An indolinedione piperazine alkaloid compound, and a preparation method and application thereof

Abstract

The application discloses an indole dione piperazine alkaloid compound and a preparation method and application thereof, and belongs to the technical field of marine natural products. The Aspergillus sp. SYPHU504 is preserved in the Guangdong Microbial Culture Collection Center on August 26, 2024, and the preservation number is GDMCC No: 65055. The indole dione piperazine alkaloid compound and a pharmaceutically acceptable salt, isomer or solvate thereof are prepared from a fermentation culture of the Aspergillus sp. SYPHU504. It is found through experiments that the indole dione piperazine alkaloid compound can significantly inhibit the proliferation of leukemia cell strains K562 and RS4:11 in a dose-dependent manner. Therefore, the indole dione piperazine alkaloid compound is expected to be developed into a safe and effective novel anti-leukemia drug, and has important significance for the development of Chinese marine drug resources.

Description

Technical Field

[0001] This invention relates to the field of marine pharmaceutical technology, and more specifically, to an indole-diketopiramate alkaloid compound, its preparation method, and its application. Background Technology

[0002] Leukemia, also known as blood cancer, is a malignant tumor of the hematopoietic system. Its main symptoms are caused by the proliferation of leukemia cells in the bone marrow and other hematopoietic tissues, which inhibits normal hematopoietic function, leading to anemia, infection, and bleeding tendencies. Leukemia can spread to lymph nodes, spleen, liver, central nervous system, and other organs, posing a significant threat to human life and health. Currently, chemotherapy, molecular targeted therapy, immunotherapy, and combination therapy are commonly used in clinical practice, but these treatments are expensive and carry risks such as drug resistance and relapse. Therefore, there is an urgent need to find a class of inexpensive, highly effective, low-toxicity, and broad-spectrum drugs for treating leukemia, effectively addressing the huge clinical demand for leukemia inhibitors. Summary of the Invention

[0003] The purpose of this invention is to overcome the above-mentioned defects in the prior art and provide an indole-diketopiramate alkaloid compound, its preparation method and application. This invention isolates and identifies three indole-diketopiramate alkaloids 1-4 from the ethyl acetate extract of the fermentation product of Aspergillus sp. SYPHU504, and for the first time demonstrates through pharmacological experiments that the above compounds have significant inhibitory activity against the proliferation of leukemia cell lines K562 and RS4:11 in a dose-dependent manner. Therefore, this invention provides ideal candidate compounds for the development of novel anti-leukemia drugs.

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

[0005] An indole-diketopiramine alkaloid compound and its pharmaceutically acceptable salts, isomers or solvates, having the structural formula shown in formula (1) or (2) or (3) or (4):

[0006]

[0007] The present invention also discloses an Aspergillus sp. SYPHU504, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 26, 2024, with accession number GDMCCNo: 65055.

[0008] The present invention also discloses a method for preparing the indole-dikepiperazine alkaloids and their pharmaceutically acceptable salts, isomers or solvates as described above, wherein the indole-dikepiperazine alkaloids and their pharmaceutically acceptable salts, isomers or solvates are prepared by isolation from the fermentation culture of Aspergillus sp. SYPHU504.

[0009] Optionally, the preparation method specifically includes:

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

[0011] (2) The fermentation product was successively extracted, sonicated and concentrated under reduced pressure to obtain crude extract;

[0012] (3) The crude extract was subjected to silica gel column chromatography with gradient elution using a mixture of dichloromethane and methanol as the elution solvent. The obtained fraction was subjected to ODS chromatography with gradient elution using a mixture of methanol and water as the mobile phase. Then, it was separated by HPLC-UV chromatography with a mixture of methanol and water as the mobile phase to obtain the indole-diketopiramate alkaloid compound.

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

[0014] Using culture dishes containing PDA medium, inoculate the mycelium of Aspergillus sp. SYPHU504 and incubate upside down at 27°C for 5 to 7 days as the inoculum.

[0015] The bacterial strain was inoculated into the seed culture medium and cultured in a shaker at 27°C and 180-190 rpm for 3-5 days. This culture was used as the fermentation seed culture.

[0016] Solid-state fermentation was used. 20 mL of the fermentation seed liquid was inoculated into rice culture medium, and a total of 100 bottles were inoculated. The culture was then placed at room temperature for 30 to 32 days.

