CN114835594B - Tri-cation quaternary ammonium salt antibacterial peptide mimic with antibacterial activity and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of pharmaceutical chemistry, and discloses a tri-cation quaternary ammonium salt antibacterial peptide mimic with antibacterial activity and a preparation method thereof. Has the following structural general formulas I-III. In-vitro antibacterial activity experiments prove that partial compounds of the series show better activity on gram-positive bacteria staphylococcus aureus and enterococcus faecalis, gram-negative bacteria escherichia coli and stenotrophomonas maltophilia, and the compounds have excellent broad-spectrum antibacterial activity; meanwhile, in-vitro erythrocyte hemolytic data show that the compounds have smaller hemolytic toxicity and better selectivity.

Description

Tricationic quaternary ammonium salt antibacterial peptide mimetic with antibacterial activity and preparation method thereof

Technical field

The invention belongs to the technical field of medicinal chemistry and discloses a tricationic quaternary ammonium salt antibacterial peptide mimetic with antibacterial activity and a preparation method thereof.

Background technique

The discovery and widespread use of antibiotics has an indelible significance in the development of human civilization. According to different mechanisms, they are divided into aminoglycosides, tetracyclines, chloramphenicol, macrolides, and lincomycin that affect bacterial protein synthesis. β-lactams that interfere with bacterial cell wall synthesis; polymyxins that damage bacterial cell membranes; sulfonamides and trimethoprim that interfere with folic acid metabolism; quinolones that affect nucleic acid metabolism, etc. However, with the rapid development of society and economy, antibiotic resistance (Antimicrobial resistance, AMR) has become a major threat to public health caused by bacteria since the antibiotic era. Bacterial resistance is mainly divided into intrinsic resistance and acquired resistance. Acquired resistance is mainly caused by bacteria undergoing certain genetic mutations or acquiring exogenous resistance genes under the action of antibiotics (Cell, 2007, 128(6):1037-1050.). It is mainly divided into several categories: bacteria produce inactivating enzymes, which make antibiotics ineffective; bacteria produce specific efflux pumps, which promote the efflux of drugs and reduce the drug concentration inside the bacteria, thereby causing drug inactivation; the reduction of bacterial permeability to antibiotics causes Antibiotics cannot enter bacterial cells to work; bacteria modify or mutate the targets of existing antibiotics, resulting in unclear targets of antibiotics, thereby reducing activity. For example, methicillin-resistant Staphylococcus aureus (MRSA) reduces the binding ability of β-lactam antibiotics to penicillin-binding proteins by producing a special penicillin-binding protein (PBP2a), thereby causing it to lose its antibacterial activity. As the “last line of defense” against Gram-positive bacteria, vancomycin has also shown a tendency to become resistant (Science, 2008, 321, 356-361). Therefore, there is an urgent need to develop new antibacterial drugs that are effective against drug-resistant bacteria.

In order to solve the problem of bacterial resistance, researchers from the 1960s to the 1980s discovered antimicrobial peptides (AMPs) with broad-spectrum antibacterial effects. As a class of bioactive peptides with antibacterial activity, AMPs have mostly different molecular sizes, structures and compositions (generally containing 5-50 amino acids). Their exposed cationic and hydrophobic residues make them amphipathic. The secondary structure of antimicrobial peptides is the same as that of general polypeptides, including α-helix, β-sheet or cyclic structure. These structural characteristics enable AMPs to destroy the integrity of bacterial cell membranes, and the destruction of cell membranes also makes it difficult for bacteria to develop corresponding resistance mechanisms. This is also the main reason why antimicrobial peptides are not easy to develop resistance. However, as antimicrobial peptides are widely studied as a new type of antibacterial drugs, more and more problems are exposed to researchers, especially the development of drugs, which is limited by its own inherent shortcomings, such as large molecular weight, It has high production cost, is easily degraded by proteases, has poor activity in the presence of salts, has high cytotoxicity to host cells and poor pharmacokinetics. Based on the amphipathic structural characteristics of natural antimicrobial peptides, researchers synthesized antimicrobial peptide mimics to overcome the above shortcomings. For example, Cai's research group used nitrofurantoin as the core to synthesize a series of compounds with hydrophobic alkane chains and positive charges, which showed good activity (J. Med. Chem., 2017, 60, 8456); Haldar's research group designed and synthesized a For a series of symmetrical quaternary ammonium salt compounds, the activity and toxicity can be adjusted by changing the length of the intermediate alkane and side chain to obtain compounds with better activity (J. Med. Chem., 2016, 59, 1075).

Based on the existing research on antimicrobial peptide mimetics, it is necessary to further design and develop small molecule antimicrobial peptide mimetics with high efficiency, low toxicity and low induced resistance.

Contents of the invention

Based on the current state of the art, the purpose of the present invention is to provide a series of tricationic quaternary ammonium salt antimicrobial peptide mimetics with high antibacterial activity and low toxicity, which is beneficial to the development of new antibacterial drugs; another purpose is to provide a preparation method thereof.

In order to achieve the purpose of the present invention, the present invention uses the dimethyl quaternary ammonium salt structure as a hydrophilic group, designs molecules by changing the structure of the hydrophobic region and the number of cationic groups, and verifies its antibacterial activity and effectiveness through in vitro activity experiments and mechanism experiments. Potential mode of action, toxicity verified by in vitro cytotoxicity and in vivo toxicity. The specific technical solutions are as follows:

This compound has the following structural formula I-III:

Features: This structure uses phloroglucinol as the middle connecting part, the middle chain length m=3, 4, 5; the side chain length n=3, 5, 7, 9, 11. The following compounds are preferred:

3a: m=3, n=7;

3b: m=3, n=9;

3c: m=3, n=11;

3d: m=4, n=3;

3e: m=4, n=5;

3f: m=4, n=7;

3g: m=4, n=9;

3h: m=4, n=11;

3i: m=5, n=3;

3j: m=5, n=5;

3k: m=5, n=7;

3l: m=5, n=9;

3m: m=5, n=11;

Features: This structure uses phloroglucinol as the middle connecting part and the hydrophobic group of phenylene ether as the side chain. The middle chain length m=3, 4, 5; the side chain length n=3, 4, 5. It is the following compound:

4a: m=3, n=3;

4b: m=3, n=4;

4c: m=3, n=5;

4d: m=4, n=3;

4e: m=4, n=4;

4f: m=4, n=5;

4g: m=5, n=3;

4h: m=5, n=4;

4i: m=5, n=5;

Features: This structure uses phloroglucinol as the middle connecting part and the phenyl hydrophobic group as the side chain. The length of the middle chain is m=3, 4, 5; the length of the side chain is n=3, 4. It is the following compound:

5a: m=3, n=3;

5b: m=3, n=4;

5c: m=4, n=3;

5d: m=4, n=4;

5e: m=5, n=3;

5f: m=5, n=4;

The route for synthesizing the tricationic quaternary ammonium salt antibacterial peptide mimetics (3a–3m, 4a–4i, 5a–5f) of the present invention is as follows:

Reaction conditions: a) 3-bromo-1-propanol, acetone, potassium carbonate, reflux at 65°C, 24 hours; b) phosphine tribromide, dichloromethane, room temperature, 6 hours; c) N, N-dimethyl Formamide, potassium carbonate, room temperature, 24 hours.

Route 1 Preparation of Intermediates 1a–1c

Reaction conditions: d) N, N-dimethylformamide, potassium carbonate, room temperature, 24 hours; e) ethanol, potassium carbonate, room temperature, 12 hours.

Preparation of Route 2 Intermediates 2a–2f

Reaction conditions: f) ethanol, pressure-resistant tube, 90°C, 96-168 hours.

Preparation of final products 3a–3m, 4a–4i and 5a–5f of route three

This is specifically achieved through the following steps:

(1) Phloroglucinol reacts with 3-bromo-1-propanol in acetone through the action of potassium carbonate. After the reaction, the system was concentrated, and extracted with ethyl acetate and water. After concentration, the product was reacted with phosphine tribromide in dichloromethane to generate intermediate 1a; phloroglucinol was dissolved in N, N-dimethylformamide. , add potassium carbonate solid, and react with the corresponding dibromoalkane to obtain intermediates 1b and 1c.

(2) Dissolve phenol in N,N-dimethylformamide, add solid potassium carbonate, and react with dibromopentane to obtain intermediate 2a; add phenoxy bromide and potassium carbonate of different chain lengths to In ethanol, dimethylamine aqueous solution is then added to react to generate intermediates 2b–2d; phenyl bromide and potassium carbonate of different chain lengths are added to ethanol, and then dimethylamine aqueous solution is added to react to generate intermediates 2e and 2f.

(3) React intermediates 1a–1c with dimethylalkylamines of different lengths, intermediates 2b–2d, and intermediates 2e–2f in ethanol in a pressure-resistant tube at 90°C to obtain the target final products. 3a–3m, 4a–4i and 5a–5f.

