Application of small-molecule terpenoid in preparation of medicine for treating tumors

Abstract

The invention belongs to the technical field of biological medicines, and relates to an application of a small-molecule terpenoid in preparation of a medicine for treating tumors, and the small-molecule terpenoid is a compound with a structure shown in the specification or pharmaceutically acceptable salt of the compound, the small molecule compound provided by the invention can effectively inhibit growth and metastasis of liver cancer, colorectal cancer, melanoma, bile duct cancer, kidney cancer, glioma, stomach cancer, esophageal cancer, prostatic cancer, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer and sarcoma, especially liver cancer, colorectal cancer or breast cancer, has good tumor treatment potential, and can be used for preparing medicines for treating tumors. The compound can be used for preparing anti-tumor drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a small molecule terpenoid compound in the preparation of a drug for treating tumors. Background Art

[0002] In 2020, there were 19.29 million new cancer cases worldwide, of which 4.57 million were in China, accounting for 23.7% of the world's total. As China is the world's most populous country, the number of new cancer cases far exceeds that of other countries in the world. In 2020, there were 3 million cancer deaths in China, which has become a major disease that seriously endangers the health of the people. Deaths from malignant tumors in my country account for 30% of all deaths among residents, and cancer has become the leading cause of death in my country. With the increase in the number of cancer patients (the incidence of tumors increases at a rate of 3%-5% per year), the 5-year relative survival rate of cancer patients in my country is about 40.5%. Nearly 60% of cancer patients cannot achieve clinical cure. Lung cancer, breast cancer, liver cancer, colorectal cancer and other tumors are the most common types of tumors. 83% of colon cancer patients are in the middle and late stages, and 44% of patients have metastases to the liver, lungs and other parts, which brings great challenges to treatment and prognosis, and brings a huge burden to patients and society.

[0003] Small molecule drugs play an important regulatory role in immune cell metabolism, so the development of small molecule immunomodulatory drugs is of great value. After the cell surface receptors are activated by ligands in the microenvironment, the signals of some receptors are transmitted to some common key pathways and proteins downstream. Antibody therapeutic drugs represented by immune checkpoints can target a group of receptors and ligands on the surface of immune cells. After other receptors are activated, they can still maintain the transmission of related signals, forming a compensation mechanism, resulting in poor treatment effects. It is difficult for antibody molecules to enter the cell to bind to key molecules in the signal pathway, block the corresponding signal pathway, and inhibit signal compensation. However, due to their small molecular weight, small molecule compounds can directly pass through the cell plasma membrane, enter the cell and bind to metabolism-related proteins and key biological macromolecules in the signal pathway that regulates metabolism, thereby blocking multiple signal compensation mechanisms and receptor signals, and more effectively regulating cell metabolism and function.

[0004] The following compounds are a series of small molecule terpenoid compounds, and the prior art does not disclose the relevant activities of the compounds.

[0005] Summary of the invention

[0006] In order to overcome the defects of the prior art, the technical problem solved by the present invention is to provide the use of small molecule terpenoid compounds in the preparation of drugs for treating tumors.

[0007] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0008] The present invention provides the use of a small molecule terpenoid compound in the preparation of a drug for treating tumors. The small molecule terpenoid compound is a compound shown in the following structural formula or a pharmaceutically acceptable salt thereof.

[0009]

[0010] The tumors are liver cancer, colorectal cancer, melanoma, bile duct cancer, kidney cancer, glioma, gastric cancer, esophageal cancer, prostate cancer, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, and sarcoma.

[0011] Preferably, the tumor is liver cancer, colorectal cancer or breast cancer.

[0012] Furthermore, the small molecule terpenoid compound can be prepared into a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient.

[0013] The small molecule terpenoid compound or the composition thereof described in the present invention exerts anti-tumor effect by promoting apoptosis of tumor cells.

[0014] The small molecule terpenoid compound or the composition thereof of the present invention exerts an anti-tumor effect by inhibiting the formation of tumor cell colonies.

[0015] The small molecule terpenoid compound or the composition thereof described in the present invention exerts an anti-tumor effect by inhibiting the migration of tumor cells.

[0016] The small molecule terpenoid compound or the composition thereof of the present invention exerts an anti-tumor effect by reducing the generation of reactive oxygen species in tumor cells.

