Chemiluminescent probe targeting ab oligomer and preparation method and application thereof

Abstract

The application discloses a chemiluminescent probe for targeting A beta oligomer and a preparation method and application thereof, and a structural general formula of the chemiluminescent probe for targeting A beta oligomer is shown as I. The chemiluminescent probe for targeting A beta oligomer is successfully constructed for the first time, so that early diagnosis and treatment of AD can be realized. An external light source is not needed, background signals caused by excitation light can be effectively reduced, and therefore the detection sensitivity is higher. The chemiluminescent probe for targeting A beta oligomer can promote selective oxygenation of beta amyloid through chemical excitation, neutralize toxicity of A beta oligomer, has certain treatment potential, and is expected to develop into an effective method for treating amyloidosis.

Description

TECHNICAL FIELD

[0001] The present application relates to a chemiluminescent probe targeting Aβ oligomer and its preparation method and application, and belongs to the field of chemiluminescent probes, BACKGROUND

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease, the main pathological features are the progressive loss of neurons and the formation of amyloid plaques in the brain, and the clinical manifestations are cognitive and memory function decline, which causes heavy economic burden to patients and society. Unfortunately, there is no treatment that can effectively prevent or reverse the course of AD. Among the many hypotheses of AD etiology, the β amyloid (AmyLoid beta, Aβ) cascade hypothesis is widely recognized.

[0003] Aβ hypothesis believes that the deposition of Aβ in the brain is the main cause of AD, and Aβ protein includes Aβ monomer, oligomer and fiber, which plays a key role in the occurrence and development of AD. Amyloid Precursor Protein (APP) is dissociated by β-secretase and γ-secretase to form Aβ monomer, mainly Aβ40 and Aβ42 two subtypes. Under normal conditions, Aβ monomer remains in a random state and has no neurotoxicity, and under the induction of external environment such as pH and temperature, it will undergo conformational transition to form a β sheet structure, then interact through the hydrophobic region (Aβ16-22), self-aggregate, form dimer, trimer, hexamer and dodecamer, etc. The soluble Aβ oligomer has stronger neurotoxicity than the insoluble deposit plaque. On the one hand, the smaller Aβ oligomer has a large contact area with neurons and can enter the synaptic cleft, aggregate on the cell membrane and lipid valve, leading to membrane splitting and damage; on the other hand, Aβ oligomer binds to the receptor on the cell membrane, induces endocytosis into the cell, affects synaptic signal transmission, and ultimately leads to synaptic loss and memory impairment. Therefore, Aβ oligomer plays an important role in the pathogenesis of AD and is one of the key targets in current AD research and treatment strategies. A highly sensitive Aβ oligomer probe can achieve early diagnosis of AD and help to conduct in-depth research on the pathogenesis of AD, achieve early detection and early intervention, and has important scientific value and clinical significance.

[0004] Currently, the development of therapeutic drugs based on the Aβ protein hypothesis is difficult, and the main reasons are two key points. First, the timing of therapeutic intervention is too late, resulting in the formation of senile plaques, and toxic forms of Aβ have caused irreversible damage to nerve cells. Second, before Aβ forms fibers, its soluble oligomers are more toxic forms, and there is currently a lack of efficient and specific Aβ oligomer inhibitors. Therefore, early diagnosis of Aβ oligomers is of great significance for the treatment of AD. At the same time, effectively inhibiting the formation of Aβ oligomers and their toxic effects is an important direction in AD treatment strategies. Recent literature reports that the imidazopyrazine skeleton can promote the degradation of Aβ without external light irradiation. Specifically, the luciferin analogue dmCLA-X undergoes a chemical reaction through self-oxidation to selectively oxygenate amyloid in the dark, effectively reducing the aggregation efficiency and cytotoxicity of oxygen-containing Aβ. This chemical excitation strategy can effectively treat amyloidosis by reducing the accumulation of pathogenic amyloid in the deep brain, especially in the deep brain where light is difficult to reach.

[0005] Due to the heterogeneity and transience of Aβ oligomers, it is quite difficult to selectively detect these species. Previous studies have shown that most probes targeting Aβ oligomers are fluorescent probes or nanometer contrast agents, and chemiluminescent probes targeting Aβ oligomers have not been reported. However, traditional optical imaging (fluorescence imaging, near-infrared fluorescence imaging, etc.) has strong tissue autofluorescence, low tissue penetration depth, and low signal-to-noise ratio, limiting its application in living organisms. Chemiluminescence (CL) is a process in which light is generated through a chemical reaction without external light or other energy. Chemiluminescence has the advantages of high sensitivity, no need for external light, high signal-to-noise ratio, etc., providing a new method and approach for the further development of AD imaging technology. In 2020, a coelenterazine analogue chemiluminescent core was first developed and applied to Aβ imaging. This new type of chemiluminescent probe greatly broadens the application range of chemiluminescence imaging, but its wavelength is short and it is difficult to reach deep tissue sites. SUMMARY

[0006] The first object of the present application is to provide a chemiluminescent probe targeting Aβ oligomers. The second object of the present application is to provide a preparation method for the chemiluminescent probe targeting Aβ oligomers. The third object of the present application is to provide the application of the chemiluminescent probe targeting Aβ oligomers in the preparation of drugs for diagnosing or treating Alzheimer's disease.

