CN112641781B

CN112641781B - SARMs compounds containing ester-based aromatic propionamide and application of metabolites thereof in preparation of anti-new coronavirus drugs

Info

Publication number: CN112641781B
Application number: CN202110022301.4A
Authority: CN
Inventors: 朱新法; 曹长清; 朱然
Original assignee: Changchun Genescience Pharmaceutical Co Ltd
Current assignee: Changchun Genescience Pharmaceutical Co Ltd
Priority date: 2021-01-08
Filing date: 2021-01-08
Publication date: 2022-07-12
Anticipated expiration: 2041-01-08
Also published as: WO2022147917A1; CN112641781A; CN116829189A

Abstract

The invention belongs to the technical field of medicines, and particularly discloses an application of SARMs compound EG-017 containing ester-based aromatic propionamide and a metabolite EG-2 thereof in preparation of anti-new coronavirus medicines, inhibition of multi-organ injuries of lung, heart and the like caused by new coronavirus and treatment of pulmonary fibrosis. IFA is applied to evaluate the activity and cytotoxicity of the compound against the novel coronavirus (SARS-CoV-2) in vitro, and the obvious inhibition activity of EG-017 on the replication of the novel coronavirus beta CoV/KOR/KCDC03/2020 strain in Vero cells is well verified, which shows that the compound has application prospect in preparing anti-novel coronavirus medicines; experiments also show that EG-017 can well inhibit the cytotoxicity of high-concentration rapamycin to cells; the MTT method proves that EG-017 can obviously improve the survival rate of rat myocardial cells. The potential application value of EG-017 and metabolites thereof in treating lung diseases such as lung fibrosis caused by new coronavirus and lung diseases such as lung fibrosis caused by other reasons is verified by an experiment on the influence of EG-017 on a TGF-beta 1 induced A549 lung cancer cell fibrosis process.

Description

SARMs compounds containing ester-based aromatic propionamide and application of metabolites thereof in preparation of anti-new coronavirus drugs

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an SARMs compound containing ester-based aromatic propionamide and application of a metabolite thereof in preparation of a new coronavirus resistant medicine.

Background

At the initial stage, COVID-19 mainly shows fever, dry cough, hypodynamia and the like, and a few patients are accompanied with upper respiratory tract and digestive tract symptoms such as nasal obstruction, nasal discharge, diarrhea and the like, so that severe cases are developed; in the early stage, lung diseases are mainly caused and further spread to heart diseases and other organs of the whole body; the clinical symptom is dyspnea, and severe patients rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis, coagulation dysfunction and multiple organ failure. The main transmission route of the novel coronavirus is respiratory droplet transmission and contact transmission, and epidemiological investigation shows that many cases can track the close contact condition with the diagnosed cases. Currently, no effective drug is available for control.

Disclosure of Invention

The invention aims to provide an application of SARMs (selective antigen receptors modulators) compound containing ester-based aromatic propionamides and a metabolite thereof in preparing anti-new coronavirus medicines.

In order to achieve the purpose, the invention adopts the technical scheme that:

SARMs compounds containing ester-based aromatic propionamide and application of metabolites thereof in preparing anti-new coronavirus medicines.

Preferably, the SARMs compound containing ester-based aromatic propionamide is EG-017 and has the chemical formula C₂₅H₁₇F₃N₄O₄。

Preferably, the metabolite of the ester group-containing aromatic propanamide compound is EG-2.

Preferably, the ester group-containing aromatic propionamide compound and the metabolite thereof are used for inhibiting the cytotoxicity of rapamycin on cells.

Preferably, EG-017 and its metabolites are used in the manufacture of a medicament for treating lung lesions caused by new corona viruses.

Preferably, EG-017 and metabolites thereof are potentially useful for the preparation of a medicament for the treatment of pulmonary fibrosis lung diseases.

Preferably, EG-017 and its metabolites are used in the preparation of a medicament for treating diseases caused by adriamycin damage to cardiomyocytes.

