WO2020238375A1 - 一种与Aβ寡聚体具有亲和力的荧光化合物及制备方法与应用 - Google Patents

一种与Aβ寡聚体具有亲和力的荧光化合物及制备方法与应用 Download PDF

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WO2020238375A1
WO2020238375A1 PCT/CN2020/081290 CN2020081290W WO2020238375A1 WO 2020238375 A1 WO2020238375 A1 WO 2020238375A1 CN 2020081290 W CN2020081290 W CN 2020081290W WO 2020238375 A1 WO2020238375 A1 WO 2020238375A1
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compound
formula
preparation
oligomer
compound represented
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李玉艳
曾繁天
徐云根
杨剑
刘正世
李小芳
任陇飞
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中国药科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/022—Boron compounds without C-boron linkages
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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    • C09K2211/1003—Carbocyclic compounds
    • C09K2211/1007—Non-condensed systems
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    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1096—Heterocyclic compounds characterised by ligands containing other heteroatoms

Definitions

  • the invention belongs to the field of specific molecular recognition diagnostic reagents, and specifically relates to a fluorescent compound having affinity with A ⁇ oligomers, and preparation and application thereof.
  • AD Alzheimer’s disease
  • AD Alzheimer’s disease
  • cognitive and memory functions impaired activities of daily living, and accompanied by various neuropsychiatric symptoms and behavioral disorders.
  • AD patients There are about 47 million AD patients worldwide. Due to the aging population, the number is expected to reach 131 million by 2050. The cause is unknown so far.
  • effective drugs or methods can reverse the course of AD and can only partially improve the clinical symptoms.
  • Clinical diagnosis is mainly based on evaluating the cognitive impairment of patients. At this time, most of the confirmed patients have entered the middle and late stages of the disease course and delayed treatment. The lack of effective detection methods has become a major obstacle to the early diagnosis and treatment of AD. Therefore, the development of an effective and reliable diagnostic method for early diagnosis and early treatment of AD to improve the survival rate of patients has high clinical application value.
  • the smaller A ⁇ oligomers have a large contact area with neurons and can enter the synaptic cleft and gather in On the cell membrane and lipid valve, leading to membrane division and destruction; on the other hand, A ⁇ oligomers bind to receptors on the cell membrane, induce endocytosis into the cell, affect synaptic signal transduction, and ultimately lead to loss of synapses and impaired memory .
  • a ⁇ oligomers can also cause abnormal depolarization of the mitochondrial cell membrane and release the apoptotic factor cytochrome C, which has been shown to be closely related to synaptic dysfunction and memory impairment.
  • the near-infrared fluorescence imaging technology has the advantages of: (1) High sensitivity, which can realize the detection of weak signals; (2) The detection is safe, without contact with radioactive elements; (3) No time-consuming, real-time imaging during data acquisition; (4) Moderate cost, no need for expensive equipment and skilled personnel; (5) After the NIRF probe is combined with the target protein, the fluorescence characteristics (such as fluorescence intensity, emission wavelength and quantum yield) will change significantly, without the need to connect a reporter base Group, you can directly detect the interaction between the probe and the target protein.
  • probes targeting insoluble plates such as CRANAD-2, AOI987, NIAD-16, DANIR-2C and DBA-SLOH.
  • this application provides a fluorescent compound that can selectively bind to soluble A ⁇ oligomers, and provides its preparation method and application.
  • R1 is -NRaRb, wherein Ra and Rb are independently selected from hydrogen, C1-6 alkyl or C1-6 hydroxyalkyl, but Ra and Rb are not hydrogen at the same time;
  • R 2 is phenyl, C3-6 cycloalkyl or C 1-4 alkyl
  • R3 is hydrogen, C1-6 alkyl, wherein R3 represents mono- or di-substituted (for example, 3,5-disubstituted);
  • X represents C atom or N atom
  • n 1, 2, 3, 4.
  • the C 1-6 alkyl group in Ra , R b and R 3 is preferably a C 1-4 alkyl group, and further, the C 1-4 alkyl group is preferably a methyl, ethyl, or n-propyl group. , Isopropyl, n-butyl, isobutyl or tert-butyl.
  • C3-6 cycloalkyl group in R2 is preferably cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.
  • R2 is preferably hydrogen, 3-ethyl or 2,5-dimethyl.
  • R1 is preferably N,N-dimethylamino, N,N-diethylamino, and N,N-methylhydroxyethylamino.
  • the compound described in this application is selected from any of the following compounds:
  • the present invention also discloses a preparation method of the compound represented by formula I, which comprises the following steps: in an organic solvent, under the catalysis of a base, in the presence of a dehydrating agent, the compound represented by formula 3 is combined with the compound represented by formula 4
  • the compound undergoes condensation reaction, the reaction formula is as follows:
  • the organic solvent may be a conventional organic solvent for this type of reaction in the art, preferably a benzene solvent, among which, the benzene solvent is preferably toluene.
  • the volume molar ratio of the compound represented by formula 3 and formula 4 is 1:1.
  • the base can be a conventional base for such reactions in the art, preferably tetrahydroisoquinoline (ie, 1,2,3,4-tetrahydroisoquinoline).
  • the amount of the base may not be specifically limited, and is generally a catalytic amount.
  • the molar ratio of the base to the compound represented by formula 3 is 0.1-0.9:1, more preferably 0.1-0.3:1.
  • the dehydrating agent may be a conventional dehydrating agent for such reactions in the art, preferably glacial acetic acid.
  • the amount of the dehydrating agent can be a conventional amount for such reactions in the art, and preferably the molar ratio of the dehydrating agent to the compound represented by formula 3 is 0.1-0.9:1, more preferably 0.2-0.5:1.
  • the molar ratio of the compound represented by Formula 3 to the compound represented by Formula 4 may not be specifically limited as long as it does not affect the progress of the reaction, and is preferably 1:1.
  • the temperature of the condensation reaction can be a conventional temperature for this type of reaction in the art, preferably room temperature (0-30°C).
  • the progress of the condensation reaction can be monitored using conventional detection methods for organic synthesis reactions in the art (for example, TLC, GC, HPLC or NMR, etc.), and the end of the reaction is usually when the compound shown in formula 4 disappears.
  • the time for the condensation reaction is preferably 2-6 hours (for example, 3 hours).
  • the compound represented by the above formula 4 can be prepared by the following method steps:
  • the invention also discloses the application of the compound in the preparation of NIRF probes.
  • the compound is used in the preparation of NIRF probes targeting soluble A ⁇ oligomers.
