Penicillium as well as compound separated from fermentation product of penicillium and application of penicillium

Abstract

The invention discloses penicillium, a compound separated from a fermentation product of the penicillium and application of the penicillium. The compound provided by the invention is shown as a formula (V). The invention also protects the application of any one of the compounds or the pharmaceutically acceptable salt thereof: (a1) the application in preparation of an influenza virus inhibitor; (a2) application in preparation of drugs for treating and / or preventing influenza virus infection. The invention also protects a strain of penicillium, and the preservation registration number is CGMCC (China General Microbiological Culture Collection Center) No.40910. The invention also protects a preparation method of the compound, and the preparation method comprises the following steps: carrying out fermentation culture on the penicillium to obtain the compound. The invention has important application value in prevention and control of influenza virus, and can be used in the field of medicines, clinical treatment and public health.

Description

Technical Field

[0001] The present invention relates to a Penicillium strain and compounds separated from its fermentation products and uses. Background Art

[0002] Influenza, commonly known as the flu, is a highly contagious acute respiratory infection caused by the influenza virus and one of the most common upper respiratory tract illnesses in humans. It is highly contagious, spreads rapidly, is prone to mutation, can spread across species, and has a high morbidity rate, posing a serious threat to human health.

[0003] Influenza viruses belong to the Orthomyxoviridae family, a type of RNA virus. Based on differences in the antigenicity of their nucleoproteins and matrix proteins, they can be divided into four types: type A (A), type B (B), type C (C), and type D (D). Of these four types, type A (IAV) is the most common influenza virus strain, infecting the widest range of hosts and potentially causing human influenza pandemics.

[0004] Currently, the main clinical approaches to preventing and treating influenza are vaccination and antiviral drugs. However, the variability of influenza virus antigens and the emergence of seasonal influenza strains severely limit the immune protection provided by influenza vaccines. Furthermore, varying degrees of resistance have developed to commonly used antiviral drugs in clinical treatment. These challenges pose a significant challenge to preventing or treating influenza. Therefore, the search for novel antiviral drugs is crucial. Summary of the Invention

[0005] The purpose of the present invention is to provide a Penicillium strain and compounds separated from its fermentation products and uses.

[0006] The compound provided by the present invention is shown in formula (V);

[0007]

[0008] Specifically, the compound is represented by formula (I), formula (II), formula (III), or formula (IV).

[0009]

[0010]

[0011] The compound represented by formula (I) is compound 1.

[0012] The compound represented by formula (II) is compound 2.

[0013] The compound represented by formula (III) is compound 3.

[0014] The compound represented by formula (IV) is compound 4.

[0015] The present invention also protects the use of any of the above compounds or pharmaceutically acceptable salts thereof, which are as follows (a1) and / or (a2):

[0016] (a1) Use in the preparation of influenza virus inhibitors;

[0017] (a2) Use in the preparation of a medicament for treating and / or preventing influenza virus infection.

[0018] The present invention also protects a product containing any one of the above compounds or pharmaceutically acceptable salts thereof;

[0019] The product is (b1) and / or (b2) as follows:

[0020] (b1) influenza virus inhibitors;

[0021] (b2) Medications for the treatment and / or prevention of influenza virus infection.

[0022] The present invention also protects a strain of Penicillium, namely Penicillium sp. CPCC 401065, which was deposited on November 8, 2023 in the General Microbiology Center of the China Culture Collection Administration (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with the deposit registration number CGMCC No. 40910.

[0023] The present invention also protects a bacterial agent comprising the Penicillium or / and a culture of the Penicillium or / and a metabolite of the Penicillium.

[0024] The Penicillium culture is a substance obtained by culturing Penicillium CPCC 401065. The culturing may specifically be performed using a culture medium. The culture medium may specifically be a microbial culture medium. The culture medium may specifically be a fermentation medium. The culture may specifically be a fermentation product obtained by fermenting Penicillium CPCC 401065 using the culture medium. When the culture medium is a liquid culture medium, the fermentation product is a fermentation broth. When the culture medium is a solid culture medium, the fermentation product is a solid fermentation product. The fermentation product may include Penicillium CPCC 401065 and substances secreted into the culture medium during the culturing of Penicillium CPCC 401065.

[0025] The metabolite of Penicillium can be a fermentation broth or fermentation product of Penicillium CPCC 401065. The fermentation broth of Penicillium CPCC 401065 can be prepared by culturing Penicillium CPCC 401065 in a liquid fermentation medium and collecting the fermentation broth (containing Penicillium CPCC 401065 and substances secreted into the liquid medium). The fermentation product of Penicillium CPCC 401065 can be prepared by culturing Penicillium CPCC 401065 in a solid fermentation medium and collecting the fermentation product (containing Penicillium CPCC 401065 and substances secreted into the solid medium).

[0026] The active ingredients of the above-mentioned microbial agent may also contain other biological components or non-biological components. Those skilled in the art can determine the other active ingredients of the above-mentioned microbial agent based on the effects of the microbial agent.

[0027] In the above-mentioned microbial agent, the microbial agent may further include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0028] The above-mentioned microbial agents may be in various dosage forms, including but not limited to liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule, etc.

[0029] The present invention also protects the use of the Penicillium in preparing the compound.

[0030] The present invention also protects the use of the Penicillium and / or the bacterial agent in preparing products;

[0031] The product is (b1) and / or (b2) as follows:

[0032] (b1) influenza virus inhibitors;

[0033] (b2) Medications for the treatment and / or prevention of influenza virus infection.

[0034] The present invention also protects a product containing the Penicillium and / or the bacterial agent;

[0035] The product is (b1) and / or (b2) as follows:

[0036] (b1) influenza virus inhibitors;

[0037] (b2) Medications for the treatment and / or prevention of influenza virus infection.

[0038] The present invention also protects a method for preparing a product, comprising the following steps: using the Penicillium or / and the bacterial agent as components of the product to obtain the product;

[0039] The product is (b1) and / or (b2) as follows:

[0040] (b1) influenza virus inhibitors;

[0041] (b2) Medications for the treatment and / or prevention of influenza virus infection.

[0042] The present invention also protects a preparation method of the compound, which comprises the following steps: fermenting and culturing the Penicillium to obtain the compound.

[0043] Specifically, the preparation method comprises the following steps: fermenting and culturing the Penicillium in a culture medium to obtain a fermentation product, and obtaining the compound from the fermentation product.

[0044] The culture medium can specifically be a microbial culture medium.

[0045] The culture medium may specifically be a fermentation medium.

