CN105902546A - Application of substituted piperazine-1,4-diamide compound in preparation of medicines for treating and/or preventing inflammation reaction - Google Patents

Abstract

The invention relates to an application of a substituted piperazine-1,4-diamide compound in medicine production, wherein the substituted piperazine-1,4-diamide compound can be used as a SIRT1 activator to excite nuclear receptors LXR[alpha] and LXR[beta], regulate expression of target protein ABCA1/G1, promote excretion of lipids and cholesterol, regulate expression of an inflammation key protein p65 and regulate expression of senescence associated genes such as p66shc, etc., thereby achieving regulation effects on metabolism of the cholesterol, inflammation associated genes, and senescence genes or proteins. The compound is used for treating cardiovascular and cerebrovascular diseases, regulating blood fat, treating atherosclerosis disease, resisting inflammation reaction and resisting senescence.

Description

The application in the medicine of preparation treatment and/or prevention of inflammation reaction of the substituted piperazine-1,4-diamide compound

The application is the divisional application of the patent application of Application No. 201410224804.X, invention entitled " application in pharmacy of the substituted piperazine-1,4-diamide compound ".

Technical field

The present invention relates to the purposes of substituted piperazine-Isosorbide-5-Nitrae-diamide compound, particularly relate to the application in the medicine of preparation treatment and/or prevention of inflammation reaction.

Background technology

Inherited genetic factors, diet and aging all have a certain impact for the homeostasis of internal cholesterol and fat.Increasing research shows, limit (calorie restriction, CR) by heat and reduce triglyceride, T-CHOL and low-density lipoprotein cholesterol, increase HDL-C, the sickness rate of cardiovascular disease with slow down aging, can be reduced.CR is uniquely can be prolonged long-life control measures in yeast, anthelmintic, fruit bat and mammal by the universally recognized one of scientific circles.It is reported; CR is by regulation silent message regulatory factor 2 (silent information regulator 2; Sir2) signal path plays a role, and has the effects such as atherosclerosis, antiinflammatory, defying age, oxidative stress, anti-apoptotic, regulation energy metabolism, cardiovascular and cerebrovascular vessel protection, blood lipid regulation.

Sir2 is the important gene being regulated cell survival by chromosome Silencing Mechanisms and cellular energy metabolism mechanism that first a class finds in yeast, has histone deacetylase activity, and the aging with various kinds of cell is closely related.Mammal Siruins family includes 7 albumen altogether, is respectively designated as SIRT1-7, and wherein SIRT1 and yeast Sir2 homology are the highest, the most studied at most.It is on the one hand by modifying histone, and deacetylation H1K26, H3K9 and H4K16 maintain chromatin to be in silence state and genome is stable；On the other hand numerous nonhistones by deacetylation, participate in the energy metabolism of regulating cell, propagation, apoptosis, aging and tumor and occur.The deacetylation substrate of SIRT1 has p53 albumen, jaw transcription factor (forkhead-O-box transcription factors; FOXOs), the PPAR gamma co-activation factor-1 α (peroxisome-proliferated activated receptor c coactivator; PGC-1 α), Sterol regulatory element binding protein (sterol regulatory element binding protein; and liver X receptor (liver X receptor, LXR) etc. SREBP-1c).

LXRs is a member in nuclear receptor family, during by ligand activation, the antiport of cholesterol can be promoted, promote the secretion of bile inner cholesterol, the absorption of suppression enteral cholesterol, have in maintaining intracellular and body cholesterol homeostasis and be of great significance, during reverse cholesterol transport, play extremely important effect.So-called reverse cholesterol transport, it may be assumed that by cholesterol transport to liver, the form by liver metabolism or with bile is discharged, and this process is an important physiological process of the cholesterol tissue accumulation around preventing excess.Document is reported; SIRT1 with LXRs interacts and brings it about deacetylation (432 lysines of LXR α, 433 lysines of LXR β) and activate; the expression of regulation LXRs downstream target gene, in SIRT1 Gene-Deficient Mice body, cholesterol level significantly raises.

Cardiovascular disease is occurred development to have certain protective effect by SIRT1.Studying discovery, the nitricoxide synthase (eNOS) in SIRT1 mono-aspect deacetylation vascular endothelial cell the earliest, activation eNOS suppresses angiotensin receptor AT1；On the other hand, the aging of suppression vascular endothelial cell, delay atherosclerotic generation.Studying discovery subsequently, SIRT1 passes through deacetylation use, activation LXRs and FXR (farnesoid X receptor) regulation lipid and cholesterol metabolism; promote that high density lipoprotein is combined with cholesterol; reduce cholesterol deposits, regulate blood lipid level, affect atherosclerotic formation.Additionally, SIRT1 can also suppress the expression of angiotensin receptor AT1, the pathology of prevention heart is loose.SIRT1 can regulate liver carbohydate metabolism and lipid metabolism.In short-term fasted mice liver, knock out SIRT1 gene and the expression of liver fat acid beta-oxidation gene can be caused to reduce.

A series of inside and outsides it is experimentally confirmed that SIRT1 to inflammation gene expression express and tissue inflammatory damage there is significant depression effect.Mice RAW264.7 macrophage at SIRT1 gene knockout, lipopolysaccharide (lipopolysaccharide, LPS) the Nuclear factor kappa B (nuclear factor κ B, NF-κ B) induced activates and the expression of the multiple proinflammatory cytokine such as TNF-α, IL-1 β, IL-6 is significantly increased.More and more researchs show, atheromatous plaque does not contain only lipid, and have a large amount of inflammatory cell infiltration, gather substantial amounts of mononuclear cell with blood vessel wall and lymphocyte is characterized, this prompting inflammation status in mediation atherosclerosis generation, evolution, also embodies the internal relation between SIRT1, atherosclerosis and inflammation and important function.

Formula 1 is known in piperazine-1,4-diamide compound expression formula

In the piperazine-1,4-diamides of above-mentioned formula 1, R1 and R2 can be the substituent group shown in table 1 respectively.

Summary of the invention

It is an object of the invention to provide the new application of substituted piperazine-Isosorbide-5-Nitrae-diamide compound, i.e. application in pharmacy.

In particular it relates to substituted piperazine-Isosorbide-5-Nitrae-diamide compound is as the application in the medicine of preparation treatment and/or prevention of arterial atherosclerotic disease.

Relate to substituted piperazine-1,4-diamide compound as the application in the medicine of preparation treatment and/or prevention cardiovascular and cerebrovascular disease.

Relate to the application in the medicine of preparation treatment and/or prevention regulation blood fat of the substituted piperazine-1,4-diamide compound.

