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CN116115768B - An antibacterial agent and its preparation method and application - Google Patents

An antibacterial agent and its preparation method and application Download PDF

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Publication number
CN116115768B
CN116115768B CN202211531556.4A CN202211531556A CN116115768B CN 116115768 B CN116115768 B CN 116115768B CN 202211531556 A CN202211531556 A CN 202211531556A CN 116115768 B CN116115768 B CN 116115768B
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antibacterial
micelle
cip
antibacterial agent
room temperature
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CN116115768A (en
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罗建斌
姚永超
肖际鹏
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Southwest Minzu University
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Abstract

The invention discloses an antibacterial agent, a preparation method and application thereof, wherein the antibacterial agent comprises small organic molecules and antibacterial drugs, the small organic molecules are connected with the antibacterial drugs through ester bonds, and the small organic molecules are carboxylic acid betaine or mannose. According to the invention, organic micromolecular substance carboxylic betaine or mannose is connected with an antibacterial drug through an ester bond, and can self-assemble in water to form a micelle with a pH induced charge turnover function, wherein the micelle presents negative charges in a normal physiological environment, and when the micelle presents positive charges on the surface of the micelle in a bacterial acidic environment, the micelle with the pH induced function is beneficial to in-vivo long circulation, and interaction between bacteria and the antibacterial drug can be enhanced through electrostatic adsorption. The micelle can effectively penetrate and eliminate biological films, and further effectively inhibit bacterial growth, and is an intelligent nano drug-carrying system with a very good prospect.

Description

Antibacterial agent and preparation method and application thereof
Technical Field
The invention relates to the technical field of antibacterial agents, in particular to an antibacterial agent and a preparation method and application thereof.
Background
Bacteria are unique among prokaryotes and play an important role in maintaining the balance of our living environment, most of them do not cause infection, but a small part of them cause infection and disease, and bacterial infection caused by pathogenic bacteria, if not diagnosed and treated in time, will cause irreversible damage, and many serious diseases such as septicemia, bacteremia, pneumonia, endocarditis and even death have been caused. Antibiotics are considered to be the most effective drugs for the treatment of bacterial infections, and they reduce morbidity and mortality due to bacterial infections and even save the lives of the infected subjects. However, high doses of antibiotics are frequently used to maintain therapeutic levels, causing serious side effects to the liver and other normal tissues. Moreover, antibiotic therapy lacks specificity and is rapidly metabolized and excreted through the circulatory system in the body before reaching the site of infection, resulting in low bioavailability of the antibiotic. The presence of resistant strains due to excessive use of antibiotics reduces the effectiveness of clinical antibacterial agents. Thus, solving bacterial resistance is a significant challenge in the world today.
The bacteria in the biofilm encapsulate themselves in a shielding extracellular polymeric matrix (EPS) consisting essentially of proteins, extracellular bacterial DNA, extracellular polysaccharides and enzymes. The extracellular polysaccharide matrix can act as a protective barrier, protecting the bacteria from the host's innate immune cells and preventing the penetration of antibiotics. The amount of antibiotic used for the treatment of biofilm infection is 100-1000 times that of ordinary planktonic bacteria, and the use of a large amount of antibiotic results in a greater probability of occurrence of drug-resistant bacteria. Because antibiotics have the defects of low bioavailability, low penetrability to biological membranes, short half-life, high toxicity and the like in the treatment of biological membrane infection.
It is therefore very interesting to study an antimicrobial agent that has a penetrating effect on the biofilm and thus improves the bactericidal properties.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide an antibacterial agent, a preparation method and application thereof, so as to solve the problems of poor cell membrane penetration effect and the like of the existing antibiotics.
The technical scheme for solving the technical problems is as follows, the antibacterial agent comprises small organic molecules and antibacterial drugs, the small organic molecules are connected with the antibacterial drugs through ester bonds, and the small organic molecules are carboxylic acid betaine or mannose.
The invention has the beneficial effects that organic micromolecular substance carboxylic acid betaine or mannose is connected with an antibacterial drug through ester bonds to form the antibacterial agent, the antibacterial agent can be self-assembled to form a micelle with a pH induced charge turnover function, the surface of the micelle presents negative charges in a normal physiological environment (pH 7.4), positive charges are presented on the surface of the micelle in a bacterial acidic environment (pH 5.5) when the micelle is in a bacterial acidic environment, the biological membrane can be effectively permeated and accumulated, the antibacterial drug is transferred into the biological membrane, and the ester bonds are degraded by lipase secreted by bacteria to release the antibacterial drug, so that the sterilization performance on planktonic bacteria and bacteria in the biological membrane is improved. The micelle with the pH induction function is beneficial to long circulation in vivo, and interaction between bacteria and antibacterial drugs can be enhanced through electrostatic adsorption.
Further, the antibacterial agent is quinolone substance, penicillin substance or cephalosporin substance.
