Cannabinoid phenolic antibacterial peptide simulant with broad-spectrum antibacterial activity as well as preparation method and application of cannabinoid phenolic antibacterial peptide simulant

Abstract

The invention discloses a cannabinoid phenol antibacterial peptide simulant with broad-spectrum antibacterial activity as well as a preparation method and application of the simulant. The simulant has a structure as shown in a formula (I): # imgabs0 # formula (I). The preparation method provided by the invention is simple, effective and high in yield. An in-vitro activity result proves that the compound shown in the formula (I) has the characteristics of broad-spectrum antibacterial activity, rapid sterilization, difficulty in generating drug resistance to bacteria and the like. Therefore, the compound is expected to be developed into a novel broad-spectrum antibacterial agent to resist bacterial infection.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and relates to a cannabigerol antibacterial peptide mimic with broad-spectrum antibacterial activity, its preparation method and use. Background Art

[0002] Cannabigerol (CBG) is a non-psychoactive cannabinoid that naturally exists in cannabis plants in very small amounts and has great application prospects in drug development (Jentsch et al. , 2020).

[0003] Recent studies have shown that cannabigerol has a wide range of pharmacological effects. Specifically, CBG has therapeutic potential in the treatment of neurological diseases (Valdeolivas et al. , 2015) and inflammatory bowel disease (Borrelli et al. , 2013), and has been proven effective against glaucoma, psoriasis, Sjogren's syndrome and pain. Searching for antibacterial lead compounds from plant natural products has attracted great research interest among researchers. It has been reported that CBG has an inhibitory effect on methicillin-resistant Staphylococcus aureus and an inhibitory effect on the planktonic growth of Streptococcus mutans (Feldman et al ., 2021). In addition, CBG has an interfering effect on quorum sensing-mediated processes in Vibrio harveyi at concentrations that do not affect the growth of plankton (Aqawi et al ., 2020).

[0004] It is worth noting that cannabigerol mainly inhibits the growth of Gram-positive bacteria and has a poor antibacterial effect on Gram-negative bacteria. Just as many antibacterial compounds cannot fight against Gram-negative pathogens due to the permeability barrier, the low permeability of the outer membrane may be the reason for the poor effect of cannabigerol (Farha et al ., 2020). In terms of the structural modification in its antibacterial field, there is very little research on CBG. And CBG has poor water solubility, which brings difficulties in drug development. Summary of the Invention

[0005] Aiming at the deficiencies and needs of the prior art, the purpose of the present invention is to provide a cannabigerol antibacterial peptide mimic with broad-spectrum antibacterial activity, its preparation method and application.

[0006] Specifically, the present invention is realized through the following technical solutions: In one aspect of the present invention, the present invention provides a cannabigerol antibacterial peptide mimic with broad-spectrum antibacterial activity, and the mimic is a compound having the structure shown in formula (I) or a pharmaceutically acceptable salt of the compound: Formula (I).

[0007] In other aspects of the present invention, there is provided a method for preparing cannabigerol antibacterial peptide mimics with broad-spectrum antibacterial activity, the method comprising the following steps: (1) Reacting oleanol and geraniol in a molar ratio of 1.5 - 2:1 - 1.2 to prepare intermediate 1; (2) Reacting intermediate 1 with 1,2-dibromoethane in a molar ratio of 1:10 - 15 to obtain intermediate 2; (3) Reacting intermediate 2 with 2-(dimethylamino)- N -hexylacetamide in a molar ratio of 1:3 - 6 to generate the target compound.

[0008] Preferably, the reaction in step (1) is carried out in the presence of a catalyst; the amount of the catalyst is 1.5 - 2.5 g per millimole of geraniol; the catalyst is selected from one or more of aluminum oxide, zinc chloride, magnesium sulfate, p-toluenesulfonic acid, boron trifluoride diethyl etherate.

[0009] Preferably, the reaction in step (1) is carried out in a solvent selected from one or more of tetrahydrofuran, acetonitrile, chloroform, dichloromethane, 1,4-dioxane, methanol, 1,2-dichloroethane.

[0010] Preferably, the reaction in step (2) is carried out in the presence of an acid-binding agent, the acid-binding agent being an inorganic base; the molar ratio of intermediate 1 to the acid-binding agent is 1:4 - 6.

