Photosensitizer with high reactive oxygen generation capacity as well as preparation method and application of photosensitizer

Abstract

The invention discloses a photosensitizer with high active oxygen generation capacity, the structure of the photosensitizer is shown as a formula (I), D is an electron donor, namely an R-substituted triphenylamine structure, A1 and A2 are electron acceptors and are respectively an X-substituted benzodiazole ring and an onium bromine compound with trimethylamine at the tail end, the two electron acceptors are connected through a double bond, and the structural formula (I) is shown in the description. Compared with a single-acceptor photosensitizer, the double-acceptor photosensitizer connected through double bonds has higher donor-acceptor strength, so that the reduction of delta EST is facilitated, and further, the efficient ROS generation capability is realized. # imgabs0 # is represented by formula (I).

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical materials, and in particular to a photosensitizer with high active oxygen generation capability, a preparation method and application thereof. Background Art

[0002] Photodynamic antimicrobial therapy (PDA) is an emerging antimicrobial therapy that utilizes light and photosensitizers to generate reactive oxygen species (ROS). Due to its noninvasive nature, negligible resistance, low toxicity, and minimal side effects, PDA shows great potential for treating drug-resistant bacterial infections. To achieve effective PDA, the design of photosensitizers is crucial. Ideal photosensitizers should possess efficient ROS generation for bacterial killing, negligible dark toxicity, and good photostability. However, most conventional photosensitizers have relatively low ROS generation capacity, which severely hinders their antimicrobial applications. The structure-activity relationship between the molecular structure of photosensitizers and their ROS generation capacity requires further investigation.

[0003] The ability of photosensitizers to generate ROS depends largely on the energy gap (ΔE) between the triplet state (T1) and the singlet state (S1) of the molecule. ST ), a smaller ΔE can be achieved by separating the donor-acceptor effect ST , which is conducive to the generation of ROS. For example, the research group of Liu Bin from the National University of Singapore introduced a benzene ring between the donor and the acceptor to effectively separate the donor and the acceptor, thereby reducing ΔE ST, enhanced ROS generation (Wu Wenbo, et al. A Highly Efficient and PhotostablePhotosensitizer with Near-Infrared Aggregation-Induced Emission for Image-Guided Photodynamic Anticancer Therapy. Adv. Mater. 2017, 29, 1700548. doi:10.1002 / adma.201700548.). Tang Benzhong's team prepared a series of donor-acceptor interactive photosensitizers and evaluated their structure-function relationships (Kang Miaomiao, et al. Evaluation of Structure-Function Relationships of Aggregation-Induced Emission Luminogens forSimultaneous Dual Applications of Specific Discrimination and EfficientPhotodynamic Killing of Gram-Positive Bacteria. J. Am. Chem. Soc. 2019, 141,16781-16789. doi: 10.1021 / jacs.9b07162.). Among them, the photosensitizer TTPy can effectively generate ROS under white light irradiation due to its small ΔEST. TTPy can effectively kill Gram-positive bacteria and is also effective in a rat wound model infected with Staphylococcus aureus. In addition, Tang Benzhong's team also changed the donor-acceptor effect by cationization. This cationization can greatly enhance the electron-withdrawing ability of the pyridine part, thereby reducing ΔE ST To promote ROS production (Liu Shanshan, et al. Cationization-Enhanced Type I and Type II ROS Generation for Photodynamic Treatment of Drug-Resistant Bacteria. ACS Nano 2022, 16, 9130-9141. doi: 10.1021 / acsnano.2c01206.). Further light exposure led to a significant improvement in antibacterial properties, surpassing that of vancomycin antibiotics.

[0004] In summary, while donor-acceptor photosensitizers have made significant progress, they are still in their exploratory stages. The ROS generation capacity of photosensitizers prepared solely through the traditional isolated donor-acceptor effect remains unsatisfactory. Therefore, it is necessary to design and synthesize photosensitizer materials with higher ROS generation capacity to achieve effective antibacterial applications. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a photosensitizer with high active oxygen generation ability, which is obtained by introducing two electron acceptors (A1 and A2), and connecting A1 and A2 through a double bond to obtain a compound represented by formula (I), so as to reduce ΔE ST , thereby achieving efficient ROS generation capabilities.

