Cationic compound with long-acting high antibacterial activity and preparation method thereof

Abstract

The invention discloses a cationic compound with long-acting high antibacterial activity and a preparation method thereof. The molecular formula of the cationic compound is as shown in formula 1. The cationic compound can destroy cell membrane integrity of bacteria to cause bacterial death, and especially has good antibacterial activity on escherichia coli, staphylococcus aureus and methicillin-resistant staphylococcus aureus.

Description

Technical Field

[0001] This application belongs to the field of antibacterial technology, specifically relating to a cationic compound with long-lasting and high antibacterial activity, its preparation method, and its application. Background Technology

[0002] Bacterial infections and related diseases have severely impacted human quality of life and safety. Traditional antibiotics for bacterial infections have significant side effects, and the overuse of antibiotics in recent years has led to an increase in bacterial resistance and a escalating problem of drug-resistant infections globally. The World Health Organization (WHO) reports that at least 700,000 people die annually from antibiotic-resistant bacterial diseases. Addressing the enormous threat posed to public health security systems by bacterial infections, especially diseases caused by drug-resistant strains, has become a core issue in the fields of biomedicine and health. Developing novel antimicrobial drugs and proposing effective strategies to combat bacterial resistance resulting from long-term antibiotic use are cutting-edge research hotspots in the field of infectious diseases.

[0003] The emergence of antimicrobial peptides and antimicrobial metal / metal oxide nanoparticles has provided new ideas for the development of novel antimicrobial compounds. However, antimicrobial peptides have high production costs, significant potential in vivo toxicity, and are sensitive to in vivo environmental conditions such as pH and proteases, making them prone to inactivation. Metal / metal oxide nanoparticles are also highly toxic and their toxicity is greatly affected by their size, shape, and composition. These drawbacks limit their widespread application.

[0004] Studies have shown that cationic polymers possess the following advantages: i) wide applicability, simple preparation processes, and low toxicity; ii) a combination of the structural designability of small molecule ionic compounds and the excellent properties of polymers; iii) the positive charge of the cationic structure facilitates its binding with relevant bacteria. Furthermore, cationic compounds typically exhibit good water solubility and have wide applications in biology, medicine, chemistry, and industry. With scientific advancements, the application prospects of cationic compounds continue to expand, and they are rapidly gaining traction in the biological field as drug delivery carriers, in vivo framework materials, and in vivo wetting agents. The development of novel cationic antibacterial compounds is highly feasible and of great significance in the field of antibacterial research. Summary of the Invention

[0005] The purpose of this application is to provide a long-lasting bactericidal cationic compound that can be used in daily life and its preparation method. The specific technical solution is as follows:

[0006] This application relates to a cationic compound whose molecular structure contains 3,5-dimethoxyphenoxy and two quaternary ammonium groups.

[0007] Furthermore, the molecular formula of the cationic compound is shown in Formula 1.

[0008]

[0009] Where n is selected from integers from 2 to 20, m is selected from integers from 2 to 12, and R, R', R”, R”’, R”” are independently H, F, Cl, Br, I, NO2, CF3, OCH3, CN, NH2, COOH, (CH2) respectively. x And any of the aromatic rings, where x is an integer selected from 1 to 20.

[0010] Furthermore, in the molecular formula, n = 6, 8, 10, 12 or 16.

[0011] Furthermore, in the molecular formula, m = 6.

[0012] Furthermore, in the molecular formula, R is H.

[0013] Furthermore, in the molecular formula, R, R', R”, R”’, and R”” are all H.

[0014] Furthermore, the molecular formula of the cationic compound is shown in Formula 2.

[0015]

[0016] Another aspect of this application relates to a method for preparing a cationic compound, comprising the following steps:

[0017] 2,6-Dimethoxy-4-hydroxybenzaldehyde was dissolved in N,N-dimethylformamide, and dibromoalkane and potassium carbonate were added. After reaction and purification, solid 4-bromoalkoxy-2,6-dimethoxybenzaldehyde was obtained.

[0018] Benzyl chloride was added dropwise to an acetone solution of tetramethylalkyldiamine, and the reaction yielded benzyl-(dimethylamino)-dimethylalkylamine.

[0019] The cationic compound was obtained by dissolving 4-bromoalkoxy-2,6-dimethoxybenzaldehyde and benzyl-(dimethylamino)-dimethylalkylamine in acetonitrile and then purifying the mixture after the reaction.

[0020] Furthermore, in the preparation method, the molar ratio of 2,6-dimethoxy-4-hydroxybenzaldehyde, dibromoalkane, and potassium carbonate is 1:2-100:1-10.

