CN114835629B - 一类咔唑苯并[cd]吲哚鎓盐及其制备方法与应用 - Google Patents
一类咔唑苯并[cd]吲哚鎓盐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,具体涉及一类咔唑苯并[cd]吲哚鎓盐及其制备方法与应用,咔唑苯并[cd]吲哚鎓盐具有通式Ⅰ所示结构:该咔唑苯并[cd]吲哚鎓盐可以利用ICT原理,在肿瘤组织酸性微环境下激活,选择性地在肿瘤部位快速产生pH敏感的近红外荧光,具体实施方法是将本发明咔唑苯并[cd]吲哚鎓盐的溶液喷洒或局部注射于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检测。
Description
技术领域
本发明属于生物医药领域,涉及一类咔唑苯并[cd]吲哚鎓盐及其制备方法与应用,尤其涉及一类pH敏感的近红外成像的咔唑苯并[cd]吲哚鎓盐及其制备方法与应用。
背景技术
癌症是世界范围内的一种主要疾病,目前每年导致超过700万人死亡,预计在未来20年内将成为一个更严重的问题。一个显著影响癌症死亡率的因素是难以获得准确的早期诊断,因为此时肿瘤更容易治疗。因此,肿瘤早期诊断的研究具有重要的医学意义。
近红外(NIR)分子荧光探针具有较长的激发和发射波长,较低的能量,更深的组织渗透性以及减少荧光背景干扰等优点,在细胞成像和动物成像中有着巨大的应用前景。pH值是一个重要的生理参数,在细胞和组织稳态中起着关键作用。肿瘤细胞快速糖酵解产生的乳酸致使肿瘤微环境呈酸性,因此利用较低的pH来设计低pH响应的肿瘤诊断剂至关重要。已有文献报道可通过控制pH敏感键(如腙键、亚胺键或缩醛等)的断裂来控制荧光,以达到“开关”效果,但该类荧光探针不具备光可逆性且共价键的断裂需要时间,续而不能实现对肿瘤进行快速准确的诊断。虽然一些基于NIR荧光分子吲哚菁绿(ICG)和亚甲蓝(MB)的荧光探针已被报道用于肿瘤诊断,但由于它们处于“always on”状态,故也不具有肿瘤选择性荧光特性。
发明内容
针对以上问题,本发明提供了一类咔唑苯并[cd]吲哚鎓盐及其制备方法与应用,该咔唑苯并[cd]吲哚鎓盐为pH敏感的可近红外成像的咔唑苯并[cd]吲哚鎓盐荧光探针,可制备成通过单光子和/或双光子激发、酸性pH刺激响应,可应用于体内外肿瘤选择性荧光成像的试剂,该试剂可用于引导手术切除和/或药物治疗,该医药用途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:
一类咔唑苯并[cd]吲哚鎓盐,具有通式Ⅰ所示结构:
其中,R选自H、NO、NO2和NH2中的一种。
表1通式Ⅰ部分化合物代号及其对应的结构
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)-2-(2-(9-乙基-9H-咔唑-3-基)乙烯基)-1-甲基苯并[cd]吲哚-1-六氟磷酸盐;
I2:(E)-2-(2-(9-乙基-6-硝基-9H-咔唑-3-基)乙烯基)-1-甲基苯并[cd]吲哚-1-碘盐;
I3:(E)-2-(2-(6-氨基-9-乙基-9H-咔唑-3-基)乙烯基)-1-甲基苯并[cd]吲哚-1-碘盐。
本发明的另一目的在于提供本发明通式Ⅰ所示结构的化合物的如下制备方法:
合成路线如下所示:
将6-R-9-乙基-9H-咔唑-3-甲醛(1)与1,2-二甲基苯并[cd]吲哚-1-Y鎓盐(2)在催化量哌啶条件下,加热回流,发生Knoevenagel缩合反应获得通式Ⅰ所示结构的化合物(化合物I);
合成路线如下所示:
R为H、NO和NO2中的一种,Y-代表卤负离子、六氟磷酸根负离子、对甲苯磺酸负离子和甲磺酸负离子中的一种;
本发明还提供了化合物Ib(通式Ⅰ中R为NH2)的制备方法,具体包括:
先根据上述制备方法,制备得到化合物Ia(上述制备方法中,通式Ⅰ中R为NO2)
然后,将化合物Ia在铁粉和氯化铵的甲醇溶剂下,加热回流,经还原反应获得化合物Ib;化合物Ia和Ib均属于通式I。
合成路线如下式所示:
Y-代表卤负离子、六氟磷酸根负离子、对甲苯磺酸负离子和甲磺酸负离子中的一种。
本发明还提供了上述咔唑苯并[cd]吲哚鎓盐在制备pH响应、实现肿瘤组织或肿瘤细胞的选择性荧光成像的试剂中的应用。
本发明还提供了上述咔唑苯并[cd]吲哚鎓盐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。
将试剂喷洒或局部注射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。
进一步的,所述试剂由咔唑苯并[cd]吲哚鎓盐溶于助溶剂/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,助溶剂的含量为1~30%,表面活性剂的含量为1~30%。
