Responsive near-infrared fluorescent probe targeting butyrylcholine esterase and beta amyloid protein as well as preparation method and application of responsive near-infrared fluorescent probe

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a response type near-infrared fluorescent probe targeting butyrylcholine esterase and beta amyloid protein as well as a preparation method and application of the response type near-infrared fluorescent probe. The response type near-infrared fluorescent probe is formed by connecting positively charged hemicyanine serving as a strong electron withdrawing group, carbon-carbon double bonds and thiophene serving as bridging units and phenylcyclopropionic acid carboxylate with a BChE recognition group, so that an organic small molecular fluorescent compound with a typical D-pi-A structure is formed. The fluorescent compound not only prolongs a conjugated structure to reach a near-infrared region, but also shows good biocompatibility, has no obvious toxic or side effect, is combined with a main marker BChE of AD and is converted into a fluorescence mode from a non-fluorescence mode, and in order to overcome the defect of single-target response, a fluorescent mode probe is further combined with A beta protein, so that the specific response is enhanced; the problem of false positive of a single target spot is effectively solved.

Description

Technical Field

[0001] This application belongs to the technical field of fluorescent probes, and particularly relates to a responsive near-infrared fluorescent probe targeting butyrylcholinesterase and β-amyloid, and its preparation method and application. Background Art

[0002] Alzheimer's Disease (AD) is a progressive central nervous system degenerative disease. This disease has a strong concealment, and the incubation period can be as long as more than ten years. Since the early symptoms of AD are not obvious, the disease has often developed to a very serious stage when it is discovered, which often delays treatment. Therefore, the early diagnosis of Alzheimer's disease is particularly important. It can help patients detect and treat the disease earlier, effectively control the condition, and delay the speed of disease deterioration, which is of great significance for protecting people's physical health and improving the quality of life. Among the current clinical diagnostic methods, cerebrospinal fluid (CSF) detection has high accuracy, but the collection process of cerebrospinal fluid puncture is invasive, so it is not widely accepted.

[0003] Imaging techniques mainly include fluorescence imaging (FI), computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and single photon emission computed tomography (SPECT), etc. At present, the diagnosis and treatment effects of AD in clinical practice need to be evaluated through multiple reexaminations and long-term observations. These imaging techniques cannot achieve real-time monitoring and evaluation.

[0004] Due to the complex etiology of AD, although research on it has been carried out for more than 100 years, so far, the exact etiology of AD has not been fully understood. This has led to the fact that in the past few decades of research, the AD drug development strategy based on a single target has not been successful. Obviously, single-target drugs are not sufficient to improve the pathological condition of AD or even delay its progression. Therefore, the complex mechanism of this disease and the interaction between multiple factors make it necessary to develop diagnostic and treatment methods targeting multiple targets.

[0005] At present, the developed molecular probes have the phenomenon of poor selectivity and are prone to false positive results. For example, there are two hydrolytic enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), in the brain at the same time, and the structures and functions of AChE and BChE are very similar, resulting in low selectivity of many probes for them. Probes targeting butyrylcholinesterase have been reported in AD, but abnormal levels of cholinesterase in the brain are also related to other diseases such as Parkinson's disease, frontotemporal dementia, and Lewy body dementia, etc., which are easy to be confused. Therefore, the accuracy of dual-target probes is higher. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a responsive near-infrared fluorescent probe targeting butyrylcholinesterase and β-amyloid protein in view of the deficiencies of the above-mentioned existing technologies. The probe binds to the main marker BChE of AD, converting from a non-fluorescent state to a fluorescent mode. In the fluorescent mode, the probe further binds to Aβ protein, enhancing the specific response and effectively solving the problem of false positives of single targets.

[0007] The technical solution adopted by the present invention to solve the above-mentioned problems is as follows:

[0008] A responsive near-infrared fluorescent probe targeting butyrylcholinesterase and β-amyloid protein has the following general structural formula: Wherein, R is selected from methyl or ethyl.

