Pancreatic cancer jAK2 and STAT3 dual targeting inhibitor containing phenoxy acetyl hydrazine compound and application

Abstract

The application discloses a pancreatic cancer JAK2 and STAT3 dual-target inhibitor containing a phenoxy acetic hydrazine compound and application thereof, and the dual-target inhibitor comprises a compound containing a phenoxy acetic hydrazine structure. The dual-target inhibitor comprises a pharmaceutically acceptable salt. The pancreatic cancer JAK2 and STAT3 dual-target inhibitor containing the phenoxy acetic hydrazine compound is applied to the preparation of a drug for treating pancreatic cancer. The drug has a new use in treating pancreatic cancer by inhibiting tumor proliferation, migration and invasion, angiogenesis, epithelial mesenchymal transition, blocking a cell cycle and promoting cell apoptosis. The pharmaceutical preparation of the drug composition comprises tablets, capsules, syrup, suspensions and injections. A drug for treating pancreatic cancer comprises the pancreatic cancer JAK2 and STAT3 dual-target inhibitor containing the phenoxy acetic hydrazine compound and a pharmaceutically acceptable carrier. The application has the beneficial effect that the pancreatic cancer JAK2 and STAT3 have a better inhibiting effect, the experimental results show that the inhibiting effect is good, the application prospect is wider, and the safety is better.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology for the treatment of pancreatic cancer, specifically to a dual-targeting inhibitor of pancreatic cancer JAK2 and STAT3 containing phenoxyacetylhydrazine compounds and its application. Background Technology

[0002] Pancreatic cancer is a malignant solid tumor with an extremely poor prognosis, and its incidence is increasing year by year both domestically and internationally. While much pioneering basic research has been conducted on the pathogenesis of pancreatic cancer, clinical translational research on key targets within these mechanisms, including research on treatment strategies, is still progressing relatively slowly. More translational research is needed to advance clinical diagnosis and treatment and prolong the survival of pancreatic cancer patients.

[0003] Signal transducer and activator of transcription (STAT) proteins are a family of transcription factors found in the cytoplasm of cells. STAT3, in particular, is involved in cell proliferation, differentiation, apoptosis, and inflammatory responses. Furthermore, STAT3-related signaling pathways are abnormally overactivated in many types of cancer and are closely associated with poor prognosis in cancer patients. In addition, overactivation of STAT3 in tumor cells and other cells within the tumor microenvironment (TME) mediates a series of extracellular signals, enhancing the immune-inflammatory response in the TME, driving tumor cell proliferation, invasion, and metastasis, and strongly suppressing anti-tumor immune responses, thus forming an immunosuppressive microenvironment.

[0004] The STAT3 protein comprises six domains, with SH2 being the most conserved STAT domain, playing a crucial role in signal transduction by binding to specific phosphorylated tyrosine motifs. Based on the important biological functions of STAT3 in tumors, STAT3 has been investigated as a potential next clinical therapeutic target for cancer, with some inhibitor studies underway. In recent years, the importance of the SH2 domain of STAT3 has been increasingly recognized, and several small-molecule inhibitors directly targeting STAT3 are under investigation, their functions confirmed in preclinical studies of various cancers. Some natural compounds have also shown antitumor activity and have demonstrated inhibitory effects on STAT3 in in vitro and in vivo experiments.

[0005] A small number of studies have screened individual inhibitors that directly target the STAT3 protein in other tumors, and related biological experiments and a limited number of clinical trials have shown that direct targeting of STAT3 is a promising treatment for tumors, including solid tumors such as pancreatic cancer, exhibiting relatively stable and comprehensive anti-tumor effects. However, the screening of drugs that directly target STAT3 is still in its early stages, with few experimentally validated drugs and most research being conducted by foreign research institutions. Much more work is needed to explore more promising compounds to expedite preclinical research and clinical translation, thereby improving the prognosis of cancer patients. Summary of the Invention

[0006] The purpose of this invention is to provide a compound containing a phenoxyacetylhydrazine structure, which has a good inhibitory effect on JAK2 and STAT3 in pancreatic cancer. Experimental results show that the inhibitory effect is good, and the compound has good application prospects and good safety. This invention is a dual-target inhibitor of JAK2 and STAT3 in pancreatic cancer and its application.

[0007] This invention relates to a dual-targeting inhibitor of pancreatic cancer JAK2 and STAT3 containing phenoxyacetylhydrazine compounds. The dual-targeting inhibitor includes compounds containing a phenoxyacetylhydrazine structure, and the structural formula of the compound is as follows:

[0008] .

[0009] A dual-targeting inhibitor of pancreatic cancer JAK2 and STAT3 containing phenoxyacetylhydrazine compounds, characterized in that the dual-targeting inhibitor comprises a pharmaceutically acceptable salt, wherein the pharmaceutically acceptable salt is an acid addition salt formed by a compound containing a phenoxyacetylhydrazine structure with any of the following acids: hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or ferulic acid.

[0010] The application of the pancreatic cancer JAK2 and STAT3 dual-targeting inhibitor containing phenoxyacetylhydrazine compounds in the preparation of drugs for treating pancreatic cancer.

[0011] A novel application of this technology in the treatment of pancreatic cancer: inhibiting tumor proliferation, migration and invasion, angiogenesis, and epithelial-mesenchymal transition; arresting the cell cycle; and promoting apoptosis.

[0012] The pharmaceutical preparations of the pharmaceutical composition include tablets, capsules, syrups, suspensions, and injections.

[0013] A drug for treating pancreatic cancer, comprising a pancreatic cancer JAK2 and STAT3 dual-targeting inhibitor containing a phenoxyacetylhydrazine compound and a pharmaceutically acceptable carrier.

[0014] The beneficial effects of this invention are:

[0015] 1) Compounds containing phenoxyacetylhydrazine can inhibit the phosphorylation levels of JAK2 and STAT3 proteins, respectively. JAK2 is a crucial promoter of STAT3 phosphorylation; therefore, inhibiting JAK2 phosphorylation further weakens STAT3 phosphorylation. This synergistic effect with the compound's direct targeting and inhibition of STAT3 protein phosphorylation ultimately results in reduced activation of JAK2 and STAT3 proteins in pancreatic cancer cells, suppressing the malignant biological functions of the tumor. Using this compound alone can exert a dual inhibitory effect on both JAK2 and STAT3, reducing the dosage, improving therapeutic efficacy, decreasing drug resistance, and reducing toxic side effects. Furthermore, this compound can also produce a more significant tumor-suppressive effect by inhibiting tumor proliferation, migration, invasion, angiogenesis, and epithelial-mesenchymal transition; arresting the cell cycle; and promoting apoptosis, thus expanding its novel applications in pancreatic cancer treatment.

[0016] 2) Compounds containing phenoxyacetylhydrazine structure have good inhibitory effects on pancreatic cancer JAK2 and STAT3. Experimental results show that the inhibitory effect is good, the application prospects are more extensive, and the safety is good. Attached Figure Description

[0017] Figure 1 The expression levels of STAT3 mRNA (a) and protein (b) in different pancreatic / pancreatic cancer cell lines of this invention.

[0018] Figure 2 SPR spectrum of the compound;

[0019] Figure 3 The results of the CETSA experiment for compound CETSA;

[0020] Figure 4 Changes in cell proliferation after treatment with different concentrations of compounds;

[0021] Figure 5 Changes in cell apoptosis after treatment with different concentrations of compounds for 24 hours;

[0022] Figure 6 Cell cycle changes after 24 hours of treatment with different concentrations of compounds;

[0023] Figure 7 Changes in cell proliferation and apoptosis-related proteins after 24 hours of treatment with different concentrations of compounds;

[0024] Figure 8 Changes in JAK2 and STAT3-related proteins in cells after 24 hours of treatment with different concentrations of compounds;

[0025] Figure 9Changes in VEGF protein and CDK2-related protein in cells after 24 hours of treatment with different concentrations of compounds;

[0026] Figure 10 Changes in EMT pathway-related proteins in cells after 24 hours of treatment with different concentrations of compounds;

[0027] Figure 11 Changes in tumor weight in CDX mice with pancreatic cancer after treatment with different concentrations of compounds;

[0028] Figure 12 HE staining of heart, liver, and kidney tissues from CDX mice with pancreatic cancer treated with different concentrations of compounds (200X).