  • the probe can be used for early diagnosis of Alzheimer's disease.
  • the present invention discloses a fluorescent compound with a novel structure and further discloses its preparation method.
  • the results of in vivo imaging show that near-infrared fluorescence imaging of amyloid in the brains of 4-month-old AD mice can be used to distinguish 4-month-old AD mice from normal mice. It can be seen that it is suitable for preparation of NIRF probes, especially NIRF probes targeting soluble A ⁇ oligomers, which can be used for early diagnosis of Alzheimer's disease.
  • Figure 1 is the fluorescence emission spectra before and after mixing the most preferred fluorescent compound of the present invention with A ⁇ 42 monomer, A ⁇ 40 monomer, A ⁇ 42 oligomer, polymer, and BSA;
  • Figure 2 is a graph of MTT cell viability test of the most preferred fluorescent compound of the present invention.
  • Figure 3 is the binding constant of the most preferred fluorescent compound of the present invention and A ⁇ 42 oligomer
  • Figure 4 shows the stability of the most preferred fluorescent compound of the present invention and mouse serum
  • Figure 5 is a near-infrared imaging diagram of live small animals of the most preferred fluorescent compounds
  • Figure 6 is a transmission electron microscope image.
  • the compound represented by formula I of the present invention can be prepared by the following steps:
  • the method is the same as in Example 2, except that 3-(6-(diethylamino)pyridin-3-yl)acrolein is used instead of 6-diethylamino-3-pyridinaldehyde to obtain an orange solid product with a yield : 28%.
  • the method is the same as that in Example 5, except that 5-iodo-m-xylene is used instead of 1-ethyl-4-iodobenzene to obtain a yellow solid product with a yield of 23%.
  • the method is the same as in Example 5, except that 1-iodo-3-methylbenzene is used instead of 1-ethyl-4-iodobenzene to obtain a yellow solid product.
  • the yield is 30%.
  • the method is the same as that in Example 5, except that hexane-2,4-dione is used instead of 1-cyclopropyl-1,3,-butanedione to obtain a yellow solid product.
  • the yield is 30%.
  • the method is the same as in Example 9, except that 1-cyclopropyl-2-(3,5-dimethylphenyl)-1,3-butanedione is used instead of 1-cyclopropyl-2-(4- (Ethylphenyl)-1,3-butanedione to obtain a white solid product, yield: 80%.
  • Example 11 The synthesis method of 4-cyclopropyl-2,2-difluoro-6-methyl-5-(m-tolyl)-dioxaborane is the same as that of Example 9, except that 1- Cyclopropyl-2-(m-tolyl)butane-1,3-dione replaces 1-cyclopropyl-2-(4-ethylphenyl)-1,3-butanedione to obtain a white solid product , Yield: 81%.
  • the method is the same as in Example 13, except that 3-(6-(diethylamino)pyridin-3-yl)acrolein is used instead of 6-diethylamino-3-pyridinaldehyde to obtain a green solid product with a yield : 40%.
  • the method is the same as that in Example 13, except that 5-(4-(dimethylamino)phenyl)penta-2,4-dienal is used instead of 6-diethylamino-3-pyridine aldehyde to obtain a green solid Product, yield: 39%.
  • the method is the same as in Example 13, except that 5-(6-(diethylamino)pyridin-3-yl)penta-2,4-dienal is used instead of 6-diethylamino-3-pyridinaldehyde. A green solid product was obtained, yield: 21%.
  • the method is the same as that in Example 17, except that 5-(4-(dimethylamino)phenyl)penta-2,4-dienal is used instead of 6-diethylamino-3-pyridine aldehyde to obtain a green solid Product, yield: 35%.
  • Example 20 5-6-(6-cyclopropyl-5-(3,5-dimethylphenyl)-2,2-difluoro-2-dioxan-4-yl)hexa-1 ,3,5-Trien-1-yl)-N,N-diethylpyridin-2-amine synthesis
  • the method is the same as in Example 17, except that 5-(6-(diethylamino)pyridin-3-yl)penta-2,4-dienal is used instead of 6-diethylamino-3-pyridinaldehyde. A green solid product was obtained, yield: 21%.
  • a ⁇ 42/A ⁇ 40 monomer The purchased A ⁇ 42/A ⁇ 40 monomer (DgPeptides Co, Ltd., Hang Zhou City, China) was further purified by HPLC and then stored in hexafluoroisopropanol (HFIP). The particle size is measured by Zetasizer Nano (Malvern). 10 ⁇ L of A ⁇ 42/A ⁇ 40 monomer (25 ⁇ M) stock solution was vacuum centrifuged to evaporate the solvent and re-dissolved in 1mL of distilled water. After the particle size test, the particle size of the aqueous solution of the A ⁇ 42/A ⁇ 40 monomer is lower than the lowest value that can be measured by the instrument, that is, there is no large particle size.
  • a ⁇ 42 monomer (0.1 mg) was dissolved in 40 ⁇ L of hexafluoroisopropanol (Hexafluoroisopropanol, HFIP), incubated at room temperature for 10-20 minutes, and then added with 886 ⁇ L of double-distilled water, and incubated at room temperature for 20 minutes. Centrifuge in a 14,000x G centrifuge for 15 minutes, remove the supernatant and dry the HFIP with N2, and stir at 22°C for 24 hours.
  • HFIP hexafluoroisopropanol
  • Compound and compound and A ⁇ monomer binding experiment add 900 ⁇ L of PBS buffer to a 1.0mL cuvette and perform fluorescence measurement as a blank control; add 40 ⁇ L of compound solution (12.5 ⁇ M) to the above cuvette, Determine the self-fluorescence intensity of the compound; add 60 ⁇ L of A ⁇ 42 monomer HFIP solution (25 ⁇ M) to the above compound solution, and immediately measure the fluorescence properties.
  • Compound and compound and A ⁇ oligomer binding experiment add 900 ⁇ L PBS buffer (pH7.4) to a 1ml cuvette, perform fluorescence measurement as a blank control; add 40 ⁇ L compound solution (12.5 ⁇ M) to the above In a cuvette, determine the autofluorescence properties of the compound; add 60 ⁇ L of A ⁇ 42 oligomer solution (25 ⁇ M) to the above compound solution to determine the fluorescence properties.
  • Transmission electron microscope imaging select a specific time point for protein culture (2 hours for A ⁇ 42 monomer; 1 day for A ⁇ 42 oligomer; 3 days for A ⁇ 42 polymer), transfer a small amount of sample to the copper grid, let it stand for 5 minutes, and drop Add 2% tungsten phosphate formic acid, and use Hitachi HT7700 transmission electron microscope for detection and imaging. The results are shown in Fig. 6, a is A ⁇ 42 monomer, b is A ⁇ 42 oligomer, and c is A ⁇ 42 polymer.
