Quinoline-malononitrile derivative, preparation method thereof and application of quinoline-malononitrile derivative as near-infrared AIE fluorescent probe of targeted lysosome

Abstract

The invention discloses a quinoline-malononitrile derivative, a preparation method thereof and application of the quinoline-malononitrile derivative as a near-infrared AIE fluorescent probe of a targeted lysosome, and belongs to the technical field of fluorescent probes. According to the quinoline-malononitrile derivative, quinoline-malononitrile serves as a core fluorophore, a thiophene group and a morpholine group are introduced for conjugate connection, an alkyl sulfonic acid group is further modified, and the quinoline-malononitrile derivative is a near-infrared AIE fluorescent probe, has good biocompatibility and stability, has targeting performance on lysosome, shows bright fluorescence in MCF-7, and can be used as a fluorescent probe. The quinoline-malononitrile derivative shows weak fluorescence in HUVEC, can generate fluorescence response according to the fluctuation of lysosome viscosity and can realize accurate imaging of a tumor microenvironment, and the structure of the quinoline-malononitrile derivative is as follows: # imgabs0 #.

Description

Technical Field

[0001] The present invention relates to a compound, a preparation method and application thereof, and in particular to a quinoline-malononitrile derivative, a preparation method thereof and application thereof as a near-infrared AIE fluorescent probe targeting lysosomes, 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. Lysosome viscosity in cancer cells is significantly higher than that in normal cells.

[0004] Lysosomes are important degradation centers within cells, and their physical and chemical environment (especially viscosity) is closely related to cellular function. Abnormal metabolic activity in cancer cells leads to the accumulation of macromolecules in lysosomes, increasing internal viscosity, making them potential biomarkers for distinguishing cancerous states and understanding tumor progression.

[0005] Existing fluorescent viscosity probes have limitations in detecting viscosity at the cellular level, such as insufficient photostability, high background fluorescence in aqueous environments, and lack of comprehensive validation in in vivo tumor models. 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 lysosome targeting, sensitive lysosomal viscosity 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: A quinoline-malononitrile derivative, the structure of which is shown below: .

[0008] The preparation method of the aforementioned quinoline-malononitrile derivative comprises the following steps: (1) Compound 1 and compound 2 were added to a mixed solvent of acetonitrile / methanol in a molar ratio of 1:1, and a catalytic amount of piperidine was added. The mixture was refluxed at 85°C for 12 h under argon protection. (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 normal silica gel column chromatography and dried in vacuo to obtain the aforementioned quinoline-malononitrile derivative; The structures of compound 1 and compound 2 are shown below: .

[0009] Preferably, in step (1), the preparation method of compound 1 is as follows: 5-bromothiophene-2-carboxaldehyde and 4-morpholinophenylboronic acid are weighed in a molar ratio of 5:3, placed in a reaction vessel, potassium carbonate aqueous solution, tetrahydrofuran and tetrakis(triphenylphosphine)palladium are added in sequence, the air is evacuated and replaced with argon, refluxed at 80° C. for 8 h, the solvent is dried, extraction, water removal, and reduced pressure distillation are performed in sequence to remove the solvent to obtain a crude product, the crude product is purified and separated by normal silica gel column chromatography, and vacuum dried to obtain compound 1.

[0010] Preferably, in step (1), the preparation method of compound 2 is as follows: 3-(2-methylquinolin-1-yl)propane-1-sulfonic acid inner salt and malononitrile are weighed in a molar ratio of 10:3, dissolved in anhydrous ethanol, and sodium ethoxide is added dropwise at 0°C with stirring for 15±1h, the pH value of the solution is adjusted to 7-8, the solvent is dried by rotary evaporation, and extraction, water removal, and reduced pressure distillation are performed in sequence to obtain a crude product, the crude product is purified and separated by normal silica gel column chromatography, and vacuum dried to obtain compound 2.

[0011] Preferably, in step (2), extraction is performed with dichloromethane and saturated brine.

