Near-infrared fluorescence / photoacoustic bimodal probe as well as preparation method and application thereof

Abstract

The invention discloses a near-infrared fluorescence / photoacoustic bimodal probe as well as a preparation method and application thereof. The imaging probe is a compound MB-ClO. Test results of the applicant show that the probe MB-ClO has good water solubility and low toxicity, can accurately target mitochondria, and has the potential of accurately monitoring mitochondrial HClO fluctuation in real time in living cells. The fluorescent probe shows high selectivity and high sensitivity (LOD = 5.1 nM) on HClO, and can quickly respond within 10 seconds, and the fluorescence intensity is enhanced by about 53 times. Moreover, the probe MB-ClO can be used for detecting exogenous HClO in a living mouse through infrared fluorescence and photoacoustic bimodal imaging, and also can specifically respond to endogenous HClO in a rheumatoid arthritis mouse model. Therefore, the probe MB-ClO disclosed by the invention has the potential of imaging other HClO-related inflammations in an infrared fluorescence and photoacoustic bimodal manner in vivo.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a near-infrared fluorescence / photoacoustic dual-modality probe and a preparation method and application thereof. Background Art

[0002] Hypochlorous acid (HClO) is an important reactive oxygen species (ROS) produced in various physiological processes. It is mainly generated by the specific catalytic reaction of hydrogen peroxide and chloride ions mediated by myeloperoxidase, and mitochondria are its main production site. As a key representative of ROS, hypochlorous acid plays a vital role in both pathological and physiological processes. In the immune process, hypochlorous acid helps to enhance immune defense, resist microbial invasion and pathogen clearance. In addition, it regulates intracellular redox balance and participates in multiple signal transduction pathways. However, abnormal hypochlorous acid levels can lead to mitochondrial dysfunction and imbalance of cellular redox homeostasis, thereby affecting cell signal transduction pathways and metabolism, and ultimately leading to cell death. Excessive hypochlorous acid levels are closely related to a variety of diseases, such as Alzheimer's disease, cancer, rheumatoid arthritis, etc., which have a high incidence and prevalence. Therefore, the development of methods that can quickly, sensitively and specifically monitor the dynamic changes of hypochlorous acid in mitochondria is of great significance for the research and early diagnosis of hypochlorous acid-related diseases.

[0003] Traditional methods for detecting hypochlorous acid mainly focus on in vitro detection, including colorimetry, spectrophotometry, electrochemical analysis, etc. However, the need for real-time monitoring of endogenous hypochlorous acid in biological systems remains a huge challenge. In the medical field, biomedical optical imaging technology has become an effective tool for hypochlorous acid detection due to its unique advantages. In particular, fluorescence imaging, as a non-invasive imaging method, has attracted widespread attention due to its application in in vivo detection of hypochlorous acid. This technology has excellent spatial and temporal resolution, sensitivity and selectivity. In recent years, many organic small molecule fluorescent probes capable of detecting hypochlorous acid have been developed, including probes based on fluorophores such as naphthylimide, BODIPY, rhodamine and coumarin. However, most of these fluorescent probes are limited by the short emission wavelength, which greatly limits their application in biological systems. Recently, a class of organic small molecule fluorescent probes with methylene blue (MB) as a fluorophore has emerged, which can effectively detect hypochlorous acid in biological systems. It is worth noting that MB has been approved by the US Food and Drug Administration (FDA) for clinical applications and has good biocompatibility and a unique near-infrared (NIR) emission wavelength, making it an ideal fluorophore. Although most reported MB-based probes have high sensitivity and rapid response to hypochlorous acid, most lack mitochondrial targeting ability. F. Yang et al. prepared a hypochlorous acid probe with a structure as shown in the following formula (I), which was excited at 495 nm and reached a maximum fluorescence response within 10 seconds. The detection limit of the fluorescence titration method was 21.4 nanomoles. The probe can specifically target mitochondria, but it can only perform fluorescence imaging but not photoacoustic imaging (F. Yang, X. Song, M. Zhang, etc., Mitochondria-targeting phenothiazine-based rati ometric fluorescent probe for visual and rapid detection of hypochlorous acid in li ving cells and zebrafish, Dyes and Pigments 229 (2024) 112258.):

[0004]

[0005] Purely optical imaging modalities are limited in their imaging depth due to strong optical scattering within turbid biological tissues. This limitation highlights the urgent need for complementary or alternative imaging modalities with the ability to penetrate deep into biological tissues. Photoacoustic (PA) imaging constructs images by detecting light-induced acoustic signals. Photoacoustic imaging combines the high contrast of optical imaging with the deep penetration ability of acoustic waves, making it a powerful tool for exploring the complexity of biological structures and functions. Nevertheless, photoacoustic imaging also has limitations, mainly in terms of relatively low sensitivity. To this end, a NIRF / PA dual-modality imaging strategy was proposed to merge the advantages of both modalities and enhance the imaging depth while maintaining high sensitivity.

[0006] After searching, no probes have been found that use MB as a fluorophore to detect hypochlorous acid and hypochlorous acid-related inflammation with high sensitivity and selectivity through NIRF / PA dual-modality imaging. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a mitochondrial-targeted near-infrared fluorescence / photoacoustic dual-modality probe with high sensitivity and high selectivity for hypochlorous acid imaging, and a preparation method and application thereof.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A near-infrared fluorescence / photoacoustic dual-modal probe, denoted as compound MB-ClO, has a structure as shown below:

[0010]

[0011] The preparation method of the above-mentioned near-infrared fluorescence / photoacoustic dual-modality probe comprises the following steps:

[0012] 1) Compound MB-Cl and 4-(aminomethyl)pyridine are placed in an organic solvent, and amidation reaction is performed with or without a catalyst to obtain an intermediate compound MB-BD;

[0013]

[0014] 2) Compound MB-BD and iodomethane are reacted in an organic solvent to obtain a crude target product.

[0015] In step 1) of the above preparation method, the addition of a catalyst can increase the yield of the target compound. The catalyst can be selected from tripotassium phosphate, sodium hydroxide, potassium hydroxide, calcium hydroxide, cesium hydroxide, cesium carbonate, potassium carbonate, sodium carbonate, potassium tert-butoxide, sodium tert-butoxide, pyridine, 4-dimethylaminopyridine, triethylamine and N,N-diisopropylethylamine, or a combination of two or more thereof. Potassium carbonate, sodium carbonate, 4-dimethylaminopyridine or triethylamine are further preferred. The amount of the catalyst can be 0.1 to 10 times the molar amount of the compound MB-Cl. When 4-dimethylaminopyridine is selected as the catalyst, its amount is usually 0.1 to 0.2 times the molar amount of the compound MB-Cl; when the catalyst is other options, its amount is usually 1 to 10 times the molar amount of the compound MB-Cl.

