Cytochalasin compound with wide antibacterial activity as well as preparation method and application of cytochalasin compound

Abstract

The invention discloses a cytochalasin compound with wide antibacterial activity as well as a preparation method and application thereof. According to the present invention, the cytochalasin polymer is obtained by separation from the fermentation product derived from the Aspergillus izzukae (Aspergillus izzukae), such that the cytochalasin polymer can be obtained, the preservation number of the Aspergillus izzukae PJ03-11 is CGMCC (China General Microbiological Culture Collection Center) No.40601, and the preservation number of the Aspergillus izzukae PJ03-11 is CGMCC No.40601. The compound has a structure of a cytochalasin mother nucleus and an epiccine mother nucleus, and shows good activity in the aspects of inhibiting or killing staphylococcus aureus, methicillin-resistant staphylococcus aureus, multidrug-resistant staphylococcus aureus, streptococcus pneumoniae, staphylococcus epidermidis and vancomycin-resistant enterococcus faecalis; the compound can be used for preparing novel agricultural or medical antibacterial drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical microbial chemistry, and in particular relates to cytochalasin compounds with broad antibacterial activity and a preparation method and application thereof. Background Art

[0002] Antimicrobial resistance (AMR) is becoming increasingly serious, seriously endangering people's health. The World Bank predicts that by 2050, the annual increase in global medical investment for AMR will reach 1 trillion US dollars. "ESKAPE" refers to Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacter species, which are considered to be the six most deadly clinical drug-resistant pathogens. Among them, methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common pathogens in community-acquired and hospital-acquired infections, with high morbidity and mortality. The mortality rate of its infection is 64% higher than that of non-resistant bacteria, and has become a major global medical problem. In Europe and the United States, the mortality rate caused by MRSA exceeds the combined mortality rate of HIV and tuberculosis infections. In the first list of priority pathogens urgently in need of new antibiotics published by the WHO in 2017, the development of new antibiotics to treat MRSA infections was listed as a high urgency level.

[0003] The treatment of MRSA infection is one of the most difficult clinical problems. The key is that it has multiple resistance to many antibiotics. Vancomycin is currently the only antibiotic with proven efficacy in the treatment of MRSA in clinical practice, and it has been used for more than 30 years. Once MRSA breaks through the blockade of antibiotics, it will be incurable. Therefore, exploring new antibacterial targets and effective inhibitors is a direction that medicinal chemistry needs to focus on, and the development of new antibacterial drugs requires long-term and continuous research.

[0004] Microbial natural products are the main source of anti-infective drugs. After years of excavation and development of terrestrial microbial resources, it has become difficult to obtain new antibiotics. In recent years, with the continuous development of deep-sea operations, extraction and separation, marine biology and genetic engineering, some new structural drug candidates and lead molecules have been discovered from marine fungi, providing new ideas for solving the problem of insufficient "drug source molecules". In recent decades, many lead compounds have been excavated from marine organisms, which have brought countless surprises to the pharmaceutical industry through structural modification and chemical synthesis. As of early 2023, more than 20 marine-derived drugs have been successfully approved and used in clinical practice. Marine fungi are important microbial resources that can produce secondary metabolites with unique structures and excellent activities, providing important resources for screening new antibiotics. It is of great significance to screen new natural products with bacteriostatic effects from marine fungi. Summary of the invention

[0005] One object of the present invention is to provide an Aspergillus ibizarcus strain and application thereof.

[0006] The present invention provides Aspergillus iizukae PJ03-11. Aspergillus iizukae PJ03-11 was deposited in the General Microbiological Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on April 10, 2023, and the deposit registration number is CGMCC No.40601.

[0007] Another object of the present invention is to provide cytochalasin compounds having broad antibacterial activity and a preparation method thereof.

[0008] The cytochalasin compounds with broad antibacterial activity provided by the present invention have a structure of one or two cytochalasin (Formula A) mother nuclei and one or two epicoccine (Formula B) mother nuclei;

[0009] Wherein, carbon 19 and carbon 20 in formula A are simultaneously connected to carbon 1 and carbon 8, or carbon 8 and carbon 1, or carbon 2 and carbon 4, or carbon 4 and carbon 2 in formula B respectively through a carbon-carbon double bond;

[0010]

[0011] In formula A, there is a single bond or a double bond between carbon 17 and carbon 18; R1 is selected from any of the following groups: -H, α-OH, β-OH, =O; R2 is selected from any of the following groups: -H, α-OH, β-OH, =O;

[0012] In formula B, there are single bonds or double bonds between carbon 1 and carbon 2, carbon 2 and carbon 3, carbon 3 and carbon 4, carbon 4 and carbon 5, carbon 5 and carbon 6, carbon 6 and carbon 7, and carbon 7 and carbon 8; in particular, carbon 3 and carbon 4 may be connected in a COC manner; R3 is selected from any of the following groups: α-OH, β-OH, =O; R4 is selected from any of the following groups: α-OH, β-OH, =O; R5 is selected from any of the following groups: α-OH, β-OH, =O.

[0013] Preferably, the cytochalasin compounds having broad antibacterial activity are compounds 1-12, as shown in Formula I to Formula XII:

[0014]

[0015]

[0016]

[0017]

[0018] The present invention also provides a method for preparing the compounds represented by the above formula I to formula XII.

[0019] The preparation method of the compounds represented by Formula I to Formula XII provided by the present invention comprises the following steps:

[0020] 1) fermenting Aspergillus yizhugaye PJ03-11 to obtain a fermentation product; extracting the fermentation product with ethyl acetate, collecting the organic phase, and concentrating it to obtain a crude extract; suspending the crude extract with water, and then extracting it with n-hexane and ethyl acetate in sequence, and collecting the ethyl acetate extraction part, which is the fermentation product of Aspergillus yizhugaye;

[0021] 2) The ethyl acetate portion was subjected to normal phase silica gel column chromatography according to the following elution procedure: dichloromethane-acetone (D / A, v / v, 100 / 0, 50 / 1, 20 / 1, 10 / 1, 8 / 1, 5 / 1, 3 / 1), dichloromethane-methanol (D / M v / v, 5 / 1, 3 / 1, 2 / 1, 1 / 1, 0 / 1) for gradient elution, and the elution volume of each ratio was 5 times the column volume. After TLC thin layer chromatography detection, the fractions with the same main point were combined to obtain 10 components F1-F10; among them, F5 was the elution component using the dichloromethane-acetone volume ratio of 3 / 1 as the eluent, and F6 was the elution component using the dichloromethane-methanol volume ratio of 5 / 1 as the eluent;

[0022] 3) F5 (D / A, v / v 3 / 1) was subjected to ODS reverse C18 column chromatography, and gradient elution was performed with methanol-water as the eluent, wherein the volume ratio of methanol in the eluent was 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, and each ratio was eluted for 3 times the column volume. After TLC thin layer chromatography detection, the fractions with the same main point were combined to obtain 20 fractions F5_1-F5_20;

[0023] F5_13 (60% methanol / water) was separated by Sephadex LH-20, eluted with 5 column volumes of methanol, and each 0.5 column volume was combined into a subfraction, for a total of 10 subfractions (F5_13_1–F5_13_10);

[0024] F5_13_5 was separated by reverse phase preparative chromatography RP-HPLC, 89% acetonitrile / water (containing 1‰ trifluoroacetic acid), flow rate 2.0mL / min, and 6 (4.5mg, t R =15.4min) and 7 (7.5mg, t R =21.1min);

[0025] 4) F6 (D / M, v / v 5 / 1) was subjected to ODS reverse C18 column chromatography, and gradient elution was performed with methanol-water as the eluent, wherein the volume ratio of methanol in the eluent was 20%, 30%, 40%, 50%, 70%, 90%, and 100% in sequence, and each ratio was eluted for 3 times the column volume. After TLC thin layer chromatography detection, the fractions with the same main point were combined to obtain 7 sub-fractions (F6_1-F6_7);

[0026] F6_4 (50% methanol / water) was further separated by Sephadex LH-20 column, eluted with 3 column volumes of methanol, and combined into one subfraction every 0.3 column volumes, resulting in 10 subfractions (F6_4_1–F6_4_10);

[0027] F6_4_4 was separated by semi-preparative C18 reverse phase chromatography with 53% acetonitrile / water (containing 1‰ trifluoroacetic acid) at a flow rate of 2.0 mL / min to obtain compound 1 (t R =38.5min);

[0028] F6_4_5 was separated by semi-preparative C18 reverse phase chromatography using 70% acetonitrile / water (containing 1‰ trifluoroacetic acid) at a flow rate of 2.0 mL / min to obtain compound 2 (t R =30.2min)、3(t R =33.5min), 4(t R =36.6min) and 5(t R=40.1min);

[0029] F6_4_6 was separated by semi-preparative C18 reverse phase chromatography using 85% acetonitrile / water (containing 1‰ trifluoroacetic acid) at a flow rate of 2.0 mL / min to give compound 8 (t R =20.2min)、9(t R =25.5min) and 10(t R =28.7min);

[0030] F6_5 (70% methanol / water) was separated by semi-preparative C18 reverse phase chromatography column, and eluted with 72% acetonitrile / water (containing 1‰ trifluoroacetic acid) at a flow rate of 2.0 mL / min to obtain compound 11 (t R =23.1min) and 12(t R =24.4min).

[0031] Preferably, in the step 1), the fermentation condition is static culture at 28° C. for 4 weeks;

[0032] The culture medium used in the fermentation was prepared according to the following method: 80 g of oats and 120 mL of distilled water, sterilized at 115° C. for 30 min.

[0033] Preferably, in the step 2), the filler in the normal phase silica gel column has a size of 200 to 300 meshes.

[0034] Preferably, in the step 3), the filler specification in the ODS reverse C18 column is 50 μm.

[0035] Preferably, in the step 4), the filler specification in the ODS reverse C18 column is 50 μm.

[0036] The present invention also provides the application of the above-mentioned cytochalasin compounds having broad antibacterial activity.

[0037] The application provided by the present invention is the application of the above-mentioned cytochalasin compounds with broad antibacterial activity (especially the compounds represented by formula I to formula XII) in the preparation of antibacterial drugs.

[0038] In the above application, the antibacterial drug can inhibit the activity of Gram-positive bacteria.

[0039] In the above application, the antibacterial drug may be a drug for resisting multidrug resistant bacterial infection.