[0012] Preferably, in step (3), the eluent is a mixture of dichloromethane and methanol, V 二氯甲烷 :V 甲醇 =22:1.

[0013] The aforementioned quinoline-malononitrile derivatives are used, specifically, as near-infrared aggregation-induced emission fluorescent probes targeting lysosomes.

[0014] The benefits of the present invention lie in that the quinoline-malononitrile derivative YL-134 provided by the present invention can specifically target lysosomes and has sensitive responsiveness to lysosomal viscosity, and has excellent AIE properties, near-infrared emission, large Stokes shift, good pH stability, photostability, plasma stability and low cytotoxicity. It can achieve specific recognition and imaging of cancer cells, can be used for in vivo tumor imaging, provides a research basis for accurate tumor diagnosis, and has great potential as a guide agent in tumor surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a flow chart for the preparation of quinoline-malononitrile derivative YL-134; Figure 2 is compound 1 1 H NMR spectrum; Figure 3 is compound 1 13 C NMR spectrum; Figure 4 is the HR-MS spectrum of compound 1; Figure 5 is compound 2 1 H NMR spectrum; Figure 6 is compound 2 13 C NMR spectrum; Figure 7 is the HR-MS spectrum of compound 2; Figure 8 is compound 3 1 H NMR spectrum; Figure 9 is compound 3 13 C NMR spectrum; Figure 10 is the HR-MS spectrum of compound 3; Figure 11 This is the cell imaging result of YL-134 in HUVEC and MCF-7; Figure 12 is the calculation result of the relative fluorescence intensity of YL-134 in HUVEC and MCF-7; Figure 13 This is the cell imaging result of YL-134 in MCF-7 cells pretreated with Cu²⁺ or dexamethasone; Figure 14 This is the calculation result of the relative fluorescence intensity of YL-134 in MCF-7 pretreated with Cu²⁺ or dexamethasone; Figure 15 This is the fluorescence spectrum of YL-134 in different solvents; Figure 16 This is the fluorescence spectrum of YL-134 at different viscosities; Figure 17 is the linear relationship between fluorescence intensity and viscosity; Figure 18 This is the AIE effect test result of YL-134; Figure 19 This is the result of the ion selectivity experiment of YL-134; Figure 20 This is the pH stability test result of YL-134; Figure 21 This is the photostability test result of YL-134; Figure 22 This is a graph showing the results of a plasma stability experiment of YL-134; Figure 23 This is the result of the cytotoxicity experiment of YL-134; Figure 24 This is a cell imaging image showing the co-localization of YL-134 and lysosomes; Figure 25 is the fluorescence intensity curve of YL-134 and lysosomal green fluorescent probe; Figure 26 This is the result of in vivo fluorescence imaging of tumor-bearing mice; Figure 27 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

[0016] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0017] 1. Structure of quinoline-malononitrile derivatives The structure of the quinoline-malononitrile derivative provided by the present invention is shown below: .

[0018] 2. Preparation method of quinoline-malononitrile derivatives like Figure 1 As shown, the preparation method of the above-mentioned quinoline-malononitrile derivative provided by the present invention is as follows: (1) Weigh 5-bromothiophene-2-carboxaldehyde (191.1 mg, 1 mmol) and 4-morpholinophenylboronic acid (207.1 mg, 0.6 mmol) and place them in a 100 mL round-bottom flask. Then, add potassium carbonate aqueous solution (concentration of 0.5 M, 4 mL), tetrahydrofuran (8 mL) and tetrakis(triphenylphosphine)palladium (11.6 mg, 0.01 mmol) in sequence. Then, use a vacuum pump to evacuate the air in the system and replace it with argon. Reflux at 80 ° C for 8 h. The solvent is dried by rotary drying. The system is extracted with dichloromethane and saturated brine. The lower layer of dichloromethane is separated and anhydrous sodium sulfate is added to stand and remove water. Then, the crude product is distilled under reduced pressure on a rotary evaporator to obtain a crude product. The crude product is purified and separated by normal silica gel column chromatography (the eluent is a mixture of petroleum ether and ethyl acetate, V 石油醚 :V 乙酸乙酯 =5:1), and vacuum drying to obtain compound 1 as a golden solid with a yield of 91.1%.

