Preparation method and application of hypobromous acid ratiometric fluorescent probe capable of targeting mitochondria

Abstract

The invention relates to a preparation method and application of a hypobromous acid ratiometric fluorescent probe capable of targeting mitochondria. The probe can selectively and quickly react with HOBr through a specifically designed molecular structure, and generates ratio type fluorescence signal change, so that high-sensitivity detection of endogenous / exogenous HOBr of mitochondria in cells is realized. The long emission wavelength (gt; 600nm), low cytotoxicity and excellent co-localization performance (the mitochondrial localization coefficient is greater than or equal to 0.97), and is suitable for dynamic imaging analysis of living cells. The invention also provides a simple synthesis method of the probe.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probes, specifically relating to a hypobromite ratio fluorescent probe that can target mitochondria and its application in measuring, screening or detecting hypobromite; this invention also provides a method for preparing the fluorescent probe. Background Technology

[0002] Hypobromic acid (HOBr) is a reactive oxygen species (ROS) that is catalyzed in vivo by myeloperoxidase or eosinophil peroxidase. - It is produced by reacting with hydrogen peroxide (H2O2). Appropriate amounts of HOBr play an important role in antibacterial and immune defense functions. However, excessively high concentrations of HOBr in the body may cause inflammation, cardiovascular disease, and even cancer. Therefore, monitoring changes in HOBr concentration in living cells can further reveal its physiological and pathological functions.

[0003] Organic small molecule fluorescent probes have attracted much attention due to their advantages such as tunable structure, simple operation, high sensitivity, real-time in-situ detection, and application in bioimaging. Compared with other ROSs, HOBr has received less attention, and research on its detection has been relatively slow. Although HOBr has similar properties to HOCl, its concentration in the human body is significantly lower than that of HOCl, making detection more challenging. Compared to HOCl, there are limited fluorescent probes available for detecting HOBr; therefore, constructing novel HOBr fluorescent probes and applying them to monitor changes in intracellular HOBr concentration is of great significance.

[0004] Currently, several HOBr fluorescent probes have been developed, mainly based on the following mechanisms: (1) oxidation of HOBr; (2) substitution reaction induced by HOBr; and (3) cyclization reaction between amino and methylthio groups induced by HOBr. Due to the limited detection mechanisms and the large number of groups requiring protection during preparation, most of the reported probes exhibit problems such as fluorescence quenching, low sensitivity, poor stability, and complex synthesis, making it difficult to meet the needs of practical applications. In addition, ratiometric fluorescent probes can effectively eliminate interference from factors such as their own concentration, microenvironment, and excitation intensity through self-calibration, making them more suitable for accurate quantitative detection of target substances in complex biological systems. Therefore, developing ratiometric fluorescent probes for the detection of HOBr is more meaningful.

[0005] Currently, to study the role of HOBr in subcellular organelles, several HOBr fluorescent probes targeting mitochondria, lysosomes, endoplasmic reticulum, and Golgi apparatus have been developed. However, most probes exhibit fluorescence enhancement or quenching, with ratiometric fluorescence being relatively rare. Mitochondria are the main organelle producing ROSs, but currently only one ratiometric HOBr fluorescent probe targeting mitochondria exists, and it suffers from short excitation and emission peak wavelengths. Therefore, achieving efficient, specific, and ratiometric detection of HOBr in mitochondria remains a major challenge in current research. Designing a mitochondrial-targeting HOBr fluorescent probe that is easy to synthesize, has a longer wavelength, and exhibits ratiometric response characteristics is of significant theoretical and applied value for revealing the relationship between fluctuations in mitochondrial HOBr concentration and related diseases. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a ratiometric fluorescent probe for HOBr that can target mitochondria, as well as the preparation method and application of the probe. It has the characteristics of simple synthesis, long wavelength, good specificity, high sensitivity and rapid response, and can be applied to ratiometric fluorescence imaging analysis of endogenous and exogenous HOBr in cellular mitochondria.

