Marine fungus-derived bisindole derivative and application of marine fungus-derived bisindole derivative as anti-tumor lead compound

Abstract

The invention relates to a marine fungus-derived bisindole derivative and application of the marine fungus-derived bisindole derivative as an anti-tumor lead compound. Bisindole derivatives having structures represented by compounds 1-6: # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of marine natural product chemistry, and particularly relates to a bis-indole derivative derived from marine fungi and its application as an anti-tumor lead compound. Background Art

[0002] Aspergillus is a typical filamentous fungus, mainly divided into Aspergillus fumigatus, A. versicolor, A. flavus, A. ustus, A. sydowii, etc. In 1992, Numata first isolated Fumiquinazolines from marine Aspergillus, opening the door for the study of the metabolites of marine Aspergillus. Recent studies have found that many organic compounds with unique structures have been discovered in marine Aspergillus. These compounds include terpenoids, alkaloids, and polyketones, showing many physiological activities such as cytotoxicity, antibacterial, antifungal, antiviral, anti-inflammatory, and enzyme inhibitory activities, which have attracted extensive attention from many scholars. Among them, some alkaloid compounds have inhibitory effects on a variety of viruses, such as hepatitis virus, influenza virus, HIV virus, etc. Aspergillus terreus N4-9 is a mangrove-derived fungus isolated in our laboratory. Previous studies have found that its secondary metabolites have strong antibacterial and anti-tumor activities. The present invention further studies N4-9 and obtains a series of anti-tumor active bis-indole derivatives with different structures. Summary of the Invention

[0003] The marine fungus N4-9 of the present invention 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 N4-9 of the present invention is characterized in that its strain preservation information is as follows: Name of the preservation unit: China General Microbiological Culture Collection Center; Address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Preservation date: April 6, 2023; Preservation number: CGMCC No. 40555; Taxonomic nomenclature: Aspergillus terreus.

[0004] Another embodiment of the present invention provides a bis-indole derivative, its tautomer or its pharmaceutically acceptable salt, characterized in that the bis-indole derivative has the structures shown in Compounds 1-6:

[0005]

[0006] Another embodiment of the present invention provides a preparation method of the above-mentioned bis-indole derivatives 1, 2, 3, 4, 5, and / or 6, characterized by including the following steps:

[0007] (1) Cultivate the marine fungus N4-9 in a strain culture medium to obtain a seed solution;

[0008] (2) Inoculate the seed solution obtained in step (1) into a fermentation medium and perform fermentation culture to obtain a fermentation product;

[0009] (3) Separate the fermentation broth and the bacterial cells in the fermentation product obtained in step (2). Extract the fermentation broth with organic solvent A 2 to 4 times, and concentrate the combined extracts under reduced pressure to obtain a fermentation broth extract; Extract the bacterial cells with organic solvent B 2 to 4 times, and concentrate the combined extracts under reduced pressure to obtain a bacterial cell extract; Combine the fermentation broth extract and the bacterial cell extract to obtain a crude extract;

[0010] (4) The crude extract obtained in step (3) is subjected to chromatographic separation to obtain bis-indole derivatives 1, 2, 3, 4, 5, and 6. In the above preparation method of the present invention, the strain culture conditions, fermentation conditions, etc. (such as parameters such as medium selection, temperature, time, etc.) 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 35-38 °C, the shake flask rotation speed is preferably 120-150 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 rice solid medium, and the preferred formula (per 1 L conical flask) is: rice 40 g, seawater 20 mL; The inoculum amount in step (2) is preferably 5-10 mL inoculated in each 1 L conical flask, the fermentation culture temperature is from room temperature to 38 °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. The chromatographic separation in step (4) is preferably one or a combination of several 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 preferred scheme: The crude extract is subjected to vacuum silica gel column chromatography, and petroleum ether-ethyl acetate and ethyl acetate-methanol are first used as eluents for gradient elution. The elution gradients are respectively petroleum ether: ethyl acetate of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 0:100, and ethyl acetate: methanol of 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 11 components according to the polarity. After the eluent obtained when the gradient of petroleum ether: ethyl acetate is 60:40 and 50:50 is combined and concentrated, it is 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, and after vacuum concentration, it is then subjected to reverse phase column chromatography (ODS), and the mobile phase is MeOH:H 2O = 80:20 gives bis(indole) derivative 1; the eluents obtained with gradients of petroleum ether:ethyl acetate of 20:80 and 10:90 are combined and concentrated, and after normal-phase silica gel column chromatography, the mobile phase is ethyl acetate:petroleum ether = 1:10 to 1:4, eluting for 6 - 8 column volumes, and then through reverse-phase column chromatography (ODS), the mobile phase is MeOH:H 2 O = 80:20 to 85:15 gives bis(indole) derivatives 3, 4, 6; the eluents obtained with gradients of petroleum ether:ethyl acetate of 0:100 and ethyl acetate:methanol of 90:10 are combined and concentrated, and after Sephadex LH-20 gel column chromatography, the eluent is CH 2 Cl 2 / MeOH, v / v, 1:1 mixed solvent, eluting for 3 - 4 column volumes, concentrating under reduced pressure and then through reverse-phase column chromatography (ODS), the mobile phase is MeOH:H 2 O = 80:20 to 85:15 gives bis(indole) derivatives 2, 5. The obtained compounds 1 - 6 are optionally purified by semi-preparative HPLC. The ratios of the eluents or mobile phases described in the present invention are all volume ratios.

