Compounds with antibacterial properties, methods of making and using the same

Abstract

The application relates to the field of antibacterial materials and discloses a compound with antibacterial performance and a preparation method and application thereof, wherein the compound has the structure shown in the following formula I. The compound with antibacterial performance provided by the application can have excellent killing effect on staphylococcus aureus and escherichia coli, and the sterilization rate can be up to 99.99%. The preparation method of the compound is simple and efficient, is suitable for industrial popularization, and moreover, the compound with antibacterial performance provided by the application has stable physical properties, can be recycled, has biological safety, can reduce the infection degree of a wound, and can promote the healing of the wound.

Description

Technical Field

[0001] This invention relates to the field of antibacterial materials technology, specifically to a compound with antibacterial properties, its preparation method, and its application. Background Technology

[0002] The environment contains a large number of pathogenic and mold-causing harmful microorganisms that can threaten human health through various pathways. Using antimicrobial materials containing antimicrobial substances is the simplest, safest, and most effective way to prevent the growth, reproduction, spread, and transmission of harmful microorganisms.

[0003] Traditional antibacterial materials contain antibacterial substances such as heavy metals, quaternary ammonium salts, and triclosan, but these substances suffer from drawbacks such as high cost, weak antibacterial ability, and low bactericidal efficiency. Halogenated amine antibacterial substances have been widely used in recent years due to their broad antibacterial spectrum, rapid bactericidal action, regenerable antibacterial activity, and lack of toxic byproducts. However, the preparation of antibacterial materials based on halogenated amine antibacterial agents involves numerous reactants, expensive raw materials, harsh reaction conditions, complex processes, and high production costs, making them unsuitable for industrial-scale promotion. Furthermore, the antibacterial properties and morphological stability of these halogenated amine antibacterial substances still need improvement.

[0004] Therefore, there is an urgent need to develop a uniform, stable, and highly effective antibacterial compound and antibacterial material. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of uneven antibacterial agents and poor stability in the prior art, and to provide a compound with antibacterial properties, its preparation method and application.

[0006] To achieve the above objectives, the present invention provides a compound with antibacterial properties, the compound having the structure shown in Formula I:

[0007]

[0008] A second aspect of the present invention provides a method for preparing a compound with antibacterial properties, the method comprising:

[0009] (1) In the presence of a solvent and a basic catalyst, the compound shown in formula a is brought into contact with the compound shown in formula b to carry out a substitution reaction;

[0010] (2) Chlorinate the product of step (1) by contacting it with hypochlorite.

[0011] A third aspect of the present invention provides the use of the compounds described above in the preparation of agents for killing bacteria.

[0012] A fourth aspect of the present invention provides the use of the compounds described above in the preparation of medicaments for promoting wound healing.

[0013] The beneficial technical effects achieved by the present invention through the above technical solutions include at least the following:

[0014] 1) The compounds with antibacterial properties provided in this invention have an effective chlorine content of more than 2 wt%, and have excellent killing effects on Staphylococcus aureus and Escherichia coli, with a sterilization rate of up to 99.99%;

[0015] 2) The method for preparing compounds with antibacterial properties provided in this invention can synthesize compounds with antibacterial properties in a simple and efficient manner, which is suitable for industrial promotion. Furthermore, the antibacterial activity of the compounds and the antibacterial agents prepared therefrom can be regenerated by simple halogenation treatment, and the antibacterial ability can be quickly restored in situ.

[0016] 3) The gel prepared using the antibacterial compound provided by this invention has stable physical properties, is recyclable, has biosafety, and can reduce the degree of wound infection and promote wound healing. Attached Figure Description

[0017] Figure 1 These are the infrared spectra of 3TDETA and 3TDETA-Cl synthesized in Example 1;

[0018] Figure 2 These are the sterilization results of 3TDETA-Cl prepared in Example 1 at different concentrations;

[0019] Figure 3 The results show the sterilization effects of the control group, 3TDETA, and 3TDETA-Cl.

[0020] Figure 4 The cell morphology of Escherichia coli after sterilization with 3TDETA and 3TDETA-Cl is shown in the control group.

[0021] Figure 5 These are state diagrams of HA-DA and HA-DA@3TDETA-Cl prepared in Example 1;

[0022] Figure 6 The microstructures of HA-DA@3TDETA-Cl with different 3TDETA-Cl contents;

[0023] Figure 7 These are the performance test results of HA-DA, HA-DA@3TDETA, and HA-DA@3TDEA-Cl gels; among them, (A), (B), (C), and (D) are the variation laws of G and G” with angular frequency, time (strain constant at 1%), strain, and time (strain at 1% and 25%), respectively; (E) is the law of viscosity variation with shear rate; and (F) is the result of injectability test.

[0024] Figure 8 The data pertains to the wound condition of mice in the three experimental groups during days 1-7 of the experiment.

[0025] Figure 9 This is a graph showing the changes in the wound condition of mice in the three experimental groups from day 1 to day 7 of the experiment;

[0026] Figure 10 The results of in vivo tests on the control group, HA-DA@3TDETA, and HA-DA@3TDEA-Cl are shown below. (A) is a graph showing the wound diameter of mice in the three experimental groups during days 1-7 of the experiment; (B) is a graph showing the weight of mice in the three experimental groups during days 1-7 of the experiment; (C) is a graph showing the in vitro hemolysis test results of different gels; (D) is a graph showing the test results of inflammation-related hematological parameters of different gels; and (E) is a graph showing the histological characteristics of the wounds of mice in the three experimental groups during day 6 of the experiment.