Further, the quinolone substance is ciprofloxacin or a substance having the following structural formula:
wherein R1 is C 2H5、C3H5、C2H4 F or ;
R2 is H or NH 2 or CH 3;
R3 is F or H, or is absent, meaning that R3 is absent when Y is N, etc.;
r4 is 、、、、、、、、Or (b);
R5 is H, F, CH 3 O or F 2 CHO, or is absent, the absence meaning that R5 is absent when Y is N, etc.;
X is N or C;
Y is N or C.
Further, the structural formula of the penicillin is as follows:
Wherein R1 is 、、、、、、、、、Or (b)。
Further, the cephalosporin substance has the following structural formula:
Wherein R1 is 、、、CH 2 CN or;
R2 is、、、CH3、Cl、H、Or (b)。
The preparation method of the antibacterial agent comprises the following steps when the organic small molecule is carboxylic betaine:
(1) Dissolving an antibacterial drug, adding di-tert-butyl dicarbonate, and reacting at room temperature under an inert gas atmosphere to obtain a Boc-protected antibacterial drug;
(2) Mixing the antibacterial drug protected by Boc, N-dimethylaminoethyl glycol, TBTU and DIPEA according to the molar ratio of 1:2-4:3-5:6-8, adding the mixture into a solvent, and stirring the mixture at room temperature for reaction for 10-15h to obtain a product;
(3) Dissolving the product obtained in the step (2), and then adding tert-butyl bromoacetate and alkali to react for 0.5-1.5h at 45-55 ℃ in an inert gas atmosphere to obtain the product;
(4) And (3) performing tert-butyl removal reaction on the product obtained in the step (3) in a mixed solvent of dichloromethane and trifluoroacetic acid, then precipitating and filtering to obtain the antibacterial agent.
Further, in the step (3), the alkali is anhydrous sodium methoxide, anhydrous potassium carbonate or anhydrous sodium carbonate.
Further, the reaction condition of the tert-butyl ester removal in the step (4) is room temperature, and the reaction lasts for 1.5 to 3.5 hours.
The preparation method of the antibacterial agent comprises the following steps when the organic small molecule is mannose:
(1) Dissolving mannose under ice bath condition, adding acetic anhydride, reacting for 25-35min under ice bath condition, and reacting at room temperature for 2-4h to obtain acetylmannose;
(2) Dissolving acetylmannose and TEG, then adding a catalyst, and reacting for 34-38 hours at room temperature to obtain a product, wherein the molar ratio of the acetylmannose to the TEG to the catalyst is 1:3-4:8-10;
(3) Dissolving an antibacterial drug, adding di-tert-butyl dicarbonate, and reacting at room temperature under an inert gas atmosphere to obtain a Boc-protected antibacterial drug;
(4) Mixing the antibacterial drug protected by Boc, the product obtained in the step (2), TBTU and DIPEA according to the molar ratio of 1:2-3:3-5:5-7, adding the mixture into a solvent, and stirring the mixture for 10-14h at room temperature to obtain the product;
(5) Dissolving the product obtained in the step (4), then adding alkali, and reacting for 10-20min at room temperature to obtain the product;
(6) And (3) dissolving the product obtained in the step (5), then carrying out tert-butyl removal reaction in a mixed solvent of dichloromethane and trifluoroacetic acid, then precipitating, and filtering to obtain the antibacterial agent.
Further, the catalyst in the step (2) is boron trifluoride diethyl etherate.
Further, in the step (5), the alkali is anhydrous sodium methoxide, anhydrous potassium carbonate or anhydrous sodium carbonate.
Further, the reaction condition of the tert-butyl ester removal in the step (6) is room temperature, and the reaction lasts for 1.5 to 3.5 hours.
The antibacterial agent has good application prospect in the aspect of penetrating or removing bacterial biofilms or inhibiting bacterial growth.
The invention has the following beneficial effects:
According to the invention, organic micromolecular substance carboxylic betaine or mannose is connected with an antibacterial drug through an ester bond, and can self-assemble in water to form a micelle with a pH induced charge turnover function, the micelle presents negative charge on the surface in a normal physiological environment (pH 7.4), and positive charge is presented on the surface of the micelle in an acidic environment (pH 5.5) of bacteria.
The antibacterial agent in the antibacterial agent can be quinolone substances, penicillin substances or cephalosporin substances. Taking ciprofloxacin in quinolone substances as an example, and adopting carboxylic acid betaine for modification, the prepared micelle has good bactericidal effect on staphylococcus aureus (S.aureus) and escherichia coli (E.coil) compared with the free medicine ciprofloxacin. More importantly, as the bacterial biofilm is a microbial community with a complex structure, the bacterial biofilm has stronger resistance to traditional antibiotics. The micelle has good penetrating effect on the biological film in an acidic environment, and is beneficial to eliminating the biological film. The result shows that the carboxylic acid betaine modified ciprofloxacin small molecule micelle is a very promising intelligent nano drug carrying system.
Taking ciprofloxacin in quinolone substances as an example, mannose modification is adopted, micelle with multivalent sugar form is formed in solution, and antibacterial experiments prove that the antibacterial activity of ciprofloxacin is improved due to the enhancement of the action effect of multivalent sugar and escherichia coli, and then the antibiotic modified by mannose can prevent the fixation of escherichia coli and reduce the formation of a biological film through scanning electron microscope and laser confocal verification.
