Fluorescent probe for detecting amyloid protein oligomer as well as preparation and application of fluorescent probe

Abstract

The invention provides a fluorescent probe for detecting amyloid oligomers and preparation and application thereof, and belongs to the technical field of A beta imaging agents, the fluorescent probe is a chalcone derivative, and is particularly developed based on optimization of the process of molecular torsion limited charge transfer and excited state proton transfer of a chalcone skeleton. The probe can be used as a specific fluorescence chemical probe for detecting and imaging the A beta aggregate, can dynamically monitor the A beta aggregate in an in-vitro fiber forming process, and has important significance on A beta related pathology 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, and the fluorescent probe is a novel chalcone derivative. Background Art

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

[0003] Amyloid proteins and peptides can form multiple different assembly states, and the key question is which assembly state is more closely related to the pathogenesis of the disease. For a long time, plaque deposition caused by Aβ fibrillization has been considered the main cause of AD neurodegeneration. However, recent studies have shown that Aβ oligomers (composed of 2-12 Aβ monomers), which are mainly present in the initial lag and growth stages of Aβ fibrils, are the main drivers of AD pathogenesis, and their concentration levels play a more important role in disease progression. The concept of Aβ intermediates participating in the development of AD can explain why amyloid pathology defined based on Aβ plaque load is weakly correlated with clinical AD manifestations, suggesting that amyloid plaques may be a relatively non-toxic aggregated form of Aβ. Therefore, it is urgent to develop detection methods that can recognize multiple morphological forms of Aβ peptides.

[0004] Chalcone is a compound containing 1,3-diphenylpropenone structure in the molecule. It is a precursor of open-chain flavonoids and isoflavonoids found in edible plants. In recent years, it has attracted increasing attention due to its various potential pharmacological applications. Chalcone and its derivatives exhibit a wide range of pharmacological activities, and chemical derivatization provides a high degree of structural diversity, which has been shown to help develop new drugs with higher efficacy 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 the first aspect, the present invention provides a novel chalcone derivative, the structural formula of which is shown below: .

[0007] In a second aspect, the present invention provides a method for preparing the above-mentioned chalcone derivative, comprising the following steps: S1, 2-bromo-5-hydroxyacetophenone reacts with methyl iodide under alkaline conditions to obtain a methylated product of 2-bromo-5-methoxyacetophenone; S2, subjecting the methylated product of 2-bromo-5-methoxyacetophenone 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; S3, dissolving the intermediate product 1, 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; Among them, the structural formula of intermediate product 1 is as follows: ; The structural formula of intermediate 2 is shown below: .

[0008] Preferably, in the above preparation method, the reaction in step S2 uses 1,4-dioxane as solvent.

[0009] 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.

[0010] Preferably, in the above preparation method, the reaction temperature of step S2 is 100-105°C.

[0011] In some embodiments of the present invention, the palladium catalyst used in step S2 is specifically Pd(PPh3)2Cl2.

[0012] 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).

[0013] Preferably, in the above preparation method, the reaction temperature of step S3 is 25-35°C.

[0014] 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).

[0015] In a third aspect, the present invention provides the use of the above-mentioned chalcone derivatives, which is at least any of the following: a) As a fluorescent probe to detect Aβ aggregates; b) Preparation of diagnostic products for Alzheimer's disease; c) Preparation of Aβ aggregate imaging reagents.

[0016] In some embodiments of the present invention, the Aβ aggregates are Aβ42 monomers, oligomers and aggregates.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention uses the chalcone structure as the core of the Aβ42 imaging agent for the first time, designs and synthesizes a new chalcone derivative, the dual rotation axis characteristics of which increase its sensitivity to the protein microenvironment and can more effectively sense the protein aggregation state. At the same time, the excited state proton transfer (ESIPT) and twisted intramolecular charge transfer (TICT) characteristics of the derivative further improve its Stokes shift and fluorescence signal intensity. Experimental data show that the derivative has a good effect when used as a probe for Aβ42 aggregate detection, and has demonstrated dynamic monitoring of in vitro fiber formation in an in vitro model of Alzheimer's disease, which has potential application prospects for tracking and monitoring the Aβ protein aggregation process.

