Aryl urea-imidazole salt compounds, synthesis method and antitumor activity thereof

Abstract

The application discloses an aryl urea-imidazole salt compound, a synthesis method and antitumor activity thereof, and belongs to the field of medicinal chemistry. The aryl urea-imidazole salt compound has a general structure as shown in the specification, and is obtained by using L-amino acid as raw material, adopting a multi-step continuous reaction of reduction, upper protection group, substitution, deprotection and coupling, and the like, to obtain the bioactive aryl urea-imidazole salt compound. Research shows that the compound has high efficiency and selectivity on breast cancer MCF-7 cells, and the IC 50 The optimal value is 0.67 μM. The stability of the drug affinity reaction target, cell thermal shift experiment, molecular docking and micro-thermal mobility technology confirm that ERK1 / 2 is the target point; and the potential active skeleton structure is provided for further development of breast cancer drugs.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to the synthesis of arylurea-imidazolium salts and their antitumor activity. Background Technology

[0002] Cancer is one of the leading causes of death worldwide, and the number of cases continues to rise annually. Despite advancements in treatment, the side effects of traditional cancer treatments such as chemotherapy, radiation therapy, and surgical resection severely limit their clinical application. This necessitates the search for new anticancer drugs. Small molecule drugs exhibit excellent drug metabolism and tolerability, are readily absorbed by tumor tissue, and thus exert their therapeutic effects. In recent years, the application of small molecule drugs in tumor treatment has been continuously expanding, and several small molecule antitumor drugs have already been approved for marketing. Therefore, the search for antitumor drugs with novel structural types is of great significance.

[0003] Urea compounds all contain the urea group as their active structure, and therefore most possess a variety of biological activities such as antiviral, antibacterial, and antitumor activity. N-heterocyclic compounds have attracted great interest from medicinal chemists due to their widespread presence in bioactive natural products and drug molecules. Among them, imidazole salts have garnered significant attention due to their antitumor activity. Based on this, this invention designed and synthesized a series of arylurea-imidazolium salt compounds and studied their biological activities. Furthermore, modifications were made to three sites on these compounds to design and synthesize this class of arylurea-imidazolium salt compounds with novel structures, and their antitumor activity was investigated. Summary of the Invention

[0004] To overcome the aforementioned technical deficiencies, this invention provides an arylurea-imidazolium salt compound and evaluates its antitumor activity. Using L-amino acids as raw materials, a multi-step continuous reaction involving reduction, adding a protecting group, substitution, deprotection, and coupling was employed to obtain a bioactive arylurea-imidazolium salt compound. This arylurea-imidazolium salt compound exhibits high efficacy against MCF-7 cells, inhibiting their migration and invasion, and inducing cell cycle arrest and apoptosis. ERK1 / 2 was identified as its target using drug affinity target stability assays (DARTS), cell thermal displacement assays (CETSA), molecular docking, and microthermophoresis (MST). In vivo, the arylurea-imidazolium salt compound significantly reduces tumor volume in mice and can be used to prepare antitumor drugs, showing broad application prospects.

[0005] The arylurea-imidazolium salt compound of the present invention has the following general structural formula:

[0006]

[0007] Wherein: R1 is selected from benzyl, phenyl, isopropyl, methyl; R2 is selected from hydrogen, methyl, phenyl, trifluoromethyl; R3 is selected from phenyl or naphthyl; Ar is selected from 3,5-bistrifluoromethylphenyl, phenyl, 4-methoxyphenyl, 3,5-dimethylphenyl.

[0008] Furthermore, in the above technical solution, the arylurea-imidazolium salt compound preferably has the following structure:

[0009]

[0010] The present invention also provides a method for synthesizing the above-mentioned arylurea-imidazolium salt compound, comprising the following steps: using compound 1 as a raw material, reducing it with sodium borohydride to obtain intermediate 2; then protecting it with (Boc)₂O to obtain intermediate 3; then reacting triphenylphosphine with NBS to obtain intermediate 4; then reacting it with an imidazolium derivative in NaH / DMF to obtain intermediate 5; deprotecting the Boc with trifluoroacetic acid to obtain intermediate 6; reacting it with an aryl isocyanate to obtain arylurea-imidazolium derivative 7; and finally reacting it with a benzyl bromide compound to obtain arylurea-imidazolium salt derivative 8.

[0011] The synthetic route of this invention is represented by the following reaction equation:

[0012]

[0013] Furthermore, in the first step of the above technical solution, the reaction is carried out in methanol solvent; the reaction temperature is selected from -10℃ to 10℃; the molar ratio of compound 1 to NaBH4 is 1:2.5.

[0014] Furthermore, in the second step of the above technical solution, the reaction is carried out in tetrahydrofuran solvent, and the reaction temperature is selected from -10℃ to 10℃; the molar ratio of intermediate 2 to (Boc)2O is 1:1.1.