Drawings
FIG. 1 is a graph showing characterization results of CIP-CB micelles.
FIG. 2 is a graph of the results of antibacterial tests on CIP-CB micelles, wherein each group of bar graphs in each panel is respectively 81.8ppm, 41.9ppm, 20.5ppm, 10.2ppm, 5.10ppm, 2.55ppm, 1.28ppm and a control group from left to right, and the time from left to right of each group of bar graphs is 2h, 4h, 8h, 16h and 24h.
FIG. 3 shows the bacteriostatic effect of CIP-CB micelles on E.coli and Staphylococcus aureus at different concentrations and at different pH.
FIG. 4 shows the antibacterial effect of CIP-CB micelles on drug-resistant bacteria at different concentrations and different pH values.
FIG. 5 is a graph showing the results of CIP-CB anti-biofilm testing using standard plate count methods.
FIG. 6 shows the results of verifying CIP-CB anti-biofilm by a laser confocal microscope.
FIG. 7 is a graph showing the hemolysis ratio of CIP-CB and the result of centrifugation after 2h of co-culture with erythrocytes.
FIG. 8 is a DLS (A) and a transmission electron microscope image (B) of a Man-TEG-CIP micelle.
FIG. 9 is a critical micelle concentration result of Man-TEG-CIP micelle.
FIG. 10 is a laser confocal plot of E.coli biofilms after different times of culture with Man-TEG-CIP micelles.
FIG. 11 is a scanning electron microscope image of E.coli biofilm after different times of culture with Man-TEG-CIP micelles.
FIG. 12 is a graph showing the hemolysis ratio of Man-TEG-CIP micelles and the results after 2h centrifugation in co-culture with erythrocytes.
Detailed Description
The examples given below are only intended to illustrate the invention and are not intended to limit the scope thereof. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1:
an antibacterial agent is CIP-CB, and the synthetic route is as follows:
The method comprises the following specific steps:
(a) Ciprofloxacin (1.0 g,3.00 mmol) was added to 4.5 mL of 1n NaOH and 40mL of dioxane: water=1:1 (volume ratio), followed by di-tert-butyl dicarbonate Boc 2 O (1.0 g,4.6 mmol), reacted at room temperature under nitrogen atmosphere for 17h to completion, the white solid was filtered, then rinsed clean with water and acetone, respectively, and dried in vacuo. Developing TLC (DCM: meOH=9:1) and the product was a white to pale yellow solid (Boc-CIP, 98%) with nuclear magnetic data of ：1H NMR (400 MHz, CDCl3)δ(ppm): 8.76 (s, 1H), 8.02 (d,J= 12.9 Hz, 1H), 7.36 (d,J= 7.1 Hz, 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).
(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), DIPEA (3.0 mmol,0.388 g) were added to a dry dichloromethane solution, after reaction was detected by stirring 12. 12 h at room temperature, the solvent was dried by spinning, the light yellow precipitate was obtained by stirring with diethyl ether, the catalyst was removed by three extractions with 100 mL water after dissolution with 100 mL dichloromethane, and the product Boc-CIP-DMAEE (87%) was obtained by chromatography after drying and spinning, which had nuclear magnetic data of ：1H NMR (400 MHz,CDCl3)δ(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= 4.76 Hz, 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).13C NMR (400 MHz,CDCl3)δ(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 C28H40FN4O6 +(M+H)+547.29264, found 547.29309.
(C) Boc-CIP-DMAEE (0.015 mol,8.19 g) was dissolved in 20mL acetonitrile and then tert-butyl bromoacetate (0.022 mol,4.34 g) and a small amount of anhydrous sodium carbonate as catalyst were added thereto, and after reaction at 50℃under nitrogen atmosphere, 1: 1h was detected and after completion of the detection, boc-CIP-OB-tBu (99%) was obtained by precipitation with diethyl ether, whose nuclear magnetic data were ：1H NMR (400 MHz, CDCl3)δ( 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).13C NMR (400 MHz,CDCl3)δ(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 C34H50FN4O8 +(M)+661.36072, found 661.36102.
(D) Boc-CIP-OB-tBu was dissolved in dichloromethane/trifluoroacetic acid (1:1, v/v) solution, reacted at room temperature for 2 h, the tert-butyl ester was removed, precipitated in diethyl ether, stirred and filtered to give the product CIP-CB (95%) with nuclear magnetic data of ：1H NMR (400 MHz, MeOD-d4)δ(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).13C NMR(400 MHz, MeOD-d4)δ(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 C25H34FN4O6 +(M)+505.24569, found 505.24597.
The CIP-CB prepared by the method is prepared into micelles, and the specific process is that the uniform distribution of micelle concentration is 1 mg/mL after the solution of 20 mg CIP-CB in 20mL pH =7.4 PBS is stirred for 2h at room temperature.
