A bodipy-based mitochondrial-targeting antioxidant and a preparation method thereof

Abstract

The application provides a BODIPY-based mitochondrion-targeting antioxidant and a preparation method thereof, and belongs to the technical field of biological medicines. A novel mitochondrion-targeting antioxidant BD-Q10 is synthesized by taking a mitochondrion-targeting group (BODIPY) as a carrier and 2,3-dimethyl-1,4-benzoquinone modified by a flexible carbon chain as an antioxidant, and the antioxidant has strong mitochondrion-targeting positioning capacity and a fluorescence visual function.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a BODIPY-based mitochondrial-targeting antioxidant and its preparation method. Background Technology

[0002] Mitochondria are the "powerhouses" of the cell, providing energy for cellular physiological activities and participating in metabolic processes such as lipid metabolism, calcium ion regulation, redox signaling, steroid hormone biosynthesis, and apoptosis. Mitochondria are the main source of reactive oxygen species (ROS) in eukaryotic cells. However, under most oxidative stress conditions, the cell's endogenous antioxidant system is insufficient to remove excess ROS. Imbalances in ROS metabolism in mitochondria can lead to structural and functional disorders, subsequently triggering the development and progression of many diseases, such as neurodegenerative diseases, metabolic diseases, heart failure, ischemia-reperfusion injury, cancer, and aging. Due to the unique double-membrane structure of mitochondria, many compounds with ROS-scavenging capabilities have difficulty entering the mitochondria to exert their effects. Therefore, developing antioxidants with mitochondrial-targeting functions holds promise for solving the problem of difficult removal of reactive oxygen species within mitochondria.

[0003] The most effective way for antioxidants to enter disease treatment is by binding to carriers (such as lipophilic cations, liposomes, or peptides), enabling their bioactive components to be targeted and transported into the mitochondria. This targeted delivery allows antioxidants to accumulate in high concentrations in cells and mitochondria, thereby protecting cells and tissues from oxidative damage through various mechanisms.

[0004] Real-time monitoring and visualized therapy have garnered increasing attention in disease diagnosis and treatment. Fluorescence spectroscopy, in addition to its high selectivity and sensitivity, offers advantages such as convenient sampling, rapid response, minimal interference, simple operation, low cost, and real-time detection, making it a field of interest for many researchers. Therefore, combining mitochondrial-targeted fluorescent probes with antioxidants to develop novel fluorescently visualized mitochondrial-targeted antioxidants is of great significance for the detection and treatment of related diseases.

[0005] 4,4-Difluoro-4-boron-3a,4a-diaza-s-indane (BODIPY) is a functional dye. BODIPY possesses advantages such as high molar absorptivity, high fluorescence quantum yield, low photobleaching properties, and stable chemical structure. Furthermore, its photophysical properties can be improved by modifying the molecular structure of BODIPY. Therefore, mitochondrial targeting groups can enable antioxidant pharmacophores to cross the cell membrane and mitochondrial membrane into the mitochondria, where they accumulate, thereby enhancing the protection of cells and tissues from oxidative damage. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned problems in existing technologies by providing a BODIPY-based mitochondrial-targeting antioxidant that can be used for fluorescence-visualized therapy, demonstrating the potential of this antioxidant for fluorescence-visualized therapy.

[0007] A novel mitochondrial-targeting antioxidant, BD-Q10, was synthesized using a mitochondrial targeting group (BODIPY) as a carrier and 2,3-dimethyl-1,4-benzoquinone modified with a flexible carbon chain as an antioxidant. The compound was characterized by NMR and mass spectrometry. MTT assays showed that BD-Q10 exhibited low cytotoxicity and good biocompatibility. Laser confocal microscopy was used to investigate the cell imaging and mitochondrial co-localization capabilities of BD-Q10, revealing good cell imaging with clear morphology and bright fluorescence, and good mitochondrial co-localization. A series of in vitro antioxidant experiments showed that BD-Q10 possesses good antioxidant performance, which increases with increasing concentration. These studies demonstrate that we have synthesized an antioxidant with good biocompatibility, strong mitochondrial targeting capability, and fluorescent visualization.

