A compound and its application in detection of a beta amyloid 1-42 aggregate and early diagnosis of Alzheimer's disease

Abstract

This application provides a compound and its application in Aβ. 1‑42 Applications in aggregate detection and early diagnosis of Alzheimer's disease: The compound has the structure shown in Formula I: Formula I: wherein R1, R2, R3, and R4 are each independently selected from C1-C5 alkyl and C1-C5 hydroxyalkyl; W and X are each independently selected from benzene ring and naphthalene ring. The compound emits light in the near-infrared region and has good near-infrared fluorescence characteristics, and is effective for Aβ... 1‑42 The aggregates exhibit high sensitivity, high responsiveness, high selectivity, and high affinity, enabling rapid, efficient, and accurate imaging. They can specifically detect β-amyloid protein, are not easily affected by biological matrices and impurities, and show a significant enhancement in fluorescence signal after binding to β-amyloid protein. They can effectively cross the blood-brain barrier and can be used as near-infrared fluorescent probes for the detection of β-amyloid protein and the early diagnosis of Alzheimer's disease.

Description

Technical Field

[0001] This application relates to the field of near-infrared fluorescent probe technology, specifically to a compound and its application in Aβ. 1-42 Application of aggregate detection in early diagnosis of Alzheimer's disease Background Technology

[0002] Any discussion of prior art throughout the specification should not be construed as an admission that such prior art is well-known or constitutes part of common general knowledge in the art.

[0003] Alzheimer's disease (AD) is a common neurodegenerative disease in the elderly. Its main symptoms are memory decline and progressive cognitive impairment. The exact pathogenesis of AD is not fully understood, but it is mostly believed to be the result of a combination of factors including aging, genetics, and environment. Currently, several theories exist, among which the β-amyloid cascade hypothesis is widely accepted, suggesting that the abnormal accumulation of Aβ in various brain tissues and the formation of β-amyloid plaques lead to the development and progression of AD. However, the latest evidence suggests that Aβ oligomers are the true neurotoxins that induce AD. Specifically, Aβ… 1-42 Aggregates are oligomeric forms of β-amyloid protein (Aβ) composed of 42 amino acids and play an important role in the pathogenesis of Alzheimer's disease (AD). 1-42 Aggregates can cause neuronal damage and death, leading to a decline in cognitive function.

[0004] Currently, Aβ 1-42 Aggregates are primarily used in the research and diagnosis of Alzheimer's disease (AD). They are obtained by detecting Aβ in cerebrospinal fluid or blood. 1-42 Levels can be used to assess the risk and progression of AD. By studying Aβ... 1-42 The structure, dynamics, and toxicity mechanisms of aggregates can reveal the pathophysiological processes of Alzheimer's disease (AD) and help identify potential therapeutic targets. Detection of Aβ... 1-42Methods for detecting Aβ aggregates include various techniques such as immunology, molecular biology, biochemistry, and physicochemical methods. Immunological methods, such as ELISA and Western blot, require highly specific antibodies; however, current antibodies often cannot distinguish between different forms of Aβ aggregates, such as monomers, oligomers, and filaments. Furthermore, immunological methods are affected by various factors such as sample processing, signal detection, and data analysis, leading to low reproducibility and accuracy. Molecular biological methods, such as immunofluorescence, can directly observe the distribution and morphology of Aβ in cells or tissues, but also require highly specific antibodies and are limited by fluorescence signal attenuation, background noise, and optical resolution. Biochemical methods, such as mass spectrometry, can accurately analyze the amino acid sequence and modifications of Aβ, but require complex sample preparation, instrument operation, and data processing, and cannot reflect the spatial structure and aggregation state of Aβ. Physicochemical methods, such as nuclear magnetic resonance (NMR), can obtain the three-dimensional structure and dynamic information of Aβ, but require large amounts of purified protein and are challenged by sample stability, signal-to-noise ratio, and spectral resolution. In summary, the detection of Aβ... 1-42 Each aggregate-based approach has its own advantages and disadvantages, and no single method can fully meet the needs of early diagnosis of Alzheimer's disease (AD). Therefore, it is necessary to combine multiple methods for comprehensive analysis, or to develop new methods to improve the sensitivity and specificity of detection.

[0005] Near-infrared fluorescence imaging has been widely studied due to its high safety, long fluorescence lifetime, high penetration depth, and low autofluorescence interference. However, the probes reported so far still have problems such as poor targeting, low affinity for β-amyloid protein, short emission wavelength, poor biocompatibility, and difficulty in crossing the blood-brain barrier, which makes it difficult to enter clinical research. Summary of the Invention

[0006] This invention provides a compound and its application in Aβ. 1-42 Applications in aggregate detection and early diagnosis of Alzheimer's disease. The compound described in this invention has a "D-π-A-π-D" configuration, exhibits good fluorescence properties, and can be used as a fluorescent probe. This type of probe is effective against Aβ. 1-42 The aggregates exhibit high sensitivity, high responsiveness, high selectivity, and high affinity, enabling rapid, efficient, and accurate imaging. They are less susceptible to interference from biological matrices and impurities, and are compatible with Aβ. 1-42 The fluorescence signal is significantly enhanced after the aggregates bind, and they can effectively cross the blood-brain barrier, making them suitable for the detection of β-amyloid protein and the early diagnosis of Alzheimer's disease.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] In a first aspect of the invention, a compound or a pharmaceutically acceptable salt thereof is provided having a structure as shown in Formula I:

[0009]

[0010] R1, R2, R3, and R4 are each independently selected from C1-C5 alkyl and C1-C5 hydroxyalkyl groups;

[0011] W and X are each independently selected from benzene rings and naphthalene rings.

