CN108558682B - Aromatic phenol quaternary ammonium salt antibacterial peptide mimic with antibacterial activity and preparation method thereof - Google Patents
Aromatic phenol quaternary ammonium salt antibacterial peptide mimic with antibacterial activity and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of pharmaceutical chemistry, and discloses an aromatic phenol antibacterial peptide mimic with anti-drug-resistant bacteria activity and no obvious toxicity and a preparation method thereof. The invention obtains the target product through 3-4 steps of reaction, and the main structure is shown as follows. In-vitro antibacterial activity experiments prove that most of compounds in the series have good activity on gram-positive bacteria staphylococcus aureus, 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 medicine has low toxicity and good selectivity. Some compounds also show excellent antibacterial activity against "superbacteria" including methicillin-resistant staphylococcus aureus (MRSA), clinical strains producing NDM-1 and KPC-2 enzymes, and the like. Therefore, the series of compounds are expected to be used as new antibacterial candidate drugs.
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Description
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry, and discloses an aromatic phenol quaternary ammonium salt antibacterial peptide mimic with anti-drug-resistant bacteria activity and no obvious toxicity and a preparation method thereof.
Background
The discovery and wide use of antibiotics have the significance of being unmixable in the development process of human civilization, and are divided into aminoglycosides, tetracyclines, chloramphenics, macrolides and lincomycins which influence the synthesis of bacterial proteins according to different mechanisms; beta-lactams that interfere with bacterial cell wall synthesis; polymyxins that damage bacterial cell membranes and sulfonamides and trimethoprim that interfere with folate metabolism; quinolones which affect the metabolism of nucleic acids, and the like. However, due to the non-standard use of antibiotics, severe bacterial resistance problems arise, and even cross-resistance and multi-resistance are prevalent. Bacteria are confronted with antibiotics in order to survive under the selective pressure of antibiotics through various resistance mechanisms, which are mainly classified into the following groups: affecting the cell membrane osmosis and further hindering the entry of antibacterial drugs; producing inactivated enzymes or inactivating enzymes to destroy antibiotic activity; accelerating the active efflux of bacteria and discharging antibiotics in the bacteria; changing the target of antibiotic action, etc. (NatureReviews Microbiology,2015,13, 42-51). Such as methicillin-resistant staphylococcus aureus (MRSA), by producing beta-lactamase enzymes that hydrolyze the beta-lactam ring in the penicillin structure, thereby rendering it non-antibacterial. Vancomycin also appears to have a tendency to resist drugs as a "last line of defense" against gram-positive bacteria (Science,2008,321, 356-. Therefore, there is an urgent need to develop novel antibacterial agents effective against drug-resistant bacteria.
In order to solve the problem of bacterial resistance, researchers made many attempts, and Boman, a swedish scientist, first discovered in 1981, that a polypeptide substance with antibacterial activity, cecropin, was produced by inducing Japanese silkworm to bear the problem with Bacillus cereus (Nature,198l,292, 246-. Subsequently, a variety of natural antimicrobial peptides have been developed. The research shows that the antibacterial peptide not only has good antibacterial activity, but also the bacteria hardly have drug resistance to the antibacterial peptide. The antibacterial peptide is mainly combined with anions on the surface of the bacterial membrane through positively charged groups, and hydrophobic groups penetrate into the bacterial membrane to destroy the integrity of the bacterial membrane, so that the aim of killing bacteria is fulfilled. However, the natural antibacterial peptide has the disadvantages of high production cost, incapability of batch production, easy degradation in vivo, high toxicity of part of the antibacterial peptide, weak selectivity and the like. There are many researchers expecting to synthesize antimicrobial peptide mimetics to overcome the above-mentioned disadvantages based on the amphiphilic structural characteristics of natural antimicrobial peptides. Like the Jianfengcai project group, a series of compounds with hydrophobic alkane chains and positive charges are synthesized by taking nitrofurantoin as a mother nucleus, and the compound shows good activity (Journal of medical chemistry,2017,60, 8456-8465); further, a series of symmetric quaternary ammonium compounds designed and synthesized as in the JayantaHaldar project group can be used to adjust the activity and toxicity by changing the lengths of the intermediate alkane and side chain, so as to obtain compounds with good activity and low toxicity (Journal of medical chemistry,2016,59, 10750-10762).
Through extensive literature analysis, we hope to take advantage of the structural features of antimicrobial peptides: the compound has positive charges and hydrophobic alkane chains, a series of aromatic phenol antibacterial peptide mimics are designed and synthesized, the antibacterial activity of the aromatic phenol antibacterial peptide mimics is verified through in vitro activity experiments, and the compound with good activity, low toxicity and anti-drug resistance can be expected to be obtained.
Disclosure of Invention
The invention aims to provide a series of aromatic phenol quaternary ammonium salt antibacterial peptide mimics which have broad-spectrum anti-drug-resistance activity and no obvious toxicity, and are beneficial to research and development of new antibacterial drugs; another object is to provide a process for the preparation thereof.
In order to realize the purpose of the invention, the technical scheme is as follows:
the compound has the following structural general formulas I-III:
is characterized in that: the structure takes p-hydroxyphenol as an intermediate connecting part, and the intermediate chain length n is 2,3, 4 and 5; the two-sided chain length q is 5, 7, 9, 11. Namely the following compounds:
2a：n＝2，q＝5；
2b：n＝2，q＝7；
2c：n＝2，q＝9；
2d：n＝2，q＝11；
2e：n＝3，q＝5；
2f：n＝3，q＝7；
2g：n＝3，q＝9；
2h：n＝3，q＝11；
2i：n＝4，q＝5；
2j：n＝4，q＝7；
2k：n＝4，q＝9；
2l：n＝4，q＝11；
2m：n＝5，q＝5；
2n：n＝5，q＝7；
2o：n＝5，q＝9；
2p：n＝5，q＝11。
is characterized in that: the structure takes phenol as a parent nucleus, the middle chain length is 3, and the side chain length q is 5, 7, 9 and 11. Namely the following compounds:
3a：q＝5；
3b：q＝7；
3c：q＝9；
3d：q＝11；
is characterized in that: the structure takes 1, 6-dihydroxynaphthalene or 2, 3-dihydroxynaphthalene as an intermediate connecting part, and the intermediate chain length is 3; when 1, 6-dihydroxynaphthalene is the middle part, the chain length q on both sides is 5, 7, 9; when 2, 3-dihydroxynaphthalene is the middle moiety, the chain length q on both sides is 7, 9.
1, 6-dihydroxynaphthalene:
3e：q＝5；
3f：q＝7；
3g：q＝9。
2, 3-dihydroxynaphthalene:
3h：q＝7；
3i：q＝9。
the cationic antibacterial peptide mimics (2a-2p, 3a-3i) of the invention were synthesized as follows:
reaction conditions are as follows: a) carbon tetrabromide, triphenylphosphine, acetonitrile, room temperature; b) refluxing dibromoalkane, potassium carbonate and acetone;
route preparation of intermediates 1a-1f
c) Ethanol, high-temperature high-pressure reaction at 85-90 ℃;
preparation of the final product 2a-2p, 3a-3i of route two
The method is realized by the following steps:
(1)1, 4-bis (2-hydroxyethoxy) benzene is put in acetonitrile solution to generate a compound 1a under the action of carbon tetrabromide and triphenylphosphine; in potassium carbonate and acetone solution, hydroquinone or 1, 6-dihydroxynaphthalene or 2, 3-dihydroxynaphthalene and dibromoalkane are heated, refluxed and stirred under the protection of nitrogen to generate a disubstituted reaction to generate a compound 1b-1 f.
(2) The compounds 1a-1f and N, N-dimethyl alkane tertiary amine react in ethanol in a reaction kettle at the temperature of 85-90 ℃ to obtain a series of target compounds 2a-2p, 3a-3 i.