[0016] The crude compound MB-BD is obtained in the above step 1). It is preferred that the crude compound MB-BD is purified before being used in step 2). The crude MB-BD is usually purified by silica gel column chromatography. The eluent used in the chromatography can be a mixed solvent composed of petroleum ether and ethyl acetate in a volume ratio of 10:1 to 1:1, or a mixed solvent composed of dichloromethane and methanol in a volume ratio of 20:1 to 40:1.

[0017] In step 1) and step 2) of the above preparation method, the organic solvent involved can be one or a combination of two or more selected from dichloromethane, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, ether, benzene and toluene. More preferably, ethyl acetate, acetonitrile or toluene. The amount of the organic solvent can be determined as needed, usually to fully dissolve the raw materials to be reacted. Specifically, based on 0.1 mmol of compound MB-Cl or compound MB-BD, the total amount of organic solvent used for all raw materials is usually 2 to 6 mL.

[0018] In step 1) and step 2) of the above preparation method, the reaction involved can be carried out under heating or without heating, that is, the reaction temperature can be room temperature, or between room temperature and the boiling point of the organic solvent. Preferably, it is between 34° C. and the boiling point of the organic solvent. TLC tracking detection is performed until the reaction is complete. In order to obtain a higher yield, the reaction is preferably carried out under heating and reflux reaction.

[0019] The crude product of compound MB-ClO is obtained by the above method, and the method also includes the step of purifying the crude product of the target compound. Specifically, the crude product of compound MB-ClO can be purified by conventional purification methods to improve the purity of compound MB-ClO, such as purifying the crude product by silica gel column chromatography. The eluent used in chromatography can be a mixed solvent composed of petroleum ether and ethyl acetate in a volume ratio of 10:1 to 1:1, or a mixed solvent composed of dichloromethane and methanol in a volume ratio of 20:1 to 40:1.

[0020] In the preparation method of the present invention, the compound MB-Cl involved can be synthesized with reference to existing literature (P. Wei, L Liu, Y. Wen, etc., Release of Amino-or Carboxy-Containing Compounds Triggered by HOCl: Application for Imaging and Drug Design, Angewandte Chemie 58 (14) (2019) 4547-4551. or W. Zheng, J. Yang, Y. Shen, etc., The near-infrared fluores cent probes based on phenoxazine for the rapid detection of hypochlorous acid, Dy es and Pigments 179 (2020) 108404. or P. Wei, W. Yuan, F. Xue, etc., Deformylation reaction-based probe for in vivo imaging of HOCl, Chemical science 9 (2) (2018) 495-501.).

[0021] The applicant found through experiments that the probe MB-ClO of the present invention exhibits excellent selectivity and ultra-high sensitivity to HClO (LOD = 5.1nM), accompanied by a fast fluorescence switch response (<10s). Moreover, MB-ClO is one of the few probes that can simultaneously detect hypochlorous acid and hypochlorous acid-related inflammation using NIRF and PA imaging modes. In vitro NIRF / PA imaging demonstrated that MB-ClO has linear hypochlorous acid detection capabilities. MB-ClO can be successfully applied to NIRF imaging of intracellular and endogenous hypochlorous acid in zebrafish. It was further found that MB-ClO not only detected exogenous hypochlorous acid in mice through NIRF and PA imaging, but also specifically responded to endogenous hypochlorous acid in a mouse model of rheumatoid arthritis. This highlights the ability of MB-ClO to detect endogenous hypochlorous acid and its related inflammation (such as arthritis, rheumatoid arthritis, peritonitis, acute liver and kidney injury, atherosclerosis, etc.) using NIRF and PA imaging, indicating its potential for detecting other hypochlorous acid-related inflammations.

[0022] Based on the above findings, the present invention includes the use of the above near-infrared fluorescence / photoacoustic dual-modality probe in qualitative or quantitative detection of hypochlorous acid content, where the hypochlorous acid content refers to the hypochlorous acid content outside the body. Based on the discovery that the above probe can specifically target mitochondria, the present invention also includes the use of the above near-infrared fluorescence / photoacoustic dual-modality probe in imaging hypochlorous acid in cells, mitochondria or in vivo.

[0023] Furthermore, the near-infrared fluorescence / photoacoustic dual-modality probe can be used in one or more of the following processes:

[0024] Used to observe and monitor the dynamic changes and distribution of hypochlorous acid in vitro; or

[0025] Used to observe and monitor the dynamic changes and distribution of endogenous hypochlorous acid or exogenous hypochlorous acid in cells or organisms; or

[0026] Used to visualize the dynamic changes and distribution of hypochlorous acid within cells or organisms.

[0027] As a specific application example, the above-mentioned near-infrared fluorescence / photoacoustic dual-modality probe is used in the imaging detection of exogenous hypochlorous acid in vivo.

[0028] As another specific application example, the above-mentioned near-infrared fluorescence / photoacoustic dual-modality probe is used in the imaging detection of endogenous hypochlorous acid in rheumatoid arthritis in vivo.

[0029] Furthermore, during cell imaging, the probe MB-TPP was excited at 633 nm, and its fluorescence emission intensity was collected in the range of 650 to 750 nm to measure the fluorescence intensity.

