Heavy-atom-free BODIPY compound as well as preparation method and application thereof

Abstract

The invention discloses a heavy-atom-free BODIPY (boron dipyrromethene) compound as well as a preparation method and application thereof, the compound can be activated only in H2O2 overexpressed cancer cells and cannot be activated in normal cells, so that the problem of self toxicity of a heavy-atom BODIPY sound-sensitive agent can be effectively solved; the compound disclosed by the invention can realize lipid droplet targeting after entering cancer cells, and can induce mass production of active oxygen in lipid droplets through ultrasound, so that ferroptosis and immunogenic death of tumor cells are induced, and therefore, the compound disclosed by the invention can effectively inhibit proliferation of stratigenic tumor cells and has a wide application scene.

Description

Technical Field

[0001] The present invention relates to a heavy atom-free fluoroboron dipyrrole compound induced by H2O2, and also relates to a preparation method of the fluoroboron dipyrrole compound and application of the compound as a sonosensitizer in sonodynamic therapy. Background Art

[0002] Among many new cancer treatments, photodynamic therapy (PDT) has attracted widespread attention due to its advantages of being non-invasive, low in drug resistance, low in toxicity, and capable of eliminating invisible tumor lesions. However, due to the difficulty of light source penetrating deep tissues, PDT is not effective in treating deep tumors. Ultrasound (US), especially low-power ultrasound (1 MHz, 0.5-2.5 W cm -2 ) has been widely used in biomedical and clinical fields due to its high tissue penetration ability, biosafety and effectiveness.

[0003] Compared with photodynamic therapy, sonodynamic therapy (SDT) not only has a greater tissue penetration depth, but can also trigger the rapid release of drugs to achieve synergistic sonochemical therapy. The principle of sonodynamic tumor treatment is that the cavitation effect generated by low-frequency ultrasound stimulates the sonosensitizer to produce reactive oxygen species that can kill tumor cells. However, existing organic sonosensitizer molecules used in sonodynamic therapy, such as porphyrins, phthalocyanines, or inorganic substances such as TiO2, have the disadvantages of low tumor retention rate, insufficient reactive oxygen production after sonosensitization, or self-bioincompatibility (Angew. Chem. Int. Ed, 2022, 61, e202113506.), which limits their application in sonodynamic therapy.

[0004] BODIPY dyes with π-electron conjugation have the potential to be used as sonosensitizers in sonodynamic therapy, increasing the production of reactive oxygen species. However, these heavy-atom BODIPY sonosensitizers often exhibit strong ultrasound toxicity, killing cancer cells while also having significant side effects on normal cells (Eur. J. Med. Chem, 2024, 264, 116035). Even if current BODIPY sonosensitizers overcome their inherent toxicity, most can only generate type II reactive oxygen species and are unable to overcome the constraints of the hypoxic environment of tumors. Summary of the Invention

[0005] Purpose of the Invention: This invention aims to provide a fluoroboron dipyrrole compound that is activated only in cancer cells overexpressing H2O2 and not in normal cells, thereby effectively addressing the inherent toxicity of heavy-atom BODIPY sonosensitizers. The invention also generates type I reactive oxygen species to overcome hypoxia and enhance therapeutic efficacy. Another purpose of the invention is to provide a method for preparing the aforementioned fluoroboron dipyrrole compound and its use as a sonosensitizer in sonodynamic therapy.

[0006] Technical solution: The H2O2-induced heavy atom-free fluoroborane dipyrrole compound of the present invention has the following structural formula:

[0007]

[0008] The preparation method of the above-mentioned fluoroborane dipyrrole compound comprises the following steps:

[0009] (1) 3-Quinolinecarboxaldehyde was dissolved in anhydrous tetrahydrofuran, and 2,4-dimethylpyrrole and trifluoroacetic acid were added sequentially in an ice bath under an inert atmosphere, and the mixture was reacted at room temperature overnight;

[0010] (2) In an ice bath, 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), triethylamine (acid binding agent) and boron trifluoride ethyl ether (providing boron source coordination) are sequentially added to the system of step (1) to fully react; after the reaction is completed, the solvent is removed by rotary evaporation, and the crude product is separated and purified by column chromatography to obtain a fluoroboron dipyrrole compound modified with a quinoline group;

[0011] (3) dissolving the fluoroboron dipyrrole compound modified with a quinoline group and 4-bromomethylphenylboronic acid pinacol ester obtained in step (2) in an organic solvent, stirring and refluxing the mixture under an inert gas atmosphere; after the reaction, removing the solvent by rotary evaporation, and separating and purifying the crude product by column chromatography to obtain a lipid droplet-targeting fluoroboron dipyrrole compound free of heavy atoms, named BDP-Bpin;

[0012] Among them, the structural formula of the fluoroboron dipyrrole compound modified with a quinoline group is:

[0013]

[0014] Wherein, in step (1), the molar ratio of 3-quinolinecarboxaldehyde and 2,4-dimethylpyrrole added is 1:2.