[0027] Figure 11 This is the 1H NMR spectrum of 3TDETA obtained in Example 1. Detailed Implementation

[0028] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0029] The first aspect of this invention provides a compound with antibacterial properties, the compound having the structure shown in Formula I:

[0030]

[0031] In Formula I, m and n are each independently 1-6, each NX is selected from N-Cl and / or NH and at least one NX is N-Cl, the structure of R is as shown in Formula II (each R can be the same or different), and X1 and X2 are each independently halogens.

[0032] In a preferred embodiment of the present invention, X1 and X2 are chlorine.

[0033] Preferably, m and n are each independently 1-3.

[0034] To obtain compounds with higher antibacterial properties, more preferably, m and n are 2.

[0035] In a preferred embodiment of the present invention, the content of N-Cl (available chlorine) in Formula I is 0.2-2 mol / kg, more preferably 0.5-0.8 mol / kg.

[0036] A second aspect of the present invention provides a method for preparing a compound (formulation) with antibacterial properties, the method comprising:

[0037] (1) In the presence of a solvent and a basic catalyst, the compound shown in formula a is brought into contact with the compound shown in formula b to carry out a substitution reaction.

[0038]

[0039] Among them, X a X b and X c Each can be a halogen independently; m and n can each be 1-6 independently;

[0040] (2) Chlorinate the product of step (1) by contacting it with hypochlorite.

[0041] According to the present invention, the chlorination mainly occurs on the -NH- group, and after chlorination, at least part of the -NH- is converted to -NCl-.

[0042] In a preferred embodiment of the present invention, X a X b and X c Each is independently chlorine (i.e., the compound shown in formula a is cyanuric chloride).

[0043] Preferably, m and n are each independently 1-3.

[0044] To obtain compounds with higher antibacterial properties, more preferably, m and n are 2 (i.e., the compound shown in formula b is diethylenetriamine).

[0045] In this invention, the molar ratio of the compound shown in formula a to the compound shown in formula b can be adjusted according to the specific value of n in formula b. The molar ratio can be less than n+1, equal to n+1, or greater than n+1. According to a specific embodiment of this invention, the molar ratio of the compound shown in formula a to the compound shown in formula b can be (1-5):1, for example, it can be 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, and 5:1, as well as any range between any two values. However, in order to obtain better physical properties and bactericidal effect, it is preferred to be (2-4):1.

[0046] In this invention, the alkaline catalyst can be a substance commonly used in the art for catalyzing substitution reactions, such as at least one of alkali metal carbonates, alkali metal bicarbonates, alkali metal hydroxides and organic amines, preferably at least one of sodium carbonate, sodium bicarbonate, sodium hydroxide and 1,8-diazabicyclo[5.4.0]undec-7-ene, more preferably sodium hydroxide.

[0047] Preferably, the molar ratio of the alkaline catalyst to the compound shown in formula b is (1-5):1, more preferably (2-4):1.

[0048] According to the present invention, the alkaline catalyst can be directly mixed with the reactants in solid (including powder) form, or it can be dissolved in water and provided in solution form. When the alkaline catalyst is provided in solution form, its concentration can be a concentration commonly used in the art for catalyzing substitution reactions, which can be 1-50 wt%, for example, a range or value within any two of the following values: 1 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, and 50 wt%. In order to further promote the substitution reaction and reduce side reactions, the concentration of the alkaline catalyst is preferably 20-45 wt%.

[0049] In this invention, the solvent can be a solvent commonly used in the art, preferably at least one of dichloromethane, tetrahydrofuran and butanone, and more preferably dichloromethane.

[0050] According to the present invention, the weight ratio of the compound and solvent shown in Formula a can be 1:(10-50), for example, it can be 1:10, 1:12, 1:13, 1:14, 1:15, 1:20, 1:30 and 1:50 or any two values ​​within the range and values ​​within the range. In order to promote the substitution reaction, the weight ratio of the compound and solvent shown in Formula a is preferably 1:(10-20), more preferably 1:(12-16).

[0051] According to the present invention, in order to make the substitution reaction proceed more fully and reduce the formation of byproducts, the compound represented by formula a can be dissolved in a solvent before the reaction occurs.

[0052] According to the present invention, the order in which the compound of formula b, the basic catalyst, and the compound of formula a are mixed is not limited. The compound of formula b may be mixed with the basic catalyst first and then contacted with the compound of formula a, or the compound of formula b and the basic catalyst may be contacted with the compound of formula a sequentially. However, in order to make the reaction more complete, it is preferable to mix the compound of formula a with the basic catalyst first, and then add the compound of formula b to the mixture of the compound of formula a and the basic catalyst.

[0053] In this invention, the temperature of the substitution reaction can be from -10°C to 40°C, for example, it can be any two of the following ranges: -10°C, -5°C, -4°C, -3°C, -2°C, -1°C, 0°C, 5°C, 10°C, 20°C, 30°C, 40°C, or values ​​within that range. To control the reaction rate and reduce the formation of byproducts, the temperature of the substitution reaction is preferably from -5°C to 5°C. To better control the reaction temperature, before the substitution reaction occurs, the temperatures of the basic catalyst, the compound shown in formula b, and the compound shown in formula a can be adjusted to near the temperature of the substitution reaction. According to this invention, the temperature can be achieved using an ice bath.

[0054] In this invention, the substitution reaction time can be 1-48h, for example, it can be any two of the following values: 1h, 5h, 10h, 15h, 20h, 25h, 30h, 35h, 40h, 45h, 48h, or a value within the range of these values, preferably 2-36h, more preferably 2-4h.

