CN109369633B - 一种可靶向线粒体的近红外二区荧光化合物及其制备方法与应用 - Google Patents
一种可靶向线粒体的近红外二区荧光化合物及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种可靶向线粒体的近红外二区荧光化合物及其制备方法与应用。其发射波长从600到1200纳米,经过不同官能团的修饰,其发射波长可以到达近红外二区(900‑1200纳米)。该化合物可用于线粒体成像、光热治疗、骨肉瘤、全身骨成像等生物领域。增加的可修饰位点可用于连接不同的生物活性基团,进而改善其水溶性、生物相容性以及靶向性,还可用于多种疾病标志物的体外检测以及乳腺癌、脑胶质瘤、结肠癌、肝癌等肿瘤的在体早期诊断等。本发明的荧光化合物具有结构新颖、合成步骤简单、抗光漂白,无毒,生物相容性好等优点,具有极好的工业生产价值以及生物医学应用前景。
Description
技术领域
本发明属于近红外二区荧光探针染料领域,具体涉及一种新型噻喃鎓结构的近红外荧光化合物,其发射波长位于600-1200nm。经过不同官能团的修饰,其发射波长可以达到近红外二区(900-1600nm),可以用于线粒体成像以及生物医学材料领域中光热治疗、癌症检测、肿瘤成像以及手术导航。
背景技术
癌症(又称恶性肿瘤)严重威胁着人类健康。由于医疗技术水平的限制,目前缺乏对晚期癌症的有效治疗手段,所以癌症的早期诊断对患者来说尤为重要,若能尽早发现并及时采取治疗,可以显著提高癌症患者的存活率。非侵入式的活体动物荧光成像技术等分子影像技术的出现为癌症的早期诊断开拓了新的发展道路。
近红外二区(NIR-II,1000-1700nm)中的荧光成像已经广泛用于生物和生物医学研究,因为在这个波长区域的光线具有很深的穿透深度,较高的空间分辨率以及较低的生物自发荧光等优点。最初的近红外二区小分子荧光探针都是以苯并双噻二唑(BBTD)作为电子受体(A)以及三苯胺作为电子给体(D)组成供体-受体-供体(D-A-D)分子骨架构建,尽管最近已经发现了一系列近红外二区小分子荧光探针,但已报道的近红外二区小分子探针基本都是以苯并双噻二唑为母核构建,其结构较为单一(Nat.Mater.,2016,15,235-242;Chem.Sci.,2016,7,6203-6207)。因此寻找具有良好的生物相容性,低毒性和高光稳定性的新型近红外二区小分子探针的骨架势在必行。
作为具有正电荷的杂环型发光化合物的重要结构组成,吡喃鎓盐广泛用作光敏剂,分子开关剂和近红外染料。目前,还没有用硫原子将吡喃鎓盐中的氧原子取代,从而得到噻喃鎓盐的报道。
发明内容
为了解决现有技术存在的不足,本发明的目的在于提供一类可修饰的、光稳定性高的、生物兼容性好的新型D-A结构的近红外二区荧光有机小分子染料,并将其用作线粒体成像、骨肉瘤等癌症的早期诊断以及光热治疗。
为实现上述发明目的,本发明提供如下技术方案:
第一方面,提供一种可修饰的近红外二区荧光化合物,具有通式(I)所述的结构:
其中:n=0~1;
所述的具有通式(I)所示的结构的荧光化合物,其荧光发射波长为600~1200nm。
第二方面,提供一种通式(I)所述的荧光化合物的制备方法,反应路线如下:
其中:n=0~1;
反应条件为:
(1)将化合物2溶于20mL乙醇中,加入20%KOH溶液并在室温下反应10分钟,然后将化合物3加入到混合物中,并将反应在室温下搅拌过夜;冷却至室温后,用2M HCl溶液将反应溶液调节至pH=3,过滤收集形成的黄色中间体化合物4;