[0007] Technical scheme: The chemiluminescent probe targeting Aβ oligomers provided by the present application has the structural general formula as shown in I:

[0008]

[0009] wherein n1 is 1 or 2; n2 is 1 or 2; A is

[0010] B1 is H,

[0011] B2 is H,

[0012] R is

[0013] Further, the chemiluminescent probe targeting Aβ oligomer is:

[0014]

[0015] A preparation method of the chemiluminescent probe targeting Aβ oligomer according to the present application, comprising the following synthesis route:

[0016]

[0017] wherein n1 is 1 or 2; n2 is 1 or 2; A is B1 is H,

[0018] B2 is H,

[0019] R is

[0020] Further, synthesized according to the following route:

[0021]

[0022] Further, comprising the following steps:

[0023] (1) Synthesis of intermediate compound 1: anhydrous potassium carbonate, L-proline, cuprous iodide, acetylacetone are dissolved in dimethyl sulfoxide, and reacted under nitrogen protection, and then iodobenzene is slowly added, and the reaction is continued, extracted with ethyl acetate, dried, concentrated, and purified by column chromatography to obtain intermediate 1;

[0024] (2) Synthesis of intermediate 2: intermediate 1 is added to anhydrous dichloromethane solvent, stirred under ice bath under nitrogen protection, and then trifluoroboron ethyl ether solution is slowly added, and the reaction is carried out under ice bath under nitrogen protection, and then the reaction is carried out at room temperature, and then quenched with water, extracted with dichloromethane, dried, concentrated, and purified by column chromatography to obtain intermediate 2;

[0025] (3) Synthesis of intermediate compound 3: intermediate 2 and 4-dimethylaminobenzaldehyde are added to anhydrous ethanol, stirred at room temperature, a solution of piperidine in anhydrous tetrahydrofuran is slowly added, oil bath reaction, dichloromethane extraction, drying, concentration, column chromatography purification to obtain intermediate 3;

[0026] (4) Synthesis of intermediate compound 4: 2-amino-5-bromopyrazine and 4-formylphenylboronic acid are dissolved in a mixture of toluene and ethanol, Na2CO3 is added, after degassing, Pd(PPh3)4 is added, heated reaction, dichloromethane extraction, drying, concentration, column chromatography purification to obtain intermediate 4;

[0027] (5) Synthesis of intermediate compound 5: intermediate 3 and intermediate 4 are added to anhydrous ethanol, a solution of piperidine in anhydrous ethanol is added, oil bath heating reaction under nitrogen protection, dichloromethane extraction, drying, filtration, concentration, column chromatography purification to obtain intermediate 5;

[0028] (6) Synthesis of chemiluminescence probe ADBF: intermediate compound 5 and methylglyoxal are added to anhydrous ethanol, vacuum nitrogen protection, stirring at room temperature, slowly adding HCl, continue stirring, oil bath heating reaction, cooling to room temperature, adding EA to form a precipitate, vacuum filtration, washing, vacuum drying to obtain chemiluminescence probe ADBF.

[0029] Further, in step (1), the reaction temperature is 80-100℃ under nitrogen protection, and the reaction time is 12-18h.

[0030] Further, in step (2), the ice bath reaction time is 30-60min, and the room temperature reaction time is 6-8h.

[0031] Further, in step (3), the oil bath reaction temperature is 30-50℃, and the oil bath reaction time is 6-8h.

[0032] Further, in step (4), the heating reaction temperature is 75-95℃, and the heating reaction time is 10-12h.

[0033] Further, in step (5), the oil bath heating reaction temperature is 70-90℃, and the oil bath heating reaction time is 6-8h.

[0034] Further, in step (6), the stirring time at room temperature is 3-10min, the continue stirring time is 5-10min, the oil bath heating reaction temperature is 80℃, and the oil bath heating reaction time is 8-10h.

[0035] The chemiluminescence probe targeting Aβ oligomers according to the application is used in the preparation of a medicament for relieving or treating Alzheimer's disease.

[0036] Further, the dosage form of the drug is one of a pill, a paste, a tablet, an oral liquid, a subcutaneous injection, and an intravenous injection.

[0037] Still further, the drug further comprises a stabilizing agent, a buffer, a cosolvent, an emulsifying agent, an excipient, a diluent, and / or an isotonic agent.

