CN113735839B - 二氢呫吨/苯并[cd]吲哚杂合物荧光探针及其制备方法与应用 - Google Patents
二氢呫吨/苯并[cd]吲哚杂合物荧光探针及其制备方法与应用 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域，具体涉及二氢呫吨/苯并[cd]吲哚杂合物荧光探针及其制备方法与应用，具有通式Ⅰ所示结构：
本发明制备的二氢呫吨/苯并[cd]吲哚杂合物荧光探针表现出pH敏感荧光成像特性，能够在肿瘤酸性微环境下选择性活化，发出近红外荧光，有利于通过其pH敏感特性对肿瘤组织和细胞进行选择性荧光成像。利用本发明化合物制备成溶液喷洒到在体或离体肿瘤组织及其淋巴结上，可对肿瘤组织进行快速检测和精准成像，引导手术切除和/或药物治疗，进而对于癌症的诊断和治疗具有重要应用意义。
Description
技术领域
本发明涉及生物医药技术领域，具体涉及二氢呫吨/苯并[cd]吲哚杂合物荧光探针及其制备方法与应用。
背景技术
肿瘤是国际高发性疾病。由于环境污染、不健康作息、食品安全卫生等原因，越来越多的人成为肿瘤的“目标”，恶性肿瘤已变成危及人类健康生命的一大杀手。因此，研究如何对肿瘤进行早期诊断成为医药领域的重要内容。
近红外(NIR)分子荧光成像技术是一种在分子、细胞和组织水平上对生物事件进行可视化和量化的无创方法。不同于传统成像技术，分子荧光成像技术因其高度空间分辨率而在疾病诊断中具有强大的应用潜力。从原理上讲，荧光是荧光分子吸收光能后，激发到单重态激发态，恢复到基态所伴随的发光现象。荧光探针是能选择性识别某种特定物质，将分析对象的信号转变成光学信号，通过分析测量仪器以荧光形式呈现的一类分子检测器。尽管NIR荧光分子吲哚菁绿(ICG)和亚甲蓝(MB)已被批准用于临床，但它们都不具有肿瘤选择性荧光特性，一个重要的原因它们处于“always on”状态，不能够在肿瘤组织中选择性发光。虽然目前也有针对肿瘤微环境(如较低的pH)的“开关”型探针，但它们大多数都利用pH敏感的腙键或缩醛基团，不具备荧光可逆性，这些基团本身稳定性较差，且需要一定的响应时间，无法实施快速诊断。现阶段荧光成像探针的研究主要集中在小分子探针和纳米材料两类：纳米材料利用量子点、聚合物、磁性载体以及碳材料，发挥包括肿瘤靶向、细胞摄取、荧光成像在内的成像诊断作用，但纳米材料自身存在一定的局限性，主要体现在生物相容性、免疫性、可代谢性以及制备和质量可控性等方面。相比之下，小分子荧光探针具有生物相容性较好、制备较为简便、产品均一、质量可控性高等优势，具有重要意义和实际应用价值。
发明内容
针对以上问题，本发明提供了二氢呫吨/苯并[cd]吲哚杂合物荧光探针及其制备方法与应用，通过pH响应进行体内外肿瘤选择性荧光成像的医药用途，以引导手术切除和/或药物治疗，进而对于癌症的快速诊断和治疗具有重要应用意义。
为了实现上述目的，本发明采用的技术方案如下：
二氢呫吨/苯并[cd]吲哚杂合物荧光探针，具有通式Ⅰ所示结构：
其中，R选自H、F、Cl、Br、I中的一种。
上述通式Ⅰ部分化合物代号及其对应的化合物名称如下：
I1:(E)-4-(2-(苯并[cd]吲哚-2-基)乙烯基)-2,3-二氢-1H-呫吨-6-醇；
I2:(E)-4-(2-(6-碘代苯并[cd]吲哚-2-基)乙烯基)-2,3-二氢-1H-呫吨-6-醇。
优选地，R＝H，或者R＝I。
本发明另一目的在于提供二氢呫吨/苯并[cd]吲哚杂合物荧光探针的制备方法，将6-甲氧基-2,3-二氢-1H-呫吨-4-甲醛1与6-R-2-甲基苯并[cd]吲哚(2)在催化量甲磺酸条件下，加热回流，发生Knoevenagel缩合反应得到中间体3；最后中间体3在三溴化硼作用下脱去甲基获得二氢呫吨/苯并[cd]吲哚杂合物Ⅰ；
合成路线如下所示：
其中，R选自H、F、Cl、Br、I中的一种。