[0009] According to the above scheme, when R is selected from methyl or ethyl, the responsive near-infrared fluorescent probes are abbreviated as HCy-Me or HCy-Et respectively, and the specific structures are as follows:

[0010]

[0011] In the present invention, the responsive near-infrared fluorescent probe uses the positively charged hemicyanine part as the electron-withdrawing part, and the phenylcyclopropanecarboxylate part as the recognition group of BChE. The two are connected by a carbon-carbon double bond to the bridging unit thiophene group. The maximum ultraviolet absorption wavelengths of HCy-Me and HCy-Et in DMSO solution are about 475 nm and about 480 nm respectively, and the emission wavelengths are about 647 nm and about 622 nm respectively. The maximum ultraviolet absorption wavelengths of HMy-OH and HEy-OH in DMSO solvent are about 515 nm and about 520 nm respectively, and the emission wavelengths are about 652 nm and about 653 nm respectively.

[0012] The synthesis method of the above-mentioned responsive near-infrared fluorescent probe includes the following steps:

[0013] 1) 2,3,3-Trimethyl-3H-indole reacts with iodide to introduce a substituent on N to synthesize the electron acceptor part, that is, the positively charged hemicyanine, with the general structural formula R is methyl or ethyl;

[0014] 2) 4-Hydroxybenzeneboronic acid and 5-bromothiophene-2-carbaldehyde undergo a Suzuki coupling reaction to obtain the electron donor part, that is, 5-(4-hydroxyphenyl)thiophene-2-carbaldehyde, with the general structural formula

[0015] 3) The products of steps 1) and 2) are subjected to a Knovengel condensation reaction to obtain an intermediate compound R is methyl or ethyl; wherein, when R is methyl, the intermediate compound is HMy-OH; when R is ethyl, the intermediate compound is HEy-OH, and their structures are as follows:

[0016]

[0017] 4) Nucleophilic substitution reactions are respectively carried out on the compounds HMy-OH and HEy-OH with cyclopropylcarbonyl chloride to obtain responsive near-infrared fluorescent probes with structures of HCy-Me or HCy-Et respectively.

[0018] According to the above scheme, in step 1), the iodide is selected from one or two of methyl iodide or ethyl iodide; the molar ratio between 2,3,3-trimethyl-3H-indole and the iodide can undergo the substitution reaction in step 1) within the range of (1-3):3; the time of the substitution reaction is 10-16 h, and the temperature is 55-85 °C.

[0019] According to the above scheme, in step 1), acetonitrile is used as the reaction solvent, and the concentration of the iodide in acetonitrile is preferably 0.5-3 mol / L; after the substitution reaction is completed, cold diethyl ether (0 °C) is added to precipitate the solid product, and it is washed with diethyl ether, and then filtered and dried.

[0020] According to the above scheme, in step 2), the molar ratio between 4-hydroxybenzeneboronic acid and 5-bromothiophene-2-carbaldehyde can undergo the Suzuki coupling reaction in step 2) within the range of (1-3):1; the time of the Suzuki coupling reaction is 10-22 h and the temperature is 50-90 °C.

[0021] According to the above scheme, in step 2), the Suzuki coupling reaction uses tetrakis(triphenylphosphine)palladium as the catalyst, tetrahydrofuran as the reaction solvent, and a protective atmosphere such as nitrogen or inert gas or vacuum; the dosage of the catalyst in the reaction solvent is 3-8 mg / mL; 4-hydroxybenzeneboronic acid and 5-bromothiophene-2-carbaldehyde are first dissolved in tetrahydrofuran under the protective atmosphere respectively to obtain a 4-hydroxybenzeneboronic acid solution with a concentration of 0.5-1.5 mmol / mL and a 5-bromothiophene-2-carbaldehyde solution with a concentration of 0.5-1.5 mmol / mL; then the 5-bromothiophene-2-carbaldehyde solution is injected into the 4-hydroxybenzeneboronic acid solution (the catalyst is added to the 4-hydroxybenzeneboronic acid solution in advance), and then an aqueous sodium carbonate solution is added to adjust the pH to 8.5-9, and the temperature is raised to 70-90 °C and reacted for 10-22 hours; after the Suzuki coupling reaction is completed, extraction is carried out with a water and dichloromethane system, and then column chromatography is carried out with an eluent prepared by mixing petroleum ether and ethyl acetate in a volume ratio of (4-6):1 to obtain the product of step 2).

