CN114644598B - 二氢吩嗪衍生物及其用途 - Google Patents
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Abstract
本发明公开了一种二氢吩嗪衍生物,具有以下结构通式:本发明还提供了一种所述二氢吩嗪衍生物在制备用于活体脑血管或肝脏脂滴的三光子荧光成像剂中的应用。本发明制备的二氢吩嗪衍生物具有三光子荧光发射信号,相较于单光子/双光子荧光成像,能够增加生物活体组织成像深度,显著提高信噪比。
Description
技术领域
本发明属于活体三光子荧光成像技术领域,具体地说,涉及一种二氢吩嗪衍生物及其用途。
背景技术
脂滴(LDs)是一种存在于所有真核细胞中的细胞器。顾名思义,它们充满脂肪并被单层磷脂包围(与大多数其他细胞器周围的经典双层膜相反)。LDs主要含有甘油三酯和胆固醇酯,作为动态细胞器,参与许多细胞功能,包括脂质代谢、膜合成与传递、信号转导、蛋白质降解等。近年来,由于积累已发现外周组织中LDs与许多代谢疾病有关,包括非酒精性脂肪肝、糖尿病、肥胖和动脉粥样硬化。传统的成像技术,如透射电子显微镜、拉曼显微镜和免疫荧光显微镜已应用于脂滴成像,但这些方法具有复杂的程序和降低的细胞通透性,可能会干扰细胞功能。同时,脂肪肝是慢性肝病的主要原因。虽然超声和磁共振成像(MRI)等影像学检查可以检测肝脂肪变性,并且越来越多地被接受作为组织学检查的替代方法,不过肝活检仍然是目前脂肪肝的诊断标准。但肝活检创伤大,患者难以接受,临床上并不将肝活检作为常规检查。因此,开发用于直接和选择性脂滴可视化和监测含有活细胞和组织的生物样品的有效方法至关重要。
荧光成像是一种广泛用于显示细胞和组织中脂质的技术。它具有灵敏度高、采集速度快、可靠性好、分辨率高的关键优势。最近,已经开发了用于定位LD的荧光探针。虽然目前最广泛用于LDs标记的商业荧光染料是油红O、尼罗红和BODIPY 505/515,但它们显示出强背景和小斯托克斯位移的一些致命缺点。由于它们的高疏水性,只能用于体外细胞和固定组织染色。并且,严重的散射和吸收将高分辨率光学成像限制在薄层或表层,使得在生物组织深处获得高质量的光学图像具有挑战性。
由于传统的近红外(NIR,700-1000nm)激发激光在生物组织中表现出较少的光子吸收,双光子荧光显微镜(2PFM)成像可以大大提高穿透深度。此外,一些荧光团,如三苯胺(TPA)和萘,已被用于设计用于LD标记的双光子发射荧光团。然而,由于激发光束在生物基质中的光散射,2PF成像通常仅限于平面图像切片,并且信噪比很低。因此,近年来三光子荧光显微镜(3PFM)成像逐渐被开发并应用于脑血管成像。与2PFM成像相比,3PFM成像的FS激发波长通常在NIR-II范围内(1000-1700nm)。减少光子散射的长波长飞秒激光也可以有效穿透生物基质,聚焦在目标生物组织,产生明亮的三光子荧光(3PF)用于深层组织成像。已经有报道开发出用于脂质体内三光子成像的NIR发射荧光团,用于检测动脉粥样硬化。因此,三光子荧光显微镜是监测和观察活体动物器官脂质体的强大而理想的工具。
基于体内三光子荧光成像的优势,选择还原吩嗪作为分子设计的核心,以丰富具有三光子荧光成像能力的有机染料体系。还原吩嗪是吩嗪天然产物的人工衍生物。许多常见的染料如中性红、克林丹宁蓝等都是以吩嗪为基础的。与常规的电子供体三苯胺相比,还原的1,5-二氢吩嗪有两个sp2杂化氮原子,未配对的电子对与苯环的电子形成p-π共轭,增加了环内电子云的密度。因此,它具有更强的给电子能力,可以促进具有供体-受体(D-A)结构的小分子的长波长发射。此外,还原吩嗪具有许多修饰位点、优异的稳定性和生物相容性,其衍生物在荧光方面得到了广泛的研究。
发明内容
本发明的第一个目的是提供一种用于三光子脂滴成像的结构新颖的二氢吩嗪衍生物。
