Application of coumarin derivative in preparation of staphylococcus aureus inhibition drugs

Abstract

The invention provides an application of a coumarin derivative in preparation of a medicine for inhibiting staphylococcus aureus. The coumarin structure is subjected to bromine substitution, oxygen-containing group modification and halogen modification at specific positions, and shows a good inhibition effect on staphylococcus aureus; the minimum inhibitory concentration (MIC) on a staphylococcus aureus strain is 64 [mu] g / mL, the formation of a bacterial biofilm can be reduced, and certain cell compatibility is displayed; the specific antibacterial activity and application potential are realized.

Description

Technical Field

[0001] This application belongs to the fields of medicinal chemistry and anti-infective technology, and in particular relates to the application of a coumarin derivative in the preparation of a drug for inhibiting Staphylococcus aureus. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus Staphylococcus aureus (S. aureus) is an important group of Gram-positive opportunistic pathogens that can colonize various tissues, including the skin, nasal cavity, and respiratory tract. When the host's immunity is weakened or its barrier function is impaired, this bacterium readily invades and causes infection. Its pathogenic spectrum includes a variety of serious diseases such as superficial purulent skin infections, pneumonia, endocarditis, osteomyelitis, arthritis, and sepsis. Staphylococcus aureus infection is characterized by high incidence, serious complications, and poor prognosis, and is particularly dangerous in the elderly, immunocompromised patients, those in intensive care units, and patients undergoing implanted medical device treatment. It has become one of the most important pathogens threatening public health and medical safety worldwide.

[0003] Against the backdrop of an escalating global crisis of antibiotic resistance, there is an urgent need to develop antibacterial candidates with novel structures, unique mechanisms of action, and excellent safety profiles to overcome the limitations of existing drugs. Natural products and their derivatives, due to their rich chemical diversity and good biocompatibility, have always been an important source for the development of novel antibacterial drugs. Among them, coumarin (2H-1-benzopyran-2-one) skeletons are a class of heterocyclic compounds widely found in nature, derived from various plant metabolites. Numerous studies have shown that coumarins and their derivatives possess broad-spectrum activities in antibacterial, antiviral, anti-inflammatory, antitumor, and anticoagulant activity, demonstrating excellent development potential.

[0004] However, existing research on the antibacterial properties of coumarins still has significant shortcomings. First, the publicly disclosed coumarin derivatives have relatively limited structural types, with research mainly focusing on a few natural compounds with hydroxyl or methoxy substitutions, lacking systematic expansion into areas such as multi-site substitution, nitrogen-containing cationization modification, and heteroaryl or heterocyclic substituents. Second, the inhibitory effects of existing coumarin derivatives on Staphylococcus aureus are generally limited, with their minimum inhibitory concentrations (MICs) often at high levels, failing to meet clinical treatment needs. Third, some coumarin derivatives suffer from insufficient water solubility, poor stability, and unsatisfactory in vivo pharmacokinetic properties, severely restricting their further development as drug candidates. Furthermore, in-depth research on specific applications of coumarin compounds in medical device antibacterial coatings, biofilm inhibition, and local drug delivery systems is still lacking, and existing technologies have failed to provide comprehensive and feasible solutions.

[0005] While existing technologies have demonstrated the potential of coumarin derivatives in the antibacterial field to some extent, they have not yet effectively addressed key issues such as insufficient antibacterial activity, poor pharmacokinetic performance, and limitations in clinical application. Therefore, how to rationally modify the coumarin skeleton to obtain a class of novel compounds that possess potent antibacterial activity, excellent selectivity, safety, and favorable formulation properties is a crucial technical problem urgently needing to be solved in the current field of antibacterial drug development. Summary of the Invention

[0006] This application provides an application of a coumarin derivative in the preparation of a drug for inhibiting Staphylococcus aureus, in order to solve the problems existing in the related technology. The technical solution is as follows: In a first aspect, embodiments of this application provide the application of a coumarin derivative in the preparation of a drug for inhibiting Staphylococcus aureus, wherein the coumarin derivative has the structure shown in Formula I: ; R1 is a halogen atom, and R2 is an oxygen-containing group.

