High-sensitivity ratio type fluorescent probe for detecting hypochlorous acid as well as preparation method and application of high-sensitivity ratio type fluorescent probe

Abstract

The invention discloses a high-sensitivity ratio-type fluorescent probe for detecting hypochlorous acid as well as a preparation method and application of the high-sensitivity ratio-type fluorescent probe. The N '-(4'-(6, 6-dimethyl-4, 5, 6, 7-tetrahydro-1H-5, 7-bridge methylene indazole-3-yl)-[1, 1 '-biphenyl]-4-methylene) malononitrile prepared by using a natural renewable forest resource beta-pinene derivative nopinone as a raw material can selectively react with hypochlorous acid, and the fluorescent color of the N'-(4 '-(6, 6-dimethyl-4, 5, 6, 7-tetrahydro-1H-5, 7-bridge methylene indazole-3-yl)-[1, 1'-biphenyl]-4-methylene) malononitrile is changed from yellow to cyan, so that the effect of the N '-(4'-(6, 6-dimethyl-4, 5, 6, 7 the maximum fluorescence emission wavelength is changed from 460 nm to 375 nm, and the compound can be used as a specific ratio type fluorescent probe for detecting hypochlorous acid and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent probes, and particularly to a highly sensitive ratio-type fluorescent probe for detecting hypochlorous acid, a preparation method thereof, and an application thereof. Background Art

[0002] Hypochlorous acid (HClO), as an acidic compound with strong oxidizing properties, plays an important role in environmental disinfection, water purification, food processing, and biomedical fields. The accurate detection of its concentration is directly related to the evaluation of disinfection effects, water quality safety monitoring, and the diagnosis of oxidative stress-related diseases in organisms. Currently, the mainstream detection methods include chemical titration, colorimetry, electrochemistry, spectrophotometry, etc. However, these techniques generally have limitations such as complex operations, insufficient sensitivity, weak anti-interference ability, strong instrument dependence, and long detection time. Therefore, it is very important to develop a rapid, convenient, highly sensitive, and highly selective detection method for detecting hypochlorous acid.

[0003] In recent years, fluorescent probe technology has gradually become a research hotspot due to its high sensitivity, rapid response, and visual detection advantages. However, existing single-emission fluorescent probes still face problems such as low quantum yield, long synthesis steps, and susceptibility to interference by probe concentration and environmental factors. Ratio-type fluorescent probes achieve quantitative analysis by constructing a dual-emission system (reference signal and response signal), and have core advantages such as built-in calibration function, high sensitivity, visual detection, and wide dynamic range.

[0004] Currently reported patents on the synthesis research of fluorescent probes and their use for hypochlorous acid detection usually include fluorescent probes with luminescent structures such as rhodamine, BODIPY, hemicyanine, fluorescein, coumarin, etc. according to the different structures of the luminescent groups of the fluorescent probes. Most of these fluorescent probes require the participation of organic solvents to detect hypochlorous acid and are mostly quenching-type or enhancing-type fluorescent probes. Summary of the Invention

[0005] Aiming at the deficiencies in the prior art, the technical problems to be solved by the present invention are to provide a highly sensitive ratio-type fluorescent probe for detecting hypochlorous acid, which can specifically react with hypochlorous acid, and under ultraviolet light irradiation, the yellow fluorescence of its aqueous solution rapidly turns into cyan fluorescence, and can be used for qualitative and quantitative analysis of hypochlorous acid. Another technical problem to be solved by the present invention is to provide a preparation method of a highly sensitive ratio-type fluorescent probe for detecting hypochlorous acid. Another technical problem to be solved by the present invention is to provide an application of a highly sensitive fluorescent probe.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is: A highly sensitive ratiometric fluorescence probe for detecting hypochlorous acid, with the chemical name N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene) malononitrile, and the structural formula is: 。

[0007] The preparation method of this highly sensitive fluorescence probe has the following synthetic route: The specific synthesis includes the following technological steps: 1) Norpinone and methyl 4-bromobenzoate undergo a Claisen condensation reaction to obtain 3-(4-bromobenzoyl)norpinone (Compound I); 2) 3-(4-bromobenzoyl)norpinone and hydrazine hydrate undergo a cyclization reaction to obtain 3-(4-bromophenyl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazole (Compound II); 3) 3-(4-bromophenyl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazole and 4-formylphenylboronic acid undergo a coupling reaction to obtain 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde (Compound III); 4) 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde and malononitrile undergo a condensation reaction to obtain N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene) malononitrile.