Innovative points and advantages of the present invention: Phloroglucinol is used as the middle connecting part, and alkyl chain, phenyl ether hydrophobic group and phenyl hydrophobic group are used as side chains. By changing the length of the middle chain and side chain, the intermediate carbon The length of chain and side chain as well as the influence of phenyl ether and phenyl hydrophobic groups on the activity and toxicity of this type of compounds. Experiments have confirmed that some of the tricationic quaternary ammonium salt antibacterial peptide mimics described in the present invention show good activity against both Gram-positive bacteria and Gram-negative bacteria, indicating that this series of compounds have excellent antibacterial activity. In particular, compounds 3a, 3f, 3j, 3k, 4g, 4h, 4j, and 5f have MIC values (minimum inhibitory effects) against four standard bacteria: Staphylococcus aureus, Escherichia coli, Enterococcus faecalis and Stenotrophomonas maltophilia. bacteria concentration) between 0.5-4μg/mL. HC ₅₀ (half hemolytic concentration) results show that it has lower hemolytic toxicity and higher selectivity. Through cytotoxicity and in vivo toxicity studies, it was found that compound 3f has lower toxicity. In addition, taking compound 3f as an example to explore the antibacterial mechanism of this type of compound, it was clearly found that this type of compound has the bactericidal characteristics of targeting the PG component of the cell membrane, and can stimulate bacteria to produce reactive oxygen species, aggravate membrane damage, and then cause bacterial cell damage. The contents leak out and the bacteria eventually die. Therefore, the tricationic quaternary ammonium salt antibacterial peptide mimetic provided by the present invention is expected to be further studied as a new antibacterial candidate drug, and is of great significance to solving the problem of drug-resistant bacteria currently facing the world.

Description of drawings

Figure 1 is an image under an optical microscope of GES cells treated with compound 3f of the present invention; wherein, (a) blank group (negative control); (b) compound 3f was treated with 64×MIC _{S. aureus} for 24 hours; (c) 0.1% methyl Pull through X-100 for 24 hours (positive control); scale bar 200 μm.

Figure 2 is an image of double staining of live and dead cells in GES cells treated with compound 3f of the present invention under a fluorescence microscope; wherein, (ab) blank group (negative control); (cd) compound 3f was treated with 64×MIC _{S. aureus} for 24 hours; (ef ) 0.1% Triton X-100 for 24 hours (positive control); scale bar 200 μm.

Figure 3 shows the plasma stability and body fluid bactericidal activity of Compound 3f of the present invention; wherein, (a) the changes in the minimum inhibitory concentration of Compound 3f and vancomycin against S. aureus after interacting with plasma for a period of time. (b) Changes in the minimum bactericidal concentrations of compound 3f and vancomycin against S. aureus in different body fluids.

Figure 4 is the bactericidal kinetics experiment of compound 3f of the present invention on S. aureus and E. coli; wherein, compound 3f (6×MIC) and vancomycin (6×MIC) act on (a) the early stage of logarithmic growth and ( b) S. aureus at the end of logarithmic growth stage. Compound 3f (6×MIC) and laoxime (6×MIC) act on E.coli in (a) early logarithmic growth stage and (b) late logarithmic growth stage. Control: sterile water; "------": detection limit (100CFU/mL).

Figure 5 shows the depolarization experiment of compound 3f of the present invention on the cell membranes of S. aureus (a) and E. coli (b); in the figure, Control: blank control.

Figure 6 shows the effects of different components of exogenous bacterial cell membranes (walls) on the MIC values of compounds, including (a) S. aureus; (b) E. coli;

Figure 7 shows the effects of different concentrations of compound 3f of the present invention and melittin on the production of reactive oxygen species in S. aureus (a) and E. coli (b), where NAC is a ROS quencher, and N-acetyl-L-semi- Cystine.

Figure 8 is SEM imaging of compound 3f of the present invention acting on S. aureus and E. coli; wherein, (a) normal S. aureus group; (b) S. aureus treated with compound 3f (8×MIC, 4 μg/mL). aureus group; (c) normal E.coli group; (d) E.coli group treated with compound 3f (8×MIC, 16 μg/mL). The ruler is shown in the figure.

Figure 9 shows the nucleic acid leakage experiment of compound 3f of the present invention, melittin and polymyxin B on S. aureus (a) and E. coli (b).

Figure 10 shows the induced resistance experiment of compound 3f; wherein, (a): the induced resistance tendency experiment of compound 3f and norfloxacin on S. aureus; (b): the induced resistance tendency experiment of compound 3f and colistin on E.coli Experiment on induced resistance tendency.

Figure 11 shows the blood routine and blood biochemistry experiments of compound 3f; wherein, the blood routine indicators: (a) white blood cell count (WBC); (b) hematocrit (HCT); (c) red blood cell count (RBC); (d) Hemoglobin (HGB); (e) mean corpuscular volume (MCV) and (f) platelet count (PLT). Blood biochemical indicators: (g) albumin (ALB); (h) urea (UREA); (i) creatinine (CREA).

Figure 12 is a HE staining picture of mouse organs (liver, kidney and spleen), in which (a) scale bar: 50 μm; (b) scale bar: 100 μm.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. These examples are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. The percentages mentioned below are all mass percentages unless otherwise specified.

The instruments used to characterize the synthesized compounds: NMR spectrum was measured using a Swedish Bruker DPX-400 superconducting nuclear magnetic resonance instrument; high-resolution mass spectrometry was measured using a Waters-Micromass Q-Tof mass spectrometer.

Example 1 Preparation of Compound 1a:

In a round-bottomed flask (100mL), dissolve phloroglucinol (7.93mmol, 1eq.) in acetone (20mL), then add solid potassium carbonate (35.68mmol, 4.5eq.), stir evenly and then add 3-bromo -1-Propanol (35.68mmol, 4.5eq.), heated to reflux at 65°C, and reacted for 24 hours. After the reaction, the solvent was concentrated to remove, ethyl acetate (100 mL) and water (100 mL) were added to dissolve and dilute the solid, and the organic phase was washed three times with water (100 mL). The organic phase was retained and washed with saturated sodium chloride solution (10 mL) for 1- 3 times) and dry with anhydrous sodium sulfate, filter after drying, and concentrate the organic phase. Weigh the concentrated product (3g, 1eq.), transfer it to a clean reactor, add dichloromethane (50mL) to dissolve, and add phosphine tribromide (19.98mmol, 2eq.) dropwise into the system under an ice-water bath. , after the addition is completed and stirred evenly, react at room temperature for 12 hours. After the reaction is completed, add ice water dropwise to the system under ice-water bath conditions to quench the reaction, and then extract with dichloromethane (100 mL) and water (100 mL) to retain the organic phase. Wash 1-3 times with saturated sodium chloride solution (10 mL), dry over anhydrous sodium sulfate, filter, concentrate, and use column separation to purify the product (petroleum ether: ethyl acetate = 20:1, V:V).

1a: Yield 45%. ¹ H NMR (400MHz, Chloroform-d) δ6.10 (s, 3H), 4.07 (t, J = 5.8Hz, 6H), 3.59 (t, J = 6.5Hz, 6H), 2.30 (m, 6H). ¹³ C NMR (101MHz, Chloroform-d) δ160.57,94.24,65.39,32.31,29.94.

Example 2 Preparation of Compound 1b:

In a round bottom flask (100 mL), phloroglucinol (7.93 mmol, 1 eq.) was dissolved in N,N-dimethylformamide (20 mL), then potassium carbonate solid (35.68 mmol, 4.5 eq.) was added , stir evenly, add dibromobutane (35.68mmol, 4.5eq.), and react at room temperature for 24h. After the reaction, add ethyl acetate (100 mL) and water (100 mL) to dilute the system, extract, retain the organic phase, wash the organic phase 3-5 times with water (100 mL), and wash 1-5 times with saturated sodium chloride solution (10 mL). 3 times) and anhydrous sodium sulfate to dry the organic phase, filter and concentrate the organic phase, and use column separation to purify the product (petroleum ether: ethyl acetate = 20:1, V: V).

1b: Yield 49%. ¹ H NMR(400MHz,Chloroform-d)δ6.05(s,3H),3.95(t,J＝6.0Hz,6H),3.48(t,J＝6.6Hz,6H),2.10–2.01(m,6H ),1.93(m,6H). ¹³ C NMR(101MHz,Chloroform-d)δ160.72,93.92,66.87,33.42,29.46,27.84.

Preparation of compound 1c in Example 3: The preparation method is the same as in Example 2, and the dibromoalkane used is dibromopentane.

1c: Yield 47%. ¹ H NMR(400MHz,Chloroform-d)δ6.05(s,3H),3.92(t,J＝6.3Hz,6H),3.43(t,J＝6.8Hz,6H),1.98–1.88(m,6H ),1.84–1.74(m,6H),1.66–1.57(m,6H). ¹³ CNMR(101MHz,Chloroform-d)δ160.81,93.88,67.59,33.61,32.47,28.39,24.86.

Example 4 Preparation of Compound 2a:

In a round-bottomed flask (100 mL), dissolve phenol (10.63 mmol, 1 eq.) in N,N-dimethylformamide (DMF, 10 mL), add potassium carbonate (21.25 mmol, 2 eq.) and stir evenly. Then dibromopentane (15.94mmol, 1.5eq.) was added dropwise into the reaction system, and the reaction was carried out at room temperature for 24h. After the reaction, add ethyl acetate (50 mL) and water (50 mL) to dilute the system, then extract, retain the organic phase, and then wash the organic phase with water (50 mL) 3-5 times. After the washing is completed, use saturated sodium chloride solution ( 10 mL) and then washed 1-3 times and dried over anhydrous sodium sulfate. After drying, it was filtered and concentrated, and the product was purified by column chromatography (petroleum ether:ethyl acetate=50:1, V:V).

