Subplenodomus fungus, antibacterial compound as well as preparation method and application of antibacterial compound

Abstract

The invention discloses a Subplenodomus fungus, an antibacterial compound as well as a preparation method and application of the Subplenodomus fungus and the antibacterial compound. According to the invention, a Subplenodomus fungus is found by taking a medicinal plant endophytic fungus as a research object, a potent antibacterial compound with a novel structure is found in a fermentation product of the Subplenodomus fungus, and the Subplenodomus fungus particularly has strong antibacterial activity on a drug-resistant strain, has very good application prospect and research and development value, and provides a foundation for subsequent research and development of antibacterial drugs.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 202211291210.1, application date October 17, 2022, and invention name “A Subplenodomus fungus, antibacterial compound, preparation method and application thereof”. Technical Field

[0002] The present invention relates to the technical field of chemical medicine, and in particular to a Subplenodomus fungus, an antibacterial compound, and a preparation method and application thereof. Background Art

[0003] Bacterial infection is one of the main factors that endanger human health. Bacterial infectious diseases refer to diseases caused by bacterial infection. Among the common pathogens in clinical practice, there are more Gram-negative bacteria, such as Escherichia, Enterobacter and Pseudomonas, and there are also Gram-positive bacteria, such as Staphylococcus and Enterococcus.

[0004] At present, most of the drugs commonly used for bacterial infectious diseases are antibiotics and broad-spectrum antibacterial drugs, such as imipenem, vancomycin, teicoplanin, etc., which have great side effects, and large-scale use will produce drug resistance, leading to the emergence of super bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus aureus (MRSE), vancomycin-resistant enterococci (VRE), etc., which have become important pathogens of hospital infections. Bacterial resistance makes the challenges facing global public health more severe.

[0005] In order to alleviate the public health crisis caused by antibiotic-resistant bacteria, the need to develop new antimicrobial drugs is particularly urgent.

[0006] Subplenodomus belongs to the phylum Ascomycota, class Doctromycetes, order Lachnosporales, family Microsphaeriaceae, and is mainly isolated from plants of the family Gentianaceae. There are no reports on the secondary metabolites of strains of this genus. Summary of the invention

[0007] In view of this, the present invention provides a Subplenodomus fungus, an antibacterial compound, and a preparation method and application thereof.

[0008] In a first aspect of the present invention, a fungus of the genus Subplenodomus is provided.

[0009] Specifically, the ITS gene sequence of the Subplenodomus fungus strain is shown in SEQ ID NO:1.

[0010] Specifically, the characteristics of the Subplenodomus fungus include: the colony grows on a PDA medium with a flat edge, a bulge in the middle, white aerial hyphae that are relatively developed; the mycelium is light yellow to gray, matte and dry; the reverse side has a white edge and is brown to brown in the middle.

[0011] In one embodiment of the present invention, the Subplenodomus fungus is an endophyte from Gentiana straminea Maxim.; specifically, the Gentiana straminea is from a swamp, wet grassland, riverside, hillside or forest edge; the Gentiana straminea is from Tibet, Yunnan, Guizhou, Shanxi, Shaanxi, Gansu, Inner Mongolia and the like.

[0012] Preferably, the ITS gene sequence of the Subplenodomus fungus strain has 100% similarity with the strain Subplenodomus drobnjacensis PRG11-03 in ITS sequence; 89% similarity with Comoclathris incompta isolate CH-12 in tublin sequence; and 100% similarity with Subplenodomus drobnjacensis PRG11-03 in LSU sequence. Based on the morphological characteristics and ITS gene sequence of the strain, the strain is determined to be Subplenodomus sp.

[0013] Most preferably, the Subplenodomus fungus is CPCC 401465, which has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on April 25, 2022, with the deposit number CGMCC No.40181.

[0014] In a second aspect of the present invention, a composition is provided, comprising the Subplenodomus fungus and / or its metabolites as described in the first aspect.

[0015] Specifically, the metabolite can be a culture, a bacterial suspension, a lysate, etc. of the above-mentioned Subplenodomus fungus.

[0016] In some embodiments of the present invention, the culture is a fermentation product of the above-mentioned Subplenodomus fungus, such as a fermentation broth, a clarified liquid of the fermentation broth (such as a supernatant, a filtrate, etc., which no longer contains solid matter (such as bacteria) in the fermentation broth), a concentrate of the fermentation broth, an extract of the fermentation broth, etc. Preferably, the fermentation product of the Subplenodomus fungus is obtained by the following fermentation method.

[0017] In the third aspect of the present invention, a fermentation method of the Subplenodomus fungus described in the first aspect is provided.

[0018] Specifically, the fermentation method comprises the step of fermenting the Subplenodomus fungus in a culture medium.

[0019] Specifically, the culture medium is a fungal fermentation medium, which can be a solid culture medium, a semi-solid culture medium or a liquid culture medium; more specifically, the culture medium can be a rice culture medium, a potato dextrose agar culture medium or other fungal fermentation culture medium well known to professionals in the field.

[0020] Preferably, the rice culture medium comprises rice and water. More preferably, the rice culture medium further comprises one or more of inorganic salts and nitrogen sources.

[0021] Specifically, the rice culture medium may be made of rice and water, or may be made of rice, water and inorganic salts, or may be made of rice, water and a nitrogen source, or may be made of rice, water, a nitrogen source and inorganic salts.

[0022] The rice can be rice or brown rice. Rice or brown rice are both products of rice. Rice refers to the fruit without removing the husk of rice, and rice is composed of husk, pericarp, seed coat, endosperm, endosperm, endosperm and embryo. Brown rice refers to the product of removing the husk of rice and retaining other parts of rice; rice refers to the product of only retaining the endosperm and removing all other parts of rice.

[0023] Preferably, the potato dextrose agar medium comprises potatoes, glucose and agar; more preferably, the potato dextrose agar medium further comprises one or more of water, inorganic salts and nitrogen sources;

[0024] Specifically, the potato dextrose agar medium can be made of potatoes, glucose, agar and water, or potatoes, glucose, agar, water and inorganic salts, or potatoes, a carbon source, agar, water and a nitrogen source, or potatoes, a carbon source, agar, a nitrogen source and inorganic salts.

[0025] Specifically, the fungal fermentation medium known to professionals in the art can be made of one or more fast-acting or slow-acting carbon sources, one or more fast-acting or slow-acting nitrogen sources, water and / or inorganic salts.

[0026] In some embodiments of the present invention, the culture medium is a rice culture medium, in particular a rice culture medium.

[0027] Specifically, the mass ratio of rice to water in the rice culture medium can be 1:0.8-1.2 (e.g., 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2), especially 1:1.

