Hypochlorous acid fluorescent probe based on phenothiazine skeleton and preparation and application thereof

Abstract

The invention provides a hypochlorous acid fluorescent probe based on a phenothiazine skeleton as well as preparation and application of the hypochlorous acid fluorescent probe. The probe is prepared by taking phenothiazine and xanthene as a core skeleton and carrying out Knoevenagel condensation reaction on phenothiazine and xanthene and 4-cyanopyridine. The probe has the advantages of good selectivity, high sensitivity (the detection limit is 8.9 nM), transient response, high fluorescence quantum yield and the like in response to hypochlorous acid. Meanwhile, the probe provided by the invention not only realizes the detection of hypochlorous acid at different levels in living cells and the change of the hypochlorous acid level in ferroptosis and drug-induced liver injury models, but also realizes the quantitative detection of hypochlorous acid in food.

Description

Technical Field

[0001] The invention belongs to the technical field of organic small molecule fluorescent probes and biosensors, and specifically relates to a method for preparing a fluorescent probe for ultrafast and ultrasensitive detection of hypochlorous acid, as well as detection and quantification of hypochlorous acid levels in an inflammation model, a liver injury model and food. Background Art

[0002] Hypochlorous acid (HClO) is widely used in food and beverage processing, animal husbandry, agriculture, health care and pharmaceutical science. In addition, due to its strong oxidizing property, HClO is also an indispensable component of endogenous reactive oxygen species (ROS) ( Anal. Chem. 2024, 96 , 4129−4137). HClO is mainly produced in the body by the peroxidation reaction of chloride ions catalyzed by myeloperoxidase (MPO) secreted by immune cells during inflammatory responses. Normal levels of HClO often act as an antibacterial and anti-inflammatory agent to protect the innate immune system from invasion by microorganisms, bacteria, and pathogens. Once HClO is overproduced, it will cause oxidative stress and damage to the body, thereby inducing a series of diseases such as rheumatoid arthritis, cardiovascular disease, and cancer ( Anal. Chem. 2024, 96 , 5992-6000; ACS Appl. Bio Mater. 2022, 5 , 1683−1691; Anal. Chem. 2024, 96 , 11581-11587). Therefore, developing effective detection tools to track the changes in HClO levels in living cells in real time and efficiently is particularly important for the prevention and diagnosis of diseases.

[0003] Fluorescent probes have become one of the most attractive molecular imaging technologies due to their non-invasiveness, high sensitivity, ease of operation, and ability to achieve in situ real-time detection at the cellular level in vivo. In recent years, many fluorescent probes have been developed for the detection of HClO, mainly including the following categories: (1) oxidized p-methoxyphenols, oximes, benzoyl hydrazides, thioethers, and selenoethers ( Org.Lett. 2008, 10 , 2171−2174; Chem. - Eur. J. 2009, 15 , 2305−2309; Chem.Sci. 2022, 13 , 11140−11149; J. Am. Chem. Soc. 2011, 133 , 5680−5682; Org. Lett. 2013,15 , 878-881); (2) Oxidative cleavage of C=C or C=N bonds ( ACS Appl. Mater. Interfaces 2020, 12 , 45822−45829; Chem. Commun. 2011, 47 , 12691−12693); (3) Deprotection of N,N-dimethylthioformamide and thioacetal ( Anal. Chem. 2016, 88 , 12532−12538; Chem. Commun. 2018, 54 , 8522-8525); (4) Desulfurization reaction of C=S bond ( ACS Appl. Mater. Interfaces 2021, 13 , 13949-13957; Sensors Actuators B: Chem. 2018, 255 , 2223-2231), etc. Among them, phenothiazine, as a classic skeleton that responds to HClO, has been widely used to design HClO fluorescent probes, attributed to the presence of sulfur atoms in the molecular structure that are easily oxidized by HClO. In addition, there is an electron-rich nitrogen atom in the skeleton. Most of the nitrogen atoms in the currently designed phenothiazine-type fluorescent skeletons are tertiary amines, and whether changes in the order of nitrogen atoms will affect their optical properties has not been studied. Based on this, this patent develops a new type of HClO fluorescent probe based on a phenothiazine skeleton to further explore its ability to detect HClO in real time and with high sensitivity, as well as its ability to detect changes in hypochlorous acid levels in living cells, inflammatory models, drug-induced liver injury models, and ferroptosis. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a novel fluorescent probe that can detect hypochlorous acid in real time and quantitatively, which realizes the detection of different levels of hypochlorous acid in living cells and the changes in hypochlorous acid levels in ferroptosis and drug-induced liver injury models, and also realizes the quantitative detection of hypochlorous acid in food.

