A fluorescent probe for detecting acetylcholinesterase, its preparation method and application

Abstract

This invention provides a fluorescent probe for detecting acetylcholinesterase, its preparation method, and its application, belonging to the field of detection and analysis technology. The fluorescent probe is particularly suitable for detecting acetylcholinesterase in vivo. It is constructed by connecting an electron donor, diphenylamineoxanthracene, to an electron acceptor, benzoindolesulfonate, via an ethylene bridge, with the following structural formula: The fluorescent probe of this invention has advantages such as high detection sensitivity, good selectivity, and fast response time. This invention also marks the first successful in vivo imaging analysis of changes in AChE in the brains of depressed rats, demonstrating significant practical application value.

Description

Technical Field

[0001] This invention belongs to the field of detection and analysis technology, specifically a fluorescent probe for detecting acetylcholinesterase, its preparation method, and its application. Background Technology

[0002] Depression, as a mental illness, has an extremely high incidence and mortality rate, posing a serious threat to human health. To effectively prevent and treat depression, a thorough understanding of its occurrence and development is essential. However, depression remains a major challenge in neuroscience due to its complex causative factors, unclear etiology, and unknown pathogenesis. Fluorescence imaging, with its excellent temporal and spatial resolution and the ability to be performed with simple instruments and equipment, has been widely used in various research fields. Fluorescence imaging technology offers significant advantages such as high sensitivity, non-invasiveness, and high spatiotemporal resolution, leading to the development of various fluorescent probes for imaging detection and research of live cells and enzymes in vivo.

[0003] Previous studies have shown a close association between acetylcholinesterase (AChE) and mental illnesses such as depression, with patients exhibiting higher levels of acetylcholine in their brains than healthy controls. AChE is a key enzyme in the breakdown of acetylcholine. Therefore, exploring the role of acetylcholinesterase in the development and progression of depression is crucial for understanding its pathogenesis. However, no non-invasive tools for detecting acetylcholinesterase in vivo have yet been reported. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a fluorescent probe for detecting acetylcholinesterase (AChE), its preparation method, and its applications. It is particularly suitable for in vivo detection of AChE. This invention's AChE fluorescent probe possesses advantages such as high detection sensitivity, good selectivity, and fast response time. This invention is the first to successfully achieve in vivo imaging analysis of changes in AChE in the brains of depressed rats, demonstrating significant practical application value.

[0005] To achieve the above objectives, the present invention employs the following technical solutions:

[0006] In a first aspect, the present invention provides an AIE fluorescent probe for detecting acetylcholinesterase, said AIE fluorescent probe being composed of cyclohexanone, 4-bromo-2-hydroxybenzaldehyde, 4-methoxydiphenylamine, compound B1, and dimethylamino, and having the following structural formula:

[0007]

[0008] The structural formula of compound B1 is:

[0009]

[0010] Furthermore, cyclohexanone, 4-bromo-2-hydroxybenzaldehyde, 4-methoxydiphenylamine, and compound B1 serve as fluorescent groups, and dimethylamino serves as an AChE recognition group. Studies have confirmed that the AIE probe prepared by this invention is not limited by the complex biological background of the brain and the presence of the blood-brain barrier, and can directly and rapidly detect the content of AChE in the living (rat) brain. Under 808 nm near-infrared II excitation, the fluorescence emission intensity of the AIE probe prepared by this invention at 950 nm increases with increasing AChE concentration, and the probe can quickly reflect changes in AChE activity in cells and in vivo.

[0011] A second aspect of the present invention provides a method for preparing the above-mentioned AIE fluorescent probe, the method specifically comprising the following steps:

[0012] Step 1: CHCl3 was reacted with DMF, PBr3, cyclohexanone and NaHCO3. After the reaction was completed, the mixture was purified to obtain a yellow oily compound 1.