2a: Yield 48%. ¹ H NMR (400MHz, Chloroform-d) δ7.31–7.26(m,2H),7.00–6.83(m,3H),3.97(t,J＝6.3Hz,2H),3.44(t,J＝6.8Hz ,2H),1.99–1.90(m,2H),1.86–1.77(m,2H),1.69–1.58(m,2H). ¹³ C NMR(101MHz,Chloroform-d)δ158.94,129.43,120.62,114.47,77.21 ,67.43,33.61,32.51,28.47,24.87.

Example 5 Preparation of Compound 2b:

In a round-bottomed flask (100 mL), add 3-phenoxypropyl bromide (4.36 mmol, 1 eq.) and potassium carbonate (6.55 mmol, 1.5 eq.) to ethanol (20 mL), stir evenly, and then add 40% dihydrogen Methylamine aqueous solution (1.11mL, 8.73mmol, 2eq.), react at room temperature, use a rotary evaporator to evaporate the system to dryness after 12 hours, then add water (100mL) to dissolve the solid, and then use dichloromethane (50mL) to extract 3-5 times, retain the organic phase, wash the organic phase 1-3 times with saturated sodium chloride solution (10 mL), and dry over anhydrous sodium sulfate. After drying, filter and concentrate, and the product is purified by column chromatography (dichloromethane:methanol=20:1, V:V).

2b: Yield 75%. ¹ H NMR (400MHz, Chloroform-d) δ7.30–7.24(m,2H),6.98–6.86(m,3H),4.01(t,J＝6.4Hz,2H),2.45(t,J＝7.3Hz ,2H),2.26(s,6H),2.00–1.91(m,2H). ¹³ C NMR(101MHz,Chloroform-d)δ158.98,129.39,120.55,114.49,66.10,56.45,45.52,27.60.

Preparation of compound 2c in Example 6: The preparation method is the same as in Example 5, and the phenoxyalkyl bromide used is 4-phenoxybutyl bromide.

2c: Yield 78%. ¹ H NMR (400MHz, Chloroform-d) δ7.30–7.25(m,2H),7.01–6.82(m,3H),3.99(t,J＝6.0Hz,2H),2.56–2.46(m,2H) ,2.37(s,6H),1.94–1.65(m,4H). ¹³ C NMR(101MHz,Chloroform-d)δ158.90,129.43,120.63,114.47,67.37,59.08,44.89,27.03,23.69.

Preparation of compound 2d in Example 7: The preparation method is the same as in Example 5, and the phenoxy bromide alkyl used is 2a (5-phenoxy pentane bromide).

2d: Yield 79%. ¹ H NMR (400MHz, Chloroform-d) δ7.30–7.24(m,2H),6.95–6.87(m,3H),3.96(t,J＝6.5Hz,2H),2.31–2.25(m,2H) ,2.22(s,6H),1.85–1.76(m,2H),1.59–1.43(m,4H). ¹³ C NMR(101MHz,Chloroform-d)δ159.05,129.38,120.47,114.47,67.71,59.76,45.55, 29.26,27.55,24.01.

Example 8 Preparation of Compound 2e:

In a round-bottomed flask (100 mL), add 3-phenyl bromide propane (4.69 mmol, 1 eq.) and potassium carbonate (7.04 mmol, 1.5 eq.) to ethanol (20 mL), stir evenly, and then add 40% dimethyl Amine aqueous solution (1.19mL, 9.38mmol, 2eq.), react at room temperature for 12h. The post-treatment and purification methods are the same as in Example 5.

2e: Yield 76%. ¹ H NMR (400MHz, Chloroform-d) δ7.30–7.26(m,2H),7.21–7.14(m,3H),2.68–2.60(m,2H),2.34–2.29(m,2H),2.24( s,6H),1.86–1.75(m,2H). ¹³ C NMR(101MHz,Chloroform-d)δ142.18,128.36,128.31,125.74,59.24,45.40,33.65,29.34.

Preparation of compound 2f in Example 9: The preparation method is the same as in Example 8, and the phenyl bromide alkane used is 4-phenyl bromide butane.

2f: Yield 73%. ¹ H NMR (400MHz, Chloroform-d) δ7.30–7.24(m,2H),7.20–7.14(m,3H),2.63(t,J＝7.6Hz,2H),2.30–2.23(m,2H) ,2.20(s,6H),1.69–1.59(m,2H),1.55–1.44(m,2H). ¹³ C NMR(101MHz,Chloroform-d)δ142.50,128.39,128.24,125.65,59.73,45.51,35.86, 29.31,27.40.

Example 10 Preparation of Compound 3a:

Intermediate 1a (613.44 μmol, 1 eq.) and N,N-dimethyloctylamine (2.76 mmol, 4.5 eq.) were added to ethanol (5 mL), and the reaction was carried out in a pressure-resistant tube at 90°C for 96-168 hours. After the reaction is completed, concentrate the system to obtain a yellow oily viscous substance. Add a small amount of methanol to completely dissolve the oily substance, and then add a large amount of ethyl acetate for recrystallization. At this time, a yellow-white solid will precipitate. Place the system in the refrigerator for a period of time to promote crystal precipitation. The upper layer of ethyl acetate was then poured. Repeat the above recrystallization operation until the impurities are removed as much as possible. During this period, more impurities in the yellow-white oily product can be dissolved in ethyl acetate through ultrasound. The crude product was then dried and purified by column chromatography (dichloromethane:methanol=10:1~5:1, V:V).

3a: Yield 48%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ6.15 (s, 3H), 4.01 (t, J = 6.0Hz, 6H), 3.51–3.41 (m, 6H), 3.32–3.30 (m, 6H), 3.07(s,18H),2.16–2.08(m,6H),1.70–1.63(m,6H),1.32–1.23(m,30H),0.88–0.84(m,9H). ¹³ C NMR(101MHz,DMSO -d ₆ )δ160.46,94.75,65.24,63.43,60.76,50.57,31.62,28.94,28.93,26.22,22.65,22.50,22.16,14.43.HR-MS(ESI):m/z calculated for C ₄₅ H ₉₀ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3:240.2322, found 240.2327.

Preparation of compound 3b in Example 11: The preparation method is the same as in Example 10, and the dimethylalkylamine used is N,N-dimethyldecylamine.

3b: Yield 45%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ6.15 (s, 3H), 4.01 (t, J = 6.0Hz, 6H), 3.50–3.43 (m, 6H), 3.32–3.30 (m, 6H), 3.08(s,18H),2.16–2.09(m,6H),1.70–1.63(m,6H),1.29–1.23(m,42H),0.87–0.83(m,10H). ¹³ C NMR(101MHz,DMSO -d ₆ )δ160.09,94.41,64.89,63.08,60.40,50.19,31.37,28.99,28.93,28.75,28.60,25.86,22.18,21.81,14.05.HR-MS(ESI):m/z calculated for C ₅₁ H ₁₀₂ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3:268.2635, found 268.2642.

Preparation of compound 3c in Example 12: The preparation method is the same as in Example 10, and the dimethylalkylamine used is N,N-dimethyldodecylamine.

3c: Yield 48%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ6.15 (s, 3H), 4.01 (t, J = 6.0Hz, 6H), 3.50–3.43 (m, 6H), 3.32–3.30 (m, 6H), 3.08(s,18H),2.13(m,6H),1.67(m,6H),1.28–1.24(m,54H),0.87–0.83(m,9H). ¹³ C NMR(101MHz,DMSO-d ₆ ) Δ160.09,94.39,63.07, 60.38,5.19,39.59,37,29.10, 29.03,28.93,28.80,25.85,228,22.180,14.04.hr-MS (ESI): M/Z CAL. Culied for C ₅₇ H ₁₁₄ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3:296.2948,found296.2953.

Preparation of compound 3d in Example 13: The preparation method is the same as in Example 10, and the raw materials used are intermediate 1b and N,N-dimethylbutylamine.

3d: Yield 30%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ6.12 (s, 3H), 3.97 (t, J = 6.1Hz, 6H), 3.42–3.37 (m, 6H), 3.33–3.25 (m, 6H), 3.04(s,18H),1.86–1.57(m,18H),1.29(m,6H),0.91(m,9H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ160.71,94.40,67.19,63.20 ,62.85,50.44,25.97,24.15,19.65,19.20,13.97.HR-MS(ESI):m/zcalculated for C ₃₆ H ₇₂ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3:198.1852,found 198.1857 .

Preparation of compound 3e in Example 14: The preparation method is the same as in Example 10, and the raw materials used are intermediate 1b and N,N-dimethylhexylamine.

3e: Yield 35%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ6.12 (s, 3H), 3.97 (t, J = 6.1Hz, 6H), 3.41–3.36 (m, 6H), 3.33–3.26 (m, 6H), 3.05(s,18H),1.84–1.61(m,18H),1.27(m,18H),0.90–0.82(m,9H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ160.27,93.94,66.73 ,62.90,62.36,49.98,30.67,25.53,25.46,21.91,21.67,18.76,13.85.HR-MS(ESI):m/z calculated forC ₄₂ H ₈₄ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ / 3:226.2165,found226.2171.

Preparation of compound 3f in Example 15: The preparation method is the same as in Example 10, and the raw materials used are intermediate 1b and N,N-dimethyloctylamine.