Relate to the application in the medicine of preparation treatment and/or prevention of inflammation reaction of the substituted piperazine-1,4-diamide compound.

Relate to the application in preparation treatment and/or pre-anti-aging medicine of the substituted piperazine-1,4-diamide compound.

Substituted piperazine-Isosorbide-5-Nitrae-the diamide compound of the present invention can be administered with itself, or is administered with the form of pharmaceutical composition.The Pharmaceutical composition of the present invention includes the compounds of this invention of effective dose or its officinal salt and one or more pharmaceutically suitable carrier physiologically accepted.

The pharmaceutical composition of the present invention can be prepared in the usual way, uses one or more physiologically acceptable carriers, excipient and auxiliary agent, is conducive to reactive compound is processed into pharmaceutical preparations.Suitable preparation depends on selected route of administration, can be prepared according to method well known in the art.

Substituted piperazine-1,4-the diamide compound of the present invention or its pharmaceutical salts can be discharged to patient by various route of administration or mode.The route of administration being suitable for includes but not limited to suction, transdermal, oral, rectum, through mucous membrane, enteral and parenteral, and parenteral includes intramuscular, subcutaneous and intravenous injection.

Accompanying drawing explanation

Fig. 1 is resveratrol Activation Activity amount effect curve on SIRT1 activator screening model.

Fig. 2 is the effect schematic diagram of micromolecular compound atherosclerosis, antiinflammatory, defying age, Adjust-blood lipid.

Fig. 3 is the checking of compound (I) intracellular deacetylation functional activity.Wherein, the Western blot figure of intracellular p53 and Ac-p53 of U2OS after a is compound (I) effect in Fig. 3；In Fig. 3, b is that albumen gray scale is quantitatively schemed.

Fig. 4 is compound (I) and SIRT1 protein molecular interphase interaction, specifically E123 Yu SIRTI-N-CC-C protein-interacting sensing figure.

Fig. 5 is the compound (I) impact on LXRs deacetylation degree.Wherein, the Western blot figure of HepG2 intracellular acetylation LXRs (specially LXR α, LXR β) after a is compound (I) effect in Fig. 5；In Fig. 5, b is that albumen gray scale is quantitatively schemed.

Fig. 6 is the exciting LXRs activity amount effect curve of compound (I).

Fig. 7 is compound (I) regulation ABCA1 promoter transcription activity.

Fig. 8 is ABCA1 and ABCG1 protein expression in compound (I) regulation RAW264.7 cell.Wherein, the Western blot figure of intracellular ABCA1 and ABCG1 of RAW264.7 after a is compound (I) effect in Fig. 8；In Fig. 8, b is that albumen gray scale is quantitatively schemed.

Fig. 9 is that compound (I) regulates HepG2 intracellular ABCG5/G8 albumen.Wherein, the Western blot figure of intracellular ABCG5 and ABCG8 of HepG2 after a is compound (I) effect in Fig. 9；In Fig. 9, b is that ABCG5 albumen gray scale is quantitatively schemed；In Fig. 9, c is that ABCG8 albumen gray scale is quantitatively schemed)

Figure 10 is that compound (I) promotes to arrange outside RAW264.7 cellular cholesterol.Wherein, in Figure 10, a is the cholesterol efflux that compound (I) promotes RAW264.7 intracellular HDL mediation；In Figure 10, b is the cholesterol efflux that compound (I) promotes RAW264.7 intracellular Apo-AI mediation.

Figure 11 is that compound (I) suppresses macrophage foam cell formation carburetion red O dyeing microscopic examination photo.Wherein, a is blanc cell (being not added with Ox-LDL)；B is solvent control (80 μ g/ml Ox-LDL)；C is 10 μMs of compounds (I)+80 μ g/ml Ox-LDL；D is positive control (10 μMs of 9CRA+80 μ g/ml Ox-LDL).

Figure 12 is the compound (I) impact on RAW264.7 intracellular total cholesterol level.

Figure 13 is the expression of compound (I) suppression inflammation key protein p65.Wherein, the Western blot figure of the intracellular p65 of THP-1 after a is compound (I) effect in Figure 13；In Figure 13, b is that albumen gray scale is quantitatively schemed.

Figure 14 is that compound (I) is to apoE^-/-The impact of speckle in mouse aorta total length.Aorta total length oil red coloration result shows, compound (I) significantly reduces apoE^-/-The formation of mouse aorta plaque, reduces cholesterol and lipid accumulation.Wherein a is blank group (normal diet)；B is model group (high fat diet)；C is compound (I) 20mg/kg (high fat diet).

Figure 15 is that compound (I) is to apoE^-/-The impact of mouse heart efferent tract.Oil red coloration result display compound (I) of heart efferent tract can significantly reduce apoE^-/-The area of speckle and quantity in mouse heart efferent tract, reduce cholesterol and lipid accumulation.Wherein, a is blank group (normal diet)；B is high fat diet model group；C is compound (I) 20mg/kg (high fat diet).

Figure 16 is that the mrna expression of C57BL/6 mouse aorta SAG p66shc, PAI-1, p21 is affected by compound (I).

Detailed description of the invention

In order to be more clearly understood that the essence of the present invention, the activity that first substituted piperazine-Isosorbide-5-Nitrae-diamide compound is activated SIRT1 by us is measured, and the results are shown in Table 1.From table 1 it follows that substituted piperazine-Isosorbide-5-Nitrae-diamide compound has the activity of activation SIRT1 in various degree.With result, the application in pharmacy of the substituted piperazine-1,4-diamide compound is deeply described with the pathology test of the substituted piperazine-1,4-diamide compound (referred to as compound (I)) of label " E1231 " below.

First, it is successfully obtained destination protein SIRT1 by escherichia expression system.Based on homogeneous phase time discrimination fluorescence technology, utilize activated albumen to set up high flux SIRT1 activator screening model, after model optimization, utilize known generally acknowledged positive drug resveratrol that model is verified, EC₅₀Value is consistent with document report, sees Fig. 1, shows that expressed albumen is highly active destination protein, and screening model is believable.Combination of compounds storehouse (country new drug (microorganism) screening experiment room) is carried out screening bioactive compounds, it is thus achieved that several reactive compounds, and measure all compounds activation to SIRT1 albumen.Representative reactive compound (I) is selected to carry out further activity research; confirm that compound (I) enables to the deacetylation degree increase of the target protein p53 of SIRT1 albumen, the activator of strictly SIRT1 albumen on a cellular level.The K between compound (I) and Protein S IRT1 has been recorded in surface plasma resonance is tested_DValue is 9.61 μMs.Finally; compound (I) has been carried out the research in terms of molecular pharmacology; find that compound (I) can make the deacetylation degree of LXRs increase; exciting nuclear receptor LXR α and LXR β; the expression of regulation target protein ABCA1/G1, promotes intracellular cholesterol efflux, suppresses macrophage foam cell formation; raise ABCG5/G8 to express, promote cholesterol secretion.Inquire into compound (I) to the effect of inflammatory factor associated metabolic path in foam cell, found that it can suppress the expression of inflammation transcription factor NF-κ B key protein p65.Have studied the compound (I) impact on mouse aorta Aging-associated gene expression, find that it can suppress the mrna expression of p66shc, PAI-1, p21.

