CN114573465B - Honokiol antibacterial peptide mimics with broad-spectrum antibacterial activity and preparation method thereof - Google Patents

Honokiol antibacterial peptide mimics with broad-spectrum antibacterial activity and preparation method thereof Download PDF

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CN114573465B
CN114573465B CN202210223350.9A CN202210223350A CN114573465B CN 114573465 B CN114573465 B CN 114573465B CN 202210223350 A CN202210223350 A CN 202210223350A CN 114573465 B CN114573465 B CN 114573465B
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王印虎
刘方全
陈军节
薛洁
朱冰雪
周焕焕
李超
韦桂云
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Liaocheng University
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Abstract

The invention belongs to the technical field of pharmaceutical chemistry, and discloses a honokiol antibacterial peptide analogue with broad-spectrum antibacterial activity and a preparation method thereof. The invention obtains the target product through 3-5 steps of reaction. In vitro activity results prove that the honokiol antibacterial peptide mimics have the characteristics of broad-spectrum antibacterial activity, rapid sterilization and difficult generation of drug resistance. The chemical structural formula of the honokiol antibacterial peptide analogue is shown as the following, wherein X, q is defined in the specification.

Description

Honokiol antibacterial peptide mimics with broad-spectrum antibacterial activity and preparation method thereof
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry, and relates to a preparation method of an antibacterial peptide mimic with a honokiol structure and application thereof in antibacterial.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
The constant generation and spread of bacterial resistance is a serious threat to public health worldwide, and mortality rates caused by it have increased year by year. This trend is particularly pronounced by the emergence of multiple resistant bacteria represented by "ESKAPE" pathogens (enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa and enterobacter bacteria). The number of people dying from bacterial infection worldwide is about 70 ten thousand at present. Finding and developing new antibacterial drugs with new structures and new targets is one of the most effective methods for solving the increasingly severe problem of bacterial drug resistance at present.
The outer membrane of gram-negative bacteria is composed of a complex of lipopolysaccharide, phospholipid, protein, lipoprotein, etc., and forms an asymmetric membrane structure due to the presence of the outer membrane, thus forming a permeation barrier against hydrophilic and hydrophobic substances. Antibacterial peptides are positively charged hydrophilic lipophilic natural peptides widely existing in multicellular organisms, and are the first line of defense of the immune system against microbial infection. Antibacterial peptides share common structural features-a hydrophilic positive charge and a lipophilic side chain that spatially form a spatial conformation of the hydrophilic moiety on one side and the lipophilic moiety on the other side of the same molecule. This spatial structure is considered to be the basis for the antibacterial activity of the antibacterial peptide. It is believed that positively charged antimicrobial peptides accumulate on the surface of negatively charged microbial membranes by electrostatic attraction. When the aggregated antimicrobial peptide reaches a critical concentration or above, its hydrophobic moiety will intercalate into the hydrophobic moiety of the bacterial lipid membrane, causing rupture of the bacterial membrane, resulting in leakage of intracellular material, membrane depolarization and cell death. Compared with the traditional antibacterial agent, the antibacterial peptide has the characteristics of novel action mechanism, strong antibacterial performance, high sterilization speed, broad antibacterial spectrum, difficult generation of drug resistance and the like. Therefore, antimicrobial peptides have great potential as a novel class of potent antimicrobial agents, and have received widespread attention from global researchers. Although the antibacterial peptide shows excellent antibacterial activity against both gram-positive bacteria and gram-negative bacteria, it has some drawbacks, thereby restricting its clinical application. First, the potential toxicity of antibacterial peptides. The antibacterial peptide has the function target of bacterial cell membrane, and can produce hemolytic toxic and side effects on normal mammals while playing antibacterial property. Second, the antibacterial peptide has poor stability under physiological conditions. Because of the extensive presence of proteolytic enzymes in the human body, antibacterial peptides that enter the human body are very susceptible to degradation by these enzymes and lose antibacterial activity.
In addition, the relatively high synthetic cost is also a factor limiting the clinical application of antimicrobial peptides. Aiming at the defects of the antibacterial peptide, scientific researchers design and synthesize a series of low-molecular antibacterial peptide mimics according to the unique molecular structure of the antibacterial peptide, and simulate the antibacterial peptide in terms of chemical structure, physicochemical property and biological function. To date, only vancomycin, daptomycin and polymyxin have been used in clinical batches, whereas 3 of the mimics advanced to the clinical study stage are PMX-30063, LTX-109 and CA-13, respectively. Aiming at the problems of antibacterial peptide drugs, pharmaceutical chemists try to realize the aims of high activity, low cytotoxicity, high protease stability and low synthesis cost through the research of antibacterial peptide mimics. Over ten years of research, a series of small molecules with structural features of antibacterial peptides have been found to also possess properties similar to those of antibacterial peptides. The small molecular mimic of the antibacterial peptide with a hydrophilic side chain and a lipophilic skeleton can form a space structure similar to that of the antibacterial peptide, and realize antibacterial effect similar to that of the antibacterial peptide.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a series of novel honokiol antibacterial peptide mimics with strong antibacterial performance, high sterilization speed and wide antibacterial spectrum and a preparation method thereof.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a honokiol antibacterial peptide analogue or a pharmaceutically acceptable salt thereof having antibacterial activity, the compound structure being as follows:
wherein X is selected from CH 2 、CONH；q＝0、1、2。
According to the invention, the amphiphilic structure characteristic of the antibacterial peptide is constructed by utilizing the structural framework of honokiol, a series of honokiol antibacterial peptide mimics are designed and synthesized, and the antibacterial activity of the honokiol antibacterial peptide mimics is verified through an in-vitro activity experiment, so that a novel antibacterial medicament which has strong antibacterial performance, high sterilization speed, broad antibacterial spectrum and difficult generation of drug resistance is obtained.