[0030] Compared with the prior art, the present invention provides a novel mitochondrial-targeted NIRF / PA dual-modal probe MB-ClO, which cleverly uses a molecular amide bond as a response group for hypochlorous acid. MB-ClO has good water solubility and low toxicity, and has the ability to linearly detect and image HClO in vitro by infrared fluorescence and photoacoustic dual-modal imaging methods, and exhibits significant selectivity, rapid response (<10 seconds) and ultrasensitivity (LOD=5.1nM) to hypochlorous acid. Since the inner membrane of mitochondria is rich in negative charges, it exhibits a strong affinity for cations. This property promotes the effective attraction and enrichment of cationic MB-ClO in mitochondria, thereby giving MB-ClO mitochondrial targeting ability. This mitochondrial targeting function enables the probe to accurately monitor the fluctuation of mitochondrial hypochlorous acid in living cells in real time. MB-ClO has achieved excellent NIRF imaging of hypochlorous acid in cells and zebrafish models. In addition, MB-ClO has also achieved NIRF / PA dual-modal imaging of chloric acid in mice, especially for rheumatoid arthritis. MB-ClO shows unique advantages and broad application prospects in the biological detection of hypochlorous acid, especially in mitochondrial detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The response mechanism and spectral characteristics of MB-ClO to HClO. (a) Schematic diagram of the response mechanism of MB-ClO to HClO. (b) Absorption spectra of MB-ClO (10μM) at different concentrations of HClO (0~10μM). (c) Fluorescence spectra of MB-ClO (10μM) at different concentrations of HClO (0~10μM). (d) Linear relationship between fluorescence intensity at 683nm and HClO concentration (0~10μM). (e) Changes of fluorescence intensity of MB-ClO (10μM) at 683nm over time in the presence of HClO (0,10μM). (f) Fluorescence changes of MB-ClO (10μM) in the presence of HClO (0,10μM) at different pH values. (g) Fluorescence intensity of MB-ClO (10μM) in the presence of various reactive oxygen / reactive nitrogen (ROS / RNS) (10μM). λ ex / λ em =620 / 683nm. Slit width: 10×10nm. (h) Photoacoustic image of MB-ClO (1mM) after adding HClO, λ ex = 680 nm. (i) Photoacoustic intensity of the image in (h). Data are expressed as mean ± standard error (n = 3).

[0032] Figure 2The selectivity test of MB-ClO (10 μM) to HClO in PBS solution (pH=7.4, containing 0.1% DMSO). (a) MB-ClO in various anions (10 μM, from A to U: Control, Fe 3+ , Fe 2+ Mg 2+ 、Na + , K + , Hg 2+ , Ca 2+ 、Al 3+ 、Zn 2+ ,I - 、NO2 - 、NO3 - 、CO3 2- 、HCO3 - 、COOH - 、SO4 2- , Cl - OH - 、HSO4 2- (a) Fluorescence intensity of MB-ClO (10 μM) in the presence of a series of amino acids (10 μM, from A to R: Control, Hcy, GSH, Leu, Glu, Gly, Gln, Lys, Ala, FBS, Pro, Asp, Thr, Ser, Val, Ile, Met, HClO). ex / λ em =620 / 683 nm. Error bars represent mean ± standard deviation (n=3). Slit: 10×10 nm.

[0033] Figure 3 The color changes of MB-ClO (10 μM) after adding different ions (10 μM).

[0034] Figure 4Figure 2 is the fluorescence image of MB-ClO (10 μM) in RAW 264.7 cells. (a) From left to right, the experimental groups are: Control: MB-ClO incubation for 30 min. HClO (10 μM): pre-incubation with HClO (10 μM) for 30 min, and then pre-incubation with MB-ClO for 30 min. LPS+PMA: LPS (1 μg / mL) + PMA (1 μg / mL) stimulation for 1 h, and MB-ClO stimulation for 30 min. LPS+PMA+ABAH: LPS (1 μg / mL) + PMA (1 μg / mL) pre-incubation for 1 hour, and then ABAH (200 μM) and MB-ClO pre-incubation for 30 minutes. (b) Relative fluorescence intensity in (a). Data represent the mean ± standard error (n=5). (c) CCK8 assay of MB-ClO cytotoxicity to RAW 264.7 cells. Data represent the mean ± standard error (n=5). (d) Co-localization experiment of RAW 264.7 cells treated with 10 μM MB-ClO and 500 nM commercial mitochondrial dye (Mito-Tracker Green) (MB-ClO treated RAW 264.7 cells showed red fluorescence signals, Mito-Tracker Green treated RAW 264.7 cells showed green fluorescence signals). (e) is the fluorescence intensity correlation diagram of (d). Scale bar: 10 μm. *p<0.05, ****p<0.0001.

[0035] Figure 5 Fluorescence imaging and fluorescence intensity of MB-ClO (10 μM) in zebrafish. (a) Fluorescence imaging of MB-ClO (10 μM) in zebrafish. The experimental groups from left to right are: Control: zebrafish were incubated with MB-ClO for 1 h. HClO: zebrafish were incubated with HClO (10 μM) for 1 h, and then incubated with MB-ClO for 1 h. LPS+PMA: zebrafish were pretreated with LPS (1 μg / mL) + PMA (1 μg / mL) for 4 h, and then pretreated with MB-ClO for 1 h. LPS+PMA+ABAH: zebrafish were pretreated with LPS (1 μg / mL) + PMA (1 μg / mL) for 4 h, ABAH (200 μM) for 1 h, and MB-ClO for 1 h. Scale bar: 0.44 mm. (b) is the relative fluorescence intensity of the image in (a). Error bars represent mean ± SD (n = 3). *p<0.05,***p<0.001,****p<0.0001.

[0036] Figure 6NIRF / PA images of exogenous HClO in the peritoneal cavity of mice. (a) NIRF imaging of mice after intraperitoneal injection of HClO (100 μL, 1 mM, left image in a) or PBS (100 μL, right image in a) and then injection of MB-ClO (100 μL, 1 mM). ex =600±20nm,λ em =680±20nm. (b) Fluorescence intensity in (a). (c) PA imaging in (a), PA detection depth is about 12mm. (d) PA intensity in (c). Error bars represent mean±SD (n=3). ****p<0.0001.

[0037] Figure 7 NIRF / PA images of mice with rheumatoid arthritis. (a) NIRF imaging of rheumatoid arthritis (right ankle joint) at various time points (0-10 min) after injection of MB-ClO (1 mM, 100 μL). The left ankle joint served as a healthy control group. ex =600±20nm,λ em =680±20nm. (b) Fluorescence intensity of NIRF imaging in (a). (c) Imaging of PA in rheumatoid arthritis 10min after injection of MB-ClO (1mM, 100μL), PA detection depth is about 6mm. (d) PA intensity in (c) image. Error bars represent mean±SD (n=3), ns P>0.05, **P<0.01, ****P<0.0001.

[0038] Figure 8 For MB-BD 1 H NMR spectrum (600 MHz, CDCl3).

[0039] Fig. 9 For MB-BD 13 C NMR spectrum (600 MHz, CDCl3).

[0040] Fig.10 HRMS of MB-BD (calculated C 23 H 25 N5OS[M+H] + 420.1853, HRMS found 420.1835).

[0041] Fig.11 For MB-ClO 1 H NMR spectrum (600 MHz, DMSO-d6).