[0028] Specifically, the inoculum amount of the fermentation can be 1-10% (e.g., 1%, 2%, 4%, 5%, 6%, 7%, 8%, 10%), especially 5-8%.

[0029] Specifically, the fermentation temperature can be 20-35°C (e.g., 20, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35°C), in particular 28-30°C.

[0030] Specifically, the fermentation time can be 1-40 days (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40 days), in particular 25-35 days.

[0031] Specifically, the fermentation is static culture fermentation.

[0032] Specifically, the above method may further include the step of expanding the culture of the bacterial strain (before fermentation), for example, expanding the culture of the bacterial strain activated by the slant culture medium in a seed culture medium.

[0033] Specifically, the temperature of the expanded culture can be 20-35°C (e.g., 20, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35°C), in particular 28°C.

[0034] Specifically, the time for expanded culture can be 1-5 days, especially 1-3 days.

[0035] Specifically, the expansion culture is performed under shaking conditions (eg, 200 rpm).

[0036] Specifically, the seed culture medium can be any suitable fungal culture medium, such as Sabouraud medium, potato dextrose medium, etc.;

[0037] In one embodiment of the present invention, the seed culture medium is a potato glucose liquid culture medium, which is composed of potato powder, glucose and water, for example, it contains 4 g / L potato powder and 20 g / L glucose. Specifically, the pH value of the seed culture medium can be 5-7 (for example, 5, 5.4, 5.6, 5.8, 6, 6.5, 7).

[0038] In a fourth aspect of the present invention, a compound and a pharmaceutically acceptable salt, stereoisomer, prodrug or solvate thereof are provided, wherein the compound has the following structure:

[0039]

[0040] in,

[0041] represents a single bond or a double bond;

[0042] X and Y are independently selected from:

[0043] R1 and R2 are independently selected from: H, -OH, -O-(C 1-3 Alkyl), -OCO-(C 1-3 alkyl);

[0044] R3 and R4 are independently selected from: H, -OH, -O-(C 1-3 Alkyl), -OCO-(C 1-3 Alkyl), C 1-3 alkyl;

[0045] R5 and R6 are independently selected from: H, -OH, -O-(C 1-3 Alkyl), -OCO-(C 1-3 Alkyl), C 1-3 alkyl;

[0046] R7 and R8 are independently selected from: H, -OH, -O-(C 1-3 Alkyl), -OCO-(C 1-3 Alkyl), C 1-3 alkyl;

[0047] R9 and R 10 Independently selected from: H, -OH, -O-(C 1-3 Alkyl), -OCO-(C 1-3 Alkyl), C 1-3 alkyl;

[0048] R 11 To R 14 Independently selected from: H, -OH, -O-(C 1-3 Alkyl), -OCO-(C 1-3 Alkyl), C 1-3 Alkyl; or, R 11 and R 13 Together with the carbon atom to which it is attached, it forms a heterocyclic group (e.g. ), and / or, R 12 and R 14 Together with the carbon atom to which it is attached, it forms a heterocyclic group (e.g. ).

[0049] In one embodiment of the present invention, the compound has the following structure:

[0050]

[0051] in,

[0052] represents a single bond or a double bond;

[0053] X and Y are independently selected from:

[0054] R1 and R2 are independently selected from: H, -OH, -O-(C 1-3 Alkyl), -OCO-(C 1-3 alkyl).

[0055] Specifically, the compound may have the following structure:

[0056]

[0057] Specifically, R1 can be selected from: H, -OH.

[0058] Specifically, R2 can be selected from: H, -OH.

[0059] Specifically, X can be selected from:

[0060] Specifically, Y can be selected from:

[0061] More specifically, the compound may have the following structure:

[0062]

[0063]

[0064] In some embodiments of the present invention, the stereoisomers of the compound have the following structures:

[0065]

[0066] In another embodiment of the present invention, the compound has the following structure:

[0067]

[0068] in,

[0069] represents a single bond or a double bond;

[0070] Y is selected from: R2 is selected from: H, -OH, -O-(C 1-3 Alkyl), -OCO-(C 1-3 alkyl);

[0071] R5 is selected from: H, -OH, -O-(C 1-3 Alkyl), -OCO-(C 1-3 Alkyl), C 1-3 alkyl.

[0072] Specifically, R2 can be selected from: H, -OH.

[0073] Specifically, R5 can be selected from: -OH, -OCH3, -OCO-CH3.

[0074] Specifically, the compound may have the following structure:

[0075]

[0076] More specifically, the compound may have the following structure:

[0077]

[0078] In some embodiments of the present invention, the stereoisomers of the compound have the following structures:

[0079]

[0080] In another embodiment of the present invention, the compound has the following structure:

[0081]

[0082] in,

[0083] R5 and R6 are independently selected from: H, -OH, -O-(C 1-3 Alkyl), -OCO-(C 1-3 alkyl).

[0084] Specifically, R5 can be selected from: -OH, -OCH3, -OCO-CH3.

[0085] Specifically, R6 can be selected from: -OH, -OCH3, -OCO-CH3.

[0086] Specifically, the compound may have the following structure:

[0087]

[0088] More specifically, the compound may have the following structure:

[0089]

[0090] In a fifth aspect of the present invention, provided are compounds selected from the following A1 to A13 and structural analogs thereof:

[0091]

[0092] Specifically, the above structural analogs are compounds that are structurally modified and transformed based on the basic core of compounds A1 to A13, such as other compounds or stereoisomers (except A1 to A13) covered by the general formula I described in the fourth aspect of the present invention. The structural modification and transformation methods can be methods well known to those skilled in the art.

[0093] In a sixth aspect of the present invention, a method for preparing the compound described in the fifth aspect is provided, which comprises the steps of extracting and separating the fermentation product of the Subplenodomus fungus described in the first aspect.

[0094] Specifically, the fermentation product can be prepared by the fermentation method described in the third aspect of the present invention.

[0095] Specifically, the extraction and separation comprises:

[0096] (1) extracting the fermentation product with an organic solvent I to obtain a crude extract;

[0097] (2) dispersing the crude extract obtained in step (1), and extracting with an organic solvent II to obtain an extract;

[0098] (3) subjecting the extract obtained in step (2) to adsorption chromatography and collecting the eluate;

[0099] Optionally, (4) extracting and separating the eluate obtained in step (3).

[0100] In one embodiment of the present invention, the extraction in step (1) is ultrasonic extraction.

[0101] Specifically, the extraction temperature in step (1) is 20-30°C, such as room temperature.

[0102] Specifically, the extraction time in step (1) can be 1-5 days, for example 3 days.

[0103] Specifically, the extraction in step (1) can be performed once or multiple times, for example, three times.

