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 in pharmacy for the piperazine -1,4- diamide compound replacing

Technical field

The present invention relates to the purposes of the piperazine-Isosorbide-5-Nitrae-diamide compound replacing, more particularly, in pharmaceutical field Application.

Background technology

Inherited genetic factorss, diet and aging all have a certain impact for the homeostasiss of internal cholesterol and fat.More next More research shows, limits (calorie restriction, cr) by heat and reduces triglyceride, T-CHOL and low Density lipoprotein-cholesterol, increases HDL-C, with slow down aging, can reduce the sickness rate of cardiovascular disease. Cr be uniquely can be prolonged in yeast, anthelmintic, fruit bat and mammal by the universally recognized one kind of scientific circles long-life Control measures.It is reported that, cr be by adjust silent message regulatory factor 2 (silent information regulator2, Sir2) signal path to be playing a role, and has atherosclerosiss, antiinflammatory, defying age, oxidative stress, anti-apoptotic, a tune Amount of energy saving metabolism, cardiovascular and cerebrovascular vessel protection, blood lipid regulation etc. act on.

Sir2 is being adjusted by chromosome Silencing Mechanisms and cellular energy metabolism mechanism of finding first in yeast of a class The important gene in ganglion cell's life-span, has histone deacetylase activity, closely related with the aging of various kinds of cell.Suckling is moved Thing siruins family includes 7 albumen altogether, is respectively designated as sirt1-7, wherein sirt1 and yeast sir2 homology highest, mesh Front also studied most.On the one hand it pass through to modify histone, deacetylation h1k26, h3k9 and h4k16, maintains at chromatin Stable in silence state and genome；On the other hand pass through deacetylation numerous nonhistones, participate in the energy generation of regulating cell Thank, breed, apoptosis, aging and tumor occur.The deacetylation substrate of sirt1 has p53 albumen, jaw transcription factor (forkhead-o-box transcription factors, foxos), PPAR gamma co-activation because Sub -1 α (peroxisome-proliferated activated receptor c coactivator, pgc-1 α), sterin are adjusted Section element conjugated protein (sterol regulatory element binding protein, srebp-1c) and liver x receptor (liver x receptor, lxr) etc..

Lxrs is a member in nuclear receptor family, during by ligand activation, can promote the antiport of cholesterol, promotes gallbladder The secretion of juice inner cholesterol, the absorption of suppression enteral cholesterol, have very in maintaining intracellular and body cholesterol homeostasis Important meaning, plays extremely important effect during reverse cholesterol transport.So-called reverse cholesterol transport is it may be assumed that by gallbladder Sterin is transported to liver, discharges by liver metabolism or in the form of bile, this process is to prevent excessive cholesterol group around Knit an important physiological process of accumulation.Document report, sirt1 and lxrs interacts and brings it about deacetylation (lxr α 432 lysines, 433 lysines of lxr β) and activate, adjust the expression of lxrs downstream target gene, lack in sirt1 gene Lose in mice body, cholesterol level significantly raises.

Sirt1 has certain protective effect to the development that occurs of cardiovascular disease.Research finds earliest, sirt1 mono- side Nitricoxide synthase (enos) in the deacetylation vascular endothelial cell of face, activation enos suppression angiotensin receptor at1；Separately On the one hand, suppress the aging of vascular endothelial cell, delay atherosclerotic generation.Subsequently research finds, sirt1 passes through to go Acetylation is used, and activation lxrs and fxr (farnesoid x receptor) adjusts lipid and cholesterol metabolism, promotes high density fat Albumen is combined with cholesterol, reduces cholesterol depositss, adjusts blood lipid level, affects atherosclerotic formation.Additionally, Sirt1 can also suppress the expression of angiotensin receptor at1, and the pathology of prevention heart is loose.Sirt1 can adjust liver sugar Class metabolism and lipid metabolism.In short-term fasted mice liver, knock out sirt1 gene and can lead to liver fat acid beta-oxidation gene Expression reduce.

A series of inside and outsides it is experimentally confirmed that sirt1 damages to inflammation gene expression expression and tissue inflammatory has significant suppression Effect processed.In the mice raw264.7 macrophage of sirt1 gene knockout, lipopolysaccharide (lipopolysaccharide, lps) lures Nuclear factor kappa b (nuclear factor κ b, the nf- κ b) activation led and multiple proinflammatory cytokine such as tnf- α, il-1 β, il-6 Expression significantly increase.More and more researchs show, do not contain only lipid in atheromatous plaque, and have a large amount of inflammation Cellular infiltration, gathers substantial amounts of mononuclear cell with blood vessel wall and lymphocyte is characterized, and this prompting inflammation is athero- in mediation tremulous pulse Hardening occurs, the status in evolution, also embody internal relation between sirt1, atherosclerosiss and inflammation with 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.

Content of the invention

It is an object of the invention to provide the new application of the piperazine-Isosorbide-5-Nitrae-diamide compound replacing, that is, in pharmacy Application.

In particular it relates to piperazine-Isosorbide-5-Nitrae-the diamide compound replacing is dynamic as preparation treatment and/or prevention Application in the medicine of pulse atherosclerosis disease.

Piperazine -1,4- the diamide compound being related to replace as preparation treatment and/or prevents cardiovascular and cerebrovascular disease Application in medicine.

Piperazine -1,4- the diamide compound being related to replace is adjusted in the medicine of blood fat in preparation treatment and/or prevention Application.

It is related to the piperazine -1,4- diamide compound replacing in the medicine of preparation treatment and/or prevention of inflammation reaction Application.

Be related to replace piperazine -1,4- diamide compound preparation treatment and/or pre- anti-aging medicine in application.