[0022] According to the above - mentioned scheme, in step 3), the product of step 1) and the product of step 2) undergo a Knoevenagel condensation reaction in a molar ratio of 1:(1 - 3). The reaction time is 1.5 - 3 h, and the temperature is 50 - 80 °C.

[0023] According to the above - mentioned scheme, in step 3), ethanol is used as the reaction solvent, and there is a small amount of piperidine in the reaction solvent. The volume of piperidine is 2 - 3% of that of ethanol. The concentration of the product of step 2) in ethanol is 0.02 - 0.05 mmol / mL. A protective atmosphere such as nitrogen, inert gas, or vacuum is used during the reaction. After the reaction, dichloromethane and methanol are mixed in a volume ratio of (15 - 25):1 to form an eluent for column chromatography.

[0024] According to the above - mentioned scheme, in step 4), the molar ratio of the intermediate compound obtained in step 3) to cyclopropylcarbonyl chloride is (0.5 - 1.5):2. The reaction time is 1.5 - 3 h, and the temperature is - 5 °C - 0 °C.

[0025] According to the above - mentioned scheme, in step 4), dichloromethane is used as the reaction solvent. The dosing concentration of cyclopropylcarbonyl chloride in the reaction solvent is 0.03 - 0.05 mmol / mL, and a certain amount of triethylamine needs to be added. The concentration of triethylamine in the reaction solvent is 2 - 5 mg / mL. A protective atmosphere such as nitrogen, inert gas, or vacuum is used during the reaction. After the reaction, dichloromethane and methanol are mixed in a volume ratio of (15 - 25):1 to form an eluent for column chromatography.

[0026] The technical concept of the present invention is as follows:

[0027] The responsive near - infrared fluorescent probes involved in the present invention include HCy - Me and HCy - Et, whose initial fluorescence is weak or non - fluorescent. After contacting with butyrylcholinesterase (BChE), the ester bonds are rapidly hydrolyzed and broken, turning back to HMy - OH and HEy - OH (which can be called hydrolysis fluorescent probes at this time), and the fluorescence instantaneously increases. Then, during the binding process of the probes with the marker β - amyloid protein, the hydroxyl groups of HMy - OH and HEy - OH respectively form 2 or 1 hydrogen bond with the active site F20 on β - amyloid protein. The force of the hydrogen bond restricts the torsional motion of the probe molecule, thereby inhibiting the intramolecular charge transfer (ICT) process. The ICT process usually leads to fluorescence quenching, so its inhibition will significantly enhance the fluorescence. In the brains of AD patients, the probes HCy - Me and HCy - Et can sensitively detect the main marker BChE, converting from non - fluorescent to fluorescent mode. Then, the fluorescent - mode probes further bind to Aβ protein and specifically respond with enhanced fluorescence, which can help in the early detection and treatment of AD patients and effectively control the condition.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] 1. The present invention provides a responsive near-infrared fluorescent probe targeting butyrylcholinesterase and β-amyloid protein. Structurally, it first reacts a semi-cyanine structure with iodide to introduce a substituent on N, forming an electron acceptor with stronger electron-withdrawing ability. The middle of the molecular structure is composed of vinyl and heterocycles to expand the conjugated system; the binding group of BChE is mainly composed of phenylcyclopropionate carboxylate, and the cyclopropionate carboxylate is a leaving group, and finally a phenolic hydroxyl group is formed as an electron-donating group. Such fluorescent compounds can expand the conjugation degree of the original structure and cause its wavelength to redshift.

