Application of quinazoline compound ZJCK-6-68 in preparation of antibacterial, anti-inflammatory and anti-skin infection drugs

Abstract

The invention relates to the technical field of medicine, in particular to application of a quinazoline compound ZJCK-6-68 in preparation of antibacterial, anti-inflammatory and anti-skin infection drugs, and the ZJCK-6-68 provided by the invention has the advantages of antibacterial property, low cytotoxicity, low hemolytic toxicity, good in-vivo biological safety and low drug resistance tendency. An antibacterial mechanism research shows that the ZJCK-6-68 can inhibit the formation of a biological membrane, reduce the generation of golden pigment and destroy cell membranes. This disruption results in membrane depolarization, cytoplasmic DNA leakage and DNA synthesis inhibition, ultimately accelerating bacterial death. The ZJCK-6-68 can inhibit the expression of inflammatory factors TNF-alpha, IL-6 and NO, and can play an anti-inflammatory role by inhibiting a DYRK1A signal channel. It is worth noting that ZJCK-6-68 has anti-MRSA activity and anti-inflammatory activity in a mouse skin infection model, and has great clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of a quinazoline compound ZJCK-6-68 in the preparation of antibacterial, anti-inflammatory, and anti-skin infection drugs. Background Technology

[0002] Skin and soft tissue infections (SSTIs) are caused by microorganisms invading the skin and surrounding tissues. They are among the most common infections seen in outpatients, manifesting as boils, carbuncles, abscesses, or cellulitis. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens. MRSA is not only highly invasive but also strongly activates the host's immune system by releasing various virulence factors (such as hemolysins, leukocidins, and superantigens), leading to excessive or persistent inflammatory responses, exacerbating tissue damage, and hindering healing. Due to the irrational use of antibiotics, the situation of microbial resistance is becoming increasingly serious, posing a serious threat to global public health. MRSA, as a typical example of "superbugs," was listed as a high-priority pathogen by the WHO in 2024. Against this backdrop, there is an urgent need to develop novel antibacterial and anti-inflammatory drugs to address the increasingly serious problem of MRSA resistance.

[0003] Compared to traditional antibiotics, natural products are receiving increasing attention due to their strong pharmacological activity, fewer side effects, easy availability, and low cost. Camel thorn ( Peganum harmala *L.* (L.) is a perennial herb belonging to the genus *L.* of the family Zygophyllaceae. It is a commonly used medicinal material among the Uyghur, Kazakh, Mongolian, and Tibetan peoples in Northwest my country and is included in the *Pharmaceutical Standards of the People's Republic of China: Uyghur Medicines*. Modern pharmacological studies have shown that it possesses various effects, including anti-infection, anti-inflammation, anti-tumor, anti-diabetic, antihypertensive, antidepressant, anti-Parkinson's disease, and anti-Alzheimer's disease. *L.* alkaloids are mainly classified into two types based on their structural characteristics: β-carboline alkaloids and quinazoline alkaloids. It is noteworthy that β-carboline alkaloids have attracted considerable attention from the academic community due to their significant pharmacological activity, resulting in a wealth of research findings. In contrast, systematic research on quinazoline alkaloids remains relatively limited.

[0004] Based on the above situation, this application focuses on the development and utilization of quinazoline alkaloids, aiming to provide a novel compound with antibacterial, anti-inflammatory and anti-skin infection potential, and to provide a new candidate drug for dealing with MRSA and related infections. Summary of the Invention

[0005] This invention proposes the application of a quinazoline compound ZJCK-6-68 in the preparation of antibacterial, anti-inflammatory, and anti-skin infection drugs, with the aim of providing a novel compound with antibacterial, anti-inflammatory, and anti-skin infection potential.

[0006] To achieve the above objectives, the present invention provides the following solution: The first aspect of this invention discloses the application of a quinoline compound, ZJCK-6-68, in the preparation of antibacterial, anti-inflammatory, and anti-skin infection drugs. The structural formula of compound ZJCK-6-68 is as follows: .

[0007] Furthermore, the compound ZJCK-6-68 can be used in the preparation of antibacterial drugs by inhibiting biofilm formation.

[0008] Furthermore, the antibacterial spectrum of the compound ZJCK-6-68 includes MRSA, Enterococcus faecalis, Enterococcus faecium, and Streptococcus pneumoniae.

[0009] Furthermore, the compound ZJCK-6-68 can be used in the preparation of anti-inflammatory drugs by inhibiting the expression of inflammatory factors TNF-α, IL-6 and NO in a RAW 264.7 cell inflammation model.

