CN120081836A

CN120081836A - A responsive near-infrared fluorescent probe targeting butyrylcholinesterase and beta-amyloid protein, and its preparation method and application

Info

Publication number: CN120081836A
Application number: CN202510188189.XA
Authority: CN
Inventors: 潘洁; 望雯静; 冀士悦; 马思洁
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2025-02-20
Filing date: 2025-02-20
Publication date: 2025-06-03
Anticipated expiration: 2045-02-20
Also published as: CN120081836B

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a response near infrared fluorescent probe targeting butyrylcholine esterase and beta amyloid, a preparation method and application thereof. The response near-infrared fluorescent probe is formed by taking half cyanine with positive charges as a strong electron withdrawing group, carbon-carbon double bond and thiophene as bridging units and connecting the bridging units with phenyl cyclopropane carboxylic ester with a BChE recognition group, so that an organic small molecule fluorescent compound with a typical D-pi-A structure is formed. The fluorescent compound not only prolongs the conjugated structure to reach the near infrared region, but also shows good biocompatibility, has no obvious toxic or side effect, is combined with an AD main marker BChE, and is converted into a fluorescent mode from non-fluorescence, so that the fluorescent mode probe is further combined with Abeta protein, the specific response is enhanced, and the problem of single-target false positive is effectively solved.

Description

Response near infrared fluorescent probe targeting butyrylcholinesterase and beta amyloid, and preparation method and application thereof

Technical Field

The application belongs to the technical field of fluorescent probes, and particularly relates to a response near infrared fluorescent probe targeting butyrylcholine esterase and beta amyloid, and a preparation method and application thereof.

Background

Alzheimer's Disease (AD) is a progressive central nervous system degenerative Disease. The disease has strong concealment, and the incubation period can be as long as ten years. Because the early symptoms of AD are not obvious, the disease state is very serious when the disease state is developed, and the treatment is often delayed, the early diagnosis of Alzheimer disease is particularly important, the early diagnosis of Alzheimer disease can help patients to discover and treat early, effectively control the disease state, delay the disease state deterioration speed, and has important significance for guaranteeing the physical health of people and improving the life quality. In current clinical diagnostic methods, cerebrospinal fluid (CSF) testing has high accuracy, but the collection process of cerebrospinal fluid puncture is invasive and therefore not widely accepted.

Imaging techniques mainly include Fluorescence Imaging (FI), computed Tomography (CT), magnetic Resonance Imaging (MRI), positron Emission Tomography (PET), single Photon Emission Computed Tomography (SPECT), and the like. At present, diagnosis and treatment effects of AD in clinic need to be evaluated through repeated check and long-term observation. These imaging techniques are not practical in real-time monitoring and evaluation.

Because of the complex etiology of AD, although its research has been conducted for over 100 years, the exact etiology of AD has not been fully understood so far. This has led to the lack of success in single target-based AD drug development strategies in the last decades of research. Clearly, single-target drugs are not sufficient to improve AD pathology and even delay its progression. Thus, the complex mechanisms of the disease and the interactions between various factors make it necessary to develop diagnostic methods for multiple targets.

The molecular probes developed at present have poor selectivity and are easy to generate false positive results. For example, two hydrolases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are present in the brain at the same time, and the structure and function of AChE and BChE are very similar, resulting in a number of probes that are not highly selective for them. Probes targeting butyrylcholinesterase have been reported in AD, but abnormalities in brain cholinesterase levels are equally associated with other diseases such as Parkinson's disease, frontotemporal dementia, dementia with Lewy bodies, and the like, and are easily confused. Thus the accuracy of the double-target probe is higher.

Disclosure of Invention

The invention aims to solve the technical problems of providing a response near infrared fluorescent probe targeting butyrylcholine esterase and beta amyloid, combining the probe with an AD main marker BChE, converting from non-fluorescence to fluorescence mode, further combining with Abeta protein by the probe in the fluorescence mode, enhancing specific response and effectively solving the problem of single-target false positive.

