Spiropyran-modified bodipy tumor phototherapeutic agents and methods of making the same

Abstract

A spiropyran modified BODIPY tumor phototherapeutic agent and a preparation method thereof, and relate to a phototherapeutic agent for tumor treatment and a preparation method thereof, the present application takes 1,1,3-trimethyl-1,3-dihydrospiro[benzo[e]indole-2,2'-chromene]-6'-formaldehyde as a functional building block, which is covalently coupled with fluoroboron dipyrromethene core through condensation reaction. Realize the integration of photothermal therapy and ROS fluorescence tracing. Utilize the efficient photothermal conversion of BODIPY and the electronic sensitive properties of closed ring spiropyran. Under near infrared light, BODIPY is used for tumor thermal ablation, and spiropyran is used for in-situ tracing by responding to promote ROS generation and fluorescence change, breaking through the traditional "treatment only, difficult to monitor" limitation. The present application effectively inhibits the generation of unstable open ring product, while retaining the original optical advantages of the two types of molecules, and still can promote the generation of active oxygen under light, and realize the fluorescence visualization tracing of active oxygen in the treatment process.

Description

Technical Field

[0001] This invention relates to a tumor phototherapy agent and its preparation method, and particularly to a spiropyran-modified BODIPY tumor phototherapy agent and its preparation method. Background Technology

[0002] Botryolean dipyrrole (BODIPY) dyes have become a research hotspot in optoelectronics and biofluorescence due to their excellent optical properties, including high molar extinction coefficients, narrow-band absorption / emission, and favorable Stokes shift. However, these dyes still have some shortcomings in practical applications, such as easy aggregation in aqueous media leading to fluorescence quenching (ACQ effect), weak cell penetration, and a small multiphoton absorption cross-section. These defects limit their further expansion in application scenarios. Therefore, strategies such as molecular structure modification and functionalized material construction are needed to systematically optimize the optical properties of BODIPY dyes. In particular, in the research and development of near-infrared BODIPY dyes, expanding the molecular π-conjugated framework and rationally screening electron donor groups have proven to be a core and efficient design approach for improving their optical properties and constructing high-performance near-infrared BODIPY fluorescent dyes.

[0003] Photochromic organic molecules have attracted much scientific attention due to their ability to undergo reversible isomerization transitions between multiple absorption states in the visible light band (J. Am. Chem. Soc. 2017, 139, 13429−13441). These molecules can undergo structural rearrangement under specific wavelengths of light, leading to significant changes in their ultraviolet-visible absorption spectra. In some systems, the fluorescence emission behavior is simultaneously and reversibly modulated along with the absorption spectrum change, thus providing an ideal platform for the construction of multifunctional photoresponsive materials. However, most spiropyran-based photochromic molecules generate colored anthocyanins after ring-opening under light. These ring-opening products have poor stability and are prone to non-specific side reactions, greatly limiting their practical value in biophototherapy and near-infrared imaging.

[0004] In recent years, studies have attempted to couple spiropyrans with other photosensitive molecules or introduce them into supramolecular assembly systems to expand their photoresponsive functions. BODIPY dyes are renowned for their high fluorescence quantum yield, good photostability, and tunable excitation / emission wavelengths. If they can be organically combined with spiropyran structures, it is hoped that a new class of photosensitive diagnostic and therapeutic agents can be developed. However, molecular design strategies that covalently couple fluoroboron dipyrrole (BODIPY) units with photochromic materials are rarely reported in the literature (Dye. and Pig. 2012, 94, 175-182).

[0005] No existing technology employs a molecular design strategy that introduces electron-pulling groups at the meso position of the BODIPY core and simultaneously bonds a spiropyran ring at the 3,5-position via a condensation reaction. This strategy effectively addresses the core issue of poor stability in photochromic ring-opening, providing a novel design pathway for constructing stable photoresponsive near-infrared diagnostic and therapeutic reagents. Summary of the Invention

[0006] The purpose of this invention is to provide a spiropyran-modified BODIPY tumor phototherapy agent and its preparation method. This invention uses spiropyran as a synergistic functional group and combines it with the photothermal properties of BODIPY to develop a new type of phototherapy agent that combines spiropyran with fluoroboropyrrole for ring-opening inhibition and synergistic photothermal and photodynamic therapy.

[0007] The technical solution of the present invention is as follows: A spiropyran-modified BODIPY tumor phototherapy agent, the phototherapy agent having the general formula shown in Formula I: ; In Formula I, R is selected from different electron-donating and electron-withdrawing groups R1-R9.

