Pancreatic cancer jAK2 and STAT3 dual targeting inhibitors containing compounds of zinc218238819 and applications

Abstract

This invention relates to a dual-targeting inhibitor of JAK2 and STAT3 in pancreatic cancer containing the compound ZINC218238819 and its application. The dual-targeting inhibitor includes the compound ZINC218238819 and comprises a pharmaceutically acceptable salt. The invention also relates to the application of this dual-targeting inhibitor of JAK2 and STAT3 in the preparation of drugs for treating pancreatic cancer. It utilizes this inhibitor to inhibit tumor proliferation, migration, invasion, angiogenesis, and epithelial-mesenchymal transition; to arrest the cell cycle; and to promote apoptosis in the treatment of pancreatic cancer. A drug for treating pancreatic cancer is also described, comprising a dual-targeting inhibitor of JAK2 and STAT3 in pancreatic cancer containing the compound ZINC218238819 and a pharmaceutically acceptable carrier. The beneficial effects of this invention are: it has a good inhibitory effect on JAK2 and STAT3 in pancreatic cancer, experimental results show a good inhibitory effect, broad application prospects, and good safety.

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 JAK2 and STAT3 in pancreatic cancer containing the compound ZINC218238819 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 identified as a potential target for future clinical treatment, leading to some inhibitor studies. However, most current inhibitors targeting STAT3 indirectly inhibit its biological function by blocking upstream signaling mechanisms, including IL-6 inhibitors, JAK inhibitors, and various growth factor receptor inhibitors. Furthermore, the importance of the SH2 domain of STAT3 has been increasingly recognized in recent years, and several small-molecule inhibitors directly targeting STAT3 are under investigation, with 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 ZINC218238819, which has a good inhibitory effect on JAK2 and STAT3 in pancreatic cancer. Experimental results show that it has a good inhibitory effect, a promising application prospect, and a good safety profile. It is a dual-target inhibitor of JAK2 and STAT3 in pancreatic cancer and its application.

[0007] A dual-targeting inhibitor of JAK2 and STAT3 in pancreatic cancer containing the compound ZINC218238819. The dual-targeting inhibitor includes the compound ZINC218238819, whose structural formula is as follows: .

[0008] A dual-targeting inhibitor of JAK2 and STAT3 in pancreatic cancer containing the compound ZINC218238819. The dual-targeting inhibitor includes a pharmaceutically acceptable salt, which is an acid addition salt formed by compound ZINC218238819 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.

[0009] The application of the pancreatic cancer JAK2 and STAT3 dual-targeting inhibitor containing the compound ZINC218238819 in the preparation of drugs for treating pancreatic cancer.

[0010] 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.

[0011] The pharmaceutical preparations of the pharmaceutical composition include tablets, capsules, syrups, suspensions, and injections.

[0012] A drug for treating pancreatic cancer, comprising a dual-targeting inhibitor of pancreatic cancer JAK2 and STAT3 containing the compound ZINC218238819 and a pharmaceutically acceptable carrier.

[0013] The beneficial effects of this invention are: 1) This compound 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, combined 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 provides 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.

[0014] 2) This compound has a good inhibitory effect on JAK2 and STAT3 in pancreatic cancer. Experimental results show that the inhibition is good, indicating a promising application prospect and good safety. Attached Figure Description

[0015] Figure 1 The expression levels of STAT3 mRNA and protein in different pancreatic / pancreatic cancer cell lines of this invention; Figure 2 SPR results for the compound; Figure 3 The results of the CETSA experiment for compound CETSA; Figure 4 Changes in cell proliferation after treatment with different concentrations of compounds; Figure 5 Changes in cell apoptosis after treatment with different concentrations of compounds for 24 hours; Figure 6 Cell cycle changes after 24 hours of treatment with different concentrations of compounds; Figure 7 Changes in cell proliferation and apoptosis-related proteins after 24 hours of treatment with different concentrations of compounds; Figure 8 Changes in JAK2 / STAT3-related proteins in cells after 24 hours of treatment with different concentrations of compounds; Figure 9 Changes in VEGF protein and CDK2-related protein in cells after 24 hours of treatment with different concentrations of compounds; Figure 10 Changes in EMT pathway-related proteins in cells after 24 hours of treatment with different concentrations of compounds. Detailed Implementation

[0016] The present invention will be further described below with reference to the accompanying drawings.

