Quinoxaline Fluorescent Probes, Methods and Applications

Abstract

The present invention discloses quinoxaline-based fluorescent probes, methods and applications. The quinoxaline-based fluorescent probes have the characteristics of strong affinity for Tau protein aggregates, fast response speed, good specificity, high selectivity and low cytotoxicity, and can be directly used as fluorescent probes for detecting neurofibrillary tangles in living cells and tissue samples, thereby bringing help and breakthroughs to the clinical diagnosis of neurodegenerative diseases with neurofibrillary tangle characteristics including AD.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, specifically quinoxaline-based fluorescent probes, methods and applications. Background Art

[0002] Alzheimer's disease (AD) is a lethal neurodegenerative disease that is relatively common in the elderly population, and there is no effective treatment yet. Early diagnosis of AD can provide the possibility of therapeutic intervention for this devastating disease. Neuronal fibrillary tangles (Tau-PHF) caused by abnormal aggregation and deposition of Tau protein in brain tissue are one of the important pathological features of AD and have been proven to be closely related to the progression and severity of the disease. Therefore, the development of non-invasive fluorescence imaging technology targeting Tau protein aggregates may promote the early diagnosis of AD and Tau protein-related diseases.

[0003] Currently, the technical means of positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging can achieve early detection of diseases, "real-time" monitoring and evaluation of treatment effects. However, PET and SPECT are limited by exposure to radioactivity, expensive equipment, highly skilled personnel, and relatively poor spatial resolution.

[0004] Fluorescence detection is one of the most sensitive photochemical signal detection methods currently recognized and is widely used in the field of bioanalysis. In recent years, fluorescence imaging technology has also been used for visual detection of Tau protein aggregation at the cellular and tissue levels due to its advantages such as high sensitivity, high resolution, high cost performance, and real-time imaging. Some small molecule fluorescent probes have been used to selectively label Tau protein aggregates. However, their general ability to specifically recognize Tau protein aggregates is not strong. Therefore, there is an urgent need to develop fluorescent imaging probes with higher selectivity and affinity for Tau protein aggregates. Summary of the Invention

[0005] The present invention provides quinoxaline-based fluorescent probes, methods and applications, which bring help and breakthroughs to the clinical diagnosis of neurodegenerative diseases with neurofibrillary tangle characteristics, including AD.

[0006] To achieve this purpose, the present invention provides the following technical solutions:

[0007] In the first aspect of the present invention, a quinoxaline-based fluorescent probe is provided, and the fluorescent probe has the following structural formula:

[0008]

[0009] Among them, n is 1, 2 or 3.

[0010] Preferably, when n is 1, the probe is a yellow solid; when n is 2, the probe is a dark red solid; when n is 3, the probe is a black solid.

[0011] In the second aspect of the present invention, there is provided the use of the quinoxaline fluorescent probe described in the present invention in detecting Tau protein aggregates in a sample.

[0012] Preferably, the concentration of the fluorescent probe is not less than 0.1 μM.

[0013] Preferably, the sample includes cells and tissues.

[0014] Preferably, the maximum excitation wavelength of the fluorescent probe is: 400 - 600 nm, and the maximum emission wavelength is 500 - 750.

[0015] Preferably, the fluorescent probe has the following structural formula:

[0016]

[0017] Among them, n is 1, 2 or 3.

[0018] Preferably, when n is 1, the probe is a yellow solid; when n is 2, the probe is a dark red solid; when n is 3, the probe is a black solid.

[0019] In the third aspect of the present invention, there is provided a fluorescent probe kit, including the fluorescent probe described in the present invention.

[0020] Preferably, the fluorescent probe has the following structural formula:

[0021]

[0022] Among them, n is 1, 2 or 3.

[0023] Preferably, when n is 1, the probe is a yellow solid; when n is 2, the probe is a dark red solid; when n is 3, the probe is a black solid.