[0011] Preferably, the reaction in step (2) is carried out in a solvent selected from one or more of tetrahydrofuran, acetonitrile, dichloromethane, 1,4-dioxane, methanol, acetone.

[0012] Preferably, the reaction in step (3) is carried out in a solvent selected from one or more of tetrahydrofuran, acetonitrile, ethanol, methanol, 1,4-dioxane.

[0013] In other aspects of the present invention, there is also provided the use of the cannabigerol antibacterial peptide mimics with broad-spectrum antibacterial activity in the preparation of antibacterial drugs.

[0014] Preferably, the antibacterial drug is a drug that inhibits Gram-positive bacteria and Gram-negative bacteria.

[0015] In other aspects of the present invention, there is also provided a broad-spectrum antibacterial agent comprising an effective dose of the cannabigerol antibacterial peptide mimics.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The method for preparing the compound of the present invention is simple, effective and has a high yield. The in vitro activity results confirm that the compound has characteristics such as broad-spectrum antibacterial activity, fast bactericidal speed and low tendency to develop drug resistance. Therefore, the compound is expected to be developed into a broad-spectrum antibacterial agent to combat bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Time-kill curves of the compound of the present invention against Staphylococcus aureus and Escherichia coli; wherein, a. Staphylococcus aureus ATCC 29213; b. Escherichia coli ATCC 25922; Figure 2 Drug resistance evaluation of the compound of the present invention against Staphylococcus aureus and Escherichia coli; wherein, a. Staphylococcus aureus ATCC 29213; b. Escherichia coli ATCC 25922. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturers.

[0019] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described herein are only for demonstration purposes.

[0020] Example 1 Preparation of a cannabinol antibacterial peptide mimic with broad-spectrum antibacterial activity. The synthetic route is as follows:

[0021] Reagents and reaction conditions used in the synthetic route: (a) acidic alumina, 1,2-dichloroethane, 83 °C, 6 h; (b) 1,2-dibromoethane, potassium carbonate, acetone, 57 °C, 24 h; (c) ethanol, 85 °C, 10 h.

[0022] The specific preparation process includes the following steps: (1) Preparation of cannabinol (Intermediate 1) Dissolve oleyl alcohol (1.5 mmol) and geraniol (1 mmol) in 12 mL of 1,2-dichloroethane, then add 2 g of acidic alumina and react under reflux at 83 °C for 6 h. Monitor the reaction by TLC until completion. Filter under reduced pressure, distill off 1,2-dichloroethane, dilute the reaction solution with 15 mL of ethyl acetate, wash it 3 times with distilled water (3 × 15 mL), then wash it once with saturated brine (1 × 15 mL). Dry the organic phase with anhydrous magnesium sulfate, filter under reduced pressure, evaporate and concentrate. The crude product is purified by silica gel chromatography column (200 -300 mesh), using petroleum ether:dichloromethane = 10:1 as the eluent to obtain a white solid product.

[0023] (2) (E) Preparation of -1,3-bis(2-bromoethoxy)-2-(3,7-dimethylocta-2,6-dien-1-yl)-5-pentylbenzene (Intermediate 2) Dissolve Intermediate 1 (mmol) and 1,2-dibromoethane (mmol) successively in 10 mL of acetone, then add potassium carbonate. Stir at 57 °C for 24 h. Monitor the reaction by TLC until completion. Filter under reduced pressure, evaporate the solvent to dryness. The crude product is purified by silica gel chromatography column (200 -300 mesh), using petroleum ether:ethyl acetate = 250:1 as the eluent to obtain a pale yellow oil.

[0024] (3) (E) -[[]]END]] N,N' -(((2-(3,7-dimethylocta-2,6-dien-1-yl)-5-pentyl-1,3-phenylene)bis(oxy))bis(ethane-2,1-diyl))bis(2-(hexylamino)- N,N -dimethyl-2-oxoethane-1-ammonium) bromide (Compound of Formula (I)) In a pressure-resistant bottle, dissolve Intermediate 2 (mmol) and 2-(dimethylamino)-N-hexylethanamide (mmol) in 6 mL of absolute ethanol, stir at 85 °C for 48 h, and monitor the reaction by TLC until the raw materials are completely reacted. Cool the system to room temperature, and then concentrate by vacuum evaporation to obtain the crude product. Purify and separate using a silica gel chromatography column (200 -300 mesh), using dichloromethane:methanol = 20:1 as the eluent to obtain a white solid. HRMS (ESI) C 45 H 82 Br 2 N 4 O 4 [M − 2Br] / 2+ calcd = 371.3163;found = 371.3154.

