N-(3 / 4-((6-substituted pyrimidine-4-yl) amino) phenyl) amide derivative and application thereof

Abstract

The invention discloses an N-(3 / 4-((6-substituted pyrimidine-4-yl) amino) phenyl) amide derivative and application thereof, and belongs to the field of medical chemistry. The structural formula of the compound is as follows: R1 is 4-methyl-1H-imidazole-1-group, 3-methyl-1H-pyrazole-1-group or 4-methyl-1H-pyrazole-1-group, R2 is 4-methyl-1H-imidazole-1-group, and R3 is 3-methyl-1H-pyrazole-1-group; r2 is a linear alkyl group, a cycloalkyl group, a 1-methylpiperidine-4-group, a morpholinyl ethyl group, a substituted or unsubstituted phenyl group, a substituted or unsubstituted styryl group, a substituted or unsubstituted phenethyl group, a phenylpropyl group, a phenylbutenyl group, a 6-bromo-pyridine-2-group, a (benzo [d] [1, 3] dioxol-5-group) vinyl group or a (thiophene-2-group) methyl group. The invention relates to a novel Nur77 targeted giant vesicle type death inducer which is suitable for single-drug treatment of lung cancer or is combined with other targeted / immune / chemotherapeutic drugs to improve the curative effect and reduce the drug resistance risk. And the Nur77 can be expanded to breast cancer, liver cancer and other Nur77 related tumors.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry, and more particularly to a... N -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivatives and their applications. Background Technology

[0002] Currently, molecularly targeted drugs are one of the main treatments for lung cancer. Although significant progress has been made in targeted therapy for lung cancer, targeted drugs often lead to primary or secondary drug resistance. Therefore, developing novel small-molecule targeted drugs for lung cancer treatment has important scientific and practical significance. Previous studies have shown that Nur77 has been proven to be a key regulatory molecule in the occurrence and development of lung cancer. Clinical studies have found that elevated Nur77 protein expression is associated with poor histopathological differentiation (P=0.034), advanced clinical stage (P=0.001), and cancer recurrence (P=0.033), and lung cancer patients with high Nur77 expression have lower overall survival (OS) and progression-free survival (PFS). In cell models, Nur77 has been found to play an important role in the proliferation, migration, and invasion of lung cancer cells. In addition, various Nur77-targeting small molecules have been shown to have certain activities in lung cancer models. In summary, Nur77 is a novel target for lung cancer treatment, and Nur77-targeted anticancer small molecules are an effective strategy for lung cancer treatment. Summary of the Invention

[0003] The purpose of this invention is to solve the above-mentioned problems in the prior art and provide a solution. N -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivatives and their applications.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A sort of N -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivatives, with the following structural formula:

[0006]

[0007] Wherein, R1 is 4-methyl-1H-imidazol-1-yl, 3-methyl-1H-pyrazol-1-yl or 4-methyl-1H-pyrazol-1-yl; R2 is a straight-chain alkyl, cycloalkyl, 1-methylpiperidin-4-yl, morpholinylethyl, substituted or unsubstituted phenyl, substituted or unsubstituted styryl, substituted or unsubstituted phenethyl, phenylpropyl, phenylbutenyl, 6-bromopyridin-2-yl, (benzo[d][1,3]dioxacyclopenten-5-yl)vinyl or (thiophen-2-yl)methyl.

[0008] Preferably, the one described above N-(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivatives, including at least one of the following compounds:

[0009] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

[0010] The aforementioned N Use of -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivatives in the preparation of Nur77-related tumor therapeutic compositions.

[0011] The Nur77-related tumor is lung cancer.

[0012] Preferably, the N -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivatives are selected from at least one of the following compounds:

[0013] , , , , .

[0014] More preferably, the N -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivatives are:

[0015] .

[0016] A Nur77-related tumor therapeutic composition comprising the aforementioned N -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivative.

[0017] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0018] 1. This invention has excellent anti-proliferative activity: it shows significant inhibitory effects on a variety of lung cancer cell lines, which is superior to the positive control 12A; it can achieve high-efficiency inhibition at low concentrations, and the long-term proliferation inhibition effect is further confirmed by colony formation experiments.

[0019] 2. This invention has good selectivity: it has low toxicity to HBE cells (human bronchial epithelial cells) and HHL-5 cells (human hepatocytes) and a high selectivity index.

[0020] 3. The target of this invention is clearly defined: thermodynamic migration experiments directly confirm the stable binding of the compound to the target protein Nur77-LBD, enhancing the thermal stability of the complex; reporter gene experiments confirm that the compound inhibits the transcriptional activity of Nur77; Nur77 knockdown experiments demonstrate that the activity of the compound is highly dependent on the target, avoiding non-specific toxicity.

[0021] 4. The pharmacodynamic mechanism of this invention is comprehensive: it activates AMPK phosphorylation, inhibits endocytosis and autophagy, induces macrovesicular cell death in a concentration-dependent manner, and effectively blocks tumor cell proliferation, migration and invasion.