[0042] Fig.12 For MB-ClO 13 C NMR (150 MHz, CDCl3).

[0043] Fig.13 HRMS of MB-ClO (calculated C 24 H 28 N5OS + [M] + was 434.2009, HRMS found 434.2011). DETAILED DESCRIPTION

[0044] In order to better explain the technical solution of the present invention, the present invention is further described in detail below in conjunction with embodiments, but the embodiments of the present invention are not limited thereto.

[0045] The compound MB-Cl involved in the following examples was prepared according to the following synthetic route:

[0046]

[0047] (a) Na2S2O4, Na2CO3, dichloromethane, water, room temperature, nitrogen protection, 10 minutes. (b) Triphosgene, Na2CO3, triethylamine, dichloromethane, room temperature, 1 hour, yield 66.4%.

[0048] Preparation of compound MB-Cl: Methylene blue (1918.9 mg, 6.0 mmol), sodium carbonate (1260.0 mg, 18.0 mmol) and sodium dithionite (2089.3 mg, 12.0 mmol) were placed in a double-necked flask, followed by the addition of dichloromethane (50 ml) and deionized water (30 ml) as solvents. After the solvent was added, a vacuum pump was used to remove oxygen immediately, followed by the rapid introduction of nitrogen for protection. Under the continuous protection of nitrogen, the stirring device was started to stir the mixture vigorously at room temperature. As the reaction proceeded, it was observed that the solution gradually turned into a bright yellow color, indicating that methylene blue had been successfully reduced to its reduced state MB-1. At this time, the stirring speed was appropriately slowed down. After slowing down the stirring, it was clearly seen that the organic layer and the water layer separated naturally, wherein the dichloromethane layer containing the reduced methylene blue MB-1 settled to the lower layer, forming an obvious stratification effect (reduced methylene blue MB-1 dissolved in the dichloromethane layer). Subsequently, triethylamine (0.52 ml, 3.8 mmol) was added to the above reaction system at 0°C, followed by the slow addition of a solution of triphosgene (819.2 mg, 3.0 mmol) dissolved in dichloromethane (3.0 ml). The resulting mixture was stirred at room temperature for 1 hour under nitrogen protection. After the reaction was completed, the mixture was poured into ice water (100 ml) and extracted three times with dichloromethane (40 ml each time). The dichloromethane obtained by extraction was combined, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio of 1:20) to obtain compound MB-Cl (light gray-blue solid, 1380.0 mg, 66.4%). 1 HNMR (600MHz, DMSO-d6) δ7.41 (d, J = 8.9 Hz, 2H), 6.78 (d, J = 2.8 Hz, 2H), 6.70 (dd, J = 9.0, 2.8 Hz, 2H), 2.92 (s, 12H). 13 C NMR (150MHz, DMSO-d6) δ149.25,148.50,129.67,127.04,124.22,110.79,109.82,40.08. HRMS(ESI + ): Calculate C 17 H 18 ClN3OS[M+H] + :348.0932,HRMS found:348.0940.

[0049] Example 1: Preparation of compound MB-ClO

[0050]

[0051] (c) Dichloromethane, triethylamine, room temperature for 1 hour, yield is 72.6%. (d) Methyl iodide, acetonitrile, 80°C, reflux for 1 hour, yield is 70.0%.

[0052] 1) Preparation of intermediate compound MB-BD: MB-Cl (300.0 mg, 0.78 mmol) and 4-(aminomethyl)pyridine (150 μL, 1.45 mmol) were dissolved in dichloromethane (10 mL). Triethylamine (0.1 mL, 3.25 mmol) was then added as a catalyst for the amidation reaction. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain a crude product of MB-ClO. The crude product of MB-ClO was purified by silica gel column chromatography (dichloromethane / methanol, v / v=30:1) to obtain compound MB-BD (light gray-blue solid, 279.0 mg, 72.6%). 1 H NMR (600MHz, CDCl3) δ8.49-8.45(m,2H),7.35(d,J=8.8Hz,2H),7.14(d,J=5.0Hz,2H),6.69(d,J=2.8Hz,2 H),6.60(dd,J=8.9,1.4Hz,2H),5.48(q,J=5.2,4.7Hz,1H),4.39(d,J=6.1Hz,2H),2.90(d,J=1.9Hz,12H)( Figure 8 ). 13 C NMR (150MHz, CDCl3) δ156.32,149.79,149.09,148.70,134.32,128.13,127.23,121.98,111.25,110.91,43.54,40.65( Fig. 9 ). C 23 H 25 Calculated molecular weight of N5OS [M+H] + :420.1853, HRMS spectrum shows:420.1835( Fig.10 ).

[0053] 2) Preparation of compound MB-ClO: Compound MB-BD (220.0 mg, 0.525 mmol) and iodomethane (35 μL, 0.788 mmol) were dissolved in acetonitrile (15 mL). The mixture was refluxed and stirred at 80°C for 1 hour. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain a crude product of MB-ClO. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, v / v=30:1) to obtain compound MB-ClO (light gray-blue solid, 159.6 mg, 70.0%). The melting point was 195-205°C. 1H NMR (600MHz, CDCl3) δ8.75(d,J=6.3Hz,2H),7.79(d,J=6.3Hz,2H),7.32(d,J=8.8Hz,2H),6.67(d,J=2.8Hz ,2H),6.62(dd,J=9.0,2.9Hz,2H),6.04(t,J=6.0Hz,1H),4.55(d,J=5.8Hz,2H),4.32(s,3H),2.90(s,12H)( Fig.11 ). 13 C NMR (150MHz, CDCl3) δ160.79,156.80,149.52,149.43,144.94,134.50,127.96 ,127.62,126.12,125.73,111.84,111.17,111.02,48.89,44.47,41.00,29.97( Fig.12 ). C 24 H 28 N5OS + Calculated molecular weight [M] + :434.2009, HRMS spectrum shows:434.2011( Fig.13 ).

[0054] Example 2: Preparation of compound MB-ClO

[0055] Example 1 was repeated, except that in step 2), dichloromethane was used instead of acetonitrile, and the reaction was carried out at 25° C. until the reaction was complete.

[0056] Finally, 125.4 mg of light gray-blue solid was obtained with a yield of 55%. The compound was identified as MB-ClO by characterization of H-NMR, C-NMR and high-resolution mass spectrometry.

