A self-amplified activation type reactive oxygen species probe, its preparation method and application

Abstract

The present invention discloses a self-amplified activation type reactive oxygen species probe, a preparation method thereof, and an application. The structural formula of the reactive oxygen species probe is shown in Formula I. This reactive oxygen species probe has great application potential in aspects such as disease early warning diagnosis and precise monitoring of biomarkers. Especially in the early diagnosis of diseases, by highly sensitively detecting low levels of reactive oxygen species, the diagnostic accuracy rate can be greatly improved, so as to achieve earlier therapeutic intervention and improve the cure rate of patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a self-amplified activation type reactive oxygen species probe and a preparation method thereof. Background Art

[0002] Bioactive small molecules such as reactive oxygen species, reactive nitrogen species, and reactive sulfur species are closely related to the life activities in cells and in vivo, and play a very important role in maintaining various normal life activities of organisms and the occurrence of various diseases. Reactive oxygen species are by-products of biological aerobic metabolism and are a general term for a class of oxygen-containing and highly reactive substances. Their content is usually related to tumors, inflammatory diseases, and immune diseases, etc. Research shows that reactive oxygen and nitrogen species play a very important role in the physiological and pathological processes of the life system. Among them, reactive oxygen species, as highly bioactive chemical substances, can exert toxic effects on biomolecules and are thus considered to be the main culprits causing various diseases such as cardiovascular diseases, neurodegenerative diseases, and even cancer.

[0003] In recent years, the development of fluorescent probes has been particularly rapid. With their visualization, real-time monitoring ability, good sensitivity, and easy operation and many other advantages, they have played a crucial role in the early diagnosis of clinical diseases. Currently, all the studied fluorescent probes can only activate one molecule of fluorescent group per molecule of stimulation signal, which leads to the situation that when the concentration of reactive oxygen species is low, the fluorescent probe cannot detect the content change in time, resulting in the delay of the disease condition.

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

[0005] In order to overcome the deficiencies of the prior art, the present invention provides a self-amplified activation type reactive oxygen species probe, a preparation method thereof, and an application thereof. The present invention realizes a one-to-many response mode by introducing a self-degrading linker, and is expected to improve the detection ability of the reactive oxygen species probe. It has great application potential in aspects such as disease early warning diagnosis and accurate monitoring of biomarkers.

[0006] The purpose of the present invention is achieved by at least one of the following technical solutions:

[0007] In the first aspect of the present invention, a self-amplified activation type reactive oxygen species probe is provided, and its structural formula is as shown in Formula I:

[0008] .

[0009] In the second aspect of the present invention, a preparation method of the self-amplified activation type reactive oxygen species probe is provided, including the following steps:

[0010] 2,6 - bis(hydroxymethyl) - m - cresol is mixed with tert - butyldimethylchlorosilane for a substitution reaction to obtain compound 2;

[0011] Using the compound 2 and 4 - (bromomethyl)phenylboronic acid pinacol ester as raw materials for a substitution reaction to obtain compound 3;

[0012] Using the compound 3 and tetrabutylammonium fluoride as raw materials for a nucleophilic substitution reaction to obtain compound 4;

[0013] Isophorone is mixed with malononitrile for a Knoevenagel condensation reaction to obtain compound 1;

[0014] Using compound 1 and 3 - chloro - 4 - hydroxybenzaldehyde as raw materials for a substitution reaction to obtain intermediate 1;

[0015] Mixing intermediate 1 with p - nitrophenyl chloroformate for a substitution reaction to obtain compound 5;

[0016] Using the compound 4 and the compound 5 as raw materials for a substitution reaction to obtain the compound shown in formula I;

[0017] The synthetic route is:

[0018] .

[0019] As a preferred embodiment of the present invention, the molar ratio of 2,6 - bis(hydroxymethyl) - m - cresol to tert - butyldimethylchlorosilane is 1:2 to 2.2;

[0020] The molar ratio of the compound 2 and 4 - (bromomethyl)phenylboronic acid pinacol ester is 1:1 to 1.1;

[0021] The molar ratio of the compound 3 to tetrabutylammonium fluoride is 1:5 to 10;

[0022] The molar ratio of isophorone to malononitrile is 1:1 to 1.2;

[0023] The molar ratio of the compound 1 and 3 - chloro - 4 - hydroxybenzaldehyde is 1:1 to 1.2;

[0024] The molar ratio of intermediate 1 to p - nitrophenyl chloroformate is 1:2 to 6;

[0025] The molar ratio of the compound 4 to the compound 5 is 1:2 to 2.2.

[0026] As a preferred embodiment of the present invention, the reaction of 2,6 - bis(hydroxymethyl) - m - cresol with tert - butyldimethylchlorosilane is stirred at room temperature for 4 h to 6 h;

[0027] The reaction of the said compound 2 and 4-(bromomethyl)phenylboronic acid pinacol ester is carried out with stirring at 85 °C to 95 °C for 8 h to 10 h;

[0028] The reaction of the said compound 3 and tetrabutylammonium fluoride is carried out with stirring at room temperature for 8 h to 10 h;

[0029] The reaction of the said isophorone and malononitrile is carried out with stirring at 80 °C to 85 °C for 8 h to 10 h;

[0030] The reaction of the said compound 1 and 3-chloro-4-hydroxybenzaldehyde is carried out with stirring at 80 °C to 85 °C for 6 h to 8 h;

[0031] The reaction of the said intermediate 1 and p-nitrophenyl chloroformate is carried out with stirring in the dark at room temperature for 6 h to 8 h;

[0032] The reaction of the said compound 4 and compound 5 is carried out with stirring at room temperature for 7 h to 10 h.