[0046] The fermentation culture conditions may specifically be: static culture at 15-30° C. for 20-60 days.

[0047] The fermentation culture conditions may specifically be: static culture at 20-30° C. for 30-50 days.

[0048] The fermentation culture conditions may specifically be: static culture at 25-28° C. for 40 days.

[0049] Any of the above-mentioned substratums can be rice culture medium, potato dextrose agar culture medium or other fungal fermentation medium well known to professionals in the art, etc. The rice culture medium can be made of rice and water, or can be made of rice, water and inorganic salts, or can be made of rice, water and a nitrogen source, or can be made of rice, water, a nitrogen source and inorganic salts. The potato dextrose agar culture medium can be made of potato, glucose, agar and water, or can be made of potato, glucose, agar, water and inorganic salts, or can be made of potato, a carbon source, agar, water and a nitrogen source, or can be made of potato, a carbon source, agar, a nitrogen source and inorganic salts. The fungal fermentation medium well known to professionals in the art can be made of one or more quick-acting or slow-acting carbon sources, one or more quick-acting or slow-acting nitrogen sources, water and / or inorganic salts, etc.

[0050] The rice may be rice or brown rice. Both rice and brown rice are products of rice. Rice refers to the fruit without removing the rice husk, and rice is composed of husk, pericarp, seed coat, endosperm, endosperm, endosperm and embryo. Brown rice refers to the product of rice with the husk removed and the other parts of the rice retained; rice refers to the product of only retaining the endosperm and removing all other parts of the rice. Carbon source is a type of nutrient for microbial growth, which is a carbon-containing compound, including fast-acting and slow-acting carbon sources such as sugars, oils, organic acids and organic acid esters and small molecule alcohols. Nitrogen source refers to a substance that provides nitrogen elements required for microbial nutrition, including fast-acting and slow-acting nitrogen sources such as peanut cake powder, soybean cake powder, yeast powder, protein chen, ammonia water, ammonium salt and nitrate.

[0051] Specifically, the fermentation medium can be prepared by taking 60-90 g of rice and 90-100 mL of water, and soaking them at 26-30° C. for 6-10 hours.

[0052] Specifically, the fermentation medium can be prepared by taking 80 g of rice and 100 mL of water, and soaking them at 28° C. for 8 hours.

[0053] The fermentation medium described above can be placed in a conical flask.

[0054] A flask containing fermentation medium can be inoculated with 10 mL of the spore suspension of Penicillium.

[0055] Specifically, the concentration of the spore suspension can be 10 6 -10 8 pieces / mL.

[0056] Specifically, the concentration of the spore suspension can be 10 7 pieces / mL.

[0057] The method for obtaining the compound from the fermentation product comprises the following steps:

[0058] (1) extracting the fermentation product with ethyl acetate, collecting the ethyl acetate phase, filtering and collecting the filtrate, and evaporating the solvent to obtain a crude ethyl acetate extract;

[0059] (2) taking the crude ethyl acetate extract from step (1) and performing silica gel column chromatography to obtain fractions 6-9;

[0060] (3) Fractions 6-9 were concentrated and evaporated to dryness, then dissolved in methanol. The samples were then mixed with C18 reverse-phase silica gel (20-35 μm) and loaded onto the solid phase for medium-pressure ODS liquid phase preparative separation to obtain fractions Fr.13 and Fr.14.

[0061] (4) Fraction Fr.13 was separated using SB-C18 liquid phase preparative separation to obtain fractions Fr.13.1 and Fr.13.2;

[0062] (5) Fraction Fr.13.1 was taken and subjected to preparative separation using SB-C18 liquid phase to obtain compound 1;

[0063] (6) Fraction Fr.13.2 was taken and subjected to preparative separation using SB-C18 liquid phase to obtain compound 2;

[0064] (7) Fraction Fr.14 was taken and subjected to preparative separation using SB-C18 liquid phase to obtain fractions Fr.14.1 and Fr.14.2;

[0065] (8) Fraction Fr.14.1 was taken and subjected to preparative separation using SunFire-C18 liquid phase to obtain fraction Fr.14.1.1;

[0066] (9) Fraction Fr.14.1.1 was taken and preparative separation was performed using SilGreen-C18 liquid phase to obtain compound 3;

[0067] (10) Fraction Fr.14.2 was taken and subjected to preparative separation using a SHIMADZU C18 liquid phase to obtain compound 4.

[0068] The silica gel column in step (2) has a size of 10*60 cm and a dead volume of 3400 mL.

[0069] The silica gel column filler in the step (2) is 200-300 mesh column chromatography silica gel.

[0070] The elution procedure in the step (2) is as follows: ① first elute with dichloromethane for 3 column volumes; ② then sequentially elute with 95:5 solution (95 parts by volume of dichloromethane and 5 parts by volume of ethyl acetate), 90:10 solution (90 parts by volume of dichloromethane and 10 parts by volume of ethyl acetate), 85:15 solution (85 parts by volume of dichloromethane and 15 parts by volume of ethyl acetate), 80:20 solution (80 parts by volume of dichloromethane and 20 parts by volume of ethyl acetate), 75:25 solution (75 parts by volume of dichloromethane and 25 parts by volume of ethyl acetate), 70:30 solution (70 parts by volume of dichloromethane and 30 parts by volume of ethyl acetate), 65:35 solution (65 parts by volume of dichloromethane and 35 parts by volume of ethyl acetate), 60:40 solution (60 parts by volume of dichloromethane and 40 parts by volume of ethyl acetate), 50:5 ③ Then, elute with 90:10 solution (90 parts by volume of dichloromethane and 10 parts by volume of methanol), 85:15 solution (85 parts by volume of dichloromethane and 15 parts by volume of methanol), 80:20 solution (80 parts by volume of dichloromethane and 20 parts by volume of methanol), 75:25 solution (75 parts by volume of dichloromethane and 25 parts by volume of methanol), 70:30 solution (70 parts by volume of dichloromethane and 30 parts by volume of methanol), 65:35 solution (65 parts by volume of dichloromethane and 35 parts by volume of methanol), 60:40 solution (60 parts by volume of dichloromethane and 40 parts by volume of methanol), 50:50 solution (50 parts by volume of dichloromethane and 50 parts by volume of methanol), and methanol, for 2 column volumes each.

[0071] In the step (2), the eluate after column with a retention volume of 18000ml-26000ml is fractions 6-9.