[0008] The mitochondrial-targeting antioxidant based on BODIPY is abbreviated as BD-Q10, and its general structural formula is as follows:

[0009]

[0010] The preparation method of BODIPY-based mitochondrial-targeting antioxidants includes the following steps:

[0011] (1) Add 300 mL of water to a 1000 mL three-necked flask, and slowly add 150 mL of concentrated sulfuric acid dropwise under ice-water bath conditions, controlling the temperature to be less than 40 °C; after the addition is complete, add 216 g of sodium dichromate in batches, controlling the temperature to be less than 40 °C, and stir for 30 minutes until it is completely dissolved and clear, to obtain Jones' reagent for later use; add 44.2 g of 2,3-xylenol and 400 mL of diethyl ether to another 2000 mL three-necked flask, and cool to 0 °C; add the Jones' reagent prepared above, controlling the dropping temperature to 0-10 °C, and after the dropping is complete, let it return to room temperature naturally and stir for 24 h; TLC monitoring (PE; EA = 10:1) shows that the 2,3-xylenol reaction is complete; extract with diethyl ether, dry with anhydrous sodium sulfate, remove the solvent on a rotary evaporator, perform rapid column chromatography on a silica gel column, and concentrate under vacuum to obtain yellow crystalline compound A;

[0012] (2) Add 240 mL of acetonitrile solution with a 1:1 ratio of acetonitrile to water to the reaction flask. Mix 5.3 g of compound A, 10.4 g of bromoundecanoic acid, and 3.98 g of silver nitrate evenly and raise the temperature to 75 °C. Slowly add the ammonium persulfate solution formed by mixing 8.95 g of ammonium persulfate and 60 mL of water to the reaction flask, controlling the dropping rate so that the dropping time is 3 h. After the dropping is completed, stir for another 30 min and monitor the reaction of the raw materials by TLC until the reaction is complete. After extraction, drying and concentration, perform rapid column chromatography on a silica gel column and evaporate to dryness to obtain compound B, which is a yellow oily liquid.

[0013] (3) Synthesis of pyridine BODIPY: Add an appropriate amount of organic solvent to a 1000ml flask, add 8mmol of 2,4-dimethylpyrrole and 4mmol of 4-methylpyridine, and then add 2 drops of trifluoroacetic acid as a catalyst. After reacting for 8 hours, add 4mmol of DDQ, continue to react for 2 hours, add 10ml of triethylamine, stir for half an hour, add 15ml of boron trifluoride diethyl ether, stir at room temperature for 10 hours, add 350ml of saturated sodium bicarbonate aqueous solution, stir at room temperature for half an hour to extract, take the lower organic phase, filter the extract, concentrate, and purify by silica gel column chromatography to obtain compound C;

[0014] (4) Compound B220 mg and compound C367 mg were dissolved in 5 mL of dry acetonitrile and stirred overnight at 83 °C. After the reaction was completed, the product was purified by column chromatography to obtain an orange-yellow solid mitochondrial-targeting antioxidant based on BODIPY.

[0015] The fluorescent targeting group is pyridine (BODIPY).

[0016] The antioxidant pharmacophore is 2,3-dimethyl-1,4-benzoquinone.

[0017] The antioxidant pharmacophore is modified by oxidative decarboxylation coupling of bromoundecanoic acid.

[0018] BODIPY-based mitochondrial-targeting antioxidants can be applied in the field of biomarkers.

[0019] BODIPY-based mitochondrial-targeting antioxidants can be used for live-cell mitochondrial labeling and imaging.

[0020] When detecting mitochondrial morphology changes using BODIPY-based mitochondrial targeted antioxidants, confocal imaging technology is used to detect these morphological changes.

[0021] BODIPY-based mitochondrial-targeting antioxidants can be used in in vitro antioxidant experiments.

[0022] BODIPY-based mitochondrial-targeting antioxidants have at least the following effects:

[0023] The method for synthesizing the mitochondrial-targeted antioxidant that can be used for fluorescence-visualized therapy prepared by the present invention is simple;

[0024] The mitochondrial-targeting antioxidant based on BODIPY prepared by the present invention can specifically target mitochondria;

[0025] The BODIPY-based mitochondrial-targeting antioxidant prepared according to the present invention has good cell membrane penetration and can be highly selectively enriched in mitochondria;

[0026] The BODIPY-based mitochondrial-targeting antioxidant prepared according to the present invention has good cell compatibility and is essentially non-cytotoxic to cells at a concentration of 100 μmol / L.

