Fluorescent probe compound for simultaneously detecting HClO and ONOO <-> based on rhodamine skeleton as well as preparation and application of fluorescent probe compound

Abstract

The invention provides a fluorescent probe compound for simultaneously detecting HClO and ONOO <-> at two sites as well as preparation and application of the fluorescent probe compound. According to the probe compound, N, N-dimethylaminothioformyl ester is used as a response group of HClO, and the fluorescent probe RhB-ClO for simultaneously detecting HClO and ONOO <-> is constructed by expanding a conjugated structure of rhodamine. The probe shows excellent selectivity, high sensitivity (the detection limits are 9.9 nM and 13.1 nM respectively), good light stability and real-time responsiveness (the response time is 30 s and 150 s respectively) on HClO and ONOO <->. The probe not only can realize dual-channel imaging of HClO and ONOO <-> under oxidative stress conditions, but also can visualize the ferroptosis process of cells, a Parkinson's disease model and changes of HClO and ONOO <-> levels in an APAP-induced liver injury model. In addition, RhB-ClO can also be used for screening potential candidate drugs for nerve injury and liver injury, and it is proved that luteolin and piplongumine can be used as candidate antioxidant drugs.

Description

Technical Field

[0001] The present invention belongs to the technical fields of organic small molecule fluorescent probes and biosensing technology, and specifically relates to a rhodamine skeleton-based fluorescent probe compound for simultaneous detection of HClO and ONOO – and its preparation and application. Background Art

[0002] HClO and ONOO⁻ have high reactivity and can directly modify a variety of biomolecules through reactions such as oxidation, nitration, and halogenation ( Free Radic. Biol. Med., 2022 , 193, 474 - 484; Proteins Struct. Funct. Bioinform. , 2020, 88 , 166 - 174.). These modification effects play important roles in cell signal transduction, immune defense, and maintaining physiological balance ( Molecules , 2023, 28 , 7976.; Curr. Opin. Toxicol. , 2018, 7 , 73 - 80.). HClO and ONOO⁻ play dual roles in living organisms, being both important messenger molecules and potential disease-inducing factors ( Anal. Chem. , 2022, 94 , 5744 - 5751.; Anal. Chem. , 2024, 96 , 17657 - 17664.). Normal concentrations of HClO and ONOO⁻ play key roles in regulating redox homeostasis, signal transduction, and normal immune defense; however, excessive HClO and ONOO⁻ can disrupt the intracellular redox balance, over-oxidize biomolecules, and thus trigger various pathological processes such as neurodegenerative diseases, cardiovascular and cerebrovascular diseases, cancer, inflammatory diseases, and autoimmune diseases ( Int. J. Biochem. Cell Biol. , 2007, 39, 44 - 84.; Sens. Actuators B Chem. , 2022, 359, 131565.; Angew. Chem. Int. Ed. , 2022, 134, e202206894.). For example, high levels of HClO and ONOO⁻ damage cells, induce inflammation and neuronal dysfunction, and then induce liver injury and nerve injury ( Talanta , 2025, 283, 127194.; Sens. Actuators B Chem. , 2024, 415 , 135995.; Chem. Eng. J. , 2023, 464, 142496.). Therefore, observing the levels of HClO and ONOO⁻ in these two disease models is of great significance for a deeper understanding of their pathological mechanisms.

[0003] Due to their non-invasive nature, high sensitivity, ease of operation, and ability to achieve in-situ real-time detection at the cellular level in vivo, fluorescent probes have become one of the most attractive molecular imaging techniques. In recent years, a large number of fluorescent probes have been used to selectively detect HClO and ONOO⁻ respectively, but only a few fluorescent probes can detect both HClO and ONOO⁻ simultaneously. In particular, probes that can continuously respond to HClO and ONOO⁻ have not been reported. Based on this, this patent utilizes the oxidizing properties of HClO and ONOO – to design a "double-lock and double-key" type fluorescent probe for the simultaneous detection of HClO and ONOO⁻ with sequential activation. This probe first reacts with HClO to produce a strong fluorescence emission at 700 nm; subsequently, it reacts with ONOO⁻, and the fluorescence signal is significantly enhanced at 570 nm. Therefore, this probe can achieve continuous response to HClO and ONOO⁻ through two completely separate emission channels (130 nm). And it is used to explore the changes in the levels of HClO and ONOO⁻ during the detection of oxidative stress, nerve injury, liver injury, and ferroptosis, etc. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a novel fluorescent probe that can continuously respond to HClO and ONOO⁻, realizing the detection of HClO and ONOO⁻ in living cells, as well as the changes in the levels of HClO and ONOO⁻ during oxidative stress, nerve injury, liver injury, and ferroptosis. At the same time, it is also used to screen potential antioxidant drugs during nerve injury and liver injury processes.

