Acetylcholinesterase fluorescent probe based on non-broken bond mechanism as well as preparation method and application of acetylcholinesterase fluorescent probe

Abstract

The invention provides an acetylcholin esterase fluorescent probe based on a non-broken bond mechanism and a preparation method and application thereof, and belongs to the technical field of acetylcholin esterase detection.The fluorescent probe is designed based on a naphthalimide structure, and the preparation process comprises intermediate synthesis, final product preparation and column chromatography purification. Reagents such as solvents, alkali and acyl chloride are easy to obtain, reaction conditions are mild, and preparation is simple and convenient. The fluorescent probe realizes non-broken bond recognition of AChE through a PET mechanism: a carbamate fragment in a probe structure quenches fluorescence of a fluorophore, and when acting with AChE, the fragment enters a hydrophobic pocket of an enzyme to break a PET process so as to recover fluorescence. Experiments show that the probe is sensitive and rapid in response to AChE, the fluorescence intensity is positively correlated with the concentration of AChE, and the probe has good specificity to AChE and is slightly interfered by common zwitterions and amino acids; the fluorescent probe can be used for detecting acetylcholin esterase in acetylcholin esterase indications.

Description

Technical Field

[0001] This invention belongs to the field of acetylcholinesterase detection technology, specifically relating to a class of fluorescent probes for acetylcholinesterase based on a non-bond-breaking mechanism, their preparation methods, and applications. Background Technology

[0002] Acetylcholinesterase (AChE) is a key enzyme in biological neurotransmission, primarily found in the synaptic cleft of cholinergic nerve endings, particularly concentrated in the folds of the postsynaptic membrane of motor nerve endplates; it is also present in cholinergic neurons and erythrocytes. Its physiological function is to rapidly hydrolyze acetylcholine into choline and acetic acid, thereby terminating nerve impulses and maintaining the high timeliness of synaptic transmission. AChE activity imbalance is closely related to various pathological conditions, including neurodegenerative diseases such as Alzheimer's disease, organic or drug-induced cholinergic crises, and poisoning by organophosphates or carbamates. Therefore, establishing accurate, sensitive, and rapid-response AChE detection techniques is of great significance for elucidating disease mechanisms, conducting drug screening, performing toxicological assessments, and achieving early clinical warning.

[0003] Currently, research on small molecule fluorescent probes targeting AChE is quite active, with "self-destruction-triggered" reactive probes being the most widely used. These probes typically introduce ester, carbonate, or thioester units that are easily hydrolyzed by AChE into the molecule; when enzymatic bond cleavage occurs, it triggers self-destruction (such as p-aminobenzyl alcohol cleavage) to release the fluorophore or remove quenching, thereby generating a detectable fluorescent signal. This strategy possesses certain selectivity and amplification effects, and is therefore widely adopted. However, bond-crack triggered probes have inherent limitations. Their signal generation involves at least multiple steps, including enzyme-substrate binding, chemical bond cleavage, and possible self-destruction. The overall rate is limited by both substrate diffusion and chemical cleavage kinetics, with typical responses taking several minutes to over ten minutes, making it difficult to meet the "near real-time" monitoring requirements for rapid neural events or transient regulatory processes. Furthermore, these probes are consumed and converted into new molecules during the reaction, and the system is difficult to restore to its initial state through simple displacement, which is detrimental to dynamic reversible monitoring and long-term in vivo imaging.

[0004] Based on the above shortcomings, there is an urgent need to develop AChE probes that do not rely on chemical bond breaking in order to achieve better identification of AChE. Summary of the Invention

