A benzopyridazine derivative and its preparation method and application in photodynamic antibacterial drugs

Abstract

The present invention relates to the technical field of biochemical materials, and provides a benzopyridazine derivative and a preparation method thereof and an application thereof in a photodynamic antibacterial drug. The benzopyridazine derivative provided by the present invention has a structure shown in Formula I, is positively charged, can effectively bind to bacteria, has an aggregation-induced emission (AIE) effect, has a strong ability to generate active oxygen, has a good photodynamic sterilization effect, and has good biocompatibility; the results of the embodiment show that the benzopyridazine derivative of the present invention is used to photodynamically kill Staphylococcus aureus, and the sterilization rate is close to 100% when the concentration is 0.6 μM.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical materials, and in particular to a benzopyridazine derivative and a preparation method thereof and application thereof in photodynamic antibacterial drugs. Background Art

[0002] Photodynamic sterilization is a new non-thermal sterilization technology that uses photosensitizers to produce reactive oxygen species (ROS) under the irradiation of appropriate excitation light sources, causing oxidative damage to surrounding biological molecules (such as lipids, proteins, and nucleic acids), thereby killing pathogenic microorganisms.

[0003] Photosensitizers play a vital role in photodynamic sterilization. The performance of photosensitizers determines their absorption wavelength, light absorption efficiency, ROS yield, etc. At present, most photosensitizers are colored compounds that can absorb visible light to produce ROS. Commonly used photosensitizers include porphyrin compounds, phenothiazine compounds, riboflavin and curcumin. These compounds all have certain photodynamic sterilization properties, but the sterilization ability needs to be improved.

[0004] Benzopyridazine compounds are a class of derivatives formed with benzopyridazine as the core. This type of derivative can be used in herbicides. At the same time, because the pyridazine structure has a strong electron-deficient characteristic, it is also often used to synthesize photoelectric materials. However, there are currently very few reports on benzopyridazine derivatives with photodynamic antibacterial effects. Summary of the invention

[0005] In view of this, the present invention provides a benzopyridazine derivative and a preparation method thereof and application thereof in photodynamic antibacterial drugs. The benzopyridazine derivative provided by the present invention has high efficiency in generating reactive oxygen species (ROS), good antibacterial activity and has broad application prospects in photodynamic sterilization.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A benzopyridazine derivative, characterized in that it has a structure shown in Formula I:

[0008]

[0009] The present invention also provides a method for preparing the benzopyridazine derivatives described in the above scheme, comprising the following steps:

[0010] A compound having a structure shown in Formula II, diphenylacetylene, an iridium catalyst, an inorganic base, a compound containing a non-coordinating tetrafluoroborate, and an organic solvent are mixed, and a cyclization reaction is carried out under a protective atmosphere to obtain the benzopyridazine derivative;

[0011]

[0012] Preferably, the method for preparing the compound having the structure shown in Formula II comprises the following steps:

[0013] The p-bromoaniline, chloroalkane solvent and catalyst are mixed to carry out oxidative dimerization reaction to obtain a compound having a structure shown in Formula III; the catalyst is potassium permanganate and ferrous sulfate;

[0014]

[0015] The compound having the structure shown in formula III, diphenylamine, a palladium catalyst, tri-tert-butylphosphine tetrafluoroborate, an organic base and a benzene solvent are mixed to carry out a Buchwald-Hartwig coupling reaction to obtain a compound having the structure shown in formula II.

[0016] Preferably, the molar ratio of potassium permanganate to ferrous sulfate is 1:1-1.2; the molar ratio of p-bromoaniline to potassium permanganate is 1:2.5-3.5.

[0017] Preferably, the molar ratio of the compound having the structure shown in formula III to diphenylamine is 1:2-2.5; the molar ratio of the compound having the structure shown in formula III to the palladium catalyst is 1:0.05-0.08; the molar ratio of the compound having the structure shown in formula III to tri-tert-butylphosphine tetrafluoroborate is 1:0.08-0.12; the molar ratio of the compound having the structure shown in formula III to the organic base is 1:2.5-3.5.

[0018] Preferably, the temperature of the oxidative dimerization reaction is 30-50° C., and the time is 6-8 h; the temperature of the Buchwald-Hartwig coupling reaction is 95-105° C., and the time is 20-25 h.

