WO2011107023A1 - 一种防治代谢性疾病的化合物及其用途 - Google Patents

一种防治代谢性疾病的化合物及其用途 Download PDF

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WO2011107023A1
WO2011107023A1 PCT/CN2011/071446 CN2011071446W WO2011107023A1 WO 2011107023 A1 WO2011107023 A1 WO 2011107023A1 CN 2011071446 W CN2011071446 W CN 2011071446W WO 2011107023 A1 WO2011107023 A1 WO 2011107023A1
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betulin
cholesterol
srebp
cells
mice
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French (fr)
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宋保亮
唐静洁
李家贵
李培山
戚炜
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中国科学院上海生命科学研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention belongs to the field of pharmacy; more specifically, the present invention relates to a compound for preventing and treating metabolic diseases such as hyperlipemia, atherosclerosis and type II diabetes and uses thereof. Background technique
  • Metabolic diseases such as atherosclerosis and type 2 diabetes have become increasingly serious worldwide health problems, and their occurrence is known to be closely related to hyperlipidemia.
  • hypercholesterolemia which in turn induces atherosclerosis.
  • Atherosclerosis is the most common cause of cardiovascular diseases such as coronary heart disease, stroke, and myocardial infarction; blood fatty acids and glycerol Excessive levels of esters can cause hypertriglyceridemia, which is the leading cause of insulin resistance and type 2 diabetes.
  • Due to the pathogenicity of hyperlipidemia lipid-lowering is an important component in the treatment and prevention of metabolic diseases.
  • a key factor in the regulation of cholesterol and fatty acid synthesis in mammals is known to be a class of transcription factor proteins: alcohol response element binding protein (SREBP) (Goldstein, J. L. et al. (2006). Protein sensors for membrane sterols. Cell 124, 35-46).
  • SREBP alcohol response element binding protein
  • the precursors of this class of proteins are first synthesized on the endoplasmic reticulum (ER), which is transported to the Golgi by SREBP cleavage-activating protein (SCAP) and then passed through two proteases (site- 1 protease (S IP) and site- 2 protease (S2P)) digests, releases its N-terminal active domain, enters the nucleus to act as a transcription factor, and binds to the SREBP response element (SRE) of the target gene promoter region to initiate expression of downstream genes.
  • SCAP SREBP cleavage-activating protein
  • SIP site- 1 protease
  • S2P site- 2 protease
  • SRE SREBP response element
  • the shear maturation of the SREBP protein is strictly regulated by the level of intracellular alcohol (cholesterol or oxidized alcohol such as 25-hydroxycholesterol).
  • SCAP binds to the Insig protein on the endoplasmic reticulum to retain the SREBP precursor in the ER, reducing the expression of cellular lipid synthesis genes. Conversely, the active form of SREBP in the nucleus increases, promoting cellular lipid synthesis.
  • SREBP-la SREBP-la
  • - lc SREBP-2
  • SREBP-2 SREBP-2 mainly regulates the expression of genes in the cholesterol synthesis pathway and low-density lipoprotein receptor gene expression.
  • SREBP pathway-specific inhibitors can be used as a potential hypolipidemic substance (Yang, J. et al. (2001). Decreased lipid synthesis in livers of mice with disrupted Site-1 protease. Proc. Natl. Acad. Sci. US A 98, 13607-13612; Matsuda, M. et al. (2001). SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation. Genes Dev. 75, 1206-1216).
  • SCAP SREBP cleavage-activating protein
  • LXR is another class of alcohol-regulated nuclear receptor factors, and its endogenous agonists mainly include 24,25-epoxycholesterol and 25-hydroxycholesterol (Repa, J ⁇ et al. (2000). The role of orphan nuclear Receptors in the regulation Of cholesterol homeostasis. Annu. Rev. Cell Dev. Biol. 16, 459-481). It activates the expression of genes involved in cholesterol efflux in cells including ABCAl, ABCG5/8 and Cyp7al, which has the function of promoting blood lipid levels. Therefore, the use of pharmacological ligands to activate LXR can effectively lower serum cholesterol levels and provide good anti-atherosclerotic effects.
  • LXR also has the effect of activating SREBP-l c, which will eventually lead to liver necrosis and hypertriglyceridemia caused by fatty liver (Schultz J.R. et al. (2000). Role of LXRs in control of lipogenesis. Genes Dev. 14, 2831 -2838).
  • the composition is a drug.
  • R is independently selected from the group consisting of hydrogen, hydroxy, C ⁇ C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 decyloxy, halogen.
  • the compound has the structure shown in Formula III -
  • the metabolic diseases include: type II diabetes, hyperlipidemia, hypercholesterolemia, fatty liver, insulin resistance, obesity, atherosclerosis, coronary heart disease, stroke, myocardial infarction Wait. Replacement page (Article 26)
  • the hyperlipidemia is including, but not limited to, atherosclerosis or type II diabetes.
  • the composition is further used to:
  • SREBP sterol response element binding protein
  • SCAPED Insig protein insulin-induced gene
  • composition is further used to:
  • composition is further used to:
  • HDL-c blood high-density lipoprotein cholesterol
  • a method of preparing a medicament for preventing or treating a metabolic disease comprising: administering an effective amount of a compound having a core structure as shown in Formula I Or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier
  • a method of reducing cellular cholesterol and fatty acid biosynthesis in vitro wherein a compound having a parent core structure as shown in Formula I or a pharmaceutically acceptable salt thereof is administered to a cell:
  • the cell is a liver cell.
  • Figure 1 Screening of compounds that modulate the transcriptional activity of the SRE promoter.
  • Betulin has an effective reduction of luciferase activity in Huh-7/SRE-Luc cells.
  • Huh-7/SRE-Luc cells were treated in a medium containing 10% delipoprotein serum, ⁇ lovastatin and 50 ⁇ M mevalonate for 16 hours, which was effective in up-regulating the endogenous active form of the cells.
  • SREBP-2 The cells were replaced with medium containing 10% delipoprotein serum, ⁇ ⁇ lovastatin, 50 ⁇ M mevalonate and different candidate substances (in which the concentration of different compounds was in the range of 0.5-5 ⁇ ), after 6 hours of culture. Changes in cytoluciferase activity were measured. Substances that are effective in reducing (less than 50%) cytoluciferase activity are potential SREBP pathway inhibitors compared to controls. In the present invention, by testing 2000 candidate substances, it is finally found that the compound white mixed alcohol can effectively reduce
  • Betulin ester specifically inhibits the SREBP pathway.
  • HMGCR and Insigl expression plasmids were co-transfected into CHO cells by FuGENE6 transfection. After 24 hours of transfection, the cells were treated with medium E for 16 hours, and then 25-hydroxycholesterol or betulinic acid was added at different concentrations, and 10 mM mevalonate was added. After treating the cells for 6 hours, the whole protein of the cells was collected. Lysate, protein immunohybridization detects changes in the amount of exogenous HMGCR protein in cells. Immunoblotting was performed using an anti-T7 antibody (anti-HMGCR) or an anti-Myc antibody (anti-Insig-1). The results showed that, unlike 25-hydroxycholesterol, betulinol specifically inhibited the SREBP pathway and had no effect on the stability of HMGCR protein.
  • the density of X 10 5 per 60-mm dish was inoculated in medium C. After 24 hours, the cells were changed to medium B and transfected with FuGENE 6 reagent (Roche). The total transfection amount of plasmid DNA was 3 g per plate of cells. Not enough to use pTK empty or pcDNA3 to fill. After 8 hours of transfection, cells were exchanged for medium and 1% CDX was added. Culture at 37 ° C 1 small one 4 1
  • SREBP-2, SCAP and Insig 1 expression plasmids were co-transfected into SRD-13A cells by FuGENE6 transfection. After transfection for 24 hours, the cells were treated with medium E for 16 hours. After treated with betulin for 6 hours, the protein was co-immunoprecipitated with exogenous Insig protein, and the exogenous SCAP combined with it was detected by protein immunohybridization. The amount of protein changes. The lysate was immunoprecipitated with an anti-Myc antibody; the immunoblot was an anti-SCAP antibody or an anti-Myc antibody (anti-Insig-1). The results showed that betulin can significantly promote the interaction between SCAP and Insig protein, thereby inhibiting the shear maturation of SREBP protein.
  • Betulin ester specifically inhibits the production of SREBP-2 in the nuclear form of the active form.
  • CHO-7 cells cells were first treated with medium E for 16 hours, and cells were treated with betulin and its structural analogs for 6 hours.
  • Cell whole protein lysates were collected using IgG-7D4 (anti-SREBP-2). The antibody was subjected to a protein immunoblot assay and the results showed that the compound betulinol specifically reduced the active form of SREBP-2 in the nucleus.
  • Betulin ester inhibits cellular cholesterol synthesis and reduces cellular cholesterol levels by reducing SREBP in the active form of the nucleus.
  • CHO-7 cells the cells were cultured for 10 days with medium B containing 5% delipoprotein serum and different concentration gradients of betulin, and the cells were fixed in absolute ethanol, and the cells were grown by crystal violet staining. It is fixed and crystal violet stained to show purple. The results showed that: with the increase in the concentration of betulin, the cells died, and the supplementation of exogenous cholesterol allowed the cells to survive, suggesting that betulin esters caused cell death by lowering the level of cellular cholesterol.
  • the compound betulin ester specifically inhibits the expression of genes and enzymes in cellular cholesterol synthesis and fatty acid synthesis pathways, thereby significantly inhibiting cellular lipid synthesis, lowering cellular cholesterol levels and levels of fatty acids and neutral lipids.
  • CRL-1601 cells were treated in a medium containing 10% delipoprotein de-fatty acid serum, 1 ⁇ L lovastatin and a small amount of mevalonate (50 ⁇ ) for 16 hours, followed by different concentration gradients of betulin. After treating the cells for 6 hours, the cellular RNA was collected. The cDNA was obtained by reverse transcription, and the expression regulation of a series of SREBP target genes in the cholesterol synthesis pathway and the fatty acid synthesis pathway was detected by real-time quantitative PCR. The results showed that betulinol significantly reduced the expression of a series of SREBP target genes (AB) in the cholesterol synthesis pathway and fatty acid synthesis pathway.
  • AB SREBP target genes
  • CRL-1601 cell culture was carried out in a medium containing 10% delipoprotein de-fatty acid serum, 1 ⁇ L of lovastatin and a small amount of mevalonate (50 ⁇ M) simultaneously with different concentration gradients of betulin to treat cells 16 After an hour, the cells were replaced with medium A containing 10% delipoprotein de-fatty acid serum, simultaneously with different concentration gradients of the medium of betulin, and a certain concentration of 14 C-labeled acetate was added to the cells as cholesterol.
  • a substrate synthesized from de novo fatty acids traces the synthesis of cholesterol and fatty acids, and collects cells after 2 hours of labeling.
