Application of 4-(4-aminophenoxy) pyridine carboxamide compound in preparation of antitumor drugs and pharmaceutical composition

Abstract

The invention belongs to the technical field of medicine organic matters, and discloses application of a 4-(4-aminophenoxy) pyridine carboxamide compound in preparation of an anti-tumor medicine and a medicine composition. The invention provides an application of a 4-(4-aminophenoxy) pyridine carboxamide compound in medicine preparation. The compound is used for preparing an anti-tumor medicine. The invention also provides a pharmaceutical composition containing the compound, and the pharmaceutical composition has antitumor activity, and especially can be used for targeted therapy of tumor diseases and acute myelogenous leukemia caused by abnormal Flt3 and c-Met signal pathways.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical organic matter, and specifically relates to the application of 4-(4-aminophenoxy)picolinamide compounds in the preparation of anti-tumor drugs and a pharmaceutical composition. Background Art

[0002] Studies have shown that the incidence and mortality of cancer are increasing year by year. In addition to cardiovascular disease, cancer has become the main cause of death in humans. Although surgery, radiotherapy and chemotherapy play a vital role in cancer treatment, they have great limitations. Acute myeloid leukemia (AML) is the most common malignant clonal disease in adult leukemia. It originates from the abnormal differentiation and proliferation of myeloid hematopoietic stem cells, and is characterized by the rapid proliferation of abnormal bud cells in the bone marrow, which affects the production of normal hematopoietic cells. Its clinical manifestations are fatal infections, bleeding, and organ infiltration. Receptor tyrosine kinases (RTKs) are a subclass of tyrosine kinases, which can be divided into 19 subfamilies according to the homology of their N-terminal extracellular domain (EC). RTKs are closely related to cell growth, proliferation and metastasis. However, abnormal activation of RTKs often leads to the occurrence, invasion and metastasis of various cancers. A large number of studies have shown that receptor tyrosine kinase inhibitors (TKIs) can effectively block some tumor cell signaling pathways, inhibit the metastasis of malignant tumor cells, and effectively reduce the spread of tumor cells. As targeted anti-tumor drugs, TKIs have important clinical significance and broad application prospects.

[0003] FMS-like tyrosine kinase 3 (Flt3) belongs to type III receptor tyrosine kinase and plays a definite role in the growth and differentiation of hematopoietic progenitor cells. Under normal physiological conditions, when there is no Flt3 ligand (FL), the near-membrane structure of Flt3 inhibits the formation of Flt3 dimerization. When FL is present, it binds to the extracellular receptor region to cause Flt3 dimerization, causing structural changes in the kinase region, phosphorylating downstream signaling proteins such as PI3K / AKT, Ras / MAKP, etc., thereby activating downstream signaling pathways, and regulating cell growth and proliferation through a series of signal transduction. In 1996, Nakao et al. found that Flt3-ITD mutations were detected in up to 30% of patient samples. Subsequently, Yamamoto et al. first discovered in 2001 that Flt3-TKD point mutations were present in about 5% of patients, mainly located at the D835 site on the activation loop (AL). Both mutations can activate Flt3 kinase independently of FL binding. In addition to the approved PKC412, more than 20 small molecule compounds have been found to have Flt3 kinase inhibitory activity, many of which are in clinical research and have achieved good therapeutic effects. c-Met is a member of the RTK family and a mesenchymal epithelial transition factor, also known as hepatocyte growth factor receptor (HGFR). c-Met binds to its unique ligand (HGF) and activates multiple complex signaling pathways related to cell proliferation, angiogenesis, invasion and migration. Normal cell growth and function depend on the regulation of the HGF / c-Met signaling pathway. This pathway plays a key role in embryogenesis, organ development and regeneration. HGF / SF overexpression, abnormal expression of c-Met, gene amplification or rearrangement can lead to abnormal activation of the c-Met signaling pathway in various human cancers. Therefore, how to block this abnormal activation and use small molecule inhibitors to block the transformation of the signaling pathway by targeting specific sites (including RTKs), thereby inducing apoptosis and necrosis of cancer cells, has become a key research issue. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide the use of 4-(4-aminophenoxy)picolinamide compounds in the preparation of anti-tumor drugs and pharmaceutical compositions, specifically using the following technical solutions: According to a first aspect of the present invention, a use of a 4-(4-aminophenoxy)picolinamide compound in the preparation of an anti-tumor drug is provided. Preferably, the tumor cell is A549 / MCF-7 / HeLa.

[0005] The 4-(4-aminophenoxy)picolinamide compound used in the present invention is selected from any one of the following compounds (Compound 20, Compound 27, Compound 31, Compound 36, Compound 46), and its structural formula is as follows: .