[0013] Step 2: At room temperature, compound 1 was mixed with Cs2CO3 and 4-bromo-2-hydroxybenzaldehyde in DMF and reacted. The reaction solution was then purified and eluted to obtain compound 2 as a yellow powder.

[0014] Step 3: Under argon atmosphere, compound 2, 4-methoxydiphenylamine, Cs2CO3, Pd2(OAc)2 and tri-tert-butylphosphine were reacted in toluene, and purified and eluted to obtain compound 3 as an orange powder.

[0015] Step 4: Under nitrogen atmosphere, compound 3 and compound B1 were catalytically refluxed in ethanol, and purified by elution to obtain blue-green solid compound 4;

[0016] Step 5: Compound 4 is reacted with BBr3 in dichloromethane, and the reaction solution is purified and eluted to obtain compound DPXBI as a blue-green powder.

[0017] Step 6: Under argon atmosphere, dissolve compound DPXBI and cesium carbonate in dichloromethane and react. After the reaction is complete, add dimethylaminochlorine to the mixture. Finally, purify and elute the resulting mixture to obtain a blue-green powder, namely compound DDPXBI.

[0018] Furthermore, the specific process of step 1 is as follows:

[0019] (1) Mix CHCl3 and DMF, then slowly add PBr3 to the mixture while keeping the solution temperature at 0°C. After injecting PBr3 for 1 hour, inject cyclohexanone into the mixture and stir the mixture at room temperature for 18 hours.

[0020] (2) After stirring, the mixture was poured into ice water, and then solid NaHCO3 was slowly added until pH=7. Finally, the aqueous phase was extracted with dichloromethane, and the combined organic layers were dried with anhydrous Na2SO4 and concentrated under reduced pressure to obtain yellow oily compound 1.

[0021] Furthermore, the specific process of step 2 is as follows:

[0022] (1) At room temperature, compound 1 and Cs2CO3 were mixed in DMF, and then the mixture was added to a solution of 4-bromo-2-hydroxybenzaldehyde in DMF;

[0023] (2) Stir the mixture obtained in step (2) at room temperature for 48 hours until a strong yellow spot appears on the reaction TLC (TLC monitoring: n-hexane / ethyl acetate (v / v = 4:1)). The reaction is then complete. Filter the precipitate, wash it twice with water, and extract it with ethyl acetate.

[0024] (3) The combined organic layers were dried with anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by silica gel chromatography using hexane to dichloromethane in a volume ratio of 5:1 to 0:1 as the eluent. The purified elution yielded compound 2 as a yellow powder.

[0025] Furthermore, the specific process of step 3 is as follows:

[0026] (1) Under argon atmosphere, compounds 2, 4-methoxydiphenylamine, Cs2CO3, Pd2(OAc)2 and tritert-butylphosphine were mixed in toluene;

[0027] (2) The mixture was stirred at 120°C for 48 h until orange spots appeared on the reaction TLC (TLC monitoring: n-hexane / ethyl acetate (v / v = 4:1)). After cooling to room temperature, the solvent was removed by vacuum distillation. The residue was redissolved in dichloromethane, washed three times with saturated brine, and the organic layer was dried on anhydrous Na2SO4 and dried under reduced pressure. The crude product was purified by silica gel chromatography with n-hexane and dichloromethane in a volume ratio of 1:1 as the eluent to obtain compound 3 in the form of orange powder.

[0028] Furthermore, the specific process of step 4 is as follows:

[0029] Compound 3 and compound B1 were mixed in ethanol under nitrogen atmosphere. 0.1 mL of piperidine was added dropwise to the mixture, and the mixture was refluxed for 24 h under the catalysis of piperidine. After reflux, the mixture was cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography, and the blue-green solid compound 4 was obtained by using dichloromethane and methanol in a volume ratio of 20:1 as the eluent.

[0030] Furthermore, the specific process of step 5 is as follows:

[0031] (1) Mix compound 4 with dichloromethane at -50℃, then add BBr3 dropwise to the mixture. After the addition is complete, return the solution to room temperature and stir overnight.