  • the cell cholesterol and fatty acid components were obtained by organic solvent extraction, and the lipid components were separated by thin layer chromatography.
  • Nile-red staining method was used to analyze the effect of betulin on cell neutral lipid levels.
  • CRL-1601 cells cells were first treated with serum-free medium for 16 hours, and cells were exchanged for 5% delipoprotein serum, ⁇ lovastatin, 500 ⁇ M mevalonate medium, and different concentrations. After 12 hours of treatment with Betulin, cells were fixed in 4% PFA and stained with Nile red. The results showed that betulin significantly reduced the level of intracellular neutral lipids.
  • Figure 5 Effect of the compound betulin on the lipid levels of blood and liver adipose tissue in mice at the animal level.
  • mice Eight-week-old C57BL/6 mice were divided into 4 groups, 5-7 in each group, fed with basic feed (Chow), high cholesterol and high fat feed CWD) + normal saline, WD + lovastatin C 30 mg/kg/day. , WD + betulin ester C30mg / kg / day). After 6 weeks, the mice were sacrificed for the next experiment.
  • mice fed the basal diet Compared with the mice fed the basal diet, the body weight of the high cholesterol and high fat mice increased significantly with time. Both lovastatin and betulinic alcohol significantly decreased the body weight gain of the mice.
  • mice Eight-week-old C57BL/6 mice were divided into 4 groups, 5-7 in each group, fed with basic feed (Chow), high cholesterol and high fat feed CWD) + normal saline, WD + lovastatin C 30 mg/kg/day. , WD + betulin ester C30mg / kg / day). After 8 weeks, glucose tolerance and insulin resistance tests were performed. Before the test, the mice were fasted for 12 hours, and the mice were injected with 2 g/kg glucose or 0.75 U/kg insulin by intraperitoneal injection. , blood glucose levels were measured at each time point. L: lovastatin; D: betulin. Three days later, the mice were sacrificed and the blood glucose levels and blood insulin levels were again measured.
  • betulinol significantly improved glucose tolerance in mice.
  • betulinol significantly improved insulin resistance in mice.
  • Betulin significantly reduces blood glucose levels and increases insulin sensitivity.
  • betulin can significantly improve glucose metabolism, increase insulin-sensitive glucose tolerance, and lower blood glucose levels and insulin levels.
  • Figure 7 Expression of compound betulin at the animal level for expression of lipid metabolism genes in liver and adipose tissue of mice.
  • mice Eight-week-old C57BL/6 mice were divided into 4 groups, 5-7 in each group, fed with basic feed (Chow), high cholesterol and high fat feed CWD) + normal saline, WD + lovastatin C 30 mg/kg/day. , WD + betulin ester C30mg / kg / day). After 6 weeks, the mice were sacrificed to isolate liver tissue and white adipose tissue, and RNA was prepared and quantified by fluorescence.
  • PCR quantitative PCR
  • the mRNA levels of genes and enzymes (such as HMGCR, HMGCS, SS, etc.) of the cholesterol synthesis pathway in the betulinol treatment group were down-regulated by an average of 31-65%.
  • the adiponectin, LPL and PPAR- ⁇ genes were up-regulated by 2-3.5 fold in the betulin-treated group, and the up-regulation of these genes was known to have anti-diabetic and anti-inflammatory effects.
  • Figure 8 Effect of detection of betulin on the formation of atherosclerosis in mice at the animal level.
  • mice Eight-week-old LDLR- ⁇ mice were divided into two groups, fed with high cholesterol and high fat diet (WD) + normal saline, WD + betulin (30 mg/kg/day), and the mice were sacrificed 10 weeks later.
  • WD high cholesterol and high fat diet
  • Betulin ester specifically binds to the SCAP protein.
  • Photo-affinity labeling probe based on betulin alcohol synthesis (Compound 1) and in vitro photoaffinity labeling protocol.
  • CHO-7 cells were cultured in conventional medium containing 5% LPDS, 10 ⁇ compactin and P 10 ⁇ mevalonate for 16 hours. After treatment with betulin for 6 hours, cell membrane fractions and nuclear fractions were collected to detect SREBP. -2 shear regulation.
  • Betulin at a significant reduction in blood lipid levels is SCAP protein dependent.
  • C57BL/6J mice were given normal saline, lovastatin (30 mg/kg/day) or betulin C 30 mg/kg/day by gavage, while feeding high-cholesterol and high-fat diets, 4 weeks later.
  • the mice were injected with adenovirus in the tail vein according to the infection amount of 5 X 10 8 pfu per mouse. After that, the mice were sacrificed for 5 days, and the mice were sacrificed. Blood and liver tissues were taken for detection of liver gene expression changes (A) and blood lipid levels (BE).
  • betulinol is a specific inhibitor of the SREBP pathway, which specifically promotes the interaction between SCAP and Insig, inhibits the shear maturation of SREBP proteins, and down-regulates a series of genes for cholesterol, fatty acid, and triglyceride synthesis pathways. And the expression of enzymes, it is therefore known that a class of compounds consistent with the parental structure of betulinic alcohol is useful for the prevention and treatment of metabolic diseases.
  • the present invention provides an effective way to develop a multifunctional drug that targets SREBP.
  • alkyl refers to a straight or branched saturated aliphatic hydrocarbon group containing from 1 to 4 carbon atoms, preferably from 1 to 2 carbon atoms.
  • mercapto groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl.
  • alkenyl as used herein includes both straight-chain and branched hydrocarbon groups containing at least one carbon-carbon double bond and 2-4 carbon atoms, preferably 2-3 carbon atoms.
  • alkynyl as used herein includes both straight-chain and branched hydrocarbon groups containing at least one carbon-carbon triple bond and 2-4 carbon atoms, preferably 2-3 carbon atoms.
  • halogen refers to! ⁇ , Cl, Br, or I.
  • a mercapto group, an alkenyl group, an alkynyl group and the like may or may not have a substituent.
  • they may be one containing 1-3 (more preferably 1-2) substituents selected from (but not limited to):.. CM alkyl, C 2 4 alkenyl, C 2 4 alkynyl group, halogen Replaced by the group.
  • isomer as used herein includes: geometric isomers, enantiomers, diastereomers (e.g., cis isomers, conformational isomers).
  • the present invention first provides a compound having a parent core structure as shown in (I):
  • the present inventors conducted experiments on betulin and its derivatives, and found that CH 2 OH at the 17 position is a key site for inhibiting SREBP. Therefore, other sites can be modified while still retaining their activity.
  • the parent core structure of the formula (I) forms a suitable compound spatial configuration, and the compound having the structure can be targeted by the SREBP pathway, and interacts with the related protein of the SREBP pathway, thereby preventing and controlling the SREBP pathway.
  • the purpose of metabolic diseases That is, the compound having the parent core structure as shown in Structural Formula (I) is an inhibitor of the SREBP pathway.
  • the compound has a structure represented by the formula (II):
  • R is independently selected from the group consisting of: hydrogen, hydroxy, C 1 -C 4 alkyl, C 2 -C 4 alkenyl, C 2 -C 4 alkynyl, C 1 -C 4 decyloxy, halogen.
  • R is independently selected from the group consisting of: hydrogen, hydroxy, C 1 -C 2 fluorenyl, C 2 -C 3 alkenyl, C 2 -C 3 alkynyl.
  • a representation such as " R ⁇ " is well known to those skilled in the art and indicates that the group R can be substituted for any one or more positions that can be substituted on the ring. Also, the choice of R can be different at different substitution locations.
  • the present invention also includes pharmaceutically acceptable salts, hydrates or precursors of the above compounds as long as they also have an effect of preventing metabolic diseases.
  • pharmaceutically acceptable salt refers to a salt formed by reacting a compound with an inorganic acid, an organic acid, an alkali metal or an alkaline earth metal.
  • These salts include, but are not limited to: (1) salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts with organic acids such as acetic acid, oxalic acid, succinic acid, tartaric acid , methanesulfonic acid, maleic acid, or arginine.
  • Other salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, in the form of esters, carbamates, or other conventional "prodrugs".
  • precursor of a compound means a compound which, when taken by a suitable method, is converted into a parent structure having a structure of the formula (I) by undergoing a metabolic or chemical reaction in a patient, or A salt or a solution of a compound of the parent core structure represented by the formula (I).
  • the present invention also includes isomers and racemates of the above compounds as long as they also have an effect of preventing metabolic diseases.
  • the compound has one or more asymmetric centers.
  • these compounds may exist as racemic mixtures, as individual enantiomers, as individual diastereomers, as mixtures of diastereomers, as cis or trans isomers.
  • the compounds of the present invention can be obtained by a variety of methods well known in the art, using known starting materials, such as chemical synthesis or from organisms (such as animals or plants). The method of extraction, which is included in the present invention.
  • the compounds having the parent core structure of formula (I) can also be extracted, isolated and purified from organisms such as animals or plants.
  • the compound of the formula (III) (beiferyl alcohol) is a kind of pentacyclic triterpenoid which is abundant in nature, and can be carried out from birch bark by a simple separation method (separation yield is more than 30%). Extract preparation.
  • the present invention provides the use of a compound having a parent core structure represented by formula (I) or an isomer thereof, a racemate, a pharmaceutically acceptable salt, a hydrate or a precursor thereof. , a medicament (or composition) for preparing a metabolic disease.
  • the metabolic disease refers to a disease caused by abnormal metabolism.
  • the pathological basis of metabolic diseases is the abnormal metabolism of sugar, fat and protein.
  • metabolic diseases are not directly life-threatening, they can cause other life-threatening diseases.
  • the main indicators for determining metabolic diseases are: blood sugar, triglycerides, total cholesterol (TC), unsaturated free fatty acids (NFFA) and uric acid; if the above indicators exceed normal levels, they may have metabolic diseases.
  • Metabolic diseases mainly include: insulin resistance, hyperinsulinemia, diabetes (especially type II diabetes), impaired glucose tolerance, obesity, hypercholesterolemia, abnormal lipid metabolism, arteriosclerosis, coronary heart disease, hypertension, hyperuric acid
  • the main clinical manifestations of blood and gout are abdominal obesity, atherosclerotic dyslipidemia, elevated blood pressure, insulin resistance (with or without impaired glucose tolerance), and embolism and inflammatory response.
  • Atherosclerosis is caused by the deposition of cholesterol and cholesterol esters under the arterial wall. It is the main cause of coronary heart disease and stroke. Lowering blood cholesterol levels is the main method for clinical treatment of such diseases.
  • Abnormal metabolism of fatty acids and triglycerides directly causes insulin resistance, which is a key cause of type 2 diabetes.
  • betulin ester significantly reduced blood and tissue lipid levels in mice, reduced body weight gain, and increased insulin sensitivity.
  • LDLR low-density lipoprotein receptor
  • the mechanism of action of the compound of the present invention is to specifically block the maturation of SREBP and inhibit its activity.
