CN114671851A - 萘二甲酰亚胺-四嗪类化合物及其制备方法与应用 - Google Patents

萘二甲酰亚胺-四嗪类化合物及其制备方法与应用 Download PDF

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CN114671851A
CN114671851A CN202011556194.5A CN202011556194A CN114671851A CN 114671851 A CN114671851 A CN 114671851A CN 202011556194 A CN202011556194 A CN 202011556194A CN 114671851 A CN114671851 A CN 114671851A
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王永成
田育林
杨虹
李想
滕雨
尹大力
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Abstract

本发明属于荧光探针领域，公开了萘二甲酰亚胺‑四嗪类化合物及其制备方法与应用，涉及一种由下述通式I表示的萘二甲酰亚胺‑四嗪类荧光探针，及其相应的与环辛炔(BCN)或反式环辛烯(TCO)发生生物正交反应后的产物。点击反应后的探针分子大部分同时具有生物正交荧光增强(Turn‑on)效应和聚集诱导发光(AIE)效应。该类荧光探针具有优良的光物理性质，能够克服在复杂生物体系中由于聚集产生的荧光减弱甚至淬灭的现象，可以快速、高效、特异性标记目标生物蛋白分子。本发明可应用于体外蛋白标记、活细胞成像和组织的标记成像，具有广泛的应用前景。

Description

萘二甲酰亚胺-四嗪类化合物及其制备方法与应用
技术领域
本发明属于荧光探针领域，具体涉及一类具有聚集诱导发光(AIE)性质的萘二甲酰亚胺-四嗪类生物正交增强型(Turn-on)荧光探针的合成和应用。
背景技术
小分子荧光探针具有灵敏度高，选择性强等特点，运用小分子荧光探针对生物大分子进行标记得到了快速的发展，已成为当前的研究热点。然而，传统的小分子荧光基团大多存在体系背景高、灵敏度差、易产生自身荧光等问题，在对蛋白质等生物大分子进行标记时具有一定的限制。针对这一问题，“Turn on”点亮型荧光增强探针因其荧光强度可随体系反应前后变化而显著增强，可以排除光漂白或其他背景干扰因素，实现活体状态下对生命活动和病理过程的实时可视化，在化学生物学和医药学研究领域里发挥着重要作用。
生物正交反应(Bioorthogonal Reactions)是一类在生理条件下能够特异性与目标生物分子发生的快速、高效的化学反应，现已被广泛应用于生物分子的标记。在目前已知的生物正交反应类型中，基于环张力的反式环辛烯(TCO)、环辛炔(BCN)与四嗪(tetrazines)的逆电子需求的狄尔斯-阿尔德反应(Inverse electron demand Diels-Alder,IEDDA)具有极高的反应速率、反应正交性和生物兼容性，已成为目前最有效和最普遍的生物正交反应类型。因此，将这类生物正交反应与有机荧光小分子结合，设计合成“Turn-on”型荧光增强探针，可以对生物大分子进行实时精准标记、成像及活体状态下生命活动现象的可视化等。
在小分子荧光化合物中，1,8-萘二甲酰亚胺类化合物具有刚性环系共轭结构，可形成D-π-A类电子结构，其具有斯托克斯位移大、荧光量子产率高、稳定性好、化学结构易修饰、荧光发射波长可控等优点，目前已广泛应用于荧光探针，细胞成像，荧光传感器，生物荧光标记等领域。
大多数小分子荧光探针具有聚集诱导淬灭(aggregation caused quenching,ACQ)的效果，从而使得探针分子在生物体系下的应用受到较大限制。ACQ主要是由于聚集状态下分子间π-π堆积，增加了分子间的非辐射跃迁衰变，进而导致荧光猝灭。2001年唐本忠院士课题组首次发现了聚集诱导发光(Aggregation Induce Emission，AIE)效应，即在溶液中发光很弱，但在聚集状态或者固态下发光却明显增强。具有AIE性质的化合物从根本上克服了聚集导致淬灭(ACQ)的难题，受到了广泛关注。AIE现象的主要机制是聚集状态下分子内运动受限(restriction of intramolecular motion,RIM)，包括分子内旋转受限(Restriction of Intramolecular Rotations,RIR)和分子内震动受限(Restriction ofIntramolecular Vibrations,RIV)，即分子在聚集过程中分子内的自由旋转运动以及原子或基团间震动受到限制，减弱了非辐射跃迁，激发态分子只能通过辐射衰变的方式回到基态，以荧光形式释放，从而产生AIE效应。AIE现象的发现使得荧光探针的应用由有机环境体系逐渐向水相、固相环境体系转移，极大地扩大了小分子荧光探针的应用范围。与传统的ACQ荧光探针相比，AIE型荧光探针往往还具有更高的灵敏度，拥有更广泛的应用前景。
目前具有AIE效应的生物正交“Turn-on”型荧光探针鲜有报道，基于研究现状，本发明旨在提供一类具有AIE效应的萘二甲酰亚胺-四嗪类生物正交“Turn-on”型荧光探针及其制备方法和生物学应用。
发明内容
本发明的目的是提供一类新型的具有聚集诱导发光(AIE)性质的萘二甲酰亚胺-四嗪类生物正交荧光增强型(Turn-on)荧光探针及其制备方法和在光学成像和生物学中的应用。
本发明技术方案的第一方面在于：提供一种由下述通式I表示的萘二甲酰亚胺-四嗪类小分子探针，及其相应的与环辛炔BCN[(1R,8S,9S)-Bicyclo[6.1.0]non-4-yn-9-ylmethanol]或反式环辛烯TCO[(4E)-4-Cycloocten-1-ol]发生生物正交反应后的产物，
Figure BDA0002858696760000021
其中，X为如下结构片段的一种：
Figure BDA0002858696760000022
其中，R1选自C1-C6的烷基、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
R2选自C1-C6的烷基、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
L选自氧原子、硫原子、氮原子、亚甲基、乙烯基、含有1-3个氮、氧、硫原子的五元或六元芳杂环、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
其中，Y选自C1-C6的烷基、含氮、氧、硫杂原子的烷基、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
优选的化合物为通式I-1所述的化合物，
Figure BDA0002858696760000023
其中，R1选自C1-C6的烷基、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
Y选自C1-C6的烷基、含氮、氧、硫杂原子的烷基、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
优选的化合物选自通式I-2所述的化合物，
Figure BDA0002858696760000031
其中，Y选自C1-C6的烷基、含氮、氧、硫杂原子的烷基、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
R2选自C1-C6的烷基、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
L选自氧原子、硫原子、氮原子、亚甲基、乙烯基、含有1-3个氮、氧、硫原子的五元或六元芳杂环、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
优选的化合物为通式I-3所述的化合物，
Figure BDA0002858696760000032
其中，Y选自C1-C6的烷基、含氮、氧、硫原子的烷基、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
R2选自C1-C6的烷基、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
R3和R4分别选自氢、甲基、甲氧基、三氟甲基、卤素原子、硝基、氰基、二甲氨基，其中卤素选自氟、氯、溴和碘；
更优选的化合物为下述的化合物，其特征在于，所述的化合物选自：
Figure BDA0002858696760000041
Figure BDA0002858696760000051
本发明技术方案的第二方面在于提供了第一方面所述化合物的制备方法，其合成路线如下：
(1)探针分子TZ-1和TZ-2的合成路线：
Figure BDA0002858696760000061
(2)探针分子TZ-3的合成路线：
Figure BDA0002858696760000071
(3)探针分子TZ-4的合成路线：
Figure BDA0002858696760000072
(4)探针分子TZ-5～TZ-12的合成路线：
Figure BDA0002858696760000073
(5)探针分子TZBCN-1～TZBCN-12的制备方法：
Figure BDA0002858696760000074
(6)探针分子TZTCO-1～TZTCO-3的制备方法：
Figure BDA0002858696760000081
本发明技术方案的第三方面是通过荧光性质测试阐明了第一方面所述化合物具有生物正交反应“Turn on”效应和AIE效应的属性。
本发明技术方案的第四方面是提供了第一方面所述化合物对BCN修饰的胎牛血清白蛋白(bovine serum albumin,BSA)实现了高选择性的原位荧光标记。
本发明具有如下优点和有益效果：
本发明将四嗪作为生物正交反应基团与萘二甲酰亚胺结合,设计了一类萘二甲酰亚胺-四嗪的新型荧光增强Turn-on荧光探针。该类荧光探针具有优良的光物理性质，不仅可以实现对目标生物蛋白分子的高效、特异性原位标记，而且具有明显的聚集诱导发光AIE性质，能够克服在复杂生物体系中由于聚集产生的荧光减弱甚至淬灭的现象。该研究有效地扩展了生物正交化学荧光探针分子工具库，为蛋白质等生物大分子的荧光标记和可视化研究提供了新的思路，具有较好的应用前景。
附图说明
附图是用来提供对本发明的进一步阐述，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本发明，但并不构成对本发明的限制；
图1 TZ-1和TZBCN-1探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(80％)，激发波长为340nm，探针浓度为10μM；
图2 TZBCN-1探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为340nm，探针浓度为10μM；
图3 TZ-2和TZBCN-2探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(99％)，激发波长为340nm，探针浓度为10μM；
图4 TZBCN-2探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为340nm，探针浓度为10μM；
图5 TZ-3和TZBCN-3探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(99％)，激发波长为365nm，探针浓度为10μM；图6TZBCN-3探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为365nm，探针浓度为10μM；