[0015] In step (2), the molar ratio of 2,3-dichloro-5,6-dicyanobenzoquinone, boron trifluoride etherate and triethylamine is 1:49:43; after adding 2,3-dichloro-5,6-dicyanobenzoquinone, triethylamine and boron trifluoride etherate are added in sequence at an interval of not less than 7 hours.

[0016] In step (3), the reflux reaction temperature is 60° C. and the reaction time is 24 h. The molar ratio of the fluoroboron dipyrrole compound modified with a quinoline group to 4-bromomethylphenylboronic acid pinacol ester is 1:3, and the compound is named BDP-QL.

[0017] The above-mentioned fluoroboron dipyrrole compound is used as a sonosensitizer in sonodynamic therapy.

[0018] Principle of the Invention: The phenylboronic acid ester structure of the compound of this invention is broken down in cancer cells overexpressing H2O2, forming a quinoline nucleus that is inactive in normal cells. The compound of this invention carries a positive charge, making it more soluble than the neutral quinoline nucleus. Upon entering cancer cells, the quinoline nucleus is released, enhancing aggregation. Under ultrasound, type I reactive oxygen species are generated, leading to ferroptosis and immunogenic cell death. Furthermore, the compound of this invention can target lipid droplets, thereby inducing mitochondrial dysfunction and inhibiting tumor cell proliferation.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The compound of the present invention can effectively solve the problem of the self-toxicity of heavy atom BODIPY sonosensitizers and has good biocompatibility; (2) The sonosensitizer of the present invention can achieve lipid droplet targeting after entering cancer cells, and can induce the production of a large amount of type I reactive oxygen species in lipid droplets through ultrasound, thereby overcoming the disadvantage of insufficient therapeutic effect caused by the hypoxic environment in the tumor; (3) The large amount of reactive oxygen species produced can induce ferroptosis and immunogenic death of tumor cells, so the compound of the present invention can effectively inhibit the proliferation of tumor cells, has good anti-tumor effects, and has a wide range of application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the H NMR spectrum of the compound BDP-QL prepared in Example 1;

[0021] Figure 2 This is the C NMR spectrum of the compound BDP-QL prepared in Example 1;

[0022] Figure 3 This is the H NMR spectrum of the compound BDP-Bpin prepared in Example 1;

[0023] Figure 4 This is the C NMR spectrum of the compound BDP-Bpin prepared in Example 1;

[0024] Figure 5 The specific responses of the compounds BDP-Bpin and BDP-QL prepared in Example 1 to H2O2;

[0025] Figure 6 The compounds BDP-Bpin, BDP-QL and [Ru(bpy)3] 2+ Total reactive oxygen species production capacity in vitro;

[0026] Figure 7 The EPR patterns of the active oxygen species generated by the compounds BDP-Bpin and BDP-QL prepared in Example 1 include hydroxyl radicals, superoxide anions, and singlet oxygen;

[0027] Figure 8 is the cell survival rate of the compound BDP-Bpin prepared in Example 1 in HeLa cells after ultrasound, wherein a is the ultrasound group and b is the non-sound group;

[0028] Figure 9 This is a confocal fluorescence imaging image of the subcellular organelle distribution of the compound BDP-Bpin prepared in Example 1 in HeLa cells;

[0029] Figure 10 This is a confocal fluorescence imaging image of the cell uptake of the compound BDP-Bpin prepared in Example 1 in HeLa cells;

[0030] Figure 11 The cell death mode induced by the compound BDP-Bpin prepared in Example 1 in HeLa cells;

[0031] Figure 12 The compound BDP-Bpin prepared in Example 1 induced ATP release in HeLa cells;

[0032] Figure 13 This is an immunoblot protein image of the compound BDP-Bpin prepared in Example 1 that induces ferroptosis in HeLa cells after ultrasound;

[0033] Figure 14 This is a quantitative bar graph of the GSH / GSSG content of the compound BDP-Bpin prepared in Example 1 in HeLa cells after ultrasound;