Therefore, with the compound (I) the substituted piperazine-1 as representative, 4-diamide compound can be as the activator of SIRT1, play cholesterol metabolism, inflammation, the regulation effect (see Fig. 2) of gene of diseases associated with senescence, can be used for atherosclerosis, inflammation, aging, cardio-cerebrovascular diseases prevention and/or treatment.

The embodiment 1. substituted piperazine-1,4-diamide compound mensuration to SIRT1 Activation Activity

1) protein expression

First, obtain the catalytic domain of people SIRT1 from plasmid pcDNA3.1-SIRT1 (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences Liu Depei teaches seminar's present) amplification and comprise N end, the cDNA of C end portion sequence, be connected order-checking correctly with cloning vehicle pBlunt-Simple Vector after, with coli expression carrier pET-30a (+) be connected, the named pET-SIRT1 of recombinant expression plasmid built, convert escherichia coli expression host BL21 (DE3), picking monoclonal, order-checking is identified.By single bacterium colony shake-flask culture correct for order-checking, at OD₆₀₀When value is for 0.6-0.8, add the isopropyl-β-D-thiogalactoside (IPTG) of final concentration of 0.5mM, 15 DEG C, 200rpm, collects bacterium solution after cultivating 24h.It is centrifuged 30min with 12,000rpm after ultrasonic disruption thalline.Collect supernatant, through 0.45 μM of membrane filtration.UseExplorer system carries out protein purification work, uses 1ml prepacked column HisTrapFF crud as purification media.Its principle is the His-tag and the Chelating Sepharose in post that destination protein passes through its N end^TMOn Ni²⁺Ion chelating, and then absorption is on pillar, and other foreign proteins are because of can not be with Ni²⁺Ion chelating, directly flows out pillar with sample-loading buffer, then with elution buffer eluting destination protein, thus obtain highly purified destination protein SIRT1.

2) compound mensuration to SIRT1 Activation Activity

This determination of activity uses Cisbio company man's SIRT1 activator screening reagent box to carry out the mensuration of compound activity.

Measuring principle:

Detection system is as follows: recombined human SIRT1 albumen, and substrate substrate-d2 is the little fragments of peptides of p53 of fluorophor d2 labelling, NAD⁺Playing the necessary cofactor of enzymatic activity for SIRT1, anti-acetyl-cryptate is Eu²⁺The cryptate of labelling.If the compound added has activation to enzyme, substrate deacetylation degree increases, and the distance between substrate and cryptate becomes remote, is not enough to produce Resonance energy transfer, shows as fluorescence reading and reduces.

Assay method:

(1) in Reaction buffer, add the DTT of final concentration of 1mM, be configured to Enzymatic buffer.

(2) recombiant protein, substrate substrate-d2 and coenzyme NAD are diluted successively with Enzymatic buffer⁺So that it is final concentration be respectively 0.5mU/ μ l, 6nM, 150 μMs.

(3) by Enzymatic buffer diluted compounds to certain concentration.

(4) 2 μ l testing compounds, 2 μ lNAD it are sequentially added into⁺, 4 μ l substrates to 384 orifice plates, be eventually adding 2 μ l recombiant proteins, start enzymatic reaction.Arrange and be not added with the No enzyme group of albumen and be not added with the Negative control group of compound, often 3 parallel holes of group.

(5) incubated at room 120min.

(6) Anti-acetyl-cryptate of the 10 final concentration of 7.5nM of μ l is added, incubated at room 5h or overnight, measure fluorescent value (Ex:340nm, Em:665/615nm) by microplate reader.

(7) computational methods:

Ratio:(665nm/615nm)×10⁴

Substrate deacetylation ratio (%): 100-(Ratio_Sample/Ratio_{No enzyme}×100)

Rise rate (%)=deacetylation ratio_Sample/ deacetylation ratio_{Negative control}× 100%

If it is positive that rise rate is considered as primary dcreening operation more than or equal to 150%, the multiple sieve and the amount effect curve that carry out next step measure.

Read fluorescence reading, after calculating according to formula, be vertical coordinate using the logarithm value of compound concentration as abscissa, the deacetylation degree of substrate, with GraphPad Prism software matched curve, obtain EC₅₀Value and maximum rise rate, the results are shown in Table 1.

In this measurement result explanation present invention, substituted piperazine-1,4-diamide compound has significant deacetylation activation to SIRT1.Wherein, the maximum rise rate of active best compound (I) is 384.09%, EC₅₀Value is 0.43 μM.Due to SIRT1 in participating in cholesterol metabolism and the physiological process such as fatty acid, carbohydate metabolism significant, therefore the substituted piperazine-Isosorbide-5-Nitrae-diamide compound in this result explanation present invention can play a role in regulation and control relate to the disease of Relevant Physiological Courses by activating SIRT1.

The cultivation of embodiment 2. cell

HepG2, ABCA1-LUC HepG2, U2OS and HEK293 cell is attached cell, and about 48h passes on once.After cell covers with, abandon old culture medium, discard after PBS rinsing cell, adding appropriate pancreatin, at room temperature peptic cell about 2min, abandons Digestive system, add the culture medium containing 10%FBS immediately, to suppress trypsin vigor, blow and beat culture bottle inner cell the most gently with elbow straw, at the bottom of making cell completely disengage from bottle and piping and druming is allowed to be separated into individual cells suspension.Again in 1:3 ratio inoculating cell suspension in new cell bottle, or discard appropriate cell suspension, then add appropriate complete medium, put into incubator and continue to cultivate.Condition of culture: 37 DEG C, 5%CO₂。

Mouse monokaryon macrophage RAW264.7 is that half attached cell, about 48h are passed on once.Trypsinization 5min, is passed in 1:4-1:6 ratio, uses DMEM complete medium.Condition of culture: 37 DEG C, 5%CO₂。

People Acute Monocytic Leukemia Cell Line THP-1 is that suspension cell, about 48h pass on once.Treat that cell density is (3-5) × 10⁶Individual/ml time, with elbow straw blow and beat culture bottle inner cell, be allowed to be separated into individual cells suspension.Again in 1:4 ratio inoculating cell suspension in new cell bottle, or discard appropriate cell suspension, then add appropriate complete medium, put into incubator and continue to cultivate.Condition of culture: 37 DEG C, 5%CO₂。

The impact of acetylation p53 protein expression level in the human osteosarcoma U2OS cell that doxorubicin is induced by embodiment 3. compound (I).