图7 TZ-4和TZBCN-4探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(99％)，激发波长为340nm，探针浓度为10μM；图8TZBCN-4探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为340nm，探针浓度为10μM；
图9 TZ-5和TZBCN-5探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(99％)，激发波长为340nm，探针浓度为10μM；图10TZBCN-5探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为340nm，探针浓度为10μM；
图11 TZ-6和TZBCN-6探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(99％)，激发波长为340nm，探针浓度为10μM；
图12 TZBCN-6探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为340nm，探针浓度为10μM；
图13 TZ-7和TZBCN-7探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(99％)，激发波长为340nm，探针浓度为10μM；
图14 TZBCN-7探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为340nm，探针浓度为10μM；
图15 TZ-8和TZBCN-8探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(99％)，激发波长为340nm，探针浓度为10μM；
图16 TZBCN-8探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为340nm，探针浓度为10μM；
图17 TZ-9和TZBCN-9探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(99％)，激发波长为340nm，探针浓度为10μM；
图18 TZBCN-9探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为340nm，探针浓度为10μM；
图19 TZ-10和TZBCN-10探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(99％)，激发波长为340nm，探针浓度为10μM；
图20 TZBCN-10探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为340nm，探针浓度为10μM；
图21 TZ-11和TZBCN-11探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(99％)，激发波长为365nm，探针浓度为10μM；
图22 TZBCN-11探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为365nm，探针浓度为10μM；
图23 TZ-12和TZBCN-12探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(50％)，激发波长为365nm，探针浓度为10μM；
图24 TZBCN-12探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为365nm，探针浓度为10μM；
图25 TZ-3和TZTCO-1探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(95％)，激发波长为365nm，探针浓度为10μM；
图26 TZTCO-1A探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为365nm，探针浓度为10μM；
图27 TZTCO-1B探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为365nm，探针浓度为10μM；
图28 TZ-5和TZTCO-2探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(90％)，激发波长为340nm，探针浓度为10μM；
图29 TZTCO-2A探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为340nm，探针浓度为10μM；
图30 TZTCO-2B探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为340nm，探针浓度为10μM；
图31 TZ-10和TZTCO-3探针分子的发射光谱，溶剂体系为DMSO和水/DMSO(99％)，激发波长为340nm，探针浓度为10μM；
图32 TZTCO-3A探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为340nm，探针浓度为10μM；
图33 TZTCO-3B探针分子的发射光谱，溶剂体系为不同比例的水/DMSO，激发波长为340nm，探针浓度为10μM；
图34 TZ-6与BCN的二级反应动力学曲线，溶剂体系为DMSO，TZ-6浓度为25μM，BCN浓度为250μM；
图35 TZ-6与TCO的二级反应动力学曲线，溶剂体系为DMSO，TZ-6浓度为25μM，TCO浓度为250μM；
图36 TZ-11与BCN的二级反应动力学曲线，溶剂体系为DMSO，TZ-11浓度为25μM，BCN浓度为250μM；
图37 TZ-12与BCN的二级反应动力学曲线，溶剂体系为DMSO，TZ-12浓度为25μM，BCN浓度为250μM；
图38萘二甲酰亚胺-四嗪类探针分子标记含有BCN修饰的胎牛血清白蛋白(BSA)的SDS-PAGE效果图，探针分子终浓度为0.5mM；BCN修饰的BSA蛋白终浓度为15μM。
具体实施方式
为了使本发明的目的，技术方案和优点更加清楚明白，下面结合实例对本发明作进一步的详细说明，本发明的实施方式及其说明仅用于解释本发明，但这些实施例和说明并不限制本发明的范围。
实施例1：TZ-1的合成
Figure BDA0002858696760000101
(1)中间体1的合成：
将4-溴-1,8-萘二甲酸酐(277mg,1.00mmol)，联硼酸频那醇酯(380mg，1.50mmol),Pd(dppf)Cl2(73mg,0.10mmol),KOAc(294mg,3.00mmol)加入二氧六环(10mL)中，氩气保护，90℃加热搅拌反应6小时。TLC监测反应完全，将反应液浓缩拌样，flash柱层析分离(PE/EA＝4:1)得白色固体240mg,收率74％。1H NMR(400MHz,CDCl3)δ9.23(dd,J＝8.4,1.2Hz,1H),8.63(dd,J＝7.2,1.2Hz,1H),8.59(d,J＝7.6Hz,1H),8.35(d,J＝7.6Hz,1H),7.84(dd,J＝8.4,7.2Hz,1H),1.46(s,12H).
(2)中间体2的合成：
将中间体1(240mg,0.74mmol)加入乙醇(10mL)中，然后滴加正丁胺(59mg,0.81mmol)，80℃回流反应3小时。TLC监测反应完全，将反应液浓缩拌样，flash柱层析分离(PE/EA＝4:1)得白色固体186mg,收率86％。1H NMR(400MHz,CDCl3)δ9.10(dd,J＝8.4,1.2Hz,1H),8.60(dd,J＝7.2,1.2Hz,1H),8.56(d,J＝7.6Hz,1H),8.29(d,J＝7.6Hz,1H),7.77(dd,J＝8.4,7.2Hz,1H),4.18(t,J＝7.6Hz,2H),1.77–1.67(m,2H),1.51–1.39(m,14H),0.98(t,J＝7.6Hz,3H).
(3)中间体3的合成：
将中间体2(600mg,1.58mmol)和高碘酸钠(1016mg,4.75mmol)加入丙酮(10mL)中，然后滴加2N盐酸水溶液(2mL)，室温搅拌反应1小时。TLC监测反应完全，向反应液中加入冰水30mL，抽滤得产物白色固体390mg,收率83％。1H NMR(400MHz,DMSO-d6)δ8.80(d,J＝8.4Hz,1H),8.51–8.42(m,2H),8.05(d,J＝7.2Hz,1H),7.90–7.83(m,1H),4.12–3.97(m,2H),1.74–1.56(m,2H),1.40–1.31(m,2H),0.93(t,J＝7.6Hz,3H)；HRMS(ESI)m/zcalculated for C16H17O4NB[M+H]+:298.1245,found:298.1231.
(4)中间体4的合成：
将硫代卡巴肼(2120mg,20mmol)和4-溴甲基联苯(4940mg,20mmol)加入乙醇(50mL)中，氩气保护下60℃搅拌反应过夜。将反应液冷至室温，加入30mL乙醚，将所得白色固体过滤干燥得产物4750mg,收率67％。1H NMR(400MHz,Methanol-d4)δ7.67–7.60(m,4H),7.52–7.48(m,2H),7.47–7.41(m,2H),7.38–7.32(m,1H),4.29(s,2H).
(5)中间体5的合成：
将中间体4(1414mg,4.00mmol)和原乙酸三乙酯(972mg,6.00mmol)加入乙醇(20mL)中，室温搅拌10min,加入三乙胺(404mg,4.00mmol)，80℃加热回流2h,然后向反应液中加入亚硝酸钠(552mg,8.00mmol),小心滴加2N盐酸水溶液至无气泡产生，乙酸乙酯萃取，有机相浓缩拌样，flash柱层析分离(PE/EA＝4:1)得酒红色固体880mg,收率75％。1H NMR(400MHz,CDCl3)δ7.58–7.57(m,1H),7.57–7.54(m,5H),7.46–7.41(m,2H),7.37–7.32(m,1H),4.58(s,2H),2.98(s,3H)；HRMS(ESI)m/z calculated for C16H15N4S[M+H]+:295.1012,found:295.1008.
(6)TZ-1的合成
将中间体5(35mg,0.12mmol),中间体3(71mg,0.24mmol),Pd(dppf)Cl2(7mg,0.01mmol),和氧化银(68mg,0.30mmol)加入DMF(3mL)中，60℃氩气保护，搅拌反应3h，TLC监测反应完全。将反应液直接浓缩拌样，flash柱层析分离(PE/EA＝2:1)得红色固体21mg,收率50％。1H NMR(400MHz,CDCl3)δ9.04(dd,J＝8.8,0.8Hz,1H),8.79(d,J＝7.6Hz,1H),8.71(dd,J＝7.2,0.8Hz,1H),8.59(d,J＝7.6Hz,1H),7.87(dd,J＝8.8,7.2Hz,1H),4.22(t,J＝7.6Hz,2H),3.23(s,3H),1.82–1.70(m,2H),1.51–1.44(m,2H),1.00(t,J＝7.6Hz,3H)；13CNMR(101MHz,CDCl3)δ167.35,166.30,164.03,163.70,134.81,131.89,131.85,130.82,130.40,129.42,129.04,128.61,125.73,123.33,40.65,30.33,21.52,20.54,13.99；HRMS(ESI)m/z calculated for C19H18O2N5[M+H]+:348.1455,found:348.1444.
