Piperazine coumarin derivatives, methods of synthesis and use thereof

Abstract

The present application relates to the technical field of detection, and discloses a piperazine coumarin derivative, a synthesis method and application thereof. The piperazine coumarin derivative provided by the present application takes coumarin as a matrix, can be used for double-channel double-emission area distinguishing detection of hydrazine and sulfur dioxide, and has high selectivity, high sensitivity and excellent stability. The piperazine coumarin derivative provided by the present application can detect hydrazine (N2H4) and sulfur dioxide (SO2) at a cell level; also has the ability of rapidly detecting N2H4 in different soil samples and test papers, and has great potential in the analysis and monitoring of environmental pollutants.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection, in particular to a piperazine coumarin derivative and a synthesis method and application thereof. BACKGROUND

[0002] Sulfur dioxide (SO2) is a colorless, irritating gas. SO2 is easily absorbed by the moist mucous membranes of the body to form sulfurous acid and sulfuric acid. SO2 can be endogenously produced 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 can cause diseases such as asthma, allergic reactions, cardiovascular diseases, and neurological disorders. In addition, SO2 is also an environmental pollutant, which can easily react with water in the atmosphere to produce acid rain, causing harm to the ecological environment. Due to its importance to human health and the environment, it has received great attention.

[0003] Hydrazine (N2H4) is an important chemical raw material, which inevitably poses potential threats to the environment and human health during production and use. At the same time, N2H4 is also a highly toxic substance, and 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 central nervous system of the human body, and even organ failure and death in severe cases. 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 efficient and sensitive detection of N2H4 and SO2 in environmental and biological samples.

[0005] Fluorescent probe technology, as a new type of molecular recognition and detection technology, has the advantages of high sensitivity, good selectivity, and fast response speed, and has been widely used in the fields of biomedicine, environmental monitoring, etc. Near-infrared fluorescent probes have great application potential in the fields of biological imaging, medical diagnosis and environmental monitoring due to their deep tissue penetration ability, low background interference and high light stability. Although so far, a large number of fluorescent probes have been reported for the detection of SO2 and N2H4 in biological and environmental systems, most of them can only detect one analyte, and few fluorescent probes can simultaneously detect SO2 and N2H4. Multifunctional fluorescent probes break through the limitation of single fluorescent analyte only detecting single analyte, and have more applications and detection efficiency. At the same time, the differential detection of SO2 and N2H4 can avoid the light crosstalk, metabolism and different positioning of multiple probes in the body, while maintaining cost-effectiveness, less time consumption and non-invasive imaging. SUMMARY

[0006] The present application aims to overcome the problems existing in the prior art, and provides a piperazine coumarin derivative, a synthesis method and application thereof. The piperazine coumarin derivative provided by the present application takes coumarin as a mother body, and can be used for dual-channel dual-emission area distinguishing detection of hydrazine and sulfur dioxide.

[0007] In order to achieve the above-mentioned purpose, the present application provides a piperazine coumarin derivative in a first aspect, wherein the structure of the piperazine coumarin derivative is as follows:

[0008]

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

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

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

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

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

[0014] The present application provides a piperazine coumarin derivative prepared by the preparation method according to the second aspect in a third aspect.

[0015] The present application provides an application 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 reagent in a fourth aspect.

[0016] The present application provides a detection method using the piperazine coumarin derivative according to the first aspect or the third aspect in a fifth aspect, wherein the method comprises the following steps:

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

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

[0019] (3) Determining the ultraviolet spectrum and fluorescence spectrum of the mixed solution, and determining the content of hydrazine and sulfur dioxide in the sample to be tested according to the ultraviolet absorption and fluorescence intensity at the corresponding wavelength obtained by testing through the corresponding linear observation.

[0020] Through the above technical solutions, the beneficial technical effects obtained by the present application are as follows:

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

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

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

[0024] Figure 1 is the nuclear magnetic hydrogen spectrum of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present application.

[0025] Figure 2 is the nuclear magnetic carbon spectrum of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present application.

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

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

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

[0029] Figure 6 is the fluorescence intensity graph of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present application interacting with hydrazine and sulfur dioxide at different pH.

[0030] Figure 7 is the fluorescence intensity graph of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present application with various analytes.

