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

Abstract

The present invention provides a phenothiazine-based fluorescent probe for hypochlorous acid, as well as its preparation and application. The probe, which uses a phenothiazinyl xanthene as its core skeleton, is prepared via a Knoevenagel condensation reaction with 4-cyanopyridine. The probe exhibits advantages in its response to hypochlorous acid, including good selectivity, high sensitivity (detection limit of 8.9 nM), instantaneous response, and high fluorescence quantum yield. Furthermore, the probe provided by the present invention not only enables the detection of varying levels of hypochlorous acid in living cells and changes in hypochlorous acid levels in models of ferroptosis and drug-induced liver injury, but also enables the quantitative detection of hypochlorous acid in food.

Description

Technical Field

[0001] The present invention belongs to the field of organic small molecule fluorescent probes and biosensor technology, and specifically relates to a method for preparing a fluorescent probe for ultrafast and ultrasensitive detection of hypochlorous acid, as well as the detection and quantification of hypochlorous acid levels in inflammation models, liver injury models and foods. 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 primarily produced in the body through the peroxidation of chloride ions catalyzed by myeloperoxidase (MPO), which is secreted by immune cells during inflammatory responses. Normal levels of HClO often act as an antibacterial and anti-inflammatory agent, protecting the innate immune system from invasion by microorganisms, bacteria, and pathogens. However, excessive HClO production can trigger oxidative stress and damage in the body, leading to the development of a range 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 efficiently track changes in HClO levels in living cells in real time is particularly important for disease prevention and diagnosis.

[0003] Fluorescent probes have become one of the most attractive molecular imaging technologies due to their advantages of being non-invasive, highly sensitive, easy to operate, and capable of in situ real-time detection at the cellular level in vivo. In recent years, many fluorescent probes for detecting HClO have been developed, mainly including the following categories: (1) oxidized p-methoxyphenols, oximes, benzoylhydrazides, sulfides, 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. This is 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 the 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 response to the shortcomings of the existing technology, 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, as well as 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, based on a phenothiazinoxanthene core structure and prepared via a Knoevenagel condensation reaction with 4-cyanopyridine, exhibits excellent selectivity, high sensitivity (detection limit of 8.9 nM), instantaneous response, and high fluorescence quantum yield in response to hypochlorous acid.

[0006] The present invention provides a fluorescent probe of hypochlorous acid based on a phenothiazine skeleton, whose English name is (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, named DMPTC, and has the following structural formula:

[0007]

[0008] 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:

[0009] (1) Under argon atmosphere, 2-methoxyphenothiazine, bromobutyl, 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;

[0010] 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.

[0011] (2) Add N,N-dimethylformamide (DMF) to phosphorus oxychloride at 0-5°C under argon atmosphere, stir and react at 0-5°C for 20-40 min, then add a solution of 10-n-butyl-2-methoxyphenothiazine in N,N-dimethylformamide to the above reaction system, and continue heating at 75-85°C for 4-6 h. After the reaction is completed, pour into ice water to quench, neutralize with sodium bicarbonate solution, extract with dichloromethane, dry with anhydrous sodium sulfate, and remove the residual solvent by vacuum spin drying. Purify by column chromatography to obtain compound 2.

[0012] The structural formula of compound 2 is:

[0013] 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.

[0014] (3) Under argon atmosphere 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.

[0015] The structural formula of compound 3 is: ; The molar ratio of compound 2 to boron tribromide is 1:2.5.

[0016] (4) Phosphorus tribromide (PBr3) was slowly added dropwise to a mixed solution of DMF and dichloromethane at 0-5°C. After reacting at room temperature for 0.5-1.5 h, the mixture was 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, and the pH was adjusted to neutral. The mixture was then extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain compound 4.

[0017] The structural formula of compound 4 is: ; The molar ratio of cyclohexanone, phosphorus tribromide and DMF is 1:3:3.

[0018] (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.

[0019] The structural formula of compound 5 is: ; The molar ratio of compound 3, compound 4 and Cs2CO3 is 1:5:3.

[0020] (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, and the solvent was removed under reduced pressure. The obtained crude product was separated and purified by column chromatography to obtain compound 6.