[0055] According to the present invention, after the substitution reaction is completed, the system can be post-treated to remove the solvent and residual or generated inorganic salts. The solvent can be removed by evaporation, and the evaporation temperature can be adjusted conventionally according to the solvent used, for example, from 30°C to 120°C. In one embodiment of the present invention, the evaporation method is rotary evaporation at a temperature of 35-55°C. The inorganic salts can be removed by water washing, which can be performed before or after solvent removal.

[0056] According to the present invention, after removing the solvent and inorganic salts, the product can be subjected to a first drying process, which can be freeze drying, supercritical carbon dioxide drying, or vacuum drying, preferably vacuum drying. The conditions for vacuum drying can include: a pressure of -0.08 MPa to -0.04 MPa (gauge pressure), a temperature of 40-70°C, preferably 50-60°C, and a time of 5-20 h, preferably 10-15 h.

[0057] According to the present invention, in order to obtain a product with higher purity, the substituted product can be ground into a fine powder before post-processing.

[0058] To further improve the purity of the substitution reaction product, the substitution product can be washed. The solvent used for washing can be a solvent capable of dissolving the reactants, such as at least one of dichloromethane, tetrahydrofuran, and butanone, preferably tetrahydrofuran. The washing can be performed at any time after the substitution reaction is completed, and the order of washing, evaporation, and water washing is not strictly limited. However, to further improve the purity of the substitution product, the washing is performed after evaporation and water washing.

[0059] According to the present invention, a second drying can be performed after washing. The conditions for the second drying may include: a pressure of -0.08 MPa to -0.04 MPa (gauge pressure); a temperature of 40-70°C, preferably 50-60°C; and a time of 1-20 h, preferably 10-15 h.

[0060] In this invention, common hypochlorite can be used to chlorinate the substituted product, but preferably, the hypochlorite is sodium hypochlorite.

[0061] In this invention, the weight ratio of hypochlorite to the substitution product of step (1) is preferably 1:(0.2-2), for example, it can be any two values ​​forming a range or value within the range of 1:0.2, 1:0.3, 1:0.5, 1:0.55, 1:0.6, 1:0.65, 1:1 and 1:2 or above, more preferably 1:(0.2-1), and even more preferably 1:(0.3-0.8).

[0062] According to the present invention, the hypochlorite can be directly mixed with the reactants in solid (including powder) form, or it can be dissolved in water and provided in solution form. When the hypochlorite is provided in solution form, its concentration can be a concentration commonly used in chlorination reactions in the art, which can be 0.1-20 wt%, for example, it can be any two values ​​forming a range or a value within a range of 0.1 wt%, 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 4 wt%, 6 wt%, 8 wt%, 10 wt%, 12 wt%, 14 wt%, 16 wt%, 18 wt%, and 20 wt%, or more. In order to further improve the antibacterial properties of the compound, the concentration of the hypochlorite solution is preferably 0.5-5 wt%, more preferably 0.5-3 wt%.

[0063] In this invention, the chlorination temperature can be 20-60°C, for example, it can be any two values ​​of 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, 35°C, 40°C, 50°C, and 60°C or above, or a value within the range. However, in order to reduce the generation of by-products, the temperature is more preferably 20-30°C.

[0064] In this invention, the chlorination time can be 1-12 hours, for example, it can be any two values ​​of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours and 12 hours or more, and values ​​within the range. In order to further improve the antibacterial properties of the compound, the chlorination time is more preferably 3-8 hours.

[0065] According to the present invention, in order to obtain compounds with better bactericidal properties, the product of the chlorination reaction can be further subjected to water washing and a third drying. The number of water washings can be flexibly adjusted according to specific circumstances, for example, 1-10 times. The third drying can be any commonly used drying method in the art. However, in order to further improve the antibacterial properties of the compound, the conditions for the third drying can include: pressure (gauge pressure) of -0.08 MPa to -0.04 MPa; temperature of 35-70°C, for example, any range of 35°C, 37°C, 39°C, 40°C, 41°C, 43°C, 45°C, 50°C, 55°C, 60°C, and 70°C, preferably 35-45°C; and time of 1-20 hours.

[0066] To achieve the best bactericidal effect, according to a preferred embodiment of the present invention, the method for preparing the compound includes:

[0067] (1) In the presence of solvent dichloromethane and alkaline catalyst NaOH, cyanuric chloride and diethylenetriamine were contacted at -5℃ to 5℃ to carry out a substitution reaction for 2.5-3.5h. The weight ratio of cyanuric chloride to solvent was 1:(12-16), and the molar ratio of cyanuric chloride, diethylenetriamine and alkaline catalyst was (2.5-3.5):1:(2.5-3.5). After the reaction was completed, the product was subjected to rotary evaporation, water washing and organic solvent washing.

[0068] (2) Add the substance obtained in step (1) to a 0.5-1.5 wt% sodium hypochlorite solution and react at 20-30℃ for 5-7 h. The amount of sodium hypochlorite solution used is such that the weight ratio of sodium hypochlorite to the substance obtained in step (1) is 1:(0.5-0.65). Wash and dry.

[0069] This invention also provides a formulation prepared by the above method. This formulation mainly contains the aforementioned compounds, and the available chlorine content is as described above. Due to limitations in raw material composition and separation conditions, the product (formulation) containing an antibacterial compound prepared by the above method may inevitably contain, in addition to the antibacterial compound provided by this invention, (partially) the reactants used to synthesize the compound (including the compound shown in formula a, the compound shown in formula b, and substances that have not been chlorinated after their substitution reaction), (partially) reaction byproducts, and (partially) impurities from the raw materials used to synthesize the compound. However, as long as the compound provided by this invention is obtained or used, it falls within the protection scope of this invention.