[0017] Example 1. A dual-targeting inhibitor of JAK2 and STAT3 in pancreatic cancer containing the compound ZINC218238819. The dual-targeting inhibitor includes the compound ZINC218238819, whose structural formula is as follows: .

[0018] A dual-targeting inhibitor of JAK2 and STAT3 in pancreatic cancer containing the compound ZINC218238819. The dual-targeting inhibitor includes a pharmaceutically acceptable salt, which is an acid addition salt formed by compound ZINC218238819 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.

[0019] The application of the pancreatic cancer JAK2 and STAT3 dual-targeting inhibitor containing the compound ZINC218238819 in the preparation of drugs for treating pancreatic cancer.

[0020] 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.

[0021] The pharmaceutical preparations of the pharmaceutical composition include tablets, capsules, syrups, suspensions, and injections.

[0022] A drug for treating pancreatic cancer, comprising a dual-targeting inhibitor of pancreatic cancer JAK2 and STAT3 containing the compound ZINC218238819 and a pharmaceutically acceptable carrier.

[0023] Experimental Example 2. Determination of STAT3 mRNA expression levels in various pancreatic cancer cell lines 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: The differences in STAT3 mRNA levels between normal pancreatic cells and different pancreatic cancer cell lines were determined using RT-PCR. 1) Add 50-100 mg of TRIzol to the tissue, homogenize, and let stand at room temperature; 2) Add chloroform, shake to mix, let stand, and then centrifuge; 3) Take the supernatant and add an equal amount of isopropanol, invert to mix, let stand at room temperature and then centrifuge; 4) Discard the supernatant, add 75% ethanol, and centrifuge at 4°C; 5) Discard the supernatant and allow to dry at room temperature; 6) Reverse transcription kits and real-time fluorescence kits are used to detect the expression levels of target genes.

[0024] The differences in STAT3 mRNA levels between different pancreatic cancer cell lines and normal pancreatic cells were determined. Results are as follows: Figure 1 As shown in Figure a, 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.

[0025] Experimental Example 3. Determination of STAT3 protein expression levels in various pancreatic cancer cell lines 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 using the following method. A brief description of the method follows: Western blot was used to determine the differences in STAT3 protein expression levels between normal pancreatic cells and different pancreatic cancer cell lines.

[0026] 1) Select ASPC-1, PANC-1, BxPC-3, SW1990 cancer cells and HPDE6 cells in the logarithmic growth phase, centrifuge them, and collect the precipitate; 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; 3) Protein concentration was determined using the BCA method; 4) The target protein was separated by SDS-PAGE; 5) Transfer the target protein onto a PVDF membrane; 6) Incubated with a specific primary antibody and an HRP-labeled secondary antibody; 7) ECL staining, Image J semi-quantitative analysis of target protein expression levels.

[0027] 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 1 As shown in b, the expression levels of STAT3 protein in the four pancreatic cancer cell lines (ASPC-1, PANC-1, BxPC-3, and SW1990) were all higher than those in normal pancreatic cells (HPDE6). PANC-1 cells showed the highest STAT3 protein expression level, followed by BxPC-3 cells. Based on the STAT protein expression levels of different pancreatic cancer cell lines, PANC-1 and BxPC-3 cells were selected for subsequent experiments.

[0028] Experimental Example 4. IC50 determination of compounds 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.

[0029] Experimental steps: 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.

[0030] 1) After trypsin digestion, BxPC-3 and PANC-1 cells were adjusted to a cell density of 5 × 10⁵ cells / ml using RPMI-1640 or DMEM complete medium. 100 μl of cell suspension was seeded into each well of a 96-well plate, with a blank control group included. The culture plates were then incubated at 37°C in a 5% CO₂ incubator. 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. 3) After the detection time point, add 10 μL of CCK8 solution to each well (do not generate bubbles); 4) Wrap the culture plate with aluminum foil, place it in an incubator, and incubate in the dark for 2 hours; 5) Measure the absorbance at 450 nm using an ELISA reader. Turn on the instrument, create a new sample, shake the plate for 10 seconds, shake it again for 10 seconds, 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%; 6) After obtaining the data, use SPSS27 to calculate IC50.