[0019] Compound 1 1 H NMR spectrum, 13 C NMR spectrum and HR-MS spectrum are shown in Figure 2 、 Figure 3 、 Figure 4 , 1 H NMR, 13 The characterization results of C NMR and HR-MS are as follows: 1 H NMR (600MHz, CDCl3) δ 9.86 (s, 1H), 7.71 (d, J =3.9Hz, 1H), 7.60(d, J =8.8Hz, 2H), 7.30(d, J =3.9Hz, 1H), 6.93(d, J =8.8Hz, 2H), 3.90-3.87 (m, 4H), 3.27-3.24 (m, 4H).

[0020] 13 C NMR (151MHz, CDCl3) δ 182.60, 137.86, 127.50, 122.47, 115.19, 66.69, 48.35.

[0021] HR-MS (ESI): [M+H] + C 15 H 16 NO2S + Calculated value 274.08963, theoretical value 274.13700.

[0022] (2) 3-(2-Methylquinolin-1-yl)propane-1-sulfonic acid inner salt (185.7 mg, 0.7 mmol) and malononitrile (120 μL, 0.21 mmol) were dissolved in anhydrous ethanol (100 mL). Sodium ethoxide (1 mL) was added dropwise at 0°C with stirring and the mixture was reacted for 15±1 h. The pH value of the solution was adjusted to 7-8 with 1 M HCl. The solvent was dried by rotary evaporation. The solution was extracted with dichloromethane and saturated brine. The lower organic phase was separated and anhydrous sodium sulfate was added and allowed to stand. The crude product was obtained by vacuum distillation. The crude product was purified and separated by normal silica gel column chromatography (eluent was a mixture of dichloromethane and methanol, V 二氯甲烷 :V 甲醇 =8.5:1), and vacuum drying to obtain compound 2 as a yellow solid with a yield of 37.1%.

[0023] Compound 2 1 H NMR spectrum, 13 C NMR spectrum and HR-MS spectrum are shown in Figure 5 、 Figure 6 、 Figure 7 . 1 H NMR, 13 The characterization results of C NMR and HR-MS are as follows: 1 H NMR (600MHz, D2O) δ 8.04 (d, J =8.0Hz,1H),7.63(dd, J =39.9, 7.8Hz, 2H), 7.22 (t, J =7.2Hz, 1H), 6.30 (s, 1H), 4.37-4.08 (m, 2H), 3.30-2.98 (m, 3H), 2.46 (s, 3H), 2.07 (s, 2H).

[0024] 13 C NMR (151MHz, D2O) δ 152.07, 150.41, 137.30, 133.61, 121.65, 120.52, 120.10, 117.04, 109.93, 44.85, 29.80, 23.13, 23.08.

[0025] HR-MS (ESI): [MH] - C 16 H 14 N3O3S - Calculated value 328.07614, theoretical value 328.07593.

[0026] (3) Compound 1 (41 mg, 0.15 mmol) and compound 2 (49 mg, 0.15 mmol) were added to an acetonitrile / methanol mixed solvent (4 mL of acetonitrile and 4 mL of methanol), and 2-3 drops of piperidine (catalyst) were added. The mixture was refluxed at 85°C for 12 h under argon protection. After the reaction was completed, extraction (dichloromethane and saturated brine) and water removal (adding anhydrous sodium sulfate and allowing to stand) were performed in sequence. The solvent was removed by vacuum distillation to obtain a crude product. The crude product was purified and separated by normal silica gel column chromatography (the eluent was a mixture of dichloromethane and methanol, V 二氯甲烷 :V 甲醇 =22:1) and vacuum dried to obtain compound 3 (quinoline-malononitrile derivative, denoted as YL-134) as a dark red solid with a yield of 36%.