[0032] (2) Then, the reaction solution was quenched at 0°C with a saturated solution of NaHCO3 and extracted with dichloromethane. The organic layer was washed three times with saturated brine, dried with anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by silica gel chromatography with dichloromethane and methanol in a volume ratio of 15:1 as the eluent to obtain DPXBI as a blue-green powder.

[0033] Furthermore, the specific process of step 6 is as follows:

[0034] Under argon atmosphere, compound DPXBI and cesium carbonate were dissolved in dichloromethane. The mixture was stirred at room temperature for 30 min. After stirring, dimethylaminochloride was added to the mixture. Finally, the resulting mixture was purified by preparative thin-layer chromatography on silica gel GF254 with dichloromethane and methanol in a volume ratio of 15:1 as the eluent to obtain a blue-green powder, namely compound DDPXBI.

[0035] Furthermore, the synthesis route of the fluorescent probe is as follows:

[0036]

[0037] A third aspect of the present invention provides the application of the above-described AIE fluorescent probe in the preparation of reagents for detecting and identifying acetylcholinesterase in biological samples.

[0038] The application of the aforementioned AIE fluorescent probe in the preparation of reagents for detecting drugs for depression is also provided.

[0039] Applications of the aforementioned AIE fluorescent probes are also provided, including the detection and identification of acetylcholinesterase in biological samples;

[0040] The biological samples include biological organs, biological tissues, and biological cells;

[0041] The biological organs are the brain, heart, liver, spleen, lungs, kidneys, intestines, and stomach of a living organism (rat).

[0042] Compared with the prior art, the present invention has the following advantages:

[0043] This invention reports for the first time a fluorescent probe for drug development evaluation that specifically detects AChE. The detection process is simple, requires no additional chemicals, has a long half-life, and low cytotoxicity, which is beneficial for the detection of endogenous AChE at both cellular and in vivo levels. It exhibits good selectivity, showing no reaction to substances other than AChE; it is fast, requiring no prolonged incubation; and its large Stokes shift helps reduce interference from excitation light during imaging. Using this fluorescent probe, in vivo imaging analysis of changes in AChE in the brains of depressed rats was achieved, demonstrating significant practical value. Attached Figure Description

[0044] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0045] Figure 1 This is the mass spectrum of compound 2 during the preparation of the fluorescent probe in Example 1 of this invention;

[0046] Figure 2 This is the mass spectrum of compound 3 during the preparation of the fluorescent probe in Example 1 of this invention;

[0047] Figure 3 This is the mass spectrum of compound 4 during the preparation of the fluorescent probe in Example 1 of the present invention;

[0048] Figure 4 This is the mass spectrum of compound DPXBI during the preparation of the fluorescent probe in Example 1 of this invention;

[0049] Figure 5 This is the mass spectrum of DDPXBI, the final product in the fluorescent probe preparation process of Example 1 of the present invention;

[0050] Figure 6 This is a performance testing diagram of the fluorescent probe in Example 1 of the present invention;

[0051] Figure 7 This is an experimental diagram showing the dependence of fluorescence intensity of the fluorescent probe on AChE activity in Example 1 of this invention;

[0052] Figure 8 This is an experimental diagram illustrating the specificity of the fluorescent probe for AChE in Example 1 of this invention;

[0053] Figure 9 To detect the cytotoxicity of fluorescent probes in this invention, PC12 cells were incubated with different concentrations of fluorescent probes.

[0054] Figure 10The image shows a cell imaging diagram of the fluorescent probe of the present invention. PC12 cells treated with DDPXBI, DAPI and Merge and PC12 cells treated with corticosterone and PC12 cells with added AChE were incubated.

[0055] Figure 11 This is a subcellular organelle localization map of the fluorescent probes of the present invention. DDPXBI and different subcellular localization probes were incubated with PC12 cells.

