Leporin compounds, methods of making and use thereof in the preparation of anti-influenza virus drugs

Abstract

The application discloses Leporin compounds, a preparation method thereof and application of the Leporin compounds in preparation of anti-influenza virus drugs. Leporins B and C are separated from a solid fermentation extract of a strain CPCC 401592, and a new compound of formula III is obtained by microbial transformation of Leporin B. The application further discloses a method for separating Leporins B and C from the strain and a method for obtaining the Leporin compound of formula III by microbial transformation of Leporin B. The Leporin compounds provided by the application all show strong inhibitory effects on influenza A virus, and the inhibitory activities are higher than those of a positive control drug, so the Leporin compounds have application prospects in preparation of drugs or preparations for preventing or treating influenza virus infection.

Description

Technical Field

[0001] This invention relates to leporin compounds, particularly to leporin compounds isolated from strain CPCC 401592 and new leporin compounds obtained by microbial transformation thereof. The invention further relates to the use of leporin compounds in the preparation of medicaments for the prevention and / or treatment of influenza virus infection, belonging to the field of leporin compounds and their use in the preparation of antiviral drugs for influenza. Background Technology

[0002] Influenza, or influenza, is a highly contagious acute respiratory infectious disease caused by the influenza virus. It is characterized by its high infectivity, rapid spread, high mutation rate, cross-species transmission, and high morbidity, posing a serious threat to human health. Influenza viruses belong to the Orthomyxoviridae family and are a type of RNA virus, classified into four strains: type A, type B, type C, and type D. Among these, influenza A virus (IAV) is the most common influenza virus strain, infecting the widest range of hosts.

[0003] 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.

[0004] To date, there have been no reports of antiviral activity in Leporin-like compounds. Summary of the Invention

[0005] One objective of this invention is to provide Leporin-like compounds with anti-influenza virus activity;

[0006] A second objective of this invention is to apply the aforementioned Leporin-like compounds to the preparation of drugs or formulations for treating influenza viruses.

[0007] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0008] One aspect of this invention provides two Leporin-like compounds isolated from the polar fungus CPCC 401592, with structural formulas shown in Formula I and Formula II, respectively:

[0009]

[0010] Another aspect of the present invention is to obtain a glycosylated transformation product by microbial transformation of the compound of formula I using Mucor rouxianus, the structure of which is shown in formula III:

[0011]

[0012] All pharmaceutically acid-additive salts, solvates, or prodrugs of the compounds described above are also included in this invention.

[0013] The salt of the compound formed by acid addition is preferably a pharmaceutically acceptable suitable acid (e.g., hydrochloric acid, acetic acid, or sulfuric acid) to form a non-toxic salt. Other salts besides pharmaceutically acceptable salts are also included in this invention.

[0014] The solvent in the solvate described in this invention can be ethanol, water, etc.; and it may contain different amounts of water, such as monohydrate, hemihydrate, monihydrate, dihydrate, or trihydrate, etc.

[0015] The present invention also includes prodrugs of the above-mentioned compounds. According to the present invention, prodrugs are derivatives of the above-mentioned compounds, which may have weak or no activity on their own, but are converted into the corresponding biologically active form under physiological conditions (e.g., through metabolism, solvation or other means) after administration.

[0016] Another aspect of the present invention provides a method for preparing a compound of formula I or formula II, the method comprising:

[0017] (1) The polar fungus CPCC 401592 was fermented and cultured; (2) The fermentation product was extracted with 100% ethyl acetate and concentrated under reduced pressure to obtain an extract; (3) The ethyl acetate extract was separated by column chromatography to obtain compounds of formula I or formula II.

[0018] In a preferred embodiment of the present invention, in step (1), the polar fungus CPCC 401592 is fermented on rice culture medium, wherein the fermentation conditions are preferably: the fermentation temperature is 15°C and the fermentation time is 60 days.

[0019] In a preferred embodiment of the present invention, the column chromatography described in step (3) includes, but is not limited to, normal phase silica column chromatography, reversed phase silica column chromatography, or semi-preparative high performance liquid chromatography.

[0020] In a preferred embodiment of the present invention, in step (3), the ethyl acetate extract is separated by column chromatography using the following separation method to obtain compounds of formula I and formula II respectively: the extract is separated by silica gel column chromatography, and eluted with dichloromethane-methanol gradient (100:0→0:100) to obtain five elution fractions: F1, F2, F3, F4 and F5; wherein, fraction F1 is separated by reversed-phase silica gel column chromatography, and eluted with acetonitrile-water gradient (10:90→100:0) to obtain 23 fractions; fractions 20-22 are recrystallized to obtain compound I; fractions 18-19 are separated by semi-preparative high performance liquid chromatography (mobile phase: methanol / water = 55:45) to obtain compound II.