The invention mainly takes phenol as an intermediate connecting part, and is used for researching the influence of different side chains and intermediate alkane chain lengths on the activity and toxicity by changing the lengths of the intermediate alkane chain and the side chains. Meanwhile, the intermediate connecting part is also changed, hydroquinone is changed into 2, 3-dihydroxynaphthalene or 1, 6-dihydroxynaphthalene, and the activity of the intermediate connecting part is evaluated.
The experiment proves that: the novel aromatic phenol antibacterial peptide mimic disclosed by the invention shows good activity on gram-positive bacteria and gram-negative bacteria, and the series of compounds have excellent antibacterial activity. Particularly, the MIC results of the compounds 2b, 2f, 2j, 2n, 3f and 3h on staphylococcus aureus, escherichia coli, enterococcus faecalis and stenotrophomonas maltophilia are between 0.25 and 4 mu g/mL, and the MIC results of part of the compounds are obviously superior to those of a positive control vancomycin. 6 Compounds HC mentioned above50The results are all more than 300 mug/mL, which shows no obvious toxicity and high selectivity. Therefore, the novel aromatic phenol antibacterial peptide mimics provided by the invention are expected to be used as new antibacterial candidate drugs for deep research and have important significance for solving the problem of drug-resistant bacteria facing the world at present.
Drawings
FIG. 1 is a graph showing the MBC values of Compound 2n of the present invention against Staphylococcus aureus in plasma for various periods of time (a) and the MBC values of Compound 2n against methicillin-resistant Staphylococcus aureus in various body fluids (b).
FIG. 2 shows the number of bacteria surviving in E.coli at various concentrations of compound 2n according to the invention.
FIG. 3 shows the bactericidal rate (a) of compound 2n of the present invention against E.coli cultured for 2 hours; the sterilization rate of the compound 2n on Escherichia coli cultured for 5h (b); the clarity (c) of the bacterial liquid after the compound 2n acts on the escherichia coli cultured for 2h for 6 h; and (d) the clarity of the bacterial liquid after the compound 2n acts on the escherichia coli cultured for 5h for 24 h.
FIG. 4 is a mechanism of action of the compounds of the present invention. In the figure, (a) and (b) are experiments of cell membrane depolarization of staphylococcus aureus and escherichia coli, and the concentration is 10 mug/mL; (c) and (d) a permeabilization test of the inner membrane of Staphylococcus aureus and Escherichia coli at a concentration of 10. mu.g/mL; (e) for the E.coli outer membrane permeabilization test, the concentration was 10. mu.g/mL.
FIG. 5 is a photomicrograph of Hela cells after 24 hours of action of Compound 2n of the present invention; in the figure, (a) is no drug (negative control); (b) the concentration of the 2n medicine is 16 mug/mL; (c) the concentration of the 2n medicine is 4 mug/mL; (d) HeLa cells were treated with 0.1% Triton X (positive control) on a 10 μm scale.
FIG. 6 is a fluorescent photograph of Hela cells stained with calcein and pyridine iodide after 24 hours of the action of compound 2n of the present invention. (a-c) no drug (negative control); (d-f) the concentration of the 2n drug is 32 mug/mL; (g-i) the concentration of 2n of the drug was 4. mu.g/mL. (j-l) Hela cells were treated with 0.1% Triton X (positive control).
Detailed Description
The invention will be further illustrated with reference to the following specific examples. These examples are intended to be illustrative of the invention only and are not intended to limit the scope of the invention as claimed.
Characterization of the synthesized compounds the instrument used: NMR spectra were measured using a Bruker DPX-400 model superconducting nuclear magnetic resonance apparatus, Sweden, with TMS as an internal standard; high resolution mass spectra were determined using a Waters-Micromass Q-Tof mass spectrometer.
EXAMPLE 1 preparation of Compound 1a
In a round-bottomed flask (500mL), 1, 4-bis (2-hydroxyethoxy) benzene (5g, 25mmol) and triphenylphosphine (15.7g, 60mmol) were weighed out and dissolved in anhydrous acetonitrile (120mL), and carbon tetrabromide (19.9g, 60mmol) was slowly added to the system while maintaining the temperature at 0 ℃. Then the temperature is raised to 25 ℃ and the mixture is stirred for 4 hours under the protection of nitrogen. After the reaction was completed, ice water (200mL) was added to the system to precipitate a product, and the solid was filtered and washed 3-4 times with methanol/water (3:2, 3X 100 mL). The crude product is recrystallized in methanol for further purification to obtain a pure product.
1a:1H NMR(400MHz,CDCl3)6.86(s,4H),4.24(t,J＝6.3Hz,4H),3.61(t,J＝6.3Hz,4H).13C NMR(101MHz,CDCl3)152.85,116.12,68.74,29.25.
EXAMPLE 2 preparation of Compound 1b
In a round bottom flask (250mL), hydroquinone (8g, 72.65mmol, 1eq) was dissolved in acetone (150mL), dibromopropane (44g, 217.96mmol, 3eq) and potassium carbonate (45.18g, 326.94mmol, 4.5eq) were added and stirred to dissolve the compound, magnetons were added and the mixture was heated under reflux for 24 hours under nitrogen. After the reaction is finished, the system is cooled to room temperature, filtered, and filter residues are washed with dichloromethane for 3-4 times. The filtrate was washed with water 3-4 times, then with saturated aqueous sodium chloride solution 3-4 times, and finally the solution was dried over anhydrous sodium sulfate, filtered, evaporated to dryness with a rotary evaporator, and separated with a silica gel column (petroleum ether: ethyl acetate: 30:1) to give a white solid.
1b:1H NMR(400MHz,CDCl3)6.84(s,4H),4.05(t,J＝5.8Hz,4H),3.60(t,J＝6.5Hz,4H),2.33–2.25(m,4H).13C NMR(101MHz,CDCl3)153.09,115.60,66.05,32.49,30.08.
Example 3 preparation of compound 1 c: the dibromoalkane used was dibromobutane and was prepared by silica gel column separation (petroleum ether: ethyl acetate: 30:1) as in example 2.
1c:1H NMR(400MHz,CDCl3)6.81(s,4H),3.93(t,J＝6.1Hz,4H),3.48(t,J＝6.7Hz,4H),2.11–2.00(m,4H),1.96–1.83(m,4H).13C NMR(101MHz,CDCl3)153.11,115.44,77.41,77.09,76.77,67.49,33.55,29.54,28.03.
Example 4 preparation of compound 1 d: the dibromoalkane used was dibromopentane, which was prepared by silica gel column separation (petroleum ether: ethyl acetate: 30:1) in the same manner as in example 2.
1d:1H NMR(400MHz,CDCl3)6.84(s,4H),3.94(t,J＝6.3Hz,4H),3.46(t,J＝6.8Hz,4H),2.01–1.91(m,4H),1.86–1.77(m,4H),1.68–1.59(m,4H).13C NMR(101MHz,CDCl3)152.13,114.43,67.21,32.59,31.50,27.53,23.84.
Example 5 preparation of compound 1 e: the raw materials used were dibromopropane and 1, 6-dihydroxynaphthalene, and the preparation was performed by silica gel column separation (petroleum ether: ethyl acetate: 200:1) as in example 2.
1e:1H NMR(400MHz,CDCl3)9.09(d,J＝9.9Hz,1H),8.32–8.24(m,2H),8.11-8.00(m,2H),7.65(d,J＝6.4Hz,1H),5.21(t,J＝5.7Hz,2H),5.16(t,J＝5.8Hz,2H),4.65(t,J＝6.5Hz,2H),4.60(t,J＝6.4Hz,2H),3.40(dt,J＝12.1,6.0Hz,2H),3.33(dt,J＝12.1,6.1Hz,2H).13C NMR(101MHz,CDCl3)151.96,149.30,130.67,121.46,118.42,115.65,114.27,112.47,101.52,97.84,60.26,60.11,27.28,27.15,24.86,24.79.
