A phenothiazine fluorescent probe compound for detecting hypochlorous acid and sulfur dioxide and synthesis and application thereof

Abstract

The application provides a fluorescent probe compound for simultaneously detecting HClO and SO2 at double sites and synthesis and application thereof. The probe compound takes phenothiazine as a response group of HClO, expands the conjugated structure of phenothiazine, and introduces a response group of SO2 and an organelle positioning group on the near-infrared fluorescent skeleton, thereby constructing an endoplasmic reticulum-targeted fluorescent probe BQTPA for simultaneously detecting HClO and SO2. The probe can realize dual-channel simultaneous detection of HClO and SO2 through two completely separated emission channels (λem=598 nm and 448 nm), and exhibits excellent selectivity, high sensitivity (detection limits are 7.9 nM and 33.8 nM, respectively) and good light stability to the response of HClO and SO2. The probe is successfully used for monitoring the changes of exogenous and endogenous HClO and SO2 levels in cells, further reveals the dynamic changes of the two in the process of drug-induced liver injury and ferroptosis, and clarifies the role of ferroptosis in liver injury.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry and biosensing technology, specifically relating to an organic small molecule fluorescent probe, and more particularly to a phenothiazine fluorescent probe compound capable of simultaneously detecting hypochlorous acid (HClO) and sulfur dioxide (SO2), its synthesis method, and its application in food detection, environmental monitoring, and bioimaging. Background Technology

[0002] Health and well-being are eternal themes for humankind, but in recent years, environmental pollution and food safety issues have seriously threatened human health. Studies have shown that excessive intake of food additives can increase the risk of diseases such as cancer. Therefore, strictly controlling the use of food additives and the emission of environmental pollutants are effective means of preventing diseases such as cancer.

[0003] Sulfur dioxide (SO2) is not only a toxic environmental pollutant but also an important gaseous messenger molecule in living organisms. It acts as an antioxidant, scavenging excess reactive oxygen species and playing a crucial role in maintaining cellular redox homeostasis. J. Am. Chem. Soc. 2020, 142 , 6324-6331; J. Am. Chem. Soc. 2017, 139 , 3181-3185.). Furthermore, due to its excellent antibacterial, bacteriostatic, and antioxidant properties, SO2 is widely used as a preservative in the food, wine, and pharmaceutical industries. J. Agric. Food Chem. 2022, 70 , 10899-10906; Sens. Actuator B Chem. 2025, 429 , 137276.). Studies have shown that abnormal SO2 levels are closely related to the occurrence and development of respiratory diseases, central nervous system disorders, and lung cancer, among other diseases. Coord. Chem. Rev. 2019, 388 , 310-333; Anal. Chem. 2018, 90 Hypochlorous acid (HClO), as an important reactive oxygen species, is widely used in food disinfection, beverage processing, and pharmaceutical science due to its excellent bactericidal properties. Anal. Chem. 2024, 96HClO is mainly produced in the body through the peroxidation of chloride ions catalyzed by myeloperoxidase (MPO) secreted by immune cells during inflammatory responses. Normal levels of HClO 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 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.).

[0004] In summary, HClO and SO2, as important reactive oxygen species and reactive sulfur species, are not only key messenger molecules and biomarkers in organisms, but also important food preservatives. Furthermore, their respective oxidative and antioxidant functions determine their close relationship in the biological environment; once the balance between them is disrupted, oxidative stress and oxidative damage will be induced in the body. Therefore, developing efficient detection tools to simultaneously identify HClO and SO2 and achieve real-time monitoring of their kinetic interactions is of great value for disease diagnosis and treatment.

[0005] Fluorescence imaging has advantages such as simple operation, high sensitivity, good selectivity, non-invasiveness, and high spatiotemporal resolution, and is considered an effective tool for monitoring bioactive molecules. Chem 2018, 4 , 1609-1628; Chem. Rev. 2019, 119 (10403-10519). To date, many fluorescent probes for recognizing HClO or SO2 have been developed, but only a few can simultaneously detect HClO and SO2. However, these single-fluorescent molecules that simultaneously recognize HClO and SO2 have the following drawbacks: (1) When using a single reaction site to simultaneously recognize HClO and SO2, the competitive reaction between them will lead to deviations in the detection results; (2) The wavelength intervals for detecting HClO and SO2 are very close, which will cause mutual interference between their respective imaging channels. Therefore, it is crucial to develop a dual-reaction site probe for HClO and SO2 with high sensitivity and sufficient fluorescence signal interval.

[0006] Based on this, this patent designs a single fluorescent probe for the simultaneous detection of HClO and SO2 by expanding the conjugated structure of the phenthiazide skeleton of the HClO responsive group and introducing SO2 responsive groups and organelle localizing groups onto this near-infrared fluorescent skeleton. After reacting with HClO, the probe produces strong fluorescence emission at 598 nm, and after reacting with SO2, the fluorescence signal is significantly enhanced at 448 nm. Therefore, this probe can achieve simultaneous dual-channel detection of HClO and SO2 through two completely separate emission channels (150 nm). Its application in the quantitative detection of HClO and SO2 levels in food, as well as in detecting changes in HClO and SO2 levels during liver injury and ferroptosis, will be investigated. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a novel phenothiazine fluorescent probe compound and its synthesis method. This probe can simultaneously detect HClO and SO2 with high selectivity and high sensitivity through two completely independent fluorescence channels.