[0056] Figure 12 This is a pharmacodynamic diagram of different drugs detected by a fluorescent probe in Example 1 of the present invention. Detailed Implementation

[0057] Example 1

[0058] The synthesis steps of the probe of this invention are as follows:

[0059]

[0060] In another specific embodiment of the present invention, the specific steps for synthesizing compound 1 are as follows:

[0061] (1) PBr3 (9 mL, 94 mmol) was slowly added to a solution of CHCl3 (50 mL) and DMF (12 mL, 155 mmol) at 0 °C. One hour after the injection of PBr3, cyclohexanone (4 mL, 39 mmol) was added to the mixture, and the mixture was stirred at room temperature for 18 hours.

[0062] (2) The resulting mixture was poured into ice water, and then solid NaHCO3 was slowly added until pH = 7. The aqueous phase was extracted with dichloromethane, and the combined organic layers were dried with anhydrous Na2SO4 and concentrated under reduced pressure to give a yellow oily compound 1.

[0063] In another specific embodiment of the present invention, the specific steps for synthesizing compound 2 are as follows:

[0064] (1) Compound 1 (0.26 g, 1.26 mmol) and Cs2CO3 (1.2 g, 3.68 mmol) were mixed in dry DMF (10 mL) at room temperature, and the mixture was then added to a solution of 4-bromo-2-hydroxybenzaldehyde (0.49 g, 2.56 mmol) in DMF (1 mL);

[0065] (2) Stir the mixture obtained in step (2) at room temperature for 48 h until a strong yellow spot appears on the reaction TLC (TLC monitoring: n-hexane / ethyl acetate (v / v = 4:1)). At the end of the reaction, filter off this precipitate, wash twice with water, and extract with ethyl acetate;

[0066] (3) The combined organic layers were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by silica gel chromatography using n-hexane / dichloromethane (v / v = 5:1 to 0:1) as the eluent to obtain compound 2 (a yellow powder). Figure 1 ). ( Figure 1 a) 1 HNMR(600MHz,Chloroform-d)δ10.18(s,1H),7.42(s,1H),7.30(dd,J=8.2,1.6Hz,1H),7.13(d ,J=8.2Hz,1H),6.86(s,1H),2.67-2.64(m,2H),2.51(t,J=6.1Hz,2H),1.78(q,J=6.2Hz,2H).( Figure 2 b) 13 CNMR(101MHz,DMSO-d6)δ187.37,152.13,130.11,128.85,127.31,126.34,122.54,120.64,118.63,113.37,29.59,21.50,and20.13.

[0067] In another specific embodiment of the present invention, the specific steps for synthesizing compound 3 are as follows:

[0068] (1) Under argon atmosphere, compound 2 (100 mg, 0.34 mmol), 4-methoxydiphenylamine (103 mg, 0.52 mmol), Cs2CO3 (167 mg, 0.53 mmol), Pd2(OAc)2 (10 mg, 0.3 mmol) and tri-tert-butylphosphine (8 mg, 0.3 mmol) were mixed in toluene (15 mL);

[0069] (2) The reaction mixture was stirred at 120°C for 48 h until an orange spot appeared on the reaction TLC (TLC monitoring: n-hexane / dichloromethane (v / v = 1:2). After cooling to room temperature, the solvent was removed by vacuum distillation.

[0070] (3) The residue was redissolved in dichloromethane, washed three times with saturated brine, and the organic layer was dried on anhydrous Na2SO4 under reduced pressure. The crude product was purified by silica gel chromatography using n-hexane / dichloromethane (1:1, v / v) as the eluent to obtain compound 3 (3) as an orange powder. Figure 2 ). ( Figure 2 a) 1HNMR(500MHz,DMSO-d6)δ10.11(s,1H),7.37-7.33(m,2H),7.19(d,J=8.2Hz,1H),7.13-7.09(m,5H),7.00-6.97(m,2H ),6.93(s,1H),6.58(d,J=9.4Hz,2H),3.77(s,3H),2.56(d,J=5.5Hz,2H),2.27(d,J=5.9Hz,2H),1.63-1.59(m,2H).( Figure 2 b) 13 CNMR(126MHz,DMSO-d6)δ186.70,160.58,157.32,152.92,146.77,130.18,128.62, 128.21,125.17,124.50,115.88,115.76,111.93,105.64,55.76,29.51,and20.58.( Figure 2 c) HRMS(ESI): m / zcalcdforC 27 H 23 NO3[M+H] + ,409.1678;found,410.1751.

