CN104151287B - The agonist and purposes of the liver X receptor of one group of substituted benzamides - Google Patents

The agonist and purposes of the liver X receptor of one group of substituted benzamides Download PDF

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CN104151287B
CN104151287B CN201310178186.5A CN201310178186A CN104151287B CN 104151287 B CN104151287 B CN 104151287B CN 201310178186 A CN201310178186 A CN 201310178186A CN 104151287 B CN104151287 B CN 104151287B
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lxr
cholesterol
agonist
liver
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CN104151287A (en
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司书毅
李霓
李东升
许艳妮
李永臻
刘畅
冯婷婷
姜威
刘伟
王智敏
贺晓波
巫晔翔
王潇
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Institute of Medicinal Biotechnology of CAMS
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Abstract

The present invention relates to the new compound of one group of agonist as liver X receptor and containing the pharmaceutical composition of such compound or pharmaceutically acceptable salt thereof, the method that further relates to prepare such compound and combinations thereof.Purposes the invention discloses such compound as pharmacological active substance, especially in angiocardiopathies such as treatment atherosclerosis, hypertension, hyperlipidemia, hypercholesterolemia, coronary heart disease, with obesity, diabetes, metabolic syndrome, the neurodegenerative diseases and immune correlated disease etc. such as Alzheimer have important use.

Description

The agonist and purposes of the liver X receptor of one group of substituted benzamides
Technical field
The present invention relates to one group of substituted benzamide compounds and preparation method, more particularly to it is as liver X receptor Agonist and purposes.
The invention further relates to the pharmaceutical compositions of the compound.
Background technology
Liver X receptor (Liver X receptors, LXRs) is the transcription factor by ligand activation, belongs to nuclear receptor and surpasses house Family member.LXRs is initially by Willy in isolated (P.J.Willy et al., LXR, a from human liver cDNA library in 1994 nuclear receptor that defines a distinct retinoid response pathway,Genes Dev9 (1995)：1033-1045.), heterodimer is formed with retinoid X receptor (Retinoid X receptor, RXR), It when with ligand binding, is combined by the regulatory region with target gene, the expression of activation target gene.Due to originally not finding The native ligand of LXRs, LXRs are also referred to as " orphan receptor ".LXR subfamilies include LXR α (NR1H3) and LXR β (NR1H2) two A isomers, the two function are similar and no matter in DNA binding domain (DNA binding domain, DBD) or in ligand knot Close domain (ligand binding domain, LBD), all have on amino acid sequence 78% similitude (S.M.Ulven et al., LXR is crucial in lipid metabolism, Prostaglandins Leukot Essent Fatty Acids73 (2005)：59-63.).
The endogenous agonist of LXR is the derivative of oxidation category cholesterol, also referred to as oxygen sterol (oxysterol), such as 24 (S), 25-EC and 22 (R)-HC (J.M.Lehmann et al., Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway,J Biol Chem272(1997)：3137- 3140. and J.L.Collins et al., Identification of a nonsteroidal liver X receptor Agonist through parallel array synthesis of tertiary amines, J Med Chem45 (2002)：1963-1966.).Most of oxygen sterol has the agonist characteristics of both LXR α and LXR β.In addition to natural excitement Except agent, class compound such as TO901317 and GW3965 are synthesized, they equally have agonist activity on LXR α and LXR β (J.R.Schultz et al., Role ofLXRs in control of lipogenesis, Genes Dev14 (2000)： 2831-2838. and J.L.Collins et al., Identification of a nonsteroidal liver X receptor Agonist through parallel array synthesis of tertiary amines, J Med Chem45 (2002)：1963-1966.).LXR regulation and control participate in the expression of the relevant many target genes of cholesterol metabolic in various kinds of cell, have become For the crucial sensing element of intracellular sterol levels.When cholesterol overloads, LXR triggers various adaptation mechanisms to adjust body Interior cholesterol levels, including promote reverse cholesterol transport, promote the secretion of biliary cholesterol, inhibits enteral for courage in food The absorption of sterol, and inhibit the de novo formation etc. of cholesterol, for maintaining body cholesterol homeostasis to play weight The effect wanted.In addition, LXRs also participates in adjusting body other physiological activities, including glycometabolism, fatty acid metabolism, macrophage The innate immunity and inflammatory reaction etc..
Adjustings of the LXR to cholesterol transport and metabolism
Make cholesterol eliminate almost all in body to complete by liver, extra cholesterol is necessary in hetero-organization It is transported to and liver and then is secreted into bile by high-density lipoprotein (HDL) and the apolipoprotein for not carrying lipid, this process Referred to as reverse cholesterol transport (reverse cholesterol transport, RCT).It is originally found LXR and is able to maintain that body Cholesterol homeostasis has indicated that LXR can regulate and control reverse cholesterol transport process (S.M.Ulven et al., LXR is crucial In lipid metabolism, Prostaglandins Leukot Essent FattyAcids73 (2005)：59-63.). Large result confirms that the regulation and control of LXR are almost related to entire RCT processes.First, ATP-binding cassette transport protein A1 (ABCA1) and Two transport proteins of ABCG1 arrange cholesterol from cell China and foreign countries, and ABCA1 is mainly by cholesterol and phosphatide from cell membrane transporter to nothing The aPoA-I (apolipoproteinA-I, apoA-I) of lipid, this process is for HDL particles newborn in liver It is formed most important.In addition, ABCG1 can be by cholesterol transport to HDL (C.Cavelier et al., Lipid efflux by The ATP-binding cassette transporters ABCA1and ABCG1, Biochim Biophys Acta1761 (2006)：655-666).In the mankind and rodent, LXR α and LXR β can raise ABCA1 and ABCG1 bases by LXRE Expression (P.Costet et al., Sterol-dependent transactivation of the ABC1promoter by of cause the liver X receptor/refinoid X receptor,J Biol Chem275(2000)：28240-28245.). In the mankind, the mutation of ABCA1 genes leads to lipoprotein profile (the Singaraja RR, Burnham of height Atherogenic LR, Visscher H, Kastelein JJ, Hayden MREfflux and atherosclerosis：the clinical and biochemical impact of variations in the ABCA1gene.Arterioscler Thromb Vasc Biol23(2003)：1322-1332), Tangier disease and relevant early stage artery congee are caused in the case of most serious Sample hardens.This rare inherited disorder is characterized in that the level of high-density lipoprotein (HDL) is extremely low, cholesterol esters huge Phagocyte is accumulated and the danger of atherosclerosis disease dramatically increases (M.Bodzioch et al., The gene Encoding ATP-binding cassette transporter1is mutated in Tangier disease, Nat Genet22(1999)：352-355).During Atheromatosis is managed, macrophage largely swallows low-density lipoprotein Ox-LDL, the accumulation of lipid for causing intracellular a large amount of cholesterol to be stored in the form of cholesteryl ester, to form foamed cell (J.W.Gofman, F.Lindgren, The role oflipids and lipoproteins in atherosclerosis, Science111(1950)：166-171.).Result of study show by activate LXR α, up-regulation protein called membrane transporters ABCA1 and The expression of ABCG1 can speed up cholesterol efflux in macrophage, inhibit macrophage foam cell formation, to inhibit Atherosclerosis The formation of change.
