A beta plaque probe as well as preparation method and application thereof

Abstract

The invention relates to the field of fluorescent dyes, in particular to an A beta plaque probe as well as a preparation method and application thereof. The invention provides an A beta plaque probe. The A beta plaque probe has a structural formula as shown in a formula I: # imgabs0 # (I), wherein R1 is selected from the group consisting of # imgabs 1 #; r2 is selected from the group consisting of # imgabs2, # imgabs3 or # imgabs4. The A beta plaque probe provided by the invention has large Stokes shift, excellent blood brain barrier penetrability, rapid metabolism and dynamic imaging capability, provides a chemical tool with a wide prospect for brain imaging, and is suitable for application scenes such as early diagnosis, disease monitoring and disease process tracking of Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent dyes, and in particular to an Aβ plaque probe and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disease characterized by memory impairment, cognitive decline, and neuropsychiatric symptoms. With the advent of an aging society and the rising incidence of AD, the disease has become a major public health and social care challenge worldwide. Due to its insidious onset, early and accurate diagnosis is essential for effective treatment and management. Therefore, in order to better understand brain function and study disease-related processes, it is particularly important to develop reliable chemical tools and advanced diagnostic equipment.

[0003] Amyloid-β (Aβ) is the main pathological marker of Alzheimer's disease. In the amyloidogenic pathway, Aβ peptides are produced by cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase, generating monomeric Aβ peptides of different lengths. These monomeric Aβ peptides aggregate into higher-order structures, including oligomers, fibrils, and plaques. In addition, Aβ plaques are often found in the deep cortical layers (>500 μm depth) of Alzheimer's disease mouse models, which also emphasizes the importance of deep tissue imaging technology for the study of Alzheimer's disease (e.g., early stages) and its progression.

[0004] Fluorescence molecular imaging has been gradually recognized as a versatile means to study the pathogenesis and basic research of Alzheimer's disease due to its high sensitivity, rapid response, simple technology and ability to non-invasively monitor subcellular processes. These fluorescent probes can effectively stain Aβ plaques for microscopic imaging of brain tissues. For example, compounds such as Thioflavin T (ThT) and Thioflavin S (ThS) are widely used. Through radioactive labeling, these probes can break through the blood-brain barrier (BBB). However, current fluorescent small molecule probes still face many challenges, such as they usually have short excitation wavelengths, which limits their penetration depth, and are accompanied by strong background fluorescence.

[0005] In contrast, near-infrared (NIR) fluorescent probes have gradually become an important choice for early diagnosis of Alzheimer's disease due to their advantages such as lower autofluorescence, stronger tissue penetration, less light scattering, and lower photobleaching. However, existing near-infrared fluorescent probes (such as Chinese patent applications CN110615808A and CN114656447A) have problems such as large dosage (such as more than 2 mg / kg) and low penetration efficiency (such as more than 15 minutes) when applied to in vivo imaging. In other words, existing near-infrared fluorescent probes still face problems such as poor blood-brain barrier penetration and low blood-brain barrier penetration efficiency, making it difficult to achieve efficient in vivo imaging of Alzheimer's disease biomarkers (such as β-amyloid protein). Summary of the invention

[0006] In a first aspect, the present invention provides an Aβ plaque probe, characterized in that the probe has a structural formula shown in Formula I: (I); Among them, R 1 Selected from ; R 2 Selected from , or .

[0007] In some embodiments, the probe has the structure of the following formula: .

[0008] In some embodiments, the probe has the structure of the following formula: .

[0009] In some embodiments, the probe has the structure of the following formula: .

[0010] In some embodiments, R 1 It can also be .

[0011] In some embodiments, the probe has the structure of the following formula: .

[0012] In a second aspect, the present invention provides a method for preparing the above-mentioned Aβ plaque probe, characterized in that it comprises the following steps: S101 Compound W166 and the first reagent are dissolved in toluene, glacial acetic acid and piperidine are added, and the mixture is heated and stirred to obtain compound 1; In some embodiments, the first reagent comprises p-dimethylaminobenzaldehyde, or 4-dimethylaminocinnamaldehyde, or 5-[4-(dimethylamino)phenyl]thiophene-2-carbaldehyde.

