H-aggregating porphyrin derivative, and preparation method and application thereof
By designing the donor-receptor structure of H-aggregated pyrrole derivatives, stable H-aggregates are formed through self-assembly, which solves the problem of low efficiency of photodynamic and photothermal therapy in hypoxic tumor treatment and realizes efficient synergistic treatment in hypoxic environments.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTH CHINA UNIV OF TECH
- Filing Date
- 2026-03-13
- Publication Date
- 2026-06-05
AI Technical Summary
Existing photodynamic and photothermal therapies are inefficient and limited in the treatment of hypoxic tumors. Traditional photosensitizers are not effective in hypoxic environments, and fluorescence quenching in the aggregated state affects their performance. The lack of a single-component photosensitizer design that combines near-infrared absorption, type I reactive oxygen species generation, and photothermal properties makes it difficult to achieve.
A H-aggregate pyrrole derivative was designed. Through molecular design of donor-acceptor structure, it self-assembles into a stable H-aggregate with near-infrared absorption capability. It can efficiently generate type I reactive oxygen species and carry out photothermal conversion in hypoxic or normoxic environments, thereby achieving synergistic enhancement of photodynamic and photothermal performance.
The integration of type I photodynamic and photothermal properties on a single material has been achieved, enabling highly efficient synergistic photodynamic-photothermal therapy in hypoxic tumor treatment. This overcomes oxygen dependence limitations and provides a highly efficient anti-hypoxic tumor treatment solution.
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Figure CN122145485A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical materials technology, and in particular to an H-aggregated pyrrole derivative, its preparation method, and its application. Background Technology
[0002] Phototherapy, including photodynamic therapy and photothermal therapy, has become an important research direction in the field of tumor treatment due to its advantages such as non-invasiveness, spatiotemporal controllability, and low systemic toxicity.
[0003] Photodynamic therapy (PDT) has garnered significant attention in cancer treatment due to its advantages such as non-invasiveness, high spatiotemporal controllability, and low systemic toxicity. PDT relies on photosensitizers (PSs) to generate cytotoxic type I (superoxide anion O2) under light irradiation. - and hydroxyl radicals OH) and type II (singlet oxygen) 1 O2) Reactive oxygen species (ROS). Currently, type II PDT, which is dominant, involves a photosensitizer transferring excited-state energy (EET) to ambient oxygen to generate singlet oxygen. However, the tumor environment is hypoxic, severely impacting its therapeutic efficacy. Type I PDT, on the other hand, generates O2 by transferring electrons from a photosensitizer to ambient oxygen or the substrate. - It can generate more toxic hydrogen peroxide (H2O2) and hydroxyl radicals within cells through superoxide dismutase (SOD) reaction. (OH) and simultaneously achieve oxygen regeneration, thus effectively overcoming the hypoxia dilemma. Therefore, the development of type I photosensitizers with low oxygen dependence is of great significance. Photothermal therapy utilizes photothermal converters to convert light energy into heat energy, ablating tumors through local high temperature. This method is not limited by oxygen concentration and is effective for hypoxic tumors. However, single hyperthermia has certain limitations: heat can easily diffuse into surrounding normal tissues, causing damage, which may induce heat resistance in tumors, and it is not thorough in clearing micrometastases. Therefore, the development of photosensitizers that combine near-infrared absorption, photodynamic activity, and photothermal properties is considered an ideal way to achieve highly efficient anti-hypoxia synergistic phototherapy, but its design and synthesis still face many challenges.
[0004] Furthermore, the aggregation state of the photosensitizer has a decisive influence on its performance. Typically, most organic photosensitizers aggregate in the aggregated state, leading to fluorescence quenching. H-aggregation is a form in which molecules are stacked "face-to-face," with a high degree of overlap between the molecules. The planar shape allows for significant overlap of the molecular electronic orbitals, resulting in excellent intermolecular charge transfer and separation capabilities, thus facilitating the generation of type I reactive oxygen species (ROS). Simultaneously, based on exciton coupling theory, H-aggregation can suppress the fluorescence emission of the aggregates, thereby promoting overall thermal relaxation and providing possibilities for the generation of type I ROS and photothermal properties. However, how to precisely control and stably form this H-aggregate state with functional enhancement effects through rational molecular design remains a key scientific problem that urgently needs to be solved in the field of materials chemistry.
[0005] Therefore, there is an urgent need in this field for a novel molecular design strategy that enables the construction of a single-component photosensitizer that absorbs in the near-infrared region, can self-assemble into a stable H-aggregate state, and can simultaneously generate highly efficient type I reactive oxygen species and photothermal effects, in order to overcome the bottleneck of synergistic phototherapy for hypoxic tumors. Summary of the Invention
[0006] The purpose of this invention is to overcome the problems in the prior art and provide an H-aggregated pyrrole derivative, its preparation method, and its application.
