A strain of Penicillium, compounds isolated from its fermentation products, and their uses.

Abstract

This invention discloses a Penicillium strain, compounds isolated from its fermentation products, and their uses. The compounds provided by this invention are shown in formula (V). This invention also protects the use of any of the above-described compounds or their pharmaceutically acceptable salts: (a1) in the preparation of influenza virus inhibitors; (a2) in the preparation of medicaments for treating and / or preventing influenza virus infection. This invention also protects a Penicillium strain with accession number CGMCC No. 40910. This invention also protects a method for preparing the compounds, comprising the following steps: fermenting the Penicillium strain to obtain the compounds. This invention has significant application value for influenza virus prevention and control, and can be used in the pharmaceutical field, clinical treatment, and public health.

Description

Technical Field

[0001] This invention relates to a Penicillium strain, compounds isolated from its fermentation products, and their uses. Background Technology

[0002] Influenza, commonly known as the flu, is a highly contagious acute respiratory infectious disease caused by the influenza virus. It is one of the most common upper respiratory tract diseases in humans. It is characterized by its high infectivity, rapid spread, high variability, ability to spread across species, and high incidence rate, seriously threatening human health.

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

[0004] Currently, the main methods for preventing and treating influenza in clinical practice are vaccination and antiviral drugs. However, the high variability of influenza virus antigens and the emergence of seasonal influenza virus strains severely limit the immunoprotective effect of influenza vaccines. At the same time, commonly used antiviral drugs have developed varying degrees of drug resistance. These problems pose a serious challenge to the prevention and treatment of influenza. Therefore, the search for novel antiviral drugs is crucial. Summary of the Invention

[0005] The object of this invention is to provide a Penicillium strain, compounds isolated from its fermentation products, and uses thereof.

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

[0007]

[0008] Specifically, the compounds are as shown in formula (I), (II), (III), or (IV).

[0009]

[0010]

[0011] The compound shown in formula (Ⅰ) is compound 1.

[0012] The compound shown in formula (Ⅱ) is compound 2.

[0013] The compound shown in formula (Ⅲ) is compound 3.

[0014] The compound shown in formula (Ⅳ) is compound 4.

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

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

[0017] (a2) Use in the preparation of medicines for the treatment and / or prevention of influenza virus infection.

[0018] This invention also protects a product containing any of the compounds described above or a pharmaceutically acceptable salt thereof;

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

[0020] (b1) Influenza virus inhibitors;

[0021] (b2) Medications used to treat and / or prevent influenza virus infection.

[0022] This invention also protects a Penicillium strain, namely Penicillium sp. CPCC 401065, which was deposited on November 8, 2023, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCCNo.40910.

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

[0024] The Penicillium culture is a substance obtained by culturing Penicillium CPCC 401065. Specifically, culturing can be performed using a culture medium. The culture medium can be a microbial culture medium, specifically a fermentation culture medium. The culture product can be a fermentation product obtained by fermenting Penicillium CPCC 401065 using a culture medium. When the culture medium is a liquid medium, the fermentation product is a fermentation broth. When the culture medium is a solid 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 cultivation of Penicillium CPCC 401065.

[0025] The metabolites of *Penicillium* can be the 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 agents may also contain other biological or non-biological components. The other active ingredients of the above-mentioned microbial agents can be determined by those skilled in the art based on the effects of the microbial agents.

[0027] The aforementioned microbial agent may further include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0028] The above-mentioned microbial agents can be in various dosage forms, including but not limited to liquids, emulsions, suspensions, powders, granules, wettable powders, or water-dispersible granules.

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

[0030] This invention also protects the use of the Penicillium and / or the fungal agent in the preparation of products;

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

[0032] (b1) Influenza virus inhibitors;

[0033] (b2) Medications used to treat and / or prevent influenza virus infection.

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

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

[0036] (b1) Influenza virus inhibitors;

[0037] (b2) Medications used to treat and / or prevent influenza virus infection.

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

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

[0040] (b1) Influenza virus inhibitors;

[0041] (b2) Medications used to treat and / or prevent influenza virus infection.

