断和治疗中的应用的一种优选方案，其中乳腺癌诊断通过相对定量检测细胞中雌激素受体含量进行，具体步骤如下：(1)荧光探针与雌激素受体阳性乳腺癌细胞作用一段时间，直至荧光强度稳定；(2)使用流式细胞仪检测探针与细胞作用之后荧光强度增强倍数；(3)通过分子生物学手段测定细胞中雌激素受体含量；(4)根据荧光强度增强倍数与细胞中雌激素受体含量的相关线性关系，对细胞中雌激素受体含量进行相对定量测定。
作为优选方案，所述荧光探针与细胞共同孵育时间为0.5～5h，荧光探针浓度为0.1～10μM。
本发明的靶向雌激素受体荧光探针能够用于肿瘤细胞成像，具有较好的荧光量子产率，部分探针荧光光谱红移至近红外区(大于650nm)，可望用于临床肿瘤组织诊断与治疗的可视化研究。
附图说明
图1是靶向雌激素受体荧光探针对MCF-7细胞的荧光共聚焦成像；
图2是靶向雌激素受体荧光探针对U-87细胞的荧光共聚焦成像。
具体实施案例
实施例1：3-溴-2,5-双(2-氟-4-甲氧基苯基)噻吩(1)的制备
称取2,3,5-三溴噻吩(0.32g,1mmol)和对甲氧基苯硼酸(0.33g,2.2mmol)于50mL的圆底烧瓶中，加入磷酸三钾(0.64g,3mmol)、四(三苯基膦)钯(0.0058g,0.005mmol)、四丁基溴化铵(0.032g,0.1mmol)、5mL二氧六环、甲苯和5mL水，于110℃反应24h，TLC监测反应完全，反应结束之后用50mL乙酸乙酯萃取三次，有机层用无水硫酸钠干燥，过滤旋干得粗品，经柱层析纯化后得黄色固体产物，产率为78％。1H NMR(400MHz,CDCl3)δ7.46(dt,J＝12.9,8.9Hz,2H),7.32(s,1H),6.82-6.67(m,4H),3.85(s,3H),3.82(s,3H).13C NMR(101MHz,CDCl3)δ163.89(d,J＝248.0Hz),163.05(d,J＝247.9Hz),158.39(d,J＝10.1Hz),156.83(d,J＝9.7Hz),138.57,133.07(d,J＝10.1Hz),132.99,128.85(d,J＝10.0Hz),127.87,118.30(d,J＝3.4Hz),117.48(d,J＝3.3Hz),109.99,107.56(d,J＝21.7Hz),106.93(d,J＝21.6Hz),99.66(d,J＝25.9Hz,99.54(d,J＝25.9Hz),99.73(d,J＝11.4Hz),99.47(d,J＝11.4Hz),55.81,55.72.
实施例2：3-溴-2,5-双(2-氟-4-甲氧基苯基)硒吩(2)的制备
参照化合物1的制备方法，黄色固体产物，产率为81％。1H NMR(400MHz,CDCl3)δ7.51-7.28(m,3H),6.73-6.54(m,4H),3.73(s,3H),3.71(s,3H).
实施例3：3-溴-2,5-双(2-氟-4-羟基苯基)噻吩(3)的制备
称取化合物1(0.41g,1mmol)于50mL的圆底烧瓶中，加入5mL二氯甲烷，于0℃缓慢加入三溴化硼(0.75g,3mmol),保持该反应温度反应8h，TLC监测反应完全，反应结束之后加入冰水淬灭反应，用50mL乙酸乙酯萃取三次，有机层用无水硫酸钠干燥，过滤旋干得粗品，经柱层析纯化后得白色固体产物，产率为88％。1H NMR(400MHz,acetone-d6)δ9.24(s,2H),7.52(t,J＝8.8Hz,1H),7.41-7.29(m,2H),6.88-6.67(m,4H).13C NMR(101MHz,Acetone)δ160.79(d,J＝247.7Hz,1H),160.15(d,J＝248.4Hz,1H),160.32(d,J＝11.6Hz,1H),159.54(d,J＝12.0Hz,1H),138.21(d,J＝3.4Hz,1H),133.24(d,J＝4.2Hz,1H),131.31(d,J＝4.8Hz,1H),129.47(d,J＝5.0Hz,1H),127.86(d,J＝5.4Hz,1H),112.85(d,J＝2.7Hz,1H),112.47(d,J＝13.1Hz,1H),112.16(d,J＝2.9Hz,1H),111.36(d,J＝15.4Hz,0H),110.43(d,J＝1.4Hz,0H),103.81(d,J＝23.4Hz,1H),103.57(d,J＝23.2Hz,1H).
