An N-aryl imidazole compound and its application in the treatment of colorectal cancer

Abstract

This invention belongs to the pharmaceutical field, specifically disclosing an N-arylimidazolium compound and its application in the treatment of colorectal cancer. This invention provides a compound having the structure shown in formula (A) or formula (B), which specifically inhibits the phosphorylation of p38α MAPK and the activation of its downstream HSP27, thereby blocking the MAPK signaling pathway. Specifically, compound CIN-3 exhibits significant inhibitory activity against HT-29 and HCT-116 colon cancer cells, inducing G1 / S phase arrest in tumor cells, inhibiting DNA synthesis, downregulating migration ability, and inducing apoptosis. In a CT26 mouse homologous xenograft model, a dose of 30 mg / kg of CIN-3 achieved a tumor inhibition rate of 82.1±2.15%, with good safety, demonstrating promising drug development potential and providing a new candidate drug for targeted therapy of colorectal cancer.

Description

Technical Field This invention relates to the field of biomedicine, specifically to an N-arylimidazolium compound and its application in the treatment of colorectal cancer. Background Technology

[0001] Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies worldwide, with persistently high incidence and mortality rates, posing a serious threat to human life and health. According to statistics from the International Agency for Research on Cancer (IARC) in 2025, there were approximately 18.7 million new cancer cases and 9.7 million deaths globally in 2022; colorectal cancer accounted for 10% of new cases and 9% of deaths, ranking third in incidence and second in mortality among malignant tumors. Despite some progress in clinical diagnosis and treatment of colorectal cancer (such as screening, surgery, and radiotherapy / chemotherapy), the prognosis for patients with advanced stages remains poor, with high rates of recurrence and distant metastasis. Furthermore, existing treatment regimens generally suffer from strong drug resistance and significant side effects, greatly limiting the improvement of efficacy. Therefore, developing drugs with anti-colon cancer effects not only has significant clinical value but also provides new treatment strategies for colorectal cancer.

[0002] Targeted therapy is a precise treatment strategy that acts on specific molecular targets of tumor cells. Related drugs can specifically bind to tumor cells and inhibit their growth and metastasis by blocking proliferation signaling pathways. Identifying and intervening in the core signaling pathways driving tumor proliferation is the core of targeted therapy research. p38 mitogen-activated protein kinase (MAPK), as an important member of the MAPK family, is widely involved in the regulation of growth, development, and pathological homeostasis, and is closely related to physiological processes such as cell cycle, apoptosis, gene expression, and stress response. There are four subtypes of p38 MAPK (p38α, p38β, p38γ, and p38δ), among which p38α has been studied most extensively and has been proven to be closely related to various malignant tumors, making it a highly promising therapeutic target. Summary of the Invention

[0003] Based on pharmacophore modeling and molecular docking analysis, this invention rationally designed and synthesized two series of 32 N-arylimidazolium compounds; the specific synthetic routes are as follows. Figure 1 As shown; all synthesized compounds were confirmed by 1H NMR, 1C NMR, and mass spectrometry, and their purity was determined by high-performance liquid chromatography. The in vitro cytotoxicity of the compounds against HT-29, HCT116, and normal cell lines was evaluated using the MTT assay, and the half-maximal inhibitory concentration (IC50) was calculated. 50 The compound exhibits enhanced biological activity through structural modification, particularly in its inhibitory effect on the p38 MAPK signaling pathway. Specifically, it includes the following: In a first aspect, the present invention provides an N-arylimidazolium compound or a pharmaceutically acceptable salt thereof, said compound having the structure shown in formula (A) or formula (B):

[0004] Formula (A);

[0005] Formula (B); Wherein, R1 is selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted naphthyl; the substituted substituent is selected from halogen, hydroxyl, hydroxymethyl, trifluoromethyl, methoxy; R2 is selected from piperazine-1-methyl ketone, piperazine-1-sulfonone, and 1-(piperazine-1-yl)ethyl-1-one.

[0006] Preferably, R1 is selected from phenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 3,5-dimethoxyphenyl, 2,4-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 4-hydroxymethylphenyl, pyridine, pyrimidine, naphthyl, 6-hydroxynaphthyl, and 6-methoxynaphthyl.

[0007] Preferably, the compound has the structure shown in formula (A); wherein R1 is selected from phenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 3,5-dimethoxyphenyl, 2,4-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 4-hydroxymethylphenyl, pyridine, pyrimidine, naphthyl, 6-hydroxynaphthyl, 6-methoxynaphthyl.

[0008] Preferably, the compound is a compound with any of the following structures: , , , , , , , , , , , , , , , , , , , , , , .

[0009] Preferably, the compound is: .

[0010] Preferably, the compound has the structure shown in formula (B); wherein R1 is selected from naphthyl, 6-methoxynaphthyl, and 2,4-dimethoxyphenyl.

[0011] Preferably, the compound is a compound with any of the following structures: , , , , , , , , .

[0012] In a second aspect, the present invention provides the use of the N-arylimidazolium compounds described in the first aspect above, or pharmaceutically acceptable salts thereof, in the preparation of p38α MAPK inhibitors.

[0013] Thirdly, the present invention provides the use of the N-arylimidazolium compounds or pharmaceutically acceptable salts thereof described in the first aspect above in the preparation of antitumor drugs.

[0014] Preferably, the tumor is colorectal cancer.

[0015] Fourthly, the present invention provides a method for preparing the N-arylimidazolium compounds described in the first aspect above, wherein the synthetic route of the N-arylimidazolium compounds is as follows: , Where i is CDI, DCM, rt 4h; ii is Fe, NH4Cl, H2O, 80℃, reflux 1h; iii is HCTU, Et3N, DMF, rt 20h; iv is Cu(AcO)2, Et3N, DMF, 80℃, O2, reflux 3h; or, , Where i is HCTU, DMF, Et3N, rt 20h; ii is LiOH·H2O, MeOH / H2O, HCl; iii is EDCI, HoBt, DIPEA, DCM; iv is Cu(AcO)2, DMF, Et3N, 80℃, O2, reflux 3h.

[0016] The beneficial effects of this invention are as follows: This invention constructed two series of 32 N-arylimidazolium compounds via Chan-Lam coupling and amide condensation reactions, and their structures were confirmed by spectroscopic analysis. Bioactivity tests showed that these compounds specifically inhibited the phosphorylation of p38α MAPK and the activation of its downstream HSP27, thereby blocking the MAPK signaling pathway. Among them, the representative compound CIN-3 exhibited significant inhibitory activity against the proliferation of HT-29 and HCT-116 colon cancer cells. Further mechanistic studies showed that CIN-3 can induce G1 / S phase arrest in tumor cells, inhibit DNA synthesis, downregulate migration, and induce apoptosis. In the CT26 mouse homologous xenograft model, a dose of 30 mg / kg of CIN-3 achieved a tumor inhibition rate of 82.1±2.15%, and safety evaluation showed a high safety window. The compounds of this invention have novel structures, excellent activity, and good selectivity, showing promising drug development prospects and providing new candidate drugs for targeted therapy of colorectal cancer. Attached Figure Description

[0017] Figure 1 Synthetic routes for N-arylimidazolium compounds.

[0018] Figure 2 Representative images of colony formation in HT-29 cells treated with different concentrations of CIN-3 or the positive control SB203580, and quantitative results of relative colony numbers.

[0019] Figure 3 Compound CIN-3 inhibited the proliferation of HT-29 cells and induced G0 / G1 phase arrest; A shows the EdU assay for DNA synthesis: Hoechst 33342 stained cell nuclei (blue), EdU-labeled newly synthesized DNA (green), combined image (scale bar = 100 μm); B shows a representative histogram of cell cycle distribution analyzed by flow cytometry; C shows the quantitative analysis of the proportion of EdU-positive cells; D shows the stacked bar graph of cell distribution in G0 / G1, S, and G2 / M phases in each group; data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, and ***p<0.001 compared with the control group.

[0020] Figure 4Effects of compound CIN-3 on the migration ability and MMP-9 protein expression of HT-29 cells; where A represents the wound closure of HT-29 cells (magnification: 10×, scale bar = 100 μM); B represents the transmembrane migration ability of HT-29 cells in each group as assessed by Transwell migration assay (crystal violet staining, magnification: 10×, scale bar = 100 μm); C represents the quantitative analysis of the wound healing rate of cells in each group at 24 and 48 hours; D represents the quantitative statistics of the relative migration rate of cells in each group; E represents the protein expression level of MMP-9 detected by Western blot and corresponding grayscale quantitative analysis; data are expressed as mean ± standard deviation, and compared with the control group, *p<0.05, **p<0.01 and ***p<0.001.

[0021] Figure 5 Results of Giemsa staining morphological observation.

[0022] Figure 6 Results of Hoechst 33342 / PI double staining fluorescence experiment.

[0023] Figure 7 The results were quantitatively detected by Annexin V-FITC / PI double staining flow cytometry.

[0024] Figure 8 Effects of compound CIN-3 on reactive oxygen species (ROS) levels and mitochondrial membrane potential in HT-29 cells; A shows the intracellular ROS levels in HT-29 cells treated with control, positive control SB203580 (8 μM), and CIN-3 gradient concentrations (4, 6, 8 μM) using the DCFH-DA fluorescent probe (green fluorescence, magnification: 20×, scale bar = 100 μM); B shows the mitochondrial membrane potential (ΔΨm) levels in each group of HT-29 cells detected by the TMRE fluorescent probe (red fluorescence, magnification: 20×, scale bar = 100 μm); C shows the quantitative statistics of intracellular relative ROS content and fluorescence intensity histograms detected by flow cytometry; D shows the quantitative statistics of mitochondrial membrane potential and fluorescence intensity histograms detected by flow cytometry; data are expressed as mean ± standard deviation, and compared with the control group, *p<0.05, **p<0.01, and ***p<0.001.

[0025] Figure 9The regulatory effect of compound CIN-3 on the p38 MAPK signaling pathway in HT-29 cells and target validation; A shows the protein expression levels of p38, p-p38, HSP27, and p-HSP27 in HT-29 cells treated with gradient concentrations of CIN-3 (4, 6, 8 μM) by Western blot, with GAPDH as an internal reference; B shows the gray-scale quantitative analysis of p-p38 / p38 and p-HSP27 / HSP27 protein expression; C shows the 3D binding mode of CIN-3 to p38α protein (PDB ID: 4KIN) simulated by molecular docking; D shows the two-dimensional interaction diagram of amino acid residues of CIN-3 and p38α protein; E shows the hydrophobic surface distribution of the CIN-3 binding capsule; F shows the matching diagram of CIN-3 with the optimal pharmacophore Hypo1.

[0026] Figure 10 The inhibitory effect of compound CIN-3 on the growth of CT26 xenograft tumors in mice; A is the dynamic change curve of tumor volume in each group of mice during the administration period; B is a representative photograph of the dissected tumor tissue in each group at the end of administration; C is the statistical analysis results of tumor weight in each group at the end of administration; D is the change curve of body weight in each group of mice during the administration period. Detailed Implementation

[0027] To better understand the present invention, the following detailed description of the above-mentioned contents of the present invention is provided through specific embodiments. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods.

