A fluorescent probe for detecting amyloid oligomers and preparation and application thereof

Abstract

The application provides a fluorescent probe for detecting amyloid oligomers and preparation and application thereof, and belongs to the technical field of A beta imaging agents, wherein the fluorescent probe is a chalcone derivative, and is specifically a chalcone skeleton-based molecular twist limited charge transfer and excited state proton transfer process optimization and development, which can be used as a specific fluorescent chemical probe for detecting and imaging A beta aggregates, and can dynamically monitor A beta aggregates in an in-vitro fiber formation process, and has important significance for A beta related pathological detection and research.

Description

Technical Field

[0001] The present invention relates to the technical field of Aβ imaging agents, and in particular to a fluorescent probe for detecting amyloid protein oligomers, a preparation method and application thereof, wherein the fluorescent probe is a novel chalcone derivative. Background Art

[0002] Protein misfolding diseases are a class of disorders associated with the tissue deposition of β-sheet-rich, filamentous protein aggregates, commonly referred to as amyloid fibrils. Alzheimer's disease (AD), one of the most extensively studied protein misfolding diseases, is characterized by the aggregation of β-amyloid (Aβ) in the brain and the formation of extracellular neuritic plaques. AD currently affects over 35 million people worldwide, a number expected to increase significantly as the population ages.

[0003] Amyloid proteins and peptides can form multiple distinct assembly states, and a key question is which assembly state is most closely associated with disease pathogenesis. Plaque deposition, resulting from Aβ fibrillization, has long been considered the primary cause of neurodegeneration in AD. However, recent studies suggest that Aβ oligomers (composed of 2-12 Aβ monomers), primarily present in the initial lagging and growing stages of Aβ fibrils, are the primary drivers of AD pathogenesis, with their concentration levels playing a more crucial role in disease progression. The concept of Aβ intermediates involved in AD development could explain the weak correlation between amyloid pathology defined by Aβ plaque burden and clinical AD manifestations, suggesting that amyloid plaques may represent a relatively nontoxic aggregated form of Aβ. Therefore, the development of assays capable of recognizing multiple Aβ peptide morphologies is urgently needed.

[0004] Chalcone, a compound containing a 1,3-diphenylpropenone structure, is a precursor to open-chain flavonoids and isoflavonoids found in edible plants. In recent years, it has attracted increasing attention due to its diverse potential pharmacological applications. Chalcone and its derivatives exhibit a wide range of pharmacological activities, and chemical derivatization offers a high degree of structural diversity, which has been shown to facilitate the development of novel drugs with higher potency and lower toxicity. Summary of the Invention

[0005] In order to develop a more effective Aβ imaging agent, the present invention uses the chalcone structure as the core of the imaging agent for the first time, designs and synthesizes a chalcone derivative and successfully applies it to the detection of Aβ aggregates. The compound has obvious advantages in the detection of Aβ aggregates.

[0006] In a first aspect, the present invention provides a novel chalcone derivative, the structural formula of which is shown below:

[0007] .

[0008] In a second aspect, the present invention provides a method for preparing the above-mentioned chalcone derivative, comprising the following steps:

[0009] S1, 2-bromo-5-hydroxyacetophenone reacts with iodomethane under alkaline conditions to obtain a methylated product of 2-bromo-5-methoxyacetophenone;

[0010] S2, the methylated product of 2-bromo-5-methoxyacetophenone is subjected to a Suzuki coupling reaction with 4-(dimethylamino)phenylboronic acid in the presence of a palladium catalyst and an inorganic base to obtain an intermediate 1;

[0011] S3, dissolving the intermediate product 1, 4-(dimethylamino)benzaldehyde in methanol, adding potassium hydroxide and reacting to obtain the intermediate product 2;

[0012] S4, intermediate 2 reacts with boron tribromide to obtain the target product;

[0013] Among them, the structural formula of intermediate product 1 is as follows:

[0014] ;

[0015] The structural formula of intermediate product 2 is shown below:

[0016] .

[0017] Preferably, in the above preparation method, the reaction in step S2 uses 1,4-dioxane as solvent.

[0018] Preferably, in the above preparation method, the molar ratio of the methylated product, 4-(dimethylamino)phenylboronic acid and the inorganic base is (1-1.1):(1-1.1):(1-1.2), wherein the inorganic base is selected from cesium carbonate, potassium carbonate and sodium hydroxide.

[0019] Preferably, in the above preparation method, the reaction temperature in step S2 is 100-105°C.

[0020] In some embodiments of the present invention, the palladium catalyst used in step S2 is specifically Pd(PPh3)2Cl2.

[0021] Preferably, in step S3 of the above preparation method, the molar ratio of the intermediate product 1, 4-(dimethylamino)benzaldehyde and potassium hydroxide is (1-1.1):(1-1.1):(1-1.1).

[0022] Preferably, in the above preparation method, the reaction temperature in step S3 is 25-35°C.

[0023] Preferably, in the above preparation method, the reaction temperature of step S4 is 25-35° C., and the molar ratio of the intermediate product 2 to boron tribromide is (1-1.1):(1-1.5).