本发明另一目的在于提供二氢呫吨/苯并[cd]吲哚杂合物在制备pH响应的荧光探针、实现肿瘤组织或肿瘤细胞的选择性荧光成像的应用。
其中，二氢呫吨/苯并[cd]吲哚杂合物荧光探针配制成的溶液通过喷洒或局部注射的方式，实现肿瘤的快速、实时检测和成像。
优选地，二氢呫吨/苯并[cd]吲哚杂合物溶液的配制，在溶剂体系下加以助溶剂和/或表面活性剂溶解该化合物，具体如下：
以H2O作为溶剂，以1,2-丙二醇，DMSO或乙醇作为助溶剂，以吐温20，吐温40或吐温80作为表面活性剂；其中，1,2-丙二醇的体积百分数为1～30％，DMSO的体积百分数为1～30％，乙醇的体积百分数为1～30％，吐温20的体积百分数为1～30％，吐温40的体积百分数为1～30％，吐温80的体积百分数为1～30％。
其中，二氢呫吨/苯并[cd]吲哚杂合物荧光探针在肿瘤细胞和组织下进行pH敏感的肿瘤荧光成像试剂中的应用，可实现肿瘤细胞和组织pH敏感荧光成像作用。
其中，二氢呫吨/苯并[cd]吲哚杂合物荧光探针，将喷洒或局部注射化合物溶液于术前或术中肿瘤病灶部位及周围的组织上，利用荧光腔镜或活体成像仪对肿瘤病灶组织进行快速、选择性荧光成像和示踪，以指导手术和/或药物治疗。
其中，上述肿瘤包括肝癌、结肠癌、胰腺癌、乳腺癌、肺癌、宫颈癌肿瘤。
本发明有益效果：
本发明公开了一类非季铵盐形式的二氢呫吨/苯并[cd]吲哚杂合物，不同于以往的季铵盐型含有酚羟基的二氢呫吨类化合物(在激发光下一直亮着，无“开-关”效应荧光、无肿瘤选择性荧光成像)，本发明化合物可以利用肿瘤组织酸性微环境激活，选择性地在肿瘤部位快速产生近红外荧光，具体实施方法是将喷洒或局部注射本发明化合物溶液于术前或术中肿瘤病灶部位及周围的组织上，利用荧光腔镜或活体成像仪对肿瘤病灶组织进行快速、实时、选择性荧光成像和示踪，具有较高的穿透深度和较低的背景荧光干扰，能够对肿瘤进行精确诊断，以指导手术和/或药物治疗。
附图说明
图1是本发明荧光探针部分化合物I1的紫外吸收光谱图，横坐标为波长，纵坐标为吸光度值；
图2是本发明荧光探针部分化合物(左为I1，右为I2)不同pH的荧光发射光谱图光谱图，横坐标为波长，纵坐标为荧光强度；
图3是本发明荧光探针部分化合物I1实现体内外肿瘤细胞的选择性荧光成像的应用示意图；
图4是本发明荧光探针部分化合物I1对离体肿瘤组织选择性荧光成像试验示意图。
图5是本发明荧光探针部分化合物I2对临床结肠肿瘤组织选择性荧光成像试验示意图。
具体实施方式
下面结合附图将对本发明实施例中的技术方案进行清楚、完整地描述，以使本领域的技术人员能够更好的理解本发明的优点和特征，从而对本发明的保护范围做出更为清楚的界定。本发明所描述的实施例仅是本发明一部分实施例，而不是全部的实施例，基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：(E)-4-(2-(苯并[cd]吲哚-2-基)乙烯基)-2,3-二氢-1H-呫吨-6-醇(I1)的制备
将6-甲氧基-2,3-二氢-1H-呫吨-4-甲醛(484mg,2mmol)与2-甲基苯并[cd]吲哚(334mg,2mmol)用乙醇溶解，加入催化量甲磺酸，加热回流，待反应结束后，减压浓缩，柱层析分离，获得目标化合物I1，产率85％。
(I1)谱图数据为：1HNMR(400MHz,DMSO)δ8.81(m,1H,ArH),8.39(d,J＝8.2Hz,1H,ArH),7.95(m,2H,2ArH),7.81(m,1H,ArH),7.75(s,1H,ArH),7.61(s,1H,ArH),7.24(d,J＝15.9Hz,1H,ArH),7.10(m,1H,ArH),6.84(d,J＝2.4Hz,1H,CH＝),6.61(m,1H,CH＝),6.52(s,1H,CH＝),2.62(t,J＝5.8Hz,2H,CH2),2.52(m,2H,CH2),1.78(m,2H,CH2).