[0029] Figure 13 Ki-67 levels in tumor cells of CDX mice with pancreatic cancer after treatment with different concentrations of compounds;

[0030] Figure 14 Tunel levels in pancreatic cancer cells after treatment with different concentrations of compounds in CDX mice with orthotopic pancreatic cancer;

[0031] Figure 15 STAT3 levels in tumor cells of CDX mice with pancreatic cancer after treatment with different concentrations of compounds;

[0032] Figure 16 The p-JAK2 level in tumor cells of CDX mice with pancreatic cancer after treatment with different concentrations of compounds;

[0033] Figure 17 The p-CDK2 level in tumor cells of pancreatic cancer orthotopic CDX mice after treatment with different concentrations of compounds;

[0034] Figure 18 VEGF levels in tumor cells of CDX mice with pancreatic cancer after treatment with different concentrations of compounds;

[0035] Figure 19 The levels of EMT pathway-related proteins in pancreatic cancer cells after treatment with different concentrations of compounds in CDX mice were determined. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings.

[0037] Example 1.

[0038] A dual-targeting inhibitor of JAK2 and STAT3 in pancreatic cancer containing phenoxyacetylhydrazine compounds. The dual-targeting inhibitors include compounds containing phenoxyacetylhydrazine structures, with the following structural formulas:

[0039] .

[0040] A dual-targeting inhibitor of pancreatic cancer JAK2 and STAT3 containing phenoxyacetylhydrazine compounds, characterized in that the dual-targeting inhibitor comprises a pharmaceutically acceptable salt, wherein the pharmaceutically acceptable salt is an acid addition salt formed by a compound containing a phenoxyacetylhydrazine structure with any of the following acids: hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or ferulic acid.

[0041] The application of the pancreatic cancer JAK2 and STAT3 dual-targeting inhibitor containing phenoxyacetylhydrazine compounds in the preparation of drugs for treating pancreatic cancer.

[0042] A novel application of this technology in the treatment of pancreatic cancer: inhibiting tumor proliferation, migration and invasion, angiogenesis, and epithelial-mesenchymal transition; arresting the cell cycle; and promoting apoptosis.

[0043] The pharmaceutical preparations of the pharmaceutical composition include tablets, capsules, syrups, suspensions, and injections.

[0044] A drug for treating pancreatic cancer, comprising a pancreatic cancer JAK2 and STAT3 dual-targeting inhibitor containing a phenoxyacetylhydrazine compound and a pharmaceutically acceptable carrier.

[0045] Experimental Example 2.

[0046] Determination of STAT3 mRNA expression levels in various pancreatic cancer cell lines

[0047] The STAT3 mRNA levels in normal pancreatic cells (HPDE6) and human pancreatic cancer cell lines (ASPC-1, PANC-1, BxPC-3, SW1990) were determined using the following method, which is briefly described below:

[0048] The differences in STAT3 mRNA levels between normal pancreatic cells and different pancreatic cancer cell lines were determined using RT-PCR.

[0049] 1) Add 50-100 mg of TRIzol to the tissue, homogenize, and let stand at room temperature;

[0050] 2) Add chloroform, shake to mix, let stand, and then centrifuge;

[0051] 3) Take the supernatant and add an equal amount of isopropanol, invert to mix, let stand at room temperature and then centrifuge;

[0052] 4) Discard the supernatant, add 75% ethanol, and centrifuge at 4°C;

[0053] 5) Discard the supernatant and allow to dry at room temperature;

[0054] 6) Reverse transcription kits and fluorescence quantitative kits are used to detect the expression level of target genes.

[0055] The differences in STAT3 mRNA levels between different pancreatic cancer cell lines and normal pancreatic cells were measured, and the results are as follows: Figure 1 As shown in a, Figure 1 In the figure, *: p<0.05; ***: p<0.001, it can be seen that the STAT3 mRNA levels in the four pancreatic cancer cell lines were higher than those in normal pancreatic cells (HPDE6). Among them, BxPC-3 cells had the highest STAT3 mRNA expression level, followed by PANC-1 cells.

[0056] Experimental Example 3.

[0057] Determination of STAT3 protein expression levels in various pancreatic cancer cell lines

[0058] The expression level of STAT3 protein in normal pancreatic cells (HPDE6) and human pancreatic cancer cell lines (ASPC-1, PANC-1, BxPC-3, SW1990) was determined by the following method, which is briefly described below:

[0059] The differences in STAT3 protein expression levels between normal pancreatic cells and different pancreatic cancer cell lines were determined using Western blot.

[0060] 1) Select ASPC-1, PANC-1, BxPC-3, SW1990 cancer cells and HPDE6 cells in the logarithmic growth phase, centrifuge and collect the precipitate;

[0061] 2) Add RIPA lysis buffer and cocktail protease inhibitor to an appropriate amount of sample, and lyse on ice; centrifuge at 4°C and collect the supernatant;

[0062] 3) Protein concentration was determined using the BCA method;

[0063] 4) The target protein was separated by SDS-PAGE;

[0064] 5) Transfer the target protein onto a PVDF membrane;

[0065] 6) Incubated with a specific primary antibody and an HRP-labeled secondary antibody;

[0066] 7) ECL staining, Image J semi-quantitative analysis of target protein expression levels.

[0067] The expression levels of STAT3 protein in different pancreatic cancer cell lines and normal pancreatic cells were measured, and the results are as follows: Figure 1As shown in b, the expression level of STAT3 protein in the four pancreatic cancer cell lines (ASPC-1, PANC-1, BxPC-3, and SW1990) was higher than that in normal pancreatic cells (HPDE6). Among them, PANC-1 cells had the highest STAT3 protein expression level, followed by BxPC-3 cells. Based on the STAT3 protein expression levels of different pancreatic cancer cell lines, PANC-1 and BxPC-3 cells were selected for subsequent experiments.

[0068] Experimental Example 4.

[0069] IC50 determination of compounds

[0070] The 24-hour half-maximal inhibitory concentration (IC50) of this compound on the proliferation inhibition of human pancreatic cancer cell lines (PANC-1, BxPC-3) was determined by the following method.

[0071] Experimental steps:

[0072] The half-maximal inhibitory concentration (IC50) of this compound in inhibiting the proliferation of two pancreatic cancer cell lines was determined using the CCK8 assay.

[0073] 1) After digesting BxPC-3 and PANC-1 cells with trypsin, the cell density was adjusted to 5×105 cells / ml with RPMI-1640 complete medium or DMEM complete medium. 100 μl of cell suspension was seeded in each well of a 96-well plate and a blank control group was set up. The culture plate was placed in a constant temperature incubator with 5% CO2 at 37℃.

[0074] 2) After culturing for 24 hours, remove the culture plate, wash each well with 100 μL of PBS, dilute the compound (40 mM) to 10, 20, 30 and 40 μM with RPMI-1640 or DMEM serum-free medium, and add 100 μL / well to the cells. Use untreated cells as a control. Each group has 4 replicates and is incubated at 37°C.

[0075] 3) After the detection time point, add 10 μL of CCK8 solution to each well (do not generate bubbles);

[0076] 4) Wrap the culture plate with aluminum foil, place it in an incubator, and incubate in the dark for 2 hours;

[0077] 5) Measure the absorbance at 450nm using an ELISA reader. Turn on the reader, create a new record, shake the plate for 10s, shake it again for 10s, and measure the absorbance (Optical Density, OD). Inhibition rate = (OD value of control group - OD value of experimental group) / OD value of control group × 100%.

[0078] 6) After obtaining the data, use SPSS27 to calculate IC50.

