CN114349814B

CN114349814B - Friedelane type compound and preparation method and application thereof

Info

Publication number: CN114349814B
Application number: CN202210068659.5A
Authority: CN
Inventors: 许少华; 徐伟; 范若兰
Original assignee: Fujian University of Traditional Chinese Medicine
Current assignee: Fujian University of Traditional Chinese Medicine
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2023-02-24
Anticipated expiration: 2042-01-20
Also published as: CN114349814A

Abstract

The invention discloses a friedelane type compound and a preparation method and application thereof, belonging to the technical field of medicines. The compound can obviously inhibit STAT3 phosphorylation of colon tumor cells and downstream proteins thereof, has good affinity with human STAT3 recombinant proteins, can be used for preparing STAT3 small-molecule inhibitors, and is used for preparing medicines for preventing and/or treating diseases related to STAT3 activity. Compared with tripterine, the compound provided by the invention has the advantages that the effect of inhibiting STAT3 protein phosphorylation is obviously improved, and the activity of inhibiting HCT-116 colorectal cancer cell proliferation is stronger. The compound can promote HCT-116 colorectal cancer cell apoptosis and block cell cycle, has stronger inhibiting effect on the activity of colorectal cancer organoid than oxaliplatin as a positive drug, has basically no toxicity on normal colorectal organoids, and can be used for preparing the drug for treating colorectal cancer. The preparation method of the compound is green and environment-friendly, has high yield and is suitable for expanded production.

Description

Friedelane type compound and preparation method and application thereof

Technical Field

The invention relates to a friedelane type compound and a preparation method and application thereof, belonging to the technical field of medicines.

Background

STAT3 is a cytoplasmic transcription factor that regulates biological functions including cell proliferation, differentiation, survival, angiogenesis, and immune response by regulating the transcription of a variety of genes. STAT3 is an important member of transcription signal transduction and activation factors, and continuous activation thereof can up-regulate cell cycle regulatory factors and anti-apoptotic proteins in tumor cells, promote abnormal proliferation and malignant transformation of tumors, and exert important effects in the occurrence and development of various cancers. Recent studies have shown that under normal physiological conditions, the activation of STAT3 protein is tightly controlled; the protein is over-activated and expressed at high level in cells of various tumors (such as breast cancer, ovarian cancer, head and neck squamous cell carcinoma, prostatic cancer, malignant melanoma, multiple myeloma, lymphoma, brain tumor, non-small cell lung cancer, colorectal cancer, various leukemia and the like).

Among them, colorectal cancer (CRC) is the most common malignant tumor in gastrointestinal tract, and in recent years, with the improvement of living standard of people, the change of dietary habits and the continuous increase of colorectal cancer incidence rate, it has become one of the main tumors threatening the life health of the middle-aged and elderly people. Colorectal cancer has a high risk of metastasis, and the search for an effective treatment is urgent.

Tripterygium wilfordii is a traditional Chinese medicinal material, namely a plant in the genus Tripterygium in the family Celastraceae, in China, and one of effective components of the tripterygium wilfordii is as follows: tripterine is proved to have wide pharmacological effects. Clinically, tripterine is used as a traditional medicine for treating various inflammatory diseases and autoimmune diseases, such as allergic asthma, amyotrophic lateral sclerosis, rheumatoid arthritis and the like. The research reports that the tripterine can inhibit the generation of NO, IL-6, TNF-alpha and IL-1 beta induced by LPS, and effectively inhibit the expression of JAK1, p-JAK1, JAK2, p-JAK2, STAT3 and p-STAT3 (the research on the synergistic anti-inflammatory effect of the tripterine and indomethacin on lipopolysaccharide-induced RAW264.7 cells, zhongnan pharmacy, no. 11/11). In addition, with the progress of research, people gradually find that the tripterine can also inhibit the occurrence and development of various tumors, such as breast cancer, liver cell liver cancer, bladder cancer, gastric cancer, prostate cancer, colorectal cancer and the like (the research on the mechanism of the tripterine for inhibiting the growth of colorectal cancer, the paper of doctor research student in Beijing, together with hospitals, 2017).

However, the inhibition effect of tripterine on STAT3 cannot meet the demand, and the development of a new compound having a more excellent inhibition effect on STAT3 is of great significance in the treatment of diseases associated with STAT3 activity.

Disclosure of Invention

The invention aims to provide a friedelane type compound with a novel structure, and a preparation method and application thereof.