[0031] 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 against the two cell lines are similar. Based on this, the standard dose and treatment time for subsequent administration of the compound were set to 15 μM / 24h.

[0032] Table 1. IC50 of the compounds on the proliferation inhibition of human pancreatic cancer cells (PANC-1, BxPC-3). Experimental Example 5. Surface plasmon resonance (SPR) analysis of compounds 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 assay method is shown below: Experimental steps: 1) Protein chip preparation: Before injection, 400 mM EDC and 100 mM NHS were mixed to prepare the activator. The CM5 sensor chip was activated at a flow rate of 10 μL / min for 420 s. Then, the 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 then 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 STAT3 step was not injected into the Fc1 control channel. 2) Multi-cycle flow method for small molecule compounds: Small molecule compounds are diluted to the appropriate concentration using flow buffer. The small molecule compound is injected into the channel at a flow rate of 30 μL / min, forming binding and dissociation phases. Both binding and dissociation processes are performed in the run buffer, and the affinity between the small molecule and the protein is analyzed using Biacore 8K.

[0033] The affinity between the compound and the humanized STAT protein was determined, and the data analysis is shown in Table 2 and Figure 2. It can be seen that the affinity (KD value) between the compound and the humanized STAT3 protein is 2.44 × 10⁻⁴ M. Generally speaking, the affinity between small molecules and proteins is moderate. This compound exhibits a moderate binding strength to the humanized STAT3 protein, suggesting its potential as a STAT3-targeting drug. The SPR spectrum of this compound is shown below. Figure 2 As shown.

[0034] Table 2. SPR analysis results of compounds and humanized STAT3 protein. Experimental Example 6. Cellular Thermal Shift Assay (CETSA) 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.

[0035] Experimental steps: 1) 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. 2) Extract STAT3 protein for Western blot (WB) experiments (specific experimental protocol as described above); 3) Based on the STAT3 protein level at different temperatures, use Origin2022 to plot protein dissolution curves.

[0036] 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 3 As shown in the figure, 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.

[0037] Experimental Example 7. Determination of the inhibitory effect of different concentrations of compounds on the proliferation of pancreatic cancer cells 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.

[0038] Experimental steps: 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.

[0039] 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 into 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℃. 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. 3) After the detection time point, add 10 μL of CCK8 solution to each well (do not generate bubbles); 4) Wrap the culture plate with aluminum foil, place it in an incubator, and incubate in the dark for 2 hours; 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%. 6) After obtaining the data, use GraphPad Prism8 to plot it.

[0040] The inhibitory effects of different concentrations of the 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 It can be seen that this compound exerted a concentration-dependent inhibitory effect on the proliferation of human pancreatic cancer cell lines (PANC-1, BxPC-3), and the effect was significant (p<0.001). Furthermore, the inhibitory effect on cell proliferation increased with concentration at 24h, 48h, and 72h.

[0041] Experimental Example 8. Determination of the effect of different concentrations of compounds on apoptosis in pancreatic cancer cells.

[0042] 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: 1) The cells of the blank control group were divided into four groups (with binding solution only, Annexin V-FITC single staining, PI single staining, and PI double staining respectively). Multiple 15ml centrifuge tubes were labeled in advance according to the experimental design. The growth of pancreatic cancer cells in each group was observed under a microscope. When the confluence of tumor cells was about 70-80%, the original culture base of pancreatic cancer cells in each group was collected into the pre-labeled 15ml centrifuge tubes. After washing with 1×PBS, the cells in each group were digested with trypsin (without EDTA). The trypsin was discarded, the cells were collected a second time, and transferred to the corresponding centrifuge tubes and centrifuged at 1000g for 5min. 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. 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; 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.