(2)将化合物5和四氢吡咯溶于苯中,将溶液加热至100℃并搅拌4小时。冷却至室温后,减压旋除苯。然后将化合物4和无水的1,4-二氧六环加入上述反应液中,将溶液回流加热6小时,反应完全后,将水加入混合物中,并用乙酸乙酯萃取3次,之后,将有机层用饱和盐水洗涤。将合并的有机层用无水硫酸镁干燥,过滤并浓缩。然后真空除去溶剂,粗产物通过硅胶柱色谱纯化,得到中间体化合物6;
(3)将化合物6溶于***中并搅拌10分钟,向该混合物中加入硫代乙酸和三氟化硼醚在回流下反应3小时。冷却至室温后,将反应混合物用水淬灭,向溶液中加入过量的***,然后在室温下搅拌混合物以沉淀得到浅黄色中间体化合物7;
(4)在反应容器中加入化合物7,原料8和乙酸酐,70℃搅拌2h,加入大量***,析出粗产物,通过硅胶色谱柱纯化得到最终产物1,即通式(I)所述的荧光化合物。
优选地,上述步骤(1)所述的化合物2、3的摩尔比为1:1~3;步骤(2)所述的化合物4、四氢吡咯和5的摩尔比为1~5:2~5:1~10;步骤(3)所述的化合物6、硫代乙酸和三氟化硼***的摩尔比为1~5:3~10:4~20;步骤(4)所述的化合物7、原料8的摩尔比为1~3:2~10。
第三方面,提供一种用于生物体内成像的近红外二区荧光成像探针,具体为在通式(I)所示的化合物可修饰位点修饰聚乙二醇、多肽、蛋白、核酸适配体、叶酸及其衍生物。
第四方面,提供上述近红外二区荧光成像探针在制备用于细胞线粒体成像的试剂中的应用。
第五方面,提供上述近红外二区荧光成像探针在制备用于肿瘤诊断的试剂中的应用。
优选地,所述肿瘤指骨肉瘤、结肠癌、肝癌、脑胶质瘤、乳腺癌、***癌、黑色素瘤、食道癌、***、卵巢癌。
第六方面,提供上述近红外二区荧光成像探针在制备用于骨肉瘤成像的试剂中的应用。
第七方面,提供上述近红外二区荧光成像探针在制备用于骨肉瘤光热治疗的药物中的应用。
第八方面,提供上述近红外二区荧光成像探针在制备用于在生物体内成像的试剂中的应用。
本发明的近红外二区荧光化合物,由市售的化合物2、3在碱性条件下经过羟醛缩合反应得到化合物4,在四氢吡咯作用下,与5发生迈克尔加成反应,得到化合物6,随后在硫代乙酸和三氟化硼***共同作用下关环生成主体分子结构7,最后与原料8反应得到终产物1,即如通式(I)所述的终产物,为具有D-A结构的全新荧光化合物,其荧光发射波长位于600~1200纳米,从实施例中可知该方案合成路线简单,反应效率高,产率高,后处理简单,毒性小,生物兼容性好,易被生物体吸收和代谢。经不同后修饰,可以诊断及检测不同的肿瘤,具有较高的工业生产价值。
本发明的创造性在于引入了噻喃鎓盐结构母核,通过改变不同的基团可以使荧光红移,最终得到易制备的近红外二区荧光化合物。而且,是第一个可以用于线粒体成像的近红外二区染料。左侧环上引入可修饰基团,增加的可修饰位点可用于连接不同的生物活性官能团或者靶向基团,进而增加荧光探针的应用前景、改善其水溶性和生物相容性以及提高对不同肿瘤的靶向性。在生物医学成像实验中发现该荧光探针成像效果非常好,具有广阔的应用前景。
附图说明
图1为具体实施方式中近红外二区荧光成像探针1aa~1ag的合成路线。
图2为近红外二区荧光化合物1ag的H谱。
图3为近红外二区荧光化合物1ag的C谱。
图4为近红外二区荧光成像探针1aa~1ag的最高占据分子轨道和最低未占分子轨道图能级图。
图5为化合物1aa~1af的吸收光谱图。
图6为化合物1aa~1af的发射光谱图。
图7为近红外二区荧光化合物1ag在不同溶剂中的吸收谱图。
图8为近红外二区荧光化合物1ag在不同溶剂中的发射谱图。
图9为近红外二区成像探针1ag(10nM-100μM)在143B细胞内的线粒体成像图。
