A fluorescent probe for detecting tau protein and a preparation method and application thereof

Abstract

The application provides a fluorescent probe for detecting Tau protein and a preparation method and application thereof, the fluorescent probe is a phenothiazine compound with a specific conjugated structure, has characteristic absorption in an ultraviolet-visible light region, produces stable fluorescent emission in a visible light to near infrared wave band, can be selectively combined with Tau protein aggregates and produce a significant fluorescent signal change, thereby realizing high-sensitivity detection and pathological evaluation of Tau protein abnormal deposition.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a fluorescent probe for detecting Tau protein, its preparation method, and its application. Background Technology

[0002] Tau proteins are microtubule-associated proteins that play a crucial role in maintaining neuronal cytoskeleton stability and axonal transport. In various neurodegenerative diseases, Tau proteins undergo abnormal phosphorylation and further aggregate to form neurofibrillary tangles (NFTs), leading to neuronal dysfunction and cognitive impairment. Therefore, abnormal Tau protein deposition is considered one of the important pathological features of diseases such as Alzheimer's disease, frontotemporal degeneration, and progressive supranuclear palsy.

[0003] Early identification and dynamic monitoring of abnormal Tau protein deposition are crucial for the early diagnosis, disease progression assessment, and treatment optimization of neurodegenerative diseases. Currently, Tau protein detection methods mainly include immunological detection, biochemical analysis, and nuclear medicine imaging; however, these methods generally suffer from limitations such as complex operation, high cost, or difficulty in achieving real-time in vivo imaging. In recent years, small-molecule fluorescent probes have attracted widespread attention in Tau protein imaging research due to their advantages of tunable structure, simple synthesis, and high imaging sensitivity. However, existing probes still generally suffer from problems such as short fluorescence emission wavelengths, limited tissue penetration, insufficient selectivity for Tau proteins, and the need for improvement in photostability and adaptability to physiological conditions, making it difficult to simultaneously meet the multi-level application requirements of in vitro detection, cell imaging, and in vivo brain imaging. Summary of the Invention

[0004] The purpose of this invention is to provide a fluorescent probe for detecting Tau protein, its preparation method, and its application, so as to solve the problems existing in the prior art and achieve highly sensitive detection and pathological assessment of abnormal Tau protein deposition.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention provides a fluorescent probe for detecting Tau protein. This fluorescent probe is a phenothiazine compound with a specific conjugated structure, exhibiting characteristic absorption in the ultraviolet-visible region and stable fluorescence emission in the visible to near-infrared band. It can selectively bind to Tau protein aggregates and produce significant changes in fluorescence signal, thereby enabling the identification and imaging of abnormal Tau protein deposition. The structural formula of the fluorescent probe is as follows: , or ; in: R2 is -CH3, -CH2CH2OH, -CH2CH2NH2, -CH2CH2NHCH3 or -CH2COOH; X is a pharmaceutically acceptable anion; n is selected from 0, 1, or 2; The Ar is selected from the following structural formula: .

[0006] Preferably, the fluorescent probe for detecting Tau protein has one of the following structural formulas: Compound (1); Compound (2); Compound (3); Compound (4); Compound (5).

[0007] In this invention, different Ar molecules exhibit different specificities for binding to tau protein. For example, if the compounds have structural formulas (1) and (2), the binding site between the compounds and tau protein aggregates (tau protein) is mainly the hydrophobic core region of the β-sheet of tau protein filaments. This region is a characteristic structural domain of neurofibrillary tangles (NFTs) formed by the abnormal aggregation of tau protein. This binding mode allows the probe to form a precise spatial match with the hydrophobic pocket of tau protein. At the same time, by means of π-π conjugation, hydrophobic interaction, and hydrogen bonding, the binding affinity of the probe to tau protein aggregates is significantly improved, and the dissociation constant (Kd) can reach the micromolar level. It also effectively avoids non-specific binding to other misfolded proteins such as Aβ fibers, achieving highly selective recognition of tau protein.

[0008] If the structure of the fluorescent probe used to detect Tau protein is compound (3), the binding sites of the fluorescent probe to Tau protein are mainly the hydrophilic sites on the surface of Tau protein aggregates and side chain amino acid residues, and the binding interaction is mainly electrostatic interaction and weak hydrophobic interaction. In this binding mode, the probe can quickly bind to Tau protein aggregates and generate a fluorescent signal response, with faster binding kinetics, which is suitable for rapid in vivo imaging detection of abnormal Tau protein deposition. However, the binding affinity is slightly lower than that of quinoline probes with compounds (1) or (2) mentioned above, and the fluorescence enhancement factor is relatively mild.

