Applications of 6-{[(3-ethylbenzene)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid

Abstract

This invention discloses the application of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-en-1-carboxylic acid in the preparation of drugs that enhance autophagy activity, and its structural formula is (I). This invention mainly explores the application of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-en-1-carboxylic acid in the preparation of drugs that inhibit bone marrow mesenchymal stem cell aging, combat senile osteoporosis, prevent and treat senile fatty liver, and prevent and treat senile renal fibrosis by enhancing autophagy activity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology. More specifically, this invention relates to the application of 6-{[(3-ethylbenzene)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid. Background Technology

[0002] Rapamycin is currently the standard drug used to enhance autophagy activity. However, clinical trials have shown that the drug has serious side effects (such as megaloblastic anemia, abnormal liver and kidney function, lactic acidosis, etc.) and is not suitable for long-term, high-dose administration. Therefore, there is an urgent need for safer new drugs to promote autophagy activity. Summary of the Invention

[0003] This invention provides the application of 6-{[(3-ethylbenzene)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid, which enhances autophagy activity and is used in the preparation of drugs that inhibit bone marrow mesenchymal stem cell aging, combat osteoporosis in the elderly, prevent and treat fatty liver in the elderly, and prevent and treat renal fibrosis in the elderly.

[0004] To achieve these objectives and other advantages of the present invention, the use of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-en-1-carboxylic acid in the preparation of drugs that enhance autophagy activity is provided, the structural formula of which is (I):

[0005]

[0006] Preferably, the drug that enhances autophagy activity includes drugs used in the preparation of drugs that inhibit bone marrow mesenchymal stem cell aging and / or combat senile osteoporosis and / or prevent senile fatty liver and / or prevent senile renal fibrosis.

[0007] Preferably, the drug for inhibiting bone marrow mesenchymal stem cell aging and / or anti-aging osteoporosis enhances the autophagy activity of bone marrow mesenchymal stem cells by adding it to them.

[0008] Preferably, the drug for preventing and treating fatty liver and / or renal fibrosis in the elderly is administered via intramuscular or intravenous injection to enhance the autophagy activity of liver and kidney cells.

[0009] Preferably, the drug contains a therapeutically effective amount of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid and a pharmaceutically acceptable carrier.

[0010] Preferably, pharmaceutically acceptable carriers include solvents, diluents, solubilizers, and buffers.

[0011] Preferably, the drug is formulated as a pharmaceutically permissible injectable preparation.

[0012] Preferably, the injectable formulation is an injection solution or an injection microemulsion.

[0013] Preferably, the dosage of 6-{[(3-ethylbenzene)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid is not less than 10 mg / kg per dose, twice a week.

[0014] The present invention has at least the following beneficial effects:

[0015] The compounds of this invention exhibit a more efficient ability to induce autophagy than rapamycin. Comparative experiments have revealed that 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohexyl-3-en-1-carboxylic acid has the potential to replace rapamycin and be used to prepare safer autophagy inducers. Furthermore, the compounds of this invention have been verified to inhibit bone marrow mesenchymal stem cell senescence, combat osteoporosis in the elderly, prevent and treat fatty liver disease in the elderly, and prevent and treat renal fibrosis in the elderly.

[0016] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0017] Figure 1 This is a microscope image showing the effect of CXM10012 and rapamycin on autophagy activity in cells according to the present invention.

[0018] Figure 2 This is a bar graph showing the effect of CXM10012 and rapamycin on autophagy activity in cells.

[0019] Figure 3This is a staining image of β-galactosidase (SA-β-Gal) specific to BMSC cell senescence;

[0020] Figure 4 This is a staining image of p16INK4A, a molecular marker specific to BMSC cell senescence.

[0021] Figure 5 This is a graph showing the detection of reactive oxygen species in BMSCs;

[0022] Figure 6 This is a staining pattern of DNA damage in BMSCs;

[0023] Figure 7 This is a graph showing the mitochondrial membrane potential of BMSCs;

[0024] Figure 8 This is a graph showing the adipogenic differentiation of BMSCs;

[0025] Figure 9 This is a graph showing osteogenic differentiation of BMSCs;

[0026] Figure 10 This is a diagram showing the effect of the present invention CXM10012 and rapamycin in the prevention and treatment of osteoporosis in the elderly, including serum bone metabolism indicators (P1NP, CTX-1), bone mineral density, bone volume, trabecular bone and other related indicators.