[0006] A photosensitizer with high active oxygen generation ability, whose structure is shown in formula (I):

[0007] Formula (I) Where D is , R is selected from one of H, CH3, and OCH3; A1 is , X is S or Se; A2 is or , " ” is the connection location.

[0008] In the present invention, the photosensitizer is a D-A1-A2 type compound, wherein D is an electron donor, i.e., a triphenylamine structure substituted by R, and A1 and A2 are both electron acceptors, i.e., an onium bromide compound substituted by X and a terminal trimethylamine, respectively. The two electron acceptors are connected by a double bond. The donor-acceptor regulates ΔE by regulating intramolecular electron transfer. ST , thereby affecting the generation of ROS. Compared with single-acceptor photosensitizers, dual-acceptor photosensitizers connected by double bonds have a larger donor-acceptor strength that helps ΔE ST Therefore, there is a potential for more efficient ROS generation.

[0009] Preferably, the photosensitizer with high reactive oxygen species generating ability is selected from any of the following structures: 、 、 、 、 、 、 、 、 、 、 、 .

[0010] Further preferably, the photosensitizer with high active oxygen generation ability is selected from any of the following structures: 、 、 .

[0011] The present invention also provides a method for preparing the above-mentioned photosensitizer with high active oxygen generating ability, comprising the following steps: (1) Under the action of palladium catalyst and inorganic base, DB(OH)2 and Br-A1-CHO are placed in an inert gas to undergo Suzuki-Miyaura reaction to obtain compound D-A1-CHO; (2) The compounds D-A1-CHO and A2-CH3 obtained in step (1) are dissolved in a solvent and then condensed under alkaline conditions to obtain the compound represented by formula (I).

[0012] The specific routes are as follows: .

[0013] Preferably, in step (1), the palladium catalyst is at least one of Pd(PPh3)4, PdCl2, Pd(OAc)2, and Pd(dppf)Cl2.

[0014] Preferably, in step (1), the inorganic base is at least one of potassium carbonate, potassium bicarbonate, sodium carbonate and sodium bicarbonate.

[0015] Preferably, in step (1), the molar ratio of DB(OH)2, Br-A1-CHO, palladium catalyst and inorganic base is 1:1-2:0.01-0.2:5-20.

[0016] Preferably, in step (1), the temperature of the Suzuki reaction is 60-120°C, and the reaction time is 2-24 h.

[0017] Preferably, in step (2), the alkaline condition is at least one of piperidine, pyrrole, piperazine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate.

[0018] Preferably, in step (2), the solvent is at least one of ethanol, methanol, acetonitrile and tetrahydrofuran.

[0019] Preferably, in step (2), the condensation reaction temperature is 40-120°C, and the reaction time is 3-12 h.

[0020] The present invention also provides the use of the photosensitizer in the preparation of photodynamic antibacterial drugs.

[0021] Preferably, the photosensitizer has anti-Staphylococcus aureus and anti-Escherichia coli activities both before and after illumination.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention introduces two electron acceptors (A1 and A2), which are connected by a double bond to obtain a photosensitizer shown in formula (I) to reduce ΔE ST , thereby achieving efficient ROS generation. In addition, photosensitizers can also be anchored to the bacterial membrane through membrane anchoring groups (terminal quaternary ammonium salt structures) to overcome the shortcomings of the short lifespan and limited action radius of the reactive oxygen species produced by photosensitizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The absorbance changes at wavelengths of 300-800 nm were measured for Compound C1 prepared in Example 1, the single-receptor photosensitizer prepared in Comparative Example 1, and Rose Bengal (RB) when irradiated with white light for 0, 0.5, 1, 1.5, 2, and 2.5 min, respectively. (a) to (c) are the absorbance change curves of the single-receptor photosensitizer prepared in Comparative Example 1, Compound C1 prepared in Example 1, and Rose Bengal (RB), respectively, and (d) is the rate of decrease of absorbance at 378 nm.