1. The CIP-CB micelle obtained above was characterized in that the particle size and Zeta potential of the CIP-CB micelle were measured by a laser particle sizer (MASTERSIER, 2000), and the morphology was measured by a transmission electron microscope TEM (FEI TECNAI G F20), and by a field emission scanning electron microscope (FE-SEM, FEIINSPECT F; U.S.) at 20 kv. The Critical Micelle (CMC) is determined by using Nile red as fluorescent probe and spectrophotometrically, and the specific operation steps are that 1mg of Nile red is dissolved in 50 mL tetrahydrofuran solution, and after being evenly mixed, 100 microliter of Nile red is taken and added into a glass bottle, and after being naturally volatilized in a fume hood, 4 mL micelle is added, and the concentration is from 0.2 mg/mL to 110 -5 Mg/mL, shaking in a 40 ℃ constant temperature shaking table for one night, standing for half a day, and testing the fluorescence emission intensity of the micelle by using a fluorescence spectrometer, wherein the slit widths of excitation light and emission light are 20 nm, the excitation wavelength is 550 nm, and the emission range is 570-750 nm.
The characterization results are shown in fig. 1, wherein A in fig. 1 is the hydrodynamic diameter distribution result of CIP-CB micelle, B is the Zeta potential distribution result of CIP-CB micelle at different pH values, C is a TEM image of a sample dyed with 2.5% phosphotungstic acid, and D is the relation between the emission intensity of nile red at 575 nm and the concentration of CIP-CB. As can be seen from FIG. 1, the CIP-CB micelles were uniformly dispersed in water, the morphology was spherical with a core-shell structure, and then the potential changes of CIP-CB at different pH values were measured with DLS at 25 ℃. The carboxylic acid groups of CIP-CB exhibit deprotonation in normal physiological environment in vivo (pH 7.4), micelles exhibit negative charges to a large extent, carboxyl groups begin to undergo protonation as the pH value decreases gradually, and the charge of the micelles changes as shown in FIG. 1B, with a potential of about-25 mV in neutral pH 7.4 and a micelle point of about +10mV in acidic environment (pH 5.5). The micelle with the charge turning function can keep long circulation in vivo, target bacteria in an infection environment, strengthen the interaction between antibiotics and bacteria and improve the sterilization effect.
2. Antibacterial test of CIP-CB micelles
In order to verify the antibacterial effect of CIP-CB micelles, the present invention employs Staphylococcus aureus (S.aureus) and Escherichia coli (E.coil) as model bacteria. The antibacterial effect of CIP-CB was demonstrated by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).
The invention adopts three groups of different conditions of CIP, pH 7.4 and pH 5.5 to carry out experiments, and after the three groups of conditions are respectively co-cultured with bacteria for different times, the absorbance at the position of 600 nm is measured by means of an enzyme-labeled instrument to detect the propagation condition of the bacteria. As a result, as shown in FIG. 2, the antibacterial effect of CIP-CB was significantly improved as compared with ciprofloxacin hydrochloride, and the antibacterial effect at pH 5.5 was significantly improved as the neutral to acidic condition was changed.
The minimum inhibitory concentration of the red tetrazolium serving as an indicator for the micelle is determined by adopting a dilution method, as shown in table 1, MICs of CIP to escherichia coli and staphylococcus aureus are 20.5 ppm and 20.5 ppm respectively, MICs of CIP-CB to escherichia coli and staphylococcus aureus under the condition of pH 7.4 are 5.10 ppm and 10.2 ppm respectively, and MICs of CIP to escherichia coli and staphylococcus aureus under the condition of pH 5.5 are 2.55 ppm and 5.10 ppm respectively.
Table 1 CIP-MIC and MBC results of CB
The micelles were further assayed for Minimum Bactericidal Concentration (MBC) against e.coli and staphylococcus aureus using standard plate count methods. CIP, pH 7.4 and pH 5.5 are 41.9 ppm, 10.2 ppm and 5.10 ppm for E.coli, respectively, and 41.9 ppm, 10.2 ppm and 5.10 ppm for Staphylococcus aureus, respectively. The results from MIC and MBC as well as the microplate reader indicate that CIP-CB micelles improve the antibacterial effect of CIP-CB, especially CIP-CB under acidic conditions, over CIP-CB micelles under neutral conditions (fig. 3). Such results are attributed to the fact that micelles increase the bioavailability and delivery of antibiotics, and that the protonation of the carboxylic betaines imparts targeting functions to the micelles and bacteria, and that the penetration of such intelligently-responsive micelles in biological membranes is more desirable.
And (3) using multidrug resistant strains ESBL E.coll and MRSA as model bacteria, and detecting the sterilizing effect of CIP-CB on the drug resistant bacteria. The antibacterial effect of CIP, CIP-CB on both resistant strains is significantly reduced compared to planktonic bacteria at pH 7.4 and pH 5.5. However, the antibacterial effect of CIP-CB on drug-resistant bacteria was significantly improved compared to CIP (FIG. 4).
3. CIP-CB anti-biofilm test
CIP-CB was dispersed in PBS buffer at pH 7.4 and pH 5.5 to give micelles with opposite charges on the surface. Mature biofilms were obtained after seven days of continuous culture using staphylococcus aureus as model bacteria, then 200 μl of CIP-CB solution was added, and after 16 h co-culture with the biofilms at 37 ℃, the anti-biofilm activity of CIP-CB micelles was verified using standard plate counting method, laser confocal microscopy (CLSM).