In a second aspect of the present invention, a preparation method of honokiol antibacterial peptide analogue or pharmaceutically acceptable salt thereof with antibacterial activity is provided, and the specific route is as follows:
routes 1.4 a-4b,7a-7c preparation routes:
routes 2.10 a-10f,12a-12e preparation routes
Routes 3.16 a-16h preparation
In a third aspect, the invention provides an application of honokiol antibacterial peptide analogue with antibacterial activity or pharmaceutically acceptable salt thereof in preparing a medicament for treating bacterial infection.
The invention has the beneficial effects that:
(1) The honokiol antibacterial peptide mimics provided by the invention have good antibacterial activity on gram positive bacteria and gram negative bacteria, especially the compounds 10a-10c,12c and 16d have the minimum antibacterial concentration value of 0.5-16 mug/mL on staphylococcus aureus, bacillus subtilis and escherichia coli, and the antibacterial activity of part of the compounds is obviously better than that of positive control medicines vancomycin and colistin.
(2) The invention selects the compound 10b with the best activity, and further researches find that the compound has the characteristics of quick sterilization, no toxicity and difficult generation of drug resistance, and is expected to provide a new thought for solving the problem of bacterial drug resistance.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
Fig. 1: time sterilization profile of compound 10b against staphylococcus aureus and escherichia coli;
fig. 2: evaluation of induced resistance of Compound 10b to Staphylococcus aureus and Escherichia coli;
fig. 3: cytotoxicity evaluation of compound 10 b.
Fig. 4: hydrogen profile of compound 10 b.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The compound has the following structural formulas I-III:
wherein X is selected from CH 2 、CONH；q＝0、1、2；
Wherein X is selected from CH 2 、CONH；m＝1、3、5、7、9、11；n＝1、2；
Wherein R is selected from n-propyl, n-pentyl, n-heptyl, n-nonyl, n-undecyl, adamantyl, tert-butylphenyl and biphenyl;
more preferably, the compounds I, II, III are one of the following:
the route for the preparation of the honokiol antibacterial peptide mimics of the invention is as follows:
reaction conditions: (a) potassium carbonate, acetonitrile, 75 ℃; (b) hydrochloric acid, dichloromethane, room temperature; (c) ethyl bromoacetate, potassium carbonate, acetonitrile, 60 ℃; (d) sodium hydroxide, methanol/water, 60 ℃; (e) N, N-diisopropylethylamine, HATU, amino substituents; acetonitrile, room temperature; (f) trifluoroacetic acid, dichloromethane, 0 ℃.
Routes 1.4 a-4b,7a-7c preparation routes
The method is realized by the following steps:
carrying out substitution reaction on honokiol 1 and bromine-substituted amino compounds with different chain lengths in acetonitrile solution under alkaline conditions to obtain compounds 3a-3b, and removing corresponding protecting groups under acidic conditions to obtain compounds 4a-4b; and reacting honokiol with ethyl bromoacetate under alkaline conditions to obtain a compound 5, hydrolyzing the compound 5 under strong alkali to obtain a compound 6, then carrying out condensation reaction on the compound 6 and amino substituents with different chain lengths under alkaline conditions to obtain compounds 7a-7c, and finally removing Boc protecting groups to obtain target products 8a-8c.
Reaction conditions: (a) 1, 3-dibromopropane, potassium carbonate, acetone; (b) N, N-dimethylalkane, acetonitrile, refluxing;
(c) N, N-dimethylpropane diamine, N, N-diisopropylethylamine, HATU, acetonitrile, room temperature; (d) bromoalkane, acetonitrile, reflux.
Routes 2.10 a-10f,12a-12e preparation routes
The method is realized by the following steps:
reacting honokiol with 1, 3-dibromopropane under alkaline conditions to obtain a compound 9, and reacting the compound 9 with N, N-dimethyl alkane in acetonitrile at 80 ℃ in a reaction kettle to obtain target substances 10a-10f; the compound 6 is condensed with N, N-dimethyl propylene diamine under the catalysis of a condensing agent HATU to obtain a compound 11, and the compound 11 reacts with bromides with different chain lengths to obtain 12a-12e.
Reaction conditions: (a) H-Lys (Boc) -OMe, N, N-diisopropylethylamine, HATU, acetonitrile, room temperature; (b) lithium hydroxide, methanol/water, 60 ℃; (c) Amine, N, N-diisopropylethylamine, HATU, acetonitrile, room temperature; (d) trifluoroacetic acid, dichloromethane, 0 ℃.
Routes 3.16 a-16h preparation
The method is realized by the following steps:
condensing the compound 6 with H-Lys (Boc) -OMe to obtain a compound 13, hydrolyzing the compound 13 under the alkaline condition of lithium hydroxide to obtain a compound 14, then reacting the compound 14 with different amino compounds to obtain compounds 15a-15H, and finally removing protecting groups under the condition of trifluoroacetic acid to obtain compounds 16a-16H.
The invention will now be described in further detail with reference to the following specific examples, which should be construed as illustrative rather than limiting.
Example 1.
Preparation of Compounds 3a,3b
Honokiol (2.00 g,7.51 mmol), compound 2 (5.05 g,22.53 mmol) and potassium carbonate ((3.11 g,22.53 mmol)) were stirred in acetonitrile (30 mL) at 75 ℃ overnight. After cooling, the mixture was diluted with water and extracted with ethyl acetate. The combined extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel chromatography to give oil 3a (3.20 g, 77%) and 3b (2.96 g, 68%).
Example 2.
Preparation of Compounds 4a,4b
Compound 3a or 3b was dissolved in a solution of dichloromethane (6 mL) and trifluoroacetic acid (6 mL) was slowly added. The reaction solution was stirred at room temperature for 5 hours. After completion of the reaction, the residual solvent was concentrated in vacuo and the residue was subjected to C-18 reverse phase column chromatography, freeze-dried to give 4a and 4b as white solids.