[0005] In view of this, the purpose of this invention is to address the problems existing in the prior art by providing a class of fluorescent probes for acetylcholinesterase based on a non-bond-breaking mechanism, their preparation method, and applications. To achieve the above objectives, the technical solution of the present invention is as follows: A class of fluorescent probes for acetylcholinesterase based on a non-bond-breaking mechanism, wherein the fluorescent probe recognizes acetylcholinesterase via a non-bond-breaking method, and the fluorescent probe has the structure shown in Formula I: Formula I R1 and R2 are each independently selected from hydrogen, methyl, ethyl, propyl, butyl, methylene or substituted alkyl, benzyl or substituted benzyl. The second objective of this invention is to provide a method for preparing a class of fluorescent probes for acetylcholinesterase based on a non-bond-breaking mechanism, the preparation process of which is shown below: The specific preparation steps are as follows: S1. Compound M2 is prepared by reacting 4-bromo-1,8-naphthalenedicarboxylic anhydride with an amine compound in a molar ratio of 1:(1.5-1.6) in a reaction solvent and a basic catalyst; S2. Compound M2 and ethanolamine were added to the reaction solvent at a molar ratio of 1:(1.3-1.35), and the mixture was heated to reflux under nitrogen protection to prepare compound M3; S3. Dissolve compound M3 in the reaction solvent, add alkali while stirring, then add acyl chloride, and reflux under nitrogen protection. After the reaction is complete, evaporate the solvent and purify by column chromatography to obtain the corresponding target product.

[0006] Preferably, in step S1, the amine compound is dimethylamine, diethylamine, methyl ethylamine, piperidine, dibenzylamine, or its hydrochloride, hydrobromide, or hydroiodide salt; the molar ratio of the alkaline catalyst to 4-bromo-1,8-naphthalenedicarboxylic anhydride is (1.5-2):1, the reaction time is 12 hours, and the reaction temperature is 110-124℃. Preferably, in steps S1 and S2, the reaction solvent is one of anhydrous ethanol, tetrahydrofuran, toluene, DMF, DMSO, and 2-methoxyethanol. Preferably, in steps S1 and S3, the alkaline catalyst is an inorganic salt such as carbonate or bicarbonate, or triethylamine, diisopropylethylamine, or dimethylaminopyridine; in step S2, the reaction temperature of the system is 80°C, and the reaction time is 4-12 hours.

[0007] Preferably, in step S3, the reaction solvent is one of toluene, tetrahydrofuran, or ethylene glycol monomethyl ether; the molar ratio of compound M3 to the basic catalyst is 1:(2-5); and the molar ratio of compound M3 to acyl chloride is 1:(3-5).

[0008] Preferably, in step S3, the reaction temperature of the system is 70-110℃ and the reaction time is 36h-72h.

[0009] The fluorescent probe provided by this invention responds to acetylcholinesterase via a PET mechanism: The carbamate fragment in the probe structure can quench the fluorescence of the fluorophore. When it interacts with acetylcholinesterase, the carbamate fragment enters the hydrophobic pocket of the enzyme, breaking the PET of the probe structure and restoring the fluorescence of the probe.

[0010] Based on the response mechanism of the probe of this invention, the third objective of this invention is to provide applications of the fluorescent probe, including at least the following: (a) Application in the selective recognition of acetylcholinesterase; (b) Application in fluorescence imaging of acetylcholinesterase in cells or living tissues.

[0011] Beneficial effects of this invention: (1) The fluorescent probe obtained in this invention uses the non-bond-breaking PET (photoinduced electron transfer) mechanism to recognize AChE, which avoids the inherent defects of traditional "self-destruction dissociation type" probes that rely on chemical bond breaking. It does not need to go through multiple steps such as enzyme-substrate binding, bond breaking and self-destruction dissociation, and fundamentally solves the problem of slow response and difficulty in real-time monitoring of traditional probes. (2) The fluorescent probe obtained by the present invention is sensitive and fast to AChE. It can complete the response within 10 seconds and achieve a significant enhancement of the fluorescence signal. The fluorescence intensity tends to stabilize within 360 seconds, which is far better than the response time of traditional probes of several minutes to more than ten minutes. It can meet the "near real-time" monitoring needs of rapid neural events or transient regulation processes. (3) The fluorescent probe obtained in this invention is effective against cations and anions (such as CH3COO⁻, Na⁺, Mg). 2 In a system where 28 common ions (including ⁺) coexist with amino acids, it only produces a significant fluorescence response to AChE, while other substances have minimal impact on the fluorescence signal, demonstrating outstanding specificity and ensuring the accuracy of AChE detection in complex samples. (4) The fluorescent probe obtained in this invention is designed based on the structure of naphthalimide, and the molecular structure is well-defined and controllable (R1 and R2 can be flexibly selected as alkyl or substituted alkyl); the solvents and reagents used in the preparation process are readily available, the reaction conditions are mild, and high-purity products can be obtained through mature steps such as intermediate synthesis and column chromatography purification, which is easy to prepare on a large scale; (5) The fluorescent probe obtained in this invention can be used to detect acetylcholinesterase in indications such as arthritis-related diseases (rheumatoid arthritis, osteoarthritis (degenerative joint disease), ankylosing spondylitis), acute gouty arthritis, musculoskeletal pain, soft tissue injury (such as sprain, strain), back pain or neck pain, postoperative and other acute pain, cancer, etc. Attached Figure Description