Transport protein ABCG5, G8 are mainly expressed on the film of liver cell tubule, and the activation of LXR can be by raising liver The expression of middle ABCG5, ABCG8, promote cholesterol to secrete, to drive cholesterol transported into bile (L.Yu et al., Stimulation of cholesterol excretion by the liver X receptor agonist requires ATP-binding cassette transporters G5and G8, J Biol Chem278 (2003)：15565- 15570.)。
ABCG5 and ABCG8 is equally of great significance to the absorption of cholesterol in diet in the intestine.In small enteral, this two A albumen is located on the teleblem of enterocyte, they be responsible for by the cholesterol transport of absorption return to enteric cavity (D.Q.Wang et al., Regulation ofintestinal cholesterol absorption, Annu Rev Physiol69 (2007)：221- 248.).There are some researches prove in the enterocyte Caco-2 of people and the small intestine of mouse, the activation of LXR can be raised largely Expression (J.J.Repa et al., Regulation ofATP-binding the cassette sterol of ABCG5 and ABCG8 Transporters ABCG5and ABCG8by the liver X receptors alpha and beta, J Biol Chem277(2002)：18793-18800.).In addition to this, NPC1L1 (Niemann-PickC1like1) is an enteral courage The key protein of sterol absorption, expression can be inhibited in the enteral of mouse and Caco-2 cells by lxr agonist (C.Duval et al., Niemann-Pick C1like1gene expression is down-regulated by LXR Activators in the intestine, Biochem Biophys Res Commun340 (2006)：1259-1263.). Therefore, the activation of LXR can inhibit the absorption of enteral cholesterol.
Apo E (ApoE) is also extremely important for maintaining cholesterol homeostasis, is similarly subjected to the direct of LXR α and LXR β Regulation and control.The surface of albumen lipoprotein in blood plasma exists, and is that the high-affinity of low-density lipoprotein (LDL) receptor is matched Body.ApoE is liver intake chylomicron, necessary to the HDL of Ultra Low Density Lipoprotein and certain hypotypes, and equally can be with As ABCA1 by receptor (K.Wouters et al., Understanding the hyperlipidemia and of cholesterol efflux atherosclerosis：Lessons from genetically modified apoe and ldlr mice, Clin Chem Lab Med43(2005)：470-479.).Therefore, activation of the LXRs to reverse cholesterol transport process can not only pass through ABC protein families can also be completed by improving the availability of extracellular cholesterol carrier such as ApoE.
Adjustings of the LXR to fatty acid metabolism
LXR is also involved in the regulation and control of aliphatic acid synthesis other than participating in cholesterol metabolic.This process is mainly by sterol tune Element conjugated protein 1-c (SREBP-1c) to be saved mainly to control, it can regulate and control the GAP-associated protein GAP of this access as transcription factor, Such as acetyl-CoA carboxylase (acetyl-CoA carboxylase, ACC), fatty acid synthetase (fatty acid Synthase, FAS), (D.Eberle etc. such as stearoyl-coa dehydrogenase (stearoyl-CoA desaturase, SCD) People, SREBP transcription factors：Master regulators oflipid homeostasis, Biochimie86(2004)：839-848.).FAS, ACC, SCD-1 are not only regulated and controled by SREBP-1c, but also in its startup Subregion equally has LXRE, can directly be activated by LXR by independent of SREBP-1c (S.B.Joseph et al., Direct and indirect mechanisms for regulation of fatty acid synthase gene Expression by liver X receptors, J Biol Chem277 (2002)：11019-11025.).But LXR Excessive adjusting of the agonist to fatty acid synthesis gene, can lead to the excess accumulation of aliphatic acid in liver, and cause high glycerine three The side effects such as the raising of pionemia and triglycerides.Peet et al. is the study found that LXR α are the generations of major regulatory intrahepatic fat Hypotype, rather than LXR β (E.M.Quinet et al., Liver X receptor (LXR)-beta regulation in LXRalpha-deficient mice：Implications for therapeutic targeting, Mol Pharmacol70(2006)：1340-1349.).
Adjustings of the LXR to carbohydate metabolism
Other than fat metabolism and cholesterol metabolic, glucose metabolism is also by the active adjustings of LXRs.Studies have shown that In the animal model of different diabetes and insulin resistance, lxr agonist can also reduce the concentration of glucose in blood, increase Insulin sensitivity.For example for the mouse model with severe obesity and insulin resistance, lxr agonist TO901317 can Effectively reduce its internal blood sugar concentration (E.P.Calandre et al., lipoproteins and apolipoproteins A And B in epileptic patients treated with valproic acid, carbamazepine or Phenobarbital, Acta Neurol Scand83 (1991) 250-253. and M.Loffler, et al., Blood glucose-lowering nuclear receptor agonists only partially normalize hepatic Gene expression in db/db mice, J Pharmacol Exp Ther316 (2006) 797-804.).And phase therewith Instead, in normal animal but without finding such phenomenon.Another lxr agonist GW3965, in the ob/ob for lacking leptin In diabetic mice, can equally reduce the concentration of glucose in blood and insulin concentration (A.Grefhorst et al., Differential effects ofpharmacological liver X receptor activation on hepatic And peripheral insulin sensitivity in lean and ob/ob mice, Am J Physiol Endocrinol Metab289(2005)E829-838.)。
This effect essentially consists in, and LXRs agonists can lower the expression of sugared synzyme, including peroxidating in liver The object enzyme body proliferation activated receptor co-activation factor -1 (PGC-1), phosphoenolpyruvate ester carboxylation kinases (PEPCK), Portugal Grape sugar -6- phosphatases (G-6-Pase), fructose 1,6- diphosphatases (fructose-1,6-bisphosphatase, F-1,6- BP) etc., and then the endogenous for affecting glucose generates, and effectively reduces the output of glucose in liver, increases grape The utilization rate of sugar.
