Dicyanoisophorone derivative, preparation method thereof and application of dicyanoisophorone derivative as near-infrared fluorescent probe for detecting COX-2

Abstract

The invention discloses a dicyanoisophorone derivative, a preparation method thereof and application of the dicyanoisophorone derivative as a near-infrared fluorescent probe for detecting COX-2. The structure of the derivative is shown as a formula I. The derivative can respond to COX-2 with high selectivity and high sensitivity, an obvious new emission peak appears in a fluorescence spectrum in the presence of COX-2, the derivative can be used for qualitative and quantitative detection of COX-2 in an aqueous solution and cells, interference of external detection conditions can be greatly reduced, and the derivative has high detection speed and high detection precision. Formula I.

Description

Technical Field

[0001] This application relates to a dicyanoisoflurone derivative, its preparation method, and its application as a near-infrared fluorescent probe for detecting COX-2, belonging to the field of chemical sensing materials technology. Background Technology

[0002] Cyclooxygenase (COX) is a key enzyme catalyzing the conversion of arachidonic acid into prostaglandins, participating in various physiological and pathological processes. There are two structural subtypes of COX: structural cyclooxygenase 1 (COX-1) and inducible cyclooxygenase 2 (COX-2). COX-1 is consistently and stably expressed in most tissues and is responsible for regulating "housekeeper" functions, playing a crucial role in tissue homeostasis. In contrast, COX-2, as a trigger for prostaglandin synthesis, mediates pathological responses and specific physiological processes. It is usually undetectable or at extremely low levels in normal tissues, but can be induced by inflammatory stimuli, cytokines (such as IL-1 and TNF-α), growth factors, and tumor promoters, and its expression is significantly upregulated in pathological states such as inflammation and cancer. Furthermore, COX-2 is not only expressed in various cancers but also plays a role in regulating inflammatory responses, tumor cell proliferation, and apoptosis. Therefore, a thorough understanding of the pathological mechanisms of COX-2 in related inflammation and tumor progression, as well as in drug treatment evaluation, is essential.

[0003] Due to the diversity and complexity of the internal environment, developing a highly selective and sensitive analytical method for detecting COX-2 is of significant research value and importance. Currently common COX-2 detection methods such as Western blotting, ELISA, and immunohistochemistry can detect COX-2 protein levels, but they typically require sample destruction, are complex to operate, and cannot provide real-time monitoring. Fluorescent probe imaging technology, on the other hand, is not only highly sensitive and simple to operate, but also enables real-time, dynamic, and high spatiotemporal resolution visualization analysis, playing a crucial role in the field of bioanalysis. Utilizing the high sensitivity, high biocompatibility, and visualization advantages of fluorescent probes is highly significant for achieving real-time in-situ detection and analysis of COX-2.

[0004] However, current fluorescent probes for detecting COX-2 have low sensitivity, insufficient selectivity, short fluorescence signal wavelengths, and are greatly affected by complex backgrounds within organisms, thus limiting their application in the field of bioanalysis. For example, patent application CN202410759469.7 describes a ratiometric fluorescent probe, its preparation method, and its application, which uses dicyanoisoflurone as the fluorophore, morpholine as the lysosomal targeting group, and a naphthalene ring as a bridging group. This probe exhibits high cytotoxicity, with cell viability already below 70% at 30 μM; its spectral properties depend on solvent polarity, resulting in strong interference; and its fluorescence signal wavelength is short. SUMMARY

[0005] In order to solve the above problems, the present application provides a dicyanofluoro ketone derivative. The dicyanofluoro ketone derivative has high sensitivity and high selectivity, and can detect COX-2 in aqueous solution and cells in the near-infrared wavelength range.