[0010] Furthermore, the compound ZJCK-6-68 can be used in the preparation of anti-inflammatory drugs by inhibiting the DYRK1A signaling pathway.

[0011] Furthermore, the compound ZJCK-6-68 can be used in the preparation of drugs for treating skin infections through its anti-MRSA and anti-inflammatory activities.

[0012] Furthermore, the compound ZJCK-6-68 is used as an active ingredient in the preparation of antibacterial and / or anti-inflammatory and / or anti-skin infection drugs.

[0013] Furthermore, the drug may also include pharmaceutically acceptable carriers or adjuvants.

[0014] The beneficial effects of this invention are as follows: This invention reveals that compound ZJCK-6-68 exhibits significant antibacterial activity against MRSA and other Gram-positive bacteria, with a minimum inhibitory concentration (MIC) as low as 4 μg / mL for MRSA. Experimental verification shows that compound ZJCK-6-68 possesses antibacterial properties, low cytotoxicity, hemolytic toxicity, good in vivo biocompatibility, and low tendency to develop resistance. Mechanistic studies show that ZJCK-6-68 strongly inhibits biofilm formation, reduces the production of aurora pigment, and disrupts the cell membrane. This disruption of the cell membrane leads to membrane depolarization, cytoplasmic DNA leakage, and inhibition of DNA synthesis, ultimately accelerating bacterial death. Furthermore, ZJCK-6-68 can inhibit and reduce the expression of inflammatory factors TNF-α, IL-6, and NO in a RAW 264.7 cell inflammation model. Anti-inflammatory mechanism studies show that ZJCK-6-68 exerts its anti-inflammatory effect by inhibiting the DYRK1A signaling pathway. Notably, ZJCK-6-68 exhibits anti-MRSA and anti-inflammatory activities in a mouse skin infection model. By accelerating wound healing, reducing bacterial load in wounds, alleviating inflammation at the infection site, and relieving skin pathological damage, ZJCK-6-68 effectively improves skin infections caused by MRSA. This highlights its potential as a promising candidate drug for both anti-MRSA and anti-inflammatory purposes, demonstrating significant clinical application value. Attached Figure Description

[0015] Figure 1 In vitro antibacterial curves of different treatment groups of the quinazoline compound ZJCK-6-68; Figure 2 Time-antibacterial curves of different treatment groups of the quinazoline compound ZJCK-6-68; Figure 3 The graph shows the results of resistance development to quinazoline compounds ZJCK-6-68 and vancomycin. The left graph shows the test strain USA300, and the right graph shows the test strain NCTC 8325. Figure 4 Crystal violet staining showed that the quinazoline compound ZJCK-6-68 could inhibit biofilm formation. Figure 5 Fluorescence microscopy observation of biofilm results for different treatment groups of quinazoline compound ZJCK-6-68 (scale bar in the figure is 100 μm). Figure 6The images show scanning electron microscope (SEM) images of Staphylococcus aureus (MRSA) before and after treatment with the quinazoline compound ZJCK-6-68 (Figure A has a scale bar of 5 µm and is an SEM image of MRSA without treatment with ZJCK-6-68; Figure B has a scale bar of 5 µm and is an SEM image of MRSA after treatment with ZJCK-6-68; Figure C has a scale bar of 1 µm and is an SEM image of MRSA without treatment with ZJCK-6-68; Figure D has a scale bar of 1 µm and is an SEM image of MRSA after treatment with ZJCK-6-68). Figure 7 Images of USA300-GFP fluorescence microscopy after treatment with different concentrations of the quinazoline compound ZJCK-6-68 (scale bars in the figures are all 20 μm). Figure 8 The result is the leakage of intracellular DNA after treatment with the quinazoline compound ZJCK-6-68; Figure 9 The results show the cell membrane depolarization after treatment with the quinazoline compound ZJCK-6-68. Figure 10 The results of MIC changes after the addition of quinazoline compound ZJCK-6-68 and exogenous components of the cell membrane wall; Figure 11 The results show the amount of golden yellow pigment generated after treatment with the quinazoline compound ZJCK-6-68. Figure 12 The results show the levels of NO and pro-inflammatory cytokines TNF-α and IL-6 in RAW 264.7 cells after treatment with the quinazoline compound ZJCK-6-68; (where A represents NO levels after treatment with ZJCK-6-68; B represents TNF-α levels after treatment with ZJCK-6-68; and C represents IL-6 levels after treatment with ZJCK-6-68). Figure 13 The expression results of DYRK1A protein in RAW 264.7 cells were obtained from the quinazoline compound ZJCK-6-68. Figure 14 The results of MTT assay for detecting the survival rate of HEK293 T cells after ZJCK-6-68 treatment; Figure 15 The results show the hemolysis rate of 4% fresh sheep red blood cells after treatment with ZJCK-6-68. Figure 16 The images show the results of routine blood analysis and pathological sections. A shows the pathological images of the tissue structures of various organs, and B shows the blood analysis images of the whole blood cell count parameters in the control group and the ZJCK-6-68 administration group. Figure 17 The images show the in vivo therapeutic effects of the quinazoline compound ZJCK-6-68 on mouse skin infected with MRSA. (A is a schematic diagram of the process for constructing a mouse skin model caused by MRSA infection; B is a representative image of different treatment groups after treatment of infected skin wounds caused by MRSA; C is a representative image of MRSA cultured from infected skin tissue homogenate diluted and applied; D is the bacterial load in the skin tissue of mice treated with different treatment groups; E is the quantitative analysis result of wound closure rate; F is the evaluation result of infected skin wound tissue by H&E staining; G is the result of TNF-α in skin tissue; H is the result of IL-6 in skin tissue; I is the result of IL-1β in skin tissue).