实施例2：TZ-2的合成
Figure BDA0002858696760000121
(1)中间体6的合成：
将中间体4(1414mg,4.00mmol)和原苯甲酸三乙酯(1346mg,6.00mmol)加入乙醇(20mL)中，室温搅拌10min,加入三乙胺(404mg,4.00mmol)，80℃加热回流2h,然后向反应液中加入亚硝酸钠(552mg,8.00mmol),小心滴加2N盐酸水溶液至无气泡产生，乙酸乙酯萃取，有机相浓缩拌样，flash柱层析分离(PE/EA＝4:1)得酒红色固体725mg,收率51％。1H NMR(400MHz,DMSO-d6)δ8.44–8.40(m,2H),7.69–7.60(m,9H),7.46(t,J＝7.6Hz,2H),7.39–7.33(m,1H),4.73(s,2H)；13C NMR(101MHz,CDCl3)δ174.75,162.73,141.04,140.65,134.78,132.51,131.73,129.84,129.39,128.94,127.71,127.64,127.60,127.22,34.68；HRMS(ESI)m/z calculated for C21H17N4S[M+H]+:357.1168,found:357.1171.
(2)TZ-2的合成
将中间体6(71mg,0.20mmol),中间体3(119mg,0.40mmol),Pd(dppf)Cl2(15mg,0.02mmol),和氧化银(116mg,0.50mmol)加入DMF(5mL)中，60℃氩气保护反应3h，TLC监测反应完全。将反应液浓缩拌样，flash柱层析分离(PE/EA＝2:1)得红色固体36mg,收率44％。1HNMR(400MHz,CDCl3)δ9.14(d,J＝8.6Hz,1H),8.77(d,J＝7.7Hz,1H),8.74–8.60(m,4H),7.87(t,J＝7.9Hz,1H),7.75–7.57(m,3H),4.21(t,J＝7.2Hz,2H),1.81–1.69(m,2H),1.55–1.38(m,2H),1.00(t,J＝7.2Hz,3H),13C NMR(151MHz,CDCl3)δ166.09,163.98,163.64,163.42,134.69,133.51,132.03,131.81,131.29,130.75,130.37,129.64,129.40,129.02,128.60,128.56,125.69,123.27,40.62,30.31,20.53,13.99；HRMS(ESI)m/z calculatedforC24H20O2N5[M+H]+:410.1612,found:410.1615.
实施例3：TZ-3的合成
Figure BDA0002858696760000122
(1)中间体7的合成：
将4-溴-1,8-萘二甲酸酐(2770mg,10.00mmol)和正丁胺(803mg,11.00mmol)依次加入乙醇(30mL)，80℃加热回流2h,TLC监测反应完全，将反应液浓缩拌样，flash柱层析分离(PE/EA ＝4:1)得淡黄色固体2400mg,收率72％。1H NMR(400MHz,DMSO-d6)δ8.57–8.49(m,2H),8.31(d,J＝8.0Hz,1H),8.20(d,J＝8.0Hz,1H),7.98(dd,J＝8.0,7.6Hz,1H),4.02(t,J＝7.6Hz,2H),1.68–1.53(m,2H),1.40–1.31(m,2H),0.92(t,J＝7.6Hz,3H).
(2)中间体8的合成
将中间体7(996mg,3.00mmol)和氰化亚铜(538mg,6.00mmol)依次加入DMF(10mL)中，氩气保护150℃加热反应3h,TLC监测反应完全，将反应液浓缩拌样，flash柱层析分离(PE/EA＝3:1)得淡黄色固体620mg,收率74％。1H NMR(400MHz,CDCl3)δ8.72(dd,J＝7.2,1.2Hz,1H),8.63(d,J＝7.6Hz,1H),8.58(dd,J＝8.4,1.2Hz,1H),8.15(d,J＝7.6Hz,1H),7.98(dd,J＝8.4,7.2Hz,1H),4.19(t,J＝7.6Hz,2H),1.79–1.66(m,2H),1.50–1.38(m,2H),0.98(t,J＝7.4Hz,3H).
(3)中间体9的合成：
将中间体8(220mg,0.79mmol)加入8mL甲酸/水(3:1)混合溶剂中，然后加入铝镍合金(203mg,2.37mmol),氩气保护100℃反应3h,TLC监测反应完全，将反应液浓缩拌样，flash柱层析分离(PE/EA＝3:1)得淡黄色固体115mg,收率51％。1H NMR(400MHz,CDCl3)δ10.49(s,1H),9.50(dd,J＝8.8,0.8Hz,1H),8.73(d,J＝7.6Hz,1H),8.66(dd,J＝7.6,0.8Hz,1H),8.22(d,J＝7.6Hz,1H),7.92(t,J＝8.8,7.6Hz,1H),4.18(t,J＝7.6Hz,2H),1.82–1.63(m,2H),1.55–1.36(m,2H),0.98(t,J＝7.6Hz,3H).
(4)中间体10的合成：
将氰甲基膦酸二乙酯(354mg,2.00mmol),乙腈(656mg,16.00mmol),N-乙酰半胱氨酸(163mg,1.00mmol)加入乙醇(5mL)中，然后加入水合肼(1600mg,32.00mmol),40℃反应12h,加入亚硝酸钠(2208mg,32.00mmol)和水(20mL),缓慢加入2N HCl溶液至无气泡产生，将反应液用乙酸乙酯萃取，有机相浓缩拌样，flash柱层析分离(DCM/MeOH＝30:1)得红色液体260mg,收率52％。1H NMR(400MHz,CDCl3)δ4.27–4.14(m,4H),3.93(d,J＝22.4Hz,2H),3.06(s,3H),1.33(t,J＝7.2Hz,6H).
(5)TZ-3的合成
将中间体9(84mg,0.30mmol)和中间体10(88mg,0.36mmol)加入无水THF(5mL)中，冰浴下加入氢化钠(24mg,0.60mmol,60％分散于矿物油中)，搅拌反应2h,TLC监测反应完全，用10mL饱和氯化铵淬灭，乙酸乙酯萃取，有机相浓缩干燥，flash柱层析分离(PE/EA ＝4:1)得红色固体39mg,收率35％。1H NMR(400MHz,CDCl3)δ9.11(d,J＝16.0Hz,1H),8.65(t,J＝8.4Hz,3H),8.17(d,J＝7.6Hz,1H),7.85(t,J＝7.6Hz,1H),7.69(d,J＝16.0Hz,1H),4.19(t,J＝7.2Hz,2H),3.12(s,3H),1.83–1.64(m,2H),1.56–1.33(m,2H),0.98(t,J＝6.8Hz,3H)；13C NMR(101MHz,CDCl3)δ167.07,164.37,164.09,163.83,138.48,135.55,131.62,130.83,129.71,128.74,127.70,126.69,125.39,123.79,123.44,110.06,40.51,30.33,21.47,20.52,13.98；HRMS(ESI)m/z calculated for C21H20O2N5[M+H]+:374.1612,found:374.1597.
实施例4：TZ-4的合成
Figure BDA0002858696760000141
(1)中间体11的合成
将4-溴-1,8-萘二甲酸酐(166mg,0.50mmol),N-羟基邻苯二甲酰亚胺(98mg,0.60mmol)和碳酸钾(104mg,0.75mmol)加入DMSO(5mL)中，80℃加热反应6h,向反应液中加入20mL冰水，然后用2N盐酸调pH至2～3，乙酸乙酯萃取，有机相浓缩拌样，flash柱层析分离(PE/EA＝4:1)得黄色固体120mg,收率89％。1H NMR(400MHz,DMSO-d6)δ11.85(s,1H),8.54(dd,J＝8.4,1.2Hz,1H),8.48(dd,J＝7.2,1.2Hz,1H),8.36(d,J＝8.2Hz,1H),7.77(dd,J＝8.4,7.2Hz,1H),7.16(d,J＝8.2Hz,1H),4.07–3.96(m,2H),1.65–1.53(m,2H),1.39–1.28(m,2H),0.92(t,J＝7.4Hz,3H).
(2)中间体12的合成
将硫代卡巴肼(3180mg,30.00mmol)和碘甲烷(4686mg,33.00mmol)加入乙醇(100mL)中，80℃加热回流反应2h,析出白色固体，过滤干燥得产物3960mg,收率53％。1HNMR(400MHz,DMSO-d6)δ9.68(s,2H),5.31(s,4H),2.38(s,3H)。
(3)中间体13的合成
将中间体12(2000mg,8.10mmol)和原乙酸三乙酯(1968mg,12.15mmol)加入乙醇(50mL)中，室温搅拌10min,加入三乙胺(818mg,8.10mmol)，80℃加热回流2h,向反应液中加入亚硝酸钠(1118mg,16.20mmol),然后三氟乙酸(923mg,8.10mmol)，加热回流反应1h。将反应液冷至室温，加入100mL冰水，乙酸乙酯萃取，合并有机相浓缩拌样，flash柱层析分离(PE/EA＝10:1)得红色油状物510mg,收率44％。1H NMR(400MHz,CDCl3)δ2.97(s,3H),2.73(s,3H)。
(4)中间体14的合成
将中间体13(490mg,3.45mmol)加入乙醇(10mL)中，然后加入水合肼(259mg,5.18mmol)，室温反应过夜。向反应液中加入水(20mL)，乙酸乙酯萃取，合并有机相浓缩拌样，flash柱层析分离(DCM/MeOH＝10:1)得红色固体260mg,收率60％。1H NMR(400MHz,DMSO-d6)δ9.28(s,1H),4.50(s,2H),2.70(s,3H)。
(5)中间体15的合成
将中间体14(252mg,2.00mmol)加入乙酸(5mL)中，冰浴下缓慢加入溴素(416mg,2.60mmol)，有气泡产生，继续搅拌反应2h。TLC监测反应完全，向反应液中加入水(20mL),用饱和碳酸钠溶液缓慢调pH至6，然后用乙酸乙酯萃取，合并有机相浓缩拌样，flash柱层析分离(PE/EA＝5:1)得红色固体140mg，收率40％。1H NMR(400MHz,CDCl3)δ3.05(s,1H)。
(6)TZ-4的合成
将中间体11(27mg,0.10mmol)，中间体15(18mg,0.10mmol)和DIPEA(26mg,0.20mmol)依次加入DMF(3mL)，室温搅拌反应2h，TLC监测反应完全，将反应液直接浓缩拌样，flash柱层析分离(PE/EA＝4:1)得红色固体20mg，收率55％。1H NMR(400MHz,CDCl3)δ8.65(d,J＝8.0Hz,2H),8.35(dd,J＝8.4,0.8Hz,1H),7.83–7.73(m,1H),7.64(d,J＝8.0Hz,1H),4.19(t,J＝7.6Hz,2H),3.07(s,3H),1.77–1.65(m,2H),1.53–1.37(m,2H),0.98(t,J＝7.6Hz,3H)；13C NMR(101MHz,CDCl3)δ168.25,167.42,163.99,163.39,152.81,132.14,131.92,129.85,127.89,127.53,125.04,123.35,121.32,118.30,40.50,30.33,20.51,13.97；HRMS(ESI)m/z calculated for C19H18O3N5[M+H]+:364.1404,found:364.1412.