[0034] Figure 15 The graph shows the results of the compound BDP-Bpin prepared in Example 1 inducing immunogenic cell death in HeLa cells after ultrasound; wherein a and b are immunofluorescence confocal imaging images of CRT1 and HMGB1, respectively;

[0035] Figure 16 This is a confocal image of live-dead staining of the compound BDP-Bpin prepared in Example 1, which induced cell death in HeLa cells after ultrasound;

[0036] Figure 17 This is a depth test of the compound BDP-Bpin prepared in Example 1 under ultrasound and light conditions;

[0037] Figure 18 This is a diagram showing the mechanism of action of the compound BDP-Bpin prepared in Example 1 in cells. DETAILED DESCRIPTION

[0038] Example 1

[0039] The preparation method of the heavy atom-free fluoroboron dipyrrole compound (BDP-Bpin) of the present invention comprises the following steps:

[0040] (1) Synthesis of sonosensitizer BDP-QL: 3-quinolinecarboxaldehyde powder (943 mg, 6 mmol) was dissolved in anhydrous tetrahydrofuran (40 mL) and stirred under argon atmosphere; 2,4-dimethylpyrrole (1.14 mL) was added thereto under ice bath and stirred for 30 min; trifluoroacetic acid (0.1-0.2 mL) was added thereto under ice bath and reacted at room temperature overnight; 2,3-dichloro-5,6-dicyanobenzoquinone (1.362 g, 6 mmol) was dissolved in 10 mL anhydrous tetrahydrofuran and added to the above reaction system and reacted for 7 h; anhydrous triethylamine (36 mL) and boron trifluoride etherate (36 mL) were slowly added dropwise to the above reaction system under ice bath and reacted for one day; after the reaction, the solvent was removed by distillation under reduced pressure and the crude product was further purified by column chromatography to obtain an orange solid, named BDP-QL, 800 mg, with a yield of 35%, and the H NMR spectrum was as follows: Figure 1 As shown; 1 H NMR (400 MHz, Chloroform-d) δ9.82 (s, 1H), 9.02 (s, 1H), 8.83 (d, J = 8.7 Hz, 1H), 8.42 (d, J = 7.5 Hz, 1H), 8.11 (t, 1H), 7.29 (t, 1H), 6.08 (s, 1H), 5.56 (s, 1H), 2.55 (s, 3H), 1.29 (s, J = 12.1 Hz, 9H). Figure 2 shown 13 C NMR (101MHz, CDCl3) δ156.40,148.91,147.71,142.80,137.39,135.81,131.81,130.67 ,129.53,128.36,127.91,127.74,127.36,121.82,53.44,15.26,14.69,14.66,14.64.

[0041]

[0042] (2) Synthesis of sonosensitizer BDP-Bpin: BDP-QL (300 mg, 0.8 mmol) prepared in step (1) and 4-bromomethylphenylboronic acid pinacol ester (712.8 mg, 2.4 mmol) were dissolved in 10 mL of anhydrous acetonitrile and reacted at 60° C. under an argon atmosphere for 24 h. After the reaction, the solvent was removed by distillation under reduced pressure, and the crude product was further purified by column chromatography to obtain a red solid named BDP-Bpin, 88 mg, with a yield of 21%. The H NMR spectrum showed Figure 3As shown; 1 H NMR (400 MHz, Methanol-d4) δ9.97 (s, 1H), 9.55 (s, 1H), 8.81 (d, J = 9.1 Hz, 1H), 8.58 (d, J = 8.3 Hz, 1H), 8.42 (t, J = 8.0 Hz, 1H), 8.19 (t, J = 7.7 Hz, 1H), 7.68 (d, 2H), 7.43 (d, J = 12.8 Hz, 2H), 6.51 (s, 2H), 6.22 (s, 2H), 2.56 (s, 6H), 1.41 (s, 12H), 1.32 (s, 3H), 0.92 (s, J = 6.8 Hz, 3H). Figure 4 shown 13 CNMR(101MHz,MeOD)δ158.00,149.03,148.98,148.89,142.55,138.20,137.53,136.05,135.35,131.67,131.49,1 31.27,131.24,130.39,128.92,126.50,126.36,122.42,119.29,84.09,61.01,23.77,23.64,14.76,14.72,13.35.