[0024] In the fourth aspect of the present invention, there is provided a preparation method of a quinoxaline fluorescent probe, including the following steps:

[0025] S1. Dissolve 2 - methylquinoline in methanol; dissolve 4 - (dimethylamino) - 2,6 - dimethoxybenzene in sodium hydroxide; mix the solutions;

[0026] S2. Dissolve methyltrioctylammonium chloride in 2 ml of methanol; dropwise add it to the mixed solution of S1;

[0027] S3. Heat under reflux. After the reaction is completed, cool to room temperature.

[0028] S4. Extract with ethyl acetate. Dry the organic layer with anhydrous MgSO4.

[0029] S5. After sufficient stirring, filter. Rotate the filtrate under reduced pressure to obtain the crude product.

[0030] S6. Separate and extract to obtain the quinoxaline-based fluorescent probe.

[0031] Preferably, the reaction conditions in step S3 are: heat under reflux at 110 °C for 15 h.

[0032] Preferably, step S6 includes: separating and purifying the target product with silica gel, and the eluent is a mixture of petroleum ether and ethyl acetate.

[0033] Compared with the prior art, the beneficial effects and remarkable progress of the present invention are as follows: The present invention designs and synthesizes three quinoxaline-based fluorescent probes targeting Tau protein aggregates. These compounds have strong affinity for Tau protein aggregates, fast response speed, good specificity, high selectivity, and low cytotoxicity. They can not only effectively monitor the kinetic process of Tau protein aggregation in vitro, but also specifically recognize the neurofibrillary tangles formed by Tau protein aggregates in living AD cells and the brain tissues of AD model mice, thus bringing help and breakthroughs to the clinical diagnosis of neurodegenerative diseases with neurofibrillary tangle characteristics, including AD. Brief Description of the Drawings

[0034] To more clearly illustrate the technical solutions of the present invention, the drawings required for the embodiments of the present invention will be briefly introduced below.

[0035] Obviously, the drawings in the following description are only the drawings of some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings, and these other drawings also belong to the scope of the drawings required for the embodiments of the present invention.

[0036] Figure 1 1H NMR spectrum of T1 in Example 1 of the present invention;

[0037] Figure 2 13C NMR spectrum of T1 in Example 1 of the present invention;

[0038] Figure 3 ESI-MS spectrum of T1 in Example 1 of the present invention;

[0039] Figure 41H NMR spectrum of T2 in Example 2 of the present invention;

[0040] Figure 5 13C NMR spectrum of T2 in Example 2 of the present invention;

[0041] Figure 6 ESI-MS spectrum of T2 in Example 2 of the present invention;

[0042] Figure 7 1H NMR spectrum of T3 in Example 3 of the present invention;

[0043] Figure 8 13C NMR spectrum of T3 in Example 3 of the present invention;

[0044] Figure 9 ESI-MS spectrum of T3 in Example 3 of the present invention;

[0045] Figure 10 In a, the fluorescence emission spectra (λex = 440 nm) of ThS (2 μM) in Example 4 of the present invention after binding to Aβ1-42 aggregates (10 μM) and R3 aggregates (30 μM) respectively;

[0046] Figure 10 In b, the fluorescence emission spectra (λex = 440 nm) of ThS (10 μM) in Example 4 of the present invention after binding to K18 monomers (15 μg / mL) and K18 aggregates (15 μg / mL) respectively;

[0047] Figure 10 In c, the transmission electron microscopy results of R3 aggregates in Example 4 of the present invention;

[0048] Figure 10 In d, the transmission electron microscopy results of Aβ1-42 aggregates in Example 4 of the present invention;

[0049] Figure 11 In a, the fluorescence emission spectra of T1 (λex = 455 nm) in Example 4 of the present invention after binding to K18 aggregates, R3 aggregates, Aβ1-42 aggregates and BSA in PBS. The concentration of the probe is 2 μM, and the concentration of each protein is 30 μg / mL;

[0050] Figure 11 In b, the fluorescence emission spectra of T2 (λex = 470 nm) in Example 2 of the present invention after binding to K18 aggregates, R3 aggregates, Aβ1-42 aggregates and BSA in PBS. The concentration of the probe is 2 μM, and the concentration of each protein is 30 μg / mL;