[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an H-aggregated pyrrole derivative, the structure of which is shown below: or ; Wherein, R1 is independently a C1-C30 alkyl, a C1-C30 phenyl, a phenyl substituted with a C1-C30 alkyl or a phenyl substituted with a C1-C30 alkoxy; R2 is a C1-C30 alkyl group.
[0008] This invention provides a method for preparing the H-aggregated pyrrole derivative, comprising the following steps: (1) 4-R2-4H-dithiopheno[3,2-b:2,3-d]pyrrole, N,N-dimethylformamide and 1,2-dichloroethane were mixed and then phosphorus oxychloride was added for secondary mixing; then formylation reaction was carried out to obtain formylated product; (2) The formylated product, tetrahydrofuran and N-bromosuccinimide were mixed and then subjected to bromination to obtain the bromination product; (3) The brominated product, donor borate ester, 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl, palladium acetate, tripotassium phosphate and mixed solvent are mixed and reacted to obtain the coupling product; (4) The coupling product, acceptor unit and mixed solvent are mixed and subjected to condensation reaction to obtain the H-aggregated pyrrole derivative.
[0009] Preferably, in step (1), R2 in 4-R2-4H-dithiopheno[3,2-b:2,3-d]pyrrole is a C1-C30 alkyl group; In step (1), the ratio of 4-R2-4H-dithiopheno[3,2-b:2,3-d]pyrrole, N,N-dimethylformamide, 1,2-dichloroethane and phosphorus oxychloride is 1.5~2 mmol: 15~20 mmol: 10~20 mL: 5.5~6 mmol.
[0010] Preferably, the mixing temperature in step (1) is -5~5℃; The secondary mixing temperature is -5~5℃, and the time is ≥2h; In step (1), the formylation reaction is carried out at a temperature of 30~50℃ for ≥4h.
[0011] Preferably, in step (2), the ratio of the formylated product, tetrahydrofuran and N-bromosuccinimide is 0.65~0.75mmol: 5~10mL: 0.7~0.8mmol.
[0012] Preferably, the bromination reaction in step (2) is carried out at a temperature of 20~30℃ for a time of ≥1h.
[0013] Preferably, the structural formula of the donor unit borate ester in step (3) is: R1 is independently a C1-C30 alkyl, C1-C30 phenyl, phenyl substituted with a C1-C30 alkyl or phenyl substituted with a C1-C30 alkoxy; In step (3), the mixed solvent contains 1,4-dioxane and water, and the volume ratio of 1,4-dioxane to water is 4~6:1; In step (3), the ratio of brominated product, donor borate ester, 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl, palladium acetate, tripotassium phosphate, and mixed solvent is 400~600mg: 650~750mg: 20~30mg: 10~20mg: 1350~1450mg: 20~30mL; The reaction temperature in step (3) is 30~50℃ and the time is 12~16h.
[0014] Preferably, the acceptor unit in step (4) is 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonium or 2-[3-cyano-4-methyl-5-phenyl-5-trifluoromethyl-2(5H)-furanyl]malonium; The mixed solvent in step (4) contains ethanol and tetrahydrofuran; the volume ratio of ethanol to tetrahydrofuran is 1~2:1~2; In step (4), the ratio of the coupling product, acceptor unit, and mixed solvent is 50-100 mg: 50-100 mg: 10-15 mL; In step (4), the condensation reaction is carried out at a temperature of 40-60℃ for 2-4 hours.
[0015] The present invention also provides the application of the H-aggregated pyrrole derivative in the preparation of phototherapy drugs and drugs for treating hypoxic tumors.
[0016] This invention provides an H-aggregated pyrrole derivative, which is a derivative having a donor- -Receptor (D-) -A) structure molecules, with donor structures being aromatic amine derivatives. The bridging structure is dithieno[3,2-b:2,3-d]pyrrole, and the acceptor structure is 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonitrile or 2-[3-cyano-4-methyl-5-phenyl-5-trifluoromethyl-2(5H)-furanylidene]malonitrile; this invention utilizes a "strong donor-large planar" approach. The synergistic molecular design of the "bridge-strong receptor" successfully guided the self-assembly of molecules into stable H-aggregates. This unique aggregate state breaks the competitive relationship between traditional photosensitizer properties, achieving "integration" and "synergistic enhancement" of type I photodynamic and photothermal properties on a single material, thereby simultaneously triggering two highly efficient therapeutic mechanisms under single-wavelength near-infrared light irradiation. This invention provides a novel solution for highly efficient anti-hypoxic tumor treatment. In summary, this invention integrates anti-hypoxic type I photodynamic therapy, highly efficient photothermal therapy, and near-infrared-excited deep penetration capabilities, providing a novel and reliable solution to the challenging problem of treating hypoxic solid tumors.
