Use of benzindole derivatives for the preparation of antibacterial agents and antibacterial agents

Abstract

The application provides application of a benzindole derivative in preparation of an antibacterial agent and the antibacterial agent, and relates to the technical field of compound application. The application provides application of the benzindole derivative in preparation of the antibacterial agent, the benzindole derivative with the structure shown in formula I has good bactericidal activity on methicillin-resistant staphylococcus aureus; can induce excessive expression of ROS in bacteria, and then destroy bacterial cell membranes, inhibit ATP synthesis and expression of bacterial virulence factors; can inhibit biofilm formation, and can remove the formed biofilm; can effectively remove bacteria in a skin wound, reduce wound inflammatory reactions and promote wound healing.

Description

Technical Field

[0001] This invention relates to the field of compound application technology, specifically to the application of benzoindole derivatives in the preparation of antibacterial agents and antibacterial agents. Background Technology

[0002] Antibiotics are one of the greatest achievements in the history of medicine, effectively suppressing bacterial infections, saving tens of millions of lives, and significantly reducing mortality rates. However, due to the overuse and abuse of antibiotics, bacterial resistance is constantly increasing, posing a huge challenge to the livestock and healthcare industries worldwide. According to the World Health Organization, almost all infectious disease pathogens that cause death are resistant to antibiotics. To address the increasingly serious problem of bacterial resistance, there is an urgent need to find and develop bactericides to combat drug-resistant bacteria.

[0003] The development and approval of novel bactericides are difficult and time-consuming, making it difficult to alleviate the pressing problem of bacterial resistance. Repurposing existing drugs is currently an effective means to quickly address resistance. Benzo[cd]indol-2(1H)-one is an important molecular skeleton, commonly found in the structures of drugs, natural products, and bioactive compounds. The derivative 6-nitrobenzo[cd]indole-2(1H)-ketone is an intermediate for BET bromodomain inhibitors. Currently, no bactericidal activity has been reported for 6-nitrobenzo[cd]indole-2(1H)-ketone. Summary of the Invention

[0004] The purpose of this invention is to provide the application of benzoindole derivatives in the preparation of antibacterial agents and the antibacterial agents themselves. This invention is the first to test the bactericidal activity of 6-nitrobenzo[cd]indole-2(1H)-ketone (a benzoindole derivative having the structure shown in Formula I), which has good bactericidal activity against methicillin-resistant Staphylococcus aureus.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of benzoindole derivatives in the preparation of antibacterial agents, wherein the benzoindole derivatives have the structure shown in Formula I:

[0007]

[0008] Preferably, the method for preparing a benzoindole derivative having the structure shown in Formula I includes the following steps:

[0009] A nitration reaction was carried out by mixing benzo[c,d]indol-2(1H)-one, glacial acetic acid, and nitric acid to obtain the reaction system;

[0010] The reaction system was mixed with ice water, and the resulting precipitate was washed and dried sequentially to obtain a benzoindole derivative with the structure shown in Formula I.

[0011] Preferably, the mass ratio of benzo[c,d]indol-2(1H)-one to glacial acetic acid is 1:6 to 8; the mass ratio of benzo[c,d]indol-2(1H)-one to nitric acid is 1:0.1 to 1.5; and the concentration of nitric acid is 65 wt%.

[0012] Preferably, the nitration reaction is carried out at room temperature and the nitration reaction takes 20 to 24 hours.

[0013] Preferably, the washing solution used is water; the drying temperature is 40-60°C, and the drying time is 20-24 hours.

[0014] Preferably, the antibacterial agent includes a drug against Staphylococcus aureus.

[0015] Preferably, the antibacterial agent is administered via subcutaneous injection; the dosage is 0.5–1.5 mg / kg. -1 .

[0016] The present invention provides an antibacterial agent comprising an active ingredient and pharmaceutically acceptable excipients; said active ingredient comprising a benzoindole derivative having the structure shown in Formula I.

[0017] This invention provides the application of benzoindole derivatives in the preparation of antibacterial agents. The small molecule compound 6-nitrobenzo[cd]indole-2(1H)-ketone (a benzoindole derivative having the structure shown in Formula I) exhibits good bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA). The benzoindole derivative having the structure shown in Formula I can induce excessive expression of ROS in bacteria, thereby disrupting the bacterial cell membrane, inhibiting ATP synthesis, and suppressing the expression of bacterial virulence factors. The benzoindole derivative having the structure shown in Formula I can inhibit biofilm formation and can remove existing biofilms. More importantly, the benzoindole derivative having the structure shown in Formula I can effectively clear bacteria from skin wounds, reduce wound inflammation, and promote wound healing. Attached Figure Description

[0018] Figure 1 The kinetic curve of C2 is shown.