[0051] Figure 11The fluorescence emission spectra of c in Example 2 of the present invention with T3, λex = 510 nm, after binding to K18 aggregates, R3 aggregates, Aβ1-42 aggregates and BSA in PBS. The concentration of the probe is 2 μM, and the concentration of each protein is 30 μg / mL;

[0052] Figure 12 Figure a in shows the relationship between the change in fluorescence intensity and the concentration of T1 in Example 4 of the present invention. ΔF = F - F0, where F0 represents the background fluorescence value and F represents the fluorescence value after the T2 probe binds to R3 aggregates (20 μM). The solvent is PBS solution (20 mM, pH = 7.4);

[0053] Figure 12 Figure b in shows the relationship between the change in fluorescence intensity and the concentration of T2 in Example 4 of the present invention. ΔF = F - F0, where F0 represents the background fluorescence value and F represents the fluorescence value after the T2 probe binds to R3 aggregates (20 μM). The solvent is PBS solution (20 mM, pH = 7.4);

[0054] Figure 12 In c, T2 (0.5 μM) in Example 4 of the present invention can rapidly and effectively displace ThS (2.0 μM) from the ThS-R3 aggregate complex, and the fluorescence emission spectrum decreases significantly at 500 nm (λex = 440 nm). The concentration of R3 aggregates is 10 μM;

[0055] Figure 12 Figure d in shows the kinetic process of monitoring the in vitro aggregation of Tau protein by the T2 probe in Example 4 of the present invention, with λex = 480 nm;

[0056] Figure 12 Figure e in shows the relationship between the fluorescence intensity at the maximum emission wavelength (620 nm) and time in Example 4 of the present invention;

[0057] Figure 13 Figure a in shows the evaluation of the cytotoxicity of probe T1 using the CCK-8 method in Example 5 of the present invention;

[0058] Figure 13 Figure b in shows the evaluation of the cytotoxicity of probe T2 using the CCK-8 method in Example 5 of the present invention;

[0059] Figure 13 Figure c in shows the experimental results of visual detection of Tau protein aggregation in the Tau-AD cell model by probe T2 in Example 5 of the present invention. ThS labels Tau aggregates (λex = 488 nm, λem: 495 - 530 nm, green fluorescence), probe T2 labels Tau aggregates (λex = 488 nm, λem: 580 - 680 nm, red fluorescence), and DAPI is used to localize the cell nucleus (blue fluorescence);

[0060] Figure 14 In a, it is the immunofluorescence detection result of the hippocampal region of the mouse brain slice in Example 6 of the present invention. The phosphorylated and aggregated Tau protein was localized with the mouse monoclonal antibody AT8 (λex = 488 nm, λem: 500 - 550 nm, green fluorescence); the aggregated Tau protein was labeled with the probe T2 (λex = 488 nm, λem: 580 - 680 nm, red fluorescence); DAPI was used to localize the cell nucleus (blue fluorescence).

[0061] Figure 14 In b, it is the immunofluorescence detection result of the cerebral cortex region of the mouse brain slice in Example 6 of the present invention. The phosphorylated and aggregated Tau protein was localized with the mouse monoclonal antibody AT8 (λex = 488 nm, λem: 500 - 550 nm, green fluorescence); the aggregated Tau protein was labeled with the probe T2 (λex = 488 nm, λem: 580 - 680 nm, red fluorescence); DAPI was used to localize the cell nucleus (blue fluorescence). Detailed implementation manners

[0062] To make the objectives, technical solutions, beneficial effects and significant progress of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings provided in the embodiments of the present invention.

[0063] Obviously, all these described embodiments are only partial embodiments of the present invention, rather than all embodiments; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0064] For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0065] It should also be noted that the following specific embodiments can be combined with each other, and the same or similar concepts or processes may not be repeated in some embodiments.

[0066] Below, the technical solutions of the present invention will be described in detail with specific embodiments.