LXRs and inflammatory reaction
Different macrophage fundamental immunity functions are activation inflammatory signals and release inflammatory mediator.2003, Joseph etc. People has found that the agonist TO9013171 and GW3965 of LXR can inhibit to pass through lipopolysaccharides in the peritoneal macrophages of mouse The expression of a variety of proinflammatory albumen of (lipopolysaccharide, LPS) induction, such as derivable nitric oxide synthetase (inducible nitric oxidesynthase, iNOS), Transitional cell carcinomas (cyclooxygenase-2, COX-2), Bai Jie - 6 (interleukin-6, IL-6) of element, interleukin (interleukin-1 β, IL-1 β) and monocyte chemoattractant protein-1 With 3 (monocyte chemoattractant proteins1and3, MCP-1and MCP-3) etc. (S.B.Joseph et al., Reciprocal regulation ofinflammation and lipid metabolism by liver X Receptors, Nat Med9 (2003) 213-219.).
The ligand of LXRs can inhibit these genes in wild type (WT), LXR α knockouts, LXR β knock-out mice macrophages Expression, but cannot inhibit LXR α/βs knock out macrophage related inflammation gene expression, illustrate two kinds of LXRs hypotypes all With anti-inflammatory effect.Different models confirms that LXRs has anti-inflammatory effect, gives the LPS stimulations of LXR α/β knock-out mices, small The reactions such as serious systemic inflammatory response occurs in mouse, and liver cell expression iNOS, TNF-α, IL-1 β increase (A.J.Fowler et al., Liver X receptor activators display anti-inflammatory activity in irritant and allergic contact dermatitis models：liver-X-receptor-specific inhibition Of inflammation and primary cytokine production, J Invest Dermatol120 (2003) 246-255.)。
In conclusion currently, the importance of nuclear receptor LXRs signal pathways has received significant attention, but still there are many aspects Mechanism not yet illustrates.LXRs is maintaining body cholesterol homeostasis, adjusts cholesterol metabolic, maintains the stabilization of lipid metaboli, inhibition office The inflammatory reaction in portion and there is effect to the adjusting of glycometabolism, this makes us have reason fully to believe, using LXRs as the medicine of target spot Object will have good application prospect in future therapeutic atherosclerosis in terms of the diseases such as hyperlipidemia and type-2 diabetes mellitus.
In this context, the Active Regulation immunomodulator compounds of lxr receptor are found for the treatment as following particular pathologies, packet The relevant atherosclerosis of LXR activity is included, diabetes, hypercholesterolemia, increased TG is fat, cancer, inflammation, Immune disorders, disorders of lipid metabolism, neurodegenerative disease have very important significance.
Invention content
By numerous studies, inventor has invented compound (I) (II), and finds that it shows good excitement to LXR Activity.
Compound (I) described herein is same compound, the use that the two can be interchanged with compound " L-A9 ".
(1) substituted benzamide compound of the present invention, structure is such as formula (I) or as shown in logical formula (II)：
Wherein, R1、R2、R3It is each independently selected from H, C1-6Alkyl, phenyl ring, substituted phenyl ring, the heteroatomic aromatic radical of tool Group, the heteroatomic aromatic group of tool replaced；R4For H, monosubstituted or polysubstituted, substituent group is halogen, hydroxyl, alkoxy, ammonia Base, alkylamino, alkanoyloxy, alkyl amide, C1-6Alkyl ,-CH=CHNO2, sulfonic group, cyano, trifluoromethyl, nitro, carboxyl, Alkoxy carbonyl group, benzodioxane, phendioxin, seven ring of 5- dioxies, six ring of benzo, seven ring of benzo.
Preferably, wherein R1For phenyl ring, substituted phenyl ring；R2、R3It is each independently selected from H, C1-6Alkyl (be, for example ,- CH3), R4For phendioxin, seven ring of 5- dioxies.
Preferably, it is following compound：
N- methyl-N- (2- oxos -2- (2,3,4- trifluoro-benzene amido) ethyl) -3,4- dihydrobenzos -1,5- dioxane - 7- formamides.
(2) the present invention relates to pharmaceutical composition, it includes the compound of any one of (1) of the invention or its is pharmaceutically acceptable Salt, stereoisomer or solvate, and optional pharmaceutically acceptable auxiliary material, carrier or excipient.
(3) inventor is found that agonist activity of the compound (I) (II) to nuclear receptor LXR α.
(4) agonist activity of the compound (I) (II) to nuclear receptor LXR α, LXR β and RXR α is also found in inventor.
(5) by numerous studies horizontal in vitro, it is found that compound (I) (II) makees the cholesterol efflux of macrophage With apparent, and it can effectively inhibit macrophage foam cell formation, therefore compound (I) (II) has and inhibits macrophage foam cell formation Modulator effect, and to atherosclerosis have therapeutic effect.
(6) compound (I) (II) is by raising ABCG5/ABCG8 expressions, and inhibits the expression of NCP1L1 albumen Level has up-regulation ABCG5/ABCG8 modulator effects and inhibits the modulator effect of the expression of NCP1L1 albumen, shows pair Enteral cholesterol absorption and the application value having to the secretion of liver inner cholesterol.
(7) compound (I) (II) is by the expression of up-regulation ABCA1/ABCG1, and by LXR-ABCA1/ABCG1, this is logical for formation It is arranged outside the Macrophage cholesterol that road mediates, there is the expression of up-regulation ABCA1/ABCG1 and promotes the conditioning agent of cholesterol efflux Effect, and reverse cholesterol transport can be promoted.
(8) compound (I) (II) has the adjusting of up-regulation ApoE expression by the expression of up-regulation apo E (ApoE) Agent acts on, therefore can effectively maintain internal cholesterol environment stable state.
(9) composition of the compound of the present invention (I) (II) and compound has medicinal physiological activity, is especially treating The angiocardiopathies such as atherosclerosis, hypertension, hyperlipidemia, hypercholesterolemia, coronary heart disease, and obesity, diabetes, metabolism Syndrome, the neurodegenerative diseases and immune correlated disease etc. such as Alzheimer have important use.
Specifically, the present invention relates to：
(1) formula (I) (II) compound represented (I) (II) under.
(2) it is related to the preparation method of the compound of (1) of the invention：
Work as R2=R3, when being identical substituent group：
(1) with the acyl chlorides of chlorine substitution on last position carbon (being propionyl chloride when as n=2 being chloroacetic chloride, n=3, and so on) and single Replace (R1Substitution) amine reaction, obtain corresponding amide；
(2) compound that end position is amine substitution is made in amide and the ammonium hydroxide reaction replaced the last position chlorine of above-mentioned preparation；
(3) with benzoic acid and the thionyl chloride reaction with different substituents, the benzoyl with different substituents is prepared Chlorine；
(4) compound prepared by the chlorobenzoyl chloride and step (2) prepared above-mentioned steps (3) is reacted, and R is obtained2、R3It is H First aspect compound；
(5) R is made in the compound and halohydrocarbons reaction prepared above-mentioned steps (4)2、R3For identical substituent group first aspect Compound.
