Azidophilic ketone derivative in marine fungi HK1-8 and application of azidophilic ketone derivative in prevention and treatment of agricultural pathogenic bacteria

Abstract

The invention relates to an azophilic ketone derivative in marine fungi HK1-8 and application of the azophilic ketone derivative in prevention and treatment of agricultural pathogenic bacteria. The strain preservation unit name of the marine fungi HK1-8 is China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.40667. The invention further relates to a preparation method of the azophilic ketone derivative. Azophilic ketone derivative with structure as shown in formula I and formula II: # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of marine fungal secondary metabolites, and particularly relates to azotoxin derivatives in the marine fungus HK1-8 and their application in preventing and controlling agricultural pathogens. Background Art

[0002] Humicola sp. fungi are common saprophytes, widely distributed in soil, especially those rich in organic matter. Some species are also found in plant debris and wood. These fungi have attracted widespread attention due to their ability to produce specialized enzymes such as thermostable cellulases and hemicellulases. The biotransformation activities of Humicola sp. are essential for the fermentation of waste, the production of solid koji, and the fermentation of mushroom culture media. Furthermore, some of these fungi, such as Humicola spp., can cause certain diseases in livestock and humans and exhibit a certain degree of pathogenicity. While the enzyme metabolites produced by these fungi have practical value, the small molecule secondary metabolites produced by Humicola sp. are still underexplored, with few relevant research reports. Given the economic and ecological importance of this genus, research on its secondary metabolites is of great significance. The present invention discloses a Humicola sp. HK1-8 (abbreviated as HK1-8) isolated from the rhizosphere soil of Sonneratia caseolaris in Dongzhaigang Mangrove Nature Reserve in the South China Sea, its secondary metabolites, and applications in preventing and controlling agricultural pathogens. Summary of the Invention

[0003] The present invention provides a marine fungus HK1-8, isolated from the rhizosphere soil of the mangrove plant Sonneratia salsa, which is obtained from the Dongzhaigang Mangrove Nature Reserve in the South China Sea. The marine fungus HK1-8 described in the present invention is characterized by the following strain deposit information: Depository Name: General Microbiology Center, China Culture Collection Administration; Depository Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit Date: June 20, 2023; Deposit Number: CGMCC No. 40667; Taxonomic Name: Humicola sp.

[0004] Another embodiment of the present invention provides a secondary metabolite isolated from the marine fungus HK1-8, a tautomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that the secondary metabolite has a structure shown in Formula I, Formula II, Formula III, Formula IV, Formula V, or Formula VI:

[0005]

[0006] Wherein R1, R2, R3, and R4 are each independently selected from H, OH, or R1 and R2 are both O (i.e., forming a carbonyl group, =O); R5 is selected from methyl, carboxyl, hydroxyl, and hydroxymethyl; R6 is selected from C1-C3 alkyl (preferably methyl, ethyl); R6 is selected from H, C1-C3 alkyl acyl (preferably Ac).

[0007] The structure shown in the above formula I is preferably the following compound 1-4:

[0008]

[0009] The preferred compound 5-6 is represented by the structure of the above formula III:

[0010] The preferred compound 7-8 is represented by the structure of the above formula IV:

[0011]

[0012] The preferred compounds 9-10 are shown in the above formula VI:

[0013]

[0014] Another embodiment of the present invention provides a method for preparing compounds 1, 5-8, Formula II, and Formula V, characterized in that it comprises the following steps:

[0015] (1) Cultivating the marine fungus HK1-8 in a culture medium to obtain a seed solution;

[0016] (2) inoculating the seed solution obtained in step (1) into a fermentation medium for fermentation to obtain a fermentation product;

[0017] (3) Separating the fermentation broth and bacterial cells from the fermented product obtained in step (2), extracting the fermentation broth 2 to 4 times with an organic solvent A, combining the extracts and concentrating under reduced pressure to obtain a fermentation broth extract; extracting the bacterial cells with an organic solvent B 2 to 4 times, combining the extracts and concentrating under reduced pressure to obtain a bacterial cell extract; and combining the fermentation broth extract and the bacterial cell extract to obtain the crude extract.

[0018] (4) The crude extract obtained in step (3) is subjected to chromatographic separation to obtain compounds 1, 5-8, Formula II, and Formula V. The chromatographic separation is preferably performed by one or more combinations of vacuum silica gel column chromatography, normal phase silica gel column chromatography, reverse phase silica gel column chromatography, gel column chromatography, HPLC, etc. The following scheme is preferred: the crude extract is first subjected to vacuum silica gel column chromatography, and gradient elution is performed using petroleum ether-ethyl acetate as an eluent. The elution gradients are 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively. 2-3 column volumes are collected for each gradient and the components are divided into 10 fractions (Fr.1-Fr.10) according to their polarity. Among them, component Fr.3 is subjected to Sephadex chromatography. LH-20 gel column chromatography, using a 1:1 v / v mixed solvent of CH2Cl2 / MeOH as the eluent, yielded three components (Fr.3.1-Fr.3.3). Component Fr.3.2 was separated by reverse-phase column chromatography (ODS) with a mobile phase of 60% methanol-water, and then subjected to semi-preparative high-performance liquid chromatography with a mobile phase of 65% methanol-water to yield compound 1 and Formula II. Component Fr.3.3 was first separated by reverse-phase column chromatography with a mobile phase of 70% methanol-water to yield Formula V and a mixture. The mixture was then subjected to semi-preparative high-performance liquid chromatography with a mobile phase of 49% methanol-water to yield compounds 7 and 8, and a mobile phase of 52% methanol-water to yield compounds 5 and 6. The ratios of eluents or mobile phases described herein are all by volume.

