Indole diterpenoid alkaloid derived from marine fungi and application of indole diterpenoid alkaloid

Abstract

The invention relates to indole diterpenoid alkaloid derived from marine fungi and application of the indole diterpenoid alkaloid. The indole diterpenoid alkaloid compound 1 has a strong cell proliferation inhibition effect, and the IC50 value of the indole diterpenoid alkaloid compound 1 on a human gastric cancer cell strain SGC-7901 is 19.16 [mu] g / mL; and the IC50 value on a human hepatoma cell line HepG2 is 6.27 mu g / mL. The IC50 of the positive drug cis-platinum to SGC-7901 and the IC50 of the positive drug cis-platinum to HepG2 are 3.56 mu g / mL and 1.99 mu g / mL respectively. Compound 1 with structure as follows: # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of marine natural products, and particularly relates to an indole diterpenoid alkaloid derived from a marine fungus and its application. Background Art

[0002] The mangrove ecosystem is widely regarded as one of the most productive ecosystems in the world. Due to its special ecological environment, microorganisms have formed effective adaptation mechanisms, producing unique chemical substances and great biodiversity. Mangrove-related fungi have become an important source of natural products. The secondary metabolites of mangrove fungi can be divided into four categories according to their chemical structures and biosynthetic pathways: ketones, terpenoids, alkaloids, and other nitrogen-containing metabolites, and most of the metabolites have pharmacological activities such as antibacterial, antifungal, and cytotoxic activities. The present invention provides an indole diterpenoid alkaloid derived from a marine fungus and its application. Summary of the Invention

[0003] The present invention provides a marine fungus N4-3, which is isolated from the rhizosphere soil of the mangrove plant Sonneratia caseolaris. The mangrove plant Sonneratia caseolaris comes from the Dongzhai Harbor Mangrove Nature Reserve in Haikou, South China Sea. The marine fungus N4-3 of the present invention is characterized in that its strain preservation information is as follows: the name of the preservation unit: China General Microbiological Culture Collection Center; the address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the preservation date: April 6, 2023; the preservation number: CGMCC No. 40554; the taxonomic naming: Penicillium sp.

[0004] Another embodiment of the present invention provides an indole diterpenoid alkaloid, its tautomer or its pharmaceutically acceptable salt, which is characterized in that the indole diterpenoid alkaloid has the structures shown in Compounds 1, 2, 3, 4, and 5:

[0005]

[0006]

[0007] Another embodiment of the present invention provides a preparation method of the above indole diterpenoid alkaloid Compounds 1, 2, 3, 4, and / or 5, which is characterized by including the following steps:

[0008] (1) Culturing the marine fungus N4-3 in a strain culture medium to obtain a seed solution;

[0009] (2) Inoculating the seed solution obtained in step (1) into a fermentation medium and performing fermentation culture to obtain a fermentation product;

[0010] (3) Separate the fermentation broth and the bacterial cells in the fermented product obtained in step (2). Extract the fermentation broth with organic solvent A 2 to 4 times, combine the extracts and concentrate under reduced pressure to obtain the fermentation broth extract; extract the bacterial cells with organic solvent B 2 to 4 times, combine the extracts and concentrate under reduced pressure to obtain the bacterial cell extract; combine the fermentation broth extract and the bacterial cell extract to obtain the crude extract;

[0011] (4) The crude extract obtained in step (3) is subjected to chromatographic separation to obtain indole diterpenoid alkaloid compounds 1, 2, 3, 4 and / or 5.