[0024] Figure 2 The absorbance changes at wavelengths of 300-800 nm were measured for Compound C1 prepared in Example 1, the single-receptor photosensitizer prepared in Comparative Example 1, and Rose Bengal (RB) when irradiated at a wavelength of 520 nm for 0, 0.5, 1, 1.5, 2, and 2.5 min, respectively. (a) to (c) are the absorbance change curves of the single-receptor photosensitizer prepared in Comparative Example 1, Compound C1 prepared in Example 1, and Rose Bengal (RB), respectively, and (d) is the rate of decrease of absorbance at 378 nm.

[0025] Figure 3 This is a fluorescent image of Escherichia coli co-stained with DAPI (4′,6-diamidino-2-phenylindole) and compound C1 prepared in Example 1.

[0026] Figure 4 These are survival rate test graphs of compound C1 prepared in Example 1 and commercial photosensitizer RB in Escherichia coli and Staphylococcus aureus, wherein (a) and (c) are test graphs of compound C1 prepared in Example 1 and commercial photosensitizer RB in Escherichia coli and Staphylococcus aureus, respectively, and (b) and (d) are quantitative statistical graphs of (a) and (c), respectively. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited to the following examples.

[0028] The raw materials used in the present invention are all commercially available.

[0029] Example 1: Preparation of Compound C1

[0030] (1) Under nitrogen protection, the compound 4-boronic acid triphenylamine (500 mg, 2.057 mmol), 7-bromo-4-formaldehyde benzo[C][1,2,5]thiadiazole (714 mg, 2.468 mmol), tetrakis(triphenylphosphine)palladium (119 mg, 0.103 mmol) and potassium carbonate (1420 mg, 10.3 mmol) were dissolved in a mixed solution of 1,4-dioxane (15 mL) and deionized water (3 mL). The mixture was then stirred at 85 °C for 6 hours under nitrogen protection. The reaction system was cooled to room temperature, and the reaction solution was extracted with dichloromethane and water. The organic phase was collected, washed with water, dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation on a rotary evaporator to obtain a crude product. The crude product was separated and purified by silica gel column chromatography to obtain a solid product I-A1-CHO (748 mg, yield 89.2%).

[0031] (2) Compound I-A1-CHO (200 mg, 0.4908 mmol) and 4-methyl-1-(3-(trimethylammonium)propyl)pyridin-1-ium bromide (240 mg, 0.6777 mmol) were dissolved in 20 mL of ethanol, and 4 mg of piperidine was added as a catalyst. The mixture was reacted at 80 °C for 8 h. The reaction system was cooled to room temperature, dried by rotary evaporation, and separated by column chromatography to obtain a solid product C1 (170 mg, 46.6% yield). 1 H NMR (600 MHz, DMSO- d 6 ) δ 9.20 (d, J = 6.3 Hz, 2H), 8.48 (d, J = 6.5Hz, 2H), 8.35 (d, J = 16.2 Hz, 1H), 8.16 (d, J = 7.4 Hz, 1H), 8.10 – 7.92 (m,4H), 7.37 (t, J = 7.3 Hz, 4H), 7.16 – 7.08 (m, 8H), 4.71 (t, J = 7.2 Hz, 2H), 3.56 – 3.50 (m, 2H), 3.14 (s, 9H), 2.52 (d, J = 12.0 Hz, 2H). Example 2: Synthesis of Compound C2

[0032] (1) Under nitrogen protection, the compound (4-(di-p-toluamino)phenyl)boronic acid (650 mg, 2.057 mmol), 7-bromo-4-formaldehydebenzo[C][1,2,5]thiadiazole (714 mg, 2.468 mmol), tetrakis(triphenylphosphine)palladium (119 mg, 0.103 mmol) and potassium carbonate (1420 mg, 10.3 mmol) were dissolved in a mixed solution of 1,4-dioxane (15 mL) and deionized water (3 mL). The mixture was then stirred at 85 °C for 6 hours under nitrogen protection. The reaction system was cooled to room temperature, and the reaction solution was extracted with dichloromethane and water. The organic phase was collected, washed with water, dried over anhydrous sodium sulfate, and the organic solvent was removed by reduced pressure distillation on a rotary evaporator to obtain a crude product. The crude product was separated and purified by silica gel column chromatography to obtain a solid product II-A1-CHO (732 mg, yield 81.7%).

