Dicyanoisophorone fluorescent probe for detecting brain β-amyloid protein, preparation method and application thereof

Abstract

The present invention relates to the field of biomedicine technology, and specifically discloses a dicyanoisophorone fluorescent probe for detecting brain β-amyloid protein, its preparation method and application. The fluorescent probe of the present invention is based on the dicyanoisophorone structure, and a probe with dual recognition sites is formed by introducing a carbamate group and an N,N-dimethylamino group. Compared with probes with a single recognition site, probes with dual recognition sites have a more sensitive fluorescent response to Aβ aggregates in vitro and show higher binding affinity and selectivity. The probe can effectively cross the blood-brain barrier and show high reactivity in the brains of Alzheimer's disease model mice. In addition, the probe can monitor the Aβ levels in the brains of Alzheimer's disease mice of different ages. It has great potential in the early diagnosis of neurodegenerative diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a dicyanoisophorone fluorescent probe for detecting brain beta-amyloid protein, and a preparation method and application thereof. Background Art

[0002] Neurodegenerative diseases are major brain disorders caused by the progressive loss of function or structure of nerve cells in the central nervous system. They primarily include Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Currently, Alzheimer's disease (AD) is considered the most common neurodegenerative disease. Early onset manifests as cognitive decline, apathy, and depression. As the disease progresses, symptoms such as loss of spatial and temporal cognition, language impairment, and loss of self-care skills develop. With the increasing aging of society, the number of AD patients continues to rise, prompting increasing attention to the disease.

[0003] Numerous studies have shown that the spontaneous aggregation of β-amyloid protein (Aβ) is one of the key factors in the development of AD. Aβ is produced by the hydrolysis of amyloid precursor protein (APP) by hydrolases, but APP will produce different products when hydrolyzed by different hydrolases. During normal hydrolysis, β-secretase hydrolyzes it into αAPPs and C83, and then into P342 and P340 by γ-secretase. This pathway does not produce Aβ; when APP is hydrolyzed by another route, it is first hydrolyzed into βAPPs and C99 by β-secretase, and then into Aβ by γ-secretase. 1-42 and Aβ 1-40 Aβ 1-42 than Aβ 1-40 The presence of two more amino acids makes Aβ more neurotoxic and more prone to forming insoluble aggregates. 1-42 It can induce oxidative stress in the patient's brain, causing metabolic disorders, inducing inflammation, leading to aggravated neurofibrillary tangles, and promoting neuronal cell apoptosis. These diseases will promote Aβ 1-42 Therefore, effective detection of Aβ aggregates is of great significance for the early diagnosis and relief of AD.

[0004] Currently, most fluorescent probes used to detect Aβ aggregates rely on common recognition groups, such as N,N-dimethylamino, N,N-diethylamino, and piperidinyl. While these traditional probes can recognize Aβ aggregates to a certain extent, their sensitivity and selectivity remain limited. Therefore, developing novel fluorescent probes with dual recognition sites to improve the sensitivity and selectivity of Aβ aggregate detection holds significant research value and promise. Summary of the Invention

[0005] To address the challenges of Aβ fluorescent probe detection, the present invention provides a dicyanoisophorone fluorescent probe with dual recognition sites. This probe is capable of achieving a fluorescent response to Aβ aggregates. Furthermore, this fluorescent probe can cross the blood-brain barrier, enabling imaging of Aβ in the brains of AD mice and monitoring Aβ levels in the brains of AD mice of different ages.

[0006] In order to achieve the purpose of the present invention, the technical solution of the present invention is as follows:

[0007] The present invention provides a dicyanoisophorone fluorescent probe for detecting brain β-amyloid protein. The structural formula of the fluorescent probe is as follows:

[0008]

[0009] They were named CarbCN, NTCN and NCarbCN respectively.

[0010] The dicyanoisophorone fluorescent probe for detecting brain β-amyloid protein of the present invention is characterized by the following specific preparation steps:

[0011] Step S1: Isophorone and malononitrile were added to ethanol. Piperidine was slowly added dropwise to the reaction system and heated under reflux. After the reaction was completed, the solvent was removed under reduced pressure. Solid Compound 1 was obtained by purification by column chromatography.

[0012] Step S2: Salicylaldehyde, tert-butyl-3-bromopropylcarbamate, and potassium carbonate were added to acetonitrile and heated to reflux. After completion of the reaction, the solvent was removed under reduced pressure. The crude product was extracted with dichloromethane and water. After the solvent was removed under reduced pressure, the product was purified by column chromatography to obtain Compound 2 as an oil.