[0042] The present invention also protects a method for preparing the compound, comprising the following steps: fermenting and culturing the Penicillium mold to obtain the compound.

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

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

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

[0046] The specific conditions for fermentation culture are: static culture at 15-30℃ for 20-60 days.

[0047] The specific conditions for fermentation culture are: static culture at 20-30℃ for 30-50 days.

[0048] The specific conditions for fermentation culture are: static culture at 25-28℃ for 40 days.

[0049] Any of the above-mentioned culture media can be rice culture medium, potato dextrose agar medium, or other fungal fermentation media well known to those skilled in the art. The rice culture medium can be made from rice and water, or from rice, water, and inorganic salts, or from rice, water, and a nitrogen source, or from rice, water, a nitrogen source, and inorganic salts. The potato dextrose agar medium can be made from potato, glucose, agar, and water, or from potato, glucose, agar, water, and inorganic salts, or from potato, carbon source, agar, water, and a nitrogen source, or from potato, carbon source, agar, nitrogen source, and inorganic salts. Other fungal fermentation media well known to those skilled in the art can be made from 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.

[0050] The rice mentioned can be either white rice or brown rice. Both white rice and brown rice are products of paddy rice. Paddy rice refers to the fruit of the rice plant with the husk intact, consisting of the husk, pericarp, seed coat, outer endosperm, aleurone layer, endosperm, and embryo. Brown rice refers to paddy rice with the husk removed, retaining all other parts of the rice; white rice refers to paddy rice with only the endosperm retained, while all other parts of the rice have been removed. Carbon sources are nutrients for microbial growth, consisting of carbon-containing compounds, including sugars, oils, organic acids and esters, and small-molecule alcohols, which are both readily available and slowly available carbon sources. Nitrogen sources are substances that provide the nitrogen element required for microbial nutrition, including readily available and slowly available nitrogen sources such as peanut meal, soybean meal, yeast powder, protein powder, ammonia, ammonium salts, and nitrates.

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

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

[0053] The fermentation medium described above can be contained in Erlenmeyer flasks.

[0054] A suspension of Penicillium spores can be inoculated into an Erlenmeyer flask containing fermentation medium, with a volume of 10 mL.

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

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

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

[0058] (1) Take the fermentation product, extract it with ethyl acetate, collect the ethyl acetate phase, filter and collect the filtrate, evaporate the solvent to dryness, and obtain crude ethyl acetate extract.

[0059] (2) Take the crude ethyl acetate extract from step (1) and perform silica gel column chromatography to obtain fraction 6-9.

[0060] (3) Take fractions 6-9, concentrate and evaporate to dryness, dissolve in methanol, then mix with C18 reversed silica gel (20-35μm) and load solid sample for medium-pressure ODS liquid phase preparation and separation to obtain fractions Fr.13 and Fr.14.

[0061] (4) Take fraction Fr.13 and separate it using SB-C18 liquid phase to obtain fractions Fr.13.1 and Fr.13.2;

[0062] (5) Take fraction Fr.13.1 and perform preparative separation using SB-C18 liquid chromatography to obtain compound 1;

[0063] (6) Take fraction Fr.13.2 and perform preparative separation using SB-C18 liquid chromatography to obtain compound 2;

[0064] (7) Take fraction Fr.14 and perform preparative separation using SB-C18 liquid phase to obtain fractions Fr.14.1 and Fr.14.2;

[0065] (8) Take fraction Fr.14.1 and perform preparative separation using SunFire-C18 liquid chromatography to obtain fraction Fr.14.1.1;

[0066] (9) Take fraction Fr.14.1.1 and perform preparative separation using SilGreen-C18 liquid chromatography to obtain compound 3;

[0067] (10) Take fraction Fr.14.2 and prepare and separate it using SHIMADZU C18 liquid phase to obtain compound 4.

[0068] The silica gel column specifications in step (2) are: 10*60cm, and the dead volume of the chromatography column is 3400mL.

