Aspergillus 5,6-dihydropyran-2-one derivative 3-acetamidoaspergillus pyrone A, preparation method and application thereof

Abstract

The present invention belongs to the field of natural medicine technology, and particularly relates to an Aspergillus 5,6-dihydropyran-2-one derivative, 3-acetamido Aspergillus pyrone A, a preparation method, and applications thereof. The Aspergillus 5,6-dihydropyran-2-one derivative, 3-acetamido Aspergillus pyrone A, of the present invention has a structural formula of #imgabs0# and is named 3-acetamido-5-hydroxy-6-[(7-vinyl-15-hydroxy)-pentadecyl]-5,6-dihydropyran-2-one. 3-acetamido Aspergillus pyrone A can be used in the preparation of antibacterial drug precursors, laying a material foundation for the further biopharmaceutical development and application of antibacterial ingredients.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural medicines, and particularly relates to an Aspergillus 5,6-dihydropyran-2-one derivative 3-acetamidoaspergillus pyrone A, a preparation method and application thereof. Background Art

[0002] The discovery and application of antibiotics are outstanding contributions to the history of human medicine. However, in recent years, with the widespread use of antibiotics worldwide, sensitive strains have gradually been eliminated, and drug-resistant strains have become the dominant bacterial population, and drug resistance can be transmitted between different species. The situation of drug-resistant bacterial infections is becoming increasingly severe, and there is a serious shortage of clinically available antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA) is a common and highly toxic bacterium in clinical practice. Since its discovery, MRSA infections have spread almost worldwide and have become one of the important pathogens of nosocomial and community infections. The therapeutic effect of conventional antibiotics on infections caused by drug-resistant MRSA has been greatly reduced, leading to serious clinical complications and death.

[0003] Strategies for curbing bacterial resistance focus on two main areas: first, maintaining the effectiveness of existing antimicrobial drugs, including infection prevention, rational drug use, combination therapy, and improved dosage forms; and second, promoting the development of new antimicrobial drugs, including accelerating the development of suitable new drugs and vaccines. New antimicrobial drugs differ from traditional antibiotics in their structures and targets, resulting in improved efficacy against drug-resistant bacteria. The development of new antimicrobial drugs shows considerable potential for treating infections caused by drug-resistant bacteria. Fungal antibiotics, such as cephalosporins and penicillins, exhibit significant antibacterial effects and low human toxicity. Studies have shown that compounds with long, lipophilic side chains can bind to the cell membranes of drug-resistant bacteria, exerting potent antimicrobial activity (J Med Chem, 2015, 58, 739-752; J Med Chem, 2016, 59, 171-193). Therefore, accelerating the research of new antibacterial drugs, discovering fungal-derived antibacterial substances with new structures, studying their antibacterial effects on drug-resistant bacteria, and providing clinically with drug precursor compounds with rich structures and significant efficacy are issues that technical personnel in this field urgently need to solve. Summary of the Invention

[0004] In response to the deficiencies of the prior art, the present invention provides a 5,6-dihydropyran-2-one derivative, 3-acetamido aspergillus pyrone A, and a preparation method thereof. This compound provides an effective drug precursor for the development of antibacterial drugs and also lays a material foundation for the next step of efficacy research.

[0005] To achieve the above object, the present invention provides the following technical solution: a 5,6-dihydropyran-2-one derivative of Aspergillus niger 3-acetamidopyrone A, characterized in that the structural formula of the compound is

[0006]

[0007] The compound was named 3-acetamido-5-hydroxy-6-[(7-vinyl-15-hydroxy)-pentadecyl]-5,6-dihydropyran-2-one.

[0008] The present invention also provides the use of the Aspergillus 5,6-dihydropyran-2-one derivative 3-acetamidoaspergillus pyrone A in inhibiting the growth of Gram-positive human pathogenic bacteria Staphylococcus aureus standard strains, Bacillus subtilis, and Bacillus subtilis, inhibiting the growth of Gram-negative human pathogenic bacteria Escherichia coli, inhibiting the growth of human pathogenic bacteria Staphylococcus aureus clinical strains, and inhibiting the human pathogenic drug-resistant strain methicillin-resistant Staphylococcus aureus (MRSA).