[0021] Another aspect of the present invention provides a method for obtaining a compound of formula III by microbial transformation using a compound of formula I, the method comprising the following steps:

[0022] (1) Add the compound of formula I to the liquid fermentation culture of Mucor rouxianus for fermentation transformation; (2) Centrifuge the fermentation product and collect the mycelium; (3) Extract the mycelium with 100% methanol and combine it with the bacterial liquid, then extract with ethyl acetate and concentrate under reduced pressure to obtain the extract; (4) Separate the obtained extract by column chromatography to obtain the compound of formula III.

[0023] In a preferred embodiment of the present invention, in step (1), the mold Mucor rouxianus is inoculated into PDB liquid culture medium, fermented in a shaker at 28°C for 2 days, and then compound of formula I is added. The fermentation culture is continued to carry out fermentation transformation to obtain fermented product.

[0024] In a preferred embodiment of the present invention, the column chromatography in step (4) includes: reverse silica gel column chromatography, semi-preparative high performance liquid chromatography, etc.

[0025] In a preferred embodiment of the present invention, in step (4), the obtained extract is separated from the microbial transformation fermentation product by column chromatography using the following separation method: the extract is separated by reversed-phase silica gel column chromatography and eluted with acetonitrile-water gradient (10:90→100:0) to obtain 11 fractions; fraction 7 is separated by semi-preparative high performance liquid chromatography (mobile phase: acetonitrile / water = 40:60) to obtain compound III.

[0026] This invention determined the in vitro inhibitory activity of three compounds, represented by Formulas I, II, and III, against influenza A virus. The results showed that compounds of Formulas I, II, and III all exhibited inhibitory activity against influenza A virus strain A / WSN / 33, with IC50 values ​​exceeding 100%. 50The values ​​ranged from 0.19 to 3.8 μM, and compounds I and II showed no significant cytotoxicity at a concentration of 100 μM. In addition, compounds I, II, and III all exhibited higher inhibitory effects on influenza virus than the positive control.

[0027] Another aspect of the present invention is to use compounds of formula I, formula II and formula III to prepare drugs or preparations for treating influenza viruses; wherein the influenza virus may be influenza A virus, influenza B virus, influenza C virus or influenza D virus, preferably influenza A virus.

[0028] In a preferred embodiment, the present invention provides a pharmaceutical composition for the prevention and / or treatment of influenza virus infection. This pharmaceutical composition comprises a preventatively or therapeutically effective amount of a compound of formula I, II, or III, or a pharmaceutically acceptable salt thereof, combined with a pharmaceutically acceptable carrier. The pharmaceutically acceptable amount of the compound of formula I, II, or III is combined with a pharmaceutically acceptable carrier or excipient and then prepared into any suitable pharmaceutical composition according to conventional formulation methods in the art. This composition is generally suitable for oral and injectable administration, and is also suitable for other methods of administration. The composition may be in liquid formulation form such as tablets, capsules, powders, granules, lozenges, suppositories, or oral solutions. Depending on the method of administration, the pharmaceutical composition of the present invention may contain 0.1%-99% by weight, preferably 10-60% by weight, of the compound of formula I, II, or III.

[0029] The excipients may be antioxidant complexing agents, fillers, matrix materials, etc.; the pharmaceutically acceptable carriers are one or more of xylitol, mannitol, lactose, fructose, dextran, glucose, polyvinylpyrrolidone, low molecular weight dextran, sodium chloride, calcium gluconate or calcium phosphate, preferably mannitol or lactose. Attached Figure Description

[0030] Figure 1 This is the hydrogen spectrum of compound I.

[0031] Figure 2 This is the carbon spectrum of compound I.

[0032] Figure 3 This is the hydrogen spectrum of compound II.

[0033] Figure 4 This is the carbon spectrum of compound II.

[0034] Figure 5 This is the proton NMR spectrum of compound III.