[0021] Furthermore, in the preparation method, the molar ratio of benzyl chloride to tetramethylalkyldiamine is 1:2-100.

[0022] Furthermore, in the preparation method, the molar ratio of 4-bromoalkoxy-2,6-dimethoxybenzaldehyde to benzyl-(dimethylamino)-dimethylalkylamine is 0.9-1.2:1.

[0023] Furthermore, in the preparation method, the molecular formula of the dibromoalkane is shown in Formula 3.

[0024]

[0025] Where n is an integer selected from 2 to 20.

[0026] Furthermore, in the preparation method, the tetramethylalkyldiamine has the molecular formula shown in Formula 4.

[0027]

[0028] Where m is selected from integers from 2 to 12.

[0029] Furthermore, in the preparation method, the molecular formula of benzyl chloride is shown in Formula 5.

[0030]

[0031] Wherein, R, R', R”, R”', R”” are independently H, F, Cl, Br, I, NO2, CF3, OCH3, CN, NH2, COOH, (CH2) respectively. x And any of the aromatic rings, where x is an integer selected from 1 to 20.

[0032] This application also relates to the application of the cationic compound or the cationic compound prepared by the preparation method in antibacterial applications.

[0033] More preferably, the application is in the form of an antibacterial agent.

[0034] Furthermore, the antibacterial agent is a long-acting antibacterial agent.

[0035] Furthermore, the antibacterial properties are defined as inactivation or inhibition of bacteria and fungi.

[0036] Furthermore, the bacteria are selected from Gram-negative bacteria, Gram-positive bacteria, or methicillin-resistant Staphylococcus aureus.

[0037] More preferably, the Gram-negative bacterium is Escherichia coli.

[0038] More preferably, the Gram-positive bacterium is Staphylococcus aureus.

[0039] This application also relates to an antibacterial agent comprising the cationic compound or a cationic compound prepared by the preparation method.

[0040] More preferably, the antibacterial agent is a long-acting antibacterial agent.

[0041] The effects of the invention

[0042] The cationic compound provided in this application has a simple preparation process, low cost, and strong adaptability. It has a disinfecting effect on Gram-negative, Gram-positive, and methicillin-resistant Staphylococcus aureus, which solves the limitation of antibiotics in being unable to target drug-resistant bacteria and promotes the application of cationic compounds in the field of antibacterial technology. Attached Figure Description

[0043] Figure 1 For the cationic compounds of this application 1 H-NMR spectrum, solvent is d-CDCl3.

[0044] Figure 2 This is a curve showing the bactericidal effect of compound 3 of this application on three types of bacteria.

[0045] Figure 3 This is a diagram showing the hemolytic activity of compounds 1-5 of this application in rabbit blood.

[0046] Figure 4 This is a diagram showing the cytotoxicity of compounds 1-5 of this application on mouse fibroblasts.

[0047] Figure 5 Images of mouse skin before and after spraying.

[0048] Figure 6 The images show the morphology of Escherichia coli cells treated with compound 3 of this application (right) and the morphology of untreated Escherichia coli cells (left).

[0049] Figure 7 The images show the morphology of Staphylococcus aureus cells treated with compound 3 of this application (right) and the morphology of Staphylococcus aureus cells without treatment (left).

[0050] Figure 8 This is a graph showing the change in mouse body weight after injection.

[0051] Figure 9 This is a graph showing the organ weight distribution of different organs in mice after injection (the left side is the PBS group).

[0052] Figure 10 This is a graph showing the concentration of ions in the blood of mice after injection.

[0053] Figure 11 This is a blood biochemical graph showing liver function indicators in mice after injection.

[0054] Figure 12 This is a blood biochemical index of renal function in mice after injection. Detailed Implementation

[0055] The present application will now be described in further detail with reference to specific embodiments and accompanying drawings. The embodiments provided are intended to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0056] It should be noted that the terms "comprising" or "including" used throughout the specification and claims are open-ended terms and should be interpreted as "comprising but not limited to". The subsequent descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0057] This application provides a cationic compound whose molecular structure contains 3,5-dimethoxyphenoxy and two quaternary ammonium groups.

[0058] In a specific embodiment, the molecular formula of the cationic compound is shown in Formula 1.

[0059]

[0060] In embodiments of this application, n in Formula 1 is selected from integers from 2 to 20, for example, n = 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. In some specific embodiments, n = 6, 8, 10, 12, or 16.

[0061] In embodiments of this application, m in Formula 1 is selected from integers from 2 to 12, for example, m = 2, 4, 6, 8, 10, or 12. In some specific embodiments, m = 6.

