Self-amplification activation type active oxygen probe as well as preparation method and application thereof

Abstract

The invention discloses a self-amplification activation type active oxygen probe as well as a preparation method and application thereof. The structural formula of the active oxygen probe is as shown in formula I in the specification. The active oxygen probe has great application potential in the aspects of disease early warning and diagnosis, accurate monitoring of biomarkers and the like. Particularly in early disease diagnosis, high-sensitivity detection is carried out on low-level active oxygen, and the diagnosis accuracy can be greatly improved, so that earlier treatment intervention is realized, and the cure rate of a patient is improved; and # imgabs0 #.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a self-amplifying activated active oxygen probe and a preparation method thereof. Background Art

[0002] Bioactive small molecules such as reactive oxygen, reactive nitrogen, and reactive sulfur are closely related to the life activities of cells and living bodies, and play a very important role in maintaining the normal life activities of organisms and the occurrence of various diseases. Reactive oxygen is a byproduct of biological aerobic metabolism and is a general term for a class of oxygen-containing and active substances. Its content is usually related to tumors, inflammatory diseases, and immune diseases. Studies have shown that reactive oxygen and nitrogen substances play a very important role in the physiological and pathological processes of life systems. Among them, reactive oxygen, as a chemical substance with extremely strong biological activity, can have toxic effects on biological molecules, and is therefore considered to be the culprit for various diseases such as cardiovascular disease, neurodegenerative diseases, and even cancer.

[0003] In recent years, the development of fluorescent probes has been particularly rapid. With its advantages of visualization, real-time monitoring, good sensitivity and easy operation, it has played a vital role in the early diagnosis of clinical diseases. Currently, the fluorescent probes studied can only activate one fluorescent group per molecule of stimulation signal. This means that when the concentration of reactive oxygen is low, the fluorescent probe cannot detect the change in content in time, resulting in delayed treatment.

[0004] Therefore, designing and synthesizing new fluorescent probes for efficient detection of reactive oxygen species in vivo is a task of great significance. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the present invention provides a self-amplifying activated reactive oxygen species probe and its preparation method and application. The present invention realizes a one-to-many response mode by introducing a self-degradable linker, and is expected to improve the detection ability of the reactive oxygen species probe. It has great application potential in disease early warning diagnosis, accurate monitoring of biomarkers, etc.

[0006] The purpose of the present invention is achieved by at least one of the following technical solutions: In a first aspect, the present invention provides a self-amplifying activated reactive oxygen species probe, the structural formula of which is shown in Formula I: .

[0007] In a second aspect, the present invention provides a method for preparing the self-amplifying activated reactive oxygen species probe, comprising the following steps: 2,6-bis(hydroxymethyl)-m-cresol and tert-butyldimethylchlorosilane were mixed for substitution reaction to obtain compound 2; Using the compound 2 and 4-bromomethylphenylboronic acid pinacol ester as raw materials, a substitution reaction is carried out to obtain compound 3; Using the compound 3 and tetrabutylammonium fluoride as raw materials, a nucleophilic substitution reaction is carried out to obtain compound 4; Mix isophorone and malononitrile to carry out Knoevenagel condensation reaction to obtain compound 1; Using compound 1 and 3-chloro-4-hydroxybenzaldehyde as raw materials, a substitution reaction was carried out to obtain intermediate 1; The intermediate 1 is mixed with p-nitrophenyl chloroformate to carry out a substitution reaction to obtain compound 5; Using the compound 4 and the compound 5 as raw materials for a substitution reaction, a compound represented by formula I is obtained; The synthetic route is: .

[0008] As a preferred embodiment of the present invention, the molar ratio of 2,6-bis(hydroxymethyl)-m-cresol to tert-butyldimethylchlorosilane is 1:2-2.2; The molar ratio of the compound 2 to 4-bromomethylphenylboronic acid pinacol ester is 1:1-1.1; The molar ratio of the compound 3 to tetrabutylammonium fluoride is 1:5-10; The molar ratio of isophorone to malononitrile is 1:1-1.2; The molar ratio of the compound 1 to 3-chloro-4-hydroxybenzaldehyde is 1:1-1.2; The molar ratio of the intermediate 1 to p-nitrophenyl chloroformate is 1:2-6; The molar ratio of compound 4 to compound 5 is 1:2~2.2.

[0009] As a preferred embodiment of the present invention, the reaction of 2,6-bis(hydroxymethyl)-m-cresol and tert-butyldimethylchlorosilane is stirred at room temperature for 4h to 6h; The reaction of the compound 2 and 4-bromomethylphenylboronic acid pinacol ester is carried out by stirring at 85° C. to 95° C. for 8 h to 10 h; The reaction of compound 3 and tetrabutylammonium fluoride is stirred at room temperature for 8 h to 10 h; The reaction of isophorone and malononitrile is carried out by stirring at 80° C. to 85° C. for 8 h to 10 h; The reaction of the compound 1 and 3-chloro-4-hydroxybenzaldehyde is carried out by stirring at 80° C. to 85° C. for 6 h to 8 h; The reaction of the intermediate 1 with p-nitrophenyl chloroformate is carried out by stirring at room temperature in the dark for 6 h to 8 h; The reaction of compound 4 and compound 5 is carried out by stirring at room temperature for 7 h to 10 h.

[0010] As a preferred embodiment of the present invention, the reaction product of 2,6-bis(hydroxymethyl)-m-cresol and tert-butyldimethylchlorosilane is diluted with dichloromethane, washed with water, the organic layer is dried with magnesium sulfate, and the solvent is removed under reduced pressure, and the obtained crude product is purified by column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 20:1 as an eluent to obtain the compound 2; The reaction product of the compound 2 and 4-bromomethylphenylboronic acid pinacol ester is purified by column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 20:1 as an eluent to obtain the compound 3; The reaction product of the compound 3 and tetrabutylammonium fluoride was diluted with dichloromethane and washed with water. The organic layer was dried with magnesium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography using a mixture of n-hexane and ethyl acetate in a volume ratio of 2:1 as an eluent to obtain the compound 4. The reaction product of isophorone and malononitrile is purified by column chromatography using a mixture of petroleum ether and dichloromethane in a volume ratio of 1:5 as an eluent to obtain the compound 1; The reaction product of the compound 1 and 3-chloro-4-hydroxybenzaldehyde is purified by column chromatography using a mixture of petroleum ether and dichloromethane in a volume ratio of 1:1 as an eluent to obtain the intermediate 1; The reaction product of the intermediate 1 and p-nitrophenyl chloroformate was purified by column chromatography using a mixture of dichloromethane and n-hexane in a volume ratio of 4:1 as an eluent to obtain the compound 5; The reaction product of the compound 4 and the compound 5 is purified by column chromatography using a mixture of n-hexane and ethyl acetate in a volume ratio of 3:1 as an eluent to obtain the self-amplifying activated reactive oxygen probe.