[0040] Furthermore, the Gram-positive bacteria may be at least one of sensitive Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), multidrug-resistant Staphylococcus aureus, Streptococcus pneumoniae, Staphylococcus epidermidis, and vancomycin-resistant Enterococcus faecalis.

[0041] Furthermore, the multidrug-resistant bacteria are multidrug-resistant Staphylococcus aureus and / or vancomycin-resistant Enterococcus faecalis.

[0042] Another object of the present invention is to provide a drug for preventing and / or treating bacterial infection diseases.

[0043] The drugs for preventing and / or treating bacterial infections provided by the present invention include the above-mentioned cytochalasin compounds having broad antibacterial activity (especially the compounds represented by Formula I to Formula XII).

[0044] In the above application, the bacteria are Gram-positive bacteria.

[0045] Furthermore, the Gram-positive bacteria may be at least one of sensitive Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), multidrug-resistant Staphylococcus aureus, Streptococcus pneumoniae, Staphylococcus epidermidis, and vancomycin-resistant Enterococcus faecalis.

[0046] Another object of the present invention is to provide an antibacterial composition A.

[0047] The antibacterial composition A provided by the present invention comprises cytochalasin compounds having broad antibacterial activity and methicillin.

[0048] Furthermore, the cytochalasin compound having broad antibacterial activity is selected from at least one of the compounds represented by Formula I to Formula V.

[0049] Furthermore, the mass ratio of any one of the compounds represented by Formula I to Formula V to methicillin is 1:(1024-2.5), and the preferred mass ratio is 1:(256-20).

[0050] More specifically, the mass ratio of the compound represented by formula I (Compound 1) to methicillin is 1:256;

[0051] The mass ratio of the compound represented by formula II (compound 2) to methicillin is 1:80;

[0052] The mass ratio of the compound represented by formula III (compound 3) to methicillin is 1:20;

[0053] The mass ratio of the compound represented by formula IV (compound 4) to methicillin is 1:80;

[0054] The mass ratio of the compound represented by formula V (compound 5) to methicillin is 1:40.

[0055] Another object of the present invention is to provide an antibacterial composition B.

[0056] The antibacterial composition B provided by the present invention comprises cytochalasin compounds having broad antibacterial activity and kanamycin.

[0057] Furthermore, the cytochalasin compound having broad antibacterial activity is selected from at least one of the compounds represented by Formula II to Formula V.

[0058] Furthermore, the mass ratio of any one of the compounds represented by formula II to formula V to kanamycin is 1:(440-3), and the preferred mass ratio is 1:(52.5-26.25).

[0059] More specifically, the mass ratio of the compound represented by formula II (Compound 2) to kanamycin is 1:26.25;

[0060] The mass ratio of the compound represented by formula III (compound 3) to kanamycin is 1:26.25;

[0061] The mass ratio of the compound represented by formula IV (compound 4) to kanamycin is 1:52.5;

[0062] The mass ratio of the compound represented by formula V (compound 5) to kanamycin is 1:26.25.

[0063] The antibacterial composition A or B is particularly suitable for fighting multidrug-resistant bacteria infection. Further, the multidrug-resistant bacteria is multidrug-resistant Staphylococcus aureus.

[0064] In the above antibacterial composition A or B, the active ingredients of the antibacterial composition may be only "cytochalasin compounds with broad antibacterial activity and methicillin" or "cytochalasin compounds with broad antibacterial activity and kanamycin"; it may also further include other active ingredients, which can be determined by those skilled in the art according to the antibacterial effect.

[0065] In addition, antibacterial products containing the above-mentioned cytochalasin compounds with broad antibacterial activity (especially the compounds represented by Formula I to Formula XII) or containing the above-mentioned antibacterial composition also fall within the protection scope of the present invention.

[0066] In the present invention, the antibacterial product may also include pharmaceutically permitted diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, synergists, additives and solvents, etc. When preparing the antibacterial product, the compound of the effective dose may be mixed with pharmaceutically permitted diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, synergists, additives and solvents, etc. to prepare various pharmaceutical preparations. The form of the drug may be oral preparations such as tablets, capsules, soft capsules, powders, granules, fine granules, liquids, pills, emulsions or suspensions, and may also be non-oral preparations such as injections (such as powders, aqueous solutions, oils), suppositories, ointments, plasters, patches, sprays, tinctures or eye drops. These preparations may all be obtained by commonly used preparation methods known to those skilled in the art. Its route of administration may be oral, percutaneous, intravenous or intramuscular.

[0067] The present invention has the following beneficial effects:

[0068] The present invention discloses a type of cytochalasin polymer isolated from a fermentation product of a strain of Aspergillus iizukae. The compound has the structure of a cytochalasin mother nucleus and an epicoccine mother nucleus, and exhibits good activity in inhibiting or killing Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, multidrug-resistant Staphylococcus aureus, Streptococcus pneumoniae, Staphylococcus epidermidis, and vancomycin-resistant Enterococcus faecalis, and has the use of preparing new agricultural or medical antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 is the H NMR spectrum of compound 1 (solvent: DMSO-d6);

[0070] Figure 2 is the carbon NMR spectrum of compound 1 (solvent: DMSO-d6);

[0071] Figure 3is the H NMR spectrum of compound 2 (solvent: DMSO-d6);

[0072] Figure 4 is the carbon NMR spectrum of compound 2 (solvent: DMSO-d6);

[0073] Figure 5 is the H NMR spectrum of compound 3 (solvent: DMSO-d6);

[0074] Figure 6 is the carbon NMR spectrum of compound 3 (solvent: DMSO-d6);

[0075] Figure 7 is the H NMR spectrum of compound 4 (solvent: DMSO-d6);

[0076] Figure 8 is the carbon NMR spectrum of compound 4 (solvent: DMSO-d6);

[0077] Fig. 9 is the H NMR spectrum of compound 5 (solvent: DMSO-d6);

[0078] Fig.10 is the carbon NMR spectrum of compound 5 (solvent: DMSO-d6);

[0079] Fig.11 is the H NMR spectrum of compound 6 (solvent: CD3OD);

[0080] Fig.12 is the carbon NMR spectrum of compound 6 (solvent: CD3OD);

[0081] Fig.13 is the H NMR spectrum of compound 7 (solvent: DMSO-d6);

[0082] Fig.14 is the carbon NMR spectrum of compound 7 (solvent: DMSO-d6);

[0083] Fig.15 is the H NMR spectrum of compound 8 (solvent: DMSO-d6);

[0084] Fig.16 is the carbon NMR spectrum of compound 8 (solvent: DMSO-d6);

[0085] Fig.17 is the H NMR spectrum of compound 9 (solvent: DMSO-d6);

[0086] Fig.18 is the carbon NMR spectrum of compound 9 (solvent: DMSO-d6);

[0087] Fig.19 is the H NMR spectrum of compound 10 (solvent: DMSO-d6);

[0088] Fig. 20 is the carbon NMR spectrum of compound 10 (solvent: DMSO-d6);

[0089] Fig.21 is the H NMR spectrum of compound 11 (solvent: DMSO-d6);

[0090] Fig. 22 is the carbon NMR spectrum of compound 11 (solvent: DMSO-d6);

[0091] Fig.23 is the H NMR spectrum of compound 12 (solvent: DMSO-d6);

[0092] Fig.24 is the carbon NMR spectrum of compound 12 (solvent: DMSO-d6);

[0093] Fig.25 The antibacterial activity of compounds 1-5 in combination with methicillin (ae) and the antibacterial activity of compounds 2-5 in combination with kanamycin (fi) are shown. DETAILED DESCRIPTION

[0094] In order to make the invention purpose and invention content of this application more clearly presented, the applicant will clearly and completely describe the technical solution of the present invention in conjunction with specific embodiments below. The following embodiments are further explanations of the present invention rather than limitations of the present invention.

[0095] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0096] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0097] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0098] Example 1: Isolation, identification and preservation of strains

[0099] 1. Isolation of strain PJ03-11

[0100] The strain PJ03-11 was isolated from soil samples of the Red Beach Ecological Wetland Tourist Scenic Area in Panjin City, Liaoning Province. 10 fresh soil samples were added to 90 mL of sterile water to make a suspension, which was diluted 100,000 times. Then, the soil suspension was spread on a PDA medium (200 grams of potatoes, 20 grams of glucose and 20 grams of agar per liter of seawater) containing chloramphenicol (100 μg / mL) and streptomycin (100 μg / mL) and incubated at 28°C for 3-5 days. Single colonies were picked.

[0101] 2. Identification of strain PJ03-11

[0102] Morphological characteristics: The diameter of the colony is 22-52mm within 14 days, flat to wrinkled, loose, granular, light grayish yellow brown to medium brownish yellow in color, without secretions. The conidia are white, light brown to brown, radial to cylindrical, and the conidia are double-serial. No tiny conidia are observed. The stalk is mostly transparent, lighter to brown near the vesicle, darker near the base, smooth to slightly rough, segmented, septate, and usually more than 1mm in length in some strains. The conidia are spherical, subglobose to elliptical, smooth, sometimes connected to the connecting part on the free conidia, and 2.3-3.1(-3.5)mm in length. The number of conidia varies according to the strain, and they are sessile or on short, transparent micro- to medium-long conidial hyphae, usually carrying 1-4 conidia, which are spherical, subglobose, 3.7-6.0 mm in diameter, or ellipsoidal, club-shaped, often truncate, 4.5-7.5(-8) mm in size and 3.5-5.5 mm in width. Asci or ascospores are not produced.

[0103] The ITS gene sequence is shown in Sequence 1 of the sequence listing. According to the ITS phylogenetic tree analysis combined with morphological identification results, strain PJ03-11 belongs to Aspergillus iizukae.

[0104] 3. Storage of strain PJ03-11

[0105] Aspergillus iizukae PJ03-11 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on April 10, 2023, and the deposit registration number is CGMCC NO.40601.

[0106] Example 2: Preparation of Aspergillus niger fermentation product

[0107] Potato dextrose agar medium (PDA medium): Peel 200g of potatoes, cut into small strips and put into an aluminum pot, add 1000mL of water, boil for about 20 to 30 minutes until the potatoes are soft but not mushy, filter with 6 to 8 layers of gauze, take the filtrate into the pot, add water to 1000mL, add 20g of glucose, melt 18g of agar, and sterilize at 121℃ for 30min.