[0019] Figure 2 This is the result of C2-induced ROS production in bacteria;

[0020] Figure 3 A graph showing the reversal of C2-induced bacterial death by the ROS scavenger thiourea;

[0021] Figure 4 This is the result of C2 damaging the bacterial cell membrane;

[0022] Figure 5 A bar chart showing the inhibition of bacterial ATP production by C2;

[0023] Figure 6 A bar chart showing the inhibition of virulence factor expression by C2;

[0024] Figure 7 This is a result of C2 inhibiting biofilm formation;

[0025] Figure 8 The result of C2 removing the already formed biofilm;

[0026] Figure 9 This is a result of C2 promoting wound healing;

[0027] Figure 10 Effects of C2 treatment on major organs after 3 days;

[0028] Figure 11 The result of C2 clearing biofilm from the wound;

[0029] Figure 12 Effects of C2 treatment on major organs for 8 days. Detailed Implementation

[0030] This invention provides the application of benzoindole derivatives in the preparation of antibacterial agents, wherein the benzoindole derivatives have the structure shown in Formula I:

[0031]

[0032] In this invention, the antibacterial agent preferably includes a drug against Staphylococcus aureus, and more preferably a drug against methicillin-resistant Staphylococcus aureus.

[0033] In this invention, the method for preparing the benzoindole derivative having the structure shown in Formula I preferably includes the following steps:

[0034] A nitration reaction was carried out by mixing benzo[c,d]indol-2(1H)-one, glacial acetic acid, and nitric acid to obtain the reaction system;

[0035] The reaction system was mixed with ice water, and the resulting precipitate was washed and dried sequentially to obtain a benzoindole derivative with the structure shown in Formula I.

[0036] In this invention, benzo[c,d]indole-2(1H)-one, glacial acetic acid, and nitric acid are mixed and subjected to a nitration reaction to obtain the reaction system.

[0037] In this invention, the structural formula of the benzo[c,d]indole-2(1H)-one is shown in Formula II:

[0038]

[0039] In this invention, the mass ratio of benzo[c,d]indole-2(1H)-one to glacial acetic acid is preferably 1:6 to 8, more preferably 1:7; the mass ratio of benzo[c,d]indole-2(1H)-one to nitric acid is preferably 1:0.1 to 1.5, more preferably 1:0.5 to 1.0; and the concentration of nitric acid is preferably 65 wt%.

[0040] In this invention, the nitration reaction is preferably carried out at room temperature; the nitration reaction is preferably carried out for 20 to 24 hours. In this invention, the nitration reaction is preferably carried out under stirring conditions.

[0041] After obtaining the reaction system, the present invention mixes the reaction system with ice water, and the resulting precipitate is washed and dried in sequence to obtain a benzoindole derivative having the structure shown in Formula I.

[0042] In this invention, the washing solution used is preferably water, more preferably distilled water; the number of washing cycles is preferably 2 to 4, more preferably 3. The drying temperature is preferably 40 to 60°C, more preferably 50°C, and the drying time is preferably 20 to 24 hours.

[0043] In this invention, the preferred method of administration of the antibacterial agent is subcutaneous injection; the preferred dosage is 0.5–1.5 mg / kg. -1 More preferably 1 mg·kg -1 The dosage is calculated in terms of the amount of benzoindole derivative.

[0044] This invention provides an antibacterial agent comprising an active ingredient and pharmaceutically acceptable excipients; the active ingredient comprises a benzoindole derivative having the structure shown in Formula I. This invention does not impose any special requirements on the pharmaceutically acceptable excipients; any pharmaceutically acceptable excipients well known to those skilled in the art can be used.

[0045] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0046] Example 1

[0047] 4.25 g of benzo[c,d]indole-2(1H)-one (C1) was dissolved in 30 mL of glacial acetic acid, and 2.1 mL of nitric acid (65 wt%) was slowly added. The solution was then stirred at room temperature and reacted for 24 hours. After the reaction was completed, the solution was poured into 300 mL of ice water to obtain a large amount of precipitate. The precipitate was filtered under vacuum, washed three times with distilled water, and dried at 50 °C for 24 h to obtain a brown solid, which is the benzoindole derivative with the structure shown in Formula I, denoted as C2.