[0027] The mitochondrial-targeting antioxidant based on BODIPY prepared by the present invention has good cell imaging capabilities;

[0028] The mitochondrial-targeted antioxidant based on BODIPY prepared by the present invention can exert antioxidant effects at nanomolar concentrations. Attached Figure Description

[0029] Figure 1 This is the 1H NMR spectrum of BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY, in an embodiment of the present invention.

[0030] Figure 2 This is the carbon NMR spectrum of BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY, in an embodiment of the present invention.

[0031] Figure 3 This is a mass spectrometry characterization result of BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY, in an embodiment of the present invention.

[0032] Figure 4 This is a cell activity diagram of BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY, in an embodiment of the present invention.

[0033] Figure 5 This is a cell imaging image of BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY, in an embodiment of the present invention.

[0034] Figure 6 This is a graph showing the iron reduction capacity of BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY, in an embodiment of the present invention.

[0035] Figure 7 This is a graph showing the total antioxidant capacity of BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY, in an embodiment of the present invention.

[0036] Figure 8 This is a graph showing the SOD inhibition rate and SOD enzyme activity of BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY, in an embodiment of the present invention.

[0037] Figure 9 This is a graph showing the DPPH free radical scavenging ability of BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY, in an embodiment of the present invention.

[0038] Figure 10 This is a graph showing the detection of superoxide anion production in BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY, in an embodiment of the present invention.

[0039] Figure 11 This is a graph showing the hydroxyl radical scavenging ability of BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY, in an embodiment of the present invention. Detailed Implementation

[0040] The following are specific embodiments of the present invention, which are described in conjunction with the accompanying drawings. However, the present invention is not limited to these embodiments.

[0041] Example 1

[0042] In this embodiment, a BODIPY-based mitochondrial-targeting antioxidant (BD-Q10) was prepared. The specific process is as follows:

[0043] (1) Add 300 mL of water to a 1000 mL three-necked flask. Under ice-water bath conditions, slowly add 150 mL of concentrated sulfuric acid, controlling the temperature to be below 40 °C. After the addition is complete, add 216 g of sodium dichromate in batches, controlling the temperature to be below 40 °C (basically non-exothermic). Stir for 30 minutes after the addition is complete until completely dissolved and clear, and set aside for use. In another 2000 mL three-necked flask, add 44.2 g of 2,3-xylenol and 400 mL of diethyl ether, and cool to 0 °C. Add the Jones reagent prepared above, controlling the addition temperature to be 0-10 °C. After the addition is complete, allow it to return to room temperature naturally and stir for 24 h. TLC monitoring (PE; EA = 10:1) shows that the 2,3-xylenol reaction is complete. Extract with diethyl ether, dry with anhydrous sodium sulfate, remove the solvent on a rotary evaporator, perform rapid column chromatography on a silica gel column, and concentrate under vacuum to obtain 17.6 g of yellow crystals (compound A), yield 40%.

[0044] (2) Add 240 mL of a solution (acetonitrile:water = 1:1), 5.3 g of compound A, 10.4 g of bromoundecanoic acid, and 3.98 g of silver nitrate to a reaction flask. After mixing thoroughly, raise the temperature to 75 °C. Slowly add the prepared ammonium persulfate solution (8.95 g of ammonium persulfate, 60 mL of water) dropwise to the reaction flask, controlling the dropping rate (dropping time is 3 h). After the addition is complete, stir for another 30 min. Monitor the reaction by TLC (petroleum ether:ethyl acetate = 10:1 as the eluent). The reaction proceeds are complete. After extraction, drying, and concentration, perform rapid column chromatography on a silica gel column (petroleum ether:ethyl acetate = 100:1 as the eluent). After evaporation to dryness, 8.3 g of a yellow oily liquid (compound B) is obtained, with a yield of 60%.

[0045] (3) Synthesis of pyridine BODIPY: Add an appropriate amount of organic solvent to a 1000ml flask, add 8mmol of 2,4-dimethylpyrrole and 4mmol of 4-methylpyridine, and then add 2 drops of trifluoroacetic acid as a catalyst. After reacting for 8 hours, add 4mmol of DDQ, continue to react for 2 hours, add 10ml of triethylamine, stir for half an hour, add 15ml of boron trifluoride diethyl ether, stir at room temperature for 10 hours, add 350ml of saturated sodium bicarbonate aqueous solution, stir at room temperature for half an hour to extract, take the lower organic phase, filter the extract, concentrate, and purify by silica gel column chromatography to obtain compound C;

[0046] (4) Compound B220 m and compound C 367 mg were dissolved in 5 mL of dry acetonitrile and stirred overnight at 83 °C. After the reaction was completed, the mixture was purified by column chromatography (neutral alumina as the packing material, dichloromethane:methanol = 80:1-40:1 as the eluent) to obtain an orange-yellow solid (BD-Q10) with a yield of 63%.