[0031] Figure 8 is the fluorescence intensity plot of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present application versus time with hydrazine and sulfur dioxide.

[0032] Figure 9 is the UV plot of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present application in different soils and test paper strips.

[0033] Figure 10 is the cell imaging plot of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present application with hydrazine and sulfur dioxide.

[0034] Figure 11 is the cell imaging plot of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present application with different concentrations of hydrazine.

[0035] Figure 12 is the mitochondrial localization imaging of the piperazine coumarin derivative NH-LS4 prepared in Example 1 of the present application in Hela cells. DETAILED DESCRIPTION

[0036] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges are provided as example of the upper and lower limits for the ranges. Any value between the upper and lower limits of the ranges can be used as the endpoint values for the ranges. The upper and lower limits of the ranges can each be independently combined with any of the other values for the ranges to create new ranges.

[0037] The first aspect of the present application provides a piperazine coumarin derivative, wherein the structure of the piperazine coumarin derivative is as follows:

[0038]

[0039] In the present application, the above-mentioned piperazine coumarin derivative is named as NH-LS4, the Chinese name is 2-(4-(4-(7-acetoxy-2-oxo-2-H-chromen-3-carbonyl) piperazin-1-yl) phenyl)-7-(diethylamino) chromenylium, and the English name is 2-(4-(4-(7-acetoxy-2-oxo-2H-chromene-3-carbonyl) piperazin-1-yl) phenyl)-7-(diethylamino) chromenylium.

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

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

[0042] (2) subjecting 4-piperazinobenzophenone and 4-(diethylamino)-2-hydroxybenzaldehyde to a second reaction under acidic conditions; mixing the product obtained in 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 application, the molar ratio of Michler's ketone and 2,4-dihydroxybenzaldehyde in step (1) is 1:1.

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

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

[0048] In some embodiments of the present application, the product obtained in the first reaction is naturally cooled, and the precipitate is obtained by suction filtration after the precipitate is separated out.

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

[0050] In some embodiments of the present application, the acidic conditions are provided by concentrated sulfuric acid.

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

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

[0053] In some embodiments of the present application, 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 application, the molar ratio of the first compound, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, the second compound and triethylamine in step (3) is 1:1:1:1:0.2.

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

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

[0057] In some embodiments of the present application, the conditions of the third reaction include room temperature and 24 h.

[0058] In some embodiments of the present application, 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. Such an operation mode can improve the yield.

[0059] In some embodiments of the present application, the conditions of the first mixing include 0℃ and 30 min.

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

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

[0062] In some embodiments of the present application, 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 application, the solvent of the fourth reaction is anhydrous dichloromethane.

[0064] In some embodiments of the present application, the conditions of the fourth reaction are room temperature and 6 h.

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

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

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

[0068] 1) Meldrum's acid and 2,4-dihydroxybenzaldehyde were dissolved in water at a molar ratio of 1:1, the mixture was heated to 100℃, refluxed for 2h, and then naturally cooled, and a precipitate was separated out, and then the mixture was filtered to obtain a milky white solid as compound 1;

[0069] 2) 4-piperazinophenylacetone and 4-(diethylamino)-2-hydroxybenzaldehyde were slowly added into concentrated sulfuric acid at a molar ratio of 1:1, the mixture was heated to 90℃ and kept for 8h, and then the mixture was cooled to room temperature, and then the reaction mixture was slowly poured into ice water, and then HClO4 was added and stirred, and then the precipitate was washed with water, and then the mixture was dried under vacuum, and then the product was purified by column chromatography with dichloromethane-methanol (v:v = 15:1) to obtain a purple black powder as compound 2;

[0070] 3) Compound 1, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole were dissolved in anhydrous N,N-dimethylformamide at a molar ratio of 1:1:1, and then the mixture was stirred at 0℃ under argon protection for 30min, and then compound 2 and triethylamine were added at a molar ratio of 1:0.2 (the molar ratio of compound 1 to compound 2 was 1:1), and then the mixture was stirred at room temperature for 24h, and then the reaction mixture was washed with ice water after the reaction was completed, and then a black solid was obtained by filtration, and then the product was purified by column chromatography with dichloromethane-methanol (v:v = 15:1) 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 then the mixture was stirred at room temperature for 6h, and then the product was purified by column chromatography with dichloromethane-methanol (v:v = 10:1) to obtain a blue-black compound NH-LS4.