  • fluorescent compound (I) probe for live imaging of AD transgenic mice:
  • the fluorescent compound (I) solution probe (4mg/kg, 15% castor oil, 15% DMSO, 70% PBS) was injected into the AD transgene through the tail vein Mice (Tg, APP/PS1 double transfer, 4 months old) and normal mice (WT, C57BL/6 4 months old) under continuous isoflurane anesthesia at 15min, 30min, 60min, 120min, 240min, respectively ,
  • Carry out mouse animal imaging image collection PerkinElmer, US, IVIS LuminaXR, excitation wavelength is 570nm, emission wavelength is 660nm), and the results are analyzed.
  • Living Imaging Software to analyze the imaging results, the results are shown in Figure 5.
  • Quantitative analysis shows that the APP/PS1 group has a longer retention value than the WT group.
  • the APP/PS1 group is The 30th, 60th, 120th, 240th, and 360th minutes were 2.10 times, 2.00 times, 1.87 times and 1.74 times that of the WT group respectively. It shows that the fluorescent compound (I) can detect AD mice at 4 months.
  • MTT experiment method 1) Inoculate SH-SY5Y cells in logarithmic growth phase in a 96-well standard plate at a concentration of 5*103 cells/well, with a volume of 100 ⁇ L per well. 2) After being cultured in an incubator for 12 hours, add 10 ⁇ M, 1 ⁇ M and 0.1 ⁇ M drugs (100 ⁇ L per well). 3) After continuing to incubate in the incubator for a period of 24, add 20 ⁇ L of MTT solution to each well. 4) After incubating for 4 hours, aspirate the supernatant of each well. 5) Add 100 ⁇ L of DMSO to each well, place it on a micro shaker and shake for 5 minutes to dissolve the crystals.
  • Inhibition rate % (1—average absorbance value of cells in drug-added group/average absorbance value of cells in control group) ⁇ 100%.
  • the results of the MTT cell viability test are shown in Figure 2. The cell survival rate is greater than 90%, and the fluorescent compound (I) has low toxicity.
  • Kd binding constant

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Abstract

本发明公开了一种与Aβ寡聚体具有亲和力的荧光化合物，并公开了其制备方法以及应用。本发明获得的荧光化合物最大激发和荧光发射光谱值分别在550～560nm和600～700nm之间，大部分的化合物的发射光谱值＞650nm；适合用于阿尔茨海默氏病可溶性Aβ寡聚体成像的近红外荧光分子探针的特性，适合应用于制备NIRF探针，特别是靶向可溶性Aβ寡聚体的NIRF探针，该探针可用于阿尔兹海默症的早期诊断。

Description

一种与Aβ寡聚体具有亲和力的荧光化合物及制备方法与应用 技术领域
本发明属于特异性分子识别诊断试剂领域，具体涉及一种与Aβ寡聚体具有亲和力的荧光化合物及制备与应用。
背景技术
阿尔茨海默病(Alzheimer’s disease,AD)是最常见的神经系统退行性疾病，临床表现为认知和记忆功能下降，日常生活能力减退，并伴有各种神经精神症状和行为障碍。全球的AD患者约4700万，由于人口老龄化，到2050年，人数预计将达到1.31亿。病因迄今未明。目前尚有效的药物或方法逆转AD的进程，只能部分改善临床症状.临床上主要通过评价患者的认知能力损伤来诊断，而此时确诊患者多已进入病程的中晚期而延误了治疗。缺乏有效的检测手段已成为AD早期诊断和治疗的重大障碍。因此发展一种有效可靠的诊断手段，对AD进行早诊断、早治疗以提高病患的存活率，在临床上具有较高的应用价值。
在AD患者脑中，β-淀粉样蛋白斑块在脑中沉积是AD最为显著的病理性标志之一。然而越来越多的研究表明，Aβ斑块的多少与AD的严重程度缺乏相关性。大脑中有斑块不一定会发展成AD。与不溶性的沉淀斑相比可溶性的Aβ寡聚体要比斑块有着更强的神经毒性，一方面体积较小的Aβ寡聚体与神经元接触面积大，能进入到突触间隙，聚集在细胞膜和脂质阀上，导致膜分裂和破坏；另一方面，Aβ寡聚体结合细胞膜上的受体，诱发内吞进入细胞中，影响突触信号传导，最终导致突触丢失，记忆受损。此外，Aβ寡聚体还能导致非正常去极化线粒体细胞膜，释放细胞凋亡因子细胞色素C，而这被证明与突触功能障碍和记忆受损密切相关。