[0047] The reaction formula is as follows:

[0048]

[0049] In this process, step (1) yields compound A, step (2) yields compound B, step (3) yields compound C, and step (4) yields the target product.

[0050] Example 2

[0051] Cytotoxicity assay of mitochondrial-targeting antioxidant BD-Q10 based on BODIPY.

[0052] Cultured PC-3 cells were seeded into 96-well plates. After culturing in an incubator for 24 hours, the cells were drugged. BD-Q10 concentration gradients were set at 10⁻⁹, 10⁻⁸, 10⁻⁷, and 10⁻⁶ mol / L. After drug administration, cells were cultured in an incubator for another 48 hours. Cell viability was then measured using a commercially available MTT assay kit. The experiment was repeated three times. Cell growth curves were plotted with drug concentration on the x-axis and absorbance ratio on the y-axis. Figure 4 The study demonstrated the cytotoxicity of the probe, showing that BD-Q10 at a concentration of 10⁻⁹-10⁻⁶ mol / L had no significant toxicity to PC-3 cells, indicating that the compound has low cytotoxicity and good biocompatibility.

[0053] Example 3

[0054] Cell imaging capabilities of BODIPY-based mitochondrial-targeting antioxidant BD-Q10.

[0055] Cultured PC-3 cells were incubated with F-Q10 and BD-Q10 (1.00 μmol / L, 60 min), respectively, and then further incubated with a mitochondrial fluorescent probe (MTR, 1.00 μmol / L, 20 min). After washing three times with PBS, images were captured using an OLYMPUS FV3000 laser confocal fluorescence microscope. The green channel was collected at λem = 488 nm, and the red channel at λem = 561 nm. Figure 5 As shown, BD-Q10 exhibits green fluorescence, and the cell structure and morphology are relatively clear and intact, displaying bright fluorescence. Colocalization analysis showed excellent overlap between BD-Q10 and the luminescent region, with a Pearson correlation coefficient of Pr = 0.967. These experimental results demonstrate that BD-Q10 is a promising fluorescently visualized mitochondrial-targeting antioxidant, providing an important foundation for fluorescence-visualized therapy and dynamic monitoring.

[0056] Example 4

[0057] Experimental detection of iron ion reducing capacity of BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY.

[0058] BD-Q10 was prepared into solutions of different concentrations (10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶ mol / L) using PBS buffer. These solutions were then added to a 96-well plate along with the iron reducing power assay working solution prepared according to the manufacturer's instructions. The mixture was thoroughly mixed, and the absorbance was measured at 700 nm using a microplate reader within 10 minutes. The test results are as follows. Figure 6 As shown, the reducing power of BD-Q10 for iron ions gradually increases with increasing concentration.

[0059] Example 5

[0060] Determination of total antioxidant capacity of BD-Q10, a mitochondrial-targeting antioxidant, based on BODIPY.

[0061] PC-3 cells cultured with different concentrations of BD-Q10 (10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶ mol / L) were decomposed by aspirating the cell culture medium and washed twice with ice-cold PBS. The sample preparation solution provided by the ABTS kit was added at a ratio of 100-200 μL per million cells, and the cells were gently pipetted to lyse them thoroughly. The cells were centrifuged at approximately 12000 rpm for 3-5 minutes at 4°C, and the supernatant was used as the test sample. The protein concentration in the supernatant was determined using the BCA kit. The working solutions were prepared according to the kit instructions. The diluted supernatants of the cells treated with different drugs (protein concentration: 0.4 mg / mL) were added to 96-well plates, and a blank control was set up. The cells were incubated at 37°C for 30 minutes, and the absorbance was measured at 450 nm. The total antioxidant capacity was calculated using the relevant formula. (See below) Figure 7 As shown, the total antioxidant capacity of BD-Q10 gradually increases with increasing concentration.

[0062] Example 6

[0063] Detection of total SOD inhibition rate and SOD enzyme activity of BD-Q10, a mitochondrial-targeting antioxidant, based on BODIPY.