[0022] 5. This invention has significant in vivo antitumor effects: In a nude mouse xenograft model of lung cancer, continuous administration of moderate doses significantly inhibited the growth of tumor volume and weight; there was no weight loss or pathological changes in major organs, and the expression of proliferation markers PCNA and Nur77 in tumor tissue was downregulated, with the mechanism being consistent with that in vitro.

[0023] 6. The synthesis process of this invention is efficient and simple: the multi-step synthetic route starting from commercially available raw materials has a high overall yield, mild reaction conditions (room temperature to reflux, no need for precious metal catalysts), intermediates are easy to monitor and purify, NMR / HRMS data fully support structural confirmation, and it is convenient for laboratory and industrial-scale production.

[0024] 7. This invention has broad clinical application potential: as a novel Nur77-targeted giant bubble death inducer, it can be prepared into pharmaceutical salts and compositions, suitable for monotherapy of lung cancer, or combined with other targeted / immunotherapy / chemotherapy drugs to improve efficacy and reduce the risk of drug resistance; at the same time, it can be extended to other Nur77-related tumors such as breast cancer and liver cancer. Attached Figure Description

[0025] Figure 1 This is a graph showing the time (b) and concentration (a) dependence of the representative compound PBA12 in inducing vacuolization in H460 cells in Example 6 of the present invention.

[0026] Figure 2 The images show the Western blot analysis (a) and the corresponding Image J quantitative analysis (b) of the thermal migration experimental results of the representative compounds in Example 7 of this invention.

[0027] Figure 3 The results of reporter gene detection for representative compounds in Examples 8 and 9 of this invention are shown in (a) and Western blot analysis of Nur77 protein expression intervention is shown in (b).

[0028] Figure 4 This is a comparative graph showing the effects of the representative compound in Example 10 of the present invention on inducing vacuolization (a) and inhibiting cell proliferation (b) in siCtrl H460 and siNur77 H460 cells.

[0029] Figure 5 This is a graph showing the evaluation results of the intervention effect of different inhibitors on the vacuolization of lung cancer cells induced by compound PBA12 in Example 11 of the present invention.

[0030] Figure 6 This is a transmission electron microscope image showing the morphology of cavitation induced by compound PBA12 observed in Example 11 of the present invention.

[0031] Figure 7 The following are Western blot analysis diagrams showing the effects of compound PBA12 in Examples 12 and 13 of this invention on the expression of marker proteins (a) related to apoptosis, autophagy, macropinocytosis, and other processes in lung cancer cells, as well as marker proteins (b) related to macrovesicle death pathway; the following are evaluation diagrams showing the intervention effects of the JNK inhibitor SP600125 (c) and the AMPK inhibitor Dorsomorphin (d) on the vacuolization of lung cancer cells induced by compound PBA12; and the following are evaluation diagrams showing the inhibitory effects of compound PBA12 on lung cancer cells (e-f) before and after Dorsomorphin intervention.

[0032] Figure 8 The figures show the results of the colony formation experiment (a) and cell scratch experiment (b) of lung cancer cells with and without the intervention of the representative compound in Example 14 of this invention.

[0033] Figure 9 This is a diagram showing the in vivo experimental results of compound PBA12 in a nude mouse xenograft model in Example 15 of this invention. Figure 9 (a) shows representative images of H460 xenograft tumors in the control group and the drug-treated group after the experiment. Figure 9 (b) shows that PBA12 significantly inhibited the weight of H460 xenograft tumors; Figure 9 (c) shows that PBA12 significantly inhibited the growth of H460 cell xenograft tumors during the administration period; Figure 9 The middle (d) shows the expression of PCNA and Nur77 in tumor tissues of the control group and the PBA12 treatment group; Figure 9 (e) shows that PBA12 did not cause significant changes in body weight in nude mice during administration; Figure 9 (f) shows the H&E staining results of major organs in mice in the control group and the drug-treated group. Detailed Implementation

[0034] To make the technical problems, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0035] The present invention provides N The preparation method of -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivatives is as follows:

[0036] (1) Commercially available 4,6-dichloropyrimidine was reacted with 4-methylimidazole, 3-methyl-1H-pyrazole or 4-methyl-1H-pyrazole in N,N-dimethylformamide (DMF) solvent containing anhydrous potassium carbonate to give the corresponding 4-chloro-6-substituted pyrimidine intermediates (1a-c).

[0037] (2) Under nitrogen protection, 4-chloro-6-substituted pyrimidine intermediates (1a-c) and N-(3-aminophenyl)acetamide or N-(4-aminophenyl)acetamide are refluxed in N,N-diisopropylethylamine (DIPEA) and N-methylpyrrolidone (NMP) to form the corresponding intermediates (2a-d).

[0038] (3) In the methanol:water system, intermediate 2a-d is refluxed under acidic conditions to remove the acetyl group, yielding the key intermediate (3a-d).

