Biphenyl-diketopiperazine indole alkaloid hybrid compound as well as preparation method and application thereof

Abstract

The invention discloses a biphenyl-diketopiperazine indole alkaloid hybrid compound, which is separated from a fermentation product of a strain Aspergillus candidus HNNU0546, and has a skeleton molecule formed by hybridizing rare diketopiperazine indole alkaloid with biphenyl molecules through an isopentenylation branch chain, and the biphenyl compound can obviously inhibit the activity of crop disease fungi.

Description

[0001] A biphenyl-dikepiperazine-indole alkaloid hybrid compound, its preparation method and application Technical Field

[0002] This invention relates to the field of biotechnology, specifically to a biphenyl-dikepiperazine-indole alkaloid hybrid compound, its preparation method, and its application in the development of pesticides against fungal diseases affecting crops. Background Technology

[0003] Marine fungi can produce novel secondary metabolites, including alkaloids, peptides, polyketides, steroids, and terpenes, many of which possess significant antibacterial, antitumor, antiviral, and antifouling activities. The search for bioactive compounds from fungi is a hot topic in natural drug development. Marine-derived indole-dikepiperazine alkaloids, with their novel molecular structure and broad range of activities, possess excellent potential for pharmaceutical development and have attracted widespread attention from chemists.

[0004] The applicant disclosed in Chinese patent document CN120004833A (application number 202510119650.6) a fungus derived from marine cold seep sediments. Aspergillus candidus A class of biphenyl compounds was isolated from the fermentation broth of HNNU0546. These biphenyl compounds exhibited significant activity against *Pseudomonas aeruginosa* (potato early blight) and *Pseudomonas aeruginosa* (pineapple black heart rot), showing promising application prospects in the preparation of pesticides against crop diseases and fungi. The applicant, in its research... Aspergillus candidus Further research on the HNNU0546 fermentation broth yielded the biphenyl-dikepiperazine-indole alkaloid hybrid compound of this application. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a fungus of marine origin. Aspergillus white A biphenyl-dionepiperazine indole alkaloid hybrid compound isolated from the fermentation product of HNNU0546 exhibits the effect of inhibiting the growth of pathogenic fungi in crops.

[0006] The technical solution to achieve the first objective of this invention is a biphenyl-diketopiramine-indole alkaloid hybrid compound with the following structural formula: R is H or OH.

[0007] The technical solution to achieve the second objective of this invention is the application of the compound described above in inhibiting fungal diseases affecting crops.

[0008] The technical solution for achieving the third objective of this invention is a pesticide composition comprising at least one of the compounds described above, and an acceptable carrier for the pesticide.

[0009] The technical solution to achieve the fourth objective of this invention is a method for preparing the biphenyl-diketopiramine indole alkaloid hybrid compound as described above, from strains Aspergillus candidus Compound 1, with R being H, and compound 2, with R being OH, were isolated from the fermentation product of HNNU0546; Aspergillus candidus HNNU0546 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 65074, and the deposit date is August 29, 2024.

[0010] The preparation method of the biphenyl-diketopiramine indole alkaloid hybrid compound includes the following steps: ① Activation and fermentation of bacterial strains; Aspergillus candidus HNNU0546 was activated, cultured, and fermented to obtain fermentation product.

[0011] ② The fermentation product is extracted with ethyl acetate or macroporous adsorption resin, and then concentrated to obtain crude extract.

[0012] ③ The crude extract obtained in step ② was fractionated by silica gel column chromatography under normal pressure. The eluent was petroleum ether-ethyl acetate, chloroform-acetone, or chloroform-methanol solvent system, and the elution was carried out in a gradient from 100:0 to 0:100 (v / v). The fractions were tracked and combined by thin-layer chromatography (TLC). The fractions that could be developed on TLC with chloroform-acetone solvent system of 9:1 (v / v) or chloroform-methanol solvent system of 20:1 (v / v) were further subjected to silica gel column chromatography to obtain crude products. The crude products were then purified by high performance liquid chromatography to obtain compound 1 and compound 2.