过去的数几十年里，已有较多具有放射性针对Aβ斑块的分子探针进入临床试验阶段，并有相关PET成像试剂上市(Florbetapir、Florbetaben、Flutemetamol)，然而放射性显像方法的应用也受一些因素限制，比如放射性元素所发出的射线对人体具有一定的辐射损伤、需要昂贵的仪器和专业的人员。相比较之下，光学成像具有安全无放射性、数据采集时间短以及成本低廉等诸多优势，近年在医学诊断等中的应用受到广泛重视。尤其是近红外荧光成像技术，其优点在于：(1)灵 敏度高，可实现微弱信号的检测；(2)检测安全，不接触放射性元素；(3)无需耗时，数据采集过程中实时成像；(4)成本适中，无需昂贵的设备和技术高超的人员；(5)NIRF探针结合了靶蛋白后，荧光特性(如荧光强度、发射波长和量子产率)发生显著改变，无需连接报告基团，就可以直接检测探针和靶蛋白的相互作用。目前靶向不可溶性的板块的探针已有较多成功的研究工作，如CRANAD-2、AOI987、NIAD-16、DANIR-2C和DBA-SLOH等。但是，设计专一性靶向可溶性Aβ寡聚体的NIRF探针具有很大的难度。
发明内容
发明目的：针对上述现有技术存在的技术问题，本申请提供了一种可选择性地与可溶性Aβ寡聚体结合的荧光化合物，并提供了其制备方法与应用。
技术方案：本发明公开了一种如式I所示的化合物，
Figure PCTCN2020081290-appb-000001
其中，R1为-NRaRb，其中，Ra和Rb独立地选自为氢、C1-6烷基或C1-6羟烷基，但Ra和Rb不同时为氢；
R 2为苯基、C3-6环烷基或C 1-4烷基；
R3为氢、C1-6烷基，其中R3表示单取代或二取代(例如3，5-位二取代)；
X表示C原子或者N原子；
n＝1、2、3、4。
进一步的，R a、R b和R 3中所述的C 1-6烷基优选C 1-4烷基，进一步的，所述的C1-4烷基优选甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基。
进一步的，R2中所述的C3-6环烷基优选环丙基、环丁基、环戊基或环己基。更进一步的，R2优选氢、3-乙基或2，5-二甲基。
R1优选N，N-二甲基氨基、N，N-二乙基氨基、N，N-甲基羟乙氨基。
优选的，本申请所述化合物选自如下任一化合物：
Figure PCTCN2020081290-appb-000002
本发明还公开了如式I所示化合物的制备方法，包括以下步骤：有机溶剂中，在碱的催化下，脱水剂的存在下，将如式3所示的化合物与如式4所示的化合物进行缩合反应，反应式如下：
Figure PCTCN2020081290-appb-000003
所述有机溶剂可为本领域此类反应常规的有机溶剂，优选苯类溶剂，其中，苯类溶剂优选甲苯。优选式3与式4所示化合物的体积摩尔比为1∶1。
所述碱可为本领域此类反应常规的碱，优选四氢异喹啉(即1，2，3，4-四氢异喹啉)。所述的碱的用量可不做具体限定，一般为催化量，较佳地其与如式3所示的化合物的摩尔比为0.1-0.9∶1，更佳地为0.1-0.3∶1。
所述脱水剂可为本领域此类反应常规的脱水剂，优选冰乙酸。所述的脱水剂的用量可为本领域此类反应常规的用量，较佳地其与如式3所示的化合物的摩尔比为0.1-0.9∶1，更佳地为0.2-0.5∶1。
所述的如式3所示的化合物与如式4所示的化合物的摩尔比可不作具体限定，只要不影响反应进行即可，较佳地为1∶1。
所述的缩合反应的温度可为本领域此类反应常规的温度，较佳地为室温(0-30℃)。所述的缩合反应的进程可采用本领域有机合成反应常规的检测方法进行监测(例如TLC、GC、HPLC或NMR等)，通常以如式4所示的化合物消失时作为反应的终点。所述的缩合反应的时间较佳地为2-6小时(例如3小时)。
进一步优选的，上述式4所示的化合物可以通过以下方法步骤制备得到：
Figure PCTCN2020081290-appb-000004
本发明还公开了所述化合物在制备NIRF探针中的应用。
进一步的，所述化合物在制备靶向可溶性Aβ寡聚体的NIRF探针中的应用。
所述探针可用于阿尔兹海默症的早期诊断。
有益效果：本发明公开了一种结构新颖的荧光化合物，并进一步公开了其制备方法，本发明获得的荧光化合物最大激发和荧光发射光谱值分别在550～560nm和600～700nm之间，大部分的化合物的发射光谱值>650nm；适合用于阿尔茨海默氏病可溶性Aβ寡聚体成像的近红外荧光分子探针的特性；本申请还通过体内外实验证实，其体内代谢稳定、与可溶性Aβ＝寡聚体具有很好的亲和力、活体成像结果表明能够对4个月大的AD老鼠脑内淀粉样蛋白进行近红外荧光显像，能够区分4个月大的AD老鼠和正常老鼠，由此可见，其适合应用于制备NIRF探针，特别是靶向可溶性Aβ寡聚体的NIRF探针，该探针可用于阿尔兹海默症的早期诊断。
附图说明
图1是本发明最优选荧光化合物与Aβ42单体、Aβ40单体、Aβ42寡聚体、聚合物、BSA混合前后的荧光发射光谱；
图2为本发明最优选荧光化合物的MTT细胞活力测试图；
图3为本发明最优选荧光化合物与Aβ42寡聚体的结合常数；
图4为本发明最优选荧光化合物与小鼠血清的稳定性；
图5是最优选荧光化合物的活体小动物近红外成像图；
图6是透射电子显微镜成像图。
具体实施方式
下面结合具体实施例对本发明进行详细说明。
本发明式I所示化合物可通过以下步骤制备得到：
Figure PCTCN2020081290-appb-000005
实施例1：6-二乙胺基-3-吡啶醛的合成
依次将6.5mL DMF、2-溴-5-醛基吡啶(1200mg,6.45mmol)、二乙胺(6mL,58.06mmol)加入35ml的封管中，磁力搅拌，反应温度90℃，反应17h，TLC监测(PE:EA＝6:1)。后处理：二氯甲烷(DCM)萃取(3×10ml)，合并有机层，用饱和食盐水洗涤(3×10ml)，无水Na 2SO 4干燥。减压浓缩后柱层析，洗脱剂为PE:EA＝10:1(v/v)，得淡黄色固体782mg，收率68％。1H NMR(300MHz,,Chloroform-d)δ:9.68(s,1H),8.48-8.47(d,J＝2,1H),7.85-7.81(dd,J＝10Hz,1H),6.48-6.45(d,J＝15Hz,1H),3.58-3.51(m,4H),1.19-1.15(t,6H)
化学式如下：
Figure PCTCN2020081290-appb-000006
实施例2：3-(6-(二乙基氨基)吡啶-3-基)丙烯醛的合成
加入6-二乙胺基-3-吡啶醛(400mg,2.24mmol)，无水THF(16ml)，搅拌后就加入2-[(1,3)-二氧戊环-2-基]-甲基三苯基溴化瞵(1900mg,4.48mmol)，分两次加入NaH(215mg,8.96mmol)，最后加入18-冠醚-6(118mg.0.048mmol))。室温反应24h.TLC监测(PE:EA＝6:1)。后处理：反应液冷却至室温后，抽滤，滤液用乙醚萃取(3×10ml)，合并有机层，用饱和食盐水洗涤(3×10ml)，无水Na 2SO 4干燥。浓缩后加入THF和10％盐酸(42ml/17ml)的混合溶液，搅拌5h,加入10％NaOH溶液调中性。接着用DCM萃取(3×10ml)，合并有机层，用饱和食盐水洗涤(3×10ml)，无水Na 2SO 4干燥。浓缩(柱层析，洗脱剂为PE:EA＝10:1，真空干燥，得橙红色油状物固体137mg，收率30％。
1H NMR(300MHz,Chloroform))δ9.60(d,J＝7.8Hz,1H),8.29(s,1H),7.66(d,J＝9.2Hz,1H),7.35(d,J＝15.6Hz,1H),6.59–6.44(m,2H),3.58(q,J＝7.3Hz,4H),1.22(t,J＝7.3Hz,6H).