[0006] In order to achieve the above object, the present application adopts the following technical solution: The present application provides a dicyanofluoro ketone derivative, the structural formula of which is shown as formula I: Formula I The present application provides a preparation method of the above-mentioned dicyanofluoro ketone derivative, which comprises the following steps: Step 1: Dissolve dicyanofluoro ketone and p-acetylamino benzaldehyde completely in anhydrous ethanol, add a small amount of piperidine to catalyze the reaction, reflux for 5-6 hours, extract with dichloromethane, remove the solvent to obtain product A; dissolve product A obtained above in mixed solution 1 and reflux for 3-4 hours, add the reaction solution to twice the volume of ice water and adjust the pH to neutral, extract with dichloromethane, remove the solvent and separate by column chromatography to obtain a dicyanofluoro ketone fluorophore; Step 2: Dissolve the dicyanofluoro ketone fluorophore obtained in step 1 and 6-(Boc-amino) bromohexane completely in ethylene glycol methyl ether, add N,N-diisopropyl ethylamine dropwise, and reflux for 10-12 hours; remove the solvent to obtain product B; dissolve product B obtained above in mixed solution 2, stir at room temperature for 3-3.5 hours, add the reaction solution to twice the volume of ice water and adjust the pH to neutral, extract with dichloromethane and spin dry to obtain product C; dissolve product C and indometacin completely in dichloromethane, and add 4-dimethylamino pyridine and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride in an amount equal to that of product C to catalyze the reaction at room temperature for 1-2 hours; Step 3: After monitoring the completion of the reaction by thin layer chromatography, separate by column chromatography to obtain purple-red solid, which is the dicyanofluoro ketone derivative.

[0007] Preferably, in step 1, the molar ratio of dicyanofluoro ketone, p-acetylamino benzaldehyde and piperidine is 1:1:1-1:1.5:2; the volume ratio of concentrated hydrochloric acid and ethanol is 2:1-3:1; the column chromatography uses a silica gel column, and the eluent is a mixture of dichloromethane and petroleum ether in a volume ratio of 2:1.

[0008] Preferably, in step 1, the mixed solution 1 is a mixed solution prepared by mixing concentrated hydrochloric acid and ethanol in a volume ratio of 2:1-3:1.

[0009] Preferably, in step 2, the molar ratio of the dicyanoisofluoran derivative, 6-(Boc-amino) bromohexane, N,N-diisopropylethylamine and indomethacin is 1:1:1:1 ~ 1:1.5:2:1.5; the molar ratio of 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1 ~ 1.2:1.

[0010] Preferably, in step 2, the mixed solution 2 is a mixed solution prepared by mixing dichloromethane and trifluoroacetic acid in a volume ratio of 1:1 ~ 1.2:1.

[0011] Preferably, in step 3, the column chromatography is performed on a silica gel column, and the eluents used are dichloromethane and petroleum ether mixed in a volume ratio of 10:1, dichloromethane and methanol mixed in a volume ratio of 50:1, and dichloromethane and methanol mixed in a volume ratio of 100:1; the volume ratio of the above three eluents is 2:1:1 ~ 3:2:1.

[0012] The application also provides a use of the dicyanoisofluoran derivative as a near-infrared fluorescent probe for detecting COX-2.

[0013] Preferably, the detection method is as follows: the dicyanoisofluoran derivative is dissolved in dimethyl sulfoxide to prepare a 10 mM stock solution, which is stored in a 4℃ medicine refrigerator; during detection, the dicyanoisofluoran derivative stock solution is diluted with a Tris-HCl (pH 8.0) mixed solution, and the working concentration is 10 μM.

[0014] Preferably, the Tris-HCl (pH 8.0) mixed solution is a mixed solution composed of Tris-HCl (pH 8.0) buffer solution and DMSO, and the volume ratio of Tris-HCl (pH 8.0) buffer solution to DMSO is 9:1.

[0015] Preferably, the application is for detecting COX-2 in an aqueous solution.

[0016] Preferably, the application is for detecting endogenous COX-2 in a cell.

[0017] The application has the following advantages: (1) The derivative represented by formula I provided by the application has the advantages of small sample amount, high detection sensitivity, real-time in-situ detection, and great advantages in detecting COX-2 when detecting COX-2 in an aqueous solution and a cell.