[0033] 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. The obtained crude product is further purified by column chromatography using a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 20:1 as the eluent to obtain the said compound 2;

[0034] The reaction product of the said compound 2 and 4-(bromomethyl)phenylboronic acid pinacol ester is purified by column chromatography using a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 20:1 as the eluent to obtain the said compound 3;

[0035] The reaction product of the said compound 3 and tetrabutylammonium fluoride is diluted with dichloromethane, washed with water, the organic layer is dried with magnesium sulfate, and the solvent is removed under reduced pressure. The obtained crude product is further purified by column chromatography using a mixed solution of n-hexane and ethyl acetate with a volume ratio of 2:1 as the eluent to obtain the said compound 4;

[0036] The reaction product of the said isophorone and malononitrile is purified by column chromatography using a mixed solution of petroleum ether and dichloromethane with a volume ratio of 1:5 as the eluent to obtain the said compound 1;

[0037] The reaction product of the said compound 1 and 3-chloro-4-hydroxybenzaldehyde is purified by column chromatography using a mixed solution of petroleum ether and dichloromethane with a volume ratio of 1:1 as the eluent to obtain the said intermediate 1;

[0038] The reaction product of the said intermediate 1 and p-nitrophenyl chloroformate is purified by column chromatography using a mixed solution of dichloromethane and n-hexane with a volume ratio of 4:1 as the eluent to obtain the said compound 5;

[0039] The reaction product of compound 4 and compound 5 was purified by column chromatography using a mixed solution of n-hexane and ethyl acetate with a volume ratio of 3:1 as the eluent to obtain the self-amplifying activated ROS probe.

[0040] In the third aspect of the present invention, there is provided an application of the self-amplifying activated ROS probe in the preparation of a fluorescent probe.

[0041] As a preferred embodiment of the present invention, the self-amplifying activated ROS probe is used to amplify low-level ROS signals.

[0042] As a preferred embodiment of the present invention, the self-amplifying activated ROS probe is used to detect the content of ROS.

[0043] In the fourth aspect of the present invention, there is provided an application of the self-amplifying activated ROS probe in the preparation of products related to disease diagnosis, where the disease is a disease related to abnormal changes in ROS, such as cancer, cardiovascular disease, diabetes, Parkinson's disease, and Alzheimer's disease, etc.

[0044] In the fifth aspect of the present invention, there is provided an application of the self-amplifying activated ROS probe in the preparation of products for monitoring biomarkers, where the biomarker is a biomarker related to a disease caused by abnormal changes in ROS.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] The self-amplifying activated ROS probe prepared by using the compound provided by the present invention has the following characteristics:

[0047] (1) In the present invention, 2,6-bis(hydroxymethyl)-m-cresol is used as a self-degrading linker, a ROS-responsive phenylboronic acid ester group is attached, and then the hydroxyl group is modified by p-nitrophenyl chloroformate, and two fluorescent groups are attached to both sides respectively. Under the stimulation reaction, triggering the responsive group on the self-degrading linker can 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 ROS probe.

[0048] (2) Aiming at the limitation that common fluorescent probes cannot effectively diagnose early diseases, the present invention designs a ROS fluorescent probe with the ability to amplify low-level ROS signals, which can be activated by intracellular ROS and detect its own ROS level through the recovery of its own fluorescence. This ROS probe has great application potential in aspects such as disease early warning diagnosis and precise biomarker monitoring. Especially in the early diagnosis of diseases, by highly sensitively detecting low-level ROS, the diagnostic accuracy can be greatly improved, thereby enabling earlier treatment intervention and improving the cure rate of patients. Description of the Drawings

[0049] Figure 1 1H NMR spectrum of NFP-OH.

[0050] Figure 2 1H NMR spectrum of the ROS probe NFP-R.

[0051] Figure 3 Absorption and fluorescence spectra of NFP-OH.

[0052] Figure 4 Spectra of the fluorescence intensity changes of NFP-R after incubation with different concentrations of H2O2.

[0053] Figure 5 Curve of the fluorescence intensity of NFP-R vs. H2O2 concentration.

[0054] Figure 6 Fluorescence intensity of NFP-R after incubation with different interfering ions.

[0055] Figure 7 1H NMR spectrum of the self-amplified activated ROS probe NFP-DR.

[0056] Figure 8 Spectra of the fluorescence intensity changes of NFP-DR after incubation with different concentrations of H2O2.

[0057] Figure 9 Curve of the fluorescence intensity of NFP-DR vs. H2O2 concentration.

[0058] Figure 10 Fluorescence intensity of NFP-DR after incubation with different interfering ions.

[0059] Figure 11 Cytotoxicity of NFP-R and NFP-DR against 4T1 cells.

[0060] Figure 12 Cytotoxicity of NFP-R and NFP-DR against MEF cells.

[0061] Figure 13 Cytotoxicity of NFP-R and NFP-DR against HK-2 cells.

[0062] Figure 14 Confocal laser microscopy images (A) and flow cytometry quantitative analysis (B) of NFP-R and NFP-DR in 4T1 cells.

[0063] Figure 15 Confocal laser microscopy images (A) and flow cytometry quantitative analysis (B) of NFP-R and NFP-DR in MCF-7 cells.

[0064] Figure 16 For confocal laser scanning microscopy observation of the fluorescence images (A) and flow cytometry quantitative analysis (B) of NFP-DR in 4T1 cells and MEF cells.

[0065] Figure 17 For confocal laser scanning microscopy observation of the fluorescence images (A) and quantitative analysis of fluorescence signal intensity (B) of NFP-R and NFP-DR in acute kidney injury cells. Specific implementation mode

[0066] The following further illustrates the specific implementation of the present invention in conjunction with examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that for the processes not specifically described in detail below, those skilled in the art can implement or understand them with reference to the prior art. Reagents or instruments without indicating the manufacturer are regarded as conventional products that can be purchased commercially.

[0067] Example 1

[0068] A self-amplified activated reactive oxygen species probe, labeled as NFP-DR, and its structural formula is shown as follows:

[0069] .

[0070] A preparation method of a self-amplified activated reactive oxygen species probe, comprising the following steps:

[0071] 1) Take 1.4680 g of 2,6-bis(hydroxymethyl)-m-cresol and 1.3070 g of imidazole, dissolve them in 15 mL of N,N-dimethylformamide, and place them in an ice bath. Then take 2.8020 g of tert-butyldimethylchlorosilane, dissolve it in 5 mL of DMF, and drop it into the solution. The mixed solution is stirred at room temperature for 6 h. Pour the mixture into a separatory funnel, add 20 mL of dichloromethane for dilution and wash with pure water. After drying with anhydrous magnesium sulfate, distill under reduced pressure to obtain a crude product. The crude product is purified by column chromatography with a petroleum ether:ethyl acetate = 20:1 eluent to obtain compound 2, and compound 2 is a colorless transparent oil.