[0072] The chromatographic column in step (3) is: Aiershi Technology spherical C18 FLASH column (Aiershi Technology; product website: http: / / www.tjairs.com / aiershi / products / 20925717.html): diameter: 50 mm, height: 240 mm, column volume: 470 mL; filling medium: spherical-C18, filler particle size: 20-35 μm.

[0073] The mobile phase in step (3) is acetonitrile or acetonitrile aqueous solution. The mobile phase flow rate is 30 mL / min.

[0074] The elution process in step (3) is as follows: the elution time is 70 min, the volume fraction of acetonitrile in the mobile phase increases linearly, the volume fraction of acetonitrile in the mobile phase is 20% at the initial moment, and the volume fraction of acetonitrile in the mobile phase is 100% at the end moment.

[0075] In the step (3), the post-column solution with a retention time of 50-55 min is collected, i.e., fraction Fr.13.

[0076] In the step (3), the post-column solution with a retention time of 55-60 min is collected, i.e., fraction Fr.14.

[0077] The chromatographic column in step (4) is: a chromatographic column ZORBAX SB-C18 (Agilent): the filler particle size is 5 μm; the chromatographic column diameter is 9.4 mm, the height is 250 mm, and the column volume is 20 mL.

[0078] The mobile phase in step (4) is composed of 60 parts by volume of acetonitrile and 40 parts by volume of water. The flow rate of the mobile phase is 3 mL / min.

[0079] In the step (4), the post-column solution of the elution peak corresponding to the retention time of 27.8 min is collected, namely, fraction Fr.13.1.

[0080] In the step (4), the post-column solution of the elution peak corresponding to the retention time of 30.3 min is collected, namely, fraction Fr.13.2.

[0081] The chromatographic column in step (5) is: a chromatographic column ZORBAX SB-C18 (Agilent): the filler particle size is 5 μm; the chromatographic column diameter is 9.4 mm, the height is 250 mm, and the column volume is 20 mL.

[0082] The mobile phase in step (5) is composed of 70 parts by volume of acetonitrile and 30 parts by volume of water. The flow rate of the mobile phase is 3 mL / min.

[0083] In the step (5), the post-column solution of the elution peak corresponding to the retention time of 16.9 min is collected, and the solvent is evaporated to obtain compound 1.

[0084] The chromatographic column in step (6) is: a chromatographic column ZORBAX SB-C18 (Agilent): the filler particle size is 5 μm; the chromatographic column diameter is 9.4 mm, the height is 250 mm, and the column volume is 20 mL.

[0085] The mobile phase in step (6) is composed of 70 parts by volume of acetonitrile and 30 parts by volume of water. The flow rate of the mobile phase is 3 mL / min.

[0086] In the step (6), the post-column solution of the elution peak corresponding to the retention time of 18.3 min is collected, and the solvent is evaporated to obtain compound 2.

[0087] The chromatographic column in step (7) is: a chromatographic column ZORBAX SB-C18 (Agilent): the filler particle size is 5 μm; the chromatographic column diameter is 9.4 mm, the height is 250 mm, and the column volume is 20 mL.

[0088] The mobile phase in step (7) is composed of 60 parts by volume of acetonitrile and 40 parts by volume of water. The flow rate of the mobile phase is 4 mL / min.

[0089] In the step (7), the post-column solution of the elution peak corresponding to the retention time of 20.9 min is collected, namely, fraction Fr.14.1.

[0090] In the step (7), the post-column solution of the elution peak corresponding to the retention time of 26.9 min is collected, namely, fraction Fr.14.2.

[0091] The chromatographic column in step (8) is: a chromatographic column SunFire-C18 (Waters Corporation); the filler particle size is 5 μm; the chromatographic column diameter is 10 mm, the height is 250 mm, and the column volume is 20 mL.

[0092] The mobile phase in step (8) is composed of 75 parts by volume of methanol and 25 parts by volume of water. The flow rate of the mobile phase is 3 mL / min.

[0093] In the step (8), the post-column eluate of the elution peak corresponding to the retention time of 35.2 min is collected, namely, fraction Fr.14.1.1.

[0094] The chromatographic column in step (9) is: SilGreen HPLC COLUMN C18 (Green Herbs, product number GH0525046C18A; product website: http: / / www.greenherbs.com.cn / product_details / 1052912801187983360.html): the filler particle size is 5 μm; the chromatographic column has a diameter of 4.6 mm, a height of 250 mm, and a column volume of 5 mL.

[0095] The mobile phase in step (9) is methanol or methanol-water solution. The mobile phase flow rate is 1 ml / min.

[0096] The elution procedure in step (9) is as follows: the elution time is 25 min, the volume fraction of methanol in the mobile phase increases linearly, the volume fraction of methanol in the mobile phase is 50% at the initial moment, and the volume fraction of methanol in the mobile phase is 100% at the end moment.

[0097] In the step (9), the post-column solution of the elution peak corresponding to the retention time of 21.8 min is collected, and the solvent is evaporated to obtain compound 3.

[0098] The chromatographic column in step (10) is: SHIMADZU C18 (Shimadzu Corporation): the filler particle size is 5 μm; the chromatographic column has a diameter of 10 mm, a height of 250 mm, and a column volume of 20 mL.

[0099] The mobile phase in step (10) is composed of 70% by volume of acetonitrile and 30 parts by volume of water. The flow rate of the mobile phase is 4 mL / min.

[0100] In the step (10), the post-column solution of the elution peak corresponding to the retention time of 30.0 min is collected, and the solvent is evaporated to obtain compound 4.

[0101] The present invention also provides a method for inhibiting virus infection in animals.

[0102] The method for inhibiting virus infection in animals provided by the present invention comprises administering the compound or a pharmaceutically acceptable salt thereof to a recipient animal to inhibit virus infection in the animal.

[0103] The present invention also provides a method for treating and / or preventing viral infection.

[0104] The method for treating and / or preventing viral infection provided by the present invention comprises administering the compound or a pharmaceutically acceptable salt thereof to a recipient animal to treat and / or prevent viral infection.

[0105] In the present invention, the animal may be a mammal, such as a human; the animal may also be another animal infected by the virus other than a mammal, such as a bird.

[0106] Any of the above viruses may be influenza viruses.

[0107] Specifically, any of the above viruses may be influenza A virus.

[0108] Specifically, any of the above viruses can be human influenza A virus, such as the A / WSN / 33 strain.

[0109] In the above, the compound includes polymorphs, pseudopolymorphs, amorphous forms, hydrates or solvates.