[0104] Specifically, the organic solvent II in step (2) can be selected from: methanol, ethanol, ethyl acetate; in one embodiment of the present invention,

[0105] Specifically, step (1) further includes the step of filtering the extract; more specifically, step (1) further includes the step of concentrating the filtrate, and the crude extract obtained in this case is a concentrated crude extract.

[0106] Specifically, the dispersing solvent in step (2) is immiscible with the organic solvent II; in one embodiment of the present invention, the dispersing solvent in step (2) is water, such as deionized water.

[0107] Specifically, the organic solvent II in step (2) can be selected from: ethyl acetate, N,N-dimethylacetamide, isopropyl acetate, methyl acetate, methyl isobutyl ketone; in one embodiment of the present invention, the organic solvent II in step (2) is ethyl acetate.

[0108] Specifically, the extraction in step (2) can be performed once or multiple times, for example, three times.

[0109] Specifically, the adsorbent (stationary phase) of the adsorption chromatography in step (3) is silica gel, especially silica gel H.

[0110] Specifically, the mobile phase of the adsorption chromatography in step (3) is dichloromethane and / or methanol, in particular a mixture of dichloromethane and methanol, and the volume ratio thereof can be 100-0:0-100.

[0111] Specifically, the extraction and separation in step (4) includes the steps of ODS column chromatography separation and preparative HPLC separation.

[0112] In the seventh aspect of the present invention, a pharmaceutical composition is provided, which comprises the composition of the second aspect, the compound of the fourth aspect or its pharmaceutically acceptable salt, stereoisomer, prodrug, solvate, or the compound of the fifth aspect or its structural analogue, and one or more pharmaceutically acceptable excipients.

[0113] In one embodiment of the present invention, the pharmaceutical composition is used as an antimicrobial drug, particularly an antibacterial drug.

[0114] Specifically, the bacteria are pathogenic bacteria, such as, but not limited to, Staphylococcus aureus (including methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA) (e.g., vancomycin-resistant Staphylococcus aureus (VISA))), Staphylococcus epidermidis (including methicillin-sensitive Staphylococcus epidermidis (MSSE), methicillin-resistant Staphylococcus epidermidis (MRSE)), Enterococcus faecalis (including vancomycin-sensitive Enterococcus faecalis, vancomycin-resistant Enterococcus faecalis (VRE)), Enterococcus faecium (including vancomycin-sensitive Enterococcus faecium, vancomycin-resistant Enterococcus faecium (VRE)), Streptococcus pneumoniae (including penicillin-sensitive Streptococcus pneumoniae Pseudomonas aeruginosa (including multidrug-resistant Pseudomonas aeruginosa (MDR-PA), carbapenem-resistant Pseudomonas aeruginosa (CRPA)), Klebsiella pneumoniae (including third-generation cephalosporin-resistant Klebsiella pneumoniae (3GCRKP)), Escherichia coli (including third-generation cephalosporin-resistant Escherichia coli (3GCREC), fluoroquinolone-resistant Escherichia coli (FQREC), polymyxin-resistant Escherichia coli (ColR-EC)), Acinetobacter baumannii (including multidrug-resistant Acinetobacter baumannii (MDR-AB), carbapenem-resistant Acinetobacter baumannii (CRAB)), Clostridium difficile, Streptococcus pyogenes, Serratia marcescens, Micrococcus lysodeikticus, Micrococcus luteus, Salmonella, Pseudomonas aeruginosa, etc.

[0115] In particular, the bacteria are Gram-positive bacteria.

[0116] In particular, the bacteria are drug-resistant strains, such as, but not limited to, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), vancomycin-resistant Enterococcus (VRE), penicillin-resistant Streptococcus pneumoniae (PRSP), multidrug-resistant Pseudomonas aeruginosa (MDR-PA), carbapenem-resistant Pseudomonas aeruginosa (CRPA), third-generation cephalosporin-resistant Klebsiella pneumoniae (3GCRKP), third-generation cephalosporin-resistant Streptococcus pneumoniae (3GCRKP), and third-generation cephalosporin-resistant Streptococcus pneumoniae (3GCRKP). Enterobacter (3GCREC), fluoroquinolone-resistant Escherichia coli (FQREC), polymyxin-resistant Escherichia coli (ColR-EC), multidrug-resistant Acinetobacter baumannii (MDR-AB), carbapenem-resistant Acinetobacter baumannii (CRAB), etc., especially methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), vancomycin-resistant Enterococcus (VRE), and penicillin-resistant Streptococcus pneumoniae (PRSP).

[0117] In some embodiments of the present invention, the bacteria is Staphylococcus epidermidis, such as Staphylococcus epidermidis (ATCC12228, MSSE) and Staphylococcus epidermidis (19-1, MRSE).

[0118] In other embodiments of the present invention, the bacteria is Staphylococcus aureus, such as Staphylococcus aureus (ATCC 29213, MSSA), Staphylococcus aureus (ATCC 33591, MRSA), Staphylococcus aureus (ATCC 43300, MRSA), Staphylococcus aureus (ATCC 700698, MRSA, VISA), Staphylococcus aureus (15, MSSA).

[0119] In other embodiments of the present invention, the bacteria are enterococci, such as Enterococcus faecalis (ATCC 29212, VSE), Enterococcus faecalis (ATCC 51299, VRE), Enterococcus faecium (ATCC 700221, VRE), and Enterococcus faecium (20-2, VSE).

[0120] Specifically, the pharmaceutical composition can be in any suitable dosage form, such as, but not limited to, tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, electuary preparations, pills, powders, pastes, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, patches, etc., preferably injections.

[0121] Specifically, pharmaceutically acceptable excipients may be, for example, but not limited to, binders, fillers, diluents, tableting agents, lubricants, disintegrants, colorants, flavoring agents and wetting agents, etc.; suitable fillers may be, for example, cellulose, mannitol, lactose and other similar fillers; suitable disintegrants may be, for example, starch, polyvinyl pyrrolidone and starch derivatives may be, for example, sodium starch glycolate; suitable lubricants may be, for example, magnesium stearate; suitable wetting agents may be, for example, sodium lauryl sulfate.

[0122] In the eighth aspect of the present invention, there is provided the use of the Subplenodomus fungus described in the first aspect, the composition described in the second aspect, the compound described in the fourth aspect or its pharmaceutically acceptable salt, stereoisomer, prodrug, solvate, the compound described in the fifth aspect or its structural analogue in the preparation of an antibacterial agent.

[0123] Specifically, the antimicrobial agent can be used to kill or inhibit pathogens, especially pathogenic bacteria, as described in the seventh aspect of the present invention.

[0124] In particular, the antimicrobial agent can be used to kill or inhibit drug-resistant strains, as described in the seventh aspect of the present invention.

[0125] In one embodiment of the present invention, the antibacterial agent is a drug, which can be used to prevent and / or treat diseases caused by pathogen infection.

