A kind of sulfur-containing fluorine boron dipyrrin photosensitizer targeted to lysosome and preparation method and application

Abstract

The application belongs to the field of biomedical materials, and particularly relates to a lysosome-targeting sulfur-containing fluorine boron dipyrrin photosensitizer, a preparation method and application. In order to develop a non-heavy atom photosensitizer capable of efficiently generating ROS, having lysosome targeting ability and still maintaining high treatment effect under hypoxic conditions, the application takes a traditional BODIPY as a molecular skeleton, and constructs a new non-heavy atom photosensitizer platform BDPS by introducing a benzothiophene group. The design uses the spin-orbit coupling effect of sulfur atoms to improve the intersystem crossing efficiency, and realizes efficient active oxygen generation without introducing heavy elements. On this basis, the strategy of meso position morpholine targeting group modification is adopted, and the photosensitizer L-BDPS with lysosome targeting function is researched. The photosensitizer L-BDPS can be used for preparing drugs for photodynamic therapy, tumor fluorescence imaging and preclinical efficacy research.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a lysosome-targeted sulfur-containing fluoroboron dipyrrole photosensitizer, its preparation method, and its application. Background Technology

[0002] Lysosomes, as the main degradation sites within cells, are responsible for the catabolism of various biomolecules and play a crucial role in the regulation of autophagy and apoptosis signaling. Lysosomes contain more than 60 acid-hydrolyzing enzymes that can break down cells and complex macromolecules into their constituent structural units. Therefore, lysosomes are effective therapeutic targets for lysing tumors. Once the structure and permeability of lysosomes are disrupted, cancer cells rapidly initiate a suicide process.

[0003] Photodynamic therapy (PDT) is a novel non-invasive treatment method that applies the principles of photochemistry, photophysics, and photobiology to the diagnosis and treatment of diseases. However, existing photosensitizers have significant limitations in clinical applications: First, most photosensitizers (such as cyanines, phthalocyanines, and phenothiazines) perform poorly in terms of photostability, biocompatibility, and photosensitization efficiency. For example, cyanine photosensitizers have poor photostability; phthalocyanines and fluoroboron dipyrrole photosensitizers suffer from poor biocompatibility due to their large conjugated planar structures leading to aggregation and quenching in aqueous solutions; and phenothiazines (such as methylene blue) suffer from low photosensitization efficiency, high dark toxicity, and short triplet lifetime. Second, existing photosensitizers typically rely on type II photodynamic mechanisms (generating singlet oxygen through energy transfer), which significantly reduces efficacy in the hypoxic tumor microenvironment. Furthermore, the design of traditional photosensitizers generally employs heavy atom doping strategies; however, the introduction of heavy atoms leads to high synthesis costs, strong dark toxicity to cells, reduced compound stability, and shortened triplet lifetime, ultimately limiting their practical application value.

[0004] Therefore, developing a heavy atom-free photosensitizer that can efficiently generate ROS, has lysosomal targeting capabilities, and maintains high therapeutic efficacy under hypoxic conditions has become a key issue that urgently needs to be addressed in the field of photodynamic therapy. Summary of the Invention

[0005] This invention addresses the aforementioned problems by providing a lysosome-targeted sulfur-containing fluoroboron dipyrrole photosensitizer, its preparation method, and its applications. A photosensitizing platform, BDPS, was constructed by introducing a bisbenzothiophene group, aiming to expand the π-conjugated system and enhance intersystem crossing ability by utilizing the absence of heavy atoms, thereby improving the efficiency of reactive oxygen species (ROS) generation. Based on this, a novel photosensitizer, L-BDPS, combining type I / II ROS generation capability and lysosome-targeting properties, was successfully developed through functionalization modification using a morpholine targeting group introduced at the meso site. This invention includes the structural design and preparation method of the photosensitizer and systematically evaluates its photophysical properties, ROS mechanism, subcellular organelle localization, and photodynamic therapeutic effects, providing a new strategy for tumor treatment.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] This invention provides a lysosome-targeted sulfur-containing fluoroboron dipyrrole photosensitizer, the structural formula of which is:

[0008] .

[0009] The photosensitizer has the following properties:

[0010] The main absorption peak is located at 628-642nm, and the characteristic emission peak is located in the near-infrared region at 654-656nm.

[0011] Under illumination, the yield of singlet oxygen in dichloromethane is as high as 0.161.

[0012] Under light, the half-inhibitory concentration (IC50) values ​​of L-BDPS on A549 cells were very close under normal oxygen and hypoxia conditions, indicating that its photodynamic activity remained highly efficient in a hypoxic microenvironment.

[0013] Regardless of oxygen concentration, cells remained in good condition with L-BDPS concentrations ranging from 0.1 μM to 2.5 μM, indicating very low dark toxicity.

[0014] The photosensitizer fluorescence is highly colocalized with lysosomes (Pearson correlation coefficient 0.84).

[0015] This invention also provides a method for preparing a lysosome-targeted sulfur-containing fluoroboron dipyrrole photosensitizer, characterized by comprising the following steps:

[0016] Step 1: Dissolve 3-bromobenzothiophene in carbon disulfide, slowly add aluminum trichloride, stir thoroughly, and then slowly add acetyl chloride dropwise to react. After the reaction is complete, add ice water and stir, extract, dry and rotary evaporate, and purify to obtain compounds 1-2.

[0017] Step 2: Dissolve compounds 1-2, cuprous iodide, and cesium carbonate in an organic solvent. After the reaction, add ethyl isocyanate to continue the reaction. After the reaction is complete, cool to room temperature, extract, dry, and rotary evaporate to purify and separate compounds 1-3.