[0070] A third aspect of the present invention provides the use of the compounds described above in the preparation of agents for killing bacteria.

[0071] In this invention, the bacteria may be Staphylococcus aureus and / or Escherichia coli. According to some embodiments of the invention, the Staphylococcus aureus is designated ATCC 6538, and / or the Escherichia coli is designated ATCC 8099.

[0072] According to one embodiment of the present invention, the content of the compound represented by Formula I is 20-28 μg / mL, and the bacterial concentration is 5 × 10⁻⁶. 5 -5×10 6 At a concentration of CFU / mL, the sterilization rate reached 99.99%, and the sterilization rate will further increase as the concentration increases to 50 μg / mL or higher.

[0073] A fourth aspect of the present invention provides the use of the compounds described above in the preparation of medicaments for promoting wound healing.

[0074] In this invention, the drug preferably contains the compounds as described above and dopamine-modified hyaluronic acid.

[0075] According to the present invention, the hyaluronic acid is preferably provided (used) in the form of sodium hyaluronate; the purity can be greater than 50%, and in order to obtain a drug with better bactericidal effect and more stable physical properties, the purity is preferably greater than 90%.

[0076] According to the present invention, in the drug, the weight ratio of the compound as described above to dopamine-modified hyaluronic acid can be 1:(5-100), preferably 1:(25-65), and more preferably 1:(25-35).

[0077] According to the present invention, the method for preparing the drug may involve adding the aforementioned compound to dopamine-modified hyaluronic acid. According to some embodiments of the present invention, when the content of the aforementioned antibacterial compound in the drug is 0.6-1.5 mg / 30 mg of dopamine-modified hyaluronic acid, the network structure of the pores on the drug surface is optimal, which is most suitable for wound healing.

[0078] According to the present invention, the dopamine-modified hyaluronic acid can be a hydrogel material commonly prepared in the art by chemical functionalization reaction of dopamine and hyaluronic acid. In a preferred embodiment of the present invention, the method for preparing the dopamine-modified hyaluronic acid may include: polymerizing dopamine and hyaluronic acid in contact under an inert atmosphere. The conditions for the polymerization reaction may include: a temperature of 15-30°C, preferably 16-22°C; a pH of 3-7, preferably 4.5-5.5; and a reaction time of 5-15 h. According to the present invention, the inert atmosphere may be provided by a gas that does not react with the reactants, preferably nitrogen. The weight ratio of dopamine to hyaluronic acid may be (0.2-1):1, for example, 0.2:1, 0.4:1, 0.5:1, 0.55:1, 0.6:1, 0.8:1, and 1:1, or any range between any two values, preferably (0.4-0.7):1.

[0079] According to the present invention, the pH value of the polymerization reaction can be adjusted using an inorganic acid and / or an inorganic base. According to the present invention, the inorganic acid is preferably hydrochloric acid, and the inorganic base is preferably sodium hydroxide.

[0080] According to the present invention, in order to better promote the polymerization reaction, hyaluronic acid can be dissolved in water first and then contacted with other substances. Preferably, the water is deionized water, and the weight ratio of hyaluronic acid to water can be (2-50):1, preferably (5-15):1.

[0081] According to the present invention, in order to better promote the polymerization reaction, optional auxiliaries may be added during the polymerization reaction, wherein the auxiliaries may be 1-ethyl-(3-dimethylaminopropyl)carbodiimide and / or N-hydroxysuccinimide; the weight ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to hyaluronic acid may be (0.1-0.6):1, preferably (0.3-0.4):1, and the weight ratio of N-hydroxysuccinimide to hyaluronic acid may be (0.2-0.8):1, preferably (0.5-0.6):1. According to some embodiments of the present invention, the auxiliaries are 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0082] According to the present invention, the polymerization product can be dialyzed and / or subjected to a fourth drying after the polymerization reaction; wherein the dialyzed method may be: dialyzing with deionized water with pH 3-6.5 and pH 6.6-7.5 for 1-4 days and 0.5-3 days respectively. The molecular weight cutoff of the device used for the dialyzed reaction can be 6000-10000 Da. According to a specific embodiment of the present invention, the dialyzed reaction is performed in a dialysis bag. The fourth drying can be a commonly used drying method in the art, but in order to further extend the shelf life of dopamine-modified hyaluronic acid, the fourth drying is preferably carried out at a low temperature. According to some embodiments of the present invention, the conditions for the fourth drying can be: a temperature of -80°C to -50°C, preferably -75°C to -65°C, a pressure of 0.4 MPa to 0.55 MPa, and the drying time is not limited, as long as it is dried to meet the corresponding storage requirements.

[0083] According to the present invention, in order to directly obtain a drug with antibacterial properties, the dopamine-modified hyaluronic acid can be directly mixed with the antibacterial compound as described above without undergoing a fourth drying process after successful synthesis; in order to meet the requirements of industrial production and to maximize the antibacterial effect, the pre-prepared and dried dopamine-modified hyaluronic acid can also be redissolved in a pharmaceutically acceptable solvent and then mixed with the antibacterial compound as described above. The pharmaceutically acceptable solvent can be water, such as deionized water. The relative amount of dopamine-modified hyaluronic acid to solvent can be adjusted according to the actual application scenario and requirements. The weight ratio of dopamine-modified hyaluronic acid to solvent can be 1:(0.5-100), for example, 1:0.5, 1:1, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 and 1:100 and any range between any two values, preferably 1:(1-50). In order to make the final drug have better physical properties (such as pore state, self-healing performance, shear thinning properties, etc.), according to some embodiments of the present invention, the weight ratio of dopamine-modified hyaluronic acid to solvent is more preferably 1:(20-40). Similarly, if the dopamine-modified hyaluronic acid is successfully synthesized and the fourth drying step is not performed, and the antibacterial compound provided by the present invention is added directly, the dopamine-modified hyaluronic acid product at this time preferably also contains the solvent as described above. Alternatively, the dopamine-modified hyaluronic acid can be "partially dried (concentrated)" or an appropriate amount of solvent can be added so that the ratio of dopamine-modified hyaluronic acid to solvent is also the value described above.

