Chemiluminescent probe targeting A beta oligomer as well as preparation method and application of chemiluminescent probe

Abstract

The invention discloses a chemiluminescent probe of a targeted A beta oligomer as well as a preparation method and application of the chemiluminescent probe. The structural general formula of the chemiluminescent probe of the targeted A beta oligomer is shown as I in the specification. According to the present invention, the chemiluminescence probe capable of specifically targeting the A beta oligomer is successfully constructed for the first time so as to achieve the early diagnosis and treatment of AD; an additional light source is not needed, and background signals caused by exciting light can be effectively reduced, so that the detection sensitivity is higher. The chemiluminescent probe targeting the A beta oligomer can promote selective oxygenation of beta amyloid protein through chemical excitation and neutralize toxicity of the A beta oligomer, has certain treatment potential and is expected to be developed into an effective method for treating amyloidosis. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a chemiluminescent probe targeting Aβ oligomers and a preparation method and application thereof, belonging to the field of chemiluminescent probes. Background Art

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

[0003] The Aβ hypothesis holds that the deposition of Aβ in the brain is the main cause of AD. Aβ proteins include Aβ monomers, oligomers, and fibers, which play a key role in the occurrence and development of AD. Aβ precursor protein (APP) forms Aβ monomers under the dissociation of β-secretase and γ-secretase, and there are mainly two subtypes, Aβ40 and Aβ42. Under normal conditions, Aβ monomers maintain an irregular state and have no neurotoxicity. Under the induction of external environments such as pH and temperature, they undergo conformational conversion to form a β sheet structure, and then interact with the hydrophobic region (Aβ16-22) and aggregate themselves to form soluble Aβ oligomers with strong neurotoxicity, such as dimers, trimers, hexamers, and dodecamers. Compared with insoluble precipitated plaques, soluble Aβ oligomers are more neurotoxic than plaques. On the one hand, the smaller Aβ oligomers have a large contact area with neurons and can enter the synaptic cleft, aggregate on the cell membrane and lipid valves, and cause membrane fission and destruction; on the other hand, Aβ oligomers bind to receptors on the cell membrane, induce endocytosis into the cell, affect synaptic signal transduction, and ultimately lead to synaptic loss and memory impairment. Therefore, Aβ oligomers play an important role in the pathogenesis of AD and are one of the key targets in current AD research and treatment strategies. Highly sensitive Aβ oligomer probes 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 have important scientific value and clinical significance.

[0004] At present, the development of therapeutic drugs based on the Aβ protein hypothesis is difficult. The reasons for this are mainly attributed to two key points. First, the timing of therapeutic intervention is too late, resulting in the formation of senile plaques and the irreversible damage of toxic forms of Aβ 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 vital importance 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 indicate that the imidazopyrazine skeleton can promote the degradation of Aβ without external light source irradiation. Specifically, the fluorescein analog dmCLA-X undergoes a chemical reaction through self-oxidation to achieve selective oxygenation of amyloid in the dark, effectively reducing the aggregation efficacy and cytotoxicity of oxygenated Aβ. This chemical excitation strategy may effectively treat amyloidosis by reducing the accumulation of pathogenic amyloid deep in the brain, especially deep in the brain where light is difficult to reach.

[0005] Due to the heterogeneity and transient nature of Aβ oligomers, it is quite difficult to selectively detect these species. Previous research reports have shown that the probes targeting Aβ oligomers are mostly fluorescent probes or nanocontrast agents, and chemiluminescent probes targeting Aβ oligomers have not yet been reported. However, traditional optical imaging (fluorescence imaging, near-infrared fluorescence imaging, etc.) has strong tissue spontaneous luminescence, low tissue penetration depth and low signal-to-noise ratio, which limits its application in organisms. Chemiluminescence (CL) refers to the process of producing luminescence through chemical reactions in the absence of external light sources or other energy. Chemiluminescence has the advantages of high sensitivity, no need for external light sources, and high signal-to-noise ratio, providing new methods and ideas for the further development of AD imaging technology. In 2020, the coelenterazine analog chemiluminescent core was first developed for Aβ imaging. This new type of chemiluminescent probe has greatly broadened the application range of chemiluminescent imaging, but its wavelength is short and it is difficult to reach deep tissue sites. Summary of the invention

