A compound for selectively inhibiting staphylococcus aureus, and a preparation method, application and product thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a compound for selectively inhibiting staphylococcus aureus, a preparation method, application and product thereof. It is found through a bacteriostatic experiment that the compound has a stronger selective inhibiting effect on three kinds of gram-positive bacteria and three kinds of gram-negative bacteria than cannabidiol, overcomes the defect that cannabidiol has no differential inhibiting effect on staphylococcus aureus, bacillus licheniformis and enterococcus faecium, and can be used for preparing a product for selectively inhibiting staphylococcus aureus, has a good application prospect, and has excellent bacteriostatic effect. The compound is obtained through an ozone oxidation reaction of cannabidiol, the method is simple, production efficiency is high, and the compound is beneficial to industrialization and popularization.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a cannabidiol ozonolysis product and its preparation method, as well as its application and product in the preparation of selectively inhibiting Staphylococcus aureus. Background Technology

[0002] Cannabidiol (CBD) Cannabidiol CBD possesses various pharmacological properties, such as antibacterial activity. CBD has shown antimicrobial activity against Gram-positive bacteria, including Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus Streptococcus pneumoniae () Streptococcus penumoniae Furthermore, CBD exhibits selective bactericidal activity against specific subgroups of Gram-negative bacteria (such as Neisseria gonorrhoeae and Neisseria meningitidis). Literature reports that CBD inhibits three types of Gram-positive bacteria (Staphylococcus aureus, Bacillus licheniformis, and Enterococcus faecalis) but has no inhibitory effect on three types of Gram-negative bacteria (Aeromonas hydrophila, Aeromonas tempera, and Proteus vulgaris). This may be because Gram-negative bacteria possess a difficult-to-penetrate lipopolysaccharide outer membrane, making them less susceptible to drug effects than Gram-positive bacteria. Among these three Gram-positive bacteria, Staphylococcus aureus is a common pathogen, while Bacillus licheniformis and Enterococcus faecalis are both probiotics, and Enterococcus faecalis is a normal part of the human gut flora. Therefore, the indiscriminate inhibitory effect of CBD on these three bacteria may lead to an imbalance in the human gut microbiota during clinical treatment. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a compound that can selectively inhibit Staphylococcus aureus, as well as its preparation method, application and product, in order to address the deficiency of cannabidiol indiscriminately inhibiting Staphylococcus aureus, Bacillus licheniformis and Enterococcus faecalis.

[0004] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0005] In a first aspect, the present invention provides a compound that selectively inhibits Staphylococcus aureus, the chemical structure of which is as follows:

[0006] .

[0007] The compound was named systematically as: 4-((2) S 4 S 5 R 10 R )-6-hydroxy-4-methoxy-2-methyl-8-pentyl-4,5-dihydro-2,5-methanobenzo[ d[1,3]dioxepin-10-yl)butan-2-one, referred to as CBD-O3.

[0008] In a second aspect, the present application provides a preparation method of the compound, comprising the following steps: performing an ozonation reaction on cannabidiol to obtain a cannabidiol ozonation product preparation liquid, and performing separation and purification on the cannabidiol ozonation product preparation liquid to obtain the compound for selectively inhibiting Staphylococcus aureus.

[0009] In the preparation method, further, the specific operation of performing the ozonation reaction on the cannabidiol comprises the following steps: introducing ozone into a cannabidiol solution to perform the reaction, pausing the introduction of ozone into the cannabidiol solution every 25-35 min during the reaction, adding methanol into the cannabidiol solution and performing ultrasonic treatment, continuing the introduction of ozone into the cannabidiol solution to perform the reaction, and obtaining the cannabidiol ozonation product preparation liquid after the reaction is completed.

[0010] Further, the concentration of the cannabidiol solution is 50-70 mg / mL, the concentration of the ozone is 0.6-0.8 µmol / mL, and the mass ratio of the methanol to the cannabidiol is 11.30-15.82:1.

[0011] Further, the ultrasonic treatment lasts for 1-2 min, and the total reaction time is 2.0-2.5 h.

[0012] Further, the specific operation of the separation and purification comprises the following steps: performing silica gel column chromatography separation on the cannabidiol ozonation product preparation liquid to obtain a crude product, and further purifying the crude product by means of preparative chromatography to obtain the compound for selectively inhibiting Staphylococcus aureus.

[0013] Further, the adsorbent used in the silica gel column chromatography separation is 30-40 g of silica gel powder with a particle size of 100-200 mesh, and the chromatography column has a specification of φ32 mm and an effective length of 305 mm; the equipment used in the preparative chromatography is a Shimadzu LC-8A type preparative liquid chromatograph, the chromatography column is an Ultimate XB-C18, and the chromatography column has a specification of 5 µm and 21.2×250 mm.

