A cox-2 targeting fluorescent probe and a preparation method and application thereof

Abstract

The application relates to a COX-2-targeted fluorescent probe and a preparation method and application thereof. The structure of the fluorescent probe is shown in the following. The fluorescent probe in the application can distinguish normal cells and breast cancer cells through the expression level of COX-2, can be used as an important strategy for developing cancer detection and research, and has potential as a guide agent in tumor surgery.

Description

Technical Field

[0001] This invention relates to a COX-2 targeted fluorescent probe, its preparation method, and its application. Background Technology

[0002] Cancer has long been known for its low cure rate and high mortality rate, largely because it is often difficult to diagnose accurately in its early stages, causing patients to miss the optimal treatment window. Accurate early diagnosis of cancer significantly improves patient survival rates. Currently, several imaging techniques are used in cancer diagnosis, such as computed tomography (CT), magnetic resonance imaging (MRI), radionuclide scanning, and positron emission tomography (PET). While these techniques can detect abnormal masses in the body, they are insufficient for definitive cancer diagnosis based on images and have limitations, including low resolution and the use of harmful ionizing radiation. Fluorescence imaging, on the other hand, is favored due to its high sensitivity, high resolution, and minimal damage during detection and imaging. Therefore, many fluorescent probes have been developed for use in cancer imaging research.

[0003] The key to early cancer diagnosis lies in distinguishing cancer cells from normal cells. In the early stages of cancer, the morphology and structure of some cancer cells are not fully formed, making them difficult to identify by their appearance. However, significant differences have already emerged between the cells and normal cells, such as increased expression of certain enzymes, changes in intracellular pH or viscosity, and alterations in reactive oxygen species levels. Therefore, there is an urgent need for an effective method to track changes in the intracellular environment or enzymes. Summary of the Invention

[0004] To address the shortcomings of the existing technologies, this application provides a COX-2 targeted fluorescent probe, its preparation method, and its application.

[0005] A COX-2 targeting fluorescent probe, the structure of which is shown below:

[0006] The group R is selected from one of the following formulas II-IV: .

[0007] This invention also provides a method for preparing a COX-2 targeted fluorescent probe, comprising the following steps: Step 1: Dissolve 4-bromo-1,8-naphthalenedicarboxylic anhydride and 4-aminobutyric acid in anhydrous ethanol. After the reflux reaction is completed, cool the reaction solution to room temperature, remove the solvent, purify and separate to obtain compound A1. Step 2: Compound A1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, and triethylamine were dissolved in dichloromethane and reacted under N2 protection for a certain time. Celecoxib was then added. The reaction was carried out at room temperature under N2 protection. After the reaction was completed, the mixture was extracted, dehydrated, distilled under reduced pressure, and purified to obtain a white solid compound A2. Step 3: Compound A2,4-boronic acid triphenylamine, tetra(triphenylphosphine)palladium, and potassium carbonate are dissolved in a mixed solvent of methanol / toluene = 1 / 1, reacted at 85°C, and then extracted, dehydrated, distilled under reduced pressure, purified and separated to obtain fluorescent probe S1.

[0008] This invention also provides a method for preparing a COX-2 targeted fluorescent probe, comprising the following steps: Step 1: Dissolve 4-bromo-1,8-naphthalenedicarboxylic anhydride and 4-aminobutyric acid in anhydrous ethanol. After the reflux reaction is completed, cool the reaction solution to room temperature, remove the solvent, purify and separate to obtain compound A1. Step 2: Compound A1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, and triethylamine were dissolved in dichloromethane and reacted under N2 protection for a certain time. Celecoxib was then added. The reaction was carried out at room temperature under N2 protection. After the reaction was completed, the mixture was extracted, dehydrated, distilled under reduced pressure, and purified to obtain a white solid compound A2. Step 3: Compound A2 and p-aminophenol were added to N,N-dimethylformamide. After stirring at room temperature under N2 protection, sodium hydride was added and heated and stirred under N2 protection. After the reaction was completed, extraction, dehydration, vacuum distillation and purification were performed to obtain fluorescent probe S2.

[0009] This invention also provides a method for preparing a COX-2 targeted fluorescent probe, comprising the following steps: Step 1: Dissolve 4-bromo-1,8-naphthalenedicarboxylic anhydride and 4-aminobutyric acid in anhydrous ethanol. After the reflux reaction is completed, cool the reaction solution to room temperature, remove the solvent, purify and separate to obtain compound A1. Step 2: Compound A1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, and triethylamine were dissolved in dichloromethane and reacted under N2 protection for a certain time. Celecoxib was then added. The reaction was carried out at room temperature under N2 protection. After the reaction was completed, the mixture was extracted, dehydrated, distilled under reduced pressure, and purified to obtain a white solid compound A2. Step 3: Compound A2, hydroquinone, and potassium carbonate are dissolved in acetonitrile. After the reaction is completed under N2 protection, the mixture is extracted, dehydrated, distilled under reduced pressure, purified, and separated to obtain fluorescent probe S3.