[0024] In a third aspect, the present invention provides the use of the above-mentioned chalcone derivatives, which is at least any of the following:

[0025] a) As a fluorescent probe to detect Aβ aggregates;

[0026] b) preparing diagnostic products for Alzheimer's disease;

[0027] c) Preparation of Aβ aggregate imaging reagents.

[0028] In some embodiments of the present invention, the Aβ aggregates are Aβ42 monomers, oligomers, and aggregates.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This study, for the first time, utilizes a chalcone structure as the core of an Aβ42 imaging agent. A novel chalcone derivative was designed and synthesized. The derivative's dual rotation axis enhances its sensitivity to the protein microenvironment, enabling more effective sensing of protein aggregation. Furthermore, its excited-state proton transfer (ESIPT) and twisted intramolecular charge transfer (TICT) properties further enhance its Stokes shift and fluorescence signal intensity. Experimental data demonstrate that this derivative exhibits promising results as a probe for detecting Aβ42 aggregates. Dynamic monitoring of in vitro fibril formation has been demonstrated in an in vitro Alzheimer's disease model, suggesting potential applications for tracking and monitoring Aβ protein aggregation.

[0031] The synthetic route provided by the present invention ensures high yield and purity of the product by utilizing the reflux conditions of the Suzuki coupling reaction in 1,4-dioxane. This reaction pathway lays the foundation for the successful preparation of the chalcone derivatives of the present invention and provides the possibility for further applied research. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] To more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0033] Figure 1 : is a synthetic route of the chalcone derivative M3 in Example 1 of the present invention;

[0034] Figure 2 is the chalcone derivative M3 in Example 1 of the present invention1 H NMR spectrum;

[0035] Figure 3 is the chalcone derivative M3 in Example 1 of the present invention 13 C NMR spectrum;

[0036] Figure 4 The synthetic route of compounds M1 and M2 in Comparative Example 1 of the present invention is shown in FIG.

[0037] Figure 5 is the compound M1 in Comparative Example 1 of the present invention 1 H NMR spectrum;

[0038] Figure 6 is the compound M2 in Comparative Example 2 of the present invention 1 H NMR spectrum;

[0039] Figure 7 The absorption spectra (a) and fluorescence emission spectra (b) of compounds M1, M2 and M3 of the present invention are shown in FIG.

[0040] Figure 8 The fluorescence spectra of compounds M1, M2 and M3 in the present invention at different H2O / THF mixed solvent ratios;

[0041] Figure 9 The energy change curves of the ground state (S0) and excited state (S1) of compounds M1, M2 and M3 in the present invention caused by scanning the rotation of the aniline group under the TD-DFT method; wherein, a is the benzene rotation on the left side of M1-scan-TD scan, b is the benzene rotation on the right side of M2-scan-TD scan, c is the left rotation of M3-scanL-TD scan, and d is the benzene rotation on the right side of M3-scanR-TD scan;

[0042] Figure 10 Figure 5 is the energy change curve of the S0 and S1 states caused by scanning H migration of compounds M1, M2 and M3 in the present invention under the TD-DFT method; wherein, a is the M1-scanH-TD rigid scan H moving from the left to the right, b is the M2-scanH-TD rigid scan H moving from the left to the right, and c is the M3-scanH-TD rigid scan H moving from the left to the right;

[0043] Figure 11 This is the binding test of M1, M2 and M3 with Aβ oligomers in Example 2 of the present invention; wherein ac is the molecular docking result, and df is the saturation binding curve of M1, M2 and M3 with Aβ42 oligomers;

[0044] Figure 12 : The fluorescence emission spectra of M1, M2 and M3 before and after binding to Aβ42 oligomers in Example 2 of the present invention;

[0045] Figure 13 The fluorescence spectrum of M3 in different concentrations of Aβ oligomer and fibril environment in Example 3 of the present application; wherein a-c are the fluorescence spectrum in the Aβ42 oligomer environment, d-f are the fluorescence spectrum in the Aβ42 fibril environment;

[0046] Figure 14 The fluorescence imaging of color developer ThT and M3 in different Aβ aggregate environments in Example 4 of the present application, wherein a-d are the fluorescence images of ThT, and e-h are the fluorescence images of M3. DETAILED DESCRIPTION

[0047] The embodiments of the technical solutions of the present application will be described in detail below with reference to the accompanying drawings. The following examples are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, and cannot limit the protection scope of the present application.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present application; the terms "include" and "have" and any variations thereof in the specification and claims of the present application and the above description of drawings are intended to cover non-exclusive inclusion.

[0049] The aggregation of Aβ is closely related to the pathological process of Alzheimer's disease, and soluble Aβ aggregates are widely considered to be key pathogenic substances. Identifying and characterizing intermediate species formed during aggregation is crucial for understanding aggregate-mediated neuronal toxicity and its mechanism in disease progression. However, due to the heterogeneity and transience of these substances, it is extremely challenging to detect them, and the lack of reliability of existing sensors further exacerbates the problem. To solve this problem, the present application develops a specific fluorescence probe for detecting aggregates, which is based on the optimization of molecular twist restricted charge transfer and excited state proton transfer processes based on the chalcone skeleton, and its structural formula is as follows:

[0050] ;

[0051] Due to the complex molecular structure and double-rotation axis characteristics of the fluorescence probe, it has superior performance in detecting and imaging Aβ aggregates, and is expected to be used for high-sensitivity Aβ-related pathological detection and research.