[0012] Wherein, alkyl represents a saturated aliphatic group composed only of carbon and hydrogen, with the general formula C1. n H 2n+1 C1-C5 alkyl groups represent alkyl groups with 1-5 carbon atoms, such as methyl (-CH3), ethyl (-C2H5), propyl (-C3H7), butyl (-C4H9), and pentyl (-C5H5). 11 ).

[0013] C1-C5 hydroxyalkyl groups represent alkyl groups containing hydroxyl groups (-OH) with 1-5 carbon atoms; for example, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH, -CH2CH2CH2CH2CH2OH, -CH2CHOHCH3, -CH2CH(OH)CH2OH, etc.

[0014] In some embodiments of the present invention, W and X may be the same or different.

[0015] In some embodiments of the present invention, R1, R2, R3, and R4 may be the same or different.

[0016] In some embodiments of the present invention, R1, R2, R3, and R4 are each independently selected from C1-C3 alkyl and C1-C3 hydroxyalkyl.

[0017] In some embodiments of the present invention, R1, R2, R3, and R4 are the same and are C1-C5 alkyl groups, preferably C1-C3 alkyl groups, such as methyl, ethyl, etc.

[0018] In some embodiments of the present invention, at least three of R1, R2, R3, and R4 are identical and are C1-C5 alkyl groups, preferably C1-C3 alkyl groups.

[0019] In some embodiments of the present invention, at least two of R1, R2, R3, and R4 are identical and are C1-C5 alkyl groups, preferably C1-C3 alkyl groups.

[0020] In some embodiments of the present invention, W and X are the same, and at least three of R1, R2, R3, and R4 are the same and are all C1-C5 alkyl groups.

[0021] In some embodiments of the present invention, W and X are the same, and at least two of R1, R2, R3, and R4 are the same and are C1-C5 alkyl groups;

[0022] In some embodiments of the present invention, W and X are the same, and at most two of R1, R2, R3, and R4 are C1-C5 hydroxyalkyl, preferably C1-C3 hydroxyalkyl, such as -CH2OH or -CH2CH2OH.

[0023] In some embodiments of the present invention, the compound has the structure shown in Formula I1, Formula I2 or Formula I3:

[0024]

[0025] In some embodiments of the present invention, as an example, the compound comprises the following structure:

[0026]

[0027] In a second aspect of the invention, a method for preparing the compound described in the first aspect is provided, comprising: reacting a compound of formula II with a compound of formula III via a Knoevenagel reaction to generate a compound of formula I.

[0028]

[0029] Among them, W, X, R1, R2, R3, and R4 are the same as those defined in the first aspect above.

[0030] In some embodiments of the present invention, when hydroxyl groups are present in R1, R2, R3, and R4, the method further includes a step of protecting the hydroxyl groups, for example, converting the hydroxyl groups into -OTHP groups (hydroxyl groups protected by tetrahydropyran), the operation of which includes: dissolving the compound of formula II in dichloromethane, adding 3,4-dihydropyran and p-toluenesulfonate pyridinium salt, and refluxing.

[0031] In some embodiments of the present invention, the method may further include a step of obtaining a compound of formula III, comprising: reacting a compound of formula IV with a compound of formula V in a Knoevenagel reaction to generate a compound of formula III.

[0032]

[0033] Among them, W, R1, and R2 are the same as those defined in the first aspect above.

[0034] In some embodiments of the present invention, the method may further include a step of obtaining a compound of formula IV, comprising: reacting malononitrile with acetone to generate a compound of formula IV.

[0035] In a third aspect of the invention, a composition is provided comprising the compound described in the first aspect above or a pharmaceutically acceptable salt thereof.

[0036] In a fourth aspect of the invention, a formulation is provided comprising the compound described in the first aspect above, or a pharmaceutically acceptable salt thereof, or the composition described in the third aspect above, and at least one pharmaceutically acceptable carrier or excipient.