[0008] The aforementioned spiropyran-modified BODIPY tumor phototherapy agent, wherein R is trifluoromethyl, 2,4-dinitrophenyl, or 1,5-dimethoxyphenyl.

[0009] A method for preparing a spiropyran-modified BODIPY tumor phototherapy agent, the method comprising the following steps: (1) The R groups of compounds M1, M2 and M3 are selected from R1-R9; among them, compound M1 is synthesized according to the literature method;

[0010] (2) Synthesis of compounds M2 and M3: Under nitrogen protection, 2,4-dinitrobenzaldehyde (or 1,5-dimethoxybenzaldehyde) was added to a two-necked reaction flask and dissolved in tetrahydrofuran; then 2,4-dimethylpyrrole and trifluoroacetic acid were added sequentially, and the reaction was stirred at room temperature for 5–6 hours; then DDQ (2,3-dichloro-5,6-dicyanobenzoquinone) was added, and the reaction was stirred at room temperature for another 2–3 hours; after the reaction was completed, the reaction solution was extracted with dichloromethane, and the organic phase was washed with water and then subjected to rotational osmosis. After removing the solvent by rotary evaporation, the compounds were separated and purified by column chromatography to obtain the compounds. Under nitrogen protection, the compounds were dissolved in tetrahydrofuran, and then DIPEA (N,N-diisopropylethylamine) was added. After stirring for 10–15 minutes, boron trifluoride diethyl ether complex was added, and the reaction was continued to be stirred at room temperature for 2–3 hours. After the reaction was completed, the reaction solution was extracted with dichloromethane, and the organic phase was washed with water. After removing the solvent by rotary evaporation, the compounds were separated and purified by column chromatography to obtain compounds M2 and M3. (3) Synthesis of compound M4: Under nitrogen protection, compound 1,1,2-trimethyl-1H-benzoylindole was dissolved in ultra-dry dichloromethane, methyl trifluoromethanesulfonate was added, and the reaction was stirred at room temperature for 48 hours. After the reaction was completed, the solvent was removed by vacuum distillation, and the residue was washed with ethyl acetate to obtain compound M4. (4) Synthesis of compound M5: Dissolve compound M4 in water, add sodium hydroxide solution dropwise until the solution becomes clear and transparent, and continue stirring for 1 hour; after the reaction is complete, extract with diethyl ether, dry the organic phase with anhydrous sodium sulfate, and then remove the solvent by rotary evaporation to obtain product M5; (5) Synthesis of compound M6: Under nitrogen protection, compound M5 and 4-hydroxy-m-phthalaldehyde were added to a two-necked reaction flask, dissolved in anhydrous ethanol, and heated under reflux for 5 hours. After the reaction was completed, the reaction was quenched with ice water, extracted with ethyl acetate, and the organic phase was dried with anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain the crude product. The crude product was separated and purified by column chromatography to obtain compound M6.

[0011] (6) Synthesis of compounds I1, I2, and I3: Under nitrogen protection, compound M1 (or M2, M3) was placed in a single-necked reaction flask and dissolved in anhydrous toluene. Then, compound M6, piperidine, and glacial acetic acid were added sequentially. The reaction flask was transferred to an oil bath and heated to 80°C (or 100°C), and the reaction was stirred continuously for 1–2 hours. After the reaction was completed, the reaction solution was extracted with dichloromethane, and the organic phase was washed with water. After removing the solvent by rotary evaporation, the target compounds I1, I2, and I3 were separated and purified by column chromatography.

[0012]

[0013] To improve the biocompatibility of phototherapy agent I, BODIPY phototherapy agent I containing a spiropyran photochromic structure was prepared into a nano-formulation. The preparation steps were as follows: phototherapy agent I was accurately weighed and dissolved in tetrahydrofuran, dissolved by ultrasonication, and then slowly added dropwise to an aqueous solution containing DSPE-PEG2000. The solvent was evaporated by continuous stirring for 24 hours, and the nanoparticle dispersion was obtained by dialysis or filtration purification. The aforementioned nano-formulations are used as phototherapy drugs in biological therapy, and their phototherapy drugs are photodynamic-photothermal synergistic therapy drugs.

[0014] The method for preparing a spiropyran-modified BODIPY tumor phototherapy agent, wherein in step (6), piperidine and glacial acetic acid are used as catalysts, anhydrous toluene is used as the reaction solvent, and the reaction temperature is 80-100℃.

