An Aβ plaque probe, its preparation method and use

Abstract

The present invention relates to the field of fluorescent dyes, and particularly relates to an Aβ plaque probe, a preparation method thereof, and uses thereof. The present invention provides an Aβ plaque probe having a structural formula shown in Formula I: #imgabs0# (I); wherein, R1 is selected from #imgabs1#; R2 is selected from #imgabs2#, #imgabs3# or #imgabs4#. 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 chemical tool with broad prospects for brain imaging, and is applicable to application scenarios such as early diagnosis, disease monitoring, and disease progression tracking of Alzheimer's disease.

Description

Technical Field

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

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

[0003] β-amyloid (Aβ) is the main pathological marker of Alzheimer's disease. In the amyloidogenic pathway, Aβ peptides are generated by the cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase, producing monomeric Aβ peptides of different lengths. These monomeric Aβ peptides aggregate into higher-order structures, including oligomers, fibrils, and plaques. In addition, Aβ plaques usually appear in the deep cortical layer (>500 μm depth) of Alzheimer's disease mouse models, which also emphasizes the importance of deep tissue imaging techniques for the study of Alzheimer's disease (such as the early stage) and its progression.

[0004] Fluorescence molecular imaging has gradually been recognized as a versatile means for studying the pathogenesis of Alzheimer's disease and basic research due to its high sensitivity, rapid response, simple technique, 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 radiolabeling, these probes can cross the blood-brain barrier (BBB). However, current fluorescent small molecule probes still face many challenges. For example, they usually have a short excitation wavelength, which limits their penetration depth and is accompanied by strong background fluorescence.

[0005] In contrast, near-infrared (NIR) fluorescence probes have gradually become an important option for the early diagnosis of Alzheimer's disease due to their advantages such as low autofluorescence, stronger tissue penetration ability, less light scattering, and low photobleaching. However, when existing near-infrared fluorescence probes (such as Chinese patent applications CN110615808A and CN114656447A) are applied to in vivo imaging, there are problems such as a large administration dose (e.g., above 2 mg / kg) and low penetration efficiency (e.g., requiring more than 15 minutes). That is to say, existing near-infrared fluorescence 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). 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:

[0007] (I);

[0008] Wherein, R 1 is selected from ;

[0009] R 2 is selected from , or .

[0010] In some embodiments, the probe has the following structure:

[0011] .

[0012] In some embodiments, the probe has the following structure:

[0013] .

[0014] In some embodiments, the probe has the following structure:

[0015] .

[0016] In some embodiments, R 1 can also be .

[0017] In some embodiments, the probe has the following structure:

[0018] .

[0019] In a second aspect, the present invention provides a method for preparing the above Aβ plaque probe, characterized in that it includes the following steps:

[0020] S101 Dissolve compound W166 and the first reagent in toluene. After adding glacial acetic acid and piperidine, heat and stir to obtain compound 1;

[0021]

[0022] In some embodiments, the first reagent includes p-dimethylaminobenzaldehyde, or 4-dimethylaminocinnamaldehyde, or 5-[4-(dimethylamino)phenyl]thiophene-2-carbaldehyde.

[0023] In some embodiments, the molar ratio of compound W166 to the first reagent is 1:(1 - 1.5).

[0024] In some embodiments, the heating temperature in S101 is 50 - 65 °C.

[0025] In some embodiments, the heating temperature in S101 is 60 °C.

[0026] In some embodiments, the stirring time in S101 is 1.5 - 3 hours.

[0027] In some embodiments, the stirring time in S101 is 1.5 hours.

[0028] S102 Dissolve compound 1 and N-hydroxysuccinimide in dichloromethane. After adding N,N-diisopropylethylamine, stir at room temperature to obtain compound 2;

[0029]

[0030] In some embodiments, the molar ratio of compound 1 to N-hydroxysuccinimide is 1:(1 - 1.5).

[0031] In some embodiments, the stirring time in S102 is 5 - 8 hours.

[0032] In some embodiments, the stirring time in S102 is 5 hours.

