Indole diketopiperazine alkaloid compound as well as preparation method and application thereof

Abstract

The invention discloses an indole diketopiperazine alkaloid compound as well as a preparation method and application of the indole diketopiperazine alkaloid compound. Aspergillus sp. SYPHU504 is preserved in Guangdong Microbial Culture Collection Center on August 26, 2024, and the preservation number is GDMCC No: 65055. According to the invention, the indole diketopiperazine alkaloid compound and the pharmaceutically acceptable salt, isomer or solvate of the indole diketopiperazine alkaloid compound are separated and prepared from a fermentation culture of Aspergillus sp. SYPHU504. It is found through tests that the indole diketopiperazine alkaloid compound can remarkably inhibit proliferation of leukemia cell strains K562 and RS4: 11, and dose dependence is shown. Therefore, the compound is expected to be developed into a safe and effective novel anti-leukemia drug, and has important significance on development of Chinese marine drug resources.

Description

Technical Field

[0001] The present invention relates to the technical field of marine drugs, and more specifically to an indolediketopiperazine alkaloid compound and a preparation method and application thereof. Background Art

[0002] Leukemia, also known as blood cancer, is a malignant tumor of the hematopoietic system. Its main symptoms are that leukemia cells proliferate in large numbers in the bone marrow and other hematopoietic tissues, inhibiting normal hematopoietic function, leading to anemia, infection and bleeding tendency. Leukemia can spread to lymph nodes, spleen, liver, central nervous system and other organs, causing great harm to human life and health. Currently, chemotherapy, molecular targeted therapy, immunotherapy, combined therapy and other treatment methods are commonly used clinically, but the treatment period is expensive, and there are risks such as drug resistance and recurrence that are very likely to occur. Therefore, it is urgent to find a class of cheap, efficient, low-toxic, and broad-spectrum drugs for the treatment of leukemia to effectively solve the huge demand for leukemia inhibitors in the clinic. Summary of the invention

[0003] The purpose of the present invention is to overcome the above-mentioned defects of the prior art and provide an indoledione piperazine alkaloid compound and a preparation method and application thereof. The present invention separates and identifies three indoledione piperazine alkaloids 1-4 from the ethyl acetate extract of the fermentation product of Aspergillus sp. SYPHU504, and for the first time proves through pharmacological experiments that the above-mentioned compounds have significant activity in inhibiting the proliferation of leukemia cell lines K562 and RS4:11, and are dose-dependent. Therefore, the present invention provides an ideal candidate compound for the development of a new anti-leukemia drug.

[0004] To achieve the above object, the technical solution of the present invention is as follows:

[0005] An indoledione piperazine alkaloid compound and a pharmaceutically acceptable salt, isomer or solvate thereof, wherein the structural formula is as shown in formula (1) or formula (2) or formula (3) or formula (4):

[0006]

[0007] The present invention also discloses an Aspergillus sp.SYPHU504, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on August 26, 2024, with a deposit number of GDMCC No: 65055.

[0008] The present invention also discloses a method for preparing the above-mentioned indoledione piperazine alkaloid compounds and pharmaceutically acceptable salts, isomers or solvates thereof. The indoledione piperazine alkaloid compounds and pharmaceutically acceptable salts, isomers or solvates thereof are separated and prepared from the fermentation culture of Aspergillus sp. SYPHU504.

[0009] Optionally, the preparation method specifically comprises:

[0010] (1) Preparing fermentation products of Aspergillus sp.SYPHU504;

[0011] (2) extracting, ultrasonicating and concentrating the fermentation product in sequence to obtain a crude extract;

[0012] (3) The crude extract is subjected to silica gel column chromatography, with a mixed solvent of dichloromethane and methanol as the elution solvent for gradient elution, and the obtained fractions are subjected to ODS chromatography with a mixed solvent of methanol and water as the mobile phase for gradient elution, and then separated by HPLC-UV chromatography with a mixed solvent of methanol and water as the mobile phase to obtain the indolediketopiperazine alkaloid compound.

