A near-infrared fluorescent probe targeting amyloid β and its application in the diagnosis of Alzheimer's disease

Abstract

The present invention belongs to the field of fluorescent probe preparation and disease detection technology, and specifically relates to a near-infrared fluorescent probe targeting amyloid beta protein and its application in the diagnosis of Alzheimer's disease. The near-infrared fluorescent probe provided by the present invention is a type of D-π-A type fluorescent probe, which has an emission wavelength in the near-infrared region, can target and bind to amyloid beta protein, is not easily interfered by biological matrices and impurities, and has a significantly enhanced fluorescence intensity after specific binding to the beta protein, high sensitivity, low cytotoxicity, and good imaging effect, so it can be used to detect amyloid beta protein and Alzheimer's disease diagnosis, and therefore has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probe preparation and disease detection, and specifically relates to a near-infrared fluorescent probe targeting beta-amyloid protein and its application in the diagnosis of Alzheimer's disease. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Alzheimer's disease (AD) is an irreversible neurodegenerative disease with major clinical manifestations including short-term memory loss, decreased sense of direction, decreased ability to express, and personality changes. It is one of the main causes of disability and death in the elderly population and has caused serious social, medical, and economic problems. The etiology of AD is complex and the pathogenesis is unknown. The early pathological features are the deposition of amyloid-β (Aβ) in the brain, neurofibrillary tangles, and neuronal damage or apoptosis.

[0004] In order to achieve early and accurate diagnosis of AD, researchers have developed a large number of imaging detection probes for Aβ proteins based on the advantages of near-infrared fluorescent probe imaging, such as safety, high efficiency and low cost. These probes can be mainly divided into curcumin and its derivatives, cyanine derivatives, boron dipyrromethene (BODIPY) and donor-acceptor (Donor-Acceptor Architecture, DA) type probes.

[0005] However, the probes reported so far are still limited by factors such as short emission wavelength, poor targeting, low affinity to amyloid-β protein, and low sensitivity, making it difficult for them to enter subsequent clinical trial research. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a near-infrared fluorescent probe targeting amyloid beta and its application in the diagnosis of Alzheimer's disease. The near-infrared fluorescent probe provided by the present invention is a type of D-π-A type fluorescent probe, which has an emission wavelength in the near-infrared region, can target and bind to amyloid beta, is not easily interfered by biological matrices and impurities, and has a significantly enhanced fluorescence intensity after specific binding to the beta protein, high sensitivity, low cytotoxicity, and good imaging effect, so it can be used to detect amyloid beta and diagnose Alzheimer's disease. Based on the above research results, the present invention is completed.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a compound, the structural formula of which is shown in formula (I):

[0009]

[0010] Wherein, n = 0 to 2;

[0011] R1 is independently selected from: C1-C5 alkyl or C1-C5 hydroxyalkyl;

[0012] R2 is independently selected from: C1-C5 alkyl or C1-C5 hydroxyalkyl;

[0013] W is independently selected from: a benzene ring or a naphthalene ring;

[0014] R3 is independently selected from:

[0015] Here, the C1-C5 hydroxyalkyl group represents a C1-C5 alkyl group substituted by a hydroxy group.

[0016] With respect to substituents, the term "independently" used herein refers to the situation where when more than one substituent is possible, the substituents may be the same or different from each other.

[0017] The compounds also include pharmaceutically acceptable salts or esters or solvates, tautomers, meso- and racemates, racemates, stereoisomers, metabolites or prodrugs thereof.

[0018] The compound has the following structure:

[0019]

[0020] The second aspect of the present invention provides a method for synthesizing the above-mentioned compound, wherein the synthesis route of the compound represented by formula (I) is as follows:

[0021]

[0022] The third aspect of the present invention provides the use of the above compound as a fluorescent probe or in the preparation of a fluorescent probe. The emission wavelength of the compound is in the near-infrared region, and it can target and bind to amyloid β protein. After specifically binding to the β protein, the fluorescence intensity is significantly enhanced. Therefore, it can be specifically used as a near-infrared fluorescent probe.

[0023] The fourth aspect of the present invention provides a preparation comprising the above-mentioned compound and at least one pharmaceutically acceptable carrier.

[0024] A fifth aspect of the present invention provides a product for detecting amyloid β, wherein the product at least comprises the above-mentioned compound and / or preparation.

[0025] As mentioned above, the above compound, as a D-π-A type near-infrared fluorescent probe, can target and bind to β-amyloid protein, and thus can be used for the detection of Alzheimer's disease.

[0026] Therefore, the sixth aspect of the present invention provides a product for detecting Alzheimer's disease, wherein the product at least comprises the above-mentioned compound and / or preparation.

[0027] A seventh aspect of the present invention provides a method for detecting amyloid β protein, the method comprising: using the above-mentioned compounds, preparations and / or products to detect a sample to be tested.

[0028] An eighth aspect of the present invention provides a method for detecting Alzheimer's disease, the method comprising: using the above-mentioned compounds, preparations and / or products to detect a test sample of a subject.