The invention adopts the technical proposal for solving the problems that:

a response near infrared fluorescent probe for targeting butyrylcholinesterase and beta amyloid has the following structural general formula: wherein R is selected from methyl or ethyl.

According to the scheme, when R is selected from methyl or ethyl, the responsive near infrared fluorescent probe is abbreviated as HCy-Me or HCy-Et respectively, and the specific structure is as follows:

In the invention, the response near infrared fluorescent probe is formed by taking a half cyanine part with positive charges as an electron-withdrawing part and a phenyl cyclopropane carboxylic ester part as a BChE recognition group, wherein the two parts are connected with a bridging unit thiophene group through a carbon-carbon double bond. The maximum UV absorption wavelength of HCy-Me and HCy-Et in DMSO solution is about 475nm and about 480nm, respectively, and the emission wavelength is about 647nm and about 622nm, respectively. The maximum ultraviolet absorption wavelength of HMy-OH and HEy-OH in DMSO solvent is about 515nm and about 520nm, and the emission wavelength is about 652nm and about 653nm, respectively.

The synthesis method of the responsive near infrared fluorescent probe comprises the following steps:

1) 2, 3-trimethyl-3H-indole and iodide undergo substitution reaction, and a substituent group is introduced on N to synthesize an electron acceptor part, namely the half-cyanine with positive charges, and the structural general formula is shown in the specification R is methyl or ethyl;

2) The 4-hydroxy phenylboronic acid and 5-bromothiophene-2-formaldehyde are subjected to Suzuki coupling reaction to obtain an electron donor part, namely 5- (4-hydroxy phenyl) thiophene-2-formaldehyde, with the structural general formula of

3) The products of the step 1) and the step 2) are subjected to a condensation reaction of Kenaonvingel (Knovengel) to obtain an intermediate compoundR is methyl or ethyl, wherein when R is methyl, the intermediate compound is HMy-OH, and when R is ethyl, the intermediate compound is HEy-OH, and the structures are as follows:

4) And (3) carrying out nucleophilic substitution reaction on the compounds HMy-OH and HEy-OH and cyclopropyl formyl chloride respectively to obtain the response near infrared fluorescent probe with the structure of HCy-Me or HCy-Et respectively.

According to the scheme, in the step 1), the iodide is selected from one or two of methyl iodide and ethyl iodide, the molar ratio of the 2, 3-trimethyl-3H-indole to the iodide is within the range of (1-3): 3, the substitution reaction of the step 1) can occur, the time of the substitution reaction is 10-16H, and the temperature is 55-85 ℃.

According to the scheme, in the step 1), acetonitrile is adopted as a reaction solvent, the concentration of iodide in acetonitrile is preferably 0.5-3 mol/L, after the substitution reaction is finished, cold diethyl ether (0 ℃) is added to precipitate a solid product, and the solid product is washed by diethyl ether and then filtered and dried.

According to the scheme, in the step 2), the Suzuki coupling reaction of the step 2) can be carried out when the molar ratio of the 4-hydroxyphenylboric acid to the 5-bromothiophene-2-formaldehyde is within the range of (1-3): 1, wherein the time of the Suzuki coupling reaction is 10-22 h and the temperature is 50-90 ℃.

According to the scheme, in the step 2), four (triphenylphosphine) palladium is used as a catalyst in the Suzuki coupling reaction, tetrahydrofuran is used as a reaction solvent, nitrogen or inert gas or vacuum protective atmosphere is adopted, the adding amount of the catalyst in the reaction solvent is 3-8 mg/mL, 4-hydroxyphenylboric acid and 5-bromothiophene-2-formaldehyde are respectively dissolved in the tetrahydrofuran under the protective atmosphere to respectively obtain a 4-hydroxyphenylboric acid solution with the concentration of 0.5-1.5 mmol/mL and a 5-bromothiophene-2-formaldehyde solution with the concentration of 0.5-1.5 mmol/mL, then the 5-bromothiophene-2-formaldehyde solution is injected into the 4-hydroxyphenylboric acid solution (the catalyst is added into the 4-hydroxyphenylboric acid solution in advance), then sodium carbonate aqueous solution is added to adjust the pH to 8.5-9, the temperature is increased to 70-90 ℃ for 10-22 hours, water and methylene dichloride systems are respectively extracted after the Suzuki coupling reaction is finished, petroleum ether and ethyl acetate are mixed into eluent according to the volume ratio of 4-6:1, and the product of the Suzuki coupling reaction is obtained.