[0018] Step 3: Dissolve compounds 1-3 and potassium hydroxide in an organic solvent and react them. After the reaction is complete, cool to room temperature, extract, dry and rotary evaporate to purify and separate compounds 1-4.

[0019] Step 4: Dissolve compound 2-1 in an organic solvent, add potassium carbonate, stir, add 4-(2-bromoethyl)morpholine hydrobromide, heat and stir, cool to room temperature, extract, dry and rotary evaporate, and purify to obtain compound 2-2.

[0020] Step 5: Dissolve compounds 1-4 and 2-2 and trifluoroacetic acid in an organic solvent, and slowly add tetrachlorobenzoquinone, N,N-diisopropylethylamine and boron trifluoride diethyl ether dropwise. After the reaction is complete, extract, dry and rotary evaporate to purify and separate the target product.

[0021] Furthermore, in step 1, the molar ratio of 3-bromobenzothiophene, aluminum trichloride, and acetyl chloride is 1:1 to 2:1.

[0022] Furthermore, in step 2, the molar ratio of compounds 1-2, cuprous iodide, cesium carbonate, and ethyl isocyanate is 10:0.5 to 1:20:11.

[0023] Furthermore, in step 3, the molar ratio of compounds 1-3 to potassium hydroxide is 1:5~6.

[0024] Furthermore, in step 4, the molar ratio of compound 2-1, 4-(2-bromoethyl)morpholine hydrobromide, and potassium carbonate is 1:1:1.5~2.

[0025] Furthermore, in step 5, the molar ratio of compound 2-2, compound 1-4, tetrachlorobenzoquinone, N,N-diisopropylethylamine, and boron trifluoride diethyl ether is 1:2:1 to 2:18:18.

[0026] This invention also provides an application of a lysosome-targeting sulfur-containing fluoroboron dipyrrole photosensitizer for the preparation of pharmaceutical products for photodynamic therapy, tumor fluorescence imaging, and preclinical efficacy evaluation. The photosensitizer, through its unique photodynamic properties, promotes cell-selective action.

[0027] Specifically, the development of drugs for photodynamic therapy involves: activating reactive oxygen species through light irradiation to precisely kill target cells; developing drugs for tumor fluorescence imaging with excellent near-infrared fluorescence properties, enabling real-time in vivo imaging of tumor accumulation, distribution, and metabolism; and developing reagents for preclinical efficacy studies, demonstrating significant tumor-suppressive effects and high biosafety in mouse tumor models, with potential for photodynamic clearance of superficial tumors and postoperative residual lesions, showing good clinical translational potential.

[0028] Furthermore, the photosensitizer can target tumor lysosomes, generating type I and type II reactive oxygen species under light irradiation, thereby enhancing the anti-tumor effect. It can specifically bind to the lysosomes of tumor cells, achieving precise labeling and efficient destruction of tumor cells.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] This invention uses traditional BODIPY as the molecular backbone and constructs a novel heavy-atom-free photosensitizing platform, BDPS, by introducing benzothiophene groups. This design utilizes the spin-orbit coupling effect of sulfur atoms to improve intersystem crossing efficiency, achieving highly efficient reactive oxygen species (ROS) generation without introducing heavy elements. Based on this, a strategy of modifying with meso-position morpholine targeting groups was employed to design a photosensitizer, L-BDPS, with lysosomal targeting function. This photosensitizer L-BDPS possesses the following characteristics:

[0031] 1. Optical properties and reactive oxygen species generation capacity

[0032] Compared to existing technologies, L-BDPS has a major absorption peak at approximately 628-642 nm and a characteristic emission peak at 654-656 nm, exhibiting superior near-infrared absorption and emission characteristics, making it suitable for deep tissue treatment. Compared to traditional photosensitizers, L-BDPS achieves a singlet oxygen yield of 0.161 under light irradiation, significantly outperforming most photosensitizers. L-BDPS also generates superoxide anions through a type I photodynamic mechanism, significantly reducing the oxygen dependence of photodynamic therapy and maintaining highly effective photodynamic therapy even under hypoxic conditions.

[0033] 2. Lysosomal targeting ability

[0034] Compared with existing technologies, L-BDPS can rapidly and specifically target tumor cells to lysosomes, significantly improving the precision of treatment.

[0035] 3. Cytotoxicity and phototoxicity

[0036] Compared to existing photosensitizers, L-BDPS exhibits high cell viability under light-free conditions regardless of oxygen concentration, indicating extremely low dark toxicity, superior to many traditional photosensitizers (such as phenothiazine photosensitizers, which typically have high dark toxicity). Under illumination, the half-maximal inhibitory concentration (IC50) of L-BDPS against A549 cells is highly similar under both normoxic and hypoxic conditions, demonstrating that its photodynamic activity remains highly efficient in hypoxic microenvironments.

[0037] 4. In vivo therapeutic effect

[0038] L-BDPS significantly accumulated at the tumor site 12 hours after injection and was gradually cleared within 24-36 hours. The tumors essentially regressed 16 days after light exposure, with no recurrence and stable mouse weight, indicating good photodynamic efficacy and biosafety.

