CN104069109B - Application of substituted piperazine-1,4-diamide compounds in pharmacy - Google Patents

Abstract

The invention relates to an application of substituted piperazine-1,4-diamide compounds in pharmacy. The substituted piperazine-1,4-diamide compounds can be used as an activating agent of SIRT1 (Silent Information Regulator 1) for activating nuclear receptors LXRalpha and LXRbeta, regulating expression of target proteins ABCA1/G1, inflammatory key proteins p65, senescence-associated genes p662hc and the like, promoting excretion of lipid and cholesterol, regulating cholesterol metabolism, inflammation-associated and senescence-associated genes or proteins, treating cardiovascular and cerebrovascular diseases, regulating blood lipids and resisting atherosclerosis, inflammatory reaction and senescence diseases.

Description

Application of substituted piperazine-1,4-diamides in pharmaceuticals

technical field

The present invention relates to the use of substituted piperazine-1,4-diamide compounds, in particular to the application in the pharmaceutical field.

Background technique

Genetic factors, diet and aging all have certain influences on the homeostasis of cholesterol and fat in the body. More and more studies have shown that reducing triglycerides, total cholesterol and low-density lipoprotein cholesterol and increasing high-density lipoprotein cholesterol through calorie restriction (CR) can delay aging and reduce the incidence of cardiovascular diseases. CR is the only regulatory mechanism generally recognized by the scientific community that can extend lifespan in yeast, worms, Drosophila, and mammals. According to reports, CR plays a role by regulating the silent information regulator 2 (silent information regulator2, Sir2) signaling pathway, and has anti-atherosclerosis, anti-inflammation, anti-aging, oxidative stress, anti-apoptosis, energy regulation Metabolism, cardiovascular and cerebrovascular protection, blood lipid regulation and other functions.

Sir2 is a class of important genes first discovered in yeast that regulate cell lifespan through chromosome silencing mechanism and cell energy metabolism mechanism. It has histone deacetylase activity and is closely related to the aging of various cells. The mammalian Siruins family includes 7 proteins, named SIRT1-7, among which SIRT1 and yeast Sir2 have the highest homology and have been studied the most. On the one hand, by modifying histones, deacetylating H1K26, H3K9 and H4K16, it maintains chromatin in a silent state and genome stability; on the other hand, it participates in the regulation of cell energy metabolism, proliferation and apoptosis by deacetylating many non-histone proteins , aging and tumorigenesis. The deacetylation substrates of SIRT1 include p53 protein, forkhead-O-box transcription factors (FOXOs), peroxisome-proliferated activated receptor gamma coactivator-1α (peroxisome-proliferated activated receptor c coactivator, PGC-1α), sterol regulatory element binding protein (sterol regulatory element binding protein, SREBP-1c), liver X receptor (liver X receptor, LXR) and so on.

LXRs is a member of the nuclear receptor family. When activated by ligands, it can promote the reverse transport of cholesterol, promote the secretion of cholesterol in the bile, and inhibit the absorption of cholesterol in the intestine. It plays an important role in maintaining the homeostasis of cholesterol in cells and the body It plays an extremely important role in the process of cholesterol reverse transport. The so-called reverse cholesterol transport means that cholesterol is transported to the liver, metabolized by the liver or excreted in the form of bile. This process is an important physiological process to prevent excess cholesterol from accumulating in the surrounding tissues. It has been reported in the literature that SIRT1 interacts with LXRs and activates them by deacetylation (lysine 432 of LXRα, lysine 433 of LXRβ), and regulates the expression of target genes downstream of LXRs. , significantly elevated cholesterol levels.

SIRT1 has a certain protective effect on the occurrence and development of cardiovascular diseases. The earliest study found that SIRT1, on the one hand, deacetylates nitric oxide synthase (eNOS) in vascular endothelial cells, activates eNOS and inhibits angiotensin receptor AT1; on the other hand, it inhibits the aging of vascular endothelial cells and delays atherosclerosis happened. Subsequent studies have found that SIRT1 regulates lipid and cholesterol metabolism through deacetylation, activates LXRs and FXR (farnesoid X receptor), promotes the combination of high-density lipoprotein and cholesterol, reduces cholesterol deposition, regulates blood lipid levels, and affects the progression of atherosclerosis. form. In addition, SIRT1 can also inhibit the expression of angiotensin receptor AT1 and prevent pathological hypertrophy of the heart. SIRT1 can regulate liver carbohydrate metabolism and fat metabolism. In the liver of short-term fasted mice, knockout of SIRT1 resulted in reduced expression of hepatic fatty acid β-oxidation genes.

A series of in vivo and in vitro experiments confirmed that SIRT1 has a significant inhibitory effect on the expression of inflammatory genes and tissue inflammatory damage. In SIRT1 knockout mouse RAW264.7 macrophages, lipopolysaccharide (LPS)-induced nuclear factor κB (nuclear factor κB, NF-κB) activation and TNF-α, IL-1β, IL-6, etc. The expression of pro-inflammatory cytokines was significantly increased. More and more studies have shown that atherosclerotic plaques not only contain lipids, but also have a large number of inflammatory cell infiltration, which is characterized by the accumulation of a large number of monocytes and lymphocytes in the vessel wall, which suggests that inflammation plays a role in mediating atherosclerosis. The position in the occurrence and development of sclerosis also reflects the internal connection and important role of SIRT1, atherosclerosis and inflammation.

Formula 1 is the known expression in piperazine-1,4-diamides

In the piperazine-1,4-diamide of the above formula 1, R1 and R2 may be substituents shown in Table 1, respectively.

Contents of the invention

The object of the present invention is to provide a new use of substituted piperazine-1,4-diamide compounds, that is, the application in pharmacy.

Specifically, the present invention relates to the use of substituted piperazine-1,4-diamide compounds as medicines for treating and/or preventing atherosclerotic diseases.

The invention relates to the application of substituted piperazine-1,4-diamide compounds as medicines for treating and/or preventing cardiovascular and cerebrovascular diseases.

It relates to the application of substituted piperazine-1,4-diamide compounds in the preparation of medicines for treating and/or preventing blood lipid regulation.

The invention relates to the application of substituted piperazine-1,4-diamide compounds in the preparation of medicines for treating and/or preventing inflammatory reactions.

The invention relates to the application of substituted piperazine-1,4-diamide compounds in the preparation of medicines for treating and/or preventing aging.

The substituted piperazine-1,4-diamide compounds of the present invention can be administered by themselves or in the form of pharmaceutical compositions. The pharmaceutical composition of the present invention comprises an effective dose of the compound of the present invention or a pharmaceutically acceptable salt thereof and one or more physiologically acceptable pharmaceutically acceptable carriers.