[0017] The small molecule terpenoid compound of the present invention can effectively inhibit the growth and metastasis of liver cancer, colorectal cancer and breast cancer, etc., has a good tumor immunotherapy potential, and can be used to prepare anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the preparation of compound 1.

[0019] Figure 2 The chemical structures of the compounds Pabagrocybone D, Conitriol and Epiconicol are shown below.

[0020] Figure 3 The graph shows the results of the inhibitory effect of compound 1-3 on the survival of human liver cancer cell line HepG2, human breast cancer cell line MDA-MB-231, and human colorectal cancer cell line HCT116.

[0021] A: Compound 1 (Pabagrocybone D) B: Compound 2 (Conitriol) C: Compound 3 (Epiconicol).

[0022] Figure 4 The graph shows the results of the inhibitory activity fitting curve analysis and IC50 value calculation of compound 1 (Pabagrocybone D) on the survival of human liver cancer cell line HepG2, human breast cancer cell line MDA-MB-231, and human colorectal cancer cell line HCT116.

[0023] Figure 5 Flow cytometry was used to detect the effect of compound 1 (Pabagrocybone D) on apoptosis of human liver cancer cell line HepG2.

[0024] A: Scatter plot of flow cytometry detection of Annexin V / 7-AAD dual fluorescence labeling.

[0025] B: The proportion of cells positively stained with Annexin V, that is, the proportion of apoptotic cells and necrotic cells at each stage.

[0026] C: The proportion of cells double positive for Annexin V and Annexin V / 7-AAD staining, that is, the proportion of late apoptotic cells and necrotic cells.

[0027] D: The proportion of cells that are Annexin V positive and 7-AAD negative, that is, the proportion of early apoptotic cells.

[0028] Figure 6 Flow cytometry was used to detect the effect of compound 1 (Pabagrocybone D) on apoptosis of human colorectal cancer cell line HCT116.

[0029] A: Scatter plot of flow cytometry detection of Annexin V / 7-AAD dual fluorescence labeling.

[0030] B: The proportion of cells positively stained with Annexin V, that is, the proportion of apoptotic cells and necrotic cells at each stage.

[0031] C: The proportion of cells double-positive for Annexin V and 7-AAD staining, that is, the proportion of late apoptotic cells and necrotic cells.

[0032] D: The proportion of cells that are Annexin V positive and 7-AAD negative, that is, the proportion of early apoptotic cells.

[0033] Figure 7Flow cytometry was used to detect the effect of compound 1 (Pabagrocybone D) on apoptosis of human breast cancer cell line MDA-MB-231.

[0034] A: Scatter plot of flow cytometry detection of Annexin V / 7-AAD dual fluorescence labeling.

[0035] B: The proportion of cells positively stained with Annexin V, that is, the proportion of apoptotic cells and necrotic cells at each stage.

[0036] C: The proportion of cells double-positive for Annexin V and 7-AAD staining, that is, the proportion of late apoptotic cells and necrotic cells.

[0037] D: The proportion of cells that are Annexin V positive and 7-AAD negative, that is, the proportion of early apoptotic cells.

[0038] Figure 8 Compound 1 (Pabagrocybone D) significantly reduced the number of colonies formed by human liver cancer cell HepG2 in a concentration-dependent manner.

[0039] A: Colony formation experiment photographed after crystal violet staining.

[0040] B: Statistical results of colony numbers.

[0041] Fig. 9 The scratch experiment was used to detect the effect of compound 1 (Pabagrocybone D) on the migration of human liver cancer cell line HepG2. The pictures were taken under a bright field microscope at different concentrations and time treatments.

[0042] Fig.10 This is a statistical curve showing the change of scratch residual area ratio with time and concentration in the scratch experiment.

[0043] Fig.11 This is a statistical curve of the proportion of tumor cells migrating to the scratch area in the scratch experiment.

[0044] Fig.12 The effect of compound 1 (Pabagrocybone D) on the production of reactive oxygen species (ROS) in the human liver cancer cell line HepG2.

[0045] A: Peak pattern of ROS detected by flow cytometry.