[0018] The synthetic route provided by the present invention ensures high yield and purity of the product by adopting the reflux conditions of the Suzuki coupling reaction in 1,4-dioxane. This reaction path lays a foundation for the successful preparation of the chalcone derivatives of the present invention and provides the possibility for further application research. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 is a synthetic route diagram of the chalcone derivative M3 in Example 1 of the present invention; Figure 2 is the chalcone derivative M3 in Example 1 of the present invention 1 H NMR spectrum; Figure 3 is the chalcone derivative M3 in Example 1 of the present invention 13 C NMR spectrum; Figure 4 The synthetic route of compounds M1 and M2 in Comparative Example 1 of the present invention; Figure 5 is the compound M1 in Comparative Example 1 of the present invention 1 H NMR spectrum; Figure 6 is the compound M2 in Comparative Example 2 of the present invention 1 H NMR spectrum; Figure 7 The absorption spectra (a) and fluorescence emission spectra (b) of compounds M1, M2 and M3 of the present invention; Figure 8 The fluorescence spectra of compounds M1, M2 and M3 in the present invention at different H2O / THF mixed solvent ratios; Fig. 9 The energy change curves of the ground state (S0) and excited state (S1) caused by scanning the rotation of the aniline group of compounds M1, M2 and M3 in the present invention under the TD-DFT method; wherein a is the left benzene rotation of M1-scan-TD scanning, b is the right benzene rotation of M2-scan-TD scanning, c is the left rotation of M3-scanL-TD scanning, and d is the right benzene rotation of M3-scanR-TD scanning; Fig.10 : These are the energy change curves of 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 M1-scanH-TD rigid scanning H moving from the left to the right, b is M2-scanH-TD rigid scanning H moving from the left to the right, and c is M3-scanH-TD rigid scanning H moving from the left to the right; Fig.11 The binding detection 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; Fig.12 : is the fluorescence emission spectra of M1, M2 and M3 before and after binding to Aβ42 oligomers in Example 2 of the present invention; Fig.13 df is the fluorescence spectrum of M3 in Example 3 of the present invention in the environment of Aβ oligomers and fibers at different concentrations; wherein ac is the fluorescence spectrum in the environment of Aβ42 oligomers, and df is the fluorescence spectrum in the environment of Aβ42 fibers; Fig.14 This is the fluorescence imaging of the color developing agents ThT and M3 in Example 4 of the present invention in different Aβ aggregate environments, wherein ad is the fluorescence image of ThT, and eh is the fluorescence image of M3. DETAILED DESCRIPTION

[0021] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" in the specification and claims of the present invention and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0023] The aggregation of Aβ is closely related to the pathological process of Alzheimer's disease, among which soluble Aβ aggregates are widely considered to be the key pathogenic substances. Identification and characterization of intermediate species formed during the aggregation process are crucial to understanding aggregate-mediated neuronal toxicity and its mechanism of action in disease progression. However, due to the heterogeneity and transient nature of these substances, it is extremely challenging to detect them, and the lack of reliability of existing sensors further exacerbates this problem. In response to this problem, the present invention has developed a specific fluorescent probe for detecting aggregates, which is specifically developed based on the optimization of the molecular torsion-restricted charge transfer and excited-state proton transfer process of the chalcone skeleton, and its structural formula is shown below: ; Due to the complex molecular structure and dual rotation axis characteristics of this fluorescent probe, it has superior performance in detecting and imaging Aβ aggregates and is expected to be used for highly sensitive Aβ-related pathology detection and research.

[0024] 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. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0025] Example 1 This example provides a chalcone derivative (denoted as M3), the structural formula of which is shown below: .

[0026] The synthetic route of the above compound M3 is as follows Figure 1 As shown, the preparation method thereof comprises the following steps: Step 1: Using 2-bromo-5-hydroxyacetophenone as the starting material, react with iodomethane (CH3I, 1 equivalent) under appropriate alkaline conditions to obtain the methylated product of 2-bromo-5-methoxyacetophenone.

[0027] Step 2: The methylated product was subjected to 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 (100°C) was carried out in 1,4-dioxane solvent under reflux conditions for 24 hours to ensure the complete coupling reaction; after the reaction was completed, the intermediate 1 was obtained by cooling, extraction and purification steps.

[0028] Step 3: The intermediate product obtained in step S2 and 4-(dimethylamino)benzaldehyde (1 equivalent) were dissolved in methanol (CH3OH), potassium hydroxide (KOH, 1 equivalent) was added, and the mixture was stirred at room temperature for 12 hours. After the reaction, the pH was adjusted to neutral with hydrochloric acid solution (1M), and the intermediate product 2 was obtained by filtration and purification.