[0015] The method for preparing a spiropyran-modified BODIPY tumor phototherapy agent, wherein the nano-formulation is self-assembled from the phototherapy agent and the amphiphilic polymer DSPE-PEG2000.

[0016] The aforementioned spiropyran-modified BODIPY tumor phototherapy agent and its preparation method, and the application of the nano-formulation in the preparation of drugs for synergistic photothermal-photodynamic therapy of tumors.

[0017] Advantages and positive effects of the present invention: 1. This invention uses 1,1,3-trimethyl-1,3-dihydrospiro[benzo[e]indole-2,2'-chromene]-6'-carboxaldehyde as a functional building block, which is covalently coupled to a fluoroboron dipyrrole core via a condensation reaction. BODIPY is a near-infrared fluorescent dye with broad application prospects. Existing literature has not reported studies on the condensation of spiropyran with BODIPY to construct phototherapy agents. There are currently no reports on modifying the molecular structure of BODIPY using the aforementioned aldehyde-substituted spiropyran. Unlike the BODIPY phototherapy agent condensed from 1,1,3-trimethyl-1,3-dihydrospiro[benzo[e]indole-2,2'-chromene]-6'-aldehyde, which is prone to photophysical quenching when coupled at the 1,7-position, this invention innovatively selects the modification site at the 3,5-position. By utilizing its steric hindrance, intramolecular π-π stacking is effectively avoided, thus overcoming the limitations of "functional stacking" and achieving a simultaneous improvement in fluorescence quantum yield and photothermal conversion efficiency. The non-radiative transitions of this compound are beneficial for photothermal conversion, and it exhibits good stability. The compound is readily available, has a novel structure, is easy to prepare, and is readily industrialized; the target compound can be obtained through a 5-step reaction.

[0018] 2. This invention integrates photothermal therapy with ROS fluorescence tracing. It utilizes the high efficiency of BODIPY photothermal conversion and the electronic sensitivity of closed-ring spiropyran. Under near-infrared light irradiation, BODIPY is used for tumor thermal ablation, while spiropyran responsively promotes ROS generation and achieves in-situ tracing through its own fluorescence changes, overcoming the limitations of traditional methods that only treat but are difficult to monitor. It avoids spectral interference and instability caused by ring-opening. Traditional spiropyran ring-opening generates anthocyanins, resulting in a redshift and dynamic spectral changes that interfere with the photothermal signal. This invention, through molecular design, ensures that spiropyran remains closed-ring, suppressing ring-opening isomerism, guaranteeing stable absorption and emission of BODIPY, and ensuring the photothermal efficiency and the stability and repeatability of the ROS detection signal. The push-pull electronic structure of closed-ring spiropyran synergistically promotes ROS generation. The closed-ring state retains the spirocyclic skeleton and push-pull substituents, promoting intramolecular charge transfer under light irradiation, enhancing interaction with oxygen, and inducing ROS generation. By combining the warm environment generated by BODIPY photothermal activity, ROS diffusion and reaction are accelerated, forming a "photothermal-ROS" dual-modal synergistic tumor killing effect.

[0019] 3. This invention bonds spiropyran at the 3,5-position of BODIPY via a condensation reaction, preserving the conjugated system of the BODIPY core and ensuring excellent photothermal conversion efficiency. Simultaneously, the complementary hydrophobic / lipophilic properties of the two molecules overcome the weakness of BODIPY's cell permeability and enhance tumor-targeting enrichment. This invention leverages the steric hindrance effect and intramolecular electronic regulation of BODIPY's rigid conjugated backbone to constrain the photoinduced ring-opening isomerization process of spiropyran at the molecular level, effectively inhibiting the formation of unstable ring-opening products. While retaining the original optical advantages of both molecules, it can still promote the generation of reactive oxygen species under illumination, simultaneously enabling fluorescent visualization and tracking of reactive oxygen species during treatment. Attached Figure Description

