Compound, preparation method thereof and application of compound as photosensitizer

Abstract

The invention discloses a compound and a preparation method and application of the compound as a photosensitizer, the structural formula of the compound is # imgabs0, R is independent, and R is H, alkyl, unsaturated alkyl, heteroatom alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy or one or more chromophores capable of being conjugated with one or more fluorescent substances. The compound provided by the invention has an efficient ROS generation rate, and can effectively combine and kill bacteria with poor membrane permeability. The antibacterial effect of the photosensitizer in a physiological environment is not reduced by ROS quenching caused by an aggregation-induced quenching effect, and the photosensitizer has wide non-specific bactericidal ability on drug-resistant bacteria such as MRSA, KREC and P.aeruginosa.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical and biomolecular engineering, and particularly relates to a compound, a preparation method thereof, and an application as a photosensitizer. Background Art

[0002] Bacterial infections, especially those caused by drug-resistant bacteria, have become a major health crisis facing humanity. In the past few decades, the widespread and long-term abuse of antibiotics has led to the emergence of drug resistance in certain pathogens. Therefore, infections caused by drug-resistant bacteria pose a major challenge to humans, as evidenced by the increasing transmission rate and mortality. This problem has been exacerbated by the slowdown in the development of new antibiotics. It is estimated that if no immediate action is taken to discover and develop new antibiotics, the number of deaths caused by drug-resistant infections will exceed 10 million per year by 2050.

[0003] Recent efforts to revitalize antibiotic research have mainly focused on modifying compounds with antibacterial mechanisms similar to traditional antibiotics. However, this approach increases the likelihood of drug resistance because mutations in drug targets reduce the efficacy of antibiotics. In contrast, drugs with multiple antibacterial targets or those capable of physically damaging pathogens (such as photosensitizers or cationic coatings) are less likely to develop drug resistance. However, due to problems such as the effectiveness of binding to bacteria and the feasibility of in vivo delivery, the application of these methods in vivo is still limited. Therefore, there is an urgent need to design new antibiotics to meet the urgent clinical needs for treating drug-resistant bacteria.

[0004] Currently, photodynamic therapy (PDT) for the elimination of bacteria is an effective method to solve the problem of bacterial drug resistance. However, existing photosensitizers for PDT generally suffer from low singlet oxygen (ROS) production rates and poor binding ability to bacteria. Most photosensitizers used for PDT bactericidal can bind well to Gram-positive bacteria, but they cannot effectively bind to and eliminate Gram-negative bacteria with more complex cell membrane structures through PDT. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the object of the present invention is to provide a compound having two rigid hydrophobic structures and a cation (the rigid hydrophobic structure is X is S or O), which can effectively bind to various Gram-negative / positive bacteria through electrostatic and hydrophobic interactions while having a high ROS production rate, and kill the bacteria through PDT.

[0006] Another object of the present invention is to provide a preparation method of the above compound.

[0007] Another object of the present invention is to provide the use of the above compound as a photosensitizer. The compound has good water solubility, strong bacterial binding ability and high Type II reactive oxygen species (ROS) generation efficiency, and can effectively bind to some drug-resistant bacteria such as P. aeruginosa that are difficult to target due to poor membrane permeability, and has a significant photodynamic bactericidal effect.

[0008] A compound, the structural formula of which is Wherein, each R is independent, and R is H, alkyl, unsaturated alkyl, heteroatom alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy or one or more chromophores capable of conjugating with one or more fluorescent substances.

[0009] The preparation method of the above compound comprises the following steps:

[0010] S1. Preparation of compound a1: Mix the reactant, biphenyl halide, Pd(OAc) 2 , the first base and t-Bu 3 P·HBF 4 , add toluene to dissolve it, and react at 90-110 °C for 10-15 h under a nitrogen or inert gas atmosphere. After the reaction is completed, cool to room temperature, pour it into ethyl acetate, and extract with saturated brine. Dry the organic phase and evaporate to dryness to obtain a crude product. Purify the crude product to obtain a white to pale yellow solid as compound a1. Among them, in terms of the number of moles, the ratio of the reactant, biphenyl halide, Pd(OAc) 2 and the first base is 10:(10-15):(0.2-1):(10-15). In terms of the number of moles, the ratio of Pd(OAc) 2 and t-Bu 3 P·HBF 4 is 1:2. The structural formula of the reactant is as follows:

[0011] X is S or O;

[0012] The structural formula of compound a1:

[0013] In the S1, the biphenyl halide is 4-bromobiphenyl or 4-iodobiphenyl.