[0014] In a third aspect, the present application provides an application of the compound in the preparation of a product for selectively inhibiting Staphylococcus aureus.

[0015] In the application, further, the concentration of the compound in the product for selectively inhibiting Staphylococcus aureus is 1-5 mg / mL.

[0016] In a fourth aspect, the present application provides a product for selectively inhibiting Staphylococcus aureus, comprising the compound.

[0017] The product is further obtained by diluting the compound with dimethyl sulfoxide to a concentration of 1-5 mg / mL.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] 1. The present application provides a compound capable of selectively inhibiting Staphylococcus aureus, which is found by bacteriostatic experiment to have a stronger selective inhibitory effect on three kinds of gram-positive bacteria and three kinds of gram-negative bacteria than cannabidiol, and overcomes the defect of cannabidiol in indiscriminately inhibiting Staphylococcus aureus, Bacillus licheniformis and Enterococcus faecium, and can be used for preparing a product for selectively inhibiting Staphylococcus aureus, and has excellent bacteriostatic effect and good application prospect.

[0020] 2. The present application also provides a preparation method of the compound, which is obtained by cannabidiol ozonation reaction, and has the advantages of simple method, high production efficiency and facilitation of industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0022] Figure 1 The structural formula of the compound CBD-O3 obtained in the embodiments of the present application is shown in the following figure:

[0023] Figure 2 The nuclear magnetic resonance hydrogen spectrum of CBD-O3 is shown in the following figure:

[0024] Figure 3 The nuclear magnetic resonance carbon spectrum of CBD-O3 is shown in the following figure:

[0025] Figure 4 The nuclear magnetic resonance HSQC spectrum of CBD-O3 is shown in the following figure:

[0026] Figure 5 The nuclear magnetic resonance HMBC spectrum of CBD-O3 is shown in the following figure:

[0027] Figure 6 The nuclear magnetic resonance 1 H- 1 H-COSY spectrum of CBD-O3 is shown in the following figure:

[0028] Figure 7 The nuclear magnetic resonance NOESY spectrum of CBD-O3 is shown in the following figure:

[0029] Figure 8The results of the inhibition zone experiment of CBD-O3; wherein 0, 1, 5 represent the concentration (mg / mL) of CBD-O3; a: Staphylococcus aureus; b: Bacillus licheniformis; c: Enterococcus faecium; d: Aeromonas hydrophila; e: Aeromonas sobria; f: Proteus vulgaris; the diameters of the inhibition zone of Staphylococcus aureus are 6.3±0.3 mm and 8.0±0.2 mm respectively when the concentration of CBD-O3 is 1 mg / mL and 5 mg / mL. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present application, the following will be a more comprehensive and detailed description of the present application in conjunction with the drawings and preferred embodiments of the specification, but the protection scope of the present application is not limited to the following specific embodiments.

[0031] Unless otherwise defined, all the professional terms used in the following have the same meaning as that generally understood by the skilled in the art. The professional terms used in the present application are only for the purpose of describing the specific embodiments and are not intended to limit the protection scope of the present application.

[0032] Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by the existing method.

[0033] Embodiment:

[0034] A compound selectively inhibiting Staphylococcus aureus is prepared by ozonization of cannabidiol of formula (I), and its chemical structure is as formula (II), which is named as: 4-((2 S ,4 S ,5 R ,10 R )-6-hydroxy-4-methoxy-2-methyl-8-pentyl-4,5-dihydro-2,5-methanobenzo[ d ][1,3]dioxepin-10-yl)butan-2-one, abbreviated as CBD-O3;

[0035] (I) (II).

[0036] The preparation method of CBD-O3 comprises the following steps:

[0037] (1) Put 25 mL of 60 mg / mL CBD solution into a 100 mL round-bottom flask, and pass ozone with a concentration of 0.6-0.8 pmol / L into the solution using an air source ozone generator; use thin layer chromatography (TLC) to track the content of CBD in the reaction process, and finally determine that the time for the CBD reaction to be complete is about 2 h; because long-term gas passage will cause the solvent methanol to volatilize, and the unreacted CBD and the reaction product generated will be in an oily state in the flask, resulting in uneven reaction of ozone with them and a slower reaction rate, therefore, 5 mL of methanol is added to the round-bottom flask every half hour during the reaction, and ultrasonic treatment is performed for 1 min, so that the oily substance in the flask is dissolved in methanol again, and ozone is continued to be passed in; after 2 h of reaction, TLC is used to confirm that the CBD reaction is complete, and the CBD ozonation product preparation solution is obtained;

[0038] (2) The CBD ozonation preparation solution is separated by silica gel column chromatography (30 g of 100-mesh silica gel powder is uniformly filled into a chromatography column, and the chromatography column has a specification of φ32 mm and an effective length of 305 mm) to obtain a crude product, which is further purified by preparative chromatography (Shimadzu preparative liquid chromatograph LC-8A, chromatography column Ultimate XB-C18, 5 pm, 21.2*250 mm) to obtain a high-purity compound CBD-O3 monomer.