Substituted piperazine-Isosorbide-5-Nitrae-the diamide compound of the present invention can be administered with itself, or with pharmaceutical composition Form administration.The Pharmaceutical composition of the present invention include the compounds of this invention of effective dose or its officinal salt and a kind of or Multiple pharmaceutical acceptable carrier physiologically accepting.

The pharmaceutical composition of the present invention can be prepared in the usual way, using one or more physiologically acceptable load Body, excipient and auxiliary agent, are conducive to for reactive compound being processed into pharmaceutical preparations.Suitable preparation depends on selected giving Medicine approach, 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 by various route of administration or Mode discharges to patient.Suitable route of administration includes but is not limited to suction, transdermal, oral, rectum, through mucous membrane, enteral and intestinal Stomach external administration, parenteral includes intramuscular, subcutaneous and intravenous injection.

Brief description

Fig. 1 is resveratrol Activation Activity amount effect curve on sirt1 activator screening model.

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

Fig. 3 is the checking of compound () intracellular deacetylation functional activity.Wherein, Fig. 3 a is u2os after compound () effect The Western blot figure of intracellular p53 and ac-p53；Fig. 3 b is that albumen gray scale is quantitatively schemed.

Fig. 4 is compound () and sirt1 protein molecular interphase interaction, specifically e1231 and sirt1-n-cc-c albumen The sensing that interacts figure.

Fig. 5 is the impact to lxrs deacetylation degree for the compound ().Wherein, after Fig. 5 a is compound () effect The Western blot figure of hepg2 intracellular acetylation lxrs (specially lxr α, lxr β)；Fig. 5 b is that albumen gray scale is quantitatively schemed.

Fig. 6 is compound () excitement lxrs activity amount effect curve.

Fig. 7 is that compound () adjusts abca1 promoter transcription activity.

Fig. 8 is that compound () adjusts abca1 and abcg1 protein expression in raw264.7 cell.Wherein, Fig. 8 a is chemical combination The Western blot figure of intracellular abca1 and abcg1 of raw264.7 after thing () effect；Fig. 8 b is that albumen gray scale is quantitatively schemed.

Fig. 9 is that compound () adjusts hepg2 intracellular abcg5/g8 albumen.Wherein, after Fig. 9 a is compound () effect The Western blot figure of intracellular abcg5 and abcg8 of hepg2；Fig. 9 b is that abcg5 albumen gray scale is quantitatively schemed；Fig. 9 c is abcg8 albumen Gray scale is quantitatively schemed)

Figure 10 is that compound () promotes to arrange outside raw264.7 cellular cholesterol.Wherein, Figure 10 a is that compound () promotes The cholesterol efflux of raw264.7 intracellular hdl mediation；Figure 10 b is that compound () promotes the intracellular apo-ai of raw264.7 to be situated between The cholesterol efflux led.

Figure 11 is compound () suppression 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 μm of compounds ()+80 μ g/ml ox-ldl；D is Positive control (10 μm of 9cra+80 μ g/ml ox-ldl).

Figure 12 is the impact to raw264.7 intracellular total cholesterol level for the compound ().

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

Figure 14 is compound () to apoe^-/-The impact of speckle in mouse aorta total length.Aorta total length oil red dyes Result shows, compound () significantly reduces apoe^-/-The formation of mouse aorta plaque, reduces cholesterol and lipid gathers Long-pending.Wherein a is blank group (normal diet)；B is model group (high fat diet)；C is compound () e123120mg/kg (high fat Diet).

Figure 15 is compound () to apoe^-/-The impact of mouse heart efferent tract.The oil red coloration result of heart efferent tract Display compound () can significantly reduce apoe^-/-The area of speckle and quantity in mouse heart efferent tract, reduce cholesterol and fat Matter is built up.Wherein, a is blank group (normal diet)；B is high fat diet model group；C is compound () 20mg/kg (high fat drink Food).

Figure 16 is the mrna to c57bl/6 mouse aorta SAG p66shc, pai-1, p21 for the compound () Expression impact.

Specific embodiment

In order to be more clearly understood that the essence of the present invention, we are first to the piperazine-Isosorbide-5-Nitrae-diamide compound replacing The activity of activation sirt1 is measured, and the results are shown in Table 1.From table 1 it follows that piperazine-Isosorbide-5-Nitrae-diamide the chemical combination replacing Thing has the activity of different degrees of activation sirt1.Below with the substituted piperazine -1,4- diamide of label " e1231 " The pathology test of compound (referred to as compound ()) and result deeply to illustrate the piperazine -1,4- diamide compound replacing Application in pharmacy.

First, successfully obtain destination protein sirt1 by escherichia expression system.Based on homogeneous phase time discrimination fluorescence skill Art, sets up high flux sirt1 activator screening model using activated albumen, after model optimization, using known generally acknowledged sun Property medicine resveratrol is verified to model, ec₅₀Value is consistent with document report, sees Fig. 1, shows that expressed albumen is high activity Destination protein, and screening model is believable.Combination of compounds storehouse (national new drug (microorganism) screening experiment room) is entered Row screening bioactive compoundses, obtain several reactive compounds, and measure the activation to sirt1 albumen for all compounds.Select Representative reactive compound () has carried out further activity research, confirms that compound () enables on a cellular level The deacetylation degree of the target protein p53 of sirt1 albumen increases, the strictly activator of sirt1 albumen.In surface plasma altogether Jolt ramming has recorded the k between compound () and albumen sirt1 in testing_dIt is worth for 9.61 μm.Finally, compound () has been carried out point The research of sub- pharmacology's aspect, find compound () the deacetylation degree of lxrs can be made to increase, exciting nuclear receptor lxr α with Lxr β, adjusts the expression of target protein abca1/g1, promotes intracellular cholesterol efflux, suppresses macrophage foam cell formation, raises Abcg5/g8 expresses, and promotes cholesterol secretion.Inquire into compound () to inflammatory factor associated metabolic path in foam cell Effect, finds that it can suppress the expression of inflammation transcription factor nf- κ b key protein p65.Have studied compound () to mice master The impact of arterial aging related gene expression, finds that it can suppress the mrna of p66shc, pai-1, p21 to express.