[0014] In the S1, the first base is potassium tert-butoxide, sodium tert-butoxide, potassium acetate or sodium acetate.

[0015] In the S1, purification is carried out by silica gel column chromatography. The silica gel column chromatography uses a mixture of dichloromethane and n-hexane as the eluent. In terms of volume fraction, dichloromethane:n-hexane = 1:(5-15).

[0016] In S1, the ratio of the number of moles of the reactant to the volume fraction of toluene is less than or equal to 10:20. The unit of the number of moles is mmol, and the unit of the volume fraction is mL.

[0017] In S1, the ratio of the number of moles of the reactant to the volume fraction of ethyl acetate is less than or equal to 10:20. The unit of the number of moles is mmol, and the unit of the volume fraction is mL.

[0018] In S1, anhydrous sodium sulfate is used for drying.

[0019] S2. Preparation of compound a2: Mix compound a1 and DCM, and cool to 0 - 4 °C to obtain a first solution. Dissolve N-bromosuccinimide in DCM to obtain a second solution. Drop the second solution into the first solution, stir in the dark at 0 - 4 °C for 0.5 - 2 hours, warm to room temperature and stir in the dark for 10 - 15 h, quench the reaction with water, extract with DCM, wash the organic layer with water and dry it, evaporate the solvent under reduced pressure, and purify to obtain compound a2. Among them, in terms of the number of moles, the ratio of compound a1 to N-bromosuccinimide is 5:(10 - 11);

[0020] Structural formula of compound a2:

[0021] In S2, NaSO is used for drying 4 .

[0022] In S2, silica gel chromatography is used for purification, and a mixture of dichloromethane and n-hexane is used as the eluent. In terms of volume fraction, dichloromethane:n-hexane = 1:(3 - 5).

[0023] In S2, the concentration of compound a1 in the first solution is 0.1 - 0.3 mmol / mL, and the concentration of N-bromosuccinimide in the second solution is 0.3 - 0.5 mmol / mL.

[0024] S3. Preparation of compound a3: Mix compound a2, a second base, and pyridine-4-boronic acid, add Pd(PPh 3 ) 4 as a catalyst. Then, after adding an aqueous solution of 1,4-dioxane, stir at 90 - 105 °C for 10 - 15 h under the protection of nitrogen or an inert gas. After cooling to room temperature, pour it into ethyl acetate, extract with water, dry the obtained organic layer and evaporate it under reduced pressure, and purify to obtain an orange-yellow solid as compound a3. Among them, in terms of the number of moles, the ratio of compound a2 to pyridine-4-boronic acid is 2:(4 - 6), and in terms of the number of moles, the ratio of pyridine-4-boronic acid to the second base is 1:(1 - 1.5);

[0025] Structural formula of compound a3:

[0026] In S3, the second base is K 2 CO 3 , Na 2 CO 3 , potassium acetate, cesium carbonate or sodium acetate.

[0027] In S3, by the number of moles, the ratio of the compound a2 and Pd(PPh 3 ) 4 is 2:(0.1 - 0.2).

[0028] In S3, the ratio of the number of moles of the compound a2 and the volume fraction of the 1,4 - dioxane aqueous solution is less than or equal to 2:20. The unit of the number of moles is mmol, and the unit of the volume fraction is mL.

[0029] In S3, the 1,4 - dioxane aqueous solution is a mixture of 1,4 - dioxane and water. By volume fraction, the ratio of 1,4 - dioxane to water in the 1,4 - dioxane aqueous solution is (4 - 5):1.

[0030] In S3, the ratio of the number of moles of the compound a2 and the volume fraction of the ethyl acetate is less than or equal to 2:40. The unit of the number of moles is mmol, and the unit of the volume fraction is mL.

[0031] In S3, purification is carried out by silica gel chromatography, using a mixture of ethyl acetate and n - hexane as the eluent. By volume fraction, ethyl acetate:n - hexane = 1:(4 - 10).

[0032] In the above technical solution, the organic layer in S3 is dried with NaSO 4 dry.