  • the compounds significantly inhibit the expression of lipid synthesis genes such as cholesterol, fatty acids and triglycerides, reduce lipid synthesis, and reduce intracellular lipid content.
  • the compound appears to significantly inhibit the weight gain of the mouse caused by the high-fat diet, reduce the lipid level in tissues such as blood, liver and fat, and increase the sensitivity of the obese mice to insulin. Diabetes has a significant therapeutic effect.
  • the compound can significantly reduce the formation of intra-arterial atherosclerotic plaque induced by a high cholesterol diet, and can effectively increase the stability of plaque, and has a good anti-atherosclerotic effect.
  • the compounds of the invention have similar or even better therapeutic effects than lovastatin.
  • betulin can significantly lower lipid levels in blood and tissues; 2) betulin can inhibit the synthesis of fatty acids and triglycerides, while statins have no such effect. Therefore, betulin can significantly improve insulin sensitivity and have a therapeutic effect on type 2 diabetes; 3) Most diabetic patients have severe atherosclerosis, and betulin has not only improved compared with the currently used diabetes drugs. Insulin sensitivity, and has a significant effect on atherosclerotic plaque.
  • the compounds of the present invention have a remarkable effect on type 2 diabetes and atherosclerosis by a novel mechanism for regulating lipid metabolism.
  • composition of the invention is generally a pharmaceutical composition comprising a compound of the parent core structure of formula (I) or an isomer thereof, a racemate, a pharmaceutically acceptable salt, hydrated Or a precursor as an active ingredient for the prevention and treatment of metabolic diseases; and a pharmaceutically acceptable carrier or excipient.
  • the term "containing” means that the various ingredients can be used together in the mixture or composition of the present invention. Therefore, the terms “consisting mainly of” and “by .. and becoming” are included in the term “contains”.
  • a "pharmaceutically acceptable" ingredient is a substance which is suitable for use in humans and/or animals without excessive adverse side effects (e.g., toxic, irritating, and allergic), i.e., having a reasonable benefit/risk ratio.
  • a "pharmaceutically acceptable carrier” is a compound for use in the present invention having a parent core structure represented by the formula (I) or an isomer thereof, a racemate, a pharmaceutically acceptable salt, hydrated A pharmaceutically or food acceptable solvent, suspending agent or excipient that is delivered to an animal or human.
  • the carrier can be a liquid or a solid.
  • Pharmaceutically acceptable carriers suitable for use in the present invention include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the present invention also provides a method of preparing a composition for controlling a metabolic disease comprising using a compound having a core structure represented by the formula (I).
  • An effective amount of a compound of formula (I) can be combined with a pharmaceutically acceptable carrier to provide a composition of the present invention.
  • the proportion by weight of active ingredient in the composition can be, for example, 0.0001 to 50% by weight; preferably 0.001- 20 wt %.
  • composition of the present invention may also be a traditional Chinese medicine or natural product extract containing the compound having the parent core structure of the formula (I) of the present invention as an active ingredient, and extraction may be carried out by some known methods.
  • the dosage form of the pharmaceutical composition of the present invention may be various, as long as it is a dosage form capable of effectively bringing the active ingredient to the affected part of the mammal.
  • the preferred pharmaceutical composition is an oral or injectable preparation.
  • it may be selected from the group consisting of granules, tablets, capsules, solutions, or suspensions, and powders.
  • a compound having a parent core structure represented by the formula (I) or an isomer, a racemate, a pharmaceutically acceptable salt, a hydrate or a precursor thereof may be present in a suitable solid or liquid carrier or diluent .
  • the conventional compositions or adjuvants required for the preparation of different dosage forms may be added to the compositions of the present invention. , pH buffer substances, etc. These additives are well known to those skilled in the art.
  • the invention also provides a method of controlling a metabolic disease comprising the steps of: administering to a subject in need thereof an effective amount of a compound of formula (I).
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the safe and effective amount of the compound of the present invention is usually from about 0.1 ng to 100 mg/kg body weight; preferably from about 1 ng to 10 mg/kg body weight.
  • specific doses should also take into account factors such as the route of administration, the health of the user, and the like, which are within the skill of the skilled physician.
  • the compounds of the present invention may also be used with other active ingredients or therapeutic agents (e.g., other hypolipidemic drugs, cholesterol lowering drugs, diabetes drugs, etc.).
  • active ingredients or therapeutic agents e.g., other hypolipidemic drugs, cholesterol lowering drugs, diabetes drugs, etc.
  • a new class of inhibitors of the SREBP pathway which inhibits the SREBP pathway, does not activate the LXR signaling pathway, thereby providing prevention and treatment of metabolic diseases such as atherosclerosis and type II diabetes. New approach.
  • the compound of the present invention can be produced by a synthetic method at a low cost.
  • Horseradish peroxidase-conjugated donkey anti-mouse and anti-rabbit IgG obtained from the Jackson Immunological Research Laboratory.
  • Betulinol and its analogues were obtained from Shanghai Pharmaceutical Valley Biotech Co., Ltd., China. The purity of betulin and its structural analogs was determined by gas chromatography to be higher than 99%.
  • Methyl- ⁇ -cyclodextrin Obtained from Cyclodextrin Technologies Development Inc.
  • the luciferase assay kit and cell lysis buffer were obtained from Pr 0 meg a .
  • Delipidated serum (LPDS, d > 1.215 g/ml): Obtained from newborn calf serum by ultracentrifugation. Protein immunohybridization antibody
  • the primary antibody against T7 was obtained from Novagen; the primary antibody against anti-Myc (IgG-9E10) was obtained from Roche;
  • SREBP-l (IgG-H160) was obtained from Santa Cruz; primary antibody against hamster SREBP-2 (IgG-7D4), anti-hamster SCAP The primary antibody (IgG-9D5), the primary antibody against HMGCR (IgG-A9) and the primary antibody against human SREBP-2 (IgG-lD2) were prepared by conventional methods from mouse hybridoma cell line (ATCC); anti-gp78 The anti-GFP and anti-HMGCR polyclonal antibodies were obtained from rabbit antiserum by a conventional method. Plasmid
  • pSRE-Luciferase promoter region comes from
  • the medium A component was Dulbecco's modified Eagle's medium containing 100 units/ml penicillin and 100 g/ml streptomycin.
  • the Medium B component was Dulbecco's modified Eagle's medium mixed in equal proportions with F12 medium containing 100 units/ml penicillin and 100 ⁇ ⁇ / ⁇ 1 streptomycin.
  • the medium C component was medium plus 5% FBS.
  • the medium D component was medium C plus 5 ⁇ ⁇ / ⁇ 1 cholesterol, 1 mM sodium mevalonate, and 20 ⁇ L sodium oleate.
  • the medium component was medium plus 5% LPDS, 1 ⁇ L lovastatin plus 50 ⁇ M sodium mevalonate.
  • CRL-1601 McArdle RH7777 rat liver cancer cells; purchased from ATCC
  • P HuH7 human hepatoma cell line; purchased from ATCC
  • single cell layer at 37 ° C and 5% CO 2 cells grown in medium A (Dulbecco's Modified Eagle's medium containing 100 units/ml penicillin and 100 g/ml streptomycin;) plus 10% FBS.
  • medium A Dulbecco's Modified Eagle's medium containing 100 units/ml penicillin and 100 g/ml streptomycin;
  • CHO-7 (CHO-K1 cells were screened in a lipoprotein serum medium; purchased from ATCC) Single cell layer at 37 ° C and 5% CO 2 , cells were grown in medium B (Dulbecco's improved The Eagle's medium is mixed in equal proportions with the F12 medium, containing 100 units/ml penicillin and 100 ⁇ ⁇ / ⁇ 1 streptomycin plus 5% FBS.
  • SRD-13A SCAP-deficient cell line, from CHO-7; purchased from ATCC
  • Single cell layer Cells were grown in medium D at 37 ° C and 5% CO 2 .
  • the pSRE-Luciferase and pEGFP-Nl were co-transfected into Huh-7 cells at a ratio of 5:1 for 48 hours, then the cells were exchanged for medium A plus 10% FBS, 700 g/ml G418. Thereafter, the solution was changed once every 2-3 days, and an independent monoclonal was formed after 2 weeks. Monoclones were picked and their luciferase expression regulatory features were identified. Finally, a cell line with high expression and regulation was selected and named Huh-7/SRE-L UC .
  • the luciferase assay was carried out using a kit method for luciferase assay, in which the amount of green fluorescent protein was used as an internal reference.
  • Huh-7/SRE-Luc cells were treated in a medium containing delipoprotein serum, statins and a small amount of sodium mevalonate (50 ⁇ M) for 16 hours. This cholesterol removal process It can effectively upregulate the endogenous active form of SREBP.
  • the cells were exchanged for medium containing different candidate substances (in which the concentration of different compounds was in the range of 0.5-5 ⁇ ), and the change in luciferase activity was measured after 6 hours of culture.
  • SRD-13A cells CHO-7 cell-derived SCAP gene-deficient cell line were obtained by somatic cytogenetic methods; CHO-7 cells were obtained from ATCC; SRD-13 sputum cells, see Rawson RB, DeBose- Boyd R, Goldstein JL, Brown MS. Failure to cleave sterol regulatory element-binding proteins (SREBPs) causes cholesterol auxotrophy in Chinese hamster ovary cells with genetic absence of SREBP cleavage-activating protein. J Biol Chem. 1999 Oct l;274 (40 ): 28549-56) Inoculated in medium C at a density of 4 X 10 5 per 60-mm dish.
  • SREBPs sterol regulatory element-binding proteins
  • step 1) the supernatant obtained by centrifugation at 1,000 g is again 10 5 g, centrifuged at 4 ° C for 30 minutes, and then precipitated with 0.1 ml of SDS lysis buffer (10 mMTris-HCl, pH 6.8; 100 mM NaCl). 1% SDS; 1 mM EDTA; I mMEGTA) Resuspend, and shaken at room temperature for 100 minutes to obtain nuclear protein.
  • SDS lysis buffer 10 mMTris-HCl, pH 6.8; 100 mM NaCl.
  • SDS 1 mM EDTA
  • I mMEGTA I mMEGTA
  • CRL-1601 cell culture was carried out in a medium containing 10% delipoprotein de-fatty acid serum, 1 ⁇ L of lovastatin and a small amount of mevalonate (50 ⁇ M) simultaneously with different concentration gradients of betulin to treat cells 16 After an hour, the cells were replaced with medium A containing 10% delipoprotein de-fatty acid serum, with different concentration gradients of the medium of betulin, and 14 C-labeled acetate (6 ⁇ / ml) was added to the cells. As a substrate for de novo synthesis of cholesterol and fatty acids, traces the synthesis of cholesterol and fatty acids in cells, and collects cells 2 hours later.
  • the cell cholesterol and fatty acid components were obtained by organic solvent extraction, and the lipid components were separated by thin layer chromatography. Then, the synthesis of cholesterol and fatty acids under different conditions was analyzed by autoradiography.
