WO2023284249A1 - 咪唑吡嗪酮类荧光素及其制备方法 - Google Patents

咪唑吡嗪酮类荧光素及其制备方法 Download PDF

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WO2023284249A1
WO2023284249A1 PCT/CN2021/138520 CN2021138520W WO2023284249A1 WO 2023284249 A1 WO2023284249 A1 WO 2023284249A1 CN 2021138520 W CN2021138520 W CN 2021138520W WO 2023284249 A1 WO2023284249 A1 WO 2023284249A1
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electron
solvent
compound
fluorescein
tautomer
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孙钦超
于海霞
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Shenzhen Institute of Advanced Technology of CAS
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    • A—HUMAN NECESSITIES
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    • A61K49/00—Preparations for testing in vivo
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    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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    • C09K11/00—Luminescent materials, e.g. electroluminescent or chemiluminescent
    • C09K11/06—Luminescent materials, e.g. electroluminescent or chemiluminescent containing organic luminescent materials
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G01N21/763—Bioluminescence
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    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1059—Heterocyclic compounds characterised by ligands containing three nitrogen atoms as heteroatoms

Definitions

  • the invention belongs to the technical field of chemical industry, and in particular relates to an imidazole pyrazinone fluorescein with a push-pull electronic structure and a preparation method thereof.
  • Bioluminescence imaging is a new optical imaging technology in recent years. Compared with fluorescence imaging, bioluminescent imaging is characterized by high sensitivity and quantitative analysis. As an in vivo reporter source, bioluminescence does not require excitation light, and emits light through the specific action of substrates and enzymes, and the animal itself does not emit light, which makes the background of the bioluminescent signal extremely low and can obtain a high signal-to-noise ratio. In addition, the level of fluorescent signal depends on the number of luminescent cells and the intensity of excitation light. The absorption of light by biological tissues makes it difficult to measure the fluorescence intensity.
  • the detected bioluminescence intensity is linearly related to the number of labeled targets.
  • bioluminescence can be quantitatively analyzed.
  • the advantages of bio-imaging technology make it possible to establish various tumor models in the study of tumor diseases (inserting the luciferase gene into random sites in the chromatin of tumor cells and then transferring it into animals), which can be used for real-time observation under near non-invasive conditions Proliferation, growth, and metastasis of tumor cells in vivo), antitumor drug research (labeling tumor cells with luciferase, by giving different doses, different administration times, and different administration routes to mice inoculated with tumors, observe and formulate appropriate Dosage form and medication time, real-time evaluation of the therapeutic effects of various treatment methods), cell markers (labeling immune cells, observing the recognition and killing functions of immune cells on tumor cells, and evaluating the immune specificity, proliferation, migration and other functions of immune cells), It has
  • Bioluminescence belongs to the category of chemiluminescence, which is a luminescent phenomenon accompanied by the luciferase-luciferin enzymatic reaction.
  • the two key factors in the in vivo optical imaging technology based on bioluminescence are luciferase and luciferin.
  • luciferase a luminescent phenomenon accompanied by the luciferase-luciferin enzymatic reaction.
  • luciferase and luciferin luciferase and luciferin.
  • common bioluminescence is mainly divided into three categories: insect luciferases (firefly luciferase FLuc), bacterial luciferases (LuxAB) and marine luciferases (such as Renilla luciferase).
  • Enzyme RLuc Enzyme RLuc-like bioluminescent phenomena, among which bacterial luciferase is mostly used in the research of bacterial infection and antibiotic drugs, insect luciferase and marine luciferase are suitable for in vivo bioluminescence imaging technology of mammals.
  • bioluminescence based on firefly luciferase.
  • the enzymatic reaction between this type of enzyme and the corresponding substrate requires the participation of adenosine triphosphate (ATP) and magnesium ion Mg 2+ as co-factors in addition to oxygen molecules.
  • ATP adenosine triphosphate
  • magnesium ion Mg 2+ magnesium ion Mg 2+
  • this type of bioluminescent technology has the following technical problems: (1) most of the reported maximum emission wavelengths are in the range of 400-550 nm, and the tissue penetration depth is limited; (2) the bulk structure of coelenterazine fluorescein ( imidazopyrazinone) is easy to ring-open and produces autofluorescence; (3) poor water solubility cannot meet the dosage requirements of large-volume animal experiments. Therefore, increasing the tissue penetration depth of the light signal, redshifting the bioluminescent wavelength to the deep red/near-infrared region, increasing the stability of the substrate molecule, meeting the requirements of high signal-to-noise ratio for in vivo imaging, and improving the fluorescein substrate Water solubility is very necessary.
  • Intramolecular charge transfer is an effective method that can redshift the molecular luminescent spectrum.
  • the wavelength red-shift of imidazopyrazinone-based fluorescein bioluminescence increases the tissue penetration depth of in vivo imaging, improves molecular stability, and inhibits background interference caused by autoxidative luminescence.
  • hydrophilic groups to increase its water solubility, it can meet the requirements of large-area imaging.
  • the new water-soluble and red-shifted imidazole pyrazinone luciferin not only enriches the structure-activity relationship of luciferin-luciferase interaction, but also provides better application prospects for bioluminescence imaging technology in the field of medical research.
  • the present invention provides an imidazopyrazinone fluorescein with a push-pull electronic structure and a preparation method thereof.