[0126] In the ninth aspect of the present invention, there is provided use of the Subplenodomus fungus described in the first aspect, the composition described in the second aspect, the compound described in the fourth aspect or its pharmaceutically acceptable salt, stereoisomer, prodrug, solvate, the compound described in the fifth aspect or its structural analogue, and the pharmaceutical composition described in the seventh aspect in the preparation of a medicament for preventing and / or treating a disease.

[0127] In one embodiment of the present invention, the disease is a disease caused by pathogen infection.

[0128] Specifically, the pathogen may be a microorganism, a parasite (protozoa, helminth, etc.) or other agent.

[0129] Specifically, the pathogenic microorganism can be selected from one or more of: viruses, chlamydia, rickettsia, mycoplasma, bacteria, spirochetes, fungi, etc., especially bacteria.

[0130] Specifically, the bacteria are as described in the seventh aspect of the present invention.

[0131] In particular, the bacteria are drug-resistant strains, as described in the seventh aspect of the present invention.

[0132] In another embodiment of the present invention, the disease is a tumor, in particular a malignant tumor, such as lung cancer, breast cancer, liver cancer, pancreatic cancer, colorectal cancer, gastric cancer, gastroesophageal adenocarcinoma, esophageal cancer, small intestine cancer, cardia cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer, kidney cancer, bladder cancer, anal cancer, gallbladder cancer, bile duct cancer, myeloma, leukemia, lymphoma, melanoma, head and neck tumors, etc.

[0133] Specifically, the subject of the drug can be a mammal, such as a human, monkey, pig, cow, horse, sheep, dog, cat, mouse, etc., especially a human.

[0134] In the tenth aspect of the present invention, a method for preventing and / or treating a disease is provided, which comprises the step of administering to a subject in need thereof an effective amount of the compound described in the fourth aspect or a pharmaceutically acceptable salt, stereoisomer, prodrug, solvate thereof, the compound described in the fifth aspect or a structural analog thereof, or the pharmaceutical composition described in the seventh aspect.

[0135] Specifically, the disease is a disease caused by infection with pathogens, especially a disease caused by infection with bacteria (such as drug-resistant strains).

[0136] Specifically, the bacteria are as described in the seventh aspect of the present invention.

[0137] Specifically, the subject is as described in the ninth aspect of the present invention.

[0138] The present invention uses endophytic fungi of medicinal plants as the research object and discovers a strain of Subplenodomus fungus. By fermenting and separating it, a novel and potent antibacterial compound can be obtained, which has very high antibacterial activity against drug-resistant strains, especially, has very good application prospects and research and development value, and provides a basis for the subsequent research and development of antibacterial drugs.

[0139] The deposit information of the biological material of the present invention is as follows:

[0140] A Subplenodomus fungus CPCC 401465, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on April 25, 2022, with the deposit number CGMCC No.40181 and the classification name Subplenodomus sp. BRIEF DESCRIPTION OF THE DRAWINGS

[0141] Figure 1 It is a high-resolution mass spectrum of the compound A1 described in the present invention.

[0142] Figure 2 It is a high-resolution mass spectrum of compound A2 described in the present invention.

[0143] Figure 3 It is a high-resolution mass spectrum of compound A3 described in the present invention.

[0144] Figure 4 It is a high-resolution mass spectrum of compound A4 described in the present invention.

[0145] Figure 5 It is a high-resolution mass spectrum of compound A5 described in the present invention.

[0146] Figure 6 It is a high-resolution mass spectrum of compound A6 described in the present invention.

[0147] Figure 7 It is a high-resolution mass spectrum of compound A7 described in the present invention.

[0148] Figure 8 It is a high-resolution mass spectrum of compound A8 described in the present invention.

[0149] Fig. 9 It is a high-resolution mass spectrum of compound A9 described in the present invention.

[0150] Fig.10 It is a high-resolution mass spectrum of compound A10 described in the present invention.

[0151] Fig.11 This is a high-resolution mass spectrum of the compound A11 described in the present invention.

[0152] Fig.12 This is a high-resolution mass spectrum of the compound A12 described in the present invention.

[0153] Fig.13 It is a high-resolution mass spectrum of compound A13 described in the present invention.

[0154] Fig.14 The compound A1 of the present invention is dissolved in CDCl3 1 H-NMR spectrum.

[0155] Fig.15 The compound A1 of the present invention is dissolved in CDCl3 13 C-NMR spectrum

[0156] Fig.16 The compound A2 of the present invention is dissolved in CDCl3 1 H-NMR spectrum.

[0157] Fig.17 The compound A2 of the present invention is dissolved in CDCl3 13 C-NMR spectrum

[0158] Fig.18 The compound A3 of the present invention is dissolved in DMSO-d6 1 H-NMR spectrum.

[0159] Fig.19 The compound A3 of the present invention is dissolved in DMSO-d6 13 C-NMR spectrum

[0160] Fig. 20 The compound A4 of the present invention is dissolved in CDCl3 1 H-NMR spectrum

[0161] Fig.21 The compound A4 of the present invention is dissolved in CDCl3 13 C-NMR spectrum.

[0162] Fig. 22 The compound A5 of the present invention is dissolved in CDCl3 1 H-NMR spectrum.

[0163] Fig.23 The compound A5 of the present invention is dissolved in CDCl3 13 C-NMR spectrum.

[0164] Fig.24The compound A6 of the present invention is dissolved in CDCl3 1 H-NMR spectrum.

[0165] Fig.25 The compound A6 of the present invention is dissolved in CDCl3 13 C-NMR spectrum.

[0166] Fig.26 The compound A7 of the present invention is dissolved in DMSO-d6 1 H-NMR spectrum.

[0167] Fig. 27 The compound A7 of the present invention is dissolved in DMSO-d6 13 C-NMR spectrum.

[0168] Fig.28 The compound A8 of the present invention is dissolved in CDCl3 1 H-NMR spectrum.

[0169] Fig.29 The compound A8 of the present invention is dissolved in CDCl3 13 C-NMR spectrum.

[0170] Fig.30 The compound A9 of the present invention is dissolved in DMSO-d6 1 H-NMR spectrum.

[0171] Fig.31 The compound A9 of the present invention is dissolved in DMSO-d6 13 C-NMR spectrum.

[0172] Fig.32 The compound A10 of the present invention is dissolved in CDCl3 1 H-NMR spectrum.

[0173] Fig.33 The compound A10 of the present invention is dissolved in CDCl3 13 C-NMR spectrum.

[0174] Fig.34 The compound A11 of the present invention is dissolved in CDCl3 1 H-NMR spectrum.

[0175] Fig.35 The compound A11 of the present invention is dissolved in CDCl3 13 C-NMR spectrum.