[0010] Furthermore, the 4-bromo-1,8-naphthalenedicarboxylic anhydride and 4-aminobutyric acid are dissolved in anhydrous ethanol at a molar ratio of 1:1.2.

[0011] Furthermore, the compound A1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, triethylamine, and celecoxib are dissolved in dichloromethane in a molar ratio of 1:2.3:0.31:3:1.3.

[0012] Furthermore, the compound A2,4-boronic acid triphenylamine, tetra(triphenylphosphine)palladium, and potassium carbonate are dissolved in a mixed solvent of methanol / toluene in a molar ratio of 1:1.3:0.05:3.

[0013] Furthermore, compound A2 and p-aminophenol are dissolved in N,N-dimethylformamide in a molar ratio of 1:1.2.

[0014] Furthermore, the compound A2, hydroquinone, and potassium carbonate are dissolved in acetonitrile in a ratio of 1:3:6.

[0015] This invention also provides an application of a COX-2-targeting fluorescent probe in human umbilical vein endothelial cells to target COX-2 and respond to the expression levels of viscosity, hypochlorous acid, or peroxynitrite in the internal environment.

[0016] This invention also provides an application of a COX-2-targeting fluorescent probe in targeting COX-2 in breast cancer cells and responding to the expression levels of viscosity, hypochlorous acid, or peroxynitrite in the internal environment.

[0017] Beneficial Effects: Cyclooxygenase-2 (COX-2) is overexpressed in cancers such as breast cancer and liver cancer, and is closely related to tumor growth and metastasis. Novel fluorescent probes are synthesized by linking COX-2 selective inhibitors, tumor marker responsive groups, and fluorescent groups. This application uses naphthalimide as the fluorescent group, celecoxib as the targeting group, and long-chain alkyl groups as linking groups, and incorporates triphenylamine, p-aminophenol, and hydroquinone as viscosity, hypochlorous acid, and peroxynitrite responsive groups to construct three fluorescent probes with different responsive groups. All three probes exhibit good pH stability, photostability, and plasma stability. The fluorescence intensity of fluorescent probe S1 responds to changes in viscosity and polarity. Attached Figure Description

[0018] Figure 1 The intermediate product compound A1 of this invention 1 H NMR spectrum.

[0019] Figure 2 The intermediate product compound A1 of this invention 13 C NMR spectrum.

[0020] Figure 3 The intermediate product compound A2 of this invention 1 H NMR spectrum.

[0021] Figure 4 The intermediate product compound A2 of this invention 13 C NMR spectrum.

[0022] Figure 5 The fluorescent probe S1 of Example 1 of the present invention 1 H NMR spectrum.

[0023] Figure 6 The fluorescent probe S1 of Example 1 of the present invention 13 C NMR spectrum.

[0024] Figure 7 The image shows the HR-MS spectrum of the fluorescent probe S1 in Example 1 of this invention.

[0025] Figure 8 The comparative fluorescent probe S0 of this invention 1 H NMR spectrum.

[0026] Figure 9 The comparative fluorescent probe S0 of this invention 13 C NMR.

[0027] Figure 10 The image shows the HR-MS spectrum of the comparative fluorescent probe S0 of this invention.

[0028] Figure 11 The fluorescent probe S2 of Example 2 of the present invention 1 H NMR spectrum.

[0029] Figure 12 The fluorescent probe S2 of Example 2 of the present invention 13 C NMR spectrum.

[0030] Figure 13 This is the HR-MS spectrum of the fluorescent probe S2 in Example 2 of the present invention.

[0031] Figure 14 The fluorescent probe S3 in Example 3 of this invention 1 H NMR spectrum.

[0032] Figure 15 The fluorescent probe S3 in Example 3 of this invention 13 C NMR spectrum.

[0033] Figure 16 The image shows the HR-MS spectrum of the fluorescent probe S3 reaction solution in Example 3 of this invention.

[0034] Figure 17 Cell imaging and average fluorescence intensity of fluorescent probe S1 and comparative fluorescent probe S0 in Example 1 of this invention. Figure 17 A consists of HUVEC cells treated with probe S0; Figure 17 B is MCF-7 cells treated with probe S0; Figure 17 C consisted of MCF-7 cells treated with probe S1. Results are presented as mean ± SD, n = 3. Compared with group A, *** P<0.001.

[0035] Figure 18 The image shows a cell imaging pattern and average fluorescence intensity of the fluorescent probe S1 in Example 1 of this invention, with λex = 422 nm and λem = 610 nm. Results are expressed as mean ± SD, n = 3. Compared to HUVEC, *** P<0.001.