实施例4：3-溴-2,5-双(2-氟-4-羟基苯基)硒吩(4)的制备
称取化合物2(0.46g,1mmol)于50mL的圆底烧瓶中，加入5mL二氯甲烷，于0℃缓慢加入三溴化硼(0.75g,3mmol),保持该反应温度反应8h，TLC监测反应完全，反应结束之后加入冰水淬灭反应，用50mL乙酸乙酯萃取三次，有机层用无水硫酸钠干燥，过滤旋干得粗品，经柱层析纯化后得白色固体产物，产率为88％。1H NMR(400MHz,acetone-d6)δ9.28(s,2H),7.59(m,,2H),7.37(t,J＝8.6Hz,1H),6.86-6.69(m,4H).13C NMR(101MHz,acetone-d6)δ160.33(d,J＝247.5Hz),160.01(d,J＝245.2Hz),160.18(d,J＝11.6Hz,1H),159.59(d,J＝12.1Hz,1H),142.65(d,J＝2.3Hz,1H),133.04(d,J＝4.0Hz,2H),129.44(d,J＝3.8Hz,1H),129.31(d,J＝5.0Hz,1H),114.35(d,J＝12.6Hz,1H),113.48(d,J＝15.3Hz,1H),112.97(d,J＝2.6Hz,1H),112.27(d,J＝2.8Hz,0H),112.13(d,J＝2.9Hz,1H),110.85(d,J＝1.5Hz,0H),103.77(d,J＝12.9Hz,1H),103.52(d,J＝12.5Hz).
实施例5：4-(2,5-双(2-氟-4-羟基苯基)噻吩-3-)苯甲醛(5)的制备
参照化合物1的制备方法，黄色固体产物，产率为65％。1H NMR(400MHz,acetone-d6)δ9.98(s,1H),9.18(s,2H),7.83(d,J＝8.3Hz,2H),7.69-7.48(m,4H),7.22(t,J＝8.6Hz,1H),6.92–6.51(m,4H).13C NMR(101MHz,acetone-d6)δ191.53,160.15(d,J＝247.1Hz),159.61(d,J＝248.2Hz),159.45(d,J＝11.5Hz),158.64(d,J＝11.9Hz),142.55,138.57,136.98(d,J＝3.4Hz),135.01,132.78(d,J＝4.3Hz),131.95(d,J＝4.2Hz),129.51,129.14(d,J＝5.2Hz),128.56,126.24(d,J＝4.7Hz),112.74(d,J＝13.2Hz),112.28(d,J＝2.6Hz),112.07(d,J＝15.7Hz),111.93(d,J＝2.9Hz),103.43(d,J＝4.9HzH),103.18(d,J＝4.7Hz).
实施例6：4-(2,5-双(2-氟-4-羟基苯基)硒吩-3)苯甲醛(6)的制备
参照化合物1的制备方法，黄色固体产物，产率为76％。1H NMR(400MHz,acetone-d6)δ10.00(s,1H),9.26(s,2H),7.89-7.79(m,3H),7.70(t,J＝8.9Hz,1H),7.52(d,J＝8.2Hz,2H),7.18(t,J＝8.6Hz,1H),6.83-6.64(m,3H),6.59(m,1H).13C NMR(101MHz,acetone-d6)δ192.00,160.05(d,J＝246.8Hz),159.88(d,J＝247.7Hz),159.73(d,J＝11.5Hz),159.17(d,J＝12.0Hz),144.29,141.87(d,J＝2.9Hz),141.06,138.23(d,J＝7.2Hz),135.50,133.13(d,J＝4.4Hz),129.92,129.44(d,J＝5.1Hz),129.36,128.96(d,J＝3.2Hz),115.19(d,J＝12.9Hz),114.62(d,J＝15.6Hz),112.81(d,J＝2.6Hz),112.32(d,J＝2.9Hz),103.77(d,J＝4.1Hz),103.52(d,J＝4.5Hz).
实施例7：2-(4-(2,5-双(2-氟-4-羟基苯基)噻吩-3)苯亚甲基)丙二腈(7a)的制备
称取化合物5(0.048g,0.1mmol)和丙二腈(0.007g,0.1mmol)于10mL的圆底烧瓶中，加入吡啶(0.0085g,0.1mmol)、2mL无水乙醇，于910℃反应24h，TLC监测反应完全，反应结束之后用50mL乙酸乙酯萃取三次，有机层用无水硫酸钠干燥，过滤旋干得粗品，经柱层析纯化后得红色固体产物，产率为88％。熔点(mp 232-233℃)。1H NMR(400MHz,acetone-d6)δ9.30(s,2H),8.21(s,1H),7.95(d,J＝8.4Hz,2H),7.72–7.47(m,4H),7.24(t,J＝8.6Hz,1H),6.86–6.70(m,3H),6.63(m,1H).13C NMR(101MHz,acetone-d6)δ160.29(d,J＝247.0Hz),159.97,159.78(d,J＝248.1Hz),159.83(d,J＝11.5Hz),158.97(d,J＝11.9Hz),142.76,138.22,137.31(d,J＝3.3Hz),132.93(d,J＝4.3Hz),132.75(d,J＝4.1Hz),131.11,130.04,129.29(d,J＝5.2Hz),128.92,126.20(d,J＝4.6Hz),114.27,113.31,112.73(d,J＝13.2Hz),112.49(d,J＝2.6Hz),112.21(d,J＝2.8Hz),112.04(d,J＝15.7Hz),103.48(d,J＝24.8Hz),81.16.19F NMR(376MHz,acetone-d6)δ-111.93,-112.96.