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

[0070] The elution procedure in step (2) is as follows: ① First, elute with dichloromethane for 3 column volumes; ② Then, elute sequentially with 95:5 solution (95 parts dichloromethane and 5 parts ethyl acetate), 90:10 solution (90 parts dichloromethane and 10 parts ethyl acetate), 85:15 solution (85 parts dichloromethane and 15 parts ethyl acetate), 80:20 solution (80 parts dichloromethane and 20 parts ethyl acetate), 75:25 solution (75 parts dichloromethane and 25 parts ethyl acetate), 70:35 solution (70 parts dichloromethane and 30 parts ethyl acetate), 65:35 solution (65 parts dichloromethane and 35 parts ethyl acetate), 60:40 solution (60 parts dichloromethane and 40 parts ethyl acetate), and 50:5 solution. Elute for 3 column volumes each with 0 solution (composed of 50 parts dichloromethylamine and 50 parts ethyl acetate); ③ Then elute for 2 column volumes each with 90:10 solution (composed of 90 parts dichloromethylamine and 10 parts methanol), 85:15 solution (composed of 85 parts dichloromethylamine and 15 parts methanol), 80:20 solution (composed of 80 parts dichloromethylamine and 20 parts methanol), 75:25 solution (composed of 75 parts dichloromethylamine and 25 parts methanol), 70:35 solution (composed of 70 parts dichloromethylamine and 30 parts methanol), 65:35 solution (composed of 65 parts dichloromethylamine and 35 parts methanol), 60:45 solution (composed of 60 parts dichloromethylamine and 40 parts methanol), 50:50 solution (composed of 50 parts dichloromethylamine and 50 parts methanol), and methanol.

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

[0072] The chromatographic column in step (3) is an Airswell C18 FLASH column (Airswell; product website: http: / / www.tjairs.com / aiershi / products / 20925717.html): with a diameter of 50 mm, a height of 240 mm, and a column volume of 470 mL; the packing medium is spherical-C18 with a particle size of 20-35 μm.

[0073] The mobile phase in step (3) is acetonitrile or an aqueous solution of acetonitrile. The flow rate of the mobile phase 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 time and 100% at the final time.

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

[0076] In 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 ZORBAX SB-C18 (Agilent Technologies) column with a packing particle size of 5 μm, a column diameter of 9.4 mm, a height of 250 mm, and a column volume of 20 mL.

[0078] The mobile phase in step (4) consists 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 step (4), the post-column solution of the elution peak with a retention time of 27.8 min corresponding to the peak value is collected, namely fraction Fr.13.1.

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

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

[0082] The mobile phase in step (5) consists 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 step (5), the post-column solution of the elution peak with a retention time of 16.9 min corresponding to the peak value is collected, the solvent is evaporated, and compound 1 is obtained.

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

[0085] The mobile phase in step (6) consists 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 step (6), the post-column solution of the elution peak with a retention time of 18.3 min corresponding to the peak value is collected, the solvent is evaporated, and compound 2 is obtained.

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

[0088] The mobile phase in step (7) consists 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 step (7), the post-column solution of the elution peak with a retention time of 20.9 min corresponding to the peak value is collected, namely fraction Fr.14.1.

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

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

[0092] The mobile phase in step (8) consists 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 step (8), the eluent after column passing of the elution peak with a retention time of 35.2 min corresponding to the peak value is collected, namely the fraction Fr.14.1.1.

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

[0095] The mobile phase in step (9) is methanol or an aqueous methanol 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 initial volume fraction of methanol in the mobile phase is 50%, and the final volume fraction of methanol in the mobile phase is 100%.

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

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

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

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

[0101] This invention also provides a method for inhibiting viral infection in animals.

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

[0103] The present invention also provides methods for treating and / or preventing viral infections.

[0104] The present invention provides a method for treating and / or preventing viral infection, comprising administering the compound or a pharmaceutically acceptable salt thereof to a recipient animal for the treatment and / or prevention of viral infection.

[0105] In this invention, the animal may be a mammal, such as a human; the animal may also be other animals infected with viruses besides mammals, such as birds.

[0106] Any of the viruses mentioned above could be influenza viruses.

[0107] Specifically, any of the viruses mentioned above could be influenza A viruses.