[0029] Step 4: The intermediate product obtained in step 3 is reacted with boron tribromide (BBr3, 1 equivalent) at room temperature. After the reaction is completed, the reaction mixture is cooled and the target product M3 is obtained through extraction and purification steps.

[0030] M3 1 H NMR spectrum Figure 2 As shown, M3 13 C NMR spectrum Figure 3 As shown; among them, 1 H 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).

[0031] Comparative Example 1 In this example, two other compounds, M1 and M2, were designed and synthesized, and their structures are shown below: .

[0032] (1) Preparation of compound M1.

[0033] like Figure 4 As shown in a, the preparation method of compound M1 comprises the following steps: Step 1: Using 2-bromo-5-hydroxyacetophenone as the starting material, react with iodomethane (CH3I, 1 equivalent) under appropriate alkaline conditions to obtain the methylated product of 2-bromo-5-methoxyacetophenone; Step 2: The methylated product is subjected to 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 is carried out in 1,4-dioxane solvent under reflux conditions for 24 hours to ensure the complete coupling reaction; after the reaction is completed, the intermediate product 1 is obtained by cooling, extraction and purification steps.

[0034] 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 completed, the reaction mixture is cooled and subjected to extraction and purification steps to obtain the target product M1.

[0035] M1 1 H NMR spectrum Figure 5 As shown, 1 H 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).

[0036] (2) Preparation of compound M2.

[0037] like Figure 4 As shown in b, the preparation method of compound M2 comprises the following steps: At room temperature, o-hydroxyacetophenone (1 equivalent) and 4-(dimethylamino)benzaldehyde (1 equivalent) were dissolved in methanol (CH3OH), 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 target product M2 was obtained by adjusting the pH to neutral with hydrochloric acid solution (1M), filtering and purification steps.

[0038] M2 1 H NMR spectrum Figure 6 As shown, 1H 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).

[0039] The following performance tests were performed on the above compounds M1, M2 and M3: 1. Absorption spectrum and fluorescence emission spectrum.

[0040] The absorption spectra 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 spectra of the above samples, and then set the excitation wavelength according to the maximum absorption wavelength to measure the fluorescence emission spectrum.

[0041] Figure 7 a shows the normalized absorption spectra of M1, M2, and M3 in DMSO solution. The absorption peak of M1 is located at about 380 nm, the absorption peak of M2 is located at about 450 nm, and the absorption peak of M3 is further red-shifted to about 470 nm. These results show that the absorption properties of chalcone derivatives can be significantly regulated by structural modification.

[0042] Figure 7 b shows the standardized fluorescence spectra of M1, M2 and M3 in DMSO solution. The fluorescence emission peak of M1 is located at about 508 nm, the emission peak of M2 is located at about 554 nm, and the fluorescence emission peak of M3 is further red-shifted to about 620 nm. These results show that the structural modification of chalcone derivatives has a significant effect on their luminescence properties.

[0043] From the above results, it can be observed that M1, M2 and M3 all exhibit obvious Stokes shifts. For example, the absorption peak of M1 is located at about 380 nm, while its fluorescence emission peak is located at about 508 nm, showing a Stokes shift of about 128 nm. This Stokes shift can effectively separate the absorption and emission spectra and avoid signal overlap, thereby 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.

[0044] 2. Fluorescence spectrum in H2O / THF solvent.

[0045] Under different excitation wavelengths, the fluorescence spectra of M1, M2 and M3 in different H2O / THF mixed solvent ratios were detected. The detection process was as follows: by gradually adding water to THF, the water fraction (fw) in the tetrahydrofuran (THF) / water mixture was changed, and the emission characteristics of M1, M2 and M3 were tested when the degree of molecular aggregation gradually increased.

[0046] Figure 8 a shows the fluorescence spectrum of M1 in a H2O / THF mixed solvent with an excitation wavelength of 370 nm. The fluorescence emission peak is mainly distributed in the range of 500-600 nm, and the fluorescence intensity gradually increases with the increase of the THF ratio. This shows that the fluorescence properties of M1 are sensitive to changes in the solvent environment. Due to its simple structure, M1 has a relatively weak fluorescence intensity, but it shows a more obvious response when the solvent environment changes.