[0029] Beneficial technical effects of the above technical solution:

[0030] The above technical scheme designs a series of compounds, which can be used as D-π-A type near-infrared fluorescent probes. Experimental verification shows that the emission wavelength of this type of probe is in the near-infrared region, and it can target and bind to β-amyloid protein. After specific binding with β-protein, the fluorescence intensity is significantly enhanced, the sensitivity is high, the cytotoxicity is low, and the imaging effect is good. It is of great significance for the research and development of early diagnostic probes for Alzheimer's disease, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 2b(a), 2c(b), 3b(c), 3c(d), 4b(e), 4c(f) in Example 6 of the present invention

[0033] ) fluorescent probes were respectively 1-42 Fluorescence spectra of aggregates before and after incubation with BSA;

[0034] Figure 2 The fluorescent probes 2b, 2c, 3b, 3c, 4b, and 4c in Example 6 of the present invention are respectively 1-42 fluorescence enhancement factor after aggregate binding;

[0035] Figure 3 The probes 2c, 3c, and 4c in Example 6 of the present invention and different concentrations of Aβ 1-42Fluorescence spectra of aggregates (0-5 μM) after incubation ((a), (c), (e)) and the fluorescence intensity of probes 2c, 3c, and 4c and Aβ 1-42 Linear relationship between aggregate concentrations ((b), (d), (f));

[0036] Figure 4 The probe 4c and Aβ in Example 6 of the present invention 1-42 Saturation binding curves of aggregates. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0038] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0039] In a typical embodiment of the present invention, a compound is provided, the structural formula of the compound is as shown in formula (I):

[0040]

[0041] Wherein, n = 0 to 2;

[0042] R1 is independently selected from: C1-C5 alkyl or C1-C5 hydroxyalkyl;

[0043] R2 is independently selected from: C1-C5 alkyl or C1-C5 hydroxyalkyl;

[0044] W is independently selected from: a benzene ring or a naphthalene ring;

[0045] R3 is independently selected from:

[0046] Here, the C1-C5 hydroxyalkyl group represents a C1-C5 alkyl group substituted by a hydroxy group.

[0047] With respect to substituents, the term "independently" used herein refers to the situation where when more than one substituent is possible, the substituents may be the same or different from each other.

[0048] The compounds also include pharmaceutically acceptable salts or esters or solvates, tautomers, meso- and racemates, racemates, stereoisomers, metabolites or prodrugs thereof.

[0049] According to the understanding of those skilled in the art, the pharmaceutically acceptable salts include alkali metal salt forms of the above compounds (specific examples include sodium salts or potassium salts), or salts formed by the compounds with inorganic salts such as hydrochloric acid, sulfuric acid, nitric acid or hydrobromic acid, and salts formed with organic acids such as methanesulfonic acid, toluenesulfonic acid, citric acid, acetic acid or trifluoroacetic acid. The term "pharmaceutically acceptable" or "pharmaceutically acceptable" used interchangeably therewith, for example, when describing "pharmaceutically acceptable salts", means that the salt is not only physiologically acceptable to the subject, but also refers to a synthetic substance with pharmaceutical use value, such as a salt formed as an intermediate when performing chiral resolution, although the salt of this intermediate cannot be directly administered to the subject, the salt can play a role in obtaining the final product of the present invention.

[0050] In another specific embodiment of the present invention, the compound has the following structure:

[0051]

[0052] In another specific embodiment of the present invention, a method for synthesizing the above-mentioned compound is provided, wherein the synthesis route of the compound represented by formula (I) is as follows:

[0053]

[0054] In another specific embodiment of the present invention, the method comprises: dissolving the compound represented by formula (II) in a protic solvent, and subjecting the compound represented by formula (III) to a Knoevenagel reaction under the catalysis of a base to obtain the compound represented by formula (I).

[0055] Among them, the specific groups of substituents R1, R2, W and R3 are as described in the first aspect and will not be repeated here.

[0056] In another specific embodiment of the present invention, the preparation method of the compound represented by formula (II) is: dissolving the compound represented by formula (IV) and (1,3-dioxolane-2-yl)methyltriphenylphosphonium bromide in a protic solvent, and performing a Wittig reaction under the action of a base and a phase transfer catalyst to obtain;

[0057] Alternatively, the above process is included, and the product obtained by the wittig reaction is used as a raw material to replace the compound represented by formula (IV) and repeat the above process;

[0058]

[0059] When R1 or R2 is (C1-C4) hydroxyalkyl, 3,4-dihydropyran is required to protect -OH during the wittig reaction, i.e., convert it into an -OTHP group. The specific method includes: dissolving the compound represented by formula (II) in dichloromethane, adding 3,4-dihydropyran and pyridinium p-toluenesulfonate, and reflux reaction for 24-48 hours.