[0027] Figure 11 This is a microscopic image showing the effects of CXM10012 and rapamycin on fatty liver in aged mice.

[0028] Figure 12 This is a bar graph showing the effects of CXM10012 and rapamycin on fatty liver in aged mice.

[0029] Figure 13 This is a microscopic image showing the effect of CXM10012 of the present invention on renal fibrosis in aged mice with rapamycin;

[0030] Figure 14 This is a bar graph showing the effect of CXM10012 of the present invention on the renal tubular diameter of aged mice with rapamycin. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0032] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0033] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0034] This invention provides the application of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohexyl-3-en-1-carboxylic acid in the preparation of drugs that enhance autophagy activity. The main focus is on its application in the preparation of drugs that inhibit bone marrow mesenchymal stem cell aging, combat senile osteoporosis, prevent and treat senile fatty liver, and prevent and treat senile renal fibrosis by enhancing autophagy activity. The structural formula of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohexyl-3-en-1-carboxylic acid is Formula (I):

[0035] This compound is hereinafter referred to by the code CXM10012.

[0036] 1. In vitro induced autophagy activity experiment

[0037] Autophagy activity was identified using LC3II staining, a specific protein marker for autophagy activity.

[0038] Bone marrow mesenchymal stem cells (BMSCs) from a young person (donated by a 14-year-old volunteer) and an elderly person (donated by a 66-year-old volunteer) were seeded in a specialized stem cell culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (double antibiotics). The cells were cultured in culture dishes at 37°C, 5% CO2, and saturated humidity, with the medium changed every 3 days. After the cells reached confluence, they were digested with 0.25% trypsin and passaged. Cells that had reached the logarithmic growth phase were collected and reconstituted with a culture medium containing 10% fetal bovine serum and 1% double antibiotics to a concentration of 1×10⁻⁶ cells / mL. 5 Single-cell suspensions of CXM10012 were seeded at 40,000 cells per well in 12-well cell culture plates and cultured for 24 h at 37°C, 5% CO2, and saturated humidity. After cell attachment, 20 μM of CXM10012 compound was added to the experimental group; a control group was also set up, which received 20 μM rapamycin. A blank experimental group was also set up, in which only an equal volume of solvent was added to the cell culture medium. Because autophagy is a dynamic process of generation and consumption, to more accurately measure the autophagy activity induced by CXM10012, an old BMSCs+CQ group was also set up, in which chloroquine was added to the old BMSCs experiment to block autophagy consumption. When autophagy consumption was shut down, the more LC3II protein produced intracellularly, the higher the autophagy activity.

[0039] For the detection of autophagy, the autophagy marker molecule LC3II was identified using immunofluorescence staining. Culture plates were incubated at 37°C, 5% CO2, and saturated humidity for 12 hours. After fixation with 4% paraformaldehyde, the cell membrane was permeabilized with 100 μL of 0.1% permeabilization buffer for 20 minutes, followed by treatment with 2% blocking buffer for 30 minutes. Immediately afterward, the cells were incubated with LC3II protein antibody at room temperature for 2 hours, then stained with a green fluorescent antibody for 45 minutes. The cells were then removed, and the nuclei were stained with DAPI staining solution for 10 minutes. After washing, the cells were photographed under a microscope. Figure 1 As shown in the diagram, more green and stronger green indicates a higher concentration of LC3II protein, meaning stronger autophagy. Figure 2 As can be seen, compared with the control group, both Rapamycin and CXM10012 can enhance autophagy activity, but CXM10012 induces significantly more autophagy activity than Rapamycin (** indicates that the difference between the two is statistically significant).

[0040] 2. In vitro inhibition of BMSC aging experiment

[0041] Human BMSCs were seeded in a special stem cell culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (double antibiotics), and cultured in a culture dish at 37°C, 5% CO2 and saturated humidity. The medium was changed every 3 days. After the cells had grown to a confluence with the culture dish, they were digested with 0.25% trypsin and then passaged.

