Use of quinazolinyl derivatives in the preparation of anti-solid tumor drugs

Abstract

The application provides an application of quinazolin-4-yl-(4, 5-dihydro-1H-imidazol-2-yl)-amine in preparation of an anti-solid tumor drug, especially in combination with PD-1 / PD-L1 monoclonal antibody for tumor treatment. The application is proved by in-vivo and in-vitro experimental research that quinazolin-4-yl-(4, 5-dihydro-1H-imidazol-2-yl)-amine can significantly increase MHC I protein expression of colon cancer and liver cancer cells, enhance antigen presentation and killing ability of T cells to tumor cells, and the combination with PD-1 / PD-L1 monoclonal antibody has significant anti-tumor activity, thereby expanding the clinical application of chemical drug quinazolin-4-yl-(4, 5-dihydro-1H-imidazol-2-yl)-amine in solid tumor treatment. The application provides a new medical use of quinazolin-4-yl-(4, 5-dihydro-1H-imidazol-2-yl)-amine, and also provides a new idea and potential target for the development of a sensitized immune checkpoint inhibitor strategy.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to the application of quinzatinib in the preparation of anti-solid tumor drugs. Background Technology

[0002] The rapid development and widespread application of immune checkpoint inhibitors have significantly changed the landscape of malignant tumor treatment, becoming a crucial cornerstone of the current cancer therapy system. However, clinical data show that, except for melanoma and Hodgkin's lymphoma, the response rate of immune checkpoint inhibitors in the treatment of most solid tumors remains generally below 30%, making it difficult for most cancer patients to benefit from immunotherapy. Improving patient responsiveness to immunotherapy remains a key unresolved challenge in clinical cancer treatment.

[0003] Recent studies have shown that low expression or absence of Major Histocompatibility Complex Class I (MHC I) in tumor cells is one of the key factors leading to patients' inability to respond to immunotherapy. MHC I is a core molecular basis for initiating adaptive immune responses and achieving immune surveillance and elimination of tumor cells. Its fundamental role is to present endogenous antigenic peptides from tumor cells to the cell surface, which are then used by CD8+. + T cells recognize MHC I, thereby initiating a specific anti-tumor immune response. However, a high rate of low MHC I expression has been observed in various tumor types, including colorectal cancer and liver cancer. Clinical evidence also suggests that patients with high MHC I expression levels and strong antigen-presenting function generally benefit more from immune checkpoint inhibitor therapy. Conversely, patients with low MHC I expression levels and weak antigen-presenting function respond poorly to immunotherapy. Therefore, identifying small molecule drugs that can induce MHC I expression may improve tumor antigen presentation function, thereby enhancing the overall efficacy of immunotherapy.

[0004] Quizartinib is approved for the treatment of newly diagnosed adult patients with acute myeloid leukemia (AML) harboring FLT3 internal tandem repeat (FLT3-ITD) mutations. Clinical trials are currently evaluating the efficacy of quizartinib in treating FLT3-ITD mutation-negative AML patients. Its primary mechanism of action involves potently binding to the inactive conformation of FLT3, specifically inhibiting the activity of FLT3 receptor tyrosine kinase, thereby blocking FLT3-ITD-dependent cell proliferation and inducing leukemia cell differentiation and death. Because FLT3-ITD mutations are among the most common and poorly prognostic driver gene mutations in AML, current research on quizartinib focuses on AML, and there are no reports of quizartinib being used for immunotherapy in solid tumors. Summary of the Invention

[0005] The purpose of this invention is to provide the application of quinzatinib in the preparation of antitumor drugs, especially its application in combination with immune checkpoint inhibitors for the treatment of solid tumors such as colon cancer and liver cancer. Quinzatinib, chemical name N-(5-tert-butyl-isoxazol-3-yl)-N-{4-[7-(2-morpholin-4-ylethoxy)imidazo[2,1-b][1,3]benzothiazo-2-yl]phenyl}urea, molecular formula: C 29 H 32 N6O4S has a molecular weight of 560.22.