[0028] Based on pharmacophore modeling and molecular docking analysis, this invention rationally designed and synthesized two series of 32 target compounds. The synthetic routes for the designed compounds are as follows: Figure 1 As shown. Using imidazole and boric acid derivatives as substrates, the key CN link was constructed via a Chan-Lam coupling reaction. For amide bond formation, a conventional synthetic strategy was employed: the carboxyl group was first activated, followed by reaction with an amine substrate to form the corresponding amide. Carbodiimide condensing agents, urea-type condensing agents, and CDI were used to efficiently promote the amide condensation reaction. All synthesized compounds were confirmed by 1H NMR, 1C NMR, and mass spectrometry, and their purity was determined by high-performance liquid chromatography.

[0029] This invention uses the MTT assay to evaluate the in vitro cytotoxicity of the target compound against human cancer cell lines and human normal cell lines, and calculates the half-maximal inhibitory concentration (IC50). 50The cytotoxicity of these compounds varied significantly across different cell lines, exhibiting marked cell line selectivity. Among them, compound CIN-3 inhibited the proliferation of various colorectal cancer cells in a concentration-dependent manner. MTT assays showed that cell viability decreased with increasing drug concentration after treatment at concentrations ranging from 0.625 to 40 μM for 24, 48, and 72 hours. After 48 hours of treatment, CIN-3 showed the strongest activity against HT-29 cells, with an IC50 value of [value missing]. 50 The concentration was 6.05 ± 0.07 μM; at a concentration of 4 μM, cell viability was still retained at approximately 80%. Meanwhile, its IC50 value for normal intestinal epithelial cells was [not specified]. 50 At concentrations above 40 μM, there was almost no cytotoxicity. Based on the results of the MTT cytotoxicity assay, eight compounds with good activity were selected, and their in vitro inhibitory effects on p38α MAPK at a standard concentration of 30 nM were evaluated using the validated ADP-Glo ​​luminescence assay. The IC50 of the positive control drug SB203580 was... 50 The effective molecular weight (VWHM) was 37.69 ± 2.14 nM, and the inhibition rate of p38α MAPK at a concentration of 30 nM was 42.56 ± 0.68%, verifying the stability and reliability of the experimental system. Significant differences were observed in the p38α MAPK inhibitory activities of the eight tested compounds. Compound CIN-3 showed slightly better activity than the positive control, with an inhibition rate of 45.38 ± 1.26% at 30 nM, indicating its good potential as a targeted inhibitor of p38α MAPK.

[0030] This invention uses a colony formation assay to investigate the effect of CIN-3 on the proliferation of HT-29 cells. The blank control group formed numerous large and regular colonies, exhibiting strong proliferative activity. Treatment with different concentrations of CIN-3 decreased colony formation ability in a concentration-dependent manner. At the same concentration, CIN-3 showed a stronger inhibitory effect on colony formation than the positive control SB203580.

[0031] This invention uses the EdU assay to detect the effect of compounds on cellular DNA synthesis. The results showed that as the concentration of CIN-3 increased, the green fluorescent signal indicating DNA synthesis in HT-29 cells gradually weakened and eventually disappeared, indicating that CIN-3 can significantly block cellular DNA replication. Simultaneously, CIN-3 reduced the number and size of cell colonies in a concentration-dependent manner.

[0032] This invention relates to the verification of the activity of compound CIN-3 in inhibiting the proliferation of HT-29 cells. The EdU assay was used to detect the effect of the compound on cellular DNA synthesis. Results showed that as the concentration of CIN-3 increased, the green fluorescent signal indicating DNA synthesis in HT-29 cells gradually weakened and eventually disappeared, indicating that CIN-3 could significantly block cellular DNA replication. Simultaneously, CIN-3 reduced the number and size of cell colonies in a concentration-dependent manner. Flow cytometry analysis using PI staining showed that CIN-3 induced G1 / S phase arrest in HT-29 cells in a dose-dependent manner and significantly increased the proportion of cells in the G0 / G1 phase. Compared with the blank control group, after treatment with 8.0 μM CIN-3, the proportion of HT-29 cells in the G0 / G1 phase increased from 53.98% to 83.81%. At the same concentration, the positive control SB203580 only increased the proportion of cells in the G0 / G1 phase to 63.25%, significantly lower than CIN-3.

[0033] This invention further verified the inhibitory effect of compound CIN-3 on the migration ability of HT-29 cells. This study used wound healing assays and Transwell migration assays to evaluate the effects of compound CIN-3 on the wound healing and directional migration abilities of HT-29 cells, respectively. The wound healing assay results showed that CIN-3 significantly inhibited the wound healing ability of HT-29 cells in a concentration-dependent manner: the healing rate of the blank control group was (45.43±0.98)% at 24 hours after scratching, and almost complete repair at 48 hours, with a healing rate of (84.06±1.72)%. After treatment with 8 μM CIN-3, the cell healing rate decreased to (13.37±1.95)% at 24 hours and to (24.59±2.18)% at 48 hours. Furthermore, at the same concentration, the inhibitory effect of CIN-3 was significantly better than that of the positive control drug.

[0034] This invention verifies that CIN-3 inhibits HT-29 cell apoptosis, exhibiting a concentration-dependent inhibitory effect. Giemsa staining morphological observation showed that after CIN-3 treatment, HT-29 cells exhibited typical apoptotic characteristics, including cell shrinkage and rounding, reduced volume, chromatin condensation or fragmentation, and the nucleus showing a strong bright yellow stain due to chromatin condensation. With increasing drug concentration, cells further exhibited necrotic morphological changes, such as cell swelling, nuclear membrane disintegration, and cell membrane rupture. These results indicate that CIN-3 primarily induces HT-29 cell apoptosis at low to medium concentrations, while triggering cell necrosis at high concentrations.

[0035] This invention verifies that CIN-3 inhibits the abnormal activation of the p38 MAPK pathway by suppressing p38 MAPK phosphorylation and blocking the phosphorylation signal transduction of its downstream substrate HSP27. Western blot was used to detect the protein expression levels of total p38 MAPK, phosphorylated activated p-p38 MAPK and its downstream substrates HSP27 and phosphorylated activated p-HSP27 in each group, with GAPDH as an internal control. Results showed that the blank control group had high expression of p-p38 MAPK and p-HSP27, indicating continuous abnormal activation of the p38 MAPK pathway in HT-29 cells. After treatment with gradient concentrations of CIN-3, the protein expression of total p38 MAPK and total HSP27 did not change significantly, while the expression of p-p38 MAPK and p-HSP27 was significantly downregulated in a concentration-dependent manner.

[0036] This invention employs a fixed-dose method to assess the acute toxicity of CIN-3. Kunming mice were intraperitoneally injected with CIN-3 at doses of 300, 600, and 900 mg / kg. During the observation period, mice in the high-dose group (900 mg / kg) exhibited signs of poisoning, such as decreased activity, rapid breathing, and unsteady gait, but no deaths occurred. Mice in the low-dose group (300 mg / kg) and the medium-dose group (600 mg / kg) showed no signs of poisoning, and their activity, appetite, and weight gain were normal. Weight monitoring showed that the weight of mice in the high-dose group initially decreased and then increased, while there was no significant difference in weight gain between the low / medium-dose groups and the control group. Hematoxylin-eosin (HE) staining results showed that there were no significant pathological changes in organs in the 300 and 600 mg / kg groups compared to the control group; however, the liver of mice in the 900 mg / kg group showed extensive vacuolation, accompanied by edema, loose degeneration, and disordered arrangement of some hepatocytes. The experiment determined that 600 mg / kg is the safe and tolerable dose of CIN-3 in mice.

[0037] A mouse model of syngeneic colorectal cancer xenograft was established by subcutaneously inoculating CT26.WT colon cancer cells into BALB / c mice. In the model control group, tumor volume showed a continuous and rapid growth trend; however, tumor growth slowed in a concentration-dependent manner with increasing CIN-3 dosage. The 30 mg / kg high-dose group showed the most significant tumor growth inhibition, and its antitumor activity was superior to the positive control drug SB203580 at the same dose. After the dosing cycle, tumor tissue was removed, and tumor weight was measured to calculate the tumor inhibition rate: the average tumor weight in the 30 mg / kg CIN-3 group was significantly lower than that in the model control group, with a tumor inhibition rate of (82.1±2.15)%. Throughout the dosing period, no signs of toxicity such as lethargy or abnormal appetite were observed in any group. Weight fluctuations were within the normal range, with a weight change rate of less than 10% in each group, indicating that CIN-3 has no significant systemic toxicity at effective antitumor doses, consistent with the safety conclusions of previous acute toxicity experiments.

[0038] The following detailed description of specific embodiments further illustrates the above-mentioned aspects of the present invention. However, this should not be construed as a limitation of the present invention.

[0039] The cells used in the following examples were HCT-116 cells and HT-29 cells, both purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; the positive control drug was SB203580, purchased from Aladdin. Cytokine kits included: ADP-Glo™ kinase assay kit (commercially available), SFClick™ EdU-488 (Abcells) kit (commercially available), Annexin V-FITC apoptosis assay kit (commercially available), ROS assay kit (commercially available), and BCA protein assay kit (commercially available). Unless otherwise specified, all other reagents and materials were commercially available.

[0040] Example 1: Synthesis of the target compound according to Figure 1 The synthetic route shown is used to synthesize the target compound. Specifically: 1. Synthesis of Class A compounds: Synthesis of intermediate a: 3-nitro-4-methylbenzoic acid (20.0 mmol, 1.0 equiv) and N,N-carbonyldiimidazole (20.0 mmol, 1.0 equiv) were dissolved in 80 mL of DCM and refluxed at 60 °C for 3 h. After the reaction cooled to room temperature, cyclopropylamine (20.0 mmol, 1.0 equiv) was slowly added and stirred at room temperature for 2 h. After the reaction was complete as monitored by TLC, the mixture was extracted three times with ethyl acetate. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated under reduced pressure using a rotary evaporator, and the crude product was purified by column chromatography to obtain intermediate a.

[0041] Synthesis of intermediate b: Intermediate a (10 mmol, 2.0 equiv), NH4Cl (5 mmol, 1.0 equiv), and iron powder (25 mmol, 5.0 equiv) were dissolved in EtOH / H2O (1:1) = 80 mL and refluxed at 80 °C for 1 h. The iron powder was removed by filtration through diatomaceous earth, and the mixture was extracted three times with ethyl acetate. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated under reduced pressure using a rotary evaporator, and the crude product was purified by column chromatography to obtain intermediate b.

[0042] Synthesis of intermediate c: 1H-imidazolium-4-carboxylic acid (2.0 mmol, 1.0 equiv) and HCTU (2.2 mmol, 1.1 equiv) were dissolved in N,N-dimethylformamide (10 mL), followed by the addition of triethylamine (2.4 mmol, 1.2 equiv). The mixture was stirred at room temperature for 1 h, and then intermediate b (2 mmol, 1.0 equiv) was added. The reaction was stirred at room temperature for another 20 h. After the reaction was complete, the mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated under reduced pressure using a rotary evaporator, and the crude product was purified by column chromatography to obtain intermediate c.