[0033] S103 Dissolve compound 2 and 2-(2-aminoethoxy)ethoxy) in dichloromethane. After adding N,N-diisopropylethylamine, stir at room temperature to obtain compound 3;

[0034]

[0035] wherein R 2 is selected from , or .

[0036] In some embodiments, the molar ratio of the compound 2 to 2-(2-aminoethoxy)ethoxy) is 1:(1 - 1.5).

[0037] In some embodiments, the stirring time in S103 is 2 - 5 hours.

[0038] In some embodiments, the stirring time in S103 is 2.5 hours.

[0039] In a third aspect, the present invention provides the use of the above Aβ plaque probe in the preparation of a detection reagent for β-amyloid and / or aggregates formed by β-amyloid.

[0040] In some embodiments, the aggregates formed by β-amyloid include Aβ oligomers, Aβ fibrils, Aβ plaques, or a combination thereof.

[0041] In some embodiments, the aggregates formed by β-amyloid are Aβ fibrils and / or Aβ plaques.

[0042] In some embodiments, the detection reagent is used for in vitro and / or in vivo detection.

[0043] In some embodiments, the detection reagent is used to detect β-amyloid and / or aggregates formed by β-amyloid in brain tissue.

[0044] In some embodiments, the detection reagent is used to perform dynamic imaging on β-amyloid and / or aggregates formed by β-amyloid in brain tissue.

[0045] In some embodiments, the concentration of β-amyloid or the aggregates formed by β-amyloid is at least 28 nM.

[0046] In some embodiments, the concentration of β-amyloid or the aggregates formed by β-amyloid is ≥1 μM.

[0047] In some embodiments, the concentration of β-amyloid or the aggregates formed by β-amyloid is 1 - 40 μM.

[0048] In a fourth aspect, the present invention provides the use of the above Aβ plaque probe in the preparation of a detection reagent for Alzheimer's disease.

[0049] In some embodiments, the detection reagent is used for in vitro and / or in vivo detection.

[0050] In some embodiments, the detection reagent is used to detect β-amyloid and / or aggregates formed by β-amyloid in brain tissue.

[0051] In some embodiments, the detection reagent is used for dynamically imaging β-amyloid protein and / or aggregates formed by β-amyloid protein in brain tissue.

[0052] In some embodiments, the concentration of the β-amyloid protein or the aggregates formed by the β-amyloid protein is at least 28 nM.

[0053] In some embodiments, the concentration of the β-amyloid protein or the aggregates formed by the β-amyloid protein is ≥1 μM.

[0054] In some embodiments, the concentration of the β-amyloid protein or the aggregates formed by the β-amyloid protein includes 1 - 40 μM.

[0055] In some embodiments, the detection reagent is used for disease monitoring and / or early diagnosis of Alzheimer's disease.

[0056] In some embodiments, the dose of the Aβ plaque probe includes 0.1 – 1.5 mg / kg.

[0057] In some embodiments, the dose of the Aβ plaque probe includes 0.5 – 1.5 mg / kg.

[0058] Compared with the prior art, the beneficial effects of the present invention at least include the following aspects:

[0059] 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 penetrability. 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 ability, and has obvious advantages in detecting fibers and plaques in Aβ-like lesions of Alzheimer's disease.

[0060] It should be emphasized that the simultaneous setting of R 1 and R 2 successfully extends the emission wavelength of the probe to the near-infrared region, ensuring that the Aβ plaque probe provided by the present invention has a higher fluorescence turn-on ratio. Furthermore, it can provide more sensitive and accurate imaging signals, significantly improving the resolution and reliability of imaging. At the same time, it also ensures that the Aβ plaque probe provided by the present invention has excellent blood-brain barrier penetrability. On the basis of meeting the requirements of penetrating the blood-brain barrier, it can quickly and efficiently penetrate living brain tissue and selectively bind to Aβ plaques at a lower dose (such as 0.5 mg / kg) and a faster penetration speed (such as within 5 minutes), showing the ability to dynamically image Aβ plaques in living brain tissue, and providing a powerful imaging tool for the early diagnosis and dynamic monitoring of Alzheimer's disease.