  • the embodiment of the present invention provides a compound represented by formula (I), or its tautomer or salt:
  • R1 is benzyl or a benzyl-containing group
  • R2 is an electron - donating group
  • R3 is an electron-deficient group
  • R is one of the following structures :
  • R3 is one of the following structures:
  • the embodiment of the present invention also provides a method for preparing the above-mentioned compound, or its tautomer or salt, comprising the following steps:
  • Step 1 under the protection of nitrogen, the compound 2-amino-3-iodo-5-bromopyrazine and the electron-deficient group pinacol borate are dissolved in the first solvent, in the presence of a catalyst, and the second solvent is added, Reacted at 110°C for 2 hours, separated and purified after cooling to obtain intermediate 1;
  • Step 2 Under the protection of nitrogen, the above-mentioned intermediate 1 and the electron-donating group pinacol borate are dissolved in the first solvent, in the presence of a catalyst, and the second solvent is added, reacted at 110°C for 12 hours, separated and purified after cooling , to obtain intermediate 2.
  • Step 3 Under the protection of nitrogen, add intermediate 2 to the third solvent to dissolve, add concentrated hydrochloric acid dropwise, add 3-phenyl-1,1-diethoxyacetone at the same time, react at 80°C for 8 hours, and separate after cooling Purify to obtain the target product.
  • the present invention also provides a chemiluminescence method, which uses the above-mentioned compound, or its tautomer or salt as a substrate, and the luminescence wavelength is in the range of 576-652nm.
  • the beneficial effects of the present invention are: 1) The imidazole pyrazinone fluorescein synthesized by the present invention is simple to prepare, the reaction conditions are mild, and the yield is relatively good. , has a certain hydrophilicity; 2) the imidazole pyrazinone fluorescein synthesized by the present invention can be ring-opened with high-brightness luminescence under alkaline catalysis in DMSO solution, and has the property of chemiluminescence; 3) the synthetic fluorescein of the present invention
  • the imidazopyrazinone fluorescein is a coelenterazine fluorescein substrate, and under the catalysis of luciferase, bioluminescence occurs when the ring is opened, and the luminescence wavelength is red-shifted, which is of great significance for the application of bioluminescence imaging in vivo research; 4) Under the same test conditions, the chemiluminescent brightness of the synthesized substrate is comparable to that of the reported substrate reference DTZ under
  • Fig. 1 is the synthetic route of imidazole pyrazinone compound in the embodiment of the present invention
  • Fig. 2 is the normalized chemiluminescent spectrum of imidazole pyrazinone fluorescein (1-12) structural formula in the embodiment of the present invention
  • Fig. 3 is the bioluminescent spectrum of imidazole pyrazinone fluorescein (9) in the example of the present invention.
  • An embodiment of the present invention provides an imidazole pyrazinone fluorescein compound with a push-pull electronic structure or a tautomer or salt thereof, which has the following general formula:
  • R 1 is benzyl or a group containing benzyl
  • R 2 is an electron-donating group (EDG)
  • R 3 is an electron-deficient group (EWG).
  • the above-mentioned compounds can be used as luciferase substrates for bioluminescent imaging, which have the property of alkaline catalysis in DMSO solution or under the catalysis of luciferase to open the imidazolone structure and emit light.
  • the glow of the rings ranges from green to red.
  • the substituted electron-donating/deficiency groups contain N, O, and F atoms, which are easy to form hydrogen bonds with water molecules and help to increase the water solubility of the molecules.
  • R is one of the following structures:
  • R3 is one of the following structures:
  • the synthetic route includes the following three steps: Step 1: under nitrogen protection, compound 2-amino-3-iodo-5-bromopyrazine Dissolve the pinacol borate with the electron deficient group (EWG, R 3 in formula I) in the first solvent, in the presence of a catalyst, and add the second solvent, react at 110°C for 2 hours, separate and purify after cooling , to obtain intermediate 1
  • Step 1 under nitrogen protection, compound 2-amino-3-iodo-5-bromopyrazine
  • EWG, R 3 in formula I electron deficient group
  • Step 2 Under the protection of nitrogen, the above-mentioned intermediate 1 and the electron-donating group (EDG, R 2 in formula I) pinacol borate are dissolved in the first solvent, in the presence of a catalyst, and the second solvent is added , reacted at 110°C for 12 hours, separated and purified after cooling to obtain intermediate 2
  • EDG electron-donating group
  • Step 3 Under the protection of nitrogen, add intermediate 2 to the third solvent to dissolve, add concentrated hydrochloric acid dropwise, and add 3-phenyl-1,1-diethoxyacetone at the same time React at 80°C for 8 hours, separate and purify after cooling to obtain the target product, namely the compound of the above general formula I.
  • the above-mentioned first solvent is 1,4-dioxane (1,4-Dioxane)
  • the second solvent is potassium carbonate solution
  • the third solvent is ethanol
  • the catalyst is bis(triphenylphosphine) dichloride Palladium, wherein 1,4-dioxane and ethanol are reaction solvents, while providing a polar environment for the reaction, its boiling point ensures the reaction temperature.
  • Embodiment 1 Preparation of 2-amino-3-iodo-5-bromopyrazine
  • the pinacol borate that concrete uses is following structure:
  • the target product information is as follows (the corresponding structural formula is as follows):
  • table 1 has summarized the spectral properties of its chemiluminescence and photoluminescence, as can be seen from the figure and the table, by Modification of C6 and C8 electron-donating/deficient groups, the maximum emission wavelength is distributed in the green to red light region, and the fluorescence is obviously red-shifted, indicating that the push-pull electronic structure can effectively adjust the imidazole that can be used as a coelenterazine fluorescein substrate Photophysical properties of the ring-opening luminescence of pyrazinone-type fluoresceins.