实施例8：3-(4-(2,5-双(2-氟-4-羟基苯基)噻吩-3-基)苯基)-2-氰基丙烯酸乙酯(7b)的制备
参照化合物7a的制备方法，黄色固体产物，产率为91％。熔点(mp 249-250℃)。1HNMR(400MHz,acetone-d6)δ9.22(s,2H),8.30(s,1H),8.05(d,J＝8.4Hz,2H),7.69(t,J＝8.9Hz,1H),7.64(s,1H),7.56(d,J＝8.4Hz,2H),7.27(t,J＝8.6Hz,1H),6.89-6.72(m,3H),6.64(dd,J＝11.6,2.4Hz,1H),4.35(q,J＝7.1Hz,2H),1.35(t,J＝7.1Hz,3H).13C NMR(101MHz,acetone-d6)δ162.30,160.40(d,J＝246.9Hz),159.84(d,J＝248.0Hz),159.91(d,J＝11.5Hz),159.07(d,J＝11.9Hz),154.09,141.75,138.54,137.24(d,J＝3.4Hz),133.01(d,J＝4.3Hz),132.43,131.39,130.33,129.37(d,J＝5.2Hz),128.85,126.34(d,J＝4.4Hz),115.64,112.79(d,J＝13.2Hz),112.54(d,J＝2.6Hz),112.25,112.21,112.05,103.50(d,J＝24.8Hz),102.46,62.43,13.70.
实施例9：2-(2-(2-(2-甲氧基乙氧基)乙氧基)乙基)-3-(4-(2,5-双(2-氟-4-羟基苯基)噻吩-3-基)苯基)-2-氰基丙烯酸(7c)的制备
参照化合物7a的制备方法，黄色油状液体，产率为82％。1H NMR(400MHz,acetone-d6)δ9.47(s,2H),8.31(s,1H),8.05(s,1H),8.03(s,1H),7.73–7.69(m,1H),7.66–7.62(m,1H),7.58(m,2H),7.25(t,J＝8.6Hz,1H),6.84-6.71(m,3H),6.64(m,1H),4.44(dd,J＝5.4,3.9Hz,2H),3.80(dd,J＝5.5,3.9Hz,2H),3.64(dd,J＝4.4,1.8Hz,2H),3.61-3.54(m,4H),3.45(dd,J＝5.7,3.9Hz,2H),3.25(s,3H).13C NMR(101MHz,acetone-d6)δ162.53,160.59(d,J＝249.0Hz).160.01(d,J＝247.9Hz).160.10(d,J＝11.5Hz),159.27(d,J＝11.4Hz).141.98 138.71137.42(d,J＝3.3Hz),133.16(d,J＝4.4Hz),132.58(d,J＝2.7Hz),132.17(d,J＝9.9Hz),131.61,130.50,129.54(d,J＝5.1Hz),129.14,129.03(d,J＝1.9Hz),126.50(d,J＝4.4Hz),115.75,112.91(d,J＝13.3Hz),112.69(d,J＝2.7Hz),112.38(d,J＝2.8Hz),112.24(d,J＝15.7Hz),103.63(d,J＝24.8Hz),102.51,72.06,70.79,70.64,70.51,68.78,65.87,58.24.19F NMR(376MHz,acetone-d6)δ-112.09,-113.22.
实施例10：2-(4-(2,5-双(2-氟-4-羟基苯基)硒吩-3-基)苯亚甲基)丙二腈(8a)的制备
参照化合物7a的制备方法，红色固体产物，产率为88％。熔点(mp 200-202℃)。1HNMR(400MHz,acetone-d6)δ9.36(s,2H),8.25(s,1H),8.06–7.89(m,2H),7.85(s,1H),7.71(t,J＝8.9Hz,1H),7.61–7.50(m,2H),7.21(t,J＝8.6Hz,1H),6.84–6.65(m,3H),6.60(dd,J＝11.7,2.4Hz,1H).13C NMR(101MHz,acetone-d6)δ160.22,159.85(d,J＝246.9Hz),159.76(d,J＝11.6Hz),159.75(d,J＝247.8Hz)159.13(d,J＝12.0Hz),144.24,141.90(d,J＝3.0Hz),140.41,132.99(d,J＝4.2Hz),131.24,130.16,129.44,129.27(d,J＝5.1Hz),128.58(d,J＝3.1Hz),114.93(d,J＝12.9Hz),114.46,114.35(d,J＝15.6Hz),113.51,112.72(d,J＝2.6Hz),112.31(d,J＝2.9Hz),103.61(d,J＝24.8Hz),103.50(d,J＝25.2Hz),81.34.19F NMR(376MHz,acetone-d6)δ-111.65,-113.68.