[0019] Another embodiment of the present invention provides a method for preparing compounds 1-10, Formula II, and Formula V, characterized in that it comprises the following steps:

[0020] (1) Cultivating the marine fungus HK1-8 in a culture medium to obtain a seed solution;

[0021] (2) inoculating the seed solution obtained in step (1) into a fermentation medium for fermentation to obtain a fermentation product;

[0022] (3) Separating the fermentation broth and bacterial cells from the fermented product obtained in step (2), extracting the fermentation broth 2 to 4 times with an organic solvent A, combining the extracts and concentrating under reduced pressure to obtain a fermentation broth extract; extracting the bacterial cells with an organic solvent B 2 to 4 times, combining the extracts and concentrating under reduced pressure to obtain a bacterial cell extract; and combining the fermentation broth extract and the bacterial cell extract to obtain the crude extract.

[0023] (4) The crude extract obtained in step (3) is subjected to chromatographic separation to obtain compounds 1-10, Formula II, and Formula V. The chromatographic separation is preferably performed by one or more combinations of reduced pressure silica gel column chromatography, normal phase silica gel column chromatography, reverse phase silica gel column chromatography, gel column chromatography, HPLC, etc. The following scheme is preferred: the crude extract is first subjected to reduced pressure silica gel column chromatography, using petroleum ether-ethyl acetate as eluent for gradient elution, and is separated into several components (10 components (Fr.1-Fr.10)) according to polarity, and then subjected to Sephadex LH-20 gel column chromatography (eluent is CH2Cl2 / MeOH, v / v, 1:1), reverse phase column chromatography (ODS) (mobile phase: 80-40% methanol-water), semi-preparative high performance liquid chromatography (mobile phase: 40-80% methanol-water) or one or more combinations thereof to separate compounds 1-10, Formula II, and Formula V. The ratio of the eluent or mobile phase described in the present invention is all volume ratios.

[0024] The bacterial culture conditions and fermentation conditions (e.g., culture medium selection, temperature, time, and other parameters) in the preparation method of the present invention are all routine experimental operations in the art, and those skilled in the art can make reasonable choices based on experimental conditions. In order to facilitate understanding of the present invention, only one of the conventional operation methods is listed below: the bacterial culture medium in step (1) is a commonly used bacterial culture medium in the art, preferably PDB medium, and the formula (per liter of water) is preferably: 20 g / L glucose, 200 g / L potato, and 30 g / L sea salt; the bacterial culture in step (1) is selected from static culture or shaking culture, the temperature is preferably room temperature to 38°C, the shaking speed is preferably 120-150 r / min, and the culture time is preferably 2-4 days. The fermentation medium in step (2) is a commonly used fermentation medium in the art and may be the same as or different from the bacterial culture medium in step (1), preferably PDB medium, with a preferred formula (per liter of water): 20 g / L glucose, 150-300 g / L potato, and 30 g / L sea salt; the inoculation amount in step (2) is preferably 1-5%, and the fermentation culture is selected from static culture or shaking culture, the temperature is room temperature to 38°C, and the culture time is 28-30 days. The organic solvent A in step (3) is preferably one or more of ethyl acetate, dichloromethane, and chloroform; the organic solvent B is preferably one or more of methanol, ethanol, or acetone. After the fermentation broth extract and the bacterial extract are combined, a drying step is optionally performed to obtain a crude extract.

[0025] Another embodiment of the present invention provides the use of the above-mentioned marine fungus HK1-8 in preparing one or more compounds of Compounds 1-10, Formula II, and Formula V, especially in preparing one or more compounds of Compounds 1, 5-8, Formula II, and Formula V.

[0026] Another embodiment of the present invention provides a composition, characterized in that the composition uses the above-mentioned formula I, formula II, formula III formula IV, formula V, formula VI, its tautomers or pharmaceutically acceptable salts thereof as active ingredients. Preferably, compound 1-10, formula II, formula V, its tautomers or pharmaceutically acceptable salts thereof are used as active ingredients. Further preferably, compound 1, 5-8, formula II, formula V, its tautomers or pharmaceutically acceptable salts thereof are used as active ingredients. The composition also includes a pharmaceutical excipient (preferably a carrier, a diluent or an excipient, etc.). The dosage form of the composition is selected from solid preparations, liquid preparations, semi-solid preparations, etc. The composition is preferably used to prevent and treat diseases caused by agricultural pathogens. The agricultural pathogens are preferably rice sheath blight pathogens and wheat fusarium pathogens.

[0027] Another embodiment of the present invention provides the use of the above-mentioned Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, their tautomers, or pharmaceutically acceptable salts thereof in plant protection, especially in controlling agricultural pathogens. Preferred are compounds 1-10, Formula II, and Formula V, and further preferred are compounds 1, 5-8, Formula II, and Formula V. The agricultural pathogens are preferably rice sheath blight pathogens and wheat head blight pathogens.