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

[0007] Technical solution: The present invention provides a chemiluminescent probe targeting Aβ oligomers, and the general structural formula of the chemiluminescent probe targeting Aβ oligomers is shown in I:

[0008]

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

[0010] B1 is H,

[0011] B2 is H,

[0012] R is

[0013] Furthermore, the chemiluminescent probe targeting Aβ oligomers is:

[0014]

[0015] A method for preparing a chemiluminescent probe targeting Aβ oligomers according to the present invention comprises the following synthetic route:

[0016]

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

[0018] B2 is H,

[0019] R is

[0020] Further, it is 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, and acetylacetone were dissolved in dimethyl sulfoxide, reacted under nitrogen protection, iodobenzene was slowly added, the reaction was continued, and the mixture was extracted with ethyl acetate, dried, concentrated, and purified by column chromatography to obtain intermediate 1;

[0024] (2) Synthesis of intermediate 2: intermediate 1 was added to anhydrous dichloromethane solvent, stirred in an ice bath under nitrogen protection, boron trifluoride ether solution was slowly added, reacted in an ice bath under nitrogen protection, reacted at room temperature, 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 were added to anhydrous ethanol, stirred at room temperature, and piperidine in anhydrous tetrahydrofuran was slowly added, reacted in an oil bath, extracted with dichloromethane, dried, concentrated, and purified by column chromatography to obtain intermediate 3;

[0026] (4) Synthesis of intermediate compound 4: 2-amino-5-bromopyrazine and 4-formylphenylboronic acid were dissolved in a mixture of toluene and ethanol, and Na2CO3 was added. After degassing, Pd(PPh3)4 was added, and the mixture was heated for reaction. The mixture was extracted with dichloromethane, dried, concentrated, and purified by column chromatography to obtain intermediate 4.

[0027] (5) Synthesis of intermediate compound 5: Intermediate 3 and intermediate 4 were added to anhydrous ethanol, and an anhydrous ethanol solution of piperidine was added, and the mixture was heated in an oil bath under nitrogen protection, extracted with dichloromethane, dried, filtered, concentrated, and purified by column chromatography to obtain intermediate 5;

[0028] (6) Synthesis of chemiluminescent probe ADBF: The intermediate compound 5 and acetone aldehyde were added to anhydrous ethanol, evacuated for nitrogen protection, stirred at room temperature, HCl was slowly added, stirring was continued, and the reaction was heated in an oil bath. The reaction was cooled to room temperature, EA was added to form a precipitate, filtered, washed, and dried in vacuum to obtain the chemiluminescent probe ADBF.

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

[0030] Furthermore, in step (2), the reaction time in an ice bath is 30-60 minutes, and the reaction time at room temperature is 6-8 hours.

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

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

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

[0034] Furthermore, in step (6), the stirring time at room temperature is 3-10 min, the stirring time is continued for 5-10 min, the temperature of the oil bath heating reaction is 80° C., and the oil bath heating reaction time is 8-10 h.

[0035] The invention relates to the use of the chemiluminescent probe targeting Aβ oligomers in the preparation of a drug for alleviating or treating Alzheimer's disease.

[0036] Furthermore, the dosage form of the drug is one of pills, ointments, tablets, oral liquids, subcutaneous injections and intravenous injections.

[0037] Furthermore, the drug also includes a stabilizer, a buffer, a solubilizer, an emulsifier, an excipient, a diluent and / or an isotonic agent.

[0038] The present invention takes Aβ oligomers as the core target. On the one hand, it studies a new type of highly efficient Aβ chemiluminescent in vivo imaging probe, and on the other hand, it further studies the therapeutic effect of the probe, that is, inhibiting Aβ aggregation and its toxic effects. Specifically, a boron difluoride structure is used as a connecting bridge, and a targeting group is connected at one end to improve the targeting of the probe to Aβ oligomers; the other end uses an imidazopyrazine skeleton as a key component of the chemiluminescent group and oxygenation therapy to construct a series of highly sensitive chemiluminescent probes that specifically target Aβ oligomers. On this basis, the therapeutic effect of the probe is further studied, that is, using chemiluminescent oxygenation to inhibit Aβ aggregation and reduce the toxic effects of Aβ oligomers. And through a series of cell and in vitro experiments, it will be verified that it can penetrate the blood-brain barrier, tightly bind to Aβ oligomers, and significantly enhance the luminescence intensity, and can achieve imaging of Aβ oligomers in the brain of AD animal models. The chemiluminescent probe of the present invention utilizes an imidazopyrazine skeleton to degrade Aβ protein without light irradiation under oxygen-containing conditions, promotes the selective oxygenation of Aβ protein through chemical excitation, and neutralizes the toxicity of Aβ oligomer protein. It is expected to be developed into an effective method for treating amyloidosis, providing new possibilities for the treatment of AD.

