Compound for selectively inhibiting staphylococcus aureus as well as preparation method, application and product thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses a compound for selectively inhibiting staphylococcus aureus as well as a preparation method, application and a product of the compound. Bacteriostatic experiments find that the compound has a stronger selective inhibition effect on three gram-positive bacteria and three gram-negative bacteria than cannabidiol, and the defect that cannabidiol has no difference in inhibition on staphylococcus aureus, bacillus licheniformis and enterococcus faecium is overcome; the compound can be used for preparing products for selectively inhibiting staphylococcus aureus, and is good in application prospect and excellent in antibacterial effect. The compound is obtained through a cannabidiol ozonation reaction, the method is simple, the production efficiency is high, and industrial popularization is facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a cannabidiol ozonation reaction product and a preparation method thereof, as well as the application and product in the preparation of a product for selectively inhibiting Staphylococcus aureus. Background Art

[0002] Cannabidiol ( Cannabidiol , CBD) has various pharmacological properties, such as antibacterial properties. CBD has shown antimicrobial activity against Gram-positive bacteria, including Staphylococcus aureus ( Staphylococcus aureus ), Streptococcus pneumoniae ( Streptococcus penumoniae ). In addition, CBD exhibits selective bactericidal effects on specific subsets of Gram-negative bacteria (such as Neisseria gonorrhoeae and Neisseria meningitidis). Literature reports that CBD has inhibitory effects on three Gram-positive bacteria (Staphylococcus aureus, Bacillus licheniformis, and Enterococcus faecium), but has no inhibitory effects on three Gram-negative bacteria (Aeromonas hydrophila, Aeromonas sobria, and Proteus). This may be because Gram-negative bacteria have a lipopolysaccharide outer membrane that is difficult to penetrate and are more difficult to be affected by drugs than Gram-positive bacteria. Among these three Gram-positive bacteria, Staphylococcus aureus is a common pathogen, Bacillus licheniformis and Enterococcus faecium are both probiotics, and Enterococcus faecium is a normal flora in the human intestine. Therefore, the indiscriminate inhibitory effect of CBD on these three bacteria may lead to an imbalance of the human flora in 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, its preparation method, application and product, in view of the defect that cannabidiol indiscriminately inhibits Staphylococcus aureus, Bacillus licheniformis and Enterococcus faecium.

[0004] In order to solve the above technical problems, the technical solution proposed by the present invention is: In a first aspect, the present invention provides a compound that selectively inhibits Staphylococcus aureus, and its chemical structure is as follows: .

[0005] The compound is named by the 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, abbreviated as CBD-O3.

[0006] In a second aspect, the present invention provides a method for preparing the compound, comprising the following steps: subjecting cannabidiol to an ozonation reaction, and separating and purifying the obtained cannabidiol ozonation product preparation liquid to obtain the compound that selectively inhibits Staphylococcus aureus.

[0007] The above-mentioned preparation method, further, the specific operation of the ozonation reaction of cannabidiol includes the following steps: introducing ozone into the cannabidiol solution for reaction, pausing the introduction of ozone every 25-35 minutes during the reaction and adding methanol to the cannabidiol solution and ultrasonically treating it, then continuing to introduce ozone for reaction, and obtaining a cannabidiol ozonation product preparation liquid after the reaction is complete.

[0008] Furthermore, 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 cannabidiol is 11.30-15.82:1.

[0009] Furthermore, the ultrasonic treatment time is 1-2 min, and the total reaction time is 2.0-2.5 h.

[0010] Furthermore, the specific operation of the separation and purification includes the following steps: subjecting the cannabidiol ozonation product preparation liquid to silica gel column chromatography to obtain a crude product, and then further purifying it by preparative chromatography to obtain the compound that selectively inhibits Staphylococcus aureus.