[0036] Figure 19 Cell imaging and average fluorescence intensity of fluorescent probe S2 in Example 2 of this invention: (A) HUVEC cells incubated with fluorescent probe S2; (B) HUVEC cells incubated with fluorescent probe S2 followed by the addition of 100 μM hypochlorous acid; (C) HUVEC cells incubated with LPS (5 μg / mL) and fluorescent probe S2; (D) MCF-7 cells incubated with fluorescent probe S2; (E) MCF-7 cells incubated with fluorescent probe S2 followed by the addition of 100 μM hypochlorous acid; (F) MCF-7 cells incubated with LPS (5 μg / mL) and fluorescent probe S2. λex = 460 nm, λem = 560 nm. Results are expressed as mean ± SD, n = 3. Compared with group A, ** P<0.01, *** P < 0.001.

[0037] Figure 20 Cell imaging and mean fluorescence intensity of fluorescent probe S3 in Example 3 of this invention: (A) HUVEC cells incubated with probe S3; (B) HUVEC cells incubated with probe S3 and SIN-1 (500 μM); (C) MCF-7 cells incubated with probe S3; (D) MCF-7 cells incubated with probe S3 and SIN-1 (500 μM). λex = 460 nm, λem = 560 nm. Results are expressed as mean ± SD, n = 3. Compared with group A, *** P < 0.001.

[0038] Figure 21 The images show the fluorescence response spectra of the fluorescent probes in Examples 1, 2, and 3 of this invention.

[0039] Figure 22This is the fluorescence response of fluorescent probe S1 to various competing substances in Example 1 of the present invention.

[0040] Figure 23 This is the fluorescence response of fluorescent probe S2 to various competing substances in Example 2 of the present invention.

[0041] Figure 24 This is the fluorescence response of fluorescent probe S3 to various competing substances in Example 3 of the present invention.

[0042] Figure 25 Example 1 of this invention: Fluorescent probe S1 (5 μM) in methanol aqueous solution at different percentages. The fluorescence intensity in the image is λex = 422 nm and λem = 610 nm. Detailed Implementation

[0043] The 4-bromo-1,8-naphthalenedicarboxylic anhydride, 4-aminobutyric acid, celecoxib, p-aminophenol, hydroquinone, triphenylamine 4-boronic acid, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetraazole bromide were purchased from Adamas, Sanam Chemical, TCI, Aladdin, Hydroquinone, BASF, Shanghai, Shanghai Yuanye Biotechnology Co., Ltd., and Solaibio, unless otherwise specified, were all purchased from commercial suppliers and required no further purification before use. UV-vis absorption spectra were obtained on a Shimadzu spectrophotometer. Fluorescence spectra were measured on a Perkin-Elmer fluorescence spectrophotometer. pH was measured using a Mettler Toledo pH meter. TLC analysis was performed on silica gel plates, and column chromatography was performed on silica gel (200-300 mesh). ¹H and ¹³C NMR spectra were measured on a Bruker 600 MHz NMR spectrometer using tetramethylsilane (TMS) as an internal reference. Analysis was performed using Thermofisher high-resolution mass spectrometry (HRMS). High-performance liquid chromatography (HPLC) chromatograms were obtained on an Agilent high-performance liquid chromatograph.

[0044] I. Structure of the COX-2 Targeted Fluorescent Probe The structure of the COX-2 targeting fluorescent probe is shown in Formula I below:

[0045] The group R is selected from one of the following formulas II-IV: .

[0046] II. Preparation method of COX-2 targeted fluorescent probe

[0047] Example 1

[0048] A method for preparing a COX-2-targeting fluorescent probe includes the following steps: Step 1: 4-Bromo-1,8-naphthalenedicarboxylic anhydride (277 mg, 1 mmol) and 4-aminobutyric acid (123.7 mg, 1.2 mmol) were added to 5 mL of anhydrous ethanol and refluxed at 78 °C for 2 hours. After the reaction was complete, the reaction solution was cooled to room temperature, and the solvent was removed by vacuum distillation to obtain the crude product. Purification and separation by column chromatography (eluent: dichloromethane / methanol = 30 / 1, v / v) yielded a pale yellow solid compound A1 in 85% yield.

[0049] The intermediate product 1 H NMR and 13 The characterization results of C NMR are as follows: 1 H NMR (600 MHz, DMSO-d6) δ:11.99 (s, 1H), 8.53 (ddd, J = 18.1, 7.9,1.1 Hz, 2H), 8.30 (d, J = 7.9 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.98 (dd, J= 8.5, 7.3 Hz, 1H), 4.07 (t, J = 7.0 Hz, 2H), 2.31 (t, J = 7.3 Hz, 2H), 1.89(m, J = 7.2 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 174.48, 163.53, 163.48, 133.00, 132.01,131.80, 131.40, 130.27, 129.48, 129.26, 128.88, 123.37, 122.60, 40.52, 31.78,23.37. Step 2: Compound A1 (72.2 mg, 0.2 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (87.97 mg, 0.46 mmol), 4-dimethylaminopyridine (37.9 mg, 0.062 mmol), and triethylamine (60.71 mg, 0.6 mmol) were added to dichloromethane and reacted under N2 protection for 30 minutes. Celecoxib (99.06 mg, 0.26 mmol) was then added. The reaction was continued overnight at room temperature under N2 protection. After the reaction was complete, the reaction solution was extracted with saturated brine. The extracted solution was then dehydrated by adding anhydrous sodium sulfate for 30 minutes, and finally, the crude product was obtained by vacuum distillation. The crude product was purified by column chromatography (eluent: dichloromethane / methanol = 35 / 1, v / v) to obtain a white solid compound A2 in 77% yield.