[0008] Another objective of this invention is to provide the application of the above-mentioned fluorescent probe compounds in the detection of HClO and SO2, particularly in food testing, environmental water sample analysis, and monitoring of dynamic changes in HClO and SO2 levels in biological systems such as living cells and tissues.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a phenothiazine fluorescent probe compound for detecting hypochlorous acid and sulfur dioxide, with the English name (…). E )-3-(14-butyl-6H,14H-quinolino[2',3':4,5]pyrano[3,2-b]phenothiazin-3-yl)-2-cyano-N-(2-((4-methylphenyl)sulfonamido)ethyl)acrylamide, named BQPTA, has the general structural formula shown in formula (I): Equation (1) This probe compound uses phenothiazine as its core skeleton, and its conjugated structure is extended through structural modification. It employs photoinduced energy transfer (PET) and intramolecular charge transfer (ICT) as fluorescence regulation mechanisms, and is designed as a dual-site fluorescent probe for simultaneous monitoring of HClO and SO2. The probe exhibits advantages such as high selectivity, high sensitivity (detection limits of 7.9 nM and 33.8 nM, respectively), high fluorescence quantum yield, and good photostability in response to hypochlorous acid and sulfur dioxide. Furthermore, as an endoplasmic reticulum-targeting probe, it can achieve precise monitoring of HClO and SO2 in specific intracellular organelles.

[0010] The method for synthesizing the fluorescent probe compound provided by the present invention includes the following steps: (1) Under an argon atmosphere, dimethyl sulfoxide (DMSO) was added to 2-methoxyphenothiazine, bromobutane, sodium hydroxide and potassium iodide. The reaction system was reacted at 90~100℃ for 5~7 h, then cooled to room temperature, the reaction was quenched with water, and then extracted with dichloromethane. The organic phase was collected and dried with anhydrous sodium sulfate. The organic solvent was removed under reduced pressure, and the compound 1 was purified by silica gel column chromatography. The molar ratio of 2-methoxyphenothiazine to bromobutane was 1:2; the molar ratio of 2-methoxyphenothiazine to sodium hydroxide was 1:2. The structural formula of compound 1 is (2) Under an argon atmosphere, at 0-5°C, N,N-dimethylformamide (DMF) was slowly added to phosphorus oxychloride (POCl3). After stirring the reaction mixture for 0.5-1 h, a DMF solution of compound 1 was added to the above reaction system, and the mixture was heated to 75-90°C. o The reaction was continued for 5-6 hours under C conditions. After the reaction was completed, the mixture was quenched in ice water, neutralized with sodium bicarbonate (10%) solution, and extracted with dichloromethane. The organic phase was obtained and dried over anhydrous Na2SO4, concentrated under vacuum, and the crude product was purified by silica gel column chromatography to obtain compound 2; the molar ratio of compound 1 to phosphorus oxychloride was 1:4; the molar ratio of compound 1 to N,N-dimethylformamide was 1:10. The structural formula of compound 2 is (3) Under an argon atmosphere, at 0-5℃, a dichloromethane solution of BBr3 was slowly added dropwise to a dichloromethane solution of compound 2. The reaction mixture was stirred for 0.5-1 h, and then stirred overnight at room temperature. After the reaction was completed, the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated under vacuum, and the crude product was purified by silica gel column chromatography to obtain compound 3; the molar ratio of compound 2 to boron tribromide was 1:2.5. The structural formula of compound 3 is (4) Under an argon atmosphere, a DMF solution of 3-bromopropyne was slowly added dropwise to a DMF system of compound 3 and potassium carbonate. The reaction mixture was stirred at room temperature for 24-26 h, then quenched with water, extracted with dichloromethane, the organic layer was washed with water, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain compound 4; the molar ratio of compound 3 to 3-bromopropyne was 1:1.2; the molar ratio of compound 3 to potassium carbonate was 1:1.2. The structural formula of compound 4 is (5) Compound 4, 4-aminobenzyl alcohol, cuprous iodide (CuI) and lanthanum trifluoromethanesulfonate were dissolved in acetonitrile, and the reaction system was heated at 75~85°C. o After reflux at C for 5-6 h, the solid insoluble matter was removed by filtration and purified by column chromatography to obtain a pale yellow solid product 5; the molar ratio of compound 4 to 4-aminobenzyl alcohol was 1:2; the molar ratio of compound 4 to CuI was 1:1; the molar ratio of compound 4 to lanthanum trifluoromethanesulfonate was 1:0.1. The structural formula of compound 5 is (6) Under argon protection, compound 5 and manganese dioxide were added to dichloromethane. The reaction mixture was stirred at room temperature for 5-6 h. The manganese dioxide was filtered through diatomaceous earth and concentrated under vacuum. The crude product was purified by silica gel column chromatography to obtain compound 6. The molar ratio of compound 5 to manganese dioxide was 1:2. The structural formula of compound 6 is ; (7) The dichloromethane solution of p-toluenesulfonyl chloride was slowly added dropwise to the dichloromethane solution of ethylenediamine. The reaction mixture was stirred at room temperature for 1-2 h, and then the reaction mixture was washed with water. The organic phase was extracted with dichloromethane, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 7. The molar ratio of p-toluenesulfonyl chloride to ethylenediamine was 1:10. The structural formula of compound 7 is (8) Compound 7 and cyanoacetic acid were dissolved in acetonitrile (50 ml), and then N-methylimidazolium (NMI) and N,N,N',N'-tetramethylformamidinium hexafluorophosphate (TCFH) were added to the above reaction system. The reaction mixture was stirred at room temperature for 2-3 h, quenched with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated under vacuum, and the crude product was purified by silica gel column chromatography to obtain compound 8; the molar ratio of compound 7 to cyanoacetic acid was 1:1.5; the molar ratio of compound 7 to NMI was 1:3; and the molar ratio of compound 7 to TCFH was 1:1.15. The structural formula of compound 8 is (9) Add ammonium acetate to an ethanol (10 mL) solution of compounds 6 and 8, and heat at 70-80 °C. o The reaction was carried out under reflux for 5-6 hours. After the reaction was completed, the organic solvent was removed by concentration under reduced pressure. The crude product was purified by column chromatography to obtain the target product BQPTA; the molar ratio of compound 6 to compound 8 was 1:1.2.