[0072] 2) Take 1.5070 g of compound 2 and 0.6290 g of K2CO3, dissolve them in 15 mL of acetonitrile solution and place them in an ice bath. Then take 1.1283 g of 4-bromomethylphenylboronic acid pinacol ester, dissolve it in 5 mL of acetonitrile solution, and drop it into the solution. The mixed solution is stirred and refluxed at 85 °C under a nitrogen atmosphere for 8 h. Cool the mixture to room temperature and evaporate under reduced pressure. The crude product is purified by column chromatography with a petroleum ether:ethyl acetate = 20:1 eluent to obtain compound 3, and compound 3 is a colorless transparent oil.

[0073] 3) Weigh 0.1560 g of Compound 3 and dissolve it in 20 mL of tetrahydrofuran solution. Dropwise add 0.6656 g of tetrabutylammonium fluoride into the solution, and stir the mixed solution at room temperature for 8 h. After the reaction is completed, dilute the mixed solution with 20 mL of dichloromethane and wash it with pure water. After drying over anhydrous magnesium sulfate, perform vacuum distillation to obtain the 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.

[0074] 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. Stir and reflux the mixed solution under a nitrogen atmosphere at 80 °C for 8 h. Cool the mixture to room temperature and evaporate it under reduced pressure. The crude product is purified by column chromatography using petroleum ether:dichloromethane = 1:5 as the eluent to obtain Compound 1 as a pale yellow solid.

[0075] 5) Dissolve a mixture of 0.3720 g of Compound 1 and 0.3130 g of 3 - chloro - 4 - hydroxybenzaldehyde in 20 mL of ethanol and stir and reflux at 85 °C for 6 h. Then evaporate the mixture under reduced pressure. The residue is purified by column chromatography using petroleum ether:dichloromethane = 1:1 as the eluent to obtain the orange solid intermediate 1, labeled as NFP - OH.

[0076] 6) Take 0.3248 g of NFP - OH, dissolve it in 20 mL of dichloromethane, and place it in an ice bath. Dropwise add 1.2090 g of phenyl p - nitrophenyl carbonate, then dropwise add 1.0340 g of N,N - diisopropylethylamine and 0.0395 g of pyridine respectively, and stir in the dark at room temperature for 8 h. After vacuum distilling the mixture, the crude product is purified by column chromatography using dichloromethane:n - hexane = 4:1 as the eluent to obtain Compound 5.

[0077] 7) Take 0.0263 g of Compound 4 and 0.0354 g of N,N - diisopropylethylamine, dissolve them in 20 mL of dichloromethane, and place it in an ice bath. Dropwise add 0.0671 g of Compound 5, and stir at room temperature for 8 h. After vacuum distilling the mixture, the crude product is purified by column chromatography using n - hexane:ethyl acetate = 3:1 as the eluent to obtain the yellow solid NFP - DR.

[0078] The synthetic route is as follows:

[0079] 。

[0080] Example 2

[0081] A self - amplifying activated reactive oxygen species probe, labeled as NFP - DR, with the same structural formula as in Example 1. The specific preparation method includes the following steps:

[0082] 1) 1.4680 g of 2,6-bis(hydroxymethyl)-m-cresol and 1.3070 g of imidazole were dissolved in 15 mL of N,N-dimethylformamide and placed in an ice bath. Then, 2.6310 g of tert-butyldimethylchlorosilane was dissolved in 5 mL of DMF and added dropwise to the solution. The mixed solution was stirred at room temperature for 6 h. The mixture was poured into a separatory funnel, diluted with 20 mL of dichloromethane and washed with pure water. After drying over anhydrous magnesium sulfate, the crude product was obtained by distillation under reduced pressure. The crude product was purified by column chromatography with a petroleum ether:ethyl acetate = 20:1 eluent to obtain compound 2, which was a colorless transparent oil.

[0083] 2) 1.5070 g of compound 2 and 0.6290 g of K2CO3 were dissolved in 15 mL of acetonitrile solution and placed in an ice bath. Then, 1.2400 g of 4-bromomethylphenylboronic acid pinacol ester was dissolved in 5 mL of acetonitrile solution and added dropwise to the solution. The mixed solution was stirred under a nitrogen atmosphere at 85 °C for 8 h. The mixture was cooled to room temperature and evaporated under reduced pressure. The crude product was purified by column chromatography with a petroleum ether:ethyl acetate = 20:1 eluent to obtain compound 3, which was a colorless transparent oil.

[0084] 3) 0.156 g of compound 3 was dissolved in 20 mL of tetrahydrofuran solution. 0.3328 g of tetrabutylammonium fluoride was added dropwise to the solution. The mixed solution was stirred at room temperature for 8 h. After the reaction was completed, the mixed solution was diluted with 20 mL of dichloromethane and washed with pure water. After drying over anhydrous magnesium sulfate, the crude product was obtained by distillation under reduced pressure. The crude product was purified by column chromatography with a n-hexane:ethyl acetate = 2:1 eluent to obtain the oily compound 4.

[0085] 4) 0.0691 g of isophorone, 0.0330 g of malononitrile and 10 μL of glacial acetic acid were dissolved in 15 mL of ethanol, and then 2 drops of piperidine were added. The mixed solution was stirred under a nitrogen atmosphere at 80 °C for 8 h. The mixture was cooled to room temperature and evaporated under reduced pressure. The crude product was purified by column chromatography with a petroleum ether:dichloromethane = 1:5 eluent to obtain the pale yellow solid compound 1.

[0086] 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 under reflux at 85 °C for 6 h. Then the mixture was evaporated under reduced pressure. The residue was purified by column chromatography with a petroleum ether:dichloromethane = 1:1 eluent to obtain the orange solid intermediate 1, labeled as NFP-OH.

[0087] 6) Dissolve 0.3248 g of Intermediate 1 in 20 mL of dichloromethane and place it in an ice bath. Dropwise add 0.4031 g of phenyl p-nitrochloroformate, then dropwise add 1.034 g of N,N-diisopropylethylamine and 0.0395 g of pyridine respectively, and stir in the dark at room temperature for 8 h. After distilling the mixture under reduced pressure, the crude product is purified by column chromatography using dichloromethane:n-hexane = 4:1 as the eluent to obtain Compound 5.