[0013] Step S3: 4-Fluorosalicylaldehyde, 1-iodopropane (or tert-butyl-3-bromopropylcarbamate), and potassium carbonate are added to acetonitrile and heated to reflux. After completion of the reaction, the solvent is removed under reduced pressure. The crude product is extracted with dichloromethane and water. After the solvent is removed under reduced pressure, it is purified by column chromatography to obtain solid compound 3 or 4.

[0014] Step S4: Compound 3 or 4, potassium carbonate, and dimethylamine are added to DMF and water, and the mixture is heated to reflux. After the reaction is completed, the crude product is extracted with dichloromethane and water. After the solvent is removed under reduced pressure, solid compound 5 or 6 is obtained by purification by column chromatography.

[0015] Step S5: Compound 2, 5, or 6 and Compound 1 are added to ethanol. Piperidine is added dropwise to the reaction system and heated to reflux. After completion of the reaction, the solvent is removed under reduced pressure. The crude product is extracted with dichloromethane and water. The solvent is removed under reduced pressure. Purification by column chromatography yields a solid compound, CarbCN, NTCN, or NCarbCN.

[0016] The present invention also provides the use of the above-mentioned dicyanoisophorone fluorescent probe for detecting brain β-amyloid protein in the fluorescent response to Aβ aggregates in vitro, its use in staining and imaging of AD mouse brain slices in vitro, its use in imaging Aβ aggregates in the brains of AD mice in vivo, and its use in monitoring the levels of Aβ aggregates in the brains of AD mice of different ages in vivo.

[0017] The beneficial effects of the present invention are:

[0018] (1) The probe synthesized in the present invention has a large Stokes shift, effectively avoiding background interference and exhibiting good fluorescence response and concentration dependence to Aβ aggregates in solution.

[0019] (2) Among the probes synthesized by the present invention, the fluorescence response of probe NCarbCN to Aβ aggregates has the highest signal-to-noise ratio, the best recognition sensitivity, good selectivity and high binding affinity.

[0020] (3) Among the probes synthesized by the present invention, the probe NCarbCN can stain Aβ plaques in brain sections of AD mice, and its staining results can have a high colocalization result with the commercial probe ThT.

[0021] (4) The probes NTCN and NCarbCN synthesized in the present invention have low cytotoxicity and low hemolysis rate, and have good biosafety.

[0022] (5) Among the probes synthesized by the present invention, NTCN and NCarbCN can cross the blood-brain barrier and achieve imaging of Aβ in the brain of AD mice, among which the probe NCarbCN shows a stronger fluorescence signal.

[0023] (6) Among the probes synthesized by the present invention, NCarbCN was able to image Aβ levels in the brains of AD mice of different ages, which has great potential in the early diagnosis of neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the synthesis route of the dicyanoisophorone fluorescent probes CarbCN, NTCN and NCarbCN described in the present invention.

[0025] Figure 2 Spectral properties of the dicyanoisophorone fluorescent probes CarbCN, NTCN, and NCarbCN in the embodiments of the present invention; wherein, (a): normalized ultraviolet absorption intensity curves of the three probes, (b): normalized fluorescence intensity curves of the three probes.

[0026] Figure 3These are the fluorescence response spectra of the dicyanoisophorone fluorescent probes (a) CarbCN, (b) NTCN, and (c) NCarbCN to Aβ aggregates in the examples of the present invention.

[0027] Figure 4 1 and 2 are linear fitting curves of the fluorescence intensity of the dicyanoisophorone fluorescent probes (a) CarbCN, (b) NTCN, and (c) NCarbCN and the concentration of Aβ aggregates in the examples of the present invention.

[0028] Figure 5 The staining of the cortex and hippocampus of AD mouse brain slices by the dicyanoisophorone fluorescent probe NCarbCN in the examples of the present invention and its co-localization with the commercial dye ThT are shown.

[0029] Figure 6 The figures show the cytotoxicity and hemolysis rates of the dicyanoisophorone fluorescent probes NTCN and NCarbCN in the examples of the present invention; (a) the cytotoxicity of the probe NCarbCN in PC-12 cells, (b) the cytotoxicity of the probe NTCN in PC-12 cells, (c) the hemolysis experiment of the probe NCarbCN, and (d) the hemolysis experiment of the probe NTCN.