[0015] Furthermore, in the third step of the above technical solution, the reaction is carried out in tetrahydrofuran solvent, and the reaction temperature is selected from -10℃ to 10℃; the molar ratio of intermediate 3, triphenylphosphine and NBS is 1:1.2:1.2.

[0016] Furthermore, in the fourth step of the above technical solution, the reaction temperature is selected from -10℃ to 10℃; the molar ratio of intermediate 4, imidazole derivative and NaH is 1:2:3.

[0017] Furthermore, in the fifth step of the above technical solution, the reaction is carried out in dichloromethane, and the reaction temperature is selected from -10℃ to 10℃; the molar ratio of intermediate 5 to TFA is 1:5.

[0018] Furthermore, in the sixth step of the above technical solution, the reaction is carried out in dichloromethane, and the reaction temperature is selected from 0-30℃; the molar ratio of intermediate 6 to isocyanate derivative is 1:1.5.

[0019] Furthermore, in step seven of the above technical solution, the reaction is carried out in acetonitrile solvent, and the reaction temperature is selected from 0-30℃; the molar ratio of intermediate 7 to benzyl bromide compound is 1:1.5.

[0020] This invention also provides the application of the above-mentioned arylurea-imidazolium salt compounds in the preparation of antitumor drugs.

[0021] Furthermore, in the above technical solution, the anti-tumor activity is specifically anti-breast cancer (MCF-7).

[0022] Furthermore, in the above technical solution, the arylurea-imidazolium salt compound 8a exhibits high efficiency and selectivity towards MCF-7.

[0023] Furthermore, ERK1 / 2 was identified as its target using drug affinity target stability assay (DARTS), cell thermal displacement assay (CETSA), molecular docking, and microthermophoresis (MST) techniques. The arylurea-imidazolium salt compound 8a inhibited the migration and invasion of MCF-7 cells, induced cell cycle arrest and apoptosis, thereby significantly reducing tumor volume in mice.

[0024] The present invention also provides an anti-breast cancer pharmaceutical composition, the active ingredient of which includes the above-mentioned arylurea-imidazolium salt compound.

[0025] Beneficial effects of the invention

[0026] 1. This invention uses L-amino acids as a starting point and employs a seven-step synthetic method to synthesize arylurea-imidazolium salts. The synthetic route is simple, the reaction efficiency is high, and it has promising prospects for industrial application.

[0027] 2. The arylurea-imidazolium salt compound 8a of this invention exhibits strong cellular activity against MCF-7 cells and also shows significant antitumor activity in vivo.

[0028] 3. The arylurea-imidazolium salt compound 8a of this invention can target ERK1 / 2 as a promising therapeutic agent for the treatment of breast cancer. Attached Figure Description

[0029] Figure 1 This refers to the target identification-DARTS experiment in Example 4a.

[0030] Figure 2 This is a target verification-kinase activity experiment for example 4a.

[0031] Figure 3 This is the target verification-CETSA experiment for example 4a.

[0032] Figure 4 This is the target verification-MST experiment in Example 4a.

[0033] Figure 5 This is for target verification - molecular docking in Example 4a.

[0034] Figure 6 Example 5 shows that 8a inhibits the migration ability of breast cancer MCF-7 cells.

[0035] Figure 7 This demonstrates the ability of 8a in Example 6 to inhibit the invasion of breast cancer MCF-7 cells.

[0036] Figure 8 Example 7 shows that 8a promotes cell cycle arrest in breast cancer MCF-7 cells.

[0037] Figure 9 Example 8a promotes apoptosis in breast cancer MCF-7 cells.

[0038] Figure 10 Example 9 shows that 8a inhibits the growth of mouse tumor volume. Specific Implementation

[0039] The present invention will be further illustrated below with specific examples. These embodiments should be understood as illustrative only and not as limiting the scope of protection of the present invention. After reading the description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.

[0040] Example 1

[0041]