[0011] In a third aspect, the present invention provides a use of the self-amplifying activated reactive oxygen species probe in the preparation of a fluorescent probe.

[0012] As a preferred embodiment of the present invention, the self-amplifying activated reactive oxygen species probe is used to amplify low-level reactive oxygen species signals.

[0013] As a preferred embodiment of the present invention, the self-amplifying activated reactive oxygen species probe is used to detect the reactive oxygen species content.

[0014] In a fourth aspect, the present invention provides an application of the self-amplifying activated reactive oxygen species probe in the preparation of disease diagnosis-related products, wherein the disease is a disease associated with abnormal changes in reactive oxygen species, such as cancer, cardiovascular disease, diabetes, Parkinson's disease, and Alzheimer's disease.

[0015] In a fifth aspect, the present invention provides a use of the self-amplifying activated reactive oxygen species probe in the preparation of a product for monitoring a biomarker, wherein the biomarker is a biomarker associated with a disease caused by abnormal changes in reactive oxygen species.

[0016] Compared with the prior art, the beneficial effects of the present invention are: The self-amplifying activated reactive oxygen probe prepared by using the compound provided by the present invention has the following characteristics: (1) The present invention uses 2,6-bis(hydroxymethyl)-m-cresol as a self-degradable linker, connected to a phenylboronic acid ester group that responds to active oxygen, and then modifies the hydroxyl group with p-nitrophenyl chloroformate, and connects two fluorescent groups on both sides. Under stimulation, the response group on the self-degradable linker is triggered to spontaneously cause two 1,4-elimination reactions, releasing the fluorescent groups on both sides, realizing a one-to-many response mode, thereby improving the detection ability of the active oxygen probe.

[0017] (2) In view of the limitation that common fluorescent probes cannot effectively diagnose early-stage diseases, the present invention designs a reactive oxygen fluorescent probe that has the ability to amplify signals of low-level reactive oxygen species. The probe can be activated by intracellular reactive oxygen species and detect its own reactive oxygen species level through its own fluorescence recovery. The reactive oxygen probe has great potential for application in disease early warning diagnosis, accurate monitoring of biomarkers, etc. In particular, in the diagnosis of early-stage diseases, the high-sensitivity detection of low-level reactive oxygen species can greatly improve the accuracy of diagnosis, thereby achieving earlier treatment intervention and improving the cure rate of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of NFP-OH.

[0019] Figure 2 This is the nuclear magnetic resonance hydrogen spectrum of the active oxygen probe NFP-R.

[0020] Figure 3 Absorption spectrum and fluorescence spectrum of NFP-OH.

[0021] Figure 4 This is the spectrum diagram of the fluorescence intensity change after NFP-R was incubated with different concentrations of H2O2.

[0022] Figure 5 This is a curve graph showing the change of NFP-R fluorescence intensity with H2O2 concentration.

[0023] Figure 6 is the fluorescence intensity of NFP-R after incubation with different interfering ions.

[0024] Figure 7 This is the H NMR spectrum of the self-amplified activated reactive oxygen species probe NFP-DR.

[0025] Figure 8 This is the spectrum diagram of the fluorescence intensity change after NFP-DR was incubated with different concentrations of H2O2.

[0026] Fig. 9 This is a curve showing the change of NFP-DR fluorescence intensity with H2O2 concentration.

[0027] Fig.10 is the fluorescence intensity of NFP-DR after incubation with different interfering ions.

[0028] Fig.11 The cytotoxicity of NFP-R and NFP-DR to 4T1 cells.

[0029] Fig.12 The cytotoxicity of NFP-R and NFP-DR to MEF cells.

[0030] Fig.13 The cytotoxicity of NFP-R and NFP-DR to HK-2 cells.

[0031] Fig.14 Laser confocal fluorescence observation of NFP-R and NFP-DR in 4T1 cells (A) and flow cytometry quantitative analysis (B).

[0032] Fig.15 Laser confocal fluorescence observation of NFP-R and NFP-DR in MCF-7 cells (A) and flow cytometry quantitative analysis (B).

[0033] Fig.16 Laser confocal fluorescence images of NFP-DR in 4T1 cells and MEF cells (A) and flow cytometry quantitative analysis (B).

[0034] Fig.17 Laser confocal observation of the fluorescence images of NFP-R and NFP-DR in acute kidney injury cells (A) and quantitative analysis of the fluorescence signal intensity (B). DETAILED DESCRIPTION

[0035] The specific implementation of the present invention is further described below in conjunction with examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0036] Example 1 A self-amplifying activated reactive oxygen species probe, labeled as NFP-DR, has the following structural formula: .

[0037] A method for preparing a self-amplifying activated reactive oxygen species probe comprises the following steps: 1) Take 1.4680g of 2,6-bis(hydroxymethyl)-m-cresol and 1.3070g of imidazole and dissolve them in 15mL of N,N-dimethylformamide, and place them in an ice bath. Then take 2.8020g of tert-butyldimethylsilyl chloride and dissolve it in 5mL of DMF and add it dropwise into the solution. The mixed solution is stirred at room temperature for 6h. Pour the mixture into a separatory funnel, add 20mL of dichloromethane to dilute it and wash it with pure water. After drying over anhydrous magnesium sulfate, distill under reduced pressure to obtain a crude product. The crude product is purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 20:1 to obtain compound 2, which is a colorless and transparent oil.

[0038] 2) 1.5070g of compound 2 and 0.6290g of K2CO3 were dissolved in 15mL of acetonitrile solution and placed in an ice bath, and then 1.1283g of 4-bromomethylphenylboronic acid pinacol ester was dissolved in 5mL of acetonitrile solution and added dropwise to the solution. The mixed solution was stirred and refluxed at 85°C for 8h under a nitrogen atmosphere, and the mixture was cooled to room temperature and evaporated under reduced pressure. The crude product was purified by column chromatography using petroleum ether: ethyl acetate = 20:1 as the eluent to obtain compound 3, which was a colorless transparent oil.