[0047] Figure 8 b shows the fluorescence spectrum of M2 in H2O / THF mixed solvent with an excitation wavelength of 450 nm. The fluorescence emission peak is in the range of 500-700 nm, and the fluorescence intensity increases significantly with the increase of THF ratio. Compared with M1, the fluorescence intensity and emission wavelength of M2 increase, which is attributed to the expansion of the molecular conjugated system, which significantly improves the fluorescence performance.

[0048] Figure 8 c shows the fluorescence spectrum of M3 in H2O / THF mixed solvent with an excitation wavelength of 370 nm. Compared with M1, the fluorescence emission peak of M3 is also distributed in the range of 500-700 nm, but the fluorescence intensity is higher and the emission peak position is slightly different. M3 forms a double rotation axis by adding a second benzene ring. This structural design significantly enhances its fluorescence performance and makes it more sensitive to the solvent environment.

[0049] Figure 8 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 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.

[0050] The fluorescence behaviors of M1, M2, and M3 in H2O / THF mixed solvents changed 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 the fluorescence performance 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 terms of fluorescence intensity and wavelength, and its sensitivity to the solvent environment is also significantly enhanced. This solvent-dependent fluorescence enhancement property makes M3 have greater potential in applications in detecting solution environments.

[0051] 3. Analysis of energy changes caused by the rotation of the aniline group.

[0052] 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: Fig. 9 shown.

[0053] Fig. 9 a is the energy change analysis of the benzene ring rotation on the left side of M1-scan-TD. During the scanning process, the dihedral angle 21-20-17-15 changed from 146.7 degrees to 56.7 degrees. At the 19th step (dihedral angle of 89.7 degrees), the energy of the S0 state reached the highest, while the energy of the S1 state was the lowest. At this time, the S1-S0 energy difference dropped to 2.978 eV, which was the minimum value. During the rotation process, there was an energy barrier in 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 energy of the S1 state gradually decreased, and no obvious rotation energy barrier was shown, suggesting that the benzene ring tended to be stable in the S1 state. The optimized M1 molecule showed a structure in which the aniline benzene ring was almost perpendicular to the phenol benzene ring in the S1 state. In summary, there is a rotation energy barrier in the S0 state and the structure is unstable; while in the S1 state, the rotation of the benzene ring is not restricted by the energy barrier, and the structure is relatively stable, indicating that the aniline and phenol benzene rings tend to be nearly vertically arranged in the S1 state.

[0054] Fig. 9b is the energy change analysis of the rotation of the right benzene ring of M2-scan-TD. During the scanning process, the dihedral angle 17-16-13-12 changed from 0.29 degrees to 90.29 degrees. At the 30th step (dihedral angle of 90.29 degrees), the S0 state has the highest energy and the S1 state has the lowest energy. The S1-S0 energy difference is 2.425 eV, which is the minimum value. There is an energy barrier with an energy of 0.412 eV during the rotation of the S0 state, indicating that the structure of the molecule is unstable at this angle. At the 9th step (27.29 degrees), the S1 state shows a weak rotation energy barrier with an energy of 0.0105 eV, indicating that the excited state has a certain stability at this angle. In the S1 state of the optimized M2 molecule, the right benzene ring is almost perpendicular to the phenol benzene ring. The S0 state has a large rotation barrier at 90.29 degrees, indicating that the structure is unstable at this angle; while the S1 state has a weak energy barrier at 27.29 degrees, showing unique stability in the excited state. The optimized structure shows that the right benzene ring in the S1 state tends to be perpendicular to the phenol benzene ring.

[0055] Fig. 9 c is the energy change analysis of the rotation of the left benzene ring of M3-scanL-TD. During the scanning process, the dihedral angle 21-20-17-15 changed from 146 degrees to 56 degrees. At step 19 (dihedral angle 89 degrees), the energy of both 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, with a rotation energy barrier of 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. There was a rotation energy barrier at 89 degrees in both the S0 state and the S1 state, and the structure was unstable in both states. The energy could not be dissipated through the rotation of the left benzene ring, which may lead to the occurrence of fluorescence emission. The optimized structure shows that the angle between the left aniline benzene ring and the phenol benzene ring tends to decrease in the S1 state.