[0011] This invention provides the application of the fluorescent probe compound BQPTA in the preparation of detection reagents or imaging reagents for detecting hypochlorous acid and / or sulfur dioxide.

[0012] The samples tested include food samples, water samples, and biological samples. Food samples include solid or liquid foods (such as white sugar, rock sugar, dried mango, oatmeal, dried lily bulbs, raisins, kumquat slices, milk, yogurt, and green tea). Biological samples include cells. Cell testing includes monitoring changes in exogenous and endogenous HClO and SO2 levels in cells, as well as the dynamic changes in HClO and SO2 during drug-induced liver injury and ferroptosis.

[0013] This probe can achieve simultaneous dual-channel detection of HClO and SO2 through two completely separate emission channels. The excitation wavelength for HClO is 405 nm and the emission wavelength is 598 nm, while the excitation wavelength for SO2 is 375 nm and the emission wavelength is 448 nm.

[0014] The probe exhibits a detection limit of 7.9 nM for HClO and 33.8 nM for SO2, and demonstrates excellent selectivity and anti-interference capabilities in its response to HClO and SO2. It is unaffected by reactive oxygen species (such as H2O2, tBuOOH, ¹O2, O2⁻•, •OH, ONOO⁻), biothiols (such as Cys, Hcy, GSH), amino acids (such as Ser, His, Lys, Pro, Leu, Phe), and common anions and cations (such as NO2⁻, SO4²⁻, PO4³⁻, OAc⁻, NO3⁻, K⁺, Na⁺, Cu²⁺, Zn²⁺), while also possessing good photostability.

[0015] The fluorescent probe compound is used for the quantitative detection of HClO and / or SO2 residues in water and food samples. The detection method involves adding the probe to the sample, measuring its fluorescence intensity at 598 nm and / or 448 nm, and performing quantitative analysis using a standard curve.

[0016] The fluorescent probe compound is used for bioimaging, particularly live-cell imaging, to monitor the levels and dynamic changes of exogenous or endogenous HClO and SO2 within cells. The probe exhibits good cell membrane permeability and low cytotoxicity.

[0017] The fluorescent probe compounds are used to study changes in redox states in disease or pathological models. They can monitor the generation and decline of HClO and SO2 in drug-induced liver injury models (such as acetaminophen, APAP); or monitor the dynamic changes of HClO and SO2 during programmed cell death processes such as ferroptosis, thereby elucidating the relevant pathological mechanisms.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. Dual-site, dual-channel independent detection: The probe BQPTA molecule of this invention is designed with two independent reaction sites, specifically responding to HClO and SO2, respectively. After reacting with HClO, it produces strong near-infrared fluorescence enhancement at 598 nm; after reacting with SO2 / HSO3, it produces strong near-infrared fluorescence enhancement. - Following the reaction, a strong blue fluorescence enhancement was generated at 448 nm. The wavelengths of the two emission channels are spaced 150 nm apart, effectively avoiding spectral crosstalk and achieving true dual-channel, simultaneous, and independent detection.

[0019] 2. High selectivity and high sensitivity: Probe BQPTA is effective against HClO and SO2 / HSO3. - It exhibits extremely high selectivity, showing no interference from common reactive oxygen species, reactive sulfur compounds, amino acids, and metal ions. It is particularly effective against HClO and HSO3. - The detection limits are extremely low, reaching 7.9 nM and 33.8 nM respectively, and the sensitivity is high.

[0020] 3. Rapid response and good photostability: The probe's response to HClO is instantaneous, and its response to HSO3 is also rapid. - The response reaches equilibrium within 20 minutes, demonstrating a rapid response speed. Both the probe molecule itself and the products resulting from its reaction with the analyte exhibit good photostability, making it suitable for long-term imaging monitoring.

[0021] 4. Excellent biological application potential: This probe exhibits good cell membrane permeability and low cytotoxicity, enabling successful imaging of exogenous and endogenous HClO and SO2 in living cells. More importantly, it can visualize and monitor the dynamic changes in intracellular HClO and SO2 levels during drug-induced liver injury models (such as acetaminophen APAP) and ferroptosis, providing a powerful chemical tool for studying the mechanisms of related diseases.

[0022] 5. Practical food and environmental detection capabilities: The probe of this invention has been successfully applied to the quantitative detection of HClO and SO2 residues in various real samples (such as drinking water, milk, yogurt, green tea, white sugar, dried fruit, etc.), with good spiked recovery rate, indicating its practical application value in the fields of food safety and environmental monitoring. Attached Figure Description

[0023] Figure 1 The proton nuclear magnetic resonance spectrum of the fluorescent probe BQPTA prepared in Example 1 of this invention (1H NMR spectrum) 1 (H NMR) image.

[0024] Figure 2 The carbon NMR spectrum of the fluorescent probe BQPTA prepared in Example 1 of this invention ( 13(C NMR) plot.

[0025] Figure 3 This is a high-resolution mass spectrometry (HRMS) image of the fluorescent probe BQPTA prepared in Example 1 of the present invention.

[0026] Figure 4 The fluorescent probe BQPTA was reacted with different concentrations of HClO(A) and HSO3. - (B) Fluorescence intensity-time kinetic curve of the reaction.

[0027] Figure 5 The fluorescent probe BQPTA is used to detect different concentrations of HClO(A) and HSO3. - (B) Fluorescence response spectrum diagram, and the corresponding linear relationship between fluorescence intensity and concentration (C and D).

