Antibacterial composition with synergistic effect and application thereof

Abstract

The invention provides an antibacterial composition with a synergistic interaction effect and application thereof.The antibacterial composition comprises a compound QA and cephalosporin antibiotics, the activity of NDM-1 and VIM-2 can be effectively inhibited, when the compound QA and the cephalosporin antibiotics are used in a combined mode, the good synergistic antibacterial effect is achieved on CRE such as E.coli BAA2452 producing NDM-1, the infection condition of skin wounds can be effectively improved, and the antibacterial effect is good. And a new thought and direction are provided for clinical treatment of CRE infection.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to an antibacterial composition with synergistic effects and a use thereof. Background Art

[0002] Bacterial infection is a major threat to human society, and carbapenem-resistant Enterobacteriaceae (CRE) is a type of drug-resistant bacteria that poses a serious threat. The β-lactam antibiotics used clinically have broad-spectrum antibacterial activity, and CRE produces New Delhi metallo-s-lactamase-1 (NDM-1), which can hydrolyze almost all bicyclic β-lactam antibiotics, thereby producing a high degree of resistance to β-lactams.

[0003] Therefore, targeting NDM-1, a drug resistance target, and studying new β-lactamase (BLs) inhibitors to reverse the drug resistance of CRE is currently a focus of global attention. Summary of the invention

[0004] The purpose of the present invention is to provide an antibacterial composition with synergistic effects on the basis of the prior art, comprising compound QA and cephalosporin antibiotics, which can effectively inhibit the activity of NDM-1 and VIM-2. The combined use has a good synergistic antibacterial effect on NDM-1-producing CRE such as E. coli BAA2452, can effectively improve the infection condition of skin wounds, and provides new ideas and directions for the clinical treatment of CRE infection.

[0005] The second object of the present invention is to provide the use of the above antibacterial composition with synergistic effect in the preparation of drugs as β-lactamase inhibitors, especially in the preparation of drugs as metal β-lactamase inhibitors.

[0006] The third object of the present invention is to provide the use of the above-mentioned antibacterial composition with synergistic effect in inhibiting the activity of β-lactamase, especially in inhibiting the activity of New Delhi metallo-β-lactamase and VIM-2.

[0007] The fourth object of the present invention is to provide the use of the above-mentioned antibacterial composition with synergistic effect in the preparation of a drug for inhibiting wound infection.

[0008] The technical solution of the present invention is as follows:

[0009] An antibacterial composition with synergistic effect comprises a compound QA and a cephalosporin antibiotic, wherein the structural formula of the compound QA is as follows:

[0010]

[0011] For the present invention, the cephalosporin antibiotic is meropenem, cefixime, cefoperazone, cefazolin sodium, cefodizime sodium, ceftazidime hydrochloride or cefmenoxime hydrochloride.

[0012] In the present invention, the following abbreviations correspond to the following: carbapenem-resistant Enterobacteriaceae (CRE), New Delhi metallo-β-lactamase (NDM-1), β-lactamase (BLs), minimum bactericidal concentration (MBC), metallo-β-lactamase (MBLs), half inhibitory concentration (IC 50 ), minimum inhibitory concentration (MIC), MHB medium (MHB), combined inhibitory concentration index (FIC), optical density (OD) and phosphate buffered saline (PBS).

[0013] The antibacterial composition with synergistic effect provided by the present invention is used in the preparation of a β-lactamase inhibitor drug, preferably in the preparation of a metallo-β-lactamase inhibitor drug, especially in the preparation of a metallo-β-lactamase inhibitor drug. The drug can be made into a solid preparation or a liquid preparation. Further, based on the technical solution provided by the present invention, the drug can be made into an injection, an oral solution, a granule, a powder, a tablet or a capsule.

[0014] The antibacterial composition with synergistic effect provided by the present invention is used in inhibiting the activity of β-lactamase, preferably in inhibiting the activity of metallo-β-lactamase; in particular, in inhibiting the activity of New Delhi metallo-β-lactamase and VIM-2.

[0015] The antibacterial composition with synergistic effect provided by the present invention can effectively improve the infection condition of skin wounds, can be used as a drug for preparing a drug for inhibiting wound infection, and provides a new idea and direction for the clinical treatment of CRE infection. The drug can be made into a solid preparation or a liquid preparation. Furthermore, on the basis of the technical solution provided by the present invention, the drug can be made into an injection, an oral liquid, a granule, a powder, a tablet or a capsule. Pharmaceutically acceptable excipients play a key role in the development of the entire formulation technology. Excipients account for a large part of many preparations, and thus the properties of the excipients determine the properties of the preparation to a large extent. Excellent excipients can enhance the stability of the main drug and prolong the shelf life of the drug; they can regulate the release rate of the main drug in vivo and in vitro; they can change the absorption of the drug in the body and increase the bioavailability, and there is no limitation on the specific type of excipients.