[0009] When the structure of the fluorescent probe used to detect Tau protein is compounds (4) and (5), the binding sites of the fluorescent probe to Tau protein aggregates are mainly the helical domains of the Tau protein aggregates and the regions surrounding the phosphorylation sites. The heterocyclic structure of the fluorine-boron group can coordinate with the phosphate group of the Tau protein phosphorylation site. At the same time, the electronegativity of the fluorine atom can regulate the electron cloud distribution of the probe and enhance the electrostatic interaction with Tau protein. This binding mode enables the fluorescent probe to have targeted recognition characteristics for phosphorylated Tau protein aggregates, which can specifically distinguish between normal Tau protein and abnormally phosphorylated Tau protein. It is suitable for the study of pathological mechanisms related to Tau protein phosphorylation modification. In addition, the introduction of the fluorine-boron group can further redshift the fluorescence emission wavelength of the probe, improving the tissue penetration ability of in vivo imaging.

[0010] A second aspect of the present invention provides a method for preparing a fluorescent probe for detecting Tau protein as described above, comprising the following steps: (1) Dissolve the first compound in an organic solvent to obtain a mixed solution; (2) Add piperidine and the second compound to the mixed solution to remove the organic solvent from the product obtained from the reaction, and obtain the crude product; (3) The crude product was separated and purified by column chromatography to obtain the fluorescent probe; The first compound is: (Formula A); (Formula B); or, (Formula C).

[0011] The structural formula of the second compound is as follows: (Formula D) Where n is selected from 0, 1, or 2.

[0012] Preferably, when the first compound is of formula A or formula B, the reaction temperature in step (2) is 25°C-50°C and the reaction time is 2-12 hours. When the first compound is of formula C, the reaction temperature in step (2) is 80-120°C and the reaction time is 2-12 hours.

[0013] Preferably, the molar ratio of the first compound to the second compound is 1:1-2, more preferably 1:1.2. Specifically, when the amount of the first compound is 1 mmol, the amount of the second compound is 1.2 mmol, and the amount of piperidine added is 30–120 μL.

[0014] Preferably, the organic solvent includes methanol or toluene.

[0015] Preferably, the developing agent used in step (3) includes dichloromethane-methanol and petroleum ether-ethyl acetate.

[0016] Preferably, the volume ratio of dichloromethane to methanol is 50:1–10:1, and the volume ratio of petroleum ether to ethyl acetate is 5:1–1:1.

[0017] A third aspect of the invention provides the use of the fluorescent probe for detecting Tau protein as described above in the preparation of medicaments for detecting diseases of abnormal Tau protein deposition, such as in the preparation of reagents or formulations for the diagnosis, detection, or imaging of diseases related to abnormal Tau protein deposition. The diseases of abnormal Tau protein deposition are selected from: Alzheimer's disease, frontotemporal degeneration, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal ganglia degeneration, or Pick's disease. This specific phenothiazine derivative, after isotope labeling, is used for nuclear medicine imaging, including PET imaging (positron emission tomography) or SPECT imaging (single-photon emission computed tomography).

[0018] The present invention discloses the following technical effects: This invention provides a fluorescent probe for detecting Tau protein, its preparation method, and its applications. The purpose of this invention is to provide a small molecule probe with near-infrared fluorescence properties that can selectively target Tau protein aggregates, along with its preparation method and uses, to achieve highly sensitive detection and pathological assessment of abnormal Tau protein deposition. This fluorescent probe is a small molecule Tau protein-targeting fluorescent probe that combines high Tau protein selectivity and affinity, good optical stability, biocompatibility, and blood-brain barrier penetration capability, which is of great significance for molecular imaging, diagnosis, and disease assessment of neurodegenerative diseases. Specifically, the fluorescent probe is an organic small molecule with an extended conjugated structure, exhibiting characteristic absorption in the ultraviolet-visible region and fluorescence emission in the visible to near-infrared region, demonstrating good optical stability. The probe can selectively bind to Tau protein aggregates and produce significant fluorescence signal changes for the identification of abnormal Tau protein deposition. Its preparation method is simple and mild, suitable for large-scale preparation. The prepared probe can penetrate the blood-brain barrier and is suitable for Tau protein imaging at the cellular and in vivo levels, and can be used for auxiliary diagnosis, model evaluation, and drug screening of related diseases. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The UV and fluorescence spectra of the fluorescent probe compounds of Examples 1 to 5 of this invention in PBS buffer solution are shown. Figure 2 This is a diagram showing the binding ability of the fluorescent probe compound PBDM from Example 1 of the present invention to different proteins and amino acids; Figure 3 The cytotoxicity of the fluorescent probe compound PBDM in Example 1 of this invention; Figure 4 This is a cell fluorescence imaging experiment diagram of the fluorescent probe compound PBDM in Example 1 of the present invention; Figure 5 This is a graph showing the blood-brain barrier penetration ability of the fluorescent probe compound PBDM in Example 1 of the present invention. Figure 6 This study investigates the neurobehavioral pharmacodynamics of the fluorescent probe compound PBDM, as described in Example 1 of this invention. Detailed Implementation

[0021] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention. It should be understood that the terminology used herein is merely for describing particular embodiments and is not intended to limit the invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of the invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] Example 1 Reaction conditions: (a) N-bromosuccinimide, 0 °C; (b) 2-(2-methoxyethoxy)ethyl-4-methylbenzenesulfonate; (c) ultra-dry THF, n-BuLi, N-formylmorpholine; (d) 18-crown ether-6, potassium carbonate, [(1,3-dioxolane-2-yl)methyl]triphenylphosphine bromide; (e) piperidine, compound 1, 50 °C; (f) acetonitrile, iodomethane, 80 °C.

