A quinoline-malononitrile derivative, its preparation method, and its application as a near-infrared AIE fluorescent probe targeting COX-2

Abstract

The present invention discloses a quinoline malononitrile derivative, a preparation method thereof, and an application as a near-infrared AIE fluorescent probe targeting COX-2, belonging to the technical field of fluorescent probes. The quinoline malononitrile derivative uses quinoline malononitrile as a fluorophore, introduces thiophene and triphenylamine, and conjugates to form a fluorescent mother core with a strong "D-π-A" structure. On this basis, celecoxib or indomethacin is connected with an alkyl chain. It is a near-infrared AIE fluorescent probe with good biocompatibility and stability, targeting COX-2, showing bright fluorescence in MCF-7, weak fluorescence in HUVEC, and can generate a fluorescent response according to COX-2 fluctuations. It can achieve accurate imaging of the tumor microenvironment. The structure of the quinoline malononitrile derivative is as follows: #imgabs0# or #imgabs1#.

Description

Technical Field

[0001] The present invention relates to a compound and a preparation method and application thereof, in particular to a quinoline-malononitrile derivative and a preparation method thereof and application thereof as a near-infrared AIE fluorescent probe targeting COX-2, belonging to the technical field of fluorescent probes. Background Art

[0002] Cancer is one of the leading causes of mortality worldwide. Early and accurate diagnosis is key to improving patient survival. Fluorescent probes, with their high responsiveness, real-time dynamic imaging capabilities, and minimally invasive properties, offer unique advantages in tumor microenvironment monitoring, tumor identification, and intraoperative guidance, making them a potential tool for visual tumor diagnosis and treatment.

[0003] Biomarkers in the tumor microenvironment are key targets for probe design. The expression level of cyclooxygenase-2 (COX-2) in cancer cells is significantly higher than that in normal cells.

[0004] COX-2 is abnormally highly expressed in the inflammatory tumor microenvironment and is closely related to the occurrence and development of various cancers. Its expression level is closely related to the malignancy of the lesions, making it an important potential target for early cancer diagnosis.

[0005] Existing COX-2 fluorescent probes typically employ a strategy combining targeted delivery with microenvironmental responsiveness. However, the ubiquitous aggregation-induced quenching (ACQ) effect of conventional organic fluorophores limits their imaging performance in high-density biological environments, making it difficult to simultaneously achieve high fluorescence intensity, excellent photostability, high specificity, and a high signal-to-noise ratio. Summary of the Invention

[0006] To address the deficiencies of the prior art, the present invention aims to provide a novel, high-performance fluorescent probe that can simultaneously achieve specific targeting of COX-2, sensitive COX-2 activity response, near-infrared emission, aggregation-induced emission (AIE) properties, excellent photostability, and good biocompatibility, and can be effectively used in tumor imaging.

[0007] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0008] A quinoline-malononitrile derivative, the structure of which is shown below:

[0009] or .

[0010] The preparation method of the aforementioned quinoline-malononitrile derivative comprises the following steps:

[0011] (1) Compound 4, Compound 5, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in N,N-dimethylformamide at a molar ratio of 26:30:52:7, or Compound 4, indomethacin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in N,N-dimethylformamide at a molar ratio of 25:25:50:7, and stirred at room temperature for reaction under argon protection;

[0012] (2) After the reaction is completed, extraction, water removal, and reduced pressure distillation are performed in sequence to remove the solvent to obtain a crude product;

[0013] (3) The crude product is purified and separated by silica gel column chromatography and dried in vacuo to obtain the aforementioned quinoline-malononitrile derivative;

[0014] Among them, the structures of compound 4 and compound 5 are shown below:

[0015] .

[0016] Preferably, the preparation method of compound 4 is as follows: compound 2 and compound 3 are added to acetonitrile in a molar ratio of 1:1, piperidine is added dropwise, and the mixture is refluxed at 90°C for 12 hours under argon protection. After cooling to room temperature, the precipitated red precipitate is washed with acetonitrile and methanol, and vacuum dried to obtain compound 4; wherein the structures of compound 2 and compound 3 are shown below:

[0017] .

[0018] Preferably, the preparation method of compound 2 is as follows: (1) 7-methylquinoline and 2-bromoethanol are added to a reaction vessel in a molar ratio of 10:11, refluxed at 140°C for 1 hour under argon protection, cooled to room temperature after the reaction, washed the solid 3 times with cold acetonitrile for purification and separation, and vacuum dried to obtain compound 1; (2) compound 1 and malononitrile are dissolved in anhydrous ethanol in a molar ratio of 2:5, sodium ethoxide is slowly added dropwise under stirring at 0°C, stirred in an ice bath for 1 hour, and then reacted at room temperature for 6 hours. After the reaction is completed, the pH value of the solution is adjusted to 7-8, the ethanol is removed by rotary evaporation, and extracted 3 times with ethyl acetate and saturated brine. The upper ethyl acetate is poured out and anhydrous sodium sulfate is added to remove water. After filtering, the crude product is obtained by vacuum distillation, which is purified and separated by silica gel column chromatography. The eluent is a mixture of petroleum ether and ethyl acetate. V 石油醚 :V 乙酸乙酯 =1:1, vacuum drying to obtain compound 2; wherein the structure of compound 1 is as follows:

[0019] .

[0020] Preferably, the preparation method of compound 3 is as follows: 4-(diphenylamino)phenylboronic acid and 5-bromothiophene-2-carboxaldehyde are placed in a round-bottom flask in a molar ratio of 1:1, and then an aqueous potassium carbonate solution, tetrakis(triphenylphosphine)palladium and anhydrous tetrahydrofuran are added, stirred evenly, and the air in the system is evacuated with a vacuum pump and replaced with argon, and refluxed at 80°C for 10 hours. After the reaction is completed, the system is cooled to room temperature, and tetrahydrofuran is removed under reduced pressure at 40°C. The system is extracted three times with dichloromethane and saturated brine, and the organic phase is separated. The organic phase is dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated on a rotary evaporator to obtain a crude product, which is purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate as the eluent. V 石油醚 :V 乙酸乙酯 =6.5:1, and vacuum dried to obtain compound 3.