[0012] Figure 1 The 1H NMR spectrum of the acetylcholinesterase fluorescent probe disclosed in Example 1 of this invention; Figure 2 This is a high-resolution mass spectrometry of the acetylcholinesterase fluorescent probe disclosed in Example 1 of the present invention; Figure 3 The 1H NMR spectrum of the acetylcholinesterase fluorescent probe disclosed in Example 2 of this invention; Figure 4 This is a high-resolution mass spectrometry of the acetylcholinesterase fluorescent probe disclosed in Example 2 of the present invention; Figure 5 The 1H NMR spectrum of the acetylcholinesterase fluorescent probe disclosed in Example 3 of this invention; Figure 6 This is a high-resolution mass spectrometry of the acetylcholinesterase fluorescent probe disclosed in Example 3 of the present invention; Figure 7 The fluorescence spectra of the acetylcholinesterase fluorescent probe before and after the AChE response disclosed in Example 1 of this invention are shown. Figure 8 The fluorescence spectra of the acetylcholinesterase fluorescent probe before and after the AChE response disclosed in Example 2 of this invention are shown below. Figure 9 The fluorescence spectra of the acetylcholinesterase fluorescent probe before and after the AChE response disclosed in Example 3 of this invention are shown. Figure 10 This is a specificity test diagram of the acetylcholinesterase fluorescent probe and amino acids disclosed in Example 1 of the present invention. Figure 11 This is a specificity test diagram of the acetylcholinesterase fluorescent probe and its anions and cations disclosed in Example 1 of this invention; in the diagram, (1-5) AChE, CH3COO - CO2- 3, SO2- 4, F - ;(6-10)Cl - I - NO 2- S2O2- 3, OCl - ; (11-15) ClO- 4, NH+ 4, Na + Mg 2+ Al 3+ (16-20)K + Ca 2+ Fe 3+ Cu 2+ Ni 2+ (20-25) Hg 2+ Sn 2+ Fe 2+ Pb 2+ Ti4+ ;(26-28)Co 2+ Zn 2+ Cd 2+ .

[0013] Figure 12 The kinetic curve of the acetylcholinesterase fluorescent probe and AChE response disclosed in Example 1 of this invention is shown. Figure 13 HPLC analysis of the acetylcholinesterase fluorescent probe disclosed in Example 1 of this invention; Figure 14 The diagram shows the HOMO and LUMO orbital energy levels of the acetylcholinesterase fluorescent probe disclosed in Example 1 of this invention. Figure 15 This is a diagram illustrating the response mechanism of the acetylcholinesterase fluorescent probe and AChE disclosed in Example 1 of this invention. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the invention, but do not limit the invention in any way.