[0009] In a third aspect of the present invention, a method for preparing the Aspergillus 5,6-dihydropyran-2-one derivative 3-acetamido Aspergillus pyrone A is provided, comprising the following steps:

[0010] (1) activating Aspergillus cristatus to form a seed liquid, and culturing and fermenting the seed liquid in a solid culture medium;

[0011] (2) extracting the fermented product obtained in step (1) with ethyl acetate by cold soaking, and concentrating under reduced pressure to obtain a crude extract;

[0012] (3) The crude extract in step (2) is purified by silica gel column chromatography, eluted by macroporous resin column chromatography, and then 3-acetamido aspergillus pyrone A is separated and purified by high performance liquid chromatography.

[0013] Furthermore, the Aspergillus cristatus was streaked and revived, inoculated into PDA solid culture medium, and activated in a 28°C incubator for 5 days; a small piece of colony was cut from the PDA culture medium with a scalpel, inoculated into PDB liquid culture medium, and shaken and cultured on a shaker at 28°C and 120 rpm for 5 days to obtain a seed liquid.

[0014] Furthermore, the solid culture medium in step (1) is prepared by: 20-70 g of rice, 20-70 g of barley, and 70-150 mL of water, soaked overnight, and then sterilized under high pressure at 100-120° C. for 10-50 min.

[0015] Furthermore, the seed liquid was inoculated into a solid culture medium and cultured statically in a constant temperature and humidity chamber at 28° C. for 30 days.

[0016] Furthermore, in step (2), 400 mL of ethyl acetate was added to every 200 g of fermentation product.

[0017] Furthermore, in step (3), the silica gel column chromatography is used for impurity removal by eluting with petroleum ether-ethyl acetate (30:1 to 0:1).

[0018] Furthermore, the elution gradient of the macroporous resin column chromatography in step (3) is 10%, 30%, 50%, 70%, 90%, and 100% methanol-water.

[0019] Furthermore, in step (3), 3-acetamido aspergillus pyrone A is a 90% methanol-10% water section.

[0020] Furthermore, the HPLC eluent in step (3) is methanol-acid water, mobile phase A is 60% to 100% methanol, mobile phase B is 40% to 0% acid water, the acid water is selected from one of aqueous solutions of formic acid, acetic acid, phosphoric acid, and trifluoroacetic acid, the acid concentration is 0.01% to 1.0%, and the detection wavelength is 230 nm.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention proposes a novel structural type of Aspergillus 5,6-dihydropyran-2-one derivative, which has a lipophilic 15-carbon long-chain olefin side chain and may act on the bacterial cell membrane; the study found that 3-acetamido Aspergillus pyrone A can significantly inhibit the growth of three Gram-positive human pathogens, namely, standard strains of Staphylococcus aureus, Bacillus subtilis, and Bacillus subtilis, with MICs of 8.15±0.60μg / mL, 11.42±1.92μg / mL, and 24.9 3-Acetamido Aspergillus pyrone A significantly inhibited the growth of the Gram-negative human pathogen Escherichia coli, with an MIC of 36.84 μg / mL. 3-Acetamido Aspergillus pyrone A also significantly inhibited the growth of a clinical strain of the human pathogen Staphylococcus aureus, with an MIC of 12.82 μg / mL. 3-Acetamido Aspergillus pyrone A also showed significant antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), a human pathogenic strain, with an MIC of 23.61 μg / mL. 3-Acetamido Aspergillus pyrone A can be used in the preparation of antimicrobial drug precursors, laying a material foundation for the further development and application of antimicrobial ingredients in biopharmaceuticals. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The colony morphology of Aspergillus cristatus is shown.