[0005] The rhodamine skeleton-based fluorescent probe for the simultaneous detection of HClO and ONOO provided by the present invention – has the English name (E)-9-(2-carboxyphenyl)-6-(diethylamino)-4-((E)-3-(4-((dimethylcarbamothioyl)oxy)phenyl)allylidene)-1,2,3,4-tetrahydroxanthylium, named RhB-ClO, and its structural formula is as follows: .

[0006] The synthesis method of the fluorescent probe provided by the present invention includes the following steps: (1) Methyl 4-hydroxycinnamate and imidazole were slowly added to DMF at 0-5°C, followed by the addition of tert-butyldimethylsilyl chloride (TBSCl). The reaction system was stirred in an ice bath for 0-20 min and then reacted at room temperature for 1-3 h. After the reaction was completed, water was added to quench the mixture, and the mixture was extracted with ethyl acetate, washed with saturated NaCl solution, dried over anhydrous Na2SO4, concentrated in vacuo, and purified by silica gel column chromatography to obtain compound 1. The molar ratio of methyl 4-hydroxycinnamate to imidazole was 1:1-1:2; the molar ratio of methyl 4-hydroxycinnamate to TBSCl was 1:1-1:2. The structural formula of compound 1 is ; (2) Under argon conditions, compound 1 was dissolved in dichloromethane, and diisobutylaluminum hydride (DIBAL-H) was slowly added at -70~-80℃. The reaction was continued for 20~40 min and then moved to room temperature for 0.5~2 h. After the reaction was completed, the system was cooled to -1~2℃ and slowly quenched with water. The filtrate was filtered through diatomaceous earth, extracted with dichloromethane, dried over anhydrous Na2SO4, concentrated in vacuo, and subjected to silica gel column chromatography to obtain compound 2. The molar ratio of compound 1 to DIBAL-H was 1:5~1:6. The structural formula of compound 2 is ; (3) MnO2 was slowly added to a dichloromethane solution containing compound 2 and stirred at room temperature for 20 to 32 h. After the reaction was completed, the solution was filtered through diatomaceous earth and the filtrate was extracted with dichloromethane to obtain an organic phase, which was dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography to obtain compound 3; the molar ratio of compound 2 to MnO2 was 1:15 to 1:16; The structural formula of compound 3 is ; (4) Compound 3 and tetrabutylammonium fluoride (TBAF) were dissolved in DMF solution and stirred at room temperature for 0-2 h. After the reaction was completed, water was added to quench the mixture, and the mixture was extracted with ethyl acetate, dried over anhydrous Na2SO4, concentrated in vacuo, and chromatographed on a silica gel column to obtain compound 4. The molar ratio of compound 3 to TBAF was 1:1-1:3. The structural formula of compound 4 is ; (5) At 0 - 5 °C, cyclohexanone was slowly added dropwise to concentrated sulfuric acid, and then 2-(4-(diethylamino)-2-hydroxybenzoyl)benzoic acid was added to this system. The reaction mixture was reacted at 80 - 100 °C for 1 - 3 h. After the reaction ended, the reaction was cooled to room temperature and then poured into ice water, and perchloric acid was added. After filtration, compound 5 was obtained; the molar ratio of 2-(4-(diethylamino)-2-hydroxybenzoyl)benzoic acid to cyclohexanone was 1:1 - 1:2; The structural formula of compound 5 is ; (6) Under nitrogen conditions, compound 5 and compound 4 were dissolved in acetic acid and refluxed at 80 - 100 °C for 5 - 7 h. After the reaction ended, it was cooled to room temperature and concentrated under vacuum. The blue-violet solid compound RhB-OH was obtained by silica gel column chromatography; the molar ratio of compound 5 to compound 4 was 1:1 - 1:2; The structural formula of compound RhB-OH is ; (7) Compound RhB-OH and triethylenediamine (DABCO) were dissolved in dichloromethane and stirred at room temperature for 5 - 10 min, then dimethylaminothiocarbonate was added and stirred at room temperature for 20 - 32 h. After the reaction ended, water was added to quench the reaction, and a solid precipitated. After filtration and washing, the pale purple solid compound RhB-ClO was obtained; the molar ratio of compound RhB-OH to DABCO was 1:2 - 1:4; the molar ratio of compound RhB-OH to dimethylaminothiocarbonate was 1:2 - 1:4; The structural formula of compound RhB-ClO is .