[0007] In one embodiment, R1 is a Br or Cl atom; R2 is a hydroxyl group or an alkoxy group with 1-3 carbon atoms.

[0008] In one embodiment, the coumarin derivative is any one of the following compounds: ; ; ; .

[0009] In one embodiment, the Staphylococcus aureus is ATCC 29213.

[0010] In one embodiment, the minimum inhibitory concentration of the coumarin derivative with the structure shown in Formula I against Staphylococcus aureus is 64 μg / mL.

[0011] In one embodiment, the drug comprises a coumarin derivative of Formula I and a pharmaceutically acceptable carrier or excipient.

[0012] In one embodiment, the drug is any one of a tablet, capsule, injection, topical preparation, or antimicrobial coating of a medical device.

[0013] In one embodiment, the topical preparation is a topical gel containing 0.1-5% by weight of a coumarin derivative with the structure shown in Formula I.

[0014] In one embodiment, the injection comprises a coumarin derivative of Formula I with a mass concentration of 1-100 mg / mL and a pharmaceutically acceptable solvent.

[0015] In one embodiment, the antibacterial coating of the medical device is a surface coating of a urinary catheter, intravenous catheter, wound dressing, or bone cement; the surface coating comprises a coumarin derivative with the structure shown in Formula I.

[0016] The advantages or beneficial effects of the above technical solutions include at least the following: This invention significantly enhances the inhibitory effect on Staphylococcus aureus by designing and synthesizing novel coumarin derivatives, while taking into account solubility, stability and pharmaceutical feasibility, thus providing new solutions and technical support for clinical antibacterial therapy and anti-infection protection of medical devices.

[0017] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0018] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0019] Figure 1 The inhibition zone patterns of compounds 1, 2, 3, and 4 against different bacterial strains are shown. Figure 2 The inhibition zone pattern of compound 1 against Staphylococcus aureus; Figure 3 Antimicrobial zone patterns of other structural analogs of compound 1; Figure 4 Growth inhibition curves for other structural analogs of compound 1; Figure 5 The growth inhibition curve of compound 1 against Staphylococcus aureus is shown. Figure 6 The bactericidal curve of compound 1 against Staphylococcus aureus is shown. Figure 7 The membrane permeability of compound 1 to Staphylococcus aureus was detected using PI dye. Figure 8 The effect of compound 1 on the leakage of lactate dehydrogenase from Staphylococcus aureus; Figure 9The change in membrane potential of compound 1 on Staphylococcus aureus; Figure 10 The effect of compound 1 on the ATP of Staphylococcus aureus; Figure 11 The effect of compound 1 on intracellular ROS in Staphylococcus aureus; Figure 12 This is a diagram showing the effect of compound 1 on skin infection of Staphylococcus aureus in animal experiments. Detailed Implementation

[0020] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0021] This application provides the use of a coumarin derivative in the preparation of a drug for inhibiting Staphylococcus aureus, wherein the coumarin derivative has the structure shown in Formula I: ; R1 is a halogen atom, and R2 is an oxygen-containing group.

[0022] The coumarin derivatives of this application can act as antibacterial agents, penetrating the interior of Staphylococcus aureus, disrupting the integrity of the bacterial cell membrane, causing the leakage of lactate dehydrogenase, greatly impairing the permeability of the bacterial cell membrane, and leading to bacterial death. Simultaneously, the antibacterial agent of this application can effectively reduce the bacterial membrane potential, further affecting the production of bacterial ATP, causing an increase in the total bacterial ATP and a decrease in intracellular ATP, thus accelerating bacterial death.

[0023] In one embodiment, R1 is a Br or Cl atom; R2 is a hydroxyl group or an alkoxy group with 1-3 carbon atoms. Preferably, R2 is a hydroxyl group or a methoxy group with 1 carbon atom.

[0024] As one embodiment, the coumarin derivative is any one of the following compounds: ; ; ; .

[0025] In one embodiment, the *Staphylococcus aureus* is ATCC 29213. The coumarin derivatives of this application exhibit good antibacterial effects against Gram-positive bacteria, including ATCC 70021, CGMCC 1.195, and ATCC 29213, with particularly significant antibacterial effects against *Staphylococcus aureus* ATCC 29213; the inhibition zone exceeds 1 cm.

