Use of small molecule terpenoids in the preparation of medicaments for the treatment of tumors

Abstract

The application belongs to the technical field of biological medicine, and relates to application of a small molecule terpenoid compound in preparation of a medicine for treating tumors, wherein the small molecule terpenoid compound is a compound shown in the following structure or a pharmaceutically acceptable salt thereof. The small molecule compound provided by the application can effectively inhibit growth and metastasis of liver cancer, colorectal cancer, melanoma, cholangiocarcinoma, renal cancer, glioma, gastric cancer, esophageal cancer, prostate 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 an anti-tumor medicine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a small molecule terpene compound in the preparation of drugs for treating tumors. Background Technology

[0002] In 2020, there were 19.29 million new cancer cases globally, with 4.57 million in China, accounting for 23.7% of the global total. As the world's most populous country, China's number of new cancer cases far exceeds that of other countries. In 2020, 3 million people died from cancer in China, making it a major disease seriously threatening the health of the nation. Malignant tumors account for 30% of all deaths in my country, making cancer the leading cause of death. With the increasing number of cancer patients (tumor incidence is increasing at a rate of 3%-5% annually), the 5-year relative survival rate for cancer patients in my country is approximately 40.5%. Nearly 60% of cancer patients do not achieve a clinical cure. Lung cancer, breast cancer, liver cancer, and colorectal cancer are the most common types of cancer. 83% of colorectal cancer patients are in the middle or late stages, and 44% have already developed metastases to the liver, lungs, and other sites, posing significant challenges to treatment and prognosis, and placing a huge burden on patients and society.

[0003] Small molecule drugs play a crucial regulatory role in immune cell metabolism, making their development of immunomodulatory drugs of significant value. After cell surface receptors are activated by ligands in the microenvironment, some receptor signals are transmitted to downstream key pathways and proteins. Antibody therapies, exemplified by immune checkpoint inhibitors, can target a group of receptors and ligands on the surface of immune cells. However, activation of other receptors can maintain signal transmission, forming a compensatory mechanism and resulting in poor therapeutic efficacy. Antibody molecules struggle to penetrate the cell interior and bind to key molecules in signaling pathways, thus blocking these pathways and inhibiting signal compensation. In contrast, small molecule compounds, due to their small molecular weight, can directly cross the cell membrane and enter the cell interior to bind to key biomolecules in metabolic-related proteins and signaling pathways that regulate metabolism. This allows them to block multiple signal compensation mechanisms and receptor signals, more effectively regulating cellular metabolism and function.

[0004] The following compounds are a series of small molecule terpenoids, and the relevant activities of these compounds have not been disclosed in the prior art.

[0005] Summary of the Invention

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

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] This invention provides the use of small molecule terpenoids in the preparation of drugs for treating tumors, wherein the small molecule terpenoids are compounds with the following structural formula or pharmaceutically acceptable salts thereof.

[0009]

[0010] The tumors mentioned include liver cancer, colorectal cancer, melanoma, bile duct cancer, kidney cancer, glioma, stomach 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 terpenoids can be prepared into pharmaceutical compositions with pharmaceutically acceptable carriers or excipients.

[0013] The small molecule terpenoid compounds or their compositions described in this invention exert anti-tumor effects by promoting tumor cell apoptosis.

[0014] The small molecule terpenoid compounds or their compositions described in this invention exert antitumor effects by inhibiting the formation of tumor cell colonies.

[0015] The small molecule terpenoid compounds or their compositions described in this invention exert anti-tumor effects by inhibiting tumor cell migration.

[0016] The small molecule terpenoid compounds or their compositions described in this invention exert antitumor effects by reducing the production of reactive oxygen species in tumor cells.

[0017] The small molecule terpenoid compounds described in this invention can effectively inhibit the growth and metastasis of liver cancer, colorectal cancer, and breast cancer, and have great potential for tumor immunotherapy. They can be used to prepare anti-tumor drugs. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the preparation of compound 1.

[0019] Figure 2 Here are the chemical structural diagrams of compounds Pabagrocybone D, Conitriol, and Epicanonicol.

