An aggregation-induced luminescence antibacterial photosensitizer and its preparation and application

Abstract

The present invention discloses an aggregation-induced luminescence antibacterial photosensitizer, a preparation method, and an application thereof. The photosensitizer is a compound TBP-QA, and the structural formula of the compound TBP-QA is: #imgabs0# The compound TBP-QA prepared by the present invention provides a new aggregation-induced luminescence material. The compound can be used in fluorescence imaging, photodynamic therapy, preparation of Gram-positive bacterial antibacterial agents, preparation of LPS-deficient Gram-negative bacterial antibacterial agents, preparation of drugs for treating organ lesions caused by MRSAT144, preparation of drugs for treating peritonitis-sepsis, and other fields.

Description

Technical Field

[0001] The present invention relates to a simple preparation method of a photosensitizer with aggregation-induced emission (AIE) properties, to enhancing the antibacterial performance by increasing the donor-acceptor strength of the AIE photosensitizer, and to the antibacterial application of the AIE photosensitizer. Background Art

[0002] The emergence and spread of bacterial resistance weakens the therapeutic effect of antimicrobial drugs and seriously threatens global public health security. For example, the emergence and widespread spread of multidrug-resistant (MDR) Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus (MRSA), has continuously increased the medical and economic burden. MRSA, commonly known as "superbugs", is a common and highly toxic bacterium in clinical practice. Since its discovery, MRSA infections have spread almost worldwide and have become one of the main pathogens of community infections. Because MRSA has multidrug resistance to many antibiotics, the treatment of its infection is one of the most difficult problems in clinical practice.

[0003] Photodynamic therapy (PDT) utilizes a photosensitizer to absorb light, generating a singlet excited state. This singlet excited state further transfers energy to a triplet excited state through intersystem crossing (ISC), sensitizing the surrounding triplet oxygen to form destructive singlet oxygen or other reactive oxygen species (ROS). ROS can rapidly destroy microbial proteins, nucleic acids, and lipids, thereby inactivating bacteria. Furthermore, the photosensitizers used in PDT do not need to enter cells, making it difficult for bacteria to develop resistance to them. Therefore, PDT has become an alternative to antibiotic therapy due to its non-invasiveness, spatiotemporal selectivity, low toxicity, and minimal side effects.

[0004] In recent years, photosensitizers with aggregation-induced emission (AIE) properties have received widespread attention in antibacterial applications. AIE photosensitizers emit very weak light in the dissolved state, but in the aggregated state, the restricted intramolecular motion activates the radiation channel and greatly enhances the fluorescence emission. This property is not only beneficial for fluorescence imaging, but also because of the increased intersystem crossing, the generation of ROS also increases, which can effectively kill bacteria. It has the advantages of strong cell penetration ability, high photostability and good biocompatibility. In recent years, researchers have successively developed antibacterial PDT schemes mediated by AIE photosensitizers, but there is still a problem of low ability to generate ROS, resulting in low antibacterial efficiency against drug-resistant bacteria.

[0005] Therefore, it is imperative to rationally design AIE photosensitizers to enhance their ROS production efficiency. Summary of the Invention

[0006] The purpose of the present invention is to provide an AIE photosensitizer that can generate more ROS and enhance antibacterial activity.

[0007] The present invention provides a compound TBP-QA, the structural formula of the compound TBP-QA is:

[0008]

[0009] The present invention also provides a method for preparing the compound TBP-QA, comprising the following steps:

[0010] 1) Add raw material a to potassium carbonate solution Raw material b and tetrahydrofuran, stirring until it becomes clear, adding tetrakistriphenylphosphine palladium under argon protection and heating. After the reaction is completed, cooling, filtering, and concentrating the filtrate to purify it to obtain intermediate 1;

[0011] 2) Add intermediate 1 and raw material c to potassium carbonate solution and 1,4-dioxane, stirred until clear, added tetrakistriphenylphosphine palladium under argon protection and heated. After the reaction was complete, cooled, filtered, and the filter cake was washed and purified to obtain intermediate 2;

[0012] 3) Intermediate 2 and iodomethane were added to dichloroethane, heated, and reacted. Iodomethane was added three times during the reaction. After the reaction was completed, the mixture was filtered and purified to obtain compound TBP-QA.

[0013] Wherein, in the step 1), the molar ratio of raw material a: raw material b: tetrakistriphenylphosphine palladium is 20:20:1.

[0014] Wherein, in said step 1), the reaction temperature is 60° C. and the reaction time is 31 h.

[0015] Wherein, in the step 2), the molar ratio of intermediate 1: raw material c: tetrakistriphenylphosphine palladium is 20:20:1.

[0016] Wherein, in the step 2), the reaction temperature is 90° C. and the reaction time is 29 h.