实施例2：(E)-4-(2-(6-碘代苯并[cd]吲哚-2-基)乙烯基)-2,3-二氢-1H-呫吨-6-醇(I2)的制备
参照实施例1中(I1)的合成方法，由6-碘-2-甲基苯并[cd]吲哚代替方法中的2-甲基苯并[cd]吲哚，最后得到棕色固I2，产率为71％。
(I2)谱图数据为：1H NMR(400MHz,DMSO)δ8.83(d,J＝4.7Hz,1H,ArH),8.40(d,J＝8.3Hz,1H,ArH),7.98(dd,J＝15.8,12.1Hz,2H,2ArH),7.83(d,J＝4.8Hz,1H,ArH),7.76(s,1H,ArH),7.62(s,1H,ArH),7.10(d,J＝8.4Hz,1H,ArH),6.85(d,J＝2.3Hz,1H,CH＝C),6.61(dd,J＝8.4,2.4Hz,1H,CH＝C),6.52(s,1H,CH＝C),2.64(t,J＝5.8Hz,2H,CH2),2.52(d,J＝6.9Hz,2H,CH2),1.78(d,J＝5.8Hz,2H,CH2).
实施例3：本发明不同pH条件下荧光探针的紫外吸收光谱测试
将本发明荧光化合物溶于含50％的乙醇水溶液中，配制成pH＝3-8，浓度为5-20μM的检测液。采用紫外-可见分光光度计测试其紫外吸收光谱数据，结果显示本发明荧光化合物紫外最大吸收波长在450-700nm范围内。其中化合物I1在480nm左右的紫外吸收峰值随化合物I1的pH减小而减小，相反其在673nm左右的紫外吸收峰值随pH减小而增加，光谱在543nm处具有等吸收点，其其峰值相差14倍(如图1所示)；
实施例4：本发明部分化合物的pH响应的荧光光谱测试
选用本发明化合物I1、I2为代表进行pH响应的荧光光谱测试，将其配制成pH＝3～8下的1～25μM的检测液。采用荧光分光光度计测试其荧光发射光谱数据，不同pH的荧光发射光谱图如图2所示。结果显示本发明荧光化合物最大发射波长在760-840nm范围内。本发明化合物在(Em＝760-840nm)的荧光峰值随pH值减小而增加，相反其荧光峰值分别随pH值增加而减小，其峰值相差10～20倍(图2)；由此说明本发明化合物具有pH响应的荧光特性。
实施例5：采用共聚焦显微镜进行细胞成像
参照图4，采用共聚焦显微镜进行细胞成像，成像前24h，HepG2细胞、Hela细胞或LO2细胞由DEME或1640培养液培养，放于激光共聚焦皿中，再向细胞中加入1～25μM的受试化合物，将其放置置于37℃、含5％CO2的细胞培养箱中孵育半小时。接着用pH＝7.4的磷酸盐缓冲溶液洗涤3次后，将孵育好的细胞置于共聚焦显微镜的载物台上进行共聚焦荧光成像，设置受试化合物激发波长：λem＝650-700nm，λex＝760-840nm。
细胞成像结果表明，本发明化合物可以被肿瘤细胞有效吸收，表明该类荧光化合物可以选择性在多个肿瘤细胞荧光成像，而对正常肝细胞荧光成像很弱，为体内外肿瘤组织或细胞成像研究提供了一种可行的手段，应用前景广阔。
实施例6：本发明化合物对离体肿瘤组织进行喷洒模式的荧光成像试验
取HT29结肠癌模型裸鼠，将其处死，取出结肠肿瘤及主要脏器进行喷洒成像分析。将配制好的本发明化合物I1溶液(10～100μM)喷洒在组织上3～5次，用PBS清洗并用棉花吸干，荧光成像结果如图4所示，结肠癌组织的荧光强度值明显高于其他器官组织，而正常器官组织几乎看不到荧光。由此说明了本发明化合物可选择性、快速对肿瘤组织喷洒成像，以实现临床对肿瘤组织的快速检测。