[0028] Figure 6 The fluorescent probe BQPTA is used for HClO(A) and HSO3. - (B) Selectivity and anti-interference test bar chart.

[0029] Figure 7 This is a confocal fluorescence image of the fluorescent probe BQPTA used to monitor changes in exogenous HClO levels in HepG2 cells.

[0030] Figure 8 This is a confocal fluorescence image of the fluorescent probe BQPTA used to monitor changes in exogenous SO2 levels in HepG2 cells.

[0031] Figure 9 This is a confocal fluorescence image of the fluorescent probe BQPTA used to monitor changes in HClO and SO2 levels in a drug-induced liver injury model (APAP).

[0032] Figure 10 This is a confocal fluorescence image of the fluorescent probe BQPTA used to monitor changes in intracellular HClO and SO2 levels during ferroptosis. Detailed Implementation

[0033] 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.

[0034] Example 1 Synthesis of fluorescent probe BQPTA (1) Synthesis of compound 1 Under an argon atmosphere, dimethyl sulfoxide (10 mL) was added to 2-methoxyphenthiazide (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 heated to 95 °C. o After reacting at C for 6 h, the mixture was cooled to room temperature, and 100 mL of water was added to quench the reaction. The mixture was then extracted with dichloromethane, and the organic phase was collected and dried with 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 a colorless oily liquid 10-n-butyl-2-methoxyphenthiazide (compound 1) with a yield of 89%. 1 H NMR(400 MHz, CDCl3)δ(ppm) 7.15 - 7.13 (m, 2H), 7.03 (d, J = 8.4 Hz, 1H), 6.88 - 6.86 (m, 2H), 6.48(d, J = 2.3 Hz, 2H), 3.88 – 3.61 (m, 5H), 1.84 – 1.77 (m, 2H), 1.51 – 1.43 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H). 13 C NMR(151 MHz, CDCl3) δ (ppm)159.8, 146.6,144.9, 127.5, 127.4, 127.3, 127.1, 125.5, 122.5, 116.0, 115.7,106.8, 103.5,55.5, 47.3, 29.0, 20.2, 13.9. (2) Synthesis of compound 2 At 0 o Under argon atmosphere, N,N-dimethylformamide (60 mmol) was slowly added to phosphorus oxychloride (24 mmol, 2.24 mL). After stirring for 30 min, a DMF solution (10 mL) of compound 1 (6 mmol, 1.7 g) was added to the reaction system, and the mixture was stirred at 80 °C. o The reaction was continued for 5 h under C conditions. After the reaction was completed, the product was quenched in ice water and neutralized with sodium bicarbonate (10%) solution, followed by extraction with dichloromethane. Finally, the crude product was separated by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to give compound 2 (yield 87%). 1H NMR(400 MHz, CDCl3) δ(ppm)10.20 (s, 1H), 7.54 (s,1H), 7.17–7.10 (m, 2H),6.95 (d, J = 7.4 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 6.38(s, 1H), 3.94–3.88 (s, 5H), 1.86–1.79 (m, 2H), 1.52–1.45(m, 2H), 0.96 (t, J =7.4 Hz, 3H). 13 C NMR(151 MHz, CDCl3) δ (ppm)187.2, 162.6, 152.4, 143.0, 127.5,127.2, 126.9, 124.7, 123.5, 119.7, 116.0, 98.7,55.8, 47.8, 28.9, 20.1, 13.8. (3) Synthesis of compound 3 At 0 o Under argon atmosphere, a 2.5 mmol solution of BBr3 in dichloromethane was slowly added dropwise to a 1 mmol solution of compound 2 in dichloromethane. The reaction mixture was stirred for 30 min and then stirred overnight at room temperature. After the reaction was complete, 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 give compound 3 in 50% yield. 1 H NMR(600 MHz, CDCl3) δ(ppm)11.38 (s, 1H), 9.59(s, 1H), 7.18–7.14 (m, 2H),7.12–7.10 (m, 1H), 6.97 (t, J = 7.2 Hz, 1H), 6.90(d, J = 8.2 Hz, 1H), 6.37 (d, J = 1.9 Hz, 1H), 3.85 (t, J = 6.8 Hz, 2H), 1.82–1.78(m, 2H), 1.49–1.42 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). 13C NMR(151 MHz, CDCl3) δ(ppm)193.2, 162.9, 153.0, 142.3, 130.7, 127.3, 123.8, 123.6, 116.1, 115.8,114.2, 102.8, 47.8, 28.5, 19.9, 13.6. (4) Synthesis of compound 4 Under an argon atmosphere, a DMF solution of 3-bromopropyne (1.2 mmol, 0.103 mL) was slowly added dropwise to a DMF system containing compound 3 (1 mmol, 0.299 g) and potassium carbonate (1.2 mmol, 0.166 g). The reaction mixture was stirred at room temperature for 24 h, quenched with water, extracted with dichloromethane, and the organic layer was washed with water. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether / dichloromethane = 100:1) to give compound 4 in 74% yield. 