The invention provides a compound shown as a formula I or a salt, a stereoisomer, a deuterated compound and a solvate thereof:

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ Each independently selected from hydrogen and C _1～4 An alkyl group.

Further, the compound is:

the present invention also provides a process for preparing the above compound CEL-2, comprising the steps of: culturing streptomyces griseus in a culture medium until logarithmic phase, adding tripterine, continuing culturing for 2-6, ending fermentation, taking fermentation supernatant, separating and purifying to obtain a compound CEL-2.

Further, the culture medium is a soybean meal liquid culture medium; the final concentration of the tripterine is 10-20mg/250mL; the culture is shake culture at 25-30 deg.C and rotation speed of shaker of 150-200 rpm/min ^-1 (ii) a The continuous culture time is 3-5 days;

the separation and purification method comprises the following steps: and (3) taking the fermentation supernatant, extracting with ethyl acetate, reserving an organic phase, concentrating under reduced pressure, and purifying by adopting silica gel column chromatography and a semi-preparative liquid phase in sequence.

Further, the final concentration of the tripterine is 15mg/250mL; the temperature of the cultureAt 28 deg.C, the rotation speed of the shaker is 180rpm min ^-1 (ii) a The time for the continuous culture was 4 days.

Further, the soybean meal liquid medium consists of the following components in concentration: 5g/L of soybean meal, 20g/L of glucose, 5g/L of yeast extract, 5g/L of NaCl and K ₂ HPO ₄ 5g/L。

The invention also provides a pharmaceutical composition, which is a preparation prepared by taking the compound or the salt, the stereoisomer, the deuterated compound and the solvate thereof as active ingredients and adding pharmaceutically acceptable auxiliary materials.

Further, the preparation is capsule, tablet, granule, powder, pill or injection.

The invention also provides application of the compound or salt, stereoisomer, deuterated compound and solvate thereof in preparation of STAT3 inhibitor.

Further, the STAT3 inhibitor can inhibit STAT3 phosphorylation, inhibit tumor cell proliferation, promote tumor cell apoptosis, and/or arrest tumor cell cycle.

Further, the STAT3 inhibitor is a drug for preventing and/or treating tumors, preferably breast cancer, ovarian cancer, head and neck squamous cell carcinoma, prostate cancer, malignant melanoma, multiple myeloma, lymphoma, brain tumor, non-small cell lung cancer or colorectal cancer.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a friedelane type compound with a novel structure: CEL-2. The compound can obviously inhibit STAT3 phosphorylation of colon tumor cells and downstream proteins thereof, has good affinity with human STAT3 recombinant proteins, can be used for preparing STAT3 small-molecule inhibitors, and is used for preparing medicines for preventing and/or treating diseases related to STAT3 activity. Compared with tripterine, the compound provided by the invention has the advantages that the effect of inhibiting STAT3 protein phosphorylation is obviously improved, and the activity of inhibiting HCT-116 colorectal cancer cell proliferation is stronger.

The compound can promote HCT-116 colorectal cancer cell apoptosis and block cell cycle, has stronger inhibiting effect on the activity of colorectal cancer organoid than oxaliplatin as a positive drug, has basically no toxicity on normal colorectal organoids, and can be used for preparing the drug for treating colorectal cancer.

The preparation method of the compound is green and environment-friendly, has high yield and is suitable for expanded production.

It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a drawing of CEL-2, a compound of the present invention ¹ H NMR spectrum.

FIG. 2 is a drawing of CEL-2, a compound of the present invention ¹³ C NMR spectrum.

FIG. 3 is a diagram of HMBC (H → C) of CEL-2, a compound of the present invention.

FIG. 4 is a graph of HR-ESI-MS of CEL-2, a compound of the present invention.

FIG. 5 shows Western blotting to analyze STAT3 phosphorylation and inhibition of upstream proteins (JAK-2, p-JAK-2) of HCT-116 cells by drugs. Wherein Panel A is a graph of drug versus transcriptional activity results; and the graph B is a result graph of no obvious effect of the medicine on STAT3 protein upstream protein tyrosine kinase (JAK-2). (indicates that the animal has statistical difference compared with the untreated control group: ( ^* P＜0.05， ^** P is less than 0.01); (#) indicates that there is a statistical difference compared with the IL-6 group: ( ^# P＜0.05， ^## P＜0.01)。

FIG. 6 is a graph of the binding characteristics of CEL-2 and STAT3 analyzed using surface plasmon resonance. Wherein, the graph A is a reaction sensorgram of the combination of the compound CEL-2 and STAT3, and the graph B is a steady-state affinity model fitting schematic diagram of the affinity constant of the compound CEL-2 and STAT 3.