[0057] Example 3: Preparation of compound MB-ClO

[0058] Repeat Example 1, except that:

[0059] In step 1), triethylamine was not added. 159.8 mg of light gray-blue solid was obtained with a yield of 41.5%. The compound was identified as MB-BD by nuclear magnetic resonance hydrogen spectrum, carbon spectrum and high-resolution mass spectrum characterization.

[0060] In step 2), toluene was used instead of acetonitrile, and the reaction was refluxed at 100°C until the reaction was complete. Finally, 166.4 mg of a light gray-blue solid was obtained with a yield of 73%. The compound was identified as MB-ClO by characterization of H NMR, C NMR and high-resolution mass spectrometry.

[0061] Example 4: Preparation of Compound MB-ClO

[0062] Repeat Example 1, except that:

[0063] In step 1), diethyl ether was used instead of dichloromethane, and potassium carbonate was used instead of triethylamine. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, volume ratio of 3:1) to obtain 254.0 mg of a light gray-blue solid with a yield of 66.1%. The compound was identified as MB-BD by characterization of H NMR, C NMR and high-resolution mass spectrometry.

[0064] In step 2), acetonitrile was replaced with ethyl acetate, and the reaction was refluxed at 75°C until the reaction was complete. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, volume ratio of 3:1), and finally 155.9 mg of light gray-blue solid was obtained, with a yield of 68.4%. The compound was identified as MB-ClO by nuclear magnetic hydrogen spectrum, carbon spectrum and high-resolution mass spectrum characterization.

[0065] Example 5: Preparation of Compound MB-ClO

[0066] Repeat Example 1, except that:

[0067] In step 1), acetonitrile was used instead of dichloromethane, sodium hydroxide was used instead of triethylamine, and the reaction was refluxed at 80°C until the reaction was complete. 153.9 mg of a light gray-blue solid was obtained with a yield of 67.5%. The compound was identified as MB-BD by characterization of H NMR, C NMR and high-resolution mass spectrometry.

[0068] Experimental Example: Detection Limit and Imaging Capability of the NIRF / PA Dual-Mode Probe MB-ClO of the Present Invention 1. Experimental Section

[0069] 1.1 Instruments and Materials

[0070] All reaction reagents were purchased from commercial channels without further purification. MB-ClO used was prepared according to the method described in Example 1. 1 H NMR and 13C NMR spectra were obtained on a Bruker 600MHz spectrometer (Switzerland) at an operating temperature of 25°C with TMS as internal standard in DMSO-d6 and solvent peak as internal standard in CDCl3. High-resolution mass spectrometry (HRMS) analysis was performed on a Thermo Fisher Scientific LTQ FT Ultra instrument, USA. UV-visible absorption and fluorescence spectra were recorded using an Agilent Cary 3500 UV-visible spectrophotometer, USA and an Agilent Eclipse fluorescence spectrometer, USA, respectively, at room temperature. Cytotoxicity assessment was performed using a Tecan Infinite 200pro microplate spectrophotometer (Switzerland). Cell imaging was performed using a multiphoton laser confocal scanning microscope with an Olympus Fv3000 system (Japan). Zebrafish fertilized eggs were maintained in an illuminated incubator (GZX-250E) for culture. In vivo imaging of zebrafish with MB-ClO was achieved using a Leica M205FCA imaging system (Germany). In vivo near-infrared fluorescence (NIRF) imaging of mice was performed using the AniView600 multi-mode animal in vivo imaging system (China). In addition, in vivo photoacoustic imaging (PA) was performed using MB-ClO on a Fujifilm Visualsonic visualization ultrasound imaging system (USA).

[0071] 1.2 Detection of MB-ClO in solution

[0072] All optical experiments were performed in phosphate buffered saline (PBS) (pH 7.4, containing 0.1% DMSO). First, MB-ClO was dissolved in dimethyl sulfoxide (DMSO) to obtain a 1 mM stock solution. To prepare the working solution, 0.01 mL of the above stock solution was taken with a pipette and the volume was adjusted to 1 mL using phosphate buffer (pH 7.4, 10 mM). The mixture was thoroughly stirred and then 1 mL of the resulting solution was transferred to a 1 cm 3 quartz cuvette for subsequent determination of absorbance or fluorescence properties.

[0073] 1.3 Limit of detection (LOD) of MB-ClO in vitro

[0074] The determination of the limit of detection (LOD) of fluorescence titration is a popular and widely used method. The calculation formula of LOD is LOD = 3σ / k, Where, σ is the standard deviation of the blank solution measurement. The square root of the sum of the square differences of each blank measurement value and the average value (xi) of all blank measurements, divided by the number of blank measurements minus 1 (n-1), where n is the total number of blank samples tested (n=5). In addition, the slope (k) of the linear calibration curve of fluorescence intensity at 683nm and hypochlorous acid concentration is also considered in the LOD calculation to ensure scientific rigor and accuracy.

[0075] 1.4 Cell culture and activity assay

[0076] RAW264.7 cells (mouse mononuclear macrophage leukemia cells) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and an antibiotic mixture consisting of 0.5 units / ml penicillin and 0.5 g / ml streptomycin, placed in cell culture flasks, and maintained in an incubator environment at 37°C, 5% CO2 concentration and constant humidity. Cell imaging experiments were performed using a multiphoton laser confocal scanning microscope (Olympus FV3000, Japan).

[0077] The toxicity of the probe MB-ClO was evaluated using RAW 264.7 cells. First, RAW 264.7 cells were seeded in 96-well plates containing growth medium and incubated at 37°C for 24 hours to ensure proper cell attachment and initial proliferation. Afterwards, different concentrations (0, 12.5, 25, 50, 100, and 200 μM) of MB-ClO were added to each well. An equal volume of PBS solution (pH = 7.4, containing 0.1% dimethyl sulfoxide) was added to the control group to maintain experimental consistency. After the addition of MB-ClO, the cells were incubated for another 24 hours at 37°C and 5% CO2 to observe the treatment effect. Then, 10 μL of CCK-8 reagent was added to each well, and the culture plate was incubated at 37°C for another 24 hours. All sample concentrations were tested in parallel in 5 groups to enhance the robustness and reproducibility of the experimental results. Afterwards, the absorbance of each well was measured at a wavelength of 450 nm using a microplate spectrophotometer (Tecan Infinite 200Pro, Switzerland). Cell viability was calculated according to the following formula: Cell viability (%) = (average absorbance of the treatment group / average absorbance of the control group) × 100%. The absorbance measurement value was used as an indicator of the relative survival level of cells in each well. RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 0.5 units / ml penicillin and 0.5 g / ml streptomycin to maintain optimal growth conditions. The cells were placed in a humidified incubator at 37°C with 5% CO2 to promote cell recovery and proliferation.

