An antibacterial agent and its preparation method and application

Abstract

The present invention discloses an antibacterial agent and a preparation method and application thereof. The antibacterial agent includes an organic small molecule and an antibacterial drug, wherein the organic small molecule and the antibacterial drug are connected by an ester bond, and the organic small molecule is carboxylic acid betaine or mannose. The present invention connects the organic small molecule carboxylic acid betaine or mannose with the antibacterial drug by an ester bond, and can self-assemble in water to form micelles with pH-induced charge flipping function, wherein the surface of the micelle presents a negative charge in a normal physiological environment, and the surface of the micelle presents a positive charge when the micelle is in an acidic environment of bacteria. Such micelles with pH-inducing function are not only conducive to long-term circulation in the body, but also can strengthen the interaction between bacteria and antibacterial drugs by electrostatic adsorption. The micelles of the present invention can effectively penetrate and eliminate biofilms, thereby effectively inhibiting bacterial growth, and are a very promising intelligent nano drug delivery system.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial agents, and in particular to an antimicrobial agent and a preparation method and application thereof. Background Art

[0002] Bacteria are unique among prokaryotes and also play an important role in maintaining the balance of our living environment. Most bacteria do not cause infection, but a small number of bacteria can cause infection and disease. Bacterial infections caused by pathogenic bacteria, if not diagnosed and treated in time, will cause irreversible damage, and have now led to many serious diseases, such as sepsis, bacteremia, pneumonia, endocarditis, and even death. Antibiotics are considered to be the most effective drugs for treating bacterial infections, which reduce the morbidity and mortality caused by bacterial infections and even save the lives of infected people. However, frequent use of high doses of antibiotics to maintain therapeutic levels causes serious side effects on the liver and other normal tissues. In addition, antibiotic treatment lacks specificity and is rapidly metabolized and excreted before reaching the site of infection through the body's circulatory system, resulting in low antibiotic bioavailability. The overuse of antibiotics has led to the emergence of drug-resistant strains, reducing the effectiveness of clinical antimicrobial drugs. Therefore, addressing bacterial resistance is a major challenge facing the world today.

[0003] Bacteria in biofilms wrap themselves in a shielding extracellular polymer matrix (EPS), which is basically composed of proteins, extracellular bacterial DNA, exopolysaccharides and enzymes. The extracellular polysaccharide matrix can act as a protective barrier, not only protecting bacteria from the host's innate immune cells but also preventing the penetration of antibiotics. Therefore, the amount of antibiotics used to treat biofilm infections is 100-1000 times that of ordinary planktonic bacteria. The large-scale use of antibiotics leads to a greater chance of drug-resistant bacteria. This is because antibiotics have the disadvantages of low bioavailability, low penetration into biofilms, short half-life and high toxicity in the treatment of biofilm infections.

[0004] Therefore, it is very meaningful to study an antibacterial agent that can penetrate biofilm and thus improve its bactericidal performance. Summary of the invention

[0005] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an antibacterial agent and a preparation method and application thereof, so as to solve the problems that the existing antibiotics have poor cell membrane penetration effect.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: an antibacterial agent is provided, which includes an organic small molecule and an antibacterial drug, the organic small molecule and the antibacterial drug are connected by an ester bond, and the organic small molecule is carboxylic acid betaine or mannose.

[0007] The beneficial effects of the present invention are as follows: organic small molecule carboxylic acid betaine or mannose is connected to an antibacterial drug through an ester bond to form an antibacterial agent, which can self-assemble to form micelles with pH-induced charge flipping function. The micelles present a negative charge on the surface in a normal physiological environment (pH 7.4), and present a positive charge on the surface when the micelles are in the acidic environment of bacteria (pH 5.5). They can effectively penetrate and accumulate in the biofilm, deliver the antibacterial drug to the inside of the biofilm, and release the antibacterial drug by degrading the ester bond through the lipase secreted by the bacteria, thereby improving the bactericidal performance against planktonic bacteria and bacteria in the biofilm. Such micelles with pH-inducing function are not only conducive to long-term circulation in the body, but also can strengthen the interaction between bacteria and antibacterial drugs through electrostatic adsorption.

[0008] Furthermore, the antibacterial drug is a quinolone substance, a penicillin substance or a cephalosporin substance.

[0009] Furthermore, the quinolone substance is ciprofloxacin or a substance having the following structural formula:

[0010]

[0011] Wherein, R1 is C2H5, C3H5, C2H4F or ;

[0012] R2 is H or NH2 or CH3;

[0013] R3 is F or H, or does not exist, which means that R3 does not exist when Y is N, etc.;

[0014] R4 is , , , , , , , , or ;

[0015] R5 is H, F, CH3O or F2CHO, or does not exist, which means that R5 does not exist when Y is N, etc.;

[0016] X is N or C;

[0017] Y is N or C.

[0018] Further, the structural formula of penicillin is as follows:

[0019]

[0020] Among them, R1 is , , , , , , , , , or .

[0021] Furthermore, the structural formula of cephalosporins is as follows:

[0022]

[0023] Among them, R1 is , , , , CH2CN or ;

[0024] R2 is , , 、CH3、Cl、H、 or .

[0025] The preparation method of the above antibacterial agent, when the organic small molecule is carboxylic acid betaine, comprises the following steps:

[0026] (1) dissolving the antibiotic, then adding di-tert-butyl dicarbonate to react at room temperature under an inert gas atmosphere to obtain a Boc-protected antibiotic;

[0027] (2) Mixing a Boc-protected antibacterial drug, N,N-dimethylaminoethyl glycol, TBTU and DIPEA in a molar ratio of 1:2-4:3-5:6-8, adding the mixture to a solvent, and stirring the mixture at room temperature for 10-15 hours to obtain a product;

[0028] (3) dissolving the product obtained in step (2), then adding tert-butyl bromoacetate and a base to react at 45-55° C. under an inert gas atmosphere for 0.5-1.5 h to obtain the product;

[0029] (4) The product obtained in step (3) is subjected to a de-tert-butyl esterification reaction in a mixed solvent of dichloromethane and trifluoroacetic acid, followed by precipitation and filtration to obtain an antibacterial agent.

[0030] Furthermore, the base in step (3) is anhydrous sodium methoxide, anhydrous potassium carbonate or anhydrous sodium carbonate.

[0031] Furthermore, in step (4), the de-tert-butyl esterification reaction conditions are room temperature and the reaction time is 1.5-3.5 hours.

[0032] The method for preparing the antibacterial agent, when the organic small molecule is mannose, comprises the following steps:

[0033] (1) dissolving mannose in an ice bath, then adding acetic anhydride and reacting for 25-35 min in an ice bath, and then continuing to react at room temperature for 2-4 h to obtain acetyl mannose;

[0034] (2) dissolving acetyl mannose and TEG, then adding a catalyst, and reacting at room temperature for 34-38 hours to obtain a product; wherein the molar ratio of acetyl mannose, TEG, and catalyst is 1:3-4:8-10;

[0035] (3) dissolving the antibiotic, and then adding di-tert-butyl dicarbonate to react at room temperature under an inert gas atmosphere to obtain a Boc-protected antibiotic;

[0036] (4) mixing the Boc-protected antibacterial drug, the product obtained in step (2), TBTU, and DIPEA in a molar ratio of 1:2-3:3-5:5-7, adding the mixture to a solvent, and stirring at room temperature for 10-14 hours to obtain a product;

[0037] (5) dissolving the product obtained in step (4), then adding a base and reacting at room temperature for 10-20 minutes to obtain the product;

[0038] (6) The product obtained in step (5) is dissolved, and then subjected to a de-tert-butyl esterification reaction in a mixed solvent of dichloromethane and trifluoroacetic acid, followed by precipitation and filtration to obtain an antibacterial agent.