本发明的第二个目的是提供一种所述二氢吩嗪衍生物在制备用于活体脑血管或肝脏脂滴的三光子荧光成像剂中的应用。
为了实现上述目的,本发明采用的技术方案如下:
本发明的第一个方面提供了一种二氢吩嗪衍生物,具有以下结构通式:
其中,R1选自氢、C1~C5烷基;
R2选自氢、C1~C5烷基;
R3选自氢、腈基(CN);
R4选自氢、腈基(CN)、
R5选自C1~C5烷基。
较优选的,所述二氢吩嗪衍生物中,
R1选自氢、甲基、乙基;
R2选自氢、甲基、乙基;
R3选自氢、腈基(CN);
R4选自氢、腈基(CN)、
R5选自甲基、乙基、正丁基、叔丁基、异丙基。
最优选的,所述二氢吩嗪衍生物选自以下结构的一种:
本发明的第二方面提供了一种所述二氢吩嗪衍生物在制备用于活体脑血管或肝脏脂滴的三光子荧光成像剂中的应用。
由于采用上述技术方案,本发明具有以下优点和有益效果:
本发明制备的二氢吩嗪衍生物具有较大的斯托克斯位移(~200nm),有利于避免荧光的吸收,提高成像对比度。
本发明制备的二氢吩嗪衍生物的分子尺寸小,毒性小,有利于进行生物成像应用。
本发明制备的二氢吩嗪衍生物具有三光子荧光发射信号,相较于单光子/双光子荧光成像,能够增加生物活体组织成像深度,显著提高信噪比。
附图说明
图1为化合物I-1a在环己烷、甲苯、乙酸乙酯、乙腈和二甲基亚砜(10μM)中的紫外吸收光谱示意图,其中:横坐标表示的是波长(单位为纳米),纵坐标表示吸光度。
图2为化合物I-1a在环己烷、甲苯、乙酸乙酯、乙腈和二甲基亚砜(10μM)中的归一化荧光发射光谱示意图,其中:横坐标表示的是波长(单位为纳米),纵坐标表示的是归一化荧光强度。
图3为化合物I-1a的荧光强度对激发激光强度的功率依赖性示意图。插图是毛细管中DMPCN的三光子荧光图像;其中:横坐标表示的是激发激光强度的功率的对数,纵坐标表示的荧光强度的对数。
图4为化合物I-1a在飞秒激光(1300nm,4mW)照射下的光稳定性评估示意图。
图5为化合物I-1a(25μg·mL-1)和a)BODIPY 505/515(5μg·mL-1)、b)MitoTrackerGreen(2μM)或c)LysoTracker Green(2μM)染色的MCF-7细胞的共聚焦荧光图像及其对应的散点图与Pearson相关系数(R),化合物I-1a激发波长λex:490nm;发射波长λem:商业荧光团(绿色)为500-530nm,化合物I-1a(红色)为580-630nm,比例尺:25μm。
图6为化合物I-1a标记的颅窗内3PF显微脑血管成像示意图,(a-h)从100μm深度到1100μm深度的3PF显微脑血管成像;3PF图像在1300nm fs处被激发;(i-l)在300μm(b)、700μm(d)、900μm(f)、1100μm(h)成像深度处沿白线的高斯拟合轮廓;(m)重建3D 3PF脑血管成像,深度从0到1200μm。
图7为脂肪肝中化合物I-1a染色的LDs的3PF显微成像示意图,(a)2PF和(b)5X物镜下LD的3PF显微成像;2PF和3PF图像分别由920nm fs和1300nm fs激发;(c)沿着(a)和(b)中LD的白线的归一化强度分布;(d-f)25X物镜下10到40μm深度的LD的3PF显微成像;(g)沿LD白线的高斯拟合轮廓,成像深度为10μm;(h)在0到50μm的深度重建LD的3D 3PF成像。
图8为(a)脂肪肝和(b)正常肝脏中被化合物I-1a染色的LDs的活体三光子荧光成像;(c)未注射化合物I-1a的肝组织三光子荧光成像示意图,激发:1300nm fs,发射:560-700nm。
具体实施方式
为了更清楚地说明本发明,下面结合优选实施例对本发明做进一步的说明。本领域技术人员应当理解,下面所具体描述的内容是说明性的而非限制性的,不应以此限制本发明的保护范围。
实施例1