[0079] The half-maximal inhibitory concentration (IC50) of the compound against the proliferation activity of two pancreatic cancer cell lines was determined. The measured IC50 values ​​are shown in Table 1. It can be seen that the compound has an inhibitory effect on both PANC-1 and BxPC-3 cells, and the IC50 values ​​of the compound inhibiting the two cell lines are similar. Based on this, the standard dose and treatment time for subsequent administration of the compound were set to 6 μM / 24h.

[0080]

[0081] Table 1. IC50 of compounds inhibiting the proliferation of human pancreatic cancer cells (PANC-1, BxPC-3)

[0082] Experimental Example 5.

[0083] Surface plasmon resonance (SPR) analysis of compounds

[0084] To accurately assess the affinity of the compound for the STAT3 protein and preliminarily clarify its targeting and binding force, we used surface plasmon resonance (SPR) analysis to examine the affinity between the compound as a small molecule STAT3 inhibitor and the humanized STAT3 protein. The test method is shown below:

[0085] Experimental steps:

[0086] Protein chip preparation: Before injection, 400 mM EDC and 100 mM NHS were mixed to prepare an activator. The CM5 sensor chip was activated at a flow rate of 10 μL / min for 420 s. Then, SPR chip fixative was injected into the Fc2 sample channel at a flow rate of 10 μL / min to achieve a fixation level of approximately 8000-15000 RU. The chip was inactivated with 1 M Ethanolamine-HCl solution at a flow rate of 10 μL / min for 420 s. The Fc1 control channel was blocked using the same procedure as the Fc2 sample channel, but the Fc1 control channel was not injected with STAT3.

[0087] Multi-cycle flow of small molecule compounds: Small molecule compounds are diluted to the appropriate concentration using flow buffer and injected into the channel at a flow rate of 30 μL / min to form binding and dissociation phases. Both binding and dissociation processes occur within the run buffer, and the affinity between the small molecules and proteins is analyzed using Biacore8K.

[0088] The affinity between the compound and humanized STAT3 protein was determined, and the data analysis is shown in Table 2. It can be seen that the affinity (KD value) between the compound and humanized STAT3 protein is 2.15 × 10⁻⁴ M. Generally speaking, the affinity between small molecules and proteins is moderate, and the compound exhibits a moderate binding strength to humanized STAT3 protein. This compound has the potential to become a STAT3-targeting drug. The SPR spectrum of this compound is shown below. Figure 2 As shown.

[0089]

[0090] Table 2 shows the SPR analysis results of the compound and humanized STAT3 protein.

[0091] Experimental Example 6.

[0092] Cellular Thermal Shift Assay (CETSA)

[0093] Based on the characteristic that the binding of compounds with targeting functions to target proteins often alters the thermal stability of their target proteins, the expression level of STAT3 protein in the cytoplasm of the compound and human pancreatic cancer cell lines (PANC-1, BxPC-3) was measured after incubation at different fixed temperatures for corresponding times. The targeting of the compound to STAT3 protein in pancreatic cancer cells was then observed by plotting thermal melting curves. The measurement method is shown below.

[0094] Experimental steps:

[0095] PANC-1 and BxPC-3 cells were incubated with the compound at seven different temperatures (45℃, 48℃, 51℃, 54℃, 57℃, 60℃, and 63℃) for 40 min each.

[0096] Extract STAT3 protein for Western blot experiments (specific experimental protocol as described above);

[0097] Based on the STAT3 protein level at different temperatures, protein dissolution curves were plotted using Origin2022.

[0098] The expression levels of STAT3 protein in the cytoplasm of the compound and human pancreatic cancer cell lines (PANC-1 and BxPC-3) were determined after culture at different fixed temperatures for corresponding time periods. The thermolysis curves of the two cell lines were plotted based on the results. Figure 3As shown, within PANC-1 and BxPC-3 cells, the stability of STAT3 protein in different pancreatic cancer cells treated with this compound was significantly enhanced with increasing temperature compared to the control group (which only added the same dose of the candidate drug solvent DMSO). The CETSA results fully demonstrate that this compound can enter pancreatic cancer cells and target and bind to intracellular STAT3 protein, exhibiting certain pancreatic cancer cell penetration and STAT3 target binding affinity.

[0099] Experimental Example 7.

[0100] Determination of the inhibitory effect of different concentrations of compounds on the proliferation of pancreatic cancer cells

[0101] The inhibitory effects of this compound on the proliferation of human pancreatic cancer (PANC-1, BxPC-3) cell lines at high, medium, and low doses were determined at 0h, 24h, 48h, and 72h. The high, medium, and low doses were set as 2 times (12μM), 1 times (6μM), and 0.5 times (3μM) of the standard dose, respectively. The determination method is shown below.

[0102] Experimental steps:

[0103] The inhibitory effects of different concentrations of the compound on the proliferation of two pancreatic cancer cells at different time points were determined using the CCK8 assay.

[0104] 1) After digesting BxPC-3 and PANC-1 cells with trypsin, adjust the cell density to 5×105 cells / ml with RPMI-1640 complete medium or DMEM complete medium; seed 100 μl of cell suspension in each well of a 96-well plate and set up a blank control group; place the culture plate in a 37℃ constant temperature incubator containing 5% CO2.

[0105] 2) After culturing for 24 hours, remove the culture plate, wash each well with 100 μL of PBS, dilute the compound (40 mM) to 12 μM, 6 μM, and 3 μM with RPMI-1640 or DMEM serum-free medium, and add 100 μL / well to the cells. Use untreated cells as a control. Each group has 4 replicates and is incubated at 37°C for 0 h, 24 h, 48 h, and 72 h.

[0106] 3) After the detection time point, add 10 μL of CCK8 solution to each well (do not generate bubbles);

[0107] 4) Wrap the culture plate with aluminum foil, place it in an incubator, and incubate in the dark for 2 hours;

[0108] 5) Measure the absorbance at 450nm using an ELISA reader. Turn on the reader, create a new record, shake the plate for 10s, shake it again for 10s, and measure the absorbance (Optical Density, OD). Inhibition rate = (OD value of control group - OD value of experimental group) / OD value of control group × 100%.

[0109] 6) After obtaining the data, use GraphPad Prism8 to plot the graph;

[0110] The inhibitory effects of different concentrations of this compound on the proliferation of two pancreatic cancer cell lines at different time points were determined, and the results are shown in [Figure number missing]. Figure 4 , Figure 4 In the table, *: p<0.05; **: p<0.01; ***: p<0.001, it can be seen that the compound produced concentration-dependent inhibition of cell proliferation in human pancreatic cancer cell lines (PANC-1 and BxPC-3), and the effect was significant (p<0.001). Furthermore, the cell proliferation inhibition effect increased with increasing concentration at 24h, 48h, and 72h.

[0111] Experimental Example 8.

[0112] Determination of the effect of different concentrations of compounds on apoptosis in pancreatic cancer cells.

[0113] The effect of this compound on apoptosis in human pancreatic cancer cell lines (PANC-1 and BxPC-3) at high, medium, and low doses was determined using flow cytometry, as shown below:

[0114] 1) The cells from the blank control group were divided into four groups (treated with binding buffer only, Annexin V-FITC single staining, PI single staining, and PI double staining, respectively). Multiple 15ml centrifuge tubes were pre-labeled according to the experimental design. The growth of pancreatic cancer cells in each group was observed under a microscope. When the tumor cell confluence reached approximately 70-80%, the original culture medium of pancreatic cancer cells from each group was collected into the pre-labeled 15ml centrifuge tubes. After washing with 1×PBS, the cells were digested with trypsin (without EDTA). The trypsin was discarded, and the cells were collected a second time and transferred to the corresponding centrifuge tubes for centrifugation at 1000g for 5 minutes.

[0115] 2) Discard the supernatant after centrifugation, add 5 ml of pre-cooled PBS to each centrifuge tube, mix well, take 20 μl of the solution and count in a cell counter, take 8 × 10⁴ pancreatic cancer cells and centrifuge at 1000g for 5 min.