[0024] Figure 2 1H-1H COSY and key HMBC signals of 3-acetamidoaspergillus pyrone A are shown.

[0025] Figure 3 The 1H-NMR chart of 3-acetamidoasperpyrone A is shown.

[0026] Figure 4 The 13C-NMR chart of 3-acetamidoasperpyrone A is shown.

[0027] Figure 5 The 1H-1H COSY pattern of 3-acetamidoaspergillus pyrone A is shown.

[0028] Figure 6 The HSQC pattern of 3-acetamidoasperpyrone A is shown.

[0029] Figure 7 The HMBC diagram of 3-acetamidoasperpyrone A is shown.

[0030] Figure 8 The graph shows the antibacterial effect of 3-acetamidopyrone A on clinical strains of Staphylococcus aureus.

[0031] Figure 9 The graph shows the antibacterial effect of 3-acetamido aspergillus pyrone A on MRSA. DETAILED DESCRIPTION

[0032] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0033] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0034] Example 1 Fermentation of strains and extraction of extract

[0035] Aspergillus cristatus was purchased from the American Type Culture Collection (ATCC) with the accession number ATCC 22166. The strain was revived by streaking, inoculated onto PDA solid medium, and activated in a 28°C incubator for 5 days. A small colony fragment was cut from the PDA medium with a scalpel and inoculated into PDB liquid medium. The seed solution was shaken and cultured on a shaker at 28°C and 120 rpm for 5 days. The seed solution was then inoculated into a solid culture medium (40 g of rice, 40 g of barley, and 125 mL of water, soaked overnight and sterilized at 110°C for 25 minutes) and cultured in a 28°C constant temperature and humidity chamber for 30 days. The solid fermentation product was taken, and 400 mL of ethyl acetate was added to every 200 g of fermentation product. The product was cold-extracted twice, and the combined extracts were concentrated under reduced pressure until there was no ethyl acetate smell to obtain the ethyl acetate extract.

[0036] Example 2 Isolation and Identification of 3-Acetamido Aspergillus Pyrone A

[0037] The extract was segmented by silica gel column chromatography and eluted with petroleum ether-ethyl acetate (30:1-0:1) respectively. After merging, it was divided into 8 segments (AH). The CG segments were combined and placed on an MCI column, adsorbed overnight, and eluted with a gradient of 10%, 30%, 50%, 70%, 90%, and 100% methanol-water. Each gradient elution was 5 column volumes. Each gradient was combined and dried until there was no liquid to obtain 6 subcomponents. The fifth subcomponent (90% methanol-water portion) was separated and purified by semi-preparative high performance liquid chromatography. The mobile phase was 85% methanol-water + 0.1% CF3COOH, the flow rate was 4.0 mL / min, the detection wavelength was 230 nm, and the peak time was 28.6-29.4 min. The peak was taken out and evaporated to complete dryness to obtain 16.5 mg of white powder. The white powder was subjected to mass spectrometry and one-dimensional and two-dimensional nuclear magnetic resonance analysis. The results are as follows Figure 2-Figure 7 , the structure of the compound was determined to be