[0019] Preferably, the iridium catalyst is dichloro(pentamethylcyclopentadiene)iridium(III) dimer; the inorganic base is copper acetate; and the compound containing non-coordinated tetrafluoroborate is silver tetrafluoroborate.

[0020] Preferably, the molar ratio of the compound having the structure shown in Formula II to diphenylacetylene is 1:1.2-1.8; the molar ratio of the compound having the structure shown in Formula II, the iridium catalyst, the inorganic base and the compound containing non-coordinating tetrafluoroborate is 1:0.05-0.2:0.05-0.2:1.5-2.5.

[0021] Preferably, the cyclization reaction is carried out at a temperature of 120 to 135° C. and for a time of 20 to 25 hours.

[0022] The present invention also provides the use of the benzopyridazine derivatives described in the above scheme or the benzopyridazine derivatives prepared by the preparation method described in the above scheme in the preparation of photodynamic antibacterial drugs.

[0023] The present invention provides a benzopyridazine derivative having a structure shown in Formula I. The molecular structure of the benzopyridazine derivative provided by the present invention has a benzopyridazine salt structure and a tetrafluoroborate anion, which can make the derivative positively charged, while the bacterial surface is negatively charged. Due to electrostatic interaction, the derivative can be effectively combined with the bacteria, which establishes a foundation for subsequent efficient sterilization; and the present invention introduces a diphenylamino group into the structure of the benzopyridazine derivative, which is a strong electron donor and can enhance the donor-acceptor interaction within the molecule, is conducive to the generation of active oxygen, and makes the benzopyridazine derivative have better antibacterial activity; the benzopyridazine derivative provided by the present invention has a strong ability to generate active oxygen and a good photodynamic sterilization effect. At the same time, the benzopyridazine derivative provided by the present invention gradually increases its fluorescence intensity as the water volume fraction in the solution increases, has an obvious aggregation-induced emission (AIE) effect, has a higher fluorescence intensity in an aggregated state, has a higher ROS generation efficiency, and has a better photodynamic sterilization effect.

[0024] The results of the examples show that when the benzopyridazine derivatives of the present invention are mixed with the active oxygen scavenger DCFH, under white light irradiation, the fluorescence intensity of DCFH gradually increases with the increase of illumination time and the enhancement rate is relatively fast, indicating that the benzopyridazine derivatives of the present invention have a high efficiency in generating active oxygen; when the benzopyridazine derivatives provided by the present invention are used for photodynamic antibacterial, the sterilization rate is close to 100% at a concentration of 0.6 μM; at the same time, the benzopyridazine derivatives provided by the present invention also have good biocompatibility, which helps to broaden its application range and is used to construct photodynamic antibacterial drugs with safe and efficient antibacterial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The fluorescence intensity test results of TPCL-DPA in DMSO / n-hexane mixed solutions with different n-hexane volume fractions;

[0026] Figure 2 The fluorescence emission spectrum of the mixed solution of TPCL-DPA and DCFH changes with the illumination time;

[0027] Figure 3 The photodynamic killing of Staphylococcus aureus by TPCL-DPA at different concentrations;

[0028] Figure 4 The phototoxicity and dark toxicity of different concentrations of TPCL-DPA on normal cells LO2. DETAILED DESCRIPTION

[0029] The present invention provides a benzopyridazine derivative having a structure shown in Formula I:

[0030]

[0031] The present invention also provides a method for preparing the benzopyridazine derivatives described in the above scheme, comprising the following steps:

[0032] The compound having the structure shown in Formula II is mixed with diphenylacetylene, an iridium catalyst, an inorganic base, a compound containing a non-coordinating tetrafluoroborate and an organic solvent, and a cyclization reaction is carried out under a protective atmosphere to obtain the benzopyridazine derivative;

[0033]

[0034] First, the preparation method of the compound having the structure shown in Formula II is described:

[0035] In the present invention, the method for preparing the compound having the structure shown in Formula II comprises the following steps:

[0036] The p-bromoaniline, chloroalkane solvent and catalyst are mixed to carry out oxidative dimerization reaction to obtain a compound having a structure shown in Formula III; the catalyst is potassium permanganate and ferrous sulfate;

[0037]

[0038] The compound having the structure shown in formula III, diphenylamine, a palladium catalyst, tri-tert-butylphosphine tetrafluoroborate, an organic base and a benzene solvent are mixed to carry out a Buchwald-Hartwig CrossCoupling Reaction to obtain a compound having the structure shown in formula II.

