Coumarin and hemicyanine conjugated derivatives, synthesis method and application thereof

Abstract

The application provides a coumarin and hemicyanine coupling derivative, a synthesis method and application thereof, and the specific Chinese name of the derivative is 1-(3-(4-((Z)-2-cyano-3-(7-(diethylamino)-2-oxo-2H-chromen-3-yl) acryloyl) piperazin-1-yl)-3,3-dimethyl-2-((E)-2-(6-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl) benzyl) oxy)-2,3-dihydro-1H-xanthine-4-yl) ethenyl)-3H-indol-1-ium. The application also provides a synthesis method of the derivative and an application of the derivative in visual monitoring of redox dynamic changes of cells in ferroptosis. The method is based on the coumarin and hemicyanine coupling derivative, and the concentration changes of glutathione and hydrogen peroxide are detected by a fluorescence spectrophotometer in a mixed solution of phosphate buffer and dimethyl sulfoxide (pH=7.4, volume ratio 1:1). The method also realizes synchronous fluorescence visual monitoring of the concentration changes of glutathione and hydrogen peroxide with time in the process of cell ferroptosis.

Description

Technical Field

[0001] The invention relates to a hydrogen peroxide and glutathione specific fluorescent probe, in particular to a coumarin and hemicyanine coupling derivative DRSP-HYL and a synthesis method and application thereof. Background Art

[0002] Moderate redox stress positively impacts homeostasis through signaling. However, when the body is acutely injured or overly stressed, it overexpresses reactive oxygen species (ROS) or reactive sulfur species (RSS) to alleviate redox imbalance. Ferroptosis, an excessive redox metabolic process associated with diseases such as tumors and ischemic injury, involves complex signaling cascades and precise molecular mediation mechanisms, including iron accumulation, lipid peroxidation, and amino acid metabolism. It is closely related to the cystine / glutamate antiporter and glutathione peroxidase 4. This regulatory mechanism relies on the formation of a sophisticated regulatory network of ROS and RSS species.

[0003] Currently, in clinical practice, there are some typical ferroptosis agonists that disrupt the body's redox balance and promote the ferroptosis process by intervening in H2O2 or GSH levels. Therefore, revealing the dynamic correlation of H2O2 or GSH in the ferroptosis pathway will help to efficiently regulate the ferroptosis process and promote the diagnosis and treatment of diseases. However, the traditional "separation-detection" method can no longer meet the needs of in vivo, in situ, real-time, and dynamic monitoring. Therefore, the development of single-molecule fluorescent probes for synchronously monitoring the dynamic changes of H2O2 and GSH in the ferroptosis process can effectively break the current detection technology dilemma and is of great significance for the early diagnosis and treatment of diseases. Summary of the Invention

[0004] The present invention aims to provide a coumarin and hemicyanine coupling derivative DRSP-HYL and a synthesis method thereof, as well as an application of DRSP-HYL in visually monitoring redox in the process of cell ferroptosis.

[0005] The present invention provides a coumarin and hemicyanine coupled derivative DRSP-HYL, the Chinese name of which is 1-(3-(4-((Z)-2-cyano-3-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)acryloyl)piperazin-1-yl)-3,3-dimethyl-2-((E)-2-(6-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborol-2-yl)benzyl)oxy)-2,3-dihydro-1H-xanthine-4- Its English name is 1-(3-(4-((Z)-2-cyano-3-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)acryloyl)piperazin-1-yl)-3-oxopropyl)-3,3-dimethyl-2-((E)-2-(6-((4-(4,4,5,5-tetramet hyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3H-indol-1-ium. Its structural formula is:

[0006]

[0007] The synthesis method of DRSP-HYL comprises the following steps:

[0008] (1) Phosphorus oxychloride was added dropwise to DMF in a molar ratio of 1:1.0-1.5, and stirred at room temperature for 20-50 minutes; then 0.6-1.0 equivalents of cyclohexanone was added to the reaction system and stirred at room temperature for 1.5-4 hours. The reaction was quenched with water and adjusted to neutral with sodium bicarbonate; then extracted with ethyl acetate, and the organic phase was collected and concentrated to obtain a yellow oily crude product, Compound 1;

