A dicyanoketone derivative, a preparation method thereof and application of the dicyanoketone derivative as a near-infrared fluorescent probe for detecting COX-2

Abstract

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

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, composed of 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 of the Invention

[0005] To address the shortcomings of the prior art, this application provides a dicyanoisoflurone derivative. This dicyanoisoflurone derivative exhibits high sensitivity and selectivity, enabling the detection of COX-2 in aqueous solutions and intracellular cells within the near-infrared wavelength range.

[0006] To achieve the above objectives, this application adopts the following technical solution:

[0007] This application provides a dicyanoisoflurone derivative, the structural formula of which is shown in Formula I:

[0008]

[0009] This application provides a method for preparing the above-mentioned dicyanoisoflurone derivative, comprising the following steps:

[0010] 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 the obtained 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;

[0011] 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.

[0012] 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.

[0013] Preferably, 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.

[0014] Preferably, 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.

[0015] Preferably, in step 2, the molar ratio of dicyanoisoflurone fluorophore, 6-(Boc-amino)bromohexane, N,N-diisopropylethylamine, and indomethacin is 1:1:1:1 ~ 1:1.5:2:1.5; and the molar ratio of 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1 ~ 1.2:1.

[0016] Preferably, 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.

[0017] Preferably, in step 3, the column chromatography uses a silica gel column, and the eluents are, in order, dichloromethane and petroleum ether mixed at a volume ratio of 10:1, dichloromethane and methanol mixed at a volume ratio of 50:1, and dichloromethane and methanol mixed at a volume ratio of 100:1; the volume ratio of the above three eluents is 2:1:1 to 3:2:1.

[0018] This application also provides an application of a dicyanoisoflurone derivative as a near-infrared fluorescent probe for detecting COX-2.

[0019] Preferably, the detection method is as follows: the dicyanoisoflurone derivative is dissolved in dimethyl sulfoxide to prepare a 10 mM stock solution, which is stored in a pharmaceutical cooler at 4°C; during detection, the dicyanoisoflurone derivative stock solution is diluted with a Tris-HCl (pH 8.0) mixed solution to make its working concentration 10 μM.

[0020] Preferably, the Tris-HCl (pH 8.0) mixed solution is a mixture of Tris-HCl (pH 8.0) buffer solution and DMSO, with a volume ratio of Tris-HCl (pH 8.0) buffer solution to DMSO of 9:1.

[0021] Preferably, the application is in the detection of COX-2 in aqueous solution.

[0022] Preferably, the application is in the detection of endogenous COX-2 in cells.

[0023] The advantages of this application are:

[0024] (1) The derivatives of Formula I provided in this application have a small sample volume and high detection sensitivity when detecting COX-2 in aqueous solution and cells, and can realize real-time in-situ detection, which has great advantages in detecting COX-2.

[0025] (2) The derivatives shown in Formula I provided in this application have good cell permeability and biocompatibility, and can detect the expression of endogenous COX-2 in cells in real time in the near-infrared wavelength range. They have strong biological penetration ability and are widely used. Attached Figure Description

[0026] Figure 1 The figure shows the experimental results of selective detection of COX-2 by the dicyanoisoflurone derivative shown in Formula I in a mixture of DMSO and Tris-HCl (pH 8.0) buffer solution at a volume ratio of 1:9.

[0027] Figure 2 The graph shows the time-dependent fluorescence intensity change of COX-2 detected in a mixture of DMSO and Tris-HCl (pH 8.0) buffer solution at a volume ratio of 1:9, representing the dicyanoisoflurone derivative of Formula I.

[0028] Figure 3 The graph shows the fluorescence intensity changes of different concentrations of COX-2 in a mixture of DMSO and Tris-HCl (pH 8.0) buffer solution at a volume ratio of 1:9, representing the dicyanoisoflurone derivative of Formula I.

