A near-infrared fluorescence / photoacoustic dual-modal probe and its preparation method and application

Abstract

The present invention discloses a near-infrared fluorescence / photoacoustic dual-modal probe, a preparation method and an application thereof. The imaging probe is the compound MB-ClO. The applicant's experimental results show that the probe MB-ClO of the present invention has good water solubility and low toxicity, can accurately target mitochondria, and has the potential to accurately monitor mitochondrial HClO fluctuations in real time in living cells. It shows high selectivity and high sensitivity to HClO (LOD = 5.1nM), responds rapidly within 10s, and the fluorescence intensity is enhanced by about 53 times. Moreover, the probe MB-ClO can detect exogenous HClO in living mice by infrared fluorescence and photoacoustic dual-modal imaging, and can also specifically respond to endogenous HClO in a rheumatoid arthritis mouse model. Therefore, the probe MB-ClO of the present invention has the potential for infrared fluorescence and photoacoustic dual-modal imaging of other HClO-related inflammations 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 a key reactive oxygen species (ROS) generated in various physiological processes. It is primarily produced through the specific catalytic reaction of hydrogen peroxide with chloride ions, mediated by myeloperoxidase, with mitochondria being the primary site of its generation. As a key ROS agent, HClO plays a crucial role in both pathological and physiological processes. In immune processes, HClO contributes to immune defense, combating microbial invasion and clearing pathogens. Furthermore, it regulates intracellular redox homeostasis and participates in multiple signaling pathways. However, abnormal HClO levels can lead to mitochondrial dysfunction and imbalance in cellular redox homeostasis, thereby affecting cellular signaling pathways and metabolism, ultimately leading to cell death. Excessive HClO levels are closely associated with a variety of diseases, such as Alzheimer's disease, cancer, and rheumatoid arthritis, all of which have a high incidence and prevalence. Therefore, developing methods that can rapidly, sensitively, and specifically monitor the dynamic changes of HClO within mitochondria is of great significance for the research and early diagnosis of HClO-related diseases.

[0003] Traditional methods for hypochlorous acid (HOCl) detection primarily focus on in vitro detection, including colorimetry, spectrophotometry, and electrochemical analysis. However, real-time monitoring of endogenous HOCl in biological systems remains a significant challenge. In medicine, biomedical optical imaging has emerged as an effective tool for HOCl detection due to its unique advantages. In particular, fluorescence imaging, as a noninvasive imaging modality, has attracted widespread attention for its application in in vivo detection of HOCl. This technique offers excellent spatial and temporal resolution, sensitivity, and selectivity. In recent years, numerous organic small molecule fluorescent probes capable of detecting HOCl have been developed, including those based on fluorophores such as naphthalimide, BODIPY, rhodamine, and coumarin. However, most of these fluorescent probes are limited by their short emission wavelengths, which significantly restricts their application in biological systems. Recently, a class of organic small molecule fluorescent probes based on methylene blue (MB) has emerged as a fluorophore capable of effectively detecting HOCl in biological systems. Notably, MB has been approved by the US Food and Drug Administration (FDA) for clinical use. Its excellent biocompatibility and unique near-infrared (NIR) emission wavelength make it an ideal fluorophore. Although most reported MB-based probes have high sensitivity and rapid response to hypochlorous acid, most lack mitochondrial targeting capabilities. 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 nanomolar. The probe can specifically target mitochondria, but it can only perform fluorescence imaging and not photoacoustic imaging (F. Yang, X. Song, M. Zhang, etc., Mitochondria-targeting phenothiazine-based ratiometric fluorescent probe for visual and rapid detection of hypochlorous acid in living cells and zebrafish, Dyes and Pigments 229 (2024) 112258.):

[0004]

[0005] Pure 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 combine the advantages of the two modes and enhance the imaging depth while maintaining high sensitivity.