Work as R2It is not hydrogen, and R3With R2When different：
(1) with the acyl chlorides of chlorine substitution on last position carbon (being propionyl chloride when as n=2 being chloroacetic chloride, n=3, and so on) and two Replace (R1、R2Substitution) amine reaction, obtain corresponding amide；
(2) compound that end position is amine substitution is made in amide and the ammonium hydroxide reaction replaced the last position chlorine of above-mentioned preparation；
(3) with benzoic acid and the thionyl chloride reaction with different substituents, the benzoyl with different substituents is prepared Chlorine；
(4) compound prepared by the chlorobenzoyl chloride and step (2) prepared above-mentioned steps (3) is reacted, and R is obtained2Be not hydrogen, R3For the first aspect compound of H；
(5) R is made in the compound and halohydrocarbons reaction prepared above-mentioned steps (4)2It is not hydrogen, and R3With R2Difference first Aspect compound.
Work as R2For hydrogen, and R3With R2When different：
(1) with the acyl chlorides of chlorine substitution on last position carbon (being propionyl chloride when as n=2 being chloroacetic chloride, n=3, and so on) and single Replace (R1Substitution) amine reaction, obtain corresponding amide；
(2) amide (Boc) for replacing the last position chlorine of above-mentioned preparation2O reacts, and the last position chlorine substitution of N-Boc protections is made Compound；
(3) with benzoic acid and the thionyl chloride reaction with different substituents, the benzoyl with different substituents is prepared Chlorine；
(4) compound prepared by the chlorobenzoyl chloride and step (2) prepared above-mentioned steps (3) is reacted, and R is obtained2For Boc, R3 For the first aspect compound of H；
(5) R is made in the compound and halohydrocarbons reaction prepared above-mentioned steps (4)2For Boc, and R3With R2Different first Aspect compound.
(6) compound for preparing above-mentioned (5) is passed through hydrogen chloride gas and sloughs Boc protections, R is made in organic phase2For H, and R3With R2The hydrochloride of different first aspect compounds.
The same first aspect present invention of definition of wherein each substituent group.
Specifically, the preparation method of the compound of first aspect present invention is as follows：
Work as R2=R3, when being identical substituent group：
Work as R2It is not hydrogen, and R3With R2When different：
Work as R2For hydrogen, and R3With R2When different：
Wherein a is tetrahydrofuran；B is reflux；C is catalyzed for DMF, reflux；D is≤0 DEG C of dropwise addition, is stirred at room temperature；F is second Nitrile, reflux；G is dichloromethane, is stirred at room temperature；H is dichloromethane, and HC1 (gas) is stirred at room temperature.
Preferably, reaction condition is：A be tetrahydrofuran, triethylamine, 0 DEG C, 4h；B is reflux, 3h；C is catalyzed for DMF, Flow back 6h；D is≤0 DEG C of dropwise addition, and 12h is stirred at room temperature；F is acetonitrile, and flow back 2~4h；G is dichloromethane, and room temperature stirs 12h；h For dichloromethane, 8h is stirred at room temperature in HCl (gas).
The same first aspect present invention of definition of wherein each substituent group.
(3) the present invention also provides the compositions for including the compound；Provide the medicine group for including the compound Close object, it is characterised in that it contains one or more pharmacy using the above compound containing therapeutically effective amount as active constituent Upper acceptable carrier.
(4) application of compound of the present invention and composition in preparing Antiatherosclerosis medicine.
(5) the various dosage forms of pharmaceutical composition of the present invention can be prepared according to the conventional production process of pharmaceutical field, Such as active constituent is made to be mixed with one or more carriers, then it is made into required dosage form.
(6) compound (I) is used to prepare the purposes of the conditioning agent of nuclear receptor LXR α, LXR β and RXR α.
(7) compound (II) or derivatives thereof is used to prepare the purposes of nuclear receptor LXR alpha modulators.
(8) compound (I) (II) is used to prepare the cholesterol efflux for promoting macrophage or inhibits macrophage foam cell formation Drug purposes.
(9) compound (I) (II) is used to prepare the use of the expression conditioning agent of ABCG5/ABCG8 or NCP1L1 albumen On the way.
(10) compound (I) (II) is used to prepare the purposes for the drug for promoting reverse cholesterol transport.
(11) compound (I) (II) or the composition comprising compound (I) (II) be used to prepare treatment atherosclerosis, The angiocardiopathies such as hypertension, hyperlipidemia, hypercholesterolemia, coronary heart disease, and obesity, diabetes, metabolic syndrome, A Erci The purposes of the drug of the neurodegenerative diseases such as sea is silent and immune correlated disease.
Description of the drawings
The compounds for being named as " L-A9 " all in figure are heretofore described compound (I).
Fig. 1 compounds (I) N- methyl-N- (2- oxos -2- (2,3,4- trifluoro-benzenes amido) ethyl) -3,4- dihydrobenzos - 1,5- dioxane -7- formamides1H NMR figures
Fig. 2 compounds (I) N- methyl-N- (2- oxos -2- (2,3,4- trifluoro-benzenes amido) ethyl) -3,4- dihydrobenzos - The MS of 1,5- dioxane -7- formamides schemes
Amount effect relation curve of Fig. 3 compounds (I) on LXR alfa agonists screening models.
Influence of Fig. 4 compounds (I) to ABCA1 and ABCG1 protein expression levels in Human THP-1 monocyte.
Influence of Fig. 5 compounds (I) to macrophage foam cell formation.Figure is oil red O stain microscopy photo (× 400 times of amplification)
Fig. 6 compounds (I) promote to arrange outside the RAW264.7 intracellular cholesteryls mediated by ApoA-I and HDL.
Influence of Fig. 7 compounds (I) to ABCG5 and ABCG8 protein expression levels in human HepG2 cell.
The influence of Fig. 8 compounds (I) NCP1L1 protein expression levels intracellular to people Caco-2.
Influence of Fig. 9 compounds (I) to ApoE protein expression levels in Human THP-1 monocyte.
Amount effect relation curve of Figure 10 compounds (I) on LXR β and RXR α nuclear receptors agonists models.
The structure of Figure 11 compounds (I) (II).
Specific implementation mode
Embodiments described just below and embodiment detail the activity of compound, are described in detail by the following examples The present invention, but they are not interpreted as any limitation on the scope of the present invention.These embodiments are only limitted to task of explanation, this field Staff, which will be able to contemplate, to be modified and is changed according to it, these modifications and changes are also included within the essence and model of the application Enclose in attached claim scope.All open source literatures cited herein, patents and patent applications are that all purposes introduces The application is as reference.The person that is not specified actual conditions in embodiment, carries out according to conventional conditions or manufacturer's recommended conditions.Institute Production firm person is not specified with reagent or instrument, being can be with conventional products that are commercially available.