[0026] As one embodiment, the minimum inhibitory concentration of the coumarin derivative with the structure shown in Formula I against Staphylococcus aureus is 64 μg / mL.

[0027] As one embodiment, the drug comprises a coumarin derivative of Formula I and a pharmaceutically acceptable carrier or excipient.

[0028] As one embodiment, the drug is any one of tablets, capsules, injections, topical preparations, or antibacterial coatings for medical devices.

[0029] In one embodiment, the topical preparation is a topical gel containing 0.1-5% by weight of a coumarin derivative with the structure shown in Formula I.

[0030] As one embodiment, the injection comprises a coumarin derivative of Formula I with a mass concentration of 1-100 mg / mL and a pharmaceutically acceptable solvent.

[0031] As one embodiment, the antibacterial coating of the medical device is a surface coating of a urinary catheter, intravenous catheter, wound dressing, or bone cement; the surface coating comprises a coumarin derivative with the structure shown in Formula I.

[0032] The following is a further explanation using specific embodiments.

[0033] Example 1

[0034] The synthetic route of compound 1 is shown below:

[0035] A solution of 4-bromo-2-methylacetone ethyl acetate (0.95 g, 1.0 eq) and 4-bromo-3-hydroxyphenol (1.41 g, 7.5 eq) in 10 mL of CH3SO3H was stirred overnight at room temperature. The mixture was then poured into 30 mL of H2O and extracted with 100 mL of ethyl acetate. The organic layer was separated, washed with brine, dried over Na2SO4, and the solvent was removed by vacuum distillation. The crude product was then separated by column chromatography (PE / EA = 4 / 1). (The crude product was recrystallized from EA to give compound 1 (yellow powder). Yield: 235 mg, yield: 14.2%) 1 H NMR (500 MHz, DMSO- d6 ) δ 11.58 (s, 1H), 8.04 (s, 1H), 6.92 (s,1H), 6.54 (s, 1H), 4.87 (s, 2H).

[0036] Example 2

[0037] The synthetic route of compound 2 is shown below:

[0038] A solution of ethyl 4-bromo-2-methylacetone (0.95 g, 1.0 eq) and 4-bromo-3-methoxyphenol (1.52 g, 1.5 eq) in 10 mL of CH3SO3H was stirred overnight at room temperature. The mixture was then poured into 30 mL of H2O and extracted with 100 mL of EtOAc. The organic layer was separated, washed with brine, dried over Na2SO4, and the solvent was removed by vacuum distillation. The crude product was then separated by column chromatography (PE / EA = 4 / 1). (The crude product was recrystallized with EA to give compound 2 (yellow powder). Yield: 180 mg, yield 10.2%) 1 H NMR (500 MHz, DMSO- d6 ) δ 7.83 (s, 1H), 7.45 (s, 1H), 6.79 (s, 1H), 4.98 (s, 2H), 3.95 (s, 3H).

[0039] Example 3

[0040] The synthetic route of compound 3 is shown below:

[0041] A solution of ethyl 4-chloro-2-methylacetone (0.82 g, 1.0 eq) and 4-bromo-3-hydroxyphenol (1.41 g, 1.5 eq) in 10 mL of CH3SO3H was stirred overnight at room temperature. The mixture was then poured into 30 mL of H2O and extracted with 100 mL of LEtOAc. The organic layer was separated, washed with brine, and dried over Na2SO4. The solvent was removed by vacuum distillation to obtain a crude product. The crude product was recrystallized from EA to give compound 3 (white powder). Yield: 648 mg, yield: 45.1%. 1 H NMR (500 MHz, DMSO- d6 ) δ11.58 (s, 1H), 8.00 (s, 1H), 6.92 (s, 1H), 6.48 (s, 1H), 5.01 (s, 2H).