  • Filipin staining method and Nile-Red staining method were obtained by organic solvent extraction, and the lipid components were separated by thin layer chromatography. Then, the synthesis of cholesterol and fatty acids under different conditions was analyzed by autoradiography.
  • the fresh Filipin stock solution is 5 mg/ml and the working solution is 0.5 mg/ml, diluted in absolute ethanol.
  • the cells were fixed with 4% paraformaldehyde (diluted in PBS;) for 30 minutes at room temperature, washed three times with PBS, and then treated with 0.5 g/ml working solution for 30 minutes in the dark.
  • Filipin signals were observed with a Zeiss LSM 510 confocal microscope two-photon laser confocal microscope with excitation at 720 nm.
  • the fresh Nile-Red stock concentration was 0.5 mg/ml, the working solution concentration was 0.5 ⁇ ⁇ / ⁇ 1, diluted in PBS containing 10 serum, fixed in 4% paraformaldehyde room temperature, and washed with PBS 3 times. Then, the cells were treated with 0.5 g/ml Nile-Red working solution at 37 ° C for 10 minutes.
  • the Nile-Red signal was observed with a Zeiss LSM 510 confocal microscope with excitation at 523 nm. Fluorescence quantification
  • the average signal intensity of the control cells was defined as 1, and 50 cells were arbitrarily selected as quantitative objects in each experimental group in each experiment. Fluorescence was quantified using Image Pro Plus 5.02 software. Fluorescence real-time quantitative PCR (Q-PCR)
  • cDNA synthesis was performed using Promega reverse transcriptase and buffer.
  • Each dNTP system containing 4 g of the RNA 0.5 ⁇ ⁇ 01igodT (T15Vl), remove the 0.4 mM final concentration of each
  • Realtime PCR uses a 20 ⁇ system. Takara's HS Taq enzyme and buffer were used. Each system contains 0.5 U HS Taq enzyme, final concentration 0.5 X SYBR green, final concentration 1.5 mM MgCl 2 , final concentration of 62.5 ⁇ dNTP, final concentration 0.6 ⁇ primer, 1.5 ⁇ l template cDNA (reverse transcription product diluted 4 times) .
  • reaction was carried out according to the following reaction conditions: a. Pre-denaturation at 94 °C for 5 minutes, b. Denaturation at 94 °C for 30 seconds, c. Annealing at 60 °C for 30 seconds, d. 72 °C for 30 seconds, e. bd cycle 40 times, f. 72 ° C extension for 10 minutes.
  • mice Male C57BL/6J after 8 weeks of birth was obtained from Shanghai Slaccas Laboratory Animal Center. After the mice were randomly divided into cages, the mice were fed a basal diet or a high-fat, high-cholesterol diet (high-fat, high-cholesterol feed consisting of basal feed plus 1.25% cholesterol and 20% lard) of lovastatin and betulin. The dose is 30 mg/kg/day, and the administration method is gavage. The body weight and food intake of the mice were counted weekly. After 6 weeks, the content of mouse adipose tissue was statistically analyzed by NMR (Bmker, Houston) method. The mice were then fasted for 14 hours to take blood and various organ tissues.
  • a basal diet or a high-fat, high-cholesterol diet high-fat, high-cholesterol feed consisting of basal feed plus 1.25% cholesterol and 20% lard
  • the dose is 30 mg/kg/day, and the administration method is gavage.
  • the body weight and food intake of the mice
  • mice were randomly divided into basal diet or high-fat and high-cholesterol diet, and lovastatin or betulin in the same time. 30 mg/kg/day;), 8 weeks after the test of insulin and glucose tolerance.
  • mice LDL receptor-deficient mice 6 weeks after birth were removed (reversely obtained 10 times with C57BL/6J mice, purchased from Jackson Lab). After 8 weeks of feeding, the mice were randomly divided into two groups, 5 in each group. The two groups of mice were fed with normal fat or betulin (30 mg/kg/day) while feeding high-fat and high-cholesterol diet. After 10 weeks, blood and various organ tissues were taken for experiments. Determination of blood and liver lipid levels in mice
  • the liver tissue was homogenized and centrifuged, and the supernatant was taken for determination of total cholesterol and total triglyceride levels.
  • the protein quantification method was Lowry quantitative method CBIO-RAD). Histopathological analysis of mouse liver and adipose tissue
  • the liver tissue of the mice was embedded with Tissue-Tek OCT cryostat molds (Leica), and pre-cooled at -80 °C, and sections of 10- ⁇ thickness were cut out, and the cells were stained with 0.5% oil red 0 and hematoxylin.
  • the adipose tissue of the mice was embedded with Paraffin before sectioning, and 5 ⁇ thickness sections were cut into hematoxylin-eosin stains after placing multiple L-lysine coated slides.
  • the monthly fat cell size was quantified by referring to the Computer-Assisted Morphometric Analysis method. Glucose tolerance, insulin resistance test
  • mice C57BL/6 control mice (purchased from Slaccas Experimental Animal Center, Shanghai, China) were fed with basal diet.
  • the mice in the treatment group were fed with high-fat, high-cholesterol diet and given a certain concentration of normal saline and lovastatin (30 mg/kg/day).
  • Betulin 30 m g / kg / day
  • blood glucose tolerance and insulin resistance were measured after 8 weeks.
  • the mice were fasted for 12 hours.
  • the mice were injected with 2 g/kg glucose or 0.75 U/kg insulin by intraperitoneal injection. After 15, 30, 60 and 120 minutes, the tail tip was taken for blood. Blood glucose levels. After 3 days, check again Mouse blood glucose levels and blood insulin levels were measured. Effect of detection of compound betulin on the formation of atherosclerosis in mice at the animal level
  • LDLR-/- mice Eight-week-old LDLR-/- mice (purchased from Jackson Lab) were divided into two groups. Under the condition of feeding high-fat and high-cholesterol diet, normal saline and betulin (30 mg/kg/day;) were administered, 10 weeks later. The mouse aorta was taken, and after fixation, Sudan IV staining was performed for 6 minutes, and 80% ethanol was counterstained for 3 minutes. At the same time, the tissue block of the aorta near the heart was taken 10 weeks later, and tissue-Tek OCT cryostat molds were embedded and fixed to obtain tissue sections of 10- ⁇ thickness, which were analyzed by 0.5% oil red 0 and hematoxylin staining. Lipid deposition. Macrophage and muscle cell immunohistochemical staining were used to analyze the stability of the plate near the aorta. The final staining results were quantitatively analyzed by computer software. Adenovirus packaging and purification
  • the virus expression vector digested with Pad endonuclease was transfected into cells, and each well was transfected with 4 ⁇ ⁇ plasmid, and cultured at 37 ° C for 24 hours.
  • 293A cells were inoculated into 24-well plates after digestion, inoculated at 5 > ⁇ 10 4 /well, and cultured at 37 °C for 24 hours.
  • the mixture was centrifuged at 12,000 rpm for 20 minutes, the supernatant was discarded, and the precipitate was suspended in 10 ml of a CsCl solution having a density of 1.0 g/ml (solvent: 20 mM Tris-HCl, pH 8.0), 4 ° C, 7000 rpm. Centrifuge for 5 minutes and take the supernatant.
  • the CsCl gradient was prepared as follows: 2.0 ml of a CsCl solution having a density of 1.40 g/ml (solvent as above) was added, then 3.0 ml of a CsCl solution having a density of 1.30 g/ml was added, and 5 ml of the virus suspension was added. Centrifuge at 20,000 rpm for 2 hours at room temperature.
  • the present invention uses adenovirus AdEasy TM expression vector (available from Stratagene) system mediated gene silencing in mouse liver SCAP.
  • AdEasy TM expression vector available from Stratagene
  • the specific practices are as follows:
  • the mouse SCAP shRNA sequence is (SEQ ID NO: 138):
  • the negative control shRNA sequence is (SEQ ID NO: 139):
  • shRNA sequence and its upstream HI promoter sequence were digested with Xhol/Hindlll from the aforementioned vector, and subcloned into the Xhol/Hindlll site of the pShuttle vector (purchased from Stratagene).
  • the adenovirus was packaged in 293A cells and concentrated and purified by cesium chloride density gradient centrifugation.
  • mice are injected with tail vein according to the infection amount of 5 > ⁇ 10 8 pfu per mouse, and the injection volume is preferably not more than 200 ⁇ .
  • mice After 5 days, the mice were sacrificed and blood and liver tissues were collected for subsequent identification experiments.
  • Betulinol is a specific inhibitor of the SREBP pathway
  • the active form of SERBP in the nucleus initiates the expression of downstream genes by binding to the alcohol regulatory element (SRE) of the promoter region of the target gene, thereby promoting lipid biosynthesis (Wang, X. et al. (1993). Nuclear protein that binds sterol regulatory II. Purification and characterization. J. Biol. Chem. 268, 14497-14504).
  • the present inventors constructed a luciferase reporter gene system, the promoter region of which contains a sterol regulatory element (SRE), in human hepatoma cell line Huh-7 The luciferase was stably expressed in the cells, and finally Huh-7/SRE-Luc cells (Fig.
  • the present inventors compared the effects and specificities of betulin and 25-hydroxycholesterol in inhibiting SERBP.
  • 25-hydroxycholesterol has three main effects: inhibition of SREBP-2 cleavage, reduction of active forms in the nucleus
  • SREBP-2 (Fig. 2A); activates the LXR signaling pathway, up-regulates SERBP-1 transcription, constitutively up-regulates SERBP-1 in the nuclear nucleus (Fig. 2A); promotes the degradation of the cholesterol synthesis pathway HMGCR protein (Fig. 2B).
  • betulin can simultaneously inhibit the sequestration of SREBP-1 and SREBP-2 (Fig. 2A), but does not activate the LXR signaling pathway and does not promote HMGCR protein degradation (Fig. 2B).
  • transient transfection was used to detect the shear regulation of betulin in exogenous SREBP-2.
  • Fig. 3A The group is a methanol group (-CH 2 OH) which determines its inhibitory effect on the SREBP pathway.
  • Other compounds are formaldehyde (-CHO), methylhydroxy (-COOH), or methyl (-CH3).
  • protopanaxadiol (20(S)-protopanaxadiol) protopanaxatriol) (20(s)-protopanaxatriol) also did not have the above activity (Fig. 3B;).
  • betulin-specific inhibition of SREBP transcription factor shear maturation inhibition of cellular cholesterol synthesis reduces cellular cholesterol levels, and at certain concentrations, cells die due to lack of cholesterol, and cholesterol can be made to cells after cell supplementation. Therefore, the hydroxyl group at the 17 position is necessary for its activity, and -CHO, -COOH, or -CH 3 no longer have the activity of inhibiting SREBP shearing, and the other two structural analogs are the original ginseng diol, the original ginseng III. Alcohol does not have the above activity.