[0021] Preferably, the preparation method of compound 5 is as follows: celecoxib and glutaric anhydride are dissolved in dichloromethane in a molar ratio of 2:1, triethylamine is added, and the reaction is stirred at room temperature under argon protection for 15±1h. After the reaction is completed, the solvent is removed by rotary evaporation to obtain a white oily liquid, the pH is adjusted to 13, and the mixture is extracted 3 times with ethyl acetate and saturated brine. The aqueous phase is separated and the pH is adjusted to acidic, and then extraction, dehydration, and reduced pressure distillation are performed in sequence to remove the solvent, and vacuum drying is performed to obtain compound 5.

[0022] Preferably, in step (2), extraction is performed with dichloromethane and saturated brine.

[0023] Preferably, in step (3), the eluent is a mixture of dichloromethane and petroleum ether, V 二氯甲烷 :V 石油醚 =1.5:1.

[0024] The aforementioned quinoline-malononitrile derivatives are used, specifically, as near-infrared aggregation-induced emission fluorescent probes targeting COX-2.

[0025] The benefits of the present invention are that the quinoline-malononitrile derivatives YL-181 and YL-186 provided by the present invention can specifically target COX-2 and have sensitive responsiveness to COX-2 activity, and have excellent AIE properties, near-infrared emission, large Stokes shift, good pH stability, photostability, plasma stability and low cytotoxicity. They can achieve specific recognition and imaging of cancer cells and can be used for in vivo tumor imaging, providing a research basis for accurate tumor diagnosis and having great potential as guide agents in tumor surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a flow chart for the preparation of quinoline-malononitrile derivative YL-180;

[0027] Figure 2 This is a flow chart for the preparation of quinoline-malononitrile derivative YL-181;

[0028] Figure 3 This is a flow chart for the preparation of quinoline-malononitrile derivative YL-186;

[0029] Figure 4 is compound 2 1 H NMR spectrum;

[0030] Figure 5 is compound 2 13 C NMR spectrum;

[0031] Figure 6 is compound 4 1 H NMR spectrum;

[0032] Figure 7 is compound 4 13 C NMR spectrum;

[0033] Figure 8 is the HR-MS spectrum of compound 4;

[0034] Figure 9 is compound 5 1 H NMR spectrum;

[0035] Figure 10 is compound 5 13 C NMR spectrum;

[0036] Figure 11 is compound 6 1 H NMR spectrum;

[0037] Figure 12 is compound 6 13 C NMR spectrum;

[0038] Figure 13 is the HR-MS spectrum of compound 6;

[0039] Figure 14 is compound 7 1 H NMR spectrum;

[0040] Figure 15 is compound 7 13 C NMR spectrum;

[0041] Figure 16 is the HR-MS spectrum of compound 7;

[0042] Figure 17are the fluorescence spectra of YL-180, YL-181 and YL-186 in different solvents, wherein A is the fluorescence spectrum of YL-180 in different solvents, B is the fluorescence spectrum of YL-181 in different solvents, and C is the fluorescence spectrum of YL-186 in different solvents;

[0043] Figure 18 : are the AIE effect test result diagrams of YL-180, YL-181 and YL-186, wherein A is the AIE effect test result diagram of YL-180, B is the AIE effect test result diagram of YL-181, and C is the AIE effect test result diagram of YL-186;

[0044] Figure 19 Graphs showing the ion selectivity test results of YL-180, YL-181, and YL-186, wherein A is the ion selectivity test result graph of YL-180, B is the ion selectivity test result graph of YL-181, and C is the ion selectivity test result graph of YL-186;

[0045] Figure 20 Graphs showing the pH stability test results for YL-180, YL-181, and YL-186, wherein A is a graph showing the pH stability test results for YL-180, B is a graph showing the pH stability test results for YL-181, and C is a graph showing the pH stability test results for YL-186;

[0046] Figure 21 Graphs showing the photostability test results of YL-180, YL-181, and YL-186, wherein A is a graph showing the photostability test results of YL-180, B is a graph showing the photostability test results of YL-181, and C is a graph showing the photostability test results of YL-186;

[0047] Figure 22 Figures A and B are the results of cytotoxicity experiments on YL-180, YL-181, and YL-186, wherein Figure A is the result of cytotoxicity experiments on YL-180, Figure B is the result of cytotoxicity experiments on YL-181, and Figure C is the result of cytotoxicity experiments on YL-186;

[0048] Figure 23 Figures 2 and 3 are cell imaging results of YL-180, YL-181, and YL-186 in HUVEC and MCF-7, where A and B are cell imaging results of YL-180 in HUVEC and MCF-7, respectively; C and D are cell imaging results of YL-181 in HUVEC and MCF-7, respectively; and E and F are cell imaging results of YL-186 in HUVEC and MCF-7, respectively.

[0049] Figure 24Figures 1 and 2 are the calculation results of the relative fluorescence intensities of YL-180, YL-181, and YL-186 in HUVEC and MCF-7, wherein A and B are the calculation results of the relative fluorescence intensities of YL-180 in HUVEC and MCF-7, respectively; C and D are the calculation results of the relative fluorescence intensities of YL-181 in HUVEC and MCF-7, respectively; and E and F are the calculation results of the relative fluorescence intensities of YL-186 in HUVEC and MCF-7, respectively;

[0050] Figure 25 This is the cell imaging result of YL-181 in MCF-7 cells pretreated with CCB or LPS;

[0051] Figure 26 This is the calculation result of the relative fluorescence intensity of YL-181 in MCF-7 cells pretreated with CCB or LPS;

[0052] Figure 27 This is the cell imaging result of YL-186 in MCF-7 cells pretreated with CCB or LPS;

[0053] Figure 28 This is the calculation result of the relative fluorescence intensity of YL-186 in MCF-7 cells pretreated with CCB or LPS;

[0054] Figure 29 Figures A and B show the results of plasma stability experiments on YL-181 and YL-186, where A shows the results of plasma stability experiments on YL-181 and B shows the results of plasma stability experiments on YL-186.