[0005] The probe is prepared by Knoevenagel condensation reaction of phenothiazine-oxanthene as the core skeleton and 4-cyanopyridine. The probe has the advantages of good selectivity, high sensitivity (detection limit 8.9 nM), instantaneous response and high fluorescence quantum yield in response to hypochlorous acid.

[0006] The fluorescent probe of hypochlorous acid based on the phenothiazine skeleton provided by the present invention is named (Z)-7-butyl-4-(2-cyano-2-(1-methylpyridin-1-ium-4-yl)vinyl)-7-methyl-1,2,3,7-tetrahydrochromeno[2,3-b]phenothiazin-7-ium in English, named DMPTC, and has the following structural formula: The method for synthesizing a fluorescent probe of hypochlorous acid based on a phenothiazine skeleton of the present invention is characterized by comprising the following steps: (1) Under argon atmosphere, 2-methoxyphenothiazine, butyl bromide, sodium hydroxide and potassium iodide were dissolved in dimethyl sulfoxide, reacted at 90-100°C for 6-8 h, then cooled to room temperature, quenched with water, extracted with dichloromethane, and the organic phase was collected and dried over anhydrous sodium sulfate, dried under reduced pressure, and separated and purified by column chromatography to obtain 10-n-butyl-2-methoxyphenothiazine; The molar ratio of 2-methoxyphenothiazine to butyl bromide is 1:2; the molar ratio of 2-methoxyphenothiazine to sodium hydroxide is 1:2; and the molar ratio of 2-methoxyphenothiazine to potassium iodide is 65:1-70:1.

[0007] (2) Under argon conditions at 0-5°C, N,N-dimethylformamide (DMF) was added to phosphorus oxychloride, and the mixture was stirred at 0-5°C for 20-40 min. Then, a solution of 10-n-butyl-2-methoxyphenothiazine in N,N-dimethylformamide was added to the reaction system, and the mixture was heated at 75-85°C for 4-6 h. After the reaction, the mixture was poured into ice water to quench, neutralized with sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and the residual solvent was dried under reduced pressure and purified by column chromatography to obtain compound 2. The structural formula of compound 2 is: The molar ratio of 10-n-butyl-2-methoxyphenothiazine to phosphorus oxychloride is 1:4; the molar ratio of N,N-dimethylformamide to phosphorus oxychloride is 2:1~3:1.

[0008] (3) Under argon conditions at 0-5°C, a dichloromethane solution of boron tribromide (BBr3) was added dropwise to a dichloromethane solution of compound 2. The reaction mixture was stirred for 20-40 min and then stirred at room temperature for 10-15 h. The reaction mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was separated by column chromatography to obtain compound 3. The structural formula of compound 3 is: ; The molar ratio of compound 2 to boron tribromide is 1:2.5.

[0009] (4) phosphorus tribromide (PBr3) was slowly added dropwise to a mixed solution of DMF and dichloromethane at 0-5°C, reacted at room temperature for 0.5-1.5 h, and then placed in an ice bath. Subsequently, a dichloromethane solution of cyclohexanone was added dropwise to the above reaction system. The reaction mixture was stirred at room temperature for 10-15 h, quenched with a saturated sodium bicarbonate solution, the pH was adjusted to neutral, and then extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain compound 4; The structural formula of compound 4 is: ; The molar ratio of cyclohexanone, phosphorus tribromide and DMF is 1:3:3.

[0010] (5) Compound 4 and Cs2CO3 were dissolved in DMF, and then the DMF solution of compound 3 was slowly added dropwise to the above system. The reaction mixture was stirred at room temperature for 24-26 h, and then water was added to quench the reaction. The mixture was extracted with dichloromethane and dried over anhydrous sodium sulfate. The organic solvent was removed under reduced pressure, and the resulting residue was separated by column chromatography to obtain compound 5. The structural formula of compound 5 is: ; The molar ratio of compound 3, compound 4 and Cs2CO3 is 1:5:3.