[0088] 7) Dissolve 0.0263 g of Compound 4 and 0.0354 g of N,N-diisopropylethylamine in 20 mL of dichloromethane and place it in an ice bath. Dropwise add 0.0737 g of Compound 5, and stir at room temperature for 8 h. After distilling the mixture under reduced pressure, the crude product is purified by column chromatography using n-hexane:ethyl acetate = 3:1 as the eluent to obtain the yellow solid NFP-DR.

[0089] Example 3

[0090] A self-amplified activated reactive oxygen species probe, labeled as NFP-DR, with the same structural formula as in Example 1. The specific preparation method includes the following steps:

[0091] 1) Take 1.4680 g of 2,6-bis(hydroxymethyl)-m-cresol and 1.3070 g of imidazole and dissolve them in 15 mL of N,N-dimethylformamide, and place it in an ice bath. Then take 2.8940 g of tert-butyldimethylchlorosilane, dissolve it in 5 mL of DMF, and dropwise add it into the solution. The mixed solution is stirred at room temperature for 6 h. Pour the mixture into a separatory funnel, add 20 mL of dichloromethane for dilution and wash with pure water. After drying over anhydrous magnesium sulfate, distill it under reduced pressure to obtain the crude product. The crude product is purified by column chromatography using petroleum ether:ethyl acetate = 20:1 as the eluent to obtain Compound 2, and Compound 2 is a colorless transparent oil.

[0092] 2) Take 1.5070 g of Compound 2 and 0.6290 g of K2CO3 and dissolve them in 15 mL of acetonitrile solution and place it in an ice bath. Then take 1.2045 g of 4-(bromomethyl)phenylboronic acid pinacol ester, dissolve it in 5 mL of acetonitrile solution, and dropwise add it into the solution. The mixed solution is stirred and refluxed at 85 °C under a nitrogen atmosphere for 8 h. Cool the mixture to room temperature and evaporate it under reduced pressure. The crude product is purified by column chromatography using petroleum ether:ethyl acetate = 20:1 as the eluent to obtain Compound 3, and Compound 3 is a colorless transparent oil.

[0093] 3) Weigh 0.156 g of Compound 3 and dissolve it in 20 mL of tetrahydrofuran solution. Drop 0.5218 g of tetrabutylammonium fluoride into the solution, and stir the mixed solution at room temperature for 8 h. After the reaction is completed, dilute the mixed solution with 20 mL of dichloromethane and wash it with pure water. After drying over anhydrous magnesium sulfate, distill it under reduced pressure to obtain the 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.

[0094] 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. Stir and reflux the mixed solution under a nitrogen atmosphere at 80 °C for 8 h. Cool the mixture to room temperature and evaporate it under reduced pressure. The crude product is purified by column chromatography using petroleum ether:dichloromethane = 1:5 as the eluent to obtain Compound 1 as a pale yellow solid.

[0095] 5) Dissolve a mixture of 0.3720 g of Compound 1 and 0.3490 g of 3-chloro-4-hydroxybenzaldehyde in 20 mL of ethanol and stir and reflux at 85 °C for 6 h. Then evaporate the mixture under reduced pressure. The residue is purified by column chromatography using petroleum ether:dichloromethane = 1:1 as the eluent to obtain the orange solid intermediate 1, labeled as NFP-OH.

[0096] 6) Take 0.3248 g of Intermediate 1, dissolve it in 20 mL of dichloromethane and place it in an ice bath. Drop 0.8672 g of phenyl p-nitrochloroformate, then drop 1.034 g of N,N-diisopropylethylamine and 0.0395 g of pyridine respectively, and stir in the dark at room temperature for 8 h. After distilling the mixture under reduced pressure, the crude product is purified by column chromatography using dichloromethane:n-hexane = 4:1 as the eluent to obtain Compound 5.

[0097] 7) Take 0.0263 g of Compound 4 and 0.0354 g of N,N-diisopropylethylamine, dissolve them in 20 mL of dichloromethane and place it in an ice bath. Drop 0.0704 g of Compound 5 and stir at room temperature for 8 h. After distilling the mixture under reduced pressure, the crude product is purified by column chromatography using n-hexane:ethyl acetate = 3:1 as the eluent to obtain the yellow solid NFP-DR.

[0098] Example 4

[0099] A self-amplified activated reactive oxygen species probe, labeled as NFP-DR, has the same structural formula as in Example 1. The specific preparation method includes the following steps:

[0100] 1) 1.4680 g of 2,6-bis(hydroxymethyl)-m-cresol and 1.3070 g of imidazole were dissolved in 15 mL of N,N-dimethylformamide and placed in an ice bath. Then, 2.8020 g of tert-butyldimethylchlorosilane was dissolved in 5 mL of DMF and added dropwise to the solution. The mixed solution was stirred at room temperature for 4 h. The mixture was poured into a separatory funnel, diluted with 20 mL of dichloromethane and washed with pure water. After drying over anhydrous magnesium sulfate, the crude product was obtained by distillation under reduced pressure. The crude product was purified by column chromatography with a petroleum ether:ethyl acetate = 20:1 eluent to obtain Compound 2, which was a colorless transparent oil.

[0101] 2) 1.5070 g of Compound 2 and 0.6290 g of K2CO3 were dissolved in 15 mL of acetonitrile solution and placed in an ice bath. Then, 1.1285 g of 4-bromomethylphenylboronic acid pinacol ester was dissolved in 5 mL of acetonitrile solution and added dropwise to the solution. The mixed solution was stirred and refluxed at 95 °C for 8 h under a nitrogen atmosphere. The mixture was cooled to room temperature and evaporated under reduced pressure. The crude product was purified by column chromatography with a petroleum ether:ethyl acetate = 20:1 eluent to obtain Compound 3, which was a colorless transparent oil.

[0102] 3) 0.1560 g of Compound 3 was dissolved in 20 mL of tetrahydrofuran solution, and 0.6640 g of tetrabutylammonium fluoride was added dropwise to the solution. The mixed solution was stirred at room temperature for 10 h. After the reaction was completed, the mixed solution was diluted with 20 mL of dichloromethane and washed with pure water. After drying over anhydrous magnesium sulfate, the crude product was obtained by distillation under reduced pressure. The crude product was purified by column chromatography with a n-hexane:ethyl acetate = 2:1 eluent to obtain the oily Compound 4.