[0039] The present invention mixes p-bromoaniline, a chloroalkane solvent and a catalyst for oxidative dimerization reaction to obtain a compound having a structure shown in Formula III. In the present invention, the catalyst is potassium permanganate and ferrous sulfate; the ferrous sulfate is preferably ferrous sulfate heptahydrate; the molar ratio of potassium permanganate to ferrous sulfate is preferably 1:1-1.2, more preferably 1:1; the molar ratio of p-bromoaniline to potassium permanganate is preferably 1:2.5-3.5, more preferably 1:3; the chloroalkane solvent is preferably dichloromethane; the present invention preferably mixes p-bromoaniline and a chloroalkane solvent to obtain a p-bromoaniline solution, grinds and mixes potassium permanganate and ferrous sulfate, and adds the resulting mixture to the p-bromoaniline solution. In the present invention, the temperature of the oxidative dimerization reaction is preferably 30-50°C, more preferably 40°C, and the time is preferably 6-8h.

[0040] After the oxidative dimerization reaction is completed, the present invention preferably performs post-treatment on the obtained product liquid; the post-treatment preferably comprises the following steps: cooling the product liquid obtained by the oxidative dimerization reaction and diluting it with a chloroalkane solvent, filtering, concentrating and column chromatography purification of the dilution in sequence to obtain a compound having a structure shown in Formula III. In the present invention, the type of the chloroalkane used for dilution is consistent with the above scheme, and will not be repeated here; the filtration is preferably performed using diatomaceous earth, and after the filtration is completed, the diatomaceous earth is preferably rinsed with a chloroalkane solvent, the rinse liquid and the filtrate are combined, and the obtained mixed liquid is subsequently concentrated; the concentration is preferably vacuum concentration; the eluent for column chromatography purification is preferably petroleum ether; the compound having a structure shown in Formula III obtained after purification is an orange-yellow solid.

[0041] After obtaining the compound having the structure shown in formula III, the present invention mixes the compound having the structure shown in formula III, diphenylamine, a palladium catalyst, tri-tert-butylphosphine tetrafluoroborate, an organic base and a benzene solvent to carry out a Buchwald-Hartwig coupling reaction to obtain a compound having the structure shown in formula II. In the present invention, the palladium catalyst is preferably tri(dibenzylideneacetone)dipalladium; the organic base is preferably sodium tert-butoxide, and the role of the organic base is to neutralize the HBr generated by the reaction; the role of the tri-tert-butylphosphine tetrafluoroborate is a ligand of the palladium catalyst; the benzene solvent is preferably toluene; the molar ratio of the compound having the structure shown in formula III to diphenylamine is preferably 1:2 to 2.5; the molar ratio of the compound having the structure shown in formula III to the palladium catalyst is preferably 1:0.05 to 0.08; the molar ratio of the compound having the structure shown in formula III to tri-tert-butylphosphine tetrafluoroborate is preferably 1:0.05 to 0.08. The ratio is preferably 1:0.08-0.12, more preferably 1:0.1; the molar ratio of the compound having the structure shown in formula III to the organic base is preferably 1:2.5-3.5, more preferably 1:3; the present invention has no special requirements on the amount of the benzene solvent, as long as the Buchwald-Hartwig coupling reaction can proceed smoothly; the Buchwald-Hartwig coupling temperature is preferably 95-105°C, more preferably 100°C, and the time is preferably 20-25h, more preferably 24h; the Buchwald-Hartwig coupling reaction is preferably carried out under nitrogen protection.

[0042] After the Buchwald-Hartwig coupling reaction is completed, the present invention preferably performs post-treatment on the obtained product liquid; the post-treatment preferably includes the following steps: cooling the product liquid obtained by the Buchwald-Hartwig coupling reaction to room temperature and then pouring it into methanol for first precipitation, filtering to obtain a first precipitate; washing the first precipitate with methanol, disodium ethylenediaminetetraacetic acid aqueous solution, water and n-hexane in sequence, and then performing a second precipitation in a chloroform-methanol mixed solution, filtering to obtain a second precipitate, and vacuum drying the second precipitate to obtain a compound having a structure shown in formula II; the temperature of the first precipitation is preferably room temperature, and the time is preferably 3 to 5 minutes; the temperature of the second precipitation is preferably room temperature, and the time is preferably 3 to 5 minutes; the volume ratio of chloroform and methanol in the chloroform-methanol mixed solution is preferably 15 to 25:1; the compound having a structure shown in formula II is a red powder.