[0043] Synthesis of the target compound: Intermediate c (1.2 mmol, 1.2 equiv) was dissolved in 10 mL of N,N-dimethylformamide, and boric acid derivative (1.0 mmol, 1.0 equiv) and copper acetate (0.3 mmol, 0.3 equiv) were added. The reaction mixture was stirred and refluxed at 80 °C for 4 h under an oxygen atmosphere. After cooling to room temperature, the solvent was removed under reduced pressure, and the mixture was extracted three times with ethyl acetate. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated under reduced pressure using a rotary evaporator, and the crude product was purified by column chromatography to obtain the target compound.

[0044] 2. Synthesis of Class B compounds: Synthesis of intermediate d: 1H-imidazolium-4-carboxylic acid (20.0 mmol, 1.0 eq) and HCTU (22 mmol, 1.1 eq) were dissolved in N,N-dimethylformamide (80 mL), followed by the addition of triethylamine (24 mmol, 1.2 eq). The mixture was stirred at room temperature for 1 h, and then ethyl 3-amino-4-methylbenzoate (20 mmol, 1.0 eq) was added. The reaction was stirred at room temperature for another 20 h. After the reaction was complete, the mixture was extracted with EtoAc (3 × 50 mL). The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated under reduced pressure using a rotary evaporator, and the crude product was purified by column chromatography to obtain intermediate d.

[0045] Synthesis of intermediate e: Intermediate d (10 mmol, 1.0 equiv) was dissolved in 20 mL of a mixed solvent of methanol and water (MeOH:H2O = 1:1). Then, LiOH·H2O (30 mmol, 3 equiv) was added to the reaction system. The mixture was refluxed at 80 °C and stirred for 3 h. After cooling to room temperature, methanol was removed from the solvent under reduced pressure. 10 mL of water was added, and the solution was cooled to 0 - 5 °C in an ice-water bath. Dilute hydrochloric acid was added dropwise under continuous and vigorous stirring to adjust the pH of the solution to 3 - 4. The mixture was stirred for another 15 min in an ice-water bath. The mixture was then filtered using a Buchner funnel, and the filter cake was dried in a drying oven to obtain intermediate e.

[0046] Synthesis of intermediate f: In a dry round-bottom flask, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (4.5 mmol, 1.5 equiv), HOBt (4.5 mmol, 1.5 equiv), DIPEA (7.5 mmol, 2.5 equiv), and 25 mL of dichloromethane were added sequentially. The mixture was stirred in an ice bath for 1 h. Then, a piperazine derivative (3.6 mmol, 1.2 equiv) was added to the solution in a single batch. The reaction mixture was allowed to warm to room temperature naturally and stirred overnight. After the reaction was complete, the mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with saturated sodium bicarbonate (NaHCO3) solution, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated under reduced pressure using a rotary evaporator. The crude product was purified by column chromatography to obtain intermediate f.

[0047] Synthesis of the target compound: Intermediate f (1.2 mmol, 1.2 equiv) was dissolved in 10 mL of N,N-dimethylformamide, and boric acid derivative (1.0 mmol, 1.0 equiv) and copper acetate (0.3 mmol, 0.3 equiv) were added. The reaction mixture was stirred and refluxed at 80 °C for 4 h under an oxygen atmosphere. After cooling to room temperature, the solvent was removed under reduced pressure, and the mixture was extracted three times with ethyl acetate. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated under reduced pressure using a rotary evaporator, and the crude product was purified by column chromatography to obtain the target compound.

[0048] The structures of the synthesized target compounds are summarized in Table 1.

[0049] Table 1 Structures of N-arylimidazol compounds

[0050] Example 2: Spectral data of the target compound Compound CMP-1: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-phenyl-1H-imidazolium-4-carboxamide (CMP-1): White solid, yield: 33.8% 1 H NMR (400 MHz, DMSO- d 6) δ 9.58 (s, 1H), 8.45 (dd, J = 12.3, 1.4 Hz, 2H), 8.41 (d, J = 4.3 Hz, 1H), 8.16 (d, J = 1.8 Hz, 1H), 7.79 (d, J = 7.7Hz, 2H), 7.56 (t, J = 7.8 Hz, 3H), 7.43 (t, J = 7.4 Hz, 1H), 7.32 (d, J = 7.9 Hz,1H), 2.90 – 2.81 (m, 1H), 2.32 (s, 3H), 0.72 – 0.65 (m, 2H), 0.61 – 0.54 (m,2H). 13 C NMR (101 MHz, DMSO- d 6) δ 167.63, 160.60, 137.91, 136.81, 136.63,136.51, 134.89, 133.14, 130.45, 130.41, 128.20, 124.07, 123.70, 121.61,121.35, 23.54, 18.08, 6.16. ESI-MS (m / z) : calculated for C 21 H 20 N4O2[M+H] + 361.1586 found 361.1583.The purity was 98.12% by HPLC analysis.

[0051] Compound CMP-2: ; 1-(2-Bromophenyl)-N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1H-imidazolium-4-carboxamide (CMP-2): Brown solid, yield: 46.4% 1 H NMR (400 MHz, DMSO- d6) δ 9.59 (s, 1H), 8.39 (d, J = 4.3 Hz, 1H), 8.16 (d, J = 1.8 Hz, 1H), 8.10 (q, 2H), 7.90 (dd, J = 8.0, 1.3 Hz,1H), 7.67 – 7.61 (m, 1H), 7.60 – 7.55 (m, 2H), 7.54 – 7.47 (m, 1H), 7.32 (d, J = 7.9 Hz, 1H), 2.90 – 2.80 (m, 1H), 2.33 (s, 3H), 0.72 – 0.65 (m, 2H), 0.61 –0.54 (m, 2H). 13 C NMR (101 MHz, DMSO- d 6) δ 167.59, 160.58, 138.81, 136.99,136.61, 136.24, 134.97, 134.06, 133.09, 131.65, 130.45, 129.51, 129.31,124.73, 124.11, 123.75, 119.86, 23.54, 18.11, 6.16.ESI-MS (m / z): calculated for C 21 H 19 BrN4O2[M+H] + 439.0691 found 439.0681.The purity was 98.42% by HPLC analysis.

[0052] Compound CMP-3: ; 1-(3-Bromophenyl)-N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1H-imidazolium-4-carboxamide (CMP-3): Brown solid, yield: 27.3% 1 H NMR (400 MHz, DMSO- d 6) δ 9.56 (s, 1H), 8.51 –8.46 (m, 2H), 8.37 (d, J = 3.9 Hz, 1H), 8.09 (s, 2H), 7.80 (dd, J= 8.0, 1.8 Hz,1H), 7.61 – 7.57 (m, 1H), 7.56 – 7.51 (m, 1H), 7.47 (t, J = 8.1 Hz, 1H), 7.28(d, J = 8.0 Hz, 1H), 2.85 – 2.77 (m, 1H), 2.27 (s, 3H), 0.67 – 0.61 (m, 2H), 0.56 – 0.51 (m, 2H). 13 C NMR (101 MHz, DMSO- d 6) δ 167.59, 160.50, 138.07, 137.99, 136.63, 136.57, 135.10, 133.06, 132.22, 130.90, 130.45, 124.16, 123.91 , 123.87 , 123.09 , 121.64 , 120.25 , 23.54 , 18.11 , 6.17.ESI-MS (m / z): calculated for C 21 H 19 BrN4O2[M+H] + 439.0691 found 439.0678.The purity was 97.21% by HPLC analysis.

[0053] Compound CMP-4: ; 1-(4-Bromophenyl)-N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1H-imidazol-4-carboxamide (CMP-4): Brown solid, yield: 30.8% 1 H NMR (400 MHz, DMSO- d 6) δ 9.59 (s, 1H), 8.48 (dd, J = 11.2, 1.4 Hz, 2H), 8.41 (d, J = 4.3 Hz, 1H), 8.13 (d, J = 1.8 Hz, 1H), 7.77(d, J = 21.8 Hz, 4H), 7.57 (dd, J = 7.9, 1.9 Hz, 1H), 7.32 (d, J= 8.0 Hz, 1H), 2.89 – 2.81 (m, 1H), 2.35 (s, 3H), 0.71 – 0.66 (m, 2H), 0.60 – 0.55 (m, 2H). 13 C NMR (101 MHz, DMSO- d 6) δ 167.59, 160.51, 138.02, 136.57, 136.54, 135.04, 133.18, 133.08, 130.44, 124.13, 123.82, 123.36, 121.60 ,120.72 , 23.54 , 18.10 , 6.17 .ESI-MS (m / z) : calculated for C 21 H 19 BrN4O2[M+H] + 439.0691 found439.0684.The purity was 99.12% by HPLC analysis.

[0054] Compound CMP-5: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(2-hydroxyphenyl)-1H-imidazolium-4-carboxamide (CMP-5): White solid, yield: 29.7% 1 H NMR (400 MHz, DMSO- d 6) δ 10.46 (s, 1H), 9.52 (s, 1H), 8.39 (d, J = 4.3 Hz, 1H), 8.18 (d, J = 1.8 Hz, 1H), 8.09 (dd, J = 15.0, 1.3Hz, 2H), 7.56 (dd, J = 7.9, 1.8 Hz, 1H), 7.46 (dd, J = 7.9, 1.6 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.28 (dd, J = 7.8, 1.6 Hz, 1H), 7.11 (d, J = 8.1 Hz, 1H), 6.96(t, J= 7.6 Hz, 1H), 2.88 – 2.82 (m, 1H), 2.32 (s, 3H), 0.71 – 0.65 (m, 2H), 0.60 – 0.55 (m, 2H). 13 C NMR (101 MHz, DMSO- d 6) δ 167.67, 160.78, 138.40,136.71, 136.40, 134.61, 133.15, 130.44, 125.88, 124.57, 124.00, 123.45,117.61, 23.54, 18.06, 6.17.ESI-MS (m / z) : calculated for C 21 H 20 N4O3[M+H] + 377.1535 found 377.1520.The purity was 97.83% by HPLC analysis.

[0055] Compound CMP-6: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(3-hydroxyphenyl)-1H-imidazolium-4-carboxamide (CMP-6): White solid, yield: 33.8% 1 H NMR (400 MHz, DMSO- d 6) δ 9.98 (s, 1H), 9.56 (s, 1H), 8.40 (d, J = 4.2 Hz, 2H), 8.33 (d, J = 7.0 Hz, 2H), 8.16 (d, J = 1.5 Hz, 1H), 7.57 (dd, J = 7.9, 1.7 Hz, 1H), 7.37 – 7.30 (m, 2H), 7.17 (dd, J = 7.7, 1.7 Hz, 1H), 7.11 (t, J = 2.1 Hz, 1H), 6.87 – 6.82 (m, 1H), 2.91 – 2.81 (m, 1H), 0.73 –0.65 (m, 2H), 0.60 – 0.53 (m, 2H) 13 C NMR (101 MHz, DMSO- d6) δ 167.63, 160.56,159.08, 137.85, 137.70, 136.62, 134.83, 133.12, 131.25, 130.45, 124.04,123.62, 121.60, 115.21, 111.94, 108.52, 23.54, 18.07, 6.17.ESI-MS (m / z) :calculated for C 21 H 20 N4O3[M+H] + 377.1535 found 377.1530.The purity was 99.15% by HPLC analysis.