[0027] Compound 3 (YL-134) 1 H NMR spectrum, 13 C NMR spectrum and HR-MS spectrum are shown in Figure 8 、 Figure 9 、 Figure 10 . 1 H NMR, 13 The characterization results of C NMR and HR-MS are as follows: 1H NMR (600MHz, DMSO) δ 8.93 (d, J =8.4Hz,1H),8.25(d, J =8.9Hz,1H),7.91(t, J =7.7Hz, 1H), 7.73(d, J =3.4Hz, 1H), 7.67-7.56 (m, 4H), 7.45 (d, J =3.5Hz, 1H), 7.36(d, J =15.4Hz, 1H), 7.08 (s, 1H), 7.02 (d, J =8.6Hz, 2H), 4.77-4.66 (m, 2H), 3.76 (s, 4H), 3.20 (s, 4H), 2.69 (s, 2H), 2.12 (s, 2H).

[0028] 13 C NMR (151MHz, DMSO) δ 152.38, 151.47, 149.39, 147.66, 138.70, 138.02, 134.11, 133.37, 132.89, 126.92, 125.49, 125. 41, 124.25, 123.54, 121.09, 118.93, 118.25, 115.48, 106.66, 66.43, 48.21, 48.00, 46.74, 25.04.

[0029] HR-MS (ESI): [MH] - C 31 H 27 N4O4S 2- Calculated value 583.14792, theoretical value 583.14382.

[0030] 3. Cellular Imaging of Quinoline-Malononitrile Derivative (YL-134) Normal HUVEC cells and MCF-7 cancer cells were incubated with YL-134 (5 μM) for 2 h, washed with PBS, and then imaged using CLSM.

[0031] The results of cell imaging of YL-134 in HUVEC and MCF-7 are shown in Figure 11 The relative fluorescence intensity calculation results in HUVEC and MCF-7 are shown in Figure 12 .

[0032] Depend on Figure 11 and Figure 12It can be seen that the fluorescence intensity in MCF-7 is much higher than that in HUVEC, 16 times that of HUVEC, indicating that YL-134 can distinguish normal cells from cancer cells.

[0033] 4. Lysosomal viscosity response experiment of quinoline-malononitrile derivative (YL-134) To verify the responsiveness of YL-134 to changes in lysosomal viscosity, MCF-7 cells were first treated with Cu²⁺ (20 μM) to decrease lysosomal viscosity or dexamethasone (20 μM) to increase lysosomal viscosity for 3 hours. MCF-7 cells were then incubated with YL-134 (5 μM) for 2 hours. Following incubation, the cells were washed with PBS and imaged using CLSM.

[0034] The results of cell imaging of YL-134 in MCF-7 cells stimulated with Cu²⁺ or dexamethasone are shown in Figure 13 The relative fluorescence intensity calculation results of MCF-7 stimulated by Cu²⁺ or dexamethasone are shown in Figure 14 .

[0035] Depend on Figure 13 and Figure 14 It can be seen that after incubation of MCF-7 stimulated by Cu²⁺ and YL-134, the fluorescence intensity decreased by 78% compared with the control group; after incubation of MCF-7 stimulated by dexamethasone and YL-134, the fluorescence intensity increased by 87% compared with the control group.

[0036] These results suggest that YL-134 can respond to changes in lysosomal viscosity.

[0037] 5. Fluorescence properties of quinoline-malononitrile derivative (YL-134) in different solvents Accurately pipette 2 μL of YL-134 mother solution (dissolved in DMSO, concentration 10 mM) and dilute to 2 mL with glycerol, methanol, phosphate buffer, tetrahydrofuran, acetonitrile, 1,4-dioxane, dimethyl sulfoxide, dichloromethane, dimethylformamide, and ethyl acetate, respectively. Vortex mix to obtain a YL-134 test solution with a concentration of 10 μM.

[0038] The fluorescence emission spectrum of the YL-134 test solution was measured in the range of 500-850 nm using the optimal excitation wavelength of 447 nm for YL-134.