[0048]

[0049] Dissolve C2 in dimethyl sulfoxide (DMSO) to a concentration of 10 mM (mmol / L) to obtain a C2 solution for later use.

[0050] The characterization results for C2 are as follows:

[0051] 1 H NMR (400MHz, DMSO-d6) δ11.38(s,1H),8.84(d,J=8.4Hz,1H),8.60(d,J=8.0Hz, 1H), 8.15 (d, J=7.0Hz, 1H), 8.03 (dd, J=8.4, 7.1Hz, 1H), 7.09 (d, J=8.0Hz, 1H). 13 CNMR(400MHz,DMSO-d6)δ169.37,145.90,138.19,132.98,131.37,129.53,126.93,126.10,125.77,122.46,105.42.[MH] - =213.0305, found:213.0311.

[0052] Example 2

[0053] 2.1 Minimum inhibitory concentration (MIC) detection

[0054] (1) Dilute the methicillin-resistant Staphylococcus aureus (MRSA) suspension to 10. 6 cfu·mL-1 The resulting MRSA bacterial suspension was used to determine the MIC and for subsequent test results.

[0055] (2) Dilute the C2 solution prepared in Example 1 to different concentrations using LB liquid medium: 2048 μg·mL -1 1024 μg·mL -1 512 μg·mL -1 256 μg·mL -1 128 μg·mL -1 64 μg·mL -1 32 μg·mL -1 16 μg·mL -1 8 μg·mL -1 4 μg·mL -1 2 μg·mL -1 1 μg·mL -1 0.5 μg·mL -1 0.25 μg·mL -1 .

[0056] (3) Add 100 μL of the above-mentioned culture medium containing different concentrations of C2 to a 96-well plate, and then add 100 μL of bacterial suspension (10 6 cfu·mL -1 Two control groups were set up: one group with 200 μL of culture medium and the other group with 100 μL of culture medium + 100 μL of bacterial suspension. Each concentration was tested in triplicate. The mixture was incubated at 37°C with shaking for 20 hours. After incubation, the results were observed; the concentration at which no significant bacterial proliferation was observed was defined as the MIC of C2. As shown in Table 1, the MIC of C2 was 4 μg·mL⁻¹. -1 .

[0057] 2.2 Minimum bactericidal concentration (MBC) detection

[0058] Add 100 μL of C2-containing culture medium with low turbidity (10 μL) to 100 μL of bacterial suspension (10 6 cfu·mL -1 (10 μL) was evenly spread onto an agar plate. The agar plates were incubated at 37°C for 24 hours. The concentration at which no bacteria grew (no colonies) was C2 of MBC. The results are shown in Table 1. The concentration of MBC at C2 was 8 μg / mL. -1 The MBC / MIC ratio of 2 indicates that C2 exhibits bactericidal activity against MRSA.

[0059] Table 1. MIC and MBC values ​​of compound C2 for MRSA

[0060] C2 4 8 2 Vancomycin 1 2 2 Methicillin 64 128 2

[0061] Example 3

[0062] C2 bactericidal kinetics analysis

[0063] MRSA bacterial suspensions were co-incubated with different concentrations of C2 (0.5×MIC, 1×MIC, 2×MIC, and 4×MIC) for 0, 2, 4, 8, 12, 18, and 24 hours. After treatment, the treated bacterial suspensions were diluted and spread onto agar plates. After incubation at 37°C for 24 hours, the number of colonies on the plates was counted and expressed as CFU / mL. Results are as follows: Figure 1 As shown, Figure 1 The "Van 4×MIC" indicates "vancomycin 4×MIC". The C2 of 4×MIC can kill all bacteria within 2 hours.

[0064] Example 4

[0065] ROS detection

[0066] 4.1 Flow cytometry detection of ROS content in bacteria

[0067] MRSA bacterial suspension was mixed with different concentrations of C2 (0 μg·mL⁻¹). -1 4 μg·mL -1 6 μg·mL -1 8 μg·mL -1 The bacteria were incubated for 2 hours. The treated bacteria were then incubated with the ROS probe DCFH-DA (10 μM) for 20 minutes. The ROS content in the bacteria was detected by flow cytometry. Results are as follows: Figure 2 As shown in Figure A, C2 treatment significantly increased intracellular ROS.