[0011] Another embodiment of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition uses the above-mentioned bis(indole) derivatives 1, 2, 3, 4, 5, 6, their tautomers or their pharmaceutically acceptable salts as active ingredients. The pharmaceutical composition optionally contains other active ingredients with anti-tumor effects. The pharmaceutical composition also includes pharmaceutically acceptable excipients (preferably pharmaceutically acceptable carriers, diluents or excipients). The dosage form of the pharmaceutical composition is selected from solid preparations, liquid preparations, semi-solid preparations, etc., preferably injections, tablets, capsules, lyophilized powder injections, suspensions, etc. The pharmaceutical composition is used for preventing and / or treating tumors, especially gastric cancer, liver cancer or non-small cell lung cancer.

[0012] Another embodiment of the present invention provides the use of the above-mentioned bis(indole) derivatives 1, 2, 3, 4, 5, 6, their tautomers or their pharmaceutically acceptable salts in the preparation of anti-tumor drugs. Especially gastric cancer, liver cancer or non-small cell lung cancer.

[0013] Another embodiment of the present invention provides the use of the above-mentioned bis(indole) derivative 1, its tautomer or its pharmaceutically acceptable salt in the preparation of drugs for promoting cell growth and differentiation.

[0014] Another embodiment of the present invention provides the use of the above-mentioned marine fungus N4-9 in the preparation of the above-mentioned bis(indole) derivatives 1, 2, 3, 4, 5 and / or 6. Description of the Drawings

[0015] Figure 1 are the HMBC (A), NOESY (B) and ECD (C) diagrams of compound 1.

[0016] Figure 2 It is the ECD diagram of Compound 2.

[0017] Figure 3 It is that of Compound 1 1 H NMR (600 MHz, CDCl 3 ) diagram.

[0018] Figure 4 It is that of Compound 1 13 C NMR (150 MHz, CDCl 3 ) diagram.

[0019] Figure 5 It is that of Compound 2 1 H NMR (600 MHz, CDCl 3 ) diagram.

[0020] Figure 6 It is that of Compound 2 13 C NMR (150 MHz, CDCl 3 ) diagram.

[0021] Figure 7 It is that of Compound 3 1 H NMR (600 MHz, CDCl 3 ) diagram.

[0022] Figure 8 It is that of Compound 3 13 C NMR (150 MHz, CDCl 3 ) diagram. Detailed implementation mode

[0023] Example 1

[0024] (1) Cultivation of marine fungus strain N4-9

[0025] The culture medium used for culturing the marine fungus strain N4-9 is as follows: add to every 1000 mL of water: 200 g of potatoes, boil to extract the juice, 20 g of glucose, and 30 g of crude sea salt; when in use, dispense into conical flasks. Cultivate on a shaker at a temperature of 36 - 38 °C, a shaker rotation speed of 120 r / min, and a cultivation time of 3 d to obtain a seed solution.

[0026] (2) Fermentation of marine fungus N4-9

[0027] The fermentation medium used for the fermentation culture of marine fungus N4-9 is a rice solid medium. The preferred formula is: 40 g of rice and 20 mL of seawater are put into each 1-L conical flask. The seed liquid is inoculated into the medium in the conical flask, and the inoculation amount is 5-10 mL inoculated into each 1-L conical flask. The fermentation culture temperature is room temperature, and the static culture time is 30 d to obtain the fermented product. (3) Preparation of the crude extract of marine fungus N4-9

[0028] 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; after combining the fermentation broth extract and the thalli extract (optionally with further drying operation), the crude extract is obtained.