1) compound treated cells: in advance by human osteosarcoma cell U2OS by McCoy ' the s 5a culture medium containing 10%FBS with every hole 3 × 10⁵Individual cell is inoculated in 6 orifice plates, 37 DEG C, 5%CO₂Under the conditions of cultivate to logarithmic (log) phase.After cell is the most adherent, draw Cell sap, it is changed to blank McCoy ' s5a culture medium, add the doxorubicin hydrochloride (DOX) of final concentration of 5 μMs, the SIRT1 specific inhibitor EX-527 of 10 μMs and final concentration are respectively the positive compound of 0.1,1,10 μMs, 37 DEG C, 5%CO₂Under the conditions of hatch 6h.

2) preparation of albumen: after 6h, discard culture medium, rinses cell 2 times with the PBS of pre-cooling, collects cell by culture medium after trypsinization, and 800rpm is centrifuged 3min, then uses PBS suspension cell, and 800rpm is centrifuged 3min.Often pipe is separately added into the RIPA cell pyrolysis liquid (adding the PMSF of final concentration of 100 μ g/ml in every 1ml lysate) of 52 μ l, in cell lysis 30min on ice.4 DEG C, 12000 × g is centrifuged 20min.Collect supernatant, carry out quantitatively, using ddH to albumen with BCA quantification of protein test kit (Thermo company)₂Each histone sample concentration is adjusted to 2 μ g/ μ l by O.Adding a certain amount of 5 × albumen sample-loading buffer, boil 10min in boiling water bath, of short duration centrifugal after cooling, every hole loading 40 μ g albumen carries out SDS-PAGE electrophoresis.Remaining sample is in-80 DEG C of preservations.

3) Western blot method measures protein level: after SDS-PAGE electrophoresis, polyacrylamide gel and transferring film filter paper is put into and soaks balance 5min in transfering buffering liquid.Pvdf membrane is submerged initially in methanol 20s activation, is washed with water, and is placed in transfering buffering liquid immersion balance 2min.By BioRad semidry method transferring film instrument transferring film, electric current 5mA/cm is set², constant current transferring film 30min.After transferring film terminates, after cutting off respectively, put into incubated at room 1h in 1 × TBST containing 5% (W/V) defatted milk powder according to the size of destination protein.Closing terminates, and dilutes one with 1 × TBST containing 5% (W/V) defatted milk powder and resists, is placed in hybridization bag, after incubated at room 1h, and 4 DEG C of overnight incubation.Wash three times with 1 × TBST, each 10min.Dilute corresponding two with 1 × TBST containing 5% (W/V) defatted milk powder afterwards to resist, be placed in hybridization bag, incubated at room 2h.From hybridization bag, take out pvdf membrane, wash three times with 1 × TBST, each 10min.Colour developing: in the front of film, i.e. turn the mixed liquor (in 1:1 ratio matching while using) having the one side of albumen to add appropriate enhancement mode HRP (horseradish peroxidase) substrate chemiluminescence liquid (ECL) A, B liquid, be immediately placed in gel imaging instrument colour developing.

4) colour developing result, is scanned with ImageJ software, quantitative Treatment.Result is shown in Fig. 3.

Doxorubicin (Doxorubicin, DOX) can inducing cell generation DNA damage so that 382 lysine generation acetylations of p53 albumen.In order to verify compound the deacetylation function of intracellular and investigate function play dependency to SIRT1, add SIRT1 specific inhibitor EX-527.Along with the rising of compound (I) concentration, the expression of acetylation p53 albumen substantially reduces with Concentraton gradient, and the amount of intracellular total p53 albumen does not has significant change, illustrates that p53 albumen deacetylation degree adds.The expression of specific inhibitor EX-527, the Ac-p53 albumen adding SIRT1 albumen adds again.Thus disclosure sets forth compound (I) and have obvious rise effect to the substrate deacetylation effect of intracellular SIRT1, and this effect depends on SIRT1.

Embodiment 4. compound (I) measures with the intermolecular interaction of SIRT1 albumen

Buffer soln used in this mensuration system consists of: HEPES 10mmol/L, NaCl150mmol/L, DTT 1mmol/L, NAD⁺1mmol/L, glycerol 10% (V/V), DMSO2% (V/V).Buffer and sample, by 0.45 μm membrane filtration, use and front ultrasonic remove bubble removing.Completed by Biacore T200System (GE Healthcare) instrument.

Assay method:

1) pretreatment of chip surface: take out maintenance chip, is replaced by CM5 chip, selects Prime, uses wash buffer chip surface.Select Normalize, use Biacore standardizing agents (BIA normalizing solution 70%) that CM5 chip is standardized.Use two chip channel to test, SIRT1 albumen is coated required pH value condition and gropes (immobilization pH-scouting for pre-concentration).First the sodium acetate solution selecting the pH in the range of 3.5 < pH < pI (hSIRT1) carries out preenrichment, according to preenrichment result, selects pH4.0 sodium acetate solution diluted protein to 50 μ g/ml, carries out coupling.Arranging flow velocity is 10 μ l/min, and binding time is 2min, finally rinses chip surface 20s with the NaOH of 50mM and removes the SIRT1 albumen of residual.

2) albumen envelope chip surface: EDC/NHS equal-volume is mixed, with the flow velocity sample introduction 10min of 1 μ l/min, CM5 chip surface carboxyl is activated.Result is groped according to chip surface pretreatment, SIRT1 albumen is diluted to 50 μ g/ml as buffer by the sodium acetate solution selecting pH value to be 4.0, arranging flow velocity is 10 μ l/min, binding time is 20min, and the surface of CM5 chip it is coated on by amino coupled effect, arranging target coupling amount is 7000RU, concurrently sets a blank channel as comparison passage.After sample introduction terminates, adding ethanolamine, with the flow velocity sample introduction 10min of 10 μ l/min, be not associated with the pendant carboxylic group site of amino closing chip surface, final coupling result is 7056RU.

3) add compound (I) and be combined detection: CM5 chip coated for SIRT1, by compound (0,0.39,1.56,3.12,6.25, the 12.5 μ Μ) sample introduction successively of variable concentrations, compound is made to flow through chip surface with the flow velocity of 30 μ l/min, binding time is set to 60s, after Dissociation time is 120s, grope the regeneration condition of chip surface, find that compound can dissociate within a certain period of time the most naturally.