[0116] 3) Discard the supernatant, add 195 μl of Annexin V-FITC binding solution to the centrifuge tube using a 200 μl pipette tip, gently mix, add 5 μl of Annexin V-FITC and 10 μl of PI, mix well; incubate at 4°C in the dark for 20 min, add 300 μl of binding solution, place on ice and complete flow cytometry analysis within 1 hour;

[0117] 4) Flow joX.10.0.7 software was used to analyze the apoptosis status of each group, and GraphPad 7.00 software was used to draw a bar chart of the changes in apoptosis.

[0118] The effect of this compound at different concentrations on apoptosis in pancreatic cancer PANC-1 and BxPC-3 cells was determined, and the results are as follows: Figure 5 As shown, Figure 5 In the table, *: p<0.05; **: p<0.01; ***: p<0.001, it can be seen that compared with the control group, the apoptosis of both cell lines increased after administration of this compound in a concentration gradient-dependent manner.

[0119] Experimental Example 9.

[0120] Determination of the effects of different concentrations of compounds on the cell cycle of pancreatic cancer cells.

[0121] The effects of this compound on the cell cycle of human pancreatic cancer cell lines (PANC-1 and BxPC-3) at high, medium, and low doses were determined using flow cytometry, as shown below:

[0122] 1) Label centrifuge tubes according to the experimental design. When the cell confluence is about 70-80%, collect the digested cells from each group, transfer them to centrifuge tubes, and centrifuge at 1000 rpm for 5 minutes.

[0123] 2) Discard the supernatant. Add 5 ml of pre-cooled PBS (4°C) to the cell pellet of each centrifuge tube to wash the cells. Centrifuge at 1500 rpm for 5 min. Resuspend the cells and count them. After adjusting the concentration, take 1 ml of cell suspension containing 1×106 cells, centrifuge (800 rpm, 5 min), and wash twice with pre-cooled PBS.

[0124] 3) Preparation of enzyme-free water with 70% anhydrous ethanol: Measure 7 ml of 100% anhydrous ethanol and 3 ml of DEPC water and add them to a 15 ml centrifuge tube. Place the tube in a 4°C refrigerator to pre-cool.

[0125] 4) Discard the supernatant, use a 1ml pipette tip to measure 500μl of enzyme-free water in 70% anhydrous ethanol and add it to each cell pellet, gently mix and place in a 4°C refrigerator overnight.

[0126] 5) Preheat the water bath to 37°C; add 5 ml of pre-cooled PBS to each centrifuge tube, centrifuge at 800 rpm for 5 min, discard the PBS solution, resuspend in 100 µl of RNase A solution, and incubate in a water bath for 30 min; add 400 µl of propidium iodide (PI), and fix at 4°C for 30 min. Analyze the DNA content of cells in each group using flow cytometry.

[0127] 6) Use Modfit LT32 software to analyze the cell cycle status of each group, and use GraphPad 7.00 software to draw a bar chart of cell cycle changes.

[0128] The effects of different concentrations of the compound on the cell cycle of pancreatic cancer PANC-1 and BxPC-3 cells were determined, and the results are as follows: Figure 6 As shown, Figure 6 In the figures, *: p<0.05; **: p<0.01; ***: p<0.001, it can be seen that after treatment of PANC-1 cells with this compound, the high, medium, and low concentration groups showed a significant increase in the proportion of cells in the G0 / G1 phase (p<0.01) and a significant decrease in the proportion of cells in the S phase (p<0.01), but only the medium concentration group showed a decrease in the proportion of cells in the G2 / M phase (p<0.05). After treatment of BxPC-3 cells with this compound, the high, medium, and low concentration groups all showed a significant increase in the proportion of cells in the G0 / G1 phase (p<0.05) and a significant decrease in the proportion of cells in the S phase (p<0.01), and this trend was concentration-gradient dependent, but the change in the proportion of cells in the G2 / M phase was not significant. The cell cycle results after administration to the two cell lines were similar, and this compound can arrest pancreatic cancer cells in the G0 / G1 phase in a concentration-gradient dependent manner.

[0129] Experimental Example 10.

[0130] Determination of the effects of different concentrations of compounds on pancreatic cancer cell proliferation and apoptosis-related proteins.

[0131] The effects of high, medium, and low concentrations of the compound on the levels of cell proliferation-related proteins Ki67 and PCNA, as well as apoptosis-related proteins BCL-2, BAX, and Cleaved Caspase-3 in human pancreatic cancer cell lines (PANC-1 and BxPC-3) were determined using the following methods, which are briefly described below (for specific experimental techniques, please refer to the Western blot experimental procedure section of Molecular Cloning: A Laboratory Manual (Fourth Edition)).

[0132] Experimental steps:

[0133] The effects of different concentrations of the compound on the protein levels of Ki67, PCNA, BCL-2, BAX, and Cleaved Caspase-3 in PANC-1 and BxPC-3 cell lines were determined using Western blot.

[0134] 1) BxPC-3 and PANC-1 were treated with compounds at 2 times the standard dose (12 μM), 1 times the standard dose (6 μM), and 0.5 times the standard dose (3 μM) for 24 h before being collected;

[0135] 2) Add RIPA lysis buffer and cocktail protease inhibitor to an appropriate amount of sample, lyse on ice; centrifuge at 4°C and collect the supernatant;

[0136] 3) Protein concentration was determined using the BCA method;

[0137] 4) The target protein was separated by SDS-PAGE;

[0138] 5) The target protein was transferred onto a PVDF membrane;

[0139] 6) Incubated with a specific primary antibody and an HRP-labeled secondary antibody;

[0140] 7) ECL staining, Image J semi-quantitative analysis of target protein expression levels.

[0141] The effects of different concentrations of the compound on the expression levels of Ki67 and PCNA, proliferation-related proteins of pancreatic cancer PANC-1 and BxPC-3 cells, as well as apoptosis-related proteins BCL-2, BAX, and Cleaved Caspase-3, were determined. The results are as follows: Figure 7 As shown, Figure 7 In the expression *: p<0.05; **: p<0.01; ***: p<0.001, we can see that:

[0142] After treating pancreatic cancer cells PANC-1 and BxPC-3 at different concentrations for 24 hours, the expression levels of cell proliferation-related proteins Ki67 and PCNA decreased, exhibiting concentration-dependent proliferation inhibition. Conversely, the expression levels of apoptosis-related proteins BCL-2 decreased, while the expression levels of BAX and Cleaved Caspase-3 increased, showing a concentration gradient dependence and promoting apoptosis. These trends are consistent with the cell proliferation and apoptosis results obtained by flow cytometry, further confirming the effects of the compound at different concentrations on the proliferation and apoptosis of pancreatic cancer cells.

[0143] Experimental Example 11.

[0144] Determination of the effect of different concentrations of compounds on phosphorylation of JAK2 / STAT3 protein in pancreatic cancer cells.

[0145] As a candidate drug for STAT3 targeting inhibitors, determining its effect on STAT3 phosphorylation in pancreatic cancer is crucial. Human pancreatic cancer (PANC-1, BxPC-3) cell lines were treated with high (2 times the standard dose), medium (1 times the standard dose), and low (0.5 times the standard dose) concentrations to observe the effect of the standard dose on the phosphorylation of STAT3 protein and its upstream JAK2 protein. The changes in the levels of STAT3, JAK2, p-STAT3, and p-JAK2 proteins in human pancreatic cancer (PANC-1, BxPC-3) cell lines at high, medium, and low concentrations of the compound were determined using the following methods, briefly described below. 23 (For specific experimental techniques, please refer to the Western blot experimental operation section of Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0146] Experimental steps:

[0147] The effects of different concentrations of the compound on the levels of STAT3, JAK2, phosphorylated STAT3, and phosphorylated JAK2 proteins in PANC-1 and BxPC-3 cell lines were determined using Western blot.