[0039] The fluorescence spectra of YL-134 in different solvents are shown in Figure 15 .

[0040] Depend on Figure 15It can be seen that the fluorescence intensity of YL-134 in phosphate buffer, tetrahydrofuran, 1,4-dioxane and dichloromethane is very low, the fluorescence emission in glycerol is significantly enhanced, the fluorescence intensity in dimethyl sulfoxide, ethyl acetate and methanol is also enhanced, and the fluorescence intensity in dimethylformamide and acetonitrile is relatively low.

[0041] VI. Fluorescence test of quinoline-malononitrile derivative (YL-134) at different viscosities Using deionized water / glycerol as the solvent system, YL-134 stock solution was prepared at different ratios from 0 / 100 to 100 / 0 (water / glycerol, v / v) at room temperature to prepare 10 μM YL-134 test solutions with different viscosity systems.

[0042] The mixture was thoroughly mixed by vortexing for 3 min, excited with 447 nm excitation light (slit width 5 nm), and the fluorescence emission spectrum of each YL-134 test solution in the range of 500-850 nm was measured.

[0043] The fluorescence spectra of YL-134 at different viscosities are shown in Figure 16 .

[0044] Depend on Figure 16 It can be seen that as the percentage of glycerol increases, the fluorescence intensity of YL-134 at 674 nm continues to increase.

[0045] The system viscosity value (Log η) is the horizontal axis (X), the fluorescence intensity at 674nm (Log I 674 ) as the vertical axis to obtain the linear relationship between fluorescence intensity and viscosity.

[0046] The linear relationship between fluorescence intensity and viscosity is shown in the figure. Figure 17 .

[0047] Depend on Figure 17 It can be seen that there is a good linear relationship between fluorescence intensity and viscosity.

[0048] 7. Aggregation-induced emission (AIE) effect test of quinoline-malononitrile derivative (YL-134) Deionized water is used as a good solvent, and anhydrous ethanol is used as a poor solvent, and mixed solvent systems containing poor solvents in proportions ranging from 0% to 100% are prepared.

[0049] When the mixed solvent system is at room temperature, the YL-134 mother solution is measured and added to the mixed solvent system to prepare a YL-134 test solution with a concentration of 10 μM.

[0050] A fluorescence spectrophotometer was used to perform light excitation at the optimal excitation wavelength of 447 nm for YL-134, and the fluorescence emission spectrum of the YL-134 test solution was scanned within the range of 500-850 nm.

[0051] The AIE effect test results of YL-134 are shown in Figure 18 .

[0052] Depend on Figure 18 It can be seen that the fluorescence intensity of YL-134 is weak in the benign solvent deionized water. As the proportion of the poor solvent anhydrous ethanol increases, the fluorescence intensity continues to increase. When the fraction of anhydrous ethanol in the mixed solvent system is 80%, YL-134 has the highest fluorescence intensity, and then the fluorescence intensity decreases.

[0053] 8. Study on the ion selectivity of quinoline-malononitrile derivative (YL-134) 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 separately in a 100 mL volumetric flask. Add deionized water and YL-134 mother liquor to prepare a solution system with a YL-134 concentration of 10 μM and an ion concentration of 1 mM.

[0054] Accurately measure the YL-134 mother liquor and place it in a 100 mL volumetric flask. Add a mixed solvent of glycerol and water (glycerol / water = 4 / 1, v / v) to prepare a solution system with a YL-134 concentration of 10 μM and a glycerol concentration of 80% (v / v) (referred to as 80% glycerol, glycerol system).

[0055] Accurately measure the YL-134 mother solution, place it in a 100 mL volumetric flask, add deionized water, and prepare a solution system with a YL-134 concentration of 10 μM (recorded as water, blank control group).

[0056] After the solution is mixed, it is placed in a fluorescence spectrophotometer, and the fluorescence intensity at the maximum emission peak of the solution system is measured under excitation at the optimal excitation wavelength of YL-134, 447 nm.