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

[0073]

[0074] The third aspect of the present application provides a piperazine coumarin derivative prepared by the preparation method according to the second aspect.

[0075] The fourth aspect of the present application provides an application of the piperazine coumarin derivative according to the first aspect or the third aspect as a probe for detecting hydrazine and sulfur dioxide in a double channel and as a cell imaging reagent.

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

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

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

[0079] (3) determining the ultraviolet spectrum and fluorescence spectrum of the mixed solution, and determining the content of hydrazine and sulfur dioxide in the sample to be detected according to the ultraviolet absorption and fluorescence intensity at the corresponding wavelength obtained by testing through the corresponding linear observation.

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

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

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

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

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

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

[0086] In the following examples and comparative examples, the specific conditions not specified are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be obtained by market purchase.

[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 Michler's acid and 1 g of 2,4-dihydroxybenzaldehyde were dissolved in 20 mL of water, and the mixture was heated to 100°C and refluxed for 2 h, then naturally cooled, and a precipitate was obtained. After suction filtration, a white solid was obtained as compound 1;

[0090] 2) In a 25 mL round-bottom flask, 408 mg of 4-piperazinophenylacetone 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 HCIO4 was added with stirring. The precipitated suspension was washed with water and dried under vacuum. The product was purified by column chromatography with dichloromethane-methanol (v:v = 15:1) to obtain a purple-black powder, which was 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 completed, the reaction mixture was poured into ice water and washed, and a black solid was obtained by suction filtration. The product was purified by column chromatography with dichloromethane-methanol (v:v = 15:1) to obtain a deep 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 column chromatography with dichloromethane-methanol (v:v = 10:1) to obtain a blue-black compound NH-LS4 (0.028 g, with a yield of 30%).

[0093] Figure 1 The nuclear magnetic hydrogen spectrum of NH-LS4; Figure 2 The nuclear magnetic carbon spectrum of NH-LS4; Figure 3 The mass spectrum of NH-LS4.

[0094] 1 H NMR (600 MHz, DMSO-d6) δ 8.73 (d, J = 8.1 Hz, 1H), 8.65 (d, J = 8.4 Hz, 1H), 8.29 (d, J = 8.7 Hz, 2H), 8.00 (d, J = 7.9 Hz, 2H), 7.92 (d, J = 9.2 Hz, 1H), 7.66 (s, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 9.0 Hz, 1H), 7.31 (s, 1H), 7.21 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 8.3 Hz, 1H), 3.78 (d, J = 40.8 Hz, 4H), 3.72 - 3.60 (m, 8H), 2.25 (d, J = 8.1 Hz, 3H), 1.25 (s, 7H). HS-MS m / z: [M]+ calcd for 592.24; Found 592.30.

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

[0096] Test Example 1

[0097] 1400 μL of Hepes buffer solution (concentration of 10 mmol / L, pH 7.4), 600 μL of methanol and 10 μL of DMSO solution of probe NH-LS4 (concentration of 2 mmol / L) were combined in a cuvette, different concentrations of aqueous hydrazine / sodium sulfite aqueous solution were added, and the absorbance change of probe NH-LS4 was detected on a UV-Vis spectrophotometer after 15 min.

[0098] Figure 4 A shows that with the increase of hydrazine concentration, the absorbance at 425 nm is cross-over, and the absorbance at 580 nm gradually decreases. This shows that the probe NH-LS4 can be used for quantitative detection of hydrazine.

[0099] Gradually increase

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

[0101] Test Example 2

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

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

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

[0105] Test Example 3

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

[0107] Add 1400 μL of Hepes buffer solution with different pH, 600 μL of methanol and 10 μL of DMSO solution of probe NH-LS4 into a fluorescence cuvette, respectively, and then measure the pH curves of pure probe and probe plus hydrazine / sulfur dioxide on a fluorescence spectrometer, respectively, as shown in Figure 6 .