[0052] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product specifications were used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased commercially.

[0053] Example 1

[0054] This example provides a chalcone derivative (denoted as M3), the structural formula of which is shown below:

[0055] .

[0056] The synthetic route of the compound M3 is as follows: Figure 1 As shown, the preparation method comprises the following steps:

[0057] Step 1: 2-bromo-5-hydroxyacetophenone is used as the starting material and reacted with iodomethane (CH3I, 1 equivalent) under appropriate alkaline conditions to obtain the methylated product of 2-bromo-5-methoxyacetophenone.

[0058] Step 2: The methylated product was subjected to a Suzuki coupling reaction with 4-(dimethylamino)phenylboronic acid (1 equivalent) in the presence of a palladium catalyst (Pd(PPh3)2Cl2) and cesium carbonate (Cs2CO3, 1 equivalent). The reaction was carried out in 1,4-dioxane solvent at reflux conditions (100°C) for 24 hours to ensure the complete coupling reaction. After the reaction was completed, the intermediate product 1 was obtained through cooling, extraction and purification steps.

[0059] Step 3: The intermediate obtained in Step S2 and 4-(dimethylamino)benzaldehyde (1 equivalent) were dissolved in methanol (CH3OH). Potassium hydroxide (KOH, 1 equivalent) was added and the reaction was stirred at room temperature for 12 hours. After completion of the reaction, the pH was adjusted to neutral with hydrochloric acid solution (1 M), and the mixture was filtered and purified to obtain Intermediate 2.

[0060] Step 4: The intermediate obtained in Step 3 is reacted with boron tribromide (BBr3, 1 equivalent) at room temperature. After the reaction is complete, the reaction mixture is cooled and subjected to extraction and purification steps to obtain the target product M3.

[0061] M3 1 H NMR spectrum Figure 2 As shown, M3 13 C NMR spectrum Figure 3 shown; among them, 1H NMR (400 MHz, D6-DMSO) δ 13.4(s,1H), 7.94(d,1H), 7.91(d,1H), 7.61(d, 1H), 7.57(d, 1H), 7.49(d, 1H), 7.26(dd, 1H), 7.21(m, 2H), 7.17(d, 1H), 6.80(d, 1H), 6.72(s, 1H), 3.06(s, 3H), 3.02(s, 3H).

[0062] Comparative Example 1

[0063] In this example, two other compounds, M1 and M2, were designed and synthesized. Their structures are shown below:

[0064] .

[0065] (1) Preparation of compound M1.

[0066] like Figure 4 As shown in a, the preparation method of compound M1 comprises the following steps:

[0067] Step 1: 2-bromo-5-hydroxyacetophenone is used as the starting material and reacted with iodomethane (CH3I, 1 equivalent) under appropriate alkaline conditions to obtain the methylated product of 2-bromo-5-methoxyacetophenone;

[0068] Step 2: The methylated product was subjected to a Suzuki coupling reaction with 4-(dimethylamino)phenylboronic acid (1 equivalent) in the presence of a palladium catalyst (Pd(PPh3)2Cl2) and cesium carbonate (Cs2CO3, 1 equivalent); the reaction was carried out in 1,4-dioxane solvent under reflux conditions for 24 hours to ensure the complete coupling reaction; after completion of the reaction, the intermediate product 1 was obtained through cooling, extraction and purification steps.

[0069] Step 3: The intermediate product 1 obtained in step S2 is reacted with boron tribromide (BBr3, 1 equivalent) at room temperature. After the reaction is complete, the reaction mixture is cooled and subjected to extraction and purification steps to obtain the target product M1.

[0070] M1 1 H NMR spectrum Figure 5 As shown, 1H NMR (400 MHz, CD3Cl) δ 12.4 (s, 1H), 7.74(d, 1H), 7.58 (d, 2H), 7.17 (d, 1H), 7.14 (d, 1H), 6.80 (d, 1H), 6.79 (d,2H), 3.02 (s, 6H), 2.63 (s, 3H).

[0071] (2) Preparation of compound M2.

[0072] like Figure 4 As shown in b, the preparation method of compound M2 comprises the following steps:

[0073] o-Hydroxyacetophenone (1 equivalent) and 4-(dimethylamino)benzaldehyde (1 equivalent) were dissolved in methanol (CH3OH) at room temperature, followed by the addition of potassium hydroxide (KOH, 1 equivalent) as a basic catalyst. The mixture was stirred at room temperature for 12 hours to complete the condensation reaction. After the reaction, the pH was adjusted to neutral with a 1M hydrochloric acid solution, followed by filtration and purification to obtain the target product M2.

[0074] M2 1 H NMR spectrum Figure 6 As shown, 1 H NMR (400 MHz, D6-DMSO) δ 13.14 (s, 1H), 8.27 (d, 1H), 7.85-7.74 (m, 4H), 7.53 (t, 1H), 7.00-6.95 (m, 2H), 6.77 (d, 2H), 3.03 (s, 6H).

[0075] The following performance tests were performed on the above compounds M1, M2 and M3:

[0076] 1. Absorption spectrum and fluorescence emission spectrum.