[0020] Figure 3 The figure shows the results of the inhibitory effects of compounds 1-3 on the survival of human hepatocellular carcinoma 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 figure shows the results of the curve fitting analysis and IC50 value calculation of the inhibitory activity of compound 1 (Pabagrocybone D) on the survival of human hepatocellular carcinoma cell line HepG2, human breast cancer cell line MDA-MB-231, and human colorectal cancer cell line HCT116.

[0023] Figure 5 To detect the effect of compound 1 (Pabagrocybone D) on apoptosis in the human hepatocellular carcinoma cell line HepG2 by flow cytometry.

[0024] A: Scatter plot of Annexin V / 7-AAD dual fluorescent labeling detected by flow cytometry.

[0025] B: The proportion of Annexin V positive cells, i.e., the proportion of apoptotic and necrotic cells at each stage.

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

[0027] D: The proportion of Annexin V positive and 7-AAD negative cells, i.e., the proportion of cells in early apoptosis.

[0028] Figure 6 To detect the effect of compound 1 (Pabagrocybone D) on apoptosis in the human colorectal cancer cell line HCT116 by flow cytometry.

[0029] A: Scatter plot of Annexin V / 7-AAD dual fluorescent labeling detected by flow cytometry.

[0030] B: The proportion of Annexin V positive cells, i.e., the proportion of apoptotic and necrotic cells at each stage.

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

[0032] D: The proportion of Annexin V positive and 7-AAD negative cells, i.e., the proportion of cells in early apoptosis.

[0033] Figure 7To detect the effect of compound 1 (Pabagrocybone D) on apoptosis in the human breast cancer cell line MDA-MB-231 by flow cytometry.

[0034] A: Scatter plot of Annexin V / 7-AAD dual fluorescent labeling detected by flow cytometry.

[0035] B: The proportion of Annexin V positive cells, i.e., the proportion of apoptotic and necrotic cells at each stage.

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

[0037] D: The proportion of Annexin V positive and 7-AAD negative cells, i.e., the proportion of cells in early apoptosis.

[0038] Figure 8 Compound 1 (Pabagrocybone D) significantly reduced the number of colonies formed in human hepatocellular carcinoma cells HepG2 in a concentration-dependent manner.

[0039] A: A photograph taken after staining with crystal violet in a colony formation experiment.

[0040] B: Statistical results of the number of settlements.

[0041] Figure 9 Images taken under a bright-field microscope at different concentrations and treatment times to detect the effect of compound 1 (Pabagrocybone D) on the migration of the human hepatocellular carcinoma line HepG2 using a scratch assay.

[0042] Figure 10 This is a statistical curve showing the change in the proportion of residual scratch area with time and concentration in a scratch test.

[0043] Figure 11 This is a statistical curve showing the proportion of tumor cells migrating to the scratch area during the scratch assay.

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

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

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

[0047] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0048] This invention screens and verifies the antitumor activity of small molecule compounds through the following steps:

[0049] 1) Prepare small molecule compound 1 by utilizing the activity characteristics of exogenous precursor compounds in the metabolism of edible fungi;

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

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

[0052] 4) To detect the effect of compound 1 (Pabagrocybone D) on the formation of liver cancer cell colonies;

[0053] 5) To 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 reactive oxygen species production in liver cancer cells.

[0055] Example 1: Preparation of Compound 1

[0056] Compound 1 was prepared according 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 Inoculate mycelia of the fungus *Agrocybe pediades*, which lacks a precursor molecule, into a culture medium (glucose 4.0 g / L, malt extract 10.0 g / L, and yeast extract 4.0 g / L). On day 7 after inoculation, the precursor molecule GHQ synthesized in the first step is added to the culture medium, and the culture is continued for 2 days. Figure 1 )

[0062] 1.3 Two days later, the culture medium was collected, extracted with ethyl acetate, and the extract was analyzed by high-performance liquid chromatography (HPLC). Monomeric compound 1 was separated by repeated column chromatography and semi-preparative reversed-phase HPLC.

[0063] 1.4 The structure and relative stereochemical structure of the compound were determined by nuclear magnetic resonance spectroscopy and X-ray single-crystal 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] MedChemExpress orders compounds 2 and 3.