[0011] (6) piperidine was added dropwise to a solution of compound 5 and 4-pyridineacetonitrile in anhydrous ethanol, the reaction mixture was refluxed at 75-85°C for 10-15 hours, the solvent was removed under reduced pressure, and the obtained crude product was separated and purified by column chromatography to obtain compound 6; The structural formula of compound 6 is: ; The molar ratio of 4-pyridineacetonitrile and compound 5 is 1:1.5.

[0012] (7) Under argon, methyl trifluoromethanesulfonate was added to a chloroform solution of compound 6. The reaction mixture was stirred at room temperature for 24 to 26 h, and then the solvent was evaporated under reduced pressure. The residue was purified by column chromatography to obtain the target product MPTC. The structural formula of the target product MPTC is: ; The molar ratio of compound 6 and methyl trifluoromethanesulfonate is 1:2.

[0013] (8) Under argon, methyl trifluoromethanesulfonate was added to a chloroform solution of compound 6, and the reaction mixture was stirred at room temperature for 36-40 h, and then the solvent was evaporated under reduced pressure, and the residual liquid was purified by column chromatography to obtain DMPTC, a fluorescent probe of hypochlorous acid based on a phenothiazine skeleton. The molar ratio of compound 6 to methyl trifluoromethanesulfonate was 1:50.

[0014] In step (2), the silica gel column is passed with petroleum ether: ethyl acetate = 20:1 as the eluent; in step (3), the silica gel column is passed with petroleum ether: dichloromethane = 10:1 as the eluent; in step (5), the silica gel column is passed with dichloromethane as the eluent; in step (6), the silica gel column is passed with dichloromethane: ethyl acetate = 60:1 as the eluent; in step (7), the silica gel column is passed with dichloromethane: ethanol = 30:1 as the eluent; in step (8), the silica gel column is passed with dichloromethane: ethanol = 20:1 as the eluent.

[0015] The present invention provides an application of a hypochlorous acid fluorescent probe based on a phenothiazine skeleton in detecting HClO. The concentration of HClO varies in the range of 0-50 μM. As the concentration of hypochlorous acid increases, its fluorescence intensity also increases. Moreover, within a certain concentration range, there is a good linear relationship between the fluorescence intensity of the hypochlorous acid fluorescent probe and the concentration of HClO: Y=153.38X-401.0667, R 2 =0.9995, Y-fluorescence intensity, X-HClO concentration, unit, μM; the detection limit of the probe for HClO is 8.9 nM through calculation.

[0016] The present invention provides an application of a hypochlorous acid fluorescent probe based on a phenothiazine skeleton in detecting HClO. HClO, H2O2, t BuOOH, 1 O2, O2 -• ,•OH,ONOO - ,Cys,GSH,Ser,His,Lys,Pro,Leu,Phe,HSO3 - 、NO2 - 、SO4 2- PO4 3- ,OAc - 、NO3 - , K + 、Na + , Cu 2+ 、Zn 2+ Only in the presence of HClO can the probe produce strong fluorescence emission. Other reactive oxygen species, amino acids, anions and cations will not cause changes in the probe fluorescence signal, nor will they interfere with the response of HClO, thus achieving highly selective detection of HClO.

[0017] The present invention provides an application of a hypochlorous acid fluorescent probe based on a phenothiazine skeleton in detecting changes in hypochlorous acid levels in living cells, inflammation models and drug-induced liver injury models for non-diagnosis or treatment purposes.

[0018] The invention provides application of a hypochlorous acid fluorescent probe based on a phenothiazine skeleton in quantitatively detecting the content of hypochlorous acid in food and water samples.

[0019] Compared with the prior art, the present invention has the following advantages and effects: (1) The present invention provides a novel fluorescent probe based on phenothiazine. The dicationic salt DMPTC exhibits better photophysical properties and subcellular organelle mitochondrial targeting than the monocationic salt MPTC.

[0020] (2) The fluorescent probe DMPTC provided by the present invention exhibits the advantages of high sensitivity, high selectivity and instantaneous response for the detection of HClO.

[0021] (3) The fluorescent probe DMPTC provided by the present invention can detect changes in hypochlorous acid levels in living cells, inflammation models, and drug-induced liver injury models in real time.