[0020] Figure 1 The structural formula of the novel phototherapy agent of this invention is I1; Figure 2 This is the proton spectrum of structural formula I1 of the novel phototherapy agent of the present invention; Figure 3 This is the carbon spectrum of the novel phototherapy agent of the present invention, structural formula I1; Figure 4 This is the proton spectrum of the intermediate structural formula M6 of the present invention; Figure 5 This is the carbon spectrum of the intermediate structural formula M6 of the present invention; Figure 6 The absorption spectrum (λabs = 736 nm in CH2Cl2) of the novel phototherapy agent of this invention is shown in structural formula I1. Figure 7 The fluorescence spectrum (λem = 799 nm in CH2Cl2) of the novel phototherapy agent of the present invention with structural formula I1 is shown. Figure 8 This is the crystal structure of the phototherapy agent I1 of the present invention; Figure 9 This is a photothermal test diagram of the phototherapy agent I1 of the present invention; Figure 10 This is an in vitro reactive oxygen species test diagram of the phototherapy agent I1 of the present invention; Figure 11 This is a cellular reactive oxygen species test diagram of phototherapy agent I1 of the present invention; Figure 12 This is the MTT test chart of phototherapy agent I1 of the present invention; Figure 13 This is a test diagram of the live and dead cell staining experiment of the phototherapy agent I1 of the present invention. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the embodiments shown in the accompanying drawings.

[0022] The present invention relates to a BODIPY phototherapy agent condensed from 1,1,3-trimethyl-1,3-dihydrospiro[benzo[e]indole-2,2'-chromene]-6'-aldehyde, the general formula of which is shown in Formula I:

[0023] In Formula I, R is selected from different electron-donating and electron-withdrawing groups R1-R9.

[0024]

[0025] Examples of representative molecular structures are shown below:

[0026] The specific steps for preparing the phototherapy agent are as follows: (1) The R groups of compounds M1, M2, and M3 are selected from R1-R9. Among them, compound M1 was synthesized according to the literature method ( Eur. J. Org. Chem. , 2017, 34, 5074-5079).

[0027]

[0028] (2) Synthesis of compounds M2 and M3: Under nitrogen protection, 2,4-dinitrobenzaldehyde (or 1,5-dimethoxybenzaldehyde) was added to a two-necked reaction flask and dissolved in tetrahydrofuran. Then, 2,4-dimethylpyrrole and trifluoroacetic acid were added sequentially, and the reaction was stirred at room temperature for 5–6 hours. DDQ (2,3-dichloro-5,6-dicyanobenzoquinone) was then added, and the reaction was continued at room temperature for 2–3 hours. After the reaction was complete, the reaction solution was extracted with dichloromethane, and the organic phase was washed with water. After removing the solvent by rotary evaporation, the compounds were purified by column chromatography to obtain the compounds. Under nitrogen protection, the compounds were dissolved in tetrahydrofuran, and then DIPEA (N,N-diisopropylethylamine) was added. After stirring for 10–15 minutes, the boron trifluoride diethyl ether complex was added, and the reaction was continued at room temperature for 2–3 hours. After the reaction was completed, the reaction solution was extracted with dichloromethane and the organic phase was washed with water. After the solvent was removed by rotary evaporation, the organic phase was separated and purified by column chromatography to obtain compounds M2 and M3.

[0029] (3) Synthesis of compound M4: Under nitrogen protection, compound 1,1,2-trimethyl-1H-benzoylindole was dissolved in ultra-dry dichloromethane, and methyl trifluoromethanesulfonate was added. The mixture was stirred at room temperature for 48 hours. After the reaction was completed, the solvent was removed by rotary evaporation, and the solution was washed with ethyl acetate to obtain compound M4.

[0030] (4) Synthesis of compound M5: Compound M4 was dissolved in water, and sodium hydroxide solution was added until the solution became clear and transparent. Stirring was continued for 1 hour. After the reaction was completed, the reaction solution was extracted with diethyl ether, and the organic phase was dried with anhydrous sodium sulfate and the solvent was removed by rotary evaporator to obtain product M5.

[0031] (5) Synthesis of compound M6: Under nitrogen protection, compound M5 and 4-hydroxy-m-phthalaldehyde were added to a two-necked reaction flask, dissolved in anhydrous ethanol, and heated under reflux for 5 hours. After the reaction was completed, the reaction was quenched with ice water, the reaction solution was extracted with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography to obtain compound M6.

[0032]

[0033] (6) Synthesis of compounds I1, I2, and I3: Under nitrogen protection, compound M1 (or M2, M3) was placed in a single-necked reaction flask and dissolved in anhydrous toluene. Then, compound M6, piperidine, and glacial acetic acid were added sequentially. The reaction flask was transferred to an oil bath and heated to 80°C (or 100°C), and the reaction was stirred continuously for 1–2 hours. After the reaction was completed, the reaction solution was extracted with dichloromethane, and the organic phase was washed with water. After removing the solvent by rotary evaporation, the target compounds I1, I2, and I3 were separated and purified by column chromatography.

