Application of (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium in the preparation of anticancer drugs

Abstract

This invention relates to the application of (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onthium in the preparation of anticancer drugs, belonging to the field of pharmaceutical technology. The structure of (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onthium is shown in Formula I below. The (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onthium of this invention can inhibit the growth of gastric cancer cells. By promoting autophagy and mitochondrial fragmentation in gastric cancer cells AGS and HGC-27, it inhibits the proliferation, invasion, and migration of gastric cancer cells and arrests them in the G1 phase. Furthermore, in vivo experiments in mice have confirmed that ZWK-3 can inhibit tumor growth in mice in a significant dose-dependent manner, without affecting mouse body weight or visceral indices. Formula I.

Description

Technical Field

[0001] This invention relates to the application of (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium in the preparation of anticancer drugs, belonging to the field of pharmaceutical application technology. Background Technology

[0002] Cancer is a major global disease burden, with approximately 11 million new cases each year, posing a serious threat to human health. The most common cancers worldwide are lung cancer, breast cancer, and colorectal cancer, while the most common cancer deaths are from lung cancer, stomach cancer, and liver cancer. China accounts for 20.3% of global cancer incidence, with over 2.2 million new cases annually. The most common cancers in men are lung cancer, stomach cancer, liver cancer, and esophageal cancer, while in women they are breast cancer, esophageal cancer, stomach cancer, lung cancer, liver cancer, and cervical cancer. Significant progress has been made in the research of anticancer drugs in recent years. Drug therapy is a crucial component of cancer treatment; the use of effective anticancer drugs can help patients achieve longer survival times and have a chance to live. Currently, the most common anticancer drugs include chemotherapy drugs, traditional Chinese medicine, biopharmaceuticals, and targeted therapies. However, current anticancer drugs are expensive and have significant side effects, with the most common adverse reaction being bone marrow suppression. Therefore, developing drugs with fewer side effects, lower costs, and better anticancer efficacy has become a current research hotspot.

[0003] Heterocyclic compounds are important pharmacologically active substances and play a crucial role in creating specific chemical structures with pharmacological activity. Five-membered heterocyclic compounds containing oxygen, nitrogen, and sulfur are present in broad-spectrum therapeutic agents and are important in drug discovery and development. Benzothiazol (BTA) is a fused benzo[a]heterocyclic compound found in many natural products and is responsible for the medicinal, pharmacological, and pharmaceutical applications of these products.

[0004] BTA derivatives possess a broad spectrum of biological activities, including anticancer, antioxidant, anti-inflammatory, antitumor, antiviral, antibacterial, antiproliferative, antidiabetic, anticonvulsant, analgesic, antituberculosis, antimalarial, antileishmaniasis, antihistamine, and antifungal effects. However, the role of methoxybenzothiazole derivatives in the inhibition of gastric cancer has been rarely reported. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium in the preparation of anticancer drugs.

[0006] The (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium of this invention can inhibit the growth of gastric cancer cells. It inhibits the proliferation, invasion, and migration of gastric cancer cells by promoting autophagy and mitochondrial fragmentation in AGS and HGC-27 cells, and arrests gastric cancer cells in the G1 phase with minimal side effects. This invention provides the application of (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium as an anticancer drug, offering beneficial assistance in the treatment of gastric cancer and showing potential application prospects in the pharmaceutical field.

[0007] Terminology Explanation:

[0008] ZWK-3: Abbreviation for (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazole-3-onium.

[0009] This invention is achieved through the following technical solution:

[0010] The application of (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium in the preparation of anticancer drugs, wherein the structure of (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium is shown in Formula I below:

[0011]

[0012] Formula I.

[0013] According to a preferred embodiment of the present invention, the molecular formula of (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium is C 19 H 21 IN2OS, with a molecular weight of 452.04, is a purplish-red powdery solid.

[0014] According to a preferred embodiment of the present invention, the (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium is prepared by the following method:

[0015] 6-Methoxy-3-methylbenzo[d]thiazol-3-onium and 4-dimethylaminobenzaldehyde were dissolved in anhydrous ethanol, heated to reflux, and after the reaction was stopped, cooled to room temperature. The solvent was removed by vacuum distillation, and the solid was purified by silica gel column chromatography to obtain a purple-red solid.