[0029] Figure 4 This is a diagram showing the cytotoxicity study results of the dicyanoisoflurone derivative shown in Formula I on J774A.1 cells;

[0030] Figure 5 This is a laser confocal microscopy image of the dicyanoisoflurone derivative shown in Formula I detecting endogenous COX-2 in J774A.1 cells;

[0031] Figure 6 It is a dicyanoisoflurone derivative represented by Formula I. 1 H NMR spectrum;

[0032] Figure 7 It is a dicyanoisoflurone derivative represented by Formula I. 13 C NMR spectrum. Detailed Implementation

[0033] The present application will be described in detail below with reference to the accompanying drawings and specific embodiments. Example 1

[0034] A dicyanoisoflurone derivative, wherein the derivative is a small organic molecule compound based on dicyanoisoflurone derivatives, and the structure is shown in Formula I:

[0035] .

[0036] The preparation method of the dicyanoisoflurone derivative shown in Formula I above, and the synthetic route are as follows:

[0037]

[0038] The above preparation method specifically includes the following steps:

[0039] (1) Dissolve dicyanoisoflurone form IV (280 mg, 1.5 mmol) and p-acetaminobenzaldehyde (244 mg, 1.5 mmol) in anhydrous ethanol, add 250 μL piperidine dropwise and reflux for 6 h, then extract with dichloromethane to remove the solvent. Dissolve the solid in a mixed solution of concentrated hydrochloric acid and anhydrous ethanol (volume ratio 2:1) and reflux for 3 h to 4 h. Add the reaction solution dropwise to ice water and adjust the pH to neutral. After dichloromethane extraction, separate by column chromatography to obtain dicyanoisoflurone fluorophore form III.

[0040] (2) Dissolve dicyanoisoflurone fluorophore formula III (289 mg, 1 mmol) and 6-(Boc-amino)bromohexane (280 mg, 1 mmol) thoroughly in ethylene glycol methyl ether and add N,N-diisopropylethylamine (250 μL) dropwise and reflux for 12 h. After removing the solvent, dissolve the compound in dichloromethane (2 mL) and trifluoroacetic acid (2 mL) and stir at room temperature for 3 h. Adjust the pH of the above reaction by adding ice water dropwise and extract by rotary evaporation to remove the solvent to obtain compound formula II.

[0041] (3) The compound of formula II obtained in step (2) was dissolved in dichloromethane and then indomethacin (251 mg, 0.7 mmol) was added. The mixture was then reacted with 4-dimethylaminopyridine (85 mg, 0.7 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (134 mg, 0.7 mmol) at room temperature for 1 h to obtain the crude product of formula I.

[0042] (4) After the reaction is completed by thin-layer chromatography monitoring, a purple-red solid is obtained by column chromatography separation, namely the dicyanoisoflurone derivative shown in Formula I.

[0043] The nuclear magnetic resonance spectrum of the purplish-red solid is as follows:

[0044] 1 H NMR (400MHz, CDCl3-d1) δ (ppm): 7.65 (d, J =8.6Hz, 2H), 7.47 (d, J =8.6Hz, 2H), 7.34 (d, J =8.7Hz, 2H), 6.99 (d, J =15.9Hz, 1H), 6.89 (d, J =2.5Hz, 1H), 6.86 (d, J=9.0Hz, 1H), 6.78 (d, J =15.9Hz, 1H), 6.73 (s, 1H), 6.70 (dd, J =9.0, 2.5Hz, 1H), 6.57 (d, J =8.7Hz, 2H), 3.81 (s, 3H), 3.64 (s, 2H), 3.21 (t, J =6.8Hz, 2H), 3.10 (t, J =7.0Hz, 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).

[0045] 13 C NMR (400MHz, 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

[0046] Application of a dicyanoisoflurone derivative as shown in Formula I as a near-infrared fluorescent probe for detecting COX-2.

[0047] The principle of COX-2 detection is as follows:

[0048]

[0049] When COX-2 is detected using the dicyanoisoflurone derivative shown in Formula I, the indomethacin moiety of the derivative's responsive group is recognized and bound by the analyte COX-2, suppressing the PET effect of the probe and thus recovering the quenched fluorescence signal in the near-infrared fluorescence wavelength range. Specifically, when the dicyanoisoflurone derivative shown in Formula I provided in this application reacts with COX-2, the indomethacin moiety of the derivative's recognition group specifically binds to the hydrophobic pocket composed of Arg120, Tyr355, and Glu524 in COX-2, forming an unfolded conformation. This suppresses the PET effect of the derivative, and the quenched fluorescence signal is recovered in the near-infrared fluorescence wavelength range of 625 nm, thereby achieving the detection of COX-2.