[0033] S4. Preparation of the compound: Dissolve the compound a3 in ACN, under the protection of nitrogen or inert gas, add methyl iodide, stir at 70 - 80 °C for 10 - 15 h, remove the solvent under reduced pressure to obtain a crude product of an orange solid. After adding the crude product to the minimum amount of good solvent that can dissolve it, add a poor solvent to obtain an orange precipitate as the compound. Among them, by the number of moles, the ratio of the compound a3 and methyl iodide is 1:(2 - 2.5).

[0034] In S4, the ratio of the number of moles of the compound a3 and the volume fraction of the ACN is less than or equal to 1:20. The unit of the number of moles is mmol, and the unit of the volume fraction is mL.

[0035] In S4, the ratio of the number of moles of compound a3 to the volume fraction of the poor solvent is less than or equal to 1:20. The unit of the number of moles is mmol, and the unit of the volume fraction is mL.

[0036] Application of the above compound in improving the ROS generation rate as a photosensitizer.

[0037] The beneficial effects of the present invention are as follows:

[0038] The molecular structure of the compound of the present invention has a rigid hydrophobic structure, which helps the molecule to better insert into the bacterial outer membrane; in addition, the pyridinium salt structure in the compound molecule makes the molecule positively charged, which helps the molecule to combine with bacteria showing negative charge. In addition, the strong D-A interaction formed by the reactant and the pyridinium salt structure can effectively promote the separation of charges within the molecule, making the molecule have a high ROS generation rate. These characteristics lay a foundation for the efficient sterilization of photosensitizers.

[0039] In the present invention, through the combination of two rigid hydrophobic structures and cations, the obtained compound has strong membrane penetration ability, so that the obtained photosensitizer can effectively bind to and kill some bacteria with poor membrane permeability (such as P. aeruginosa); in addition, the modification of two cations makes the compound have good hydrophilicity, and will not reduce the antibacterial effect of the photosensitizer in the physiological environment due to the ROS quenching caused by the aggregation-induced quenching (ACQ) effect.

[0040] PT2PyPh and PO2PyPh obtained in the present invention can effectively generate Type II ROS under low light intensity (10 mW / cm 2 ) white light irradiation. Compared with the commercial photosensitizer Rose Bengal, almost no ROS generation was detected under the same conditions. This shows the high efficient reactive oxygen generation ability of PT2PyPh and PO2PyPh. In vitro experiments show that PT2PyPh and PO2PyPh have a broad non-specific bactericidal ability against drug-resistant bacteria such as MRSA, KREC and P. aeruginosa. Description of the Drawings

[0041] Figure 1 : A is the synthetic route of the preparation method of the compound, B is the ultraviolet-visible spectrum of the PBS solution of PT2PyPh and the PBS solution of PO2PyPh, C is the photoluminescence spectrum of the compound in the PBS solution, D is the photoluminescence spectrum of the PBS solution (red) and the PBS suspension (blue) of PT2PyPh, and E is the photoluminescence spectrum of the PBS solution (red) and the PBS suspension (blue) of PO2PyPh;

[0042] Figure 2: A is the UV-visible absorption spectra of the test solution prepared with the second mother liquor containing RB at time points of 0, 30, 60, 90, 120, 150, and 180 s; B is the UV-visible absorption spectra of the test solution prepared with the second mother liquor containing PT2PyPh at time points of 0, 30, 60, 90, 120, 150, and 180 s; C is the UV-visible absorption spectra of the test solution prepared with the second mother liquor containing PO2PyPh at time points of 0, 30, 60, 90, 120, 150, and 180 s; D is the PL spectra of the test solution prepared with the third mother liquor containing RB after being irradiated by white light for different times; E is the PL spectra of the test solution prepared with the third mother liquor containing PT2PyPh after being irradiated by white light for different times; F is the PL spectra of the test solution prepared with the third mother liquor containing PO2PyPh after being irradiated by white light for different times; G is the absorbance change at the ABDA absorption peak of 399 nm at different time points under the same light irradiation in the test solution prepared with RB, PT2PyPh, or PO2PyPh; H is the PL intensity change at the DCFH emission peak of 525 nm at different time points under the same light irradiation in the test solution prepared with RB, PT2PyPh, or PO2PyPh; I is the test solution obtained from the second mother liquor containing PT2PyPh and the second mother liquor containing PO2PyPh at 10 mW cm -2 Absorbance change of the compound absorption peak under white light irradiation;

[0043] Figure 3 : A is the confocal fluorescence images of Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa) cultured with PO2PyPh; B - D are the photodynamic bactericidal efficiency of PO2PyPh against MRSA, KREC, and Pseudomonas aeruginosa under white light irradiation. Detailed implementation manners

[0044] The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0045] The temperature of the saturated brine is 21 - 25 °C.