[0013] In step ① above, the strain Aspergillus candidus HNNU0546 was inoculated onto PDA agar medium to obtain a plate containing the bacterial strain. Then, a single colony was selected from the plate using an inoculation loop or bamboo stick and inoculated into an Erlenmeyer flask containing PDB seed culture medium. The flask was then cultured on a shaker to obtain the seed culture. The seed culture was then inoculated into bottles containing PDB fermentation medium and allowed to stand to obtain the fermentation product.

[0014] As a priority, in step ① above, the strain Aspergillus candidus HNNU0546 was inoculated into PDA agar medium supplemented with 3% artificial sea salt and activated by incubation at 28 °C for 3 days to obtain plates containing the strain. The strain was then inoculated into sterile fresh PDB seed culture medium supplemented with 3% sea salt and incubated on a shaker at 200 rpm and 28 °C for 3 days to obtain the seed culture. The seed culture was then inoculated into PDB fermentation medium supplemented with 3% sea salt and incubated statically at room temperature for 28 days.

[0015] When preparing the PDA agar medium, wash and peel 200 g of potatoes, cut them into slices and boil them in water. After the water boils, boil for 30 minutes, filter with gauze to remove the residue, add 20 g of glucose, 30 g of sea salt and 20 g of agar to the filtrate, and finally adjust the volume to 1 liter.

[0016] When preparing the PDB culture medium, wash and peel 200 g of potatoes, cut them into slices and boil them in water. After the water boils, boil for 30 minutes, filter with gauze to remove the residue, add 20 g of glucose and 30 g of sea salt to the filtrate, and finally adjust the volume to 1 L.

[0017] In step ② above, the fermentation product is extracted three times with ethyl acetate, each time for 2-3 days, and the extracts are combined and concentrated under reduced pressure to obtain crude extract.

[0018] In step ③ above, the fractions that can be developed in thin-layer chromatography using a chloroform-acetone solvent system with a volume ratio of 9:1 or a chloroform-methanol solvent system with a volume ratio of 20:1 are subjected to gradient elution using a pure chloroform-methanol system of 100:0-0:100 as the eluent, and similar fractions are combined to obtain 5 subfractions Fr.1-Fr.5.

[0019] Subfraction Fr.4 was purified by silica gel column chromatography using a chloroform-methanol solvent system of 15:1-0:100 to obtain four subfractions: Fr.4-1, Fr.4-2, Fr.4-3 and Fr.4-4.

[0020] Subfraction Fr.4-3 was subjected to silica gel column chromatography and developed in a chloroform-acetone solvent system with a volume ratio of 6:4 to obtain four subfractions: Fr.4-3-1, Fr.4-3-2, Fr.4-3-3 and Fr.4-3-4.

[0021] The sub-fraction Fr.4-3-3 was purified by high-performance liquid chromatography (HPLC) with acetonitrile-water in a mobile phase of 45:55. Compounds 2 and 1 were collected at elution times of 17 min and 31 min, respectively.

[0022] Alternatively, during silica gel column chromatography, the stationary phase can be 100-200 mesh silica gel.

[0023] This invention has positive effects: This invention is derived from marine fungi Aspergillus candidus A novel biphenyl-diketopiramate-indole alkaloid hybrid compound was isolated from the fermentation broth of HNNU0546. This compound possesses a rare biphenyl-based skeletal structure, with the diketopiramate-indole alkaloid hybridized with a biphenyl molecule via isopentenylated branches. This biphenyl compound significantly inhibits the activity of crop pathogenic fungi. Compound 1 (R = H) showed activity against *Phytophthora blight* (potato early blight pathogen). Alternaria sp.) has significant antifungal activity, EC 50 The value is 19.21µ M, compared with the positive control difenoconazole (EC) 50 = 3.22 µ Compound 2 is at the same level as M, and is effective against early blight of potato (M). Alternaria sp.) exhibits weak antibacterial activity, with an inhibition zone of 6 mm at 20 μg / disc, EC. 50 The value is 86.42 µ M. Attached Figure Description

[0024] Figure 1 NMR of compound 1 1 H spectrum.

