Bisphenol fluorene antibacterial peptide simulant containing quaternary amine cations as well as preparation method and application of bisphenol fluorene antibacterial peptide simulant

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a bisphenol fluorene antibacterial peptide simulant containing quaternary amine cations as well as a preparation method and application of the bisphenol fluorene antibacterial peptide simulant. The simulant is a compound with a structure as shown in a formula (I) or a pharmaceutically acceptable salt of the compound: # imgabs0 # formula (I). The preparation method of the compound is simple, effective and high in yield. An in-vitro activity result proves that the compound of the formula (I) has the characteristics of broad-spectrum antibacterial activity, rapid sterilization, difficulty in generating drug resistance to bacteria and the like. Therefore, the compound is expected to be developed into a novel broad-spectrum antibacterial agent to resist bacterial infection.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a bisphenol fluorene-based antimicrobial peptide mimic containing quaternary ammonium cations, and a preparation method and use thereof. Background Art

[0003] To combat the problem of bacterial resistance, it is necessary not only to modify existing drugs to develop new antibacterial drugs, but also to design new structures. Antimicrobial peptides (AMPs) have been widely recognized as a host defense system in various animals, plants, and bacteria. In recent years, AMPs have attracted extensive attention due to their unique antibacterial mechanism and broad-spectrum antibacterial activity, and are expected to become a powerful weapon to solve the problem of drug-resistant microbial infections. However, they also have some disadvantages, including unknown in vivo toxicity and low stability, which severely limit their clinical applications. Summary of the Invention

[0004] Aiming at the deficiencies and needs of the existing technology, the purpose of the present invention is to provide a bisphenol fluorene-based antimicrobial peptide mimic containing quaternary ammonium cations and a preparation method thereof, which are used for treating infectious diseases caused by bacterial infections.

[0005] In the first aspect of the present invention, there is provided a bisphenol fluorene-based antimicrobial peptide mimic containing quaternary ammonium cations, which is a compound having the structure shown in formula (I) or a pharmaceutically acceptable salt of the compound: Formula (I).

[0006] In the second aspect of the present invention, there is provided a method for preparing the bisphenol fluorene-based antimicrobial peptide mimic containing quaternary ammonium cations, which method comprises the following steps: (1) Add bisphenol fluorene to a reaction flask containing a potassium carbonate acetone solution, then slowly add 1,2-dibromoethane, heat up to 58 °C, and reflux and stir the reaction under nitrogen protection to generate intermediate 1; the molar ratio of bisphenol fluorene, 1,2-dibromoethane, and potassium carbonate is 1:10-15:3-5; (2) Mix intermediate 1 with N,N N,N-dimethylhexylamine in a molar ratio of 1:4-8 in a pressure-resistant bottle containing acetonitrile, and carry out the reaction at 85 °C to obtain the compound of formula (I).

[0007] In other aspects of the present invention, there is also provided the use of the bisphenol fluorene-based antimicrobial peptide mimic containing quaternary ammonium cations in the preparation of antibacterial drugs.

[0008] Preferably, the antibacterial agent is a drug that inhibits the growth of Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pyogenes) or Gram-negative bacteria (Acinetobacter baumannii, Salmonella typhimurium, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa).

[0009] In other aspects of the present invention, a broad-spectrum antibacterial agent is also provided, which contains an effective dose of a bisphenol fluorene antibacterial peptide mimic containing a quaternary ammonium cation.

[0010] The present invention has the following beneficial effects: The compound represented by formula (I) and its pharmaceutically acceptable salts provided by the present invention have excellent antibacterial activities against both Gram-positive bacteria (minimum inhibitory concentration MIC = 0.5 - 2 μg / mL) and Gram-negative bacteria (MIC = 2 - 8 μg / mL). It has good water solubility, shows low cytotoxicity to mammals, and can achieve rapid sterilization without generating drug resistance and other advantages. Therefore, this compound can provide a new design idea for the development of novel antibacterial agents for the treatment of drug-resistant bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the time-kill curve of the compound prepared according to the present invention; Figure 2 is the drug resistance evaluation of the compound prepared according to the present invention; Figure 3 is the in vitro cytotoxicity evaluation of the compound prepared according to the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0012] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0013] Example 1 Preparation of a bisphenol fluorene antibacterial peptide mimic containing a quaternary ammonium cation. The synthesis route is as follows: .

[0014] The reagents and reaction conditions used in the synthesis route are as follows: a. 1,2-dibromoethane, potassium carbonate, acetone, reflux; b. N,N-dimethylhexylamine, acetonitrile, reflux.

[0015] The specific preparation process includes the following steps: (1) Preparation of 9,9-bis(4-(2-bromoethoxy)phenyl)-9H-fluorene (Intermediate 1) Bisphenol fluorene (10 g, 28.53 mmol) and 1,2-dibromoethane (80.4 g, 427.95 mmol) were successively dissolved in 200 mL of acetone, and then potassium carbonate (19.71 g, 142.65 mmol) was added. The mixture was stirred at 57 °C for 48 h. The reaction was monitored by TLC and found to be complete. After suction filtration, the solvent was evaporated to dryness to obtain the crude product, which was then purified by column chromatography using petroleum ether:ethyl acetate = 30:1 as the eluent, yielding 1.1 g of a white solid product with a yield of 13.6%.