The pharmaceutical compositions of the present invention can be formulated in a conventional manner, using one or more physiologically acceptable carriers, excipients and auxiliaries, which facilitate processing the active compounds into pharmaceutically acceptable preparations. Proper formulations depend on the chosen route of administration and may be prepared according to methods well known in the art.

The substituted piperazine-1,4-diamide compound or pharmaceutically acceptable salt thereof of the present invention can be released to patients through various routes or modes of administration. Suitable routes of administration include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, enteral and parenteral administration, including intramuscular, subcutaneous and intravenous injection.

Description of drawings

Figure 1 is the dose-effect curve of resveratrol's activation activity on the SIRT1 activator screening model.

Figure 2 is a schematic diagram of the effects of small molecular compounds on anti-atherosclerosis, anti-inflammation, anti-aging, and regulating blood lipids.

Figure 3 is the verification of the intracellular deacetylation functional activity of compound (I). Among them, Fig. 3a is a Western blot image of p53 and Ac-p53 in U2OS cells treated with compound (I); Fig. 3b is a gray scale quantitative image of protein.

Fig. 4 is a sensorgram of the interaction between compound (I) and SIRT1 protein, specifically the interaction between E1231 and SIRT1-N-CC-C protein.

Fig. 5 is the effect of compound (I) on the degree of deacetylation of LXRs. Among them, Fig. 5a is a Western blot image of acetylated LXRs (specifically LXRα, LXRβ) in HepG2 cells treated with compound (I); Fig. 5b is a gray scale quantitative image of protein.

Fig. 6 is a dose-effect curve of compound (I) stimulating LXRs activity.

Fig. 7 shows that compound (I) regulates the transcriptional activity of ABCA1 promoter.

Fig. 8 shows that compound (I) regulates the expression of ABCA1 and ABCG1 proteins in RAW264.7 cells. Among them, Fig. 8a is a Western blot image of ABCA1 and ABCG1 in RAW264.7 cells treated with compound (I); Fig. 8b is a gray scale quantitative image of protein.

Fig. 9 shows that compound (I) regulates ABCG5/G8 protein in HepG2 cells. Among them, Figure 9a is the Western blot of ABCG5 and ABCG8 in HepG2 cells after the action of compound (Ⅰ); Figure 9b is the gray scale quantitative map of ABCG5 protein; Figure 9c is the gray scale quantitative map of ABCG8 protein)

Figure 10 shows that compound (I) promotes cholesterol efflux in RAW264.7 cells. Among them, Figure 10a shows that compound (I) promotes cholesterol efflux mediated by HDL in RAW264.7 cells; Figure 10b shows that compound (I) promotes cholesterol efflux mediated by Apo-AI in RAW264.7 cells.

Fig. 11 is a microscope photo of compound (I) inhibiting foaming of macrophages by oil red O staining. Among them, a is a blank cell (without Ox-LDL); b is a solvent control (80 μg/ml Ox-LDL); c is 10 μM compound (I) + 80 μg/ml Ox-LDL; d is a positive control (10 μM 9CRA + 80 μg /ml Ox-LDL).

Figure 12 is the effect of compound (I) on the total cholesterol content in RAW264.7 cells.

Figure 13 shows that compound (I) inhibits the expression of the key inflammatory protein p65. Among them, Fig. 13a is the western blot of p65 in THP-1 cells treated with compound (I); Fig. 13b is the grayscale quantification of the protein.

Fig. 14 is the effect of compound (I) on apoE ^-/- mouse aorta full-length plaques. The results of full-length oil red staining of the aorta showed that compound (I) significantly reduced the formation of aortic sclerotic plaques in apoE ^-/- mice, and reduced the accumulation of cholesterol and lipids. Wherein a is the blank group (common feed); b is the model group (high-fat diet); c is the compound (I) E123120mg/kg (high-fat diet).

Fig. 15 is the effect of compound (I) on the cardiac outflow tract of apoE ^-/- mice. The results of oil red staining of the cardiac outflow tract showed that compound (I) could significantly reduce the area and number of plaques in the cardiac outflow tract of apoE ^-/- mice, and reduce the accumulation of cholesterol and lipid. Wherein, a is blank group (common feed); b is high-fat diet model group; c is compound (I) 20 mg/kg (high-fat diet).

Fig. 16 is the effect of compound (I) on the mRNA expression of p66shc, PAI-1 and p21 genes related to aortic aging in C57BL/6 mice.

detailed description

In order to understand the essence of the present invention more clearly, we firstly measured the activity of substituted piperazine-1,4-diamide compounds in activating SIRT1, and the results are shown in Table 1. It can be seen from Table 1 that the substituted piperazine-1,4-diamide compounds have different degrees of activity to activate SIRT1. The pathological test and results of the substituted piperazine-1,4-diamide compound (abbreviated as compound (I)) with the label "E1231" are used to further illustrate the role of the substituted piperazine-1,4-diamide compound in the following aspects: Applications in pharmaceuticals.

First, the target protein SIRT1 was successfully obtained with the help of Escherichia coli expression system. Based on homogeneous time-resolved fluorescence technology, active proteins were used to establish a high-throughput SIRT1 activator screening model. After the model was optimized, the known and recognized positive drug resveratrol was used to verify the model. The EC ₅₀ value was consistent with the literature report , see Figure 1, indicating that the expressed protein is a highly active target protein, and the screening model is credible. Active substance screening was carried out on the combined compound library (National New Drug (Microbial) Screening Laboratory), several active compounds were obtained, and the activation effect of all compounds on SIRT1 protein was determined. The representative active compound (I) was selected for further activity research, and it was confirmed at the cellular level that compound (I) can increase the degree of deacetylation of the target protein p53 of the SIRT1 protein, and is indeed an activator of the SIRT1 protein. In the surface plasmon resonance experiment, the K _D value between the compound (Ⅰ) and the protein SIRT1 was measured to be 9.61 μM. Finally, compound (I) was studied in molecular pharmacology, and it was found that compound (I) can increase the degree of deacetylation of LXRs, activate nuclear receptors LXRα and LXRβ, regulate the expression of target protein ABCA1/G1, and promote cell Internal cholesterol efflux, inhibition of macrophage foaming, up-regulation of ABCG5/G8 expression, and promotion of cholesterol secretion. The effect of compound (Ⅰ) on metabolic pathways related to inflammatory factors in foam cells was explored, and it was found that it could inhibit the expression of p65, a key protein of inflammatory transcription factor NF-κB. The effect of compound (I) on the expression of aging-related genes in mouse aorta was studied, and it was found that it could inhibit the mRNA expression of p66shc, PAI-1 and p21.