FIG. 7 shows the detection of HCT-116 apoptosis and cell cycle arrest by CEL-2 compound using flow cytometry. Wherein FIGS. A1-A4 are graphs showing the results of apoptosis, and FIG. A5 is a graph showing the cycle of apoptosis; FIGS. B1-B5 are graphs showing cell cycle trends.

FIG. 8 is a graph showing the inhibition of colorectal cancer by the compound CEL-2, which was tested using 4 colorectal organoid models (CCO-1, CCO-2, CCO-3, CCO-4) cultured in vitro. Wherein Panel A is a representative microscopy image of organoids incubated with Compound CEL-2 with human colorectal cancer organoids (CCO-4) and 1 Colorectal Normal Organoid (CNO) for 6d at different concentrations; FIG. B is a dose-dependent effect of Compound CEL-2 on organoid viability; panel C is a representative microscope image of organisms incubated with L-OHP with CCO-4 and CNO for 6 days at different concentrations; panel D is the dose-dependent effect of L-OHP on organoid viability.

Detailed Description

The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.

Example 1 preparation of Compound CEL-2

The method comprises the following steps: 400mg of tripterine is dissolved in 40ml of absolute ethyl alcohol to prepare a substrate solution of 10 mg/ml. The two-step culture method is carried out on a soybean meal liquid culture medium (formula: commercially available soybean meal 5g, glucose 20g, yeast extract 5g, naCl 5g, K) ₂ HPO ₄ 5g, fixing the volume of distilled water to 1L), culturing the streptomyces griseus to a logarithmic growth phase (the culture condition is 28 ℃, and the rotating speed of a shaking table is 180 rpm/min), adding the substrate solution (the final concentration of the tripterine is controlled to be 15mg/250 mL), continuing to culture for 4 days, ending fermentation, extracting a fermentation supernatant for 3 times by adopting equal volume of ethyl acetate, and recovering the organic solvent under reduced pressure to obtain a fermentation product containing the compound CEL-2.

Step two: separating and purifying the fermentation product obtained in the step one by adopting silica gel column chromatography and combining with a semi-prepared liquid phase to obtain a compound CEL-2: column chromatography was performed using silica gel (200-300 mesh) column, eluting with dichloromethane: methanol system polarity gradient elution (dichloromethane: methanol volume ratio 98-90). The purity of the obtained compound CEL-2 is more than 99 percent, and the yield is 25 to 30 percent.

The separated and purified compound CEL-2 is subjected to a reaction based on ¹ H NMR and ¹³ c NMR method to obtain NMR spectrum and ¹ heteronuclear multiple carbon correlation spectrum of H (HMBC (H → C)), using UPLC-Q-TOF-MS based characterization, HR-ESI-MS plot was obtained. The results are shown in FIGS. 1-4.

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Experimental example 1 Effect of CEL-2 Compound on STAT 3-specific inhibitor

1. Experimental method

In the experimental example, western blotting was used to analyze the effect of CEL-2 on STAT3 phosphorylation and downstream proteins (Survivin, MCL-1) in HCT-116 cells, and on upstream proteins (JAK-2, p-JAK-2), and the specific method was as follows:

inducing inflammation of HCT-116 cells by IL-6 stimulation, culturing HCT-116 cells in RPMI/1640 medium containing 10% FBS, and when the cells grew to logarithmic growth phase, collecting the cells by trypsinization centrifugation (centrifugation at 1200rpm/min for 4 min) using 0.25% EDTA at 1: and 3, diluting and passaging.

(1) Detecting the inhibition effect of Celastrol (CEL) and CEL-2 on HCT-116 cells by CCK-8 method, and calculating the IC of CEL and CEL-2 on HCT-116 cells ₅₀ After centrifugation of HCT-116 cells for routine digestion, the cell suspension concentration was adjusted to 5X 10 cells/ml ³ Each of the cells was inoculated into a 96-well cell culture plate at 100. Mu.L/well, and cultured in a 5-% CO2 incubator at 37 ℃. Culturing normally for 24h, after the cells are completely attached to the wall, sucking off the culture medium, adding 100 μ L of culture medium and cells to the control group without adding drugs, and adding prepared liquid drugs with different concentrations to the CEL-2 group at 0.02, 0.1, 0.5, 2.5 and 12.5 μ M, each well at 100 μ L, and administering for 24h.