[0030] 2. After the responsive near-infrared fluorescent probe proposed by the present invention binds to BChE, the cyclopropionate carboxylate part of the probe hydrolyzes and leaves, converting from a non-fluorescent state to a fluorescent mode; at the same time, the hydroxybenzene group can act as an electron-donating group to form a "push-pull" structure with the indole group. This probe is very sensitive to the environmental solvent and is prone to aggregation leading to luminescence quenching (ACQ), and the fluorescence quantum yield in aqueous solution is very low. However, after this probe binds to Aβ protein, the intramolecular rotation is restricted, and due to the intramolecular charge transfer (ICT) effect, the fluorescence signal of the probe molecule is significantly enhanced, solving the problems of false positives of single targets and the defects of single-target responses. The above-mentioned novel responsive near-infrared fluorescent probe improves the accuracy of fluorescence probe diagnosis and provides new ideas for the early and accurate diagnosis of Alzheimer's disease.

[0031] 3. The responsive near-infrared fluorescent probe described in the present invention has good liposolubility, can quickly cross the blood-brain barrier, has good biocompatibility, and has successfully performed diagnostic imaging on a living Alzheimer's disease model mouse (C57BL / 6, APP / PS1), and can realize real-time visualization and specifically detect the molecular changes in the early stage of the disease.

[0032] 4. The responsive near-infrared fluorescent probe described in the present invention has high selectivity for BChE. There are differences in the active sites of BChE and acetylcholinesterase (AChE) in structure. In particular, the active cavity of BChE is larger and lacks the narrow aromatic amino acid residues (such as Tyr124 and Phe337) in AChE. In view of this structural feature, in the present invention, a group with a large steric hindrance - cyclopropyl is introduced as an identification unit to adjust the size of the binding group for BChE to construct a highly selective probe for BChE. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a synthetic route diagram of the fluorescent probe of the present invention.

[0034] Figure 2Study on the response and selectivity of the probe of the present invention to BChE. a-b are the fluorescence emission spectra of HCy-Me and HCy-Et (5 μM, final concentration after mixing) cultured with different concentrations of BChE in PBS (10 mM, pH 7.4), (HCy-Me: λex = 475 nm; HCy-Et: λex = 480 nm). c-d are the graphs of fluorescence intensity as a function of BChE concentration. e-f are the selectivity studies of HCy-Me and HCy-Et with potential competing substances (metal ions, amino acids, AChE, etc.) and BChE.

[0035] Figure 3 Study on the probe response and selectivity of Αβ fibrils of the present invention. a-b are the fluorescence emission spectra of the hydrolytic fluorescent probes HMy-OH and HEy-OH cultured with different concentrations of Αβ aggregates in PBS (10 mM, pH 7.4), (HMy-OH: λex = 515 nm; HEy-OH: λex = 520 nm). c-d are the graphs of the fluorescence intensity of HMy-OH and HEy-OH as a function of the concentration of Αβ aggregates. e is the selectivity study of HMy-OH and HEy-OH with potential competing substances and Αβ aggregates.

[0036] Figure 4 Fluorescence imaging study of the fluorescent probes HCy-Me and HCy-Et of the present invention in brain slices,

[0037] Figure 5 Fluorescence imaging study of the hydrolytic fluorescent probes HMy-OH and HEy-OH of the present invention in brain slices, (HMy-OH: λex = 515 nm, λem = 640 - 650 nm; HEy-OH: λex = 520 nm, λem = 640 - 650 nm); wherein, a, b are the in vitro fluorescence staining of brain slices of age-matched Tg mice (APP / PS1, 12 months old) using HMy-OH and ThT, d, e are brain slices of WT mice (12 months old), and c, f are their merged images. j, h are the in vitro fluorescence staining of brain slices of age-matched Tg mice (APP / PS1, 12 months old) using HEy-OH and ThT, h-k are brain slices of WT mice (12 months old), and i, l are their merged images.