[0024] Compound 1: 4-Methylquinoline (0.66 g, 4.65 mmol) and iodomethane (1.32 g, 9.3 mmol) were dissolved in acetonitrile (30 mL), and the mixture was heated under reflux overnight in a sealed tube. After cooling to room temperature, the acetonitrile was removed under reduced pressure. Anhydrous acetone was added to the residue, and the mixture was filtered. The resulting solid was washed with acetone and dried to give compound 1 (1.1 g) as a yellow solid, in 94% yield.

[0025] Synthesis of 3-bromo-10-H-phenothiazine (Compound 2): Phenothiazine (2.00 g, 10 mmol) was dissolved in 100 mL of tetrahydrofuran and cooled to 0 °C in an ice-water bath. N-bromosuccinimide (1.78 g, 10 mmol) was slowly added dropwise. After the reaction was complete, the reaction was quenched with saturated sodium chloride solution. The resulting mixture was extracted three times with ethyl acetate and dried over anhydrous sodium sulfate to give the greenish-blue solid product, Compound 2, in 83% yield.

[0026] Synthesis of 10-[2-(2-methoxyethoxy)ethyl]-10H-phenothiazine-3-bromo (Compound 3): 3-bromo-10-H-phenothiazine (2.78 g, 10 mmol), 2-(2-methoxyethoxy)ethyl-4-methylbenzenesulfonate (10 mmol), and potassium carbonate (2.76 g, 20 mmol) were dissolved in 50 mL of tetrahydrofuran. The mixture was refluxed overnight, cooled to room temperature, and the tetrahydrofuran was removed under reduced pressure. Anhydrous acetone was added to the residue, and the mixture was filtered. The resulting solid was washed with acetone and dried to give a yellow solid (2.1 g), yield 68%.

[0027] Synthesis of 10-[2-(2-methoxyethoxy)ethyl]-10H-phenthiazine-3-carboxaldehyde (compound 4): Compound 3 (1.42 g, 4.3 mmol) was dissolved in 30 mL of ultra-dry tetrahydrofuran and cooled to -80 °C. After maintaining this temperature for 10 minutes, n-butyllithium solution (4.00 mL, 6.4 mmol) was added dropwise. The reaction was allowed to proceed for 40 minutes, and then slowly raised to room temperature. N-formylmorpholine (1.00 mL, 8.6 mmol) was added, and the reaction was allowed to proceed at room temperature for 2 hours. The reaction was quenched with saturated sodium chloride solution, diluted with water, and extracted three times with ethyl acetate. After drying with anhydrous sodium sulfate, the collected organic phases were combined, the solvent was evaporated, and the mixture was separated by silica gel column chromatography. Elution was performed using ethyl acetate and petroleum ether in a volume ratio of 1:2 to give a white solid in 45% yield.

[0028] Synthesis of (E)-3-(10-(2-(2-methoxyethoxy)ethyl)-10H-phenthiazin-3-yl) acrolein (compound 5): Compound 4 (442.00 mg, 1.34 mmol) was dissolved in 10 mL of dichloromethane, and [(1,3-dioxolane-2-yl)methyl]triphenylphosphine bromide (862.00 mg, 2.01 mmol), 18-crown ether-6 (389.00 mg, 1.47 mmol), and 10 mL of saturated potassium carbonate solution were added. The reaction was carried out at 65 °C for 24 hours. A 1:2 mixture of 10% hydrochloric acid:tetrahydrofuran was added and stirred at room temperature for 2 hours. The solution was then neutralized with 10% sodium hydroxide to pH 7. The product was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, and the organic phases were combined and collected. After evaporation of the solution, the product was separated by silica gel column chromatography. Eluent was prepared by ethyl acetate and petroleum ether in a volume ratio of 1:1 to obtain an orange-yellow liquid product with a yield of 63%.

[0029] Synthesis of 4-[(1E,3E)-4-[10-[2-(2-methoxyethoxy)ethyl]-10H-phenthiazin-3-yl]but-1,3-dien-1-yl]-1-methylquinoline-1-onium compound (PBDM): Compound 5 (601.00 mL, 1.79 mmol) and 1,4-dimethylquinolineonium iodide (compound 1, 256.00 mg, 1.79 mmol) were dissolved in 15 mL of methanol, and piperidine (100 μL, 1.0 mmol) was added. The reaction was carried out overnight at 50 °C. After evaporation of ethanol, the product was separated by silica gel column chromatography. Elution was performed using a 1:10 (v / v) methanol and dichloromethane solution to give a red solid product in 77% yield.