[0012] In the above preparation method of the present invention, the strain culture conditions, fermentation conditions, etc. (such as medium selection, temperature, time and other parameters) are all conventional experimental operations in the art, and those skilled in the art can make reasonable selections according to the experimental situation. For the convenience of understanding the present invention, only one conventional operation method is listed below: The strain medium in step (1) is a commonly used strain medium in the art, preferably PDB medium, and the preferred formula (per liter of water) is: glucose 20 g / L, potato 200 g / L, sea salt 30 g / L; The strain culture in step (1) is preferably shake flask culture, the temperature is preferably from room temperature to 30 °C, the shake flask rotation speed is preferably 150 - 200 r / min, and the culture time is preferably 2 - 4 d. The fermentation medium in step (2) is a commonly used fermentation medium in the art, which can be the same as or different from the strain medium in step (1), preferably PDB medium, and the preferred formula (per liter of water) is: glucose 20 g / L, potato 150 - 300 g / L, sea salt 30 g / L; The inoculum amount in step (2) is preferably 1 - 5%, the fermentation culture temperature is from room temperature to 30 °C, and the culture time is 28 - 30 d. The organic solvent A in step (3) is preferably one or more of ethyl acetate, dichloromethane, chloroform or ether; the organic solvent B is preferably one or more of methanol, ethanol, THF or acetone. Optionally, the combined fermentation broth extract and the bacterial cell extract are subjected to a drying step to obtain the crude extract. The chromatographic separation in step (4) is preferably one or a combination of several 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 preferred scheme: The crude extract of marine fungus N4-3 is first subjected to reduced pressure silica gel column chromatography, and petroleum ether - ethyl acetate is used as the eluent for gradient elution. The elution gradients are 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 0:100 respectively. Two column volumes are collected for each gradient, and they are divided into 5 components according to the polarity. After the eluates obtained from the gradients 50:50 and 40:60 are combined and concentrated, they are subjected to Sephadex LH-20 gel column chromatography, and the eluent is CH 2 Cl 2 / MeOH, v / v, 1:1 mixed solvent, elute for 4 - 5 column volumes, concentrate under reduced pressure and then pass through reverse-phase column chromatography (ODS), the mobile phase is MeOH:H 2 O = 85:15 to obtain indole diterpenoid alkaloid compounds 1 and 4; after combining and concentrating the eluates obtained with gradients of 30:70 and 20:80, and subjecting them to normal-phase silica gel column chromatography, the mobile phase is ethyl acetate:petroleum ether = 1:8 to 1:3, elute for 6 - 8 column volumes, and then pass through Sephadex LH-20 gel column chromatography, the eluent is CH 2 Cl 2 / MeOH, v / v, 1:1 mixed solvent, elute for 4 - 5 column volumes, concentrate under reduced pressure and then pass through reverse-phase column chromatography (ODS), the mobile phase is MeOH:H 2 O = 90:10 to 85:15 to obtain indole diterpenoid alkaloid compounds 2, 3, and 5. The obtained compounds 1 - 5 are optionally purified by semi-preparative HPLC. The ratios of the eluents or mobile phases described in the present invention are all volume ratios.

[0013] Another embodiment of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition uses one or more of the above indole diterpenoid alkaloid compounds 1, 2, 3, 4, 5, their tautomers or their pharmaceutically acceptable salts as active ingredients. The pharmaceutical composition also includes pharmaceutically acceptable carriers, diluents or excipients. The dosage form of the pharmaceutical composition is selected from solid preparations, liquid preparations, semi-solid preparations, etc. The pharmaceutical composition is used for preventing and treating diseases such as human pathogenic bacteria and tumors.

[0014] Another embodiment of the present invention provides the use of one or more of the above indole diterpenoid alkaloid compounds 1, 2, 3, 4, 5, their tautomers or their pharmaceutically acceptable salts in the preparation of antibacterial drugs or anti-tumor drugs. The antibacterial drugs are preferably for diseases caused by infections such as Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Bacillus subtilis, Escherichia coli, Vibrio parahaemolyticus, etc. The anti-tumor drugs are preferably for gastric cancer, liver cancer, etc.

[0015] Another embodiment of the present invention provides the use of the above marine fungus N4-3 in the preparation of one or more of indole diterpenoid alkaloid compounds 1, 2, 3, 4, 5. Description of the Drawings

[0016] Figure 1 are the 1 H- 1 H COSY, HMBC, ROESY, NOESY diagrams of compounds 1 and 2.

[0017] Figure 2 is the HMBC correlation diagram of compound 3.

[0018] Figure 3 is the 1 H NMR (400 MHz, CDCl 3 ) spectrum.

[0019] Figure 4 is the 13 C NMR (100 MHz, CDCl 3 ) spectrum.

[0020] Figure 5 is the high-resolution mass spectrum of Compound 1.

[0021] Figure 6 is the 1 H NMR (600 MHz, CDCl 3 ) spectrum.

[0022] Figure 7 is the 13 C NMR (150 MHz, CDCl 3 ) spectrum.