实施例5：TZ-5的合成
Figure BDA0002858696760000151
(1)中间体16-1的合成
将4-溴苯腈(182mg,1.00mmol),乙腈(410mg,10.00mmol),N-乙酰半胱氨酸(82mg,0.50mmol)加入乙醇(3mL)中，然后加入水合肼(750mg,15.00mmol),40℃反应12h,加入亚硝酸钠(1035mg,15.00mmol)和水(20mL),室温搅拌反应10min,然后缓慢滴加2N盐酸溶液至无气泡产生，将反应液用乙酸乙酯萃取，合并有机相浓缩拌样，flash柱层析分离(PE/EA ＝10:1)得红色固体81mg,收率32％。1H NMR(400MHz,Chloroform-d)δ8.47(d,J＝8.8Hz,2H),7.73(d,J＝8.8Hz,2H),3.10(s,3H)；HRMS(ESI)m/z calculated for C9H8N4Br[M+H]+:250.9927,found:250.9931.
(2)TZ-5的合成
将中间体2(114mg,0.30mmol),中间体16-1(75mg,0.30mmol),Pd(dppf)Cl2(22mg,0.03mmol),Cs2CO3(195mg,0.60mmol)加入10mL二氧六环/水(9:1)混合溶剂中，氩气保护下100℃加热反应3h，TLC监测反应完全。将反应液浓缩拌样，flash柱层析分离(PE/EA＝5:1)得红色固体40mg,收率31％。1H NMR(400MHz,CDCl3)δ8.78(d,J＝8.4Hz,2H),8.69(d,J＝7.6Hz,1H),8.66(dd,J＝7.2,0.8Hz,1H),8.28(dd,J＝8.8,0.8Hz,1H),7.80–7.72(m,4H),4.22(t,J＝7.6Hz,2H),3.15(s,3H),1.82–1.69(m,2H),1.54–1.41(m,2H),1.00(t,J＝7.6Hz,3H)；13C NMR(101MHz,CDCl3)δ167.70,164.35,164.14,163.96,145.60,143.22,132.25,132.09,131.47,130.97,130.87,129.96,128.85,128.33,127.98,127.35,123.25,122.60,40.49,30.38,21.41,20.56,14.01；HRMS(ESI)m/z calculated for C25H22O2N5[M+H]+:424.1768,found:424.1766.
实施例6：TZ-6的合成
Figure BDA0002858696760000161
(1)中间体16-2的合成
中间体16-2的合成同16-1类似，得红色固体154mg,收率55％。1H NMR(400MHz,CDCl3)δ8.13(d,J＝2.0Hz,1H),8.09(dd,J＝8.4,2.0Hz,1H),7.75(d,J＝8.4Hz,1H),4.04(s,3H),3.10(s,3H)；13C NMR(101MHz,CDCl3)δ167.60,163.66,156.82,134.37,132.30,121.39,117.33,110.61,56.66,21.35；HRMS(ESI)m/z calculated for C10H10ON4Br[M+H]+:281.0033,found:281.0042.
(2)TZ-6的合成
TZ-6的合成同TZ-5类似，得红色固体39mg,收率28％。1H NMR(400MHz,CDCl3)δ8.68(d,J＝7.6Hz,1H),8.63(d,J＝7.2Hz,1H),8.39(d,J＝8.0Hz,1H),8.32(s,1H),7.98(d,J＝8.4Hz,1H),7.73(d,J＝7.6Hz,1H),7.68(t,J＝8.0Hz,1H),7.52(d,J＝7.6Hz,1H),4.22(t,J＝7.6Hz,2H),3.85(s,3H),3.15(s,3H),1.81–1.68(m,2H),1.54–1.40(m,2H),1.00(t,J＝7.6Hz,3H)；13C NMR(101MHz,CDCl3)δ167.73,164.44,164.25,163.86,157.74,142.84,133.75,132.71,132.54,132.20,131.28,130.83,130.45,128.47,126.88,123.10,122.57,120.71,110.13,56.01,40.40,30.39,21.40,20.55,14.00；HRMS(ESI)m/z calculated forC26H24O3N5[M+H]+:454.1874,found:454.1881.
实施例7：TZ-7的合成
Figure BDA0002858696760000162
(1)中间体16-3的合成
中间体16-3的合成同16-1类似，得红色固体132mg,收率47％。1H NMR(400MHz,CDCl3)δ7.82(d,J＝8.4Hz,1H),7.32(dd,J＝8.4,2.0Hz,1H),7.26(d,J＝2.0Hz,1H),3.90(s,3H),3.10(s,3H)；HRMS(ESI)m/z calculated for C10H10ON4Br[M+H]+:281.0033,found:281.0045.
(2)TZ-7的合成
TZ-7的合成同TZ-5类似，得红色固体39mg,收率34％。1H NMR(400MHz,CDCl3)δ8.71–8.62(m,2H),8.31(dd,J＝8.5,1.0Hz,1H),8.09(d,J＝7.8Hz,1H),7.81–7.71(m,2H),7.30(dd,J＝7.8,1.4Hz,1H),7.22(d,J＝1.2Hz,1H),4.22(t,J＝7.6Hz,2H),3.94(s,3H),3.15(s,3H),1.80–1.69(m,2H),1.53–1.40(m,2H),1.00(t,J＝7.6Hz,3H)；13C NMR(101MHz,CDCl3)δ166.59,165.75,164.34,164.13,158.50,145.73,143.92,132.33,132.05,131.49,130.83,130.05,128.78,127.82,127.35,123.19,122.77,122.58,122.39,113.86,56.45,40.49,30.37,21.42,20.54,14.00；HRMS(ESI)m/z calculated for C26H24O3N5[M+H]+:454.1874,found:454.1880.
实施例8：TZ-8的合成
Figure BDA0002858696760000171
(1)中间体16-4的合成
中间体16-4的合成同16-1类似，得红色固体180mg,收率61％。1H NMR(400MHz,CDCl3)δ8.29(d,J＝2.0Hz,1H),8.09(dd,J＝8.0,2.0Hz,1H),7.76(d,J＝8.0Hz,1H),3.09(s,3H),2.91(s,6H)；13C NMR(101MHz,CDCl3)δ167.47,163.82,153.02,135.09,131.82,124.26,123.11,119.67,44.29,21.33；HRMS(ESI)m/z calculated for C26H24O3N5[M+H]+:454.1874,found:454.1880.
(2)TZ-8的合成
TZ-8的合成同TZ-5类似，得红棕色固体39mg,收率32％。1H NMR(400MHz,CDCl3)δ8.67(d,J＝7.6Hz,1H),8.62(dd,J＝7.2,1.2Hz,1H),8.35(d,J＝1.6Hz,1H),8.28(dd,J＝8.0,1.6Hz,1H),8.08(dd,J＝8.4,1.2Hz,1H),7.83(d,J＝7.6Hz,1H),7.67(dd,J＝8.4,7.2Hz,1H),7.43(d,J＝8.0Hz,1H),4.21(t,J＝7.6Hz,2H),3.13(s,3H),2.53(s,6H),1.81–1.70(m,2H),1.53–1.41(m,2H),0.99(t,J＝7.6Hz,3H)；13C NMR(101MHz,CDCl3)δ167.53,164.40,164.24,164.12,152.76,146.09,134.54,133.64,132.96,131.45,131.13,129.53,128.78,128.07,126.80,123.10,122.15,120.62,117.23,110.05,43.20,40.42,30.39,21.37,20.56,14.01；HRMS(ESI)m/z calculated for C27H27O2N6[M+H]+:467.2190,found:467.2192.