[0055] Figure 30 This is the result of in vivo fluorescence imaging of tumor-bearing mice;

[0056] Figure 31 These are the in vitro imaging results of the main organs and tumor tissues of tumor-bearing mice, where 1 is the heart, 2 is the liver, 3 is the spleen, 4 is the lung, 5 is the kidney, and 6 is the tumor tissue. DETAILED DESCRIPTION

[0057] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0058] 1. Structure of quinoline-malononitrile derivatives

[0059] The structure of the quinoline-malononitrile derivative provided by the present invention is shown below:

[0060] 、 or .

[0061] 2. Preparation method of quinoline-malononitrile derivatives Example

[0062] like Figure 1 As shown, the preparation method of quinoline-malononitrile derivative YL-180 is as follows:

[0063] 7-Methylquinoline (1431 μL, 10 mmol) and 2-bromoethanol (780 μL, 11 mmol) were added to a 50 mL round-bottom flask and refluxed at 140°C for 1 h under argon protection. After the reaction, the mixture was cooled to room temperature and the solid was washed three times with cold acetonitrile for purification and separation. The solid was then dried in vacuo to obtain compound 1 as a pink solid with a yield of 52.2%.

[0064] Compound 1 (536 mg, 2 mmol) and malononitrile (355 μL, 5 mmol) were dissolved in anhydrous ethanol (100 mL). Sodium ethoxide (2-3 drops) was slowly added dropwise under stirring at 0°C. The mixture was stirred in an ice bath for 1 h and then reacted at room temperature for 6 h. After the reaction, the pH value of the solution was adjusted to 7-8 with 1 M HCl. The ethanol was removed by rotary evaporation. The solution was extracted three times with ethyl acetate and saturated brine. The upper ethyl acetate was poured out and anhydrous sodium sulfate was added to remove water for 30 min. After filtration, the crude product was obtained by vacuum distillation and purification by silica gel column chromatography (the eluent was a mixture of petroleum ether and ethyl acetate, V 石油醚 :V 乙酸乙酯 =1:1), and vacuum dried to obtain compound 2 as a yellow solid with a yield of 26%.

[0065] Compound 2 1 H NMR spectrum, 13 C NMR spectra are shown in Figure 4 、 Figure 5 , 1 H NMR and 13 The characterization results of C NMR are as follows:

[0066] 1 H NMR (600MHz, DMSO) δ 8.91 (d, J =8.4Hz, 1H), 8.06(d, J =8.9Hz,1H),7.87(t, J =7.8Hz, 1H), 7.59(t, J =7.7Hz, 1H), 6.81 (s, 1H), 5.09 (t, J =5.5Hz,1H),4.55(t, J =5.2Hz, 2H), 3.87-3.67 (m, 2H), 2.71 (s, 3H).

[0067] 13C NMR (151MHz, DMSO) δ 153.05, 152.37, 138.84, 133.66, 125.61, 125.22, 121.14, 119.70, 118.82, 109.53, 59.09, 50.19, 46.43, 22.65.

[0068] 4-(Diphenylamino)phenylboronic acid (290 mg, 1.0 mmol) and 5-bromothiophene-2-carboxaldehyde (191.1 mg, 1.0 mmol) were placed in a round-bottom flask, and then potassium carbonate aqueous solution (concentration of 0.5 M, 4 mL), tetrakis(triphenylphosphine)palladium (11.6 mg, 0.01 mmol) and anhydrous tetrahydrofuran (20 mL) were added and stirred evenly. The air in the system was evacuated with a vacuum pump and replaced with argon. The mixture was refluxed at 80°C for 10 h. After the reaction was completed, the system was cooled to room temperature, tetrahydrofuran was removed under reduced pressure at 40°C, and the mixture was extracted three times with dichloromethane and saturated brine. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated on a rotary evaporator to obtain a crude product, which was purified by silica gel column chromatography (eluent: a mixture of petroleum ether and ethyl acetate, V 石油醚 :V 乙酸乙酯 =6.5:1), and vacuum drying to obtain compound 3 as a yellow solid with a yield of 65.5%.

[0069] Compound 2 (50.2 mg, 0.2 mmol) and compound 3 (70.1 mg, 0.2 mmol) were added to acetonitrile (6 mL), and piperidine (2-3 drops) was added dropwise. The mixture was refluxed at 90°C for 12 h under argon protection. After cooling to room temperature, the precipitated red precipitate was washed (with acetonitrile and methanol) and dried in vacuo to obtain compound 4 (quinoline-malononitrile derivative, denoted as YL-180) as a dark red solid with a yield of 36%.

[0070] Compound 4 1 H NMR spectrum, 13 C NMR spectrum and HR-MS spectrum are shown in Figure 6 、 Figure 7 、 Figure 8 . 1 H NMR, 13 The characterization results of C NMR and HR-MS are as follows:

[0071] 1 H NMR (600MHz, DMSO) δ 8.93 (dd, J =8.5, 1.3Hz, 1H), 8.07 (d, J =8.9Hz, 1H), 7.90 (ddd, J=8.6, 7.0, 1.3Hz, 1H), 7.64-7.59 (m, 4H), 7.54 (d, J =3.9Hz, 1H), 7.47(d, J =3.8Hz, 1H), 7.40(d, J =15.6Hz, 1H), 7.37-7.33 (m, 4H), 7.13-7.07 (m, 6H), 7.03 (s, 1H), 7.00-6.97 (m, 2H), 5.23 (t, J =5.5Hz,1H),4.61(t, J =5.1Hz, 2H), 3.88(d, J =5.3Hz, 2H).