[0062] In the embodiments of this application, R, R', R”, R”', R”” in Formula 1 can be independently selected from H, F, Cl, Br, I, NO2, CF3, OCH3, CN, NH2, COOH, (CH2). x And any of the aromatic rings. In a specific embodiment, (CH2) X X is selected from integers from 1 to 20.

[0063] In one specific embodiment, the molecular structure of the cationic compound is shown in Formula 2.

[0064]

[0065] The cationic compounds provided in this application are simple cationic compounds. Their positive charge can interact with the negative charge on the surface of bacterial cell membranes. At the same time, in conjunction with the other hydrophobic groups in their molecular structure, they act on the cell membrane, disrupting its integrity and causing bacterial death, thereby achieving a bactericidal effect.

[0066] This application also provides a method for preparing a cationic compound, specifically including the following steps:

[0067] To generate 4-bromoalkoxy-2,6-dimethoxybenzaldehyde: 2,6-dimethoxy-4-hydroxybenzaldehyde was dissolved in N,N-dimethylformamide, dibromoalkane and potassium carbonate were added, and the mixture was purified after reaction to obtain solid 4-bromoalkoxy-2,6-dimethoxybenzaldehyde.

[0068] To generate benzyl-(dimethylamino)-dimethylalkylamine: add benzyl chloride dropwise to an acetone solution of tetramethylalkyldiamine, and the reaction yields benzyl-(dimethylamino)-dimethylalkylamine;

[0069] The cationic compound is generated by dissolving 4-bromoalkoxy-2,6-dimethoxybenzaldehyde and benzyl-(dimethylamino)-dimethylalkylamine in acetonitrile and reacting the mixture to obtain the cationic compound.

[0070] In a specific embodiment, the step of generating 4-bromoalkoxy-2,6-dimethoxybenzaldehyde includes: dissolving 2,6-dimethoxy-4-hydroxybenzaldehyde in N,N-dimethylformamide, followed by the addition of dibromoalkane and potassium carbonate. The reaction is stirred overnight at 65°C, diluted with dichloromethane, extracted, and washed with water and saturated brine. The organic phase is dried over anhydrous sodium sulfate and then rotary evaporated. The mixture is purified by column chromatography using hexane / ethyl acetate as eluent to obtain a grayish-white solid, 4-bromoalkoxy-2,6-dimethoxybenzaldehyde.

[0071] In some specific embodiments, the column chromatography purification includes: using n-hexane / ethyl acetate = 50 / 50 as the eluent, column chromatography purification, collecting the target component, and then vacuum drying at 25-30℃ and 30-40mbar for 24-72 hours.

[0072] In some specific embodiments, the molar ratio of 2,6-dimethoxy-4-hydroxybenzaldehyde, dibromoalkane, and potassium carbonate is 1:2-100:1-10.

[0073] In a specific embodiment, the step of generating benzyl-(dimethylamino)-dimethylalkylamine includes: adding benzyl chloride dropwise to an acetone solution of tetramethylalkyldiamine, stirring overnight, filtering, concentrating and drying to obtain benzyl-(dimethylamino)-dimethylalkylamine.

[0074] In some specific embodiments, the concentration and drying process includes: filtering the reaction solution through a Buchner funnel, collecting the filtrate, rotary evaporating, and placing the obtained concentrated emulsion-like substance under vacuum drying at 25-30°C and 30-40 mbar for 24-72 hours.

[0075] In some specific embodiments, the molar ratio of benzyl chloride to tetramethylalkyldiamine is 1:2-100.

[0076] In a specific embodiment, the step of generating the cationic compound includes: dissolving 4-bromoalkoxy-2,6-dimethoxybenzaldehyde and benzyl-(dimethylamino)-dimethylalkylamine in acetonitrile, stirring overnight at 65-75°C, cooling and concentrating, precipitating, and drying to obtain a cationic compound with antibacterial properties.

[0077] In some specific embodiments, the precipitation and drying process includes: adding 20 times the volume of ice-cold ether to the concentrated solution for precipitation, placing the resulting solid under vacuum drying at 25-30°C and 30-40 mbar for 24 hours, adding a small amount of water, filtering the aqueous solution, and freeze-drying to obtain the target product.

[0078] In some specific embodiments, the molar ratio of 4-bromoalkoxy-2,6-dimethoxybenzaldehyde to benzyl-(dimethylamino)-dimethylalkylamine is 0.9-1.2:1.

[0079] In the embodiments of this application, the molecular formula of the dibromoalkane is shown in Formula 3.