实施例11：3-(4-(2,5-双(2-氟-4-羟基苯基)硒吩-3-基)苯基)-2-氰基丙烯酸乙酯(8b)的制备
参照化合物7a的制备方法，黄色固体产物，产率为84％。熔点(mp 255-256℃)。1HNMR(400MHz,acetone-d6)δ9.22(s,1H),9.19(s,1H),8.30(s,1H),8.04(d,J＝8.4Hz,2H),7.85(s,1H),7.73(t,J＝8.9Hz,1H),7.54(d,J＝8.4Hz,2H),7.22(t,J＝8.6Hz,1H),6.83-6.67(m,3H),6.60(dd,J＝11.7,2.3Hz,1H),4.35(q,J＝7.1Hz,2H),1.35(t,J＝7.1Hz,3H).13C NMR(101MHz,acetone-d6)δ162.43,159.90(d,J＝246.8Hz),159.84(d,J＝247.6Hz),159.74(d,J＝11.6Hz),159.14(d,J＝12.0Hz),154.24,143.16,141.73(d,J＝3.0Hz),140.64,138.37(d,J＝7.3Hz),134.20,132.99(d,J＝4.2Hz),131.46,130.36,129.30,129.23,128.69(d,J＝3.0Hz),116.61,115.78,114.95(d,J＝12.9Hz),114.40(d,J＝15.7Hz),112.70(d,J＝2.6Hz),112.26(d,J＝2.8Hz),103.56(d,J＝24.8Hz),103.47(d,J＝25.2Hz),102.56,62.54,13.82.
实施例12：2-(2-(2-(2-甲氧基)乙氧基)乙氧基)乙基)-3-(4-(2,5-双(2-氟-4-羟苯基)硒吩-3-基)苯基)-2-氰基丙烯酸(8c)的制备
参照化合物7a的制备方法，黄色油状液体，产率为71％。1H NMR(400MHz,acetone-d6)δ8.31(s,1H),8.04(s,1H),8.02(s,1H),7.84(s,1H),7.71(t,J＝8.9Hz,2H),7.55(s,1H),7.52(s,1H),7.20(t,J＝8.6Hz,1H),6.85-6.67(m,3H),6.60(dd,J＝11.7,2.3Hz,1H),4.49-4.41(m,2H),3.83-3.77(m,2H),3.65(m,2H),3.61-3.54(m,4H),3.45(m,2H),3.25(s,3H).13C NMR(101MHz,acetone-d6)δ162.23,159.63(d,J＝246.7Hz),159.46(d,J＝247.5Hz),159.39(d,J＝11.6Hz),158.80(d,J＝12.3Hz),154.20,142.95,141.46(d,J＝3.0Hz),140.37,138.12(d,J＝7.9Hz),132.71(d,J＝4.3Hz),131.86(d,J＝10.0Hz),131.23,130.08,129.04,128.96,128.79(d,J＝12.2Hz),128.41(d,J＝2.9Hz),115.45,114.66(d,J＝12.9Hz),114.10(d,J＝15.7Hz),112.39,111.95,103.23(d,J＝25.0Hz),103.14(d,J＝25.3Hz),102.14,71.75,70.48,70.32,70.20,68.47,65.55,57.93.
实施例13：2,5-双(2-氟-4-甲氧基苯基)噻吩-3-甲醛(9)的制备
参照化合物1的制备方法，绿色固体，产率为79％。1H NMR(400MHz,CDCl3)δ9.74(s,1H),7.75(s,1H),7.54(t,J＝8.7Hz,1H),7.38(t,J＝8.4Hz,1H),6.87–6.68(m,4H),3.87(s,3H),3.84(s,3H).13C NMR(101MHz,CDCl3)δ185.57(d,J＝3.2Hz),162.10(d,J＝10.8Hz),160.74(d,J＝11.2Hz),160.10(d,J＝249.1Hz),159.79(d,J＝250.5Hz),146.79(d,J＝4.7Hz),138.04,132.84(d,J＝3.7Hz),132.09,131.99,131.91(d,J＝2.5Hz),129.04(d,J＝5.0Hz),128.52,128.40,122.90(d,J＝4.8Hz),110.74(d,J＝2.9Hz),110.58(d,J＝3.0Hz),55.76,55.66.