[0016] Example 1: Preparation method of a class of fluorescent probes for acetylcholinesterase based on non-bond-breaking mechanism: The synthetic route for the acetylcholinesterase fluorescent probe A1, in which both R1 and R2 are methyl groups, is shown below: S1: Synthesis of intermediate compound 1 of A1: Weigh 11.08 g (1 eq, 40 mmol) of 4-bromo-1,8-naphthalenedicarboxylic anhydride and 2.7 g (1.5 eq, 60 mmol) of dimethylamine into a 150 mL flask containing DMF. Then add 6.06 g (1.5 eq, 60 mmol) of triethylamine dropwise into the flask. Under nitrogen protection, heat to 110 °C and reflux with stirring for 12 h. The reaction is monitored by thin-layer chromatography (TLC) until the reaction is complete. Remove DMF by rotary evaporation under reduced pressure. Purify the product by slurrying with methanol and deionized water. Filter and collect the product. The filter cake obtained is intermediate compound A1. S2: Synthesis of intermediate compound 2 of A1: Weigh 1205 mg (1 eq, 5 mmol) of intermediate A1 compound 1 and ethanolamine (397 mg, 1.3 eq, 6.5 mmol) into a round-bottom flask containing 40 mL of anhydrous ethanol. Under nitrogen protection, heat to 80 °C and reflux with stirring for 6 h. The reaction was monitored by thin-layer chromatography (TLC) until complete. Remove the anhydrous ethanol by rotary evaporation under reduced pressure. Then purify the product by slurry mixing with methanol and deionized water. Filter and collect the product; the filter cake obtained is intermediate A1 compound 2. The product is a yellow solid with a yield of 99%.

[0017] S3: Synthesis of acetylcholinesterase fluorescent probe A1: Intermediate compound 2 (1420 mg, 1 eq, 5 mmol) and N,N-dimethylcarbamoyl chloride (1620 mg, 3 eq, 15 mmol) were added to a 50 mL THF flask. Triethylamine (1010 mg, 2 eq, 10 mmol) was then added dropwise to the flask. Under nitrogen protection, the mixture was heated to 70 °C and refluxed with stirring for 36 h. The reaction was monitored by thin-layer chromatography (TLC) until complete. THF was removed by rotary evaporation under reduced pressure, followed by purification using a silica gel column (methanol / dichloromethane) to obtain a yellow solid compound A1. The structure of A1 was identified, and its 1H NMR and high-resolution mass spectra are shown below. Figure 1 and Figure 2 As shown, the result is displayed as the target structure.

[0018] Example 2: Preparation method of a class of fluorescent probes for acetylcholinesterase based on non-bond-breaking mechanism: The synthetic route for acetylcholinesterase fluorescent probe A2, in which both R1 and R2 are methylene groups, is shown below: S1: Synthesis of intermediate compound 1 of A2: Weigh 1385 mg (1 eq, 5 mmol) of 4-bromo-1,8-naphthalenedicarboxylic anhydride and 680 mg (1.6 eq, 8 mmol) into a 30 mL methoxyethanol flask. Then, add 1010 mg (2 eq, 10 mmol) of triethylamine dropwise to the flask. Under nitrogen protection, heat to 124 °C and reflux with stirring for 12 h. The reaction is monitored by thin-layer chromatography (TLC) until complete. Cool the reaction system in an ice-water bath, add deionized water to slurry, and filter to collect the product. The filter cake obtained is intermediate compound A2. The product is brownish-yellow, with a yield of 96%.

[0019] Synthesis of S2: A2 intermediate compound 2: Weigh 843 mg (1 eq, 3 mmol) of intermediate A2 compound 1 and ethanolamine (275 mg, 1.5 eq, 4.5 mmol) into a round-bottom flask containing 30 mL of ethanol. Under nitrogen protection, heat to 80 °C and reflux with stirring for 12 hours. The reaction was monitored by thin-layer chromatography (TLC) until completion. Anhydrous ethanol was removed by rotary evaporation under reduced pressure, and the product was purified by silica gel column chromatography (MeOH / DCM = 1 / 80) to obtain approximately 890 mg of intermediate A2 compound 2 as a yellow solid, with a yield of 91.5%.

[0020] S3: Synthesis of acetylcholinesterase fluorescent probe A2: Intermediate compound 2 (324 mg, 1 eq, 1 mmol) and N,N-dimethylcarbamoyl chloride (323 mg, 3 eq, 3 mmol) were added to a 30 mL toluene-containing flask. Triethylamine (505 mg, 5 eq, 5 mmol) was then added dropwise to the flask. Under nitrogen protection, the mixture was heated to 110 °C and refluxed with stirring for 72 h. The reaction was monitored by thin-layer chromatography (TLC) until completion. Toluene was removed by rotary evaporation under reduced pressure, followed by purification using a silica gel column (MeOH / DCM = 1 / 60) to obtain a yellow solid compound A2. The structure of A2 was identified, and its 1H NMR and high-resolution mass spectra are shown below. Figure 3 and Figure 4 As shown, the result is displayed as the target structure.