[0046] B: Statistical graph of mean fluorescence intensity (MFI) of ROS. DETAILED DESCRIPTION

[0047] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0048] The present invention screens and verifies the anti-tumor activity of small molecule compounds by the following steps:

[0049] 1) preparing small molecule compound 1 by taking advantage of the activity of edible fungi in metabolizing exogenous precursor compounds;

[0050] 2) Determine the inhibitory effect of small molecule compounds 1-3 on the activity of common tumor cells and calculate the IC50 value;

[0051] 3) Detect the effect of compound 1 (Pabagrocybone D) on tumor cell apoptosis;

[0052] 4) Detecting the effect of compound 1 (Pabagrocybone D) on the colony formation of liver cancer cells;

[0053] 5) Detect the effect of compound 1 (Pabagrocybone D) on the migration of liver cancer cells;

[0054] 6) Detect the effect of compound 1 (Pabagrocybone D) on the production of reactive oxygen species in liver cancer cells.

[0055] Example 1 Preparation of Compound 1

[0056] Compound 1 was prepared with reference to the following literature:

[0057] Construction of a meroterpenoid-like compound collection by precursor-assisted biosynthesis, Org. Biomol. Chem., 2020, 18, 5850, Panlong Ren, Xinyu Miao, Ting Tang, Yueting Wu, a Jing Wang, a Ying Zeng, b Yun Li, a Kun Gao* a and Yan-Long Yang* a

[0058] The specific steps are as follows:

[0059] 1.1 Preparation of precursor molecule GHQ (geranylhydroquinone).

[0060]

[0061] 1.2 The mycelium of the fungus Agrocybe pediades lacking the precursor molecule was inoculated in a culture medium (glucose 4.0 g / L, malt extract 10.0 g / L, and yeast extract 4.0 g / L). On the 7th day after inoculation, the precursor molecule GHQ synthesized in the first step was added to the culture medium and culture was continued for 2 days. Figure 1 )

[0062] 1.3 After two days, the culture medium was collected, extracted with ethyl acetate, and the extract was analyzed by HPLC. Monomeric compound 1 was separated by repeated column chromatography and semi-preparative reverse phase HPLC.

[0063] 1.4 The structure and relative stereochemistry of the compound were determined by nuclear magnetic resonance spectroscopy and single-crystal X-ray diffraction analysis ( Figure 2 ).

[0064] Pabagrocybone D(1): 1 H NMR (600MHz, (CD3)2CO) δ6.79 (d, J=9.9Hz, 1H), 6.55 (d, J=

[0065] 8.6Hz,1H),6.52(d,J=8.6Hz,1H),5.90(dd,J=9.9,5.4Hz,1H),2.48(m,2H),2.06(s,3H),1.90(m,2H),1.50(s,3H),1.37(m,1H),1.28(s,3H). 13 C NMR (150MHz, (CD3)2CO) δ208.4,149.5,146.9,129.6,129.5,122.4,122.1,117.3,114.3,47.1,41.8,38.4,29.8,28.1,25.3,25.2.

[0066] Compounds 2 and 3 were ordered from MedChemExpress.

[0067] Conitriol(2): 1 H NMR(400MHz, CDCl3)δ6.72(d,J=8.5Hz,1H),6.65(d,J=3.0Hz,1H),

[0068] 6.58(dd,J=8.5,3.0Hz,1H),5.32(d,J=5.4Hz,1H),3.91(m,1H),2.11(m,2H),1.99(m,1H),1.70(s,3H),1.63(m,2H),1.28(s,3H),0.57(s,3H). 13 C NMR (100MHz, CDCl3) δ149.2,148.0,133.1,131.0,125.5,119.1,118.1,114.4,75.1,49.4,34.5,32.0,31.5,23.4,23.0,20.8.

[0069] Epiconicol(3): 1 H NMR (400MHz, CDCl3) δ6.75 (d, J = 2.5Hz, 1H), 6.62 (d, J = 8.7Hz, 1H),

[0070] 6.57(dd,J=8.7,2.5Hz,1H),5.84(d,J=5.5Hz,1H),3.48(d,J=5.5Hz,1H),2. 10(m,1H),1.97(m,2H),1.88(m,2H),1.69(s,3H),1.41(s,3H),1.25(s,3H). 13 C NMR (100MHz, CDCl3) δ149.3,145.9,135.4,125.8,121.9,117.9,114.8,114.4,75.7,39.5,32.6,31.1,26.6,25.5,23.7,20.0.