[0025] Figure 2 NMR of compound 1 13 C-spectrum.

[0026] Figure 3 This is the high-resolution mass spectrum of compound 1.

[0027] Figure 4 NMR of compound 2 1 H spectrum.

[0028] Figure 5 NMR of compound 2 13 C spectrum.

[0029] Figure 6 This is the high-resolution mass spectrum of compound 2.

[0030] Figure 7 Compounds 1 and 2 in the test examples, as well as the positive control difenoconazole, are effective against early blight of potato (Bacillus thuringiensis). Alternaria Comparison chart of the inhibitory effects of sp. Detailed Implementation

[0031] The following describes some of the possible embodiments of the present invention, intended to provide a basic understanding of the invention, and is not intended to identify the key or decisive elements of the invention or limit the scope of protection. It is readily understood that, based on the technical solutions of the present invention, those skilled in the art can propose other interchangeable implementations without altering the essential spirit of the invention. Therefore, the following specific embodiments are merely illustrative examples of the technical solutions of the present invention and should not be considered as the entirety of the invention or as limitations or restrictions on the technical solutions of the present invention.

[0032] (Example 1) ① Microbial activation and fermentation.

[0033] In this embodiment, the bacteria used for fermentation to produce biphenyl-diketopiramine-indole alkaloid hybrid compounds were strains isolated and identified from deep-sea cold seep sediments. Aspergillus candidus HNNU0546 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC NO: 65074.

[0034] During fermentation culture Aspergillus candidus HNNU0546 was inoculated into a culture medium suitable for Aspergillus fungi, and fermentation broth was prepared under normal fermentation conditions.

[0035] The culture media include PDA agar medium, PDB seed culture medium and PDB fermentation medium.

[0036] The PDA agar medium is prepared as follows: Wash and peel 200 g of potatoes, slice them, and boil them in water for 30 minutes after the water boils. Filter the liquid through gauze to remove residue. Add 20 g of glucose, 30 g of sea salt, and 20 g of agar to the filtrate, and finally bring the volume to 1 liter. Autoclave at 121 °C for 25 minutes and set aside.

[0037] The method for preparing the PDB seed culture medium is as follows: Wash and peel 200 g of potatoes, slice them, and boil them in water for 30 minutes after the water boils. Filter the solution through gauze to remove residue. Add 20 g of glucose and 30 g of sea salt to the filtrate, and finally adjust the volume to 1 L. PDB culture medium is then poured into 1-3 500 mL conical containers, approximately 150 mL per container, and autoclaved at 121 °C for 25 minutes.

[0038] The method for preparing the PDB fermentation medium is as follows: Wash and peel 200 g of potatoes, slice them, and boil them in water for 30 minutes after the water boils. Filter the liquid through gauze to remove the residue. Add 20 g of glucose and 30 g of sea salt to the filtrate, and finally adjust the volume to 1 L. PDB medium is then poured into 1-3 500 mL Erlenmeyer flasks, approximately 400 mL per flask, and autoclaved at 121 °C for 25 minutes.

[0039] Use an inoculation loop or bamboo skewer to remove the fungus from the slant. Aspergillus candidus The HNNU0546 strain was inoculated onto PDA agar medium and cultured at 28 °C for 3 days to obtain a plate containing the strain. Then, a single colony was selected from the plate using an inoculation loop or bamboo stick and inoculated into an Erlenmeyer flask containing 150 mL of PDB seed culture medium. The flask was then cultured on a shaker at 200 rpm and 28 °C for 3 days to obtain the seed culture. Next, 5 mL of the seed culture was inoculated into each flask containing 400 mL of PDB fermentation medium. After static culture at room temperature (28 °C) for 28 days, the flasks were collected to obtain the fermentation product.