[0030] Example 2: Reaction conditions: (g) acetonitrile, iodine ethanol 80℃; (h) ethanol, 50℃.

[0031] Compound 4 (0.26 g, 0.8 mmol), compound 6 (0.24 g, 1.1 mmol), and piperidine (0.1 mL) were dissolved in ethanol (40 mL), and the mixture was heated under reflux at 50 °C overnight. After cooling to room temperature, the ethanol was evaporated, and the mixture was separated by silica gel column chromatography using a 1:10 (v / v) methanol and dichloromethane solution to give the red solid product PLOH in 73% yield.

[0032] Example 3: The synthesis steps of compound O-PBDM are as follows: Reaction conditions: (f) Acetonitrile, iodomethane, 80℃; (h) Ethanol, 50℃.

[0033] Synthesis of 1-[(1E,3E)-4-[10-[2-(2-methoxyethoxy)ethyl]-10H-phenthiazin-3-yl]but-1,3-dien-1-yl]-2-methylisoquinoline-2-onium iodide (O-PBDM).

[0034] Compound 7: 2-Methylisoquinoline and iodomethane were dissolved in acetonitrile (30 mL), and the mixture was heated under reflux overnight in a sealed tube. After cooling to room temperature, the acetonitrile was removed under reduced pressure. Anhydrous acetone was added to the residue, and the mixture was filtered. The resulting solid was washed with acetone and dried to give compound 7 in 97% yield. Compound 5 (556.00 mg, 1.79 mmol) and compound 7 were dissolved in 15 mL of ethanol, and piperidine (100 μL, 1.0 mmol) was added. The mixture was reacted overnight at 50 °C. After evaporating the ethanol, the mixture was separated by silica gel column chromatography. Eluent was obtained by a 1:10 mixture of methanol and dichloromethane to give the solid product in 70% yield.

[0035] Example 4: The synthetic steps of compound PBF are as follows: Reaction conditions: (i) Phosphorus oxychloride, CH2Cl2, boron trifluoride ether, N,N-diisopropylethylamine, room temperature; (j) Piperidine, acetic acid, toluene, reflux overnight.

[0036] Compound 8: 2,4-Dimethylpyrrole (1.00 mL, 10 mmol) and 2-pyrrolecarboxaldehyde (1.95 g, 10 mmol) were dissolved in 15 mL of anhydrous dichloromethane and cooled to 0°C in an ice-water bath under nitrogen protection. Phosphorus oxychloride (0.85 mL, 10 mmol) was slowly added dropwise. After the addition was complete, the reaction was maintained in an ice-water bath for 3 h, and then the temperature was raised to room temperature and the reaction was continued for another 3 h. After the reaction was completed, the reaction solution was slowly poured into ice water to quench it, extracted with dichloromethane (3 × 15 mL), the organic phases were combined, washed with ice water, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain the crude condensation intermediate. The crude product was dissolved in 15 mL of anhydrous dichloromethane. Under nitrogen protection, N,N-diisopropylethylamine (DIPEA, 1.74 mL, 10 mmol) was added, and the mixture was stirred until homogeneous. Then, boron trifluoride diethyl ether (BF3.Et2O, 1.26 mL, 10 mmol) was slowly added dropwise, and the reaction was stirred at room temperature for 2 h. After removing the solvent under reduced pressure, the residue was separated by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:5, v / v) to give a black-green solid product in 95% yield.

[0037] (E)-3-[2-(5,5-difluoro-1-methyl-5H-4λ] 4 ,5λ 4 Synthesis of 1,3,2-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborhexane-3-yl)vinyl]-10-[2-(2-methoxyethoxy)ethyl]-10H-phenthiazine (PBF): Compound 4 (600.00 mg, 1.7 mmol) and compound 8 (212.00 mg, 0.95 mmol) were dissolved in 20 mL of toluene, and 0.3 mL each of piperidine and acetic acid were added. The reaction was carried out at 120 °C for 2 hours. The reaction was quenched with saturated sodium chloride solution. The mixture was extracted three times with ethyl acetate and dried over anhydrous sodium sulfate. The collected organic phases were combined, the solution was evaporated, and the mixture was separated by silica gel column chromatography. Eluent was prepared by ethyl acetate and petroleum ether in a volume ratio of 1:2 to give a black solid product with a yield of 85%.

[0038] Example 5: The synthesis steps of compound PDBF are as follows: Reaction conditions: (j) piperidine, acetic acid, toluene, reflux overnight.