[0016] In the image above, compared to the Control group, * indicates... p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤0.001, **** indicates p ≤ 0.0001; compared with the vancomycin group, # indicates p ≤ 0.05, ### indicates p ≤ 0.001. Detailed Implementation

[0017] The present invention will be described below through specific embodiments, but the present invention is not limited thereto.

[0018] The structure of the compound was determined by nuclear magnetic resonance (NMR). The NMR measurements were performed using a Bruker AVANCE-300 / 500 NMR spectrometer, and the solvent was DMSO. d 6 and CDCl3, with TMS as the internal standard. Example 1: The preparation method of the quinoline compound ZJCK-6-68 proposed in this invention comprises the following steps: 1.1 Preparation of p-Toluenesulfonate A0i Under ice-water bath conditions, commercially available 3-iodo-6-methyl-1 H -pyrrolo[2,3- b p-Toluenesulfonyl chloride (TsCl) was slowly added to an anhydrous DMF solution of pyridine, and the reaction was continued for 12 h to obtain p-toluenesulfonate A0i.

[0019] 1.2 General preparation of intermediate A38 6-Bromo-4-chloroquinazoline (1.0 equivalent) was dissolved in isopropanol, followed by the addition of 1-(3-fluorophenyl)cyclopropylamine (1.5 equivalent) and triethylamine (5.0 equivalent). The reaction was stirred at 85 °C for 6 h. After the reaction was complete, the solvent was removed by vacuum distillation, and the product was diluted with ethyl acetate and water. The organic layer was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The concentrated product required no further purification and could be used directly in subsequent reaction steps.

[0020] 1.3 Preparation of intermediate A38t (Miyaura reaction) A38 (1.0 equivalent) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxoborane) (1.2 equivalent) were dissolved in anhydrous dioxane. KOAc (3.0 equivalent) and Pd(PPh3)2Cl2 (5 mol%) were added sequentially, and the reaction was carried out at 100 °C for 12 h under N2 protection. After the reaction was complete, the solvent was carefully removed by vacuum distillation, and the mixture was diluted with ethyl acetate and water. The organic layer was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The concentrated product did not require further purification and could be used directly in subsequent reaction steps.

[0021] 1.4 Preparation of intermediate B38 (Suzuki coupling reaction) Compounds A0i (1.0 equivalent) and A38 (1.2 equivalent) were dissolved in a dioxane / water mixed solvent (4:1, v / v), followed by the sequential addition of Cs₂CO₃ (3.0 equivalent) and Pd(dppf)Cl₂ (5 mol%). The reaction was carried out at 90 °C for 6 h under N₂ protection. After the reaction was complete, the solvent was removed by vacuum distillation. The concentrated product was purified by rapid silica gel column chromatography to obtain intermediate B38.