[0148] 1) BxPC-3 and PANC-1 were treated with compounds at 2 times the standard dose (12 μM), 1 times the standard dose (6 μM), and 0.5 times the standard dose (3 μM) for 24 h before being collected;

[0149] 2) Add RIPA lysis buffer and cocktail protease inhibitor to an appropriate amount of sample, and lyse on ice; centrifuge at 4°C and collect the supernatant;

[0150] 3) Protein concentration was determined using the BCA method;

[0151] 4) The target protein was separated by SDS-PAGE;

[0152] 5) The target protein was transferred onto a PVDF membrane;

[0153] 6) Incubated with a specific primary antibody and an HRP-labeled secondary antibody;

[0154] 7) ECL staining, ImageJ semi-quantitative analysis of target protein expression levels.

[0155] The effects of different concentrations of the compound on the expression levels of specific proteins in two pancreatic cancer cell lines were measured, and the results are as follows: Figure 8It can be seen that after 24 hours of treatment with different concentrations of the compound, all concentrations of the compound can inhibit the phosphorylation level of STAT3. The inhibitory effect of the compound on STAT3 phosphorylation in PANC-1 cells is more obvious than that in BxPC-3 cells, and both show a concentration gradient dependence. This indicates that the compound has a significant inhibitory effect on STAT3 protein in pancreatic cancer cells and can produce a good inhibitory effect on the STAT3 target.

[0156] Analysis of the JAK2 protein upstream of STAT3 yielded the following results: Figure 8 As shown, Figure 8 In the table, *: p<0.05; **: p<0.01; ***: p<0.001, it can be seen that in PANC-1 cells, different concentrations of the compound significantly inhibited JAK2 phosphorylation (p<0.05) in a dose-dependent manner. In BxPC-3 cells, although all concentrations of the compound showed a decrease in JAK2 phosphorylation in a concentration gradient-dependent manner, only the high concentration group of the compound showed significant inhibition of JAK2 phosphorylation (p<0.01), indicating that the compound can also have a good target inhibitory effect on JAK2 protein.

[0157] In summary, this compound has the potential to serve as a dual-target inhibitor of JAK2 / STAT3.

[0158] Experimental Example 12.

[0159] Determination of the effects of different concentrations of compounds on VEGF protein in pancreatic cancer cells.

[0160] The effects of high, medium, and low concentrations of the compound on the levels of VEGF and CDK2 proteins in human pancreatic cancer cell lines (PANC-1 and BxPC-3) were determined using the following method. The experimental method is briefly described below (for detailed experimental techniques, please refer to the Western blot section of *Molecular Cloning: A Laboratory Manual (Fourth Edition)*).

[0161] Experimental steps:

[0162] Western blot was used to determine the effect of the compound at different concentrations on VEGF protein in PANC-1 and BxPC-3 cell lines.

[0163] 1) BxPC-3 and PANC-1 were treated with compounds at 2 times the standard dose (12 μM), 1 times the standard dose (6 μM), and 0.5 times the standard dose (3 μM) for 24 h before being collected;

[0164] 2) Add RIPA lysis buffer and cocktail protease inhibitor to an appropriate amount of sample, lyse on ice; centrifuge at 4°C and collect the supernatant;

[0165] 3) Protein concentration was determined using the BCA method;

[0166] 4) The target protein was separated by SDS-PAGE;

[0167] 5) The target protein was transferred onto a PVDF membrane;

[0168] 6) Incubated with a specific primary antibody and an HRP-labeled secondary antibody;

[0169] 7) ECL staining, Image J semi-quantitative analysis of target protein expression levels.

[0170] Since one of the functions of STAT3 in tumors is to promote angiogenesis, this study aimed to verify whether the compound could inhibit angiogenesis in pancreatic cancer by targeting STAT3. The effects of different concentrations of the compound on the expression levels of VEGF protein in two pancreatic cancer cell lines were measured. The results are as follows: Figure 9 As shown, Figure 9 In the table, *: p<0.05; **: p<0.01; ***: p<0.001, it can be seen that only in the PANC-1 cell line did the VEGF protein level decrease but not significantly. The remaining groups all indicated that the compound inhibited VEGF protein expression in cells under different concentration conditions (p<0.01), and this showed a concentration gradient dependence. In the BxPC-3 cell line, the compound significantly inhibited intracellular VEGF protein expression in different concentration groups (p<0.05), and this also showed a concentration gradient dependence.

[0171] Experimental Example 13.

[0172] Determination of the effects of different concentrations of compounds on CDK2 protein in pancreatic cancer cells.

[0173] The effects of high, medium, and low concentrations of compounds on CDK2 protein and its phosphorylation level in human pancreatic cancer cell lines (PANC-1 and BxPC-3) were determined using the following method. The experimental method is briefly described below (for detailed experimental techniques, please refer to the Western blot section of *Molecular Cloning: A Laboratory Manual (Fourth Edition)*). Experimental steps:

[0174] Western blot was used to determine the effect of the compound at different concentrations on CDK2 protein in PANC-1 and BxPC-3 cell lines.

[0175] 1) BxPC-3 and PANC-1 were treated with compounds at 2 times the standard dose (12 μM), 1 times the standard dose (6 μM), and 0.5 times the standard dose (3 μM) for 24 h before being collected;

[0176] 2) Add RIPA lysis buffer and cocktail protease inhibitor to an appropriate amount of sample, lyse on ice; centrifuge at 4°C and collect the supernatant;

[0177] 3) Protein concentration was determined using the BCA method;

[0178] 4) The target protein was separated by SDS-PAGE;

[0179] 5) The target protein was transferred onto a PVDF membrane;

[0180] 6) Incubated with a specific primary antibody and an HRP-labeled secondary antibody;

[0181] 7) ECL staining, Image J semi-quantitative analysis of target protein expression levels.

[0182] Based on the aforementioned experimental findings that the compound can arrest the cell cycle of pancreatic cancer cells in the G0 / G1 phase, the cell cycle regulator CDK2 was analyzed, and the results are as follows: Figure 9 As shown, in PANC-1 cells, the total CDK2 protein level significantly decreased in the high, medium, and low concentration groups of this compound (p<0.01) in a concentration gradient-dependent manner. In the BxPC-3 cell line, the total CDK2 protein level significantly decreased in the high concentration group of the compound (p<0.01), while the CDK2 protein levels in the remaining groups decreased in a concentration gradient-dependent manner, but not significantly.

[0183] It can be seen that in the PANC-1 cell line, the phosphorylation of CDK2 protein was significantly inhibited in both high and medium concentrations of the compound (p<0.05), and the inhibition of CDK2 phosphorylation in different concentration groups of the compound showed a concentration gradient dependence. In the BxPC-3 cell line, the high, medium and low concentrations of the compound significantly inhibited the phosphorylation of CDK2 protein (p<0.01), and showed a concentration gradient dependence. In summary, the inhibition of CDK2 protein phosphorylation and its inactivation may be related to the cell cycle arrest in the G0 / G1 phase after drug administration.

[0184] Experimental Example 14.

[0185] Determination of the effects of different concentrations of compounds on EMT-related proteins in pancreatic cancer cells.

[0186] The effects of high, medium, and low concentrations of the compound on the levels of E-Cadherin, Vimentin, and Snail proteins related to the EMT pathway in human pancreatic cancer cell lines (PANC-1 and BxPC-3) were determined using the following method. The experimental methods are briefly described below (for detailed experimental techniques, please refer to the Western blot section of *Molecular Cloning: A Laboratory Manual (Fourth Edition)*).

[0187] Experimental steps:

[0188] Western blot was used to determine the effect of the compound at different concentrations on VEGF protein in PANC-1 and BxPC-3 cell lines.