[0046] The pH of PBS in the following embodiments is 7.4.

[0047] S. aureus was purchased from ATCC (Strain designation: NCTC 8532);

[0048] E. coli was purchased from ATCC (Strain designation: HB101);

[0049] P. aeruginosa was purchased from ATCC (Strain designation: PRD-10 [CIP 103467, NCIB10421, PCI 812]);

[0050] Methicillin-resistant Staphylococcus aureus (MRSA) was purchased from ATCC (Strain designation: F-182);

[0051] As Figure 1 shown in A of

[0052]

[0053] Example 1

[0054] A method for preparing a compound, comprising the following steps:

[0055] S1. Preparation of compound a1: Add phenothiazine (1.99 g, 10 mmol), 4-iodobiphenyl (2.8 g, 10 mmol), Pd(OAc) 2 (0.5 mmol), potassium tert-butoxide (15 mmol) and t-Bu 3 P·HBF 4 (1 mmol) into a 100 mL round-bottom flask, add 50 mL of toluene to dissolve it, seal the reaction flask and protect it with nitrogen, and heat the reaction in an oil bath at 90 °C for 12 h. After the reaction is completed and cooled to room temperature, pour the solution into 100 mL of ethyl acetate, and extract the organic phase with saturated brine. The organic phase is dried with anhydrous sodium sulfate and concentrated by rotary evaporation to obtain a crude product. The crude product is purified by silica gel column chromatography, using a mixture of dichloromethane and n-hexane as the eluent (by volume fraction, dichloromethane: n-hexane = 1:10) to obtain 1.72 g of a white to pale yellow solid (compound a1), with a yield of 49%.

[0056]

[0057] S2. Preparation of compound a2: Add compound a1 (1.75 g, 5 mmol) and 30 mL of DCM into a 100 mL round-bottom flask, cool it to 0 °C in an ice bath to obtain a first solution. Dissolve N-bromosuccinimide (1.72 g, 10 mmol) in 30 mL of DCM to obtain a second solution, and add the second solution dropwise to the first solution through a dropping funnel. Stir in the ice bath at 0 °C in the dark for 1 hour, then warm to room temperature and stir in the dark for 12 h. Quench the reaction with water and extract with DCM. The organic layer is washed with water and dried over NaSO 4Dry it above. After evaporating the solvent under reduced pressure, the crude product was purified by silica gel chromatography, using a mixture of dichloromethane and n-hexane as the eluent (by volume, dichloromethane:n-hexane = 1:4), to obtain a white to pale yellow solid as compound a2, with a yield of (2.45 g, 97%).

[0058]

[0059] S3. Preparation of compound a3: Compound a2 (1010 mg, 2 mmol), K 2 CO 3 (828 mg, 6 mmol) and pyridine-4-boronic acid (541.2 mg, 4.4 mmol) were added to a 100 mL round-bottom flask. Pd(PPh 3 ) 4 (0.1 mmol) was added as a catalyst. After adding 25 mL of a 1,4-dioxane aqueous solution (by volume, the ratio of 1,4-dioxane to water in the 1,4-dioxane aqueous solution is 4:1), the flask was sealed and stirred at 90 °C for 12 h under nitrogen protection. The resulting solution was poured into 100 mL of ethyl acetate after cooling to room temperature and extracted with water. The organic layer was dried with NaSO 4 and evaporated under reduced pressure. The crude product was purified by silica gel chromatography, using a mixture of ethyl acetate and n-hexane as the eluent (by volume, ethyl acetate:n-hexane = 4:1), to obtain an orange-yellow solid as compound a3, with a yield of (787 mg, 78%).