[0103] 4) 0.0691 g of isophorone, 0.0391 g of malononitrile and 10 μL of glacial acetic acid were dissolved in 15 mL of ethanol, and then 2 drops of piperidine were added. The mixed solution was stirred and refluxed at 80 °C for 8 h under a nitrogen atmosphere. The mixture was cooled to room temperature and evaporated under reduced pressure. The crude product was purified by column chromatography with a petroleum ether:dichloromethane = 1:5 eluent to obtain the light yellow solid Compound 1.

[0104] 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 and refluxed at 80 °C for 8 h. Then the mixture was evaporated under reduced pressure. The residue was purified by column chromatography with a petroleum ether:dichloromethane = 1:1 eluent to obtain the orange solid intermediate 1, labeled as NFP-OH.

[0105] 6) Dissolve 0.3248 g of NFP-OH in 20 mL of dichloromethane and place it in an ice bath. Dropwise add 1.209 g of phenyl 4-nitrophenyl carbonate, then dropwise add 1.034 g of N,N-diisopropylethylamine and 0.0395 g of pyridine respectively, and stir in the dark at room temperature for 6 h. After distilling the mixture under reduced pressure, the crude product is purified by column chromatography using dichloromethane:n-hexane = 4:1 as the eluent to obtain Compound 5.

[0106] 7) Dissolve 0.0263 g of Compound 4 and 0.0354 g of N,N-diisopropylethylamine in 20 mL of dichloromethane and place it in an ice bath. Dropwise add 0.0705 g of Compound 5, and stir at room temperature for 10 h. After distilling the mixture under reduced pressure, the crude product is purified by column chromatography using n-hexane:ethyl acetate = 3:1 as the eluent to obtain the yellow solid NFP-DR.

[0107] Example 5

[0108] A self-amplified activated reactive oxygen species probe, labeled as NFP-DR, with the same structural formula as in Example 1. The specific preparation method includes the following steps:

[0109] 1) Take 1.4680 g of 2,6-bis(hydroxymethyl)-m-cresol and 1.3070 g of imidazole and dissolve them in 15 mL of N,N-dimethylformamide, and place it in an ice bath. Then take 2.8020 g of tert-butyldimethylchlorosilane, dissolve it in 5 mL of DMF and dropwise add it into the solution. The mixed solution is stirred at room temperature for 5 h. Pour the mixture into a separatory funnel, add 20 mL of dichloromethane for dilution and wash with pure water. After drying over anhydrous magnesium sulfate, distill under reduced pressure to obtain the crude product. The crude product is purified by column chromatography using petroleum ether:ethyl acetate = 20:1 as the eluent to obtain Compound 2, and Compound 2 is a colorless transparent oil.

[0110] 2) Take 1.5070 g of Compound 2 and 0.6290 g of K2CO3 and dissolve them in 15 mL of acetonitrile solution and place it in an ice bath. Then take 1.1285 g of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl bromide, dissolve it in 5 mL of acetonitrile solution and dropwise add it into the solution. The mixed solution is stirred and refluxed at 85 °C under a nitrogen atmosphere for 9 h. Cool the mixture to room temperature and evaporate under reduced pressure. The crude product is purified by column chromatography using petroleum ether:ethyl acetate = 20:1 as the eluent to obtain Compound 3, and Compound 3 is a colorless transparent oil.

[0111] 3) Weigh 0.1560 g of Compound 3 and dissolve it in 20 mL of tetrahydrofuran solution. Drop 0.6640 g of tetrabutylammonium fluoride into the solution, and stir the mixed solution at room temperature for 10 h. After the reaction is completed, dilute the mixed solution with 20 mL of dichloromethane and wash it with pure water. After drying over anhydrous magnesium sulfate, distill it under reduced pressure to obtain the 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.

[0112] 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. Stir and reflux the mixed solution under a nitrogen atmosphere at 85 °C for 8 h. Cool the mixture to room temperature and evaporate it under reduced pressure. The crude product is purified by column chromatography using petroleum ether:dichloromethane = 1:5 as the eluent to obtain Compound 1 as a pale yellow solid.

[0113] 5) Dissolve a mixture of 0.3720 g of Compound 1 and 0.3130 g of 3-chloro-4-hydroxybenzaldehyde in 20 mL of ethanol and stir and reflux at 80 °C for 6 h. Then evaporate the mixture under reduced pressure. The residue is purified by column chromatography using petroleum ether:dichloromethane = 1:1 as the eluent to obtain the orange solid intermediate 1, labeled as NFP-OH.

[0114] 6) Take 0.3248 g of NFP-OH and dissolve it in 20 mL of dichloromethane and place it in an ice bath. Drop in 1.209 g of phenyl p-nitrocarbonate, then drop in 1.0340 g of N,N-diisopropylethylamine and 0.0395 g of pyridine respectively, and stir in the dark at room temperature for 8 h. After distilling the mixture under reduced pressure, the crude product is purified by column chromatography using dichloromethane:n-hexane = 4:1 as the eluent to obtain Compound 5.

[0115] 7) Take 0.0263 g of Compound 4 and 0.0354 g of N,N-diisopropylethylamine and dissolve them in 20 mL of dichloromethane and place it in an ice bath. Drop in 0.0705 g of Compound 5 and stir at room temperature for 7 h. After distilling the mixture under reduced pressure, the crude product is purified by column chromatography using n-hexane:ethyl acetate = 3:1 as the eluent to obtain the yellow solid NFP-DR.

[0116] Example 6

[0117] A self-amplified activated reactive oxygen species probe, labeled as NFP-DR, has the same structural formula as in Example 1. The specific preparation method includes the following steps:

[0118] 1) 1.4680 g of 2,6-bis(hydroxymethyl)-m-cresol and 1.3070 g of imidazole were dissolved in 15 mL of N,N-dimethylformamide and placed in an ice bath. Then, 2.8020 g of tert-butyldimethylchlorosilane was dissolved in 5 mL of DMF and added dropwise to the solution. The mixed solution was stirred at room temperature for 4 h. The mixture was poured into a separatory funnel, diluted with 20 mL of dichloromethane and washed with pure water. After drying over anhydrous magnesium sulfate, the crude product was obtained by distillation under reduced pressure. The crude product was purified by column chromatography with a petroleum ether:ethyl acetate = 20:1 eluent to obtain Compound 2, which was a colorless transparent oil.