[0016] According to a preferred embodiment of the present invention, the molar ratio of 6-methoxy-3-methylbenzo[d]thiazol-3-onium to 4-dimethylaminobenzaldehyde is (1-3):(1-3).

[0017] Most preferably, the molar ratio of 6-methoxy-3-methylbenzo[d]thiazol-3-onium to 4-dimethylaminobenzaldehyde is 1:1.3.

[0018] According to a preferred embodiment of the present invention, the molar ratio of 4-dimethylaminobenzaldehyde to the volume ratio of anhydrous ethanol is (0.5-3):10, unit, mmol / mL.

[0019] Most preferably, the molar ratio of 4-dimethylaminobenzaldehyde to anhydrous ethanol is 1:10, in mmol / mL.

[0020] According to a preferred embodiment of the present invention, the heating reflux time is 10-16 hours.

[0021] According to a preferred embodiment of the present invention, the elution solvent for the silica gel column is a mixture of dichloromethane and methanol in a volume ratio of 25:1.

[0022] According to a preferred embodiment of the present invention, the cancer targeted by the anticancer drug is gastric cancer.

[0023] According to a preferred embodiment of the present invention, the drug inhibits the proliferation and migration of gastric cancer cells.

[0024] According to the present invention, the gastric cancer cells are preferably human HGC-27 cells or AGS cells.

[0025] According to a preferred embodiment of the present invention, the drug arrests gastric cancer cells in the G1 phase, inhibiting their DNA synthesis.

[0026] According to a preferred embodiment of the present invention, the concentration of (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium in the anticancer drug is 0.1-5 μM.

[0027] More preferably, the concentration of (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium in the anticancer drug is 2 μM.

[0028] An anticancer drug comprising the active ingredient (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium.

[0029] According to a preferred embodiment of the present invention, the dosage form of the drug is one or more of the following: injection, powder for injection, tablet, oral liquid, capsule, granule or powder.

[0030] According to a preferred embodiment of the present invention, the anticancer drug further includes pharmaceutically acceptable excipients.

[0031] Technical features and advantages of the present invention:

[0032] The (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium of this invention can inhibit the growth of gastric cancer cells. It inhibits the proliferation, invasion, and migration of gastric cancer cells by promoting autophagy and mitochondrial fragmentation in AGS and HGC-27 cells, and arrests gastric cancer cells in the G1 phase. Furthermore, in vivo experiments in mice confirmed that ZWK-3 can inhibit tumor growth in mice in a significant dose-dependent manner, without affecting mouse body weight or visceral indices. Attached Figure Description

[0033] Figure 1 The mass spectrum of the small molecule ZWK-3 prepared in Example 1 is shown.

[0034] Figure 2 The carbon spectrum of the small molecule ZWK-3 prepared in Example 1 is shown.

[0035] Figure 3 The effects of ZWK-3 on the survival and proliferation of gastric cancer cells were investigated. A represents the effect of ZWK-3 on the survival of HGC-27 gastric cancer cells as detected by CCK8 assay; B represents the half-maximal inhibitory concentration (IC50) of ZWK-3 on HGC-27 gastric cancer cells at 24 h and 48 h, calculated based on the CCK8 results; C represents the effect of ZWK-3 on the survival of AGS gastric cancer cells as detected by CCK8 assay; and D represents the half-maximal inhibitory concentration (IC50) of ZWK-3 on AGS gastric cancer cells at 24 h and 48 h, calculated based on the CCK8 results.

[0036] Figure 4 The effect of ZWK-3 on the proliferation of gastric cancer cells HGC-27 was detected by plate clone assay (A) and statistical analysis was performed (B).

[0037] Figure 5 The effect of ZWK-3 on the proliferation of gastric cancer cells AGS was detected by plate clone assay (A) and statistical analysis was performed (B).

[0038] Figure 6 The effect of ZWK-3 on the migration ability of gastric cancer cells HGC-27 was detected by scratch healing assay (A) and statistical analysis (B).