[0023] Figure 8 is the high-resolution mass spectrum of Compound 2.

[0024] Figure 9 is the 1 H NMR (600 MHz, CDCl 3 ) spectrum.

[0025] Figure 10 is the 13 C NMR (150 MHz, CDCl 3 ) spectrum.

[0026] Figure 11 is the high-resolution mass spectrum of Compound 3.

[0027] Figure 12 is the 1 H NMR (600 MHz, CDCl 3 ) spectrum.

[0028] Figure 13 is the HSQC (CDCl 3 ) spectrum of Compound 1.

[0029] Figure 14 is the 1 H– 1 H COSY (CDCl 3 ) spectrum.

[0030] Figure 15 is the HMBC (CDCl 3)Figure.

[0031] Figure 16 is the ROESY (CDCl of Compound 1 3 )Figure.

[0032] Figure 17 is the ECD figure of Compound 1.

[0033] Figure 18 is the HSQC (CDCl of Compound 2 3 )Figure.

[0034] Figure 19 is of Compound 2 1 H– 1 H COSY (CDCl of Compound 2 3 )Figure.

[0035] Figure 20 is the HMBC (CDCl of Compound 2 3 )Figure.

[0036] Figure 21 is the ROESY (CDCl of Compound 2 3 )Figure.

[0037] Figure 22 is the ECD figure of Compound 2. Detailed implementation manners

[0038] Example 1

[0039] (1) Cultivation of marine fungus N4-3 strain

[0040] The culture medium used for cultivating the marine fungus N4-3 strain is prepared by adding to every 1000 mL of water: 200 g of potato boiled to extract juice, 20 g of glucose, and 30 g of crude sea salt; it is dispensed into conical flasks during use. At room temperature, it is cultured on a shaker at a shaker speed of 180 r / min for 3 days to obtain the seed liquid.

[0041] (2) Fermentation of marine fungus N4-3

[0042] The fermentation medium used for the fermentation culture of marine fungus N4-3 is prepared by adding to every 1000 mL of water: 200 g of potato boiled to extract juice, 20 g of glucose, and 30 g of crude sea salt; it is dispensed into conical flasks during use (100 1-L conical flasks, each containing 400 mL of PDB medium). The seed liquid is inoculated into the medium in the conical flasks, with an inoculation amount of 1.0%, and the fermentation culture temperature is room temperature. The static culture time is 30 days to obtain the fermentation product.

[0043] (3) Preparation of the crude extract of marine fungus N4-3

[0044] Take the fermented product obtained after the fermentation culture in step (2), separate the fermentation broth and the thalli. The fermentation broth is extracted with ethyl acetate three times, and the extract is concentrated under reduced pressure to obtain the fermentation broth extract; the thalli are extracted with methanol three times, and concentrated under reduced pressure to obtain the thalli extract; the fermentation broth extract and the thalli extract are combined (optionally with a further drying operation) to obtain the crude extract.

[0045] (4) Separation and purification of compounds 1-5

[0046] First, subject the crude extract of marine fungus N4-3 obtained in step (3) to silica gel column chromatography under reduced pressure (preferably silica gel of 100-200 mesh), and use petroleum ether-ethyl acetate as the eluent for gradient elution. The elution gradients are 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 0:100. Two column volumes are collected for each gradient, and they are divided into 5 fractions according to the polarity. Among them, the eluents obtained from the gradients of 50:50 and 40:60 are combined and concentrated, and then subjected to Sephadex LH-20 gel column chromatography. The eluent is a mixed solvent of CH 2 Cl 2 / MeOH, v / v, 1:1, and elute for 4-5 column volumes. After concentration under reduced pressure, it is then subjected to reverse-phase column chromatography (ODS). The mobile phase is MeOH:H 2 O = 85:15 to obtain indole diterpenoid alkaloid compounds 1 (45.2 mg), 4 (12.3 mg); the eluents obtained from the gradients of 30:70 and 20:80 are combined and concentrated, and after normal-phase silica gel column chromatography, the mobile phase is ethyl acetate:petroleum ether = 1:8 to 1:3, and elute for 6-8 column volumes. Then, it is subjected to Sephadex LH-20 gel column chromatography. The eluent is a mixed solvent of CH 2 Cl 2 / MeOH, v / v, 1:1, and elute for 4-5 column volumes. After concentration under reduced pressure, it is then subjected to reverse-phase column chromatography (ODS). The mobile phase is MeOH:H 2 O = 90:10 to 85:15 to obtain indole diterpenoid alkaloid compounds 2 (6.2 mg), 3 (4.5 mg), 5 (28.5 mg).