[0061] 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.

[0062] 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

[0063] 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.

[0064] Figure 1 Absorption spectra of CL-07 (A) and CL-21 (B) in solvents of different polarities;

[0065] Figure 2 The emission spectra of CL-07 (A) and CL-21 (B) in solvents of different polarity;

[0066] 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;

[0067] Figure 4 Results graph of the selectivity of CL-07 (A) and CL-21 (B) for Aβ plaques;

[0068] Figure 5 Results graph of the fluorescence intensity change after the action of CL-07 with Aβ fibers at different concentrations (0 - 25 μM) (A), and the fluorescence intensity change after the action of CL-21 with Aβ fibers at different concentrations (0 - 40 μM) (B);

[0069] Figure 6 Results graph of the Aβ plaque imaging of the brain slices of Alzheimer's disease model mice with CL-21;

[0070] Figure 7 Results graph of the in vivo fluorescence imaging of CL-21 (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);

[0071] Figure 8 Results graph showing the co-localization of the results of CL-21 and THT;

[0072] Figure 9 Results graph of the fluorescence turn-on ratio of different compounds;

[0073] Figure 10 Results graph of the fluorescence intensity change after different compounds were co-incubated with Aβ fibers at the same concentration under the same conditions. Detailed implementation manners

[0074] To make the objectives, technical solutions, and advantages 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 in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0075] In this article, terms such as "upper", "lower", "inner", "outer", "front", "rear", "one end", "the other end", etc. indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be construed as a limitation of the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0076] In this article, "and / or" includes any and all combinations of one or more of the listed related items.

[0077] As used herein, "a plurality of" means two or more, i.e., it includes two, three, four, five, etc.

[0078] It should be noted that, as used herein, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or apparatus. Without further limitation, an element defined by the statement "comprising one..." does not exclude the presence of additional identical elements in the process, method, article or apparatus comprising such element.

[0079] 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.

[0080] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Thus, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that 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 the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0081] There are no particular restrictions on the sources of all raw materials of the present invention, and they can be purchased on the market or prepared according to conventional methods well-known to those skilled in the art. There are no particular restrictions on the purity of all raw materials of the present invention, and the present invention preferably adopts analytical purity or the conventional purity requirements in the field of fluorescent dyes. The trade names and abbreviations of all raw materials of the present invention are all conventional trade names and abbreviations in the art, and each trade name and abbreviation is clear and definite within the field of its relevant use. Those skilled in the art can purchase them from the market or prepare them by conventional methods according to the trade name, abbreviation, and corresponding use.

[0082] The present invention has no particular restrictions on the expression mode of the substituents, and all use the expression modes well-known to those skilled in the art. Based on common sense, those skilled in the art can correctly understand their meanings according to their expression modes.

[0083] Example 1

[0084] Preparation of Probe

[0085]

[0086] CL-07: Dissolve compound W166 (49.1 mg, 0.15 mmol) and 4-dimethylaminobenzaldehyde (i.e., p-dimethylaminobenzaldehyde, 29.1 mg, 0.195 mmol) in toluene (2.0 mL), add glacial acetic acid (28 μL) and piperidine (60 μL). Heat the mixture to 60 °C and stir for 1.5 hours, and monitor the reaction process by liquid chromatography - mass spectrometry. After the reaction is completed, cool the mixture to room temperature, remove the solvent components by a rotary evaporator to obtain a crude product, and distill off the organic solvent under reduced pressure. Purify with a neutral alumina chromatography column using methanol:dichloromethane as the eluent, and collect to obtain a blue-violet solid powder CL-04 with a yield of 70%.

[0087] Dissolve CL-04 (68.8 mg, 0.15 mmol) and N-hydroxysuccinimide (50 mg, 0.195 mmol) in dichloromethane (2.0 mL), add N,N-diisopropylethylamine (39 μL, 0.225 mmol). Stir the mixture at room temperature for 5 hours, and monitor the reaction process by liquid chromatography - mass spectrometry. After the reaction is completed, remove the solvent components by a rotary evaporator to obtain a crude product. Purify with dichloromethane:ethyl acetate (25:1) as the eluent, and collect to obtain CL-06 with a yield of 60%.