[0072] 13 C NMR (151MHz, DMSO) δ 152.67, 150.52, 148.02, 147.13, 146.28, 139.12, 138.90, 133.96, 133.32, 132.83, 130.20, 127.19, 127.1 4, 125.54, 125.43, 125.09, 124.29, 124.23, 122.97, 121.15, 120.25, 118.99, 107.09, 59.39, 51.17, 47.27.

[0073] HR-MS (ESI): [MH] - C 38 H 27 N4OS - Calculated value 587.19111, theoretical value 587.19183. Example

[0074] like Figure 2 As shown, the preparation method of quinoline-malononitrile derivative YL-181 is as follows:

[0075] Celecoxib (2000 mg, 5.25 mmol) and glutaric anhydride (1050 mg, 10.5 mmol) were dissolved in dichloromethane (10 mL). Triethylamine (360 μL) was added and the mixture was stirred at room temperature under argon for 15 ± 1 h. After completion of the reaction, the solvent was removed by rotary evaporation to obtain a white oily liquid. 1 M NaOH solution was slowly added dropwise to the system to adjust the pH to 13 (a large amount of white compound was continuously precipitated during the addition of NaOH solution). The mixture was extracted three times with ethyl acetate and saturated brine. The aqueous phase was separated and transferred to a 300 mL conical flask. 1 M HCl solution was added dropwise to adjust the pH to acidic (a large amount of white solid precipitated in the solution). The mixture was extracted three times with dichloromethane and water. The organic phase was collected and dried over anhydrous sodium sulfate to remove water. After filtration, the solvent was distilled off under reduced pressure and dried in vacuo to obtain compound 5 as a white solid with a yield of 36%.

[0076] Compound 5 1 H NMR spectrum, 13 C NMR spectra are shown in Figure 9 、 Figure 10 , 1 H NMR and 13 The characterization results of C NMR are as follows:

[0077] 1 H NMR (600MHz, CDCl3) δ 7.83 (d, J =8.7Hz, 2H), 7.45-7.42 (m, 2H), 7.17 (d, J =7.9Hz, 2H), 7.05(d, J =8.1Hz, 2H), 6.79 (s, 1H), 2.74-2.69 (m, 2H), 2.55-2.51 (m, 2H), 2.37 (s, 3H).

[0078] 13 C NMR (151MHz, CDCl3) δ 177.93, 170.51, 146.18, 144.27, 144.02, 142.37, 140.25, 139.35, 129.89, 12 8.73, 128.65, 125.31, 125.04, 121.79, 120.01, 106.64, 30.96, 28.64, 21.34.

[0079] Compound 4 (16 mg, 0.026 mmol) prepared in Example 1, compound 5 (18 mg, 0.03 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10 mg, 0.052 mmol), and 4-dimethylaminopyridine (4 mg, 0.007 mmol) were dissolved in N,N-dimethylformamide (10 mL). The mixture was stirred at room temperature for 30 min under argon protection (activation reaction system), and then stirred for 12 h. After the reaction, extraction (dichloromethane and saturated brine), dehydration (addition of anhydrous sodium sulfate), and reduced pressure distillation were performed to remove the solvent to obtain a crude product, which was purified and separated by silica gel column chromatography (eluent: a mixture of dichloromethane and petroleum ether, V 二氯甲烷 :V 石油醚 =1.5:1) and vacuum dried to obtain compound 6 (quinoline-malononitrile derivative, denoted as YL-181) as a dark red solid with a yield of 24%.

[0080] Compound 6 1 H NMR spectrum, 13 C NMR spectrum and HR-MS spectrum are shown in Figure 11 、 Figure 12 、 Figure 13 . 1 H NMR, 13 The characterization results of C NMR and HR-MS are as follows:

[0081] 1 H NMR (600MHz, DMSO) δ 8.90 (d, J =8.4Hz,1H),8.05(d, J =9.0Hz, 1H), 7.94-7.83 (m, 1H), 7.75-7.66 (m, 1H), 7.59 (dd, J =23.0, 14.1Hz, 1H), 7.51-7.44 (m, 1H), 7.41 (d, J =3.6Hz, 2H), 7.36-7.30 (m, 1H), 7.24 (d, J =15.5Hz, 2H), 7.16(t, J =6.6Hz, 1H), 7.13 (s, 1H), 7.11 (d, J =4.4Hz, 1H), 7.09 (s, 1H), 7.09-7.04 (m, 3H), 6.99 (dd, J =14.4, 7.7Hz, 4H), 4.75 (d, J =46.0Hz,1H),4.40(d, J =27.1Hz, 1H), 2.38 (dd,J =14.2, 7.2Hz, 2H), 2.25 (s, 1H).

[0082] 13 C NMR (151MHz, DMSO) δ 172.16, 152.77, 149.88, 148.01, 147.13, 146.48, 145.74, 142.69, 139.58, 138.99, 138.76, 137.36 , 134.10, 133.29, 133.13, 132.13, 131.94, 130.17, 129.87, 129.17, 129.14, 127.15, 126.29, 125.60 , 125.50, 125.05, 124.24, 124.19, 122.98, 121.00, 119.35, 118.63, 107.15, 106.71, 65.50, 62.12, 53.09, 48.21, 47.29, 31.77, 30.73, 30.47, 29.49, 29.18, 28.08, 22.57, 21.27, 19.13, 14.43, 14.03.