[0037] In a fifth aspect of the invention, a product is provided comprising the compound described in the first aspect above or a pharmaceutically acceptable salt thereof, and the product is capable of performing at least one of the following functions:

[0038] (1)Aβ 1-42 Quantitative detection of aggregates, for example, can be achieved by measuring fluorescence intensity to calculate Aβ in a sample. 1-42 The content of aggregates;

[0039] (2)Aβ 1-42 Monitoring the spatiotemporal distribution of aggregates, for example, can be achieved using fluorescence imaging techniques to observe Aβ. 1-42 The distribution of aggregates at a certain point in time, or the aggregation pattern that changes over time;

[0040] (3) Evaluation of the efficacy of drugs that inhibit Aβ aggregation, for example, detecting changes in fluorescence signals after drug treatment to determine whether the drug can effectively inhibit Aβ aggregation;

[0041] (4) Research on the formation and toxicity mechanisms of Aβ aggregates, such as using fluorescent probes to study the aggregation kinetics, stability, and cell-damaging effects of Aβ; and

[0042] (5) Early diagnosis of Alzheimer's disease, such as detecting Aβ aggregation before the onset of lesions, can help detect the disease in advance.

[0043] In some embodiments of the present invention, the product includes fluorescent probes, diagnostic kits, detection devices, and detection equipment. In one embodiment, when the product is a diagnostic kit, it further contains a solvent, such as phosphate buffer solution or physiological saline.

[0044] In a sixth aspect of the invention, the use of the compound described in the first aspect above, or a pharmaceutically acceptable salt thereof, or the composition described in the third aspect above, or the formulation described in the fourth aspect above, in the preparation of a product having at least one of the following uses is provided:

[0045] (1) Used for quantitative detection of Aβ 1-42 Aggregates;

[0046] (2) Used for monitoring Aβ 1-42The spatiotemporal distribution of aggregates;

[0047] (3) Used to evaluate the efficacy of drugs that inhibit Aβ aggregation;

[0048] (4) Used to study the formation and toxicity mechanisms of Aβ aggregates; and

[0049] (5) Early diagnosis of Alzheimer's disease.

[0050] In some embodiments of the present invention, the product is an experimental reagent, a fluorescent probe, a diagnostic kit, a detection device, and a detection equipment, etc.

[0051] In a seventh aspect of the invention, a method for detecting Aβ is provided. 1-42 Methods for aggregates, including:

[0052] The compounds described in the first aspect above, or their pharmaceutically acceptable salts, are used as fluorescent probes;

[0053] Add the fluorescent probe to a substance containing Aβ 1-42 Incubate in samples containing aggregates;

[0054] The fluorescence intensity of the sample is measured using a fluorescence spectrophotometer or imaging instrument.

[0055] Calculate Aβ in the sample based on fluorescence intensity. 1-42 The content of aggregates.

[0056] The method can be used to detect Aβ in biological samples and live animals. 1-42 Aggregates enable early, non-invasive diagnosis of Alzheimer's disease.

[0057] In an eighth aspect of the invention, a method for monitoring Aβ is provided. 1-42 Methods for spatiotemporal distribution of aggregates include:

[0058] The compounds described in the first aspect above, or their pharmaceutically acceptable salts, are used as fluorescent probes;

[0059] Aβ labeled with fluorescent probes 1-42 Aggregates;

[0060] Fluorescence imaging technology was used to continuously monitor changes in fluorescence signals in samples at different time points.

[0061] Determine Aβ based on signal differences 1-42 The distribution of aggregates in time and space.

[0062] In a ninth aspect of the invention, a method for evaluating the efficacy of a drug that inhibits Aβ aggregation is provided, comprising:

[0063] The compounds described in the first aspect above, or their pharmaceutically acceptable salts, are used as fluorescent probes;

[0064] Aβ-containing compounds, both drug-treated and untreated 1-42 The aggregate samples were labeled with fluorescent probes;

[0065] Measure the fluorescence intensity of the two samples;

[0066] The effect of drug treatment on Aβ was determined based on the difference in fluorescence intensity between the two groups. 1-42 Inhibitory effect of aggregates.

[0067] In some embodiments of the present invention, the method can be used to evaluate drug efficacy in in vitro cell models and transgenic animals.

[0068] In a tenth aspect, the present invention provides a method for early diagnosis of Alzheimer's disease, comprising:

[0069] Employing a method that specifically identifies Aβ 1-42 The fluorescent probe compound aggregates detect Aβ in bodily fluid samples (such as cerebrospinal fluid CSF) or blood samples (such as serum). 1-42 The concentration of aggregates is used to achieve non-invasive diagnosis of diseases, and the near-infrared fluorescent probe compound is as described in the first aspect of the present invention above.

[0070] In some embodiments of the present invention, the method includes:

[0071] Extracting bodily fluid samples from patients;

[0072] Add a specific fluorescent probe compound;

[0073] Measure the fluorescence intensity of the sample;

[0074] Aβ was quantitatively calculated based on fluorescence intensity. 1-42 The content of aggregates;

[0075] If the test results are compared with the standard control group and are greater than the normal value, an AD diagnosis can be made.

[0076] Generally speaking, Aβ in CSF 1-42 The normal range for Aβ is between 500-700 pg / mL. Therefore, if Aβ in CSF... 1-42 AD can be diagnosed if the concentration is <500 pg / mL or if the Aβ1-42 in CSF is reduced by more than 30%.

[0077] In embodiments of the present invention, the method enables non-invasive, early diagnosis of Alzheimer's disease.