[0042] Example 4

[0043] The synthetic route of compound 4 is shown below:

[0044] A solution of ethyl 4-chloro-2-methylacetone (0.82 g, 1.0 eq) and 4-bromo-3-methoxyphenol (1.52 g, 1.5 eq) in 10 mL of CH3SO3H was stirred overnight at room temperature. The mixture was then poured into 30 mL of H2O and extracted with 100 mL of EtOAc. The organic layer was separated, washed with brine, and dried over Na2SO4. The solvent was removed by vacuum distillation to obtain the crude product. The crude product was recrystallized from EA to give compound 4 (white powder). Yield: 785 mg, yield: 52%. 1 H NMR (500 MHz, DMSO- d6 ) δ 8.06 (s, 1H), 7.25 (s, 1H), 6.55 (s, 1H), 5.03 (s, 2H), 3.96 (s, 3H).

[0045] Example 5

[0046] Compounds 1, 2, 3, and 4 were used in antibacterial experiments against various Gram-positive bacteria (Enterococcus faecalis ATCC 70021, Staphylococcus aureus ATCC 29123, Enterococcus faecalis ATCC 29212, Enterococcus faecalis ATCC 19434, Bacillus acetitidis CGMCC 1.195) and Gram-negative bacteria (Salmonella ATCC 14028, Escherichia coli ATCC 25922). Bacterial culture images are shown below. Figure 1 As shown; the markings on the petri dish (OH-Br is compound 1, O-Br is compound 2, OH-Cl is compound 3, O-Cl is compound 4).

[0047] The inhibition zones showed that compounds 1-4 had good antibacterial effects against Gram-positive bacteria (including ATCC 70021, CGMCC1.195, and ATCC 29213), especially against Staphylococcus aureus, where the inhibition zones exceeded 1 cm. Figure 2 As shown in the figure, compound 1 exhibits the best antibacterial effect.

[0048] The size of the inhibition zone of compound 1 was statistically analyzed, and the results are shown in Table 1.

[0049] Table 1

[0050] Comparative Example 1 Thirteen compounds with structures similar to compound 1 are numbered sequentially from 5 to 17, corresponding to the following compounds: (7-hydroxy-4-methyl-2H-chromogenone-2-one, 6-bromo-7-hydroxy-2H-chromogenone-2-one, 2H-chromogenone-2-one, 4-(bromomethyl)-7-hydroxy-2H-chromogenone-2-one, 4-(bromomethyl)-8-hydroxy-2H-chromogenone-2-one, 6-bromo-4-(bromomethyl) -2H-chromogenone-2-one, 7-methoxy-4-methyl-2H-chromogenone-2-one, 4-(bromomethyl)-7-methoxy-2H-chromogenone-2-one, 6-bromo-7-methoxy-2H-chromogenone-2-one, 5-bromo-4-(bromomethyl)-8-hydroxy-2H-chromogenone-2-one, 4-methylumbelliferone, 6,7-dihydroxycoumarin, 4-hydroxycoumarin), antibacterial experiments were conducted according to the method in Example 5, and the results are as follows: Figure 3 and Figure 4 As shown.

[0051] from Figure 3 The size of the inhibition zone indicates that compounds 5-17 have no antibacterial effect. Figure 5 The growth inhibition curves showed that bacterial growth remained stable within one hour, with no obvious trend of bacterial death. The relatively high OD values ​​of compounds 13 and 15 may be due to the presence of some fluorescence in their structures, but they still did not inhibit bacterial growth.

[0052] Example 6

[0053] inhibition zone experiment of compound 1 against Staphylococcus aureus As shown in Example 2, Compound 1 exhibits significant antibacterial activity against Staphylococcus aureus. Next, serial dilutions were performed in a 96-well plate: 200 μL of the highest analyte concentration was added to the first well (A1), and subsequent wells were serially diluted. The MIC (Minimum Inhibitory Concentration) was determined by adding 10 μL of the diluted bacterial suspension to each well (A1-A12). The 96-well plate was incubated at 37°C for 16 hours. The lowest drug concentration at which no bacterial growth occurred was recorded as the minimum inhibitory concentration (MIC) against the drug. The results showed that the MIC of Compound 1 against Staphylococcus aureus was 64 μg / mL.