[0084] In this invention, the preferred dosage form of the drug is a dressing, which can be applied directly to the wound.

[0085] In this invention, all pressures are gauge pressures.

[0086] The present invention will be described in detail below through examples. Unless otherwise stated, all reagents and instruments used in the present invention are commercially available.

[0087] Hyaluronic acid: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number H107141, purity ≥95%;

[0088] Escherichia coli, ATCC 8099, purchased from Beijing BioBio Biotechnology Co., Ltd.

[0089] Staphylococcus aureus, ATCC 6538, purchased from Beijing BioBio Biotechnology Co., Ltd.

[0090] Example 1

[0091] One part by weight of cyanuric chloride (TCT) was dissolved in 14 parts by weight of dichloromethane. A 40 wt% NaOH aqueous solution and diethylenetriamine were added dropwise to the cyanuric chloride solution at 0 °C under ice bath conditions. The reaction was allowed to proceed for 3 hours, with the molar ratio of cyanuric chloride, diethylenetriamine, and NaOH being 3:1:3. The organic solvent was removed by rotary evaporation at 40 °C, and the inorganic salts were removed by washing with water. The product was then dried at -0.07 MPa (gauge pressure) and 55 °C for 12 hours. The crude product was ground into a fine powder, washed with tetrahydrofuran to remove unreacted cyanuric chloride, and dried at -0.07 MPa (gauge pressure) and 55 °C for 12 hours to obtain a white powdery solid functionalized product, 3TDETA.

[0092] 3TDETA was characterized by proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 11 As shown in the spectrum, it can be seen that: (1) the chemical shifts of the two CHs are less than 0.5, indicating that the hydrogen on the secondary amine has disappeared, that is, the hydrogen atom on the secondary amine has been replaced by cyanuric chloride; (2) the sum of the peak areas of CH is 4, and NH has only one peak with a peak area of ​​2, that is, one hydrogen atom on the primary amine has been replaced by one cyanuric chloride; In summary, the structure of 3TDETA is shown below.

[0093]

[0094] 3TDETA was added to a 1 wt% sodium hypochlorite solution, with the amount of sodium hypochlorite solution used to achieve a sodium hypochlorite to 3TDETA weight ratio of 1:0.59. The mixture was stirred and reacted at room temperature (25℃) for 6 hours. After washing three times with water, it was dried in a vacuum oven at -0.07 MPa (gauge pressure) at 40℃ for 12 hours to obtain the novel small molecule antibacterial product 3TDETA-Cl. The 3TDETA and 3TDETA-Cl powder samples were respectively mixed with KBr at a weight ratio of 1:100, ground uniformly, and then pressed into tablets. The transmittance was measured using a Fourier transform infrared spectroscopy (FTIR) spectrometer. The results are as follows: Figure 1 As shown in the spectrum of 3TDETA, at 1711 cm⁻¹ -1 and 1567cm -1 There is a broad absorption peak at 843 cm⁻¹, which is the vibrational absorption peak of the triazine ring skeleton of TCT; -1 The absorption peak at 3443 cm⁻¹ corresponds to the C-Cl absorption peak of TCT. -1 and 1650cm -1 The peak at 2914 cm⁻¹ is the absorption peak of the stretching vibration of the NH bond. -1 and 2832cm -1 The peak at 3443 cm⁻¹ is the absorption peak of -CH₂. In the spectrum of 3TDETA-Cl, the peak at 3443 cm⁻¹ is... -1 and 1650cm -1 The disappearance of the absorption peak indicates that NH was successfully chlorinated.

[0095] Test Example 1

[0096] 1. Determination of available chlorine

[0097] Take 0.0021g of 3TDETA-Cl powder in an Erlenmeyer flask, add 10mL of distilled water, and sonicate to disperse it. Add 7mL of 1wt% potassium iodide solution and 2mL of 2mol / L sulfuric acid solution, mix well, and add 5 drops of starch indicator to turn the solution blue. Titrate the solution with standardized sodium thiosulfate solution until the blue color disappears and does not fade within 30 seconds. Perform three parallel titrations and calculate the available chlorine (the oxidizing chlorine produced after the N-Cl bond breaks, which has bactericidal effect) according to the following formula. + Content (in %), where c is the weight concentration of sodium thiosulfate standard, V is the volume of sodium thiosulfate consumed, W is the weight of 3TDETA-Cl, and n(Cl) is the available chlorine content.

[0098] n(Cl) = c × V × 0.3545 / 2W

[0099] The effective chlorine content of 3TDETA-Cl was calculated to be 2.47 wt% based on the titration results.