3f: Yield 44%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ6.12 (s, 3H), 3.97 (t, J = 6.1Hz, 6H), 3.39–3.34 (m, 6H), 3.29–3.23 (m, 6H), 3.03(s,18H),1.84–1.60(m,18H),1.32–1.22(m,30H),0.90–0.84(t,J＝6.8Hz,9H). ¹³ C NMR(101MHz,DMSO-d ₆ ) δ160.35,94.03,66.80,63.08,62.55,50.08,31.21,28.53,28.51,25.87,25.63,22.09,21.77,18.84,14.01.HR-MS(ESI):m/z calculated for C ₄₈ H ₉₆ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3:254.2478, found 254.2483.

Preparation of compound 3g in Example 16: The preparation method is the same as in Example 10, and the raw materials used are intermediate 1b and N,N-dimethyldecylamine.

3g: Yield 48%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ6.12 (s, 3H), 3.97 (t, J = 6.1Hz, 6H), 3.43–3.34 (m, 6H), 3.31–3.24 (m, 6H), 3.04(s,18H),1.73(m,18H),1.26(m,42H),0.88–0.82(m,9H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ160.25,93.95,66.73,62.95 ,62.42,49.96,31.25,28.87,28.81,28.63,28.48,25.79,25.54,22.06,21.70,18.75,13.93.HR-MS(ESI):m/zcalculated for C ₅₄ H ₁₀₈ Br ₃ N ₃ O ₃ [M -3Br] ³⁺ /3:282.2791,found 282.2798.

Preparation of compound 3h in Example 17: The preparation method is the same as in Example 10, and the raw materials used are intermediate 1b and N,N-dimethyldodecylamine.

3h: Yield 46%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ6.13 (s, 3H), 3.97 (t, J = 6.1Hz, 6H), 3.42–3.35 (m, 6H), 3.32–3.25 (m, 6H), 3.04(s,18H),1.84–1.61(m,18H),1.25(m,54H),0.87–0.82(m,9H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ160.33,94.03,66.81 ,63.02,62.49,50.03,31.35,29.07,29.00,28.90,28.76,28.57,25.88,25.62,22.15,21.78,18.82,14.00.HR-MS(ESI):m/z calculated for C ₆₀ H ₁₂₀ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3:310.3104,found 310.3111.

Preparation of compound 3i in Example 18: The preparation method is the same as in Example 10, and the raw materials used are intermediate 1c and N,N-dimethylbutylamine.

3i: Yield 30%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ6.06 (s, 3H), 3.92 (t, J = 6.3Hz, 6H), 3.32–3.21 (m, 12H), 3.01 (s, 18H), 1.77– 1.58(m,18H),1.34(m,12H),0.91(m,9H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ160.87,94.10,67.61,63.26,50.40,28.56,24.15,22.94, 21.95,19.63,13.96.HR-MS(ESI):m/z calculated for C ₃₉ H ₇₈ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3:212.2009,found 212.2015.

Preparation of compound 3j in Example 19: The preparation method is the same as in Example 10, and the raw materials used are intermediate 1c and N,N-dimethylhexylamine.

3j: Yield 33%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ6.07 (s, 3H), 3.93 (t, J = 6.3Hz, 6H), 3.28 (m, 12H), 3.02 (s, 18H), 1.78–1.60 ( m,18H),1.45–1.24(m,24H),0.87(q,J＝5.3,3.8Hz,9H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ160.88,94.09,67.57,63.38,63.22 ,50.38,31.11,28.59,25.90,22.97,22.34,22.11,21.94,14.29.HR-MS(ESI):m/z calculated for C ₄₅ H ₉₀ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3 :240.2322, found 240.2327.

Preparation of compound 3k in Example 20: The preparation method is the same as in Example 10, and the raw materials used are intermediate 1c and N,N-dimethyloctylamine.

3k: Yield 42%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ6.07 (s, 3H), 3.92 (t, J = 6.3Hz, 6H), 3.32–3.21 (m, 12H), 3.02 (s, 18H), 1.78– 1.60(m,18H),1.45–1.22(m,36H),0.90–0.83(m,9H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ160.52,93.72,67.21,63.03,62.85,50.02, 31.25,28.57,28.55,28.24,25.89,22.61,22.14,21.79,21.58,14.06.HR-MS(ESI):m/z calculated forC ₅₁ H ₁₀₂ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3 :268.2635,found 268.2641.

Preparation of compound 3l in Example 21: The preparation method is the same as in Example 10, and the raw materials used are intermediate 1c and N,N-dimethyldecylamine.

3l: Yield 45%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ6.07 (s, 3H), 3.92 (t, J = 6.3Hz, 6H), 3.30–3.23 (m, 12H), 3.01 (s, 18H), 1.77– 1.61(m,18H),1.45–1.23(m,48H),0.89–0.83(m,9H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ160.88,94.08,67.57,63.39,63.21,50.39, 31.73,29.35,29.28,29.12,28.95,28.61,26.25,22.97,22.55,22.15,21.95,14.42.HR-MS(ESI):m/zcalculated for C ₅₇ H ₁₁₄ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3:296.2948, found 296.2954.

Preparation of compound 3m in Example 22: The preparation method is the same as in Example 10, and the raw materials used are intermediate 1c and N,N-dimethyldodecylamine.

3m: Yield 48%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ6.06 (s, 3H), 3.92 (t, J = 6.3Hz, 6H), 3.30–3.23 (m, 12H), 3.02 (s, 18H), 1.78– 1.60(m,18H),1.45–1.20(m,60H),0.88–0.80(m,9H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ160.52,93.72,67.22,63.02,62.84,50.03, 31.39,29.11,29.04,28.92,28.81,28.59,28.25,25.89,22.61,22.19,21.79,21.59,14.06.HR-MS(ESI):m/z calculated for C ₆₃ H ₁₂₆ Br ₃ N ₃ O ₃ [M -3Br] ³⁺ /3:324.3261, found 324.3268.

Preparation of compound 4a in Example 23: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1a and 2b.

4a: Yield 40%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.33–7.28 (m, 6H), 6.99–6.93 (m, 9H), 6.16 (s, 3H), 4.06 (t, J = 5.9Hz, 6H), 4.01 (t, J＝6.0Hz, 6H), 3.58–3.46 (m, 12H), 3.13 (s, 18H), 2.24–2.10 (m, 12H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ158. 11,129.53,120.90,114.46,94.33,64.50,60.53,50.34,22.24.HR-MS(ESI):m/z calculated for C ₄₈ H ₇₂ Br ₃ N ₃ O ₆ [M-3Br] ³⁺ /3:262.1802, found 262.1805.

Preparation of compound 4b in Example 24: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1a and 2c.

4b: Yield 41%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.31–7.26(m,6H),6.96–6.90(m,9H),6.16(s,3H),4.05–3.97(m,12H),3.51–3.39 (m,12H),3.09(s,18H),2.20–2.08(m,6H),1.91–1.83(m,6H),1.79–1.70(m,6H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ160.01,158.36,129.46,120.58,114.44,94.33,66.48,62.65,50.15,25.56,22.18,18.85.HR-MS(ESI):m/zcalculated for C ₅₁ H ₇₈ Br ₃ N ₃ O ₆ [M-3Br ] ³⁺ /3:276.1958, found 276.1962.

Preparation of compound 4c in Example 25: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1a and 2d.

4c: Yield 41%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.31–7.24(m,6H),6.95–6.89(m,9H),6.16(s,3H),4.05–3.95(m,12H),3.50–3.40 (m,6H),3.39–3.34(m,6H),3.08(s,18H),2.20–2.20(m,6H),1.82–1.72(m,12H),1.48–1.40(m 6H) ^.13 C NMR (101MHz, DMSO-d ₆ ) δ160.00,158.51,129.44,120.42,114.36,94.34,66.92,64.80,62.90,60.44,50.12,28.16,22.50,22.18,21.52.HR-MS(ESI):m/z calculated for C ₅₄ H ₈₄ Br ₃ N ₃ O ₆ [M-3Br] ³⁺ /3:290.2115,found290.2120.

Preparation of compound 4d in Example 26: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1b and 2b.

4d: Yield 41%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.32–7.28 (m, 6H), 6.97–6.93 (m, 9H), 6.13 (s, 3H), 4.04 (t, J = 5.9Hz, 6H), 3.95(t,J＝6.1Hz,6H),3.50–3.40(m,12H),3.09(s,18H),2.19–2.15(m,6H),1.83–1.68(m,6H),1.74–1.68( m,6H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ160.26,158.10,129.51,114.45,93.95,66.71,64.50,62.54,60.47,50.19,25.52,22.22,18.76.HR-MS(ESI):m /z calculated for C ₅₁ H ₇₈ Br ₃ N ₃ O ₆ [M-3Br] ³⁺ /3:276.1958,found276.1961.

Preparation of compound 4e in Example 27: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1b and 2c.

4e: Yield 44%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.28 (t, J = 7.8 Hz, 6H), 6.93 (m, 9H), 6.12 (s, 3H), 3.99 (t, J = 6.0 Hz, 6H) ,3.94(t,J＝6.0Hz,6H),3.38(m,12H),3.04(s,18H),1.88–1.66(m,24H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ160.37,158.45 ,129.58,120.70,114.50,94.00,66.54,62.65,50.18,25.66,18.95,18.81.HR-MS(ESI):m/z calculated for C ₅₄ H ₈₄ Br ₃ N ₃ O ₆ [M-3Br] ³⁺ /3:290.2115, found 290.2123.