[0060] In another specific embodiment of the present invention, the use of the above compound as a fluorescent probe or in the preparation of a fluorescent probe is provided. The emission wavelength of the compound is in the near-infrared region, and it can target and bind to amyloid β protein. After specifically binding to β protein, the fluorescence intensity is significantly enhanced. Therefore, it can be specifically used as a near-infrared fluorescent probe.

[0061] In another specific embodiment of the present invention, a preparation is provided, which comprises the above-mentioned compound and at least one pharmaceutically acceptable carrier.

[0062] The pharmaceutically acceptable carriers described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum proteins, buffer substances such as phosphates, glycerol, sorbitol, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, lanolin, etc. The content of the carrier in the preparation can be 1% by weight to 98% by weight, usually accounting for about 80% by weight. For convenience, local anesthetics, preservatives, buffers, etc. can be directly dissolved in the carrier.

[0063] In another specific embodiment of the present invention, a product for detecting amyloid β is provided, wherein the product at least comprises the above-mentioned compound and / or preparation.

[0064] The product may be a detection kit, a detection device and / or a detection equipment, which is not specifically limited here.

[0065] The product may also include substances and components commonly used in detection kits, detection devices and / or detection equipment. For example, in the detection kit, a solvent may be included, and the solvent may be a phosphate buffer solution, physiological saline, etc. In the detection device or equipment, a fluorescence spectrophotometer may also be included to detect the emitted fluorescence signal.

[0066] As mentioned above, the above compound, as a D-π-A type near-infrared fluorescent probe, can target and bind to β-amyloid protein, and thus can be used for the detection of Alzheimer's disease.

[0067] Therefore, in another specific embodiment of the present invention, a product for detecting Alzheimer's disease is provided, wherein the product at least comprises the above-mentioned compound and / or preparation.

[0068] In the present invention, the detection of Alzheimer's disease includes but is not limited to screening, (early) diagnosis, monitoring or prediction of the progression of Alzheimer's disease in patients with Alzheimer's disease.

[0069] Likewise, the product may be a detection kit, a detection device and / or a detection equipment, which is not specifically limited herein.

[0070] The product may also include substances and components commonly used in detection kits, detection devices and / or detection equipment. For example, in the detection kit, a solvent may be included, and the solvent may be a phosphate buffer solution, physiological saline, etc. In the detection device or equipment, a fluorescence spectrophotometer may also be included to detect the emitted fluorescence signal.

[0071] In another specific embodiment of the present invention, a method for detecting amyloid β is provided, the method comprising: using the above-mentioned compounds, preparations and / or products to detect a sample to be tested.

[0072] The sample to be tested is a sample containing or suspected of containing β-amyloid protein, and the sample can be a nerve cell or a nerve tissue (such as brain tissue), which is not specifically limited here.

[0073] In another specific embodiment of the present invention, a method for detecting Alzheimer's disease is provided, the method comprising: using the above-mentioned compounds, preparations and / or products to detect a test sample of a subject.

[0074] The detection of Alzheimer's disease includes but is not limited to screening, (early) diagnosis, monitoring or prediction of the progression of Alzheimer's disease in patients with Alzheimer's disease.

[0075] The subject may be an animal, especially a mammal, among which humans are most preferred.

[0076] The sample to be tested is a sample containing or suspected of containing β-amyloid protein, and the sample can be a nerve cell or a nerve tissue (such as brain tissue), which is not specifically limited here.

[0077] The present invention is further explained by the following examples, but it does not constitute a limitation of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Example 1: Preparation of intermediates

[0078] Preparation of compound 1b

[0079] Compound 1a (1 g), (1,3-dioxolane-2-yl)methyltriphenylphosphonium bromide (3.46 g), NaH (643.20 mg) and trace 18-crown ether-6 were dissolved in tetrahydrofuran. After the reaction was completed, water was added to quench, extracted with dichloromethane, the organic phase was dried with anhydrous Na2SO4, and the solvent was evaporated. The residue was dissolved in THF, and hydrochloric acid was added and stirred at room temperature. After the reaction was completed, saturated NaHCO3 solution was added to adjust the pH to 7, extracted with ethyl acetate, the organic phase was dried with anhydrous Na2SO4, and the solvent was evaporated. Purification by column chromatography gave compound 1b (624 mg).

[0080] Compound 1b: 1 H NMR (600 MHz, Chloroform-d) δ9.59 (d, J = 7.9 Hz, 1H), 7.45 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 15.6 Hz, 1H), 6.69 (d, J = 8.9 Hz, 2H), 6.54 (dd, J = 15.6, 7.9 Hz,1H),3.05(s,6H).

[0081]

[0082] Preparation of compound 1c

[0083] Referring to the synthesis method of compound 1b, compound 1c (27 mg) was prepared using compound 1b (40 mg, 0.23 mmol) as raw material.

[0084] Compound 1c: 1 H NMR (600 MHz, Chloroform-d) δ9.56 (d, J = 8.1 Hz, 1H), 7.40 (d, J = 8.8 Hz, 2H), 7.25 (dd, J = 15.0, 11.0 Hz, 1H), 6.94 (d, J = 15.3 Hz, 1H), 6.82 (dd, J=15.3, 11.0 Hz, 1H), 6.67 (d, J=8.7 Hz, 2H), 6.18 (dd, J=15.0, 8.1 Hz, 1H), 3.03 (s, 6H).