[0039] Furthermore, in step (2), the catalyst is boron trifluoride etherate.

[0040] Furthermore, the base in step (5) is anhydrous sodium methoxide, anhydrous potassium carbonate or anhydrous sodium carbonate.

[0041] Furthermore, in step (6), the de-tert-butyl esterification reaction conditions are room temperature and the reaction time is 1.5-3.5 hours.

[0042] The above antimicrobial agents have good application prospects in penetrating or removing bacterial biofilms or inhibiting bacterial growth.

[0043] The present invention has the following beneficial effects:

[0044] The present invention connects organic small molecule substances such as carboxylic acid betaine or mannose with antibacterial drugs through ester bonds, and can self-assemble in water to form micelles with pH-induced charge reversal function. The micelles have a negative charge on the surface in a normal physiological environment (pH 7.4), and a positive charge on the surface when the micelles are in the acidic environment of bacteria (pH 5.5). Such micelles with pH-inducing function are not only conducive to long-term circulation in the body, but also can enhance the interaction between bacteria and antibacterial drugs through electrostatic adsorption.

[0045] The antibacterial drug in the antibacterial agent of the present invention can be a quinolone substance, a penicillin substance or a cephalosporin substance. Taking ciprofloxacin in the quinolone substance as an example, the micelles prepared by modification with carboxylic acid betaine are more effective against Staphylococcus aureus ( S.aureus ) and Escherichia coli ( E.coil ) showed a good bactericidal effect. More importantly, since bacterial biofilm is a microbial community with a complex structure, it has a strong resistance to traditional antibiotics. The micelles of the present invention have a good penetration effect on biofilm in an acidic environment, which is conducive to eliminating biofilm. The above results show that the ciprofloxacin small molecule micelle modified with carboxylic acid betaine is a very promising intelligent nano drug delivery system.

[0046] Taking ciprofloxacin in the quinolone class as an example, it was modified with mannose to form micelles in the form of polyvalent sugars in the solution. Antibacterial experiments proved that mannose enhanced the antibacterial activity of ciprofloxacin due to its polyvalent sugar effect on Escherichia coli. Scanning electron microscopy and laser confocal microscopy then verified that the mannose-modified antibiotics could prevent the colonization of Escherichia coli and reduce the formation of biofilms. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the characterization result of CIP-CB micelles.

[0048] Figure 2 This is the antibacterial test result of CIP-CB micelles; among them, each group of bar graphs in each small figure are 81.8ppm, 41.9ppm, 20.5ppm, 10.2ppm, 5.10ppm, 2.55ppm, 1.28ppm, and control group from left to right; the time of each group of bar graphs from left to right is 2h, 4h, 8h, 16h, and 24h.

[0049] Figure 3 The antibacterial effect of CIP-CB micelles on Escherichia coli and Staphylococcus aureus at different concentrations and pH conditions.

[0050] Figure 4 The antibacterial effect of CIP-CB micelles on drug-resistant bacteria at different concentrations and pH conditions.

[0051] Figure 5 The results of CIP-CB anti-biofilm testing were performed using the standard plate count method.

[0052] Figure 6 Confocal laser scanning microscopy was used to verify the anti-biofilm results of CIP-CB.

[0053] Figure 7The graph shows the hemolysis rate of CIP-CB and the results after centrifugation for 2 hours after co-culture with red blood cells.

[0054] Figure 8 DLS (A) and transmission electron microscopy (TEM) images of Man-TEG-CIP micelles.

[0055] Figure 9 This is the critical micelle concentration result of Man-TEG-CIP micelles.

[0056] Figure 10 Laser confocal images of Escherichia coli biofilm after incubation with Man-TEG-CIP micelles for different time periods.

[0057] Figure 11 Scanning electron microscopy images of Escherichia coli biofilms after incubation with Man-TEG-CIP micelles for different time periods.

[0058] Figure 12 The graph shows the hemolysis rate of Man-TEG-CIP micelles and the results after centrifugation for 2 hours after co-culture with red blood cells. DETAILED DESCRIPTION

[0059] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0060] Embodiment 1:

[0061] An antibacterial agent, the antibacterial agent is CIP-CB, and its synthesis route is as follows:

[0062]

[0063] The specific steps are:

[0064] (a) Add 4.5 mL 1N NaOH and 40 mL dioxane: water = 1:1 (volume ratio) to ciprofloxacin (1.0 g, 3.00 mmol), then add di-tert-butyl dicarbonate Boc2O (1.0 g, 4.6 mmol), react at room temperature under nitrogen atmosphere for 17 h until the reaction is complete, filter the white solid, rinse with water and acetone, and dry in vacuum. The product is a white to light yellow solid (Boc-CIP, 98%) detected by developing solvent TLC (DCM:MeOH = 9:1), and its NMR data are: 1 H NMR (400 MHz, CDCl3) d (ppm): 8.76 (s, 1H), 8.02 (d, J= 12.9 Hz, 1H), 7.36 (d, J = 7.1Hz, 1H), 3.67 (t, J = 4.9 Hz, 4H), 3.54 (m, 1H), 3.29 (t, J = 5.2 Hz, 4H), 1.5(s, 9H), 1.39 (m, 2H), 1.20 (d, J = 3.8 Hz, 2H).

[0065] (b) Boc-CIP (0.5 mmol, 1.917 g), N,N-dimethylaminoethyl glycol DMAEE (1.0 mmol, 0.1331 g), TBTU (1.5 mmol, 0.481 g), and DIPEA (3.0 mmol, 0.388 g) were added to a dry dichloromethane solution. After stirring at room temperature for 12 h, the reaction was detected to be complete. The solvent was dried and then precipitated with ether to obtain a light yellow precipitate. The precipitate was filtered, dissolved with 100 mL of dichloromethane, and extracted three times with 100 mL of water to remove the catalyst. After drying and drying, the product Boc-CIP-DMAEE (87%) was obtained by chromatography. Its NMR data are as follows: 1 H NMR (400 MHz, CDCl3) d (ppm): 8.55(s, 1H), 8.00 (d, J = 13.1 Hz, 1H), 7.28 (d, J = 7.08 Hz, 1H), 4.46 (t, J = 4.76Hz, 2H), 3.81 (t, J = 5.0 Hz, 2H), 3.77 (t, J = 5.4 Hz, 2H), 3.65 (t, J = 4.68 Hz,4H), 3.44 (m, 1H), 3.22 (t, J = 5.0 Hz, 4H), 2.84 (t, J = 5.4 Hz, 2H), 2.52 (s,6H), 1.49 (s, 9H), 1.33 (m, 2H), 1.13 (m, 2H). 13 C NMR (400 MHz, CDCl3) d(ppm):166.92, 165.14, 154.57, 151.59, 148.17, 137.93, 131.18, 123.23, 113.55,113.32, 110.10, 109.08, 104.98, 104.96, 80.22, 80.18, 69.12, 68.96, 64.28,63.68, 63.65, 58.72, 50.32, 49.89, 45.75, 34.74, 28.39, 8.17. HRMS (ESI) m / z:calcd for C 28 H 40 FN4O6 + (M+H) + 547.29264, found 547.29309.