[0078] 1.5 Cell imaging

[0079] Cell imaging experiments were performed using a multiphoton laser confocal scanning microscope (Olympus FV3000, Japan). The probe MB-ClO was excited at 633 nm and its emission light was collected in the range of 650-750 nm, while Mito-Tracker Green was excited at 488 nm and its emission light was collected in the range of 500-560 nm.

[0080] 1.6 Zebrafish husbandry and NIRF / PA imaging

[0081] A novel biomedical research and development model using zebrafish larvae was developed at Guangxi Medical University (Nanning, China). Zebrafish embryos were first obtained and subsequently placed in a culture incubator (GZX-250E) at a temperature of 28°C to ensure optimal developmental conditions. After exactly 18 hours, 1-phenyl-2-thiourea (PTU) was added to the culture medium with the goal of reducing the pigmentation of the zebrafish. This intervention facilitated clearer visualization during subsequent experiments. The zebrafish were then placed in an incubator for long-term storage for 6 to 7 days to allow for continued development and maturation. MB-ClO in vivo imaging of zebrafish was performed using a Leica M205 FCA (Germany). 1.7 NIRF / PA imaging of mice

[0082] Female BALB / c nude mice and female C57BL / 6 mice, all 6–7 weeks old, were obtained from the Experimental Animal Center of Guangxi Medical University. This study has obtained ethical approval from the Ethics Committee of the Affiliated Cancer Hospital of Guangxi Medical University, and all animal treatment procedures were performed in strict accordance with the protocols approved by the Institute for Animal Care and Use Committee. In the in vivo experiments, nude mice were selected for NIRF imaging, while C57BL / 6 mice were used for PA imaging. Before the experiments, mice (n = 3 per group) were fasted overnight. NIRF imaging was performed using the AniView600 multimodal animal imaging system (China), which was configured with excitation and emission filters centered at 600 ± 20 nm and 680 ± 20 nm, respectively. PA imaging was performed using the Fujifilm Visualsonics Visual Ultrasound Imaging System (USA) of Shanghai University of Traditional Chinese Medicine, λ ex =680nm.

[0083] 2. Results and Discussion

[0084] 2.1. Mechanism

[0085] Based on the literature and the research results of the applicant, we have determined that the selective response of MB-ClO to hypochlorous acid originates from the amide group in its molecule. The response process involves hypochlorous acid attacking the electron-deficient carbon atom in the amide carbon group, which then triggers the cleavage of the amide bond and releases the reduced form MB-Cl. MB-Cl is rapidly oxidized to form fluorescent MB. In addition, HRMS analysis of MB-ClO after the response to hypochlorous acid confirmed that the main product is MB, verifying our theoretical mechanism ( Figure 1 a).

[0086] 2.2. Characteristic spectrum of MB-ClO probe for hypochlorous acid

[0087] First, the optical properties of MB-ClO in solution were studied. MB-ClO itself exhibits weak absorption and fluorescence. After adding hypochlorous acid, obvious absorption peaks appear at 620nm and 660nm, of which 620nm is used for fluorescence excitation ( Figure 1 b). Under simulated physiological conditions, after adding 10 micromolar hypochlorous acid, the fluorescence intensity of MB-ClO at 683 nm increased significantly (about 53 times) ( Figure 1 c). It is worth noting that there is a linear correlation between the fluorescence intensity at 683 nm and the concentration of hypochlorous acid, and the regression equation is y=55.897x+7.4249(R 2 =0.9888), and the calculated limit of detection (LOD) was 5.1 nmol / L, which indicated that MB-ClO has the potential to detect hypochlorous acid with ultrasensitive qualitative and quantitative in vitro detection ( Figure 1 d). After hypochlorous acid rapidly cleaves the amide group in MB-ClO, the maximum fluorescence response is reached within 10 seconds, indicating a rapid response. After the response, the fluorescence intensity remains stable for more than 10 minutes, showing its excellent stability ( Figure 1 e). In addition to the fluorescence change, a clear color change from colorless to distinct blue was observed after the addition of hypochlorous acid, which can be easily distinguished by the naked eye ( Figure 3 ). These findings collectively demonstrate that MB-ClO serves as a versatile probe suitable for sensitive and rapid detection of hypochlorous acid both visually and spectroscopically.

[0088] In addition, to investigate whether MB-ClO detection of hypochlorous acid is affected by pH, we examined the fluorescence changes of MB-ClO at different pH levels. In the pH range of 3.0 to 12.0, the fluorescence intensity of MB-ClO remained almost constant, indicating that pH changes do not trigger a fluorescence response. Similarly, pH changes have little effect on the fluorescence intensity of MB-ClO detection of hypochlorous acid. These results indicate that MB-ClO is robust for the detection of hypochlorous acid and its performance is not significantly affected by pH changes ( Figure 1 f).

[0089] 2.3. Selectivity of MB-ClO for hypochlorous acid

[0090] Subsequently, the specificity of MB-ClO for hypochlorous acid was rigorously evaluated. A series of reactive oxygen species / reactive nitrogen species (ROS / RNS), common ions, and amino acids were selected as competitive analytes. The effects of these substances on the fluorescence spectrum of MB-ClO in solution were carefully evaluated. Figure 1 g, after adding various ROS / RNS (10 μM) including H2O2, ONOO - 、O2 - 、ROO - 、NO、·OH、t-BuOO - After 24h, the fluorescence intensity of MB-ClO remained almost unchanged. Similarly, in the presence of a series of ions (10 μM) such as Fe 3+ , Fe 2+ Mg 2+ 、Na + , K + , Hg 2+ , Ca 2+ 、Al 3+ 、Zn 2+ ,I - 、NO2 - 、NO3 - 、CO3 2- 、HCO3 - 、COOH - 、SO4 2- , Cl - OH - 、HSO4 2- , hypochlorous acid, and amino acids (10 μM) including Hcy, GSH, Leu, Glu, Gly, Gln, Lys, Ala, FBS, Pro, Asp, Thr, Ser, Val, Ile, and Met, no significant changes were observed in the fluorescence intensity of MB-ClO ( Figure 2 ). In contrast, the fluorescence of MB-ClO was significantly enhanced after the addition of hypochlorous acid, indicating its high specificity. In addition, a clear color difference was observed, with only the solution containing hypochlorous acid turning the MB-ClO solution blue, while other competing substances did not cause any color change ( Figure 3 ). This observation further emphasizes the excellent selectivity of MB-ClO for hypochlorous acid over other interfering substances.

