CN114835681A - 一类咔唑喹啉杂合物、制备方法与应用 - Google Patents
一类咔唑喹啉杂合物、制备方法与应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及生物医药领域,涉及一类咔唑喹啉杂合物、制备方法与应用,具体涉及一类pH敏感、近红外成像的咔唑喹啉杂合物荧光探针及其制备方法与应用。
背景技术
2021年,癌症统计数据显示,癌症仍是世界范围内的重大公共卫生问题,给家庭和社会带来巨大痛苦和负担。如果可以及时检测到癌症的早期发作,绝大多数患者可以在没有复发的风险的情况下治愈。因此,肿瘤的早期诊断近年来备受关注。
肿瘤的早期发病由于其在微观分子尺度上的变化而难以被发现。由于没有外在表现,患者也忽略了肿瘤的存在。在临床前试验和临床实践中,监测组织、体液和排泄物中的肿瘤生物标志物,如活性氧(ROS)、酶和抗原有助于早期肿瘤的检测和肿瘤的预后评估。荧光成像技术凭借其固有的优势,如高时空分辨率、良好的灵敏度和生物相容性,被广泛应用于生物医学领域,在分子和细胞水平上监测生理和病理过程。特别是近红外(NIR)发射(650–900nm)荧光探针,它们能够避免自发荧光、减少光子散射和改善深层组织穿透。荧光探针具有选择性的能力使人们满意地可视化肿瘤部位和转移灶,这引起了成像引导手术(IGS)的广泛关注。目前,生物成像中使用两种有机荧光探针:“始终开启”和“可激活/响应”探针。前者的荧光信号始终存在,这导致感兴趣目标的可视化与信噪比低,不适合体内准确检测。相比之下,响应探针在被特定生物目标打开之前不会产生荧光信号,从而产生高对比度荧光成像。此外,目前大多数小分子荧光探针仅具有单光子激发特性,与这些荧光探针相比,具有双光子激发特性的荧光探针不仅能够对组织产生更深的穿透能力,而且扩大激发和发射波长差值,可有效避免组织自身荧光和光源背景的干扰。
因此为了实现能够对肿瘤进行实时、精确、快速地成像诊断,本发明研究开发的咔唑喹啉杂合物小分子荧光探针,希望结合单光子和/或双光子激发、酸性pH刺激响应的优势特点,发挥肿瘤组织体内外近红外荧光成像,将在医药用途方向发挥重要的作用。
发明内容
针对以上问题,本发明提供了一类咔唑喹啉杂合物、制备方法与应用,该咔唑喹啉杂合物为pH敏感的近红外成像的咔唑喹啉杂合物荧光探针,通过单光子和/或双光子激发、酸性pH刺激响应,可应用于进行体内外肿瘤选择性荧光成像试剂制备的医药用途,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:
一类咔唑喹啉杂合物,具有通式Ⅰ所示结构:
其中,咔唑喹啉杂合物为pH敏感、近红外成像的咔唑喹啉杂合物荧光探针,R选自H或NH2。
R=H时,化合物I1为(E)-9-乙基-3-(2-(喹啉-4-基)乙烯基)-9H-咔唑;
R=NH2时,化合物I2为(E)-9-乙基-6-氨基-3-(2-(喹啉-4-基)乙烯基)-9H-咔唑。
本发明的另一目的在于提供本发明通式Ⅰ所述化合物的制备方法:
化合物I1的制备方法具体为:
将9-乙基-9H-咔唑-3-甲醛(1)与4-甲基喹啉(2)在催化量哌啶条件下,加热回流,发生Knoevenagel缩合反应获得化合物I1;
合成路线如下所示:
化合物I2的制备方法具体为:
S1.将9-乙基-6-硝基-9H-咔唑-3-甲醛(3)与4-甲基喹啉(2)在催化量哌啶条件下,加热回流,发生Knoevenagel缩合反应获得化合物4;
S2.化合物4在铁粉和氯化铵催化下,加热回流,还原获得化合物I2:
所述制备方法的合成路线如下所示:
本发明还提供了上述咔唑喹啉杂合物在制备通过单光子和/或双光子激发的荧光成像试剂中的应用。
进一步的,所述荧光成像试剂为用于体内外肿瘤组织或肿瘤细胞的选择性荧光成像试剂;优选的,该荧光成像试剂为荧光显影剂。
进一步的,所述荧光成像试剂为通过喷洒或局部注射的方式实现肿瘤的快速、实时检测和成像的试剂。