实施例14：2,5-双(2-氟-4-羟基苯基)噻吩-3-甲醛(10)的制备
参照化合物3的制备方法，白色固体，产率为83％。1H NMR(400MHz,acetone-d6)δ9.74(s,1H),9.41(s,1H),9.23(s,1H),7.69(s,1H),7.64(t,J＝8.9Hz,1H),7.45(t,J＝8.6Hz,1H),7.03–6.47(m,4H).13C NMR(101MHz,acetone-d6)δ185.15(d,J＝3.3Hz),160.61(d,J＝246.4Hz),160.27(d,J＝248.2Hz),161.08(d,J＝11.8Hz),159.74(d,J＝11.6Hz),146.75(d,J＝4.8Hz),138.59,138.02(d,J＝3.4Hz),133.63(d,J＝3.7Hz),129.90(d,J＝5.1Hz),122.73(d,J＝4.9Hz),112.94(d,J＝2.1Hz),112.78(d,J＝2.8Hz),112.44(d,J＝13.3Hz),110.05(d,J＝15.3Hz),103.82(d,J＝24.0Hz).
实施例15：2-((2,5-双(2-氟-4-羟基苯基)噻吩-3-基)亚甲基)丙二腈(11a)的制备
参照化合物7a的制备方法，黄色固体，产率为85％。熔点(mp 262-264℃)。1H NMR(400MHz,acetone-d6)δ9.49(s,2H),8.21(s,1H),7.81(d,J＝2.0Hz,1H),7.57(t,J＝8.8Hz,1H),7.36(t,J＝8.6Hz,1H),6.82(dddd,J＝19.1,15.4,10.6,2.3Hz,4H).13C NMR(101MHz,acetone-d6)δ161.40(d,J＝11.9Hz),160.33(d,J＝247.6Hz),160.09(d,J＝248.8Hz),159.93(d,J＝12.1Hz),152.08(d,J＝2.1Hz),147.96(d,J＝3.6Hz),138.98(d,J＝3.3Hz),133.64(d,J＝3.7Hz),131.52,129.47(d,J＝4.9Hz),121.61(d,J＝6.1Hz),114.39,113.60,112.82(d,J＝2.7Hz),112.70(d,J＝2.8Hz),111.56(d,J＝13.0Hz),109.58(d,J＝15.0Hz),103.82(d,J＝4.5Hz),103.57(d,J＝4.6Hz),80.10.19F NMR(376MHz,acetone-d6)δ-112.99.
实施例16：3-(2,5-双(2-氟-4-羟基苯基)噻吩-3-基)-2-氰基丙烯酸乙酯(11b)的制备
参照化合物7a的制备方法，黄色固体，产率为86％。熔点(mp 267-268℃)。1H NMR(400MHz,acetone-d6)δ9.42(s,2H),8.35(s,1H),7.99(d,J＝2.2Hz,1H),7.58(t,J＝8.8Hz,1H),7.35(t,J＝8.6Hz,1H),6.97-6.73(m,4H),4.30(q,J＝7.1Hz,2H),1.31(t,J＝7.1Hz,6H).13C NMR(101MHz,acetone-d6)δ162.92(s,1H),160.68(d,J＝247.6Hz,1H),160.41(d,J＝248.8Hz,1H),161.40(d,J＝11.8Hz,1H),159.96(d,J＝12.0Hz,1H),146.97(d,J＝4.1Hz,1H),146.72(d,J＝2.0Hz,2H),138.85(d,J＝3.3Hz,2H),133.99(d,J＝3.7Hz,2H),132.10(s,2H),129.85(d,J＝5.0Hz,3H),122.75(d,J＝5.7Hz,3H),116.06(s,2H),113.10(d,J＝2.5Hz,1H),112.93(d,J＝2.9Hz,1H),112.37(d,J＝13.1Hz,1H),110.45(d,J＝15.0Hz,1H),104.12(d,J＝4.5Hz,1H),103.87(d,J＝4.5Hz,2H),101.63(s,1H),62.67(s,11H),14.02(s,9H).