[0071] Example 2 Evaluation of Antitumor Activity of Compounds 1-3

[0072] 2.1 Instruments and Materials

[0073] CO2 incubator (Thermo); clean bench (Airtech); microplate reader (Biotek); 4℃ centrifuge (Eppendorf); inverted microscope (Olympus); high-glucose DMEM medium (Keygen Biotechnology), fetal bovine serum (CLARK), trypsin digestion solution (VICMED), phosphate buffered saline (PBS); dimethyl sulfoxide (DMSO, VICMED); CCK8 kit (Polymerics);

[0074] Human liver cancer cell line HepG2; human breast cancer cell line MDA-MB-231; human colorectal cancer cell line HCT116; all were purchased commercially.

[0075] 2.2 Specific detection methods

[0076] 2.2.1 Cell culture

[0077] HepG2, MDA-MB-231, and HCT116 cells were cultured in a 37°C, 5% CO2 incubator in a high-glucose DMEM medium containing 10% fetal bovine serum and 1% double antibody. The cells grew in an adherent state, and the growth status was observed under an inverted microscope. When the number of cells was appropriate, they were subcultured.

[0078] 2.2.2 Cytotoxic activity screening

[0079] Set up a cell control group and a blank control group. Each concentration of the drug group was repeated 3 wells, and the cell control group and the blank control group were repeated 3 wells. HepG2, MDA-MB-231, and HCT116 cells in the logarithmic growth phase were selected and plated at 3×10 3 / well were inoculated in a 96-well culture plate and cultured at 37°C in a cell culture incubator containing 5% CO2 for 24 hours. DMSO was used to dissolve each compound of the present invention to prepare a 0.5M compound stock solution. 100 μL of the test compound was added to each well to make the final volume of each well 200 μL, and the final concentrations were 40 μM, 8 μM, 1.6 μM, 0.32 μM, and 0 μM, respectively. After continuing to culture in the incubator for 72 hours, CCK8 solution was added at a ratio of 1:10. After incubation in the incubator for 2 hours, the OD value was detected with an enzyme marker, and the excitation wavelength was 450nm.

[0080] 2.2.3 Calculation of cell viability

[0081] Cell viability (%) = [drug group - blank group] / [cell control group - blank group] × 100%

[0082] 2.2.4 Calculation of half inhibitory concentration IC50

[0083] The calculation of cell survival rate was the same as in 2.3. The IC50 was calculated and the curve was fitted using the “[Inhibitor] vs. normalized response--Variable slope” function in the “Nonlinear regression” module in the “Analyze” module of GraphPad Prism.

[0084] The results of the inhibitory effects of compounds 1-3 on the survival of human liver cancer cell line HepG2, human breast cancer cell line MDA-MB-231, and human colorectal cancer cell line HCT116 are shown in Figure 3 shown.

[0085] The results of inhibitory activity fitting curve analysis and IC50 value calculation of compound 1 (Pabagrocybone D) on the survival of human liver cancer cell line HepG2, human breast cancer cell line MDA-MB-231, and human colorectal cancer cell line HCT116 are shown in Figure 4 shown.

[0086] Figure 3 The results show that at different concentrations, the compounds Pabagrocybone D, Conitriol and Epiconicol of the present invention have different degrees of inhibitory activity on human liver cancer cell line HepG2, human breast cancer cell line MDA-MB-231, and human colorectal cancer cell line HCT116. Compound 1 (Pabagrocybone D) has the strongest inhibitory activity on all three tumor cells, and shows a strong concentration effect on the activity of tumor cells. Although compounds 2 (Conitriol) and 3 (Epiconicol) have no obvious inhibitory effect on tumor cells, the inhibitory effect is enhanced when the concentration increases. For example, the inhibitory effect of compound 2 on the human colorectal cancer cell line HCT116 and compound 3 on the three tumor cells is expected to increase when the concentration increases.

[0087] The IC50 value of the inhibitory activity of compound Pabagrocybone D on tumor cells was calculated by fitting the curve. The results showed that the IC50 values ​​of compound Pabagrocybone D on human liver cancer cell line HepG2, human breast cancer cell line MDA-MB-231, and human colorectal cancer cell line HCT116 were 1.343 μM, 1.044 μM, and 1.714 μM, respectively. From the IC50 values, it can be seen that compound Pabagrocybone D has stronger cytotoxic activity against the fast-proliferating human breast cancer cell line MDA-MB-231 ( Figure 4 ).