[0078] Compared to existing technologies, the advantages of this invention include:

[0079] The compound shown in Formula I of this invention has a "D-π-A-π-D" configuration and exhibits good fluorescence properties, particularly near-infrared fluorescence, making it suitable for use as a fluorescent probe, especially a near-infrared fluorescent probe. The compound described in this invention is effective against Aβ... 1-42 The aggregates exhibit high sensitivity, high responsiveness, high selectivity, and high affinity, enabling rapid, efficient, and accurate imaging. The compounds described in this invention can specifically detect β-amyloid protein, are not easily affected by biological matrices and impurities, and show significantly enhanced fluorescence signals after binding to β-amyloid protein. They can effectively cross the blood-brain barrier and can be used for the detection of β-amyloid protein and the early diagnosis of Alzheimer's disease. Attached Figure Description

[0080] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. The embodiments of this application will be described in detail below with reference to the accompanying drawings, wherein:

[0081] Figure 1 In the embodiments of this invention, fluorescent probe compounds 1-5 are respectively reacted with Aβ 1-42 Fluorescence spectra of aggregates and BSA before and after incubation.

[0082] Figure 2 In the embodiments of the present invention, probe compounds 1, 2, and 5 are used with different concentrations of Aβ. 1-42 Fluorescence spectra of aggregates (0-5 μM) after incubation and fluorescence intensity of the probe versus Aβ 1-42 Linear relationship between aggregate concentrations.

[0083] Figure 3 In the embodiments of this invention, probe compounds 1, 2, and 5 and Aβ 1-42 Saturation binding curve of aggregates.

[0084] Figure 4 In vivo imaging of probe compound 2 in Tg mice in this embodiment of the invention. Detailed Implementation

[0085] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this application are readily available through conventional means, and unless otherwise specified, they are used in accordance with conventional methods in the art or as per the product instructions. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this application. The preferred embodiments and materials described herein are for illustrative purposes only.

[0087] This invention provides a compound represented by Formula I, and a method for preparing the compound represented by Formula I.

[0088] Formula I:

[0089] R1, R2, R3, and R4 are each independently selected from C1-C5 alkyl and C1-C5 hydroxyalkyl groups;

[0090] W and X are each independently selected from benzene rings and naphthalene rings.

[0091] This invention provides a series of compounds, wherein compounds 1-5 are used as examples to illustrate the preparation methods, properties, and applications of the compounds.

[0092]

[0093] Example 1 Preparation of intermediates

[0094] 1. Preparation of Compound 7

[0095]

[0096] Compound 6 (500 mg) and acetone (659.40 mg) were dissolved in dichloromethane, and Al2O3 (1.08 g) was added. The mixture was reacted at room temperature for 3 h. After the reaction was completed, the insoluble matter was filtered off, the solvent was evaporated, and the mixture was purified by column chromatography to obtain compound 7 (713 mg).

[0097] Compound 7: 1 H NMR(600MHz,Chloroform-d)δ2.31(s,6H)

[0098] 2. Preparation of Compound 9

[0099]

[0100] Compound 8 (42.21 mg) and compound 7 (36 mg) were dissolved in EtOH, and 60 μL of piperidine was added. The mixture was stirred at room temperature for 3 h. After the reaction was completed, the solvent was evaporated and the mixture was purified by column chromatography to obtain compound 20 (48 mg).

[0101] Compound 9: 1 H NMR(600MHz,Chloroform-d)δ7.50(d,J=7.4Hz,2H),7.26(d,J=15.3Hz,1H),7.19(d,J=15.4Hz,1H),6.68(d,J=7.4Hz,2H),3.08(s,6H),2.42(s,3H);

[0102] 3. Preparation of Compound 12

[0103]

[0104] Compound 10 (1 g), dimethylamine (991.80 mg), and sodium metabisulfite (1.60 g) were added to water and reacted in a sealed container at 140 °C for 48 h. After the reaction was complete, the system was cooled, and ethyl acetate was added for extraction. The organic phase was washed with saturated NaHCO3 and then dried over anhydrous Na2SO4. The solid was filtered off, and the solvent was evaporated. Compound 11 (875 mg, 78%) was purified by column chromatography.

[0105] Under N2 protection conditions at -78℃, 61.46 mg of n-butyllithium was added to a THF solution of 200 mg of compound 11 and stirred for 30 min. Then, 116.89 mg of N,N-dimethylformamide was added, and the reaction mixture was transferred to room temperature and reacted for 20 min. After the reaction was complete, 1 M HCl solution was added and stirred for 30 min. Ammonia water was then added for neutralization, followed by extraction with dichloromethane. The organic phase was dried over anhydrous Na2SO4. The solid was filtered off, and the solvent was evaporated. Compound 12 (85 mg) was purified by column chromatography.

[0106] Compound 11: 1 H NMR(600MHz,Chloroform-d)δ7.82(d,J=2.0Hz,1H),7.61(d,J=9.1Hz,1H),7.52(d,J=8 .7Hz,1H),7.41(d,J=8.7Hz,1H),7.16(dd,J=9.1,2.4Hz,1H),6.86(s,1H),3.05(s,6H).