[0085]

[0086] Preparation of compound 1f

[0087] Compound 1d (200 mg) and pyridinium p-toluenesulfonate (70.10 mg) were dissolved in dichloromethane, 3,4-dihydropyran (328.56 mg) was added, and the mixture was refluxed for 28 h. After the reaction was completed, the solvent was evaporated and the compound 1e (162 mg) was purified by column chromatography.

[0088] Compound 1e (88.40 mg), (1,3-dioxolan-2-yl)methyltriphenylphosphonium bromide (288.20 mg), sodium hydride (NaH, 32.20 mg) and trace 18-crown ether-6 were dissolved in tetrahydrofuran and reacted at room temperature for 24 h. The reaction was completed.

[0089] Afterwards, water was added to quench the NaH, extracted with dichloromethane, and the organic phase was dried over anhydrous Na2SO4 and the solvent was evaporated. The residue was dissolved in THF, excess hydrochloric acid (2M) was added, and stirred at room temperature for 3 hours. Subsequently, a saturated NaHCO3 solution was added dropwise to the system to adjust the pH value to about 7, extracted with ethyl acetate, and the organic phase was dried over anhydrous Na2SO4 and the solvent was evaporated. Purification by column chromatography gave compound 1f (624 mg).

[0090] Compound 1e: 1 H NMR(600MHz,Chloroform-d)δ9.73(s,1H),7.72(d,J=8.8Hz,2H),6.75(d,J=9.0Hz,2H),4.58(t,J=3.6Hz,1H),3.95–3.91(m,1H) ,3.78–3.74(m,1H),3.69–3.60(m,3H),3.49–3.45(m,1H),3.11(s,3H),1.79–1.74(m,1H),1.70–1.66(m,1H),1.57–1.49(m,4H).

[0091] Compound 1f: 1 H NMR(600MHz,Chloroform-d)δ9.51(d,J=7.9Hz,1H),7.40(d,J=8.9Hz,2H),7.32(d,J=15.6Hz,1H),6.73( d,J=8.9Hz,2H),6.46(dd,J=15.6,7.9Hz,1H),3.84(t,J=5.6Hz,2H),3.58(t,J=5.7Hz,2H),3.07(s,3H).

[0092]

[0093] Preparation of compound 1h

[0094] According to the preparation steps of compound 1e, compound 1f (40 mg) was used as the raw material to obtain compound 1g (49 mg). According to the synthesis method of compound 1f, compound 1g (50 mg) was used as the raw material to obtain compound 1h (23 mg). Compound 1g: 1H NMR(600MHz,Chloroform-d)δ9.59(d,J=7.9Hz,1H),7.44(d,J=8.9Hz,2H),7.37(d, J=15.6Hz,1H),6.72(d,J=8.9Hz,2H),6.54(dd,J=15.6,7.9Hz,1H),4.59–4.57(m,1H ),3.94–3.90(m,1H),3.80–3.76(m,1H),3.66–3.59(m,3H),3.49–3.45(m,1H),3.08 (s,3H),1.80–1.75(m,1H),1.71–1.66(m,1H),1.58–1.53(s,2H),1.52–1.48(s,2H).

[0095] Compound 1h: 1 H NMR(600MHz,Chloroform-d)δ9.53(d,J=8.1Hz,1H),7.39(d,J=7.2Hz,2H),7.23(d,J=12.6Hz,1H),6.93(d,J=15.3Hz,1H),6.81(dd, J=14.5,11.8Hz,1H),6.74(d,J=7.3Hz,2H),6.16(dd,J=14.9,8.0Hz,1H),3.85(t,J=5.0Hz,2H),3.56(t,J=5.6Hz,2H),3.06(s,3H).

[0096]

[0097] Preparation of compound 11

[0098] Compound 1i (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 completed, 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 1j (744 mg).

[0099] According to the synthesis method of compound 1e, compound 1k (245 mg) was prepared using compound 1j (200.02 mg) as raw material.

[0100] At -78°C, under N2 protection, n-butyl lithium (66.78 mg) was added to a THF solution of compound 1k (245 mg), and N,N-dimethylformamide (76.20 mg) was added after stirring, and the mixture was transferred to room temperature and stirred for 15 min. After the reaction was completed, 1M HCl solution was added and stirred for 30 min, and then ammonia water was added to neutralize the system, and DCM was extracted. The organic phase was dried over anhydrous Na2SO4 and the solvent was evaporated, and purified by column chromatography to obtain compound 1l (85 mg).

[0101] Compound 1j: 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).

[0102] Compound 1k: 1 H 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).

[0103] Compound 11: 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).

[0104]

[0105] Preparation of compound 1n

[0106] According to the synthesis method of compound 1f, compound 1m (119 mg) was prepared using compound 1l (100 mg) as raw material.