4a： 1 H NMR(500MHz,DMSO-d 6 )δ8.44(d,J＝46.2Hz,6H),7.42–7.36(m,1H),7.32(d,J＝1.8 Hz,1H),7.10(q,J＝9.1,8.3Hz,3H),7.01(d,J＝8.5Hz,1H),5.98(dddd,J＝26.8,16.8,8.5,4.8Hz,2H),5.19–4.97(m,4H),4.25(t,J＝4.5Hz,2H),4.16(t,J＝5.4Hz,2H),3.48(d,J＝6.7Hz, 2H),3.35(d,J＝6.6Hz,2H),3.23(s,2H),3.08(s,2H).HRMS(ESI)C 22 H 29 N 2 O 2 [M+H] + calcd＝ 353.2224；found＝353.2226.
4b: 1 H NMR(500MHz,DMSO-d 6 )δ8.25(d,J＝23.9Hz,6H),7.36–7.24(m,2H),7.14–7.05(m, 2H),7.01(d,J＝8.4Hz,2H),5.96(dddd,J＝16.8,14.5,10.0,6.8Hz,2H),5.18–4.99(m,4H),4.13(t,J＝6.0Hz,2H),4.03(t,J＝6.1Hz,2H),3.35(dd,J＝19.7,6.7Hz,4H),2.99(q,J＝6.7Hz,2H), 2.85(q,J＝6.6Hz,2H),2.10(p,J＝6.3Hz,2H),1.97(p,J＝6.3Hz,2H).HRMS(ESI) C 24 H 33 N 2 O 2 [M+H] + calcd＝381.2537；found＝381.2536.
Example 3.
Preparation of Compound 5
Honokiol (2.00 g,7.51 mmol) was dissolved in anhydrous N, N-dimethylformamide (10 mL), followed by ethyl bromoacetate (3.14 g,18.77 mmol) and potassium carbonate (3.11 g,22.53 mmol). The mixture was stirred at 60℃for 6 hours.
After completion of the reaction, the solvent was removed in vacuo and purified by flash column chromatography to give 2.70g of a yellow solid in 82% yield.
Example 4.
Preparation of Compound 6
Compound 5 (2.50 g,5.70 mmol) was dissolved in a methanol/water (80 mL) mixture, sodium hydroxide (2.28 g, 57.00 mmol) was added, and the reaction was stirred at 60℃overnight. After the reaction was complete, the excess methanol was removed and the pH was adjusted to 3 by the addition of concentrated hydrochloric acid. The precipitate produced was filtered and dried to give 1.85g of a white solid in 88% yield.
Example 5.
Preparation of Compounds 7a-7c
Intermediate 6 (1.50 g,3.92 mmol) was dissolved in anhydrous acetonitrile (30 mL), HATU (3.58 g,9.41 mmol) and N, N-diisopropylethylamine (1.52 g,11.76 mmol) were added, the reaction stirred at 0deg.C for 30 min, then the corresponding amino substituent (9.41 mmol) was added and stirring continued at room temperature for an additional 2h. After completion of the reaction, the solvent was removed under reduced pressure, the residue was neutralized with 2M hydrochloric acid diluted solution and extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the crude product was further purified by silica gel column chromatography to give 7a-7c.
Example 6.
Preparation of Compounds 8a-8c
The starting materials used were intermediates 7a-7c and trifluoroacetic acid, prepared in the same manner as in example 2.
8a: 1 H NMR(500MHz,DMSO-d 6 )δ8.49(d,J＝20.4Hz,1H),8.32(t,J＝5.8Hz,1H),8.24–8.15 (m,6H),7.43(dd,J＝8.5,2.2Hz,1H),7.34(d,J＝2.1Hz,1H),7.12–7.05(m,2H),6.95(dd,J＝8.8,2.7Hz,2H),6.10–5.91(m,2H),5.16–4.99(m,4H),4.59(s,2H),4.47(s,2H),3.49(d,J＝6.6 Hz,2H),3.46–3.38(m,4H),3.34(d,J＝6.7Hz,2H),2.97–2.80(m,4H),HRMS(ESI)C 26 H 35 N 4 O 4 [M+H] + calcd＝467.2653；found＝467.2654.
8b: 1 H NMR(500MHz,DMSO-d 6 )δ8.30–8.03(m,8H),7.44(dd,J＝8.5,2.2Hz,1H),7.34(d,J＝ 2.1Hz,1H),7.15–7.04(m,2H),6.93(d,J＝8.8Hz,2H),6.00(dddt,J＝37.3,16.8,10.0,6.7Hz,2H),5.17–4.98(m,4H),4.57(s,2H),4.44(s,2H),3.48(d,J＝6.6Hz,2H),3.34(d,J＝6.7Hz, 2H),3.21(dq,J＝31.1,6.5Hz,4H),2.77(dt,J＝14.8,7.8Hz,4H),1.76(dp,J＝21.7,6.8Hz,4H).HRMS(ESI)C 28 H 39 N 4 O 4 [M+H] + calcd＝495.2966；found＝495.2966.
8c: 1 H NMR(500MHz,DMSO-d 6 )δ8.18(s,2H),8.14–8.06(m,6H),7.43(dd,J＝8.5,2.2Hz, 1H),7.33(d,J＝2.1Hz,1H),7.09(d,J＝8.1Hz,2H),6.93(dd,J＝8.3,2.0Hz,2H),6.08–5.91(m,2H),5.14–5.00(m,4H),4.55(s,2H),4.44(s,2H),3.47(d,J＝6.6Hz,2H),3.34(d,J＝6.7Hz,2H), 3.17(q,J＝6.4Hz,2H),3.11(q,J＝6.6Hz,2H),2.80–2.75(m,4H),1.56(dq,J＝22.9,7.6Hz, 8H).HRMS(ESI)C 30 H 43 N 4 O 4 [M+H] + calcd＝523.3279；found＝523.3278.