[0039] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0040] (1) The present invention successfully constructed for the first time a chemiluminescent probe that can specifically target Aβ oligomers to achieve early diagnosis and treatment of AD.

[0041] (2) The chemiluminescence of the present invention is based on the generation of light by chemical reactions and does not require an external light source. It can effectively reduce the background signal caused by the excitation light, thus increasing the detection sensitivity.

[0042] (3) The chemiluminescent probe targeting Aβ oligomers described in the present invention can promote the selective oxygenation of β-amyloid protein through chemical excitation, neutralize the toxicity of Aβ oligomers, have certain therapeutic potential, and are expected to be developed into an effective method for treating amyloidosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The H NMR spectrum of intermediate 1

[0044] Figure 2 is the H NMR spectrum of intermediate 2;

[0045] Figure 3 is the H NMR spectrum of intermediate 3;

[0046] Figure 4 is the H NMR spectrum of intermediate 4;

[0047] Figure 5 is the H NMR spectrum of intermediate 5;

[0048] Figure 6 This is the H-NMR spectrum of the chemiluminescent probe ADBF;

[0049] Figure 7 This is the high-resolution mass spectrum of the chemiluminescent probe ADBF;

[0050] Figure 8 is the chemiluminescence spectrum of the chemiluminescent probe ADBF in Example 2;

[0051] Fig. 9 The fluorescence spectra of the chemiluminescent probe ADBF in Example 3 after binding to Aβ monomers, Aβ oligomers, and Aβ aggregates, respectively;

[0052] Fig.10 The fluorescence intensity quantification result diagram of the selectivity of compound ADBF to Aβ oligomers;

[0053] Fig.11 This is a diagram showing the results of the cytotoxicity evaluation experiment in Example 4;

[0054] Fig.12 The figure is the result of the effect of ADBF on PC12 cell viability under different conditions in Example 5;

[0055] Fig.13 This is a graph showing the results of ADBF inhibiting Aβ oligomer aggregation and toxicity under different conditions in Example 5;

[0056] Fig.14 The images are of the brain imaging of mice injected with ADBF and the chemiluminescent signal images of the brain of mice at different time points after injection in Example 5;

[0057] Fig.15 This is a quantitative graph of the chemiluminescent signal in the mouse brain at different time points after ADBF injection in Example 5. DETAILED DESCRIPTION

[0058] The technical solution of the present invention is further described below through specific embodiments.

[0059] Example 1 Preparation of chemiluminescent probe ADBF

[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), and cuprous iodide (187.00 mg, 0.1 mmol) were added to the reaction flask, 6.00 mL of dimethyl sulfoxide was added as solvent, acetylacetone (1477.00 mg, 1.5 mmol) was added, a condenser was connected, vacuum was evacuated for nitrogen protection, and the reaction was carried out at 90° C. for 40 minutes. Iodobenzene (2000.00 mg, 1 mmol) was dissolved in 1.00 mL of dimethyl sulfoxide solvent, and slowly added dropwise into the reaction flask with a syringe to continue the reaction. The progress of the reaction was detected by thin layer chromatography spot plate. After the reaction was completed, the pH was adjusted to neutral, extracted three times with ethyl acetate (700 mL), the organic phases were combined, dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by PE:EA=100:1 column chromatography to obtain a white crystalline solid intermediate 1 (yield 700.00 mg, yield 40.56%).

[0062] The intermediate 1 was subjected to H NMR spectrum analysis, and the result was: 1 H NMR (300 MHz, Chloroform-d) δ7.40–7.27 (m, 3H), 7.21–7.12 (m, 2H), 1.85 (s, 6H), the spectrum is shown in 1.