[0034]

[0035] To improve the biocompatibility of phototherapy agent I, BODIPY phototherapy agent I containing a spiropyran photochromic structure was prepared into a nano-formulation. The preparation steps were as follows: phototherapy agent I was accurately weighed and dissolved in tetrahydrofuran. After ultrasonic dissolution, it was slowly added dropwise to an aqueous solution containing DSPE-PEG2000. The solvent was evaporated by continuous stirring for 24 hours. The nanoparticle dispersion was obtained by dialysis or filtration purification.

[0036] The aforementioned nano-formulations are used as phototherapy drugs in biological therapy, and the phototherapy drugs mentioned are photodynamic-photothermal synergistic therapy drugs. Example 1

[0037] The structures of the compounds described above in this invention are determined by nuclear magnetic resonance (NMR) spectra, including 1H NMR, 13C NMR, absorption spectra, fluorescence spectra, mass spectra, and single-crystal structures.

[0038] Prepare BODIPY phototherapy agent I1 by condensation of 1,1,3-trimethyl-1,3-dihydrospiro[benzo[e]indole-2,2'-chromene]-6'-aldehyde.

[0039]

[0040] (1) Compound M1 was synthesized according to the literature method (Eur. J. Org. Chem., 2017, 34, 5074-5079).

[0041] (2) Synthesis of compound M4: 1,1,2-trimethyl-1H-benzoylindole (2.09 g, 10 mmol) was dissolved in ultra-dry dichloromethane under nitrogen protection, and methyl trifluoromethanesulfonate (2.46 g, 15 mmol) was added. The mixture was stirred at room temperature for 48 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the residue was washed with ethyl acetate to obtain compound M4 (1.5 g, 6.70 mmol). The yield was 67.0%.

[0042] (3) Synthesis of compound M5: Compound M4 (1.5 g, 6.70 mmol) was dissolved in water, and sodium hydroxide solution was added dropwise until the solution became clear and transparent. The mixture was stirred for 1 hour. After the reaction was complete, the mixture was extracted with diethyl ether. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain product M5 (0.698 g, 3.13 mmol). The yield was 46.7%.

[0043] (4) Synthesis of compound M6: Under nitrogen protection, compound M5 (0.698 g, 3.13 mmol) and 4-hydroxy-isophthalaldehyde (0.427 g, 2.84 mmol) were added to a two-necked reaction flask, dissolved in anhydrous ethanol, and heated to reflux for 5 hours. After the reaction was completed, the reaction was quenched with ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was separated by silica gel column chromatography, using ethyl acetate-petroleum ether (v / v 1:10) as the eluent, to purify compound M6 (0.96 g, 2.70 mmol). The yield was 86.3%. 1H NMR (400 MHz, CDCl3) δ 9.81 (s, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.78 (dd, J = 20.8, 8.0 Hz, 2H), 7.65–7.56 (m, 2H), 7.41 (ddd, J = 8.0, 6.8, 1.2 Hz, 1H), 7.23 (ddd, J =6.8, 5.6, 1.2 Hz, 1H), 6.97 (dd, J = 9.6, 2.4 Hz, 2H), 6.74 (d, J = 8.0 Hz,1H), 5.85 (d, J = 10.4 Hz, 1H), 2.83 (s, 3H), 1.64 (s, 3H), 1.35 (s, 3H). 13CNMR (151 MHz, CDCl3) δ 190.58, 160.08, 145.69, 132.61, 129.97, 129.64,129.57, 129.50, 129.28, 129.19, 128.53, 126.49, 125.48, 121.85, 121.40,120.27, 118.98, 115.55, 110.26, 106.97, 53.71, 29.29, 24.08, 21.66. (5) Under nitrogen protection, compound M1 (0.19 g, 0.6 mmol) was placed in a single-necked reaction flask and dissolved in anhydrous toluene. Then, compound M6 (0.43 g, 1.2 mmol), piperidine (0.3 mL), and glacial acetic acid (0.3 mL) were added sequentially. The reaction flask was transferred to an oil bath and heated to 80 °C, and the reaction was stirred continuously for 12 hours. After the reaction was completed, the reaction solution was extracted with dichloromethane, and the organic phase was washed with water. After removing the solvent by rotary evaporation, the crude product was recrystallized from petroleum ether to give compound I1 (0.21 g, 0.13 mmol). The yield was 21.7%. 1H NMR (400 MHz, CDCl3) δ 8.10 (d, J =7.2 Hz, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.0 Hz, 2H), 7.76 (d, J =8.4 Hz, 2H), 7.58 (d, J = 16.4 Hz, 2H), 7.43 - 7.35 (m, 6H), 7.23 (t, J = 8.0Hz, 3H), 7.04 - 6.97 (m, 4H), 6.79 (s, 2H), 6.70 (d, J = 8.4 Hz, 2H), 5.85(d, J = 10.4 Hz, 2H), 2.85 (s, 6H), 2.36 (d, J = 3.2 Hz, 6H), 1.55 (s, 12H).13C NMR (151 MHz, CDCl3) δ 155.40, 153.39, 144.82, 139.84, 136.99, 129.28,128.99, 128.61, 128.54, 128.42, 128.00, 127.72, 125.31, 125.01, 124.61,120.62, 120.39, 118.82, 118.07, 114.52, 109.24, 105.11, 52.49, 28.69, 28.29,23.06, 20.68, 13.11. HRMS (ESI) m / z calcd for C62H52BF5N4O2Na+ (M+Na)+1013.39957, found 1013.39917. Example 2