[0017] The derivative provided by this invention simultaneously possesses near-infrared light absorption capability, type I reactive oxygen species (ROS) generation capability, and photothermal conversion capability. It exhibits strong absorption in the near-infrared region where biological tissues have good light transmittance. Under light irradiation, it can efficiently generate type I ROS, especially in hypoxic or normoxic environments, fundamentally overcoming the severe oxygen dependence of traditional type II photodynamic therapy. It also possesses highly efficient photothermal conversion capability. Through self-assembly, it forms a stable H-aggregate state, thereby synergistically enhancing the aforementioned type I photodynamic and photothermal properties. Ultimately, it achieves highly efficient and synergistic photodynamic-photothermal therapy for hypoxic tumors. Attached Figure Description
[0018] Figure 1 This is a schematic diagram of the preparation process of the H-aggregated pyrrole derivative in Example 1; Figure 2 The absorption and emission spectra of the H-aggregated pyrrole derivative in tetrahydrofuran in Example 1 are shown. Figure 3The particle size distribution and absorption spectrum of the nanoparticles in Example 1 are shown. Figure 4 The graph shows the fold increase in fluorescence intensity of the DCFH solution under 808 nm laser irradiation for nanoparticles, DCFH, and the commercial photosensitizer ICG in Example 1. Figure 5 The graph shows the fold increase in fluorescence intensity of the solution of nanoparticles, DHR123, and HPF under 808 nm laser irradiation in Example 1. Figure 6 This is a graph showing the fold increase in fluorescence intensity of the solution containing nanoparticles and ABDA under 808 nm laser irradiation in Example 1. Figure 7 The graphs show the temperature rise curve and photothermal cycle curve of the solution containing nanoparticles and the commercial photosensitizer ICG under 808 nm laser irradiation in Example 1. Figure 8 The image shows the phototoxicity and dark toxicity results of the nanoparticles in Example 1 on 4T1 cells under normoxic and hypoxic conditions. Detailed Implementation
[0019] This invention provides an H-aggregated pyrrole derivative, characterized in that the structure of the H-aggregated pyrrole derivative is as follows: or ; Wherein, R1 is independently a C1-C30 alkyl, a C1-C30 phenyl, a phenyl substituted with a C1-C30 alkyl or a phenyl substituted with a C1-C30 alkoxy; R2 is a C1-C30 alkyl group.
[0020] This invention provides a method for preparing the H-aggregated pyrrole derivative, comprising the following steps: (1) 4-R2-4H-dithiopheno[3,2-b:2,3-d]pyrrole, N,N-dimethylformamide and 1,2-dichloroethane were mixed and then phosphorus oxychloride was added for secondary mixing; then formylation reaction was carried out to obtain formylated product; (2) The formylated product, tetrahydrofuran and N-bromosuccinimide were mixed and then subjected to bromination to obtain the bromination product; (3) The brominated product, donor borate ester, 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl, palladium acetate, tripotassium phosphate and mixed solvent are mixed and reacted to obtain the coupling product; (4) The coupling product, acceptor unit and mixed solvent are mixed and subjected to condensation reaction to obtain the H-aggregated pyrrole derivative.
[0021] In this invention, R2 in 4-R2-4H-dithiopheno[3,2-b:2,3-d]pyrrole in step (1) is a C1-C30 alkyl group.
[0022] In this invention, the preferred ratio of 4-R2-4H-dithiopheno[3,2-b:2,3-d]pyrrole, N,N-dimethylformamide, 1,2-dichloroethane and phosphorus oxychloride in step (1) is 1.5~2 mmol: 15~20 mmol: 10~20 mL: 5.5~6 mmol, more preferably 1.6~1.9 mmol: 16~19 mmol: 12~18 mL: 5.6~5.9 mmol, and even more preferably 1.7~1.8 mmol: 17~18 mmol: 14~16 mL: 5.7~5.8 mmol.
[0023] In this invention, all steps in step (1) are performed in a protective atmosphere.
[0024] In this invention, the mixing temperature in step (1) is preferably -5~5℃, more preferably -4~4℃, and even more preferably -3~3℃; stirring is maintained during the mixing process, and the next step is carried out after the system is mixed evenly.
[0025] In this invention, phosphorus oxychloride is added dropwise to the system and then mixed twice. The temperature of the second mixing is preferably -5~5℃, more preferably -4~4℃, and even more preferably -3~3℃. The time is preferably ≥2h, more preferably ≥2.5h, and even more preferably ≥3h.
[0026] In this invention, the temperature of the formylation reaction in step (1) is preferably 30~50℃, more preferably 35~45℃, and even more preferably 38~42℃; the time is preferably ≥4h, more preferably ≥4.5h, and even more preferably ≥5h.