图10为近红外二区荧光成像探针1ag-PEG-PT修饰物的合成路线。
图11为为近红外二区荧光成像探针1ag-PEG-PT修饰物的Maldi TOf质谱。
图12为上侧为尾静脉注射化合物1ag-PEG-PT(150μg)修饰物在进入左后肢接种肿瘤细胞的荷瘤小鼠体内近红外二区成像效果,下侧为尾静脉注射化合物1ag-PEG-PT(150μg)修饰物和多肽PT(2mg)的混合液在左后肢接种肿瘤细胞的荷瘤小鼠体内近红外二区成像效果。
图13为荷瘤小鼠尾静脉注射注射化合物1ag-PEG-PT(150μg)修饰物在808nm激光下的光热成像图,激光照射时间为5分钟。
图14为荷瘤小鼠尾静脉注射注射化合物1ag-PEG-PT(150μg)修饰物后,在808nm激光连续照射5分钟后,14天后离体的肿瘤大小。
具体实施方式
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明,而不以任何方式限制本发明揭示的其余内容。
实施例1:化合物4a的制备
将化合物2a(1.36g,10mmol)溶于20mL乙醇中,加入20%KOH(20mL)溶液并在室温下反应10分钟。然后将苯甲醛3a(1.16g,11mmol)加入到混合物中,并将反应在室温下搅拌过夜。冷却至室温后,用2M HCl溶液将反应溶液调节至pH=3。过滤收集形成的黄色中间体查尔酮。将查尔酮(1.12g,5mmol),碳酸钾(1.52g,11mol)和丙酮(10mL)加入到反应容器中,室温搅拌10分钟,然后向混合物中加入3-溴丙炔(0.654g,5.5mol)加热回流并搅拌4小时。然后过滤混合物,减压浓缩滤液,得到产物4a(1.27g,产率97%)。
化合物4a结构测定数据如下:
1H NMR(400MHz,Acetone-d6)δ8.24–8.18(m,2H),7.92(d,J=15.6Hz,1H),7.88–7.83(m,2H),7.79(d,J=15.6Hz,1H),7.52–7.43(m,3H),7.21–7.15(m,2H),4.95(d,J=2.4Hz,2H),3.19(t,J=2.4Hz,1H).
13C NMR(101MHz,Acetone-d6)δ187.3,161.5,143.3,135.3,131.7,130.7,130.3,128.9,128.6,121.9,114.7,78.3,76.7,55.6.
实施例2:化合物6a的制备
将环戊酮(1.2g,14.3mmol)和四氢吡咯(1.02g,14.3mmol)溶于苯(20mL)中,将溶液加热至100℃并搅拌4小时。冷却至室温后,减压旋除苯。然后将化合物4a(2.5g,9.53mmol)和无水的1,4-二氧六环(20mL)加入上述反应液中,将溶液回流加热6小时。反应完全后,将水(40mL)加入混合物中,并用乙酸乙酯萃取(50mL×3)。之后,将有机层用饱和盐水洗涤。将合并的有机层用无水硫酸镁干燥,过滤并浓缩。然后真空除去溶剂,粗产物通过硅胶柱色谱纯化(2.17g,yield 66%)。
化合物6a结构测定数据如下:
1H NMR(400MHz,Chloroform-d)δ8.02–7.95(m,2H),7.28(m,1H),7.25(m,2H),7.25–7.16(m,2H),7.03–6.98(m,2H),4.74(d,J=2.5Hz,2H),3.82–3.68(m,2H),3.48–3.35(m,1H),2.55(t,J=2.4Hz,1H),2.29–2.19(m,1H),2.13(m,1H),1.92(m,1H),1.77(m,2H),1.71–1.58(m,2H).
