Preparation method and application of a coumarin derivative
By synthesizing a molecular fluorescent probe based on coumarin structure, the problems of insufficient selectivity and sensitivity in GSH detection in the prior art have been solved, realizing highly selective and highly sensitive GSH detection, which is suitable for applications in the biological environment.
Patent Information
- Application Number
- CN202610274553.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-03-06
- Publication Date
- 2026-06-26
AI Technical Summary
There is a lack of highly selective, sensitive and biocompatible methods for the detection of glutathione (GSH) in the current technology.
A molecular fluorescent probe based on coumarin structure was designed and synthesized. 2,4-dinitrobenzenesulfonate was introduced as a targeting group, and an electron-withdrawing acetyl group was introduced into the molecule to enhance the recognition ability of GSH. Coumarin derivative (II) was prepared through specific synthetic steps.
It achieves highly selective and sensitive detection of GSH with a detection limit of 0.49 μM, exhibits good biocompatibility, and is suitable for the detection of GSH in biological environments.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of organic synthesis technology and relates to a method for preparing coumarin derivatives and their applications. Background Technology
[0002] Glutathione (GSH) is the most important antioxidant naturally synthesized in human cells. It is composed of three amino acids: glutamic acid, cysteine, and glycine. The sulfhydryl group (-SH) on cysteine is the key site for its activity. The concentrations and dynamic changes of reduced glutathione (GSH) and oxidized glutathione (GSSG) in cells are key biomarkers for assessing the redox state, detoxification capacity, and overall health of cells, tissues, and even the entire body. Given the importance of glutathione in physiological and pathological processes, rapid quantitative detection of its content in the body has become a research hotspot.
[0003] Coumarins are a class of compounds with a benzo-α-pyranone skeleton. Their core structure consists of a benzene ring fused with an α-pyranone (lactone ring). They are naturally found in many plants, such as tonka bean, cinnamon, vanilla, angelica, angelica dahurica, and artemisia capillaris. Fluorescent probes using coumarin as the fluorophore have advantages such as excellent fluorescence properties, ease of modification, environmental sensitivity, and good biocompatibility.
[0004] Therefore, this invention designs and prepares a molecular fluorescent probe based on the coumarin structure, using 2,4-dinitrobenzenesulfonate as the targeting group for the recognition of GSH; and introduces an electron-withdrawing acetyl group to enhance intramolecular charge transfer (ICT). This coumarin derivative (II) exhibits superior recognition of GSH, and its synthesis is simple and yield is high. In a 0.2% DMSO-water system, after interaction with GSH, the fluorescence intensity at the maximum fluorescence emission wavelength (455 nm) increased by nearly 40 times, and the detection limit was 0.49 μM. Furthermore, this coumarin exhibits low cytotoxicity and good biocompatibility, making it suitable for the detection of GSH in biological environments. Due to its advantages of high selectivity and sensitivity, good biocompatibility, and minimal interference from other analytes, this probe has great application potential in biological and environmental detection. Summary of the Invention
[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a preparation method and application for GSH detection of coumarin derivatives.
[0006] Technical Solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: a coumarin derivative for GSH detection, the chemical structural formula of which is shown in formula (II) below:
[0007]
[0008] The above-mentioned method for preparing coumarin derivatives for detecting GSH is characterized in that it is synthesized according to the following experimental steps:
[0009] 1) Dissolve 2,4-dihydroxybenzaldehyde in a certain amount of ethanol, add ethyl acetoacetate, and then add a catalytic amount of piperidine. Heat the mixture under reflux for 18 hours. After the reaction is complete, cool to room temperature, pour into ice water to precipitate the solid, filter, wash with water, dry, and then recrystallize with ethanol to obtain compound (I).
[0010] 2) Compound (I) was dissolved in a certain amount of dry dichloromethane, and triethylamine (TEA) was added as a catalyst. A solution of 2,4-dinitrobenzenesulfonyl chloride in dichloromethane was added dropwise at 0°C. The reaction was stirred for 3 h, and the reaction was quenched with water. The organic phase was washed with brine, and the separated organic phase was dried with anhydrous sodium sulfate. The organic solvent was removed by evaporation under reduced pressure. The crude product was purified to obtain the coumarin derivative (II).