[0039] 3) Weigh 0.1560g of compound 3 and dissolve it in 20mL of tetrahydrofuran solution, add 0.6656g of tetrabutylammonium fluoride dropwise into the solution, and stir the mixed solution at room temperature for 8h. After the reaction is completed, the mixed solution is diluted with 20mL of dichloromethane and washed with pure water, dried over anhydrous magnesium sulfate, and distilled under reduced pressure to obtain a crude product. The crude product is purified by column chromatography using n-hexane: ethyl acetate = 2:1 as the eluent to obtain compound 4 as an oil.

[0040] 4) Dissolve 0.0691 g of isophorone, 0.0396 g of malononitrile and 10 μL of glacial acetic acid in 15 mL of ethanol, and then add 2 drops of piperidine. The mixed solution is stirred and refluxed at 80°C for 8 h under a nitrogen atmosphere. The mixture is cooled to room temperature and evaporated under reduced pressure. The crude product is purified by column chromatography using petroleum ether: dichloromethane = 1:5 as the eluent to obtain a light yellow solid compound 1.

[0041] 5) A mixture of 0.3720 g of compound 1 and 0.3130 g of 3-chloro-4-hydroxybenzaldehyde was dissolved in 20 mL of ethanol and stirred at 85°C for 6 h. The mixture was then evaporated under reduced pressure. The residue was purified by column chromatography using petroleum ether: dichloromethane = 1:1 as the eluent to obtain an orange solid intermediate 1, which was labeled as NFP-OH.

[0042] 6) Take 0.3248g of NFP-OH and dissolve it in 20mL of dichloromethane and place it in an ice bath, then drop 1.2090g of p-nitrophenyl chloroformate, then drop 1.0340g of N,N-diisopropylethylamine and 0.0395g of pyridine, and stir at room temperature in the dark for 8h. After the mixture is distilled under reduced pressure, the crude product is purified by column chromatography using dichloromethane:n-hexane = 4:1 as the eluent to obtain compound 5.

[0043] 7) 0.0263 g of compound 4 and 0.0354 g of N,N-diisopropylethylamine were dissolved in 20 mL of dichloromethane and placed in an ice bath, and 0.0671 g of compound 5 was added dropwise, and stirred at room temperature for 8 h. After the mixture was distilled under reduced pressure, the crude product was purified by column chromatography using n-hexane: ethyl acetate = 3:1 as the eluent to obtain yellow solid NFP-DR.

[0044] The synthetic route is as follows: .

[0045] Example 2 A self-amplifying activated reactive oxygen species probe, labeled as NFP-DR, has the same structural formula as in Example 1, and a specific preparation method comprises the following steps: 1) Take 1.4680g of 2,6-bis(hydroxymethyl)-m-cresol and 1.3070g of imidazole and dissolve them in 15mL of N,N-dimethylformamide, and place them in an ice bath. Then take 2.6310g of tert-butyldimethylsilyl chloride and dissolve it in 5mL of DMF and add it dropwise into the solution. The mixed solution is stirred at room temperature for 6h. Pour the mixture into a separatory funnel, add 20mL of dichloromethane to dilute it and wash it with pure water. After drying over anhydrous magnesium sulfate, distill under reduced pressure to obtain a crude product. The crude product is purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 20:1 to obtain compound 2, which is a colorless and transparent oil.

[0046] 2) 1.5070g of compound 2 and 0.6290g of K2CO3 were dissolved in 15mL of acetonitrile solution and placed in an ice bath. Then 1.2400g of 4-bromomethylphenylboronic acid pinacol ester was dissolved in 5mL of acetonitrile solution and added dropwise to the solution. The mixed solution was stirred and refluxed at 85°C for 8h under a nitrogen atmosphere. The mixture was cooled to room temperature and evaporated under reduced pressure. The crude product was purified by column chromatography using petroleum ether: ethyl acetate = 20:1 as the eluent to obtain compound 3, which was a colorless transparent oil.

[0047] 3) Weigh 0.156g of compound 3 and dissolve it in 20mL of tetrahydrofuran solution, add 0.3328g of tetrabutylammonium fluoride dropwise into the solution, and stir the mixed solution at room temperature for 8h. After the reaction is completed, the mixed solution is diluted with 20mL of dichloromethane and washed with pure water, dried over anhydrous magnesium sulfate, and distilled under reduced pressure to obtain a crude product. The crude product is purified by column chromatography using n-hexane: ethyl acetate = 2:1 as the eluent to obtain compound 4 as an oily substance.

[0048] 4) Dissolve 0.0691 g of isophorone, 0.0330 g of malononitrile and 10 μL of glacial acetic acid in 15 mL of ethanol, and then add 2 drops of piperidine. The mixed solution is stirred and refluxed at 80°C for 8 h under a nitrogen atmosphere. The mixture is cooled to room temperature and evaporated under reduced pressure. The crude product is purified by column chromatography using petroleum ether: dichloromethane = 1:5 as the eluent to obtain a light yellow solid compound 1.

[0049] 5) A mixture of 0.3720 g of compound 1 and 0.3750 g of 3-chloro-4-hydroxybenzaldehyde was dissolved in 20 mL of ethanol and stirred at 85°C for 6 h. The mixture was then evaporated under reduced pressure. The residue was purified by column chromatography using petroleum ether: dichloromethane = 1:1 as the eluent to obtain an orange solid intermediate 1, which was labeled as NFP-OH.

[0050] 6) 0.3248 g of intermediate 1 was dissolved in 20 mL of dichloromethane and placed in an ice bath, 0.4031 g of p-nitrophenyl chloroformate was added dropwise, and then 1.034 g of N,N-diisopropylethylamine and 0.0395 g of pyridine were added dropwise, and stirred at room temperature in the dark for 8 h. The mixture was distilled under reduced pressure, and the crude product was purified by column chromatography using dichloromethane:n-hexane = 4:1 as the eluent to obtain compound 5.

[0051] 7) 0.0263 g of compound 4 and 0.0354 g of N,N-diisopropylethylamine were dissolved in 20 mL of dichloromethane and placed in an ice bath, and 0.0737 g of compound 5 was added dropwise, and stirred at room temperature for 8 h. After the mixture was distilled under reduced pressure, the crude product was purified by column chromatography using n-hexane: ethyl acetate = 3:1 as the eluent to obtain yellow solid NFP-DR.