[0016] In the present invention, the inhibitory effect of compound QA on NDM-1 is IC 50=0.24 μM, IC of compound QA against VIM-2 50 =0.71 μM; the FIC of compound QA combined with meropenem against E.coli BAA 2452 was 0.07, which had a significant in vitro synergistic antibacterial effect; compound QA and cefoperazone, cefazolin sodium, cefixime, ceftazidime hydrochloride, cefmenoxime hydrochloride, and cefodizime sodium were used in combination to inhibit E.coli The FICs of BAA2452 were 0.25, 0.53, 1.00, 0.125, 0.09, and 0.188, respectively. Compound QA had a synergistic effect with some cephalosporin antibiotics. The MICs of compound QA for cefoperazone, cefodizime sodium, ceftazidime hydrochloride, and cefotaxime hydrochloride were increased by 8 times, 8 times, 16 times, and 32 times, respectively, at different concentrations. The minimum bactericidal concentration of compound QA and meropenem was 0.186 μg / mL. HRMS results showed that compound QA could stably chelate Zn(II) to form a molecular ion peak. After compound QA chelated Zn(II), the absorption peak at 550 nm disappeared. After compound QA responded to NDM-1, the ultraviolet absorption peak at 550 nm disappeared, indicating that compound QA successfully chelated Zn(II) at the active center of NDM-1. In the presence of 20 μL Zn(II) and 50 μL In the presence of Zn(II), the inhibitory effect of compound QA combined with meropenem on E.coliBAA2452 was weakened, indicating that the antibacterial effect of compound QA combined with meropenem is related to Zn(II).

[0017] For the present invention, the above-mentioned antibacterial composition with synergistic enhancement effect was demonstrated in enzyme inhibition activity experiments, combined drug sensitivity tests, and drug combined minimum bactericidal concentration tests, showing good metallo-β-lactamase inhibition activity. It can produce good in vitro synergistic antibacterial effects when used in combination with antibiotics such as meropenem and cephalosporin, and can effectively improve the infection condition of skin wounds, providing new ideas and directions for the clinical treatment of CRE infections.

[0018] Adopt the technical scheme of the present invention, the advantages are as follows:

[0019] The present invention provides an antibacterial composition with synergistic effects, comprising compound QA and cephalosporin antibiotics, which can effectively inhibit the activities of NDM-1 and VIM-2. When used in combination, it has a good synergistic antibacterial effect on CRE producing NDM-1, such as E. coli BAA2452, and can effectively improve the infection of skin wounds, providing a new idea and direction for the clinical treatment of CRE infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is compound A1 1 H NMR;

[0021] Figure 2 It is the compound QA 1 H NMR;

[0022] Figure 3 The IC of compound QA for inhibiting the activity of NDM-1 and VIM-2 50 curve; among them, Figure 3 a in the middle is NDM-1; Figure 3 b in the middle is VIM-2; Figure 3 c is the IC of QA against NDM-1 in the presence of 20 μM zinc ions 50 ; Figure 3 d in the figure is the IC of QA against NDM-1 in the presence of 50 μM zinc ions 50 ;

[0023] Figure 4 It is the antibacterial effect of compound QA combined with meropenem against E.coli BAA2452;

[0024] Figure 5 It is the antibacterial effect of compound QA combined with cephalosporin antibiotics on 2452;

[0025] Figure 6 The bactericidal effect of compound QA combined with meropenem on 2452; Figure 6 In a, compound QA with a concentration of 32 μg / mL was combined with 4 μg / mL of meropenem; Figure 6 Middle b: 4 μg / mL meropenem used alone; Figure 6 c was 64 μg / mL of compound QA and 2 μg / mL of meropenem; Figure 6 Middle d: 2 μg / mL meropenem alone;

[0026] Figure 7 It is the resin azure stain of E. coli 1864;

[0027] Figure 8 is the HRMS of compound QA chelated with Zn(II); HR-MS(ESI)calculated for C23H21N5O + [M+Zn 2+ -H + ]446.0948, found 466.0957;

[0028] Fig. 9 is the UV absorption spectrum of compound QA and NDM-1; where, Fig. 9 In the figure a, it is the UV absorption spectrum of the interaction between compound QA and zinc ion detected alone; Fig. 9 b is the absorption spectrum of the interaction between compound QA and NDM-1 enzyme;

[0029] Fig.10 The antibacterial effect of compound QA combined with meropenem in the presence of Zn(II) is shown in Table 1. Fig.10 Middle a is the effect of adding 20 μL of 20 μmol / L Zn(II) solution and combining compound QA with meropenem; Fig.10 Middle b shows the effect of compound QA combined with meropenem after adding 20 μL of 50 μmol / L Zn(II) solution;

[0030] Fig.11 This is the growth curve of Escherichia coli when 32μg / mL of compound QA was combined with 2μg / mL of meropenem.