[0008] In step 1), norpinone and methyl 4-bromobenzoate undergo a Claisen condensation to obtain Compound I. The specific preparation steps are as follows: (1) Add 3 mmol of norpinone, 2 - 6 mmol of methyl 4-bromobenzoate, 5 - 10 mmol of sodium hydride, and 40 - 80 mL of 1,2-dimethoxyethane into a clean and dry three-necked flask in sequence. Heat under reflux for 6 - 8 h under nitrogen protection, and monitor the reaction progress by TLC until the conversion rate of norpinone reaches over 96%; (2) After the reaction solution is cooled, add deionized water to quench the reaction, then extract with ethyl acetate three times. The combined organic phase is washed with saturated brine several times until neutral, dried over anhydrous sodium sulfate, filtered, concentrated, and the resulting solid product is washed with a small amount of ethyl acetate to obtain Compound I.

[0009] In step (2), under the catalysis of glacial acetic acid, compound I undergoes a cyclization reaction with hydrazine hydrate to obtain compound II. The specific preparation steps are as follows: (1) Add 0.5 mol of compound I, 1.5 - 2.0 mol of hydrazine hydrate, and 30 - 60 mL of ethanol to a three-necked flask, then add 1 - 2 drops of glacial acetic acid, heat under reflux for 3 - 6 h, and monitor the reaction progress by TLC until the conversion rate of compound I reaches 100%, then terminate the reaction; (2) After the reaction solution is concentrated to remove ethanol, add ethyl acetate and wash it several times with saturated brine until neutral; the organic phase is dried over anhydrous sodium sulfate, filtered, concentrated, and a white solid is precipitated. After recrystallization with ethanol and suction filtration and washing, compound II is obtained.

[0010] In step (3), using tetrakis(triphenylphosphine)palladium as a catalyst, compound II undergoes a coupling reaction with 4-formylphenylboronic acid to obtain compound III. The specific preparation steps are as follows: (1) Under nitrogen protection, add 5 mmol of compound II, 10 - 20 mmol of 4-formylphenylboronic acid, 0.2 - 0.3 mmol of tetrakis(triphenylphosphine)palladium, and 30 - 60 mL of 1,4-dioxane to a three-necked flask, then dissolve 20 - 30 mmol of potassium carbonate in 10 - 20 mL of deionized water and add it dropwise to the flask, and react at 100 °C for 12 - 24 h; (2) After the reaction solution is filtered to remove solid impurities, the filtrate is distilled under reduced pressure to remove the 1,4-dioxane solvent, add ethyl acetate, and wash it several times with saturated brine until neutral; the organic phase is dried over anhydrous sodium sulfate, filtered, and the solvent is evaporated to obtain a crude product containing compound III; (3) The crude product of compound III is separated by a silica gel column (200 - 300 mesh, eluent petroleum ether / ethyl acetate = 3 / 1, v / v) to obtain compound III.

[0011] In step (4), using piperidine as a catalyst, compound III undergoes a condensation reaction with malononitrile to obtain the final product N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile. The specific preparation steps are as follows: (1) Add 1 mmol of compound III, 1 - 3 mmol of malononitrile, 10 - 20 mL of absolute ethanol, and 1 - 3 drops of piperidine to a three-necked flask, reflux for 12 - 24 h, and monitor the reaction progress by TLC until the reaction of compound III is complete, then stop the reaction; (2)After concentrating the reaction solution to remove part of ethanol, it was cooled to precipitate solids, and the solids were filtered by suction and the filter cake was washed with cold ethanol to obtain the target compound N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile.

[0012] Application of N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile in detecting hypochlorous acid. It can selectively react with hypochlorous acid, and its aqueous solution changes from yellow fluorescence to cyan fluorescence under 365 nm ultraviolet light.

[0013] In this invention, using norpinone as the raw material, it reacts with methyl 4-bromobenzoate to generate 3-(4-bromobenzoyl)norpinone; 3-(4-bromobenzoyl)norpinone then undergoes a cyclization reaction with hydrazine hydrate to obtain 3-(4-bromophenyl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazole; 3-(4-bromophenyl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazole then undergoes a coupling reaction with 4-formylphenylboronic acid to obtain the compound 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde; 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde continues to condense with malononitrile to obtain N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile. In this case, it is found that this compound can specifically recognize hypochlorous acid and can be used as a ratiometric fluorescent probe for detecting hypochlorous acid.

[0014] Beneficial effects: Compared with the prior art, this invention utilizes the natural renewable resource β N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile prepared from the α-pinene derivative norpinone as the raw material can selectively react with hypochlorous acid and make its aqueous solution change from yellow fluorescence to cyan fluorescence, and the maximum fluorescence emission wavelength changes from 460 nm to 375 nm. Therefore, this compound can be used as a highly sensitive ratiometric fluorescent probe for specifically detecting hypochlorous acid. Description of the Drawings

[0015] Figure 11H NMR spectrum of N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile.