[0107] Compound 12: 1 H NMR (600MHz, Chloroform-d) δ10.01(s,1H),8.14(s,1H),7.82(d,J=8.8Hz,2H),7.66(d,J=8.6Hz,1H),7.17(d,J=9.1Hz,1H),6.88(s,1H),3.12(s,6H).

[0108] 4. Preparation of Compound 16

[0109]

[0110] Compound 13 (936.91 mg), 2-methylaminoethanol (3 g), and sodium metabisulfite (4 g) were added to water and reacted at 140 °C for 24 h. After the reaction was complete, the mixture was extracted with ethyl acetate, the organic phase was dried over anhydrous Na2SO4, the solvent was evaporated, and the mixture was purified by column chromatography to obtain compound 14 (744 mg).

[0111] Compound 17 (200 mg) and p-toluenesulfonic acid pyridinium salt (70.10 mg) were dissolved in dichloromethane, and 3,4-dihydropyran (328.56 mg) was added. The mixture was refluxed for 28 h. After the reaction was complete, the solvent was evaporated, and compound 15 (162 mg) was purified by column chromatography.

[0112] Under N2 protection at -78°C, 66.78 mg of n-butyllithium was added to a THF solution of 245 mg of compound 15. After stirring, 76.20 mg of N,N-dimethylformamide was added, and the mixture was transferred to room temperature and stirred for 15 min. After the reaction was complete, 1 M HCl solution was added and stirred for 30 min. Then, ammonia water was added to neutralize the system, followed by DCM extraction. The organic phase was dried over anhydrous Na2SO4 and the solvent was evaporated. The solution was purified by column chromatography to obtain 85 mg of compound 16.

[0113] Compound 14: 1 H NMR(600MHz,Chloroform-d)δ7.82(s,1H),7.60(d,J=9.1Hz,1H),7.51(d,J=8.8Hz,1H),7.42(d,J=8.8H z,1H),7.21(dd,J=9.0,2.3Hz,1H),6.93(s,1H),3.88-3.85(m,2H),3.58(t,J=5.6Hz,2H),3.06(s,3H).

[0114] Compound 15: 1H NMR(600MHz,Chloroform-d)δ7.81(s,1H),7.58(d,J=9.1Hz,1H),7.49(d,J=8.7 Hz,1H),7.39(d,J=8.7Hz,1H),7.18(d,J=9.1Hz,1H),6.85(s,1H),4.59(s,1H), 3.96–3.92(m,1H),3.82–3.78(m,1H),3.68–3.62(m,3H),3.48–3.45(m,1H),3.0 9(s,3H),1.79–1.75(m,1H),1.70–1.65(m,1H),1.56(m,2H),1.51–1.47(m,2H).

[0115] Compound 16: 1 H NMR(600MHz,Chloroform-d)δ9.99(s,1H),8.12(s,1H),7.81(d,J=8.6Hz,2H),7.64(d,J=8.6Hz ,1H),7.30–7.14(m,1H),6.93(s,1H),3.91(t,J=5.4Hz,2H),3.67(t,J=5.5Hz,2H),3.15(s,3H).

[0116] Example 2 Preparation of compounds 1, 2, 3, 4, and 5

[0117] 1. Preparation of Compound 1

[0118]

[0119] Compound 12 (25 mg) and compound 7 (6.66 mg) were dissolved in EtOH, and 50 μL of piperidine was added. The mixture was reacted at room temperature for 24 h. After the reaction was completed, the solvent was evaporated and compound 1 (9 mg) was purified by column chromatography.

[0120] Compound 1: ESI-MS (m / z): 399 [M+H] + ; 1H NMR(600MHz,Chloroform-d)δ7.48(d,J=7.3Hz,4H),7.32(d,J=15.6Hz,1H),7.30(d,J=15.6Hz,1H),7.07(d,J=15.6Hz,1H),7.05(d ,J=15.6Hz,1H),6.78(d,J=8.1Hz,2H),6.70(d,J=8.4Hz,2H),3.87(t,J=5.6Hz,2H),3.60(t,J=5.6Hz,2H),3.10(s,3H),3.07(s,6H)

[0121] 2. Preparation of Compound 2

[0122]

[0123] Compound 17 (15.12 mg) and compound 9 (20 mg) were dissolved in EtOH, and 30 μL of piperidine was added. The mixture was reacted at room temperature for 8 h. After the reaction was completed, the solvent was evaporated and compound 2 (4 mg) was purified by column chromatography.

[0124] Compound 2: ESI-MS (m / z): 399 [M+H] + ; 1 H NMR(600MHz,Chloroform-d)δ7.48(d,J=7.3Hz,4H),7.32(d,J=15.6Hz,1H),7.30(d,J=15.6Hz,1H),7.07(d,J=15.6Hz,1H),7.05(d ,J=15.6Hz,1H),6.78(d,J=8.1Hz,2H),6.70(d,J=8.4Hz,2H),3.87(t,J=5.6Hz,2H),3.60(t,J=5.6Hz,2H),3.10(s,3H),3.07(s,6H)

[0125] 3. Preparation of Compound 3

[0126]

[0127] Compound 12 (12.60 mg) and compound 9 (10 mg) were dissolved in EtOH, and 25 μL of piperidine was added. The mixture was reacted at room temperature for 16 h. After the reaction was completed, the solvent was evaporated and the mixture was purified by column chromatography to obtain compound 3 (4 mg).