[0007] The present invention provides the application of the fluorescent probe compound for detecting HClO and ONOO for non-diagnostic or therapeutic purposes. – The present invention also provides the application of the fluorescent probe compound for monitoring the changes in the levels of HClO and ONOO during oxidative stress, nerve injury, liver injury and ferroptosis for non-diagnostic or therapeutic purposes. – The fluorescent probe compound can also be used as a tool molecule for screening potential candidate drugs during nerve injury and liver injury.

[0008] In summary, the present invention uses N , N -dimethylaminothiocarbonate as the response group for HClO, and constructs the fluorescent probe RhB-ClO for simultaneously detecting HClO and ONOO by expanding the conjugated structure of rhodamine. This probe has high selectivity and sensitivity for HClO and ONOO – fluorescent probe RhB-ClO. This probe has high selectivity and sensitivity for HClO and ONOO –Exhibits excellent selectivity, high sensitivity (detection limits are 9.9 nM and 13.1 nM respectively), good photostability, and real-time responsiveness (response times are 30 s and 150 s respectively). This probe can not only achieve dual-channel imaging of HClO and ONOO – under oxidative stress conditions, but also visualize the changes in the levels of HClO and ONOO – during the ferroptosis process of cells, Parkinson's disease model, and APAP-induced liver injury model. In addition, RhB-ClO can also be used to screen potential candidate drugs for nerve injury and liver injury, and it is confirmed that luteolin, piperlongumine, and 6-gingerol can be used as candidate antioxidant drugs.

[0009] Compared with the prior art, the present invention has the following advantages and effects: (1) The present invention provides a fluorescence probe RhB-ClO of the "double-lock and double-key" type with sequential activation for simultaneous detection of HClO and ONOO – .

[0010] (2) The fluorescence probe RhB-ClO provided by the present invention can be used to monitor the changes in the levels of HClO and ONOO – during processes such as oxidative stress, nerve injury, liver injury, and ferroptosis.

[0011] (3) The fluorescence probe RhB-ClO provided by the present invention can be used as a tool molecule to screen potential candidate drugs during nerve injury and liver injury processes. Brief Description of the Drawings

[0012] Figure 1 is the 1 H NMR spectrum of the fluorescence probe; Figure 2 is the 13 C NMR spectrum of the fluorescence probe; Figure 3 is the high-resolution mass spectrum of the fluorescence probe; Figure 4 is the kinetics of the fluorescence probe reacting with different concentrations of HClO and ONOO – ; Figure 5 is the fluorescence spectrum of the fluorescence probe under different concentrations of HClO and ONOO – conditions; Figure 6 is the selectivity and anti-interference of the fluorescence probe to different substances; Figure 7 is the application of the fluorescence probe to monitor the changes in the levels of HClO and ONOO – during the oxidative stress model; Figure 8It is a fluorescent probe for HClO and ONOO in nerve injury models – Application of level changes; Figure 9 It is a fluorescent probe for HClO and ONOO in liver injury model – Application of level changes; Figure 10 It is a fluorescent probe for screening antioxidant drugs in nerve damage. DETAILED DESCRIPTION

[0013] The present invention will be further described below with reference to the embodiments and drawings, but the present invention is not limited to the following embodiments.

[0014] Example 1 Synthesis of fluorescent probe (1) Synthesis of compound 1 Methyl 4-hydroxycinnamate (11.4 g, 64 mmol) and imidazole (7 g, 100 mmol) were slowly added to DMF (20 mL) at 0°C, followed by the addition of TBSCl (15.1 g, 100 mmol). The reaction system was stirred in an ice bath for 10 min and then allowed to react at room temperature for 2 h. After completion of the reaction, the product was quenched with water, extracted with ethyl acetate, washed with saturated NaCl solution, dried over anhydrous Na₂SO₄, and concentrated in vacuo. Finally, the product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (200 / 1) as the eluent to afford compound 1 as a white solid (88% yield).