[0039] Figure 7 The effect of ZWK-3 on the migration ability of gastric cancer cells AGS was detected by scratch healing assay (A) and statistical analysis (B).

[0040] Figure 8 The effect of ZWK-3 on the migration ability of gastric cancer cells HGC-27 and AGS was detected by Transwell assay (A) and statistical analysis (B).

[0041] Figure 9The effect of ZWK-3 on the invasive ability of gastric cancer cells HGC-27 and AGS was detected by Transwell assay (A) and statistical analysis (B).

[0042] Figure 10 The effect of ZWK-3 on the cell cycle of gastric cancer cells AGS was detected by flow cytometry (A) and statistical analysis was performed (B).

[0043] Figure 11 The effect of ZWK-3 on the cell cycle of gastric cancer cells HGC-27 was detected by flow cytometry (A) and statistical analysis was performed (B).

[0044] Figure 12 The effect of ZWK-3 on the protein levels of cell cycle-related proteins P21 and Gyclin D1 in gastric cancer cells HGC-27 was detected by Western blotting.

[0045] Figure 13 The effect of ZWK-3 on the protein levels of P21 and Gyclin D1, cell cycle-related proteins, in gastric cancer cells AGS was detected by Western blotting.

[0046] Figure 14 The effect of ZWK-3 on the protein levels of autophagy-related proteins P62 and LC3 in gastric cancer cells AGS and HGC-27 by Western blotting (A) and statistical analysis (B).

[0047] Figure 15 To detect the effect of ZWK-3 on the formation of autophagosomes in gastric cancer cells AGS and HGC-27 using immunofluorescence.

[0048] Figure 16 To detect the effect of ZWK-3 on the mitochondrial morphology of gastric cancer cells AGS and HGC-27 using immunofluorescence.

[0049] Figure 17 The effect of ZWK-3 on mouse body weight was investigated in a toxicological experiment.

[0050] Figure 18 The images of nude mice in each group were used to characterize the inhibition of subcutaneous xenograft growth by ZWK-3.

[0051] Figure 19 Representative images of tumor tissue dissected from nude mice in each group are provided to characterize the inhibition of subcutaneous xenograft growth by ZWK-3.

[0052] Figure 20 Statistical analysis of tumor weight in each group showed that ZWK-3 significantly reduced the weight of subcutaneous xenografts in nude mice.

[0053] Figure 21The tumor volume growth curves of each group indicate that ZWK-3 significantly inhibits the growth rate of subcutaneous xenografts in nude mice.

[0054] Figure 22 The effect of immunohistochemical assays on the level of Ki-67 protein in mouse tumor tissues. Detailed Implementation

[0055] The following embodiments are provided to further illustrate various aspects of the invention. These embodiments are non-limiting and should not be construed as limiting any aspect of the invention. The scope of protection of the invention is limited only by the claims. Various modifications and improvements can be made to various aspects of the invention by those skilled in the art without departing from the scope of the claims, and these modifications and improvements also fall within the scope of protection of the invention.

[0056] Additionally, it should be noted that, unless otherwise specified, all materials and reagents used in the following embodiments are commonly used in the art and can be obtained through conventional commercial means; all methods used are conventional methods known to those skilled in the art.

[0057] Example 1

[0058] Preparation of (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium:

[0059] 6-Methoxy-3-methylbenzo[d]thiazol-3-onium (0.5 mmol, 153.5 mg) and 4-dimethylaminobenzaldehyde (0.5 mmol, 88.5 mg) were dissolved in 10 mL of ethanol and heated under reflux for 12 h. After cooling to room temperature, the mixture was filtered under vacuum, and the solvent was removed by distillation under reduced pressure. The mixture was then purified by silica gel column chromatography. The eluent was a mixture of dichloromethane and methanol in a volume ratio of 25:1 to give a purple-red solid with a yield of 59%, designated as ZWK-3.

[0060] The ZWK-3 mass spectrum obtained in this embodiment is shown in the figure. Figure 1 The carbon spectrum is shown below. Figure 2 This demonstrates that the present invention successfully prepared (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium.

[0061] Example 2

[0062] Application of (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium prepared in Example 1 in the preparation of anticancer drugs.

