Application of 7H-indole [2, 3-j] phenanthridine-7, 13 (8H)-diketone serving as JAK2 inhibitor and used for preparing medicine for relieving and treating pancreatic cancer

Abstract

The invention belongs to the field of medicines, and particularly discloses application of 7H-indole [2, 3-j] phenanthridine-7, 13 (8H)-diketone serving as a JAK2 inhibitor and in preparation of medicines for relieving and treating pancreatic cancer. The research shows that the 7H-indole [2, 3-j] phenanthridine-7, 13 (8H)-diketone can effectively target JAK2 protein so as to inhibit the malignant proliferation and migration ability of pancreatic cancer, and a new potential drug treatment scheme is provided for pancreatic cancer patients.

Description

Technical Field

[0001] The present invention relates to the field of medicine, in particular to the new field of JAK2 targeted inhibition. Background Art

[0002] Pancreatic cancer is one of the leading causes of cancer death worldwide and is known as the "king of cancer." It is a highly malignant solid tumor with the characteristics of increasing incidence, poor prognosis, and high mortality. Diagnosis usually occurs in the late 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 is still unsatisfactory. Currently, the main clinical methods for treating pancreatic cancer include the FOLFIRINOX regimen (a combination of chemotherapy drugs including irinotecan, fluorouracil, folinic acid, and oxaliplatin) and the gemcitabine-albumin-paclitaxel regimen. However, pancreatic cancer shows a high tendency to be resistant to these chemotherapy drugs, resulting in significant adverse reactions and greatly reducing the quality of life of patients. Therefore, there is an urgent need to develop new strategies for the treatment of pancreatic cancer.

[0003] With the development of technology, some new pancreatic cancer treatment methods have been developed in the prior art. For example, the US patent document with publication number US12116422A discloses a stapled peptide and its use in the preparation of drugs for treating pancreatic cancer. The Chinese patent document with publication number CN110312508A discloses a pharmaceutical composition for preventing and treating pancreatic cancer containing gossypol and phenformin as active ingredients. The Chinese patent document with publication number CN116515009A discloses a bamboo fungus egg polysaccharide that can treat pancreatic cancer. For another example, the Korean patent with publication number KR1020240125510A discloses a composition containing 2,4-diaminopyrimidine for preventing or treating pancreatic cancer.

[0004] In summary, although there are some treatments for pancreatic cancer in the existing technology, effective treatments for pancreatic cancer are still scarce. Summary of the invention

[0005] In view of the problems of the prior art, the first object of the present 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 new application of 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione as a JAK2 inhibitor.

[0006] The second object of the present invention is to provide the use of the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione as a JAK2 inhibitor in inhibiting pancreatic cancer cells.

[0007] The third object of the present invention is to provide the use of the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione in the preparation of relief and treatment of pancreatic cancer.

[0008] The fourth object of the present invention is to provide an active ingredient and a drug for preventing pancreatic cancer comprising 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione.

[0009] A use of 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 a structure of Formula 1 and a crystal and a pharmaceutically acceptable salt thereof;

[0010]

[0011] The innovative research of the present invention shows that the compound of formula 1 can unexpectedly target and inhibit JAK2, and can be used in JAK2-related scientific research and pharmaceutical development research.

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

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

[0014] Different cancer cells have different surface characteristics and different treatment methods. Among them, pancreatic cancer cells are easy to differentiate and migrate, and it is more difficult to effectively inhibit them. However, the present invention shows that the 7H-indole [2,3-j] phenanthridine-7,13 (8H) -dione can unexpectedly inhibit pancreatic cancer cells based on JAK2 targeted inhibition, so it can be used for pancreatic cancer cell inhibition research and drug development research.

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

[0016] In the present invention, the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione is used to prepare a component for inhibiting the growth, migration and / or tumor formation ability of pancreatic cancer cells.