[0035] Figure 6 This is the carbon spectrum of compound III. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0037] Example 1: Isolation and identification of compounds of formula I and formula II from strain CPCC 401592

[0038] 1. Cultivation and fermentation of strains

[0039] Strain CPCC 401592 was isolated from plants in the Ny-Ålesund region of the Arctic and identified by researcher Zhang Tao (Institute of Pharmaceutical Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College). The sample is preserved at the China Pharmaceutical Microbiology Culture Collection Center.

[0040] First, strain CPCC 401592 was revived from glycerol tubes to PDA slant medium and grown at 15°C for 10-14 days to obtain the slant culture. The spores or mycelia from the slant culture were inoculated into PDB liquid medium and fermented on a shaker at 15°C for 8 days to obtain the fermentation seed culture. The seed culture was then inoculated into Erlenmeyer flasks containing fermentation medium (10 mL seed culture / flask) and incubated statically at 15°C for 60 days to obtain the fermentation product (the fermentation product being the entire contents of the Erlenmeyer flask).

[0041] Erlenmeyer flasks containing fermentation medium: Take a 500mL Erlenmeyer flask, add 100g of rice and 100mL of water, soak at 28℃ for 8 hours, and then sterilize at 121℃ for 20 minutes.

[0042] 2. Extraction and separation of compounds

[0043] After harvesting the fermentation product, it was extracted with 100% ethyl acetate and concentrated under reduced pressure to obtain an extract. The extract was separated by silica gel column chromatography with a dichloromethane-methanol gradient elution (100:0→0:100) to obtain five eluting fractions: F1, F2, F3, F4, and F5. Fraction F1 was separated by reversed-phase silica gel column chromatography with an acetonitrile-water gradient elution (10:90→100:0) to obtain 23 fractions. Fractions 20-22 were recrystallized to obtain compound I. Fractions 18-19 were separated by semi-preparative high-performance liquid chromatography (mobile phase: methanol / water = 55:45) to obtain compound II.

[0044] 3. Structural identification

[0045] Two leporin-like compounds were isolated from the fermentation culture of strain CPCC 401592, and their structural identification data are as follows:

[0046] (1) Identification of compound I (Leporin B).

[0047] White powder, soluble in methanol, dimethyl sulfoxide, and dichloromethane. ESIMS: m / z 352.2 [M+H] + . 1 H-NMR (600MHz, CD3OD)δ H : 7.63(1H,s,H-3), 7.41(2H,d,J=7.2Hz,H-18,H-22), 7.34(2H,t,J=7.2Hz,H-19,H-21), 7.29(1H,t,J=7 .2Hz,H-20), 5.82(1H,dq,J=15.2,6.6Hz,H-15), 5.40(1H,ddd,J=15.2,8.3,1.8Hz,H-14), 4.91(1H,d,J =8.3Hz,H-13), 2.77(1H,m,H-7), 1.83(1H,m,H-11), 1.78(1H,m,H-12), 1.72(3H,dd,J=6.5,1.6Hz,H-16 ), 1.68(1H,m,H-8), 1.65(1H,m,H-11), 1.54(2H,m,H-10), 1.26(2H,m,H-9), 0.95(3H,d,J=6.6Hz,H-23). 13 C-NMR (150MHz, CD3OD)δ C : 160.7(C-1), 159.2(C-5), 135.1(C-17), 133.3(C-3), 132.3(C-15), 130.8(C-14), 130.4(C-18,22), 129.1(C-19,21), 128.3(C-20), 115 .2(C-4), 112.9(C-6), 79.3(C-13), 39.4(C-7), 37.3(C-12), 37.2(C-8), 36.5(C-9), 27.7(C-11), 21.8(C-10), 21.0(C-23), 17.9(C-16).

[0048] (2) Identification of compound II (Leporin C)

[0049] White powder, soluble in methanol, dimethyl sulfoxide, and dichloromethane. ESIMS: m / z 336.2 [M+H] + . 1 H-NMR (600MHz, CDCl3)δ H: 7.40(2H,dd,J=7.8,1.8Hz,H-18,H-22), 7.36(2H,t,J=7.8Hz,H-19,H-21), 7.30(1H,t,J=7.2Hz,H-20), 7.25 (1H,s,H-3), 5.75(1H,dq,J=15.0,6.6Hz,H-15), 5.37(1H,ddd,J=15.0,6.6,1.8Hz,H-14), 4.86(1H,dd,J=11. 4,8.4Hz,H-13), 2.78(1H,dd,J=10.8,3.6Hz,H-7), 1.77(3H,m,H-11,H-12), 1.72(3H,dd,J=6.6,1.2Hz,H-16) , 1.61(2H,m,H-8,H-11), 1.54(1H,m,H-10), 1.42(1H,m,H-10), 1.29(1H,m,H-9), 0.99(3H,d,J=6.6Hz,H-23). 13 C-NMR (150MHz, CDCl3)δ C :164.1(C-1), 161.1(C-5), 134.2(C-17), 131.6(C-3), 131.2(C-15), 129.4(C-14), 129.2(C-18,22), 128.3(C-19,21), 127.4(C-20), 116 .2(C-6), 111.6(C-4), 78.5(C-13), 37.2(C-7), 36.1(C-12), 35.9(C-8), 35.3(C-9), 26.6(C-11), 21.0(C-10), 20.6(C-23), 18.0(C-16).