[0060]

[0061] S4. Preparation of compound (PT2PyPh): Compound a3 (505 mg, 1 mmol) was dissolved in 50 mL of ACN and placed in a 100 mL round-bottom flask. The flask was sealed and protected with nitrogen. Then, methyl iodide (0.137 mL, 2.2 mmol) was added to this solution under nitrogen. The reaction was stirred at 70 °C for 12 h. The solvent was removed under reduced pressure to obtain a crude orange solid. After adding the crude product to the minimum amount of methanol that could dissolve it, 100 mL of diethyl ether was added to obtain an orange precipitate as the compound. The yield was (710 mg, 90%). 11H NMR (400 MHz, DMSO) δ 8.91 (d, J = 6.8 Hz, 4H), 8.40–8.37 (m, 4H), 8.10–8.06 (m, 2H), 7.93 (d, J = 2.3 Hz, 2H), 7.86–7.82 (m, 2H), 7.71 (dd, J = 8.8, 2.3 Hz, 2H), 7.66–7.62 (m, 2H), 7.58–7.54 (m, 2H), 7.50–7.44 (m, 1H), 6.27 (d, J = 8.8 Hz, 2H), 4.28 (s, 6H).

[0062]

[0063] Example 2

[0064] A method for preparing a compound, comprising the following steps:

[0065] S1. Preparation of compound a1: Add phenoxazine (1.83 g, 10 mmol), 4-iodobiphenyl (2.8 g, 10 mmol), Pd(OAc) 2 (0.5 mmol), potassium tert-butoxide (15 mmol) and t-Bu 3 P·HBF 4 (1 mmol) into a 100 mL round-bottom flask, and add 50 mL of toluene to dissolve it. Seal the reaction flask and protect it with nitrogen, and heat the reaction in an oil bath at 90 °C for 12 h. After the reaction is completed and cooled to room temperature, pour the solution into 100 mL of ethyl acetate, and extract the organic phase with saturated brine. The organic phase is dried with anhydrous sodium sulfate and concentrated by rotary evaporation to obtain a crude product. The crude product is purified by silica gel column chromatography, using a mixture of dichloromethane and n-hexane as the eluent (by volume fraction, dichloromethane: n-hexane = 1:10) to obtain 1.84 g of a white to pale yellow solid with a yield of 54%.

[0066]

[0067] S2. Preparation of compound a2: Add compound a1 (1.68 g, 5 mmol) and 30 mL of DCM into a 100 mL round-bottom flask, cool it to 0 °C in an ice bath to obtain a first solution. Dissolve N-bromosuccinimide (1.72 g, 10 mmol) in 30 mL of DCM to obtain a second solution, and add the second solution dropwise into the first solution through a dropping funnel. The solution is stirred in the dark in an ice bath at 0 °C for 1 h, then warmed to room temperature and stirred in the dark for 12 h. Quench the reaction with water, and extract with DCM. The organic layer is washed with water and dried over NaSO 4Drying. After evaporating the solvent under reduced pressure, the crude product was purified by silica gel chromatography, using a mixture of dichloromethane and n-hexane as the eluent (by volume, dichloromethane:n-hexane = 1:4), to obtain a white to pale yellow solid as compound a2, with a yield of (2.34 g, 95%).

[0068]

[0069] S3. Preparation of compound a3: Compound a2 (986 mg, 2 mmol), K 2 CO 3 (828 mg, 6 mmol) and pyridine-4-boronic acid (541.2 mg, 4.4 mmol) were added to a 100 mL round-bottom flask, and Pd(PPh 3 ) 4 (0.1 mmol) was added as a catalyst. After adding 25 mL of a 1,4-dioxane aqueous solution (by volume, the ratio of 1,4-dioxane to water in the 1,4-dioxane aqueous solution is 4:1), the flask was sealed and stirred at 90 °C for 12 h under nitrogen protection. The resulting solution was poured into 100 mL of ethyl acetate after cooling to room temperature and extracted with water. The organic layer was dried with NaSO 4 and evaporated under reduced pressure. The crude product was purified by silica gel chromatography, using a mixture of ethyl acetate and n-hexane as the eluent (by volume, ethyl acetate:n-hexane = 4:1), to obtain an orange-yellow solid as compound a3, with a yield of (665 mg, 68%).

[0070]

[0071] S4. Preparation of compound (PO2PyPh): Compound a3 (489 mg, 1 mmol) was dissolved in 50 mL of ACN and placed in a 100 mL round-bottom flask. The flask was sealed and protected with nitrogen. Then, methyl iodide (0.137 mL, 2.2 mmol) was added to this solution under nitrogen. The reaction was stirred at 70 °C for 12 h. The solvent was removed under reduced pressure to obtain a crude orange solid. After adding the crude product to the minimum amount of methanol that could dissolve it, 100 mL of ether was added to obtain an orange precipitate as the compound. The yield was (719 mg, 93%). 1 1H NMR (400 MHz, DMSO) δ 8.88 (d, J = 6.6 Hz, 4H), 8.40–8.36 (m, 4H), 8.09–8.04 (m, 2H), 7.84–7.81 (m, 2H), 7.67–7.63 (m, 2H), 7.56 (ddd, J = 12.2, 6.1, 2.9 Hz, 7H), 6.15 (d, J = 8.4 Hz, 2H), 4.28 (s, 6H).