  • CHO-7 cells were cultured in a medium containing delipoprotein serum under which the cells were unable to obtain exogenous cholesterol, and the cholesterol required for growth was derived from an endogenous synthesis pathway.
  • the cells died as the concentration of the compound was increased (Fig. 3C, upper row;). If a certain amount of exogenous cholesterol is added to the medium while giving betulin, the cells can survive at a higher concentration of betulin ( Figure 3C, lower row), indicating cell death after betulin treatment.
  • SREBP-2 is its own target gene (Sato, R. et al. (1996). Sterol-dependent transcriptional regulation of sterol regulatory element-binding protein-2. J. Biol. Chem. 277, 26461-26464), SERBP The expression of -2 was reduced by about 40% after treatment with betulin. At the same time, the inventors detected dozens of other SREBP-2 target genes involved in the cholesterol synthesis pathway such as HMGCR, HMG-CoA synthetase.
  • HMGCS P Squalene Epoxidase
  • SE P Squalene Epoxidase
  • Fig. 4A the expression levels of genes and enzymes involved in cellular fatty acid synthesis, such as SREBP-l c, fatty acid synthase (FAS), and acetyl-CoA carboxylase a (ACC), were significantly down-regulated by betulin ( Figure 4B).
  • Figure 4B the expression of target genes such as ABCG5 and ABCG8 of LXR was not affected by betulin (Fig. 4C).
  • the isotope tracer assay was used to detect the de novo synthesis of cell cholesterol and fatty acids after treatment with betulin (Figure 4D-E) and the lipid staining assay for the analysis of cellular and neutral lipids (mainly Cholesterol esters and triglycerides; changes in the amount of Figure 4F-I).
  • the results showed that the de novo synthesis of cell cholesterol and fatty acids was significantly inhibited after betulin treatment, and the cell lipid level was significantly reduced compared with the control group.
  • this result confirms that betulin can inhibit the SERBP pathway, down-regulate cholesterol and fatty acid biosynthesis, and reduce cellular lipid levels.
  • Betulinol reverses diet-induced weight gain in obese mice and improves lipid composition in blood, liver and adipose tissue of mice
  • Lovastatin is known to be a specific inhibitor of HMGCR, and its efficacy against metabolic diseases has been widely accepted.
  • the inventors compared the biological effects of betulin and lovastatin.
  • Eight-week-old C57BL/6J mice were randomly divided into 4 groups, of which group 1 mice were fed a basal diet (Chow), groups 2-4 were fed a high-fat, high-cholesterol diet (WD), and the first group was group 2
  • the mice were orally administered with normal saline, and the third group of 4 mice were intragastrically administered with lovastatin (30 mg/kg/day) and betulin (30 mg/kg/day).
  • Weight (g) 26.9 ⁇ 1.6 30.9 ⁇ 1.3 27.6 ⁇ 1.1 * 27.4 ⁇ 1.3 * Weight gain (g) 4.3 ⁇ 0.5 8.3 ⁇ 0.6 5.1 ⁇ 0.5 * 4.9 ⁇ 0.4**
  • Liver weight (g) 1.04 ⁇ 0.17 1.23 ⁇ 0.09 1.29 ⁇ 0.07 1.34 ⁇ 0.12 Spleen weight (g) 0.08 ⁇ 0.02 0.09 ⁇ 0.01 0.10 ⁇ 0.02 0.08 ⁇ 0.01
  • Heart weight (g) 0.12 ⁇ 0.02 0.12 ⁇ 0.02 0.12 ⁇ 0.01 0.12 ⁇ 0.01
  • Kidney weight (g) 0.15 ⁇ 0.02 0.16 ⁇ 0.01 0.17 ⁇ 0.02 0.16 ⁇ 0.01
  • Brain weight (g) 0.42 ⁇ 0.01 0.42 ⁇ 0.02 0.39 ⁇ 0.03 0.38 ⁇ 0.06
  • the ratio of adipose tissue to muscle tissue and the ratio of adipose tissue to body weight were analyzed by NMR spectrophotometer. The results showed that these two ratios were down-regulated after betulin treatment compared with the control group. -40% (Fig. 5C-D).
  • betulin ester significantly down-regulated fatty acid metabolism-related genes such as SREBP-1c was down-regulated by about 32%
  • Brown adipose tissue plays an important role in energy metabolism.
  • Betulin has a significant role in promoting the expression of genes such as Adiponectin, LPL and PPAR- ⁇ in brown adipose tissue (Fig. 7E), which are known to participate in a series of important biological processes. Their up-regulation has anti-diabetic and anti-inflammatory effects (Havel, PJ (2002). Control of energy homeostasis and insulin action by adipocyte hormones: leptin, acylation stimulating protein, and adiponectin. Curr. Opin. Lipidol. 13, 51 -59 Tontonoz, P. et al. (2008). Fat and beyond: the diverse biology of PP ARgamma. Annu. Rev.
  • betulin ester regulates the expression of a series of SREBP target genes in mouse liver and adipose tissue, thereby reducing the body's lipid level and increasing insulin sensitivity.
  • Example 6 Betulin has an effective effect on reducing atherosclerotic plaque formation and increasing plaque stability.
  • LDLR knockout mice Ishibashi, S. et al. (1993). Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J. Clin. Invest 92, 883-893) Effect of betulin on the formation of atherosclerotic plaque.
  • the results showed that the number and size of plaques in the aorta were significantly lower than those in the control group after betulin treatment (Fig. 8A-D). It should be noted that the plaque area at the aortic arch and thoracic artery was significantly reduced by approximately 43% and 77%, respectively, after treatment with betulinol compared to the control group (Fig. 8C-D).
  • the present inventors examined the distribution and area of the marker molecule MOMA-2 of the macrophage at the plaque and the marker molecule SMC-actin of the smooth muscle cell by immunohistochemical method, and the results showed that: compared with the control group, betulin The accumulation of macrophage plaques after alcohol treatment was significantly reduced by about 55% (Fig. 8F, H), and the amount of smooth muscle cells was significantly increased by about 21% (Fig. 8F, 1). It indicated that in LDLR knockout mice, under high cholesterol and high fat feeding conditions, betulin can significantly inhibit the formation of atherosclerotic plaque and effectively increase the stability of plaque.
  • Example 7 Specific binding of betulin to SCAP
  • the present inventors presume that the direct target protein of betulin is a SCAP protein. To verify this hypothesis, the inventors synthesized a small molecule probe based on the structural characteristics of betulin, and named it Compound 1 (Fig. 9A).
  • Compound 1 photoaffinity probe, obtained from East China Normal University
  • the protein sample can be collected, and the azide-modified reporter group can be linked by Click Chemistry for target detection, enrichment and identification.
  • Compound 1 has a biological activity similar to that of betulin (Fig. 9B).
  • the compound is covalently bound to its target protein by UV irradiation after incubation with the cell membrane protein. Subsequent click cycloaddition reactions are then carried out, and finally the target protein is fully enriched by the biotin-avidin reaction.
  • the present inventors used the adenovirus-mediated RNA interference assay to silence the expression of liver gene expression in mice and detect the liver gene expression and mouse blood. Changes in lipid levels. Adenovirus-mediated SCAP shRNA expression in the liver of mice, the expression level of liver SCAP gene was down-regulated by more than 50% (Fig. 10A), indicating that gene silencing effect is good. At the same time, consistent with the previous results, the expression of lipid synthesis pathways such as SREBP-2, HMGCS and SS was significantly down-regulated in the shRNA control group before and after treatment with betulin.
  • the SCAP shRNA treatment group was unable to enter the nucleus due to SREBP.
  • the horizontal composition was down-regulated, and no further down-regulation was observed after the treatment with betulin (Fig. 10A).
  • the results of TC, TG, LDL-c and HDL-c in the blood of mice showed that the changes of blood lipid levels before and after treatment with shRNA control group (Ad-shControl) were consistent with the previous results, while the SCAP shRNA treatment group (Ad-shSCAP) There was no significant effect after treatment with betulin (Figure 10B-E).
  • betulin has an effect of lowering lipid levels by inhibiting the SCAP-SREBP pathway in animals. Discussion
  • SREBP protein is a key transcription factor that regulates the biosynthesis of cholesterol fatty acids and other lipids.
  • the present inventors constructed a cell biology assay method for high-throughput screening of small molecules that specifically regulate the SREBP pathway and identified betulin as a specific inhibitor of the SREBP pathway.
  • Betulin has retained SREBP in the endoplasmic reticulum by promoting the interaction between SCAP and Insig, thereby inhibiting SREBP cleavage into the nucleus, down-regulating the expression of genes and enzymes in cholesterol and fatty acid synthesis pathways, inhibiting cellular lipid synthesis pathways, and reducing cells. Lipid levels. In diet-induced obese mice, betulin can reduce total cholesterol and total triglyceride levels in animals and increase insulin sensitivity.
  • betulinol is effective against atherosclerosis in atherosclerotic model mouse LDLR knockout mice.
  • betulin As a SREBP inhibitor, betulin has a good multi-faceted effect at the body level, which demonstrates that the SREBP pathway can serve as a potential drug target for metabolic diseases. Especially needle For type 2 diabetes and atherosclerosis.
  • betulin The results of screening and identification of betulin have confirmed the effectiveness of this system constructed by the inventors. More interestingly, the mechanism of action of betulin is to promote the interaction between SCAP and Insig, which is the regulation mechanism of endogenous regulators such as cholesterol or 25-hydroxycholesterol.
  • 25-hydroxycholesterol is one of the most potent of the various known SREBP inhibitors, and its potency is more than 100 times that of cholesterol, and the betulinol identified by the present inventors here is equivalent in efficacy to 25-hydroxycholesterol.
  • betulin ester does not activate the LXR signaling pathway and does not promote HMGCR degradation. Its high potency and high specificity make it significantly reduce cholesterol and fatty acids, significantly improve the body's lipid metabolism, and effectively fight atherosclerosis and type 2 diabetes.
  • Statins are currently the first-line drugs widely used to treat high cholesterol. As an inhibitor of HMGCR, statins effectively inhibit cholesterol synthesis. At the same time, inhibition of cholesterol synthesis in the liver will up-regulate the expression of SREBP, and up-regulate the expression of LDLR to promote the absorption of LDL. Through these two mechanisms, statins effectively lower serum cholesterol levels. However, statin activation of SREBP expression will result in poor expression of genes and enzymes in the liver cholesterol and fatty acid synthesis pathways (Kita, T. et al. (1980). feedback regulation of
  • betulin ester Under the conditions of administration of equal doses of lovastatin and betulin, the betulin ester showed comparable or even better effects to statins.
  • betulinol reduces lipid levels in mouse blood, liver, brown adipose tissue, and white adipose tissue better than lovastatin.
  • betulin has a better effect than lovastatin in improving insulin resistance. The reason is that betulin has an effective inhibition of the synthesis of fatty acid triglycerides while inhibiting cholesterol synthesis.