[0007] Specifically, the present invention provides a compound, said compound being the compound shown in formula (I):

[0008]

[0009] This invention also provides a ratiometric fluorescent probe that can target mitochondria, wherein the fluorescent probe is a compound of formula (I):

[0010]

[0011] The present invention also provides a method for preparing the compound of formula (I), comprising the following reaction steps:

[0012] Step (1): Compound (II) is reacted with compound (III) to prepare compound (IV), and the reaction formula is as follows:

[0014]

[0015] Step (2): Compound (IV) is reacted with compound (V) to prepare compound (I), and the reaction formula is as follows:

[0016]

[0017] In some specific embodiments of the present invention, the specific preparation steps are as follows:

[0018] Step (1): Compound (II) and compound (III) were heated under nitrogen protection in ethanol and refluxed. After the reaction was completed, a solid crude product was obtained by vacuum distillation. The solid crude product was dissolved in dichloromethane and further separated by column chromatography. A mixture of dichloromethane and methanol was used as the eluent to obtain a pure compound (IV).

[0019] Step (2): Compound (IV) and compound (V), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex and potassium carbonate were refluxed in a tetrahydrofuran and water system under anaerobic and nitrogen protection conditions. After the reaction was completed, an appropriate amount of water was added, and the mixture was extracted with dichloromethane. The product was dried with anhydrous sodium sulfate and the dichloromethane was removed by vacuum distillation to obtain a crude product containing compound (I). The crude product was further separated by column chromatography using a mixture of dichloromethane and methanol as the eluent to obtain a pure compound (I).

[0020] In some specific embodiments of the present invention, the compound of formula (II) and the compound of formula (III) are placed in anhydrous ethanol solution and stirred at 82°C for 10-12 h under nitrogen protection; when using column chromatography to separate the crude product of compound (IV), the volume ratio of dichloromethane and methanol as eluents is 25:1.

[0021] In some specific embodiments of the present invention, the compound of formula (IV) and the compound of formula (V) are refluxed in a tetrahydrofuran (THF) and water system (V / V = 3:1) under anaerobic and nitrogen protection conditions, and stirred at 80°C for 8 hours; when using column chromatography to separate the crude product of compound (I), the volume ratio of dichloromethane and methanol as eluents is 40:1.

[0022] In some specific embodiments of the present invention, the molar ratio of compound (II) to compound (III) is 1.2:1 to 1.5:1.

[0023] In some specific embodiments of the present invention, the molar ratio of compound (IV) to compound (V) is 1:1.2 to 1:1.5.

[0024] The present invention also provides a fluorescent probe composition for measuring, screening or detecting hypobromic acid, comprising a compound of formula (I).

[0025] In some specific embodiments of the present invention, the fluorescent probe composition further comprises a solvent, an acid, a base, a buffer solution, or a combination thereof.

[0026] The present invention also provides the application of the compound of formula (I) in the preparation of reagents for measuring, screening or detecting hypobromic acid.

[0027] The present invention also provides a method for detecting the presence of hypobromic acid in a sample or determining the hypobromic acid content in a sample, comprising the following steps:

[0028] a) Contact the fluorescent probe of formula (I) with the sample to form a mixture with changing fluorescence;

[0029] b) Determine the fluorescence properties of the mixture of the fluorescence changes.

[0030] In some specific embodiments of the present invention, the sample is a chemical sample.

[0031] In some specific embodiments of the present invention, the sample is a biological sample.

[0032] In some specific embodiments of the present invention, the sample is an aquatic environment sample.

[0033] The present invention also provides the use of the compound of formula (I) or the above-described fluorescent probe composition in the preparation of reagents for measuring, screening or detecting hypobromonic acid.

[0034] This invention also provides the application of the compound of formula (I) in cell fluorescence imaging.

[0035] The present invention has the following significant advantages and effects compared with the prior art:

[0036] 1. Ratio-based quantitative detection of hypobromoic acid: The fluorescent probe provided by this invention can react with hypobromoic acid to produce obvious changes in fluorescence spectrum. Moreover, the ratio of its fluorescence intensity at 610 nm and 560 nm has a good linear relationship with the concentration of hypobromoic acid, thereby achieving accurate quantitative detection of hypobromoic acid.

[0037] 2. High selectivity recognition: The fluorescent probe provided by this invention can specifically react with hypobromic acid and does not react with other reactive oxygen species (e.g., hydrogen peroxide, hypochlorous acid, nitrite peroxide, tert-butyl hydrogen peroxide, singlet oxygen, hydroxyl radicals, etc.), thiols (glutathione, cysteine, homocysteine), cations (potassium ions, sodium ions), and anions (nitro, nitroso), ensuring the accuracy and reliability of detection.