[0039] 5. Broad application potential

[0040] Compared to existing technologies, L-BDPS is not only suitable for photodynamic therapy of solid tumors, but can also serve as a tool for tumor fluorescence imaging and preclinical pharmacodynamic studies. Its near-infrared absorption and emission properties make it potentially valuable for deep tissue imaging and treatment, while traditional photosensitizers have limited effectiveness in deep tissue therapy. Attached Figure Description

[0041] Figure 1 The 1H NMR spectrum of the photosensitizer L-BDPS;

[0042] Figure 2 Carbon NMR spectrum of the photosensitizer L-BDPS;

[0043] Figure 3 High-resolution mass spectrometry of photosensitizer L-BDPS;

[0044] Figure 4 The spectra of the photosensitizer L-BDPS in different solvents are shown, where (A) is the UV-Vis absorption spectrum and (B) is the fluorescence spectrum.

[0045] Figure 5 To evaluate the reactive oxygen species generation capacity of photosensitizer L-BDPS under light irradiation, (A) is the UV-Vis absorption spectrum of L-BDPS / DPBF after light irradiation, (B) is the UV-Vis absorption spectrum of MB / DPBF after light irradiation, (C) is the curve of the UV-Vis absorption peak at 410 nm after DPBF is consumed by L-BDPS or MB over time, (D) is the fluorescence spectrum of L-BDPS / DHR123 after light irradiation, and (E) is the fluorescence spectrum of L-BDPS / HPF after light irradiation.

[0046] Figure 6The UV-Vis absorption spectrum shows the continuous consumption of NADH by the photosensitizer L-BDPS over time.

[0047] Figure 7 The photodynamic killing effect of photosensitizer L-BDPS on human lung cancer cells A549 under normal oxygen and hypoxia conditions is shown in the figure. (A) is the phototoxicity of L-BDPS on A549 cells under normal oxygen and hypoxia conditions, and (B) is the dark toxicity of L-BDPS on A549 cells under normal oxygen and hypoxia conditions.

[0048] Figure 8 Confocal fluorescence images showing the co-localization of the photosensitizer L-BDPS with the commercial Lyso / Mito Tracker probe;

[0049] Figure 9 To evaluate the generation of singlet oxygen and superoxide anion radicals by photosensitizer L-BDPS using different probes, (A) is a confocal image of L-BDPS inducing singlet oxygen generation in A549 cells using SOSG fluorescent probe, and (B) is a confocal image of L-BDPS exciting superoxide anion radicals in A549 cells using DHE fluorescent probe.

[0050] Figure 10 Confocal fluorescence imaging of A549 cells co-stained with photosensitizer L-BDPS (1.0 μM), Calcein-AM, and PI;

[0051] Figure 11 To evaluate the photodynamic therapy effect of photosensitizer L-BDPS in vivo, (A) is an in vivo fluorescence imaging image of 4T1 tumor-bearing BALB / c mice after tail vein injection of photosensitizer L-BDPS, (B) is the weight change curve of mice in different treatment groups (experimental period: 0-15 days), (C) is the relative tumor volume change of different treatment groups; (D) is the end of treatment. Detailed Implementation

[0052] To further illustrate the technical solution of the present invention, the present invention will be further described below through embodiments.

[0053] In the specific implementation of this invention, in addition to the key steps described in detail, a variety of conventional chemical reagents and standard operating procedures were used. For example, all chemical reagents and solvents used in the experiments were purchased directly from commercial suppliers, and their purity met the experimental requirements, eliminating the need for further purification. Solvents underwent necessary pretreatment steps according to solvent manuals before formal use. Furthermore, commonly used chemical reaction conditions such as TLC monitoring, vacuum rotary evaporation, and silica gel column chromatography are all standard operating procedures in the field of organic synthesis.

[0054] In particular, in the synthesis steps of the sulfur-containing fluoroboron dipyrrole photosensitizer L-BDPS of this invention, the preparation of intermediates 1-2 to 1-4 has undergone rigorous condition optimization to ensure the high purity and high yield of the target product L-BDPS. The optical properties, reactive oxygen species generation capacity, and cell experiments of the photosensitizer have been verified using standardized spectroscopic and biological methods, and the specific experimental conditions and methods are detailed in the Examples section.

[0055] The selection of these routine steps and reagents is based on their broad applicability and reliability, and does not have a critical impact on understanding the core technology of this invention, therefore they are not described in detail.

[0056] Example 1

[0057] This embodiment describes a lysosome-targeted sulfur-containing fluoroboron dipyrrole photosensitizer, whose absorption and emission are located in the near-infrared region, and its structural formula is as follows:

[0058] .

[0059] This embodiment describes a method for preparing a lysosome-targeted sulfur-containing fluoroboron dipyrrole photosensitizer, comprising the following steps:

[0060]

[0061] (1) Synthesis of compound 2-2

[0062] Under a nitrogen atmosphere, compound 2-1 (1 mmol, 122 mg) was dissolved in a dry N,N-dimethylformamide solution (DMF, 1 ml) in an organic solvent, and potassium carbonate (2 mmol, 276 mg) was added to the solution. Maintaining a nitrogen atmosphere, the mixture was stirred for 30 minutes, and then 4-(2-bromoethyl)morpholine hydrobromide (1 mmol, 274.982 mg) was added. The reaction was heated to 60 °C and stirred for 12 hours. The reaction was monitored by thin-layer chromatography (TCL). After the reaction was complete, the reaction solution was cooled to room temperature, extracted with ethyl acetate (3 × 50 mL) and brine, dried over anhydrous Na₂SO₄, and evaporated under reduced pressure to obtain a deep yellow oil. The product was then further purified by column chromatography (eluent: dichloromethane / methanol = 50:1, v / v) to obtain a light yellow oil (212 mg, 0.9 mmol, 90%), which is compound 2-2. 1H NMR (600 MHz, DMSO-d6) δ 9.86(d, J=1.7 Hz, 1H),7.85 (d, J=8.8 Hz, 2H), 7.18–7.08(m, 2H), 4.20(t, J=5.7 Hz,2H), 3.57(s, 4H), 2.71(t, J=6.0 Hz, 2H), 2.47(s, 4H). 13 C NMR (151 MHz, DMSO-d6) δ 191.73, 163.92, 132.25, 130.09, 115.44, 66.63, 66.28, 57.27, 54.05.ESI-MS: m / z calcd for the compound 2-2 (C 13 H 17 NO3, [M+H] + ): 236.1282, Found:236.1280.