[0033] (2) Compound II-A1-CHO (214 mg, 0.4908 mmol) and 4-methyl-1-(3-(trimethylammonium)propyl)pyridin-1-ium bromide (240 mg, 0.6777 mmol) were dissolved in 20 mL of ethanol, and 4 mg of piperidine was added as a catalyst. The mixture was reacted at 80 °C for 8 h. The reaction system was cooled to room temperature, dried by rotary evaporation, and separated by column chromatography to obtain a solid product C2 (156 mg, 41.3% yield). 1 H NMR (600 MHz, DMSO- d 6 ) δ 9.22 (d, J = 6.7 Hz, 2H), 8.49 (d, J = 6.4Hz, 2H), 8.38 (d, J = 16.3 Hz, 1H), 8.17 (d, J = 7.6 Hz, 1H), 8.13 – 7.95 (m,4H), 7.39 (t, 2.33 (t, J) =0.9 Hz, 6H). Example 3: Synthesis of Compound C3

[0034] (1) Under nitrogen protection, the compound 4-bis(4-methoxyphenyl)aminophenylboronic acid (720 mg, 2.057 mmol), 7-bromo-4-formaldehydebenzo[C][1,2,5]thiadiazole (714 mg, 2.468 mmol), tetrakis(triphenylphosphine)palladium (119 mg, 0.103 mmol) and potassium carbonate (1420 mg, 10.3 mmol) were dissolved in a mixed solution of 1,4-dioxane (15 mL) and deionized water (3 mL). The mixture was then stirred at 85 °C for 6 hours under nitrogen protection. The reaction system was cooled to room temperature, and the reaction solution was extracted with dichloromethane and water. The organic phase was collected, washed with water, dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation on a rotary evaporator to obtain a crude product. The crude product was separated and purified by silica gel column chromatography to obtain a solid product III-A1-CHO (735 mg, yield 76.4%).

[0035] (2) Compound III-A1-CHO (230 mg, 0.4908 mmol) and 4-methyl-1-(3-(trimethylammonium)propyl)pyridin-1-ium bromide (240 mg, 0.6777 mmol) were dissolved in 20 mL of ethanol, and 4 mg of piperidine was added as a catalyst. The mixture was reacted at 80 °C for 8 h. The reaction system was cooled to room temperature, dried by rotary evaporation, and separated by column chromatography to obtain a solid product C3 (154 mg, 39.1% yield). 1 H NMR (600 MHz, DMSO- d 6 ) δ 9.21 (d, J = 6.8 Hz, 2H), 8.47 (d, J =6.5 Hz, 2H), 8.36 (d, J = 16.1 Hz, 1H), 8.16 (d, J = 7.8 Hz, 1H), 8.12 – 7.93(m, 4H), 7.38 (t, J = 7.3 Hz, 4H), 7.16 – 7.08 (m, 6H), 4.71 (t, J = 7.4 Hz, 2H), 3.82 (s, 6H), 3.56 – 3.52 (m, 2H), 3.14 (s, 9H), 2.54 (d, J = 12.0 Hz,2H). Comparative Example 1: Single receptor photosensitizer (relative to compound C1)