[0064] PC-3 cells cultured with different concentrations of BD-Q10 (10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶ mol / L) were decomposed by aspirating the cell culture medium and washed twice with ice-cold PBS. The sample preparation solution provided by the SOD detection kit was added at a ratio of 100-200 μL per million cells, and the cells were gently pipetted to ensure complete cell lysis. The cells were centrifuged at approximately 12000 rpm for 3-5 minutes at 4°C, and the supernatant was used as the test sample. The protein concentration in the supernatant was determined using the BCA kit. The working solution was prepared according to the kit instructions. The diluted supernatant from the cells treated with different drugs (protein concentration: 0.4 mg / mL) was added to a 96-well plate, and a blank control was set up. The cells were incubated at 37°C for 30 minutes, and the absorbance was measured at 450 nm. The SOD inhibition rate and SOD enzyme activity were calculated using the relevant formulas. Figure 8 As shown, with increasing concentration, the inhibition rate of BD-Q10 on SOD and the activity of SOD enzyme also gradually increased.

[0065] Example 7

[0066] DPPH free radical scavenging rate of BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY.

[0067] PC-3 cells cultured with different concentrations of BD-Q10 (10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶ mol / L) were decanted from the cell culture medium, washed twice with ice-cold PBS, and cell lysis buffer was added at a ratio of 100-200 μL per million cells. The cells were gently pipetted to ensure complete lysis. The cells were centrifuged at approximately 12000 rpm for 3-5 minutes at 4°C, and the supernatant was used as the test sample. The protein concentration in the supernatant was determined using a BCA kit. Working solutions were prepared according to the kit instructions. The diluted supernatants from the different drug treatments (protein concentration: 0.4 mg / mL) were added to 96-well plates, and a blank control was set up. The DPPH free radical scavenging rate was calculated using the relevant formula. (See below) Figure 9 As shown, the scavenging ability of BD-Q10 for DPPH free radicals also increases with increasing concentration.

[0068] Example 8

[0069] Detection of superoxide anion production content of BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY.

[0070] PC-3 cells cultured with different concentrations of BD-Q10 (10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶ mol / L) were decanted from the cell culture medium, washed twice with pre-cooled PBS, and cell lysis buffer was added at a ratio of 100-200 μL per million cells. The cells were gently pipetted to ensure complete lysis. The cells were centrifuged at approximately 12000 rpm for 3-5 minutes at 4°C, and the supernatant was used as the test sample. The protein concentration in the supernatant was determined using a BCA assay kit. The working solutions were prepared according to the kit instructions. The diluted supernatants of the cells treated with different drugs (protein concentration: 0.4 mg / mL) were added to 96-well plates, and a blank control was set up. The microplate reader was preheated for 30 minutes, the wavelength was adjusted to 530 nm, and the absorbance was measured. The superoxide anion production was calculated using the relevant formula. (See below) Figure 10 As shown, the amount of superoxide anion produced by BD-Q10 gradually decreases with increasing concentration. This indicates that BD-Q10 has a good ability to inhibit the production of superoxide anions.

[0071] Example 9

[0072] Determination of the hydroxyl radical scavenging capacity of BD-Q10, a mitochondrial-targeting antioxidant based on BODIPY.

[0073] PC-3 cells cultured with different concentrations of BD-Q10 (10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶ mol / L) were decanted from the cell culture medium, washed twice with pre-cooled PBS, and cell lysis buffer was added at a ratio of 100-200 μL per million cells. The cells were gently pipetted to ensure complete lysis. The cells were centrifuged at approximately 12000 rpm for 3-5 minutes at 4°C, and the supernatant was used as the test sample. The protein concentration in the supernatant was determined using a BCA assay kit. The working solutions were prepared according to the kit instructions. The diluted supernatants of the cells treated with different drugs (protein concentration: 0.4 mg / mL) were added to 96-well plates, and a blank control was set up. The microplate reader was preheated for 30 minutes, and the wavelength was adjusted to 536 nm. The superoxide anion production was calculated using the relevant formula. (See below) Figure 11 As shown, the scavenging ability of BD-Q10 against hydroxyl radicals increases with increasing drug concentration.

[0074] In summary, the mitochondrial-targeted antioxidant prepared by this invention, which can be used for fluorescence-guided therapy, exhibits low cytotoxicity, good biocompatibility, and excellent cell imaging and mitochondrial co-localization capabilities. In vitro antioxidant experiments in related embodiments demonstrate that the mitochondrial-targeted antioxidant prepared by this invention possesses good antioxidant capacity and shows promising commercial application prospects.