[0013] Optionally, step (1) specifically includes the following steps:

[0014] A culture dish containing PDA medium was inoculated with mycelium of Aspergillus sp.SYPHU504 and inverted for culture at 27°C for 5 to 7 days to obtain the strain;

[0015] The bacterial strain is inoculated into a seed liquid culture medium, and cultured in a shaking incubator at 27° C. and 180 rpm to 190 rpm for 3 to 5 days, and the culture is used as a fermentation seed liquid;

[0016] Using solid fermentation method, 20 mL of fermentation seed liquid was inoculated into rice culture medium, a total of 100 bottles were inoculated, and placed at room temperature for 30 to 32 days.

[0017] Optionally, in step (1), the PDA culture medium includes the following components: 200 g of potato, 20 g of glucose, 15 g to 20 g of agar, 1000 ml of water, and natural pH.

[0018] Optionally, in step (1), the seed liquid culture medium includes the following components: 20 g of mannose, 20 g of glucose, 10 g of peptone, 5 g of yeast extract, 1 g of corn steep liquor, 0.5 g of KH2PO4, 0.3 g of MgSO4·7H2O, and 1 L of water.

[0019] Optionally, in step (1), the rice culture medium comprises the following components: 160 g of rice and 200 mL of tap water per 1 L of Erlenmeyer flask culture medium.

[0020] Optionally, in step (2), ethyl acetate is used for extraction, and the extraction is performed 3 to 5 times at a volume ratio of 1:1 between the solid phase and the organic phase, and then the extraction is performed under reduced pressure after ultrasound to obtain an ethyl acetate extract.

[0021] Optionally, in step (3), the crude extract is separated by silica gel column chromatography, gradient elution is performed with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:0 to 0:100, and fractions eluted by gradient elution with a volume ratio of dichloromethane to methanol of 100:5 to 100:10 are collected, and the obtained fractions are subjected to ODS chromatography with a mixed solvent of methanol and water as the mobile phase in a volume ratio of 10:90 to 100:0, and fractions eluted by gradient elution with a volume ratio of methanol to water of 3:7 to 6:4 are collected, and then separated by HPLC-UV chromatography, eluted with a mixed solvent of methanol and water in a volume ratio of 3:7 to 8:2 as the mobile phase, to obtain the indoledione piperazine alkaloid compound.

[0022] The present invention also discloses the use of the above-mentioned indolediketopiperazine alkaloid compound and its pharmaceutically acceptable salt, isomer or solvate in the preparation of drugs for preventing and / or treating leukemia.

[0023] The present invention also discloses an anti-leukemia drug, comprising the above-mentioned indolediketopiperazine alkaloid compound and its pharmaceutically acceptable salt, isomer or solvate, and a pharmaceutically acceptable carrier.

[0024] Implementing the embodiments of the present invention will have the following beneficial effects:

[0025] The invention provides a method for preparing indolediketopiperazine alkaloid compounds and pharmaceutically acceptable salts, isomers or solvates thereof by fermenting Aspergillus sp.SYPHU504. Three indolediketopiperazine compounds 1-4 are obtained by fermenting and separating Aspergillus sp.SYPHU504.

[0026] The present invention has found through experiments that the indole diterpene alkaloid compounds 12β-hydroxyverruculogen TR-2 (1), 6-methoxyspirotryprostatin B (2), and spirotryprostatin A (3) can significantly inhibit the proliferation of leukemia cell lines K562 and RS4:11, and are dose-dependent. Therefore, they are expected to be developed into safe and effective new anti-leukemia drugs, which are of great significance to the development of China's marine drug resources.

[0027] Biodeposit Information:

[0028] The Aspergillus sp.SYPHU504 of the present invention is deposited in Guangdong Microbiological Culture Collection Center (GDMCC); the deposit number is GDMCC No: 65055, and the deposit time is August 26, 2024; the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Postal Code: 510070. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the strain morphology of Aspergillus sp.SYPHU504.