[0047] The structural characterization data of compounds 1-5 are as follows. According to the data of one-dimensional, two-dimensional NMR, MS, ECD, etc., the structures of compounds 1-5 can be determined as follows:

[0048]

[0049] Compound 1: [α] 20 D-215 (c = 0.10, MeOH). UV (MeOH) λmax (log ε) 367 (0.5), 285 (2.3), 259 (1.6), 204 (1.3) nm. ECD (0.25 mM, MeOH) λmax (Δε) 373 (-24.6), 324 (+26.5), 281 (-13.3), 251 (+18.3), 224 (+10.9), 205 (-12.5) nm. IR (KBr) νmax 3329, 2975, 2934, 1658, 1456, 1375, 1258, 1167, 1132, 1010, 868, 534, 458. 1 1H NMR (CDCl 3 , 400 MHz) and 13 13C NMR (CDCl 3 , 100 MHz) are shown in Table 1; HRESIMS m / z 604.3042 [M+Na] + (calcd for C 37 31 43 H 5 + 10

[0050] Table 1 1H and 1 13C NMR data of compound 1 13

[0051]

[0052]

[0053] Compound 2: [α] 20 D +15 (c = 0.10, MeOH). UV (MeOH) λ max (log ε) 368 (0.3), 282 (2.7), 215 (1.3) nm. ECD (0.25 mM, MeOH) λ max (Δε) 353 (-13.6), 315 (-7.8), 280 (-61.4), 235 (+26.2), 215 (-15.2) nm. 1 1H NMR (CDCl 3 , 600 MHz) and 13 13C NMR (CDCl 3 , 150 MHz) are shown in Table 2; HRESIMS m / z 618.3187, [M+Na] + , (calcd for C 38 32 45 H​5 + , 618.3190).

[0054] Table 2 of Compound 2 1 H and 13 C NMR data

[0055]

[0056]

[0057] Compound 3: [α] 20 D -43 (c = 0.10, MeOH). UV (MeOH) λ max (logε) 309 (0.7), 242 (2.2), nm. 1 H NMR (CDCl 3 , 600 MHz) and 13 C NMR (CDCl 3 , 150 MHz) are shown in Table 3; HRESIMS m / z 650.3120, [M+Na] + , (calcd for C 38 H 45 NNaO 7 + , 650.3088).

[0058] Table 3 of Compound 1 H and 13 C NMR data

[0059]

[0060]

[0061] Compound 4: High-resolution mass spectrometry gave the signal of [M+Na] + at 620.2993 (calcd for C 37 H 43 NNaO 6 + , 620.2983), indicating that the molecular weight of this compound is 583 and the molecular formula is C 37 H 45 NO 5 , and the degree of unsaturation is 16. 1 The 1H NMR data revealed the presence of an NH signal at δ H 7.75 (H-1), and in the low-field region at δ H1.45 (H-32), 1.33 (H-37 / 38), 1.28 (H-36), 1.25 (H-39 / 40), 1.09 (H-35) and 1.06 (H-33) gave eight singly methyl signals, δ H 7.49 (H-20) and 7.03 (H-30) were its aromatic hydrogen signals, and also gave two olefin signals δ H 6.05 (H-11), 5.7 - 5.75 (H-6) as well as six methylene signals and six methine signals. Compared with compound 1, there were two more methylene signals δ H 2.84 - 2.75 (27), δ H 3.27 (H-22), and two groups of methine signals were missing. It was speculated that the two double bonds at C-22 and C-27 in rings A and B were replaced by double bonds at C-23 and C-28. By consulting the literature and comparing the data, the compound was finally determined to be the known compound shearinine O. 1 H NMR (600 MHz, CDCl 3 ) δ 7.75 (1H, d, J = 5.4 Hz, H-1), 7.49 (1H, d, J = 3.3 Hz, H-20), 7.03 (1H, d, J = 4.9 Hz, H-30), 6.05 (1H, s, H-11), 5.7 - 5.75 (1H, m, H-6), 4.24 (1H, s, H-9), 3.27 (2H, d, J = 16.9 Hz, H-22), 3.04 (1H, d, J = 11.2 Hz, H-5a), 2.84 - 2.75 (2H, m, H-27), 2.73 (1H, m, H-16), 2.50 - 2.30 (2H, m, H-5b / 17a), 2.19 - 1.92 (5H, m, H-14a / 14b / 15a / 15b / 17b), 1.45 (3H, s, H-32), 1.33 (6H, s, H-37 / 38), 1.28 (3H, s, H-36), 1.25 (6H, s, H-39 / 40), 1.09 (3H, s, H-35), 1.06 (3H, s, H-33).