[0038] The present application takes Aβ oligomers as a core target, studies a new efficient Aβ chemiluminescence imaging probe on one hand, and further studies the therapeutic effect of the probe, i.e., inhibiting Aβ aggregation and its toxic effect, on the other hand. Specifically, a boron difluoride structure is taken as a connecting bridge, one end of which is connected with a targeting group to improve the targeting of the probe to Aβ oligomers; the other end adopts an imidazopyrazine skeleton as a key component of chemiluminescence and oxygenation therapy, and a series of high-sensitivity chemiluminescence probes specifically targeting Aβ oligomers are constructed. On this basis, the therapeutic effect of the probe is further studied, i.e., the chemiluminescence oxygenation is used to inhibit Aβ aggregation and reduce the toxicity of Aβ oligomers. And through a series of cell and in vitro experiments, it is verified that the probe can penetrate the blood-brain barrier, tightly bind to Aβ oligomers, significantly enhance the luminescence intensity, and realize imaging of Aβ oligomers in the brain of an AD animal model. The chemiluminescence probe of the present application uses the imidazopyrazine skeleton to degrade Aβ protein under oxygen-containing conditions without light irradiation, promotes the selective oxygenation of Aβ protein through chemical excitation, neutralizes the toxicity of Aβ oligomer protein, and is expected to develop into an effective method for treating amyloidosis, providing a new possibility for the treatment of AD.

[0039] Advantages: Compared with the prior art, the present application has the following remarkable advantages:

[0040] (1) The present application successfully constructs a chemiluminescence probe that can specifically target Aβ oligomers for the first time, so as to realize early diagnosis and treatment of AD.

[0041] (2) The chemiluminescence of the present application is based on the generation of light through chemical reaction, without the need for an external light source, which can effectively reduce the background signal brought by excitation light, so the detection sensitivity is higher.

[0042] (3) The chemiluminescence probe for targeting Aβ oligomers of the present application can promote the selective oxygenation of β amyloid protein through chemical excitation, neutralize the toxicity of Aβ oligomers, has a certain therapeutic potential, and is expected to develop into an effective method for treating amyloidosis. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 The nuclear magnetic hydrogen spectrum of intermediate 1

[0044] Figure 2 The nuclear magnetic hydrogen spectrum of intermediate 2

[0045] Figure 3 NMR spectrum of intermediate 3;

[0046] Figure 4 NMR spectrum of intermediate 4;

[0047] Figure 5 NMR spectrum of intermediate 5;

[0048] Figure 6 NMR spectrum of chemiluminescent probe ADBF;

[0049] Figure 7 High resolution mass spectrum of chemiluminescent probe ADBF;

[0050] Figure 8 Chemiluminescence spectrum of chemiluminescent probe ADBF in Example 2;

[0051] Figure 9 Fluorescence spectrum of chemiluminescent probe ADBF in Example 3 after binding with Aβ monomer, Aβ oligomer and Aβ aggregate respectively;

[0052] Figure 10 Fluorescence intensity quantification results of compound ADBF for Aβ oligomer selectivity;

[0053] Figure 11 Cell toxicity evaluation experimental results in Example 4;

[0054] Figure 12 Influence of ADBF on PC12 cell viability under different conditions in Example 5;

[0055] Figure 13 Inhibition of Aβ oligomer aggregation and toxicity of ADBF under different conditions in Example 5;

[0056] Figure 14 Mouse brain imaging after ADBF injection and chemiluminescence signal of mouse brain at different time nodes after injection in Example 5;

[0057] Figure 15 Quantification of chemiluminescence signal of mouse brain at different time nodes after ADBF injection in Example 5. DETAILED DESCRIPTION

[0058] The technical solutions of the present application will be further described below through specific examples.

[0059] Preparation of chemiluminescent probe ADBF in Example 1

[0060] 1. Synthesis of intermediate compound 1

[0061] Anhydrous potassium carbonate (5434.00 mg, 4.00 mmol), L-proline (227.00 mg, 0.2 mmol), cuprous iodide (187.00 mg, 0.1 mmol) were added to the reaction bottle, 6.00 mL of dimethyl sulfoxide was added as a solvent, acetylacetone (1477.00 mg, 1.5 mmol) was added, a condenser was connected, vacuum was extracted and nitrogen protection was carried out, after 40 minutes of reaction at 90°C. Iodobenzene (2000.00 mg, 1 mmol) was dissolved in 1.00 mL of dimethyl sulfoxide solvent, and was slowly added into the reaction bottle with a syringe, and the reaction was continued. The degree of reaction was detected by thin layer chromatography, after the reaction was completed, the pH was adjusted to neutral, and ethyl acetate (700 mL) was extracted 3 times, the organic phase was combined and dried with anhydrous Na2SO4, and concentrated under reduced pressure, and PE:EA=100:1 column chromatography purification was carried out to obtain white crystalline solid intermediate 1 (yield 700.00 mg, yield 40.56%).

[0062] The intermediate 1 was analyzed by nuclear magnetic hydrogen spectrum, and the results were as follows: 1 H NMR (300 MHz, Chloroform-d) δ 7.40-7.27 (m, 3H), 7.21-7.12 (m, 2H), 1.85 (s, 6H), as shown in Figure 1.