[0030] Figure 2 (A) is a graph showing the inhibitory activity of compounds 1-4 against the leukemia cell line K562; (B) is a graph showing the inhibitory activity of compounds 1-4 against the leukemia cell line RS4:11. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0032] Example 1

[0033] (1) The Aspergillus sp.SYPHU504 strain from sponge tissue of South China Sea (Xuwen County, Zhanjiang City, Guangdong Province) was obtained (sequencing by ITS, Gene Bank No. PP600218, confirmed to be Aspergillus genus, see Figure 1 ) mycelium, inverted and cultured for 5 days; the activated Aspergillus sp.SYPHU504 was inoculated into the seed culture medium, and dynamically cultured at 27° C. and 190 rpm for 5 days; the above seed liquid was inoculated into the rice fermentation medium, and statically cultured at room temperature for 32 days to obtain the fermentation culture.

[0034] (2) The fermentation culture obtained in step (1) is cut into small pieces, extracted with ethyl acetate three times, and then concentrated under reduced pressure to remove ethyl acetate after ultrasonication to obtain an extract.

[0035] (3) The extract obtained in step (2) is separated by silica gel column chromatography, and gradient eluted with a mixed solvent of dichloromethane and methanol in a ratio of 100:0, 100:1, 100:3, 100:5, 100:10, 100:15, 100:20, 100:30, 100:50, 100:100; the 100:5-100:10 fraction is collected and separated by ODS chromatography, and gradient eluted with methanol-water in a ratio of 1:9, 3:7, 5:5, 7:3, 9:1 as the mobile phase; the fraction eluted with a gradient of methanol: water in a volume ratio of 3:7 to 6:4 is collected, and the fraction is purified to obtain indoledione piperazine compounds 1-4.

[0036] Based on the physicochemical properties and spectral data of new compounds 1 and 2, their structures were identified as shown in formulas (1) to (4):

[0037]

[0038] The structural identification data of compound 1 are as follows:

[0039] White powder (methanol), bismuth potassium iodide solution turns orange. 1 The H NMR (600 MHz, CD3OD) spectrum gives three aromatic hydrogen signals δ H 7.41 (1H, d, J = 8.6 Hz, H-4), 6.81 (1H, d, J = 2.2 Hz, H-7), 6.74 (1H, dd, J = 8.6, 2.2 Hz, H-5); four groups of heteroatom hydrogen signals δ H 5.92 (1H, t, J = 5.7 Hz, H-18), 5.02 (1H, s, H-8), 4.30 (1H, dd, J = 11.2, 6.0 Hz, H-12), 3.77 (1H, m, H-15a), 3.61 (1H, m, H-15b); three groups of methylene hydrogen signals δ H 2.44 (1H, m, H-19a), 2.22 (2H, m, H-13), 2.11 (1H, m, H-19b), 1.95 (2H, m, H-14); a group of methoxy hydrogen signals δ H 3.80(3H,s,6-OC H 3); Two groups of methyl hydrogen signals δ H 1.45(3H,s,H-22), 1.33(3H,s,H-21). 13 The CNMR (150MHz, CD3OD) spectrum gave a total of 21 carbon atom signals, including two carbonyl carbon signals δ C 168.9 (C-11), 167.7 (C-17); six aromatic carbon signals δ C157.7 (C-6), 138.3 (C-7a), 122.4 (C-3a), 119.1 (C-4), 110.3 (C-5), 96.0 (C-7); a group of double bond carbon signals δ C 135.4 (C-2), 107.4 (C-3); two methyl carbon signals δ C 31.4 (C-21), 29.1 (C-22); one methoxy carbon signal δ C 56.0(6-O C H3), indicating that compound YN13 is a diketopiperazine alkaloid compound. The NMR signal attribution is shown in Table 1.