[0066] (c) Boc-CIP-DMAEE (0.015 mol, 8.19 g) was dissolved in 20 mL of acetonitrile, and then tert-butyl bromoacetate (0.022 mol, 4.34 g) and a small amount of anhydrous sodium carbonate as a catalyst were added. The mixture was reacted at 50°C under a nitrogen atmosphere for 1 h. After the reaction was completed, Boc-CIP-OB-tBu (99%) was precipitated with ether. Its NMR data were as follows: 1 H NMR (400 MHz,CDCl3) d (ppm): 8.61 (s, 1H), 7.89 (d, J = 13.0 Hz, 1H), 7.33 (d, J = 7.0 Hz, 1H),4.40 (t, J = 3.6 Hz, 2H), 4.22 (s, 2H), 4.06 (m, 2H), 3.89 (m, 2H), 3.83 (m,2H), 3.9 (t, J = 5.4 Hz, 2H), 3.83 (t, J = 4.7 Hz, 2H), 3.64 (t, J = 4.7 Hz, 4H), 3.53 (m, 1H), 3.37 (s, 6H), 3.23 (t, J = 5.0 Hz, 4H), 1.49 (s, 9H), 1.45 (s, 9H), 1.35 (m, 2H), 1.15 (m, 2H). 13 C NMR (400 MHz, CDCl3) d (ppm): 172.84, 163.82,154.54, 152.25, 152.20, 151.53, 151.43, 149.05, 144.08, 142.87, 138.93,138.15, 113.09, 112.86, 110.71, 105.36, 105.33, 84.81, 80.19, 77.32, 77.00,76.68, 69.21, 65.17, 63.89, 62.45, 52.84, 49.85, 34.94, 34.92, 29.64, 28.36,27.92, 8.25. HRMS (ESI) m / z calcd for C 34 H 50 FN4O8 + (M) + 661.36072, found661.36102.

[0067] (d) Boc-CIP-OB-tBu was dissolved in a dichloromethane:trifluoroacetic acid (1:1, v / v) solution and reacted at room temperature for 2 h. After the tert-butyl ester was removed, the product CIP-CB (95%) was obtained by precipitation in ether, stirring, and filtration. Its NMR data were as follows: 1 HNMR (400 MHz, MeOD- d 4) d (ppm): 8.76 (s, 1H), 8.02 (d, J = 13.0 Hz, 1H), 7.63 (d, J = 6.8 Hz, 1H), 4.47 (t, J = 3.7 Hz, 2H), 4.33 (s, 2H), 4.01 (m, 2H), 3.89 (m,2H), 3.85 (m, 2H), 3.71 (m,1H), 3.59 (m, 4H), 3.48 (t, J = 5.0 Hz, 4H), 3.34(s, 6H), 1.40 (m, 2H), 1.18 (m, 2H). 13 C NMR (400 MHz, MeOD- d 4) d(ppm): 178.14,166.06, 154.54, 152.17, 149.14, 143.65, 143.54, 138.41, 112.11, 111.89,109.98, 108.86, 106.75, 106.54, 104.72, 68.89, 64.30, 64.20, 63.57, 63.15,61.97, 51.97, 43.26, 34.97, 26.72, 7.22. HRMS (ESI) m / z calcd for C 25 H 34 FN4O6 + (M) + 505.24569, found 505.24597.

[0068] The CIP-CB prepared above was prepared into micelles. The specific process was as follows: 20 mg CIP-CB was dissolved in 20 mL PBS with pH = 7.4 and stirred at room temperature for 2 h to obtain uniformly distributed micelles with a concentration of 1 mg / mL.

[0069] 1. The CIP-CB micelles obtained above were characterized as follows: The particle size and Zeta potential of CIP-CB micelles were detected by laser particle size analyzer (Mastersier 2000), the morphology was detected by transmission electron microscope TEM (FEI Tecnai G2 F20), and the critical micelle (CMC) was determined by spectrophotometer using Nile red as a fluorescent probe. The specific operation steps are as follows: 1 mg of Nile red was dissolved in 50 mL of tetrahydrofuran solution, mixed evenly, 100 μL was taken from it and added to a glass bottle, placed in a fume hood to evaporate the solvent naturally, and then 4 mL of micelles were added, and the concentration was increased from 0.2 mg / mL to 1 10 -5 mg / mL, and then shaken in a constant temperature shaker at 40°C overnight. After standing for half a day, the fluorescence emission intensity of the micelles was tested using a fluorescence spectrometer. The slit widths of the excitation light and emission light were both 20 nm, the excitation wavelength was 550 nm, and the emission range was 570-750 nm.

[0070] The above characterization results can be found in Figure 1 , Figure 1A is the hydrodynamic diameter distribution of CIP-CB micelles; B is the Zeta potential distribution of CIP-CB micelles at different pH values; C is a TEM image of the sample stained with 2.5% phosphotungstic acid; D is the relationship between the emission intensity of Nile red at 575 nm and the CIP-CB concentration. Figure 1 It can be seen that CIP-CB micelles are uniformly dispersed in water, and the morphology is a spherical core-shell structure. Then, DLS was used to measure the potential changes of CIP-CB at different pH at 25°C. In the normal physiological environment in the body (pH 7.4), the carboxylic acid group of CIP-CB is deprotonated, and the micelles show negative charges to a large extent. As the pH value gradually decreases, the carboxyl group begins to protonate, and the charge of the micelles also changes, such as Figure 1 As shown in Figure B, the potential is about -25 mV in a neutral environment of pH 7.4, and the micelle potential is about +10 mV in an acidic environment (pH 5.5). This type of micelle with charge reversal function can not only maintain long-term circulation in the body but also target bacteria in the infection environment, enhance the interaction between antibiotics and bacteria, and improve the bactericidal effect.

[0071] 2. Antibacterial test of CIP-CB micelles

[0072] In order to verify the antibacterial effect of CIP-CB micelles, the present invention uses Staphylococcus aureus ( S.aureus ) and Escherichia coli ( E.coil ) were used as model bacteria. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were used to demonstrate the antibacterial effect of CIP-CB.

[0073] The present invention uses three different conditions of CIP, pH 7.4 and pH 5.5 to conduct experiments. After co-culturing with bacteria for different time periods, the absorbance at 600 nm is measured by an enzyme marker to detect the growth of bacteria. Figure 2 As shown in the figure, compared with ciprofloxacin hydrochloride, the antibacterial effect of CIP-CB is significantly improved. With the transition from neutral to acidic conditions, the antibacterial effect under pH 5.5 is significantly enhanced.

[0074] The present invention uses red tetrazolium as an indicator and adopts a dilution method to determine the minimum inhibitory concentration of micelles. As shown in Table 1, the MICs of CIP to Escherichia coli and Staphylococcus aureus are 20.5 ppm and 20.5 ppm, respectively, the MICs of CIP-CB to Escherichia coli and Staphylococcus aureus at pH 7.4 are 5.10 ppm and 10.2 ppm, respectively, and the MICs of CIP-CB to Escherichia coli and Staphylococcus aureus at pH 5.5 are 2.55 ppm and 5.10 ppm, respectively.

[0075] Table 1 MIC and MBC results of CIP-CB

[0076]

[0077] The standard plate count method was used to further determine the minimum bactericidal concentration (MBC) of the micelles against Escherichia coli and Staphylococcus aureus. The MBCs of CIP, pH 7.4 and pH 5.5 against Escherichia coli were 41.9 ppm, 10.2 ppm and 5.10 ppm, respectively, and the MBCs against Staphylococcus aureus were 41.9 ppm, 10.2 ppm and 5.10 ppm, respectively. The results of MIC, MBC and microplate reader all showed that CIP-CB micelles improved the antibacterial effect of ciprofloxacin (CIP), especially the antibacterial effect of CIP-CB micelles under acidic conditions was higher than that of CIP-CB micelles under neutral conditions ( Figure 3 ). This result is attributed to the micelles improving the bioavailability and delivery effect of antibiotics, as well as the protonation of carboxylic acid betaine, which gives the micelles a targeting function to bacteria. The penetration ability of this intelligent responsive micelle in the biomembrane is more promising.