[0080]

[0081] In Equation 3, n is selected from integers between 2 and 20. For example, n = 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. In some specific implementations, n = 6, 8, 10, 12, or 16.

[0082] In the embodiments of this application, the molecular structural formula of 2,6-dimethoxy-4-hydroxybenzaldehyde is shown in Formula 6.

[0083]

[0084] In the embodiments of this application, the molecular structural formula of potassium carbonate is shown in Formula 7.

[0085]

[0086] In the embodiments of this application, the molecular structural formula of 4-bromoalkoxy-2,6-dimethoxybenzaldehyde is shown in Formula 8.

[0087]

[0088] In Equation 8, n is selected from integers between 2 and 20. For example, n = 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. In some specific implementations, n = 6, 8, 10, 12, or 16.

[0089] In the embodiments of this application, the molecular structural formula of benzyl chloride is shown in Formula 9.

[0090]

[0091] In the embodiments of this application, the molecular formula of tetramethylalkyldiamine is shown in Formula 4.

[0092]

[0093] In Equation 4, m is selected from integers between 2 and 12, for example, m = 2, 4, 6, 8, 10, or 12. In some specific implementations, m = 6.

[0094] In the embodiments of this application, the molecular formula of benzyl chloride is shown in Formula 5.

[0095]

[0096] In Equation 5, R, R', R”, R”’, R”” are independently H, F, Cl, Br, I, NO2, CF3, OCH3, CN, NH2, COOH, (CH2). x And any of the aromatic rings, where x is an integer selected from 1 to 20.

[0097] In some specific implementations, R stands for H.

[0098] In some other specific implementations, R,R',R”,R”',R”” are all H.

[0099] In some specific embodiments, the molecular structural formula of benzyl-6-(dimethylamino)-dimethylalkylamine is shown in Formula 10.

[0100]

[0101] In Equation 8, m is selected from integers between 2 and 12, for example, m = 2, 4, 6, 8, 10, or 12. In some specific implementations, m = 6.

[0102] In some specific embodiments, the cationic compounds provided in this application are prepared by the above-described preparation method.

[0103] This application also provides the application of the cationic compound or the cationic compound prepared by the above preparation method in antibacterial activity.

[0104] In a specific implementation, the application is to an antibacterial agent.

[0105] In a specific implementation, the antibacterial agent is a long-acting antibacterial agent.

[0106] In a specific implementation, the antibacterial effect is the inactivation or inhibition of bacteria and fungi.

[0107] In a specific implementation, the bacteria are selected from Gram-negative bacteria, Gram-positive bacteria, or methicillin-resistant Staphylococcus aureus.

[0108] In a specific implementation, the Gram-negative bacterium is Escherichia coli.

[0109] In a specific implementation, the Gram-positive bacterium is Staphylococcus aureus.

[0110] The cationic compounds provided in this application have good antibacterial activity against Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus.

[0111] In a specific embodiment, the cationic compound (e.g., Formula 2, n=16, m=6) can achieve an antibacterial activity of MIC=8μg / mL against Escherichia coli.

[0112] In this application, MIC stands for Minimum Inhibitory Concentration, which refers to the lowest concentration of an antimicrobial drug required to inhibit bacterial growth. MIC is an important pharmacological parameter, used to measure the ability of an antimicrobial drug to inhibit pathogenic microorganisms and to assess the sensitivity of antibiotics to specific bacteria. A lower MIC value indicates that a smaller drug concentration is required to inhibit the growth of pathogenic microorganisms, thus indicating stronger antimicrobial activity of the drug.

[0113] In this application, MBC stands for Minimum Bactericidal Concentration, referring to the lowest concentration of an antibacterial ingredient capable of killing 99.9% of bacteria or fungi. When the concentration of an antibacterial ingredient reaches the MBC, most bacteria or fungi will be killed. MBC is mainly used to evaluate the bactericidal ability of antibacterial ingredients. If the MBC of an antibacterial ingredient is low, it indicates that the ingredient has better antibacterial properties.

[0114] In a specific embodiment, the cationic compound described in this application can fully exert its effect within 6 hours.

[0115] In a specific embodiment, the cationic compound described in this application has long-lasting antibacterial properties, and the antibacterial duration of the material treated with it exceeds 120 hours.

[0116] In specific embodiments, the cationic compound can be used for antibacterial treatment of metals, inorganic non-metals, organic polymers, composite materials, etc.

[0117] This application also provides an antibacterial agent comprising the cationic compound or the cationic compound prepared by the preparation method.

[0118] In some specific embodiments, the antibacterial agent is a long-acting antibacterial agent.