[0077] The absorption and fluorescence emission spectra of M1, M2 and M3 in dimethyl sulfoxide (DMSO) solution were detected. The specific detection process was as follows: DMSO was selected as the solvent and the molar concentration of each solution was 1×10 -5 For the M1, M2 and M3 solutions of M, first test the absorption spectrum of the above samples, and then set the excitation wavelength according to the maximum absorption wavelength to measure the fluorescence emission spectrum.

[0078] Figure 7Figure (a) shows the normalized absorption spectra of M1, M2, and M3 in DMSO solution. The absorption peak of M1 is located at approximately 380 nm, the absorption peak of M2 is located at approximately 450 nm, and the absorption peak of M3 is further red-shifted to approximately 470 nm. These results demonstrate that the absorption properties of chalcone derivatives can be significantly tuned through structural modification.

[0079] Figure 7 Figure (b) shows the normalized fluorescence spectra of M1, M2, and M3 in DMSO solution. The fluorescence emission peak of M1 is located at approximately 508 nm, the emission peak of M2 is located at approximately 554 nm, and the fluorescence emission peak of M3 is further red-shifted to approximately 620 nm. These results indicate that structural modification of chalcone derivatives significantly affects their luminescence properties.

[0080] From the above results, it can be observed that M1, M2, and M3 all exhibit significant Stokes shifts. For example, the absorption peak of M1 is located at approximately 380 nm, while its fluorescence emission peak is located at approximately 508 nm, showing a Stokes shift of approximately 128 nm. This Stokes shift effectively separates the absorption and emission spectra, avoiding signal overlap and thus improving the detection performance of the fluorescent probe. M3 exhibits stronger luminescence intensity in the longer wavelength region, which may be attributed to the effective interaction between the electron donor and acceptor groups in its molecular structure, thereby improving its fluorescence quantum yield.

[0081] 2. Fluorescence spectrum in H2O / THF solvent.

[0082] Under different excitation wavelengths, the fluorescence spectra of M1, M2, and M3 in different H2O / THF mixed solvent ratios were detected. The specific detection process was as follows: by gradually adding water to THF to change the water fraction (fw) in the tetrahydrofuran (THF) / water mixture, the emission characteristics of M1, M2, and M3 were tested as the degree of molecular aggregation gradually increased.

[0083] Figure 8 Figure (a) shows the fluorescence spectrum of M1 in a H2O / THF mixture, with an excitation wavelength of 370 nm. The fluorescence emission peak is primarily distributed in the 500-600 nm range, and the fluorescence intensity gradually increases with increasing THF content. This indicates that the fluorescence properties of M1 are sensitive to changes in the solvent environment. While M1's fluorescence intensity is relatively weak due to its simple structure, it exhibits a significant response to changes in the solvent environment.

[0084] Figure 8Figure b shows the fluorescence spectrum of M2 in a H₂O / THF mixture, with an excitation wavelength of 450 nm. The fluorescence emission peak is located in the 500-700 nm range, and the fluorescence intensity increases significantly with increasing THF content. Compared to M1, the fluorescence intensity and emission wavelength of M2 increase significantly, which can be attributed to the extended molecular conjugation system, which significantly enhances the fluorescence performance.

[0085] Figure 8 Figure (c) shows the fluorescence spectrum of M3 in a H₂O / THF mixture, with an excitation wavelength of 370 nm. Compared to M1, M3's fluorescence emission peak also lies within the 500-700 nm range, but with higher intensity and a slightly different peak position. The addition of a second benzene ring to M3 creates a double rotation axis, significantly enhancing its fluorescence properties and making it more sensitive to the solvent environment.

[0086] Figure 8 Figure d shows the fluorescence spectrum of M3 in H2O / THF mixed solvent with an excitation wavelength of 450 nm. Figure 8 Compared with the spectrum of M2 in panel b, M3 exhibits higher fluorescence intensity and a slightly red-shifted emission peak at similar solvent ratios, further demonstrating the sensitivity of M3 to changes in the solvent environment.

[0087] The fluorescence behavior of M1, M2, and M3 in H2O / THF mixed solvents changes significantly with the change of solvent ratio, reflecting the different responsiveness of these dyes to the solvent environment. M1 has a simple structure, resulting in low fluorescence intensity, but is sensitive to environmental changes. M2 significantly enhances its fluorescence properties by extending the conjugated system and exhibits higher fluorescence intensity and emission wavelength in the solvent environment. The double rotation axis structure of M3 makes it superior to M1 and M2 in both fluorescence intensity and wavelength, and its sensitivity to the solvent environment is also significantly enhanced. This solvent-dependent fluorescence enhancement property gives M3 greater potential for applications in detecting solution environments.

[0088] 3. Analysis of energy changes caused by the rotation of the aniline group.

[0089] Using the TD-DFT method, the energy change curves of the S0 and S1 states caused by the rotation of the aniline groups in M1, M2 and M3 were scanned and displayed. The results are as follows: Figure 9 shown.