[0056] Fig. 9d is the energy change analysis of the rotation of the right benzene ring of M3-scanR-TD. 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 is 0.391 eV, which is similar to M2. The rotation energy barrier of the S1 state at 63.04 degrees is 0.124 eV, indicating that the S1 state has special stability at this angle. In the S1 state of the optimized M3 molecule, the angle between the right benzene ring and the phenol benzene ring is small. The S0 state has a large rotation energy barrier at 90.04 degrees, indicating that the structure is unstable at this angle; while the S1 state has a rotation energy barrier at 63.04 degrees, showing unique behavior in the excited state. Since the right-side rotation energy barrier of the S1 state is high, it is difficult for energy to dissipate through right-side rotation, thereby maintaining fluorescence emission. The optimized structure shows that the angle between the right-side aniline benzene ring and the phenol benzene ring in the S1 state tends to decrease.

[0057] 4. Analysis of energy changes caused by H migration.

[0058] Using the TD-DFT method, the energy change curves of the S0 and S1 states caused by H migration in M1, M2 and M3 were scanned and displayed. The results are as follows: Fig.10 shown.

[0059] Fig.10 a is the energy change analysis of M1-scanH-TD rigid scanning when H moves from the left to the right. During the scanning process, the dihedral angle ∠H36C27O35 changes from 23.8 degrees to 44.8 degrees. After the 16th step of the scan, the energy increases significantly because the H atom is too close to the oxygen atom on the right, so the energy change in this part can be ignored. As the H atom moves to the right, the energy of the S0 state gradually increases, indicating that the S0 state is most stable when H is on the left. The energy of the S1 state rises in the initial stage, then decreases slightly, and then increases again. The energy difference (excitation energy) between S1 and S0 gradually decreases, indicating that the movement of H from left to right is accompanied by a red shift in the ultraviolet absorption spectrum. The H migration energy barrier of the S1 state is 0.523 eV, which is significantly higher than the rotation energy barrier of the left group, so M1 is more likely to undergo torsion rather than H migration in the excited state. At the 13th step (36.8 degrees), the energy of the S0 state reaches a local maximum point, with an energy barrier of 0.824 eV relative to the initial state (step 0), and at the 14th step (37.8 degrees), the energy reaches a local minimum point, indicating that H may have successfully transferred to the right. In general, the ground state is most stable when H is on the left, and the migration of H in the excited state is accompanied by a decrease in excitation energy and a red shift in the ultraviolet absorption spectrum. However, the energy barrier for H migration in the S1 state is higher, so M1 is more likely to undergo excited state torsion rather than H migration.

[0060] Fig.10 b is the energy change analysis of M2-scanH-TD rigid scanning when H moves from the left to the right. During the scanning process, the dihedral angle ∠H36C8O35 changes from 24 degrees to 45 degrees. After the 16th step of the scan, the energy increases significantly because the H atom is too close to the oxygen atom on the right, so the energy change in this part can be ignored. As the H atom moves to the right, the energy of the S0 state gradually increases, indicating that the ground state is most stable when H is on the left. The energy of the S1 state rises in the initial stage, then decreases slightly, and then increases again. The energy difference (excitation energy) between S1 and S0 gradually decreases, indicating that the movement of H is accompanied by a red shift in the ultraviolet absorption spectrum. The H migration energy barrier of the S1 state is 0.211 eV, which is higher than the rotation energy barrier of the right group (0.0105 eV), but it is still low, indicating that both hydrogen migration and torsion processes are easier to occur in the excited state of M2. At the 14th step, the S1 state energy has a local minimum point, corresponding to the excited state structure of H migrating 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, 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 the 14th step.

[0061] Fig.10 c is the energy change analysis of M3-scanH-TD rigid scanning when H moves from the left to the right. During the scanning process, the dihedral angle ∠H36C8O35 changes from 24 degrees to 45 degrees. After the 16th step of the scan, the energy increases significantly because the H atom is too close to the oxygen atom on the right, so the energy change of this part can be ignored. As the H atom moves to the right, the energy of the S0 state gradually increases, indicating that the ground state is most stable when H is on the left. The energy of the S1 state rises in the initial stage, then decreases slightly, and finally increases again. The energy difference (excitation energy) between S1 and S0 gradually decreases, indicating that the movement of H is accompanied by a red shift in the ultraviolet absorption spectrum. The H migration energy barrier of the S1 state is 0.349 eV, which is between M1 and M2. The left-side group has a greater obstacle to H migration, while the right-side group is more favorable to H migration, which indicates that M3 has a locally stable hydrogen migration structure around the 14th step. In general, the ground state is most stable when H is on the left, and the energy gradually increases with the movement of H; in the excited state, the movement of H is accompanied by a decrease in excitation energy, and the ultraviolet absorption spectrum red-shifts. 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.