[0088] Dissolve CL-06 (16.7 mg, 0.03 mmol) and 2-(2-aminoethoxy)ethoxy) (6 mg, 0.04 mmol) in dichloromethane (1.0 mL), add N,N-diisopropylethylamine (10 μL). Stir the mixture at room temperature for 2.5 hours, and monitor the reaction process by liquid chromatography - mass spectrometry. Purify with dichloromethane:methanol as the eluent, and collect to obtain CL-07 with a yield of 70%.

[0089] 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).

[0090] 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.

[0091]

[0092] CL-21: Compound W166 (49.1 mg, 0.15 mmol) and 5-[4-(dimethylamino)phenyl]thiophene-2-carbaldehyde (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, the solvent components were removed by a rotary evaporator to obtain a crude product, and the organic solvents were removed by distillation under reduced pressure. Purification was carried out using a neutral alumina chromatography column with methanol:dichloromethane (15:1) as the eluent, and a blue-violet solid powder CL-18 was collected with a yield of 70%.

[0093] CL-18 (81.123 mg, 0.15 mmol) and N-hydroxysuccinimide (50 mg, 0.195 mmol) were added to N,N-diisopropylethylamine (39 µL, 0.225 mmol) dissolved in dichloromethane (2.0 mL). 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 components were removed by a rotary evaporator to obtain the crude product. Purification was carried out using dichloromethane:ethyl acetate (25:1) as the eluent, and CL-19 was collected with a yield of 60%.

[0094] 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. Purification was carried out using dichloromethane:methanol (15:1) as the eluent, and CL-21 was collected with a yield of 95%.

[0095] 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).

[0096] Example 2

[0097] Photophysical Properties of the Probe and Its Response to Aβ

[0098] To evaluate the optical properties of CL-07 and CL-21 probes, ultraviolet absorption and fluorescence spectroscopy tests were conducted in different solvents. Specifically, the ultraviolet 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). For the fluorescence spectroscopy test, CL-07 and CL-21 at a concentration of 1 μM were used (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 the emission wavelength and intensity in different solvents were measured.

[0099] As Figure 1 and Figure 2 shown, the ultraviolet absorption and fluorescence spectral properties of CL-07 and CL-21 in different polar solvents were measured. In terms of ultraviolet absorption, the absorption maximum of CL-07 in different solutions was between 550 nm and 650 nm, while the absorption maximum of CL-21 was in the range of 550 nm to 610 nm, indicating certain differences in the ultraviolet absorption wavelengths of the two. In the fluorescence spectroscopy test, CL-07 exhibited strong fluorescence intensity, and its emission wavelength was distributed between 620 nm and 660 nm. CL-21, on the other hand, showed a longer fluorescence emission wavelength, with an emission wavelength range of 700 nm to 750 nm and a larger Stokes shift. This property gives CL-21 a significant advantage in fluorescence imaging, enabling effective avoidance of fluorescence crosstalk problems, especially suitable for applications such as in-brain imaging that require high resolution and low interference. In addition, the solvent effect also plays an important role in the fluorescence properties. In aqueous solutions, the absorption spectrum of the probe showed a large blue shift, which may be due to the occurrence of J-aggregation in water.

[0100] Example 3

[0101] In this example, the stock solutions (1 μM) of CL-07 and CL-21 were respectively added to an appropriate amount of PBS (pH 7.4), and then Aβ fibril aggregates (25 μM) were added. After incubating at 37°C for 5 minutes, the reaction solution was transferred to a quartz fluorescence cuvette, 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, the probe itself without Aβ was prepared as a blank control and measured under the same experimental conditions for comparison.