[0119] 2) 1.5070 g of Compound 2 and 0.6290 g of K2CO3 were dissolved in 15 mL of acetonitrile solution and placed in an ice bath. Then, 1.1286 g of 4-bromomethylphenylboronic acid pinacol ester was dissolved in 5 mL of acetonitrile solution and added dropwise to the solution. The mixed solution was stirred and refluxed at 90 °C under a nitrogen atmosphere for 10 h. The mixture was cooled to room temperature and evaporated under reduced pressure. The crude product was purified by column chromatography with a petroleum ether:ethyl acetate = 20:1 eluent to obtain Compound 3, which was a colorless transparent oil.

[0120] 3) 0.1560 g of Compound 3 was dissolved in 20 mL of tetrahydrofuran solution, and 0.6640 g of tetrabutylammonium fluoride was added dropwise to the solution. The mixed solution was stirred at room temperature for 8 h. After the reaction was completed, the mixed solution was diluted with 20 mL of dichloromethane and washed with pure water. After drying over anhydrous magnesium sulfate, the crude product was obtained by distillation under reduced pressure. The crude product was purified by column chromatography with a n-hexane:ethyl acetate = 2:1 eluent to obtain the oily Compound 4.

[0121] 4) 0.0691 g of isophorone, 0.0395 g of malononitrile and 10 μL of glacial acetic acid were dissolved in 15 mL of ethanol, and then 2 drops of piperidine were added. The mixed solution was stirred and refluxed under a nitrogen atmosphere for 10 h. The mixture was cooled to room temperature and evaporated under reduced pressure. The crude product was purified by column chromatography with a petroleum ether:dichloromethane = 1:5 eluent to obtain the light yellow solid Compound 1.

[0122] 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 and refluxed at 85 °C for 8 h. Then the mixture was evaporated under reduced pressure. The residue was purified by column chromatography with a petroleum ether:dichloromethane = 1:1 eluent to obtain the orange solid Intermediate 1, labeled as NFP-OH.

[0123] 6) Dissolve 0.3248 g of NFP-OH in 20 mL of dichloromethane and place it in an ice bath. Dropwise add 1.2090 g of phenyl p-nitrocarbonate, then dropwise add 1.0340 g of N,N-diisopropylethylamine and 0.0395 g of pyridine respectively, and stir in the dark at room temperature for 7 h. After distilling the mixture under reduced pressure, the crude product is purified by column chromatography using dichloromethane:n-hexane = 4:1 as the eluent to obtain compound 5.

[0124] 7) Dissolve 0.0263 g of compound 4 and 0.0354 g of N,N-diisopropylethylamine in 20 mL of dichloromethane and place it in an ice bath. Dropwise add 0.0705 g of compound 5, and stir at room temperature for 8 h. After distilling the mixture under reduced pressure, the crude product is purified by column chromatography using n-hexane:ethyl acetate = 3:1 as the eluent to obtain the yellow solid NFP-DR.

[0125] Since the self-amplified activated ROS probes provided in Examples 1 to 6 have similar performances and effects, the present invention only takes Example 1 as an example to illustrate its performance and effects:

[0126] 1) Characterization of NFP-DR:

[0127] 1. 1H NMR spectrum of NFP-DR 1 The 1H NMR is as Figure 7 shown.

[0128] It can be Figure 7 seen that NFP-DR is successfully prepared.

[0129] 2. Determination of the ROS detection ability of NFP-DR:

[0130] Dilute the above-prepared probe mother liquor into a solution with a concentration of 10 μM using a mixed solvent of DMSO:PBS = 1:9, pH = 7.4. Take 1 mL into a 2 mL centrifuge tube, add hydrogen peroxide solutions with concentrations of 0 μM to 200 μM respectively and mix evenly. After incubation for 1 h, observe the changes in the fluorescence spectra of the solutions after adding different concentrations of H2O2 through a fluorescence spectrophotometer. The excitation wavelength is 415 nm, and the test results are as Figure 8 、 Figure 9 shown.

[0131] It can be Figure 8 、 Figure 9 seen that NFP-DR can respond to H2O2 stimulation and achieve fluorescence recovery, indicating its potential for detecting ROS. By comparing the results of NFP-R, it can be found that at lower hydrogen peroxide concentrations, the fluorescence of NFP-DR can recover, and at high concentrations, the fluorescence recovery of NFP-DR is stronger. The above results show that NFP-DR has a stronger ROS detection ability.

[0132] 5. Determination of the selectivity of the fluorescent probe

[0133] Prepare a 10 μM probe solution with a mixed solvent of DMSO:PBS = 1:9 and pH = 7.4. Take 1 mL to a 2 mL centrifuge tube, and add interference ion solutions composed of substances such as anions, cations, amino acids, and proteins at certain concentrations. After mixing evenly, incubate for 1 hour. Then, observe the changes in the fluorescence spectra of the solutions after adding different ions through a fluorescence spectrophotometer. The excitation wavelength is 415 nm, and the test results are as Figure 10 shown

[0134] As Figure 10 can be seen: When different anions, cations, amino acids, proteins, etc. are added to the 10 μM probe NFP-DR in PBS solution, only reactive oxygen species have a significant enhancing effect on the fluorescence signal, indicating that NFP-DR can achieve highly specific recognition of reactive oxygen species in complex biological systems.

[0135] Comparative example

[0136] Take the compound without introducing the self-degrading linker 2,6-bis(hydroxymethyl)-m-cresol as a comparative example. This compound is prepared according to the following steps:

[0137] 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 then add 2 drops of piperidine. The mixed solution is stirred and refluxed at 80 °C under a nitrogen atmosphere for 8 h. Cool the mixture to room temperature and evaporate 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.

[0138] 2) Dissolve a mixture of 0.3720 g of compound 1 and 0.3130 g of 3-chloro-4-hydroxybenzaldehyde in 20 mL of ethanol and stir and reflux for 6 h. Then evaporate the mixture under reduced pressure. The residue is purified by column chromatography using petroleum ether:dichloromethane = 1:1 as the eluent to obtain an orange solid intermediate 1.

[0139] 3) Dissolve 0.1018 g of intermediate 1 and 0.0522 g of K2CO3 in 15 mL of acetonitrile solution and place it in an ice bath. Then take 0.0960 g of 4-bromomethylphenylboronic acid pinacol ester dissolved in 5 mL of acetonitrile solution and add it dropwise into the solution. The mixed solution is stirred and refluxed at 90 °C under a nitrogen atmosphere for 8 h. Cool the mixture to room temperature and evaporate and concentrate 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, and its structural formula is as follows:

[0140]

[0141] The synthetic route is as follows:

[0142] .