[0043]

[0044] Example 2

[0045] The selectivity of the sonosensitizers BDP-Bpin and BDP-QL prepared in Example 1 for different types of reactive oxygen species in vitro:

[0046] Prepare 1mM H2O2 solution with 30wt.% H2O2 solution and water; prepare 1mM NaClO solution with hypochlorous acid and sodium hydroxide; prepare 1mM ·OH solution with ferrous chloride and water; prepare 1mM ·OH solution with H2O2 solution and NaClO solution; 1 O2 solution. Different reactive oxygen species concentrations were added to a 10 μM BDP-Bpin dilution to a final concentration of 100 μM, and then fluorescence spectroscopy was performed.

[0047] The results of the selectivity of sonosensitizer BDP-Bpin for different types of reactive oxygen species in vitro are as follows Figure 5 Select 1The selectivity of BDP-Bpin for reactive oxygen species was tested by using multiple reactive oxygen species including O2, H2O2, NaClO, and ·OH. BDP-QL had no specific response to the above four reactive oxygen species, and its fluorescence signal was similar to that of the control group. However, the fluorescence response signal of BDP-Bpin to H2O2 was significantly stronger than that of the other three reactive oxygen species and the control group. The results showed that BDP-Bpin had a specific response to H2O2.

[0048] Example 3

[0049] BDP-Bpin, BDP-QL and [Ru(bpy)3] 2+ Total reactive oxygen species generation capacity in vitro:

[0050] A 2 mL solution of 10 μM BDP-Bpin and 20 μM DCFH-DA was prepared in H2O at 0.6 W / cm 2 The power of ultrasound was tested every 5 minutes, for a total of half an hour.

[0051] The experimental results of the sonosensitizer BDP-Bpin on the generation of total reactive oxygen species by ultrasound in vitro are as follows: Figure 6 As shown in Figure 2, the fluorescence signal increased most rapidly in the BDP-Bpin + DCFH-DA group after ultrasound, while the signal changes in the BDP-QL + DCFH-DA and DCFH-DA groups alone were minimal. These results suggest that BDP-Bpin has a capacity second only to terpyridine ruthenium in generating total reactive oxygen species.

[0052] Example 4

[0053] Characterization of reactive oxygen species generated by BDP-Bpin and BDP-QL after ultrasonic treatment:

[0054] A 10 mM stock solution of BDP-Bpin and BDP-QL was prepared in acetonitrile, and the sample to be tested was prepared with 190 μL ultrapure water, 10 μL DMPO and 2 μL drug stock solution. The ultrasound group was operated at 1 MHz and 0.6 W / cm 2 The test was immediately performed after 5 minutes of ultrasound (hydroxyl radicals); 130 μL ultrapure water, 50 μL DMPO, 20 μL methanol and 2 μL drug concentrate were used to prepare the test sample. The ultrasound group used 1 MHz, 0.6 W / cm 2 The test was performed immediately after 5 minutes of ultrasound at a power of 100W / cm2 (superoxide anion free radicals). A 100mM concentrated stock of TEMP was prepared with acetonitrile, and the sample to be tested was prepared with 40μL of the concentrated TEMP stock, 160μL of ultrapure water, and 2μL of the drug concentrated stock. The ultrasound group was operated at 1MHz, 0.6W / cm2 2 The test (singlet oxygen) was performed immediately after 5 minutes of ultrasound at a power of 100 nm.

[0055] The experimental results related to the EPR generation of reactive oxygen species by sonosensitizers BDP-Bpin and BDP-QL are as follows Figure 7 a(·OH), 7b(O2· - )、7c( 1 No ROS signal was captured in the BDP-QL or BDP-Bpin alone group, while the BDP-QL+US and BDP-Bpin+H2O2+US groups did capture signals. This indirectly confirms that BDP-Bpin converts to BDP-QL in a H2O2 environment and demonstrates that the sonosensitizer BDP-Bpin generates type I ROS after ultrasound, demonstrating type I sonodynamic therapy capabilities, potentially enabling BDP-Bpin to overcome hypoxic tumor treatment.

[0056] Example 5

[0057] Cytotoxic activity of BDP-Bpin on different cells before and after ultrasound:

[0058] The MTT colorimetric method was used to analyze the anti-proliferative effect of BDP-Bpin. Human cervical cancer cells HeLa, human lung cancer cells A549, human breast cancer cells MDA-MB-231 and human lung fibroblast-like cells HLF were selected as cell models. 100 μL cell suspension was diluted with 5×10 4 The cells were seeded at a density of 100 cells / mL in a 96-well plate and incubated in an incubator overnight. Then, a certain concentration gradient of sonosensitizer BDP-Bpin was added to the 96-well plate and the cells were incubated at 37°C for 4 h. Then, the ultrasound group was set at 1 MHz, 0.6 W / cm 2 The cells were sonicated at 100 μL for 10 minutes and incubated for another 20 hours. The non-sonicated group was incubated for 24 hours. 20 μL of MTT was added to each well and incubated for an additional 4 hours. All culture medium was carefully removed, and 150 μL of DMSO was added. The absorbance was measured using a microplate reader, and cytotoxicity data were calculated using SPSS software.