The standard plate count results are shown in FIG. 5, where the bar graphs in FIG. 5A are CIP-CB (pH 7.4), CIP-CB (pH 5.5), CIP and Control, respectively, from left to right. As can be seen from FIG. 5, after 16 h is co-cultured with CIP-CB and the biological film, the survival rates of the biological film under the condition of pH 7.4 are respectively 16.6%, 22.3%, 24.6%, 32.8% and 35.6% respectively, and the survival rates of the biological film under the condition of pH 5.5 are respectively 26.7%, 31.9%, 37.4%, 45.4% and 56.0%, and in an acidic environment, the surfaces of the biological film take positive charges, and the CIP-CB micelles are favorable for penetrating through the biological film matrix and conveying antibiotics to bacteria wrapped in the biological film, so that the biological film removing effect is achieved.
The results of laser confocal microscopy verification are shown in FIG. 6, where CIP-CB is shown in FIG. 6 for confocal microscopy images at pH 5.5 and pH 7.4 for treatment of Staphylococcus aureus biofilms 2h (A), 16 h (B), 2h (C) 16 h (D) and 2h (E) and 16 h (F), respectively. After incubation of 7d, staining was performed with live/dead bacteria viability kit and live (green) and dead (red) bacteria were imaged with laser scanning confocal microscopy. As can be seen in fig. 6, after the untreated mature biofilm was stained with dye, a thick biofilm was seen, all with green fluorescence. The red fluorescence intensity of the biological film after 16 treatment under the condition of pH 7.4 is enhanced, and the red fluorescence of the biological film after 16 h treatment under the condition of pH 5.5 is stronger, which proves that the CIP-CB micelle has the capability of penetrating the biological film and achieves a deeper sterilization effect.
4. CIP-CB hemolytic Activity test
The effect of different concentrations of CIP-CB on mouse Red Blood Cells (RBCs) was obtained by co-culturing the CIP-CB diluted to different concentrations with PBS (ph=7.4) for 2 h with red blood cells, and measuring the absorbance at 570 nm with a microplate reader to evaluate the release of hemorrhagic hemoglobin. The hemolysis rate of the micelle detected by photographs and a microplate reader after the CIP-CB (5.11-81.8 ppm) and the red blood cells are suspended in co-culture 2 h and centrifugalized is shown as 2.15, the hemolysis rate of the micelle is generally lower, the hemolysis rate is less than 3% even when the concentration is as high as 81.8-ppm, and the sterilization effect on escherichia coli and staphylococcus aureus is also improved while the blood compatibility is ensured to be good. This is sufficient to demonstrate that CIP-CB exhibits a satisfactory result for erythrocytes and bacteria (FIG. 7).
Example 2:
The antibacterial agent of example 1 was replaced with the following quinolones, and the preparation process was the same as that of example, and the substrate and the prepared antibacterial agent had the following efficiency in removing s.aureus biofilm at a concentration of 50 ppm:
TABLE 2 efficiency of removal of S.aureus biofilm by quinolone substrates at a concentration of 50ppm
The nuclear magnetic data of the above products are as follows:
product(s) 1：1H NMR (400 MHz, MeOD)δ(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).
Product(s) 2：1H NMR (400 MHz, MeOD)δ(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).
Product(s) 3：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 4：1H NMR (400 MHz,MeOD)δ(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= 5.14 Hz, 4H), 3.34 (s, 6H), 1.42 (m, 2H), 1.19 (m, 2H).
Product(s) 5：1H NMR (400 MHz,MeOD)δ(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).
Example 3:
The antibacterial agent of example 1 was replaced with the following penicillin species, and the preparation process was the same as in example, and the substrate and the prepared antibacterial agent had the following efficiency in removing s.aureus biofilm at a concentration of 50 ppm:
TABLE 3 efficiency of removal of S.aureus biofilm by penicillin substrates at a concentration of 50ppm
The nuclear magnetic data of the above products are as follows:
product(s) 1：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 2：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 3：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 4：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 5：1H NMR (400 MHz,MeOD)δ(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).
Example 4:
The antibacterial agent of example 1 was replaced with the following cephalosporins, and the preparation process was the same as in example, and the substrate and the prepared antibacterial agent were as follows at a concentration of 50ppm for removing S.aureus biofilm:
TABLE 4 efficiency of removal of S.aureus biofilm by cephalosporin substrates at a concentration of 50ppm
The nuclear magnetic data of the above products are as follows:
product(s) 1：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 2：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 3：1H NMR (400 MHz,MeOD)δ(ppm)：8.19 (s, 1H), 5.52 (m, 1H), 4.48 (t,J= 3.56 Hz, 2H), 3.89 (s, 2H), 3.83 (m, 2H), 3.63 (m, 2H), 3.52 (m, 2H), 3.15 (s, 2H), 2.27 (s, 6H), 3.20 (m, 1H). 2.54 (m, 1H). 2.12 (s, 3H). 1.16 (m, 6H). 1.03 (m, 6H).