[0009] (2) Compound 1 and 2-hydroxy-4-methoxybenzaldehyde were dissolved in an appropriate amount of DMF in a molar ratio of 1:0.7 to 0.9, and then 2 to 3 equivalents of cesium carbonate were added and stirred at room temperature for 16 to 32 hours; the filtrate was filtered and collected, and purified by column chromatography using a mixed solvent of ethyl acetate and petroleum ether as an eluent to obtain a yellow solid compound 2;

[0010] (3) Compound 2 and boron tribromide were dissolved in a dichloromethane solution at a molar ratio of 1:5.0-10.0 and stirred at room temperature for 6 hours; the mixture was quenched with water, extracted with dichloromethane, and the organic phase was collected and concentrated. Column chromatography was performed using a mixed solvent of dichloromethane and methanol as the eluent to obtain an orange solid compound 3;

[0011] (4) Compound 3 and 1-(2-carboxyethyl)-2,3,3-trimethylindol-1-ium were dissolved in an appropriate amount of acetonitrile in a molar ratio of 1:1.0 to 1.5, and a catalytic amount of piperidine was added. The mixture was heated under reflux for 12 to 20 hours. The solvent was removed by distillation under reduced pressure, and the mixture was purified by column chromatography using a mixed solvent of dichloromethane and methanol as an eluent to obtain compound 4 as a blue-purple solid powder.

[0012] (5) Compound 4, EDCI, and HOBT were dissolved in dichloromethane in a molar ratio of 1:1 to 3:1 to 3, and the mixture was reacted at 0°C under nitrogen atmosphere for 20 to 50 minutes; 1.0 to 2.0 equivalents of 1-tert-butyloxycarbonylpiperazine were then added, and the reaction was continued at room temperature for 20 to 30 hours; the solvent was removed by distillation under reduced pressure, and the mixture was separated and purified by column chromatography using a mixed solvent of dichloromethane and methanol as an eluent to obtain a blue-green solid compound 5;

[0013] (6) Compound 5 and potassium carbonate were combined in acetonitrile in a molar ratio of 1:1.2-1.5, and reacted at 45°C in a nitrogen environment for 5-15 minutes. Then, 1.2-1.5 equivalents of potassium iodide and 1.5-2.5 equivalents of 4-bromomethylphenylboronic acid pinacol ester were added and the reaction was continued at 45°C for 3-5 hours. The solvent was removed by distillation under reduced pressure, and a mixed solvent of dichloromethane and methanol was used as an eluent for column chromatography to obtain a blue solid compound 6.

[0014] (7) Compound 6 and trifluoroacetic acid were dissolved in dichloromethane at a molar ratio of 1:50-80, stirred at room temperature for 20-50 minutes, and concentrated under reduced pressure to obtain a crude product 7, which was directly used for the next step of synthesis. Next, (Z)-2-cyano-3-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)acrylic acid, EDCI, and HOBT were dissolved in dichloromethane at a molar ratio of 1:1.0-3.0:1.0-3.0, and stirred for reaction at 0°C under a nitrogen atmosphere for 20-50 minutes; then, 1.0-2.0 equivalents of compound 7 and 0.8-1.2 equivalents of triethylamine were added to the reaction system, and the reaction was continued at room temperature for 20-30 hours; the reaction was concentrated under reduced pressure, and a mixed solvent of dichloromethane and methanol was used as an eluent for column chromatography to obtain a purple-black solid DRSO-HYL.

[0015] The synthesized DRSP-HYL is used for in vitro specific qualitative and quantitative fluorescence analysis of hydrogen peroxide and glutathione, and the derivative is used to visually monitor the redox state during ferroptosis in cells, including:

[0016] (1) Prepare equal volumes of dimethyl sulfoxide and PBS (phosphate buffer solution with pH = 7.4 prepared with sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride) mixed buffer solution (referred to as test system), prepare 2 mmol / L RSP-HYL dimethyl sulfoxide solution (referred to as probe stock solution), prepare 200 mmol / L glutathione aqueous solution, prepare 20 mmol / L hydrogen peroxide aqueous solution, prepare 10 mmol / L erastin dimethyl sulfoxide solution, prepare 0.1 mmol / L glutathione peroxidase 4 inhibitor (RSL3) dimethyl sulfoxide solution;

[0017] (2) Place 2.0 mL of the test system in a cuvette, add 10 μL of the probe stock solution, and then add 20 μL of a 20 mmol / L hydrogen peroxide solution. Monitor the fluorescence intensity at 723 nm using a fluorescence spectrophotometer with 695 nm UV-visible absorption as the excitation light source. With the addition of hydrogen peroxide, the fluorescence intensity at 723 nm increases.