[0029] (4) Isolation and purification of compounds 1-6

[0030] The crude extract of marine fungus N4-9 obtained in step (3) is subjected to silica gel column chromatography under reduced pressure. First, petroleum ether-ethyl acetate and ethyl acetate-methanol are used as eluents for gradient elution. The elution gradients are respectively: petroleum ether: ethyl acetate is 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 0:100, and ethyl acetate: methanol is 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 11 components according to the polarity. Among them, the eluents obtained with the gradient of petroleum ether: ethyl acetate being 60:40 and 50:50 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 = 80:20 to obtain bis-indole derivative 1 (8.2 mg); the eluents obtained with the gradient of petroleum ether: ethyl acetate being 20:80 and 10:90 are combined and concentrated, and after normal-phase silica gel column chromatography, the mobile phase is ethyl acetate: petroleum ether = 1:10 to 1:4, and elute for 6-8 column volumes, and then subjected to reverse-phase column chromatography (ODS). The mobile phase is MeOH:H 2 O = 80:20 to 85:15 to obtain bis-indole derivatives 3 (16.3 mg), 4 (23.0 mg), 6 (12.0 mg); the eluents obtained with the gradient of petroleum ether: ethyl acetate being 0:100 and ethyl acetate: methanol being 90:10 are combined and concentrated, and then subjected to Sephadex LH-20 gel column chromatography. The eluent is CH2 Cl 2 / MeOH, v / v, 1:1 mixed solvent, elute for 3 - 4 column volumes, concentrate under reduced pressure and then pass through reverse-phase column chromatography (ODS), the mobile phase is MeOH:H 2 O = 80:20 to 85:15 to obtain bis-indole derivatives 2 (22.8 mg), 5 (12.2 mg). The structural characterization data of compounds 1 - 6 are as follows. Based on one-dimensional, two-dimensional NMR, MS, ECD and other data, the structures of compounds 1 - 6 can be determined as follows:

[0031]

[0032]

[0033] Compound 1: [α] 20 D 11.3 (c = 0.10, MeOH). UV (MeOH) λ max (logε) 304 (1.0), 288 (1.4), 273 (1.2) 231 (0.8) nm. ECD (0.25 mM, MeOH) λ max (Δε) 317 (27.9), 285 (1.4), 266 (5.1), 234 (-22.4), 224 (-10.2), 218 (-17.4), 210 (4.9), 203 (-13.4) nm. 1 H NMR (CDCl 3 , 600 MHz) and 13 C NMR (CDCl 3 , 150 MHz) are shown in Table 1; HRESIMS m / z 815.4361, [M + Na] + , (calcd for C 52 H 60 N 2 NaO 5 + , 815.4394).

[0034] Table 1 1 H (600 MHz) and 13 C (150 MHz) NMR spectroscopic data of Compound 1

[0035]

[0036]

[0037] Compound 2: [α] 20 D75.6 (c = 0.10, MeOH). UV (MeOH) λ max (logε) 310 (0.1), 289 (0.3), 220 (1.5) 205 (1.6) nm. ECD (0.25 mM, MeOH) λ max (Δε) 312 (11.0), 276 (1.3), 253 (9.4), 231 (-66.9), 210 (-10.2), 218 (-33.3) nm. 1 H NMR (CDCl 3 , 600 MHz) and 13 C NMR (CDCl 3 , 150 MHz) see Table 2; HRESIMS m / z 485.2068, [M + H] + , (calcd for C 29 H 29 N 2 O 5 + 485.2071), 507.1892 [M + Na] + (calcd for C 29 H 28 N 2 NaO 5 + 507.1892).

[0038] Table 2 1 H (600 MHz) and 13 C (150 MHz) NMR spectroscopic data of Compound 2

[0039]

[0040]

[0041] Compound 3: [α] 20 D 14.2 (c = 0.10, MeOH). UV (MeOH) λ max (logε) 290 (0.5), 269 (0.6), 220 (1.5) 204 (1.4) nm. ECD (0.25 mM, MeOH) λ max (Δε) 316 (7.6), 266 (0), 259 (0.7), 228 (-13.4), 201 (12.8) nm. 1 H NMR (CDCl 3 , 600 MHz) and 13 C NMR (CDCl 3, 150 MHz) See Table 3; HRESIMS m / z 417.1471, [M+H] + , (calcd for C 24 H 21 N 2 O 5 + 417.1445), m / z 439.1276 [M+Na] + (calcd for C 24 H 20 N 2 NaO 5 + 439.1264).