[0062] The above results show that the optical properties of M3 are significantly improved compared with M1 and M2. Although the fluorescence emission peak of M1 shows a moderate Stokes shift, its absorption and emission wavelengths are short and the fluorescence intensity is relatively low. Compared with M1, M2 significantly increases the absorption and emission wavelengths and enhances the fluorescence intensity by extending the conjugated system of the molecule. M3 further extends the conjugated system on the basis of M2 and introduces two rotation axes to maximize its absorption and emission wavelengths and improve the fluorescence intensity. M1, M2 and M3 adjust the conjugated system length and electron distribution of the molecule by introducing different electron donor and acceptor groups, thereby affecting the characteristics of its absorption and emission spectra. The position changes of the absorption and emission peaks directly reflect the differences in the electronic structure and energy level transitions of the compounds. The dual rotation axis characteristics of M3 increase its sensitivity to the protein microenvironment and can more effectively sense the protein aggregation state. At the same time, the excited state proton transfer and twisted intramolecular charge transfer characteristics of M3 further improve its Stokes shift and fluorescence signal intensity.

[0063] Example 2 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: (1) Molecular docking simulation study.

[0064] 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 in this example. Fig.11 As shown in (a-c), 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.

[0065] In M1, a hydrogen bond interaction was observed between its benzene ring hydroxyl group and Ile28 (Ile28-NH…OH, distance 2.0Å). For M2, its hydroxyl group can form a hydrogen bond with the carbonyl group of Glu23 (Glu23-C=O···HO, distance 2.0Å). In M3, the hydroxyl group forms a hydrogen bond with the amino group of Glu23 (Glu23-NH…OH, distance 1.9Å). These docking results indicate that the hydroxyl group may be an important functional group for the binding activity of these compounds with Aβ oligomers.

[0066] By calculation, the binding free energies of M1, M2 and M3 are -30.53 kJ / mol, -20.43 kJ / mol and -33.26 kJ / mol, respectively, indicating that M3 has the highest binding affinity with Aβ oligomers. This also explains the enhancement of M3 in luminescence ability, because Aβ oligomers effectively restrict the free twisting of M3, thereby minimizing the energy dissipation caused by non-radiative transitions.

[0067] (2) Detection of the binding affinity of the compounds to Aβ42 oligomers.

[0068] In this case, the binding affinity of compounds M1, M2, and M3 to Aβ42 oligomers was evaluated using a fluorescence saturation binding assay. The ThT dissociation constant (Kd) value of Aβ42 oligomers was first measured to be 0.556 μM, which is consistent with previously reported results. Fig.11 df shows typical results of fluorescence titration of compounds M1, M2, and M3 in 4 μM Aβ42 oligomer solution. In all cases, the titration data fit well with the 1:1 binding isotherm, showing the best fit calculated curve. For Aβ42 oligomers, the Kd values ​​of compounds M1, M2, and M3 were 1.180, 0.896, and 0.683 μM, respectively. Compound M3 showed the highest binding affinity and the smallest Kd value, showing its advantage in the detection of Aβ42 oligomers.

[0069] (3) Detection of the fluorescence properties of the compounds under Aβ42 oligomer conditions.

[0070] 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. Fig.12 In the absence of Aβ42, M1 ( Fig.12 a) and M2 ( Fig.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 ( Fig.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.

[0071] Example 3 In this example, the fluorescence spectra of compound M3 in the environment of Aβ42 oligomers or Aβ42 fibers with different concentrations were detected at different excitation wavelengths. The specific detection process was as follows: a DMSO buffer solution (10 μM) of M3 was prepared and added to Aβ42 oligomers and fiber samples with concentrations of 0, 4, 8, 16 and 32 μM, respectively, and the fluorescence spectrum data at excitation wavelengths of 380, 420 and 460 nm were recorded, respectively. Fig.13 a shows the fluorescence spectra of M3 in the environment of Aβ42 oligomers with different concentrations, with an excitation wavelength of 380 nm; Fig.13 b shows the fluorescence spectra of M3 in the environment of Aβ42 oligomers with different concentrations, with an excitation wavelength of 420 nm; Fig.13 c shows the fluorescence spectra of M3 in the environment of Aβ42 oligomers with different concentrations, with an excitation wavelength of 460 nm; Fig.13 d shows the fluorescence spectra of M3 in the environment of Aβ42 fibers with different concentrations, with an excitation wavelength of 380 nm; Fig.13 e shows the fluorescence spectra of M3 in the environment of Aβ42 fibers with different concentrations, with an excitation wavelength of 420 nm; Fig.13 f shows the fluorescence spectra of M3 in the environment of Aβ42 fibers with 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.