By Fig. 4, can be combined with the dose-dependent mode SIRT1 that recombinates with people by the change visual compounds (I) of RU value, K_DValue is 9.61 μMs.Little molecule and protein bound K_DValue generally 10^-3To 10^-6M, therefore, has the strongest affinity between compound (I) and Protein S IRT1.

The embodiment 5. compound (I) mensuration to LXRs deacetylation degree

In order to investigate the compound impact on LXRs deacetylation degree, we utilize co-immunoprecipitation experiment to verify.

This experiment uses HepG2 cell, is laid in the big culture dish of a diameter of 90mm, after cell is the most adherent, is separately added into the compound (I) of 10 μMs, arranges solvent control hole, processes cell 20 to 24h.Extract Nuclear extract, jointly hatch 4h 4 DEG C of conditions with LXR α and LXR β antibody and Protein A/G agarose beads respectively, wash beads, boil sample, carry out western blot detection with acetylated-Lys antibody.After film membrane regeneration liquor after exposure is carried out the experimental procedure regeneration of gene technology company limited by Beijing Puli, then with corresponding LXRs antibody test.Result is shown in Fig. 5.

As can be seen from Figure 5; after adding compound (I); the expression substantially constant of LXR α and LXR β; the expression of acetylation LXR β has a certain degree of decline; but; the expression of acetylation LXR α declines notable, shows that SIRT1 activator compound (I) can regulate LXR α and the Acetylation Level of LXR β in hepatocyte.

The embodiment 6. compound (I) mensuration to LXRs agonist activity

This determination of activity uses the agonist screening model of LXRs to carry out the mensuration of compound activity.

Measuring principle:

The principle of this detection is, two main domains according in LXRs structure: ligand binding domains (LBD) and DNA binding structural domain (DBD), and yeast transcription factor GAL4 has the feature of nuclear receptor analog structure, the principle of application yeast two-hybrid, two plasmids of structure respectively: pBIND-LXRs plasmid contains DBD part and the LBD domain of LXRs of GAL4 gene, can change conformation when by ligand activation；Another plasmid is pGL4-GAL4, containing the luciferase reporter gene Luc of GAL4 gene promoter region response element.The present invention is by two plasmid co-transfections to HEK293 cell, to study the compound activation to LXRs.In this detection system, compound is if able to activate LXRs, and the LXRs protein conformation that expression plasmid is expressed changes, and combines with reporter plasmid, and now the expression activity of compound group luciferase is higher than the matched group not adding compound；Otherwise when compound to LXRs inactive time, then LXRs protein conformation does not changes, and does not combines with reporter plasmid, then the expression activity of compound group luciferase does not produce change.

Assay method:

1) transfecting at 96 orifice plates, day before transfection, HEK293 plating cells, density is 1.5-3.0 × 10⁵Individual/ml cell.Treat that HEK293 cell length is converged to 90% and its growth conditions is good, dilute 0.5 μ l liposome Lipofectamine according to 25 μ l/ hole Opti-MEM culture medium^TM2000Reagent, incubated at room 5min.25 μ l/ hole Opti-MEM culture medium dilution 200ng plasmid DNA, including pGL4-GAL4 and pBIND-LXR α/β, merge mixing, incubated at room 20min with dilution liposome afterwards.

2) the DNA-liposome complex solution after merging adds MEM complete medium according to every hole 100 μ l, culture medium is made fully to mix with DNA-liposome complex, now the culture medium in cell plates is sucked, mixed cell culture fluid-DNA-liposome complex solution is added in 96 orifice plates, every hole 150 μ l.96 orifice plates are placed in CO2 gas incubator 37 DEG C.

3) 6h is cultivated, sucking-off transfection media, by compound (I) from the beginning of the maximum concentration of 40 μMs, become 8 concentration with 2 times of Concentraton gradient stepwise dilutions, negative control hole adds the DMSO of 0.1% simultaneously, processes cell respectively, continues to cultivate 18h.

4) culture medium after 18h, in the every hole of sucking-off 96 orifice plate.Every hole adds 150 μ l PBS and rinses cell gently, overturns 96 orifice plates and outwells PBS and dry.Every hole adds 20 μ l 1 × CCLR cell pyrolysis liquids, 37 DEG C of cell lysis 20-30min (how much determining the time according to cell number), examines under a microscope cell and crack the most completely.Lysate being fully transferred in the fluorescence analysis respective aperture with the 96 opaque blanks in hole, notice that lysate avoids bubble as far as possible, otherwise making to affect reading (if using 96 hole clear bottom blanks, need not shift lysate).

5) in every hole, it is rapidly added 50 μ l fluorescence analysis reagent (Promega company), immediately analysis blank is put in microplate reader and detect.

6) by the equation below calculating testing sample rate of change to uciferase activity:

Rate of change (%)=A/B × 100

Wherein, A is to add cell fluorescence element enzymatic activity (RLU) measured after testing sample, and B be cell fluorescence element enzymatic activity (RLU) that addition negative control sample (DMSO) measures afterwards.Rate of change (%) is considered as the compound agonist activity to LXR, represents with % percentage ratio, or represents with multiple.Using the logarithm value of compound concentration as abscissa, rate of change is vertical coordinate, and with GraphPad Prism software matched curve, result is shown in Fig. 6.

In this measurement result explanation present invention, compound (I) has significant transcriptional activation to LXR α/β, and has obtained the compound (I) amount effect relation curve on LXR α/β agonist screening model.From fig. 6, it can be seen that compound (I) is with dosage-dependent manner excitement LXR α/β, it is 188.14% to LXR α maximum agonist activity, EC₅₀Value is 0.65 μM；It is 182.32% to LXR β maximum agonist activity, EC₅₀Value is 0.08 μM.Owing to nuclear receptor LXR is participating in during cholesterol metabolism and the physiological process such as fatty acid, carbohydate metabolism converge significant, therefore the compound (I) in this result explanation present invention can be played a role in regulation and control relate to the disease of Relevant Physiological Courses by transcribing of exciting LXR.

The embodiment 7. compound (I) mensuration to ABCA1 promoter transcription activity

This determination of activity uses adjustment screening model on ABCA1 to carry out the mensuration of compound activity.

Measuring principle:

The principle of this detection is, recombiant plasmid pGL3-ABCA1 and the pcDNA3.1 cotransfection the pure man hepatocellular carcinoma H22 cell of ABCA1 regulatory sequences will be had the reporter gene upstream of luciferase clone, stable transfected cells strain, named ABCA1-LUC HepG2 cell is obtained through G418 screening.By adding the change of the expression activity of compound test cell fluorescence element enzyme, it can be seen that the compound impact on ABCA1 promoter transcription activity, and then judge the rise rate to ABCA1.