[0063] The following section, based on cellular functional experiments and results of (E)-2-(4-(dimethylamino)styryl)-6-methoxy-3-methylbenzo[d]thiazol-3-onium, elucidates its role in effectively inhibiting the proliferation and migration of gastric cancer cells. The following experiments were conducted using cellular functional methods:

[0064] Experimental Example 1: The effect of ZWK-3 on the survival rate of gastric cancer cells

[0065] Human HGC-27 and AGS cells were used as model cells for in vitro culture.

[0066] HGC-27 and AGS cells were seeded in 96-well cell culture plates at a cell count of 3000 cells / ml per well, with cells evenly seeded and 100 μL of complete 1640 medium added. After 24 hours of cell culture in an incubator to allow cell attachment, experiments were conducted, with control and experimental groups. Control group: cultured in 1% serum with 5 μM dimethyl sulfoxide (DMSO); Experimental group: cultured in 1% serum with 0.4 μM, 0.8 μM, 1 μM, 2 μM, and 5 μM ZWK-3 medium prepared in Example 1, with each concentration gradient having 5 replicates for both control and experimental groups. After culturing at 37°C in a CO2 incubator for 24 h and 48 h, 10 μL of CCK-8 solution was added to 90 μL of basal 1640 medium per well, and the reaction was carried out in a cell culture incubator for 2 hours. After 2 hours, the culture was terminated, and the OD value of each well was measured at 450 nm using a microplate reader. A line graph was plotted using GraphPad Prism, and the half-maximal inhibitory concentration (IC50) of ZWK-3 against HGC-27 and AGS was calculated using GraphPad Prism based on the experimental data. The effect of ZWK-3 on the survival and proliferation of gastric cancer cells is shown in [reference needed]. Figure 3 .

[0067] The results showed that the growth of AGS and HGC-27 cell lines was significantly inhibited by different concentration gradients of drugs (0.4 uM, 0.8 uM, 1 uM, 2 uM, 5 uM) compared with the control group, and the inhibition was significant in a concentration-dependent manner. In addition, the drug was time-dependent, with a significant statistical difference after 48 h compared with 24 h.

[0068] Experimental Example 2: Effect of ZWK-3 on the proliferation of gastric cancer cells

[0069] HGC-27 and AGS cells were seeded into 6-well cell culture plates, with 1000 cells per well. The experiment was divided into two groups: the experimental group was cultured in 1% serum with 0.1 μM and 0.2 μM ZWK-3 media (the amount of cells seeded in this experiment was small, resulting in lower drug concentrations than in previous experiments), while the control group was cultured in 1% serum with 0.2 μM DMSO media. Each concentration gradient in both groups was repeated three times. After 12 hours of treatment, the medium was replaced with 10% serum-containing complete 1640 medium. Cells were cultured continuously at 37°C in a CO2 incubator for 10 days, and photographs were taken. Clonal clusters were counted and plotted.

[0070] The results show that: Figure 4 , Figure 5 Clonal colony formation assays showed that ZWK-3 at concentrations of 0.1 μM and 0.2 μM significantly inhibited the formation of clonal colonies in HGC-27 and AGS cells.

[0071] Experimental Example 3: ZWK-3 inhibits the invasion and migration of gastric cancer cells.

[0072] AGS and HGC-27 cells were seeded into 6-well plates. After the cells had filled the plates, they were streaked vertically using a 10 μL pipette tip. The cells were washed three times with PBS (to remove the streaked cells). Control and experimental groups were then established and the following experiments were performed: Control group: cultured in 1% serum with 2 μM dimethyl sulfoxide (DMSO); Experimental group: cultured in 1% serum with ZWK-3 at concentrations of 0.4 μM, 0.8 μM, 1 μM, and 2 μM, respectively. After continuous incubation at 37℃ in a CO2 incubator for 24 h and 48 h, the same location was observed and photographed under a high-content microscope to analyze the healing area. The test results are shown below. Figure 6 , Figure 7 .