[0030] Figure 7 This is a fluorescence imaging diagram of 8-month-old AD mice using the dicyanoisophorone fluorescent probes NTCN and NCarbCN in an example of the present invention.

[0031] Figure 8 This is a fluorescence imaging diagram of the dicyanoisophorone fluorescent probe NCarbCN on 12-month-old AD mice in an example of the present invention. DETAILED DESCRIPTION

[0032] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available. The experimental methods used are all conventional methods unless otherwise specified.

[0033] Example 1: Synthesis of probes CarbCN, NTCN, and NCarbCN

[0034] (1) Synthesis of Compound 1: Isophorone (1.38 g, 10 mmol) and malononitrile (0.66 g, 10 mmol) were added to 40 mL of ethanol and heated to reflux. A catalytic amount of piperidine was slowly added dropwise to the reaction system. After the reaction was completed, the reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. Solid Compound 1 was obtained by purification by column chromatography (petroleum ether / dichloromethane = 1:1). 1HNMR(400MHz,Chloroform-d)δ6.66–6.61(m,1H),2.53(s,2H),2.19(s,2H),2.05(s,3H),1.03(s,6H).APCI-MS m / z:[M+H] + cacld for C 12 H 14 N2,186.1157;found,187.1207.

[0035] (2) Synthesis of Compound 2: Salicylaldehyde (0.61 g, 5 mmol), tert-butyl-3-bromopropylcarbamate (1.19 g, 5 mmol), and potassium carbonate (0.69 g, 5 mmol) were added to 40 mL of acetonitrile and heated to reflux. After the reaction was completed, the reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. Purification by column chromatography (petroleum ether / ethyl acetate = 20:1) gave Compound 2 as an oil. 1 H NMR(400MHz,Chloroform-d)δ10.48(s,1H),7.84(dd,J=7.7,1.8Hz,1H),7.60–7.50(m,1H),7.06(t,J=7.5Hz,1H),7.00(d ,J=8.4Hz,1H),4.88(s,1H),4.17(t,J=6.0Hz,2H),3.38(q,J=6.1,5.6Hz,2H),2.08(p,J=6.3Hz,2H),1.46(s,9H).ESI-MS m / z:[M-Carb+H] + cacld for C 15 H 21 NO4,279.15;found,180.06.

[0036] (3) Synthesis of Compound 3: 4-Fluorosalicylaldehyde (1.0 g, 7.14 mmol), 1-iodopropane (1.21 g, 7.14 mmol), and potassium carbonate (0.99 g, 7.14 mmol) were added to 40 mL of acetonitrile and heated to reflux. After the reaction was completed, the reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. Solid Compound 3 was obtained by purification by column chromatography (petroleum ether / ethyl acetate = 50:1). 1 H NMR(400MHz,Chloroform-d)δ10.43(s,1H),7.88(dd,J=8.6,6.9Hz,1H),6.77–6.65 (m,2H),4.04(t,J=6.4Hz,2H),1.91(h,J=7.4Hz,2H),1.10(t,J=7.4Hz,3H).APCI-MS m / z:[M+H]+ cacld for C 10 H 11 FO2,182.0743; found,183.0788.

[0037] (4) Synthesis of Compound 4: 4-Fluorosalicylaldehyde (1.0 g, 7.14 mmol), tert-butyl-3-bromopropylcarbamate (1.55 g, 6.5 mmol), and potassium carbonate (0.99 g, 7.14 mmol) were added to 40 mL of acetonitrile and heated under reflux. After the reaction was completed, the reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. Solid Compound 4 was obtained by purification by column chromatography (petroleum ether / ethyl acetate = 5:1). 1 H NMR (400MHz, Chloroform-d) δ10.37(s,1H),7.87(dd,J=8.6,6.9Hz,1H),6.75(td,J=8.3,2.3Hz,1H),6.70(dd,J=10.8 ,2.2Hz,1H),4.87(s,1H),4.14(t,J=6.0Hz,2H),3.38(q,J=6.5Hz,2H),2.09(p,J=6.4,5.7Hz,2H),1.46(s,9H).ESI-MS m / z:[M-Carb+H] + cacld for C 15 H 20 FNO4,297.14;found,198.08.