The invention has the advantages that: the activity and cytotoxicity of the compound against novel coronavirus (SARS-CoV-2) in vitro are evaluated by immunofluorescence assay (IFA). The EG-017 has obvious inhibitory activity on the replication of a novel coronavirus beta CoV/KOR/KCDC03/2020 strain in Vero cells, and shows that the EG-017 has application prospect in preparing anti-novel coronavirus medicines.

Drawings

FIG. 1: histogram of cytotoxicity of EG-017 at various concentrations.

FIG. 2: different concentrations of EG-017 induced cell damage histograms.

FIG. 3: effect of different concentrations of EG-017 on Vim protein expression in a549 cells.

FIG. 4: effect of different concentrations of EG-017 on TGF-. beta.1 induced proliferation of hepatic stellate cells.

Detailed Description

EG017 as mentioned herein is a novel non-steroidal SARM of the formula C₂₅H₁₇F₃N4O4, chinese chemical name (S) -1- ((4-cyano-3- (trifluoromethyl) phenyl) amino) -3- (4-cyanophenoxy) -2-methyl-1-oxopropan-2-yl nicotinate. The preparation method and the structural formula of EG017 refer to the content described in a patent document with a patent number of CN 201410033958.0.

Mechanism of action of EG-017

Preliminary studies indicate that the new coronavirus causes damage to human body through ACE2 target; ACE2 is present in the epithelium of the nose, mouth and lungs as well as in most of the major tissues and organs of the human body, and is the entry of new coronavirus infection into the body; the new coronavirus initially causes lung infection, causes serious damage to lung epithelium and endothelial tissues, and causes heart and other important tube lesions through blood diffusion; ACE2 contains androgen receptor, SARMs has effect target as androgen receptor, and exogenous androgen receptor regulator SARMs is supplemented in large amount, so that normal biological function of lung organ containing ACE2 can be rapidly activated, lung immune system can be regulated, and lung injury caused by immune storm due to new coronavirus can be avoided; meanwhile, the exogenous sarms can be combined with the ACE2 competitive with the virus and relevant cells (so as to reduce the invasion probability of the virus to the cells and the invasion of the virus to the organism), thus indirectly enhancing the tolerance of the organism to the virus and promoting the organism to quickly recover health in the fight between the organism and the virus.

1) A large amount of exogenous non-steroidal androgen receptor modulators are utilized to regulate the activity of macrophages (containing androgen receptors) in the lung and enhance the virus removal capacity of lung tissues; 2) the combination of an exogenous non-steroidal androgen regulator and in-vivo ACE2 (containing androgen receptor) is utilized to change the activity and spatial structure of ACE2 and block the combination point of virus and ACE2, thereby reducing the harm of the virus to human body through an ACE2 channel; 3) the exogenous androgen receptor regulator is used to regulate the activity of cell nucleus, raise the self-repairing capacity of body while resisting virus.

In particular, studies have shown that the human body has a specific protein that enables the virus to infect human cells. This protein is known as angiotensin converting enzyme 2, or ACE2 is the "receptor" that provides an entry point for the coronavirus to enter and infect various human cells. ACE2 is present in many cell types and tissues, including lung, heart, blood vessels, kidney, liver, and gastrointestinal tract. It is present in epithelial cells that are in contact with certain tissue lines and form a protective barrier, and oxygen and carbon dioxide exchange between the lung and blood vessels occurs on the epithelial lining of the lung. ACE2 is present in the epithelium of the nose, mouth and lungs. In the lung, ACE2 is very abundant on type 2 pneumocytes, an important cell type, present in the chambers of the lung lungs where oxygen is absorbed and waste carbon dioxide is released.

ACE enzyme converts angiotensin I to angiotensin II. The main role of ACE2 is to break down angiotensin II into molecules to counteract the deleterious effects of angiotensin II; that is, ACE2 helps regulate many activities of a protein called angiotensin ii (ang ii), which increases blood pressure and inflammation, increases damage to the vascular lining and various types of tissue damage. ACE2 converts ANG II into other molecules to counteract the effects of ANG II.

Also, studies have shown that most closely related to COVID-19, ANG II increases inflammation and alviginolide cell death, which is critical for the introduction of oxygen into the body, and taken together, these detrimental effects of ANG II are reduced by ACE 2.