[0017] Wherein, in the step 3), the molar ratio of intermediate 1: first addition of iodomethane: additional addition of iodomethane is 2:3:7.6.

[0018] The reaction conditions in step 3) are as follows: heating the system to reflux reaction, monitoring the reaction progress by thin layer chromatography, adding iodomethane three times during the reaction, stopping the reaction after 72 hours, filtering while hot, and cooling the filtrate to precipitate solids, filtering, and drying the filter cake.

[0019] The application of the compound TBP-QA or the preparation method described above in any of the following applications should also fall within the scope of protection of the present invention:

[0020] 1) Application as aggregation-induced emission material;

[0021] 2) Application in fluorescence imaging;

[0022] 3) Application in photodynamic therapy;

[0023] 4) Application in the preparation of Gram-positive antibacterial agents;

[0024] 5) Application in the preparation of LPS-deficient Gram-negative antibacterial agents;

[0025] 6) Use in the preparation of drugs for treating organ lesions caused by MRSA T144;

[0026] 7) Use in the preparation of medicines for treating peritonitis-sepsis.

[0027] The compound TBP-QA prepared by the present invention provides a new aggregation-induced luminescence material. The compound can be used in the fields of fluorescence imaging, photodynamic therapy, preparation of Gram-positive bacteria antibacterial agents, preparation of LPS-deficient Gram-negative bacteria antibacterial agents, preparation of drugs for treating organ lesions caused by MRSA T144, and preparation of drugs for treating peritonitis-sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the synthetic route of TBP-QA.

[0029] Figure 2 : is the nuclear magnetic resonance identification diagram of intermediate 1, wherein A is the nuclear magnetic resonance hydrogen spectrum of intermediate 1, and B is the nuclear magnetic resonance carbon spectrum of intermediate 1.

[0030] Figure 3 : is the nuclear magnetic resonance identification diagram of intermediate 2, wherein A is the nuclear magnetic resonance hydrogen spectrum of intermediate 2, and B is the nuclear magnetic resonance carbon spectrum of intermediate 2.

[0031] Figure 4 The mass spectrum and nuclear magnetic resonance identification diagrams of TBP-QA, wherein A is the mass spectrum of TBP-QA, B is the hydrogen nuclear magnetic resonance spectrum of TBP-QA, and C is the carbon nuclear magnetic resonance spectrum of TBP-QA.

[0032] Figure 5 Frontier molecular orbital diagrams of TBP-1 and TBP-QA.

[0033] Figure 6 is the UV-visible absorption spectrum of TBP-QA in DMSO.

[0034] Figure 7 The luminescence properties of TBP-QA in mixed solvents of tetrahydrofuran and DMSO at different ratios are shown.

[0035] Figure 8 To identify the ability of TBP-QA to produce reactive oxygen species; where A is the ESR spectrum of singlet oxygen; B is the ESR spectrum of hydroxyl radicals; and C is the ESR spectrum of superoxide radicals.

[0036] Figure 9 The antibacterial effect of TBP-QA against Staphylococcus aureus under different light / dark conditions.

[0037] Figure 10 The antibacterial effect of TBP-QA against Bacillus cereus under different light / dark conditions.

[0038] Figure 11 The antibacterial effect of TBP-QA against MRSA T144 under different light / dark conditions.

[0039] Figure 12 These are the SEM results of bacteria after being treated with TBP-QA for different time periods; A is Staphylococcus aureus; B is Bacillus cereus.

[0040] Figure 13 The change in nucleic acid concentration of bacteria after being acted upon by TBP-QA.

[0041] Figure 14 The changes in intracellular ATP concentration of Staphylococcus aureus and Escherichia coli after the action of TBP-QA.

[0042] Figure 15 The changes in intracellular ATP concentration of Bacillus cereus and Salmonella after the action of TBP-QA.

[0043] Figure 16 The imaging results of TBP-QA for Escherichia coli (A) and Staphylococcus aureus (B).

[0044] Figure 17 Figure 3 shows the changes in the antibacterial MIC of TBP-QA against Staphylococcus aureus when different phospholipids were added. PE, CL, and PG represent phosphatidylethanolamine, cardiolipin, and phosphatidylglycerol, respectively.

[0045] Figure 18The growth of RAW264.7 and HepG2 cells after TBP-QA treatment.

[0046] Figure 19 is the therapeutic effect of TBP-QA on the peritonitis-sepsis model of mice infected with MRSA T144; A is the process of establishing the peritonitis-sepsis model of mice infected with MRSA T144; BC are the effects of TBP-QA and vancomycin treatment on the bacterial counts in the main organs of mice (n=6), respectively, where - represents the control sample, +V represents vancomycin treatment; +T represents TBP-QA treatment.