[0038] (5) Synthesis of Compound 5: Compound 3 (1.00 g, 5.49 mmol), potassium carbonate (0.75 g, 5.49 mmol), and dimethylamine (0.25 g, 5.49 mmol) were added to a mixed solvent of DMF and water and heated under reflux. After the reaction was completed, the reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. Solid Compound 5 was obtained by purification by column chromatography (petroleum ether / ethyl acetate = 10:1). 1 HNMR(400MHz,Chloroform-d)δ10.24(s,1H),7.74(d,J=8.9Hz,1H),6.31(dd,J=8.9,1.5Hz,1H),6.05(d ,J=2.3Hz,1H),4.02(t,J=6.4Hz,2H),3.08(s,6H),1.88(h,J=7.4Hz,2H),1.08(t,J=7.4Hz,3H).APCI-MS m / z:[M+H] + cacld for C 12 H 17NO2,207.1259; found,208.1300.

[0039] (6) Synthesis of compound 6: Compound 4 (1.00 g, 3.37 mmol), potassium carbonate (0.47 g, 3.37 mmol) and dimethylamine (0.15 g, 3.37 mmol) were added to a mixed solvent of DMF and water and heated under reflux. After the reaction was completed, the reaction was cooled to room temperature and the solvent was removed under reduced pressure. Solid compound 6 was obtained by purification by column chromatography (petroleum ether / ethyl acetate = 5:1). 1 HNMR(400MHz,Chloroform-d)δ10.17(s,1H),7.73(d,J=8.8Hz,1H),6.36(dd,J=8.9,2.1Hz,1H),6.12(s,1H),5. 02(s,1H),4.14(t,J=5.9Hz,2H),3.38(d,J=6.1Hz,2H),3.10(s,6H),2.07(p,J=6.3Hz,2H),1.47(s,9H).ESI-MS m / z:[M-Carb+H] + cacld forC 17 H 26 N2O4,223.13;found,322.18.

[0040] (7) Synthesis of Probe CarbCN: Compound 2 (0.38 g, 1.35 mmol) and compound 1 (0.25 g, 1.35 mmol) were added to 20 mL of ethanol. A catalytic amount of piperidine was added dropwise to the reaction system and heated to reflux. After the reaction was completed, the reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. Solid compound CarbCN was obtained by purification by column chromatography (petroleum ether / dichloromethane = 2:1). 1 H NMR(400MHz,Chloroform-d)δ7.59(dd,J=7.8,1.6Hz,1H),7.49(d,J=16.3Hz,1H),7.36–7.30(m,1H),7.06–6.98(m,2H),6.93(d,J=8.3Hz,1 H),6.84(s,1H),4.71(s,1H),4.13(t,J=6.1Hz,2H),3.38(s,2H),2.62(s,2H),2.53(s,2H),2.09(p,J=6.5Hz,2H),1.46(s,9H),1.11(s,6H). 13C NMR(101MHz,Chloroform-d)δ169.50,156.85,155.95,154.86,131.88,131.01,129.44,127.22,124.86,123.18,12 1.18,113.66,112.88,112.17,65.99,43.10,39.14,37.77,32.08,29.83,28.40,28.01.HR-MS(ESI,positive)calcd for C 27 H 33 N3O3 + ,[M+Na] + 470.2414; found,470.2414.

[0041] (8) Synthesis of probe NTCN: Compound 5 (0.40 g, 1.93 mmol) and compound 1 (0.36 g, 1.93 mmol) were added to 20 mL of ethanol. A catalytic amount of piperidine was added dropwise to the reaction system and heated to reflux. After the reaction was completed, the reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. Solid compound NTCN was obtained by purification by column chromatography (petroleum ether / dichloromethane = 1:2). 1 H NMR (400MHz, DMSO-d6) δ7.59(d,J=8.9Hz,1H),7.45(d,J=16.0Hz,1H),7.14(d,J=16.0Hz,1H),6.66(s,1H),6.37(dd,J=8.9,2.4Hz,1H),6.2 3(d,J=2.4Hz,1H),4.04(t,J=6.4Hz,2H),3.02(s,6H),2.56(s,2H),2.48(s,2H),1.81(h,J=7.2Hz,2H),1.05(t,J=7.4Hz,3H),1.01(s,6H). 13 C NMR(101MHz,Chloroform-d)δ169.20,159.24,156.44,152.88,133.59,129.07,124.48,120.90,114.59,11 3.73,105.37,95.55,69.85,43.06,40.35,39.12,31.97,28.08,22.64,10.69.HR-MS(ESI,negative)calcd for C 24 H 29 N3O - ,[MH] - 374.2238; found,374.2238.