Assay method:

The ABCA1-LUC HepG2 cell of digestion exponential phase, with culture medium dilution cell counting, with 5 × 10⁵The density of individual/ml is seeded to 96 orifice plates, and every hole adds 100 μ l single cell suspensions.After 6h, treating that cell is the most adherent, remove the culture medium containing serum, rinse cell gently once with PBS, every hole adds the serum-free RPMI-1640 culture medium 200 μ l containing different compound concentrations, adds the DMSO of respective concentration in control wells simultaneously.Removing culture medium after 18-24h, measure according to the method described in assay method (4)-(6) in embodiment 6, calculate and map, result is shown in Fig. 7.

Compound (I) increases ABCA1-LUC HepG2 cell fluorescence element enzymatic activity in dose-dependently mode.Compound (I) raises ABCA1 expression activity and reaches peak 226.67%, its EC₅₀Value is 0.25 μM.Due to ABCA1 during reverse cholesterol transport significant, therefore this result explanation the present invention in compound (I) can strengthen ABCA1 promoter transcription activity, play a role in the disease relate to Relevant Physiological Courses.

Embodiment 8. compound (I) impact on ABCA1 and ABCG1 protein expression level in RAW264.7 macrophage.

1) compound treated cells: mouse monokaryon macrophage RAW264.7 is with 4 × 10⁵Individual/ml be inoculated in 6 orifice plates adherent after, process with the compound (I) of 0.1,1 and 10 μM, using concentration for 0.1%DMSO as negative control group, compound (the I)+EX-527 group of 10 μMs, all at 37 DEG C, 5%CO be set simultaneously₂Under the conditions of cultivate 20-24h.

2) Western blot method measures ABCA1 and ABCG1 protein expression level, and uses software quantitative Treatment.Result is shown in Fig. 8.

ABCA1 and ABCG1 is the target protein of LXR direct regulation and control, the two major function in macrophage is all by intracellular unnecessary cholesterol efflux, promote reverse cholesterol transport process, prevent the OxLDL ELISA in macrophage (Ox-LDL) accumulation from causing macrophage foam cell formation.Being found out by Fig. 8, through the effect of compound (I) 20-24h, can raise the expression of ABCA1 and ABCG1 when 1-10 μM, ABCA1 maximum raises 2.17 times, and ABCG1 is up to 2.48 times dose-dependant.Thus disclosure sets forth compound (I) to macrophage promoting, cholesterol efflux associated protein has obvious rise effect.

Embodiment 9. compound (I) impact on ABCG5 and ABCG8 expression in HepG2 cell

1) compound treated cells: the compound (I) that the HepG2 cell being inoculated in 6 porocyte culture plates adds variable concentrations in advance processes, using concentration for 0.1%DMSO as negative control group, at 37 DEG C, 5%CO₂Under the conditions of cultivate 20-24h.

2) Western blot method measures ABCG5 and ABCG8 protein expression level, and uses software quantitative Treatment.

ABCG5 in liver, ABCG8 albumen is also the target protein in LXR downstream, and they are by the direct regulation and control of LXR, and the two transport protein is expressed on the film of hepatocyte tubule, thus drives cholesterol to transport expression in bile, promotes cholesterol to secrete.As it is shown in figure 9, in the present invention, compound (I) is the highest to the rise multiple of ABCG5 when 0.1 μM, up to about 1.2 times；In the range of 0.1-10 μM, raise the expression of ABCG8 dose-dependant, when 10 μMs, ABCG8 is raised and reach as high as 2.2 times.Illustrate that compound (I) likely can be expressed by raising ABCG5 and ABCG8 and promote that in liver, cholesterol is secreted.

Embodiment 10. compound (I) promotes to arrange experiment outside Macrophage cholesterol

1) mouse monokaryon-macrophage RAW264.7 DMEM-high glucose medium (500 μ l/ hole) containing 10%FBS, with 2 × 10⁵Individual/hole is inoculated on 24 porocyte culture plates, in 37 DEG C, and 5%CO₂Under the conditions of incubated overnight.

2) abandon Cell sap, be changed to the DMEM-high glucose medium (500 μ l/ hole) containing 0.2% (W/V) BSA, add 1,2-[³H] cholesterol make its final concentration of 1 μ Ci/ml, 37 DEG C, 5%CO₂Under the conditions of hatch 24h.

3) wash cell 2 times with PBS (1ml/ hole), add the mensuration culture medium (DMEM adds 0.2%BSA, 0.1%DMSO, 25mM HEPES, pH7.4) containing finite concentration compound, hatch 18～24h for 37 DEG C.

4) PBS (1ml/ hole) washes cell 2 times, adds culture medium (DMEM adds 0.2%BSA, 0.1%DMSO, 25mM HEPES, pH7.4), the apoA-I or the HDL of 50 μ g/ml of 10 μ g/ml, hatches 4h.

5) collecting culture medium, 10,000 × g is centrifuged 5min, takes supernatant to be measured.

6) with 0.1M NaOH 0.5ml lysis at room temperature cell 30min, lysate is collected to be measured.

7) measure: testing sample is transferred to respectively on 3mm filter paper, 75 DEG C of drying, the scraps of paper are placed on liquid and dodge in cup, add 10ml liquid and dodge liquid (mass concentration is 0.5%PPO and 0.05%POPOP) and the solvent mixed preparing that volume fraction is 55% dimethylbenzene and 45% glycol dimethyl ether, deposit in being placed in brown container, overnight using, liquid scintillation counter counts.Whole experimental cell is divided into matched group (but be not added with cholesterol add apoA-I/HDL, add cholesterol) and sample-adding group (being simultaneously introduced cholesterol, apoA-I/HDL and certain density testing sample).

Cholesterol Efflux rate (%)=culture fluid cpm value/total cpm value × 100%

=culture fluid cpm value/(culture fluid cpm value+cell cpm value) × 100%

Cholesterol efflux result in macrophage as shown in Figure 10, under compound (I) acts at 0.1,1,10 μMs, can promote 1,2-[³H] cholesterol flow out to cholesterol acceptor, and along with the increase of compound concentration, 1,2-[³H] Cholesterol Efflux level also increases, and flows out to the multiple of apoA-I between 1.2-1.7 times, and the multiple flowing out to HDL is higher than the multiple to apoA-I, about between 1.5-2.0 times.This result meets described in forward part of the present invention, compound (I) is by ABCA1 and ABCG1 protein expression in rise macrophage, and then promote the outer row of intracellular cholesteryl, meet the physiologically active of lxr agonist, and it is more notable as the impact of cholesterol acceptor for HDL, and ABCG1 is mainly responsible for unnecessary cholesterol transport to HDL, the inventive result of this albumen that can significantly raise ABCG1 with the compound inquired into before and mrna expression is consistent.