[0143] Performance test

[0144] 1) Characterization of NFP-R:

[0145] 1. 1H NMR spectrum of NFP-OH 1 The 1H NMR is as Figure 1 shown. The 1H NMR spectrum of NFP-R 1 The 1H NMR is as Figure 2 shown.

[0146] It can be seen from Figure 2 that NFP-R was successfully prepared.

[0147] 2. The absorption spectrum and emission spectrum of NFP-OH are as Figure 3 shown. It can be seen from Figure 3 that the maximum excitation wavelength of NFP-OH is 415 nm and the maximum emission wavelength is 660 nm.

[0148] 3. Determination of the ability of NFP-R to detect reactive oxygen species

[0149] The prepared 10 mM mother liquor was diluted to a solution with a concentration of 10 μM. The diluting solvent was a mixed solvent of DMSO:PBS = 1:9, pH = 7.4; 1 mL was taken into a 2 mL centrifuge tube, and hydrogen peroxide solutions with concentrations of 0 μM to 200 μM were added and mixed evenly. After incubation for 1 hour, the changes in the fluorescence spectra of the solutions after adding different concentrations of H2O2 were observed through a fluorescence spectrophotometer. The excitation wavelength was 415 nm, and the test results are as Figure 4 , Figure 5 shown.

[0150] It can be seen from Figure 4 , Figure 5 that NFP-R can respond to H2O2 stimulation and achieve fluorescence recovery, indicating its potential to detect reactive oxygen species. And under the condition of high concentration of H2O2, the fluorescence recovery of NFP-R is stronger.

[0151] 4. Determination of the selectivity of the fluorescent probe

[0152] A probe solution with a concentration of 10 μM was prepared using a mixed solvent of DMSO:PBS = 1:9, pH = 7.4. 1 mL was taken into a 2 mL centrifuge tube, and interference ion solutions composed of anions, cations, amino acids, proteins, etc. with certain concentrations were added. After mixing evenly, after incubation for 1 hour, the changes in the fluorescence spectra of the solutions after adding different ions were observed through a fluorescence spectrophotometer. The excitation wavelength was 415 nm, and the test results are asFigure 6 as shown

[0153] From Figure 6 it can be seen that: when different substances such as anions, cations, amino acids, and proteins are added to the PBS solution of 10 μM probe NFP-R, only reactive oxygen species have a significant enhancing effect on the fluorescence signal, indicating that NFP-R can achieve highly specific recognition of reactive oxygen species in complex biological systems.

[0154] In vitro cell experiments

[0155] 1. Cytotoxicity test

[0156] To study whether the fluorescent probes NFP-R and NFP-DR have good biocompatibility, before cell imaging, the cytotoxicity of NFP-R and NFP-DR on breast cancer tumor cells 4T1, mouse embryonic fibroblasts MEF, and human renal proximal tubular cells HK-2 was detected by the CCK-8 method. 4T1, MEF, and HK-2 cells were respectively seeded into 96-well plates at a density of 1×10 4 cells / well and cultured overnight. Serum-free media containing different concentrations of NFP-R and NFP-DR were added and cultured for 24 hours. Then, the media was discarded, and media containing 10% CCK-8 was added to each well and incubated for another half hour. The absorbance at 450 nm was measured using a microplate reader. The test results are as Figure 11 , Figure 12 and Figure 13 shown.

[0157] From Figure 11 , Figure 12 and Figure 13 it can be seen that: even when incubated with probes NFP-R and NFP-DR at a concentration as high as 20 μM for 24 hours, the survival rates of 4T1, MEF, and HK-2 cells still exceed 85%. This indicates that both NFP-R and NFP-DR have low cytotoxicity and are suitable for subsequent bioimaging applications.

[0158] 2. Intracellular endogenous reactive oxygen species detection ability

[0159] 4T1, MCF-7, or MEF cells were seeded into glass-bottom culture dishes at a density of 2×10 5 per well and cultured overnight. Serum-free media containing 1 μM NFP-DR and 1 μM NFP-R were added and cultured for 1 hour. Then, the media was replaced with fresh media and incubated for another 4 hours. Finally, the media was removed, the nuclei were labeled with Hoechst33342, and the intracellular fluorescence signal was observed through a confocal microscope. Then, 4T1, MCF-7, or MEF cells were seeded at a density of 1×10 5Inoculate at a density of per well into a 24-well plate and culture overnight. Add serum-free medium containing 1 μM NFP-DR and 1 μM NFP-R respectively and culture for 2 hours, with 5 replicates in each group. Then, replace it with fresh medium and continue to incubate for 4 hours. Finally, remove the medium, digest the cells with trypsin and collect the cells, and quantitatively analyze the intracellular fluorescence intensity by flow cytometry.

[0160] Seed HK-2 cells at a density of 2×10 5 per well into a glass-bottom culture dish and culture overnight. Divide them into four groups. Incubate two of the groups with the ROS scavenger thiourea for 2 h first, and then incubate the remaining two groups with 80 μM cisplatin solution for 5 h respectively to induce the generation of ROS. Then, add serum-free medium containing 10 μM NFP-DR to both the two groups incubated with thiourea and the two groups only incubated with cisplatin solution, and add serum-free medium containing 10 μM NFP-R to the remaining two groups incubated with thiourea and the two groups only incubated with cisplatin solution, and culture all four groups for 2 hours. Finally, remove the medium, label the cell nuclei with Hoechst33342, observe the intracellular fluorescence signal by confocal microscopy, and quantitatively analyze the fluorescence recovery in the cells.

[0161] The test results are as Figure 14 、 Figure 15 、 Figure 16 and Figure 17 shown.

[0162] It can be seen from Figure 14 that: There are obvious red fluorescence signals in both 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. Therefore, at the same concentration of reactive oxygen species, more NFP-OH can be released, so the fluorescence signal is stronger. The results of flow cytometry analysis are consistent with those of confocal observation, and the fluorescence intensity of the NFP-DR group is significantly higher than that of the NFP-R group.