[0013] In some embodiments, the molar ratio of the compound W166 to the first reagent comprises 1:(1-1.5).

[0014] In some embodiments, the heating temperature in S101 is 50-65°C.

[0015] In some embodiments, the heating temperature in S101 is 60°C.

[0016] In some embodiments, the stirring time in S101 is 1.5-3 hours.

[0017] In some embodiments, the stirring time in S101 is 1.5 hours.

[0018] S102 The compound 1 and N-hydroxysuccinimide are dissolved in dichloromethane, N,N-diisopropylethylamine is added, and the mixture is stirred at room temperature to obtain compound 2; In some embodiments, the molar ratio of the compound 1 to the N-hydroxysuccinimide comprises 1:(1-1.5).

[0019] In some embodiments, the stirring time in S102 is 5-8 hours.

[0020] In some embodiments, the stirring time in S102 is 5 hours.

[0021] S103 The compound 2 and 2-(2-aminoethoxy)ethoxy) are dissolved in dichloromethane, N,N-diisopropylethylamine is added, and the mixture is stirred at room temperature to obtain compound 3; Where R 2 Selected from , or .

[0022] In some embodiments, the molar ratio of the compound 2 to the 2-(2-aminoethoxy)ethoxy) comprises 1:(1-1.5).

[0023] In some embodiments, the stirring time in S103 is 2-5 hours.

[0024] In some embodiments, the stirring time in S103 is 2.5 hours.

[0025] In a third aspect, the present invention provides use of the above-mentioned Aβ plaque probe in preparing a detection reagent for β-amyloid protein and / or aggregates formed by β-amyloid protein.

[0026] In some embodiments, the aggregates formed by the β-amyloid protein include Aβ oligomers, Aβ fibers, Aβ plaques, or a combination thereof.

[0027] In some embodiments, the aggregates formed by the β-amyloid protein are Aβ fibers and / or Aβ plaques.

[0028] In some embodiments, the detection reagents are used for in vitro and / or in vivo detection.

[0029] In some embodiments, the detection reagent is used to detect β-amyloid protein and / or aggregates formed by β-amyloid protein in brain tissue.

[0030] In some embodiments, the detection reagent is used to perform dynamic imaging of β-amyloid protein and / or aggregates formed by β-amyloid protein in brain tissue.

[0031] In some embodiments, the concentration of the β-amyloid protein or aggregates formed by the β-amyloid protein is at least 28 nM.

[0032] In some embodiments, the concentration of the β-amyloid protein or aggregates formed by the β-amyloid protein is ≥ 1 μM.

[0033] In some embodiments, the concentration of the β-amyloid protein or the aggregates formed by the β-amyloid protein is 1-40 μM.

[0034] In a fourth aspect, the present invention provides use of the above-mentioned Aβ plaque probe in preparing a detection reagent for Alzheimer's disease.

[0035] In some embodiments, the detection reagents are used for in vitro and / or in vivo detection.

[0036] In some embodiments, the detection reagent is used to detect β-amyloid protein and / or aggregates formed by β-amyloid protein in brain tissue.

[0037] In some embodiments, the detection reagent is used to perform dynamic imaging of β-amyloid protein and / or aggregates formed by β-amyloid protein in brain tissue.

[0038] In some embodiments, the concentration of the β-amyloid protein or aggregates formed by the β-amyloid protein is at least 28 nM.

[0039] In some embodiments, the concentration of the β-amyloid protein or aggregates formed by the β-amyloid protein is ≥ 1 μM.

[0040] In some embodiments, the concentration of the β-amyloid protein or the aggregates formed by the β-amyloid protein is 1-40 μM.

[0041] In some embodiments, the detection reagent is used for disease monitoring and / or early diagnosis of Alzheimer's disease.

[0042] In some embodiments, the dosage of the Aβ plaque probe comprises 0.1 - 1.5 mg / kg.

[0043] In some embodiments, the dosage of the Aβ plaque probe comprises 0.5 - 1.5 mg / kg.