实施例9：TZ-9的合成
Figure BDA0002858696760000181
(1)中间体16-5的合成
中间体16-5的合成同16-1类似，得红色固体118mg,收率44％。1H NMR(400MHz,DMSO-d6)δ8.31(d,J＝9.6Hz,1H),8.22(d,J＝8.0Hz,1H),8.01(t,J＝7.6Hz,1H),3.01(s,3H)；13C NMR(101MHz,DMSO-d6)δ167.51,162.08,162.05,160.04,157.60,134.84,133.67,133.60,124.79,124.75,115.13,114.89,112.98,112.77,20.92；HRMS(ESI)m/zcalculated for C9H7N4BrF[M+H]+:268.9833,found:268.9830.
(2)TZ-9的合成
TZ-9的合成同TZ-5类似，得红色固体29mg,收率22％。1H NMR(400MHz,CDCl3)δ8.70(d,J＝7.6Hz,1H),8.67(dd,J＝7.2,1.0Hz,1H),8.60(dd,J＝8.0,1.6Hz,1H),8.52(dd,J＝10.4,1.6Hz,1H),8.10–8.04(m,1H),7.80(d,J＝7.6Hz,1H),7.75(dd,J＝8.4,7.2Hz,1H),7.67(t,J＝7.6Hz,1H),4.23(d,J＝7.2Hz,2H),3.17(s,3H),1.81–1.70(m,2H),1.53–1.41(m,2H),1.00(t,J＝7.2Hz,3H)；13C NMR(151MHz,CDCl3)δ168.05,164.26,164.06,163.22,163.20,161.15,159.50,139.42,134.72,134.66,133.12,133.10,132.02,131.57,130.70,130.60,130.14,128.79,128.57,127.51,124.03,124.01,123.29,123.26,115.71,115.54,40.51,30.38,21.47,20.56,14.02；HRMS(ESI)m/z calculated for C25H21O2N5F[M+H]+:442.1674,found:442.1666.
实施例10：TZ-10的合成
Figure BDA0002858696760000182
(1)中间体16-6的合成
中间体16-6的合成同16-1类似，得红色固体162mg,收率58％。1H NMR(400MHz,CDCl3)δ8.28(s,2H),3.09(s,3H),2.54(s,6H)；13C NMR(101MHz,CDCl3)δ167.40,163.93,139.77,132.97,130.13,127.34,24.22,21.33；HRMS(ESI)m/z calculated for C11H12N4Br[M+H]+:279.0240,found:279.0252.
(2)TZ-10的合成
TZ-10的合成同TZ-5类似，得红色固体36mg,收率27％。1H NMR(400MHz,CDCl3)δ8.70(d,J＝7.2Hz,1H),8.63(dd,J＝7.2,1.2Hz,1H),8.43(s,2H),7.73(dd,J＝8.4,1.2Hz,1H),7.66(dd,J＝8.4,7.2Hz,1H),7.60(d,J＝7.6Hz,1H),4.22(t,J＝7.6Hz,2H),3.13(s,3H),2.02(s,6H),1.80–1.70(m,2H),1.53–1.41(m,2H),0.99(t,J＝7.6Hz,3H)；13C NMR(101MHz,CDCl3)δ167.52,164.26,164.15,164.10,144.96,142.39,137.96,131.76,131.54,131.35,131.18,129.94,128.73,127.51,127.50,127.17,123.45,122.50,40.43,30.37,21.35,20.74,20.53,13.99；HRMS(ESI)m/z calculated for C27H26O2N5[M+H]+:452.2081,found:452.2093.
实施例11：TZ-11的合成
Figure BDA0002858696760000191
(1)中间体16-7的合成
中间体16-7的合成同16-1类似，得红色固体108mg,收率44％。1H NMR(400MHz,CDCl3)δ7.53(d,J＝3.6Hz,1H),6.60(d,J＝3.6Hz,1H),3.03(s,3H)；HRMS(ESI)m/zcalculated forC7H6ON4Br[M+H]+:240.9719,found:240.9723.
(2)TZ-11的合成
TZ-11的合成同TZ-5类似，得红色固体35mg,收率28％。1H NMR(400MHz,CDCl3)δ9.01(d,J＝8.4Hz,1H),8.67(t,J＝8.4Hz,2H),8.19(d,J＝8.0Hz,1H),7.92–7.82(m,2H),7.22(d,J＝3.6Hz,1H),4.20(t,J＝7.6Hz,2H),3.12(s,3H),1.81–1.68(m,2H),1.54–1.38(m,2H),0.99(t,J＝7.2Hz,3H)；13C NMR(101MHz,CDCl3)δ166.88,164.19,163.84,158.75,156.48,148.32,132.48,131.93,131.66,130.68,129.10,128.41,128.12,127.12,123.32,123.22,118.27,114.51,40.54,30.35,21.54,20.55,14.00；HRMS(ESI)m/z calculatedfor C23H20O3N5[M+H]+:414.1561,found:414.1569.
实施例12：TZ-12的合成
Figure BDA0002858696760000201
(1)中间体16-8的合成
中间体16-8的合成同16-1类似，得红色固体96mg,收率37％。1H NMR(400MHz,CDCl3)δ8.00(d,J＝4.0Hz,1H),7.21(d,J＝4.0Hz,1H),3.04(s,3H)；13C NMR(101MHz,CDCl3)δ167.06,161.50,137.08,132.05,131.39,120.65,21.37；HRMS(ESI)m/zcalculated for C7H6N4BrS[M+H]+:256.9491,found:256.9494.
(2)TZ-12的合成
TZ-12的合成同TZ-5类似，得红色固体31mg,收率24％。1H NMR(400MHz,CDCl3)δ8.71–8.59(m,3H),8.38(d,J＝3.9Hz,1H),7.91(d,J＝7.6Hz,1H),7.81(dd,J＝8.4,7.6Hz,1H),7.48(d,J＝3.9Hz,1H),4.21(t,J＝7.6Hz,2H),3.09(s,3H),1.81–1.68(m,2H),1.54–1.39(m,2H),0.99(t,J＝7.6Hz,3H)；13C NMR(101MHz,CDCl3)δ167.19,164.15,163.87,162.10,146.69,137.75,137.48,131.83,131.70,131.60,130.67,130.49,129.90,128.94,128.91,127.78,123.32,123.12,40.52,30.34,21.42,20.53,13.99；HRMS(ESI)m/zcalculated for C23H20O2N5S[M+H]+:430.1332,found:430.1335.
实施例13：TZBCN-1的合成
Figure BDA0002858696760000202
将TZ-1(14mg,0.04mmol)加入DCM/MeOH(5mL,4:1),然后加入BCN(12mg,0.08mmol),室温搅拌反应5min，将反应液浓缩，Pre-TLC分离纯化(DCM/MeOH＝30:1)得黄色固体12mg,收率64％。1H NMR(400MHz,CDCl3)δ8.69(t,J＝7.6Hz,1H),8.62(d,J＝7.2Hz,1H),7.80–7.61(m,3H),4.26–4.17(m,2H),3.70(dd,J＝10.0,8.4Hz,2H),3.16–2.88(m,2H),2.86(d,J＝4.2Hz,3H),2.80–2.63(m,1H),2.62–2.36(m,2H),1.95–1.84(m,1H),1.83–1.66(m,3H),1.61–1.38(m,4H),1.16–1.09(m,1H),0.99(t,J＝7.4Hz,3H),0.92–0.70(m,2H)；13C NMR(151MHz,CDCl3)δ164.24,164.05,159.15,158.90,142.22,141.94,140.89,140.27,131.94,131.59,130.68,130.45,128.32,128.10,127.47,123.23,59.32,50.94,40.51,30.35,28.64,27.94,27.45,26.99,21.68,20.84,20.75,20.53,13.99；HRMS(ESI)m/z calculated for C29H32O3N3[M+H]+:470.2438,found:470.2436.
实施例14：TZBCN-2的合成
Figure BDA0002858696760000211
TZBCN-2的合成同TZBCN-1类似，得黄色固体15mg,收率70％。1H NMR(400MHz,CDCl3)δ8.74–8.57(m,2H),7.91–7.80(m,1H),7.80–7.64(m,2H),7.60(brs,2H),7.58–7.47(m,3H),4.30–4.18(m,2H),3.72(d,J＝7.6Hz,2H),3.09–2.61(m,4H),2.51–2.15(m,2H),1.98–1.67(m,5H),1.51–1.45(m,2H),1.22–1.10(m,2H),1.01(t,J＝7.2Hz,3H)；13C NMR(101MHz,CDCl3)δ164.22,163.98,161.18,158.82,141.61,135.68,132.05,131.66,131.59,130.77,130.59,130.41,130.35,129.46,129.17,128.63,128.44,128.22,127.60,123.51,123.39,123.13,59.36,45.31,40.54,30.36,29.83,28.16,25.11,22.82,22.28,20.55,18.67,14.01；HRMS(ESI)m/z calculated for C34H34O3N3[M+H]+:532.2595,found:532.2605.