[0176] Fig.36 The compound A12 of the present invention is dissolved in DMSO-d61 H-NMR spectrum.

[0177] Fig.37 The compound A12 of the present invention is dissolved in DMSO-d6 13 C-NMR spectrum.

[0178] Fig.38 The compound A13 of the present invention is dissolved in CDCl3 1 H-NMR spectrum.

[0179] Fig.39 The compound A13 of the present invention is dissolved in CDCl3 13 C-NMR spectrum. DETAILED DESCRIPTION

[0180] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0181] The term "culture" refers to a cell group or growth of a microorganism in a certain time and space, and particularly refers to a liquid or solid culture medium in which a group of microorganisms (such as the Subplenodomus fungus CPCC 401465 described in the present invention) grows after artificial inoculation and cultivation, such as a slant culture of a microorganism, a fermentation product, etc.

[0182] The term "bacterial suspension" refers to a suspension obtained by dispersing cells of a microorganism (such as the Subplenodomus fungus CPCC 401465 described in the present invention) in a solvent (such as water).

[0183] The term "lysate" refers to a product obtained after the cells of a microorganism (such as the Subplenodomus fungus CPCC 401465 described in the present invention) are lysed.

[0184] The term "carbon source" refers to a class of nutrients that contain carbon and can be used by microorganisms for growth and reproduction, including fast-acting and slow-acting carbon sources such as sugars, oils, organic acids and organic acid esters, and small molecule alcohols.

[0185] The term "nitrogen source" refers to substances that provide nitrogen elements required for microbial nutrition, including fast-acting and slow-acting nitrogen sources such as peanut cake powder, soybean cake powder, yeast powder, peptone, ammonia water, ammonium salts and nitrates.

[0186] The term "alkyl" refers to a straight or branched hydrocarbon group that does not contain unsaturated bonds and is connected to the rest of the molecule by a single bond. The alkyl group used herein generally contains 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms (i.e., C1-10 alkyl), preferably 1 to 3 carbon atoms (i.e., C1-3 Examples of the alkyl group include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, and the like.

[0187] The pharmaceutically acceptable salts of the present invention include acid addition salts and base addition salts.

[0188] The acid addition salts include, but are not limited to, salts from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid and phosphonic acid, and salts from organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids and aliphatic and aromatic sulfonic acids. Therefore, these salts include but are not limited to sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, iodate, acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate and mesylate, also comprising amino acid salts such as arginate, gluconate, galacturonate etc. Acid addition salts can be prepared by contacting the free alkali form with a sufficient amount of the required acid to form a salt in a conventional manner. The free alkali form can be regenerated by contacting the salt form with an alkali, and the free alkali is separated in a conventional manner.

[0189] The base addition salts are salts formed with metals or amines, such as hydroxides of alkali metals and alkaline earth metals, or with organic amines. Examples of metals used as cations include, but are not limited to, sodium, potassium, magnesium, and calcium. Examples of suitable amines include, but are not limited to, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (ethane-1,2-diamine), N-methylglucamine, and procaine. Base addition salts can be prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to form a salt. The free acid form can be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner.

[0190] Stereoisomers of the present invention include enantiomers, diastereomers and geometric isomers. Some compounds of the present invention have cycloalkyl groups, which can be substituted on more than one carbon atom, in which case all geometric forms, including cis and trans, and mixtures thereof, are within the scope of the present invention. The cycloalkyl groups include alicyclic hydrocarbon groups and aryl groups, wherein the alicyclic hydrocarbon groups can be non-aromatic monocyclic, condensed rings, bridged rings or spirocyclic saturated or unsaturated cyclic hydrocarbon groups, aryl groups such as phenyl, naphthyl, phenanthrenyl, biphenyl etc.

[0191] Solvates of the present invention refer to the physical combination of a compound of the present invention and one or more solvent molecules. The physical combination includes various degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, the solvate can be separated, for example, when one or more solvent molecules are incorporated into the lattice of a crystalline solid. Solvates include solution phases and separable solvates. Representative solvates include ethanolates, methanolates, etc.

[0192] The prodrugs of the present invention refer to forms of the compounds of formula I which are suitable for administration to patients without excessive toxicity, irritation, allergic reactions, etc. and are effective for their intended use, including acetal, ester and zwitterion forms. The prodrugs are transformed in vivo, such as by hydrolysis in the blood, to obtain the parent compound.

[0193] The terms "patient" or "subject" and the like are used interchangeably herein and refer to any animal or cell thereof treated according to the methods described herein, whether in vitro or in situ. Specifically, the aforementioned animals include mammals, e.g., rats, mice, guinea pigs, rabbits, dogs, cats, pigs, cows, horses, sheep, monkeys or humans, particularly humans.

[0194] The term "treatment" as used herein refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, stopping and / or stopping one or more clinical symptoms of a disease after the onset of the disease.

[0195] The term "prevention" as used in the present invention refers to preventing, minimizing or making it difficult for a disease to occur or develop by treatment before the disease occurs.

[0196]

[00136] Various publications, patents, and published patent specifications are cited herein, the disclosures of which are incorporated by reference in their entireties.

[0197] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0198] Example 1: Isolation and identification of Subplenodomus fungus CPCC 401465

[0199] 1. Isolation of strains

[0200] The fungus of the genus Subplenodomus CPCC 401465 was isolated from the root sample of the medicinal plant Macrophylla tricolor in Mila Mountain, Gongbujiangda County, Linzhi City, Tibet Autonomous Region. The specific method is as follows: collect the whole Macrophylla tricolor plant into a ziplock bag and bring it back to the laboratory for processing. The whole plant was rinsed with 75% alcohol for 1 minute, 2% sodium hypochlorite for 3 minutes, 75% alcohol for 30 seconds, and sterile water for 3 times. After that, the surface water of the plant was dried with sterile absorbent paper, and the plant was cut into tissue blocks of about 0.5 cm in length with sterile scissors, and placed in a tube containing streptomycin sulfate (50 mg L -1 ) and tetracycline (50 mg L -1 ) on a PDA solid medium plate. Place in a 28°C incubator for culture. After 5-7 days, pick a single colony based on the color, size, shape and other characteristics of the colony, place it on a test tube slope containing PDA medium to obtain a pure culture, and store it in a refrigerator after it grows well. Take the strain numbered CPCC401465 for the following identification.

[0201] The preservation method of strain CPCC 401465 was as follows: stored in 20% glycerol cryovials at -80°C.

[0202] 2. Identification of strains

[0203] (1) Morphological characteristics

[0204] The colonies of strain CPCC 401465 grew on PDA medium with flat edges and a bulge in the middle. The aerial hyphae were white and relatively developed. The mycelium was light yellow to gray, with a dull and dry surface. The reverse side had white edges and was brown to brown in the middle.