1 H NMR(400 MHz, CDCl3)δ (ppm)10.20 (s, 1H), 7.54 (s, 1H), 7.17 – 7.12 (m, 1H),7.10 – 7.08 (m, 1H),6.95 (t, J = 7.4 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 6.54 (s, 1H), 4.81 (d, J = 2.5Hz, 2H), 3.91 – 3.87 (m, 2H), 2.61 (t, J = 2.4 Hz, 1H), 1.88 – 1.81 (m, 2H), 1.53 – 1.44 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). 13 C NMR(151 MHz, CDCl3) δ (ppm)186.9, 160.4, 151.8, 143.0, 127.5, 127.3, 126.6, 124.2, 123.5, 120.2,116.9,115.9, 100.4, 77.8, 76.7, 56.5, 48.0, 28.8, 20.1, 13.7. (5) Synthesis of compound 5 Compound 4 (1 mmol, 0.337 g), 4-aminobenzyl alcohol (2 mmol, 0.246 g), CuI (1 mmol, 0.191 g), and (CF3SO3)3La (0.1 mmol, 0.059 g) were dissolved in acetonitrile, and the reaction system was heated at 80 °C. o After reflux at C for 5 h, the solid insoluble matter was removed by filtration, the solvent was removed by vacuum, and the residue was purified by column chromatography (petroleum ether / ethyl acetate = 30 / 1) to give a pale yellow solid product 5, with a yield of 31%. 1 H NMR(400 MHz, CDCl3) δ (ppm)8.11 (s, 1H),8.00 (d, J = 8.6 Hz, 1H), 7.67 (s, 1H), 7.63 – 7.59 (m, 2H), 7.15 (t, J = 7.1 Hz, 2H), 6.93 (t, J = 7.4 Hz, 1H), 6.87 (d, J = 7.4 Hz, 1H), 6.46 (s, 1H), 5.25 (s,2H), 4.83 (s, 2H), 3.83 (t, J = 7.2 Hz, 2H), 1.85 – 1.78 (m, 2H), 1.51 – 1.44(m, 2H), 0.96 (t, J = 7.3 Hz, 3H). 13 C NMR(151 MHz, CDCl3) δ (ppm)157.5, 148.7,148.4, 147.9, 143.9, 138.4, 130.6, 129.2, 128.7, 127.4, 127.1, 124.8,124.6,124.5, 123.5, 122.7, 118.0, 117.3, 115.5, 103.9, 68.6, 64.9, 47.6, 28.8,26.9, 20.1, 13.8. (6) Synthesis of compound 6 Compound 5 (1 mmol, 0.440 g) and manganese dioxide (10 mmol, 0.870 g) were added to a reaction flask, followed by 20 mL of dichloromethane. Under argon protection, the reaction mixture was stirred at room temperature for 5 h. After the reaction was complete, the manganese dioxide was filtered through diatomaceous earth, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel chromatography using dichloromethane / ethyl acetate = 50 / 1 as the eluent (dichloromethane / ethyl acetate = 50 / 1) to give compound 6 in 77% yield. 1 H NMR(400 MHz, CDCl3) δ(ppm) 10.10 (s, 1H), 8.17 (t, J = 1.4 Hz, 1H), 8.12 – 8.08 (m, 3H), 7.87 (s,1H), 7.18 – 7.14 (m, 2H), 6.97 – 6.94(m, 1H), 6.88 – 6.86 (m, 1H), 6.44 (s,1H), 5.32 (d, J = 1.2 Hz, 2H), 3.83 (t, J = 7.2 Hz, 2H), 1.85 – 1.78 (m, 2H), 1.50 – 1.43 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). 13 C NMR(151 MHz, CDCl3) δ (ppm)191.3, 158.0, 151.2, 151.0, 149.4, 143.5, 133.3, 132.8, 131.7, 130.1, 127.4,127.2,127.1, 126.6, 125.3, 124.3, 123.7, 123.0, 118.1, 116.7, 115.6, 103.7,68.4, 47.7, 28.7, 20.1, 13.8. (7) Synthesis of compound 7 A solution of p-toluenesulfonyl chloride (10.7 mmol, 2.04 g) in dichloromethane was slowly added dropwise to a solution of ethylenediamine (100 mmol, 6.7 mmol) in dichloromethane. After stirring at room temperature for 1 h, the reaction mixture was washed with water (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100:1) to give compound 7 in 85% yield. 1H NMR (400 MHz, CDCl3) δ (ppm) 7.75 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 8.2 Hz, 2H), 2.95 (t, J = 5.7 Hz, 2H), 2.78 (t, J = 5.7 Hz, 2H), 2.42 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ (ppm) 143.2, 136.9, 129.6, 126.9, 45.3,40.9. (8) Synthesis of compound 8 Compound 7 (10 mmol, 2.143 g) and cyanoacetic acid (15 mmol, 1.275 g) were dissolved in acetonitrile (50 mL). Then, N-methylimidazole (NMI, 30 mmol, 2.46 g) and N,N,N',N'-tetramethylformamidinium hexafluorophosphate (TCFH, 11.5 mmol, 3.23 g) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h, quenched with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated under vacuum, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50:1) to give compound 8 in 86% yield. 1 H NMR (400 MHz, DMSO-) d6 ) δ (ppm) 8.26 (s, 1H),7.68 – 7.60 (m, 3H), 7.39 (d, J = 8.4 Hz, 2H), 3.57 (s, 2H), 3.13 – 3.07 (m, 2H), 2.78 – 2.72 (m, 2H), 2.37 (s, 3H). 13 C NMR (151 MHz, DMSO-) d6 ) δ (ppm) 162.4,142.8, 137.3, 129.7, 126.6, 116.1, 41.7, 39.0, 25.3, 21.0. (9) Synthesis of compound BQPTA Ammonium acetate (10% mmol, 7.7 mg) was added to a 10 mL ethanol solution of compound 6 (1 mmol, 0.438 g) and compound 8 (1.2 mmol, 0.337 g), and the solution was heated to 70 °C. o The reaction was carried out under reflux for 5 h. After the reaction was completed, the organic solvent was removed by concentration under reduced pressure. The crude product was purified by column chromatography (dichloromethane / ethanol = 20:1) to obtain the target product BQPTA in 69% yield.