[0063] (2) Synthesis of compounds 1-2

[0064] 3-Bromobenzothiophene (10 mmol, 2.13 g) was dissolved in carbon disulfide (30 mL) at 0 °C, and aluminum trichloride (20.2 mmol, 2.69 g) was slowly added. After thorough stirring, acetyl chloride solution (10.1 mmol, 793 mg) was slowly added dropwise, and the reaction was continued for about 5 hours, monitored by thin-layer chromatography (TCL). After the reaction was completed, 200 mL of ice water was added and the mixture was stirred for 20 minutes. The reaction solution was extracted three times with dichloromethane, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to give a deep yellow solid. The crude product was separated by column chromatography using petroleum ether-ethyl acetate (30:1, v / v) as eluent. The target product was collected, concentrated, and a light yellow solid, namely compounds 1-2 (1.53 g, 6 mmol, 60%), was obtained. 1 H NMR (600 MHz, DMSO-d6) δ 8.11 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 7.5 Hz, 1H), 7.62 (dt, J = 20.9, 7.7 Hz, 2H), 2.78 (s, 3H). 13CNMR (151 MHz, DMSO-d6) δ 191.89, 139.00, 129.28, 126.81, 125.72, 123.88,112.34, 30.92. ESI-MS: m / z calcd for the compound 1-2 (C 10 H7BrOS, [M+H] + ):254.9474, Found: 254.9472.

[0065] (3) Synthesis of compounds 1-3

[0066] Compounds 1-2 (5 mmol, 1.27 g), cuprous iodide (0.5 mmol, 95 mg), and cesium carbonate (10 mmol, 3.25 g) were added separately to Schlenk flasks. The flasks were evacuated three times to ensure the reaction was carried out under a nitrogen atmosphere. Dimethyl sulfoxide (3 mL) was added as an organic solvent. After reacting for 30 minutes, ethyl isocyanate (5.5 mmol, 622 mg) was added, and the reaction was maintained at 50 °C for 4 hours. The reaction was monitored by thin-layer chromatography (TCL). After the reaction was complete, the reaction mixture was extracted with dichloromethane (10 mL × 3) and saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a dark brown solid. The crude product was separated by column chromatography (petroleum ether / ethyl acetate = 30:1, v / v) and concentrated to obtain a pale yellow solid (648 mg, 2.5 mmol, 50%), namely compounds 1-3. 1 H NMR (600 MHz, CDCl3) δ8.15 (d, J = 7.9 Hz, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 2.46 (s, 3H), 1.35 (t, J= 7.1 Hz, 3H). 13 C NMR (151 MHz, CDCl3) δ 166.41, 147.62, 139.76, 131.73,129.91, 129.53, 128.29, 127.90, 126.09, 125.97, 124.29, 65.05, 19.67, 17.11.ESI-MS: m / z calcd for the compound 1-3 (C 14 H 13NO2S, [M+H] + ): 260.0740, Found:260.0739.

[0067] (4) Synthesis of compounds 1-4

[0068] Under a nitrogen atmosphere, compounds 1-3 (2 mmol, 518.6 mg) and potassium hydroxide (12 mmol, 673 mg) were dissolved in 20 mL of ethylene glycol, and the mixture was heated to 160 °C and reacted for 3.5 h (protected from light throughout). The reaction was monitored by thin-layer chromatography (TCL). After the reaction was complete, the reaction solution was cooled to room temperature, and 30 mL of an ice-water mixture was added. The mixture was extracted three times with dichloromethane, and washed with water after extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation to obtain the mixture. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 60:1, v / v), and concentrated to give a white solid (187.26 mg, 1 mmol, 50%), namely compounds 1-4. ¹H NMR (600 MHz, CDCl₃) δ 8.39 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 7.7 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.23 (t, J = 7.4 Hz, 1H), 6.87 (s, 1H), 2.31 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 141.99, 132.05, 127.33, 124.26, 123.93, 122.48, 120.45, 117.97,112.95, 11.16. ESI-MS: m / z calcd for the compound 1-4 (C11H9NS, [M+H]+ ): 188.0529, Found: 188.0527.