  • Bioluminescent properties Select imidazole pyrazinone luciferin (9) as the luciferin substrate, and use a microplate reader to test its bioluminescent properties with the new luciferase NanoLuc in a 96-well plate.
  • Figure 3 shows the enzyme-substrate It can be seen from the figure that the luminescence is obviously red-shifted, indicating that the push-pull electronic structure has an obvious regulation effect on the bioluminescent properties of the luciferin substrate.
  • the red-shifted emission wavelength of the novel luciferase-luciferin substrate interaction will help to promote the application of bioluminescent imaging in the biomedical field.

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Abstract

本发明属于化工技术领域，具体提供式(I)所示的化合物，或其互变异构体或盐：其中R1为苄基或者含有苄基的基团，R2为给电子基团，R3为缺电子基团。本发明通过对分子结构的设计，实现对材料性质的调控：一方面通过分子设计在分子本体结构的C6/C8位分别引入给电子和缺电子基团，形成推拉电子结构，调整分子内电子云分布，使得咪唑吡嗪酮类荧光素发光光谱红移且稳定性好。

Description

咪唑吡嗪酮类荧光素及其制备方法 技术领域
本发明属于化工技术领域，具体涉及一种具有推拉电子结构的咪唑吡嗪酮类荧光素及其制备方法。
背景技术
活体光学成像以其操作简便及直观性等特点成为生命科学研究中的一种重要的非侵入性成像手段，生物发光成像是近年来新兴的一种光学成像技术。生物发光成像相较于荧光成像的显著特点是灵敏度高、可定量分析。作为体内报告源，生物发光不需要激发光激发，是以底物和酶的特异作用而发光，且动物体本身不发光，这使得生物发光信号背景极低，可获得很高的信噪比。另外，荧光信号水平取决于发光细胞的数量及激发光强度，生物组织对光的吸收使得荧光强度很难计量。然而，检测到的生物发光强度与标记靶体的数量呈线性相关，通过记录单位时间、单位面积、单位角度接收的光子数进行比较，生物发光可实现定量分析。生物成像技术的优势使其在肿瘤疾病研究(将荧光素酶基因插入到肿瘤细胞染色质的随机位点后将其转入动物体内可以建立各种肿瘤模型，可用于在近无创条件下实时观察体内肿瘤细胞的增殖、生长、转移情况)、抗肿瘤药物研究(用荧光素酶标记肿瘤细胞，通过给予接种肿瘤的小鼠不同剂量、不同给药时间、不同给药途径，观察并制定合适的剂型与服药时间，实时评价各种治疗手段的治疗效果)、细胞标记(标记免疫细胞，观察免疫细胞对肿瘤细胞的识别和杀伤功能，评价免疫细胞的免疫特异性、增殖、迁移等功能)、观测目的基因表达及蛋白质相互作用等领域得到广泛应用，具有很高的科学研究价值。
生物发光属于化学发光范畴，是一种伴随荧光素酶-荧光素酶促反应而产生的发光现象，基于生物发光的活体光学成像技术的两个关键因素是荧光素酶和荧光素。按荧光素酶种类的不同，常见的生物发光主要分为三类:昆虫荧光素 酶(萤火虫荧光素酶FLuc)类、细菌荧光素酶(LuxAB)类和海洋荧光素酶(如海肾荧光素酶RLuc)类生物发光现象，其中，细菌荧光素酶多用于进行细菌侵染和抗生素药物的研究，昆虫荧光素酶和海洋荧光素酶适用于哺乳类动物的活体生物发光成像技术。目前，基于萤火虫荧光素酶的生物发光研究较多的，该类酶与相应底物的酶促反应，除氧气分子外，还需要三磷酸腺苷ATP、镁离子Mg 2+作为共因子参与。然而，ATP、Mg 2+在不同的生物环境中的含量不同影响酶的活性，且消耗ATP可能导致正常的新陈代谢紊乱；而基于腔肠素类荧光素——咪唑吡嗪酮类荧光素为催化底物，其与新型低分子量的海洋荧光素酶类荧光素酶的酶促反应不需要共因子，在氧气分子参与下即可使其生物发光在活体内表达而更具优势(ACS Chem.Biol.,2019,14,959)。
然而，该类生物发光技术存在如下技术问题：(1)目前多数该类报道的最大发射波长在400-550nm范围内，组织穿透深度有限；(2)腔肠素类荧光素的本体结构(咪唑吡嗪酮)容易开环，产生自发荧光；(3)水溶性差，不能满足大体积的动物实验的剂量需要。因此，增加光信号的组织穿透深度，将生物发光波长红移到深红/近红外光区，增加底物分子的稳定性，满足活体成像高信噪比的要求，以及改善荧光素底物的水溶性是十分必要的。