[0011] The specific synthetic route for detecting GSH coumarin derivative (II) is as follows:
[0012]
[0013] The steps are completed using the following method:
[0014] (1) Dissolve 2,4-dihydroxybenzaldehyde and ethyl acetoacetate in anhydrous ethanol, add a catalytic amount of piperidine, heat under reflux for 18 h, cool, add cold water, filter, and recrystallize with ethanol to obtain compound (I).
[0015] (2) Compound (I) and 2,4-dinitrobenzenesulfonyl chloride were added to a reaction flask, dissolved in anhydrous dichloromethane, and then triethylamine was added. The reaction mixture was reacted at 0°C for 3 h. A certain amount of cold water was added, and the organic phase was separated. The organic phase was dried with anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. The coumarin derivative (II) was then separated and purified by silica gel chromatography with dichloromethane as the eluent, with a yield of 65%.
[0016] In the above synthesis step (1), the molar ratio of 2,4-dihydroxybenzaldehyde to ethyl acetoacetate is 1:1;
[0017] In the above synthesis step (2), the molar ratio of compound (I), triethylamine, and 2,4-dinitrobenzenesulfonyl chloride is 1:1.1:1.1.
[0018] The coumarin derivative of the present invention exhibits a unique fluorescence response to GSH in a 0.2% DMSO-water system.
[0019] A 0.2% DMSO- aqueous solution (10 μM) of coumarin derivative (II) was prepared, and different concentrations of glutathione (GSH) (0-220 μM) were added dropwise. Excitation was performed at a wavelength of 380 nm, and fluorescence spectra at different concentrations of glutathione (GSH) were measured, as shown below. Figure 2 As shown.
[0020] To determine the relationship between the fluorescence intensity of coumarin derivative (II) and GSH concentration, based on Figure 2 The results showed that the fluorescence intensity at 455 nm was used as the ordinate and the drop concentration as the abscissa. Linear fitting was performed in the range of 0-150 μM to obtain the linear regression curve y = 2.005x[GSH] + 2.332, with a fitting coefficient R. 2 The value was 0.9945, and the detection limit was calculated to be 0.49 μM. The results are as follows: Figure 3 As shown, the concentration of coumarin derivative (II) is highly linearly correlated with the concentration of GSH. Therefore, this fluorescent probe can be used for the quantitative analysis and detection of GSH within a certain concentration range.
[0021] The coumarin derivative (II) described in this invention has the characteristic of stable performance under different pH conditions, such as... Figure 4 As shown, the probe pair still exhibits stable fluorescence emission for GSH recognition within a pH range of 7.0-10.0, which is beneficial for the practical application of coumarin derivative (II) under physiological conditions.
[0022] Prepare a 0.2% DMSO- aqueous solution (10 μM) of coumarin derivative (II), and add GSH (200 μM) or other different analytes (200 μM), such as Na. + Al 3+ Co 2+ Cu 2+ Ca 2+ Mg 2+ Zn 2+ K + NO3 - CO3 2- SO4 2- HSO4 - Aqueous solutions of Met, Ser, Arg, Tyr, Glu, His, Lys, Leu, Cys, and Hcy were used to study the selective recognition of different analytes by coumarin derivative (II) using fluorescence spectroscopy. The results are as follows: Figure 5As shown, by observing the intensity changes in its fluorescence emission spectrum, it was found that the coumarin derivative (II) of this invention has a certain fluorescence response to Cys and Hcy, but a significant fluorescence response to GSH. When it reacts with GSH, the fluorescence intensity is significantly enhanced; while other analytes have almost no effect on the fluorescence emission of coumarin derivative (II).