[0018] (2) The derivative shown in formula I has good cell permeability and biocompatibility, can detect the expression of endogenous COX-2 in cells in real time in the near-infrared wavelength range, has strong biological penetration ability, and has a wide application field. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a graph of the experimental results of the dicyanoisatin derivative shown in formula I for selective detection of COX-2 in a mixed solution of DMSO and Tris-HCl (pH 8.0) buffer solution mixed at a volume ratio of 1:9; Figure 2 is a graph of the time-dependent fluorescence intensity change of the dicyanoisatin derivative shown in formula I for detecting COX-2 in a mixed solution of DMSO and Tris-HCl (pH 8.0) buffer solution mixed at a volume ratio of 1:9; Figure 3 is a graph of the fluorescence intensity change of the dicyanoisatin derivative shown in formula I for detecting COX-2 of different concentrations in a mixed solution of DMSO and Tris-HCl (pH 8.0) buffer solution mixed at a volume ratio of 1:9; Figure 4 is a graph of the results of the cytotoxicity study of the dicyanoisatin derivative shown in formula I on J774A.1 cells; Figure 5 is a laser confocal microscope imaging graph of the dicyanoisatin derivative shown in formula I for detecting endogenous COX-2 in J774A.1 cells. DETAILED DESCRIPTION

[0020] The present application will be specifically described below in combination with the drawings and specific examples. Example 1

[0021] A dicyanoisatin derivative, which is an organic small molecule compound based on a dicyanoisatin derivative, has the structure shown in formula I:

[0022] Formula I.

[0023] The preparation method of the above-mentioned dicyanoisatin derivative shown in formula I, and the synthesis route graph is as follows:

[0024] The above-mentioned preparation method specifically includes the following steps: (1) Dissolve dicyanofluorene of formula IV (280 mg, 1.5 mmol) and p-acetamidobenzaldehyde (244 mg, 1.5 mmol) in anhydrous ethanol, add 250 μL of piperidine dropwise and reflux for 6 h. Remove the solvent by extraction with dichloromethane. Dissolve the solid in a mixture of concentrated hydrochloric acid and anhydrous ethanol (volume ratio 2:1) and reflux for 3-4 h. Add the reaction solution dropwise to ice water and adjust the pH to neutral. Extract with dichloromethane and separate by column chromatography to obtain the dicyanofluorene fluorophore of formula III; (2) Dissolve the dicyanofluorene fluorophore of formula III (289 mg, 1 mmol) and 6-(Boc-amino) bromohexane (280 mg, 1 mmol) in ethylene glycol methyl ether and add N,N-diisopropylethylamine (250 μL) dropwise and reflux for 12 h. Re-dissolve the solvent in dichloromethane (2 mL) and trifluoroacetic acid (2 mL) and stir at room temperature for 3 h. Add the above reaction solution dropwise to ice water, adjust the pH and extract. Remove the solvent by rotary evaporation to obtain the compound of formula II; (3) Dissolve the compound of formula II obtained in step (2) in dichloromethane and add indometacin (251 mg, 0.7 mmol). Catalyze at room temperature for 1 h with 4-dimethylaminopyridine (85 mg, 0.7 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (134 mg, 0.7 mmol) to obtain the crude product of formula I; (4) After monitoring the completion of the reaction by thin layer chromatography, separate by column chromatography to obtain a purple red solid, which is the dicyanofluorene derivative of formula I.

[0025] The nuclear magnetic resonance spectrum of the purple red solid is as follows: 1 H NMR (400 MHz, CDCl3-d1) δ (ppm): 7.65 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H), 6.99 (d, J = 15.9 Hz, 1H), 6.89 (d, J = 2.5 Hz, 1H), 6.86 (d, J = 9.0 Hz, 1H), 6.78 (d, J = 15.9 Hz, 1H), 6.73 (s, 1H), 6.70 (dd, J = 9.0, 2.5 Hz, 1H), 6.57 (d, J = 8.7 Hz, 2H), 3.81 (s, 3H), 3.64 (s, 2H), 3.21 (t, J = 6.8 Hz, 2H), 3.10 (t,J = 7.0 Hz, 2H), 2.56 (s, 2H), 2.44 (s, 2H), 2.38 (s, 3H), 1.58-1.51 (m, 2H), 1.46-1.39 (m, 2H), 1.35-1.31 (m, 2H), 1.25-1.20 (m, 2H), 1.06 (s, 6H).