[0039] Figure 1 is a schematic diagram of the carbon atom number of compound CBD-O3 formula (II), Figures 2-7 respectively, are the 1 H-NMR spectrum, 13 C-NMR spectrum, HSQC spectrum, HMBC spectrum, 1 H- 1 H-COSY spectrum, NOESY spectrum, from Figures 1-7 It can be seen that 1 the δ7.26 (1H, s) in the H-NMR spectrum is a solvent peak of CDCl3, and δ6.21 (1H, s) and δ6.19 (1H, s) are two proton signals, which are possibly two protons on the benzene ring, from 1 H- 1Both protons were correlated to the protons at δ 2.38-2.46 (2H, overlapped) in H-COSY spectrum, which indicated that there was a tetra-substituted benzene ring in the structure and the two protons were at the meta position of the benzene ring. δ 5.38 (1H, s) was a singlet signal of one hydrogen, which had no correlated carbon in HSQC spectrum. It was inferred to be the hydrogen of a phenolic hydroxyl group according to its chemical shift, which indicated that there was a phenolic hydroxyl group as a substituent on the benzene ring. δ 2.38-2.46 (2H, overlapped) was a doublet signal of two hydrogens, δ 1.54 (2H, m), δ 1.27 (2H, m), δ 1.30 (2H, m) were multiplet signals of six hydrogens, and δ 0.87 (3H, t) was a doublet of three peaks of hydrogens. From the results of H-COSY and HSQC spectra, it was inferred that these were the hydrogens of a n-pentyl group substituted on the benzene ring, which indicated that one of the substituents of the benzene ring in the structure was a n-pentyl group. According to the results of H-COSY and HSQC spectra, it was inferred that δ 3.44 (1H, d) was the hydrogen of a methine group, which was correlated to δ 105.5 (C-1′) in HMBC spectrum, indicating that C-1′ was connected to C-1 and C-6. 1 H- 1 From the results of H-COSY and HSQC spectra, it was inferred that δ 3.44 (1H, d) was the hydrogen of a methine group, which was correlated to δ 105.5 (C-1′) in HMBC spectrum, indicating that C-1′ was connected to C-1 and C-6. 13 From the chemical shift of δ 208.7 (C-3) in C-NMR spectrum, it was inferred that it might be a carbonyl group. 1 In H-NMR spectrum, δ 2.09 (3H, s) was the signal of the protons of a methyl group, 1 H- 1 There was no correlated proton signal in H-COSY spectrum, and it was inferred to be a methyl group connected to a carbonyl group according to the chemical shift, which indicated that there was a methyl ketone in the structure. In HMBC spectrum, δ 2.09 (3H, s) was correlated to δ 41.9 (C-4) remotely, so the methyl ketone was connected to C-4. 1 H- 1 In H-COSY spectrum, δ 1.32 (1H, m), δ 1.79 (1H, m) of C-5 were correlated to δ 2.39-2.48 (2H, overlapped) of C-4 and δ 2.39 (1H, ddd) of C-6, indicating that C-5 was connected to C-4 and C-6. 1 H- 1 In H-COSY spectrum and HMBC spectrum, δ 2.39 (1H, ddd) and δ 3.44 (1H, d) were correlated, and δ 105.5 (C-1′) was correlated remotely, indicating that C-1 and C-6 were connected to C-1′. 1 In H-NMR spectrum, δ 1.63 (3H, s) was the signal of the protons of a methyl group, and HSQC spectrum showed that they were both hydrogens of δ 22.0 (C-10), 1 H- 1 There was no correlated proton signal in H-COSY spectrum, and HMBC spectrum showed that it was correlated to δ 39.8 (C-6) remotely. 1 In H-NMR spectrum, δ 3.34 (3H, s) was the signal of the protons of a methyl group,1 H- 1 H-COSY spectrum no relevant proton signal, combined with its chemical shift, it is speculated that this part is methoxy. HMBC spectrum shows that δ3.34 (3H, s) is remotely related to δ109.2 (C-2). Based on the above analysis, it is speculated that the structure of CBD-O3 is formula (II), and the molecular formula is C 21 H 30 O5, the accurate molecular weight is 362.2093, and the CBD-O3 is named by systematic nomenclature: 4-((2 S ,4 S ,5 R ,10 R )-6-hydroxy-4-methoxy-2-methyl-8-pentyl-4,5-dihydro-2,5-methanobenzo[ d ][1,3]dioxepin-10-yl)butan-2-one.