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

[0018] In the application of the present invention, it can be used in combination with other anticancer active ingredients to prepare drugs for alleviating and / or inhibiting pancreatic cancer;

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

[0020] The present invention studies show that the use of the compound of formula 1 described in the present invention in combination with the other anticancer ingredients described above can achieve synergy and further enhance the therapeutic effect of pancreatic cancer.

[0021] The application of the present invention is to use the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione and a pharmaceutically acceptable excipient in combination to prepare a pharmaceutically acceptable dosage form.

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

[0023] Preferably, the other anticancer ingredients include at least one of gemcitabine, albumin, paclitaxel, fluorouracil, oxaliplatin and the like.

[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 anticancer active ingredient.

[0025] In addition, the medicine of the present invention further comprises pharmaceutically acceptable excipients and pharmaceutically acceptable dosage forms.

[0026] Beneficial Effects

[0027] The present invention study shows that the structural components of Formula 1 can unexpectedly target and inhibit JAK2, and can be used for related scientific research. In addition, the present invention study also shows that Formula 1 can achieve good inhibitory and therapeutic effects on pancreatic cancer cells based on JAK2 targeted inhibition. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1The following are the analysis of pancreatic cancer tissue transcriptome data and the molecular interaction of the 1-JAK2 complex. A is the TCGA pancreatic cancer tissue transcriptome data analysis and the GTEx normal pancreatic tissue transcriptome data analysis showing that the IL6-JAK-STAT3 signaling axis is significantly upregulated in pancreatic cancer. B is the bubble chart_GSEA_Hallmark_top 20. C is a two-dimensional interaction showing ligand-amino acid hydrogen bonds. D is the effective binding of 1 to JAK2 in the protein cavity. E is a three-dimensional representation of the interaction, showing the amino acid residues that interact with the ligand.

[0029] Figure 2 1 is a molecular dynamics simulation of the JAK2 complex of Formula 1 and the regulation of JAK2-STAT3. Wherein, A is the RMSD diagram of the JAK2 complex of Formula 1. B is the RMSF diagram of the JAK2 protein. C is the RMSF diagram of Formula 1. DE is the contact interaction of the JAK2 complex of Formula 1. F is the property of Formula 1. G is the inhibition of the JAK2-STAT3 signaling pathway by Formula 1.

[0030] Figure 3 The activity of human pancreatic cancer cells inhibited by Formula 1. Among them, AB is the comparison of the cell viability of Miapaca2 and HPAC cells treated with different concentrations of Formula 1 by CCK8 method. C is the crystal violet method to detect the inhibitory effect of Formula 1 on the growth of pancreatic cancer cells. D is the flow cytometry analysis of the treated cells and the effect of Formula 1 on the cycle distribution of pancreatic cancer cells after propidium iodide staining and culture. E is the Western blot analysis of the expression of cell cycle-related protein CyclinD1 after Formula 1 treated pancreatic cancer cells *p<0.05,**p<0.01,***p<0.001.

[0031] Figure 4 Formula 1 induces apoptosis of human pancreatic cancer cells. A is the effect of formula 1 on apoptosis of pancreatic cancer cells after Annexin V-FITC and PI staining, analyzed by flow cytometry. B is the apoptosis flow cytometry showing that after incubation with formula 1, the apoptosis rate of pancreatic cancer cells increased significantly, and the apoptosis rate = Q2 + Q3. C is the expression of apoptosis-related proteins Bcl-2, Bax, Caspase3 and Cleaved-Caspase3 detected by Western blot. D is the Bax / Bcl-2 ratio of HPAC and Miapaca2 that gradually increases with the increase of the concentration of formula 1. *p<0.05.

[0032] Figure 5Formula 1 inhibits the invasion of human pancreatic cancer cells. A, B, D, and E are Transwell experiments showing that formula 1 limits the migration ability of HPAC and Miapaca2 compared with the control. C and F are Western blots to detect the effect of formula 1 on EMT-related expressions when HPAC and Miapaca2 cells are treated for 48 hours. **p<0.01, **p<0.001.