The experiment of embodiment 11. compound (I) suppression macrophage foam cell formation effect

The mononuclear phagocyte RAW264.7 of the mice DMEM-height sugar culture fluid containing 10%FBS is cultivated on transparent 96 porocyte culture plates.After adherent, it is changed to serum-free DMEM-high glucose medium (100 μ l/ hole).Cell being divided into blank group, foam cell group and dosing group, adds the Ox-LDL of final concentration of 80 μ g/ml to foam cell group and dosing group, after 24h, dosing group adds certain density compound (I).37 DEG C, 5%CO₂Under the conditions of cultivate after 24h, carry out oil red O stain.By 96 orifice plates from CO₂Taking out in incubator, cell is fixed (15 μ l/ hole) 10min with 4% paraformaldehyde, is abandoned solution, and distilled water is washed twice, adds 60% isopropanol (150 μ l/ hole), places 5min, discards solution.Oil red O uses liquid (matching while using filters) add in each hole, and 150 μ l/ holes, dye 1h.Discarding solution, with 60% isopropanol (150 μ l/ hole) hole flushing, then wash twice with distilled water (150 μ l/ hole), last every hole adds 150 μ l distilled waters and is placed in basis of microscopic observation, takes pictures, and result is shown in Figure 11.

Only as foamed groups of cells, by oil red O stain, the coloring degree of cell will be examined under a microscope by addition Ox-LDL.As can be seen from Figure 11, a () blank group does not has red fat to drip formation, b () Ox-LDL solvent control group forms the most red fat and drips existence, (c) compound (I) process group, intracellular occur that Red oil granule significantly reduces compared with matched group, illustrate that compound (I) can reduce lipid and accumulate in macrophage, effectively suppression macrophage foam cell formation, (d) adds lipid accumulation in 10 μMs of 9CRA (LXRs agonist) cell and also significantly reduces.

Embodiment 12. compound (I) reduces intracellular T-CHOL

Utilizing BioVision company cholesterol quantification kit to recommend method to measure intracellular T-CHOL, assay method is as follows:

1) preparation cholesterol standards Concentraton gradient: 0,1,2,3,4,5 μ g/well.

2) collect cell, add 200 μ l CH₃Cl:IPA:NP-40 (7:11:01), 12,000g room temperatures are centrifuged 5min.

3) supernatant is transferred in new EP pipe, be placed in 50 DEG C of baking ovens and liquid is dried.

4) add 100 μ l cholesterol assay buffer and dissolve mixing.

5) adding 2 μ l esterase, 2 μ l substrate mix, 2 μ l cholesterol enzyme mix and 44ul sample or standard substance, 37 DEG C of lucifuges hatch 30min.Read absorbance value under 450nm wavelength and calculate.

Intracellular T-CHOL mainly includes free cholesterol and cholesteryl ester, and the amount of T-CHOL is also a principal character of cell foamed.Being found out by Figure 12, it is notable that compound (I) reduces intracellular total cholesterol level when 1 μM and 10 μMs.Illustrate that the compound added can suppress macrophage foam cell formation, reduce lipid and accumulate in macrophage.

The impact that inflammation key protein p65 in THP-1 cell is expressed by embodiment 13. compound (I)

1) compound treated cells: in advance by the THP-1 being inoculated in 6 porocyte culture plates after adding final concentration 50nM phorbol exters PMA induction 24h, it is added thereto to the E1231 of 10 μMs, hatch 2h for 37 DEG C, the LPS adding final concentration of 10ng/ml stimulates the generation of intracellular cytokine, continues 37 DEG C and hatches 18h.

2) Western blot method measures p65 protein expression level, and uses software quantitative Treatment.Result is shown in Figure 13.

Heat limit after another prominent feature is that suppression inflammatory reaction.Compound (I) should have certain inhibitory action as the activator of SIRT1, the release to inflammatory factor, has positive meaning for prevention of arterial is atherosis.As seen from Figure 13, compound (I) reduces p65 protein expression when 1 μM and 10 μMs, the highest suppresses 35%.Thus disclosure sets forth compound (I) and inflammation related proteins in macrophage is had obvious inhibitory action.

Embodiment 14. compound (I) is at apoE^-/-Pharmacodynamic evaluation in Mice Body

A)apoE^-/-The structure of rat aorta hardening model

1)apoE^-/-Mice, 7 week old, normal diet is fed one week；

2) by apoE^-/-Mice is weighed, random packet, is divided into 3 groups (blank group, model group, administration groups), and often group 6-8 is only；

3) from the beginning of 8 week old, model group and administration group feed high lipid food diet, and blank group continues to feed normal diet；

4) compound (I) sample-adding group (20mg/kg), gastric infusion；Blank group and model group are to carboxymethylcellulose sodium solution, gastric infusion；It is administered 4 weeks；

5) apoE of 4 weeks will be administered^-/-Mice fasting 6h；Pluck eyeball and take blood, collect blood with the EP pipe of heparin rinse, turn upside down, be placed on ice, 4000r/min, 4 DEG C of centrifugal 3min, supernatant is transferred in new EP pipe (dividing two parts), is placed in-20 DEG C of preservations.

6) fixing mice, cuts off thoracic cavity, cuts off breastbone, exposes heart, carries out cardiac perfusion immediately, first pour into about 1ml with the paraformaldehyde of 4%, then change PBS into and pour into about 4ml, stops after liver bleaches；

7) it is isolatable from aorta to the tremulous pulse total length of the total branch of ilium, takes out heart and tremulous pulse.After dissection, heart and aorta are put in the paraformaldehyde of 4%, 37 DEG C of fixing 2h, it is then placed in the sucrose solution of 20%, 4 DEG C are overnight.

B) mensuration of blood lipid level

Serum after being centrifuged, carrys out method described in Bioisystech Co., Ltd's test kit according to Puli and is measured.

C) manufacture method of frozen section

1) 1.5ml EP pipe is cut off from middle part, leave part with cover, by lid lid, carry out labelling；

2) OCT embedding medium is added in EP pipe, has been careful not to bubble；

3) tissue soaked in 20% sucrose is put in OCT, during heart embedding, stay about 1/3 heart, cut flat with, by apex of the heart part upward, slowly put in OCT；

4) organized for bag EP pipe is slowly put in liquid nitrogen；

5) wrap the EP pipe freezed immediately with tinfoil ,-20 DEG C keep in Dark Place, and long-term preservation is placed on-80 DEG C.