According to the scheme, in the step 3), the product of the step 1) and the product of the step 2) are subjected to a Kebrain Velcro condensation reaction according to a molar ratio of 1 (1-3), wherein the reaction time is 1.5-3 h, and the temperature is 50-80 ℃.

According to the scheme, the ethanol is adopted as the reaction solvent in the step 3), a small amount of piperidine is added in the reaction solvent, the volume of the piperidine is 2-3% of that of the ethanol, the concentration of the product in the step 2) in the ethanol is 0.02-0.05 mmol/mL, a nitrogen or inert gas or vacuum protective atmosphere is adopted in the reaction process, and after the reaction is finished, dichloromethane and methanol are mixed into eluent according to the volume ratio (15-25): 1 to carry out column chromatography.

According to the scheme, in the step 4), the molar ratio of the intermediate compound obtained in the step 3) to the cyclopropyl formyl chloride is (0.5-1.5): 2, the reaction time is 1.5-3 h, and the temperature is-5-0 ℃.

According to the scheme, in the step 4), dichloromethane is adopted as a reaction solvent, the adding concentration of cyclopropylcarboxychloride in the reaction solvent is 0.03-0.05mmol/mL, a certain amount of triethylamine is required to be added, the concentration of the triethylamine in the reaction solvent is 2-5mg/mL, nitrogen or inert gas or vacuum protective atmosphere is adopted in the reaction process, and after the reaction is finished, dichloromethane and methanol are mixed into eluent according to the volume ratio (15-25): 1 to carry out column chromatography.

The technical conception of the invention is as follows:

the responsive near infrared fluorescent probes related to the invention comprise HCy-Me and HCy-Et, the initial state of which is weak or non-fluorescent, and after contacting with butyrylcholinesterase (BChE), the probes are rapidly hydrolyzed and broken down in ester bonds, and are changed into HMy-OH and HEy-OH (which can be called hydrolysis fluorescent probes at the moment), and the fluorescence is enhanced instantaneously. And in the process of combining the probe and the marker beta amyloid, the hydroxyl groups of the HMy-OH and the HEy-OH respectively form 2 or 1 hydrogen bonds with an active site F20 on the beta amyloid, and the acting force of the hydrogen bonds limits the twisting movement of the probe molecules, so that the Intramolecular Charge Transfer (ICT) process is inhibited. ICT processes typically result in fluorescence quenching, so their inhibition results in a significant increase in fluorescence. In the brain of an AD patient, probes HCy-Me and HCy-Et can sensitively detect a main marker BChE, the non-fluorescence is converted into a fluorescence mode, and then the fluorescence mode probes are further combined with Abeta protein, and the specific response is enhanced, so that the early detection and early treatment of the AD patient can be helped, and the illness state can be effectively controlled.

Compared with the prior art, the invention has the beneficial effects that:

1. The invention provides a response near infrared fluorescent probe for targeting butyrylcholine esterase and beta amyloid, which structurally comprises the steps of firstly reacting a hemicyanine structure with iodide, introducing substituent groups on N to form an electron acceptor with stronger electron withdrawing capability, wherein the middle of a molecular structure consists of vinyl and heterocycle for expanding a conjugated system, the binding group of BChE mainly consists of phenyl cyclopropane carboxylic ester, the cyclopropane carboxylic ester is taken as a leaving group, and finally phenolic hydroxyl is formed as an electron donor group. The fluorescent compound can expand the conjugation degree of the original structure and lead the wavelength of the fluorescent compound to be red-shifted.