[0090] Figure 9a is the energy change analysis of the left benzene ring rotation in Ml-scan-TD. During the scan, dihedral angle 21-20-17-15 changed from 146.7 to 56.7 degrees. At step 19 (dihedral angle of 89.7 degrees), the S0 state energy reached the highest, while the S1 state energy was the lowest, and the S1-S0 energy gap dropped to 2.978 eV, which was the minimum. During the rotation, there was an energy barrier for the S0 state, with an energy of 0.155 eV, indicating that the molecular structure was in the most unstable state at this angle. In contrast, the S1 state energy gradually decreased and did not show a significant rotation energy barrier, suggesting that the benzene ring tended to be stable in the S1 state. The optimized Ml molecule showed that the aniline benzene ring was almost perpendicular to the phenol benzene ring in the S1 state. In summary, there was a rotation energy barrier for the S0 state and the structure was unstable; while in the S1 state, the rotation of the benzene ring was not limited by the energy barrier, and the structure was relatively stable, indicating that the aniline and phenol benzene rings tended to be close to perpendicular arrangement in the S1 state.

[0091] Figure 9 b is the energy change analysis of the right benzene ring rotation in M2-scan-TD. During the scan, dihedral angle 17-16-13-12 changed from 0.29 to 90.29 degrees. At step 30 (dihedral angle of 90.29 degrees), the S0 state energy was the highest, while the S1 state energy was the lowest, and the S1-S0 energy gap was 2.425 eV, which was the minimum. During the rotation of the S0 state, there was an energy barrier with an energy of 0.412 eV, indicating that the molecule was unstable at this angle. At step 9 (27.29 degrees), the S1 state showed a weak rotation energy barrier with an energy of 0.0105 eV, indicating that the excited state had a certain stability at this angle. The optimized M2 molecule showed that the right benzene ring was almost perpendicular to the phenol benzene ring in the S1 state. The S0 state had a large rotation energy barrier at 90.29 degrees, indicating that the structure was unstable at this angle; while the S1 state had a weak energy barrier at 27.29 degrees, showing unique stability in the excited state. The optimized structure showed that the right benzene ring tended to be perpendicular to the phenol benzene ring in the S1 state.

[0092] Figure 9c in the M3-scanL-TD left side benzene ring rotation energy change analysis. During the scanning process, the dihedral angle 21-20-17-15 changed from 146 degrees to 56 degrees. At the 19th step (dihedral angle 89 degrees), the energy of the S0 state and the S1 state reached the highest value, and the S1-S0 energy difference was 2.718 eV, which was the minimum value. The rotation energy barrier of the S0 state at 89 degrees was 0.147 eV, which was similar to M1. The S1 state also showed the highest energy at 89 degrees, and the rotation energy barrier was 0.101 eV, indicating that there was an energy barrier at this angle. The optimized M3 molecule showed a smaller angle between the left aniline benzene ring and the phenol benzene ring in the S1 state. Both the S0 state and the S1 state had a rotation energy barrier at 89 degrees, and the structure was unstable in both states. The energy could not be dissipated through the rotation of the left benzene ring, which might lead to the occurrence of fluorescence emission. The optimized structure showed that the angle between the left aniline benzene ring and the phenol benzene ring tended to decrease in the S1 state.

[0093] Figure 9 d in the M3-scanR-TD right side benzene ring rotation energy change analysis. During the scanning process, the dihedral angle 36-35-32-31 changed from 0.04 degrees to 90.04 degrees. At the 30th step (dihedral angle 90.04 degrees), the S0 state energy was the highest, and the S1-S0 energy difference was 2.538 eV, which was the minimum value; while the S1 state energy reached the highest at the 21st step (63.04 degrees). The rotation energy barrier of the S0 state at 90.04 degrees was 0.391 eV, which was similar to M2. The rotation energy barrier of the S1 state at 63.04 degrees was 0.124 eV, indicating that the S1 state had special stability at this angle. The optimized M3 molecule showed a smaller angle between the right benzene ring and the phenol benzene ring in the S1 state. The S0 state had a large rotation energy barrier at 90.04 degrees, indicating that the structure was unstable at this angle; while the S1 state had a rotation energy barrier at 63.04 degrees, showing a unique behavior in the excited state. Due to the high right rotation energy barrier of the S1 state, the energy was difficult to dissipate through the right rotation, thereby maintaining the fluorescence emission. The optimized structure showed that the angle between the right aniline benzene ring and the phenol benzene ring tended to decrease in the S1 state.

[0094] 4. Energy change analysis caused by H migration.

[0095] Using the TD-DFT method, the scanning showed the S0 and S1 state energy change curves caused by H migration in M1, M2 and M3, and the results are shown in Figure 10 .

[0096] Figure 10Figure a in the figure represents the energy change analysis of H moving from the left to the right during an M1-scan H-TD rigid scan. During the scan, the dihedral angle ∠H36C27O35 changes from 23.8 to 44.8 degrees. After step 16 of the scan, the energy rises significantly due to the close proximity of the H atom to the right oxygen atom, making this energy change negligible. As the H atom moves to the right, the S0 state energy gradually increases, indicating that the S0 state is most stable when H is on the left. The S1 state energy rises initially, then decreases slightly before rising again. The S1-S0 energy difference (excitation energy) gradually decreases, indicating that H movement from left to right is accompanied by a red shift in the UV absorption spectrum. The H migration energy barrier in the S1 state is 0.523 eV, significantly higher than the rotational energy barrier of the left-hand group. Therefore, torsional rather than H migration is more likely to occur in the excited state of M1. At step 13 (36.8 degrees), the S0 state energy reaches a local maximum, with an energy barrier of 0.824 eV relative to the initial state (step 0). At step 14 (37.8 degrees), the energy reaches a local minimum, indicating that H may have successfully transferred to the right. Overall, the ground state is most stable when H is on the left, while H migration in the excited state is accompanied by a decrease in excitation energy and a red shift in the UV absorption spectrum. However, the energy barrier for H migration in the S1 state is higher, making M1 more susceptible to excited state torsion rather than H migration.