Embodiment 1.N- methyl-N- (2- oxos -2- (2,3,4- trifluoro-benzene amido) ethyl) -3,4- dihydrobenzos -1,5- The synthesis of dioxane -7- formamides
It takes 2,3,4- benzamide trifluoroacetates (5.90g, 0.04mol) in 100mL three-necked flasks, tetrahydrofuran 40mL is added It dissolves, under the conditions of 0 DEG C, the tetrahydrofuran solution of 15mL chloro-acetyl chlorides (4.52g, 0.04mol) is added dropwise thereto, is added dropwise 4.2mL (0.06mol) triethylamine is added, 4h is stirred at room temperature；Reaction finishes, and boils off solvent, and the dissolving of 40mL dioxanes is added (Boc)212h is stirred at room temperature in O (8.73g, 0.04mol).It is dissolved with 40mL dichloromethane after boiling off solvent, 10mL ammonium hydroxide is added, 90 DEG C of back flow reaction 12h.After the completion of reaction, it is evaporated dissolving, yellow solid is obtained, is dissolved out with 5% hydrochloric acid 30ml under low temperature, second Acetoacetic ester 20ml*3 washs water phase, then saturated sodium carbonate is used to lower pH to 9 in condition of ice bath, a large amount of yellow solids are precipitated 7.24g, yield 59.5%, drying for standby.
Take 3,4- dihydrobenzo -1,5- dioxane -7- formic acid (4.66g, 0.024mol) in 100mL round-bottomed flasks, Thionyl chloride 40mL, 1 drips DMF, flows back 4 hours, excessive thionyl chloride is evaporated off, obtains 3,4- dihydrobenzos -1,5- dioxane - 7- formyl chlorides, are dissolved with dichloromethane, are instilled in the dichloromethane solution of above-mentioned amine, are added dropwise under the conditions of ice-water bath, are added Reaction 12h is stirred at room temperature in 4mL triethylamines.Post separation, dichloromethane：Methanol=9：1 elution, obtains yellow solid 6.78g, yield 58.8%
Above-mentioned solid is dissolved in 40mL acetonitriles in 100mL round-bottomed flasks, iodomethane (2g, 0.014mol) is added, is returned Stream reaction 4h.Reaction finishes, and solvent seasoning is evaporated off.Then it is placed in 100mL three-necked flasks, is passed through with the dissolving of 60mL dichloromethane Dry hydrogen chloride gas 8h obtains white solid after solvent is evaporated off, after solid 25mL water dissolutions, under condition of ice bath, With the sodium hydroxide tune pH to 10 of 1mol/L, dichloromethane 25mL*3 aqueous phase extracteds merge organic phase, are evaporated to obtain yellow targeted Close object 2.29g, yield 41.5%.
1H NMR(DMSO-d6, 500M)：10.10 (1H, m ,-CONH-), 7.67~6.96 (5H, m, Ph-H), 4.24 (6H, M ,-CH2× 3), 3.00 (1H, s ,-CH3), 2.13 (2H, m ,-CH2-)
MS(ESI)：[M+1]+395.62 [M+Na]+417.48
The culture of 2. cell of embodiment
Human liver cancer cell HepG2 and people's Colon and rectum gland cancer cell Caco-2 is attached cell, HepG2 about 48h passages one Secondary, Caco-2 about 96h passages are primary.After cell covers with, old culture medium is abandoned, is discarded after rinsing cell with PBS, it is later plus appropriate Pancreatin, HepG2 digest about 2min and discard digestive juice after Caco-2 digests 10min at 37 DEG C at room temperature, are added contain immediately The MEM complete mediums of 10%FBS gently blow and beat culture bottle inner cell repeatedly to inhibit trypsase vigor with elbow straw, Make cell completely disengage bottom of bottle and blow and beat to be allowed to be separated into individual cells suspension.1 is pressed again：3 ratio inoculating cell suspensions are in new In cell bottle, appropriate complete medium is then added, incubator is put into and continues to cultivate.Condition of culture：37 DEG C, 5%CO2。
Mouse monokaryon macrophage leukaemia cell RAW264.7 is half attached cell, and about 48h passages are primary.Pancreatin digests 5min, by 1：4-1：6 ratios pass on, and using DMEM complete mediums, other conditions are identical as HepG2 condition of culture.
People's Acute Monocytic Leukemia Cell Line THP-1 is suspension cell, and about 48h passages are primary.Wait for that cell density is 3-5 ×106When a/ml, culture bottle inner cell is blown and beaten with elbow straw, is allowed to be separated into individual cells suspension.1 is pressed again：4 ratios connect Kind of cell suspension in new cell bottle, then adding appropriate RPMI-1640 culture mediums complete medium, other conditions with HepG2 condition of culture is identical.
Measurement of 3. compound of embodiment (I) to LXR agonist activities
This determination of activity carries out the measurement of compound activity using the agonist screening model of LXR.
Measuring principle：
The principle of this detection is, according to two main domains in LXR structures：Ligand binding domains (LBD) and DNA Transcription factor GAL4 has the characteristics that the similar structure of nuclear receptor in binding structural domain (DBD) and yeast, using yeast two-hybrid Principle, respectively build two plasmids：PBIND-LXR plasmids contain the LBD structural domains of the DBD part and LXR of GAL4 genes, can To change conformation when by ligand activation；Another plasmid is pGL4-GAL4, reacts member containing GAL4 gene promoter regions The luciferase reporter gene Luc of part.We are by two plasmid co-transfections to HepG2 cells, to study compound to LXR's Activation.In the detection architecture, if compound can activate LXR, the LXR protein conformations of expression plasmid expression to change Become, be combined with reporter plasmid, the expression activity of compound group luciferase is higher than the control group for not adding compound at this time；It is on the contrary When compound is inactive to LXR, then LXR protein conformations do not change, and are not combined with reporter plasmid, then compound group The expression activity of luciferase does not generate variation.
Assay method：
(1) it is transfected in 96 orifice plates, the day before transfection, HepG2 plating cells, density is 1.5-3.0 × 105A/ml Cell.It waits for that HepG2 cells are grown to 90% to converge and growth is in good condition, according to 25 holes μ l/ Opti-MEM culture mediums dilutions 0.5 μ l liposomes LipofectamineTM2000Reagent is incubated at room temperature 5min.25 holes μ l/ Opti-MEM culture mediums dilutions 200ng Plasmid DNA, including pGL4-GAL4 and pBIND-LXR α merge mixing with dilution liposome afterwards, are incubated at room temperature 20min.
(2) MEM complete mediums are added according to 100 μ l of every hole in the DNA- liposome complexes solution after merging, make training It supports base to mix well with DNA- liposome complexes, at this time suck the culture medium in cell plates, by mixed cell culture Liquid-DNA- liposome complex solution is added in 96 orifice plates, per 150 μ l of hole.96 orifice plates are placed in 37 in carbon dioxide incubator ℃。
(3) 6h is cultivated, transfection media, the concentration model by compound (I) at final concentration of 0.001 μM to 100 μM is sucked out It encloses and is diluted in 200 μ l MEM serum free mediums, while 0.1% DMSO is added in negative control hole, handles cell respectively, continue Cultivate 18h.