[0062] Compound 5: High-resolution mass spectrometry gave [m / z 604.3048 (C 37 H 43 NNaO 5 + , Calcd. 604.3033)] indicating that the molecular weight of the compound was 581 and the molecular formula was C 37 H 43 NO 5 , and the degree of unsaturation was 17. Further analysis 1The \(^1H\) NMR data (Table 2 - 6) revealed the presence of an NH signal at \(\delta\) H 7.81 (brd, \(J = 6.4\) Hz), and eight single - methyl signals were given at high - field regions \(\delta\) H 1.54 (H - 37 / 38), 1.47 (H - 39 / 40), 1.45 (H - 36), 1.37 (H - 32), 1.24 (H - 33), and 1.19 (H - 35). Two aromatic - hydrogen signals \(\delta\) H 7.20 (H - 20) and 7.43 (H - 30) were also given, and three alkene signals \(\delta\) H 5.84 (H - 11), 6.38 (H - 22), and 6.50 (H - 27), as well as five chemically non - equivalent methylene signals and two methine signals \(\delta\) H 4.32 (H - 9), 2.79 (H - 16). 13 \(^{13}\)C NMR showed a total of 37 carbons, including eight methyl signals \(\delta\) C 16.2 (C - 32), 28.3 (C - 33), 23.2 (C - 35), 28.9 (C - 36), 31.5 (C - 37 / 38), 30.9 (C - 39 / 40), an unsaturated ketone group (\(\delta\) C 197.2, C - 10), four oxygen - connected carbons \(\delta\) C 104.5 (C - 7), 88.0 (C - 9), 73.3 (C - 24), 72.8 (C - 26), and eight alkenyl or aromatic carbons \(\delta\) C 117.7 (C - 11), 110.4 (C - 20), 121.4 (C - 22), 129.0 (C - 27), 104.2 (C - 30). The above spectral signals indicated that Compound 5 was also an indole diterpenoid compound. By comparing with the literature, it was determined to be 22,23 - dehydroshearinine A.

[0063] The 1 \(^1H\) and 13 \(^{13}\)C NMR data of Compound 5

[0064]

[0065]

[0066] Example 2 Evaluation of the anti - phytopathogenic fungal activities of Compounds 1 - 5

[0067] The inhibitory activity of the crude extract against the pathogen of rice sheath blight was determined by the mycelial growth rate method (Agricultural industry standard NY / T 1156.2 - 2006). The positive drug was fluxapyroxad.

[0068] The specific method is as follows: Dilute the test drugs (Compounds 1-5) with a solvent to a series of concentration gradient mother liquors. Under aseptic operation conditions, quantitatively add the pre-melted culture medium into a sterile conical flask according to the test treatment. Sequentially and quantitatively pipette the drug solutions from low concentration to high concentration, and add them into the above conical flasks respectively, and shake well. Then pour an equal amount into 3 Petri dishes of the same size to prepare PDA plates containing drugs at corresponding concentrations; For the cultured pathogenic bacteria, under aseptic conditions, use a sterilized punch with a diameter of 7 mm to punch out a bacterial cake from the edge of the colony, and use an inoculator to inoculate the bacterial cake in the center of the drug-containing plate. The solvent control is added with an equal amount of solvent instead. Incubate at 25 °C. When the control is about to cover the Petri dish, measure the colony diameter (cm) by the cross method, calculate and take the average value, calculate the inhibition rate, repeat three times, and obtain the results after multiple repetitions.