[0044] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects: The present invention provides an Aβ plaque probe as shown in Formula I, which is a near-infrared fluorescent probe with a large Stokes shift and excellent blood-brain barrier penetration. Compared with traditional Aβ imaging probes (such as THT), it has a higher signal-to-noise ratio, fluorescence turn-on ratio and excellent Aβ responsive imaging capability, and has obvious advantages in detecting fibers and plaques in Aβ-like lesions in Alzheimer's disease.

[0045] It should be emphasized that R 1 and R 2 At the same time, the emission wavelength of the probe is successfully extended to the near-infrared region, ensuring that the Aβ plaque probe provided by the present invention has a higher fluorescence turn-on ratio, thereby being able to provide more sensitive and accurate imaging signals, significantly improving the resolution and reliability of imaging, while also ensuring that the Aβ plaque probe provided by the present invention has excellent blood-brain barrier penetration. On the basis of meeting the requirements for penetrating the blood-brain barrier, it can quickly and efficiently penetrate living brain tissue and selectively bind to Aβ plaques at a lower dose (e.g., 0.5 mg / kg) and a faster penetration speed (e.g., within 5 minutes), showing the ability to dynamically image Aβ plaques in living brain tissue, providing a powerful imaging tool for early diagnosis and dynamic monitoring of Alzheimer's disease.

[0046] In addition, the near-infrared emission wavelength of the Aβ plaque probe provided by the present invention makes it have lower background noise and deeper imaging depth, which is particularly suitable for use in in vivo imaging. Compared with the existing near-infrared fluorescent probes for detecting Aβ plaques, the detection limit of the Aβ plaque probe provided by the present invention for β-amyloid protein or aggregates formed by β-amyloid protein is very low, which can be as low as 28nM. In other words, when applied to Aβ plaque imaging (such as detecting the presence or absence of Aβ plaques) in a large number of in vivo animal models, the present invention can efficiently, quickly and sensitively detect low levels of Aβ plaques with a lower dose and faster penetration rate, which is helpful for the early diagnosis of Alzheimer's disease and significantly saves experimental costs. In addition, by virtue of its unique dynamic imaging characteristics, the Aβ plaque probe provided by the present invention can also accurately locate and track the distribution of Aβ plaques, and can be used for long-term tracking of the distribution of Aβ plaques in the brain tissue of subjects with Alzheimer's disease in vivo.

[0047] In summary, the Aβ plaque probe provided by the present invention has a large Stokes shift, excellent blood-brain barrier penetration, rapid metabolism and dynamic imaging capabilities, providing a promising chemical tool for brain imaging, and is suitable for application scenarios such as early diagnosis of Alzheimer's disease, disease monitoring and disease progression tracking. In addition, the preparation process of the Aβ plaque probe provided by the present invention is simple, the reaction conditions are mild, and the synthesis path is short, which is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0049] Figure 1 Absorption spectra of CL-07 (A) and CL-21 (B) in solvents of different polarities; Figure 2 The emission spectra of CL-07 (A) and CL-21 (B) in solvents with different polarities; Figure 3 The graph shows the change in fluorescence intensity after the same concentration (1 μM) of CL-07, CL-21, and ThT were co-incubated with the same concentration (25 μM) of Aβ fibers under the same conditions; Figure 4 The results of the selectivity of CL-07 (A) and CL-21 (B) for Aβ plaques are shown; Figure 5 The results of the changes in fluorescence intensity after CL-07 interacted with Aβ fibers of different concentrations (0-25 μM) (A) and the changes in fluorescence intensity after CL-21 interacted with Aβ fibers of different concentrations (0-40 μM) (B) are shown; Figure 6 The results of Aβ plaque imaging in brain slices of CL-21 and Alzheimer's disease model mice; Figure 7 The in vivo fluorescence imaging results of CL-21 are shown in Figure 1 (A: in vivo fluorescence imaging of CL-21 in wild-type mice and AD model mice; B: relative fluorescence signal intensity of CL-21 in wild-type mice and AD model mice); Figure 8 The results of CL-21 and THT show co-localization results; Fig. 9 The result diagram of fluorescence on-ratio of different compounds; Fig.10 The graph shows the change in fluorescence intensity after different compounds were co-incubated with Aβ fibers of the same concentration under the same conditions. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0051] In this document, the directions or positional relationships indicated by the terms "upper", "lower", "inner", "outer", "front", "rear", "one end", "the other end", etc. are based on the directions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific direction, be constructed and operate in a specific direction, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0052] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0053] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0054] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0055] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0056] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0057] All raw materials of the present invention are not particularly limited to their sources, and can be prepared by commercial purchase or conventional methods well known to those skilled in the art. All raw materials of the present invention are not particularly limited to their purity, and the present invention preferably adopts analytically pure or conventional purity requirements in the field of fluorescent dyes. All raw materials of the present invention, their brands and abbreviations all belong to conventional brands and abbreviations in this area, and each brand and abbreviation is clear and definite in the field of its related use, and those skilled in the art can purchase from commercial or prepare by conventional methods according to the brands, abbreviations and corresponding uses.