分子内电荷转移是一种能够将分子发光光谱红移的有效方法，通过引入并调整推拉电子结构，改变分子内电子云分布来优化荧光素底物结构，为根源性地从分子结构方面改善基于咪唑吡嗪酮类荧光素生物发光的波长红移，增加活体成像的组织穿透深度，以及改善分子稳定性，抑制自氧化发光产生背景干扰。另外，鉴于当前咪唑吡嗪酮类荧光素的水溶性较差，通过引入亲水性基团，增加其水溶性，可以满足大面积成像的要求。因此，新型水溶性好的发光红移的咪唑吡嗪酮类荧光素在丰富荧光素-荧光素酶作用的构效关系的同时，为生物发光成像技术在医学研究领域提供更好的应用前景。
发明内容
为解决上述技术问题，本发明提供一种具有推拉电子结构的咪唑吡嗪酮类荧光素及其制备方法。
本发明实施例提供一种式(I)所示的化合物，或其互变异构体或盐：
Figure PCTCN2021138520-appb-000001
其中R 1为苄基或者含有苄基的基团，R 2为给电子基团，R 3为缺电子基团。
优选地，R 2为下列结构中的一种:
Figure PCTCN2021138520-appb-000002
优选地，R 3为下列结构中的一种:
Figure PCTCN2021138520-appb-000003
本发明实施例还提供一种制备上述化合物，或其互变异构体或盐的方法，包括如下步骤：
步骤一：在氮气保护下，化合物2-氨基-3-碘-5-溴吡嗪与缺电子基团频哪醇硼酸酯溶解在第一溶剂中，在存在催化剂，且加入第二溶剂，在110℃反应2小时，冷却后分离提纯，得到中间体1；
步骤二：在氮气保护下，上述中间体1与给电子基团频哪醇硼酸酯溶解在第一溶剂中，在存在催化剂，且加入第二溶剂，110℃反应12小时，冷却后分离提纯，得到中间体2。
步骤三：在氮气保护下，将中间体2加入第三溶剂中溶解，滴加浓盐酸，同时加入3-苯基-1,1-二乙氧基丙酮，80℃反应8小时，冷却后分离提纯，得到目标产物。
本发明还提供一种化学发光的方法，所述方法采用上述化合物，或其互变异构体或盐作为底物，且发光的波长在576-652nm范围。
本发明的有益效果是：1)本发明合成的咪唑吡嗪酮类荧光素制备简单，反应条件温和，产率相对较好，在修饰的给电子/缺电子取代基中引入含孤对电子原子，具有一定的亲水性；2)本发明合成的咪唑吡嗪酮类荧光素用在DMSO溶液中碱性催化下能够开环伴随高亮度发光，具有化学发光的性质；3)本发明合成的咪唑吡嗪酮类荧光素为腔肠素类荧光素底物，荧光素酶催化下开环发生生物发光现象，发光波长红移，这对生物发光成像在体研究应用方面有重要意义；4)该合成方法在相同的测试条件下，相对已报道的底物对照物DTZ，合成的底物的化学发光的亮度可比，且波长在576-652nm范围内，光谱明显红移。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例。
图1为本发明实施例中咪唑吡嗪酮类化合物的合成路线；
图2为本发明实施例中咪唑吡嗪酮类荧光素(1-12)结构式的归一化的化学 发光光谱；
图3为本发明实施例中咪唑吡嗪酮类荧光素(9)的生物发光光谱。
具体实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明实施例提供一种具有推拉电子结构的咪唑吡嗪酮类荧光素化合物或其互变异构体或盐，其具有如下通式：
Figure PCTCN2021138520-appb-000004
其中R 1为苄基或者含有苄基的基团，R 2为给电子基团(EDG)，R 3为缺电子基团(EWG)。
上述化合物可作为荧光素酶底物用于生物发光成像，其具有在DMSO溶液中碱性催化或在荧光素酶催化下咪唑酮结构开环发光的性质，给/缺电子取代基修饰后分子开环的发光在绿光到红光区间分布。取代的给/缺电子基团中含有N、O、F原子，易与水分子形成氢键，有助于增加分子的水溶性。
优选地，上述结构式中，R 2为下列结构中的一种:
Figure PCTCN2021138520-appb-000005
优选地，上述结构式中，R 3为下列结构中的一种:
Figure PCTCN2021138520-appb-000006
本发明实施例提供的上述化合物的合成路线如图1所示。从图1可以看出，该合成路线包括如下三个步骤：步骤一：在氮气保护下，化合物2-氨基-3-碘-5-溴吡嗪
Figure PCTCN2021138520-appb-000007
与缺电子基团(EWG，通式I中的R 3)频哪醇硼酸酯溶解在第一溶剂中，在存在催化剂，且加入第二溶剂，在110℃反应2小时，冷却后分离提纯，得到中间体1
Figure PCTCN2021138520-appb-000008
步骤二：在氮气保护下，上述中间体1与给电子基团(EDG，通式I中的R 2)频哪醇硼酸酯溶解在第一溶剂中，在存在催化剂，且加入第二溶剂，110℃反应12小时，冷却后分离提纯，得到中间体2
Figure PCTCN2021138520-appb-000009
步骤三：在氮气保护下，将中间体2加入第三溶剂中溶解，滴加浓盐酸，同时加入3-苯基-1,1-二乙氧基丙酮
Figure PCTCN2021138520-appb-000010
80℃反应8小时，冷却后分离提纯，得到目标产物，即上述通式I的化合物。