  • betulinol is a specific inhibitor of the SREBP pathway, which can significantly reduce lipid levels, increase insulin sensitivity, and resist the formation of atherosclerosis.
  • the above experimental data supports a view: Inhibition of the SREBP pathway is a beneficial strategy for type 2 diabetes and atherosclerosis.
  • Betulinol can be used as a potential drug for the treatment or control of metabolic diseases.

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Description

一种防治代谢性疾病的化合物及其用途
技术领域
本发明属于药物学领域； 更具体地， 本发明涉及一种防治高脂血症、动脉粥样硬化症 和 II型糖尿病等代谢性疾病的化合物及其用途。 背景技术
代谢性疾病如动脉粥样硬化症， II型糖尿病等已经成为越来越严重的世界性健康难 题， 已知其发生与高脂血症有密切关系。例如血液中胆固醇水平过高引起高胆固醇血症， 进而诱发动脉粥样硬化症， 动脉粥样硬化症又是引起冠心病、 中风、 心肌梗塞等心血管 疾病的最常见原因； 血液脂肪酸和甘油三酯水平过高将会引起高甘油三酯血症， 后者是 诱发胰岛素抵抗及 II型糖尿病的主要原因。 由于高血脂的致病性， 在代谢性疾病的治疗 与预防方案中都把降脂作为重要组成。
已知哺乳动物调控胆固醇和脂肪酸合成的关键因子是一类转录因子蛋白： 醇反应 元件结合蛋白（SREBP)(Goldstein,J.L.等（2006). Protein sensors for membrane sterols. Cell 124, 35-46)。这一类蛋白质的前体首先在内质网 (ER)上合成， 前体通过 SREBP切割激活 蛋白（SCAP)转运到高尔基体， 然后经过两种蛋白酶 (site- 1 protease (S IP)和 site-2 protease (S2P))酶切， 释放其 N端的活性结构域， 进入细胞核发挥转录因子作用， 与靶基因启动 子区的 SREBP反应元件 (SRE)结合， 启动下游基因的表达。 SREBP蛋白的剪切成熟严格 受细胞内 醇 (胆固醇或氧化型 醇如 25-羟胆固醇)水平的调控。 细胞内 醇水平过高 时， SCAP与内质网上的 Insig蛋白结合将 SREBP前体滞留在 ER， 降低细胞脂质合成基 因表达。 反之， 核内活性形式的 SREBP增多， 促进细胞脂质合成。
已知哺乳动物细胞有三种不同形式的 SREBP : SREBP- l a，- l c和 SREBP -2(Horton,J.D. 等 (2002). SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J. Clin. Invest 109, 1 125- 1 13 1) , 它们具有相同的剪切入核调控特征， 其成熟 过程均如上所述。 三种亚型的区别在于： 它们靶基因不同， SREBP- l a， - l c主要调控脂 肪酸合成途径的基因的表达， SREBP-2主要调控胆固醇合成途径的基因的表达以及低密 度脂蛋白受体基因表达。 肝脏特异性敲除 SCAP或者 S 1P基因的小鼠， 其 SREBP剪切 成熟过程受到抑制， 进而表现为其脂质合成显著下调， 血液胆固醇和甘油三酯水平明显 降低。 提示 SERBP途径特异性抑制剂可作为一种潜在的降血脂物质 (Yang,J.等 (2001). Decreased lipid synthesis in livers of mice with disrupted Site- 1 protease gene. Proc. Natl. Acad. Sci. U. S. A 98, 13607- 13612； Matsuda,M.等 (2001). SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation. Genes Dev. 75, 1206- 1216)。
LXR是另一类受 醇调控的核受体因子， 其内源性激动剂主要包括 24， 25-环氧胆固 醇禾口 25-羟胆固醇（Repa, J丄等（2000). The role of orphan nuclear receptors in the regulation of cholesterol homeostasis. Annu. Rev. Cell Dev. Biol. 16, 459-481)。 它能够活化细胞内包 括 ABCAl , ABCG5/8 及 Cyp7al在内的参与胆固醇外排的基因的表达， '这」 H使其 具有促进血液脂质水平下降的功能。 因此， 釆用药理学配体活化 LXR能够有效降低血 清胆固醇水平起到良好的抗动脉粥样硬化作用。 然而另一方面， LXR还具有激活 SREBP-l c的作用， 最终将导致脂肪肝引起的肝坏死及高甘油三酯血症 (SchultzJ.R.等 (2000). Role of LXRs in control of lipogenesis. Genes Dev. 14, 2831 -2838)。
目前已知的细胞内源性 SREBP抑制剂如 24-羟胆固醇， 25-羟胆固醇 (25-HC)和 27- 羟胆固醇， 它们也是 LXR的激动剂， 这类小分子无法作为针对高血脂相关疾病的潜在 药物组成。因此，需要寻找新的 SERBP途径的特异性抑制剂，使其特异性抑制 SERBP- 1 , SREBP-2活化， 同时不激活 LXR信号通路。 这将为高脂血症、 动脉粥样硬化症和 II型 糖尿病等代谢性疾病的预防和治疗提供新的途径。 发明内容
本发明的目的在于提供一种防治高脂血症、 动脉粥样硬化症和 II型糖尿病等代谢性 疾病的化合物及其用途。
在本发明的第一方面， 提供一种具有如式 I所示的母核结构的化合物或其药学上可 接受的盐在制备预防或治疗代谢 用途：
Figure imgf000003_0001
；
在一个优选例中， 所述的组合物是药物。
在另一优选例中， 所述的 结构,
Figure imgf000003_0002
其中， R独立地选自： 氢、 羟基、 C^C4烷基、 C2-C4链烯基、 C2-C4链炔基、 C 1 -C4 垸氧基、 卤素。
在另一优选例中 所述的化合物具有式 III所示的结构-
Figure imgf000003_0003
在另一优选例中， 所述的代谢性疾病包括： II型糖尿病、 高脂血症、 高胆固醇症、 脂肪 肝、 胰岛素抵抗、 肥胖症、 动脉粥样硬化症、 冠心病、 中风、 心肌梗塞等。 替换页 （细则第 26条) 在另一优选例中， 所述高脂血症是包括但不仅限于动脉粥样硬化症或 II型糖尿病。 在另一优选例中， 所述的组合物还用于：
特异性的抑制甾醇反应元件结合蛋白 (SREBP)途径； 或
促进 SREBP切割激活蛋白（SCAPED Insig蛋白 (胰岛素诱导基因）的相互作用。
在另一优选例中， 所述的组合物还用于：
下调甾醇反应元件结合蛋白 -l(SREBP-l), 甾醇反应元件结合蛋白 -2(SREBP-2)， β- 羟 [基] -β-甲 [基]戊二酸单酰辅酶 Α还原酶 (HMGCR)，P-羟 [基] -β-甲 [基]戊二酸单酰辅酶 A 合成酶 (HMGCS), 鲨烯环氧酶（SE)， SC4MOL(sterol-C4-methyl oxidase-like) , 24-脱氢胆 固醇还原酶 (DHCR24), FPPS , 7-脱氢胆固醇还原酶 (DHCR7), 甲羟戊酸激酶 (ΜνΚ)， 羊 毛固醇合成酶 (LSS)， FDFTl (farnesyl-diphosphate farnesyltransferase 1), 低密度脂蛋白受 体 (LDLR), 胰岛素诱导基因 (Insig), 鲨烯合成酶 (SS)， 脂肪酸合成酶 (FAS)， ATP-柠檬 酸裂解酶 (ACL), 硬脂酰辅酶 A去饱和酶 -l(SCD-l), 硬脂酰辅酶 A去饱和酶 -2(SCD-2)， 甘油 -3-磷酸转酰酶 (GP AT), 乙酰辅酶 A羧化酶 a (ACC)， 脂肪酸去饱和酶 -l (FADS-l) 或脂肪酸去饱和酶 -2(FADS-2)的表达。
在另一优选例中， 所述的组合物还用于：
下调胆固醇、 甘油三酯或脂肪酸生物合成；
降低细胞脂质水平；
降低血液和肝脏的总胆固醇和总甘油三酯水平；
降低血液低密度脂蛋白胆固醇 (LDL-c)含量， 升高血液高密度脂蛋白胆固醇 (HDL-c) 含
减小白色脂肪组织和褐色脂肪组织中脂肪细胞的大小；
改善胰岛素抵抗， 减低血糖， 或提高胰岛素敏感性；
减少动脉粥样硬化斑块面积； 或
增加动脉粥样硬化斑块稳定性。
在本发明的另一方面， 提供一种制备药物的方法， 所述的药物用于预防或治疗代谢 性疾病， 所述方法包括： 将有效量的具有如式 I所示的母核结构的化合物或其药学上可 接受的盐与药学上可接受的载体
Figure imgf000004_0001
在本发明的另一方面， 提供一种体外降低细胞胆固醇和脂肪酸生物合成的方法， 对 细胞施用具有如式 I所示的母核结构的化合物或其药学上可接受的盐：
替换页 （细则第 26条)
Figure imgf000005_0001
。
在一个优选例中， 所述细胞是肝脏细胞。
本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易见的 附图说明
图 1. 筛选调节 SRE启动子转录活性的化合物。
A. 表达质粒 pSRE-Luciferase的部分结构示意图。