Example 7.
Preparation of Compound 9
Honokiol 1 (2.00 g,7.51 mmol) was dissolved in acetone (50 mL) and potassium carbonate (2.49, 18.00 mmol) and 1, 3-dibromopropane (12.13 g,60.08 mmol) were added. The mixture was stirred at 55℃for 8 hours, then diluted with water (100 mL) and extracted with ethyl acetate (3X 20 mL). The combined extracts were washed with brine, dried, and purified by flash chromatography on silica gel to give the compound as a colorless oil 2.60g in 68% yield.
Example 8.
Preparation of Compounds 10a-10f
Compound 12 (2.54 g,5.00 mmol) was dissolved in acetonitrile, the corresponding N, N-dimethylalkylamines (150 mmol) with varying chain lengths were added, and the mixture was vigorously stirred at 80℃for 12h. After the reaction was completed, the solvent was removed under reduced pressure. The crude product was then stirred in anhydrous diethyl ether to remove excess impurities and the resulting precipitate was filtered to give the target compounds 13a-13f.
10a: 1 H NMR(500MHz,DMSO-d 6 )δ7.37–7.32(m,1H),7.28–7.25(m,1H),7.14–7.09(m,1H), 7.08–7.00(m,3H),6.08–5.89(m,2H),5.16–4.98(m,4H),4.05(dt,J＝32.8,5.6Hz,4H),3.52–3.44(m,2H),3.40(d,J＝6.5Hz,2H),3.34(s,4H),3.30(dd,J＝9.7,6.7Hz,2H),3.27–3.20 (m,2H),3.10(s,6H),3.00(s,6H),2.27–2.15(m,2H),2.06(dt,J＝9.8,6.1Hz,2H),1.68(dq,J＝15.7,8.0Hz,2H),1.52(dq,J＝15.7,7.9Hz,2H),1.34(dt,J＝14.7,7.3Hz,2H),1.25(dd,J＝14.3, 6.9Hz,2H),0.92(dt,J＝32.0,7.3Hz,6H).HRMS(ESI)C 36 H 58 Br 2 N 2 O 2 [M-2Br]/2 + calcd＝ 275.2244；found＝275.2251.
10b: 1 H NMR(500MHz,DMSO-d 6 )δ7.35(dd,J＝8.4,2.0Hz,1H),7.26(d,J＝1.9Hz,1H),7.11 (dd,J＝8.3,1.8Hz,1H),7.09–7.00(m,3H),5.98(dddt,J＝23.7,16.8,10.0,6.7Hz,2H),5.15–5.01(m,4H),4.06(dt,J＝37.0,5.6Hz,4H),3.55–3.49(m,2H),3.44–3.35(m,8H), 3.30–3.24(m,2H),3.12(s,7H),3.03(s,6H),2.21(d,J＝9.3Hz,2H),2.11–2.03(m,2H),1.72–1.66(m,2H),1.54(m,2H),1.32–1.23(m,12H),0.88(t,J＝6.2Hz,6H).HRMS(ESI)C 40 H 66 Br 2 N 2 O 2 [M-2Br]/2 + calcd＝303.2557；found＝303.2557.
10c: 1 H NMR(500MHz,DMSO-d 6 )δ7.35(dd,J＝8.4,2.0Hz,1H),7.26(d,J＝1.9Hz,1H),7.11 (dd,J＝8.3,1.9Hz,1H),7.08–6.99(m,3H),6.07–5.90(m,2H),5.14–4.99(m,4H),4.05(dt,J＝36.6,5.6Hz,4H),3.54–3.44(m,2H),3.40(d,J＝6.5Hz,2H),3.37(s,4H),3.30(s,2H),3.28–3.20 (m,2H),3.10(s,6H),3.01(s,6H),2.23–2.02(m,4H),1.61(d,J＝71.1Hz,4H),1.33–1.19(m,20H),0.87(t,J＝6.5Hz,6H).HRMS(ESI)C 44 H 74 Br 2 N 2 O 2 [M-2Br]/2 + calcd＝331.2870；found ＝331.2871.
10d: 1 H NMR(500MHz,DMSO-d 6 )δ7.35(dd,J＝8.4,2.0Hz,1H),7.26(s,1H),7.11(d,J＝8.4 Hz,1H),7.09–7.01(m,3H),6.06–5.90(m,2H),5.14–5.00(m,4H),4.10(s,2H),4.02(t,J＝5.8Hz, 2H),3.50(s,2H),3.43–3.34(m,9H),3.29–3.21(m,2H),3.12(d,J＝7.9Hz,7H),3.02(d,J＝7.6Hz,6H),2.26–2.02(m,4H),1.61(d,J＝73.1Hz,4H),1.24(m,28H),0.86(t,J＝6.2Hz,6H). HRMS(ESI)C 48 H 82 Br 2 N 2 O 2 [M-2Br]/2 + calcd＝359.3183；found＝359.3193.
10e: 1 H NMR(500MHz,DMSO-d 6 )δ7.35(d,J＝8.4Hz,1H),7.25(s,1H),7.16–7.00(m,4H), 6.07–5.89(m,2H),5.16–4.99(m,4H),4.17–3.97(m,4H),3.62–3.27(m,12H),3.06(dd,J＝48.0,14.3Hz,12H),2.28–2.01(m,4H),1.61(d,J＝74.8Hz,4H),1.23(m,36H),0.85(t,J＝6.5Hz,6H). HRMS(ESI)C 52 H 90 Br 2 N 2 O 2 [M-2Br]/2 + calcd＝387.3496；found＝387.3492.