[0067] Conitriol(2): 1 H NMR (400MHz, CDCl3) δ6.72 (d, J = 8.5 Hz, 1H), 6.65 (d, J = 3.0 Hz, 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 the antitumor activity of compounds 1-3

[0072] 2.1 Instruments and Materials

[0073] CO2 incubator (Thermo Fisher Scientific); Clean bench (Airtech); Microplate reader (Biotek); 4°C centrifuge (Eppendorf); Inverted microscope (Olympus); High-glucose DMEM medium (KGI Biotech), fetal bovine serum (CLARK), trypsin digestion solution (VICMED), phosphate-buffered saline (PBS); Dimethyl sulfoxide (DMSO, VICMED); CCK8 reagent kit (Polymerex);

[0074] Human hepatocellular carcinoma cell line HepG2; human breast cancer cell line MDA-MB-231; and human colorectal cancer cell line HCT116 were all commercially obtained.

[0075] 2.2 Specific testing methods

[0076] 2.2.1 Cell Culture

[0077] HepG2, MDA-MB-231, and HCT116 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics at 37°C in a 5% CO2 incubator. All cells grew in an adherent state, and their growth was observed under an inverted microscope. Once the cell number reached an appropriate level, the cells were passaged.

[0078] 2.2.2 Cytotoxic activity screening

[0079] A cell control group and a blank control group were set up. Each drug concentration was replicated in 3 wells, and the cell control group and blank control group were replicated in 3 wells each. HepG2, MDA-MB-231, and HCT116 cells in logarithmic growth phase were used at a concentration of 3 × 10⁻⁶. 3 The cells were seeded into 96-well plates and cultured at 37°C for 24 h in a cell culture incubator containing 5% CO2. The compounds of this invention were dissolved in DMSO to prepare 0.5 M stock solutions. 100 μL of the test compound was added to each well, bringing the final volume to 200 μL per well, resulting in final concentrations of 40 μM, 8 μM, 1.6 μM, 0.32 μM, and 0 μM, respectively. After culturing for another 72 h, CCK8 solution was added at a 1:10 ratio. After incubation for 2 h, the OD value was measured using a microplate reader at an excitation wavelength of 450 nm.

[0080] 2.2.3 Cell Viability Calculation

[0081] Cell viability (%) = [Drug group - Blank group] / [Cell control group - Blank group] × 100%

[0082] 2.2.4 Calculation of the half-maximal inhibitory concentration (IC50)

[0083] Cell viability was calculated in the same way 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 of the "Analyze" module in GraphPad Prism.

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

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

[0086] Figure 3 The results showed that, at different concentrations, the compounds Pabagrocybone D, Conitriol, and Epicanonicol of this invention exhibited varying degrees of inhibitory activity against human hepatocellular carcinoma cell line HepG2, human breast cancer cell line MDA-MB-231, and human colorectal cancer cell line HCT116. Compound 1 (Pabagrocybone D) showed the strongest inhibitory activity against all three tumor cell lines, and its activity exhibited a strong concentration-dependent effect. While compounds 2 (Conitriol) and 3 (Epiconicol) did not show significant inhibitory effects on tumor cells, their inhibitory effects increased with increasing concentration. For example, the inhibitory effects of compound 2 on human colorectal cancer cell line HCT116 and compound 3 on all three tumor cell lines are expected to increase with increasing concentration.

[0087] The IC50 values ​​of the inhibitory activity of compound Pabagrocybone D against tumor cells were calculated by fitting curves. The results showed that the IC50 values ​​of compound Pabagrocybone D against human hepatocellular carcinoma 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. The IC50 values ​​indicate that compound Pabagrocybone D exhibits stronger cytotoxic activity against the rapidly proliferating human breast cancer cell line MDA-MB-231. Figure 4 ).

[0088] Example 3: The promoting effect of compound 1 (Pabagrocybone D) on apoptosis in different tumor cells.

[0089] 3.1 HepG2 liver cancer cells, HCT116 colorectal cancer cells, and MDA-MB-231 breast cancer cells in the logarithmic growth phase were digested with trypsin, and the cells were collected into complete cell culture medium and the cell concentration was adjusted to 1x10⁻⁶. 6 Add 100 μl of cell suspension to a 96-well flat-bottom cell culture plate.

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

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

[0092] 3.4 After culturing for another 48 hours, the cells were digested with trypsin and collected into Annexin V staining buffer.

[0093] 3.5 Transfer the cells to U-96 well plates, centrifuge, and obtain cell clumps.