[0100] 2. Antibacterial performance test

[0101] Staphylococcus aureus and Escherichia coli were selected as experimental strains, and the indicator bacteria were cultured to a viable count greater than 10 using the national standard method. 7 CFU / mL bacterial culture. Transfer 1 mL of bacterial culture to a 1.5 mL centrifuge tube, centrifuge, discard the supernatant, and add sterile water to obtain a viable bacterial concentration of 10. 7 CFU / mL bacterial suspensions were prepared. 250 μg, 500 μg, 1000 μg, and 2500 μg of 3TDETA-Cl were weighed into 1.5 mL centrifuge tubes, and 1 mL of sterile water was added to each. After ultrasonic dispersion, 100 μL of each concentration was transferred to a new 1.5 mL centrifuge tube, followed by the addition of 100 μL of bacterial suspension and 800 μL of sterile water to obtain bacterial suspensions with 3TDETA-Cl concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, and 250 μg / mL. These suspensions were incubated at 37℃ for 60 min. Three parallel experiments were conducted for each concentration, and three blank control groups were included, each containing 1000 μL of sterile water. The bactericidal results of different concentrations of 3TDETA-Cl against *Escherichia coli* are shown below. Figure 2 As shown; the number of cells in each group was counted using scanning electron microscopy, and the sterilization rate was calculated;

[0102] Sterilization rate = (number of viable bacteria before sterilization - number of viable bacteria after sterilization) / number of viable bacteria before sterilization × 100%;

[0103] The antibacterial experiment against Staphylococcus aureus was conducted using the same method, except that the concentrations of 3TDETA-Cl in the final bacterial solutions were 50 μg / mL, 100 μg / mL, 250 μg / mL, and 500 μg / mL. The results are as follows. Figure 2 As shown in the figure, for *Escherichia coli*, the bactericidal rate was 99.99% when the concentration of 3TDETA-Cl was 25 μg / mL, and reached 99.999% when the concentration increased to 50 μg / mL or higher. For *Staphylococcus aureus*, the inhibition rate reached 99.99% at a concentration of 100 μg / mL. These results indicate that 3TDETA-Cl has bactericidal ability.

[0104] Next, two experimental groups were set up: 3TDETA (50 μg / mL) and 3TDETA-Cl (50 μg / mL), and a blank control group without bactericidal material. These three groups were in triplicate and cultured on plates at 37°C for 12 hours. The bactericidal effect on *E. coli* was as follows: Figure 3 As shown, the bactericidal rate of 3TDETA-Cl reached 99.99%, significantly higher than that of 3TDETA (around 40%), demonstrating that 3TDETA-Cl has a better bactericidal effect than 3TDETA. The cell morphology of each group of cells after sterilization was observed using scanning electron microscopy (SEM), as shown below. Figure 4As shown (from left to right: blank control group, 3TDETA group, and 3TDETA-Cl group), the bacteria in the blank control group had smooth surfaces, intact cell membrane structures, and good activity. The bacteria in the 3TDETA group had intact cell membrane structures, some wrinkles on the surface, and some activity. In the 3TDETA-Cl experimental group, the cell membrane structure was destroyed, and a large number of wrinkles appeared on the surface, indicating that the cells were completely dead.

[0105] Example 2

[0106] 1000 mg of hyaluronic acid (HA) was dissolved in 100 mL of deionized water. Dopamine (4-(2-aminoethyl)-1,2-benzenediol, DA, 575 mg), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 345 mg), and N-hydroxysuccinimide (NHS, 569 mg) were then added to the HA-containing solution. The pH was adjusted to 5 using HCl and NaOH. The mixture was stirred at room temperature (20 °C) under a nitrogen atmosphere for 8 h. The mixture was then transferred to a dialysis bag (molecular weight cutoff (MWCO): 8000 Da) and dialyzed against deionized water at pH 5 for two days, followed by dialyzed against deionized water at pH 7 for one day. The polymer was then freeze-dried at 0.5 MPa and -70 °C to obtain the HA-DA polymer, which was stored at 4 °C.

[0107] The structure of the HA-DA polymer was characterized by ultraviolet spectrophotometry. Its UV absorption peak appeared at 280 nm, which is different from the absorption peak of HA, proving that HA has completely reacted and HA-DA has been successfully synthesized.

[0108] Three 30 mg portions of HA-DA were dissolved in 1 mL of deionized water. Two of these portions were then mixed with 1 mg of 3TDETA and 1 mg of 3TDETA-Cl, respectively. After vortexing and thorough mixing for several minutes, HA-DA@3TDETA hydrogels and HA-DA@3TDETA-Cl hydrogels in a gel state were obtained. The states of HA-DA and HA-DA@3TDETA-Cl changed from... Figure 5 As shown in the figure, the left image represents HA-DA, and the right image represents HA-DA@3TDETA-Cl. Additionally, HA-DA@3TDETA-Cl hydrogels with 3TDETA-Cl contents of 0.5 mg / 30 mg HA-DA and 2 mg / 30 mg HA-DA were prepared. The microstructure of HA-DA@3TDETA-Cl with different 3TDETA-Cl contents was examined using SEM, as shown below. Figure 6As shown, all hydrogels containing 3TDETA-Cl exhibit a porous network structure. However, as the 3TDETA-Cl content increases, the pore size of the hydrogel gradually decreases, indicating that higher concentrations of 3TDETA-Cl can partially disrupt the hydrogel network, affecting the absorption of wound exudate and the transport of nutrients. Therefore, among the three types of hydrogels, the 3TDETA-Cl content of 1 mg / 30 mg HA-DA is the most suitable for wound healing.