实施例15：TZBCN-3的合成
Figure BDA0002858696760000212
TZBCN-3的合成同TZBCN-1类似，得黄色固体11mg,收率55％。1H NMR(400MHz,CDCl3)δ8.80(d,J＝15.6Hz,1H),8.68(d,J＝8.4Hz,1H),8.63(dd,J＝7.2,0.8Hz,1H),8.60(d,J＝7.6Hz,1H),8.04(d,J＝7.6Hz,1H),7.79(dd,J＝8.4,7.6Hz,1H),7.59(d,J＝15.6Hz,1H),4.23–4.14(m,2H),3.74(d,J＝7.6Hz,2H),3.16–2.87(m,4H),2.77(s,3H),2.50–2.33(m,2H),1.80–1.55(m,5H),1.52–1.37(m,3H),1.19–1.09(m,1H),0.98(t,J＝7.2Hz,3H)；13C NMR(101MHz,CDCl3)δ164.34,164.15,158.02,154.38,140.99,140.26,139.07,131.48,130.97,130.65,130.49,130.09,128.74,127.89,127.18,124.45,123.17,122.43,59.50,40.42,30.36,29.83,27.29,26.31,23.39,22.96,22.16,21.23,20.54,19.03,14.00；HRMS(ESI)m/z calculated for C31H34O3N3[M+H]+:496.2595,found:496.2629.
实施例16：TZBCN-4的合成
Figure BDA0002858696760000221
TZBCN-4的合成同TZBCN-1类似，得白色固体12mg,收率62％。1H NMR(400MHz,CDCl3)δ8.62(d,J＝8.0Hz,1H),8.54(d,J＝8.4Hz,1H),8.41(d,J＝8.4Hz,1H),7.77–7.68(m,1H),7.33(d,J＝8.2Hz,1H),4.21–4.13(m,2H),3.76(d,J＝7.8Hz,2H),3.25–3.11(m,1H),3.11–2.98(m,2H),2.97–2.83(m,1H),2.67(s,3H),2.47–2.33(m,2H),1.78–1.54(m,5H),1.51–1.38(m,2H),1.22–1.12(m,1H),1.01–0.88(m,5H)；13C NMR(151MHz,CDCl3)δ164.39,163.81,163.58,157.69,156.41,144.34,132.41,132.13,131.77,129.82,128.30,126.93,125.10,123.06,118.84,115.96,59.48,40.36,30.37,27.69,24.30,23.20,22.62,21.97,20.61,20.52,18.75,18.46,13.99；HRMS(ESI)m/z calculated for C29H32O4N3[M+H]+:486.2387,found:486.2382.
实施例17：TZBCN-5的合成
Figure BDA0002858696760000222
TZBCN-5的合成同TZBCN-1类似，得白色固体13mg,收率59％。1H NMR(400MHz,CDCl3)δ8.65(t,J＝7.6Hz,2H),8.34(d,J＝9.2Hz,1H),7.77–7.69(m,2H),7.68–7.59(m,4H),4.27–4.16(m,2H),3.76(d,J＝7.8Hz,2H),3.14–2.84(m,4H),2.80(s,3H),2.52–2.37(m,1H),2.37–2.21(m,1H),1.85–1.67(m,3H),1.52–1.39(m,3H),1.21–1.13(m,1H),1.07–0.91(m,5H)；13C NMR(101MHz,CDCl3)δ164.44,164.25,160.53,158.05,146.34,140.90,139.30,139.06,138.69,132.69,131.38,130.91,130.15,129.97,129.81,128.84,128.01,127.12,123.12,122.17,59.49,40.43,30.37,29.82,28.25,27.37,27.34,27.30,22.56,22.53,20.88,20.54,14.00；HRMS(ESI)m/z calculated for C35H36O3N3[M+H]+:546.2751,found:546.2757.
实施例18：TZBCN-6的合成
Figure BDA0002858696760000231
TZBCN-6的合成同TZBCN-1类似，得白色固体12mg,收率52％。1H NMR(400MHz,CDCl3)δ8.70–8.56(m,2H),8.03(d,J＝8.4Hz,1H),7.75–7.63(m,2H),7.40–7.31(m,1H),7.26–7.20(m,1H),7.15(t,J＝7.2Hz,1H),4.27–4.12(m,2H),3.79–3.68(m,5H),3.14–2.83(m,4H),2.79(s,3H),2.51–2.36(m,1H),2.36–2.18(m,1H),1.77–1.69(m,2H),1.61(s,2H),1.51–1.41(m,2H),1.21–1.15(m,1H),1.08–0.91(m,5H)；13C NMR(101MHz,CDCl3)δ164.54,164.36,160.64,158.13,156.94,143.58,141.00,140.41,139.35,133.21,131.24,130.88,130.69,128.62,128.46,127.83,126.70,122.97,122.21,121.83,121.78,112.60,59.49,55.75,40.36,32.05,30.39,29.82,28.34,28.22,27.34,22.82,22.62,20.87,20.55,14.01；HRMS(ESI)m/z calculated for C36H38O4N3[M+H]+:576.2857,found:576.2861.
实施例19：TZBCN-7的合成
Figure BDA0002858696760000232
TZBCN-7的合成同TZBCN-1类似，得白色固体14mg,收率60％。1H NMR(400MHz,CDCl3)δ8.70–8.62(m,2H),8.38(d,J＝8.4Hz,1H),7.80–7.70(m,2H),7.51–7.38(m,1H),7.23–7.17(m,1H),7.09(dd,J＝9.6,1.2Hz,1H),4.27–4.18(m,2H),3.83–3.70(m,5H),3.14–2.98(m,1H),2.98–2.83(m,2H),2.83–2.67(m,4H),2.52–2.36(m,1H),2.23–2.05(m,1H),1.81–1.71(m,3H),1.71–1.56(m,2H),1.54–1.40(m,3H),1.19–1.09(m,1H),0.99(t,J＝7.2Hz,3H)；13C NMR(101MHz,CDCl3)δ164.45,164.25,158.75,158.31,157.26,156.98,146.56,140.97,132.76,131.43,131.24,130.87,130.21,128.81,127.91,127.85,127.14,123.08,122.65,122.18,112.56,112.24,59.60,55.74,40.45,30.38,29.84,28.35,28.25,27.26,27.09,23.86,22.13,20.77,20.55,14.02；HRMS(ESI)m/z calculated forC36H38O4N3[M+H]+:576.2857,found:576.2866.
实施例20：TZBCN-8的合成
Figure BDA0002858696760000241
TZBCN-8的合成同TZBCN-1类似，得棕黄色固体15mg,收率63％。1H NMR(400MHz,CDCl3)δ8.66(d,J＝7.6Hz,1H),8.62(d,J＝6.4Hz,1H),8.18–8.12(m,1H),7.82(d,J＝7.6Hz,1H),7.69–7.63(m,1H),7.28–7.23(m,2H),7.10(td,J＝7.6,7.2,1.6Hz,1H),4.27–4.15(m,2H),3.76(d,J＝7.6Hz,2H),3.14–2.84(m,4H),2.79(s,3H),2.53–2.39(m,7H),2.37–2.23(m,1H),1.83–1.70(m,3H),1.53–1.37(m,3H),1.20–1.12(m,1H),1.08–0.92(m,5H)；13C NMR(151MHz,CDCl3)δ164.49,164.34,160.95,157.90,151.81,146.80,140.92,139.56,139.36,133.42,132.38,131.36,131.15,130.36,129.74,128.78,128.16,126.57,122.94,121.93,121.70,119.22,59.41,43.18,40.36,30.37,29.80,29.42,28.29,27.34,24.68,22.79,22.57,20.83,20.54,14.00；HRMS(ESI)m/z calculated for C37H41O3N4[M+H]+:589.3173,found:589.3159.
实施例21：TZBCN-9的合成
Figure BDA0002858696760000242
TZBCN-9的合成同TZBCN-1类似，得白色固体11mg,收率49％。1H NMR(400MHz,CDCl3)δ8.68(d,J＝7.2Hz,1H),8.65(d,J＝7.2Hz,1H),8.15–8.09(m,1H),7.80–7.70(m,2H),7.53(t,J＝7.6Hz,1H),7.48–7.37(m,2H),4.26–4.17(m,2H),3.76(d,J＝8.0Hz,2H),3.14–2.84(m,4H),2.81(s,3H),2.55–2.38(m,1H),2.38–2.23(m,1H),1.82–1.67(m,3H),1.53–1.34(m,3H),1.22–1.14(m,1H),1.08–0.90(m,5H)；13C NMR(101MHz,CDCl3)δ164.34,164.15,160.31,159.45,158.65,158.41,141.31,141.26,141.09,140.09,139.21,132.41,132.00,131.93,131.48,130.72,130.36,128.85,128.56,127.31,126.54,126.43,125.70,123.15,122.94,117.48,117.32,59.47,40.45,30.37,29.83,28.24,27.35,24.51,22.56,20.89,20.54,14.00；HRMS(ESI)m/z calculated for C35H35O3N3F[M+H]+:564.2657,found:564.2662.