实施例7：本发明化合物对临床肿瘤组织荧光成像试验
在此基础上，进一步研究本发明化合物对临床肿瘤组织的选择性成像能力。对临床结肠癌组织和癌旁组织以及正常结肠组织进行喷洒成像对比分析，将本发明化合物I2溶液均匀喷洒在结肠癌组织和癌旁组织以及正常结肠组织1～3次，3～10min后再用适当生理盐水洗去表面多余的溶液，利用活体成像仪进行荧光成像。
荧光成像结果表明，本发明化合物I2能够选择性、快速点亮临床结肠癌组织，而对周围正常组织不显色或显示较弱(图5)。由此进一步确认了本发明化合物对临床结肠肿瘤组织的选择性成像能力。
以上对本发明的实施例进行了详细说明，但所述内容仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等，均应仍归属于本发明的专利涵盖范围之内。
Claims (9)
1.二氢呫吨/苯并[cd]吲哚杂合物荧光探针，其特征在于：具有通式Ⅰ所示结构：
其中，R选自H、F、Cl、Br、I中的一种。
2.根据权利要求1所述的二氢呫吨/苯并[cd]吲哚杂合物荧光探针，其特征在于：R＝H，或者R＝I。
3.根据权利要求1-2任一项所述的二氢呫吨/苯并[cd]吲哚杂合物荧光探针的制备方法，其特征在于：将6-甲氧基-2,3-二氢-1H-呫吨-4-甲醛1与6-R-2-甲基苯并[cd]吲哚(2)在催化量甲磺酸条件下，加热回流，发生Knoevenagel缩合反应得到中间体3；最后中间体3在三溴化硼作用下脱去甲基获得二氢呫吨/苯并[cd]吲哚杂合物Ⅰ；
合成路线如下所示：
其中，R选自H、F、Cl、Br、I中的一种。
4.根据权利要求1-2任一项所述的二氢呫吨/苯并[cd]吲哚杂合物在制备pH响应的荧光探针、制备实现肿瘤组织或肿瘤细胞的选择性荧光成像试剂的应用。
5.根据权利要求1-2任一项所述的二氢呫吨/苯并[cd]吲哚杂合物荧光探针配制成通过喷洒或局部注射的方式，实现肿瘤的快速、实时检测和成像的溶液。
6.根据权利要求5所述的溶液的配制，其特征在于，在溶剂体系下加以助溶剂和/或表面活性剂溶解该化合物，具体如下：
以H2O作为溶剂，以1,2-丙二醇，DMSO或乙醇作为助溶剂，以吐温20，吐温40或吐温80作为表面活性剂；其中，1,2-丙二醇的体积百分数为1～30％，DMSO的体积百分数为1～30％，乙醇的体积百分数为1～30％，吐温20的体积百分数为1～30％，吐温40的体积百分数为1～30％，吐温80的体积百分数为1～30％。
7.根据权利要求1所述的二氢呫吨/苯并[cd]吲哚杂合物荧光探针在制备肿瘤细胞和组织下进行pH敏感的肿瘤荧光成像试剂中的应用，其特征在于，可实现肿瘤细胞和组织pH敏感荧光成像作用。
8.根据权利要求4所述的应用，其特征在于：所述实现肿瘤组织或肿瘤细胞的选择性荧光成像为将喷洒或局部注射化合物溶液于术前或术中肿瘤病灶部位及周围的组织上，利用荧光腔镜或活体成像仪对肿瘤病灶组织进行快速、选择性荧光成像和示踪，以指导手术和/或药物治疗。
9.根据权利要求7或8所述的应用，其特征在于，所述肿瘤包括肝癌、结肠癌、胰腺癌、乳腺癌、肺癌、宫颈癌肿瘤。
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