[0097] Figure 10 Figure b shows the energy change analysis of an M2-scan H-TD rigid scan of hydrogen moving from the left to the right. During the scan, the dihedral angle ∠H36C8O35 changes from 24 degrees to 45 degrees. After step 16 of the scan, the energy of the hydrogen atom increases significantly due to its proximity to the right oxygen atom, making this energy change negligible. As the hydrogen atom moves to the right, the S0 state energy gradually increases, indicating that the ground state is most stable when the hydrogen atom is located on the left. The S1 state energy initially rises, then decreases slightly, and then increases again. The S1-S0 energy difference (excitation energy) gradually decreases, indicating that hydrogen migration is accompanied by a red shift in the UV absorption spectrum. The energy barrier for hydrogen migration in the S1 state is 0.211 eV. While higher than the rotational barrier for the right-hand group (0.0105 eV), it is still relatively low, indicating that hydrogen migration and torsional processes are both more likely to occur in the excited state of M2. At step 14, the S1 state energy reaches a local minimum, corresponding to the excited state structure in which hydrogen migrates to the right. In general, the ground state is most stable when H is on the left. As H moves, the energy gradually increases. The movement of H in the excited state is accompanied by a decrease in excitation energy and a red shift in ultraviolet absorption occurs, indicating that hydrogen migration is more likely to occur in the excited state of M2, and a locally stable excited state structure is exhibited at step 14.

[0098] Figure 10Figure c in the figure shows the energy change analysis of H moving from the left to the right during an M3-scan H-TD rigid scan. During the scan, the dihedral angle ∠H36C8O35 changes from 24 degrees to 45 degrees. After step 16 of the scan, the energy rises significantly due to the close proximity of the H atom to the right oxygen atom, making this energy change negligible. As the H atom moves rightward, the S0 state energy gradually increases, indicating that the ground state is most stable when the H atom is on the left. The S1 state energy rises initially, then decreases slightly, and finally rises again. The S1-S0 energy difference (excitation energy) gradually decreases, indicating that H migration is accompanied by a red shift in the UV absorption spectrum. The H migration energy barrier for the S1 state is 0.349 eV, intermediate between that of M1 and M2. The left-side groups significantly hinder H migration, while the right-side groups favor it. This suggests that M3 has a locally stable hydrogen migration structure around step 14. Overall, the ground state is most stable when H is located on the left, with the energy gradually increasing as H moves. In the excited state, H movement is accompanied by a decrease in excitation energy, resulting in a red shift in the UV absorption spectrum. The hydrogen migration energy barrier in the S1 state of M3 is lower than that of M1 but higher than that of M2, indicating that M3 has a moderate hydrogen migration tendency.

[0099] These results demonstrate that the optical properties of M3 are significantly improved compared to those of M1 and M2. Although the fluorescence emission peak of M1 exhibits a modest Stokes shift, its absorption and emission wavelengths are short, and the fluorescence intensity is relatively low. Compared to M1, M2 significantly increases the absorption and emission wavelengths and enhances the fluorescence intensity by extending the molecule's conjugated system. M3 further extends the conjugated system and introduces two rotation axes to maximize its absorption and emission wavelengths and improve fluorescence intensity. M1, M2, and M3 modulate the conjugated system length and electron distribution of the molecules by introducing different electron donor and acceptor groups, thereby affecting the characteristics of their absorption and emission spectra. Shifts in the position of the absorption and emission peaks directly reflect differences in the electronic structure and energy level transitions of the compounds. The dual rotation axis property of M3 increases its sensitivity to the protein microenvironment, enabling more effective sensing of protein aggregation. Furthermore, M3's excited-state proton transfer and twisted intramolecular charge transfer properties further enhance its Stokes shift and fluorescence signal intensity.

[0100] Example 2

[0101] Using compounds M1 and M2 as controls, this example demonstrates the ability of compound M3 as a probe to detect Aβ oligomers, including the following aspects:

[0102] (1) Molecular docking simulation research.

[0103] In order to better understand the binding mechanism between Aβ oligomers and chalcone derivative chromophores, a non-covalent molecular docking simulation study based on the Glide program was performed. Figure 11 As shown in Figures ac, the three compounds (M1, M2, and M3) were respectively inserted into the binding pocket formed by several key amino acid residues (including Phe21, Glu23, and Ile28) of the surrounding chains of Aβ oligomers.

[0104] In M1, a hydrogen bond interaction was observed between the hydroxyl group on the phenyl ring and Ile28 (Ile28-NH…OH, distance 2.0 Å). In M2, the hydroxyl group formed a hydrogen bond with the carbonyl group of Glu23 (Glu23-C=O···HO, distance 2.0 Å). In M3, the hydroxyl group formed a hydrogen bond with the amino group of Glu23 (Glu23-NH…OH, distance 1.9 Å). These docking results suggest that the hydroxyl group may be an important functional group in the binding activity of these compounds to Aβ oligomers.