[0205] (2) Molecular Biological Identification

[0206] The strain CPCC 401465 was picked from the solid culture medium and placed in a sterile 1.5 mL centrifuge tube. Liquid nitrogen was added. After cooling, the sample was quickly ground into powder using a special glass grinding rod. 300 μL of 2×CTAB extract was added, gently mixed, and placed in a 95°C water bath for 2 min. An equal volume (300 μL) of phenol-chloroform mixture (phenol: chloroform: isoamyl alcohol (25:24:1, V / V / V)) was added, oscillated and mixed, and centrifuged at 12000 rpm for 5 min. The supernatant was transferred to a new 1.5 mL sterile centrifuge tube, an equal volume of isopropanol was added, oscillated and mixed, and transferred to a silica membrane adsorption centrifuge column. The tube was allowed to stand for 2 min and centrifuged at 12000 rpm for 30 s. The liquid was discarded, and 500 μL of EB washing solution, centrifuge at 12000rpm for 30s; centrifuge and dry the silica column at 12000rpm for 2min; move the silica membrane adsorption centrifuge column into a new 1.5mL sterile centrifuge tube, add 65℃50μL TE elution solution, centrifuge at 12000rpm for 30s; discard the silica column, collect 50μL liquid in the centrifuge tube, and obtain the crude extract of total DNA. The crude extract of the above DNA was sent to Shanghai Biotech for sequencing. The sequence determination results were submitted to NCBI and compared with the corresponding gene sequences that effectively describe the strain in the relevant genus and species to preliminarily determine the group to which the strain belongs.

[0207] The ITS gene sequence of strain CPCC 401465 is shown in SEQ ID NO: 1. It has 100% similarity with strain Subplenodomus drobnjacensis PRG11-03 in ITS sequence, 89% similarity with Comoclathris incompta isolateCH-12 in tublin sequence, and 100% similarity with Subplenodomus drobnjacensis PRG11-03 in LSU sequence. Based on the morphological characteristics and ITS gene sequence of the strain, the strain was identified as Subplenodomus sp.

[0208] The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 25, 2022, with the deposit number CGMCC No. 40181. It is hereinafter referred to as Subplenodomus fungus CPCC 401465.

[0209] Example 2: Fermentation culture

[0210] (1) Spores of the fungus species CPCC 401465 of the genus Subplenodomus were inoculated into a slant culture medium and cultured at 28°C for 5 days to obtain a slant culture.

[0211] The slant culture medium is composed of potato extract powder, glucose, agar and water. The concentrations of the above components in the slant culture medium are: 4 g / L potato extract powder, 20 g / L glucose, and 15 g / L agar; the pH value of the slant culture medium is 5.6±0.2.

[0212] (2) 100 mL of seed culture medium was placed in each of several 500 mL glass bottles, sterilized at 121°C for 20 minutes, and mycelium was picked from the slant for inoculation. The mixture was cultured at 28°C with shaking (200 rpm) for 2 days to obtain seed solution.

[0213] The seed culture medium is composed of potato powder, glucose and water. The concentrations of the above ingredients in the seed culture medium are: 4 g / L potato powder; 20 g / L glucose; and the pH value of the liquid culture medium is 5.6±0.2.

[0214] (3) 80 g of rice was placed in a 500 mL triangular flask, 80 mL of water was added for soaking, and the mixture was sterilized at 121° C. for 20 minutes to obtain a fermentation medium. The seed solution obtained in the above step (2) was inoculated into the fermentation medium (5 mL of seed solution was inoculated into each bottle of fermentation medium), and the fermentation product was obtained by static culture at 28-30° C. for 30 days.

[0215] Example 3: Isolation, purification and identification of antibacterial compounds

[0216] 1. Isolation and purification of antibacterial compounds

[0217] Adsorption chromatography:

[0218] The solid fermentation culture product of CPCC 401465 rice (4.0 kg) was poured into a 30 L barrel, soaked in methanol for 3 days, ultrasonically extracted 3 times, filtered, centrifuged and the filtrate was concentrated under reduced pressure to obtain 350.0 g of methanol crude extract; the methanol crude extract was dispersed with 3 L of deionized water, extracted with an equal volume of ethyl acetate 3 times to obtain 180.0 g of ethyl acetate extract, and the antibacterial activity was detected by the paper strip method, and it was found that the active component was the ethyl acetate extract. TLC detection and analysis were performed, and based on the results, silica gel column chromatography was performed with a dichloromethane-methanol system for separation and purification, and gradient elution (v / v, 100:0, 95:5, 90:10, 85:15, 80:10, 75:15, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 0:100). After TLC detection, 15 components, component 1 to component 15, were obtained by merging.

[0219] 500 mg of component 2 was purified by ODS pressure purification (10%-100% acetonitrile-0.01% TFA water, 25.0 mL / min flow rate) to obtain components B1-B3. Component B2 was prepared by HPLC (68% acetonitrile-0.01% TFA water, 3.5 mL / min flow rate) to obtain compounds A13 (10.8 mg, tR 14.2 min) and A6 (10.2 mg, tR 18.2 min). Components 3 (14.63 g) and 4 (33.24 g) had the same main component, so the two components were combined and separated and purified. 500 mg of component 3 was purified by ODS pressure (10%-100% acetonitrile-0.01% TFA water, 25.0 mL / min flow rate) to obtain components C1-C3. Component C1 was prepared by HPLC (60% acetonitrile-0.01% TFA water, 3.5 mL / min flow rate) to obtain A10 (14.2 mg, tR 13.2 min) and A11 (10.3 mg, tR 14.5 min); component C3 was prepared by HPLC (70% acetonitrile-0.01% TFA water, 3.5 mL / min flow rate) to obtain A4 (17.1 mg, tR 16.2 min), A5 (5.0 mg, tR 17.5 min), A1 (6.7 mg, tR 1 Fraction 5 was purified by ODS pressure (10%-100% acetonitrile-0.01% TFA water, 25.0 mL / min flow rate) to obtain fractions D1-D5, and 60 mg of D4 was directly subjected to HPLC preparation (50% acetonitrile-0.01% TFA water, 3.5 mL / min flow rate) to obtain compounds A3 (19.0 mg, tR 14.4 min) and A12 (19.8 mg, tR 12.1 min). Fraction 6 (3.50 g) was purified by ODS pressure (10%-100% acetonitrile-0.01% TFA water, 25.0 mL / min flow rate) to obtain fractions E1-E4, and fraction E4 was subjected to HPLC preparation (65% acetonitrile-0.01% TFA water, 3.5 mL / min flow rate) to obtain compound A9 (10.8 mg, tR 14.2 min). Fraction 7 (2.82 g) was purified by ODS pressure (10%-100% acetonitrile-0.01% TFA water, 25.0 mL / min flow rate) to obtain fractions F1-F4, and fraction F3 was subjected to HPLC preparation (65% acetonitrile-0.01% TFA water, 3.5 mL / min flow rate) to obtain compound A7 (6.6 mg, tR 16.2 min).Fraction 8 (5.53 g) was purified by ODS pressure (10%-100% acetonitrile-0.01% TFA water, 25.0 mL / min flow rate) to give fractions G1-G7. Fraction G5 was prepared by HPLC (55% acetonitrile-0.01% TFA water, 3.5 mL / min flow rate) to give compound A8 (13.1 mg, tR 16.6 min).