将Pd2(dba)3(0.25mmol,230mg)、Ruphos(1mmol,470mg)与叔丁醇钠(30mmol,2.9g)加入到250mL三口烧瓶中,使用双排管抽真空并用吹风机加热烘干。然后用注射器加入化合物Ⅱ-1(10mmol,2.64g)、100mL重蒸甲苯与1.8mL重蒸苯胺(20mmol),混合加热至110℃,反应14小时。过滤反应液,将滤液旋干,干法上样柱层析提纯,展开剂PE:DCM(10/1,v/v),得到1.68g白色片状固体物质即化合物III-1,产率58.3%。1H NMR(600MHz,CDCl3,ppm):δ=7.17–7.10(m,4H),7.05(s,2H),6.77(t,J=7.3Hz,2H),6.55–6.50(m,4H),5.50(s,2H),2.18(s,6H);13C NMR(151MHz,CDCl3,ppm):δ=146.05,136.40,133.46,129.32,127.67,119.16,114.70,18.67;HRMS ESI(m/z)[M+H]+:calcd.for C20H21N2289.1705;Found,289.1694.
在100mL烧瓶中加入化合物Ⅲ-1(3.47mmol,1g)、NaH(4mmol,0.504g)、30mL超干DMF,氮气保护后,搅拌10min,加入2,3-二氟苯腈(4mmol,0.556g),常温反应8小时,溶液呈紫黑色。加入乙醇淬灭反应,过滤反应液,将滤液旋干,干法上样柱层析提纯,PE:DCM(2/1,v/v),得到600mg绿色粉末状固体即化合物IV-1,产率45%。1HNMR(400MHz,DMSO-d6)δ8.11(s,1H),7.77–7.69(m,2H),7.29–7.18(m,4H),7.13–7.02(m,5H),7.01–6.92(m,3H),2.05(s,3H),1.91(s,3H).13C NMR(151MHz,DMSO)δ148.51,147.21,145.53,142.54,140.74,139.64,132.18,130.64,130.41,129.94,129.74,129.02,127.99,124.73,124.28,122.74,121.65,119.15,118.34,107.15,40.60,40.39,40.19,39.77,39.56,39.35,18.67,17.85.HRMS EI(m/z)[M]+:calcd.for C27H21N3387.1735;Found,387.1731.
将化合物IV-1(0.2mmol,77.4mg)置于史莱克管中,抽换气3次进行氩气保护,加入二氯甲烷10mL,超声溶解,置于-78℃,加入二异丁基氢化铝(1.5mol/L,2mL),有黄色液体生成,搅拌30分钟,然后置于常温环境反应过夜。加入5mL 10%NaOH溶液淬灭反应,萃取反应液并旋干,分离纯化,得到40mg浅灰绿色固体即化合物V-1,产率50%。1H NMR(400MHz,DMSO-d6)δ10.02(s,1H),8.09(s,1H),7.87–7.75(m,2H),7.23(dt,J=21.2,7.9Hz,4H),7.14–7.00(m,5H),6.93(dd,J=14.9,7.6Hz,3H),2.10(s,3H),1.91(s,3H).13C NMR(151MHz,DMSO-d6)δ192.23,149.72,147.38,145.54,142.19,140.91,139.68,133.04,132.36,130.62,129.76,129.71,129.02,127.86,127.64,127.59,124.25,124.16,122.32,121.62,117.51,40.60,40.40,40.19,39.98,39.77,39.56,39.35,18.73,17.84.HRMS ESI(m/z)[M+H]+:calcd.for C27H23N2391.1810;Found,391.1801.