[0011] Furthermore, the adsorbent used in the silica gel column chromatography separation is 30-40 g of 100-200 mesh silica gel powder, the chromatography column specification is φ32 mm, and the effective length is 305 mm; the equipment used for the preparative chromatography is a Shimadzu LC-8A preparative liquid chromatograph, the chromatographic column is Ultimate XB-C18, and the chromatographic column specification is 5 µm, 21.2×250 mm.

[0012] In a third aspect, the present invention provides a use of the compound in preparing a product for selectively inhibiting Staphylococcus aureus.

[0013] In the above application, further, in the product for selectively inhibiting Staphylococcus aureus, the concentration of the compound is 1-5 mg / mL.

[0014] In a fourth aspect, the present invention provides a product for selectively inhibiting Staphylococcus aureus, comprising the compound.

[0015] The above product is further obtained by diluting the compound with dimethyl sulfoxide to a concentration of 1-5 mg / mL.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a compound that can selectively inhibit Staphylococcus aureus. Antibacterial experiments show that the compound has a stronger selective inhibitory effect on three Gram-positive bacteria and three Gram-negative bacteria than cannabidiol, overcoming the defect of cannabidiol in indiscriminate inhibition of Staphylococcus aureus, Bacillus licheniformis and Enterococcus faecium. The compound can be used to prepare a product that selectively inhibits Staphylococcus aureus, has excellent antibacterial effect, and has good application prospects.

[0017] 2. The present invention also provides a method for preparing the compound, which is obtained through the ozonation reaction of cannabidiol. The method is simple, has high production efficiency, and is conducive to industrial promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 is the structural formula of the compound CBD-O3 obtained in the examples of the present invention; Figure 2 This is the CBD-O3 H NMR spectrum; Figure 3 This is the CBD-O3 carbon nuclear magnetic resonance spectrum; Figure 4 This is the CBD-O3 NMR HSQC spectrum; Figure 5 This is the HMBC NMR spectrum of CBD-O3; Figure 6 CBD-O3 NMR 1 H- 1 H-COSY spectrum; Figure 7 This is the CBD-O3 NMR NOESY spectrum; Figure 8 The inhibition zone test results of CBD-O3; 0, 1, and 5 represent the concentrations of CBD-O3 (mg / mL), respectively; a: Staphylococcus aureus; b: Bacillus licheniformis; c: Enterococcus faecium; d: Aeromonas hydrophila; e: Aeromonas sobria; f: Proteus; when the concentrations of CBD-O3 were 1 and 5 mg / mL, the inhibition zone diameters of Staphylococcus aureus were 6.3±0.3 mm and 8.0±0.2 mm, respectively. DETAILED DESCRIPTION

[0020] To facilitate understanding of the present invention, the present invention will be described in more comprehensive and detailed form below in conjunction with the accompanying drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0021] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0022] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0023] Example: A compound that selectively inhibits Staphylococcus aureus is prepared by ozonating cannabidiol of formula (I), and its chemical structure is as shown in formula (II), and is named by the 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, abbreviated as CBD-O3; (I) (II).

[0024] The preparation method of CBD-O3 comprises the following steps: (1) 25 mL of 60 mg / mL CBD solution was placed in a 100 mL round-bottom flask, and ozone with a concentration of 0.6-0.8 µmol / L was introduced into the solution using an air source ozone generator. The CBD content during the reaction was tracked and detected using thin layer chromatography (TLC), and it was finally determined that the time for the CBD reaction to be complete was approximately 2 h. Since the long-term introduction of gas would cause the solvent methanol to evaporate, the unreacted CBD and the generated reaction products would appear as oil in the flask, resulting in uneven reaction between ozone and CBD and a slower reaction rate. Therefore, 5 mL of methanol was added to the round-bottom flask every half an hour during the reaction, and ultrasonicated for 1 min to dissolve the oil in the flask in methanol again, and then ozone was continued to be introduced. After 2 h of reaction, TLC was used to confirm that the CBD reaction was complete, and the CBD ozonation product preparation solution was obtained. (2) The CBD ozonation preparation solution was separated by silica gel column chromatography (30 g of 100 mesh silica gel powder was evenly filled into the chromatography column, the chromatography column specification was φ32 mm, and the effective length was 305 mm) to obtain a crude product, which was further purified by preparative chromatography (Shimadzu preparative liquid chromatography LC-8A, chromatographic column Ultimate XB-C18, 5 µm, 21.2×250 mm) to obtain a high-purity monomer of the compound CBD-O3.