[0016] Figure 2 Emission fluorescence spectra of N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile after adding different analytes measured by fluorescence spectral titration analysis.

[0017] Figure 3 Emission fluorescence spectra of N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile after adding different concentrations of hypochlorous acid measured by fluorescence spectral titration analysis. Detailed implementation mode

[0018] The present invention will be further described below in conjunction with specific embodiments.

[0019] Example 1 Preparation of N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile 1) Preparation of 3-(4-bromobenzoyl)nopinone: 3 mmol of nopinone, 3.6 mmol of methyl 4-bromobenzoate, 9 mmol of sodium hydride and 60 mL of ethylene glycol dimethyl ether were successively added to a three-necked flask, and the reaction was heated under reflux under nitrogen protection. The reaction was carried out for about 8 h until the conversion rate of nopinone reached more than 96% (detected by TLC). After the reaction solution was cooled, deionized water was added to quench the reaction, and then extracted with ethyl acetate three times. The combined organic phases were washed with saturated brine several times until neutral, dried over anhydrous sodium sulfate, filtered, concentrated, and the resulting solid product was washed with a small amount of ethyl acetate to obtain 3-(4-bromobenzoyl)nopinone, with a yield of 64.2% and a purity of 99.3%. 1 H NMR (600 MHz, CDCl3) δ : 15.48 (s, 1H), 7.57 (s, 4H), 2.68–2.64 (m, 2H),2.59(t, J = 5.6 Hz, 1H), 2.57–2.53 (m, 1H), 2.30–2.27 (m, 1H), 1.45 (d, J= 9.9 Hz, 1H), 1.35 (s, 3H), 0.95 (s, 3H). 13 C NMR (150 MHz, CDCl3) δ : 209.53, 171.56, 134.21, 131.63, 129.91, 124.77, 104.19, 54.91, 39.94, 39.69, 28.39, 27.85, 25.93, 21.66.

[0020] 2) Preparation of 3-(4-bromophenyl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazole: 0.5 mol of 3-(4-bromobenzoyl)nopinone, 1.5 mol of hydrazine hydrate, and 30 mL of ethanol were added to a three-necked flask, and then 1 drop of glacial acetic acid was added. The mixture was heated under reflux for 5 h, and the reaction was monitored by TLC until the conversion rate of 3-(4-bromobenzoyl)nopinone reached 100%, and then the reaction was terminated. After the reaction solution was concentrated to remove ethanol, ethyl acetate was added, and it was washed several times with saturated brine until neutral; the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to precipitate a white solid. After recrystallization with ethanol and suction filtration and washing, 3-(4-bromophenyl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazole was obtained with a yield of 70.2% and a purity of 96.5%. 1 H NMR (600 MHz, CDCl3) δ : 7.50 (d, J = 2.4 Hz, 4H), 7.08 (s, 1H), 2.96–2.91 (m, 1H), 2.89–2.83 (m, 2H), 2.74–2.69 (m, 1H), 2.36–2.32 (m, 1H), 1.40 (s, 3H), 1.39 (s, 1H), 0.71 (s, 3H). 13 C NMR (150 MHz, CDCl3) δ : 158.61, 139.44, 132.04, 130.49, 127.31, 121.55, 109.88, 41.64, 41.49, 41.23, 32.49, 26.39, 26.19, 21.61.

[0021] 3) Preparation of 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde: 5 mmol of 3-(4-bromophenyl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazole, 10 mmol of 4-formylphenylboronic acid, 0.25 mmol of tetrakis(triphenylphosphine)palladium, and 40 mL of 1,4-dioxane were added to a three-necked flask under nitrogen protection, stirred and heated. Then, 20 mmol of potassium carbonate was dissolved in deionized water and added dropwise to the flask, and the reaction was carried out at 100 °C overnight. After the reaction solution was filtered to remove solid impurities, the filtrate was distilled under reduced pressure to remove the 1,4-dioxane solvent. Ethyl acetate was added, and the mixture was washed several times with saturated brine until neutral. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain the crude product of 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde. The crude product of 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde was subjected to column chromatography (silica gel 200 - 300 mesh, eluent petroleum ether / ethyl acetate ratio 3 / 1, v / v) to obtain 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde with a yield of 63.1% and a purity of 99.1%. 1 H NMR (400 MHz, CDCl3) δ : 10.05 (s, 1H), 7.94 (d, J = 8.1 Hz, 2H), 7.76 (t, J = 8.7 Hz, 4H), 7.66 (d, J = 8.3 Hz, 2H), 5.29 (s, 1H), 3.07–2.89 (m, 3H), 2.74 (d, J = 9.4 Hz, 1H), 2.37 (s, 1H), 1.45 (d, J = 9.6 Hz, 1H), 1.41 (s, 3H), 0.75 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ: 191.95, 159.04, 146.51, 139.50, 138.64, 135.40, 131.69, 130.46, 127.84, 127.51, 126.28, 110.20, 72.90, 60.53, 41.79, 41.55, 41.26, 32.55, 29.83, 26.44, 26.35, 21.64, 14.24。