[0022] 1.5 Preparation of N-(1-(3-fluorophenyl)cyclopropyl)-6-(6-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)quinazolin-4-amine (ZJCK-6-68) Compound B38 (150 mg, 1.0 equivalent) was dissolved in a mixed solvent of dichloromethane and methanol (1:1, v / v, 30 mL), followed by the addition of sodium hydroxide (5.0 equivalent), and the reaction was stirred at 50 °C for 2 h. After the reaction was complete, the solvent was removed by vacuum distillation, and the mixture was diluted with ethyl acetate and water. The organic layer was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The concentrated product was purified by rapid silica gel column chromatography or recrystallized from methanol to give the target compound in 70% yield as a yellow solid.

[0023] The structural formula of compound ZJCK-6-68 is: .

[0024] 1 H NMR (400 MHz, DMSO- d 6) δ 11.84 (s, 1H), 9.05 (s, 1H), 8.58 (d, J =1.9 Hz, 1H), 8.40 (d, J = 7.3 Hz, 2H), 8.19 (dd, J = 8.7, 1.9 Hz, 1H), 7.93(d, J = 2.6 Hz, 1H), 7.76 (d, J = 8.7 Hz, 1H), 7.28 (td, J = 8.0, 6.3 Hz, 1H), 7.10 (d, J = 8.2 Hz, 1H), 7.06 (dt, J = 8.1, 1.1 Hz, 1H), 7.02 – 6.98(m, 1H), 6.96 (dd, J = 9.6, 7.0 Hz, 1H), 2.57 (s, 3H), 1.53 – 1.40 (m, 4H). 13 C NMR (100 MHz, DMSO- d 6) δ 162.40 (d, J = 243.4 Hz), 159.50, 154.02, 151.51,148.92, 147.45, 147.17 (d, J = 7.1 Hz), 133.17, 131.52, 129.88 (d, J = 8.1Hz), 128.38, 128.08, 123.57, 120.54 (d, J = 2.0 Hz), 118.19, 115.97, 115.39,114.72, 113.66, 112.09 (d, J = 20.2 Hz), 111.31 (d, J = 22.2 Hz), 35.17,23.97, 19.77 (2C). 19F NMR (377 MHz, DMSO- d 6) δ -113.91. HRMS (ESI, m / z) forC 25 H 20 FN5 [M + H] + : calcd, 410.1781; found, 410.1783. HPLC purity: 95.01%, t R =2.347 min.

[0025] Example 2: Antibacterial activity of ZJCK-6-68 against different bacterial strains The MIC was determined using the two-fold dilution method. The specific experimental procedure was as follows: the strain was inoculated into CA-MHB liquid medium and cultured at 37℃ and 220 rpm until the logarithmic growth phase. The bacterial concentration was then adjusted to 1×10⁻⁶ using sterile CA-MHB medium. 6 CFU / mL. In a 96-well microtiter plate, the compound was diluted with CA-MHB medium to prepare serially double dilutions, with a final volume of 100 μL. 100 μL of the adjusted bacterial suspension was then inoculated into each well, mixed thoroughly, and incubated at 37°C for 24 h. The lowest concentration result, where the well showed a clear liquid with no bacterial precipitate, was defined as the MIC value. The results are shown in Table 1.

[0026] Table 1. Antibacterial activity of ZJCK-6-68 against different bacterial strains

[0027] Table 1 shows that ZJCK-6-68 has a MIC of 4-16 μg / mL against MRSA, 32 μg / mL against Enterococcus faecalis, and 64 μg / mL against Streptococcus pneumoniae. The MIC values ​​of ZJCK-6-68 against Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Bacillus subtilis, and Candida albicans are all greater than or equal to 128 μg / mL. Therefore, this concentration of MIC is considered meaningless for antibacterial activity screening, meaning it has no antibacterial activity.

[0028] Example 3: Growth inhibitory effect of ZJCK-6-68 on MRSA USA300 samples were incubated overnight, then adjusted to an OD of approximately 0.3 using TSB. Different concentrations of ZJCK-6-68 were added to the culture medium, with the group without ZJCK-6-68 serving as a control. Samples were incubated at 37°C on a shaker at 180 rpm. At different time points, the OD value of each well was measured at a wavelength of 600 nm using an automated universal microplate reader. The results are shown below. Figure 1 .

[0029] like Figure 1 As shown, ZJCK-6-68 can continuously inhibit the growth of the strain. Compared with the control group, different concentrations of ZJCK-6-68 prolong the logarithmic growth phase and increase the time required for the bacteria to reach the plateau phase. Moreover, the inhibitory effect on the growth of the strain is more obvious with the increase of ZJCK-6-68 concentration.