[0027] In this invention, after the formylation reaction in step (1) is completed, the reaction solution is cooled to room temperature. A saturated sodium acetate aqueous solution is slowly added to the reaction solution to make the solution neutral, and stirring continues for 2 hours at room temperature. The mixture is then transferred to a separatory funnel and extracted with chloroform. The combined organic phases are washed once with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product is purified by silica gel column chromatography. First, a mixed solvent of petroleum ether / tetrahydrofuran = 100:1 (v / v) is used to wash away the less polar starting material, then a gradient solvent system of petroleum ether / ethyl acetate 20~10:1 (v / v) is switched to elute the target product. The fraction containing the target product is collected, concentrated under reduced pressure, and the formylated product is obtained.
[0028] In this invention, the preferred ratio of the formylated product, tetrahydrofuran and N-bromosuccinimide in step (2) is 0.65~0.75mmol:5~10mL:0.7~0.8mmol, more preferably 0.66~0.74mmol:6~9mL:0.72~0.78mmol, and even more preferably 0.68~0.72mmol:7~8mL:0.74~0.76mmol.
[0029] In this invention, all steps in step (2) are performed in a protective atmosphere.
[0030] In this invention, the formylated product is first dissolved in tetrahydrofuran and cooled to 0°C in an ice-water bath. Then, N-bromosuccinimide is added. After all the product has been added, the ice bath is removed, and the bromination reaction is carried out.
[0031] In this invention, the temperature of the bromination reaction in step (2) is preferably 20~30℃, more preferably 22~28℃, and even more preferably 24~26℃; the time is preferably ≥1h, more preferably ≥1.5h, and even more preferably ≥2h.
[0032] In this invention, after the bromination reaction in step (2) is completed, water is added to quench the reaction solution. The mixture is transferred to a separatory funnel and extracted with chloroform. The combined organic phases are washed once with water, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure in a water bath at 30°C to obtain a crude oil product. The crude product is purified by vacuum silica gel column chromatography using chloroform as the eluent. The target fraction is collected, concentrated under reduced pressure, and the bromination product is obtained.
[0033] In this invention, the structural formula of the donor unit borate ester in step (3) is as follows: R1 is independently a C1-C30 alkyl, C1-C30 phenyl, phenyl substituted with a C1-C30 alkyl or phenyl substituted with a C1-C30 alkoxy; In this invention, the mixed solvent in step (3) comprises 1,4-dioxane and water, and the volume ratio of 1,4-dioxane to water is preferably 4~6:1, more preferably 4.5~5.5:1, and even more preferably 4.8~5.2:1.
[0034] In this invention, the preferred ratio of the brominated product, donor borate ester, 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl, palladium acetate, tripotassium phosphate, and mixed solvent in step (3) is 400~600mg:650~750mg:20~30mg:10~20mg:1350~1450mg:20~30mL, more preferably 450~550mg:660~740mg:22~28mg:12~18mg:1360~1440mg:22~28mL, and even more preferably 480~520mg:680~720mg:24~26mg:14~16mg:1380~1420mg:24~26mL.
[0035] In this invention, all steps in step (3) are carried out in a protective atmosphere.
[0036] In this invention, the bromide product, the donor unit borate ester, 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl, palladium acetate and tripotassium phosphate are mixed, the system is purged with nitrogen, and then a mixed solvent is added to carry out the reaction.
[0037] In this invention, the reaction temperature in step (3) is preferably 30~50℃, more preferably 35~45℃, and even more preferably 38~42℃; the reaction time is preferably 12~16h, more preferably 13~15h, and even more preferably 13.5~14.5h; stirring is maintained during the reaction.
[0038] In this invention, after the reaction in step (3) is completed, the reaction solution is cooled to room temperature and concentrated under reduced pressure to remove most of the solvent. A suitable amount of dichloromethane is added to the residue to dissolve it, and the resulting solution is washed with saturated brine. The organic layer is dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to obtain the crude product. The crude product is purified by silica gel column chromatography. First, a mixed solvent of dichloromethane / n-hexane = 10:1 (v / v) is used for elution to remove the less polar borate ester raw material and its tailing impurities. Then, a gradient solvent system of ethyl acetate / n-hexane = 10:1 to 5:1 (v / v) is switched to elute the target product. The fraction containing the target product is collected, combined, and concentrated under reduced pressure to obtain a preliminarily purified oil. The oil is recrystallized to obtain the coupling product.
[0039] In this invention, the acceptor unit in step (4) is 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonitrile or 2-[3-cyano-4-methyl-5-phenyl-5-trifluoromethyl-2(5H)-furanyl]-malonitrile.
[0040] In this invention, the mixed solvent in step (4) comprises ethanol and tetrahydrofuran; the volume ratio of ethanol to tetrahydrofuran is preferably 1~2:1~2, more preferably 1.2~1.8:1.2~1.8, and even more preferably 1.4~1.6:1.4~1.6.