[0059] The experimental results of the toxicity of sonosensitizer BDP-Bpin to cells are as follows: Figure 8 shown. Figure 8 aThe results showed that in the non-ultrasound group, BDP-Bpin had almost no toxicity to cancer cells and normal cells. In the ultrasound group, BDP-Bpin showed concentration-dependent toxicity to cancer cells, but had almost no toxicity to normal cells ( Figure 8 b) These results indicate that the sonosensitizer BDP-Bpin has good sonotoxicity to HeLa cells and low toxicity to normal cells, indicating that it has selective killing effect on cancer cells.

[0060] Example 6

[0061] Subcellular organelle localization of sonosensitizer BDP-Bpin:

[0062] HeLa cells were seeded in 35 mm Corning laser confocal culture dishes. When the cell density grew to 70%, 50 μM BDP-Bpin was added. After incubation for 4 h, ultrasound was applied at 1 MHz and 0.6 W / cm 2 Ultrasound was performed at a power of 1000 nm for 10 minutes and the cells were incubated for 20 hours. The culture medium was aspirated and washed twice with PBS. 500 μL of the commercial lipid droplet deep red fluorescent probe and commercial mitochondrial red probe were added in the dark. The cells were incubated in a 37°C incubator for 30 minutes, then the probes were aspirated, washed twice with PBS, and replaced with fresh preheated serum-free medium. The cells were then immediately observed using a confocal microscope (A1, Nikon, Japan). Commercial lipid droplet deep red fluorescent probe Lipi-Deep Red and commercial mitochondrial red probe Mito-Tracker Red CMXRos: λ ex =640nm,λ em =650-700nm; Commercial Mitochondria Red Probe: λ ex =579nm,λ em =599nm.

[0063] The results of subcellular organelle localization experiments are as follows Figure 9 As shown, the fluorescence signal of the sonosensitizer BDP-Bpin hardly overlaps with the mitochondrial probe signal, but almost completely overlaps with the lipid droplet probe signal, indicating that BDP-Bpin is mainly distributed in lipid droplets and has excellent lipid droplet targeting ability.

[0064] Example 7

[0065] Application of the sonosensitizer BDP-Bpin prepared in Example 1 to the cellular uptake of HeLa cells:

[0066] HeLa cells were seeded in 35 mm Corning laser confocal culture dishes. When the cell density reached 70%, 10 μM BDP-Bpin was added. The cells were incubated for different time periods (0 min, 10 min, 30 min, and 50 min). The BDP-Bpin was aspirated, the cells were washed twice with PBS, and then replaced with fresh pre-warmed serum-free medium. The cells were then immediately observed using a confocal microscope. ex =488nm,λ em =525nm.

[0067] The experimental results of the cellular uptake of sonosensitizer BDP-Bpin are as follows Figure 10 As shown, as time goes by, the fluorescence of BDP-Bpin in cancer cells (HeLa cells) changes significantly compared with that in normal cells (HLF cells), indicating that the sonosensitizer can be specifically activated by excessive H2O2 in cancer cells.

[0068] Example 8

[0069] Mode of cell death induced by ultrasound-treated BDP-Bpin:

[0070] 100 μL of cell suspension was diluted with 5×10 4 Cells were seeded at a density of 100 cells / mL in a 96-well plate and incubated in an incubator overnight. Apoptosis inhibitors, ferroptosis inhibitors, necrosis inhibitors, autophagy inhibitors, and pyroptosis inhibitors were added, respectively. After incubation for 1 hour, the inhibitors were precipitated. A certain concentration gradient of sonosensitizer BDP-Bpin was added to the 96-well plate, and the cells were incubated at 37°C for 4 hours. Then, ultrasound was applied at 1 MHz, 0.6 W / cm 2 Ultrasonicate at 1000W for 10 minutes and continue incubating for 20 hours. Add 20 μL MTT to each well and incubate for another 4 hours. Carefully remove all culture medium, add 150 μL DMSO, measure the absorbance with a microplate reader, and calculate the cell absorbance using SPSS software.