[0039] (4) The key intermediate (3a-d) undergoes N-acylation reaction with different types of carboxylic acids to obtain the target compounds (PBA1-27, PBB1-3, PBC1-3 and PBD1-3).

[0040] The specific reaction route is shown below:

[0041]

[0042] Example 1: Preparation of intermediate 4-chloro-6-(4-methyl-1H-imidazol-1-yl)pyrimidine (1a)

[0043] To a 250 mL dry reaction flask, 4,6-dichloropyrimidine (10.00 g, 67.12 mmol), anhydrous potassium carbonate (27.78 g, 201.36 mmol), and N,N-dimethylformamide (DMF, 60 mL) were added sequentially. Then, 4-methylimidazole (5.51 g, 67.12 mmol) was added with stirring. After the addition was complete, the reaction was allowed to proceed at room temperature for 24 hours. The reaction was stopped when TLC showed equilibrium. 50 mL of water was added to the reaction mixture, and the mixture was filtered, collecting the filter cake. The filter cake was recrystallized from methanol-water (1:1, v / v) and dried to give 8.6 g of a white solid, namely intermediate 1a: 4-chloro-6-(4-methyl-1H-imidazol-1-yl)pyrimidine (yield: 66%).

[0044] Similarly, by replacing the 4-methylimidazolium in the above steps with 3-methyl-1H-pyrazole or 4-methyl-1H-pyrazole, the corresponding intermediates can be obtained: 4-chloro-6-(3-methyl-1H-pyrazole-1-yl)pyrimidine (1b) and 4-chloro-6-(4-methyl-1H-pyrazole-1-yl)pyrimidine (1c).

[0045] Example 2: Intermediate N Preparation of -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)acetamide (2a)

[0046] Add the following ingredients sequentially to a 50 mL dry reaction flask: N -(3-aminophenyl)acetamide (1.84 g, 12.26 mmol), intermediate 1a (2.00 g, 10.22 mmol), N,N-diisopropylethylamine (DIPEA, 3.6 mL, 20.44 mmol), and N-methylpyrrolidone (NMP, 5 mL). After addition, the mixture was refluxed at 120 °C under nitrogen protection overnight, and the reaction was monitored by TLC until complete. The reaction solution was added to 50 mL of water, and the pH was adjusted to weakly acidic (pH 5-6) with stirring at 0 °C, resulting in the precipitation of a large amount of solid. The mixture was filtered, the filter cake was collected, and dried to obtain a white solid, namely intermediate 2a. N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)acetamide (Yield: 70%)

[0047] Similarly, in the above experimental steps:

[0048] 1) By replacing intermediate 1a with 1b or 1c respectively, the corresponding intermediates can be obtained: N -(3-((6-(3-methyl-1H-pyrazol-1-yl)pyrimidin-4-yl)amino)phenyl)acetamide (2b) and N-(3-((6-(4-methyl-1H-pyrazol-1-yl)pyrimidin-4-yl)amino)phenyl)acetamide (2c).

[0049] 2) N By replacing -(3-aminophenyl)acetamide with N-(4-aminophenyl)acetamide, the intermediate can be prepared: N -(4-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)acetamide (2d).

[0050] Example 3: Intermediate N 1 Preparation of -(6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)phenyl-1,3-diamine (3a)

[0051] Add 2.5 g of intermediate 2a and a methanol-water mixture (30 mL, 1:1, v:v) to a 100 mL reaction flask, and add concentrated hydrochloric acid (5 equiv) dropwise while stirring. After the addition is complete, reflux at 80 °C for 4–6 hours under nitrogen protection, and monitor the reaction for completeness by TLC. Add the reaction solution to 50 mL of water, and adjust the pH to weakly alkaline (pH 7–8) while stirring at 0 °C, resulting in the precipitation of a large amount of solid. Filter, collect the filter cake, and dry to obtain a white solid, i.e., intermediate 3a. N 1 -(6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)phenyl-1,3-diamine (yield: 75%).

[0052] Similarly, in the above experimental steps, replacing intermediate 2a with 2b, 2c, or 2d respectively will yield the corresponding intermediates: N 1 -(6-(3-methyl-1H-pyrazol-1-yl)pyrimidin-4-yl)phenyl-1,3-diamine (3b) N 1 -(6-(4-methyl-1H-pyrazol-1-yl)pyrimidin-4-yl)phenyl-1,3-diamine (3c) and N 1 -(6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)phenyl-1,4-diamine (3d).