[0110] In the above, the viral inhibitor or the drug, in addition to containing the compound or its pharmaceutically acceptable salt, may also contain a suitable carrier or excipient. The carrier material here includes but is not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, water-soluble carrier materials are preferred. These materials can be made into a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a conventional preparation, sustained-release preparation, controlled-release preparation and various particulate delivery systems. In order to make the unit dosage form into tablets, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate with citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets. To prepare the unit dosage form into a pill, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, gelucine, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginates, sodium lauryl sulfate, methylcellulose, and ethylcellulose. To prepare unit dosage forms as suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, and semi-synthetic glycerides. To prepare unit dosage forms as injectable preparations, such as solutions, emulsions, lyophilized powder injections, and suspensions, all diluents commonly used in the art can be used, including water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters.In addition, to prepare an isotonic injection solution, an appropriate amount of sodium chloride, glucose, or glycerol may be added to the injectable formulation. Conventional solubilizers, buffers, pH adjusters, and the like may also be added. Furthermore, colorants, preservatives, fragrances, flavoring agents, sweeteners, or other materials may be added to the pharmaceutical formulation as needed. The above dosage forms can be administered via injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections; cavitary administration, such as rectal and vaginal; respiratory tract administration, such as nasal; and mucosal administration.

[0111] The present invention has significant application value for influenza virus prevention and control, and can be used in the pharmaceutical field, clinical treatment, and public health.

[0112] Description of biological material deposit

[0113] Classification and nomenclature of biological materials: Penicillium

[0114] Latin name of biomaterial: Penicillium sp.

[0115] Strain name of biological material: CPCC 401065

[0116] Full name of the depository: General Microbiology Center of China Culture Collection Administration of Microorganisms

[0117] Abbreviation of depository unit: CGMCC

[0118] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0119] Deposit date: November 8, 2023

[0120] Deposit number: CGMCC No.40910 BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1 is the mass spectrum of compound 1.

[0122] Figure 2 is the mass spectrum of compound 2.

[0123] Figure 3 is the mass spectrum of compound 3.

[0124] Figure 4 is the mass spectrum of compound 4.

[0125] Figure 5 This is the ROESY spectrum of compound 1 dissolved in DMSO-d6.

[0126] Figure 6 This is the ROESY spectrum of compound 2 dissolved in DMSO-d6.

[0127] Figure 7 This is the ROESY spectrum of compound 3 dissolved in DMSO-d6.

[0128] Figure 8 This is the ROESY spectrum of compound 4 dissolved in DMSO-d6. DETAILED DESCRIPTION

[0129] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0130] Unless otherwise noted, the experimental methods in the following examples are conventional methods and were performed according to the techniques and conditions described in literature in the field or according to product specifications. The materials and reagents used in the following examples are commercially available unless otherwise noted. Unless otherwise noted, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged. Human influenza A virus strain A / WSN / 33: Chinese Center for Disease Control and Prevention.

[0131] PDA medium (5.6±0.2): contains 4 g / L potato extract powder, 20 g / L glucose and 15 g / L agar, with the balance being water.

[0132] 293T-Gluc cells are described in the following literature: Gao Q, Wang Z, Liu Z, et al. A cell-based high-throughput approach to identify inhibitors of influenza A virus[J]. ActaPharmaceutica Sinica B, 2014, 4(4): 301-306.

[0133] Example 1: Isolation, Identification, and Preservation of Penicillium CPCC 401065

[0134] 1. Isolation of strains

[0135] Seawater samples: collected near Fildes Peninsula, Antarctica.

[0136] Seawater samples were filtered through a 24mm sterile polyethersulfone filter and then inoculated onto PDA plates containing 50 mg / L streptomycin sulfate and 50 mg / L tetracycline. The plates were incubated at 28°C for 5-7 days. Individual colonies were selected based on their color, size, and shape and inoculated onto slants on the PDA medium. Several pure cultures of bacterial strains were obtained, one of which was strain CPCC 401065.

[0137] The strain was stored in 20% glycerol cryovials at -80°C.

[0138] 2. Identification of strains

[0139] 1. Morphological identification of strain CPCC 401065

[0140] The hyphae are thick and long, the colonies are large and not limited. The colonies are loose in texture, dry in appearance, opaque, and appear to be tight or loose spider-web-like, fluffy or cotton-like. The colonies are tightly connected to the culture medium and are not easy to pick up.

[0141] 2. Molecular identification of strain CPCC 401065

[0142] Total DNA was extracted and PCR amplified using genomic DNA as a template using a primer pair consisting of ITS1F and ITS4 (ITS1F: 5'-CTTGGTCATTTAGAGTAA-3'; ITS4: 5'-TCCTCCGCTTAGATATGC-3'). The amplified product of approximately 500-600 bp was recovered and sequenced. The sequencing result is shown in SEQ ID NO: 1.

[0143] Molecular identification of fungal strains was based on base sequence similarity (i.e., the percentage of nucleic acid sequences similar to reference sequences) and phylogenetic tree analysis. Comparison of SEQ ID NO: 1 with the GenBank database revealed a 99.8% sequence similarity to the ITS gene of Penicillium allii strain MH210.

[0144] According to the results of morphological characteristics and molecular identification, strain CPCC 401065 belonged to the genus Penicillium sp., so it was named Penicillium sp. CPCC 401065.

[0145] 3. Preservation of strains

[0146] Penicillium sp. CPCC 401065 was deposited on November 8, 2023 at the General Microbiology Center of China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with the deposit registration number CGMCC No. 40910.

[0147] Example 2: Fermentation of compounds by Penicillium CPCC 401065

[0148] 1. Fermentation culture

[0149] 1. Inoculate spores of Penicillium CPCC 401065 onto a slant of PDA medium and culture at 28°C for 7 days to obtain a slant culture.

[0150] 2. Inoculate the spores on the slant onto a PDA medium plate and culture at 28°C for 10 days. Collect the spores on the plate with sterile water to obtain a spore suspension (spore concentration of 10 7 / mL).

[0151] 3. The spore suspension obtained in step 2 was inoculated into a triangular flask containing fermentation medium (10 mL of spore suspension / flask), and cultured at 25-28°C for 40 days (shaken occasionally during the culture period to ensure the entry of air during the culture process) to obtain a fermentation product (the fermentation product is the entire content of the triangular flask).

[0152] Flask containing fermentation medium: Take a 500mL triangular flask, add 80g of rice and 100mL of water, soak at 28℃ for 8 hours, and then sterilize at 121℃ for 20 minutes.

[0153] Multiple repeated treatments were performed simultaneously to obtain 4 kg of fermentation material.