[0063] From the above analysis, the structure of the intermediate 1 is as follows:

[0064]

[0065] 2. Synthesis of intermediate compound 2

[0066] Intermediate 1 (255.00 mg, 1 mmol) was added to the reaction bottle, vacuum was extracted and nitrogen protection was carried out, 6.00 mL of anhydrous dichloromethane solvent was added to the reaction bottle, and stirred in an ice bath, and a solution of boron trifluoride in ether (0.731 mL, 2 mmol) was slowly added with a syringe, and reacted for 30 minutes in an ice bath, and then reacted at room temperature, and the reaction was monitored by TLC, and after the reaction was completed, 10 mL of water was added to quench the reaction. Dichloromethane (20 mL) was extracted 3 times, the organic phase was combined and dried with anhydrous Na2SO4, and concentrated under reduced pressure, and PE:EA=50:1 column chromatography purification was carried out to obtain white solid intermediate 2 (yield 230.00 mg, yield 70.8%).

[0067] The intermediate 2 was analyzed by nuclear magnetic hydrogen spectrum, and the results were as follows: 1 H NMR (300 MHz, Chloroform-d) δ 7.52-7.39 (m, 3H), 7.22-7.14 (m, 2H), 2.11 (s, 6H), as shown in Figure 2.

[0068] From the above analysis, the structure of intermediate 2 is as follows:

[0069]

[0070] 3. Synthesis of intermediate compound 3

[0071] Intermediate 2 (112.00 mg, 1 mmol) and 4-dimethylaminobenzaldehyde (67.00 mg, 1 mmol) were weighed into a reaction bottle, vacuumed and protected by nitrogen, 4.00 mL of anhydrous ethanol was added, and stirred at 40°C for 5 min. 1 mL of anhydrous tetrahydrofuran was added to a 1 mL EP tube, 1 drop of piperidine was added, and the reagent in the EP tube was slowly added into the reaction system, and the reaction was continued at 40°C. TLC point plate monitoring reaction, after the reaction was completed, dichloromethane (20 mL) was extracted 3 times, the organic phase was combined and dried with anhydrous Na2SO4, concentrated under reduced pressure, and purified by PE:EA = 10:1 column chromatography to obtain red solid intermediate 3 (yield 73.00 mg, yield 41.1%).

[0072] The nuclear magnetic hydrogen spectrum analysis of intermediate 3 is as follows: 1 H NMR (300 MHz, Chloroform-d) δ 8.08 (d, J = 15.1 Hz, 1H), 7.46 (qd, J = 5.1, 1.8 Hz, 3H), 7.34 - 7.28 (m, 2H), 7.25 - 7.17 (m, 2H), 6.68 - 6.51 (m, 2H), 6.12 (d, J = 15.1 Hz, 1H), 3.05 (s, 6H), 2.07 (s, 3H), spectrum as shown in 3.

[0073] From the above analysis, the structure of intermediate 3 is as follows:

[0074]

[0075] 4. Synthesis of intermediate compound 4

[0076] 2-amino-5-bromopyrazine (500.00 mg, 1 mmol) and 4-formylphenylboronic acid (474.00 mg, 1 mmol) were dissolved in toluene and ethanol (10 mL, toluene:methanol = 4:1), then Na2CO3 solution (2 mol / mL, 6 mL) was added, degassed, and Pd(PPh3)4 (165.00 mg, 0.4 mmol) was added. The reaction was stirred at 85°C for 12 hours. TLC point plate monitoring reaction, after the reaction was completed, dichloromethane (20 mL) was extracted 3 times, the organic phase was combined and dried with anhydrous Na2SO4, concentrated under reduced pressure, and purified by column chromatography to obtain yellow solid intermediate 4 (yield 540.00 mg, yield 93.9%).

[0077] The intermediate 4 was analyzed by nuclear magnetic hydrogen spectrum, and the results were as follows: 1 H NMR (400 MHz, Chloroform-d) δ 10.01 (s, 1H), 8.67 (d, J = 1.4 Hz, 1H), 8.18 - 8.14 (m, 2H), 8.01 (d, J = 1.5 Hz, 1H), 7.96 - 7.92 (m, 2H), 6.85 (s, 2H), 3.35 (s, 1H), the spectrum was as shown in 4.

[0078] From the above analysis, the structure of the intermediate 4 was as shown below:

[0079]

[0080] 5, synthesis of intermediate compound 5

[0081] Intermediate 3 (100.00 mg, 1 mmol) and intermediate 4 (151.00 mg, 1 mmol) were weighed and added to a reaction bottle, vacuumized and protected by nitrogen, 5.00 ml of anhydrous ethanol solvent was added and stirred at room temperature. 1.00 ml of anhydrous ethanol was added to an EP tube, and 1 drop of piperidine was added, and the reagent in the EP tube was slowly added into the reaction bottle. The reaction system was moved into an oil bath, and the reaction was carried out at 80°C, and TLC point plate was used to monitor the reaction. After the reaction was completed, dichloromethane (20 ml) was extracted for 3 times, the organic phase was combined and dried with anhydrous Na2SO4, and concentrated under reduced pressure, and column chromatography was used for purification to obtain a dark blue solid intermediate 5 (yield was 70.00 mg, yield was 46.4%).