[0053] Example 4: Preparation of target products PBA1-27, PBB1-3, PBC1-3 and PBD1-3

[0054] In a dry 25 mL reaction flask, DMF-DCM (10 mL, 1:1, v:v), the corresponding carboxylic acid (1 equiv), and HATU (2 equiv) were added sequentially at room temperature, and the mixture was stirred and activated for 5 minutes. Subsequently, intermediate amines (3a, 3b, 3c, or 3d, 1 equiv) and N,N-diisopropylethylamine (DIPEA, 3 equiv) were added to the reaction mixture, and the reaction was carried out at room temperature for 3–8 hours. After TLC monitoring of the reaction equilibrium, 15 mL of water was added, and the mixture was concentrated under reduced pressure to remove DCM, precipitating a solid. The solid was washed with a methanol-water mixture (1:1, v / v) and dried to obtain the corresponding target products: PBA1-27 (based on intermediate 3a), PBB1-3 (based on intermediate 3b), PBC1-3 (based on intermediate 3c), and PBD1-3 (based on intermediate 3d).

[0055] The target products PBA1-27, PBB1-3, PBC1-3, and PBD1-3 are respectively: N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)hexanoamide (PBA1), N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)octamide (PBA2) N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)cyclopentaneformamide (PBA3) N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)cyclohexaneformamide (PBA4), 1-methyl- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)piperidine-4-carboxamide (PBA5) N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)-3-morpholinopropionamide (PBA6) N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)-3-phenylpropionamide (PBA7) N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)-4-phenylbutyramide (PBA8), 3-(3-bromophenyl)- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)propionamide (PBA9), 3-(4-bromophenyl)- N-(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)propionamide (PBA10) N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)cinnamamide (PBA11), (E)-3-(3-bromophenyl)- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)acrylamide (PBA12), (E)-3-(3-fluorophenyl)- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)acrylamide (PBA13), (E)-3-(3-chlorophenyl)- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)acrylamide (PBA14), (E)- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)-3-(m-tolyl)acrylamide (PBA15), (E)-3-(3-methoxyphenyl)- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)acrylamide (PBA16), (E)- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)-3-(3-nitrophenyl)acrylamide (PBA17), (E)- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)-3-(p-tolyl)acrylamide (PBA18), (E)-3-(4-methoxyphenyl)- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)acrylamide (PBA19), (E)- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)-3-(o-tolyl)acrylamide (PBA20), (E)-3-(2-methoxyphenyl)- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)acrylamide (PBA21), (E)-3-(2,6-dichlorophenyl)- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)acrylamide (PBA22) N-(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)-3-(trifluoromethyl)benzamide (PBA23), 6-bromo- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)pyridinecarboxamide (PBA24), (2E,4E)- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)-5-phenylpentan-2,4-dieneamide (PBA25), (E)-3-(benzo[d][1,3]dioxacyclopenten-5-yl)- N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)acrylamide (PBA26) N -(3-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)-2-(thiophen-2-yl)acetamide (PBA27), (E)-3-(3-bromophenyl)- N -(3-((6-(3-methyl-1H-pyrazol-1-yl)pyrimidin-4-yl)amino)phenyl)acrylamide (PBB1), (E)- N -(3-((6-(3-methyl-1H-pyrazol-1-yl)pyrimidin-4-yl)amino)phenyl)-3-(m-tolyl)acrylamide (PBB2) N -(3-((6-(3-methyl-1H-pyrazol-1-yl)pyrimidin-4-yl)amino)phenyl)-3-(trifluoromethyl)benzamide (PBB3), (E)-3-(3-bromophenyl)- N -(3-((6-(4-methyl-1H-pyrazol-1-yl)pyrimidin-4-yl)amino)phenyl)acrylamide (PBC1), (E)- N -(3-((6-(4-methyl-1H-pyrazol-1-yl)pyrimidin-4-yl)amino)phenyl)-3-(m-tolyl)acrylamide (PBC2) N -(3-((6-(4-methyl-1H-pyrazol-1-yl)pyrimidin-4-yl)amino)phenyl)-3-(trifluoromethyl)benzamide (PBC3), (E)-3-(3-bromophenyl)- N -(4-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)acrylamide (PBD1), (E)- N -(4-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)-3-(m-tolyl)acrylamide (PBD2) N-(4-((6-(4-methyl-1H-imidazol-1-yl)pyrimidin-4-yl)amino)phenyl)-3-(trifluoromethyl)benzamide (PBD3).

[0056] Table 1. Structure and proton NMR spectrum of the compounds described in this invention 1 ¹H NMR and high-resolution mass spectrometry (HRMS)

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] Example 5: MTT assay to evaluate the anti-lung cancer cell activity of the compound of the present invention.

[0069] 1) Cell line and culture conditions

[0070] Human non-small cell lung cancer cell lines H460, H1975, and A549, human bronchial epithelial cells HBE, and human embryonic hepatocytes HHL-5 were all cultured in RPMI-1640 medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) at 37 ℃ in a 5% CO2 saturated humidity incubator.

[0071] 2) Compound preparation

[0072] The test compound was dissolved in DMSO to prepare a 10 mM stock solution, which was stored at -20 °C for later use. During the experiment, the solution was diluted with culture medium to the required concentration, with the final DMSO concentration below 0.1%.