13C NMR(101MHz,Chloroform-d)δ220.8,197.7,161.4,142.5,131.0,130.5,128.6,128.6,126.8,114.7,77.9,76.3,55.9,53.2,41.3,40.7,39.8,27.2,20.7.
实施例3:化合物7a的制备
将化合物6a(1.2g,3.46mmol)溶于***(10mL)中并搅拌10分钟,向该混合物中加入硫代乙酸(659mg,8.66mmol)和三氟化硼醚(2.46g,17.32mmol)在回流下加热3小时。冷却至室温后,将反应混合物用水淬灭,向溶液中加入过量的***。然后在室温下搅拌混合物以沉淀浅黄色固体(400mg,yield 50%)。
化合物7a结构测定数据如下:
1H NMR(400MHz,Acetonitrile-d3)δ8.63(s,1H),8.01(d,J=9.1Hz,2H),7.85–7.76(m,2H),7.76–7.63(m,3H),7.29(d,J=8.9Hz,2H),4.92(d,J=2.4Hz,2H),3.67(t,J=7.6Hz,2H),3.34(t,J=7.4Hz,3H),2.95(t,J=2.5Hz,1H),2.37(p,J=7.5Hz,2H).
13C NMR(101MHz,Acetonitrile-d3)δ174.5,165.7,161.9,159.4,149.5,137.2,132.5,131.7,130.3,129.3,129.2,127.3,116.6,77.8,76.8,56.2,38.2,34.1,24.6.
HRMS(ESI)Calcd for:C23H19OS+([M-BF4]+):343.1151,found:343.1155.
实施例4:化合物1aa-1ag的制备
在反应容器中加入化合物7a(1eq),原料8a(1.3eq)以及醋酸酐2mL,70℃搅拌2h。加入大量***,析出粗产物,通过硅胶色谱柱纯化得到最终产物1aa-1ag(产量:1a 48%;1b52%;1c 58;1d 49;1e 52%;1f 54%;1g 50%)。
1aa 1H NMR(400MHz,Acetonitrile-d3)δ8.47(s,1H),8.03(d,J=8.9Hz,2H),7.77(d,J=7.0Hz,2H),7.76(d,J=7.8Hz,2H),7.72(d,J=7.2Hz,2H),7.70–7.68(m,2H),7.54(d,J=7.5Hz,2H),7.29(d,J=8.8Hz,2H),5.47(s,1H),4.92(d,J=2.4Hz,2H),3.50–3.43(m,2H),3.39–3.33(m,2H),2.95(t,J=2.4Hz,1H).
HRMS(ESI)Calcd for:C30H23OS+([M-BF4]+):431.15,found:431.21.
1ab 1H NMR(600MHz,Acetonitrile-d3)δ8.48(s,1H),8.04(d,J=8.8Hz,2H),7.80(s,1H),7.80–7.74(m,4H),7.69(d,J=7.0Hz,2H),7.31(dd,J=8.8,1.9Hz,4H),4.93(d,J=2.5Hz,2H),3.52–3.45(m,2H),3.41–3.34(m,2H),2.94(t,J=2.5Hz,1H),2.32(s,3H).13C NMR(151MHz,Acetonitrile-d3)δ171.0,163.9,154.2,143.9,138.8,135.1,134.6,133.7,133.3,133.0,132.7,132.0,131.6,131.1,130.6,130.5,129.2,124.5,124.4,118.4,79.5,78.5,58.0,31.6,31.1,22.0.
HRMS(ESI)Calcd for:C31H25O3S+([M-BF4]+):489.1519,found:489.1509.