[0078] Multidrug resistant strains ESBL E.coli and MRSA As a model bacteria, the bactericidal effect of CIP-CB on drug-resistant bacteria was tested. Compared with planktonic bacteria, the antibacterial effects of CIP and CIP-CB on these two drug-resistant strains were significantly reduced under pH 7.4 and pH 5.5 environments. However, the antibacterial effect of CIP-CB on drug-resistant bacteria was significantly improved compared with CIP ( Figure 4 ).

[0079] 3. CIP-CB anti-biofilm test

[0080] CIP-CB was dispersed in PBS buffer solutions at pH 7.4 and pH 5.5 to obtain micelles with opposite charges on the surface. Staphylococcus aureus was used as a model bacterium, and mature biofilms were obtained after seven consecutive days of culture. Then 200 μL of CIP-CB solution was added and co-cultured with the biofilm at 37°C for 16 h. The anti-biofilm activity of CIP-CB micelles was verified by standard plate counting method and confocal laser scanning microscopy (CLSM).

[0081] Standard plate count results are shown in Figure 5 , Figure 5 The bar graphs in A in Figure 1 are CIP-CB (pH 7.4), CIP-CB (pH 5.5), CIP and Control from left to right. Figure 5It can be seen that after CIP-CB was co-cultured with biofilm for 16 h, the survival rates of biofilms in CIP-CB micelles with different concentrations (163.7 ppm, 81.8 ppm, 41.9 ppm, 20.5 ppm, 10.2 ppm) at pH 7.4 were 16.6%, 22.3%, 24.6%, 32.8%, 35.6%, respectively, and the survival rates of biofilms at pH 5.5 were 26.7%, 31.9%, 37.4%, 45.4%, 56.0%, respectively. In an acidic environment, the micelle surface presents a positive charge, and CIP-CB micelles are conducive to penetrating the biofilm matrix and delivering antibiotics to bacteria encapsulated in the biofilm, thereby achieving the effect of clearing the biofilm.

[0082] The results of laser confocal microscopy verification are shown in Figure 6 , Figure 6 Confocal microscopy images of S. aureus biofilms treated with CIP-CB at pH 5.5, pH 7.4 for 2 h (A), 16 h (B), 2 h (C), 16 h (D), and 2 h (E) and 16 h (F). After 7 days of incubation, the biofilms were stained with a live / dead bacterial viability kit, and live (green) and dead (red) bacteria were imaged using a laser scanning confocal microscope. Figure 6 It can be seen that after the untreated mature biofilm is stained with dye, a thick layer of biofilm can be seen, all of which is green fluorescence. The red fluorescence intensity of the biofilm treated for 16 h under pH 7.4 conditions is enhanced, and the red fluorescence of the biofilm treated for 16 h under pH 5.5 conditions is even stronger, which proves that CIP-CB micelles have the ability to penetrate the biofilm and achieve a deeper degree of bactericidal effect.

[0083] 4. CIP-CB hemolytic activity test

[0084] CIP-CB was diluted to different concentrations with PBS (pH=7.4) and co-cultured with red blood cells for 2 h. The absorbance at 570 nm was measured with an enzyme-labeled instrument to evaluate the release of hemoglobin, and the effects of different concentrations of CIP-CB on mouse red blood cells (RBCs) were obtained. The photographs of different concentrations of CIP-CB (5.11~81.8 ppm) suspended with red blood cells for 2 h of co-culture and centrifugation and the hemolysis rate detected by the enzyme-labeled instrument are shown in Figure 2.15. The hemolysis rate of micelles is generally low, and even at a high concentration of 81.8 ppm, the hemolysis rate is less than 3%. While ensuring good blood compatibility, the bactericidal effect on Escherichia coli and Staphylococcus aureus is also increased. This is sufficient to prove that CIP-CB shows a satisfactory result against red blood cells and bacteria ( Figure 7 ).

[0085] Embodiment 2:

[0086] The antibacterial drug in Example 1 was replaced by the following quinolone substances, and the preparation process was the same as in Example 1. The substrate and the antibacterial agent prepared were able to remove S.aureus The efficiency of the biofilm is as follows:

[0087]

[0088] Table 2 Quinolone substrate removal at a concentration of 50 ppm S.aureus Efficiency of biofilm

[0089]

[0090] The NMR data of the above product are as follows:

[0091] Product 1: 1 H NMR (400 MHz, MeOD) d (ppm): 9.24 (s, 1H), 7.48 (d, J = 13.0 Hz,1H), 6.35 (d, J = 7.0 Hz, 1H), 4.47 (t, J = 3.7 Hz, 2H), 4.01 (m, 2H), 3.89 (m,2H), 3.85 (m, 2H), 3.71 (m,1H), 3.59 (m, 4H), 3.48 (t, J = 5.0 Hz, 4H), 3.34(s, 6H), 3.17 (s, 2H), 1.40 (m, 2H), 1.18 (t, 3H).

[0092] Product 2: 1 H NMR (400 MHz, MeOD) d (ppm): 9.10 (s, 1H), 7.25 (d, J = 12.7 Hz,1H), 6.63 (m, 4H) 6.45 (d, J = 7.0 Hz, 1H), 4.47 (t, J = 3.7 Hz, 2H), 4.01 (m,2H), 3.89 (m, 2H), 3.80 (m, 2H), 3.71 (m,1H), 3.34 (s, 6H), 3.17 (s, 2H),1.42 (m, 2H), 1.17 (t, 3H).

[0093] Product 3: 1 H NMR (400 MHz, MeOD) d (ppm): 9.25 (s, 1H), 7.46 (d, J = 7 Hz, 1H), 4.48 (t, J = 3.66Hz, 2H), 4.36 (s, 2H), 4.14 (m,2H), 4.09 (m, 2H), 3.79(m, 2H), 3.89 (m, 2H), 3.50 (m, 4H), 3.48 (t, J = 5.14 Hz, 4H), 1.40 (m, 2H),1.39 (t, 2H).

[0094] Product 4: 1 H NMR (400 MHz, MeOD) d (ppm): 8.79 (s, 1H), 8.12 (d, J = 13.04 Hz,1H), 7.69 (d, J = 7 Hz, 1H), 4.48 (t, J = 3.46Hz, 2H), 4.36 (s, 2H), 4.01 (m,2H), 3.79 (m, 2H), 3.85 (m, 2H), 3.71 (m,1H), 3.59 (m, 4H), 3.48 (t, J = 5.14Hz, 4H), 3.34 (s, 6H), 1.42 (m, 2H), 1.19 (m, 2H).

[0095] Product 5: 1 H NMR (400 MHz, MeOD) d (ppm): 8.70 (s, 1H), 7.74 (d, J = 13.04 Hz,1H), 4.57 (t, J = 3.56Hz, 2H), 4.34 (s, 2H), 4.15 (m, 2H) 4.11 (m, 2H), 3.89(m, 2H), 3.86 (m, 2H), 3.71 (m,1H), 3.59 (m, 4H), 3.49 (t, J = 5.14 Hz, 4H), 3.23 (s, H), 2.26 (d, 1H), 1.19 (m, 2H).