[0107] According to the synthesis method of compound 1f, compound 1n (107 mg) was prepared using compound 1m (261.91 mg) as raw material.

[0108] Compound 1m: 1 H NMR(600MHz,Chloroform-d)δ10.00(s,1H),8.14(s,1H),7.83–7.79(m,2H),7.64(d,J=8.5Hz,1H),7.22(d,J=9.1Hz,1 H),6.90(s,1H),4.59(s,1H),3.99–3.95(m,1H),3.80–3.65(m,4H),3.49–3.45(m,1H),3.16(s,3H),1.72–1.49(m,6H).

[0109] Compound 1n: 1 H NMR(600MHz,Chloroform-d)δ9.67(d,J=7.8Hz,1H),7.80(s,1H),7.72(d,J=9.1Hz,1H),7.62(d,J=8.6Hz,1H),7.56(d,J=8.6Hz,1H),7.52(d ,J=15.4Hz,1H),7.21(d,J=9.1Hz,1H),6.93(s,1H),6.71(dd,J=15.7,7.8Hz,1H),3.90(t,J=5.5Hz,2H),3.65(t,J=5.6Hz,2H),3.13(s,3H).

[0110]

[0111] Preparation of compound 1p

[0112] According to the synthesis method of compound 1e, compound 1o (172 mg) was prepared using compound 1n (300 mg) as raw material.

[0113] According to the synthesis method of compound 1f, compound 1p (72 mg) was prepared using compound 1o (300 mg) as raw material.

[0114] Compound 1o: 1H NMR(600MHz,Chloroform-d)δ9.70(d,J=7.8Hz,1H),7.81(s,1H),7.71(d,J=9.1Hz,1H),7.61(d ,J=8.6Hz,1H),7.58–7.55(m,2H),7.19(d,J=9.1Hz,1H),6.88(s,1H),6.74(dd,J=15.7,7.8Hz,1 H),4.60(t,J=3.4Hz,1H),3.98–3.95(m,1H),3.82–3.78(m,1H),3.74–3.69(m,2H),3.67–3.64( m,1H),3.49–3.45(m,1H),3.14(s,3H),1.78–1.65(m,2H),1.59–1.54(m,2H),1.52–1.46(m,2H).

[0115] Compound 1p: 1 H NMR(600MHz,Chloroform-d)δ9.61(d,J=8.0Hz,1H),7.73(s,1H),7.70(d,J=8.7Hz,1H), 7.62(d,J=8.2Hz,1H),7.58(d,J=8.5Hz,1H),7.31(dd,J=15.1,10.8Hz,1H),7.21(d,J=7 .8Hz,1H),7.12(d,J=15.5Hz,1H),7.03(dd,J=15.5,10.8Hz,1H),6.98(d,J=8.0Hz,1H), 6.26(dd,J=15.1,8.0Hz,1H),3.89(t,J=5.4Hz,2H),3.63(t,J=5.4Hz,2H),3.12(s,3H).

[0116]

[0117] Preparation of compound 1r

[0118] Compound 1p (150 mg) and methylglyoxal (100 mg) were dissolved in EtOH, and 4M HCl solution was added to react at room temperature for 4 h. After the reaction was completed, saturated NaHCO3 solution was added to adjust the pH value to 7, extracted with ethyl acetate, and the organic phase was dried over anhydrous Na2SO4 and the solvent was evaporated. Purification by column chromatography gave compound 1r (139 mg).

[0119] Compound 1r: 1 H NMR(600MHz,Chloroform-d)δ8.75(s,1H),2.77(s,3H).

[0120]

[0121] Example 2: Preparation of Compounds 2a, 2b, and 2c

[0122] Preparation of compound 2a

[0123] Compound 1a (31.05 mg), 1e (25 mg) and piperidine were dissolved in EtOH and reacted at room temperature. After the reaction was completed, the solvent was evaporated and the residue was purified by column chromatography to obtain compound 2a (26 mg).

[0124] Compound 2a: ESI-MS (m / z): 276 [M+H] + ; 1 H NMR (600MHz, Chloroform-d) δ8.89(s,1H),8.05(d,J=15.9Hz,1H),7.84(d,J=8.6Hz,2H),7.66(d,J=8.7Hz,2H),7.28(d,J=15.9Hz,1H),3.37(s,6H).

[0125]

[0126] Preparation of compound 2b

[0127] According to the preparation method of compound 2a, compound 2b (18 mg) was prepared using compound 1e (54.60 mg) and 1b (79.70 mg) as raw materials.

[0128] Compound 2b: ESI-MS (m / z): 302 [M+H] + ; 1 H NMR(600MHz,Chloroform-d)δ8.60(s,1H),7.83(dd,J=15.0,11.1Hz,1H),7.42(d,J=8.8Hz,2H),7.01(d ,J=15.2Hz,1H),6.87(dd,J=15.3,11.2,1H),6.69(d,J=8.9Hz,2H),6.55(d,J=15.0Hz,1H),3.04(s,6H).