In vitro cell experiment results show that the effects (efficiency Max Dose) of EG02 and EG017 on HEK293 with androgen receptor transferred by external source are all more than 70%; on human prostate cancer cell LNCaP endogenously expressing an androgen receptor, EG02 and EG017 showed good tissue selectivity, and the highest Dose Efficacy (Efficacy Max Dose) EG02 was 66.7%; and 47.2% for EG 017. The results show that both EG017 and EG02 have tissue selectivity, show good selective androgen receptor regulation effect, and particularly have more remarkable EG017 effect.

Human metabolism studies have shown that EG-017 is metabolized to EG-2 in vivo, with concentrations reaching maximum in vivo for 1.5-3hrs and 6-7hrs, respectively.

In order to verify the effect of EG-017 on treating the pathological changes of organs such as the lung caused by the new coronavirus, the following specific examples are combined to demonstrate the beneficial effects of EG-017.

Test and control compounds

(1) Test compounds

Name: EG-017

The source is as follows: pharmaceutical source medicinal chemistry (Shanghai) Co., Ltd

Batch number: r191095

The preparation method comprises the following steps: 120mM stock solution prepared in 100% DMSO

(2) Test Compound 2

Name: rapamycin

The source is as follows: medchemiexpress

Batch number: 61925

The preparation method comprises the following steps: 40mM stock solution prepared in 100% DMSO

(3) Control Compound 1

Name: remdesivir, Reidesvir

The source is as follows: medchemiexpress

Batch number: CS-0028115

The preparation method comprises the following steps: preparation of 10mM stock solution in 100% DMSO

(4) Control Compound 2

Name: lopinavir, Lopinavir

The source is as follows: selleckchem

Batch number: s138003

The preparation method comprises the following steps: made up in 10mM stock solution in 100% DMSO

(5) Control Compound 3

Name: chloroquinone diphosphate salt, Chloroquinone, Chloroquine diphosphate, Chloroquine phosphate

The source is as follows: Sigma-Aldrich

Batch number: 109M4003V

The preparation method comprises the following steps: 30mM stock solution was prepared in 100% DMSO

Viral strains

The novel coronavirus strain β CoV/KOR/KCDC03/2020 was provided by Korea Centers for Disease Control and preservation (KCDC), having the sequence number NCCP 43326.

Cells

African green monkey kidney (Vero) cells were obtained from the American Type Culture Collection (ATCC) under the accession number CCL-81. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, WelGene) supplemented with 10% fetal bovine serum (Gibco) and 1% double antibody (Gibco). DMEM medium supplemented with 2% fetal bovine serum (Gibco) and 1% double antibody (Gibco) was used as the experimental medium.

Main instrument and reagent

(1) The main apparatus is as follows: the main instruments used in this project were a high content imaging analyzer (PerkinElmer, model Operetta), an automatic dispenser (ThermoFisher, model Multidrop comb), a plate washer (BioTek, model ELx406) and a liquid workstation (CyBio, model CyBi-HummingWell).

(2) The main reagents are as follows: the main reagents used in the project comprise novel coronavirus N protein anti-Human SARS-CoV-2N protein antibody (Beijing Yiqian Shenzhou science and technology Co., Ltd., product No. 40143-T62), ｃA sheep anti-rabbit IgG secondary antibody (Molecular Probes, product No. MOP-A-11034) marked by AlexｃA Fluor 488, and ｃA cell nucleus staining solution Hoechst 33342(Molecular Probes, product No. MOP-H-3570).

Test method

IFA experiments are used in the research to evaluate the in vitro antiviral activity of a tested compound on the new coronavirus beta CoV/KOR/KCDC03/2020 strain in Vero cells. In addition to testing the antiviral activity of the test compounds EG-017 and rapamycin when treated individually, the combined antiviral activity of EG-017 in the presence of 100. mu.M rapamycin was also tested. Redesivir, lopinavir and chloroquine phosphate were used as positive control compounds. Test compounds and control compounds were tested at 10 concentration points. The test concentrations are shown in Table 1.