[0021] The structural formula of compound 6 is: ; The molar ratio of 4-pyridineacetonitrile and compound 5 is 1:1.5.

[0022] (7) Under argon, methyl trifluoromethanesulfonate was added to a chloroform solution of compound 6. The reaction mixture was stirred at room temperature for 24-26 h, and then the solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography to obtain the target product MPTC.

[0023] The structural formula of the target product MPTC is: ; The molar ratio of compound 6 and methyl trifluoromethanesulfonate is 1:2.

[0024] (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 resulting residue was purified by column chromatography to obtain DMPTC, a fluorescent probe for hypochlorous acid based on a phenothiazine skeleton. The molar ratio of compound 6 to methyl trifluoromethanesulfonate was 1:50.

[0025] 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.

[0026] The present invention provides a phenothiazine skeleton-based hypochlorous acid fluorescent probe for use in detecting HClO. The concentration of HClO varies within the range of 0-50 μM. As the concentration of hypochlorous acid increases, its fluorescence intensity also increases. Moreover, within a certain concentration range, 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 detection limit of the probe for HClO was calculated to be 8.9 nM.

[0027] 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.

[0028] The present invention provides the use of a phenothiazine skeleton-based hypochlorous acid fluorescent probe for detecting changes in hypochlorous acid levels in living cells, inflammation models, and drug-induced liver injury models for non-diagnostic or therapeutic purposes.

[0029] The present invention provides application of a phenothiazine skeleton-based hypochlorous acid fluorescent probe in quantitatively detecting the hypochlorous acid content in food and water samples.

[0030] Compared with the prior art, the present invention has the following advantages and effects:

[0031] (1) The present invention provides a new fluorescent probe based on phenothiazine. The double positive ion salt DMPTC exhibits better photophysical properties and subcellular organelle mitochondrial targeting than the single positive ion salt MPTC.

[0032] (2) The fluorescent probe DMPTC provided by the present invention exhibits advantages such as high sensitivity, high selectivity, and instantaneous response for the detection of HClO.

[0033] (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.

[0034] (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

[0035] Figure 1 It is a fluorescent probe 1 H NMR spectrum;

[0036] Figure 2 It is a fluorescent probe 13 C NMR spectrum;

[0037] Figure 3 It is a high-resolution mass spectrum of the fluorescent probe;

[0038] Figure 4 is the fluorescence spectrum of the fluorescent probe reacting under different concentrations of hypochlorous acid;

[0039] Figure 5 is the kinetics of the reaction between the fluorescent probe and different concentrations of hypochlorous acid;

[0040] Figure 6 It is the selectivity and anti-interference property of fluorescent probes to different substances;

[0041] Figure 7 It is the application of fluorescent probes to monitor endogenous HClO levels in cells;

[0042] Figure 8It is the application of fluorescent probes to monitor changes in HClO levels in inflammation models;

[0043] Figure 9 This is an application of fluorescent probes to detect changes in HClO levels in drug-induced liver injury models. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0045] Example 1 Synthesis of fluorescent probe

[0046] (1) Synthesis of compound 1

[0047]

[0048] Under argon atmosphere, dimethyl sulfoxide (10 mL) was added to 2-methoxyphenothiazine (5 mmol, 1.145 g), bromobutane (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, then cooled to room temperature and quenched by the addition of 100 mL of water. The reaction was then extracted with dichloromethane, and the organic phase was collected and dried over anhydrous sodium sulfate. The organic solvent was removed under reduced pressure, and the resulting sample was separated by column chromatography (petroleum ether as eluent) to obtain 10-n-butyl-2-methoxyphenothiazine (compound 1) as a colorless oily liquid in an 89% yield.

[0049]

[0050] 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 The reaction was continued at 4°C for 5 h. After completion, the product was poured into ice water to quench the reaction, neutralized with 10% sodium bicarbonate solution, and extracted with dichloromethane. Finally, the crude product was separated on a silica gel column (petroleum ether / ethyl acetate = 20 / 1) to obtain compound 2 (80% yield).