[0082] The intermediate 5 was analyzed by nuclear magnetic hydrogen spectrum, and the results were as follows: 1 H NMR (400 MHz, Chloroform-d) δ 8.43 (s, 1H), 8.16 - 8.03 (m, 2H), 7.98 (d, J = 15.4 Hz, 1H), 7.84 (d, J = 8.0 Hz, 2H), 7.58 - 7.39 (m, 5H), 7.41 - 7.28 (m, 4H), 6.70 - 6.47 (m, 3H), 6.25 (d, J = 15.3 Hz, 1H), 4.82 (s, 2H), 3.06 (s, 6H), the spectrum was as shown in 5.

[0083] From the above analysis, the structure of the intermediate 5 was as shown below:

[0084]

[0085] 6, synthesis of compound ADBF

[0086] Intermediate compound 5 (200 mg, 1 mmol) and methylglyoxal (161.00 mg, 3 mmol) were weighed into a reaction flask, 3 mL of anhydrous ethanol was added, a three-way stopcock was connected, vacuum was drawn and nitrogen protection was performed, and stirring was performed at room temperature for 5 minutes. 400 μL of 6 mol / mL HCl was slowly added to the reaction system, and stirring was performed for 5-10 minutes. The reaction flask was moved into an oil bath at 80°C and reacted for 10-12 hours. After the reaction was completed, it was cooled to room temperature, EA was added to form a precipitate, vacuum filtration was performed, washing was performed, and vacuum drying was performed to obtain a dark solid, which was the chemiluminescence probe ADBF (yield 50 mg, yield 22.7%).

[0087] The chemiluminescence probe ADBF was analyzed by nuclear magnetic hydrogen spectrum, carbon spectrum and mass spectrum, and the results were as follows: 1H NMR (400 MHz, DMSO-d6) δ = 7.92-7.86 (m, 2H), 7.62-7.58 (dd, J = 5.0, 2.1, 2H), 7.55-7.46 (m, 4H), 7.44-7.37 (m, 4H), 7.37-7.33 (m, 2H), 7.24-7.20 (m, 2H), 6.69-6.62 (d, J = 8.8, 2H), 6.52-6.45 (m, 1H), 6.21-6.14 (m, 1H), 2.98-2.94 (s, 6H), 2.36-2.30 (s, 3H), as shown in FIG. 6.

[0088] The chemiluminescence probe ADBF was analyzed by high-resolution mass spectrometry, and the results were as follows: HRMS: C 34 H 29 BF2N4O3, [M] 590.23833, [M+H]+, calculated 591.2399, found 591.2399, consistent with the theoretical calculation value, the accuracy is within 2.76 ppm. The experiment was performed in positive ion mode using an electrospray ionization source (ESI). The signal intensity indicates that the sample purity is high, and the background noise level is low, as shown in FIG. 7.

[0089] From the above analysis, the structure of the chemiluminescence probe ADBF is as follows:

[0090]

[0091] Example 2: Chemiluminescence wavelength determination of chemiluminescence probe ADBF

[0092] The standard solution of chemiluminescent probe ADBF prepared in Example 1 (chemiluminescent probe ADBF prepared in Example 1 was configured into a 10 mmol / mL standard stock solution with DMSO, and then diluted with PBS to 5 μmol / mL to obtain a chemiluminescent probe ADBF standard solution) was added to a black 96-well plate, respectively, and the chemiluminescence wavelength of the compound ADBF was measured by a small animal in vivo imaging instrument (IVIS Lumia II). The grating settings were Excitation: Block, Emission: 520 nm, 570 nm, 620 nm, 670 nm, 710 nm, 790 nm, 845 nm. The results are shown in Figure 8 . Figure 8 The chemiluminescence spectrum of the chemiluminescent probe ADBF in Example 2 is shown in Figure 8 It can be seen that the chemiluminescence wavelength of the chemiluminescent probe ADBF is about 720 nm, which is relatively long and has good biological tissue penetration ability, so ADBF can be used for detection in deep tissue or in vivo.