[0040] The structure of compound 2 is identified as follows:

[0041] White powder (methanol), bismuth potassium iodide solution turns orange. 1 In the H NMR (600 MHz, CD3OD) spectrum, three aromatic hydrogen signals δ H 7.07 (d, J = 8.4 Hz, 1H, H-4), 6.59 (dd, J = 8.4, 2.3 Hz, 1H, H-5), 6.49 (d, J = 2.3 Hz, 1H, H-7); two double bond hydrogen signals δ H 5.86 (s, 1H, H-8), 5.18 (d, J = 8.4 Hz, 1H, H-19); three groups of heteroatom-connected methine hydrogen signals δ H 3.75 (m, 1H, H-12), 5.36 (d, J = 8.4 Hz, 1H, H-18), 3.55 (m, 2H, H-15); a group of methoxy hydrogen signals δ H 3.79(s,3H,6-OC H 3); Two groups of methylene hydrogen signals δ H 2.22 (m, 2H, H-13), 2.05 (m, 2H, H-14); two groups of methyl hydrogen signals δ H 1.55(s,3H,H-21), 1.45(s,3H,H-22). 13 C-NMR (150 MHz, CD3OD) gave three carbonyl carbon signals δ C 181.1 (C-2), 164.5 (C-11), 156.8 (C-17); six aromatic carbon signals δ C 162.5 (C-6), 143.8 (C-7a), 130.6 (C-4), 121.1 (C-3a), 108.3 (C-5), 98.3 (C-7); two groups of double bond carbon signals δ C138.6 (C-20), 138.3 (C-9), 128.8 (C-19), 120.7 (C-8); two methyl carbon signals δ C 25.5(C-21), 18.4(C-22); and six sp 3 Hybridized carbon signal δ C 65.4 (C-18), 64.4 (C-12), 63.4 (C-3), 45.8 (C-15), 33.1 (C-13), 28.1 (C-14). The attribution of NMR signals is shown in Table 2.

[0042] The structure of compound 3 is identified as follows:

[0043] White powder (methanol), bismuth potassium iodide solution turns orange. 1 The HNMR (600 MHz, CD3OD) spectrum gives three aromatic hydrogen signals δ H 7.49 (1H, d, J = 8.5 Hz, H-4), 6.78 (1H, dd, J = 8.5, 2.2 Hz, H-5), 6.79 (1H, d, J = 2.2 Hz, H-7); two connected heteroatom hydrogen signals δ H 6.65 (1H, d, J = 9.7 Hz, H-18), 4.82 (1H, s, H-8); two double bond hydrogen signals δ H 5.08 (1H, m, H-24), 5.54 (1H, m, H-19); two groups of methoxy hydrogen signals δ H 3.82(3H,s,6-OC H 3), 3.38(3H,s,8-OC H 3); a methine hydrogen signal δ H 4.28 (1H, dd, J = 9.9, 6.9 Hz, H-12); four groups of methylene hydrogen signals δ H 4.64 (2H, m, H-23), 3.74 (2H, m, H-15), 2.43 (2H, m, H-13), 2.09 (2H, m, H-14); four groups of methyl hydrogen signals δ H 1.99(3H,s,H-21), 1.75(3H,s,H-26), 1.73(3H,s,H-27), 1.85(3H,s,H-22).

[0044] 13 C NMR (150MHz, CD3OD) spectrum gives two carbonyl carbon signals δ C 168.3 (C-11), 167.6 (C-17); six aromatic carbon signals δ C157.7 (C-6), 138.2 (C-7a), 119.8 (C-4), 123.4 (C-3a), 110.7 (C-5), 94.7 (C-7); three groups of double bond carbon signals δ C 138.9 (C-20), 135.8 (C-25), 134.9 (C-2), 126.0 (C-19), 121.8 (C-24), 106.1 (C-3); two methoxy carbon signals δ C 57.6(8-O C H3), 56.0(6-O C H3); four methyl carbon signals δ C 25.7 (C-27), 26.1 (C-22), 18.7 (C-21), 18.3 (C-26). See Table 3 for detailed data.