[0051]

[0052] at 0 oUnder argon atmosphere at C, a solution of BBr (2.5 mmol) in dichloromethane was slowly added dropwise to a solution of compound 2 (1 mmol) in dichloromethane. The reaction mixture was stirred for 30 minutes and then allowed to react overnight at room temperature. After the reaction, the residue was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent removed under reduced pressure. The crude product was separated by column chromatography (petroleum ether / dichloromethane = 10:1) to afford compound 3 in a 70% yield.

[0053]

[0054] 0 o C. 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 above reaction system. The reaction mixture is stirred at room temperature for 12 h, quenched with saturated sodium bicarbonate solution, and the pH is adjusted to neutral. The mixture is then extracted with dichloromethane and washed with saturated brine. The organic phase is dried over anhydrous sodium sulfate, and the solvent is evaporated under reduced pressure and directly proceeded to the next step without purification.

[0055]

[0056] 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, quenched by adding water, extracted with dichloromethane, dried over anhydrous Na2SO4, and the organic solvent was removed under reduced pressure. The resulting residue was separated by column chromatography (dichloromethane) to obtain compound 5 (yield 80%).

[0057]

[0058] Compound 5 (1.5 mmol) and 4-pyridineacetonitrile (1 mmol) were dissolved in anhydrous ethanol, and then piperidine (10% mmol) was added dropwise to the reaction mixture. The reaction mixture was stirred at 80 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%).

[0059]

[0060] 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 the solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography (dichloromethane / ethanol = 30:1) to obtain the target product MPTC with a yield of 74%.

[0061]

[0062] 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 the solvent was evaporated under reduced pressure. The resulting residue was purified by column chromatography (dichloromethane / ethanol = 20:1) to obtain the target product DMPTC with a yield of 90%.

[0063] 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).

[0064] 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.

[0065] Its high-resolution mass spectrometry is as follows Figure 3 : HRMS (ESI) m / z calcd for C33H33N3OS (M 2+ ):259.6167. Found: 259.6167.

[0066] Example 2 Responsiveness of the fluorescent probe DMPTC to different concentrations of hypochlorous acid

[0067] Figure 4 The following is a fluorescence spectrum of the fluorescent probe DMPTC after reacting with different concentrations of HClO. 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: pre-configured hypochlorous acid of different concentrations is added to the DMPTC PBS buffer system in sequence, and after mixing evenly, the fluorescence spectrum changes in the wavelength range of 400-700 nm (λex = 370 nm, λem = 460 nm) are immediately measured using a fluorescence spectrophotometer to obtain the relationship between the fluorescence spectrum and the hypochlorous acid concentration. Figure 4 As can be seen from A, 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 Figure B shows the linear fluorescence response of the probe to different HClO concentrations. The fluorescence intensity of the probe exhibits a good linear relationship with the HClO concentration within a certain range. The detection limit of the probe for HClO was calculated to be 8.9 nM. These results indicate that the DMPTC probe can detect HClO with high sensitivity.

[0068] Example 3 Response Kinetics of Fluorescent Probe DMPTC to Different Concentrations of Hypochlorous Acid

[0069] Figure 5To measure the time 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.

[0070] Example 4 Selectivity and Anti-interference of Fluorescent Probe DMPTC

[0071] 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 - , SO4 2- , 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 histogram of different analytes were obtained. Figure 6 As shown, the probe produces strong fluorescence emission only in the presence of HClO. Other reactive oxygen species, amino acids, anions, and cations do not cause changes in the DMPTC fluorescence signal, nor do they interfere with the HClO response. Therefore, the probe DMPTC is capable of highly selective detection of HClO.

[0072] Example 5 Application of the fluorescent probe DMPTC to monitor endogenous HClO levels in cells

[0073] 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 hour, and then the probe DMPTC (10 μM) was added and the cells were incubated for another 30 minutes. After the culture medium was discarded, 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 changes in HClO content in cells.

[0074] Example 6 Application of the fluorescent probe DMPTC to monitor changes in HClO levels in an inflammation model

[0075] Macrophages RAW264.7 were selected for confocal microscopy imaging, and an inflammatory model was established by inducing RAW264.7 with lipopolysaccharide (LPS) and phorbol methyl paraformaldehyde (PMA). 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, 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, 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 2. 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.