[0033] Figure 6 Formula 1 inhibits the migration of human pancreatic cancer cells. AB is the effect of Formula 1 on the expression of Emt in HPAC and Miapaca2 cells detected by Western blot. CD is the wound healing experiment showing that the migration ability of HPAC and Miapaca2 is restricted by Formula 1. **p<0.01.

[0034] Figure 7 Formula 1 can reduce the tumor growth of xenograft mice derived from the Miapaca2 cell line. Wherein, A is the image of the xenograft tumor after treatment with Formula 1. B is the weight of the nude mouse after treatment with Formula 1. CD is the weight and volume of the tumor after treatment with Formula 1. E is the 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 has a better therapeutic effect. Among them, AB is Formula 1, gemcitabine and combined drug use have limited growth capacity of HPAC and Miapaca2. ***p<0.001. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

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

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

[0040] The Western Blot method was used to verify the inhibitory effect of Formula 1 on JAK2-STAT3 signaling to confirm that Formula 1 downregulated JAK2-STAT3 signaling in PDAC cells.

[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 proteins such as p-JAK2 and p-STAT3 are detected using the Western Blot method.

[0042] The results showed that compared with the control group, Formula 1 significantly downregulated p-JAK2 and p-STAT3 (Y705), showing 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: (1) The molecular simulation docking of formula 1 and JAK2 protein is obtained by virtual screening technology and molecular docking technology. (2) Growth inhibition is evaluated using the CCK-8 method (YEASN, 40203ES60). HPAC cells and Miapaca2 cells (3000 cells / well) were inoculated in 96-well plates, respectively, 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 were used as control cells. After 48 hours of treatment, 10 μL of CCK-8 solution was added to the cells. After incubation for another 4 hours, the optical density (OD) value at 450 nm was determined using an enzyme reader. Three experiments were performed for each group, and cell viability was calculated as a value relative to the control group. (3) Human pancreatic cancer cells (HPAC and Miapaca2) were inoculated in 6-well plates (6000-8000 cells / well) and treated with different concentrations (final concentration 1μM and 2μM) of Formula 1 for 1 day. Then, the culture medium without Formula 1 was replaced for 7-10 days, and the old culture medium was replaced every 2 days. Finally, after washing twice with PBS, the colonies were stained with 0.05% crystal violet solution for 10 minutes, then washed with water, dried, and photographed. (4) Experiments such as subcutaneous xenograft mouse model oral administration evaluated the inhibitory effect of Formula 1 (5 mg / kg / day) on PDAC cell proliferation. Nude mice were maintained in a specific pathogen-free environment. The cells were subcutaneously injected into 5-week-old male nude mice. The tumors were randomly divided into two groups. Drug treatment conditions: Tumors were treated with Formula 1 (5 mg / kg) or control agent. The formula for calculating tumor volume is length × (width) 2 / 2. At the end of the study, the tumors were surgically removed, weighed and processed. The heart, liver, spleen, lung and kidney organs of the mice were collected, fixed with 4% formalin, and the tissues were embedded in paraffin. Further hematoxylin-eosin (H&E) staining was performed according to the method reported in the literature. All animal experiments were performed in accordance with the guidelines of the Chinese FDA. The protocol was reviewed and approved by the Department of Experimental Animals 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 growth of pancreatic cancer cells in vivo and in vitro and is an alternative drug for the treatment of pancreatic cancer. (3) Formula 1 has little effect on the body weight and heart, liver, spleen, lung and kidney organs of mice, shows less toxicity, and has a certain safety.