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

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

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

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

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

[0022] The present invention also discloses the use of the indole-dikepiperazine alkaloids as described above and their pharmaceutically acceptable salts, isomers or solvates in the preparation of drugs for the prevention and / or treatment of leukemia.

[0023] The present invention also discloses an anti-leukemia drug comprising the above-mentioned indole-dikepiperazine alkaloid compounds and their pharmaceutically acceptable salts, isomers or solvates, and a pharmaceutically acceptable carrier.

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

[0025] This invention provides a method for preparing indole-dione-piperazine alkaloids and their pharmaceutically acceptable salts, isomers or solvates by fermentation with Aspergillus sp. SYPHU504. Three indole-dione-piperazine compounds 1-4 were isolated by fermentation with Aspergillus sp. SYPHU504.

[0026] This invention, through experiments, has found that indole diterpenoid alkaloids 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 in a dose-dependent manner. Therefore, they hold promise for development into safe and effective novel anti-leukemia drugs, which is of great significance for the development of marine drug resources in China.

[0027] Biological Preservation Information:

[0028] The Aspergillus sp. SYPHU504 of this invention is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC); accession number: GDMCC No: 65055, deposit date: August 26, 2024; deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China. Attached Figure Description

[0029] Figure 1 The strain morphology of Aspergillus sp. SYPHU504.

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

[0031] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0032] Example 1

[0033] (1) Aspergillus sp. SYPHU504, a strain derived from sponge tissue in the South China Sea (Xuwen County, Zhanjiang City, Guangdong Province), was identified as belonging to the Aspergillus genus by ITS sequencing (Gene Bank number PP600218). (See [link to relevant documentation]). Figure 1 The mycelium of Aspergillus sp. SYPHU504 was inoculated into seed culture medium and cultured dynamically at 27°C and 190 rpm for 5 days. The above seed culture was inoculated into rice fermentation medium and cultured statically at room temperature for 32 days to obtain fermentation culture.

[0034] (2) The fermentation culture obtained in step (1) was cut into small pieces, extracted with ethyl acetate three times, and concentrated under reduced pressure after sonication to remove ethyl acetate, thus obtaining the extract.

[0035] (3) The extract obtained in step (2) was separated by silica gel column chromatography with gradient elution of dichloromethane and methanol at ratios of 100:0, 100:1, 100:3, 100:5, 100:10, 100:15, 100:20, 100:30, 100:50, and 100:100. The fractions of 100:5 to 100:10 were collected and separated by ODS chromatography with gradient elution of methanol-water at ratios of 1:9, 3:7, 5:5, 7:3, and 9:1. The fractions eluted by gradient elution of methanol:water at volume ratios of 3:7 to 6:4 were collected, and the fractions were purified to obtain indole-dione-piperazine compounds 1-4.

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

[0037]

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

[0039] White powder (methanol), turns orange when mixed with potassium bismuth iodide solution. 1 The 1H NMR (600MHz, 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 hydrogen signals δ of linked heteroatoms H 5.92 (1H,t,J=5.7Hz,H-18), 5.02 (1H,s,H-8), 4.30 (1H,dd,J=11.2,6.0Hz,H-12), 3.77 (1H,m,H-15a), 3.61 (1H,m,H-15b); three sets 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 set of methoxy hydrogen signals δ H 3.80(3H,s,6-OC H 3); Two sets of methyl hydrogen signals δ H 1.45(3H,s,H-22), 1.33(3H,s,H-21). 13 The CNMR (150MHz, CD3OD) spectrum yielded signals for 21 carbon atoms, 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 set of double-bonded 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); a methoxy carbon signal δ C 56.0(6-O C H3) indicates that compound YN13 is a diketopiperazine alkaloid compound, and the NMR signal assignments are shown in Table 1.

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

[0041] White powder (methanol), turns orange in potassium bismuth iodide solution. 1 The 1H NMR (600MHz, CD3OD) spectrum gives 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); δ signals of methylene hydrogen atoms in three groups of heteroatoms H 3.75 (m, 1H, H-12), 5.36 (d, J = 8.4 Hz, 1H, H-18), 3.55 (m, 2H, H-15); a set of methoxy hydrogen signals δ H 3.79(s,3H,6-OC H 3); Two sets of methylene hydrogen signals δ H 2.22 (m, 2H, H-13), 2.05 (m, 2H, H-14); two sets of methyl hydrogen signals δ H 1.55(s,3H,H-21), 1.45(s,3H,H-22). 13 C-NMR (150MHz, CD3OD) yielded 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 sets 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 The NMR signal attributions are shown in Table 2: 65.4 (C-18), 64.4 (C-12), 63.4 (C-3), 45.8 (C-15), 33.1 (C-13), 28.1 (C-14).