[0096] Embodiment 3:

[0097] The antibacterial drug in Example 1 was replaced by the following penicillin-like substance, and the preparation process was the same as in Example 1. The substrate and the antibacterial agent prepared were able to remove S.aureus The efficiency of the biofilm is as follows:

[0098]

[0099] Table 3 Penicillin substrates removed at a concentration of 50 ppm S.aureus Efficiency of biofilm

[0100]

[0101] The NMR data of the above product are as follows:

[0102] Product 1: 1 H NMR (400 MHz, MeOD) d (ppm): 7.28 (m, 8H), 6.93 (m, 2H), 5.94 (s, 1H), 4.83 (m, 1H), 4.65 (m, 1H), 4.47 (t, J = 3.57 Hz, 2H), 4.33 (s, 2H), 4.11 (m, 2H), 3.89 (m, 2H), 3.89 (m, 2H), 3.86 (m, 2H), 3.71 (m, 1H), 3.60(m, 4H), 3.48 (t, J = 5.04 Hz, 4H), 3.4 (s, 1H), 3.34 (s, 6H), 1.56 (m, 6H).

[0103] Product 2: 1 H NMR (400 MHz, MeOD) d (ppm): 7.18 (m, 5H), 4.87 (m, 1H), 4.63 (m, 1H), 4.42 (t, J = 3.56Hz, 2H), 4.23 (s, 2H), 4.11 (m, 2H), 3.89 (m, 2H), 3.78 (m, 2H), 3.86 (m, 2H), 3.25 (s, 1H), 3.21 (s, 1H), 3.11 (s, 6H), 1.54(m, 6H).

[0104] Product 3: 1 H NMR (400 MHz, MeOD) d(ppm): 7.45 (m, 1H), 6.68 (m, 2H), 4.78 (m, 1H), 4.45 (m, 1H), 4.47 (t, J = 3.68 Hz, 2H), 4.13 (s, 2H), 4.01 (m, 2H), 3.89 (m, 2H), 3.87 (m, 2H), 3.45 (m, 2H), 3.52 (s, 6H), 3.27(s, 1H), 3.16 (s,1H), 3.25 (s, 6H), 1.56 (m, 6H).

[0105] Product 4: 1 H NMR (400 MHz, MeOD) d (ppm): 7.58 (m, 1H), 7.39 (m, 2H), 4.65 (m, 1H), 4.55 (m, 1H), 4.37 (t, J = 3.56 Hz, 2H), 4.23 (s, 2H), 4.11 (m, 2H), 3.79 (m, 2H), 3.88 (m, 2H), 3.86 (m, 2H), 3.37 (s, 1H), 3.25 (s, 1H), 3.14(s, 6H), 2.28 (s, 3H), 1.56 (m, 6H).

[0106] Product 5: 1 H NMR (400 MHz, MeOD) d (ppm): 7.54 (m, 4H), 7.29 (m, 1H), 4.65 (m, 1H), 4.65 (m, 1H), 4.47 (t, J = 3.56 Hz, 2H), 4.33 (s, 2H), 4.11 (m, 2H), 3.69 (m, 2H), 3.58 (m, 2H), 3.55 (m, 2H), 3.48 (s, 1H), 3.21 (s, 1H), 3.14(s, 6H), 2.38 (s, 3H),1.56 (m, 6H).

[0107] Embodiment 4:

[0108] The antibacterial drug in Example 1 was replaced by the following cephalosporin substances, and the preparation process was the same as in Example 1. The substrate and the antibacterial agent prepared were able to remove S.aureus The efficiency of the biofilm is as follows:

[0109]

[0110] Table 4 Cephalosporin substrate removal at a concentration of 50 ppm S.aureus Efficiency of biofilm

[0111]

[0112] The NMR data of the above product are as follows:

[0113] Product 1: 1 H NMR (400 MHz, MeOD) d (ppm): 9.46 (s, 1H), 5.52 (m, 1H), 4.65 (s, 1H), 4.47 (t, J = 3.46 Hz, 2H), 3.62 (s, 2H), 3.79 (s, 2H), 3.86 (m, 2H), 3.53 (m, 2H), 3.55 (m, 2H), 3.26 (s, 2H), 2.28 (s, 6H), 2.69 (s, 3H).

[0114] Product 2: 1 H NMR (400 MHz, MeOD) d (ppm): 8.75 (m, 1H), 8.34 (m, 4H), 7.45 (m, 1H), 6.84 (m, 1H), 6.83 (m, 1H), 5.72 (s, 1H), 5.52 (m, 1H), 4.47 (t, J =3.56 Hz, 2H), 3.72 (s, 2H), 3.87 (s, 2H), 3.85 (m, 2H), 3.64 (m, 2H), 3.51(m, 2H), 3.26 (s, 2H), 2.27 (s, 6H).

[0115] Product 3: 1 H NMR (400 MHz, MeOD) d (ppm): 8.19 (s, 1H), 5.52 (m, 1H), 4.48(t, J2.54 (m, 1H). 2.12 (s, 3H). 1.16 (m, 6H). 1.03 (m, 6H).

[0116] Product 4: 1 H NMR (400 MHz, MeOD) d (ppm): 8.67 (m, 2H), 8.33 (m, 1H), 7.31(m, 4H), 7.28 (m, 1H), 5.5(m, 1H), 4.85 (s,1H), 4.47 (t, J = 3.56 Hz, 2H), 3.89(m, 2H), 3.85 (m, 2H), 3.62 (s, 2H), 3.51 (s, 2H), 3.35 (m, 2H), 3.26 (s,2H), 2.27 (s, 6H), 2.09 (s, 3H).

[0117] Product 5: 1 H NMR (400 MHz, MeOD) d (ppm): 8.22 (m, 1H), 5.57 (m, 1H), 4.85 (s, 1H), 4.37 (t, J = 3.56Hz, 2H), 3.79 (s, 2H), 3.65 (m, 2H), 3.43 (m, 2H), 3.41 (m, 2H), 3.30 (s, 2H), 3.16 (s, 2H), 2.27 (s, 6H), 2.21 (s, 3H).

[0118] The antibacterial agent in Examples 1-4 is obtained by modifying carboxylic acid betaine on the antibacterial drug through an ester bond, and then forming an amphiphilic small molecule, which self-assembles into micelles in water. The micelles are negatively charged under physiological conditions, which reduces the cytotoxicity of the antibacterial drug and is conducive to long circulation in the body. On the contrary, under acidic conditions, the carboxylic acid betaine is deprotonated, and the micelle surface is converted into a positive charge, which can effectively penetrate and accumulate in the biofilm, deliver the antibacterial drug to the inside of the biofilm, and degrade the ester bond through the lipase secreted by the bacteria to release the antibacterial drug, thereby improving the bactericidal performance against planktonic bacteria and bacteria in the biofilm.

[0119] Embodiment 5:

[0120] An antibacterial agent, the antibacterial agent is Man-TEG-CIP, and its synthesis route is as follows:

[0121]

[0122] (a) Synthesis of acetyl mannose (Ac-Man): 15 g of mannose (add within 15-20 min) was slowly added to 60 ml of pyridine in batches under ice bath conditions. After continued stirring to dissolve, 100 mL of acetic anhydride was slowly added. After the reaction was continued for 30 min, the ice bath was removed and the reaction was continued at room temperature for 3 h. The plate (V 乙酸乙酯 :V 石油醚 =1:3) After confirming that the reaction is complete, dissolve it in DCM (about 100 mL), wash it with 5% hydrochloric acid aqueous solution, 0.5M NaOH aqueous solution, and saturated brine in sequence, dry the organic layer with anhydrous sodium sulfate, filter, and concentrate to obtain acetyl mannose (Ac-Man). Ac-Man: 1 H NMR (400 MHz, CDCl3, d ): d 6.08 (d, J =1.88Hz, 1H, 1-H), 5.28-5.32 (m, 2 H, 3-H and 4-H), 5.23 (m, 1H, 2-H), 4.08-4.27 (m, 2H, CH2OAc), 4.00-4.05 (m,1H, 5-H), 2.19 (s, 3H, C(O)CH3), 2.13(s,3H, C(O)CH3), 2.07 (s, 3H, C(O)CH3), 2.06 (s, 3H, C(O)CH3), 2.02 (s, 3H, C(O)CH3).