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

[0109] In the above text, the compounds include polymorphs, pseudopolymorphs, amorphous forms, hydrates, or solvates.

[0110] In the above text, the virus 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 materials here include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred. Using these materials, various dosage forms can be formulated, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. To formulate unit-dose dosage forms 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, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste, etc.; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc. To formulate unit-dose dosage forms into suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. To formulate unit-dose dosage forms into injectable formulations, such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc.In addition, to prepare isotonic injection solutions, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added. Furthermore, if necessary, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical formulation. The above dosage forms can be used for administration via injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections; cavity administration, such as rectal and vaginal; respiratory administration, such as nasal administration; and mucosal administration.

[0111] This 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] Instructions for the Preservation of Biological Materials

[0113] Classification and nomenclature of biological materials: Penicillium

[0114] The Latin scientific name of the biological material is Penicillium sp.

[0115] Strains of the biological material: CPCC 401065

[0116] Full name of the depository: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0117] Abbreviation of depositary institution: CGMCC

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

[0119] Deposit date: November 8, 2023

[0120] Accession number: CGMCC No. 40910 Attached Figure Description

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

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

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

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

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

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

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

[0128] Figure 8 The ROESY spectrum of compound 4 dissolved in DMSO-d6. Detailed Implementation

[0129] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

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

[0131] PDA medium (5.6±0.2): contains 4 g / L potato starch, 20 g / L glucose and 15 g / L agar, with the remainder 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]. Acta Pharmaceutica Sinica B, 2014, 4(4): 301-306.

[0133] Example 1: Isolation, identification and preservation of Penicillium CPCC 401065

[0134] I. Isolation of bacterial strains

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

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

[0137] Preservation method for the strain: Store in 20% glycerol cryovials at -80℃.

[0138] II. Identification of the strain

[0139] 1. Morphological identification of strain CPCC 401065

[0140] The hyphae are relatively thick and long, and the colonies are large and unrestricted. The colonies are loose in texture, dry in appearance, and opaque, appearing as either tight or loose spider webs, fluff, or cotton wool. 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 amplification was performed using genomic DNA as a template with primers consisting of ITS1F and ITS4 (ITS1F: 5'-CTTGGTCATTTAGAGTAA-3'; ITS4: 5'-TCCTCCGCTTAGATATGC-3'). Approximately 500-600 bp of the amplified product was recovered and sequenced. The sequencing results are 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 a reference sequence) and phylogenetic tree analysis. SEQ ID NO: 1 was compared with the GenBank database, showing 99.8% sequence similarity to the ITS gene of the strain *Penicillium allii* strain MH210.

[0144] Based on the results of morphological and molecular identification, strain CPCC 401065 belongs to the genus Penicillium sp., and is therefore named Penicillium sp. CPCC 401065.

[0145] III. Preservation of bacterial strains

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

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

[0148] I. Fermentation Culture

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

[0150] 2. Streak the spores from the slant culture onto PDA agar plates and incubate at 28°C for 10 days. Collect the spores from the plates with sterile water to obtain a spore suspension (spore concentration of 10). 7 (units / mL).

[0151] 3. Inoculate the spore suspension obtained in step 2 into an Erlenmeyer flask containing fermentation medium (10 mL spore suspension / flask), and incubate at 25-28℃ for 40 days (shake occasionally during the incubation period to ensure air entry during the incubation process) to obtain fermentation product (fermentation product is all the contents of the Erlenmeyer flask).

[0152] Erlenmeyer flasks containing fermentation medium: Take a 500mL Erlenmeyer 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 processes were performed simultaneously to obtain 4 kg of fermented product.

[0154] II. Separation and Purification of Compounds

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

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

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

[0158] Packing material: 200-300 mesh column chromatography silica gel (Qingdao Marine Chemical Plant Branch).

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

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

[0161] The eluent after column chromatography, with a volume of 18,000 ml to 26,000 ml, is fraction 6-9.

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

[0163] Aires Technology Spherical C18 FLASH Column (Aires Technology; product website: http: / / www.tjairs.com / aiershi / products / 20925717.html): Diameter 50mm, height 240mm, column volume 470mL; filling medium is spherical-C18, filler particle size 20-35μm.