Product(s) 4：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 5：1H NMR (400 MHz,MeOD)δ(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).
The antibacterial agent in examples 1-4 is prepared by modifying carboxylic betaine on an antibacterial agent through an ester bond, forming amphiphilic small molecules, self-assembling in water to form micelles, wherein the micelles have negative charges in a physiological environment, so that cytotoxicity of the antibacterial agent is reduced, long circulation in vivo is facilitated, conversely, the carboxylic betaine is deprotonated under an acidic condition, the surface of the micelles is converted into positive charges, effective permeation and accumulation can be performed on a biological membrane, the antibacterial agent is transferred into the biological membrane, and the ester bond is degraded by lipase secreted by bacteria to release the antibacterial agent, so that the antibacterial performance on planktonic bacteria and bacteria in the biological membrane is improved.
Example 5:
an antibacterial agent is Man-TEG-CIP, and the synthetic route is as follows:
(a) Synthesis of Acetylmannose (Ac-Man) by slowly adding 15g of mannose (added within 15-20 min) into 60ml of pyridine in batch under ice bath condition, continuously stirring for dissolving, slowly adding 100mL acetic anhydride, continuously reacting for 30min, removing ice bath, reacting at room temperature for 3 h point plate (V acetic acid ethyl ester ：V petroleum ether =1:3), determining that the reaction is complete, dissolving with DCM (100 mL), washing with 5% hydrochloric acid aqueous solution, 0.5M NaOH aqueous solution, saturated saline solution, drying the organic layer with anhydrous sodium sulfate, filtering, concentrating to obtain Acetylmannose （Ac-Man）.Ac-Man：1H NMR (400 MHz, CDCl3,δ):δ6.08 (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).
(B) Synthesis of Compound Ac-Man-TEG 5.7 g (0.07 mol) acetylmannose, 26 mL (0.21 mol) triethylene glycol (TEG) were dissolved in 150 ml anhydrous dichloromethane, 60 mL (0.56 mol) boron trifluoride diethyl ether was slowly added to the above solution at 0℃under nitrogen, after the reaction was completed by 36. 36 h plates at room temperature, each was washed with 2X 100 mL saturated sodium bicarbonate solution, 2X 100 mL distilled water, 100 mL saturated saline until no bubbles were generated, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure, and purified by silica gel chromatography (TLC=EA: meOH=6:1) as eluent with ethyl acetate and n-hexane to give Compound Ac-Man-TEG.Ac-Man-TEG：1H NMR (400 MHz, CDCl3,δ):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, 4HAc) ppm.
(C) Synthesis of Compound Boc-CIP CIP ciprofloxacin (1.0 g,3.00 mmol) was added to 4.5 mL of 1N NaOH and 40 mL dioxane: water=1:1 (volume ratio), followed by di-tert-butyl dicarbonate Boc 2 O (1.0 g,4.6 mmol), reacted at room temperature under nitrogen atmosphere for 17h until the reaction was complete, the white solid was filtered, then rinsed clean with water and acetone, respectively, and dried in vacuo. Developer TLC (DCM: meoh=9:1) detection, product was a white to pale yellow solid （Boc-CIP,98%）1H NMR (400 MHz, CDCl3)δ(ppm): 8.76 (s, 1H), 8.02 (d,J= 12.9 Hz, 1H), 7.36 (d,J= 7.1 Hz, 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).
(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）,DIPEA（26.7 mmol,3.45 g,2.0 eq） after completion of the reaction by stirring 12: 12 h under room temperature conditions in a dry dichloromethane solution, extraction with 100 mL water three times, drying over anhydrous sodium sulfate, filtration, evaporation of the solvent under reduced pressure, purification by chromatography (TLC=CH 2Cl2: meOH=20:1) eluting with dichloromethane and methanol to give the product Ac-Man-TEG-Boc-CIP.Ac-Man-TEG-Boc-CIP：1H NMR (400 MHz,CDCl3)δ8.54 (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, 4HAc), 1.49(s, 9H), 1.32(m, 2H), 1.13(m, 2H). HRMS (ESI) m/z calcd for C42H56FN3O17Na+(M+Na)+916.34860, found 916.34900.
(E) Synthesis of the Compound Man-TEG-Boc-CIP 0.3 g Ac-Man-TEG-Boc-CIP was dissolved in 10 ml of anhydrous methanol, anhydrous sodium methoxide (0.06 wt% Na) 3 mL was slowly added dropwise, after fifteen minutes of reaction at room temperature, the reaction was detected to be complete, neutralization was performed with acetic acid solution, and solvent evaporation was performed with silica gel column chromatography to obtain the product Man-TEG-Boc-CIP.Man-TEG-Boc-CIP：1H NMR (400 MHz,MeOD)δ8.74 (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 C34H48FN3O13Na+(M+Na)+748.30634,found 748.30652.