[0042] Table 3 1 H (600 MHz) and 13 C (150 MHz) NMR spectroscopic data of Compound 3

[0043]

[0044]

[0045] Compound 4: 1 H NMR (400 MHz, DMSO-d 6 ) δ 11.63 (1H, s, NH), 7.60 (1H, d, J = 2.4 Hz, H-2'), 7.46 (1H, t, J = 8.0 Hz, H-4'), 7.44 (1H, d, 8.0 Hz, H-7'), 7.14 (1H, t, J = 8.0 Hz, H-6'), 7.05 (1H, t, J = 8.0 Hz, H-5'), 3.75 (3H, s, 3-OCH 3 ). HRESIMS m / z 399.135, [M+H] + , (calcd for C 24 H 19 N 2 O4 + 399.1339), m / z 421.1161 [M+Na] + (calcd for C 24 H 18 N 2 NaO 4 + 421.1159).

[0046] Compound 5: 11H NMR (400 MHz, Chloroform-d) δ 8.61 (1H, s, H-1'), 8.17 (1H, s, H-1”), 7.59 (1H, d, J = 2.4 Hz, H-2'), 7.56 (1H, J = 8.0 Hz, H-7'), 7.43 (1H, d, J = 8.0 Hz, H-4'), 7.33 (1H, d, J = 8.0 Hz, H-7”), 7.30 (1H, d, J = 8.0 Hz, H-4”), 7.23 (1H, m, H-6'), 7.19 (1H, m, H-6”), 7.16 (1H, m, H-5'), 7.10 (1H, t, J = 7.4 Hz, H-5”), 6.11 (1H, dd, J = 17.4, 10.5 Hz, H-11'), 5.20 (1H, d, J = 17.4 Hz, H-12”a), 5.14 (1H, d, J = 10.5 Hz, H-12”b), 3.80 (3H, s, OCH 3 -3), 3.70 (3H, s, OCH3-6), 1.25 (6H, s, CH 3 -3” / 14”).

[0047] Compound 6: 1 1H NMR (400 MHz, Chloroform-d) δ H 8.58 (1H, brs, H-1'), 8.18 (1H, brs, H-1”), 7.58 (1H, d, J = 2.6 Hz, H-2'), 7.43 (1H, d, J = 8.0 Hz, H-4'), 7.32 (1H, d, J = 8.0 Hz, H-7”), 7.30 (1H, d, J = 8.0 Hz, H-4”), 7.17 (1H, m, H-6'), 7.15 (1H, m, H-6”), 7.10 (1H, m, H-5'), 7.06 (1H, m, H-5”), 6.11 (1H, dd, J = 17.4 10.5 Hz, H-11”), 5.22 (1H, d, J = 17.4 Hz, H-12”a), 5.17 (1H, d, J = 10.5 Hz, H-12”b), 3.81 (3H, s, OCH 3 -3), 3.69 (3H, s, OCH 3 -6), 3.61 (1H, d, J = 6.9 Hz, H-10'), 1.85 (3H, s, CH 3 -14'), 1.81 (3H, s, CH 3 -13'), 1.26 (6H, s, CH 3 -13 / 14”). 13 13C NMR (CDCl 3, 100MHz) δ C 184.3 (C, C-1), 183.7 (C, C-4), 156.4 (C, C-6), 154.7 (C, C-3), 145.6 (C, C-2″), 142.3 (CH, C-11″), 135.1 (C, C-8′), 134.7 (C, C-9″), 133.8 (C, C-12′), 130.1 (CH, C-2′), 127.6 (C, C-5), 126.9 (C, C-2), 124.4 (C, C-10″), 122.4 (C, C-9′), 122.3 (C, C-7′), 122.1 (C, C-11′), 122.0 (CH, C-6′), 120.8 (CH, C-5′), 120.7 (CH, C-5″), 120.5 (CH, C-7″), 119.5. (CH, C-6″), 118.9 (CH, C-4′), 118.5 (C, C-8″), 110.8 (C, C-3″), 105.9 (C, C-3′), 101.9 (CH 2 , C-12″), 61.0 (CH 3 , C-3-OCH 3 ), 60.3 (CH 3 , C-6-OCH 3 ), 31.0 (CH, C-10′), 27.3 (CH 3 , C-14″), 27.0 (CH 3 , C-13″), 26.0 (CH 3 , C-13′), 18.2 (CH 3 , C-14′).