(4) after 18h, the culture medium during 96 orifice plates are sucked out per hole.150 μ l PBS are added per hole and gently rinse cell, overturn 96 orifice plates are outwelled PBS and are dried.Per being added 20 μ l1 × CCLR cell pyrolysis liquids in hole, 37 DEG C of lytic cell 20-30min (according to How much cell number determines the time), observe whether cell cracks completely under the microscope.Lysate is fully transferred to fluorescence point It analyses in the corresponding aperture with the opaque blank in 96 holes, notices that lysate avoids bubble as possible, influence reading otherwise can be made (if used 96 hole clear bottom blanks need not then shift lysate).
(5) 50-70 μ l fluorescence analysis reagents (Promega companies) are rapidly added in every hole to be immediately put into analysis blank It is detected in microplate reader.
(6) with following equation calculation sample to be tested to the rate of change of uciferase activity：
Rate of change (%)=A/B × 100%
Wherein, A is that the cell fluorescence element enzymatic activity (RLU) measured after sample to be tested is added, and B is that negative control sample is added (DMSO) the cell fluorescence element enzymatic activity (RLU) measured afterwards.Rate of change (%) is considered as agonist activity of the compound to LXR, uses % Percentage indicates, or is indicated with multiple.
It being found by measuring, A is the RLU that compound (I) is added and measures afterwards, and B is the RLU being added after DMSO negative controls, After rate of change is calculated according to formula, using the logarithm of compound concentration as abscissa, rate of change is ordinate, uses Graphpad Prism software matched curves, are as a result shown in Fig. 3.
The measurement result illustrates that compound in the present invention (I) has significant transcriptional activation to LXR α, and obtains Amount effect relation curve of the compound (I) on LXR alfa agonists screening models.From figure 3, it can be seen that compound (I) is 12.5 μM when for LXR α, to show highest agonist activity be 180%, ECso values is 0.16 μM.Since nuclear receptor LXR α are solid in participation courage Alcohol is metabolized to be of great significance in the remittance of the physiology courses such as aliphatic acid, carbohydate metabolism, therefore the result illustrates the change in the present invention Closing object (I) can be by activating the transcription of LXR α to play a role in the disease that regulation and control are related to Relevant Physiological Courses.Embodiment 4. Measurement of the derivative of compound (II) to LXR α agonist activities
This determination of activity carries out the measurement of compound activity using the agonist screening model of LXR α.
Assay method：
It is transfected in 96 orifice plates, the day before transfection, HepG2 plating cells, density is 1.5-3.0 × 105A/ml is thin Born of the same parents.It waits for that HepG2 cells are grown to 90% to converge and growth is in good condition, according to 25 holes μ l/ Opti-MEM culture mediums dilutions 0.5 μ l liposomes LipofectamineTM2000Reagent is incubated at room temperature 5min.25 holes μ l/ Opti-MEM culture mediums dilute 200ng Plasmid DNA, including pGL4-GAL4 and pBIND-LXR α merge mixing with dilution liposome afterwards, are incubated at room temperature 20min.It will merge MEM complete mediums are added according to 100 μ l of every hole in DNA- liposome complexes solution afterwards, keep culture medium multiple with DNA- liposomes It closes object to mix well, at this time suck the culture medium in cell plates, by mixed cell culture fluid-DNA- liposome complexes Solution is added in 96 orifice plates, per 150 μ l of hole.96 orifice plates are placed in carbon dioxide incubator 37 DEG C.6h is cultivated, transfection training is sucked out Base is supported, compound is diluted to final concentration 2 μ g/ml and 10 μ g/ml respectively in 200 μ l MEM serum free mediums, while cloudy Property control wells be added 0.1% DMSO, handle cell respectively, continue cultivate 18h.After 18h, according to the method in embodiment 3, inspection The activity of luciferase is surveyed, rate of change (%) is calculated.It the results are shown in Table 1.
Table 1
Influence of 5. compound of embodiment (I) in person monocytic cell THP-1 to ABCA1 and ABCG1 protein expression levels.
(1) compound handles cell：It will be inoculated in the THP-1 in 6 porocyte culture plates in advance, final concentration 200nM is being added After phorbol exters PMA inductions for 24 hours, handled with 1 μM and 10 μM of compound (I), using a concentration of 0.1%DMSO as negative control group (4 con groups in figure), using 1 μM of compound TO901317 (4 T1317 groups in figure) as positive controls, at 37 DEG C, 5% CO2Under the conditions of cultivate 18h.
(2) preparation of albumen：After 18h, culture medium is discarded, cell is rinsed 2 times with the PBS of precooling, is used after pancreatin digestion Culture medium collect cell, 900rpm centrifuge 4min, then use PBS suspension cells, 900rpm centrifugation 4min.Often pipe is separately added into 60 μ l RIPA cell pyrolysis liquids (PMSF that final concentration of 100 μ g/ml are added in per 1ml lysates), in lytic cell on ice 30min.4 DEG C, 12000 × g centrifuges 20min.Supernatant is collected, with BCA quantification of protein kit (Thermo companies) to albumen It is quantified, ddH is used in combination2Each group protein sample is adjusted to 2 μ g/ μ l concentration by O.A certain amount of 5 × albumen loading buffer is added Liquid, boils 10min in boiling water bath, and of short duration centrifugation after cooling carries out SDS-PAGE electrophoresis per 40 μ g albumen of hole loading.Remaining sample In -80 DEG C of preservations.
(3) Western blot methods measure protein level：After SDS-PAGE electrophoresis, by polyacrylamide gel and filter paper It is put into transfering buffering liquid and impregnates balance 5min.Pvdf membrane is submerged initially in methanol activation, is washed with water, is placed in transfering buffering liquid and soaks Bubble balance 2min.With BioRad semidry method transferring film instrument transferring films, setting electric current 5mA/cm2, constant current transferring film 30min.After transferring film, It is cut off respectively according to the size of destination protein, is put into methanol and infiltrates 1min, placed into 1 × TBST and rinse.Later by purpose Band is respectively put into rocked at room temperature in 1 × TBST containing 5% skimmed milk power (W/V) and closes 1h.Closing terminates, with containing 5% degreasing The TBST (W/V) of milk powder dilutes primary antibody, is placed in hybridization bag, after being incubated at room temperature 1h, 4 DEG C of overnight incubations.It is washed later with 1 × TBST Three times, each 10min.Later goat is marked with TBST (W/V) the dilution horseradish peroxidases (HRP) containing 5% skimmed milk power Anti-rabbit IgG (H+L) secondary antibody is placed in hybridization bag, is incubated at room temperature 2h.Pvdf membrane is taken out from hybridization bag, is washed three times with 1 × TBST, Each 10min.Colour developing：In the front of film, that is, turn have the one side of albumen that appropriate enhanced HRP (horseradish peroxidase) bottom is added The mixed liquor (matching while using) of object chemical luminescence for liquid (ECL) A, B liquid, is immediately placed in gel imager and develops the color.