[0041] In this invention, the preferred ratio of the amount of coupling product, acceptor unit and mixed solvent in step (4) is 50~100mg:50~100mg:10~15mL, more preferably 60~90mg:60~90mg:11~14mL, and even more preferably 70~80mg:70~80mg:12~13mL.
[0042] In this invention, the temperature of the condensation reaction in step (4) is preferably 40~60℃, more preferably 45~55℃, and even more preferably 48~52℃; the time is preferably 2~4h, more preferably 2.5~3.5h, and even more preferably 2.8~3.2h; and stirring is maintained during the reaction.
[0043] In this invention, after the reaction in step (4) is completed, the reaction solution is cooled to room temperature and concentrated under reduced pressure to completely remove the solvent. A mixed solvent of dichloromethane / methanol is added to the obtained residue for recrystallization. The precipitated solid is collected by filtration, washed with a small amount of cold solvent, and dried under vacuum to obtain the final product, H-aggregated pyrrole derivative.
[0044] The present invention also provides the application of the H-aggregated pyrrole derivative in the preparation of phototherapy drugs and drugs for treating hypoxic tumors.
[0045] This invention also provides a method for preparing photosensitizers using the H-aggregated pyrrole derivative, comprising the following steps: dissolving the H-aggregated pyrrole derivative and an amphiphilic polymer together in a good solvent to form a mixed solution; injecting the mixed solution into a poor solvent to induce co-self-assembly, ultimately forming nanoparticles with the H-aggregated pyrrole derivative as the core and the amphiphilic polymer as the shell, which is the H-aggregated type I photosensitizer.
[0046] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0047] Example 1
[0048] Under nitrogen protection, 1.83 mmol (1.0 equivalent) of 4-(deca-7-yl)-4H-dithiopheno[3,2-b:2,3-d]pyrrole and 18.3 mmol (10.0 equivalent) of N,N-dimethylformamide (DMF) were dissolved in 15 mL of anhydrous 1,2-dichloroethane. The reaction mixture was cooled to 0 °C in an ice-water bath. Phosphorus oxychloride (0.53 mL, 5.87 mmol, 3.2 equivalent) was slowly added dropwise using a constant-pressure dropping funnel while stirring at 0 °C. After the addition was complete, the reaction mixture was stirred at 0 °C for 2 hours. Subsequently, the ice bath was removed, the reaction mixture was heated to 40 °C, and stirred at this temperature for 4 hours. After the reaction was complete, the reaction solution was cooled to room temperature. A saturated sodium acetate aqueous solution (50 mL) was slowly added to the reaction solution to neutralize it, and the mixture was stirred at room temperature for 2 hours. The mixture was then transferred to a separatory funnel and extracted with chloroform (3 × 30 mL). The combined organic phases were washed once with water (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography. First, the less polar starting material was removed by washing with a mixed solvent of petroleum ether / tetrahydrofuran = 100:1 (v / v), and then the target product was eluted using a gradient solvent system of petroleum ether / ethyl acetate = 20:1 to 10:1 (v / v). The fraction containing the target product was collected, concentrated under reduced pressure, and the intermediate N-CHO (formylated product) was obtained as a yellow oil (641 mg, yield 92.66%). This product showed yellow fluorescence under UV light.
[0049] b. Under nitrogen protection, the formylated product obtained in the previous step (0.30 g, 0.69 mmol, 1.0 equivalent) was dissolved in ultra-dry tetrahydrofuran (8 mL). The reaction solution was cooled to 0°C in an ice-water bath. N-bromosuccinimide (NBS) (0.14 g, 0.76 mmol, 1.1 equivalent) was slowly added to this solution. After the addition was complete, the ice bath was removed, and the reaction solution was allowed to rise naturally to 25°C and stirred at this temperature for 1 hour. After the reaction was completed, water (10 mL) was added to quench the reaction solution. The mixture was transferred to a separatory funnel and extracted with chloroform (3 × 20 mL). The combined organic phases were washed once with water (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure in a 30°C water bath to obtain a crude oil product. The crude product was purified by reduced-pressure silica gel column chromatography using chloroform as the eluent. The target fraction was collected and concentrated under reduced pressure to obtain the intermediate Br-N-CHO (bromination product), which was a yellow oil (0.35 g, yield 96.9%).
