Piperazine coumarin derivative as well as synthesis method and application thereof

Abstract

The invention relates to the technical field of detection, and discloses a piperazine coumarin derivative as well as a synthesis method and application thereof. The piperazine coumarin derivative provided by the invention takes coumarin as a parent, can be used for dual-channel dual-emission distinguishing detection of hydrazine and sulfur dioxide, and has high selectivity, high sensitivity and excellent stability. The piperazine coumarin derivative provided by the invention can be used for detecting hydrazine (N2H4) and sulfur dioxide (SO2) at a cellular level; in different soil samples and test paper, the kit also has the capability of rapidly detecting N2H4, and has great potential in analysis and monitoring of environmental pollutants.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and in particular to a piperazine coumarin derivative and a synthesis method and application thereof. Background Art

[0002] Sulfur dioxide (SO2) is a colorless gas with a pungent odor. After being inhaled into the human body, SO2 is easily absorbed by the moist mucous membrane surface to produce sulfurous acid and sulfuric acid. SO2 can be produced endogenously in the body and has a unique physiological regulatory effect in the cardiovascular system. However, excessive SO2 can have toxic effects on humans and animals, causing adverse reactions and diseases. Excessive intake of SO2 may cause diseases such as asthma, allergic reactions, cardiovascular diseases and neurological disorders. In addition, SO2 is also an environmental pollutant. It easily reacts with water in the atmosphere to produce acid rain, which harms the ecological environment. Due to its importance to human health and the environment, it has received great attention.

[0003] As an important chemical raw material, hydrazine (N2H4) will inevitably pose a potential threat to the environment and human health during its production and use. At the same time, N2H4 is also a highly toxic substance. The human body does not produce endogenous N2H4, but N2H4 can enter the human body through the skin, respiratory tract or digestive tract, causing damage to the liver, lungs, kidneys and the human central nervous system. In severe cases, it can lead to organ failure and threaten life. The residual amount of N2H4 in drinking water should also be strictly controlled.

[0004] Therefore, it is of great significance to develop a dual-functional method for the efficient and sensitive detection of N2H4 and SO2 in environmental and biological samples.

[0005] As a new type of molecular recognition and detection technology, fluorescent probe technology has the advantages of high sensitivity, good selectivity, and fast response speed. It has been widely used in biomedicine, environmental monitoring and other fields. Near-infrared fluorescent probes have great application potential in bioimaging, medical diagnosis and environmental monitoring due to their deep tissue penetration ability, low background interference and high photostability. Although a large number of fluorescent probes have been reported to detect SO2 and N2H4 in biological and environmental systems, most of them can only detect one analyte, and few fluorescent probes can detect SO2 and N2H4 at the same time. Multifunctional fluorescent probes break through the limitation that a single fluorescent analyte can only detect a single analyte, and have more applications and detection efficiency. Simultaneous identification and detection of SO2 and N2H4 can avoid optical crosstalk, metabolism and different localization of multiple probes in the body, while maintaining cost-effectiveness, less time consumption and non-invasive imaging. Summary of the invention

[0006] The purpose of the present invention is to overcome the problems existing in the prior art and provide a piperazine coumarin derivative and its synthesis method and application. The piperazine coumarin derivative provided by the present invention uses coumarin as a parent and can be used for dual-channel dual-emission differential detection of hydrazine and sulfur dioxide.

[0007] In order to achieve the above object, the first aspect of the present invention provides a piperazine coumarin derivative, wherein the structural formula of the piperazine coumarin derivative is as follows:

[0008]

[0009] The second aspect of the present invention provides a method for synthesizing the piperazine coumarin derivative according to the first aspect, wherein the method comprises the following steps:

[0010] (1) subjecting Michaelis' acid and 2,4-dihydroxybenzaldehyde to a first reaction to obtain a first compound;

[0011] (2) subjecting 4-piperazineacetophenone and 4-(diethylamino)-2-hydroxybenzaldehyde to a second reaction under acidic conditions; mixing the product obtained by the second reaction with perchloric acid to obtain a second compound;

[0012] (3) subjecting the first compound, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, the second compound and triethylamine to a third reaction in a protective atmosphere to obtain a third compound;

[0013] (4) subjecting the third compound, acetyl bromide and triethylamine to a fourth reaction to obtain the piperazine coumarin derivative.