[0108] Preparation of fungal liquid culture medium: Glucose (20 g), mannose (20 g), yeast extract (50 g), peptone (10 g), corn steep liquor (1 g), KH2PO4 (0.5 g), and MgSO4·7H2O (0.3 g) were dissolved in each liter of distilled water and sterilized at 115°C for 30 min.

[0109] Preparation of fermentation medium: 80 g oats and 120 mL distilled water, sterilize at 115 °C for 30 min.

[0110] The strain PJ03-11 was inoculated on a PDA medium and cultured at 28°C for 7 days. The strain spores on the plate were collected with a 0.01% sterile Tween 80 solution, and inoculated in a fungal liquid culture medium for culture. Each bottle contained 200 mL of the culture medium and cultured at 28°C for 7 days. At this time, each bottle contained 25-40 mycelium balls with a diameter of 0.5-1.5 cm, and thus a seed solution was obtained. The seed solution was inoculated in 120 bottles of fermentation medium (each bottle contained 80 g of oats and 120 mL of distilled water) for large-scale fermentation. Each bottle was inoculated with 10 mL of the seed solution and cultured at 28°C for 4 weeks. The fermented product (i.e., the content in the fermentation bottle) was extracted with 2 times the volume of ethyl acetate, and ultrasonic-assisted extraction was performed four times (each time the amount of ethyl acetate used was 2 times the volume of the fermented product), and the organic phase was combined. The organic phase was concentrated by a rotary evaporator at 37°C to obtain a crude extract. The crude extract was suspended with water (the volume ratio of crude extract to water was 1:2), and then extracted three times with n-hexane and ethyl acetate respectively (in each extraction, the volume of the extractant to the suspension was 1:1), and concentrated under reduced pressure to obtain n-hexane part (515g) and ethyl acetate part (202g). The ethyl acetate part is the fermentation product of Aspergillus niger.

[0111] Example 3: Preparation of Compound 1-12

[0112] The ethyl acetate portion (202 g) was subjected to normal phase silica gel column chromatography (200-300 mesh, 500 g) according to the following elution procedure: dichloromethane-acetone (D / A, v / v, 100 / 0, 50 / 1, 20 / 1, 10 / 1, 8 / 1, 5 / 1, 3 / 1), dichloromethane-methanol (D / Mv / v, 5 / 1, 3 / 1, 2 / 1, 1 / 1, 0 / 1) were sequentially gradient eluted, and the elution volume of each ratio was 5 times the column volume. After TLC thin layer chromatography detection, the fractions with the same main point were combined to obtain 10 components F1-F10; among them, F5 was the elution component using the dichloromethane-acetone volume ratio of 3 / 1 as the eluent, and F6 was the elution component using the dichloromethane-methanol volume ratio of 5 / 1 as the eluent;

[0113] 3) F5 (23.9 g, D / A, v / v 3 / 1) was chromatographed on an ODS reverse C18 column (50 μm, 100 g) with methanol-water as the eluent for gradient elution. The methanol content in the eluent was 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, respectively. Each ratio was eluted with 3 column volumes. The fractions with the same main point were combined after TLC thin layer chromatography detection to obtain 20 fractions F5_1–F5_20.

[0114] F5_13 (2.3 g, 60% methanol / water) was separated by Sephadex LH-20, eluted with 5 column volumes of methanol, and each 0.5 column volume was combined into a sub-fraction, a total of 10 sub-fractions (F5_13_1-F5_13_10).

[0115] F5_13_5 (132.5 mg) was eluted by reverse phase preparative chromatography RP-HPLC with 89% acetonitrile / water (containing 1‰ trifluoroacetic acid) at a flow rate of 2.0 mL / min to give 6 (4.5 mg, t R =15.4min) and 7 (7.5mg, t R =21.1min);

[0116] 4) F6 (20.1 g, D / M, v / v 5 / 1) was subjected to ODS reverse C18 column chromatography (50 μm, 100 g) with methanol-water as the eluent, and the elution ratio was 20%, 30%, 40%, 50%, 70%, 90%, 100% gradient elution, and each ratio was eluted for 3 times the column volume. The fractions with the same main point were combined by TLC thin layer chromatography to obtain 7 sub-fractions (F6_1-F6_7);

[0117] F6_4 (2.0 g, 50% methanol / water) was further separated by Sephadex LH-20 column, eluted with 3 column volumes of methanol, and combined into one subfraction every 0.3 column volumes, obtaining 10 subfractions (F6_4_1–F6_4_10);

[0118] F6_4_4 (320.5 mg) was separated by semi-preparative reverse phase chromatography on a Kromasil C18 column, 53% acetonitrile / water (containing 1‰ trifluoroacetic acid), flow rate 2.0 mL / min, to give compound 1 (30.0 mg, t R =38.5min);

[0119] F6_4_5 (298.5 mg) was separated by semi-preparative reverse phase chromatography on a Kromasil C18 column, eluted with 70% acetonitrile / water (containing 1‰ trifluoroacetic acid) at a flow rate of 2.0 mL / min to give compound 2 (102.0 mg, t R =30.2min), 3(14.2mg,t R =33.5min), 4(26.7mg,t R =36.6min) and 5 (10.5mg, t R =40.1min);

[0120] F6_4_6 (152.5 mg) was separated by semi-preparative reverse phase chromatography on a Kromasil C18 column at a flow rate of 2.0 mL / min and 85% acetonitrile / water (containing 1‰ trifluoroacetic acid) to give compound 8 (3.2 mg, t R =20.2min), 9(3.9mg,t R =25.5min) and 10(11.5mg,t R =28.7min);

[0121] F6_5 (285.6 mg, 70% methanol / water) was separated by semi-preparative reverse phase chromatography on a Kromasil C18 column, using a flow rate of 2.0 mL / min and 72% acetonitrile / water (containing 1‰ trifluoroacetic acid) to give compound 11 (2.7 mg, t R =23.1min) and 12 (1.6mg, t R =24.4min).

[0122] Example 3: Structural Identification and Physicochemical Properties of Compounds 1-12

[0123] The structure of the compound is analyzed using high-resolution mass spectrometry, ultraviolet spectroscopy, optical rotation chromatography, infrared chromatography, circular dichroism, X-Ray single crystal diffraction and nuclear magnetic resonance data. In short, the molecular weight of the compound is determined by high-resolution mass spectrometry, and the molecular formula of the compound is determined by combining the compound's nuclear magnetic hydrogen spectrum and carbon spectrum. 1 H NMR and 13 C NMR was used to preliminarily determine the structural fragments of the compound. HSQC was also used to confirm the direct carbon-hydrogen relationship of the compound. 1 H- 1 H COSY determines the correlation between protons and hydrogens in the coupling system of the compound, and HMBC determines the coupling information between protons and carbons in 2-3 bonds in the compound. The planar structure of the compound is determined, and the relative configuration is determined by ROESY. The structure and absolute configuration of the compound are further determined by X-RAY single crystal diffraction, calculated nuclear magnetic resonance, biosynthesis analysis, etc.

[0124] Compound 1, iizuchalasin A, white amorphous powder. [α] 25 D -71.99(c 0.05,MeOH); UV(MeOH)λ max (logε): 205(1.38),230(sh,0.65)nm; CD(c 0.2mg / ml,MeOH)λ max (Δε): 203(-37.7),232(8.15),268(0.78),285(1.61)nm; IR(neat)ν max： 3334,2958,2933,2869,2849,1754,1694,1454,1373,1234,1153,1108,1073,1044,936,901; positive ion mode HRESIMS m / z1181.5542 [M+Na] + , (calculated value C 66 H 82 N2O 16 Na,1181.5563), thus determining the molecular formula of the compound to be C 66 H 82 N2O 16 ; 1H NMR (500 MHz, DMSO-d6) δ8.43s (H-2 or 2'), 3.06o (H-3 or 3'), 2.36dd (J = 6.3, 1.9 Hz, H-4 or 4'), 2.43m (H-5 or 5'), 5.26s (H-7 or 7'), 3.04o (H-8 or 8'), 1.00m (H-10 or 10'), 1.13d (J = 7.0 Hz, H-11 or 11'), 1.69s (H-12 or 12'), 6.08d (J = 10.8 Hz, H-13 or 13'), 2.08m (H-15 or 15'), 1.86o (H-15 or 15'), 1.88o (H-16 or 16'), 1.33m (H-16 or 16'), 3.79t ( J=9.0Hz,H-17 or 17'),6.22s(17- or 17'-OH),4.42s(H-18 or 18'),1.91o(H-19 or 19'),3.55d(J=5.4Hz,H-20 or 20'),1.57m(H-22 or 22'),0.80d(J=6.6Hz,H-23 or 23'),0. 80d (J = 6.6 Hz, H-24 or 24'), 1.38s (H-25 or 25'), 4.76d (J = 5.4 Hz, H-1" or 1'"), 3.85s (H-8" or 8'"), 1.19s (H-9" or 9'"), 5.91s (H-3"- or 3'"-OH), 8.83s (H-4"- or 4'"-OH); 13C NMR (125 MHz, DMSO-d6) δ 174.6 (C-1 or 1'), 49.6 (C-3 or 3'), 52.8 (C-4 or 4'), 34.9 (C-5 or 5'), 139.2 (C-6 or 6'), 125.1 (C-7 or 7'), 44.1 (C-8 or 8'), 68.4 (C-9 or 9'), 50.0 (C-10 or 10'), 13.0 (C-11 or 11'), 19.4 (C-12 or 12'), 124.3 (C-13 or 13'), 135.5 (C-14 or 14'), 34.1 (C-15 or 15'), 28.3 (C-16 or 16'), 72.5 (C-17 or 17'), 76. 9(C-18 or 18'),41.7(C-19 or 19'),48.6(C-20 or 20'),214.7(C-21 or 21'),23.8(C-22 or 22'),21.6(C-23 or 23'),23.4(C-24 or 24'),15.1(C-25 or 25'),73.0(C-1" or 1'"),56.6(C-2" or 2'"),95.6(C-3" or 3'"),81.3(C-4" or 4'"),203.1(C-5" or 5'"),49.7(C-6" or 6'"),59.7(C-7" or 7'"),78.0(C-8" or 8'"),9.9(C-9" or 9'"). The single crystal structure of compound 1 was obtained by slow evaporation of MeOH-H2O (10:1) solvent in a sealed vial at 25°C. The single crystal data of the compound was deposited in the Cambridge Crystallographic Data Centre with the number CCDC2235068.