进一步的,所述肿瘤包括肺癌、乳腺癌、结肠癌、肝癌、宫颈癌和胰腺癌肿瘤中的一种。
与现有技术相比,本发明具有的应用效果:本发明化合物利用ICT原理,通过单光子和/或双光子激发,在肿瘤组织酸性微环境下激活,选择性地在肿瘤部位快速产生pH敏感的近红外荧光,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、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是本发明荧光探针部分化合物I1-I3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I1-I3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图;
图4是本发明荧光探针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是本发明荧光探针部分化合物对离体肿瘤组织选择性荧光成像试验示意图;
图6是本发明荧光探针部分化合物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)-2-(2-(9-乙基-9H-咔唑-3-基)乙烯基)-1-甲基苯并[cd]吲哚-1-六氟磷酸盐(I1)的制备
将9-乙基-9H-咔唑-3-甲醛(500mg,1.0mmol)和1,2-二甲基苯并[cd]吲哚-1-六氟磷酸盐(692.29mg,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86%。
(I1)谱图数据为:1H NMR(400MHz,DMSO)δ9.37(d,J=7.4Hz,1H,ArH),9.13(s,1H,ArH),9.06(d,J=15.8Hz,1H,CH),8.67(d,J=8.1Hz,1H,ArH),8.40(d,J=8.6Hz,1H,ArH),8.27(m,3H,ArH),8.17(m,1H,ArH),8.03(d,J=15.7Hz,1H,CH),7.94(m,1H,ArH),7.87(m,1H,ArH),7.75(d,J=8.2Hz,1H,ArH),7.59(m,1H,ArH),7.39(m,1H,ArH),4.55(q,J=7.2Hz,2H,CH2),4.32(s,3H,CH3),1.39(t,J=7.1Hz,3H,CH3).
实施例2:(E)-2-(2-(9-乙基-6-硝基-9H-咔唑-3-基)乙烯基)-1-甲基苯并[cd]吲哚-1-碘盐(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-甲醛和1,2-二甲基苯并[cd]吲哚-1-碘盐代替(I1)的合成方法中的9-乙基-9H-咔唑-3-甲醛和1,2-二甲基苯并[cd]吲哚-1-六氟磷酸盐,最后得到化合物I2,产率为82%。
(I2)谱图数据为:1H NMR(400MHz,DMSO)δ8.89(d,J=2.0Hz,1H,ArH),8.71(d,J=15.7Hz,1H,CH),8.48(d,J=8.5Hz,1H,ArH),8.31(m,1H,ArH),8.28–8.21(m,2H,ArH),8.16(d,J=8.9Hz,1H,ArH),7.98(d,J=15.7Hz,1H,CH),7.83(m,1H,ArH),7.77–7.71(m,2H,ArH),7.65(m,1H,ArH),7.44–7.38(m,1H,ArH),4.26(s,2H,CH2),2.93(s,3H,CH3),2.08(s,3H,CH3).
实施例3:(E)-2-(2-(6-氨基-9-乙基-9H-咔唑-3-基)乙烯基)-1-甲基苯并[cd]吲哚-1-碘盐(I3)的制备
将化合物I2(500mg,1.0mmol)、铁粉(273.73mg,4.0mmol)、氯化铵(522.92mg,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I3,产率为73%。
(I3)谱图数据为:1H NMR(400MHz,DMSO)δ9.37(d,J=7.4Hz,1H,ArH),9.13(s,1H,ArH),9.07(d,J=15.8Hz,1H,CH),8.67(d,J=8.0Hz,1H,ArH),8.40(d,J=8.7Hz,1H,ArH),8.27(m,2H,ArH),8.17(m,1H,ArH),8.03(d,J=15.7Hz,1H,CH),7.94(m,1H,ArH),7.87(m,1H,ArH),7.75(d,J=8.2Hz,1H,CH),7.59(m,1H,ArH),7.39(m,1H,ArH),4.55(q,J=7.2Hz,2H,CH2),4.32(s,2H,NH2),1.38(q,J=8.1Hz,3H,CH3).