[0042] Compound 6 (6 mmol) (Eur. J. Med. Chem. 2019, 177, 374-385) and 1-isocyanate-3,5-bis(trifluoromethyl)benzene (9 mmol, 1.5 eq) were added to the reaction flask and reacted in DCM solution for 5 minutes. After the reaction was completed, the mixture was filtered, concentrated, and column-sected to give (S)-1-(1-(1H-benzo[d]imidazol-1-yl)-3-phenylprop-2-yl)-3-(3,5-bis(trifluoromethyl)phenyl)urea 7 (520 mg), with a yield of 62%. 1H NMR (400MHz, CDCl3) δ9.62(s,1H),7.79(d,J=1.7Hz,2H),7.66(s,1H),7.47-7.40(m,3H),7.35(t,J=7.6Hz,1H),7.32-7.23(m,4H),7.08(dd ,J=2.4,7.1Hz,2H),6.46(d,J=7.1Hz,1H),4.66-4.50(m,2H),4.42(dd,J=5.2,14.5Hz,1H),2.99-2.89(m,1H),2.65(dd,J=8.6,14.2Hz,1H); 13 C NMR(101MHz, CDCl3)δ155.25,143.36,141.97,141.20,136.55,134.53,132.14(q,J=33.2Hz),129.08, 128.98,127.29,12432,123.49,123.32(q,J=272.8Hz),117.89,115.38,110.87,51.52,47.94,38.23; 19 F NMR(377MHz, CDCl3)δ-63.01; HRMS(ESI)calcd.for C 25 H 21 F6N4O([M+H)) + ):507.15,found:507.1614.

[0043] Example 2

[0044]

[0045] Compound 7 (2 mmol), 2-(bromomethyl)naphthalene (3 mmol, 1.5 eq), and 5 mL of acetonitrile were added to the reaction flask and mixed. The temperature was maintained at 85 °C. After the reaction was completed, the mixture was filtered, concentrated, and column filtered to give (S)-1-(2-(3-(3,5-bis(trifluoromethyl)phenyl)ureido)-3-phenylpropyl)-3-(naphth-1-ylmethyl)-1H-benzo[d]imidazolium-3-bromium 8a (279 mg), with a yield of 77%. 1HNMR(400MHz, CDCl3)δ10.98(s,1H),9.39(s,1H),7.94(s,2H),7.81(s,1H),7.6 8(ddd,J=8.4,14.0,15.6Hz,3H),7.58-7.46(m,4H),7.45-7.33(m,8H),7.27(d, J=8.1Hz,1H),6.03-5.66(m,2H),4.89(dd,J=10.2,14.0Hz,1H),4.59(ddd,J=4. 1,11.7,17.0Hz,2H),3.40(dd,J=5.4,13.7Hz,1H),3.02(dd,J=9.5,13.7Hz,1H); 13 C NMR (101MHz, CDCl3) δ155.02,142.78,141.19,136.88,133.19,132.93,13 1.71(q,J=32.7Hz),131.26,130.90,129.59,129.20,129.10,127.82,127 .66,127.63,127.51,127.37,127.34,127.04,126.90,124.25,123.30(d, J=272.9Hz),117.96,114.93,113.69,112.68,51.84,51.34,49.37,39.84; 19 FNMR(377MHz, CDCl3)δ-62.84; HRMS(ESI)calcd.for C 36 H 29 F6N4O([M-Br)) + ):647.2240,found:647.2232.

[0046] Example 3

[0047] The CCK8 assay was used to evaluate the inhibitory effect of the compound on tumor cell growth.

[0048] Experimental Principle: CCK-8 (Cell Counting Kit-8) is a simple and accurate reagent for cell proliferation and toxicity analysis. The basic principle is that WST-8 (chemical name 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt) is reduced by dehydrogenases in cells to a highly water-soluble yellow formazanye product in the presence of dimethyl 1-methoxy-5-methylphenazine sulfate (1-Methoxy PMS). The amount of formazanye produced is directly proportional to the number of viable cells; therefore, this property can be used to directly analyze cell proliferation and toxicity.

[0049] Experimental methods: Tumor cells were cultured in 10% fetal bovine serum (FBS) at 37°C under a humidified environment of 5% CO2. A 1% penicillin-streptomycin solution (10000 U / mL penicillin and 10000 μg / mL streptomycin) was added to the culture medium to prevent potential contamination. Cells were seeded in 96-well plates (5 × 10⁶ cells / well). 3 Cells were cultured at 100 cells / well and incubated overnight at 37°C under humid conditions. Cells were treated with different concentrations (1–50 μM) of each compound. Cell viability was determined using a CCK-8 assay after 48 hours. CCK-8 assay products were measured at 450 nm and quantified using a Synergy Neo2 multimode microplate reader. The cytotoxicity of each compound was expressed as an IC50 value. 50 The value represents IC. 50 The value represents the drug concentration required to induce 50% inhibition of tumor cell growth.

[0050] Experimental results: Compound 8a showed high efficiency and selectivity against MCF-7 breast cancer cells, with an IC50 concentration of 100%. 50 =0.67μM.