[0189] 1) BxPC-3 and PANC-1 were treated with compounds at 2 times the standard dose (12 μM), 1 times the standard dose (6 μM), and 0.5 times the standard dose (3 μM) for 24 h before being collected;

[0190] 2) Add RIPA lysis buffer and cocktail protease inhibitor to an appropriate amount of sample, lyse on ice; centrifuge at 4°C and collect the supernatant;

[0191] 3) Protein concentration was determined using the BCA method;

[0192] 4) The target protein was separated by SDS-PAGE;

[0193] 5) The target protein was transferred onto a PVDF membrane;

[0194] 6) Incubated with a specific primary antibody and an HRP-labeled secondary antibody;

[0195] 7) ECL staining, ImageJ semi-quantitative analysis of target protein expression levels.

[0196] Since activation of the EMT pathway is one of the key reasons for the invasion and metastasis of malignant tumors, to verify whether the target of this compound to STAT3 can affect the invasion and metastasis of tumor cells, the effects of different concentrations of this compound on the expression levels of EMT pathway-related proteins in pancreatic cancer cells PANC-1 and BxPC-3 were measured. The results are as follows: Figure 10 As shown, after 24 hours of treatment with different concentrations of compounds, pancreatic cancer cells PANC-1 and BxPC-3 showed increased expression of E-Cadherin and decreased expression of Vimentin and Snail, which were related to the EMT pathway, in a concentration gradient-dependent manner, indicating concentration-dependent inhibition of the EMT pathway.

[0197] Experimental Example 15.

[0198] Compound anti-pancreatic cancer activity assay

[0199] The treatment drug was this compound, and the cell line was the human pancreatic cancer cell line PANC-1 cultured in DMEM medium containing 10% fetal bovine serum. The test animals were SPF-grade BALB / c-nu nude mice; 5-6 weeks old females; 5 mice per group. Drug dosage settings are shown in Table 3.

[0200]

[0201] Table 3 Drug Dosage Configuration

[0202] Drug dosing concentration design:

[0203] In the preliminary study, the mouse LD50 of a similar compound to the reference compound was 232 mg / kg. Based on a 1 / 10 LD50, 20 mg / kg and 30 mg / kg were used as candidate concentrations. Three 8-9 week old BALB / C-nu female nude mice were selected from each group for the preliminary experiment. The compound was administered via intraperitoneal injection. It was found that mice in the 30 mg / kg group showed reduced activity on the 4th day of continuous administration, while mice in the 20 mg / kg group only began to show reduced activity after the 8th day. Therefore, 20 mg / kg was selected as the high-dose concentration of the compound for subsequent experiments.

[0204] Drug preparation method:

[0205] High-dose compound group: Weigh 2.8 mg of the compound, take 50 μL of DMSO solution, dissolve it, add 1.95 ml of physiological saline to dilute, prepare 2 ml of 1.4 mg / ml compound solution (DMSO concentration 2.5%), and administer intraperitoneally at a volume of 0.2 ml / 14 g.

[0206] Medium-dose group of the compound: Take 1 ml of the 1.4 mg / ml compound solution from the high-dose group above, add 1 ml of physiological saline to form 2 ml of 0.7 mg / ml compound solution (DMSO concentration 1.25%), and administer intraperitoneally. The administration volume is 0.2 ml / 14 g.

[0207] Low-dose group of the compound: Take 1 ml of the 0.7 mg / ml compound solution from the medium-dose group above, add 1 ml of physiological saline to form 2 ml of 0.35 mg / ml compound solution (DMSO concentration 0.625%), and administer intraperitoneally at a volume of 0.2 ml / 14 g.

[0208] The specific method for constructing a mouse model of pancreatic cancer xenograft (CDX) is as follows:

[0209] First, a subcutaneous pancreatic cancer xenograft model was constructed in nude mice. PANC-1 cells with high STAT3 expression were selected and cultured in vitro until the cell density reached 80%-90%. The cells were then digested with trypsin to prepare a cell suspension for later use. 4-6 week old female BALB / C-nu nude mice were subcutaneously injected with the prepared PANC-1 cell suspension in the axilla, with each mouse receiving 2-3 × 10⁶ cells. After injection, the mice were fed normally. After about 3 weeks, the tumor diameter reached 1 cm. The mice were then euthanized by intraperitoneal injection of an excessive amount of sodium pentobarbital. The subcutaneous tumor was removed and cut into 1 mm³ pieces in sterile cell culture medium for later use.

[0210] We continued to construct a mouse model of orthotopic pancreatic cancer xenograft (CDX) using human tumor cell lines. Five- to six-week-old female BALB / c-nu nude mice were anesthetized with isoflurane. The mice were placed on the operating table in a right-sided oblique position. The surgical area on the upper left abdomen was disinfected with iodine solution using a cotton swab. An oblique incision was made under the left rib to expose the abdominal muscles. The spleen was visible through the abdominal wall. The abdominal wall was incised at the surface of the spleen tail. The spleen tail was gently grasped with forceps and slightly pulled out of the body. The thin membrane between the stomach and spleen was carefully separated, and the pancreas was visible by turning it downwards. The capsule of the pancreas was gently cut open with forceps, and the cut tumor fragment was inserted into the capsule and pushed towards the pancreas, taking care not to damage the capsule. After confirming the tumor fragment was correctly positioned and would not slip out, the spleen was returned to the abdominal cavity. The abdominal wall and skin were sutured together with 6-0 silk sutures. The wound was disinfected with iodine solution after skin-to-skin closure. Anesthesia was removed, and the mice were fed normally after recovery.

[0211] Experimental methods:

[0212] After the orthotopic CDX model of pancreatic cancer was established in mice and they were fed for 3 weeks, they were randomly divided into 8 groups of 5 mice each. Specifically, the control group (no intervention, conventional feeding), the DMSO group (intraperitoneal injection of physiological saline containing an equal volume of DMSO as in the high-dose group), the high-dose group (intraperitoneal injection of 20 mg / kg compound), the medium-dose group (intraperitoneal injection of 10 mg / kg compound), and the low-dose group (intraperitoneal injection of 5 mg / kg compound). After 7 consecutive days of administration, the mice were fed conventionally for 14 days. The mice were then euthanized by intraperitoneal injection of an excessive amount of sodium pentobarbital. The orthotopic pancreatic tumor tissue was obtained by dissection, weighed, recorded, and frozen in liquid nitrogen for later use. At the same time, the heart, liver, and kidneys of the mice were obtained by dissection, fixed in 10% formaldehyde, and used for later use.

[0213] Experimental results show that: Figure 11 Compared with the blank group (0.92±0.11g) and the DMSO group (0.88±0.07g), the mass of in situ pancreatic tumor tissue in mice was significantly reduced in the high-dose group (0.22±0.14g, p<0.001), medium-dose group (0.48±0.24g, p<0.001), and low-dose group (0.54±0.10g, p<0.001), and this reduction was in concentration gradient dependence.

[0214] Experimental Example 16.

[0215] Compound toxicity assay

[0216] Immunohistochemical hematoxylin and eosin (HE) staining was used to observe the pathological changes in the heart, liver, and kidney tissues of CDX mice with pancreatic cancer after treatment with the compound. The experimental method is shown below:

[0217] 1) Paraffin embedding, sectioning, and mounting;

[0218] 2) Dewaxing and hydration of paraffin sections: 25 min each in xylene I and II, 10 min each in different gradients of ethanol (100%, 95%, 90%, 85%, 80%, 70%), and rinsed with tap water.

[0219] 3) Hematoxylin staining: stain with hematoxylin for 5 min, then rinse with running water for 2 min;

[0220] 4) Differentiation: 1% hydrochloric acid ethanol for 2 seconds, then wash with water for 2 minutes;

[0221] 5) Blueing: Blueing for 2 minutes (using warm water at around 80℃);

[0222] 6) Eosin staining: Stain with 0.5% eosin for 5 min, then wash with water for 2 min;

[0223] 7) Dehydration and clearing: 70%, 5 min; 80%, 5 min; 85%, 5 min; 90%, 5 min; 95%, 10 min; 100%, 10 min; Xylene I and II, 25 min each;

[0224] 8) Mounting: Neutral resin for mounting.