B. 白桦酯醇能有效降低 Huh-7/SRE-Luc细胞的荧光素酶活性。 Huh-7/SRE-Luc细胞 在含有 10%去脂蛋白血清， Ι μΜ洛伐他汀及 50μΜ甲羟戊酸的培养基 Α中处理 16小时， 这一过程能够有效上调细胞内源性活性形式的 SREBP-2。再给细胞换成含有 10%去脂蛋 白血清， Ι μΜ洛伐他汀， 50μΜ甲羟戊酸及不同候选物质的培养基 Α (其中不同化合物作 用浓度在 0.5-5μΜ范围内）， 培养 6小时后测定细胞荧光素酶活性变化。 与对照相比， 能 够有效降低 (低于 50%)细胞荧光素酶活性的物质即为潜在的 SREBP途径抑制剂。本发明 中， 通过对 2000种候选物质进行检测， 最终发现化合物白拌酯醇能有效降低
Huh-7/SRE-Luc细胞的荧光素酶活性。
图 2. 白桦酯醇特异性抑制 SREBP途径。
Α.白桦酯醇对内源性 SREBP蛋白剪切的影响。 CRL-1601细胞在含有 10%去脂蛋白 去脂肪酸血清， 1 μΜ洛伐他汀及微少量的甲羟戊酸 (50 μΜ)的培养基 Α中处理 16小时， 再加入不同浓度梯度的白桦酯醇或 1 g/ml 25-HC处理细胞 6小时后， 收集细胞的全蛋 白裂解液， 蛋白免疫杂交检测细胞内源性 SREBP-1， SREBP-2蛋白的剪切调控的变化。 结果显示： 白桦酯醇能够显著减少细胞核内活性形式的内源性 SREBP-l(n-SERBP-l), SREBP-2蛋白（n-SERBP-2)。
B.白桦酯醇对 HMGCR蛋白稳定性的影响。 采用 FuGENE6转染方法向 CHO细胞中 共转染 HMGCR、 Insigl表达质粒。 转染 24小时后给细胞换培养基 E处理 16小时， 再 加入不同浓度梯度的 25-羟胆固醇或者白桦酯醇同时均加入 10 mM甲羟戊酸， 处理细胞 6小时后， 收集细胞的全蛋白裂解液， 蛋白免疫杂交检测细胞外源性 HMGCR蛋白量的 变化。 免疫印迹采用抗 -T7抗体 (抗 HMGCR)或抗 -Myc抗体 (抗 Insig-1)。 结果显示： 与 25羟胆固醇不同， 白桦酯醇特异性抑制 SREBP途径， 对 HMGCR蛋白的稳定性没有影 响。
C.白桦酯醇对外源性 SREBP蛋白剪切的影响。 转染前一天将 SRD-13A细胞按照 4
X 105 每 60-mm 培养皿的密度接种在培养基 C中。 24小时后给细胞换培养基 B并采用 FuGENE 6 试剂 (Roche)进行转染。 质粒 DNA总转染量是 3 g每盘细胞。 不足用 pTK 空载或 pcDNA3补平。 转染 8小时后给细胞换培养基 Ε再加上 1% CDX。 37°C培养 1小 一 4一
替换页 （细则第 26条） 时， PBS洗细胞两次， 换培养基 E再加上 醇或者白桦酯醇， 对照细胞加 DMSO (所有 细胞中 DMSO终浓度为 0. 1%)。 换液 6小时后， PBS洗细胞并收集细胞蛋白， 蛋白免疫 杂交检测细胞外源性 SREBP- 1 , SREBP-2蛋白的剪切调控的变化。 免疫印迹采用抗 -SREBP-2抗体、 抗 SCAP抗体或抗 -Myc抗体 (抗 Insig- 1)。 结果显示： 白桦酯醇能够显 著减少细胞核内活性形式的外源性 SREBP-2蛋白， 且这一抑制效果是 Insig蛋白依赖性 的。
D.白桦酯醇抑制 SREBP途径的作用机制。采用 FuGENE6转染方法向 SRD- 13A细胞 中共转染 SREBP-2、 SCAP、 Insig 1表达质粒。 转染 24小时后给细胞换培养基 E处理 16 小时， 再加入白桦酯醇处理细胞 6小时后， 蛋白免疫共沉淀外源性 Insig蛋白， 再通过 蛋白免疫杂交检测与之结合的外源性 SCAP蛋白量的变化。裂解物用抗 -Myc抗体免疫沉 淀； 免疫印迹采用抗 SCAP抗体或抗 -Myc抗体 (抗 Insig- 1)。 结果显示： 白桦酯醇能够显 著促进 SCAP与 Insig蛋白相互作用， 从而抑制细胞 SREBP蛋白剪切成熟。
图 3. 对化合物白桦酯醇构效关系的研究。
A. 白桦酯醇及其类似物的结构。
B. 白桦酯醇特异性抑制细胞核内活性形式的 SREBP-2的产生。 在 CHO-7细胞中， 首先利用培养基 E处理细胞 16小时， 再采用白桦酯醇及其结构类似物处理细胞 6小时 后， 收集细胞全蛋白裂解液， 用 IgG-7D4 (抗 -SREBP-2抗体)进行蛋白免疫杂交实验， 结 果显示： 化合物白桦酯醇特异性减少细胞核内活性形式的 SREBP-2。
C. 白桦酯醇通过减少细胞核内活性形式的 SREBP而抑制细胞胆固醇合成， 降低细 胞胆固醇水平。 CHO-7细胞中， 用含有 5%去脂蛋白血清和不同浓度梯度的白桦酯醇的 培养基 B培养细胞 10天后， 无水乙醇固定细胞， 结晶紫染色检测细胞生长情况， 正常 生长的细胞可以被固定并结晶紫染色后显示紫色。 结果显示： 随着白桦酯醇处理浓度的 升高， 细胞死亡， 补充了外源胆固醇能够使细胞存活， 提示白桦酯醇通过降低细胞胆固 醇水平使细胞死亡。
图 4. 化合物白桦酯醇特异性抑制细胞胆固醇合成及脂肪酸合成途径的基因和酶类 的表达，从而显著抑制细胞脂质合成， 降低细胞胆固醇水平和脂肪酸及中性脂质的水平。
A-C.白桦酯醇对胆固醇合成途径及脂肪酸合成途径中一系列基因的表达调控。
CRL- 1601细胞在含有 10%去脂蛋白去脂肪酸血清， 1 μΜ洛伐他汀及微少量的甲羟戊酸 (50 μΜ)的培养基 Α中处理 16小时，再加入不同浓度梯度的白桦酯醇处理细胞 6小时后， 收集细胞 RNA。 逆转录得到 cDNA， 通过荧光实时定量 PCR方法检测胆固醇合成途径 及脂肪酸合成途径中一系列 SREBP靶基因的表达调控。 结果显示： 白桦酯醇能够显著 减少胆固醇合成途径及脂肪酸合成途径中一系列 SREBP靶基因的表达 (A-B)。 采用同样 的方法， 还分析了白桦酯醇对 LXR信号通路基因的表达调控。 结果显示： 白桦酯醇特 异性减少胆固醇合成途径及脂肪酸合成途径中一系列 SREBP靶基因的表达， 但是对于 对 LXR信号通路基因的表达基本上没有影响， 说明其调控具有特异性 (C)。 D-E. 白桦酯醇对细胞胆固醇合成及脂肪酸合成过程的调控。 CRL-1601细胞培养在 含有 10%去脂蛋白去脂肪酸血清， 1 μΜ洛伐他汀及微少量的甲羟戊酸 (50 μΜ)的培养基 Α中同时加入不同浓度梯度的白桦酯醇处理细胞 16小时后， 给细胞换成含有 10%去脂 蛋白去脂肪酸血清的培养基 A,同时不同浓度梯度的白桦酯醇的培养基，并向细胞中加入 一定浓度的 14C标记的乙酸盐作为胆固醇和脂肪酸从头合成的底物， 示踪细胞胆固醇和 脂肪酸的合成， 标记 2个小时后收集细胞。 采用有机溶剂抽提的方法获得细胞胆固醇和 脂肪酸组分， 通过薄层层析方法分离各个脂质组分， 然后利用放射自显影方法分析不同 条件下细胞胆固醇和脂肪酸的合成情况。 结果显示： 随着白桦酯醇处理浓度的增加， 细 胞胆固醇和脂肪酸的从头被显著抑制。 而他汀仅能够抑制胆固醇合成， 不能够抑制脂肪 酸合成。
F、 H. Fillipin染色方法分析白桦酯醇对细胞胆固醇水平的影响。在 CRL-1601细胞中， 首先利用含有 5%去脂蛋白血清 Ι μΜ 洛伐他汀， 50 μΜ 甲羟戊酸的培养基 Α处理细胞 16小时， 给细胞换含有 5%去脂蛋白血清， 500 μΜ 甲羟戊酸的培养基 Α， 同时采用不 同浓度的白桦酯醇处理细胞 12小时后， 4%PFA固定细胞，进行 Fillipin染色，结果显示： 白桦酯醇显著降低细胞内胆固醇水平。
G、 I. Nile-red 染色方法分析白桦酯醇对细胞中性脂质水平的影响。 在 CRL-1601细 胞中， 首先利用无血清培养基处理细胞 16小时， 给细胞换含有 5%去脂蛋白血清， Ι μΜ 洛伐他汀， 500 μΜ 甲羟戊酸的培养基 Α， 同时采用不同浓度的白桦酯醇处理细胞 12小 时后， 4%PFA固定细胞， 进行 Nile red染色， 结果显示： 白桦酯醇显著降低细胞内中性 脂质的水平。
图 5. 在动物水平上检测化合物白桦酯醇对小鼠血液及肝脏脂肪组织脂质水平的影 响。
8周龄的 C57BL/6小鼠分为 4组， 每组 5-7只， 分别喂养基础饲料 (Chow)、 高胆固 醇高脂肪饲料 CWD)+生理盐水、 WD+洛伐他汀 C30mg/kg/天)、 WD+白桦酯醇 C30mg/kg/天）。 6周后处死小鼠进行下一步实验。
A. 各处理组中小鼠的食物摄入量没有明显异常。
B. 与喂养基础饲料的小鼠相比， 高胆固醇高脂肪组小鼠的体重随时间有显著上升趋 势， 洛伐他汀和白桦酯醇均能显著降低小鼠的体重增加值。
C D. 核磁共振 (MRI)分析小鼠脂肪组织与肌肉组织的百分比值变化。 结果显示： 与 基础饲料组相比， 高胆固醇高脂肪加生理盐水的处理组小鼠脂肪组织与肌肉组织百分比 值显著增加而洛伐他汀和白桦酯醇均能显著降低小鼠的这一比值的增加 (C)，而且小鼠脂 肪组织占总体重的百分比也呈现同样变化趋势即： 与对照组相比， 高胆固醇高脂肪加生 理盐水的处理组小鼠脂肪组织占总体重的百分比显著增加， 而洛伐他汀和白桦酯醇均能 显著降低小鼠的脂肪组织占总体重比例的增加值 (D)。
E-H. 分析小鼠血液总胆固醇 (TC；)、 总甘油三酯 (TG；)、 LDL胆固醇 (LDL-c)和 HDL- 胆固醇 (HDL-c)水平的变化。 结果显示： 白桦酯醇显著降低血液 TC、 TG、 LDL-c, 并显 著增加 HDL-c水平。
I-J. 分析小鼠肝脏总胆固醇、 总甘油三酯水平的变化。 结果显示： 白桦酯醇显著降 低高胆固醇高脂肪喂养条件下小鼠肝脏脂质水平。
K. 对小鼠白色脂肪组织 (WAT)、 褐色脂肪组织 (BAT)切片进行 HE染色。 肝脏组织 切片用油红 0染色。 结果显示： 与对照组相比， 高胆固醇高脂肪加生理盐水的处理组小 鼠脂肪细胞显著增生肥大， 而洛伐他汀和白桦酯醇均能显著抑制其细胞增生和肥大。
图 6. 在动物水平上检测化合物白桦酯醇对小鼠胰岛素抵抗的作用效果。