[0072] according to Fig.13 a and Fig.13 It can be seen from the data that the fluorescence characteristics of M3 in the Aβ42 oligomer and fiber environment 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 has a significant deviation.

[0073] according to Fig.13 b and Fig.13 e It can be seen that the fluorescence characteristics of M3 in the Aβ42 oligomer and fiber environment are as follows: in the Aβ oligomer environment, M3 mainly emits long-wavelength signals, and the fluorescence intensity gradually increases with the increase of Aβ concentration; in the Aβ42 fiber environment, 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 distinction capabilities for Aβ42 oligomers and fibers.

[0074] according to Fig.13 c and Fig.13f It can be seen 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.

[0075] From the above results, it can be seen that with the increase of Aβ oligomer and fiber concentration, the fluorescence intensity of M3 showed an increasing trend, indicating that these compounds can effectively recognize and bind to Aβ aggregates. Due to its complex molecular structure and dual rotation axis characteristics, it shows significant environmental sensitivity and good aggregation state differentiation ability in different Aβ aggregate environments.

[0076] Example 4 This example detects the fluorescence imaging of the commonly used colorimetric agents ThT and M3 in the environment of Aβ42 oligomers and fibers at different concentrations. The specific detection process is as follows: ThT (30 μM in DMSO) and M3 (10 μM in DMSO) were used to perform time-dependent fluorescence imaging of Aβ42 (40 μM) using a confocal laser scanning microscope (excitation wavelength = 480 nm; emission wavelength = 620 nm, scale bar 100 μm).

[0077] Test results such as Fig.14 The specific analysis is as follows: Fig.14 ab showed that the commercial probe molecule Thioflavin T (ThT) showed some scattered fluorescent spots in the environment of Aβ42 monomers and Aβ42 oligomers, indicating that ThT could not detect low levels of Aβ42 oligomers. Fig.14 c shows that the number of fluorescent spots of ThT slightly increased in the environment of Aβ42 oligomers. Fig.14 d shows that in the Aβ42 fiber sample, ThT exhibited more fluorescence aggregation, confirming the weak binding and poor detection ability of ThT to Aβ42 oligomers.

[0078] Fig.14 e shows that in the environment of Aβ42 monomers, the fluorescence signal of M3 is weak, indicating that the background signal is small at low Aβ42 aggregate concentrations. Fig.14 fg shows that in the Aβ42 oligomer environment, the fluorescence signal of M3 is significantly enhanced, indicating that its detection ability increases with the increase in the aggregation degree of Aβ42, proving its strong binding ability to Aβ42 oligomers. Fig.14h 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.

[0079] Fluorescence imaging experiments showed that M3 produced significant fluorescence signals in Aβ42 oligomers and fiber environments with different aggregation levels, especially in the A42β fiber environment, where M3 exhibited excellent fluorescence imaging properties. This indicates that M3 has superior performance in detecting and imaging Aβ42 fibers and may be used for more sensitive Aβ42-related pathology detection and research.

[0080] 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.

[0081] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and embodiments having substantially the same structure as the technical idea and exerting the same effects within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, within the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments, and other methods of combining some of the constituent elements in the embodiments to construct 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 as claimed in claim 1 as a fluorescent probe in detecting Aβ aggregates.

3. Use of the chalcone derivative as claimed in claim 1 in the preparation of a diagnostic product for Alzheimer's disease.

4. Use of the chalcone derivative according to claim 1 in the preparation of an Aβ aggregate imaging agent.

5. A method for preparing the chalcone derivative according to claim 1, characterized in that: The following steps are involved: S1, 2-bromo-5-hydroxyacetophenone reacts with methyl iodide under alkaline conditions to obtain a methylated product of 2-bromo-5-methoxyacetophenone; S2, subjecting the methylated product of 2-bromo-5-methoxyacetophenone 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; 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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