[0093] Example 3 Selectivity of chemiluminescent probe ADBF

[0094] In order to explore the ability of chemiluminescent probe ADBF to detect Aβ oligomers in vivo, the chemiluminescent probe ADBF prepared in Example 1 was configured into a 10 mmol / mL standard stock solution with DMSO, and then diluted with PBS to 5 μmol / mL to obtain a chemiluminescent probe ADBF standard solution. Preparation of Aβ monomer: 0.25 mg of Aβ protein treated with trifluoroacetate was dissolved in 100 μL of hexafluoroisopropanol, and 900 μL of DDH2O was added, noting that it was prepared immediately. Preparation of Aβ oligomer: 0.25 mg of Aβ protein treated with hexafluoroisopropanol was dissolved in 100 μL of hexafluoroisopropanol, and 900 μL of DDH2O was added, and the constant oscillation was continued for 24 hours at 22°C and 100 rpm. Preparation of Aβ aggregate: 0.25 mg of Aβ protein treated with trifluoroacetate was dissolved in 1000 μL of buffer solution (containing 10 mmol / mL NaH2PO4, 1 mmol / mL EDTA in DDH2O), and the constant oscillation was continued for 48 hours at 37°C and 120 rpm. First, the fluorescence curve of the PBS buffer solution was measured by F-47000 fluorescence spectrophotometer (slit 10 / 10, response 0.5 s, PMT 700 V). Then 1900 μL of the chemiluminescent probe ADBF standard solution (5 μmol / mL) was taken with a pipette, and the fluorescence curve was measured, and then 100 μL of Aβ monomer, Aβ oligomer, Aβ aggregate solution (solution concentration was 50 μmol / mL) was added, and the fluorescence response was measured. The calculation formula of fluorescence enhancement multiple is as follows: FL enhancement = (FL Test- FL PBS ) / (FL probe - FL PBS ), wherein FL enhancement is the fluorescence enhancement fold, FL Test is the fluorescence intensity of the test, FL PBS is the fluorescence intensity of pure PBS, FL probe is the fluorescence intensity of the probe standard solution. The results are shown in Figure 9

[0095] Figure 9 are the fluorescence spectra of the chemiluminescent probe ADBF after binding with Aβ monomer, Aβ oligomer and Aβ aggregation respectively in Example 3; wherein, 5uM probe (red curve) is the fluorescence image of 5 μmol / mL pure probe ADBF standard solution, 5uM probe + 2.5uM monomer (black curve) is the fluorescence image of the mixed solution of 5 μmol / mL probe ADBF + 2.5 μmol / mL Aβ monomer, 5uM probe + 2.5uM aggergation (purple curve) is the fluorescence image of the mixed solution of 5 μmol / mL probe ADBF + 2.5 μmol / mL Aβ aggregation, 5uM probe + 2.5uM oligomer (green curve) is the fluorescence image of the mixed solution of 5 μmol / mL probe ADBF + 2.5 μmol / mL Aβ oligomer. It is shown from Figure 9 that the selectivity of the chemiluminescent probe ADBF for Aβ oligomer is obviously better than that for Aβ monomer and Aβ aggregation, indicating that the probe has good targeting property and can be effectively used for Aβ oligomer imaging in vivo. This finding confirms the potential of the compound ADBF in selectivity, and provides a scientific basis for its application in biomedical research, especially in the early diagnosis and treatment monitoring of Alzheimer's disease.

[0096] Figure 10 is the fluorescence intensity quantification result graph of the selectivity of the compound ADBF for Aβ oligomer, Figure 10 Quantitative analysis shows that the fluorescence intensity of the chemiluminescent probe ADBF after binding with Aβ oligomer is enhanced by 16 times, and that after binding with Aβ aggregation is enhanced by about 10 times, and the blue shift of the maximum absorption peak occurs.

[0097] Example 4 Cell toxicity test of chemiluminescent probe ADBF

[0098] ​To verify the biocompatibility of the chemiluminescent probe ADBF for Aβ oligomers at the cellular level, the standard CCK-8 experiment protocol was followed, and the PC12 cell, a type of nerve cell closely related to Alzheimer's disease, was selected to evaluate the in vitro cytotoxicity of the chemiluminescent probe ADBF. The PC12 cells were resuspended and plated in a 96-well plate, with 4000 cells per well. After 24 hours of incubation at 37°C in a 5% CO2 incubator, the supernatant in the wells was aspirated, and high-sugar DMEM culture solution containing the chemiluminescent probe ADBF (ADBF concentrations were 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, and 40 μM, respectively) was added, and the plate was placed in the incubator for further incubation. After 24 hours, the supernatant was again aspirated, and 100 μL of incomplete DMEM (high-sugar) culture medium containing 10% CCK8 was added, and the plate was incubated at 37°C in a 5% CO2 incubator for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and the cytotoxicity of the chemiluminescent probe ADBF was calculated. Each experiment was repeated three times.

[0099] The following formula was used to calculate the cell growth viability: Viability (%) = (average absorbance of test wells - average absorbance of medium control wells) / (average absorbance of untreated wells - average absorbance of medium control wells) x 100%. The results are shown in Figure 11 . Figure 11 The results of the cytotoxicity evaluation experiment in Example 4 are shown in the graph Figure 11 The results show that the chemiluminescent probe ADBF exhibits low toxicity to the PC12 cell. In PC12 cells, the survival rate is more than 85%, which indicates that the chemiluminescent probe ADBF has good biocompatibility. Therefore, the chemiluminescent probe ADBF has the potential to further explore its application value in the treatment of neurodegenerative diseases.