[0047] Figure 20 Figure 3 Histopathological analysis of different organs in a mouse peritonitis-sepsis model after treatment with PBS, TBP-QA, and vancomycin. DETAILED DESCRIPTION

[0048] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0049] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0050] In the following examples, the raw material a is 4,7-dibromo-2,1,3-benzothiadiazole, the structural formula of which is Purchased from Merck (Cat. No. 693847).

[0051] Raw material b is N,N-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)aniline, the structural formula of which is Purchased from Maclean (cat. no. MFCD05663854).

[0052] Raw material c is N,N-Diphenyl-4-(4,4,5,5-Tetramethyl-1,3,2-Dioxaborolan-2-yl)Aniline, with the structural formula It was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (catalog number: S97671).

[0053] Tetrakistriphenylphosphine palladium, molecular formula Pd[P(C6H5)3]4, was purchased from Merck (Cat. No. 216666).

[0054] TBP-1 is N,N-diphenyl-4-(7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazol-4-yl)aniline, with the structural formula (Shi et al., 2020). Dichloroethane, iodomethane, 1,4-dioxane, and potassium carbonate were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0055] Staphylococcus aureus, Bacillus cereus, Staphylococcus epidermidis, Staphylococcus chromogenes, MRSAT144, Escherichia coli ATCC 25922, Salmonella ATCC H9812, Acinetobacter baumannii 176, Acinetobacter baumannii 176ΔLPS, Acinetobacter baumannii 7-2, Acinetobacter baumannii 7-2ΔLPS (Song et al., 2020).

[0056] RAW264.7 and HepG2JUN were purchased from Beyotime Biotechnology Co., Ltd. (catalog numbers: C7505 and C6346, respectively).

[0057] Example 1 Synthesis of photosensitizer

[0058] The preparation formula and structure of the photosensitizer TBP-QA prepared in this example are as follows: Figure 1 As shown, the photosensitizer TBP-QA uses triphenylamine as the core structure of AIE. Triphenylamine has a typical propeller structure, in which the rotational motion of the benzene ring helps to dissipate the excited state energy in the solution state, providing AIE characteristics and photodynamic antibacterial activity; 2,1,3-benzothiadiazole and a benzene ring are connected in sequence after the triphenylamine. Both of them can act as co-receptors and can combine with triphenylamine to form a donor-acceptor structure, so that the emission wavelength of the molecule is red-shifted, providing red fluorescence and improving the signal-to-noise ratio; the benzene ring also acts as a π bridge; then, a quaternary ammonium salt is connected after the benzene ring. On the one hand, the quaternary ammonium salt is positively charged and can capture bacteria through electrostatic action; in addition, the quaternary ammonium salt itself has certain antibacterial properties, which can synergize with the photodynamic activity of AIE and enhance the antibacterial properties of the molecule.

[0059] Described TBP-QA is synthesized by the following method:

[0060] Step 1: Add 41.5 g (0.3002 mol) of potassium carbonate and 150 mL of water to a 3000 mL three-necked flask and stir until the solution is clear. Then, add 44.12 g (0.1501 mol) of raw material a, 38.95 g (0.1576 mol) of raw material b, and 1500 mL of tetrahydrofuran in sequence and stir until the solution is clear. Replace the air with argon twice, then add 8.67 g (0.007503 mol) of tetrakistriphenylphosphine palladium. Under argon protection, heat the reaction to 60°C, where reflux is evident. Continue the reaction for 31 hours before stopping (the reaction solution is brownish red).

[0061] Post-treatment: The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure, and then an appropriate amount of water was added and stirred. The organic layer was concentrated and dried to obtain 85g of brown-red solid crude product. Then, 11g of intermediate 1 was obtained by column chromatography using a dichloromethane and petroleum ether (1:3) system. 1 H-NMR and 13 C-NMR, the structure is correct. 1 H NMR (400MHz, DMSO-D6) δ8.05(d,J=7.6Hz,1H),7.97(s,10H),7.90(d,J=9.0Hz,2H),7.6 8(d,J=7.6Hz,1H),6.87(d,J=8.8Hz,2H),3.35(s),3.00(s,6H,N-CH3),2.51(s,DMSO). 13 CNMR (DMSO-D6) δ 152.76, 133.27, 130.48, 126.58, 113.57, 112.22, all C on the benzene ring, 40.60 (DMSO). The structural formula of the intermediate 1 is

[0062] Step 2: To a 1000mL three-necked flask, add 8.39g (0.0607mol) of potassium carbonate and 30.37mL of water and stir until the solution is clear. Then, add 10.15g (0.030368mol) of intermediate 1, 11.27g (0.030368mol) of starting material c, and 300mL of 1,4-dioxane in sequence. Stir until the solution is clear, then replace the air with argon three times. Then, add 1.75g ​​(0.001514mol) of tetrakistriphenylphosphine palladium. Under argon protection, heat the reaction to 90°C and continue the reaction for 29 hours before stopping the reaction (the reaction solution turns brownish red).