Therefore, the substituted piperazine-1,4-diamide compounds represented by compound (I) can be used as SIRT1 activators to regulate genes related to cholesterol metabolism, inflammation, and aging-related diseases (see Figure 2) , can be used for the prevention and/or treatment of diseases such as atherosclerosis, inflammation, aging, cardiovascular and cerebrovascular diseases.

Example 1. Determination of the activation activity of substituted piperazine-1,4-diamides on SIRT1

1) Protein expression

First, from the plasmid pcDNA3.1-SIRT1 (a gift from the research group of Professor Liu Depei, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), the catalytic domain of human SIRT1 and the cDNA including the N-terminal and C-terminal partial sequences were amplified, and the cloning vector pBlunt-Simple After the Vector connection sequence is correct, it is connected with the Escherichia coli expression vector pET-30a(+), the constructed recombinant expression plasmid is named pET-SIRT1, transformed into the Escherichia coli expression host BL21(DE3), single clones are picked, and sequenced for identification. Cultivate the correct sequenced single colony shake flask, when the _OD600 value is 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.5mM, 15°C, 200rpm, culture After 24 hours, the bacterial liquid was collected. Centrifuge at 12,000rpm for 30min after disrupting the cells with ultrasonic waves. The supernatant was collected and filtered through a 0.45 μM membrane filter. use The explorer system was used for protein purification, using 1ml prepacked column HisTrapFF crud as the purification medium. The principle is that the target protein chelates with the Ni ²⁺ ions on the Chelating Sepharose ^TM in the column through the His-tag at its N-terminus, and then adsorbs on the column, while other miscellaneous proteins cannot be chelated with Ni ²⁺ ions, and are directly attached to the column. The loading buffer flows out of the column, and then the target protein is eluted with the elution buffer, thereby obtaining the high-purity target protein SIRT1.

2) Determination of compound activation activity on SIRT1

In this activity determination, the human SIRT1 activator screening kit from Cisbio Company was used to determine the activity of the compound.

Measuring principle:

The detection system is as follows: recombinant human SIRT1 protein, substrate substrate-d2 is a small p53 peptide fragment labeled with fluorescent group d2, NAD ⁺ is a cofactor necessary for SIRT1 to exert enzymatic activity, and anti-acetyl-cryptate is a hole labeled with Eu ²⁺ compound. If the added compound has an activation effect on the enzyme, the degree of deacetylation of the substrate increases, and the distance between the substrate and cryptate becomes longer, which is not enough to generate resonance energy transfer, which is manifested by a decrease in fluorescence readings.

test methods:

(1) Add DTT with a final concentration of 1mM to the reaction buffer to prepare Enzymatic buffer.

(2) Dilute the recombinant protein, substrate substrate-d2 and coenzyme NAD ⁺ sequentially with Enzymatic buffer, so that the final concentrations are 0.5mU/μl, 6nM, 150μM respectively.

(3) Dilute the compound to a certain concentration with Enzymatic buffer.

(4) Add 2 μl of the compound to be tested, 2 μl of NAD ⁺ , and 4 μl of substrate to the 384-well plate in sequence, and finally add 2 μl of recombinant protein to start the enzymatic reaction. Set up the No enzyme group without protein and the Negative control group without compound, with 3 parallel wells for each group.

(5) Incubate at room temperature for 120 minutes.

(6) Add 10 μl of Anti-acetyl-cryptate with a final concentration of 7.5nM, incubate at room temperature for 5 hours or overnight, and measure the fluorescence value (Ex: 340nm, Em: 665/615nm) with a microplate reader.

(7) Calculation method:

Ratio: (665nm/615nm)×10 ⁴

Substrate deacetylation ratio (%): 100-(Ratio _Sample /Ratio _{No enzyme} ×100)

Up-regulation rate (%) = deacetylation ratio _Sample / deacetylation ratio _{Negative control} × 100%

If the up-regulation rate is greater than or equal to 150%, it is considered positive in the primary screening, and the next step of re-screening and dose-effect curve determination is carried out.

Read the fluorescence readings, calculate according to the formula, take the logarithmic value of the compound concentration as the abscissa, and the degree of deacetylation of the substrate as the ordinate, and use the GraphPad Prism software to fit the curve to obtain the EC ₅₀ value and the maximum up-regulation rate. The results are shown in Table 1.

The assay results show that the substituted piperazine-1,4-diamide compounds of the present invention have significant deacetylation and activation effects on SIRT1. Among them, the compound (I) with the best activity had a maximum up-regulation rate of 384.09% and an EC ₅₀ value of 0.43 μM. Since SIRT1 is of great significance in participating in physiological processes such as cholesterol metabolism and fatty acid and carbohydrate metabolism, this result shows that the substituted piperazine-1,4-diamide compounds in the present invention can regulate related physiological processes by activating SIRT1. play a role in the disease process.

Example 2. Culture of cells

HepG2, ABCA1-LUC HepG2, U2OS and HEK293 cells are all adherent cells, which are passaged every 48 hours. After the cells are full, discard the old medium, rinse the cells with PBS and discard, add an appropriate amount of trypsin, digest the cells at room temperature for about 2 minutes, discard the digestion solution, and immediately add the medium containing 10% FBS to inhibit trypsin Vitality, gently blow and tap the cells in the culture flask with an elbow pipette to make the cells completely detach from the bottom of the bottle and disperse them into a single cell suspension by blowing and blowing. Then inoculate the cell suspension in a new cell flask at a ratio of 1:3, or discard an appropriate amount of cell suspension, then add an appropriate amount of complete medium, and put it in an incubator to continue culturing. Culture conditions: 37°C, 5% CO ₂ .

Mouse mononuclear macrophage RAW264.7 is a semi-adherent cell, which is passaged every 48 hours. Digest with trypsin for 5 minutes, pass passage according to the ratio of 1:4-1:6, and use DMEM complete medium. Culture conditions: 37°C, 5% CO ₂ .

Human acute monocytic leukemia cells THP-1 are suspension cells, which are passaged once every 48 hours. When the cell density is (3-5)×10 ⁶ cells/ml, blow and beat the cells in the culture flask with an elbow pipette to disperse them into a single cell suspension. Then inoculate the cell suspension in a new cell flask at a ratio of 1:4, or discard an appropriate amount of cell suspension, then add an appropriate amount of complete medium, and put it in an incubator to continue culturing. Culture conditions: 37°C, 5% CO ₂ .

Example 3. The effect of compound (I) on the expression level of acetylated p53 protein in human osteosarcoma U2OS cells induced by doxorubicin.

1) Compound-treated cells: Human osteosarcoma cells U2OS were inoculated in 6-well plates at 3×10 ⁵ cells per well in McCoy’s 5a medium containing 10% FBS in advance, and cultured at 37°C and 5% CO ₂ until Logarithmic period. After the cells were completely adhered to the wall, the cell solution was aspirated, replaced with blank McCoy's5a medium, and added with a final concentration of 5 μM doxorubicin hydrochloride (DOX), 10 μM SIRT1-specific inhibitor EX-527 and a final concentration of 0.1, 1, 10 μM positive compounds were incubated at 37° C., 5% CO ₂ for 6 hours.