化学式如下：
Figure PCTCN2020081290-appb-000007
实施例3：5-(4-(二甲基氨基)苯基)戊-2,4-二烯醛的合成
方法同实施例2，所不同的是用3-(4-(二甲基氨基)苯基)丙烯醛代替6-二乙胺基-3-吡啶醛，得到棕黄色固体产物，收率：41％
1H NMR(300MHz,Chloroform-d)δ9.50(dt,J＝7.1,0.9Hz,1H),7.46–7.39(m,2H),7.15(ddd,J＝14.3,7.3,1.0Hz,1H),7.05(d,J＝14.0Hz,1H),6.82–6.68(m,3H),6.09(ddd,J＝14.1,7.2,0.7Hz,1H),3.01(s,5H).化学式如下：
Figure PCTCN2020081290-appb-000008
实施例4：5-(6-(二乙基氨基)吡啶-3-基)戊-2,4-二烯醛的合成
方法同实施例2，所不同的是用3-(6-(二乙基氨基)吡啶-3-基)丙烯醛代 替6-二乙胺基-3-吡啶醛，得到橙色固体产物，收率：28％.
1H NMR(300MHz,Chloroform-d)δ9.50(d,J＝7.1,0.9Hz,1H),8.35(d,J＝1.9Hz,1H),7.49(dd,J＝8.4,1.8Hz,1H),7.14(ddd,J＝14.2,7.3,1.0Hz,1H),7.00(d,J＝14.1Hz,1H),6.81(ddt,J＝14.1,7.3,0.8Hz,1H),6.73(d,J＝8.4Hz,1H),6.13(ddd,J＝14.3,7.1,0.7Hz,1H),3.47(q,J＝7.2Hz,4H),1.17(t,J＝7.2Hz,6H).
化学式如下：
Figure PCTCN2020081290-appb-000009
实施例5：1-环丙基-2-(4-乙基苯基)-1,3-丁二酮的合成
依次将无水K 2CO 3(2.76g,20mmol)、L-脯氨酸(230mg,2mmol)、CuI(95mg,0.5mmol)、加入100ml三颈反应瓶中，N2保护，通过注射器向其中加入DMSO(20ml)、1-环丙基-1,3,-丁二酮(0.59ml,5mmol)、1-乙基-4-碘苯(0.724ml,5mmol)磁力搅拌，反应温度90℃，反应18h，TLC监测(PE:EA＝20:1)。后处理：反应液冷却至室温后，抽滤，滤液用乙酸乙酯(EA)萃取(3×10ml)，合并有机层，用饱和食盐水洗涤(3×10ml)，无水Na 2SO 4干燥。浓缩后柱层析，洗脱剂为PE:EA＝70:1，真空干燥，得黄色固体160mg，收率14％。
1H NMR(300MHz,Chloroform-d)δ16.88(s,1H),7.27–7.09(m,4H),2.67(q,J＝7.6Hz,2H),1.71(tt,J＝8.0,4.5Hz,1H),1.48(q,J＝3.9Hz,2H),1.46–1.31(m,6H),1.04(dq,J＝7.6,3.9Hz,2H)
化学式如下：
Figure PCTCN2020081290-appb-000010
实施例6：1-环丙基-2-(3,5-二甲基苯基)-1,3-丁二酮的合成
方法同实施例5，所不同的是用5-碘间二甲苯代替1-乙基-4-碘苯，得到黄色固体产物，收率：23％。
1H NMR(300MHz,Chloroform-d)δ7.08(s,1H),6.94(s,2H),2.05(s,6H),1.89(s,3H),1.70(tt,J＝8.0,4.5Hz,1H),1.49(q,J＝3.9Hz,2H),1.05(dq,J＝7.6,3.9Hz,2H).
化学式如下：
Figure PCTCN2020081290-appb-000011
实施例7：1-环丙基-2-(间甲苯基)丁烷-1,3-二酮的合成
方法同实施例5，所不同的是用1-碘-3-甲基苯代替1-乙基-4-碘苯，得到黄色固体产物，收率：30％。
1H NMR(300MHz,Chloroform-d)δ7.08(s,1H),6.94(s,2H),2.05(s,3H),1.89(s,3H),1.70(tt,J＝8.0,4.5Hz,1H),1.49(q,J＝3.9Hz,2H),1.05(dq,J＝7.6,3.9Hz,2H).
化学式如下：
Figure PCTCN2020081290-appb-000012
实施例8：3-(3,5-二甲基苯基)己烷-2,4-二酮的合成
方法同实施例5，所不同的是用己烷-2,4-二酮代替1-环丙基-1,3,-丁二酮，得到黄色固体产物，收率：30％。
1H NMR(300MHz,Chloroform-d)δ16.55(s,1H),7.49(d,J＝9.0Hz,1H),6.79(d,J＝1.5Hz,2H),2.45–2.35(m,8H),1.35–1.20(m,6H).
化学式如下：
Figure PCTCN2020081290-appb-000013
实施例9：4-环丙基-5-(4-乙基苯基)-2,2-二氟-6-甲基-二氧杂硼烷的合成
将1-环丙基-2-(4-乙基苯基)-1,3-丁二酮(160mg，0.78mmol)、硼酸三丁酯(0.47ml，1.57mmol)加入25ml反应瓶中，橡胶塞封口并插上N2气球针管，缓慢滴加三氟化硼乙醚(0.47ml，1.57mmol)，室温搅拌5h，TLC监测(PE:EA＝6:1)。后处理：缓慢加入水淬灭三氟化硼乙醚，析出白色固体，继续加入石油醚析出更多的固体，抽滤得白色固体153mg，收率78％。
1H NMR(300MHz,Chloroform-d)δ16.88(s,1H),7.27–7.09(m,4H),2.67(q,J＝7.6Hz,2H),1.71(tt,J＝8.0,4.5Hz,1H),1.48(q,J＝3.9Hz,2H),1.46–1.31(m,6H),1.04(dq,J＝7.6,3.9Hz,2H).