[0050] c. Under nitrogen protection, in a dry, double-necked, round-bottom reaction flask, add the intermediate Br-N-CHO (500 mg, 1.0 equivalent) and the donor borate unit sequentially. The following compounds were added: 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl (S-Phos) (26.93 mg, 5 mol%), palladium(II) acetate (Pd(OAc)2) (15.6 mg, 5 mol%), and tripotassium phosphate (K3PO4) (1387.49 mg, 5.0 equivalents). After purging the system three times with nitrogen, a degassed mixed solvent of 1,4-dioxane / water = 5:1 (v / v) (total volume 24 mL) was added. The reaction mixture was heated to 40 °C and stirred at this temperature for 14 hours. After the reaction was complete, the reaction solution was cooled to room temperature and concentrated under reduced pressure to remove most of the solvent. The residue was dissolved in dichloromethane, and the resulting solution was washed with saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography. First, elution was performed using a mixed solvent of dichloromethane / n-hexane = 10:1 (v / v) to remove the less polar borate ester feedstock and its tailing impurities. Then, the target product was eluted using a gradient solvent system of ethyl acetate / n-hexane = 10:1 to 5:1 (v / v). The fractions containing the target product were collected, combined, and concentrated under reduced pressure to obtain a preliminarily purified oil. Recrystallization of this oil yielded the intermediate TNCHO (Suzuki coupling product), an orange-yellow oil (yield approximately 90%).
[0051] d. The intermediate TNCHO (100 mg, 1.0 equivalent) obtained in the previous step and the acceptor unit 2-[3-cyano-4-methyl-5-phenyl-5-trifluoromethyl-2(5H)-furanide]-malonadionitrile (55 mg) were dissolved in a mixed solvent (13 mL) of ethanol / tetrahydrofuran (1:1, v / v). The reaction mixture was heated to 50 °C and stirred at this temperature for 3 hours, monitored by thin-layer chromatography (TLC) until the starting material was substantially eliminated. After the reaction was complete, the reaction solution was cooled to room temperature and concentrated under reduced pressure to completely remove the solvent. The residue was recrystallized by adding a mixed solvent of dichloromethane / methanol. The precipitated solid was collected by filtration, washed with a small amount of cold solvent, and dried under vacuum to give the final product TNCF3-2C6 (yield approximately 85%).
[0052] The preparation process of the H-aggregated pyrrole derivative in this embodiment is as follows: Figure 1 As shown.
[0053] The H-aggregate pyrrole derivative prepared in this embodiment was dissolved in tetrahydrofuran and prepared to a concentration of 10. The absorbance of a solution with concentration M was measured at room temperature using a Shimadzu UV-2600 ultraviolet spectrophotometer (Japan). Data were collected from 400 to 1000 nm. The fluorescence emission was measured at room temperature using a Shimadzu Horiba Fluoromax-4 fluorescence spectrometer (Japan), with data collected from 800 to 1300 nm. The obtained absorption and emission spectra are shown below. Figure 2 As shown, the TNCF3-2C6 molecule exhibits a maximum wavelength absorption of 766 nm, with its absorption tail extending to 950 nm, demonstrating excellent near-infrared absorption capabilities. The emission of the TNCF3-2C6 molecule is located at 992 nm.
[0054] 1 mg of the H-aggregated pyrrole derivative prepared in this example and 2 mg of DSPE-PEG2000 were dissolved in 1 mL of tetrahydrofuran. After ultrasonic treatment for 30 minutes to ensure complete dissolution, the THF solution was added to 9 mL of ultrapure water under probe ultrasonic conditions, and ultrasonic treatment was continued for 2 minutes. The mixture was then placed in a fume hood and stirred overnight in the dark to evaporate the tetrahydrofuran. The solution was filtered through a 0.22 µm polyvinylidene fluoride (PVDF) needle filter to obtain a transparent nanoparticle solution. The particle size and distribution of the nanoparticles were determined using a Zetasizer Nano-S90 dynamic light scattering spectrometer from Malvern Instruments, UK. Absorption was measured using a UV-2600 ultraviolet spectrophotometer from Shimadzu, Japan, with data collected from 400 to 1000 nm. The particle size distribution and absorption spectrum of the nanoparticles are shown below. Figure 3 As shown, the nanoparticles exhibit a narrow particle size distribution with an average particle size of 30.00 nm. After being encapsulated into nanoparticles, the TNCF3-2C6 molecule absorbs at 744 nm, which is a significant blue shift of 22 nm compared to the wavelength of the monomer molecule. At the same time, the absorption wavelength waveform of TNCF3-2C6 NPs also shifts to the left, indicating the formation of H-aggregates.
[0055] The ROS generation capacity of the nanoparticles prepared in this embodiment under 808 nm laser irradiation was detected using the 2′,7′-dichlorodihydrofluorescein (DCFH) probe method to determine the total ROS generation. First, DCFH-DA was dissolved in anhydrous ethanol to prepare a 10 mM stock solution, which was then hydrolyzed under alkaline conditions (0.01 M NaOH) to generate DCFH, and diluted with PBS to the working concentration (50 mM). M). During the test, the photosensitizer (final concentration 10) was placed in a quartz cuvette. M) and DCFH (final concentration 50) M) was mixed with PBS, and the total volume of the system was 2 mL. An 808 nm laser (1.0 W / cm²) was used. 2Irradiation was performed for a total duration of 300 seconds, with fluorescence intensity measured every 60 seconds on a Horiba Fluoromax-4 fluorescence spectrometer (excitation wavelength 488 nm, emission wavelength 525 nm, slit width 1 nm). Testing with the commercial photosensitizer ICG was conducted under identical conditions, and the results are as follows: Figure 4 As shown, under 808nm laser irradiation, TNCF3-2C6 nanoparticles exhibit a total ROS generation capacity far exceeding that of the commercial photosensitizer ICG.