[0021] Example 3: Preparation method of a class of fluorescent probes for acetylcholinesterase based on non-bond-breaking mechanism: The synthetic route for the fluorescent acetylcholinesterase probe A3, in which both R1 and R2 are benzyl groups, is shown below: S1: Synthesis of intermediate compound 1 of A3: Weigh 2771 mg (1 eq, 10 mmol) of 4-bromo-1,8-naphthalenedicarboxylic anhydride and 2950 mg (1.5 eq, 15 mmol) into a 40 mL flask containing DMF. Then add 1518 mg (1.5 eq, 15 mmol) of triethylamine dropwise into the flask. Under nitrogen protection, heat to 110 °C and reflux with stirring for 12 h. Monitor the reaction for completion by thin-layer chromatography (TLC). Remove DMF by rotary evaporation under reduced pressure, and purify by silica gel column chromatography (pure DCM column chromatography) to obtain approximately 396 mg of intermediate compound A3, with a yield of approximately 10%.

[0022] Synthesis of S2: A3 intermediate compound 2: Weigh 290 mg (1 eq, 0.74 mmol) of intermediate A3 compound 1 and ethanolamine (61 mg, 1.35 eq, 1 mmol) into a round-bottom flask containing 20 mL of ethanol. Under nitrogen protection, heat to 80 °C and reflux with stirring for 4 h. The reaction was monitored by thin-layer chromatography (TLC) until complete. Anhydrous ethanol was removed by rotary evaporation under reduced pressure, and the mixture was purified by silica gel column chromatography (MeOH / DCM = 1 / 80) to give approximately 309 mg of intermediate A3 compound 2, with a yield of 96.6%.

[0023] S3: Synthesis of intermediate compound 3 of A3: Intermediate compound 2 of A3 (309 mg, 1 eq, 0.71 mmol) and N,N-dimethylcarbamoyl chloride (384 mg, 5 eq, 3.55 mmol) were added to a 20 mL toluene-containing flask. Triethylamine (360 mg, 5 eq, 3.55 mmol) was then added dropwise to the flask. Under nitrogen protection, the mixture was heated to 110 °C and refluxed with stirring for 36 h. The reaction was monitored by thin-layer chromatography (TLC) until completion. Toluene was removed by rotary evaporation under reduced pressure, followed by purification using a silica gel column (EA / PE = 3 / 1) to obtain approximately 58 mg of a yellow solid compound A3, with a yield of approximately 16.2%. The structure of A3 was identified, and its 1H NMR and high-resolution mass spectra are shown below. Figure 5 and Figure 6 As shown, the result is displayed as the target structure.

[0024] Example 4: Study on the response performance of acetylcholinesterase fluorescent probes A1, A2, and A3 prepared in Examples 1-3 of this invention to AChE: At room temperature, the stock solutions of acetylcholinesterase fluorescent probes A1, A2, and A3 were added to PBS buffer solution at pH 7.35 to make the concentrations of A1, A2, and A3 in the buffer solution 10 μM, respectively. Different concentrations (0, 5, 10, 15, and 20 U / mL) of AChE were added to the system, and the mixture was placed in a shaker at 37°C for 30 min. The reaction was then performed using a fluorescence spectrometer with an excitation wavelength of 440 nm.

[0025] Experimental results are as follows Figure 7 , Figure 8 , Figure 9 As shown, when no AChE was added to the system, the fluorescence signals of compounds A1, A2 and A3 were weak; however, with the increase of AChE concentration, the fluorescence emission in the 510-560 nm range was significantly enhanced, and the fluorescence intensity was significantly positively correlated with the AChE concentration.

[0026] The results of this experiment show that the acetylcholinesterase fluorescent probe obtained in this invention exhibits excellent response performance to AChE.