[0125] Based on the above data, the structure of the compound is determined as shown in Formula I. Its H NMR spectrum and C NMR spectrum (solvent: DMSO-d6) are shown in Figure 1 and 2 .

[0126]

[0127] Compound 2, asperflavipine C, white amorphous powder. [α] 25 D -51.99(c 0.05,MeOH); UV(MeOH)λ max (logε): 205(3.38)nm; CD(c 0.2mg / ml, MeOH)λ max (Δε): 232(4.15),258(-37.8),305(+20.12)nm; IR(neat)νmax： 3431,2958,2933,2870,2849,1761,1690,1634,1564,1454,1333,1073; positive ion mode HRESIMS m / z 1181.5562 [M+Na] + , (calculated value C 66 H 82 N2O 16 Na,1181.5563), thus determining the molecular formula of the compound to be C 66 H 82 N2O 16 ; 1 H NMR(500MHz,DMSO-d6)δ8.34s(H-2),3.08o(H-3),2.35m(H-4),2.46o(H-5),5.24s(H-7),3.06o(H-8),1.06m (H-10),1.14d(J=7.0Hz,H-11),1.69s(H-12),6.00d(J=10.9Hz,H-13),2.05m(H-15),1.84o(H-15),1.84o(H- 16),1.31m(H-16),3.68o(H-17),4.54o(H-18),1.90o(H-19),3.79d(J=5.5Hz,H-20),1.60o(H-22),0.83d(J= 6.5Hz,H-23),0.85d(J=6.5Hz,H-24),1.38s(H-25),5.21m(H-1”),4.53o(H-8”),1.60s(H-9”),8.10s(H-2’), 2.90m(H-3'),2.13o(H-4'),2.47o(H-5'),5.40s(H-7'),3.37m(H-8'),1.21m(H-10'),1.06m(H-10'),1.14d (J=7.0Hz,H-11'),1.73s(H-12'),5.89d(J=11.0Hz,H-13'),1.92o(H-15'),1.80o(H-15'),1.11m(H-16'),3. 70o(H-17'),2.60dd(J=11.7,4.0Hz,H-18'),2.13o(H-19'),4.08d(J=5.5Hz,H-20'),1.70o(H-22'),0.97d(J =6.5Hz,H-23'),0.88d(J=6.5Hz,H-24'),1.38s(H-25'),4.54o(H-1"'),5.09d(J=5.5H-8"'),1.66s(H-9"'); 13CNMR(125MHz,DMSO-d6)δ174.6(C-1),49.9(C-3),53.4(C-4),34.9(C-5),139.2(C-6),125.2(C-7),44.1(C-8),68. 7(C-9),50.2(C-10),13.1(C-11),19.5(C-12),124.7(C-13),135.2(C-14),34.3(C-15),28.4(C-16),73.3(C-17),7 5.8(C-18),41.7(C-19),50.2(C-20),214.0(C-21),23.9(C-22),23.7(C-23),21.3(C-24),15.0(C-25),74.7(C-1”) ,51.1(C-2”),91.9(C-3”),186.3(C-4”),147.5(C-5”),121.5(C-6”),66.3(C-7”),83.3(C-8”),16.7(C-9”),174.0( C-1'),51.3(C-3'),54.7(C-4'),35.7(C-5'),138.8(C-6'),125.8(C-7'),42.7(C-8'),67.3(C-9'),49.2(C-10'),1 3.6(C-11'),20.1(C-12'),124.1(C-13'),135.4(C-14'),36.0(C-15'),29.7(C-16'),69.9(C-17'),76.1(C-18'),5 3.0(C-19'),47.1(C-20'),213.7(C-21'),24.6(C-22'),23.6(C-23'),21.8(C-24'),16.0(C-25'),81.7(C-1"'),71 .9(C-2"'),192.8(C-3"'),91.1(C-4"'),194.0(C-5"'),131.6(C-6"'),160.7(C-7"'),80.1(C-8"'),12.5(C-9"').

[0128] Based on the above data, the structure of the compound is determined as shown in Formula II, and its H NMR spectrum and C NMR spectrum (solvent: DMSO-d6) are as follows Figure 3 and 4 shown.

[0129]

[0130] Compound 3, asperflavipine D, white amorphous powder. [α] 25D -71.99(c 0.05,MeOH); UV(MeOH)λ max (logε): 205(4.38)nm; CD(c 0.2mg / ml, MeOH)λ max (Δε): 232(3.15),258(-35.8),305(+18.12)nm; IR(neat)ν max： 3435,2956,2933,2870,2849,1763,1691,1454,1383,1314,1251,1204,1051; positive ion mode HRESIMS m / z 1179.5411 [M+Na] + , (calculated value C 66 H 80 N2O 16 Na,1179.5406), thus determining the molecular formula of the compound to be C 66 H 80 N2O 16 ; 1H NMR(500MHz,DMSO-d6)δ3.08o(H-3),2.30m(H-4),2.45o(H-5),5.24o(H-7),3.06o(H-8),1.04m(H-10),1.14d(J=7.0Hz,H-11),1.69s(H-12),6.03d(J=10.9Hz,H-13),2.05o(H-15),1.84o(H-15),1.84o(H-16),1.32m(H-16),3.68m(H-17),4.49s(H-18),1.90t(J=5.5Hz,H-19),3.79o(H-20),1.60o(H-22),0.84d(J=6.5Hz,H-23),0.85d(J=6.5Hz,H-24),1.39s(H-25),5.23o(H-1”),4.50s(H-8”),1.60s(H-9”),3.12m(H-3'),2.41o(H-4'),2.44o(H-5'),5.15s(H-7'),2.90d(J=10.4Hz,H-8'),1.12m(H-10'),0.94m(H-10'),1.14d(J=7.0Hz,H-11'),1.66s(H-12'),5.85d(J=10.4Hz,H-13'),2.08o(H-15'),1.78m(H-15'),1.96m(H-16'),1.73m(H-16'),3.79o(H-17'),3.35dd(J=6.4,5.0Hz,H-19'),4.63d(J=6.4Hz,H-20'),1.57m(H-22'),0.88d(J=6.5Hz,H-23'),0.85d(J=6.5Hz,H-24'),1.39s(H-25'),4.59s(H-1”'),5.34d(J=5.5Hz,H-8”'),1.39s(H-9”'); 13CNMR(125MHz,DMSO-d6)δ174.5(C-1),49.8(C-3),53.6(C-4),34.9(C-5),139.3(C-6),125.1(C-7),44.1(C-8),68.7 (C-9),50.3(C-10),13.1(C-11),19.5(C-12),124.6(C-13),135.2(C-14),34.2(C-15),28.4(C-16),73.3(C-17),75 .9(C-18),41.6(C-19),50.1(C-20),214.5(C-21),23.8(C-22),23.5(C-23),21.1(C-24),14.9(C-25),74.6(C-1”), 51.4(C-2”),92.0(C-3”),186.5(C-4”),147.4(C-5”),121.7(C-6”),65.2(C-7”),83.1(C-8”),16.8(C-9”),172.7(C -1'),49.3(C-3'),53.1(C-4'),34.5(C-5'),138.6(C-6'),125.6(C-7'),43.3(C-8'),68.6(C-9'),49.6(C-10'),13 .2(C-11'),19.3(C-12'),123.5(C-13'),135.8(C-14'),31.5(C-15'),30.9(C-16'),76.5(C-17'),205.5(C-18'),5 8.7(C-19'),46.7(C-20'),215.2(C-21'),24.0(C-22'),23.2(C-23'),22.7(C-24'),16.5(C-25'),81.9(C-1"'),71 .8(C-2"'),191.7(C-3"'),91.0(C-4"'),193.9(C-5"'),130.9(C-6"'),159.0(C-7"'),79.6(C-8"'),11.5(C-9"').

[0131] Based on the above data, the structure of the compound is determined as shown in Formula III, and its H NMR spectrum and C NMR spectrum (solvent: DMSO-d6) are shown in Figure 5 and 6 .

[0132]

[0133] Compound 4, iizuchalasin B, white amorphous powder. [α] 25D -66.99(c 0.05,MeOH); UV(MeOH)λ max (logε): 205(1.48),240(sh,0.88)nm; CD(c 0.2mg / ml, MeOH)λ max (Δε): 203(-10.47), 231(-0.88), 259(-7.25), 306(3.81)nm; IR(neat)ν max： 3368,2959,2926,2868,1769,1693,1445,1385,1202,1140,1080; positive ion mode HRESIMS m / z 1157.5581 [M+H] + , (calculated value C 66 H 81 N2O 16 ,1157.5586), thus determining the molecular formula of the compound to be C 66 H 80 N2O 16 ; 1H NMR(500MHz,DMSO-d6)δ8.39s(H-2),3.07o(H-3),2.34dd(J=6.1,2.2Hz,H-4),2.43o(H-5),5.24s(H-7),3.07o(H-8),0.97o(H-10),1.06o(H-10),1.13o(H-11),1.70o(H-12),6.02d(J=10.9Hz,H-13),2.06o(H-15),1.85o(H-15),1.85o(H-16),1.34o(H-16),3.70t(J=9.0Hz,H-17),4.57s(H-18),1.92t(J=5.4Hz,H-19),3.83d(J=5.4Hz,H-20),1.58o(H-22),0.79d(J=6.6Hz,H-23),0.84d(J=6.4Hz,H-24),1.40s(H-25),5.27d(J=5.4Hz,H-1”),4.36s(H-8”),1.56s(H-9”),4.57s(17-OH),8.86s(5”-OH),8.00s(H-2'),2.87m(H-3'),2.12t(J=4.3Hz,H-4'),2.47o(H-5'),5.33s(H-7'),3.09o(H-8'),1.06o(H-10'),1.17o(H-10'),1.13o(H-11'),1.69o(H-12'),5.97d(J=11.1Hz,H-13'),2.47o(H-15'),1.82o(H-15'),2.80dd(J=16.5,12.3Hz,H-16'),2.02o(H-16'),2.72dd(J=11.1,4.3Hz,H-18'),2.58m(H-19'),4.42d(J=4.6Hz,H-20'),1.73o(H-22'),0.94d(J=6.7Hz,H-23'),0.88d(J=6.5Hz,H-24'),1.38s(H-25'),4.39s(H-1”'),5.29d(J=5.4Hz,H-8”'),1.61s(H-9”'),5.55d(J=4.3Hz,18'-OH),5.51s(4”'-OH); 13C NMR(125MHz,DMSO-d6)δ174.5(C-1),49.8(C-3),53.4(C-4),35.0(C-5),139.3(C-6),125.1(C-7),44.1(C-8),68.7 (C-9),50.5(C-10),12.9(C-11),19.5(C-12),124.7(C-13),135.2(C-14),34.3(C-15),28.4(C-16),73.3(C-17),75 .8(C-18),41.9(C-19),50.1(C-20),214.5(C-21),23.8(C-22),23.9(C-23),20.8(C-24),15.6(C-25),74.8(C-1”), 51.1(C-2”),91.9(C-3”),186.3(C-4”),147.6(C-5”),120.7(C-6”),66.0(C-7”),83.4(C-8”),16.4(C-9”),173.1(C -1'),50.7(C-3'),54.1(C-4'),35.9(C-5'),139.1(C-6'),125.6(C-7'),45.0(C-8'),66.0(C-9'),50.1(C-10'),13 .5(C-11'),19.9(C-12'),124.0(C-13'),135.4(C-14'),32.7(C-15'),35.0(C-16'),209.4(C-17'),77.5(C-18'),4 8.4(C-19'),46.2(C-20'),211.8(C-21'),24.7(C-22'),23.8(C-23'),22.0(C-24'),15.0(C-25'),80.7(C-1"'),71 .5(C-2"'),191.7(C-3"'),91.2(C-4"'),193.7(C-5"'),131.6(C-6"'),159.5(C-7"'),80.3(C-8"'),12.4(C-9"').