[0067] Synthesis of the compound T1 of (E)-3,5-dimethoxy-N,N-dimethyl-4-(2-(quinoxalin-2-yl)vinyl)aniline in Example 1

[0068] Synthesis method: Dissolve 2-methylquinoline (288.7 mg, 2.0 mmol) in 2 ml of methanol. Dissolve 4-(dimethylamino)-2,6-dimethoxybenzaldehyde (209.2 mg, 1.0 mmol) in sodium hydroxide (5.0 M, 15 ml), and add it to a round-bottom flask. Dissolve methyltrioctylammonium chloride (aliquat336, 43.2 mg, 0.1 mmol) in 2 ml of methanol, and add it dropwise to the round-bottom flask. Then, heat under reflux at 110 °C for 15 h. After the reaction is completed, cool to room temperature. Extract with 40 mL of ethyl acetate three times, and dry the organic layer with anhydrous MgSO4; after stirring well, filter, and rotary evaporate the filtrate under reduced pressure to obtain the crude product; separate and purify the target product by silica gel, and use a petroleum ether:ethyl acetate mixture as the eluent.

[0069] Synthesis product: Yellow solid T1, 33.2 mg, yield 19.8%.

[0070] The 1H NMR spectrum of compound T1 is as Figure 1 shown, and the 13C NMR spectrum of T1 is as Figure 2 shown, and the ESI-MS spectrum of T1 is as Figure 3 shown. 1 1H NMR (600 MHz, methanol-d4) δ 8.98 (s, 1H), 8.25 (s, 1H), 7.94 (d, J = 7.5 Hz, 2H), 7.74 (d, J = 7.1 Hz, 1H), 7.67 - 7.63 (m, 2H), 5.97 (s, 2H), 3.95 (s, 6H), 3.05 (s, 6H). 13C NMR (600 MHz, DMSO-d6) δ 160.97, 153.38, 152.78, 146.25, 142.57, 140.84, 130.57, 129.18, 128.84, 128.66, 128.50, 121.75, 102.58, 88.85, 56.02. HRMS (ESI) Calcd. for C20H21N3O2 [M + H]+: 336.1712; Found: 336.1700.

[0071] Example 2 Synthesis of 3,5-dimethoxy-N,N-dimethyl-4-((1E,3E)-4-(quinoxalin-2-yl)buta-1,3-dien-1-yl)aniline compound T2

[0072] Synthesis method: The same as the method in Example 1.

[0073] Synthesis product: T2 is a dark red solid, 25.6 mg, yield 7.1%.

[0074] The 1H NMR spectrum of compound T2 is as shown in Figure 4 below, and the 13C NMR spectrum of T2 is as shown in Figure 5 below, and the ESI-MS spectrum of T2 is as shown in Figure 6 below. 1 1H NMR (600 MHz, Methanol-d4) δ 9.01 (s, 1H), 7.99 - 7.93 (m, 2H), 7.77 - 7.67 (m, 3H), 7.36 - 7.27 (m, 2H), 6.69 (d, J = 15.4 Hz, 1H), 5.94 (s, 2H), 3.89 (s, 6H), 3.02 (s, 6H). 13C NMR (600 MHz, DMSO-d6) δ 160.29, 152.28, 152.09, 145.51, 142.45, 141.55, 140.78, 130.95, 130.67, 129.18, 128.95, 126.80, 124.95, 55.97. HRMS (ESI) Calcd. for C22H24N3O2 [M+H] + : 362.1869; Found: 362.1856.

[0075] Example 3 Synthesis of 3,5-dimethoxy-N,N-dimethyl-4-((1E,3E,5E)-6-(quinoxalin-2-yl)hexa-1,3,5-trien-1-yl)aniline Compound T3

[0076] Synthesis method: The same as the method in Example 1.

[0077] Synthesis product: T3 is a black solid, 17.8 mg, yield 4.6%.