[0006] Preferably, the 4-(4-aminophenoxy)picolinamide compound is selected from the following compounds, and its structural formula is as follows: .

[0007] 4-(4-Aminophenoxy)picolinamide compounds, such as compound 46, can effectively inhibit the proliferation of A549 / MCF-7 / HeLa tumor cells.

[0008] According to the second aspect of the present invention, the present invention also provides a use of the above-mentioned 4-(4-aminophenoxy)picolinamide compounds or pharmaceutically acceptable salts thereof in the preparation of Flt3 inhibitors and c-Met inhibitors.

[0009] Preferably, the Flt3 inhibitor and the c-Met inhibitor can be used to prepare drugs for treating related diseases caused by abnormal Flt3 and c-Met signaling pathways. More preferably, the related diseases include tumor diseases and acute myeloid leukemia.

[0010] The above diseases include but are not limited to diseases caused by the following reasons: abnormal expression, gene amplification or rearrangement of Flt3 and c-Met can lead to abnormal activation of Flt3 and c-Met signaling pathways in various human cancers.

[0011] According to the third aspect of the present invention, the present invention also provides a pharmaceutical composition for treating tumors, comprising the above-mentioned 4-(4-aminophenoxy)picolinamide compound and pharmaceutical excipients. The pharmaceutical excipients include excipients, and the excipients are at least one of gum arabic, syrup, lanolin, and starch.

[0012] Preferably, the pharmaceutical composition is in the form of injections, tablets, capsules, aerosols, suppositories, films, pellets, ointments, controlled-release or sustained-release preparations, and nanoformulations.

[0013] The beneficial effects of the present invention are as follows: the present invention provides a new application of 4-(4-aminophenoxy)picolinamide compounds in the preparation of anti-tumor drugs. The compounds have anti-tumor activity, especially targeted treatment of tumor diseases and acute myeloid leukemia caused by abnormal Flt3 and c-Met signaling pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1The graphs show the inhibition of tumor cell proliferation by compound 46 at different concentrations; (A) blank; (B) cabozantinib drug concentration is 5 μM; (C) compound 46 concentration is 2.5 μM; (D) compound 46 concentration is 5 μM; (E) compound 46 concentration is 10 μM; Figure 2 The graphs show the effects of different concentrations of compound 46 and cabozantinib on the cell cycle process; (A) blank; (B) cabozantinib concentration of 5 μM; (C) compound 46 concentration of 2.5 μM; (D) compound 46 concentration of 5 μM; (E) compound 46 concentration of 10 μM; (F) integrated graph of all concentrations of compound 46; Figure 3 The blood concentration of compound 46 was measured. DETAILED DESCRIPTION

[0015] The following will be combined with the embodiments and drawings to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, scheme and effect of the present invention. It should be noted that the embodiments and features in the embodiments of this application can be combined with each other without conflict.

[0016] Example 1 This example is an experiment on the inhibition of tumor cell proliferation by compound 46, comprising the following steps: Three cell lines, A549, Hela and MCF-7, were placed in each well (about 5×10 3 Cells) were added to the 96-well plate to be tested and cultured in a cell culture incubator (37°C, 5% CO2) for 24 h to allow the cells to adhere to the wall. Different concentrations of the derivatives (100, 33.33, 11.11, 3.70, 1.23 g / mL) and the positive control cabozantinib were added to the 96-well plate and placed in the incubator for 72 h. After 72 h, the culture medium in the 96-well plate was poured out. Then, the prepared 5 mg / mL MTT solution was added to the plate at 20 μL / well and cultured in a 5% CO2 incubator at 37°C for 3.5 h. Next, MTT was aspirated from the 96-well plate with a 1 mL needle, 150 μL DMSO was added to each well, and the enzyme-linked immunosorbent assay (ELISA) was set to medium-speed vibration for 10 seconds. The absorbance value of each well was measured by wavelength method at 492 nm. As shown in Table 1, the experimental results show that several compounds exhibited moderate to excellent antiproliferative activity against three different cell lines. Among them, compounds 20, 27, 36, and 46 had stronger inhibitory activity against one or more cell lines than the positive control cabozantinib. Among them, the best compound 46 had an IC of 1.04 for A549, Hela, and MCF-7 cells. 50They were 0.26±0.04 μM, 0.82±0.04 μM and 2.35±0.07 μM respectively.