[0042] When examining the effect of drugs on cell senescence, cells grown to the logarithmic growth phase in the culture medium were used to prepare a solution with a concentration of 1×10⁻⁶ cells / mL using a culture medium containing 10% fetal bovine serum and 1% penicillin-dextrose anhydride. 5 A single-cell suspension of 40,000 cells per well was seeded into 12-well cell culture plates at a density of 1 mL / mL and cultured at 37°C, 5% CO2, and saturated humidity for 24 h. After cell attachment, 20 μM of the CXM10012 compound of this invention and 600 μM hydrogen peroxide were added to the cell culture medium to form the experimental group. A negative control group and a positive control group were also set up. The negative control group received only 600 μM hydrogen peroxide, while the positive control group received 20 μM rapamycin. The hydrogen peroxide treatment time was 2 h, followed by culturing in half fresh medium for another 12 h.

[0043] 2.1 For the detection of cell senescence, SA-β-Gal staining was used for identification. The culture plate was incubated at 37℃, 5% CO2, and saturated humidity for 12 hours. 500 μL of SA-β-Gal staining working solution was added to each well, and the plate was incubated at 37℃ for 2 hours. After incubation, the cell nuclei were stained with nuclear-specific staining solution (DAPI) for 10 minutes, then washed and photographed under a fluorescence microscope. Figure 3 As shown, in the top row of staining, SA-β-Gal staining only stained senescent cells blue-green; the more blue-green cells there are, the more senescent cells there are. In the bottom row of staining, DAPI staining only stained the cell nuclei, indicating that the total number of cells in each experiment remained consistent. Therefore, CXM10012 is more effective than rapamycin in inhibiting the expression of senescence-specific β-galactosidase in BMSCs.

[0044] 2.2 For the detection of molecular markers of cellular senescence, specific antibodies were used to identify intracellular p16INK4a molecules through immunofluorescence staining. After aspirating the culture medium from the wells, 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 a 1% BSA solution (50 μL) diluted p16INK4a antibody (1:200) at 4°C for 12 h. After washing with PBS buffer, the cells were incubated with green fluorescent antibody at room temperature for 1 h. Finally, the cell nuclei were stained with DAPI (blue) for 10 min, washed, and photographed under a fluorescence microscope. Figure 4 As shown in the figure, more green indicates more senescence-specific proteins expressed by the cells. DAPI represents cell localization, one blue dot represents one cell, and Merge represents a composite image of the p16INK4a molecule expression and cell localization. Therefore, CXM10012 is more effective than rapamycin in inhibiting the expression of senescence-specific proteins in BMSCs.

[0045] 2.3 For the detection of reactive oxygen species (ROS), specific ROS probes were used to identify intracellular ROS molecules through fluorescent staining. First, the ROS probe working solution (DCFH-DA) was diluted 1:1000 with serum-free culture medium to a final concentration of 10 μM. Then, the cell culture medium was removed, and 300 μL of DCFH-DA working solution was added to each well. The cells were incubated at 37°C for 20 min. The cells were then washed three times with serum-free cell culture medium to thoroughly remove any uninfiltrated DCFH-DA, and finally photographed under a fluorescence microscope. Figure 5 As shown in the figure, the more green there is, the more ROS in BMSCs. Therefore, it can be concluded that CXM10012 is more effective than rapamycin in eliminating ROS accumulation caused by BMSC aging.

[0046] 2.4 For the detection of DNA damage, immunofluorescence staining of intracellular γH2A.X molecules with specific antibodies was used for identification. After aspirating the culture medium from the wells, 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 a 1% BSA solution (50 μL) diluted γH2A.X antibody (1:200) at 4°C for 12 h. After washing with PBS buffer, the cells were incubated with red fluorescent antibody at room temperature for 1 h. Finally, the cell nuclei were stained with DAPI (blue) for 10 min, washed, and photographed under a microscope. Figure 6 As shown in the figure, more red indicates more DNA-damaging molecules expressed by the cells. DAPI represents cell localization, one blue line represents one cell, and Merge represents a composite image of the γH2A.X molecule expression and cell localization. Therefore, CXM10012 is more effective than rapamycin in maintaining the DNA stability of BMSCs.