[0129]

[0130] Preparation of compound 2c

[0131] According to the preparation method of compound 2b, compound 2c (11m) was prepared using compound 1c (40 mg) and 1r (62.70 mg) as raw materials.

[0132] Compound 2c: ESI-MS (m / z): 328 [M+H] + ; 1 H NMR(600MHz,Chloroform-d)δ8.72(s,1H),7.78(dd,J=14.8,10.9Hz,1H),7.62(d,J=8.2Hz,2H),7.52(d,J=8.2Hz,2H) ,7.00(dd,J=15.4,10.9Hz,1H),6.88(dd,J=14.5,11.3Hz,1H),6.80(d,J=15.4Hz,1H),6.73–6.68(m,2H),3.34(s,6H).

[0133]

[0134] Example 3: Preparation of compounds 3a, 3b, 3c

[0135] Preparation of compound 3a

[0136] Compounds 1d (33.51 mg) and 1r (50 mg) were dissolved in EtOH, and 23 μL of piperidine was added. The system was reacted at room temperature. After the reaction was completed, the solvent was evaporated and purified by column chromatography to obtain compound 3a (46 mg).

[0137] Compound 3a: ESI-MS (m / z): 306 [M+H] + ; 1 H NMR(600MHz,Chloroform-d)δ8.64(s,1H),7.93(d,J=15.7Hz,1H),7.52(d,J=7.9Hz,2H),6.88(d ,J=15.6Hz,1H),6.78(d,J=7.9Hz,2H),3.88(t,J=5.3Hz,2H),3.62(t,J=5.5Hz,2H),3.12(s,3H).

[0138]

[0139] Preparation of compound 3b

[0140] According to the preparation method of compound 3a, compound 3b (27 mg) was prepared using compound 1r (22.33 mg) and 1f (26.50 mg) as raw materials.

[0141] Compound 3b: ESI-MS (m / z): 332 [M+H] + ; 1H NMR(600MHz,Chloroform-d)δ8.60(s,1H),7.82(dd,J=14.9,11.2Hz,1H),7.41(d,J=8.8Hz,2H),7.00(d,J=15.1Hz,1H),6.87(d d,J=15.2,11.2Hz,1H),6.75(d,J=8.8Hz,2H),6.57(d,J=14.9Hz,1H),3.86(t,J=5.5Hz,2H),3.58(t,J=5.7Hz,2H),3.08(s,3H).

[0142]

[0143] Preparation of compound 3c

[0144] According to the preparation method of compound 3a, compound 3c was prepared using compound 1r (22.31 mg) and 1h (17.90 mg) as raw materials to obtain compound 3c (10 mg).

[0145] Compound 3c: ESI-MS (m / z): 358 [M+H] + ; 1 H NMR(600MHz,Chloroform-d)δ9.04(s,1H),7.77(dd,J=15.1,11.5Hz,1H),7.38(d,J=8.8Hz,2H),6.99(dd,J=14.3,11.0Hz,1H),6.88(dd,J=15.1,11.0 Hz,1H),6.77(dd,J=15.1,11.9Hz,2H),6.69(d,J=8.9Hz,2H),6.62(dd,J=1 4.3,11.6Hz,1H),3.54(t,J=5.7Hz,2H),3.44(t,J=6.1Hz,2H),2.98(s,3H).

[0146]

[0147] Example 4: Preparation of Compounds 4a, 4b, and 4c

[0148] Preparation of compound 4a

[0149] Compound 11 (20.12 mg) and 1r (25.14 mg) were dissolved in EtOH, and 15 μL of piperidine was added. The mixture was stirred at room temperature to react. After the reaction was completed, the solvent was evaporated and the mixture was purified by column chromatography to obtain compound 4a (10 mg).

[0150] Compound 4a: ESI-MS (m / z): 356 [M+H]+ ; 1 H NMR (400MHz, Chloroform-d) δ8.67(s,1H),7.99(d,J=15.7Hz,1H),7.74(s,1H),7.58(d,J=9.1Hz,1H),7.53(d,J=8.6Hz,1H),7.49(d ,J=8.6Hz,1H),7.09(d,J=6.9Hz,1H),7.03(d,J=15.7Hz,1H),6.79(s,1H),3.69(t,J=6.0Hz,2H),3.51(t,J=6.1Hz,2H),3.02(s,3H).

[0151]

[0152] Preparation of compound 4b

[0153] According to the preparation method of compound 4a, compound 4a (25 mg) was prepared using compound 1r (77.06 mg) and 1n (54.60 mg) as raw materials.

[0154] Compound 4b: ESI-MS (m / z): 382 [M+H] + ; 1 H NMR (600MHz, DMSO-d6) δ9.13(s,1H),7.90(dd,J=15.4,9.6Hz,2H),7.82(s,1H),7.73(d,J=9.2Hz,1H),7.67(d,J=8.7Hz,1H),7.63(d,J=8.7Hz ,1H),7.34–7.24(m,2H),7.21(d,J=9.2Hz,1H),6.90(s,1H),6.88(d,J= 15.3Hz,1H),3.60(t,J=5.6Hz,3H),3.53(t,J=6.0Hz,3H),3.06(s,3H).