[0043] The effects of the compound at different concentrations on apoptosis in pancreatic cancer PANC-1 and BxPC-3 cells were determined, and the results are as follows: Figure 5 As shown, compared to the control group, the apoptosis of both cell lines increased after administration of this compound in a concentration gradient-dependent manner.

[0044] Experimental Example 9. Determination of the effects of different concentrations of compounds on the cell cycle of pancreatic cancer cells.

[0045] 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. The assay methods are shown below: 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.

[0046] 2) Discard the supernatant, add 5 ml of pre-cooled PBS at 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, adjust the concentration and take 1 ml of cell suspension containing 1×106 cells, centrifuge (800 rpm, 5 min) and wash twice with pre-cooled PBS.

[0047] 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.

[0048] 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.

[0049] 5) Preheat the water bath to 37°C; add 5ml of pre-cooled PBS to each centrifuge tube, centrifuge at 800rpm for 5min, discard the PBS solution, resuspend in 100µl of RNase A solution, and incubate in a water bath for 30min; add 400µl of propidium iodide (PI), fix in a refrigerator at 4°C for 30min, and then use a flow cytometer to detect the DNA content of each group of cells.

[0050] 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.

[0051] 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 in the figure, treatment of PANC-1 cells with this compound resulted in 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) across the high, medium, and low concentration groups. However, both the high and medium concentration groups showed a decrease in the proportion of cells in the G2 / M phase (p<0.001). Treatment of BxPC-3 cells with this compound also resulted in 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) across the high, medium, and low concentration groups, with this trend showing a concentration gradient dependence. However, the change in the proportion of cells in the G2 / M phase was not significant. The cell cycle results after administration to both cell lines were similar, indicating that this compound can arrest pancreatic cancer cells in the G0 / G1 phase in a concentration gradient dependence.

[0052] Experimental Example 10. Determination of the effects of different concentrations of compounds on pancreatic cancer cell proliferation and apoptosis-related proteins.

[0053] The effects of high, medium, and low concentrations of the compound on the levels of Ki67 and PCNA (cell proliferation-related proteins) and BCL-2, BAX, and Cleaved Caspase-3 (cell apoptosis-related proteins) in human pancreatic cancer cell lines (PANC-1 and BxPC-3) were determined using the following method. A brief description of the experimental methods is as follows: Experimental steps: Western blot was used to determine 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.

[0054] 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; 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; 3) Protein concentration was determined using the BCA method; 4) The target protein was separated by SDS-PAGE; 5) The target protein was transferred onto a PVDF membrane; 6) Incubated with a specific primary antibody and an HRP-labeled secondary antibody; 7) ECL staining, Image J semi-quantitative analysis of target protein expression levels.

[0055] 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. Results are as follows: Figure 7 As shown, it can be seen that: 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, showing concentration-dependent proliferation inhibition. The expression levels of apoptosis-related proteins BCL-2 decreased, while the expression levels of BAX and Cleaved Caspase-3 increased, showing concentration gradient dependence and concentration-dependent apoptosis promotion. These trends are consistent with the cell proliferation and apoptosis results obtained by flow cytometry, which can indirectly reflect the effects of different concentrations of the compound on the proliferation and apoptosis of pancreatic cancer cells.

[0056] Experimental Example 11. Determination of the effect of different concentrations of compounds on phosphorylation of JAK2 / STAT3 protein in pancreatic cancer cells.

[0057] 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 STAT3, JAK2, p-STAT3, and p-JAK2 protein levels in human pancreatic cancer (PANC-1, BxPC-3) cell lines at high, medium, and low concentrations of the compound 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)*).

[0058] Experimental steps: Western blot was used to determine 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.

[0059] 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; 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; 3) Protein concentration was determined using the BCA method; 4) The target protein was separated by SDS-PAGE; 5) The target protein was transferred onto a PVDF membrane; 6) Incubated with a specific primary antibody and an HRP-labeled secondary antibody; 7) ECL staining, Image J semi-quantitative analysis of target protein expression levels.

