6-{[(3-Ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid and its preparation method and uses

Abstract

The present invention discloses 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid, its preparation method and uses, and a preparation method of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid, comprising the following steps: 1,4-diphenyl-1,3-butadiene reacts with maleic anhydride in xylene and an alkaline solution to obtain a first product; the first product reacts with an aniline derivative in a weak acid environment to obtain the target compound. The compound prepared by the present invention has the effects of inhibiting the growth of various cancer cells, resisting cell senescence, protecting the genomic stability of cells and inhibiting aging-induced cardiac fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. More specifically, the present invention relates to 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid and its preparation method and uses. Background Art

[0002] Existing studies (Chen MS, Lee RT, Garbern JC. Senescence mechanisms and targets in the heart. Cardiovasc Res. 2022 Mar 25;118(5):1173-1187. doi:10.1093 / cvr / cvab161.) have confirmed that the senescence of heart cells is the main factor leading to functional degeneration of various cardiovascular diseases such as atherosclerosis, myocardial infarction, and cardiac fibrosis. Cardiac fibrosis is a hallmark event of heart aging, which increases with age. The reduction of cardiac fibrosis is beneficial to maintaining the normal function of the heart organ. In addition, there are also studies (1) Zhao Y, Simon M, Seluanov A, Gorbunova V. DNA damage and repair in age-related inflammation. Nat Rev Immunol. 2023 Feb;23(2):75-89. doi:10.1038 / s41577-022-00751-y. (2) Schumacher B, Pothof J, Vijg J, Hoeijmakers JHJ. The central role of DNA damage in the ageing process. Nature. 2021 Apr;592(7856):695-703. doi:10.1038 / s41586-021-03307-7.) that have confirmed that the accumulation of DNA damage is considered the main initiating factor inducing cell death, senescence, and tissue function degeneration. Therefore, maintaining the stability of the cell genome is beneficial to fundamentally inhibiting cell senescence from the cell fate and promoting the senescent organs to return to the track of normal function.

[0003] Currently, no commercially available drugs for treating cardiac fibrosis have been found to have both the functions of protecting the stability of the cell genome and resisting cell senescence. Summary of the Invention

[0004] An object of the present invention is to provide a compound 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid, which is a newly synthesized compound after the structural optimization and upgrading of the compound described in Application No. 202210976338.5. This compound has the effects of protecting the stability of cell genome, resisting cardiomyocyte senescence and preventing and treating cardiac fibrosis, and can prevent cardiovascular diseases.

[0005] Another object of the present invention is to provide a preparation method of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid. The synthesis route of the compound of the present invention adopts a two-step synthesis method, which has the characteristics of simplicity, high efficiency, greenness and low toxicity. The raw materials and auxiliary materials used have only a weak pungent smell and have no obvious harm to human health and the ecological environment.

[0006] Still another object of the present invention is to provide the application of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid. This compound has the effects of inhibiting the growth of various tumor (liver cancer, highly metastatic liver cancer, lung cancer, acute monocytic leukemia, colon cancer, prostate cancer) cells, protecting the stability of cell genome and treating cardiac fibrosis.

[0007] To achieve these objects and other advantages of the present invention, 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid is provided, and its chemical structural formula is shown as formula (I):

[0008]

[0009] The preparation method of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid includes the following steps:

[0010] S1. 1,4-Diphenyl-1,3-butadiene reacts with maleic anhydride in xylene vapor and an alkaline solution to obtain product one;

[0011] S2. Product one reacts with an aniline derivative in a weak acid environment to obtain the target compound.

[0012] Preferably, in the preparation method of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid, the aniline derivative is one or more of 3-methylaniline, 3-ethylaniline, 3,5-dimethylaniline, p-methylaniline or p-ethylaniline.

[0013] Preferably, in the preparation method of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid, the reaction conditions in S1 are 180 °C, humidity 65%, and the reaction time is not less than 10 h.

[0014] Preferably, in the preparation method of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid, the reaction conditions in S2 are weak acid and a dry environment, and the reaction time is 20 - 30 min.

[0015] Preferably, in the preparation method of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid, the basic solution is one or more of potassium hydroxide solution and sodium hydroxide solution.

[0016] Preferably, in the preparation method of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid, the weak acid is one or more of glacial acetic acid and carbonic acid.

[0017] Use of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid in the preparation of a drug for inhibiting cell senescence and protecting genomic stability.

[0018] Use of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid in the preparation of drugs for treating liver cancer, highly metastatic liver cancer, lung cancer, acute monocytic leukemia, colon cancer, and prostate cancer.

[0019] Use of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid in the preparation of a drug for treating cardiac fibrosis.

[0020] The present invention at least includes the following beneficial effects:

[0021] The compound prepared by the present invention has the effects of inhibiting the growth of various tumor (liver cancer, highly metastatic liver cancer, lung cancer, acute monocytic leukemia, colon cancer, prostate cancer) cells, inhibiting cell senescence, protecting the genomic stability of cardiomyocytes, and inhibiting cardiac fibrosis.