[0123] (b) Synthesis of compound Ac-Man-TEG: 5.7 g (0.07 mol) of acetyl mannose and 26 mL (0.21 mol) of triethylene glycol (TEG) were dissolved in 150 ml of anhydrous dichloromethane, and 60 mL (0.56 mol) of boron trifluoride ether was slowly added to the above solution at 0°C under nitrogen. After reacting at room temperature for 36 h, the reaction was confirmed to be complete by spotting the plate. The mixture was then washed with 2×100 mL of saturated sodium bicarbonate solution, 2×100 mL of distilled water, and 100 mL of saturated brine respectively until no bubbles were generated. The organic layer was collected and dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure, and purified by silica gel chromatography (TLC=EA:MeOH=6:1) with ethyl acetate and n-hexane as the eluent to obtain compound Ac-Man-TEG. Ac-Man-TEG: 1H NMR (400 MHz, CDCl3, d ):5.37 (dd, J =10.08 Hz, 1H, H3), 5.31-5.28 (m, 1H, H4), 5.27 (dd, J =3.44Hz, 1H,H2), 4.87 (d, J =1.64Hz, 1H, H1), 4.28 (dd, J =12.68Hz, 1H, H6), 4.11(d, J =2.4Hz,1H, H6'), 4.08 (ddd, 1H, H5), 3.83 (m, 2H, CH2CH2O), 3.73 (m, 2H, CH2CH2O), 3.68-3.64 (m, 6H, CH2CH2O), 3.61-3.58 (m, 2H, CH2CH2O), 2.14, 2.09, 2.03, 1.98(4s, 12H, 4 HAc) ppm.

[0124] (c) Synthesis of compound Boc-CIP: Add 4.5 mL 1N NaOH and 40 mL dioxane: water = 1:1 (volume ratio) to ciprofloxacin (1.0 g, 3.00 mmol), then add di-tert-butyl dicarbonate Boc2O (1.0 g, 4.6 mmol), react at room temperature under nitrogen atmosphere for 17 h until the reaction is complete, filter the white solid, then rinse with water and acetone, and dry in vacuum. The product is a white to light yellow solid (Boc-CIP, 98%) detected by developing solvent TLC (DCM: MeOH = 9:1). 1 H NMR (400 MHz, CDCl3) d (ppm): 8.76 (s, 1H), 8.02 (d, J = 12.9 Hz, 1H), 7.36 (d, J = 7.1Hz, 1H), 3.67 (t, J = 4.9 Hz, 4H), 3.54 (m, 1H), 3.29 (t, J = 5.2 Hz, 4H), 1.5(s, 9H), 1.39 (m, 2H), 1.20 (d, J = 3.8 Hz, 2H).

[0125] (d) Synthesis of compound Ac-Man-TEG-Boc-CIP: Boc-CIP (4.45 mmol, 1.917 g, 0.5 eq), Ac-Man-TEG (8.9 mmol, 4.278 g, 1.0 eq), TBTU (13.35 mmol, 4.28 g, 1.5 eq), and DIPEA (26.7 mmol, 3.45 g, 2.0 eq) were added to a dry dichloromethane solution and stirred at room temperature for 12 h. After the reaction was completed, the solution was extracted three times with 100 mL of water, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The product was purified by chromatography (TLC = CH2Cl2: MeOH = 20:1) with dichloromethane and methanol as the eluent to obtain the product Ac-Man-TEG-Boc-CIP. Ac-Man-TEG-Boc-CIP: 1 H NMR (400 MHz,CDCl3) d 8.54 (s, 1H), 7.99 (d, J =13.16Hz, 1H),7.31(d, J =30.56 Hz, 1H), 5.37 (dd, J =10.06Hz, 1H, H3), 5.31-5.28 (m, 1H, H4),5.26 (dd, J =3.34Hz, 1H, H2), 4.86 (d, J =1.6Hz, 1H, H1), 4.46(m, 2H), 4.26 (dd, J =12.42Hz, 1H, H6), 4.10(d, J =2.4Hz, 1H, H6'), 4.07 (ddd, 1H, H5), 3.87-3.79(m, 2H, H2, H6), 3.84(m, 4H), 3.73-3.65(m, 12H), 3.43(m, 1H), 3.21(t, 4H),2.14, 2.09, 2.03, 1.97 (4s, 12H, 4 HAc), 1.49(s, 9H), 1.32(m, 2H), 1.13(m,2H). HRMS (ESI) m / z calcd for C 42 H 56 F3O 17 Na + (M+Na) + 916.34860, found 916.34900.

[0126] (e) Synthesis of compound Man-TEG-Boc-CIP: 0.3 g of Ac-Man-TEG-Boc-CIP was dissolved in 10 ml of anhydrous methanol, and 3 mL of anhydrous sodium methoxide (0.06 wt% Na) was slowly added dropwise. After reacting at room temperature for fifteen minutes, the reaction was detected to be complete, and the mixture was neutralized with acetic acid solution. The solvent was evaporated and the product Man-TEG-Boc-CIP was obtained by silica gel column chromatography. Man-TEG-Boc-CIP: 1 H NMR (400 MHz, MeOD) d 8.74 (s, 1H), 7.89 (d, J =13.24Hz, 1H), 7.54(d, J =7.2Hz, 1H), 4.83 (d, J =1.52Hz, 1H), 4.52 (m, 2H), 3.82 (t, 4H), 3.73-3.60(m, 12H), 3.56 (m, 1H), 3.29 (t, 4H), 1.49 (s, 9H), 1.37 (m, 2H), 1.13 (m,2H). HRMS (ESI) m / z calcd for C 34 H 48 F3O 13 Na + (M+Na) + 748.30634, found 748.30652.

[0127] (f) Synthesis of compound Man-TEG-CIP: 0.5 g Man-TEG-Boc-CIP was dissolved in 10 ml dichloromethane, and then 10 ml trifluoroacetic acid was added. After stirring at room temperature for two hours to ensure that the tert-butyl group was removed, the solvent was decompressed and dried, and then precipitated in ether to obtain the product Man-TEG-CIP. Man-TEG-CIP: 1 H NMR (400 MHz,MeOD) d 8.71(s, 1H), 7.86(d, J =13.12Hz, 1H), 7.56 (d, J =7.04Hz, 1H), 4.44 (m, 2H), 3.83(t, 4H), 1.37 (m, 2H), 1.13 (m, 2H). HRMS (ESI) m / z calcd for C 29 H 41 F3O11 + (M+H) + 626.27196, found 626.27191.

[0128] The Man-TEG-CIP prepared above was prepared into micelles. The specific process was as follows: 20 mg of Man-TEG-CIP was dissolved in 20 mL of PBS with pH = 7.4 and stirred at room temperature for 2 h to obtain uniformly distributed micelles with a concentration of 1 mg / mL.

[0129] The Man-TEG-CIP micelles prepared above were characterized as follows:

[0130] 1. The above antibacterial agent is a small amphiphilic molecule micelle formed by combining mannose and ciprofloxacin in the form of an ester bond. It can self-assemble to form micelles in an aqueous solution. The particle size of Man-TEG-CIP is measured by dynamic light scattering (DLS) at 25°C, which is about 100nm and evenly dispersed. Then, the micelles are dropped on a copper mesh, stained with phosphotungstic acid, and the morphology of the micelles is observed by transmission electron microscopy. Figure 8 As shown, the micelles are evenly dispersed and have a good core-shell structure.