[0052] Example 3 A self-amplifying activated reactive oxygen species probe, labeled as NFP-DR, has the same structural formula as in Example 1, and a specific preparation method comprises the following steps: 1) Take 1.4680g of 2,6-bis(hydroxymethyl)-m-cresol and 1.3070g of imidazole and dissolve them in 15mL of N,N-dimethylformamide, and place them in an ice bath. Then take 2.8940g of tert-butyldimethylsilyl chloride and dissolve it in 5mL of DMF and add it dropwise into the solution. The mixed solution is stirred at room temperature for 6h. Pour the mixture into a separatory funnel, add 20mL of dichloromethane to dilute it and wash it with pure water. After drying over anhydrous magnesium sulfate, distill under reduced pressure to obtain a crude product. The crude product is purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 20:1 to obtain compound 2, which is a colorless and transparent oil.

[0053] 2) 1.5070g of compound 2 and 0.6290g of K2CO3 were dissolved in 15mL of acetonitrile solution and placed in an ice bath. Then 1.2045g of 4-bromomethylphenylboronic acid pinacol ester was dissolved in 5mL of acetonitrile solution and added dropwise to the solution. The mixed solution was stirred and refluxed at 85°C for 8h under a nitrogen atmosphere. The mixture was cooled to room temperature and evaporated under reduced pressure. The crude product was purified by column chromatography using petroleum ether: ethyl acetate = 20:1 as the eluent to obtain compound 3, which was a colorless transparent oil.

[0054] 3) Weigh 0.156g of compound 3 and dissolve it in 20mL of tetrahydrofuran solution, add 0.5218g of tetrabutylammonium fluoride dropwise into the solution, and stir the mixed solution at room temperature for 8h. After the reaction is completed, the mixed solution is diluted with 20mL of dichloromethane and washed with pure water, dried over anhydrous magnesium sulfate, and distilled under reduced pressure to obtain a crude product. The crude product is purified by column chromatography using n-hexane: ethyl acetate = 2:1 as the eluent to obtain compound 4 as an oil.

[0055] 4) Dissolve 0.0691 g of isophorone, 0.0358 g of malononitrile and 10 μL of glacial acetic acid in 15 mL of ethanol, and then add 2 drops of piperidine. The mixed solution is stirred and refluxed at 80°C for 8 h under a nitrogen atmosphere. The mixture is cooled to room temperature and evaporated under reduced pressure. The crude product is purified by column chromatography using petroleum ether: dichloromethane = 1:5 as the eluent to obtain a light yellow solid compound 1.

[0056] 5) A mixture of 0.3720 g of compound 1 and 0.3490 g of 3-chloro-4-hydroxybenzaldehyde was dissolved in 20 mL of ethanol and stirred at 85°C for 6 h. The mixture was then evaporated under reduced pressure. The residue was purified by column chromatography using petroleum ether: dichloromethane = 1:1 as the eluent to obtain an orange solid intermediate 1, which was labeled as NFP-OH.

[0057] 6) 0.3248 g of intermediate 1 was dissolved in 20 mL of dichloromethane and placed in an ice bath, 0.8672 g of p-nitrophenyl chloroformate was added dropwise, and then 1.034 g of N,N-diisopropylethylamine and 0.0395 g of pyridine were added dropwise, and stirred at room temperature in the dark for 8 h. The mixture was distilled under reduced pressure, and the crude product was purified by column chromatography using dichloromethane:n-hexane = 4:1 as the eluent to obtain compound 5.

[0058] 7) 0.0263 g of compound 4 and 0.0354 g of N,N-diisopropylethylamine were dissolved in 20 mL of dichloromethane and placed in an ice bath, and 0.0704 g of compound 5 was added dropwise, and stirred at room temperature for 8 h. After the mixture was distilled under reduced pressure, the crude product was purified by column chromatography using n-hexane: ethyl acetate = 3:1 as the eluent to obtain yellow solid NFP-DR.

[0059] Example 4 A self-amplifying activated reactive oxygen species probe, labeled as NFP-DR, has the same structural formula as in Example 1, and a specific preparation method comprises the following steps: 1) Take 1.4680g of 2,6-bis(hydroxymethyl)-m-cresol and 1.3070g of imidazole and dissolve them in 15mL of N,N-dimethylformamide, and place them in an ice bath. Then take 2.8020g of tert-butyldimethylsilyl chloride and dissolve it in 5mL of DMF and add it dropwise into the solution. The mixed solution is stirred at room temperature for 4h. Pour the mixture into a separatory funnel, add 20mL of dichloromethane to dilute it and wash it with pure water. After drying over anhydrous magnesium sulfate, distill under reduced pressure to obtain a crude product. The crude product is purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 20:1 to obtain compound 2, which is a colorless and transparent oil.

[0060] 2) 1.5070g of compound 2 and 0.6290g of K2CO3 were dissolved in 15mL of acetonitrile solution and placed in an ice bath, and then 1.1285g of 4-bromomethylphenylboronic acid pinacol ester was dissolved in 5mL of acetonitrile solution and added dropwise to the solution. The mixed solution was stirred and refluxed at 95°C for 8h under a nitrogen atmosphere, and the mixture was cooled to room temperature and evaporated under reduced pressure. The crude product was purified by column chromatography using petroleum ether: ethyl acetate = 20:1 as the eluent to obtain compound 3, which was a colorless transparent oil.

[0061] 3) Weigh 0.1560g of compound 3 and dissolve it in 20mL of tetrahydrofuran solution, add 0.6640g of tetrabutylammonium fluoride dropwise into the solution, and stir the mixed solution at room temperature for 10h. After the reaction is completed, the mixed solution is diluted with 20mL of dichloromethane and washed with pure water, dried over anhydrous magnesium sulfate, and distilled under reduced pressure to obtain a crude product. The crude product is purified by column chromatography using n-hexane: ethyl acetate = 2:1 as the eluent to obtain compound 4 as an oil.

[0062] 4) Dissolve 0.0691 g of isophorone, 0.0391 g of malononitrile and 10 μL of glacial acetic acid in 15 mL of ethanol, and then add 2 drops of piperidine. The mixed solution is stirred and refluxed at 80°C for 8 h under a nitrogen atmosphere. The mixture is cooled to room temperature and evaporated under reduced pressure. The crude product is purified by column chromatography using petroleum ether: dichloromethane = 1:5 as the eluent to obtain a light yellow solid compound 1.

[0063] 5) A mixture of 0.3720 g of compound 1 and 0.3130 g of 3-chloro-4-hydroxybenzaldehyde was dissolved in 20 mL of ethanol and stirred at 80°C for 8 h. The mixture was then evaporated under reduced pressure. The residue was purified by column chromatography using petroleum ether: dichloromethane = 1:1 as the eluent to obtain an orange solid intermediate 1, which was labeled as NFP-OH.