[0108] From Figure 6 it can be seen that the fluorescence intensity of probe NH-LS4 does not change obviously in the range of pH 2-10 without hydrazine and sulfur dioxide. When hydrazine Figure 6 (A, B) and sulfur dioxide Figure 6 (C, D) are added, the emission of probe NH-LS4 increases significantly with the change of pH, and reaches the maximum value under neutral conditions.

[0109] Test Example 4

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

[0111] Add 1400 μL of Hepes buffer solution with pH 7.4, 600 μL of methanol and 10 μL of DMSO solution of probe NH-LS4 into a fluorescence cuvette, respectively, and then add 100 equivalents (100 times the amount of probe) of other analytes: 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+ NH-LS4, N2H4, SO2, detected on a fluorescence spectrometer, and the fluorescence intensity column chart at 445 nm corresponding to different analytes was drawn.

[0112] From Figure 7 It can be seen that both hydrazine and sulfur dioxide make the detection system significantly increase in fluorescence intensity at 445 nm, while other analytes do not cause significant changes in 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 hydrazine aqueous solution of 10 mmol / L, and prepare a sodium sulfite aqueous solution of 20 mmol / L.

[0115] Add 1400 μL of the Hepes buffer solution, 600 μL of methanol, and 10 μL of the DMSO solution of the probe NH-LS4 into a fluorescence cuvette, respectively, and measure the time-varying fluorescence intensity curves of the pure probe and the probe added with hydrazine and sulfur dioxide on a fluorescence spectrometer, respectively.

[0116] From Figure 8 It can be seen that after adding hydrazine, the fluorescence intensity of the probe NH-LS4 at 447 nm is significantly enhanced (A); while after adding sulfur dioxide, the fluorescence intensity rapidly decreases (B). Moreover, 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, and prepare a DMSO solution of 2 mmol / L probe NH-LS4, and prepare a hydrazine aqueous solution of 10 mmol / L.

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

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

[0121] The results show different fluorescence patterns of red and blue under UV light. This indicates that the probe NH-LS4 can qualitatively analyze the presence of hydrazine in soil and test paper under UV light.

[0122] Test Example 7

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

[0124] First, 2 mL of the Hepes buffer solution was added to the HeLa cells, and then 10 μL of the DMSO solution of the probe NH-LS4 was added to the HeLa cells. After the HeLa cells were placed at 37°C for 20 min, the cells were washed with the Hepes buffer solution three times, and then imaging was performed under the fluorescence confocal microscope, and the probe showed double-channel fluorescence (blue channel and red channel). Next, the cells incubated with the probe were added with hydrazine and sulfur dioxide so that the concentrations of hydrazine and sulfur dioxide were 5 μM and 10 μM, respectively, and the fluorescence changes were observed under the fluorescence confocal microscope (see Figure 10 ).

[0125] When hydrazine was added, the system showed obvious enhancement of blue and red channel fluorescence under the fluorescence confocal microscope, and when SO2 was added, the red channel gradually weakened.

[0126] Test Example 8

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

[0128] First, 2 mL of the Hepes buffer solution was added to the HeLa cells, and then 10 μL of the DMSO solution of the probe NH-LS4 was added to the HeLa cells. After the HeLa cells were placed at 37°C for 20 min, the cells were washed with the Hepes buffer solution three times, and then imaging was performed under the fluorescence confocal microscope, and the probe showed double-channel fluorescence. Next, the cells incubated with the probe were added with the hydrazine aqueous solution so that the concentration of hydrazine was 0 μM, 2 μM, and 5 μM, respectively, and the fluorescence changes were observed under the fluorescence confocal microscope (see Figure 11 ). As the concentration of hydrazine added increased, the system showed gradual enhancement of blue and red channel fluorescence under the fluorescence imaging instrument.

[0129] Test Example 9

[0130] HeLa cells were added with 500 μM Mito-Tracker Green, and after being placed in a thermostat at 37℃ for 20 min, the cells were washed with Hepes buffer solution for three times, and then 10 μL of DMSO solution of probe NH-LS4 was added into the cells, and after being placed for 20 min, the cells were washed with Hepes buffer solution for three times, and then imaging was performed under a fluorescence confocal microscope (see Figure 12 ) The red channel was the probe, and the green channel was Mito-Tracker Green, and the results showed that the probe had good targeting property to mitochondria (Pearson coefficient was 0.87), which indicated that the probe could specifically stain mitochondria in living cells.