[0030] Example 4: Antimicrobial kinetics of ZJCK-6-68 against MRSA Compound ZJCK-6-68 was added to a Staphylococcus aureus suspension containing TSB medium (concentration approximately 5 × 10⁻⁶). 5 -10 6 ZJCK-6-68 was added to a solution of CFU / mL to achieve final concentrations of 1×MIC, 4×MIC, and 8×MIC. Samples without ZJCK-6-68 were used as a control group. Samples were incubated at 37℃ with shaking at 40 rpm, and plate colony counts were performed at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h. Results are shown in [Figure number missing]. Figure 2 .

[0031] like Figure 2 As shown, different concentrations of ZJCK-6-68 were observed to have antibacterial effects against MRSA compared to the control group, and the antibacterial effect against MRSA was more obvious with the increase of ZJCK-6-68 concentration.

[0032] Example 5: ZJCK-6-68 bacterial resistance test This embodiment uses USA300 and NCTC 8325 as test strains and vancomycin as a control to test the bacterial resistance of ZJCK-6-68. The specific steps are as follows: USA300 and NCTC 8325 were cultured in MHB culture medium at 37°C for 6 hours, and the bacterial concentration was adjusted to 1×10⁻⁶. 6 CFU / mL, the MIC of the compound was determined according to the steps in Example 1 above, with vancomycin as the control group; this was generation 0. Take a 10 mL ep culturing tube, add 5 mL of MHB medium, select the first well of a 96-well plate where bacteria have grown (1 / 2 × MIC), mix thoroughly by pipetting, and add 50 μL of bacterial suspension to the medium. Place the plate in a shaker at 37°C for subculturing. The MIC of the subcultured bacterial suspension was determined. The above steps were repeated for further subculturing, for a total of 30 generations. Bacterial growth was observed daily, and changes in the MIC of the compound were detected to determine drug resistance. Results are shown in [Table missing]. Figure 3 .

[0033] like Figure 3 As shown, regardless of whether USA300 or NCTC8325 was used as the test strain, the MIC of compound ZJCK-6-68 increased only twofold within 30 days, indicating a lower degree of resistance development compared to the Vancomycin control group.

[0034] Example 6: ZJCK-6-68 Bacterial Anti-Biofilm Test During the intermediate growth stage of strain USA300, the bacterial culture was inoculated into TSB medium containing 0.5% glucose, and the bacterial concentration was adjusted to 10 in 96-well plates. 6 CFU / mL. Different concentrations of ZJCK-6-68 were added to each well, with an equal volume of TSB medium added as a control (CON). Biofilm formation was detected using crystal violet staining, and the absorbance of each well was measured at 562 nm. All experiments were independently repeated three times, with at least three replicates for each sample in each experiment. Results are shown below. Figure 4 .

[0035] like Figure 4 As shown, crystal violet quantification of biofilms formed in the absence and presence of different concentrations of ZJCK-6-68 (1 / 16×MIC to MIC) showed dose-dependent antibiofilm activity.

[0036] Example 7: ZJCK-6-68 Bacterial Adhesion Experiment Bacterial adhesion assays were performed in glass-bottomed cell culture dishes. The concentration of the USA300-GFP bacterial suspension was adjusted to 10. 7 CFU / mL, cultured in TSB medium containing 1×, 1 / 2×, and 1 / 4× MICZJCK-6-68, respectively, with a control culture without ZJCK-6-68 added. All cultures were incubated statically at 37°C for 24 hours, followed by washing the culture dishes three times with PBS to remove unattached airborne bacteria. Finally, the glass-bottomed cell culture dishes were scanned and observed using a fluorescence microscope. Results are shown in [Figure number missing]. Figure 5 .

[0037] like Figure 5 As shown, fluorescence microscopy observations revealed that ZJCK-6-68 could inhibit the formation of MRSA biofilms within the concentration range of 1 / 16×MIC to MIC, and the inhibitory effect was dose-dependent.

[0038] Example 8: Scanning electron microscopy observation of bacterial morphology after ZJCK-6-68 treatment USA300 bacterial culture was inoculated into MH broth medium (concentration 10). 8(CFU / mL) Experimental groups were set up with 1×MIC of ZJCK-6-68 added simultaneously, and a control group without drug addition. The samples were incubated at 220 rpm for 2 hours. Subsequently, the samples were observed using a scanning electron microscope. Results are shown below. Figure 6 .