[0056] Compound CMP-7: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(4-hydroxyphenyl)-1H-imidazolium-4-carboxamide (CMP-7): Brown solid, yield: 36.4% 1 H NMR (400 MHz, DMSO- d 6) δ 9.82 (s, 1H), 9.48 (s, 1H), 8.35 (d, J = 4.3 Hz, 1H), 8.24 – 8.19 (m, 2H), 8.13 (d, J = 1.8 Hz, 1H), 7.50 (t, J = 8.5 Hz, 3H), 7.27 (d, J = 7.9 Hz, 1H), 6.86 (d, J = 8.8 Hz, 2H), 2.85– 2.76 (m, 1H), 2.27 (s, 3H), 0.68 – 0.60 (m, 2H), 0.56 – 0.50 (m, 2H). 13 C NMR (101 MHz, DMSO-) d6) δ 167.64 , 160.67 , 157.55 , 137.45 , 136.65 , 136.48 ,134.67 , 133.12 , 130.42 , 128.71 , 123.94 , 123.49 , 123.15 , 121.91 ,116.56 , 23.54 , 18.07 , 6.17 .ESI-MS (m / z): calculated for C 21 H 20 N4O3[M+H] + 377.1535 found 377.1528.The purity was 98.85% by HPLC analysis.

[0057] Compound CMP-8: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(2-(trifluoromethyl)phenyl)-1H-imidazol-4-carboxamide (CMP-8): Brown solid, yield: 23.7%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.63 (s, 1H), 8.40 (d, J = 4.3 Hz, 1H), 8.14 (d, J = 1.8 Hz, 1H), 8.10 (s, 2H), 8.01 (d, J = 7.9 Hz,1H), 7.94 – 7.88 (m, 1H), 7.82 (t, J = 7.7 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.58 (dd, J = 7.9, 1.9 Hz, 1H), 7.33 (d, J = 7.9 Hz, 1H), 2.89 – 2.81 (m, 1H), 2.32 (s, 3H), 0.71 – 0.65 (m, 2H), 0.60 – 0.55 (m, 2H) 13 C NMR (101 MHz, DMSO- d6) δ 167.56, 160.56, 139.58, 136.97, 136.57, 135.14, 134.71, 134.54, 133.03,130.94, 130.72, 130.46, 127.66(q, 2 J C-F = 29.8 Hz), 127.61, 125.80 (q, 3 J C-F = 5.6 Hz),124.20, 123.86, 23.53, 18.12, 6.17.ESI-MS (m / z): calculated for C 22 H 19 F3N4O2[M+H] + 429.1460 found 429.1445.The purity was 96.94% by HPLC analysis.

[0058] Compound CMP-9: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(3-(trifluoromethyl)phenyl)-1H-imidazol-4-carboxamide: brown solid, yield: 27.4%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.62 (s, 1H), 8.62 – 8.60 (m, 2H), 8.40 (d, J = 4.3 Hz, 1H), 8.24 (s, 1H), 8.14 (dd, J = 7.4, 1.9 Hz, 2H),7.82 – 7.80 (m, 1H), 7.79 (d, J = 1.1 Hz, 1H), 7.58 (dd, J = 7.9, 1.9 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 2.88 – 2.82 (m, 1H), 2.32 (s, 3H), 0.71 – 0.65 (m,2H), 0.60 – 0.55 (m, 2H). 13 C NMR (101 MHz, DMSO- d6) δ 167.59, 160.52, 138.13,137.42, 136.77, 136.58, 135.18, 133.09, 131.64, 131.29, 130.97, 130.45(q, 2 J C-F = 32.4 Hz), 125.28, 124.64 (q, 1 J C-F =272.2 Hz), 124.19, 123.96(q, 3 J C-F = 3.6 Hz),121.83, 118.15, 23.54, 18.11, 6.16.ESI-MS (m / z): calculated for C 22 H 19 F3N4O2[M+H] + 429.1460 found 429.1436.The purity was 99.52% by HPLC analysis.

[0059] Compound CMP-10: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(4-(trifluoromethyl)phenyl)-1H-imidazol-4-carboxamide: brown solid, yield: 34.1%. 1 H NMR (400 MHz, Chloroform- d ) δ 9.63 (s, 1H), 8.60 (d, J = 13.1 Hz, 2H), 8.41 (d, J = 3.7 Hz, 1H), 8.13 (s, 1H), 8.00 (dd, J = 51.5, 8.4 Hz, 4H), 7.58 (d, J = 7.7 Hz, 1H), 7.32 (d, J = 7.9 Hz, 1H), 2.90 – 2.80 (m,1H), 2.31 (s, 3H), 0.73 – 0.65 (m, 2H), 0.62 – 0.54 (m, 2H). 13 C NMR (101 MHz, DMSO-d 6) δ 167.59, 160.47, 139.85, 138.29, 136.73, 136.55, 135.20, 133.09,130.46, 128.42(q, 2 J C-F = 29.4 Hz), 128.10, 127.62 (q, 3 J C-F = 3.7 Hz), 125.75, 124.224 (q, 1 J C-F = 271.5 Hz), 123.97, 123.04, 121.79, 121.58, 23.54, 18.10, 6.16. ESI-MS (m / z) : calculated for C 22 H 19 F3N4O2[M+H] + 429.1460 found 429.1445.The purity was 99.36% by HPLC analysis.

[0060] Compound CMP-11: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(2-methoxyphenyl)-1H-imidazol-4-carboxamide: white solid, yield: 42.1%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.53 (s, 1H), 8.39 (d, J =4.3 Hz, 1H), 8.19 (s, 1H), 8.08 (dd, J = 10.4, 1.3 Hz, 2H), 7.57 (d, J = 7.9 Hz, 1H), 7.52 (dd, J = 7.8, 1.6 Hz, 1H), 7.51 – 7.45 (m, 1H), 7.34 – 7.28 (m, 2H),7.12 (t, J= 7.6 Hz, 1H), 3.86 (s, 3H), 2.90 – 2.81 (m, 1H), 2.33 (s, 3H), 0.72 – 0.66 (m, 2H), 0.60 – 0.55 (m, 2H). 13 C NMR (101 MHz, DMSO- d 6) δ 167.66,160.69, 152.75, 138.64, 136.68, 136.61, 134.65, 133.15, 130.43, 130.33,126.36, 125.72, 124.31, 123.95, 123.51, 121.45, 113.45, 56.55, 23.54, 18.06,6.17.ESI-MS (m / z): calculated for C 22 H 22 N4O3[M+H] + 391.1692 found 391.1668.The purity was 99.18% by HPLC analysis.

[0061] Compound CMP-12: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(3-methoxyphenyl)-1H-imidazol-4-carboxamide: white solid, yield: 38.9%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.55 (s, 1H), 8.45 (d, J =2.3 Hz, 2H), 8.37 (d, J = 4.2 Hz, 1H), 8.11 (d, J = 1.4 Hz, 1H), 7.53 (dd, J = 7.9, 1.6 Hz, 1H), 7.42 (t, J = 8.1 Hz, 1H), 7.36 – 7.30 (m, 2H), 7.29 (d, J = 8.0 Hz, 1H), 6.96 (dd, J= 8.2, 2.2 Hz, 1H), 3.83 (s, 3H), 2.86 – 2.77 (m, 1H), 2.28 (s, 3H), 0.68 – 0.62 (m, 2H), 0.56 – 0.51 (m, 2H). 13 C NMR (101 MHz, DMSO- d 6) δ167.60, 160.84, 160.60, 137.87, 137.79, 136.61, 136.54, 135.00, 133.08, 131.26, 130.44, 124.08, 123.77 , 121.71 , 114.01 , 113.16 , 106.98, 56.09 , 23.54 , 18.10 , 6.18 .ESI-MS (m / z): calculated for C 22 H 22 N4O3[M+H] + 391.1692 found 391.1665.The purity was 98.89% by HPLC analysis.

[0062] Compound CMP-13: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(4-methoxyphenyl)-1H-imidazol-4-carboxamide: white solid, yield: 33.2%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.55 (s, 1H), 8.39 (d, J =4.1 Hz, 1H), 8.34 (dd, J = 6.8, 1.2 Hz, 2H), 8.17 – 8.15 (m, 1H), 7.72 – 7.66(m, 2H), 7.56 (dd, J = 7.9, 1.5 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.10 (d, J = 9.0Hz, 2H), 3.82 (s, 3H), 2.89 – 2.81 (m, 1H), 2.31 (s, 3H), 0.72 – 0.65 (m,2H), 0.60 – 0.54 (m, 2H).13 C NMR (101 MHz, DMSO- d 6) δ 167.62, 160.64, 159.10, 137.60, 136.65, 136.51, 134.77, 133.11, 130.43, 130.09, 123.98, 123.58, 122.97 , 121.88 , 115.40 , 56.03 , 23.54 , 18.08 , 6.17 .ESI-MS (m / z): calculated for C 22 H 22 N4O3[M+H] + 391.1692 found 391.1677.The purity was97.41% by HPLC analysis. Compound CMP-14: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(3,5-dimethoxyphenyl)-1H-imidazolium-4-carboxamide: brown solid, yield: 23.6%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.55 (s, 1H), 8.47 (s, 2H), 8.37 (d, J = 2.6 Hz, 1H), 8.10 (s, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.28 (d, J =8.0 Hz, 1H), 6.94 (d, J = 2.1 Hz, 2H), 6.51 (t, J = 2.1 Hz, 1H), 3.80 (s, 6H), 2.85 – 2.78 (m, 1H), 2.28 (s, 3H), 0.69 – 0.60 (m, 2H), 0.57 – 0.50 (m, 2H). 13 C NMR (101 MHz, DMSO- d6) δ 161.80 , 160.61 , 138.37 , 137.70 , 136.61 ,136.57 , 135.03 , 133.09 , 130.43 , 124.09 , 123.81 , 121.77 , 99.95, 99.57, 56.21, 23.54, 18.10, 6.17.ESI-MS (m / z): calculated for: C 23 H 24 N4O4[M+H] + 421.1798 found 421.1775.The purity was 98.93% by HPLC analysis.

[0063] Compound CMP-15: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(2,4-dimethoxyphenyl)-1H-imidazol-4-carboxamide: brown solid, yield: 30.6%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.51 (s, 1H), 8.39 (d, J =4.1 Hz, 1H), 8.18 (s, 1H), 7.97 (d, J = 7.1 Hz, 2H), 7.56 (d, J = 6.6 Hz, 1H), 7.42 (d, J = 8.7 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 6.81 (d, J = 2.5 Hz, 1H), 6.66(dd, J = 8.7, 2.5 Hz, 1H), 3.84 (s, 6H), 2.90 – 2.80 (m, 1H), 2.32 (s, 3H), 0.72 – 0.65 (m, 2H), 0.60 – 0.55 (m, 2H). 13 C NMR (101 MHz, DMSO- d6) δ 167.64 ,154.01 , 138.77 , 136.69 , 136.42 , 134.58 , 133.12 , 130.41 , 127.29 ,124.61 , 123.88 , 123.44 , 105.67 , 100.18 , 56.60 , 56.16 , 23.54 , 18.06 ,6.18 .ESI-MS (m / z) : calculated for : C 23 H 24 N4O4[M+H] + 421.1798 found421.1764.The purity was 95.90 % by HPLC analysis.