[0063] From the above analysis, it can be seen that the structure of intermediate 1 is as follows:

[0064]

[0065] 2. Synthesis of intermediate compound 2

[0066] The intermediate 1 (255.00 mg, 1 mmol) was added to the reaction flask, and the evacuation was performed for nitrogen protection. 6.00 mL of anhydrous dichloromethane solvent was added to the reaction flask, and the mixture was stirred in an ice bath. Boron trifluoride ether solution (0.731 mL, 2 mmol) was slowly added dropwise with a syringe. After 30 minutes of ice bath reaction, the mixture was reacted at room temperature. The reaction was monitored by TLC spot plate. After the reaction was completed, 10 mL of water was quenched. Dichloromethane (20 mL) was extracted 3 times, and the organic phases were combined and dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by PE:EA=50:1 column chromatography to obtain a white solid intermediate 2 (yield of 230.00 mg, yield of 70.8%).

[0067] The results of H NMR analysis of intermediate 2 are as follows: 1 H NMR (300 MHz, Chloroform-d) δ7.52–7.39 (m, 3H), 7.22–7.14 (m, 2H), 2.11 (s, 6H), the spectrum is shown in Figure 2.

[0068] From the above analysis, it can be seen that the structure of intermediate 2 is as follows:

[0069]

[0070] 3. Synthesis of intermediate compound 3

[0071] Weigh intermediate 2 (112.00 mg, 1 mmmol) and 4-dimethylaminobenzaldehyde (67.00 mg, 1 mmol) and add them to the reaction bottle, evacuate the nitrogen protection, add 4.00 mL of anhydrous ethanol, and stir in an oil bath at 40°C for 5 min. Add 1 mL of anhydrous tetrahydrofuran to a 1 mL EP tube, add 1 drop of piperidine, slowly add the reagent in the EP tube into the reaction system, and continue the reaction at 40°C. Monitor the reaction by TLC spot plate. After the reaction is completed, extract with dichloromethane (20 mL) 3 times, combine the organic phases, dry with anhydrous Na2SO4, concentrate under reduced pressure, and purify by PE:EA=10:1 column chromatography to obtain red solid intermediate 3 (yield 73.00 mg, yield 41.1%).

[0072] The intermediate 3 was analyzed by nuclear magnetic hydrogen spectrum, and the result was: 1 H NMR (300 MHz, Chloroform-d) δ8.08 (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). The spectrum is shown in 3.

[0073] From the above analysis, it can be seen that 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), and then Na2CO3 solution (2 mol / mL, 6 mL) was added. After degassing, Pd(PPh3)4 (165.00 mg, 0.4 mmol) was added. The reactants were stirred at 85°C for 12 hours. The reaction was monitored by TLC spot plate. After the reaction was completed, dichloromethane (20 mL) was extracted 3 times, the organic phases were combined and dried over 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 subjected to H NMR spectrum analysis, and the result was: 1 H NMR (400 MHz, Chloroform-d) δ10.01 (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 is shown in Figure 4.

[0078] From the above analysis, it can be seen that the structure of intermediate 4 is as follows:

[0079]

[0080] 5. Synthesis of intermediate compound 5

[0081] Weigh intermediate 3 (100.00 mg, 1 mmol) and intermediate 4 (151.00 mg, 1 mmol), add to the reaction bottle, evacuate for nitrogen protection, add 5.00 ml of anhydrous ethanol solvent, and stir at room temperature. Add 1.00 mL of anhydrous ethanol to the EP tube, add 1 drop of piperidine, and slowly drip the reagent in the EP tube into the reaction bottle. Move the reaction system into an oil bath pot, react at 80 ° C, and monitor the reaction with a TLC spot plate. After the reaction is completed, extract with dichloromethane (20 mL) 3 times, combine the organic phases, dry with anhydrous Na2SO4, concentrate under reduced pressure, and purify by column chromatography to obtain a dark blue solid intermediate 5 (yield 70.00 mg, yield 46.4%).

[0082] The intermediate 5 was subjected to H NMR spectrum analysis, and the result was: 1 H NMR (400 MHz, Chloroform-d) δ8.43 (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 is shown in 5.

[0083] From the above analysis, it can be seen that the structure of intermediate 5 is as follows:

[0084]

[0085] 6. Synthesis of compound ADBF

[0086] Weigh the intermediate compound 5 (200 mg, 1 mmol) and methylglyoxal (161.00 mg, 3 mmol) and add them to the reaction bottle, add 3 mL of anhydrous ethanol, connect the three-way, evacuate the system for nitrogen protection, and stir at room temperature for 5 minutes. Slowly drop 400 μL of 6 mol / mL HCl into the reaction system and stir for 5 to 10 minutes. Move the reaction bottle into an oil bath at 80 ° C for 10-12 hours. After the reaction is completed, cool to room temperature, add EA to form a precipitate, filter, wash, and vacuum dry to obtain a dark solid, which is the chemiluminescent probe ADBF (yield 50 mg, yield 22.7%).