[0069]

[0070] The results showed that when the concentration of Compounds 1-5 was 200 mg / L, the inhibition rates against the pathogenic bacteria of rice sheath blight were all above 10%. The inhibition rate of the positive control fluxapyroxad against the pathogenic bacteria of rice sheath blight was 100% at a concentration of 20 mg / L.

[0071] Example 3 Antibacterial Activity Test against Human Pathogenic Bacteria

[0072] The antibacterial activity of the compounds was determined using the microdilution method. There were 9 test strains, including 7 Gram-positive bacteria: Staphylococcus aureus (Staphylococcus aureus ATCC 43300, S. aureus ATCC 33591, S. aureus ATTCC 25923, S. aureus ATCC 29213), Enterococcus faecalis (Enterococcus faecalis ATCC 51299), Enterococcus faecium (Enterococcus faecium ATCC 35667), Bacillus subtilis (Bacillus subtilis ATCC 19659) and 2 Gram-negative bacteria: Escherichia coli (Escherichia coli ATTCC 25922), Vibrio parahaemolyticus (Vibrio parahaemolyticus ATCC 17802). Vancomycin hydrochloride and oxacillin sodium were used as positive controls, the negative control was DMSO, and the blank control was the bacterial culture medium. Incubate at a constant temperature of 28 °C for 24 h.

[0073] Prepare LB medium, evenly dispense it into 250 mL conical flasks, and sterilize it by high-pressure steam for later use. Take out the cryopreservation tube of the bacterial strain to be tested from the -80 °C refrigerator, restore its activity at room temperature, use a pipette to aspirate 200 μL of the bacterial solution and add it to the prepared medium, and culture it at 180 r / min and 37 °C for a period of time. Accurately weigh 1 mg of the test compound, put it into a 2 mL EP tube, add a certain amount of DMSO and dissolve it fully. For areas that do not dissolve, sonicate to make the concentration of the compound 10 μg / μL. Accurately weigh an appropriate amount of the positive drugs vancomycin hydrochloride and oxacillin sodium, and dissolve them with DMSO to make their concentration 2.5 μg / μL.

[0074] First, conduct a preliminary screening of antibacterial activity. Dilute the bacterial solution in a laminar flow hood, and aspirate 100 μL of the bacterial solution for dilution and standby. Take out a 96-well plate, aspirate 200 μL of blank LB medium liquid into B1-D1 as a blank control, aspirate 198 μL of the medium containing the bacterial solution into E1-G1, and add 2 μL of DMSO as a negative control. Aspirate 198 μL of the medium containing the bacterial solution into B2-G11, and aspirate 2 μL of the test sample or positive drug into B2-G11 as the sample group. The final sample concentration is 100 μg / mL. Seal it with a sealing film to prevent subsequent bacterial contamination. Do not shake it during the culture period to prevent cross-infection. Use the same plate for one type of bacteria, and take the average of three repetitions. After the operation is completed, place it in a constant temperature incubator, culture it at 37 °C for 24 h, and then read the OD 600 value and calculate the inhibition rate. Screen samples with an inhibition rate greater than 50% for testing by the microdilution method.

[0075]

[0076] The blank control is to add 200 μL of blank LB medium liquid into B1-D1, aspirate 198 μL of the medium containing the bacterial solution into E1-G1, and add 2 μL of DMSO as a negative control. Use a pipette to aspirate 198 μL of the medium containing the bacterial solution into A2-A4, and add 2 μL of the test sample or positive drug, and blow and suck evenly. Aspirate 100 μL of the medium containing the bacteria into B2-H10, aspirate 100 μL from A2-A4 to B2-B4, blow and suck evenly, and dilute it successively down to the H row. Aspirate and discard 100 μL from the H row, and then aspirate 100 μL of the medium containing the bacteria to fill the A-H rows. The final test concentrations are 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5625 μg / mL, 0.78125 μg / mL respectively. Only test one strain on one 96-well plate. Seal it with a sealing film and place it in a 37 °C constant temperature incubator for 24 h. Then, read the OD 600 value and calculate the growth inhibition rate, and use the software GraphPad Prism 8 to calculate the IC 50 value.

[0077]

[0078] a Date are expressed in IC 50 values. b O = oxacillin sodium. c V = VancomycinHCl.