10f: 1 H NMR(500MHz,DMSO-d 6 )δ7.35(d,J＝8.4Hz,1H),7.28–7.23(m,1H),7.14–7.08(m, 1H),7.08–7.01(m,3H),6.0–5.89(m,2H),5.13-5.00(m,4H),4.14–3.98(m,4H),3.58–3.43(m,2H),3.42–3.33(m,8H),3.29–3.19(m,2H),3.11(d,J＝18.9Hz,6H),3.02(d,J＝18.3Hz,6H), 2.26–2.01(m,4H),1.61(d,J＝76.8Hz,4H),1.23(m,44H),0.90–0.81(m,6H).HRMS(ESI)C 56 H 98 Br 2 N 2 O 2 [M-2Br]/2 + calcd＝415.3809；found＝415.3846.
Example 9.
Preparation of Compound 11
The raw materials used are compound 6 and N, N-dimethylpropanediamine, and the preparation method is the same as that of example 5. 1 H NMR(500MHz,DMSO-d 6 ) δ7.93(t,J＝5.7Hz,1H),7.69(t,J＝5.6Hz,1H),7.40(dd,J＝8.4,2.2Hz,1H),7.34(d,J＝2.2Hz,1H),7.08(dd,J＝4.3,2.1Hz,2H),6.96–6.90(m,2H),5.99(dddt,J＝38.1,16.8,10.0,6.7Hz,2H), 5.15–4.99(m,4H),4.52(s,2H),4.41(s,2H),3.47(d,J＝6.7Hz,2H),3.34(d,J＝6.7Hz,2H),3.20(q,J＝6.6Hz,2H),3.12(q,J＝6.6Hz,2H),2.23(t,J＝7.0Hz,2H),2.17(t,J＝7.0Hz,2H), 2.11(s,6H),2.06(s,6H),1.62–1.47(m,4H).HRMS(ESI)C 32 H 47 N 4 O 4 [M+H] + calcd＝551.3592； found＝551.3600.
Example 10.
Preparation of Compounds 12a-12e
The raw materials used are intermediate 11 and halogenated compounds with different chain lengths, and the preparation method is the same as that of example 8.
12a: 1 H NMR(500MHz,DMSO-d 6 )δ8.13(dt,J＝31.3,5.7Hz,2H),7.47–7.41(m,1H),7.36(s, 1H),7.09(d,J＝8.2Hz,2H),6.97–6.89(m,2H),6.09–5.90(m,2H),5.15–5.01(m,4H),4.58(s,2H),4.48(s,2H),3.48(d,J＝6.5Hz,2H),3.34(d,J＝6.4Hz,6H),3.21(dt,J＝27.2,5.1Hz,8H), 3.00(s,12H),1.93–1.77(m,4H),1.68–1.51(m,4H),1.28(h,J＝7.2Hz,4H),0.91(t,J＝7.3Hz,6H).HRMS(ESI)C 40 H 64 Br 2 N 4 O 4 [M-2Br]/2 + calcd＝332.2458；found＝332.2459.
12b: 1 H NMR(500MHz,DMSO-d 6 )δ8.12(dt,J＝31.1,5.8Hz,2H),7.44(dd,J＝8.4,2.1Hz, 1H),7.36(d,J＝2.0Hz,1H),7.09(d,J＝7.8Hz,2H),6.96–6.90(m,2H),6.00(dddt,J＝45.3,16.8, 10.0,6.8Hz,2H),5.16–5.01(m,4H),4.58(s,2H),4.47(s,2H),3.48(d,J＝6.6Hz,2H),3.34(d,J＝4.9Hz,4H),3.29–3.20(m,10H),3.00(s,12H),1.85(dp,J＝17.1,6.0Hz,4H),1.68–1.53(m, 4H),1.26(d,J＝14.7Hz,12H),0.86(t,J＝6.4Hz,6H).HRMS(ESI)C 44 H 72 Br 2 N 4 O 4 [M- 2Br]/2 + calcd＝360.2771；found＝360.2764.
12c: 1 H NMR(500MHz,DMSO-d 6 )δ8.10(dt,J＝23.5,5.8Hz,2H),7.43(dd,J＝8.4,2.0Hz,1H), 7.36(d,J＝1.8Hz,1H),7.09(d,J＝8.6Hz,2H),6.92(dd,J＝8.3,4.1Hz,2H),6.09–5.89(m,2H),5.16–5.00(m,4H),4.57(s,2H),4.46(s,2H),3.48(d,J＝6.6Hz,2H),3.34(d,J＝6.5Hz,8H), 3.22–3.16(m,6H),2.98(s,12H),1.83(dtt,J＝22.0,15.9,7.9Hz,4H),1.67–1.52(m,4H),1.30–1.20(m,20H),0.86(t,J＝6.8Hz,6H).HRMS(ESI)C 48 H 80 Br 2 N 4 O 4 [M-2Br]/2 + calcd＝ 388.3084；found＝388.3064.
12d: 1 H NMR(500MHz,DMSO-d 6 )δ8.22–8.03(m,2H),7.50–7.40(m,1H),7.36(d,J＝1.9Hz, 1H),7.09(d,J＝8.2Hz,2H),6.93(d,J＝8.5Hz,2H),5.99(dddt,J＝45.7,16.8,9.9,6.8Hz,2H),5.17–4.98(m,4H),4.58(s,2H),4.47(s,2H),3.48(d,J＝6.6Hz,2H),3.34(d,J＝5.3Hz,4H), 3.21(dd,J＝26.8,6.1Hz,10H),3.00(s,12H),1.83(ddt,J＝22.7,12.6,8.3Hz,4H),1.68–1.52(m,4H),1.24(m,28H),0.85(t,J＝6.8Hz,6H).HRMS(ESI)C 52 H 88 Br 2 N 4 O 4 [M-2Br]/2 + calcd＝ 416.3397；found＝416.3402.