[0018] (3) Place 2.0 mL of the test system in a cuvette, add 10 μL of the probe stock solution, and then add 20 μL of a 200 mmol / L glutathione aqueous solution. Monitor the fluorescence intensity at 485 nm using a fluorescence spectrophotometer with 380 nm UV-visible absorption as the excitation light source. With the addition of glutathione, the fluorescence intensity at 485 nm increases.

[0019] (4) Place 10 μL of the probe stock solution and 2.0 mL of the test system in a cuvette, add 20 μL of a 20 mmol / L aqueous hydrogen peroxide solution, and monitor the fluorescence intensity at 723 nm over time using a fluorescence spectrophotometer with 695 nm UV-visible absorption as the excitation light source. The fluorescence intensity of the probe DRSP-HYL stabilizes after approximately 30 minutes of reaction with hydrogen peroxide.

[0020] (5) Place 10 μL of the probe stock solution in a cuvette containing 2.0 mL of the test system, add 20 μL of a 200 mmol / L glutathione aqueous solution, and monitor the change in fluorescence intensity at 485 nm over time using a fluorescence spectrophotometer with 380 nm UV-visible absorption as the excitation light source. The probe DRSP-HYL reacts immediately with glutathione, and the fluorescence intensity tends to stabilize.

[0021] (6) Take 10 μL of probe stock solution and 2.0 mL of test system in a cuvette, and add 2 μL, 3 μL, 3.5 μL, 4 μL, 5 μL, 6 μL, and 7 μL of 20 mmol / L hydrogen peroxide solution, respectively. After 30 minutes, monitor the fluorescence intensity at 723 nm on a fluorescence spectrophotometer using 695 nm ultraviolet-visible absorption as the excitation light source. As the hydrogen peroxide concentration increases, the fluorescence intensity at 723 nm gradually increases;

[0022] (7) Take 10 μL of probe stock solution and 2.0 mL of test system in a cuvette, and add 0 μL, 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, 12 μL, 14 μL, 16 μL, 18 μL, and 20 μL of 200 mmol / L glutathione aqueous solution respectively. After 30 seconds, use 380 nm UV-visible absorption as the excitation light source on a fluorescence spectrophotometer to monitor the change in fluorescence intensity at 485 nm. As the glutathione concentration increases, the fluorescence intensity at 485 nm increases;

[0023] (8) 10 μL of probe stock solution was dissolved in 2.0 mL of PBS (final probe concentration was 10 μmol / L) and used to incubate cervical cancer cells (Hela cells) for 20 minutes. The PBS solution was then removed and washed twice with PBS. The cells were placed on a fluorescence confocal microscope and the excitation light sources were 405 nm and 633 nm. The dual-channel fluorescence intensity in the wavelength range of 455-515 nm and 693-753 nm was monitored, respectively.

[0024] (9) Cervical cancer cells Hela were incubated with 2.0mL DMEM culture medium containing 20μL 0.1 mmol / L RSL3 stock solution for 6h, 12h, 18h and 24h, respectively, and then washed twice with PBS. 10μL probe stock solution was dissolved in 2.0mL PBS (the final concentration of the probe was 10μmol / L) and used to incubate Hela cells for 20 minutes. After that, the cells were placed on a fluorescence confocal microscope, and 405nm and 633nm were used as excitation light sources to monitor the dual-channel fluorescence intensity in the wavelength range of 455-515nm and 693-753nm respectively. The fluorescence intensity of the two channels was compared and analyzed over time, showing that the fluorescence intensity of the 693-753nm channel gradually increased over time, and the fluorescence intensity of the 455-515nm channel gradually increased over time;