[0064] Compound CMP-16: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-imidazolium-4-carboxamide: white solid, yield: 42.4%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.56 (s, 1H), 8.44 (dd, J = 16.7, 1.1 Hz, 2H), 8.36 (d, J = 4.1 Hz, 1H), 8.09 – 8.04 (m, 1H), 7.52 (dd, J = 7.9, 1.5 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.04 (s, 2H), 3.84 (s, 6H), 3.64(s, 3H), 2.86 – 2.74 (m, 1H), 2.26 (s, 3H), 0.67 – 0.60 (m, 2H), 0.55 – 0.49(m, 2H). 13 C NMR (101 MHz, DMSO- d6) δ 167.61, 160.70, 154.07, 137.61, 137.05, 136.64, 135.17, 133.07, 132.70, 130.44, 124.10, 123.94 ,122.13 , 99.33 , 60.62 , 56.83 , 23.54 , 18.13 , 6.18 ..ESI-MS (m / z) :calculated for : C 24 H 26 N4O5[M+H] + 451.1903 found 451.1875.The purity was 98.16% by HPLC analysis.

[0065] Compound CMP-17: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(4-(hydroxymethyl)phenyl)-1H-imidazolium-4-carboxamide: white solid, yield: 31.7%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.53 (s, 1H), 8.41 (d, J =1.2 Hz, 1H), 8.39 – 8.34 (m, 2H), 8.12 (d, J = 1.4 Hz, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.53 (dd, J = 7.9, 1.5 Hz, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.0 Hz, 1H), 5.30 (t, J = 5.7 Hz, 1H), 4.53 (d, J = 5.6 Hz, 2H), 2.87 – 2.78 (m, 1H), 2.28 (s, 3H), 0.69 – 0.61 (m, 2H), 0.58 – 0.51 (m, 2H). 13 C NMR (101 MHz, DMSO- d6) δ 167.62 , 160.60 , 142.71 , 137.80 , 136.62 , 136.45 , 135.35 , 134.88 ,133.11 , 130.43 , 128.19 , 124.04 , 123.67 , 121.62 , 121.04 , 62.67 , 23.54, 18.09 , 6.17 .ESI-MS (m / z) : calculated for : C 22 H 22 N4O3[M+H] + 391.1692 found391.1680.The purity was 99.75 % by HPLC analysis.

[0066] Compound CMB-1: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(pyridin-4-yl)-1H-imidazol-4-carboxamide: white solid, yield: 36.8%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.63 (s, 1H), 8.71 (d, J = 1.1 Hz, 1H), 8.69 (dd, J = 4.8, 1.4 Hz, 2H), 8.65 (d, J = 1.1 Hz, 1H), 8.37 (d, J = 4.1 Hz, 1H), 8.07 (d, J = 1.5 Hz, 1H), 7.90 (dd, J = 4.8, 1.5 Hz, 2H), 7.55 (dd, J = 7.9, 1.6 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 2.86 – 2.77 (m, 1H), 2.28 (s, 3H), 0.69 – 0.62 (m, 2H), 0.57 – 0.51 (m, 2H). 13 C NMR (101 MHz, DMSO- d6) δ 167.55 ,160.36 , 151.93 , 143.06 , 138.52 , 136.49 , 135.37 , 133.07 , 130.46 ,124.30 , 124.10 , 120.85 , 114.74 , 23.54 , 18.12 , 6.17 .ESI-MS (m / z) :calculated for : C 20 H 19 N5O2[M+H] + 362.1539 found 362.1541.The purity was 98.26% by HPLC analysis.

[0067] Compound CMB-2: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(pyridin-3-yl)-1H-imidazol-4-carboxamide: brown solid, yield: 40.8%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.62 (s, 1H), 9.07 (d, J = 2.5 Hz, 1H), 8.64 (d, J = 4.6 Hz, 1H), 8.55 (s, 2H), 8.41 (d, J = 4.0 Hz, 1H), 8.25 (d, J =7.3 Hz, 1H), 8.13 (s, 1H), 7.62 (dd, J = 8.3, 4.8 Hz, 1H), 7.59 – 7.55 (m, 1H),7.33 (d, J = 7.9 Hz, 1H), 2.90 – 2.82 (m, 1H), 2.32 (s, 3H), 0.72 – 0.65 (m,2H), 0.60 – 0.54 (m, 2H). 13 C NMR (101 MHz, DMSO- d6) δ 167.59, 160.49, 149.14, 142.65, 138.19, 136.69, 136.56, 135.13, 133.48, 133.09, 130.45, 129.05, 124.93 , 124.18 , 123.90 , 121.74 , 23.54 , 18.10 , 6.17 .ESI-MS (m / z) : calculated for : C 20 H 19 N5O2[M+H] + 362.1539 found 362.1536.The purity was99.33% by HPLC analysis.

[0068] Compound CMB-3: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(pyrimidin-5-yl)-1H-imidazol-4-carboxamide: 1 HNMR (400 MHz, DMSO- d 6) δ 9.66 (s, 1H), 9.34 (s, 2H), 9.24 (s, 1H), 8.62 (q, J =1.4 Hz, 2H), 8.41 (d, J = 4.1 Hz, 1H), 8.11 (d, J = 1.5 Hz, 1H), 7.59 (dd, J = 7.9, 1.6 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 2.88 – 2.81 (m, 1H), 2.31 (s, 3H), 0.71 – 0.65 (m, 2H), 0.60 – 0.55 (m, 2H). 13 C NMR (101 MHz, DMSO- d6) δ 167.57 ,160.39 , 157.42 , 149.86 , 138.41 , 136.73 , 136.49 , 135.34 , 133.07 ,132.15 , 130.46 , 124.30 , 124.09 , 121.64 , 23.54 , 18.11 , 6.17 .ESI-MS (m / z) : calculated for : C 19 H 18 N6O2[M+H] + 363.1491 found 363.1550.The purity was99.74% by HPLC analysis.

[0069] Compound CIN-1: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(naphth-2-yl)-1H-imidazol-4-carboxamide: brown solid, yield: 42.4%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.62 (s, 1H), 8.62 (d, J = 1.4 Hz,1H), 8.58 (s, 1H), 8.39 (t, J = 4.1 Hz, 2H), 8.17 (s, 1H), 8.13 (d, J = 8.9 Hz,1H), 8.05 – 7.96 (m, 3H), 7.64 – 7.55 (m, 3H), 7.33 (d, J = 7.9 Hz, 1H), 2.90 –2.82 (m, 1H), 0.72 – 0.66 (m, 2H), 0.61 – 0.56 (m, 2H). 13 C NMR (101 MHz, DMSO- d6) δ 167.64, 160.62, 138.04, 136.69, 136.64, 134.94, 134.24, 133.62, 133.15,132.32, 130.46, 130.38, 128.40, 128.28, 127.83, 127.06, 124.09, 123.74,121.75, 120.13, 118.80, 23.55, 18.10, 6.17.ESI-MS (m / z): calculated for:C 25 H 22 N4O2[M+H] + 411.1743 found 411.1726.The purity was 98.45% by HPLC analysis.

[0070] Compound CIN-2: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(6-hydroxynaphth-2-yl)-1H-imidazol-4-carboxamide: brown solid, yield: 27.4%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.93 (s, 1H), 9.58 (s, 1H), 8.51 (d, J = 1.4 Hz, 1H), 8.47 (d, J = 1.4 Hz, 1H), 8.38 (d, J = 4.3 Hz, 1H), 8.21(d, J = 2.2 Hz, 1H), 8.17 (d, J = 1.8 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.84 (d, J =8.8 Hz, 1H), 7.81 (dd, J = 8.8, 2.3 Hz, 1H), 7.57 (dd, J = 7.9, 1.9 Hz, 1H), 7.33(d, J= 7.9 Hz, 1H), 7.24 – 7.17 (m, 2H), 2.90 – 2.82 (m, 1H), 2.33 (s, 3H), 0.72 – 0.66 (m, 2H), 0.61 – 0.54 (m, 2H). 13 C NMR (101 MHz, DMSO- d 6) δ 167.65,160.66, 156.41, 137.80, 136.66, 136.60, 134.83, 134.08, 133.15, 131.67,130.45, 130.01, 128.37, 127.98, 123.63, 121.79, 120.53, 120.47, 119.06,109.26, 23.54, 18.09, 6.17.ESI-MS (m / z): calculated for: C 25 H 22 N4O3[M+H] + 427.1692 found 427.1666.The purity was 99.10% by HPLC analysis.

[0071] Compound CIN-3: ; N-(5-(cyclopropylcarbamoyl)-2-methylphenyl)-1-(6-methoxynaphth-2-yl)-1H-imidazol-4-carboxamide: brown solid, yield: 28.6%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.57 (s, 1H), 8.50 (dd, J =14.7, 1.2 Hz, 2H), 8.37 (d, J = 4.2 Hz, 1H), 8.27 (d, J = 2.1 Hz, 1H), 8.16 (s,1H), 7.99 (d, J = 8.9 Hz, 1H), 7.91 – 7.85 (m, 2H), 7.55 (dd, J = 7.9, 1.6 Hz, 1H), 7.42 (d, J = 2.4 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.26 (dd, J= 9.0, 2.5 Hz,1H), 3.89 (s, 3H), 2.90 – 2.78 (m, 1H), 2.31 (s, 3H), 0.70 – 0.64 (m, 2H),0.59 – 0.53 (m, 2H). 13 C NMR (101 MHz, DMSO- d 6) δ 167.64, 160.64, 158.23,137.89, 136.66, 136.61, 134.84, 133.79, 133.15, 132.39, 130.45, 129.90,129.03, 128.88, 124.04, 123.65, 121.76, 120.62, 120.48, 118.98, 106.53,55.80, 23.54, 18.09, 6.17.ESI-MS (m / z): calculated for: C 26 H 24 N4O3[M+H] + 441.1848 found 441.1876.The purity was 98.39% by HPLC analysis.

[0072] Compound JPMN-1: ; N-(5-(4'-formyl-[1,1'-piperazine]-4-carbonyl)-2-methylphenyl)-1-(naphth-2-yl)-1H-imidazol-4-carboxamide: white solid, yield: 26.5%. 1 H NMR (400 MHz, Chloroform- d ) δ 9.12 (s,1H), 8.39 (s, 1H), 8.11 (s, 2H), 8.02 (d, J = 8.8 Hz, 1H), 7.95 (s, 1H), 7.92(t, J = 6.7 Hz, 2H), 7.89 (d, J = 2.1 Hz, 1H), 7.61 (q, J = 6.1 Hz, 2H), 7.55 (dd, J = 8.8, 2.3 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.21 (d, J= 7.7 Hz, 1H), 3.65 (s, 4H), 3.48 (s, 4H), 2.47 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ 165.75, 156.24,155.50, 133.35, 130.68, 129.09, 128.65, 128.51, 127.89, 126.28, 125.85,125.09, 123.26, 123.04, 122.41, 118.69, 116.97, 115.21, 115.17, 115.01,40.94, 24.53, 12.96.ESI-MS (m / z): calculated for C 27 H 25 N5O3 [M+H] + 468.2030found 468.2035;[M+Na] + 490.1850 found 490.1851.The purity was 98.26% by HPLC analysis.