[0014] The third aspect of the present invention provides a piperazine coumarin derivative obtained according to the preparation method described in the second aspect.

[0015] The fourth aspect of the present invention provides the use of the piperazine coumarin derivative according to the first aspect or the third aspect as a probe for dual-channel detection of hydrazine and sulfur dioxide and a cell imaging agent.

[0016] The fifth aspect of the present invention provides a method for detecting the piperazine coumarin derivative according to the first aspect or the third aspect, wherein the method comprises the following steps:

[0017] (1) mixing a dimethyl sulfoxide solution of a piperazine coumarin derivative, a Hepes buffer and methanol to obtain a detection solution;

[0018] (2) adding the sample to be tested to the detection solution obtained in step (1) to obtain a mixed solution;

[0019] (3) Determine the ultraviolet spectrum and fluorescence spectrum of the mixed solution, obtain the ultraviolet absorption and fluorescence intensity at the corresponding wavelengths according to the test, and determine the content of hydrazine and sulfur dioxide in the sample through corresponding linear observation.

[0020] Through the above technical solution, the beneficial technical effects achieved by the present invention are as follows:

[0021] (1) The piperazine coumarin derivative provided by the present invention is a dual-site near-infrared fluorescent probe for detecting hydrazine and sulfur dioxide through dual-channel dual-emission differentiation, and has high selectivity, high sensitivity and excellent stability;

[0022] (2) The piperazine coumarin derivatives provided by the present invention have low cytotoxicity and high cell permeability, can visualize hydrazine and sulfur dioxide in living cells, and have good mitochondrial targeting ability;

[0023] (3) The piperazine coumarin derivatives provided by the present invention can detect hydrazine (N2H4) and sulfur dioxide (SO2) at the cellular level; they also have the ability to quickly detect N2H4 in different soil samples and test papers, and have great potential in the analysis and monitoring of environmental pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the H NMR spectrum of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present invention.

[0025] Figure 2 This is the NMR carbon spectrum of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present invention.

[0026] Figure 3 It is the mass spectrum of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present invention.

[0027] Figure 4 This is an ultraviolet spectrum of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present invention reacting with hydrazine and sulfur dioxide.

[0028] Figure 5 This is a fluorescence spectrum of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present invention reacting with hydrazine and sulfur dioxide.

[0029] Figure 6 This is a fluorescence intensity diagram of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present invention, hydrazine and sulfur dioxide under different pH conditions.

[0030] Figure 7 It is a fluorescence intensity diagram of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present invention and various analytes.

[0031] Figure 8 This is a graph showing the fluorescence intensity of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present invention, hydrazine and sulfur dioxide changing with time.

[0032] Fig. 9 It is the UV image of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present invention in different soils and test strips.

[0033] Fig.10 This is a cell imaging diagram of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present invention measured by the addition of hydrazine and sulfur dioxide.

[0034] Fig.11 The figures are cell imaging diagrams of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present invention when different concentrations of hydrazine were added.

[0035] Fig.12 The piperazine coumarin derivative prepared in Example 1 of the present invention is NH-LS4, which is imaged for mitochondrial localization in Hela cells. DETAILED DESCRIPTION

[0036] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0037] The first aspect of the present invention provides a piperazine coumarin derivative, wherein the piperazine coumarin derivative has the following structural formula:

[0038]

[0039] In the present invention, the piperazine coumarin derivative is named NH-LS4, its Chinese name is 2-(4-(4-(7-acetoxy-2-oxo-2-H-chromene-3-carbonyl)piperazin-1-yl)phenyl)-7-(diethylamino) salt, and its English name is 2-(4-(4-(7-acetoxy-2-oxo-2H-chromene-3-carbonyl)piperazin-1-yl)phenyl)-7-(die thylamino)chromenylium.