TABLE 1 test concentrations of test and control Compounds

The average antiviral inhibition and average cell viability rates of the compounds at the above different concentrations are shown in the following table:

cell plating

Vero cells were trypsinized and diluted to 480,000 cells per ml with the experimental medium. The diluted cells were added to 384-well cell assay plates using an automated dispenser, 25. mu.l per well, 12,000 cells. Cells in 5% CO₂And cultured overnight in an incubator at 37 ℃.

Compound treatment and viral infection

Compounds were diluted with DMSO and then the diluted compounds were added to the wells of test cells using a liquid station. Then 25. mu.l of the experimental culture medium was added to each well to dilute the SARS-CoV-2 virus, MOI 0.0125. Cell controls (cells, no compound treatment or viral infection) and no compound treatment controls (cells infected with virus, no compound treatment, 0.5% DMSO added) were set. The final volume of cell culture broth per well was 50. mu.l. Cells in 5% CO₂Then, the culture was continued in an incubator at 37 ℃ for 24 hours.

Immunofluorescence staining

(1) 24 hours after viral infection, 17. mu.l of 16% paraformaldehyde was added to each well. Then standing for 30 minutes at room temperature;

(2) the supernatant was aspirated off and the plate washed twice with DPBS;

(3) adding 25 mul of 0.25 percent TritonX-100 into each hole, and standing for 20 minutes at room temperature;

(4) 0.25% TritonX-100 was aspirated off, and the plate was washed twice with DPBS;

(5) mu.l of diluted primary antibody (1:3000 times diluted) was added to each well and incubated at 37 ℃ for 1 hour;

(6) absorbing the primary antibody, and washing the plate twice by using DPBS;

(7) mu.l of diluted secondary antibody Alexa Fluor 488-labeled goat anti-rabbit IgG (1: 2000-fold dilution) and 2.5. mu.g/ml (1: 4000-fold dilution) of Hoechst 33342 were added to each well and incubated at 37 ℃ for 1 hour;

(8) absorbing the secondary antibody and Hoechst, and washing the plate twice by using DPBS;

(9) using a high content imaging analyzer Operetta reading plate, instrument settings: 488/405emission,20 times objective, 5 fields per well.

Data analysis

The total number of cells (Hoechst stained cells) and the number of cells infected with the novel coronavirus (Alexa Fluor 488-labeled cells) in the image obtained by plate reading with the high content imaging analyzer were quantitatively analyzed using Columbus software. The proportion of infected cells and total cell number data were used for compound antiviral activity and cytotoxicity assays.

The calculation formula is as follows:

inhibition (%) of 100 ═ 100 — (test well infected cell ratio-cell control well average infected cell ratio)/(no-compound-treated control well average infected cell ratio-cell control well average infected cell ratio) × 100

Cell viability (%). Total cell number in test well/average Total cell number in control well without Compound treatment X100

Non-linear fit analysis of inhibitory and cellular viability of compounds using XLFit 4 software and calculation of IC for compounds₅₀And CC₅₀The fitting method is "Sigmoidal dose-response". IC (integrated circuit)₅₀And CC₅₀The calculation formula of (2) is as follows: y ═ Bottom + (Top Bottom)/(1+ (IC)₅₀/X) Hillslope). Wherein, IC₅₀Indicating a 50% inhibitory concentration.

Results and conclusions

The results of the single drug test of test and control compounds for inhibiting the replication activity of the novel coronavirus in vitro are shown in table 2. The experiment was performed 1 time.

The results show that the control compounds chloroquine phosphate, lopinavir and ridciclovir all show the activity of resisting novel coronary virus, IC₅₀The values were 4.3. mu.M, 14.1. mu.M and 5.82. mu.M, respectively, in agreement with literature data (Sangeun J., et al. 2020). None of the three control compounds showed significant cytotoxicity, CC, at the tested concentrations₅₀Values greater than the highest tested concentration are shown in table 2.

The tested compounds EG-017 and rapamycin have obvious inhibitory activity on the replication of the novel coronavirus beta CoV/KOR/KCDC03/2020 strain in Vero cells, and the IC50 values are respectively 17.09 mu M and 9.34 mu M. The test compound EG-017 showed no significant cytotoxicity on Vero cells with CC50 values greater than the highest tested concentration, >300 μ M. rapamycin showed significant cytotoxicity with a CC50 value of 29.5 μ M.