Then 10. Mu.L of CCK-8 enhanced solution per well was added, and since the amount of CCK-8 added per well was small, the medium containing 10% CCK-8 was prepared directly and added as a change solution. Care was taken not to generate bubbles in the wells so as not to affect the detection of absorbance. After culturing at 37 ℃ for 4h in a 5% CO2 incubator, the OD value of absorbance at 450nm was measured with a microplate reader. Calculating the cell proliferation inhibition rate according to the following formula: inhibition = [ (control-dose)/(control-blank) ] × 100%.

(2) Removing cell culture solution from HCT-116 cells, washing twice a 6-well plate with precooled PBS, adding 100 μ L/well of RIPA lysate pre-prepared with 1% PMSF and 1% phosphatase inhibitor, lysing on ice for 30min, sucking all liquid into a clean 1.5mL EP tube, centrifuging at 4 ℃, 12000rpm for 15min, sucking supernatant, and filling into another EP tube to obtain total protein; the BCA kit was used for protein quantification, proteins were added to a protein loading buffer according to 1. Blocking in TBST buffer solution containing 5% skimmed milk powder for 2h, incubating at 4 deg.C overnight, washing membrane with TBST for 5min 3 times, incubating in TBST diluted secondary antibody for 2h at room temperature, washing membrane with TBST for 5min 3 times, and scanning with gel imaging system.

(3) Western blotting was used to detect the inhibition of STAT3 phosphorylation by CEL-2 on HCT-116 cells. HCT-116 cells are subjected to CEL-2 administration 24h, IL-6 stimulation for 30min to generate inflammatory reaction, then the cells are cracked and collected, and western blot is used for detecting the expression levels of p-STAT3, survivin and Mcl-1 proteins.

Tripterine (CEL) was used as a control drug. The results are shown in FIG. 5.

2. Results of the experiment

CCK-8 results indicate inhibitory Activity on HCT-116 cell proliferation, CEL-2 (IC) ₅₀ =0.66 +/-0.02) is obviously stronger than CEL (IC) ₅₀ ＝1.40±0.21)。

From FIG. 5A, it is clear that CEL-2 at 0.5. Mu.M, 0.75. Mu.M, and 1. Mu.M can inhibit phosphorylation of STAT3 protein to various degrees, and exhibits concentration dependence; it is clear from FIG. 5B that CEL-2 has no inhibitory effect on the activation of its upstream protein JAK-2. CEL-2 was demonstrated to be a specific inhibitor of STAT 3.

In addition, as seen from fig. 5A, the effect of CEL-2 of the compound of the present invention in inhibiting STAT3 protein phosphorylation was significantly improved and significantly different from CEL at the same concentration.

The experiment results show that CEL-2 provided by the invention can obviously inhibit the proliferation activity of HCT-116 colorectal cancer cells and inhibit STAT3 phosphorylation of the colorectal cancer cells. In addition, compared with tripterine, the compound CEL-2 has stronger activity of inhibiting HCT-116 colorectal cancer cell proliferation, and the effect of inhibiting STAT3 protein phosphorylation is obviously improved.

EXAMPLE 2 test of binding dissociation constant of Compounds CEL-2 and STAT3

1. Experimental method

In the experimental example, the surface plasmon resonance technology is used for analyzing the interaction mode of CEL-2 and STAT3, and the specific method is as follows:

(1) The optimal concentration of human STAT3 recombinant protein and the optimal pH condition are selected according to ligand pre-enrichment, STAT3 protein is dissolved in buffer solutions with different pH values, and can be combined to carboxyl on the surface of a CM5 chip through electrostatic adsorption when flowing through the surface of the chip, 500 mu g/mL STAT3 protein and sodium acetate buffer solutions (10 mM, pH4.5,5.0 and 5.5) with different pH values are uniformly mixed, so that the final concentration of the protein is 10 mu g/mL, and bubbles can be removed by centrifugation if the bubbles exist. Protein solutions prepared by sodium acetate buffers with different pH values are injected onto a CM5 sensing chip, the binding curve of the protein solutions and the chip is observed, 50mM NaOH is used for regeneration, and the optimal pH value of STAT3 protein is finally determined to be 5.5.