[0023] The coumarin derivative (II) described in this invention exhibits low cytotoxicity. MTT assay results show that after H9c2 cells were co-incubated with 15 μM coumarin derivative (II) for 48 hours, the cell viability remained above 90%. Figure 6 As shown, this demonstrates that the coumarin derivatives described in this invention can be used for the detection of GSH in cell systems.
[0024] Beneficial effects of the present invention
[0025] (1) The coumarin derivative (II) has a simple synthetic route, mild reaction conditions, easy operation, and high yield; (2) The coumarin derivative (II) has good recognition of GSH, good selectivity, strong anti-interference ability, high sensitivity, and a detection limit of 0.49 μM; (3) The coumarin derivative (II) has low cytotoxicity, good biocompatibility, and can be used for the detection of GSH in living cells through bioimaging. Therefore, the coumarin derivative (II) of this invention has broad application prospects in analytical chemistry, environmental monitoring, and biological systems. Attached Figure Description
[0026] Figure 1 High-resolution mass spectrum of coumarin derivative (II);
[0027] Figure 2 The figure shows the fluorescence titration of glutathione (GSH) (0-220 μM) in a 0.2% DMSO-water solution of the coumarin derivative (II) (10 μM) of this invention. The vertical axis represents fluorescence emission intensity, and the horizontal axis represents emission wavelength (wavelength), with the unit being nm. The excitation wavelength is 380 nm.
[0028] Figure 3 The graph shows the linear relationship between different concentrations of GSH (μM) on the x-axis and the fluorescence intensity of coumarin derivative (II) at 455 nm on the y-axis.
[0029] Figure 4 This is a graph showing the effect of different pH values on the fluorescence intensity of coumarin derivative (II) and its recognition of GSH.
[0030] Figure 5Fluorescence intensity diagrams of coumarin derivative (II) (10 μM) after adding GSH (200 μM) or other analytes (200 μM) to a 0.2% DMSO- aqueous solution;
[0031] Figure 6 This is the MTT plot of coumarin derivative (II) in H9c2 cells (rat cardiomyocytes) in Example 6 of the present invention; Detailed Implementation
[0032] The invention will be further explained below with reference to specific examples.
[0033] use 1 The structure of coumarin derivative (II) was characterized and confirmed by 1H NMR and high-resolution mass spectrometry. The instruments used for detection were: Bruker ARX600 NMR spectrometer (TMS as internal standard, deuterated chloroform as solvent) and TECAN Infinite M200 Pro multi-functional microplate reader.
[0034] Example 1
[0035] Preparation of compound (I)
[0036] 1.5 g (0.011 mol) of 2,4-dihydroxybenzaldehyde was dissolved in 20 mL of ethanol. Ethyl acetoacetate (1.362 mL, 0.011 mol) was added, followed by a catalytic amount of piperidine (0.01 mL). The mixture was heated under reflux for 18 h. After the reaction was complete, the mixture was cooled to room temperature and poured into ice water to precipitate the solid. The crude product was filtered off, washed with water, dried, and recrystallized from ethanol to give compound 1 (1.48 g, 70% yield). H NMR (400MHz, DMSO-d6) 2.55 (3H, s), 8.60 (1H, s), 7.79 (1H, d, J = 8.6Hz), 6.86 (1H, dd, J = 8.6, 2.2Hz), 6.76 (1H, d, J = 2.2Hz)
[0037] Example 2
[0038] Preparation of coumarin derivative (II)
[0039] Compound (I) (84 mg, 0.41 mmol) was dissolved in a certain amount of dry dichloromethane (15 mL), and triethylamine (Et3N, 62 μL, 0.45 mmol) was added. A dichloromethane solution of 2,4-dinitrobenzenesulfonyl chloride (120 mg, 0.41 mmol) was added dropwise at 0 °C. The mixture was stirred for 3 h, and the reaction was quenched with water. The organic phase was washed with brine, and the water-organic phase was dried with anhydrous sodium sulfate. The organic solvent was removed by evaporation under reduced pressure. The crude product was purified by silica gel chromatography using dichloromethane as the eluent to obtain compound (I) (105 mg, yield 65%). 1 H NMR (400MHz, DMSO-d6) δ9.13 (d, J=2.3Hz, 1H), 8.75-8.55 (m, 2H), 8.33 (d, J=8.7Hz, 1H), 8.05 (d, J=8.6Hz, 1H), 7.45 (d, J=2.3Hz, 1H), 7.26 (dd, J=8.6, 2.4Hz, 1H), 2.57 (s, 3H). ESI-HRMS: m / z=456.9929, Calcd.for C 17 H 10 N2O 10 SNa, [M+Na] + ] + =457.0056; m / z=435.0116, Calcd.for C 17 H l1 N2O 10 S, [M+H + ] + =435.0056.( Figure 1 )
[0040] Example 3
[0041] Effect of different concentrations of glutathione (GSH) on the fluorescence intensity of coumarin derivative (II)
[0042] A 0.2% DMSO- aqueous solution of 10 μM coumarin derivative (II) was prepared, and different concentrations of GSH solution (0–220 μM) were added. The excitation wavelength was 380 nm, and the fluorescence spectra of the coumarin derivative (II) solution at different GSH concentrations were measured. Figure 2 ).