[0068] 4.2 Detection of ROS content in bacteria using fluorescence microscopy

[0069] MRSA bacterial suspension was mixed with different concentrations of C2 (0 μg·mL⁻¹). -1 4 μg·mL -1 8 μg·mL -1 The bacteria were incubated for 2 hours. The treated bacteria were then incubated with the ROS probe DCFH-DA (10 μM) for 20 minutes. The ROS content within the bacteria was detected using a fluorescence microscope. The results are as follows: Figure 2 As shown in Figure B, the green fluorescence in the cells was significantly enhanced after C2 treatment, indicating that C2 treatment significantly increased ROS in the bacteria.

[0070] Example 5

[0071] The effect of ROS scavengers on bacterial viability

[0072] MRSA bacterial suspension was mixed with C2 (4 μg·mL) -1 The bacterial cultures were co-incubated with thiourea (10 mM) and C2+ thiourea for 24 hours each. The bacterial suspensions were then diluted and spread onto agar plates. After incubation at 37°C for 24 hours, colonies were counted and expressed as CFU / mL. Results are as follows: Figure 3 As shown, Figure 3 "Thiourea" represents thiourea, and "C2+thiourea" represents C2+thiourea. The ROS scavenger thiourea reversed C2-induced bacterial death, indicating that C2 kills bacteria by inducing ROS production.

[0073] Example 6

[0074] Bacterial cell membrane analysis

[0075] 6.1 Cell membrane integrity analysis (SYTO9 / PI staining analysis)

[0076] C2 (0 μg·mL) at different concentrations -1 2 μg·mL -1 4 μg·mL -1 8 μg·mL -1 MRSA bacterial suspension was treated for 6 hours, and bacteria were collected. The bacteria were stained with propidium iodide (PI, 20 μM) and SYTO9 (3.34 μM) in the dark for 15 minutes and imaged using a confocal microscope. Results are as follows: Figure 4 As shown in Figure A, "Merge" indicates color merging. The red fluorescence (PI) gradually increases with increasing C2 concentration, indicating that C2 can significantly disrupt bacterial cell membranes.

[0077] 6.2 Cell membrane potential analysis

[0078] C2 (0 μg·mL) at different concentrations -1 2 μg·mL -1 4 μg·mL -1 8 μg·mL -1 MRSA bacterial suspension was treated for 6 hours, and bacteria were collected. The bacteria were stained with a DiBAC4(3) fluorescent probe in the dark for 30 minutes, and the fluorescence intensity was detected using a fluorescent microplate reader. Results are as follows: Figure 4 Figure B shows that the fluorescence intensity gradually decreases with increasing C2 concentration, indicating that C2 causes a change in the membrane potential of the cell membrane, further demonstrating that C2 disrupts the bacterial cell membrane.

[0079] Example 7

[0080] ATP detection

[0081] MRSA bacterial suspension was mixed with different concentrations of C2 (0 μg·mL⁻¹).-1 2 μg·mL -1 4 μg·mL -1 8 μg·mL -1 The bacteria were incubated for a total of 6 hours. The ATP level in the bacteria was detected using an ATP assay kit. The results are as follows: Figure 5 As shown, C2 can significantly inhibit the production of ATP in bacteria.

[0082] Example 8

[0083] Virulence factor detection

[0084] 8.1 RNA Extraction and Reverse Transcription

[0085] MRSA bacterial suspension was mixed with C2 (8 μg·mL) -1 Incubate for 10 hours. Extract total RNA from MRSA using an RNA extraction kit. Reverse transcribe 1 μg of total RNA into cDNA using a reverse transcription kit.

[0086] 8.2 Real-time quantitative PCR (qPCR)

[0087] The real-time quantitative PCR amplification system is shown in Table 2.

[0088] Table 2 Real-time quantitative PCR amplification system

[0089]

[0090] The amplification primer sequences are shown in Table 3.

[0091] Table 3 Amplification Primer Sequences

[0092]

[0093] Reaction procedure: ① 94℃ for 30 seconds; ② 94℃ for 5 seconds; ③ 60℃ for 30 seconds; ②-③ repeat for 40 cycles.