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

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

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

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

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

[0099] Inducing apoptosis in tumor cells is an important mechanism by which small molecule drugs targeting tumor cells kill them and control tumor growth. In this invention, the small molecule compound Pabagrocybone D was used to treat different types of human tumor cell lines (including HepG2 liver cancer cells, HCT116 colorectal cancer cells, and MDA-MB-231 breast cancer cells). Apoptosis in different tumor cell lines was analyzed, and the direct killing effect and mechanism of Pabagrocybone D on tumor cells were analyzed and evaluated. Results are as follows: Figure 5-7 As shown.

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

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

[0102] 4.1 After digesting HepG2 human liver cancer cells in the logarithmic growth phase with trypsin, a single-cell suspension was prepared. After counting, the cell concentration was adjusted to 300 cells / ml, and 1 ml of cell suspension was added to a 12-well plate.

[0103] 4.2 The cells were cultured in a cell culture incubator. After the cells adhered, the small molecule compound Pabagrocybone D was added at concentrations of 0, 0.25, 0.5, 1.25, 2.5 and 5 μM.

[0104] 4.3 Observe the cell growth status daily using an inverted microscope.

[0105] 4.4 Culture until colony formation (day 8), discard the cell culture medium, and wash the cells with PBS.

[0106] 4.5 Add paraformaldehyde for fixation for 30 minutes.

[0107] 4.6 After discarding paraformaldehyde, add crystal violet for staining.

[0108] 4.7 Discard the crystal violet dye and wash the cells. Count the number of tumor cell colonies. Take a photograph.

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

[0110] The tumor cell colony formation assay is an important technique used to detect cell proliferation, invasiveness, and sensitivity to cytotoxic agents. When a single cell proliferates for more than six generations in vitro, the resulting cell population forms a colony or clone. The cell colony formation rate, also known as the cell seeding survival rate, indicates the number of adherent cells that survive and form clones after seeding. Not every adherent cell will proliferate and form a clone, but cells that form clones must be adherent and proliferative. The colony formation assay indicates the proliferative capacity of treated cells by assessing their colony formation ability on cell culture plates; it evaluates the sensitivity of different cytotoxic agents (drugs, etc.) to tumor cell proliferation or population dependence; and it assesses tumorigenicity in vivo. Cancer cells do not necessarily form tumors in vivo, but stronger in vitro clonal ability indicates stronger tumorigenicity in vivo, making it an in vitro experiment that simulates in vivo tumorigenicity.

[0111] like Figure 8 As shown, the small molecule compound Pabagrocybone D, in this invention, significantly reduced the number of HepG2 liver cancer cells in a concentration-dependent manner after treatment. At a concentration of 1.25 μM, the number of colonies decreased to half that of the control group; at 2.5 μM, only a few tumor cells could form colonies; and at 5 μM, tumors could hardly form any colonies. This demonstrates the potent antitumor activity of the small molecule compound Pabagrocybone D.

[0112] Example 5: Compound 1 (Pabagrocybone D) inhibits the migration ability of tumor cells.

[0113] 5.1 Cells in the logarithmic growth phase were digested with trypsin into a single-cell suspension and seeded into 6-well culture plates; the cell seeding density was 60,000 cells / well, and the seeding principle was that the confluence rate reached 100% after overnight incubation, with a final total culture medium volume of 2 mL per well.

[0114] 5.2 Cells were cultured at 37°C in a 5% CO2 incubator for 24 hours;

[0115] 5.3 Scratches: The next day, use a 200 microliter nozzle to scratch the lines vertically, without tilting it;

[0116] 5.4 Rinse the cells three times with PBS to remove the scribbled cells;

[0117] 5.5 Take a picture under a microscope to ensure that the scratch is centered and perpendicular (the photometer is 0h at this time);

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

[0119] 5.7 Take photos every 12 hours.

[0120] 5.8 Results Analysis: After opening the image using ImageJ software, 6 to 8 horizontal lines were randomly drawn, and the mean area of ​​the cell scratches was calculated.