[0040] ② The fermentation product was extracted three times with 50L ethyl acetate, each time for 2-3 days. The extracts were combined and concentrated under reduced pressure (temperature 40~50℃, pressure -0.06 MPa to -0.15MPa) to obtain 20 g of crude extract.

[0041] Alternatively, it can be extracted using macroporous adsorption resin and then concentrated to obtain a crude extract.

[0042] ③Purification and preparation.

[0043] The crude extract obtained in step ② was fractionated by silica gel column chromatography under normal pressure, with 100-200 mesh normal-phase silica gel as the stationary phase.

[0044] Gradient elution was performed successively using petroleum ether-ethyl acetate (90:10-50:50), chloroform-methanol (90:10-0:100), and chloroform-acetone (90:10-0:100) as eluents, and the combined components were tracked by thin-layer chromatography analysis.

[0045] The initial sample could be collected by elution using a chloroform-methanol system (20:1) or a chloroform-acetone system (9:1). TLC analysis showed that when developed using a chloroform-methanol system (20:1 v / v), the R values ​​of compounds 1-2 were... f The values ​​are 0.4–0.7. When developed using pure chloroform or chloroform-acetone (9:1), the R values ​​for compounds 1–2 are... f The value was 0.3. In other systems, chloroform-acetone (8:2) or chloroform-methanol (9:1) showed significant tailing of compounds 1-2 after development, indicating poor spot formation, making them unsuitable for elution. After solvent development, the sample was observed under a 254 nm UV lamp. Subsequent development with 5% concentrated sulfuric acid-vanillin revealed distinct purple spots at the sample location. The initial sample containing samples 1-2 was further prepared using high-performance liquid chromatography (HPLC) and eluted with an acetonitrile-water solvent system to obtain compounds 1-2.

[0046] The further separation and purification operations in this embodiment are as follows: The fraction (18.6 g) that could be developed on thin-layer chromatography using a chloroform-methanol solvent system with a volume ratio of 20:1 was subjected to silica gel column chromatography (stationary phase: 100-200 mesh silica gel; alternatively, the fraction that could be developed on a chloroform-acetone solvent system with a volume ratio of 9:1 could be further subjected to silica gel column chromatography). Gradient elution was performed using a pure chloroform-methanol system (100:0-0:100) as the eluent, collecting approximately 500 mL fractions each time. Similar fractions were combined by TLC to obtain five subfractions Fr.1-Fr.5. For TLC analysis, a chloroform-methanol mixed solvent with a volume ratio of 9:1 was used as the developing solvent.

[0047] Subfraction Fr.4 was subjected to silica gel column chromatography using a chloroform-methanol solvent system (15:1-0:100) (fractions were collected in approximately 500 mL increments, and similar fractions were combined by TLC analysis using a chloroform-methanol mixed solvent with a volume ratio of 9:1 as the developing solvent), yielding four subfractions: Fr.4-1, Fr.4-2, Fr.4-3, and Fr.4-4.

[0048] Subfraction Fr.4-3 was subjected to silica gel column chromatography, developed with a chloroform-acetone solvent system at a volume ratio of 6:4. Fractions were collected in increments of approximately 500 mL each time, and similar fractions were combined by TLC analysis. During TLC analysis, a chloroform-methanol mixed solvent at a volume ratio of 9:1 was used as the developing solvent, yielding four subfractions: Fr.4-3-1, Fr.4-3-2, Fr.4-3-3, and Fr.4-3-4.

[0049] The subfraction Fr.4-3-3 was purified by high-performance liquid chromatography (HPLC) and prepared by HPLC (detection wavelengths of 210 / 230 / 254 / 280 nm, flow rate of 2 mL / min, column of Agilent Eclipse XDB-C18 (9.4 × 250 mm, 5 µm), mobile phase of acetonitrile-water at a volume ratio of 45:55). Compounds 2 and 1 were collected at elution times of 17 min and 31 min, respectively.