[0073] 3) Initial screening: Inhibitory effect of the compound at a concentration of 10 μM on lung cancer cell proliferation.

[0074] Logarithmic-phase cells were seeded into 96-well plates (~4000 cells / well) and cultured overnight to allow cell adhesion. The test compound was added to a final concentration of 10 μM, with three replicates per group. A proportionally diluted DMSO was used as a control. After 48 h of culture, 10 μL of MTT solution (5 mg / mL, prepared in PBS, pH 7.4) was added to each well, and incubation continued for another 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well. The plates were shaken slowly for 10 min to fully dissolve the formazan. The absorbance (OD) was measured at 490 nm using a microplate reader. Experimental data are expressed as follows: Cell viability was calculated. The results (Table 2) showed that several compounds significantly inhibited the proliferation of lung cancer cells at a concentration of 10 μM. In H460 cells, the cell viability after treatment with compounds PBA2, PBA5, PBA12, PBA13, PBA14, and PBA15 were 14.21±0.43%, 16.17±1.89%, and 19.04±3.71%, respectively. The effective rates of PBA12, PBA13, PBA14, and PBA15 after treatment with H1975 were 11.36±3.66%, 20.48±0.25%, 14.99±1.60%, and 8.65±0.75%, respectively. In A549 cells, the survival rates after treatment with PBA12, PBA15, PBA13, and PBA14 were 31.45±5.10%, 37.10±2.59%, 30.74±1.42%, and 31.25±3.40%, respectively. In summary, PBA12, PBA13, PBA14, and PBA15 showed superiority over the positive control group 12A (a previously reported anticancer active macrovesicle cell death inducer). [1] It exhibits anti-lung cancer proliferation activity.

[0075] Reference [1]: Wu J., Hu HY, Ao MT, et. al . Design, synthesis, and biological evaluation of 5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-Indole-2-Carbohydrazide derivatives: the methuosis inducer 12A as a Novel and selective anticancer agent. Journal of Enzyme Inhibition and MedicinalChemistry, 2021, 36(1): 1436-1453.

[0076] 4) The preferred compound's half-maximal inhibitory concentration (IC50) for inhibiting lung cancer cell proliferation. 50 ) Measurement

[0077] The MTT assay was used to test the inhibition rates of the selected compounds PBA12, PBA14, and PBA15 after treatment with H460, H1975, and A549 cells at different concentration gradients for 24 h, 48 h, or 72 h. The IC50 was calculated using GraphPad Prism software. 50 The values ​​are shown in Table 3.

[0078] After 72 h of treatment, the IC50 values ​​of PBA12, PBA14, and PBA15 on H460 cells were [not specified]. 50 The IC values ​​are 1.29 ± 0.49 µM, 1.98 ± 0.11 µM, and 6.62 ± 0.82 µM, respectively. The IC values ​​for PBA12 and PBA15 are... 50 The concentration was less than 2 µM, indicating strong inhibitory activity. Subsequently, PBA12 and PBA15 were selected as preferred compounds, and their inhibitory effects on different lung cancer cells and normal human cells were tested (Table 2). The IC50 values ​​of PBA12 after 48 h of treatment with H460, H1975, and A549 cells were... 50 The values ​​were all less than 4.5 μM, and the inhibitory activity was significantly stronger than that of PBA15 (IC50). 50 >6 µM). In normal cytotoxicity evaluation, PBA12 and PBA15 showed little inhibitory effect on HBE and HHL-5 cells, IC50 >6 µM. 50 (48 h) > 25 µM and IC 50 (72 h) > 14 µM.

[0079] Table 2. Effects of 10 μM compound on lung cancer cell proliferation

[0080]

[0081]

[0082] When three or more vacuoles appear in a single cell, the cell is considered to have vacuolated: vacuolation rate = vacuolated cells in the field of view / all cells × 100%; effect of vacuolation: -, none; ,weak; ,middle; ,powerful)

[0083] Table 3. Inhibitory effects of preferred compounds on human lung cancer cells and normal human cells (IC50) 50 (µM)

[0084]

[0085] Example 6: Evaluation of the effect of compounds on inducing vacuolization in H460 cells

[0086] To evaluate the ability of compounds to induce vacuolation in tumor cells, H460 cells were treated with a working concentration of 1 μM for 24 hours, and cell morphological changes were then observed under a microscope. Cells with three or more vacuoles in their cytoplasm were defined as vacuolated cells. The vacuolation rate was calculated by counting the proportion of vacuolated cells to the total number of cells in the field of view. The vacuolation induction effect of each compound was comprehensively evaluated by considering the number and size of intracellular vacuoles. The results are shown in Table 2. All tested compounds induced vacuolation in H460 cells, with most compounds showing better cell vacuolation rates (>90%) and vacuole area ratios (>50%) than the positive compound 12A. Further studies showed that the representative compound PBA12 can induce cell vacuolation in a time- and concentration-dependent manner. Figure 1 ).