2) Protein preparation: After 6 hours, discard the medium, rinse the cells twice with pre-cooled PBS, trypsinize and collect the cells with the medium, centrifuge at 800rpm for 3min, suspend the cells with PBS, and centrifuge at 800rpm for 3min. Add 52 μl of RIPA cell lysate (PMSF with a final concentration of 100 μg/ml per 1 ml of lysate) to each tube, and lyse the cells on ice for 30 min. Centrifuge at 12000×g for 20 minutes at 4°C. The supernatant was collected, and the protein was quantified with a BCA protein quantification kit (Thermo Company), and the concentration of each histone sample was adjusted to 2 μg/μl with ddH ₂ O. Add a certain amount of 5× protein loading buffer, cook in a boiling water bath for 10 min, and then centrifuge briefly after cooling, load 40 μg protein per well for SDS-PAGE electrophoresis. The remaining samples were stored at -80°C.

3) Determination of protein level by Western blot method: after SDS-PAGE electrophoresis, put the polyacrylamide gel and transfer membrane filter paper into the transfer buffer and soak for 5 minutes to balance. The PVDF membrane was first activated by immersing in methanol for 20s, then washed with water, and soaked in transfer buffer for 2 minutes to balance. The membrane was transferred with a BioRad semi-dry transfer apparatus, the current was set at 5mA/cm ² , and the membrane was transferred at a constant current for 30min. After the membrane transfer, cut them according to the size of the target protein and put them into 1×TBST containing 5% (W/V) skimmed milk powder for incubation at room temperature for 1 h. After blocking, the primary antibody was diluted with 1×TBST containing 5% (W/V) skimmed milk powder, placed in a hybridization bag, incubated at room temperature for 1 hour, and then incubated overnight at 4°C. Wash three times with 1×TBST, 10 min each time. Afterwards, the corresponding secondary antibody was diluted with 1×TBST containing 5% (W/V) skimmed milk powder, placed in a hybridization bag, and incubated at room temperature for 2 hours. Take out the PVDF membrane from the hybridization bag and wash it three times with 1×TBST for 10 min each time. Color development: Add an appropriate amount of enhanced HRP (horseradish peroxidase) substrate chemiluminescence solution (ECL) A and B mixture on the front side of the membrane, that is, the side where the protein is transferred (1:1 ratio for current use) Now prepared), immediately placed in a gel imager for color development.

4) The results of color development were scanned with ImageJ software and processed quantitatively. The results are shown in Figure 3.

Doxorubicin (DOX) can induce DNA damage in cells, resulting in acetylation of lysine 382 of p53 protein. In order to verify the intracellular deacetylation function of the compound and investigate the dependence of the functional effect on SIRT1, SIRT1-specific inhibitor EX-527 was added. As the concentration of compound (I) increased, the expression of acetylated p53 protein decreased significantly with the concentration gradient, while the amount of total p53 protein in cells did not change significantly, indicating that the degree of deacetylation of p53 protein increased. Adding EX-527, a specific inhibitor of SIRT1 protein, the expression of Ac-p53 protein increased again. Therefore, the present invention demonstrates that compound (I) has obvious up-regulation effect on deacetylation of intracellular SIRT1 substrate, and this effect is dependent on SIRT1.

Example 4. Determination of the intermolecular interaction between compound (I) and SIRT1 protein

The buffer solution used in this assay system consists of: HEPES10mmol/L, NaCl150mmol/L, DTT1mmol/L, NAD ⁺ 1mmol/L, glycerol 10% (V/V), DMSO2% (V/V). The buffer solution and samples were filtered through a 0.45 μm filter membrane, and the air bubbles were removed by ultrasonic before use. Performed by a Biacore T200System (GE Healthcare) instrument.

test methods:

1) Pretreatment of the chip surface: Take out the maintenance chip, replace it with a CM5 chip, select Prime, and rinse the chip surface with buffer. Select Normalize, and use Biacore normalizing reagent (BIA normalizing solution 70%) to normalize the CM5 chip. Experiments were carried out using two chip channels to explore the pH conditions required for SIRT1 protein coating (immobilization pH-scouting for pre-concentration). First, select a sodium acetate solution with a pH within the range of 3.5<pH<pI(hSIRT1) for pre-enrichment, and select a pH 4.0 sodium acetate solution to dilute the protein to 50 μg/ml according to the pre-enrichment results for coupling. Set the flow rate to 10 μl/min, the binding time to 2 min, and finally rinse the surface of the chip with 50 mM NaOH for 20 s to remove residual SIRT1 protein.

2) Protein-coated chip surface: EDC/NHS were mixed in equal volumes, injected at a flow rate of 1 μl/min for 10 minutes, and the carboxyl groups on the surface of the CM5 chip were activated. According to the pretreatment results of the chip surface, sodium acetate solution with a pH value of 4.0 was selected as the buffer solution to dilute the SIRT1 protein to 50 μg/ml, the flow rate was set to 10 μl/min, and the binding time was 20 minutes. Coated on the surface of the CM5 chip, set the target coupling amount to 7000RU, and set a blank channel as a control channel. After the injection, ethanolamine was added, and the sample was injected at a flow rate of 10 μl/min for 10 minutes to block the active carboxyl sites on the surface of the chip that were not bound to amino groups. The final coupling result was 7056RU.

3) Compound (I) was added for binding detection: for SIRT1-coated CM5 chips, different concentrations of compounds (0, 0.39, 1.56, 3.12, 6.25, 12.5 μM) were sequentially injected, and the compound was injected at a flow rate of 30 μl/min. After flowing through the surface of the chip, setting the binding time to 60s and dissociation time to 120s, the regeneration conditions of the chip surface were explored, and it was found that the compound could be completely and naturally dissociated within a certain period of time.

From Figure 4, it can be seen from the change of RU value that compound (I) can bind to human recombinant SIRT1 in a dose-dependent manner, with a _KD value of 9.61 μM. The K _D value of the small molecule binding to the protein is generally 10 ^-3 to 10 ^-6 M, therefore, the compound (I) has a strong affinity with the protein SIRT1.

Example 5. Deacetylation of LXRs by Compound (I)

In order to investigate the effect of compounds on the degree of deacetylation of LXRs, we used co-immunoprecipitation experiments to verify.