[0064] 6) Take 0.3248g of NFP-OH and dissolve it in 20mL of dichloromethane and place it in an ice bath, then drop 1.209g of p-nitrophenyl chloroformate, then drop 1.034g of N,N-diisopropylethylamine and 0.0395g of pyridine, and stir at room temperature in the dark for 6h. After the mixture is distilled under reduced pressure, the crude product is purified by column chromatography using dichloromethane:n-hexane = 4:1 as the eluent to obtain compound 5.

[0065] 7) 0.0263 g of compound 4 and 0.0354 g of N,N-diisopropylethylamine were dissolved in 20 mL of dichloromethane and placed in an ice bath, and 0.0705 g of compound 5 was added dropwise, and stirred at room temperature for 10 h. After the mixture was distilled under reduced pressure, the crude product was purified by column chromatography using n-hexane: ethyl acetate = 3:1 as the eluent to obtain yellow solid NFP-DR.

[0066] Example 5 A self-amplifying activated reactive oxygen species probe, labeled as NFP-DR, has the same structural formula as in Example 1, and a specific preparation method comprises the following steps: 1) Take 1.4680g of 2,6-bis(hydroxymethyl)-m-cresol and 1.3070g of imidazole and dissolve them in 15mL of N,N-dimethylformamide, and place them in an ice bath. Then take 2.8020g of tert-butyldimethylsilyl chloride and dissolve it in 5mL of DMF and add it dropwise into the solution. The mixed solution is stirred at room temperature for 5h. Pour the mixture into a separatory funnel, add 20mL of dichloromethane to dilute it and wash it with pure water. After drying over anhydrous magnesium sulfate, distill under reduced pressure to obtain a crude product. The crude product is purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 20:1 to obtain compound 2, which is a colorless and transparent oil.

[0067] 2) 1.5070g of compound 2 and 0.6290g of K2CO3 were dissolved in 15mL of acetonitrile solution and placed in an ice bath, and then 1.1285g of 4-bromomethylphenylboronic acid pinacol ester was dissolved in 5mL of acetonitrile solution and added dropwise to the solution. The mixed solution was stirred and refluxed at 85°C for 9h under a nitrogen atmosphere, and the mixture was cooled to room temperature and evaporated under reduced pressure. The crude product was purified by column chromatography using petroleum ether: ethyl acetate = 20:1 as the eluent to obtain compound 3, which was a colorless transparent oil.

[0068] 3) Weigh 0.1560g of compound 3 and dissolve it in 20mL of tetrahydrofuran solution, add 0.6640g of tetrabutylammonium fluoride dropwise into the solution, and stir the mixed solution at room temperature for 10h. After the reaction is completed, the mixed solution is diluted with 20mL of dichloromethane and washed with pure water, dried over anhydrous magnesium sulfate, and distilled under reduced pressure to obtain a crude product. The crude product is purified by column chromatography using n-hexane: ethyl acetate = 2:1 as the eluent to obtain compound 4 as an oil.

[0069] 4) Dissolve 0.0691 g of isophorone, 0.0395 g of malononitrile and 10 μL of glacial acetic acid in 15 mL of ethanol, and then add 2 drops of piperidine. The mixed solution is stirred and refluxed at 85°C for 8 h under a nitrogen atmosphere. The mixture is cooled to room temperature and evaporated under reduced pressure. The crude product is purified by column chromatography using petroleum ether: dichloromethane = 1:5 as the eluent to obtain a light yellow solid compound 1.

[0070] 5) A mixture of 0.3720 g of compound 1 and 0.3130 g of 3-chloro-4-hydroxybenzaldehyde was dissolved in 20 mL of ethanol and stirred at 80°C for 6 h. The mixture was then evaporated under reduced pressure. The residue was purified by column chromatography using petroleum ether: dichloromethane = 1:1 as the eluent to obtain an orange solid intermediate 1, which was labeled as NFP-OH.

[0071] 6) Take 0.3248g of NFP-OH and dissolve it in 20mL of dichloromethane and place it in an ice bath, then drop 1.209g of p-nitrophenyl chloroformate, then drop 1.0340g of N,N-diisopropylethylamine and 0.0395g of pyridine, and stir at room temperature in the dark for 8h. After the mixture is distilled under reduced pressure, the crude product is purified by column chromatography using dichloromethane:n-hexane = 4:1 as the eluent to obtain compound 5.

[0072] 7) 0.0263 g of compound 4 and 0.0354 g of N,N-diisopropylethylamine were dissolved in 20 mL of dichloromethane and placed in an ice bath, and 0.0705 g of compound 5 was added dropwise, and stirred at room temperature for 7 h. After the mixture was distilled under reduced pressure, the crude product was purified by column chromatography using n-hexane: ethyl acetate = 3:1 as the eluent to obtain yellow solid NFP-DR.

[0073] Example 6 A self-amplifying activated reactive oxygen species probe, labeled as NFP-DR, has the same structural formula as in Example 1, and a specific preparation method comprises the following steps: 1) Take 1.4680g of 2,6-bis(hydroxymethyl)-m-cresol and 1.3070g of imidazole and dissolve them in 15mL of N,N-dimethylformamide, and place them in an ice bath. Then take 2.8020g of tert-butyldimethylsilyl chloride and dissolve it in 5mL of DMF and add it dropwise into the solution. The mixed solution is stirred at room temperature for 4h. Pour the mixture into a separatory funnel, add 20mL of dichloromethane to dilute it and wash it with pure water. After drying over anhydrous magnesium sulfate, distill under reduced pressure to obtain a crude product. The crude product is purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 20:1 to obtain compound 2, which is a colorless and transparent oil.

[0074] 2) 1.5070g of compound 2 and 0.6290g of K2CO3 were dissolved in 15mL of acetonitrile solution and placed in an ice bath, and then 1.1286g of 4-bromomethylphenylboronic acid pinacol ester was dissolved in 5mL of acetonitrile solution and added dropwise to the solution. The mixed solution was stirred and refluxed at 90°C for 10h under a nitrogen atmosphere, and the mixture was cooled to room temperature and evaporated under reduced pressure. The crude product was purified by column chromatography using petroleum ether: ethyl acetate = 20:1 as the eluent to obtain compound 3, which was a colorless transparent oil.