[0119] In some specific embodiments, the antibacterial agent is an aqueous solution of the cationic compound.

[0120] In some specific embodiments, the concentration of the cationic compound in the antibacterial agent is 100–2000 μg / mL.

[0121] The cationic compounds provided in this application exhibit rapid bactericidal action and long-lasting antibacterial effects against Gram-negative, Gram-positive, and methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, these compounds demonstrate good stability, exhibiting no significant cytotoxicity or in vitro / in vivo toxicity, and showing no acute skin irritation or corrosiveness. They represent a potential antibiotic alternative and lay the foundation for the discovery of novel antibacterial compounds.

[0122] Example

[0123] This application provides a general and / or specific description of the materials and methods used in the experiments. In the examples below, unless otherwise specified, all original reagents were purchased from Sigma-Aldrich, McLean, or Energie. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0124] Example 1: Preparation of cationic compounds

[0125] (1) 2,6-Dimethoxy-4-hydroxybenzaldehyde (5.0 g, 1.0 equiv., 27.5 mmol) was dissolved in 200 mL of N,N-dimethylformamide, followed by the addition of dibromoalkanes (1,6-dibromohexane, 1,8-dibromooctane, 1,10-dibromodecane, 1,12-dibromododecane, 1,16-dibromohexadecane, 10 equiv., 275 mmol) and potassium carbonate (4.5 g, 1.2 equiv., 33 mmol). The reaction was stirred overnight (12–16 h) at 65 °C, diluted with dichloromethane, extracted, and washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and evaporated. Using hexane / ethyl acetate (50:50) as eluent, the sample was purified by column chromatography and dried (after collecting the target component, it was vacuum dried at 25-30℃ and 30-40 mbar for 24-72 hours) to obtain a grayish-white solid, 4-bromoalkoxy-2,6-dimethoxybenzaldehyde. The specific synthetic reaction is shown in formula (1).

[0126]

[0127] The yield of 4-[(6-bromohexyl)oxy]-2,6-dimethoxybenzaldehyde was 81%.

[0128] 4-[(8-bromooctyl)oxy]-2,6-dimethoxybenzaldehyde yield 79%;

[0129] 4-[(10-bromodecyl)oxy]-2,6-dimethoxybenzaldehyde yield 83%;

[0130] 4-[(12-bromododecyl)oxy]-2,6-dimethoxybenzaldehyde, yield 79%;

[0131] 4-[(16-bromohexadecyl)oxy]-2,6-dimethoxybenzaldehyde yield was 73%.

[0132] (2) Benzyl chloride (10.0 g, 1.0 equiv., 79.0 mmol) was added dropwise to an acetone solution of N,N,N′,N′-tetramethyl-1,6-hexanediamine (136.1 g, 10.0 equiv., 790.0 mmol), stirred overnight, filtered to remove solid impurities, and the obtained organic solution was concentrated and dried to obtain benzyl-6-(dimethylamino)-dimethylhexane-1-amine. The specific concentration and drying operation was as follows: the reaction solution was filtered through a Buchner funnel, the filtrate was collected, and the concentrated emulsion was dried under vacuum at 25-30°C and 30-40 mbar for 24-72 hours. The specific synthetic reaction formula is given in formula (2), with a yield of 92%.

[0133]

[0134] (3) The 4-bromoalkoxy-2,6-dimethoxybenzaldehyde (1.05 equiv., 2.0 mmol) obtained in step (1) and the benzyl-6-(dimethylamino)-dimethylhexane-1-amine (1.0 equiv., 1.9 mmol, 0.76 g) obtained in step (2) were dissolved in 10 mL of acetonitrile and stirred overnight (12-16 hours) at 65-75 °C. The mixture was then cooled and concentrated. The concentrate was placed in ice-cold diethyl ether to precipitate the precipitate. After drying, the cationic compound (Formula 2) was obtained. The specific precipitation and drying operations were as follows: 20 times the volume of ice-cold diethyl ether was added to the concentrated solution for precipitation. The resulting solid was vacuum dried at 25-30 °C and 30-40 mbar for 24 hours. A small amount of water was added, and the filtered aqueous solution was freeze-dried to obtain the target product. The specific synthetic reaction formula is shown in Formula (3).

[0135]

[0136] The products obtained above include compound 1 (n=6, m=6 in Formula 2), compound 2 (n=8, m=6 in Formula 2), compound 3 (n=10, m=6 in Formula 2), compound 4 (n=12, m=6 in Formula 2), and compound 5 (n=16, m=6 in Formula 2).