[0094] Triple replicates were used, and 16S rRNA mRNA levels were used as internal controls in all experiments. Relative expression levels were determined using the Ct assay. Results are as follows: Figure 6 As shown, "Ctrl" represents the control group DMSO. C2 significantly inhibited the expression of virulence factors α-haemolysin and agrA.

[0095] Example 9

[0096] Biofilm formation analysis

[0097] 9.1 Biofilm formation

[0098] (1) The MRSA bacterial suspension was cultured in LB liquid medium overnight at 180 rpm and 37°C.

[0099] (2) Dilute the cultured bacterial solution and add it to a 24-well glass plate with a flat bottom. Incubate at 37°C for 48 hours to form a biofilm. Remove the old culture medium and add fresh culture medium every 24 hours.

[0100] 9.2 Quantitative Analysis of Biofilms

[0101] (1) Mix MRSA bacterial suspension with different concentrations (0 μg·mL) -1 1 μg·mL -1 2 μg·mL -1 4 μg·mL -1 8 μg·mL -1 16 μg·mL -1 The biofilms were co-incubated with C2 for different times (0 h, 2 h, 4 h, 6 h, 12 h, 24 h, 36 h, 48 h), with three replicates for each experiment. The biofilms were washed three times with PBS buffer at pH 7.4.

[0102] (2) Add 500 μL of glutaraldehyde solution (2.5 vol%) to fix for 10 minutes. Discard the glutaraldehyde solution and wash the biofilm with PBS buffer at pH 7.4.

[0103] (3) Add 500 μL of 0.1 wt% crystal violet solution and incubate for 10 minutes. Remove the crystal violet solution, wash with PBS buffer at pH 7.4, and air dry at room temperature.

[0104] (4) Add 500 μL of 30 vol% acetic acid and incubate for 30 minutes. Detect the absorbance (OD value) of the solution at 590 nm using a microplate reader.

[0105] The results are as follows Figure 7 As shown in Figures A through B, “Ctrl” represents the control group DMSO. Compound C2 significantly inhibited biofilm formation, exhibiting a concentration- and time-dependent effect.

[0106] 9.3 Crystal violet staining of biomembranes

[0107] MRSA bacterial suspension and different concentrations (0 μg·mL) were prepared. -1 2 μg·mL -1 4 μg·mL -1 8 μg·mL -1 16 μg·mL -1The biofilm was incubated with C2 for 48 hours. It was washed three times with PBS buffer (pH 7.4). 0.1 wt% crystal violet solution was added, and the biofilm was incubated for 10 minutes. The crystal violet solution was removed, and the biofilm was washed with PBS buffer (pH 7.4). The biofilm was observed and photographed under a microscope. Results are as follows: Figure 7 As shown in Figure C, compound C2 can significantly inhibit biofilm formation.

[0108] 9.4 Analysis of bacterial count in biofilms

[0109] MRSA bacterial suspension and C2 (4 μg·mL) -1 The cells were incubated for a total of 48 hours. The biofilm was washed three times with PBS buffer (pH 7.4). After washing, 200 μL of PBS buffer (pH 7.4) was added to each well, and the biofilm was separated by rotating the tip of a 200 μL pipette 15 times in each well. The biofilm was repeatedly pipetted to evenly disperse the scraped biofilm. The solution was serially diluted. Finally, 100 μL of the biofilm suspension was evenly spread onto an agar plate. The plate was placed in a 37°C incubator. After 24 hours, the number of colonies on the plate was counted and expressed as CFU / mL. The results are as follows: Figure 7 As shown in D, C2 treatment significantly inhibited the amount of bacteria in the biofilm.

[0110] Example 10

[0111] Biomembrane analysis

[0112] Biofilm formation

[0113] (1) The MRSA bacterial suspension was cultured in LB liquid medium overnight at 180 rpm and 37°C.

[0114] (2) Dilute the cultured bacterial solution and add it to a 24-well glass plate with a flat bottom. Incubate at 37°C for 36 hours to form a biofilm. Remove the old culture medium and add fresh culture medium every 24 hours.

[0115] 10.1 Quantitative Analysis of Biofilms

[0116] (1) The biofilm after 36 hours was subjected to different concentrations (0 μg·mL⁻¹) -1 1 μg·mL -1 2 μg·mL -1 4 μg·mL -1 8 μg·mL -1 16 μg·mL -1 The biofilm was incubated with C2 for 12 hours. Three replicates were set up for each concentration. The biofilm was washed three times with PBS buffer at pH 7.4.