Therefore, using compound (), the substituted piperazine-Isosorbide-5-Nitrae-diamide compound as representative can swashing as sirt1 Live agent, play to cholesterol metabolism, inflammation, the gene of diseases associated with senescence adjustment effect (see Fig. 2), can be used for tremulous pulse athero- Hardening, inflammation, aging, cardio-cerebrovascular diseases prevention and/or treatment.

The mensure to sirt1 Activation Activity for the substituted piperazine -1,4- diamide compound of embodiment 1.

1) protein expression

First, from plasmid pcdna3.1-sirt1, (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences Liu Depei teaches problem Group present) amplification obtain the catalytic domain of people sirt1 and comprise n end, the cdna of c end sub-sequence, with cloning vehicle pblunt- Simple vector connect sequencing correct after, with coli expression carrier pet-30a (+) be connected, the recombinant expressed matter of structure Grain is named as pet-sirt1, conversion escherichia coli expression host bl21 (de3), picking monoclonal, sequencing identification.Just will be sequenced True single bacterium colony shake-flask culture, in od₆₀₀When being worth for 0.6-0.8, add the thio gala of isopropyl-β-d- of final concentration of 0.5mm Glucosides (iptg), 15 DEG C, 200rpm, collects bacterium solution after culture 24h.It is centrifuged with 12,000rpm after ultrasonic disruption thalline 30min.Collect supernatant, through 0.45 μm of membrane filtration.UsingExplorer system carries out protein purification work, uses 1ml prepacked column histrapff crud is as purification media.Its principle is in his-tag and the post that destination protein passes through its n end Chelating sepharose^tmOn ni²⁺Ion chelating, and then adsorb on pillar, and other foreign proteins are because can not be with ni²⁺Ion chelating, directly flows out pillar with sample-loading buffer, then with elution buffer eluting destination protein, thus obtaining high-purity The destination protein sirt1 of degree.

2) mensure to sirt1 Activation Activity for the compound

This determination of activity carries out the mensure of compound activity using cisbio company man's sirt1 activator screening reagent box.

Measuring principle:

Detection system is as follows: recombined human sirt1 albumen, and substrate substrate-d2 is that the p53 of fluorophor d2 labelling is little Fragments of peptides, nad⁺Play the necessary cofactor of enzymatic activity for sirt1, anti-acetyl-cryptate is eu²⁺Labelling cave-shaped Compound.If the compound adding has activation to enzyme, substrate deacetylation degree increases, between substrate and cryptate Distance becomes remote, is not enough to produce Resonance energy transfer, shows as fluorescence reading and reduces.

Assay method:

(1) add the dtt of final concentration of 1mm in reaction buffer, be configured to enzymatic buffer.

(2) recombiant protein, substrate substrate-d2 and coenzyme nad are diluted successively with enzymatic buffer⁺, make Its final concentration be respectively 0.5mu/ μ l, 6nm, 150 μm.

(3) enzymatic buffer diluted compounds are used to certain concentration.

(4) 2 μ l testing compounds, 2 μ lnad are sequentially added⁺, 4 μ l substrates to 384 orifice plates, be eventually adding 2 μ l restructuring eggs In vain, start enzymatic reaction.Setting is not added with the no enzyme group of albumen and is not added with the negative control group of compound, Every group of 3 parallel holes.

(5) incubated at room 120min.

(6) add the anti-acetyl-cryptate of the final concentration of 7.5nm of 10 μ l, incubated at room 5h or overnight, use enzyme Mark instrument measures fluorescent value (ex:340nm, em:665/615nm).

(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 rise rate is considered as more than or equal to 150%, and primary dcreening operation is positive, carries out the secondary screening of next step and amount effect curve measures.

Read fluorescence reading, after being calculated according to formula, using the logarithm value of compound concentration as abscissa, substrate go second Acylated degree is vertical coordinate, with graphpad prism software matched curve, obtains ec₅₀Value and maximum rise rate, the results are shown in Table 1.

This measurement result illustrates that the piperazine -1,4- diamide compound replacing in the present invention has significantly to sirt1 Deacetylation activation.Wherein, best compound () the maximum rise rate of activity is 384.09%, ec₅₀It is worth for 0.43 μm. Because sirt1 is participating in significant, therefore this result in the physiological process such as cholesterol metabolism and fatty acid, carbohydate metabolism Illustrate that the substituted piperazine -1,4- diamide compound in the present invention can be related to related life by activating sirt1 in regulation and control Play a role in the disease of reason process.

The culture of embodiment 2. cell

Hepg2, abca1-luc hepg2, u2os and hek293 cell is attached cell, and about 48h passes on once.Treat After cell covers with, abandon old culture medium, discarded with after pbs rinsing cell, plus appropriate pancreatin, peptic cell about 2min at room temperature, Abandon Digestive system, add the culture medium containing 10%fbs immediately, to suppress trypsin vigor, repeatedly gently blown and beaten with elbow straw Culture bottle inner cell, make cell completely disengage from bottom of bottle and blow and beat be allowed to be separated into individual cells suspension.Thin in the inoculation of 1:3 ratio again Born of the same parents' suspension is in new cell bottle, or discards appropriate cell suspension, then adds appropriate complete medium, puts into incubator and continues Culture.Condition of culture: 37 DEG C, 5%co₂.