[0102] The results are asFigure 3 As shown, the responsiveness of CL-07 and CL-21 to Aβ fibers was measured. Due to the TICT effect, they emit low-intensity fluorescence in the solvent, which is beneficial for constructing a fluorescence probe with a high signal-to-noise ratio. When the probe binds to Aβ fibers, the fluorescence intensity increases significantly. After incubation with 25 μM 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 indicate that CL-07 and CL-21 are very suitable molecular tools for detecting Aβ fibers.

[0103] Example 4

[0104] In this example, to verify the selectivity of CL-07 and CL-21 probes for Aβ protofibrils, two probes, CL-07 and CL-21, with a concentration of 1 μM were used in the experiment. The experimental design was as follows: The CL-07 (1 μM) and CL-21 (1 μM) probes were incubated with Aβ protofibrils (25 μM) or bovine serum albumin (BSA, 10 μM), and all samples in the reaction system were incubated at room temperature for 30 minutes. After incubation, the fluorescence intensity changes of the samples were measured using a fluorescence spectrometer. The specific fluorescence detection conditions were as follows: For CL-07: the excitation wavelength (ex) was 555 nm, the emission wavelength (em) was 580 nm, and the spectral bandwidth (slit) was set to 5 nm. For CL-21: the excitation wavelength (ex) was 575 nm, the emission wavelength (em) was 595 nm, and the spectral bandwidth (slit) was set to 5 nm.

[0105] As Figure 4 shown, when the CL-07 and CL-21 probes were co-incubated with Aβ protofibrils, both showed significant fluorescence enhancement. In contrast, the fluorescence signals were relatively weak when CL-07 and CL-21 were co-incubated with BSA. The weak fluorescence emission observed for all probes in the BSA samples confirmed their high selectivity for Aβ protofibrils. This result further supports the potential of CL-07 and CL-21 as specific probes for Aβ protofibril detection.

[0106] Example 5

[0107] In this example, to study the fluorescence response changes of CL-07 and CL-21 probes when reacting with different concentrations of Aβ fibers, CL-07 with a concentration of 1 μM was used with Aβ fibers in the concentration range of 1 - 30 μM, and CL-21 with a concentration of 1 μM was used with Aβ fibers in the concentration range of 1 - 40 μM. 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 using a fluorescence spectrometer, and the fluorescence intensity was recorded within a specific wavelength range. The specific fluorescence detection conditions were as follows:

[0108] 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.

[0109] As Figure 5 shown, with the increase in the concentration of Aβ fibers, the fluorescence intensities of both CL-07 and CL-21 probes showed a significant increasing trend. This result indicates that the CL-07 and CL-21 probes can sensitively respond to the changes in different concentrations of Aβ fibers, and their fluorescence intensities increase with the increase in the concentration of Aβ fibers, which shows that the CL-07 and CL-21 probes have good sensitivity and specificity within the concentration range of Aβ fibers. After calculation, the detection limit of CL-07 for Aβ fibers is 28 nM (i.e., 0.028 μm), and the detection limit of CL-21 for Aβ fibers is 60 nM (i.e., 0.060 μm).

[0110] Example 6

[0111] Probes for Aβ imaging studies in mice

[0112] The mice were anesthetized to a deep anesthetic state and subjected to transcardial perfusion with PBS and 4% formalin as the perfusion fluid. The mouse brains were removed and fixed in 4% formalin for 24 hours, then transferred to 30% sucrose solution and stored at 4°C until the tissues sank. The brains were sectioned into consecutive slices with a thickness of 25 μm using a cryostat. The cryosections 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 overnight at 4°C with a monoclonal mouse antibody (6E10, 1:1000, Biolegend, SIG-39320). After washing, the sections were incubated with a fluorescent secondary antibody (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 Biochemical Technology Co., Ltd., Shanghai, China) for 1 hour, incubated at room temperature, washed with PBS, then stained with CL-21 for 2 hours, and washed with 40% ethanol and PBS. After sucking off the residual liquid with lint-free paper, the sections were mounted on a coverslip containing DAPI. The 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.

[0113] As Figure 6 shown, based on the positive results of CL-21 and CL-07 on Aβ fibrils in vitro, this example further investigated their specific imaging ability and practical application in the brain tissues of APP / PS1 model mice. Compared with the wild-type control group, CL-21 was able to well image the Aβ plaques in the brain sections of Alzheimer's disease model mice, and an obvious red fluorescence was observed.