[0038] Figure 6 In vivo fluorescence imaging of BChE of the present invention, (HCy-Me: λex = 475 nm, λem = 640 - 650 nm; HCy-Et: λex = 480 nm, λem = 620 - 640 nm); wherein, a, b are the fluorescence images of wild-type mice and APP / PS1 mice (12 months old) at different time points before and after tail vein injection of HCy-Me and HCy-Et, respectively. Detailed implementation mode

[0039] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with embodiments, but the present invention is not limited only to the following embodiments.

[0040] Example 1

[0041] Referring to Figure 1 As shown, a method for synthesizing a responsive near-infrared fluorescent probe (abbreviated as HCy-Me) targeting butyrylcholinesterase and β-amyloid specifically includes the following steps:

[0042] (1) Synthesis of Compound 1:

[0043] Methyl iodide (0.93 mL, 15 mM) and 2,3,3-trimethyl-3H-indole (1.6 mL, 10 mM) were taken, and then acetonitrile (15 mL) was added as the reaction solvent. They were placed in an oil bath and refluxed at 80 °C with vigorous stirring for 12 h to obtain a pink solution. Stirring was stopped, and the pink solution was naturally cooled to room temperature. Excess cold ether was added to precipitate the solid. After vacuum filtration, it was washed 3 times with ether to obtain a filter cake. The filter cake was placed in a vacuum drying oven to dry to obtain a clean product, namely Compound 1, and this product did not require further purification. Compound 1 was a pink solid with a yield of 78.0% (1.7 g).

[0044] (2) Synthesis of Compound 3:

[0045] Tetrakis(triphenylphosphine)palladium (0.1 g) was placed in a three-necked flask. 4-Hydroxybenzeneboronic acid (10 mmol) was dissolved in 10 mL of tetrahydrofuran and injected into the three-necked flask under nitrogen protection. Then, 5-bromothiophene-2-carbaldehyde (10 mmol) dissolved in 10 mL of tetrahydrofuran was injected into the above three-necked flask using a syringe. After that, sodium carbonate (2.76 g) dissolved in water (6 mL) was injected. At this time, the pH was 8.5 - 9, and the temperature was raised to 80 °C and stirred for 16 h. After the reaction ended, the temperature was lowered to room temperature, then 100 mL of water was added, and it was extracted with dichloromethane (3 × 50 mL). The obtained organic phase was dried with anhydrous sodium sulfate, filtered and evaporated to obtain a crude product. The crude product was eluted with petroleum ether and ethyl acetate (volume ratio 5 / 1) and obtained yellow solid Compound 3 (136 mg, 67%) by column chromatography.

[0046] (3) Synthesis of Compound HMy-OH:

[0047] Compound 1 (162.5 mg, 0.93 mmol) and compound 3 (300 mg, 1.40 mmol) were mixed and dissolved in ethanol (30 mL) and piperidine (4 drops), and then heated to 60 °C and stirred for 2 h under nitrogen. After the reaction was completed, it was filtered and evaporated to obtain a crude product. The crude product was eluted with DCM / MeOH (20 / 1, volume ratio of dichloromethane and methanol) and purified by column chromatography to obtain a dark red solid compound HMy-OH (134 mg, 40%).

[0048] (4) Synthesis of compound HCy-Me:

[0049] Compound HMy-OH (100 mg, 0.27 mmol) and cyclopropylcarbonyl chloride (56 mg, 0.54 mmol) were dissolved in dichloromethane (15 mL), triethylamine (54 mg) was slowly added dropwise, and then the mixture was stirred at 0 °C for 2 h under a nitrogen atmosphere. After the reaction was completed, it was filtered and evaporated to obtain a crude product. The crude product was eluted with DCM / MeOH (20 / 1, volume ratio) and purified by column chromatography to obtain a dark red solid compound HCy-Me (86 mg, 75%), which is a responsive near-infrared fluorescent probe targeting butyrylcholinesterase and β-amyloid (abbreviated as HCy-Me).