[0117] (2) Add 500 μL of glutaraldehyde solution (2.5 vol%) to fix for 10 minutes. Discard the glutaraldehyde solution and wash the biofilm with PBS buffer at pH 7.4.

[0118] (3) Add 500 μL of 0.1 wt% crystal violet solution and incubate for 10 minutes. Remove the crystal violet solution, wash with PBS buffer at pH 7.4, and air dry at room temperature.

[0119] (4) Add 500 μL of 30 vol% acetic acid and incubate for 30 minutes. Detect the absorbance (OD value) of the solution at 590 nm using a microplate reader.

[0120] The results are as follows Figure 8 As shown in A, compound C2 can significantly reduce biofilm content, indicating that C2 can reduce the amount of biofilm that has already formed.

[0121] 10.2 Crystal violet staining of biomembranes

[0122] The biofilm after 36 hours was subjected to different concentrations (0 μg·mL⁻¹). -1 4 μg·mL -1 8 μg·mL -1 16 μg·mL -1 The biofilm was incubated with C2 for 12 hours. It was then washed three times with PBS buffer (pH 7.4). 0.1 wt% crystal violet solution was added, and the biofilm was incubated for 10 minutes. The crystal violet solution was removed, and the biofilm was washed with PBS buffer (pH 7.4). The biofilm was observed and photographed under a microscope. Results are as follows: Figure 8 As shown in B, compound C2 can significantly scavenge biofilms.

[0123] 10.3 Diacetic acid fluorescein (FDA) staining of biomembranes

[0124] Biofilms aged 36 hours were subjected to different concentrations (0 μg·mL⁻¹). -1 4 μg·mL -1 8 μg·mL -1 16 μg·mL -1 Incubate with C2 for 12 hours. Wash the biofilm three times with PBS buffer (pH 7.4). Add 500 μL of 20 μg / mL solution. -1 The FDA solution was incubated in the dark for 30 minutes. After washing twice with PBS buffer (pH 7.4), the three-dimensional structure of the biomembrane was observed and recorded under a confocal microscope. Results are as follows: Figure 8 The C-values ​​show that compound C2 can significantly reduce the volume of biofilms.

[0125] 10.4 Analysis of bacterial count in biofilms

[0126] The 36-hour-old biofilm was then treated with C2 (8 μg·mL⁻¹). -1 The biofilm was co-incubated for 12 hours. The biofilm was washed three times with PBS buffer (pH 7.4). After washing, 200 μL of PBS buffer (pH 7.4) was added to each well, and the biofilm was separated by rotating the tip of a 200 μL pipette 15 times in each well. The biofilm was repeatedly pipetted to evenly disperse the scraped biofilm. The solution was serially diluted. Finally, 100 μL of the biofilm suspension was evenly spread onto an agar plate. The plate was placed in a 37°C incubator. After 24 hours, the colonies on the plate were counted and expressed as CFU / mL. The results are as follows: Figure 8 As shown in D, C2 treatment can significantly inhibit the amount of bacteria encapsulated in the biofilm.

[0127] Example 11

[0128] Wound healing experiment

[0129] (1) Male ICR mice weighing approximately 20.0g were housed in a standardized animal room. After one week of acclimatization, a wound approximately 1 cm long was made on the back of the mouse using sterile surgical scissors, and 10 mg of iodine was injected into the wound. 7 A wound infection model was constructed using methicillin-resistant Staphylococcus aureus (MRSA).

[0130] (2) Mice with wounds were randomly divided into four groups (n=6). Then, the mice were subcutaneously injected with PBS buffer (pH 7.4, negative control), C2 solution, and vancomycin solution (positive control) (50 μL, 1 mg / kg). -1 The medication was administered once daily for three consecutive days. Wound changes were recorded daily using a camera. After three days, the mice were euthanized, and wound tissue and major organ tissues (heart, liver, spleen, lung, and kidney) were collected. Wound changes were recorded as follows. Figure 9 As shown in A, C2 can significantly accelerate wound healing.

[0131] (3) Add PBS buffer (pH 7.4) to the removed wound tissue and homogenize. Dilute the homogenate and spread it on agar plates for incubation. After 24 hours, count the colonies on the plates and express the colony count as CFU / mL. The results are as follows: Figure 9 As shown in B to C, treatment C2 can significantly inhibit the amount of bacteria in the wound.