[0083] HR-MS (ESI): [MH] - C 59 H 43 F3N7O5S2 - Calculated value 1050.27247, theoretical value 1050.27197. Example

[0084] like Figure 3 As shown, the preparation method of quinoline-malononitrile derivative YL-186 is as follows:

[0085] Compound 4 (30 mg, 0.05 mmol) prepared in Example 1, indomethacin (18 mg, 0.05 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (20 mg, 0.1 mmol), and 4-dimethylaminopyridine (8 mg, 0.014 mmol) were dissolved in N,N-dimethylformamide (10 mL), and the mixture was stirred at room temperature under argon protection. After the reaction, extraction (dichloromethane and saturated brine) and dehydration (addition of anhydrous sodium sulfate) were carried out in sequence, and the solvent was removed by reduced pressure distillation to obtain a crude product, which was purified and separated by silica gel column chromatography (eluent: a mixture of dichloromethane and petroleum ether, V 二氯甲烷 :V 石油醚 =1:1) and vacuum dried to obtain compound 7 (quinoline-malononitrile derivative, denoted as YL-186) as a dark red solid with a yield of 92%.

[0086] Compound 7 1 H NMR spectrum, 13 C NMR spectrum and HR-MS spectrum are shown in Figure 14 、 Figure 15 、 Figure 16 . 1 H NMR, 13 The characterization results of C NMR and HR-MS are as follows:

[0087] 1 H NMR (600MHz, CDCl3) δ 9.09 (dd, J=8.5, 1.1Hz, 1H), 7.72-7.67 (m, 1H), 7.60-7.57 (m, 2H), 7.55 (d, J=8.7Hz, 1H), 7.46-7.41 (m, 5 H), 7.37 (d, J=15.3Hz, 1H), 7.29 (t, J=7.9Hz, 4H), 7.23 (d, J=3.8Hz, 1H), 7.19 (d, J=3.8Hz, 1H), 7.14 (d, J= 7.6Hz, 4H), 7.10-7.04 (m, 4H), 6.99 (s, 1H), 6.80 (dd, J=21.1, 12.2Hz, 2H), 6.70 (d, J=2.4Hz, 1H), 6.62 (d d, J=9.0, 2.5Hz, 1H), 4.59 (d, J=5.5Hz, 2H), 4.55 (d, J=5.5Hz, 2H), 3.68 (s, 2H), 3.64 (s, 3H), 2.31 (s, 3H).

[0088] 13 C NMR (151MHz, CDCl3) δ 170.66, 168.07, 155.91, 153.16, 148.33, 147.92, 147.37, 147.13, 139.41, 138.19, 138.10, 136 .06, 133.54, 133.33, 133.28, 132.57, 131.17, 130.65, 130.09, 129.44, 129.12, 127.14, 126.79 , 126.73, 124.94, 124.77, 123.62, 123.16, 122.84, 121.30, 119.89, 118.82, 116.88, 115.62, 115.08, 111.40, 111.19, 107.17, 100.98, 61.22, 55.48, 52.59, 46.27, 30.20, 29.69, 14.12, 13.27.

[0089] HR-MS (ESI): [MH]- C 57 H 41 ClN5O4S - Calculated value 926.25733, theoretical value 926.25836.

[0090] 3. Fluorescence properties of quinoline-malononitrile derivatives (YL-180, YL-181, YL-186) in different solvents

[0091] Accurately pipette 2 μL of YL-180 mother solution (dissolved in DMSO, concentration of 10 mM), YL-181 mother solution (dissolved in DMSO, concentration of 10 mM), or YL-186 mother solution (dissolved in DMSO, concentration of 10 mM), and dilute to 2 mL with dimethyl sulfoxide, methanol, acetonitrile, ethanol, dimethylformamide, tetrahydrofuran, ethyl acetate, dichloromethane, or water, respectively. Vortex to mix to obtain a 10 μM YL-180 test solution, YL-181 test solution, or YL-186 test solution.

[0092] The fluorescence emission spectrum of the YL-180 test solution was measured in the range of 500-850 nm using the optimal excitation wavelength of 452 nm for YL-180.

[0093] The fluorescence emission spectrum of the YL-181 test solution was measured in the range of 500-850 nm using the optimal excitation wavelength of 454 nm for YL-181.

[0094] The fluorescence emission spectrum of the YL-186 test solution was measured in the range of 500-850 nm using the optimal excitation wavelength of 447 nm for YL-186.

[0095] Fluorescence spectra of YL-180, YL-181 and YL-186 in different solvents are shown in Figure 17 .

[0096] Depend on Figure 17 It can be seen that YL-180, YL-181 and YL-186 all have a "D-π-A" structure, and the maximum wavelength and emission intensity of fluorescence emission in different solvents are also different. The fluorescence intensity is enhanced in polar solvents and is lower in non-polar solvents.

[0097] 4. Aggregation-induced emission (AIE) effect test of quinoline-malononitrile derivatives (YL-180, YL-181, YL-186)

[0098] Tetrahydrofuran (THF) was used as a good solvent and deionized water was used as a poor solvent to prepare mixed solvent systems containing 0% to 100% of the poor solvent.

[0099] When the mixed solvent system is at room temperature, the YL-180 mother liquor, YL-181 mother liquor or YL-186 mother liquor is measured and added to the mixed solvent system to prepare a YL-180 test solution, YL-181 test solution or YL-186 test solution with a concentration of 10 μM.

[0100] A fluorescence spectrophotometer was used to perform light excitation at the optimal excitation wavelength of YL-180, 452 nm (slit width 5 nm), and the fluorescence emission spectrum of the YL-180 test solution was scanned in the range of 500-850 nm.

[0101] A fluorescence spectrophotometer was used to perform light excitation at the optimal excitation wavelength of YL-181, 454 nm (slit width 5 nm), and the fluorescence emission spectrum of the YL-181 test solution was scanned in the range of 500-850 nm.

[0102] A fluorescence spectrophotometer was used to perform light excitation at the optimal excitation wavelength of YL-186, 447 nm (slit width 5 nm), and the fluorescence emission spectrum of the YL-186 test solution was scanned in the range of 500-850 nm.

[0103] The AIE effect test results of YL-180, YL-181 and YL-186 are shown in Figure 18 .