[0038] Low-resolution mass spectrometry in positive ion mode revealed [M+Na]+ m / z 418.3, [M+K]+ m / z 434.3, and negative ion mode [M+Cl]- m / z 430.4, indicating a molecular weight of 395 and a molecular formula of C22H37NO5. Its 1H and 13C NMR data are shown in Table 1. 1H and 13C NMR data revealed the presence of two carbonyl groups (C-2 and C-22), four olefinic carbons (C-3, C-4, C-7, and C-8), three oxygen-linked quaternary carbons (C-5, C-6, and C-15), 11 methylene groups (C-9, C-10, C-11, C-12, C-13, C-14, C-16, C-17, C-18, C-19, and C-20), and two methyl groups (C-21 and C-23). ​​Signal assignment was performed using two-dimensional NMR data, including 1H-1H COSY (homonuclear correlation spectroscopy), HSQC (heteronuclear single quantum correlation), and HMBC (multi-bond carbon-hydrogen correlation spectroscopy). The pyrone ring is confirmed by 1H-1H COSY signals from H-4 / H-5 / H-6, and HMBC signals from H-4 to C-2 / C-3 / C-5 / C-6, H-5 to C-3 / C-4 / C-6, and H-6 to C-5. The 3-acetamido substitution is confirmed by key HMBC signals from the amide hydrogen to C-2 / C-4 / C-22 and from H3-23 to C-22. The 15-carbon long side chain at position 6 was confirmed by 1H-1H COSY signals at H-7 / H-8 / H-9 / H-10, H-10 / H-11 / H-12, H-13 / H-14 / H-15 / H-16 / H-17, H-17 / H-18 / H-19 / H-20 / H-21, as well as key HMBC signals at H-7 to C-8 / C-9, H-8 to C-9 / C-10, H-15 to C-13 / C-14 / C-16 / C-17, and H3-21 to C-19 / C-20. The 15-carbon long side chain substitution at position C-6 was confirmed by key HMBC signals at H-6 to C-7 / C-8, H-7 to C-5 / C-6, and H-8 to C-6. The structure of the compound was thus determined. The new structural compound proposed in the present invention is a 5,6-dihydropyran-2-one derivative in which the 3-position is substituted by an acetamido group and the 6-position is substituted by a 15-carbon long-chain olefin. Its structure is 3-acetamido-5-hydroxy-6-[(7-vinyl-15-hydroxy)-pentadecanyl]-5,6-dihydropyran-2-one, and its common name is 3-acetamidopyranone A.

[0039] Table 1 Compounds 1 H(500MHz) and 13 C (125MHz) NMR data (CDCl3)

[0040]

[0041]

[0042] Test Example 1 Inhibition experiment on three common strains of Gram-positive human pathogenic bacteria

[0043] The human pathogenic bacteria used in the antibacterial experiments were standard strains of Staphylococcus aureus (ATCC 25922), Bacillus subtilis (ATCC 6633), and Bacillus altitudinis. The bacteria were activated on beef extract peptone plates at 37°C for 24 hours. Colonies were picked and added to Mueller-Hinton broth (MH broth) and incubated with shaking for 6 hours. The culture was then diluted to a concentration of 1.0×10⁴ to 1.0×10⁵ CFU / mL. 3-Acetamidoaspergillus pyrone A was dissolved in DMSO and diluted in MH broth to concentrations of 200.0, 100.0, 50.0, 25.0, and 12.5 μg / mL. The blank control group received 200 μL of MH culture medium, the positive control group received 100 μL of penicillin G sodium solution and 100 μL of bacterial suspension, the test group received 100 μL of the test sample 3-acetamidopyrone A solution and 100 μL of bacterial suspension, and the growth group received 100 μL of MH culture medium and 100 μL of bacterial suspension. After incubation at 37°C for 24 hours, the OD value at 530 nm was measured using a microplate reader, with three replicates per group. The MIC (minimum inhibitory concentration) was the drug concentration that reduced the bacterial OD value by half. GraphPad Prism 8 software was used to statistically analyze and calculate the MIC value and ±SD (standard deviation). The results showed that 3-acetamidopyrone A had significant antibacterial activity against three strains of human pathogenic Gram-positive bacteria, with MICs ranging from 8.15 to 24.93 μg / mL. Studies have shown that 3-acetamidopyrone A has a lipophilic 15-carbon long side chain. This type of compound can destroy the bacterial cell membrane and thus exert an antibacterial effect.