(F) Synthesis of the Compound Man-TEG-CIP 0.5 g Man-TEG-Boc-CIP was dissolved in 10ml of dichloromethane, 10mL trifluoroacetic acid was added, after stirring for two hours at RT, the tert-butyl group was determined to be removed, and after drying the solvent under reduced pressure, it was precipitated in diethyl ether to give the product Man-TEG-CIP.Man-TEG-CIP：1H NMR (400 MHz,MeOD)δ8.71 (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 C29H41FN3O11 +(M+H)+626.27196, found 626.27191.
The Man-TEG-CIP prepared in the above way is prepared into micelle, and the specific process is that the uniform distribution micelle concentration is 1 mg/mL after the preparation of the Man-TEG-CIP is weighed to be 20 mg Man-TEG-CIP and dissolved in PBS of 20 mL pH =7.4 and stirred for 2 h at room temperature.
The Man-TEG-CIP micelles prepared above were characterized as follows:
1. The antibacterial agent is an amphiphilic small molecular micelle formed by combining mannose and ciprofloxacin in an ester bond mode, the micelle can be formed by self-assembly in an aqueous solution, the particle size of Man-TEG-CIP is measured by a dynamic light scattering instrument (DLS) at 25 ℃, the particle size is about 100nm, the particle size is uniformly dispersed, the micelle is dripped on a copper mesh, the morphology of the micelle is observed by a transmission electron microscope after being dyed by phosphotungstic acid, and the micelle is uniformly dispersed and has a good core-shell structure as shown in figure 8.
2. The critical micelle concentration of the micelle is determined by adopting nile red as fluorescent molecules, as shown in fig. 9, the critical micelle concentration of the Man-TEG-CIP is 6.98X10 -4 mg/mL, the concentration is above CMC and exists in a micelle form, mannose is exposed outside, ciprofloxacin is in an inner shell, mannose of a micelle shell presents a multivalent state, the affinity to escherichia coli is high, and the interaction between the Man-TEG-CIP and bacteria can be enhanced.
3. Man-TEG-CIP prevents biofilm colonization
After co-culturing Man-TEG-CIP micelles with different concentrations with escherichia coli for different times, the conditions of the escherichia coli biofilms were observed through laser confocal, and the adhesion conditions of 24h, 48 h and 72 h biofilms were respectively explored as shown in FIG. 10. The biological film on the surface of the membrane is gradually increased and the thickness is also gradually increased along with the increase of the blank control group along with the time, and a compact biological film is formed after 72 h. After the escherichia coli is co-cultured with the Man-TEG-CIP for 24h hours, only a small amount of bacteria adhere to the surface of the membrane, after the time delay is 48. 48 h, biofilm colonization starts to appear on the membrane after the membrane is cultured with the 5.10 ppm Man-TEG-CIP micelle, a small amount of biofilm colonization also appears in the membrane at 10.2 ppm, and a trace amount of biofilm appears under the condition of 20.5 ppm. After 24h of co-culture of escherichia coli and Man-TEG-CIP, colonization of the membrane begins to occur, because the micelle is degraded by enzymes in a bacterial environment, so that the multivalent sugar form micelle begins to be lysed, and the effect on bacteria is reduced.
To further demonstrate that Man-TEG-CIP prevented biofilm colonization, we observed with Scanning Electron Microscopy (SEM) after the same procedure, as shown in fig. 11, the blank group proliferated in large numbers with time, e.g. and colonized the membrane with FimH lectin to form a dense biofilm. With the addition of Man-TEG-CIP, the biofilm on the membrane began to develop low density, dispersed colonies, with the increased Man-TEG-CIP concentration, the integrity of the biofilm being more affected, consistent with the laser confocal data.
4. Hemolytic Activity test
The haemocompatibility of Man-TEG-CIP was assessed by a haemolysis test, specifically by co-culturing with erythrocytes 2h after dilution of Man-TEG-CIP to different concentrations with PBS (ph=7.4), and by measuring the absorbance at 570 nm with a microplate reader to assess the release of haemoglobin, the effect of Man-TEG-CIP at different concentrations on mouse erythrocytes (RBCs) was obtained. The hemolysis rate of the micelle detected by photographs and a microplate reader after the different concentrations of Man-TEG-CIP (5.11-81.8 ppm) and red blood cells are suspended in co-culture 2h and centrifuged is shown as 12, the hemolysis rate of the micelle is generally lower, the hemolysis rate is less than 5% even when the concentration is up to 81.8 ppm, and the effect on escherichia coli is greatly improved while the blood compatibility is good.
Example 6:
The antibacterial agent of example 5 was replaced with the following quinolones, and the preparation process was the same as that of example, and the substrate and the prepared antibacterial agent had the following efficiency in removing E.coil biofilm at a concentration of 50 ppm:
TABLE 5 efficiency of E.coil biofilm removal of quinolone substrates at a concentration of 50ppm
The nuclear magnetic data of the above products are as follows:
Product(s) 1：1H NMR (400 MHz, MeOD)δ(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).
Product(s) 2：1H NMR (400 MHz, MeOD)δ(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).
Product(s) 3：1H NMR (400 MHz, MeOD)δ(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).