[0072]

[0073] PT2PyPh and PO2PyPh are photosensitizers designed and synthesized for photodynamic therapy (PDT). The photosensitizer molecules are obtained by a "ring fixation" strategy of introducing S and O atoms on the TPP-based luminescent skeleton.

[0074] Among them, the structure of the TPP-based luminescent skeleton is:

[0075] PT2PyPh and PO2PyPh were respectively dissolved in DMSO to prepare a first mother liquor with a compound concentration of 10 mM. 1 μL of the first mother liquor was taken and added to 999 μL of PBS to prepare 1 mL of a PBS solution with a compound concentration of 10 μM, and the photophysical properties of the PBS solution were tested. Due to the good water solubility of PT2PyPh and PO2PyPh, both PT2PyPh and PO2PyPh can be well dissolved in PBS. As Figure 1 shown in B of, the PBS solutions of PT2PyPh and PO2PyPh both showed a relatively wide absorption spectrum between 300 - 600 nm, with peak values of 488 nm (PT2PyPh) and 495 nm (PO2PyPh), respectively.

[0076] The fluorescence emission of the PBS solution of PT2PyPh and the PBS solution of PO2PyPh was tested by an FS5 fluorescence spectrometer. As Figure 1 shown in C of, when dissolved in PBS, the maximum emission wavelength of PT2PyPh was 645 nm, and the maximum emission wavelength of PO2PyPh was 689 nm.

[0077] 10 6 CFU of Staphylococcus aureus was added to the PBS solution of PT2PyPh and the PBS solution of PO2PyPh respectively and mixed evenly to obtain a PBS suspension. As Figure 1 shown in D of and Figure 1 shown in E of, compared with the PBS solution ( Figure 1 PT2PyPh in D of and Figure 1 PO2PyPh in E of), the intensity of the emission peak of the PBS suspension ( Figure 1 "PT2PyPh + S. aureus" in D of and Figure 1 "PO2PyPh + S. aureus" in E of) was significantly enhanced. This was mainly due to the fluorescence enhancement caused by the restriction of intramolecular rotation after the compound molecules bind to bacteria, indicating that the compounds have a strong affinity for bacteria.

[0078] Dissolve RB (Rose Bengal) or the compound in DMSO to prepare a second mother liquor with a concentration of 10 mM. Take 1 μL of the second mother liquor and add it to 999 μL of PBS (pH = 7.4) containing 50 μM 9,10-anthracenediyl-bis(methylene)dimalonic acid (ROS indicator, ABDA) to prepare 1 mL of the test solution. The final concentration of RB or the compound in the test solution is 10 μM, and the final concentration of the ROS indicator is approximately 50 μM. The compound is PT2PyPh or PO2PyPh. Irradiate the test solution with a white light source of 10 mW / cm 2 . Test the absorption of the test solution with a UV-Vis spectrometer at time points of 0, 30, 60, 90, 120, 150, and 180 s respectively. As can be seen from Figure 2 A-C and G, under the same conditions, the absorption peak of the reactive oxygen species indicator ABDA in the test solutions containing PT2PyPh and PO2PyPh decreased rapidly, while the absorption peak of ABDA in the test solution containing RB did not show an obvious decrease, indicating that PT2PyPh and PO2PyPh have a high ROS production rate. It was observed that under white light irradiation of 10 mW / cm 2 , the intensity of the absorption peak (488 nm) of PT2PyPh in the test solution gradually decreased, while the intensity of the absorption peak (495 nm) of PO2PyPh did not change significantly under the same conditions, indicating that PO2PyPh has better photostability ( Figure 2 I).