将丙二腈(1mmol,66mg)、醋酸铵(1mmol,77mg)和化合物Ⅴ-1(1mmol,390mg)混合溶解在10mL醋酸中,在氩气保护下,回流搅拌6小时。降至室温,将混合物倒入水中,抽滤析出红棕色固体,使用硅胶柱层析纯化,展开剂DCM:EtOH(20/1,v/v)。得到100mg红色固体即化合物I-1a,产率22.8%。1H NMR(400MHz,DMSO-d6)δ8.50(s,1H),8.18(d,J=1.8Hz,1H),7.85(dd,J=8.6,1.8Hz,1H),7.73(d,J=8.6Hz,1H),7.32(t,J=7.8Hz,2H),7.26–7.18(m,4H),7.14(t,J=7.3Hz,1H),7.07(d,J=7.9Hz,1H),7.04–6.93(m,4H),2.06(s,3H),1.83(s,3H).13C NMR(151MHz,DMSO-d6)δ158.29,150.45,148.03,144.41,140.56,139.91,138.05,132.51,129.47,129.40,129.29,129.17,128.89,127.37,127.11,125.97,125.69,125.25,122.82,120.37,119.06,114.51,113.46,78.90,77.35,77.03,76.72,53.43,19.73,17.97.HRMS ESI(m/z)[M+H]+:calcd.for C30H23N4439.1923;Found,439.1915.
实施例2
将化合物Ⅴ-1(0.256mmol,100mg)、对二甲基吡啶盐(0.256mmol,60mg)加入到50mL烧瓶中,加入10mL乙腈,1~2滴哌啶抽真空,90℃回流6小时,溶液呈深棕色,使用厚制备板进行纯化,展开剂DCM:EtOH(10/1,v/v),得到10mg黄棕色固体即化合物I-1b,产率6.4%。1H NMR(400MHz,DMSO-d6)δ8.87(d,J=6.6Hz,2H),8.22(d,J=6.5Hz,2H),8.10(d,J=16.2Hz,1H),8.05(s,1H),7.77(d,J=8.4Hz,1H),7.68(dd,J=8.2,1.4Hz,1H),7.63(d,J=16.3Hz,1H),7.19(dd,J=16.1,8.5Hz,4H),7.11(q,J=7.9Hz,2H),7.03(d,J=7.9Hz,2H),6.95(dd,J=7.4,5.2Hz,3H),6.88(t,J=7.2Hz,1H),4.26(s,3H),2.13(s,3H),1.99(s,3H).13C NMR(100MHz,DMSO-d6)δ:157.0,146.3,145.9,133.5,133.4,133.3,132.6,131.1,130.5,129.6,127.4,126.8,125.7,124.8,124.1,123.5,118.0,49.1,17.9.HRMSESI(m/z)[M]+:calcd.for C34H30N3 +480.2434;Found,480.2419.
实施例3
将化合物Ⅴ-1(0.256mmol,100mg)、2,3,3-三甲基-1-(3-磺丙基)-3H-吲哚盐(0.256mmol,72mg)加入到50mL烧瓶中,加入10mL乙腈,1~2滴哌啶抽真空,90℃回流6h,溶液呈紫黑色,使用厚制备板进行纯化,展开剂DCM:EtOH(10/1,v/v),得到10mg紫黑色固体即化合物I-1c,产率6.4%。1H NMR(400MHz,DMSO-d6)δ8.53(s,1H),8.31(d,J=9.8Hz,1H),8.01(d,J=5.9Hz,1H),7.92–7.86(m,1H),7.68(d,J=7.4Hz,1H),7.64–7.60(m,2H),7.31(t,J=7.5Hz,2H),7.23(t,J=9.1Hz,4H),7.11(t,J=5.4Hz,4H),7.05(d,J=7.8Hz,3H),6.93(d,J=7.5Hz,2H),4.88(t,J=8.2Hz,2H),2.15(s,3H),1.99(d,J=9.1Hz,2H),1.87(s,3H),1.84(s,6H),1.18–1.12(m,2H).13C NMR(100MHz,DMSO-d6)δ:173.1,145.9,141.2,138.1,135.8,133.3,132.6,128.3,126.8,125.7,125.0,124.1,123.5,120.1,118.0,113.3,111.0,55.9,54.9,51.0,24.7,20.0,125.7.HRMS ESI(m/z)[M+Na]+:calcd.forC41H39N3O3SNa 676.2310;Found,676.2300.