化学式如下：
Figure PCTCN2020081290-appb-000014
实施例10：4-环丙基-5-(3,5-二甲基苯基)-2,2-二氟-6-甲基二氧杂硼烷的合成
方法同实施例9，所不同的是用1-环丙基-2-(3,5-二甲基苯基)-1,3-丁二酮代替1-环丙基-2-(4-乙基苯基)-1,3-丁二酮，得到白色固体产物，收率：80％。
1H NMR(300MHz,Chloroform-d)δ7.08(s,1H),6.94(s,2H),2.05(s,6H),1.89(s,3H),1.70(tt,J＝8.0,4.5Hz,1H),1.49(q,J＝3.9Hz,2H),1.05(dq,J＝7.6,3.9Hz,2H)
化学式如下：
Figure PCTCN2020081290-appb-000015
实施例11：4-环丙基-2,2-二氟-6-甲基-5-(间甲苯基)-二氧杂硼烷的合成方法同实施例9，所不同的是用1-环丙基-2-(间甲苯基)丁烷-1,3-二酮代替1-环丙基-2-(4-乙基苯基)-1,3-丁二酮，得到白色固体产物，收率：81％。
1H NMR(300MHz,Chloroform-d)δ7.08(s,1H),6.94(s,2H),2.05(s,3H),1.89(s,3H),1.70(tt,J＝8.0,4.5Hz,1H),1.49(q,J＝3.9Hz,2H),1.05(dq,J＝7.6,3.9Hz,2H)
化学式如下：
Figure PCTCN2020081290-appb-000016
实施例12：5-(3,5-二甲基苯基)-4-乙基-2,2-二氟-6-甲基--二氧杂硼烷的合成
方法同实施例9，所不同的是用3-(3,5-二甲基苯基)己烷-2,4-二酮代替1-环丙基-2-(4-乙基苯基)-1,3-丁二酮，得到白色固体产物，收率：85％。 1H NMR(300MHz,Chloroform-d)δ7.14(d,J＝7.6Hz,2H),6.45(d,J＝9.1Hz,2H),2.76(q,J＝7.6Hz,2H),2.37(q,J＝7.5Hz,2H),1.34(t,J＝7.6Hz,3H),1.34–1.19(m,6H).
化学式如下：
Figure PCTCN2020081290-appb-000017
实施例13：5-(2-(6-环丙基-5-(4-乙基苯基)-2,2-二氟-二氧杂环-4-基)乙烯基)-N，N-二乙基-2-胺的合成
向25ml三颈烧瓶中依次加入4-环丙基-5-(4-乙基苯基)-2,2-二氟-6-甲基-二氧杂硼烷(1mmol，277mg)，甲苯(6ml)溶解后，再依次加入6-二乙胺基-3-吡啶醛(204mg,1mmol)，乙酸(37μl，0.648mmol)，四氢异喹啉(27μl,0.216mmol,)，磁力搅拌，室温反应6h。TLC板监测(DCM)，反应完毕，旋蒸除去溶剂得粗品，过柱纯化(洗脱剂：DCM)，得到红色粉末状产物收率187mg,40.4％.
1H NMR(300MHz,Chloroform-d)δ8.25(d,J＝2.4Hz,1H),7.94(d,J＝15.3Hz,1H),7.39(dd,J＝9.1,2.5Hz,1H),7.34–7.16(m,4H),6.42(d,J＝9.1Hz,1H),6.19(d,J＝15.3Hz,1H),3.55(q,J＝7.1Hz,4H),2.74(q,J＝7.6Hz,2H),1.68(tt,J＝8.1,4.5Hz,1H),1.47(q,J＝3.9Hz,2H),1.32(t,J＝7.6Hz,3H),1.19(t,J＝7.1Hz,6H),1.03(dq,J＝7.6,3.8Hz,2H).HRMS(ESI)C 25H 29BF 2N 2O 2,[M+H] +calculated＝439.2363；found＝439.2379.
化学式如下：
Figure PCTCN2020081290-appb-000018
实施例14：4-(6-环丙基-5-(4-乙基苯基)-2,2-二氟-2二氧杂环-4-基)丁-1,3-二烯-1-基)-N，N-二乙基吡啶-2-胺的合成
方法同实施例13，所不同的是用3-(6-(二乙基氨基)吡啶-3-基)丙烯醛代替6-二乙胺基-3-吡啶醛，得到绿色固体产物，收率：40％。
1H NMR(400MHz,Chloroform-d)δ8.21(d,J＝2.4Hz,1H),7.84(dd,J＝14.5,11.5Hz,1H),7.55(dd,J＝9.1,2.5Hz,1H),7.32(d,J＝8.1Hz,2H),7.22(d,J＝8.0Hz,2H),6.95(d,J＝15.2Hz,1H),6.61(dd,J＝15.2,11.5Hz,1H),6.45(d,J＝9.0 Hz,1H),3.57(q,J＝7.1Hz,4H),2.76(q,J＝7.6Hz,2H),1.72–1.66(m,1H),1.50(p,J＝3.9Hz,2H),1.34(t,J＝7.6Hz,3H),1.21(t,J＝7.1Hz,6H),1.05(dt,J＝7.5,3.8Hz,2H).
化学式如下：
Figure PCTCN2020081290-appb-000019
实施例15：4-4-(6-环丙基-5-(4-乙基苯基)-2,2-二氟-2-二氧杂环-4-基)丁-1,3-二烯-1-基)-N，N-二甲基苯胺的合成
方法同实施例13，所不同的是用5-(4-(二甲基氨基)苯基)戊-2,4-二烯醛代替6-二乙胺基-3-吡啶醛，得到绿色固体产物，收率：39％。
1H NMR(300MHz,CDCl3)δ7.88(dd,J＝14.4,11.6Hz,1H),7.38(d,J＝9.4Hz,4H),7.25(d,J＝7.7Hz,2H),7.02(d,J＝15.1Hz,1H),6.69(t,J＝10.3Hz,3H),5.90(d,J＝14.5Hz,1H),3.07(s,6H),2.79(q,J＝7.6Hz,2H),1.79–1.64(m,1H),1.52(t,J＝3.9Hz,2H),1.37(t,J＝7.7Hz,3H),1.07(dq,J＝7.7,3.8Hz,2H).HRMS(ESI)C 26H 28BF 2NO 2,[M+H] +calculated＝435.2289；found＝436.2267.
化学式如下：
Figure PCTCN2020081290-appb-000020
实施例16：5-6-(6-环丙基-5-(4-乙基苯基)-2,2-二氟-2-二氧杂环-4-基)六-1，3,5-三烯-1-基)-N，N-二乙基吡啶-2-胺的合成
方法同实施例13，所不同的是用5-(6-(二乙基氨基)吡啶-3-基)戊-2,4-二烯醛代替6-二乙胺基-3-吡啶醛，得到绿色固体产物，收率：21％。
1H NMR(300MHz,Chloroform-d)δ8.22(d,J＝9.3Hz,1H),7.92–7.62(m,1H),7.55(d,J＝8.9Hz,1H),7.42–7.15(m,4H),7.01–6.44(m,4H),6.41–6.02(m,1H),5.87(d,J＝14.4Hz,1H),3.67–3.48(m,3H),2.75(q,J＝7.6Hz,2H),1.69(s,1H),1.51(s,2H),1.31–1.16(m,9H),1.07(s,2H).
