7H-Indole[2,3-j]phenanthridine-7,13(8H)-dione is used as a JAK2 inhibitor and in the preparation of drugs for alleviating and treating pancreatic cancer.

Abstract

This invention belongs to the field of pharmaceuticals, specifically disclosing the application of 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione as a JAK2 inhibitor and in the preparation of drugs for alleviating and treating pancreatic cancer. This study demonstrates that 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione can effectively target the JAK2 protein, thereby inhibiting the malignant proliferation and migration of pancreatic cancer, providing a new potential drug treatment option for pancreatic cancer patients.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, specifically to the novel field of JAK2-targeted inhibition association. Background Technology

[0002] Pancreatic cancer is one of the leading causes of cancer death worldwide, often referred to as the "king of cancers." It is a highly malignant solid tumor characterized by a rising incidence rate, poor prognosis, and high mortality. Diagnosis usually occurs at an advanced stage, with a median survival of 3-6 months for most patients and a 5-year survival rate of less than 5%. Although surgical resection, immunotherapy, adjuvant chemotherapy, and radiotherapy are used to treat pancreatic cancer, their efficacy remains unsatisfactory. Currently, the main clinical approaches to treating pancreatic cancer include the FOLFIRINOX regimen (a combination of chemotherapy drugs, including irinotecan, fluorouracil, leucovorin, and oxaliplatin) and the gemcitabine-albumin-paclitaxel regimen. However, pancreatic cancer exhibits a high tendency to resist these chemotherapy drugs, leading to significant adverse reactions and greatly reducing patients' quality of life. Therefore, there is an urgent need to develop new treatment strategies for pancreatic cancer.

[0003] With technological advancements, existing technologies have led to the development of novel treatments for pancreatic cancer. For instance, US Patent Publication No. US12116422A discloses a chelating peptide and its application in the preparation of drugs for treating pancreatic cancer. Chinese Patent Publication No. CN110312508A discloses a pharmaceutical composition for the prevention and treatment of pancreatic cancer containing gossypol and phenformin as active ingredients. Chinese Patent Publication No. CN116515009A discloses a bamboo fungus polysaccharide capable of treating pancreatic cancer. Furthermore, Korean Patent Publication No. KR1020240125510A discloses a composition containing 2,4-diaminopyrimidine for the prevention or treatment of pancreatic cancer.

[0004] In summary, although there are some existing technologies for treating pancreatic cancer, effective treatments for pancreatic cancer are still relatively scarce. Summary of the Invention

[0005] To address the problems of the prior art, the primary objective of this invention is to provide an application of 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione as a JAK2 inhibitor, aiming to provide a novel application of 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione as a JAK2 inhibitor.

[0006] The second objective of this invention is to provide the application of the aforementioned 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione as a JAK2 inhibitor in the inhibition of pancreatic cancer cells.

[0007] A third objective of this invention is to provide the application of the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione in the preparation of drugs for the relief and treatment of pancreatic cancer.

[0008] The fourth objective of this invention is to provide an active ingredient and a drug containing 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione for anti-pancreatic cancer.

[0009] Application of a 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione as a JAK2 inhibitor, wherein the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione is a compound having the structure of Formula 1 and its crystals and pharmaceutically acceptable salts;

[0010]

[0011] This invention innovatively demonstrates that compounds of Formula 1 can unexpectedly target and inhibit JAK2, and can be used for JAK2-related scientific research and pharmaceutical development.

[0012] The present invention demonstrates that the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione can be used to prepare components that inhibit and / or phosphorylation of STAT3.

[0013] The present invention also provides the application of the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione in the preparation of a component for inhibiting pancreatic cancer cells.

[0014] Different cancer cells have different surface characteristics and require different treatment approaches. Pancreatic cancer cells, in particular, are prone to differentiation and migration, making effective inhibition more challenging. However, this invention demonstrates that the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione can unexpectedly inhibit pancreatic cancer cells through a JAK2-targeted inhibition mechanism, thus enabling its application in pancreatic cancer cell inhibition research and drug development.

[0015] In this invention, the pancreatic cancer cells include human pancreatic cancer cells; for example, HPAC cells.

[0016] In this invention, the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione is used to prepare a component that inhibits the growth, migration, and / or tumorigenesis of pancreatic cancer cells.

[0017] The present invention also provides the application of the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione in the preparation of drugs for alleviating and / or inhibiting pancreatic cancer.