[0038] 3. Fast reaction speed and high sensitivity: The fluorescent probe provided by this invention reacts with hypobromoic acid quickly (less than 1 minute) and has high sensitivity (detection limit of 200 nM), which can quickly respond to and capture the presence of hypobromoic acid, which is beneficial for timely detection and analysis.

[0039] 4. Good stability: The fluorescent probe provided by this invention has good stability, can be stored and used for a long time, and is not easily degraded by changes in external conditions, thus ensuring the stability and reliability of detection.

[0040] 5. Simple synthesis, conducive to commercialization: The fluorescent probe provided by this invention has a simple synthesis process, is easy to mass-produce and commercialize, reduces production costs and improves market competitiveness.

[0041] 6. The fluorescent probe provided by this invention can accurately target mitochondria in cells, and its colocalization coefficient with commercial mitochondrial fluorescent dyes is 0.97.

[0042] In summary, the hypobromic acid fluorescent probe provided by this invention has multiple beneficial technical effects, including quantitative detection, high selectivity recognition, high reaction sensitivity, good stability, simple synthesis, and the ability to target intracellular mitochondria, providing an effective tool and method for the detection and analysis of hypobromic acid. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 For compounds of formula (IV) 1 H-NMR spectrum;

[0045] Figure 2 For the fluorescent probe of formula (I) 1 H-NMR spectrum;

[0046] Figure 3 Here is the high-resolution mass spectrum of the fluorescent probe of formula (I);

[0047] Figure 4 The response time spectra of hypobromic acid fluorescent probe (10 μM) before and after the addition of hypobromic acid (50 μM);

[0048] Figure 5 Fluorescence spectra of hypobromic acid (0-50 μM) before and after adding hypobromic acid to the hypobromic acid fluorescent probe (10 μM);

[0049] Figure 6 The graph shows the linear relationship between the ratio of fluorescence intensity of hypobromic acid fluorescent probe (10 μM) at 610 nm and 560 nm and hypobromic acid (5-25 μM).

[0050] Figure 7 This is a selectivity graph for the hypobromic acid fluorescent probe; the bar chart represents the ratio of the fluorescence intensity of the probe at 610 nm and 560 nm in the presence of different analytes.

[0051] Figure 8The cytotoxicity diagram of the fluorescent probe of formula (I) is shown.

[0052] Figure 9 Colocalization fluorescence imaging of the formula (I) fluorescent probe and the commercial mitochondrial red probe Mito-Tracker Red CMXROS;

[0053] Figure 10 Fluorescence imaging of the fluorescent probe of formula (I) for exogenous hypobromite in cellular mitochondria;

[0054] Figure 11 Fluorescence imaging of the fluorescent probe of formula (I) for endogenous hypobromite in cellular mitochondria; Detailed Implementation

[0055] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0056] Example 1: Synthesis of compound (IV)

[0057] The synthetic design route is as follows:

[0058]

[0059] Implementation Plan 1:

[0060] 4-Bromo-5-amino-1,8-naphthalene anhydride (175 mg, 0.60 mmol) and 3-aminopropyl(triphenyl)phosphine bromide (200 mg, 0.5 mmol) were dissolved in 15 mL of anhydrous ethanol. The solution was refluxed at 82 °C for 10 hours under nitrogen protection. After cooling to room temperature, the ethanol was removed by vacuum distillation to obtain the crude product of compound (IV). The crude product was separated by column chromatography using a mixture of dichloromethane and methanol as eluent (V / V = 25:1) to obtain 269 mg of pure compound (IV), with a yield of 80%.

[0061] Implementation Plan 2:

[0062] 4-Bromo-5-amino-1,8-naphthalene anhydride (218 mg, 0.75 mmol) and 3-aminopropyl(triphenyl)phosphine bromide (200 mg, 0.5 mmol) were dissolved in 15 mL of anhydrous ethanol. The solution was refluxed at 82 °C for 12 hours under nitrogen protection. After cooling to room temperature, the ethanol was removed by vacuum distillation to obtain the crude product of formula (IV). The crude product was separated by column chromatography using a mixture of dichloromethane and methanol as eluent (V / V = 25:1) to obtain 279 mg of pure formula (IV) compound, with a yield of 83%.