[0022] 4) Preparation of N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile: Add 1 mmol of 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde, 1 mmol of malononitrile, 10 mL of absolute ethanol, and 3 drops of piperidine into a three-necked flask, and reflux for 12 - 24 h. Monitor the reaction progress by TLC until the reaction of 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde is complete, then stop the reaction. Concentrate the reaction solution to remove part of the ethanol, then cool to precipitate a solid, filter by suction and wash the filter cake with cold ethanol to obtain N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile, with a yield of 93.2% and a purity of 99.4%. 1 H NMR(600 MHz, DMSO- d 6) δ 12.55 (s, 1H), 8.56 (s, 1H), 8.06 (d, J = 8.5 Hz, 2H), 8.02 (d, J = 8.3 Hz, 2H), 7.87 (dd, 4H), 2.99 (dd, J = 15.7, 3.0 Hz, 1H), 2.88(dd, J = 15.6, 2.6 Hz, 1H), 2.80 (s, 1H), 2.71 (dt, J = 9.3, 5.8 Hz, 1H), 2.34 – 2.29 (m, 1H), 1.39 (s, 3H), 1.29 (d, J = 9.3 Hz, 1H), 0.66 (s, 3H). 1313C NMR (150MHz, DMSO- d 6) δ : 161.63, 159.24, 147.02, 141.64, 140.28, 130.73, 129.76, 127.91, 127.89, 127.49, 127.45, 113.93, 101.29, 82.81, 50.77, 47.16, 41.55, 30.31, 24.07, 22.03. Example 2 Dissolve N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile in DMF to prepare a solution with a concentration of 1 mM, and then dilute it to 10 μM with a mixed solution of PBS buffer: ethanol = 9:1 (pH = 7.4, 10 mM) for use. Dissolve different interfering substances (ClO − , HCO3 − , CO3 − , HSO3 − , SO3 2− , SO4 2− , NO3 − , Cl − , Br − , AcO − , ONOO − , O2 − , 1 O2, N2H4, Cys, Hcy, GSH) in deionized water to prepare a solution with a concentration of 1 mM and add it to the mixed solution containing the probe compound to make its concentration 10 μM. The fluorescence spectra of different interfering substances on N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile were measured by fluorescence spectrometry, as shown in Figure 2 shown. The results show that compared with other interfering substances, only hypochlorous acid can cause obvious changes in the fluorescence spectrum of the probe compound, indicating that the compound N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile can be used as a highly selective fluorescence probe for detecting hypochlorous acid.

[0023] Example 3 Dissolve N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile in DMF to prepare a solution with a concentration of 1 mM, and then dilute it to 10 μM with a mixed solution of PBS buffer:ethanol = 9:1 (pH = 7.4, 10 mM) for use. Similarly, dissolve hypochlorous acid in PBS buffer to prepare solutions with concentrations of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μM. The fluorescence spectra of N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile at different concentrations of hypochlorous acid were measured by fluorescence spectral titration analysis, as Figure 3 shown. The results show that as the concentration of added hypochlorous acid increases, the maximum fluorescence intensity of the probe solution at 460 nm gradually decreases, while the fluorescence intensity at 375 nm gradually increases, indicating that the compound N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene)malononitrile can be used as a highly sensitive fluorescence probe for detecting hypochlorous acid.

Claims

1. A highly sensitive ratiometric fluorescent probe for detecting hypochlorous acid, characterized in that, Its chemical name is N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-ylmethylene) malononitrile, and its structural formula is as follows: .