[0026] 13 C NMR (400 MHz, CDCl3-d1) δ (ppm): 169.98, 169.36, 168.49, 156.37, 155.36, 149.98, 139.77, 138.27, 136.45, 133.62, 131.32, 131.05, 130.42, 129.75, 129.36, 124.86, 124.56, 121.62, 115.22, 114.30, 113.52, 113.04, 112.92, 112.41, 101.02, 76.02, 55.8, 43.48, 43.12, 39.36, 32.38, 32.09, 29.55, 29.18, 28.16, 26.48, 13.36. Example 2

[0027] The application of a dicyanoisatin derivative as shown in formula I as a near-infrared fluorescent probe for detecting COX-2.

[0028] The principle of detecting COX-2 is shown as follows:

[0029] When the dicyanoisatin derivative as shown in formula I is used to detect COX-2, the response group indomethacin part of the derivative is recognized and combined by the detected COX-2, so that the PET effect of the probe is inhibited, thereby causing the quenched fluorescent signal to be restored in the near-infrared fluorescent wavelength range. Specifically, when the dicyanoisatin derivative as shown in formula I provided by the present application reacts with COX-2, the recognition group indomethacin part of the derivative specifically binds to the hydrophobic pocket composed of Arg120, Tyr355 and Glu524 in COX-2, forming an unfolded conformation, which inhibits the PET effect of the derivative, and the quenched fluorescent signal is restored in the near-infrared fluorescent wavelength range of 625 nm, thereby realizing the detection of COX-2.

[0030] The carboxyl group of indomethacin is replaced by an amide structure, which eliminates the inhibitory activity of COX-1 and retains the selectivity of COX-2. The affinity of the derivative to the tested enzyme COX-2 is much higher than that to other similar enzymes (such as COX-1), which can effectively distinguish between the two, and this "on-off" response mode greatly improves the signal-to-noise ratio and sensitivity of the detection. The combination of the present application makes the probe specific and reduces the influence of other interfering ions in the living body.

[0031] A mixed solution (volume ratio 9:1) of Tris-HCl buffer solution and dimethyl sulfoxide (DMSO) was used as the reaction system, and the final concentration of the derivative represented by formula I was 10 μM. The fluorescence intensity was detected at an excitation wavelength of 525 nm. Then COX-2 (dissolved in ultrapure water with a concentration of 10 μg / mL) was added to the above mixed solution, and the final concentration of the derivative was 10 μM, and the final concentration of COX-2 was 0.3 μg / mL. The maximum fluorescence emission wavelength under 525 nm excitation was 625 nm, and the fluorescence intensity at 625 nm near-infrared fluorescence wavelength at different time points (0 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min) was collected.

[0032] The reaction time and the fluorescence intensity at 625 nm wavelength were plotted as the abscissa and the ordinate respectively, and the time-dependent results of the probe for COX-2 detection were shown in Figure 1 . It can be seen from Figure 1 that the derivative reacts with COX-2 within 60 min and reaches a stable equilibrium. Therefore, the fluorescence intensity can reach saturation only after 60 min of reaction, and the derivative represented by formula I can realize rapid and sensitive detection of COX-2.