[0073] AGS and HGC-27 cells were seeded in 6-well plates. After cell adhesion, control and experimental groups were established for the following experiments. Control group: cultured in 1% serum with 0.5 μM (HGC-27) and 1 μM (AGS) in dimethyl sulfoxide (DMSO); Experimental group: cultured in 1% serum with 0.5 μM (HGC-27) and 1 μM (AGS) in ZWK-3 medium. After continuous induction at 37℃ in a CO2 incubator for 24 h, cells in each group were treated with trypsin, the reaction was terminated with complete culture medium, and the cells were centrifuged at 800 rpm for 5 min. The supernatant was discarded, and culture medium (serum-free) was added and mixed well. The cell density was adjusted to 3 × 10⁶ cells / well. 5 (Migration), 5×10 5(Invasion) 200 μL of cell suspension from both the control and experimental groups was added to a Transwell chamber, and medium containing 10% FBS was added to the bottom of a 24-well plate. The chambers were incubated at 37°C with CO2 for 24 h. The chambers were then removed and washed twice with PBS. The chambers were transferred to another 24-well plate containing 700 μL of 4% formaldehyde and fixed at room temperature for 30 min, followed by two washes with PBS. 0.1% crystal violet was added to the 24-well plate, and the chambers were transferred to this well and stained for 20 min. The chambers were then washed three times with PBS, and the chambers were removed from the plate, carefully removing any excess cells from the surface. Five fields of view were randomly selected to count the number of cells that had migrated through the membrane. The invasion experiment followed the same procedure, except that a layer of Matrigel basement membrane was pre-laid inside the chamber, and 50 mg / L Matrigel was added to the bottom membrane of the migration chamber (1:7 dilution). The chambers were air-dried at 37°C, taking care to avoid air bubbles. The test results are shown in [link to results]. Figure 8 , Figure 9 .

[0074] The results showed that the scratch assay demonstrated that ZWK-3 significantly inhibited the migration ability of gastric cancer cells AGS and HGC-27 in a concentration-dependent manner; the Transwell assay demonstrated that ZWK-3 significantly inhibited the invasion and migration ability of gastric cancer cells AGS and HGC-27.

[0075] Experiment Example 4: ZWK-3 arrests the cell cycle of gastric cancer cells HGC-27 and AGS

[0076] AGS and HGC-27 cells were seeded in 6-well plates. After cell adhesion, control and experimental groups were established for the following experiments. Control group: cultured in 1% serum with 1 μM dimethyl sulfoxide (DMSO); Experimental group: cultured in 1% serum with 1 μM ZWK-3. After induction at 37℃ in a CO2 incubator for 24 h, cells in each group were treated with trypsin, the reaction was terminated with complete culture medium, and the cells were centrifuged at 800 rpm for 5 min. The supernatant was discarded, and the cell pellet was collected. Cells were washed once with PBS (300×g, centrifuged for 5 min), and the cell concentration was adjusted to 1×10⁶ cells / well. 6 / mL. Take 1 mL of single-cell suspension, centrifuge at 300×g for 5 min, discard the supernatant, and collect the cell pellet. Add 500 μL of pre-chilled 70% ethanol to the cell pellet, mix well by pipetting, and fix overnight at -20℃. On the second day, take the fixed cells, centrifuge at 300×g for 5 min, remove the 70% ethanol supernatant, wash the cells once with PBS (300×g, centrifuge for 5 min), discard the supernatant, and collect the cell pellet. Prepare the staining working solution by mixing RNase A and PI working solution at a volume ratio of 1:9. Add 500 μL of the prepared 500 μL PI / RNase A staining working solution to each sample, mix well by pipetting, incubate at room temperature in the dark for 30 min, sieve through a 200-mesh sterile filter, and analyze by flow cytometry. The test results are shown in the figure. Figure 10 , Figure 11 .

[0077] Human HGC-27 and AGS cells were seeded in 6 cm diameter culture dishes, and control and experimental groups were set up for the following experiments. Control group: cultured with 2 μM dimethyl sulfoxide (DMSO) under 1% serum; Experimental group: cultured with ZWK-3 at concentrations of 0.4 μM, 0.8 μM, 1 μM, and 2 μM under 1% serum. After culturing at 37℃ in a CO2 incubator for 24 h, the protein levels of P21 and Cyclin D1 were detected by Western blotting. The results are shown below. Figure 12 , Figure 13 .