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

[0043] White powder (methanol), turns orange when mixed with potassium bismuth iodide solution. 1 The HNMR (600MHz, 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); δ signals of two connected heteroatoms (hydrogen atoms). 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 sets of methoxy hydrogen signals δ H 3.82(3H,s,6-OC H 3), 3.38(3H,s,8-OC) H 3); A methylene hydrogen signal δ H 4.28 (1H, dd, J = 9.9, 6.9 Hz, H-12); four sets 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 The 150 MHz C NMR (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 sets 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), turns orange when mixed with potassium bismuth iodide solution. 1 The 1H NMR (600MHz, CDCl3) spectrum gives four aromatic hydrogen signals δ H 7.57 (1H,d,J=7.9Hz,H-4), 7.15 (1H,ddd,J=8.0,7.9,1.3Hz,H-5), 7.18 (1H,ddd,J=8.0,7.9,1.3Hz,H-6), 7.35 (1H,d,J=8.0Hz,H-7); δ signals of three bonded heteroatoms of hydrogen H 6.07 (1H, d, J = 9.6 Hz, H-18), 4.38 (1H, m, H-9), 3.82 (1H, m, H-12); a double bond hydrogen signal δ H 4.92 (1H, d, J = 9.6 Hz, H-19); three sets 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 sets of methyl hydrogen signals δ H 2.01(3H,s,H-21), 1.66(3H,s,H-22). 13 The 150 MHz, CDCl3 NMR spectrum (150 MHz, CDCl3) 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 sets 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), see Table 4 for detailed data.

[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 bioactivity of indole-dione-piperazine compounds 1-4 against leukemia cell lines K562 and RS4:11

[0058] The effects of indole-diketopiramate compounds 1-4 on the viability of leukemia cell lines K562 and RS4:11 were detected using the MTT assay. Specific experiments are as follows:

[0059] (1) Cell resuscitation:

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

[0061] b) After complete thawing, wipe the outside of the cryovial with 75% alcohol to sterilize it, then open the cap and remove the cell suspension from the cryovial.

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

[0063] d) After discarding the supernatant, add 1 mL of RPMI 1640 medium containing 10% FBS and pipette to form a single-cell suspension. Then transfer the suspension to a T25 culture flask containing 4 mL of culture medium, shake gently, and label the cell name, passage number, and time.

[0064] e) Incubate in a 37°C incubator containing 5% CO2. After 24 hours, replace with fresh culture medium and remove dead cells. Change the medium every two days. When the cells reach 80-90% cell growth, passage them.

[0065] (2) Cell passage:

[0066] a) Prepare RPMI 1640 medium with 10% FBS, PBS, trypsin, and other relevant laboratory supplies. Ensure that all supplies are ready and have been properly sterilized and treated.

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

[0068] c) Centrifugation and resuspension of cells: Centrifuge the collected cell suspension at 1000 rpm for 5 min, then remove the supernatant, add 1 mL of 10% FBS RPMI 1640 medium and pipette to mix the cell pellet evenly.

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

[0070] e) Culture and monitoring: After inoculation, the cells are placed in an incubator containing 5% CO2 at 37°C and observed regularly to ensure healthy cell growth.

[0071] (3) MTT analysis

[0072] MTT (3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide) is a reducing chemical dye, commercially known as thiazolium blue, or simply MTT. In living cells, mitochondria succinate dehydrogenase reduces exogenous MTT to insoluble blue-violet crystals, which then deposit within the cells. Dead cells lack this reducing ability and therefore do not form these blue-violet crystals. DMSO dissolves the blue-violet crystals in the cells, and the absorbance at 490 nm is measured using a microplate reader. This absorbance indirectly reflects the number of living cells. Within a certain cell number range, the amount of MTT crystals formed is directly proportional to the cell number.