[0017] Table 1 Example 2 This example is an experiment to explore the mechanism of compound 46 in inhibiting tumor cells, comprising the following steps: (1) Add the required cells to a six-well culture plate and incubate for 24 hours. Prepare the compound and culture medium to the required concentration and culture the cells for 24 hours. After 24 hours, take about 60,000 suspended cells, centrifuge and discard the culture medium. Rinse the cells three times with PBS. Then add 500 μL PBS after centrifugation, mix well, and wrap the tube wall with tin foil; add 5.0 μL Annexin V-FITC and 10.0 μL iodide to the two blank pairs respectively, add the mixture to the remaining tubes, and mix gently. Incubate at room temperature in the dark for 30 minutes and detect by flow cytometry. The measurement is best controlled within 1 hour. Figure 1 As shown, the experimental results showed that compound 46 induced apoptosis of A549 cells in a dose-dependent manner. In addition, the effect of compound 46 (7.31%) in inducing apoptosis of A549 cells was better than that of cabozantinib (6.58%) at the same concentration (5 μM).

[0018] (2) The dosing procedure for the cell cycle is the same as that for the apoptosis assay. 24 hours after dosing, the adherent cells were digested with trypsin, transferred to a 5.0 mL eppendorf (EP) tube, collected by centrifugation, washed three times with pre-cooled PBS, and centrifuged sequentially. Finally, 500 μL of pre-cooled PBS was added to each tube and mixed evenly. Then, while shaking the EP tube, 70% ice ethanol was slowly dripped into the tube to make the suspension uniform. The suspension was fixed in a 4°C refrigerator overnight or even longer; the 70% ice ethanol was removed by centrifugation and washed again three times with pre-cooled PBS. Then, 500 μL of PBS was added to each tube. The suspension was gently blown open with a pipette, and the tube wall was wrapped with tin foil. A mixed staining solution of RNase A and propidium iodide (PI) was added to each EP tube. The tubes were incubated in the dark at room temperature for 30 min, and the cell cycle was detected by flow cytometry. Figure 2 As shown, compound 46 induced apoptosis of A549 cells mainly in the G0 / G1 phase. With the increase of compound 46 concentration, the blocking rate of cells in the G0 / G1 phase increased from 55.95% to 58.67%. Compared with the positive control drug cabozantinib, compound 46 has a stronger blocking ability on A549 cells in the G0 / G1 phase. All the results show that the inhibitory effect of compound 46 on the proliferation of A549 cells in the G0 / G1 phase is dose-dependent.

[0019] Example 3 This example is a blood concentration determination experiment of compound 46, comprising the following steps: (3) Three male rats were orally administered the drug. Blood was collected from the tail at 0.25, 0.33, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 hours. The blood was centrifuged as quickly as possible (8000 r, 10 min). The blood drug concentration was determined by LC-MS / MS. See Table 2 and Figure 3 As shown, the AUC value is 3351 μg / L*h and the elimination half-life is 4.5 h. The experimental results show that compound 46 has good pharmacokinetic properties.

[0020] Table 2 Although the description of the present invention has been quite detailed and specifically describes several described embodiments, it is not intended to be limited to any of these details or embodiments or any particular embodiment, but should be regarded as providing a broad possible interpretation of these claims in view of the prior art by reference to the appended claims, thereby effectively covering the intended scope of the present invention. In addition, the above description of the present invention is based on the embodiments foreseeable by the inventor, and its purpose is to provide a useful description, and those non-substantial changes to the present invention that have not yet been foreseen may still represent equivalent changes to the present invention.

Claims

1. Application of 4-(4-aminophenoxy)picolinamide compounds in the preparation of anti-tumor drugs.

2. The use according to claim 1, characterized in that: The 4-(4-aminophenoxy)picolinamide compound is selected from any one of the following compounds (Compound 20, Compound 27, Compound 31, Compound 36, Compound 46), and its structural formula is as follows: 。 3. The use according to claim 2, characterized in that: The 4-(4-aminophenoxy)pyridinecarboxamide compound is selected from the following compounds, and its structural formula is as follows: 。 4. The use according to claim 1, characterized in that: The tumor cells are A549 / MCF-7 / HeLa.

5. Use of the 4-(4-aminophenoxy)picolinamide compound or a pharmaceutically acceptable salt thereof according to any one of claims 2 or 3 in the preparation of a Flt3 inhibitor and a c-Met inhibitor.

6. The use according to claim 5, characterized in that: The Flt3 inhibitor and c-Met inhibitor can be used to prepare drugs for treating related diseases caused by abnormal Flt3 and c-Met signal pathways.

7. The use according to claim 6, characterized in that: The related diseases include tumor diseases and acute myeloid leukemia.

8. A pharmaceutical composition for treating tumors, characterized in that: The pharmaceutical composition comprises the 4-(4-aminophenoxy)picolinamide compound according to any one of claims 2 or 3.

9. The pharmaceutical composition according to claim 8, characterized in that Also contains pharmaceutical excipients.

10. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition may be in the form of injection, tablet, capsule, aerosol, suppository, film, pellet, ointment, controlled-release or sustained-release preparation and nano preparation.

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