[0050] The intermediate product 1 H NMR and 13 The characterization results of C NMR are as follows: 1 H NMR (600 MHz, DMSO-d6) δ:12.16 (s, 1H), 8.55 (d, J = 7.9 Hz, 2H), 8.32 (d, J = 7.8 Hz, 1H), 8.22 (d, J = 7.9 Hz, 1H), 8.00 (t, J = 7.9 Hz, 1H),7.97 – 7.94 (m, 2H), 7.59 – 7.54 (m, 2H), 7.20 (s, 1H), 7.17 (s, 4H), 3.99(t, J = 6.8 Hz, 2H), 2.33 (t, J = 7.4 Hz, 2H), 2.25 (s, 3H), 1.82 (m, J = 7.1Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 171.77, 163.52, 163.48, 145.82, 142.95,142.82, 142.70, 139.70, 139.59, 133.07, 132.08, 131.83, 131.47, 130.31,129.87, 129.51, 129.29, 129.24, 129.19, 128.93, 126.45, 125.66, 123.38,122.61, 120.83, 106.74, 40.52, 33.52, 22.83, 21.22. Step 3: Compound A2 (144.8 mg, 0.2 mmol), triphenylamine 4-borate (75.18 mg, 0.26 mmol), tetrakis(triphenylphosphine)palladium (11.56 mg, 0.01 mmol), and potassium carbonate (82.92 mg, 0.6 mmol) were added to a 1 / 1 mixture of methanol and toluene and reacted at 85°C for 8 hours. After the reaction, the solution was extracted with saturated brine. The extracted solution was then distilled under reduced pressure to obtain the crude product. Purification and separation were performed by column chromatography (eluent: dichloromethane / methanol = 40 / 1, v / v) to obtain the orange fluorescent probe S1 in 67% yield.

[0051] The final product 1 H NMR and 13 The characterization results of C NMR are as follows: 1 H NMR (600 MHz, CDCl3) δ:8.61 – 8.57 (m, 2H), 8.40 (dd, J = 8.5, 1.2Hz, 1H), 8.12 – 8.08 (m, 2H), 7.72 – 7.64 (m, 2H), 7.50 – 7.44 (m, 2H), 7.33(ddd, J = 8.5, 4.7, 2.4 Hz, 6H), 7.23 – 7.18 (m, 6H), 7.16 (d, J = 8.0 Hz,2H), 7.12 – 7.07 (m, 4H), 6.72 (s, 1H), 4.16 (t, J = 6.4 Hz, 2H), 2.36 (d, J= 7.8 Hz, 5H), 2.07 (m, J = 6.3 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 170.69, 164.89, 164.73, 148.51, 147.41,147.25, 145.30, 143.97, 143.26, 139.83, 138.27, 133.44, 131.69, 131.49,130.80, 129.92, 129.77, 129.53, 129.51, 128.81, 128.70, 127.74, 126.75,125.66, 125.11, 123.74, 122.36, 122.22, 120.47, 106.39, 106.38, 39.22, 34.08,23.82, 21.31. HR-MS (ESI): m / z [M] calcd for C 51 H 38 F3N5O5S: 889.2545; found:889.2528. Cell imaging of fluorescent probe S1 Methanol and water were selected as the good and bad solvents for the fluorescent probe S1, respectively. Methanol-water solutions with different percentages were prepared, and the fluorescence intensity of the different solutions was measured after adding the fluorescent probe S1. Figure 25 As shown, the fluorescence intensity is essentially zero in pure methanol solution, but gradually increases with the percentage of water, a poor solvent. Therefore, its fluorescence generation mechanism is aggregation-induced emission (AIE).

[0052] Human umbilical vein endothelial cells (HUVECs) and breast cancer cells (MCF-7) were incubated with the fluorescent probe S1 (5 μM), respectively. Figure 18 As shown, the fluorescence intensity in MCF-7 cells was significantly higher than that in HUVEC cells, indicating that the fluorescent probe S1 has the ability to distinguish between normal cells and cancer cells.