[0063] Post-treatment: The reaction solution was cooled to room temperature, filtered, and the filter cake was washed twice with 1,4-dioxane and dried to obtain 11g of red powder crude product. The product was dissolved by heating with 200mL of dichloromethane and 100mL of tetrahydrofuran, cooled to room temperature, and filtered to obtain orange-red crystalline powder intermediate 2, which was sent for inspection. 1 H-NMR and 13 C-NMR, the structure is correct. 1H NMR (400 MHz, Chloroform-D) δ 7.94-7.97 (m, 2H), 7.90 (dt, J = 9.2, 2.3 Hz, 2H), 7.74 (d, J = 2.4 Hz, 2H), 7.30-7.34 (m, 4H), 7.23 (t, J = 8.6 Hz, 6H), 7.09 (t, J = 7.2 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 20 H atoms in total on the benzene ring, 3.05-3.11 (m, 6H, N-CH3). 13 C NMR (101MHz, Chloroform-D) δ147.54, 131.37, 130.00, 129.83, 129.33, 127.66, 126.40, 124.81, 123.19, 123.08, 112.38, all C in the benzene ring structure, 77.34 (CHCl3), 40.49 (-CH3). The structural formula of the intermediate 2 is

[0064] Step 3: Add 5.5g (0.01103mol) of intermediate 2, 250mL of dichloroethane and 2.35g (0.016545mol) of iodomethane to a 500mL three-necked reaction bottle, stir, and heat the system to reflux reaction. Monitor the reaction progress by thin layer chromatography. Add iodomethane three times during the reaction, 6g each time. Stop the reaction after 72h of reaction, filter while hot, and solid precipitate after the filtrate is cooled. Filter and dry the filter cake to obtain 3.6g of brownish-red solid. The obtained 3.6g solid was refluxed with 100mL of chloroform, then stirred at room temperature overnight, filtered, and the filter cake was rinsed twice with chloroform and dried to obtain a dark purple-red fine scaly crystalline powder, which is TBP-QA, and its structural formula is The molecular weight of TBP-QA is known to be 513.68, and the molecular ion peak displayed in positive ion mode is 513.3, which is consistent with the known molecular weight, indicating that TBP-QA was successfully synthesized. Characteristic hydrogen and carbon shift peaks also indicate that TBP-QA was successfully synthesized. 1 H NMR (400 MHz, DMSO-D6) δ8.32 (s, 1, active H), 8.26 (s, 2H), 8.17 (s, 2H), 8.09-8.03 (1H), 7.98 (s, 3H), 7.37 (d, J = 8.1 Hz, 4H), 7.11-7.14 (m, 8H), a total of 20 H in the benzene ring, measured 20H, 3.70 (s, 9H, N-CH3),, 3.35 (s, water peak), 2.51 (s, DMSO). 13C NMR (101 MHz, DMSO-D6) δ 147.27, 130.83, 130.73, 130.19, 125.09, 124.18, 122.55, 121.23, all C in the benzene ring structure, 79.63 (-CH3), 56.93 (-CH3), 40.60 (DMSO).

[0065] The structures of intermediate 1, intermediate 2 and TBP-QA were verified by nuclear magnetic resonance and high-resolution mass spectrometry, respectively. Figure 2-4 As shown, the structures and high purity of intermediate 1, intermediate 2 and TBP-QA were demonstrated.

[0066] Example 2

[0067] ROS are produced when the photosensitizer absorbs light under light conditions, generating a singlet excited state (S1). S1 further transfers energy to the triplet excited state (T1), sensitizing the surrounding triplet oxygen to form destructive singlet oxygen or other ROS. The occurrence of the above-mentioned ISC process is closely related to the frontier molecular orbital of the molecule. Among the frontier orbitals, the electrons on the HOMO have the highest energy and are least bound, so they are the most active and easy to change, while the LUMO has the lowest energy among all unoccupied orbitals and is most likely to accept electrons. The energy difference between the two orbitals (Δ E ) can be used to measure whether a molecule is easily excited; the smaller the difference, the more easily excited the molecule. Reducing the difference between the two orbitals can promote their spatial separation, accelerate the ISC process, thereby generating more ROS and enhancing antibacterial activity. In order to enhance the ROS-generating ability of AIE photosensitizers and thus enhance their antibacterial activity, the present invention attempts to increase the ISC rate by introducing a co-receptor into the AIE structure to enhance the donor-acceptor relationship.

[0068] The present invention uses TBP-1 as a comparison, introduces a co-receptor and a π bridge, which can promote a better separation of the molecular HOMO and LUMO, reduce the repulsion of the valence electrons with opposite spins in the S1 state, and help reduce Δ EST , enhance the ISC process and enhance the ROS generation ability. The frontier orbitals of TBP-QA and TBP-1 were calculated and analyzed by density functional theory. The results are as follows Figure 5 As shown, the TBP-1 molecule Δ E is 3.43, while the Δ E is 3.39. Obviously, the Δ E The value is smaller. Of course, compared with TBP-1, TBP-QA has higher ROS production activity and thus higher antibacterial activity. According to the structural design and theoretical calculation results, TBP-QA will have good performance as a photosensitizer.