[0078] The 1 1H NMR spectrum of compound T3 is as shown in Figure 7 below, and the 13C NMR spectrum of T3 is as shown in Figure 8 below, and the ESI-MS spectrum of T3 is as shown in Figure 9As shown. 1H NMR (600 MHz, Chloroform-d) δ 8.81 (d, J = 8.1 Hz, 1H), 7.95 - 7.89 (m, 2H), 7.65 - 7.59 (m, 2H), 7.57 - 7.50 (m, 2H), 7.33 - 7.25 (m, 1H), 7.00 (d, J = 15.5 Hz, 1H), 6.74 - 6.62 (m, 2H), 6.46 - 6.40 (m, 1H), 5.79 (s, 2H), 3.80 (s, 6H), 2.93 (s, 7H). 13C NMR (600 MHz, DMSO-d6) δ 160.30, 160.02, 151.94, 151.66, 145.61, 142.98, 142.42, 140.93, 138.37, 130.80, 129.23, 129.01, 128.86, 128.68, 127.46, 126.80, 126.46, 124.94, 103.33, 89.06, 55.91. HRMS (ESI) Calcd. for C24H26N3O2 [M + H]+: 388.2025; Found: 388.2010.

[0079] Example 4 Evaluation of the potential of quinoline compounds T1, T2, and T3 as Tau aggregation probes at the molecular level

[0080] 4.1 Evaluation of the ability of quinoline compounds T1, T2, and T3 to selectively recognize Tau aggregates by fluorescence spectroscopy

[0081] Recombinant protein K18 is a truncated fragment of Tau protein containing four repeat unit domains and is produced from Escherichia coli; the R3 polypeptide (residues 306 - 336) is the third repeat unit fragment of Tau protein and is also a key core fragment for the formation of Tau aggregates. Heparin-induced K18 or R3 aggregates are commonly used to mimic clinical pathological Tau aggregates.

[0082] First, the above protein aggregates were confirmed by the fluorescence spectrum and transmission electron microscopy of thioflavin S (ThS).

[0083] The results are as Figure 10 shown, Figure 10 In a, the fluorescence emission spectra (λex = 440 nm) of ThS (2 μM) binding to Aβ1 - 42 aggregates (10 μM) and R3 aggregates (30 μM) respectively; Figure 10 In b, the fluorescence emission spectra (λex = 440 nm) of ThS (10 μM) binding to K18 monomer (15 μg / mL) and K18 aggregates (15 μg / mL) respectively; Figure 10 In c, the transmission electron microscopy results of R3 aggregates;Figure 10 In which, d is the transmission electron microscopy result of Aβ1-42 aggregates.

[0084] Furthermore, the fluorescence spectra of the quinoline compounds T1-T3 synthesized in Examples 1-3 interacting with Tau aggregates, Aβ42 aggregates and bovine serum albumin (BSA) were studied to evaluate the ability of the quinoline compounds T1, T2, T3 as fluorescence probes for Tau aggregates.

[0085] The results are as Figure 11 shown, Figure 11 In a, T1, λex = 455 nm, Figure 11 In b, T2, λex = 470 nm, Figure 11 In c, T3, λex = 510 nm, are the fluorescence emission spectra after binding with K18 aggregates, R3 aggregates, Aβ1-42 aggregates and BSA in PBS. The concentration of the probe is 2 μM, and the concentration of each protein is 30 μg / mL. It can be seen that the prepared T1-T3 probes have almost no fluorescence in phosphate buffered saline (PBS). However, when they are incubated with K18 aggregates, the most significant increase in fluorescence intensity can be observed. After T1, T2, T3 bind to K18 aggregates, the fluorescence intensities increase by 30.1, 26.3 and 7.5 times respectively, which is much higher than the increase when they interact with Aβ aggregates and BSA. This indicates that the probes T1, T2, T3 can selectively recognize Tau polymers.

[0086] 4.2 Detection of the affinity between the quinoline compound T1, T2, T3 probes and Tau aggregates

[0087] To quantify the binding affinity between T2 and Tau aggregates, an in vitro fluorescence saturation binding assay was performed in the present invention.

[0088] The results are as Figure 12 shown in a and b. As the probe concentration increases, the fluorescence intensities of T1 and T2 with R3 aggregates gradually increase. From the dissociation constant Kd calculation formula: Y = Bmax*X / (Kd+X), it can be calculated that the Kd value of T1 with R3 aggregates is about 203.1 nM. Similarly, it can be calculated that the Kd value of T2 with R3 aggregates is about 234.7 nM. The affinity ranking of the two with R3 aggregates is: T2 < T1. Since the fluorescence signal is poor when T3 binds to R3 aggregates, the affinity of T3 with R3 aggregates cannot be determined by this method.