[0163] It can be seen from Figure 15 that: Verifying the above conclusion at the MCF-7 cell level, the results are consistent with those observed in 4T1 cells, and the fluorescence intensity of the NFP-DR group is significantly higher than that of the NFP-R group. Combining the above experimental results shows that NFP-DR can amplify the intracellular reactive oxygen species signal in tumor cells through its self-degradation property.

[0164] It can be seen from Figure 16It can be seen that the NFP-OH fluorescence signal in 4T1 cells after co-incubation with NFP-DR was significantly stronger than that in MEF cells. This is mainly because the level of reactive oxygen species (ROS) in tumor cells is much higher than that in normal cells. Therefore, more NFP-OH was activated in 4T1 cells. The results of flow cytometry quantitative analysis also showed that the CyOH fluorescence intensity in 4T1 cells was higher. The above experimental results indicate that NFP-DR can specifically image tumor cells with high ROS levels, which will be beneficial for its use in tumor diagnosis.

[0165] It can be seen from Figure 17 that: compared with the group of NFP-DR pre-loaded with thiourea, the fluorescence intensity of the other group was higher, indicating that NFP-DR can specifically image ROS, which will be beneficial for its use in the diagnosis of in vivo ROS levels. Moreover, the fluorescence signal of the NFP-DR group was significantly higher than that of NFP-R, indicating that NFP-DR can amplify the ROS stimulation signal in cells and has the potential for early detection of diseases.

Claims

1. A self-amplifying activated reactive oxygen species probe, characterized in that, Its structural formula is shown in Formula I: The self-amplifying and activatable reactive oxygen species (ROS) probe is used for preparing a fluorescent probe, and the fluorescent probe is used for amplifying the ROS signal and real-time monitoring of the change of ROS.

2. The preparation method of the self-amplifying activation type reactive oxygen species probe according to claim 1, characterized in that, It includes the following steps: Mix 2,6-bis(hydroxymethyl)-m-cresol with tert-butyldimethylchlorosilane for a substitution reaction to obtain Compound 2; Use Compound 2 and 4-(bromomethyl)phenylboronic acid pinacol ester as raw materials for a substitution reaction to obtain Compound 3; Use Compound 3 and tetrabutylammonium fluoride as raw materials for a nucleophilic substitution reaction to obtain Compound 4; Mix isophorone with malononitrile for a Knoevenagel condensation reaction to obtain Compound 1; Use Compound 1 and 3-chloro-4-hydroxybenzaldehyde as raw materials for a substitution reaction to obtain Intermediate 1; Mix Intermediate 1 with p-nitrophenyl chloroformate for a substitution reaction to obtain Compound 5; Use Compound 4 and Compound 5 as raw materials for a substitution reaction to obtain the compound shown in Formula I; The synthetic route is:

3. The preparation method of the self-amplifying and activatable ROS probe according to claim 2, wherein the molar ratio of 2,6-bis(hydroxymethyl)-m-cresol to tert-butyldimethylchlorosilane is 1:2 to 2.2; the molar ratio of Compound 2 to 4-(bromomethyl)phenylboronic acid pinacol ester is 1:1 to 1.1; the molar ratio of Compound 3 to tetrabutylammonium fluoride is 1:5 to 10; the molar ratio of isophorone to malononitrile is 1:1 to 1.2; the molar ratio of Compound 1 to 3-chloro-4-hydroxybenzaldehyde is 1:1 to 1.2; the molar ratio of Intermediate 1 to p-nitrophenyl chloroformate is 1:2 to 6; the molar ratio of Compound 4 to Compound 5 is 1:2 to 2.

2.

4. The preparation method of the self-amplifying and activatable ROS probe according to claim 3, wherein the reaction of 2,6-bis(hydroxymethyl)-m-cresol with tert-butyldimethylchlorosilane is stirred at room temperature for 4 h to 6 h; the reaction of Compound 2 and 4-(bromomethyl)phenylboronic acid pinacol ester is stirred in a nitrogen atmosphere 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 stirred in a nitrogen atmosphere at 80 °C to 85 °C for 8 h to 10 h; the reaction of Compound 1 and 3-chloro-4-hydroxybenzaldehyde is stirred at 80 °C to 85 °C for 6 h to 8 h; the reaction of Intermediate 1 and p-nitrophenyl chloroformate is stirred in the dark at room temperature for 6 h to 8 h; the reaction of Compound 4 and Compound 5 is stirred at room temperature for 7 h to 10 h.

5. The preparation method of the self-amplifying and activatable ROS probe according to claim 4, wherein after the reaction product of 2,6-bis(hydroxymethyl)-m-cresol and tert-butyldimethylchlorosilane is diluted with dichloromethane, washed with water, and the obtained organic layer is dried, the obtained crude product is further purified by column chromatography using a mixed solution of petroleum ether and ethyl acetate at a volume ratio of 20:1 as the eluent to obtain Compound 2; The reaction product of the said compound 2 and 4-(bromomethyl)phenylboronic acid pinacol ester was purified by column chromatography using a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 20:1 as the eluent to obtain the said compound 3; The reaction product of the said 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 mixed solution of n-hexane and ethyl acetate with a volume ratio of 2:1 as the eluent to obtain the said compound 4; The reaction product of the said isophorone and malononitrile was purified by column chromatography using a mixed solution of petroleum ether and dichloromethane with a volume ratio of 1:5 as the eluent to obtain the said compound 1; The reaction product of the said compound 1 and 3-chloro-4-hydroxybenzaldehyde was purified by column chromatography using a mixed solution of petroleum ether and dichloromethane with a volume ratio of 1:1 as the eluent to obtain the said intermediate 1; The reaction product of the said intermediate 1 and p-nitrophenyl chloroformate was purified by column chromatography using a mixed solution of chloroform and n-hexane with a volume ratio of 4:1 as the eluent to obtain the said compound 5; The reaction product of the said compound 4 and compound 5 was purified by column chromatography using a mixed solution of n-hexane and ethyl acetate with a volume ratio of 3:1 as the eluent to obtain the said self-amplifying activated reactive oxygen species probe.

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

7. The application according to claim 6, wherein The said fluorescent probe is used for amplifying the reactive oxygen species signal.

8. The application according to claim 7, wherein The said self-amplifying activated reactive oxygen species probe is used for detecting the content of reactive oxygen species.

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

10. 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 said biomarker is a biomarker related to a disease caused by abnormal changes in reactive oxygen species.

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