[0025] Figure 1 This is a schematic diagram of the carbon atom numbering of compound CBD-O3 formula (II), Figure 2-Figure 7 CBD-O3 1 H-NMR spectrum, 13 C-NMR spectrum, HSQC spectrum, HMBC spectrum, 1 H- 1 H-COSY spectrum, NOESY spectrum, by Figure 1-Figure 7 It can be seen that 1 In the H-NMR spectrum, δ7.26 (1H, s) is the solvent peak of CDCl3, and δ6.21 (1H, s) and δ6.19 (1H, s) are two proton signals, which may be the two protons on the benzene ring. 1 H- 1 The H-COSY spectrum shows that these two protons are related to the protons on δ2.38‒2.46 (2H, overlapped), which indicates that there is a tetrasubstituted benzene ring in the structure, and the two protons are located in the meta position on the benzene ring. δ5.38 (1H, s) is a singlet signal of hydrogen. The HSQC spectrum shows that it has no connected carbon. Combined with its chemical shift, it is speculated that it may be a hydrogen on a phenolic hydroxyl group, indicating that one of the substituents on the benzene ring is a phenolic hydroxyl group. δ2.38‒2.46 (2H, overlapped) is a doublet signal of two hydrogens, δ1.54 (2H, m), δ1.27 (2H, m), δ1.30 (2H, m) are multiplet signals of six hydrogens, and δ0.87 (3H, t) is a doublet signal of three peaks. 1 H- 1 The results of H-COSY and HSQC spectra suggest that this is the hydrogen of the n-pentyl group substituted on the benzene ring, indicating that one of the substituents of the benzene ring in the structure is n-pentyl. 13 The chemical shift of δ208.7 (C-3) in the C-NMR spectrum suggested that it might be a carbonyl group. 1 In the H-NMR spectrum, δ2.09 (3H, s) is the signal of the proton on the methyl group. 1 H- 1The absence of relevant proton signals in the H-COSY spectrum suggests a methyl group attached to a carbonyl group, based on the shift, indicating a methyl ketone in the structure. The HMBC spectrum shows a long-range correlation between δ2.09 (3H, s) and δ41.9 (C-4), suggesting that the methyl ketone is attached to C-4. 1 H- 1 In the H-COSY spectrum, δ1.32 (1H, m) and δ1.79 (1H, m) of C-5 are correlated with δ2.39‒2.48 (2H, overlapped) of C-4 and δ2.39 (1H, ddd) of C-6, indicating that C-5 is connected to C-4 and C-6. 1 H- 1 The H-COSY and HMBC spectra showed correlations at δ2.39 (1H, ddd) and δ3.44 (1H, d), and a long-range correlation at δ105.5 (C-1′), indicating that C-1 is connected to C-6 and C-1′. 1 In the H-NMR spectrum, δ1.63 (3H, s) is the signal of the proton on the methyl group, and the HSQC spectrum shows that they are all hydrogens on δ22.0 (C-10). 1 H- 1 The HCOSY spectrum showed no relevant proton signal, while the HMBC spectrum showed a long-range correlation with δ39.8 (C-6). 1 In the H-NMR spectrum, δ3.34 (3H, s) is the proton signal of the methyl group. 1 H- 1 The H-COSY spectrum showed no relevant proton signal. Combined with its chemical shift, it was speculated that this part was a methoxy group. The HMBC spectrum showed that δ3.34 (3H, s) was remotely correlated with δ109.2 (C-2). Based on the above analysis, it can be inferred that the structure of CBD-O3 is formula (II), and the molecular formula is C 21 H 30 O5, the exact molecular weight is 362.2093, and CBD-O3 is named using the 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.