[0040] The second aspect of the present invention provides a method for synthesizing the piperazine coumarin derivative according to the first aspect, wherein the method comprises the following steps:

[0041] (1) subjecting Michaelis' acid and 2,4-dihydroxybenzaldehyde to a first reaction to obtain a first compound;

[0042] (2) subjecting 4-piperazineacetophenone and 4-(diethylamino)-2-hydroxybenzaldehyde to a second reaction under acidic conditions; mixing the product obtained by the second reaction with perchloric acid to obtain a second compound;

[0043] (3) subjecting the first compound, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, the second compound and triethylamine to a third reaction in a protective atmosphere to obtain a third compound;

[0044] (4) subjecting the third compound, acetyl bromide and triethylamine to a fourth reaction to obtain the piperazine coumarin derivative.

[0045] In some embodiments of the present invention, the molar ratio of Michaelis acid to 2,4-dihydroxybenzaldehyde in step (1) is 1:1.

[0046] In some embodiments of the present invention, the solvent of the first reaction is water.

[0047] In some embodiments of the present invention, the conditions of the first reaction include: heating to reflux; temperature of 100° C.; time of 2 h.

[0048] In some embodiments of the present invention, the product obtained by the first reaction is naturally cooled, and the precipitate is filtered out to obtain the first compound.

[0049] In some embodiments of the present invention, the molar ratio of 4-piperazineacetophenone to 4-(diethylamino)-2-hydroxybenzaldehyde in step (2) is 1:1.

[0050] In some embodiments of the invention, the acidic condition is provided by concentrated sulfuric acid.

[0051] In some embodiments of the present invention, the conditions of the second reaction include: temperature of 90° C.; time of 8 h.

[0052] In some embodiments of the present invention, the product obtained from the second reaction is mixed with perchloric acid in ice water, and the obtained suspension is washed with water, dried in vacuo, and then purified by column chromatography to obtain the second compound.

[0053] In some embodiments of the present invention, the mobile phase used in the column chromatography purification is dichloromethane and methanol in a volume ratio of 15:1.

[0054] In some embodiments of the present invention, in step (3), the molar ratio of the first compound, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, the second compound and triethylamine is 1:1:1:1:0.2.

[0055] In some embodiments of the present invention, the solvent for the third reaction is anhydrous N,N-dimethylformamide.

[0056] In some embodiments of the present invention, the gas of the protective atmosphere is argon.

[0057] In some embodiments of the present invention, the conditions of the third reaction include: room temperature; time is 24 hours.

[0058] In some embodiments of the present invention, the first compound, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole are first mixed in a solvent, and then the second compound and triethylamine are added. This operation method can improve the yield.

[0059] In some embodiments of the present invention, the first mixing conditions include: temperature of 0° C.; time of 30 min.

[0060] In some embodiments of the present invention, the product of the third reaction is washed with water, filtered, and then purified by column chromatography to obtain a third compound.

[0061] In some embodiments of the present invention, the mobile phase used in the column chromatography purification is dichloromethane and methanol in a volume ratio of 15:1.

[0062] In some embodiments of the present invention, the molar ratio of the third compound, acetyl bromide and triethylamine in step (4) is 1:1:0.2.

[0063] In some embodiments of the present invention, the solvent for the fourth reaction is anhydrous dichloromethane.

[0064] In some embodiments of the present invention, the conditions of the fourth reaction are: temperature is room temperature; time is 6 hours.

[0065] In some embodiments of the present invention, the product of the fourth reaction is purified by column chromatography to obtain the piperazine coumarin derivative.

[0066] In some embodiments of the present invention, the mobile phase used in the column chromatography purification is dichloromethane and methanol in a volume ratio of 10:1.

[0067] According to a particularly preferred embodiment of the present invention, a method for synthesizing a piperazine coumarin derivative NH-LS4, wherein the method comprises the following steps:

[0068] 1) Melech's acid and 2,4-dihydroxybenzaldehyde were dissolved in water at a molar ratio of 1:1, and the mixture was heated to 100°C, refluxed for 2 hours, and cooled naturally to obtain a precipitate. The precipitate was filtered to obtain an off-white solid as compound 1;