[0105] Calculated binding free energies for M1, M2, and M3 were -30.53 kJ / mol, -20.43 kJ / mol, and -33.26 kJ / mol, respectively, indicating that M3 has the highest binding affinity for Aβ oligomers. This also explains the enhanced luminescence of M3, as Aβ oligomers effectively restrict the free torsion of M3, thereby minimizing energy dissipation caused by non-radiative transitions.

[0106] (2) Detection of the binding affinity of the compound to Aβ42 oligomers.

[0107] In this study, a fluorescence saturation binding assay was used to evaluate the binding affinity of compounds M1, M2, and M3 to Aβ42 oligomers. The ThT dissociation constant (Kd) for Aβ42 oligomers was determined to be 0.556 μM, consistent with previously reported results. Figure 11 The df in the figure shows typical fluorescence titration results for compounds M1, M2, and M3 in a 4 μM Aβ42 oligomer solution. In all cases, the titration data were well fitted to a 1:1 binding isotherm, indicating the best-fit calculated curve. For Aβ42 oligomers, the Kd values ​​for compounds M1, M2, and M3 were 1.180, 0.896, and 0.683 μM, respectively. Compound M3 exhibited the highest binding affinity and the smallest Kd value, demonstrating its superiority in the detection of Aβ42 oligomers.

[0108] (3) Detection of the fluorescence properties of the compounds under Aβ42 oligomer conditions.

[0109] In this example, the fluorescence characteristics of compounds M1, M2, and M3 were detected in PBS (10 μM, pH = 7.4) and in the presence of Aβ42 oligomers. The results are shown in Figure 2. Figure 12 In the absence of Aβ42, M1 ( Figure 12 a) and M2 ( Figure 12 b) showed high background fluorescence, which is unfavorable for ultrasensitive detection of Aβ42 aggregates; moreover, M1 and M2 did not show significant fluorescence response after binding to Aβ42 oligomers. In contrast, M3 ( Figure 12 (c) After binding to Aβ42 oligomers, the fluorescence intensity in the emission spectrum increased significantly by 11.0 times, further demonstrating its superiority in the detection of Aβ42 oligomers.

[0110] Example 3

[0111] In this example, the fluorescence spectra of compound M3 in the presence of Aβ42 oligomers or Aβ42 fibers at different concentrations were detected at different excitation wavelengths. The detection process was as follows: a DMSO buffer solution of M3 (10 μM) was prepared and added to Aβ42 oligomer and fiber samples at concentrations of 0, 4, 8, 16, and 32 μM, respectively. The fluorescence spectrum data were recorded at excitation wavelengths of 380, 420, and 460 nm, respectively.

[0112] Figure 13 (a) shows the fluorescence spectra of M3 in the environment of Aβ42 oligomers with different concentrations, with an excitation wavelength of 380 nm; Figure 13 b shows the fluorescence spectra of M3 in the environment of Aβ42 oligomers with different concentrations, with an excitation wavelength of 420 nm; Figure 13 Figure c shows the fluorescence spectra of M3 in the environment of Aβ42 oligomers with different concentrations, with an excitation wavelength of 460 nm; Figure 13 (d) shows the fluorescence spectra of M3 in the environment of Aβ42 fibers with different concentrations, with an excitation wavelength of 380 nm; Figure 13 e shows the fluorescence spectra of M3 in the environment of Aβ42 fibers with different concentrations, with an excitation wavelength of 420 nm; Figure 13 Figure f shows the fluorescence spectra of M3 in the presence of Aβ42 fibrils at different concentrations, with an excitation wavelength of 460 nm. In the above results, the fluorescence emission peaks are all located in the range of 500-700 nm.

[0113] according to Figure 13 a and Figure 13As can be seen from d in, the fluorescence characteristics of M3 in Aβ42 oligomer and fiber environments are as follows: in the Aβ42 oligomer environment, with the increase of Aβ42 concentration, M3 exhibits dual-emission fluorescence characteristics, and the fluorescence intensity gradually increases, indicating that M3 can effectively recognize and bind to Aβ42 oligomers; in the Aβ42 fiber environment, M3 exhibits dual-emission fluorescence characteristics, and the fluorescence intensity also increases with the increase of Aβ42 concentration, but compared with the oligomer environment, its dual-emission change trend deviates significantly.

[0114] according to Figure 13 b and Figure 13 From the figure e, we can see that the fluorescence characteristics of M3 in the environment of Aβ42 oligomers and fibers are as follows: in the environment of Aβ oligomers, M3 mainly emits signals with long wavelengths, and the fluorescence intensity gradually increases with the increase of Aβ concentration; in the environment of Aβ42 fibers, M3 exhibits dual-emission fluorescence characteristics, and the fluorescence intensity change trend is similar to that in the oligomer environment, indicating that M3 has good recognition and discrimination capabilities for Aβ42 oligomers and fibers.