[0058] The present invention has no particular limitation on the expression of the substituents, and all expressions familiar to those skilled in the art are adopted. Based on common sense, those skilled in the art can correctly understand the meanings according to the expressions.

[0059] Embodiment 1 Preparation of probes CL-07: Compound W166 (49.1 mg, 0.15 mmol) and 4-dimethylaminobenzaldehyde (i.e., p-dimethylaminobenzaldehyde, 29.1 mg, 0.195 mmol) were dissolved in toluene (2.0 mL), and glacial acetic acid (28 µL) and piperidine (60 µL) were added. The mixture was heated to 60°C and stirred for 1.5 hours, and the progress of the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the mixture was cooled to room temperature, and the solvent component was removed by rotary evaporation to obtain a crude product, and the organic solvent was evaporated under reduced pressure. Purification was performed using a neutral alumina column with methanol: dichloromethane as the eluent, and a blue-purple solid powder CL-04 was collected with a yield of 70%.

[0060] CL-04 (68.8 mg, 0.15 mmol) and N-hydroxysuccinimide (50 mg, 0.195 mmol) were dissolved in dichloromethane (2.0 mL), and N,N-diisopropylethylamine (39 µL, 0.225 mmol) was added. The mixture was stirred at room temperature for 5 hours, and the progress of the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the solvent component was removed by rotary evaporation to obtain a crude product. Purification was performed using dichloromethane: ethyl acetate (25:1) as the eluent, and CL-06 was collected with a yield of 60%.

[0061] CL-06 (16.7 mg, 0.03 mmol) and 2-(2-aminoethoxy)ethoxy) (6 mg, 0.04 mmol) were dissolved in dichloromethane (1.0 mL) and N,N-diisopropylethylamine (10 µL) was added. The mixture was stirred at room temperature for 2.5 hours and the progress of the reaction was monitored by liquid chromatography-mass spectrometry. CL-07 was collected for purification using dichloromethane:methanol as the eluent with a yield of 70%.

[0062] 1 H NMR (400 MHz, Chloroform- d ) δ 7.98 (d, J = 15.2 Hz, 1H), 7.89 (s,1H), 7.77 (d, J = 8.2 Hz, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.57 (d, J = 8.7 Hz, 2H),7.13 (d, J = 15.2 Hz, 1H), 6.99 (s, 1H), 6.70 (d, J = 8.8 Hz, 2H), 3.71 (d,J =10.2 Hz, 11H), 3.64 (d, J = 4.6 Hz, 2H), 3.09 (s, 6H), 1.76 (s, 7H). 13 C NMR (101 MHz, CDCl 3 ) δ 176.09, 169.51, 167.02, 152.79, 147.56,145.47, 142.10, 131.70, 131.67, 127.58, 122.92, 121.52, 115.41, 112.01,111.09, 101.30, 72.68, 70.54, 70.51, 70.18, 61.87, 52.40, 40.24, 40.05,22.58. CL-21: Compound W166 (49.1 mg, 0.15 mmol) and 5-[4-(dimethylamino)phenyl]thiophene-2-carboxaldehyde (44.6 mg, 0.18 mmol) were dissolved in toluene (2.0 mL), and glacial acetic acid (28 µL) and piperidine (60 µL) were added. The mixture was heated to 60°C and stirred for 2 hours, and the progress of the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the mixture was cooled to room temperature, and the solvent component was removed by rotary evaporation to obtain a crude product, and the organic solvent was evaporated under reduced pressure. Purification was performed using a neutral alumina column with methanol: dichloromethane (15:1) as the eluent, and a blue-purple solid powder CL-18 was collected with a yield of 70%.