[0069] 2) 4-piperazineacetophenone and 4-(diethylamino)-2-hydroxybenzaldehyde were slowly added to concentrated sulfuric acid at a molar ratio of 1:1, and the mixture was heated to 90°C for 8 hours. After cooling to room temperature, the reaction mixture was slowly poured into ice water, and HClO4 was added and stirred. The precipitated suspension was washed with water and dried in vacuo; the product was purified by dichloromethane-methanol (v:v=15:1) column chromatography to obtain a purple-black powder, namely compound 2;

[0070] 3) Compound 1, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole were dissolved in anhydrous N,N-dimethylformamide in a molar ratio of 1:1:1, and the mixture was stirred at 0°C for 30 min under argon protection, and then compound 2 and triethylamine were added in a molar ratio of 1:0.2 (the molar ratio of compound 1 to compound 2 was 1:1), and the mixture was stirred at room temperature for 24 h. After the reaction was complete, the reaction mixture was poured into ice water for washing, and filtered to obtain a black solid. The product was purified by dichloromethane-methanol (v:v=15:1) column chromatography to obtain a dark purple compound NH;

[0071] 4) Compound NH, acetyl bromide and triethylamine were dissolved in anhydrous dichloromethane at a molar ratio of 1:1:0.2, and the mixture was stirred at room temperature for 6 h; the product was purified by dichloromethane-methanol (v:v=10:1) column chromatography to obtain a blue-black compound NH-LS4.

[0072] The synthetic route of NH-LS4 is as follows:

[0073]

[0074] The third aspect of the present invention provides a piperazine coumarin derivative obtained according to the preparation method described in the second aspect.

[0075] The fourth aspect of the present invention provides the use of the piperazine coumarin derivative according to the first aspect or the third aspect as a probe for dual-channel detection of hydrazine and sulfur dioxide and a cell imaging agent.

[0076] The fifth aspect of the present invention provides a method for detecting the piperazine coumarin derivative according to the first aspect or the third aspect, wherein the method comprises the following steps:

[0077] (1) mixing a dimethyl sulfoxide solution of a piperazine coumarin derivative, a Hepes buffer and methanol to obtain a detection solution;

[0078] (2) adding the sample to be tested to the detection solution obtained in step (1) to obtain a mixed solution;

[0079] (3) Determine the ultraviolet spectrum and fluorescence spectrum of the mixed solution, obtain the ultraviolet absorption and fluorescence intensity at the corresponding wavelengths according to the test, and determine the content of hydrazine and sulfur dioxide in the sample through corresponding linear observation.

[0080] In the present invention, when quantitatively detecting hydrazine, the corresponding wavelengths of ultraviolet spectrum detection can be 425nm and 580nm, and the corresponding wavelengths of fluorescence spectrum detection can be 447nm and 633nm; when quantitatively detecting sulfur dioxide, the corresponding wavelengths of ultraviolet spectrum detection can be 425nm and 580nm, and the corresponding wavelengths of fluorescence spectrum detection can be 445nm and 640nm. The corresponding linear observation refers to the standard curve obtained by ultraviolet or fluorescence titration.

[0081] In some embodiments of the present invention, in the dimethyl sulfoxide solution of the piperazine coumarin derivative, the concentration of the piperazine coumarin derivative is 2 mmol / L.

[0082] In some embodiments of the present invention, the pH of the Hepes buffer is 7.4.

[0083] In some embodiments of the present invention, the concentration of the Hepes buffer is 10 mmol / L.

[0084] In some embodiments of the present invention, the volume ratio of the dimethyl sulfoxide solution of the piperazine coumarin derivative, the Hepes buffer and methanol is 0.05:7:3.

[0085] The present invention will be described in detail below through examples.

[0086] In the following examples and comparative examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if no manufacturer is specified, are all conventional products that can be obtained through commercial channels.

[0087] Example 1

[0088] This example is used to illustrate the synthesis of the target compound NH-LS4.

[0089] 1) In a 50 mL round-bottom flask, 1 g of Michaelis acid and 1 g of 2,4-dihydroxybenzaldehyde were dissolved in 20 mL of water. The mixture was heated to 100° C. and refluxed for 2 h. The mixture was cooled naturally to obtain a precipitate. The precipitate was filtered to obtain an off-white solid as compound 1.