[0134] Based on the above data, the structure of the compound is determined to be as shown in Formula IV. Its H NMR spectrum and C NMR spectrum (solvent: DMSO-d6) are shown in Figure 7 and 8 .

[0135]

[0136] Compound 5, iizuchalasin C, white amorphous powder. [α] 25D -71.99(c 0.05,MeOH); UV(MeOH)λ max (logε): 205(1.38),250(sh,0.94)nm; CD(c 0.2mg / ml,MeOH)λ max (Δε): 203(-8.99), 229(-0.35), 261(-6.11), 307(2.89)nm; IR(neat)ν max： 3368,2957,2928,2868,1774,1693,1444,1384,1298,1249,1201,1115,1079; positive ion mode HRESIMS m / z 1141.5621 [M+H] + , (calculated value C 66 H 81 N2O 15 ,1141.5637), thus determining the molecular formula of the compound to be C 66 H 80 N2O 15 ; 1H NMR(500MHz,DMSO-d6)δ8.32s(H-2),3.07o(H-3),2.30dd(J=6.1,2.3Hz,H-4),2.47o(H-5),5.24o(H-7),3.06o(H-8),1.06m(H-10),1.14d(J=7.0Hz,H-11),1.70s(H-12),6.03d(J=11.2Hz,H-13),2.07o(H-15),1.84o(H-15),1.85o(H-16),1.34 0(H-16),3.68t(J=8.6Hz,H-17),4.51br.s(H-18),1.89t(J=5.3Hz,H-19),3.80d(J=5.3Hz,H-20),1.60m(H-22),0.88d(J=6.5Hz,H-23),0.85d(J=6.5Hz,H-24),1.39s(H-25),5.23d(J=5.3Hz,H-1”),4.65s(H-8”),1.59s(H-9”),5.29s(17-OH),7.46s(3”-OH),7.93s(H-2'),3.08o(H-3'),2.44o(H-4'),2.44o(H-5'),5.17s(H-7'),2.88d(J=10.3Hz,H-8'),1.14m(H-10'),0.98m(H-10'),1.14d(J=7.0Hz,H-11'),1.68s(H-12'),5.96d(J=10.3Hz,H-13'),2.02o(H-15'),1.83o(H-15'),2.39o(H-16'),2.14m(H-16'),1.84o(H-17'),1.71o(H-17'),3.24t(J=5.6Hz,H-19'),4.70d(J=6.0Hz,H-20'),1.60m(H-22'),0.84d(J=6.5Hz,H-23'),0.85d(J=6.5Hz,H-24'),1.37s(H-25'),4.54s(H-1”'),5.64d(J=5.1Hz,H-8”'),1.33s(H-9”'),5.34br s(4”'-OH),8.81s(5”-OH); 13C NMR(125MHz,DMSO-d6)δ174.5(C-1),50.0(C-3),53.6(C-4),34.9(C-5),139.3(C-6),125.1(C-7),44.1(C-8),68.7 (C-9),50.2(C-10),13.1(C-11),19.5(C-12),124.6(C-13),135.1(C-14),34.3(C-15),28.4(C-16),73.3(C-17),75 .9(C-18),41.6(C-19),49.9(C-20),214.7(C-21),23.8(C-22),23.6(C-23),21.1(C-24),14.9(C-25),74.7(C-1”), 51.4(C-2”),92.0(C-3”),186.6(C-4”),147.3(C-5”),121.2(C-6”),65.2(C-7”),82.2(C-8”),16.7(C-9”),172.5(C -1'),49.5(C-3'),52.8(C-4'),34.6(C-5'),139.0(C-6'),125.6(C-7'),43.1(C-8'),68.5(C-9'),49.5(C-10'),13 .3(C-11'),19.5(C-12'),125.5(C-13'),135.0(C-14'),37.9(C-15'),43.2(C-16'),21.8(C-17'),203.8(C-18'),6 0.8(C-19'),47.3(C-20'),214.3(C-21'),24.0(C-22'),23.4(C-23'),22.5(C-24'),15.3(C-25'),83.2(C-1"'),71 .7(C-2"'),191.5(C-3"'),91.0(C-4"'),193.6(C-5"'),130.8(C-6"'),159.5(C-7"'),78.5(C-8"'),11.8(C-9"').

[0137] Based on the above data, the structure of the compound is determined to be as shown in Formula V. Its H NMR spectrum and C NMR spectrum (solvent: DMSO-d6) are shown in Fig. 9 and 10 .

[0138]

[0139] Compound 6,17'R-hydroxy-amichalasine B, white amorphous powder. [α]25 D -47.99(c0.05,MeOH); UV(MeOH)λ max (logε): 205(1.74),260(sh,1.24)nm; CD(c 0.2mg / ml,MeOH)λ max (Δε): 250(6.86),283(1.14),307(2.03)nm; IR(neat)ν max： 3368,2958,1693,1444,1385,1248,1204,1157,1124,1074; positive ion mode HRESIMS m / z 1001.5151 [M+Na] + , (calculated value C 57 H 74 N2O 12 Na,1001.51405), thus determining the molecular formula of the compound to be C 57 H 74 N2O 12 ; 1H NMR(500MHz,CD3OD)δ3.41t(J=7.0Hz,H-3),2.66d(J=7.0Hz,H-4),2.57o(H-5),5.35s(H-7),3.13d(J=10.9Hz,H-8),1.26o(H-10),1.14o(H-10),1.26d(J=7.3Hz,H-11),1.75s(H-12),6.05d(J=10.9Hz,H-13),2.18m(H-15),1.94m(H-15),2.08m(H-16),1.44m(H-16),3.91dd(J=10.1,7.5Hz,H-17),4.39s(H-18),2.28o(H-19),3.75d(J=3.3Hz,H-20),1.67o(H-22),0.97d(J=6.5Hz,H-23),0.96d(J=6.5Hz,H-24),1.49s(H-25),2.95m(H-2'),2.44o(H-3'),2.40m(H-5'),5.21s(H-7'),2.57o(H-8'),1.70o(H-10'),0.98o(H-10'),1.18s(H-11'),1.75s(H-12'),6.44d(J=10.4Hz,H-13'),2.28o(H-15'),2.09o(H-15'),2.44o(H-16'),1.72o(H-16'),4.64d(J=8.8Hz,H-17'),3.26d(J=7.4Hz,H-19'),5.44d(J=7.4Hz,H-20'),1.72o(H-22'),0.94d(J=6.5Hz,H-23'),0.91d(J=6.5Hz,H-24'),1.19s(H-25'),5.10d(J=5.6Hz,H-1”),4.67s(H-8”),1.83s(H-9”); 13C NMR(125MHz,CD3OD)δ177.5(C-1),51.7(C-3),53.5(C-4),36.1(C-5),140.9(C-6),126.5(C-7),45 .6(C-8),69.9(C-9),50.7(C-10),13.7(C-11),19.5(C-12),124.2(C-13),139.3(C-14),34.8(C-15 ),30.3(C-16),75.2(C-17),82.8(C-18),40.6(C-19),58.5(C-20),211.1(C-21),25.8(C-22),23.6 (C-23),22.5(C-24),16.1(C-25),176.1(C-1'),53.2(C-2'),53.9(C-3'),37.2(C-5'),143.0(C-6' ),126.3(C-7'),46.5(C-8'),67.9(C-9'),48.5(C-10'),14.5(C-11'),20.4(C-12'),126.5(C-13' ),138.5(C-14'),38.2(C-15'),30.6(C-16'),71.8(C-17'),210.9(C-18'),49.1(C-19'),50.0(C-2 0'),211.1(C-21'),26.1(C-22'),24.8(C-23'),21.3(C-24'),14.9(C-25'),77.8(C-1”),64.2(C-2 ”),104.6(C-3”),86.9(C-4”),197.5(C-5”),126.8(C-6”),162.5(C-7”),81.6(C-8”),11.6(C-9”).

[0140] Based on the above data, the structure of the compound is determined to be as shown in Formula VI. Its H NMR spectrum and C NMR spectrum (solvent: CD3OD) are shown in Fig.11 and 12 .