Example 6 preparation of compound 1 f: the raw materials used were dibromopropane and 2, 3-dihydroxynaphthalene, and the preparation was the same as in example 2, using silica gel column separation (petroleum ether: ethyl acetate: 150: 1).
1f:1H NMR(400MHz,CDCl3)7.60(dd,J＝6.1,3.3Hz,2H),7.26(dd,J＝6.2,3.2Hz,2H),7.09(s,2H),4.18(t,J＝5.9Hz,4H),3.59(t,J＝6.4Hz,4H),2.35(p,J＝6.1Hz,4H).13CNMR(101MHz,CDCl3)148.88,129.35,126.35,124.36,108.54,66.29,32.30,30.08.
EXAMPLE 7 preparation of Compound 2a
Dissolving the intermediate 1a (300mg, 1eq) in 10mL of ethanol in a high-temperature high-pressure reaction kettle, then dissolving N, N-dimethylhexylamine (359.01mg, 3eq) in a system, screwing down the cover of the reaction kettle, heating at 85 ℃ for reaction for 24 hours, evaporating the system to dryness after the reaction is finished to obtain a viscous substance, dissolving the viscous substance by using a small amount of acetone, then adding a large amount of ethyl acetate or diethyl ether to separate out a white substance, standing in a refrigerator for a period of time to obtain a white precipitate, and if the white precipitate is not obtained, carrying out ultrasound treatment on the system for a period of time. Filtration gave a white precipitate which was washed several times with ethyl acetate or diethyl ether. Drying to obtain the final product.
2a:1H NMR(400MHz,DMSO)7.05(s,4H),4.46(s,4H),3.89–3.80(m,4H),3.54–3.43(m,4H),3.22(s,12H),1.86-1.70(m,4H),1.41-1.31(m,12H),0.94(t,J＝6.7Hz,6H).13C NMR(101MHz,DMSO)152.03,115.69,64.01,61.97,61.88,50.82,30.66,25.40,21.84,21.78,13.81.Calculated for C44H84Br2N4O4:366.3247,found:366.3256.
Example 8 preparation of compound 2 b: the starting materials used were N, N-dimethyloctylamine and intermediate 1a, and the preparation was carried out as in example 7.
2b:1H NMR(400MHz,DMSO)6.99(s,4H),4.41(s,4H),3.80(s,4H),3.52–3.39(m,4H),3.18(s,12H),1.72(s,4H),1.39–1.15(m,20H),0.87(t,J＝6.5Hz,6H).13C NMR(101MHz,DMSO)152.04,115.71,64.01,62.04,61.86,50.83,31.13,28.44,25.76,22.00,21.85,13.91.Calculated for C36H68Br2N4O4:310.2621,found:310.2621.
Example 9 preparation of compound 2 c: the starting materials used were N, N-dimethyl pelargonimine and intermediate 1a, and the preparation method was the same as in example 7.
2c:1H NMR(400MHz,DMSO)6.98(s,4H),4.43-4.34(m,4H),3.86–3.69(m,4H),3.45-3.35(m,4H),3.15(s,12H),1.79-1.63(m,4H),1.37-1.17(m,28H),0.86(t,J＝6.8Hz,6H).13C NMR(101MHz,DMSO)152.05,115.70,64.04,62.00,61.91,50.84,31.24,28.85,28.78,28.61,28.48,25.76,22.05,21.83,13.92.Calculated for C26H50Br2N2O2:211.1937,found:211.1937.
Example 10 preparation of compound 2 d: the starting materials used were N, N-dimethyldodecylamine and intermediate 1a, prepared in the same manner as in example 7.
2d:1H NMR(400MHz,DMSO)6.99(s,4H),4.47-4.33(m,4H),3.88-3.70(m,4H),3.50–3.37(m,4H),3.17(s,12H),1.80-1.60(m,4H),1.45-1.11(m,36H),0.86(t,J＝6.6Hz,6H).13C NMR(101MHz,DMSO)152.05,115.70,64.01,62.03,61.87,50.83,31.26,30.66,28.98,28.91,28.67,28.50,25.77,22.06,21.85,13.91.Calculated for C30H58Br2N2O2:239.2250,found:239.2250.
Example 11 preparation of compound 2 e: the starting materials used were N, N-dimethylhexylamine and intermediate 1b, and the preparation was carried out in the same manner as in example 7.
2e:1H NMR(400MHz,DMSO)6.91(s,4H),4.00(t,J＝5.2Hz,4H),3.58–3.42(m,4H),3.36(t,J＝7.8Hz,4H),3.11(s,12H),2.22-2.20(m,4H),1.76-1.58(m,4H),1.39-1.19(m,12H),0.88(t,J＝6.8Hz,6H).13C NMR(101MHz,DMSO)152.44,115.54,65.14,62.94,60.40,50.14,30.62,25.37,22.28,21.84,21.62,13.79.Calculated for C34H66Br2N2O2:267.2563,found:267.2561.
Example 12 preparation of compound 2 f: the starting materials used were N, N-dimethyloctylamine and intermediate 1b, prepared in the same manner as in example 7.
2f:1H NMR(400MHz,DMSO)6.90(s,4H),3.99(t,J＝5.8Hz,4H),3.57–3.40(m,4H),3.34–3.25(m,4H),3.06(s,12H),2.18-2.10(m,4H),1.80-1.54(m,4H),1.42–1.15(m,20H),0.87(t,J＝6.8Hz,6H).13C NMR(101MHz,DMSO)152.92,115.97,65.59,63.38,60.84,50.64,31.64,31.18,28.94,26.23,22.76,22.52,22.15,14.44.Calculated forC38H74Br2N2O2:295.2876,found:295.2874.
Example 13 preparation of compound 2 g: the raw materials used were N, N-dimethyl pelargonimine and intermediate 1b, and the preparation method was the same as in example 7.
2g:1H NMR(400MHz,DMSO)6.90(s,4H),3.99(t,J＝5.7Hz,4H),3.46(dd,J＝9.7,6.5Hz,4H),3.33-3.26(m,4H),3.08(s,12H),2.21–2.04(m,4H),1.75-1.59(m,4H),1.36-1.16(m,28H),0.86(t,J＝6.6Hz,6H).13C NMR(101MHz,DMSO)152.45,115.52,65.15,62.94,60.39,50.14,31.24,28.86,28.78,28.62,28.46,25.73,22.30,22.05,21.67,13.91.Calculated for C28H54Br2N2O2:225.2093,found:225.2090.
Example 14 preparation of compound 2 h: the starting materials used were N, N-dimethyldodecylamine and intermediate 1b, prepared in the same manner as in example 7.
2h:1H NMR(400MHz,DMSO)6.90(s,4H),3.99(t,J＝5.6Hz,4H),3.55–3.40(m,4H),3.35–3.29(m,4H),3.08(s,12H),2.19–2.06(m,4H),1.74-1.59(m,4H),1.36-1.16(m,36H),0.86(t,J＝6.6Hz,6H).13C NMR(101MHz,DMSO)152.45,115.52,65.15,62.93,60.37,50.14,31.26,28.98,,28.91,28.79,28.66,28.47,25.73,22.30,22.06,21.67,13.91.Calculated for C32H62Br2N2O2:253.2406,found:253.2404.
Example 15 preparation of compound 2 i: the starting materials used were N, N-dimethylhexylamine and intermediate 1c, and the preparation was carried out in the same manner as in example 7.