[0027] Example 5: Specificity test and analysis of the AChE response of the acetylcholinesterase fluorescent probe A1 obtained in Example 1 of this invention: The stock solution of acetylcholinesterase fluorescent probe A1 was added to PBS buffer solution at pH 7.35 to make the concentration of A1 in the buffer solution 10 μM. AChE (20 U / mL), anions and cations, and amino acids (50 μM / L) were added to the buffer system, and the fluorescence intensity at 535 nm was measured by fluorescence spectroscopy.

[0028] Experimental results are as follows Figure 10 , Figure 11 As shown, when AChE is not added to the system or only various anions, cations and amino acids are added, the fluorescence signal of the buffer system is weak; when AChE is added, the fluorescence signal is greatly enhanced.

[0029] This demonstrates that the acetylcholinesterase fluorescent probe A1 obtained in this invention exhibits good specificity for the response to AChE, is minimally affected by common anions, cations, and amino acids, and can ensure the accuracy of AChE detection in complex systems.

[0030] Example 6: Kinetic analysis of the response of the acetylcholinesterase fluorescent probe A1 obtained in Example 1 of this invention to AChE: The stock solution of acetylcholinesterase fluorescent probe A1 was added to PBS buffer solution at pH 7.35 to make the concentration of A1 in the buffer solution 10 μM. Then, AChE (concentration of 20 U / mL) was added to the system, the excitation wavelength was 440 nm, and the fluorescence intensity change at 535 nm was monitored in real time.

[0031] In the experiment, a PBS buffer solution of acetylcholinesterase fluorescent probe A1 (concentration 10 μM) was first prepared, at which point the fluorescence intensity was low. Then, AChE was added, and the mixture was shaken vigorously for 2-3 seconds. The fluorescence intensity at 535 nm was measured immediately after approximately 10 seconds, and a significant increase in fluorescence intensity was observed. Subsequently, the fluorescence intensity at 535 nm was measured every 120 seconds (i.e., the fluorescence intensity at 535 nm was measured at 120s, 240s, and 360s).

[0032] The results are as follows Figure 12 As shown, the fluorescence intensity increases significantly from 0 to 10 s, and tends to stabilize from 10 to 360 s.

[0033] This demonstrates that the acetylcholinesterase fluorescent probe A1 obtained in Example 1 of this invention is sensitive and rapid in its response to AChE, and can complete the response to AChE within 10 seconds.

[0034] Example 7: Explanation of the mechanism of the acetylcholinesterase fluorescent probe A1 obtained in Example 1 of the present invention in response to AChE: Considering the extremely rapid response of the acetylcholinesterase fluorescent probe A1 to AChE, it is clearly not a cleavage-type release response. It is hypothesized that the probe embeds into AChE, interrupting PET, thus leading to enhanced luminescence. Therefore, control groups of A1 and (A1+AChE) were designed. Both groups were shaken on a shaker at 37℃ for 1.5 hours, then filtered before HPLC analysis. Since the size of (A1+AChE) is much larger than that of A1, after filtration, the A1 content in the AChE-responsive group should be lower than that in the group without AChE in the HPLC results.

[0035] like Figure 13 As shown, the A1 content in the AChE-responsive group was lower than that in the group without AChE, consistent with our prediction. Furthermore, no new peaks or significant enhancement peaks were observed in the HPLC, thus ruling out a cut-off release response.

[0036] like Figure 14 As shown, calculations using Gausssion 09W and GaussView show that the HOMO and LUMO of the donor and acceptor portions of the acetylcholinesterase fluorescent probe A1 meet the theoretical requirements of PET.

[0037] refer to Figure 15 As shown, the response mechanism of the acetylcholinesterase fluorescent probe A1 to AChE is PET (photoinduced electron transfer): the carbamate fragment in the probe structure enters the hydrophobic pocket of AChE, interrupting the original photoinduced electron transfer process, so that the fluorescence of the fluorophore can be restored.