[0051] Example 4

[0052] Given the strong antitumor effect of 8a on MCF-7 cells, label-free DARTS and LC-MS / MS proteomics techniques were used to identify the direct targets of 8a. Figure 1As shown, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and intact protein bands were obtained by Coomassie brilliant blue staining. The study found that the ~42 kDa protein band in the drug group was significantly darker than that in the control group, indicating that the ~42 kDa protein band has a specific affinity for 8a, which may represent its target protein. The ~42 kDa band was then excised from the gel for subsequent gel digestion and LC-MS / MS analysis. Based on its relative molecular mass, the identified peptide sequences were matched to databases for approximately 42 kDa. In total, five proteins with a molecular weight of approximately 42 kDa were identified. Notably, ERK1 / 2 matched a large number of high-scoring peptide fragments among the five proteins; therefore, ERK1 / 2 may be an 8a target, such as... Figures 2-5 As shown, ERK1 / 2 was verified as a direct target of 8a by kinase activity assay, CETSA, MST and molecular docking.

[0053] Example 5

[0054] To determine migration ability, MCF-7 cells were cultured to complete confluence. Wounds were created by scraping the cell monolayer with the tip of a 200 μL sterile pipette. A reference image was specified on the platform, and an image at time 0 hours was obtained. Cells were cultured with the specified dose of compound 8a for 48 hours, and images were captured using a fluorescence microscope, as shown below. Figure 6 As shown.

[0055] Example 6

[0056] Cell invasion capability was assessed using Matrigel (BD Biosciences, USA) transwell plates. Each transwell chamber was then spread with 80 μL of 1:8 Matrigel and seeded with 1 × 10⁶ cells. 5 Cells were then added to 100 μL of serum-free DMEM medium and treated with 8a for 48 hours. The lower chamber was then incubated with intact medium for 48 hours. Cells in the upper chamber were then swabbed, fixed with 4% paraformaldehyde for 15 minutes, and stained with crystal violet for 10 minutes. Each membrane was then imaged and counted using a Leica DMIL fluorescence microscope (Leica, Germany). Figure 7 As shown.

[0057] Example 7

[0058] MCF-7 cells (4×10) 5Cells were seeded (1 cell / well) into six-well plates and allowed to adhere overnight. Cells were then treated with compound 8a and incubated at 37°C with 5% CO2. After 48 hours of treatment, cells were collected and fixed in ice-cold 70% ethanol for 24 hours. Cells were then treated with RNase (30 mL, 1 mg / mL) and stained with PI solution (50 mL, 500 mg / mL) in the dark for 30 minutes. The DNA content of the stained cells was recorded and analyzed by flow cytometry (BD Bioscience). Figure 8 As shown.

[0059] Example 8

[0060] MCF-7 cells were seeded in 6-well dishes (4 × 10⁶ cells / well). 5 Cells were then exposed to different concentrations of 8α for 48 hours and stained with FITC-conjugated Annexin V and PE. Apoptotic cells were then recorded and analyzed by flow cytometry (BDBioscience). Figure 9 As shown.

[0061] Example 9

[0062] To further evaluate the in vivo anti-breast cancer activity of 8a, its in vivo antitumor efficacy was assessed in female BALB / c mice carrying 4T1 xenograft tumors. Ten mice in each group were administered 8a orally (25 mg / kg and 50 mg / kg) once daily and the positive control drug BVD-523 (50 mg / kg) intraperitoneally for 16 days. Figure 10 As shown, the results indicated that at doses of 25 mg / kg and 50 mg / kg, the tumor growth inhibition (TGI) values ​​of 8a were 40.72% and 54.86%, respectively, which were significantly higher than those of BVD-523 (26.89%).

[0063] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An aryl urea-imidazolium compound characterized in that, The specific structure is: 。 2. The method for synthesizing the arylurea-imidazolium salt compound as described in claim 1, characterized in that, It comprises the following steps: To a reaction flask was added 6 mmol of compound 6 and 9 mmol of 1- isocyanate-3,5-bis(trifluoromethyl)benzene, reacted in dichloromethane solution for 5 minutes, filtered after the reaction was complete, concentrated, and columned to obtain S )-1-(1-(1 H -phenyl-1H- d -imidazol-1-yl)-3-phenylpropan-2-yl)-3-(3,5-bis(trifluoromethyl)phenyl)urea 7; To the reaction bottle was added 2 mmol of compound 7, 3 mmol of 2- (bromomethyl) naphthalene and 5 mL of acetonitrile, the temperature was maintained at 85°C, and after the reaction was completed, it was filtered, concentrated and columned to obtain S -1- (2- (3- (3, 5-bis (trifluoromethyl) phenyl) ureido) -3- phenylpropyl) -3- (naphthalen-1-ylmethyl) -1 H - benzo [d] imidazole-3-bromonium 8a.

3. Use of the aryl urea-imidazole salt compound of claim 1 in the preparation of an anticancer drug.

4. Use of the aryl urea-imidazole salt compound according to claim 3 for the preparation of an anticancer drug, characterized in that: The anticancer is an anti-breast cancer.

5. An anti-breast cancer pharmaceutical composition, characterized by: The active ingredient thereof comprises the compound described in claim 1.

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