具体地，上述第一溶剂为1,4-二氧六烷(1,4-Dioxane)，第二溶剂为碳酸钾溶液，第三溶剂为乙醇；催化剂为二(三苯基膦)二氯化钯，其中1,4-二氧 六烷、乙醇是反应溶剂，在提供反应的极性环境的同时其沸点保证反应温度。
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1：2-氨基-3-碘-5-溴吡嗪的制备
向反应瓶中称取2g 2-氨基-5-溴吡嗪，3.1g N-碘代丁二酰亚胺，加入20mL1,4-二氧六烷，在氮气保护下，80℃反应4小时，反应液冷却至室温后倒入20mL饱和硫代硫酸钠水溶液中，乙酸乙酯萃取三次，收集的有机相用盐水洗，无水硫酸钠干燥，真空旋转蒸发浓缩，柱层析分离纯化，得3.8g白色固体，为目标化合物，产率58％。 1H NMR(400MHz,DMSO-d6)δ8.04(s,1H),6.76(brs,2H)。
实施例2：缺电子基团取代的中间体-1的制备
采用Suzuki偶联反应合成不同缺电子基团取代的中间体-1：具体操作如下：反应瓶中称取实施例1制备的2-氨基-3-碘-5-溴吡嗪(1当量(equiv.))(当量浓度)，频哪醇硼酸酯(1.1当量)，催化剂二(三苯基膦)二氯化钯(5-7mol％)，加入1,4-二氧六烷/1M碳酸钾水溶液(3当量)，在氮气保护下，110℃反应2小时，反应液冷却至室温后倒入水中，乙酸乙酯萃取三次，收集的有机相经无水硫酸钠干燥，真空旋转蒸发浓缩，柱层析分离纯化，得中间体-1。具体使用的频哪醇硼酸酯为如下结构：
Figure PCTCN2021138520-appb-000011
上述结构式对应的产物信息如下：
2-氨基-3-吡啶基-5-溴吡嗪， 1H NMR(400MHz,CD3OD)δ8.67(d,J＝6.4Hz,2H),8.11(s,1H),7.81(d,J＝6.4Hz,2H)，黄色固体，产率80％。
2-氨基-3-(3-甲基吡啶基)-5-溴吡嗪， 1H NMR(400MHz,CD 3OD)δ8.55(d,J＝5.2Hz,2H),8.12(s,1H),7.68(s,1H),7.61(dd,J1＝5.2Hz,J2＝1.4Hz,1H), 2.65(s,3H)，黄色固体，产率85％。
2-氨基-3-(4-甲基吡啶基)-5-溴吡嗪， 1H NMR(400MHz,CD3OD)δ8.77(d,J＝2.0Hz,2H),8.12(dd,J 1＝2.4Hz,J 2＝4.2Hz,1H),8.08(s,1H),7.46(d,J＝8.0Hz,1H),2.63(s,3H)，黄色固体，产率82％。
2-氨基-3-(4-氟代吡啶基)-5-溴吡嗪， 1H NMR(400MHz,CD3OD)δ8.32(d,J＝5.2Hz,1H),8.13(s,1H),7.69(dt,J 1＝1.6Hz,J 2＝5.2Hz,1H),7.44(s,1H)，淡黄色固体，产率80％。
2-氨基-3-(3,4,5-三氟代苯基)-5-溴吡嗪， 1H NMR(400MHz,CD 3OD)δ8.07(s,1H),8.13(s,1H),7.53(d,J＝6.8Hz,1H),7.51(d,J＝6.8Hz,1H)，淡黄色固体，产率85％。
2-氨基-3-(3-氰基吡啶基)-5-溴吡嗪， 1H NMR(400MHz,CD 3OD)δ8.80(d,J＝4.8Hz,1H),8.25(q,J＝0.8Hz,1H),8.16(s,1H),8.05(dd,J1＝1.6Hz,J2＝5.2Hz,1H)，黄色固体，产率80％。
2-氨基-3-(5-嘧啶基)-5-溴吡嗪， 1H NMR(400MHz,CD 3OD)δ9.20(s,1H),9.14(s,2H),8.12(s,1H)，淡黄色固体，产率85％。
2-氨基-3-(2-氯-5-嘧啶基)-5-溴吡嗪， 1H NMR(400MHz,CD 3OD)δ9.04(s,1H),8.12(s,1H)，淡黄色固体，产率80％。
实施例3：给电子基团取代的中间体-2的制备
采用Suzuki偶联反应合成不同给电子基团取代的中间体-2：反应瓶中称取2-氨基-3-碘-5-溴吡嗪(1当量)，频哪醇硼酸酯(1.5当量)，二(三苯基膦)二氯化钯(5-7mol％)，加入1,4-二氧六烷/1M碳酸钾水溶液(3当量)，在氮气保护下，110℃反应12小时，反应液冷却至室温后倒入水中，乙酸乙酯萃取三次，收集的有机相经无水硫酸钠干燥，真空旋转蒸发浓缩，柱层析分离纯化，得中间体-2，具体使用的频哪醇硼酸酯结构如下所示：
Figure PCTCN2021138520-appb-000012
上述结构式对应的产物信息如下：
2-氨基-3-吡啶基-5-(4-N,N-二甲氨基苯基)吡嗪， 1H NMR(400MHz,CD3OD)δ8.68(d,J＝6.0Hz,2H),8.43(s,1H),7.93(d,J＝6.0Hz,2H),7.84(d,J＝8.0Hz,2H),6.84(d,J＝8.0Hz,2H),2.99(s,6H)，黄色固体，产率70％。