[0006] After searching, no probes were 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, as well as 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 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 carried out 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 one or a combination of two or more of 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. Potassium carbonate, sodium carbonate, 4-dimethylaminopyridine, or triethylamine are more 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 used as the catalyst, its amount is typically 0.1 to 0.2 times the molar amount of the compound MB-Cl. When other catalysts are used, their amount is typically 1 to 10 times the molar amount of the compound MB-Cl.

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

[0017] In steps 1) and 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, diethyl ether, benzene, and toluene. Ethyl acetate, acetonitrile, or toluene is more preferred. The amount of the organic solvent used can be determined as needed, generally sufficient to 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 generally 2 to 6 mL.

[0018] In steps 1) and 2) of the above preparation method, the reactions involved can be carried out with 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, the reaction temperature is between 34°C and the boiling point of the organic solvent. TLC monitoring is performed until the reaction is complete. To obtain a higher yield, the reaction is preferably carried out under reflux with heating.

[0019] The above method produces a crude compound MB-ClO, which also includes a purification step for the crude target compound. Specifically, conventional purification methods can be used to improve the purity of compound MB-ClO, such as silica gel column chromatography. The eluent used in the chromatography can be a mixed solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 10:1 to 1:1, or a mixed solvent consisting 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 discovered through experiments that the probe MB-ClO of the present invention exhibits excellent selectivity and ultra-high sensitivity to HClO (LOD = 5.1 nM), accompanied by a rapid fluorescence switching 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 endogenous hypochlorous acid in cells and zebrafish. It was further discovered 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 rheumatoid arthritis mouse model. 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 aforementioned near-infrared fluorescence / photoacoustic dual-modality probe for the qualitative or quantitative detection of hypochlorous acid content, where the hypochlorous acid content refers to hypochlorous acid content in vitro. Based on the discovery that the aforementioned probe can specifically target mitochondria, the present invention also includes the use of the aforementioned near-infrared fluorescence / photoacoustic dual-modality probe for imaging hypochlorous acid within cells, mitochondria, or organisms.