[0225] HE staining results are as follows Figure 12 Compared with the heart, liver, and kidney tissues of normal mice, no pathological changes were observed in the heart, liver, and kidney tissues of mice after administration. No toxic effects on the heart, liver, or kidneys were observed in pancreatic cancer CDX mice treated with high, medium, and low concentrations of the compound.

[0226] Experimental Example 17.

[0227] Determination of the effect of different concentrations of compounds on the proliferation of pancreatic cancer tumor cells.

[0228] The effects of high, medium, and low doses of the compound on the levels of the proliferation-related protein Ki-67 in orthotopic CDX mouse tumor tissues of pancreatic cancer were determined using the following experimental method:

[0229] 1) Paraffin embedding, sectioning, and mounting;

[0230] 2) Dewaxing and hydration of paraffin sections: 25 min each in xylene I and II, 10 min each in different gradients of ethanol (100%, 95%, 90%, 85%, 80%, 70%), and rinsed with tap water.

[0231] 3) Hematoxylin staining: stain with hematoxylin for 5 min, then rinse with running water for 2 min;

[0232] 4) Differentiation: 1% hydrochloric acid ethanol for 2 seconds, then wash with water for 2 minutes;

[0233] 5) Blueing: Blueing for 2 minutes (using warm water at around 80℃);

[0234] 6) Eosin staining: Stain with 0.5% eosin for 5 min, then wash with water for 2 min;

[0235] 7) Dehydration and clearing: 70%, 5 min; 80%, 5 min; 85%, 5 min; 90%, 5 min; 95%, 10 min; 100%, 10 min; Xylene I and II, 25 min each;

[0236] 8) Mounting: Neutral resin for mounting.

[0237] The expression levels of the proliferation-related protein Ki-67 in tumor tissues of CDX mice with pancreatic cancer were measured after treatment with different doses. The results are as follows: Figure 13 As shown, Figure 13 In the table, *: p<0.05; **: p<0.01; ***: p<0.001, it can be seen that compared with the blank group, the Ki67 level in the solvent group did not change significantly, while the Ki67 level in the low concentration group was significantly reduced (p<0.05), and the Ki67 levels in the high and medium concentration groups were significantly reduced (p<0.001). This indicates that the compound can inhibit the growth of pancreatic cancer cells in vivo.

[0238] Experimental Example 18.

[0239] Determination of the effect of different concentrations of compounds on apoptosis in pancreatic cancer tumor tissue cells.

[0240] The effects of high, medium, and low doses of the compound on apoptosis levels in CDX mouse tumor tissues with pancreatic cancer were determined. The experimental methods are as follows:

[0241] 1) Paraffin embedding, sectioning, and mounting;

[0242] 2) Immerse paraffin-coated tissue sections in xylene at room temperature for 5 minutes, repeating once to completely remove the paraffin. 2. Immerse sections in 100% ethanol at room temperature for 5 minutes, repeating once.

[0243] 3) Wash once each with gradient ethanol (90%, 80%, 70%) at room temperature, for 3 minutes each time;

[0244] 4) Gently rinse the slide with PBS and carefully blot away excess liquid around the sample on the slide with filter paper. At this point, use a wax pen or hydrophobic pen to outline the sample distribution around the slide to facilitate downstream permeability processing and equilibration labeling. During the experiment, never allow the sample to dry out; keep the processed sample moist in a humidified chamber.

[0245] 5) Add 100 μl of Proteinase K working solution to each sample to ensure complete coverage, and incubate at room temperature for 20 min. Note: Proteinase K helps tissues and cells permeate to staining reagents in subsequent steps. Excessive incubation time increases the risk of tissue sections detaching from the slide during subsequent washing steps, while insufficient permeability treatment may affect labeling efficiency and result in suboptimal results. It may be necessary to optimize the Proteinase K incubation time.

[0246] 6) Rinse the sample with PBS solution, gently remove excess liquid, and carefully blot the liquid around the sample on the slide with filter paper. After treatment, place the sample in a humidified chamber to keep it moist.

[0247] 7) Dilute 5×Equilibration Buffer with deionized water at a ratio of 1:5. Add 100 μl of 1×Equilibration Buffer to each sample to completely cover the sample area. Incubate at room temperature for 10-30 min. Alternatively, place the slide in a container containing 1×Equilibration Buffer, ensuring the buffer completely covers the sample. While equilibrating the cells, thaw the FITC-12-dUTP Labling Mix on ice. Prepare sufficient TdT incubation buffer for all experiments and optional positive control reactions. For a standard reaction with an area less than 5 cm², the volume is 50 μl. Multiply 50 μl by the number of experiments and positive control reactions to determine the total volume of TdT incubation buffer required. For samples with larger surface areas, the reagent volume can be increased proportionally.

[0248] 8) After equilibration, wash away most of the 100 μl 1×Equilibration Buffer around the area with absorbent paper. Then, add 50 μl TdT incubation buffer to a 5 cm² area of ​​cells. Do not let the cells dry out. After this operation, the slide should be protected from light.

[0249] 9) Cover the cells with a plastic coverslip to ensure even distribution of reagents. Place a paper towel moistened with water at the bottom of the humidified chamber, place the slide inside the humidified chamber, and incubate at 37°C for 60 min. Wrap the humidified chamber with aluminum foil to protect it from light. Note: The plastic coverslip can be cut in half before use. Fold up the edges of the coverslip for easy removal and handling.

[0250] 10) Remove the plastic coverslip and incubate the slide in PBS solution at room temperature for 5 min;

[0251] 11) Gently remove excess liquid, replace with fresh PBS solution, and incubate at room temperature for 5 min. Repeat once.

[0252] 12) Gently wipe away the PBS solution around and on the back of the sample with filter paper. Note: In order to reduce the background, after washing the slide with PBS once, you can wash it three more times with PBS containing 0.1% Triton X-100 and 5 mg / ml BSA, each time for 5 min. This will remove the free unreacted label.

[0253] 13) The sample was stained in a staining jar. In the dark, the slide was immersed in a staining jar containing DAPI solution (2 μg / ml, freshly prepared and diluted with PBS) and left at room temperature for 5 min.

[0254] 14) Wash the sample by immersing the slide in deionized water and leaving it at room temperature for 5 minutes. Repeat twice for a total of 3 washes. Tap the slide to dry any excess water and wipe the area around the cells with absorbent paper.

[0255] 15) Analyze the sample immediately under a fluorescence microscope. Observe the green fluorescence at 520±20 nm using a standard fluorescence filter. Observe the blue DAPI at 460 nm. If necessary, the slide can be stored overnight in the dark at 4°C. DAPI can stain both apoptotic and non-apoptotic cells blue. Only in the nuclei of apoptotic cells is there green fluorescence localized by FITC-12-dUTP incorporation.

[0256] TUNEL immunofluorescence staining was used to assess changes in apoptosis levels in tumor tissues of pancreatic cancer orthotopic CDX mice after treatment with high, medium, and low doses of the compound. The quantitative results are as follows: Figure 14 As shown, Figure 14In the table, *: p<0.05; **: p<0.01; ***: p<0.001, it can be seen that the TUNEL fluorescence level in the solvent group did not change significantly compared with the blank group. The TUNEL fluorescence levels of the high, medium and low concentration groups of this compound increased significantly (p<0.01), and showed a concentration gradient dependence. This indicates that this compound can promote apoptosis of pancreatic cancer cells in vivo.

[0257] Experimental Example 19.

[0258] Determination of the effect of different concentrations of compounds on STAT3 / p-STAT3 expression in pancreatic cancer tumor cells

[0259] Based on the aforementioned anti-tumor activity of this compound in vivo, the effect of this compound on the phosphorylation level of STAT3 in CDX mouse tumors of pancreatic cancer at different dose concentrations was further determined.