[0026] Inhibition zone test: To demonstrate the antibacterial effect of compound CBD-O3, this experiment used compound CBD-O3 to conduct an inhibition zone experiment on target strains. The target strain types included three Gram-positive bacteria (Staphylococcus aureus, Bacillus licheniformis, and Enterococcus faecium) and three Gram-negative bacteria (Aeromonas hydrophila, Aeromonas sobria, and Proteus). The experimental method is as follows: Bacteria frozen at -80°C were inoculated onto the corresponding solid culture medium for activation. Single colonies were then picked and inoculated onto fresh solid culture medium for further culture. Aeromonas sobriae was incubated at 27°C, while the other bacteria were incubated at 37°C. After 24 hours of incubation, single colonies of each bacterial species were picked from the culture medium and mixed thoroughly with sterile 0.85% NaCl solution. The bacterial suspension was adjusted to a concentration of 0.5 McFarland. The adjusted bacterial suspension was diluted 100-fold with the corresponding liquid culture medium for each bacterial species. On a clean bench, 400 µL of the 100-fold diluted bacterial suspension was pipetted onto a solid culture plate and evenly spread using a sterile triangular rod. After the suspension on the solid culture plate dried, sterile filter paper discs (6 mm in diameter) were gently placed onto the plate using sterile tweezers. Nine filter paper discs were placed on each plate, arranged in three columns and three rows. A 5 µL DMSO solution was dripped onto the paper disc in the first column as a blank. CBD-O3 was diluted with DMSO to 1 mg / mL and 5 mg / mL, and 5 µL was dripped onto the paper discs in the second and third columns, respectively. After the dripped solution dried, Aeromonas sobria was placed in a 27°C incubator, while the other bacterial species were placed in a 37°C incubator. After 24 hours of incubation, the size of the inhibition zone was measured using the cross-hatch method. The average diameter of the inhibition zone was used to evaluate the antibacterial activity of the sample against the test species.

[0027] After antibacterial tests on various Gram-negative and Gram-positive bacteria, the results of the inhibition zone test are as follows Figure 8 As shown, CBD-O3 only has an inhibitory effect on Staphylococcus aureus among the target strains, and has no inhibitory effect on Bacillus licheniformis, Enterococcus faecium, Aeromonas hydrophila, Aeromonas sobria, Proteus, etc., proving that CBD-O3 has a selective inhibitory effect on Staphylococcus aureus.

Claims

1. A compound that selectively inhibits Staphylococcus aureus, characterized in that: Its chemical structure is as follows: 。 2. A method for preparing the compound according to claim 1, characterized in that: The method comprises the following steps: performing an ozonation reaction on cannabidiol, separating and purifying the obtained cannabidiol ozonation product preparation liquid to obtain the compound that selectively inhibits Staphylococcus aureus.

3. The preparation method according to claim 2, wherein The specific operation of the ozonation reaction of cannabidiol includes the following steps: introducing ozone into the cannabidiol solution for reaction, pausing the introduction of ozone every 25-35 minutes during the reaction, adding methanol to the cannabidiol solution and ultrasonically treating it, and then continuing to introduce ozone for reaction, and obtaining a cannabidiol ozonation product preparation liquid after the reaction is complete.

4. The preparation method according to claim 3, wherein 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 preparation method according to claim 3, wherein The duration of the ultrasonic treatment is 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 includes the following steps: subjecting the cannabidiol ozonation product preparation liquid to silica gel column chromatography to obtain a crude product, and then further purifying it through preparative chromatography to obtain the compound that selectively inhibits Staphylococcus aureus.

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

8. The 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 selectively inhibiting Staphylococcus aureus, characterized in that: The invention relates to a compound according to claim 1 or a compound obtained by the preparation method according to any one of claims 2 to 6.

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

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