[0071] The experimental results of ultrasound-treated BDP-Bpin-induced cancer cell death are as follows: Figure 11 As shown in Figure 2, treatment of HeLa cells with apoptosis inhibitors (Z-VAD-FMK), necrosis inhibitors (Nec-1), autophagy inhibitors (3-MA), and pyroptosis inhibitors (NSA) did not significantly improve cell survival. However, treatment with a ferroptosis inhibitor (Fer-1) did improve cancer cell survival. The results indicate that ferroptosis is the primary mode of death for BDP-Bpin cells after ultrasound.

[0072] Example 9

[0073] BDP-Bpin releases extracellular ATP in HeLa cells after sonication:

[0074] HeLa cells were seeded at a density of 10 5 The cells were cultured in a 12-well culture dish with a concentration of 100 cells / mL until the cells grew to 70%. After that, the cells were incubated with BDP-Bpin-containing culture medium for 4 hours. The ultrasound group was used at 1 MHz and 0.6 W / cm 2 Ultrasonication was performed at 1000W power for 10 minutes, followed by incubation for 20 hours; ATP concentration was then measured. Cells were washed three times with PBS, followed by the addition of 100 μM lysis reagent. The cell lysate was centrifuged at 12,000g at 4°C for 5 minutes. Cells were cultured in a 96-well microplate according to the manufacturer's instructions. The supernatant of the cell lysate was added to the ATP working solution. Luminescence from each well was immediately detected using a multi-function microplate reader.

[0075] The extracellular ATP release of sonosensitizer BDP-Bpin in HeLa cells after ultrasound Figure 12As shown in Figure 3 . Compared with the control group, the drug-only group, and the ultrasound-only group, the BDP-Bpin + US ultrasound group showed a significant ATP release effect, and the release effect was more pronounced with increasing BDP-Bpin concentration. These results indicate that ultrasound treatment with this sonosensitizer induces the release of extracellular ATP from HeLa cells, a key indicator of immunogenic cell death.

[0076] Example 10

[0077] Application of BDP-Bpin in immunoblotting of ferroptosis induced by ultrasound in HeLa cells:

[0078] Western blotting (WB) was used to detect changes in ferroptosis protein content. Pre-prepared cell culture medium containing 50 μM and 100 μM BDP-Bpin complexes was added to a 100 mm culture dish filled with HeLa cells. After incubation for 4 hours, ultrasound was applied at 1 MHz and 0.6 W / cm 2 Ultrasonication was performed at 1000 nm power for 10 minutes and incubated for 20 hours. Cells were digested with T+E and terminated with 10% DMEM. Cells were harvested by centrifugation, washed twice with PBS, and lysed in 200–400 μL of RIPA strong lysis buffer containing PMSF. The cells were vortexed every 5 minutes at 4°C for five times. After lysis, the cells were centrifuged at 13,400 rpm for 20 minutes at 4°C. The supernatant was the target protein solution. Protein concentrations were determined using a BCA protein assay kit. SDS-PAGE loading buffer was added to each sample and boiled at 95°C for 15 minutes to denature the proteins. For gel preparation, an equal amount of protein solution was added to each well and electrophoresed for 1.5 hours. The proteins were then transferred to a PVDF membrane and blotted on ice for 1 hour. Block with 5% skim milk powder for 0.5–1 hour. Prepare the primary antibody with 5% skim milk powder according to the primary antibody instructions and incubate overnight at 4°C. Wash with PBST (5 × 6 min / each). Prepare the secondary antibody with 5% skim milk powder according to the secondary antibody instructions and incubate at room temperature for 2 hours. Wash with PBST (5 × 6 min / each). Prepare an equal volume of ECL developer and add it dropwise to the PVDF membrane. Wait 2 minutes before imaging using a chemiluminescence imaging system.

[0079] The results of immunoblotting of proteins related to the sonosensitizer BDP-Bpin inducing ferroptosis in HeLa cells after ultrasound are as follows: Figure 13 The results showed that compared with the control group without drug treatment or ultrasonic treatment, the ultrasound treatment of cells with the sonosensitizer BDP-Bpin promoted a significant downregulation of GPX4 protein, indicating that the sonosensitizer BDP-Bpin can induce ferroptosis in HeLa cells.