[0025] (10) Hela cells were incubated with 2.0 mL DMEM culture medium containing 8 μL of 10 mmol / L Erastin stock solution for 6 h, 12 h, 18 h, and 24 h, respectively. Then, they were washed twice with PBS. 10 μL of the probe stock solution was dissolved in 2.0 mL PBS (the final concentration of the probe was 10 μmol / L) and used to incubate the Hela cells for 20 minutes. After that, the cells were placed on a fluorescence confocal microscope. With 405 nm and 633 nm as the excitation light sources, the fluorescence intensity of the two channels in the wavelength range of 455-515 nm and 693-753 nm was monitored respectively. The fluorescence intensity of the two channels was compared and analyzed over time. It was shown that the fluorescence intensity of the 693-753 nm channel gradually increased over time, while the fluorescence intensity of the 455-515 nm channel first decreased and then increased over time.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The synthesis of the coumarin and hemicyanine coupled derivative DRSP-HYL of the present invention is convenient and easy to operate;

[0028] 2. As a fluorescent probe, DRSP-HYL has the characteristics of single-molecule, dual-site simultaneous detection of H2O2 and GSH, which solves the shortcomings of previous molecular probes that cannot detect at the same time and space, and improves the accuracy of imaging the dynamic changes of H2O2 and GSH;

[0029] 3. The detection method is simple. Fluorescence spectroscopy enables qualitative and quantitative detection of H2O2 and GSH in vitro. Confocal microscopy allows real-time imaging monitoring of the dynamic changes of H2O2 and GSH during ferroptosis. Spectroscopic experiments and cell imaging demonstrate that the probe DRSP-HYL is an excellent sensor for detecting H2O2 and GSH, providing a reliable tool for accurately analyzing the real-time changes of H2O2 and GSH during ferroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 H NMR spectrum of DRSP-HYL

[0031] Figure 2 C-NMR spectrum of DRSP-HYL

[0032] Figure 3 Mass spectrum of DRSP-HYL

[0033] Figure 4 Fluorescence spectrum changes of DRSP-HYL reacting with GSH and H2O2 respectively

[0034] Figure 5 The fluorescence intensity changes of DRSP-HYL with GSH and H2O2 over time

[0035] Figure 6 Linear function relationship diagram of DRSP-HYL reacting with different concentrations of GSH and H2O2

[0036] Figure 7 DRSP-HYL imaging is stimulated by glutathione peroxidase 4 inhibitor (RSL3) for different time periods corresponding to the intensity changes of H2O2 and GSH fluorescence channels

[0037] Figure 8 DRSP-HYL imaging shows the intensity changes of H2O2 and GSH fluorescence channels at different times of stimulation by Erastin. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the embodiments and drawings, but the protection scope of the present invention is not limited to the following embodiments.

[0039] Example 1

[0040] Preparation and characterization of DRSP-HYL:

[0041]

[0042] (1) DMF (8.0 mL, 64.8 mmol) was placed in a 50 mL round-bottom flask and cooled to 0°C. Phosphorus oxychloride (POCl3, 8.0 mL, 85.2 mmol) was added dropwise and stirred at room temperature for 30 minutes. Cyclohexanone (5.0 g, 50.9 mmol) was then added dropwise and stirred at room temperature for 2 hours. The reaction mixture was then quenched with 200 mL of water and the pH was adjusted to 7.0 with solid sodium bicarbonate. The mixture was extracted with ethyl acetate (3 times, 200 mL of ethyl acetate each time). The organic phase was collected and dried over anhydrous sodium sulfate. The mixture was concentrated by distillation under reduced pressure to obtain a yellow oily crude product, Compound 1 (yield 93%).

[0043] (2) Compound 1 (4.0 g, 27.76 mmol), 2-hydroxy-4-methoxybenzaldehyde (3.52 g, 23.12 mmol), and cesium carbonate (22.6 g, 69.33 mmol) were dissolved in 100 mL of DMF and stirred at room temperature for 24 hours. The mixture was filtered and the filter cake was washed with dichloromethane. The filtrate was concentrated by vacuum distillation and purified by column chromatography using a mixed solvent of ethyl acetate and petroleum ether (volume ratio 3:1) as the eluent to obtain compound 2 as a yellow solid (yield 78%).