In the combined antiviral activity test of 100 mu M Rapamycin and EG-017, the inhibition activity of the combined drug on viruses cannot be distinguished due to obvious cytotoxicity of the Rapamycin at the concentration of 100 mu M; however, the combined administration result shows that EG-017 can inhibit the toxicity of Rapamycin to cells at the concentration of 300 mu M-11.11 mu M; the efficacy of the combination of the two against viruses requires further evaluation by lowering the test concentration of Rapamycin.

TABLE 2 anti-New coronavirus Activity of test and control Compounds

Compound (I)	Unit of	IC₅₀	CC₅₀
				EG-017	μM	17.09	>300
rapamycin	μM	9.34	29.5
				Chloroquine	μM	4.30	>150
Lopinavir	μM	14.10	>50
				Remdesivir	μM	5.82	>50

The MTT method is adopted to further verify that EG-017 and metabolites thereof repair cytotoxicity caused by adriamycin to myocardial cells

Subject: H9C2 rat cardiomyocytes

The experimental method comprises the following steps: MTT method

The experimental principle is as follows:

the MTT method is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-violet crystalline Formazan (Formazan) and deposit the Formazan in the cells, and dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and an enzyme linked immunosorbent assay detector is used for measuring the light absorption value of formazan at the wavelength of 550nm, so that the quantity of living cells can be indirectly reflected. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number. The method is widely used for screening antitumor drugs and cytotoxicity tests.

Experimental procedure

1. Cytotoxicity of EG-017 at various concentrations

(1) Logarithmic phase H9C2 cells were collected and cell suspension concentration was adjusted to 5X 10⁴Per mL, 100ul per well, plates to density the test cells to 5000 per well, and fill the marginal wells with sterile PBS.

(2)5％CO₂Incubate at 37 ℃ until the cell monolayer is well-bottom (96-well flat bottom plate), add the concentration gradient drug, plate the day before afternoon, and add the drug the next day in the morning. EG-017 sets up six gradients of 0, 1, 10, 50, 100 and 500uM, and 100ul of each hole sets up 4 ~ 6 compound holes. And setting a zero setting hole (culture medium, MTT and dimethyl sulfoxide) and a control hole (cells, a drug dissolving medium with the same concentration, a culture solution, MTT and dimethyl sulfoxide).

(3)5％CO₂Incubated at 37 ℃ for 24 hours and observed under an inverted microscope.

(4) 20ul MTT solution (5mg/ml, i.e. 0.5% MTT), 5% CO was added per well₂The incubator was incubated at 37 ℃ for 4 hours.

(5) The culture was terminated and the culture medium in the wells was carefully aspirated.

(6) 150ul of dimethyl sulfoxide was added to each well, and the mixture was shaken on a shaker at a low speed for 10min to dissolve the crystals sufficiently. The absorbance of each well was measured at OD 490nm in an ELISA detector.

The experimental results are shown in fig. 1, wherein a: the MTT method is used for detecting the survival condition of the cells with the EG-017 action concentration of 1, 10, 50, 100 and 500M. P <0.05 compared to control group; p <0.01 compared to control group; p <0.001 compared to control group; p <0.0001 compared to control. The results show that A.EG-017 can obviously promote the proliferation of the myocardial cells when the concentration is 100 and 500M.

2. Different concentrations of EG-017 induce cell damage

(2)5％CO₂Incubate at 37 ℃ until the cell monolayer is well-bottom (96-well flat bottom plate), add the concentration gradient drug, plate the day before afternoon, and add the drug the next day in the morning. EG-017 sets up six gradients of 0, 1, 10, 50, 100 and 500uM, and 100ul of each hole sets up 4 ~ 6 compound holes. After 2 hours of pretreatment, culture was continued by changing the medium to which DOX (1uM) was added, while setting the zero-setting wells (medium, MTT, DMSO) and the control wells (cells, drug-dissolving medium of the same concentration, culture solution, MTT, DMSO).

(3)5％CO₂Incubated at 37 ℃ for 48 hours and observed under an inverted microscope.