[0128] Compound 3: ESI-MS (m / z): 418 [M+H] + ; 1H NMR(600MHz,Chloroform-d)δ7.81(s,1H),7.74(d,J=8.2Hz,1H),7.67(s,1H),7.51(d,J=8.7Hz,1H),7.54-7.44(m,5H),7.39(d,J= 15.6Hz,1H),7.32(d,J=15.5Hz,1H),7.13(d,J=15.6Hz,1H),7.06(d,J=15.7Hz,1H),6.70(d,J=8.2Hz,2H),3.13(s,6H),3.06(s,6H)

[0129] 4. Preparation of Compound 4

[0130]

[0131] Compound 16 (14.21 mg) and compound 9 (9.80 mg) were dissolved in EtOH, and 25 μL of piperidine was added. The reaction was carried out at room temperature for 10 h. After the reaction was complete, the solvent was evaporated, and compound 4 (4 mg) was purified by column chromatography.

[0132] Compound 4: ESI-MS (m / z): 448 [M+H] + ; 1 H NMR(600MHz,Chloroform-d)δ7.80(s,1H),7.72(d,J=9.0Hz,1H),7.69–7.64(m,2H),7.51(d,J=8.7Hz,2H),7.47(d,J=15.9Hz,1H),7.39(d,J=15.5Hz 1H),7.26–7.21(m,2H),7.13(d,J=15.6Hz,1H),6.98(s,1H),6.70(d,J=8.7Hz ,2H),3.91(t,J=5.6Hz,2H),3.66(t,J=6.1Hz,2H),3.14(s,3H),3.08(s,6H).

[0133] 5. Preparation of Compound 5

[0134]

[0135] Compound 16 (26 mg) and compound 7 (6.66 mg) were dissolved in EtOH, and 50 μL of piperidine was added. The mixture was reacted at room temperature for 24 h. After the reaction was completed, the solvent was evaporated and compound 5 (8 mg) was purified by column chromatography.

[0136] Compound 5: Data MS m / z 304 (M+1); 1H NMR(600MHz,Chloroform-d)δ7.80(s,2H),7.70(d,J=9.1Hz,2H),7.667(d,J=8.8Hz,2H),7.63(d,J=8.7 Hz,2H),7.53(d,J=15.7Hz,2H),7.29(d,J=15.7Hz,2H),7.19(d,J=8.3Hz,2H),6.91(s,2H),3.15(s,6H).

[0137] Example 3 Evaluation of the optical properties of fluorescent probes

[0138] Evaluation of fluorescence spectral properties:

[0139] Compounds 1-5 prepared in Example 2 were formulated into a stock solution with a concentration of 2 mM. The stock solution was diluted with different solvents such as dichloromethane (DCM), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), methanol (MeOH) and phosphate buffered saline (PBS). The excitation and emission wavelengths of the probe compounds were measured using an RF-4600 fluorescence spectrophotometer. The results are shown in Table 1.

[0140] Table 1. Fluorescence spectral data of fluorescent probes

[0141]

[0142]

[0143] Where: λ em Indicates the maximum emission wavelength of the probe; λ ex This indicates the maximum absorption wavelength of the probe; Stokes shift refers to the difference between the strongest wavelengths of the same electronic transition in the absorption and emission spectra.

[0144] Table 1 shows that the emission wavelengths of the compounds are all above 600 nm, belonging to red fluorescence. Among them, compounds 3, 4, and 5 have emission wavelengths in the near-infrared region (650-900 nm) in some solvents, exhibiting good near-infrared fluorescence characteristics. Compounds 1-5 can be used as fluorescent probes; therefore, in the following experiments, compounds 1-5 are also referred to as probe compounds 1-5.

[0145] Example 4 Bioactivity evaluation of fluorescent probes

[0146] 1. Fluorescent probe and Aβ 1-42 In vitro binding assay of aggregates

[0147] Purchase of Aβ 1-42TFA monomer was dissolved in 1 mL of 1% ammonia water. 100 μL of the solution was taken and 900 μL of PBS was added to make the concentration 100 μM. The solution was then placed in a shaker and incubated at 37 °C and 100 rpm / min for 7 days.

[0148] Prepare PBS solutions of compounds 1-5 (prepared according to Example 2), and add the Aβ obtained from incubation respectively. 1-42 Aggregates, with a final probe concentration of 1 μM, Aβ 1-42 The final concentration of the aggregates was 5 μM. After incubation in a shaker for 30 min, the excitation and emission spectra were recorded and measured using a fluorescence spectrophotometer.