[0039] like Figure 6 As shown, the ultrastructural changes of MRSA cells treated with ZJCK-6-68 were observed using scanning electron microscopy to investigate the damaging effect of this drug on the USA300 strain. The results showed that... Figure 6 In the control group (A), which received no drugs, the USA300 bacteria exhibited intact morphology, clear cell membrane and cell wall structures, and a smooth surface; while... Figure 6 After treatment with ZJCK-6-68, BC cells showed obvious morphological damage, including cell wall rupture, perforation, and cell membrane invagination. This suggests that ZJCK-6-68 may exert its antibacterial effect by disrupting the structural integrity of bacterial cells.

[0040] Example 9: Observation of the ratio of dead to live cells using confocal laser scanning microscopy The USA300-GFP bacterial culture in the logarithmic growth phase was diluted to 10. 6 CFU / mL of ZJCK-6-68 (4×, 2×, and 1×MIC) were added in equal volumes and incubated at 37°C for 4 hours. A control group containing an equal amount of DMSO was also prepared under the same conditions. Subsequently, the bacterial culture was co-incubated with propidium iodide (PI) for 30 minutes. Finally, images were acquired using a confocal laser scanning microscope. The results are shown below. Figure 7 .

[0041] like Figure 7 As shown, treatment with ZJCK-6-68 significantly enhanced MRSA uptake of PI, and the number of dead bacteria increased in a dose-dependent manner. This phenomenon indicates that the integrity of the bacterial cell membrane was disrupted, confirming that ZJCK-6-68 possesses membrane-disrupting capabilities.

[0042] Example 10: Bacterial DNA Leakage Experiment Adjust the USA300 bacterial solution to 10. 6 CFU / mL was resuspended in PBS, and ZJCK-6-68 was added to achieve final concentrations of 8×, 4×, 2×, and 1×MIC, respectively. A control group containing an equal amount of DMSO was also included, and experiments were conducted under the same conditions. Each group was incubated at 37℃ and 200 rpm with shaking for 6 hours, with samples centrifuged hourly. The DNA concentration in the supernatant was measured using a micro-spectrophotometer. The results are shown below. Figure 8 .

[0043] like Figure 8 As shown, the time- and dose-dependent increase in intracellular DNA leakage confirms that ZJCK-6-68 can disrupt the integrity of bacterial cell membranes.

[0044] Example 11: Bacterial membrane depolarization experiment USA300 was cultured in TSB medium for 5 hours. Bacterial cells were resuspended 1:100 in 8 mL TSB, and 3,3'-dipropylthiocyanoiodic acid (DiSC) (10 mM, 30 μL) was added. After 1 hour of culture, the cells were washed three times with PBS and resuspended in the same volume of PBS before being added to a 96-well plate containing the compound. Fluorescence intensity was measured and recorded at an excitation wavelength of 622 nm and an emission wavelength of 670 nm. The results are shown in [Figure number missing]. Figure 9 .

[0045] like Figure 9 As shown, the cell membrane potential increased in a dose-dependent manner with respect to ZJCK-6-68, confirming that ZJCK-6-68 can disrupt the bacterial cell membrane, leading to cell membrane depolarization.

[0046] Example 12: Experiment on the addition of exogenous components to the cell membrane The effect of bacterial cell membrane components on the anti-MRSA activity of ZJCK-6-68 was investigated using the checkerboard broth dilution method. Phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and cardiolipin (CL) were diluted with CA-MHB at different concentrations (128, 64, 32, 16, 8, 4, 2, and 1 μg / mL). The test compounds were prepared with sterile water at a stock solution of 1024 μg / mL. First, different concentrations of ZJCK-6-68, PE, PG, and CL were added to 96-well plates. Then, 100 μL LUSA300 (1×10⁻⁶) was added. 6 (CFU / mL), incubate at 37℃ for 16-18 hours, and then read the results. See [link to results]. Figure 10 .

[0047] like Figure 10 As shown, with the increase of PG and PE concentrations, the inhibitory activity of ZJCK-6-68 against methicillin-resistant Staphylococcus aureus (MRSA) gradually decreased. This indicates that ZJCK-6-68 interacts with MRSA membrane components, and the damage to the cell membrane may be related to the competitive binding with membrane components phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), ultimately leading to membrane damage.

[0048] The results of the studies in Examples 6-12 show that the damage to the cell membrane caused by compound ZJCK-6-68 leads to membrane depolarization, cytoplasmic DNA leakage, and DNA synthesis inhibition, ultimately accelerating bacterial death.