In this experiment, HepG2 cells were used, and they were placed in a large culture dish with a diameter of 90 mm. After the cells were completely adhered to the wall, 10 μM of compound (I) was added, and solvent control wells were set up, and the cells were treated for 20 to 24 hours. The nuclear protein was extracted, incubated with LXRα and LXRβ antibodies and Protein A/G agarose beads at 4°C for 4 hours, washed the beads, boiled, and detected by western blot with acetylated-Lys antibody. After exposure, the membrane was regenerated with the membrane regeneration solution according to the experimental procedure of Beijing Pulilai Gene Technology Co., Ltd., and then detected with the corresponding LXRs antibody. The results are shown in Figure 5.

It can be seen from Figure 5 that after adding compound (Ⅰ), the expression levels of LXRα and LXRβ were basically constant, and the expression level of acetylated LXRβ decreased to a certain extent. However, the expression level of acetylated LXRα decreased significantly, indicating that SIRT1 activator Compound (I) can regulate the acetylation levels of LXRα and LXRβ in hepatocytes.

Example 6. Determination of compound (I) agonistic activity on LXRs

In this activity determination, the agonist screening model of LXRs is used to measure the activity of the compound.

Measuring principle:

The principle of this assay is based on the two main domains in the structure of LXRs: Ligand Binding Domain (LBD) and DNA Binding Domain (DBD), and the fact that the transcription factor GAL4 in yeast has a similar structure to nuclear receptors. According to the principle of yeast two-hybrid, two plasmids are constructed respectively: pBIND-LXRs plasmid contains the DBD part of the GAL4 gene and the LBD domain of LXRs, which can change conformation when activated by ligand; the other plasmid is pGL4-GAL4, which contains the GAL4 gene Luciferase reporter gene Luc from the promoter region response element. In the present invention, two plasmids are co-transfected into HEK293 cells to study the activation effect of the compound on LXRs. In this detection system, if the compound can activate LXRs, the conformation of the LXRs protein expressed by the expression plasmid will change and combine with the reporter plasmid. At this time, the expression activity of luciferase in the compound group is higher than that of the control group without the compound; otherwise, when the compound When there is no activity on LXRs, the conformation of LXRs protein does not change, and it does not combine with the reporter plasmid, so the expression activity of luciferase in the compound group does not change.

test methods:

1) Transfection was carried out in a 96-well plate. One day before transfection, HEK293 cells were plated at a density of 1.5-3.0×10 ⁵ cells/ml. When the HEK293 cells grew to 90% confluence and their growth status was good, dilute 0.5 μl liposome Lipofectamine ^TM 2000 Reagent according to 25 μl/well Opti-MEM medium, and incubate at room temperature for 5 minutes. Dilute 200 ng of plasmid DNA, including pGL4-GAL4 and pBIND-LXRα/β, in 25 μl/well of Opti-MEM medium, combine with diluted liposomes, and incubate at room temperature for 20 minutes.

2) Add 100 μl of MEM complete medium to the combined DNA-liposome complex solution per well to fully mix the medium and DNA-liposome complex, and then suck out the medium in the cell plate , add the mixed cell culture fluid-DNA-liposome complex solution to a 96-well plate, 150 μl per well. Place the 96-well plate in a carbon dioxide incubator at 37°C.

3) Incubate for 6 hours, suck out the transfection medium, and dilute the compound (I) from the highest concentration of 40 μM to 8 concentrations step by step with a 2-fold concentration gradient. At the same time, add 0.1% DMSO to the negative control wells to treat the cells respectively, Continue to cultivate for 18h.

4) After 18 hours, suck out the culture medium in each well of the 96-well plate. Add 150 μl PBS to each well to gently rinse the cells, invert the 96-well plate to pour off the PBS and shake dry. Add 20 μl of 1×CCLR cell lysate to each well, lyse the cells at 37° C. for 20-30 min (the time is determined according to the number of cells), and observe under the microscope whether the cells are completely lysed. Transfer all the lysate to the corresponding wells of the 96-well opaque white plate for fluorescence analysis, and pay attention to avoid air bubbles in the lysate as much as possible, otherwise it will affect the reading (if using a 96-well transparent bottom white plate, it is not necessary to transfer the lysate).

5) Quickly add 50 μl of fluorescence analysis reagent (Promega Company) to each well, and immediately put the analysis white plate into a microplate reader for detection.

6) Calculate the rate of change of the luciferase activity of the sample to be tested with the following equation:

Change rate (%) = A/B × 100

Wherein, A is the cell luciferase activity (RLU) measured after adding the test sample, and B is the cell luciferase activity (RLU) measured after adding the negative control sample (DMSO). The rate of change (%) is regarded as the agonistic activity of the compound on LXR, and it can be expressed as a percentage or as a multiple. The logarithmic value of the compound concentration was used as the abscissa, and the rate of change was used as the ordinate, and the curve was fitted with GraphPad Prism software, and the results are shown in Figure 6.

The assay results show that the compound (I) of the present invention has a significant transcriptional activation effect on LXRα/β, and the dose-effect relationship curve of the compound (I) on the LXRα/β agonist screening model has been obtained. It can be seen from Figure 6 that compound (Ⅰ) stimulates LXRα/β in a dose-dependent manner, the maximum agonistic activity on LXRα is 188.14%, and the EC ₅₀ value is 0.65μM; the maximum agonistic activity on LXRβ is 182.32%, and the EC ₅₀ value is 0.08 μM. Since the nuclear receptor LXR is of great significance in participating in physiological processes such as cholesterol metabolism and fatty acid and sugar metabolism, this result shows that the compound (I) in the present invention can regulate diseases involving related physiological processes by activating the transcription of LXR play a role in.

Example 7. Determination of the transcriptional activity of the ABCA1 promoter by the compound (I)

In this activity determination, the ABCA1 up-regulator screening model is used to measure the activity of the compound.

Measuring principle:

The principle of this assay is to co-transfect the recombinant plasmid pGL3-ABCA1 with the upstream regulatory sequence of the ABCA1 gene cloned upstream of the luciferase reporter gene and pcDNA3.1 into human liver cancer cells HepG2 cells, and obtain stable transfected cells by G418 screening strain, named ABCA1-LUCHepG2 cells. By adding the compound to detect the change in the expression activity of the luciferase in the cell, we can see the effect of the compound on the transcriptional activity of the ABCA1 promoter, and then determine the up-regulation rate of the ABCA1.

test methods:

ABCA1-LUC HepG2 cells in the logarithmic growth phase were digested, diluted with culture medium and counted, seeded into a 96-well plate at a density of 5×10 ⁵ cells/ml, and 100 μl of single-cell suspension was added to each well. After 6 hours, when the cells were fully attached to the wall, remove the serum-containing medium, gently rinse the cells once with PBS, add 200 μl of serum-free RPMI-1640 medium containing different compound concentrations to each well, and add corresponding concentrations of RPMI-1640 medium to each well. DMSO. After 18-24 hours, the culture medium was removed, and measured, calculated and plotted according to the methods described in the measurement methods (4)-(6) in Example 6, and the results are shown in FIG. 7 .