[0031] The control group was a bacterial solution without any treatment; the Mer group was added with meropenem at a concentration of 2 μg / mL; the QA group was added with compound QA at a concentration of 32 μg / mL; the Mer+QA group was added with compound QA at a concentration of 32 μg / mL and meropenem at a concentration of 2 μg / mL respectively;

[0032] Fig.12 are the results of the ELISA test of the biofilm after treatment with different concentrations of QA compounds;

[0033] Fig.13 This is a diagram showing the effect of compound QA combined with meropenem in treating mouse wounds; Fig.13 Middle A shows the healing status of wounds at different days; Fig.13 B is a graph showing the weight changes of mice. DETAILED DESCRIPTION

[0034] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0035] 1. Materials and Methods

[0036] 1 Materials

[0037] 1.1 Bacterial strains

[0038] E. coli BAA 2452.

[0039] 1.2 Reagents

[0040] 8-Aminoquinoline; bromoacetyl bromide; DCM; Et3N; petroleum ether; acetonitrile; methanol; potassium carbonate; dimethylpyridinium; cefixime; cefoperazone; cefazolin sodium; cefodizime sodium; ceftazidime hydrochloride; cefmenoxime hydrochloride; meropenem; MHB medium; Tris; NaCl; Triton X-100; NDM-1; VIM-2; culture dish; LB agar medium.

[0041] 1.3 Instruments

[0042]

[0045] 2. Materials and Methods

[0046] 2.1 Synthesis of compound QA, the specific synthesis route is as follows:

[0047]

[0048] Synthesis of compound A1: 8-aminoquinoline (0.60 g, 4.16 mmol / L), triethylamine (0.40 g, 3.95 mmol / L), and dichloromethane (20 mL) were added to a 50 mL flask in sequence, stirred and mixed at 20-30 ° C, and bromoacetyl bromide (1.20 g, 5.94 mmol / L) was added dropwise using a dropping funnel, and detected using TLC (petroleum ether: dichloromethane = 1:4, V / V). After the reaction was completed for 12 h, silica gel was added and evaporated to dryness using a rotary evaporator. The silica gel powder containing the compound was added to the sample column, and a rapid liquid phase separation chromatography system was used. At a wavelength of 254 nm and a column temperature of 40 ° C, the chromatographic column was activated, and the products were separated according to different polarities, and separated using an eluent (petroleum ether: dichloromethane = 1:4, V / V). Petroleum ether and dichloromethane were evaporated to dryness at 49 ° C using a rotary evaporator to obtain 1.20 g of white powder. The yield was 66.7%. 1 H NMR (400MHz, chloroform-d) δ10.74 (s, 1H), 8.86 (dd, J = 4.3, 1.6 Hz, 1H), 8.74 (dd, J = 5.3, 3.6 Hz, 1H), 8.18 (dd, J = 8.3, 1.5 Hz, 1H), 7.57-7.55 (m, 2H), 7.50-7.47 (m, 1H), 4.15 (d, J = 1.2 Hz, 2H). See related spectra for details. Figure 1 .

[0049] Synthesis of compound QA: Compound A1 (0.50 g, 1.9 mmol / L), dimethylpyridinamine (0.38 g, 1.9 mmol / L), potassium carbonate (0.26 g, 1.9 mmol / L) and 20 mL of solvent acetonitrile were added to a 50 mL reaction bottle and the reaction was monitored by TLC. After 12 h, the reaction was completed, silica gel was added, and the solvent was evaporated under reduced pressure. The silica gel powder containing the compound was added to the sample column, and a rapid liquid phase separation chromatography system was used. At a wavelength of 254 nm and a column temperature of 40 ° C, the chromatographic column was activated, and the products were separated according to different polarities, and the eluent (dichloromethane: methanol = 100: 1, V / V) was used for separation. The brown oil compound QA 0.35 g was obtained by evaporation at 39 ° C using a rotary evaporator, and the yield was 40%. 1 H NMR (400 MHz, DMSO-d6) δ11.47 (s, 1H), 9.06 (dd, J = 4.2, 1.5 Hz, 1H), 8.62 (d, J = 7.6 Hz, 1H), 8.48 (dd, J = 4.8, 0.7 Hz, 2H), 8.42 (dd, J = 8.3, 1.4 Hz, 1H), 7.88 (d, J = 7.8 Hz, 2H), 7.79 (td, J = 7.6, 1.6 Hz, 2H), 7.68 (dd, J = 11.3, 6.9 Hz, 2H), 7.56 (t, J = 7.9 Hz, 1H), 7.24 (dd, J = 7.3, 5.0 Hz, 2H), 3.95 (s, 4H), 3.52 (s, 2H). See related spectra for details. Figure 2 .