[0075] 3) Weigh 0.1560g of compound 3 and dissolve it in 20mL of tetrahydrofuran solution, add 0.6640g of tetrabutylammonium fluoride dropwise into the solution, and stir the mixed solution at room temperature for 8h. After the reaction is completed, the mixed solution is diluted with 20mL of dichloromethane and washed with pure water, dried over anhydrous magnesium sulfate, and distilled under reduced pressure to obtain a crude product. The crude product is purified by column chromatography using n-hexane: ethyl acetate = 2:1 as the eluent to obtain compound 4 as an oil.

[0076] 4) Dissolve 0.0691 g of isophorone, 0.0395 g of malononitrile and 10 μL of glacial acetic acid in 15 mL of ethanol, and then add 2 drops of piperidine. The mixed solution is stirred and refluxed for 10 h under a nitrogen atmosphere. The mixture is cooled to room temperature and evaporated under reduced pressure. The crude product is purified by column chromatography using petroleum ether: dichloromethane = 1:5 as the eluent to obtain a light yellow solid compound 1.

[0077] 5) A mixture of 0.3720 g of compound 1 and 0.3130 g of 3-chloro-4-hydroxybenzaldehyde was dissolved in 20 mL of ethanol and stirred at 85°C for 8 h. The mixture was then evaporated under reduced pressure. The residue was purified by column chromatography using petroleum ether: dichloromethane = 1:1 as the eluent to obtain an orange solid intermediate 1, which was labeled as NFP-OH.

[0078] 6) Take 0.3248g of NFP-OH and dissolve it in 20mL of dichloromethane and place it in an ice bath, then drop 1.2090g of p-nitrophenyl chloroformate, then drop 1.0340g of N,N-diisopropylethylamine and 0.0395g of pyridine, and stir at room temperature in the dark for 7h. After the mixture is distilled under reduced pressure, the crude product is purified by column chromatography using dichloromethane:n-hexane = 4:1 as the eluent to obtain compound 5.

[0079] 7) 0.0263 g of compound 4 and 0.0354 g of N,N-diisopropylethylamine were dissolved in 20 mL of dichloromethane and placed in an ice bath, and 0.0705 g of compound 5 was added dropwise, and stirred at room temperature for 8 h. After the mixture was distilled under reduced pressure, the crude product was purified by column chromatography using n-hexane: ethyl acetate = 3:1 as the eluent to obtain yellow solid NFP-DR.

[0080] Since the self-amplified activated reactive oxygen species probes provided in Examples 1 to 6 have similar performance and effects, the present invention only takes Example 1 as an example to illustrate its performance and effects: 1) Characterization of NFP-DR: 1. NMR hydrogen spectrum of NFP-DR 1 HNMR Figure 7 shown.

[0081] Depend on Figure 7 It can be seen that NFP-DR was successfully prepared.

[0082] 2. Determination of the active oxygen detection ability of NFP-DR: The probe mother solution prepared above was diluted into a solution with a concentration of 10 μM using a mixed solvent of DMSO:PBS=1:9, pH=7.4. A 1mL to 2mL centrifuge tube was taken and 0μM~200μM hydrogen peroxide solution was added to each tube and mixed evenly. After incubation for 1 hour, the fluorescence spectra of the solutions after adding different concentrations of H2O2 were observed by a fluorescence spectrophotometer. The excitation wavelength was 415nm. The test results are as follows: Figure 8 , Fig. 9 shown.

[0083] Depend on Figure 8 , Fig. 9 It can be seen that NFP-DR can respond to H2O2 stimulation and achieve fluorescence recovery, indicating that it has the potential to detect reactive oxygen species. Comparing the results of NFP-R, it can be found that at lower hydrogen peroxide concentrations, the fluorescence of NFP-DR can be restored, and at high concentrations, the fluorescence recovery of NFP-DR is stronger. The above results show that NFP-DR has a stronger ability to detect reactive oxygen species.

[0084] 5. Determination of fluorescent probe selectivity Prepare a 10μM probe solution with a mixed solvent of DMSO:PBS=1:9, pH=7.4. Take 1mL to 2mL centrifuge tubes, add a certain concentration of interfering ion solutions composed of anions, cations, amino acids, proteins and other substances, mix well, incubate for 1 hour, and observe the changes in the fluorescence spectrum of the solution after adding different ions using a fluorescence spectrophotometer. The excitation wavelength is 415nm. The test results are as follows: Fig.10 shown.

[0085] Depend on Fig.10 It can be seen that when different anions, cations, amino acids, proteins and other substances are added to the PBS solution of 10 μM probe NFP-DR, only reactive oxygen species has a significant enhancement effect on the fluorescence signal, indicating that NFP-DR can achieve highly specific recognition of reactive oxygen species in complex biological systems.

[0086] Comparative Example A compound without the introduction of the self-degradable linker 2,6-bis(hydroxymethyl)-m-cresol was used as a comparative example. The compound was prepared according to the following steps: 6) Dissolve 0.0691 g of isophorone, 0.0396 g of malononitrile and 10 μL of glacial acetic acid in 15 mL of ethanol, and add 2 drops of piperidine. The mixed solution is stirred and refluxed at 80°C for 8 h under a nitrogen atmosphere. The mixture is cooled to room temperature and evaporated under reduced pressure. The crude product is purified by column chromatography using petroleum ether: dichloromethane = 1:5 as the eluent to obtain a light yellow solid compound 1.

[0087] 2) A mixture of 0.3720 g of compound 1 and 0.3130 g of 3-chloro-4-hydroxybenzaldehyde was dissolved in 20 mL of ethanol and stirred under reflux for 6 h. The mixture was then evaporated under reduced pressure. The residue was purified by column chromatography using petroleum ether: dichloromethane = 1:1 as the eluent to obtain an orange solid intermediate 1.

[0088] 3) Dissolve 0.1018g of intermediate 1 and 0.0522g of K2CO3 in 15mL of acetonitrile solution and place in an ice bath. Then, dissolve 0.0960g of 4-bromomethylphenylboronic acid pinacol ester in 5mL of acetonitrile solution and add dropwise to the solution. The mixed solution is stirred and refluxed at 90°C for 8h under a nitrogen atmosphere. The mixture is cooled to room temperature and evaporated and concentrated under reduced pressure. The crude product is purified by column chromatography using n-hexane:ethyl acetate = 2:1 as the eluent to obtain an orange solid NFP-R, whose structural formula is as follows:

[0089] The synthetic route is as follows: .

[0090] Performance Testing 1) Characterization of NFP-R: 1. NMR hydrogen spectrum of NFP-OH 1 HNMR Figure 1 As shown, the H NMR spectrum of NFP-R 1 HNMR Figure 2 shown.