[0071] In another specific embodiment of the present invention, the specific steps for synthesizing compound 4 are as follows:

[0072] Compound 3 (82 mg, 0.2 mmol) and compound B1 (100 mg, 0.3 mmol) were mixed in dry ethanol under nitrogen atmosphere. 0.1 mL of piperidine was added dropwise to the mixture, and the mixture was refluxed for 24 h under the catalysis of piperidine. After the reflux was complete, the mixture was cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography, eluting with dichloromethane / methanol (v / v = 20:1) to give a blue-green solid compound 4 (… Figure 3 ). ( Figure 3 a) 1HNMR(500MHz,DMSO-d6)δ8.51(d,J=14.9Hz,1H),8.21-8.13(m,3H),8.00(d,J=9.0Hz,1H),7.76 -7.73(m,1H),7.61(dd,J=8.2,6.9Hz,1H),7.44(q,J=8.8,8.4Hz,4H),7.26-7.23(m,5H),7.08( d,J=8.9Hz,2H),6.78-6.73(m,2H),6.68(d,J=2.2Hz,1H),4.64(t,J=7.8Hz,2H),3.84-3.83(m, 3H),2.72(s,4H),2.60(t,J=6.6Hz,2H),2.10(t,J=7.5Hz,2H),1.85(s,6H),1.84-1.79(m,2H).( Figure 3 b) 13 CNMR(126MHz,DMSO-d6)δ177.69,160.31,157.45,153.81,151.28,145.50,143.44,13 9.18,138.05.135.02,133.25,132.13,130.76,130.10,129.89,128.69,128.44,127. 07,125.80,125.23,124.98,122.09,116.58,115.40,115.18,114.27,112.41,103.70 ,103.62,55.47,51.57,47.51,43.92,28.39,27.34,27.02,24.07,23.65,and20.16.( Figure 3 c) HRMS(ESI): m / zcalcdforC 45 H 42 N₂O₅S[M+H] + ,722.2814; found,723.2891.

[0073] In another specific embodiment of the present invention, the specific steps for synthesizing the compound DPXBI are as follows:

[0074] (1) Compound 4 (72 mg, 0.1 mmol) was mixed with dichloromethane (5 mL) at -50 °C, and then BBr3 (10 equivalents) was added dropwise to the mixture. After the addition was complete, the solution was returned to room temperature and stirred overnight.

[0075] (2) Then, the resulting reaction solution was quenched at 0℃ with a saturated solution of NaHCO3, extracted with dichloromethane, and the organic layer was washed three times with saturated brine. The mixture was dried with anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by silica gel chromatography using dichloromethane / methanol (v / v = 15:1) as the eluent to obtain a blue-green powder of DPXBI. Figure 4 ). ( Figure 4 a) 1 HNMR(600MHz,DMSO-d6)δ9.73(s,1H),8.51(d,J=14.9Hz,1H),8.22(d,J=8.6Hz,1H),8.17(d,J=8.9Hz,1H), 8.13(d,J=8.3Hz,1H),7.99(d,J=8.9Hz,1H),7.76-7.74(m,1H),7.62-7.60(m,1H),7.48-7.39(m,4H),7.25( d,J=8.3Hz,3H),7.15-7.11(m,2H),6.93-6.88(m,2H),6.77-6.72(m,2H),6.64(d,J=2.1Hz,1H),4.66-4.61( m,2H),2.71(s,4H),2.61(dd,J=8.3,4.8Hz,2H),2.10(p,J=7.1Hz,2H),1.85(s,6H),1.80(d,J=6.4Hz,2H).( Figure 4 b) 13 C NMR (126MHz, DMSO-d6) δ177.58,160.47,156.03,153.85,151.57,145.51,139.18,136 .40,134.97,133.50,132.57,132.08,130.74,130.08,129.84,128.72,128.10,127.0 9,126.52,126.03,125.73,125.15,124.91,122.25,116.69,114.93,114.20,112.35, 103.49,103.15,51.54,47.53,43.86,28.35,27.06,25.79,24.04,21.65,and20.18.( Figure 4 c) HRMS(ESI): m / zcalcdforC 44 H 40 N₂O₅S[M+H] + ,708.2658;found,709.2731.