[0050] 2.2 Mother liquor preparation

[0051] 2.2.1 Preparation of MHB and LB agar media

[0052] Weigh 4.20 g of MHB medium powder with a 1 / 10,000 analytical balance, dissolve in 250 mL of distilled water, and sterilize at high temperature for later use. Weigh 4.00 g of LB agar medium with a 1 / 10,000 analytical balance, dissolve in 100 mL of distilled water, and sterilize at high temperature for later use.

[0053] 2.2.2 Preparation of compound QA stock solution

[0054] Weigh 2.56 mg of compound QA using a 1 / 10,000 analytical balance and add it to a 2 mL EP tube. Add 10 μL DMSO to dissolve compound QA, and add 1 mL of sterilized MHB liquid medium to fully dissolve it for later use.

[0055] 2.2.3 Preparation of meropenem stock solution

[0056] Weigh 2.56 mg of meropenem using a 1 / 10,000 analytical balance, add 1 mL of sterilized MHB liquid culture medium, and dissolve it fully for later use.

[0057] 2.2.4 Preparation of Cephalosporin Antibiotic Stock Solution

[0058] Use a 1 / 10,000 analytical balance to weigh 8.19 mg of cephalosporin, add 1 mL of sterilized MHB liquid culture medium, and dissolve it fully for later use.

[0059] 2.2.5 Preparation of zinc sulfate mother liquor

[0060] Weigh 14.38 mg of zinc sulfate heptahydrate using a 1 / 10,000 analytical balance, pour into a 2 ml EP tube, dissolve in 1 ml MHB culture medium to a concentration of 50 mmol / L, and dilute 1000 times to 50 μmol / L for later use.

[0061] Weigh 5.75 mg of zinc sulfate heptahydrate using a 1 / 10,000 analytical balance, pour into a 2 ml EP tube, and dissolve in 1 mL of MHB culture medium to a concentration of 20 mmol / L. Dilute 1000 times to 20 μmol / L for later use.

[0062] 2.2.6 Preparation of Resin Azure Mother Solution

[0063] Weigh 1 mg of resin azure using a 1 / 10,000 analytical balance, pour into a 2 ml EP tube, add 1 ml of MHB culture medium, and dissolve fully for later use.

[0064] 2.3 Bacterial Activation

[0065] Take out E.coli BAA 2452 bacterial solution from -80℃ refrigerator, take 20μL of bacterial solution after melting, add it to 5mL EP tube, then add 2mL culture medium, keep constant temperature at 37℃ and 180rpm for 16-18h. Take 200μL and dilute it ten times, and measure OD by UV-Vis spectrophotometer. 600 =0.170.

[0066] 2.4 Compound enzyme inhibition activity test

[0067] 2.4.1 Buffer preparation

[0068] Take Tris (1.21 g, 20 mmol / L), NaCl (5.84 g, 200 mmol / L) and ultrapure water to adjust the pH to 7.5, make up to 500 mL, and add 1‰ Triton X-100 when using.

[0069] 2.4.2 VIM-2 and NDM-1 Configuration

[0070] The stock solutions of VIM-2 and NDM-1 are 200 nmol / L. Take 12 μL of the stock solution and add it into 2 mL of buffer, the concentration is 1.20 nmol / L.

[0071] 2.4.3 Compound configuration

[0072] Weigh 1 mg of compound QA and add 725 μL Tris-HCl buffer to a concentration of 3.6 mmol / L. Add three volumes of Tris-HCl buffer to dilute to 900 μmol / L, and dilute 9 times in sequence, each time by 3 times, that is, the concentrations are 300 μmol / L, 100 μmol / L, 33.33 μmol / L, 11.11 μmol / L, 3.70 μmol / L, 1.23 μmol / L, 0.411 μmol / L, 0.14 μmol / L, and 0.05 μmol / L.

[0073] 2.4.4 Preparation of fluorescent substrate FC-5 stock solution

[0074] The fluorescent substrate FC-5 (2mmol / L) was shaken well when used. 10μL of substrate solution was taken and prepared with 657μL buffer to a solution with a concentration of 30μmol / L (the final concentration of the substrate during the test was 5μmol / L).

[0075] 2.4.5 Filling the black 96-well plate

[0076] In two rows of duplicate wells, add 40 μL of compound to wells 1-10, from high to low concentrations, add 40 μL buffer to well 11, and add 50 μL buffer to well 12. Add 10 μL VIM-2 or NDM-1 to each well in wells 1-11. Incubate for 30 minutes. When ready to test, add 10 μL FC-5 to all wells.

[0077] 2.4.6 Enzyme inhibition activity assay

[0078] After adding 10 μL FC-5, the fluorescence intensity was measured using a multifunctional microplate reader in the kinetic mode with the excitation light λ ex is 380nm, and the emission light λ em The fluorescence intensity was measured at 460 nm over time, and the IC was calculated using GraphPad Prism. 50 .