1ac 1H NMR(600MHz,Acetonitrile-d3)δ8.37(s,1H),8.00(d,J=8.9Hz,2H),7.78–7.73(m,3H),7.71(d,J=8.9Hz,2H),7.69–7.66(m,3H),7.28(d,J=8.9Hz,2H),7.09(d,J=8.8Hz,2H),4.91(d,J=2.4Hz,2H),3.89(s,3H),3.46–3.43(m,2H),3.33–3.30(m,2H),2.94(t,J=2.4Hz,1H).13C NMR(151MHz,Acetonitrile-d3)δ163.8,163.7,141.5,138.9,136.8,135.3,134.8,133.1,132.1,131.8,131.6,131.3,131.2,131.0,130.6,129.8,129.2,118.3,116.7,79.5,78.5,58.0,57.2,34.3,31.6.
HRMS(ESI)Calcd for:C31H25O2S+([M-BF4]+):461.1570,found:461.1555.
1ad 1H NMR(600MHz,Acetonitrile-d3)δ8.50(s,1H),8.06(d,J=8.9Hz,2H),7.82(s,1H),7.81–7.76(m,4H),7.73–7.68(m,3H),7.47(d,J=8.5Hz,2H),7.31(d,J=8.9Hz,2H),4.94(d,J=2.3Hz,2H),3.55–3.49(m,2H),3.42–3.38(m,2H),3.29(s,3H),2.95(t,J=2.4Hz,1H),1.30(s,3H).13C NMR(151MHz,Acetonitrile-d3)δ174.6,163.6,147.8,143.9,138.5,135.2,134.9,133.8,133.5,133.1,133.0,132.2,131.7,131.1,131.0,130.8,130.4,128.9,118.1,117.7,79.2,78.3,57.7,34.2,31.4,30.8,23.4.
HRMS(ESI)Calcd for:C33H28NO2S+([M-BF4]+):502.1835,found:502.1855.
1ae 1H NMR(500MHz,Acetonitrile-d3)δ8.40(s,1H),8.00(d,J=8.9Hz,2H),7.76–7.71(m,3H),7.69–7.65(m,3H),7.63(d,J=8.5Hz,2H),7.37(d,J=8.5Hz,2H),7.27(d,J=8.9Hz,2H),4.90(d,J=2.4Hz,2H),3.46–3.42(m,2H),3.34–3.30(m,2H),2.91(t,J=2.4Hz,1H),2.53(s,3H).13C NMR(126MHz,Acetonitrile-d3)δ162.1,143.8,141.2,137.2,134.4,131.5,131.4,131.2,130.5,130.3,130.1,129.6,129.5,129.3,129.2,128.8,127.5,125.7,116.6,77.8,76.8,56.2,32.7,30.0,19.6.
HRMS(ESI)Calcd for:C31H25OS2+([M-BF4]+):477.1341,found:477.1330.
1af 1H NMR(500MHz,Acetonitrile-d3)δ8.05(s,1H),7.90(d,J=8.8Hz,2H),7.72(s,1H),7.71–7.68(m,2H),7.66–7.62(m,5H),7.24(d,J=8.8Hz,2H),6.84(d,J=9.0Hz,2H),4.89(d,J=2.4Hz,2H),3.38–3.33(m,2H),3.28–3.22(m,2H),3.11(s,6H),2.93(t,J=2.4Hz,1H).13C NMR(126MHz,Acetonitrile-d3)δ170.4,161.1,156.0,154.8,153.0,147.2,138.2,137.6,136.4,134.5,130.8,129.4,129.3,129.2,128.6,127.7,123.5,116.2,112.6,77.9,76.7,56.1,39.5,31.9,30.2.
HRMS(ESI)Calcd for:C32H28NOS+([M-BF4]+):474.1886,found:474.1895.