实施例4
将化合物Ⅴ-1(0.256mmol,100mg)、1,4-二甲基喹啉-1碘化物(0.256mmol,72.96mg)加入到50mL烧瓶中,加入10mL乙腈,1~2滴哌啶抽真空,90℃回流6小时,溶液呈棕黑色,使用厚制备板进行纯化,展开剂DCM:EtOH(10/1,v/v),得到20mg棕色固体即化合物I-1d,产率11.8%。1H NMR(400MHz,DMSO-d6)δ:9.01(d,J=7.5Hz,1H),8.60(d,1H),8.42(d,J=7.0Hz,1H),8.23(t,J=7.8Hz,1H),8.10(t,J=7.3Hz,1H),7.94(d,J=7.5Hz,1H),7.43(t,J=7.1Hz,1H),7.24(m,4H),7.08(m,5H),7.01(m,3H),6.95(m,2H),6.64(s,1H),4.39(s,3H),2.12(s,6H).13C NMR(100MHz,DMSO-d6)δ:157.0,146.3,145.9,139.8,136.0,133.3,132.6,131.1,130.5,129.6,126.8,125.7,124.8,124.2,123.5,122.1,118.6,118.0,45.3,25.7.HRMS ESI(m/z)[M]+:calcd.for C38H32N3 +530.2951;Found,530.2913.
实施例5
将化合物Ⅴ-1(0.256mmol,100mg)、1-丁基-4-甲基喹啉-1-碘化物(0.256mmol,83.71mg)加入到50mL烧瓶中,加入10mL乙腈,1~2滴哌啶抽真空,90℃回流6小时,溶液呈深棕色,使用厚制备板进行纯化,展开剂DCM:EtOH(10/1,v/v),得到15mg棕色固体即化合物I-1e,产率10.3%。1H NMR(400MHz,DMSO-d6)δ:9.03(d,J=7.5Hz,1H),8.58(d,1H),8.41(d,J=7.0Hz,1H),8.22(t,J=7.8Hz,1H),8.13(t,J=7.3Hz,1H),7.91(d,J=7.5Hz,1H),7.39(t,J=7.1Hz,1H),7.22(m,4H),7.05(m,5H),7.02(m,3H),6.96(m,2H),6.62(s,1H),5.01(t,J=7.2Hz,2H),2.13(s,6H),2.01(m,2H),1.30(m,2H),1.15(t,J=7.2Hz,3H).13C NMR(100MHz,DMSO-d6)δ:157.2,145.9,145.7,139.7,136.3,133.2,132.4,131.0,130.4,129.3,126.7,125.5,124.6,124.3,123.6,122.0,118.5,118.1,60.2,32.1,21.0,17.9,13.8.HRMS ESI(m/z)[M]+:calcd.for C41H38N3 +572.3060;Found,572.3045.
实施例6
化合物I-1a在不同溶剂中的紫外吸收-荧光发射光谱图
配置10mL化合物I-1a的DMSO溶液(10μM),分别取30μL加入到3mL环己烷、甲苯、乙酸乙酯、乙腈和DMSO中,进行紫外吸收-荧光发射光谱测试。如图1和图2所示,图1为化合物I-1a在环己烷、甲苯、乙酸乙酯、乙腈和二甲基亚砜(10μM)中的紫外吸收光谱示意图,其中:横坐标表示的是波长(单位为纳米),纵坐标表示吸光度。图2为化合物I-1a在环己烷、甲苯、乙酸乙酯、乙腈和二甲基亚砜(10μM)中的归一化荧光发射光谱示意图,其中:横坐标表示的是波长(单位为纳米),纵坐标表示的是归一化荧光强度。在不同溶剂中,化合物I-1a的最大吸收波长几乎不变,最大发射波长处于650nm到750nm范围内,并随着溶剂极性增加而发生红移。这些结果表明化合物I-1a具有较长的发射波长,有利于其应用于近红外荧光成像。
实施例7
化合物I-1a的三光子性能测试