[0050] Example 2: Microbial transformation of compound I to obtain compound III and identification of compound III.

[0051] 1. Microbial transformation

[0052] First, strain Mucor rouxianus CGMCC 3.2545 (purchased from the China General Microbiological Culture Collection Center) was revived from glycerol tubes to PDA slant medium and grown at 28°C for 7-10 days to obtain a slant culture. The spores from the slant culture were inoculated into PDB liquid medium and fermented on a shaker at 28°C for 2 days. Then, compound I was added, and the culture was continued for 3 days to obtain the fermentation broth.

[0053] 2. Extraction and separation

[0054] After harvesting the fermentation broth obtained in the above steps, centrifuge it. Extract the mycelium with 100% methanol and combine it with the bacterial broth. Then extract with ethyl acetate and concentrate under reduced pressure to obtain the extract. (3) Separate the obtained extract by reversed-phase silica gel column chromatography as follows: take the extract and separate it by reversed-phase silica gel column chromatography, elute with acetonitrile-water gradient (10:90→100:0), and obtain 11 fractions. Fraction 7 is separated by semi-preparative high performance liquid chromatography (mobile phase: acetonitrile / water = 40:60) to obtain compound III.

[0055] 3. Structural identification

[0056] A novel transformation product was obtained by microbial transformation of the compound of formula I using the strain Mucor rouxianus. The structural identification data of the product are as follows:

[0057] Identification of compound of formula III: Leporin B-2-O-β-D-glucoside.

[0058]

[0059] White powder, soluble in methanol, dimethyl sulfoxide, and dichloromethane. ESIMS: m / z 514.2 [M+H] + The molecular ion peak m / z given by (+)HRESIMS is 514.2472 [M+H]. + (calcd for 514.2441) determines its molecular formula to be C. 28 H 34 NO8 has an unsaturation degree of 12. This transformed product has a molecular weight increase of 162 amu compared to the substrate leporin B, presumably due to the introduction of a glucose atom. Comparison of compound III and compound I (substrate) is also provided. 1 H-NMR and 13 C-NMR data revealed that the chemical shifts of the two compounds on the phenylpyridinone core were very similar, but compound III had an additional set of typical glycosyl signals compared to compound I: 4.93 (1H, d, J = 8.0 Hz, H-1′), 3.65 (1H, ddd, J = 12.0, 6.0, 2.0 Hz, H-6′), 3.45 (1H, ddd, J = 12.0, 6.0, 6.0 Hz, H-6′), 3.26 (1H, m, H-5′), 3.25 (1H, m, H-3′), 3.13 (1H, td, J = 8.0, 3.0 Hz, H-2′), and 3.07 (1H, ddd, J = 9.0, 9.0, 5.0 Hz, H-4′). Further analysis using... 1 H- 1The HCOSY spectrum showed correlations between the H-1′ / H-2′ / H-3′ / H-4′ / H-5′ / H-6′ spin coupling system and the HMBC spectrum showed correlations between H-1′ and C-2′, C-3′, and C-5′, and between H-4′ and C-2′, C-3′, C-5′, and C-6′, confirming the presence of a glucose group in the compound. The coupling constant J = 8.0 Hz of the terminal hydrogens identified the sugar type as O-β-D-glucose. In the NOESY spectrum, the correlation between H-1′ and H-3 confirmed that the sugar group was attached to an amino hydroxyl group; the correlations between H-7 and H-12 and H-23, and between H-8 and H-13 determined the relative configuration of the compound. Ultimately, compound III was identified as Leporin B-2-O-β-D-glucoside.