[0131] 2. Use Nile red as the fluorescent molecule to determine the critical micelle concentration of micelles, such as Figure 9 As shown in Figure 2, the critical micelle concentration of Man-TEG-CIP is 6.98×10 -4 mg / mL, and concentrations above CMC exist in the form of micelles, with mannose exposed on the outside and ciprofloxacin in the inner shell. The mannose in the outer shell of the micelle is multivalent and has a strong affinity for Escherichia coli, which can enhance the interaction between Man-TEG-CIP and bacteria.

[0132] 3. Man-TEG-CIP prevents biofilm colonization

[0133] After co-culturing Man-TEG-CIP micelles with different concentrations for different periods of time, the biofilm of E. coli was observed by laser confocal microscopy. Figure 10As shown in the figure, the attachment of biofilms at 24 h, 48 h, and 72 h was investigated. As time went on, the biofilm on the surface of the membrane gradually increased in the blank control group, and the thickness also gradually increased, and a dense biofilm was formed after 72 h. After E. coli was co-cultured with Man-TEG-CIP for 24 h, only a small amount of bacteria adhered to the surface of the membrane. When the time was delayed to 48 h, biofilm colonization began to appear on the membrane after being cultured with Man-TEG-CIP micelles at a concentration of 5.10 ppm, and a small amount of biofilm colonization also appeared at 10.2 ppm, and trace biofilm appeared under 20.5 ppm conditions. After E. coli was co-cultured with Man-TEG-CIP for 24 h, biofilm colonization began to appear on the membrane. This is because the micelles will be degraded by enzymes in the bacterial environment, causing the multivalent sugar-shaped micelles to begin to lyse, and the effect on bacteria is reduced.

[0134] To further prove that Man-TEG-CIP prevents biofilm colonization, we used the same operation and observed it using a scanning electron microscope (SEM). Figure 11 As shown in the figure, in the blank control group, E. coli multiplied in large numbers over time and colonized on the membrane through FimH lectin to form a dense biofilm. With the addition of Man-TEG-CIP, low-density, dispersed colonies began to appear in the biofilm on the membrane. As the concentration of Man-TEG-CIP increased, the integrity of the biofilm was more affected, which was consistent with the data of laser confocal microscopy.

[0135] 4. Hemolytic activity test

[0136] The blood compatibility of Man-TEG-CIP was evaluated by a hemolysis test. Specifically, Man-TEG-CIP was diluted to different concentrations with PBS (pH=7.4) and co-cultured with red blood cells for 2 h. The absorbance at 570 nm was measured with an enzyme-labeled instrument to evaluate the release of hemoglobin, so as to obtain the effect of different concentrations of Man-TEG-CIP on mouse red blood cells (RBCs). The photos of different concentrations of Man-TEG-CIP (5.11~81.8 ppm) suspended with red blood cells and centrifuged for 2 h and the hemolysis rate detected by the enzyme-labeled instrument are shown in Figure 12. The hemolysis rate of micelles is generally low, and even at a high concentration of 81.8 ppm, the hemolysis rate is less than 5%. While having good blood compatibility, the effect on Escherichia coli is also greatly improved.

[0137] Embodiment 6:

[0138] The antibacterial drug in Example 5 was replaced by the following quinolone substances, and the preparation process was the same as in Example 5. The substrate and the antibacterial agent prepared were able to remove E.coil The efficiency of the biofilm is as follows:

[0139]

[0140] Table 5 Quinolone substrate removal at a concentration of 50 ppm E.coil Efficiency of biofilm

[0141]

[0142] The NMR data of the above product are as follows:

[0143] Product 1: 1 H NMR (400 MHz, MeOD) d (ppm): 7.84 (s, 1H), 6.20 (d, J = 13.0 Hz,1H), 6.12 (d, J = 7.0 Hz, 1H), 3.83 (t, 4H), 3.80-3.60 (m, 5H), 3.57 (m, 2H), 3.51 (m, 2H), 3.60 (m, 2H), 3.47 (m, 2H), 3.44 (m, 6H), 3.42 (m, 4H), 2.69(m, 2H), 1.47 (m, 2H), 1.17 (m, 2H).

[0144] Product 2: 1 H NMR (400 MHz, MeOD) d (ppm): 9.15 (s, 1H), 7.25 (d, J = 13.7 Hz,1H), 6.62 (m, 4H), 6.50 (d, J = 7.0 Hz, 1H), 3.70 (m, 2H), 3.80-3.60 (m, 5H), 3.57-3.55 (m, 4H), 3.54 (m, 6H), 3.52 (m, 4H), 3.17 (s, 2H), 1.40 (m, 2H), 1.17 (t, 3H).

[0145] Product 3: 1 H NMR (400 MHz, MeOD) d (ppm): 9.33 (s, 1H), 7.34 (d, J= 7 Hz,1H), 3.90-3.72 (m, 5H), 3.70 (m, 2H), 3.60-3.56 (m, 4H), 3.54 (m, 6H), 3.51(m, 4H), 3.17 (s, 2H), 1.41 (m, 2H), 1.18 (t, 3H).

[0146] Product 4: 1 H NMR (400 MHz,MeOD) d (ppm): 8.72 (s, 1H), 7.87 (d, J =13.12 Hz,1H), 7.56 (d, J =7.04 Hz, 1H), 4.45 (m, 2H), 3.83 (t, 4H), 3.80-3.60 (m, 5H), 3.57 (m, 2H), 1.37 (m, 2H), 1.13 (m, 2H).

[0147] Product 5: 1 H NMR (400 MHz, MeOD) d (ppm): 8.79 (s, 1H), 7.69 (d, J = 13.04 Hz,1H), 4.45 (m, 2H), 3.90-3.60 (m, 5H), 3.51 (m, 2H), 3.23 (s, 3H), 2.26 (d,1H), 1.20 (m, 2H).

[0148] Embodiment 7:

[0149] The antibacterial drug in Example 5 was replaced by the following penicillin-like substance, and the preparation process was the same as in Example 5. The substrate and the antibacterial agent prepared were able to remove E.coil The efficiency of the biofilm is as follows:

[0150]

[0151] Table 6 Penicillin substrates removed at a concentration of 50 ppm E.coil Efficiency of biofilm

[0152]

[0153] The NMR data of the above product are as follows:

[0154] Product 1: 1 H NMR (400 MHz,MeOD) d(ppm): 7.28 (m, 8H), 6.94 (m, 2H), 5.94(s, 1H), 4.87 (m, 1H), 4.66 (m, 1H), 3.85 (m, 2H), 3.71 (m, 2H), 3.70 - 3.57(m, 5H), 3.51 (m, 2H), 3.47 (m, 2H), 3.72 (m,1H), 3.49 (m, 4H),3.44 (m, 6H),3.42 (m, 4H), 3.31 (s, 1H), 1.55 (m, 6H).

[0155] Product 2: 1 H NMR (400 MHz, MeOD) d (ppm): 7.28 (m, 5H), 4.82 (m, 1H), 4.66(m, 1H), 3.89 (m, 2H), 3.71 (m, 2H), 3.70 - 3.60 (m, 5H), 3.56 (m, 2H), 3.54(m, 6H), 3.52 - 3.50 (m, 6H), 3.38 (s, 1H), 3.41 (s, 1H), 1.56 (m, 6H).

[0156] Product 3: 1 H NMR (400 MHz, MeOD) d (ppm): 7.49 (m, 1H), 6.48 (m, 2H), 4.82(m, 1H), 4.65 (m, 1H), 3.78 (m, 2H), 3.90 - 3.65 (m, 5H), 3.62 (s, 6H), 3.60(m, 2H), 3.57 (m, 2H), 3.54 (m, 6H), 3.52 - 3.50 (m, 6H), 3.27 (s, 1H), 3.14(s, 1H), 1.55 (m, 6H).