[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 to existing technologies, the present invention provides a novel mitochondrial-targeted NIRF / PA dual-modal probe, MB-ClO, which cleverly utilizes an intramolecular amide bond as a hypochlorous acid (HOClO) response group. MB-ClO exhibits excellent water solubility and low toxicity, and is capable of linear detection and imaging of HClO in vitro using infrared fluorescence and photoacoustic dual-modal imaging methods. It also exhibits remarkable selectivity, rapid response (<10 seconds), and ultrasensitivity (LOD = 5.1 nM) for HOCl. Because the mitochondrial inner membrane is rich in negative charges, it exhibits a strong affinity for cations. This property promotes the efficient attraction and accumulation of cations within mitochondria, thereby endowing MB-ClO with mitochondrial targeting capabilities. This mitochondrial targeting capability enables the probe to accurately monitor mitochondrial HOCl fluctuations in living cells in real time. MB-ClO has demonstrated excellent NIRF imaging of HOCl in cells and zebrafish models. Furthermore, MB-ClO has demonstrated NIRF / PA dual-modal imaging of HOCl in mice, with particular application in 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 (10 μM) in the presence of different HClO concentrations (0-10 μM). (c) Fluorescence spectra of MB-ClO (10 μM) in the presence of different HClO concentrations (0-10 μM). (d) Linear relationship between fluorescence intensity at 683 nm and HClO concentration (0-10 μM). (e) Changes in the fluorescence intensity of MB-ClO (10 μM) at 683 nm over time in the presence of HClO (0, 10 μM). (f) Fluorescence changes of MB-ClO (10 μM) in the presence of HClO (0, 10 μM) at different pH values. (g) Fluorescence intensity of MB-ClO (10 μM) in the presence of various reactive oxygen species / reactive nitrogen species (ROS / RNS) (10 μM). 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) for HClO in PBS solution (pH = 7.4, containing 0.1% DMSO). (a) MB-ClO in various ions (10 μM, from A to U are: 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- (b) 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 4Fluorescence images of MB-ClO (10 μM) in RAW 264.7 cells. (a) From left to right, experimental groups are: Control: Incubation with MB-ClO for 30 minutes. HClO (10 μM): Preincubation with HClO (10 μM) for 30 minutes, followed by preincubation with MB-ClO for 30 minutes. LPS+PMA: Stimulation with LPS (1 μg / mL) and PMA (1 μg / mL) for 1 hour, followed by stimulation with MB-ClO for 30 minutes. LPS+PMA+ABAH: Preincubation with LPS (1 μg / mL) and PMA (1 μg / mL) for 1 hour, followed by preincubation with ABAH (200 μM) and MB-ClO for 30 minutes. (b) Relative fluorescence intensity in (a). Data represent the mean ± standard error (n = 5). (c) CCK8 cytotoxicity assay of MB-ClO against RAW 264.7 cells. Data represent the mean ± standard error (n = 5). (d) Colocalization assay of RAW 264.7 cells treated with 10 μM MB-ClO and 500 nM of a commercial mitochondrial dye (Mito-Tracker Green). (MB-ClO-treated RAW 264.7 cells show red fluorescence signals, while Mito-Tracker Green-treated RAW 264.7 cells show green fluorescence signals. (e) Fluorescence intensity correlation graph 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. Experimental groups from left to right are: Control: zebrafish incubated with MB-ClO for 1 hour. HClO: zebrafish incubated with HClO (10 μM) for 1 hour, followed by MB-ClO for 1 hour. LPS+PMA: zebrafish pretreated with LPS (1 μg / mL) + PMA (1 μg / mL) for 4 hours, followed by MB-ClO for 1 hour. LPS+PMA+ABAH: zebrafish pretreated with LPS (1 μg / mL) + PMA (1 μg / mL) for 4 hours, followed by ABAH (200 μM) for 1 hour, and then MB-ClO for 1 hour. Scale bar: 0.44 mm. (b) 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 mouse peritoneal cavity. (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) followed by injection of MB-ClO (100 μL, 1 mM). ex =600±20nm,λ em = 680 ± 20 nm. (b) Fluorescence intensity of the image in (a). (c) PA imaging of the image in (a), with PA detection depth of approximately 12 mm. (d) PA intensity of the image in (c). Error bars represent mean ± SD (n = 3). ****p < 0.0001.

[0037] Figure 7 NIRF / PA images of rheumatoid arthritis mice. (a) NIRF images 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 ± 20 nm. (b) Fluorescence intensity from the NIRF imaging in (a). (c) Imaging of PA in rheumatoid arthritis 10 minutes after injection of MB-ClO (1 mM, 100 μL). PA detection depth is approximately 6 mm. (d) PA intensity from the image in (c). 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] Figure 9 For MB-BD 13 C NMR spectrum (600 MHz, CDCl3).

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

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

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

[0043] Figure 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 with reference to examples, 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 atmosphere, 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 addition of the solvent, oxygen was immediately removed using a vacuum pump, and then nitrogen was quickly introduced for protection. Under the continuous protection of nitrogen, the stirring device was started and the mixture was vigorously stirred at room temperature. As the reaction proceeded, it was observed that the solution gradually turned bright yellow, indicating that the 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 aqueous layer separated naturally, wherein the dichloromethane layer containing the reduced methylene blue MB-1 settled to the lower layer, forming a clear stratification effect (the reduced methylene blue MB-1 was 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: 72.6% (d) Methyl iodide, acetonitrile, 80°C, reflux for 1 hour, yield: 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 to initiate an amidation reaction. The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the solvent was removed by distillation under reduced pressure to obtain crude MB-ClO. The crude MB-ClO was purified by silica gel column chromatography (dichloromethane / methanol, v / v = 30:1) to yield compound MB-BD (a 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( Figure 9 ). C 23 H 25 Calculated molecular weight of N5OS [M+H] + :420.1853, HRMS spectrum found:420.1835( Figure 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 stirred at reflux at 80°C for 1 hour. After completion of the reaction, the solvent was removed by distillation under reduced pressure to obtain crude 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)( Figure 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( Figure 12 ). C 24 H 28 N5OS + Calculated molecular weight [M] + :434.2009, HRMS spectrum found:434.2011( Figure 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 a light gray-blue solid was obtained with a yield of 55%. Characterization by H NMR, C NMR, and high-resolution mass spectrometry confirmed the compound to be MB-ClO.