[0089] Compared with the Kd values of other fluorescent probes targeting Tau protein reported in the literature, such as curcumin derivative 1c (0.77 μM), ThS (1.9 μM), difluoroboron β-diketonate probe 2e (1.41 μM), 3,5-dimethoxy-N,N-dimethylaniline-4-yl derivatives 3g (0.89 μM) and 3h (1.50 μM), the Kd values of the quinoline compound probes T1 and T2 synthesized in this invention are significantly smaller than the literature values, indicating that the affinity of this invention for Tau protein aggregates is higher than that reported in the literature.

[0090] It is worth noting that in the in vitro fluorescence competitive binding experiment (T2 competes with ThS to inhibit fluorescence intensity), it takes about 5 minutes for T2 to displace ThS from the ThS-R3 aggregation complex, significantly weakening the intensity of the characteristic fluorescence emission peak of ThS at 440 nm ( Figure 12 in c). This shows that the response speed of this invention to detect Tau protein aggregates is very fast, and it also shows again that T2 is an excellent fluorescent probe for detecting Tau aggregates.

[0091] 4.3 Fluorescence kinetic characteristics of quinoline compound T2 probe and Tau aggregates

[0092] Previous studies mostly used fluorescence analysis methods based on ThT or ThS to study the kinetic process of Tau protein aggregation. Next, this invention explored whether the T2 probe can be used to monitor the kinetic process of Tau protein aggregation in vitro.

[0093] The results are shown in Figure 12 d and e. As can be seen from d and e in Figure 12 , the kinetic data of R3 aggregation monitored by the T2 probe show an exponential growth characteristic: first, it experiences a rapid elongation period, and then reaches a plateau (~60 minutes). There is no obvious lag period in the kinetic process of R3 aggregation. This shows that T2 can be used as a new type, highly efficient, and high-affinity fluorescent probe for monitoring the kinetics of Tau protein aggregation in vitro.

[0094] Example 5 Evaluate the potential of quinoline compounds T1 and T2 as Tau aggregation probes at the cellular level

[0095] 5.1 Evaluation of the cytotoxicity of quinoline compound probes

[0096] Low cytotoxicity is an essential characteristic of a good in vivo diagnostic reagent. In this invention, in human neuroblastoma cell line SK-N-SH cells, after treating the cells with different concentrations of compounds T1 and T2 for 24 hours, the CCK-8 method was used to evaluate the cytotoxicity of probes T1 and T2.

[0097] The results are shown in Figure 13As shown by a and b in [description], the above experimental results prove that the cell viability of all experimental groups is higher than 90%. That is to say, the probes T1 and T2 have relatively low cytotoxicity.

[0098] 5.2 Application of quinoline compound T2 probe for visual detection of Tau protein aggregation in Tau-AD cell model

[0099] In our previous study, pre-aggregated Tau protofibrils were introduced into the human neuroblastoma cell line SK-N-SH cells, and a cell model of Tau-AD was successfully constructed. In the present invention, to evaluate whether probe T2 can detect intracellular Tau protein aggregates, SK-N-SH cells were treated with 2.0 μM R3 aggregates at 37 °C for 36 h to induce endogenous Tau protein aggregation in cells, and a cell model of Tau-AD was established. Then, it was incubated with T2 for 2 h. ThS is a general dye for detecting Tau protein aggregates, and the positive signal shows obvious green fluorescence.

[0100] The results are as Figure 13 shown in c in [description]. Compared with the normal cell control group, the Tau-AD cell model group showed clear green fluorescence, indicating that R3 aggregates can successfully induce endogenous Tau protein aggregation in living cells. The red fluorescence signal of probe T2 co-localizes with the green fluorescence signal of ThS, confirming that probe T2 can specifically detect intracellular Tau protein aggregates.