[0053] Example 2

[0054] A method for preparing a COX-2 targeted fluorescent probe includes the following three steps. Steps 1 and 2 are the same as steps 1 and 2 in Example 1, the only difference being step 3: Step 3: Compound A2 (144.8 mg, 0.2 mmol) and p-aminophenol (26.2 mg, 0.24 mmol) were added to 5 mL of N,N-dimethylformamide (DMF). The mixture was stirred at room temperature under N2 protection for 15 minutes. Then, 60% sodium hydride (9.6 mg, 0.24 mmol) was added, and the mixture was heated and stirred at 100°C for 12 hours under N2 protection. After the reaction was complete, the mixture was extracted with saturated brine. After extraction, anhydrous sodium sulfate was added to remove water for 30 minutes. Finally, the crude product was obtained by vacuum distillation. After purification and separation by column chromatography (eluent: dichloromethane / methanol = 30 / 1, v / v), the final product, fluorescent probe S2, was obtained with a yield of 63%.

[0055] The final product 1 H NMR and 13 The characterization results of C NMR are as follows: 1 H NMR (600 MHz, DMSO-d6) δ:7.85 (dd, J = 8.4, 1.6 Hz, 1H), 7.67 (dd,J = 7.4, 1.7 Hz, 1H), 7.53 – 7.47 (m, 2H), 7.16 – 7.11 (m, 2H), 7.02 (m, J =8.0, 1.8 Hz, 1H), 6.73 (d, J = 8.4 Hz, 2H), 6.34 (d, J = 14.9 Hz, 5H), 6.14 –6.09 (m, 2H), 6.01 (dd, J = 8.5, 1.5 Hz, 1H), 5.89 – 5.83 (m, 2H), 3.14 (t, J= 6.8 Hz, 2H), 1.48 (t, J = 7.4 Hz, 2H), 1.40 (s, 3H), 0.96 (t, J = 7.1 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 171.69, 164.09, 163.40, 161.18, 147.28,145.81, 144.12, 143.21, 142.96, 142.85, 142.71, 142.45, 139.66, 139.58,133.46, 131.85, 129.85, 129.42, 129.26, 129.18, 128.76, 127.18, 126.47,125.66, 124.39, 123.29, 122.61, 122.50, 122.10, 120.83, 115.52, 115.48,109.73, 106.72, 79.65, 39.05, 33.49, 22.98, 21.20. HR-MS (ESI): m / z [M+H] + calcd for C 39 H 30 F3N5O6S: 754.1942; found: 754.1942. Cell imaging of fluorescent probe S2 Incubation of HUVEC cells with fluorescent probe S2 (20 μM) showed low fluorescence intensity, while incubation of MCF-7 cells with fluorescent probe S2 showed significant fluorescence, indicating that fluorescent probe S2 can distinguish between normal cells and cancer cells. Figure 19 (A, D). The ability to detect endogenous and exogenous hypochlorous acid was then investigated. After incubation of the fluorescent probe S2 (20 μM) with cells, 100 μM hypochlorous acid was added to HUVEC cells and MCF-7 cells, and incubation was carried out for ten minutes. The results showed that the fluorescence intensity increased in both HUVEC and MCF-7 cells, indicating that the probe has the ability to detect exogenous hypochlorous acid. Figure 19 (B, E). The ability of fluorescent probe S2 to detect endogenous hypochlorous acid was then verified by incubating HUVEC and MCF-7 cells with LPS (5 μg / mL) and fluorescent probe S2 (20 μM). Figure 19 (C, F) Both normal and cancer cells produced significant fluorescence intensity. Compared with A, the experimental results of each group showed significant differences. Therefore, the fluorescent probe S2 can not only distinguish between normal and cancer cells, but also detect endogenous and exogenous hypochlorous acid.

[0056] Example 3

[0057] A method for preparing a COX-2 targeted fluorescent probe includes the following three steps. Steps 1 and 2 are the same as steps 1 and 2 in Example 1, the only difference being step 3: Step 3: Compound A2 (144.8 mg, 0.2 mmol), hydroquinone (66 mg, 0.6 mmol), and potassium carbonate (165.6 mg, 1.2 mmol) were added to 5 mL of acetonitrile and reacted at 90 °C under N2 protection for 24 hours. After the reaction was complete, ethyl acetate and saturated brine were added for extraction. The upper layer of ethyl acetate was poured off, and anhydrous sodium sulfate was added for dehydration for 30 minutes. After completion, the crude product was obtained by vacuum distillation, and purified by column chromatography (eluent: dichloromethane / methanol = 40 / 1, v / v) to obtain the final product, fluorescent probe S3, with a yield of 55%.