[0050] The structures of the prepared compounds were confirmed, and the nuclear magnetic resonance (NMR) of compound 1 was analyzed. 1 See H spectrum Figure 1 Nuclear magnetic resonance 13 See the C spectrum. Figure 2 High-resolution mass spectra can be found in [the image]. Figure 3 Nuclear magnetic resonance of compound 2 1 See H spectrum Figure 4 Nuclear magnetic resonance 13 See the C spectrum. Figure 5 High-resolution mass spectra can be found in [the image]. Figure 6 The proton and carbon NMR spectra of compounds 1-2 are shown in Tables 1 and 2, respectively. Their structural analyses are as follows.

[0051] Table 1. Compound 1 1 H NMR (700 MHz) and 13 C NMR (175 MHz) data (DMSO- d 6) The structural analysis of the new compound 1 is as follows: pass Figure 3 High-resolution mass spectrometry (HRESIMS) in m / z805.2874 [M+H] + The presence of a quasi-molecular ion peak indicates that the molecular formula of compound 1 is C1. 47 H 40 N4O9. 1 H-NMR showed signals from three active amino protons ( δ H 11.2, 7.80, 6.80), 3 active hydroxyl proton signals ( δ H 9.80, 9.60, 6.06), 16 olefin proton signals ( δ H 7.55, 7.45, 7.31, 7.30, 7.19, 7.17, 7.05, 7.03, 7.00, 6.87, 6.75, 6.72, 6.67, 6.55), 3 methylene proton signals ( δ H 5.34, 4.68, 4.50), 3 methylene proton signals ( δ H 3.82 / 3.63, 3.38 / 3.11, 2.46 / 1.85), 2 methoxy proton signals ( δ H 3.96, 3.75), 1 methyl proton signal ( δ H 1.75). 13 C-NMR and DEPT135 show two amide carbons ( δ C 169.8, 167.7), 34 unsaturated carbons ( δ C 156.8, 151.0, 148.9, 148.8, 148.4, 143.6, 141.8, 136.0, 133.9, 133.5, 131.1, 131.0, 130.4×2, 129.0, 128.5, 128.4, 124.4, 124.1, 122.5, 120.2, 119.3(C-1''), 118.8, 118.7, 118.4, 117.8, 115.2 ×2, 113.6, 111.0, 110.1, 109.9, 105.5, 97.8), 2 oxygen-linked quaternary carbons ( δ C 88.1, 85.9), 3 methines ( δ C 84.0, 58.7, 55.1), 2 methoxy groups ( δC 60.7, 55.9), 3 methylene groups ( δ C 44.4, 41.4, 24.8), 1 methyl group ( δ C 26.2). In the HMBC spectrum, H-9' is correlated with C-7' / C-11', H-10' with C-8' / C-12', H-11' with C-7' / C-9', H-5' with C-3' / C-4' / C-7' / C-12', H-3' with C-4' / C-5' / C-12' / C-13', H-2' with C-3' / C-4' / C-13', and H-2 with C... -3 / C-13 is correlated, H-32 is correlated with C-2 / C-4 / C-5 / C-12 / C-13, H-6 is correlated with C-4 / C-8 / C-10, H-7 is correlated with C-5 / C-9, H-8 is correlated with C-6 / C-10, H-9 is correlated with C-5 / C-7, and H-12 is correlated with C-2 / C-3 / C-4 / C-5 / C-10 / C-13', thus yielding the indole-dione-piperazine unit. Two sets of equivalent aromatic proton signals are present in the proton spectrum ( δ H 7.45, 6.87) indicates the presence of a 1,4-para-substituted aromatic unit. H-14'' is associated with C-6" / C-16" / C-18"; H-5'' is associated with C-3" / C-4" / C-6" / C-7" / C-13"; 19"-OMe is associated with C-4"; 20"-OMe is associated with C-7"; H-10'' is associated with C-2" / C-12"; H2-18' is associated with C-1" / C-2" / C-12" / C-15' / C-16' / C-17'; H-17' is associated with C-15' / C-16' / C-18'; H-15' is associated with C-8' / C-14' / C-16' / C-17' / C-18'; H-14' is associated with C-7'. The correlation between C-9', C-15', and C-16', combined with the remaining two degrees of unsaturation, yields a biphenyl unit, which is attached to the C-8' atom. In the COSY spectrum, H-2 is correlated with H2-3, H-6 / H-7 / H-8 / H-9, H-2' with H2-3', H-5' with H-6', H-9' / H-10' / H-11', H-14' with H-15', H-14'' with H-15'', and H-17'' with H-18''. Its stereoconfiguration was determined through theoretical chemical calculations, DP4+ analysis, and ECD calculations. Compound 1 is a brown solid, readily soluble in chloroform, acetone, and DMSO, and sparingly soluble in water.