Product(s) 4：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 5：1H NMR (400 MHz, MeOD)δ(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).
Example 7:
The antibacterial agent of example 5 was replaced with the following penicillin substances, and the preparation process was the same as in example, and the substrate and the prepared antibacterial agent had the following efficiency in removing E.coil biofilm at a concentration of 50 ppm:
TABLE 6 efficiency of removal of E.coil biofilm from penicillin substrates at a concentration of 50ppm
The nuclear magnetic data of the above products are as follows:
product(s) 1：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 2：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 3：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 4：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 5：1H NMR (400 MHz,MeOD)δ(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).
Example 8:
The antibacterial agent of example 5 was replaced with the following cephalosporins, and the preparation process was the same as in example, and the substrate and the prepared antibacterial agent had the following efficiency in removing E.coil biofilm at a concentration of 50 ppm:
TABLE 7 efficiency of removal of E.coil biofilm from cephalosporin substrates at a concentration of 50ppm
The nuclear magnetic data of the above products are as follows:
product(s) 1：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 2：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 3：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 4：1H NMR (400 MHz,MeOD)δ(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).
Product(s) 5：1H NMR (400 MHz,MeOD)δ(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).
The antibacterial agent in examples 5-8 is prepared by modifying mannose on an antibacterial drug through an ester bond, forming micelle with multivalent sugar form in solution, testing the structure and morphology of the micelle, and verifying that mannose enhances the antibacterial activity of ciprofloxacin due to the enhancement of the action effect of multivalent sugar and escherichia coli through an antibacterial experiment, and then verifying that mannose-modified antibiotics can prevent the colonization of escherichia coli and reduce the formation of a biological film through a scanning electron microscope and laser confocal.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (3)

1. The preparation method of the antibacterial agent is characterized in that the antibacterial agent comprises small organic molecules and antibacterial drugs, the small organic molecules and the antibacterial drugs are connected through ester bonds, the small organic molecules are carboxylic acid betaine or mannose, the antibacterial drug is ciprofloxacin, and the structural formula of the antibacterial agent is as follows:
Or (b) ;
When the organic small molecule is a carboxylic betaine, the method comprises the following steps:
(1) Dissolving an antibacterial drug, adding di-tert-butyl dicarbonate, and reacting at room temperature under an inert gas atmosphere to obtain a Boc-protected antibacterial drug;
(2) Mixing the antibacterial drug protected by Boc, N-dimethylaminoethyl glycol, TBTU and DIPEA according to the molar ratio of 1:2-4:3-5:6-8, adding the mixture into a solvent, and stirring the mixture at room temperature for reaction for 10-15h to obtain a product;
(3) Dissolving the product obtained in the step (2), and then adding tert-butyl bromoacetate and alkali to react for 0.5-1.5h at 45-55 ℃ in an inert gas atmosphere to obtain the product;
(4) Performing tert-butyl removal reaction on the product obtained in the step (3) in a mixed solvent of dichloromethane and trifluoroacetic acid, then precipitating and filtering to obtain an antibacterial agent;
when the organic small molecule is mannose, the method comprises the following steps:
(1) Dissolving mannose under ice bath condition, adding acetic anhydride, reacting for 25-35min under ice bath condition, and reacting at room temperature for 2-4h to obtain acetylmannose;
(2) Dissolving acetylmannose and TEG, then adding a catalyst, and reacting for 34-38 hours at room temperature to obtain a product, wherein the molar ratio of the acetylmannose to the TEG to the catalyst is 1:3-4:8-10;
(3) Dissolving an antibacterial drug, adding di-tert-butyl dicarbonate, and reacting at room temperature under an inert gas atmosphere to obtain a Boc-protected antibacterial drug;
(4) Mixing the antibacterial drug protected by Boc, the product obtained in the step (2), TBTU and DIPEA according to the molar ratio of 1:2-3:3-5:5-7, adding the mixture into a solvent, and stirring the mixture for 10-14h at room temperature to obtain the product;
(5) Dissolving the product obtained in the step (4), then adding alkali, and reacting for 10-20min at room temperature to obtain the product;
(6) And (3) dissolving the product obtained in the step (5), then carrying out tert-butyl removal reaction in a mixed solvent of dichloromethane and trifluoroacetic acid, then precipitating, and filtering to obtain the antibacterial agent.
2. The method for preparing the antibacterial agent according to claim 1, wherein when the organic small molecule is carboxylic betaine, the alkali in the step (3) is anhydrous sodium methoxide, anhydrous potassium carbonate or anhydrous sodium carbonate, and the reaction condition for removing tert-butyl ester in the step (4) is room temperature, and the reaction is carried out for 1.5-3.5 hours.
3. The method for preparing the antibacterial agent according to claim 1, wherein when the organic small molecule is mannose, boron trifluoride diethyl etherate is used as the catalyst in the step (2), anhydrous sodium methoxide, anhydrous potassium carbonate or anhydrous sodium carbonate is used as the base in the step (5), and the reaction condition of t-butyl removal in the step (6) is room temperature and the reaction time is 1.5-3.5 hours.
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