Compound (I) increases the luciferase activity of ABCA1-LUC HepG2 cells in a dose-dependent manner. Compound (I) up-regulated the expression activity of ABCA1 to the highest value of 226.67%, and its EC ₅₀ value was 0.25 μM. Since ABCA1 plays an important role in cholesterol reverse transport, the results indicate that the compound (I) of the present invention can enhance the transcriptional activity of ABCA1 promoter and play a role in diseases involving related physiological processes.

Example 8. The effect of compound (I) on the expression levels of ABCA1 and ABCG1 proteins in RAW264.7 macrophages.

1) Compound-treated cells: mouse mononuclear macrophage RAW264.7 was inoculated at 4×10 ⁵ cells/ml on a 6-well plate and treated with 0.1, 1 and 10 μM compound (I) at a concentration of 0.1 %DMSO was used as a negative control group, and a 10 μM compound (I)+EX-527 group was set at the same time, all cultured at 37° C. and 5% CO ₂ for 20-24 hours.

2) The protein expression levels of ABCA1 and ABCG1 were measured by Western blot method, and quantitatively processed by software. The results are shown in Figure 8.

ABCA1 and ABCG1 are the target proteins directly regulated by LXR. Their main functions in macrophages are to expel excess cholesterol in the cell, promote the process of reverse cholesterol transport, and prevent the oxidation of low-density lipoprotein (LDL) in macrophages. Ox-LDL) accumulation causes foaming of macrophages. It can be seen from Fig. 8 that the expression of ABCA1 and ABCG1 can be dose-dependently up-regulated at 1-10 μM after 20-24 h of compound (I), and the maximum up-regulation of ABCA1 is 2.17 times, while that of ABCG1 can reach 2.48 times. Therefore, the present invention illustrates that the compound (I) has obvious up-regulation effect on promoting cholesterol efflux-related proteins in macrophages.

Example 9. Effect of compound (I) on the expression levels of ABCG5 and ABCG8 in HepG2 cells

1) Compound-treated cells: pre-treated HepG2 cells seeded in 6-well cell culture plates with different concentrations of compound (I), with a concentration of 0.1% DMSO as a negative control group, at 37°C and 5% CO ₂ Cultivate for 20-24h.

2) The protein expression levels of ABCG5 and ABCG8 were measured by Western blot method, and quantitatively processed by software.

The ABCG5 and ABCG8 proteins in the liver are also downstream target proteins of LXR. They are directly regulated by LXR. These two transport proteins are expressed on the membrane of hepatocyte tubules, thereby driving the expression of cholesterol transport to bile and promoting cholesterol secretion. As shown in Figure 9, compound (I) in the present invention has the highest up-regulation factor of ABCG5 at 0.1 μM, up to about 1.2 times; it dose-dependently up-regulates the expression of ABCG8 in the range of 0.1-10 μM, and up-regulates ABCG8 at 10 μM Up to 2.2 times. It shows that compound (I) may promote the secretion of cholesterol in the liver by up-regulating the expression of ABCG5 and ABCG8.

Example 10. Compound (I) promotes macrophage cholesterol efflux experiment

1) Mouse monocyte-macrophage RAW264.7 was inoculated on 24-well cell culture plate with 2×10 ⁵ cells/well in DMEM-high glucose medium (500 μl/well) containing 10% FBS, at 37 °C, 5% CO ₂ overnight.

2) Discard the cell solution, replace with DMEM-high glucose medium (500 μl/well) containing 0.2% (W/V) BSA, add 1,2-[ ³ H] cholesterol to make the final concentration 1 μCi/ml, Incubate for 24 hours at 37°C and 5% CO ₂ .

3) Wash cells twice with PBS (1ml/well), add assay medium (DMEM with 0.2% BSA, 0.1% DMSO, 25mM HEPES, pH7.4) containing a certain concentration of compound, and incubate at 37°C for 18-24h.

4) Wash the cells twice with PBS (1ml/well), add medium (DMEM with 0.2% BSA, 0.1% DMSO, 25mM HEPES, pH7.4), 10μg/ml apoA-I or 50μg/ml HDL, and incubate for 4h .

5) Collect the medium, centrifuge at 10,000×g for 5 min, and take the supernatant for testing.

6) Cells were lysed with 0.5 ml of 0.1M NaOH at room temperature for 30 min, and the lysate was collected for testing.

7) Determination: Transfer the samples to be tested to 3mm filter paper, dry at 75°C, put the paper in the liquid scintillation cup, add 10ml liquid scintillation liquid (mass concentration is 0.5% PPO and 0.05% POPOP) and volume fraction Prepare by mixing 55% xylene and 45% ethylene glycol dimethyl ether as a solvent, store it in a brown container, use it overnight, and count with a liquid scintillation counter. The whole experimental cells were divided into control group (without adding cholesterol but adding apoA-I/HDL, adding cholesterol) and adding sample group (adding cholesterol, apoA-I/HDL and a certain concentration of the sample to be tested at the same time).

Cholesterol efflux rate (%) = cpm value of culture medium/total cpm value × 100%

=Cpm value of culture medium/(cpm value of culture medium+cpm value of cells)×100%

The results of cholesterol efflux in macrophages are shown in Figure 10. Compound (Ⅰ) can promote the efflux of 1,2-[ ³ H]cholesterol to the cholesterol acceptor under the action of 0.1, 1, and 10 μM, and with the compound As the concentration increases, the efflux level of 1,2-[ ³ H] cholesterol also increases, the multiple of efflux to apoA-I is between 1.2-1.7 times, the multiple of efflux to HDL is higher than the multiple of apoA-I, about Between 1.5-2.0 times. This result is in line with what was stated in the previous part of the present invention. Compound (I) promotes the efflux of intracellular cholesterol by up-regulating the expression of ABCA1 and ABCG1 proteins in macrophages, which is consistent with the physiological activity of LXR agonists and accepts HDL as cholesterol. The effect of the body is more significant, while ABCG1 is mainly responsible for transporting excess cholesterol to HDL, which is consistent with the findings that the previously discussed compounds can significantly up-regulate the protein and mRNA expression of ABCG1.