[0045] The structure of compound 4 was identified as follows:

[0046] Yellow powder (methanol), bismuth potassium iodide solution turns orange. 1 The H NMR (600 MHz, CDCl3) spectrum gives four aromatic hydrogen signals δ H 7.57 (1H, d, J = 7.9 Hz, H-4), 7.15 (1H, ddd, J = 8.0, 7.9, 1.3 Hz, H-5), 7.18 (1H, ddd, J = 8.0, 7.9, 1.3 Hz, H-6), 7.35 (1H, d, J = 8.0 Hz, H-7); three connected heteroatom hydrogen signals δ H 6.07 (1H, d, J = 9.6 Hz, H-18), 4.38 (1H, m, H-9), 3.82 (1H, m, H-12); one double bond hydrogen signal δ H 4.92 (1H, d, J = 9.6 Hz, H-19); three groups of methylene hydrogen signals δ H 3.68 (2H, m, H-15), 2.08 (2H, m, H-13), 1.95 (2H, m, H-14); two groups of methyl hydrogen signals δ H 2.01(3H,s,H-21), 1.66(3H,s,H-22). 13 C NMR (150MHz, CDCl3) spectrum gives two carbonyl carbon signals δ C 171.0 (C-11), 164.0 (C-17); six aromatic carbon signals δ C136.7 (C-7a), 126.7 (C-3a), 122.2 (C-6), 120.1 (C-5), 118.2 (C-4), 111.4 (C-7); two groups of double bond carbon signals δ C 134.6 (C-20), 133.0 (C-2), 123.6 (C-19), 103.8 (C-3); two methyl carbon signals δ C 25.8 (C-22), 18.3 (C-21), detailed data see Table 4.

[0047] Table 1 Compound 1 1 H-NMR and 13 C-NMR (CD3OD) data

[0048]

[0049] Table 2 Compound 2 1 H-NMR and 13 C-NMR (CD3OD) data

[0050]

[0051] Table 3 Compound 3 1 H-NMR and 13 C-NMR (CD3OD) data

[0052]

[0053] Table 4 Compound 4 1 H-NMR and 13 C-NMR (CD3Cl) data

[0054]

[0055]

[0056] Example 2

[0057] Antiproliferative biological activities of indoledione piperazines 1-4 against leukemia cell lines K562 and RS4:11

[0058] The MTT method was used to detect the effects of indolediketopiperazine compounds 1-4 on the viability of leukemia cell lines K562 and RS4:11. The specific experiment is as follows:

[0059] (1) Cell recovery:

[0060] a) Quickly take out the K562 and RS4:11 cell cryovials from the liquid nitrogen tank and quickly place them in a 37°C water bath, shaking them occasionally to allow them to melt rapidly within 2 minutes.

[0061] b) After complete thawing, wipe the outside of the cryopreservation tube with 75% alcohol to sterilize it, open the lid and take out the cell suspension from the cryopreservation tube.

[0062] c) Transfer the cells in the cryopreservation tube to a centrifuge tube containing 9 mL of PBS solution, mix well, and centrifuge at 1000 rpm for 4 minutes.

[0063] d) After discarding the supernatant, add 1 mL of RPMI1640 culture medium containing 10% FBS, and pipette to form a single cell suspension. Then transfer it to a T25 culture flask containing 4 mL of culture medium, shake it slowly, and then mark the cell name, generation number, and time.

[0064] e) Place the cells in a 37°C incubator containing 5% CO2 and culture. After 24 hours, replace the culture medium with fresh one to remove dead cells. The medium can be changed every two days. When the cells have grown to 80-90%, they can be passaged.