[0076] Example 7 Application of the fluorescent probe DMPTC to monitor changes in HClO levels in a drug-induced liver injury model

[0077] 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 and the cells were incubated for another 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 species inhibitor NAC were added and incubated for 2 h, and then the cells were incubated with the probe DMPTC for 30 min. The culture medium was then discarded, the cells were washed with PBS buffer solution, and the images were taken using a confocal microscope. The experimental results are shown in Figure 2. Figure 9 As shown in Figure 2, as the concentration of APAP gradually increases, the fluorescence intensity of the green channel gradually increases ( Figure 9 BE), when the inhibitor was added, the green fluorescence signal was significantly weakened ( Figure 9 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.

[0078] Example 8 Quantitative Detection of HClO Content in Water Samples and Foods Using Fluorescent Probe DMPTC

[0079] Lake water samples were collected from Ruyi Lake at Northwest Normal University. Drinking water, milk, yogurt, and green tea were obtained from a local supermarket (Lanzhou, Gansu). Milk and yogurt samples were prepared as follows: 50 µL of trichloroacetic acid was added to 5 mL of milk or yogurt. After protein sedimentation, the supernatant was centrifuged (9000 rpm) and the pH was adjusted to 7.4. The pH-adjusted supernatant was then diluted to 100 mL with PBS buffer (10 mM, pH 7.4, containing 30% DMSO) to prepare the test sample. Lake water, tap water, drinking water, and green tea samples were prepared as follows: the pH of each sample was adjusted to 7.4. 1 mL of each pH-adjusted sample was then diluted to 10 mL to prepare the test solution. After preparing the test samples, 3 mL of each sample was added to a cuvette. The probe DMPTC (10 μM) was then added to measure the fluorescence spectrum. Fluorescence signal changes were also measured after adding different concentrations of HClO (0, 10, 20, 30, 40, and 50 μM). All experiments were repeated three times. The results are shown in Table 1.

[0080]

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, wherein: The following steps are involved: (1) Under argon atmosphere, 2-methoxyphenothiazine, bromobutyl, 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) Add N,N-dimethylformamide (DMF) to phosphorus oxychloride at 0-5°C under argon atmosphere, stir and react at 0-5°C for 20-40 min, then add a solution of 10-n-butyl-2-methoxyphenothiazine in N,N-dimethylformamide to the above reaction system, and continue heating at 75-85°C for 4-6 h. After the reaction is completed, pour into ice water to quench, neutralize with sodium bicarbonate solution, extract with dichloromethane, dry with anhydrous sodium sulfate, and remove the residual solvent by vacuum spin drying. Purify by column chromatography to obtain compound 2. The structural formula of compound 2 is (3) Under argon atmosphere 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. After reacting at room temperature for 0.5-1.5 h, the mixture was 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, and the pH was adjusted to neutral. The mixture was 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, and the solvent was removed under reduced pressure. 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 24-26 h, and then the solvent was evaporated under reduced pressure. The resulting 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 resulting residue was purified by column chromatography to obtain DMPTC, a fluorescent probe for hypochlorous acid based on a phenothiazine skeleton.

3. The synthesis method according to claim 2, wherein 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; in step (2), 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, wherein 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, wherein 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; and in step (8), the molar ratio of compound 6 to methyl trifluoromethanesulfonate is 1:

50.

6. Use of the phenothiazine skeleton-based hypochlorous acid fluorescent probe according to claim 1 in detecting HClO for non-diagnostic or therapeutic purposes.

7. Use of the phenothiazine skeleton-based hypochlorous acid fluorescent probe according to claim 6 in detecting HClO for non-diagnostic or therapeutic purposes, 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 substituted into the linear relationship to calculate the concentration of the HClO solution to be tested, thus achieving quantitative detection.

8. The use of the phenothiazine skeleton-based hypochlorous acid fluorescent probe according to claim 6 in detecting HClO for non-diagnostic or therapeutic purposes, 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 + 、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.

9. Use of the phenothiazine skeleton-based hypochlorous acid fluorescent probe according to 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 phenothiazine skeleton-based hypochlorous acid fluorescent probe according to claim 1 in quantitatively detecting the hypochlorous acid content in food and water samples.

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