[0050] Example 2

[0051] Reference Figure 1 As shown, a method for synthesizing a responsive near-infrared fluorescent probe targeting butyrylcholinesterase and β-amyloid (abbreviated as HCy-Et) specifically includes the following steps:

[0052] (1) Synthesis of compound 2:

[0053] Iodoethane (1.20 mL, 15 mM) and 2,3,3-trimethyl-3H-indole (1.6 mL, 10 mM) were added, and then toluene (15 mL) was used as the reaction solvent. They were placed in an oil bath and refluxed at 80 °C with vigorous stirring for 12 h to obtain a pink solution. Stirring was stopped, and the pink solution was naturally cooled to room temperature. Excess cold ether was added to precipitate the solid. After suction filtration, it was washed 3 times with ether to obtain a filter cake. The filter cake was placed in a vacuum drying oven to dry to obtain a clean product, compound 2, which did not require further purification. Compound 2 was a pink solid with a yield of 75.0% (1.7 g).

[0054] (2) Synthesis of compound 3: The same as in Example 1.

[0055] (3) Synthesis of compound HEy-OH:

[0056] Compound 2 (100 mg, 0.53 mmol) and compound 3 (163 mg, 0.8 mmol) were mixed and dissolved in ethanol (30 mL) and piperidine (4 drops), and then heated and stirred at 60 °C for 2 h under a nitrogen atmosphere. After the reaction, filtration and evaporation were carried out to obtain the crude product. The crude product was eluted with DCM / MeOH (20 / 1, volume ratio) by column chromatography to obtain the dark red solid compound HEy-OH (79 mg, 40%).

[0057] (4) Synthesis of compound HCy-Et:

[0058] Compound HEy-OH (180 mg, 0.48 mmol) and cyclopropylcarbonyl chloride (100 mg, 0.96 mmol) were dissolved in dichloromethane (20 mL), triethylamine (97 mg) was slowly added dropwise, and then the mixture was stirred at 0 °C for 2 h under a nitrogen atmosphere. After the reaction, filtration and evaporation were carried out to obtain the crude product. The crude product was eluted with DCM / MeOH (20 / 1, volume ratio) by column chromatography to obtain the dark red solid compound HCy-Et (160 mg, 75%), namely the responsive near-infrared fluorescent probe targeting butyrylcholinesterase and β-amyloid (abbreviated as HCy-Et).

[0059] Application Example 1

[0060] The responsive near-infrared fluorescent probes HCy-Me and HCy-Et prepared in Examples 1-2 were subjected to application tests to test the response ability and specificity of the probes to BChE.

[0061] The specific operation for testing the response ability of the probes to BChE was as follows: BChE (0 - 1.6 U / mL, this concentration refers to the final concentration in PBS, the same below) was mixed with HCy-Me and HCy-Et (5 μM) in PBS (pH 7.4) respectively, vortexed for 10 seconds and then incubated in a non-shaking constant temperature incubator at 37 °C for 60 minutes, and their fluorescence emission spectra were measured. The specific operation for testing the specificity of the probes to BChE was as follows: The probes HCy-Me and HCy-Et (5 μM, this concentration refers to the final concentration after mixing, the same below) were mixed with 1: Na + (10 μM), 2: Mg 2+ (10 μM), 3: K + (10 μM), 4: Fe 2+ (10 mM), 5: Cu 2+ (10 μM), 6: Ca 2+ (10 μM), 7: Zn 2+(10 μM), 8: Ser. (10 μM), 9: Lys (10 μM), 10: Arg. (10 μM), 11: GSH (10 μM), 12: Gly. (10 μM), 13: Phe. (10 μM), 14: Cys (10 μM), 15: Ala. (10 μM), 16: Pepsin (10 mg / mL), 17: HSA (10 mg / mL), 18: BSA (10 mg / mL), 19: AChE (1 U / mL), 20: BChE (1 U / mL) were mixed in PBS (pH 7.4), placed in an incubator at 37 °C for 30 minutes, and then their fluorescence emission spectra were measured. All samples were prepared in triplicate as their own control groups.