[0063] CL-18 (81.123 mg, 0.15 mmol) and N-hydroxysuccinimide (50 mg, 0.195 mmol) were dissolved in dichloromethane (2.0 mL) with N,N-diisopropylethylamine (39 µL, 0.225 mmol). The mixture was stirred at room temperature for 4 hours, and the progress of the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the solvent component was removed by rotary evaporation to obtain a crude product. Purification was performed using dichloromethane: ethyl acetate (25:1) as the eluent, and CL-19 was collected with a yield of 60%.

[0064] CL-19 (9.5 mg, 0.015 mmol) and 2-(2-aminoethoxy)ethoxy) (3 mg, 0.02 mmol) were dissolved in dichloromethane (1.0 mL) and N,N-diisopropylethylamine (4 µL) was added. The mixture was stirred at room temperature for 2 hours and the progress of the reaction was monitored by liquid chromatography-mass spectrometry. CL-21 was collected for purification using dichloromethane:methanol (15:1) as the eluent with a yield of 95%.

[0065] 1 H NMR (400 MHz, Chloroform- d ) δ 8.08 (d, J = 14.9 Hz, 1H), 7.91 (d, J =1.6 Hz, 1H), 7.78 (dd, J = 8.3, 1.7 Hz, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.58 –7.51 (m, 2H), 7.35 (d, J = 4.0 Hz, 1H), 7.19 (d, J = 4.0 Hz, 1H), 7.02 (d, J = 14.9Hz, 1H), 6.77 – 6.68 (m, 2H), 3.78 – 3.59 (m, 12H), 3.03 (s, 6H), 1.77 (s, 6H). Embodiment 2 Photophysical properties of the probe and its response to Aβ In order to evaluate the optical properties of the CL-07 and CL-21 probes, UV absorption and fluorescence spectroscopy tests were performed in different solvents. Specifically, the UV absorption spectra of CL-07 and CL-21 were prepared at a concentration of 10 μM in different polar solvents (water, methanol, dichloromethane, tetrahydrofuran, and 50% tetrahydrofuran). The fluorescence spectroscopy test used 1 μM concentrations of CL-07 and CL-21 (the measurement conditions for CL-07 were: excitation wavelength 555 nm, emission wavelength 580 nm, and bandwidth set to 5; the measurement conditions for CL-21 were: excitation wavelength 575 nm, emission wavelength 595 nm, and bandwidth set to 5), and measured their emission wavelengths and intensities in different solvents.

[0066] like Figure 1 and Figure 2As shown in the figure, the UV absorption and fluorescence spectral characteristics of CL-07 and CL-21 in different polar solvents were measured. In terms of UV absorption, the absorption maximum of CL-07 in different solutions is between 550nm and 650nm, while the absorption maximum of CL-21 is between 550nm and 610nm, indicating that there are certain differences in the UV absorption wavelength between the two. In the fluorescence spectrum test, CL-07 showed a strong fluorescence intensity, and its emission wavelength was distributed between 620nm and 660nm. CL-21 showed a longer fluorescence emission wavelength, ranging from 700nm to 750nm, and had a larger Stokes shift. This feature gives CL-21 a significant advantage in fluorescence imaging, which can effectively avoid the problem of fluorescence crosstalk, and is particularly suitable for applications such as brain imaging that require high resolution and low interference. In addition, solvent effects also play an important role in fluorescence characteristics. In aqueous solution, the absorption spectrum of the probe shows a large blue shift, which may be due to the J aggregation phenomenon in water.