[0154] 2. Isolation and purification of compounds

[0155] 1. Take 4 kg of the fermentation product obtained in step 1, ultrasonically extract it with ethyl acetate 5 times, collect and combine the ethyl acetate phases, filter through four layers of emery cloth, collect the filtrate, and evaporate the solvent to obtain 90 g of ethyl acetate crude extract.

[0156] 2. Take 90 g of the crude ethyl acetate extract obtained in step 1 and perform silica gel column chromatography.

[0157] Silica gel column specifications: 10*60cm, chromatography column dead volume is 3400mL.

[0158] Filler: 200-300 mesh column chromatography silica gel (Qingdao Ocean Chemical Plant Branch).

[0159] Elution procedure: ① first elute with dichloromethane for 3 column volumes; ② then sequentially elute with 95:5 solution (95 parts by volume of dichloromethane and 5 parts by volume of ethyl acetate), 90:10 solution (90 parts by volume of dichloromethane and 10 parts by volume of ethyl acetate), 85:15 solution (85 parts by volume of dichloromethane and 15 parts by volume of ethyl acetate), 80:20 solution (80 parts by volume of dichloromethane and 20 parts by volume of ethyl acetate), 75:25 solution (75 parts by volume of dichloromethane and 25 parts by volume of ethyl acetate), 70:30 solution (70 parts by volume of dichloromethane and 30 parts by volume of ethyl acetate), 65:35 solution (65 parts by volume of dichloromethane and 35 parts by volume of ethyl acetate), 60:40 solution (60 parts by volume of dichloromethane and 40 parts by volume of ethyl acetate), 50:50 solution ( ③ Then, use 90:10 solution (90 parts by volume of dichloromethane and 10 parts by volume of methanol), 85:15 solution (85 parts by volume of dichloromethane and 15 parts by volume of methanol), 80:20 solution (80 parts by volume of dichloromethane and 20 parts by volume of methanol), 75:25 solution (75 parts by volume of dichloromethane and 25 parts by volume of methanol), 70:30 solution (70 parts by volume of dichloromethane and 30 parts by volume of methanol), 65:35 solution (65 parts by volume of dichloromethane and 35 parts by volume of methanol), 60:40 solution (60 parts by volume of dichloromethane and 40 parts by volume of methanol), 50:50 solution (50 parts by volume of dichloromethane and 50 parts by volume of methanol), and methanol to elute for 2 column volumes each.

[0160] The post-column solution was continuously collected during the entire elution process.

[0161] The eluate after column passage with a retention volume of 18,000 ml to 26,000 ml is fractions 6 to 9.

[0162] 3. Take all fractions 6-9, concentrate and evaporate to dryness by rotary evaporation, then dissolve in 30 ml of methanol, and then mix the sample with 5 g of C18 reverse phase silica gel (20-35 μm, spherical) for solid loading, and then perform medium-pressure ODS liquid phase preparative separation.

[0163] Aiers Technologies spherical C18 FLASH column (Aiers Technologies; product website: http: / / www.tjairs.com / aiershi / products / 20925717.html): diameter 50 mm, height 240 mm, column volume 470 mL; packed with spherical-C18 medium, particle size 20-35 μm.

[0164] Mobile phase: acetonitrile or acetonitrile-water solution. Mobile phase flow rate: 30 mL / min.

[0165] Elution process: the elution time is 70 min, the volume fraction of acetonitrile in the mobile phase increases linearly, the volume fraction of acetonitrile in the mobile phase is 20% at the initial moment, and 100% at the end moment.

[0166] The post-column solution with a retention time of 50-55 min was collected and named Fr.13.

[0167] The post-column solution with a retention time of 55-60 min was collected and named Fr.14.

[0168] 4. Take all Fr.13 and use SB-C18 liquid phase preparation for separation.

[0169] Chromatographic column ZORBAX SB-C18 (Agilent): the filler particle size is 5 μm; the chromatographic column diameter is 9.4 mm, the height is 250 mm, and the column volume is 20 mL.

[0170] Mobile phase: 60 parts by volume of acetonitrile and 40 parts by volume of water. Mobile phase flow rate: 3 mL / min.

[0171] The post-column solution of the elution peak corresponding to the retention time of 27.8 min was collected and named Fr.13.1.

[0172] The post-column solution of the elution peak corresponding to the retention time of 30.3 min was collected and named Fr.13.2.

[0173] 5. Take all of Fr.13.1 and perform preparative separation using SB-C18 liquid phase.

[0174] Chromatographic column ZORBAX SB-C18 (Agilent): the filler particle size is 5 μm; the chromatographic column diameter is 9.4 mm, the height is 250 mm, and the column volume is 20 mL.

[0175] Mobile phase: 70 parts by volume of acetonitrile and 30 parts by volume of water. Mobile phase flow rate: 3 mL / min.

[0176] The post-column solution of the elution peak corresponding to the retention time of 16.9 min was collected, and the solvent was evaporated to dryness to obtain 7 mg of compound 1.

[0177] 6. Take all Fr.13.2 and perform preparative separation using SB-C18 liquid phase.

[0178] Chromatographic column ZORBAX SB-C18 (Agilent): the filler particle size is 5 μm; the chromatographic column diameter is 9.4 mm, the height is 250 mm, and the column volume is 20 mL.

[0179] Mobile phase: 70 parts by volume of acetonitrile and 30 parts by volume of water. Mobile phase flow rate: 3 mL / min.

[0180] The post-column solution of the elution peak corresponding to the retention time of 18.3 min was collected, and the solvent was evaporated to dryness to obtain 15 mg of compound 2.

[0181] 7. Take all Fr.14 and use SB-C18 liquid phase for preparative separation.

[0182] Chromatographic column ZORBAX SB-C18 (Agilent): the filler particle size is 5 μm; the chromatographic column diameter is 9.4 mm, the height is 250 mm, and the column volume is 20 mL.

[0183] Mobile phase: 60 parts by volume of acetonitrile and 40 parts by volume of water. Mobile phase flow rate: 4 mL / min.

[0184] The post-column solution of the elution peak corresponding to the retention time of 20.9 min was collected and named Fr.14.1.

[0185] The post-column solution of the elution peak corresponding to the retention time of 26.9 min was collected and named Fr.14.2.

[0186] 8. Take all of Fr.14.1 and perform preparative separation using SunFire-C18 liquid phase.

[0187] Chromatographic column SunFire-C18 (Waters Corporation): the filler particle size is 5 μm; the chromatographic column diameter is 10 mm, the height is 250 mm, and the column volume is 20 mL.