用不同功率(10、15、20、25、30、35、40、45、50、55、60、65、70、75、80mW)的波长为1300nm(扫描速度为2.2μs/pixe)的飞秒激光扫描装有化合物I-1a(0.1mg mL-1)的毛细管,并通过过滤器(560–700nm)记录荧光信号并计算以进行比较。如图3和图4所示,图3为化合物I-1a的荧光强度对激发激光强度的功率依赖性示意图。插图是毛细管中DMPCN的三光子荧光图像;其中:横坐标表示的是激发激光强度的功率的对数,纵坐标表示的荧光强度的对数。图4为化合物I-1a在飞秒激光(1300nm,4mW)照射下的光稳定性评估示意图。化合物I-1a的荧光强度对激发激光强度的功率依赖性符合三光子荧光信号特征,并且在照射20min后,化合物I-1a的光稳定性仍保持在良好水平。这些结果说明化合物I-1a在波长为1300nm的飞秒激光的激发下能够产生640nm左右的三光子荧光发射信号,并且在激光的连续照射下,仍具有良好的稳定性,具有生物活体三光子成像的潜力。
实施例8
化合物I-1a对细胞内脂滴的共定位成像
将细胞接种在共聚焦细胞培养皿中并在培养箱中培养。当细胞汇合度达到70%左右时,将含有化合物I-1a(5μg mL-1)的新鲜培养基加入细胞培养皿中。孵育1小时后,用PBS洗涤细胞,并与BODIPY 505/515(5μg·mL–1)或MitoTracker Green(2μM)或LysoTrackerGreen(2μM)共染色30分钟。之后,用PBS洗涤细胞并进行共聚焦荧光成像(Nikon A1R)。激发激光为450nm,绿色荧光团(BODIPY 505/515、MitoTracker Green和LysoTracker Green)的发射在510-550nm内收集,化合物I-1a的发射在590-650nm内收集。皮尔逊相关系数R由ImageJ计算。结果见图5所示,图5为化合物I-1a(25μg·mL-1)和a)BODIPY 505/515(5μg·mL-1)、b)MitoTracker Green(2μM)或c)LysoTracker Green(2μM)染色的MCF-7细胞的共聚焦荧光图像及其对应的散点图与Pearson相关系数(R),化合物I-1a激发波长λex:490nm;发射波长λem:商业荧光团(绿色)为500-530nm,化合物I-1a(红色)为580-630nm,比例尺:25μm。结果表明,化合物I-1a对脂滴的共定位效果要明显好于其他两种亚细胞器,显示出良好的脂滴标记能力,这为活体肝脏组织脂滴的成像奠定了基础。
实施例9
化合物I-1a对小鼠脑部血管和脂肪肝内脂滴成像
在商用扫描布鲁克显微镜上进行多光子荧光脑血管成像。激光源是非共线光学参量放大器(NOPA)激光器。双光子显微镜的波长可以调整为920nm,三光子显微镜的波长可以调整为1300nm。激光通过水浸显微镜物镜(XLPLN25XWMP2,Olympus,25X 1.05NA)聚焦后,通过光电倍增管(H7422-40,Hamamatsu)在红色通道(560-700纳米)进行成像。扫描速度为2.2μs/pixe。为了在5X物镜下对脂肪肝中的LD成像,应用了空气物镜(LSM03,WD=25.1mm,Thorlabs)。其他条件与多光子荧光脑血管成像一致。
如图6、图7和图8所示,图6为化合物I-1a标记的颅窗内3PF显微脑血管成像示意图,(a-h)从100μm深度到1100μm深度的3PF显微脑血管成像;3PF图像在1300nm fs处被激发;(i-l)在300μm(b)、700μm(d)、900μm(f)、1100μm(h)成像深度处沿白线的高斯拟合轮廓;(m)重建3D 3PF脑血管成像,深度从0到1200μm。图7为脂肪肝中化合物I-1a染色的LDs的3PF显微成像示意图,(a)2PF和(b)5X物镜下LD的3PF显微成像;2PF和3PF图像分别由920nm fs和1300nm fs激发;(c)沿着(a)和(b)中LD的白线的归一化强度分布;(d-f)25X物镜下10到40μm深度的LD的3PF显微成像;(g)沿LD白线的高斯拟合轮廓,成像深度为10μm;(h)在0到50μm的深度重建LD的3D 3PF成像。图8为(a)脂肪肝和(b)正常肝脏中被化合物I-1a染色的LDs的活体三光子荧光成像;(c)未注射化合物I-1a的肝组织三光子荧光成像示意图,激发波长:1300nm fs,发射波长:560-700nm。以上结果表明,3PF荧光成像显示出优异的轴向分辨率,并且对于信噪比(SBR),50-700μm不同深度的脑血管显示较高的SBR(>5)(图6a-d)。即使小到2.3μm(图6i)和2.4μm(图6j)的血管可以很好地区分。在900μm的深度,仍能观察到3.1μm的脑血管,SBR相对较高(>3)。即使是在1.1毫米的成像深度,也可以观察到7.5μm的脑血管(图6l)。此外,图6m中的3D高分辨率重建血管图像(0-1200μm)展示了丰富生动的脑血管结构,强有力的证明了化合物I-1a的脑部血管成像能力。