[0069] (5) Synthesis of the target compound L-BDPS

[0070] Under a nitrogen atmosphere, compounds 2-2 (1 mmol, 235 mg) and 1-4 (2 mmol, 374.52 mg) were dissolved in dichloromethane (100 mL), and trifluoroacetic acid (10 µL) was added. The mixture was stirred at room temperature in the dark for 18 h. Tetrachlorobenzoquinone (1.2 mmol, 295 mg) was dissolved in dichloromethane (50 mL), and the resulting solution was slowly added dropwise to the reaction flask. After stirring at room temperature for 2 h, N,N-diisopropylethylamine (18 mmol, 2.33 g) was slowly added to the system through a dropping funnel. Stirring was continued for 1 h, and boron trifluoride diethyl ether (18 mmol, 2.55 g) was added dropwise in the same manner. After reacting for 30 min, the reaction was terminated by adding deionized water, and the mixture was extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a dark blue solid. The crude product was purified by column chromatography (dichloromethane / methanol = 100:1, v / v), the target fraction was collected, and after concentration, a dark blue solid (191 mg, 0.3 mmol, 30%) was obtained, which is the target compound L-BDPS. 1 H NMR (600 MHz, CDCl3) δ 8.76 (s, 2H), 7.68 (s, 2H), 7.49 (s, 2H), 7.44 (s, 2H), 7.32 (s, 2H), 7.11 (s, 2H), 2.21 (s, 2H), 2.01 (s, 5H), 1.42 (s, 3H), 0.89 (s, 8H). 13 C NMR (151 MHz, DMSO) δ152.48, 147.26, 141.38, 137.64, 136.63, 133.71, 132.76, 130.12, 126.52,125.51, 125.00, 124.32, 122.18, 120.84, 115.99, 66.56, 54.11, 49.06, 19.30,14.42. ESI-MS: m / z calcd for the compound L-BDPS (C 35 H 30 BF2N3O2S2, [M+H + ):638.1914, Found: 638.1922.

[0071] Example 2

[0072] This embodiment explores the photophysical properties and mechanism of the photosensitizer L-BDPS prepared in Example 1.

[0073] (1) Preparation for spectral testing

[0074] Compounds L-BDPS, Cy5, MB, DPBF, HPF, and DHR123 were prepared into standard solutions of different concentrations using chromatographic grade DMSO as the solvent (L-BDPS, Cy5, and MB: 2 mM; DPBF, HPF, and DHR123: 10 mM). NADH was dissolved in ultrapure water to prepare a 10 mM aqueous solution.

[0075] (2) Photophysical property testing

[0076] Add 4 μL of L-BDPS and 2 mL of solvent to a quartz cuvette, and measure its UV-Vis absorption spectrum and fluorescence emission spectrum, respectively.

[0077] The fluorescence quantum yield of L-BDPS was determined by a relative method using Cy5 (Φf = 0.15 in methanol) as the reference compound.

[0078] Experimental results are as follows Figure 4 As shown, L-BDPS exhibits a maximum absorption wavelength of 630 nm and a maximum emission wavelength of 656 nm in dichloromethane (DCM), with a molar absorptivity (ε) of 6.7 × 10⁻⁶. 4 L·mol -1 ·cm -1 The maximum absorption wavelength in dimethyl sulfoxide (DMSO) is 628 nm, the maximum emission wavelength is 654 nm, and the molar absorptivity (ε) is 7.7 × 10⁻⁶. 4 L·mol -1 ·cm -1 The maximum absorption wavelength in PBS is 642 nm, and the molar absorptivity (ε) is 3.5 × 10⁻⁶. 4 L·mol -1 ·cm -1 The fluorescence quantum yield in methanol was 0.143, indicating that L-BDPS has excellent fluorescence imaging potential.

[0079] Example 3

[0080] This example investigates the generation of reactive oxygen species in the photosensitizer L-BDPS prepared in Example 1.

[0081] (1) Detection of singlet oxygen in solution

[0082] Appropriate amounts of 1,3-diphenylisobenzofuran (DPBF) and L-BDPS solutions were added to a cuvette containing 3 mL of DMSO solution, and their absorbance values ​​were adjusted to 1.0 and 0.2, respectively. After thorough mixing, the mixture was irradiated using a 660 nm LED light source (2 mW·cm⁻²). The absorbance at 410 nm was dynamically acquired at 10 s intervals using a UV-Vis spectrometer, and the singlet oxygen yield was calculated.

[0083] (2) Detection of superoxide anion free radicals in solution

[0084] Using dihydrorhodamine 123 (DHR123) as O2 •− The probe was tested. DHR123 is non-fluorescent under normal conditions. After being oxidized by superoxide anions, it generates rhodamine 123, which has strong green fluorescence. The fluorescence intensity is proportional to the superoxide anion level. L-BDPS (4 μM) standard solution and DHR123 (10 μM) were added to a cuvette containing 3 mL of PBS solution, mixed thoroughly, and then tested using a 660 nm LED light source (20 mW·cm⁻¹). -2 Irradiation was applied. Fluorescence intensity changes at 545 nm were dynamically acquired at 10-second intervals using a fluorescence spectrometer. The excitation wavelength was 495 nm, and the slit width was 5 / 5 nm.

[0085] (3) Detection of hydroxyl radicals in solution

[0086] Hydroxyphenyl fluorescein (HPF) was used as a specific fluorescent probe to detect hydroxyl radicals (•OH) generated during photoexcitation. HPF can selectively oxidize •OH to generate a strongly fluorescent product with a maximum excitation wavelength of 490 nm and an emission wavelength of 535 nm. The photosensitizer L-BDPS (10 μM) and HPF (10 μM) were co-dissolved in phosphate-buffered saline (PBS, 3 mL), thoroughly mixed, and transferred to a quartz cuvette (1 cm path length). A 660 nm LED light source (20 mW·cm²) was used. -2 The sample was vertically irradiated, and the fluorescence intensity at 535 nm was dynamically acquired at 10-second intervals using a fluorescence spectrometer. The fluorescence intensity change curve over time was recorded. All experiments were repeated three times, and the average value was used for analysis.