[0075] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A method for preparing a BODIPY-based mitochondrial-targeting antioxidant, characterized in that, Includes the following steps: (1) Add 300 mL of water to a 1000 mL three-necked flask, and slowly add 150 mL of concentrated sulfuric acid dropwise under ice-water bath conditions, controlling the temperature to be less than 40 °C; after the addition is complete, add 216 g of sodium dichromate in batches, controlling the temperature to be less than 40 °C, and stir for 30 minutes until it is completely dissolved and clear, to obtain Jones' reagent for later use; add 44.2 g of 2,3-xylenol and 400 mL of diethyl ether to another 2000 mL three-necked flask, and cool to 0 °C; add the Jones' reagent prepared above, controlling the dropping temperature to 0-10 °C, and after the dropping is complete, let it return to room temperature naturally and stir for 24 h; TLC monitoring (PE; EA = 10:1) shows that the 2,3-xylenol reaction is complete; extract with diethyl ether, dry with anhydrous sodium sulfate, remove the solvent on a rotary evaporator, perform rapid column chromatography on a silica gel column, and concentrate under vacuum to obtain yellow crystalline compound A; (2) Add 240 mL of acetonitrile solution with a 1:1 ratio of acetonitrile to water to the reaction flask. Mix 5.3 g of compound A, 10.4 g of bromoundecanoic acid, and 3.98 g of silver nitrate evenly and raise the temperature to 75 °C. Slowly add the ammonium persulfate solution formed by mixing 8.95 g of ammonium persulfate and 60 mL of water to the reaction flask, controlling the dropping rate so that the dropping time is 3 h. After the dropping is completed, stir for another 30 min and monitor the reaction of the raw materials by TLC until the reaction is complete. After extraction, drying and concentration, perform rapid column chromatography on a silica gel column and evaporate to dryness to obtain compound B, which is a yellow oily liquid. (3) Synthesis of pyridine BODIPY: Add an appropriate amount of organic solvent to a 1000ml flask, add 8mmol of 2,4-dimethylpyrrole and 4mmol of 4-methylpyridine, and then add 2 drops of trifluoroacetic acid as a catalyst. After reacting for 8 hours, add 4mmol of DDQ, continue to react for 2 hours, add 10ml of triethylamine, stir for half an hour, add 15ml of boron trifluoride diethyl ether, stir at room temperature for 10 hours, add 350ml of saturated sodium bicarbonate aqueous solution, stir at room temperature for half an hour to extract, take the lower organic phase, filter the extract, concentrate, and purify by silica gel column chromatography to obtain compound C; (4) Compound B220 mg and compound C367 mg were dissolved in 5 mL of dry acetonitrile and stirred overnight at 83 °C. After the reaction was completed, the product was purified by column chromatography to obtain an orange-yellow solid mitochondrial-targeting antioxidant based on BODIPY.

2. The method for preparing a BODIPY-based mitochondrial-targeting antioxidant according to claim 1, characterized in that, The antioxidant pharmacophore in BODIPY-based mitochondrial-targeting antioxidants is 2,3-dimethyl-1,4-benzoquinone.

3. The method for preparing a BODIPY-based mitochondrial-targeting antioxidant according to claim 1, characterized in that, The antioxidant pharmacophore is modified by oxidative decarboxylation coupling of bromoundecanoic acid.

4. The method for preparing a BODIPY-based mitochondrial-targeting antioxidant according to claim 1, characterized in that, BODIPY-based mitochondrial-targeting antioxidants can be applied in the field of biomarkers.

5. The method for preparing a BODIPY-based mitochondrial-targeting antioxidant according to claim 1, characterized in that, BODIPY-based mitochondrial-targeting antioxidants can be used for live-cell mitochondrial labeling and imaging.

6. The method for preparing a BODIPY-based mitochondrial-targeting antioxidant according to claim 1, characterized in that, When detecting mitochondrial morphology changes using BODIPY-based mitochondrial targeted antioxidants, confocal imaging technology is used to detect these morphological changes.

7. The method for preparing a BODIPY-based mitochondrial-targeting antioxidant according to claim 1, characterized in that, BODIPY-based mitochondrial-targeting antioxidants can be used in in vitro antioxidant experiments.

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