[0080] Example 11

[0081] Changes in GSH / GSSG content in HeLa cells after ultrasound treatment with the sonosensitizer BDP-Bpin prepared in Example 1:

[0082] HeLa cells were cultured at a density of 5 × 10 5 The cells were inoculated into 12-well culture dishes and cultured until the cells grew to 70%. The cell culture medium containing BDP-Bpin was added to the HeLa cells with good morphology and normal growth. After the drug was incubated for 4 hours, the ultrasound group was used at 1 MHz, 0.6 W / cm 2 Ultrasonication was performed at 1000 nm for 10 minutes and incubation was continued for 20 hours. The drug-treated cells were harvested and homogenized. Total glutathione levels were measured using glutathione reductase and 5,5-dithiobis(2-nitrobenzoic acid). The thiol group of GSH reacts with DTNB to form yellow 5-thio-2-nitrobenzoic acid (TNB), with an absorbance of 405-414 nm. TNB (A405) can be detected by enzyme labeling, and reduced GSH levels are calculated by subtracting GSSG levels from total GSH (GSH = total GSH - 2 × GSSG).

[0083] The changes of GSH / GSSG content induced by sonosensitizer BDP-Bpin in HeLa cells after ultrasound Figure 14 Compared with the control group without drug addition or ultrasound, the GSH / GSSG ratio decreased after treatment with the sonosensitizer BDP-Bpin, indicating that the sonosensitizer BDP-Bpin disrupted redox homeostasis after ultrasound and aggravated the ferroptosis process.

[0084] Example 12

[0085] BDP-Bpin induces immunogenic cell death after sonication in HeLa cells:

[0086] Immunofluorescence of CRT and HMGB1 was detected by confocal microscopy. HeLa cells were plated at a density of 5×10 5 The cells were inoculated into 35mm Corning laser confocal culture dishes. When the cell density grew to 70%, the cell culture medium containing different concentrations of BDP-Bpin was added to the HeLa cells with good morphology and normal growth. After incubation with drugs for 4 hours, the ultrasound group was irradiated at 1MHz, 0.6W / cm 2The cells were ultrasonically treated at a power of 100 nm for 10 min and incubated for 20 h. After washing with PBS, the cells were fixed with 4% paraformaldehyde fixative for 15 min, washed with PBS, and 0.2% Triton X-100 was added to promote permeation for 30 min. The cells were washed with PBS. The primary antibody was diluted with 5% skim milk powder according to the instructions for use of the antibody, and the cells were incubated with primary antibody incubation solution at 4°C overnight. The cells were washed twice with PBS, and the cells were incubated with diluted TRITC secondary antibody at room temperature for 1 h. The cells were washed twice with PBS, and 0.3 μM DAPI was added to stain the nucleus for 10 min. The cells were washed twice with PBS and then observed immediately with a confocal microscope.

[0087] The results of sonosensitizer BDP-Bpin inducing immunogenic cell death in HeLa cells after ultrasound are shown in Figure 2. Figure 15 Immunofluorescence showed that compared with the control group, HMGB1 fluorescence was weakened and CRT fluorescence was enhanced, indicating the upregulation of calreticulin CRT, the release of high-mobility group protein B1 HMGB1, and the efflux of ATP. The results showed that the sonosensitizer BDP-Bpin can effectively induce the release of damage-associated molecular patterns (DAMPs) after ultrasound treatment, causing immunogenic death of HeLa cells.

[0088] Example 13

[0089] Application of BDP-Bpin in inhibiting the growth of 3D cell spheroids:

[0090] HeLa cells were cultured at a density of 2×10 5 100 μg / mL was seeded in an ultra-low adhesion 96-well round-bottom microplate (Corning). After 24 hours, HeLa MCTSs with a diameter of 500 μm were formed. Drug treatment was started 4 days later. Ultrasonic treatment was applied every two days. After 4 hours of drug treatment, the ultrasound group was ultrasonically treated at 1 MHz and 0.6 W / cm 2 Spheroids were sonicated at 1000W for 10 min and incubated continuously. Spheroid growth was monitored using live cell phase contrast microscopy. Spheroids were then washed twice with PBS, stained with calcein AM / PI according to the manufacturer's instructions, and fixed in 4% paraformaldehyde. Spheroids were placed in glass-bottomed dishes and imaged using a confocal scanning microscope system (PI:λ). ex =561nm,λ em range 570-620 nm) were imaged at different depths (z-stacking).

[0091] Application of sonosensitizer BDP-Bpin in inhibiting the growth of 3D cell spheroids Figure 16 Compared with the control group without drug treatment and ultrasound, the red fluorescence inside the cells was enhanced after the cells were treated with the sonosensitizer BDP-Bpin, indicating cell death.