[0090] 2) In a 25 mL round-bottom flask, 408 mg of 4-piperazineacetophenone and 386 mg of 4-(diethylamino)-2-hydroxybenzaldehyde were slowly added to 10 mL of concentrated sulfuric acid, and the mixture was heated to 90°C for 8 h. After cooling to room temperature, the reaction mixture was slowly poured into 200 mL of ice water, and 1 mL of HClO4 was added and stirred. The precipitated suspension was washed with water and dried in vacuo; the product was purified by dichloromethane-methanol (v:v=15:1) column chromatography to obtain a purple-black powder, namely compound 2;

[0091] 3) In a 25 mL round-bottom flask, 103 mg of compound 1, 380 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 270 mg of 1-hydroxybenzotriazole were dissolved in 15 mL of anhydrous N,N-dimethylformamide, and the mixture was stirred at 0°C for 30 min under argon protection, then 230 mg of compound 2 and 200 μL of triethylamine were added, and the mixture was stirred at room temperature for 24 h. After the reaction was complete, the reaction mixture was poured into ice water for washing, and a black solid was obtained by suction filtration. The product was purified by dichloromethane-methanol (v:v=15:1) column chromatography to obtain a dark purple compound NH;

[0092] 4) In a 25-mL round-bottom flask, 88 mg of compound NH, 40 mg of acetyl bromide and 45 μL of trimethylamine were dissolved in 5 mL of anhydrous dichloromethane, and the mixture was stirred at room temperature for 6 h; the product was purified by dichloromethane-methanol (v:v=10:1) column chromatography to obtain a blue-black compound NH-LS4 (0.028 g, yield 30%).

[0093] Figure 1 is the H NMR spectrum of NH-LS4; Figure 2 is the NMR carbon spectrum of NH-LS4; Figure 3 This is the mass spectrum of NH-LS4.

[0094] 1 H NMR (600MHz, DMSO-d6) δ8.73(d,J=8.1Hz,1H),8.65(d,J=8.4Hz,1H),8.29(d,J=8.7Hz ,2H),8.00(d,J=7.9Hz,2H),7.92(d,J=9.2Hz,1H),7.66(s,1H),7.50(d,J=8.4Hz,1H), 7.37(d,J=9.0Hz,1H),7.31(s,1H),7.21(d,J=8.6Hz,2H),7.07(d,J=8.3Hz,1H),3.78( d,J=40.8Hz,4H),3.72–3.60(m,8H),2.25(d,J=8.1Hz,3H),1.25(s,7H).HS-MSm / z:[M]+ calcd for592.24;Found 592.30.

[0095] In the following tests, sodium sulfite solution was used as the sulfur dioxide donor.

[0096] Test Example 1

[0097] Take 1400 μL Hepes buffer solution (concentration of 10 mmol / L, pH 7.4), 600 μL methanol and 10 μL DMSO solution of probe NH-LS4 (concentration of 2 mmol / L) and combine them in a cuvette, add different concentrations of hydrazine aqueous solution / sodium sulfite aqueous solution, and detect the absorbance change of probe NH-LS4 on a UV-visible absorption spectrophotometer after 15 minutes.

[0098] Figure 4 A shows that as the concentration of hydrazine increases, the absorbance at 425 nm overlaps and the absorbance at 580 nm gradually decreases, which indicates that the probe NH-LS4 can be used for the quantitative detection of hydrazine.

[0099] Gradually increase

[0100] Figure 4 B shows that as the concentration of sulfur dioxide increases, the absorbance at 425 nm gradually increases and the absorbance at 580 nm gradually decreases. This indicates that the probe NH-LS4 can be used for the quantitative detection of sulfur dioxide.

[0101] Test Example 2

[0102] 1400 μL Hepes buffer solution (concentration of 10 mmol / L, pH 7.4), 600 μL methanol and 10 μL DMSO solution of probe NH-LS4 (concentration of 2 mmol / L) were combined in a cuvette, and different concentrations of hydrazine aqueous solution / sodium sulfite aqueous solution were added. After 15 minutes, the fluorescence intensity of the probe NH-LS4 was measured on a fluorescence spectrometer.