[0087] Experimental results are as follows Figure 5 In section A, using a 660 nm LED light source (2 mW·cm²), -2 After illumination, the intensity of the characteristic absorption peak of DPBF at 410 nm continuously decreased, indicating that a substance was generated in the system. 1O2. Using methylene blue (MB) as a reference compound ( Figure 5 (B) Based on the linear relationship of the attenuation curve ( Figure 5 (C) The singlet oxygen yield of compound L-BDPS was calculated to be 0.161. Subsequently, the O2 production of L-BDPS under photoexcitation was monitored. •− The ability to generate •OH. For example... Figure 5 In the D region, under illumination, the fluorescence intensity in the 500-600 nm area significantly increases with increasing illumination time, indicating that O2 is generated in the system. •− Under the same illumination conditions, the change in fluorescence intensity of HPF is negligible. Figure 5 (E).

[0088] In summary, L-BDPS possesses both Type I and Type II photodynamic mechanisms, which will be further confirmed in subsequent content.

[0089] (4) Detection of reduced coenzyme I (NADH)

[0090] NADH and photosensitizer L-BDPS were dissolved together in 3 mL of chromatographic grade DMSO solution and mixed thoroughly. An appropriate amount of solution was taken, and its initial absorbance (A0) at 340 nm was measured using a UV-Vis spectrophotometer with a 660 nm LED light source (20 mW·cm⁻¹). -2 The irradiation system was used. The absorbance at 340 nm was dynamically collected at 20-second intervals. The conversion of NADH to NAD by L-BDPS per unit time under illumination was calculated. + The efficiency (TOF).

[0091] Experimental results are as follows Figure 6 With increasing illumination time (0 s–120 s), the absorption peak of NADH at 340 nm gradually decreased, while the absorption peak at 260 nm significantly increased. This confirms that L-BDPS can catalyze the oxidation of NADH to NAD+ under photoexcitation conditions. The calculated catalytic conversion efficiency (TOF) of L-BDPS was 7.73 min. -1 .

[0092] Example 4

[0093] This example investigated the cytotoxicity assessment of the photosensitizer L-BDPS.

[0094] Under normal oxygen conditions:

[0095] Phototoxicity experimental group: The experimental procedure was as follows: A549 cells were seeded at a density of approximately 8000-10000 cells per well in a 96-well cell culture plate, with 100 μL of cell suspension per well. The plates were cultured for 24 hours at 37 ℃, 21% O2, and 5% CO2. After washing three times with PBS, the medium was replaced with a gradient concentration of the photosensitizer L-BDPS, with six replicates per concentration. A control group was also included. The plates were cultured for another 12 hours, after which the drug-containing medium was removed, washed with PBS, and replaced with fresh complete medium. The 96-well plates were then placed under a 660 nm light source (20 mW·cm²). -2 Irradiate the cells under aerobic conditions for 20 minutes, then incubate for 24 hours. Afterward, add 100 μL of MTT solution (final concentration 5 mg / mL) to each well and incubate for another 4 hours. Remove the culture medium, add 100 μL of DMSO to each well, shake at room temperature for 5 minutes, and measure the absorbance at 550 nm using a microplate reader. Calculate the relative cell viability of each normoxic experimental group and the control group.

[0096] Dark toxicity experimental group: The experimental procedure is the same as that for phototoxicity, and no light-avoidance treatment is required.

[0097] Under hypoxic conditions:

[0098] Phototoxicity Experiment Group: A549 cells were seeded at a density of approximately 8000-10000 cells per well in 96-well cell culture plates, with 100 μL of cell suspension per well. The plates were initially cultured in an incubator (37 ℃, 21% O2, 5% CO2) for 12 hours to allow cell adhesion. The plates were then transferred to a pre-set hypoxic incubator (2% O2), and the oxygen concentration was maintained at 2% in real-time for 12 hours. The plates were then washed three times with PBS and replaced with hypoxic-equilibrated medium containing gradient concentrations of the photosensitizer L-BDPS, with six replicates per concentration. A corresponding hypoxic control group (no drug) was also included. The plates were incubated under hypoxic conditions in the dark for 12 hours. After incubation, the drug-containing medium was removed, and the plates were washed with PBS. Under hypoxic conditions, the 96-well plates were then placed under a 660 nm laser light source (20 mW·cm⁻¹). -2 Under light, the cells were exposed to light for 20 minutes. After light treatment, they were cultured for another 24 hours. Then, 100 μL of MTT solution (final concentration 5 mg / mL) was added to each well, and incubation continued for 4 hours. The culture medium was then discarded, and 100 μL of DMSO was added to each well. The cells were shaken at room temperature for 5 minutes, and the absorbance at 550 nm was measured using a microplate reader. The relative cell viability of each hypoxic experimental group and the control group was calculated.

[0099] Dark toxicity experimental group: The experimental procedure is the same as that for phototoxicity, and no light-shielding treatment is required.

[0100] Experimental results are as follows Figure 7As shown in Figure A, under illumination, the half-maximal inhibitory concentrations (IC50) of L-BDPS against A549 cells under normal oxygen and hypoxic conditions were 0.085 μM and 0.091 μM, respectively. These values ​​were highly similar, indicating that its photodynamic activity remained highly efficient in a hypoxic microenvironment. Further analysis of dark toxicity (DDT) was conducted. Figure 7 Regardless of oxygen concentration, cells remained in good condition when L-BDPS concentrations ranged from 0.1 μM to 2.5 μM. This sufficiently demonstrates that L-BDPS exhibits good biocompatibility.

[0101] Example 5

[0102] This embodiment investigated the intracellular enrichment region of the photosensitizer L-BDPS and its photodynamic therapy effect.