[0088] Example 3 Effect of Compound 1 (Pabagrocybone D) on the Apoptosis of Different Tumor Cells

[0089] 3.1 HepG2, HCT116, and MDA-MB-231 were digested with trypsin in the logarithmic phase and collected into complete cell culture medium. The cell concentration was adjusted to 1x10 6 / ml, add 100 μl of cell suspension to a 96-well flat-bottom cell culture plate.

[0090] 3.2 Place the cells in a 37°C, 5% CO2 incubator.

[0091] 3.3 After 24 hours of culture, 100 μL of compound Pabagrocybone D was added to each well, so that the final volume of each well was 200 μL, and the final concentrations were 0 μM, 1 μM, 5 μM, and 10 μM, respectively.

[0092] 3.4 After culturing for 48 hours, trypsinize the cells and collect the cells into Annexin V staining buffer.

[0093] 3.5 Transfer the cells to a U-96 well plate and centrifuge to obtain cell pellets.

[0094] 3.6 Add 50 μL of prepared Annexin V dye solution and mix gently by pipetting.

[0095] 3.7 Protect from light and stain at 4°C for 20 minutes.

[0096] 3.8 Wash twice with Annexin V staining buffer.

[0097] 3.9 Resuspend the cells to 150 μL using a buffer containing the nucleic acid dye 7-aminoactinomycin D (7-AAD).

[0098] 3.10 Immediately analyze using a flow cytometer (Canto II, BD).

[0099] Inducing apoptosis of tumor cells is an important mechanism for small molecule drugs targeting tumor cells to kill tumor cells and achieve control of tumor growth. The small molecule compound Pabagrocybone D of the present invention was used to treat different types of human tumor cell lines (including liver cancer cell HepG2, colorectal cancer cell HCT116, breast cancer cell MDA-MB-231), analyze the apoptosis of different tumor cells, and analyze and evaluate the direct killing effect and mechanism of Pabagrocybone D on tumor cells. The results are as follows Figure 5-7 shown.

[0100] The results showed that Pabagrocybone D induced significant apoptosis in three different tumor cell lines. Among them, Pabagrocybone D had the most significant effect on the fastest growing breast cancer cell line, specifically, 1uM Pabagrocybone D could induce apoptosis in breast cancer cells. As the concentration of Pabagrocybone D increased, the proportion of apoptotic cells also increased. At a concentration of 5uM, Pabagrocybone D induced apoptosis in almost all breast cancer cells, of which 80% were in late stage apoptosis (i.e., 7AAD+Annexin V+)( Figure 7). For the fast-growing colorectal cancer cell line HCT116, Pabagrocybone D induced 50% of the cells to undergo apoptosis at a concentration of 10uM, and the apoptotic cells were all in the late stage of apoptosis ( Figure 6 Compared with the first two tumor cell lines, Pabagrocybone D has a milder effect on the slower growing HepG2 cells. At a concentration of 10uM, only 10% of the cells underwent apoptosis ( Figure 5 ).

[0101] Example 4 Compound 1 (Pabagrocybone D) inhibits the colony formation of tumor cells

[0102] 4.1 After trypsin digestion of human liver cancer cells HepG2 in the logarithmic growth phase, prepare a single cell suspension, adjust the cell concentration to 300 cells / ml after counting, and add 1 ml of cell suspension to a 12-well plate.

[0103] 4.2 Place the cells in a cell culture incubator for culture. After the cells adhere to the wall, add the small molecule compound Pabagrocybone D at treatment concentrations of 0, 0.25, 0.5, 1.25, 2.5 and 5uM.

[0104] 4.3 Use an inverted microscope to observe the cell growth status every day.

[0105] 4.4 Culture until colonies are formed (Day 8), discard the cell culture medium, and wash the cells with PBS.

[0106] 4.5 Add paraformaldehyde and fix for 30 minutes.

[0107] 4.6 After discarding the paraformaldehyde, add crystal violet stain.

[0108] 4.7 Discard the crystal violet dye, wash the cells, count the number of tumor cell colonies, and take photos.

[0109] 4.8 Use GraphPad software for graphing and statistical analysis.