12e: 1 H NMR(500MHz,DMSO-d 6 )δ8.14(dt,J＝33.2,5.7Hz,2H),7.44(dd,J＝8.4,2.0Hz,1H), 7.36(d,J＝1.9Hz,1H),7.09(d,J＝8.1Hz,2H),6.93(d,J＝8.4Hz,2H),6.12–5.87(m,2H),5.17–5.00(m,4H),4.58(s,2H),4.48(s,2H),3.48(d,J＝6.6Hz,2H),3.34(d,J＝9.2Hz,6H), 3.27–3.21(m,8H),3.00(s,12H),1.82(ddt,J＝22.9,15.3,6.3Hz,4H),1.66–1.52(m,4H),1.24(m,36H),0.85(t,J＝6.7Hz,6H).HRMS(ESI)C 56 H 96 Br 2 N 4 O 4 [M-2Br]/2 + calcd＝444.3710；found ＝444.3696.
Example 11.
Preparation of Compound 13
The starting materials used were intermediate 6 and H-Lys (Boc) -OMe, prepared in the same manner as in example 5.
Example 12.
Preparation of Compound 14
To a solution of compound 15 (2.50 g,2.88 mmol) in methanol (20 mL) was added lithium hydroxide (8 mL, 2M aqueous solution) at 0deg.C. After stirring for 6 hours, the reaction mixture was acidified with dilute hydrochloric acid and the white solid was collected by filtration and the filter cake was washed with cold methanol. The product was dried to give 1.96g of a white solid in 81% yield.
Example 13.
Preparation of Compounds 16a-16h
Intermediate 14 (0.84 g,1.00 mmol) was dissolved in anhydrous acetonitrile (30 mL) and HATU (0.91 g,2.40 mmol) and N, N-diisopropylethylamine (0.39 g,3.00 mmol) were added. The mixture was stirred at 0 ℃ for 30 min, then the corresponding amine (2.40 mmol) was added and the mixture was stirred at room temperature for an additional 10 hours. After the starting material was consumed, the solvent was removed and the residue was neutralized with 2N diluted hydrochloric acid and washed with saturated sodium chloride. The organic extract was dried and concentrated under reduced pressure and the residue was subjected to flash column chromatography to give crude product 15a-15h, which was dissolved in dichloromethane (10 mL) and trifluoroacetic acid (8 mL) was slowly added. After the reaction is completed, the solvent is concentrated under reduced pressure, and the residue is purified by a C-18 column and freeze-dried to obtain the target product.
16a: 1 H NMR(500MHz,CD 3 OD)δ7.32(dd,J＝8.4,2.2Hz,1H),7.24(d,J＝2.2Hz,1H),7.03(d, J＝8.2Hz,2H),6.89(dd,J＝14.9,8.3Hz,2H),5.92(dddt,J＝45.2,16.8,10.0,6.5Hz,2H),5.04–4.91(m,4H),4.55(d,J＝2.9Hz,2H),4.44(d,J＝14.9Hz,1H),4.39–4.33(m,2H),4.25(dd, J＝8.2,5.7Hz,1H),3.44(qd,J＝13.7,12.1,4.8Hz,2H),3.27(d,J＝6.5Hz,2H),3.08(d,J＝3.6Hz,4H),2.81–2.60(m,4H),1.79–1.57(m,4H),1.54–1.44(m,4H),1.41–1.35(m,4H),1.22(ddd,J ＝28.7,14.5,7.2Hz,8H),0.85–0.79(t,6H).HRMS(ESI)C 42 H 65 N 6 O 6 [M+H] + calcd＝749.4960； found＝749.4961.
16b: 1 H NMR(500MHz,CD 3 OD)δ7.42(dd,J＝8.4,2.2Hz,1H),7.34(s,1H),7.13(d,J＝8.2Hz,2H),6.99(dd,J＝14.8,8.3Hz,2H),6.11–5.92(m,2H),5.13–5.01(m,4H),4.65(d,J＝1.4Hz, 2H),4.55–4.34(m,4H),3.49(d,J＝7.3Hz,4H),3.20–3.16(m,4H),2.81(dtt,J＝12.2,8.2,4.7Hz,4H),1.89–1.67(m,4H),1.61(ddt,J＝17.1,8.7,5.5Hz,4H),1.52–1.47(m,4H),1.30(q,J＝8.7, 7.2Hz,16H),0.91–0.88(t,6H).HRMS(ESI)C 46 H 73 N 6 O 6 [M+H] + calcd＝805.5586；found＝ 805.5585.
16c: 1 H NMR(500MHz,CD 3 OD)δ7.32(dd,J＝8.4,2.1Hz,1H),7.24(d,J＝1.9Hz,1H),7.03(d, J＝8.6Hz,2H),6.89(dd,J＝13.9,8.3Hz,2H),5.92(dddt,J＝44.2,16.8,10.0,6.5Hz,2H),5.06–4.92(m,4H),4.56(s,2H),4.47–4.23(m,4H),3.45(ddt,J＝20.4,15.5,7.4Hz,2H),3.27(d,J ＝6.6Hz,2H),3.09(ddd,J＝10.7,7.4,4.0Hz,4H),2.75(tt,J＝12.2,5.6Hz,4H),1.80–1.60(m,4H),1.58–1.50(m,4H),1.44–1.36(m,4H),1.24–1.17(m,24H),0.80(t,J＝7.0Hz,6H).HRMS (ESI)C 50 H 81 N 6 O 6 [M+H] + calcd＝861.6212；found＝861.6212.