[0149] Fluorescent probes and Aβ 1-42 Fluorescence spectra of aggregates before and after incubation are shown below. Figure 1 As shown, probe compounds 1-5 exhibit weak fluorescence intensity in PBS solution. When the probe compounds react with Aβ... 1-42 The aggregates exhibited significant fluorescence enhancement upon binding, along with a certain degree of blue shift in emission wavelength (due to λ). em 1 Blue shift to λ em 2 ), probe compounds 1-5 and Aβ 1-42 Table 2 shows the fluorescence spectra before and after aggregate binding. Probe compounds 1-5 can all bind to Aβ. 1-42 The fluorescence enhancement effect produced after aggregate binding indicates that probe compounds 1-5 all have a certain degree of recognition and labeling of Aβ. 1-42 The ability of the aggregate to form compounds. Probe compound 3 showed the highest fluorescence enhancement, reaching 61-fold, and the emission wavelength decreased from 610 nm to 529 nm, indicating its affinity for Aβ. 1-42 Significant fluorescence changes occurred after the aggregates bound. Probe compounds 2 and 5 also showed high fluorescence enhancement factors, 17-fold and 27-fold respectively, accompanied by amplitude changes in emission wavelength.

[0150] The above experimental results demonstrate that probe compounds 1-5 are effective against Aβ. 1-42 The aggregates exhibit a strong fluorescence response, making them valuable for the early diagnosis of Alzheimer's disease (AD).

[0151] Table 2 Probe compounds 1-5 and Aβ 1-42 Fluorescence spectral data before and after aggregate binding

[0152]

[0153] Where: λem 1 Indicates the emission wavelength of the probe in PBS; λem 2 Indicates the probe and Aβ 1-42 The emission wavelength after aggregation; fold indicates the probe and Aβ1-42 The fluorescence enhancement factor produced after aggregate binding.

[0154] 2. In vitro binding experiment of probe to bovine serum albumin (BSA)

[0155] Prepare PBS solutions of compounds 1-5 (prepared according to Example 2) and record their fluorescence intensity respectively; add BSA (final concentration of 23 μg / mL) to PBS buffer containing fluorescent probes (final concentration of 1 μM each), incubate on a shaker for 30 min and then record and measure their fluorescence intensity using a fluorescence spectrophotometer.

[0156] Fluorescence spectra of the fluorescent probe before and after incubation with BSA are shown below. Figure 1 As shown, the data indicate that probe compounds 1 and 2, when co-incubated with BSA, only produced weak fluorescence response signals, and the fluorescence enhancement factor was much smaller than that with Aβ. 1-42 The fluorescence is enhanced by the binding of aggregates; the fluorescence response signals produced by probe compounds 3, 4, and 5 with BSA are relatively strong, but not as strong as those produced by Aβ. 1-42 The fluorescence enhancement resulting from aggregate binding was still not significant. Experimental results demonstrate that probe compounds 1-5 can avoid fluorescence signal interference caused by interaction with BSA.

[0157] 3. Probe fluorescence titration spectroscopy experiment:

[0158] Take a certain amount of probe compound solution and a certain concentration gradient of Aβ 1-42 Mix the aggregate solutions and add PBS buffer to a total volume of 1 mL (final concentration of probe compound is 1 μM, Aβ). 1-42 The final concentration of the aggregate was 0-5 μM. The probe compound was then mixed with different concentrations of Aβ. 1-42 After the aggregates were co-incubated for about 30 minutes, their fluorescence spectra were measured and recorded using a fluorescence spectrophotometer.

[0159] Taking probe compounds 1, 2, and 5 as examples, the experimental results are as follows: Figure 2 As shown, in Aβ 1-42 When the aggregate concentration is in the range of 0-5 μM, probe compounds 1, 2, and 5 react with Aβ. 1-42 The fluorescence enhancement resulting from aggregate binding increases linearly with increasing concentration, demonstrating that probe compounds 1, 2, and 5 can achieve Aβ inhibition within a certain concentration range. 1-42 The ability to monitor changes in aggregate concentration.

[0160] 4. Saturation bonding test

[0161] A probe solution with a certain concentration gradient and a certain amount of Aβ were added. 1-42Aggregate solutions were mixed (the final concentrations of the probe in the mixed solutions were 0, 2.5, 5, 10, 50, 100, 150, 500, 1000, and 2000 nM, respectively), Aβ 1-42 The final concentration of the aggregate was 10 μg / mL. PBS was added to bring the final volume of the mixture to 1 mL. The probe was then mixed with Aβ. 1-42 After incubating the aggregates with shaking for about 30 minutes, the fluorescence intensity was measured using a microplate reader. GraphPad Prism 5.0 was used to perform nonlinear fitting and plot the relationship between fluorescence intensity and probe concentration to calculate the binding constant.

[0162] Taking probe compounds 1, 2, and 5 as examples, the results are as follows: Figure 3 As shown, the data indicate that probe compounds 1, 2, and 5 are related to Aβ. 1-42 The binding constant (K) of aggregates d The concentrations were 50.07±3.88 nM, 16.07±4.23 nM, and 47.05±4.12 nM, respectively, especially probe 2 against Aβ. 1-42 It has higher affinity, enabling probes to target Aβ. 1-42 A highly efficient combination.