[0103] (1) Colocation experiment

[0104] A549 cells were co-incubated with L-BDPS, Lyso, and Mito in DMEM medium for 30 minutes each. After incubation, the cells were washed three times with PBS, followed by confocal imaging. For the green channel, the collection wavelength was 550-600 nm (excitation wavelength: 488 nm); for the red channel, the collection wavelength was 635-690 nm (excitation wavelength: 633 nm). By calculating the Pearson correlation coefficient and colocalization coefficient, the enrichment of photosensitizers in the two organelles could be quantitatively assessed, thereby quantitatively evaluating the colocalization of L-BDPS with each organelle probe.

[0105] (2) Live / dead cell co-staining imaging

[0106] A549 cells were placed in confocal cell culture dishes at a density of 1 × 10⁶ cells per dish. 5 The cells were inoculated at a density of [number] cells / units. After culturing at 37 °C and 21% O2 for 24 hours, the cells were washed three times with PBS, and DMEM medium containing 1 μM L-BDPS was added. The cells were then cultured for another 2 hours. The experimental group used a 660 nm LED light source (20 mW·cm²). -2 Irradiate the cells for 20 minutes, while the control group remains in darkness. Prepare freshly made DMEM medium containing 5 μM Calcein AM and 1 μM PI, and protect from light. Aspirate the cell culture medium, add an appropriate amount of staining working solution, ensuring complete coverage of the cells. Incubate at 37 ℃ in the dark for 30 minutes. Aspirate the staining solution again, wash the cells with PBS, and observe them under a confocal microscope. Calcein AM is excited at a wavelength of 488 nm, and emission light is collected at 500-550 nm; PI is excited at a wavelength of 561 nm, and emission light is collected at 570-620 nm.

[0107] (3) Construction and imaging of BALB / c breast cancer mouse model

[0108] Female BALB / c mice aged 4-6 weeks (purchased from Beijing Spaford Biotechnology Co., Ltd.) were used, and 4T1 cells (2×10⁻⁶) were injected. 6 Inject the mice subcutaneously into the right back region with a solution of 50 μL PBS (cells / 50 μL). Observe the mice daily and measure the tumor periodically using calipers, according to the formula V = 0.5 × a × b. 2 Tumor volume was calculated. Once the tumor volume reached approximately 100 mm³, the tumor-bearing mice were randomly divided into four groups: PBS without light, PBS with light, L-BDPS without light, and L-BDPS with light. The dosage administered via intratumoral injection was 25 μL / 50 mm³. 3 Subsequently, the tumor sites of mice in the treatment group were irradiated with a specific wavelength laser (660 nm) to implement photodynamic therapy. After treatment, the intensity of fluorescence signals within the tumor was monitored regularly using a small animal in vivo imaging system to assess drug distribution and metabolism, and changes in tumor volume and mouse weight were continuously tracked and measured to comprehensively evaluate the tumor-suppressing effect and safety of the treatment.

[0109] Experimental results are as follows Figure 8 As shown, the fluorescence signals of L-BDPS and LysoTracker exhibit a high degree of overlap. Quantitative analysis revealed a Pearson correlation coefficient (R) of 0.84 and a co-localization coefficient of 0.91, indicating a significant correlation between the two and confirming that L-BDPS is primarily enriched in the lysosomal region. In contrast, the co-localization of L-BDPS with Mito Tracker was lower, with a Pearson correlation coefficient (R) of 0.49, and the fluorescence signal overlap was not significant.

[0110] like Figure 9 As shown in Figure A, no green signal was detected before illumination, but the green fluorescence increased after illumination. Next, a DHE probe was used to evaluate O2. •− The generation situation. For example... Figure 9 As shown in Figure B, A549 cells incubated with DHE and L-BDPS exhibited significantly enhanced red fluorescence after light irradiation. These experimental results are consistent with findings in in vitro testing, further confirming that L-BDPS still possesses both Type I and Type II photodynamic therapy mechanisms in cells.

[0111] like Figure 10 As shown, in the unilluminated group, Calcein-AM exhibited bright green fluorescence, while the red fluorescence signal was weak and negligible. L-BDPS did not significantly affect cell membrane integrity or cell viability. However, after illumination, the green fluorescence signal weakened, while the red fluorescence significantly increased. This indicates that PI enters the cell and binds to DNA, leading to cell death.

[0112] like Figure 11 As shown in Figure A, a significant fluorescence signal was detected at the tumor site 12 hours after injection. At 36 hours post-injection, the fluorescence intensity at the tumor site significantly decreased, indicating a gradual metabolic clearance trend. At 24 hours post-injection, a 660 nm LED light source (250 mW·cm⁻¹) was used. -2 The tumor site was continuously irradiated for 30 minutes. The mouse body weight was monitored every other day during the treatment period. Figure 11 (B) and tumor volume ( Figure 11 Changes in (C) were observed. During treatment, the body weight of mice in all groups increased slightly, with no significant difference between groups, indicating that L-BDPS-mediated photodynamic therapy did not cause significant systemic toxicity. The tumor volume of mice in the PBS-free group, PBS-lit group, and L-BDPS-free group continued to increase, with similar growth rates, confirming that L-BDPS did not have a tumor-suppressing effect under light-free conditions. However, the tumors in the L-BDPS-lit group shrank significantly after treatment and did not recur during the treatment period. Tumor tissue was removed on day 16 (…). Figure 11 (D). The above experimental results show that L-BDPS has good PDT efficacy.

[0113] The above results indicate that L-BDPS fluorescence is highly colocalized with lysosomes (correlation coefficient 0.84), and has good photodynamic therapeutic effects and biosafety.