Mouse monokaryon macrophage raw264.7 is half attached cell, and about 48h passes on once.Pancreatin digests 5min, by 1: 4-1:6 ratio passes on, using dmem complete medium.Condition of culture: 37 DEG C, 5%co₂.

People Acute Monocytic Leukemia Cell Line thp-1 is suspension cell, and about 48h passes on once.Treat that cell density is (3- 5)×10⁶During individual/ml, blow and beat culture bottle inner cell with elbow straw, be allowed to be separated into individual cells suspension.Again in 1:4 ratio Inoculating cell suspension is in new cell bottle, or discards appropriate cell suspension, then adds appropriate complete medium, puts into culture Case continues culture.Condition of culture: 37 DEG C, 5%co₂.

Acetylation p53 protein expression in the human osteosarcoma u2os cell that embodiment 3. compound () induces to doxorubicin The impact of level.

1) compound treated cells: in advance by human osteosarcoma cell u2os with mccoy ' the s5a 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 completely adherent, Draw Cell sap, be changed to blank mccoy ' s5a culture medium, add final concentration of 5 μm of doxorubicin hydrochloride (dox), 10 μm Sirt1 specific inhibitor ex-527 and the positive compound of respectively 0.1,1,10 μm of final concentration, 37 DEG C, 5%co₂Condition Lower incubation 6h.

2) preparation of albumen: after 6h, discard culture medium, with the pbs of pre-cooling rinsing cell 2 times, with culture after pancreatin digestion Cell collected by base, and 800rpm is centrifuged 3min, then uses pbs suspension cell, and 800rpm is centrifuged 3min.Often pipe is separately added into 52 μ l's Ripa cell pyrolysis liquid (adds the pmsf of final concentration of 100 μ g/ml) in every 1ml lysate, in cell lysis 30min on ice.4 DEG C, 12000 × g is centrifuged 20min.Collect supernatant, it is fixed with bca quantification of protein test kit (thermo company), albumen to be carried out Amount, uses ddh₂Each group protein sample concentration is adjusted to 2 μ g/ μ l by o.Add a certain amount of 5 × albumen sample-loading buffer, in boiling water 10min is boiled in bath, of short duration centrifugation after cooling, every hole loading 40 μ g albumen carries out sds-page electrophoresis.Remaining sample is protected in -80 DEG C Deposit.

3) western blot method measures protein level: after sds-page electrophoresis, by polyacrylamide gel and transferring film Filter paper is put into and is soaked balance 5min in transfering buffering liquid.Pvdf film is submerged initially in methanol 20s activation, is washed with water, and is placed in transfer slow Rush and in liquid, soak balance 2min.With biorad semidry method transferring film instrument transferring film, electric current 5ma/cm is set², constant current transferring film 30min.Turn After film terminates, put into room temperature in 1 × tbst containing 5% (w/v) defatted milk powder after cutting off respectively according to the size of destination protein and incubate Educate 1h.Closing terminates, and is resisted with the 1 × tbst dilution one containing 5% (w/v) defatted milk powder, is placed in hybridization bag, incubated at room 1h Afterwards, 4 DEG C of overnight incubation.Washed three times with 1 × tbst, each 10min.Dilute with 1 × tbst containing 5% (w/v) defatted milk powder afterwards Release corresponding two to resist, be placed in hybridization bag, incubated at room 2h.Take out pvdf film from hybridization bag, wash three times with 1 × tbst, every time 10min.Colour developing: in the front of film, that is, turn the one side having albumen and add appropriate enhancement mode hrp (horseradish peroxidase) substrate Learn the mixed liquor (in 1:1 ratio matching while using) of luminescent solution (ecl) a, b liquid, be immediately placed on colour developing in gel imaging instrument.

4) develop the color result, is scanned with imagej software, quantitative Treatment.Result is shown in Fig. 3.

Doxorubicin (doxorubicin, dox) can induce cell and occurs dna to damage so that 382 bad ammonia of p53 albumen There is acetylation in acid.In order to verify that compound in the deacetylation function of intracellular and investigates the performance of function to sirt1 Dependency, add sirt1 specific inhibitor ex-527.With the rising of compound () concentration, acetylation p53 albumen Expression substantially reduces with Concentraton gradient, and the amount of intracellular total p53 albumen does not have significant change, illustrates that p53 albumen goes second Acylated degree increased.Add the specific inhibitor ex-527 of sirt1 albumen, the expression of ac-p53 albumen increased again. Thus disclosure sets forth compound () and have obvious rise effect, and this to the effect of intracellular sirt1 substrate deacetylation Effect depends on sirt1.

Embodiment 4. compound () is measured with the intermolecular interaction of sirt1 albumen

In this mensure system, buffer soln used consists of: hepes10mmol/l, nacl150mmol/l, Dtt1mmol/l, nad⁺1mmol/l, glycerol 10% (v/v), dmso2% (v/v).Buffer and sample are filtered by 0.45 μm Membrane filtration, ultrasonic removes bubble removing using front.Completed by biacore t200system (ge healthcare) instrument.

Assay method:

1) pretreatment of chip surface: take out maintenance chip, be replaced by cm5 chip, select prime, with buffering Liquid rinses chip surface.Select normalize, using biacore standardizing agents (bia normalizing Solution70%) cm5 chip is standardized.Tested using two chip channel, sirt1 albumen is coated required The ph value condition wanted is groped (immobilization ph-scouting for pre-concentration).Select 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, is coupled.Setting flow velocity be 10 μ l/min, binding time be 2min, finally with The naoh of 50mm rinses the sirt1 albumen that chip surface 20s removes residual.