2i:1H NMR(400MHz,DMSO)6.88(s,4H),3.95(t,J＝5.9Hz,4H),3.37(d,J＝13.7Hz,4H),3.32–3.25(m,4H),3.04(s,12H),1.88-1.76(m,4H),1.76-1.68(m,6.0Hz,4H),1.67-1.57(m,4H),1.38-1.17(m,12H),0.87(t,J＝6.4Hz,6H).13C NMR(101MHz,DMSO)152.53,115.37,67.07,62.91,62.45,50.00,30.61,25.63,25.41,21.84,21.62,18.79,13.79.Calculated for C36H70Br2N2O2:281.2719,found:281.2717.
Example 16 preparation of compound 2 j: the starting materials used were N, N-dimethyloctylamine and intermediate 1c, and the preparation was carried out as in example 7.
2j:1H NMR(400MHz,DMSO)6.88(s,4H),3.95(t,J＝6.0Hz,4H),3.31–3.24(m,4H),3.04(s,12H),1.87–1.76(m,4H),1.75-1.68(m,4H),1.68-1.58(m,4H),1.32-1.18(m,20H),0.87(t,J＝6.8Hz,6H).13C NMR(101MHz,DMSO)157.77,120.59,72.30,68.15,67.68,55.26,36.38,33.69,33.66,31.01,30.86,27.26,26.92,24.04,19.17.Calculated forC40H78Br2N2O2:309.3032,found:309.3033.
Example 17 preparation of compound 2 k: the starting materials used were N, N-dimethyl pelargonimine and intermediate 1c, and the preparation method was the same as in example 7.
2k:1H NMR(400MHz,DMSO)6.88(s,4H),3.95(t,J＝5.9Hz,4H),3.35(t,J＝8.1Hz,4H),3.31–3.23(m,4H),3.03(s,12H),1.87-1.77(m,4H),1.77-1.67(m,4H),1.67-1.58(m,4H),1.34-1.18(m,28H),0.86(t,J＝6.7Hz,6H).13C NMR(101MHz,DMSO)152.54,115.35,67.07,62.92,62.46,50.01,31.25,28.86,28.78,28.62,28.45,25.76,25.64,22.05,21.67,18.80,13.92.Calculated for C30H58Br2N2O2:239.2250,found:239.2248.
Example 18 preparation of compound 2 l: the starting materials used were N, N-dimethyldodecylamine and intermediate 1c, prepared in the same manner as in example 7.
2l:1H NMR(400MHz,DMSO)6.87(s,4H),3.95(t,J＝5.9Hz,4H),3.34(t,J＝8.1Hz,4H),3.30–3.21(m,4H),3.02(s,12H),1.87-1.75(m,4H),1.76–1.68(m,4H),1.68-1.58(m,4H),1.33-1.17(m,36H),0.86(t,J＝6.6Hz,6H).13C NMR(101MHz,DMSO)152.54,115.35,67.07,62.93,62.47,50.02,31.26,28.97,28.90,28.77,28.66,28.45,25.76,25.64,22.05,21.66,18.81,13.92.Calculated for C34H66Br2N2O2:267.2563,found:267.2565.
Example 19 preparation of compound 2 m: the starting materials used were N, N-dimethylhexylamine and intermediate 1d, and the preparation was carried out in the same manner as in example 7.
2m:1H NMR(400MHz,DMSO)6.85(s,4H),3.92(t,J＝6.3Hz,4H),3.28(dd,J＝16.8,11.7Hz,8H),3.02(s,12H),1.81–1.68(m,8H),1.68-1.58(m,4H),1.48–1.36(m,4H),1.36-1.22(m,12H),0.88(t,J＝6.6Hz,6H).13C NMR(101MHz,DMSO)152.59,115.28,67.52,62.97,62.81,49.92,30.62,28.21,25.42,22.53,21.84,21.63,21.48,13.78.Calculatedfor C38H74Br2N2O2:295.2876,found:295.2876.
Example 20 preparation of compound 2 n: the starting materials used were N, N-dimethyloctylamine and intermediate 1d, prepared in the same manner as in example 7.
2n:1H NMR(400MHz,DMSO)6.85(s,4H),3.91(t,J＝6.3Hz,4H),3.26(dd,J＝17.0,12.4Hz,8H),3.01(s,12H),1.83–1.67(m,8H),1.67–1.59(m,4H),1.49–1.36(m,4H),1.36–1.18(m,20H),0.87(t,J＝6.9Hz,6H).13C NMR(101MHz,DMSO)152.60,115.28,67.53,62.96,62.79,49.93,31.13,28.43,28.41,28.21,25.78,22.53,22.01,21.69,21.50,13.91.Calculated for C42H82Br2N2O2:323.3189,found:323.3189.
Example 21 preparation of compound 2 o: the raw materials used were N, N-dimethyl pelargonimine and intermediate 1d, and the preparation method was the same as in example 7.
2o:1H NMR(400MHz,DMSO)6.86(s,4H),3.92(t,J＝5.7Hz,4H),3.33(dd,J＝17.0,5.0Hz,8H),3.05(s,12H),1.81–1.68(m,8H),1.68-1.57(m,4H),1.48-1.37(m,4H),1.35-1.16(m,28H),0.85(t,J＝6.7Hz,6H).13C NMR(101MHz,DMSO)152.59,115.27,67.53,62.96,62.79,49.92,31.25,28.86,28.79,28.63,28.46,28.22,25.78,22.53,22.05,21.69,21.50,13.91.Calculated for C32H62Br2N2O2:253.2406,found:253.2406.
Example 22 preparation of compound 2 p: the starting materials used were N, N-dimethyldodecylamine and intermediate 1d, prepared in the same manner as in example 7.
2p:1H NMR(400MHz,DMSO)6.85(s,4H),3.91(t,J＝6.2Hz,4H),3.29(dd,J＝16.7,12.6Hz,8H),3.03(s,12H),1.81-1.68(m,8H),1.68-1.57(m,4H),1.47–1.37(m,4H),1.35-1.16(m,36H),0.86(t,J＝6.6Hz,6H).13C NMR(101MHz,DMSO)152.60,115.26,67.53,62.94,62.78,49.93,31.26,28.98,28.90,28.78,28.67,28.46,28.22,25.77,22.53,22.06,21.68,21.49,13.91.Calculated for C36H70Br2N2O2:281.2719,found:281.2717.
Example 23 preparation of compound 3 a: the starting materials used were 3-bromobispropyl ether and N, N-dimethylhexylamine, prepared in the same manner as in example 7.
3a:1H NMR(400MHz,DMSO)7.31(t,J＝8.0Hz,2H),6.99-6.91(m,3H),4.06(t,J＝5.9Hz,2H),3.54–3.42(m,2H),3.37-3.29(m,2H),3.08(s,6H),2.16(td,J＝11.6,5.8Hz,2H),1.75-1.59(m,2H),1.36-1.22(m,6H),0.88(t,J＝6.7Hz,3H).13C NMR(101MHz,DMSO)158.09,129.52,120.85,114.46,64.47,62.90,60.31,50.15,31.14,28.43,25.73,22.19,22.01 21.66,13.93.Calculated for C21H37BrN2O2:349.2855,found:349.2858.
Example 24 preparation of compound 3 b: the starting materials used were 3-bromobispropyl ether and N, N-dimethyloctylamine, the preparation being carried out in the same manner as in example 7.
3b:1H NMR(400MHz,DMSO)7.35–7.27(m,2H),6.99–6.92(m,3H),4.06(t,J＝5.9Hz,2H),3.54–3.40(m,2H),3.32-3.26(m,2H),3.07(s,6H),2.16(td,J＝11.6,5.9Hz,2H),1.74-1.60(m,2H),1.35–1.22(m,10H),0.87(t,J＝6.8Hz,3H).13C NMR(101MHz,DMSO)158.09,129.52,120.85,114.47,64.47,62.90,60.31,50.14,30.63,25.37,22.20,21.85,21.61,13.80.Calculated for C22H39BrN2O2:363.3012,found:363.3013.
Example 25 preparation of compound 3 c: the raw materials used were 3-bromophenyl propyl ether and N, N-dimethyl pelargylamine, and the preparation method was the same as in example 7.