[0079] In vitro synergistic antibacterial experiment of 2.5 compounds combined with meropenem

[0080] ①Take out the activated E.coli BAA 2452 bacterial solution, pipette 100μL of bacterial solution and 1600μL of MHB medium, and dilute 2452 to OD 600 =0.1, and then dilute the bacterial solution 100 times with MHB medium.

[0081] ② Add the diluted bacterial solution to a 96-well plate, with 200 μL added to well A1, 180 μL added to wells A2-A12 and B1-H1, and 160 μL added to the remaining wells.

[0082] ③ Add the diluted compound QA of different concentrations to B1-B12, C1-C12, and so on until H1-H12 are added.

[0083] ④The steps are the same as the third step, and different concentrations of meropenem are added vertically.

[0084] ⑤ Place the added 96-well plate in a 37°C incubator for 12-16 hours.

[0085] 2.6 Resin Azure Staining

[0086] ①Take out the activated E.coli 1864 bacterial solution, take 100μL of bacterial solution and 1600μL of MHB medium, and dilute 2452 to OD 600 =0.1, and then dilute the bacterial solution 100 times with MHB medium.

[0087] ② Add the diluted bacterial solution to a 96-well plate, with 200 μL added to well A1, 180 μL added to wells A2-A12 and B1-H1, and 160 μL added to the remaining wells.

[0088] ③ Add the diluted compound QA of different concentrations to B1-B12, C1-C12, and so on until H1-H12 are added.

[0089] ④The steps are the same as the third step, and different concentrations of meropenem are added vertically.

[0090] ⑤ Place the added 96-well plate in a 37°C incubator for 12-16 hours.

[0091] ⑥ Pipette 20 μL of 1 mg / mL resin azure and add it to the 96-well plate after culture, and place it in a 37°C incubator to wait for color development.

[0092] 2.7 Compounds combined with cephalosporin antibiotics

[0093] Repeat step 2.5, replace meropenem with cefixime, cefoperazone, cefazolin sodium, cefmenoxime hydrochloride, cefodizime sodium, and ceftazidime hydrochloride, respectively. Adjust the initial concentration of antibiotics to 8.192 mg / mL, and dilute ten times by two times.

[0094] 2.8 Calculation of combined inhibition fraction (FIC)

[0095]

[0096] When FIC ≤ 0.5, 0.5 < FIC ≤ 1, 1 < FIC < 2, and 2 ≤ FIC, they represent synergistic, additive, irrelevant, and antagonistic effects respectively.

[0097] 2.9 MBC determination experiment of the compound

[0098] For MBC determination, the agar dilution method was selected. Concentrations higher than MIC were chosen, including compounds at 32 μg / mL with meropenem at 4 μg / mL and compounds at 64 μg / mL with meropenem at 2 μg / mL. Meropenem at 2 μg / mL and 4 μg / mL was selected as the control group, and the preparation steps are as follows.

[0099] ① Add 100 μL of the compound QA with a concentration of 25.6 mg / mL and 100 μL of meropenem with a concentration of 1.6 mg / mL to a 50 mL centrifuge tube, and then add 40 mL of LB agar medium. Other concentrations can be prepared in the same way.

[0100] ② Pour about 20 mL of LB agar medium containing the compound QA and meropenem into each petri dish. After cooling and solidifying, add 50 μL of E. coli BAA 2452 bacterial solution and spread it evenly with a cotton swab.

[0101] ③ Put the inoculated petri dishes into an incubator at 37 °C for 12 - 16 h, then take them out and observe the number of colonies.

[0102] 2.10 UV - visible absorption spectrum determination of the co - incubation of the compound and NDM - 1

[0103] Take 10 μM / L and 20 μM / L of the compound QA, 20 μM / L of Zn(II) solution, 10 μM / L and 20 μM / L of NDM - 1, and use a UV - visible spectrophotometer to measure the UV - visible absorption spectra of the compound QA, the compound QA and Zn(II), NDM - 1, and NDM - 1 and the compound QA respectively. The wavelength range is 200 nm - 800 nm.

[0104] 2.11 Synergistic experiment of the compound and meropenem in the presence of Zn(II)

[0105] ① Take out the activated E. coli BAA2452 bacterial solution, pipette 100 μL of the bacterial solution and 1600 μL of MHB medium, dilute E. coli BAA 2452 to OD 600 = 0.1, and then dilute the bacterial solution 100 - fold with MHB medium.

[0106] ② Add the diluted bacterial solution to a 96-well plate, with 200 μL added to well A1, 160 μL added to wells A2-A12 and B1-H1, and 140 μL added to the remaining wells. Add 20 μL of 50 μmol / L Zn(II) solution to each well except well A1. Repeat the above steps and add 20 μL of Zn(II) solution (20 μmol / L).