[0044] (3) Compound 2 (3.0 g, 12.40 mmol) was dissolved in 50 mL of dichloromethane, and boron tribromide (9.65 mL, 99.2 mmol) was added dropwise at 0 °C, and stirring was continued at room temperature for 6 hours. The reaction was quenched with 200 mL of distilled water and extracted with 200 mL of dichloromethane. The organic phase was washed with water three times (200 mL each), and the organic phase was collected and dried with anhydrous sodium sulfate, and the filtrate was collected after filtration and concentrated in vacuo. Column chromatography was performed with dichloromethane and methanol (20:1 by volume) as an eluent to obtain compound 3 as an orange solid powder (97% yield);

[0045] (4) Compound 3 (2.5 g, 10.96 mmol) and 1-(2-carboxyethyl)-2,3,3-trimethylindol-1-ium (3.1 g, 13.34 mmol) were dissolved in 50 mL of acetonitrile, and then 100 μL of piperidine was added, and heating was continued at reflux for 16 hours. After the completion of the reaction, the solvent was removed by distillation under reduced pressure, and column chromatography was performed with dichloromethane and methanol (15:1 by volume) as an eluent to obtain compound 4 as a blue-purple solid powder (57% yield);

[0046] (5) Compound 4 (2.2 g, 4.97 mmol), EDCI (1.9 g, 9.91 mmol), and HOBT (1.34 g, 9.91 mmol) were dissolved in 20 mL of dichloride, and the reaction was continued for 30 min at 0 °C under nitrogen atmosphere. Then, 1-tert-butoxycarbonylpiperazine (1.4 g, 7.51 mmol) was added, and the reaction was continued at room temperature for 24 hours. After the completion of the reaction, the solvent was removed by distillation under reduced pressure, and column chromatography was performed with dichloromethane and methanol (20:1 by volume) to obtain compound 5 as a blue-green solid powder (62% yield). 1 H NMR (600 MHz, DMSO) δ 8.20-8.11 (m, 1H), 7.60 (s, 1H), 7.52 (s, 1H), 7.38 (s, 1H), 7.34 (s, 1H), 7.24 (s, 1H), 7.15 (s, 1H), 6.59 (s, 1H), 6.44 (s, 1H), 6.14-5.99 (m, 1H), 4.27 (s, 1H), 3.46-3.43 (m, 1H), 3.26 (s, 1H), 2.85-2.77 (m, 1H), 2.69-2.65 (m, 1H), 2.65-2.61 (m, 1H), 1.78 (d, J = 5.0 Hz, 1H), 1.66 (s, 1H), 1.38 (s, 1H). 13C NMR (151 MHz, DMSO) δ 168.59, 159.35, 156.79, 153.79, 142.43, 140.22, 137.93, 129.92, 128.36, 123.71, 122.27, 115.55, 115.38, 110.27, 102.29, 79.17, 48.13, 44.59, 40.91, 29.67, 28.01, 27.97, 27.59, 23.89, 20.58. HR-MS: Calc. for C 37 H 44 N3O5 + [M+H] + 610.32755, found 610.32790;

[0047] (6) Compound 5 (1.5 g, 2.46 mmol) and potassium carbonate (K2CO3, 0.45 g, 3.28 mmol) were dissolved in 30 mL of acetonitrile and reacted at 45 °C for 10 min under nitrogen atmosphere, then potassium iodide (KI, 0.54 g, 3.28 mmol) and 4-bromomethylphenylboronic acid pinacol ester (1.5 g, 4.92 mmol) were added and the reaction was continued at 45 °C for 4 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and column chromatography was performed using dichloromethane and methanol at a volume ratio of 20:1 to purify the product, thereby obtaining compound 6 (52%) as a blue solid;