[0087] Example 7: Heat Transfer Experiment

[0088] To investigate the interaction between the selected compounds and Nur77 protein, a thermomigration assay was used. H460 cells were treated with 1 µM PBA12, PBA15, or DMSO for 6 h, respectively. After washing with PBS, cells were collected in EP tubes and centrifuged at 300 g for 3 min. The supernatant was discarded, and 1.5 mL of lysis buffer containing 250 mM NaCl was added. Lysis was performed on ice for 2 h. Subsequently, the cells were centrifuged at 12000 g for 10 min, and the supernatant was aliquoted into 200 µL PCR tubes. Different temperature programs were set in the PCR instrument, and incubation was performed at different temperature gradients for 3 min. After incubation at room temperature for 3 min, the cells were centrifuged at 12000 g for 10 min. The supernatant was added to loading buffer, heated for denaturation, and then subjected to Western blotting for detection. The experimental results are shown below. Figure 2 As shown in the figure, the experimental results indicate that Nur77 protein in each group degraded to varying degrees with increasing temperature; however, compared with the DMSO control group, the thermal stability of Nur77 was significantly improved after treatment with PBA12 or PBA15. This result suggests that PBA12 and PBA15 can bind to Nur77, thereby enhancing the thermal stability of the protein.

[0089] Example 8: Reporter gene experiment of preferred compounds

[0090] 293T cells in logarithmic growth phase were seeded into 48-well plates and cultured overnight. Each well was co-transfected with 60 ng pBind-Nur77-LBD and 60 ng pG5-Luciferase plasmids, and cultured for another 24 h. After treatment with the selected compound for 24 h, the supernatant was discarded, and 50 μL of 1×Lysis buffer was added to each well. The cells were then incubated at -80°C overnight for lysis. The activities of Firefly and Renilla luciferases were measured using a dual-luciferase reporter assay kit (Promega, #E1910), and the ratio of these two activities was plotted. See [link to documentation]. Figure 3 (a) The results showed that, similar to the positive control drug triptolide, compounds PBA12 and PBA15 significantly inhibited the transcriptional activity of Nur77.

[0091] Example 9: Regulatory effect of preferred compounds on Nur77 protein expression

[0092] The effect of the selected compounds on Nur77 protein levels was detected by Western blotting. H460 cells were treated with 1 μM and 2 μM PBA12 or PBA15 for 24 hours, respectively. Cells were collected and lysed, and the whole-cell lysates were analyzed by Western blotting. The results are shown below. Figure 3 As shown in (b). Experimental results show that the preferred compounds PBA12 and PBA15 can both downregulate the protein expression level of Nur77.

[0093] Example 10: Preferred compounds induce vacuolization and inhibit proliferation of lung cancer cells in a Nur77-dependent manner.

[0094] To investigate the dependence of the preferred compound on Nur77, control cells (siCtrl) and Nur77 knockdown cells (siNur77) H460 were treated with DMSO, 1 μM PBA12, and 2 μM PBA15, respectively. The cells were observed and photographed under a microscope after 2 hours. Results are as follows: Figure 4 As shown in (a), both PBA12 and PBA15 (after 2 hours of treatment) significantly induced vacuolation in siCtrl H460 cells; however, neither induced vacuolation in siNur77 H460 cells. This indicates that PBA12 and PBA15 induce vacuolation in H460 cells in a Nur77-dependent manner.

[0095] The inhibitory effect of the selected compounds on the proliferation of siCtrl H460 and siNur77 H460 cells was further detected using the MTT assay. See [link to MTT assay]. Figure 4In Figure (b), the experimental results showed that the IC50 value of PBA12 after 72 hours of treatment was 1.47 ± 0.22 μM in siCtrl H460 cells and 2.63 ± 0.08 μM in siNur77 H460 cells; the IC50 value of PBA15 after 72 hours of treatment was 4.99 ± 0.01 μM in siCtrl H460 cells and 16.55 ± 1.87 μM in siNur77 H460 cells. These results indicate that compounds PBA12 and PBA15 inhibit lung cancer cell proliferation in a Nur77-dependent manner.

[0096] Example 11: Preferred compound PBA12 induces macrovesicular death in lung cancer cells

[0097] To investigate the effects of different cell death pathway inhibitors on PBA12-induced vacuolization in lung cancer cells, H460 cells were treated with 50 μM working concentration of each of the following inhibitors: Z-VAD-FMK (a caspase inhibitor), Necrostatin-1 (Nec-1), chloroquine (CQ), 3-methyladenine (3-MA), and EIPA (a macropinocytosis inhibitor). Cell morphology was observed and recorded under a microscope after 2, 4, 6, and 8 hours of treatment. Figure 5 The results showed that EIPA completely inhibited PBA12-induced vacuolation within 2h, 4h, and 6h, with only a small number of vacuoles appearing after 8 hours; CQ also significantly reduced vacuole volume and number. In contrast, 3-MA, Z-VAD-FMK, and Nec-1 had no significant inhibitory effect on PBA12-induced vacuolation. These results indicate that PBA12-induced vacuolation is closely related to macropinocytosis, but not to apoptosis, autophagy, or necroptosis.