[0188] Mobile phase: 75 parts by volume of methanol and 25 parts by volume of water. Mobile phase flow rate: 3 mL / min.

[0189] The post-column eluate of the elution peak corresponding to the retention time of 35.2 min was collected and named Fr.14.1.1.

[0190] 9. Take all of Fr.14.1.1 and perform preparative separation using SilGreen-C18 liquid phase.

[0191] Chromatographic column SilGreen HPLC COLUMN C18 (Green Herbs Company, product number GH0525046C18A; product website: http: / / www.greenherbs.com.cn / product_details / 1052912801187983360.html): filler particle size 5 μm; chromatographic column diameter 4.6 mm, height 250 mm, column volume 5 mL.

[0192] Mobile phase: methanol or methanol-water solution. Mobile phase flow rate: 1 ml / min.

[0193] Elution procedure: the elution time is 25 min, the volume fraction of methanol in the mobile phase increases linearly, the volume fraction of methanol in the mobile phase is 50% at the initial moment, and the volume fraction of methanol in the mobile phase is 100% at the end moment.

[0194] The post-column solution of the elution peak corresponding to the retention time of 21.8 min was collected, and the solvent was evaporated to dryness to obtain 3.0 mg of compound 3.

[0195] 10. Take all of Fr.14.2 and perform preparative separation using SHIMADZU C18 liquid phase.

[0196] Chromatographic column SHIMADZU C18 (Shimadzu Corporation): the filler particle size is 5 μm; the chromatographic column diameter is 10 mm, the height is 250 mm, and the column volume is 20 mL.

[0197] Mobile phase: 70% by volume acetonitrile and 30% by volume water. Mobile phase flow rate: 4 mL / min.

[0198] The post-column solution of the elution peak corresponding to the retention time of 30.0 min was collected, and the solvent was evaporated to dryness to obtain 3.5 mg of compound 4.

[0199] 3. Identification and Characterization of Compounds

[0200] The NMR data of the compound ( 1 H-NMR, 600 MHz; 13 The results of C-NMR (150 MHz) are shown in Tables 1 and 2.

[0201] Table 1

[0202]

[0203]

[0204] Table 2

[0205]

[0206]

[0207] 1. Identification and characterization of compound 1 The (+)-HRESIMS mass spectrum showed that its [M+H]+ peak was m / z 659.21112, suggesting that its molecular formula was C 36 H 34 O 12 The hydrogen spectrum shows two olefinic hydrogen signals, δ H6.79 (1H, s) and 6.62 (1H, s); four oxygen-linked methine signals, δ H 5.19 (1H, s), 4.15 (1H, d, J = 8.4 Hz), 4.11 (1H, s), 3.63 (1H, d, J = 8.4 Hz) and 3.63 (1H, s); two methine signals, δ H 2.94 (1H, m) and 2.31 (1H, m); two oxygen-linked methylene signals, δ H 5.08 (1H, d, J = 14.4 Hz), 5.06 (1H, d, J = 14.4 Hz), 3.93 (1H, t, J = 8.4 Hz) and 3.46 (1H, t, J = 8.4 Hz); one methylene signal δ H 2.18 (1H, d, J = 15.6 Hz) and 1.94 (1H, dd, J = 15.6, 8.4 Hz); six methyl signals, δ H 2.68 (1H, s), 2.54 (1H, s), 2.13 (1H, s), 2.08 (1H, s), 0.97 (1H, d, J = 6.6 Hz) and 0.84 (1H, d, J = 7.2 Hz). The carbon spectrum shows 36 carbon signals, including 19 quaternary carbon signals (δ C 191.4,169.8,169.1,167.6,163.4,161.4,150.5,149.6,148.2,145.3,135.5,119.9,116.1,109.6,104.5,101.8,101.3,87.3,51.1), 8 methine carbon signals (δ C 120.0, 117.3, 77.4, 66.6, 66.3, 56.8, 43.8, 33.2), 3 methylene carbon signals (δ C 73.5, 70.4, 33.5) and 6 methyl signals (δ CIn the HMBC spectrum, it can be observed that H-1′ is correlated with C-8, C-3′, C-3′b, C-9′, C-9′a; H-8′ is correlated with C-7, C-8, C-6′a, C-7′, C-9′, C-9′a; H-9′ is correlated with C-7, C-8, C-3′b, C-7′, C-8′, C-11′; H-10′ is correlated with C-5′, C-6′, C-6′a; H-12′ is correlated with C-11′; 4′-OH is correlated with C-3′a, C-4′, C-5′; H-1 is correlated with C-3b, C-9, C-9a; H-5 and C-3, C-3a, C-4, C-6a, and C-15 are related. H-8 is related to C-6a, C-7, C-9, C-9a, C-1′, C-3′b, and C-9′a. H-15 is related to C-5, C-6, and C-6a. H-16 is related to C-7. 4-OH is related to C-3a, C-4, and C-5. H-10 is related to C-11, C-12, C-13, C-14, and C-17. H-12 is related to C-10, C-11, C-13, and C-18. H-14 is related to C-1, C-9a, C-10, and C-13. 1 H- 1In the H COSY spectrum, H-9′ and H-11′ were correlated, H-1 was correlated with H-14, H-10 was correlated with H-11 and H-17, and H-11 was correlated with H-10, H-12, and H-18. These data, combined with the molecular formula, confirmed the planar structure of compound 1 as shown in Formula (I). In the NOESY spectrum, H-8′ is correlated with H-9′ and H-15, H-8 is correlated with H-16 and H-1′, H-9′ is correlated with Hα-1′, H-17 is correlated with H-11, H-18 is correlated with H-10 and Hα-12, H-11 is correlated with Hβ-12, H-17 is correlated with Hα-14 and Hβ-14, and H-10 is not correlated with Hα-14and Hβ-14. Further combined with the coupling constants of H-5 and H-6 (J=0 Hz), the coupling constants of H-1 and Hα-14 (J=9.0 Hz), and the coupling constants of H-11 and Hβ-14 (J=0 Hz), the relative configuration of compound 1 was determined. In addition, the correctness of this configuration was verified by the computational NMR method reported in the literature (Grimblat Nicolás, Zanardi M.María, Sarotti M.Ariel.Beyond DP4:an improved probability for the stereochemical assignment of isomeric compounds using quantum chemical calculations of NMR shifts[J].The Journal of Organic Chemistry 2015,80:12526-12534.). Based on the calculation results of computational NMR, the relative configuration of compound 1 was further confirmed. In the ECD spectrum of compound 1, a positive Cotton effect was shown at 253 nm, and a negative Cotton effect was shown at 214 nm and 299 nm. Based on similar ECD data reported in the literature (Wang Minghui, Yang Longhe, Feng Liubin, et al. Verruculosins AB, new oligophenalenone dimers from the soft coral-derived fungus Talaromyces verruculosus [J]. Marine Drugs, 2019, 17: 516.), the absolute configuration of compound 1 was determined. Finally, compound 1 was determined to have the structure shown in formula (I), which has not been reported and is a new compound.