[0103] Figure 5 A and B show that the fluorescence intensity at 447 nm and 633 nm gradually increases with the increase of hydrazine concentration, which indicates that the probe NH-LS4 can be used for the quantitative detection of hydrazine.

[0104] Figure 5 C and D show that as the concentration of sulfur dioxide increases, the fluorescence intensity at 445 nm gradually increases, while the fluorescence intensity at 640 nm gradually decreases. This indicates that the probe NH-LS4 can be used for the quantitative detection of sulfur dioxide.

[0105] Test Example 3

[0106] Prepare Hepes buffer solutions with different pH values ​​and concentrations of 10 mmol / L, prepare 2 mmol / L DMSO solution of probe NH-LS4, prepare 10 mmol / L hydrazine aqueous solution, and prepare 20 mmol / L sodium sulfite aqueous solution.

[0107] 1400 μL of Hepes buffer solution with different pH values, 600 μL of methanol and 10 μL of DMSO solution of probe NH-LS4 were added to the fluorescence cuvettes, and the pH curves of the pure probe and the probe plus hydrazine / sulfur dioxide were measured on the fluorescence spectrometer, as shown in Fig. Figure 6 .

[0108] from Figure 6 It can be seen that in the absence of hydrazine and sulfur dioxide, the fluorescence intensity of the probe NH-LS4 did not change significantly in the pH range of 2-10. Figure 6 A, B) and sulfur dioxide ( Figure 6 C, D), the emission of probe NH-LS4 increased significantly with the change of pH and reached a maximum value under neutral conditions.

[0109] Test Example 4

[0110] Prepare a Hepes buffer solution with a pH of 7.4 and a concentration of 10 mmol / L, prepare a DMSO solution of 2 mmol / L probe NH-LS4, prepare a 10 mmol / L hydrazine aqueous solution, and prepare a 20 mmol / L sodium sulfite aqueous solution.

[0111] 1400 μL of pH 7.4 Hepes buffer solution, 600 μL of methanol and 10 μL of DMSO solution of probe NH-LS4 were added to the fluorescence cuvette, and then 100 equivalents (100 times the amount of the probe) of other analytes were added: NO3 - ,I - 、SO3 2- 、S2O3 2- Br - , Cl - 、F - 、NO2 - 、CH3COO - OH - 、O2 - 、ONOO - , Cys, Glycine, GSH, H2O2, Hcy, L-Cystine, L-Glutamate, L-Lysine, L-Proline; SO4 2- , SNP, K + 、Na + Mg 2+ , Ca2+ , N2H4, SO2, and detected on a fluorescence spectrometer, and a histogram of the fluorescence intensity at 445 nm corresponding to different analytes was drawn.

[0112] from Figure 7 It can be seen that both hydrazine and sulfur dioxide significantly increase the fluorescence intensity of the detection system at 445 nm, while other analytes basically do not cause significant changes in the fluorescence intensity of the detection system. This shows that the probe NH-LS4 can selectively recognize hydrazine and sulfur dioxide.

[0113] Test Example 5

[0114] Prepare a Hepes buffer solution with a pH of 7.4 and a concentration of 10 mmol / L, prepare a DMSO solution of 2 mmol / L probe NH-LS4, prepare a 10 mmol / L hydrazine aqueous solution, and prepare a 20 mmol / L sodium sulfite aqueous solution.

[0115] 1400 μL Hepes buffer solution, 600 μL methanol and 10 μL DMSO solution of probe NH-LS4 were added to the fluorescence cuvette, and the fluorescence intensity curves of the pure probe and the probe with hydrazine and sulfur dioxide changing with time were measured on a fluorescence spectrometer.

[0116] from Figure 8 It can be seen that after adding hydrazine, the fluorescence intensity of the probe NH-LS4 at 447nm increased significantly (A); while after adding sulfur dioxide, the fluorescence intensity decreased rapidly (B). In addition, the probe NH-LS4 has good stability over time.

[0117] Test Example 6

[0118] Prepare a Hepes buffer solution with a pH of 7.4 and a concentration of 10 mmol / L, prepare a DMSO solution of 2 mmol / L probe NH-LS4, and prepare a 10 mmol / L hydrazine aqueous solution.