[0044] Synthesis of compound M2: Under nitrogen protection, 2,4-dinitrobenzaldehyde (0.78 g, 4 mmol) was added to a two-necked flask and dissolved in 20 mL of tetrahydrofuran (THF). Then, 2,4-dimethylpyrrole (1 mL, 10 mmol) and trifluoroacetic acid (0.20 mL, 2.8 mmol) were added sequentially, and the mixture was stirred at room temperature for 5–6 hours. DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) (0.91 g, 4 mmol) was then added, and the mixture was stirred at room temperature for another 2–3 hours. After the reaction was complete, the reaction mixture was extracted with dichloromethane, and the organic phase was washed with water. The solvent was removed by rotary evaporation, and the compound was purified by column chromatography. Under nitrogen protection, the compound was dissolved in 20 mL of tetrahydrofuran (THF), followed by the addition of DIPEA (N,N-diisopropylethylamine) (0.85 g, 5 mmol). After stirring for 10–15 minutes, the boron trifluoride diethyl ether complex (0.71 g, 5 mmol) was added, and the reaction was continued with stirring at room temperature for 2–3 hours. After the reaction was complete, the reaction solution was extracted with dichloromethane, and the organic phase was washed with water. The solvent was removed by rotary evaporation, and the compound was purified by column chromatography to give compound M2 (0.55 g, 1.34 mmol). The yield was 33.4%. 1H NMR (400 MHz, CDCl3) δ 8.995 (d, J = 2.4 Hz, 1H), 8.623 (dd, J= 8.4, 2.4 Hz, 1H), 7.745 (d, J = 8.4 Hz, 1H), 6.034 (s, 2H), 2.573 (s, 6H),1.376 (s, 6H). 13C NMR (151 MHz, CDCl3) δ 157.44, 156.43, 148.95, 148.54,141.25, 136.30, 133.05, 130.04, 127.91, 122.28, 122.11, 120.34, 118.32, 113.60, 14.75, 14.17.