[0110] The tumor cell colony formation test is an important technical method used to detect cell proliferation ability, invasiveness, sensitivity to killing factors and other items. When a single cell proliferates in vitro for more than 6 generations, the cell group composed of its offspring becomes a colony or clone. The cell clone formation rate is the cell inoculation survival rate, which indicates the number of cells that survive and form clones after cell inoculation. Not every cell that adheres to the wall can proliferate and form clones, but cells that form clones must be cells that adhere to the wall and have proliferation activity. The colony formation experiment indicates the proliferation ability of treated cells by the colony formation ability of cells on the cell culture plate after treatment; evaluates the sensitivity of different killing factors (drugs, etc.) to the proliferation ability or group dependence of tumor cells; evaluates the tumorigenicity of cells in vivo. Not all cancer cells can form tumors in vivo, but the stronger the in vitro cloning ability, the stronger the in vivo tumorigenicity. It is an in vitro experiment that simulates in vivo tumor formation.

[0111] like Figure 8 As shown, after treating human liver cancer cells HepG2 with the small molecule compound Pabagrocybone D of the present invention, the number of colonies formed by HepG2 cells was significantly reduced in a concentration-dependent manner. At a concentration of 1.25uM, the number of colonies was reduced to half of that of the control group. At a concentration of 2.5uM, only a few tumor cells could form colonies, and at a concentration of 5uM, the tumor could hardly form colonies. This indicates that the small molecule compound Pabagrocybone D has a strong anti-tumor activity.

[0112] Example 5 Compound 1 (Pabagrocybone D) inhibits tumor cell migration

[0113] 5.1 Cells in the logarithmic growth phase were digested with trypsin into a single cell suspension and inoculated into a 6-well culture plate; 60,000 cells were plated per well, and the inoculation principle was that the fusion rate reached 100% overnight, and the final total volume of culture medium per well was 2 mL;

[0114] 5.2 The cells were cultured in a 37°C, 5% CO2 incubator for 24 h;

[0115] 5.3 Scratch: On the second day, use a 200 μL pipette tip to scratch vertically without tilting the tip;

[0116] 5.4 Rinse the cells 3 times with PBS and remove the scratched cells;

[0117] 5.5 Take a photo under a microscope to ensure that the scratch is centered and vertical (the photo at this time is counted as 0h);

[0118] 5.6 Add the small molecule compound Pabagrocybone D to make the final concentrations 0, 1.25, 2.5 and 5uM;

[0119] 5.7 Take photos every 12 hours.

[0120] 5.8 Result analysis: After opening the image using Image J software, 6 to 8 horizontal lines were randomly drawn to calculate the mean cell scratch area.

[0121] 5.9 Data Processing:

[0122] Cell migration rate (wound healing rate) = [(initial scratch area - scratch area at time T)] / [initial scratch area] × 100%

[0123] The cell scratch test is a common method for studying cell migration in vitro. This method is often used to study the invasion and metastasis ability of tumor cells. The specific operation is to draw a straight line in the middle of densely grown adherent cells, rinse the scratch area, put the cells into a microscope imaging device that can be cultured and observed for a long time, take pictures and record them at regular intervals, and obtain a sequence of pictures at multiple time points. The scratch test simulates the process of cell migration in vivo to a certain extent. The change in scratch area is mainly used to reflect the speed of migration. Figure 9-11 As shown, after the small molecule compound Pabagrocybone D is used in the present invention, the migration ability of the liver cancer cells is significantly inhibited. At a low concentration of 1.25uM, the migration ability of tumor cells can be effectively inhibited. Fig. 9 This is a microscopic photograph of the scratch, which directly shows the change in the scratch area when treated with different drug concentrations and time. Fig.10 The statistics of the reduction ratio of the scratch area are: Fig.11 is the ratio of cell migration area calculated based on the scratch area.

[0124] Metastasis, the growth of cancer cells in organs far from their site of origin, is the final and most lethal manifestation of cancer. The vast majority of cancer patients die from metastatic disease rather than the primary tumor. Metastasis involves a series of processes, including cells from the primary tumor gradually becoming more invasive; implanting through the blood, lymphatic vessels or through direct infiltration of adjacent structures; colonizing distant organs and eventually proliferating in distant organs. Pabagrocybone D's strong inhibitory effect on the migration ability of tumor cells indicates that Pabagrocybone D has the activity of inhibiting tumor metastasis.