2-氨基-3-(3-甲基吡啶基)-5-(4-N,N-二甲氨基苯基)吡嗪， 1H NMR(400MHz,CD3OD)δ8.56(d,J＝5.6Hz,2H),8.43(s,1H),7.85(d,J＝6.0Hz,2H),7.84(d,J＝8.4Hz,2H),7.79(s,1H),7.73(d,J＝5.4Hz,1H),6.86(d,J＝9.2Hz,1H),3.01(s,6H),2.66(s,3H)，黄色固体，产率70％。
2-氨基-3-(4-甲基吡啶基)-5-(4-N,N-二甲氨基苯基)吡嗪， 1H NMR(400MHz,CD3OD)δ8.85(d,J＝1.6Hz,1H),8.36(s,1H),8.19(dd,J1＝2.8Hz,J2＝7.6Hz,2H),7.81(d,J＝8.8Hz,2H),7.45(d,J＝7.2Hz,1H),6.83(d,J＝8.0Hz,1H),6.86(d,J＝9.2Hz,1H),2.99(s,6H),2.62(s,3H)，黄色固体，产率70％。
2-氨基-3-(4-氟代吡啶基)-5-(4-N,N-二甲氨基苯基)吡嗪， 1H NMR(400MHz,CD3OD)δ8.45(s,1H),8.33(d,J＝6.8Hz,1H),7.93-7.75(m,3H),7.56(s,1H),6.84(d,J＝8.4Hz,2H),3.00(s,6H)，黄色固体，产率70％。
2-氨基-3-(3,4,5-三氟代苯基)-5-(4-N,N-二甲氨基苯基)吡嗪， 1H NMR(400MHz,CD3OD)δ8.37(s,1H),7.82(d,J＝9.2Hz,2H),7.64(d,J＝6.4Hz,1H),7.62(d,J＝6.4Hz,1H),6.84(d,J＝9.2Hz,2H),2.99(s,6H)，黄色固体，产率70％。
2-氨基-3-(3-氰基吡啶基)-5-(4-N,N-二甲氨基苯基)吡嗪， 1H NMR(400MHz,CD3OD)δ8.81(d,J＝5.2Hz,1H),8.48(s,1H),8.37(s,1H),8.19(dd,J1＝1.6Hz,J2＝5.2Hz,1H),7.86(d,J＝8.8Hz,2H),6.85(d,J＝8.8Hz,2H),
3.00(s,6H)，黄色固体，产率70％。2-氨基-3-(5-嘧啶基)-5-(4-N,N-二甲氨基苯基)吡嗪， 1H NMR(400MHz,CD3OD)δ9.25(s,2H),9.20(s,1H),8.44 (s,1H),7.84(d,J＝8.8Hz,2H),6.84(d,J＝8.8Hz,2H),2.99(s,6H)，黄色固体，产率70％。
2-氨基-3-吡啶基-5-(4-羟基苯乙烯基)吡嗪， 1H NMR(400MHz,CD3OD)δ8.71(d,J＝6.0Hz,2H),8.14(s,1H),7.90(d,J＝6.0Hz,2H),7.79-7.73(m,2H),7.68-7.61(m,2H),7.49-7.39(m,3H),7.04(d,J＝16.0Hz,1H),6.80(d,J＝9.2Hz,1H)，黄色固体，产率80％。
2-氨基-3-吡啶基-5-(4-N,N-二甲氨基苯乙烯基)吡嗪， 1H NMR(400MHz,CD3OD)δ8.71(d,J＝6.4Hz,2H),8.13(s,1H),7.90(d,J＝6.0Hz,2H),7.47-7.39(m,3H),6.98(m,2H),7.49-7.39(m,3H),6.98(d,J＝16.0Hz,1H),6.78(d,J＝8.8Hz,2H),2.99(s,6H)，黄色固体，产率80％。
2-氨基-3-(3-氰基吡啶基)-5-(4-羟基苯乙烯基)吡嗪， 1H NMR(400MHz,CD3OD)δ8.79(d,J＝5.2Hz,2H),8.57(s,1H),8.15(s,1H),8.02(dd,J1＝1.2Hz,J2＝4.8Hz,1H),7.50-7.41(m,3H),7.03(d,J＝16.0Hz,1H),6.80(d,J＝8.0Hz,2H)，黄色固体，产率80％。
2-氨基-3-(3-氰基吡啶基)-5-(4-N,N-二甲氨基苯乙烯基)吡嗪， 1H NMR(400MHz,CD3OD)δ8.74(d,J＝4.8Hz,1H),8.68(s,1H),8.15(s,1H),7.97(dd,J＝4.8Hz,J＝1.6Hz,1H),8.53-8.42(m,4H),6.94(d,J＝16.0Hz,1H),3.02(s,6H)，黄色固体，产率80％。
2-氨基-3-(3-氰基吡啶基)-5-(4-氨基苯乙烯基)吡嗪， 1H NMR(400MHz,CD3OD)δ8.83(d,J＝5.7Hz,1H),8.35(s,1H),8.18-8.14(m,1H),7.70-7.64(m,1H),7.61-7.54(m,1H),7.41(d,J＝16.0Hz,1H),7.36(d,J＝8.0Hz,1H),6.96(d,J＝16.0Hz,1H),6.72(d,J＝8.0Hz,2H)，黄色固体，产率75％。
实施例4：咪唑吡嗪酮类荧光素(1-12)的制备
采用不同取代基修饰的中间体-2与3-苯基-1,1-二乙基-丙酮反应关环制备对应的目标分子：反应瓶中称取中间体-2(1当量)，3-苯基-1,1-二乙基-丙酮(2当量)，溶解在乙醇中，在氮气保护下滴加浓盐酸(7当量)，80℃反应8小时，反应液冷却至室温后真空旋转蒸发浓缩，柱层析分离纯化，得目标化合物。