[0016] (2) N,N' -((((9H-Fluorene-9,9-diyl)bis(4,1-phenylene))bis(oxy))bis(ethane-2,1-diyl))bis( N,N -dimethylhexan-1-aminium) bromide (the compound shown in formula (I)) Intermediate 1 (0.25 g, 0.44 mmol) and N,N-dimethylhexylamine (0.34 g, 2.64 mmol) were dissolved in a pressure-resistant bottle containing 6 mL of anhydrous acetonitrile and reacted continuously at 85 °C for 24 h. The reaction was monitored by TLC until the raw materials were completely reacted. The system was cooled to room temperature and then concentrated by vacuum evaporation to obtain the crude product. Excess ethyl acetate was added to the crude product, and a solid precipitated after standing. After filtration and vacuum drying, 0.27 g of a white solid was obtained with a yield of 92.7%. HRMS (ESI) C 45 H 62 Br 2 N 2 O 2 [M−2Br] / 2 + calcd = 331.2400; found = 371.3154.

[0017] Example 2. In vitro antibacterial activity assay of the compound shown in formula (I) Experimental method: According to the standards of the Clinical and Laboratory Standards Institute (CLSI) of the United States, the MIC value of the compound shown in formula (I) was detected by the microbroth dilution method. The compound was dissolved in DMSO or water and then diluted in MH medium to prepare a stock solution. The compound was serially diluted two-fold with MH medium (100 μL) to obtain concentrations of 0.25 to 128 μg / mL in a 96-well plate. A monoclonal colony on the plate was picked and suspended in MH medium, and then the bacterial solution was adjusted to about 1×10 6CFU / mL. 100 μL of the bacterial suspension was added to a 96-well plate containing serially two-fold diluted compounds and incubated at 37 °C for 18 - 24 h. Vancomycin, sodium oxacillin, and colistin were used as positive control drugs, a drug-free bacterial solution (200 μL) was used as the negative control, and MH medium (200 μL) was used as the blank control. Finally, the growth of bacteria in each well was observed, and the lowest concentration of the compound that completely inhibited bacterial growth was set as the MIC value. The above experiments were effectively repeated 3 times.

[0018] Table 1. In vitro antibacterial activity of the compounds shown by formula (I) (unit μg / mL) ;

[0019] Note: a S.a. : Staphylococcus aureus ATCC 12600, b MRSA: Staphylococcus aureus ATCC 43300, c E.f. : Enterococcus faecalis ATCC 29212, d S.p. : Streptococcus pyogenes ATCC 12344, e A.b. : Acinetobacter baumannii ATCC19606, f S.e. Salmonella typhimurium SL1344, g K.p. : Klebsiella pneumoniae ATCC 13883, h E.c. : Escherichia coli UPEC, i P.a. : Pseudomonas aeruginosa PAO1, j ND: Not determined. As can be seen from Table 1, the compounds shown by formula (I) exhibited excellent antibacterial activity against both Gram-positive and Gram-negative bacteria. Its minimum inhibitory concentration range against Gram-positive bacteria was 0.5 - 2 μg / mL, and the minimum inhibitory concentration range against Gram-negative bacteria was 2 - 8 μg / mL.

[0020] Example 3. Time-kill kinetic test of the compounds shown by formula (I) A single colony of Staphylococcus aureus ATCC 43300 was selected and placed in 1 mL of MHB, incubated in a shaker (200 rpm, 37 °C) for 16 - 18 h, and then the bacterial cell concentration was adjusted to 1 × 10 6CFU / mL. Subsequently, different concentrations of the compound of formula (I) (1 ×, 2 ×, 4 ×, 8 × MIC) were added to the bacterial solution, and incubation was continued at 37°C. Colony counts were determined at 0, 0.5, 1, 2, 4, 8, and 24 h. Vancomycin (4 ×, 8 × MIC) was used as a positive control drug, and no drug was added as a blank control. The experimental results are as Figure 1 shown.

[0021] The time-kill kinetics of the compound shown in formula (1) against Staphylococcus aureus ATCC 43300 was determined using the plate colony counting method. First, the bacteria were cultured in TSB medium at 37°C and 200 rpm for 16 - 18 h, and diluted with MH medium to a final concentration of 5×10 5 CFU / mL for use. Different concentrations of the compound shown in formula (1) (1 × MIC, 2 × MIC, 4 × MIC, 8 × MIC) and vancomycin (4 × MIC, 8 × MIC) were added to the bacterial solution. At 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h of culture, 20 μL of the bacterial solution was taken out, serially diluted 10-fold in a 96-well plate with sterile PBS, then the bacterial solution was placed on a TSB solid plate, spread using a sterile spreading rod, and incubated at 37°C for 16 - 18 h. The bacterial solution without drug treatment was used as a blank control, and each group was repeated 3 times.