[0054] Example 7

[0055] Effect of compound 1 on the time growth of Staphylococcus aureus Staphylococcus aureus was transferred to LB broth and incubated at a suitable temperature until the optimal bacterial concentration for the experiment was reached (approximately 10⁻⁶). 8 CFU / mL). Adjusting bacterial concentration: Adjust the initial bacterial concentration to 10 by dilution. 6 CFU / mL. Compound 1 at concentrations of 32 μg / mL, 64 μg / mL, 320 μg / mL, and 10 μg / mL were added to the bacterial-containing broth, with three replicates for each drug. The broth was incubated at 37°C and 200 rpm in a shaker. The PBS group served as a negative control. 100 μL of culture was collected at 0 min, 15 min, 30 min, 45 min, 1 h, and 2 h after the addition of the antibiotics, serially diluted in PBS, and plated for counting. Results are as follows: Figure 5 As shown.

[0056] Figure 5 The experimental results showed that after co-culturing compound 1 with Staphylococcus aureus for 12 hours, the bacterial growth trend was stable. Compound 1 at 32ug / mL and 64ug / mL had significant inhibitory effects on bacteria. The antibacterial effect of compound 1 at 320ug / mL on Staphylococcus aureus was comparable to that of vancomycin, indicating that compound 1 has good antibacterial activity against Staphylococcus aureus.

[0057] Example 8

[0058] Effect of Compound 1 on the bactericidal curve of Staphylococcus aureus A single colony of *S. aureus* ATCC 29213 was picked and incubated in 1 mL of culture medium at 37°C and 200 rpm for 3 h to obtain bacteria in the logarithmic growth phase. The concentration of *S. aureus* ATCC 29213 was adjusted to a McFarland turbidity of 0.5, and then diluted 100-fold to 5 mL of prepared MHB broth to obtain a bacterial concentration of approximately 10⁻⁶. 6 CFU / mL bacterial culture was prepared. Vancomycin (10 μg / mL), Compound 1 (320 μg / mL), and Compound 1 (640 μg / mL) were added to the bacterial broth, with three replicates for each drug. The cultures were incubated at 37°C and 200 rpm in a shaker. The PBS group served as a negative control. 100 μL of culture was collected at 0, 1, 2, 4, and 8 h after antibiotic addition, serially diluted in PBS, and plated for bacterial count. Results are as follows: Figure 6 As shown.

[0059] Experimental results showed that the antibacterial effect of compound 1 at 320 μg / mL against Staphylococcus aureus was comparable to that of vancomycin. Furthermore, the bactericidal effect was even higher at 640 μg / mL, indicating that the bactericidal effect of compound 1 against Staphylococcus aureus was dose-dependent. Example 9

[0060] Effect of Compound 1 on Staphylococcus aureus membrane permeability Changes in bacterial cell membrane permeability are determined by two indicators: propidium iodide (for DNA staining) and extracellular lactate dehydrogenase (LDH) content.

[0061] Propidium iodide (PI) assay: *S. aureus* ATCC 29213 was cultured overnight, centrifuged, and resuspended in PBS to OD600 = 0.5. The fluorescent probe PI was added to a final concentration of 10 μM. After incubation for 10 min, different compounds were added (final concentrations of 0 μg / mL, 32 μg / mL, 64 μg / mL, and 320 μg / mL), or vancomycin (10 μg / mL), or nisin (nisin, a natural bioactive antimicrobial peptide). 100 μg / mL of nisin was used as a positive control. The assay was performed using a multi-functional microplate reader, maintaining a constant temperature of 37℃. Fluorescence values ​​were measured every 2 min (excitation wavelength 535 nm, emission wavelength 615 nm), starting 10 min before PI addition, and continued for 2 h. Results are as follows: Figure 7 As shown.

[0062] PI is a membrane-impermeable nucleic acid dye. Only when the cell membrane barrier is disrupted can the PI dye enter the cell membrane. The higher the membrane permeability, the stronger the fluorescence intensity. Figure 7 The results showed that the permeability of compound 1 to the cell membrane was dose-dependent, and when the concentration of compound 1 was 32 ug / mL, compound 1 was more damaging to the cell membrane than vancomycin.