[0067] Embodiment 3 In this example, the stock solutions (1 μM) of CL-07 and CL-21 were added to an appropriate amount of PBS (pH 7.4), and then Aβ fiber aggregates (25 μM) were added. After incubation at 37°C for 5 minutes, the reaction solution was transferred to a quartz fluorescent dish, and its fluorescence intensity was measured using a fluorescence spectrometer. The measurement conditions for ThT were: excitation wavelength 440 nm, emission wavelength 455 nm, and bandwidth set to 2.9; the measurement conditions for CL-07 were: excitation wavelength 555 nm, emission wavelength 580 nm, and bandwidth set to 5; the measurement conditions for CL-21 were: excitation wavelength 575 nm, emission wavelength 595 nm, and bandwidth set to 5. At the same time, a probe without Aβ itself was prepared as a blank control, and measurements were performed under the same experimental conditions for comparison.

[0068] The results are as follows Figure 3 As shown, the responsiveness of CL-07 and CL-21 to Aβ fibers was measured. They emitted low-intensity fluorescence in the solvent, possibly due to the TICT effect, which is conducive to the construction of fluorescent probes with high signal-to-noise ratio. When the probes bind to Aβ fibers, the fluorescence intensity increases significantly. After incubation with 25μM Aβ fibers, CL-07 showed a 56.5-fold fluorescence enhancement, CL-21 showed a 29.3-fold fluorescence enhancement, and THT showed an 11.2-fold fluorescence enhancement. These results show that CL-07 and CL-21 are very suitable molecular tools for detecting Aβ fibers.

[0069] Embodiment 4 In this example, in order to verify the selectivity of CL-07 and CL-21 probes for Aβ fibrils, two probes, CL-07 and CL-21, were used in the experiment, both at a concentration of 1 μM. The experimental design is as follows: CL-07 (1 μM) and CL-21 (1 μM) probes were incubated with Aβ fibrils (25 μM) or bovine serum albumin (BSA, 10 μM), and all samples in the reaction system were kept at room temperature for 30 minutes. After the incubation, the fluorescence intensity change of the sample was tested using a fluorescence spectrometer. The specific fluorescence detection conditions are as follows: CL-07: excitation wavelength (ex) is 555 nm, emission wavelength (em) is 580 nm, and spectral bandwidth (slit) is set to 5 nm. CL-21: excitation wavelength (ex) is 575 nm, emission wavelength (em) is 595 nm, and spectral bandwidth (slit) is set to 5 nm.

[0070] like Figure 4 As shown in the figure, when CL-07 and CL-21 probes were co-incubated with Aβ fibrils, they all showed obvious fluorescence enhancement. In contrast, the fluorescence signals of CL-07 and CL-21 when co-incubated with BSA were relatively weak. The weak fluorescence emission observed for all probes in the BSA sample confirmed their high selectivity for Aβ fibrils. This result further supports the potential of CL-07 and CL-21 as specific probes for Aβ fibril detection.

[0071] Embodiment 5 In this example, in order to study the changes in fluorescence response when CL-07 and CL-21 probes react with Aβ fibers of different concentrations, 1 μM concentration of CL-07 and Aβ fibers in the concentration range of 1-30 μM, and 1 μM concentration of CL-21 and Aβ fibers in the concentration range of 1-40 μM were used respectively. All experiments were carried out at room temperature, and the incubation time of the probe and Aβ was 1 minute. The fluorescence signal was measured by a fluorescence spectrometer, and the fluorescence intensity was recorded within a specific wavelength range. The specific fluorescence detection conditions are as follows: For the reaction system of CL-07 and Aβ fibers: the excitation wavelength (ex) is 555 nm, the emission wavelength (em) is 570 nm, and the spectral bandwidth (slit) is set to 1.9 nm. For the reaction system of CL-21 and Aβ fibers: the excitation wavelength (ex) is 555 nm, the emission wavelength (em) is 570 nm, and the spectral bandwidth (slit) is set to 1.9 nm.

[0072] like Figure 5As shown in the figure, with the increase of Aβ fiber concentration, the fluorescence intensity of CL-07 and CL-21 probes showed a significant increase trend. This result shows that CL-07 and CL-21 probes can sensitively respond to the changes of Aβ fiber concentrations, and their fluorescence intensity increases with the increase of Aβ fiber concentration, which shows that CL-07 and CL-21 probes have good sensitivity and specificity within the range of Aβ fiber concentration. It is calculated that the detection limit of CL-07 for Aβ fiber is 28nM (i.e. 0.028 μm), and the detection limit of CL-21 for Aβ fiber is 60nM (i.e. 0.060 μm).