[0033] Selective detection of COX-2 in weak alkaline aqueous solution by dicyanoisatin derivative represented by formula I and spectral response

[0034] The dicyanoisatin derivative represented by formula I (dissolved in DMSO) and the tested ions (SO4 2- , Cl - , K + , S2O3 2- , Mg 2+ , Al 3+ , Na + , Ca 2+ , HSO3 - , HSO4 -, Vc, L-Arg, L-Cys, Gly, L-Ala, trypsin, BSA, ovalbumin, COX-1, COX-2 were dissolved in ultrapure water and added to a mixed solution prepared by mixing DMSO and Tris-HCl (100 mM, pH = 8.0) at a volume ratio of 1:9, wherein the final concentration of the derivative was 10 μM, the final concentration of SO4 2- , Cl - , K + , S2O3 2- , Mg 2+ , Al 3+ , Na + , Ca 2 + , HSO3 - , HSO4 - , the final concentration of Vc, L-Arg, L-Cys, Gly, L-Ala was 500 μM, the final concentration of trypsin, BSA, ovalbumin, COX-1 was 10 μg / mL, and the final concentration of COX-2 was 0.5 μg / mL. The corresponding solutions were respectively recorded as 1-21, and the solution without adding any test substance and only adding the derivative was recorded as 0. The fluorescence intensity at the maximum fluorescence emission wavelength of 625 nm was detected under an excitation wavelength of 525 nm.

[0035] The results of the selective detection of the dicyanofurone derivative represented by Formula I on COX-2 are shown in Figure 2 . It can be seen from Figure 2 that when COX-2 is added to the mixed solution prepared by mixing DMSO and Tris-HCl (100 mM, pH = 8.0) at a volume ratio of 1:9, the fluorescence intensity of the derivative at 625 nm is significantly enhanced; and when other possible interfering ions, molecules, common amino acids and proteases are added to the mixed solution prepared by mixing DMSO and Tris-HCl (100 mM, pH = 8.0) at a volume ratio of 1:9, the fluorescence intensity of the derivative at 625 nm hardly changes. Therefore, it is shown that the dicyanofurone derivative represented by Formula I can realize high selective detection of COX-2 in weakly basic aqueous solution.

[0036] The dicyanofurone derivative represented by Formula I (dissolved in DMSO) and COX-2 (dissolved in ultrapure water) were added to a mixed solution prepared by mixing DMSO and Tris-HCl (100 mM, pH = 8.0) at a volume ratio of 1:9. The final concentration of the derivative was 10 μM, and the final concentration of COX-2 was 0 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, respectively. The fluorescence intensity was detected under an excitation wavelength of 525 nm.

[0037] The results of fluorescence intensity detection are shown in [the table]. Figure 3 .Depend on Figure 3 It was found that when only the derivative shown in Formula I was present in the mixed solution prepared by mixing DMSO and Tris-HCl (100 mM, pH=8.0) at a volume ratio of 1:9, a weak fluorescence signal was generated at 625 nm under an excitation wavelength of 525 nm. When COX-2 was further added to the mixed solution, a stronger fluorescence signal was generated at 625 nm under an excitation wavelength of 525 nm, and the fluorescence signal significantly increased with increasing COX-2 concentration. Therefore, the dicyanoisoflurone derivative shown in Formula I has the purpose of highly selective and sensitive detection of COX-2 in vitro, especially for the detection and analysis of COX-2 in weakly alkaline aqueous solutions.

[0038] Experimental verification of the cell biocompatibility of the near-infrared probe of the dicyanoisoflurone derivative shown in Formula I.

[0039] The biocompatibility and safety of the dicyanoisoflurone derivative shown in Formula I were assessed using the Cell Counting Kit-8 (CCK-8) reagent, with human and mouse macrophage J774A.1 cells as the experimental cell line. After cells adhered to 96-well plates for 12 h, different concentrations of the fluorescent probe shown in Formula I (0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, and 45 μM) were added to multiple groups of cells and incubated for 6 h. Subsequently, 10 μL of CCK-8 solution was added to each well and incubated for 2 h. The absorbance of the samples at 450 nm was measured using a microplate reader, and the difference in absorbance reflected the cell viability.