[0063] Lactate dehydrogenase (LDH) assay: *S. aureus* ATCC 29213 was cultured overnight, centrifuged, and resuspended in PBS to OD600 = 0.5. Different concentrations of compound 1 (final concentrations of 0 μg / mL, 32 μg / mL, 64 μg / mL, and 320 μg / mL) or vancomycin (10 μg / mL) were added and incubated for 2 hours. The supernatant was then collected by centrifugation, and reaction solution was added to the supernatant. After incubation for 30 minutes, the absorbance at 490 nm was measured using a multi-mode microplate reader. Results are as follows: Figure 8 As shown.

[0064] LDH is a stable enzyme present in the cytoplasm of almost all animals, plants, and microorganisms. When the cell membrane is damaged, LDH in the cytoplasm leaks into the extracellular fluid. The integrity of the cell membrane can be determined by detecting the amount of LDH leaked out. Figure 8 The results showed that the concentration of compound 1 was dose-dependent on LDH leakage, indicating that compound 1 could disrupt bacterial cell membranes.

[0065] Example 10

[0066] Effect of compound 1 on bacterial membrane potential After culturing *S. aureus* ATCC 29213 to the logarithmic growth phase, centrifuged, washed three times with HEPES, and resuspended to OD600=0.5. The pH-sensitive fluorescent probe BCECF-AM was added to a final concentration of 10 μM, and incubated at 37℃ for 30 min. The cells were then washed twice with HEPES to remove any unloaded probe. 190 μL of probe-loaded bacterial culture was added to a 96-well plate, followed by 10 μL of compound 1 (final concentrations of 0 μg / mL, 32 μg / mL, 64 μg / mL, or 320 μg / mL) or vancomycin (10 μg / mL) and CCCP (50 μM CCCP served as a positive control; CCCP is a proton carrier that disrupts the electrochemical gradient (ΔΨm) of the mitochondrial inner membrane, preventing mitochondria from synthesizing ATP). A multi-mode microplate reader was used in long-term measurement mode, maintaining a constant temperature of 37℃, and measuring fluorescence values ​​every 2 min at an excitation wavelength of 490 nm and an emission wavelength of 535 nm. Detection began 10 minutes before the addition of BCECF-AM and continued for 2 hours. Results are as follows: Figure 9 As shown.

[0067] Figure 9 The results showed that the concentration of compound 1 was dose-dependent on changes in bacterial membrane potential, and that compound 1 was more effective than vancomycin in reducing membrane potential. This indicates that compound 1 can disrupt bacterial membrane potential, which in turn affects bacterial ATP formation.

[0068] Example 11

[0069] Effect of compound 1 on intracellular and extracellular ATP in Staphylococcus aureus S. aureus ATCC 29213 was cultured to the logarithmic growth phase, centrifuged, and resuspended in PBS to OD600 = 0.5. Different concentrations of compound 1 (final concentrations of 0 μg / mL, 32 μg / mL, 64 μg / mL, and 320 μg / mL) or vancomycin (10 μg / mL) were added for 2 h, followed by centrifugation for 2 min to collect bacterial cells and supernatant. The supernatant was added to the reaction solution according to the kit instructions, and the extracellular ATP content was measured using a multi-mode microplate reader in chemiluminescence mode. For bacterial pellets, lysostaphin was added to each tube to a final concentration of 1 μg / mL, centrifuged, and the supernatant was collected. The reaction solution was added according to the kit instructions, and the intracellular ATP content was measured using a multi-mode microplate reader in chemiluminescence mode. Results are as follows: Figure 10 As shown.

[0070] Figure 10The results showed that after compound 1 disrupted the integrity of the bacterial cell membrane, it further reduced the bacterial membrane potential. Membrane potential is the core of bacterial energy metabolism. Reducing the membrane potential affects the production of bacterial ATP, causing a decrease in intracellular ATP and an increase in extracellular ATP, resulting in an increase in the total amount of ATP and accelerating bacterial death.