Preparation of compound 4f in Example 28: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1b and 2d.

4f: Yield 47%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.31–7.25 (m, 6H), 6.94–6.89 (m, 9H), 6.13 (s, 3H), 3.97 (t, J = 6.2Hz, 12H), 3.40–3.34(m,6H),3.33–3.27(m,6H),3.04(s,18H),1.84–1.67(m,24H),1.46–1.38(m,6H). ¹³ C NMR (101MHz, DMSO -d ₆ )δ160.26,158.51,129.44,120.42,114.35,66.90,49.98,28.14,25.54,22.52,21.49,18.75.HR-MS(ESI):m/zcalculated for C ₅₇ H ₉₀ Br ₃ N ₃ O ₆ [ M-3Br] ³⁺ /3:304.2271, found 304.2279.

Preparation of compound 4g in Example 29: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1c and 2b.

4g: Yield 40%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.33–7.27 (m, 6H), 6.98–6.93 (m, 9H), 6.08 (s, 3H), 4.05 (t, J = 5.9Hz, 6H), 3.93(t,J＝6.3Hz,6H),3.51–3.43(m,6H),3.40–3.35(m,6H),3.08(s,18H),2.20–2.12(m,6H),1.79–1.70( m,12H),1.45–1.36(m,6H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ160.42,158.08,129.52,120.87,114.45,93.66,67.11,64.49,62.80,60.44,50.16,28.17,22 .46 ,22.20,21.49.HR-MS(ESI):m/z calculated for C ₅₄ H ₈₄ Br ₃ N ₃ O ₆ [M-3Br] ³⁺ /3:290.2115, found 290.2121.

Preparation of compound 4h in Example 30: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1c and 2c.

4h: Yield 42%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.31–7.26 (m, 6H), 6.95–6.90 (m, 9H), 6.07 (s, 3H), 4.01 (t, J = 6.0Hz, 6H), 3.92(t,J＝6.3Hz,6H),3.40–3.34(m,6H),3.33–3.27(m,6H),3.04(s,18H),1.85–1.68(m,24H),1.43–1.37( m,6H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ160.41,158.35,129.46,120.57,114.41,93.65,67.11,66.43,62.82,62.51,50.00,28.14,25.55,22.48,21.48,1 8.83.HR- MS(ESI):m/z calculated for C ₅₇ H ₉₀ Br ₃ N ₃ O ₆ [M-3Br] ³⁺ /3:304.2271, found 290.2279.

Preparation of compound 4i in Example 31: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1c and 2d.

4i: Yield 45%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.31–7.25 (m, 6H), 6.94–6.89 (m, 9H), 6.07 (s, 3H), 3.98 (t, J = 6.3Hz, 6H), 3.92(t,J＝6.3Hz,6H),3.32–3.24(m,12H),3.02(s,18H),1.82–1.68(m,24H),1.48–1.36(m,12H). ¹³ C NMR( 101MHz, DMSO-d ₆ )δ160.42,158.52,129.44,120.42,114.35,93.66,67.12,66.90,62.87,49.94,28.13,22.53,21.49.HR-MS(ESI):m/z calculated for C ₆₀ H ₉₆ Br ₃ N ₃ O ₆ [M-3Br] ³⁺ /3:318.2428,found 318.2437.

Preparation of compound 5a in Example 32: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1a and 2e.

5a: Yield 40%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.36–7.19 (m, 15H), 6.13 (s, 3H), 3.98 (t, J = 5.9Hz, 6H), 3.49–3.43 (m, 6H), 3.40–3.36(m,6H),3.08(s,18H),2.61(t,J＝7.8Hz,6H),2.16–1.99(m,12H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ159. 99,140.29,128.41,128.28,126.21,94.25,64.72,62.64,60.37,50.27,31.64,23.63,22.14.HR-MS(ESI):m/z calculated forC ₄₈ H ₇₂ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3:246.1858, found 246.1859.

Preparation of compound 5b in Example 33: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1a and 2f.

5b: Yield 40%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.32–7.26 (m, 6H), 7.24–7.16 (m, 9H), 6.16 (s, 3H), 4.01 (t, J = 5.9Hz, 6H), 3.49–3.44(m,6H),3.42–3.36(m,6H),3.07(s,18H),2.63(t,J＝7.7Hz,6H),2.17–2.09(m,6H),1.76–1.68m ,6H),1.63–1.54(m,6H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ160.00,141.47,128.33,128.22,125.88,94.35,64.79,62.76,60.42,50.13,34.39,27.71,22. 16, 21.39.HR-MS(ESI):m/z calculated for C ₅₁ H ₇₈ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3:260.2009, found 260.2012.

Preparation of compound 5c in Example 34: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1b and 2e.

5c: Yield 40%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.33–7.20 (m, 15H), 6.13 (s, 3H), 3.95 (t, J = 6.0Hz, 6H), 3.38–3.36 (m, 6H), 3.33–3.28(m,6H),3.04(s,18H),2.61(t,J＝7.7Hz,6H),2.04–1.95(m,6H),1.83–1.65(m,12H). ¹³ C NMR ( 101MHz, DMSO-d ₆ )δ160.26,140.28,128.39,128.27,126.20,93.92,66.70,62.61,50.11,31.67,25.51,23.60,18.73.HR-MS(ESI):m/zcalculated for C ₅₁ H ₇₈ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3:260.2009, found 260.2014.

Preparation of compound 5d in Example 35: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1b and 2f.

5d: Yield 43%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.31–7.27(m,6H),7.23–7.16(m,9H),6.13(s,3H),3.95(t,J＝6.1Hz,6H), 3.36–3.33(m,6H),3.31–3.28(m,6H),3.01(s,18H),2.61(t,J＝7.6Hz,6H),1.82–1.74(m,6H),1.73–1.64( m,12H),1.62–1.52(m,6H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ160.27,141.45,128.33,128.20,125.89,93.91,66.69,62.67,62.45,50.02,34.36,27.71,25 .51 ,21.36,18.74.HR-MS(ESI):m/z calculated for C ₅₄ H ₈₄ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3:274.2165, found 274.2173.

Preparation of compound 5e in Example 36: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1c and 2e.

5e: Yield 41%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.33–7.21 (m, 15H), 6.02 (s, 3H), 3.92 (t, J = 6.3Hz, 6H), 3.32–3.27 (m, 12H), 3.02(s,18H),2.60(t,J＝7.7Hz,6H),2.00–1.96(m,6H),1.73–1.65(m,12H),1.41–1.36(m,6H). ¹³ C NMR( 101MHz, DMSO-d ₆ )δ160.42,140.32,128.39,128.30,126.19,93.66,62.79,62.51,50.08,31.65,28.12,23.63,22.45,21.44.HR-MS(ESI):m/z calculated for C ₅₄ H ₈₄ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3:274.2165,found274.2173.

Preparation of compound 5f in Example 37: The preparation method is the same as in Example 10, and the raw materials used are intermediates 1c and 2f.

5f: Yield 43%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.31–7.27(m,6H),7.25–7.15(m,9H),6.07(s,3H),3.92(t,J＝6.3Hz,6H), 3.32–3.23(m,12H),3.01(s,18H),2.63(t,J＝7.6Hz,6H),1.81–1.64(m,18H),1.62–1.54(m,6H),1.42–1.32( m,6H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ160.42,141.45,128.32,128.23,125.87,93.66,67.11,62.79,62.67,49.98,34.33,28.13,27.67,22.48,21.46,2 1.32.HR- MS(ESI): m/z calculated for C ₅₇ H ₉₀ Br ₃ N ₃ O ₃ [M-3Br] ³⁺ /3: 288.2322, found 288.2332. Application example 1 In vitro antibacterial activity test

(1) Preparation of antibacterial drug storage solution: The concentration of the antibacterial drug stock solution prepared is 25600 μg/mL. Antibacterial drugs with low solubility can be slightly lower than the above concentration. The solvent for dissolving the drug should be ultrapure water or dimethyl sulfoxide.

(2) Preparation of bacteria to be tested: Use an inoculating loop to pick a single colony on the MH (A) culture dish that was cultured overnight and place it in the MH (B) culture medium. Calibrate it to a 0.5 McFarland turbidimetric standard, containing approximately 1 bacteria. ×10 ⁸ CFU/mL, and then dilute it 100 times to obtain a bacterial solution containing approximately 1 × 10 ⁶ CFU/mL, which is ready for use.

(3) Dilute the antibacterial drug stock solution (25600 μg/mL) 100 times with ultrapure water to obtain an antibacterial drug solution with a concentration of 256 μg/mL. Take a sterile 96-well plate, add 200 μL of antibacterial drugs to the first well, add 100 μL of MH(B) broth culture medium to the second to tenth wells, add 100 μL from the first well to the second well, mix well, and then Pipette 100 μL into the third well, and so on. Pipette 100 μL into the tenth well and discard. At this time, the drug concentration in each well is: 128, 64, 32, 16, 8, 4, 2, 1, 0.5 μg/mL. Add 200 μL bacterial solution (positive control) to the eleventh well, and add 200 μL MH to the twelfth well. (B) Medium (negative control). Vancomycin and meropenem were also used as quality control drugs.