[0015] (2) Synthesis of compound 2 Under argon, compound 1 (30 mmol, 8.49 g) was dissolved in dichloromethane (150 mL). DIBAL-H (180 mmol, 30.2 mL) was slowly added at -78 °C. The reaction was continued at -78 °C for 30 min and then at room temperature for 1 h. After completion, the reaction was cooled to 0 °C and quenched by the slow addition of water. The mixture was filtered through celite, extracted with dichloromethane, dried over anhydrous Na₂SO₄, and concentrated in vacuo. Finally, the crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (50 / 1) as the eluent to afford compound 2 as a brown-yellow oil (yield 64%).

[0016] (3) Synthesis of compound 3 MnO2 (20.01 g, 230.17 mmol) was slowly added to a solution of compound 2 (3.8 g, 14.39 mmol) in dichloromethane (100 mL), and the mixture was stirred at room temperature for 24 h. After the reaction was completed, it was filtered through diatomaceous earth, and the filtrate was extracted with dichloromethane. The organic phase was dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50 / 1) to obtain compound 3 as a yellow oil (yield 63%).

[0017] (4) Synthesis of compound 4: Compound 3 (20 mmol, 5.2 g) and TBAF (40 mmol, 25.24 g) were dissolved in DMF solution (50 mL) and stirred at room temperature for 1 h. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with ethyl acetate, dried over anhydrous Na2SO4, and concentrated under vacuum. Finally, silica gel column chromatography was carried out using petroleum ether / ethyl acetate = 50 / 1 as the eluent to obtain compound 4 as a yellow solid (yield 52%).

[0018] (5) Synthesis of compound 5: At 0 °C, cyclohexanone (25.48 mmol, 2.64 mL) was slowly added dropwise to concentrated sulfuric acid (28 mL), and then 2-(4-(diethylamino)-2-hydroxybenzoyl)benzoic acid (12.8 mmol, 4 g) was added to the system. The reaction mixture was reacted at 90 o °C for 2 h. After the reaction was completed, the reaction was cooled to room temperature. Then it was poured into ice water, and 70% perchloric acid (2.8 mL) was added. After filtration, red solid 5 was obtained (yield 71%).

[0019] (6) Synthesis of compound RhB-OH: Under nitrogen atmosphere, compound 5 (2.11 g, 5.6 mmol) and compound 4 (0.754 g, 6.72 mmol) were dissolved in acetic acid (30 mL) and refluxed at 90 o °C for 6 h. After the reaction was completed, it was cooled to room temperature and concentrated under vacuum. Finally, silica gel column chromatography was carried out using dichloromethane / ethanol = 30 / 1 as the eluent to obtain compound RhB-OH as a blue-violet solid (yield 40%).

[0020] (7) Synthesis of compound RhB-ClO: The compound RhB-OH (1.13 g, 2.24 mmol) and DABCO (0.753 g, 6.72 mmol) were dissolved in dichloromethane (10 mL), stirred at room temperature for 5 min, and then dimethylaminothiocarbonate (0.90 g, 6.72 mmol) was added and stirred at room temperature for 24 h. After the reaction was completed, the reaction was quenched with water, and a solid precipitated. Then it was filtered and washed to obtain the light purple solid compound RhB-ClO (yield 62%).