[0028] Another embodiment of the present invention provides the use of the marine fungus HK1-8 in plant protection, especially in controlling agricultural pathogens, preferably rice sheath blight pathogens and wheat head blight pathogens.

[0029] Another embodiment of the present invention provides the use of the above-mentioned Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, their tautomers, or pharmaceutically acceptable salts thereof in the preparation of a medicament for controlling agricultural pathogens. Preferred are compounds 1-10, Formula II, and Formula V, and further preferred are compounds 1, 5-8, Formula II, and Formula V.

[0030] Another embodiment of the present invention provides the use of the marine fungus HK1-8 in preparing one or more compounds of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, their tautomers, or pharmaceutically acceptable salts thereof. Preferably, one or more compounds of Compounds 1-10, Formula II, and Formula V are used, and further preferably, one or more compounds of Compounds 1, 5-8, Formula II, and Formula V are used. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a flow chart for further separation and purification of the crude extract of marine fungus HK1-8.

[0032] Figure 2 It is the NOESY related signal diagram of compound 1.

[0033] Figure 3is the measured ECD spectrum of compound 1.

[0034] Figure 4 is the key HMBC of compound 1, 1 H- 1 H COSY correlation signal diagram.

[0035] Figure 5 It is the NOESY related signal diagram of the compound of formula II.

[0036] Figure 6 It is the ECD spectrum of compound 1 and formula II.

[0037] Figure 7 is the key HMBC of the compound of formula II, 1 H- 1 H COSY correlation signal diagram.

[0038] Figure 8 This is a possible biosynthetic pathway diagram of compound 1.

[0039] Figure 9 This is the key HMBC signal diagram of compound 5.

[0040] Figure 10 This is a molecular cluster diagram of the fungus HK1-8 azotoxin.

[0041] Figure 11 This is the secondary mass spectrometry fragmentation analysis diagram of compound 1.

[0042] Figure 12 This is the secondary mass spectrometry characteristic fragment analysis diagram of azotoxin compounds 2–4.

[0043] Figure 13 This is the structure prediction and analysis diagram of sesquiterpenes.

[0044] Figure 14 is compound 1 1 H NMR (600 MHz, CDCl3) chart.

[0045] Figure 15 is compound 1 13 C NMR (150 MHz, CDCl3) chart.

[0046] Figure 16 This is the high-resolution mass spectrum of compound 1.

[0047] Figure 17 is a compound of formula II 1 H NMR (600 MHz, CDCl3) chart.

[0048] Figure 18 is a compound of formula II 13C NMR (150 MHz, CDCl3) chart.

[0049] Figure 19 It is a high-resolution mass spectrum of the compound of formula II.

[0050] Figure 20 is compound 5 1 H NMR (600 MHz, CDCl3) chart.

[0051] Figure 21 is compound 5 13 C NMR (150 MHz, CDCl3) chart.

[0052] Figure 22 This is the high-resolution mass spectrum of compound 5.

[0053] Figure 23 is compound 6 1 H NMR (600 MHz, CDCl3) spectrum.

[0054] Figure 24 is compound 6 13 C NMR (150 MHz, CDCl3) spectrum. DETAILED DESCRIPTION

[0055] Example 1

[0056] (1) Cultivation of marine fungus strain HK1-8

[0057] The culture medium for the marine fungus HK1-8 is: 200g of boiled potato juice, 20g of glucose, and 30g of coarse sea salt per 1000mL of water. The mixture is divided into Erlenmeyer flasks. The culture is shaken at 35-38°C and 120 rpm for 3 days to obtain the seed solution.

[0058] (2) Fermentation of marine fungus HK1-8

[0059] The fermentation medium used for the fermentation of marine fungus HK1-8 consists of the following: 200g of boiled potato juice, 20g of glucose, and 30g of coarse sea salt per 1000mL of water. The mixture is divided into 50 1L Erlenmeyer flasks, each containing 400mL of PDB medium. The seed solution is inoculated into the medium in the Erlenmeyer flasks at an inoculum level of 1.0-1.5%. The fermentation is carried out at room temperature and incubated for 30 days to obtain a fermented product.

[0060] (3) Preparation of crude extract of marine fungus HK1-8

[0061] The fermented product obtained after fermentation and culturing in step (2) is taken, the fermentation broth and the bacterial cells are separated, the fermentation broth is extracted three times with ethyl acetate, and the extract is concentrated under reduced pressure to obtain a fermentation broth extract; the bacterial cells are extracted three times with methanol, and concentrated under reduced pressure to obtain a bacterial cell extract; the fermentation broth extract and the bacterial cell extract are combined to obtain the crude extract.