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

[0165] Elution process: The elution time was 70 min. The volume fraction of acetonitrile in the mobile phase increased linearly. At the initial moment, the volume fraction of acetonitrile in the mobile phase was 20%, and at the final moment, the volume fraction of acetonitrile in the mobile phase was 100%.

[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 prepare and separate it using SB-C18 liquid phase.

[0169] ZORBAX SB-C18 column (Agilent Technologies): packing particle size 5μm; column diameter 9.4mm, height 250mm, column volume 20mL.

[0170] Mobile phase: Composed of 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 with a retention time of 27.8 min was collected and named Fr.13.1.

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

[0173] 5. Take all Fr.13.1 and prepare and separate it using SB-C18 liquid chromatography.

[0174] ZORBAX SB-C18 column (Agilent Technologies): packing particle size 5μm; column diameter 9.4mm, height 250mm, column volume 20mL.

[0175] Mobile phase: Composed of 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 with a retention time of 16.9 min was collected, the solvent was evaporated, and 7 mg of compound 1 was obtained.

[0177] 6. Take all of Fr.13.2 and prepare and separate it using SB-C18 liquid chromatography.

[0178] ZORBAX SB-C18 column (Agilent Technologies): packing particle size 5μm; column diameter 9.4mm, height 250mm, column volume 20mL.

[0179] Mobile phase: Composed of 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 with a retention time of 18.3 min was collected, the solvent was evaporated, and 15 mg of compound 2 was obtained.

[0181] 7. Take all Fr.14 and prepare and separate it using SB-C18 liquid chromatography.

[0182] ZORBAX SB-C18 column (Agilent Technologies): packing particle size 5μm; column diameter 9.4mm, height 250mm, column volume 20mL.

[0183] Mobile phase: Composed of 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 with a retention time of 20.9 min was collected and named Fr.14.1.

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

[0186] 8. Take all Fr.14.1 and prepare and separate it using SunFire-C18 liquid chromatography.

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

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

[0189] The eluent after column percolation of the elution peak with a retention time of 35.2 min corresponding to the peak value was collected and named Fr.14.1.1.

[0190] 9. Take all of Fr.14.1.1 and prepare and separate it using SilGreen-C18 liquid chromatography.

[0191] The chromatographic column is a SilGreen HPLC COLUMN C18 (Greenherbs Co., Ltd., product number GH0525046C18A; product website: http: / / www.greenherbs.com.cn / product_details / 1052912801187983360.html): the packing particle size is 5μm; the column diameter is 4.6mm, the height is 250mm, and the column volume is 5mL.

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

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

[0194] The post-column solution of the elution peak with a retention time of 21.8 min was collected, the solvent was evaporated, and 3.0 mg of compound 3 was obtained.

[0195] 10. Take all of Fr.14.2 and prepare and separate it using SHIMADZU C18 liquid chromatography.

[0196] SHIMADZU C18 chromatographic column: packing particle size 5μm; column diameter 10mm, height 250mm, column volume 20mL.

[0197] Mobile phase: Composed of 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 with a retention time of 30.0 min was collected, the solvent was evaporated, and 3.5 mg of compound 4 was obtained.