[0157] Product 4: 1 H NMR (400 MHz, MeOD) d(ppm): 7.65 (m, 1H), 7.39 (m, 2H), 4.82 (m, 1H), 4.65 (m, 1H), 3.90-3.60 (m, 9H), 3.57 (m, 2H), 3.54 (m, 6H), 3.53-3.51 (m, 6H), 3.27(s, 1H), 3.42 (s, 1H), 2.38 (s, 3H), 1.55(m, 6H).

[0158] Product 5: 1 H NMR (400 MHz, MeOD) d (ppm): 7.54 (m, 4H), 7.48 (m, 1H), 4.82(m, 1H), 4.66 (m, 1H), 3.90-3.60 (m, 9H), 3.67 (m, 2H), 3.54 (m, 6H), 3.51(m, 6H), 2.38 (s, 1H), 3.34 (s, 1H), 2.48 (s, 3H), 1.55 (m, 6H).

[0159] Embodiment 8:

[0160] The antibacterial drug in Example 5 was replaced by the following cephalosporin substances, and the preparation process was the same as in Example 5. The substrate and the antibacterial agent prepared were able to remove E.coil The efficiency of the biofilm is as follows:

[0161]

[0162] Table 7 Cephalosporin substrate removal at a concentration of 50 ppm E.coil Efficiency of biofilm

[0163]

[0164] The NMR data of the above product are as follows:

[0165] Product 1: 1 H NMR (400 MHz, MeOD) d (ppm): 9.46 (s, 1H), 5.55 (m, 1H), 4.64 (s,1H), 3.90-3.68 (m, 7H), 3.62 (s, 2H), 3.57 (m, 2H), 3.53 (m, 6H), 3.51 (m,6H), 3.16 (s, 2H), 2.68 (s, 3H).

[0166] Product 2: 1 H NMR (400 MHz, MeOD) d (ppm): 8.75 (m, 1H), 8.25 (m, 4H), 7.36 (m, 1H), 6.95 (m, 1H), 6.85 (m, 1H), 5.76 (s,1H), 5.52 (m, 1H), 3.90-3.75 (m,5H), 3.72 (s, 2H), 3.71 (m, 2H), 3.57 (m, 2H), 3.54 (m, 6H), 3.51 (m, 6H), 3.45 (s, 1H), 1.56 (m, 6H).

[0167] Product 3: 1 H NMR (400 MHz, MeOD) d (ppm): 7.49 (m, 1H), 6.53 (m, 2H), 4.82(m, 1H), 4.66 (m, 1H), 3.90-3.75 (m, 7H), 3.73 (s, 6H), 3.71 (m, 2H), 3.58(m, 2H), 3.54 (m, 6H), 3.52-3.51 (m, 6H), 3.38 (s, 1H), 3.42 (s, 1H), 1.56(m, 6H).

[0168] Product 4: 1 H NMR (400 MHz, MeOD) d (ppm): 8.78 (m, 2H), 8.33 (m, 1H), 7.32 (m, 4H), 7.39 (m, 1H), 5.52 (m, 1H), 4.86 (s,1H), 3.90-3.70 (m, 7H), 3.65 (s,2H), 3.57 (m, 2H), 3.54 (m, 6H), 3.53 (m, 4H), 3.51 (m, 2H), 3.12 (s, 2H), 2.19 (s, 3H).

[0169] Product 5: 1 H NMR (400 MHz, MeOD) d(ppm): 8.33 (m, 1H), 5.58 (m, 1H), 4.85 (s,1H), 3.90-3.70 (m, 5H), 3.68 (m, 2H), 3.57 (m, 2H), 3.54 (m, 6H), 3.52 (m,6H), 3.30 (s, 2H), 3.16 (s, 2H), 2.12 (s, 3H).

[0170] The antibacterial agent in Examples 5-8 is obtained by modifying mannose on the antibacterial drug through an ester bond, forming micelles in the form of polyvalent sugars in the solution, and testing its structure and morphology. Antibacterial experiments have shown that mannose enhances the antibacterial activity of ciprofloxacin due to its polyvalent sugar effect on Escherichia coli, and then scanning electron microscopy and laser confocal microscopy have verified that the mannose-modified antibiotics can prevent the colonization of Escherichia coli and reduce the formation of biofilms.

[0171] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an antibacterial agent, characterized in that: The antibacterial agent includes an organic small molecule and an antibacterial drug, wherein the organic small molecule and the antibacterial drug are connected via an ester bond, the organic small molecule is carboxylic acid betaine or mannose, and the antibacterial drug is ciprofloxacin. The structural formula of the antibacterial agent is as follows: or ; When the organic small molecule is carboxylic acid betaine, the method comprises the following steps: (1) dissolving the antibiotic, then adding di-tert-butyl dicarbonate to react at room temperature under an inert gas atmosphere to obtain a Boc-protected antibiotic; (2) Mixing a Boc-protected antibacterial drug, N,N-dimethylaminoethyl glycol, TBTU and DIPEA in a molar ratio of 1:2-4:3-5:6-8, adding the mixture to a solvent, and stirring the mixture at room temperature for 10-15 hours to obtain a product; (3) dissolving the product obtained in step (2), then adding tert-butyl bromoacetate and a base to react at 45-55° C. under an inert gas atmosphere for 0.5-1.5 h to obtain the product; (4) subjecting the product obtained in step (3) to a de-tert-butyl esterification reaction in a mixed solvent of dichloromethane and trifluoroacetic acid, followed by precipitation and filtration to obtain an antibacterial agent; When the organic small molecule is mannose, the method comprises the following steps: (1) dissolving mannose in an ice bath, then adding acetic anhydride and reacting for 25-35 min in an ice bath, and then continuing to react at room temperature for 2-4 h to obtain acetyl mannose; (2) dissolving acetyl mannose and TEG, then adding a catalyst, and reacting at room temperature for 34-38 hours to obtain a product; wherein the molar ratio of acetyl mannose, TEG, and catalyst is 1:3-4:8-10; (3) dissolving the antibiotic, and then adding di-tert-butyl dicarbonate to react at room temperature under an inert gas atmosphere to obtain a Boc-protected antibiotic; (4) mixing the Boc-protected antibacterial drug, the product obtained in step (2), TBTU, and DIPEA in a molar ratio of 1:2-3:3-5:5-7, adding the mixture to a solvent, and stirring at room temperature for 10-14 hours to obtain a product; (5) dissolving the product obtained in step (4), then adding a base and reacting at room temperature for 10-20 minutes to obtain the product; (6) The product obtained in step (5) is dissolved, and then subjected to a de-tert-butyl esterification reaction in a mixed solvent of dichloromethane and trifluoroacetic acid, followed by precipitation and filtration to obtain an antibacterial agent.

2. The method for preparing the antibacterial agent according to claim 1, characterized in that: When the organic small molecule is carboxylic acid betaine, the base in step (3) is anhydrous sodium methoxide, anhydrous potassium carbonate or anhydrous sodium carbonate; and the de-tert-butyl esterification reaction conditions in step (4) are room temperature and the reaction time is 1.5-3.5 hours.

3. The method for preparing the antibacterial agent according to claim 1, characterized in that: When the organic small molecule is mannose, the catalyst in step (2) is boron trifluoride etherate; the base in step (5) is anhydrous sodium methoxide, anhydrous potassium carbonate or anhydrous sodium carbonate; and the de-tert-butyl esterification reaction conditions in step (6) are room temperature and the reaction time is 1.5-3.5 hours.

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