实施例17：2-(2-(2-(2-甲氧基)乙氧基)乙氧基)乙基(E)-3-(2,5-双(2-氟-4-羟基苯基)噻吩-3-基)-2-氰基丙烯酸酯(11c)的制备
参照化合物7a的制备方法，黄色固体体，产率为82％。熔点(mp 121-123℃)。1HNMR(400MHz,acetone-d6)δ9.44(s,2H),8.37(s,1H),8.01(d,J＝2.0Hz,1H),7.60(t,J＝8.8Hz,1H),7.37(t,J＝8.6Hz,1H),6.98-6.70(m,4H),4.48-4.30(m,2H),3.80-3.73(m,2H),3.64-3.59(m,2H),3.59-3.54(m,4H),3.46(m,2H),3.27(s,3H).13C NMR(101MHz,acetone-d6)δ163.03,161.48(d,J＝11.7Hz),160.73(d,J＝247.4Hz),160.46(d,J＝248.6Hz),160.03(d,J＝12.1Hz),147.18(d,J＝3.4Hz),146.90(d,J＝2.0Hz),138.90,134.06(d,J＝3.7Hz),132.15,129.91(d,J＝5.0Hz),122.77(d,J＝5.8Hz),116.05,113.16(d,J＝2.4Hz),113.01(d,J＝2.8Hz),112.38(d,J＝12.9Hz),110.43(d,J＝15.1Hz),104.17(d,J＝2.1Hz),103.92(d,J＝2.1Hz),101.56,72.30,71.04,70.92,70.73,69.04,66.14,58.45.19F NMR(376MHz,acetone-d6)δ-112.92,-113.05.
实施例18：2-(4-(2-(2,5-双(2-氟-4-羟基苯基)噻吩-3-基)乙烯基)-3-氰基-5,5-二甲基-2(5H)-亚甲基)丙二腈(11d)的制备
参照化合物7a的制备方法，褐色固体，产率为57％。熔点(mp 188-190℃)。1H NMR(400MHz,acetone-d6)δ9.61(s,1H),9.46(s,1H),8.04(s,1H),7.82(m,1H),7.66(m,1H),7.38(d,J＝8.5Hz,1H),7.21(d,J＝16.2Hz,1H),6.87-6.76(m,4H),1.78(s,3H),1.71(s,3H).13C NMR(101MHz,acetone-d6)δ185.17,177.28,177.12,175.70,161.01(d,J＝11.8Hz),160.36(d,J＝247.1Hz),160.18(d,J＝248.2Hz),159.80(d,J＝12.0Hz),143.37(d,J＝4.8Hz),140.18(d,J＝2.1Hz),138.52(d,J＝3.4Hz),135.52,133.47(d,J＝4.0Hz),129.85(d,J＝5.0Hz),122.31(d,J＝3.9Hz),115.71,112.92(d,J＝3.1Hz),112.88(d,J＝2.9Hz),112.59,112.36(d,J＝13.2Hz),111.92(d,J＝19.2Hz),111.29(d,J＝20.5Hz),110.71(d,J＝15.2Hz),109.86,104.01(d,J＝24.9Hz),103.73(d,J＝24.8Hz),101.25,99.07),98.30,55.57,25.18,23.49,13.95.
实施例19：3-(3-氟-4-甲氧基苯基)-7-甲氧基-1-萘醛(12)的制备
参照化合物1的制备方法，绿色固体，产率为87％。1H NMR(400MHz,acetone-d6)δ10.44(s,1H),8.72(d,J＝2.5Hz,1H),8.40(t,J＝2.5Hz,2H),7.99(d,J＝9.0Hz,1H),7.73-7.56(m,2H),7.38–7.19(m,2H),3.97(s,3H),3.95(s,3H).13C NMR(101MHz,CDCl3)δ193.69,160.45,152.56(d,J＝243.9Hz),147.24(d,J＝10.7Hz),136.65,133.63(d,J＝1.8Hz),132.38(d,J＝6.5Hz),131.97(d,J＝10.0Hz),131.66,130.93,130.40,129.91,129.58,128.47(d,J＝12.2Hz),122.41(d,J＝3.3Hz),120.09,114.41(d,J＝19.1Hz),113.62,103.20,56.18,55.36.
实施例20：3-(3-氟-4-羟基苯基)-7-羟基-1-萘醛(13)的制备
参照化合物3的制备方法，黄色固体，产率为53％。1H NMR(400MHz,acetone-d6)δ10.40(s,1H),9.13(s,1H),8.90(d,J＝1.4Hz,1H),8.69(d,J＝2.4Hz,1H),8.38(t,J＝1.9Hz,2H),7.99(d,J＝8.9Hz,1H),7.65(m,1H),7.58-7.50(m,1H),7.28(m,1H),7.15(t,J＝8.8Hz,1H).13C NMR(101MHz,acetone-d6)δ194.68,159.27,152.71(d,J＝240.3Hz),145.45(d,J＝13.1Hz),137.77,134.18,132.51(d,J＝6.2Hz),132.40),131.82),131.56,131.44,130.29,123.71(d,J＝3.1Hz),120.21,119.17(d,J＝3.1Hz),115.09(d,J＝19.4Hz),107.62.