[0062] (4) Purification of compounds of formula I, formula II, formula III, formula IV, formula V, and formula VI

[0063] The crude extract obtained in step (3) was first subjected to reduced pressure silica gel column chromatography, using petroleum ether-ethyl acetate as eluent for gradient elution, the elution gradients were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 0:100, and 2-3 column volumes were collected for each gradient. The product was divided into 10 components (Fr.1-Fr.10) according to polarity, of which component Fr.3 was purified by Sephadex LH-20 gel column chromatography, with the eluent being a mixed solvent of CH2Cl2 / MeOH, v / v, 1:1, obtained three components (Fr.3.1-Fr.3.3). Component Fr.3.2 was separated by reverse phase column chromatography (mobile phase 60% methanol-water) and then by semi-preparative high performance liquid chromatography (mobile phase: 65% methanol-water) to obtain compound 1 (20.5 mg) and formula II (16.8 mg); component Fr.3.3 was first separated by reverse column chromatography (mobile phase 70% methanol-water) to obtain formula V (6.8 mg) and a mixture, and the mixture was then separated by semi-preparative high performance liquid chromatography, mobile phase: 49% methanol-water to obtain compounds 7 (5.8 mg) and 8 (5.6 mg), and mobile phase: 52% methanol-water to obtain compounds 5 (6.2 mg) and 6 (5.5 mg).

[0064] The structural characterization data of compounds 1, 5-8, Formula II, and Formula V are as follows. Based on one-dimensional and two-dimensional NMR, MS, ECD and other data, the structures of compounds 1, 5-8, Formula II, and Formula V can be determined as follows:

[0065]

[0066] Compound 1: It is a brown oil with obvious blue fluorescent spots under 365nm fluorescence. It turns green when heated with sulfuric acid-vanillin. High-resolution mass spectrometry shows HRESIMS m / z: [M+H] + =565.2796,[M+Na] + =587.2615, indicating that the molecular weight of the compound is 564.2 and the molecular formula is C 33 H 40O8, the degree of unsaturation is 14. The hydrogen spectrum signal of this compound shows two aromatic hydrogen signals or olefinic hydrogen signals δ H 6.77 (1H, s), 6.61 (1H, s); a group of mutually coupled low-field methylene signals 3.11 (1H, d, J = 17.2 Hz), 2.68 (1H, dd, J = 17.2, 9.6 Hz); a group of mutually coupled methylene and methyl signals 1.64 (2H, q, J = 7.2 Hz), 0.93 (3H, t, J = 7.2 Hz); a methylene singlet signal 3.47 (2H, s); two groups of methyl singlet signals 2.13 (3H, s), 1.00 (3H, s); its carbon spectrum signal shows two carbonyl signals δ C 209.8,198.3; three low-field olefin carbon signals 158.6,145.5,144.0; three olefin carbon signals 120.7,112.5,106.0; one oxygen-linked carbon signal 72.9; eight high-field carbon signals 44.7,40.3,39.2,21.2,19.9,19.5,17.5,13.9; through literature research, it was found that the hydrogen and carbon spectrum data of this compound are highly similar to those of Chermesinone A, and it is speculated that it is a nitrogen-acid ketone compound with a similar skeleton. The analysis of the two-dimensional spectrum further confirmed our guess: in 1 H- 1 In the H COSY spectrum, H-12 and H-13 are coupled to each other, and H-13 is coupled to H-12 and H-14 respectively, which indicates the existence of a n-propyl structural fragment; at the same time, in the HMBC spectrum, H-12 is coupled to C-11, and H-13 is also coupled to C-11, which indicates that the 11-position carbon of the keto carbonyl is connected to the 12-position carbon of the n-propyl; 1 H- 1 The H COSY spectrum shows that H-7 and H-10 are coupled to each other. At the same time, in the HMBC spectrum, H-7 and H-10 are coupled to C-6, and H-17 is coupled to C-6 and C-7, respectively. Thus, the structural fragment of -CH3CCHCH2- is determined. In the HMBC spectrum, H-1 is coupled to C-7 and C-8, respectively. At the same time, H-7 is coupled to C-6 and C-8, respectively. Thus, it is inferred that C-1 is directly connected to C-8, and C-8 is directly connected to C-7. In the HMBC spectrum, H-3 is coupled to C-2 and C-15, respectively, and H-15 is coupled to C-2, thus it is inferred that the 15-position carbon is directly connected to the 2-position carbon. Based on the above inferred structural fragments and further detailed analysis 1 H- 1The planar structure of compound 1 was confirmed using H COSY, HSQC, and HMBC spectra. Interestingly, the H-16 integral value was 1 when assigning the H-spectral data, indicating that the compound's structure is symmetrical about the C-16 axis, resulting in a half-valued H-signal integral, which is consistent with the high-resolution mass spectrometry data.

[0067] The relative configuration was determined by NOESY spectra. No correlation signals were found between H-7 and H-17, indicating that H-7 and H-17 were not in the same plane. However, correlation signals were found between H-10 and H-17, indicating that H-10 and H-17 were in the same plane, thus confirming the relative configuration of compound 1. The absolute configuration was determined by ECD comparison. Literature research identified a known compound with a similar structure, Mycoleptone A. Comparison of the measured ECD spectrum of compound 1 with this compound revealed a very similar ECD spectrum, leading to the determination of the absolute configuration of compound 1 as (6R, 7S, 6′R, 7′S), which was named Humicone A.