[0043] Example 4
[0044] The linear relationship between the fluorescence intensity of coumarin derivative (II) and GSH concentration was determined.
[0045] A 0.2% DMSO- aqueous solution of 10 μM coumarin derivative (II) was prepared, and different concentrations of GSH solution (0–220 μM) were added. The excitation wavelength was 380 nm, and the fluorescence intensity of the emitted light from the coumarin derivative (II) solution at 455 nm was measured at different GSH concentrations. The fluorescence intensity of coumarin derivative (II) at 455 nm increased with increasing GSH concentration (0–150 μM). When the GSH concentration exceeded 150 μM, the fluorescence intensity of coumarin derivative (II) remained almost unchanged. The results are as follows: Figure 3 As shown; using fluorescence intensity as the ordinate and concentration as the abscissa, a linear regression equation was obtained by linear fitting: y = 2.005x[GSH] + 2.332, with a fitting coefficient R. 2 The value was 0.9945, and the detection limit was 0.49 μM; the linear relationship results are as follows: Figure 3 As shown, the coumarin derivative (II) has a high linear correlation with the concentration change of GSH. Therefore, the coumarin derivative (II) can be used for the quantitative analysis and detection of GSH within a certain concentration range.
[0046] Example 4
[0047] The effect of different pH values on the recognition of GSH by coumarin derivatives (II):
[0048] To investigate the optimal pH range for the detection of GSH by coumarin derivative (II), the changes in fluorescence intensity of coumarin derivative (II) at different pH values were studied in the presence and absence of GSH. Figure 4 As shown, without the addition of GSH, the fluorescence intensity was weak and almost unchanged in the pH range of 2.0–8.0; however, the fluorescence intensity increased slightly in the pH range of 8.0–11.0, due to partial hydrolysis under alkaline conditions, but the fluorescence intensity remained weak. When GSH was added, the fluorescence response was minimal in the pH range of 2.0–6.0, significantly enhanced in the pH range of 7.0–10.0, and reached its maximum value above pH 7.6, remaining unchanged in the range of 7.6–9.3. Therefore, this coumarin derivative (II) has a good detection effect on GSH in the pH range of 7–10, indicating that coumarin derivative (II) exhibits excellent performance in the physiological pH range, which is conducive to improving the practical application of coumarin derivative (II).
[0049] Example 5
[0050] Selectivity study of coumarin derivatives (II) for different analytes
[0051] Prepare a 0.2% DMSO- aqueous solution of 10 μM coumarin derivative (II), and add GSH (200 μM) or other analyte solutions (200 μM), such as Na+. + Al 3+ Co 2+ Cu 2+ Ca 2+ Mg 2+ Zn 2+ K + NO3 - CO3 2- SO4 2- HSO4 - Met, Ser, Arg, Tyr, Glu, His, Lys, Leu, Cys, and Hcy were used to study the selective recognition of different analytes by coumarin derivatives (II) using fluorescence spectroscopy. The results are as follows: Figure 5 As shown in 5a and 5b, besides GSH, Hcy and Cys have some influence, while other analytes have almost no effect on the fluorescence emission spectrum of the probe, and the fluorescence emission spectrum does not change significantly. However, when GSH is added, the fluorescence emission intensity is significantly enhanced. The above results indicate that the coumarin derivative (II) described in this invention has a significant fluorescence response to GSH, and the fluorescence intensity is significantly enhanced after interacting with GSH, so it can be used as a specific fluorescent probe for detecting GSH.