[0069] Example 3 Photophysical properties of photosensitizers and ROS production

[0070] In this example, the UV-visible absorption spectrum of TBP-QA in DMSO was tested (whether there was a ratio of TBP-QA to DMSO during the test), and the results were as follows: Figure 6 As shown, TBP-QA exhibits maximum absorption peaks at 368 nm and 430 nm, and absorbs the visible light region from 300 nm to 600 nm, so it can be effectively activated by ordinary white light.

[0071] The luminescence performance of TBP-QA in different ratios of tetrahydrofuran and DMSO mixed solvents (the volume ratio of tetrahydrofuran in the mixed solvent is 0%, 20%, 40%, 60%, 80%, 90%, 95%, 99%) was tested. The results are as follows Figure 7 As shown in the figure, the AIE properties of TBP-QA were measured in DMSO / THF mixed solvent. They showed very weak emission in DMSO solution. (四氢呋喃) gradually increases, the fluorescence intensity of TBP-QA is increasing, and when fw (四氢呋喃) When the fluorescence intensity rises to 99%, the emission peak is at 665 nm. Compared to the single-molecule state in pure DMSO solution, the formation of aggregates results in restricted intramolecular motion, activating radiative decay and significantly enhancing fluorescence, with the fluorescence intensity increasing by approximately 23 times. Furthermore, the quantum yields of TBP-QA in the dispersed and aggregated states are 1.29 and 5.69, respectively. The fluorescence quantum yield in the aggregated state is 5.3 times that of the dispersed state, indicating that TBP-QA molecules exhibit enhanced luminescence in the aggregated state and a distinct AIE property. The fluorescence lifetimes of TBP-QA in the dispersed and aggregated states were measured to be 7.3 and 38.5, respectively. The fluorescence lifetime in the aggregated state is 4.4 times that of the dispersed state. This is due to the restricted intramolecular motion in the aggregated state, which effectively suppresses the rate of non-radiative transitions and increases the fluorescence lifetime, demonstrating that TBP-QA exhibits aggregation-enhanced luminescence.

[0072] ROS is a key factor in the function of photosensitizer. The present invention uses electron spin resonance technology to detect ROS generated by TBP-QA during illumination, including singlet oxygen ( 1 O2), hydroxyl radicals (·OH) and superoxide radicals (·O2 - ). The result is as follows Figure 8 As shown in the figure, TBP-QA does not generate the above ROS in the dark, but generates obvious ESR signals after only 2 minutes of light exposure. 1The ESR spectrum of O2 shows a typical triplet peak characteristic of 1:1:1 (8-A), and the ESR spectrum of ·OH shows a typical quartet peak characteristic of 1:2:2:1 (8-B). - The ESR spectrum of TBP-QA showed a typical 1:1:1:1 quartet characteristic (8-C). 1 The ESR signals of O2 and ·OH are higher than those of ·O2 - signal, indicating that TBP-QA produces 1 The above results indicate that TBP-QA molecules have strong ROS production ability, and the higher ROS production ability makes them have potential photodynamic antibacterial activity.

[0073] Example 4 Study on the antibacterial properties of AIE photosensitizer

[0074] After demonstrating the ability of TBP-QA to produce ROS, their antibacterial effects on Gram-positive bacteria (G(+)) and Gram-negative bacteria (G(-)) were further studied. The antibacterial activity of TBP-QA was determined according to the CLSI 2020 standard, and it was found that it had a moderate antibacterial effect on Gram-positive bacteria. As shown in Table 1, the MIC of TBP-QA against a variety of Gram-positive bacteria, including Staphylococcus aureus, Bacillus cereus, Staphylococcus epidermidis, and Staphylococcus chromogenes, was 4-8μg / mL; among them, TBP-QA had the same antibacterial activity against MRSAT144, with an MIC value of 4μg / mL. However, TBP-QA has basically no antibacterial activity against a variety of Gram-negative bacteria such as Escherichia coli and Salmonella, with an MIC greater than 128μg / mL. This result shows that TBP-QA has specific antibacterial activity against Gram-positive bacteria.

[0075] Table 1 MICs of TBP-QA against Gram-positive and Gram-negative bacteria

[0076]

[0077] The above experimental results show that TBP-QA has excellent antibacterial effect on Gram-positive bacteria, and TBP-QA itself has a high ROS yield, which can be used to achieve antibacterial purposes through photodynamics.