[0071] Example 12

[0072] Effect of compound 1 on intracellular ROS in Staphylococcus aureus The accumulation of intracellular ROS in bacteria was determined using the Beyotime ROS detection kit. The specific steps are as follows: *S. aureus* ATCC 29213 was cultured to the logarithmic growth phase, centrifuged, washed three times with PBS, and resuspended to OD600 = 0.5. The fluorescent probe DCFH-DA was added to a final concentration of 10 μM, and incubated at 37°C for 30 min. The cells were then washed twice with PBS to remove any unloaded probe. 190 μL of probe-loaded bacterial culture was added to a 96-well plate, followed by 10 μL of compound 1 (final concentrations of 0 μg / mL, 32 μg / mL, 64 μg / mL, or 320 μg / mL) or vancomycin (10 μg / mL). The plate was immediately used in long-term assay mode on a multi-mode microplate reader, maintaining a constant temperature of 37°C. Fluorescence values ​​were measured every 2 min at an excitation wavelength of 490 nm and an emission wavelength of 535 nm (or excitation wavelength of 488 nm and emission wavelength of 535 nm, depending on the manufacturer's instructions). Detection began 10 minutes before the addition of DCFH-DA and continued for 2 hours. Results are as follows: Figure 11 As shown.

[0073] Figure 11 The results showed that the concentration of compound 1 was dose-dependent on the production of intracellular ROS. When the concentration of compound 1 was 32 ug / mL, it was comparable to the effect of vancomycin in promoting ROS growth in the intracellular environment, indicating that compound 1 caused the production of ROS in the bacterial cells, leading to the rapid death of the bacteria.

[0074] Example 13

[0075] Effect of compound 1 on mouse skin experiments Staphylococcus aureus was incubated in a constant temperature incubator for 12 hours, and the Staphylococcus aureus was adjusted to a concentration of 1×10⁻⁶ using a multi-functional microplate reader. 8 Each milliliter. After one week of acclimatization, 24 mice were randomly divided into 4 groups of 6. Each mouse was anesthetized by intraperitoneal injection of 4% chloral hydrate (0.1 mL / 10 g), and a skin wound approximately 10 mm in diameter was prepared using surgical instruments. 100 μL of chloral hydrate containing 10 g was injected into each mouse's skin wound. 7CFU of Staphylococcus aureus. One hour after bacterial infection, mice in each group were treated with either 64 mg / kg of compound 1 or 10 mg / kg of vancomycin in their wounds. The mice were observed for 10 consecutive days, and the wound healing process was recorded by photography. Results are as follows: Figure 12 As shown.

[0076] Figure 12 As shown, compound 1 exhibits antibacterial effects compared to the Staphylococcus aureus model, significantly reducing the degree of infection in mouse wounds. Compared to vancomycin, the infected skin area in mice is significantly smaller, indicating that compound 1 has a significant inhibitory effect on Staphylococcus aureus.

[0077] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0078] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0079] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. The application of a coumarin derivative in the preparation of a drug for inhibiting Staphylococcus aureus, characterized in that, The coumarin derivative has the structure shown in Formula I: ； R1 is a halogen atom, and R2 is an oxygen-containing group.

2. The application according to claim 1, characterized in that, R1 is a Br or Cl atom; R2 is a hydroxyl group or an alkoxy group with 1-3 carbon atoms.

3. The application according to claim 1, characterized in that, The coumarin derivative is any one of the following compounds: ； ； ； 。 4. The application according to claim 1, characterized in that, The Staphylococcus aureus in question is ATCC 29213.

5. The application according to claim 1, characterized in that, The minimum inhibitory concentration of the coumarin derivative with the structure shown in Formula I against Staphylococcus aureus is 64 μg / mL.

6. The application according to claim 1, characterized in that, The drug comprises a coumarin derivative of Formula I and a pharmaceutically acceptable carrier or excipient.

7. The application according to claim 1, characterized in that, The drug is any one of tablets, capsules, injections, topical preparations, or antibacterial coatings for medical devices.

8. The application according to claim 7, characterized in that, The topical preparation is a topical gel containing 0.1-5% by weight of a coumarin derivative with the structure shown in Formula I.

9. The application according to claim 7, characterized in that, The injection comprises a coumarin derivative of Formula I with a mass concentration of 1-100 mg / mL and a pharmaceutically acceptable solvent.

10. The application according to claim 7, characterized in that, The antibacterial coating of the medical device is a surface coating of a urinary catheter, intravenous catheter, wound dressing, or bone cement; the surface coating comprises a coumarin derivative with the structure shown in Formula I.

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