16d: 1 H NMR(500 MHz,CD 3 OD)δ7.32(dd,J＝8.4,2.2 Hz,1H),7.25(d,J＝2.2 Hz,1H),7.03(d, J＝8.0 Hz,2H),6.89(dd,J＝14.4,8.3 Hz,2H),6.03–5.82(m,2H),5.05–4.91(m,4H),4.59–4.52(m,2H),4.47–4.23(m,4H),3.44(qd,J＝13.1,10.9,4.1 Hz,2H),3.27(q,J＝5.6,4.4 Hz,2H),3.08 (s,4H),2.73(tq,J＝14.1,5.7,4.4 Hz,4H),1.79–1.59(m,4H),1.57–1.47(m,4H),1.40(q,J＝12.2,7.1 Hz,4H),1.34–1.14(m,24H),1.00(t,J＝7.3 Hz,8H),0.79(t,J＝7.0 Hz,6H).HRMS(ESI) C 54 H 89 N 6 O 6 [M+H] + calcd＝917.6838；found＝917.6841.
16e: 1 H NMR(500 MHz,CD 3 OD)δ7.32(dt,J＝8.4,2.9 Hz,1H),7.24(s,1H),7.02(d,J＝6.9 Hz, 2H),6.94–6.82(m,2H),6.02–5.81(m,2H),5.08–4.90(m,4H),4.54(s,2H),4.47–4.22(m,4H),3.44(tt,J＝18.2,9.2 Hz,2H),3.26(d,J＝6.9 Hz,2H),3.08(s,4H),2.80–2.59(m,4H),1.79–1.57 (m,4H),1.56–1.45(m,4H),1.43–1.36(m,4H),1.18(d,J＝10.6 Hz,38H),0.79(t,J＝7.0 Hz,6H).HRMS(ESI)C 58 H 97 N 6 O 6 [M+H] + calcd＝973.7464；found＝973.7461.
16f: 1 H NMR(500 MHz,CD 3 OD)δ7.41(dd,J＝8.4,2.2 Hz,1H),7.34(d,J＝2.2 Hz,1H),7.12(d, J＝7.6 Hz,2H),6.99(dd,J＝16.4,8.6 Hz,2H),6.01(dddt,J＝47.3,16.8,10.1,6.6 Hz,2H),5.17–5.01(m,4H),4.65(s,2H),4.59–4.39(m,4H),3.53(tq,J＝14.6,7.3,6.9 Hz,2H),3.36(d,J＝ 6.6 Hz,2H),3.17(q,J＝7.3 Hz,4H),2.84(dq,J＝12.4,5.0,4.1 Hz,4H),1.93(m,6H),1.90(d,J＝9.6 Hz,8H),1.79–1.70(m,8H),1.67–1.62(m,8H),1.54–1.47(m,12H).HRMS(ESI)C 56 H 81 N 6 O 6 [M+H] + calcd＝933.6212；found＝933.6207.
16g: 1 H NMR(500 MHz,CD 3 OD)δ7.31–7.19(m,6H),7.09(d,J＝8.1 Hz,4H),7.04–6.97(m, 2H),6.87(dd,J＝8.3,3.5 Hz,2H),5.90(dddt,J＝41.9,16.8,10.0,6.6 Hz,2H),5.03–4.90(m,4H),4.55(s,2H),4.46–4.29(m,4H),4.24(d,J＝5.1 Hz,4H),3.41(qt,J＝14.6,6.9 Hz,2H),3.26(d,J ＝6.8 Hz,2H),2.82–2.66(m,4H),1.79–1.61(m,4H),1.58–1.48(m,4H),1.36–1.22(m,4H),1.17(d,J＝5.5 Hz,18H).HRMS(ESI)C 56 H 77 N 6 O 6 [M+H] + calcd＝929.5899；found＝929.5898.
16h: 1 H NMR(500 MHz,CD 3 OD)δ7.53–7.36(m,8H),7.35–7.15(m,12H),7.03–6.93(m,2H), 6.90–6.79(m,2H),6.03–5.74(m,2H),5.07–4.87(m,4H),4.54(s,2H),4.41(dd,J＝15.0,9.1 Hz,2H),4.37–4.25(m,6H),3.40(dq,J＝17.4,8.1,7.2 Hz,2H),3.30–3.21(m,2H),2.67(qq,J＝12.5, 7.0 Hz,4H),1.78–1.58(m,4H),1.50(ddq,J＝22.3,16.5,9.0,8.3 Hz,4H),1.28(dp,J＝22.3,7.3Hz,4H).HRMS(ESI)C 60 H 69 N 6 O 6 [M+H] + calcd＝969.5273；found＝969.5276.
Application example 1.
In vitro antibacterial Activity test
Micro broth dilution method: fresh nutrient broth culture medium is added into a 96-well plate, a certain amount of sterile water or DMSO solution (2560 mug/mL, m/V) of a compound to be tested and a control drug is sequentially added into the No. 1 holes of each row of the 96-well plates A-H and diluted by a double dilution method, then a proper amount of bacteria solution with a certain turbidity is inoculated, after incubation for 24 hours at the constant temperature of 37 ℃, the bacterial growth condition of each hole is observed, and the Minimum Inhibitory Concentration (MIC) of the drug is read. The MIC of the test strain was determined by visual inspection as the lowest concentration tube of the drug with no bacterial growth.
Experimental results:
TABLE 1 antibacterial Activity of honokiol antibacterial peptide mimics
MIC values (2 gram-negative bacteria, 4 gram-positive bacteria) of the corresponding compounds for six clinically common bacteria are given in table 1. In general, the target compounds have broad spectrum antimicrobial activity, but based on MIC data, these compounds were found to have better antimicrobial activity against gram-positive bacteria than gram-negative bacteria. Compounds 10a-10c,12c,16d all showed broad-spectrum bactericidal activity, especially with MIC values of 0.5-2. Mu.g/mL for gram-positive bacteria. At the same time they also show better activity against gram-negative bacteria, especially the lowest inhibitory activity of compound 10b against E.coli ATCC25922 can reach 4. Mu.g/mL, all compounds show poor or moderate bacteriostatic activity against P.aeruginosa ATCC 27853.