[0087] The chemiluminescent probe ADBF was analyzed by hydrogen nuclear magnetic spectrum, carbon nuclear magnetic spectrum and mass spectrometry. The results were: 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). The spectrum is shown in Figure 6.

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

[0089] From the above analysis, it can be seen that the structure of the chemiluminescent probe ADBF is as follows:

[0090]

[0091] Example 2 Determination of chemiluminescent wavelength of chemiluminescent probe ADBF

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

[0093] Example 3 Selectivity of chemiluminescent probe ADBF

[0094] In order to explore the ability of the chemiluminescent probe ADBF to detect Aβ oligomers in vivo, the chemiluminescent probe ADBF prepared in Example 1 was prepared into a 10 mmol / mL standard stock solution with DMSO, and then diluted to 5 μmol / mL with PBS to obtain a chemiluminescent probe ADBF standard solution. Preparation of Aβ monomers: 0.25 mg of Aβ protein treated with trifluoroacetate was dissolved in 100 μL of hexafluoroisopropanol, and 900 μL of DDH2O was added. Note that it should be prepared and used immediately. Preparation of Aβ oligomers: 0.25 mg of Aβ protein treated with hexafluoroisopropanol was dissolved in 100 μL of hexafluoroisopropanol, and 900 μL of DDH2O was added. It was shaken continuously for 24 hours at a constant shaking condition of 22°C and 100 rpm. Preparation of Aβ aggregates: 0.25 mg of trifluoroacetate-treated Aβ protein was dissolved in 1000 μL buffer solution (DDH2O containing 10 mmol / mL NaH2PO4, 1 mmol / mL EDTA), and the solution was shaken continuously for 48 hours at 37°C and 120 rpm. First, the fluorescence curve of the PBS buffer solution was measured using an F-47000 fluorescence spectrophotometer (slit 10 / 10, response 0.5s, PMT 700V). Then, 1900 μL of the chemiluminescent probe ADBF standard solution (5 μmol / mL) was taken with a pipette, the fluorescence curve was measured, and then 100 μL of Aβ monomer, Aβ oligomer, and Aβ aggregate solution (the solution concentration was 50 μmol / mL) was added to measure the fluorescence response. The calculation formula for the fluorescence enhancement factor is as follows: FL enhancement = (FL Test-FL PBS ) / (FL probe -FL PBS ), where FL enhancement is the fluorescence enhancement factor, 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. Fig. 9 shown.

[0095] Fig. 9 The fluorescence spectra of the chemiluminescent probe ADBF in Example 3 after binding to Aβ monomer, Aβ oligomer and Aβ aggregate respectively; wherein, 5uM probe (red curve) is the fluorescence image of 5μmol / mL pure probe ADBF standard solution, 5uMprobe+2.5uM monomer (black curve) is the fluorescence image of the mixed solution of 5μmol / mL probe ADBF+2.5μmol / mAβ monomer, 5uM probe+2.5uM aggergation (purple curve) is the fluorescence image of the mixed solution of 5μmol / mL probe ADBF+2.5μmol / mAβ aggregate, 5uM probe+2.5uM oligomer (green curve) is the fluorescence image of the mixed solution of 5μmol / mL probe ADBF+2.5μmol / mAβ oligomer. Fig. 9 The results showed that the selectivity of the chemiluminescent probe ADBF for Aβ oligomers is significantly better than that for Aβ monomers and Aβ aggregates, indicating that the probe has good targeting and can be effectively used for Aβ oligomer imaging in animals. This discovery confirms the potential of the compound ADBF in terms of 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] Fig.10 This is the fluorescence intensity quantification result of the selectivity of compound ADBF for Aβ oligomers. Fig.10 Quantitative analysis showed that the fluorescence intensity of the chemiluminescent probe ADBF increased 16 times after binding to Aβ oligomers and about 10 times after binding to Aβ aggregates, and a blue shift occurred in the maximum absorption peak.