[0048] Example 2 Cytotoxic Activity Test

[0049] (1) Resuscitation of Tumor Cells

[0050] The cytotoxic activities of Compounds 1-6 were tested using the MTT method, with the human gastric cancer cell line SGC-7901 as the test tumor cell line and cisplatin as the positive control drug.

[0051] First, thaw the cancer cells, centrifuge for 5 min and 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 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.

[0052] (2) MTT Assay

[0053] Human gastric cancer cells were added to a 96-well plate, 100 μL of suspension was added to each well, and it was observed after culturing in a 5% CO 2 incubator at 37 °C for one day; the test compound and the positive drug needed to be added to each well, and 100 mL of culture medium was added to the well to form the negative control group and the blank control group respectively; after a short period of time, 20 μL of MTT was added to each well, and it was cultured in the incubator for another 4 hours; the excess liquid was aspirated out, and 100 μL of DMSO was continuously added to dissolve it, and it was gently shaken by hand for about 10 minutes, and finally the absorbance was read at a wavelength of 490 nm. The following is the formula for calculating the proliferation inhibition rate:

[0054]

[0055] Inhibition rate determination criteria: having a strong proliferation inhibitory effect, IC50 value < 10 μg / mL; having an obvious proliferation inhibitory effect, IC 50 value 10 - 20 μg / mL; having a certain proliferation inhibitory effect, IC 50 value 20 - 50 μg / mL; having no obvious proliferation inhibitory activity, IC 50 value > 50 μg / mL.

[0056] The inhibition rates were tested at five concentrations (50 μg / mL, 20 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL), and finally the IC 50 was calculated, and cisplatin (1 μg / mL) was used as the positive drug. The results are shown in the following table.

[0057]

[0058]

[0059] The cytotoxicity results showed that Compounds 2 - 6 had strong cytotoxic activities; Compound 1 not only had no proliferation inhibitory effect at a concentration of 50 μg / mL, but instead showed a certain promoting effect on cell proliferation (the proliferation inhibition rate was negative). This indicates that N-H in indole may be the active group, and the introduction of the steroid structure loses cytotoxicity but brings the effect of promoting cell proliferation, and it is expected to be developed into a drug for promoting growth and differentiation.

[0060] (3) Further test the cytotoxic activities of Compounds 2 - 6 against human hepatoma cell line HepG2 and human non-small cell lung cancer cell line A549, and the results are shown in the following table.

[0061]

Claims

1. A marine fungus N4-9, characterized in that The name of the strain preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration; the preservation number is CGMCC No.40555.

2. A bisindole derivative, a tautomer thereof or a pharmaceutically acceptable salt thereof, characterized in that The bisindole derivatives have the structures shown in compounds 1 and 2:

3. A method for preparing bisindole derivatives 1, 2, 3, 4, 5 and / or 6, characterized in that The steps include: (1) Cultivating the marine fungus N4-9 according to claim 1 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 a crude extract; (4) subjecting the crude extract obtained in step (3) to chromatographic separation to obtain the bisindole derivatives 1, 2, 3, 4, 5 and / or 6; The structures of the bisindole derivatives 1, 2, 3, 4, 5, and 6 are as follows:

4. A pharmaceutical composition, characterized in that The pharmaceutical composition uses the bisindole derivatives 1, 2, their tautomers or pharmaceutically acceptable salts thereof as claimed in claim 2 as active ingredients.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition optionally contains other active ingredients having anti-tumor effects.

6. The pharmaceutical composition according to any one of claims 4 to 5, characterized in that The pharmaceutical composition also contains pharmaceutically acceptable excipients.

7. The pharmaceutical composition according to any one of claims 4 to 6, characterized in that The dosage form of the pharmaceutical composition is selected from solid preparations, liquid preparations, semisolid preparations, etc., preferably injections, tablets, capsules, lyophilized powder injections, suspensions, etc.

8. Use of the bisindole derivatives 2, 3, 4, 5, 6, their tautomers or pharmaceutically acceptable salts thereof according to claim 3 in the preparation of antitumor drugs.

9. Use of the bisindole derivative 1, its tautomer or a pharmaceutically acceptable salt thereof according to claim 2 in the preparation of a drug for promoting cell growth and differentiation.

10. Use of the marine fungus N4-9 according to claim 1 in the preparation of the bisindole derivatives 1, 2, 3, 4, 5 and / or 6 according to claim 3.

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