[0076] In another specific embodiment of the present invention, the specific steps for synthesizing the compound DDPXBI are as follows:

[0077] (1) Under argon atmosphere, compound DPXBI (50 mg, 70 μmol) and cesium carbonate (23 mg, 70 μmol) were dissolved in dry dichloromethane (5 mL). The reaction mixture was stirred at room temperature. After 30 minutes, dimethylaminochloride (151.7 mg, 1.41 mmol) was added to the solvent.

[0078] (2) The final mixture was purified by preparative thin-layer chromatography on silica gel GF254 using dichloromethane / methanol (15:1) as the eluent to obtain a blue-green powder. Figure 5 Subsequently, the probe was subjected to performance testing, and it showed an absorption peak in the 750-950 nm range. Figure 6 Furthermore, the fluorescence intensity increases with increasing AChE concentration. Figure 7 The probe was incubated with other cholinergic substances, but only after incubation with AChE did the PL spectral intensity increase, indicating that the probe has strong specificity only with AChE. Figure 8 ). ( Figure 5 a) 1 HNMR(500MHz,DMSO-d6)δ8.51(d,J=14.9Hz,1H),8.22(d,J=8.5Hz,1H),8.18(d,J=8.9Hz,1H),8 .13(d,J=8.2Hz,1H),8.01(d,J=8.9Hz,1H),7.71(t,J=7.6Hz,1H),7.61(t,J=7.5Hz,1H),7.49- 7.41(m,4H),7.29-7.19(m,7H),6.84-6.72(m,3H),4.72-4.62(m,2H),3.05(s,3H),2.89(s,3H) ,2.75-2.68(m,4H),2.62(t,J=6.4Hz,2H),2.15-2.07(m,2H),1.85(s,6H),1.84-1.78(m,2H).( Figure 5 b) 13CNMR(126MHz,DMSO-d6)δ178.52,160.49,154.44,154.20,151.25,145.98,144.08,142.8 5,139.59,135.84,133.24,132.67,131.25,130.51,130.33,129.21,128.46,127.56,127 .42,127.32,126.34,126.07,125.72,123.92,122.81,117.98,117.20,116.25,114.79,112.93,105.46,104.64,52.23,47.99,44.46,36.82,34.58,27.54,27.47,24.58,20.61. Figure 5 c) HRMS(ESI): m / zcalcdforC 44 H 40 N₂O₅S[M+H] + ,779.3029;found,780.3107.