[0045] Synthesis of compound I2: Under nitrogen protection, compound M2 (0.25 g, 0.6 mmol) was dissolved in anhydrous toluene in a single-necked reaction flask, followed by the sequential addition of compound M6 (0.43 g, 1.2 mmol), piperidine (0.3 mL), and glacial acetic acid (0.3 mL). The reaction flask was transferred to an oil bath and heated to 100 °C, with continuous stirring for 12 hours. After the reaction was complete, the reaction solution was extracted with dichloromethane, and the organic phase was washed with water. The solvent was removed by rotary evaporation, and the crude product was recrystallized from petroleum ether to give compound I2 (0.12 g, 0.11 mmol). The yield was 18.1%. 1H NMR (400 MHz, CDCl3) δ8.995 (d, J = 2.4 Hz, 1H), 8.623 (dd, J = 8.4, 2.4 Hz, 1H), 7.745 (d, J = 8.4Hz, 1H), 6.034 (s, 2H), 2.573 (s, 6H), 1.376 (s, 6H). 13C NMR (151 MHz, CDCl3) δ 157.44, 156.43, 148.95, 148.54, 141.25, 136.30, 133.05, 130.04,127.91, 122.28, 122.11, 120.34, 118.32, 113.60, 14.75, 14.17. Example 3 Synthesis of compound M3: Under nitrogen protection, 1,5-dimethoxybenzaldehyde (0.665 g, 4 mmol) was added to a two-necked reaction flask and dissolved in 20 mL of tetrahydrofuran (THF). Then, 2,4-dimethylpyrrole (1 mL, 10 mmol) and trifluoroacetic acid (0.20 mL, 2.8 mmol) were added sequentially, and the mixture was stirred at room temperature for 5–6 hours. DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) (0.91 g, 4 mmol) was then added, and the mixture was stirred at room temperature for another 2–3 hours. After the reaction was complete, the reaction mixture was extracted with dichloromethane, and the organic phase was washed with water. The solvent was removed by rotary evaporation, and the compound was purified by column chromatography to obtain the final product. Under nitrogen protection, the compound was dissolved in tetrahydrofuran (THF), followed by the addition of DIPEA (N,N-diisopropylethylamine) (0.85 g, 5 mmol). After stirring for 10–15 minutes, the boron trifluoride diethyl ether complex (0.71 g, 5 mmol) was added, and the reaction was continued with stirring at room temperature for 2–3 hours. After the reaction was complete, the reaction solution was extracted with dichloromethane, and the organic phase was washed with water. The solvent was removed by rotary evaporation, and the compound was purified by column chromatography to give compound M3 (0.59 g, 0.13 mmol). The yield was 38.6%. 1H NMR (400 MHz, CDCl3) δ 6.97 (dd, J = 8.8, 2.8 Hz, 1H), 6.91 (d, J =9.2 Hz, 1H), 6.73 (d, J = 2.8 Hz, 1H), 5.97 (s, 2H), 3.74 (d, J = 14.4 Hz,6H), 2.55 (s, 6H), 1.49 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 155.07, 154.50,150.41, 142.61, 138.51, 131.45, 124.40, 120.89, 115.64, 114.83, 112.47, 56.11, 55.93, 14.60, 13.83.

[0046] Synthesis of compound I3: Under nitrogen protection, compound M3 (0.23 g, 0.6 mmol) was dissolved in anhydrous toluene in a single-necked reaction flask, followed by the sequential addition of compound M6 (0.43 g, 1.2 mmol), piperidine (0.3 mL), and glacial acetic acid (0.3 mL). The reaction flask was transferred to an oil bath and heated to 100 °C, with continuous stirring for 12 hours. After the reaction was complete, the reaction solution was extracted with dichloromethane, and the organic phase was washed with water. The solvent was removed by rotary evaporation, and the crude product was recrystallized from petroleum ether to give compound I3 (0.104 g, 0.10 mmol). The yield was 16.5%. 1H NMR (400 MHz, CDCl3) δ6.97 (dd, J = 8.8, 2.8 Hz, 1H), 6.91 (d, J = 9.2 Hz, 1H), 6.73 (d, J = 2.8Hz, 1H), 5.97 (s, 2H), 3.74 (d, J = 14.4 Hz, 6H), 2.55 (s, 6H), 1.49 (s, 6H).13C NMR (101 MHz, CDCl3) δ 155.07, 154.50, 150.41, 142.61, 138.51, 131.45,124.40, 120.89, 115.64, 114.83, 112.47, 56.11, 55.93, 14.60, 13.83. The above description, in conjunction with specific optimized embodiments, provides a further detailed explanation of the present invention and should not be construed as limiting the specific implementation of the invention to these descriptions. For those skilled in the art, based on the core concept of the present invention, several simple deductions and substitutions can be made, all of which should be considered within the scope of protection of the present invention. Furthermore, for those skilled in the art, based on the present invention, several simple inferences can be made to derive other applications of the compounds of the present invention, all of which should be considered within the scope of protection of the present invention.

Claims

1. A spiropyran-modified BODIPY tumor phototherapy agent, characterized in that, The phototherapy agent has the general formula structure shown in Formula I: ； In Formula I, R is selected from different electron-donating and electron-withdrawing groups R1-R9; 。 2. The spiropyran-modified BODIPY tumor phototherapy agent according to claim 1, characterized in that, R is trifluoromethyl, 2,4-dinitrophenyl, or 1,5-dimethoxyphenyl.