实施例22：TZBCN-10的合成
Figure BDA0002858696760000251
TZBCN-10的合成同TZBCN-1类似，得白色固体13mg,收率56％。1H NMR(400MHz,CDCl3)δ8.69(d,J＝7.2Hz,1H),8.63(dd,J＝7.2,1.2Hz,1H),7.80(dd,J＝8.4,1.2Hz,1H),7.68–7.62(m,1H),7.60(d,J＝7.6Hz,1H),7.29(d,J＝7.6Hz,2H),4.27–4.16(m,2H),3.76(d,J＝7.6Hz,2H),3.14–2.82(m,4H),2.78(s,3H),2.50–2.35(m,1H),2.35–2.17(m,1H),1.94(s,6H),1.78–1.71(m,2H),1.61(s,2H),1.52–1.42(m,2H),1.20–1.12(m,1H),1.07–0.90(m,5H)；13C NMR(101MHz,CDCl3)δ164.39,164.29,160.91,157.89,145.73,140.80,139.30,138.36,138.04,136.61,136.53,131.90,131.48,131.20,130.31,128.64,127.82,127.33,123.29,122.18,59.48,58.51,53.56,40.41,30.38,28.25,27.29,24.47,22.44,20.82,20.69,20.54,18.55,14.00；HRMS(ESI)m/z calculated for C37H40O3N3[M+H]+:574.3064,found:574.3097.
实施例23：TZBCN-11的合成
Figure BDA0002858696760000252
TZBCN-11的合成同TZBCN-1类似，得黄色固体11mg,收率51％。1H NMR(400MHz,CDCl3)δ8.87(dd,J＝8.8,1.2Hz,1H),8.63(dd,J＝7.2,1.2Hz,1H),8.61(d,J＝8.0Hz,1H),8.01(d,J＝8.0Hz,1H),7.79(dd,J＝8.8,7.2Hz,1H),7.31(d,J＝3.6Hz,1H),7.11(d,J＝3.6Hz,1H),4.23–4.12(m,2H),3.73(d,J＝8.0Hz,2H),3.20(s,2H),3.10–2.99(m,1H),2.98–2.85(m,1H),2.77(s,3H),2.51–2.35(m,2H),1.85–1.66(m,4H),1.52–1.36(m,3H),1.20–1.10(m,1H),1.10–0.89(m,5H)；13C NMR(151MHz,CDCl3)δ164.21,163.87,157.85,153.87,152.81,150.70,141.49,138.92,133.72,132.00,131.48,130.83,129.05,128.17,127.46,125.99,123.15,122.05,114.26,59.39,40.43,30.31,29.80,27.70,27.32,24.15,22.80,21.10,20.51,13.98；HRMS(ESI)m/z calculated for C33H34O4N3[M+H]+:536.2544,found:536.2533.
实施例24：TZBCN-12的合成
Figure BDA0002858696760000261
TZBCN-12的合成同TZBCN-1类似，得黄色固体12mg,收率54％。1H NMR(400MHz,CDCl3)δ8.73(dd,J＝8.4,0.8Hz,1H),8.65(dd,J＝7.2,0.8Hz,1H),8.62(d,J＝7.6Hz,1H),7.89(d,J＝7.6Hz,1H),7.78(dd,J＝8.4,7.2Hz,1H),7.44(d,J＝3.6Hz,1H),7.37(d,J＝3.6Hz,1H),4.26–4.14(m,2H),3.76(d,J＝7.7Hz,2H),3.20(brs,2H),3.08(dd,J＝14.5,8.7Hz,1H),2.95–2.83(m,1H),2.77(s,3H),2.52–2.37(m,2H),1.78–1.70(m,2H),1.68–1.52(m,2H),1.52–1.39(m,3H),1.23–1.17(m,1H),1.08–0.95(m,4H)；13C NMR(101MHz,CDCl3)δ164.31,164.04,157.64,154.24,142.87,142.20,141.40,139.04,138.73,132.39,131.55,130.78,129.99,129.07,128.96,128.74,128.70,127.44,123.14,122.34,59.49,40.46,32.05,31.57,30.35,29.83,28.14,27.41,24.99,22.82,20.92,20.54,14.00；HRMS(ESI)m/z calculated for C33H34O3N3S[M+H]+:552.2315,found:552.2316.
实施例25：TZTCO-1的合成
Figure BDA0002858696760000262
将TZ-3(17mg,0.04mmol)加入DCM/MeOH(5mL,4:1),然后加入TCO(10mg,0.08mmol),室温搅拌反应3min，将反应液浓缩，Pre-TLC分离纯化(DCM/MeOH＝30:1)得黄色固体(TLC较小极性产物点)TZTCO-1A:6mg,收率32％；黄色固体(TLC较大极性产物点)TZTCO-1B:6mg,收率32％。
TZTCO-1A:1H NMR(400MHz,CDCl3)δ8.87(d,J＝15.6Hz,1H),8.68(d,J＝8.4Hz,1H),8.64(dd,J＝7.2,0.8Hz,1H),8.60(d,J＝7.6Hz,1H),8.04(d,J＝7.6Hz,1H),7.83–7.76(m,1H),7.54(d,J＝15.6Hz,1H),4.25–4.12(m,2H),3.68–3.56(m,1H),3.19–3.07(m,1H),3.00–2.83(m,3H),2.75(s,3H),2.23–2.13(m,1H),2.06–1.96(m,1H),1.92–1.82(m,1H),1.77–1.67(m,5H),1.50–1.40(m,2H),0.99(t,J＝7.6Hz,3H)；13C NMR(151MHz,CDCl3)δ164.35,164.15,157.55,153.67,140.89,138.31,137.36,131.52,130.96,130.85,130.49,130.12,128.75,127.34,127.24,124.52,123.20,122.53,71.48,40.45,38.29,36.21,30.37,27.19,24.93,22.83,20.68,20.56,14.02；HRMS(ESI)m/z calculated forC29H32O3N3[M+H]+:470.2438,found:470.2433.
TZTCO-1B:1H NMR(400MHz,CDCl3)δ8.88(d,J＝15.6Hz,1H),8.69(d,J＝8.4Hz,1H),8.67–8.63(m,1H),8.61(d,J＝7.6Hz,1H),8.04(d,J＝7.6Hz,1H),7.81(dd,J＝8.4,7.6Hz,1H),7.51(d,J＝15.6Hz,1H),4.20(t,J＝7.6Hz,2H),3.80–3.70(m,1H),3.03–2.98(m,2H),2.86–2.75(m,4H),2.18–2.07(m,1H),2.05–1.96(m,1H),1.90–1.78(m,2H),1.78–1.69(m,3H),1.66–1.58(m,2H),1.52–1.40(m,2H),0.99(t,J＝7.6Hz,3H)；13C NMR(151MHz,CDCl3)δ164.36,164.16,157.38,153.80,140.90,138.90,137.16,131.53,130.97,130.86,130.51,130.13,128.77,127.28,127.25,124.52,123.20,122.54,71.71,40.45,37.61,35.11,30.37,29.85,25.89,25.53,23.72,20.56,14.02；HRMS(ESI)m/z calculated forC29H32O3N3[M+H]+:470.2438,found:470.2435.
实施例26：TZTCO-2的合成
Figure BDA0002858696760000271
TZTCO-2的合成同TZTCO-1类似，Pre-TLC分离纯化(DCM/MeOH＝30:1)得白色固体(TLC较小极性产物点)TZTCO-2A:6mg,收率28％；白色固体(TLC较大极性产物点)TZTCO-2B:7mg,收率33％。TZTCO-2A:1H NMR(400MHz,CDCl3)δ8.66(t,J＝7.2Hz,2H),8.33(d,J＝8.4Hz,1H),7.78–7.70(m,2H),7.68–7.59(m,4H),4.27–4.17(m,2H),3.77–3.66(m,1H),3.03–2.94(m,1H),2.91(t,J＝6.4Hz,2H),2.87–2.80(m,1H),2.79(s,3H),2.07–1.95(m,2H),1.89–1.81(m,1H),1.80–1.70(m,5H),1.68–1.63(m,1H),1.52–1.43(m,2H),1.00(t,J＝7.6Hz,3H)；13C NMR(151MHz,CDCl3)δ164.44,164.25,160.32,158.01,146.26,139.21,138.66,138.35,138.04,132.64,131.40,130.90,130.13,130.07,129.57,128.83,128.03,127.16,123.11,122.19,71.64,40.46,39.39,35.90,30.38,29.84,27.15,24.43,24.18,20.56,14.02；HRMS(ESI)m/z calculated for C33H34 O3N3[M+H]+:520.2595,found:520.2593.
TZTCO-2B:1H NMR(400MHz,CDCl3)δ8.66(t,J＝7.6Hz,2H),8.34(dd,J＝8.4,1.2Hz,1H),7.78–7.70(m,2H),7.67–7.59(m,4H),4.27–4.18(m,2H),3.75–3.64(m,1H),3.11–3.00(m,1H),2.91–2.85(m,2H),2.83(s,3H),2.26–2.14(m,1H),1.89–1.81(m,2H),1.78–1.63(m,7H),1.51–1.45(m,2H),1.00(t,J＝7.6Hz,3H)；13C NMR(151MHz,CDCl3)δ164.45,164.27,160.40,157.84,146.28,139.20,138.31,137.78,132.66,131.41,130.91,130.14,130.03,129.57,128.85,128.04,127.16,123.12,122.19,71.88,40.46,37.20,35.84,30.39,29.85,27.43,26.49,24.10,20.56,14.03；HRMS(ESI)m/z calculated forC33H34 O3N3[M+H]+:520.2595,found:520.2589.