[0155]

[0156] Preparation of compound 4c

[0157] According to the preparation method of compound 4a, compound 4c was prepared using compound 1r (27.56 mg) and 1p (26.90 mg) as raw materials to obtain compound 4c (7 mg).

[0158] Compound C-3: ESI-MS (m / z): 408 [M+H] + ; 1H NMR(600MHz,DMSO-d6)δ9.09(s,1H),7.81(dd,J=15.1,11.5Hz,2H),7.77(s,1H),7.69(d,J=9 .2Hz,1H),7.61(s,2H),7.60(d,J=8.7Hz,2H),7.20(d,J=9.1Hz,2H),7.15(dd,J=15.3,11.0Hz ,2H),7.06(dd,J=14.4,11.1Hz,2H),6.98(d,J=15.4Hz,1H),6.89(s,1H),6.85(d,J=15.2Hz, 1H), 6.73 (dd, J=14.3, 11.6Hz, 2H), 3.60 (t, J=5.7Hz, 3H), 3.53 (t, J=6.0Hz, 3H), 3.05 (s, 3H).

[0159]

[0160] Example 5: Evaluation of the optical properties of fluorescent probes.

[0161] Accurately weigh a certain mass of probes 2a-2c, 3a-3c, 4a-4c, dissolve them in DMF to prepare a mother solution with a concentration of 2 mM, and store them at -20°C in the dark for later use; during the experiment, dilute the probe mother solution in a certain proportion according to the requirements.

[0162] Fluorescence spectroscopy property evaluation:

[0163] Each probe stock solution was diluted to a concentration of 10 μM with dichloromethane (DCM), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), methanol (MeOH), and PBS, respectively. The fluorescence spectra of each probe molecule in different solvents were measured using a fluorescence spectrophotometer, and the excitation wavelength and emission wavelength were recorded.

[0164] Table 1 Basic spectral properties of fluorescent probes

[0165]

[0166]

[0167] in:

[0168] λ em represents the maximum emission wavelength of the probe;

[0169] λ ex represents the maximum absorption wavelength of the probe;

[0170] Table 1 shows that probes 2a-2c, 3a-3c and 4a-4c all exhibit significant solvatochromic phenomena in different solvents, which can produce different degrees of blue shift or red shift. The emission wavelengths of probes 2a-2c, 3a-3c and 4a-4c are in the range of 568-715nm. With the increase of the number of double bonds, the fluorescence excitation wavelength and emission wavelength of the probes increase significantly, and the ultraviolet maximum absorption wavelength, absorbance and molar absorptivity of probes 2a-2c also increase accordingly; the emission wavelengths of probes 2b, 2c, 3b, 3c, 4b and 4c are all in the near-infrared range (650-900nm), among which the maximum emission wavelengths of probes 2c and 4c in DMSO can reach 720 and 727nm, and the emission wavelengths of compound 4c in five solvents of different polarity are all above 700nm, indicating that this type of probe has good near-infrared fluorescence properties.

[0171] Example 6: Evaluation of biological activity of fluorescent probes

[0172] 1. Aβ 1-42 Preparation of aggregates

[0173] Weigh 1.0 mg Aβ1-42TFA monomer and dissolve it in 1.0 mL 1% ammonia water. Pipette 100 μL and add 900 μL PBS (1 mM, pH = 7.4) to prepare a final concentration of 100 μg / mL Aβ 1-42 The monomer solution was incubated in a shaker at 37 °C for 7 days.

[0174] 2. Probe and Aβ 1-42 In vitro fluorescence response experiment of aggregates

[0175] First, each probe stock solution was diluted with PBS to a concentration of 1 μM, and its fluorescence emission spectrum was measured using a fluorescence spectrophotometer; then, 184 μL Aβ 1-42 The aggregate solution was added with 536 μL PBS buffer and 80 μL probe ethanol solution; it was placed in a shaker and incubated for about 30 min, and the fluorescence emission spectrum was measured using a fluorescence spectrophotometer. During the experiment, the fluorescence spectrum of PBS buffer was measured using the same parameters, and all spectra were finally corrected using blank controls to calculate the probe and Aβ 1-42 Fluorescence enhancement factor after aggregate binding.

[0176] Fluorescent probes and Aβ 1-42 The spectral data before and after incubation of the aggregates are shown in Table 2, and the fluorescence spectra are shown in Figure 1 The fluorescence change is shown in Figure 2 The data showed that compound 4c and Aβ 1-42 The fluorescence intensity increased 28 times after the aggregates were bound. Probes 3b and 3c were 1-42After binding, the fluorescence enhancement times were very significant, about 73 times and 60 times respectively, indicating that this series of probes can effectively inhibit Aβ 1-42 It exhibits strong fluorescence response performance and is a relatively ideal probe.