[0119] Mix 10 μL of DMSO solution of probe NH-LS4 with the soil, and then observe the fluorescence color under UV light (see Fig. 9 , A). Then add the hydrazine solution to the soil mixed with the probe and observe the fluorescence color under ultraviolet light again (see Fig. 9 , B).

[0120] In the test strip experiment, 10 μL of the DMSO solution of the probe NH-LS4 was dissolved in a water sample (from Yunzhong River, Xinzhou, Shanxi Province), and the color of the trace left by the liquid on the filter paper was observed under ultraviolet light (see Fig. 9 , C), then add hydrazine solution to the liquid mixed with the probe, and then observe the color of the trace left by the liquid on the filter paper under ultraviolet light (see Fig. 9 , D).

[0121] The results showed different fluorescence patterns of red and blue under UV light, which indicated that the probe NH-LS4 could be used for qualitative analysis of the presence of hydrazine in soil and test paper under UV light.

[0122] Test Example 7

[0123] Prepare a Hepes buffer solution with a pH of 7.4 and a concentration of 10 mmol / L, prepare a DMSO solution of 2 mmol / L probe NH-LS4, prepare a 10 mmol / L hydrazine aqueous solution, and prepare a 20 mmol / L sodium sulfite aqueous solution.

[0124] First, add 2mL of Hepes buffer solution to the HeLa cells, and then add 10μL of DMSO solution of probe NH-LS4 to the HeLa cells. After placing the HeLa cells at 37℃ for 20min, rinse the cells three times with Hepes buffer solution, and then take images under a fluorescence confocal microscope. The probe shows dual-channel fluorescence (blue channel and red channel). Next, add hydrazine and sulfur dioxide to the cells incubated with the probe to make the concentrations reach 5μM and 10μM respectively, and observe the fluorescence changes under a fluorescence confocal microscope (see Fig.10 ).

[0125] When hydrazine was added, the fluorescence of the blue and red channels of the system was significantly enhanced under a fluorescence confocal microscope, while when SO2 was added, the red channel gradually weakened.

[0126] Test Case 8

[0127] Prepare a Hepes buffer solution with a pH of 7.4 and a concentration of 10 mmol / L, prepare a DMSO solution of 2 mmol / L probe NH-LS4, and prepare a 10 mmol / L hydrazine aqueous solution.

[0128] First, add 2mL of Hepes buffer solution to the HeLa cells, and then add 10μL of DMSO solution of probe NH-LS4 to the HeLa cells. After placing the HeLa cells at 37℃ for 20min, rinse the cells three times with Hepes buffer solution, and then image and photograph under a fluorescence confocal microscope. The probe exhibits dual-channel fluorescence. Next, add hydrazine aqueous solution to the cells incubated with the probe to make the concentration reach 0μM, 2μM, and 5μM respectively, and observe the fluorescence changes under a fluorescence confocal microscope (see Fig.11 ). As the concentration of hydrazine increases, the fluorescence of the blue and red channels of the system gradually increases under the fluorescence imaging instrument.

[0129] Test Example 9

[0130] 500 μM Mito-Tracker Green was added to HeLa cells, and the cells were placed in a 37°C incubator for 20 min, then rinsed three times with Hepes buffer solution, and then 10 μL of DMSO solution of probe NH-LS4 was added to the cells, and the cells were placed for 20 min, then rinsed three times with Hepes buffer solution, and then imaged and photographed under a fluorescence confocal microscope (see Fig.12 ). The red channel is the probe, and the green channel is Mito-Tracker Green. The results show that the probe has good targeting to mitochondria (Pearson coefficient is 0.87), indicating that the probe can specifically stain mitochondria in living cells.

[0131] The above experimental results indicate that the probe NH-LS4 is a good candidate for dual-channel differential detection of changes in hydrazine and sulfur dioxide in mitochondria.