[0022] (4) The fluorescent probe DMPTC provided by the present invention can quantitatively detect the content of hypochlorous acid in food and water samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a fluorescent probe 1 H NMR spectrum; Figure 2 It is a fluorescent probe 13 C NMR spectrum; Figure 3 It is a high-resolution mass spectrum of the fluorescent probe; Figure 4 It is the fluorescence spectrum of the fluorescent probe under different concentrations of hypochlorous acid; Figure 5 is the kinetics of the reaction between the fluorescent probe and different concentrations of hypochlorous acid; Figure 6 It is the selectivity and anti-interference of fluorescent probes to different substances; Figure 7 It is the application of fluorescent probes to monitor endogenous HClO levels in cells; Figure 8 It is the application of fluorescent probes to monitor changes in HClO levels in inflammation models; Fig. 9 It is the application of fluorescent probe to detect changes in HClO levels in drug-induced liver injury model. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0025] Example 1 Synthesis of fluorescent probe (1) Synthesis of compound 1 Under argon atmosphere, dimethyl sulfoxide (10 mL) was added to 2-methoxyphenothiazine (5 mmol, 1.145 g), bromobutyl (10 mmol, 1.36 g), sodium hydroxide (10 mmol, 0.4 g) and potassium iodide (0.072 mmol, 12 mg). The reaction system was reacted at 95 °C for 6 h and then cooled to room temperature. 100 mL of water was added to quench the reaction and then extracted with dichloromethane. The organic phase was collected and dried over anhydrous sodium sulfate. The organic solvent was removed under reduced pressure and the obtained sample was separated by column chromatography (petroleum ether as eluent) to obtain colorless oily liquid 10-n-butyl-2-methoxyphenothiazine (compound 1) with a yield of 89%.

[0026] At 0 o Under argon atmosphere at 80 °C, N,N-dimethylformamide (60 mmol) was slowly added to phosphorus oxychloride (24 mmol, 2.24 mL). After stirring for 30 min, a solution of compound 1 (6 mmol, 1.7 g) in DMF (10 mL) was added to the reaction system and stirred at 80 °C. o C for 5 h. After the reaction, it was poured into ice water to quench, neutralized with sodium bicarbonate (10%) solution, and extracted with dichloromethane. Finally, the crude product was separated by silica gel column (petroleum ether / ethyl acetate = 20 / 1) to obtain compound 2 (yield 80%).

[0027] At 0 o Under argon atmosphere, a dichloromethane solution of BBr3 (2.5 mmol) was slowly added dropwise to a dichloromethane solution of compound 2 (1 mmol). The reaction mixture was stirred for 30 min and then stirred at room temperature overnight. After the reaction, the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was separated by column chromatography (petroleum ether / dichloromethane = 10:1) to obtain compound 3 with a yield of 70%.

[0028] 0 oC. Slowly add PBr3 (3 mmol) dropwise to a mixed solution of DMF (3 mmol) and dichloromethane (5 mL). After reacting at room temperature for 1 h, place the mixture in an ice bath. Add 1 mL of cyclohexanone (1 mmol) in dichloromethane dropwise to the reaction system. Stir the reaction mixture at room temperature for 12 h, quench with saturated sodium bicarbonate solution, adjust the pH to neutral, extract with dichloromethane, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, spin dry the solvent under reduced pressure, and proceed to the next step without purification.

[0029] Compound 4 (5 mmol) and Cs2CO3 (3 mmol) were dissolved in DMF, and then the DMF solution of compound 3 (1 mmol) was slowly added dropwise to the above system. The reaction mixture was stirred at room temperature for 48 h, and then water was added to quench the reaction. The mixture was extracted with dichloromethane and dried over anhydrous Na2SO4. The organic solvent was removed under reduced pressure and the resulting residue was separated by column chromatography (dichloromethane) to obtain compound 5 (yield 80%).

[0030] Compound 5 (1.5 mmol) and 4-pyridineacetonitrile (1 mmol) were dissolved in anhydrous ethanol, and piperidine (10% mmol) was then added dropwise to the reaction mixture. o The reaction was refluxed at C overnight, and the solvent was removed under reduced pressure to obtain a crude product which was separated and purified by column chromatography (dichloromethane:ethyl acetate = 60:1) to obtain compound 6 (yield 79%).