实施例21：2-((3-(3-氟-4-羟基苯基)-7-羟基萘-1-基)亚甲基)丙二腈(14a)的制备
参照化合物7a的制备方法，红色固体，产率为79％。熔点(mp 189-192℃)。1H NMR(400MHz,acetone-d6)δ9.25(s,1H),9.03(s,1H),8.98(s,1H),8.50(d,J＝1.4Hz,1H),8.34(s,1H),7.99(d,J＝8.9Hz,1H),7.57(m,1H),7.52(d,J＝2.1Hz,1H),7.48(m,1H),7.32(m,1H),7.19-7.07(m,1H).13C NMR(101MHz,acetone-d6)δ159.04,157.84,152.13(d,J＝238.0Hz),145.13(d,J＝12.7Hz),133.67(d,J＝1.9Hz),132.46,131.82(d,J＝6.3Hz),131.53,131.28,129.25,127.60,127.30,123.13(d,J＝3.1Hz),120.12,118.67(d,J＝3.2Hz),114.52(d,J＝19.3Hz),114.18,113.75,105.31,84.63.19F NMR(376MHz,acetone-d6)δ-137.42.
实施例22：2-氰基-3-(3-(3-氟-4-羟基苯基)-7-羟基萘-1-基)丙烯酸(14b)的制备
参照化合物7a的制备方法，红色固体，产率为62％。熔点(mp 184-186℃)。1H NMR(400MHz,DMSO-d6)δ10.67(s,1H),10.25(s,1H),8.71(s,1H),8.25(s,1H),8.18(s,1H),7.90(d,J＝9.0Hz,1H),7.56(dd,J＝12.7,2.1Hz,1H),7.42(dd,J＝8.4,1.6Hz,1H),7.37(s,1H),7.19(d,J＝9.0Hz,1H),7.10(t,J＝8.8Hz,1H).13C NMR(101MHz,DMSO-d6)δ176.65,156.68,151.45(d,J＝235.2Hz),144.63(d,J＝12.2Hz),132.44,131.91,131.22(d,J＝6.4Hz),130.87,128.56,128.20,127.53,125.41,122.59(d,J＝2.6Hz),119.63,118.89(d,J＝2.5Hz),118.39(d,J＝2.7Hz),114.12(d,J＝18.9Hz),114.12(d,J＝18.9Hz),104.78.
实施例23：2-氰基-3-(3-(3-氟-4-羟基苯基)-7-羟基萘-1-基)丙烯酸乙酯(14c)的制备
参照化合物7a的制备方法，黄色固体，产率为87％。熔点(mp 216-218℃)。1H NMR(400MHz,acetone-d6)δ8.98(s,3H),8.51(s,1H),8.28(s,1H),7.97(d,J＝8.9Hz,1H),7.57(d,J＝12.6Hz,1H),7.48(d,J＝8.4Hz,1H),7.37(s,1H),7.28(dd,J＝9.0,1.7Hz,1H),7.14(t,J＝8.8Hz,1H),4.42(q,J＝7.1Hz,2H),1.41(t,J＝7.1Hz,3H).13C NMR(101MHz,acetone-d6)δ162.50,157.64,152.73,152.37(d,J＝240.5Hz),145.17(d,J＝12.9Hz),134.01(d,J＝1.8Hz),133.00,132.55(d,J＝6.1Hz),131.86,130.45,129.54,127.73,127.69,123.46(d,J＝3.0Hz),120.08,118.90(d,J＝2.9Hz),116.09,114.82(d,J＝19.4Hz),106.28,105.17,62.89,14.10.
实施例24：2-(2-(2-(2-甲氧基)乙氧基)乙氧基)乙基-2-氰基-3-(3-(3-氟-4-羟基苯基)-7-羟基萘-1-基)丙烯酸乙酯(14d)的制备
参照化合物7a的制备方法，黄色油状液体，产率为69％。1H NMR(400MHz,acetone-d6)δ9.65(s,1H),9.24(s,1H),9.02(s,1H),8.53(s,1H),8.29(s,1H),7.97(d,J＝8.9Hz,1H),7.75-7.72(m,1H),7.63(m,1H),7.56(m,1H),7.33-7.30(m,1H),7.18(m,1H),4.52-4.47(m,2H),3.86-3.78(m,2H),3.68(m,2H),3.63-3.55(m,4H),3.46(m,2H),3.24(s,3H).13C NMR(101MHz,acetone-d6)δ182.25,162.49,157.88,152.99,152.35(d,J＝235.8Hz),145.31(d,J＝13.1Hz),132.55(d,J＝2.7Hz),132.31,132.21,129.11(d,J＝12.0Hz),127.61(d,J＝4.1Hz),123.28,114.71(d,J＝19.3Hz),105.53(d,J＝83.0Hz),72.15,70.91,70.77,70.62,68.91,66.07,58.30.