[0098] To further confirm whether PBA12 induces macrovesicle death by intervening in macropinocytosis, transmission electron microscopy was used to observe the cell ultrastructure. H460 cells were seeded in 60 mm culture dishes, and when the cell density reached 70%, they were treated with 0.3 μM PBA12 for 24 hours. Cells were collected, washed with PBS, fixed with 2.5% glutaraldehyde, and electron microscopy sections were prepared for observation and photography. Figure 6 As shown, numerous single-membrane-enclosed vacuoles were observed within the cells. No damaged organelles were seen within the vacuoles, the nucleus was not shrunken, the mitochondria (red arrows) were morphologically normal, and the endoplasmic reticulum (yellow arrows) was not swollen. These morphological characteristics are consistent with the typical manifestations of macrovesicular cell death, suggesting that PBA12 can induce macrovesicular cell death in lung cancer cells.

[0099] Example 12: Preferred compound PBA12 inhibits autophagy in lung cancer cells

[0100] Macropinocytosis and autophagy form a synergistic regulatory network in tumor cells, jointly maintaining nutritional homeostasis and stress adaptation. Studies have shown that aberrant activation of the macropinocytosis pathway can induce macrovesicular cell death and may be accompanied by changes in autophagic flux. To investigate the effect of PBA12 on autophagy during PBA12-induced macrovesicular cell death, H460 cells were treated with different concentrations (0.625, 1.25, 2.50 μM) of PBA12 for 24 hours. Cell lysates were collected, and the expression changes of autophagy and apoptosis-related proteins were detected by Western blot. The results are as follows: Figure 7 (a) Experimental results showed that PBA12 at concentrations of 2.50 μM and below did not significantly induce PARP cleavage, indicating that apoptosis was not triggered. Simultaneously, the expression levels of Beclin-1, a key protein regulating autophagosome formation and maturation, and STX17, involved in autophagosome-lysosome fusion, did not change significantly. However, PBA12 treatment significantly upregulated the expression of the lysosome-associated membrane protein LAMP1, accompanied by increased levels of the autophagy marker LC3-II and accumulation of the autophagy substrate p62, suggesting increased autophagosome formation but inhibited degradation, thus suppressing autophagic flux. These results indicate that PBA12, while inducing macrovesicular cell death, did not activate apoptosis but significantly inhibited autophagy.

[0101] Example 13: Preferred compound PBA12 induces macrovesicle death via the AMPK signaling pathway

[0102] To investigate the molecular mechanism of PBA12-induced vesicular cell death, H460 cells were treated with different concentrations (0.3125 μM, 0.625 μM, 1.25 μM, and 2.50 μM) of PBA12 for 24 hours. Cell lysates were collected, and changes in protein phosphorylation levels in vesicular cell death-related signaling pathways were detected by Western blot. The results are as follows: Figure 7 As shown in (b), PBA12 treatment had no significant effect on the phosphorylation level of mTOR, but significantly upregulated JNK phosphorylation (≥ 0.625 μM) and AMPK phosphorylation (≥ 0.3125 μM).

[0103] Further intervention experiments were conducted using specific inhibitors. The results were as follows: Figure 7 As shown in (c), the JNK inhibitor SP600125 (10 μM) had no significant inhibitory effect on PBA12-induced vacuolation; while the AMPK inhibitor Dorsomorphin (20 nM) significantly inhibited PBA12-induced vacuolation. (See also...) Figure 7 (d); and simultaneously significantly attenuated the inhibitory effect of PBA12 on the proliferation of lung cancer cells, see [reference]. Figure 7(e) and (f). The above results indicate that PBA12 induces giant bubble death by activating the AMPK signaling pathway.

[0104] Example 14: Inhibitory effect of preferred compounds on lung cancer cell colony formation and migration.

[0105] The colony formation assay was used to evaluate the long-term inhibitory effect of the selected compounds on the proliferation of H460 cells. H460 cells were seeded in 12-well plates (~500 cells / well) and cultured for 24 hours. Different concentrations of PBA12 (0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM) or PBA15 (0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM) were added for further culture for 10 days. The culture medium was discarded, and the cells were washed three times with PBS and fixed with methanol at room temperature for 15 minutes. The methanol was discarded, and the cells were washed three more times with PBS and air-dried. The cells were then stained with 0.5% crystal violet solution for 10-15 minutes. After discarding the stain, the cells were washed with ultrapure water to remove the background color. Colony formation was observed and photographed under a microscope. Results are shown below. Figure 8 As shown in (a), both PBA12 and PBA15 inhibited H460 cell colony formation in a concentration-dependent manner.