[0132] (4) After surgical resection, the wound tissue and organ (liver, spleen, kidney, heart, lung) tissues were fixed overnight in 4% paraformaldehyde fixative, embedded in paraffin, sectioned (4 μm), and stained with hematoxylin and eosin (H&E). The tissues were observed and photographed under an optical microscope. Results are as follows: Figure 9 D, H&E tissue images show that C2 can significantly accelerate wound healing.

[0133] (5) Add PBS buffer (pH 7.4) to the removed wound tissue and homogenize. Centrifuge at 3000 rpm for 5 minutes, collect the supernatant, and use an ELISA kit to detect the levels of inflammatory factors TNF-α and IL-6 in the supernatant. Results are as follows: Figure 9 As shown in E-F, C2 can reduce the expression of inflammatory factors around the wound, indicating that C2 can reduce the inflammatory response around the wound.

[0134] (6) After surgical resection, tissues from the major organs (liver, spleen, kidney, heart, and lungs) of the mice were fixed overnight in 4% paraformaldehyde fixative, embedded in paraffin, sectioned (4 μm), and stained with hematoxylin and eosin (H&E). The tissues were observed and photographed under an optical microscope. Results are as follows: Figure 10 C2 treatment had virtually no effect on the major organs of mice.

[0135] Example 12

[0136] Wound biofilm removal experiment

[0137] (1) Male ICR mice weighing approximately 20.0g were housed in a standardized animal room. After one week of acclimatization, a wound approximately 1 cm long was made on the back of the mouse using sterile surgical scissors, and 10 mg of iodine was injected into the wound. 8 A model of wound infection with a biofilm-covered wound was created by a methicillin-resistant Staphylococcus aureus (MRSA) strain two days later.

[0138] (2) Mice were randomly divided into four groups (n=6). Then, mice were subcutaneously injected with PBS buffer (pH 7.4, negative control), C2 solution, and vancomycin solution (positive control) (50 μL, 1 mg / kg). -1 The medication was administered once daily for 8 consecutive days. Wound changes were recorded daily using a camera. After 8 days, the mice were euthanized, and wound tissue and major organ tissues (heart, liver, spleen, lungs, and kidneys) were harvested. Wound images are shown below. Figure 11 As shown in A, C2 can significantly remove biofilm and accelerate wound healing.

[0139] (3) After surgical resection, the wound tissue was fixed overnight in 4% paraformaldehyde fixative, embedded in paraffin, sectioned (4 μm), and stained with hematoxylin and eosin (H&E). The tissue was observed and photographed under an optical microscope. Results are as follows: Figure 11 B, H&E tissue images show that C2 can significantly accelerate wound healing.

[0140] (4) Add PBS buffer (pH 7.4) to the removed wound tissue and homogenize. Dilute the homogenate and spread it on agar plates for incubation. After 24 hours, count the colonies on the plates and express the colony count as CFU / mL. Results are as follows: Figure 11As shown in C to D, C2 can remove bacteria from the wound biofilm.

[0141] (5) Add PBS buffer (pH 7.4) to the removed wound tissue and homogenize. Centrifuge at 3000 rpm for 5 minutes, collect the supernatant, and use an ELISA kit to detect the levels of inflammatory factors TNF-α and IL-6 in the supernatant. Results are as follows: Figure 11 As shown in E-F, C2 can reduce the expression of inflammatory factors around the wound, indicating that C2 can reduce the inflammatory response around the biofilm-covered wound.

[0142] (6) After surgical resection, tissues from the major organs (heart, liver, spleen, lung, and kidney) were fixed overnight in 4% paraformaldehyde fixative, embedded in paraffin, sectioned (4 μm), and stained with hematoxylin and eosin (H&E). The tissues were observed and photographed under an optical microscope. Results are as follows: Figure 12 C2 treatment had virtually no effect on the major organs of mice.

[0143] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of benzoindole derivatives in the preparation of antibacterial agents, wherein the antibacterial agent is a drug against methicillin-resistant Staphylococcus aureus; The benzoindole derivative has the structure shown in Formula I: Equation I.

2. The application according to claim 1, characterized in that, The antibacterial agent is administered via subcutaneous injection; the dosage is 0.5~1.5 mg / kg. -1 .

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