[0097] Example 4 Cytotoxicity test of chemiluminescent probe ADBF

[0098] In order to verify the biocompatibility of the needle chemiluminescent probe ADBF for Aβ oligomers at the cellular level, the standard CCK-8 experimental protocol was followed and the neuronal cell type closely related to Alzheimer's disease, PC12 cells, was selected to evaluate the in vitro cytotoxicity of the chemiluminescent probe ADBF. PC12 cells were resuspended and plated in a 96-well plate, with 4000 cells per well. After 24 hours of culture in a 37°C, 5% CO2 constant temperature incubator, the supernatant in the well was aspirated, and the high-glucose DMEM culture medium of the chemiluminescent probe ADBF was added (the concentrations of ADBF were 0μM, 5μM, 10μM, 15μM, 20μM, 25μM, 30μM, 35μM, and 40μM, respectively), and then placed in the incubator for continued culture. After 24 hours, the supernatant was aspirated again, and 100μL of incomplete DMEM (high glucose) medium containing 10% CCK8 was added, and incubated in a 37°C, 5% CO2 constant temperature incubator for 2 hours. The absorbance at 450 nm was measured using an enzyme reader to calculate the cytotoxicity of the chemiluminescent probe ADBF. Each experiment was repeated 3 times.

[0099] The cell growth activity was calculated using the following formula: Activity (%) = (average absorbance of test wells - average absorbance of culture medium control wells) / (average absorbance of untreated wells - average absorbance of culture medium control wells) × 100%. The results are shown in Fig.11 Place. Fig.11 This is a diagram showing the results of the cytotoxicity evaluation experiment in Example 4. Fig.11 The results showed that the chemiluminescent probe ADBF showed low toxicity to neuroblastoma PC12 cells. In PC12 cells, the survival rate exceeded 85%, which showed that the chemiluminescent probe ADBF had 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 oxygenation efficacy of the compound ADBF of the present invention

[0101] Photooxygenation of Aβ can be performed in the presence of cells by using a selective catalyst riboflavin attached to an Aβ binding peptide and irradiated with 500nm visible light. Compared with native Aβ, oxygenated Aβ exhibits significantly lower aggregation efficacy and neurotoxicity. The present invention uses riboflavin as a control to evaluate the photooxygenation efficacy of the compound ADBF.

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

[0103] Fig.12 The results showed that the viability of PC12 cells treated with 10 μmol / mL Aβ oligomers alone was only 50%, while about 80% of the cells survived after co-incubation with 10 μmol / mL Aβ oligomers and compound ADBF, and whether or not light was irradiated had little effect on the viability of cells co-incubated with compound ADBF, indicating that compound ADBF has the efficacy of oxygenation therapy and does not require light activation.

[0104] Compound ADBF has the effect of oxygenation therapy. In order to verify whether the oxygenation therapy of compound ADBF is concentration-dependent, the following experimental groups were set up: ① Blank group (black): nothing was added, only DMEM medium. ② Aβ oligomer treatment group (oligomer): 10 μmol / mL 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 Aβ oligomer and 0.5 μmol / mL 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 Aβ oligomer and 3 μmol / mL 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 Aβ oligomer and 6 μmol / mL compound ADBF were added to each well at the same time. The concentrations set in the above groups are the final concentrations in each well. Fig.13 As shown in the results, the concentration dependence of oxygenated Aβ was observed (54.0% activity at 0.5 μmol / mL, 72.3% at 3 μmol / mL, and 83.1% at 6 μmol / mL), which inhibited the cytotoxicity of Aβ oligomers. Therefore, co-treatment of Aβ oligomers with the compound ADBF significantly inhibited the aggregation efficiency and cytotoxicity of Aβ oligomers.

[0105] APP / PSI transgenic AD mice (APP) and control wild-type mice (WT) of different ages were subjected to local head hair removal and anesthetized with 10% chloral hydrate (4 μL / g) by intraperitoneal injection. We injected the chemiluminescent probe ADBF at a dose of 4 mg / Kg (10% DMSO, 15% Tween20, 75% PBS) (the specific dosage was calculated based on the body weight of each mouse) into the APP / PS1 transgenic AD mouse model (APP) and control wild-type mice (WT) through the tail vein, and then imaged the mouse brains with a small animal in vivo imaging system (IVIS LumiaⅡ), and collected the chemiluminescent signals of the mouse brains before and at different time points after the probe injection. The results are shown in the figure. Fig.14 As shown in the figure, 1 minute after the probe ADBF was injected into the mouse, both APP mice and WT mice produced enhanced chemiluminescence signals, which reached a maximum at the 10th minute and then gradually decreased. The chemiluminescence intensity of the mouse brain was quantitatively analyzed using the living Image analysis software corresponding to the small animal living imaging system. Fig.15Quantitative analysis showed that the chemiluminescence intensity of APP mice was 1.74 times that of WT mice at the first minute, and reached 1.84 times at the 15th minute. The results of in vivo imaging showed that the probe could effectively pass through the blood-brain barrier and enter the mouse brain to bind to Aβ, thereby producing a significant enhancement of the chemiluminescence signal, which could clearly distinguish APP mice from WT mice.