[0065] (2) Cell passaging:

[0066] a) Prepare RPMI1640 medium with 10% FBS, PBS, trypsin and related experimental supplies. Make sure they are all ready and properly disinfected and sterilized.

[0067] b) Collect old culture medium: Transfer the culture medium in the culture flask to a 10 mL centrifuge tube.

[0068] c) Centrifugation and resuspending cells: The collected cell suspension was centrifuged at 1000 rpm for 5 min, and then the supernatant was removed. 1 mL of RPMI1640 medium containing 10% FBS was added and the cell pellet was pipetted evenly.

[0069] d) Inoculation of cells: Transfer the cell suspension to a new culture flask and add an additional 4 mL of fresh culture medium.

[0070] e) Cultivation and monitoring: The inoculated cells were cultured in a 37°C incubator containing 5% CO2 and observed regularly to ensure healthy cell growth.

[0071] (3) MTT analysis

[0072] MTT tetrazolium salt is a reduced chemical dye, its chemical name is 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide, its trade name is thiazolium blue, referred to as MTT. Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to insoluble blue-purple crystals and deposit them in the cells, while dead cells have no reducing ability and therefore cannot generate blue-purple crystals. DMSO can dissolve the blue-purple crystals in the cells, and its absorbance at a wavelength of 490nM is measured by an enzyme marker. By measuring its absorbance, the number of living cells can be indirectly reflected. Within a certain range of cell numbers, the amount of MTT crystals formed is proportional to the number of cells.

[0073] a) Cell plating: The cells were cultured to the logarithmic growth phase, K562 and RS4:11 cells were counted, and the cells were added to a 96-well plate at a density of 3*10000 / ml, 100 μL per well, and no cells were added to well A1.

[0074] b) Cell administration: The administration volume per well was 10 μL, and the drug was prepared in serum-free medium. Compounds 1-4 were prepared at concentrations of 1, 25, 50, and 100 μM, respectively. The control group was given 10 μL of serum-free medium. Six replicate wells were set for each concentration. Placed in a carbon dioxide incubator and cultured at 37 degrees for 72 hours. After 72 hours of culture, 100 μL of MTT was added to each well and incubated at 37 degrees for 3-4 hours. Centrifuge and shake the plate, remove the supernatant, absorb the excess supernatant with gauze, add 100 μL of DMSO to each well, gently shake to dissolve MTT, and measure the absorbance after 10 minutes.

[0075] c) Microplate reader determination: Measure the absorbance at 490nm, set the test well and control well, check, and save the experimental results. Cell survival rate = (experimental group / blank group mean) × 100%, draw a graph with the concentration variable of the compound as the horizontal axis and the cell survival rate as the vertical axis.

[0076] (4) Statistical methods

[0077] All data were analyzed using SPSS (13.0) statistical software package. The results were expressed as mean ± standard error to evaluate the overall difference. The mean between groups was analyzed for variance homogeneity using One-Way ANOVA analysis method, and compared between groups using Dunnett's test analysis method. The Levene test was used for the homogeneity of variance test for multiple samples. When p>0.05, the variance was homogeneous, and the Dunnett's two-sided T test was used to test the differences in the means between multiple groups. When p<0.05, the variance was unequal, and the Dunnett T3 test was used to test the differences in the means between multiple groups.

[0078] (5) Activity results

[0079] The results of in vitro activity evaluation showed that compared with the blank group, compounds 1-4 could significantly reduce the expression of two cell lines K562 ( Figure 2 A) RS4:11( Figure 2 B) survival rate, and showed a certain dose dependence, and has the potential to be developed into an anti-leukemia drug.

[0080] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An indolediketopiperazine alkaloid compound and a pharmaceutically acceptable salt, isomer or solvate thereof, characterized in that: Its structural formula is shown in formula (1) or formula (2) or formula (3) or formula (4):

2. An Aspergillus sp.SYPHU504, characterized in that: The Aspergillus sp.SYPHU504 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 26, 2024, with the deposit number GDMCC No: 65055.