2) albumen envelope chip surface: edc/nhs equal-volume is mixed, with the flow velocity sample introduction 10min of 1 μ l/min, to cm5 Chip surface carboxyl is activated.According to the result of groping of chip surface pretreatment, the sodium acetate solution for 4.0 for the ph value is selected to make For buffer, sirt1 albumen is diluted to 50 μ g/ml, setting flow velocity is 10 μ l/min, binding time is 20min, and passes through ammonia Base coupled action is coated on the surface of cm5 chip, and setting target coupling amount is 7000ru, concurrently sets a blank channel As comparison passage.After sample introduction terminates, add ethanolamine, with the flow velocity sample introduction 10min of 10 μ l/min, to close chip surface not In conjunction with the pendant carboxylic group site of amino, the final result that is coupled is 7056ru.

3) compound () is added to be combined detection: for sirt1 coated cm5 chip, by the compound of variable concentrations (0,0.39,1.56,3.12,6.25,12.5 μ μ) sample introduction successively, makes compound flow through chip surface with the flow velocity of 30 μ l/min, Binding time is set to 60s, after Dissociation time is 120s, gropes the regeneration condition of chip surface, finds compound certain Can naturally dissociate completely in time.

By Fig. 4, can be with dose-dependent mode and people's restructuring sirt1 knot by change visual compounds () of ru value Close, k_dIt is worth for 9.61 μm.Small molecule and protein bound k_dValue generally 10^-3To 10^-6M, therefore, compound () and albumen There is between sirt1 very strong affinity.

The mensure to lxrs deacetylation degree for embodiment 5. compound ()

In order to investigate the impact to lxrs deacetylation degree for the compound, we are tested using co-immunoprecipitation experiment Card.

This experiment adopts hepg2 cell, is laid in the big culture dish of a diameter of 90mm, after cell is completely adherent, point Not Jia Ru 10 μm of compound (), solvent control hole is set, processes cell 20 to 24h.Extract Nuclear extract, respectively with lxr α and lxr β antibody and protein a/g agarose beads are incubated 4h in 4 DEG C of conditions jointly, wash beads, boil sample, use Acetylated-lys antibody is carrying out western blot detection.Film after exposure membrane regeneration liquor is by Beijing Puli come gene After the experimental procedure regeneration of Technology Co., Ltd., then with corresponding lxrs antibody test.Result is shown in Fig. 5.

From fig. 5, it can be seen that after adding compound (), the expression substantially constant of lxr α and lxr β, acetylation lxr β's Expression has a certain degree of decline, however, the expression of acetylation lxr α declines significantly, shows sirt1 activation immunomodulator compounds () can adjust the Acetylation Level of lxr α and lxr β in hepatocyte.

The mensure to lxrs agonist activity for embodiment 6. compound ()

This determination of activity carries out the mensure of compound activity using the agonist screening model of lxrs.

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 in yeast, transcription factor gal4 has the characteristics that nuclear receptor analog structure, application yeast is double The principle of hybridization, builds two plasmids: dbd part and the lbd knot of lxrs that pbind-lxrs plasmid contains gal4 gene respectively Structure domain, can change conformation when by ligand activation；Another plasmid is pgl4-gal4, containing gal4 gene promoter area The luciferase reporter gene luc of domain response element.The present invention by two plasmid co-transfections to hek293 cell, with research The activation to lxrs for the compound.In this detection system, if compound can activate lxrs, the lxrs of expression plasmid expression Protein conformation changes, and combines with reporter plasmid, and now the expression activity of compound group luciferase is higher than not add chemical combination The matched group of thing；Otherwise when compound is inactive to lxrs, then lxrs protein conformation does not change, not with reporter plasmid phase In conjunction with, then the expression activity of compound group luciferase does not produce change.

Assay method:

1) transfected in 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, according to 25 μ l/ hole opti-mem culture medium dilutions 0.5 μ l liposome lipofectamine^tm2000reagent, incubated at room 5min.25 μ l/ hole opti-mem culture medium dilutions 200ng plasmid dna, including pgl4-gal4 and pbind-lxr α/β, merges mixing, incubated at room with dilution liposome afterwards 20min.

2) the dna- liposome complex solution after merging is added mem complete medium according to every hole 100 μ l, make culture Base is fully mixed with dna- liposome complex, now sucks the culture medium in cell plates, by 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 37 DEG C in CO2 gas incubator.

3) cultivate 6h, suction out transfection media, by compound () from the beginning of 40 μm of maximum concentration, with 2 times of Concentraton gradient Stepwise dilution becomes 8 concentration, and negative control hole adds 0.1% dmso simultaneously, processes cell respectively, continues culture 18h.

4), after 18h, suction out the culture medium in the every hole of 96 orifice plates.Every hole adds 150 μ l pbs gently to rinse cell, overturns 96 Orifice plate is outwelled pbs and is dried.20 μ l1 × cclr cell pyrolysis liquid is added, 37 DEG C of cell lysis 20-30min are (according to thin in every hole How many born of the same parents' numbers determines the time), examine under a microscope whether cell cracks completely.Lysate is fully transferred to fluorescence analysiss With the corresponding in the hole of the opaque blank in 96 holes, noticing that lysate avoids bubble as far as possible, otherwise making impact reading (if using 96 Hole clear bottom blank does not then need to shift lysate).

5) it is rapidly added 50 μ l fluorescence analysis reagents (promega company) in every hole, immediately analysis blank is put into enzyme Detect in mark instrument.