(2) STAT3 is coupled to a CM5 chip by an amino coupling method, and 100 mu L of NHS (N-hydroxysuccinimide) and 100 mu L of EDC (ethyl [3- (dimethylamino) propyl ] carbodiimide hydrochloride) are mixed in an amino coupling kit; mixing 4 μ L STAT3 protein and 196 μ L sodium acetate with pH of 5.5 in an EP tube; then, 140. Mu.L of ethanolamine was taken from the amino-coupled kit into an EP tube to block the non-protein-bound region on the surface of the CM5 sensor chip. Equilibrate to room temperature around 25 ℃, place the EP tube on the sample holder and set the flow rate at 10 μ L/min for 420s. The binding value of STAT3 protein to the chip was calculated to be 14937RU.

(3) CEL-2 was diluted with PBS containing 5% DMSO to 7 gradients of 3.91. Mu.M, 7.81. Mu.M, 15.63. Mu.M, 31.25. Mu.M, 62.5. Mu.M, 125. Mu.M and 250. Mu.M, respectively, and the samples were sequentially injected from low to high concentrations, and the 3.91. Mu.M set was repeated to determine whether the system was stable, with a binding time of 80s, a flow rate of 30. Mu.L/min, and a dissociation time of 180s. The obtained experimental result is fitted by a computer and is subjected to steady state analysis to obtain the affinity K of the protein _D The values, results are shown in FIG. 6.

2. Results of the experiment

As is clear from FIG. 6, CEL-2 specifically binds STAT3 in a concentration-dependent manner, and the dissociation constant K of the binding _D Is 4.096X 10 ^-5 M。

EXAMPLE 3 Effect of CEL-2 test Compound on inhibiting growth of colorectal cancer cells

In the experimental example, annexin V-FITC/PI staining is combined with a flow cytometer to detect the apoptosis and cell cycle retardation of CEL-2 on HCT-166 cells; the specific operation steps are as follows:

(1) Culturing HCT-116 cells in vitro, digesting, centrifuging and collecting cells when the cells grow to logarithmic growth phase, and diluting the cells to 2 × 10 ⁵ Per ml, 2ml per well were inoculated in 6-well plates and, at 37 ℃,5% CO ₂ Culturing for 24h in an incubator, sucking off the supernatant, replacing with STAT3 (0, 0.661,1.65,3.3,5 μ M) with different concentrations for culturing for 48h, centrifuging to collect cell precipitate, washing the cells with precooled PBS twice, discarding the supernatant, adding 1 × Binding Buffer working solution to resuspend the cells until the cell concentration reaches 1 × 10 ⁶ And/ml, sucking 100 mu l of cell suspension, subpackaging into new tubes, adding Annexin V-FITC/PI into the tubes respectively for staining, incubating for 15min at room temperature in a dark place, adding 1 × Binding Buffer working solution, uniformly mixing, detecting by using a flow cytometer at 488nm, analyzing by using Novoexpress software, drawing a double-dispersion dot diagram, and obtaining a result shown in figure 7.

(2) The culture and administration mode is the same as that in step (1), the cells are washed by precooled PBS after cell precipitation is collected by centrifugation, the supernatant is discarded, precooled 70% ethanol is added to blow and mix uniformly, after 12h of fixation at 4 ℃, centrifugation is carried out at 1000rpm for 3min, PI is added, incubation is carried out at 37 ℃ in the dark for 30min, and the fluorescence intensity is measured at 488nm by using a flow cytometer, and the result is shown in figure 7.

2. Results of the experiment

As can be seen from FIG. 7, CEL-2 can promote apoptosis of HCT-116 cells, and compared with the blank control group, HCT-116 cells to which CEL-2 was administered for interference increase, and the apoptosis percentage increases with the increase of concentration, thus confirming that CEL-2 inhibits the activity of colorectal cancer by inducing apoptosis of colorectal cancer cells.

It is also shown in FIG. 7 that CEL-2 interferes with the normal cell cycle of HCT-116, and it can be seen from FIGS. B1-B4 that CEL-2 increases G0/G1 cells and arrests S phase cell cycle compared to the control group, further indicating that CEL-2 blocks cell cycle to induce apoptosis, thereby inhibiting colorectal cancer growth.

The experimental results show that the compound CEL-2 can induce the apoptosis of colorectal cancer cells and inhibit the growth of the colorectal cancer cells.