[0079] In cell experiments, this invention uses fluorescent probes of different concentrations incubated with PC12 to test their cytotoxicity. Figure 9 PC12 cells were stimulated with corticosterone to induce oxidative stress. Imaging with an AChE fluorescent probe revealed that, compared to the control group, corticosterone-stimulated cells exhibited strong fluorescence, indicating an increased AChE concentration. Treatment of corticosterone-stimulated cells with an AChE inhibitory enzyme resulted in decreased fluorescence, indicating a reduced AChE concentration. These imaging results demonstrate that the probe can detect changes in AChE in living cells. Figure 10 PC12 cells were co-incubated with the AIE probe and a commercial dye (Fbfp) targeting the endoplasmic reticulum (ER), lysosomes (Lysosomes), mitochondria (Mito), and lipid droplets (LD). The superimposed imaging of the AIE probe (mCherry) and the green channels of the ER / lysosomes / mitochondria / lipid droplets was observed through fluorescence imaging. Figure 11 As shown, when using Lyso, the red-green channels overlap well, exhibiting good co-localization, indicating that the probe can effectively target lysosomes. However, when using ER-P and Mito-P, the red-green channel overlap is poor. This demonstrates that after entering the cell, the AIE probe targets lysosomes and has poor binding to the endoplasmic reticulum and mitochondria.

[0080] In an in vivo experiment, rats in the experimental groups were injected with venlafaxine (6 weeks) and toludecvenlafaxine (2 weeks), respectively. Fluorescent probes were then injected via the tail vein, and the fluorescence probe content in the rat brain tissue was detected. The results showed that compared to the experimental group, the levels of fluorescent probes in the brains of the treated rats were significantly reduced, indicating that the effects of venlafaxine administered for 6 weeks and toludecvenlafaxine administered for 2 weeks were similar. Figure 12 Through the above experiments, we analyzed for the first time the levels of fluorescent probes in the brain tissues of rats in the drug-treated group, normal group, and experimental group. The results suggest that this fluorescent probe can be used as a tool for drug development evaluation.

[0081] Matters not covered in this invention are common knowledge.

[0082] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

[0083] Contents not described in detail in this specification are prior art known to those skilled in the art. Although illustrative specific embodiments of the invention have been described above to facilitate understanding by those skilled in the art, it should be understood that the invention is not limited to the scope of the specific embodiments. Various modifications are readily apparent to those skilled in the art as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims, and all inventions utilizing the concept of this invention are protected.

Claims

1. An AIE fluorescent probe for detecting acetylcholinesterase, characterized in that, The structure of the AIE fluorescent probe is as follows: 。 2. The AIE fluorescent probe for detecting acetylcholinesterase according to claim 1, characterized in that, Dimethylamino is the recognition group.

3. A method for preparing an AIE fluorescent probe for detecting acetylcholinesterase according to claim 1 or 2, characterized in that, The preparation method of the AIE fluorescent probe specifically includes the following steps: Step 1: CHCl3 was reacted with DMF, PBr3, cyclohexanone and NaHCO3. After the reaction was completed, the mixture was purified to obtain a yellow oily compound 1. Step 2: At room temperature, compound 1 was mixed with Cs2CO3 and 4-bromo-2-hydroxybenzaldehyde in DMF and reacted. The reaction solution was then purified and eluted to obtain compound 2 as a yellow powder. Step 3: Under argon atmosphere, compound 2, 4-methoxydiphenylamine, Cs2CO3, Pd2(OAc)2 and tri-tert-butylphosphine were reacted in toluene, and purified and eluted to obtain compound 3 as an orange powder. Step 4: Under nitrogen atmosphere, compound 3 and compound B1 were catalytically refluxed in ethanol, and purified by elution to obtain blue-green solid compound 4; Step 5: Compound 4 is reacted with BBr3 in dichloromethane, and the reaction solution is purified and eluted to obtain compound DPXBI as a blue-green powder. Step 6: Under argon atmosphere, compound DPXBI and cesium carbonate are dissolved in dichloromethane and reacted. After the reaction is complete, dimethylaminochloride is added to the mixture. Finally, the resulting mixture is purified and eluted to obtain a blue-green powder, namely compound DDPXBI. The synthesis route of the fluorescent probe is as follows: 。 4. The method for preparing an AIE fluorescent probe for detecting acetylcholinesterase according to claim 3, characterized in that, The specific process of step 1 is as follows: (1) Mix CHCl3 and DMF, and then slowly add PBr3 to the mixture. During this process, control the solution temperature at 0°C. After injecting PBr3 for 1 hour, inject cyclohexanone into the mixture. Stir the mixture at room temperature for 18 hours. (2) After stirring, pour the mixture into ice water, then slowly add solid NaHCO3 until pH=7. Finally, extract the aqueous phase with dichloromethane, dry the combined organic layer with anhydrous Na2SO4, concentrate under reduced pressure, and obtain yellow oily compound 1.