化学式如下：
Figure PCTCN2020081290-appb-000021
实施例17：4-4-(6-环丙基-5-(3,5-二甲基苯基)-2,2-二氟-2-二氧杂环-4-基)丁-1,3-二烯-1-基)-N，N-二甲基苯胺的合成
向25ml三颈烧瓶中依次加入4-环丙基-5-(3,5-二甲基苯基)-2,2-二氟-6-甲基-二氧杂硼烷(1mmol，277mg)，甲苯(6ml)溶解后，再依次加入6-二乙胺基-3-吡啶醛(204mg,1mmol)，乙酸(37μl，0.648mmol)，四氢异喹啉(27μl,0.216mmol,)，磁力搅拌，室温反应6h。TLC板监测(DCM)，反应完毕，旋蒸除去溶剂得粗品，过柱纯化(洗脱剂：DCM)，得到红色粉末状产物收率187mg,40.4％.
1H NMR(500MHz,Chloroform-d)δ8.32(d,J＝2.4Hz,1H),7.97(d,J＝15.4Hz,1H),7.55(d,J＝9.0Hz,1H),7.11(s,1H),6.85(d,J＝1.5Hz,2H),6.60(s,1H),6.25(d,J＝15.4Hz,1H),3.68(s,4H),2.46–2.36(m,8H),1.29(t,J＝7.1Hz,6H),1.18(t,J＝7.4Hz,3H).HRMS(ESI)C 24H 29BF 2N 2O 2,[M+H] +calculated＝427.2363；found＝427.2382
化学式如下：
Figure PCTCN2020081290-appb-000022
实施例18：4-4-(6-环丙基-5-(3,5-二甲基苯基)-2,2-二氟-2-二氧杂环-4-基)丁-1,3-二烯-1-基)-N，N-二甲基苯胺的合成
方法同实施例17，所不同的是用5-(4-(二甲基氨基)苯基)戊-2,4-二烯醛
代替6-二乙胺基-3-吡啶醛，得到绿色固体产物，收率：39％。 1H NMR(500MHz,Chloroform-d)δ7.45–7.35(m,3H),7.04(d,J＝15.5Hz,1H),6.99–6.92(m,3H),6.75–6.68(m,2H),6.54(ddd,J＝15.5,7.4,1.0Hz,1H),6.04(dd,J＝14.7,7.4Hz,1H),3.71(p,J＝6.4Hz,1H),3.03(s,5H),2.31(s,6H),1.39(d,J＝6.4Hz,4H).
化学式如下：
Figure PCTCN2020081290-appb-000023
实施例19：4-4-(6-环丙基-5-(3,5-二甲基苯基)-2,2-二氟-2-二氧杂环-4-基)丁-1,3-二烯-1-基)-N，N-二甲基苯胺的合成
方法同实施例17，所不同的是用5-(4-(二甲基氨基)苯基)戊-2,4-二烯醛代替6-二乙胺基-3-吡啶醛，得到绿色固体产物，收率：35％。
1H NMR(500MHz,Chloroform-d)δ8.31(d,J＝1.9Hz,1H),7.49(dd,J＝8.4,1.9Hz,1H),7.43–7.33(m,2H),7.01–6.94(m,3H),6.69(d,J＝8.5Hz,1H),6.59(ddd,J＝14.1,7.3,0.9Hz,1H),6.21(dd,J＝14.7,7.4Hz,1H),3.72(p,J＝6.4Hz,1H),3.47(q,J＝7.2Hz,4H),2.31(s,6H),1.39(d,J＝6.4Hz,4H),1.10–1.03(m,6H).
化学式如下：
Figure PCTCN2020081290-appb-000024
实施例20：5-6-(6-环丙基-5-(3,5-二甲基苯基)-2,2-二氟-2-二氧杂环-4-基)六-1，3,5-三烯-1-基)-N，N-二乙基吡啶-2-胺的合成
方法同实施例17，所不同的是用5-(6-(二乙基氨基)吡啶-3-基)戊-2,4-二烯醛代替6-二乙胺基-3-吡啶醛，得到绿色固体产物，收率：21％。
1H NMR(500MHz,Chloroform-d)δ8.29(d,J＝1.9Hz,1H),7.43(dd,J＝8.4,1.8Hz,1H),7.40–7.32(m,1H),7.31(d,J＝14.0Hz,1H),7.00–6.93(m,3H),6.72–6.62(m,2H),6.56–6.39(m,3H),3.71(p,J＝6.4Hz,1H),3.47(q,J＝7.3Hz,4H),2.31(s,6H),1.39(d,J＝6.5Hz,4H),1.10–1.03(m,6H).