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

[0058] Repeat Example 1, except that:

[0059] In step 1), triethylamine was omitted. 159.8 mg of a light gray-blue solid was obtained with a yield of 41.5%. Characterization by H-NMR, C-NMR, and high-resolution mass spectrometry confirmed the product to be compound MB-BD.

[0060] In step 2), toluene was used instead of acetonitrile, and the reaction was refluxed at 100°C until completion. This yielded 166.4 mg of a pale gray-blue solid with a 73% yield. Characterization by H-NMR, C-NMR, and high-resolution mass spectrometry confirmed the product to be compound MB-ClO.

[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 3:1) to yield 254.0 mg of a light gray-blue solid in 66.1% yield. Characterization by H NMR, C NMR, and high-resolution mass spectrometry confirmed the product to be compound MB-BD.

[0064] In step 2), acetonitrile was replaced with ethyl acetate, and the reaction was refluxed at 75°C until completion. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, volume ratio 3:1) to yield 155.9 mg of a light gray-blue solid in 68.4% yield. Characterization by H NMR, C NMR, and high-resolution mass spectrometry confirmed the product to be compound MB-ClO.

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

[0066] Repeat Example 1, except that:

[0067] In step 1), acetonitrile was used instead of dichloromethane, and sodium hydroxide was used instead of triethylamine. The reaction was refluxed at 80°C until completion. This yielded 153.9 mg of a light gray-blue solid in 67.5% yield. Characterization by H NMR, C NMR, and high-resolution mass spectrometry confirmed the product to be compound MB-BD.

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

[0069] 1. Experimental

[0070] 1.1 Instruments and Materials

[0071] All reaction reagents were purchased from commercial sources without further purification. MB-ClO was prepared according to the method described in Example 1. 1 H NMR and 13C NMR spectra were acquired on a Bruker 600 MHz spectrometer (Switzerland) at 25°C, using TMS as the internal standard in DMSO-d6 and the solvent peak as the internal standard in CDCl3. High-resolution mass spectrometry (HRMS) analysis was performed on a Thermo Fisher Scientific LTQ FT Ultra instrument. UV-visible absorption and fluorescence spectra were recorded at room temperature using an Agilent Cary 3500 UV-visible spectrophotometer and an Agilent Eclipse fluorescence spectrometer, respectively. Cytotoxicity was assessed 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 and cultured in an illuminated incubator (GZX-250E). In vivo imaging of MB-ClO in zebrafish was achieved using a Leica M205FCA imaging system (Germany). In vivo near-infrared fluorescence (NIRF) imaging of mice was performed using an AniView600 multimode animal in vivo imaging system (China). In addition, in vivo photoacoustic imaging (PA) was performed using MB-ClO on a Fujifilm Visualsonic ultrasound imaging system (USA).

[0072] 1.2 Detection of MB-ClO in solution

[0073] 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 a working solution, 0.01 mL of this stock solution was pipetted and the volume was adjusted to 1 mL using phosphate buffer (pH 7.4, 10 mM). The mixture was stirred thoroughly, 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.