[0100] Example 5 Evaluation of the photo-oxidation efficacy of the compound ADBF of the present application

[0101] Photo-oxidation of Aβ can be carried out in the presence of cells by using a selective catalyst, riboflavin, linked to an Aβ-binding peptide and irradiation with 500 nm visible light. Oxygenated Aβ exhibits considerably lower aggregation potency and neurotoxicity compared to native Aβ. The present application uses riboflavin as a control to evaluate the photo-oxidation efficacy of the compound ADBF.

[0102] First, PC12 cells were resuspended with DMEM medium and evenly spread in two 96-well plates, ensuring that each well contained 4000 cells. Incubate at 37°C in a 5% CO2 constant temperature incubator for 24 hours, carefully aspirate the culture supernatant in each well. Next, according to the experimental design, the following experimental groups are set up: ① Aβ oligomer treatment group (oligomer): add 10 μmol / mL of Aβ oligomer to each well. ② Riboflavin treatment group (Riboflavin): add 5 μmol / mL of riboflavin to each well. ③ Compound ADBF treatment group (ADBF): add 5 μmol / mL of compound ADBF to each well. ④ Aβ oligomer and riboflavin co-treatment group (Riboflavin+oligomer): add 10 μmol / mL of Aβ oligomer and 5 μmol / mL of riboflavin to each well. ⑤ Aβ oligomer and compound ADBF co-treatment group (ADBF+oligomer): add 10 μmol / mL of Aβ oligomer and 5 μmol / mL of compound ADBF to each well. ⑥ Blank group (black): nothing. The concentrations of the above groups are all the final concentrations in each well. After setting up the groups, one of the hole plates was subjected to 500 nm wavelength light treatment, while the other hole plate was placed in the dark environment. By measuring the cell viability under light and dark conditions, it is evaluated whether the therapeutic effect of compound ADBF is dependent on light activation. It helps to explore the effect of compound ADBF on PC12 cell viability under different conditions, and its potential oxygenation treatment. The results are shown in Figure 12 .

[0103] Figure 12 It is shown that the PC12 cell viability after treatment with 10 μmol / mL Aβ oligomer alone is only 50%, while about 80% of the cells survive after co-incubation with 10 μmol / mL Aβ oligomer and compound ADBF, and whether the light has little effect on the cell viability of compound ADBF co-incubation, indicating that compound ADBF has oxygenation treatment effect, and does not need light activation.

[0104] To verify whether the oxygenation therapy of compound ADBF is concentration-dependent, the following experimental groups were set: ① black: nothing was added, only DMEM medium. ② Aβ oligomer treatment group (oligomer): 10 μmol / mL of Aβ oligomer was added to each well. ③ Aβ oligomer and 0.5 μmol / mL compound ADBF co-treatment group (0.5): 10 μmol / mL of Aβ oligomer and 0.5 μmol / mL of compound ADBF were added to each well at the same time. ④ Aβ oligomer and 3 μmol / mL compound ADBF co-treatment group (3): 10 μmol / mL of Aβ oligomer and 3 μmol / mL of compound ADBF were added to each well at the same time. ⑤ Aβ oligomer and 6 μmol / mL compound ADBF co-treatment group (6): 10 μmol / mL of Aβ oligomer and 6 μmol / mL of compound ADBF were added to each well at the same time. The concentrations of the above groups were all the final concentrations in each well. The results are shown in Figure 13 As shown in the figure, the concentration-dependent of oxygen-containing Aβ (54.0% at 0.5 μmol / mL, 72.3% at 3 μmol / mL, and 83.1% at 6 μmol / mL) was observed, and the cytotoxicity of Aβ oligomer was inhibited. Therefore, the co-treatment of Aβ oligomer and compound ADBF significantly inhibited the aggregation potency and cytotoxicity of Aβ oligomer.

[0105] APP / PSI transgenic AD mice (APP) and control wild-type mice (WT) of different months of age were subjected to head partial depilation, and were anesthetized by intraperitoneal injection of 10% chloral hydrate (4 μL / g). We injected the chemiluminescent probe ADBF into the APP / PS1 transgenic AD mouse model (APP) and control wild-type mice (WT) at a dose of 4 mg / Kg (10% DMSO, 15% Tween20, 75% PBS) (the specific dose was calculated according to the body weight of each mouse), and then imaged the brain of the mice by a small animal in vivo imaging system (IVIS Lumia II), and collected the chemiluminescent signals of the brain of the mice at different time points before and after the injection of the probe. The results are shown in Figure 14 As shown in the figure, the chemiluminescent signal of the APP mice and the WT mice was enhanced 1 minute after the injection of the probe ADBF into the mice, the chemiluminescent intensity reached the maximum at the 10th minute, and the chemiluminescent intensity gradually decreased at subsequent times. The chemiluminescent intensity of the brain of the mice was quantitatively analyzed by the corresponding analysis software living Image of the small animal in vivo imaging system. Figure 15The quantitative analysis of the chemiluminescence intensity of the mice showed that the chemiluminescence intensity of the APP mice was 1.74 times of that of the WT mice at the first minute, and reached 1.84 times at the 15th minute. The results of the in vivo imaging showed that the probe could effectively pass through the blood-brain barrier, enter the brain of the mice, and bind with the Aβ to produce an obvious enhancement of the chemiluminescence signal, so that the APP mice and the WT mice could be clearly distinguished.