[0107] ③ Dilute 2.56mg / mL meropenem twice ten times to concentrations of 1.28mg / mL, 0.64mg / mL, 0.32mg / mL, 0.16mg / mL, 0.08mg / mL, 0.04mg / mL, 0.02mg / mL, 0.01mg / mL, 0.005mg / mL, 0.0025mg / mL. Add to 96-well plates, add columns 2-12 from low to high concentrations. 20μL per well.

[0108] ④ Dilute the 2.56 mg / mL compound QA six times, with concentrations of 1.28 mg / mL, 0.64 mg / mL, 0.32 mg / mL, 0.16 mg / mL, 0.08 mg / mL, and 0.04 mg / mL. Add them to the 96-well plate, and add them to the BH rows from low to high concentrations, with 20 μL added to each well.

[0109] ⑤ Add 20μmol / L and 50μmol / L to all wells except well A1.

[0110] ⑥ Place the added 96-well plate in a 37°C incubator for 12-16 hours.

[0111] 2.12 Biosafety Tests of QA Compounds

[0112] Weigh 4 mg of compound QA with a 1 / 10,000 balance, add a small amount of DMSO, add 1 mL of saline, dilute 10 times to 400 μg / mL, and then dilute 2 times to 200 μg / mL and 100 μg / mL. Divide the wax moth into four groups, one group is injected with saline blank control, and the remaining groups of 10 wax moths are injected with 10 μL of different concentrations of compound QA, and the survival of wax moths is continuously observed.

[0113] 3. Materials and Methods

[0114] 1. Inhibition of enzyme activity by compound QA

[0115] When compound QA reacts with NDM-1 as an inhibitor, after incubation for 30 min, the higher the concentration of compound QA, the stronger the inhibitory effect on NDN-1. The IC value of the inhibitory effect of compound QA on NDM-1 obtained by fitting the steady-state enzyme inhibition kinetic data 50 =0.24μM, the fitting curve is as follows Figure 3As shown in a.

[0116] When compound QA reacts with VIM-2 as an inhibitor, after incubation for 30 min, the higher the concentration of compound QA, the stronger the inhibitory effect on VIM-2. The IC value of compound QA inhibitory effect on VIM-2 obtained by fitting the steady-state enzyme inhibition kinetic data 50 =0.71μM, the fitting curve is as follows Figure 3 As shown in b.

[0117] When compound QA was added as an inhibitor and reacted with NDM-1 at 20 μM zinc ions, the inhibitory effect of compound QA on NDN-1 weakened after 30 min of incubation. The IC value of the inhibitory effect of compound QA on NDM-1 obtained by fitting the steady-state enzyme inhibition kinetic data was 50 =52μM, the fitting curve is as follows Figure 3 As shown in c.

[0118] When compound QA was added as an inhibitor and reacted with NDM-1 at 50 μM zinc ions, the inhibitory effect of compound QA on NDN-1 was further weakened after incubation for 30 min. The IC value of the inhibitory effect of compound QA on NDM-1 obtained by fitting the steady-state enzyme inhibition kinetic data was 50 =160μM, the fitting curve is as follows Figure 3 As shown in d.

[0119] 2. Antibacterial effect of compounds

[0120] 2.1 Antibacterial effect of compound QA combined with meropenem on E. coli BAA2452

[0121] like Figure 4 As shown, the minimum inhibitory concentration (MIC) of compound QA is greater than 256 μg / mL, the MIC of meropenem is 64 μg / mL, and the combined inhibitory index (FIC) of compound QA and meropenem is 0.07, indicating that compound QA and meropenem have a synergistic effect. Compared with single use, the MIC of meropenem in the case of combined use is 0.5 μg / mL, which is increased by 128 times.

[0122] 2.2 Antibacterial effect of compound QA combined with cephalosporin antibiotics against E. coli BAA2452