[0091] Depend on Figure 2 It can be seen that NFP-R was successfully prepared.

[0092] 2. The absorption and emission spectra of NFP-OH are as follows: Figure 3 As shown. Figure 3 It can be seen that the maximum excitation wavelength of NFP-OH is 415 nm, and the maximum emission wavelength is 660 nm.

[0093] 3. Determination of the reactive oxygen species detection capability of NFP-R The prepared 10mM stock solution was diluted to a solution with a concentration of 10μM. The solvent used for dilution was a mixed solvent of DMSO:PBS=1:9, pH=7.4. Take 1mL to 2mL centrifuge tubes, add 0μM~200μM hydrogen peroxide solution respectively, mix well, incubate for 1 hour, and observe the changes in the fluorescence spectrum of the solution after adding different concentrations of H2O2 by fluorescence spectrophotometer. The excitation wavelength is 415nm. The test results are as follows: Figure 4 , Figure 5 shown.

[0094] Depend on Figure 4 , Figure 5 It can be seen that NFP-R can respond to H2O2 stimulation and achieve fluorescence recovery, indicating that it has the potential to detect reactive oxygen species. In addition, under high concentration H2O2 conditions, the fluorescence recovery of NFP-R is stronger.

[0095] 4. Determination of fluorescent probe selectivity Use DMSO:PBS=1:9, pH=7.4 mixed solvent to prepare a probe solution with a concentration of 10μM. Take 1mL to 2mL centrifuge tubes and add a certain concentration of interfering ion solutions composed of anions, cations, amino acids, proteins and other substances. After mixing evenly, incubate for 1 hour, and observe the changes in the fluorescence spectrum of the solution after adding different ions using a fluorescence spectrophotometer. The excitation wavelength is 415nm. The test results are as follows: Figure 6 shown.

[0096] Depend on Figure 6 It can be seen that when different anions, cations, amino acids, proteins and other substances are added to the PBS solution of 10 μM probe NFP-R, only reactive oxygen species has a significant enhancement effect on the fluorescence signal, indicating that NFP-R can achieve highly specific recognition of reactive oxygen species in complex biological systems.

[0097] In vitro cell experiments 1. Cytotoxicity test In order to study whether the fluorescent probes NFP-R and NFP-DR have good biocompatibility, the cytotoxicity of NFP-R and NFP-DR on breast cancer tumor cells 4T1, mouse embryonic fibroblasts MEF and human renal proximal tubule cells HK-2 was detected by CCK-8 method before cell imaging. 4T1, MEF and HK-2 cells were respectively cultured at 1×10 4 The cells were inoculated into a 96-well plate at a density of 10 cells / well and cultured overnight. Serum-free culture medium containing different concentrations of NFP-R and NFP-DR was added for 24 hours. Then, the culture medium was discarded and culture medium containing 10% CCK-8 was added to each well to continue incubation for half an hour. The absorbance at 450nm was measured using an enzyme marker. The test results are as follows: Fig.11 , Fig.12 and Fig.13 shown.

[0098] Depend on Fig.11 , Fig.12 and Fig.13 It can be seen that even if the probe NFP-R and NFP-DR were incubated with a concentration of up to 20μM for 24 hours, the survival rate of 4T1, MEF and HK-2 cells was still over 85%. This shows that both NFP-R and NFP-DR have low cytotoxicity and are suitable for subsequent biological imaging applications.

[0099] 2. Capability of detecting endogenous reactive oxygen species in cells 4T1, MCF-7 or MEF cells were cultured at 2×10 5 The cells were seeded at a density of 1 μM NFP-DR and 1 μM NFP-R per well in a glass-bottomed culture dish and cultured overnight. Serum-free culture medium containing 1 μM NFP-DR and 1 μM NFP-R was added for 1 hour. Then, fresh culture medium was replaced and incubated for another 4 hours. Finally, the culture medium was removed, the cell nuclei were labeled with Hoechst33342, and the intracellular fluorescence signal was observed by confocal microscopy. 4T1, MCF-7 or MEF cells were then cultured at 1×10 5 The cells were inoculated into 24-well plates at a density of 1 μM per well and cultured overnight. Serum-free medium containing 1 μM NFP-DR and 1 μM NFP-R was added for 2 hours, with 5 replicates per group. Then, fresh medium was replaced and incubated for 4 hours. Finally, the medium was removed, the cells were digested with trypsin and collected, and the intracellular fluorescence intensity was quantitatively analyzed by flow cytometry.

[0100] HK-2 cells were cultured at 2×10 5The density of each well was inoculated into a glass-bottomed culture dish and cultured overnight. Divided into four groups, two of which were first incubated with the reactive oxygen scavenger thiourea for 2 hours, and the remaining two groups were incubated with 80μM cisplatin solution for 5 hours to induce the generation of reactive oxygen. Serum-free culture medium containing 10μM NFP-DR was added to the two groups incubated with thiourea and only added with cisplatin solution, and serum-free culture medium containing 10μM NFP-R was added to the remaining two groups incubated with thiourea and only added with cisplatin solution. All four groups were cultured for 2 hours. Finally, the culture medium was removed, the cell nucleus was labeled with Hoechst33342, and the intracellular fluorescence signal was observed by confocal microscopy, and the fluorescence recovery in the cells was quantitatively analyzed.

[0101] The test results are as follows Fig.14 , Fig.15 , Fig.16 and Fig.17 shown.

[0102] Depend on Fig.14 It can be seen that there are obvious red fluorescence signals in 4T1 cells and MCF-7 cells co-incubated with NFP-R and NFP-DR, indicating that both NFP-R and NFP-DR can respond to intracellular ROS and release NFP-OH. However, compared with the NFP-R group, the fluorescence signal of the NFP-DR group is significantly stronger, so at the same reactive oxygen concentration, more NFP-OH can be released, so the fluorescence signal is stronger. The results of flow cytometry analysis are consistent with confocal observations, and the fluorescence intensity of the NFP-DR group is significantly higher than that of the NFP-R group.

[0103] Depend on Fig.15 It can be seen that the above conclusions were verified at the MCF-7 cell level, and the results were consistent with those observed in 4T1 cells, with the fluorescence intensity of the NFP-DR group being significantly higher than that of the NFP-R group. The above experimental results show that NFP-DR can amplify the reactive oxygen signal in tumor cells through its self-degradation properties.