[0043] In the present invention, the reaction formula of the oxidative dimerization reaction and the Buchwald-Hartwig coupling reaction is as follows:

[0044]

[0045] The preparation method of the benzopyridazine derivative is described in detail below:

[0046] The compound having the structure shown in formula II is mixed with diphenylacetylene, an iridium catalyst, an inorganic base, a compound containing non-coordinating tetrafluoroborate and an organic solvent, and a cyclization reaction is carried out under a protective atmosphere to obtain a benzopyridazine derivative having the structure shown in formula I. In the present invention, the iridium catalyst is preferably dichloro(pentamethylcyclopentadiene)iridium(III) dimer; the inorganic base is preferably copper acetate; the compound containing non-coordinating tetrafluoroborate is preferably silver tetrafluoroborate; the molar ratio of the compound having the structure shown in formula II to diphenylacetylene is preferably 1:1.2-1.8, more preferably 1:1.5; the molar ratio of the compound having the structure shown in formula II, the iridium catalyst, the inorganic base and the compound containing non-coordinating tetrafluoroborate is preferably 1:0.05-0.2:0.05-0.2:1.5-2.5, more preferably 1:0.1:0.1:2.

[0047] In the present invention, the temperature of the cyclization reaction is preferably 120-135° C., more preferably 125-130° C., and the time of the cyclization reaction is preferably 24 h; the cyclization reaction is preferably carried out in a thick-walled reaction bottle.

[0048] In a specific embodiment of the present invention, it is preferred to first add a compound having a structure shown in Formula II, diphenylacetylene, an iridium catalyst and an inorganic base into a thick-walled reaction bottle, then evacuate the reaction bottle, add a compound containing a non-coordinating tetrafluoroborate and an organic solvent under nitrogen protection, then tighten the mouth of the thick-walled reaction bottle, evacuate the bottle again, and then react at the cyclization reaction temperature.

[0049] In the present invention, the reaction formula of the cyclization reaction is as follows:

[0050]

[0051] In the present invention, after the cyclization reaction is completed, it is preferred to further include post-treatment of the obtained product liquid, and the post-treatment preferably includes the following steps: filtering, concentrating, and column chromatography of the product liquid in sequence to obtain a benzopyridazine derivative having a structure shown in Formula I; in the present invention, preferably after the reaction is completed, the product liquid is cooled to room temperature, and then filtered using diatomaceous earth, and the diatomaceous earth is rinsed with dichloromethane after the filtration is completed, and the rinse liquid and the filtrate are combined, and the combined liquid is used as the final subsequent concentration; the column chromatography eluent is preferably a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol in the mixture is preferably 20:1; after the column chromatography is completed, the present invention preferably removes the solvent in the column chromatography product. The present invention does not specifically limit the method of removing the solvent, and a conventional method of removing the solvent can be used, such as rotary evaporation.

[0052] The present invention also provides the use of the benzopyridazine derivatives described in the above scheme in the preparation of photodynamic antibacterial drugs; in the present invention, the benzopyridazine derivatives are preferably used as photosensitizers; the photodynamic antibacterial drugs are preferably anti-Gram-positive bacteria drugs; the Gram-positive bacteria are preferably Staphylococcus aureus; the excitation wavelength of the photodynamic antibacterial drugs is preferably 450-500nm. The benzopyridazine derivatives provided by the present invention have aggregation-induced emission (AIE) effect, can emit stronger fluorescence after aggregation in aqueous solution, and have strong reactive oxygen species (ROS) production ability. Under illumination conditions, a large amount of reactive oxygen species can be generated to photodynamically kill bacteria; and the benzopyridazine derivatives provided by the present invention have good biocompatibility and good safety.