[0035] Its proton spectrum is as follows Figure 1 : 1 H NMR (400 MHz, DMSO- d6 ) δ 8.41 (s, 1H), 8.34 (s,1H), 8.22 (s, 2H), 8.16 (s, 1H), 8.03 (d, J = 8.8 Hz, 1H), 7.88 (s, 1H), 7.68(d, J = 8.0 Hz, 3H), 7.37 (d, J = 8.2 Hz, 2H), 7.22 – 7.15 (m, 2H), 7.04 – 6.94(m, 2H), 6.58 (s, 1H), 5.38 (s, 2H), 3.85 (s, 2H), 3.28 (s, 2H), 2.90 (d, J =6.4 Hz, 2H), 2.30 (s, 3H), 1.67 – 1.63(m, 2H), 1.40 – 1.36 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H). Its carbon spectrum is as follows Figure 2 : 13 C NMR (151 MHz, DMSO- d6 ) δ 160.9, 157.9, 149.9,149.5, 148.8, 143.0, 142.6, 137.5, 132.2, 132.1, 129.6, 129.3, 129.0,128.7,127.6, 127.1, 126.7, 126.5, 125.4, 123.0, 123.0, 122.7, 116.5, 116.4, 116.3,116.2, 105.7, 104.0, 67.7, 46.6, 41.6, 28.2, 20.9, 19.4, 13.6. Its high-resolution mass spectrum is as follows Figure 3 HRMS (ESI) m / z calcd for C 39 H 35 N5O4S2[M+H] + :702.2203. Found: 702.2202, error: 0.1 ppm. Example 2: Response kinetics of fluorescent probe BQPTA to different concentrations of HClO and SO2 Figure 4 A represents the time response of the fluorescent probe BQPTA to HClO. Fluorescence spectral changes at different time gradients were measured by mixing different concentrations of HClO (5, 10, and 20 μM) with the probe BQPTA. Figure 4 As can be seen from A, the probe molecule BQPTA reacts immediately upon mixing with HClO, and the fluorescence intensity at 598 nm remains unchanged, indicating that the probe BQPTA provided by this invention can detect changes in HClO levels in real time. Figure 4 B represents the fluorescent probe BQPTA reacting with different concentrations of HSO3. - Fluorescence spectra after the reaction (10, 30, and 50 μM). Figure 4 As can be seen from B, the probe molecule BQPTA reacts with HSO3. - After mixing, the fluorescence intensity at 448 nm gradually increased with the extension of reaction time, and reached equilibrium within 20 min, indicating that the probe BQPTA provided by this invention can rapidly detect changes in HClO levels.

[0036] Example 3: Response of the fluorescent probe BQPTA to different concentrations of HClO and SO2 Figure 5 A and 5B are fluorescent probes BQPTA reacting with different concentrations of HClO and HSO3. - The fluorescence spectrum after the reaction shows that the concentration of probe BQPTA was 10 μM, the concentration of HClO varied from 0 to 30 μM, and HSO3... - The concentration varied within the range of 0-80 μM, and the buffer solution used was PBS solution containing 50% 1,4-dioxane (pH = 7.4). The test method was as follows: different concentrations of HClO or HSO3 were prepared in advance. - BQPTA was added sequentially to the PBS buffer system, and after thorough mixing, the fluorescence spectrum changes in the wavelength range of 300-800 nm were immediately measured using a fluorescence spectrophotometer. The excitation and emission wavelengths in response to HClO were 405 nm and 598 nm, respectively, while the response to HSO3... - The excitation and emission wavelengths were 375 nm and 448 nm, respectively, and the fluorescence spectra were obtained as a function of HClO and SO2 concentrations. Figure 5 As shown in A and 5B, the fluorescence intensity at 598 nm is positively correlated with the HClO concentration as the HClO concentration increases sequentially; however, with the increase of HSO3... - With increasing concentration, the fluorescence intensity at 448 nm correlates with that of HSO3. - The concentrations showed a positive correlation. This indicates that the probe BQPTA provided by this invention can respond efficiently to HClO and HSO3. - It also exhibits good concentration dependence. Figure 5 C and 5D are probes for different concentrations of HClO and HSO3. - The fluorescence linear response diagram shows the fluorescence intensity of the probe versus HClO and HSO3. - The concentrations of HClO and HSO3 showed a good linear relationship within a certain range. Calculations yielded results showing the probe's response to HClO and HSO3. - The detection limits were 7.9 nM and 33.8 nM, respectively. From the above conclusions, it can be concluded that the probe BQPTA can detect HClO and HSO3 with high sensitivity. - .

[0037] Example 4: Selectivity and anti-interference properties of the fluorescent probe BQPTA Figure 6 A and 6B represent the selectivity and anti-interference properties of the fluorescent probe BQPTA for HClO and SO2, respectively. The compatibility of probe BQPTA (10 μM) with HClO (30 μM) and reactive oxygen species (H2O2) was measured. t BuOOH, 1 O2, O2 –• , • OH, ONOO – Biothiols (Cys, Hcy, GSH), amino acids (Ser, His, Lys, Pro, Leu, Phe), and anions (NO2) - SO4 2- PO4 3- ,OAc - NO3 - ) and cations (K) + Na + Cu 2+ Zn 2+ The fluorescence spectrum changes in response to (200 μM) were analyzed, and bar charts were obtained showing the fluorescence intensity at 598 and 448 nm versus different analytes. Figure 6 From A and 6B, we know that only the presence of HClO can cause the probe to produce strong fluorescence emission at 598 nm; only HSO3... -In its presence, the probe exhibits strong fluorescence emission at 448 nm. Various reactive oxygen species, biothiols, amino acids, anions, and cations do not cause changes in the BQPTA fluorescence signal, nor do they affect HClO and HSO3. - The response does not produce any interference. Therefore, the probe BQPTA is capable of detecting HClO and HSO3. - Highly selective detection.