[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 assay is a common method for studying cell migration ability in vitro. This method is frequently used to investigate the invasive and metastatic capabilities of tumor cells. The procedure involves drawing a straight line down the middle of densely growing adherent cells, washing the scratched area, and then placing the cells in a microscopic imaging device capable of long-term culture and observation. Images are taken at regular intervals to obtain a sequence of images at multiple time points. The scratch assay, to some extent, simulates the in vivo cell migration process. The speed of migration is primarily reflected by changes in the scratch area. Figure 9-11 As shown, the use of the small molecule compound PabagrocyboneD in liver cancer cells significantly inhibits the migration ability of liver cancer cells. At a low concentration of 1.25 μM, it can effectively inhibit the migration ability of tumor cells. Figure 9 These are microscopic photographs of the scratches, which visually demonstrate the changes in the scratch area under different drug concentrations and treatment times. Figure 10 Statistics on the reduction ratio of scratch area. Figure 11 This represents the proportion 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 deadly manifestation of cancer. The vast majority of cancer patients die from metastatic disease, not from the primary tumor. Metastasis involves a series of processes, including cells from the primary tumor gradually acquiring greater invasiveness; implantation via the bloodstream, lymphatic vessels, or direct infiltration through adjacent structures; colonization in distant organs; and eventual proliferation in those distant organs. The strong inhibitory effect of Pabagrocybone D on tumor cell migration indicates that Pabagrocybone D possesses activity in inhibiting tumor metastasis.

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

[0126] 6.1 Perform cell plating the day before the test to ensure that the cell density reaches 50-70% at the time of the test.

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

[0128] 6.3 After treating the cells for 12 hours, add H2O2 (0.25 μM) and treat for 1 hour.

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

[0130] 6.5 Discard the supernatant, wash once with PBS, add ROS probe (1000x) and treat at 37 degrees Celsius for 30 min (1640 serum-free and antibiotic-free).

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

[0132] 6.6 Wash once with PBS, add 150 μL of 7AAD (400x) to each well in 2% FACS Buffer, mix well, filter into flow cytometry tubes, and perform detection on a Canto II flow cytometer. Analyze using Flowjo software.

[0133] Under normal circumstances, reactive oxygen species (ROS), as a natural byproduct of oxygen metabolism, are present at low levels in the body. They act as "redox messengers," participating in intracellular signal transduction and regulation, and playing a crucial role in maintaining cell cycle, gene expression, and homeostasis. However, when the body is stimulated, such as by ultraviolet radiation, radiation, hypoxia, or heat exposure, ROS levels increase dramatically, exceeding the body's own clearance capacity. This leads to an imbalance in the body's oxidation-antioxidant processes, resulting in oxidative stress, which in turn causes DNA damage, lipid peroxidation, and alterations in protein structure and function. Furthermore, damage to these macromolecules is also associated with the pathogenesis of cancer, aging, inflammation, and various human diseases (neurodegenerative diseases, cardiovascular diseases, and diabetes).

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

[0135] The detection results of this invention are as follows Figure 12 As shown.

[0136] The results showed that treatment with compound 1 (Pabagrocybone D) significantly reduced 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 species levels in liver cancer, colorectal cancer, and breast cancer tumor cells, with compound 1 being particularly potent.

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

Claims

1. The application of small molecule terpenoids in the preparation of drugs for treating tumors, wherein the small molecule terpenoids are compounds with the following structural formula or pharmaceutically acceptable salts thereof, and the tumor is liver cancer, breast cancer or colorectal cancer; 。 2. The application of small molecule terpenoids in the preparation of drugs for treating tumor metastasis, wherein the small molecule terpenoids are compounds with the following structural formula or pharmaceutically acceptable salts thereof, and the tumor is liver cancer; 。 3. The application according to claim 1 or 2, characterized in that, The compound is prepared into a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient.

4. The application according to claim 1 or 3, characterized in that, The compound or its pharmaceutical composition exerts its anti-tumor effect by promoting tumor cell apoptosis.

5. The application according to claim 1 or 3, characterized in that, The compound or its pharmaceutical composition exerts its antitumor effect by inhibiting the formation of tumor cell colonies.

6. The application according to claim 2 or 3, characterized in that, The compound or its pharmaceutical composition exerts an anti-tumor metastasis effect by inhibiting tumor cell migration.

7. The application according to claim 1 or 3, characterized in that, The compound or its pharmaceutical composition exerts its antitumor effect by reducing the production of reactive oxygen species in tumor cells.

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