[0063] The structural characterization data of the compounds of formula (IV) prepared according to implementation schemes 1 and 2 are as follows (the proton NMR spectra are attached). Figure 1 ): 1 HNMR (400MHz, DMSO-d6): δ (ppm): 8.24 (d, J=8.0Hz, 1H), 8.15 (d, J=8.0Hz, 1H), 8.12 (s, 1H), 7.91-7.88 (m, 3H), 7.80-7.78(m,5H),7.77-7.75(m,8H),6.37(s,2H),4.20(t,J=8.0Hz,2H),3.80-3.72(m,2H),2.00-1.96(m,2H).

[0064] Example 2: Synthesis of Formula (I) fluorescent probe

[0065] The synthetic design route is as follows:

[0066]

[0067] Implementation Plan 1:

[0068] Compound (IV) (102 mg, 0.15 mmol), 2-methylthiophenylboronic acid (30.2 mg, 0.18 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex (30 mg, 0.037 mmol), and potassium carbonate (209 mg, 1.5 mmol) were dissolved in a mixture of tetrahydrofuran and water (8 mL, V / V = 3:1). The reaction was carried out under anaerobic, nitrogen-protected conditions at 80 °C for 8 hours under reflux. After cooling to room temperature, 50 mL of water was added, followed by three fractions of extraction with 20 mL of dichloromethane. The extracts were collected, dried over anhydrous sodium sulfate, and rotary evaporated to obtain the crude product of formula (I). The crude product was separated by column chromatography using a mixture of dichloromethane and methanol as the eluent (V / V = 40:1), yielding 42.9 mg of pure compound (I) in a yield of 40%.

[0069] Implementation Plan 2:

[0070] Compound (IV) (84.3 mg, 0.13 mmol), 2-methylthiophenylboronic acid (31.6 mg, 0.19 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex (25 mg, 0.03 mmol), and potassium carbonate (173 mg, 1.25 mmol) were dissolved in a mixture of tetrahydrofuran and water (8 mL, V / V = 3:1). The reaction was carried out under anaerobic and nitrogen protection at 80 °C for 8 hours under reflux. After the reaction was cooled to room temperature, 50 mL of water was added, and the mixture was extracted three times with 20 mL of dichloromethane each time. The extracts were collected and dried with anhydrous sodium sulfate. The solution was then rotary evaporated to obtain the crude product of formula (I).

[0071] The crude product was separated by column chromatography using a mixture of dichloromethane and methanol as eluent (V / V = 40:1) to obtain 38.3 mg of pure compound (I), with a yield of 41%.

[0072] The structural characterization data of the compounds of formula (I) prepared according to implementation schemes 1 and 2 are as follows (the proton NMR spectra are attached). Figure 2 The high-resolution mass spectrum is attached. Figure 3 ): 1 H NMR (400MHz, DMSO-d6): δ (ppm): 8.13 (s, 1H), 8.08-8.06 (m, 1H), 7.82-7.80 (m, 1H), 7.80-7.76 (m, 4H), 7.57-7.46 (m, 13H), 7.37- 7.35(m,1H),7.25-7.22(m,1H),7.14-7.12(m,1H),5.29(s,2H),4.13(t,J=8.0Hz,2H),2.33(s,3H),1.85-1.79(m,2H).ESI-HRMS calcd for C 40 H 34 N2O2SP[M] + ,637.2079; found,637.2062;

[0073] Example 3: Testing the time dynamics of a fluorescent probe

[0074] A 10 mL test system with a fluorescent probe concentration of 10 μM was prepared. Then, 50 μM hypobromochloric acid was added to the test system, and after shaking thoroughly, the fluorescence intensity change was immediately measured using a fluorescence spectrometer. The above determination was performed in an acetonitrile:water (V / V = 2:3, containing 10 mM PBS, pH 7.4) system. The probe used was the one prepared in Example 2, and the fluorescence spectrum was measured at 25°C. Unless otherwise specified, all spectral data are within the range of λ.ex Data was acquired at 470nm, slit size: W ex =W em =10nm.

[0075] Test results are as follows Figure 4 As shown, by Figure 4 It can be clearly seen that when hypobromous acid is added, the ratio of the fluorescence intensity of the probe at 610 nm and 560 nm reaches its maximum value within 40 seconds and remains unchanged. This indicates that the probe reacts rapidly with hypobromous acid and can provide a rapid analytical method for the determination of hypobromous acid.