[0038] Example 5: Quantitative detection of HClO and SO2 content in water and food samples using the fluorescent probe BQPTA. Drinking water, milk, yogurt, and green tea were all sourced from a local supermarket (Lanzhou, Gansu). Milk and yogurt samples were processed as follows: 50 µL of trichloroacetic acid was added to 5 mL of milk or yogurt. After protein sedimentation, the supernatant was collected by centrifugation (9000 rpm), and its 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 50% 1,4-dioxane) to prepare the test sample.

[0039] The processing methods for tap water, drinking water, and green tea samples are as follows: Adjust the pH of each sample to 7.4. Then, take 1 mL of each pH-adjusted sample and dilute it to 10 mL to prepare the test solution. After preparing the test samples, add 3 mL of each sample to a cuvette, then add the probe BQPTA (10 μM) to measure its fluorescence spectrum change. Simultaneously, add different concentrations of HClO (0, 5, 10, 15, and 20 μM) and HSO3. - The changes in fluorescence signal were measured after (0, 10, 20, 30, and 40 μM). All experiments were repeated three times. The results are shown in Table 1.

[0040] The food sample processing method is as follows: For granulated sugar and rock sugar samples, weigh 1.5 g directly, dissolve in 10 mL of PBS buffer solution, and adjust the pH to 7.4 to prepare the test sample; for solid food samples such as dried mango, oatmeal, dried lily bulbs, raisins, and kumquat slices, cut appropriately, weigh 1.5 g, and extract with PBS buffer solution by sonication for 2 hours, then filter to prepare the test solution. Adjust the pH of the test solution to 7.4, then take 1 mL of the test solution and dilute it with PBS buffer solution (10 mM, pH = 7.4, containing 50% 1,4-dioxane) to 10 mL to prepare the test sample. After preparing the test sample, take 3 mL of the sample and add it to a cuvette, then add the probe BQPTA (10 μM) to measure its fluorescence spectrum change, and at the same time, add different concentrations of HSO3. -The changes in fluorescence signal were measured after (0, 10, 20, 30, and 40 μM). All experiments were repeated three times. The results are shown in Table 2.

[0041] The probe BQPTA was applied to the quantitative detection of HClO and SO2 residues in drinking water, milk, yogurt, green tea and various solid foods (Tables 1 and 2). The spiked recovery rate was good, which proves its practical value in the fields of food safety and environmental monitoring.

[0042] Example 6: Application of the fluorescent probe BQPTA in monitoring changes in exogenous HClO and SO2 levels HepG2 cells were selected for confocal microscopy imaging using different concentrations of HClO (20, 50, 100, and 200 μM) and HSO3. - Cells were incubated with (50, 100, 250, and 500 μM) for 1 hour, then incubated for another 30 minutes with the addition of probe BQPTA (10 μM). After discarding the culture medium, the cells were washed with PBS buffer and imaged using a confocal microscope. The experimental results are shown below. Figure 7 and 8 As shown. By Figure 7 It can be seen that when HepG2 cells are incubated with BQPTA alone, only a weak red fluorescence signal is observed. Figure 7 A) As the concentration of added HClO increases sequentially, the fluorescence signal in the red channel gradually strengthens. Figure 7 BE). Similarly, a weak blue fluorescence signal was observed when cells were incubated with BQPTA alone. Figure 8 A) As the concentration of added SO2 increases sequentially, the blue fluorescence signal gradually strengthens. Figure 8 (BE). The above results indicate that the probe BQPTA can monitor changes in the levels of HClO and SO2 in cells.

[0043] Example 7: Application of the fluorescent probe BQPTA in monitoring changes in HClO and SO2 levels in a drug-induced liver injury model. HepG2 liver cancer cells were selected for confocal microscopy imaging to construct a liver injury model induced by acetaminophen (APAP). HepG2 cells were incubated with different concentrations of APAP (0.2, 0.5, and 1 mM) for 8 h, followed by incubation with the probe BQPTA (10 μM) for another 30 min. For the inhibitor group, cells were incubated with APAP (1 mM) for 8 h, then incubated with the reactive oxygen species inhibitor NAC or the myeloperoxidase inhibitor ABH for 2 h, followed by incubation with the probe BQPTA for 30 min. The culture medium was then discarded, cells were washed with PBS buffer, and images were obtained using a confocal microscope. The experimental results are shown below. Figure 9 As shown. By Figure 9 It can be seen that after incubating HepG2 cells with different concentrations of APAP, the fluorescence intensity of the blue and red channels increases sequentially with the increase of APAP concentration. Figure 9 (AD); when the inhibitors NAC or ABH are added, the enhancement of fluorescence signal can be significantly reversed. These results demonstrate that the probe provided by this invention can monitor changes in HClO and SO2 levels in a drug-induced liver injury model.

[0044] Example 8: Application of the fluorescent probe BQPTA in monitoring changes in HClO and SO2 levels during ferroptosis. HepG2 cells were selected for confocal microscopy imaging. HepG2 cells were incubated for 12 h with different concentrations of the ferroptosis inducer Erastin (10, 30, 50 μg / mL), followed by incubation for 30 min with the probe BQPTA (10 μM). For the inhibitor group, cells were incubated for 2 h with the ferroptosis-specific inhibitor Fer-1 and the iron chelator DFP, followed by incubation with the probe BQPTA for 30 min. The culture medium was then discarded, and the cells were washed with PBS buffer. Confocal microscopy imaging was performed, and the experimental results are shown below. Figure 10 As shown. By Figure 10 As can be seen, when cells were treated with BQPTA, only a weak fluorescence signal was observed (10A). When cells were pre-incubated with different concentrations of Erastin, the fluorescence in both the blue and red channels was significantly enhanced (10B-D). When cells were pre-incubated with Erastin and then incubated with Fer-1 and DFP, the fluorescence intensity was significantly weaker compared to 10D (10E and F). These results demonstrate that the fluorescent probe BQPTA provided by this invention can monitor changes in HClO and SO2 levels during ferroptosis.