[0060] Its mass spectrometry and NMR data are as follows:

[0061] ESIMS: m / z 514.2 [M+H] + . 1 H-NMR (500MHz, DMSO-d6)δ H:7.75(1H,s,H-3),7.43(2H,dd,J=7.0,1.5Hz,H-18,22),7.37(2H,dd,J=7.5,7.0Hz,H-19,21),7.31(1H,tt,J=7.2,1.5Hz,H-20),5.82(1H,dq,J=15.5,6.5Hz,H-15),5.72(1H,d,J=3.0Hz,2′-OH),5.42(1H,m,H-14),5.20(1H,d,J=5.0Hz,3′-OH),5.08(1H,d,J=5.0Hz,4′-OH),4.93(1H,d,J=8.0Hz,H-1′),4.91(1H,dd,J=11.5,8.0Hz,H-13),4.64(1H,t,J=6.0Hz,6′-OH),3.65(1H,ddd,J=12.0,6.0,2.0Hz,H-6′),3.45(1H,ddd,J=12.0,6.0,6.0Hz,H-6′),3.26(1H,m,H-5′),3.25(1H,m,H-3′),3.13(1H,td,J=8.0,3.0Hz,H-2′),3.07(1H,ddd,J=9.0,9.0,5.0Hz,H-4′),2.65(1H,dd,J=11.0,4.0Hz,H-7),1.75(1H,m,H-12),1.69(3H,dd,J=6.5,1.5Hz,H-16),1.68(1H,m,H-9),1.67(1H,m,H-11),1.63(1H,m,H-8),1.56(1H,m,H-11),1.45(2H,m,H-10),1.17(1H,m,H-9),0.87(3H,d,J=6.5Hz,H-23)。 13 C-NMR(125MHz,DMSO-d6)δ C:158.7(C-1), 157.9(C-5), 134.9(C-3), 133.3(C-17), 131.1(C-15), 129.5(C-14), 129. 3(C-18,22), 128.1(C-19,21), 127.3(C-20), 112.2(C-4), 111.4(C-6), 107.3(C-1′), 77 .7(C-13), 77.6(C-5′), 75.8(C-3′), 71.9(C-2′), 69.4(C-4′), 61.0(C-6′), 37.5(C-7), 35.2(C-8), 35.1(C-9), 35.0(C-12), 26.2(C-11), 20.6(C-23), 20.2(C-10), 17.6(C-16).

[0062] Test Example 1: Antiviral Activity and Cytotoxicity Tests of Leporin-like Compounds

[0063] 1. Test materials

[0064] Test compounds: Compounds of formulas I, II, and III prepared in Examples 1 and 2. The test compounds were dissolved in DMSO to a concentration of 10 mM, i.e., the test compound stock solution.

[0065] The positive control was ribavirin. The ribavirin solution was obtained by dissolving and diluting ribavirin in DMSO.

[0066] 2. Test methods

[0067] (1) Antiviral activity (EC) 50 (Value) Determination Method

[0068] The anti-influenza virus activity of the tested compounds was determined using the Gaussian luciferase reporter system. 293T-Gluc cells were resuspended in DMEM medium containing 10% FBS to achieve a cell concentration of 2.0 × 10⁻⁶ cells / year. 5Cell suspension was prepared by inoculating the cells at a density of 100 μL / mL in 96-well plates. The test wells, positive control wells, and negative control wells were all inoculated with the prepared cell suspension (100 μL / well). Blank control wells were inoculated with DMEM medium containing 10% FBS (100 μL / well) and incubated for 24 hours. The test wells were then inoculated with the test compound solution (1 μL / well), the positive control wells with ribavirin solution (1 μL / well), and the negative control wells with DMSO (1 μL / well), and incubated for 2 hours. The concentrations of the test compound in the test wells were 0.01, 0.02, 0.1, 0.625, 1.25, 2.5, 5, 10, 20, 50, and 100 μM, respectively. The concentrations of ribavirin in the positive control wells were 0.01, 0.02, 0.1, 0.625, 1.25, 2.5, 5, 10, 20, 50, and 100 μM, respectively. Add human influenza A virus strain A / WSN / 33 (MOI = 0.3), incubate for 24 hours, then 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 half-maximal effective concentration (EC50). 50 .

[0069] The experiment was repeated three times, and the average value of the results was taken. The results are shown in Table 1.