3c:1H NMR(400MHz,DMSO)7.35–7.27(m,2H),6.99–6.92(m,3H),4.06(t,J＝5.9Hz,2H),3.53–3.41(m,2H),3.32-3.26(m,2H),3.07(s,6H),2.16(td,J＝11.6,5.8Hz,2H),1.74-1.59(m,2H),1.33-1.20(m,14H),0.86(t,J＝6.8Hz,3H).13C NMR(101MHz,DMSO)158.10,129.51,120.86,114.48,64.49,62.94,60.34,50.17,31.24,28.86,28.77,28.62,28.46,25.72,22.20,22.05,21.65,13.91.Calculated for C17H30BrNO:264.2328,found:264.2327.
Example 26 preparation of compound 3 d: the starting materials used were 3-bromobispropyl ether and N, N-dimethyldodecylamine, the preparation being carried out in the same manner as in example 7.
3d:1H NMR(400MHz,DMSO)7.35–7.27(m,2H),6.99–6.92(m,3H),4.06(t,J＝5.9Hz,2H),3.53–3.43(m,2H),3.33-3.29(m,2H),3.08(s,6H),2.16(td,J＝11.6,5.8Hz,2H),1.73-1.60(m,2H),1.33-1.20(m,18H),0.86(t,J＝6.8Hz,3H).13C NMR(101MHz,DMSO)158.11,129.50,120.84,114.48,64.49,62.93,60.32,50.17,31.25,28.97,28.91,28.78,28.67,28.46,25.72,22.21,22.05,21.66,13.91.Calculated for C19H34BrNO:292.2641,found:292.2639.
Example 27 preparation of compound 3 e: the starting materials used were N, N-dimethylhexylamine and intermediate 1e, prepared in the same manner as in example 7.
3e:1H NMR(400MHz,DMSO)8.15(d,J＝9.1Hz,1H),7.40(s,2H),7.33(d,J＝1.7Hz,1H),7.13(dd,J＝9.1,2.0Hz,1H),6.85(dd,J＝5.3,2.9Hz,1H),4.20(dd,J＝13.0,6.2Hz,4H),3.31-3.35(m,4H),3.66–3.46(m,4H),3.11(d,J＝11.5Hz,12H),2.26(d,J＝22.4Hz,4H),1.80-1.62(m,4H),1.40-1.20(m,12H),0.96-0.80(m,6H).13C NMR(101MHz,DMSO)156.48,153.82,135.52,126.98,123.47,120.00,119.39,117.38,106.92,103.42,64.80,64.63,62.94,60.51,60.30,50.22,30.66,30.63,25.38,22.29,22.15,21.84,21.68,21.62,13.81,13.78.Calculated for C32H56Br2N2O2:250.2172,found:250.2172.
Example 28 preparation of compound 3 f: the starting materials used were N, N-dimethyloctylamine and intermediate 1e, prepared in the same manner as in example 7.
3f:1H NMR(400MHz,DMSO)8.18(d,J＝9.2Hz,1H),7.46-7.36(m,2H),7.34(d,J＝1.6Hz,1H),7.14(dd,J＝9.1,1.9Hz,1H),6.85(d,J＝6.0Hz,1H),4.21(dd,J＝12.2,5.8Hz,4H),3.68–3.59(m,2H),3.59–3.49(m,2H),3.48–3.37(m,4H),3.14(d,J＝11.9Hz,12H),2.40-2.28(m,2H),2.28-2.15(m,2H),1.80-1.56(m,4H),1.39-1.17(m,20H),0.87(t,J＝6.3Hz,6H).13C NMR(101MHz,DMSO)156.48,153.82,135.51,126.93,123.52,120.00,119.38,117.41,106.91,103.38,64.81,64.64,62.88,60.45,60.24,50.20,31.14,31.13,28.45,25.75,22.33,22.19,21.01,21.76,13.92.Calculated for C36H64Br2N2O2:278.2485,found:278.2485.
Preparation of 3g of the compound of example 29: the raw materials used were N, N-dimethyl pelargonimine and intermediate 1e, and the preparation method was the same as in example 7.
3g:1H NMR(400MHz,DMSO)8.17(d,J＝9.1Hz,1H),7.45-7.36(m,2H),7.34(s,1H),7.14(d,J＝8.9Hz,1H),6.85(d,J＝5.8Hz,1H),4.29–4.14(m,4H),3.66-3.58(m,2H),3.58-350(m,2H),3.45-3.38(m,4H),3.13(d,J＝11.6Hz,12H),2.37-2.27(m,2H),2.27-2.17(m,2H),1.78-1.62(m,4H),1.37-1.18(m,28H),0.92-0.78(m,6H).13C NMR(101MHz,DMSO)156.49,153.83,135.52,126.92,123.50,120.02,119.39,117.40,106.92,103.39,64.82,64.65,62.95,60.48,60.27,50.21,31.24,28.86,28.79,28.62,28.48,25.75,22.33,22.21,22.05,21.75,21.70,13.91.Calculated for C40H72Br2N2O2:306.2798,found:306.2796.
Example 30 preparation of compound 3 h: the starting materials used were N, N-dimethyloctylamine and intermediate 1f, and the preparation was carried out in the same manner as in example 7.
3h:1H NMR(400MHz,DMSO)7.77(dd,J＝6.0,3.3Hz,2H),7.42(s,2H),7.34(dd,J＝6.1,3.2Hz,2H),4.21(t,J＝5.8Hz,4H),3.67–3.49(m,4H),3.48–3.39(m,4H),3.16(s,12H),2.28(s,4H),1.70(s,4H),1.33-1.17(m,20H),0.86(t,J＝6.7Hz,6H).13C NMR(101MHz,DMSO)148.00,128.99,126.25,124.14,108.51,65.48,63.04,60.34,50.18,31.14,28.51,28.46,25.79,22.22,22.01,21.74,13.91.Calculated for C38H66Br2N4O4:321.2543,found:321.2544.
Example 31 preparation of compound 3 i: the raw materials used were N, N-dimethyl pelargonimine and intermediate 1f, and the preparation method was the same as in example 7.
3i:1H NMR(400MHz,DMSO)7.69(dd,J＝6.0,3.3Hz,2H),7.34(s,2H),7.26(dd,J＝6.1,3.2Hz,2H),4.13(t,J＝5.9Hz,4H),3.57–3.42(m,4H),3.38-3.30(m,4H),3.08(s,12H),2.28–2.13(m,4H),1.62(s,4H),1.24-1.11(m,28H),0.78(t,J＝6.7Hz,6H).13C NMR(101MHz,DMSO)148.01,128.99,126.24,124.12,108.50,65.48,63.03,60.35,50.18,31.26,28.89,28.65,28.52,25.80,22.23,22.06,21.75,13.91.Calculated forC40H70Br2N4O4:335.2699,found:335.2696.
Application example 1 in vitro antibacterial Activity test
1. Experimental methods
Broth microdilution method:
(1) preparing an antibacterial medicament stock solution: the concentration of the prepared antibacterial agent stock solution is 2560 mug/mL, and the antibacterial agent with low solubility can be slightly lower than the concentration. The amount of the antibacterial agent solution or the amount of the powder required can be calculated by a formula. The prepared antibacterial drug stock solution should be stored in an environment below-20 ℃ and the storage life is not more than 6 months.
(2) Preparing bacteria to be detected: single colonies on overnight MH (A) plates were picked up with an inoculating loop in MH (B) medium, calibrated to a 0.5M turbidimetric standard, with an approximate bacterial count of 1X 108CFU/mL, and then diluting 100 times to obtain about 1 × 10 bacteria-containing number6CFU/mL of bacterial liquid for later use.