[0079] To further verify this conclusion, another reactive oxygen species indicator was selected for testing. Dissolve RB (Rose Bengal) or the compound in DMSO to prepare a third mother liquor with a concentration of 10 mM. Take 1 μL of the third mother liquor and add it to 999 μL of PBS containing 50 μM 2`,7`-dichlorodihydrofluorescein (DCFH) to prepare 1 mL of the test solution. DCFH will be oxidized by ROS to produce DCF and emit green fluorescence; the fluorescence intensity will gradually increase with the increase of ROS in the test solution. Therefore, use an FS5 fluorescence spectrometer to test the fluorescence of the test solution irradiated with white light (10 mW cm -2 ) at different time intervals (0, 30, 60, 90, 120, 150, and 180 s) to evaluate the ROS generation efficiency of the test solution. As shown in Figure 2 D, E, and F. As can be seen from Figure 2 D-F and H, under the same conditions, the fluorescence in the test solutions containing PT2PyPh and PO2PyPh increased significantly, while the fluorescence intensity in the test solution containing RB increased less.

[0080] Furthermore, the binding ability of PO2PyPh to Gram-positive bacteria / Gram-negative bacteria was studied by confocal microscopy. To verify whether PO2PyPh has good bacterial binding ability, a bacterium P. aeruginosa with poor membrane penetration and difficult imaging was also selected as the research object of this experiment. 1 μL of the PO2PyP DMSO mother liquor with a concentration of 10 mM was added to 1 mL of PBS containing 10 8 CFU of S. aureus, E. coli or P. aeruginosa respectively, so that the final concentration of PO2PyPh was 10 μM, and the test solutions were obtained. After incubation for 5 minutes, 30 μL of the test solution was taken from each group and dropped on a glass slide, sealed with a coverslip, and the imaging effect was observed with a confocal microscope. As can be seen from Figure 3 A, PO2PyPh can clearly image S. aureus, E. coli and P. aeruginosa, and the imaging edges of the bacteria are clear, with good contrast. This shows that PO2PyPh can effectively bind to bacteria.

[0081] In view of the good photostability, high ROS generation efficiency and good bacterial binding ability of PO2PyPh, its photodynamic bactericidal effect was further evaluated. Methicillin-resistant Staphylococcus aureus (MRSA), kanamycin-resistant Escherichia coli (KREC) and P. aeruginosa were selected as the research targets. 1 μL of the PO2PyP DMSO mother liquor with a concentration of 10 mM was added to 1 mL of PBS containing 10 6 CFU of the drug-resistant bacteria respectively, so that the final concentrations of PO2PyPh were 0.1, 0.2, 0.5, 1, 2, 5, 10 and 20 μM, and multiple first incubation solutions with different PO2PyPh concentrations were obtained. In addition, 1 mL of PBS containing 10 6 CFU of the drug-resistant bacteria was prepared as the second incubation solution. After incubating the second incubation solution and multiple first incubation solutions for 5 min each, the mixed solutions of each group were irradiated with a white light source of 30 mW / cm 2 for 10 min. After the irradiation, 30 μL was taken from each mixed solution, and the number of colonies in the mixed solution was counted by the plate counting method. The results are shown in Figure 3 B-D. The results show that with the increase of the concentration of PO2PyPh, the number of surviving bacteria in the mixed solution decreased significantly, indicating that PO2PyPh has a photodynamic killing effect on MRSA, KREC and P. aeruginosa.

[0082] The diagnosis of bacterial infections remains a major challenge in the medical field. Despite the development of many contrast agents for bacterial imaging, their clinical impact has been minimal. This is mainly due to their inability to detect small amounts of bacteria and image Gram-negative or drug-resistant bacteria with low membrane permeability. Pseudomonas aeruginosa is a bacterium that can cause severe and often life-threatening infections, especially in immunocompromised individuals or patients with cystic fibrosis. Infections caused by Pseudomonas aeruginosa are often challenging due to its poor membrane permeability and biofilm formation. This makes it resistant to most antimicrobial therapies and immune responses, leading to its evasion of treatment. PT2PyPh and PO2PyPh, as photosensitizers, can improve membrane penetration ability through rational molecular design, effectively label and kill Pseudomonas aeruginosa.

[0083] The above provides an exemplary description of the present invention. It should be noted that any simple deformation, modification, or equivalent substitution that can be made by those skilled in the art without creative effort falls within the protection scope of the present invention without departing from the core of the present invention.

Claims

1. A compound, characterized in that, its structural formula is: Wherein each R is independent, and R is H, alkyl, unsaturated alkyl, heteroatom-containing alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy or one or more chromophores capable of conjugating with one or more fluorescent substances.