[0068] Assignment of NMR spectrum data of compound 1 (600, 150 MHz, CDCl3, TMS, δ ppm)

[0069]

[0070]

[0071]

[0072]

[0073] Compound II: A monomeric compound of compound 1, also a brown oil. It also exhibits a relatively obvious blue fluorescent spot at 365 nm. It turns light brown when heated with sulfuric acid-vanillin. High-resolution mass spectrometry shows HRESIMS m / z: [M+H] + =277.1433,[M+Na] + =299.1245, indicating that the molecular weight of the compound is 276.1 and the molecular formula is C 16 H 20 O4, the degree of unsaturation is 7. The hydrogen spectrum and carbon spectrum data of the compound of formula II are very similar to those of compound 1, and it is speculated that they are both azotoxin compounds. The hydrogen spectrum data of this compound show three olefinic hydrogen signals δ H6.88 (1H, s), 6.03 (1H, s), 5.46 (1H, s), one more olefinic hydrogen signal of 5.46 (1H, s) than compound 1, it is speculated that there is no substituent at the 4-carbon position; a group of mutually coupled low-field methylene signals 3.12 (1H, d, J = 20.4 Hz), 2.70 (1H, dd, J = 20.4, 10.2 Hz); two groups of high-field methylene signals 2.49 (2H, m), 1.62 (2H, m); three groups of methyl singlet signals 2.15 (3H, s), 1.18 (3H, s), 0.91 (3H, s); its carbon spectrum signal shows two carbonyl signals δ C 209.8,199.1; three low-field olefin carbon signals 159.4,149.5,145.7; three olefin carbon signals 120.9,107.7,104.5; one oxygen-linked carbon signal 72.8; seven high-field carbon signals 44.7,40.6,39.3,21.4,19.5,17.4,13.8; at the same time, in the HMBC spectrum, H-4 is coupled with C-3, C-5, C-8, and C-9, proving that there is no substituent at carbon 4. Based on the above inference and further detailed analysis 1 H- 1 The planar structure of the compound of formula II was determined based on H COSY, HSQC and HMBC spectra.

[0074] The relative configuration was determined by NOESY spectroscopy. No correlation signals were observed between H-7 and H-16, indicating that they were not coplanar. However, correlation signals were observed between H-10 and H-16, indicating that they were coplanar. This confirmed the relative configuration of the compound of Formula II. Its absolute configuration was determined by ECD comparison and further analysis of the biosynthetic pathway to be (6R, 7S), and it was named Humicone B.

[0075] The NMR spectrum data of the compound of formula II are assigned (600, 150 MHz, CDCl3, TMS, δ ppm)

[0076]

[0077]

[0078]

[0079] Compound 5: light yellow oil; high-resolution mass spectrometry showed HRESIMS m / z: [M+H] + =261.1109, indicating that the molecular weight of the compound is 260 and the molecular formula is C 15 H 16 O4, the degree of unsaturation is 8.1 The H NMR spectrum showed four methine signals δ H 4.50 (2H, t, J = 6.0 Hz), 3.52 (2H, m), 2.95 (2H, m), 2.90 (2H, t, J = 17.4 Hz), two methyl signals δ H 2.21 (3H, s), 1.43 (3H, s), its carbon spectrum gave six aromatic carbon signals, indicating that there may be a benzene ring in the structure, and a carboxyl carbon signal δ C 182.2 (C-15), a carbonyl carbon signal δ C 166.2 (C-7). After consulting the literature, it was found that the NMR data of this compound were very similar to those of echinolactone D. The only difference was that the methyl group at position 15 was oxidized to a carboxyl group. Combining high-resolution mass spectrometry data with two-dimensional NMR data, the planar structure of compound 5 was determined. Compound 5 contains a chiral carbon (C-12). Since compound 5 may be derived from the oxidation of C-15 of the isolated compound echinolactone D (6), it is speculated based on the biosynthetic pathway that compound 5 may have a 12S configuration. The new compound was named echinolactone E.

[0080] NMR spectrum data of compound 5 (600, 150 MHz, CDCl3, δ ppm)

[0081]

[0082]

[0083] Compound 6: light yellow oil. The hydrogen spectrum data of this compound gave three groups of chemically equivalent methine signals δ H 4.48 (2H, t, J = 6.0 Hz), 3.54 (2H, s), 2.94 (2H, t, J = 6.0 Hz), two groups of chemically unequal methine signals δ H 2.97 (1H, d, J = 16.8 Hz, H-1), 2.64 (1H, d, J = 16.8 Hz, H-1), 2.95 (1H, d, J = 16.2 Hz, H-11), 2.71 (1H, d, J = 16.2 Hz, H-11), two methyl signals δ H 2.19 (3H, s), 1.18 (3H, s), its carbon spectrum gave six aromatic carbon signals, indicating that there may be a benzene ring in the structure. After consulting the literature, it was determined that the structure was the known compound echinolactone D.

[0084]

[0085] Compound 7: Transparent oil. The hydrogen spectrum of this compound gave two methyl signals δ H 1.68 (3H, s), 0.92 (3H, s), four methine signals δ H 2.58 (2H, m), 2.25 (2H, m), 2.25 (2H, m), 1.49 (2H, m), and its carbon spectrum data gave a carbonyl carbon signal δ C 196.9 (C-8), a carboxyl carbon signal δ C 175.2 (C-11), two olefin carbon signals δ C 158.2 (C-4), 130.2 (C-9). After consulting the literature and comparing the spectral data, it was found that the structure is similar to the skeleton of organic acids 5-oxo-2-cyclohexenecarboxylic acid and 5-oxo-1-cyclohexenecarboxylic acid. In addition to the change in the position of the double bond, the structure also has additional side chains. By analyzing the structure 1 H- 1 The H COSY spectrum revealed that the hydrogens at positions C-1 and C-2, and C-2 and C-3 were mutually coupled, indicating that the structure may have a -CH2CH2CH3 side chain. Analysis of its HMBC spectrum revealed that C-4 was mutually coupled with the hydrogen at position 10, C-9 was mutually coupled with the hydrogen at position 3, and C-4 was mutually coupled with the hydrogens at positions 2 and 3. This determined the positions of the two side chains and the planar structure of compound 7.