[0114] In summary, L-BDPS, as a novel photosensitizer possessing both type I and type II reactive oxygen species (ROS) generation capabilities, not only exhibits excellent photochemical properties and ROS generation capacity, but also demonstrates lysosomal targeting, low cytotoxicity, and high photodynamic activity even in hypoxic microenvironments during tumor treatment. It holds the potential to be developed into a highly effective photodynamic therapy drug, and its properties include:

[0115] 1. Optical Properties and Reactive Oxygen Species Generation Capacity: L-BDPS exhibits maximum absorption in the 628-642 nm range, within the near-infrared region. It possesses strong tissue penetration and a fluorescence quantum yield of 0.143 in methanol, demonstrating excellent imaging performance. Under illumination, L-BDPS efficiently generates singlet oxygen with a yield as high as 0.161, showcasing its superior photochemical properties. L-BDPS can also generate superoxide anions, a characteristic that allows it to maintain highly effective photodynamic therapy even in hypoxic tumor environments.

[0116] 2. Subcellular targeting capability: L-BDPS are specifically enriched in lysosomes, with a Pearson correlation coefficient of 0.84 and a colocalization coefficient of 0.91, indicating excellent targeting selectivity and improved therapeutic specificity.

[0117] 3. Cytotoxicity and Phototoxicity: L-BDPS exhibits low cytotoxicity under dark conditions, indicating good biocompatibility. Under light, L-BDPS demonstrates significant cytotoxicity; higher concentrations result in lower cell viability, indicating a significant photodynamic effect.

[0118] 4. In vivo therapeutic effect: L-BDPS significantly accumulated at the tumor site 12 hours after injection and was gradually cleared within 24-36 hours. The tumors basically regressed 16 days after light irradiation, with no recurrence and stable mouse weight, indicating its good photodynamic efficacy and biosafety.

[0119] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A lysosome-targeted sulfur-containing fluoroboron dipyrrole photosensitizer, characterized in that, The structural formula of the photosensitizer is: 。 2. The method for preparing a lysosome-targeted sulfur-containing fluoroboron dipyrrole photosensitizer according to claim 1, characterized in that, Includes the following steps: Step 1: Dissolve 3-bromobenzothiophene in carbon disulfide, slowly add aluminum trichloride, stir thoroughly, and then slowly add acetyl chloride dropwise to react. After the reaction is complete, add ice water and stir, extract, dry and rotary evaporate, and purify to obtain compounds 1-2. Step 2: Dissolve compounds 1-2, cuprous iodide, and cesium carbonate in an organic solvent. After the reaction, add ethyl isocyanate to continue the reaction. After the reaction is complete, cool to room temperature, extract, dry, and rotary evaporate to purify and separate compounds 1-3. Step 3: Dissolve compounds 1-3 and potassium hydroxide in an organic solvent and react them. After the reaction is complete, cool to room temperature, extract, dry and rotary evaporate to purify and separate compounds 1-4. Step 4: Dissolve compound 2-1 in an organic solvent, add potassium carbonate, stir, add 4-(2-bromoethyl)morpholine hydrobromide, heat and stir, cool to room temperature, extract, dry and rotary evaporate, and purify to obtain compound 2-2. Step 5: Dissolve compounds 1-4 and 2-2 and trifluoroacetic acid in an organic solvent, and slowly add tetrachlorobenzoquinone, N,N-diisopropylethylamine and boron trifluoride diethyl ether dropwise in sequence. After the reaction is complete, extract, dry and rotary evaporate to purify and separate the target product. The structural formulas of compounds 1-2 are as follows: ; The structural formulas of compounds 1-3 are as follows: ; The structural formulas of compounds 1-4 are as follows: ; The structural formula of compound 2-1 is: ; The structural formula of compound 2-2 is: .

3. The method for preparing a lysosome-targeted sulfur-containing fluoroboron dipyrrole photosensitizer according to claim 2, characterized in that, In step 1, the molar ratio of 3-bromobenzothiophene, aluminum trichloride, and acetyl chloride is 1:1 to 2:

1.

4. The method for preparing a lysosome-targeted sulfur-containing fluoroboron dipyrrole photosensitizer according to claim 2, characterized in that, In step 2, the molar ratio of compounds 1-2, cuprous iodide, cesium carbonate, and ethyl isocyanate is 10:0.5 to 1:20:

11.

5. The method for preparing a lysosome-targeted sulfur-containing fluoroboron dipyrrole photosensitizer according to claim 2, characterized in that, In step 3, the molar ratio of compounds 1-3 to potassium hydroxide is 1:5~6.

6. The method for preparing a lysosome-targeted sulfur-containing fluoroboron dipyrrole photosensitizer according to claim 2, characterized in that, In step 4, the molar ratio of compound 2-1, 4-(2-bromoethyl)morpholine hydrobromide, and potassium carbonate is 1:1:1.5~2.

7. The method for preparing a lysosome-targeted sulfur-containing fluoroboron dipyrrole photosensitizer according to claim 2, characterized in that, In step 5, the molar ratio of compound 2-2, compound 1-4, tetrachlorobenzoquinone, N,N-diisopropylethylamine, and boron trifluoride diethyl ether is 1:2:1 to 2:18:

18.

8. The application of the lysosome-targeted sulfur-containing fluoroboron dipyrrole photosensitizer as described in claim 1, characterized in that, Pharmaceutical products used for photodynamic therapy, tumor fluorescence imaging, and preclinical efficacy evaluation.

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