[0040] The results of the study on the cytotoxicity of the dicyanoisoflurone derivative shown in Formula I against J774A.1 cells are as follows: Figure 4 As shown. By Figure 4 It can be seen that the dicyanoisoflurone derivative shown in Formula I has low toxicity to J774A.1 cells. Even at a derivative concentration of 10 μM, the survival rate of J774A.1 cells can still reach over 90%. In cell experiments, when using the derivative shown in Formula I at a working concentration of 10 μM and for a short treatment time, the effect of the probe on cell survival is negligible. In other words, the derivative has high safety and good biocompatibility for cells.

[0041] The near-infrared probe of dicyanoisoflurone derivative shown in Formula I is used for the detection of intracellular COX-2.

[0042] Using the mouse mononuclear macrophage cell line J774A.1 as an example, the dicyanoisoflurone derivative shown in Formula I was used to detect endogenous COX-2 in the cells.

[0043] Group a: the dicyanoisatone derivative of formula I (dissolved in DMSO, concentration 10 mM) was added to J774A.1 cells, and the final concentration of the dicyanoisatone derivative of formula I was 10 μM, and incubated at 37 °C for 60 min.

[0044] Group b: lipopolysaccharide (LPS, to further trigger the production of endogenous COX-2) was added to J774A.1 cells, and the final concentration of LPS was 1 μg / mL, and incubated at 37 °C for 24 h, and then washed with PBS buffer solution (10 mM, pH = 7.40), and then the dicyanoisatone derivative of formula I (dissolved in DMSO, concentration 10 mM) was added, and the final concentration of the dicyanoisatone derivative of formula I was 10 μM, and incubated at 37 °C for 60 min.

[0045] Group c: lipopolysaccharide (LPS, to stimulate the occurrence of inflammation and further trigger the production of endogenous COX-2) was added to J774A.1 cells, and the final concentration of LPS was 1 μg / mL, and incubated at 37 °C for 24 h, and then dexamethasone (DEX, to treat inflammation and reduce the production of endogenous COX-2) was added after 1 h of LPS incubation, and the final concentration of DEX was 500 μM, and incubated at 37 °C for 24 h, and then washed with PBS buffer solution (10 mM, pH = 7.40) after incubation, and then the dicyanoisatone derivative of formula I (dissolved in DMSO, concentration 10 mM) was added, and the final concentration of the dicyanoisatone derivative of formula I was 10 μM, and incubated at 37 °C for 60 min.

[0046] The laser confocal microscope imaging of each group of J774A.1 cells was detected, and the excitation wavelength was 515 nm, and the fluorescence collection wavelength was 575 nm-675 nm.

[0047] The confocal microscope imaging results are shown in Figure 5The imaging results show that when J774A.1 cells are only incubated with the dicyanoisatin derivative shown in formula I, a fluorescent signal can appear in the cells (group a), indicating that the dicyanoisatin derivative shown in formula I can detect endogenous COX-2 in cells; when LPS is added to J774A.1 cells, and then the dicyanoisatin derivative shown in formula I is added, it is observed that the fluorescent signal in J774A.1 cells is significantly enhanced compared with the fluorescent signal of the experimental control group a (group b); after the inflammation of the cells is treated with anti-inflammatory agent DEX after LPS stimulation, the fluorescent signal in the cells is significantly lower than that of group b. This shows that after LPS stimulates cell inflammation, the expression of COX-2 in the cells increases, and after the inflammation is treated, the level of COX-2 in the cells decreases, further proving that the dicyanoisatin derivative shown in formula I can detect different levels of endogenous COX-2 in cells.

[0048] Therefore, it is shown that the dicyanoisatin derivative shown in formula I provided in the present application can realize the monitoring of COX-2 in cells, and has good cell permeability and biological applicability.

[0049] In summary, the dicyanoisatin derivative fluorescent probe shown in formula I of the present application can be applied to the rapid and sensitive detection of low-concentration COX-2 in aqueous solution and cells, and the detection of COX-2 is carried out by directly combining the response group with COX-2, which has stronger specificity, and the change in spectrum is completely dependent on the content of COX-2 and is not affected by the solvent and the intracellular environment, which has important significance for the rapid detection of COX-2.

[0050] It should be noted that the above examples do not limit the present application in any form, and any technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present application.