[0047] 2.5. For the detection of mitochondrial membrane potential in BMSCs, a mitochondrial membrane potential change kit was used for identification. First, 300 μL of JC-1 staining working solution was added to the prepared cell culture medium, mixed thoroughly, and incubated at 37°C for 20 min. The supernatant was removed, and the cells were washed twice with JC-1 staining buffer. Then, 300 μL of cell culture medium was added, and the cells were observed under a fluorescence microscope. Figure 7 As shown, red indicates that the JC-1 probe aggregates in the mitochondrial matrix in polymer form, indicating normal mitochondrial membrane potential, i.e., normal mitochondrial function; green indicates that the JC-1 probe is free in monomeric form outside the mitochondria, indicating decreased mitochondrial membrane potential, i.e., impaired mitochondrial function. Therefore, CXM10012 can more significantly inhibit the loss of mitochondrial membrane potential caused by BMSC aging than rapamycin, thereby maintaining mitochondrial function.

[0048] 2.6. For the detection of adipogenic differentiation of BMSCs, Oil Red O staining solution, a specific stain for adipogenic differentiation, was used for identification. BMSCs were treated with adipogenic differentiation induction culture medium. After 7 days, they were fixed with 4% paraformaldehyde, stained with Oil Red O solution for 15 minutes, washed, and photographed under a microscope. Figure 8 As shown in the figure, the more red granules there are, the more BMSCs differentiate into adipocytes. Therefore, CXM10012 significantly inhibits the adipocyte differentiation characteristics of BMSCs than rapamycin.

[0049] 2.7. For the detection of osteogenic differentiation in BMSCs, alizarin red staining solution, a specific stain for osteogenic differentiation, was used for identification. BMSCs were treated with osteogenic differentiation induction culture medium. After 7 days, the cells were fixed with anhydrous ethanol for 10 min, then stained with alizarin red staining solution for 15 min, washed, and photographed under a microscope. Figure 9 As shown in the figure, the more red cells there are, the more BMSCs differentiate into osteoblasts. Therefore, CXM10012 is more effective than rapamycin in promoting osteogenic differentiation of BMSCs.

[0050] 3. In vivo experiments on anti-osteoporosis in the elderly, and in vivo experiments on inhibiting fatty liver and renal fibrosis in the elderly.

[0051] 3.1 Mice enter senescence at 14 months of age, therefore 14-month-old mice were used as the starting point for the experiment. The experiment included an injection control group and a drug administration group. In the control group, mice received an intramuscular injection of DMSO at a dose equal to the drug volume (10 μL). In the drug administration group, mice received intramuscular injections of compound CXM10012 and rapamycin at a dose of 10 mg / kg, respectively, twice weekly until 18 months of age. DMSO was used as the solvent to dissolve all drugs. For ease of description, in the analysis of data on the efficacy in preventing and treating osteoporosis in the elderly, the control group was labeled "Old," the mice injected with compound CXM10012 were labeled "OC," and the mice injected with rapamycin were labeled "OR."

[0052] 3.2 For the detection of osteoporosis assessment indicators, enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of type I procollagen N-terminal peptide (P1NP) and type I collagen C-terminal peptide (CTX-1) in serum. P1NP and CTX-1 are clinically recognized indicators for assessing bone synthesis and bone resorption activity in vivo. Comparison revealed that the serum P1NP level in mice injected with CXM10012 was significantly higher than that in the control group (see...). Figure 10 A) indicates that CXM10012 can promote bone synthesis and metabolism in mice, increasing bone formation. However, detection of CTX-1 revealed that CXM10012 significantly reduced serum CTX-1 levels in mice, indicating that the drug inhibits bone resorption activity in mice (see A). Figure 10 B). Therefore, CXM10012 can both promote bone formation and inhibit bone resorption in mice, making it a potential drug for treating osteoporosis in the elderly.