[0123] like Figure 5 As shown in a, the MIC of compound QA is greater than 256 μg / mL, the MIC of cefoperazone is 1024 μg / mL, and the FIC of compound QA and cefoperazone is 0.25, indicating that compound QA and cefoperazone have a synergistic effect. Compared with the use of cefoperazone alone, the MIC of cefoperazone in the combined use is 128 μg / mL, which is 8 times higher. Figure 5As shown in Figure b, the MIC of compound QA is greater than 256 μg / mL, the MIC of cefixime is 2048 μg / mL, and the FIC of compound QA and cefixime is 1.00, indicating that compound QA and cefixime have an additive effect. Figure 5 The MIC of compound QA shown in c is greater than 256 μg / mL, the MIC of cefazolin sodium is 4096 μg / mL, and the FIC of compound QA and cefazolin sodium is 0.53, indicating that compound QA and cefazolin sodium have an additive effect. Figure 5 d shows that the MIC of compound QA is greater than 256 μg / mL, the MIC of cefodizime sodium is 2048 μg / mL, and the FIC of compound QA and cefodizime sodium is 0.188, indicating that compound QA and cefodizime sodium have synergistic effects. Compared with the use of cefodizime sodium alone, the MIC of cefodizime sodium in the combined use is 256 μg / mL, which is 8 times higher. Figure 5 As shown in Figure e, the MIC of compound QA is greater than 256 μg / mL, the MIC of ceftazidime hydrochloride is 2048 μg / mL, and the FIC of compound QA and ceftazidime hydrochloride is 0.125, indicating that compound QA and ceftazidime hydrochloride have a synergistic effect. Compared with the use of ceftazidime hydrochloride alone, the MIC of ceftazidime hydrochloride in the combined use is 128 μg / mL, which is increased by 16 times. Figure 5 As shown in Figure f, the MIC of compound QA is greater than 256 μg / mL, the MIC of cefmenoxime hydrochloride is 1024 μg / mL, and the FIC of compound QA and cefmenoxime hydrochloride is 0.09, indicating that compound QA and cefmenoxime hydrochloride have a synergistic effect. Compared with the use of cefmenoxime hydrochloride alone, the MIC of cefmenoxime hydrochloride in the combined use is 32 μg / mL, which is increased by 32 times.

[0124] 2.3 Minimum bactericidal concentration of compound QA combined with meropenem against E. coli BAA2452

[0125] Depend on Figure 6 As shown, when the concentration of compound QA was 32 μg / mL and the concentration of meropenem was 4 μg / mL, the bactericidal effect on E. coli BAA2452 was the strongest, and no colonies were visible to the naked eye. The MBC at this time was 0.186 μg / mL.

[0126] 2.4 Resin Azure Staining

[0127] Depend on Figure 7As shown, the synergistic antibacterial effect of meropenem combined with QA on E.Coli 1864 was investigated. Bacteria stained with resin azure: blue indicates bacterial inhibition, and red indicates a large amount of bacterial growth. In a 96-well plate, the MIC of meropenem alone was 16 μg / mL. After adding 32 μg / mL of compound QA, the MIC dropped to 0.5 μg / mL. By calculating the FIC, the FIC was 0.16 at this time, which showed a synergistic effect.

[0128] 3 Mechanism of action of compound QA

[0129] 3.1 HRMS of compounds chelated with Zn(II)

[0130] like Figure 8 As shown, the HRMS results showed that compound QA could stably chelate Zn(II) to form a molecular ion peak.

[0131] 3.2 UV-visible absorption spectrum experiment

[0132] like Fig. 9 As shown in a, compound QA has a maximum UV absorption peak at 266nm, and after reacting with Zn(II), it has a maximum UV absorption peak at 261nm. Fig. 9 As shown in b, NDM-1 has a maximum UV absorption peak at 211nm, and after reacting with compound QA, it has a maximum UV absorption peak at 214nm. More importantly, Fig. 9 a, After compound QA binds to Zn(II), the ultraviolet absorption peak around 550nm disappears. Fig. 9 b. After compound QA was co-incubated with NDM-1, the UV absorption peak at 550 nm disappeared, indicating that compound QA chelated with Zn(II), thereby depriving the active center of NDM-1 of Zn(II), thereby exerting an inhibitory effect on NDM-1.

[0133] 3.3 Synergistic antibacterial experiment of compounds and meropenem in the presence of Zn(II)

[0134] like Fig.10 As shown in a, after adding 20 μg / mL Zn(II), the MIC of compound QA was greater than 256 μg / mL, and the MIC of meropenem was 128 μg / mL. After adding Zn(II), the FIC of compound QA and meropenem was 0.07, indicating that after adding 20 μmol / L Zn(II), the synergistic effect of compound QA and meropenem was weakened. Fig.10 As shown in b, after adding 50 μmol / L Zn(II), the MIC of compound QA was greater than 256 μg / mL, and the MIC of meropenem was 128 μg / mL. After adding Zn(II), the FIC of compound QA and meropenem was 0.26, which was similar to Figure 3In comparison, the synergistic effect of compound QA and meropenem was reduced, indicating that compound QA inhibited the activity of E. coli BAA2452 by chelating Zn(II), and the addition of exogenous Zn(II) could counteract the synergistic effect of compound QA and meropenem.

[0135] 4 Bacterial growth curve experiment

[0136] Experimental plan: E. coli BAA 2452 was cultured overnight at 37°C and 180 rpm and transferred into LB medium at a ratio of 1:100, and divided into the control group, the Mer group with a final concentration of 2 μg / mL meropenem, the compound QA group with a concentration of 64 μg / mL, and the Mer+QA group with 2 μg / mL meropenem combined with 64 μg / mL QA, and added to 96-well plates. Cultured at 37°C, OD was measured at 0, 2, 4, 6, 8, 10, 12, 14, and 24 h. 600 , sort the values ​​and plot them using Graph Prism.