[0006] Experiments have confirmed that quinzartinib (10 μM) significantly increases MHC I expression in colon and liver cancer cells in vitro, as observed by Western blotting and flow cytometry. In an allogeneic xenograft mouse model (colon cancer model) constructed from MC38 cells and a c-Myc-MCL1-driven orthotopic liver cancer model, quinzartinib significantly enhances the antitumor activity of PD-1 / PD-L1 monoclonal antibodies, exhibiting a synergistic effect.

[0007] The drug is made from quezartinib and pharmaceutically acceptable excipients, and is formulated as an oral dosage form, once daily.

[0008] Quezatinib is a selective FLT3 inhibitor approved for the treatment of newly diagnosed adult patients with FLT3 internal tandem repeat (FLT3-ITD) mutations. Its application in solid tumors and its potential in immunosensitizing therapy have not yet been reported. This invention demonstrates through in vitro and in vivo studies that quezartinib significantly upregulates MHC I expression in various tumor cells and exhibits significant antitumor activity when combined with PD-1 / PD-L1 monoclonal antibodies, thereby expanding the application of the marketed drug quezartinib in the immunotherapy of solid tumors. The significance of this invention lies not only in clarifying the possibility of using quezartinib for the treatment of solid tumors but also in providing new ideas and promising candidate drugs for rapidly translating clinical tumor immunotherapy responses.

[0009] In their preliminary research, the inventors systematically screened a library of clinically used drugs and discovered that quezartinib, a drug used clinically to treat AML, significantly upregulates MHC I expression in various tumor cells and exerts significant anti-tumor activity in synergistic effect with immune checkpoint inhibitors. Currently, there are no studies on quezartinib upregulating MHC I expression in tumor cells, nor are there any reports on its use in immunotherapy for solid tumors. This invention provides a marketed small molecule drug with the potential for rapid clinical translation into clinical tumor immunotherapy, offering a new disease application for quezartinib. Attached Figure Description

[0010] Figure 1Quezatinib increases the expression of total MHC I protein in colorectal cancer and liver cancer cells in a concentration-dependent manner. These are Western blot images of the effects of quezartinib (2.5 μM, 5 μM and 10 μM) on SW480 colorectal cancer cells and HepG2 liver cancer cells.

[0011] Figure 2 Quezatinib increases the expression of MHC I protein and antigen presentation on the surface of tumor cells. This is a flow cytometry bar graph showing the effect of quezatinib (10 μM) on tumor cells.

[0012] Figure 3 This shows that quezartinib can enhance the killing effect of T cells on tumor cells. The figure shows the experimental results of the killing function of quezartinib (10 μM) on T cells in a T cell-tumor cell co-incubation model.

[0013] Figure 4 This image shows how quezartinib enhances the antitumor activity of PD-1 / PD-L1 monoclonal antibodies. It also shows the tumor-suppressive effect of quezartinib (5 mg / kg) in combination with PD-1 / PD-L1 monoclonal antibodies in MC38 allogeneic xenograft models (colon cancer model) and c-Myc-MCL1-driven liver cancer models. Detailed Implementation

[0014] The present invention will be further described in conjunction with the accompanying drawings and embodiments. The following embodiments are not intended to limit the invention; any improvements or substitutions based on the basic content of these embodiments shall still fall within the scope of protection of the claims of the present invention.

[0015] Example 1: Quizatinib concentration-dependently increases the expression of total MHC I protein in colon cancer and liver cancer cells.

[0016] The specific steps are as follows: SW480 colon cancer cells and HepG2 liver cancer cells were selected, and 1.0 × 10⁻⁶ cells were used. 6 Cells were seeded at a density of 6-well plates and cultured overnight at 37°C with 5% CO2. The following day, cells were treated with quinzartinib (2.5 μM, 5 μM, and 10 μM) for 24 h. Cells were then collected, lysed, and proteins were extracted. Western blotting was used to detect MHC I expression in the cells. The effect of quinzartinib on increasing MHC I protein expression in colon cancer and liver cancer cells is shown in [the table below]. Figure 1 Experimental results showed that quezartinib increased MHC I expression in tumor cells in a concentration-dependent manner.

[0017] Example 2: Quizatinib increases the expression of MHC I protein on the cell membrane surface and the level of antigen presentation in tumor cells.