[0062] As can be seen from Figure 2 a, b, the fluorescence of the probes HCy-Me and HCy-Et increased with the increase in the concentration of BChE, indicating that the probes HCy-Me and HCy-Et can sensitively recognize BChE. Figure 2 c, d show the functional relationship between the fluorescence intensity of the probe and the concentration of BChE, and it can be seen that there is a good linear relationship between the two. As can be seen from Figure 2 e, f, no significant increase or quenching of fluorescence emission was observed for the probe in the presence of some metal ions, amino acids, and AChE, while a significant increase in fluorescence intensity was shown after binding to the biomarker BChE, indicating that the probe has specific responsiveness to the biomarker BChE.

[0063] Application Example 2

[0064] The hydrolytic fluorescent probes HMy-OH and HEy-OH prepared in Examples 1-2 were subjected to application tests to measure their responsiveness and specificity to Aβ fibrils.

[0065] To measure the responsiveness of the probe to Aβ 42 The specific operation was as follows: The incubated Aβ 42 aggregates (0 - 10 μM, this concentration refers to the final concentration after mixing, the same below) were mixed with HMy-OH and HEy-OH (10 μM) respectively. After vortexing for 10 seconds, they were placed in an incubator at 37 °C without shaking for 30 minutes, and their fluorescence emission spectra were measured. To select the specificity of the probe for Aβ fibrils, the fluorescent probes HMy-OH and HEy-OH (10 μM) were mixed with Aβ monomers, Aβ oligomers, and Aβ aggregates in PBS (pH 7.4) respectively, placed in an incubator at 37 °C for 30 minutes, and then their fluorescence emission spectra were measured. All samples were prepared in triplicate as their own control groups.

[0066] As can be seen from Figure 3As can be seen from a and b, the fluorescence of probes HMy-OH and HEy-OH increases with the increase in the concentration of Aβ, indicating that probes HMy-OH and HCy-OH can sensitively recognize Aβ. Figure 3 c and d show the functional relationship between the fluorescence intensity of the probe and the concentration of Aβ. It can be seen from the figure that there is a good linear relationship between the two. Figure 3 From the data in e and f, it can be known that no enhancement or quenching of fluorescence emission was observed for the probe in the environment of Aβ monomers and Aβ oligomers, while a significant increase in fluorescence intensity was shown after binding to the marker Aβ aggregates, indicating that the probe indeed has a specific response to the marker Aβ.

[0067] Application Example 3

[0068] The in vitro section staining effects of the fluorescent probes HCy-Me and HCy-Et prepared in Examples 1-2 were tested. The specific process was as follows: The brain tissues of 8-month-old APP / PS1 mice and normal mice of the same age were removed by perfusion. After the brain tissues were sectioned, adjacent two layers of sections were respectively infiltrated with HCy-Me (10 μM, prepared with PBS at pH 7.4) and HCy-Et (10 μM, prepared with PBS at pH 7.4) to fully infiltrate the tissues, and incubated together at room temperature for 15 minutes, and then the tissue sections were washed with physiological saline. The results are as Figure 4 shown. No fluorescence signal was observed in normal mice; in adjacent brain sections of AD model mice, the fluorescence intensity increased significantly, thus proving that both probes HCy-Me and HCy-Et have the ability to specifically recognize BChE.

[0069] Application Example 4

[0070] The in vitro section staining effects of the hydrolytic fluorescent probes HMy-OH and HEy-OH prepared in Examples 1-2 were tested. The specific process was as follows: The brain tissues of 8-month-old APP / PS1 mice and normal mice of the same age were removed by perfusion. After the brain tissues were sectioned, adjacent two layers of sections were respectively infiltrated with HMy-OH (10 μM, prepared with PBS at pH 7.4) and HCy-OH (10 μM, prepared with PBS at pH 7.4) for staining to infiltrate the tissues, and incubated together at room temperature for 15 minutes, and then the tissue sections were washed with physiological saline, and then infiltrated with ThT (10 μM, prepared with PBS at pH 7.4) for staining, incubated together at room temperature for 15 minutes, and the tissue sections were washed with physiological saline again. The results are as Figure 5 shown. No fluorescence signal was observed in normal mice; in adjacent brain sections of AD model mice, the staining areas of ThT and the hydrolytic fluorescent probes HMy-OH and HCy-OH overlapped. It was proved that both the hydrolytic fluorescent probes HMy-OH and HCy-OH have the ability to specifically target Aβ.