[0036] 4-Diphenylaminobenzaldehyde (100 mg, 0.3658 mmol) and 4-methyl-1-(3-(trimethylammonium)propyl)pyridin-1-ium bromide (108 mg, 0.3049 mmol) were dissolved in 4 mL of ethanol. One drop of piperidine was added as a catalyst and the reaction was carried out at 100°C for 5 hours. The reaction system was cooled to room temperature, dried by spin drying, and separated by column chromatography to obtain a solid product (103 mg, 46.2% yield). 1 H NMR (400 MHz, DMSO- d 6 ) δ 9.19 (d, J = 6.5 Hz, 2H), 8.29 (d, J = 6.5Hz, 2H), 8.10 (d, J = 16.2 Hz, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.48 – 7.34 (m,5H), 7.17 (t, J = 7.4 Hz, 2H), 7.14 – 7.08 (m, 4H), 6.93 (d, J = 8.4 Hz, 2H), 4.71 (t, J = 7.4 Hz, 2H), 3.65 – 3.55 (m, 2H), 3.20 (s, 9H), 2.53 (d, J =16.4 Hz, 2H). Sample analysis Active oxygen production capacity test (1) Test of active oxygen production capacity under composite light: Compound C1 prepared in Example 1, the single receptor photosensitizer prepared in Comparative Example 1, and Rose Bengal (RB) were treated with 9,10-anthracenediyl-bis(methylene)dicarboxylic acid (ABDA) at white light irradiation times of 0, 0.5, 1, 1.5, 2, and 2.5 min, respectively. The changes in absorbance at wavelengths of 300-800 nm were measured, and the rates of decrease of absorbance at 378 nm were compared.

[0037] Figure 1The absorbance changes at wavelengths of 300-800 nm are measured for Compound C1 prepared in Example 1, the single receptor photosensitizer prepared in Comparative Example 1, and Rose Bengal (RB) when irradiated with white light for 0, 0.5, 1, 1.5, 2, and 2.5 min, respectively. (a) to (c) are the absorbance change curves of the single receptor photosensitizer prepared in Comparative Example 1, Compound C1 prepared in Example 1, and Rose Bengal (RB), respectively, and (d) is the rate of decrease of absorbance at 378 nm. As shown in the figure, under white light irradiation, the absorbance of the single receptor photosensitizer prepared in Comparative Example 1 remains almost unchanged with the extension of irradiation time, while the absorbance of Compound C1 prepared in Example 1 and Rose Bengal (RB) gradually decreases with the extension of irradiation time ( Figure 1 (a) to (c) in the figure). The rate of decrease of absorbance at 378 nm is significantly greater than that of the comparative example and RB, indicating that C1 has a greater ability to produce active oxygen than the comparative example and RB under white light irradiation ( Figure 1 (d) in the figure).

[0038] (2) Active oxygen species under single wavelength light Compound C1 prepared in Example 1, the single receptor photosensitizer prepared in Comparative Example 1, and Rose Bengal (RB) were irradiated at a wavelength of 520 nm for 0, 0.5, 1, 1.5, 2, and 2.5 min, respectively, and reacted with 9,10-anthracenediyl-bis(methylene)dicarboxylic acid (ABDA). The changes in absorbance at wavelengths of 300-800 nm were measured, and the rates of decrease of absorbance at 378 nm were compared. Figure 2 The absorbance changes at wavelengths of 300-800 nm were measured for Compound C1 prepared in Example 1, the single receptor photosensitizer prepared in Comparative Example 1, and Rose Bengal (RB) when irradiated at a wavelength of 520 nm for 0, 0.5, 1, 1.5, 2, and 2.5 min, respectively. (a) to (c) are the absorbance change curves of the single receptor photosensitizer prepared in Comparative Example 1, Compound C1 prepared in Example 1, and Rose Bengal (RB), respectively, and (d) is the rate of decrease of absorbance at 378 nm. As shown in the figure, under single wavelength light, the absorbance of the single receptor photosensitizer prepared in Comparative Example 1 remains almost unchanged with the extension of irradiation time, while the absorbance of Compound C1 prepared in Example 1 and Rose Bengal (RB) gradually decreases with the extension of irradiation time ( Figure 2 (a) to (c) in the figure). The rate of decrease of absorbance at 378 nm is significantly greater than that of the comparative example and RB, indicating that C1 has a greater ability to produce active oxygen than the comparative example and RB under white light irradiation ( Figure 2 (d) in the figure).

[0039] 2. Bacterial localization of compound C1 Fluorescence images of Escherichia coli co-stained with DAPI (4′,6-diamidino-2-phenylindole) and C1 were observed using a confocal laser scanning microscope (CLSM).