[0137] The yields of compounds were as follows: compound 1 (91%), compound 2 (93%), compound 3 (87%), compound 4 (86%), and compound 5 (94%).

[0138] The NMR results of the above compounds are as follows Figure 1 As shown. The experiments in the following examples were carried out using compounds 1-5 as described above.

[0139] Example 2 Antibacterial Performance Test

[0140] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the micro-dilution method according to the Clinical and Laboratory Standards Intitude (CLSI) guidelines. The specific experimental method is as follows:

[0141] The bacteria were grown overnight in LB medium (Thermo Fisher Scientific) at 37°C. Bacteria in the logarithmic growth phase (Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus MRSR) were selected for testing (bacterial suspension OD600 between 0.4 and 0.5). Procedure: 1. Dilute the bacteria in LB medium to 1×10⁻⁶. 5 CFU / mL; 2. Dissolve compounds 1-5 obtained in Example 1 and the antibiotic (kanamycin) separately in water (concentration of 20 mg / mL); 3. Add 1×10 5CFU / mL bacterial cultures were incubated in 96-well plates with different concentrations of compounds or antibiotics (512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL) at 37°C and 200 rpm for 16–18 hours. Growth inhibition was determined by measuring the absorbance of each well at 600 nm (OD600) using an enzyme analyzer; the lowest polymer concentration at which no bacterial growth occurred was defined as the MIC. The bacterial cultures from the MIC, 2×MIC, and 4×MIC wells were mixed by pipetting, and 100 μL of each was aspirated onto pre-labeled agar plates. The plates were capped, and the bacterial culture was gently shaken back and forth or spread using a spreader until the plates were fully covered. The plates were incubated overnight at 37°C; the lowest concentration at which no colonies formed was defined as the MBC. The specific results are shown in Table 1.

[0142] Table 1. MIC and MBC test results for different compounds

[0143]

[0144] The results showed that compounds 1-5 all exhibited good antibacterial properties, and the MIC and MBC values ​​of these compounds gradually decreased with increasing carbon chain length, indicating a trend of enhanced antibacterial activity. Specifically, against *Escherichia coli*, the MIC value of BTA-16 (compound 5) decreased to 8 μg / mL, and its antibacterial activity reached that of the positive control kanamycin. Against MRSR, the antibacterial properties of several compounds were superior to the positive control.

[0145] Example 3: Sterilization Curve Test

[0146] Using the same method as in Example 2, the bacteria were grown overnight in LB medium at 37°C, and bacteria in the logarithmic growth phase were selected for testing (bacterial suspension OD600 between 0.4 and 0.5). Steps: 1. Dilute the bacteria in LB medium to 10... 5 CFU / mL; 2. Dissolve the cationic antibacterial compound (compound 3) in water; 3. Add 10 5CFU / mL bacterial culture was added to 96-well plates and then mixed with compounds or antibiotic solutions at concentrations of 0.5 times (0.5×MIC), 1 times (1×MIC), 2 times (2×MIC), and 4 times (4×MIC), respectively (the control group consisted of samples without antibiotics or compounds). After thorough mixing, 10 μL of sample was taken at 0, 1, 2, 3, 4, 5, 6, 7, 8, 14, and 24 hours, diluted in sterile PBS, and plated onto LB agar plates. The plates were incubated at 37°C for 12 hours, and the colony count was calculated to determine the bacterial count at each time point. The antibacterial curves obtained from the example treatment are shown below. Figure 2 As shown, it achieved a highly effective bactericidal effect within 6 hours. These findings indicate that the cationic compound of this application possesses excellent antibacterial properties and exhibits highly effective bactericidal activity against both common Gram-negative and Gram-positive bacteria.

[0147] Example 4: Long-lasting bactericidal ability test

[0148] Inside the clean bench, a solution of compounds 1-5 (1 g / L) was sprayed onto the surfaces of polystyrene petri dishes, nitrile gloves, metal scissors, and glass petri dishes. After 120 hours, a 1×10⁻⁶ solution was used. 5 Materials treated with and untreated with the cationic compound were contaminated with CFU / mL concentrations of Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. One hour after contamination, residual liquid was extracted from the surface of the contaminated materials, diluted in sterile PBS, and plated onto LB agar plates. The plates were incubated at 37°C for 12 hours, and the colony count was calculated to determine the antibacterial efficiency. The results are shown in Table 2.

[0149] Antibacterial efficiency (%) = 1 - (number of colonies on plate treated material / number of colonies on plate untreated material) × 100%

[0150] Table 2. Antibacterial effects of each material after 120 hours of compound solution treatment.