[0039] (1E,3E)-3-[4-(5,5-difluoro-1-methyl-5H-4λ] 4 ,5λ 4Synthesis of 1,3,2-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborhexane-3-yl)but-1,3-dien-1-yl]-10-[2-(2-methoxyethoxy)ethyl]-10H-phenthiazine (PDBF).

[0040] Compound 8 (194.00 mg, 0.55 mmol) and compound 5 (80.00 mg, 0.36 mmol) were dissolved in 10 mL of toluene, and 0.2 mL each of piperidine and acetic acid were added. The mixture was reacted at 120 °C for 2 hours, and the reaction was quenched with saturated sodium chloride solution. The mixture was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, and the collected organic phases were combined. After evaporation of the solution, the mixture was separated by silica gel column chromatography. Eluent was prepared by ethyl acetate and petroleum ether in a volume ratio of 1:2 to give a black solid product with a yield of 90%.

[0041] Example 6: Determination of the optical properties of the compound I. Experimental Procedure: Absorption spectroscopy: The compounds prepared in Examples 1-5 were accurately prepared into 100 μM DMSO solutions and PBS buffer solutions (50 mM, pH=7.4). The UV absorption and fluorescence spectra of the compounds were measured to obtain the maximum absorption wavelength (Aabs, nm). The maximum absorption wavelengths of the compounds are shown in Table 1. Figure 1 The left side shows the UV spectra of each compound in PBS buffer solution. Figure 1 The right side shows the fluorescence spectra of each compound in PBS buffer solution. Figure 1 In the right figure, PBDF is the same as PDBF.

[0042] II. Experimental Results: Table 1 shows that the emission wavelengths of the five compounds red-shift with increasing solvent polarity, which is due to the solvation effect. Furthermore, the maximum emission wavelengths in PBS solution are all greater than or near 650 nm, and they exhibit a large Stokes shift, making them suitable for near-infrared fluorescence imaging.

[0043] In Table 1, a represents the maximum UV absorption peak of the compound in PBS buffer, and b represents the maximum fluorescence emission peak under excitation at the maximum UV absorption wavelength.

[0044] Example 7: In vitro binding experiment of compound PBDM with Tau aggregates I. Experimental Procedure: (1) A 100 nM fluorophore solution (compound PBDM) was mixed with different proteins, amino acids and cations (the final volume of each mixture was 15 μL), and incubated in 25 mM phosphate buffer (pH=7.4) at room temperature for 15 min. The preparation process specifically included: A. Preparation of cation stock solution: Dissolve the corresponding metal salts (such as AlCl3, BaCl3, FeCl3, KCl, MgCl2, NaCl, CuCl2, MnCl2, etc.) in PBS buffer to prepare a high-concentration stock solution (concentration of 100 mmol / L), and filter it through a 0.22 μm filter membrane for sterilization.

[0045] B. Working Solution Dilution: During the experiment, dilute the stock solution with the same buffer to the required working concentration, ensuring that the concentrations of all cations are consistent. Specifically, the concentration of the cation working solution is 10 μmol / L or 100 μmol / L. Additionally, amino acids are not labeled with additional concentrations (equimolar to protein), the fluorophore solution is 100 nM, and Tau filaments / Aβ filaments / BSA are all 100 nM.

[0046] C. System incubation: The mixing ratio is: fluorophore solution: each analyte solution (Tau fiber / Aβ fiber / BSA / amino acid / cation) = 1:1 (volume ratio). The mixing volume of each analyte system is 15 μL. Incubate at room temperature or 37°C for a certain time (e.g., 10-30 minutes), and then perform fluorescence intensity detection.

[0047] (2) The excitation and emission spectra of each test system solution and the blank control solution (PBS solution) were scanned using a fluorescence spectrophotometer to determine the maximum emission wavelength of the compound. At this wavelength, the fluorescence intensity of each system was measured, and the fluorescence enhancement factor of the compound binding to the protein was calculated using the following formula: Fluorescence enhancement factor = FI 探针+Tau / FI 探针 ; FI 探针+Tau FI represents the fluorescence intensity of the compound after binding with Tau fibers. 探针 The fluorescence intensity of the compound in its PBS solution is given.

[0048] II. Experimental Results: The binding affinity of compound PBDM to different proteins, amino acids, and cations is as follows: Figure 2As shown in the figure. The results indicate that the fluorescence enhancement effect of the system is most significant when PBDM is co-incubated with Tau fibers, which is much higher than the fluorescence enhancement level after interaction with Aβ fibers, BSA, various amino acids and different cations. This suggests that the compound has selective binding properties to Tau fibers and its affinity is significantly better than that of other tested proteins and amino acids.