实施例27：TZTCO-3的合成
Figure BDA0002858696760000281
TZTCO-3的合成同TZTCO-1类似，Pre-TLC分离纯化(DCM/MeOH＝30:1)得白色固体(TLC较小极性产物点)TZTCO-3A:6mg,收率27％；白色固体(TLC较大极性产物点)TZTCO-3B:6mg,收率27％。TZTCO-3A:1H NMR(400MHz,CDCl3)δ8.69(d,J＝7.6Hz,1H),8.63(d,J＝7.2Hz,1H),7.80(d,J＝7.6Hz,1H),7.66(t,J＝7.6Hz,1H),7.61(d,J＝7.2Hz,1H),7.30(d,J＝2.8Hz,2H),4.22(d,J＝7.6Hz,2H),3.78–3.65(m,1H),3.05–2.94(m,1H),2.94–2.79(m,3H),2.77(s,3H),2.09–1.96(m,2H),1.94(s,6H),1.88–1.81(m,1H),1.79–1.72(m,4H),1.69–1.59(m,2H),1.53–1.43(m,2H),1.00(t,J＝7.6Hz,3H)；13C NMR(151MHz,CDCl3)δ164.40,164.29,160.67,157.82,145.68,138.41,138.14,138.11,137.86,136.71,136.62,131.88,131.50,131.20,130.30,128.71,128.45,128.38,127.82,127.35,123.30,122.20,71.74,40.42,39.35,35.98,30.39,27.11,24.45,24.21,20.69,20.55,20.42,14.01；HRMS(ESI)m/z calculated for C35H38O3N3[M+H]+:548.2908,found:548.2905.
TZTCO-3B:1H NMR(400MHz,CDCl3)δ8.69(d,J＝7.6Hz,1H),8.63(dd,J＝7.2,1.2Hz,1H),7.80(dd,J＝8.4,1.2Hz,1H),7.66(dd,J＝8.4,7.2Hz,1H),7.61(d,J＝7.6Hz,1H),7.28(s,2H),4.26–4.18(m,2H),3.72–3.63(m,1H),3.09–2.98(m,1H),2.91–2.85(m,2H),2.80(s,3H),2.26–2.14(m,1H),1.94(s,6H),1.89–1.80(m,2H),1.78–1.66(m,6H),1.52–1.43(m,2H),1.00(t,J＝7.6Hz,3H)；13C NMR(101MHz,CDCl3)δ164.40,164.30,160.76,157.66,145.69,138.95,138.12,138.09,137.59,136.65,136.56,131.89,131.50,131.20,130.31,128.71,128.45,128.38,127.83,127.35,123.30,122.20,71.90,40.42,37.20,35.95,30.39,27.46,26.56,24.10,20.69,20.56,20.47,14.02；HRMS(ESI)m/zcalculated for C35H38O3N3[M+H]+:548.2908,found:548.2903.
实验例28：实施例中化合物探针分子的荧光性质测试
1.探针分子的吸收和发射光谱测定
(1)配制实施例中化合物探针分子的DMSO储备液(10mM),然后用DMSO稀释至终浓度为10μM的探针分子测试液。
(2)将上述探针分子测试液加入96孔板中，复孔，每孔200μL,然后用TecanSparkTM10M Multimode Microplate Reader多模式酶标仪测试化合物的吸收光谱，并记录每个探针分子的最大吸收波长，如表1所示。
(3)将DMSO储备液(10mM)，用DMSO或相应的水/DMSO溶剂体系稀释至终浓度为10μM，根据上述所得最大吸收波长，分别选择340nm或365nm激发测试探针分子的发射光谱，如图1～图33所示；并记录每个探针分子的最大发射波长，如表1所示。
2.探针分子的摩尔消光系数和荧光量子产率的测定
(1)将上述探针分子储备液用乙腈稀释至终浓度为10μM的探针分子测试液，用石英比色皿在酶标仪上测试探针分子的吸光度，然后计算摩尔消光系数，如表1所示。
(2)以3μg/ml的0.1N硫酸为参比溶液，分别测试探针分子的吸光度(调节浓度使吸光度<0.05)和峰面积，然后通过参比法计算各探针分子的荧光量子产率，如表1所示。
表1探针分子的光物理常数
Figure BDA0002858696760000291
3.探针分子的AIE性质测试
(1)将探针分子储备液配制成终浓度为10μM的不同含水量的DMSO/H2O溶液，含水量依次为10％，20％，30％，40％，50％，60％，70％，80％，85％，90％，95％，99％。
(2)根据探针分子的最大吸收波长，分别选择340nm或365nm激发，测试探针分子在上述不同含水量体系中的发射光谱，如图1～图33所示，并分析探针分子的AIE性质。
实验例29：萘二甲酰亚胺-四嗪类探针分子点击反应的二级动力学常数测定
(1)配制1mM的四嗪探针DMSO测试液，10mM的BCN和TCO的DMSO测试液。
(2)将5μL的上述四嗪探针测试液和190μLDMSO加入96孔板中，然后快速加入5μL的上述BCN或TCO，探针分子终浓度为25μM，BCN或TCO终浓度为250μM，选择四嗪小分子探针的最大吸收波长和相应的点击反应后产物的最大发射波长作为测试波长，用酶标仪测试荧光强度随时间的变化，用graphpad prism 8软件拟合曲线，并计算反应的二级反应动力学常数，如图34～图37所示。
实验例30：萘二甲酰亚胺-四嗪类探针分子的生物学应用
(1)将探针储备液配制成1mM四嗪探针的乙腈溶液，配制30μM含有BCN基团的胎牛血清白蛋白(BSA)的PBS溶液。
(2)将10μL上述探针储备液和10μL含有BCN的胎牛血清白蛋白(BSA)的PBS溶液加入离心管中，离心后静置1h，然后沸水加热10min,取出离心；每个离心管中加入2.5μL的5*SDS loading buffer，离心。
(3)在BeyogelTM SDS-PAGE预制胶中加入上述测试液，100V,80min电泳，取出水洗，进行胶内荧光观察，随后用考马斯亮兰染色成像，观察蛋白条带，如图38所示。

Claims (9)

1.通式I所示的萘二甲酰亚胺-四嗪类化合物及其相应的与环辛炔BCN[(1R,8S,9S)-Bicyclo[6.1.0]non-4-yn-9-ylmethanol]或反式环辛烯TCO[(4E)-4-Cycloocten-1-ol]发生生物正交反应后的产物，
Figure FDA0002858696750000011
其中，X选自如下结构片段：
Figure FDA0002858696750000012
R1选自C1-C6的烷基、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
R2选自C1-C6的烷基、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
L选自氧、硫、氮杂原子、亚甲基、乙烯基、含有1-3个氮、氧、硫杂原子的五元或六元芳杂环、取代或未取代的苯基，所述取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
Y选自C1-C6的烷基、含氮、氧、硫杂原子的烷基、取代或未取代的苯基，所述取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基。
2.根据权利要求1所述的化合物，其特征在于，所述的化合物选自通式I-1A、I-1B或I-1C：
Figure FDA0002858696750000013
其中，R1选自C1-C6的烷基、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
Y选自C1-C6的烷基、含氮、氧、硫杂原子的烷基、取代或未取代的苯基，所述取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基。
3.根据权利要求1所述的化合物，其特征在于，所述的化合物选自通式I-2所示的化合物：
Figure FDA0002858696750000021
其中，Y选自C1-C6的烷基、含氮、氧、硫杂原子的烷基、取代或未取代的苯基，所述取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
R2选自C1-C6的烷基、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
L选自氧、硫、氮杂原子、亚甲基、乙烯基、含有1-3个氮、氧、硫原子的五元或六元芳杂环、取代或未取代的苯基，所述取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基。
4.根据权利要求3所述的化合物，其特征在于，所述的化合物选自通式I-3所示的化合物：
Figure FDA0002858696750000022
其中，Y选自C1-C6的烷基、含氮、氧、硫杂原子的烷基、取代或未取代的苯基，所述取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
R2选自C1-C6的烷基、取代或未取代的苯基，所述取代或未取代的苯基中的取代基选自甲基、甲氧基、三氟甲基、氰基、卤素原子、硝基、二甲氨基；
R3和R4分别选自氢、甲基、甲氧基、三氟甲基、卤素原子、硝基、氰基、二甲氨基，其中卤素选自氟、氯、溴和碘。
5.根据权利要求1～4任一项所述的萘二甲酰亚胺-四嗪类化合物，其特征在于，所述的化合物选自：
Figure FDA0002858696750000031
Figure FDA0002858696750000041
6.权利要求1～2所述化合物的制备方法，其特征在于，通过以下合成路线进行制备：
(1)探针分子TZ-1和TZ-2的合成路线：
Figure FDA0002858696750000051
(2)探针分子TZ-3的合成路线：
Figure FDA0002858696750000061
(3)探针分子TZ-4的合成路线：
Figure FDA0002858696750000062
(4)探针分子TZ-5～TZ-12的合成路线：
Figure FDA0002858696750000063
(5)探针分子TZBCN-1～TZBCN-12的制备方法：
Figure FDA0002858696750000064
(6)探针分子TZTCO-1～TZTCO-3的制备方法：
Figure FDA0002858696750000071
7.权利要求1～5任一项所述的化合物具有生物正交荧光增强效应和聚集诱导发光效应，及其在制备光学成像中的应用。
8.权利要求1～5任一项所述的化合物在制备荧光标记成像中的应用。
9.权利要求1～5任一项所述的化合物在制备荧光探针中的应用。
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