[0073] Embodiment 6 Probes for Aβ imaging studies in mice Mice were anesthetized to a deep state of anesthesia and perfused transcardially with PBS and 4% formalin. The brains of mice were removed and fixed in 4% formalin for 24 hours, then transferred to 30% sucrose solution and stored at 4°C until the tissue settled. The brains were cut into serial sections with a thickness of 25 μm using a freezing microtome. The frozen sections were blocked in a blocking solution containing 5% goat serum, 3% BSA, and 0.4% Triton X-100. After blocking for 2 hours at room temperature, the sections were incubated with monoclonal mouse antibodies (6E10, 1:1000, Biolegend, SIG-39320) at 4°C overnight. After washing, the sections were incubated with fluorescent secondary antibodies (Alexa488 goat anti-rabbit antibody, A11070) for 2 hours at room temperature. After immunofluorescence staining, the sections were stained with CL-21 for 2 hours and washed with PBS. To better confirm the co-staining effect, adjacent sections were taken and stained with ThT (10 μM, 10% ethanol / water, Shanghai Aladdin Bio, Shanghai, China) for 1 h, incubated at room temperature, washed with PBS, stained with CL-21 for 2 h, and washed with 40% ethanol and PBS. After removing the residual liquid with dust-free paper, the sections were mounted on coverslips containing DAPI. Brain sections were observed using a confocal laser fluorescence microscope (Leica Stellaris 5). The excitation wavelength of CL-21 was 596 nm and the emission wavelength was 650 nm; the excitation wavelength of the secondary antibody was 488 nm and the emission wavelength was 595 nm; the excitation wavelength of ThT was 405 nm and the emission wavelength was 485 nm.

[0074] like Figure 6 As shown, based on the positive results of CL-21 and CL-07 on Aβ fibers in vitro, this example further studies its specific imaging ability and practical application in the brain tissue of APP / PS1 model mice. Compared with the wild-type control group, CL-21 can well image Aβ plaques in the brain slices of Alzheimer's model mice, and an obvious red fluorescence is observed.

[0075] Embodiment 8 To evaluate the potential binding capacity of CL-21 in vivo, near-infrared fluorescence (NIRF) imaging was performed in 5-month-old APP / PS1 transgenic mice (n = 3) and C57 mice (n = 3). Animals were anesthetized with 1.5% isoflurane in oxygen. CL-21 was injected via the tail vein at a dose of 0.5 mg / kg (vehicle 30% dimethylsulfoxide (DMSO), 30% propylene glycol, 40% PBS). The hair on the head of the animals was shaved to avoid light absorption by black hair. No toxic effects were observed at this dose. In vivo NIRF imaging was performed using an IVIS imaging system (IVIS Lumina LT system, PerkinElmer, USA) using appropriate filters (excitation wavelength λex of 600 nm and emission wavelength λem of 700 nm). To uniformly illuminate the entire animal, fluorescence excitation was performed using light at a wavelength of 640 nm. NIRF images were acquired at multiple time points from 1 minute to 1500 minutes (25 hours) after injection. Data were analyzed using Living Imaging software (Caliper Lifescience, USA) and quantitatively assessed using the region of interest (ROI) analysis tool. Autofluorescence was measured immediately before drug administration and showed negligible autofluorescence.

[0076] like Figure 7 As shown, in vivo fluorescence imaging was performed using CL-21 mice. The probe quickly crossed the blood-brain barrier and showed fluorescence differences after 5 minutes, with the fluorescence of CL-21 increasing by 1 times. In addition, the fluorescence signal in the brain of AD model mice was significantly higher than that in wild-type (WT) mice.

[0077] Embodiment 9 Nineteen-month-old mice and their corresponding littermates were sacrificed 180 minutes after CL-21 injection and perfused with PBS and 4% formalin. The brains were removed and embedded in optimal cutting temperature compound (OCT). The brains were cut into 25 µm thick sections and stained with ThT (10 μM, 10% ethanol / water, Shanghai Aladdin Bio, Shanghai, China) for 1 hour, incubated at room temperature, and then washed with PBS.