(6) 150ul of dimethyl sulfoxide is added into each hole, and the mixture is placed on a shaking bed to be shaken at low speed for 10min so as to fully dissolve crystals. The absorbance of each well was measured at OD 490nm in an ELISA detector.

The results are shown in FIG. 2, in which A: the MTT method is used for detecting the survival condition of the cells with the EG-017 action concentration of 1, 10, 50, 100 and 500M under the condition that the adriamycin is 1M. P <0.05 compared to control group C; p <0.01 compared to control group C; p <0.001 compared to control group C; p <0.0001 compared to control C. # P <0.05 compared to control group D; # P <0.01 compared to control group D; # P <0.001 compared to control group D; # P <0.0001 compared to control group D. In the figure, C is control; d, control + DOX (1M).

The results show that the A.EG-017 can obviously improve the survival rate of the myocardial cells under DOX treatment when the concentration is 100 and 500M.

In order to further verify the effectiveness of EG-017 in preparing anti-new coronavirus medicines, experiments are carried out to verify the influence of EG-017 on the process of TGF-beta 1 inducing A549 lung cancer cell fibrosis.

Subject: a549 lung cancer cell

The experimental method comprises the following steps: WB method

The experimental principle is as follows:

western blot, a Western blot technique, is a protein detection technique in which the total proteins of cells or tissues after electrophoretic separation are transferred from a gel to a solid support NC membrane or PVDF membrane, and then a specific antigen is detected with a specific antibody, and the position of the band staining and the depth of staining of the specific protein are obtained by analysis to know the expression of the specific protein in the cells or tissues.

Transforming growth factor-beta (TGF-beta 1) is the most critical cytokine for liver fibrosis. TGF-beta 1 is used for inducing A549 lung cancer cell fibrosis in vitro, the wavy protein (Vim) in the fibrotic lung cancer cell is obviously expressed, and the expression of the cadherin E (E-Cad) is obviously reduced. And detecting protein expression levels of Vim and E-Cad by using a WB technology, and verifying the fibrosis level of A549 cells.

Experimental procedure

Western blot is used for detecting the influence of EG-017 with different concentrations on the expression of Vim and E-Cad proteins in cells,

1. protein sample preparation

(1) A549 cells were cultured to log phase and plated in six well plates after digestion. A blank control group and a dosing group were set. After 24h, when the cell monolayer is uniformly attached to about 80% of the bottom of the plate, adding 2ml of culture medium containing TGF-beta 1 and having the concentration of 20ng/ml, wherein the serum concentration of the culture medium is 2%, adding the original culture medium after 48h, discarding the original culture medium, adding culture medium containing EG-017 with different concentrations, and placing the culture medium in an incubator for 48 h.

(2) And (3) extracting total cell protein: after 48h, the six-well plate was taken out, the medium was aspirated off, washed with PBS for 2 times, pancreatin was added to digest the cells, the cells were collected and centrifuged, the supernatant was discarded, washed with PBS twice, then the prepared RIPA lysate was added, shaken back and forth or blown with a gun several times to allow the lysate to fully contact the cells, lysed on ice for 30min, and shaken every 5 min. After the lysis is finished, the EP tube is placed under a low-temperature centrifuge at 12000rpm for centrifugation for 5min, and colorless transparent supernatant is the total protein of the cells. The supernatant after centrifugation was concentrated by BCA protein assay, and the diluted protein sample was mixed with 5 XSDS loading buffer as 4: adding into the mixture at a ratio of 1, boiling in boiling water for 5min to completely denature protein, and storing at-70 deg.C.

2. SDS-PAGE electrophoretic separation of proteins

Preparing separation gel and concentrated gel according to the instruction of a gel preparation kit and the molecular weight of target protein, adding each extracted protein into a sample adding hole by using a liquid transfer gun, adding 10 mu L of each sample, adding 3 mu L of protein marker, assembling an electrophoresis apparatus, adjusting the electrophoresis apparatus to 80V, adjusting the electrophoresis apparatus to 120V to continue gel running when a sample strip runs to the vicinity of the boundary limit of the compressed gel and the separation gel, and stopping gel running until the bottom reaches 1 cm. After electrophoresis is finished, cutting out a needed strip according to the marker indication.