[0022] Other advantages, objectives, and features of the present invention will be partially reflected by the following description, and partially will also be understood by those skilled in the art through the research and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the NMR diagram of the target compound of the present invention;

[0024] Figure 2It is a test chart of the OD values of the activities of multiple cancer cells;

[0025] Figure 3 It is a staining identification chart of myocardial senescent cells before and after intervention;

[0026] Figure 4 It is a staining identification chart of mouse heart fibrosis before and after intervention;

[0027] Figure 5 It is a physical picture of the compound of the present invention. Detailed implementation manners

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, so that those skilled in the art can implement it according to the description in the specification.

[0029] It should be noted that the experimental methods described in the following implementation schemes are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0030] Example 1

[0031] 6-{[(3-Ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid, the chemical structural formula is shown in formula (I):

[0032] This compound is hereinafter represented by the code CXM10012.

[0033] The physical picture of the prepared compound is as Figure 5 shown.

[0034] The preparation method of 6-{[(3-Ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid includes the following steps:

[0035] S1. Mix 1,4-diphenyl-1,3-butadiene and maleic anhydride in a mass ratio of 1:2.5, react in xylene vapor and a potassium hydroxide (KOH) solution with a mass fraction of 65% for 10 hours, and then cool to room temperature to obtain Product 1;

[0036]

[0037] S2. Mix Product 1 and 3-ethylaniline in a mass ratio of 2:1, and react in a weak acid environment for 20 - 30 minutes to obtain the target compound.

[0038]

[0039] In the preparation method of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid, the reaction conditions in S1 are 180 °C and a humidity of 65%.

[0040] In the preparation method of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid, the reaction conditions in S2 are room temperature, a dry and well-ventilated environment, and the reaction time is 20 - 30 minutes.

[0041] Application of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid in the preparation of drugs for protecting the genomic stability of cardiomyocytes and inhibiting cardiac senescence.

[0042] Application of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid in the preparation of drugs for treating liver cancer, highly metastatic liver cancer, colon cancer, lung cancer, acute monocytic leukemia, and prostate cancer.

[0043] Application of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid in the preparation of drugs for treating senile cardiac fibrosis.

[0044] Experiment

[0045] 1. In vitro anti-tumor activity experiment

[0046] Using the MTT method, in vitro cytotoxicity assays were performed on human liver cancer Hep3B cells, highly metastatic liver cancer HCCLM3 cells, colon cancer HCT116 cells, lung cancer A549 cells, monocytic leukemia THP-1 cells, and prostate cancer DU145 cells with reference to the experimental operations in the patent application number 202210976338.5.

[0047] After culturing the culture plate under the conditions of 37 °C, 5% CO2, and saturated humidity for 12 h, 10 μL of CCK-8 solution was added to each well, and the culture was continued for 2 h. Then, it was placed on a shaker and shaken at a low speed for 5 min. The OD value of each well was detected with an enzyme-linked immunosorbent assay instrument (the light source was at a wavelength of 450 nm). The higher the OD value, the more cells survived, and the lower the OD value, the fewer cells survived. The measured results are as Figure 2 shown. Figure 2The figure in the upper left corner shows the cell viability measured at 12 h and 24 h under different drug concentrations (0, 5, 10, 20, 30, 50 μM). It can be seen that the viability of liver cancer cells decreased significantly after being treated with this compound for 12 h, that is, the compound described in the present invention has the effect of inhibiting the growth of liver cancer cells. Therefore, when testing the viability of the above-mentioned other cells, only the cell viability after the compound is treated for 12 h needs to be measured to evaluate the regulatory effect of the compound on cell viability. Finally, the drug IC50 concentration was obtained by converting the cell viability of each cell under the treatment of the compound for 12 h, and the results are shown in Table 1.

[0048] Table 1 Drug IC50 concentration values corresponding to each cell

[0049] Cell line IC50 (μM) Hep3B 62.5 HCCLM3 80 HCT116 22.5 A549 65 THP-1 8.5 DU145 12

[0050] As can be seen from Table 1, the compound CXM10012 of the present invention has a similar effect of inhibiting cancer cell growth as the compound CXM102 described in Application No. 202210976338.5, but the inhibitory effect is more obvious.

[0051] 2. Experiments on inhibiting cell senescence and maintaining genomic stability of human cardiomyocytes in vitro

[0052] Identification was carried out by double staining of the cell senescence-specific protein marker p16INK4a and the cell genomic damage γH2AX.

[0053] Human cardiomyocytes were seeded in a special stem cell culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (double antibody), and cultured in a culture dish at 37 °C, 5% CO2 and saturated humidity. The medium was changed every 3 days. After the cells grew to confluence in the culture dish, they were digested with 0.25% trypsin and then subcultured.