[0049] Example 8: In vitro saturation binding experiment I. Experimental Procedure: To investigate the binding affinity of compound PBDM to Tau fibers, a fluorescence titration method was used. 100 nM PBDM solution was mixed with different concentrations (100 nM, 200 nM, 500 nM, 1 μM, 2 μM, 4 μM, 10 μM, 20 μM, 40 μM, and 100 μM) of Tau fibers (the final volume after mixing was 15 μL for each concentration), and incubated in 25 mM phosphate buffer at room temperature for 15 min. The emission spectra of the mixtures were measured using a fluorescence spectrometer, and the dissociation constant (Kd) was calculated using the saturation method.

[0050] II. Experimental Results: The results of the fluorescence titration experiment showed that the dissociation constant Kd of compound PBDM with Tau fibers was 15 μM, indicating that the probe has a strong specific binding effect with Tau fibers, which is far superior to some existing Tau fluorescent probes (most of which have Kd > 50 μM), and has the potential to be used as a high-affinity Tau protein detection probe. When the Tau fiber concentration was > 40 μM, the fluorescence intensity of the probe reached saturation, indicating that the binding of the probe with Tau fibers is a quantitative and specific binding.

[0051] Example 9: Cytotoxicity Experiment I. Experimental Procedure The SHSY-5Y cells (human neuroblastoma cell line) to be tested were loaded with 5 × 10⁻⁶ cells. 4 Cells were seeded at a density of 1 / mL in 96-well plates, and different concentrations of the test compound PBDM were added. After cell adhesion, the drug was administered, with three replicates for each concentration. After 24 hours of drug treatment, 10 μL of 5 mg / mL MTT (thiazolyl blue) was added to each well, and the cells were cultured for another 4 hours. The supernatant was discarded, and 100 μL of DMSO was added to each well. The cells were shaken for 10 min to dissolve the crystals completely. The absorbance of each well was measured at 570 nm using a microplate reader to calculate the inhibition rate for each drug concentration. Inhibition rate = (1 - drug group / blank control group) × 100%. Compounds without significant cytotoxic activity were selected for further screening.

[0052] II. Experimental Results like Figure 3The curve trend shows that when the concentration of compound PBDM reaches 200 μmol / L (logarithm of compound concentration ≈ -3.7), the cell viability remains above 80%, with no significant cytotoxicity; when the concentration continues to increase, the cell viability does not decrease significantly. Therefore, the median lethal concentration (LC50) of compound PBDM can be determined. 50 The concentration is greater than 200 μmol / L, which is much higher than the actual application concentration of the probe (nM~μM level in cell / in vivo experiments). This proves that the probe has no cytotoxicity at the effective detection concentration, meets the application requirements of molecular probes, and can be directly used for subsequent experimental research in mouse models.

[0053] Example 10: Cell fluorescence imaging experiment I. Experimental Procedure Experiments were performed using standard SHSY-5Y and SHSY-5Y treated with okadaic acid. Cells were cultured in wells at a concentration of 7000 cells / mL for 48 hours. Cells were treated with the probe PBDM for half an hour, then fixed with 4% PFA (paraformaldehyde) for 15 minutes, followed by washing twice with PBS buffer. Cells were then infiltrated with 0.1% PBST for 15 minutes, followed by cell blocking with 1% BSA buffer for 1 hour. Cells were incubated with specific primary antibodies against ThS (thioflavin S) and phosphorylated Tau protein for 2 hours, respectively. After 2 hours, ThS and primary antibodies were removed, cells were washed twice with PBS buffer, and then co-incubated with p-tau secondary antibody for 1 hour. Finally, cells were washed twice with PBS buffer, and coverslips were mounted on glass slides. Intracellular probe activity was monitored using confocal laser scanning microscopy. The experiment was repeated three times.

[0054] II. Experimental Results Figure 4 In the figure, DAPI is the nuclear staining agent, displayed in pseudo-color red. PBDM is red fluorescence, and the primary antibody (p-tau primary antibody) is green fluorescence. The Merge column combines the fluorescence signals of DAPI, PBDM, and the primary antibody to observe the co-localization relationship between PBDM and phosphorylated Tau protein. Figure 4Qualitative observation (Merge plot): In the Merge plot, the red (PBDM) and green (MAPT) fluorescent signals highly overlap (appearing yellow), indicating that the probe (PBDM) and the antibody against phosphorylated Tau protein (MAPT) are spatially highly overlapping, suggesting that the probe can target Tau fibromas within neurons. Quantitative verification (right block analysis plot): The colocalization coefficient R values ​​are all greater than 0.67, with some approaching 0.81, indicating a significant degree of colocalization between PBDM and MAPT, further confirming that the probe can target phosphorylated Tau protein fibromas. Cell integrity confirmation (DAPI): DAPI staining shows clear cell nuclei, excluding interference from anucleate cell debris, ensuring that the observed cells are intact, and improving the reliability of the conclusions. Figure 4 It can be seen that the probe (PBDM) is mainly located in neurons and has a significant co-localization relationship with neuronal structure. The probe can enter the cell and has good specificity. In SH-SY5Y cells treated with okadaic acid, its fluorescence is highly co-localized with the fluorescence of Tau fiber antibody, which can be monitored in real time and specifically bind to Tau fibers.