[0048] (7) Compound 6 (0.83 g, 1.0 mmol) was dissolved in a mixed solvent of dichloromethane and trifluoroacetic acid at a volume ratio of 1:1 (5 mL each), and stirred at room temperature for 30 min. Then, the solvent was removed by distillation under reduced pressure, and the crude product 7 was used directly for the next step. (Z)-2-cyano-3-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)acrylic acid (0.16 g, 0.51 mmol), EDCI (0.19 g, 1.0 mmol), and HOBT (0.14 g, 1.0 mmol) were combined and dissolved in 15 mL of dichloromethane, and stirred at 0 °C for 30 min under nitrogen atmosphere. Then, compound 7 (0.55 g, 0.75 mmol) and 80 μL of triethylamine were added to the reaction system, and the reaction was continued at room temperature. After 24 h, the solvent was removed by distillation under reduced pressure, and column chromatography was performed using dichloromethane and methanol at a volume ratio of 20:1 to purify the product, thereby obtaining DRSP-HYL (23%) as a purple-black solid. 1H NMR(600MHz,DMSO)δ8.64(s,1H),8.57(d,J=14.8Hz,1H),7.85(d,J=7.8Hz,2H),7.75(d,J=7.4Hz,1H),7.70(s,1H),7.66(d,J=8.0Hz,1H),7.55(dt,J=15.7,9.9Hz,3H),7.51(s,1H),7.46(dd,J=13.4,7.6Hz,3H),7.17(d,J=1.6Hz,1H),7.08(dd,J=8.6,2.1Hz,1H),6.81(dd,J=8.9,1.6Hz,1H),6.63(d,J=15.0Hz,2H),5.30(d,J=7.0Hz,2H),4.60(t,J=6.9Hz,2H),3.57(s,4H),3.52(dt,J=14.0,5.9Hz,9H),3.00(t,J=7.0Hz,2H),2.75–2.70(m,2H),2.67(d,J=5.0Hz,2H),1.83(s,2H),1.76(s,6H),1.23(s,14H),1.15(t,J=7.0Hz,7H)( Figure 1 )。 13 C NMR(151MHz,DMSO)δ177.63,168.22,162.79,161.48,160.61,160.07,157.09,153.72,152.82,144.99,144.33,143.06,142.00,141.30,137.97,134.33,133.28,131.66,128.89,128.79,127.05,126.95,126.76,122.62,116.46,115.58,114.14,113.68,113.21,110.42,107.61,104.67,102.48,101.96,96.55,70.21,50.37,44.49,41.36,40.06,31.24,30.23,28.97,28.40,27.47,23.62,22.04,19.90,13.90,12.35( Figure 2 )。HR-MS:Calc.for C 62 H 67 BN5O8 + [M+H] + 1020.50772,found 1020.50910( Figure 3 )。

[0049] 实施例2

[0050] Prepare equal volumes of dimethyl sulfoxide and PBS (phosphate buffer solution with pH = 7.4 prepared with sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride) mixed buffer solution (referred to as test system), and prepare 2mmol / LDRSP-HYL dimethyl sulfoxide solution (referred to as probe stock solution).

[0051] Example 3

[0052] Take 2.0mL of the test system in a cuvette, add 10μL of the probe mother solution, add 20μL of a 200mmol / L glutathione aqueous solution, and monitor the changes in fluorescence intensity at 485nm on a fluorescence spectrophotometer using 380nm UV-visible absorption as the excitation light source. With the addition of glutathione, the fluorescence intensity at 485nm increases and the intensity remains unchanged ( Figure 4 a). Take 2.0mL of the test system in a cuvette, add 10μL of the probe stock solution, add 20μL of a 20mmol / L hydrogen peroxide solution, and monitor the change in fluorescence intensity at 723nm on a fluorescence spectrophotometer using 695nm UV-visible absorption as the excitation light source. With the addition of hydrogen peroxide, the fluorescence intensity at 723nm increases ( Figure 4 b).