[0031] Under argon, methyl trifluoromethanesulfonate (2 mmol) was added to a chloroform solution of compound 6 (1 mmol). The reaction mixture was stirred at room temperature for 24 h and then the solvent was dried under reduced pressure. The residue was purified by column chromatography (dichloromethane / ethanol = 30:1) to obtain the target product MPTC with a yield of 74%.

[0032] Under argon, methyl trifluoromethanesulfonate (50 mmol) was added to a chloroform solution of compound 6 (1 mmol). The reaction mixture was stirred at room temperature for 36 h and then the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (dichloromethane / ethanol = 20:1) to obtain the target product DMPTC with a yield of 90%.

[0033] Its hydrogen spectrum is as follows Figure 1 : 1 H NMR (400 MHz, DMSO-d6 ) δ 8.86 (d, J = 6.8 Hz, 2H),8.63 (s, 1H), 8.27 (d, J = 6.4 Hz, 2H), 8.07 (s, 1H), 8.03 (d, J = 7.6 Hz, 1H),7.85 (t, J = 7.8 Hz, 1H), 7.83 – 7.76 (m, 2H), 7.45 (t, J = 7.4 Hz, 1H), 7.27 (s,1H), 4.39 – 4.26 (m, 5H), 3.03 – 2.94 (m, 5H), 2.69(t, J = 5.0 Hz,2H), 1.91 –1.78 (m, 4H), 1.57 – 1.48 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H). Its carbon spectrum is as follows Figure 2 : 13 C NMR (151 MHz, DMSO- d6 ) δ 156.6, 155.3, 149.8,144.5, 142.8, 142.2, 140.3, 134.4, 131.4, 130.1, 128.5, 126.9, 123.7,121.2,121.1, 119.1, 117.8, 117.0, 111.7, 106.6, 104.2, 102.1, 97.7, 47.1, 46.3,30.6, 27.8, 27.7, 24.8, 19.7, 19.1, 13.1. Its high-resolution mass spectrometry is shown in Figure 3 : HRMS (ESI) m / z calcd for C33H33N3OS (M 2+ ):259.6167. Found: 259.6167. Example 2 Responsiveness of fluorescent probe DMPTC to different concentrations of hypochlorous acid Figure 4The fluorescence spectrum of the fluorescent probe DMPTC after reacting with different concentrations of HClO, where the concentration of the probe DMPTC is 10 μM, the concentration of HClO varies in the range of 0-50 μM, and the buffer solution used is a PBS solution containing 30% DMSO (pH = 7.4). The test method is: add pre-configured hypochlorous acid of different concentrations to the PBS buffer system of DMPTC in sequence, mix evenly, and immediately use a fluorescence spectrophotometer to measure the fluorescence spectrum changes in the wavelength range of 400-700 nm (λex = 370 nm, λem = 460 nm), and obtain a graph showing the relationship between the fluorescence spectrum and the hypochlorous acid concentration. Figure 4 It can be seen from A that as the concentration of HClO increases, the fluorescence intensity at 460 nm is positively correlated with the concentration of HClO, which indicates that the probe DMPTC provided by the present invention can efficiently respond to HClO and exhibits good concentration dependence. Figure 4 B is the fluorescence linear response diagram of the probe to different concentrations of HClO. There is a good linear relationship between the fluorescence intensity of the probe and the concentration of HClO within a certain range. The detection limit of the probe for HClO is 8.9 nM through calculation. From the above conclusions, it can be seen that the probe DMPTC can detect HClO with high sensitivity.

[0034] Example 3 Response kinetics of fluorescent probe DMPTC to different concentrations of hypochlorous acid Figure 5 To investigate the temporal response of the fluorescent probe DMPTC to HClO, different concentrations of HClO (10, 30, and 50 μM) were mixed with the probe DMPTC to measure the changes in fluorescence spectra at different time gradients. Figure 5 It can be seen that once the probe molecule DMPTC is mixed with HClO, it reacts immediately, and the fluorescence intensity at 460 nm no longer changes, indicating that the probe DMPTC provided by the present invention can detect changes in HClO levels in real time.