[0057] The results of the ion selectivity experiments of YL-134 are shown in Figure 19 .

[0058] Depend on Figure 19 It can be seen that compared with the blank control group, the fluorescence intensity of YL-134 changed less after adding various interfering ions or biothiols, while it showed significant fluorescence enhancement in the glycerol system (glycerol / water = 4 / 1, v / v).

[0059] IX. pH stability test of quinoline-malononitrile derivative (YL-134) Deionized water 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.

[0060] YL-134 mother solution was added to the prepared solutions of different pH values, and the mixture was mixed to prepare solutions containing 10 μM YL-134 at different pH values.

[0061] YL-134 solutions with 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 447 nm for YL-134.

[0062] The pH stability test results of YL-134 are shown in Figure 20 .

[0063] 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 intensity of YL-134 remains stable.

[0064] 10. Photostability test of quinoline-malononitrile derivative (YL-134) Accurately pipette the YL-134 stock solution into the premixed solvent system (deionized water / glycerol = 1 / 1, v / v) to prepare a YL-134 test solution with a final concentration of 10 μM.

[0065] After vortex mixing, the YL-134 test solution was placed under normal lighting conditions and placed in a fluorescence spectrophotometer to measure the fluorescence intensity every 10 minutes (for a total of 90 minutes). The fluorescence intensity was measured at the maximum emission wavelength using the optimal excitation wavelength of 447 nm for YL-134.

[0066] The photostability test results of YL-134 are shown in Figure 21 .

[0067] Depend on Figure 21 It can be seen that the fluorescence intensity of YL-134 did not change significantly within 90 minutes of illumination.

[0068] 11. Plasma stability test of quinoline-malononitrile derivative (YL-134) 900 μL of treated rat plasma was placed in a 2 mL centrifuge tube, and YL-134 stock solution (final concentration 10 μM) was added. The tube was then transferred to a 37°C water bath and incubated on a shaker. Samples were collected at various time points between 0 and 120 minutes. After sampling, the samples were vortex-extracted with acetonitrile. After centrifugation, the supernatant was filtered through a membrane and the peak area of YL-134 was determined using high-performance liquid chromatography.

[0069] The results of the plasma stability test of YL-134 are shown in Figure 22 .

[0070] Depend on Figure 22 It can be seen that YL-134 can still be effectively detected after incubation in plasma for 120 minutes, and its chromatographic peak area has no obvious change.

[0071] 12. Cytotoxicity Study of Quinoline-Malononitrile Derivative (YL-134) 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, the cells were seeded at a density of 5 × 10 cells / well in a 96-well plate. Serum-containing medium was added to 200 µL per well and incubated at 37°C in a 5% CO2 incubator for 24 hours. Subsequently, YL-134 stock solution was added to a concentration gradient of 0 µM (blank control), 2.5 µM, 5 µM, 10 µM, 20 µM, and 40 µM. The cells were incubated for an additional 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. Absorbance was measured at 570 nm using a microplate reader to calculate cell viability.

[0072] The results of the cytotoxicity test of YL-134 are shown in Figure 23 .

[0073] Depend on Figure 23 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-134 at a concentration of up to 40μM for 24 hours.

[0074] 13. Cellular Colocalization of Quinoline-Malononitrile Derivative (YL-134) YL-134 (5 μM) was co-incubated with MCF-7 cancer cells for 1.5 hours, followed by staining with Lyso-Tracker Green (50 nM), a lysosomal green fluorescent probe, for 30 minutes. After washing with PBS, the cells were imaged using confocal laser scanning microscopy (CLSM).

[0075] Cell imaging of YL-134 co-localizing with lysosomes is shown in Figure 24 , the fluorescence intensity curve of YL-134 and lysosomal green fluorescent probe is shown in Figure 25 .