[0018] In the applications described in this invention, it can be used in combination with other anticancer active ingredients to prepare drugs that alleviate and / or inhibit pancreatic cancer;

[0019] Preferably, the anticancer active ingredient includes at least one of gemcitabine, albumin-bound paclitaxel, fluorouracil, oxaliplatin, etc.

[0020] The present invention demonstrates that using the compound of Formula 1 described in this invention in combination with other anticancer components can achieve synergy and further enhance the therapeutic effect on pancreatic cancer.

[0021] The application described in this invention involves combining the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione with pharmaceutically acceptable excipients to prepare a pharmaceutically acceptable dosage form.

[0022] The present invention discloses an anticancer active ingredient for alleviating and treating pancreatic cancer, comprising the aforementioned 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione; and also contains other anticancer components;

[0023] Preferably, the other anticancer components include at least one of gemcitabine, albumin, paclitaxel, fluorouracil, oxaliplatin, etc.

[0024] The present invention also provides an anticancer drug for alleviating and treating pancreatic cancer, comprising an effective amount of 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione; and further comprising a pharmaceutically effective amount of the aforementioned anticancer active ingredient.

[0025] Furthermore, the medicament described in this invention also comprises pharmaceutically acceptable excipients. Additionally, a pharmaceutically acceptable dosage form is permitted.

[0026] Beneficial effects

[0027] This invention demonstrates that the structural components of Formula 1 can unexpectedly target and inhibit JAK2, which can be used for related scientific research. Furthermore, this invention also shows that Formula 1 can achieve good inhibitory and therapeutic effects on pancreatic cancer cells based on its JAK2-targeting inhibitory effect. Attached Figure Description

[0028] Figure 1This section presents transcriptomic data analysis of pancreatic cancer tissues and the molecular interactions of the Formula 1-JAK2 complex. A shows a significant upregulation of the IL6-JAK-STAT3 signaling axis in pancreatic cancer, as revealed by TCGA and GTEx normal pancreatic tissue transcriptomic data analysis. B shows the top 20 bubble charts from the GSEA Hallmark. C shows two-dimensional interactions demonstrating ligand-amino acid hydrogen bonds. D shows the effective binding of Formula 1 to JAK2 within the protein lumen. E shows a three-dimensional representation of the interactions, displaying the amino acid residues interacting with the ligand.

[0029] Figure 2 Molecular dynamics simulations of the Formula 1-JAK2 complex and the regulation of JAK2-STAT3 are presented. In the diagrams, A is the RMSD diagram of the Formula 1-JAK2 complex; B is the RMSF diagram of the JAK2 protein; C is the RMSF diagram of Formula 1; DE represents the contact interactions of the Formula 1-JAK2 complex; F represents the properties of Formula 1; and G represents the inhibition of the JAK2-STAT3 signaling pathway by Formula 1.

[0030] Figure 3 Formula 1 was used to inhibit the viability of human pancreatic cancer cells. Specifically, AB represents the comparison of cell viability of Miapaca2 and HPAC cells treated with different concentrations of Formula 1 using the CCK8 assay. C shows the inhibitory effect of Formula 1 on pancreatic cancer cell growth as detected by crystal violet assay. D represents the effect of Propidium iodide staining followed by flow cytometry analysis of the treated cells and the effect of Formula 1 on the cell cycle distribution of pancreatic cancer cells. E represents the Western blot analysis of the expression of the cell cycle-related protein Cyclin D1 after treatment of pancreatic cancer cells with Formula 1. *p<0.05, **p<0.01, ***p<0.001.

[0031] Figure 4 Formula 1 was used to induce apoptosis in human pancreatic cancer cells. In formula A, flow cytometry analysis of the effect of Formula 1 on pancreatic cancer cell apoptosis after Annexin V-FITC and PI staining was performed. In formula B, flow cytometry analysis showed that incubation with Formula 1 significantly increased the apoptosis rate of pancreatic cancer cells (apoptosis rate = Q2 + Q3). In formula C, Western blot analysis of the expression of apoptosis-related proteins Bcl-2, Bax, Caspase 3, and Cleaved-Caspase 3 was performed. In formula D, the Bax / Bcl-2 ratio of HPAC and Miapaca2 gradually increased with increasing Formula 1 concentration. *p<0.05.

[0032] Figure 5Formula 1 was used to inhibit the invasion of human pancreatic cancer cells. In the formula, A, B, D, and E represent Transwell assays showing that, compared to the control, Formula 1 limited the migration ability of HPAC and Miapaca2 cells. C and F represent Western blot analysis of the effect of Formula 1 on EMT-related expression in HPAC and Miapaca2 cells after 48 h of treatment. **p<0.01,**p<0.001.