Example 11. Experiment of Compound (I) Inhibiting Foaming of Macrophages

The mouse mononuclear macrophage RAW264.7 was cultured on a transparent 96-well cell culture plate with DMEM-high glucose medium containing 10% FBS. After adhering to the wall, replace with serum-free DMEM-high glucose medium (100 μl/well). The cells were divided into blank control group, foam cell group and drug-dosed group, and Ox-LDL with a final concentration of 80 μg/ml was added to the foam cell group and drug-dosed group. After 24 hours, a certain concentration of compound (I) was added to the drug-dosed group . After culturing for 24 hours at 37°C and 5% CO ₂ , oil red O staining was carried out. Take the 96-well plate out of the _CO2 incubator, fix the cells with 4% paraformaldehyde (15 μl/well) for 10 min, discard the solution, wash twice with double distilled water, then add 60% isopropanol (150 μl/well), and place 5min, discard the solution. Add Oil Red O solution (ready to use, filter) into each well, 150 μl/well, and stain for 1 hour. Discard the solution, wash the wells with 60% isopropanol (150 μl/well), then wash twice with double distilled water (150 μl/well), and finally add 150 μl double distilled water to each well, observe and take pictures under a microscope, the results are shown in Figure 11.

Only Ox-LDL was added as the foamed cell group, stained with Oil Red O, and the staining degree of the cells was observed under a microscope. It can be seen from Figure 11 that (a) no red lipid droplets were formed in the blank group, (b) obvious red lipid droplets were formed in the Ox-LDL solvent control group, and (c) red lipid droplets appeared in the cells in the compound (I) treatment group Compared with the control group, the oily particles were significantly reduced, indicating that compound (Ⅰ) can reduce lipid accumulation in macrophages and effectively inhibit macrophage foaming. obviously decrease.

Example 12. Compound (I) reduces intracellular total cholesterol

Use the method recommended by the BioVision Cholesterol Quantification Kit to determine the total intracellular cholesterol. The determination method is as follows:

1) Prepare cholesterol standard concentration gradient: 0, 1, 2, 3, 4, 5 μg/well.

2) Collect the cells, add 200 μl of CH ₃ Cl:IPA:NP-40 (7:11:01), and centrifuge at 12,000 g for 5 min at room temperature.

3) Transfer the supernatant to a new EP tube, and dry the liquid in an oven at 50°C.

4) Add 100 μl cholesterol assay buffer to dissolve and mix.

5) Add 2 μl esterase, 2 μl substrate mix, 2 μl cholesterol enzyme mix and 44ul sample or standard, and incubate at 37°C in the dark for 30min. The light absorption value was read and calculated at a wavelength of 450nm.

Intracellular total cholesterol mainly includes free cholesterol and cholesteryl ester, and the amount of total cholesterol is also a main characteristic of cell foaming. It can be seen from Figure 12 that compound (I) significantly reduced the total intracellular cholesterol content at 1 μM and 10 μM. It shows that the added compound can inhibit the foaming of macrophages and reduce the accumulation of lipids in macrophages.

Example 13. Effect of Compound (I) on the Expression of Key Inflammation Protein p65 in THP-1 Cells

1) Compound-treated cells: THP-1 inoculated in a 6-well cell culture plate was induced by adding a final concentration of 50 nM phorbol ester PMA for 24 h, then 10 μM of E1231 was added to it, incubated at 37°C for 2 h, and then added with a final concentration of 10ng/ml LPS stimulated the production of intracellular cytokines, and continued to incubate at 37°C for 18h.

2) The Western blot method was used to measure the expression level of p65 protein, and it was quantitatively processed by software. The results are shown in Figure 13.

Another notable feature after caloric restriction is the suppression of inflammatory responses. As an activator of SIRT1, compound (I) should have a certain inhibitory effect on the release of inflammatory factors, and has positive significance for the prevention of atherosclerosis. It can be seen from Fig. 13 that compound (I) reduces the expression of p65 protein at 1 μM and 10 μM, and can inhibit up to 35%. Therefore, the present invention illustrates that compound (I) has obvious inhibitory effect on inflammation-related proteins in macrophages.

Example 14. Pharmacodynamic evaluation of compound (I) in apoE ^-/- mice

A) Construction of apoE ^-/- mouse model of arteriosclerosis

1) apoE ^-/- mice, 7 weeks old, were fed with normal feed for one week;

2) The apoE ^-/- mice were weighed, randomly grouped, and divided into 3 groups (blank control group, model group, and drug administration group), with 6-8 mice in each group;

3) From the age of 8 weeks, the model group and the treatment group were fed with a high-fat feed diet, and the blank group continued to be fed with a common feed;

4) Compound (I) sample addition group (20mg/kg), intragastric administration; blank control group and model group were given carboxymethylcellulose sodium solution, intragastric administration; administration for 4 weeks;

5) The apoE ^-/- mice administered for 4 weeks were fasted for 6 hours; the eyeballs were removed to collect blood, and the blood was collected in an EP tube washed with heparin, turned upside down, placed on ice, 4000r/min, and centrifuged at 4°C for 3min , transfer the supernatant to a new EP tube (in two parts), and store at -20°C.

6) Fix the mouse, cut open the thorax, cut off the sternum, expose the heart, and immediately perfuse the heart with about 1ml of 4% paraformaldehyde, then replace it with PBS and pour about 4ml, stop after the liver turns white ;

7) The entire length of the artery from the aorta to the common branch of the ilium was separated, and the heart and artery were taken out. After dissection, the heart and aorta were fixed in 4% paraformaldehyde at 37°C for 2h, and then placed in 20% sucrose solution at 4°C overnight.

B) Determination of blood lipid levels

The serum after centrifugation was determined according to the method described in the kit of Prilai Biotechnology Co., Ltd.

C) Method for making frozen sections

1) Cut off the 1.5ml EP tube from the middle, leave the part with the cover, cover the cover, and mark it;

2) Add the OCT embedding agent to the EP tube, be careful not to have air bubbles;

3) Put the tissue soaked in 20% sucrose into the OCT. When embedding the heart, leave about 1/3 of the heart, cut it flat, put the apex of the heart upwards, and slowly put it into the OCT;

4) Slowly put the organized EP tube into the liquid nitrogen;

5) Immediately wrap the frozen EP tube with tin foil, store it in the dark at -20°C, and store it at -80°C for long-term storage.

D) Oil red O staining of aorta

1) Take out the aorta from 20% sucrose, and wash once with PBS;

2) Under a dissecting microscope, the aorta is longitudinally dissected;

3) Wash the cut aorta 3 times with distilled water;

4) Soak in 60% isopropanol for 10 minutes for synchronization;

5) Put the synchronized product into the newly prepared oil red working solution (oil red storage solution configuration, use within 1-2 hours of filtration), 30min;

6) Put in 60% isopropanol for color separation for 1 min;

7) Wash with double distilled water for 3 times;

8) Spread the dissected aorta on the black wax, and immediately take pictures with the camera.