[0053] Example 4

[0054] Take 10 μL of probe mother solution and place it in a cuvette containing 2.0 mL of test system, add 20 μL of 200 mmol / L glutathione aqueous solution, use 380 nm ultraviolet-visible absorption as the excitation light source on the fluorescence spectrophotometer, and monitor the change of fluorescence intensity at 485 nm over time. The probe DRSP-HYL reacts immediately with glutathione and the fluorescence intensity tends to be stable. Take 10 μL of probe mother solution and 2.0 mL of test system in a cuvette, add 20 μL of 20 mmol / L hydrogen peroxide aqueous solution, use 695 nm ultraviolet-visible absorption as the excitation light source on the fluorescence spectrophotometer, and monitor the change of fluorescence intensity at 723 nm over time. The probe DRSP-HYL reacts with hydrogen peroxide for about 30 minutes and the fluorescence intensity tends to be stable ( Figure 5 a)( Figure 5 b).

[0055] Example 5

[0056] Take 10 μL of probe mother solution and 2.0 mL of test system in a cuvette, and add 0 μL, 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, 12 μL, 14 μL, 16 μL, 18 μL, and 20 μL of 200 mmol / L glutathione aqueous solution respectively. Use 380 nm ultraviolet-visible absorption as the excitation light source on a fluorescence spectrophotometer, and monitor the change of fluorescence intensity at 485 nm after 30 seconds. As the concentration of glutathione increases, the fluorescence intensity at 485 nm increases ( Figure 6 a). Take 10 μL of probe stock solution and 2.0 mL of test system in a cuvette, and add 2 μL, 3 μL, 3.5 μL, 4 μL, 5 μL, 6 μL, and 7 μL of 20 mmol / L hydrogen peroxide solution respectively. After 30 minutes, monitor the fluorescence intensity at 723 nm on a fluorescence spectrophotometer with 695 nm UV-visible absorption as the excitation light source. As the hydrogen peroxide concentration increases, the fluorescence intensity at 723 nm gradually increases ( Figure 6 b).

[0057] Example 6

[0058] 10 μL of probe stock solution was dissolved in 2.0 mL of PBS (final probe concentration was 10 μmol / L) and used to incubate cervical cancer cells (Hela cells) for 20 minutes. The PBS solution was then removed and washed twice with PBS. The cells were placed on a fluorescence confocal microscope and the excitation light sources were 405 nm and 633 nm. The dual-channel fluorescence intensity in the wavelength range of 455-515 nm and 693-753 nm was monitored respectively. Figure 7 A1 and a1); Cervical cancer cells Hela were incubated with 2.0mL DMEM culture medium containing 20μL 0.1 mmol / L RSL3 mother solution for 6h, 12h, 18h and 24h, respectively, and then washed twice with PBS. 10μL probe mother solution was dissolved in 2.0mL PBS (the final concentration of the probe was 10μmol / L) and used to incubate Hela cells for 20 minutes. After that, the cells were placed on a fluorescence confocal microscope, and 405nm and 633nm were used as excitation light sources to monitor the dual-channel fluorescence intensity in the wavelength range of 455-515nm and 693-753nm, respectively. The fluorescence intensity of the two channels was compared and analyzed over time, showing that the fluorescence intensity of the 693-753nm channel gradually increased over time, and the fluorescence intensity of the 455-515nm channel gradually increased over time ( Figure 7 ).

[0059] Example 7

[0060] Hela cells were incubated with 2.0mL DMEM culture medium containing 8μL of 10mmol / L Erastin stock solution for 6h, 12h, 18h and 24h, then washed twice with PBS. 10μL of probe stock solution was dissolved in 2.0mL PBS (the final concentration of the probe was 10μmol / L) and used to incubate Hela cells for 20 minutes. The cells were then placed on a fluorescence confocal microscope, with 405nm and 633nm as excitation light sources, and the dual-channel fluorescence intensity in the wavelength range of 455-515nm and 693-753nm was monitored respectively. The fluorescence intensity of the two channels was compared over time and analyzed, showing that the fluorescence intensity of the 693-753nm channel gradually increased over time, while the fluorescence intensity of the 455-515nm channel first decreased and then increased over time ( Figure 8 ).

[0061] The above experimental results indicate that DRSP-HYL is a good tool for detecting GSH and H2O2 in vitro and in cells.