[0053] 3.3 For the detection of bone mineral density and bone volume in osteoporosis, microCT scanning was used. Mice were anesthetized at 14 months of age, and a full-segment scan of the femur was performed using microCT. Mice were then fed until 18 months of age. MicroCT scans were performed every month during this period, and the femoral mineral density (BMD), bone volume (BV / TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), and trabecular bone spacing (Tb.Sp) were analyzed using 3D imaging software. Figure 10 As shown, Figure 10 C is a three-dimensional image of the mouse femur. Figure 10 DH is a parameter for osteoporosis in the elderly. Therefore, CXM10012 is more effective than rapamycin in preventing and treating osteoporosis in the elderly.

[0054] 3.4 Effects of the drug on the liver and kidneys in the body

[0055] The liver and kidneys are the main metabolic organs after drugs enter the body. By observing their tissue morphology, the toxic effects of CXM10012 and rapamycin on mice can be determined.

[0056] The experimental mice were grouped and treated with the drugs as described in (3.1) above. Mice in each group were randomly selected, sacrificed, and their livers and kidneys were harvested. The livers and kidneys were soaked in 4% paraformaldehyde for 48 hours, and then soaked in 30% sucrose until the tissue blocks settled from suspension. The tissue blocks were then frozen sectioned and stained for analysis. In addition, livers and kidneys before drug administration were used as blank controls.

[0057] To observe liver tissue morphology, the sections were soaked in 4% paraformaldehyde for 20 minutes, washed with PBS, and then stained with 1.5% Oil Red O-isopropanol solution. After 10 minutes, they were washed with PBS and photographed under a microscope. Figure 11 As shown, the more red particles there are, the more fat granules formed in the liver tissue. Figure 12 As shown, CXM10012 significantly reduced fatty granules in mouse livers more than rapamycin (** indicates a significant difference between the two). Therefore, CXM10012 is more effective than rapamycin in preventing and treating fatty liver disease in the elderly.

[0058] To observe the morphology of kidney tissue, the sections were soaked in 4% paraformaldehyde for 20 minutes, washed with PBS, stained with Masson's stain, washed with PBS after 10 minutes, and photographed under a microscope. Figure 13 As shown in the diagram, the more blue there is, the more severe the renal fibrosis; the more red there is, the more intact the renal tissue muscle fiber structure. Figure 14As shown, pathological analysis revealed that CXM10012 significantly reduced the incidence of renal fibrosis compared to rapamycin, and CXM10012 significantly increased renal tubular diameter compared to rapamycin (** indicates a significant difference between the two). Therefore, CXM10012 is more effective than rapamycin in preventing and treating renal fibrosis in the elderly.

[0059] The above experimental results indicate that CXM10012 has a more effective effect than rapamycin in inhibiting bone marrow mesenchymal stem cell aging, resisting senile osteoporosis, preventing and treating senile fatty liver and senile renal fibrosis, and also suggests that CXM10012 has lower drug toxicity than rapamycin.

[0060] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0061] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

The application of 1,6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohexyl-3-en-1-carboxylic acid in the preparation of drugs that enhance autophagy activity, characterized in that, The structural formula of the 6-{[(3-ethylbenzene)amino]carbonyl}-2,5-diphenylcyclohex-3-en-1-carboxylic acid is formula (I): （I）； The drug that enhances autophagy activity is used to treat osteoporosis in the elderly and / or to prevent fatty liver in the elderly and / or to prevent renal fibrosis in the elderly. The drugs for preventing and treating fatty liver and / or renal fibrosis in the elderly are administered via intramuscular or intravenous injection to enhance the autophagy activity of liver and kidney cells. The drug is formulated into a pharmaceutically permissible injectable preparation, which is an injection solution or an injectable microemulsion. The dosage of 6-{[(3-ethylbenzene)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid in the drug is not less than 10 mg / kg each time, twice a week.

2. The application as described in claim 1, characterized in that, The drug contains a therapeutically effective amount of 6-{[(3-ethylphenyl)amino]carbonyl}-2,5-diphenylcyclohex-3-ene-1-carboxylic acid and a pharmaceutically acceptable carrier.

3. The application as described in claim 2, characterized in that, Pharmaceutically acceptable carriers include solvents, diluents, solubilizers, and buffers.

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