[0141]

[0142] Compound 7, iizuchalasin B, white amorphous powder. [α] 25 D -233.96(c 0.05,MeOH); UV(MeOH)λ max (logε): 205 (1.89), 230 (sh, 1.74), 350 (sh, 0.88) nm; CD (c 0.2mg / ml, MeOH) λmax (Δε): 224(-13.74),238(-7.23),250(-9.08),270(-1.51),289(-11.06),301(-8.76),309(-9.22)nm; IR(neat)ν max： 3367,2955,2933,2867,1776,1741,1689,1626,1604,1455,1340,1231,1139,1108,1052,1026; HRESIMS in positive ion mode m / z 910.3665 [M+H-H2O] + , (calculated value C 50 H 56 NO 15 ,910.3650), thus determining the molecular formula of the compound to be C 50 H 57 NO 16 ; 1 H NMR(500MHz,DMSO-d6)δ8.04s(H-2),2.86dt(J=11.0,3.5Hz,H-3),1.93dd(J=4.9,3.5Hz,H-4),2.38m(H-5),5.30br.s(H- 7),3.19m(H-8),1.78m(H-10),0.98m(H-10),1.10d(J=7.0Hz,H-11),1.70s(H-12),5.84d(J=10.0Hz,H-13),1.96m(H-15), 1.89m(H-15),1.43m(H-16),1.37m(H-16),3.79m(H-17),4.03m(H-18),1.88m(H-19),4.08d(J=5.4Hz,H-20),1.64m(H-22) ,0.89d(J=6.5Hz,H-23),0.83d(J=6.5Hz,H-24),1.28s(H-25),6.34d(J=6.5Hz,H-1'),5.82s(H-8'),2.06s(H-9'),6.10br s(4'-OH),4.94s(H-3”),3.76d(J=6.5Hz,H-5”),6.30s(H-11”),6.54s(H-13 ”),2.33s(H-15”),3.23s(H-2”-OCH3),3.31s(H-7”-OCH3),11.05s(10”-OH); 13C NMR(125MHz,DMSO-d6)δ174.2(C-1),51.1(C-3),52.9(C-4),35.4(C-5),139.6(C-6),12 5.2(C-7),43.7(C-8),68.1(C-9),46.9(C-10),13.4(C-11),19.9(C-12),124.7(C-13),1 35.0(C-14),36.6(C-15),30.0(C-16),72.3(C-17),75.8(C-18),48.2(C-19),52.5(C-20 ),214.1(C-21),24.1(C-22),23.9(C-23),21.2(C-24),15.5(C-25),82.9(C-1'),64.3(C -2'),197.0(C-3'),91.7(C-4'),196.5(C-5'),131.6(C-6'),152.0(C-7'),84.0(C-8'), 14.2(C-9'),86.8(C-1”),164.1(C-2”),105.1(C-3”),189.9(C-4”),51.3(C-5”),60.2(C -6”),169.7(C-7”),191.1(C-8”),104.0(C-9”),173.0(C-10”),109.8(C-11”),152.7(C- 12”),103.0(C-13”),156.9(C-14”),22.2(C-15”),56.8(C-2”-OCH3),52.9(C-7”-OCH3).

[0143] Based on the above data, the structure of the compound is determined as shown in Formula VII, and its H NMR spectrum and C NMR spectrum (solvent: DMSO-d6) are shown in Fig.13 and 14 .

[0144]

[0145] Compound 8, iizuchalasin C, white amorphous powder. [α] 25 D -230.96(c 0.05,MeOH); UV(MeOH)λ max (logε): 210(1.70),280(1.24)nm; CD(c 0.2mg / ml, MeOH)λ max(Δε): 202(12.16),224(-7.45),239(-5.56),250(-6.66),272(0.47),294(-8.05)nm; IR(neat)ν max： 3420,2953,2933,2866,1776,1715,1688,1622,1455,1339,1333,1056,1031; positive ion mode HRESIMS m / z 944.3706 [M+H] + , (calculated value C 50 H 58 NO 17 ,944.3705), thus determining the molecular formula of the compound to be C 50 H 57 NO 17 ; 1 HNMR (500MHz, DMSO-d6) δ8.04s (H-2), 2.86dt (J=11.0, 3.5Hz, H-3), 1.93dd (J=4 .9,3.5Hz,H-4),2.36m(H-5),5.30s(H-7),3.19m(H-8),1.78m(H-10),0.98ddd(J =13.5,10.0,3.5Hz,H-10),1.10d(J=7.0Hz,H-11),1.70s(H-12),5.85d(J=10.0H z,H-13),1.96m(H-15),1.89m(H-15),1.43m(H-16),1.37m(H-16),3.80m(H-17), 4.05m(H-18),1.88m(H-19),4.08d(J=5.4Hz,H-20),1.64m(H-22),0.89d(J=6.5H z,H-23),0.83d(J=6.5Hz,H-24),1.28s(H-25),6.36d(J=6.5Hz,H-1'),5.85s(H- 8'),2.15s(H-9'),6.12s(4'-OH),4.96s(H-3”),3.76d(J=6.5Hz,H-5”),6.50s(H -11”),2.38s(H-15”),3.26s(H-2”-OCH3),3.31s(H-7”-OCH3),11.05s(10”-OH); 13CNMR(125MHz,DMSO-d6)δ174.2(C-1),51.0(C-3),52.9(C-4),35.4(C-5),139.6(C-6),12 5.1(C-7),43.7(C-8),68.1(C-9),47.0(C-10),13.4(C-11),19.9(C-12),124.8(C-13),1 35.0(C-14),36.7(C-15),30.0(C-16),72.5(C-17),75.8(C-18),48.2(C-19),52.4(C-20 ),213.7(C-21),24.1(C-22),23.9(C-23),21.3(C-24),15.5(C-25),82.9(C-1'),64.2(C -2'),197.0(C-3'),91.8(C-4'),196.5(C-5'),131.9(C-6'),151.6(C-7'),83.9(C-8'), 14.5(C-9'),87.9(C-1”),163.3(C-2”),105.1(C-3”),189.7(C-4”),51.4(C-5”),60.2(C -6”),169.5(C-7”),190.6(C-8”),105.7(C-9”),155.4(C-10”),111.6(C-11”),105.5(C- 12”),149.4(C-13”),167.9(C-14”),20.4(C-15”),56.8(C-2”-OCH3),52.9(C-7”-OCH3).

[0146] Based on the above data, the structure of the compound is determined to be as shown in Formula VIII, and its H NMR spectrum and C NMR spectrum (solvent: DMSO-d6) are shown in Fig.15 and 16 .

[0147]

[0148] Compound 9, iizuchalasin D, light green powder. [α] 25 D -161.97(c 0.05,MeOH); UV(MeOH)λ max (logε): 210(1.83),280(1.51)nm; CD(c 0.2mg / ml, MeOH)λ max(Δε): 204(11.67),227(-6.34),236(-5.65),245(-5.89),272(0.36),297(-9.1)nm; IR(neat)ν max： 3371,2954,2932,2864,1770,1732,1688,1614,1531,1455,1234,1025; positive ion mode HRESIMS m / z978.3304 [M+H] + , (calculated value C 50 H 57 ClNO 17 ,978.3315), thus determining the molecular formula of the compound to be C 50 H 56 ClNO 17 ; 1 H NMR(500MHz,DMSO-d6)δ8.05s(H-2),2.86dt(J=11.0,3.5Hz,H-3),1.93dd(J =4.9,3.5Hz,H-4),2.38m(H-5),5.30s(H-7),3.18m(H-8),1.78m(H-10),0.9 8ddd(J=13.5,10.0,3.5Hz,H-10),1.11d(J=7.0Hz,H-11),1.70s(H-12),5.8 dd(J=10.4Hz,H-13),1.96m(H-15),1.89m(H-15),1.43m(H-16),1.37m(H-16) ,3.79m(H-17),4.05m(H-18),1.88m(H-19),4.08d(J=5.4Hz,H-20),1.64m(H -22),0.89d(J=6.5Hz,H-23),0.86d(J=6.5Hz,H-24),1.28s(H-25),6.36d(J =6.5Hz,H-1'),5.84s(H-8'),2.14s(H-9'),6.11s(4'-OH),4.96s(H-3”),3. 77d(J=6.5Hz,H-5”),2.48s(H-15”),3.25s(H-2”-OCH3),3.34s(H-7”-OCH3); 13C NMR(125MHz,DMSO-d6)δ174.2(C-1),51.0(C-3),53.0(C-4),35.4(C-5),139.6(C-6),12 5.2(C-7),43.7(C-8),68.2(C-9),47.0(C-10),13.4(C-11),19.9(C-12),124.8(C-13),1 35.0(C-14),36.7(C-15),30.0(C-16),72.7(C-17),75.4(C-18),48.3(C-19),52.5(C-20 ),213.7(C-21),24.1(C-22),23.9(C-23),21.3(C-24),14.6(C-25),83.0(C-1'),64.3(C -2'),197.0(C-3'),91.8(C-4'),196.5(C-5'),132.0(C-6'),151.5(C-7'),83.9(C-8'), 15.5(C-9'),88.2(C-1”),162.8(C-2”),105.2(C-3”),189.6(C-4”),51.4(C-5”),60.0(C -6”),169.4(C-7”),191.3(C-8”),106.6(C-9”),150.5(C-10”),146.3(C-11”),107.0(C- 12”),115.7(C-13”),166.0(C-14”),18.9(C-15”),57.1(C-2”-OCH3),53.3(C-7”-OCH3).

[0149] Based on the above data, the structure of the compound is determined as shown in Formula IX. Its H NMR spectrum and C NMR spectrum (solvent: DMSO-d6) are shown in Fig.17 and 18 .