[0104] Depend on Figure 18 It can be seen that the fluorescence intensity of YL-180, YL-181 and YL-186 is weak in the benign solvent THF, but the fluorescence intensity continues to increase when the proportion of deionized water, a poor solvent, is continuously increased. The maximum fluorescence emission of YL-180 and YL-186 occurs at 70% deionized water, and the maximum fluorescence emission of YL-181 occurs at 80% deionized water, after which the fluorescence intensity weakens.

[0105] 5. Study on the ion selectivity of quinoline-malononitrile derivatives (YL-180, YL-181, YL-186)

[0106] Accurately weigh sodium chloride, potassium carbonate, magnesium sulfate, copper sulfate, ferrous sulfate, ferric sulfate, calcium chloride, zinc chloride, sodium nitrite, sodium nitrate, sodium bisulfite, sodium hypochlorite, sodium bicarbonate, sodium hydrogen phosphate, cysteine, and glutathione, and place them in a 100 mL volumetric flask. Then, add tetrahydrofuran and YL-180 mother liquor, YL-181 mother liquor, or YL-186 mother liquor to prepare a solution system with a YL-180, YL-181, or YL-186 concentration of 10 μM and an ion concentration of 1 mM.

[0107] Accurately weigh the YL-180 mother liquor, YL-181 mother liquor or YL-186 mother liquor and place it in a 100 mL volumetric flask. Add deionized water to prepare a solution system with a YL-180, YL-181 or YL-186 concentration of 10 μM (referred to as the H2O group and the blank control group).

[0108] The solution system containing YL-180 was mixed and placed in a fluorescence spectrophotometer, and the fluorescence intensity at the maximum emission peak was measured under the optimal excitation wavelength of YL-180, 452 nm.

[0109] The solution system containing YL-181 was mixed and placed in a fluorescence spectrophotometer, and the fluorescence intensity at the maximum emission peak was measured under excitation at the optimal excitation wavelength of YL-181, 454 nm.

[0110] The solution system containing YL-186 was mixed and placed in a fluorescence spectrophotometer, and the fluorescence intensity at the maximum emission peak was measured under the optimal excitation wavelength of YL-186, 447 nm.

[0111] The ion selectivity test results of YL-180, YL-181 and YL-186 are shown in Figure 19 .

[0112] Depend on Figure 19 It can be seen that compared with the blank control group, the fluorescence intensity of YL-180, YL-181 and YL-186 remained basically unchanged after the addition of various interfering ions or biological thiols.

[0113] 6. pH stability test of quinoline-malononitrile derivatives (YL-180, YL-181, YL-186)

[0114] Tetrahydrofuran / deionized water (3 / 7, v / v) was adjusted to the target pH gradient (4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0) using 1 mM HCl solution or NaOH solution.

[0115] YL-180 mother liquor, YL-181 mother liquor or YL-186 mother liquor was added to the prepared solutions of different pH values, and after mixing, solutions containing 10 μM YL-180 at different pH values, solutions containing 10 μM YL-181 at different pH values, and solutions containing 10 μM YL-186 at different pH values ​​were prepared respectively.

[0116] YL-180 solutions of different pH values ​​were placed in a fluorescence spectrophotometer for measurement. The fluorescence intensity at the maximum emission wavelength was measured using the optimal excitation wavelength of 452 nm for YL-180.

[0117] YL-181 solutions of different pH values ​​were placed in a fluorescence spectrophotometer for measurement. The fluorescence intensity at the maximum emission wavelength was measured using the optimal excitation wavelength of 454 nm for YL-181.

[0118] YL-186 solutions of different pH values ​​were placed in a fluorescence spectrophotometer for measurement. The fluorescence intensity at the maximum emission wavelength was measured using the optimal excitation wavelength of YL-186, 447 nm.

[0119] The pH stability test results of YL-180, YL-181 and YL-186 are shown in Figure 20 .

[0120] Depend on Figure 20 It can be seen that when the pH value of the solution system is in the range of 4.0 to 10.0, the fluorescence intensities of YL-180, YL-181 and YL-186 remain stable.

[0121] 7. Photostability test of quinoline-malononitrile derivatives (YL-180, YL-181, YL-186)

[0122] Accurately pipette the YL-180 stock solution, YL-181 stock solution, or YL-186 stock solution into a premixed solvent system (tetrahydrofuran / deionized water = 3 / 7, v / v) to prepare a final concentration of 10 μM YL-180 test solution, YL-181 test solution, or YL-186 test solution, respectively.

[0123] After vortex mixing, the YL-180 test solution, the YL-181 test solution, and the YL-186 test solution were placed under normal lighting conditions and placed in a fluorescence spectrophotometer to measure the fluorescence intensity every 10 minutes (90 minutes in total).

[0124] The optimal excitation wavelength of YL-180 is 452 nm, and the fluorescence intensity of the YL-180 test solution at the maximum emission wavelength is measured.

[0125] The fluorescence intensity of the YL-181 test solution at the maximum emission wavelength was measured using the optimal excitation wavelength of 454 nm for YL-181.

[0126] The optimal excitation wavelength of YL-186, 447 nm, was used to excite the solution, and the fluorescence intensity at the maximum emission wavelength of the YL-186 solution was measured.

[0127] The photostability test results of YL-180, YL-181 and YL-186 are shown in Figure 21 .

[0128] Depend on Figure 21 It can be seen that the fluorescence intensity of YL-180, YL-181 and YL-186 remains almost unchanged within 90 minutes of illumination.

[0129] 8. Cytotoxicity Study of Quinoline-Malononitrile Derivatives (YL-180, YL-181, YL-186)

[0130] Human umbilical vein endothelial cells (HUVEC) and breast cancer cells (MCF-7) in good logarithmic growth phase were grown to a cell density of >80%. After trypsinization, centrifugation, and counting, cells were seeded into 96-well plates at a density of 5 × 10 cells / well. Serum-containing medium (200 μL / well) was added to each well and incubated at 37°C in a 5% CO2 incubator for 24 hours. Subsequently, YL-180, YL-181, or YL-186 stock solutions were added to achieve a concentration gradient of 0 μM (blank control), 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM. Incubation continued for 24 hours. MTT (5 mg / mL, 20 μL / well) was then added and incubated for 3 hours. The supernatant was aspirated, and DMSO (150 μL / well) was added with shaking to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader, and the cell viability was calculated.