Application example 2.
Sterilization kinetics test
Culturing Staphylococcus aureus and Escherichia coli overnight, diluting to a certain multiple, collecting a certain amount of bacterial liquid, and culturing in shaking table
Bacteria are grown to the logarithmic phase, and test compounds or control drugs with different concentrations are added. After dosing for 0h, 1h, 3h, 5h, 7 h, 9h, 100 μl each was taken in groups of different compounds and different concentrations into 96 well plates, centrifuged for 3min, the supernatant was discarded, 100 μl of 1×pbs resuspended bacteria, and ten-fold gradient diluted, 10 μl was taken and dropped onto MHA agar plates, three of each concentration in parallel, the agar plates were incubated in an incubator for 24h, and the colony count was calculated from the dilution.
Compound 10b, which was superior in primary antibacterial activity, was selected as a subject to study its bactericidal efficacy against staphylococcus aureus and escherichia coli at different concentrations over time, as shown in fig. 1. The result shows that the compound 13b has a rapid sterilization effect, and can kill staphylococcus aureus and escherichia coli completely within 3 hours at the concentration of 8 mug/mL and 32 mug/mL.
Application example 3.
Drug resistance inducibility test
The MIC values of the compounds and positive control drugs (norfloxacin and polymyxin) for Staphylococcus aureus and Escherichia coli, respectively, were first determined. The compound and the control were added to the agar solution to a final concentration of 1/2MIC. Staphylococcus aureus and escherichia coli were then cultured on agar plates containing different drugs, respectively, and the surviving bacteria were inoculated into the next new plate for several days, and then MIC values of the compounds and positive drugs for the bacteria on the agar plates were determined.
Compound 10b was also selected for evaluation in terms of resistance induction against staphylococcus aureus and escherichia coli. The result shows that the compound 10b is excellent in inhibiting bacterial drug resistance, and through 15 generations of drug resistance induction of bacteria at the concentration of MIC/2 of the drug, the MIC of the compound 10b to staphylococcus aureus and escherichia coli is basically kept stable, and the MIC of the control drugs norfloxacin and colistin to bacteria is respectively increased by 256 times and 64 times, as shown in figure 2, the compound has the characteristic that the antibacterial peptide is not easy to be resistant to bacteria.
Application example 4.
Cytotoxicity test
Sulfonyl rhodamine B assay: cell proliferation of adherent cells was determined by the sulfonylrhodamine B assay (SRB). Cells were seeded in 96-well plates and then treated with different concentrations of drug. After 72 hours of incubation, the cells were fixed with 10% trichloroacetic acid at 4℃for 1 hour, washed 5 times with sterile water and air-dried. Surviving cells were stained with 0.4% (w/v) sulforhodamine B for 20 min at room temperature and washed 5 times with 1% acetic acid. Bound sulfonylrhodamine B was dissolved with 10mM Tris and absorbance was measured at 540 nm.
Compound 13b was selected and assayed for toxicity to mammalian cell HepG2 at various concentrations of drug. As shown in fig. 3, the compound had no inhibitory effect on the cells at low concentrations, but only at high concentrations, indicating that the compound had lower cytotoxicity.
Finally, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited to the above-mentioned embodiments, but may be modified or substituted for some of them by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (3)

1. A honokiol antibacterial peptide analogue or a pharmaceutically acceptable salt thereof with antibacterial activity, characterized in that the compound structure is as follows:
2. a process for the preparation of a honokiol antibacterial peptide analogue having antibacterial activity as claimed in claim 1 or a pharmaceutically acceptable salt thereof, which comprises the following steps:
condensing the compound 6 with N, N-dimethylpropanediamine under the catalysis of a condensing agent HATU to obtain a compound 11, and reacting the compound 11 with bromides with different chain lengths to obtain 12b-12e;
wherein, the structural formula of the compound 6 is as follows:
3. the use of a honokiol antibacterial peptide mimetic having antibacterial activity as claimed in claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of bacterial infections which are manifested by infections caused by staphylococcus aureus, bacillus subtilis, escherichia coli.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108558682A (en) * 2018-05-21 2018-09-21 郑州大学 Fragrant phenol quaternary ammonium salt antibacterial peptide mimics with antibacterial activity and preparation method thereof
CN108794343A (en) * 2018-05-21 2018-11-13 郑州大学 Amides fragrance phenol antibacterial peptide mimics with antibacterial activity and preparation method thereof
CN113024404A (en) * 2021-03-10 2021-06-25 郑州大学 Quaternary ammonium salt type honokiol/magnolol derivative and preparation method and application thereof

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Publication number Priority date Publication date Assignee Title
CN108558682A (en) * 2018-05-21 2018-09-21 郑州大学 Fragrant phenol quaternary ammonium salt antibacterial peptide mimics with antibacterial activity and preparation method thereof
CN108794343A (en) * 2018-05-21 2018-11-13 郑州大学 Amides fragrance phenol antibacterial peptide mimics with antibacterial activity and preparation method thereof
CN113024404A (en) * 2021-03-10 2021-06-25 郑州大学 Quaternary ammonium salt type honokiol/magnolol derivative and preparation method and application thereof

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De Novo Design of Flavonoid-Based Mimetics of Cationic Antimicrobial Peptides: Discovery, Development, and Applications;Lin, Shuimu et al.;《Accounts of Chemical Research》;第54卷(第1期);第104-119页 *
Design, synthesis, and biological evaluation of membrane-active honokiol derivatives as potent antibacterial agents;Wang, Yinhu et al.;《 European Journal of Medicinal Chemistry》;第240卷;第114593页 *
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