[0044] Table 2 MICs of 3-acetamidopyrone A against three Gram-positive human pathogens (μg / mL)

[0045]

[0046] Test Example 2: Antibacterial effect on a Gram-negative human pathogen

[0047] The Escherichia coli (E. coli ATCC 25922) strain was activated on beef extract peptone plates at 37°C for 24 hours. Colonies were picked and added to MH broth, incubated with shaking for 6 hours, and then diluted for later use. 3-Acetamidopyrone A was dissolved in DMSO and diluted with MH broth to test concentrations of 200.0, 100.0, 50.0, 25.0, and 12.5 μg / mL. The test group was treated with 100 μL of 3-Acetamidopyrone A solution at varying concentrations and 100 μL of bacterial suspension. The positive control group was treated with 100 μL of the positive drug streptomycin sulfate and 100 μL of bacterial suspension. After incubation at 37°C for 24 hours, the OD value at 530 nm was measured using a microplate reader. Three replicates were set up for each group. The results showed that 3-Acetamidopyrone A had a significant inhibitory effect on E. coli, with an MIC of 36.84 μg / mL. The MIC value of streptomycin sulfate against this strain of Escherichia coli was 1.08 μg / mL.

[0048] Test Example 3: Antibacterial Effect on Clinical Strain of Staphylococcus aureus Pathogenic to Human Body

[0049] The clinical strain of Staphylococcus aureus (S.aureus clinical isolate) was activated on a beef extract peptone medium plate at 37°C for 24 hours, the colonies were picked and added to MH culture medium, shaken and cultured for 6 hours, and the bacterial solution was diluted for use. 3-acetamido aspergillus pyrone A was dissolved in DMSO and diluted with MH culture medium to 200.0, 100.0, 50.0, 25.0, and 12.5 μg / mL of the test solution. The test group was added with 100 μL of 3-acetamido aspergillus pyrone A solution of different concentrations and 100 μL of bacterial suspension, with tigecycline as the positive drug. After culturing at 37°C for 24 hours, the OD value at 530 nm was measured with an enzyme-linked microplate reader, and 3 parallels were set for each group. The results showed that ( Figure 8 ), the inhibition rate of 12.5μg / mL 3-acetamido aspergillus pyrone A against the clinical strain of Staphylococcus aureus reached 47.7%, and the inhibition rate of 25.0μg / mL 3-acetamido aspergillus pyrone A against the clinical strain of Staphylococcus aureus was 77.6%. Based on this, the MIC of 3-acetamido aspergillus pyrone A against the clinical strain of Staphylococcus aureus was calculated to be 12.82μg / mL. The MIC value of the positive drug tigecycline against the clinical strain of Staphylococcus aureus was 1.25μg / mL, and the MIC value of penicillin G sodium against the clinical strain of Staphylococcus aureus was 60.53μg / mL. This strain has strong resistance to penicillin G sodium. 3-acetamido aspergillus pyrone A has a significant antibacterial effect on the clinical strain of Staphylococcus aureus that is pathogenic to humans.

[0050] Test Example 4: Antibacterial Effect on MRSA Pathogenic Resistant Strain in Human Body

[0051] Methicillin-resistant Staphylococcus aureus (MRSA) was activated on a beef extract peptone medium plate at 37°C for 24 hours, colonies were picked and added to MH culture medium, shaken and cultured for 6 hours, and the bacterial solution was diluted for use. 3-acetamidoaspergillus pyrone A was dissolved in DMSO and diluted with MH culture medium to 200.0, 100.0, 50.0, 25.0, and 12.5 μg / mL test solutions, with tigecycline as the positive drug. 100 μL of compound solutions of different concentrations and 100 μL of bacterial suspension were added to the test group. After culturing at 37°C for 24 hours, the OD value at 530 nm was measured with an enzyme reader, and 3 parallels were set for each group. The results showed that ( Figure 9 ), the inhibition rate of 3-acetamidopyrone A against MRSA reached 82.2% at 50.0 μg / mL, and 46.8% at 25.0 μg / mL. Based on this, the calculated MIC of 3-acetamidopyrone A against MRSA was 23.61 μg / mL. The MIC value of the positive drug tigecycline against this MRSA strain was 6.25 μg / mL, and the MIC value of penicillin G sodium against this clinical strain of Staphylococcus aureus was greater than 100 μg / mL, indicating that this MRSA strain was completely resistant to penicillin G sodium. 3-acetamidopyrone A showed significant antibacterial activity against human pathogenic and drug-resistant MRSA strains.