[0125] Example 6 Compound 1 (Pabagrocybone D) reduces the production of reactive oxygen species in tumor cells

[0126] 6.1 Plate cells one day before the test to ensure that the cell density reaches 50-70% during the test.

[0127] 6.2 Add the small molecule compound Pabagrocybone D at a treatment concentration of 5uM.

[0128] 6.3 After the cells were treated for 12 hours, H2O2 (0.25 μM) was added and treated for 1 hour.

[0129] 6.4 Discard the supernatant, add PBS to wash once, resuspend in 200ul 2% FACS Buffer per well in a 96-well U-bottom plate, and centrifuge at 2000rpm for 4min.

[0130] 6.5 Discard the supernatant, wash once with PBS, add ROS probe (1000x) and treat at 37 degrees for 30 minutes (1640 serum-free and without double antibody)

[0131] (Probe preparation: dilute DCFH-DA with serum-free medium at 1:1000 to a final concentration of 10 μM)

[0132] 6.6 Wash once with PBS, add 150ul of 7AAD (400x) to each well of 2% FACS Buffer, mix well, filter into flow tube, detect on Canto II flow cytometer, and analyze with Flowjo software.

[0133] Under normal circumstances, ROS, as a natural byproduct of oxygen metabolism, is at a low level in the body. As a "redox messenger", it participates in intracellular signal transmission and regulation, and plays an important role in maintaining the cell cycle, gene expression, and the homeostasis of the body's internal environment. However, when the body is stimulated, such as ultraviolet rays, radiation, hypoxia, heat exposure, etc., the level of ROS will increase sharply, exceeding the body's own clearance and processing capacity, and the body's oxidation-antioxidation function will be unbalanced, and oxidative stress will occur, leading to DNA damage, lipid peroxidation, and changes in protein structure and function. In addition, damage to these macromolecules is also related to the pathogenesis of cancer, aging, inflammation, and a variety of human diseases (neurodegenerative diseases, cardiovascular diseases, and diabetes).

[0134] ROS level is an important marker of cellular oxidative damage caused by normal physiological functions of cells and environmental factors. Therefore, it is very necessary to reliably measure the concentration or relative level of ROS. Detecting the intracellular ROS level is of great significance for understanding cell signal transduction and studying the potential mechanism of disease.

[0135] The test results of the present invention are as follows Fig.12 shown.

[0136] The results showed that compound 1 (Pabagrocybone D) treatment could significantly reduce the level of reactive oxygen species in tumor cells.

[0137] In summary, compounds 1-3 of the present invention can significantly inhibit the proliferation, survival, migration, colony formation and reactive oxygen levels in liver cancer, colorectal cancer and breast cancer tumor cells, especially compound 1, which has a stronger effect.

[0138] The present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the specification and drawings should be regarded as illustrative rather than restrictive.

Claims

1. Use of a small molecule terpenoid compound in the preparation of a drug for treating tumors, wherein the small molecule terpenoid compound is a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof:

2. Use of a small molecule terpenoid compound in the preparation of a drug for treating tumor metastasis, wherein the small molecule terpenoid compound is a compound represented by the following structural formula or a pharmaceutically acceptable salt thereof:

3. The use according to claim 1 or 2, characterized in that: The tumor is liver cancer, colorectal cancer, melanoma, bile duct cancer, kidney cancer, glioma, gastric cancer, esophageal cancer, prostate cancer, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer or sarcoma.

4. The use according to claim 1 or 2, characterized in that: The compound is prepared into a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient.

5. The use according to claim 3, characterized in that: The compound is prepared into a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient.

6. The use according to any one of claims 1 to 5, characterized in that: The compound or its pharmaceutical composition exerts anti-tumor effect by promoting apoptosis of tumor cells.

7. The use according to any one of claims 1 to 5, characterized in that: The compound or its pharmaceutical composition exerts anti-tumor effect by inhibiting the formation of tumor cell colonies.

8. The use according to any one of claims 1 to 5, characterized in that: The compound or its pharmaceutical composition exerts anti-tumor effect by inhibiting tumor cell migration.

9. The use according to any one of claims 1 to 5, characterized in that: The compound or its pharmaceutical composition exerts an anti-tumor effect by reducing the generation of reactive oxygen species in tumor cells.

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