1ag 1H NMR(800MHz,Acetonitrile-d3)δ8.12(s,1H),7.95(s,1H),7.91(d,J=8.9Hz,2H),7.70(dd,J=6.8,2.9Hz,2H),7.68–7.66(m,3H),7.58(d,J=3.9Hz,1H),7.56(d,J=8.7Hz,2H),7.44(d,J=3.9Hz,1H),7.25(d,J=8.9Hz,2H),6.68(d,J=8.7Hz,2H),4.91(d,J=2.4Hz,2H),3.48–3.28(m,2H),3.23–3.11(m,2H),2.95(s,6H),2.92(t,J=2.4Hz,1H).13C NMR(151MHz,Acetonitrile-d3)δ168.1,161.3,158.1,156.0,155.5,151.2,148.4,137.7,137.0,130.8,129.8,129.5,129.3,129.2,129.0,129.0,128.5,127.2,126.9,122.8,120.0,116.1,111.9,77.6,76.5,55.9,34.9,31.3,29.2.
HRMS(ESI)Calcd for:C23H19OS+([M-BF4]+):556.18,found:556.42.
化合物1a的合成路线如图1所示。化合物1ag的核磁共振氢谱见图2,核磁共振碳谱见图3,证明其结构正确。
实施例5:荧光化合物1ag在143B细胞中的线粒体成像
在培养瓶中贴壁生长的U87-MG细胞,消化,共3mL;取0.5mL细胞悬浮液于EP管中,加入1.5mL新鲜培养基稀释;涡旋,使细胞分散均匀,在荧光小皿中加入1mL细胞悬浮液;培养24h后,使细胞贴壁生长,加入荧光化合物1g(10nM~100μM),孵育6h;6h后,弃去含有化合物的培养基,用PBS反复清洗细胞3次;配好100nM的Mito-Tracker Green溶液,取1μL至1mL培养基中,摇匀加入荧光小皿中,染色30min;染色结束后,再次用PBS清洗细胞3次,尽量除去除贴壁生长细胞外的其他物质;加入新鲜培养基,可直接用于激光共聚焦显微镜的观察。参见图9,通过用Mito-Tracker Green和1ag分别对细胞的线粒体染色,发现两者几乎完全重合,证明了化合物1ag对线粒体的靶向。本发明所述材料在近红外二区线粒体染色方面具有较好的应用前景。
实施例6:化合物PEG-PT的制备
在氩气氛围下,将化合物COOH-PEG-N3(2.0mg,0.0024mmol),EDCI(0.55mg,0.00288mmol)和NHS(0.33mg,0.00288mmol)溶解于500μL重蒸的DMF中,室温搅拌30分钟。然后将肽PPSHTPT(2.1mg,0.00288mmol)和DIPEA(50μL)加入到上述反应混合物中,并将溶液在室温下搅拌过夜。粗产物在冷***中沉淀。将粗产物溶于水中并通过HPLC纯化。化合物PEG-PT修饰物的结构测定数据如下:MALDI-TOF-MS Calcd for:1537.3([M-BF4]+),Measured M.W.1538.9.