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

Rate of change (%)=a/b × 100

Wherein, a is cell fluorescence element enzymatic activity (rlu) of mensure after addition testing sample, and b is to add negative control sample (dmso) cell fluorescence element enzymatic activity (rlu) measuring afterwards.Rate of change (%) is considered as the agonist activity to lxr for the compound, uses % Percentage ratio represents, or is represented with multiple.Using the logarithm value of compound concentration as abscissa, rate of change is vertical coordinate, With graphpad prism software matched curve, result is shown in Fig. 6.

In this measurement result explanation present invention, compound () has significant transcriptional activation to lxr α/β, and Arrive amount effect relation curve on lxr α/β agonist screening model for the compound ().From fig. 6, it can be seen that compound () with Dosage-dependent manner excitement lxr α/β, is 188.14%, ec to lxr α maximum agonist activity₅₀It is worth for 0.65 μm；To lxr β Big agonist activity is 182.32%, ec₅₀It is worth for 0.08 μm.Because nuclear receptor lxr is participating in cholesterol metabolism and fatty acid, saccharide The physiological process such as metabolism are significant in converging, and therefore this result illustrates that the compound () in the present invention can be by excitement The transcription of lxr plays a role in the disease that regulation and control are related to Relevant Physiological Courses.

The mensure to abca1 promoter transcription activity for embodiment 7. compound ()

This determination of activity carries out the mensure of compound activity using adjustment screening model on abca1.

Measuring principle:

The principle of this detection is that will have abca1 regulatory sequences in the reporter gene upstream of luciferase clone Recombiant plasmid pgl3-abca1 and pcdna3.1 cotransfection to human liver cancer cell hepg2 cell, stablized through g418 screening Transfection cell strain, is named as abca1-luchepg2 cell.By adding the expression activity of compound test cell fluorescence element enzyme Change it can be seen that the impact to abca1 promoter transcription activity for the compound, and then judge the rise rate to abca1.

Assay method:

The abca1-luc hepg2 cell of digestion exponential phase, with culture medium dilution simultaneously cell counting, with 5 × 10⁵ The density of individual/ml is seeded to 96 orifice plates, and every hole adds 100 μ l single cell suspensions.After 6h, treat that cell is fully adherent, remove containing blood Clear culture medium, gently rinses cell once with pbs, and every hole adds the serum-free rpmi-1640 training containing different compound concentrations Foster base 200 μ l, the dmso of comparison in the hole addition respective concentration simultaneously.Remove culture medium after 18-24h, measure according in embodiment 6 Method described in method (4)-(6) measures, calculates and map, and result is shown in Fig. 7.

Compound () increases abca1-luc hepg2 cell fluorescence element enzymatic activity in dose-dependently mode.Compound () is raised abca1 expression activity and is reached peak 226.67%, its ec₅₀It is worth for 0.25 μm.Because abca1 is in reverse cholesterol transport During significant, therefore this result illustrate the present invention in compound () can strengthen abca1 promoter transcription live Property, play a role in the disease be related to Relevant Physiological Courses.

Embodiment 8. compound () shadow to abca1 and abcg1 protein expression level in raw264.7 macrophage Ring.

1) compound treated cells: mouse monokaryon macrophage raw264.7 is with 4 × 10⁵It is adherent that individual/ml is inoculated in 6 orifice plates Afterwards, processed with 0.1,1 and 10 μm of compound (), using concentration for 0.1%dmso as negative control group, arrange 10 μm simultaneously Compound ()+ex-527 group, all at 37 DEG C, 5%co₂Under the conditions of cultivate 20-24h.

2) western blot method measures abca1 and abcg1 protein expression level, and uses software quantitative Treatment.Result See Fig. 8.

Abca1 and abcg1 is the target protein of lxr direct regulation and control, and the two major function in macrophage is all will be thin The unnecessary cholesterol efflux of intracellular, promotes reverse cholesterol transport process, prevents the OxLDL ELISA in macrophage (ox-ldl) accumulation causes macrophage foam cell formation.Found out by Fig. 8, through the effect of compound () 20-24h, can be in 1-10 μm when dose-dependant ground raise the expression of abca1 and abcg1, abca1 is maximum to raise 2.17 times, and abcg1 is up to 2.48 times.Cause And disclosure sets forth compound () and have obvious rise effect to promotion cholesterol efflux associated protein in macrophage.

Embodiment 9. compound () impact to abcg5 and abcg8 expression in hepg2 cell

1) compound treated cells: in advance the hepg2 cell being inoculated in 6 porocyte culture plates is added variable concentrations Compound () is processed, 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, they be subject to lxr direct regulation and control, this two Transport protein is expressed on the film of hepatocyte tubule, thus driving cholesterol to transport expression in bile, promotes cholesterol to secrete. As shown in figure 9, compound () rise multiple highest to abcg5 when 0.1 μm in the present invention, up to 1.2 times about；? In 0.1-10 μ m, dose-dependant ground raises the expression of abcg8, when 10 μm, abcg8 is raised and reaches as high as 2.2 times.Explanation Compound () is possible to promote cholesterol secretion in liver by raising abcg5 and abcg8 expression.

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

1) the dmem- high glucose medium (500 μ l/ hole) containing 10%fbs for the mouse monokaryon-macrophage raw264.7, with 2 ×10⁵Individual/hole is inoculated in 24 porocyte culture plates, in 37 DEG C, 5%co₂Under the conditions of incubated overnight.

2) abandon Cell sap, be changed to the dmem- high glucose medium containing 0.2% (w/v) bsa (500 μ l/ hole), add 1,2- [³H] cholesterol make its final concentration of 1 μ ci/ml, 37 DEG C, 5%co₂Under the conditions of be incubated 24h.