(4) Then add 50 μL of the previously prepared bacterial solution to each of wells 1 to 10, so that the final bacterial solution concentration in each tube is approximately 5×10 ⁵ CFU/mL, and the drug concentrations from wells 1 to 11 are 128 and 64 respectively. , 32, 16, 8, 4, 2, 1, 0.5, 0.25μg/mL. Place the 96-well plate in a 37°C incubator for 24 hours and then observe the growth of the bacterial solution.

(5) Result judgment and interpretation: Before reading and reporting the MIC of the tested strain, you should check whether the bacterial growth in the positive control tube is good, whether the negative control is contaminated, and whether the MIC value of the quality control drug is within the quality control range. Observe with the naked eye, the lowest drug concentration corresponding to the clear well is the MIC of the test bacteria. Application example 2 In vitro red blood cell hemolysis test

(1) PBS buffer: PBS phosphate is prepared into 1×PBS with ultrapure water, and then sterilized by high pressure.

(2) Preparation of 5% red blood cell suspension: Take 300 μL of blood into a 10 mL EP tube, add 5700 μL of 1×PBS, mix evenly, and centrifuge at low temperature (4°C, 3500 rpm, 10 min). Broken blood cells will cause the supernatant to become Red, discard the supernatant at this time, add 5700 μL 1×PBS, mix evenly, and centrifuge at low temperature. Repeat this operation until the supernatant is colorless and discard the supernatant. Finally, resuspend the blood cells at the bottom of the EP tube with 5700 μL 1×PBS to obtain a 5% red blood cell suspension. Use 1×PBS to prepare 0.1% Triton X-100 solution as a positive control.

(3) Preparation of sample solution: Dissolve the drug to be tested with a small amount of DMSO (the final concentration of DMSO cannot be greater than 0.5%), and use the same volume of DMSO as a negative control. The dissolved drug solution to be tested is diluted with PBS, and the concentration in the first hole is 1280 μg/mL. At this time, the drug in this EP tube is the initial drug. Then take nine 1.5mL EP tubes in parallel and place them in the test tube rack, and add 200 μL of PBS (numbered No. 2, No. 3, No. 4...No. 10) respectively. All drugs operate in parallel like this. Finally, draw 200 μL of the drug solution from the initial drug EP tube and add it to the No. 2 EP tube. After repeated purging, draw 200 μL into the No. 3 EP tube. Repeat the purging... Repeat the operation until the No. 10 EP tube. So, dilute the medicine well.

(4) Plate: Take a 96-well plate and write down the experiment number, drug code, and date. Adjust the pipette to 150 μL, mix the prepared 5% red blood cell suspension by gently inverting it up and down, and then pipet it into a 96-well plate (6×10). Then add the prepared drugs into the 96-well plate correspondingly, with one drug in three duplicate wells. After adding, place it in a 37°C incubator and incubate for 1 hour.

(5) Post-processing: Take out the 96-well plate from the incubator and place it in a centrifuge at 4°C for centrifugation (3500 rpm, 5 min). After centrifugation, take a new 96-well plate corresponding to each plate. Compare the labeled and centrifuged plates. Then draw 100 μL of supernatant accordingly (corresponding to the wells). After the absorption is completed, measure the OD value with a microplate reader, analyze the data, and obtain HC ₅₀ .

Application example 3 Minimum bactericidal concentration (MIC) determination experiment in plasma

(1) Preparation of bacteria to be tested: Use an inoculation loop to pick a single colony on the MH (A) culture dish that was cultured overnight and place it in the MH (B) culture medium. Calibrate it to a 0.5 McFarland turbidimetric standard, containing approximately 1 bacteria. ×10 ⁸ CFU/mL, and then dilute it 100 times to obtain a bacterial solution containing approximately 1 × 10 ⁶ CFU/mL, which is ready for use.

(2) Preparation of plasma: Take fresh sterile defibrinated sheep blood in a 10mL EP tube, and centrifuge in a centrifuge (3500rpm, 5min). Carefully aspirate the supernatant and obtain the plasma.

(3) Preparation of drug solution: First, use DMSO to prepare the drug to be tested into a large concentration stock solution of 25600 μg/mL (the final concentration of DMSO cannot be greater than 0.5%). Then use sterile ultrapure water and freshly prepared plasma as solvents to dilute to 256 μg/mL (the volume ratio of sterile ultrapure water and freshly prepared plasma is 1:1). Place eight 4mL EP tubes (numbered No. 1, No. 2, No. 3, No. 4... No. 8) on the EP tube rack. First, add 1500 μL of sterile ultrapure water and newly prepared plasma to each EP tube. Mix the solution (the volume ratio of sterile ultrapure water and fresh plasma is 1:1), then add 1500 μL of 256 μg/mL drug solution to EP tube No. 1, rinse repeatedly, then pipet 1500 μL into EP tube No. 2, and blow repeatedly Wash...repeat until EP tube No. 8. (The corresponding drug concentrations for each EP tube are 128, 64, 32, 16, 8, 4, 2, and 1 μg/mL) In this way, the drug is diluted well.

(4) Plate: Take a 96-well plate and write down the experiment number, drug code, and date. Adjust the pipette to 150 μL, draw 150 μL of drugs of different concentrations from the corresponding EP tubes, and add them to the 96-well plate to cover 9 rows, and each row is a drug gradient. Place in a 37°C incubator and incubate for 2 hours. Use a pipette to draw 50 μL of the bacterial solution to be tested from the first three rows and add it to the 96-well plate containing the drug. No bacterial solution is added to the last six rows. After the addition, place it in a 37°C incubator and incubate for 2 hours. Use a pipette to draw 50 μL of the bacterial solution to be tested from the middle three rows and add it to the 96-well plate containing the drug. Do not add the bacterial solution to the last three rows. After the addition is completed, place it in a 37°C incubator and incubate for 2 hours. Use a pipette in the last three rows to draw 50 μL of the bacterial solution to be tested and add it to the 96-well plate containing the drug. According to the duration of action of the drug and plasma, the shortest action time of the first three rows is 2 hours, the middle three rows are 4 hours, and the last three rows are 6 hours. After all the bacterial solution is added, place it in a 37°C incubator for incubation.

(5) After incubating the 96-well plate in a constant temperature incubator at 37°C for 24 hours, read the MIC value.

Application Example 4 Determination Experiment of Minimum Bactericidal Concentration (MBC) in Body Fluids

(1) Preparation of body fluids: Take fresh sterile defibrinated sheep blood in a 10mL EP tube, and centrifuge at 3500rpm/10min in a centrifuge. The supernatant was carefully drawn to become plasma; whole blood was fresh sterile defibrinated sheep blood; serum was purchased Zeta Life fetal calf serum.

(2) Preparation of bacteria to be tested: Use an inoculation loop to pick a single colony of MRSA on the MH (A) culture dish that was cultured overnight and place it in the MH (B) culture medium. Calibrate it to a 0.5 McFarland turbidimetric standard, containing approximately the number of bacteria. 1×10 ⁸ CFU/mL, and then dilute the bacterial solution to 10 ⁵ CFU/mL with 50% body fluid (whole blood, plasma or serum) and 50% MHB culture medium, and set aside. .

(3) Preparation of drug solution: First, use DMSO to prepare the drug to be tested into a large concentration stock solution of 25600 μg/mL (the final concentration of DMSO cannot be greater than 0.5%). The drug to be tested was diluted with sterile ultrapure water to 512, 256, 128, 64, 32, 16, 8, 4, and 2 μg/mL.

(4) Plating: Use a pipette to pipette 150 μL of the bacterial liquid diluted with different body fluids and add it to a 96-well plate. Plate the bacterial liquid diluted in each body fluid in 3 rows for parallel testing. Then add 50 μL of the diluted drug to be tested.

(5) Agar plate counting: After incubating the 96-well plate in a 37°C constant-temperature incubator for 24 hours, read the MIC value, and select 2 to 4 gradient concentrations greater than the MIC result as the MBC concentration to be tested. From the concentration to be tested, Take 20 μL of bacterial solution and apply it on the MH agar plate with a coating rod, dry it in a biological safety cabinet, and culture it in a 37°C constant-temperature incubator for 24 hours. The number of colonies on the MH agar plate is ≤5. The corresponding well concentration is the MBC of the compound against the corresponding strain.

Application Example 5 Time Sterilization Kinetics Experiment

Pick a single colony on the MH(A) agar plate in a clean bench and add it to 1mL of MH(B) culture medium. Then incubate it in a constant temperature shaker (37°C, 200rpm) for 12 hours and then dilute it 10,000 times. Take 2mL of bacterial liquid. The culture was continued in a constant-temperature shaker (37°C, 200 rpm) for 2 h (the early stage of bacterial logarithmic growth) and 5 h (the end of bacterial logarithmic growth). Then add expected concentrations of compounds, positive control drugs vancomycin, and laxamethasone, and add PBS to the negative control. Then, 100 μL of bacterial liquid was taken from each group at time points of 0h, 2h, 4h, 6h, 8h and 12h, centrifuged (4°C, 3500rpm, 5min), and resuspended in 1×PBS. The resuspended bacterial solution was diluted tenfold with PBS in a 96-well plate, and 10 μL was dropped from each well onto the agar medium. Perform three parallel operations, and then place it in a 37°C constant-temperature incubator for 24 hours. After the incubation, Perform colony counting and calculate the bacterial concentration of the initial bacterial solution.