[0021] Its hydrogen spectrum is as follows Figure 1 : 1 H NMR(600 MHz, CDCl3) δ 8.09 (d, J J = 7.8 Hz, 1H), 7.59(t, J J = 7.5 Hz, 1H), 7.52 (t, J J = 7.5 Hz, 1H), 7.49 (d, J J = 9.0 Hz, 3H), 7.10 –7.06 (m, 3H), 7.02 (d, J J = 8.4 Hz, 2H), 6.75 (s, 1H), 6.66 (dd, J J = 9.6 Hz, 2.4Hz, 1H), 6.54 (d, J = 12.0 Hz, 1H), 3.50 – 3.47 (m, 4H), 3.45 (s, 3H), 3.34(s, 3H), 2.68 – 2.59(m, 2H), 2.23 – 2.20 (m, 1H), 1.93 – 1.89 (m, 1H), 1.72 –1.64 (m, 2H), 1.23 (t, J J = 7.2 Hz, 6H). 11H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 11.7 Hz, 1H), 7.62 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.45 (d, J = 8.6 Hz, 2H), 7.21 (d, J = 15.6 Hz, 1H), 7.08 – 6.96 (m, 5H), 6.79 – 6.68 (m, 2H), 3.55 (d, J = 8.0 Hz, 4H), 3.44 (s, 3H), 3.33 (s, 3H), 2.64 (s, 2H), 2.34 – 2.07 (m, 2H), 1.67 (d, J = 20.5 Hz, 2H), 1.26 (t, J = 7.1 Hz, 6H). Its carbon spectrum is as follows Figure 2 : 13 13C NMR (151 MHz, CDCl3) δ 187.6, 169.9, 153.5, 152.8, 149.8, 148.4, 135.2, 134.6, 134.0, 129.6, 129.2, 128.6, 128.2, 127.5, 127.3, 126.0, 125.5, 124.6, 123.8, 123.0, 121.3, 109.4, 105.9, 97.1, 44.5, 43.2, 38.7, 25.9, 23.4, 21.9, 12.5. Its high-resolution mass spectrum is as follows Figure 3 : HRMS (ESI) m / z calcd for C 36 H 37 N2O4S (M): 593.2469. Found: 593.2470, error: 0.3 ppm. Example 2. Response kinetics of the fluorescent probe RhB-ClO to different concentrations of HClO and ONOO – Figure 4 For the time response of the fluorescent probe RhB-ClO to HClO, different concentrations of HClO (5, 10, 15, 20 μM) were mixed with the probe RhB-ClO, and the fluorescence spectral changes were measured at different time gradients. From Figure 4 ​As can be seen, once the probe molecule RhB-ClO is mixed with HClO, the reaction is completed within 30 s, and the fluorescence intensity at 700 nm no longer changes; different concentrations of ONOO – (10, 20, 30, 40 μM) are added to the system that has responded to HClO, and the reaction is completed within 150 s, and the fluorescence intensity at 570 nm no longer changes. This indicates that the probe RhB-ClO provided by the present invention can detect the changes in the levels of HClO and ONOO – in real time.

[0022] Example 3 Responsiveness of the fluorescent probe RhB-ClO to different concentrations of hypochlorous acid and ONOO – Figure 5 The fluorescence spectra after the reaction of the fluorescent probe RhB-ClO with different concentrations of HCl and ONOO – are shown in the figure. The concentration of the probe RhB-ClO is 10 μM, the concentration of HClO varies in the range of 0 - 20 μM, and the concentration of ONOO – varies in the range of 0 - 40 μM. The buffer solution used is PBS solution containing 30% MeOH (pH = 7.4). The test method is as follows: Different concentrations of hypochlorous acid prepared in advance are added to the PBS buffer system of RhB-ClO in sequence. After mixing evenly, the fluorescence spectral changes in the wavelength range of 600 - 800 nm are measured with a fluorescence spectrophotometer (λex = 594 nm, λem = 700 nm) to obtain the relationship diagram of the fluorescence spectrum changing with the concentration of hypochlorous acid. Subsequently, different concentrations of ONOO – prepared in advance are added to the system that has responded to HClO. After mixing evenly, the fluorescence spectral changes in the wavelength range of 480 - 600 nm are measured with a fluorescence spectrophotometer (λex = 480 nm, λem = 570 nm). As can be seen from Figure 5 Figures 5A and 5B, as the concentration of HClO increases in sequence, the fluorescence intensity at 700 nm is positively correlated with the concentration of HClO; as the concentration of ONOO – increases in sequence, the fluorescence intensity at 570 nm is positively correlated with the concentration of ONOO – . This indicates that the probe RhB-ClO provided by the present invention can efficiently respond to HClO and shows good concentration dependence. Figure 5 Figures 5C and 5D are the fluorescence linear response diagrams of the probe to different concentrations of HClO and ONOO – . There is a good linear relationship between the fluorescence intensity of the probe and the concentrations of HClO and ONOO – within a certain range. By calculation, the detection limits of the probe for HClO and ONOO –The detection limits are 9.9 nM and 13.1 nM. From the above conclusions, it can be seen that the probe RhB-ClO can detect HClO and ONOO with high sensitivity. – .