E) Oil red O staining of frozen sections (cardiac outflow tract)

1) Place the slices at room temperature for 30 minutes, and dry the frozen slices;

2) Put the slice into 4% paraformaldehyde and fix it for 10 min;

3) Discard the paraformaldehyde solution, add double distilled water, wash 3 times, 3 minutes each time;

4) Soak slices in 60% isopropanol for 3 minutes for synchronization;

5) Then put the synchronized slices into the newly prepared oil red working solution (oil red storage solution configuration, use within 1-2 hours of filtration), 30min;

6) Put the slice into 60% isopropanol for color separation, and observe under the microscope at any time;

7) Wash with double distilled water for 3 times;

8) Stain with hematoxylin for 2-3 minutes, and counterstain the cell nucleus;

9) After washing with double distilled water for 3 times, place the slices in distilled water;

10) Water-based mounting agent for sealing (9 parts of medical glycerin + 1 part of double distilled water);

Apply nail polish around the sealed film, dry it in the shade, and take a picture immediately.

Table 2 shows the results of determination of blood lipid levels in apoE ^-/- mice. It can be seen from Table 2 that the apoE ^-/- mice fed with high-fat diet, after compound (I) administration, the total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL- C) and high-density lipoprotein cholesterol (HDL-C) content. Compared with the model group, the administration group has a certain effect of reducing TC, TG, LDL-C, and increasing the content of HDL-C. It shows that compound (I) can regulate blood lipid level.

The full length of apoE ^-/- mouse aorta was stained with oil red, and the results are shown in Figure 14. It can be seen from Fig. 14b that obvious arteriosclerotic plaques appeared in the whole aorta of the high-fat diet model group, indicating that the arteriosclerosis model was successfully constructed. Compared with the high-fat diet model group, the aortic plaques in the administration group (Fig. 14c) were significantly less and smaller, indicating that compound (I) can play a positive role in the treatment of arteriosclerosis.

Figure 15 shows the results of frozen sections of apoE ^-/- mouse hearts. It can be seen from Figure 15 that in the high-fat diet model group (Figure 15b) a large area of arteriosclerotic plaque appeared in the entire aorta of the cardiac outflow tract, and the lipid droplets were large and dense. Smaller arterial plaques could be seen in the blank control group (Fig. 15a), which may be spontaneously generated in apoE ^-/- mice. Compared with the high-fat diet group, the compound (I) group (Fig. 15c) had significantly reduced arterial plaque area.

Based on the results of Figures 14-15, compound (I) can significantly reduce the plaque area and number in the full length of aorta and cardiac flow in apoE ^-/- mice. The results in apoE ^-/- mice all show that the compound (I) has good anti-atherosclerosis effect.

Example 15. Detection of mRNA levels of aging-related marker molecules in mouse aorta

A) Breeding of mice: C57BL/6 mice, 7 weeks old, were kept in an SPF grade animal room with clean drinking water and free access to food. The negative control group was fed with sodium carboxymethyl cellulose, and the treatment group was fed with E1231, 20 mg/kg, by intragastric administration. Feed for 16 weeks.

B) RT-PCR method to detect the effect of compound (I) on the mRNA levels of p66shc, PAI-1, and p21 in the aorta

1) Extraction of RNA:

Take the centrifuge tube of the tissue sample out of the liquid nitrogen tank and put it into a liquid nitrogen pre-cooled mortar, add a certain amount of Trizol, and grind it repeatedly with a grinding rod until uniform. Extract the total RNA of cells according to the instructions, and measure the ratio of OD ₂₆₀ and OD ₂₈₀ to evaluate the quality of the extracted RNA (OD ₂₆₀ /OD ₂₈₀ can be between 1.8-2.0). After quantification, each group was purified with DEPC water RNA was adjusted to the same concentration.

2) Reverse transcription-cDNA synthesis

The RevertAid ^™ First Strand cDNA Synthesis Kit (Fermentas) was used. The RNA obtained in step 1) was reverse-transcribed into cDNA (200ng/μl), and stored at -20°C or continued with the following PCR reaction.

3) RT-PCR reaction

The cDNA template (50ng/μl) and all cDNA samples obtained by reverse transcription in the dilution step 2) were respectively configured for RT-PCR reaction system. Roche's FastStart Universal SYBR Green Master (ROX) kit was used to prepare a PCR reaction system, which was then placed on a RT-PCR (Biorad) instrument for PCR reaction. Each set of primers (synthesized by Invitrogen) was subjected to melting curve experiments. The target genes (p66shc, PAI-1, p21) and housekeeping genes (β-actin) of each sample were subjected to RT-PCR reaction respectively. The fold adjustment was calculated based on the Ct value of the gene of interest and the Ct value of its housekeeping gene for each sample. Up-regulation fold = 2 ^{- [(Ct value of the target gene of the sample - Ct value of the internal reference of the sample) - (Ct value of the blank target gene - Ct value of the blank internal reference)]}

Mouse p66shc primer sequence:

F3: 5'-AAGTACAACCCACTTCGGAATGA-3'

R3: 5'-GGGTCCCAGGGATGAAG-3'

Mouse PAI-1 primer sequence:

F1: 5'-CTCCGAGAATCCCACACAG-3'

R1: 5'-ACTTTGAATCCCATAGCATC-3'

Mouse p21 primer sequence:

F2: 5'-TCTCAGGGCCGAAAACGGAG-3'

R2: 5'-ACACAGAGTGAGGGCTAAGG-3'

Mouse β-actin primer sequence:

F4:5'-CCTTCCTTCTTGGGTATGGAATC-3'

R4:5'-AGCACTGTGTTGGCATAGAGGT-3'

As shown in Figure 16, compared with the sodium carboxymethylcellulose control mice, the mice fed the compound (I) had significantly decreased p66Shc, PAI-1, and p21 mRNA levels in the aorta. It shows that the compound (I) has the effects of delaying vascular aging and anti-aging in vivo.

Table 1 Compounds and activities of different substituents

Table 2 Blood lipid levels in apoE ^-/- mice after administration

(# indicates that the model group is compared with the blank group #P<0.05, * indicates that the drug treatment group is compared with the model group *P<0.05)

Claims (3)

1. Application of substituted piperazine-1,4-diamide compounds in the preparation of medicines for treating and/or preventing atherosclerotic diseases; wherein, the substituted piperazine-1,4-diamide compounds As shown in formula 1; In formula 1, R1 and R2 are: or 2. The application of substituted piperazine-1,4-diamide compounds in the preparation of medicines for the treatment and/or prevention of cardiovascular and cerebrovascular diseases; wherein, the substituted piperazine-1,4-diamide compounds such as Formula 1 shows; In formula 1, R1 and R2 are: or 3. The application of substituted piperazine-1,4-diamide compounds in the preparation of drugs for the treatment and/or prevention of blood lipid regulation; wherein, the substituted piperazine-1,4-diamide compounds are as shown in formula 1 shown; In formula 1, R1 and R2 are: or