[0058] The final product 1 H NMR and 13 The characterization results of C NMR are as follows: 1 H NMR (600 MHz, DMSO-d6) δ:9.63 (s, 1H), 8.69 (d, J = 8.4 Hz, 1H), 8.51 (d, J = 7.3 Hz, 1H), 8.34 (d, J = 8.3 Hz, 1H), 7.96 (d, J = 8.7 Hz, 2H),7.89 – 7.85 (m, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.18 (d, J = 9.8 Hz, 5H), 7.12(d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 8.3 Hz, 1H), 6.56(s, 1H), 3.98 (t, J = 6.9 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 2.25 (s, 3H), 1.80 (m, J = 7.2 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 172.36, 170.15, 164.04, 163.35, 160.67,155.70, 150.20, 146.42, 145.76, 142.88, 142.63, 142.50, 140.55, 139.55,133.36, 131.87, 129.84, 129.40, 129.17, 129.14, 128.69, 127.28, 126.31,125.69, 124.41, 123.33, 122.63, 122.56, 122.52, 120.84, 117.15, 116.11,115.86, 110.01, 106.64, 39.19, 33.97, 23.22, 21.20. HR-MS (ESI): m / z [M+H] + calcd for C 39 H 29 F3N4O7S: 755.1781; found: 755.1752. As "off-on" fluorescent probes, S2 and S3, after the addition of hypochlorous acid and peroxynitrite respectively, react with hypochlorous acid to remove the aniline unit, and react with peroxynitrite to remove the phenol unit, thereby blocking the PET effect. The probe fluorescence is excited, and the oxygen anion generated by the reaction will form a new intramolecular charge transfer (ICT) with the carbonyl group, which further enhances the fluorescence intensity (reaction mechanism as follows).

[0059]

[0060] Cell imaging of fluorescent probe S3 Cells were incubated with the fluorescent probe S3 (10 μM). The fluorescence intensity in MCF-7 cells was significantly higher than that in normal cells, indicating that the fluorescent probe S3 can distinguish between normal cells and cancer cells. Figure 20 (A, C). To verify that the fluorescent probe S3 can detect endogenous peroxynitrite, cells were incubated with the peroxynitrite inducer SIN-1 (500 μM) and probe S3 (10 μM). Compared with the imaging results of cells not incubated with SIN-1, the fluorescence intensity in cells incubated with SIN-1 increased, such as... Figure 20 (B, D) indicates that the fluorescent probe S3 can be used to detect changes in peroxynitrite levels in vivo.

[0061] Comparative Example A method for preparing a COX-2 fluorescent probe includes the following steps: Compound A1 (72.2 mg, 0.2 mmol), prepared in the example, triphenylamine 4-borate (75.18 mg, 0.26 mmol), tetrakis(triphenylphosphine)palladium (11.56 mg, 0.01 mmol), and potassium carbonate (82.92 mg, 0.6 mmol) were added to a 1 / 1 mixture of methanol and toluene and reacted at 85°C for 8 hours. After the reaction, the reaction solution was extracted with saturated brine. The extracted solution was then distilled under reduced pressure to obtain the crude product. Purification and separation by column chromatography (eluent: dichloromethane / methanol = 25 / 1, v / v) yielded an orange solid compound S0 in 73% yield.

[0062] The final product 1 H NMR and 13 The characterization results of C NMR are as follows: 1 H NMR (600 MHz, DMSO-d6) δ:8.53 (dd, J = 10.7, 7.4 Hz, 2H), 8.38 (d,J = 8.3 Hz, 1H), 7.90 – 7.84 (m, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.50 – 7.44(m, 2H), 7.42 – 7.34 (m, 4H), 7.22 – 7.02 (m, 8H), 4.11 (t, J = 7.0 Hz, 2H), 2.31 (t, J = 7.3 Hz, 2H), 1.91 (m, J = 7.2 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 164.10, 163.87, 148.20, 147.26, 146.21,132.78, 131.88, 131.49, 131.21, 130.92, 130.25, 129.72, 128.67, 128.24,127.84, 125.28, 124.30, 123.04, 122.39, 121.40, 40.54, 31.96, 23.53. HR-MS(ESI): m / z [MH] － calcd for C 34 H 26 N2O4: 525.1820; found: 525.1823. The preparation methods of the COX-2 targeted fluorescent probes in Examples 1, 2, and 3 above, as well as the comparative fluorescent probes, are shown below:

[0063] COX-2 targeting performance study of fluorescent probes in Example 1 and comparative examples The targeting ability of celecoxib was demonstrated using cellular imaging experiments with fluorescent probe S0 from the comparative example and fluorescent probe S1 from Example 1. Human umbilical vein endothelial cells (HUVECs) and breast cancer cells (MCF-7) were incubated with fluorescent probe S0 (5 μM), while MCF-7 cells were incubated with fluorescent probe S1 (5 μM). The experimental results are as follows: Figure 17 As shown, fluorescent probe S0 did not produce obvious fluorescence in normal cells and cancer cells, but fluorescent probe S1 showed strong fluorescence in cancer cells, indicating that the probe modified with celecoxib has COX-2 targeting ability.

[0064] Study on the response performance of fluorescent probes in Examples 1, 2, and 3 The response fluorescence spectra of S1, S2, and S3 in the measurement examples were analyzed. Appropriate amounts of the fluorescent probe compound solids from Examples 1, 2, and 3 were weighed and diluted in DMSO to a final concentration of 10 mM. The probe stock solution was diluted with ultrapure water to the experimental concentration, then co-incubated with the desired compound, and the fluorescence intensity was measured. All samples used for fluorescence testing were prepared using this method, including fluorescence selectivity testing, interference testing, and stability testing.