5. The method for preparing an AIE fluorescent probe for detecting acetylcholinesterase according to claim 3, characterized in that, The specific process of step 2 is as follows: (1) At room temperature, compound 1 and Cs2CO3 were mixed in DMF, and then the mixture was added to a solution of 4-bromo-2-hydroxybenzaldehyde in DMF; (2) Stir the mixture obtained in step (2) at room temperature for 48 hours until yellow spots appear on the reaction TLC. The reaction is then complete. Filter the precipitate, wash it twice with water, and extract it with ethyl acetate. (3) The combined organic layers were dried with anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by silica gel chromatography using hexane to dichloromethane in a volume ratio of 5:1 to 0:1 as the eluent. The purified elution yielded compound 2 as a yellow powder.

6. The method for preparing an AIE fluorescent probe for detecting acetylcholinesterase according to claim 3, characterized in that, The specific process of step 3 is as follows: (1) Under argon atmosphere, compounds 2, 4-methoxydiphenylamine, Cs2CO3, Pd2(OAc)2 and tri-tert-butylphosphine were mixed in toluene; (2) The mixture was stirred at 120°C for 48 h until orange spots appeared on the reaction TLC. After cooling to room temperature, the solvent was removed by vacuum distillation. The residue was redissolved in dichloromethane, washed three times with saturated brine, and the organic layer was dried on anhydrous Na2SO4 and dried under reduced pressure. The crude product was purified by silica gel chromatography with hexane and dichloromethane in a volume ratio of 1:1 as the eluent to obtain compound 3 in orange powder form.

7. The method for preparing an AIE fluorescent probe for detecting acetylcholinesterase according to claim 3, characterized in that, The specific process of step 4 is as follows: Compound 3 and compound B1 were mixed in ethanol under nitrogen atmosphere. 0.1 mL of piperidine was added dropwise to the mixture, and the mixture was refluxed for 24 h under the catalysis of piperidine. After the reflux was completed, the mixture was cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography, and the blue-green solid compound 4 was obtained by using dichloromethane and methanol in a volume ratio of 20:1 as the eluent.

8. The method for preparing an AIE fluorescent probe for detecting acetylcholinesterase according to claim 3, characterized in that, The specific process of step 5 is as follows: (1) Mix compound 4 with dichloromethane at -50℃, then add BBr3 dropwise to the mixture. After the addition is complete, return the solution to room temperature and stir overnight. (2) Then, the reaction solution was quenched at 0°C with a saturated solution of NaHCO3 and extracted with dichloromethane. The organic layer was washed three times with saturated brine, dried with anhydrous Na2SO4 and evaporated under reduced pressure. The residue was purified by silica gel chromatography with dichloromethane and methanol in a volume ratio of 15:1 as the eluent to obtain blue-green powder DPXBI.

9. The method for preparing an AIE fluorescent probe for detecting acetylcholinesterase according to claim 3, characterized in that, The specific process of step 6 is as follows: Under argon atmosphere, compound DPXBI and cesium carbonate were dissolved in dichloromethane. The mixture was stirred at room temperature for 30 min. After stirring, dimethylaminochloride was added to the mixture. Finally, the resulting mixture was purified by preparative thin-layer chromatography on silica gel GF254 with dichloromethane and methanol in a volume ratio of 15:1 as the eluent to obtain a blue-green powder, namely compound DDPXBI.

10. The application of the AIE fluorescent probe according to claim 1 or 2 in the preparation of reagents for detecting and identifying acetylcholinesterase in biological samples.

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