目标产物信息如下(对应结构式如下)：
2-苄基-6-(4-N,N-二甲氨基苯基)-8-吡啶基-咪唑并[1,2-a]吡嗪-3-(7H)-酮(No.1)， 1H NMR(400MHz,CD3OD)δ9.40(d,J＝6.4Hz,2H),8.99(d,J＝6.4Hz,2H),8.78-8.73(m,1H),8.32(dd,J1＝2.4Hz,J2＝8.0Hz,2H),7.74(d,J＝8.0Hz,2H),7.39-7.27(m,4H),7.20(t,J＝7.2Hz,1H),4.26(s,2H),3.38(s,6H)，产率80％。。
2-苄基-6-(4-N,N-二甲氨基苯基)-8-(3-甲基吡啶基)-咪唑并[1,2-a]吡嗪-3-(7H)-酮(No.2)， 1H NMR(400MHz,CD3OD)δ9.31-9.28(m,2H),8.89-8.81(m,2H),8.43(d,J＝8.0Hz,2H),7.75(d,J＝8.0Hz,2H),7.40-7.25(m,1H),8.32(dd,J1＝2.4Hz,J2＝8.0Hz,2H),7.74(d,J＝8.0Hz,2H),7.39-7.27(m,4H),7.19(t,J＝7.2Hz,1H),4.28(s,2H),3.36(s,6H),2.93(s,3H)，产率75％。
2-苄基-6-(4-N,N-二甲氨基苯基)-8-(4-甲基吡啶基)-咪唑并[1,2-a]吡嗪-3-(7H)-酮(No.3)， 1H NMR(400MHz,CD3OD)δ9.94(s,1H),9.53(dd,J1＝1.6Hz,J2＝8.4Hz,2H),8.78(s,1H),8.35(d,J＝8.4Hz,2H),8.10(d,J＝8.4Hz,1H),7.85(d,J＝8.4Hz,2H),7.39-7.25(m,4H),7.21(t,J＝7.2Hz,1H),4.27(s,2H),3.37(s,6H),2.92(s,3H)，产率80％。
化合物4：2-苄基-6-(4-N,N-二甲氨基苯基)-8-(4-氟代吡啶基)-咪唑并[1,2-a]吡嗪-3-(7H)-酮(No.4)， 1H NMR(400MHz,CD3OD)δ8.91(s,1H),8.43(d,J＝5.4Hz,1H),8.39(d,J＝8.8Hz,2H),8.28(s,1H),8.13(d,J＝5.2Hz,1H),7.71(d,J＝8.4Hz,2H),7.37-7.28(m,4H),7.26-7.21(m,1H),4.29(s,2H),3.34(s,6H)，产率80％。
化合物5：2-苄基-6-(4-N,N-二甲氨基苯基)-8-(3,4,5-三氟代苯基)-咪唑并[1,2-a]吡嗪-3-(7H)-酮(No.5)， 1H NMR(400MHz,CD3OD)δ8.71(s,1H),8.27(d,J＝8.4Hz,1H),8.16(t,J＝7.6Hz,2H),7.60(d,J＝8.4Hz,2H),7.35-7.28(m,4H),7.26-7.19(m,1H),4.28(s,2H),3.30(s,6H)，产率80％。。
2-苄基-6-(4-N,N-二甲氨基苯基)-8-(3-氰基吡啶基)-咪唑并[1,2-a]吡嗪-3-(7H)-酮(No.6)， 1H NMR(400MHz,CD3OD)δ8.96(s,1H),8.92(d,J＝4.8Hz,1H),8.79(dd,J1＝1.2Hz,J2＝4.8Hz,1H),8.75(s,1H),8.34(d,J＝8.4Hz,2H), 7.63(d,J＝8.4Hz,2H),7.35-7.28(m,4H),7.21(t,J＝6.8Hz,1H),4.27(s,2H),3.32(s,6H)，产率60％。
2-苄基-6-(4-N,N-二甲氨基苯基)-8-(5-嘧啶基)-咪唑并[1,2-a]吡嗪-3-(7H)-酮(No.7)， 1H NMR(400MHz,CD3OD)δ9.73(s,1H),9.38(s,1H),8.87(s,1H),8.33(d,J＝8.4Hz,2H),7.70(d,J＝8.4Hz,2H),7.35-7.29(m,4H),7.21(t,J＝6.8Hz,1H),4.28(s,2H),3.33(s,6H)，产率60％。
2-苄基-6-(4-羟基苯乙烯基)-8-吡啶基-咪唑并[1,2-a]吡嗪-3-(7H)-酮(No.8)， 1H NMR(400MHz,CD3OD)δ9.34(d,J＝6.4Hz,2H),9.04(d,J＝6.4Hz,2H),8.03(s,1H),7.76(d,J＝16Hz,1H),7.51(d,J＝8.4Hz,2H),7.37-7.29(m,4H),7.21(t,J＝6.8Hz,1H),7.14(d,J＝16Hz,1H),6.84(d,J＝8.4Hz,2H),4.28(s,2H)，产率80％。
2-苄基-6-(4-N,N-二甲氨基苯乙烯基)-8-吡啶基-咪唑并[1,2-a]吡嗪-3-(7H)-酮(No.9)， 1H NMR(400MHz,CD3OD)δ9.52(t,J＝6.4Hz,2H),9.04(d,J＝6.8Hz,2H),8.41(s,1H),7.95-7.89(m,3H),7.73(d,J＝8.4Hz,2H),7.50(d,J＝16Hz,1H),7.37-7.26(m,4H),7.21(t,J＝6.8Hz,1H),7.14(d,J＝16Hz,1H),6.84(d,J＝8.4Hz,2H),4.27(s,2H),3.36(s,6H)，产率85％。