[0038] The above description is not limited to the examples above. Technical features not described in this invention can be implemented by or using existing technology, and will not be repeated here. The above embodiments are only used to illustrate the technical solutions of this invention and are not intended to limit this invention. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of this invention do not depart from the spirit of this invention and should also fall within the scope of protection of the claims of this invention.

Claims

1. A class of acetylcholinesterase fluorescent probes based on the non-breakage mechanism, characterized in that, The fluorescent probe for acetylcholinesterase recognizes acetylcholinesterase via a non-bond-breaking mechanism, and has the structure shown in Formula I: Formula I R1 and R2 are each independently selected from hydrogen, methyl, ethyl, propyl, butyl, methylene or substituted alkyl, benzyl or substituted benzyl.

2. A method for preparing a class of acetylcholinesterase fluorescent probes based on the non-broken bond mechanism, characterized in that, The preparation process is as follows: The steps are as follows: S1. Compound M2 is prepared by reacting 4-bromo-1,8-naphthalenedicarboxylic anhydride with an amine compound in a molar ratio of 1:(1.5-1.6) in a reaction solvent and a basic catalyst; S2. Compound M2 and ethanolamine were added to the reaction solvent at a molar ratio of 1:(1.3-1.35), and the mixture was heated to reflux under nitrogen protection to prepare compound M3; S3. Dissolve compound M3 in the reaction solvent, add alkali while stirring, then add acyl chloride, and reflux under nitrogen protection. After the reaction is complete, evaporate the solvent and purify by column chromatography to obtain the corresponding target product.

3. A method of preparing a class of non-bond-cleavage mechanism based acetylcholinesterase fluorescent probes according to claim 2, characterized in that, In step S1, the amine compound is dimethylamine, diethylamine, methyl ethylamine, piperidine, dibenzylamine, or hydrochloride, hydrobromide, or hydroiodide of these amines; the molar ratio of the alkaline catalyst to 4-bromo-1,8-naphthalenedicarboxylic anhydride is (1.5-2):1, the reaction time is 12 hours, and the reaction temperature of the system is 110-124℃.

4. A method of preparing a class of non-bond-cleavage mechanism based acetylcholinesterase fluorescent probes according to claim 2, characterized in that, In steps S1 and S2, the reaction solvent is one of anhydrous ethanol, tetrahydrofuran, toluene, DMF, DMSO, and 2-methoxyethanol; in step S2, the reaction temperature of the system is 80℃ and the reaction time is 4-12 hours.

5. A method of preparing a class of non-bond-cleavage mechanism based acetylcholinesterase fluorescent probes according to claim 2, characterized in that, In steps S1 and S3, the alkaline catalyst is an inorganic salt such as carbonate or bicarbonate, or triethylamine, diisopropylethylamine, or dimethylaminopyridine.

6. A method of preparing a class of non-bond-cleavage mechanism based acetylcholinesterase fluorescent probes according to claim 2, characterized in that, In step S3, the reaction solvent is one of toluene, tetrahydrofuran, or ethylene glycol monomethyl ether; the molar ratio of compound M3 to the basic catalyst is 1:(2-5); and the molar ratio of compound M3 to acyl chloride is 1:(3-5).

7. A method of preparing a class of non-bond-cleavage mechanism based acetylcholinesterase fluorescent probes according to claim 2, characterized in that, In step S3, the reaction temperature of the system is 70-110℃, and the reaction time is 36h-72h.

8. The fluorescent probe prepared according to claim 1 or the method for preparing the fluorescent probe according to claim 2. Application of fluorescent probes in the selective recognition of acetylcholinesterase.

9. The fluorescent probe prepared according to claim 1 or the method for preparing the fluorescent probe according to claim 2. The response of the fluorescent probe to acetylcholinesterase is the mechanism of PET: The carbamate fragment in the probe structure can quench the fluorescence of the fluorophore. When it interacts with acetylcholinesterase, the carbamate fragment enters the hydrophobic pocket of the enzyme, breaking the PET of the probe structure and restoring the fluorescence of the probe.

10. The fluorescent probe prepared according to claim 1 or the method for preparing the fluorescent probe according to claim 2. Application of fluorescent probes in fluorescence imaging of acetylcholinesterase in cells or living tissues.

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