[0150]

[0151] Compound 10, iizuchalasin E, white amorphous powder. [α] 25 D -96.98(c 0.05,MeOH); UV(MeOH)λ max (logε): 210(1.81),280(1.48)nm; CD(c 0.2mg / ml, MeOH)λ max (Δε): 223(-7.06), 275(1.62), 314(-2.98)nm; IR(neat)ν max：3390,2955,2933,2864,1747,1688,1622,1455,1220,1057; positive ion mode HRESIMS m / z 960.3662 [M+H] + , (calculated value C 50 H 58 NO 18 ,960.3654), thus determining the molecular formula of the compound to be C 50 H 57 NO 18 ; 1 H NMR (500MHz, DMSO-d6) δ8.11s (H-2), 2.80dt (J=11.0, 3.5Hz, H-3), 1.85dd (J=4. 5,3.5Hz,H-4),2.41m(H-5),5.30br.s(H-7),3.16m(H-8),1.59m(H-10),0.92m(H -10),1.09d(J=7.0Hz,H-11),1.70s(H-12),5.77d(J=11.0Hz,H-13),2.03m(H-1 5),1.93m(H-15),1.65m(H-16),1.50m(H-16),3.83m(H-17),4.33br.s(H-18),1. 95m(H-19),4.52d(J=5.0Hz,H-20),1.65m(H-22),0.89d(J=6.5Hz,H-23),0.84d (J=6.5Hz,H-24),1.28s(H-25),4.13s(18-OH),6.76d(J=6.5Hz,H-1'),5.73s(H- 8'),2.26s(H-9'),8.24s(4'-OH),5.23s(H-3”),3.79d(J=6.5Hz,H-5”),6.51s(H -11”),2.38s(H-15”),3.30s(H-2”-OCH3),3.33s(H-7”-OCH3),11.43s(10”-OH); 13C NMR(125MHz,DMSO-d6)δ174.0(C-1),51.3(C-3),54.2(C-4),35.6(C-5),140.0(C-6),125 .2(C-7),44.0(C-8),68.3(C-9),47.1(C-10),13.5(C-11),20.0(C-12),125.1(C-13),13 4.7(C-14),37.3(C-15),29.6(C-16),73.0(C-17),75.4(C-18),46.2(C-19),55.3(C-20) ,214.2(C-21),24.2(C-22),23.9(C-23),21.0(C-24),15.2(C-25),84.5(C-1'),59.0(C- 2'),166.6(C-3'),100.3(C-4'),190.8(C-5'),132.5(C-6'),147.0(C-7'),86.3(C-8'), 16.9(C-9'),87.8(C-1”),161.9(C-2”),105.9(C-3”),189.5(C-4”),52.9(C-5”),59.9(C -6”),169.5(C-7”),190.8(C-8”),105.7(C-9”),155.5(C-10”),111.7(C-11”),105.5(C- 12”),149.4(C-13”),167.7(C-14”),20.4(C-15”),56.8(C-2”-OCH3),53.2(C-7”-OCH3).

[0152] Based on the above data, the structure of the compound is determined to be as shown in Formula X. Its H NMR spectrum and C NMR spectrum (solvent: DMSO-d6) are shown in Fig.19 and 20 .

[0153]

[0154] Compound 11, iizuchalasin F is a white amorphous powder. [α] 25 D -90.98(c 0.05,MeOH); UV(MeOH)λ max (logε): 210(1.81),280(1.48)nm; CD(c 0.2mg / ml, MeOH)λ max (Δε): 223(-6.85), 274(1.37), 314(-2.91)nm; IR(neat)ν max：3368,2955,2933,2864,1734,1685,1624,1436,1203,1138,1058,1032; positive ion mode HRESIMS m / z 974.3816 [M+H] + , (calculated value C 51 H 60 NO 18 ,974.3811), thus determining the molecular formula of the compound to be C 51 H 59 NO 18 ; 1 H NMR (500MHz, DMSO-d6) δ8.21s (H-2), 2.84dt (J=11.0, 3.5Hz, H-3), 1.83dd (J=4.5, 3.5Hz,H-4),2.41m(H-5),5.31br.s(H-7),3.16m(H-8),1.53m(H-10),0.93m(H-10) ,1.11d(J=7.0Hz,H-11),1.71s(H-12),5.78d(J=11.0Hz,H-13),2.01m(H-15),1.9 3m(H-15),1.68m(H-16),1.51m(H-16),3.87m(H-17),4.44br.s(H-18),2.01m(H-19 ),4.60d(J=4.2Hz,H-20),1.68m(H-22),0.90d(J=6.5Hz,H-23),0.87d(J=6.5Hz,H -24),1.27s(H-25),4.44s(17-OH),4.15s(18-OH),6.81d(J=6.5Hz,H-1'),5.74s(H -8'),2.21s(H-9'),3.47(H-4'-OCH3),5.28s(H-3”),3.81d(J=6.5Hz,H-5”),6.51s (H-11”),2.38s(H-15”),3.32s(H-2”-OCH3),3.34s(H-7”-OCH3),11.45s(10”-OH); 13C NMR(125MHz,DMSO-d6)δ174.0(C-1),51.3(C-3),54.0(C-4),35.6(C-5),140.0(C-6),125.0 (C-7),44.3(C-8),68.2(C-9),47.1(C-10),13.5(C-11),20.0(C-12),125.0(C-13),135.0( C-14),37.1(C-15),29.4(C-16),72.5(C-17),76.3(C-18),46.2(C-19),52.5(C-20),212.4 (C-21),24.0(C-22),23.9(C-23),21.0(C-24),15.2(C-25),84.3(C-1'),59.9(C-2'),165. 4(C-3'),103.2(C-4'),189.0(C-5'),133.5(C-6'),147.3(C-7'),86.6(C-8'),16.7(C-9') ,52.5(C-4'-OCH3),87.8(C-1”),162.1(C-2”),106.0(C-3”),189.3(C-4”),52.9(C-5”),59 .5(C-6”),169.4(C-7”),190.6(C-8”),105.6(C-9”),155.5(C-10”),111.7(C-11”),105.5( C-12”), 149.5 (C-13”), 167.7 (C-14”), 20.4 (C-15”), 56.9 (C-2”-OCH3), 53.2 (C-7”-OCH3).

[0155] Based on the above data, the structure of the compound is determined to be as shown in Formula XI. Its H NMR spectrum and C NMR spectrum (solvent: DMSO-d6) are shown in Fig.21 and 22 .

[0156]

[0157] Compound 12, iizuchalasin G, light green powder. [α] 25 D -83.99(c 0.05,MeOH); UV(MeOH)λ max (logε): 205 (1.74), 230 (sh, 1.30), 280 (sh, 1.18) nm; CD (c 0.2mg / ml, MeOH) λ max(Δε): 223(-3.49), 270(1.27), 300(-2.55)nm; IR(neat)ν max： 3375,2957,2993,1682,1615,1532,1434,1205,1136,1027,1006; HRESIMS in positive ion mode m / z 1016.3089 [M+Na] + , (calculated value C 50 H 56 ClNO 18 Na,1016.3083), thus determining the molecular formula of the compound to be C 50 H 56 ClNO 18;8.11s(H-2),2.80dt(J=11.0,3.5Hz,H-3),1.85dd(J=4.5,3.5Hz,H-4),2.41m(H-5),5.30br.s(H-7),3.16d(J=12.0Hz,H-8),1.60m(H-10),0.92m(H-10),1.09d(J=7.0Hz,H-11),1.70s(H-12),5.77d(J=11.0Hz,H-13),2.03m(H-15),1.93m(H-15),1.66m(H-16),1.51m(H-16),3.83m(H-17),4.34br.s(H-18),1.95m(H-19),4.52m(H-20),1.65m(H-22),0.88d(J=6.5Hz,H-23),0.84d(J=6.5Hz,H-24),1.28s(H-25),4.14s(18-OH),6.78d(J=6.5Hz,H-1'),5.75s(H-8'),8.25s(4'-OH),2.26s(H-9'),5.26s(H-3”),3.83d(J=6.5Hz,H-5”),2.48s(H-15”),3.30s(H-2”-OCH3),3.36s(H-7”-OCH3),11.68s(10”-OH),174.0(C-1),51.3(C-3),54.2(C-4),35.6(C-5),140.0(C-6),125.2(C-7),43.9(C-8),68.3(C-9),47.1(C-10),13.5(C-11),20.0(C-12),125.0(C-13),134.7(C-14),37.3(C-15),29.6(C-16),73.0(C-17),75.4(C-18),46.2(C-19),55.3(C-20),214.2(C-21),24.2(C-22),23.9(C-23),21.0(C-24),15.2(C-25),84.5(C-1'),58.9(C-2'),166.6(C-3'),100.3(C-4'),189.4(C-5'),132.5(C-6'),146.9(C-7'),86.2(C-8'),16.9(C-9'),87.9(C-1”),161.4(C-2”),106.0(C-3”),189.4(C-4”),53.0(C-5”),59.7(C-6”),169.5(C-7”),190.8(C-8”),106.0(C-9”),157.8(C-10”),165.7(C-14”),18.8(C-15”),56.8(C-2”-OCH3),53.3(C-7”-OCH3). .

[0158] Based on the above data, the structure of the compound is determined to be as shown in Formula XII. Its H NMR spectrum and C NMR spectrum (solvent: DMSO-d6) are shown in Fig.23 and 24 .

[0159]

[0160] Example 4: Determination of antibacterial activity of compounds 1-12

[0161] The minimal inhibitory concentration (MIC) was determined using the microbroth dilution method recommended by the American Association for Clinical and Laboratory Standards. 100 μl of DMSO solution of the sample to be tested, which was serially diluted, was added to a 96-well plate, and then an equal volume of diluted bacterial suspension was added (the final concentration of the bacterial suspension was about 5×10 5 CFU / ml), and finally, 2μl of 0.1% resazurin aqueous solution was added to each well. The experiment also set up a blank control of the culture medium, a bacterial control (negative control), and a common anti-Gram-positive drug in the clinic as a positive drug control. The results were observed after incubation at 37°C for 16 to 20 hours. The bacterial solution in the well turned red for no antibacterial activity, blue for antibacterial activity, and the lowest antibacterial drug concentration at which the bacterial solution remained blue was recorded as MIC. Each sample was operated in parallel 3 times, and the results were averaged. All operations were performed under sterile conditions.

[0162] The strains evaluated in this example are: Staphylococcus aureus is a sensitive Staphylococcus aureus, purchased from the ATCC Biological Standard Resource Center, USA, strain number ATCC6538, Staphylococcus aureus8325-4 is a methicillin-resistant Staphylococcus aureus, donated by Professor Hanne Ingmer of the University of Copenhagen (reference doi:10.3389 / fmicb.2018.00255), Staphylococcus aureus (MRSA-B) is a clinical strain of multidrug-resistant Staphylococcus aureus isolated from a hospital, Enterococcus faecalis (VRE) is a vancomycin-resistant Enterococcus faecalis isolated from a hospital, and Streptococcus pneumoniae was a gift from Professor Yang Jinghua of the Institute of Microbiology, Chinese Academy of Sciences (reference doi: 10.1128 / JB.01731-14), with a serotype of 42, and Staphylococcus epidermidis was purchased from the China General Microbiological Culture Collection Center, with a strain number of CGMCC3472. The evaluation results are shown in Table 1.