3) wash cell 2 times with pbs (1ml/ hole), (dmem adds the mensure culture medium adding containing finite concentration compound 0.2%bsa, 0.1%dmso, 25mm hepes, ph7.4), 37 DEG C of incubation 18～24h.

4) pbs (1ml/ hole) washes cell 2 times, and (dmem adds 0.2%bsa, 0.1%dmso, 25mm to add culture medium Hepes, ph7.4), the hdl of the apoa-i of 10 μ g/ml or 50 μ g/ml, it is incubated 4h.

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

6) use 0.1m naoh0.5ml lysis at room temperature cell 30min, collect lysate to be measured.

7) measure: testing sample is transferred to respectively on 3mm filter paper, 75 DEG C of drying, the scraps of paper is placed on liquid to dodge in cup, adds It is 55% dimethylbenzene and 45% ethylene glycol with volume fraction that 10ml liquid dodges liquid (mass concentration is 0.5%ppo and 0.05%popop) The solvent mixed preparing of dimethyl ether, is placed in brown container and deposits, overnight use, and liquid scintillation counter counts.Whole experimental cell It is divided into matched group (but be not added with cholesterol add apoa-i/hdl, add cholesterol) and sample-adding group (to be 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%

As shown in Figure 10, compound () is under 0.1,1,10 μm of effects, permissible for cholesterol efflux result in macrophage Promotion 1,2- [³H] cholesterol flow out to cholesterol acceptor, and the increase with compound concentration, 1,2- [³H] cholesterol Runout level also increases, and, between 1.2-1.7 times, the multiple flowing out to hdl is than to apoa-i's for the multiple flowing out to apoa-i Multiple is high, about between 1.5-2.0 times.This result meets described in forward part of the present invention, and compound () passes through to raise huge biting Intracellular abca1 and abcg1 protein expression, and then promote the outer row of intracellular cholesteryl, the physiology meeting lxr agonist lives Property, and more notable as the impact of cholesterol acceptor for hdl, and abcg1 is mainly responsible for by unnecessary cholesterol transport extremely Hdl, the inventive result of this albumen that can significantly raise abcg1 with the compound inquired into before and mrna expression is consistent.

Embodiment 11. compound () suppresses the experiment of macrophage foam cell formationization effect

The mononuclear phagocyte raw264.7 of the mice high sugar culture fluid of the dmem- containing 10%fbs is in transparent 96 hole cells Cultivate on culture plate.After adherent, it is changed to serum-free dmem- high glucose medium (100 μ l/ hole).By cell be divided into blank control group, Foam cell group and dosing group, the ox-ldl adding final concentration of 80 μ g/ml is to foam cell group and dosing group, after 24h, dosing Group adds certain density compound ().37 DEG C, 5%co₂Under the conditions of culture 24h after, carry out oil red o dyeing.By 96 orifice plates from co₂Take out in incubator, cell is fixed (15 μ l/ hole) 10min, abandoned solution, distilled water is washed twice, adds with 4% paraformaldehyde 60% isopropanol (150 μ l/ hole), places 5min, discards solution.Oil red o is used liquid (matching while using filters) to add each hole In, 150 μ l/ holes, dye 1h.Discard solution, with 60% isopropanol (150 μ l/ hole) hole flushing, then use distilled water (150 μ l/ hole) Wash twice, finally 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.

To only add ox-ldl as foamed groups of cells, and be dyeed by oil red o, examine under a microscope the coloring of cell Degree.It can be seen from fig. 11 that (a) blank group does not have red fat drips to be formed, (b) ox-ldl solvent control group is formed significantly Red fat drips exist, (c) compound () treatment group, and intracellular Red oil granule and significantly reduced compared with matched group, says Bright compound () can be reduced lipid and be accumulated in macrophage, effectively suppression macrophage foam cell formation, and (d) adds 10 μ In m9cra (lxrs agonist) cell, lipid accumulation also significantly reduces.

Embodiment 12. compound () reduces intracellular T-CHOL

Method is recommended to measure intracellular T-CHOL using biovision company cholesterol quantification kit, assay method is such as Under:

1) prepare 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 temperature centrifugation 5min.

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

4) 100 μ l cholesterol assay buffer dissolving mixing are added.

5) add 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 are incubated 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 cell foamed One principal character.Found out by Figure 12, it is notable that compound () reduces intracellular total cholesterol level in 1 μm and 10 μm.Illustrate to add The compound entering can suppress macrophage foam cell formation, reduce lipid and accumulate in macrophage.

The impact to inflammation key protein p65 expression in thp-1 cell for embodiment 13. compound ()

1) compound treated cells: in advance the thp-1 being inoculated in 6 porocyte culture plates is being added final concentration 50nm Buddhist After ripple ester pma induction 24h, it is added thereto to 10 μm of e1231,37 DEG C of incubation 2h, adds the lps of final concentration of 10ng/ml Stimulate the generation of intracellular cytokine, continue 37 DEG C of incubation 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 () is as the activator of sirt1, right The release of inflammatory factor should have certain inhibitory action, has positive meaning for prevention of arterial is atherosis.By Figure 13 Can be seen that, compound () reduces p65 protein expression in 1 μm and 10 μm, highest can suppress 35%.Thus disclosure sets forth Compound () has obvious inhibitory action to inflammation related proteins in macrophage.