[0055] Example 11: Determination of blood-brain barrier penetration ability I. Experimental Procedure Transgenic mice (Tau-P301S, 30-40 g) of different ages (3 months, 9 months, 12 months) and wild-type mice (WT, 30-40 g) of corresponding ages were selected and fasted for 12 hours before the experiment. WT and TG mice of each age were randomly divided into groups (I), (II), (III) and a control group, with 5 mice in each group. WT and TG mice were injected intravenously with 100 μL of PBDM fluorescent probe (concentration 5 mg / kg) dissolved in 25 mM phosphate buffer solution. Mice were anesthetized using a mouse anesthesia machine. Direct fluorescence imaging of the mouse brain was performed using a small animal in vivo optical three-dimensional imaging system. Figure 5 Representative images of 6-month-old wild-type (WT) mice are used for comparison and illustration.

[0056] II. Experimental Results like Figure 5 By comparing wild-type (WT) and TG mice at time points of 20 / 40 / 60 / 90 / 120 minutes, the time-dynamic characteristics of compound PBDM's penetration of the blood-brain barrier were observed, indicating that PBDM can penetrate the blood-brain barrier. Comparison of changes in fluorescence signal intensity in the brains of WT and TG mice showed that PBDM can target Tau fibers in transgenic mice (P301S), repeated three times. This demonstrates that compound PBDM can trace changes in Tau fiber aggregation in the in vivo brain.

[0057] Example 12: Pharmacodynamic Study Based on Neurobehavioral Therapy I. Experimental Procedure Ten-month-old tau-overexpressing transgenic mice (P301S, 30-40 g) and age-matched wild-type mice (WT, 30-40 g) were selected to evaluate the neurobehavioral efficacy of compound PBDM. The mice were randomly divided into three groups: a normal group (wild-type), a transgenic group, and a PBDM compound test group (5.0 mg / kg, 10.0 mg / kg), with six mice in each group. After three days of acclimatization, the mice were randomly assigned to groups based on their body weight. The normal control group and the transgenic group received a single intraperitoneal injection of saline. The test compound group received oral administration starting on day 2, once daily for eight weeks. Mouse weight was recorded at the end of the experiment. Learning and memory tests were conducted on the mice, including the Morris water maze, Y-maze, mine test, and platform jump test. The neurobehavioral efficacy was obtained by observing and recording the behavioral responses and memory duration of the mice in the normal group, the treated group, and the transgenic group.

[0058] II. Experimental Results like Figure 6 The results showed that transgenic mice exhibited significant learning and memory impairments and behavioral abnormalities, while the mice in the treatment group showed significant improvement in learning and memory functions and autonomous behavior in a dose-dependent manner. The 10.0 mg / kg dose group showed even better improvement, with all indicators approaching the levels of the normal group. This confirms that the compound PBDM can effectively improve the neurobehavioral abnormalities in Tau-overexpressing transgenic mice and has potential therapeutic intervention effects on Tau-related neurodegenerative diseases, providing key experimental evidence for its application in the treatment of related diseases. Figure 6 The first image in the first row shows a comparison of spontaneous alternation rates in the Y-maze. Spontaneous alternation rate reflects the working memory ability of mice; a higher value indicates better working memory. Grouping and Results: Wild-type control group (WT control): Spontaneous alternation rate remained at a high level. Tau transgenic model control group (Tau control): Spontaneous alternation rate was significantly lower than that of the WT group (…). p<0.0001, indicating that tau overexpression significantly impaired working memory in mice. The spontaneous alternation rate was significantly higher in the 5 mg / kg and 10 mg / kg (PBDM treatment groups) than in the tau control group. (p<0.05), and the effect of the 10 mg / kg group was closer to the WT level, indicating that PBDM can effectively improve working memory deficit in tau mice.

[0059] The second image in the first row shows a comparison of latency in the jumping platform experiment. Latency is the time it takes for mice to jump off the platform. A longer latency indicates a deeper memory of the electric shock and stronger passive memory. Grouping and Results: Wild-type control group (WT control): Longer latency, normal memory. Tau transgenic model control group (Tau control): Significantly shorter latency than the WT group (…). p<0.001 indicates that passive memory in tau mice is significantly impaired. At 5 mg / kg and 10 mg / kg (PBDM treatment groups): the latency was significantly longer than in the tau control group. (p<0.05), and the 10 mg / kg group showed better results, approaching the WT level, indicating that PBDM can improve the passive memory ability of tau mice.