[0076] Example 4: Testing the fluorescence spectra of a fluorescent probe for different concentration gradients of hypobromic acid

[0077] Multiple parallel samples with a probe concentration of 10 μM were prepared in 10 mL colorimetric tubes. Then, hypobromic acid of different concentrations (0-50 μM) was added to the test system. After shaking and mixing, the mixture was allowed to stand for 10 minutes. The ratio of fluorescence intensity at 610 nm to 560 nm was measured using a fluorescence spectrometer. The above determinations were performed in an acetonitrile:water (V / V = 2:3, containing 10 mM PBS, pH 7.4) system. The probe used was the one prepared in Example 2, and all spectral measurements were taken at 25 °C. The test results are as follows: Figure 5 and 6 As shown.

[0078] from Figure 5 It can be clearly seen that as the concentration of hypobromic acid increases, the fluorescence intensity at 610 nm gradually increases, while the fluorescence at 560 nm gradually decreases. Furthermore, from... Figure 6 As can be seen, after the probe (10 μM) is added to hypobromic acid (5-25 μM), the ratio of fluorescence intensity at 610 nm and 560 nm shows a good linear relationship with the concentration of hypobromic acid, which proves that this fluorescent probe can be used to quantitatively analyze hypobromic acid.

[0079] Example 5: Selectivity test of fluorescent probe for hypobromic acid

[0080] The analytes were: blank, hypochlorous acid, hydrogen peroxide, tert-butyl hydrogen peroxide, singlet oxygen, nitrite peroxide, hydroxyl radical, tert-butyl radical, glutathione (5 mM), cysteine ​​(200 μM), homocysteine ​​(200 μM), nitric oxide, nitrate ion, nitrite ion, and hypobromic acid (50 μM) (unless otherwise specified, all other analyte concentrations were 100 μM). The bar chart represents the ratio of the fluorescence intensity of the probe at 610 nm and 560 nm in the presence of different analytes. The above measurements were performed in an acetonitrile:water (V / V = 2:3, containing 10 mM PBS, pH 7.4) system. The probe used was the probe prepared in Example 2, and all spectral measurements were taken at 25 °C. Specifically, multiple parallel samples with a probe concentration of 10 μM were prepared in 10 mL colorimetric tubes, then a certain amount of analyte was added, shaken well, and allowed to stand for 10 minutes before measuring the fluorescence intensity. The results are as follows Figure 7 As shown.

[0081] from Figure 7 It can be seen that the probe of the present invention has high selectivity for hypobromoic acid and can react specifically with hypobromoic acid.

[0082] Example 6: Cytotoxicity test of fluorescent probe

[0083] The cytotoxicity of different concentrations of the probe to md-231 cells was detected using a cell counting kit (CCK-8). The probe concentrations were 0 μM, 1 μM, 5 μM, 10 μM, 15 μM, and 20 μM, and the cells were incubated with the probe for 24 h.

[0084] from Figure 8 It is clear that the probe has low toxicity and can be used for long-term real-time detection of hypobromic acid in cell samples.

[0085] Example 7: Test of the mitochondrial target capability of fluorescent probes

[0086] After co-incubating md-231 cells with the fluorescent probe of formula (I) (5 μM) and the commercial mitochondrial red probe Mito-Tracker Red CMXROS (1 μM) for 15 minutes, the cells were washed three times with PBS, and then imaged under a fluorescence microscope after adding 2 mL of PBS solution. The excitation wavelength of the fluorescent probe of formula (I) was 425 nm, and the acquisition wavelength was 500–570 nm. The excitation wavelength of Mito-Tracker Red CMXROS was 579 nm, and the acquisition wavelength was 585–600 nm.

[0087] from Figure 9It can be clearly seen that the colocalization coefficient of the formula (I) probe and Mito-Tracker Red CMXROS is very high, reaching 0.97, which proves that it can effectively target intracellular mitochondria.