2-苄基-6-(4-羟基苯乙烯基)-8-(3-氰基吡啶基)-咪唑并[1,2-a]吡嗪-3-(7H)-酮(No.10)， 1H NMR(400MHz,CD3OD)δ8.95(d,J＝4.4Hz,2H),8.73(s,1H),8.51(s,1H),8.25(d,J＝4.0Hz,1H),7.81(d,J＝16Hz,1H),7.53(d,J＝8.4Hz,2H),7.37-7.19(m,6H),6.84(d,J＝8.4Hz,2H),4.32(s,2H)，产率55％。
2-苄基-6-(4-N,N-二甲氨基苯乙烯基)-8-(3-氰基吡啶基)-咪唑并[1,2-a]吡嗪-3-(7H)-酮(No.11)， 1H NMR(400MHz,CD3OD)δ8.94(d,J＝4.8Hz,1H),8.86(s,1H),8.50(s,1H),8.41(d,J＝4.4Hz,1H),7.98-7.85(m,3H),7.68(d,J＝8.4Hz,2H),7.51(d,J＝16Hz,1H),7.40-7.19(m,5H),4.41(s,2H),3.34(s,6H)，红色固体，产率50％。
2-苄基-6-(4-氨基苯乙烯基)-8-(3-氰基吡啶基)-咪唑并[1,2-a]吡嗪-3-(7H)-酮(No.12)， 1H NMR(400MHz,CD3OD)δ8.97(d,J＝5.0Hz,1H),8.82(m,1H), 8.65-8.59(m,1H),8.42(s,1H),7.93-7.82(m,3H),7.51-7.41(m,3H),7.38-7.28(m,5H),7.26-7.22(m,1H),4.29(s,2H)，产率55％。
上述化合物1-12结构分别如下：
Figure PCTCN2021138520-appb-000013
实施例5：咪唑吡嗪酮类荧光素(1-12)开环发光的光物理性质测试
用酶标仪测试合成的咪唑吡嗪酮类荧光素(1-12)在含痕量氢氧化钠(0.25％)的二甲基亚砜溶液中的化学发光和光致发光的光谱性质。图2给出了归一化的咪唑吡嗪酮类荧光素(1-12)的化学发光光谱，表1汇总了其化学发光和光致发光的光谱性质，从图、表中可以看出，通过C6、C8位给/缺电子基团的修饰，最大发射波长分布在绿光到红光区，荧光明显红移，说明推拉电子结构能有效调节可用作腔肠素类荧光素底物的咪唑吡嗪酮类荧光素的开环发光的光物理性质。
生物发光性质选用咪唑吡嗪酮类荧光素(9)作为荧光素底物，用酶标仪测试在96孔板中测试其与新型荧光素酶NanoLuc生物发光性质，图3给出酶-底物作用的生物发光光谱，从图中可以看出，发光明显红移，说明推拉电子结构对荧光素底物的生物发光性质具有明显的调节作用。
新型荧光素酶-荧光素底物的相互作用发射波长红移，将有助于促进生物发光成像在生物医学领域的应用。
Figure PCTCN2021138520-appb-000014
表1咪唑吡嗪酮类荧光素(1-12)光致发光的光物理性质数据表
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (7)

  1. 式(I)所示的化合物，或其互变异构体或盐：
    Figure PCTCN2021138520-appb-100001
    其中R 1为苄基或者含有苄基的基团，R 2为给电子基团，R 3为缺电子基团。
  2. 如权利要求1所述的化合物，或其互变异构体或盐，其特征在于，所述R 2为下列结构中的一种:
    Figure PCTCN2021138520-appb-100002
  3. 如权利要求1或2所述的化合物，或其互变异构体或盐，其特征在于，所述R 3为下列结构中的一种:
    Figure PCTCN2021138520-appb-100003
  4. 一种制备如权利要求1-3中任一项所述的化合物，或其互变异构体或盐的方法，包括如下步骤：
    步骤一：在氮气保护下，化合物2-氨基-3-碘-5-溴吡嗪与缺电子基团频哪醇 硼酸酯溶解在第一溶剂中，在存在催化剂，且加入第二溶剂，在110℃反应，冷却后分离提纯，得到中间体1；
    步骤二：在氮气保护下，上述中间体1与给电子基团频哪醇硼酸酯溶解在第一溶剂中，在存在催化剂，且加入第二溶剂，110℃反应，冷却后分离提纯，得到中间体2；
    步骤三：在氮气保护下，将中间体2加入第三溶剂中溶解，滴加浓盐酸，同时加入3-苯基-1,1-二乙氧基丙酮，80℃反应，冷却后分离提纯，得到目标产物。
  5. 如权利要求4所述的方法，其特征在于，所述第一溶剂为1,4-二氧六烷，第二溶剂为碳酸钾溶液，第三溶剂为乙醇。
  6. 如权利要求4或5所述的方法，其特征在于，所述催化剂为二(三苯基膦)二氯化钯。
  7. 一种化学发光的方法，采用如权利要求1-3中任一项所述的化合物，或其互变异构体或盐作为底物，且发光的波长在576-652nm范围。
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