[0078] The inhibitory effect of TBP-QA on Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus and MRSAT144) under light and dark conditions was further investigated, and two groups of action time (10 minutes or 30 minutes) were set for comparison. Figure 9As shown in the figure, TBP-QA has a significantly higher bactericidal efficiency against Staphylococcus aureus in the light group than in the dark group, regardless of whether the exposure time is 10 minutes or 30 minutes. After 10 minutes of exposure, the bactericidal efficiency of the light group TBP-QA concentration at 1×MIC can reach about 90%, while the bactericidal efficiency of the dark group is only about 10%; the bactericidal efficiency of the light group TBP-QA concentration at 2×MIC reaches more than 95%, while the bactericidal efficiency of the dark group is less than 60%; as the exposure time is extended to 30 minutes, the bactericidal efficiency of both the light and dark groups increases. Taking Bacillus cereus as an example, Figure 10 As shown, similar to Staphylococcus aureus, the bactericidal efficiency of TBP-QA against Bacillus cereus in the light group was significantly higher than that in the dark group, regardless of whether the action time was 10 minutes or 30 minutes. After 10 minutes of action, the bactericidal efficiency of TBP-QA in the light group reached about 90% at a concentration of 2×MIC, while the bactericidal efficiency of the dark group was only about 50%; as the action time was extended to 30 minutes, the bactericidal efficiency of both the light and dark groups increased. However, compared with Staphylococcus aureus in general, the antibacterial activity of TBP-QA against Bacillus cereus was slightly weaker. In view of the excellent antibacterial activity of TBP-QA against Staphylococcus aureus, its antibacterial effect on MRSAT144 was further studied. Figure 11 As shown, similar to Staphylococcus aureus, TBP-QA showed significantly higher bactericidal efficiency against MRSAT144 in the light-exposed group than in the dark-exposed group, regardless of whether the exposure time was 10 or 30 minutes. At 10 minutes, the bactericidal efficiency of TBP-QA in the light-exposed group reached over 90% at a concentration of 1×MIC, while the bactericidal efficiency in the dark-exposed group was less than 10%. At a concentration of 2×MIC, the bactericidal efficiency in the light-exposed group reached over 99%, while the bactericidal efficiency in the dark-exposed group was approximately 40%. As the exposure time increased to 30 minutes, the bactericidal efficiency in both the light-exposed and dark-exposed groups increased.

[0079] Example 5 Discussion on the sterilization mechanism

[0080] The above results show that TBP-QA has excellent antibacterial properties against Gram-positive bacteria. Therefore, the antibacterial mechanism of TBP-QA was studied using Staphylococcus aureus and Bacillus cereus as model bacteria. First, the morphological changes of Staphylococcus aureus and Bacillus cereus after TBP-QA treatment were observed by SEM. Figure 12 As shown, the surfaces of Staphylococcus aureus and Bacillus cereus in the control group were smooth and structurally intact. However, after 5 minutes of TBP-QA treatment, the surfaces of the membranes of Staphylococcus aureus and Bacillus cereus were no longer smooth, but wrinkled and ruptured in some areas. As the TBP-QA treatment time was extended to 10 minutes, obvious cavities appeared on the surface of the bacterial membranes, the structures collapsed, and the bacterial membranes were completely destroyed and no longer intact.

[0081] We further explored the leakage of four representative bacterial intracellular substances (nucleic acid substances and ATP) after TBP-QA treatment to characterize the integrity of their structure. First, we targeted nucleic acid substances, such as Figure 13 As shown in the results, after treatment with TBP-QA, the extracellular nucleic acid concentrations of two Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus, increased with increasing TBP-QA concentrations, indicating that nucleic acid leakage occurs in Gram-positive bacteria after treatment with TBP-QA. This further demonstrates that TBP-QA disrupts the integrity of Gram-positive bacterial membranes, causing leakage of bacterial contents. In contrast, the extracellular nucleic acid concentrations of two Gram-negative bacteria, Escherichia coli and Salmonella, remained almost unchanged after treatment with TBP-QA, indicating that nucleic acid leakage does not occur after treatment with TBP-QA, further demonstrating that TBP-QA has little antibacterial effect against Gram-negative bacteria.

[0082] Then, the intracellular ATP leakage of four representative bacteria after TBP-QA was investigated. Figure 14 and 15 As shown, after treatment with TBP-QA, the intracellular ATP concentration of two Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus, decreased with increasing TBP-QA concentration, indicating that ATP leaked from the bacteria after TBP-QA treatment, further confirming that TBP-QA damages the integrity of Gram-positive bacteria and causes the leakage of bacterial contents. In contrast, the intracellular ATP concentration of two Gram-negative bacteria, Escherichia coli and Salmonella, remained almost unchanged after treatment with TBP-QA, indicating that intracellular ATP did not leak after TBP-QA treatment and that the bacterial structure was intact, further confirming that TBP-QA has no antibacterial activity against Gram-negative bacteria.