[0033] Figure 6 Formula 1 was used to inhibit the migration of human pancreatic cancer cells. In this formula, AB represents the effect of Western blot analysis on Emt-related expression in HPAC and Miapaca2 cells. CD represents a wound healing assay showing that the migration ability of HPAC and Miapaca2 cells was limited by Formula 1. **p<0.01.

[0034] Figure 7 Formula 1 reduces tumor growth in xenograft mice derived from the Miapaca2 cell line. In the formula, A shows an image of the xenograft tumor after treatment with Formula 1. B shows the body weight of the nude mouse after treatment with Formula 1. CD shows the weight and volume of the tumor after treatment with Formula 1. E shows HE staining of the heart, liver, spleen, lung, and kidney of the nude mouse. ***p<0.001.

[0035] Figure 8 Formula 1 combined with gemcitabine showed better efficacy. AB indicates that Formula 1, gemcitabine, and the combination therapy limited the growth capacity of HPAC and Miapaca2. ***p<0.001. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0038] The reagents and raw materials used in this invention are as shown in Formula 1. All are commercially available.

[0039] Example 1 Formula 1 Downregulates JAK2-STAT3 signaling in PDAC cells

[0040] Western blotting was used to verify the inhibitory effect of Equation 1 on JAK2-STAT3 signaling, in order to clarify the downregulation of JAK2-STAT3 signaling in PDAC cells by Equation 1.

[0041] The specific steps are as follows: different PDAC cell lines are treated with Formula 1 (final concentrations of 1 μM and 2 μM), and then the p-JAK2 and p-STAT3 proteins are detected by Western Blot.

[0042] The results showed that, compared with the control group, Equation 1 significantly downregulated p-JAK2 and p-STAT3 (Y705), demonstrating a significant inhibitory effect on JAK2-STAT3 signaling.

[0043] Example 2 Formula 1 inhibits the malignant proliferation of PDAC cells by targeting JAK2.

[0044] The specific scheme is as follows: (1) The molecular simulation docking of Formula 1 with JAK2 protein was obtained through virtual screening and molecular docking techniques. (2) Growth inhibition was evaluated using the CCK-8 assay (YEASN, 40203ES60). HPAC cells and Miapaca2 cells (3000 cells / well) were seeded in 96-well plates and treated with different concentrations (0.3, 0.6, 1.2, 2.4, 4.8 and 9.6 μM) of Formula 1. Cells treated with 0.01% DMSO served as control cells. After 48 h of treatment, 10 μL of CCK-8 solution was added to the cells. After another 4 h of incubation, the optical density (OD) value at 450 nm was measured using a microplate reader. Each group was tested three times, and cell viability was calculated as the value relative to the control group. (3) Human pancreatic cancer cells (HPAC and Miapaca2) were seeded in 6-well plates (6000-8000 cells / well) and treated with different concentrations (final concentrations of 1 μM and 2 μM) of Formula 1 for 1 day. Afterward, the culture medium was replaced with a medium without Formula 1 for 7-10 days, with the old medium replaced every 2 days. Finally, the cells were washed twice with PBS, stained with 0.05% crystal violet solution for 10 minutes, washed with water, dried, and photographed. (4) Experiments such as subcutaneous xenograft mouse model gavage administration evaluated the inhibitory effect of Formula 1 (5 mg / kg / day) on PDAC cell proliferation. Nude mice were kept in a specific pathogen-free environment. Cells were subcutaneously injected into 5-week-old male nude mice. Tumors were randomly divided into two groups. Drug treatment conditions: tumors were treated with Formula 1 (5 mg / kg) or a control. Tumor volume was calculated as length × (width). 2 / 2. At the end of the study, the tumor was surgically removed, weighed, and processed. The heart, liver, spleen, lung, and kidney organs of mice were collected, fixed with 4% formalin, embedded in paraffin, and further stained with hematoxylin and eosin (H&E) according to the methods reported in the literature. All animal experiments were conducted in accordance with the guidelines of the Chinese FDA. The protocol was reviewed and approved by the Department of Laboratory Animal Science of Central South University. The results showed that: (1) Formula 1 can target JAK2 and is a potential competitive binding drug for JAK2 protein. (2) Formula 1 can effectively inhibit the in vivo and in vitro growth of pancreatic cancer cells and is an alternative drug for the treatment of pancreatic cancer. (3) Formula 1 has little effect on the weight of mice and the heart, liver, spleen, lung, and kidney organs, showing low toxicity and certain safety.