Claims

1. A dicyanoisoflurone derivative, characterized in that, The structure of the dicyanoisoflurone derivative is shown in Formula I below: Formula I.

2. The method for preparing a dicyanoisoflurone derivative as described in claim 1, characterized in that, The method includes the following steps: Step 1: Dissolve dicyanoisoflurone and p-acetaminobenzaldehyde completely in anhydrous ethanol, add a small amount of piperidine to catalyze the reaction and reflux for 5-6 hours, then extract with dichloromethane to remove the solvent and obtain product A; dissolve product A completely in mixed solution 1 and reflux for 3-4 hours, add the reaction solution dropwise to twice the volume of ice water and adjust the pH to neutral, extract with dichloromethane to remove the solvent, and separate by column chromatography to obtain the dicyanoisoflurone fluorophore; Step 2: The dicyanoisoflurone fluorophore and 6-(Boc-amino)bromohexane obtained in Step 1 are completely dissolved in ethylene glycol methyl ether, and N,N-diisopropylethylamine is added dropwise and refluxed for 10-12 hours. After removing the solvent, product B is obtained. Product B is completely dissolved in mixed solution 2 and stirred at room temperature for 3-3.5 hours. The above reaction solution is adjusted to neutral pH by adding twice the volume of ice water. Dichloromethane is extracted and evaporated to dryness to obtain product C. Product C and indomethacin are completely dissolved in dichloromethane, and 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in an equimolar amount as product C are added and catalyzed at room temperature for 1-2 hours. Step 3: After the reaction is completed by thin-layer chromatography monitoring, the purple-red solid, namely dicyanoisoflurone derivative, is obtained by column chromatography separation.

3. The method for preparing the dicyanoisoflurone derivative as described in claim 2, characterized in that, In step 1, the molar ratio of dicyanoisoflurone, p-acetaminobenzaldehyde, and piperidine is 1:1:1 to 1:1.5:2; the volume ratio of concentrated hydrochloric acid to ethanol is 2:1 to 3:1; the column chromatography uses a silica gel column, and the eluent is a mixture of dichloromethane and petroleum ether in a volume ratio of 2:

1.

4. The method for preparing the dicyanoisoflurone derivative as described in claim 3, characterized in that, In step 1, the mixed solution 1 is a mixed solution prepared by concentrated hydrochloric acid and ethanol in a volume ratio of 2:1 to 3:

1.

5. The method for preparing the dicyanoisoflurone derivative as described in claim 2, characterized in that, In step 2, the molar ratio of the dicyanoisoflurone fluorophore, 6-(Boc-amino)bromohexane, N,N-diisopropylethylamine, and indomethacin is 1:1:1:1 ~ 1:1.5:2:1.5; the molar ratio of 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1 ~ 1.2:

1.

6. The method for preparing the dicyanoisoflurone derivative as described in claim 5, characterized in that, In step 2, the mixed solution 2 is a mixed solution prepared by dichloromethane and trifluoroacetic acid in a volume ratio of 1:1 to 1.2:

1.

7. The method for preparing the dicyanoisoflurone derivative as described in claim 2, characterized in that, In step 3, the column chromatography uses a silica gel column, and the eluents are used in the following order: dichloromethane and petroleum ether are mixed at a volume ratio of 10:1, dichloromethane and methanol are mixed at a volume ratio of 50:1, and dichloromethane and methanol are mixed at a volume ratio of 100:1; the volume ratio of the above three eluents is 2:1:1 to 3:2:

1.

8. The application of the dicyanoisoflurone derivative as described in claim 1 as a near-infrared fluorescent probe for detecting COX-2.

9. The application of the dicyanoisoflurone derivative as described in claim 8 as a near-infrared fluorescent probe for detecting COX-2, characterized in that, The application is in the detection of COX-2 in aqueous solution.

10. The application of the dicyanoisoflurone derivative as described in claim 8 as a near-infrared fluorescent probe for detecting COX-2, characterized in that, The application is in the detection of endogenous COX-2 in cells.

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