[0035] Example 4 Selectivity and anti-interference of fluorescent probe DMPTC Figure 6 The selectivity and anti-interference of the fluorescent probe DMPTC to HClO were measured. t BuOOH, 1 O2, O2 -• , •OH, ONOO - ), biothiols (Cys and GSH), amino acids (Ser, His, Lys, Pro, Leu, Phe), anions (HSO3 - , NO2 - , SO42- , PO4 3- , OAc - , NO3 - ) and cations (K + , Na + , Cu 2+ , Zn 2+ ) (200 μM) in response to the fluorescence spectrum changes, and the fluorescence intensity at 460 nm and the bar graph of different analytes were obtained. Figure 6 It can be seen that only in the presence of HClO can the probe produce strong fluorescence emission, and other reactive oxygen species, amino acids, anions and cations will not cause changes in the DMPTC fluorescence signal, nor will they interfere with the response of HClO. Therefore, the probe DMPTC can achieve highly selective detection of HClO.

[0036] Example 5 Application of fluorescent probe DMPTC to monitor endogenous HClO levels in cells HeLa cells were selected for confocal microscopy imaging. HeLa cells were incubated with different concentrations of HClO (20, 30, 50, 100 and 200 μM) for 1 h, and then the probe DMPTC (10 μM) was added to continue incubating the cells for 30 min. After discarding the culture medium, the cells were washed with PBS buffer solution and imaged with a confocal microscope. The experimental results are shown in Figure 2. Figure 7 After adding the probe to the cells, a weak fluorescence signal can be observed in the green channel ( Figure 7 A), when the concentration of added HClO gradually increases, the green fluorescence signal gradually increases ( Figure 7 BF). These results indicate that the probe DMPTC can monitor the changes in HClO content in cells.

[0037] Example 6 Application of fluorescent probe DMPTC to monitor changes in HClO levels in an inflammatory model Macrophages RAW264.7 were selected for confocal microscopy imaging, and lipopolysaccharide (LPS) and phorbol 3-myristate monophosphate (PMA) were used to induce RAW264.7 to establish an inflammatory model. The specific experimental process is as follows: (1) RAW264.7 cells were incubated with DMPTC (10 μM) for 30 min, (2) RAW264.7 cells were incubated with LPS (2 μg / mL) for 2 h, and then with DMPTC (10 μM) for 30 min, (3) RAW264.7 cells were incubated with PMA (2 μg / mL) for 2 h, and then with DMPTC for 30 min; (4) RAW264.7 cells were co-incubated with LPS and PMA for 2 h, and then with DMPTC for 30 min; (5) RAW264.7 cells were incubated with LPS and PMA for 2 h, and then with the myeloperoxidase inhibitor ABH (1 mM) for 1 h, and then with DMPTC for 30 min; (6) RAW264.7 cells were incubated with LPS and PMA for 2 h, and then with the anti-inflammatory drug methotrexate (0.2 mM) for 1 h, and then with DMPTC for 30 min. After the incubation, the culture medium was discarded, the cells were washed with PBS, and the changes in fluorescence signals were observed using a confocal fluorescence microscope. The experimental results are shown in Figure 8 As shown. After incubating cells with the probe DMPTC alone, only a weak fluorescence signal was shown ( Figure 8 A), after incubation with LPS and PMA, the green fluorescence signal of cells was significantly enhanced ( Figure 8 BD), when the inhibitor ABH and the anti-inflammatory drug MTX were added, the fluorescence signal was significantly weakened. The above experimental results show that the probe provided by the present invention can monitor the changes in HClO levels during the inflammatory process.

[0038] Example 7 Application of fluorescent probe DMPTC to monitor changes in HClO levels in drug-induced liver injury model HepG2 hepatoma cells were selected for confocal microscopy imaging, and a liver injury model was established by inducing HepG2 with acetaminophen (APAP). HepG2 cells were incubated with different concentrations of APAP (0.1, 0.2, 0.3 and 1 mM) for 8 h, and then the probe DMPTC (10 μM) was added to continue incubating the cells for 30 min; for the inhibitor group, after incubating the cells with APAP (1 mM) for 8 h, the myeloperoxidase inhibitor ABH and the reactive oxygen inhibitor NAC were added to incubate the cells for 2 h, and then the probe DMPTC was incubated for 30 min. The culture medium was then discarded, the cells were washed with PBS buffer solution, and the images were taken with a confocal microscope. The experimental results are shown in Fig. 9 As shown in Figure 2, as the concentration of APAP increases, the fluorescence intensity of the green channel increases gradually ( Fig. 9 BE), when the inhibitor was added, the green fluorescence signal was significantly weakened ( Fig. 9FH). The above results indicate that the probe provided by the present invention can monitor the changes in HClO levels in drug-induced liver injury models.