[0056] Superoxide cation generation test: Detection of superoxide anion (O2) using the dihydrorhodamine 123 (DHR123) probe method The formation of -) was as follows: First, DHR123 was dissolved in DMF to prepare a 10 mM stock solution, which was then diluted with ultrapure water to a 50 μM working solution. In a quartz cuvette, 20 μL of the photosensitizer stock solution (10 μM) and the DHR123 working solution were added sequentially, and then brought to a total volume of 2 mL with PBS, so that the final concentrations of the photosensitizer and DHR123 were 10 μM and 20 μM, respectively. An 808 nm laser (1.0 W / cm²) was used. 2 Irradiation was performed for 300 seconds, during which the fluorescence intensity was measured every 30 seconds using a Horiba Fluoromax-4 fluorescence spectrometer (excitation wavelength 488 nm, emission wavelength 525 nm, slit width 1 nm).
[0057] Hydroxyl radical generation test: Detection of hydroxyl radicals (OH) using the hydroxyphenyl fluorescein (HPF) probe method The formation of HPF involves first dissolving HPF in DMF to prepare a 5 mM stock solution, then diluting it with ultrapure water to 50%. M working solution. In a quartz cuvette, add the photosensitizer stock solution (10...) sequentially. M) and HPF working solution total 20 L, and add PBS to bring the total volume to 2 mL, so that the final concentrations of photosensitizer and HPF are 10 L and 10 mL respectively. M and 10 M. 808 nm laser (1.0 W / cm) 2 Irradiation was performed for 300 seconds, during which the fluorescence intensity was measured every 30 seconds using a Horiba Fluoromax-4 fluorescence spectrometer (excitation wavelength 480 nm, emission wavelength 515 nm, slit width 1 nm).
[0058] Singlet oxygen production test: 9,10-Anthracenediylbis(methylene)-dimalonic acid (ABDA) was used as an indicator, and its ability to generate singlet oxygen (1O2) was detected by the attenuation of its ultraviolet absorption. First, ABDA was dissolved in DMF to prepare a 10 mM stock solution, which was then diluted with ultrapure water to a 50 μM working solution. During detection, 20 μL of the 10 μM photosensitizer stock solution was mixed with 10 μL of the ABDA working solution in a quartz cuvette to achieve a final photosensitizer concentration of 10 μM. An 808 nm laser (1.0 W / cm²) was used. 2 Irradiation was performed for 5 minutes, and the absorption spectrum was recorded every minute using a Shimadzu UV-2600 UV spectrophotometer (Japan). Simultaneously, the intensity change of the characteristic absorption peak of ABDA at 380 nm was monitored, and background interference from the photosensitizer's own absorption was subtracted.
[0059] The fluorescence intensity of the solution of nanoparticles, DHR123, and HPF increased by a factor of [number missing] under 808 nm laser irradiation. Figure 5 As shown; the fluorescence intensity of the solution containing nanoparticles and ABDA increased by a factor of [number] under 808 nm laser irradiation. Figure 6 As shown; from Figure 5 and Figure 6 It can be seen that the ROS generated by nanoparticles are mainly superoxide anions and hydroxyl radicals.
[0060] To test the photothermal conversion capability of the nanoparticles in this embodiment, a 10 μM aqueous solution of TNCF3-2C6 nanoparticles was placed in a 96-well plate. The laser spot diameter was adjusted to 10 mm to ensure it covered the area of the nanoparticle solution. Subsequently, the laser power density was adjusted to 1.0 W / cm². 2 The thermocouple probe was placed inside the container. Temperature changes before and after laser irradiation were recorded, and the results are as follows: Figure 7 As shown, the photothermal heating curve of ICG is consistent with that of TNCF3-2C6.
[0061] The tumor cell killing ability of the nanoparticles in this embodiment was tested to verify their cytotoxicity and photodynamic therapy effects. Cytotoxicity experiments were conducted using 4T1 cells as a model. Cells were cultured at 2.5 × 10⁶ cells per well. 3 In the phototoxicity group, cells were seeded at a density of [number] cells / well in 96-well plates and incubated for 24 hours. The medium was replaced with different concentrations of TNCF3-2C6, and after a total incubation of 12 hours, the medium was discarded, and the cells were washed three times with PBS. Subsequently, an 808 nm laser (0.2 W / cm²) was used. 2Irradiate for 10 minutes, then continue culturing for 12 hours. Dark toxicity group: Replace with medium containing different concentrations of TNCF3-2C6, and co-incubate for 24 hours. Hypoxic environment group: Treatment steps are the same as the phototoxicity and dark toxicity groups, except that cells are cultured and incubated under hypoxic conditions. Detection method: After treatment, cells in each group are washed three times with PBS, and medium containing 0.5 mg / mL MTT is added to each well, and incubated in the dark for 3 hours. After carefully aspirating the medium, 100 mg / mL MTT is added to each well. L DMSO was shaken for 5 minutes using a multi-functional microplate reader, and the absorbance at 570 nm was measured. The results are as follows: Figure 8 As shown, the near-infrared photosensitizer TNCF3-2C6 achieved good killing of 4T1 cancer cells under both normoxic and hypoxic conditions, but the killing effect under hypoxia showed a slightly weaker trend than that under normoxic conditions.