[0045] Example 3 Formula 1 inhibits migration and invasion of PDAC cells

[0046] The specific scheme is: the inhibitory effect of formula 1 (final concentration 1μM and 2μM) on PDAC cell migration and invasion was investigated 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 using a 10μL pipette tip. The cells were then washed with PBS and serum-free medium was added. Subsequently, the cells were incubated in a 5% CO 2 The cells were cultured in a humidified incubator with 95% air (v / v) at 37°C. Cell images were taken with 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 × 10 4 4000 cells / mL were added to the upper compartment of the Transwell, that is, the cells diluted with serum-free DMEM and the mixture of formula 1 were added vertically and evenly to the bottom of the top chamber of the Transwell chamber (4000 cells / well). Then 600 μL of DMEM medium containing 10% FBS was added to the lower chamber as a chemical attractant. After 48 hours, the cells were fixed with 4% paraformaldehyde, stained with 0.05% crystal violet, washed with water to remove the floating color, 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 the cycle progression of PDAC cells and promotes cell apoptosis

[0049] The specific scheme is as follows: (1) cells (1×10 5Cells (1×10 cells / well) were inoculated in 6-well plates and treated with 1 μM and 2 μM formula 1 for 12 h, respectively. The cells were then washed and frozen with 70% ethanol. After incubation at 4°C overnight, the cells were washed twice with PBS and incubated with 1 ml of a mixture of propidium iodide (100 μg / ml) and RNase (10 μg / ml) at 37°C in the dark for 30 min. The samples were then acquired and analyzed by flow cytometry. (2) Cells (1×10 5 Cells were inoculated into 6-well plates with 1 μM and 2 μM formula 1 for 12 h, and cell 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 minutes, and the samples were acquired 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 and inhibited the cell cycle progression. (2) Compared with the treated cells, the low-dose group (1 μM) Formula 1 had no significant promoting effect on the apoptosis of pancreatic cancer cells, while the high-dose group (2 μM) Formula 1 had a significant promoting effect on the apoptosis of pancreatic cancer cells, and is an alternative drug for the treatment of pancreatic cancer. The above-mentioned example results show that Formula 1 of the present invention has a significant effect on the malignant proliferation of pancreatic cancer and can be used as a candidate drug for the clinical treatment of pancreatic cancer patients.

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

[0052] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A use of 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione as a JAK2 inhibitor, characterized in that: The 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione is a compound having a structure of Formula 1 and its crystals and pharmaceutically acceptable salts; 2. The use according to claim 1, characterized in that The 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione is used to prepare a component for inhibiting and / or phosphorylating STAT3.

3. Use of the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione described in claim 1 or 2 for preparing a component for inhibiting pancreatic cancer cells.

4. The use according to claim 3, characterized in that The pancreatic cancer cells include human pancreatic cancer cells; for example, HPAC cells.

5. The use according to claim 3 or 4, characterized in that The 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione is used to prepare a component for inhibiting the growth, migration and / or tumor formation ability of pancreatic cancer cells.

6. Use of the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione described in the application of claim 1 or 2 in the preparation of a drug for alleviating and / or inhibiting pancreatic cancer.

7. The use according to claim 6, characterized in that It is used in combination with other anticancer active ingredients to prepare drugs for alleviating and / or inhibiting pancreatic cancer; preferably, the anticancer active ingredients include at least one of gemcitabine, albumin paclitaxel, fluorouracil, oxaliplatin, etc.

8. The use according to claim 6 or 7, characterized in that The 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione and a pharmaceutically acceptable adjuvant are used in combination to prepare a pharmaceutically acceptable dosage form.

9. An anticancer active ingredient for alleviating and treating pancreatic cancer, characterized in that: The invention comprises the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione described in any one of claims 1 to 2; and further comprises other anticancer ingredients; preferably, the other anticancer ingredients include at least one of gemcitabine, albumin paclitaxel, fluorouracil, oxaliplatin, etc.

10. An anticancer drug for alleviating and treating pancreatic cancer, characterized in that: A method comprising a pharmaceutically effective amount of the 7H-indole[2,3-j]phenanthridine-7,13(8H)-dione described in any one of claims 1 to 2; preferably, comprising the anticancer active ingredient described in claim 9; preferably, further comprising a pharmaceutically acceptable excipient; preferably, having a pharmaceutically acceptable dosage form.

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