[0074] 1.3 Limit of detection (LOD) of MB-ClO in vitro

[0075] 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 LOD is calculated as the square root of the sum of the squared differences between each blank measurement and the mean of all blank measurements (xi), divided by the number of blank measurements minus 1 (n-1), where n is the total number of blank samples tested (n=5). Furthermore, the slope (k) of the linear calibration curve of fluorescence intensity at 683 nm versus hypochlorous acid concentration is also factored into the LOD calculation to ensure scientific rigor and accuracy.

[0076] 1.4 Cell culture and activity assay

[0077] RAW264.7 cells (mouse monocytic macrophage leukemia cells) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and an antibiotic cocktail consisting of 0.5 units / ml penicillin and 0.5 g / ml streptomycin. They were placed in cell culture flasks and maintained in an incubator at 37°C, 5% CO₂, and a constant humidity. Cell imaging experiments were performed using a multiphoton laser confocal scanning microscope (Olympus FV3000, Japan).

[0078] 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. Subsequently, different concentrations of MB-ClO (0, 12.5, 25, 50, 100, and 200 μM) were added to each well. An equal volume of PBS (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 an additional 24 hours at 37°C and 5% CO2 to observe the effects of the treatment. Then, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for an additional 24 hours. All sample concentrations were tested in parallel in five groups to enhance the robustness and reproducibility of the experimental results. The absorbance of each well was then measured at a wavelength of 450 nm using a microplate spectrophotometer (Tecan Infinite 200Pro, Switzerland). Cell viability was calculated using the following formula: Cell viability (%) = (average absorbance of the treated group / average absorbance of the control group) × 100%. This absorbance measurement serves as an indicator of the relative cell viability level 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. Cells were placed in a humidified incubator at 37°C with 5% CO2 to promote cell recovery and proliferation.

[0079] 1.5 Cell Imaging

[0080] 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 650-750 nm range; while Mito-Tracker Green was excited at 488 nm, and its emission light was collected in the 500-560 nm range.

[0081] 1.6 Zebrafish husbandry and NIRF / PA imaging

[0082] 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 precisely 18 hours, 1-phenyl-2-thiourea (PTU) was added to the culture medium to reduce the zebrafish's pigmentation. This intervention facilitated clearer visualization during subsequent experiments. The zebrafish were then placed in an incubator for 6-7 days to allow for continued development and maturation. MB-ClO zebrafish in vivo imaging was performed using a Leica M205FCA (Germany).

[0083] 1.7 NIRF / PA Imaging in Mice

[0084] Female BALB / c nude mice and female C57BL / 6 mice, all 6 to 7 weeks old, were obtained from the Experimental Animal Center of Guangxi Medical University. This study has been approved by the Ethics Committee of the Affiliated Cancer Hospital of Guangxi Medical University, and all animal treatment procedures were carried out 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 experiment, 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.

[0085] 2. Results and Discussion

[0086] Mechanism

[0087] Based on the literature and our research, we have determined that the selective response of MB-ClO to hypochlorous acid originates from the amide group within its molecule. The response process involves hypochlorous acid attacking the electron-deficient carbon atom within 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. Furthermore, HRMS analysis of MB-ClO after the reaction with hypochlorous acid confirmed that the main product is MB, validating our theoretical mechanism ( Figure 1 (a)

[0088] 2.2. Characteristic Spectrum of MB-ClO Probe for Hypochlorous Acid

[0089] 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 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 be an ultrasensitive qualitative and quantitative detector of hypochlorous acid in vitro ( 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, demonstrating its excellent stability ( Figure 1 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.

[0090] 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 unchanged, 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).

[0091] 2.3. Selectivity of MB-ClO for hypochlorous acid

[0092] 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 As shown in g, after adding various ROS / RNS (10 μM) including H2O2, ONOO - 、O2 - 、ROO - 、NO、·OH、t-BuOO - After 4 h, 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 causing the MB-ClO solution to turn 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 relative to other interfering substances.