实施例7:化合物1ag-PEG-PT修饰物的制备及肿瘤成像效果
在反应容器中加入1ag(1.2mg,0.00186mmol),PEG-PT(2.86mg,0.00186mmol)和DMF/PBS(300μL/300μL),然后在上述溶液中加入50μL催化剂溶液(10mM CuSO4·5H2O,在20mM PBS中的50mM抗坏血酸钠),混合物在室温下在氩气保护下搅拌过夜。粗产物在冷***中沉淀。将粗产物溶于水中并通过HPLC纯化。化合物1ag-PEG-PT修饰物的结构测定数据如下:MALDI-TOF-MS Calcd for:2094.3([M-BF4]+),Measured M.W.2094.6。化合物1ag-PEG-PT修饰物的质谱图见图11,分子量与预期的相符,其结构正确。
通过尾静脉注射如上所述制作的近红外二区荧光染料1ag-PEG-PT 150μg进入左后肢接种肿瘤细胞的荷瘤小鼠体内,近红外二区相机拍摄小鼠肿瘤部位图,参见图12,小鼠肿瘤部位清晰可见。本发明所述材料在肿瘤早期诊断方面具有较好的应用前景。
通过尾静脉注射如上所述制作的近红外二区荧光染料1ag-PEG-PT 200μg进入左后肢接种肿瘤细胞的荷瘤小鼠体内,12小时后,用808nm激光连续照射荷瘤小鼠肿瘤部位5分钟,参见图13,肿瘤部位温度随时间的变化清晰可见。光热治疗成像效果参见图14,注射了1ag-PEG-PT染料并且肿瘤用激光照射5分钟的荷瘤小鼠,在14天后肿瘤明显减小甚至消失。本发明所述材料在肿瘤光热治疗方面具有较好的应用前景。
Claims (10)
2.一种如权利要求1所述的荧光化合物的制备方法,其特征在于,反应路线如下:
反应条件为:
(1)将化合物2a溶于20mL乙醇中,加入20%KOH溶液并在室温下反应10分钟,然后将化合物3a加入到混合物中,并将反应在室温下搅拌过夜;冷却至室温后,用2M HCl溶液将反应溶液调节至pH=3,过滤收集形成的黄色中间体,然后将得到的中间体溶于丙酮,加入碳酸钾,与原料溴丙炔在碱性条件下回流反应4小时,得到化合物4a;
(2)将化合物5a和四氢吡咯溶于苯中,将溶液加热至100℃并搅拌4小时,冷却至室温后,减压旋除苯,然后将化合物4a和无水的1,4-二氧六环加入上述反应液中,将溶液回流加热6小时,反应完全后,将水加入混合物中,并用乙酸乙酯萃取3次,之后,将有机层用饱和盐水洗涤,将合并的有机层用无水硫酸镁干燥,过滤并浓缩,然后真空除去溶剂,粗产物通过硅胶柱色谱纯化,得到中间体化合物6a;
(3)将化合物6a溶于***中并搅拌10分钟,向该混合物中加入硫代乙酸和三氟化硼***在回流下反应3小时,冷却至室温后,将反应混合物用水淬灭,向溶液中加入过量的***,然后在室温下搅拌混合物以沉淀得到浅黄色中间体化合物7a;
(4)在反应容器中加入化合物7a,原料8a和乙酸酐,70℃搅拌2h,加入大量***,析出粗产物,通过硅胶色谱柱纯化得到最终产物1ag荧光化合物。
3.根据权利要求2所述的制备方法,其特征在于,步骤(1)所述的化合物2a、3a的摩尔比为1:1~3;步骤(2)所述的化合物4a、四氢吡咯和5a的摩尔比为1~5:2~5:1~10;步骤(3)所述的化合物6a、硫代乙酸和三氟化硼***的摩尔比为1~5:3~10:4~20;步骤(4)所述的化合物7a、原料8a的摩尔比为1~3:2~10。
4.一种用于生物体内成像的近红外二区荧光成像探针,其特征在于,所述探针为在权利要求1的近红外二区荧光化合物可修饰位点修饰聚乙二醇、多肽、蛋白、核酸适配体、叶酸。
5.权利要求4所述的近红外二区荧光成像探针在制备用于细胞线粒体成像的试剂中的应用。
6.权利要求4所述的近红外二区荧光成像探针在制备用于肿瘤诊断的试剂中的应用。
7.根据权利要求6所述的应用,其特征在于,所述肿瘤指骨肉瘤、结肠癌、肝癌、脑胶质瘤、乳腺癌、***癌、黑色素瘤、食道癌、***、卵巢癌。
8.权利要求4所述的近红外二区荧光成像探针在制备用于骨肉瘤成像的试剂中的应用。
9.权利要求4所述的近红外二区荧光成像探针在制备用于骨肉瘤光热治疗的药物中的应用。
10.权利要求4所述的近红外二区荧光成像探针在制备用于在生物体内成像的试剂中的应用。
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