[0163] 5. Determination of the lipid-water partition coefficient

[0164] A certain amount of n-octanol (water-saturated) solution of probe compounds 1, 2, 3, 4, and 5 was measured into an Erlenmeyer flask. A certain amount of distilled water and pH 7.4 buffer solution (saturated with n-octanol) were added. Two parallel operations were performed. After sealing the flasks, they were shaken for 1 hour (25°C). The flasks were then transferred to a separatory funnel and allowed to stand to separate the aqueous and organic phases. The organic and aqueous layers were taken for liquid chromatography analysis to detect the sample concentration. The lipid-water partition coefficient was calculated according to the formula.

[0165] Calculations showed that the lipid-water partition coefficients of probe compounds 1, 2, 3, 4, and 5 were 6.45, 3.45, 4.23, 2.49, and 2.44, respectively, indicating that the introduction of hydroxyl groups can increase the hydrophilicity of the compounds. Among them, probe compounds 2, 4, and 5 had lipid-water partition coefficients between 2.0 and 3.5, exhibiting more ideal biocompatibility and potentially crossing the blood-brain barrier.

[0166] 6. In vivo near-infrared imaging experiment

[0167] Prior to background imaging, Tg mice were intravenously injected with a compound (0.1 mg / kg, 20% DMSO, 80% propylene glycol, 50 μL). Fluorescent signals from the brain were recorded at different time points following the intravenous injection of the compound. During imaging, the mice were anesthetized with isoflurane gas under oxygen flow to maintain them in the imaging phase. The imaging data were analyzed, and regions of interest (ROIs) were plotted around the brain regions.

[0168] Taking compound 2 as an example, the results are as follows: Figure 4 As shown, compound 2 can effectively penetrate the blood-brain barrier in Tg mice and has a certain binding affinity to Aβ plaques, thus enabling the labeling of Aβ plaques in Tg mice.

[0169] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, having the following structure: 、 。 2. The process for the preparation of a compound according to claim 1, wherein, The preparation method of compound 2 is as follows: 15.12 mg of compound 17 and 20 mg of compound 9 were dissolved in ethanol, 30 μL of piperidine was added, and the reaction was carried out at room temperature for 8 h; after the reaction was completed, the solvent was evaporated, and compound 2 was purified by column chromatography. The reaction formula for compound 2 is: ； The preparation method of compound 5 is as follows: 26 mg of compound 16 and 6.66 mg of compound 7 were dissolved in ethanol, 50 μL of piperidine was added, and the reaction was carried out at room temperature for 24 h. After the reaction was completed, the solvent was evaporated and compound 5 was purified by column chromatography. The reaction formula for compound 5 is: 。 3. The method of making according to claim 2, wherein, The preparation method of compound 9 is as follows: 42.21 mg of compound 8 and 36 mg of compound 7 were dissolved in ethanol, 60 μL of piperidine was added, the mixture was stirred at room temperature for 3 h, after the reaction was completed, the solvent was evaporated, and compound 9 was purified by column chromatography. The reaction formula for compound 9 is: 。 4. The method of making according to claim 2, wherein, The preparation method of compound 7 is as follows: 500 mg of compound 6 and 659.40 mg of acetone were dissolved in dichloromethane, 1.08 g of Al2O3 was added, and the reaction was carried out at room temperature for 3 h. After the reaction was completed, the insoluble matter was filtered off, the solvent was evaporated, and the compound 7 was purified by column chromatography. The reaction formula for compound 7 is: 。 5. A composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof.

6. A formulation comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof or the composition of claim 5 and at least one pharmaceutically acceptable carrier.

7. A product comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof, and capable of performing at least one of the following functions: (1) Αβ 1-42 quantitative detection of aggregates; (2) Αβ 1-42 Monitoring of the spatiotemporal distribution of aggregates; (3) Evaluation of the efficacy of drugs that inhibit Aβ aggregation; (4) Research on the formation and toxicity mechanisms of Aβ aggregates; and (5) Early diagnosis of Alzheimer's disease.

8. The product of claim 7, wherein, The products include fluorescent probes and detection devices.

9. The product of claim 8, wherein, The product in question is a diagnostic reagent kit.

10. The product of claim 9, wherein, When the product is a diagnostic kit, it also contains a solvent.

11. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof, or the composition of claim 5, or the formulation of claim 6, in the preparation of a product having at least one of the following uses: (1) for quantitative detection of Αβ 1-42 aggregates; (2) for monitoring Abeta 1-42 spatiotemporal distribution of aggregates; (3) Used to evaluate the efficacy of drugs that inhibit Aβ aggregation; (4) Used to study the formation and toxicity mechanisms of Aβ aggregates; and (5) Early diagnosis of Alzheimer's disease.

12. Use according to claim 11, characterized in that, The products are laboratory reagents, fluorescent probes, and detection devices.

13. The use according to claim 11, characterized in that, The product in question is a diagnostic reagent kit.

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