(3) The stock solutions of the antibacterial agent stock solutions (2560. mu.g/mL) were each diluted 10-fold to give an antibacterial agent solution having a concentration of 256. mu.g/mL. Taking a sterile 96-well plate, adding 200 mu L of antibacterial agent into the first well, adding 100 mu L of MH broth into the second to ten wells respectively, sucking 100 mu L from the first well, adding into the second well, mixing uniformly, sucking 100 mu L to the third well, repeating the steps, sucking 100 mu L from the tenth well, and discarding. The drug concentration in each well is as follows: 128. 64, 32, 16, 8, 4, 2, 1, 0.5. mu.g/mL, 200. mu.L of the inoculum (positive control) was added to the eleventh well, and 200. mu.L of the LMH (B) medium (negative control) was added to the twelfth well.
(4) Then 50 μ L of the previously prepared bacterial suspension was added to each of the wells 1 to 10, so that the final concentration of bacterial suspension per tube was about 5X 105CFU/mL, and the drug concentrations of the 1 st to 11 th wells are 128, 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25. mu.g/mL respectively. And (3) placing the inoculated 96-well plate in an incubator at 37 ℃ for culture, and observing the growth condition of the bacterial liquid for 24 hours. Meanwhile, standard strains are used for quality control.
(5) And (3) judging and explaining a result: before reading and reporting the MIC of the tested strain, the growth of the bacteria in the growth control tube should be checked for good condition, and the subculture condition of the inoculum should be checked to determine whether the inoculum is contaminated, and the MIC value of the quality control strain is in the quality control range. And (4) observing by naked eyes, wherein the lowest concentration tube of the medicament has no bacteria growth, namely the MIC of the tested bacteria.
Application example 2 in vitro erythrocyte hemolytic test
(1) Experimental materials: 10mLEP tube, 96-well plate, fresh defatted sheep blood.
(2) PBS buffer: 500mL, 4g of sodium chloride, 100mg of potassium chloride, 1.49g of sodium dihydrogen phosphate dihydrate, 100mg of anhydrous potassium dihydrogen phosphate, and a constant volume of deionized water to 490mL, adjusting the pH value to 7.2-7.4, sterilizing, dissolving 900mg of glucose in 10mL of sterilized ultrapure water, and adding the dissolved glucose into the PBS solution.
(3) Preparation of 5% red blood cell suspension: freezing fresh defibered sheep blood in a refrigerator, and placing the prepared PBS buffer solution in a water bath kettle at 37 ℃ to be taken immediately. Two 10mL EP tubes were placed in a test tube rack, PBS was taken out of the water bath at 37 ℃ and sprayed with alcohol together with the refrigerated fresh sheep blood, which was then placed on a clean bench. Respectively sucking 5700 microliters of PBS by using a pipette gun, adding the PBS into the two EP tubes, respectively sucking 300 microliters of goat blood, slowly adding the goat blood into the PBS solution, covering a cover, slowly turning upside down, uniformly mixing, putting into a centrifugal machine for 1500 revolutions, centrifuging for 10min, taking out the EP tubes, carefully sucking a supernatant, and removing the supernatant. And adding 5-7 mL of PBS solution again, slowly reversing the solution from top to bottom, uniformly mixing, and centrifuging for 10min at 1500 rpm. The operation is repeated until the supernatant is not turbid after centrifugation. After the last centrifugation, the supernatant is skimmed off, and the erythrocyte sediment is left for later use.
Several 10mL EP tubes were placed on a test tube rack, and 5700. mu.L of PBS (37 ℃) was added to each EP tube, followed by 300. mu.L of erythrocyte sediment. The mixture was slowly turned upside down to mix, and thus a 5% suspension of erythrocytes was prepared.
(4) Preparation of sample solution: the test drug was dissolved in a small amount of DMSO (final DMSO concentration could not be greater than 0.5%), and the same volume of DMSO was used as a negative control. The solubilized drug solution to be tested was diluted with PBS and the first well concentration was 1280 μ g/mL, at which time the drug in this EP tube was the initial drug. Nine 1.5mL EP tubes were then placed in parallel in a tube rack and 200. mu.L of PBS (Nos. 2,3, 4, … … 10) was added. All drugs were operated in parallel as such. Finally, 200. mu.L of the drug solution was pipetted from the initial drug EP tube into the No. 2 EP tube, 200. mu.L was pipetted into the No. 3 EP tube after repeated purging, and the operation was repeated by repeating the purging … … until reaching the No. 10 EP tube. Thus, the drug is diluted.
(5) Plate paving: and (4) taking a 96-well plate, and writing an experiment number, a medicine code and a date. The pipette is adjusted to 150. mu.L, the prepared 5% erythrocyte suspension is mixed up and down gently and reversely, and the mixture is sequentially sucked and spread into a 96-well plate (6X 10). The prepared drugs are correspondingly added into a 96-well plate, and one drug is added into three multiple wells. After the addition, the mixture is placed in a 37 ℃ incubator for incubation for 1 h.
(6) And (3) post-treatment: the 96-well plate was taken out of the incubator and centrifuged in a 4 ℃ centrifuge (3500rpm, 5 min). After centrifugation, a new 96-well plate is taken for each plate. Plate controls after labeling and centrifugation. Then 100 μ L of supernatant was aspirated correspondingly (well to well). After the absorption is finished, measuring the OD value with a microplate reader, and analyzing the data to obtain HC50。
Application example 3 Minimum Bactericidal Concentration (MBC) determination experiment in plasma
(1) Preparing bacteria to be detected: single colonies on overnight MH (A) plates were picked up with an inoculating loop in MH (B) medium, calibrated to a 0.5M turbidimetric standard, with an approximate bacterial count of 1X 108CFU/mL, and then diluting 100 times to obtain about 1 × 10 bacteria-containing number6CFU/mL of bacterial liquid for later use.
(2) Preparation of plasma: fresh sterile defibrinated sheep blood was placed in a 10mL EP tube and centrifuged at 3500rpm/10min in a centrifuge. The supernatant was carefully aspirated to obtain plasma.
(3) Preparation of a drug solution: the drug to be tested is first prepared into large-concentration stock solution 25600 mu g/mL (the final concentration of DMSO cannot be more than 0.5%) by DMSO. Then, the mixture was diluted to 256. mu.g/mL (volume ratio of sterile ultrapure water to fresh plasma: 1) with sterile ultrapure water and fresh plasma as a solvent. The method comprises placing 84 mL EP tubes (Nos. 1, 2,3, 4 … … 8) on an EP tube holder, adding 1500. mu.L of a mixed solution of sterile ultrapure water and fresh plasma (the volume ratio of sterile ultrapure water to fresh plasma is 1:1) to each EP tube, adding 1500. mu.L of a drug solution to each EP tube 1, repeatedly purging, sucking 1500. mu.L to each EP tube 2, and repeatedly purging … … until reaching the EP tube 8. (concentrations of 128, 64, 32, 16, 8, 4, 2, 1. mu.g/mL of drug per EP tube) thus the drug was diluted.
(4) Plate paving: and (4) taking a 96-well plate, and writing an experiment number, a medicine code and a date. The pipette was adjusted to 150 μ L, 150 μ L of different concentrations of drug was aspirated from the corresponding EP tube and added to a 96-well plate, which was flooded with 9 rows, each row having a drug gradient. And (3) placing the mixture in a 37 ℃ incubator for incubation for 2h, sucking 50 mu L of the bacteria solution to be detected by the first three rows of liquid transfer guns, adding the bacteria solution into a 96-well plate containing the medicine, and adding no bacteria solution into the last 6 rows. After the addition, the mixture is placed in a 37 ℃ incubator for incubation for 2 hours, 50 mu L of bacteria liquid to be detected is absorbed by three rows of pipette guns in the middle and added into a 96-well plate containing the medicine, and the bacteria liquid is not added in the last 3 rows. After the addition, the mixture is placed in a constant temperature box at 37 ℃ for incubation for 2h, and finally, 50 mu L of bacteria liquid to be detected is absorbed by three rows of pipette guns and added into a 96-well plate containing the medicine. According to the action time of the medicine and the blood plasma, the action time of the first three rows is shortest 2 hours, the action time of the middle three rows is 4 hours, and the action time of the last three rows is 6 hours. After the bacterial liquid is completely added, putting the mixture into a constant temperature box at 37 ℃ for incubation for 24 h.