[0260] The effect of different concentrations of this compound on STAT3 phosphorylation in tumor tissue was observed using immunofluorescence. The experimental method is as follows:

[0261] 1) Paraffin embedding, sectioning, and mounting;

[0262] 2) Dewaxing and hydration of paraffin sections: 25 min each in xylene I and II, and 10 min each in different gradients of ethanol (100%, 95%, 90%, 85%, 80%, and 70%).

[0263] 3) Rinse twice with tap water; (avoid excessive rinsing force during rinsing to prevent damage to the tissue slide).

[0264] 4) Wash with PBS for 5 min x 3 times (avoid excessive force during washing to prevent tissue from sticking to the slide);

[0265] 5) Repair: Heat repair with sodium citrate twice, 5 min each time; wash with PBS for 5 min x 3 times;

[0266] 6) Normal serum blocking: Remove the slide from the staining jar, wipe off the moisture on the back of the slide and the moisture around the tissue on the front of the slide (keep the tissue moist), add normal goat serum (serum of an animal homologous to the second antibody), and incubate at 37°C for 30 min;

[0267] 7) Add primary antibody (1:100): Blot away the serum with filter paper, do not wash, add the primary antibody directly, and incubate overnight in a humidified chamber at 4°C.

[0268] 8) Recover at 37℃ for 35 min, then wash with PBS for 5 min x 3 times;

[0269] 9) Add goat anti-rabbit secondary antibody labeled with 488 wavelength (1:500), incubate at 37℃ for 60 min, and wash with PBS for 5 min x 3 times;

[0270] 10) Add the second primary antibody (1:100): Add the primary antibody, incubate at 37°C for 60 min, and wash with PBS for 5 min x 3 times;

[0271] 11) Add goat anti-mouse secondary antibody labeled with cy3 fluorescent (1:500), incubate at 37℃ for 60 min, and wash with PBS for 5 min x 3 times;

[0272] 12) Add DAPI staining solution and stain at 37°C for 10 min; wash with PBS for 5 min x 3 times.

[0273] 13) Mounting: Use anti-fluorescence quenching mounting medium to mount the slide, and try to avoid the formation of air bubbles;

[0274] 14) Observation results under a fluorescence microscope: green fluorescence indicates expression of the first target protein, red fluorescence indicates expression of the second target protein, and blue fluorescence indicates staining of the cell nucleus. Specific fluorescence labels are shown in the results section.

[0275] The effect of different concentrations of this compound on STAT3 phosphorylation in tumor tissue was observed using immunofluorescence. Quantitative results are shown below. Figure 15 As shown, Figure 15 In the table, *: p<0.05; **: p<0.01; ***: p<0.001, it can be observed that there was no significant difference in the total STAT3 expression level among the groups of this compound, but the p-STAT3 / STAT3 ratio varied considerably among the groups, indicating that the phosphorylation degree of STAT3 protein was not the same in each group. Compared with the blank group, there was no significant difference in STAT3 protein phosphorylation in the solvent group. However, after treating CDX mice with pancreatic cancer in situ with this compound, the phosphorylation of STAT3 protein in the mouse tumor tissue was significantly inhibited (p<0.05), and the inhibition trend showed a concentration gradient dependence, indicating that this compound has a good in vivo inhibitory effect on STAT3 phosphorylation, which warrants further investigation.

[0276] Experimental Example 20.

[0277] Determination of the effect of different concentrations of compounds on p-JAK2 expression in pancreatic cancer tumor cells.

[0278] To further determine the changes in the JAK2 / STAT3 pathway, the phosphorylation level of JAK p-CDK2 protein in tumor tissue cells of pancreatic cancer orthotopic CDX mice was measured after treatment with different concentrations of compounds. The specific experimental procedures were consistent with those described in "Experimental Example 17".

[0279] The measurement results are as follows Figure 16 As shown, compared with the blank group, the p-JAK2 level in the solvent group did not change significantly. The p-JAK2 expression level in the low concentration group of the compound decreased but not significantly. The p-JAK2 expression level in the tumor tissue of the high and medium concentration groups was significantly decreased (p<0.05). Moreover, the decrease in p-JAK2 expression in cells of different concentration groups of the compound was concentration gradient dependent, indicating that the compound also inhibited the expression of p-JAK2 in vivo.

[0280] Experimental Example 21.

[0281] Determination of the effect of different concentrations of compounds on p-CDK2 expression in pancreatic cancer tumor cells.

[0282] To further determine the changes in other pathways after targeting the STAT3 protein, the changes in the phosphorylation level of CDK2 protein in tumor tissue cells of pancreatic cancer orthotopic CDX mice were measured after treatment with different concentrations of compounds. The specific experimental procedures were consistent with those described in "Experimental Example 17".

[0283] The measurement results are as follows Figure 17 As shown, compared to the blank group, the p-CDK2 level in the solvent group was decreased with statistical significance (p<0.01). Treatment of mice with different concentrations of this compound significantly inhibited p-CDK2 expression in tumor tissues (p<0.001), and the inhibition trend showed a concentration gradient dependence. The trend of p-CDK2 expression changes after administration was basically consistent with the results of cell experiments. Although the solvent group also showed a statistically significant decrease in p-CDK2 expression, the degree of decrease in p-CDK2 expression in each administration group was significantly greater than that in the solvent group, indicating that this compound also inhibited p-CDK2 expression.

[0284] Experimental Example 22.

[0285] Determination of the effect of different concentrations of compounds on VEGF expression in pancreatic cancer tumor cells.

[0286] Based on the aforementioned cell experiments, it was found that VEGF expression, which is related to tumor angiogenesis, decreased after drug administration. Further measurements were conducted to determine the changes in VEGF expression in pancreatic cancer tumor cells after treatment with different concentrations of the compound. The specific experimental procedures were consistent with those described in "Example 16".

[0287] The measurement results are as follows Figure 18 As shown, compared with the blank group, the VEGF level in the solvent group increased slightly but there was no statistical difference. After administration of high, medium and low concentrations of the compound, the expression of VEGF in mouse tumor tissue was significantly inhibited (p<0.001), and the inhibition trend showed a concentration gradient dependence, indicating that the compound also inhibited the expression of VEGF in vivo.

[0288] Experimental Example 23.

[0289] Determination of the effects of different concentrations of compounds on the expression of EMT markers in pancreatic cancer tumor cells.

[0290] Based on the aforementioned cell experiments, it was found that targeting STAT3 can reduce the expression levels of EMT pathway-related markers. Further verification was conducted in animals using different concentrations of compounds to observe changes in the expression levels of EMT pathway-related proteins in tumor tissues. The experimental methods were as shown in "Biological Evaluation Experiment 16".

[0291] The measurement results are as follows Figure 19 As shown, after in vivo injection of different concentrations of the compound, the expression level of E-Cadherin in the tumor tissue of CDX mice with pancreatic cancer increased and the expression level of Snail decreased, and the EMT pathway in the tumor tissue was inhibited. The relevant in vivo study results are consistent with the trend of cell experiment results.

Claims

1. The application of a JAK2 and STAT3 dual-targeting inhibitor containing a phenoxyacetylhydrazine structure in the preparation of a drug for treating pancreatic cancer, characterized in that, The structure of the dual-targeting inhibitor is as follows: .

2. The use of the JAK2 and STAT3 dual-targeting inhibitor containing a phenoxyacetylhydrazine structure as described in claim 1 in the preparation of a drug for treating pancreatic cancer, characterized in that: The dual-target inhibitor is used to treat pancreatic cancer by inhibiting tumor proliferation, migration and invasion, angiogenesis, epithelial-mesenchymal transition, arresting the cell cycle, and promoting apoptosis.

3. The use of the JAK2 and STAT3 dual-targeting inhibitor containing a phenoxyacetylhydrazine structure as described in claim 1 in the preparation of a drug for treating pancreatic cancer, characterized in that: The pharmaceutical formulations of the dual-target inhibitors include tablets, capsules, syrups, suspensions, and injections.

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