[0114] Example VIII

[0115] To evaluate the potential binding ability of CL-21 in vivo, 5-month-old APP / PS1 transgenic mice (n = 3) and C57 mice (n = 3) were studied using near-infrared fluorescence (NIRF) imaging technology. The 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 (the vehicle was 30% dimethyl sulfoxide (DMSO), 30% propylene glycol, 40% PBS). To avoid light absorption caused by black hair, the hair on the animal's head was shaved. No toxic reaction was observed at this dose. In vivo NIRF imaging was performed using an IVIS imaging system (IVIS Lumina LT system, PerkinElmer, USA), with appropriate filters (excitation wavelength λex = 600 nm, emission wavelength λem = 700 nm). To uniformly irradiate the whole animal, light with a wavelength of 640 nm was used for fluorescence excitation. NIRF images were acquired at multiple time points during the period from 1 minute to 1500 minutes (25 hours) after injection. The data were analyzed using Living Imaging software (Caliper Lifescience, USA), and quantitative evaluation was performed using the region of interest (ROI) analysis tool. Autofluorescence was measured immediately before drug administration, and the results showed that autofluorescence was negligible.

[0116] As Figure 7 shown, in vivo fluorescence imaging was performed using CL-21 in mice. The probe rapidly crossed the blood-brain barrier and showed a fluorescence difference after 5 min, with the fluorescence of CL-21 increasing by 1 fold. Moreover, the fluorescence signal in the brains of AD model mice was significantly higher than that in wild-type (WT) mice.

[0117] Example 9

[0118] Nineteen-month-old mice and corresponding littermates were sacrificed 180 minutes after injection of CL-21 and perfused with PBS and 4% formalin. After removing the brains, they were embedded using 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 Biochemical Technology Co., Ltd., Shanghai, China) for 1 hour, and then washed with PBS after incubation at room temperature.

[0119] Experimental results ( Figure 8)indicated 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. These results indicate that CL-21, as a near-infrared fluorescent probe, can effectively label Aβ plaques and has good imaging ability, showing the same performance as the traditional dye THT in the Alzheimer's disease model.

[0120] Example Ten

[0121] In this example, the stock solutions (1 μM) of CL-01, CL-05, CL-07, CL-11, and CL-21 were respectively added to an appropriate amount of PBS (pH 7.4), and then Aβ fiber aggregates (25 μM) were added. After incubating at 37 °C for 5 minutes, the reaction solution was transferred to a quartz fluorescence cuvette, 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, the probe itself without Aβ was prepared as a blank control and measured under the same experimental conditions for comparison.

[0122]

[0123] The results are as Figure 9 and Figure 10 shown. After incubation with 25 μM Aβ fibers, CL-01 and CL-11 emitted low-intensity fluorescence. The above results indicate that when CL-07 and CL-21 do not have the structure of R 1 , they show extremely low fluorescence enhancement and are difficult to be used as near-infrared fluorescent probes for detecting Aβ plaques. While CL-05, CL-07, and CL-21 with the groups of R 1 and R 2 can be used as near-infrared fluorescent probes for detecting Aβ plaques.

[0124] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the present invention and the claims. These all belong to the protection scope 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 .

2. The method for preparing the Aβ plaque probe according to claim 1, 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 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 .

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

4. The use according to claim 3, characterized in that The detection reagent is used for in vitro and / or in vivo detection.

5. Use of the Aβ plaque probe according to claim 1 in preparing a detection reagent for Alzheimer's disease.

6. The use according to claim 5, characterized in that The detection reagent is used for in vivo imaging of beta-amyloid protein and / or aggregates formed by beta-amyloid protein in brain tissue.

7. The use according to claim 5, characterized in that The detection reagent is used for disease monitoring and / or early diagnosis of Alzheimer's disease.

8. The use according to claim 5, characterized in that The detection reagent is used for in vitro and / or in vivo detection.

Images