[0208] 2. Identification and characterization of compound 2

[0209] The (+)-HRESIMS mass spectrum showed that its [M+H]+ peak was m / z 659.21161, suggesting that its molecular formula was C 36 H 34 O 12 . The structures of compound 2 and compound 1 are very similar. Comparison of their hydrogen and carbon spectrum data revealed that H-10 and H-11 of compound 2 shifted 0.64 and 0.69 ppm to the high field compared with compound 1, and C-10 and C-11 of compound 2 shifted 4.4 and 4.0 ppm to the low field, respectively. It is speculated that the configuration of C-10 and C-11 has changed. Further comprehensive analysis of one-dimensional and two-dimensional nuclear magnetic spectra determined the planar structure of compound 2, which is the same as the planar structure of compound 1. In the NOESY spectrum, H-11 is correlated with H-17, H-10 is correlated with H-18, H-10 is correlated with Hα-14 and Hβ-14, and H-17 is not correlated with Hα-14 and Hβ-14, which determined the relative configuration of compound 2. In addition, the correctness of this configuration was verified by the computational NMR method reported in the literature (Grimblat Nicolás, Zanardi M.María, Sarotti M.Ariel.Beyond DP4:an improved probability for the stereochemical assignment of isomeric compounds using quantum chemical calculations of NMR shifts[J].The Journal of Organic Chemistry 2015,80:12526-12534.). Based on the calculation results of computational NMR, the relative configuration of compound 2 was further confirmed. In the ECD spectrum of compound 2, a positive Cotton effect was shown at 260 nm, and a negative Cotton effect was shown at 215 nm and 304 nm. Based on similar ECD data reported in the literature (Wang Minghui, Yang Longhe, Feng Liubin, et al. Verruculosins AB, new oligophenalenone dimers from the soft coral-derived fungus Talaromyces verruculosus [J]. Marine Drugs, 2019, 17: 516.), the absolute configuration of compound 2 was determined. Finally, compound 2 was determined to have the structure shown in formula (II), which has not been reported and is a new compound.

[0210] 3. Identification and characterization of compound 3

[0211] The (+)-HRESIMS mass spectrum showed that its [M+H]+ peak was m / z 659.20917, suggesting that its molecular formula was C 36 H 34 O 12The structures of compound 3 and compound 2 are very similar. The planar structure of compound 3 was determined by comprehensive analysis of one-dimensional and two-dimensional NMR spectra, which is the same as the planar structure of compound 2. The specific coupling constants of its H-1 proton and other protons (J = 11.4, 6.0, 1.8 Hz) are consistent with those reported for Verruculosin A in the literature (Wang Minghui, Yang Longhe, Feng Liubin, et al. Verruculosins AB, new oligophenalenone dimers from the soft coral-derived fungus Talaromyces verruculosus [J]. Marine Drugs, 2019, 17: 516.), so the configuration of H-1 was determined. In the NOESY spectrum, H-11 and H-17 are correlated, and H-10 and H-18 are correlated, and the relative configuration of H-17 and H-18 is determined. The relative configuration of compound 3 was further determined by the calculated NMR method reported in the literature (Grimblat Nicolás, Zanardi M.María, Sarotti M.Ariel.Beyond DP4:an improved probability for the stereochemical assignment of isomeric compounds using quantum chemical calculations of NMRshifts[J].The Journal of Organic Chemistry 2015,80:12526-12534.). The relative configuration of compound 3 was confirmed based on the calculated NMR results. In the ECD spectrum of compound 3, a positive Cotton effect was shown at 258 nm, and a negative Cotton effect was shown at 217 nm and 305 nm. Based on similar ECD data reported in the literature (Wang Minghui, Yang Longhe, Feng Liubin, et al. Verruculosins AB, new oligophenalenone dimers from the soft coral-derived fungus Talaromyces verruculosus [J]. Marine Drugs, 2019, 17: 516.), the absolute configuration of compound 3 was determined. Finally, compound 3 was determined to have the structure shown in formula (III), which has not been reported and is a new compound.

[0212] 4. Identification and characterization of compound 4

[0213] The (+)-HRESIMS mass spectrum showed that its [M+H]+ peak was m / z 659.2106, suggesting that its molecular formula was C 36 H 34 O 12The structures of compound 4 and compound 3 are very similar. Comparison of their hydrogen and carbon spectra revealed that H-10 and H-11 of compound 4 shifted by 0.42 and -0.46 ppm, respectively, compared to compound 3. This suggests that the configurations of C-10 and C-11 have changed. Further analysis of the one-dimensional and two-dimensional NMR spectra confirmed the planar structure of compound 2, which is identical to that of compound 4 and compound 3. The specific coupling constants of its H-1 proton and other protons (J=11.4, 4.8, 1.8 Hz) are consistent with those reported for Verruculosin A in the literature (Wang Minghui, Yang Longhe, Feng Liubin, et al. Verruculosins AB, new oligophenalenone dimers from the soft coral-derived fungus Talaromyces verruculosus[J]. Marine Drugs, 2019, 17: 516.), so the configuration of H-1 was determined. In the NOESY spectrum, H-11 and H-17 were correlated, and H-10 and H-18 were correlated, and the relative configurations of H-17 and H-18 were determined. The relative configuration of compound 4 was further determined by the NMR method reported in the literature (Grimblat Nicolás, Zanardi M.María, Sarotti M.Ariel.Beyond DP4: an improved probability for the stereochemical assignment of isomeric compounds using quantum chemical calculations of NMR shifts[J].The Journal of Organic Chemistry 2015,80:12526-12534.). The relative configuration of compound 4 was confirmed based on the calculation results of NMR.In the ECD spectrum of compound 4, a positive Cotton effect was shown at 258 nm, and a negative Cotton effect was shown at 211 nm and 299 nm. Based on similar ECD data reported in the literature (Wang Minghui, Yang Longhe, Feng Liubin, et al. Verruculosins AB, new oligophenalenone dimers from the soft coral-derived fungus Talaromyces verruculosus [J]. Marine Drugs, 2019, 17: 516.), the absolute configuration of compound 4 was determined. Finally, compound 4 was determined to have the structure shown in formula (IV), which has not been reported and is a new compound.