[0060] The third image in the first row shows a comparison of the number of errors in the platform jumping experiment. The number of errors represents the number of times the mice jumped off the platform within the specified time; the fewer the errors, the stronger the memory consolidation ability. Grouping and Results: Wild-type control group (WT control): Fewer errors, normal memory consolidation ability. Tau transgenic model control group (Tau control): Significantly more errors than the WT group (…). p<0.05 indicates that tau mice have poor memory consolidation ability. The 5 mg / kg and 10 mg / kg (PBDM treatment groups) showed significantly fewer errors than the tau control group. (p<0.05), and the 10 mg / kg group was closer to the WT level, indicating that PBDM can reduce memory errors in tau mice and improve memory consolidation ability.

[0061] The first figure in the bottom row shows a comparison of mouse trajectories in the open field experiment. The red lines represent the mouse movement trajectories, and the white boxes represent the central area, visually demonstrating the spatial activity patterns of mice in the open field. Grouping and Results: Wild-type control group (WT control): Trajectories were evenly distributed, covering both the central and peripheral areas, with normal behavioral patterns. Tau transgenic model control group (Tau control): Trajectories were more biased towards the central area, consistent with the result of "prolonged central dwell time" in the Tau control group (the second figure in the bottom row), indicating abnormal behavior. 5 mg / kg and 10 mg / kg (PBDM treatment groups): Trajectories significantly reduced entry into the central area, more closely resembling the behavioral pattern of WT mice, further validating the reversal effect of PBDM on abnormal behavior in tau mice.

[0062] The second figure from the bottom row shows a comparison of dwell time in the central region during the open field experiment. Dwell time in the central region reflects the anxiety level and exploratory behavior of the mice; a longer dwell time generally indicates lower anxiety or enhanced exploratory behavior. Grouping and Results: Wild-type control group (WT control): Dwell time in the central region was within the normal range. Tau transgenic model control group (Tau control): Dwell time in the central region was significantly longer than that of the WT group. p<0.05, suggesting that tau overexpression leads to abnormal behavior in mice, manifested as increased exploration of the central region or altered anxiety levels. At 5 mg / kg and 10 mg / kg (PBDM treatment groups): the central residence time was significantly shorter than in the Tau control group. (p<0.01), and the 10 mg / kg group was closer to the WT level, indicating that PBDM can reverse the abnormal behavior of tau mice, restoring their anxiety level and exploration pattern to a state close to wild type.

[0063] therefore, Figure 6 Overall results of the behavioral experiments showed that tau overexpression led to impaired working memory, decreased passive memory, and abnormal behavioral patterns in mice; while the compound PBDM (especially at a dose of 10 mg / kg) could significantly improve these defects, restoring the behavioral performance of tau mice to levels close to those of wild-type mice.

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A fluorescent probe for detecting Tau protein, characterized in that, The structural formula of the fluorescent probe is as follows: , or ; in: R2 is -CH3, -CH2CH2OH, -CH2CH2NH2, -CH2CH2NHCH3 or -CH2COOH; X is a pharmaceutically acceptable anion; n is selected from 0, 1, or 2; The Ar is selected from one of the following structural formulas: 。 2. The fluorescent probe for detecting Tau protein as described in claim 1, characterized in that, The fluorescent probe has one of the following structural formulas: 、 、 、 、 。 3. A method for preparing a fluorescent probe for detecting Tau protein as described in any one of claims 1-2, characterized in that, Includes the following steps: (1) Dissolve the first compound in an organic solvent to obtain a mixed solution; (2) Add piperidine and the second compound to the mixed solution to react and obtain the crude product; (3) The crude product was separated and purified by column chromatography to obtain the fluorescent probe; The first compound is one of the following structural formulas: ; Where R2 is -CH3, -CH2CH2OH, -CH2CH2NH2, -CH2CH2NHCH3 or -CH2COOH; The structural formula of the second compound is as follows: ; Where n is selected from 0, 1, or 2.

4. The method for preparing a fluorescent probe for detecting Tau protein as described in claim 3, characterized in that, The first compound is or hour, The reaction temperature in step (2) is 25℃ - 50℃; If the first compound is hour, The reaction temperature in step (2) is 80-120℃.

5. The method for preparing a fluorescent probe for detecting Tau protein as described in claim 3, characterized in that, The molar ratio of the first compound to the second compound is 1:1-2.

6. The method for preparing a fluorescent probe for detecting Tau protein as described in claim 3, characterized in that, The developing solvent used in the column chromatography in step (3) includes: dichloromethane-methanol and / or petroleum ether-ethyl acetate.

7. The method for preparing a fluorescent probe for detecting Tau protein as described in claim 6, characterized in that, The volume ratio of dichloromethane to methanol is 50:1–10:1, and the volume ratio of petroleum ether to ethyl acetate is 5:1–1:

1.

8. The use of the fluorescent probe for detecting Tau protein as described in any one of claims 1-2 in the preparation of a medicament for detecting diseases caused by abnormal deposition of Tau protein.

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