[0092] Example 14

[0093] Example 1 Effect of tissue depth on the generation of reactive oxygen species by sonosensitizer BDP-Bpin prepared by ultrasound or light irradiation:

[0094] The pork was cut into rectangular blocks with a length of 12 cm, a width of 2 cm, and a height of 2 cm. A mixed solution of BDP-Bpin probe and CellROX Deep Red probe was added every 2 cm. The ultrasound group placed the ultrasound probe on the left side of the pork and used 1 MHz, 1 W / cm 2 The cells were ultrasonicated at 100 nm power for 10 minutes and then photographed using a fluorescence imager for three consecutive times. The cells in the illumination group were irradiated with 530 nm laser light at the same position for 10 minutes and then photographed for three consecutive times.

[0095] Application of sonosensitizer BDP-Bpin on ultrasound depth and light depth Figure 17 As shown in Figure 2, fluorescence intensity indicates that the ultrasound group penetrated to a depth of 7 cm, while the light group only penetrated to a depth of 1 cm. These results suggest that BDP-Bpin is a promising sonosensitizer with the potential to treat deep tumors.

[0096] Therefore, the heavy-atom-free, lipid-droplet-targeted BODIPY sonosensitizer of the present invention exhibits excellent biocompatibility and tissue penetration depth. Upon entry into cancer cells, BDP-Bpin is activated by overexpressed H₂O₂ in the cells to form BDP-QL. Low-power ultrasound then generates large amounts of type I and II reactive oxygen species, disrupting cellular redox homeostasis and causing lipid peroxidation, leading to ferroptosis. Simultaneously, it produces DAMPs and induces immunogenic cell death, effectively inhibiting tumor cell proliferation and generating immunity. This has potential application in the treatment of deep-seated hypoxic tumors.

Claims

1. A heavy atom-free fluoroborane dipyrrole compound, characterized in that: Its structural formula is shown below:

2. The method for preparing the fluoroboron dipyrrole compound according to claim 1, wherein The steps include: (1) 3-Quinolinecarboxaldehyde is dissolved in an organic solvent, and 2,4-dimethylpyrrole and trifluoroacetic acid are added sequentially thereto in an inert atmosphere in an ice bath, and the reaction is carried out at room temperature overnight; (2) In an ice bath, 2,3-dichloro-5,6-dicyanobenzoquinone, triethylamine and boron trifluoride etherate are sequentially added to the system of step (1) to fully react; after the reaction, the solvent is removed by rotary evaporation, and the crude product is separated and purified by column chromatography to obtain a fluoroboron dipyrrole compound modified with a quinoline group; (3) dissolving the fluoroboron dipyrrole compound modified with a quinoline group obtained in step (2) and 4-bromomethylphenylboronic acid pinacol ester in an organic solvent, stirring and refluxing the mixture under an inert gas atmosphere; after the reaction, removing the solvent by rotary evaporation, and separating and purifying the crude product by column chromatography to obtain a fluoroboron dipyrrole compound free of heavy atoms that is lipid droplet-targeted; Among them, the structural formula of the fluoroboron dipyrrole compound modified with a quinoline group is:

3. The preparation method according to claim 2, wherein: In step (1), the molar ratio of 3-quinolinecarboxaldehyde to 2,4-dimethylpyrrole is 1:1.9-2.

4. The preparation method according to claim 2, wherein: In step (1), the organic solvent is anhydrous tetrahydrofuran.

5. The preparation method according to claim 2, wherein: In step (2), the molar ratio of 2,3-dichloro-5,6-dicyanobenzoquinone, boron trifluoride etherate and triethylamine is 1:49-50:42-43.

6. The preparation method according to claim 2, wherein: In step (2), after adding 2,3-dichloro-5,6-dicyanobenzoquinone, triethylamine and boron trifluoride etherate are added in sequence at intervals of not less than 7 hours.

7. The preparation method according to claim 2, characterized in that: In step (3), the organic solvent is anhydrous acetonitrile.

8. The preparation method according to claim 2, wherein: In step (3), the temperature of the reflux reaction is 60-65° C., and the reaction time is 22-24 h.

9. The preparation method according to claim 2, wherein: In step (3), the molar ratio of the fluoroboron dipyrrole compound modified with a quinoline group to 4-bromomethylphenylboronic acid pinacol ester is 1:3 to 3.

5.

10. Use of the fluoroboron dipyrrole compound according to claim 1 as a sonosensitizer in sonodynamic therapy.

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