[0045] Example 3 Formula 1 Inhibits the Migration and Invasion of PDAC Cells

[0046] The specific scheme is as follows: the inhibitory effect of formula 1 (final concentrations of 1 μM and 2 μM) on the migration and invasion of PDAC cells was detected by wound healing assay and Transwell assay. (1) Wound healing assay: Cells were cultured in 6-well plates. When the cells covered the entire surface, the cells were scraped off using the tip of a 10 μL pipette. The cells were then washed with PBS and serum-free medium was added. Subsequently, the cells were cultured at 37°C in a humidified incubator containing 5% CO2 / 95% air (v / v). Cell images were taken using an inverted microscope at 0, 24, and 48 h. (2) Transwell assay: The cells were resuspended in serum-free DMEM medium and the cell count was 4 × 10⁶ cells / 10⁻ ... 4 Cells were added to the upper compartment of a Transwell chamber at a concentration of 4000 cells / well. This involved vertically and evenly adding the serum-free DMEM diluted cells and Formula 1 mixture to the bottom of the upper compartment of the Transwell chamber. Then, 600 μL of DMEM medium containing 10% FBS was added to the lower compartment as a chemical inducer. After 48 h, cells were fixed with 4% paraformaldehyde, stained with 0.05% crystal violet, washed with water to remove excess stain, air-dried, and photographed.

[0047] The results showed that Formula 1 can effectively inhibit the migration and invasion of pancreatic cancer cells and is an alternative drug for the treatment of pancreatic cancer.

[0048] Example 4 Formula 1 Inhibits cell cycle progression and promotes apoptosis in PDAC cells

[0049] The specific plan is as follows: (1) Place cells (1×10) 5Cells (1 × 10⁶ cells / well) were seeded in 6-well plates and treated with 1 μM and 2 μM Formula 1 for 12 h, respectively. Cells were then washed and fixed with 70% ethanol. After incubation overnight at 4 °C, cells were washed twice with PBS and incubated in the dark at 37 °C for 30 min with a mixture of 1 ml propidium iodide (100 μg / ml) and RNase (10 μg / ml). Samples were then obtained and analyzed by flow cytometry. (2) Cells (1 × 10⁶ cells / well) were seeded in 6-well plates and treated with 1 μM and 2 μM Formula 1 for 12 h, respectively. 5 Cells were seeded in 6-well plates at different concentrations (1 μM and 2 μM) of Formula 1 for 12 h, and apoptosis was observed. The collected cells were then centrifuged and washed twice with pre-cooled PBS. They were then stained with FITC-annexin V and propidium iodide (PI) at room temperature for 15 min, and samples were obtained and analyzed by flow cytometry.

[0050] The results showed that: (1) Compared with the treated cells, Formula 1 treatment significantly inhibited HPAC and Miapaca2 in the G1 phase, thus inhibiting cell cycle progression. (2) Compared with the treated cells, the low-dose group (1 μM) of Formula 1 had no significant promoting effect on pancreatic cancer cell apoptosis, while the high-dose group (2 μM) of Formula 1 significantly promoted pancreatic cancer cell apoptosis, making it an alternative drug for the treatment of pancreatic cancer. The results of the above examples indicate that Formula 1 of the present invention has a significant anti-malignant proliferation effect on pancreatic cancer and can be used as a candidate drug for the clinical treatment of pancreatic cancer patients.

[0051] In summary, the research of this invention shows that Formula 1 can unexpectedly target and inhibit JAK2, and can be used for JAK2-related scientific research (such as non-therapeutic and diagnostic research applications). In addition, related drugs can be applied based on this mechanism, such as pharmaceutical development research.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of a 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione in the preparation of drugs for alleviating and / or inhibiting pancreatic cancer, characterized in that, The 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione is a compound having the structure of Formula 1 or a pharmaceutically acceptable salt; Formula 1.

2. The application as described in claim 1, characterized in that, The 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione was used to prepare a component that inhibits the growth, migration, and / or tumorigenesis of pancreatic cancer cells.

3. The application as described in claim 2, characterized in that, Pancreatic cancer cells include human pancreatic cancer cells.

4. The application as described in claim 2, characterized in that, Pancreatic cancer cells are HPAC cells.

5. The application as described in claim 1, characterized in that, It is used in combination with other anticancer active ingredients to prepare drugs that alleviate and / or inhibit pancreatic cancer; wherein the anticancer active ingredients include gemcitabine.

6. The application as described in any one of claims 1 to 5, characterized in that, The 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione was combined with pharmaceutically acceptable excipients to prepare a pharmaceutically acceptable dosage form.

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