[0078] The results showed that flow cytometry experiments demonstrated that ZWK-3 arrested the cell cycle of gastric cancer cells AGS and HGC-27 in the G1 phase and inhibited the expression of cell cycle-related proteins P21 and Cyclin D1 in gastric cancer cells HGC-27 and AGS.

[0079] Experimental Example 5: ZWK-3 induces autophagy in gastric cancer cells HGC-27 and AGS

[0080] Human HGC-27 and AGS cells were seeded in 6 cm diameter culture dishes, and control and experimental groups were set up for the following experiments. Control group: cultured with 2 μM dimethyl sulfoxide (DMSO) under 1% serum; experimental group: cultured with 1 μM and 2 μM ZWK-3, respectively, under 1% serum. After culturing at 37℃ in a CO2 incubator for 12 h, the cells were lysed, the cell lysate was collected, centrifuged at 12000 g for 30 min at 4℃, and the supernatant was collected. Western blotting was used to detect changes in the expression levels of P62 and LC3 proteins in the cells, and GraphPad Prism was used for statistical analysis of P62 and LC3 I / II. The results are shown in the figure below. Figure 14 .

[0081] Human HGC-27 and AGS cells 6×10 4 Cells were seeded at 1 / ml in confocal microplates and incubated overnight at 37°C in a CO2 incubator. Control and experimental groups were then established for the following experiments: Control group: cultured with 2 μM dimethyl sulfoxide (DMSO) under 1% serum conditions; Experimental group: cultured with 2 μM ZWK-3 under 1% serum conditions. After 12 h of continuous induction at 37°C in a CO2 incubator, the liquid was aspirated, and cells were fixed with ice-cold methanol at -20°C for 15 min. The fixative was aspirated, and the cells were washed three times with 1×PBS for 5 min each time. The samples were blocked at room temperature in 3% BSA solution for 60 min. The primary antibody was diluted 1:300 with 1% BSA. The blocking solution was aspirated, and the diluted primary antibody was added. The cells were incubated overnight at 4°C. On the second day, the cells were washed three times with 1×PBS for 5 min each time. The samples were then incubated at room temperature in the dark with CoraLite488-labeled goat anti-rabbit secondary antibody (diluted in 1% BSA) for 1 h. After rinsing in 1X PBS, images were taken under a confocal microscope. The test results are shown below. Figure 15 .

[0082] The results showed that WB and LC3 immunofluorescence assays confirmed that ZWK-3 induced autophagy in gastric cancer cells HGC-27 and AGS.

[0083] Experimental Example 6: ZWK-3 induces mitochondrial fragmentation in gastric cancer cells HGC-27 and AGS.

[0084] Human HGC-27 and AGS cells 6×10 4 Cells were seeded at 1 / ml in confocal microscopy dishes and incubated overnight at 37°C in a CO2 incubator. Control and experimental groups were then established for the following experiments: Control group: cultured with 2 μM dimethyl sulfoxide (DMSO) under 1% serum; Experimental group: cultured with 2 μM ZWK-3 solution under 1% serum, incubated at 37°C in a CO2 incubator for 12 h. The cell culture medium was removed, and the pre-prepared Mito-Tracker Deep Red FM stock solution (200 μM) was diluted 1:20000 with serum-free medium to prepare a 20 nM working solution. Cells were incubated in the prepared working solution at 37°C in a CO2 incubator for 30 min, followed by confocal microscopy imaging. Figure 16 .

[0085] The results showed that ZWK-3 can induce mitochondrial fragmentation in gastric cancer cells HGC-27 and AGS.

[0086] Experimental Example 7: ZWK-3 showed no significant toxicity to mice and inhibited tumor growth in mice.