[0065] like Figure 21 As shown, the fluorescence spectrum excited by probe S1 at 422 nm has a certain fluorescence intensity in pure aqueous solution. With the continuous increase of glycerol percentage, the fluorescence intensity of the probe solution at 610 nm continuously increases. Figure 21 A), fluorescence intensity and system viscosity show a good linear relationship (R). 2 = 0.9814) Figure 21 B). The fluorescence spectrum of probe S2 excited at 460 nm showed weak fluorescence in solution, and the fluorescence intensity at 560 nm was significantly enhanced after the addition of hypochlorous acid (560 μM). Figure 21 The detection limit for S2 was 0.44 μM (3σ / k), and the probe S2 showed a good correlation with the concentration of hypochlorous acid (R). 2 = 0.9902)( Figure 21 D). The fluorescence intensity of probe S3 solution significantly increased with the gradual addition of peroxynitrite ions. Figure 21 Linear regression analysis of E revealed a strong correlation between the fluorescence intensity of probe S3 and the concentration of peroxynitrite (R² = 0.9897). Figure 21F). The probe response experiment results show that fluorescent probe S1 has a good response to the viscosity of the system, and fluorescent probes S2 and S3 can produce good responses to changes in the concentration levels of hypochlorous acid and peroxynitrite, respectively. Therefore, they can be used as a visualization tool for selectively labeling tumor cells.

[0066] Toxicity studies of fluorescent probes in Examples 1, 2, and 3 Studying the cytotoxicity of probes is an important method for predicting whether they can exist normally in cells or organisms without affecting their normal growth. MCF-7 or HUVEC cells in good growth condition with a growth density of 70% were taken, digested with trypsin, and then reconstituted with the appropriate culture medium. After centrifugation at 1500 rpm for 5 minutes, the cells were counted using a cell counting chamber at a value of 5 × 10⁻⁶. 3 The target concentration was diluted with culture medium at 100 μL. Six concentration gradient groups of the three probes from Examples 1-3 were set up: 0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM. It was found that, whether in normal cells or cancer cells, even after adding 40 μM probe and culturing for 24 hours, the cell survival rate was above 80%, indicating that the fluorescent probes of this application are non-cytotoxic.

[0067] Selectivity of fluorescent probes in Examples 1, 2, and 3 The fluorescence enhancement of the probe in this application is selective. We measured the fluorescence response of S1, S2, and S3 in Examples 1-3 to various competing substances, including Na+ ions. + K + Mg2 + Cu2 + Fe2 + Fe3 + Ca2 + NO3 - CO3 2- HCO3 - SO3 2- and HPO4 - ), ROS ( 1 O2, ONOO - , . OH, HClO and H2O2) and RSS (Hcy and GSH). Figure 22 The results showed that after adding a high concentration of interfering substance (100 μM), the change in fluorescence intensity of the fluorescent probe S1 solution (5 μM) was not significantly different from that in water, indicating that the interfering ions had no interfering ability with the fluorescent probe S1. Figure 23 , 24As shown, after adding various ions and biomolecules (100 μM) to the fluorescent probe S2 and S3 solutions (5 μM), the fluorescence intensity of the solutions remained basically unchanged except for hypochlorous acid and peroxynitrite. This indicates that the fluorescent probes S2 and S3 only respond to hypochlorous acid and peroxynitrite, respectively, and have excellent selectivity, which is beneficial for the detection of hypochlorous acid and peroxynitrite in complex environments.

[0068] This application constructs three fluorescent probes with different responsive groups, using naphthalimide as the fluorescent group, celecoxib as the targeting group, and long-chain alkyl groups as linking groups, and incorporating triphenylamine, p-aminophenol, and hydroquinone as viscosity, hypochlorous acid, and peroxynitrite responsive groups. All three probes exhibit good pH stability, photostability, and plasma stability. Fluorescent probe S1 responds to changes in viscosity and polarity, with fluorescence generation via aggregation-induced emission (AIE). Fluorescent probe S2 exhibits excellent hypochlorous acid selectivity, and fluorescent probe S3 exhibits sensitive peroxynitrite selectivity; fluorescence generation via photoinduced electron transfer (PET).

[0069] The unmodified celecoxib S0 in this application showed no significant fluorescence in HUVEC and MCF-7 cells with both low and high COX-2 expression, while fluorescent probes S1, S2, and S3 all showed bright fluorescence in MCF-7 cells and weak fluorescence in HUVEC cells. Cell imaging experiments with probe S1 showed that celecoxib has significant COX-2 targeting ability, and probe S1 can distinguish between normal cells and cancer cells. Furthermore, the off-on fluorescent probes S2 and S3 exhibited resistance to induced and exogenously produced ClO₂. － and ONOO － All three probes possess detection capabilities. Cell imaging experiments with probe S2 showed that it can distinguish between normal and cancer cells, and also detect endogenous and exogenous hypochlorous acid. Cell imaging experiments with probe S3 showed that it can distinguish between normal and cancer cells, and also detect changes in cellular peroxynitrite levels. In summary, we have developed three different fluorescent probes that can distinguish between normal cells and breast cancer cells based on COX-2 expression levels. Therefore, they can serve as important strategies for developing cancer detection and research, and have the potential to act as guiding agents in tumor surgery.