3. Rotary film

(1) Sufficient transfer buffer (100mL of 10 × transfer solution +200mL of methanol +700mL of ultrapure water) was prepared for use.

(2) After electrophoresis, the glass plate was taken out and gently pried open with tweezers. And (3) soaking the target strip by using the membrane transferring solution, cutting the target strip according to the position of a protein marker, making a mark, and putting the mark into the membrane transferring solution. The gel was transferred to transfer buffer. And (3) cutting out a PVDF membrane with the size consistent with that of the cut gel, soaking the PVDF membrane in methanol for 5min for activation, and then putting the PVDF membrane into a transfer buffer solution for balancing for 2min for later use.

(3) Making a sandwich structure: in the electrotransfer liquid, opening the clamp, horizontally placing the clamp, padding a layer of sponge on the black clamp, and rolling the black clamp by using a glass rod to drive bubbles; then putting thick filter paper, fixing, removing bubbles by using a glass rod, then putting gel, covering the gel with a PVDF film, upwards sequentially covering three layers of filter paper and sponge, and finally closing and clamping the white clamp.

(4) The clamp is placed in a transfer tank, glue is applied to the negative electrode, and the membrane is applied to the positive electrode.

(5) Film transferring conditions: constant current of 200mA for 100min, heat generation during electric conversion, and film transfer in ice bath.

4. Immune response

(1) Sealing milk: after the membrane transfer was completed, the PVDF membrane was taken out and placed in an incubation box containing 5% skim milk prepared with 5ml of LTBST on a shaker and sealed at room temperature for 4 hours.

(2) Primary anti-incubation: with 1 × TBST at 1: diluting GAPDH, Vim and E-Cad antibody 1000, pouring off the blocking solution after blocking, adding the prepared primary anti-dilution solution to make the primary anti-dilution solution to cover the PVDF membrane, standing overnight at 4 ℃, taking out the solution the next day, and rinsing with 1 × TBST for 3 times, 15min each time.

(3) And (3) secondary antibody incubation: using 1 × TBST at 1: 10000 dilution of secondary antibody, recovery of primary antibody dilution after night, addition of secondary antibody dilution, placing on a shaking table, incubation for 2h at room temperature, and rinsing with TBST for 3 times, 15min each time.

5. ECL luminescence detection

(1) Mixing the two reagents of the solution A and the solution B in the luminescence kit in equal volume in a centrifuge tube.

(2) The PVDF membrane is placed on a sample tray, and then the mixed luminescent liquid is uniformly dropped on the PVDF membrane.

(3) Placed in the instrument and the instrument is manipulated to expose it.

The results of the effect of EG-017 at different concentrations on Vim protein expression in A549 cells are shown in FIG. 3, and the WB method is used for detecting the Vim protein expression in cells after EG-017 action concentrations are 0.4, 2 and 10M for treating the cells for 48h, and the results show that EG-017 inhibits Vim protein expression after treating the cells for 48 h. The results of the effect of different concentrations of EG-017 on TGF-beta 1-induced proliferation of hepatic stellate cells are shown in FIG. 4, and the WB method is used for detecting the expression condition of the E-cad protein in the cells after the cells are treated for 48h by using EG-017 action concentrations of 50, 100 and 200. The results show that EG-017 promotes the expression of the E-cad protein.

Finally, the experiments show that EG-017 can inhibit A549 cell fibrosis induced by TGF-beta 1 and shows certain dose dependence.

The invention is capable of other embodiments and its several details are capable of modifications in various obvious respects, all without departing from the invention.

Claims

1. Application of SARMs compound containing ester-based aromatic propionamide in preparation of anti-new coronavirus medicines, wherein the SARMs compound containing ester-based aromatic propionamide is EG-017 and has a chemical formula of C₂₅H₁₇F₃N₄O₄The chemical name is (S) -1- ((4-cyano-3- (trifluoromethyl) phenyl) amino) -3- (4-cyanophenoxy) -2-methyl-1-oxoprop-2-yl nicotinate.