[0220] 2. Identification of antibacterial compounds

[0221] The compounds A1 to A13 obtained above were identified as follows:

[0222] (1) Appearance: Compound A1, Compound A2, Compound A3, Compound A4, Compound A5, Compound A6, Compound A7, Compound A8, Compound A9, Compound A10, Compound A11, Compound A12, and Compound A13 were light yellow solid powders.

[0223] (2) Solubility: Compound A1, Compound A2, Compound A3, Compound A4, Compound A5, Compound A6, Compound A7, Compound A8, Compound A9, Compound A10, Compound A11, Compound A12 and Compound A13 are all soluble in dichloromethane, ethyl acetate, methanol and acetonitrile.

[0224] (3) Mass spectrometry: Figure 1 is the HRESIMS mass spectrum of compound A1, showing its [MH] - The peak is m / z 543.12964, indicating that its molecular formula is C 30 H 24 O 10 . Figure 2 is the HRESIMS mass spectrum of compound A2, showing that its [MH] - The peak is m / z 543.12946, indicating that its molecular formula is C 30 H 24 O 10 . Figure 3 is the HRESIMS mass spectrum of compound A3, showing that its [MH] - The peak is m / z 545.14465, indicating that its molecular formula is C 30 H 26 O 10 . Figure 4 is the HRESIMS mass spectrum of compound A4, showing that its [MH] - The peak is m / z 543.13007, indicating that its molecular formula is C 30 H 24 O 10 . Figure 5is the HRESIMS mass spectrum of compound A5, showing that its [MH] - The peak is m / z 543.12958, indicating that its molecular formula is C 30 H 24 O 10 . Figure 6 is the HRESIMS mass spectrum of compound A6, showing that its [MH] - The peak is m / z 541.11407, indicating that its molecular formula is C 30 H 22 O 10 . Figure 7 is the HRESIMS mass spectrum of compound A7, showing that its [MH] - The peak is m / z 561.14093, indicating that its molecular formula is C 30 H 26 O 11 . Figure 8 is the HRESIMS mass spectrum of compound A8, showing that its [MH] - The peak is m / z 547.16174, indicating that its molecular formula is C 30 H 28 O 11 . Fig. 9 is the HRESIMS mass spectrum of compound A9, showing that its [MH] - The peak is m / z 565.17218, indicating that its molecular formula is C 30 H 30 O 11 . Fig.10 is the HRESIMS mass spectrum of compound A10, showing its [MH] - The peak is m / z 545.11490, indicating that its molecular formula is C 30 H 26 O 10 . Fig.11 is the HRESIMS mass spectrum of compound A11, showing its [MH] - The peak is m / z 543.12958, indicating that its molecular formula is C 30 H 24 O 10 . Fig.12 is the HRESIMS mass spectrum of compound A12, showing that its [MH] - The peak is m / z 545.14490, indicating that its molecular formula is C 30 H 26 O 10 . Fig.13 is the HRESIMS mass spectrum of compound A13-1, showing that its [MH] -The peak is m / z 541.11475, indicating that its molecular formula is C 30 H 22 O 10 The HRESIMS test was performed using a Thermo Scientific LTQ Orbitrap XL mass spectrometer system with methanol as the solvent.

[0225] (4) Nuclear Magnetic Resonance Spectroscopy: Figure 14-15 are respectively 1 H-NMR, 13 C-NMR spectrum, Figure 16-17 are compound A2 1 H-NMR, 13 C-NMR spectrum, Figure 18-19 are respectively 1 H-NMR, 13 C-NMR spectrum, Figure 20-21 are respectively 1 H-NMR, 13 C-NMR spectrum, Figure 22-23 is compound A5 1 H-NMR, 13 C-NMR nuclear magnetic spectrum. Figure 24-25 is compound A6 1 H-NMR, 13 C-NMR nuclear magnetic spectrum. Figure 26-27 is compound A7 1 H-NMR, 13 C-NMR nuclear magnetic spectrum. Figure 28-29 is compound A8 1 H-NMR, 13 C-NMR nuclear magnetic spectrum. Figure 30-31 is compound A9 1 H-NMR, 13 C-NMR nuclear magnetic spectrum. Figure 32-33 is compound A10 1 H-NMR, 13 C-NMR nuclear magnetic spectrum. Figure 34-35 is compound A11 1 H-NMR, 13 C-NMR nuclear magnetic spectrum. Figure 36-37 is compound A12 1 H-NMR, 13 C-NMR nuclear magnetic spectrum. Figure 38-Figure 39 is compound A13-1 1 H-NMR, 13 C-NMR spectrum. The NMR signals of the compounds were assigned as shown in the following table.

[0226] Table 1.1 NMR data of compounds A1-A6 ( 1 H NMR 600 MHz; 13 C NMR 151MHz; CDCl3)

[0227]

[0228] Table 1.2 NMR data of compounds A7-A11 ( 1 H NMR 600 MHz; 13 C NMR 151MHz; CDCl3)

[0229]

[0230]

[0231] Table 1.3 NMR data of compounds A12-A13 ( 1 H NMR 600 MHz; 13 C NMR 151MHz; CDCl3)

[0232]

[0233] The chemical structures of compounds A1-A13 are 1 H-NMR, 13 C-NMR, HSQC, HMBC, DEPT, 1 H- 1 The structures of compounds A1-A13 were determined by H COSY and NOE spectra as follows:

[0234]

[0235]

[0236] Example 4: Detection of antibacterial activity of compounds

[0237] The MIC of compounds A1-A13 was detected using the microplate method. The specific steps are as follows:

[0238] According to the standards of the American Committee for Clinical Laboratory Standards, the isolated compounds were tested for in vitro activity against clinically resistant bacteria using the microdilution method: the test bacteria were streaked from the cryopreserved tubes onto nutrient agar plates for activation, cultured overnight at 35±2°C, 3-5 single colonies were picked out and transferred to nutrient broth medium, and cultured at 35±2°C for 6-8 hours. The compound to be tested is diluted to 256 μg / mL using CAMHB medium, 100 μL of the diluted compound is added to each well in the second column of the 96-well plate, 100 μL of CAMHB medium is added to each well in the remaining columns, 100 μL of CAMHB medium is added to each well in the second column by using a pistol, and mixed evenly by blowing repeatedly, 100 μL is added to each well in the third column in parallel, and mixed evenly by blowing again, 100 μL of the mixture is added to each well in the fourth column, and so on, double dilution is made to the penultimate column, 100 μL is taken out from the penultimate column and discarded, and the final concentrations of the drugs in the second column to the penultimate column are: 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μg / mL, and the final volume of the system is 100 μl. The first and last columns of the 96-well plate are used as the blank control of the culture medium and the growth control of the strain (only the culture medium or bacterial solution is added), respectively. The cultured bacterial suspension was adjusted to 0.5McF (approximately 1×10 8 CFU / mL), and then diluted 20 times with CAMHB medium to a concentration of about 5×10 6 CFU / mL bacterial suspension. Take the diluted bacterial suspension and add it to the wells in the second to twelfth columns of rows 1 to 7, 10 μL of bacterial solution in each well. The eighth row is used as the blank control of the drug. The final bacterial concentration in each well is about 5×10 5 CFU / mL. The 96-well plate was placed at 35±2℃ for 16-18 hours and the results were observed. The minimum concentration of the compound contained in the wells where no strain growth was observed (no turbidity was observed by the naked eye) was the minimum inhibitory concentration (MIC). The antibacterial activity test strains used in the experiment were preserved in the Pathogen (Virus) Collection Center of the Chinese Academy of Medical Sciences, Beijing, 100054. The information is: Staphylococcus epidermidis (ATCC 12228, MSSE), Staphylococcus aureus (ATCC 29213, MSSA), Staphylococcus aureus (ATCC 43300, MRSA), Staphylococcus aureus (ATCC 700698, MRSA, VISA), Enterococcus faecalis (ATCC 29212, VSE), Enterococcus faecalis (ATCC 51299, VRE), Enterococcus faecium (ATCC 700221, VRE). The experimental results are shown in the table below.

[0239] Table 2.1 Antibacterial activity of compounds A1-A7

[0240]

[0241]

[0242] Table 2.2 Antibacterial activity of compounds A8-A13

[0243]

[0244] The above data show that compounds A1-A13 have inhibitory effects on Staphylococcus epidermidis (ATCC 12228, MSSE), Staphylococcus aureus (ATCC 29213, MSSA), Staphylococcus aureus (ATCC 43300, MRSA), Staphylococcus aureus (ATCC700698, MRSA, VISA), Enterococcus faecalis (ATCC 29212, VSE), Enterococcus faecium (ATCC 700221, VRE), and Enterococcus faecalis (ATCC 51299, VRE), especially Enterococcus faecium and Staphylococcus epidermidis. They have good antibacterial activity and are even better than the positive control drug levofloxacin.

[0245] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0246] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience and preference of those skilled in the art.

[0247] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

Claims

1. A compound or a pharmaceutically acceptable salt, stereoisomer, prodrug or solvate thereof, wherein the compound has the following structure: in, represents a single bond or a double bond; X and Y are independently selected from: R1 and R2 are independently selected from: H, -OH, -O-(C 1-3 Alkyl), -OCO-(C 1-3 alkyl); R5 and R6 are independently selected from: H, -OH, -O-(C 1-3 Alkyl), -OCO-(C 1-3 alkyl).

2. The compound according to claim 1, characterized in that The compound has the following structure:

3. The compound according to claim 1, characterized in that The compound is selected from the following structures:

4. The compound according to claim 1, characterized in that The stereoisomers of the compound are selected from the following structures:

5. The method for preparing the compound according to claim 3 or 4, characterized in that: The preparation method comprises fermenting a Subplenodomus fungus, and extracting and separating the fermentation product to obtain the compound; the ITS gene sequence of the Subplenodomus fungus is shown in SEQ ID NO: 1; Preferably, the deposit number of the fungus is CGMCC No.40181.

6. The preparation method according to claim 5, characterized in that: The extraction and separation comprises: (1) extracting the fermentation product with an organic solvent I to obtain a crude extract; (2) dispersing the crude extract obtained in step (1), and extracting with an organic solvent II to obtain an extract; (3) subjecting the extract obtained in step (2) to adsorption chromatography and collecting the eluate; Optionally, (4) extracting and separating the eluate obtained in step (3).

7. The preparation method according to claim 6, characterized in that: The organic solvent I in step (1) is selected from one or more of methanol, ethanol, and ethyl acetate, preferably methanol; and / or the organic solvent II in step (2) is selected from one or more of ethyl acetate, N,N-dimethylacetamide, isopropyl acetate, methyl acetate, and methyl isobutyl ketone, preferably ethyl acetate; Preferably, step (1) further comprises the step of filtering the extract; More preferably, step (1) further comprises the step of concentrating the filtrate; Preferably, the solvent used for dispersing in step (2) is water; Preferably, the adsorbent for the adsorption chromatography in step (3) is silica gel; Preferably, the mobile phase of the adsorption chromatography in step (3) is dichloromethane and / or methanol.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt, stereoisomer, prodrug or solvate thereof, and one or more pharmaceutically acceptable excipients.

9. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt, stereoisomer, prodrug or solvate thereof in the preparation of an antibacterial agent.

10. The use according to claim 9, characterized in that The antibacterial agent is used to treat diseases caused by pathogen infection; Preferably, the pathogen is a microorganism or a parasite, and the pathogenic microorganism is selected from: one or more of viruses, chlamydia, rickettsia, mycoplasma, bacteria, spirochetes, fungi, etc., especially bacteria; More preferably, the bacterium is a Gram-positive bacterium or a Gram-negative bacterium, for example, selected from: one or more of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, Clostridium difficile, Streptococcus pyogenes, Serratia marcescens, Micrococcus lysodeikticus, Micrococcus luteus, Salmonella, and Pseudomonas aeruginosa; More preferably, the bacteria is a drug-resistant strain, preferably selected from: methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), vancomycin-resistant Enterococcus (VRE), penicillin-resistant Streptococcus pneumoniae (PRSP), multidrug-resistant Pseudomonas aeruginosa (MDR-PA), carbapenem-resistant Pseudomonas aeruginosa (CRPA), third-generation cephalosporin-resistant Klebsiella pneumoniae (3GCRKP), third-generation cephalosporin-resistant Escherichia coli (3GCREC), fluoroquinolone-resistant Escherichia coli (FQREC), polymyxin-resistant Escherichia coli (ColR-EC), multidrug-resistant Acinetobacter baumannii (MDR-AB), and carbapenem-resistant Acinetobacter baumannii (CRAB). One or more.

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