3. A method for preparing the indoledione piperazine alkaloid compound and its pharmaceutically acceptable salt, isomer or solvate as claimed in claim 1, characterized in that: The indolediketopiperazine alkaloid compound and its pharmaceutically acceptable salt, isomer or solvate are separated and prepared from the fermentation culture of Aspergillus sp.SYPHU504.

4. The preparation method according to claim 3, characterized in that: The preparation method specifically comprises: (1) Preparing fermentation products of Aspergillus sp.SYPHU504; (2) extracting, ultrasonicating and concentrating the fermentation product in sequence to obtain a crude extract; (3) The crude extract is subjected to silica gel column chromatography, with a mixed solvent of dichloromethane and methanol as the elution solvent for gradient elution, and the obtained fractions are subjected to ODS chromatography with a mixed solvent of methanol and water as the mobile phase for gradient elution, and then separated by HPLC-UV chromatography with a mixed solvent of methanol and water as the mobile phase to obtain the indolediketopiperazine alkaloid compound.

5. The preparation method according to claim 3, characterized in that: Step (1) specifically includes the following steps: A culture dish containing PDA medium was inoculated with mycelium of Aspergillus sp.SYPHU504 and inverted for culture at 27°C for 5 to 7 days to obtain the strain; The bacterial strain is inoculated into a seed liquid culture medium, and cultured in a shaking incubator at 27° C. and 180 rpm to 190 rpm for 3 to 5 days, and the culture is used as a fermentation seed liquid; Using solid fermentation method, 20 mL of fermentation seed liquid was inoculated into rice culture medium, a total of 100 bottles were inoculated, and placed at room temperature for 30 to 32 days.

6. The preparation method according to claim 5, characterized in that: In step (1), the PDA medium comprises the following components: 200 g of potato, 20 g of glucose, 15 g to 20 g of agar, 1000 ml of water, and natural pH; In step (1), the seed liquid culture medium comprises the following components: 20 g of mannose, 20 g of glucose, 10 g of peptone, 5 g of yeast extract, 1 g of corn steep liquor, 0.5 g of KH2PO4, 0.3 g of MgSO4·7H2O, and 1 L of water; In step (1), the rice culture medium comprises the following components: 160 g of rice and 200 mL of tap water per 1 L of Erlenmeyer flask culture medium.

7. The preparation method according to claim 5, characterized in that: In step (2), ethyl acetate is used for extraction, and the extraction is performed 3 to 5 times at a volume ratio of 1:1 between the solid phase and the organic phase, and then the ethyl acetate extract is concentrated under reduced pressure after ultrasonication.

8. The preparation method according to claim 5, characterized in that: In step (3), the crude extract is separated by silica gel column chromatography, gradient elution is performed with a mixed solvent of dichloromethane and methanol in a volume ratio of 100:0 to 0:100, and fractions eluted with a gradient ratio of dichloromethane to methanol in a volume ratio of 100:5 to 100:10 are collected, and the obtained fractions are subjected to ODS chromatography with a mixed solvent of methanol and water as a mobile phase in a volume ratio of 10:90 to 100:0, and fractions eluted with a gradient ratio of methanol to water in a volume ratio of 3:7 to 6:4 are collected, and then separated by HPLC-UV chromatography, and eluted with a mixed solvent of methanol and water in a volume ratio of 3:7 to 8:2 as the mobile phase to obtain the indoledione piperazine alkaloid compound.

9. Use of the indoledione piperazine alkaloid compound and its pharmaceutically acceptable salt, isomer or solvate as claimed in claim 1 in the preparation of a drug for preventing and / or treating leukemia.

10. An anti-leukemia drug, characterized in that: The invention comprises the indoledione piperazine alkaloid compound as claimed in claim 1 and a pharmaceutically acceptable salt, isomer or solvate thereof, and a pharmaceutically acceptable carrier.

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