实施例25：靶向雌激素受体荧光探针对雌激素受体的相对亲和力测定
目标化合物与ERα和ERβ的亲和力通过荧光偏振法进行测定，化合物的亲和力是内源性E2亲和力的相对值，设定E2与受体亲和力的值RBA＝100％。在384孔板中，加入20μL由0.8μM ERα或ERβ蛋白、150nM荧光配体和2.4μg牛免疫球蛋白的磷酸钾缓冲液后，再加入20μL目标化合物溶液，化合物浓度梯度为：3.16×10-4M,1×10-4M,3.16×10-5M,1×10-5M,3.16×10-6M,1×10-6M,3.16×10-7M,1×10-7M,3.16×10-8M,1×10-8M,3.16×10-9M。避光、室温下放置2小时后，在酶标仪上读板，选取485nm处波长为主波长，528nm处波长为参照波长，分析实验结果，根据公式受体亲和力RBA＝测试物Ki/雌二醇Ki×100计算出每个化合物的RBA值。
表1.探针与雌激素受体亚型α和β的相对亲和力数据
Figure BDA0001352160740000121
Figure BDA0001352160740000131
实施例26：靶向雌激素受体荧光探针对雌激素受体的转录活性测定
目标化合物的转录活性是通过磷酸钙转染法在HEK 293T细胞中进行测定。在48孔板中，每孔加入150ng 3×ERE的荧光素酶、1ng CMV质粒、50ng ERα或ERβ质粒和12.5μL2×HBS，转染24h后。之后吸去每孔液体，然后再加入100μL目标化合物溶液，化合物浓度梯度为：1×10-5M,1×10-6M,1×10-7M,1×10-8M,1×10-9M,1×10-10M。置于37℃、5％CO2培养箱中孵育24小时。用细胞裂解液裂解细胞，离心后取上清液在酶标仪上测试双荧光值，分析实验结果，并计算出化合物的EC50、IC50值和Eff值。
表2.探针对雌激素受体亚型α和β的转录活性数据
Figure BDA0001352160740000132
实施例27：靶向雌激素受体荧光探针对MCF-7乳腺癌细胞的抗肿瘤活性测试
MCF-7细胞在含10％胎牛血清的有酚红DMEM液体培养基中培养。细胞密度至80％～90％时，消化细胞，并用含10％CS的无酚红DMEM培养基将细胞悬浮液铺至96孔细胞培养板中。待细胞完全贴壁后，弃去原培养液，每孔加入100μl新鲜的用含10％CS的DMEM培养基配制的化合物溶液，化合物浓度梯度为：1×10-6M,5×10-5M,1×10-5M,5×10-4M,1×10-4M。药物处理培养3天后，取出培养板，每孔加入20μl 5mg/ml MTT工作液，置于37℃、5％CO2培养箱中孵育4小时。之后吸去每孔液体，然后每孔加入100μL二甲亚砜(DMSO)，放在微量搅拌器上震荡10～15分钟使结晶物充分溶解。在酶标仪上读板，选取490nm处波长为主波长，630nm处波长为参照波长，分析实验结果，并计算出IC50。
表3.探针对MCF-7细胞的抗增殖活性测试(IC50,μM)
Figure BDA0001352160740000141
实施例28：靶向雌激素受体荧光探针的光谱数据测试
以二氯甲烷为溶剂分别测试了探针紫外吸收光谱和荧光光谱，具体数据如表4。
表4.探针的光谱数据
Figure BDA0001352160740000142
实施例29：靶向雌激素受体荧光探针对细胞的荧光共聚焦成像
乳腺癌细胞(MCF-7)和脑胶质瘤细胞(U-87)均购自与中国医学科学院基础医学研究所细胞资源中心，成像前以每皿1×104个细胞接种到共聚焦玻璃小皿中，每皿加入培养基1.5mL，在37℃含5％CO2的培养箱中培养12h，跟换新的培养基，加入一定浓度的探针，共同孵育一定时间后，移去培养基，加入PBS，以探针的最大激发波长观察细胞成像状况。具体成像状况请参考图1和图2。
上述结果表明本发明的荧光探针能够用于雌激素受体相关的癌症成像，具有诊断与治疗功效。

Claims (3)

1.一类靶向雌激素受体荧光探针，其特征在于，所述靶向雌激素受体荧光探针为以下化合物：
Figure FDA0002591202560000011
或
Figure FDA0002591202560000012
或
Figure FDA0002591202560000013
或
Figure FDA0002591202560000014
或
Figure FDA0002591202560000015
或
Figure FDA0002591202560000016
或
Figure FDA0002591202560000021
或
Figure FDA0002591202560000022
或
2-氰基-3-(3-(3-氟-4-羟基苯基)-7-羟基萘-1-基)丙烯酸乙酯。
2.根据权利要求1所述的一类靶向雌激素受体荧光探针用于制备诊断或治疗肿瘤药物的用途。
3.根据权利要求2所述的用途，其特征在于，所述诊断或治疗肿瘤药物还包括一种或多种药学上可接受的载体或赋形剂。
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