[0199] III. Identification and Characterization of Compounds

[0200] NMR data of the compound ( 1 H-NMR, 600MHz; 13 The results of C-NMR (150MHz) 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 a [M+H]+ peak at m / z 659.21112, indicating that its molecular formula is C. 36 H 34 O 12 The 1H NMR spectrum shows two olefinic hydrogen signals, δ H6.79 (1H, s) and 6.62 (1H, s); four oxygen-bound 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-bound 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); a 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 The δ values ​​were 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 showed 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 methylene 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 C(50.6, 22.1, 21.2, 20.8, 12.0, 8.9). In the HMBC spectrum, correlations can be observed between H-1′ and C-8, C-3′, C-3′b, C-9′, C-9′a; between H-8′ and C-7, C-8, C-6′a, C-7′, C-9′, C-9′a; between H-9′ and C-7, C-8, C-3′b, C-7′, C-8′, C-11′; between H-10′ and C-5′, C-6′, C-6′a; between H-12′ and 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; and H-5 is correlated with... 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; and H-14 is related to C-1, C-9a, C-10, and C-13. 1 H- 1In the 1H COSY spectrum, H-9′ and H-11′ are correlated, H-1 and H-14 are correlated, H-10 is correlated with H-11 and H-17, and H-11 is correlated with H-10, H-12 and H-18. These data, combined with the molecular formula, determined 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-14 and Hβ-14. Further analysis using the coupling constants of H-5 and H-6 (J = 0 Hz), H-1 and Hα-14 (J = 9.0 Hz), and H-11 and Hβ-14 (J = 0 Hz) determined the relative configuration of compound 1. Furthermore, the correctness of this configuration was verified by the NMR calculation 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 NMR calculation results, the relative configuration of compound 1 was further confirmed. In the ECD spectrum of compound 1, a positive Cotton effect was observed at 253 nm, and negative Cotton effects were observed 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). This compound was previously unreported and is a novel compound.

[0208] 2. Identification and characterization of compound 2

[0209] The (+)-HRESIMS mass spectrum shows its [M+H]+ peak at m / z 659.21161, indicating that its molecular formula is C. 36 H 34 O 12 Compound 2 and compound 1 have very similar structures. Comparison of their proton and carbon NMR spectra revealed that H-10 and H-11 in compound 2 shifted to higher fields by 0.64 and 0.69 ppm, respectively, while C-10 and C-11 shifted to lower fields by 4.4 and 4.0 ppm, respectively, suggesting a change in the configuration of C-10 and C-11. Further analysis of one-dimensional and two-dimensional NMR spectra confirmed the planar structure of compound 2, which is identical to that of compound 1. In the NOESY spectrum, H-11 and H-17 were correlated, H-10 and H-18 were correlated, H-10 was correlated with Hα-14 and Hβ-14, while H-17 was not correlated with Hα-14 and Hβ-14, thus determining the relative configuration of compound 2. Furthermore, the correctness of this configuration was verified by calculating NMR values ​​as 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 calculated NMR results, the relative configuration of compound 2 was further confirmed. In the ECD spectrum of compound 2, a positive Cotton effect was observed at 260 nm, and a negative Cotton effect was observed 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). This compound has not been previously reported and is a novel compound.

[0210] 3. Identification and characterization of compound 3

[0211] The (+)-HRESIMS mass spectrum shows its [M+H]+ peak at m / z 659.20917, indicating that its molecular formula is C. 36 H 34 O 12Compound 3 and compound 2 have very similar structures. Through comprehensive analysis of one-dimensional and two-dimensional NMR spectra, the planar structure of compound 3 was determined, which is identical to that of compound 2. The specific coupling constants of the proton at position H-1 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-derivedfungus Talaromyces verruculosus[J]. Marine Drugs, 2019, 17: 516.), thus determining the configuration of H-1. In the NOESY spectrum, H-11 and H-17 are correlated, and H-10 and H-18 are correlated, determining the relative configurations of H-17 and H-18. The relative configuration of compound 3 was further determined using the NMR calculation 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.). Based on the NMR calculation results, the relative configuration of compound 3 was confirmed. In the ECD spectrum of compound 3, a positive Cotton effect was observed at 258 nm, and negative Cotton effects were observed 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). This compound has not been previously reported and is a novel compound.

[0212] 4. Identification and characterization of compound 4

[0213] The (+)-HRESIMS mass spectrum shows its [M+H]+ peak at m / z 659.2106, indicating that its molecular formula is C. 36 H 34 O 12Compound 4 and compound 3 have very similar structures. Comparison of their proton and carbon NMR spectra revealed that the H-10 and H-11 spectral shifts in compound 4 were 0.42 and -0.46 ppm, respectively, compared to compound 3, suggesting a change in the configurations of C-10 and C-11. Further analysis of 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 (J = 11.4, 4.8, 1.8 Hz) of the proton at position H-1 and other protons are consistent with those reported by 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.), thus determining the configuration of H-1. In the NOESY spectrum, H-11 and H-17 are correlated, and H-10 and H-18 are correlated, thus determining the relative configurations of H-17 and H-18. The relative configuration of compound 4 was further determined by calculating the NMR values ​​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 calculated NMR results, the relative configuration of compound 4 was confirmed.In the ECD spectrum of compound 4, a positive Cotton effect was observed at 258 nm, and negative Cotton effects were observed 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). This compound was previously unreported and is a novel compound.