Claims

1. A coumarin and hemicyanine coupled derivative DRSP-HYL, characterized in that: The structural formula is: 。 2. The method for synthesizing a coumarin and hemicyanine coupled derivative DRSP-HYL according to claim 1, wherein: The steps include: (1) Phosphorus oxychloride was added dropwise to N,N-dimethylformamide in a molar ratio of 1:1.0-1.5, and the mixture was stirred at room temperature for 20-50 minutes. Then, 0.6-1.0 equivalents of cyclohexanone were added, and the mixture was stirred for 1.5-4 hours. The reaction was quenched with water and adjusted to neutral with sodium bicarbonate. The mixture was then extracted with ethyl acetate, and the organic phase was collected and concentrated to obtain a yellow oily compound 1, whose structural formula is: ; (2) Compound 1 and 2-hydroxy-4-methoxybenzaldehyde were dissolved in DMF at a molar ratio of 1:0.7-0.9, and then 2-3 equivalents of cesium carbonate were added and stirred at room temperature for 16-32 hours; the filtrate was filtered and collected, and purified by column chromatography using a mixed solvent of ethyl acetate and petroleum ether to obtain compound 2, whose structural formula is: ; (3) Compound 2 and boron tribromide were dissolved in a dichloromethane solution at a molar ratio of 1:5.0-10.0 and stirred at room temperature for 6 hours; the reaction was quenched with water, extracted with dichloromethane, and the organic phase was collected and concentrated; and purified by column chromatography using a mixed solvent of dichloromethane and methanol to obtain compound 3, whose structural formula is: ; (4) Compound 3 and 1-(2-carboxyethyl)-2,3,3-trimethylindol-1-ium were dissolved in acetonitrile at a molar ratio of 1:1.0-1.5, and a catalytic amount of piperidine was added. The mixture was heated under reflux for 12-20 hours. The mixture was concentrated under reduced pressure and purified by column chromatography using a mixed solvent of dichloromethane and methanol to obtain compound 4, whose structural formula is: ; (5) Compound 4, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBT) were dissolved in dichloromethane at a molar ratio of 1:1-3:1-3 and reacted at 0°C in a nitrogen atmosphere for 20-50 minutes; 1.0-2.0 equivalents of 1-tert-butyloxycarbonylpiperazine were added and reacted at room temperature for 20-30 hours; the mixture was concentrated under reduced pressure and purified by column chromatography using a mixed solvent of dichloromethane and methanol to obtain compound 5, whose structural formula is: ; (6) Compound 5 and potassium carbonate were combined in acetonitrile at a molar ratio of 1:1.2-1.5, stirred at 45°C in a nitrogen atmosphere for 5-15 minutes, and then 1.2-1.5 equivalents of potassium iodide and 1.5-2.5 equivalents of 4-bromomethylphenylboronic acid pinacol ester were added and the reaction was continued at 45°C for 3-5 hours. The mixture was concentrated under reduced pressure and purified by column chromatography using a mixed solvent of dichloromethane and methanol to obtain compound 6, whose structural formula is: ; (7) Compound 6 and trifluoroacetic acid were dissolved in dichloromethane at a molar ratio of 1:50-80, stirred at room temperature for 20-50 minutes, and concentrated under reduced pressure to obtain a crude product, compound 7, for later use. Subsequently, (Z)-2-cyano-3-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)acrylic acid, EDCI, and HOBT were dissolved in dichloromethane at a molar ratio of 1:1.0-3.0:1.0-3.0, and stirred for reaction at 0°C under a nitrogen atmosphere for 20-50 minutes. Then, 1.0-2.0 equivalents of compound 7 and 0.8-1.2 equivalents of triethylamine were added, and the reaction was continued at room temperature for 20-30 hours. The mixture was concentrated under reduced pressure, and purified by column chromatography using a mixed solvent of dichloromethane and methanol to obtain DRSP-HYL. The structural formula of the compound 7 is: .

3. Use of the DRSP-HYL according to claim 1 in the spatiotemporal synchronous detection of hydrogen peroxide (H2O2) and glutathione (GSH) for non-disease diagnosis or treatment.

4. Use of the DRSP-HYL according to claim 1 in preparing a single-molecule fluorescent probe for spatiotemporally synchronously detecting the dynamic changes of GSH and H2O2 in the process of cell ferroptosis.

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