[0101] Example 6 Evaluation of the potential of quinoline compound T2 as a Tau aggregation probe at the animal level

[0102] Okadaic acid (OA) is a protein phosphatase inhibitor that can rapidly initiate hyperphosphorylation of Tau protein in the mouse brain, and then aggregate into neurofibrils, leading to neuronal apoptosis. It is widely used in the establishment of AD mouse models. In this study, 4-month-old C57BL / 6 wild-type mice were selected, and 50 ng OA (DMSO as the solvent) was injected into the bilateral hippocampal regions respectively to establish an AD mouse model.

[0103] The results are as Figure 14 shown. In the control group brain slices (only the group injected with DMSO), whether it is the cerebral cortex ( Figure 14 in b) or the hippocampal region ( Figure 14In a) above, the green fluorescence signals shown by AT8 antibody immunofluorescence staining were all less. In the OA-induced AD model group, obvious AT8 positive signals could be observed in the cerebral cortex and hippocampal region, indicating that OA injection induced obvious hyperphosphorylation of Tau protein in the mouse brain and aggregation into neurofibrils, and the model was successfully established. In addition, the green fluorescence of AT8 was co-localized with the red fluorescence of probe T2, and clear yellow fluorescence could be seen in the merged image. Under the same imaging settings, very weak background signals were shown by AT8 and T2 in the cerebral cortex and hippocampal region of control group mice. These results indicate that probe T2 can replace expensive antibodies to histologically stain Tau protein aggregates formed by hyperphosphorylation, and T2 has broad prospects in the diagnosis of AD and the research on related Tau protein lesions.

[0104] During the description process of the above specification:

[0105] The descriptions of terms such as "this embodiment", "the embodiments of the present invention", "as shown in...", "further", "further improved technical sub-schemes", etc. mean that the specific features, structures, materials or characteristics described in this embodiment or example are included in at least one embodiment or example of the present invention; in this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example, and moreover, the specific features, structures, materials or characteristics described can be combined or combined in a suitable manner in any one or more embodiments or examples; in addition, on the premise of not generating contradictions, those of ordinary skill in the art can combine or combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0106] Finally, it should be noted that:

[0107] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it;

[0108] Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. The non-essential improvements, adjustments or replacements made by those skilled in the art according to the content of this specification all fall within the scope claimed by the present invention.

Claims

1. A quinoxaline-based fluorescent probe, characterized in that, The fluorescent probe has the following structural formula: ， Wherein, n is 2.

2. The quinoxaline fluorescent probe according to claim 1, wherein When n is 2, the probe is a dark red solid.

3. The quinoxaline fluorescent probe according to claim 1 is used in the preparation of a reagent for detecting Tau protein aggregates in a test sample.

4. The application according to claim 3, characterized in that The concentration of the fluorescent probe is not less than 0.1 μM.

5. The application according to claim 3, wherein The sample includes cells and tissues.

6. The application according to claim 3, characterized in that, The maximum excitation wavelength of the fluorescent probe is 400 - 600 nm, and the maximum emission wavelength is 500 - 750 nm.

7. A fluorescent probe kit, characterized in that, It includes the fluorescent probe according to claim 1 or 2.

8. A preparation method of a quinoxaline-based fluorescent probe, characterized in that, It includes the following steps: S1. Dissolve 2-methylquinoline in methanol; dissolve 4-(dimethylamino)-2,6-dimethoxyphenylacetaldehyde in sodium hydroxide; mix the solutions. S2. Dissolve methyltrioctylammonium chloride in 2 ml of methanol; add dropwise to the mixed solution in S1. S3. Heat under reflux, and after the reaction is completed, cool to room temperature. S4. Extract with ethyl acetate, and dry the organic layer with anhydrous MgSO4. S5. After sufficient stirring, filter, and rotary evaporate the filtrate under reduced pressure to obtain the crude product. S6. Isolate and extract to obtain the quinoxaline fluorescent probe.

9. The method according to claim 8, characterized in that, The reaction conditions for step S3 are: heat under reflux at 110 °C for 15 h.

10. The method according to claim 8, wherein Step S6 includes: separating and purifying the target product with silica gel, and the eluent is a mixed solution of petroleum ether and ethyl acetate.

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