2. After the response near infrared fluorescent probe is combined with BChE, the cyclopropane carboxylic ester part of the probe is hydrolyzed and separated, and is converted into a fluorescent mode from a non-fluorescent state, and meanwhile, a hydroxyl phenyl group can be used as an electron donating group to form a push-pull structure with an indole group. Such probes are very sensitive to environmental solvents, are prone to aggregation resulting in luminescence quenching (ACQ), and have low fluorescence quantum yields in aqueous solutions. However, after the probe is combined with the Abeta protein, the rotation in the molecule is limited, and the fluorescent signal of the probe molecule is obviously enhanced due to the Intramolecular Charge Transfer (ICT) effect, so that the problem of single-target false positive and the defect of single-target response are solved. The novel response near infrared fluorescent probe increases the accuracy of fluorescent probe diagnosis and provides a new idea for early accurate diagnosis of Alzheimer's disease.

3. The response near infrared fluorescent probe has good fat solubility, can quickly pass through a blood brain barrier, has good biocompatibility, can successfully perform diagnostic imaging on a living Alzheimer disease model mouse (C57 BL/6, APP/PS 1), and can realize real-time visualization and specific detection of early molecular changes of diseases.

4. The response near infrared fluorescent probe has high selectivity to BChE, and the active site of the BChE is structurally different from that of acetylcholinesterase (AChE), especially the active cavity of the BChE is large, and the active cavity lacks narrow aromatic amino acid residues (such as Tyr124 and Phe 337) in the AChE. Aiming at the structural characteristics, the high-selectivity probe aiming at the BChE is constructed by introducing a group-cyclopropyl with larger steric hindrance as an identification unit and adjusting the size of a binding group for the BChE.

Drawings

FIG. 1 is a synthetic route diagram of a fluorescent probe of the present invention.

FIG. 2 shows the response and selectivity studies of the probes of the invention to BChE. a-b are fluorescence emission spectra of HCy-Me and HCy-Et (5 μm, final concentration after mixing) after incubation with different concentrations of BChE in PBS (10 mm, ph 7.4), (HCy-Me: λex=475nm; HCy-Et: λex=480 nm). c-d is a plot of fluorescence intensity as a function of BChE concentration. e-f is a selective study of HCy-Me and HCy-Et with potentially competing materials (metal ions, amino acids, AChE, etc.) and BChE.

Figure 3 is a probe response and selectivity study of aβ fibrils of the present invention. a-b are fluorescence emission spectra of hydrolyzed fluorescent probes HMy-OH and HEy-OH after incubation with different concentrations of aβ aggregates in PBS (10 mm, ph 7.4) (HMy-OH: λex=515 nm; HEy-OH: λex=520 nm). c-d is a plot of fluorescence intensity of HMy-OH and HEy-OH as a function of Abeta aggregate concentration. e is a selective study of HMy-OH and HEy-OH with potential competitors and aβ aggregates.

FIG. 4 is a fluorescence imaging study of the fluorescent probes HCy-Me and HCy-Et of the invention in brain sections,

FIG. 5 shows the fluorescence imaging studies of hydrolyzed fluorescent probes HMy-OH and HEy-OH in brain sections, (HMy-OH: λex=515 nm, λem=640-650 nm; HEy-OH: λex=520 nm, λem=640-650 nm), where a, b is the in vitro fluorescent staining of brain sections of age-matched Tg mice (APP/PS 1,12 months of age) using HMy-OH and thT, d, e is WT mice (12 months of age), and c, f are their combined plots. j, h is in vitro fluorescent staining of brain sections of age-matched Tg mice (APP/PS 1,12 months old) using HEy-OH and ThT, h-k is WT mice (12 months old), i, l is a combined plot of them.

FIG. 6 shows 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), where a, b are fluorescence images of wild-type mice and APP/PS1 mice (12 months old) at different time points before and after tail intravenous injection of HCy-Me and HCy-Et, respectively.