[0151]

[0152] The results show that the cationic compound provided in this application, as an antibacterial agent, still has high antibacterial activity within 120 hours after the material is disinfected, and the elimination effect on bacteria is >98%, which has a long-lasting antibacterial function.

[0153] Example 5 Hemolytic Test

[0154] First, add 95 mL of sterile 0.9% sodium chloride solution to 5 mL of fresh rabbit blood, shake well, and centrifuge at 1000-1500 rpm for 15 minutes. Remove the supernatant, and wash the precipitated red blood cells 2-3 times with 0.9% sodium chloride solution as described above, until the supernatant no longer appears red. Then, prepare a 5% suspension of the obtained red blood cells with 0.9% sodium chloride solution. Compounds 1-5 were dissolved in water (concentration 20 mg / mL); finally, the erythrocyte suspension was incubated in 96-well plates with different concentrations of compounds (1024 μg / mL, 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL) or 0.2% Triton-X-100 at 37°C and 60 rpm for 1 hour. After incubation, the cells were centrifuged at 1000-1500 rpm for 10 minutes, and the supernatant was placed in a new 96-well plate to record the absorbance at 540 nm. Calculation formula:

[0155] Hemolysis% = [(OD 测试样品组 -OD 阴性对照组 ) / (OD 阳性对照组 -O 阴性对照组 )]×100%

[0156] The test sample group was supplemented with compounds 1-5, the negative control group was supplemented with PBS, and the positive control group was supplemented with 2% Triton-X-100.

[0157] Results of erythrocyte hemolytic activity test as follows: Figure 3 As shown ( Figure 3 In the bar chart for each concentration, compounds 1-5 are shown from left to right. The results indicate that the cationic compounds of this application do not have a significant destructive effect on erythrocytes at the cellular level.

[0158] Example 6 Cytotoxicity Test

[0159] Cytotoxicity was assessed using the Cell Counting Kit-8 (CCK-8) assay. In a 96-well plate, 1 × 10⁶ cells were seeded in each well. 4 Mouse fibroblast 3T3 cells (100 μL) were incubated for 4 hours with different concentrations of compounds 1-5 (256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL) and co-cultured at 37°C for 24 hours (with the control group not receiving the compounds). After the incubation period, 10 μL of CCK8 reagent was added, and the absorbance at 450 nm was measured using a microplate reader within 15 min to calculate cell viability, further evaluating the toxicity. The calculation formula is as follows:

[0160] Cell viability = [(A s -A b [(Ac-Ab)]×100%

[0161] Among them, A s This is the absorbance value of the sample group, A. b This is the absorbance value of the blank group, A. c This is the absorbance value of the control group.

[0162] Cytotoxicity test results as follows Figure 4 As shown ( Figure 4 The bar chart for each concentration shows compounds 1-5 from left to right. The results indicate that the cationic compounds of this application have no significant toxicity at the cellular level.

[0163] Example 7 Skin Irritation Test

[0164] Female mice were used. After removing hair from their abdomens, an aqueous solution (5 g / L) of compound 3 was sprayed onto their surface (distilled water was used as a control group). Skin symptoms were observed one hour later. Results were as follows: Figure 5 As shown in the figure. The results showed that no abnormalities such as redness or swelling were observed on the skin surface of mice in both the drug administration group and the ultrapure water group, indicating that the spray sterilizer using the cationic compound of this application had no obvious skin irritation. Skin irritation is an important indicator for assessing the discomfort or inflammatory response caused by a compound on the skin. Based on the experimental results, the cationic compound of this application did not cause significant adverse skin irritation under the experimental conditions.

[0165] Example 8: Study on antibacterial mechanism

[0166] Experimental methods: 1. Compound 3 was dissolved in PBS buffer (pH 7.4) to a concentration of 1 mg / mL. After sterilization by UV irradiation for 15-30 min, it was mixed 1:1 with a bacterial suspension of a certain concentration and placed in a 1.5 mL centrifuge tube. 2. Centrifuge tubes without any polymer solution were inoculated as a control group. 3. All samples and controls were incubated at 37°C for 1 hour. 4. After incubation, samples were prepared, and their morphology was observed using SEM. The results of the examples are as follows. Figure 6-7 As shown in the image, the surface of bacteria treated with compound 3 underwent deformation. This is likely due to changes in the bacterial cell membrane caused by the compound, leading to leakage of internal substances and ultimately bacterial death. These findings provide important evidence for further elucidating the antibacterial mechanism of this type of compound and its application in antibacterial therapy.