2. A method for preparing a compound, characterized in that, it comprises the following steps: S1. Preparation of compound a1: Mix the reactant, biphenyl halide, Pd(OAc) 2 , the first base and t-Bu 3 P·HBF 4 . Add toluene to dissolve it. Under a nitrogen or inert gas atmosphere, react at 90 - 110 °C for 10 - 15 h. After the reaction, cool to room temperature, pour it into ethyl acetate, and extract with saturated brine. Dry the organic phase and rotary evaporate to obtain the crude product. Purify the crude product to obtain a white to light yellow solid as compound a1. Among them, calculated by the number of moles, the ratio of the reactant, biphenyl halide, Pd(OAc) 2 and the first base is 10:(10 - 15):(0.2 - 1):(10 - 15). Calculated by the number of moles, the ratio of Pd(OAc) 2 and t-Bu 3 P·HBF 4 is 1:

2. The structural formula of the reactant is as follows: X is S or O; S2. Preparation of compound a2: Mix compound a1 and DCM, cool to 0 - 4 °C to obtain a first solution, dissolve N-bromosuccinimide in DCM to obtain a second solution, add the second solution dropwise to the first solution, stir in the dark at 0 - 4 °C for 0.5 - 2 hours, warm to room temperature and stir in the dark for 10 - 15 h, quench the reaction with water, extract with DCM, wash the organic layer with water and dry, evaporate the solvent under reduced pressure, and purify to obtain compound a2, wherein, calculated by the number of moles, the ratio of compound a1 to N-bromosuccinimide is 5:(10 - 11); S3. Preparation of compound a3: Mix compound a2, the second base, and pyridine-4-boronic acid, add Pd(PPh 3 ) 4 as a catalyst, then after adding an aqueous solution of 1,4-dioxane, stir at 90-105 °C for 10-15 h under the protection of nitrogen or an inert gas. After cooling to room temperature, pour it into ethyl acetate, extract with water, dry the obtained organic layer and evaporate under reduced pressure, and purify to obtain an orange-yellow solid as compound a3. Among them, by the number of moles, the ratio of compound a2 to pyridine-4-boronic acid is 2:(4-6), and by the number of moles, the ratio of pyridine-4-boronic acid to the second base is 1:(1-1.5); S4. Preparation of the compound: Dissolve compound a3 in ACN, under the protection of nitrogen or inert gas, add methyl iodide, stir at 70 - 80 °C for 10 - 15 h, remove the solvent under reduced pressure to obtain a crude orange solid, add the crude product to the minimum amount of good solvent that can dissolve it, and then add a poor solvent to obtain an orange precipitate as the compound, wherein, calculated by the number of moles, the ratio of compound a3 to methyl iodide is 1:(2 - 2.5).

3. The preparation method according to claim 2, characterized in that, in S1, the biphenyl halide is 4-bromobiphenyl or 4-iodobiphenyl.

4. The preparation method according to claim 2, characterized in that, in S1, the first base is potassium tert-butoxide, sodium tert-butoxide, potassium acetate or sodium acetate.

5. The preparation method according to claim 2, characterized in that, In S3, the second base is K 2 CO 3 , Na 2 CO 3 , potassium acetate, cesium carbonate or sodium acetate.

6. The preparation method according to claim 2, characterized in that, In S3, by the number of moles of the components, the ratio of the compound a2 to Pd(PPh 3 ) 4 is 2:(0.1 - 0.2).

7. The preparation method according to claim 2, characterized in that, in S3, the 1,4-dioxane aqueous solution is a mixture of 1,4-dioxane and water, and calculated by volume, the ratio of 1,4-dioxane to water in the 1,4-dioxane aqueous solution is (4 - 5):

1.

8. The preparation method according to claim 2, characterized in that, in S2, the concentration of compound a1 in the first solution is 0.1 - 0.3 mmol / mL, and the concentration of N-bromosuccinimide in the second solution is 0.3 - 0.5 mmol / mL.

9. The preparation method according to claim 2, characterized in that, in S3, the ratio of the number of moles of compound a2 to the volume of the 1,4-dioxane aqueous solution is less than or equal to 2:20, the unit of the number of moles is mmol, and the unit of the volume is mL.

10. Use of one of the compound according to claim 1 and the compound obtained by the preparation method according to claims 2 - 9 as a photosensitizer to increase the ROS generation rate.

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