[0093] 2.4. In vitro photoacoustic (PA) imaging of hypochlorous acid using MB-ClO

[0094] Given MB-ClO's excellent response to hypochlorous acid and its near-infrared excitation wavelength, it is 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, the photoacoustic intensity was observed to gradually increase ( 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 These results indicate that MB-ClO has the potential to serve as an effective photoacoustic probe for the quantitative detection of hypochlorous acid.

[0095] NIRF Imaging of MB-ClO in Cells

[0096] 2.5.1. Cytotoxicity of MB-ClO

[0097] Before MB-ClO is used in cell experiments, its safety in vivo must be ensured, which prompted us to conduct a comprehensive evaluation of MB-ClO's cytotoxicity. 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)

[0098] 2.5.2. Visualization of Hypochlorous Acid Detection in Living Cells by MB-ClO

[0099] 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 exhibit a strong fluorescence response, whereas the presence of hypochlorous acid significantly enhanced the fluorescence intensity of MB-ClO-treated RAW 264.7 cells. Given that phorbol ester (PMA) and lipopolysaccharide (LPS) can stimulate cells to produce endogenous hypochlorous acid, we observed a significant increase in fluorescence intensity in RAW 264.7 cells incubated with MB-ClO compared to the control group. This initial observation provided preliminary evidence that MB-ClO was capable of imaging and detecting endogenous hypochlorous acid. To conclusively verify that the fluorescence enhancement was indeed due to elevated endogenous hypochlorous acid levels, we used ABAH, a compound capable of reducing endogenous hypochlorous acid levels, to treat RAW 264.7 cells that had previously been 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 the increase in endogenous hypochlorous acid levels ( Figure 4 Middle a, Figure 4(middle b). The results of this study not only highlight the potential of MB-ClO as a sensitive and specific probe for cellular hypochlorous acid detection but also demonstrate its feasibility for visualizing the dynamic changes of intracellular and exogenous hypochlorous acid via fluorescence imaging. This work provides a powerful tool for gaining a deeper understanding of the functions and mechanisms of hypochlorous acid in biological systems.

[0100] 2.5.3. Mitochondrial targeting of MB-ClO

[0101] 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 displayed 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, providing a basis for the probe to accurately monitor the dynamic changes of hypochlorous acid in mitochondria. Figure 4 Middle d, Figure 4 (e)

[0102] NIRF imaging of MB-ClO in zebrafish

[0103] Based on the above satisfactory experimental results, we evaluated the ability of MB-ClO to detect hypochlorous acid in vivo. Figure 5 As shown, zebrafish fed only MB-ClO exhibited little fluorescence, whereas exposure to exogenous hypochlorous acid significantly increased fluorescence, validating the probe's ability to image hypochlorous acid in vivo. Subsequently, we evaluated the ability of MB-ClO to image endogenous hypochlorous acid in vivo. Zebrafish treated with LPS and PMA exhibited a strong red fluorescence signal. These findings highlight the utility of MB-ClO for visualizing endogenous hypochlorous acid in vivo. To conclusively verify that the observed fluorescence increase in zebrafish was indeed due to elevated endogenous hypochlorous acid levels, we conducted another experiment using ABAH. When zebrafish previously exposed to PMA and LPS were treated with ABAH, the fluorescence signal 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 demonstrate the feasibility of MB-ClO as a fluorescence imaging tool for visualizing the dynamics of hypochlorous acid in vivo.

[0104] NIRF / PA Imaging of MB-ClO in Mice

[0105] 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 abdominal 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 abdominal 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 fluorescence produced 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 rheumatoid arthritis mouse model, which is an inflammatory disease associated with endogenous hypochlorous acid production. The inflammatory 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 shown Figure 7 A and Figure 7 As shown in (b), after MB-ClO injection, a difference in fluorescence intensity gradually emerged between the two ankle joints. After 5 minutes, a significant fluorescence difference emerged, with the right ankle joint (inflamed joint) of the rheumatoid arthritis mouse exhibiting stronger fluorescence than the left ankle joint of the control. These findings demonstrate that MB-ClO can detect exogenous and endogenous hypochlorous acid via near-infrared fluorescence imaging in living mice, highlighting its potential for bioimaging applications.