[0109] Test Example 2

[0110] The rheological properties of HA-DA, HA-DA@3TDETA with a 3TDETA content of 1 mg / 30 mg HA-DA, and HA-DA@3TDEA-Cl with a 3TDETA-Cl content of 1 mg / 30 mg HA-DA were compared using a 20 mm diameter parallel plate rheometer (TA DHR-1). The storage modulus (G') and loss modulus (G”) of the hydrogels were tested at 25 °C as a function of angular frequency (frequency range 0.1–100 Hz, constant strain 1%), time (under constant strain and frequency of 1 Hz, and at a constant frequency of 1 Hz between 1% and 25% strain), strain (range 1–1000%, constant frequency 1 Hz), and shear rate (ls). -1 up to 1000s -1 The changing pattern of ) and the results are as follows Figure 7 .

[0111] like Figure 7 As shown in (A), G' is greater than G' for all hydrogels, indicating hydrogel formation. Figure 7 As shown in (B), all hydrogels exhibited rapid gel formation, and their storage moduli were 364.51 Pa, 1922.61 Pa, and 719.19 Pa, respectively. These values ​​indicate that all hydrogels can form a gel rapidly. Figure 7 As shown in (C), the G' of all hydrogels decreases rapidly under high strain. The strain intersection points of G' and G” for HA-DA@3TDETA and HA-DA@3TDEA-Cl hydrogels are 155% and 18.8%, respectively, indicating that the hydrogels collapsed under this strain. Figure 7 As shown in (D), at a strain of 25%, the G' of the HA-DA@3TDETA-Cl hydrogel is lower than that of G”, indicating that the hydrogel network structure is destroyed under high strain. When the strain value is 1%, the G' and G” of the HA-DA@3TDETA-Cl hydrogel are almost completely recovered within a few seconds. The above data demonstrate that HA-DA@3TDETA-Cl has good self-healing properties. Figure 7 As shown in (E), the shear rate starts from 1 s. -1 Increase to 1000s-1 During the process, the viscosity of the HA-DA@3TDETA-Cl hydrogel decreased significantly, indicating that the hydrogel exhibits good shear-thinning properties.

[0112] In addition, after staining the HA-DA@3TDETA-Cl hydrogel with methyl orange solid staining agent, the plunger was pressed evenly using a syringe with a 1mL orifice to observe the hydrogel extrusion process to determine its injectability. The results are as follows: Figure 7 As shown in (F), the HA-DA@3TDETA-Cl hydrogel extrudate has a stable shape and extrusion speed, with no obvious large particles. It can be continuously injected without clogging, further highlighting its shear-thinning properties.

[0113] Test Example 3

[0114] Male BALB / c mice were purchased from the Experimental Animal Center of Inner Mongolia University. After being housed in the animal laboratory for 7 days to acclimatize, the mice were anesthetized with 10 wt% chloral hydrate, and a 4 mm diameter wound was made on each mouse using a punch. 10 μL of Escherichia coli (10 wt% chloral hydrate) was then inoculated into each mouse. 6 Mice were randomly divided into 4 groups, with 3 groups serving as experimental groups and 3 replicates per group. On the first day of the experiment, the wound surface was covered with PBS (control group), HA-DA@3TDETA (3TDETA content of 1 mg / 30 mg HA-DA), and HA-DA@3TDETA-Cl (3TDETA-Cl content of 1 mg / 30 mg HA-DA) hydrogels, and the mice were fed under the same conditions for 8 days.

[0115] The mice were weighed daily, and wound healing was observed by taking digital photographs. Figure 8 As shown, red, orange, yellow, green, and blue were used to represent the wound condition on days 0, 1, 3, 5, and 7, respectively. The wounds from each day were overlaid to observe the wound healing process. The results are as follows: Figure 9 As shown, the wound area was measured. The wound healing rate was calculated using the following formula, where A0 and A... t The wound areas on day 0 and day t are respectively:

[0116] Wound healing rate (%) = (1-A) t / A0)×100%.

[0117] Figure 8 and Figure 9 As shown, compared with the control group, the HA-DA@3TDETA-Cl group significantly reduced wound area and promoted wound healing. Furthermore, through... Figure 8It was also found that HA-DA@3TDETA mainly promotes wound healing in the early stage (within 0-3 days), while the wound healing rate of HA-DA@3TDETA-Cl increases in the later stage (days 3-7).

[0118] Figure 10 (A) shows that the wound area in the HA-DA@3TDETA-Cl hydrogel decreased at a significantly faster rate than in the other two groups. On the 7th day after surgery, the wound healing rate in the HA-DA@3TDETA-Cl group (78% ± 2.13%) was significantly higher than that in the control group (50% ± 4.24%). Figure 10 (B) shows that the mice's body weight steadily increased over 7 days.

[0119] In vitro hemolysis assay: 200 μL of mouse whole blood was centrifuged at 2000 rpm for 10 min to separate red blood cells. The cells were then washed with sterile PBS to remove fragmented red blood cells, yielding the cell stock solution. 100 μL of PBS solution containing the test antimicrobial agent (1 mg / mL), 150 μL of the red blood cell stock solution, and 750 μL of 0.9 wt% NaCl solution were mixed and incubated at 37°C with shaking at 100 rpm for 1 h. A positive control group (0.1 wt% Triton X-100) and a negative control group (an equal volume of PBS buffer) were also included. After incubation, the test substance was removed, and the remaining red blood cell stock solution was centrifuged at 2000 rpm for 10 min to obtain the supernatant. 100 μL of the supernatant was added to a 96-well plate, and the absorbance was measured at 540 nm using a microplate reader. The hemolysis rate of the test substance was calculated.