实施例4: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1-20μM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450-650nm范围内(图1)。其中化合物I1在413nm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30倍,光谱在446nm处具有等吸收点;化合物I2在454nm左右的紫外吸收峰值随化合物I2的pH减小而减小,相反其在565nm左右的紫外吸收峰值随pH减小而增加,光谱在491nm处具有等吸收点,其峰值相差53倍,光谱在492nm处具有等吸收点;化合物I3在415nm左右的紫外吸收峰值随化合物I3的pH减小而减小,相反其在570nm左右的紫外吸收峰值随pH减小而增加,其峰值相差265倍,光谱在475nm处具有等吸收点(图1)。
实施例5:本发明部分化合物的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600-750nm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;化合物I2在662nm左右的荧光峰值随化合物I2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25倍;化合物I3在708nm左右的荧光峰值随化合物I3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23倍(图2)。
实施例6: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I和对照化合物Ru(bpy)3 2+从930nm到1060nm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990nm具有最大的双光子吸收截面(δmax=171GM)(图3)。
实施例7:采用共聚焦显微镜进行细胞成像
参照图4,采用结肠癌细胞(HT29)、正常结肠上皮细胞(CCD841)、肝癌细胞(HepG2)或乳腺癌细胞(MCF7)进行共聚焦显微镜进行细胞成像,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=450-650nm,λex=600-750nm。
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,而对正常结肠上皮细胞荧光成像很弱(图4),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8: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A549肺癌模型裸鼠,将其处死,取出肺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50μM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5所示,肺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9: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3~5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6),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 (10)
1.一类咔唑苯并[cd]吲哚鎓盐,其特征在于:具有如下通式所示结构:
所述咔唑苯并[cd]吲哚鎓盐为pH敏感的近红外成像的咔唑苯并[cd]吲哚鎓盐荧光探针;R选自H、NO2和NH2中的一种,Y-代表卤负离子、六氟磷酸根负离子、对甲苯磺酸负离子和甲磺酸负离子中的一种。
2.根据权利要求1所述的咔唑苯并[cd]吲哚鎓盐,其特征在于,R、Y选自如下组合:
R=H,Y=PF6;
或者R=NO2,Y=I;
或者R=NH2,Y=I。
3.一种咔唑苯并[cd]吲哚鎓盐的制备方法,其特征在于:将6-R-9-乙基-9H-咔唑-3-甲醛与1,2-二甲基苯并[cd]吲哚-1-Y鎓盐在催化量哌啶条件下,加热回流,发生Knoevenagel缩合反应获得如下通式所示结构的咔唑苯并[cd]吲哚鎓盐;
所述制备方法的合成路线如下所示:
其中,R为H或NO2,Y-代表卤负离子、六氟磷酸根负离子、对甲苯磺酸负离子和甲磺酸负离子中的一种。
4.一种咔唑苯并[cd]吲哚鎓盐的制备方法,其特征在于,化合物Ia在铁粉和氯化铵的甲醇溶剂下,加热回流,经还原反应获得化合物Ib;化合物Ia为通式Ⅰ中R为NO2时对应的咔唑苯并[cd]吲哚鎓盐,化合物Ib为通式I中R为NH2时对应的咔唑苯并[cd]吲哚鎓盐;
所述制备方法的合成路线如下:
其中Y-代表卤负离子、六氟磷酸根负离子、对甲苯磺酸负离子和甲磺酸负离子中的一种。
5.权利要求1所述的咔唑苯并[cd]吲哚鎓盐在制备pH响应、实现肿瘤组织或肿瘤细胞的选择性荧光成像的试剂中的应用。
6.权利要求1所述的咔唑苯并[cd]吲哚鎓盐在制备通过单光子和/或双光子激发荧光成像的试剂中的应用。
7.根据权利要求5所述的应用,其特征在于,所述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。
8.根据权利要求5所述的应用,其特征在于,所述试剂由咔唑苯并[cd]吲哚鎓盐溶于助溶剂/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。
9.根据权利要求8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%。
10.根据权利要求5或7~8任一项所述的应用,其特征在于,所述肿瘤包括肺癌、乳腺癌、结肠癌、肝癌、宫颈癌和胰腺癌肿瘤中的一种。
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