[0070] This invention can be summarized in other specific forms that do not depart from the spirit or essential features of the invention. Therefore, in all respects, the above embodiments of the invention should be considered illustrative only and not limiting, while the claims define the scope of the invention. The foregoing description does not define the scope of the invention; therefore, any changes within the meaning and scope equivalent to the claims should be considered to be included within the scope of the claims.

Claims

1. A COX-2 targeting fluorescent probe, characterized in that, The structure of the fluorescent probe is shown below: ； The group R is selected from one of the following formulas II-IV: 。 2. A method for preparing the COX-2 targeted fluorescent probe as described in claim 1, characterized in that, Includes the following steps: Step 1: Dissolve 4-bromo-1,8-naphthalenedicarboxylic anhydride and 4-aminobutyric acid in anhydrous ethanol. After the reflux reaction is completed, cool the reaction solution to room temperature, remove the solvent, purify and separate to obtain compound A1. Step 2: Compound A1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, and triethylamine were dissolved in dichloromethane and reacted under N2 protection for a certain time. Celecoxib was then added. The reaction was carried out at room temperature under N2 protection. After the reaction was completed, the mixture was extracted, dehydrated, distilled under reduced pressure, and purified to obtain a white solid compound A2. Step 3: Compound A2,4-boronic acid triphenylamine, tetra(triphenylphosphine)palladium, and potassium carbonate are dissolved in a mixed solvent of methanol / toluene = 1 / 1, reacted at 85°C, and then extracted, dehydrated, distilled under reduced pressure, purified and separated to obtain fluorescent probe S1.

3. A method for preparing the COX-2 targeted fluorescent probe as described in claim 1, characterized in that, Includes the following steps: Step 1: Dissolve 4-bromo-1,8-naphthalenedicarboxylic anhydride and 4-aminobutyric acid in anhydrous ethanol. After the reflux reaction is completed, cool the reaction solution to room temperature, remove the solvent, purify and separate to obtain compound A1. Step 2: Compound A1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, and triethylamine were dissolved in dichloromethane and reacted under N2 protection for a certain time. Celecoxib was then added. The reaction was carried out at room temperature under N2 protection. After the reaction was completed, the mixture was extracted, dehydrated, distilled under reduced pressure, and purified to obtain a white solid compound A2. Step 3: Compound A2 and p-aminophenol were added to N,N-dimethylformamide. After stirring at room temperature under N2 protection, sodium hydride was added and heated and stirred under N2 protection. After the reaction was completed, extraction, dehydration, vacuum distillation and purification were performed to obtain fluorescent probe S2.

4. A method for preparing the COX-2 targeted fluorescent probe as described in claim 1, characterized in that, Includes the following steps: Step 1: Dissolve 4-bromo-1,8-naphthalenedicarboxylic anhydride and 4-aminobutyric acid in anhydrous ethanol. After the reflux reaction is completed, cool the reaction solution to room temperature, remove the solvent, purify and separate to obtain compound A1. Step 2: Compound A1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, and triethylamine were dissolved in dichloromethane and reacted under N2 protection for a certain time. Celecoxib was then added. The reaction was carried out at room temperature under N2 protection. After the reaction was completed, the mixture was extracted, dehydrated, distilled under reduced pressure, and purified to obtain a white solid compound A2. Step 3: Compound A2, hydroquinone, and potassium carbonate are dissolved in acetonitrile. After the reaction is completed under N2 protection, the mixture is extracted, dehydrated, distilled under reduced pressure, purified, and separated to obtain fluorescent probe S3.

5. The method for preparing the COX-2 targeted fluorescent probe according to any one of claims 2-4, characterized in that, The 4-bromo-1,8-naphthalenedicarboxylic anhydride and 4-aminobutyric acid were dissolved in anhydrous ethanol at a molar ratio of 1:1.

2.

6. The method for preparing the COX-2 targeted fluorescent probe according to any one of claims 2-4, characterized in that, The compound A1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, triethylamine, and celecoxib were dissolved in dichloromethane in a molar ratio of 1:2.3:0.31:3:1.

3.

7. The use of the COX-2 targeting fluorescent probe as described in claim 1 in the preparation of a reagent for targeting COX-2 in human umbilical vein endothelial cells and responding to the expression levels of viscosity, hypochlorous acid, or peroxynitrite in the internal environment.

8. The use of the COX-2 targeting fluorescent probe as described in claim 1 in the preparation of a reagent for targeting COX-2 in breast cancer cells and responding to the expression levels of viscosity, hypochlorous acid, or peroxynitrite in the internal environment.

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