[0040] Bacteriostatic circle experiment:

[0041] In order to prove the bacteriostatic effect of compound CBD-O3, the compound CBD-O3 was used for bacteriostatic circle experiment on target strains, including three kinds of gram-positive bacteria (staphylococcus aureus, bacillus licheniformis and enterococcus faecium) and three kinds of gram-negative bacteria (aeromonas hydrophila, aeromonas sobria and proteus vulgaris), and the experimental method is as follows:

[0042] After the bacteria frozen at -80℃ were inoculated on the corresponding solid medium and activated, single colonies were picked and inoculated on new solid medium for further culture. Aeromonas sobria was placed in a 27℃ incubator, and other bacteria were placed in a 37℃ incubator. After 24 h of culture, single colonies of each strain were picked on the medium, mixed in sterile 0.85% NaCl solution, and the concentration of the bacterial suspension was adjusted to 0.5 McFarland. The bacterial suspension with the adjusted concentration was diluted 100-fold with the corresponding liquid medium for each bacterium. On the clean bench, 400 μL of the diluted bacterial suspension was taken with a pipette and added to the solid plate, and the bacterial suspension was evenly spread with a sterile triangular stick. After the bacterial suspension on the solid plate dried, sterile filter paper pieces with a diameter of 6 mm were gently placed on the plate, 9 pieces per plate, arranged in 3 columns and 3 rows. 5 μL of DMSO solution was added to the paper pieces in the first column as a blank, and CBD-O3 was diluted with DMSO to 1 mg / mL and 5 mg / mL, and 5 μL was added to the paper pieces in the second and third columns, respectively. After the added solution dried, Aeromonas sobria was placed in a 27℃ incubator, and other bacteria were placed in a 37℃ incubator. After 24 h of culture, the size of the inhibition zone was measured by cross method, and the average diameter of the inhibition zone was used to evaluate the antibacterial activity of the sample on the tested bacteria.

[0043] After the antibacterial test of various gram-negative and gram-positive bacteria, the inhibition zone experiment results are shown in Table 1. Figure 8 As shown in Table 1, CBD-O3 only has inhibitory effect on Staphylococcus aureus among the target strains, and has no inhibitory effect on Bacillus licheniformis, Enterococcus faecium, Aeromonas hydrophila, Aeromonas sobria, and Proteus vulgaris, proving that CBD-O3 has selective inhibitory effect on Staphylococcus aureus.

Claims

1. A compound that selectively inhibits Staphylococcus aureus, characterized in that, The chemical structure is as follows: 。 2. A process for the preparation of a compound as claimed in claim 1, characterized in that, The method comprises the following steps: performing an ozonation reaction on a cannabidiol solution, wherein the solvent of the cannabidiol solution is methanol, and the cannabidiol ozonation product preparation liquid obtained is separated and purified to obtain the compound for selectively inhibiting Staphylococcus aureus.

3. The production method according to claim 2, characterized by, The specific operation of the ozonation reaction on the cannabidiol comprises the following steps: introducing ozone into the cannabidiol solution to perform the reaction, pausing the introduction of ozone into the cannabidiol solution every 25-35 min during the reaction, adding methanol into the cannabidiol solution and performing ultrasonic treatment, and then continuing to introduce ozone into the cannabidiol solution to perform the reaction, and obtaining the cannabidiol ozonation product preparation liquid after the reaction is completed.

4. The production method according to claim 3, characterized by, The concentration of the cannabidiol solution is 50-70 mg / mL, the concentration of the ozone is 0.6-0.8 μmol / mL, and the mass ratio of the methanol to the cannabidiol is 11.30-15.82:

1.

5. The production method according to claim 3, characterized by, The ultrasonic treatment lasts for 1-2 min, and the total reaction time is 2.0-2.5 h.

6. The preparation method according to claim 2, characterized in that, The specific operation of the separation and purification comprises the following steps: performing silica gel column chromatography separation on the cannabidiol ozonation product preparation liquid to obtain a crude product, and further purifying the crude product by preparative chromatography to obtain the compound for selectively inhibiting Staphylococcus aureus.

7. Use of the compound of claim 1 or the compound obtained by the preparation method of any one of claims 2-6 in the preparation of a product for selectively inhibiting Staphylococcus aureus.

8. Use according to claim 7, characterized in that, In the product for selectively inhibiting Staphylococcus aureus, the concentration of the compound is 1-5 mg / mL.

9. A product for the selective inhibition of Staphylococcus aureus, characterized in that, The product comprises the compound of claim 1 or the compound obtained by the preparation method of any one of claims 2-6.

10. The product according to claim 9, characterized in that The product is obtained by diluting the compound of claim 1 or the compound obtained by the preparation method of any one of claims 2-6 with dimethyl sulfoxide to a concentration of 1-5 mg / mL.

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