[0073] Compound JPMN-2: ; N-(5-(4'-formyl-[1,1'-piperazine]-4-carbonyl)-2-methylphenyl)-1-(6-methoxynaphthyl-2-yl)-1H-imidazol-4-carboxamide: white solid, yield: 31.6%. 1 H NMR (400 MHz, Chloroform- d ) δ9.12 (s, 1H), 8.39 (s, 1H), 8.10 (d, J = 16.0 Hz, 2H), 7.89 (d, J = 9.4 Hz, 2H),7.83 – 7.78 (m, 2H), 7.50 (dd, J = 8.7, 2.2 Hz, 1H), 7.34 – 7.27 (m, 2H), 7.23– 7.18 (m, 2H), 3.96 (s, 3H), 3.65 (s, 4H), 3.48 (s, 4H), 2.46 (s, 3H). 13 C NMR (101 MHz, Chloroform- d) δ 160.98, 158.67, 134.14, 133.24, 131.04, 129.46,129.14, 120.86, 120.51, 119.98, 105.83, 55.48, 29.71, 17.77.ESI-MS (m / z) :calculated for C 28 H 27 N5O4[M+H] + 498.2136 found 498.2210;[M+Na] + 520.2015 found520.2037.The purity was 98.47% by HPLC analysis.

[0074] Compound JPMN-3: ; 1-(2,4-Dimethoxyphenyl)-N-(5-(4'-formyl-[1,1'-piperazine]-4-carbonyl)-2-methylphenyl)-1H-imidazol-4-carboxamide: pale yellow solid, yield: 47.2%. 1 H NMR (400 MHz, Chloroform- d ) δ9.12 (s, 1H), 8.38 (s, 1H), 8.11 (s, 1H), 7.84 (d, J = 1.3 Hz, 1H), 7.66 (d, J =1.3 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 7.21 (t, J = 8.8 Hz, 2H), 6.62 (d, J = 2.5Hz, 1H), 6.57 (dd, J = 8.6, 2.5 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.64 (s,4H), 3.47 (s, 4H), 2.45 (s, 3H). 13 C NMR (101 MHz, Chloroform- d) δ 170.57,161.05, 160.98, 160.68, 153.80, 137.61, 136.53, 133.20, 130.99, 126.34,124.15, 119.77, 119.01, 104.58, 99.85, 55.89, 55.75, 29.72, 17.69.ESI-MS (m / z): calculated for C 25 H 27 N5O5[M+H] + 478.2085 found 478.2067.The purity was96.59% by HPLC analysis.

[0075] Compound YPMN-1: ; N-(5-(4'-acetyl-[1,1'-piperazine]-4-carbonyl)-2-methylphenyl)-1-(naphth-2-yl)-1H-imidazol-4-carboxamide: pale yellow solid, yield: 36.4%. 1 H NMR (400 MHz, Chloroform- d ) δ 9.12 (s,1H), 8.37 (s, 1H), 8.11 (s, 1H), 8.02 (d, J = 8.7 Hz, 1H), 7.95 (s, 1H), 7.93(t, J = 6.6 Hz, 2H), 7.89 (d, J = 2.2 Hz, 1H), 7.65 – 7.58 (m, 2H), 7.55 (dd, J =8.8, 2.3 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.22 (s, 1H), 3.69 (s, 4H), 3.55(s, 4H), 2.46 (s, 3H), 2.14 (s, 3H). 13 C NMR (101 MHz, Chloroform- d) δ 169.23,135.45, 133.44, 132.65, 130.96, 130.59, 128.02, 127.79, 127.15, 121.69,119.96, 119.77, 29.71, 21.45, 17.70.ESI-MS (m / z) : calculated for C 28 H 27 N5O3[M+H] + 481.2237, found 482.2262;[M+Na] + 504.2057, found 504.2082.The purity was99.31% by HPLC analysis.

[0076] Compound YPMN-2: ; N-(5-(4'-acetyl-[1,1'-piperazine]-4-carbonyl)-2-methylphenyl)-1-(6-methoxynaphth-2-yl)-1H-imidazol-4-carboxamide: brown solid, yield: 33.7%. 1 H NMR (400 MHz, Chloroform- d ) δ9.11 (s, 1H), 8.37 (s, 1H), 8.07 (d, J = 1.4 Hz, 1H), 7.91 – 7.87 (m, 2H), 7.80(dd, J = 5.7, 3.4 Hz, 2H), 7.49 (dd, J = 8.7, 2.3 Hz, 1H), 7.32 – 7.26 (m, 2H), 7.20 (d, J = 2.6 Hz, 2H), 3.96 (s, 3H), 3.69 (s, 4H), 3.55 (s, 4H), 2.45 (s, 3H), 2.13 (s, 3H). 13 C NMR (101 MHz, Chloroform- d) δ 169.22, 160.37, 158.58,138.03, 135.49, 134.04, 133.43, 132.05, 130.94, 129.43, 129.07, 128.71,121.79, 120.78, 120.55, 119.86, 105.82, 55.47, 29.29, 21.45, 17.69.ESI-MS (m / z): calculated for C 29 H 29 N5O4[M+Na] + 534.2112, found 534.2190.The purity was97.93% by HPLC analysis.

[0077] Compound YPMN-3: ; N-(5-(4'-acetyl-[1,1'-piperazine]-4-carbonyl)-2-methylphenyl)-1-(2,4-dimethoxyphenyl)-1H-imidazol-4-carboxamide: brown solid, yield: 37.3%. 1 H NMR (400 MHz, Chloroform- d ) δ9.10 (s, 1H), 8.36 (s, 1H), 7.83 (d, J = 1.3 Hz, 1H), 7.66 (d, J = 1.3 Hz, 1H), 7.28 (d, J = 7.7 Hz, 1H), 7.21 (d, J = 8.6 Hz, 2H), 6.61 (d, J = 2.5 Hz, 1H), 6.56(dd, J = 8.6, 2.6 Hz, 1H), 3.86 (s, 3H), 3.83 (s, 3H), 3.68 (s, 4H), 3.54 (s,4H), 2.44 (s, 3H), 2.12 (s, 3H). 13 C NMR (101 MHz, Chloroform- d) δ 169.21,161.04, 160.67, 153.80, 137.60, 136.55, 133.37, 130.93, 129.59, 126.34,124.13, 119.88, 119.01, 104.58, 99.85, 55.88, 55.74, 29.71, 21.46, 17.69.ESI-MS (m / z): calculated for C 26 H 29 N5O5[M+H] + 491.2169, found 492.2197. The purity was 97.45 by HPLC analysis.

[0078] Compound HPMN-1: ; N-(2-methyl-5-(4'-(methanesulfonyl)-[1,1'-piperazine]-4-carbonyl)phenyl)-1-(naphth-2-yl)-1H-imidazol-4-carboxamide: white solid, yield: 29.6%. 1 H NMR (400 MHz, Chloroform- d ) δ 9.13(s, 1H), 8.39 (d, J = 1.7 Hz, 1H), 8.11 (d, J = 1.4 Hz, 1H), 8.03 (d, J = 8.8 Hz,1H), 7.95 (s, 1H), 7.95 – 7.90 (m, 2H), 7.89 (d, J = 2.2 Hz, 1H), 7.64 – 7.58(m, 2H), 7.55 (dd, J = 8.8, 2.3 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.20 (dd, J =7.7, 1.7 Hz, 1H), 3.79 (d, J = 57.5 Hz, 5H), 3.31 (s, 5H), 2.83 (s, 3H), 2.47(s, 3H). 13 C NMR (101 MHz, Chloroform- d) δ 170.42, 160.31, 138.20, 135.46,133.88, 133.41, 133.19, 132.65, 131.01, 130.60, 129.74, 128.03, 128.00,127.80, 127.17, 123.39, 121.69, 119.95, 119.86, 119.78, 34.76, 29.71,17.70.ESI-MS (m / z): calculated for C 27 H 27 N5O4S[M+H] + 518.1857, found518.1826.The purity was 99.09% by HPLC analysis.

[0079] Compound HPMN-2: ; 1-(6-methoxynaphthyl-2-yl)-N-(2-methyl-5-(4'-(methanesulfonyl)-[1,1'-piperazine]-4-carbonyl)phenyl)-1H-imidazol-4-carboxamide: yellow solid, yield: 28.5%. 1 H NMR (400 MHz, Chloroform- d )δ 9.19 (s, 1H), 8.35 (s, 1H), 8.09 (s, 1H), 7.95 (s, 1H), 7.90 (d, J = 8.7 Hz,1H), 7.83 – 7.78 (m, 2H), 7.50 (dd, J = 8.7, 2.3 Hz, 1H), 7.33 – 7.27 (m, 2H), 7.20 (dd, J = 7.8, 1.9 Hz, 2H), 3.96 (s, 3H), 3.80 (d, J = 59.0 Hz, 4H), 3.30 (s, 4H), 2.83 (s, 3H), 2.46 (s, 3H). 13 C NMR (101 MHz, Chloroform- d) δ 170.42,158.64, 135.92, 134.10, 133.15, 131.01, 129.93, 129.44, 129.11, 128.71,123.43, 120.84, 120.54, 120.00, 119.94, 105.82, 55.48, 34.75, 29.72,17.74.ESI-MS (m / z): calculated for C 28 H 29 N5O5S[M+H] + 548.1962, found548.1938.The purity was 98.51% by HPLC analysis.

[0080] Compound HPMN-3: ; 1-(2,4-Dimethoxyphenyl)-N-(2-Methyl-5-(4'-(methanesulfonyl)-[1,1'-piperazine]-4-carbonyl)phenyl)-1H-imidazol-4-carboxamide: brown solid, yield: 39.6%. 1 H NMR (400 MHz, Chloroform- d )δ 9.11 (s, 1H), 8.36 (d, J = 1.7 Hz, 1H), 7.83 (d, J = 1.3 Hz, 1H), 7.66 (d, J =1.3 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.21 (d, J = 8.7 Hz, 1H), 7.17 (dd, J = 7.7, 1.7 Hz, 1H), 6.61 (d, J = 2.5 Hz, 1H), 6.56 (dd, J = 8.6, 2.6 Hz, 1H), 3.86 (s, 3H), 3.83 (s, 3H), 3.71 (s, 4H), 3.29 (s, 4H), 2.82 (s, 3H), 2.44 (s, 3H). 13 CNMR (101 MHz, Chloroform- d) δ 170.47, 161.06, 160.65, 153.80, 137.62, 136.50,136.06, 133.11, 130.97, 129.66, 126.36, 124.14, 123.21, 119.74, 118.98,104.59, 99.85, 55.90, 55.75, 34.73, 17.70.ESI-MS (m / z): calculated forC 25 H 29 N5O6S[M+H] + 528.1839, found 528.1816.The purity was 99.61% by HPLC analysis.

[0081] Example 3 Anti-tumor cell activity 1. Antitumor activity assay - MTT assay Logarithmic growth phase HCT-116 and HT-29 cells were seeded at appropriate densities in 96-well plates and cultured in a CO2 incubator until fully adherent. Different concentrations (150 μM, 75 μM, 32.5 μM, 16.25 μM, 8.125 μM, 4.0625 μM) of the test compound were added to the drug-treated groups, while an equal volume of PBS was added to the blank control group. SB203580 was used as the positive control. After 48 hours of incubation, 10 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued for another 4 hours in the dark. The supernatant was discarded, and 100 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader (TECAN Spark, Switzerland).