Detailed Description

For a better understanding of the present invention, the following examples are set forth to illustrate the invention further, but are not to be construed as limiting the invention.

Example 1

Referring to fig. 1, a synthesis method of a butyrylcholinesterase-beta amyloid targeted response near infrared fluorescent probe (abbreviated as HCy-Me) specifically comprises the following steps:

(1) Synthesis of Compound 1:

Methyl iodide (0.93 mL,15 mM) and 2, 3-trimethyl-3H-indole (1.6 mL,10 mM) were added, followed by acetonitrile (15 mL) as a reaction solvent. They were placed in an oil bath and reacted at 80℃under vigorous stirring under reflux for 12 hours to give a pink solution. Stopping stirring, naturally cooling the pink solution to room temperature, adding excessive cold diethyl ether to precipitate solid, filtering under reduced pressure, and washing with diethyl ether for 3 times to obtain a filter cake. The filter cake was dried in a vacuum oven to give a clean product, compound 1, which was not further purified. Compound 1 was a pink solid in 78.0% (1.7 g) yield.

(2) Synthesis of Compound 3:

Tetra (triphenylphosphine) palladium (0.1 g) is placed in a three-neck flask, 4-hydroxyphenylboric acid (10 mmol) is dissolved in 10mL of tetrahydrofuran and is injected into the three-neck flask under the protection of nitrogen, 5-bromothiophene-2-formaldehyde (10 mmol) is dissolved in 10mL of tetrahydrofuran by using a syringe and is injected into the three-neck flask, sodium carbonate (2.76 g) is dissolved in water (6 mL) and is injected, the pH is 8.5-9 at the moment, and the temperature is raised to 80 ℃ and the mixture is stirred and reacted for 16h. After the completion of the reaction, the temperature was lowered to room temperature, 100mL of water was added, and extracted with methylene chloride (3X 50 mL), and the obtained organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to give a crude product. The crude product was subjected to column chromatography using petroleum ether and ethyl acetate (volume ratio 5/1) as eluent to give compound 3 (136 mg, 67%) as a yellow solid.

(3) Synthesis of Compound HMy-OH:

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℃under nitrogen and stirred for 2h. After the reaction, the mixture was filtered and evaporated to give a crude product. The crude product was column chromatographed using DCM/MeOH (20/1, volume ratio of dichloromethane and methanol) as eluent to give the dark red solid compound HMy-OH (134 mg, 40%).

(4) Synthesis of Compound HCy-Me:

The compound HMy-OH (100 mg,0.27 mmol) and cyclopropylcarbonyl chloride (56 mg,0.54 mmol) were dissolved in dichloromethane (15 mL) and triethylamine (54 mg) was slowly added dropwise followed by stirring under nitrogen atmosphere at 0℃for 2h. After the reaction, the mixture was filtered and evaporated to give a crude product. The crude product was subjected to column chromatography using DCM/MeOH (20/1, volume ratio) as eluent to give a dark red solid compound HCy-Me (86 mg, 75%), namely a responsive near infrared fluorescent probe (abbreviated as HCy-Me) targeting butyrylcholinesterase and amyloid beta.

Example 2

Referring to FIG. 1, a method for synthesizing a responsive near infrared fluorescent probe (abbreviated as HCy-Et) targeting butyrylcholinesterase and amyloid beta specifically comprises the following steps:

(1) Synthesis of Compound 2:

Iodoethane (1.20 mL,15 mm) and 2, 3-trimethyl-3H-indole (1.6 mL,10 mm) were combined and toluene (15 mL) was added as a reaction solvent. They were placed in an oil bath and reacted at 80℃under vigorous stirring under reflux for 12 hours to give a pink solution. Stopping stirring, naturally cooling the pink solution to room temperature, adding excessive cold diethyl ether to precipitate solid, filtering under reduced pressure, and washing with diethyl ether for 3 times to obtain a filter cake. The filter cake was dried in a vacuum oven to give a clean product, which gave compound 2 without further purification. Compound 2 was a pink solid in 75.0% yield (1.7 g).