[0025] The chemical formula of the compound shown in formula (I) is C 45 H 82 Br 2 N 4 O 4 , with the Chinese name (E)-N,N'-(((2-(3,7-dimethylocta-2,6-dien-1-yl)-5-pentyl-1,3-phenylene)bis(oxy))bis(ethane-2,1-diyl))bis(2-(hexylamino)-N,N-dimethyl-2-oxoethan-1-aminium) bromide and the English name (E)-N,N'-(((2-(3,7-dimethylocta-2,6-dien-1-yl)-5-pentyl-1,3-phenylene)bis(oxy))bis(ethane-2,1-diyl))bis(2-(hexylamino)-N,N-dimethyl-2-oxoethan-1-aminium) bromide.

[0026] Pharmaceutically acceptable salts of the compound shown in formula (I) include inorganic acid salts such as hydrochloride, sulfate, hydrobromide, phosphate and nitrate; organic acid salts such as acetate, propionate, oxalate, succinate, lactate, tartrate, citrate, maleate, mesylate, p-toluenesulfonate, benzenesulfonate and ascorbate; inorganic base salts such as sodium salt, potassium salt, magnesium salt, zinc salt, calcium salt and aluminum salt; and organic base salts such as arginine salt, benzathine salt, choline salt, diethylamine salt, diolamine salt, glycine salt, tromethamine salt, ethanolamine salt and lysine salt.

[0027] Example 2. Determination of the in vitro antibacterial activity of the compound shown in formula (I) Experimental method: The minimum inhibitory concentration (MIC) of cannabigerol, the compound of formula (I), vancomycin and colistin against the selected test strains was detected by the microbroth dilution method specified in the guidelines of the Clinical and Laboratory Standards Institute (CLSI). First, the compound to be tested was dissolved in DMSO / H 2 O to prepare a stock solution with a final concentration of 5120 mg / L. The stock solution was diluted to the required concentration with MHB, and then serially diluted 2-fold on a 96-well plate with MHB, with concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0 mg / L. The concentration of the bacterial cell suspension was adjusted to about 1 × 10 6 CFU / mL. These suspensions were added to the diluted solutions containing the samples. The OD 600 value was measured after incubation at 37 °C for 18 - 24 h. The MIC value was defined as the lowest concentration at which there was no significant growth compared to the control group. The experimental results are listed in Table 1.

[0028] Table 1. In vitro antibacterial activity of the test compounds (unit: μg / mL) ; Note: a S. a. : Staphylococcus aureus ATCC 29213, b MRSA: Staphylococcus aureus ATCC 43300, c E. f. : Enterococcus faecalis ATCC 29212, d B. s. : Bacillus subtilis ATCC 6051, e A. b. : Acinetobacter baumannii ATCC19606, f S. e. Salmonella typhimurium SL1344, g K. p. : Klebsiella pneumoniae ATCC 13883, h E. c. : Escherichia coli ATCC 25922. i P. a. : Pseudomonas aeruginosa PAO1, j ND: Not determined.

[0029] As can be seen from Table 1, the compound shown in formula (I) exhibits excellent antibacterial activity against both Gram-positive and Gram-negative bacteria. Its minimum inhibitory concentration range against Gram-positive bacteria is 1 - 2 μg / mL, and against Gram-negative bacteria is 2 - 8 μg / mL. Compared with the parent compound cannabigerol, the antibacterial effect of the compound shown in formula (1) against Gram-negative bacteria has been significantly improved.

[0030] Example 3. Time-kill kinetics test of the compound shown in formula (I) Single colonies of Staphylococcus aureus and Escherichia coli were selected and placed in 1 mL of MHB, incubated in a shaker (200 rpm, 37 °C) for 16 - 18 h, and then the bacterial cell concentration was adjusted to 1 × 10 6 CFU / mL. Subsequently, different concentrations of the compound shown in formula (I) (1 ×, 2 ×, 4 ×, 8 × MIC) were added to the bacterial suspension and incubated at 37 °C. The colony counts were determined at 0, 0.5, 1, 2, 4, 8, 24 h. Vancomycin and colistin (4 ×, 8 × MIC) were used as positive control drugs, and no drug was added as the blank control. The experimental results are as Figure 1 shown.