[0082] The results are shown in Table 2: This series of compounds exhibited significant in vitro cytotoxicity against colorectal cancer cell lines, with most candidates showing an IC50 value of [missing information]. 50 The values ​​were in the range of 50-60 μM. Notably, compound CIN-3 exhibited excellent anti-proliferative activity, showing an effect on the IC50 of HT-29 cells. 50 The value was 6.05±0.07 μM, and the IC50 value for HCT-116 cells was 6.05±0.07 μM. 50 The value was 7.80±0.47 μM, which was better than the positive control drug SB203580.

[0083] Table 2. Effects of the target compound and SB203580 on HT-29, HCT116 and HIEC cells.

[0084] The above experimental results show that the compounds of the present invention exhibit certain in vitro cytotoxicity against colorectal cancer cell lines. Among them, compared with the positive control, compounds CMP-1, CMP-2, CMP-3, CMP-4, CMP-7, CMP-9, CMP-10, CMP-11, CMP-14, CIN-1, CIN-2, CIN-3 and JPMN-2 have better anti-colorectal cancer activity; and compound CIN-3 has more significant anti-colorectal cancer activity.

[0085] 2. In vitro p38α MAPK inhibitory activity assay Based on the results of the MTT cytotoxicity assay, eight compounds with good activity were selected. Stock solutions of these compounds were prepared and diluted to 10 μM. The inhibitory activity against p38α mitogen-activated protein kinase was detected using the ADP-Glo™ kinase assay kit. The kinase, substrate, and ATP were thawed on ice and diluted with kinase buffer (40 mM Tris, pH 7.5, 20 mM MgCl2, 0.1 mg / mL BSA, 50 μM DTT). The assay was performed in 384-well plates. 1 μL of the compound, 2 μL of diluted p38α kinase, and 2 μL of substrate-ATP mixture were added to each well. Blank and solvent control groups were set up. After incubation at 25°C, μL of ADP-Glo™ reagent and 10 μL of kinase assay reagent were added sequentially, and incubation continued. After incubation, the chemiluminescence signal was detected using a multi-mode microplate reader, and the relative light units (RLU) value was recorded. The inhibition rate is calculated as follows: Inhibition rate (%) = [(RLU{compound} - RLU{solvent}) / (RLU{solvent} - RLU{blank})] × 100%.

[0086] The results are shown in Table 3: IC50 of the positive control drug SB203580 50 The effective value was 37.69 ± 2.14 nM, and the inhibition rate of p38α MAPK at a concentration of 30 nM was 42.56 ± 0.68%, verifying the stability and reliability of the experimental system. Significant differences were found in the p38α MAPK inhibitory activities of the eight tested compounds. Among them, compound CIN-3 showed slightly better activity than the positive control, with an inhibition rate of 45.38 ± 1.26% at 30 nM, indicating its good potential as a targeted inhibitor of p38α MAPK. Combined with the previous MTT cytotoxicity assay results, compound CIN-3 possesses both strong antitumor activity and p38α MAPK inhibitory ability, suggesting that its antitumor effect may be mediated by targeted inhibition of the p38α MAPK pathway.

[0087] Table 3. Inhibition rate of the compound against p38αMAPK at 30 nM

[0088] 3. EdU incorporation experiment HT-29 cells cultured to the logarithmic growth phase were seeded at a density of 6000 cells / well in 96-well plates and incubated overnight at 37°C in a CO2 incubator. Cells were treated with different concentrations of compound CIN-3, while a blank control group was given 0.1% DMSO. Incubation was then continued for 48 hours at a constant temperature. EdU incorporation was detected using the SFClick™ EdU-488 (Abcells) kit according to the manufacturer's instructions. Results were observed and recorded using an inverted fluorescence microscope.

[0089] The results are as follows Figure 2 and Figure 3 As shown: Treatment of HT-29 cells with different concentrations of CIN-3 or the positive control SB203580 inhibited colony formation. Figure 2 As shown in the figure, compound CIN-3 reduces the number and size of cell colonies in a concentration-dependent manner. With increasing concentrations of compound CIN-3, the green fluorescent signal indicating DNA synthesis in HT-29 cells gradually weakens and eventually disappears. Figure 3 As shown in Figures A and C), this indicates that compound CIN-3 can significantly block cellular DNA replication. Simultaneously, compound CIN-3 inhibits HT-29 cell proliferation and induces G0 / G1 phase arrest (…). Figure 3 (As shown in B and D).

[0090] 4. Wound healing experiment HT-29 cells were seeded in 6-well plates and cultured overnight. When cell confluence reached 90%, a 10 μL sterile pipette tip was used to create a scratch on the cell monolayer. Exfoliated cell debris was gently washed away with PBS. Subsequently, serum-free medium containing different concentrations of compounds CIN-3 and SB203580 was added to each well. Cell migration was observed and recorded using an optical microscope at 0 h, 24 h, and 48 h. The experimental results were analyzed using ImageJ software.

[0091] The results of the wound healing experiment are as follows Figure 4 As shown in Figures A and C, compound CIN-3 significantly inhibited the wound healing ability of HT-29 cells in a concentration-dependent manner: the healing rate of the blank control group was (45.43±0.98)% at 24 hours after scratching, and almost complete repair was achieved at 48 hours, with a healing rate of (84.06±1.72)%. After treatment with 8 μM CIN-3, the cell healing rate decreased to (13.37±1.95)% at 24 hours and to (24.59±2.18)% at 48 hours. Moreover, at the same concentration, the inhibitory effect of CIN-3 was significantly better than that of the positive control drug.

[0092] 5. Transwell migration experiments HT-29 cells were pretreated with serum-free medium for 5 hours by starvation at 1×10⁻⁶. 5 Cells were seeded at a density per well in the upper chamber of a 24-well Transwell chamber, with 200 μL of serum-free medium containing a specified concentration of compound CIN-3 or SB203580 added. The lower chamber was filled with 700 μL of complete medium containing 10% FBS, and incubated at 37°C for 48 hours. After removing unmigrated cells, the remaining cells were fixed with 4% paraformaldehyde for 30 minutes, stained with crystal violet for 10 minutes, and air-dried. Three to five fields of view were randomly selected for photographing and quantitative analysis.

[0093] Transwell experimental results are as follows: Figure 4 As shown in Figures B and D: as the concentration of compound CIN-3 increases, the number of HT-29 cells crossing the polycarbonate membrane gradually decreases, confirming that compound CIN-3 inhibits the transmembrane migration ability of cells in a concentration-dependent manner.

[0094] 6. Giemsa staining observation experiment HT-29 cells were loaded at 2×10 4 Cells were seeded at a density of 8 μM / well in 6-well plates. After cell attachment, the treatment groups were treated with CIN-3 at final concentrations of 8 μM, 4 μM, and 2 μM, respectively. The positive control group was treated with SB203580 at a final concentration of 8 μM. After incubation for 48 hours, the culture medium was discarded, and the cells were washed 2-3 times with PBS. The cells were fixed with 4% paraformaldehyde for 30 minutes, stained with Giemsa stain for 30 minutes, and rinsed with distilled water. Cell morphology was carefully observed using an optical microscope (Moticam Pro 282B, China).

[0095] Giemsa staining morphological observations, such as Figure 5 As shown, after CIN-3 treatment, HT-29 cells exhibited typical apoptotic characteristics, including cell shrinkage and rounding, reduced volume, chromatin condensation or fragmentation, and the nucleus showing a strong bright yellow stain due to chromatin condensation. With increasing drug concentration, the cells further exhibited necrotic morphological changes, such as cell swelling, nuclear membrane disintegration, and cell membrane rupture. These results indicate that CIN-3 primarily induces apoptosis in HT-29 cells at low to medium concentrations, while triggering cell necrosis at high concentrations.

[0096] 7. Hoechst 33342 / PI double staining experiment HT-29 cells were loaded at 2×10 4Cells were seeded at a density of 8 μM / well in 6-well plates. After cell attachment, the treatment groups were treated with CIN-3 at final concentrations of 8 μM, 4 μM, and 2 μM, respectively, while the positive control group was treated with 8 μM SB203580. After 48 hours of incubation, the culture medium was discarded, and the cells were washed 2-3 times with PBS. Subsequently, sufficient Hoechst 33342 / PI staining solution was added to cover the cells, and the cells were incubated at room temperature in the dark for 10-20 minutes. The staining solution was discarded, and the cells were washed 2-3 times with PBS. Observation and photography were performed using an inverted fluorescence microscope.

[0097] The results of the Hoechst 33342 / PI double staining fluorescence experiment are as follows: Figure 6 As shown, with increasing CIN-3 concentration, the proportion of apoptotic nuclei exhibiting bright blue strong fluorescence gradually increased, while the proportion of PI-positive cells exhibiting red fluorescence also increased synchronously, further confirming that CIN-3 induces apoptosis and necrosis in HT-29 cells in a concentration-dependent manner.

[0098] 8. Apoptosis detection experiment The apoptosis rate of HT-29 cells was determined using the Annexin V-FITC apoptosis detection kit (Solarbio, Cat# CA1020) according to the manufacturer's instructions. HT-29 cells were cultured at a rate of 2 × 10⁻⁶. 5 Cells were seeded at a density of 6-well plates. After cell attachment, the cells were treated with the appropriate concentrations of CIN-3 and SB203580 and incubated for 48 hours. Cells were collected by digestion with EDTA-free trypsin and washed with pre-chilled PBS. The cells were then gently resuspended in binding buffer. 100 μL of the cell suspension was mixed with 5 μL of Annexin V-FITC and 5 μL of PI, and incubated at room temperature in the dark for 8–10 minutes. Finally, flow cytometry analysis was performed using an Agilent NovoCyte Penteon (Singapore) instrument.

[0099] The results of quantitative detection by Annexin V-FITC / PI double staining flow cytometry are as follows: Figure 7 The results showed that the total apoptosis rate in the blank control group remained at an extremely low level. With the increase of CIN-3 treatment concentration, the early apoptosis rate, late apoptosis rate and total apoptosis rate of HT-29 cells all increased significantly in a concentration-dependent manner, and at the same concentration, the pro-apoptotic effect of CIN-3 was stronger than that of the positive control SB203580.

[0100] 9. Reactive Oxygen Species (ROS) Detection Experiment The level of reactive oxygen species in HT-29 cells was detected using a ROS detection kit. HT-29 cells were cultured at a concentration of 2 × 10⁻⁶. 5Cells were seeded at a density of [insert density here] in 6-well plates. After cell attachment, cells were treated with appropriate concentrations of CIN-3 and SB203580 and incubated for 48 hours. Cells were collected by digestion with EDTA-free trypsin and washed with pre-chilled PBS. Then, cells were incubated with 10 μmol / L DCFH-DA probe solution in the dark for 20–30 minutes. After incubation, the probe solution was discarded, and cells were washed 2–3 times with PBS. Finally, cells were resuspended in 1 mL PBS and analyzed using flow cytometry and fluorescence microscopy.

[0101] The results of the reactive oxygen species detection experiment are as follows: Figure 8 As shown in Figures A and C, the blank control group HT-29 cells maintained basal levels of ROS, supporting tumor cell proliferation and survival. With increasing CIN-3 concentration, intracellular ROS accumulated explosively in a concentration-dependent manner.