[0050] The substitution of the carboxyl group in indomethacin with an amide structure eliminates the inhibitory activity against COX-1 while retaining selectivity for COX-2. The derivative exhibits a significantly higher affinity for the analyte COX-2 than other similar enzymes (such as COX-1), effectively distinguishing between the two. Furthermore, this "off-on" response mode greatly improves the signal-to-noise ratio and sensitivity of the detection. This binding mechanism in this application results in high probe specificity and reduces the influence of other interfering ions in the body.

[0051] A mixed solution of Tris-HCl buffer and dimethyl sulfoxide (DMSO) (volume ratio 9:1) was used as the reaction system. The final concentration of the derivative shown in Formula I was 10 μM, and the fluorescence intensity was detected at an excitation wavelength of 525 nm. Then, COX-2 (dissolved in ultrapure water, concentration 10 μg / mL) was added to the above mixed solution, where 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 the 625 nm near-infrared fluorescence wavelength was collected at different time points (0 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min).

[0052] Plotting reaction time and fluorescence intensity at 625 nm as the x and y axes respectively, the time dependence of the probe on COX-2 detection is shown in the figure. Figure 1 .Depend on Figure 1 It can be seen that the derivative reacts completely with COX-2 within 60 minutes and reaches a stable equilibrium. This indicates that the fluorescence intensity can reach saturation in just 60 minutes, and the derivative shown in Formula I can achieve rapid and sensitive detection of COX-2.

[0053] Selectivity and spectral response of dicyanoisoflurone derivatives in weakly alkaline aqueous solutions as shown in Formula I

[0054] The dicyanoisoflurone derivative shown in Formula I (dissolved in DMSO) and the analyte ion (SO4) were then mixed. 2- Cl - K + S2O3 2- Mg 2+ Al 3+ Na + Ca 2+ HSO3 - HSO4 - Vitamin C, L-Arg, L-Cys, Gly, L-Ala, trypsin, BSA, ovalbumin, COX-1, and COX-2 (dissolved in ultrapure water) were added to a mixed solution of DMSO and Tris-HCl (100 mM, pH=8.0) at a volume ratio of 1:9, wherein the final concentration of the derivatives was 10 μM, and SO42- 2- Cl - K + S2O3 2- Mg 2+ Al 3+ Na + Ca 2 + HSO3 - HSO4 - The final concentrations of Vc, L-Arg, L-Cys, Gly, and L-Ala were 500 μM; the final concentrations of trypsin, BSA, ovalbumin, and COX-1 were 10 μg / mL; and the final concentration of COX-2 was 0.5 μg / mL. The corresponding solutions were labeled 1–21, and the solution with no analyte and only the derivative was added was labeled 0. The fluorescence intensity at the maximum fluorescence emission wavelength of 625 nm was detected at an excitation wavelength of 525 nm.

[0055] The detection results of the selectivity of the dicyanoisoflurone derivative shown in Formula I for COX-2 are shown in the figure. Figure 2 .Depend on Figure 2 It was found that when COX-2 was added to a mixed solution of 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 was significantly enhanced. However, when other potentially interfering ions, molecules, common amino acids, and proteases were added to the mixed solution of 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 remained almost unchanged. This indicates that the dicyanoisoflurone derivative shown in Formula I can achieve highly selective detection of COX-2 in weakly alkaline aqueous solutions.

[0056] The dicyanoisoflurone derivative (dissolved in DMSO) and COX-2 (dissolved in ultrapure water) shown in Formula I 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 concentrations of COX-2 were 0 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, and 0.5 μg / mL, respectively. The fluorescence intensity was detected at an excitation wavelength of 525 nm.

[0057] 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.

[0058] Experimental verification of the cell biocompatibility of the near-infrared probe of the dicyanoisoflurone derivative shown in Formula I.

[0059] 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.

[0060] 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 4It 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.

[0061] The near-infrared probe of dicyanoisoflurone derivative shown in Formula I is used for the detection of intracellular COX-2.