[0039] Example 8 Quantitative detection of HClO content in water samples and foods using fluorescent probe DMPTC Lake water samples were collected from Ruyi Lake of Northwest Normal University, and drinking water, milk, yogurt, and green tea were obtained from a local supermarket (Lanzhou, Gansu). The processing method for milk and yogurt samples was as follows: 50 µL of trichloroacetic acid was added to 5 mL of milk or yogurt, and after the protein was precipitated, the supernatant was centrifuged (speed 9000 rpm), and its pH was adjusted to 7.4, and then the pH-adjusted supernatant was diluted to 100 mL with PBS buffer (10 mM, pH = 7.4, containing 30% DMSO) to prepare the test sample. The processing method for lake water, tap water, drinking water, and green tea samples was as follows: the pH of each sample was adjusted to 7.4, and then 1 mL of the pH-adjusted sample was taken and diluted to 10 mL to prepare the test solution. After the samples to be tested were prepared, 3 mL of the samples were added to the cuvettes, and then the probe DMPTC (10 μM) was added to measure the changes in its fluorescence spectrum. At the same time, different concentrations of HClO (0, 10, 20, 30, 40 and 50 μM) were added to measure the changes in its fluorescence signal. All experiments were repeated three times, and the experimental results are shown in Table 1.

[0040]

Claims

1. Fluorescent probe of hypochlorous acid based on phenothiazine skeleton, the structural formula is as follows: 。 2. The method for synthesizing a fluorescent probe of hypochlorous acid based on a phenothiazine skeleton according to claim 1, characterized in that: The following steps are involved: (1) Under argon atmosphere, 2-methoxyphenothiazine, butyl bromide, sodium hydroxide and potassium iodide were dissolved in dimethyl sulfoxide, reacted at 90-100°C for 6-8 h, then cooled to room temperature, quenched with water, extracted with dichloromethane, and the organic phase was collected and dried over anhydrous sodium sulfate, dried under reduced pressure, and separated and purified by column chromatography to obtain 10-n-butyl-2-methoxyphenothiazine; (2) Under argon conditions at 0-5°C, N,N-dimethylformamide (DMF) was added to phosphorus oxychloride, and the mixture was stirred at 0-5°C for 20-40 min. Then, a solution of 10-n-butyl-2-methoxyphenothiazine in N,N-dimethylformamide was added to the reaction system, and the mixture was heated at 75-85°C for 4-6 h. After the reaction, the mixture was poured into ice water to quench, neutralized with sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and the residual solvent was dried under reduced pressure and purified by column chromatography to obtain compound 2. The structural formula of compound 2 is (3) Under argon conditions at 0-5°C, a dichloromethane solution of boron tribromide (BBr3) was added dropwise to a dichloromethane solution of compound 2. The reaction mixture was stirred for 20-40 min and then stirred at room temperature for 10-15 h. The reaction mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was separated by column chromatography to obtain compound 3. The structural formula of compound 3 is (4) phosphorus tribromide (PBr3) was slowly added dropwise to a mixed solution of DMF and dichloromethane at 0-5°C, reacted at room temperature for 0.5-1.5 h, and then placed in an ice bath. Subsequently, a dichloromethane solution of cyclohexanone was added dropwise to the above reaction system. The reaction mixture was stirred at room temperature for 10-15 h, quenched with a saturated sodium bicarbonate solution, the pH was adjusted to neutral, and then extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain compound 4; The structural formula of compound 4 is (5) Compound 4 and Cs2CO3 were dissolved in DMF, and then the DMF solution of compound 3 was slowly added dropwise to the above system. The reaction mixture was stirred at room temperature for 24-26 h, and then water was added to quench the reaction. The mixture was extracted with dichloromethane and dried over anhydrous sodium sulfate. The organic solvent was removed under reduced pressure, and the resulting residue was separated by column chromatography to obtain compound 5. The structural formula of compound 5 is (6) piperidine was added dropwise to a solution of compound 5 and 4-pyridineacetonitrile in anhydrous ethanol, the reaction mixture was refluxed at 75-85°C for 10-15 hours, the solvent was removed under reduced pressure, and the obtained crude product was separated and purified by column chromatography to obtain compound 6; The structural formula of compound 6 is (7) Under argon, methyl trifluoromethanesulfonate was added to a chloroform solution of compound 6. The reaction mixture was stirred at room temperature for 24 to 26 h, and then the solvent was evaporated under reduced pressure. The residue was purified by column chromatography to obtain the target product MPTC. The structural formula of the target product MPTC is ; (8) Under argon, methyl trifluoromethanesulfonate was added to a chloroform solution of compound 6. The reaction mixture was stirred at room temperature for 36-40 h, and then the solvent was evaporated under reduced pressure. The residual liquid was purified by column chromatography to obtain DMPTC, a fluorescent probe of hypochlorous acid based on a phenothiazine skeleton.