[0102] Mitochondrial membrane potential (ΔΨm) level as Figure 8 As shown in Figure B, the fluorescence intensity histogram of flow cytometry is as follows: Figure 8 As shown in Figure D, compared with the control group, the fluorescence curves of all CIN-3 treatment groups showed a concentration-dependent rightward shift, indicating a comprehensive increase in intracellular ROS levels. Excessive ROS accumulation exacerbates oxidative damage to cellular DNA, thereby triggering tumor cell apoptosis.

[0103] 10. Western blot analysis After treatment with the appropriate concentration of drug, HT-29 cells were collected by trypsin digestion and transferred to centrifuge tubes. After centrifugation and discarding the culture medium, the cells were washed 2-3 times with PBS. Subsequently, the cells were lysed on ice using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentration was determined using a BCA protein assay kit. An equal volume of 4× protein loading buffer was added, and the sample was diluted and heated in a 100°C water bath for 30 minutes. An equal volume of protein (60 μg) was separated by 10% SDS-PAGE and transferred to a 0.45 μm PVDF membrane. The membrane was blocked with TBST buffer containing 5% skim milk for 2 hours, followed by incubation overnight at 4°C with primary antibody (dilution ratio 1:500-1:1000). After washing, the membrane was incubated with secondary antibody (goat anti-rabbit IgG H+L, 1:3000, Affinity Bioscience) at room temperature for 2 hours. The membrane was then washed with TBST, scanned and developed using an ECL chemiluminescence system and a Tanon imaging system, and protein levels were analyzed using ImageJ software.

[0104] The results are as follows Figure 9As shown in Figures A and B: the blank control group showed high expression of p-p38 MAPK and p-HSP27, indicating persistent abnormal activation of the p38 MAPK pathway in HT-29 cells. After treatment with gradient concentrations of CIN-3, the expression of total p38 MAPK and total HSP27 proteins did not change significantly, while the expression of p-p38 MAPK and p-HSP27 was significantly downregulated in a concentration-dependent manner. These results indicate that CIN-3 inhibits the abnormal activation of the p38 MAPK pathway by suppressing p38 MAPK phosphorylation and blocking the phosphorylation signal transduction of its downstream substrate HSP27.

[0105] 11. Molecular docking experiment Molecular docking experiments were performed using Discovery Studio (DS) software. The molecular structures of the compounds were constructed directly in DS, and the Ligand Preparation module was used for structure optimization, charge adjustment, hydrogenation, and conformational optimization to minimize compound energy. The 3D structures of the target proteins were downloaded from the RCSB protein database (https: / / www.rcsb.org / ). The Protein Preparation module was used to remove water molecules, add polar hydrogen, and repair missing amino acid residues. Binding sites were defined based on the binding pockets of the original co-crystallized ligands. Molecular docking was performed using the CDOCKER algorithm. The docking results were visualized and analyzed using PyMOL software.

[0106] The results are as follows Figure 9 As shown in the middle CF diagram, CIN-3 stably inserts into the ATP-binding pocket of the p38α protein, occupying the ATP-binding active site composed of Lys53, Arg67, Arg70, Glu71, and Asp168. The binding free energy is -19.4995 kcal / mol. CIN-3 forms multiple stable hydrogen bonds with key amino acid residues in the active pocket (Glu71, Asp168, Thr106, His107, Leu108, Asn115, and Gly110), providing the core driving force for the conformational stability of the ligand-receptor complex. Simultaneously, the cyclopropylamine portion of CIN-3 is embedded in the hydrophobic cavity formed by Leu74, Phe169, and Leu171, and interacts with residues such as Lys53, Leu75, Leu104, Ile84, Leu167, and Ala51 through hydrophobic interactions, further enhancing binding stability. Furthermore, the spatial conformation of CIN-3 perfectly matches the optimal pharmacophore model for p38α MAPK inhibitors.

[0107] Example 4 In vivo activity experiment 1. Acute toxicity test of compound CIN-3 in mice Animal preparation: Twenty-four Kunming mice (weighing 19-24 g) were obtained from the GLP Laboratory Animal Center of Lanzhou University. All animals were housed under 12-hour light / dark cycles at a temperature maintained at 25°C and a relative humidity of approximately 70%, and were freely fed according to the Institutional Animal Care and Use Committee (IACUC) guidelines. All animals were acclimatized to the environment for three days prior to the experiments. All animal experiments were conducted in accordance with the Guidelines for In Vivo Research Animal Studies and were approved by the Ethics Committee of Lanzhou University.

[0108] Acute toxicity test: After 7 days of acclimatization, mice were randomly divided into four groups: saline group, low-dose CIN-3 group (300 mg / kg), medium-dose CIN-3 group (600 mg / kg), and high-dose CIN-3 group (900 mg / kg). After 12 hours of fasting and water restriction, six mice in each group were administered different doses of CIN-3 or saline via gavage. During the subsequent 14-day observation period, the general condition of the mice was closely monitored, including skin, fur, respiration, and spontaneous activity, and their weight was measured regularly. At the end of the observation period, the mice were euthanized humanely, and the heart, liver, spleen, lungs, kidneys, stomach, and intestines were dissected, fixed in 4% paraformaldehyde, and embedded in paraffin. Subsequently, the mouse tissues were stained with hematoxylin and eosin (HE) to evaluate the toxic effects of CIN-3 on various organs of the mice.

[0109] During the observation period, mice in the high-dose group (900 mg / kg) exhibited signs of poisoning such as decreased activity, rapid breathing, and unsteady gait, but no deaths occurred. Mice in the low-dose group (300 mg / kg) and the medium-dose group (600 mg / kg) showed no signs of poisoning, and their activity, appetite, and weight gain were normal. Weight monitoring showed that the weight of mice in the high-dose group initially decreased and then increased, while there was no significant difference in weight gain between the low / medium-dose groups and the control group. Hematoxylin-eosin (HE) staining results showed that the organs in the 300 and 600 mg / kg groups had no significant pathological changes compared to the control group; however, the livers of mice in the 900 mg / kg group showed extensive vacuolation, accompanied by edema, loose degeneration, and disordered arrangement of some hepatocytes. These results indicate that 600 mg / kg is a safe and tolerable dose of CIN-3 in mice. Following general safety factor principles for in vivo pharmacological dose design, a conservative safety factor of 1 / 20 was used to determine the subsequent in vivo dose as 30 mg / kg. This dose is well below the toxicity threshold, posing no risk of acute toxicity, while ensuring that the drug reaches an effective pharmacological concentration.

[0110] 2. Mouse homologous xenograft model Animal preparation: SPF-grade female BALB / c mice were obtained from the GLP Laboratory Animal Center of Lanzhou University. All animals were housed under 12-hour light / dark cycles at a temperature maintained at 25°C and a relative humidity of approximately 70%, and were freely fed according to the Institutional Animal Care and Use Committee (IACUC) guidelines. All animals were acclimatized to the environment for 3 days prior to experiments. All animal experiments were conducted in accordance with the Guidelines for In Vivo Research and were approved by the Ethics Committee of Lanzhou University.

[0111] After 7 days of acclimatization feeding, administer 1×10 7 One CT26 cell was suspended in 150 μL of physiological saline and subcutaneously injected into the right axilla of mice. When the tumor volume reached approximately 50-100 mm³, all tumor-bearing mice were randomly divided into four groups: solvent group, compound CIN-3 15 mg / kg group, compound CIN-3 30 mg / kg group, and SB203580 30 mg / kg group. Administration was continued for 14 days. During the administration period, the mental status and activity of mice in each group were observed daily, and their body weight was recorded. Tumor size, tumor damage, and number of deaths were recorded every two days. After the last administration, mice were euthanized humanely, and the weight and volume of the tumor tissue were weighed and measured.

[0112] like Figure 10 As shown in Figure A: In the model control group, tumor volume showed a continuous and rapid growth trend; with increasing CIN-3 dosage, tumor growth slowed down in a concentration-dependent manner. The 30 mg / kg high-dose group showed the most significant tumor growth inhibition, and its antitumor activity was superior to the positive control drug SB203580 at the same dose. After the dosing cycle, tumor tissue was resected ( Figure 9 (B) Weigh the tumor and calculate the tumor inhibition rate (B). Figure 9 (C) The average tumor weight in the 30 mg / kg CIN-3 group was significantly lower than that in the model control group, with a tumor inhibition rate of (82.1±2.15)%.

[0113] In summary, the compounds provided by this invention possess certain inhibitory activity against colorectal cancer. Specifically, compound CIN-3 exhibits significant inhibitory activity against HT-29 and HCT-116 colorectal cancer cells, inducing G1 / S phase arrest in tumor cells, inhibiting DNA synthesis, downregulating migration ability, and inducing apoptosis. In the CT26 mouse homologous xenograft model, a dose of 30 mg / kg of CIN-3 achieved a tumor inhibition rate of 82.1 ± 2.15%, with good safety profile, demonstrating promising drug development potential and providing a novel candidate drug for targeted therapy of colorectal cancer.

Claims

1. An N-arylimidazolium compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound has the structure shown in formula (A) or formula (B): Formula (A); Formula (B); Wherein, R1 is selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted naphthyl; the substituted substituent is selected from halogen, hydroxyl, hydroxymethyl, trifluoromethyl, methoxy; R2 is selected from piperazine-1-methyl ketone, piperazine-1-sulfonone, and 1-(piperazine-1-yl)ethyl-1-one.

2. The N-arylimidazolium compound or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, The compound has the structure shown in formula (A); wherein R1 is selected from phenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 3,5-dimethoxyphenyl, 2,4-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 4-hydroxymethylphenyl, pyridine, pyrimidine, naphthyl, 6-hydroxynaphthyl, 6-methoxynaphthyl.

3. The N-arylimidazolium compound or a pharmaceutically acceptable salt thereof as described in claim 2, characterized in that, The compound is a compound with any of the following structures: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 4. The N-arylimidazolium compound or a pharmaceutically acceptable salt thereof as described in claim 3, characterized in that, The compound is: .

5. The N-arylimidazolium compound as described in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound has the structure shown in formula (B); R1 is selected from naphthyl, 6-methoxynaphthyl, and 2,4-dimethoxyphenyl.

6. The N-arylimidazolium compound or a pharmaceutically acceptable salt thereof as described in claim 5, characterized in that, The compound is a compound with any of the following structures: 、 、 、 、 、 、 、 、 。 7. Use of the N-arylimidazolium compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1–6 in the preparation of p38α MAPK inhibitors.

8. The use of the N-arylimidazolium compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1–6 in the preparation of an antitumor drug.

9. The application as described in claim 8, characterized in that, The tumor is colorectal cancer.

10. The N-arylimidazolium compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1–6, characterized in that, The synthetic route for the N-arylimidazolium compounds is shown below: ， Where i is CDI, DCM, rt 4h; ii is Fe, NH4Cl, H2O, 80℃, reflux 1h; iii is HCTU, Et3N, DMF, rt 20h; iv is Cu(AcO)2, Et3N, DMF, 80℃, O2, reflux 3h; or, ； Where i is HCTU, DMF, Et3N, rt 20h; ii is LiOH·H2O, MeOH / H2O, HCl; iii is EDCI, HoBt, DIPEA, DCM; iv is Cu(AcO)2, DMF, Et3N, 80℃, O2, reflux 3h.

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