[0049] Example 13: ZJCK-6-68 has the ability to reduce golden pigment. The effect of ZJCK-6-68 on pigment production was characterized. Staphylococcus aureus SA77 was co-cultured with different concentrations of ZJCK-6-68 (1×, 1 / 2×, 1 / 4×, 1 / 8×, and 16× MIC). After incubation at 37°C for 24 hours, the culture dishes were washed three times with PBS. Then, the bacterial cells were added to 1 mL of 99% methanol and incubated at 55°C in the dark with shaking for 30 minutes until the precipitate became colorless. The optical density of each well was measured at 450 nm using an automated ELx800 microplate reader. The half-maximum inhibitory concentration (IC50) was calculated by plotting OD450 data against the normal dose inhibition curve. The results are shown below. Figure 11 .

[0050] like Figure 11 As shown, ZJCK-6-68 can inhibit the formation of golden carotenoid pigments in a concentration-dependent manner.

[0051] Example 13: ZJCK-6-68 can inhibit and reduce the expression of inflammatory factors TNF-α, IL-6 and NO in a RAW 264.7 cell inflammation model. An LPS-induced inflammation model of RAW264.7 cells was used to detect the inflammatory factors TNF-α, IL-6, and NO. The results are as follows: Figure 12 As shown, ZJCK-6-68 can inhibit and reduce NO expression in a RAW 264.7 cell inflammation model, with an IC50 of 0.4595 μM, while dexamethasone has an IC50 greater than 5 μM. Figure 12 A). ZJCK-6-68 can inhibit and reduce the expression of TNF-α and IL-6 in a RAW 264.7 cell inflammation model, and is superior to dexamethasone ( Figure 12 B,C).

[0052] Example 14: ZJCK-6-68 exerts its anti-inflammatory effect by inhibiting the DYRK1A signaling pathway. To verify the binding efficiency of ZJCK-6-68 to the target protein DYRK1A, CETSA was used. CETSA is a widely used technique in drug target binding studies; ligand binding can enhance or reduce the thermal stability or protease stability of proteins. The results are as follows: Figure 13 The image shows the changes in the thermostability of DYRK1A protein in RAW264.7 cells after ZJCK-6-68 treatment. The expression of DYRK1A in the ZJCK-6-68-treated group was significantly higher than that in the DMSO-treated group, indicating that ZJCK-6-68 can enhance the thermostability of DYRK1A, suggesting a possible direct interaction between ZJCK-6-68 and DYRK1A, which may be a target of ZLWH-38.

[0053] Example 15: ZJCK-6-68 exhibits low cytotoxicity HEK293T cell line was used and treated with different concentrations (1-256 μg / mL) of ZJCK-6-68, and cell viability was detected by MTT assay. The OD values ​​of each well were measured at 490 nm. Results are shown below. Figure 14 .

[0054] like Figure 14 As shown, within the effective antibacterial concentration range, the survival rate of HEK293T remains above 80%.

[0055] Example 16: ZJCK-6-68 has low hemolytic toxicity The hemolytic activity of ZJCK-6-68 was assessed using fresh sheep red blood cells. First, the red blood cells were washed three times with PBS and prepared into a 4% (v / v) suspension. Then, equal volumes of the red blood cell suspension were mixed with 1% Triton X-100 (positive control), DMSO (solvent control), or different concentrations of ZJCK-6-68 (16–256 μg / mL), respectively. After incubation at 37°C for 1 hour, the supernatant was collected by centrifugation, and the absorbance was measured at 562 nm. The results are shown below. Figure 15 .

[0056] like Figure 15 As shown, ZJCK-6-68 did not induce significant hemolytic reactions within the range of 16-256 μg / mL.

[0057] Example 16: In vivo safety test To assess the in vivo safety of the compound, 20 μL of ZJCK-6-68 at a concentration of 16 μg / mL (4 times the minimum inhibitory concentration) was injected intradermally into mice for 7 days. An equal volume of dimethyl sulfoxide (DMSO) dissolved in physiological saline served as a negative control. The drug and physiological saline were applied to the wounds twice daily. On day 8, blood and internal organs were collected from the mice for routine blood analysis and pathological sections. Results are shown below. Figure 16 .

[0058] like Figure 16 As shown. No significant pathological changes were observed in the structure of any of the organs and tissues. Figure 16 A). There was no statistically significant difference in complete blood cell count parameters between the control group and the ZJCK-6-68 administration group. Figure 16 B). The above results indicate that ZJCK-6-68 has good in vivo safety.

