Dicyanoisophorone fluorescent probe for detecting brain beta-amyloid protein as well as preparation method and application of dicyanoisophorone fluorescent probe

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a dicyanoisophorone fluorescent probe for brain beta-amyloid protein detection as well as a preparation method and application of the dicyanoisophorone fluorescent probe. The fluorescent probe is based on a dicyanoisophorone structure, and the probe with double recognition sites is formed by introducing a carbamate group and N, N-dimethylamino. Compared with a probe with a single recognition site, the probe with the double recognition sites has more sensitive fluorescence response to the A beta aggregate in vitro and shows higher binding affinity and selectivity. The probe can effectively penetrate through the blood brain barrier and shows high reactivity in the brain of an Alzheimer disease model mouse. And the probe can be used for monitoring Abeta levels of brains of mice with Alzheimer's disease at different ages. And the method has huge potential in the aspect of 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 diseases caused by the progressive loss of function or structure of nerve cells in the central nervous system. They mainly include Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. At present, Alzheimer's disease (AD) is considered to be the most common neurodegenerative disease. Its early symptoms are cognitive decline, emotional indifference and depression. As the patient's condition worsens, symptoms such as time and space dislocation, language disorders, and loss of self-care ability will occur. With the intensification of the aging society, the number of AD patients continues to increase, and people are paying more and more attention to the disease.

[0003] A large number of 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, it is hydrolyzed by β-secretase into αAPPs and C83, and then hydrolyzed by γ-secretase into P342 and P340. This pathway does not produce Aβ; when APP is hydrolyzed by another route, it is first hydrolyzed by β-secretase into βAPPs and C99, and then hydrolyzed by γ-secretase into Aβ. 1-42 and Aβ 1-40 . Aβ 1-42 Aβ 1-40 The addition of two amino acids makes Aβ more neurotoxic and more likely to form 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 apoptosis of neuronal cells. 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] At present, most fluorescent probes used to detect Aβ aggregates rely on some common recognition groups, such as N,N-dimethylamino, N,N-diethylamino and piperidine. Although these traditional probes can recognize Aβ aggregates to a certain extent, their sensitivity and selectivity are still limited. Therefore, the development of new fluorescent probes with dual recognition sites to improve the detection sensitivity and selectivity of Aβ aggregates has important research value and application prospects. Summary of the invention

[0005] In order to solve the problems existing in the detection of Aβ fluorescent probes, the purpose of the present invention is to provide a dicyanoisophorone fluorescent probe with dual recognition sites. The probe can achieve a fluorescent response to Aβ aggregates. At the same time, the fluorescent probe can cross the blood-brain barrier to achieve imaging of Aβ in the brain of AD mice, and can monitor the Aβ levels in the brains of AD mice of different ages.

[0006] In order to achieve the object 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, and 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 in that the specific preparation steps are:

[0011] Step S1: adding isophorone and malononitrile to ethanol. Slowly adding piperidine dropwise to the reaction system, heating to reflux. After the reaction is completed, removing the solvent under reduced pressure. Purifying by column chromatography to obtain solid compound 1.

[0012] Step S2: salicylaldehyde, tert-butyl-3-bromopropylcarbamate and potassium carbonate are added to acetonitrile and heated to reflux. After the reaction is completed, the solvent is removed under reduced pressure. The crude product is extracted with dichloromethane and water. After the solvent is removed under reduced pressure, the oily compound 2 is purified by column chromatography.

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

[0014] Step S4: Compound 3 or 4, potassium carbonate and dimethylamine are added to DMF and water, and 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 or 5 or 6 and compound 1 are added to ethanol. Piperidine is added dropwise to the reaction system and heated to 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. Solid compound CarbCN or NTCN or NCarbCN is obtained by purification by column chromatography.

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

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

[0018] (1) The probe synthesized by the present invention has a large Stokes shift, which effectively avoids background interference and has 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 the 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 slices of AD mice, and its staining results can have a high co-localization result with the commercial probe ThT.

[0021] (4) The probes NTCN and NCarbCN synthesized by the present invention have low cytotoxicity and low hemolysis rate and 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 exhibits a stronger fluorescence signal.

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

[0024] Figure 1 The invention discloses a synthetic route for the dicyanoisophorone fluorescent probes CarbCN, NTCN and NCarbCN.

[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 31 and 2 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 colocalization with the commercial dye ThT are shown.