[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. An H-aggregated pyrrole derivative, characterized in that, The structure of the H-aggregate pyrrole derivative is shown below: or ; Wherein, R1 is independently a C1-C30 alkyl, a C1-C30 phenyl, a phenyl substituted with a C1-C30 alkyl or a phenyl substituted with a C1-C30 alkoxy; R2 is a C1-C30 alkyl group.
2. The method for preparing the H-aggregated pyrrole derivative according to claim 1, characterized in that, Includes the following steps: (1) 4-R2-4H-dithiopheno[3,2-b:2,3-d]pyrrole, N,N-dimethylformamide and 1,2-dichloroethane were mixed and then phosphorus oxychloride was added for secondary mixing; then formylation reaction was carried out to obtain formylated product; (2) The formylated product, tetrahydrofuran and N-bromosuccinimide were mixed and then subjected to bromination to obtain the bromination product; (3) The brominated product, donor borate ester, 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl, palladium acetate, tripotassium phosphate and mixed solvent are mixed and reacted to obtain the coupling product; (4) The coupling product, acceptor unit and mixed solvent are mixed and subjected to condensation reaction to obtain the H-aggregated pyrrole derivative.
3. The method for preparing the H-aggregated pyrrole derivative as described in claim 2, characterized in that, In step (1), R2 in 4-R2-4H-dithiopheno[3,2-b:2,3-d]pyrrole is a C1-C30 alkyl group; In step (1), the ratio of 4-R2-4H-dithiopheno[3,2-b:2,3-d]pyrrole, N,N-dimethylformamide, 1,2-dichloroethane and phosphorus oxychloride is 1.5~2 mmol: 15~20 mmol: 10~20 mL: 5.5~6 mmol.
4. The method for preparing the H-aggregated pyrrole derivative as described in claim 3, characterized in that, The mixing temperature in step (1) is -5~5℃; The secondary mixing temperature is -5~5℃, and the time is ≥2h; In step (1), the formylation reaction is carried out at a temperature of 30~50℃ for ≥4h.
5. The method for preparing the H-aggregated pyrrole derivative as described in claim 4, characterized in that, In step (2), the ratio of the formylated product, tetrahydrofuran and N-bromosuccinimide is 0.65~0.75mmol: 5~10mL: 0.7~0.8mmol.
6. The method for preparing the H-aggregated pyrrole derivative as described in claim 5, characterized in that, In step (2), the bromination reaction is carried out at a temperature of 20~30℃ for a time of ≥1h.
7. The method for preparing the H-aggregated pyrrole derivative as described in claim 6, characterized in that, The structural formula of the donor unit borate ester in step (3) is as follows: R1 is independently a C1-C30 alkyl, C1-C30 phenyl, phenyl substituted with a C1-C30 alkyl or phenyl substituted with a C1-C30 alkoxy; In step (3), the mixed solvent contains 1,4-dioxane and water, and the volume ratio of 1,4-dioxane to water is 4~6:1; In step (3), the ratio of brominated product, donor borate ester, 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl, palladium acetate, tripotassium phosphate, and mixed solvent is 400~600mg: 650~750mg: 20~30mg: 10~20mg: 1350~1450mg: 20~30mL; The reaction temperature in step (3) is 30~50℃ and the time is 12~16h.
8. The method for preparing the H-aggregated pyrrole derivative as described in claim 7, characterized in that, In step (4), the acceptor unit is 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malonitrile or 2-[3-cyano-4-methyl-5-phenyl-5-trifluoromethyl-2(5H)-furanyl]malonitrile; The mixed solvent in step (4) contains ethanol and tetrahydrofuran; the volume ratio of ethanol to tetrahydrofuran is 1~2:1~2; In step (4), the ratio of the coupling product, acceptor unit, and mixed solvent is 50-100 mg: 50-100 mg: 10-15 mL; In step (4), the condensation reaction is carried out at a temperature of 40-60℃ for 2-4 hours.
9. The use of the H-aggregated pyrrole derivative of claim 1 in the preparation of phototherapy drugs and drugs for treating hypoxic tumors.