Claims

1. A chemiluminescent probe targeting Aβ oligomers, characterized in that, The structure of the targeting Aβ oligomer chemiluminescent probe is shown as formula I: wherein n1 is 1 or 2; n2 is 1 or 2; A is B1is H, B2 is H, R is 2. The chemiluminescent probe for targeting Aβ oligomer according to claim 1, wherein, The targeting Aβ oligomer chemiluminescent probe is:

3. A method for preparing the chemiluminescent probe for targeting an Aβ oligomer according to claim 1 or 2, characterized by, The synthesis route comprises the following steps: wherein n1 is 1 or 2; n2 is 1 or 2; A is B1is H, B2is H, R is 4. The preparation method according to claim 3, characterized in that, The synthesis route comprises the following steps:

5. The method of claim 3, wherein: The synthesis route comprises the following steps: (1) Synthesis of intermediate compound 1: anhydrous potassium carbonate, L-proline, cuprous iodide, acetylacetone are dissolved in dimethyl sulfoxide, and reacted under nitrogen protection, and iodobenzene is slowly added, and the reaction is continued, and extracted with ethyl acetate, and dried, concentrated, and purified by column chromatography to obtain intermediate 1; (2) Synthesis of intermediate 2: intermediate 1 is added to anhydrous dichloromethane solvent, and stirred in an ice bath under nitrogen protection, and boron trifluoride in ether solution is slowly added, and reacted in an ice bath under nitrogen protection, and reacted at room temperature, and quenched with water, and extracted with dichloromethane, and dried, filtered, concentrated, and purified by column chromatography to obtain intermediate 2; (3) Synthesis of intermediate compound 3: intermediate 2 and 4-dimethylaminobenzaldehyde are added to anhydrous ethanol, and stirred at room temperature, and a solution of piperidine in anhydrous tetrahydrofuran is slowly added, and reacted in an oil bath, and extracted with dichloromethane, and dried, filtered, concentrated, and purified by column chromatography to obtain intermediate 3; (4) Synthesis of intermediate compound 4: 2-amino-5-bromopyrazine and 4-formylphenylboronic acid are dissolved in a mixture of toluene and ethanol, and Na2CO3 is added, and degassed, and Pd(PPh3)4 is added, and heated to react, and extracted with dichloromethane, and dried, concentrated, and purified by column chromatography to obtain intermediate 4; (5) Synthesis of intermediate compound 5: intermediate 3 and intermediate 4 are added to anhydrous ethanol, and a solution of piperidine in anhydrous ethanol is added, and reacted in an oil bath under nitrogen protection, and extracted with dichloromethane, and dried, concentrated, and purified by column chromatography to obtain intermediate 5; (6) Synthesis of chemiluminescent probe ADBF: intermediate compound 5 and methylglyoxal are added to anhydrous ethanol, and vacuumized to perform nitrogen protection, and stirred at room temperature, and HCl is slowly added, and continued to stir, and reacted in an oil bath, and cooled to room temperature, and EA is added to form a precipitate, and filtered, and washed, and vacuum dried to obtain chemiluminescent probe ADBF.

6. The production method according to claim 5, characterized by, In step (1), the reaction temperature under nitrogen protection is 80-100℃, and the reaction time is 12-18h; in step (2), the ice bath reaction time is 30-60min, and the room temperature reaction time is 6-8h.

7. The preparation method according to claim 5, characterized in that, In step (3), the oil bath reaction temperature is 30-50℃, and the oil bath reaction time is 6-8h; in step (4), the heating reaction temperature is 75-95℃, and the heating reaction time is 10-12h.

8. The preparation method according to claim 5, characterized in that, In step (5), the oil bath heating reaction temperature is 70-90℃, and the oil bath heating reaction time is 6-8h; in step (6), the room temperature stirring time is 3-10min, the continued stirring time is 5-10min, the oil bath heating reaction temperature is 80℃, and the oil bath heating reaction time is 8-10h.

9. The targeting Aβ oligomer chemiluminescent probe of claim 1 or 2 in the preparation of a medicament for alleviating or treating Alzheimer's disease.

10. Use according to claim 9, characterized in that, The dosage form of the drug is one of a pill, a paste, a tablet, an oral liquid, a subcutaneous injection, and an intravenous injection, and the drug further includes a stabilizing agent, a buffer, a cosolvent, an emulsifying agent, an excipient, a diluent, and / or an isotonic agent.

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