[0052] Table 2. Compound 2 1H NMR (600 MHz) and 13 C NMR (150 MHz) data (DMSO- d 6) The structure of compound 2 is analyzed as follows: pass Figure 6 High-resolution mass spectrometry (HRESIMS) in m / z 821.2816 [M+H] + The presence of a quasi-molecular ion peak indicates that the molecular formula of compound 2 is C2. 47 H 40 N4O 10 . 1 The H-NMR spectrum showed two active amino proton signals ( δ H 11.12, 7.81), 4 active hydroxyl proton signals ( δ H 9.80, 9.06×2, 6.06), 15 olefin proton signals ( δ H 7.54, 7.31, 7.30, 7.29, 7.18, 7.17, 7.03, 7.02, 7.00, 6.89, 6.82, 6.75, 6.72, 6.66, 6.54), 3 methylene proton signals ( δ H 5.32, 4.68, 4.50), 3 methylene proton signals ( δ H 3.81 / 3.62, 3.38 / 3.10, 2.45 / 1.85), 2 methoxy proton signals ( δ H 3.95, 3.74), 1 methyl proton signal ( δ H 1.74). 13 C-NMR and DEPT135 show two amide carbons ( δ C 169.8, 167.7), 34 unsaturated carbons ( δ C151.0, 148.9, 148.7, 148.4, 144.9×2, 143.6, 141.8, 136.0, 133.9, 133.5, 131.3, 131.1, 129.0×2, 128.4, 124.4, 124.1, 122.5, 120.4, 120.2, 119.2, 118.9, 118.7, 118.4, 117.8, 116.8, 115.5, 113.5, 111.1, 110.1, 109.9, 105.5, 97.8), 2 oxygen-linked quaternary carbons ( δ C 88.1, 85.9), 3 methylene carbons ( δ C 84.0, 58.7, 55.1), 2 methoxy groups ( δ C 60.7, 55.8), 3 methylene groups ( δ C 44.4, 41.4, 24.8), 1 methyl group ( δ C 26.1). Compound 2 is very similar to compound 1, except that it has one more hydroxyl group ( ). δ H 9.06), HMBC spectroscopy showed a correlation between H-14'' and C-17'', and H-15'' also a correlation between H-15'' and C-17'', indicating that compound 2 is a derivative of compound 1 in which the C-17'' is substituted with a hydroxyl group. Its stereoconfiguration was determined by theoretical chemical calculations, DP4+ analysis, and ECD calculations. Compound 2 is a brown solid, readily soluble in chloroform, acetone, and DMSO, and sparingly soluble in water.

[0053] In summary, the structural formulas of compounds 1 and 2 are determined as follows: .

[0054] In the above chemical formulas, compound 1 and compound 2 have the same main structure. Compound 1 has an H atom at the C-17'' position, while compound 2 has a hydroxyl group replaced at the C-17'' position.

[0055] (Example 2, Compound 1 granules) Granules of 6% Compound 1: 16% Compound 1, 1.0% vegetable oil-based surfactant, 0.6% alkyl succinate sulfonate, 0.2% colorant, and bentonite filler to make up to 100%.

[0056] The granules of compound 2 were prepared according to the above formulation.

[0057] In addition to granules, compounds 1 and 2 can also be formulated into other pesticide formulations, such as suspensions, wettable powders, or dispersible oils. The carriers for each formulation are conventional carriers in the field, and they are prepared according to conventional formulation methods.