D) aorta oil red O stain

1) being taken out from 20% sucrose by aorta, PBS washes once；

2) under anatomic microscope, aorta is longitudinally cut open；

3) the aorta distillation cut off is washed 3 times；

4) 60% isopropanol soaks 10min, carries out synchronization；

5) good for synchronization is put into (oil red stores liquid configuration, uses in filtering 1-2h), 30min in the oil red working solution newly prepared；

6) 60% isopropanol color separation 1min is put into；

7) distilled water washes 3 times；

8) being laid on black wax by the aorta that solution is cut open, photographing unit is taken a picture immediately.

E) frozen section oil red O stain (heart efferent tract)

1) section is at room temperature placed 30min, dry up frozen section；

2) section is put in 4% paraformaldehyde, fixing 10min；

3) abandon paraformaldehyde solution, add distilled water, wash 3 times, each 3min；

4) 60% isopropanol soaks section 3min, carries out synchronization；

5) then the section that synchronization is good is put into (oil red stores liquid configuration, uses in filtering 1-2h), 30min in the oil red working solution newly prepared；

6) section is put into 60% isopropanol color separation, examine under a microscope at any time；

7) distilled water washes 3 times；

8) hematoxylin dye 2-3min, redyes nucleus；

9), after distilled water washes 3 times, section is placed in distilled water；

10) aqueous mountant mounting (+1 part of distilled water of 9 parts of medical glycerol)；

The slice, thin piece surrounding sealed is coated with nial polish, dries in the shade at cool place, take a picture immediately.

apoE^-/-The measurement result of lipid of mice level such as table 2.The apoE of High-fat diet as can be seen from Table 2^-/-Mice, after compound (I) is administered, T-CHOL (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and the content of HDL-C (HDL-C) in blood fat.Administration group, compared with model group, has the effect necessarily reducing TC, TG, LDL-C, and raises the content of HDL-C.Illustrate that compound (I) can regulate blood lipid level.

apoE^-/-Mouse aorta total length carries out oil red dyeing, result such as Figure 14.From Figure 14 b it can be seen that obvious arteriosclerosis plaque occurs in the whole aorta of high fat diet model group, the success of arteriosclerosis model construction is described.Compared with high fat diet model group, administration group (Figure 14 c) mouse aorta speckle is the most less, reduction, illustrates that the treatment of arteriosclerosis can be played positive role by compound (I).

By apoE^-/-Mouse heart frozen section result such as Figure 15.As can be seen from Figure 15, there is large-area arteriosclerosis plaque in the whole aorta of high fat diet model group (Figure 15 b) heart efferent tract, and fat drips big and closely knit.Blank group (Figure 15 a), it can be seen that less artery plaque, is likely to be due to apoE^-/-The spontaneous generation of mice.Compared with high fat diet group, compound (I) group (Figure 15 c) artery plaque area significantly reduces.

Complex chart 14-15 result, compound (I) can substantially reduce apoE^-/-Mouse aorta total length and the plaque area of heart stream and quantity.apoE^-/-Result in Mice Body all shows that compound (I) has good anti-atherosclerotic effect.

Old and feeble associated tagged molecular mRNA level in-site detection in embodiment 15. mouse aorta

A) Mouse feeder: C57BL/6 mice, 7 week old, raise in SPF level Animal House, cleaning drinking-water, freely take food.Negative control group feeds sodium carboxymethyl cellulose, and administration group feeds E1231,20mg/kg, gavage.Feed 16 weeks.

B) RT-PCR method detection compound (I) is to the mRNA level in-site of p66shc, PAI-1, p21 in aorta

1) extraction of RNA:

The centrifuge tube of tissue sample is taken out in the mortar putting into Liquid nitrogen precooler from liquid nitrogen container, adds a certain amount of Trizol, be repeatedly ground to grinding rod uniformly.Carry out the extraction of cell total rna to specifications, and measure OD₂₆₀And OD₂₈₀Ratio, in order to evaluate the quality (OD of extracted RNA₂₆₀/OD₂₈₀Between 1.8-2.0), quantitatively after with DEPC water, each group of RNA be adjusted to same concentration.

2) synthesis of reverse transcription-cDNA

Use RevertAid^TMFirst Strand cDNA Synthesis Kit test kit (Fermentas) is carried out.By step 1) the RNA reverse transcription of gained becomes cDNA (200ng/ μ l), preserves or proceed following PCR reaction in-20 DEG C.

3) RT-PCR reaction

By dilution step 2) in the cDNA template (50ng/ μ l) that obtains of reverse transcription and all cDNA samples be respectively configured RT-PCR reaction system.Use FastStart Universal SYBR Green Master (ROX) the test kit preparation PCR reaction system of Roche, be then placed on the reaction of RT-PCR (Biorad) instrument enterprising performing PCR.Respectively organize primer (synthesis of Invitrogen company) and carry out solubility curve experiment respectively.The genes of interest (p66shc, PAI-1, p21) of each sample and house-keeping gene (β-actin) carry out RT-PCR reaction respectively.The Ct value of the genes of interest according to each sample and the Ct value of its house-keeping gene calculate regulation multiple.Raise multiple=2^{-[( The genes of interest of sample Ct Value - The internal reference of sample Ct Value )-( Blank genes of interest Ct Value - Blank internal reference Ct Value )]}

Mus p66shc primer sequence:

F3:5 '-AAGTACAACCCACTTCGGAATGA-3 '

R3:5 '-GGGTCCCAGGGATGAAG-3 '

Mus PAI-1 primer sequence:

F1:5 '-CTCCGAGAATCCCACACAG-3 '

R1:5 '-ACTTTGAATCCCATAGCATC-3 '

Mus p21 primer sequence:

F2:5 '-TCTCAGGGCCGAAAACGGAG-3 '

R2:5 '-ACACAGAGTGAGGGCTAAGG-3 '

Mus β-actin primer sequence:

F4:5’-CCTTCCTTCTTGGGTATGGAATC-3’

R4:5’-AGCACTGTGTTGGCATAGAGGT-3’

By Figure 16 compared with sodium carboxymethyl cellulose control mice, the mice that compound (I) is fed, in aorta, p66Shc, PAI-1, p21mRNA level is decreased obviously.Illustrate that compound (I) has the effect delaying vessel aging, defying age in vivo.

The compound of table 1 different substituents and activity

Table 2 apoE^-/-Blood lipid level after mice administration

(# represents that model group #P ＜ 0.05, * compared with blank group represent administration group * P ＜ 0.05 compared with model group).

Claims (1)

1. The most substituted piperazine-1,4-diamide compound reacts in preparation treatment and/or prevention of inflammation Application in medicine.