[0053] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] Example 1

[0055] (1) Preparation of the compound having the structure shown in Formula III

[0056] Add p-bromoaniline (1mmol, 172.0mg) to an eggplant-shaped bottle and dissolve it in dichloromethane. Then, grind KMnO4 solid (3mmol, 474.1mg) and FeSO4·7H2O solid (3mmol, 834.0mg) in an agate mortar as a catalyst and add them to the eggplant-shaped bottle. Stir at 40°C for 6h. After the reaction is completed, cool it and dilute it with dichloromethane. The mixture is filtered through a diatomaceous earth pad. The diatomaceous earth pad is washed with dichloromethane (30mL), and the filtrate is concentrated under vacuum. Petroleum ether is used as an eluent, and the concentrate is subjected to column chromatography to obtain an orange-yellow solid, which is a compound having the structure shown in formula III, with a yield of 70%. 1 HNMR (500MHz, CDCl3) δ7.82 (d, J = 8.7 Hz, 4H), 7.68 (d, J = 8.7 Hz, 4H).

[0057] (2) Preparation of the compound having the structure shown in Formula II

[0058] In a two-necked round-bottom flask, a mixture of a compound having a structure shown in Formula III (1mmol, 340.0mg), diphenylamine (2mmol, 338.4mg), tri(dibenzylideneacetone)dipalladium (0.05mmol, 45.8mg), tri-tert-butylphosphine tetrafluoroborate (0.1mmol, 29.0mg), and sodium tert-butoxide (3mmol, 288.3mg) was added and dissolved in toluene (10mL), and reacted at 100°C for 24h under a nitrogen atmosphere. After cooling to room temperature, the reaction mixture was poured into methanol (200mL), and the precipitate was separated by filtration, washed with methanol, ethylenediaminetetraacetic acid disodium salt aqueous solution, water and n-hexane, reprecipitated from chloroform / methanol, and vacuum dried to obtain a red powder, i.e., a compound having a structure shown in Formula III, with a yield of 94%. 1 H NMR (500MHz, CDCl3) δ7.74 (d, J = 8.9Hz, 4H), 7.34–7.29 (m, 8H), 7.20–7.14 (m, 8H), 7.13–7.07 (m, 8H).

[0059] (3) Preparation of benzopyridazine derivatives of the structure shown in Formula I

[0060] The compound having the structure shown in formula II (1mmol, 516.6mg), diphenylacetylene (1.5mmol, 267.3mg), dichloro(pentamethylcyclopentadiene)iridium(III) dimer ([Cp*IrCl2]2, 0.1mmol, 79.7mg) and copper acetate (Cu(OAc)2, 0.1mmol, 18.2mg) were added to a thick-walled reaction bottle, and then the reaction device was ventilated. Silver tetrafluoroborate (AgBF4, 2mmol, 389.3mg) and 1,2-dichloroethane (10mL) were added under nitrogen protection, the thick-walled reaction bottle was tightened, and the venting operation was performed again, and it was stirred at 130°C for 24h. After the reaction was completed and cooled to room temperature, the mixture was filtered with diatomaceous earth, the diatomaceous earth was rinsed with dichloromethane (30mL), and the filtrate was concentrated. The concentrate was subjected to column chromatography using dichloromethane / methanol (20:1, v / v) as eluent to obtain the target product as a dark red solid with a yield of 50%. The structural identification data of the product are as follows:

[0061] 1 H NMR(500MHz,MeOD)δ8.30(d,J=9.7Hz,1H),7.80(dd,J=9.7,2.6Hz,1H),7.50(t,J=7.8Hz,4H),7.41-7.35(m,6H),7.35-7.28(m, 7H),7.24-7.20(m,7H),7.14(dd,J=10.4,4.5Hz,4H),7.02(dd,J=8.5,1.0Hz,4H),6.88(d,J=9.0Hz,2H),6.65(d,J=2.6Hz,1H). 13 C NMR (151MHz, CDCl3) δ155.15,149.07,146.65,145.01,143.55,140.20,137.54,133.00,132.11,131.83,131.22,131.16,1 30.23,129.97,129.52,129.21,128.62,128.53,128.18,128.04,127.65,127.61,126.89,125.22,124.16,121.40,107.07.

[0062] According to the above characterization data, the structural formula of the obtained product is as shown in Formula I, recorded as TPCL-DPA.