[0078] Experimental results ( Figure 8) showed that in wild-type mice, neither THT nor CL-21 showed obvious fluorescence signals. In Alzheimer's disease (AD) model mice, THT successfully detected Aβ plaques, and CL-21 was also able to effectively detect Aβ plaques, and showed good co-localization with the results of THT. This result shows that CL-21, as a near-infrared fluorescent probe, can effectively label Aβ plaques, and its performance in the Alzheimer's disease model is consistent with that of the traditional dye THT, and has good imaging capabilities.

[0079] Embodiment 10 In this example, the stock solutions (1 μM) of CL-01, CL-05, CL-07, CL-11 and CL-21 were added to an appropriate amount of PBS (pH 7.4), and then Aβ fiber aggregates (25 μM) were added. After incubation at 37°C for 5 minutes, the reaction solution was transferred to a quartz fluorescent dish, and its fluorescence intensity was measured using a fluorescence spectrometer. The measurement conditions for CL-01, CL-05, CL-07 and CL-11 were: excitation wavelength 560 nm, emission wavelength 580 nm, and bandwidth set to 3; the measurement conditions for ThT were: excitation wavelength 440 nm, emission wavelength 460 nm, and bandwidth set to 3; the measurement conditions for CL-21 were: excitation wavelength 575 nm, emission wavelength 595 nm, and bandwidth set to 5. At the same time, a probe without Aβ itself was prepared as a blank control, and measurements were performed under the same experimental conditions for comparison.

[0080] The results are as follows Fig. 9 and Fig.10 As shown in Figure 2, after incubation with 25 μM Aβ fibers, CL-01 and CL-11 emitted low-intensity fluorescence. 1 When the structure is R 1 and R 2 The group's CL-05, CL-07 and CL-21 can be used as near-infrared fluorescent probes for detecting Aβ plaques.

[0081] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. An Aβ plaque probe, characterized in that: The probe has the structural formula shown in Formula I: （I）； Wherein R1 is selected from ; R2 is selected from , or .

2. The probe according to claim 1, characterized in that The probe has the following structure: 。 3. The probe according to claim 1, characterized in that The probe has the following structure: 。 4. The method for preparing the Aβ plaque probe according to any one of claims 1 to 3, characterized in that: The following steps are involved: S101 Compound W166 and a first reagent are dissolved in toluene, glacial acetic acid and piperidine are added, and the mixture is heated and stirred to obtain Compound 1; the first reagent includes p-dimethylaminobenzaldehyde, or 4-dimethylaminocinnamaldehyde, or 5-[4-(dimethylamino)phenyl]thiophene-2-carboxaldehyde; S102 The compound 1 and N-hydroxysuccinimide are dissolved in dichloromethane, N,N-diisopropylethylamine is added, and the mixture is stirred at room temperature to obtain compound 2; S103 The compound 2 and 2-(2-aminoethoxy)ethoxy) are dissolved in dichloromethane, N,N-diisopropylethylamine is added, and the mixture is stirred at room temperature to obtain compound 3; Wherein R2 is selected from , or .

5. Use of the Aβ plaque probe according to any one of claims 1 to 3 in preparing a detection reagent for β-amyloid protein and / or aggregates formed by β-amyloid protein.

6. The use according to claim 5, characterized in that The concentration of the β-amyloid protein or the aggregates formed by the β-amyloid protein is at least 28 nM.

7. Use of the Aβ plaque probe according to any one of claims 1 to 3 in preparing a detection reagent for Alzheimer's disease.

8. The use according to claim 7, characterized in that The detection reagent is used for dynamic imaging of beta-amyloid protein and / or aggregates formed by beta-amyloid protein in brain tissue.

9. The use according to claim 7, characterized in that The detection reagent is used for disease monitoring and / or early diagnosis of Alzheimer's disease.

10. The use according to claim 7, characterized in that The dosage of the Aβ plaque probe includes 0.1 - 1.5 mg / kg.

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