[0023] Example 4 Selectivity and anti-interference of the fluorescent probe RhB-ClO Figure 6 The selectivity and anti-interference of the fluorescent probe RhB-ClO for HClO and ONOO – were investigated. The fluorescence spectral changes of the probe RhB-ClO (10 μM) in response to HClO (50 μM), reactive oxygen species (H2O2, t BuOOH, 1 O2, O2 -• , • OH, ONOO – ), amino acids (Cys, GSH, 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) were measured, and the bar graphs of the fluorescence intensities at 700 nm and 570 nm versus different analytes were obtained. As Figure 6 can be seen, only in the presence of HClO can the probe produce strong fluorescence emission at 700 nm; in the system after responding to HClO, when the above-mentioned reactive oxygen species, amino acids, anions, and cations are added, only in the presence of ONOO – can the probe produce strong fluorescence emission at 570 nm. Other various reactive oxygen species, amino acids, anions, and cations will not cause changes in the fluorescence signal of RhB-ClO, nor will they interfere with the response to HClO and ONOO – . Therefore, the probe RhB-ClO can achieve highly selective detection of HClO and ONOO – .

[0024] Example 5 Application of the fluorescent probe RhB-ClO in monitoring the changes in the levels of HClO and ONOO during the oxidative stress process – Macrophage RAW264.7 cells were selected for confocal microscopy imaging, and 2-Me (dimethoxyestradiol) was used to induce RAW264.7 to establish an oxidative stress model. The specific experimental process is as follows: (1) Cells were incubated with RhB-ClO (10 μM) for 30 minutes. (2) Cells were incubated with 2-Me (2 μg / mL) for 2 hours, and then incubated with RhB-ClO (10 μM) for 30 minutes. (3) After incubating cells with 2-Me, the cells were incubated with ONOO – The cells were incubated with the scavenger UA (0.5 mM), the myeloperoxidase inhibitor ABH (1 mM), and the reactive oxygen species inhibitor NAC (0.5 mM) for 2 h, and finally incubated with RhB-ClO (10 μM) for 30 min. After the incubation period, the culture medium was discarded, the cells were washed with PBS, and the changes in fluorescence signal were monitored using a confocal fluorescence microscope. The experimental results are shown in Figure 2. Figure 7 As shown. The probe RhB-ClO alone showed only a weak fluorescence signal after incubation of cells ( Figure 7 A) After incubation with 2-Me, the red and green fluorescence signals of cells were significantly enhanced ( Figure 7 B, C). When the inhibitors UA, ABH and NAC were added after incubation with 2-Me, the fluorescence signal was significantly weakened ( Figure 7 D, E). The above experimental results show that the probe provided by the present invention can monitor the process of HClO and ONOO in the oxidative stress model. – Changes in levels.

[0025] Example 6 Fluorescent probe RhB-ClO monitoring of HClO and ONOO in a 6-hydroxydopamine (6-OHDA)-induced PC12 cell nerve injury model – Application of level changes Confocal microscopy was performed on the neuronal cell line PC12. A neuronal injury model was established by inducing 6-hydroxydopamine (6-OHDA). PC12 cells were incubated with different concentrations of 6-OHDA (100, 150 μM) for 24 h, and then the probe RhB-ClO (10 μM) was added for another 30 min. For the inhibitor group, cells were incubated with 6-OHDA (150 μM) for 24 h, and then ONOO – The cells were incubated with the scavenger UA (0.5 mM), the myeloperoxidase inhibitor ABH (1 mM), and the reactive oxygen species inhibitor NAC (0.5 mM) for 2 h, and then incubated with the probe RhB-ClO (10 μM) for 0.5 h. The culture medium was then discarded, the cells were washed with PBS buffer solution, and imaged using a confocal microscope. The experimental results are shown in Figure 2. Figure 8 As shown in Figure 2, as the concentration of 6-OHDA gradually increased, the fluorescence intensity of the red and green channels gradually increased ( Figure 8B, C), after adding the inhibitor, its red and green fluorescence signals were significantly weakened ( Figure 8 D, E, and F). The above results indicate that the probe provided by the present invention can monitor the changes in the levels of HClO and ONOO – in the 6-OHDA-induced nerve injury model.