[0059] Example 17: In vivo wound healing study BALB / c mice were randomly divided into three groups (n=5). After shaving the hair on their backs, a circular incision was made in the skin on their backs using a 10 mm diameter biopsy needle. 20 μL of USA300 bacterial suspension (1×10⁻⁶) was inoculated into the incision site. 7 CFU (dissolved in PBS) was administered, and the inoculation was repeated 12 hours later. Treatment began 24 hours later and continued for 4 days: mice in the treatment group received either ZJCK-6-68 or vancomycin (0.4 mg / kg) via wound instillation, while the control group received an equal volume of sterile PBS. All treatments were administered twice daily in 20 μL volumes. Wound area was measured on day 7 post-infection, and a portion of the wound skin tissue was homogenized to determine bacterial load. The concentrations of IL-1β, IL-6, and TNF-α in the skin homogenate were detected using an ELISA kit. The remaining skin tissue was paraffin-embedded, sectioned, and stained with H&E for histopathological observation. Results are shown below. Figure 17 .

[0060] like Figure 17 As shown, compared with the control group, treatment with 0.4 mg / kg ZJCK-6-68 or vancomycin significantly promoted wound healing and effectively reduced the bacterial load in wound tissue. Figure 17 B, C, D, F). Histopathological observation showed that the subcutaneous tissue of the control group mice exhibited full-thickness necrosis (purple arrow), accompanied by extensive inflammatory cell infiltration (orange arrow), necrotic fragments, and abnormal epidermal structure (yellow arrow); the loose arrangement of fibroblasts (green arrow) and neovascularization (cyan arrow) also indicated impaired tissue repair. Both ZJCK-6-68 and vancomycin treatment groups significantly alleviated the above-mentioned pathological damage ( Figure 17 E). Furthermore, ZJCK-6-68 also exhibits significant anti-inflammatory effects, reducing the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the local infected tissue. Figure 17 In summary, ZJCK-6-68 can improve skin infections caused by MRSA.

[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a quinoline compound ZJCK-6-68 in the preparation of antibacterial, anti-inflammatory, and anti-skin infection drugs, characterized in that, The structural formula of compound ZJCK-6-68 is: 。 2. The application of the quinoline compound ZJCK-6-68 according to claim 1 in the preparation of antibacterial, anti-inflammatory, and anti-skin infection drugs, characterized in that, The compound ZJCK-6-68 is used in the preparation of antibacterial drugs by inhibiting biofilm formation.

3. The application of the quinoline compound ZJCK-6-68 according to claim 2 in the preparation of antibacterial, anti-inflammatory, and anti-skin infection drugs, characterized in that, The antibacterial spectrum of the compound ZJCK-6-68 includes MRSA, Enterococcus faecalis, Enterococcus faecium, and Streptococcus pneumoniae.

4. The application of the quinoline compound ZJCK-6-68 according to claim 1 in the preparation of antibacterial, anti-inflammatory, and anti-skin infection drugs, characterized in that, The compound ZJCK-6-68 is used in the preparation of anti-inflammatory drugs by inhibiting the expression of inflammatory factors TNF-α, IL-6 and NO in a RAW 264.7 cell inflammation model.

5. The application of the quinoline compound ZJCK-6-68 according to claim 1 in the preparation of antibacterial, anti-inflammatory, and anti-skin infection drugs, characterized in that, The compound ZJCK-6-68 is used in the preparation of anti-inflammatory drugs by inhibiting the DYRK1A signaling pathway.

6. The application of the quinoline compound ZJCK-6-68 according to claim 1 in the preparation of antibacterial, anti-inflammatory, and anti-skin infection drugs, characterized in that, The compound ZJCK-6-68 can be used in the preparation of drugs for treating skin infections through its anti-MRSA and anti-inflammatory activities.

7. The use of the quinoline compound ZJCK-6-68 according to any one of claims 1-6 in the preparation of antibacterial, anti-inflammatory, and anti-skin infection drugs, characterized in that, The compound ZJCK-6-68 is used as an active ingredient in the preparation of antibacterial and / or anti-inflammatory and / or anti-skin infection drugs.

8. The application of the quinoline compound ZJCK-6-68 according to claim 7 in the preparation of antibacterial, anti-inflammatory, and anti-skin infection drugs, characterized in that, The drug may also include pharmaceutically acceptable carriers or adjuvants.

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