[0063] Performance Test:

[0064] (1) AIE property test of TPCL-DPA: n-Hexane is a poor solvent for TPCL-DPA. DMSO solution (1 mM) of TPCL-DPA was added to DMSO / n-hexane mixed solutions with different n-hexane volume fractions to obtain TPCL-DPA solutions with a concentration of 10 μM. The fluorescence intensity of TPCL-DPA in the mixed solutions with different n-hexane volume fractions was measured.

[0065] Figure 1 The fluorescence intensity test results of TPCL-DPA in DMSO / n-hexane mixed solutions with different n-hexane volume fractions (the corresponding n-hexane volume fractions from low to high are 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and the excitation light wavelength is 390nm). Figure 1 It can be seen that with the increase of n-hexane volume fraction, the fluorescence intensity of TPCL-DPA gradually increases, indicating that TPCL-DPA has AIE characteristics.

[0066] (2) Test of TPCL-DPA's ability to produce active oxygen: prepare the DMSO stock solution of the sample to be tested, take 20 μL of the sample to be tested and 50 μL of DCFH- and add them to a cuvette containing 2 mL of PBS buffer solution, so that the final test concentration of TPCL-DPA is 10 μM and the final concentration of DCFH is 40 μM. Use FL-4700 fluorescence spectrometer for testing, set the excitation wavelength to 489 nm, first measure the fluorescence spectrum in the absence of light (0 min), and then measure the fluorescence spectrum at the same interval (interval time is 5 s) to obtain the ratio of real-time luminescence intensity to initial luminescence intensity; at the same time, use a single DCFH solution (concentration of 40 μM) and a single TPCL-DMA solution (concentration of 10 μM) as controls. The results are as follows. Figure 2 shown.

[0067] Figure 2 The fluorescence emission spectrum of the mixed solution of TPCL-DPA and DCFH changes with the illumination time; Figure 2 It can be seen that with the increase of illumination time, the fluorescence intensity of the mixed solution gradually increases, indicating that TPCL-DPA has a strong ability to produce active oxygen.

[0068] (3) Test of the photodynamic killing ability of TPCL-DPA on Staphylococcus aureus. The specific steps are as follows:

[0069] The Staphylococcus aureus strain was revived and inoculated into LB liquid medium and cultured in a shaker at 37°C and 220 r / min for 16 h. At this time, the concentration of Staphylococcus aureus in the bacterial suspension was about 2×10 9CFU / mL; 1 mL of cultured Staphylococcus aureus was taken, the culture medium was removed, and it was washed with PBS; different volumes of DMSO solution of TPCL-DPA were added to make the final concentration of TPCL-DPA 0 μM, 0.2 μM, 0.4 μM, and 0.6 μM, respectively. The bacterial suspension was incubated in a shaker at 37°C and 220 r / min for 10 min, and then the bacterial suspension was illuminated for 20 min using a solar simulator equipped with a 400 nm filter; after the illumination, the bacterial suspension was gradiently diluted to 1×10 7 The cells were plated on LB agar solid medium and cultured in a 37°C incubator for 16 h before colony counting and survival rate calculation, which was used as the phototoxicity group. The cells were cultured and counted without light treatment, which was used as the dark toxicity group.

[0070] Figure 3 The photodynamic killing of Staphylococcus aureus by TPCL-DPA at different concentrations. Figure 3 It can be seen that with the increase of TPCL-DPA concentration, the survival rate of Staphylococcus aureus in the phototoxic group decreased significantly. When the TPCL-DPA concentration was 0.4 μM, the survival rate of Staphylococcus aureus in the phototoxic group was less than 5%. When the TPCL-DPA concentration was 0.6 μM, Staphylococcus aureus in the phototoxic group was basically killed. The survival rate of Staphylococcus aureus in the dark toxic group decreased slightly, indicating that TPCL-DPA has a certain photodynamic killing effect on Staphylococcus aureus.