[0167] Example 9: In vivo toxicity study of the compound

[0168] Eight BALB / c mice were used and divided into two groups of four each: a PBS control group (administered intravenously with an equal volume of distilled water) and an experimental group (administered intravenously with 20 mg / kg of compound 3). All behavioral characteristics of the mice were observed after injection. Behavioral characteristics were continuously observed and recorded at 4, 24, 48, 72, 96, 7, and 12 days post-injection. At the end of the observation period (12 days), the mice were sacrificed, and blood and major organs were collected for analysis. The results of this example are as follows. Figure 8-12 As shown in the figure, the results indicated that a dose of 20 mg / kg did not have significant adverse effects on mouse body weight, organ specific gravity, blood inorganic ion concentration, or liver and kidney function. This cationic antibacterial compound exhibited good biocompatibility in vivo and did not show significant in vivo toxicity at the dose used. These findings provide strong support for further research and development of this compound as a potential antibacterial agent.

[0169] Those skilled in the art will understand that the above-described embodiments are specific examples of implementing this application, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of this application. Any person skilled in the art can make their own modifications and alterations without departing from the spirit and scope of this application; therefore, the scope of protection of this application should be determined by the scope defined in the claims.

Claims

1. A cationic compound comprising, in its molecular structure, 3,5-dimethoxyphenoxy and two quaternary ammonium groups.

2. The cationic compound according to claim 1, wherein the molecular formula of the cationic compound is shown in Formula 1. in, n is an integer selected from 2 to 20, m is an integer selected from 2 to 12, and R, R', R”, R”’, R”” are independently H, F, Cl, Br, I, NO2, CF3, OCH3, CN, NH2, COOH, (CH2) respectively. x And any of the aromatic rings, where x is an integer selected from 1 to 20.

3. The cationic compound according to claim 2, wherein, n = 6, 8, 10, 12 or 16.

4. The cationic compound according to claim 2, wherein, m＝6。 5. The cationic compound according to claim 2, wherein, R is H.

6. The cationic compound according to claim 2, wherein, The molecular formula of the cationic compound is shown in Formula 2.

7. A method for preparing a cationic compound, comprising the following steps: 2,6-Dimethoxy-4-hydroxybenzaldehyde was dissolved in N,N-dimethylformamide, and dibromoalkane and potassium carbonate were added. After reaction and purification, solid 4-bromoalkoxy-2,6-dimethoxybenzaldehyde was obtained. Benzyl chloride was added dropwise to an acetone solution of tetramethylalkyldiamine, and the reaction yielded benzyl-(dimethylamino)-dimethylalkylamine. 4-Bromoalkoxy-2,6-dimethoxybenzaldehyde and benzyl-(dimethylamino)-dimethylalkylamine were dissolved in acetonitrile, and the cationic compound was obtained after the reaction. Preferably, the molar ratio of 2,6-dimethoxy-4-hydroxybenzaldehyde, dibromoalkane, and potassium carbonate is 1:2-100:1-10; Preferably, the molar ratio of benzyl chloride to tetramethylalkyldiamine is 1:2-100; More preferably, the molar ratio of 4-bromoalkoxy-2,6-dimethoxybenzaldehyde to benzyl-(dimethylamino)-dimethylalkylamine is 0.9-1.2:

1.

8. The preparation method according to claim 7, wherein, The molecular formula of the dibromoalkane is shown in Formula 3. Where n is an integer selected from 2 to 20; Preferably, the molecular formula of the tetramethylalkyldiamine is shown in Formula 4. Where m is selected from integers from 2 to 12; Preferably, the molecular formula of the benzyl chloride is shown in Formula 5. Wherein, R, R', R”, R”', R”” are independently H, F, Cl, Br, I, NO2, CF3, OCH3, CN, NH2, COOH, (CH2) respectively. x And any of the aromatic rings, where x is an integer selected from 1 to 20.

9. The application of the cationic compound according to any one of claims 1 to 6 or the cationic compound prepared by the preparation method according to claim 7 or 8 in antibacterial applications, preferably in antibacterial agents.

10. The application according to claim 9, wherein, The antibacterial agent is a long-acting antibacterial agent, preferably, the antibacterial action is to inactivate or inhibit bacteria and fungi.

11. The application according to claim 10, wherein, The bacteria are selected from Gram-negative bacteria, Gram-positive bacteria, or methicillin-resistant Staphylococcus aureus. Preferably, the Gram-negative bacteria are Escherichia coli, and the Gram-positive bacteria are Staphylococcus aureus.

12. An antibacterial agent comprising the cationic compound according to any one of claims 1 to 6 or the cationic compound prepared by the preparation method according to claim 7 or 8, preferably, the antibacterial agent is a long-acting antibacterial agent.

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