[0106] The effect of the selected compound PBA12 on cell migration ability was further investigated using a scratch assay. H460 cells were seeded in 6-well plates and cultured to a density of approximately 95%. Uniform scratches were made on the cell monolayer using a 1 mL pipette tip, and DMSO and different concentrations (1 μM and 2 μM) of PBA12 were added for treatment. After 24 hours of culture, the healing of the scratches was observed and photographed under a microscope. The results are as follows: Figure 8 As shown in (b), compared with the DMSO control group, the PBA12 treatment group showed significantly slower scratch healing, indicating that PBA12 can effectively inhibit the migration ability of H460 cells.

[0107] Example 15: The preferred compound PBA12 can inhibit the growth of lung cancer H460 cell xenograft tumors in nude mice.

[0108] Ten female BALB / c nude mice aged 4-6 weeks were selected and housed in the SPF-grade animal facility of the Experimental Animal Center of Xiamen University. After one week of acclimatization, H460 cells in the logarithmic growth phase were collected and prepared into a concentration of 1.5 × 10⁻⁶ cells. 6 Cell suspensions were inoculated subcutaneously into the right anterior axillary region of nude mice. Tumors were inoculated when they grew to approximately 80–100 mm in size. 3The tumor volume was calculated using the formula: length × width × width / 2. Tumor-bearing mice were randomly divided into a control group and a treatment group, with 5 mice in each group, ensuring that the average tumor volume was consistent across groups. Mice in the treatment group received a daily intraperitoneal injection of PBA12 solution (25 mg / kg, 200 μL), while mice in the control group received an equal volume of blank solvent. Treatment continued for 14 days, and the length, width, and weight of the tumors were measured and recorded daily. After treatment, nude mice were euthanized by cervical dislocation, and the subcutaneous tumor tissue was removed, weighed, and photographed. On day 14 after treatment, nude mice were euthanized by cervical dislocation, and the subcutaneous tumor was removed, weighed, and photographed for comparison. The tumor inhibition rate (TGI) was calculated using the formula: TGI (%) = (1 - (T - T0) / (V - V0) × 100%), where T and V are the average tumor volumes on day 14 for the treatment group and the control group, respectively, and T0 and V0 are the average tumor volumes on day 1 for the treatment group and the control group, respectively.

[0109] In addition, major organs such as the heart, liver, spleen, lungs, and kidneys, as well as some tumor tissues, were isolated, fixed with 4% paraformaldehyde, and then subjected to H&E staining to assess the in vivo toxicity of the compound. The remaining tumor tissues were used for immunohistochemical detection and protein lysis buffer preparation, and the expression of related proteins was analyzed by Western blotting.

[0110] Experimental results are as follows Figure 9 As shown: PBA12 (25 mg / kg) significantly inhibited the growth of H460 cell xenograft tumors. See [link to relevant documentation]. Figure 9 In the treatment group (a~c), the tumor inhibition rate reached 77%; compared with the control group, the expression of cell proliferation marker PCNA and target protein Nur77 in tumor tissue was significantly downregulated, see [reference needed]. Figure 9 (d); No significant weight loss was observed in mice during the administration period, see [reference needed]. Figure 9 In the middle (e), there was no obvious damage to the major organs, see Figure 9 (f)

Claims

1. A kind N -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivative, characterized in that, Its structural formula is: Wherein, R1 is 4-methyl-1H-imidazol-1-yl, 3-methyl-1H-pyrazol-1-yl or 4-methyl-1H-pyrazol-1-yl; R2 is a straight-chain alkyl, cycloalkyl, 1-methylpiperidin-4-yl, morpholinylethyl, substituted or unsubstituted phenyl, substituted or unsubstituted styryl, substituted or unsubstituted phenethyl, phenylpropyl, phenylbutenyl, 6-bromopyridin-2-yl, (benzo[d][1,3]dioxacyclopenten-5-yl)vinyl or (thiophen-2-yl)methyl.

2. The one described in claim 1 N -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivative, characterized in that, Includes at least one of the following compounds: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 3. The claim 1 or 2 N Use of -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivatives in the preparation of Nur77-related tumor therapeutic compositions.

4. The use as described in claim 3, characterized in that, The N -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivatives are selected from at least one of the following compounds: 、 、 、 、 。 5. The use as described in claim 4, characterized in that, The N -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivatives are: 。 6. The use as described in any one of claims 3 to 5, characterized in that: The Nur77-related tumor is lung cancer.

7. A Nur77-related tumor therapeutic composition, characterized in that: Including those described in claims 1-2 N -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivative.

8. The Nur77-related tumor therapeutic composition as described in claim 7, characterized in that: The N -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivatives are selected from at least one of the following compounds: 、 、 、 、 。 9. A Nur77-related tumor therapeutic composition as described in claim 8, characterized in that, The N -(3 / 4-((6-substituted pyrimidin-4-yl)amino)phenyl)amide derivatives are: 。 10. The Nur77-related tumor therapeutic composition according to any one of claims 7 to 9, characterized in that: The Nur77-related tumor is lung cancer.

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