[0058] (Experimental example) Compounds 1 and 2 prepared in Example 1 were tested for resistance to the crop disease fungus pineapple black heart disease. Curvularia australiensis early blight of potatoes Alternaria sp., mango anthracnose fungus Colletotrichum Asian HNM 408, anthracnose fungus of rubber trees Colletotrichum acutatum HNMRC 178, the fungus causing banana wilt Fusarium oxysporum HNM 1003, rice blast fungus *Pyricularia oryzae* HNM 1003, *Fusarium moniliforme* Fusarium moniliforme bio-52799 and Fusarium solani Fusarium solani bio-80814 activity screening experiment.

[0059] 1. Compound 1, compound 2 and the positive control difenoconazole were dissolved in DMSO and prepared to a concentration of 10 mg / L.

[0060] 2. After activation, the eight types of fungi causing crop diseases were diluted with a 0.02% soil-temperature-80 aqueous solution, and the spore concentration was found to be approximately 10. 6 -10 7 spores / mL.

[0061] 3. Add 100 µL of diluted bacterial solution to fresh PDA solid medium and spread it evenly.

[0062] 4. Place filter paper discs on the coated plate, loading 5µL of sample onto each disc. Perform three replicates, using DMSO as a negative control and difenoconazole as a positive control.

[0063] 5. After 3 days of culture, observe and measure the size of the inhibition zone.

[0064] Antimicrobial experiments on eight types of crop pathogenic fungi showed that compound 1 was effective against early blight of potato (Pseudomonas aeruginosa). Alternaria Compound 1 exhibits antibacterial activity against *Pseudomonas aeruginosa* sp., with an inhibition zone diameter of 15 mm at 100 µg / disc. Further testing of compound 1's antibacterial activity against *Pseudomonas aeruginosa* sp. was conducted using a 2:1 dilution method. Alternaria sp.) EC 50 The concentration was 19.21 µM, the same level as the positive control difenoconazole (2.5 µM), showing a significant inhibitory effect. Compound 2 showed an inhibitory effect on *Pseudomonas aeruginosa* (potato early blight pathogen). Alternariasp.) exhibits weak antibacterial activity, with an inhibition zone of 6 mm at 20 μg / disc, EC. 50 The value is 86.42 µ M.

[0065] Compound 1, Compound 2, and the positive control difenoconazole are effective against the crop pathogen *Phytophthora indica* (potato early blight). Alternaria See the comparison photos of the inhibition effect of sp.) Figure 7 .

[0066] Obviously, the above embodiments are merely examples to clearly illustrate the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, these obvious variations or modifications derived from the spirit of the present invention are still within the scope of protection of the present invention.

Claims

1. A biphenyl-diketopiramine-indole alkaloid hybrid compound with the following structural formula: R is H or OH.

2. The use of the compound of claim 1 in inhibiting pathogenic fungi that cause crop diseases.

3. A pesticide composition comprising at least one of the compounds of claim 1, and an acceptable carrier in the pesticide.

4. A method for preparing the biphenyl-diketopiramine indole alkaloid hybrid compound according to claim 1, characterized in that: From strain Aspergillus candidus Compound 1, with R being H, and compound 2, with R being OH, were isolated from the fermentation product of HNNU0546; Aspergillus candidus HNNU0546 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 65074.

5. The method for preparing the biphenyl-diketopiramine indole alkaloid hybrid compound according to claim 4, characterized in that... Includes the following steps: ① Activation and fermentation of bacterial strains; Aspergillus candidus HNNU0546 was activated, cultured, and fermented to obtain fermentation product; ② The fermentation product is extracted with ethyl acetate or macroporous adsorption resin, and then concentrated to obtain crude extract; ③ The crude extract obtained in step ② was fractionated by silica gel column chromatography under normal pressure. The eluent was petroleum ether-ethyl acetate, chloroform-acetone, or chloroform-methanol solvent system, and the elution was carried out in a gradient from 100:0 to 0:100 (v / v). The fractions were tracked and combined by thin-layer chromatography (TLC). The fractions that could be developed on TLC with chloroform-acetone solvent system of 9:1 (v / v) or chloroform-methanol solvent system of 20:1 (v / v) were further subjected to silica gel column chromatography to obtain crude products. The crude products were then purified by high performance liquid chromatography to obtain compound 1 and compound 2.