[0083] The above research results show that TBP-QA has a broad spectrum of bacterial imaging capabilities, but has specific Gram-positive antibacterial ability, so its specific antibacterial mechanism was analyzed and studied. Unlike Gram-positive bacteria, Gram-negative bacteria have an outer membrane structure outside the cell wall, which separates the bacteria from the external environment and acts as a permeability barrier for Gram-negative bacteria. This prevents most antibacterial molecules from passing through the outer membrane and accumulates inside the bacteria, resulting in no antibacterial activity against Gram-negative bacteria. [147,148] Although the outer membranes of different types of Gram-negative bacteria are somewhat different, their composition is basically the same, mainly composed of lipopolysaccharide (LPS) and various phospholipid components. [149,150] We speculate that the presence of LPS, the main component of the bacterial outer membrane, may prevent TBP-QA from entering the interior of Gram-negative bacteria, thereby causing bacterial damage. We first used laser confocal microscopy to investigate whether TBP-QA enters the interior of bacteria. The results are as follows Figure 16As shown, when TBP-QA was used to image E. coli, only the outer membrane of the bacteria showed bright fluorescence, indicating that TBP-QA stained only the outer membrane of Gram-negative bacteria and did not penetrate into the interior of the bacteria. However, when imaging Gram-positive bacteria, the results showed bright fluorescence also inside the bacteria, indicating that TBP-QA can penetrate and image Gram-positive bacteria.

[0084] To further verify whether the outer membrane of Gram-negative bacteria plays a protective role, the antibacterial properties of TBP-QA against two strains of Acinetobacter baumannii and LPS-deficient A. baumannii were investigated using the MIC as a standard. As shown in Table 2, the MICs of TBP-QA against the two strains of A. baumannii were greater than 128 μg / mL, indicating that TBP-QA had no antibacterial effect against them. However, the MICs against the two LPS-deficient A. baumannii were 8 and 4 μg / mL, respectively, indicating that TBP-QA had good antibacterial activity. These results indicate that LPS plays a protective role against Gram-negative bacteria, hindering TBP-QA from entering the bacterial interior and rendering TBP-QA inactive against Gram-negative bacteria.

[0085] Table 2 MIC of TBP-QA against Acinetobacter baumannii

[0086]

[0087]

[0088] Table Notes: a: △LPS represents strains lacking LPS

[0089] In addition, the present invention explored the possible targets of TBP-QA against Gram-positive bacteria. The plasma membrane of Gram-positive bacteria is composed of amphiphilic phospholipid molecules. The plasma membrane of most Gram-positive bacteria, such as Staphylococcus, is mainly composed of negatively charged PG (50%-60%) and CL, and a few bacteria contain a certain amount of PE. In order to further study the specific location of TBP-QA binding to the bacterial plasma membrane, the antibacterial activity of TBP-QA against Staphylococcus aureus was determined when the above-mentioned main bacterial cell membrane components were additionally added. The results are as follows. Figure 17 As shown, the addition of cell membrane components PE and CL barely altered the MIC of TBP-QA against S. aureus. However, the addition of membrane component PG increased the MIC of TBP-QA against S. aureus by fourfold. This result suggests that the addition of membrane components PE and CL had little effect on the antibacterial activity of TBP-QA, while the addition of membrane component PG effectively inhibited the antibacterial activity of TBP-QA against S. aureus. Therefore, it can be concluded that TBP-QA primarily targets PG in Gram-positive bacteria to specifically recognize Gram-positive bacteria and exert its specific antibacterial effect.

[0090] Based on the above mechanism research results, the specific antibacterial mechanism of TBP-QA can be summarized as follows: TBP-QA specifically targets the membrane component PG of bacteria, and produces toxic effects on bacteria when a large amount of ROS is generated under light, causing cell structure damage, leakage of intracellular substances, and ultimately bacterial death.

[0091] Example 6 Cytotoxicity Analysis

[0092] Good biosafety is the basis for the in vivo application of TBP-QA. The present invention uses a cell counting kit to measure the effect of TBP-QA on the viability of RAW264.7 and HepG2 cells to evaluate the biosafety of the molecule. Figure 18 As shown in the results, after 24 hours of co-incubation with TBP-QA, HepG2 cell viability remained essentially unchanged at concentrations up to 4× the MIC, while RAW264.7 cell viability decreased slightly, but still maintained approximately 78% of its original value. These results demonstrate that TBP-QA has excellent biosafety and can be further applied for in vivo antimicrobial applications.