[0071] Application Example 5

[0072] In vivo imaging was performed on the fluorescent probes HCy-Me and HCy-Et prepared in Examples 1-2. The specific operation was as follows: The probes HCy-Me (concentration 10 mM, 100 μL, prepared with PBS at pH 7.4) and HCy-Et (10 mM, 100 μL, prepared with PBS at pH 7.4) were respectively injected into the tail veins of mice. Fluorescence images were collected using an IVIS system. The excitation wavelength was 450 ± 10 nm, the emission wavelength was 620 ± 10 nm, and the acquisition time was 2 s. The results are as Figure 6 shown. As can be seen from the figure, at 1 h after injection of the fluorescent probes HCy-Me ( Figure 6 , a) and HCy-Et ( Figure 6 , b), the fluorescence reached the highest, while there was no obvious change in the fluorescence signal in the brains of normal mice in the control group.

[0073] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, shall be similarly included in the patent protection scope of the present invention.

Claims

1. A responsive near-infrared fluorescent probe targeting butyrylcholinesterase and amyloid β protein, characterized in that The responsive near-infrared fluorescent probe has the following general structural formula: Wherein, R is selected from methyl or ethyl.

2. The responsive near-infrared fluorescent probe according to claim 1, characterized in that: The responsive near-infrared fluorescent probe comprises the following compounds:

3. The method for preparing the responsive near-infrared fluorescent probe according to claim 2, characterized in that: The main steps are as follows: 1) 2,3,3-trimethyl-3H-indole reacts with iodide to introduce a substituent group on N to synthesize the electron acceptor part, i.e., a positively charged hemicyanine, with the general structural formula: R is methyl or ethyl; 2) 4-Hydroxyphenylboronic acid and 5-bromothiophene-2-carboxaldehyde undergo Suzuki coupling reaction to obtain the electron donor part, namely 5-(4-hydroxyphenyl)thiophene-2-carboxaldehyde, with the general structural formula: 3) The products of step 1) and step 2) are subjected to Knoevenagel condensation reaction to obtain an intermediate compound having the general structural formula: R is methyl or ethyl; 4) The intermediate compound obtained in step 3) undergoes a nucleophilic substitution reaction with cyclopropylcarbonyl chloride to obtain a responsive near-infrared fluorescent probe having a structure of HCy-Me or HCy-Et.

4. The method for preparing a responsive near-infrared fluorescent probe according to claim 3, characterized in that: In step 1), the iodide is selected from one or both of methyl iodide and ethyl iodide; the molar ratio between 2,3,3-trimethyl-3H-indole and iodide is in the range of (1-3):3; the substitution reaction time is 10-16h, and the temperature is 55-85°C.

5. The method for preparing a responsive near-infrared fluorescent probe according to claim 3, characterized in that: In step 2), the molar ratio of 4-hydroxyphenylboronic acid to 5-bromothiophene-2-carboxaldehyde is in the range of (1-3):1; the time of the Suzuki coupling reaction is 10-22 hours and the temperature is 50-90°C.

6. The method for preparing a responsive near-infrared fluorescent probe according to claim 3, characterized in that: In step 3), the molar ratio of the product of step 1) to the product of step 2) is 1:(1-3); the reaction time is 1.5-3h, and the temperature is 50-80°C.

7. The method for preparing a responsive near-infrared fluorescent probe according to claim 3, characterized in that: In step 4), the molar ratio of the intermediate compound obtained in step 3) to cyclopropylcarbonyl chloride is (0.5-1.5):2, the reaction time is 1.5-3h, and the temperature is -5-0°C.

8. Use of the responsive near-infrared fluorescent probe according to claim 1 as a diagnostic agent for Alzheimer's disease.

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