(5) Counting agar plates: after a 96-well plate is cultured in a 37-DEG C constant-temperature incubator for 24 hours, an MIC value is read, the concentration of 2-4 gradients which are larger than the MIC result is selected as the MBC concentration to be detected, 20 microliters of bacterial liquid is absorbed from the concentration to be detected and coated on an MH agar plate by a coating rod, the MH agar plate is dried in a biological safety cabinet, the MH agar plate is cultured in the 37-DEG C constant-temperature incubator for 24 hours, and the number of colonies on the MH agar plate is less than or equal. The corresponding well concentration is the MBC of the compound against the corresponding strain.
Application example 4 Minimum Bactericidal Concentration (MBC) determination experiment in body fluid
(1) Preparation of body fluid: fresh sterile defibrinated sheep blood was centrifuged in a 10mL EP tube at 3500rpm/10min centrifuge. Carefully sucking the supernatant to obtain blood plasma; whole blood adopts fresh sterile defibrinated sheep blood; serum was purchased Zeta Life fetal bovine serum;
(2) preparing bacteria to be detected: single colonies of MRSA from overnight-grown MH (A) dishes were picked up by an inoculating loop in MH (B) medium, calibrated to a 0.5M turbidimetric standard, with a count of about 1X 108CFU/mL, then diluting the bacterial liquid with 50% of body fluid (whole blood, blood plasma or blood serum) and 50% of MHB culture medium to 10%5CFU/mL, spare.
(3) Preparation of a drug solution: the drug to be tested is first prepared into large-concentration stock solution 25600 mu g/mL (the final concentration of DMSO cannot be more than 0.5%) by DMSO. The medicine to be tested is diluted to 512, 256, 128, 64, 32, 16, 8, 4 and 2 mu g/mL by using sterile ultrapure water.
(4) Plate paving: the above-mentioned bacterial liquids diluted with different body fluids were pipetted 150. mu.L into a 96-well plate using a pipette gun, and 3 rows of parallel tests were performed on each of the bacterial liquids diluted with body fluids. Then 50. mu.L of diluted test drug was added. After 24h at 37 ℃ in an incubator, MBC values were obtained by counting on agar plates as described above.
Application example 5 biofilm lysis experiment
(1) Colony counting method: diluting E.coli (M9 culture medium with 0.02% of casein and 0.5% of glycerol) growing for 4-6 h to 10%5CFU/mL, 100 μ L of the bacterial suspension was added to a 96-well plate, incubated for a certain period of time (s. aureus 24h, e. coli 24h), centrifuged at 3500rpm for 5min at 4 ℃, the suspension removed, and washed once with 1 × PBS. 100. mu.L of a 1 XPBS-diluted solution (128, 64, 32, 16, 8, 4, 2. mu.g/mL) of the test compound was added to a 96-well plate, and 100. mu.L of the control group was addedμ L of 1 XPBS. After 24h the supernatant was removed by centrifugation at 3500rpm for 5min at 4 ℃ and washed once with 1 XPBS. 100 μ L of 1 XPBS was added for resuspension, and the bacterial suspension was diluted 10-fold in a gradient and added dropwise to solid medium agar plates. Placing at 37 deg.C for 24 hr, counting colonies, and using log10(CFU/well) expression results.
Application example 6 time Sterilization kinetics experiment
After shaking overnight at 37 ℃ in a shaking table at 225rpm, diluting the E.coli by 10000 times by using an MHB culture medium, then shaking for 2h (initial logarithmic growth) and 5h (middle logarithmic growth) at 37 ℃ at 225rpm, adding the drug to be detected, wherein the drug concentrations in the initial logarithmic growth stage are respectively 2 mu g/mL, 3 mu g/mL and 4 mu g/mL, the drug concentrations in the middle logarithmic growth stage are respectively 6 mu g/mL, 8 mu g/mL and 12 mu g/mL, and the E.coli is compared by using moxalactam, and a blank group without drug is reserved. Adding drug, centrifuging each group at 3500rpm in 96-well plate at 4 deg.C for 3min, removing supernatant, adding 100 μ L1 × PBS solution, resuspending, diluting with ten times gradient of 1 × PBS solution, dropping 10 μ L diluted bacteria onto MH agar plate, dropping three drops per concentration, culturing in 37 deg.C constant temperature incubator overnight, counting colony number, and unit log10CFU/mL, plot. The number of bacteria in the middle logarithmic growth phase was also determined at 16h, 18h, 20h, and 24 h.
In order to intuitively illustrate the sterilization effect of the micromolecular cationic compound, bacteria liquid and blank groups which are 6 hours after the medicine acts are compared with those of the bacteria liquid and blank groups which are 24 hours after the medicine acts, and then photographing and turbidity comparison are carried out.
Application example 7 antibacterial mechanism research experiment
(1) Depolarization of the cytoplasmic membrane: s.aureus and e.coli grown for 6h (mid-log) were centrifuged at 3500rpm, 4 ℃ for 5min, resuspended in 1 × PBS (s.aureus) or treated with 5mM HEPES: 5mM glucose: 100mM KCl ═ 1: 1:1 (e. coli), re-centrifuged and re-resuspended. 150 μ L of bacterial suspension (10) was taken8CFU/mL) was added to a 96-well bottom clear blackboard, followed by the addition of dye DiSC3(5) (10. mu.M, 50. mu.L) and incubation for a period of time in the absence of light, Staphylococcus aureus for 30min, E.coli 40min (additionally 50. mu.L of 200. mu.M EDTA solution), and then the fluorescence intensity was measured at an excitation wavelength of 622nm (slit width: 10nm) and a cut-off wavelength of 670nm (slit width: 5nm), and the fluorescence intensity was measured 8min before the measurement at intervals of 2 min. Then the suspension of the bacteria is taken out and added on another blackboard containing 10 mu L of the drug to be detected (420 mu g/mL, solvent sterile ultrapure water), the blank group is added with 50 mu L of sterile ultrapure water, and the fluorescence intensity is continuously monitored on a microplate reader for 12min after the addition, and the time interval is 2 min.
(2) Outer membrane permeabilization experiments: the outer membrane permeability test of the compounds to be tested was carried out using NPN as dye. Coli grown for 6h was centrifuged at 3500rpm, 4 ℃ for 5min and the mixture was washed with 5mM HEPES: 5mM glucose: 100mM KCl ═ 1: 1:1, then centrifuged to remove the supernatant, and resuspended. 150 μ L of bacterial suspension (10) was taken8CFU/mL) was added to a 96-well bottom clear blackboard, followed by the dye NPN (10 μ M, 50 μ L) and incubation for 40min in the absence of light, followed by incubation at excitation wavelength 350nm (slit width: 10nm) and a cutoff wavelength of 420nm (slit width: 5nm), taking a point every 2min, and measuring the fluorescence intensity 8min before. Then the suspension of the bacteria is taken out and added on another blackboard containing 10 mu L of the drug to be detected (420 mu g/mL, solvent sterile ultrapure water), the blank group is added with 50 mu L of sterile ultrapure water, and the fluorescence intensity is continuously monitored on a microplate reader for 12min after the addition, and the time interval is 2 min.