[0040] Figure 3 This is a fluorescent image of Escherichia coli stained with DAPI (4′,6-diamidino-2-phenylindole) and compound C1 prepared in Example 1. The interior of the E. coli is clearly stained blue by DAPI. The bacterial membrane is stained green, with almost no green inside the cell, indicating that C1 is only anchored to the bacterial membrane and does not penetrate the interior of the bacteria. The merged image further confirms the efficient anchoring of C1 to the bacterial membrane. This may contribute to its high photodynamic antibacterial efficacy, as it overcomes the limitations of the short lifetime and limited radius of action of the reactive oxygen species generated by photosensitizers.

[0041] 3. Photodynamic antibacterial research The photodynamic antibacterial activity of compound C1 prepared in Example 1 and the commercial photosensitizer RB was studied using the traditional colony forming unit (CFU) counting method in the presence or absence of light.

[0042] Figure 4 The following are test graphs of the survival rates of compound C1 prepared in Example 1 and commercial photosensitizer RB in Escherichia coli and Staphylococcus aureus, wherein (a) and (c) are test graphs of compound C1 prepared in Example 1 and commercial photosensitizer RB in Escherichia coli and Staphylococcus aureus, respectively, and (b) and (d) are quantitative statistical graphs of (a) and (c) respectively. Figure 4 As shown, after 3 minutes of exposure to white light, the activity of Escherichia coli and Staphylococcus aureus treated with different photosensitizers rapidly decreased. C1 exhibited an antibacterial rate of 100% against Staphylococcus aureus and 96.2% against E. coli, respectively. Compared to commercial RB, C1 exhibited superior antibacterial activity against E. coli. Both RB and C1 exhibited an antibacterial rate of 100% against Staphylococcus aureus.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A photosensitizer with high active oxygen generation ability, characterized in that The structure of the photosensitizer is shown in formula (I):

2. Formula (I) in, D is , R is selected from one of H, CH3, and OCH3; A1 is , X is S or Se; A2 is or , " ” is the connection location.

3. The photosensitizer with high active oxygen generating ability according to claim 1, characterized in that The photosensitizer is selected from any of the following structures: 、 、 。 4. The method for preparing a photosensitizer with high active oxygen generating ability according to any one of claims 1 or 2, characterized in that: The following steps are involved: (1) Under the action of palladium catalyst and inorganic base, DB(OH)2 and Br-A1-CHO are placed in an inert gas to undergo Suzuki reaction to obtain compound D-A1-CHO; (2) The compounds D-A1-CHO and A2-CH3 obtained in step (1) are dissolved in a solvent and then condensed under alkaline conditions to obtain the compound represented by formula (I).

5. The method for preparing a photosensitizer with high active oxygen generating ability according to claim 3, characterized in that: In step (1), the palladium catalyst is at least one of Pd(PPh3)4, PdCl2, Pd(OAc)2, and Pd(dppf)Cl2, and the inorganic base is at least one of potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate.

6. The method for preparing a photosensitizer with high active oxygen generating ability according to claim 3, characterized in that: In step (1), the molar ratio of DB(OH)2, Br-A1-CHO, palladium catalyst and inorganic base is 1:1~2:0.01~0.2:5~20.

7. The method for preparing a photosensitizer with high active oxygen generating ability according to claim 3, characterized in that: In step (1), the temperature of the Suzuki reaction is 60-120°C, and the reaction time is 2-24 h.

8. The method for preparing a photosensitizer with high active oxygen generating ability according to claim 3, characterized in that: In step (2), the alkaline condition is at least one of piperidine, pyrrole, piperazine, potassium carbonate, potassium bicarbonate, sodium carbonate, and sodium bicarbonate.

9. The method for preparing a photosensitizer with high active oxygen generating ability according to claim 3, characterized in that: In step (2), the condensation reaction temperature is 40-120°C, and the reaction time is 3-12 h.

10. Use of the photosensitizer with high active oxygen generation ability according to any one of claims 1 or 2 in the preparation of photodynamic antibacterial drugs.

11. The use according to claim 9, characterized in that The photosensitizer has anti-Staphylococcus aureus and anti-Escherichia coli activities both before and after illumination.

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