[0086]

[0087] Compound 8: Transparent oil. The TLC and HPLC patterns of this compound are highly consistent with those of compound 7, which indicates that the structure of this compound is highly similar to that of compound 7. The hydrogen spectrum data of this compound showed the same two methyl signals δ H 1.06 (3H, m), 0.85 (3H, m), four methine signals δ H 2.58 (2H, m), 2.54 (2H, m), 2.26 (2H, m), 2.20 (2H, m), and its carbon spectrum data showed a carbonyl carbon signal δ C 196.6 (C-7), a carboxyl carbon signal δ C 175.1 (C-11), two olefin carbon signals δ C 158.2(C-3),130.2(C-8). Combined 1 H- 1The H COSY spectrum revealed that hydrogen at positions C-1 and C-2, and C-9 and C-10 were mutually coupled, indicating that the structure may have two -CH2CH3 fragments. Analysis of its HMBC spectrum revealed that C-3 was mutually coupled with hydrogen at positions 2 and 9, and C-8 was also mutually coupled with hydrogen at positions 2 and 9. This determined the positions of the two -CH2CH3 fragments and the planar structure of compound 8.

[0088] NMR spectrum data of compounds 7 and 8 (600, 150 MHz, CDCl3, δ ppm)

[0089]

[0090]

[0091]

[0092] Compound of formula V: light yellow oil, high resolution mass spectrometry shows HRESIMS m / z: [MH] + =211.0976, indicating that the molecular weight of the compound is 212 and the molecular formula is C 11 H 16 O4, the degree of unsaturation is 4. 1 The H NMR spectrum showed two methine signals δ H 1.91 (2H, m), 1.42 (2H, m), one methyl signal δ H 0.96 (3H, t, J = 7.2 Hz). Its carbon spectrum signal shows a carbonyl carbon signal δ C 172.5 (C-1), four olefin carbon signals δ C149.7(C-3),139.2(C-10),132.1(C-2),115.9(C-11). Analysis of its HSQC spectrum confirmed the presence of a pair of chemically inequivalent hydrogen signals at positions 8 and 11, respectively. Further analysis of its HMBC spectrum revealed mutual coupling between H-10 and H-11 and C-11, C-10, and C-9, respectively. The HH COSY spectrum showed coupling between H-10, H-11, and H-9, but no coupling between H-11 and H-9. H-9 and H-8 showed mutual coupling, indicating a double bond between C-10 and C-11, which is located at the end of the branch. Furthermore, H-7, H-6, and H-5 showed pairwise coupling, as well as coupling between H-7 and C-6 and C-5, H-6 and C-7, C-5, and C-4, and H-5 and C-7, C-6, C-4, and C-3, indicating the presence of a -CH2CH2CH3 fragment in the structure. Based on these characteristics, further literature review confirmed the planar structure of compound V.

[0093] NMR spectrum data of the compound of formula V (600, 150 MHz, CDCl3, δ ppm)

[0094]

[0095]

[0096] Example 2 Structural analysis and prediction of key molecular clusters

[0097] (1) Structural analysis and prediction of azotoxin molecular clusters

[0098] Using the isolated dimer humicone A (1) as a "seed compound," we discovered a small humicone cluster formed with its parent ion node, consisting of six nodes. Based on the characteristic fragmentation signals of the secondary mass spectrometry of each node, we analyzed and inferred the structures of the humicone corresponding to some of the nodes in this cluster and successfully predicted the structures of three new humicone compounds (2-4).

[0099] MS / MS fragmentation peaks of azotoxin compounds

[0100]

[0101] Taking the isolated compound humicone A(1) as an example, the secondary fragmentation patterns of azotoxin structures were analyzed. First, the fragment peak at m / z 289(2c) has the highest relative abundance. This fragment is half of a complete symmetrical structure, indicating that this type of structure usually splits into two after impacting an inert gas, and the fragments are relatively stable. m / z 299(2b) is the peak of Na addition of half of this symmetrical structure, and m / z 271(2d) is the product of 2c after removing a molecule of water. These three fragment peaks are characteristic peaks of this type of structure and appear in the secondary mass spectra of the remaining nodes. The remaining fragment peaks with lower relative abundance are fragments of some methylmethine groups. The dropping of these fragments is highly random and therefore not characteristic.

[0102] Referring to the MS / MS fragmentation pattern of humicone A(1), the MS / MS characteristic peaks of the three nodes of this molecular cluster, m / z 587.262, 603.256, and 605.272, were compared and analyzed. By observing the secondary mass spectra, we can find that all three groups of spectra have a set of highly retained stable characteristic fragment peaks at m / z 299, 289, and 271, indicating that all three compounds have a monomer that is the same as humicone A(1), and the difference occurs in the side chain of the other monomer. Based on the mass differences between the nodes and the biosynthetic pathway of azotoxin compounds, the chemical structures of these three nodes were finally determined and named humicones C–E.