[0131] The results of cytotoxicity experiments of YL-180, YL-181 and YL-186 are shown in Figure 22 .

[0132] Depend on Figure 22 It can be seen that whether it is normal cells (HUVEC) or cancer cells (MCF-7), the cell survival rate is above 80% after incubation with YL-180, YL-181 or YL-186 at a concentration of up to 40μM for 24 hours.

[0133] IX. Cellular Imaging of Quinoline-Malononitrile Derivatives (YL-180, YL-181, YL-186)

[0134] Normal HUVEC cells and MCF-7 cancer cells were incubated with YL-180 (5 μM), YL-181 (5 μM), or YL-186 (5 μM) for 2 hours. After incubation, the cells were washed with PBS and then imaged using CLSM.

[0135] The cell imaging results of YL-180, YL-181 and YL-186 in HUVEC and MCF-7 are shown in Figure 23 The relative fluorescence intensity calculation results in HUVEC and MCF-7 are shown in Figure 24 .

[0136] Depend on Figure 23 and Figure 24It can be seen that the fluorescence intensity of YL-180 in MCF-7 is similar to that in HUVEC; the fluorescence intensity of YL-181 in MCF-7 is much higher than that in HUVEC, which is 22 times that of HUVEC; the fluorescence intensity of YL-186 in MCF-7 is much higher than that in HUVEC, which is 4 times that of HUVEC.

[0137] The above shows that YL-180 has basically no discriminatory effect on MCF-7 and HUVEC, while YL-181 and YL-186 can distinguish MCF-7 and HUVEC.

[0138] 10. COX-2 Response Experiment of Quinoline-Malononitrile Derivatives (YL-181, YL-186)

[0139] To evaluate the responsiveness of YL-181 and YL-186 to COX-2, MCF-7 cancer cells were pretreated with the COX-2 inhibitor celecoxib (CCB, 20 μM) for 3 hours or the COX-2 inducer lipopolysaccharide (LPS, 5 μg / mL) for 3 hours. MCF-7 cells were then incubated with YL-181 (5 μM) or YL-186 (5 μM) for 2 hours. Following incubation, the cells were washed with PBS and imaged using CLSM.

[0140] The results of cell imaging of YL-181 in MCF-7 cells pretreated with CCB or LPS are shown in Figure 25 The relative fluorescence intensity calculation results of MCF-7 cells pretreated with CCB or LPS are shown in Figure 26 .

[0141] Depend on Figure 25 and Figure 26 It can be seen that after incubation of MCF-7 pretreated with CCB and YL-181, the fluorescence intensity decreased significantly by 97% compared with the control group; after incubation of MCF-7 pretreated with LPS and YL-181, the fluorescence intensity increased by 135% compared with the control group.

[0142] The results of cell imaging of YL-186 in MCF-7 cells pretreated with CCB or LPS are shown in Figure 27 The relative fluorescence intensity calculation results of MCF-7 cells pretreated with CCB or LPS are shown in Figure 28 .

[0143] Depend on Figure 27 and Figure 28It can be seen that after incubation of MCF-7 pretreated with CCB and YL-186, the fluorescence intensity decreased significantly by 44% compared with the control group; after incubation of MCF-7 pretreated with LPS and YL-181, the fluorescence intensity increased by 18% compared with the control group.

[0144] The above results fully demonstrate that YL-186 has a slight response to COX-2 fluctuations; YL-181 is highly sensitive to COX-2 fluctuations and can significantly reflect changes in COX-2 activity. COX-2 can be used as a biomarker for early diagnosis of cancer.

[0145] 11. Plasma stability test of quinoline-malononitrile derivatives (YL-181, YL-186)

[0146] 900 μL of treated rat plasma was placed in a 2 mL centrifuge tube. YL-181 or YL-186 stock solution (final concentration: 10 μM) was added and the tube was incubated in a 37°C water bath with a shaker. Samples were collected at various time points between 0 and 120 minutes. After vortex extraction with acetonitrile, the supernatant was centrifuged and filtered through a membrane. The peak area changes of YL-181 or YL-186 were determined by high-performance liquid chromatography.

[0147] The results of plasma stability tests for YL-181 and YL-186 are shown in Figure 29 .

[0148] Depend on Figure 29 It can be seen that YL-181 can still be effectively detected after incubation in plasma for 120 minutes, and its chromatographic peak area has no obvious change; YL-186 can still be effectively detected after incubation in plasma for 90 minutes, and its chromatographic peak area has no obvious change, and it is degraded by about 50% after incubation in plasma for 120 minutes.

[0149] 12. Animal Imaging Experiments with Quinoline-Malononitrile Derivative (YL-181)

[0150] A tumor-bearing mouse model was established by subcutaneously inoculating MCF-7 cells in the right forelimb of Balb / c nude mice. When the tumor reached an appropriate size, YL-181 solution (200 μM, 50 μL) was injected orthotopically into the tumor tissue and contralateral normal subcutaneous tissue.

[0151] In vivo fluorescence imaging was performed using the IVIS® Lumina III system (excitation wavelength 460 nm, emission wavelength 710 nm). Mice were then sacrificed, and major organs and tumor tissues were removed for ex vivo imaging.

[0152] The results of in vivo fluorescence imaging of tumor-bearing mice are shown in Figure 30 , the in vitro imaging results of major organs and tumor tissues are shown in Figure 31 .