(3) Inner membrane permeabilization experiments: s.aureus and e.coli grown for 6h (mid-log) were centrifuged at 3500rpm, 4 ℃ for 5min, resuspended in 1 × PBS (s.aureus) or treated with 5mM HEPES: 5mM glucose: 100mM KCl ═ 1: 1:1 (e. coli), re-centrifuged and re-resuspended. 150 μ L of bacterial suspension (10) was taken8CFU/mL) was added to a 96-well bottom transparent blackboard, then dyes PI (10 μ M, 50 μ L), s.aureus and e.coli were added and left under dark conditions for 30min and 40min, respectively, and then the resultant was left to stand at an excitation wavelength of 535nm (slit width: 10nm) and a cutoff wavelength of 617nm (slit width: 5nm), taking a point every 2min, and measuring the fluorescence intensity 8min before. The bacterial suspension was then removed and added to another block containing 10. mu.L of the drug to be testedOn a blackboard (420 mu g/mL, solvent sterile ultrapure water), a blank group is added with 50 mu L of sterile ultrapure water, and after the addition is finished, the fluorescence intensity is continuously monitored on a microplate reader for 12min, and the time interval is 2 min.
Application example 8 cytotoxicity test
(1) Observation experiment with optical microscope: after the HeLa cells were grown over the entire dish, they were digested with pancreatin digest, counted, and then blown up with DMEM medium (containing 10% fetal bovine serum) and diluted to about 5X 10 cells per 100. mu.L3～6×103Cells, 1mL of cell suspension per well in 12-well plates, and so on for a period of more than ten hours, after the cells were attached to the wall, the supernatant was aspirated off and the cells were incubated for 24h in a 37 ℃ incubator (diluted with medium in 4mL EP tubes). After completion, the old medium was pipetted into a 4mL EP tube, washed twice with 1 XPBS, and then added to the EP tube, and 700. mu.L of 1 XPBS solution was added to a 12-well plate (to avoid dry cell death), and photographed under an inverted microscope (see the state of the cells for the first time).
(2) Live and dead cell double staining experiment: the living and dead cells are stained by a color developing agent, and the shape of the drug-affected cells is visually checked by taking a picture by a fluorescence microscope. A4 mL EP tube (containing dead cells) loaded with old medium from procedure (1) above was centrifuged at 1500 Rcf. times.g for 5min, washed twice with 1 XPBS, the supernatant was decanted, and 500. mu.L of a mixture containing the dyes Calcein-AM (2. mu.M) and propidium iodide (PI, 4.5. mu.M) 1:1 (volume ratio) of 1 XPBS solution, after being blown and beaten uniformly, the 1 XPBS solution in the corresponding hole of the 12-hole plate is sucked off, and the staining solution containing dead cells is added. Culturing in 37 deg.C incubator for 15min, imaging under fluorescence microscope with 20 Xeyepiece, and taking pictures.
The experimental results are as follows:
table one: MIC (mu g/mL) results of target compound 2a-2p, 3a-3i on gram-negative and gram-positive sensitive bacteria and in vitro erythrocyte hemolytic HC50Results (. mu.g/mL)
aVancomycinbMeropenem.
Table two: MIC (μ g/mL) results for some compounds against 10 clinical strains of non-duplicate MRSA
Table three: results of MIC (mu g/mL) of partial compound for 5 clinical strains with no-repeat NDM-1 enzyme production and 1 clinical strain with no-repeat KPC-2 enzyme production
As can be seen from the table I, most of the synthesized compounds 2a-2p, 3a-3i have good activity on gram-positive bacteria staphylococcus aureus, enterococcus faecalis, gram-negative bacteria escherichia coli and stenotrophomonas maltophilia, and the compounds have obvious broad-spectrum antibacterial activity; meanwhile, in vitro erythrocyte hemolytic data show that the medicine has low toxicity and good selectivity.
MIC and HC according to Table I50As a result, it was found that 2b, 2f, 2j, 2n, 3f, 3h had good activity and low toxicity, and then the activity of these 6 compounds against drug-resistant bacteria was measured, and the results are shown in tables two and three: it can be seen that the compounds show good antibacterial activity for MRSA and clinical strains producing NDM-1 and KPC-2 enzymes. Therefore, the compounds have better patent medicine prospect.
In addition, the stability of the compound in different body fluids is tested by taking the compound 2n as an example. As can be seen from fig. 1 (a), the MBC value of compound 2n in plasma did not change significantly with longer action time, indicating that compound 2n has good plasma stability. As can be seen from FIG. 1 (b), the MBC value of compound 2n in 50% by mass of plasma was 16. mu.g/mL, while the MBC value in 50% by mass of serum and whole blood was 32. mu.g/mL, indicating that compound 2n has excellent stability in body fluid.
It was confirmed from FIG. 2 that Compound 2n had a biofilm-disrupting action, and that different concentrations of Compound 2n were applied to E.coli, and that the residual bacterial count was 10 when the compound concentration was 2. mu.g/mL7CFU/mL, and blank 107.47CFU/mL, 1/3 reduction in bacterial count compared to blank; when the concentration of the medicine is 8 mug/mL, the number of bacteria is obviously reduced compared with 4 mug/mL; when the concentration of the drug is 64 mug/mL and 128 mug/mL, the number of bacteria is 0, which indicates that the compound 2n has good capability of removing bacterial membranes.
To verify the bactericidal rate of compound 2n, bacteria were counted after different time of drug action to obtain a line graph and final bacterial fluid clarity, see fig. 3. As can be seen from (a) in FIG. 3, after 3. mu.g/mL and 4. mu.g/mL of compound 2n act on Escherichia coli cultured for 2h for 4h, the bacteria can be completely killed, and the bacteria have the same bactericidal capacity as that of a control drug, latamoxef; as can be seen from (b) in FIG. 3, when the concentration of the compound 2n is 12 mug/mL, the compound can completely kill the escherichia coli cultured for 5 hours after 3 hours, and when the concentration is 6 mug/mL and 8 mug/mL, the compound can completely kill the bacteria after 6 hours, while the bacteria still remain in the reference drug latamoxef, which indicates that the sterilization speed of the compound 2n in the escherichia coli cultured for 5 hours is obviously superior to that of the latamoxef.
6 representative compounds 2b, 2f, 2j, 2n, 3f, 3h of the present invention were selected for the study of the mechanism of action of the aromatic phenol quaternary ammonium salt antibacterial peptide mimetics. In fig. 4, (a) and (b) cell membrane depolarization experiments of staphylococcus aureus and escherichia coli show that 6 compounds all have good cell membrane depolarization ability, and the depolarization ability of compounds 2f, 2n and 3f is better than that of compounds 2b, 2j and 3h relative to bacteria staphylococcus aureus; in FIG. 4 (c) and (d) the inner membrane permeabilization test of Staphylococcus aureus and Escherichia coli, most of the compounds showed good inner membrane permeabilization ability, but a small portion of the compounds showed weak inner membrane permeabilization ability; shows good permeabilization capacity to all compounds in escherichia coli outer membrane permeabilization test.
To visually demonstrate the toxicity of compound 2n to Hela cells, the state of the cells after drug action was photographed, see fig. 5. By comparing the negative control with the photographs of the drug groups at different concentrations, it can be seen that compound 2n is less cytotoxic to Hela. Meanwhile, a fluorescent dye is used for indicating the living and dead states of cells, and calcein can stain the living cell membranes and shows green. Pyridine iodide stains the nucleus of the cell through a ruptured cell membrane, shows a red color, and can be used to indicate a dead cell. In FIG. 6, (d), (e) cells were stained green, and no red color appeared, indicating that Compound 2n was very little toxic even at a concentration of 32. mu.g/mL.
Claims (1)
1. A phenolic antimicrobial peptidomimetic having antimicrobial activity, characterized in that the compound has the following structural formula:
2b：n = 2，q = 7；
2f：n = 3，q = 7；
2g：n = 3，q = 9；
2i：n = 4，q = 5；
2j：n = 4，q = 7；
2m：n = 5，q = 5；
2n：n = 5，q = 7。
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