(4) colour developing with Quantity One softwares as a result, be scanned, quantitative Treatment.
ABCA1 and ABCG1 is the target protein of LXR, and major function of the two in macrophage is all will be intracellular extra Cholesterol efflux, promote reverse cholesterol transport process, prevent the OxLDL ELISA (ox-LDL) in macrophage from storing Manufacture into macrophage foam cell formation.By compound (I) effect in 18 hours, as shown in Figure 4, compound (I) can be in 1 He The expression of 10 μM of apparent up-regulation ABCA1 and ABCG1 albumen, in 10 μM of compound (I), up to 2.84 times of ABCA1 up-regulations, and When compound (I) is 1 μM, ABCG1 raises 2.23 times.Thus disclosure sets forth compounds (I) to promoting courage in macrophage GAP-associated protein GAP is arranged outside sterol has apparent up-regulation to act on.
6. compound of embodiment (I) inhibits the experiment of macrophage foam cell formationization effect
The mononuclear macrophage RAW264.7 of mouse is with the sugared culture solutions of the DMEM- high containing 10%FBS in transparent 96 hole cell It is cultivated on culture plate.After adherent, it is changed to serum-free DMEM- high glucose mediums (100 holes μ l/).By cell be divided into blank control group, Foam cells group and dosing group, the Oxided low-density lipoprotein (ox-LDL) that final concentration of 60 μ g/ml are added arrive foam cells Group and dosing group, for 24 hours after, certain density compound (I) is added in dosing group.37 DEG C, 5%CO2Under the conditions of cultivate for 24 hours after, into Row oil red O stain.By 96 orifice plates from CO2It is taken out in incubator, cell fixes (15 holes μ l/) 10min with 4% paraformaldehyde, abandons molten Liquid, distilled water are washed twice, and 60% isopropanol (150 holes μ l/) is added, and are placed 5min, are discarded solution.Oil red O is (existing using liquid With now matching, filter) it is added in each hole, 150 holes μ l/, dye 1h.Solution is discarded, with 60% isopropanol (150 holes μ l/) hole flushing, so It is washed twice with distilled water (150 holes μ l/) afterwards, it is last to add 150 μ l distilled waters to be placed in microscopically observation, take pictures per hole, as a result see Fig. 5.
Ox-LDL will only be added as foamed groups of cells, by oil red O stain, observe the coloring of cell under the microscope Degree.From figure 5 it can be seen that (A) blank group is formed without red fat drips, (B) ox-LDL positive controls are formed significantly Red fat drips exist, and (C)-(G) compound (I) processing group Red oil particle occurs and obviously subtracts compared with the control group into the cell It is few, and as concentration is in dose-dependant reduction, illustrates that compound (I) can reduce lipid and be accumulated in macrophage, effectively press down Macrophage foam cell formation processed, (H) are added lipid accumulation in 1 μM of T1317 cell and also significantly reduce.
7. compound of embodiment (I) promotes to arrange experiment outside Macrophage cholesterol
(1) mouse monokaryon macrophage RAW264.7 is changed in 96 porocyte culture plates cultures after cell is completely adherent The cholesterol of final concentration of 5 μM of fluorescent marker is added in DMEM culture mediums containing 0.2% (w/v) bovine serum albumin(BSA) (BSA) 22-NBD-cholesterol (Invitrogen companies), 37 DEG C, 5%CO2Under the conditions of be incubated for 24 hours.
(2) after for 24 hours, culture medium is sucked out, PBS washes cell twice, and the measurement culture medium of compound containing a certain concentration (I) is added (0.2%BSA, 0.1%DMSO is added in DMEM), 37 DEG C of incubations.
(3) HDL or ApoA-I, 37 DEG C of incubation 6h are directly added into after 18h.Supernatant is transferred in 96 orifice plate of black later, In its exciting light it is 485nm with microplate reader, transmitting light measures its fluorescent value when being 535nm, records.
(4) cell is washed twice with PBS, loss cell of trying not 100 μ l PBS is added per hole, with microplate reader at it Exciting light is 485nm, and transmitting light measures its fluorescent value when being 535nm, records.
(5) the cholesterol operation part involved in experiment should be protected from light operation.Experiment is divided into a- blank groups：It is not added with cholesterol； B- control groups：Be added cholesterol, without HDL, without ApoA-I groups, c- sample-adding groups：Cholesterol is added, HDL, ApoA-I but chemical combination is added Object various concentration.The final concentration of final concentration of 50 μ g/ml of 10 μ g/ml, ApoA-I of HDL.
The calculation formula of Cholesterol Efflux rate：
Cholesterol Efflux rate %=supernatant fluorescent value/(supernatant fluorescent value+intracellular Fluorescence value) × 100%=(in sample-adding group Clearly-blank group supernatant value)/(sample-adding group is intracellular-and blank group is intracellular+sample-adding group supernatant-blank group supernatant) × 100%
The results are shown in Figure 6 for cholesterol efflux in macrophage, in the Cholesterol Efflux experiment mediated for ApoA-I, When being not added with compound (I), Cholesterol Efflux rate is about 7%, and with the increase of compound concentration, dose-dependant is presented in discharge rate Relationship rises, and significant difference is presented with compound group is not added with, at 10 μM, discharge rate reaches maximum i.e. 22%.HDL is mediated Cholesterol Efflux experiment in, be about 13% being not added with compound (I) Cholesterol Efflux rate, with the increase of compound concentration, Discharge rate equally increases with concentration and is increased, and significant difference is presented with compound group is not added with, at 10 μM, discharge rate reaches maximum i.e. 26%.The result meets before the present invention described in part, and compound (I), which passes through, raises ABCA1 and ABCG1 albumen in macrophage Expression, and then promote the outer row of intracellular cholesteryl, meet the physiologically active of lxr agonist.
Influence of 8. compound of embodiment (I) to ABCG5 and ABCG8 expressions
(1) compound handles cell：Various concentration is added in the HepG2 cells being inoculated in 6 porocyte culture plates in advance Compound (I) processing, using a concentration of 0.1%DMSO as negative control group (the con groups in Fig. 7), with 1 μM of compound TO901317 (the T1317 groups in Fig. 7) is used as positive controls, at 37 DEG C, 5%CO2Under the conditions of cultivate 18h.