3. The synthesis method according to claim 2, characterized in that In step (1), the molar ratio of 2-methoxyphenothiazine to butyl bromide is 1:2; the molar ratio of 2-methoxyphenothiazine to sodium hydroxide is 1:2; the molar ratio of 2-methoxyphenothiazine to potassium iodide is 65:1-70:1; the molar ratio of 10-n-butyl-2-methoxyphenothiazine to phosphorus oxychloride is 1:4; and the molar ratio of N,N-dimethylformamide to phosphorus oxychloride is 2:1-3:

1.

4. The synthesis method according to claim 2, characterized in that In step (3), the molar ratio of compound 2 to boron tribromide is 1:2.5; in step (4), the molar ratio of cyclohexanone, phosphorus tribromide and DMF is 1:3:3; in step (5), the molar ratio of compound 3, compound 4 and Cs2CO3 is 1:5:

3.

5. The synthesis method according to claim 2, characterized in that In step (6), the molar ratio of 4-pyridineacetonitrile to compound 5 is 1:1.5; in step (7), the molar ratio of compound 6 to methyl trifluoromethanesulfonate is 1:2; in step (8), the molar ratio of compound 6 to methyl trifluoromethanesulfonate is 1:

50.

6. Use of the hypochlorous acid fluorescent probe based on a phenothiazine skeleton as claimed in claim 1 in detecting HClO.

7. The use of the hypochlorous acid fluorescent probe based on the phenothiazine skeleton in detecting HClO according to claim 6, characterized in that: When the concentration of HClO is in the range of 0-50 μM, the fluorescence intensity of the hypochlorous acid fluorescent probe has a good linear relationship with the concentration of HClO: Y=153.38X-401.0667, R 2 =0.9995, Y-fluorescence intensity, X-HClO concentration, unit: μM. The fluorescence intensity of the HClO solution to be tested is detected, and the concentration of the HClO solution to be tested is calculated by substituting it into the linear relationship to achieve quantitative detection.

8. The use of the hypochlorous acid fluorescent probe based on a phenothiazine skeleton in detecting HClO according to claim 6, characterized in that: In the hypochlorous acid fluorescent probe solution, HClO, H2O2, t BuOOH, 1 O2, O2 -• ,•OH,ONOO - ,Cys,GSH,Ser,His,Lys,Pro,Leu,Phe,,HSO3 - 、NO2 - 、SO4 2- PO4 3- ,OAc - 、NO3 - , K + 、 + , Cu 2+ 、Zn 2+ Only in the presence of HClO can the probe produce strong fluorescence emission. Other reactive oxygen species, amino acids, anions and cations will not cause changes in the probe fluorescence signal, nor will they interfere with the response of HClO, thus achieving highly selective detection of HClO.

9. Use of the phenothiazine skeleton-based hypochlorous acid fluorescent probe as claimed in claim 1 in detecting changes in hypochlorous acid levels in living cells, inflammation models and drug-induced liver injury models for non-diagnostic or therapeutic purposes.

10. Use of the hypochlorous acid fluorescent probe based on a phenothiazine skeleton as claimed in claim 1 in quantitatively detecting the content of hypochlorous acid in food and water samples.

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