[0042] (9) Synthesis of probe NCarbCN: Compound 6 (0.40 g, 1.24 mmol) and compound 1 (0.23 g, 1.24 mmol) were added to 20 mL of ethanol. A catalytic amount of piperidine was added dropwise to the reaction system and heated to reflux. After the reaction was completed, the reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. Solid compound NCarbCN was obtained by purification by column chromatography (petroleum ether / dichloromethane = 1:2). 1 H NMR(400MHz,Chloroform-d)δ7.48(d,J=8.9Hz,1H),7.44(d,J=15.9Hz,1H),6.88(d,J=16.0Hz,1H),6.75(s,1H),6.38(dd,J=8.8,2.3Hz,1H),6.18(s,1H ),4.75(s,1H),4.12(t,J=6.2Hz,2H),3.39(q,J=6.6Hz,2H),3.07(s,6H),2. 58(s,2H),2.49(s,2H),2.10(p,J=5.4,5.0Hz,2H),1.46(s,9H),1.09(s,6H). 13 C NMR(101MHz,Chloroform-d)δ169.26,158.74,156.30,155.99,132.97,130.93,128.69,124.53,120.98,114.49,113.71,105.66 ,95.50,71.80,65.89,43.10,40.40,39.22,37.85,32.03,29.88,28.41,28.07,28.06,19.18,1.03.HR-MS(ESI,negative)calcd for C 29 H 37 N3O3 - ,[MH] - 489.2871; found, 489.2868.

[0043] Example 2: Spectral properties of probes CarbCN, NTCN, and NCarbCN

[0044] The probes CarbCN, NTCN and NCarbCN were dissolved in dimethyl sulfoxide (DMSO) to prepare a working concentration of 10 μM, and their UV-visible absorption spectra and fluorescence emission spectra were tested using a UV-visible spectrophotometer and a fluorescence spectrophotometer, respectively. Figure 2As shown in Figure 3, the maximum absorption and emission wavelengths of the probe CarbCN are 418 nm and 562 nm, respectively. The probes NTCN and NCarbCN have the same maximum absorption and emission wavelengths, which are 540 nm and 680 nm, respectively.

[0045] like Figure 3 As shown in Figures ac, the fluorescence responses of the probes CarbCN, NTCN, and NCarbCN to Aβ aggregates were tested. After incubation with Aβ aggregates, the probes CarbCN, NTCN, and NCarbCN all showed concentration-dependent fluorescence enhancement. After the addition of Aβ aggregates, the fluorescence intensity of the probes showed a good linear correlation with the concentration of Aβ aggregates ( Figure 4 ), the detection limits of the probe CarbCN, the probe NTCN, and the probe NCarbCN were calculated to be 226.7 nM, 120.8 nM, and 80.8 nM, respectively.

[0046] Example 3: In vitro staining of brain sections using the probe NCarbCN

[0047] Paraffin-embedded 5μm brain sections of APPswe / PSEN1 or wild-type mice were taken for in vitro fluorescent staining. The sections were dewaxed by soaking in xylene for 5 minutes. Then washed with ethanol for 2 minutes and then washed with water for 5 minutes. The sections were first incubated with 40μL 1mg / mL ThT for 10 minutes and then washed with 50% ethanol for 13 minutes. Incubated with 40μL 10μM probe NCarbCN for 30 minutes and washed with 50% ethanol for 15 minutes. , anti-fluorescence quencher was added and the sections were sealed with neutral resin. Brain sections were imaged under a laser scanning confocal microscope (Leica SP8). The results are shown in Figure 2. Figure 5 As shown, Aβ plaques stained with the probe NCarbCN (red) and the commercial dye ThT (green) have a good co-localization effect.

[0048] Example 4: Cytotoxicity and hemolysis experiments of probes NTCN and NCarbCN

[0049] Cytotoxicity and hemolysis rate tests are commonly used to evaluate the biocompatibility of probes. Therefore, the cytotoxicity of the probes NTCN and NCarbCN was studied using the standard MTT assay. After incubation of the probes with PC-12 cells for 24 hours, the cell viability was still above 80%, indicating that the probes had low cytotoxicity ( Figure 6 The hemolysis rate test showed that the hemolysis rate of the probes NTCN and NCarbCN was less than 5% at a concentration of 50 μM ( Figure 6 This indicates that the probe has a low hemolysis rate and is suitable for biological research.