[0062] 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.

[0063] Group a: Add the dicyanoisoflurone derivative shown in Formula I (dissolved in DMSO at a concentration of 10 mM) to J774A.1 cells and incubate at 37°C for 60 min.

[0064] Group b: Lipopolysaccharide (LPS) was added to J774A.1 cells to further trigger the generation of endogenous COX-2. The final concentration of LPS was 1 μg / mL. The cells were incubated at 37°C for 24 h. After incubation, the cells were washed with PBS buffer (10 mM, pH=7.40). Then, the dicyanoisoflurone derivative shown in Formula I (dissolved in DMSO, concentration 10 mM) was added. The final concentration of the dicyanoisoflurone derivative shown in Formula I was 10 μM. The cells were incubated at 37°C for 60 min.

[0065] Group C: Lipopolysaccharide (LPS) was added to J774A.1 cells to stimulate inflammation and further trigger the production of endogenous COX-2. The final concentration of LPS was 1 μg / mL. After incubation at 37°C for 24 h, dexamethasone (DEX) was added after 1 h of LPS incubation. The final concentration of DEX was 500 μM. After incubation at 37°C for 24 h, the cells were washed with PBS buffer (10 mM, pH=7.40). Then, dicyanoisoflurone derivative (dissolved in DMSO, concentration 10 mM) as shown in Formula I was added. The final concentration of dicyanoisoflurone derivative as shown in Formula I was 10 μM. After incubation at 37°C for 60 min, the cells were incubated.

[0066] Laser confocal microscopy was used to examine the imaging of J774A.1 cells in each group. The excitation wavelength was 515 nm, and the fluorescence collection wavelength was 575 nm to 675 nm.

[0067] Confocal microscopy imaging results are shown in Figure 5 Imaging results showed that when J774A.1 cells were incubated only with the dicyanoisoflurone derivative shown in Formula I, intracellular fluorescence signals appeared (group a), indicating that the dicyanoisoflurone derivative shown in Formula I can detect endogenous COX-2 in cells. When LPS was added to J774A.1 cells, followed by the addition of the dicyanoisoflurone derivative shown in Formula I, the fluorescence signal in J774A.1 cells was significantly enhanced compared to the fluorescence signal in the experimental control group a (group b). When LPS stimulated cell inflammation and then treated with the anti-inflammatory reagent DEX, the intracellular fluorescence signal was significantly lower than that in group b. This indicates that LPS stimulation of cell inflammation leads to an increase in intracellular COX-2 expression, while the intracellular COX-2 level decreases after the inflammation is treated, further proving that the dicyanoisoflurone derivative shown in Formula I can detect different levels of endogenous COX-2 in cells.

[0068] This demonstrates that the dicyanoisoflurone derivative of Formula I provided in this application can achieve COX-2 monitoring in cells and has good cell permeability and biocompatibility.

[0069] In summary, the dicyanoisoflurone derivative fluorescent probe shown in Formula I of this application can be applied to the rapid and sensitive detection of low concentrations of COX-2 in aqueous solutions and cells. It detects COX-2 by directly binding to COX-2 with the responder group, which has higher specificity. The change in spectrum depends entirely on the COX-2 content and is not affected by solvent or intracellular environment, which is of great significance for the rapid detection of COX-2.

[0070] It should be noted that the above embodiments do not limit this application in any way, and all technical solutions obtained by means of equivalent substitution or equivalent transformation fall within the protection scope of this application.

Claims

1. A dicyanoisoflurone derivative, characterized in that, The structure of the dicyanoisoflurone derivative is shown in Formula I below: 。 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 mixed solution 1 is a mixed solution prepared by concentrated hydrochloric acid and ethanol in a volume ratio of 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 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.

5. The method for preparing the dicyanoisoflurone derivative as described in claim 4, 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.

6. 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.

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

8. The application of the dicyanoisoflurone derivative as described in claim 7 in the preparation of a near-infrared fluorescent probe for detecting COX-2, characterized in that, The application is in the detection of COX-2 in aqueous solution.

9. The application of the dicyanoisoflurone derivative as described in claim 7 in the preparation of 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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