[0163] Table 1 Evaluation of antibacterial activity of compounds 1-12 (MIC, μg / mL)

[0164]

[0165] Through the antibacterial activity evaluation, compounds 1-12 showed different degrees of inhibitory activity against the evaluated strains. It is worth noting that the inhibitory effect of compounds 1-12 on sensitive Staphylococcus aureus is weaker than that of vancomycin, methicillin and kanamycin, but it has good antibacterial activity against multidrug-resistant MRSA-B. Compounds 1-5 and compounds 8 and 9 have good antibacterial activity against vancomycin-resistant Enterococcus faecalis. Compounds 1-12 have moderate inhibitory activity against Streptococcus pneumoniae, but weak inhibitory activity against Staphylococcus epidermidis.

[0166] Example 5: Evaluation of the antibacterial activity of compound 1-12 combined with methicillin or kanamycin

[0167] According to the results in Example 4, the checkerboard method was used to evaluate the combined antibacterial activities of Compounds 1-12 with methicillin or kanamycin. The test strain was Staphylococcus aureus (MRSA-B). According to the MIC values of Compounds 1-12 against MRSA-B, stock solutions with certain concentrations were respectively prepared. Methicillin or kanamycin and Compounds 1-12 were added to each well of a 96-well plate. By using the two-fold dilution method, the final concentration range of methicillin was 5-640 μg / mL, the concentration range of kanamycin was 6.6-840 μg / mL, and the concentration range of the compounds was 2-64 μg / mL. The fractional inhibitory concentration index (FICI) was calculated based on the MIC values. FICI = (MIC of Drug A in combination / MIC of Drug A alone) + (MIC of Drug B in combination / MIC of Drug B alone). Among them, FICI ≤ 0.5 indicates a synergistic effect, 0.5 < FICI ≤ 1 indicates an additive effect, 1 < FICI ≤ 2 indicates an indifferent effect, and FICI > 2 indicates an antagonistic effect. The results of the activity evaluation are shown in Table 2 and Fig.25 .

[0168] Table 2 Evaluation of the antibacterial activities of Compounds 1-5 alone and in combination with methicillin or kanamycin

[0169]

[0170] Note: The unit of MIC is μg / mL, where MIC c , MIC m and MIC m respectively represent the MIC (μg / mL) of the compound, methicillin, and kanamycin in the combined use.

[0171] The experimental results showed that by combining with methicillin, the MIC values of Compounds 1-5 and methicillin against Staphylococcus aureus (MRSA-B) were significantly reduced, and the FICI values were less than 0.5, showing a synergistic effect. By combining with kanamycin, Compounds 2-5 showed obvious synergistic antibacterial activities. However, Compound 1 showed an indifferent effect.

Claims

1. A cytochalasin compound having broad antibacterial activity, which has one or two cytochalasin core structures represented by formula A and one or two epicoccine core structures represented by formula B; in, Carbon 19 and carbon 20 in formula A are simultaneously connected to carbon 1 and carbon 8, or carbon 8 and carbon 1, or carbon 2 and carbon 4, or carbon 4 and carbon 2 in formula B respectively through a carbon-carbon double bond; In the formula A, there is a single bond or a double bond between carbon 17 and carbon 18; R1 is selected from any of the following groups: -H, α-OH, β-OH, =O; R2 is selected from any of the following groups: -H, α-OH, β-OH, =O; In the formula B, there are single bonds or double bonds between carbon 1 and carbon 2, carbon 2 and carbon 3, carbon 3 and carbon 4, carbon 4 and carbon 5, carbon 5 and carbon 6, carbon 6 and carbon 7, and carbon 7 and carbon 8; in particular, carbon 3 and carbon 4 may be connected in a COC manner; R3 is selected from any of the following groups: α-OH, β-OH, =O; R4 is selected from any of the following groups: α-OH, β-OH, =O; R5 is selected from any of the following groups: α-OH, β-OH, =O.

2. The compound according to claim 1, characterized in that: The cytochalasin compound having broad antibacterial activity is a compound represented by any one of the following formulas I to XII:

3. The method for preparing the compounds of formula I to formula XII according to claim 2, comprising the following steps: fermenting Aspergillus iodendron PJ03-11 to obtain a fermentation product; and separating the fermentation product to obtain the compounds of formula I to formula XII.

4. The preparation method according to claim 3, characterized in that: The preparation method specifically comprises the following steps: 1) fermenting Aspergillus iizukae PJ03-11 to obtain a fermentation product; the Aspergillus iizukae PJ03-11 has a deposit registration number of CGMCC No.40601; extracting the fermentation product with ethyl acetate, collecting an organic phase, and concentrating the phase to obtain a crude extract; suspending the crude extract with water, and then extracting with n-hexane and ethyl acetate in sequence, collecting the ethyl acetate extraction portion to obtain the Aspergillus iizukae fermentation product; 2) The ethyl acetate portion was subjected to normal phase silica gel column chromatography, and gradient elution was performed using dichloromethane-acetone and dichloromethane-methanol as eluents, respectively. The fractions with the same main point were combined after TLC thin layer chromatography detection to obtain 10 components F1-F10; the volume ratio of the two in the dichloromethane-acetone eluent was: 100 / 0, 50 / 1, 20 / 1, 10 / 1, 8 / 1, 5 / 1, 3 / 1; the volume ratio of the two in the dichloromethane-methanol eluent was: 5 / 1, 3 / 1, 2 / 1, 1 / 1, 0 / 1, and the elution volume of each ratio of eluent was 5 times the column volume; wherein, component F5 was an elution component using a dichloromethane-acetone volume ratio of 3 / 1 as an eluent, and component F6 was an elution component using a dichloromethane-methanol volume ratio of 5 / 1 as an eluent; 3) The component F5 was subjected to ODS reverse C18 column chromatography, and gradient elution was performed with methanol-water as the eluent. The fractions with the same main point were combined after TLC thin layer chromatography detection to obtain 20 fractions, namely F5_1-F5_20; the volume ratio of methanol in the eluent was 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% respectively, and the elution volume of the eluent of each ratio was 3 times the column volume; wherein, the fraction F5_13 was the elution component of the eluent with a methanol content of 60%; The fraction F5_13 was separated by Sephadex LH-20, eluted with 5 column volumes of methanol, and each 0.5 column volume was combined into a sub-fraction, a total of 10 sub-fractions, namely F5_13_1-F5_13_10; The sub-fraction F5_13_5 is separated by reverse phase preparative chromatography RP-HPLC to obtain the compound of formula VI and the compound of formula VII; the mobile phase is an aqueous solution of 89% by volume of acetonitrile containing 1‰ trifluoroacetic acid; 4) subjecting the component F6 to ODS reverse C18 column chromatography, using methanol-water as the eluent for gradient elution, wherein the volume ratio of methanol in the eluent is 20%, 30%, 40%, 50%, 70%, 90%, and 100% in sequence, and the elution volume of the eluent in each ratio is 3 times the column volume, and the fractions with the same main point are combined by TLC thin layer chromatography to obtain 7 sub-fractions, namely F6_1-F6_7; wherein, sub-fraction F6_4 is an elution component with an eluent containing 50% methanol, and sub-fraction F6_5 is an elution component with an eluent containing 70% methanol; The sub-fraction F6_4 was separated by a Sephadex LH-20 column, eluted with methanol, and each 0.3 column volume was combined into one sub-fraction to obtain 10 sub-fractions, namely F6_4_1-F6_4_10; F6_4_4 was separated by a semi-preparative C18 reverse phase chromatography column to obtain a compound represented by formula I; the mobile phase used was an aqueous solution of 53% by volume of acetonitrile containing 1‰ trifluoroacetic acid; F6_4_5 was separated by a semi-preparative C18 reverse phase chromatography column to obtain compounds represented by formula II, III, IV and V; the mobile phase used was an aqueous solution with a volume content of 70% acetonitrile and 1‰ trifluoroacetic acid; F6_4_6 was separated by a semi-preparative C18 reverse phase chromatography column to obtain a compound represented by formula VIII, a compound represented by formula IX and a compound represented by formula X; the mobile phase used was an aqueous solution of 85% by volume of acetonitrile containing 1‰ trifluoroacetic acid; The sub-fraction F6_5 is separated by a semi-preparative C18 reverse phase chromatography column to obtain a compound represented by formula XI and a compound represented by formula XII; the mobile phase used is an aqueous solution with a volume content of 72% acetonitrile and 1‰ trifluoroacetic acid.

5. Use of the compound according to claim 1 or 2 in the preparation of antibacterial drugs.

6. The use according to claim 5, characterized in that: The antibacterial drug is capable of inhibiting the activity of Gram-positive bacteria; Or, the antibacterial drug is a drug for fighting multidrug-resistant bacterial infection; Further, the Gram-positive bacteria is at least one of sensitive Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), multidrug-resistant Staphylococcus aureus, Streptococcus pneumoniae, Staphylococcus epidermidis, and vancomycin-resistant Enterococcus faecalis; Furthermore, the multidrug-resistant bacteria are multidrug-resistant Staphylococcus aureus and / or vancomycin-resistant Enterococcus faecalis.

7. A drug for preventing and / or treating bacterial infection diseases, comprising the compound according to claim 1 or 2; Preferably, the bacteria are Gram-positive bacteria; Furthermore, the Gram-positive bacteria is at least one of sensitive Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), multidrug-resistant Staphylococcus aureus, Streptococcus pneumoniae, Staphylococcus epidermidis, and vancomycin-resistant Enterococcus faecalis.

8. An antibacterial composition A, comprising a cytochalasin compound having broad antibacterial activity and methicillin; Furthermore, the cytochalasin compound having broad antibacterial activity is selected from at least one of the compounds represented by Formula I to Formula V; Furthermore, the mass ratio of any one of the compounds represented by Formula I to Formula V to methicillin is 1:(1024-2.5), and the preferred mass ratio is 1:(256-20).

9. An antibacterial composition B, comprising a cytochalasin compound having broad antibacterial activity and kanamycin; Furthermore, the cytochalasin compound having broad antibacterial activity is selected from at least one of the compounds represented by Formula II to Formula V; Furthermore, the mass ratio of any one of the compounds represented by formula II to formula V to kanamycin is 1:(440-3), and the preferred mass ratio is 1:(52.5-26.25).

10. The Aspergillus iizukae PJ03-11 has a deposit registration number of CGMCC No.40601.

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