[0214]

[0215] Example 3. Antiviral activity and cytotoxicity of compounds

[0216] Test compound: Compound 1, Compound 2, Compound 3 or Compound 4 prepared in Example 2.

[0217] The test compound was dissolved in DMSO to a concentration of 10 mM, which was the test compound stock solution.

[0218] The positive control is ribavirin. The ribavirin solution is prepared by dissolving and diluting ribavirin in DMSO.

[0219] 1. Antiviral activity (IC 50 value)

[0220] The anti-influenza virus activity of the test compounds was determined based on the Gaussia luciferase reporter system.

[0221] 1. Resuspend 293T-Gluc cells in DMEM medium containing 10% FBS to a cell concentration of 2.0×10 5 The cell suspension is 100 cells / mL.

[0222] 2. Take a 96-well plate and inoculate the test wells, positive control wells and negative control wells with the cell suspension prepared in step 1 (100 μL / well), and inoculate the blank control wells with DMEM medium containing 10% FBS (100 μL / well) and culture for 24 hours.

[0223] 3. After completing step 2, take the 96-well plate, add the test compound solution (1 μL / well) to the test wells, add ribavirin solution (1 μL / well) to the positive control wells, and add DMSO (1 μL / well) to the negative control wells, and incubate for 2 hours.

[0224] The test compound solution was prepared by diluting the test compound stock solution with DMSO. After adding the test compound, the test compound concentration in the liquid phase system of the test well was 0.01, 0.02, 0.1, 0.625, 1.25, 2.5, 5, 10, 20, 50, 100, or 200 μM.

[0225] After the addition of the ribavirin solution, the ribavirin concentration in the liquid phase system of the positive control wells was 0.01, 0.02, 0.1, 0.625, 1.25, 2.5, 5, 10, 20, 50, 100 or 200 μM.

[0226] 4. After completing step 3, take the 96-well plate, add human influenza A virus A / WSN / 33 strain (MOI=0.3), and culture for 24 hours.

[0227] 5. After completing step 4, sample 10 μL of supernatant from each well and measure the luciferase activity using a multifunctional microplate reader (Berthold Centro LB 960). Calculate the inhibition rate of each sample and calculate the IC 50 (Concentration required to inhibit the virus by 50%).

[0228] The test was repeated three times and the results were averaged. The results are shown in Table 3.

[0229] 2. Cytotoxicity (CC 50 value)

[0230] 1. Suspend 293T-Gluc cells in DMEM medium containing 10% FBS to a cell concentration of 2.0×10 5 The cell suspension is 100 cells / mL.

[0231] 2. Take a 96-well plate and inoculate the test wells, positive control wells and negative control wells with the cell suspension prepared in step 1 (100 μL / well), and inoculate the blank control wells with DMEM medium containing 10% FBS (100 μL / well) and culture for 24 hours.

[0232] 3. After completing step 2, take the 96-well plate, add the test compound solution (1 μL / well) to the test wells, add ribavirin solution (1 μL / well) to the positive control wells, and add DMSO (1 μL / well) to the negative control wells, and incubate at 37°C for 48 hours.

[0233] The test compound solution was prepared by diluting the test compound stock solution with DMSO. After adding the test compound, the concentration of the test compound in the liquid phase system of the test well was 5, 10, 20, 50, 100 or 200 μM.

[0234] After the ribavirin solution was added, the ribavirin concentration in the liquid phase system of the positive control wells was 5, 10, 20, 50, 100 or 200 μM.

[0235] 4. After completing step 3, take the 96-well plate, add 10 μL of CCK-8 reagent to each well, incubate at 37°C for 1-2 hours, and then use a multifunctional microplate reader (Berthold Centro LB 960) to measure the absorbance value of each well at a wavelength of 450 nm to calculate the half cytotoxic concentration (CC) 50 (Drug concentration that causes 50% cell death).

[0236] The test was repeated three times and the results were averaged. The results are shown in Table 3.

[0237] IC of the test compound against human influenza A virus A / WSN / 33 50 Value and CC of the cell 50 The values ​​are shown in Table 3. The results showed that compounds 1, 2, 3 and 4 all had inhibitory activity against human influenza A virus strain A / WSN / 33, with IC 50 The values ​​ranged from 72.77 to 5197 nM, and there was no significant cytotoxicity at a concentration of 100 μM. The four compounds provided by the present invention all had higher inhibitory effects on influenza virus than the positive control, especially compound 1, which had an activity more than 200 times that of the positive control.

[0238] Table 3

[0239]

[0240]

[0241] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A compound represented by formula (V); 2. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof, which is as follows (a1) and / or (a2): (a1) Use in the preparation of influenza virus inhibitors; (a2) Use in the preparation of a medicament for treating and / or preventing influenza virus infection.

3. A product comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof; The product is (b1) and / or (b2) as follows: (b1) influenza virus inhibitors; (b2) Medications for the treatment and / or prevention of influenza virus infection.

4. Penicillium, wherein the Penicillium is Penicillium sp. CPCC 401065, and its deposit registration number is CGMCC No. 40910.

5. A bacterial agent comprising the Penicillium according to claim 4 or / and a culture of the Penicillium according to claim 4 or / and a metabolite of the Penicillium according to claim 4.

6. Use of the Penicillium according to claim 4 in the preparation of the compound according to claim 1.

7. Use of the Penicillium according to claim 4 or / and the bacterial agent according to claim 5 in preparing a product; The product is (b1) and / or (b2) as follows: (b1) influenza virus inhibitors; (b2) Medications for the treatment and / or prevention of influenza virus infection.

8. A product comprising the Penicillium according to claim 4 and / or the bacterial agent according to claim 5; The product is (b1) and / or (b2) as follows: (b1) influenza virus inhibitors; (b2) Medications for the treatment and / or prevention of influenza virus infection.

9. A method for preparing a product, comprising the following steps: using the Penicillium described in claim 4 or / and the microbial agent described in claim 5 as ingredients of the product to obtain the product; The product is (b1) and / or (b2) as follows: (b1) influenza virus inhibitors; (b2) Medications for the treatment and / or prevention of influenza virus infection.

10. A method for preparing the compound according to claim 1, comprising the steps of: fermenting and culturing the Penicillium according to claim 4 to obtain the compound according to claim 1.

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