[0132] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A piperazine coumarin derivative, characterized in that The structural formula of the piperazine coumarin derivative is as follows:

2. A method for synthesizing the piperazine coumarin derivative according to claim 1, characterized in that: The method comprises the following steps: (1) subjecting Michaelis' acid and 2,4-dihydroxybenzaldehyde to a first reaction to obtain a first compound; (2) subjecting 4-piperazineacetophenone and 4-(diethylamino)-2-hydroxybenzaldehyde to a second reaction under acidic conditions; mixing the product obtained by the second reaction with perchloric acid to obtain a second compound; (3) subjecting the first compound, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, the second compound and triethylamine to a third reaction in a protective atmosphere to obtain a third compound; (4) subjecting the third compound, acetyl bromide and triethylamine to a fourth reaction to obtain the piperazine coumarin derivative.

3. The method according to claim 2, wherein: In step (1), the molar ratio of Michaelis acid to 2,4-dihydroxybenzaldehyde is 1:1; Preferably, the solvent of the first reaction is water; Preferably, the conditions of the first reaction include: heating to reflux; temperature is 100° C.; time is 2 h; Preferably, the product obtained by the first reaction is naturally cooled, and the precipitate is filtered out to obtain the first compound.

4. The method according to claim 2 or 3, wherein: In step (2), the molar ratio of 4-piperazineacetophenone to 4-(diethylamino)-2-hydroxybenzaldehyde is 1:1; Preferably, the acidic condition is provided by concentrated sulfuric acid; Preferably, the conditions of the second reaction include: temperature of 90°C; time of 8h; Preferably, the product obtained by the second reaction is mixed with perchloric acid in ice water, and the obtained suspension is washed with water, dried under vacuum, and then purified by column chromatography to obtain the second compound; Preferably, the mobile phase used in the column chromatography purification is dichloromethane and methanol in a volume ratio of 15:

1.

5. The method according to any one of claims 2 to 4, wherein: In step (3), the molar ratio of the first compound, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, the second compound and triethylamine is 1:1:1:1:0.2; Preferably, the solvent for the third reaction is anhydrous N,N-dimethylformamide; Preferably, the protective atmosphere gas is argon; Preferably, the conditions of the third reaction include: room temperature; time is 24h; Preferably, the first compound, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole are first mixed in a solvent, and then the second compound and triethylamine are added; Preferably, the first mixing conditions include: temperature of 0°C; time of 30 min; Preferably, the product of the third reaction is washed with water, filtered and then purified by column chromatography to obtain a third compound; Preferably, the mobile phase used in the column chromatography purification is dichloromethane and methanol in a volume ratio of 15:

1.

6. The method according to any one of claims 2 to 5, wherein: In step (4), the molar ratio of the third compound, acetyl bromide and triethylamine is 1:1:0.2; Preferably, the solvent for the fourth reaction is anhydrous dichloromethane; Preferably, the conditions of the fourth reaction are: room temperature; time is 6h; Preferably, the product of the fourth reaction is purified by column chromatography to obtain the piperazine coumarin derivative; Preferably, the mobile phase used in the column chromatography purification is dichloromethane and methanol in a volume ratio of 10:

1.

7. A piperazine coumarin derivative obtained by the preparation method according to any one of claims 2 to 6.

8. Use of the piperazine coumarin derivative according to claim 1 or 7 as a probe for dual-channel detection of hydrazine and sulfur dioxide and a cell imaging agent.

9. A method for detecting the piperazine coumarin derivative according to claim 1 or 7, characterized in that: The method comprises the following steps: (1) mixing a dimethyl sulfoxide solution of a piperazine coumarin derivative, a Hepes buffer and methanol to obtain a detection solution; (2) adding the sample to be tested to the detection solution obtained in step (1) to obtain a mixed solution; (3) Determine the ultraviolet spectrum and fluorescence spectrum of the mixed solution, obtain the ultraviolet absorption and fluorescence intensity at the corresponding wavelengths according to the test, and determine the content of hydrazine and sulfur dioxide in the sample through corresponding linear observation.

10. The method according to claim 9, wherein: In the dimethyl sulfoxide solution of the piperazine coumarin derivative, the concentration of the piperazine coumarin derivative is 2 mmol / L; Preferably, the pH of the Hepes buffer is 7.4; Preferably, the concentration of the Hepes buffer is 10 mmol / L; Preferably, the volume ratio of the dimethyl sulfoxide solution of the piperazine coumarin derivative, the Hepes buffer and methanol is 0.05:7:3.

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