[0022] The results are as Figure 1 shown. The compound shown in formula (1) can completely kill Staphylococcus aureus ATCC 43300 within 4 h at a concentration of 4 × MIC, and only needs 0.5 h to completely kill Staphylococcus aureus ATCC 43300 at a concentration of 8 × MIC. However, the positive control vancomycin requires at least 12 h to completely kill the bacteria at both 4 × MIC and 8 × MIC concentrations. The results show that compared with the traditional antibiotic vancomycin, the compound shown in formula (1) can rapidly kill bacteria, and has time- and concentration-dependence, shortening the treatment time of bacterial infection, which can effectively avoid the development of bacterial drug resistance.

[0023] Example 4. Resistance study of the compound of formula (I) The MIC values of the compound of formula (I) and norfloxacin against Staphylococcus aureus ATCC 43300 were determined according to the method described in Example 2. Then, the bacteria incubated with the compound at a sub-inhibitory concentration (1 / 2 MIC) were prepared into a bacterial suspension for the next MIC determination. Incubation was carried out at 37°C for 24 h, and the new MIC value was determined. This experiment was continuously repeated for 24 days.

[0024] The results are asFigure 2 As shown, the MIC value of the compound shown in formula (I) did not change after 24 consecutive passages, indicating that the compound shown in formula (I) is not easily induced to develop drug resistance in Staphylococcus aureus ATCC 43300. In contrast, the positive control norfloxacin rapidly induced drug resistance in Staphylococcus aureus ATCC 43300 within a short period of time. After the 24th passage, its MIC value increased by 64-fold. The results showed that the compound shown in formula (I) is a potential highly effective bactericide, with a rapid bactericidal effect and antibacterial drug resistance. At the same time, different from traditional antibiotics that act on specific targets and are prone to drug resistance, the compound shown in formula (I) may have a mechanism of action similar to that of antimicrobial peptides, and can achieve a bactericidal effect by disrupting the bacterial cell membrane. The experimental results are as Figure 2 shown.

[0025] Example 5. In vitro cytotoxicity experiment of the compound shown in formula (I) The in vitro cytotoxicity of the compound shown in formula (1) was determined using human hepatoma cells (Hep3B), human renal carcinoma cells (Hek292), and mouse hippocampal neuron cells (HT22). First, the cells were evenly seeded in a 96-well plate and incubated overnight in an incubator at 37°C containing 5% CO 2 ₂. A series of gradient concentrations of the compound shown in formula (1) (128 - 0.25 μg / mL) were added thereto. The 96-well plate was treated in an incubator at 37°C containing 5% CO 2 ₂ for 24 h. Then, 10 μL of CCK-8 reagent was added to each well and incubated for 1 h. The absorbance of each well at 450 nm was measured using a microplate reader. The cell suspension plus CCK-8 reagent was used as the positive control, and the culture medium plus CCK-8 reagent was used as the negative control. The experiment was repeated at least twice to achieve the reproducibility of biological experiments.

[0026] The results are as Figure 3 shown. At a concentration of 8 μg / mL (16 × MIC), the survival rates of the three types of cells, Hep3B, Hek293, and HT22, were 76.92 ± 1.37%, 73.21 ± 5.00%, and 82.11 ± 5.29%, respectively, showing low cytotoxicity. And the concentration of 8 μg / mL is much higher than the MIC value of the compound against Staphylococcus aureus ATCC43300. Considering its good antibacterial activity, it indicates that the compound shown in formula (1) has low cytotoxicity to mammalian cells.

Claims

1. A bisphenol fluorene antimicrobial peptide mimetic containing a quaternary ammonium cation, characterized in that: The mimetic is a compound having a structure shown in formula (I) or a pharmaceutically acceptable salt of the compound: Formula (I).

2. A method for preparing the bisphenol fluorene antimicrobial peptide mimetic containing quaternary ammonium cations according to claim 1, characterized in that: The following steps are involved: (1) Add bisphenol fluorene to a reaction bottle containing a potassium carbonate acetone solution, then slowly add 1,2-dibromoethane, raise the temperature to 58°C, and reflux and stir under nitrogen protection to react to generate intermediate 1; the molar ratio of bisphenol fluorene, 1,2-dibromoethane and potassium carbonate is 1:10-15:3-5; (2) Intermediate 1 and N,N -Dimethylhexylamine is mixed in a pressure bottle containing acetonitrile at a molar ratio of 1:4-8, and reacted at 85°C to obtain the compound of formula (I).

3. Use of the bisphenol fluorene antimicrobial peptide mimetic containing quaternary ammonium cations according to claim 1 in the preparation of antibacterial drugs.

4. The use according to claim 3, characterized in that: The antibacterial drug is a drug that inhibits Gram-positive bacteria and Gram-negative bacteria.

5. A broad-spectrum antibacterial agent, characterized in that: The invention comprises an effective dose of the bisphenol fluorene antimicrobial peptide mimetic according to claim 1.

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