[0054] Cells growing to the logarithmic growth phase in the culture medium were prepared into a single cell suspension with a concentration of 1×10 5 / mL with a culture medium containing 10% fetal bovine serum and 1% double antibody, and inoculated into a 12-well cell culture plate at 10,000 cells per well, and cultured under the conditions of 37 °C, 5% CO2 and saturated humidity for 24 h. After the cells adhered to the wall, the compound in Example 1 and hydrogen peroxide with a concentration of 600 μM were added to the experimental group, as the experimental group; among them, for the addition of the anti-cell senescence compound in the experimental group, 2 concentration gradients (10 μM, 20 μM) were set. At the same time, a negative control group was set, and the negative control group only added 600 μM hydrogen peroxide and did not add the compound in Example 1. Among them, the cells were treated with hydrogen peroxide for 2 h, and then half of the fresh medium was changed and the cells were cultured for another 12 h.

[0055] (2) For the identification of molecular markers of cellular senescence and cellular genomic damage, immunofluorescence staining was used to simultaneously identify the expression levels of intracellular molecules with antibodies against the cellular senescence-specific molecule p16INK4a and the genomic damage-specific molecule γH2AX. After sucking the culture medium dry from the well plate, the cells were washed once with PBS buffer, treated with 4% paraformaldehyde (500 μL per well) for 20 min, then treated with 0.1% Triton solution (100 μL) for 10 min, and then incubated with the diluted antibodies of molecules p16INK4a and γH2AX (1:200) diluted with 1% BSA (50 μL) at 4 °C for 12 h. After washing with PBS buffer, the cells were incubated with fluorescent antibodies at room temperature for 1 h, and finally stained with DAPI solution for 10 min, then washed and photographed under a microscope. As Figure 3 shown, in the left column, Control represents the cells not treated with the compound of Example 1 of the present invention. The middle column and the right column, CXM10012, respectively represent the cells treated with 10 μM and 20 μM of the compound of Example 1. In the figure, green represents the cellular senescence-specific molecular marker p16INK4a, and the more green indicates the more cellular senescence. Red represents the cellular genomic damage-specific molecular marker γH2AX, and the more red indicates the more cellular genomic damage. Blue represents cellular localization staining (DAPI), and one blue represents one cell. Merge represents the composite image of the pictures expressing p16INK4a and γH2AX molecules and cellular localization, which is convenient for observing the distribution of p16INK4a and γH2AX expressed in senescent cells. Thus, it can be seen that the compound of the present invention can inhibit the senescence of MSCs and promote the repair of cells after genomic damage.

[0056] 3. Experiment on inhibiting myocardial fibrosis in aged mice in vivo

[0057] To detect the myocardial fibrosis condition, the heart tissue sections were stained with masson stain.

[0058] The experiment was set up with a young control group, an aged mouse DMSO injection control group, and an aged mouse drug administration experimental group. The experimental group was administered to 16-month-old C57BL6 mice by intramuscular injection at a dose of 10 mg / kg, twice a week. The control group was intramuscularly injected with the same volume of the drug (10 μL). After 3 months, the mice were sacrificed and the kidneys were taken, soaked in 4% paraformaldehyde for 48 h, and then soaked in 30% sucrose until the heart tissue blocks sank to the bottom from the suspended state, and then frozen sections were made. During staining, the sections were soaked in 4% paraformaldehyde for 20 min again, washed with PBS, stained with masson stain, washed with PBS after 10 min, and photographed under a microscope. As Figure 4As shown, the more blue in the figure indicates the more severe the cardiac fibrosis. It can be seen from this that compared with young mice (such as Figure 4 shown in the first row), the heart of old mice (such as Figure 4 shown in the second row) has undergone severe fibrotic changes. After treatment with the compound of Example 1 of the present invention, the cardiac fibrosis of old mice is significantly reduced (such as Figure 4 shown in the third row), indicating that the compound of the present invention can inhibit cardiac fibrosis in old mice.

[0059] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated and described examples here.

Claims

1. 6-{[(3-Ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid, characterized in that, The chemical structural formula is shown in Formula (I): （I）。 2. Process for the preparation of 2,6-{(3-ethylphenyl)amino}carbonyl-2,5-diphenylcyclohex-3-ene-1-carboxylic acid, characterized in that, It includes the following steps: S1. 1,4-diphenyl-1,3-butadiene reacts with maleic anhydride in xylene vapor and an alkaline solution to obtain Product 1; S2. Product 1 reacts with 3-ethylaniline in a weak acid environment to obtain the target compound.

3. The preparation method of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid according to claim 2, characterized in that, The reaction conditions in S1 are 180 °C, humidity 65%, and the reaction time is not less than 10 h.

4. The preparation method of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid according to claim 2, characterized in that, The reaction conditions in S2 are a weak acid and a dry environment, and the reaction time is 20 - 30 min.

5. The preparation method of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid according to claim 2, wherein, The alkaline solution is one or more of potassium hydroxide solution and sodium hydroxide solution.

6. The preparation method of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid according to claim 2, characterized in that, The weak acid is one or more of glacial acetic acid and carbonic acid.

7. Use of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid as claimed in claim 1 in the preparation of a drug for inhibiting cell senescence and protecting genomic stability.

8. Use of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid as claimed in claim 1 in the preparation of a drug for treating liver cancer, highly metastatic liver cancer, lung cancer, acute monocytic leukemia, colon cancer, and prostate cancer.

9. Use of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid as claimed in claim 1 in the preparation of a drug for treating cardiac fibrosis.

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