[0087] Nude mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. were housed in an SPF-grade barrier environment at the Medical Science and Technology Innovation Center of Shandong First Medical University. Cultured HGC-27 gastric cancer cells were collected under a clean bench and diluted with PBS to a cell count of 5 × 10⁶ cells / mL. 6 Cell suspensions of 10 cells / ml were transported to the Experimental Animal Center of the Medical Technology Innovation Center of Shandong First Medical University using an ice pack and introduced into an SPF-grade barrier environment. The axillary skin of the experimental animals was first disinfected with alcohol. After mixing the prepared cell suspension, 0.2 ml was drawn using a 1 ml syringe and injected subcutaneously into the scapula of nude mice. To prevent leakage from the needle hole, a zigzag injection method was used. A total of 15 mice were successfully inoculated for the experiment. After tumor implantation, the mice's mental state, weight, and tumor growth were monitored daily. After the tumors grew to a visible size (10 days), the mice were randomly divided into three groups (n=5): a PBS group, a low-dose ZWK-3 group (0.1 ml of ZWK-3 injected intraperitoneally, dose 1 mg / kg / d), and a high-dose ZWK-3 group (0.1 ml of ZWK-3 injected intraperitoneally, dose 2 mg / kg / d). (The drug treatment group's preparation regimen was: 10% ZWK-3 stock solution + 40% PEG300 + 5% Tween-80 + 45% PBS). The PBS group received intraperitoneal injections of equal volumes of PBS, PEG300, and Tween-80 every 2-3 days until day 21. The mice's weight, mental state, and tumor size were observed every 2-3 days. Tumor volume was measured using calipers, measuring the longest diameter (a) and shortest diameter (b). The volume calculation formula was: V = π / 6 × L (longest diameter) × W² (shortest diameter).

[0088] After the experiment, nude mice were euthanized by cervical dislocation. After photographing the whole nude mice, they were dissected. Tumors in the axilla of the nude mice, as well as tissues of the heart, liver, spleen, lungs, and kidneys and tumor tissues were quickly removed. Organ weight was measured and organ coefficients were calculated. At the same time, the tumors were paraffin-embedded and sectioned, and Ki67 expression was detected by immunohistochemistry. The effect of ZWK-3 on the organ index of mice was detected by toxicology experiments, as shown in Table 1.

[0089] The results showed that ZWK-3 had no significant effect on mouse body weight. Figure 17 This indicates that its overall tolerability is good. Subcutaneous xenograft experiments showed that ZWK-3 significantly inhibited the growth of xenografts in the axillary region of nude mice, manifested as a significant decrease in both tumor volume and weight. Figures 18-21 The expression of Ki67 was detected by immunohistochemistry in tumor tissue, and it showed a certain concentration dependence. To further verify the antitumor effect of ZWK-3, the expression of Ki67 was detected by immunohistochemistry. Figure 22The results showed that ZWK-3 treatment significantly reduced the expression of Ki67 in tumors, suggesting that it can effectively inhibit tumor cell proliferation.

[0090] Statistical processing of experimental data:

[0091] Experimental data are expressed as mean ± standard error. A t-test was performed: P < 0.05 indicates a significant difference; P < 0.01 indicates a highly significant difference.

[0092] In summary, ZWK-3 in Example 1 can inhibit the growth of gastric cancer cells by promoting autophagy and mitochondrial fragmentation in AGS and HGC-27 gastric cancer cells, thereby inhibiting their proliferation, invasion, and migration, and arresting them in the G1 phase. Furthermore, in vivo experiments in mice confirmed that ZWK-3 can inhibit tumor growth in mice in a significant dose-dependent manner, and neither of the two doses of ZWK-3 affected the mouse's body weight or visceral indices.

Claims

1. The application of the compound shown in Formula I in the preparation of an anticancer drug, wherein the anticancer drug targets gastric cancer. ， Formula I.

2. The application according to claim 1, characterized in that, Gastric cancer cells are human-derived HGC-27 and AGS cells.

3. The application according to claim 1, characterized in that, The drug inhibits the proliferation and migration of gastric cancer cells, arresting them in the G1 phase and inhibiting their DNA synthesis.

4. An anticancer drug, said anticancer drug comprising the active ingredient of a compound represented by Formula I.

5. The anticancer drug according to claim 4, characterized in that, The dosage form of the drug includes one or more of the following: injection, powder for injection, tablet, oral liquid, capsule, granule or powder. The anticancer drug also includes pharmaceutically acceptable excipients.

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