3. A method for preparing a spiropyran-modified BODIPY tumor phototherapy agent, characterized in that, The method includes the following steps: (1) The R groups of compounds M1, M2 and M3 are selected from R1-R9; among them, compound M1 is synthesized according to the literature method; ； (2) Synthesis of compounds M2 and M3: Under nitrogen protection, 2,4-dinitrobenzaldehyde (or 1,5-dimethoxybenzaldehyde) was added to a two-necked reaction flask and dissolved in tetrahydrofuran; then 2,4-dimethylpyrrole and trifluoroacetic acid were added sequentially, and the reaction was stirred at room temperature for 5–6 hours; then DDQ (2,3-dichloro-5,6-dicyanobenzoquinone) was added, and the reaction was stirred at room temperature for another 2–3 hours; after the reaction was completed, the reaction solution was extracted with dichloromethane, and the organic phase was washed with water and then subjected to rotational osmosis. After removing the solvent by rotary evaporation, the compounds were separated and purified by column chromatography to obtain the compounds. Under nitrogen protection, the compounds were dissolved in tetrahydrofuran, and then DIPEA (N,N-diisopropylethylamine) was added. After stirring for 10–15 minutes, boron trifluoride diethyl ether complex was added, and the reaction was continued to be stirred at room temperature for 2–3 hours. After the reaction was completed, the reaction solution was extracted with dichloromethane, and the organic phase was washed with water. After removing the solvent by rotary evaporation, the compounds were separated and purified by column chromatography to obtain compounds M2 and M3. (3) Synthesis of compound M4: Under nitrogen protection, compound 1,1,2-trimethyl-1H-benzoylindole was dissolved in ultra-dry dichloromethane, methyl trifluoromethanesulfonate was added, and the reaction was stirred at room temperature for 48 hours. After the reaction was completed, the solvent was removed by vacuum distillation, and the residue was washed with ethyl acetate to obtain compound M4. (4) Synthesis of compound M5: Dissolve compound M4 in water, add sodium hydroxide solution dropwise until the solution becomes clear and transparent, and continue stirring for 1 hour; after the reaction is complete, extract with diethyl ether, dry the organic phase with anhydrous sodium sulfate, and then remove the solvent by rotary evaporation to obtain product M5; (5) Synthesis of compound M6: Under nitrogen protection, compound M5 and 4-hydroxy-m-phthalaldehyde were added to a two-necked reaction flask, dissolved in anhydrous ethanol, and heated under reflux for 5 hours. After the reaction was completed, the reaction was quenched with ice water, extracted with ethyl acetate, and the organic phase was dried with anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain the crude product. The crude product was separated and purified by column chromatography to obtain compound M6. ； (6) Synthesis of compounds I1, I2 and I3: Under nitrogen protection, compound M1 (or M2, M3) was placed in a single-necked reaction flask and dissolved in anhydrous toluene. Then, compound M6, piperidine and glacial acetic acid were added sequentially. The reaction flask was transferred to an oil bath and heated to 80°C (or 100°C), and the reaction was stirred continuously for 1–2 hours. After the reaction was completed, the reaction solution was extracted with dichloromethane and the organic phase was washed with water. After removing the solvent by rotary evaporation, the target compounds I1, I2 and I3 were separated and purified by column chromatography. ； To improve the biocompatibility of phototherapy agent I, a BODIPY-type phototherapy agent I containing a spiropyran photochromic structure was prepared into a nano-formulation. The preparation steps were as follows: phototherapy agent I was accurately weighed and dissolved in tetrahydrofuran, and after ultrasonic dissolution, it was slowly added dropwise to a solution containing DSPE-PEG. 2000 The solvent was continuously stirred in an aqueous solution for 24 hours to evaporate, and the nanoparticle dispersion was obtained by dialysis or filtration purification. The aforementioned nano-formulations are used as phototherapy drugs in biological therapy, and their phototherapy drugs are photodynamic-photothermal synergistic therapy drugs.

4. The method for preparing a spiropyran-modified BODIPY tumor phototherapy agent according to claim 3, characterized in that, In step (6), piperidine and glacial acetic acid are used as catalysts, anhydrous toluene is used as the reaction solvent, and the reaction temperature is 80-100℃.

5. The method for preparing a spiropyran-modified BODIPY tumor phototherapy agent according to claim 3, characterized in that, The nano-formulation consists of a phototherapy agent and the amphiphilic polymer DSPE-PEG. 2000 It is self-assembled.

6. A spiropyran-modified BODIPY tumor phototherapy agent and its preparation method according to claim 1 or 3, characterized in that, The application of the nano-formulation in the preparation of drugs for the synergistic photothermal-photodynamic therapy of tumors.

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