[0103] (2) Structural analysis and prediction of sesquiterpene molecular clusters

[0104] The overall molecular network diagram shows a sesquiterpenoid cluster with three nodes. The MS / MS fragmentation analysis of the sesquiterpenoid structure was performed using the node with a charge-mass ratio of m / z 231 as an example. Secondary mass spectrometry revealed that this structure initially fragments at the C-8 position and then decarboxylates, yielding a fragment with m / z 185. This fragment may then undergo multiple C1 and C2 fragments, ultimately forming a relatively stable fragment containing a benzene ring. The structural predictions for the remaining two nodes were based on the node with a charge-mass ratio of m / z 231. This cluster contains two new compounds (9-10).

[0105] MS / MS fragment peaks of sesquiterpenoids 9-11

[0106]

[0107] Example 3 Evaluation of activity against plant pathogenic fungi

[0108] The mycelial growth rate method was used to test the antifungal activity of compounds of Formula I, Formula II, Formula III, Formula IV, Formula V, and Formula VI (preferably 1-10, Formula II, and Formula V, and further preferably 1, 5-8, Formula II, and Formula V) against rice sheath blight pathogens and wheat fusarium pathogens. The blank control was performed using PDA medium supplemented with 2 mL of DMSO.

[0109] Specific steps:

[0110] Prepare the plate: Dissolve the sample to be tested in DMSO to prepare a 5 mg / mL solution, then add it to a certain amount of sterilized PDA culture medium to obtain a PDA culture medium with a drug concentration of 200 mg / L. At the same time, prepare a blank culture medium with DMSO added. After preparation, pour the solution into 90 mm diameter culture dishes while it is hot, about 8 mL per culture dish, and wait for solidification before use.

[0111] Initial screening: Place a 5 mm diameter cake of the plant pathogenic fungus to be tested in the center of a spare treated petri dish and a blank control petri dish in triplicate. Incubate at (25 ± 0.5)°C for 2–7 days. When the cake in the blank control petri dish has completely grown, measure the mycelial diameter of each dish using the cross-hatch method and calculate the inhibition rate.

[0112] Inhibition rate (%) = [(control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter)] × 100%

[0113] The results showed that at a concentration of 200 mg / L, all test compounds exhibited strong antibacterial activity against rice sheath blight pathogen and wheat fusarium spp. pathogen (inhibition rates were all greater than 15%), especially compound 1, which had an inhibition rate of 100% against rice sheath blight pathogen and wheat fusarium spp. pathogen.

[0114] Rescreening: Compound 1 was rescreened and its concentration was prepared into 50 mg / L, 25 mg / L, and 10 mg / L PDA culture medium, and the inhibition rate of compound 1 on mycelium was measured again by the above method, and repeated three times.

[0115] The results showed that when the concentration was 10 mg / L, the inhibition rate against rice sheath blight pathogen was 45.12%, and the inhibition rate against wheat fusarium spp. pathogen was 40.23%. When the concentration was 25 and 50 mg / L, the growth of rice sheath blight pathogen and wheat fusarium spp. pathogen was completely inhibited (inhibition rate 100%).

Claims

1. A marine fungus HK1-8, characterized in that The name of the strain preservation unit is: General Microbiology Center of China Microorganism Culture Collection Administration, and the preservation number is: CGMCC No.40667.

2. A secondary metabolite, a tautomer thereof or a pharmaceutically acceptable salt thereof, characterized in that The secondary metabolites have the structures shown in Formula I, Formula II, Formula III, Formula IV, Formula V, and Formula VI: Wherein R1, R2, R3, and R4 are each independently selected from H, OH, or R1 and R2 are both O (i.e., forming a carbonyl group, =O); R5 is selected from methyl, carboxyl, hydroxyl, and hydroxymethyl; R6 is selected from C1-C3 alkyl (preferably methyl, ethyl); R6 is selected from H, C1-C3 alkyl acyl (preferably Ac).

3. The secondary metabolite, its tautomer or its pharmaceutically acceptable salt according to claim 2, characterized in that The structure shown in formula I is preferably the following compound 1-4: The preferred compound 5-6 is shown in formula III: The preferred compound 7-8 is shown in Formula IV: Preferred compounds 9-10 are represented by the structure of formula VI:

4. A method for preparing compounds 1-10, formula II, and formula V according to any one of claims 2-3, characterized in that The steps include: (1) Cultivating the marine fungus HK1-8 in a culture medium to obtain a seed solution; (2) inoculating the seed solution obtained in step (1) into a fermentation medium for fermentation to obtain a fermentation product; (3) Separating the fermentation broth and bacterial cells from the fermented product obtained in step (2), extracting the fermentation broth 2 to 4 times with an organic solvent A, combining the extracts and concentrating under reduced pressure to obtain a fermentation broth extract; extracting the bacterial cells with an organic solvent B 2 to 4 times, combining the extracts and concentrating under reduced pressure to obtain a bacterial cell extract; and combining the fermentation broth extract and the bacterial cell extract to obtain the crude extract. (4) The crude extract obtained in step (3) is subjected to chromatographic separation to obtain compounds 1-10, Formula II, and Formula V. The chromatographic separation is preferably performed by one or more combinations of reduced pressure silica gel column chromatography, normal phase silica gel column chromatography, reverse phase silica gel column chromatography, gel column chromatography, HPLC, etc. The following scheme is preferred: the crude extract is first subjected to reduced pressure silica gel column chromatography, using petroleum ether-ethyl acetate as eluent for gradient elution, and is separated into several components (10 components (Fr.1-Fr.10)) according to polarity, and then subjected to Sephadex LH-20 gel column chromatography (eluent is CH2Cl2 / MeOH, v / v, 1:1), reverse phase column chromatography (ODS) (mobile phase 80-40% methanol-water), semi-preparative high performance liquid chromatography (mobile phase: 40-80% methanol-water) or one or more combinations thereof to separate compounds 1-10, Formula II, and Formula V.

5. A method for preparing compounds 1, 5-8, formula II, and formula V according to any one of claims 2 to 3, characterized in that The steps include: (1) Cultivating the marine fungus HK1-8 in a culture medium to obtain a seed solution; (2) inoculating the seed solution obtained in step (1) into a fermentation medium for fermentation to obtain a fermentation product; (3) Separating the fermentation broth and bacterial cells from the fermented product obtained in step (2), extracting the fermentation broth 2 to 4 times with an organic solvent A, combining the extracts and concentrating under reduced pressure to obtain a fermentation broth extract; extracting the bacterial cells with an organic solvent B 2 to 4 times, combining the extracts and concentrating under reduced pressure to obtain a bacterial cell extract; and combining the fermentation broth extract and the bacterial cell extract to obtain the crude extract. (4) The crude extract obtained in step (3) is subjected to chromatographic separation to obtain compounds 1, 5-8, Formula II, and Formula V. The chromatographic separation is preferably performed by one or more combinations of vacuum silica gel column chromatography, normal phase silica gel column chromatography, reverse phase silica gel column chromatography, gel column chromatography, HPLC, etc. The following scheme is preferred: the crude extract is first subjected to vacuum silica gel column chromatography, and gradient elution is performed using petroleum ether-ethyl acetate as an eluent. The elution gradients are 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively. 2-3 column volumes are collected for each gradient and the components are divided into 10 fractions (Fr.1-Fr.10) according to their polarity. Among them, component Fr.3 is subjected to Sephadex chromatography. LH-20 gel column chromatography, with the eluent being a mixed solvent of CH2Cl2 / MeOH, v / v, 1:1, obtained three components (Fr.3.1-Fr.3.3). Component Fr.3.2 was separated by reverse phase column chromatography (ODS) with a mobile phase of 60% methanol-water, and then subjected to semi-preparative high performance liquid chromatography with a mobile phase of 65% methanol-water to obtain compound 1 and formula II; component Fr.3.3 was first separated by reverse column chromatography with a mobile phase of 70% methanol-water to obtain formula V and a mixture, and the mixture was then subjected to semi-preparative high performance liquid chromatography with a mobile phase of 49% methanol-water to obtain compounds 7 and 8, and compounds 5 and 6 were separated by a mobile phase of 52% methanol-water.

6. Use of the marine fungus HK1-8 according to claim 1 in the preparation of one or more compounds 1-10 according to any one of claims 2-3, or compounds of formula II or formula V, especially in the preparation of one or more compounds of compounds 1, 5-8, or formula II or formula V.

7. A composition characterized in that The composition uses Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, or tautomers thereof or pharmaceutically acceptable salts thereof according to claim 2 as an active ingredient. Preferably, Compound 1-10, Formula II, Formula V, or tautomers thereof or pharmaceutically acceptable salts thereof according to claim 3 are used as active ingredients. Further preferably, Compound 1, 5-8, Formula II, Formula V, or tautomers thereof or pharmaceutically acceptable salts thereof according to claim 3 are used as active ingredients. The composition further includes a pharmaceutical excipient (preferably a carrier, a diluent, or an excipient, etc.). The dosage form of the composition is selected from solid preparations, liquid preparations, semisolid preparations, etc. The composition is preferably used to prevent and treat diseases caused by agricultural pathogens. The agricultural pathogens are preferably rice sheath blight pathogens and wheat fusarium pathogens.

8. Use of the compound of formula I, formula II, formula III, formula IV, formula V, formula VI, their tautomers, or pharmaceutically acceptable salts thereof according to claim 2 in plant protection, especially in controlling agricultural pathogens. Application in the preparation of medicines for preventing and controlling agricultural pathogens. Preferred Compounds 1-10, Formula II, and Formula V according to claim 3 are further preferably compounds 1, 5-8, Formula II, and Formula V.

9. Use of the marine fungus HK1-8 according to claim 1 in plant protection, especially in controlling agricultural pathogens.

10. Use of the marine fungus HK1-8 according to claim 1 in preparing the compounds of formula I, formula II, formula III, formula IV, formula V, formula VI, or their tautomers or pharmaceutically acceptable salts according to claim 2. Compounds 1-10, formula II, and formula V are preferred, and compounds 1, 5-8, formula II, and formula V are further preferred.

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