[0029] Figure 6 The cytotoxicity and hemolysis rate of the dicyanoisophorone fluorescent probes NTCN and NCarbCN in the examples of the present invention are shown; among them, (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 can be obtained from commercial sources. The experimental methods used are 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, heated to reflux, and a catalytic amount of piperidine was slowly added dropwise to the reaction system. After the reaction was completed, the reaction 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 was cooled to room temperature and the solvent was removed under reduced pressure. The oily compound 2 was obtained by column chromatography (petroleum ether / ethyl acetate = 20:1). 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 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 to reflux. After the reaction was completed, the reaction 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 to reflux. After the reaction was completed, the reaction 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 to 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, and a catalytic amount of piperidine was added dropwise to the reaction system, and heated to reflux. After the reaction was completed, the reaction 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, and a catalytic amount of piperidine was added dropwise to the reaction system, and heated to reflux. After the reaction was completed, the reaction 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 was cooled to room temperature and the solvent was removed under reduced pressure. The 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) and prepared to a working concentration of 10 μM. Their UV-visible absorption spectra and fluorescence emission spectra were tested using a UV-visible spectrophotometer and a fluorescence spectrophotometer, respectively. Figure 2As shown, 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 response of probes CarbCN, NTCN, and NCarbCN to Aβ aggregates was tested. After incubation with Aβ aggregates, probes CarbCN, NTCN, and NCarbCN all showed concentration-dependent fluorescence enhancement. After adding 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 brain slice staining with 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 they were 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, the Aβ plaques stained with the probe NCarbCN (red) and the staining results of 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 often used to evaluate the biocompatibility of probes. Therefore, the cytotoxicity of the probes NTCN and NCarbCN was studied using the standard MTT method. After the probes were incubated with PC-12 cells for 24 hours, the cell viability was still above 80%, indicating that the cytotoxicity of the probes was low ( Figure 6 a and 6b). The hemolysis rate test showed that the hemolysis rates of the probes NTCN and NCarbCN were less than 5% at a concentration of 50 μM ( Figure 6 c and 6d). 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 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 the probe NTCN or NCarbCN was injected through the tail vein, the fluorescence signal in the mouse brain was captured ( Figure 7 ). The fluorescence intensity in the mouse brain 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 brain of AD mice was always higher than that of wild mice due to the binding of the probe to Aβ plaques in the brain of AD mice. 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 responds better to Aβ.

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

[0053] The 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 fluorescent imaging on the brains of elderly AD mice (12 months old). Figure 8 As shown, the fluorescence signal in the brain of AD mice reached a maximum at 20 minutes. The brain fluorescence signal 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: They are CarbCN, NTCN and NCarbCN respectively.

2. A method for preparing a dicyanoisophorone fluorescent probe for detecting brain β-amyloid protein, 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. after the reaction is completed, removing the solvent under reduced pressure. purifying by column chromatography to obtain solid compound 1. Step S2: salicylaldehyde, tert-butyl-3-bromopropylcarbamate and potassium carbonate are added to acetonitrile and heated to reflux. After the reaction is completed, the solvent is removed under reduced pressure. The crude product is extracted with dichloromethane and water. After the solvent is removed under reduced pressure, the oily compound 2 is purified by column chromatography. Step S3: 4-fluorosalicylaldehyde, 1-iodopropane (or tert-butyl-3-bromopropylcarbamate) and potassium carbonate are added to acetonitrile and heated to reflux. After the reaction is completed, the solvent is removed under reduced pressure. The crude product is extracted with dichloromethane and water. After the solvent is removed under reduced pressure, solid compound 3 or 4 is obtained by purification by column chromatography. Step S4: Compound 3 or 4, potassium carbonate and dimethylamine are added to DMF and water, and 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. Step S5: Compound 2 or 5 or 6 and compound 1 are added to ethanol. Piperidine is added dropwise to the reaction system and heated to 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. Solid compound CarbCN or NTCN or NCarbCN is obtained by purification by column chromatography.

3. A dicyanoisophorone fluorescent probe NCarbCN for detecting brain β-amyloid protein according to claim 1, characterized in that: Both carbamate groups and N,N-dimethylamino groups are introduced into the structure.

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

5. A use of the dicyanoisophorone fluorescent probes NTCN and NCarbCN for detecting brain β-amyloid protein according to claim 1, characterized in that: The probe is able to effectively cross the blood-brain barrier.

6. A use of the dicyanoisophorone fluorescent probes NTCN and NCarbCN for detecting brain β-amyloid protein according to claim 1, characterized in that: Used to detect or monitor Aβ levels in the brains of Alzheimer's disease mice.

7. A use of the dicyanoisophorone fluorescent probe NCarbCN for detecting brain β-amyloid protein according to claim 1, characterized in that: Used to detect or monitor Aβ levels in the brains of Alzheimer's disease mice of different ages.

8. A use of the dicyanoisophorone fluorescent probe NCarbCN for detecting brain β-amyloid protein according to claim 1, characterized in that: Used to specifically stain Aβ plaques in brain sections of Alzheimer's disease mice.

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

10. A use of the dicyanoisophorone fluorescent probe NCarbCN 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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