此外,由于图5的结果已经证明了化合物I-1a良好的脂滴标记能力,还研究了化合物I-1a在体内标记脂肪肝LDs的能力。如图7a-c所示,LDs的3PF成像比2PF成像表现出更高的SBR。然后,在25倍放大率下对LDs进行了三光子荧光显微成像。如图8a所示,只有注射了化合物I-1a的脂肪肝表现出较强的3PF信号,而图8b中注射了化合物I-1a的正常肝组织和图8c中未注射化合物I-1a的脂肪肝均未表现出明显的3PF信号。此外,在0-50μm的不同深度对LDs进行成像(图7d-f)。在10μm的深度可以观察到直径为2.4μm的小脂滴并且具有较高的SBR(图7g)。图7h中的3D重建图像(0-50μm)显示了精确的LDs体积和形态。本发明首次报道肝组织LDs的3D重建3PF成像。化合物I-1a如此出色的3PF成像能力使其成为一种在深部组织中实现脂滴可视化的有效手段,为脂肪肝等代谢类疾病的诊断提供了新的可能性。
对比例1
文献报道化合物DCPE-TPA用于三光子脑部成像深度仅为300μm(H.Tian,D.Li,X.Tang,Y.Zhang,Z.Yang,J.Qian,Y.Q.Dong and M.Han,Mater.Chem.Front.,2020,1634-1642.)。
本发明中的化合物I-1a可以达到1200μm的成像深度,极大地提高了深层脑部成像的可能性。更重要的是,DCPE-TPA不具有脂滴成像能力,而本发明的化合物I-1a具有优良的脂滴成像能力,在细胞和活体肝组织中能够对脂滴进行成像,为脂肪肝等代谢类疾病的诊断提供了新的可能性。
以上所述仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,虽然本发明已以较佳实施例揭露如上,然而并非用以限定本发明,任何熟悉本专利的技术人员在不脱离本发明技术方案范围内,当可利用上述提示的技术内容作出些许更动或修饰为等同变化的等效实施例,但凡是未脱离本发明技术方案的内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明方案的范围内。
Claims (4)
1.一种二氢吩嗪衍生物,其特征在于,具有以下结构通式:
其中,R1选自氢、C1~C5烷基;
R2选自氢、C1~C5烷基;
R3选自氢、腈基;
R4选自腈基、
R5选自C1~C5烷基。
2.根据权利要求1所述的二氢吩嗪衍生物,其特征在于,所述二氢吩嗪衍生物中,
R1选自氢、甲基、乙基;
R2选自氢、甲基、乙基;
R3选自氢、腈基;
R4选自腈基、
R5选自甲基、乙基、正丁基、叔丁基、异丙基。
3.根据权利要求2所述的二氢吩嗪衍生物,其特征在于,所述二氢吩嗪衍生物选自以下结构的一种:
4.一种权利要求1至3任一项所述的二氢吩嗪衍生物在制备用于活体脑血管或肝脏脂滴的三光子荧光成像剂中的应用。
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Colorimetric and Ratiometric Near-Infrared Fluorescent CyanideChemodosimeter Based on Phenazine Derivatives;Lin Yang,等;ACS Appl. Mater. Interfaces;第5卷;1317-1326 * |
Controlled emission colors and singlet–triplet energy gaps of dihydrophenazine-based thermally activated delayed fluorescence emitters;Jiyoung Lee,;: J. Mater. Chem. C;第3卷;2175–2181 * |
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