[0071] (4) TPCL-DPA phototoxicity and dark toxicity test on normal cell LO2, the specific steps are as follows:

[0072] LO2 cells were seeded on 96-well plates and cultured in a humidified incubator at 37°C, 5% CO2, and 20% O2 in DMEM medium (containing 10% FBS and 1% penicillin / streptomycin). 3 The cells were seeded in a 96-well plate at a density of 10 cells and incubated for 24 hours. Subsequently, the culture medium was replaced with fresh culture medium containing different concentrations of TPCL-DPA (final concentrations were 0 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1 μM, 1.2 μM, respectively). After further incubation for 20 hours, the cells were exposed to white light irradiation for 30 minutes. At the same time, cells incubated with TCPL-DPA without white light irradiation were used as a dark control group. After further incubation for 4 hours, the culture medium was removed and washed 3 times with PBS. The cells were then incubated in the dark with fresh serum-free medium containing 10% CCK-8 for 1 hour. Finally, the absorbance of the product was measured at a wavelength of 450 nm using an enzyme reader. The results are expressed as the survival percentage of cells after different treatments relative to control cells without any treatment.

[0073] Figure 4 The phototoxicity and dark toxicity of TPCL-DPA at different concentrations on normal cells LO2. It can be seen from the figure that TPCL-DPA has no toxicity to normal cells LO2 under both light and dark conditions, and the cell survival rate is above 90%, indicating that TPCL-DPA is safe for normal cells.

[0074] It can be seen from the above examples that the benzopyridazine derivatives provided by the present invention have simple synthesis steps, have AIE properties, and have efficient active oxygen generation capabilities, have good photodynamic killing effects on Gram-positive bacteria, and also have good biocompatibility, and have broad application prospects in photodynamic antibacterial therapy.

[0075] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The use of benzopyridazine derivatives in the preparation of photodynamic antibacterial drugs, characterized in that: The benzopyridazine derivative has a structure shown in Formula I: The excitation wavelength of the photodynamic antibacterial drug is 450-500nm.

2. The use according to claim 1, characterized in that: The preparation method of the benzopyridazine derivative comprises the following steps: A compound having a structure shown in Formula II, diphenylacetylene, an iridium catalyst, an inorganic base, a compound containing a non-coordinating tetrafluoroborate, and an organic solvent are mixed, and a cyclization reaction is carried out under a protective atmosphere to obtain the benzopyridazine derivative; 3. The use according to claim 2, characterized in that: The method for preparing the compound having the structure shown in Formula II comprises the following steps: The p-bromoaniline, chloroalkane solvent and catalyst are mixed to carry out oxidative dimerization reaction to obtain a compound having a structure shown in Formula III; the catalyst is potassium permanganate and ferrous sulfate; The compound having the structure shown in formula III, diphenylamine, a palladium catalyst, tri-tert-butylphosphine tetrafluoroborate, an organic base and a benzene solvent are mixed to carry out a Buchwald-Hartwig coupling reaction to obtain a compound having the structure shown in formula II.

4. The use according to claim 3, characterized in that: The molar ratio of the potassium permanganate to ferrous sulfate is 1:1-1.2; the molar ratio of the p-bromoaniline to potassium permanganate is 1:2.5-3.

5.

5. The use according to claim 3, characterized in that: The molar ratio of the compound having the structure shown in formula III to diphenylamine is 1:2-2.5; the molar ratio of the compound having the structure shown in formula III to the palladium catalyst is 1:0.05-0.08; the molar ratio of the compound having the structure shown in formula III to tri-tert-butylphosphine tetrafluoroborate is 1:0.08-0.12; the molar ratio of the compound having the structure shown in formula III to the organic base is 1:2.5-3.

5.

6. The use according to claim 3, characterized in that: The temperature of the oxidative dimerization reaction is 30-50° C., and the time is 6-8 hours; the temperature of the Buchwald-Hartwig coupling reaction is 95-105° C., and the time is 20-25 hours.

7. The use according to claim 2, characterized in that: The iridium catalyst is dichloro(pentamethylcyclopentadiene)iridium(III) dimer; the inorganic base is copper acetate; and the compound containing non-coordinated tetrafluoroborate is silver tetrafluoroborate.

8. The use according to claim 2 or 3, characterized in that: The molar ratio of the compound having the structure shown in formula II to diphenylacetylene is 1:1.2-1.8; the molar ratio of the compound having the structure shown in formula II, iridium catalyst, inorganic base and compound containing non-coordinating tetrafluoroborate is 1:0.05-0.2:0.05-0.2:1.5-2.

5.

9. The use according to claim 2, characterized in that: The temperature of the cyclization reaction is 120-135° C. and the time is 20-25 hours.

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