[0050] Example 5: Fluorescence imaging of the brain of 8-month-old AD mice using the probes NTCN and NCarbCN

[0051] Real-time brain fluorescence imaging was performed using 8-month-old AD (APP / PS1) mice and wild-type mice. Immediately after injection of the probe NTCN or NCarbCN via the tail vein, the fluorescence signal in the mouse brain was captured ( Figure 7 ). The fluorescence intensity in the mouse brain first gradually increased and then decreased within 90 minutes. In the first 20 minutes, the uptake rate of the probe in the brain was higher than the clearance rate, resulting in the enrichment of the probes NTCN and NCarbCN, and the fluorescence signal in the mouse brain was enhanced. Subsequently, due to the existence of brain clearance protection mechanisms, the fluorescence in the mouse brain gradually weakened. At all time points, the fluorescence in the AD mouse brain was always higher than that in the wild mouse due to the binding of the probe to Aβ plaques in the AD mouse brain. The maximum fluorescence signal in the brain of AD mice injected with the probe NTCN (at 20 minutes) was 1.5 times higher than that of wild mice. The maximum fluorescence signal in the brain of AD mice injected with the probe NCarbCN was 1.9 times higher than that of wild mice, indicating that it responded better to Aβ.

[0052] Example 6: Fluorescence imaging of the brain of 12-month-old AD mice using the probe NCarbCN

[0053] Researchers believe that as AD mice age, the level of Aβ aggregates in the brain also increases. Therefore, they attempted to use the probe NCarbCN to perform fluorescence imaging on the brains of elderly AD mice (12 months old). Figure 8 As shown in Figure 2, the fluorescence signal in the brain of AD mice reached its maximum at 20 minutes. The fluorescence signal in the brain of AD mice was 2.1 times that of wild-type mice.

Claims

1. A dicyanoisophorone fluorescent probe for detecting brain β-amyloid protein, characterized in that The structural formula of the fluorescent probe is as follows: 。 2. A method for preparing a fluorescent probe as claimed in claim 1, characterized in that The specific steps are: Step S1: adding isophorone and malononitrile to ethanol, adding piperidine dropwise to the reaction system, heating to reflux, removing the solvent under reduced pressure after the reaction, and purifying by column chromatography to obtain solid compound 1 ; Step S2: 4-fluorosalicylaldehyde, and potassium carbonate were added to acetonitrile, heated to reflux, and after the reaction was completed, the solvent was removed under reduced pressure, and the crude product was extracted with dichloromethane and water. After the solvent was removed under reduced pressure, solid compound 4 was obtained by column chromatography. ; Step S3: Compound 4, potassium carbonate and dimethylamine were added to DMF and water, heated to reflux, and after the reaction was completed, the crude product was extracted with dichloromethane and water, and the solvent was removed under reduced pressure, and then purified by column chromatography to obtain solid compound 6 ; Step S4: Compound 6 and compound 1 are added to ethanol, piperidine is added dropwise to the reaction system, and the mixture is heated under reflux. After the reaction is completed, the solvent is removed under reduced pressure, the crude product is extracted with dichloromethane and water, the solvent is removed under reduced pressure, and the fluorescent probe is purified by column chromatography to obtain the fluorescent probe.

3. A use of the dicyanoisophorone fluorescent probe for detecting brain β-amyloid protein according to claim 1, characterized in that: Application in the preparation of fluorescence response detection reagents for Aβ aggregates in solution.

4. A use of the dicyanoisophorone fluorescent probe for detecting brain β-amyloid protein according to claim 1, characterized in that: Application in the preparation of reagents for detecting or monitoring Aβ levels in the brains of Alzheimer's disease mice.

5. A use of the dicyanoisophorone fluorescent probe for detecting brain β-amyloid protein according to claim 1, characterized in that: Application in the preparation of reagents for specific staining of Aβ plaques in brain sections of Alzheimer's disease mice.

6. A use of the dicyanoisophorone fluorescent probe for detecting brain β-amyloid protein according to claim 1, characterized in that: Application in the preparation of reagents for brain Aβ detection.

7. Use of the dicyanoisophorone fluorescent probe for detecting brain β-amyloid protein according to claim 1, characterized in that: Application in the preparation of diagnostic reagents for Alzheimer's disease.

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