[0091] 2.4. In vitro photoacoustic (PA) imaging of hypochlorous acid using MB-ClO

[0092] Given MB-ClO's excellent response to hypochlorous acid and its near-infrared excitation wavelength, it becomes an ideal candidate for photoacoustic imaging. We studied the photoacoustic imaging of MB-ClO in the presence of different concentrations of hypochlorous acid. As the concentration of hypochlorous acid increased, a gradual increase in the photoacoustic intensity ( Figure 1 h). There is a linear correlation between the photoacoustic intensity and the hypochlorous acid concentration (0-10 μM), following the linear equation y=0.1012x+0.4595, R 2 =0.9764( Figure 1 i) These results indicate that MB-ClO has the potential to serve as an effective photoacoustic probe for quantitative detection of hypochlorous acid.

[0093] NIRF imaging of MB-ClO in cells

[0094] 2.5.1. Cytotoxicity of MB-ClO

[0095] Before MB-ClO is applied to cell experiments, its safety in vivo must be ensured, which prompted us to conduct a comprehensive evaluation of the cytotoxicity of MB-ClO. MB-ClO had no significant toxic effect on RAW 264.7 cells at concentrations up to 200 μM, indicating that its cytotoxicity is low. This finding provides important safety evidence for the application of MB-ClO in subsequent intracellular studies ( Figure 4 c).

[0096] 2.5.2. Visualization of HOCl detection in living cells by MB-ClO

[0097] After obtaining satisfactory results regarding cytotoxicity, we investigated the ability of MB-ClO to image intracellular hypochlorous acid. RAW 264.7 cells incubated with MB-ClO alone did not show a strong fluorescence response, while the presence of hypochlorous acid significantly enhanced the fluorescence intensity of RAW 264.7 cells treated with MB-ClO. Given that phorbol esters (PMA) and lipopolysaccharide (LPS) can stimulate cells to produce endogenous hypochlorous acid, we observed a significant increase in the fluorescence intensity of RAW 264.7 cells incubated with MB-ClO compared to the control group. This initial observation provides preliminary evidence that MB-ClO is capable of imaging and detecting endogenous hypochlorous acid. To finally verify that the fluorescence enhancement was indeed due to increased endogenous hypochlorous acid levels, we used ABAH, a compound that can reduce endogenous hypochlorous acid levels, to treat RAW 264.7 cells that had been previously exposed to PMA and LPS. After ABAH treatment, the fluorescence signal of RAW 264.7 cells was significantly attenuated, which further supports the hypothesis that the enhanced MB-ClO fluorescence is triggered by increased endogenous hypochlorous acid levels ( Figure 4a, 4b). The results of this study not only highlight the potential of MB-ClO as a sensitive and specific probe for HOCl detection at the cellular level, but also demonstrate its feasibility for visualizing the dynamic changes of intracellular and exogenous HOCl by fluorescence imaging. This work provides a powerful tool for a deeper understanding of the functions and mechanisms of HOCl in biological systems.

[0098] 2.5.3. Mitochondrial targeting of MB-ClO

[0099] To evaluate the ability of MB-ClO to target mitochondria, we performed colocalization experiments on RAW 264.7 cells using MB-ClO and a commercial mitochondrial dye (Mito-Tracker Green). RAW 264.7 cells showed red fluorescence signals from MB-ClO and green fluorescence signals from Mito-Tracker Green, and the fluorescence signals overlapped well, with a Pearson correlation coefficient of 0.91. These results indicate that MB-ClO can selectively accumulate in mitochondria in living cells, thus providing a basis for the probe to accurately monitor the dynamic changes of hypochlorous acid in mitochondria of cells ( Figure 4 d,4e).

[0100] NIRF imaging of MB-ClO in zebrafish

[0101] Based on the above satisfactory experimental results, we evaluated the ability of MB-ClO to detect hypochlorous acid in vivo. Figure 5 As shown, almost no fluorescence was observed in zebrafish fed only MB-ClO, while fluorescence was significantly enhanced after exposure to exogenous hypochlorous acid, thus verifying the ability of this probe to image hypochlorous acid in vivo. Subsequently, we evaluated the ability of MB-ClO to image endogenous hypochlorous acid in vivo. After treatment with LPS and PMA, strong red fluorescence signals were present in zebrafish. These findings highlight the practicality of MB-ClO for visualizing endogenous hypochlorous acid in vivo. To finally verify that the fluorescence enhancement observed in zebrafish was indeed derived from the increase in endogenous hypochlorous acid levels, we performed another experiment using ABAH. When zebrafish that had been previously exposed to PMA and LPS were treated with ABAH, the fluorescence signal in the corresponding zebrafish was significantly reduced, further confirming the conclusion that MB-ClO can be used to reliably image endogenous hypochlorous acid levels in vivo. In summary, the results of this study confirm the feasibility of MB-ClO as a fluorescence imaging tool to visualize the dynamic changes of hypochlorous acid in vivo.

[0102] 2.7. NIRF / PA Imaging of MB-ClO in Mice

[0103] After obtaining satisfactory results from the above studies, we further evaluated the ability of MB-ClO to visualize hypochlorous acid in vivo using mouse near-infrared fluorescence imaging technology. Specifically, we studied the response of MB-ClO to exogenous and endogenous hypochlorous acid in mice. First, hypochlorous acid solution was dissolved in PBS and injected into the peritoneal cavity of nude mice, while PBS alone was injected as a control. Subsequently, equal amounts of MB-ClO and PBS buffer were injected into the peritoneal cavity of mice, respectively. Figure 6 As shown in Figures 6a and 6b, obvious fluorescence emission was observed in the abdominal region of the experimental mice compared with the blank control group, indicating that MB-ClO can quickly detect exogenous hypochlorous acid in living mice and the generated fluorescence has strong tissue penetration. Next, we studied the fluorescence response of MB-ClO to endogenous hypochlorous acid in living mice. To this end, we used a mouse model of rheumatoid arthritis, an inflammatory disease associated with endogenous hypochlorous acid production. The inflammation inducer λ-carrageenan was injected into the right ankle joint of the experimental mice, while as a control, an equal volume of PBS buffer was injected into the contralateral left ankle joint. Four hours later, equal doses of MB-ClO were injected into the ankle joints on both sides of the mice. As Figure 7 As shown in a and 7b, after the injection of MB-ClO, the fluorescence intensity between the two ankle joints gradually showed a difference. After 5 minutes, a significant fluorescence difference appeared, and the right ankle joint (inflamed joint) of the rheumatoid arthritis mouse showed stronger fluorescence than the left ankle joint of the control. These findings indicate that MB-ClO can detect exogenous and endogenous hypochlorous acid by near-infrared fluorescence imaging in living mice, highlighting its potential in biological imaging applications.