[0076] Depend on Figure 24 It can be seen that the red fluorescence of YL-134 and the green fluorescence of the lysosomal green fluorescent probe show a high degree of overlap in the cells, and the merged image appears as a distinct yellow color, which intuitively indicates that YL-134 successfully targets the lysosomes in living cells.

[0077] Depend on Figure 25It can be seen that the red fluorescence of YL-134 and the green fluorescence of the lysosomal green fluorescent probe highly overlap, and the Pearson colocalization coefficient is 0.914.

[0078] 14. Animal Imaging Experiments with Quinoline-Malononitrile Derivative (YL-134) 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-134 solution (200 μM, 50 μL) was injected orthotopically into the tumor tissue and contralateral normal subcutaneous tissue.

[0079] 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.

[0080] The results of in vivo fluorescence imaging of tumor-bearing mice are shown in Figure 26 , the in vitro imaging results of major organs and tumor tissues are shown in Figure 27 .

[0081] Depend on Figure 26 It can be seen that the fluorescence signal in the tumor area is 2.16 times stronger than that in the control area. Figure 27 It can be seen that tumor tissue has obvious fluorescence, while the fluorescence of other organs can be ignored.

[0082] In summary, the quinoline-malononitrile derivative YL-134 provided by the present invention can be used as a fluorescent probe. It uses quinoline-malononitrile as the core fluorophore, introduces an electron-rich thiophene group and conjugates it with a weakly basic morpholine group targeting lysosomes, and further modifies an alkyl sulfonic acid group as a viscosity-responsive group. The fluorescent probe has good biocompatibility and stability (pH stability, photostability, and plasma stability), is targeted to lysosomes, displays bright fluorescence in MCF-7, and exhibits weak fluorescence in HUVEC. It can generate a fluorescent response according to fluctuations in lysosomal viscosity, achieve precise 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.

[0083] 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: 。 2. The method for preparing the quinoline-malononitrile derivative according to claim 1, wherein The following steps are involved: (1) Compound 1 and compound 2 were added to a mixed solvent of acetonitrile / methanol in a molar ratio of 1:1, and a catalytic amount of piperidine was added. The mixture was refluxed at 85°C for 12 h under argon protection. (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 normal silica gel column chromatography and dried in vacuo to obtain the quinoline-malononitrile derivative according to claim 1; The structures of compound 1 and compound 2 are shown below: 。 3. The preparation method according to claim 2, characterized in that In step (1), the preparation method of compound 1 is: 5-Bromothiophene-2-carboxaldehyde and 4-morpholinophenylboronic acid were weighed in a molar ratio of 5:3 and placed in a reaction vessel. Aqueous potassium carbonate solution, tetrahydrofuran, and tetrakis(triphenylphosphine)palladium were added in sequence. The air was evacuated and replaced with argon. The mixture was refluxed at 80°C for 8 hours. The solvent was dried by spin drying. Extraction, water removal, and reduced pressure distillation were performed in sequence to remove the solvent to obtain a crude product. The crude product was purified and separated by normal silica gel column chromatography and dried in vacuo to obtain compound 1.

4. The preparation method according to claim 2, characterized in that In step (1), the preparation method of compound 2 is: 3-(2-Methylquinolin-1-yl)propane-1-sulfonic acid inner salt and malononitrile were weighed in a molar ratio of 10:3 and dissolved in anhydrous ethanol. Sodium ethoxide was added dropwise at 0°C with stirring for 15±1h. The pH value of the solution was adjusted to 7-8. The solvent was dried by rotary evaporation, and extraction, water removal, and reduced pressure distillation were performed to remove the solvent to obtain a crude product. The crude product was purified and separated by normal silica gel column chromatography and dried in vacuo to obtain compound 2.

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

6. The preparation method according to claim 2, characterized in that In step (3), the eluent is a mixture of dichloromethane and methanol, V 二氯甲烷 :V 甲醇 =22:

1.

7. The use of the quinoline-malononitrile derivative according to claim 1, characterized in that: As a near-infrared aggregation-induced emission fluorescent probe targeting lysosomes.

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