6. The method for preparing the biphenyl-diketopiramine indole alkaloid hybrid compound according to claim 5, characterized in that: In step ①, the strain Aspergillus candidus HNNU0546 was inoculated onto PDA agar medium to obtain a plate containing the bacterial strain. Then, a single colony was selected from the plate using an inoculation loop or bamboo stick and inoculated into an Erlenmeyer flask containing PDB seed culture medium. The flask was then cultured on a shaker to obtain the seed culture. The seed culture was then inoculated into bottles containing PDB fermentation medium and allowed to stand to obtain the fermentation product.

7. The method for preparing the biphenyl-diketopiramine indole alkaloid hybrid compound according to claim 6, characterized in that: In step ①, the strain Aspergillus candidus HNNU0546 was inoculated into PDA agar medium supplemented with 3% artificial sea salt and activated by incubation at 28 °C for 3 days to obtain plates containing the strain; then the strain was inoculated into sterile fresh PDB seed culture medium supplemented with 3% sea salt and incubated on a shaker at 200 rpm and 28 °C for 3 days to obtain the seed culture. The seed culture was then inoculated into PDB fermentation medium with 3% sea salt and cultured at room temperature for 28 days. When preparing the PDA agar medium, wash and peel 200 g of potatoes, cut them into slices and boil them in water. After the water boils, boil for 30 minutes. Filter the liquid through gauze to remove the residue. Add 20 g of glucose, 30 g of sea salt and 20 g of agar to the filtrate and finally adjust the volume to 1 liter. When preparing the PDB culture medium, wash and peel 200 g of potatoes, cut them into slices and boil them in water. After the water boils, boil for 30 minutes, filter with gauze to remove the residue, add 20 g of glucose and 30 g of sea salt to the filtrate, and finally adjust the volume to 1 L.

8. The method for preparing the biphenyl-diketopiramine indole alkaloid hybrid compound according to claim 5, characterized in that: In step ②, the fermentation product is extracted three times with ethyl acetate, each time for 2-3 days. The extracts are then combined and concentrated under reduced pressure to obtain the crude extract.

9. The method for preparing the biphenyl-diketopiramine indole alkaloid hybrid compound according to claim 5, characterized in that: In step ③, the fractions that can be developed in thin-layer chromatography using a chloroform-acetone solvent system with a volume ratio of 9:1 or a chloroform-methanol solvent system with a volume ratio of 20:1 are subjected to gradient elution using a pure chloroform-methanol system of 100:0-0:100 as the eluent, and similar fractions are combined to obtain 5 subfractions Fr.1-Fr.5; Subfraction Fr.4 was purified by silica gel column chromatography using a chloroform-methanol solvent system at a ratio of 15:1 to 0:100 to obtain four subfractions: Fr.4-1, Fr.4-2, Fr.4-3, and Fr.4-4. Subfraction Fr.4-3 was subjected to silica gel column chromatography and developed in a chloroform-acetone solvent system with a volume ratio of 6:4 to obtain four subfractions: Fr.4-3-1, Fr.4-3-2, Fr.4-3-3 and Fr.4-3-4. The sub-fraction Fr.4-3-3 was purified by high-performance liquid chromatography (HPLC) with acetonitrile-water in a mobile phase of 45:

55. Compounds 2 and 1 were collected at elution times of 17 min and 31 min, respectively.

10. The method for preparing the biphenyl-diketopiramine indole alkaloid hybrid compound according to claim 5, characterized in that: When performing silica gel column chromatography, the stationary phase is 100-200 mesh silica gel.

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