Claims

1. A phenothiazine fluorescent probe compound for detecting hypochlorous acid and sulfur dioxide, characterized in that, The chemical structure of the fluorescent probe compound is as follows: 。 2. The method for synthesizing the fluorescent probe compound according to claim 1, characterized in that, Includes the following steps: (1) Under an argon atmosphere, dimethyl sulfoxide was added to 2-methoxyphenthiazide, bromobutane, sodium hydroxide and potassium iodide. The reaction system was reacted at 90~100℃ for 5~7 h and then cooled and quenched. Compound 1 was obtained by extraction and purification. The structural formula of compound 1 is: ； (2) Under an argon atmosphere and at 0-5℃, N,N-dimethylformamide was added to phosphorus oxychloride and stirred for 0.5-1 h. Then, N,N-dimethylformamide solution of compound 1 was added. The reaction was carried out at 75-90℃ for 5-6 h, and then quenched and neutralized. Compound 2 was obtained by extraction and purification. The structural formula of compound 2 is: ； (3) Under an argon atmosphere and at 0-5℃, a dichloromethane solution of boron tribromide was added dropwise to a dichloromethane solution of compound 2, and the mixture was stirred for 0.5-1 h before reacting at room temperature. Compound 3 was then extracted and purified. The structural formula of compound 3 is: ； (4) Under an argon atmosphere, the N,N-dimethylformamide solution of 3-bromopropyne was added dropwise to the N,N-dimethylformamide system of compound 3 and potassium carbonate. The reaction was stirred at room temperature for 24-26 h and then quenched. The mixture was then extracted and purified to obtain compound 4. The structural formula of compound 4 is: ； (5) Compound 4, 4-aminobenzyl alcohol, cuprous iodide and lanthanum trifluoromethanesulfonate were dissolved in acetonitrile and refluxed at 75~85℃ for 5~6h. The mixture was then filtered and purified to obtain compound 5. The structural formula of compound 5 is: ； (6) Under argon protection, compound 5 and manganese dioxide were added to dichloromethane and stirred at room temperature for 5-6 h. The mixture was then filtered and purified to obtain compound 6. The structural formula of compound 6 is: ； (7) Add the dichloromethane solution of p-toluenesulfonyl chloride dropwise to the dichloromethane solution of ethylenediamine, stir at room temperature for 1-2 h, and wash and extract to obtain compound 7; The structural formula of compound 7 is: (8) Compound 7 and cyanoacetic acid were dissolved in acetonitrile, N-methylimidazolium and N,N,N',N'-tetramethylformamidinium hexafluorophosphate were added, the mixture was stirred at room temperature for 2-3 h and then quenched, and the mixture was extracted and purified to obtain compound 8; The structural formula of compound 8 is: ； (9) Add ammonium acetate to the ethanol solution of compound 6 and compound 8, reflux at 70~80℃ for 5~6 h, concentrate and purify to obtain the target product phenothiazine fluorescent probe compound BQPTA.

3. The synthesis method according to claim 2, characterized in that, The molar ratios of the raw materials in each step meet the following conditions: In step (1), 2-methoxyphenthiazide: bromobutane: sodium hydroxide = 1:2:2; In step (2), compound 1: phosphorus oxychloride: N,N-dimethylformamide = 1:4:10; In step (3), compound 2: boron tribromide = 1:2.5; In step (4), compound 3: 3-bromopropyne: potassium carbonate = 1:1.2:1.2; In step (5), compound 4: 4-amino Benzyl alcohol:cuprous iodide:lanthanum trifluoromethanesulfonate = 1:2:1:0.1; in step (6) compound 5:manganese dioxide = 1:2; in step (7) p-toluenesulfonyl chloride:ethylenediamine = 1:10; in step (8) compound 7: cyanoacetic acid:N-methylimidazolium:N,N,N',N'-tetramethylformamidinium hexafluorophosphate = 1:1.5:3:1.15; in step (9) compound 6:compound 8 = 1:1.

2.

4. The use of the fluorescent probe compound according to claim 1 in the preparation of a detection reagent or imaging reagent for detecting hypochlorous acid and / or sulfur dioxide.

5. The application according to claim 4, characterized in that, The fluorescent probe compound enables simultaneous detection of hypochlorous acid and sulfur dioxide through two completely separate emission channels, with an emission wavelength of 598 nm in response to HClO and 448 nm in response to SO2.

6. The application according to claim 4, characterized in that, The objects being tested include food samples, water samples, and biological samples.

7. The application according to claim 6, characterized in that, The food samples include solid or liquid foods; the biological samples include cells; biological sample detection includes monitoring changes in exogenous and endogenous HClO and SO2 levels in cells, as well as the dynamic changes in HClO and SO2 during drug-induced liver injury and ferroptosis.

8. The application according to claim 4, characterized in that, The probe compound has a detection limit of 7.9 nM for HClO and 33.8 nM for SO2, and exhibits excellent selectivity and anti-interference ability in its response to HClO and SO2, unaffected by reactive oxygen species, biothiols, amino acids, and common anions and cations.

9. A method for detecting hypochlorous acid and / or sulfur dioxide, characterized in that, Using the fluorescent probe compound of claim 1 as a detection reagent, qualitative or quantitative detection of hypochlorous acid and / or sulfur dioxide in a sample can be achieved by measuring the change in fluorescence intensity at 598 nm and / or 448 nm.

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