(2) The synthesis of compound 3 was identical to that of example 1.

(3) Synthesis of compound HEy-OH:

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 reacted under nitrogen atmosphere with stirring at 60℃for 2h. After the reaction, the mixture was filtered and evaporated to give a crude product. The crude product was column chromatographed with DCM/MeOH (20/1, vol.) as eluent to give the compound HEy-OH (79 mg, 40%) as a dark red solid.

(4) Synthesis of Compound HCy-Et:

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 the reaction was stirred under nitrogen at 0℃for 2h. After the reaction, the mixture was filtered and evaporated to give a crude product. The crude product was column chromatographed with DCM/MeOH (20/1, vol%) as eluent to give the dark red solid compound HCy-Et (160 mg, 75%), a responsive near infrared fluorescent probe (abbreviated as HCy-Et) targeting butyrylcholinesterase and amyloid β.

Application example 1

Application tests are carried out on the response type near infrared fluorescent probes HCy-Me and HCy-Et prepared in the examples 1-2, and the response capacity and the specificity of the probes to BChE are tested.

The test probe was specifically operated for its response to BChE by mixing BChE (0-1.6U/mL, which refers to the final concentration in PBS, hereinafter the same) with HCy-Me and HCy-Et (5. Mu.M) respectively in PBS (pH 7.4), vortexing for 10 seconds, incubating in a shaking-free incubator at 37℃for 60 minutes, and testing to obtain their fluorescence emission spectra. Specific selection of test probes for BChE specific procedures probes HCy-Me and HCy-Et (5. Mu.M, which refers to the final concentration after mixing, the same applies hereinafter) were mixed with 1:Na⁺(10μM),2:Mg²⁺(10μM),3:K⁺(10μM),4:Fe²⁺(10mM),5:Cu²⁺(10μM),6:Ca²⁺(10μM),7:Zn²⁺(10μM),8:Ser.(10μM),9:Lys(10μM),10:Arg.(10μM),11:GSH(10μM),12:Gly.(10μM),13:Phe.(10μM),14:Cys(10μM),15:Ala.(10μM),16:Pepsin(10mg/mL),17:HSA(10mg/mL),18:BSA(10mg/mL),19:AChE(1U/mL),20:BChE(1U/mL) each in PBS (pH 7.4), incubated in a 37℃incubator for 30 minutes, and then tested for fluorescence emission spectra. All samples were configured in triplicate as self-control.

As can be seen from FIGS. 2a, b, the fluorescence of probes HCy-Me and HCy-Et increased with increasing concentration of BChE, indicating that probes HCy-Me and HCy-Et were able to sensitively recognize BChE. FIGS. 2c, d are graphs showing the relationship between probe fluorescence intensity and BChE concentration, and it can be seen that there is a good linear relationship between the two. As can be seen from fig. 2e and f, the probe does not observe the phenomenon of fluorescence emission enhancement or quenching in the environment of part of metal ions, amino acids and AChE, but shows the phenomenon of obvious enhancement of fluorescence intensity after being combined with the marker BChE, which indicates that the probe has specific response to the marker BChE.

Application example 2

Application tests are carried out on the hydrolysis fluorescent probes HMy-OH and HEy-OH prepared in the examples 1-2, and the response capability and the specificity of the probes to Abeta fibrils are tested.

The test probe specifically works for the response ability to Abeta ₄₂ by mixing incubated Abeta ₄₂ aggregates (0-10. Mu.M, which means the final concentration after mixing, the same applies hereinafter) with HMy-OH and HEy-OH (10. Mu.M), respectively, vortexing for 10 seconds, placing in a vibration-free incubator at 37℃for 30 minutes, and testing to obtain their fluorescence emission spectra. Specific selection of assay for Abeta fibers specific procedures were performed by mixing fluorescent probes HMy-OH and HEy-OH (10. Mu.M) with Abeta monomers, abeta oligomers and Abeta aggregates, respectively, in PBS (pH 7.4), incubating in a 37℃incubator for 30 minutes, and then testing the fluorescence emission spectra thereof. All samples were configured in triplicate as self-control.