化学式如下：
Figure PCTCN2020081290-appb-000025
下面是实施例15制备所得化合物(I)(4-4-(6-环丙基-5-(4-乙基苯基)-2,2-二氟-2-二氧杂环-4-基)丁-1,3-二烯-1-基)-N，N-二甲基苯胺)的生物活性研究。
采用F-4500荧光分光光度计在PBS(pH 7.4)条件下分别测试目标化合物(I)、目标化合物(I)与Aβ42单体作用后、Aβ42寡聚体和Aβ4寡聚体作用后的荧光强度值。
实验方法：
Aβ42/Aβ40单体的制备：购买的Aβ42/Aβ40单体(DgPeptides Co,Ltd.,HangZhou City,China))经HPLC进一步纯化后储存于六氟异丙醇(HFIP)中。粒度经由Zetasizer Nano(Malvern)测定。10μL的Aβ42/Aβ40单体(25μM)储 备液经真空离心挥干溶剂，重新溶于1mL蒸馏水中。经粒度测试该Aβ42/Aβ40单体的水溶液粒度大小低于仪器所能测定的最低值，即无大粒径颗粒。
Aβ42寡聚体的制备：将Aβ42单体(0.1mg)溶于40μL六氟异丙醇(Hexafluoroisopropanol，HFIP)，室温孵育10-20min，然后加入886μL双蒸水，室温孵育20min。14,000x G离心机离心15min，移取上清液并用N2吹干HFIP，22℃搅拌24h。
采用荧光分光光度计(日立F-4600)，参数设置：狭缝宽度(激发/发射)＝10/10；PMT＝700V；扫描速度＝1200nm/min；响应速度为自动模式。测定化合物最大激发波长及最大发射波长；后在最大激发波长下，测定化合物与Aβ蛋白结合后的荧光性质，具体方法如下：
化合物及化合物与Aβ单体的结合实验：将900μL PBS缓冲液加入到1.0mL的比色皿中，进行荧光测定，作为空白对照；将40μL化合物溶液(12.5μM)加入到上述比色皿中，测定化合物自身荧光强度；在上述化合物溶液中加入60μL的Aβ42单体的HFIP溶液(25μM)，立即测定荧光性质。
化合物及化合物与Aβ寡聚体的结合实验：将900μL PBS缓冲液(pH7.4)加入到1ml的比色皿中，进行荧光测定，作为空白对照；将40μL化合物溶液(12.5μM)加入到上述比色皿中，测定化合物自身荧光性质；在上述化合物溶液中加入60μL的Aβ42寡聚体溶液(25μM)，测定荧光性质。
测定结果如图1所示，根据图示可见，化合物I对Aβ42寡聚体具有选择性，FI(Aβ42寡聚体)/FI(Aβ42聚合物)＝2.1倍；FI(Aβ42寡聚体)/FI(Aβ42单体)＝7.0；FI(Aβ42寡聚体)/FI(Aβ40单体)＝5.25。
透射电子显微镜成像(TEM)：选取蛋白培养特定时间点(Aβ42单体2小时；Aβ42寡聚体1天；Aβ42聚合物3天)，吸取少量样品转移至铜网格，静置5min，然后滴加2％磷酸钨甲酸，采用日立HT7700透射电镜进行检测成像。结果如图6所示，a为Aβ42单体、b为Aβ42寡聚体、c为Aβ42聚合物。
荧光化合物(I)探针应用于AD转基因小鼠活体成像：将荧光化合物(I)溶液探针(4mg/kg，15％蓖麻油、15％DMSO、70％PBS)由尾静脉注射入AD转基因小鼠(Tg，APP/PS1双转，4月龄)和正常小鼠(WT，C57BL/6 4月龄)体内，在异氟烷的持续麻醉下分别在15min、30min、60min、120min、240min、进行小鼠 动物成像图像采集(PerkinElmer，U.S，IVIS LuminaXR，激发波长取570nm，发射波长取660nm)，分析结果。采用Living Imaging Software进行成像结果分析，结果如图5所示，在定量分析表明APP/PS1组相比WT组有更长的保留值，静脉注射荧光化合物(I)后，APP/PS1组分别在第30、60、,120、240、360分钟时分别为WT组的2.10倍，2.00倍，1.87倍和1.74倍。表明荧光化合物(I)能够对4个月的AD小鼠进行检测。
MTT实验方法：1)将对数生长期SH-SY5Y细胞按5*103个/孔的浓度接种于96孔标板内，每孔内体积为100μL。2)置于孵箱内培养12h后，分别加入10μM、1μM和0.1μM的药物(每孔体积为100μL)。3)继续置于孵箱内培养一段时间24后，每孔加入20μL MTT溶液。4)继续孵育4h后将每孔的上清液吸净。5)每孔加入100μL DMSO，放于微型振荡器上振动5min使结晶溶解。6)待结晶完全溶解后将其放于酶标仪上用570nm波长检测。7)将检测后的吸光值整理后按下面的公式计算药物对细胞的生长抑制率。抑制率％＝(1-加药组细胞平均吸光值/对照组细胞平均吸光值)×100％。MTT细胞活力测试结果如图2所示，细胞存活率大于90％，荧光化合物(I)毒性小。
结合常数的测定(Kd)：荧光化合物(I)和各种浓度的Aβ42寡聚物/聚集体的液进行荧光光谱记录。570nm处的发射读数用于线性回归拟合。通过GraphPad Prism测定结果，如图3所示，Kd(Aβ42寡聚物)＝284.3+49.6nM；Kd(Aβ42聚合物)＝2309+288nM。
血浆稳定性实验：荧光化合物(I)制成10μM，取50μL配好的溶液加入到装有300μL小鼠血清的EP管中37℃下孵育0min,30min,60min加入500μL冰乙腈离心(5000rmp、15min、4℃)取少量上清液。HPLC分析(乙腈/水＝80％：20％，流速1mL/min,紫外检测器320nm。如图4所示，荧光化合物(I)与小鼠血清共孵育60分钟后，化合物仍然含有84％的量。

Claims (10)

  1. 一种如式I所示的化合物，
    Figure PCTCN2020081290-appb-100001
    其中，R1为-NRaRb，Ra和Rb独立地选自为氢、C1-6烷基或C1-6羟烷基，但Ra和Rb不同时为氢；
    R 2为苯基、C3-6环烷基或C 1-4烷基；
    R3为氢、C1-6烷基，其中R3表示单取代或二取代；
    X为C原子或者N原子；
    n＝1、2、3、4。
  2. 根据权利要求1所述的化合物，其特征在于，R a、R b和R 3中所述的C 1-6烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基。
  3. 根据权利要求1所述的化合物，其特征在于，R2中所述的C3-6环烷基为环丙基、环丁基、环戊基或环己基。
  4. 根据权利要求1所述的化合物，其特征在于，R1选自N，N-二甲基氨基、N，N-二乙基氨基、N，N-甲基羟乙氨基。
  5. 根据权利要求1所述的化合物，其特征在于，所述化合物选自如下任一化合物：
    Figure PCTCN2020081290-appb-100002
  6. 权利要求1-5中任一所述化合物的制备方法，其特征在于，包括以下步骤：在有机溶剂中，碱的催化下，脱水剂的存在下，将如式3所示的化合物与如式4所示的化合物进行缩合反应，反应式如下：
    Figure PCTCN2020081290-appb-100003
  7. 根据权利要求6所述的制备方法，其特征在于，所述有机溶剂为苯类溶剂，所述碱为四氢异喹啉，所述脱水剂为冰乙酸。
  8. 根据权利要求6所述的制备方法，其特征在于，所述碱与如式3所示的化合物的摩尔比为0.1-0.9∶1，所述脱水剂与如式3所示的化合物的摩尔比为0.1-0.9∶1。
  9. 根据权利要求6所述的制备方法，其特征在于，所述式3所示的化合物可以通过以下方法制备得到：
    Figure PCTCN2020081290-appb-100004
  10. 权利要求1-5中任一所述化合物在制备NIRF探针中的应用。
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