Embodiment 14. compound () is in 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 control group, model group, administration groups), 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 () sample-adding group (20mg/kg), gastric infusion；Blank control group and model group are to carboxymethyl cellulose Sodium solution, gastric infusion；Administration 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, on Lower reverse, it is placed on ice, 4000r/min, 4 DEG C of centrifugation 3min, supernatant is transferred in new ep pipe (dividing two parts), be 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 with 4% poly Formaldehyde pours into about 1ml, then changes pbs into and pours 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, take out heart and tremulous pulse.After dissection, by heart and master Tremulous pulse is put in 4% paraformaldehyde, 37 DEG C of fixing 2h, is then placed in 20% sucrose solution, 4 DEG C overnight.

B) mensure of blood lipid level

By the serum after centrifugation, carry out Bioisystech Co., Ltd's test kit methods described according to Puli and be 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 soaking in 20% sucrose is put in oct, during heart embedding, stay about 1/3 heart, cut flat with, will Apex of the heart part upward, is slowly put in oct；

4) slowly put into wrapping organized ep pipe in liquid nitrogen；

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

D) aorta oil red o dyeing

1) aorta is taken out from 20% sucrose, pbs washes once；

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

3) by the aorta cut off distillation washing 3 times；

4) 60% isopropanol soaks 10min, synchronizing；

5) good for synchronization is put into (oil red storing liquid configures, and filters in 1-2h and uses) in the oil red working solution newly preparing, 30min；

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

7) distilled water washes 3 times；

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

E) frozen section oil red o dyeing (heart efferent tract)

1) will cut into slices and place 30min at room temperature, 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, synchronizing；

5) and then by the good section of synchronization (configuration of oil red storing liquid, filtration 1- in the oil red working solution newly preparing are put into Use in 2h), 30min；

6) 60% isopropanol color separation is put in section, 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) aqueouss mountant mounting (+1 part of distilled water of 9 parts of medical glycerol)；

The slice, thin piece sealed surrounding is applied nial polish, shady place is dried in the shade, and takes 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^-/-Little Mus, after compound () administration, T-CHOL (tc), triglyceride (tg), low-density lipoprotein cholesterol (ldl- in blood fat C) and HDL-C (hdl-c) content.Administration group compared with model group, have certain reduce tc, tg, The effect of ldl-c, and raise the content of hdl-c.Illustrate that compound () can adjust blood lipid level.

apoe^-/-Mouse aorta total length carries out oil red dyeing, result such as Figure 14.Can be seen that high fat diet from Figure 14 b Obvious arteriosclerosis plaque in the whole aorta of model group, and the success of arteriosclerosis model construction is described.With high fat diet Model group is compared, and administration group (Figure 14 c) mouse aorta speckle is substantially less, reduction, illustrates that compound () can be to arteriosclerosis Treatment play positive role.

By apoe^-/-Mouse heart frozen section result such as Figure 15.As can be seen from Figure 15, high fat diet model group (figure 15b) large-area arteriosclerosis plaque in the whole aorta of heart efferent tract, and fat drips are big and closely knit.Blank control group (Figure 15 a) can see less artery plaque, because of apoe^-/-The spontaneous generation of mice.Compared with high fat diet group, change Compound () group (Figure 15 c) artery plaque area significantly reduces.

Complex chart 14-15 result, compound () can substantially reduce apoe^-/-Mouse aorta total length and the speckle of heart stream Area and quantity.apoe^-/-Result in mice body all shows that compound () has good anti-atherosclerotic effect.

Old and feeble associated tagged molecular mrna horizontal 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 Matched group feeds sodium carboxymethyl cellulose, and administration group feeds e1231,20mg/kg, gavage.Feed 16 weeks.

B) the mrna level to p66shc, pai-1, p21 in aorta for the rt-pcr method detection compound ()

1) extraction of rna:

The centrifuge tube of tissue sample is taken out from liquid nitrogen container in the mortar putting into Liquid nitrogen precooler, add a certain amount of Trizol, is ground to uniformly repeatedly with grinding rod.Carry out the extraction of the total rna of cell to specifications, and measure od₂₆₀And od₂₈₀ Ratio, in order to evaluate the quality (od of extracted rna₂₆₀/od₂₈₀Between 1.8-2.0), with depc water by each group after quantitation Rna is adjusted to same concentration.

2) synthesis of reverse transcription-cdna

Using revertaid^tmFirst strand cdna synthesis kit test kit (fermentas) is carried out.Will 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.Faststart universal sybr green master (rox) test kit using roche is joined Pcr reaction system processed, is then placed on and carries out pcr reaction on rt-pcr (biorad) instrument.(invitrogen is public for each group primer Department's synthesis) 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 calculates with the ct value of its house-keeping gene Adjust multiple.Raise multiple=2^{- [(the internal reference ct value of the genes of interest ct value-sample of sample)-(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 () is fed, in aorta P66shc, pai-1, p21mrna level is decreased obviously.Illustrate that compound () has in vivo and delay vessel aging, defying age Effect.

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 (3)

1. substituted piperazine -1,4- diamide compound is in the medicine of preparation treatment and/or prevention of arterial atherosclerotic disease Application；Wherein, described substituted piperazine-Isosorbide-5-Nitrae-diamide compound is as shown in Equation 1；

In formula 1, r1 and r2 is respectively:

Or

2. substituted piperazine -1,4- diamide compound answering in the medicine of preparation treatment and/or prevention cardiovascular and cerebrovascular disease With；Wherein, described substituted piperazine-Isosorbide-5-Nitrae-diamide compound is as shown in Equation 1；

In formula 1, r1 and r2 is respectively:

Or

3. substituted piperazine -1,4- diamide compound adjusts the application in the medicine of blood fat in preparation treatment and/or prevention；Its In, described substituted piperazine-Isosorbide-5-Nitrae-diamide compound is as shown in Equation 1；

In formula 1, r1 and r2 is respectively:

Or