Application Example 6 Antibacterial Mechanism Research Experiment

(1) Plasma membrane depolarization experiment: Pick a single colony on the MH(A) agar plate in a clean bench into 1mL of MH(B) culture medium, and then culture it in a constant temperature shaker (37°C, 200rpm) After 6 hours, centrifuge (4°C, 3500 rpm, 5 min) and resuspend in 1×PBS. Add 150 μL of bacterial solution to a 96-well black culture plate, then add 40 μL of fluorescent dye DiSC ₃ (5) (10 μM) in the dark, and place it in a 37°C constant-temperature incubator for 30 min. Use a microplate reader to test the change in fluorescence value at an excitation wavelength of 622nm and a cutoff wavelength of 670nm. The test frequency is once a minute. After continuous testing for 8 minutes, take 10 μL of the diluted compound, melittin (positive control), Qingda Mycin (negative control) and PBS (blank control) were added, and the fluorescence value was measured for the next 16 consecutive minutes.

(2) Effect of different components of cell membrane on antibacterial activity: Prepare a bacterial suspension with a final concentration of approximately 1.0×10 ⁵ CFU/mL. Use the checkerboard dilution method to add 50 μL of PG (phosphatidylglycerol), PE (phosphatidylethanolamine), CL (cardiolipin) and LPS (lipopolysaccharide) in the 96-well plate with a concentration range of 512-0 μg/mL, and then add 50 μL , compounds with a concentration range of 512-2 μg/mL, and finally 100 μL of bacterial suspension was added. After incubating for 18 hours in a 37°C constant-temperature incubator, read the MIC value.

(3) Reactive oxygen species (ROS) generation experiment: Prepare a bacterial suspension with a concentration of approximately 1.0×10 ⁸ CFU/mL. Then centrifuge (4°C, 3500rpm, 5min) and resuspend in 1×PBS. Mix with an equal volume of fluorescent dye DCF-DA in PBS (20mM). After incubating for 30 minutes in a 37°C constant-temperature incubator, centrifuge (4°C, 3500rpm, 5min), resuspend in 1×PBS, and wash away the fluorescent dye that has not entered the bacteria. Add 180 μL of bacterial suspension to the 96-well plate, then add 20 μL of compounds of different concentrations, 1×PBS (blank control), melittin (positive control) and quality control drug NAC (100mM). After incubating for 40 minutes in a 37°C constant-temperature incubator, use a microplate reader to test the fluorescence value under the conditions of an excitation wavelength of 488nm and a cutoff wavelength of 530nm.

(4) Scanning electron microscopy experiment: Pick a single colony on the MH(A) agar plate in a clean bench and add it to 1mL of LB culture medium, then culture it in a constant temperature shaker (37°C, 200rpm) for 12 hours and then dilute it 1000 times. After adding the compound at the expected concentration, the culture was continued for 4 hours in a constant temperature shaker (37°C, 200 rpm). The blank control was LB broth without compound. After the culture, centrifuge (4°C, 3500rpm, 5min), resuspend three times in 1×PBS, add 2.5% glutaraldehyde solution to avoid light, resuspend, freeze at -20°C overnight and send to Beijing Zhongke Company for testing.

(5) Nucleic acid leakage test: Prepare a bacterial suspension with a concentration of approximately 1.0×10 ⁸ CFU/mL. Then centrifuge (4°C, 3500rpm, 5min) and resuspend in 1×PBS. Different concentrations of compounds, PBS (blank control), Melittin (positive control), and Polymyxin B (negative control), were added. Centrifuge (4°C, 3500rpm, 5min) at 30min, 60min, 120min, 180min and 240min respectively, take the supernatant and test the OD _260nm value with an ultra-micro spectrophotometer.

Application Example 7 Cytotoxicity Test

(1) Preparation of compound solution: The compound is prepared with DMSO into a solution with a concentration of 25600 μg/mL, and then diluted twice with complete culture medium into test solutions of different concentrations, so that the final concentrations are 128, 64, 32, 16, 8, 4, 2, 1μg/mL.

(2) Determination of cell survival rate: When the cells are passed to 3-6 generations, the state of the cells is relatively stable. After digesting the cells from the culture dish, count them with a hemocytometer, and dilute the cell suspension according to the number of cells in 100 μL per well of a 96-well plate, which is approximately 1 × 10 ⁵ . After the diluted cell solution is added to the 96-well plate, 100 μL of test solution of different concentrations is added, and complete culture medium is used as a negative control. After adding the test solution, place the 96-well plate in a cell culture incubator (37°C, 5% CO ₂ by volume) for 24 hours. Add 5 μL of CCK-8 solution to each well in the dark, and then place it in a cell culture incubator for culture. (37°C, 5% CO ₂ by volume) After 4 hours of culture, the OD ₄₅₀ value was measured. The negative control was a survival rate of 100%. Calculate the cell survival rate under each concentration of the test solution. The IC ₅₀ value can be obtained by simulating the concentration of the test solution and cell viability using SPSS software.

(3) Optical microscope experiment: Add 500 μL of diluted cell suspension into a 12-well plate, place it in a cell culture incubator (37°C, 5% CO ₂ by volume) and incubate for 24 hours. Discard the supernatant and use 1 Wash off dead cells with ×PBS, then add 1 mL of different concentrations of the test solution and 0.1% Triton X-100 solution, place it in a cell culture incubator (37°C, 5% CO ₂ by volume) and incubate for 24 hours. Observe the cell morphology under an optical microscope and take pictures with the correct field of view.

(4) Live-dead cell double-staining experiment: Add 500 μL of diluted cell suspension into a 12-well plate, place it in a cell culture incubator (37°C, 5% CO ₂ by volume) and incubate for 24 hours, then discard the supernatant. , wash away dead cells with 1×PBS, then add 1 mL of different concentrations of the test solution and 0.1% Triton X-100 solution, and place it in a cell incubator (37°C, 5% CO ₂ by volume) for 24 hours. Afterwards, discard the supernatant, and then add 500 μL of calcein-AM (Calcein-AM, 2 μM) and propidium iodide (PI, 4.5 μM) mixture in a cell culture incubator (37°C, 5% CO ₂ ) Incubate for 15 minutes, observe and take pictures with a fluorescence microscope.

Application Example 8 Induced Resistance Experiment

First test the MIC value of the compound. MHA agar plates and MHB medium containing compounds at 1/2 MIC concentration were then prepared. After sterilizing the MHA agar solution with flowing steam, wait until it is cooled to a temperature that is not hot to the touch, and then add the compound solution to make the final concentration 1/2 MIC. The compound solution was directly diluted with MHB medium to prepare a medium containing the compound at 1/2 MIC concentration. Pick a single colony on the MHA agar plate in a clean bench and add it to 1 mL of MHB culture fluid containing a compound with a concentration of 1/2 MIC. After culturing for 4-6 hours in a constant-temperature shaker (37°C, 200 rpm), inoculate the bacterial solution into Place the MHA agar plate containing a compound with a concentration of 1/2 MIC in a 37°C constant-temperature incubator and incubate it for 16-24 hours. Repeat the above operation after a single colony grows on the agar plate. Test the MIC value for each operation for 20 seconds. Second-rate.

Application Example 9 Acute Toxicity Test

Select SPF grade BALB/c female mice aged 6-8 weeks and weighing about 20-25g, and randomly divide the mice into groups of 6 mice in each group. A total of 4 dose groups were set up: 50mg/kg, 30mg/kg, 20mg/kg and 10mg/kg, a normal saline blank control group, and polymyxin E as an antibiotic group control. Physiological saline was used as the solvent for the compound. After each mouse was weighed, 100 μL was injected intraperitoneally. The survival status and survival rate of the mice were observed for 72 hours. During the observation period, drinking water and feed were provided normally. The maximum tolerated dose of the mice was obtained based on the survival rate after 72 hours. 24 hours after the maximum tolerated dose was injected into the mice, blood was immediately taken from the eyeballs and 20 μL was taken out for routine blood and blood biochemical tests to verify the toxicity of the compound to the blood. Routine blood test items include red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), white blood cells (WBC), platelets (PLT), and mean corpuscular volume (MCV). The blood biochemical test items are albumin (ALB), urea (UREA), and creatinine (CREA). After collecting the blood, the mice were sacrificed by dislocation and dissected. The liver, kidneys and spleen were removed and fixed with 4% paraformaldehyde fixative. HE staining was performed and pathological sections were used to observe the toxicity of the compounds to the organs.

Experimental results

Table 1. MIC (μg/mL) results and in vitro erythrocyte hemolytic HC ₅₀ (μg/mL) results of target compounds 3a–3m against Gram-negative and positive sensitive bacteria

Note: a: vancomycin; b: meropenem; ND: not tested

Table 2. MIC (μg/mL) results of target compounds 4a–4i, 5a–5f against Gram-negative and positive sensitive bacteria and in vitro erythrocyte hemolytic HC ₅₀ (μg/mL) results

Note: a: vancomycin; b: meropenem; ND: not tested.