From FIGS. 3a, b, the fluorescence of the probes HMy-OH and HEy-OH increases with increasing concentration of Abeta, indicating that the probes HMy-OH and HCy-OH can sensitively recognize Abeta. FIGS. 3c and d show the relationship between fluorescence intensity of the probe and Abeta concentration, and it can be seen that there is a good linear relationship between the two. From the data in fig. 3e and f, it can be seen that the probe does not observe a phenomenon of fluorescence emission enhancement or quenching in the environment of aβ monomers and aβ oligomers, but shows a phenomenon of significantly enhanced fluorescence intensity after binding to the marker aβ aggregates, indicating that the probe does have a specific response to the marker aβ.

Application example 3

The in vitro section staining effect was tested on the fluorescent probes HCy-Me and HCy-Et prepared in examples 1-2. The specific process is that brain tissue of 8 month old APP/PS1 mice and normal mice of the same age are taken out under perfusion, after brain tissue sections, two adjacent layers of sections are respectively fully infiltrated with HCy-Me (10 mu M, prepared by PBS with pH of 7.4) and HCy-Et (10 mu M, prepared by PBS with pH of 7.4), the tissues are incubated for 15 minutes at room temperature, and then the tissue sections are washed by normal saline. As shown in FIG. 4, no fluorescence signal was seen in the normal mice, and fluorescence intensity was significantly increased in adjacent brain sections of the A D model mice, thus demonstrating that both probes HCy-Me and HCy-Et have the ability to specifically recognize BChE.

Application example 4

The in vitro section staining effect of the hydrolyzed fluorescent probes HMy-OH and HEy-OH prepared in examples 1-2 was tested by taking out brain tissue perfusion of 8 month old APP/PS1 mice and normal mice of the same age, staining the infiltrated tissue with HMy-OH (10. Mu.M, formulated with PBS at pH 7.4) and HCy-OH (10. Mu.M, formulated with PBS at pH 7.4), incubating for 15 min at room temperature, washing the tissue sections with physiological saline, staining the infiltrated tissue with ThT (10. Mu.M, formulated with PBS at pH 7.4), incubating for 15 min at room temperature, and washing the tissue sections again with physiological saline. As shown in FIG. 5, none of the normal mice showed fluorescent signals, and in brain sections adjacent to the AD model mice, the stained areas of ThT and the hydrolysis fluorescent probes HMy-OH and HCy-OH were coincident. The hydrolysis fluorescent probe HMy-OH and HCy-OH are proved to have the capability of specifically targeting Abeta.

Application example 5

The fluorescent probes HCy-Me and HCy-Et prepared in examples 1-2 were imaged in vivo. Specifically, the probes HCy-Me (10 mM,100uL in PBS at pH 7.4) and HCy-Et (10 mM,100uL in PBS at pH 7.4) were injected into the tail vein of mice, respectively. And (3) collecting fluorescent images by adopting an IVIS system, wherein the excitation wavelength is 450+/-10 nm, the emission wavelength is 620+/-10 nm, and the collecting time is 2s. The results are shown in FIG. 6. As can be seen from the figure, at 1h after injection of the fluorescent probes HCy-Me (FIG. 6, a) and HCy-Et (FIG. 6, b), fluorescence reached the highest, whereas normal mouse brains of the control group had no significant change in fluorescence signal.

The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.

Claims

1. A responsive near-infrared fluorescent probe targeting butyrylcholinesterase and β-amyloid 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 is 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 to 3):3; the substitution reaction time is 10 to 16 hours, and the temperature is 55 to 85°C.

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

6. The method for preparing a responsive near-infrared fluorescent probe according to claim 3 is 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 is 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.