Experimental example 4 verification of inhibitory Effect of CEL-2 on colorectal cancer Using in vitro culture colorectal organoid model

1. Experimental method

In the experimental example, the inhibition effect of the compound CEL-2 on the colorectal cancer is verified by adopting an in vitro culture colorectal organoid model, and the specific operation is as follows:

(1) Human Colorectal Cancer Organoids (CCOs) were obtained from cryopreserved organoids banks and resuspended in matrigel and complete medium (about 2. The complete medium was DMEM/F12 supplemented with R-spondin-1 recombinant protein (500 ng mL) ^-1 ) Noggin recombinant protein (100 ng. ML) ^-1 ),EGF(50ng·mL ^-1 ) HEPES buffer, glutamine additive, N ₂ Additive (1X), N-acetylcysteine (1 mM), nicotinamide (10 mM), A83-01 (500 nM), Y-27632 (10. Mu.M), SB202190 (3. Mu.M), B27 additive (1X); replacing the culture medium once in 48 hours, carrying out passage once in 10-14 days, and using 1-2 mL TrypLE ^TM Express enzymatic digestion passage.

(2) CCOs were seeded in 96-well plates and when organoid growth confluency reached 50%, different concentrations of L-OHP (0.64. Mu.M, 3.2. Mu.M, 16. Mu.M, 80. Mu.M, 400. Mu.M) and CEL-2 (0.0562. Mu.M, 0.2808. Mu.M, 1.404. Mu.M, 7.02. Mu.M, 35.1. Mu.M) were administered separately, DMSO was used as a blank control, cell viability was measured by CCK-8 method 6d after administration, and organoid morphological characteristic changes after administration were recorded by photography. Wherein oxaliplatin (L-OHP) is a positive control drug.

2. Results of the experiment

According to the experimental results of 4 colorectal cancer organoid models (CCO-1, CCO-2, CCO-3 and CCO-4) from patients and 1 organoid model (CNO) of normal colorectal tissues established above (figure 8), the compound CEL-2 has stronger inhibiting effect on the activity of the colorectal cancer organoid than oxaliplatin (L-OHP), and is basically nontoxic to the normal colorectal organoids of human.

The experiment results show that the compound CEL-2 can effectively inhibit the activity of colorectal cancer organoid and can be used as a medicine for treating colorectal cancer.

In conclusion, the invention provides a friedelane type compound and a preparation method and application thereof. The compound can obviously inhibit STAT3 phosphorylation of colon tumor cells and downstream proteins thereof, has good affinity with human STAT3 recombinant proteins, can be used for preparing STAT3 small-molecule inhibitors, and is used for preparing medicines for preventing and/or treating diseases related to STAT3 activity. Compared with tripterine, the compound provided by the invention has the advantages that the effect of inhibiting STAT3 protein phosphorylation is obviously improved, and the activity of inhibiting HCT-116 colorectal cancer cell proliferation is stronger. The compound can promote HCT-116 colorectal cancer cell apoptosis and block cell cycle, has stronger inhibiting effect on the activity of colorectal cancer organoid than oxaliplatin which is a positive medicine, has basically no toxicity on colorectal normal organoids, and can be used for preparing medicines for treating colorectal cancer. The preparation method of the compound is green and environment-friendly, has high yield and is suitable for expanded production.

Claims

1. A method of preparing the compound CEL-2, comprising the steps of: culturing streptomyces griseus in a culture medium until logarithmic phase, adding tripterine, continuing culturing for 2-6, ending fermentation, taking fermentation supernatant, separating and purifying to obtain a compound CEL-2;

the culture medium is a bean pulp liquid culture medium, and the preparation method of the bean pulp liquid culture medium comprises the following steps: taking 5g of soybean meal, 20g of glucose, 5g of yeast extract, 5g of NaCl ₂ HPO ₄ 5g, adding distilled water to a constant volume of 1L; the final concentration of the tripterine is 10-20mg/250mL; the culture is shake culture at 25-30 deg.C and rotation speed of shaker of 150-200 rpm/min ^-1 (ii) a The continuous culture time is 3-5 days;

the separation and purification method comprises the following steps: taking fermentation supernatant, extracting with ethyl acetate, retaining an organic phase, concentrating under reduced pressure, and purifying by silica gel column chromatography and semi-preparative liquid phase in sequence; and in the silica gel column chromatography, performing polar gradient elution by using a dichloromethane and methanol system, wherein dichloromethane: the volume ratio of methanol is 98.

2. The method of claim 1, wherein the final concentration of celastrol is 15mg/250mL; the culture temperature is 28 ℃, and the rotation speed of a shaking table is 180 rpm-min ^-1 (ii) a The time for the continuous culture was 4 days.