[0052] In summary, the present invention proposes a novel structure of Aspergillus pyrone substituted with an acetamido group at the 2nd position and a 15-carbon long-chain olefin at the 5th position. The study found that 3-acetamido Aspergillus pyrone A can significantly inhibit the growth of three Gram-positive human pathogens, Staphylococcus aureus standard strain, Bacillus subtilis, and Bacillus subtilis, with MICs of 8.15±0.60μg / mL, 11.42±1.92μg / mL, and 24.9 3-Acetamido Aspergillus pyrone A significantly inhibited the growth of the Gram-negative human pathogen Escherichia coli, with an MIC of 36.84 μg / mL. 3-Acetamido Aspergillus pyrone A also significantly inhibited the growth of a clinical strain of the human pathogen Staphylococcus aureus, with an MIC of 12.82 μg / mL. 3-Acetamido Aspergillus pyrone A also showed significant antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), a human pathogenic strain, with an MIC of 23.61 μg / mL. 3-Acetamido Aspergillus pyrone A can be used in the preparation of antimicrobial drug precursors, laying a material foundation for the further development and application of antimicrobial ingredients in biopharmaceuticals.

[0053] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0054] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An Aspergillus 5,6-dihydropyran-2-one derivative, characterized in that The structural formula of the compound is 。 2. Use of the Aspergillus 5,6-dihydropyran-2-one derivative according to claim 1 in the preparation of a drug for inhibiting standard strains of Staphylococcus aureus, Bacillus subtilis, Bacillus thuringiensis, Escherichia coli, clinical strains of Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus.

3. The method for preparing the Aspergillus 5,6-dihydropyran-2-one derivative according to claim 1, wherein: The following steps are involved: (1) The Aspergillus cristatus strain with the accession number ATCC 22166 was streaked and revived, inoculated into PDA solid culture medium, and activated in a 28°C incubator for 5 days; a small piece of the colony was cut from the PDA culture medium with a scalpel, inoculated into PDB liquid culture medium, and cultured on a shaker at 28°C and 120 rpm for 5 days to obtain seed liquid, which was inoculated into solid culture medium and cultured in a constant temperature and humidity chamber at 28°C for 30 days; (2) extracting the fermented product obtained in step (1) with ethyl acetate by cold soaking, and concentrating under reduced pressure to obtain a crude extract; (3) The crude extract in step (2) was subjected to silica gel column chromatography, eluted with 30:1 to 0:1 petroleum ether-ethyl acetate to remove impurities, and then subjected to a macroporous resin column with 10%, 30%, 50%, 70%, 90%, and 100% methanol-water gradient elution, and the 90% methanol-10% water section was separated and purified by high performance liquid chromatography; The high performance liquid chromatography eluent is methanol-acid water, mobile phase A is 60% to 100% methanol, and mobile phase B is 40% to 0% acid water, wherein the acid water is selected from one of aqueous solutions of formic acid, acetic acid, phosphoric acid, and trifluoroacetic acid, and the acid concentration is 0.01% to 1.0%, and the detection wavelength is 230 nm.

4. The method for preparing the Aspergillus 5,6-dihydropyran-2-one derivative according to claim 3, characterized in that: The solid culture medium in step (1) is prepared as follows: 20-70 g of rice, 20-70 g of barley, and 70-150 mL of water, which are then soaked overnight and then sterilized at 100-120 °C under high pressure for 10-50 min.

5. The method for preparing the Aspergillus 5,6-dihydropyran-2-one derivative according to claim 3, characterized in that: In step (2), 400 mL of ethyl acetate was added for every 200 g of fermentation product.

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