[0026] Example 7 Application of the fluorescence probe RhB-ClO in monitoring the changes in the levels of HClO and ONOO – in the drug-induced liver injury model Hepatocarcinoma cells HepG2 were selected for confocal microscopy imaging to construct a liver injury model by incubating HepG2 with acetaminophen (APAP). HepG2 cells were incubated with different concentrations of APAP (100, 500, 1000 μM) for 8 h, and then the probe RhB-ClO (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 ONOO – scavenger UA (0.5 mM), myeloperoxidase inhibitor ABH (1 mM), and reactive oxygen species inhibitor NAC (0.5 mM) were added and the cells were incubated for 2 h, and then the probe RhB-ClO (10 μM) was used to incubate the cells for 0.5 h. Subsequently, the medium was discarded, the cells were washed with PBS buffer solution, and confocal microscopy imaging was performed. The experimental results are shown in Figure 11. As the concentration of APAP gradually increased, the fluorescence intensities of the red and green channels gradually increased ( Figure 9 B-D), after adding the inhibitor, its red and green fluorescence signals were significantly weakened ( Figure 9 E-J). The above results indicate that the probe provided by the present invention can monitor the changes in the levels of HClO and ONOO – in the drug-induced liver injury model.

[0027] Example 8 Screening of antioxidant drugs in nerve injury and liver injury using fluorescence probes The screening objects include: (1) natural flavonoid drugs, including: 3-hydroxyflavone, 5-hydroxyflavone, 6-hydroxyflavone, 3,7-dihydroxyflavone, 3,5,7-trihydroxyflavone, 3,5,7-trihydroxy-4'-methoxyflavone, 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, 2',3,4',5,7-pentahydroxyflavone, 5,7-dihydroxyflavanone; (2) natural products, including: luteolin, tea polyphenols, camptothecin, piperlongumine, curcumin, resveratrol, 6-gingerol, dehydro-6-gingerol; (3) commercially available drug molecules, including: clofibrate, indomethacin, fenofibrate, carprofen, tiopronin. Incubate the cells with the medium containing various drugs (100 μM) for 12 h, then incubate the cells with the medium containing 6-OHDA (150 μM) for 24 h, and finally incubate the cells with the medium containing RhB-ClO (10 μM) for 30 min. Aspirate the medium and wash 3 times with PBS. Add 2 mL of PBS buffer solution and perform fluorescence imaging after light-shielding treatment. The fluorescence imaging images are obtained by a Leica DM 400B fluorescence microscope (100-fold magnification). The screening results are as Figure 10 shown. In the 6-OHDA-induced nerve injury model, luteolin showed the most significant antioxidant effect, and at the same time, piperlongumine and 6-gingerol also showed good effects. These screening results provide important experimental basis for the development of potential antioxidant drugs for nerve injury and drug-induced liver injury.

Claims

1. Rhodamine-based Fluorescent Probe for Simultaneous Detection of HClO and ONOO – The structural formula of the fluorescent probe compound is as follows: 。 2. The synthesis method of the fluorescent probe compound as described in claim 1 includes the following steps: (1) At 0 - 5 °C, methyl 4-hydroxycinnamate and imidazole are slowly added to DMF, and then tert-butyldimethylchlorosilane (TBSCl) is added. After the reaction system is stirred in an ice bath for 0 - 20 min, it is then reacted at room temperature for 1 - 3 h; after the reaction is completed, water is added to quench the reaction, extracted with ethyl acetate, washed with saturated NaCl solution, dried over anhydrous Na2SO4, concentrated under vacuum, and purified by silica gel column chromatography to obtain compound 1; The structural formula of compound 1 is ； (2) Under argon atmosphere, compound 1 is dissolved in dichloromethane, and diisobutylaluminum hydride (DIBAL-H) is slowly added at -70 - 80 °C. After reacting for 20 - 40 min, it is transferred to room temperature for reaction for 0.5 - 2 h; after the reaction is completed, the system is cooled to -1 - 2 °C and water is slowly added to quench the reaction. It is filtered through diatomaceous earth, the filtrate is extracted with dichloromethane, dried over anhydrous Na2SO4, concentrated under vacuum, and compound 2 is obtained by silica gel column chromatography; The structural formula of compound 2 is ； (3) MnO2 is slowly added to the dichloromethane solution dissolving compound 2, and stirred at room temperature for 20 - 32 h. After the reaction is completed, it is filtered through diatomaceous earth, the filtrate is extracted with dichloromethane, the organic phase is obtained and dried over anhydrous Na2SO4, concentrated under vacuum, and the crude product is purified by silica gel column chromatography to obtain compound 3; The structural formula of compound 3 is ； (4) Compound 3 and tetrabutylammonium fluoride (TBAF) are dissolved in a DMF solution and stirred at room temperature for 0 - 2 h. After the reaction is completed, water is added to quench the reaction, extracted with ethyl acetate, dried over anhydrous Na2SO4, concentrated under vacuum, and compound 4 is obtained by silica gel column chromatography; The structural formula of compound 4 is ； (5) At 0 - 5 °C, cyclohexanone is slowly added dropwise to concentrated sulfuric acid, and then 2-(4-(diethylamino)-2-hydroxybenzoyl)benzoic acid is added to this system. The reaction mixture is reacted at 80 - 100 °C for 1 - 3 h. After the reaction is completed, the reaction is cooled to room temperature and then poured into ice water, and perchloric acid is added. After filtration, compound 5 is obtained; The structural formula of compound 5 is ； (6) Under nitrogen atmosphere, compound 5 and compound 4 are dissolved in acetic acid and refluxed and heated at 80 - 100 °C for 5 - 7 h. After the reaction is completed, it is cooled to room temperature, concentrated under vacuum, and the blue-violet solid compound RhB-OH is obtained by silica gel column chromatography separation; The structural formula of compound RhB-OH is ； (7) Compound RhB-OH and triethylenediamine (DABCO) are dissolved in dichloromethane and stirred at room temperature for 5 - 10 min, then dimethylaminothiocarbonate is added, and stirred at room temperature for 20 - 32 h. After the reaction is completed, water is added to quench the reaction, a solid precipitates, and the light purple solid compound RhB-ClO is obtained by filtration and washing; The structural formula of compound RhB-ClO is 。 3. The synthesis method according to claim 2, characterized in that, In step (1), the molar ratio of methyl 4-hydroxycinnamate to imidazole is 1:1 - 1:2; the molar ratio of methyl 4-hydroxycinnamate to TBSCl is 1:1 - 1:

2.

4. The synthesis method according to claim 2, wherein In step (2), the molar ratio of compound 1 to DIBAL-H is 1:5 to 1:6; in step (3), the molar ratio of compound 2 to MnO2 is 1:15 to 1:16; in step (4), the molar ratio of compound 3 to TBAF is 1:1 to 1:

3.

5. The synthesis method according to claim 2, characterized in that, In step (5), the molar ratio of 2-(4-(diethylamino)-2-hydroxybenzoyl)benzoic acid to cyclohexanone is 1:1 to 1:2; in step (6), the molar ratio of compound 5 to compound 4 is 1:1 to 1:2; in step (7), the molar ratio of compound RhB-OH to DABCO is 1:2 to 1:4; the molar ratio of compound RhB-OH to dimethylaminothiocarbonate is 1:2 to 1:

4.

6. Use of the fluorescent probe compound according to claim 1 for detecting HClO and ONOO – for non-diagnostic or therapeutic purposes.

7. The application according to claim 6, characterized in that: The fluorescent probe compound was separately mixed with HClO, reactive oxygen species (H2O2, t BuOOH, 1 O2, O2 -• , • OH, ONOO – ), amino acids (Cys, GSH, Ser, His, Lys, Pro, Leu, Phe), anions (HSO3 - , NO2 - , SO4 2- , PO4 3- , OAc - , NO3 - ), cations (K + , Na + , Cu 2+ , Zn 2+ ). Only in the presence of HClO can the probe compound produce strong fluorescence emission at 700 nm. In the system that has responded to HClO, reactive oxygen species (H2O2, t BuOOH, 1 O2, O2 -• , • OH, ONOO – ), amino acids (Cys, GSH, Ser, His, Lys, Pro, Leu, Phe), anions (HSO3 - , NO2 - , SO4 2- , PO4 3- , OAc - , NO3 - ), cations (K + , Na + , Cu 2+ , Zn 2+ ) were added respectively. Only the addition of ONOO – can make the probe produce strong fluorescence emission at 570 nm.

8. Use of the fluorescent probe compound according to claim 1 for monitoring the changes in the levels of HClO and ONOO – during the processes of oxidative stress, nerve injury, liver injury, and ferroptosis for non-diagnostic or therapeutic purposes.

9. Use of the fluorescent probe compound according to claim 1 in screening potential candidate drugs in the process of nerve injury and liver injury.

10. The application according to claim 9, wherein: Luteolin, piperlongumine, and 6-gingerol as candidate antioxidant drugs.

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