[0070] (2) Cytotoxicity (CC) 50 (Value) Determination Method

[0071] 293T-Gluc cells were suspended in DMEM medium containing 10% FBS to achieve a cell concentration of 2.0 × 10⁻⁶ cells / year. 5 Cells were cultured at a concentration of 100 μL / mL to form a cell suspension. Test wells, positive control wells, and negative control wells were seeded with the prepared cell suspension (100 μL / well), while blank control wells were seeded with DMEM medium containing 10% FBS (100 μL / well) and incubated for 24 hours. The test compound solution (1 μL / well) was added to the test wells, ribavirin solution (1 μL / well) to the positive control wells, and DMSO (1 μL / well) to the negative control wells. Incubation was carried out at 37°C for 48 hours. The concentrations of the test compound in the liquid phase of the test wells were 5, 10, 20, 50, and 100 μM, respectively. The concentrations of ribavirin in the liquid phase of the positive control wells were 5, 10, 20, 50, and 100 μM, respectively. Add 10 μL of CCK-8 reagent to each well and incubate at 37°C for 1-2 hours. Then, use a multi-mode microplate reader (Berthold Centro LB 960) to detect the absorbance at 450 nm in each well to calculate the half-maximal cytotoxic concentration (MCC). 50 .

[0072] The experiment was repeated three times, and the average value of the results was taken.

[0073] 3. Test Results

[0074] The test compound's effect on the EC of influenza A virus 50 Values ​​and CC cytotoxicity 50 The values ​​are shown in Table 1.

[0075] Table 1. Antiviral activity and cytotoxicity results of the tested compounds.

[0076]

[0077] The results showed that compounds of formula I, formula II, and formula III all exhibited significant inhibitory activity against influenza A virus strain A / WSN / 33, with IC50 values ​​of [missing information]. 50 The values ​​ranged from 0.19 to 3.8 μM, and compounds of formula I and II showed no significant cytotoxicity at a concentration of 100 μM. In addition, the three compounds provided by this invention showed significantly higher inhibitory effects on influenza virus than the positive control drug.

Claims

1. A leporin-like compound with anti-influenza virus activity, characterized in that, Its structural formula is shown in Formula III:

2. The acid addition salt, solvate, or prodrug of the Leporin-type compound according to claim 1.

3. The acid-addition salt, solvate, or prodrug according to claim 2, characterized in that, The acid-added salt is a non-toxic salt formed by the addition of hydrochloric acid, acetic acid, or sulfuric acid to a compound; the solvate is a solvate formed using ethanol or water as a solvent for the compound.

4. The method for preparing the Leporin-like compound according to claim 1, characterized in that, include: (1) Compound of Formula I was added during the liquid fermentation culture of Mucor rouxianus to carry out fermentation transformation; (2) Centrifuge the fermentation product and collect the mycelium; (3) Extract the mycelium with 100% methanol and combine it with the bacterial solution for extraction with ethyl acetate, and concentrate under reduced pressure to obtain an extract; (4) Separate the obtained extract by column chromatography to obtain compound III.

5. The preparation method according to claim 4, characterized in that, In step (1), the Mucor rouxianus strain was inoculated into PDB liquid medium, fermented in a shaker at 28°C for 2 days, and then compound I was added to continue fermentation culture to obtain fermentation product; The column chromatography described in step (4) includes: reverse silica gel column chromatography and semi-preparative high performance liquid chromatography.

6. The preparation method according to claim 4, characterized in that, In step (4), the obtained extract was separated from the microbial transformation fermentation product using the following separation method by column chromatography: the extract was separated by reversed-phase silica gel column chromatography, and 11 fractions were obtained by elution with acetonitrile-water gradient. The acetonitrile-water gradient elution program was 10:90→100:

0. Fraction 7 was separated by semi-preparative high performance liquid chromatography to obtain compound III. The conditions for semi-preparative high performance liquid chromatography separation were: mobile phase: acetonitrile / water = 40:

60.

7. Use of the Leporin-like compound of claim 1 in the preparation of drugs for the prevention or inhibition of influenza viruses.

8. Use of Leporin-like compounds represented by Formula I or Formula II in the preparation of drugs for the prevention or inhibition of influenza viruses:

9. The use according to claim 7 or 8, characterized in that, The influenza virus mentioned is influenza A, influenza B, influenza C, or influenza D.

10. A pharmaceutical composition for the prevention or treatment of influenza virus infection, characterized in that, The pharmaceutical composition comprises a prophylactic or therapeutically effective amount of a compound of formula I, II or III, or a pharmaceutically acceptable salt thereof, combined with a pharmaceutically acceptable carrier.

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