2. A preparation method of a highly sensitive ratio-type fluorescent probe for detecting hypochlorous acid as described in claim 1, characterized in that, It includes the following steps: 1) Perform a Claisen condensation reaction between norpinone and methyl 4-bromobenzoate to obtain 3-(4-bromobenzoyl)norpinone; 2) Perform a cyclization reaction between 3-(4-bromobenzoyl)norpinone and hydrazine hydrate to obtain 3-(4-bromophenyl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazole; 3) Perform a coupling reaction between 3-(4-bromophenyl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazole and 4-formylphenylboronic acid to obtain 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde; 4) Perform a condensation reaction between 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde and malononitrile to obtain N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-methylene)malononitrile.

3. The preparation method of the highly sensitive ratio-type fluorescent probe for detecting hypochlorous acid according to claim 2, wherein In step 1), the specific steps of the Claisen condensation reaction between norpinone and methyl 4-bromobenzoate are as follows: S1: Add 3 mmol of norpinone, 2 - 6 mmol of methyl 4-bromobenzoate, 5 - 10 mmol of sodium hydride, and 40 - 80 mL of 1,2-dimethoxyethane into a dry three-necked flask in sequence, heat under reflux for 6 - 8 h under nitrogen protection, and monitor the reaction progress by TLC until the conversion rate of norpinone reaches over 96%; S2: After the reaction solution is cooled, add deionized water to quench the reaction, then extract with ethyl acetate three times. The combined organic phases are washed with saturated brine until neutral, dried over anhydrous sodium sulfate, filtered, concentrated, and the resulting solid product is washed with ethyl acetate to obtain 3-(4-bromobenzoyl)norpinone.

4. The preparation method of the highly sensitive ratio-type fluorescence probe for detecting hypochlorous acid according to claim 2, wherein In step 2), the specific steps of the cyclization reaction between 3-(4-bromobenzoyl)norpinone and hydrazine hydrate are as follows: S1: Add 0.5 mol of compound I, 1.5 - 2.0 mol of hydrazine hydrate, and 30 - 60 mL of ethanol into a three-necked flask, then add 1 - 2 drops of glacial acetic acid, heat under reflux for 3 - 6 h, and monitor the reaction progress by TLC until the conversion rate of compound I reaches 100% and then terminate the reaction; S2: After the reaction solution is concentrated to remove ethanol, add ethyl acetate and wash with saturated brine several times until neutral. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated, and a white solid precipitates. After recrystallization with ethanol and suction filtration and washing, 3-(4-bromophenyl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazole is obtained.

5. The preparation method of the highly sensitive ratio fluorescent probe for detecting hypochlorous acid according to claim 2, wherein, In step 3), the specific steps of the coupling reaction between 3-(4-bromophenyl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazole and 4-formylphenylboronic acid are as follows: S1: Under nitrogen protection, 5 mmol of compound II, 10 - 20 mmol of 4-formylphenylboronic acid, 0.2 - 0.3 mmol of tetrakis(triphenylphosphine)palladium, and 30 - 60 mL of 1,4-dioxane were added to a three-necked flask. Then, 20 - 30 mmol of potassium carbonate dissolved in 10 - 20 mL of deionized water was added dropwise to the flask, and the reaction was carried out at 100 °C for 12 - 24 h; S2: After the reaction solution was filtered to remove solid impurities, the filtrate was distilled under reduced pressure to remove the 1,4-dioxane solvent. Ethyl acetate was added, and the mixture was washed with saturated brine several times until neutral. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain a crude product containing 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde; S3: The crude product was separated by a silica gel column with a mesh size of 200 - 300, and the eluent was petroleum ether / ethyl acetate with a volume ratio of 3 / 1 to obtain 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde.

6. The preparation method of a highly sensitive ratiometric fluorescence probe for detecting hypochlorous acid according to claim 2, characterized in that, In step 4), 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde undergoes a condensation reaction with malononitrile. The specific steps are as follows: S1: 1 mmol of 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde, 1 - 3 mmol of malononitrile, 10 - 20 mL of absolute ethanol, and 1 - 3 drops of piperidine were added to a three-necked flask, and the reaction was refluxed for 12 - 24 h. The reaction progress was monitored by TLC until the reaction of 4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-carbaldehyde was complete, and then the reaction was stopped; S2: After the reaction solution was concentrated to remove part of the ethanol, it was cooled and solid precipitated. The solid was filtered and the filter cake was washed with cold ethanol to obtain N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-methylene)malononitrile.

7. Use of a highly sensitive ratiometric fluorescence probe as described in claim 1 for detecting hypochlorous acid.

8. The application according to claim 7, wherein The N'-(4'-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-5,7-methanoindazol-3-yl)-[1,1'-biphenyl]-4-methylene)malononitrile can specifically react with hypochlorous acid, and its aqueous solution changes from yellow fluorescence to cyan fluorescence under irradiation with 365 nm ultraviolet light.

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