[0031] FromFigure 1 As can be seen, the compound shown in formula (I) can completely kill Staphylococcus aureus ATCC 29213 (A) and Escherichia coli ATCC 25922 (B) within 0.5 h at 4 × MIC. In contrast, the positive control drugs vancomycin and colistin failed to completely kill these two strains within 4 h at 4 × MIC. Only when vancomycin and colistin reached 8 × MIC could they completely kill Staphylococcus aureus and Escherichia coli within 4 h. The results indicate that the compound shown in formula (I) has high killing efficiency and speed, and can effectively avoid the generation of bacterial drug resistance.

[0032] Example 4. Study on drug resistance of the compound shown in formula (I) The MIC values of the compound shown in formula (I) and norfloxacin against Staphylococcus aureus ATCC 29213, and the MIC values of the compound shown in formula (I) and colistin against Escherichia coli ATCC 25922 were determined according to the method described in Example 2. Briefly, bacteria grown in a medium containing 0.5 × MIC were used to prepare the bacterial suspension for the next MIC determination. After culturing at 37 °C for 24 h, the new MIC value was determined, and this process was repeated for 20 days. All determinations were performed with at least two biological replicates. The experimental results are as Figure 2 shown.

[0033] As Figure 2 can be seen, after 20 consecutive passages, the MIC values of the compound shown in formula (I) against Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 did not change at all. In contrast, norfloxacin and colistin could rapidly induce bacterial drug resistance. After 20 days, the MIC value of norfloxacin against Staphylococcus aureus ATCC 29213 increased from 1 μg / mL to 256 μg / mL, and the MIC value of colistin against Escherichia coli ATCC 25922 increased from 0.5 μg / mL to 32 μg / mL. The results indicate that the compound shown in formula (I) is not likely to generate drug resistance in bacteria.

Claims

1. A cannabinoid antimicrobial peptide mimetic with broad-spectrum antimicrobial activity, characterized in that: The mimetic is a compound having a structure shown in formula (I) or a pharmaceutically acceptable salt of the compound: Formula (I).

2. A method for preparing the cannabinoid antimicrobial peptide mimetic with broad-spectrum antimicrobial activity according to claim 1, characterized in that: The following steps are involved: (1) olivetol and geraniol are reacted at a molar ratio of 1.5-2:1-1.2 to prepare intermediate 1; (2) Intermediate 1 reacts with 1,2-dibromoethane at a molar ratio of 1:10-15 to obtain intermediate 2; (3) Intermediate 2 and 2-(dimethylamino)- N -hexyl acetamide reacts at a molar ratio of 1:3-6 to generate the target compound.

3. The method according to claim 2, characterized in that The reaction in step (1) is carried out in the presence of a catalyst; the amount of the catalyst used is 1.5-2.5 g per millimole of geraniol; the catalyst is selected from one or more of aluminum oxide, zinc chloride, magnesium sulfate, p-toluenesulfonic acid, and boron trifluoride etherate.

4. The method according to claim 2, characterized in that: The reaction of step (1) is carried out in a solvent, and the solvent is selected from one or more of tetrahydrofuran, acetonitrile, chloroform, dichloromethane, 1,4-dioxane, methanol, and 1,2-dichloroethane.

5. The method according to claim 2, characterized in that: The reaction in step (2) is carried out in the presence of an acid-binding agent, which is an inorganic base; the molar ratio of the intermediate 1 to the acid-binding agent is 1:4-6.

6. The method according to claim 5, characterized in that: The reaction in step (2) is carried out in a solvent, and the solvent is selected from one or more of tetrahydrofuran, acetonitrile, dichloromethane, 1,4-dioxane, methanol, and acetone.

7. The method according to claim 2, characterized in that The reaction of step (3) is carried out in a solvent; the solvent is selected from one or more of tetrahydrofuran, acetonitrile, ethanol, methanol, and 1,4-dioxane.

8. Use of the cannabinoid antimicrobial peptide mimetics with broad-spectrum antimicrobial activity as claimed in claim 1 in the preparation of antibacterial drugs.

9. The use according to claim 8, characterized in that The antibacterial drug is a drug that inhibits Gram-positive bacteria and Gram-negative bacteria.

10. A broad-spectrum antibacterial agent, characterized in that: The antibacterial agent comprises an effective dose of the cannabinoid antibacterial peptide mimetic according to claim 1.

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