[0137] Experimental results: Fig.11 It can be seen that the bacterial growth in the QA group was similar to that in the Control group. The Mer group could inhibit bacterial growth within 8 hours. After 8 hours, meropenem lost its antibacterial activity due to bacterial decomposition, and bacterial growth resumed. In the Mer+QA group, QA inhibited the hydrolysis of meropenem by bacteria, and meropenem played a good antibacterial role within 24 hours.

[0138] 5 Biofilm removal experiment

[0139] Experimental plan: E. coli BAA 2452 was cultured at 37°C and 180 rpm overnight, and the bacterial solution was diluted to OD 600 =0.1, dilute 100 times, take 200μL of bacterial solution and add it to a 96-well plate, and culture for 24 hours. Aspirate the culture medium, take 200μL of QA of different concentrations and add it to a 96-well plate, incubate together for 25 minutes, aspirate the antibacterial agent, wash each well with 300μL of double distilled water twice, and dry for 5 minutes. Add 200μL of crystal violet stain and incubate for 25 minutes. Then the staining solution was aspirated, and each well was washed 3 times with 300μL of double distilled water, and then dried for 5 minutes. Add 200μL of 95% ethanol to each well and incubate at room temperature for 20 minutes. Use a multifunctional microplate reader to read OD 570 The absorbance values ​​of all samples were calculated and the biofilm removal rate was calculated.

[0140] Experimental results: Fig.12 It can be seen that QA can effectively remove the biofilm formed by E. coli at concentrations of 4, 8, and 16 μg / mL, indicating that QA has good biofilm removal activity.

[0141] 6 Mouse wound healing experiment

[0142] Wounds with a diameter of 8 mm were made on the back of mice, and the mice were divided into 4 groups: control group, meropenem group (Mer), compound QA group (QA), and combined drug group (Mer+QA). Each group was given 20 μL of E. coli BAA 1864 (1*10 8 CFU), the model was built continuously for 3 days, and when abscess appeared in the wound, the control group was treated with PBS, the meropenem group was intraperitoneally injected with 10 mg / kg meropenem, the QA group was given 20 μL of QA compound (concentration was 64 μg / mL), and the combined group was given 20 μL of QA compound (concentration was 64 μg / mL) and intraperitoneal injection of 10 mg / kg meropenem to the mouse wound. The drug administration was continued and the wound healing was recorded.

[0143] Experimental results: Fig.13 A shows that compared with the control, QA and meropenem alone can improve wound infection to a certain extent. When QA and meropenem are used together, they can effectively inhibit bacterial growth and accelerate wound healing. This shows that QA can effectively improve the infection of mouse skin wounds by removing biofilm and its combined antibacterial effect with meropenem; Fig.13 B shows that during the experiment, the weight of the mice did not change significantly, which indirectly illustrates the biosafety of the QA compound.

[0144] 7. Biosafety of Compound QA

[0145] Compound QA of different concentrations was dissolved in physiological saline, and 10 μL was injected into each wax moth. The moth was observed for 7 days to see whether it changed color and whether it responded to stimulation, and the conclusion was drawn.

[0146] Table 1 Survival of G. mellonella after injection of different concentrations of compound QA

[0147]

[0148] As shown in Table 1, after observing the wax moth for several days, only one wax moth treated with 200 μg / mL of compound QA died, which is within the error range. Therefore, compound QA has good biosafety.

[0149] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that it is still possible to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on some of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibacterial composition with synergistic effect, characterized in that: The antibacterial composition comprises a compound QA and a cephalosporin antibiotic, wherein the structural formula of the compound QA is as follows:

2. The antibacterial composition with synergistic effect according to claim 1, characterized in that: The cephalosporin antibiotic is meropenem, cefixime, cefoperazone, cefazolin sodium, cefodizime sodium, ceftazidime hydrochloride or cefmenoxime hydrochloride.

3. Use of the antibacterial composition with synergistic effect as claimed in claim 1 in the preparation of a β-lactamase inhibitor drug.

4. The use according to claim 3, wherein the antibacterial composition is used in the preparation of a drug as a metallo-β-lactamase inhibitor.

5. Use of the antibacterial composition with synergistic effect as claimed in claim 1 in inhibiting the activity of β-lactamase.

6. The use according to claim 5, wherein the antibacterial composition is used for inhibiting the activity of metallo-β-lactamase.

7. The use according to claim 6, wherein the antibacterial composition is used for inhibiting the activity of New Delhi metallo-β-lactamase and VIM-2.

8. Use of the antibacterial composition with synergistic effect as claimed in claim 1 in the preparation of a medicament for inhibiting wound infection.

9. The use according to claim 8, characterized in that: The medicine is prepared into a liquid preparation or a solid preparation.

10. The use according to claim 9, characterized in that: The medicine is prepared into injection, oral liquid, granules, powder, tablets or capsules.

Images