Claims

1. A chemiluminescent probe targeting Aβ oligomers, characterized in that: The general structural formula of the chemiluminescent probe targeting Aβ oligomers is shown in I: Where n1 is 1 or 2; n2 is 1 or 2; A is B1 is H, B2 is H, R is 2. The chemiluminescent probe targeting Aβ oligomers according to claim 1, characterized in that: The chemiluminescent probe targeting Aβ oligomers is:

3. A method for preparing a chemiluminescent probe targeting Aβ oligomers according to claim 1 or 2, characterized in that: The following synthetic routes are included: Where n1 is 1 or 2; n2 is 1 or 2; A is B1 is H, B2 is H, R is 4. The preparation method according to claim 3, characterized in that: Synthesized according to the following route:

5. The preparation method according to claim 3, characterized in that: The following steps are involved: (1) Synthesis of intermediate compound 1: Anhydrous potassium carbonate, L-proline, cuprous iodide and acetylacetone were dissolved in dimethyl sulfoxide, reacted under nitrogen protection, iodobenzene was slowly added, the reaction was continued, and the mixture was extracted with ethyl acetate, dried, concentrated and purified by column chromatography to obtain intermediate 1; (2) Synthesis of intermediate 2: intermediate 1 was added to anhydrous dichloromethane solvent, stirred in an ice bath under nitrogen protection, and a boron trifluoride ether solution was slowly added. The reaction was carried out in an ice bath under nitrogen protection, and then reacted at room temperature. Water was added to quench the reaction, and the mixture was extracted with dichloromethane. The mixture was dried, filtered, concentrated, and purified by column chromatography to obtain intermediate 2. (3) Synthesis of intermediate compound 3: intermediate 2 and 4-dimethylaminobenzaldehyde were added to anhydrous ethanol, stirred at room temperature, and piperidine in anhydrous tetrahydrofuran was slowly added, reacted in an oil bath, extracted with dichloromethane, 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 were dissolved in a mixture of toluene and ethanol, and Na2CO3 was added. After degassing, Pd(PPh3)4 was added, and the mixture was heated for reaction. The mixture was extracted with dichloromethane, dried, concentrated, and purified by column chromatography to obtain intermediate 4. (5) Synthesis of intermediate compound 5: Intermediate 3 and intermediate 4 were added to anhydrous ethanol, and an anhydrous ethanol solution of piperidine was added, and the reaction was heated in an oil bath under nitrogen protection, extracted with dichloromethane, dried, concentrated, and purified by column chromatography to obtain intermediate 5; (6) Synthesis of chemiluminescent probe ADBF: The intermediate compound 5 and acetone aldehyde were added to anhydrous ethanol, evacuated for nitrogen protection, stirred at room temperature, HCl was slowly added, stirring was continued, and the reaction was heated in an oil bath. The reaction was cooled to room temperature, EA was added to form a precipitate, filtered, washed, and dried in vacuum to obtain the chemiluminescent probe ADBF.

6. The preparation method according to claim 5, characterized in that: In step (1), the reaction temperature under nitrogen protection is 80-100° C., and the reaction time is 12-18 h. In step (2), the reaction time in an ice bath is 30-60 min, and the reaction time at room temperature is 6-8 h.

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

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

9. Use of the chemiluminescent probe targeting Aβ oligomers according to claim 1 or 2 in the preparation of a drug for alleviating or treating Alzheimer's disease.

10. The use according to claim 9, characterized in that: The dosage form of the drug is one of pills, ointments, tablets, oral liquids, subcutaneous injections and intravenous injections, and the drug also includes stabilizers, buffers, cosolvents, emulsifiers, excipients, diluents and / or isotonic agents.

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