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

[0132] The above detailed the preferred embodiments of the present application, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including that each technical feature is combined in any other suitable manner, and these simple modifications and combinations should also be considered as the disclosed content of the present application, and all fall within the protection scope of the present application.

Claims

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

2. A method of synthesizing the piperazine coumarin derivative according to claim 1, characterized in that, The method comprises the following steps: (1) subjecting Michler's acid and 2,4-dihydroxybenzaldehyde to a first reaction to obtain a first compound; (2) subjecting 4-piperazine acetophenone and 4-(diethylamino)-2-hydroxybenzaldehyde to a second reaction under acidic conditions; mixing the product obtained in 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 of claim 2, wherein, In step (1), the molar ratio of Michler's acid and 2,4-dihydroxybenzaldehyde is 1:1; And / or, the solvent of the first reaction is water; And / or, the conditions of the first reaction include: heating to reflux; the temperature is 100℃; the time is 2h; And / or, the product obtained in the first reaction is naturally cooled, and the precipitate is obtained by suction filtration to obtain the first compound.

4. The method of claim 2 or 3, wherein, In step (2), the molar ratio of 4-piperazine acetophenone and 4-(diethylamino)-2-hydroxybenzaldehyde is 1:1; And / or, the acidic conditions are provided by concentrated sulfuric acid; And / or, the conditions of the second reaction include: the temperature is 90℃; the time is 8h; And / or, the product obtained in the second reaction is mixed with perchloric acid in ice water, and the obtained suspension is washed with water, vacuum dried and then subjected to column chromatography purification to obtain the second compound.

5. The method of claim 4, wherein, The mobile phase used in the column chromatography purification is dichloromethane and methanol at a volume ratio of 15:

1.

6. The method of claim 2, 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; And / or, the solvent of the third reaction is anhydrous N,N-dimethylformamide; And / or, the gas of the protective atmosphere is argon; And / or, the conditions of the third reaction include: room temperature; the time is 24h.

7. The method of claim 2, wherein, 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.

8. The method of claim 7, wherein, The conditions of the first mixing include: the temperature is 0℃; the time is 30min.

9. The method of claim 2, wherein, The product of the third reaction is subjected to column chromatography purification after being washed with water and suction filtration to obtain the third compound.

10. The method of claim 9, wherein, The mobile phase used in the column chromatography purification is dichloromethane and methanol at a volume ratio of 15:

1.

11. The method of claim 2, wherein, In step (4), the molar ratio of the third compound, acetyl bromide and triethylamine is 1:1:0.2; And / or, the solvent of the fourth reaction is anhydrous dichloromethane; And / or, the conditions of the fourth reaction are: the temperature is room temperature; the time is 6h; And / or, the product of the fourth reaction is subjected to column chromatography purification to obtain the piperazine coumarin derivative.

12. The method of claim 11, wherein, The mobile phase used in the column chromatography purification is dichloromethane and methanol at a volume ratio of 10:

1.

13. Use of the piperazine coumarin derivative of claim 1 as a probe for detecting hydrazine and sulfur dioxide in a dual channel and as a cell imaging reagent.

14. A method for detection using the piperazine coumarin derivative according to claim 1, characterized by, The method comprises the following steps: (1) mixing a dimethyl sulfoxide solution of the piperazine coumarin derivative, a Hepes buffer and methanol to obtain a detection solution; (2) adding a sample to be tested to the detection solution obtained in step (1) to obtain a mixed solution; (3) measuring the ultraviolet spectrum and fluorescence spectrum of the mixed solution, and determining the content of hydrazine and sulfur dioxide in the sample to be tested according to the ultraviolet absorption and fluorescence intensity at the corresponding wavelength obtained by testing.

15. The method of claim 14, wherein, In the dimethyl sulfoxide solution of the piperazine coumarin derivative, the concentration of the piperazine coumarin derivative is 2 mmol / L; and / or, the pH of the Hepes buffer is 7.4; and / or, the concentration of the Hepes buffer is 10 mmol / L; and / or, 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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