[0018] The specific steps are as follows: Select colon cancer cells SW480, at a concentration of 1.0 × 10⁻⁶.6 Cells were seeded at a density of 1 / well in 6-well plates and cultured overnight at 37°C with 5% CO2. The following day, cells were treated with quinzartinib (10 μM) for 24 h, and then collected. MHC I expression on the cell membrane surface was detected by flow cytometry. Colon cancer cells MC38-OVA were selected and seeded at 1.0 × 10⁻⁶ cells / well. 6 Cells were seeded at a density of 1 / well in 6-well plates and cultured overnight at 37°C with 5% CO2. The following day, cells were treated with quinzartinib (10 μM) for 24 hours, and then collected for antigen presentation analysis using flow cytometry. The effect of quinzartinib on increasing MHC I expression on tumor cell membranes is shown in [the table below]. Figure 2 A, the effect on antigen presentation ability is shown in [see section A]. Figure 2 B. The results showed that quezartinib can effectively increase the expression of MHC I on the surface of tumor cell membranes and increase the level of antigen presentation.

[0019] Example 3: Quizatinib enhances the killing effect of T cells on tumor cells.

[0020] The specific steps are as follows: MC38-OVA colon cancer cells were selected and treated with quinzartinib (2.5 μM) for 8 h, followed by co-incubation with T cells for 12 h. The supernatant was collected, and the secretion of IFN-γ and TNF-α was detected using an ELISA kit. Crystal violet staining was used to assess the survival of tumor cells. The effect of quinzartinib on the tumor-killing effect of T cells is discussed in [the following section]. Figure 3 Experimental results showed that quezartinib could significantly enhance the killing ability of T cells against tumor cells and induce the secretion of IFN-γ and TNF-α.

[0021] Example 4: Quezatinib enhances the antitumor effect of PD-1 / PD-L1 monoclonal antibodies.

[0022] The specific steps are as follows: MC38 cells were cultured at 5×10⁻⁶... 5 Cells per mouse were subcutaneously injected into the axilla of C57BL / 6J mice to construct an allogeneic xenograft model of colon cancer. The tumors were allowed to grow to 50-100 mm. 3 Mice were then randomly divided into a control group, a quezartinib (5 mg / kg) treatment group, a PD-1 monoclonal antibody group (10 mg / kg), and a combination group. Six mice were in each group. Tumor growth was measured every two days using calipers. Tumor volume (TV) was calculated as 0.5 × tumor long axis × tumor short axis. 2Calculations were performed. Quezatinib (5 mg / kg) was administered daily by gavage, and the PD-L1 monoclonal antibody (10 mg / kg) was administered twice weekly via tail vein injection. c-MYC (8 μg) and MCL1 plasmid (5 μg) were injected into FVB female mice via high-pressure tail vein injection to establish an orthotopic hepatocellular carcinoma model. One week later, mice were randomly assigned to a control group, a quezartinib (5 mg / kg) treatment group, a PD-L1 monoclonal antibody group (10 mg / kg), and a combination group. Five mice were in each group. Quezartinib (5 mg / kg) was administered daily by gavage, and the PD-L1 monoclonal antibody (10 mg / kg) was administered twice weekly via tail vein injection. For specific effects, see [link to relevant documentation]. Figure 4 Experimental results showed that quezartinib combined with PD-1 / PD-L1 monoclonal antibody could significantly inhibit tumor growth in mouse models of colorectal cancer and in situ liver cancer models.

Claims

1. The use of quinzatinib in the preparation of an anti-solid tumor drug, wherein quinzatinib, chemically named N-(5-tert-butyl-isoxazol-3-yl)-N-{4-[7-(2-morpholin-4-ylethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea, has the molecular formula: C 29 H 32 N6O4S, with a molecular weight of 560.22, is characterized by, Application in the preparation of drugs for treating solid tumors.

2. The application according to claim 1, characterized in that, The solid tumors mentioned are colon cancer and liver cancer.

3. The application according to claim 1, characterized in that, The application described is that quezartinib significantly increases the expression of MHC I in tumor cells and enhances the sensitivity of colon cancer and liver cancer to PD-1 / PD-L1 monoclonal antibodies.

4. The application according to claim 1, characterized in that, The drug is made from quezartinib and pharmaceutically acceptable excipients, and is formulated as an oral dosage form.

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