[0120] Where A represents the absorbance of the experimental group, and A0 represents the absorbance of the positive control Triton X-100. Results regarding hemolysis are as follows: Figure 10 As shown in (C), the supernatants of the HA-DA@3TDETA and HA-DA@3TDETA-Cl groups were colorless, similar to the PBS group, and the hemolysis rate of all three experimental groups was less than 5%. These results indicate that the hydrogel has good blood compatibility.

[0121] After the experiment, the following inflammation-related hematological parameters were measured using a complete blood count (CBC) instrument, including the cell counts of white blood cells, lymphocytes, and neutrophils. The results are as follows: Figure 10 As shown in (D). There were no significant differences in any parameters between the experimental group and the healthy group, indicating that HA-DA@3THB-Cl has effective in vivo anti-infective capabilities.

[0122] On day 6 of the experiment, one mouse wound epidermis was randomly selected from each group for pathological sectioning and fixed in 10 vol% paraformaldehyde. The sections were embedded in paraffin, sectioned to a thickness of 4 μm, and then sequentially immersed in xylene for 20 min, fresh xylene for 20 min, anhydrous ethanol for 10 min, fresh anhydrous ethanol for 10 min, 95 vol% ethanol for 5 min, 90 vol% ethanol for 5 min, 80 vol% ethanol for 5 min, 70 vol% ethanol for 5 min, and washed with distilled water. Then, the cell nuclei were stained with hematoxylin: sections were stained with hematoxylin for 3-8 min, washed with tap water, differentiated for a few seconds with 1 wt% hydrochloric acid alcohol, rinsed with tap water, blued with 0.6 wt% ammonia solution, and rinsed with running water. Finally, the cytoplasm was stained with eosin: sections were stained with eosin for 1-3 min. Dehydration and mounting: The sections were sequentially immersed in 95 vol% ethanol for 5 min, then in fresh 95 vol% ethanol for 5 min, then in anhydrous ethanol for 5 min, then in xylene I for 5 min, and finally in fresh xylene for 5 min to dehydrate until clear. The sections were then removed from the xylene and allowed to air dry slightly before mounting with neutral resin. Finally, the sections were examined under a microscope, and images were acquired and analyzed. The obtained tissues were stained, and the histological characteristics of wound healing in different treatment groups were observed. The results are as follows: Figure 10 As shown in (E), in the PBS group (control group), there was no granulation tissue formation, significant edema, hemorrhage, fibrin exudation, or diffuse neutrophil infiltration in the central wound area. In contrast, the wound tissue in the HA-DA@3TDETA group showed mild hyperemia and reduced diffuse neutrophil infiltration. In the HA-DA@3TDETA-Cl group, the wound was filled with mature granulation tissue and showed complete epidermal regeneration, demonstrating excellent wound healing. These results indicate that HA-DA@3TDETA-Cl has excellent biocompatibility and safety.

[0123] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A compound with antibacterial properties, characterized in that, The compound has the structure shown in Formula I: In Equation I, m and n are each independently 1-6, each NX is selected from N-Cl and / or NH and at least one NX is N-Cl, the structure of R is as shown in Equation II, and X1 and X2 are each independently halogens.

2. The compound according to claim 1, wherein, X1 and X2 are chlorine; And / or, m and n are each independently 1-3; And / or, the N-Cl content in Formula I is 0.2-2 mol / kg.

3. The compound according to claim 1, wherein, m and n are both 2; And / or, the N-Cl content in Formula I is 0.5-0.8 mol / kg.

4. A method for preparing a compound with antibacterial properties, characterized in that, The method includes: (1) In the presence of a solvent and a basic catalyst, the compound shown in formula a is brought into contact with the compound shown in formula b to carry out a substitution reaction. Among them, X a X b and X c Each is an independent halogen; m and n are each independent of 1-6; (2) Chlorinate the product of step (1) by contacting it with hypochlorite.

5. The method according to claim 4, wherein, X a X b and X c Each is independently chlorine; And / or, m and n are each independently 1-3.

6. The method according to claim 4, wherein, m and n are both 2.

7. The method according to claim 4, wherein, The molar ratio of the compound shown in formula a to the compound shown in formula b is (1-5):1, preferably (2-4):1; And / or, the alkaline catalyst is at least one of alkali metal carbonate, alkali metal bicarbonate, alkali metal hydroxide and organic amine, preferably at least one of sodium carbonate, sodium bicarbonate, sodium hydroxide and 1,8-diazabicyclo[5.4.0]undec-7-ene, more preferably sodium hydroxide; And / or, the molar ratio of the alkaline catalyst to the compound shown in formula b is (1-5):1, preferably (2-4):1; And / or, the solvent is at least one of dichloromethane, tetrahydrofuran, and butanone, preferably dichloromethane; And / or, the temperature of the substitution reaction is from -10°C to 40°C, preferably from -5°C to 5°C; And / or, the substitution reaction takes 1-48 hours, preferably 2-36 hours.

8. The method according to claim 4, wherein, The hypochlorite is sodium hypochlorite; And / or, the weight ratio of hypochlorite to the substituted product of step (1) is 1:(0.2-2), preferably 1:(0.2-1); And / or, the chlorination temperature is 20-60°C, preferably 20-30°C; And / or, the chlorination time is 1-12 hours, preferably 3-8 hours.

9. Use of the compound according to any one of claims 1-3 in the preparation of an agent for killing bacteria.

10. The application according to claim 9, wherein, The bacteria are Staphylococcus aureus and / or Escherichia coli.

11. Use of the compound according to any one of claims 1-3 in the preparation of a medicament for promoting wound healing.

12. The application according to claim 11, wherein, The drug is in the form of a dressing; And / or, the drug contains the compound and dopamine-modified hyaluronic acid.

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