[0052] Example 6
[0053] Cytotoxicity studies of coumarin derivative (II)
[0054] The cytotoxicity of coumarin derivative (II) in H9c2 cells was determined by the MTT assay. H9c2 cells were seeded in 96-well plates, and 110 μL of culture medium was added to each well along with different concentrations of coumarin derivative (II) (0, 3, 6, 9, 12, and 15 μM) for 24 and 48 hours. After incubation, 110 μL of medium containing 5 mg / mL MTT was added to each well for an additional 1.5 hours of incubation. The culture medium was carefully removed, and 100 μL of DMSO was added to each well. After mixing, the absorbance at 630 nm and 550 nm was measured using a microplate reader to calculate cell viability. 24-hour viability was calculated as follows: Figure 6 As shown in a, the 48-hour survival rate is as follows: Figure 6 As shown in b, all exceed 90%.
[0055] Cell Viablity = OD 550nm-630nm Experimental group / OD 550nm-630nm control group × 100%
[0056] In this context, the OD experimental group represents the absorbance of the cells in the experimental group; the OD control group represents the absorbance of the cells in the control group.
Claims
1. A coumarin derivative, characterized in that, Its structural formula is shown in equation (II) below:
2. A process for the preparation of a coumarin derivative (II) according to claim 1, characterized in that, The process is achieved through the following steps: using 2,4-dihydroxybenzaldehyde as a raw material, it reacts with ethyl acetoacetate to obtain compound (I), and then reacts with 2,4-dinitrobenzenesulfonyl chloride to obtain a coumarin derivative (II). The reaction formula for this preparation process is as follows:
3. A process for the preparation of a coumarin derivative (II) according to claim 2, characterized in that The following steps are required: (1) Dissolve 2,4-dihydroxybenzaldehyde in a certain amount of ethanol, add ethyl acetoacetate, then add a catalytic amount of piperidine, reflux the mixture for 18 h, cool to room temperature after the reaction is complete, pour into ice water to precipitate solid, filter the product, wash with water, dry, and recrystallize with ethanol to obtain compound (I); (2) Compound (I) was dissolved in a certain amount of dry dichloromethane, and triethylamine (TEA) was added as a catalyst. A solution of 2,4-dinitrobenzenesulfonyl chloride in dichloromethane was added dropwise at 0°C. The mixture was stirred for 3 hours, and the reaction was quenched with water. The organic phase was washed with brine to separate the organic phase, and dried with anhydrous sodium sulfate. The organic solvent was then removed by evaporation under reduced pressure. The crude product was separated by chromatography to obtain compound (I).
4. A process for the preparation of a coumarin derivative (II) according to claim 3, characterized in that In step 1), the molar ratio of 2,4-dihydroxybenzaldehyde to ethyl acetoacetate is 1:
1.
5. A process for the preparation of a coumarin derivative (II) according to claim 3, characterized in that In step (1), the molar ratio of compound (I), TEA, and 2,4-dinitrobenzenesulfonyl chloride is 1:1.1:1.
1.
6. The application of the coumarin derivative according to claim 1 in the detection of GSH.
7. Use of a coumarin derivative (II) as claimed in claim 6, characterized in that Coumarin derivative (II) can be used for the detection and identification of GSH. The detection is a fluorescent detection. This fluorescent probe shows excellent detection performance for GSH with a detection limit of 0.49 μM. It also has low toxicity to H9c2 cells and can be used for the detection of GSH in biological systems.