进一步的,荧光成像试剂由咔唑喹啉杂合物溶于助溶剂/表面活性剂/溶剂体系得到;所述助溶剂/表面活性剂/溶剂体系中,助溶剂为1,2丙二醇、DMSO和乙醇中的一种或几种;溶剂为水;表面活性剂为吐温20、吐温40和吐温80中的一种或几种。
进一步的,所述助溶剂/表面活性剂/溶剂体系中,按体积百分比计,所述助溶剂的含量为1~30%,所述表面活性剂的含量为1~30%。
进一步的,所述肿瘤为肝癌、结肠癌、乳腺癌、肺癌和宫颈癌肿瘤中的一种。
与现有技术相比,本发明具有的应用效果:本发明公开了一类非季铵盐形式的咔唑喹啉杂合物,不同于以往的季铵盐型喹啉鎓盐类化合物(在激发光下一直亮着,无酸性pH响应的“开-关”效应荧光、无肿瘤选择性荧光成像),本发明化合物在肿瘤组织酸性微环境下激活,利用ICT原理,通过单光子和/或双光子激发,选择性地在肿瘤部位快速产生pH敏感的荧光。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、选择性荧光成像和示踪,具有较高的肿瘤组织荧光成像选择性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是本发明部分化合物不同pH的紫外吸收光谱图,横坐标为波长,纵坐标为吸光度值;
图2是本发明部分化合物不同pH的荧光发射光谱图,横坐标为波长,纵坐标为荧光强度;
图3是本发明部分化合物的双光子吸收截面图;
图4是本发明部分化合物实现体内外肿瘤细胞的选择性荧光成像的应用示意图;
图5是本发明部分化合物对离体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)-9-乙基-3-(2-(喹啉-4-基)乙烯基)-9H-咔唑(I1)的制备
将9-乙基-9H-咔唑-3-甲醛(500mg,1.0mmol)和4-甲基喹啉(320mg,1.0mmol)加入单口瓶中,用无水乙醇(10ml)溶解,随后加入1-2滴哌啶,85℃回流12h,经TLC监测反应结束后,抽滤,再次重结晶纯化得到化合物I1,产率为85%.
(I1)谱图数据为:1H NMR(400MHz,DMSO-d6)δ9.39(d,J=6.3Hz,1H,ArH),9.15(d,J=8.5Hz,1H,ArH),8.78(s,1H,ArH),8.55–8.46(m,3H,ArH),8.42(m,2H,ArH),8.34(d,J=15.6Hz,1H,CH),8.26(m,1H,ArH),8.17(d,J=8.7Hz,1H,ArH),8.05(m,2H,ArH),7.84(s,1H,ArH),7.45(m,1H,CH),4.53(q,2H,CH2),1.33(t,J=6.0Hz,3H,CH3).
实施例2:(E)-9-乙基-6-(2-(喹啉-4-基)乙烯基)-9H-咔唑-3-胺(I2)的制备
参照实施例1中(I1)的合成方法,由9-乙基-6-硝基-9H-咔唑-3-甲醛代替方法中的9-乙基-9H-咔唑-3-甲醛,最后得到化合物4;再将化合物4(500mg,1.0mmol)、铁粉(273mg,4.0mmol)、氯化铵(523mg,8.0mmol)加入单口瓶中,用无水乙醇(20ml)溶解,80℃回流5h,经TLC监测反应结束后,抽滤,将滤液旋干,经柱层析纯化,得到化合物I2,产率为72%。
(I2)谱图数据为:1H NMR(400MHz,DMSO-d6)δ9.35(d,J=6.8Hz,1H,ArH),9.12(m,1H,ArH),8.82(s,1H,ArH),8.58–8.44(m,4H,ArH),8.33(d,J=15.6Hz,1H,CH),8.24(m,1H,ArH),8.16(m,1H,ArH),8.02(m,2H,ArH),7.85(s,1H,ArH),7.41(m,1H,CH),4.52(m,2H,CH2),1.31(t,J=6.0Hz,3H,CH3).