[0115] according to Figure 13 c and Figure 13 From the f in , we can see that the fluorescence characteristics of M3 in the Aβ42 oligomer and fiber environment are as follows: in the Aβ42 oligomer environment, M3 only emits long-wavelength signals, the fluorescence emission peak is in the range of 500-700 nm, and the fluorescence intensity significantly increases with the increase of Aβ42 concentration; in the Aβ42 fiber environment, the fluorescence intensity change trend of M3 is similar to that in the oligomer environment, but the fluorescence signal-to-noise ratio is low, indicating that M3 exhibits different fluorescence response capabilities in different Aβ42 aggregate environments.

[0116] The above results show that the fluorescence intensity of M3 increases with increasing concentrations of Aβ oligomers and fibrils, indicating that these compounds can effectively recognize and bind to Aβ aggregates. Due to their complex molecular structure and dual rotation axis characteristics, they exhibit significant environmental sensitivity and good ability to distinguish between different aggregation states in different Aβ aggregate environments.

[0117] Example 4

[0118] This example examines the fluorescence imaging of the commonly used chromogens ThT and M3 in the presence of varying concentrations of Aβ42 oligomers and fibrils. The assay involves time-dependent fluorescence imaging of Aβ42 (40 μM) using confocal laser scanning microscopy (excitation wavelength = 480 nm; emission wavelength = 620 nm, scale bar, 100 μm) using ThT (30 μM in DMSO) and M3 (10 μM in DMSO).

[0119] Test results such as Figure 14 The specific analysis is as follows:

[0120] Figure 14 The ab in the figure shows that in the environment of Aβ42 monomers and Aβ42 oligomers, the commercial probe molecule Thioflavin T (ThT) shows some scattered fluorescent spots, indicating that ThT cannot detect low levels of Aβ42 oligomers. Figure 14 Panel c shows that the number of ThT fluorescent spots slightly increased in the environment of Aβ42 oligomers. Figure 14 Panel d shows that in the Aβ42 fiber sample, ThT exhibited more fluorescent aggregation, confirming its weak binding and poor detection ability for Aβ42 oligomers.

[0121] Figure 14 Figure 5e shows that the fluorescence signal of M3 is weak in the environment of Aβ42 monomers, indicating that the background signal is small at low Aβ42 aggregate concentrations. Figure 14 Figure 5f shows that in the environment of Aβ42 oligomers, the fluorescence signal of M3 is significantly enhanced, indicating that its detection ability increases with the increase in the degree of Aβ42 aggregation, proving its strong binding ability to Aβ42 oligomers. Figure 14 Figure 3h shows that in the environment of Aβ42 fibers, M3 exhibits the strongest fluorescence intensity and displays an obvious fibrillar structure, indicating that M3 performs best in detecting and imaging Aβ fibers.

[0122] Fluorescence imaging experiments showed that M3 produced significant fluorescence signals in the context of Aβ42 oligomers and fibrils at varying degrees of aggregation, with M3 exhibiting particularly excellent fluorescence imaging properties in the context of Aβ42β fibrils. This suggests that M3 has superior performance in detecting and imaging Aβ42 fibrils and has the potential to be used for more sensitive detection and research of Aβ42-related pathologies.

[0123] In summary, the chalcone derivative M3 provided by the present invention can specifically recognize Aβ42 oligomers, which is of great significance for AD detection and related research.

[0124] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the present invention, other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A chalcone derivative, characterized in that The structural formula of the chalcone derivative is shown below: 。 2. Use of the chalcone derivative according to claim 1 as a fluorescent probe in the preparation of a reagent for detecting β-amyloid protein aggregates.

3. Use of the chalcone derivative according to claim 1 in the preparation of a product for diagnosing Alzheimer's disease.

4. Use of the chalcone derivative according to claim 1 in the preparation of a β-amyloid aggregate imaging reagent.

5. A method for preparing the chalcone derivative according to claim 1, characterized in that: The following steps are involved: S1, 4-bromo-2-hydroxyacetophenone reacts with iodomethane under alkaline conditions to obtain a methylated product of 4-bromo-2-methoxyacetophenone; S2, the methylated product of 4-bromo-2-methoxyacetophenone is reacted with 4-(dimethylamino)phenylboronic acid in the presence of a palladium catalyst and an inorganic base to obtain an intermediate 1; S3, dissolving the intermediate product 1 and 4-(dimethylamino)benzaldehyde in methanol, adding potassium hydroxide and reacting to obtain the intermediate product 2; S4. The intermediate product 2 reacts with boron tribromide to obtain the target product.

6. The method according to claim 5, characterized in that The reaction in step S2 uses 1,4-dioxane as solvent.

7. The method according to claim 6, characterized in that The palladium catalyst is Pd(PPh3)2Cl2.

8. The method according to claim 6, characterized in that The molar ratio of the methylated product, 4-(dimethylamino)phenylboronic acid and the inorganic base is (1-1.1):(1-1.1):(1-1.2), wherein the inorganic base is cesium carbonate, potassium carbonate or sodium hydroxide.

9. The method according to claim 6, characterized in that The reaction temperature in step S2 is 100-105°C.

10. The method according to claim 5, characterized in that In step S3, the molar ratio of the intermediate product 1, 4-(dimethylamino)benzaldehyde and potassium hydroxide is (1-1.1):(1-1.1):(1-1.1).

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