(2) Western blot methods measure ABCG5 and ABCG8 protein expression levels, and software quantitative Treatment is used in combination.
ABCG5 in liver, ABCG8 albumen are also the target protein in the downstreams LXR, they by LXR direct regulation and control, the two Transport protein is expressed on the film of liver cell tubule, to drive cholesterol to transport expression into bile, cholesterol is promoted to secrete (L. Yu et al., Stimulation of cholesterol excretion by the liver X receptor Agonist requires ATP-binding cassette transporters G5and G8, J Biol Chem278 (2003)：15565-15570.).As shown in fig. 7, in the present invention compound (I) can within the scope of 0.01-10 μM dose-dependant Up-regulation ABCG5 and ABCG8 expression, in 1 μM of L-A9, ABCG5 up-regulation up to 1.72 times, ABCG8 is up to 1.79 times.It says Bright compound (I) is possible to cholesterol in liver can be promoted to secrete by raising ABCG5 and ABCG8 and expressing.
Influence of 9. compound of embodiment (I) to NPC1L1 protein expression levels
Compound handles cell：Various concentration is added in the Caco-2 cells being inoculated in 6 porocyte culture plates in advance Compound (I) processing, using a concentration of 0.1%DMSO as negative control group (the con groups in Fig. 8), with 1 μM of compound TO901317 (the T1317 groups in Fig. 8) is used as positive controls, at 37 DEG C, 5%CO2Under the conditions of cultivate 18h.Western Blot methods measure NPC1L1 protein expression levels, and software quantitative Treatment is used in combination.
By being found to the detection of NPC1L1 protein levels in Caco-2 cells, as shown in figure 8, it is considered herein that compound (I) in 1 and 10 μM of transcriptional activity by exciting LXR, the expression of NCP1L1 albumen can obviously be inhibited, thus with inhibition intestines The important function that inner cholesterol absorbs.
Influence of 10. compound of embodiment (I) in person monocytic cell THP-1 to ApoE protein expression levels
By THP-1 cells after final concentration 200nM phorbol exters PMA inductions for 24 hours are added, with 1 μM and 10 μM of compound (I) Processing, using a concentration of 0.1%DMSO as negative control group (the con groups in Fig. 9), with 1 μM of compound TO901317 (in Fig. 9 T1317 groups) be used as positive controls, at 37 DEG C, 5%CO2Under the conditions of cultivate 18h.Western blot methods measure huge Software quantitative Treatment is used in combination in ApoE protein expression levels in phagocyte.
Apolipoprotein E is also extremely important for maintaining cholesterol homeostasis, is similarly subjected to the direct tune of LXR α and LXR β Control.After handling THP-1 cells 18h using compound (I) in the present invention, from fig. 9, it can be seen that compound is in 1 and 10 μM of concentration The expression of ApoE can be raised down.
Determination experiment of 11. compound of embodiment (I) to nuclear receptor LXR β and RXR α agonist activities
This determination of activity carries out the measurement of compound activity using the agonist screening model of LXR β and RXR α.
Measuring principle is identical with the LXR alfa agonists models in embodiment 3.
Assay method：
It is transfected in 96 orifice plates, the day before transfection, HepG2 plating cells, density is 1.5-3.0 × 105A/ml is thin Born of the same parents.It waits for that HepG2 cells are grown to 90% to converge and growth is in good condition, according to 25 holes μ l/ Opti-MEM culture mediums dilutions 0.5 μ l liposomes LipofectamineTM2000Reagent is incubated at room temperature 5min.25 holes μ l/ Opti-MEM culture mediums dilutions 200ng Plasmid DNA, including pGL4-GAL4 and pBIND-LXR β (pBIND-RXR α) merge mixing, room with dilution liposome afterwards Temperature is incubated 20min.MEM complete mediums are added according to 100 μ l of every hole in DNA- liposome complexes solution after merging, make training It supports base to mix well with DNA- liposome complexes, at this time suck the culture medium in cell plates, by mixed cell culture Liquid-DNA- liposome complex solution is added in 96 orifice plates, per 150 μ l of hole.96 orifice plates are placed in 37 in carbon dioxide incubator ℃.6h is cultivated, transfection media is sucked out, compound (I) is diluted in final concentration of 0.001 μM to 100 μM of concentration range 200 μ l MEM serum free mediums, while 0.1% DMSO being added in negative control hole, cell is handled respectively, continues to cultivate 18h.After 18h, according to the method in embodiment 3, the activity of luciferase is detected, calculates rate of change (%)
LXR α and LXR β are hypotypes different in nuclear receptor LXR families, and RXR α and LXR can form heterodimer, Activate the transcription of downstream target gene.Measurement result in Figure 10 illustrates that compound (I) equally has LXR β and RXR α in the present invention There is transcriptional activation, and has obtained amount effect relation curve of the compound (I) on two models.Therefore the result illustrates this Compound (I) in invention equally can be by activating the transcription of LXR β and RXR α regulating and controlling to be related to the disease of Relevant Physiological Courses In there is potential effect.

Claims (6)

1. a kind of agonist of liver X receptor, which is characterized in that the agonist is N- methyl-N- (2- oxos -2- (2,3,4- Trifluoro-benzene amido) ethyl) -3,4- dihydrobenzo -1,5- dioxane -7- formamides, structure is shown in following formula (I)：
2. application of the agonist described in claim 1 in preparing liver X receptor agonists, which is characterized in that the liver X receptor It is nuclear receptor LXR α, LXR β or RXR α.
3. application of the agonist described in claim 1 in preparing molecular biology experiment preparation, the function of the preparation It is any or combinations thereof as follows：
(1) inhibit macrophage foam cell formation；
(2) enhancing Macrophage cholesterol outflow；
(3) ATP combination box protein ABCG5 and ABCG8 are raised, and inhibits the expression of NCP1L1 albumen；
(4) up-regulation apo E (ApoE).
4. the application of agonist according to claim 1 in medicine preparation, which is characterized in that the function of the drug For：
(1) steady to adjust cholesterol by promoting cholesterol efflux, adjusting enteral cholesterol absorption and liver inner cholesterol to secrete State；
(2) antiatherosclerosis；
(3) angiocardiopathy is treated, the angiocardiopathy is hypertension, hyperlipidemia, hypercholesterolemia, coronary heart disease；
(4) treatment metabolism class disease, obesity that the metabolism class disease is diabetes, Metabolic syndrome is sought peace；
(5) Alzheimer's disease is treated；
(6) immune correlated disease is treated.
5. including the pharmaceutical composition of agonist described in claim 1.
6. pharmaceutical composition according to claim 5, which is characterized in that described pharmaceutical composition contains therapeutically effective amount Agonist described in claim 1 is active constituent, and contains one or more pharmaceutically acceptable carriers.
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