Table 3 IC ₅₀ value of compound 3f against normal cells

Table 4 MIC values of compound 3f and drugs against clinically isolated MRSA

Note: a: ciprofloxacin; b: rifampicin; c: gentamicin; d: doxycycline; e: erythromycin;

Table 5 MIC values of compound 3f and drugs against drug-resistant bacteria containing NDM-1 gene

Note: a: ciprofloxacin; b: chloramphenicol; c: amikacin; d: meropenem; e: gentamicin;

Table 6 Survival rate of mice after injection of different doses of 3f and polymyxin E

It can be seen from Table 1 and Table 2 that among the synthesized compounds 3a-3m, 4a-4i and 5a-5f, some compounds (such as 3a, 3f, 3j, 3k, 4g, 4h, 4j, 5f) are Gram-positive Bacteria Staphylococcus aureus (S.aureus) and Enterococcus faecalis (E.faecalis), Gram-negative bacteria Escherichia coli (E.coli) and Stenotrophomonas maltophilia (S.maltophilia) all exhibit The better activity shows that this type of compound has significant broad-spectrum antibacterial activity; at the same time, its in vitro red blood cell hemolysis data shows that it is less toxic and has better selectivity.

After measuring the cytotoxicity of the compound, it was found that compound 3f has low cytotoxicity, and its IC ₅₀ values for human gastric mucosal cells (GES) and human normal liver cells (LO2) are 152.737±2.184μg/mL and 51.69± respectively. 1.731μg/mL. In order to intuitively demonstrate the toxicity of compound 3f to GES cells, the state of the cells after the drug action was photographed, and the results are shown in Figure 1. After compound 3f acted on GES cells for 24 hours (Figure 1b), compared with the untreated GES cell line (Figure 1a), the cells still maintained normal morphology. In contrast, 0.1% Triton X-100-treated cells showed extensive cell damage (Fig. 1c). It can be seen that compound 3f has little cytotoxicity. At the same time, fluorescent dyes are also used to indicate the living and dead status of cells. Calcein can stain the membrane of living cells and appear green. Pyridinium iodide (PI) can stain the cell nucleus through the ruptured cell membrane, showing a red color, which can be used to represent dead cells. The results are shown in Figure 2. The Calcein staining (Figure 2a) of the blank group showed green fluorescence, and the PI staining (Figure 2b) had no red color, indicating that the cells were basically alive. Figure 2c and d show GES cells treated with compound 3f with 64×MIC S. aureus, which showed only green fluorescence like the blank group. However, GES cells treated with 0.1% Triton X-100 only showed red fluorescence, indicating that the cells were dead. Therefore, compound 3f has no obvious toxicity to GES cells at the tested concentration.

As can be seen from Tables 4 and 5, compound 3f, which has high antibacterial activity, low hemolytic toxicity and low cytotoxicity, shows good antibacterial activity against both MRSA and NDM-1-producing clinical strains. Other commercially available antibiotics have shown varying degrees of resistance.

In addition, taking compound 3f of the present invention as an example, the stability of this type of compound in different body fluids was tested. In this experiment, vancomycin was selected as a positive antibiotic control to compare the stability and bactericidal activity of compound 3f with vancomycin in body fluids. The experimental results are shown in Figure 3. The compound, like vancomycin, does not affect its antibacterial activity after interacting with plasma for a period of time (the MIC value does not change, 0.5 μg/mL), indicating that the compound has good stability in plasma. sex. In different body fluids, especially in 50% plasma, the MBC values of the compound and vancomycin increased compared with the blank group, which shows that plasma has a certain impact on the bactericidal ability of compound 3f, which is also consistent with the control drug vancomycin. Consistent. In summary, compound 3f can maintain good stability in plasma and body fluids.

In order to verify the bactericidal speed of compound 3f, the bacteria after drug action for different times were counted and a line chart was obtained, as shown in Figure 4. It can be seen from Figure 4a and b that the concentration of S. aureus in the early stage of logarithmic growth is about 10 ⁵ CFU/mL. Under the action of compound 3f (6×MIC, 3μg/mL), the bacteria can be killed to the detection level in 4 hours. below the concentration limit. Vancomycin (6×MIC, 6μg/mL) requires 12h. The concentration of S. aureus at the end of logarithmic growth phase is about 10 ⁸ CFU/mL. Under the action of compound 3f (6×MIC, 3 μg/mL), the bacteria can be killed to a concentration below the detection limit in 6 hours. However, vancomycin (6×MIC, 6 μg/mL) left about 10 ³ CFU/mL of bacteria that were not completely killed at 12 hours. Overall, compound 3f showed rapid bactericidal effect on S. aureus at the early and late growth stages, and its bactericidal effect and bactericidal time were better than those of vancomycin. It can be seen from Figure 4c and d that the concentration of E.coli in the early stage of logarithmic growth is about 10 ⁶ CFU/mL. Under the action of compound 3f (6×MIC, 3μg/mL), it takes 6 hours to kill the bacteria. The concentration is below the detection limit. On the other hand, it takes 12 hours for Laoxocephalosporin (6MIC, 6μg/mL) to complete the killing of bacteria. The concentration of E.coli at the end of logarithmic growth phase is about 10 ⁸ CFU/mL. Under the action of compound 3f (6×MIC, 3 μg/mL), the bacteria can be killed to a concentration below the detection limit in 8 hours. On the other hand, the remaining bacteria with Laoxan Cephalosporin (6×MIC, 6 μg/mL) at 12 hours were about 10 ⁴ CFU/mL and were not completely killed. Overall, compound 3f showed a rapid bactericidal effect on E.coli in both the early and late stages of growth, and its bactericidal effect was better than that of oxycephalosporin in terms of sterilization time.

Taking compound 3f of the present invention as an example, the action mechanism of this type of antibacterial peptide mimetic was studied. From the cell membrane depolarization experiments of Staphylococcus aureus and Escherichia coli in Figure 5, it can be seen that compound 3f has good cell membrane depolarization ability. In Figure 6, the influence of different components of exogenous bacterial cell membrane (wall) on the MIC value of the compound can be seen from the experiment. Whether it is for Gram-positive bacteria or Gram-negative bacteria, the addition of exogenous PG makes the compound The MIC value of bacteria produced a concentration-dependent increase. This may be due to the competitive interaction of exogenous PG with the compound, causing the compound to no longer bind to PG on the bacterial cell membrane and exert an antibacterial effect, proving that this type of The target of the compound may be the PG component on the bacterial cell membrane. In Figure 7, it can be seen from the experiment that different concentrations of compound 3f affect the production of bacterial reactive oxygen species. Compound 3f stimulates bacteria to produce ROS, regardless of whether it is Gram-positive bacteria or Gram-negative bacteria. At the MIC concentration, the ROS level is significant. Increased to about 3 times the normal level, and is concentration-dependent below the MIC concentration. The decrease in fluorescence intensity at concentrations above the MIC level may be attributed to the killing effect of high concentrations of compounds on bacteria, resulting in Some bacteria die and ROS production is relatively reduced. When the compound at the MIC concentration was used together with the ROS quencher (NAC, N-acetyl-L-cysteine), it was clearly observed that the bacterial ROS level returned to around the normal level again. Therefore, it can be inferred that this type of compound will stimulate bacteria to produce endogenous ROS, and then further aggravate membrane damage through the accumulation of ROS, eventually leading to the death of bacteria. In Figure 8, the SEM imaging results after the compound acts on S. aureus and E. coli can intuitively see the damage of compound 3f to bacterial morphology, especially the damage to the cell membrane. In Figure 9, the nucleic acid leakage experiment after the compound acts on bacteria can be seen that this type of compound, like the control drug Melittin, can cause the leakage of nucleic acid in bacterial intracellular substances at a certain concentration, and has a concentration-dependent relationship. This may further accelerate the death of the bacteria and further confirms the compound's damaging effect on the outer membrane of the bacterial cell.

In addition, compound 3f of the present invention was used as an example to study the tendency of this type of antimicrobial peptide mimetics to induce resistance. The results are shown in Figure 10. As the number of culture generations increases, the MIC value of compound 3f remains stable for both Gram-positive and Gram-negative bacteria, while the MIC values of the control antibiotics norfloxacin and colistin will increase and produce a certain degree of drug resistance. The results proved that compound 3f is not easy to induce bacterial resistance and is significantly better than the control antibiotics norfloxacin and colistin.

In order to further evaluate the toxicity of representative compound 3f, acute toxicity experiments in mice were performed. The results of intraperitoneal injection of different concentrations of compound 3f on the survival rate of mice are shown in Table 6. The changes in blood routine and blood biochemical indicators of mice after injection of the maximum tolerated dose are shown in Figure 11. The HE staining results of important organs of mice after maximum tolerated dose injection are shown in Figure 12. The final results showed that compound 3f did not cause death in mice at a concentration of 20mg/kg, which was better than the control antibiotic polymyxin E. The results of mouse blood routine, blood biochemistry and pathological sections also proved that compound 3f was at the maximum tolerated dose. There is almost no toxicity in the body.

Claims (3)

1. A tricationic quaternary ammonium salt antimicrobial peptide mimetic, characterized in that the compound has the following general formula I structure: Specifically the following compounds: 3a: m=3, n=7; 3f: m=4, n=7; 3j: m=5, n=5; 3k: m=5, n=7. 2. Tricationic quaternary ammonium salt antimicrobial peptide mimetic, characterized in that the compound has the following general formula II structure: Specifically the following compounds: 4g: m=5, n=3; 4h: m=5, n=4; 4i: m=5, n=5. 3. Tricationic quaternary ammonium salt antimicrobial peptide mimetic, characterized in that the compound has the following general formula III structure: Specifically, it is the following compound: 5f: m=5, n=4.