[0106] 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 comparison with the control group receiving an equal volume of PBS, we observed that the photoacoustic signal in the abdominal area of ​​the mice injected with hypochlorous acid was significantly enhanced ( Figure 6 Middle C and Figure 6 (d) This result preliminarily confirmed the sensitive photoacoustic imaging capability of MB-ClO for hypochlorous acid in vivo. Subsequently, we evaluated the photoacoustic imaging capability of MB-ClO for endogenous hypochlorous acid produced in mice. Using a 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 7 Middle C and Figure 7As shown in center (d), the photoacoustic signal intensity in the affected ankle joint was significantly increased compared to the background signal detected in the contralateral control joint. Notably, the photoacoustic signal was evenly distributed and deeply penetrated within the ankle joint. These findings not only confirm the feasibility of MB-ClO for in vivo hypochlorous acid photoacoustic imaging but also demonstrate its potential as a probe for detecting hypochlorous acid-related inflammation, such as rheumatoid arthritis.

[0107] In summary, MB-ClO demonstrated robust near-infrared fluorescence / photoacoustic dual-modality imaging capabilities in vivo, enabling precise detection and visualization of the dynamic changes in hypochlorous acid. MB-ClO provides a novel and powerful tool for the diagnosis and monitoring of inflammatory diseases, including rheumatoid arthritis.

[0108] 3. Conclusion

[0109] In summary, we designed and synthesized a novel mitochondria-targeted near-infrared fluorescence (NIRF) / photoacoustic (PA) dual-modality probe, MB-ClO, for highly sensitive and selective imaging of hypochlorous acid (HOCl) and HOCl-related inflammation in vivo. Compared with previously reported fluorescent probes (Table 1), MB-ClO offers several advantages, including ultrahigh sensitivity (LOD = 5.1 nM), rapid response (<10 seconds), mitochondrial targeting, near-infrared emission wavelength, excellent selectivity, low toxicity, and superior water solubility. Notably, MB-ClO is one of the few probes capable of detecting HOCl and HOCl-related inflammation using both NIRF and PA imaging modalities. In vitro NIRF / PA imaging demonstrated MB-ClO's linear HOCl detection capability. MB-ClO has been successfully applied to NIRF imaging of endogenous HOCl in cells and in zebrafish. Encouragingly, MB-ClO not only detected exogenous HOCl in mice using NIRF and PA imaging but also specifically responded to endogenous HOCl in a mouse model of rheumatoid arthritis. This study highlights the ability of MB-ClO to detect endogenous hypochlorous acid and its associated inflammation (rheumatoid arthritis) using NIRF and PA imaging, and suggests its potential for detecting other hypochlorous acid-related inflammatory conditions. As a powerful tool for NIRF / PA dual-modality imaging visualization, MB-ClO demonstrates great potential for monitoring and studying the dynamics of hypochlorous acid in physiological and pathological biological systems, making an invaluable contribution to biomedical research.

[0110] Table 1:

[0111]

[0112]

[0113]

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Claims

1. A near-infrared fluorescence / photoacoustic dual-modality probe, denoted as compound MB-ClO, has the following structure: 。 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 carried out 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 selected from one or a combination of two or more of 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, wherein In step 1) and step 2), the organic solvent is selected from one or a combination of two or more 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, wherein: 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, wherein the use is for non-diagnostic or non-therapeutic purposes.

9. Use of the near-infrared fluorescence / photoacoustic dual-modality probe according to claim 1 in imaging hypochlorous acid in cells or in vivo, wherein the use is for non-diagnostic or non-therapeutic purposes.

10. The use according to claim 9, characterized in that: The near-infrared fluorescence / photoacoustic dual-modality probe can be used in one or both of the following processes: 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