[0153] Depend on Figure 30 It can be seen that the fluorescence signal in the tumor area is enhanced by 1.83 times compared with the control area. Figure 31 It can be seen that tumor tissue has obvious fluorescence, while the fluorescence of other organs can be ignored.

[0154] In summary, the quinoline-malononitrile derivatives YL-181 and YL-186 provided by the present invention can be used as fluorescent probes. Both use quinoline-malononitrile as a fluorophore and introduce electron-rich groups thiophene and triphenylamine. The quinoline-malononitrile fluorophore, thiophene and triphenylamine are conjugated to form a fluorescent core with a strong "D-π-A" structure. On this basis, celecoxib (targeting group) or indomethacin (targeting group) is connected with an alkyl chain as a COX-2 response group. The fluorescent probe has good biocompatibility and stability (pH stability, photostability, plasma stability), is targeted to COX-2, displays bright fluorescence in MCF-7, and exhibits weak fluorescence in HUVEC. It can generate a fluorescent response according to COX-2 fluctuations, can achieve accurate imaging of the tumor microenvironment, and effectively distinguish normal cells from cancer cells. The fluorescence generation mechanism is aggregation-induced emission (AIE), and it is a near-infrared aggregation-induced emission (AIE) fluorescent probe.

[0155] It should be noted that the above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art would be able to make other variations or modifications based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. A quinoline-malononitrile derivative, characterized in that The structure of the quinoline-malononitrile derivative is shown below: or .

2. The method for preparing the quinoline-malononitrile derivative according to claim 1, wherein The following steps are involved: (1) Compound 4, compound 5, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in N,N-dimethylformamide at a molar ratio of 26:30:52:7, and stirred at room temperature under argon protection to obtain quinoline-malononitrile derivative YL-181; alternatively, compound 4, indomethacin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in N,N-dimethylformamide at a molar ratio of 25:25:50:7, and stirred at room temperature under argon protection to obtain quinoline-malononitrile derivative YL-186; (2) After the reaction is completed, extraction, water removal, and reduced pressure distillation are performed in sequence to remove the solvent to obtain a crude product; (3) The crude product is purified and separated by silica gel column chromatography and vacuum dried to obtain the quinoline-malononitrile derivative according to claim 1; Among them, the structures of compound 4 and compound 5 are shown below: 。 3. The preparation method according to claim 2, characterized in that In step (1), the preparation method of compound 4 is: Compound 2 and compound 3 were added to acetonitrile in a molar ratio of 1:1, piperidine was added dropwise, and the mixture was refluxed at 90°C for 12 h under argon protection. After cooling to room temperature, the precipitated red precipitate was washed with acetonitrile and methanol, and dried in vacuo to obtain compound 4; Among them, the structures of compound 2 and compound 3 are shown below: 。 4. The preparation method according to claim 3, characterized in that The preparation method of compound 2 is: (1) 7-Methylquinoline and 2-bromoethanol were added to a reaction vessel in a molar ratio of 10:11, and refluxed at 140°C for 1 h under argon protection. After the reaction, the mixture was cooled to room temperature, and the solid was washed with cold acetonitrile three times for purification and separation, and then dried in vacuum to obtain compound 1; (2) Compound 1 and malononitrile were dissolved in anhydrous ethanol at a molar ratio of 2:5, and sodium ethoxide was slowly added dropwise at 0°C with stirring, and stirred in an ice bath for 1 hour, followed by reaction at room temperature for 6 hours. After the reaction, the pH value of the solution was adjusted to 7-8, and the ethanol was removed by rotary evaporation. The solution was extracted three times with ethyl acetate and saturated brine. The upper ethyl acetate was poured out and anhydrous sodium sulfate was added to remove water. After filtration, the crude product was obtained by vacuum distillation, and purified and separated by silica gel column chromatography. The eluent was a mixture of petroleum ether and ethyl acetate. V 石油醚 :V 乙酸乙酯 =1:1, vacuum dried to obtain compound 2; The structure of compound 1 is shown below: 。 5. The preparation method according to claim 3, characterized in that The preparation method of compound 3 is: 4-(Diphenylamino)phenylboronic acid and 5-bromothiophene-2-carboxaldehyde were placed in a round-bottom flask in a molar ratio of 1:1, and then potassium carbonate aqueous solution, tetrakis(triphenylphosphine)palladium and anhydrous tetrahydrofuran were added and stirred evenly. The air in the system was evacuated with a vacuum pump and replaced with argon. The mixture was refluxed at 80°C for 10 hours. After the reaction was completed, the system was cooled to room temperature, and tetrahydrofuran was removed under reduced pressure at 40°C. The mixture was extracted three times with dichloromethane and saturated brine, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated on a rotary evaporator to obtain a crude product, which was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate as the eluent. V 石油醚 :V 乙酸乙酯 =6.5:1, and dried in vacuum to obtain compound 3.

6. The preparation method according to claim 2, characterized in that In step (1), the preparation method of compound 5 is: Celecoxib and glutaric anhydride were dissolved in dichloromethane in a molar ratio of 2:1, triethylamine was added, and the mixture was stirred at room temperature under argon protection for 15±1h. After the reaction was completed, the solvent was removed by rotary evaporation to obtain a white oily liquid, which was adjusted to pH 13 and extracted three times with ethyl acetate and saturated brine. The aqueous phase was separated and the pH was adjusted to acidic. Then, extraction, dehydration, and reduced pressure distillation were performed in sequence, and the mixture was dried in vacuo to obtain compound 5.

7. The preparation method according to claim 2, characterized in that In step (2), extraction is performed with dichloromethane and saturated brine.

8. The preparation method according to claim 2, characterized in that In step (3), the eluent is a mixture of dichloromethane and petroleum ether, V 二氯甲烷 :V 石油醚 =1.5:

1.

9. The use of the quinoline-malononitrile derivative according to claim 1, characterized in that: As a near-infrared aggregation-induced emission fluorescent probe targeting COX-2, the application is not for the purpose of disease diagnosis or treatment.

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