[0093] Example 7 Study on the antibacterial properties of photosensitizers in vivo

[0094] Given that TBP-QA has good biosafety and excellent antibacterial effect against MRSA in vitro, the present invention further applied it to treat MRSA-infected mouse peritonitis-sepsis and back wound models to evaluate its in vivo antibacterial effect. First, the effect of TBP-QA in treating MRSA-infected mouse peritonitis-sepsis model was evaluated. Each group of mice was injected intraperitoneally with MRSAT144 suspension (10 8 CFU / mouse), and 1 h after infection, mice were treated by intraperitoneal injection of TBP-QA (16 mg / kg), PBS (negative control), and vancomycin (positive control, 20 mg / kg). Figure 19 -A). The results showed that all mice treated with PBS died within 48 hours, indicating that the injected MRSAT144 was sufficient to kill the mice. When mice were treated with TBP-QA or vancomycin, all mice survived within 48 hours. The number of bacteria in different organs of mice treated with PBS, TBP-QA and vancomycin was then counted using tissue grinding and plate counting methods. The results showed that the number of bacteria in various organs of mice treated with TBP-QA and vancomycin was significantly reduced (by about 2 orders of magnitude) ( Figure 19 The above results show that TBP-QA can significantly reduce the bacterial load of organs in the MRSA-infected mouse peritonitis-sepsis model and improve the survival rate of mice within 48 hours, and the therapeutic effect is comparable to that of vancomycin.

[0095] To further understand the therapeutic effect of TBP-QA on the mouse peritonitis-sepsis model, pathological analysis of mouse organs in the TBP-QA and vancomycin treatment groups in the mouse infection model was performed. Figure 20 As shown, pathological sections of the liver, spleen, lungs, and kidneys of the infected group (treated with PBS) exhibited varying degrees of pathological changes, with the exception of the heart. The liver showed enlarged hepatocytes with vacuolar infiltration, lymphocyte infiltration, and blurred hepatic cords; the spleen showed lymphocyte infiltration and splenic atrophy; alveolar wall rupture and fusion of adjacent alveoli; and glomerular enlargement and dilation of capillary lumens. However, these typical pathological changes in these organs were effectively alleviated in the TBP-QA and vancomycin treatment groups. This suggests that TBP-QA has a significant therapeutic effect on organ lesions caused by MRSAT144 infection in mice and can effectively treat peritonitis-sepsis in mice.

[0096] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A compound TBP-QA, characterized in that The structural formula of the compound TBP-QA is:

2. The method for preparing the compound TBP-QA according to claim 1, characterized in that: The steps include: 1) Add raw material a to potassium carbonate solution Raw material b and tetrahydrofuran, stirred until clear, under argon protection, added tetratriphenylphosphine palladium and heated. After the reaction was completed, cooled, filtered, and the filtrate was concentrated and purified to obtain intermediate 1 2) Add intermediate 1 to potassium carbonate solution Raw material c and 1,4-dioxane, stirred until clear, and under argon protection, added tetratriphenylphosphine palladium and heated. After the reaction was completed, cooled, filtered, and the filter cake was washed and purified to obtain intermediate 2 3) Intermediate 2 and iodomethane were added to dichloroethane, the temperature was raised, and the reaction was carried out. During the reaction, iodomethane was added three times. After the reaction was completed, the mixture was filtered and purified to obtain compound TBP-QA.

3. The preparation method according to claim 2, characterized in that In the step 1), the molar ratio of raw material a: raw material b: tetrakistriphenylphosphine palladium is 20:20:

1.

4. The preparation method according to claim 2, characterized in that In the step 1), the reaction temperature is 60° C. and the reaction time is 31 h.

5. The preparation method according to claim 2, characterized in that In the step 2), the molar ratio of intermediate 1: raw material c: tetrakistriphenylphosphine palladium is 20:20:

1.

6. The preparation method according to claim 2, characterized in that In the step 2), the reaction temperature is 90° C. and the reaction time is 29 h.

7. The preparation method according to claim 2, characterized in that In the step 3), the molar ratio of intermediate 2: first addition of iodomethane: additional addition of iodomethane is 2:3:7.

6.

8. The preparation method according to any one of claims 2 to 7, characterized in that: The reaction conditions in step 3) are as follows: heating the system to reflux reaction, monitoring the reaction progress by thin layer chromatography, adding iodomethane three times during the reaction, stopping the reaction after 72 hours, filtering while hot, cooling the filtrate to precipitate solids, filtering, and drying the filter cake.

9. Use of the compound TBP-QA according to claim 1 or the compound TBP-QA prepared by the method according to any one of claims 2 to 7 in any of the following: 1) Application as aggregation-induced emission material; 2) Application in the preparation of fluorescent imaging reagents; 3) Application in the preparation of photodynamic therapy drugs; 4) Application in the preparation of Gram-positive antibacterial agents; 5) Application in the preparation of LPS-deficient Gram-negative antibacterial agents; 6) Use in the preparation of drugs for treating organ lesions caused by MRSA T144; 7) Use in the preparation of medicines for treating peritonitis-sepsis.

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