[0079] Example 4 Cytotoxic Activity Test

[0080] (1) Resuscitation of Tumor Cells

[0081] The cytotoxic activity of Compound 1 was tested using the MTT method, with the human gastric cancer cell line SGC-7901 and the liver cancer cell line HepG2 as the test tumor cell lines, and cisplatin as the positive control drug.

[0082] First, thaw the cancer cells, centrifuge for 5 min to discard the supernatant, and culture the cancer cells in an incubator at 37°C and 5% CO 2 Observe the growth status of the cancer cells daily. Then add 2 - 3 mL of PBS solution, and at the same time add 0.1 - 1 mL of 0.25% trypsin containing EDTA for digestion. When the cells become round, add 3 mL of medium to prepare a cell suspension for subculture.

[0083] (2) MTT Assay

[0084] Add human gastric cancer cells SGC-7901 and liver cancer cells HepG2 to 96-well plates respectively, add 100 μL of suspension to each well, and observe after culturing in an incubator at 5% CO 2 and 37°C for one day; Add the test compound and the positive drug to each well, and add 100 mL of medium to the wells to form the negative control group and the blank control group respectively; After a short period of time, add 20 μL of MTT to each well and culture in the incubator for another 4 hours; Aspirate the excess liquid, continue to add 100 μL of DMSO to dissolve, gently shake by hand for about 10 minutes, and finally read the absorbance at a wavelength of 490 nm. The following is the formula for calculating the proliferation inhibition rate:

[0085]

[0086] The results showed that Compound 1 had a strong inhibitory effect on cell proliferation, and its IC 50 value for the human gastric cancer cell line SGC-7901 was 19.16 μg / mL; the IC 50 value for the human liver cancer cell line HepG2 was 6.27 μg / mL. The IC of the positive drug cisplatin for SGC-7901 and HepG250 They are 3.56 and 1.99 μg / mL respectively.

Claims

1. A marine fungus N4-3, characterized in that Its deposit number is CGMCC No.40554.

2. An indole diterpene alkaloid, a tautomer thereof or a pharmaceutically acceptable salt thereof, characterized in that The indole diterpene alkaloids have the structures shown in compounds 1, 2, and 3:

3. A method for preparing indole diterpene alkaloids 1, 2, 3, 4 and / or 5, characterized in that The steps include: (1) Cultivating the marine fungus N4-3 in a bacterial culture medium to obtain a seed solution; (2) inoculating the seed liquid obtained in step (1) into a fermentation medium for fermentation to obtain a fermentation product; (3) separating the fermentation broth and bacterial cells in the fermented product obtained in step (2), extracting the fermentation broth with organic solvent A for 2 to 4 times, combining the extracts and concentrating under reduced pressure to obtain a fermentation broth extract; extracting the bacterial cells with organic solvent B for 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 indole diterpene alkaloid compounds 1, 2, 3, 4 and / or 5.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises one or more of the indole diterpene alkaloids 1, 2, 3, their tautomers or pharmaceutically acceptable salts thereof as claimed in claim 2 as active ingredients. The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient. The dosage form of the pharmaceutical composition is selected from solid preparations, liquid preparations, semi-solid preparations and the like.

5. Use of one or more of the indole diterpene alkaloids 1, 2, 3, 4, 5, their tautomers or pharmaceutically acceptable salts thereof as claimed in claim 3 in plant protection, especially in controlling agricultural pathogens.

6. Use of one or more of the indole diterpene alkaloids 1, 2, 3, 4, 5, their tautomers or pharmaceutically acceptable salts thereof as claimed in claim 3 in the preparation of drugs for preventing and controlling agricultural pathogens, human pathogens and tumors.

7. Use of one or more of the indole diterpene alkaloid compounds 1, 2, 3, 4, 5, their tautomers or pharmaceutically acceptable salts thereof as claimed in claim 3 in the preparation of antibacterial drugs or antitumor drugs. The antibacterial drugs are preferably used to treat diseases caused by infections such as Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Bacillus subtilis, Escherichia coli, Vibrio parahaemolyticus, etc. The antitumor drugs are preferably used to treat gastric cancer, liver cancer, etc.

8. Use of the marine fungus N4-3 according to claim 1 in the preparation of one or more of the indole diterpene alkaloid compounds 1, 2, 3, 4, and 5.

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