[0177] Table 2 Fluorescent probes and Aβ 1-42 Fluorescence spectral data before and after aggregate binding

[0178]

[0179] in:

[0180] λem 1 represents the emission wavelength of the probe in PBS;

[0181] λem 2 Indicates the probe and Aβ in PBS 1-42 The emission wavelength after the aggregates are bound;

[0182] Fold indicates the probe and Aβ in PBS 1-42 The fluorescence intensity of the aggregates after binding is increased by a factor of fold compared to that of the unbound aggregates.

[0183] 3. In vitro binding experiment of probe and BSA

[0184] The fluorescence intensity of the probe at 1 μM in PBS solution and the fluorescence intensity of the probe after incubation with BSA for 30 min were recorded using a fluorescence spectrophotometer. All fluorescence spectra were calibrated using the blank fluorescence spectrum of PBS.

[0185] The fluorescence spectra of the fluorescent probe before and after incubation with BSA are shown in Figure 1 The data show that the fluorescence enhancement of probes 2c, 3c, and 4c after incubation with BSA is much smaller than that after incubation with Aβ 1-42 The fluorescence enhancement produced after the aggregates bind indicates that this type of probe has good selectivity and anti-interference ability, and can effectively avoid fluorescence signal interference caused by the interaction with BSA during in vivo imaging.

[0186] 4. Fluorescence titration spectrum experiment of probe:

[0187] Accurately pipette 0, 46, 93, 139, 185, 231 μL of Aβ 1-42 The aggregate solution was added to 100 μL of an ethanol solution of the probe (10 μM), followed by the addition of PBS buffer (1 mM, pH = 7.4) to a final volume of 1 mL. 1-42 The aggregates were incubated in a shaker for about 30 min, and their emission spectra were measured using a fluorescence spectrophotometer.

[0188] Probes and different concentrations of Aβ 1-42Fluorescence spectrum after incubation and the fluorescence intensity of the probe and Aβ 1-42 The current relationship between aggregate concentrations is Figure 3 As shown in the experimental results, probes 2c, 3c, and 4c have almost no fluorescence emission in PBS, which is similar to Aβ 1-42 The fluorescence intensity changes significantly after the aggregates bind and changes with Aβ 1-42 The fluorescence intensity increased significantly with the increase of aggregate concentration. 1-42 The concentration of the aggregates was in the range of 0-5 μM and showed a good linear relationship, indicating that probes 2c, 3c, and 4c had a good affinity for Aβ 1-42 The concentration of aggregates has good sensitivity and can track Aβ 1-42 The degree of aggregate aggregation and the ability to distinguish the course of AD disease.

[0189] 5. Experiment for determination of probe binding constant:

[0190] To study the relationship between probe and Aβ 1-42 The binding affinity of the probe to Aβ was determined by saturation binding method. 1-42 The binding constant (K d ) was measured. 1-42 A probe solution with a certain concentration gradient was added to the aggregate solution, and then PBS buffer was added to make the final volume 600 μL. 1-42 The aggregate mixed solution was placed in a shaker at 37°C and 100 rpm for about 30 min, then transferred to a 96-well plate and its fluorescence intensity was measured using an ELISA reader. GraphPad Prism 7.0 was used to perform nonlinear fitting on the experimental results and calculate the binding constant.

[0191] Probe and Aβ 1-42 The saturation binding curve of the aggregate is shown in Figure 4 As shown. Probe 4c for Aβ 1-42 K of the aggregate d The binding affinity of probe 4c was 8.56±3.36nM, indicating that probe 4c can be used to bind Aβ 1-42 Efficient binding of aggregates.

[0192] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. Use of a compound in the preparation of any of the following reagents: (1) Reagents for detecting amyloid β; (2) Reagents for detecting Alzheimer's disease; The compound has a structural formula as shown in formula (I): in, n=0~2, n is not 0; R1 is independently selected from: C1-C5 alkyl or C1-C5 hydroxyalkyl; R2 is independently selected from: C1-C5 alkyl or C1-C5 hydroxyalkyl; W is independently selected from: a benzene ring or a naphthalene ring; R3 is independently selected from:

2. The use according to claim 1, characterized in that The compounds also include pharmaceutically acceptable salts thereof.

3. The use according to claim 1 or 2, characterized in that: The compound has the following structure:

4. The use according to claim 1, characterized in that The synthetic route of the compound represented by formula (I) is as follows:

5. The use according to claim 4, characterized in that include: The compound represented by formula (II) is dissolved in a protic solvent and then reacted with the compound represented by formula (III) under the catalysis of a base to produce a compound represented by formula (I).

6. The use according to claim 1, characterized in that The detection of Alzheimer's disease includes diagnosing, monitoring or predicting the progression of Alzheimer's disease in patients with Alzheimer's disease.

7. The use according to claim 1, characterized in that The detection of Alzheimer's disease includes early diagnosis, monitoring or prediction of the progression of Alzheimer's disease in patients with Alzheimer's disease.

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