实施例3:本发明实施例1得到的化合物I1在不同pH条件下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450-600nm范围内。其中化合物I1在490nm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530nm左右的紫外吸收峰值随pH减小而增加,其峰值相差40倍(图1)。
实施例4:本发明实施例1制备得到的化合物I1的pH响应的荧光光谱测试
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的检测液。采用荧光光谱仪测试其荧光发射光谱数据,结果显示本发明荧光化合物最大发射波长在650-750nm范围内。其中化合物I1在680nm左右的荧光峰值随化合物I1的pH减小而增加,相反其荧光峰值随pH增加而减小,其峰值相差14倍(图2)。
实施例5:采用飞秒荧光测量技术检测不同波长下的双光子吸收截面
将本发明化合物I1溶于pH=4.0的PBS缓冲液中(5μM),检测本发明化合物从920nm到1020nm的双光子激发下的荧光强度。利用公式:δ=δr×(Fs×фr×nr)/(Fr×фs×ns),其中,δ,F,ф和n分别是双光子吸收截面,光谱积分面积,量子产率和浓度;s和r分别代表了本发明化合物和对照化合物。计算不同波长下该还原产物的双光子吸收截面,计算结果表明,本发明化合物I1在960nm具有最大的双光子吸收截面(δmax=109GM)(图3)。
实施例6:采用共聚焦显微镜进行细胞成像
参照图4,采用共聚焦显微镜对乳腺癌细胞(Mcf-7)、结肠癌细胞(HT29)、正常胚肺成纤维细胞(HFL-1)或肺癌细胞细胞(A549)进行细胞成像,由DEME或1640培养液培养24h,向细胞中加入1~10μM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=450-600nm,λex=650-750nm。
细胞成像结果表明,本发明化合物可以在多个肿瘤细胞产生清晰的荧光成像,而对正常肺成纤维细胞荧光成像很弱,由此说明本发明化合物能够对多个肿瘤细胞选择性地荧光成像,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。
实施例7: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Hela宫颈癌移植瘤模型裸鼠,将其处死,取出宫颈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I2溶液50μM喷洒在组织上3~5次,用PBS清洗并用棉花吸干,5~10min后进行荧光成像,结果如图5所示,宫颈癌组织的荧光强度值明显高于其他器官组织,而正常器官组织荧光相对较弱。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。
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)
4.权利要求1所述的咔唑喹啉杂合物在制备通过单光子和/或双光子激发的荧光成像试剂中的应用。
5.根据权利要求4所述的应用,所述荧光成像试剂为可实现肿瘤选择性荧光成像试剂,所述肿瘤为肿瘤组织或肿瘤细胞。
6.根据权利要求5所述的应用,其特征在于,所述荧光成像试剂为通过喷洒或局部注射的方式实现肿瘤的快速、实时检测和成像的试剂。
7.根据权利要求4所述的应用,其特征在于,所述荧光成像试剂由所述咔唑喹啉杂合物溶于助溶剂/表面活性剂/溶剂体系得到;所述助溶剂/表面活性剂/溶剂体系中,助溶剂为1,2丙二醇、DMSO和乙醇中的一种或几种;溶剂为水;表面活性剂为吐温20、吐温40和吐温80中的一种或几种。
8.根据权利要求7所述的应用,其特征在于,所述助溶剂/表面活性剂/溶剂体系中,按体积百分比计,助溶剂的含量为1~30%,表面活性剂的含量为1~30%。
9.根据权利要求5-8任一项所述的应用,其特征在于,所述肿瘤为结肠癌、肺癌、胰腺癌、乳腺癌、肝癌和宫颈癌肿瘤中的一种。
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