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

[0074] b) Cell drug administration: 10 μL of drug was administered to each well using serum-free medium to prepare compounds 1-4 at concentrations of 1, 25, 50, and 100 μM. The control group received 10 μL of serum-free medium. Six replicates were set up for each concentration. The cells were incubated at 37°C for 72 hours. After 72 hours of incubation, 100 μL of MTT was added to each well, and the cells were incubated at 37°C for 3-4 hours. The cells were centrifuged, the supernatant was discarded, and excess supernatant was aspirated with gauze. 100 μL of DMSO was added to each well, and the MTT was gently dissolved by shaking. The absorbance was measured after 10 minutes.

[0075] c) Microplate reader assay: Measure absorbance at 490 nm, set up test wells and control wells, check and save the experimental results. Cell viability = (mean of experimental group / blank group) × 100%. Plot a graph with the concentration of the compound on the x-axis and the cell viability on the y-axis.

[0076] (4) Statistical methods

[0077] All data were analyzed using SPSS (13.0) statistical software. Results are expressed as mean ± standard error. To assess overall differences, one-way ANOVA was used to analyze homogeneity of variance between groups, combined with Dunnett's test for inter-group comparisons. The Levene test was used to test homogeneity of variance in multiple samples. When p > 0.05, the variances were homogeneous; Dunnett's two-tailed t-test was used to assess differences in means among groups. When p < 0.05, the variances were unequal; Dunnett's T3 test was used to assess differences in means among groups.

[0078] (5) Activity Results

[0079] In vitro activity evaluation results showed that, compared with the blank group, compounds 1-4 significantly reduced the activity of both cell lines K562 (… Figure 2 A) RS4:11 ( Figure 2 B) has a high survival rate and exhibits a certain dose-dependent effect, showing the potential to be developed into an anti-leukemia drug.

[0080] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing an indole-diketopiramate alkaloid compound and its pharmaceutically acceptable salt, characterized in that, Its structural formula is shown in equation (1), equation (2), equation (3), or equation (4): ； The indole-diketopiperazine alkaloids and their pharmaceutically acceptable salts were isolated and prepared from the fermentation culture of Aspergillus sp. SYPHU504; The preparation methods specifically include: (1) Preparation of fermentation products of Aspergillus sp. SYPHU504; (2) The fermentation product was successively extracted, sonicated and concentrated under reduced pressure to obtain a crude extract; (3) The crude extract was subjected to silica gel column chromatography with gradient elution using a mixture of dichloromethane and methanol as the elution solvent. The obtained fraction was subjected to ODS chromatography with gradient elution using a mixture of methanol and water as the mobile phase. Then, it was separated by HPLC-UV chromatography with a mixture of methanol and water as the mobile phase to obtain the indole-diketopiramate alkaloid compound.

2. The preparation method according to claim 1, characterized in that, Step (1) specifically includes the following steps: Using a culture dish containing PDA medium, inoculate the mycelium of Aspergillus sp. SYPHU504 and incubate it upside down at 27°C for 5 to 7 days as the inoculum. The bacterial strain was inoculated into the seed culture medium and cultured in a shaker at 27°C and 180-190 rpm for 3-5 days. This culture was used as the fermentation seed culture. Solid-state fermentation was used. 20 mL of the fermentation seed liquid was inoculated into rice culture medium, and a total of 100 bottles were inoculated and placed at room temperature for 30 to 32 days.

3. The preparation method according to claim 2, characterized in that, 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. In step (1), the seed 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. In step (1), the rice culture medium consists of the following components: 160 g of rice and 200 mL of tap water per 1 L Erlenmeyer flask.

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

5. The preparation method according to claim 2, characterized in that, In step (3), the crude extract is separated by silica gel column chromatography, and gradient elution is performed with a mixed solvent of dichloromethane and methanol at a volume ratio of 100:0 to 0:

100. The fractions eluted by gradient elution of dichloromethane and methanol at a volume ratio of 100:5 to 100:10 are collected. The obtained fractions are then subjected to ODS chromatography, and gradient elution is performed with a mixed solvent of methanol and water at a volume ratio of 10:90 to 100:

0. The fractions eluted by gradient elution of methanol:water at a volume ratio of 3:7 to 6:4 are collected. The fractions are then separated by HPLC-UV chromatography, and eluted with a mixed solvent of methanol and water at a volume ratio of 3:7 to 8:2 to obtain the indole-diketopiramate alkaloid compound.

6. The use of an indole-diketopiramate alkaloid compound prepared by the method described in claim 1 and its pharmaceutically acceptable salt in the preparation of drugs for the prevention and / or treatment of leukemia.

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