[0104] Depend on Fig.16 It can be seen that the fluorescence signal of NFP-OH in 4T1 cells after co-incubation with NFP-DR is significantly stronger than that in MEF cells. This is mainly because the level of reactive oxygen in tumor cells is much higher than that in normal cells, so more NFP-OH is activated in 4T1 cells. The results of flow cytometry quantitative analysis also show that the fluorescence intensity of CyOH in 4T1 cells is higher. The above experimental results show that NFP-DR can specifically image tumor cells with high levels of reactive oxygen, which will be beneficial for its use in tumor diagnosis.

[0105] Depend on Fig.17It can be seen that compared with the NFP-DR group preloaded with thiourea, the other group has a higher fluorescence intensity, indicating that NFP-DR can specifically image reactive oxygen species, which will be beneficial for its use in the diagnosis of ROS levels in vivo. In addition, the fluorescence signal of the NFP-DR group is significantly higher than that of NFP-R, indicating that NFP-DR can amplify the reactive oxygen stimulation signal in cells and has the potential to be used for early detection of diseases.

Claims

1. A self-amplifying activated active oxygen probe, characterized in that: Its structural formula is shown in Formula I: 。 2. A method for preparing the self-amplifying activated reactive oxygen species probe according to claim 1, characterized in that: The following steps are involved: 2,6-bis(hydroxymethyl)-m-cresol and tert-butyldimethylchlorosilane were mixed for substitution reaction to obtain compound 2; Using the compound 2 and 4-bromomethylphenylboronic acid pinacol ester as raw materials, a substitution reaction is carried out to obtain compound 3; Using the compound 3 and tetrabutylammonium fluoride as raw materials, a nucleophilic substitution reaction is carried out to obtain compound 4; Mix isophorone and malononitrile to carry out Knoevenagel condensation reaction to obtain compound 1; Using compound 1 and 3-chloro-4-hydroxybenzaldehyde as raw materials, a substitution reaction was carried out to obtain intermediate 1; The intermediate 1 is mixed with p-nitrophenyl chloroformate to carry out a substitution reaction to obtain compound 5; Using the compound 4 and the compound 5 as raw materials for a substitution reaction, a compound represented by formula I is obtained; The synthetic route is: 。 3. The method for preparing the self-amplifying activated reactive oxygen species probe according to claim 2, characterized in that: The molar ratio of 2,6-bis(hydroxymethyl)-m-cresol to tert-butyldimethylchlorosilane is 1:2-2.2; The molar ratio of the compound 2 to 4-bromomethylphenylboronic acid pinacol ester is 1:1-1.1; The molar ratio of the compound 3 to tetrabutylammonium fluoride is 1:5-10; The molar ratio of isophorone to malononitrile is 1:1-1.2; The molar ratio of the compound 1 to 3-chloro-4-hydroxybenzaldehyde is 1:1-1.2; The molar ratio of the intermediate 1 to p-nitrophenyl chloroformate is 1:2-6; The molar ratio of compound 4 to compound 5 is 1:2~2.

2.

4. The method for preparing the self-amplifying activated reactive oxygen species probe according to claim 3, characterized in that: The reaction of 2,6-bis(hydroxymethyl)-m-cresol and tert-butyldimethylchlorosilane is stirred at room temperature for 4 h to 6 h; The reaction of the compound 2 and 4-bromomethylphenylboronic acid pinacol ester is carried out by stirring at 85° C. to 95° C. for 8 h to 10 h in a nitrogen atmosphere; The reaction of compound 3 and tetrabutylammonium fluoride is stirred at room temperature for 8 h to 10 h; The reaction of isophorone and malononitrile is carried out by stirring at 80° C. to 85° C. for 8 h to 10 h in a nitrogen atmosphere; The reaction of the compound 1 and 3-chloro-4-hydroxybenzaldehyde is carried out by stirring at 80° C. to 85° C. for 6 h to 8 h; The reaction of the intermediate 1 with p-nitrophenyl chloroformate is carried out by stirring at room temperature in the dark for 6 h to 8 h; The reaction of compound 4 and compound 5 is carried out by stirring at room temperature for 7 h to 10 h.

5. The method for preparing the self-amplifying activated reactive oxygen species probe according to claim 4, characterized in that: The reaction product of 2,6-bis(hydroxymethyl)-m-cresol and tert-butyldimethylsilyl chloride was diluted with dichloromethane, washed with water, and the obtained organic layer was dried. The obtained crude product was then purified by column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 20:1 as an eluent to obtain the compound 2; The reaction product of the compound 2 and 4-bromomethylphenylboronic acid pinacol ester is purified by column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 20:1 as an eluent to obtain the compound 3; The reaction product of the compound 3 and tetrabutylammonium fluoride was diluted with dichloromethane, washed with water, and the obtained organic layer was dried. The obtained crude product was then purified by column chromatography using a mixture of n-hexane and ethyl acetate in a volume ratio of 2:1 as an eluent to obtain the compound 4; The reaction product of isophorone and malononitrile is purified by column chromatography using a mixture of petroleum ether and dichloromethane in a volume ratio of 1:5 as an eluent to obtain the compound 1; The reaction product of the compound 1 and 3-chloro-4-hydroxybenzaldehyde is purified by column chromatography using a mixture of petroleum ether and dichloromethane in a volume ratio of 1:1 as an eluent to obtain the intermediate 1; The reaction product of the intermediate 1 and p-nitrophenyl chloroformate was purified by column chromatography using a mixture of methyl chloride and n-hexane in a volume ratio of 4:1 as an eluent to obtain the compound 5; The reaction product of the compound 4 and the compound 5 is purified by column chromatography using a mixture of n-hexane and ethyl acetate in a volume ratio of 3:1 as an eluent to obtain the self-amplifying activated reactive oxygen probe.

6. Use of the self-amplifying activated reactive oxygen species probe according to claim 1 in preparing a fluorescent probe.

7. The use according to claim 6, characterized in that: The fluorescent probe is used to amplify active oxygen signals.

8. The use according to claim 7, characterized in that: The self-amplifying activated active oxygen probe is used to detect the active oxygen content.

9. Use of the self-amplifying activated reactive oxygen species probe according to claim 1 in the preparation of disease diagnosis-related products, characterized in that: The disease is a disease associated with abnormal changes in active oxygen.

10. A use of the self-amplifying activated reactive oxygen species probe according to claim 1 in the preparation of a product for monitoring biomarkers, characterized in that: The biomarker is a biomarker associated with a disease caused by abnormal changes in reactive oxygen species.

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