[0088] Example 8: Fluorescence microscopy imaging of exogenous HOBr in MD-231 cells using fluorescent probes

[0089] MD-231 cells were divided into four groups: Group A served as the control group, incubated with the probe (10 μM) for 15 min; Group B incubated with HOBr (20 μM) for 20 min in addition to the treatment of Group A cells; Group C was incubated with NaBr (100 μM) for 30 min, followed by incubation with the probe (10 μM) for 15 min; and Group D was incubated with NAC (200 μM, a ROS scavenger that can eliminate HOBr produced by cells) and NABr (100 μM) for 30 min, followed by incubation with the probe (10 μM) for 15 min. Confocal microscopy was then performed on all four groups of cells. Figure (e) represents a bar graph showing the ratio of fluorescence intensity in the red and green channels. The excitation light was 450 nm, with the red channel collected at 600-700 nm and the green channel at 525-575 nm.

[0090] from Figure 10 It can be clearly seen that this probe can detect exogenous hypobromonic acid in the mitochondria of MD-231 cells.

[0091] Example 9: Fluorescence microscopy imaging of endogenous HOBr in MD-231 cells using fluorescent probes

[0092] MD-231 cells were divided into four groups: Group A served as the control group, incubated with a probe (10 μM) for 15 min; Group B, in addition to the treatment of Group A cells, was pre-incubated with NAC (200 μM) for 30 min; Group C was incubated with PMA (100 ng / mL, stimulating cell production of reactive oxygen species) for 2 h, followed by incubation with a probe (10 μM) for 15 min; Group d was incubated with PMA (100 ng / mL) for 2 h, followed by incubation with NABr (100 μM) for 30 min, and then incubated with a probe (10 μM) for 15 min. Finally, confocal microscopy was performed on the four groups of cells, where (e) represents a bar graph showing the ratio of fluorescence intensity in the red and green channels. The excitation light was 450 nm, with the red channel acquired at 600-700 nm and the green channel at 525-575 nm.

[0093] from Figure 11 It can be clearly seen that this probe can detect endogenous hypobromonic acid in the mitochondria of MD-231 cells. Experiments have demonstrated that this probe can be applied to the detection of hypobromonic acid in biological samples.

Claims

1. A compound having the structure shown in formula (I):

2. A method for preparing the compound of claim 1, characterized in that, The process includes the following steps: reacting compound (II) with compound (III) to prepare compound (IV), and reacting compound (IV) with compound (V) to prepare compound (I), with the reaction formulas as follows:

3. The preparation method according to claim 2, characterized in that, Includes the following steps: Step (1): Compound (II) and compound (III) were heated under nitrogen protection in ethanol and refluxed. After the reaction was completed, a solid crude product was obtained by vacuum distillation. The solid crude product was dissolved in dichloromethane and further separated by column chromatography. A mixture of dichloromethane and methanol was used as the eluent to obtain a pure compound (IV). Step (2): Compound (IV) and compound (V), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex and potassium carbonate were refluxed in a tetrahydrofuran and water system under anaerobic and nitrogen protection conditions. After the reaction was completed, an appropriate amount of water was added, and the mixture was extracted with dichloromethane. The product was dried with anhydrous sodium sulfate and the dichloromethane was removed by vacuum distillation to obtain a crude product containing compound (I). The crude product was further separated by column chromatography using a mixture of dichloromethane and methanol as the eluent to obtain a pure compound (I).

4. The preparation method according to claim 3, characterized in that, The molar ratio of compound (II) to compound (III) is 1.2:1 to 1.5:1; the molar ratio of compound (IV) to compound (V) is 1:1.2 to 1:1.

5.

5. A fluorescent probe composition for measuring, detecting, or screening hypobromic acid, characterized in that, It includes the compound described in claim 1.

6. The fluorescent probe composition according to claim 5, characterized in that, The fluorescent probe composition further comprises a solvent, an acid, a base, a buffer solution, or a combination thereof.

7. A method for detecting the presence of hypobromic acid in a sample or determining the content of hypobromic acid in a sample, characterized in that, Includes the following steps: a) Contacting the compound of claim 1 with the sample to form a mixture with changing fluorescence; b) Determine the fluorescence properties of the mixture of the fluorescence changes.

8. The method as described in claim 7, characterized in that, The sample is a chemical sample, a biological sample, or a water environment sample.

9. The use of the compound of claim 1 or the fluorescent probe composition of any one of claims 5-6 in the preparation of reagents for measuring, screening or detecting hypobromic acid.

10. The use of the compound of claim 1 in cell fluorescence imaging.

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