[0214]

[0215] Example 3: Antiviral activity and cytotoxicity of the compound

[0216] Test compounds: 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 is the test compound stock solution.

[0218] The positive control was ribavirin. The ribavirin solution was obtained by dissolving and diluting ribavirin in DMSO.

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

[0220] The anti-influenza virus activity of the tested compounds was determined using the Gaussian luciferase reporter system.

[0221] 1. Resuspend 293T-Gluc cells in DMEM medium containing 10% FBS to achieve a cell concentration of 2.0 × 10⁻⁶ cells / year. 5 Cells / mL is the cell suspension.

[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). Inoculate the blank control wells with DMEM medium containing 10% FBS (100 μL / well) and incubate 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 obtained 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 0.01, 0.02, 0.1, 0.625, 1.25, 2.5, 5, 10, 20, 50, 100 or 200 μM.

[0225] After adding ribavirin solution, the concentrations of ribavirin in the liquid phase system of the positive control wells were 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 strain A / WSN / 33 (MOI=0.3), and incubate for 24 hours.

[0227] 5. After completing step 4, take 10 μL of supernatant from each well and measure the luciferase activity using a multi-functional microplate reader (Berthold Centro LB 960). Calculate the inhibition rate of each sample and the IC50. 50 (The concentration required to suppress the virus by 50%).

[0228] The experiment was repeated three times, and the average value of the results was taken. The results are shown in Table 3.

[0229] II. Cytotoxicity (CC) 50 value)

[0230] 1. Suspend 293T-Gluc cells in DMEM medium containing 10% FBS to achieve a cell concentration of 2.0 × 10⁻⁶ cells / year. 5 Cells / mL is the cell suspension.

[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). Inoculate the blank control wells with DMEM medium containing 10% FBS (100 μL / well) and incubate 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 obtained 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 adding ribavirin solution, the concentration of ribavirin 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 multi-functional microplate reader (Berthold Centro LB 960) to detect the absorbance value of each well at a wavelength of 450 nm to calculate the half-maximal cytotoxic concentration (MCC). 50 (Drug concentration that causes 50% cell death).

[0236] The experiment was repeated three times, and the average value of the results was taken. The results are shown in Table 3.

[0237] IC50 of the test compound against human influenza A virus A / WSN / 33 50 Values ​​and CC values ​​of cells 50 The values ​​are shown in Table 3. The results showed that compounds 1, 2, 3, and 4 all exhibited inhibitory activity against human influenza A virus strain A / WSN / 33, with IC50 values ​​of [missing value]. 50 The values ​​ranged from 72.77 to 5197 nM, and no significant cytotoxicity was observed at a concentration of 100 μM. The four compounds provided by this invention exhibited higher inhibitory effects against influenza virus than the positive control, especially compound 1, whose activity was more than 200 times that of the positive control.

[0238] Table 3

[0239]

[0240]

[0241] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A compound, as shown in formula (V); Formula (V).

2. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof is as follows (a1) and / or (a2): (a1) Application in the preparation of influenza virus inhibitors; (a2) Use in the preparation of medicines for the treatment and / or prevention of influenza virus infection.

3. A product containing the compound of claim 1 or a pharmaceutically acceptable salt thereof; The product is as follows (b1) and / or (b2): (b1) Influenza virus inhibitors; (b2) Medications used to treat and / or prevent influenza virus infection.

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

5. An inoculum comprising the Penicillium of claim 4 and / or a culture of the Penicillium of claim 4.

6. The use of the Penicillium fungus of claim 4 in the preparation of the compound of claim 1.

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

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