[0104] Given the significant advantages of photoacoustic imaging technology in terms of high resolution, high contrast, and deep tissue penetration, as well as the successful application of MB-ClO in in vitro photoacoustic imaging, we further expanded its application boundaries and explored the potential of MB-ClO for photoacoustic imaging of hypochlorous acid in living mice. In in vivo photoacoustic imaging, we adopted the mouse model construction method used in the above-mentioned near-infrared fluorescence imaging study. By intraperitoneal injection of hypochlorous acid and comparing with the control group receiving an equal volume of PBS, we observed that the photoacoustic signal in the abdominal area of ​​mice injected with hypochlorous acid was significantly enhanced ( Figure 6 c and 6d). This result preliminarily confirmed the sensitive photoacoustic imaging ability of MB-ClO for hypochlorous acid in vivo. Subsequently, we evaluated the photoacoustic imaging ability of MB-ClO for endogenous hypochlorous acid produced in mice. Using the previously established standardized rheumatoid arthritis model, we injected equal volumes of MB-ClO solution into the bilateral ankle joints of mice induced with rheumatoid arthritis. Figure 7As shown in Figs. c and 7d, the photoacoustic signal intensity of the diseased ankle joint increased significantly compared with the background signal detected in the contralateral control joint. It is noteworthy that the photoacoustic signal is evenly distributed and deeply penetrated in the ankle joint. The above findings not only confirm the feasibility of MB-ClO for in vivo hypochlorous acid photoacoustic imaging, but also indicate its potential as a probe for detecting hypochlorous acid-related inflammation (such as rheumatoid arthritis).

[0105] In summary, MB-ClO exhibits powerful near-infrared fluorescence / photoacoustic dual-modality imaging capabilities in vivo, which can accurately detect and visualize the dynamic changes of hypochlorous acid in vivo. MB-ClO provides a novel and powerful tool for the diagnosis and monitoring of inflammatory diseases including rheumatoid arthritis.

[0106] 3. Conclusion

[0107] In summary, this application designed and synthesized a novel mitochondrial-targeted near-infrared fluorescence (NIRF) / photoacoustic (PA) dual-modality probe MB-ClO for high-sensitivity and high-selectivity imaging of hypochlorous acid and hypochlorous acid-related inflammation in vivo. Compared with the fluorescent probes reported in the previous literature (Table 1), MB-ClO has multiple advantages, including ultra-high sensitivity (LOD = 5.1nM), rapid response (<10 seconds), mitochondrial targeting ability, near-infrared emission wavelength, excellent selectivity, low toxicity and excellent water solubility. It is worth noting that MB-ClO is one of the few probes that can detect hypochlorous acid and hypochlorous acid-related inflammation using both NIRF and PA imaging modes. In vitro NIRF / PA imaging demonstrated that MB-ClO has linear hypochlorous acid detection capabilities. MB-ClO has been successfully applied to NIRF imaging of endogenous hypochlorous acid in cells and zebrafish. Encouragingly, MB-ClO not only detected exogenous hypochlorous acid in mice by NIRF and PA imaging, but also specifically responded to endogenous hypochlorous acid in a mouse model of rheumatoid arthritis. This highlights the ability of MB-ClO to detect endogenous hypochlorous acid and its associated inflammation (rheumatoid arthritis) using NIRF and PA imaging, and indicates its potential for detecting other hypochlorous acid-related inflammatory diseases. As a powerful tool for NIRF / PA dual-modality imaging visualization, MB-ClO has shown great potential in monitoring and studying the dynamics of hypochlorous acid in physiological and pathological biological systems, thus making an immeasurable contribution to biomedical research.

[0108] Table 1:

[0109]

[0110]

[0111]

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Claims

1. A near-infrared fluorescence / photoacoustic dual-modal probe, denoted as compound MB-ClO, has a structure as shown below:

2. The method for preparing the near-infrared fluorescence / photoacoustic dual-modality probe according to claim 1, comprising the following steps: 1) Compound MB-Cl and 4-(aminomethyl)pyridine are placed in an organic solvent, and amidation reaction is performed with or without a catalyst to obtain an intermediate compound MB-BD; 2) Compound MB-BD and iodomethane are reacted in an organic solvent to obtain a crude target product.

3. The preparation method according to claim 2, characterized in that: In step 1), the catalyst is one or a combination of two or more selected from tripotassium phosphate, sodium hydroxide, potassium hydroxide, calcium hydroxide, cesium hydroxide, cesium carbonate, potassium carbonate, sodium carbonate, potassium tert-butoxide, sodium tert-butoxide, pyridine, 4-dimethylaminopyridine, triethylamine and N,N-diisopropylethylamine.

4. The preparation method according to claim 2, characterized in that: In step 1) and step 2), the organic solvent is one or a combination of two or more selected from the group consisting of dichloromethane, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, diethyl ether, benzene and toluene.

5. The preparation method according to claim 2, characterized in that: In step 1) and step 2), the reaction temperature is room temperature, or between room temperature and the boiling point of the organic solvent.

6. The preparation method according to claim 2, characterized in that: In step 1), the obtained compound MB-BD is purified and then used in the subsequent steps.

7. The preparation method according to any one of claims 2 to 6, characterized in that: The method also includes a step of purifying the obtained crude target product.

8. Use of the near-infrared fluorescence / photoacoustic dual-modality probe according to claim 1 in the qualitative or quantitative detection of hypochlorous acid content.

9. Use of the near-infrared fluorescence / photoacoustic dual-modality probe according to claim 1 in imaging hypochlorous acid in cells or organisms.

10. The use according to claim 9, characterized in that: The near-infrared fluorescence / photoacoustic dual-modality probe can be used in one or more of the following processes: Used to observe and monitor the dynamic changes and distribution of hypochlorous acid in vitro; or Used to observe and monitor the dynamic changes and distribution of endogenous hypochlorous acid or exogenous hypochlorous acid in cells or organisms; or Used to visualize the dynamic changes and distribution of hypochlorous acid within cells or organisms.

Images