[0060] The effects of different concentrations of the compound on the expression levels of specific proteins in two pancreatic cancer cell lines were measured. Results are as follows: Figure 8 It can be seen that after 24 hours of treatment with different concentrations of the compound on pancreatic cancer cells, all concentrations of the compound were able to inhibit STAT3 protein phosphorylation. Moreover, the inhibitory effect of the compound on STAT3 phosphorylation in BxPC-3 cells was more significant than that in PANC-1 cells, and both showed 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 STAT3 target inhibition effect.

[0061] Analysis of the JAK2 protein upstream of STAT3 yielded the following results: Figure 8 It can be seen that in PANC-1 cells, different concentrations of the compound significantly inhibited JAK2 phosphorylation (p<0.05), and this effect was dose-dependent. 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.

[0062] In summary, this compound has the potential to serve as a dual-target inhibitor of JAK2 / STAT3.

[0063] Experimental Example 12. Determination of the effects of different concentrations of compounds on VEGF protein in pancreatic cancer cells.

[0064] 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)*).

[0065] Experimental steps: 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.

[0066] 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; 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; 3) Protein concentration was determined using the BCA method; 4) The target protein was separated by SDS-PAGE; 5) The target protein was transferred onto a PVDF membrane; 6) Incubated with a specific primary antibody and an HRP-labeled secondary antibody; 7) ECL staining, Image J semi-quantitative analysis of target protein expression levels.

[0067] Since one of the functions of STAT3 in tumors is to promote angiogenesis, to verify whether targeting STAT3 with this compound could affect its angiogenesis effect on tumors, the effects of different concentrations of the compound on the expression levels of VEGF and CDK2 proteins in two pancreatic cancer cell lines were measured. The results are as follows: Figure 9 As shown, different concentrations of this compound significantly inhibited VEGF protein expression in both pancreatic cancer cell lines (p<0.05), and this inhibition was concentration-dependent.

[0068] Experimental Example 13. Determination of the effects of different concentrations of compounds on CDK2 protein in pancreatic cancer cells.

[0069] The effects of high, medium, and low concentrations of the compound 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)*).

[0070] Experimental steps: 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.

[0071] 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; 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; 3) Protein concentration was determined using the BCA method; 4) The target protein was separated by SDS-PAGE; 5) The target protein was transferred onto a PVDF membrane; 6) Incubated with a specific primary antibody and an HRP-labeled secondary antibody; 7) ECL staining, Image J semi-quantitative analysis of target protein expression levels.

[0072] 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 content was significantly decreased in the high, medium, and low concentration groups of this compound (p<0.01) and showed a concentration gradient dependence. In the BxPC-3 cell line, the total CDK2 protein content was significantly decreased in the high and medium concentration groups of this compound (p<0.01), while the VEGF protein level was decreased but not significantly in the low concentration group.

[0073] It can be seen that in PANC-1 and BxPC-3 cell lines, the phosphorylation of CDK2 protein was significantly inhibited at high, medium, and low concentrations of this compound (p<0.05), and this inhibition showed a concentration gradient dependence. In summary, the inhibition of CDK2 protein phosphorylation and its inactivation may be related to cell cycle arrest at the G0 / G1 phase after drug administration.

[0074] Experimental Example 14. Determination of the effects of different concentrations of compounds on EMT-related proteins in pancreatic cancer cells.

[0075] 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)*).

[0076] Experimental steps: 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.

[0077] 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; 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; 3) Protein concentration was determined using the BCA method; 4) The target protein was separated by SDS-PAGE; 5) The target protein was transferred onto a PVDF membrane; 6) Incubated with a specific primary antibody and an HRP-labeled secondary antibody; 7) ECL staining, Image J semi-quantitative analysis of target protein expression levels.

[0078] Since EMT pathway activation 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.

Claims

1. The application of a JAK2 and STAT3 dual-targeting inhibitor containing the ZINC218238819 compound 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 application of the JAK2 and STAT3 dual-targeting inhibitor containing the ZINC218238819 compound 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 application of the JAK2 and STAT3 dual-target inhibitor containing the ZINC218238819 compound 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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