8周龄的 C57BL/6小鼠分为 4组， 每组 5-7只， 分别喂养基础饲料 (Chow)、 高胆固 醇高脂肪饲料 CWD)+生理盐水、 WD+洛伐他汀 C30mg/kg/天)、 WD+白桦酯醇 C30mg/kg/天）。 8周后， 进行葡萄糖耐受及胰岛素耐受实验， 测试前， 小鼠禁食 12小时， 采取腹腔注射 方式给小鼠注射 2g/kg葡萄糖或者 0.75U/kg胰岛素， 不同时间点尾尖取血， 检测各时间 点血液葡萄糖水平。 L: 洛伐他汀； D: 白桦酯醇。 3天后， 处死小鼠并再次检测小鼠血 液葡萄糖水平和血液胰岛素水平。
A. 与生理盐水组相比， 白桦酯醇显著改善小鼠葡萄糖耐受。
B. 根据 (A)结果， 统计曲线下面积。
C. 与生理盐水组相比， 白桦酯醇显著改善小鼠胰岛素耐受。
D. 根据 (C)结果， 统计曲线下面积。
E-F. 白桦酯醇显著降低血液葡萄糖水平并提高胰岛素敏感性。
糖耐量这些结果显示： 白桦酯醇能显著改善糖代谢， 增加胰岛素敏感性糖耐量、 降 低血液中的葡萄糖水平和胰岛素水平。
图 7. 在动物水平上检测化合物白桦酯醇对小鼠肝脏及脂肪组织脂质代谢基因的表 达调控。
8周龄的 C57BL/6小鼠分为 4组， 每组 5-7只， 分别喂养基础饲料 (Chow)、 高胆固 醇高脂肪饲料 CWD)+生理盐水、 WD+洛伐他汀 C30mg/kg/天)、 WD+白桦酯醇 C30mg/kg/天）。 6周后处死小鼠分离肝脏组织和白色脂肪组织， 制备 RNA， 采用荧光实时定量
PCR(Q-PCR)方法统计分析小鼠肝脏胆固醇代谢、 脂肪酸代谢相关基因、 载脂蛋白类基 因、 糖代谢相关基因及白色脂肪组织中能量代谢相关基因的表达变化。
A.与对照组相比， 白桦酯醇处理组胆固醇合成途径的基因和酶类 (如 HMGCR, HMGCS , SS等;)的 mRNA水平平均下调 31-65%。
B.与对照组相比， 白桦酯醇处理组脂肪酸合成途径的基因和酶类， 如 SREBP-lc 下 调约 32%， FAS 约 39%， SCD-1 约〜 20%。 同时脂质分解相关酶如 Malic酶和 PPARa 分别上调约 31%和 59%。
C.与对照组相比， 白桦酯醇处理组肝酯酶 （hepatic lipase, HL) 与载脂蛋白 ApoE均 有上调效果。 D.与对照组相比， 白桦酯醇处理组 G-6-PD基因有 1.2倍的上调效果， 而 PEPCK, IRS-1 , IRS-2表达基本上没有变化， 提示白桦酯醇有促进肝脏糖代谢的功能。
E.与对照组相比， 白桦酯醇处理组 adiponectin, LPL和 PPAR-γ基因均有 2-3.5倍上 调变化， 已知这些基因的上调具有抗糖尿病抗炎症的作用。
图 8. 在动物水平上检测白桦酯醇对小鼠动脉粥样硬化形成的作用效果。
8周龄 LDLR- Λ小鼠分成 2组， 分别喂养高胆固醇高脂肪饲料 (WD)+生理盐水、 WD+ 白桦酯醇 (30mg/kg/天)， 10周后处死小鼠进行实验。
A.取小鼠主动脉， 固定后苏丹 IV染色 6分钟， 80%乙醇复染 3分钟。
B-E.根据 A中染色结果， 对动脉粥样硬化斑块的定量。 结果显示： 与对照组相比， 白桦酯醇能够显著减少饮食引起的 LDLR基因敲除小鼠动脉粥样硬化斑块的数量和大 小。
F.取小鼠近心脏处主动脉组织块， 固定后进行组织切片染色及采用巨噬细胞标志分 子 MOMA-2和肌肉细胞标志分子 α-actin免疫组化， 分析比较小鼠动脉粥样硬化斑块处 中性脂质沉积情况及巨噬细胞和肌肉细胞的分布情况。
G-I.根据 F中染色结果， 对动脉粥样硬化斑块的定量。 结果显示， 与对照组相比， 白 桦酯醇能够显著降低小鼠动脉粥样硬化的斑块处脂质沉积和巨噬细胞增多， 能够显著增 加斑块的稳定性。
图中， 所有数据代表平均值 ±标准差； 每组包括 5只小鼠。
图 9. 白桦酯醇特异性结合 SCAP蛋白。
A. 基于白桦酯醇合成的光亲和标记探针 （化合物 1) 及体外光亲和标记实验流程示 意图。
B. CHO-7细胞在含有 5% LPDS, 10 μΜ compactin 禾 P 10 μΜ mevalonate 的常规培养 基中培养 16小时后， 白桦酯醇处理细胞 6小时后， 收集细胞膜组分和核组分， 检测 SREBP-2剪切调控。
C. CHO-7细胞饥饿培养 16小时后， 收集细胞膜组分， 光亲和标记探针的靶蛋白， 然后通过 click反应使偶联上生物素基团， 利用生物素亲和素结合反应富集靶蛋白。 WB 检测 SCAP和转铁蛋白受体 (transferrin receptor)。其中， Betulin指白桦酯醇； Compoundl 指化合物 1。
图 10. 白桦酯醇显著减低血液脂质水平是 SCAP蛋白依赖性的。 C57BL/6J小鼠在喂 养高胆固醇高脂肪饲料的同时分别通过灌胃的方法给予生理盐水、 洛伐他汀 (30 mg/kg/day)或者白桦酯醇 C30 mg/kg/day) , 4周后按照每只小鼠 5 X 108 pfu的感染量对小 鼠采用尾静脉注射腺病毒。 此后继续灌胃 5天， 处死小鼠， 取血液和肝脏组织检测肝脏 基因表达变化 (A)和血液脂质水平变化 (B-E)。 具体实施方式 本发明人经过广泛而深入的研究， 首次揭示具有式（I)所示的母核结构的化合物对于 代谢性疾病的防治具有极其显著的效果。 本发明人鉴定出白桦酯醇是 SREBP途径的特 异性抑制剂， 它特异性地促进 SCAP和 Insig的相互作用， 抑制 SREBP蛋白剪切成熟， 下调胆固醇、 脂肪酸和甘油三酯合成途径的一系列基因和酶类的表达， 因此可知与白桦 酯醇母核结构一致的一类化合物对于代谢性疾病的防治是有用的。 本发明为开发以调节 SREBP为靶点的多功能药物提供了有效途径。 化合物
本文所用的术语 "烷基"指直链或支链饱和的、 含有 1-4个碳原子 （较佳地 1 -2个碳 原子） 的脂族烃类基团。 例如， 垸基包括但不限于甲基， 乙基， 正丙基， 异丙基， 正丁 基， 异丁基， 叔丁基。
本文所用的术语 "链烯基" 包括含有至少一个碳碳双键和 2-4个碳原子 （较佳地 2-3 个碳原子） 的直链和支链烃基。
本文所用的术语 "链炔基" 包括含有至少一个碳碳三键和 2-4个碳原子 （较佳地 2-3 个碳原子） 的直链和支链烃基。
本文所用的术语 "卤素" 指！^、 Cl、 Br、 或 I。
其中， 垸基、 链烯基、 链炔基等均可以含有或不含有取代基。 例如， 它们可以被含 有 1 -3个 (更佳地 1-2个)选自 (但不限于)： CM的烷基， C2.4链烯基， C2.4链炔基， 卤素的 基团所取代。
本文所用的术语 "异构体" 包括： 几何异构体、 对映异构体、 非对映异构体 （如顺 反异构体， 构象异构体)。
本发明首先提供了一种具有如 (I)所示母核结构的化合物：
Figure imgf000010_0001
。
本发明人对白桦酯醇及其衍生物进行了实验， 发现其 17位上的 CH2OH乃是抑制 SREBP的关键性位点。 因此， 可以对其它位点进行修饰， 而仍然保留其活性。 结构式 (I) 所示母核结构形成一种适合的化合物空间构型， 具有该结构的化合物可以以 SREBP途 径为靶点， 通过与 SREBP途径的相关蛋白发生相互作用， 从而通过抑制 SREBP途径实 现防治代谢性疾病的目的。 也即： 所述的具有如结构式 (I)所示母核结构的化合物是一种 SREBP途径的抑制剂。
作为本发明的优选方式， 所述的化合物具有式 (II)所示的结构：
— Q—
替换页 （细则第 26条)
Figure imgf000011_0001
其中， R独立地选自： 氢、 羟基、 C 1 -C4烷基、 C2-C4链烯基、 C2-C4链炔基、 C 1 -C4 垸氧基、 卤素。
较佳地， 式 (II)化合物中， R独立地选自： 氢、 羟基、 C 1 -C2垸基、 C2-C3链烯基、 C2-C3链炔基。 如 "R~ "的表示方法是本领域人员熟知的， 其表示基团 R可以取代在环上的任 意一个或多个可被取代的位置。 并且， 在不同的取代位置上， R的选择可以是不同的。
本发明还包括上述化合物的药学上可接受的盐、 水合物或前体， 只要它们也具有防 治代谢性疾病的作用。 所述的 "药学上可接受的盐"是指一类化合物与无机酸、 有机酸、 碱金属或碱土金属等反应生成的盐。 这些盐包括 （但不限于)： ( 1 ) 与如下无机酸形成的 盐： 如盐酸、 硫酸、 硝酸、 磷酸； （2) 与如下有机酸形成的盐， 如乙酸、 草酸、 丁二酸、 酒石酸、 甲磺酸、 马来酸、 或精氨酸。 其它的盐包括与碱金属或碱土金属 (如钠、 钾、 钙 或镁)形成的盐， 以酯、 氨基甲酸酯， 或其它常规的 "前体药物" 的形式。
所述的 "化合物的前体"指当用适当的方法服用后， 该化合物的前体在病人体内进 行代谢或化学反应而转变成具有结构式 (I)所示母核结构的一种化合物， 或结构式 (I)所示 母核结构的一个化合物所组成的盐或溶液。
本发明还包括上述化合物的异构体、 外消旋体， 只要它们也具有防治代谢性疾病的 作用。化合物具有一个或多个不对称中心。所以， 这些化合物可以作为外消旋的混合物、 单独的对映异构体、 单独的非对映异构体、 非对映异构体混合物、 顺式或反式异构体存 在。
作为本发明的一种优选 ) 所示的结构。
Figure imgf000011_0002
本领域人员应理解， 在得知了本发明化合物的结构以后， 可通过多种本领域熟知的 方法、 利用公知的原料， 来获得本发明的化合物， 比如化学合成或从生物 （如动物或植 物） 中提取的方法， 这些方法均包含在本发明中。
合成化学改造、 保护官能团方法学 (保护或去保护)对合成应用化合物是很有帮助的， 并且是现有技术中公知的技术， 如 R. Larock, Comprehensive Organic Transformations, VCH Publishers ( 1989)； T.W. Greene and P.G.M. Wuts, Protective Groups in Organic 替换页 （细则第 26条) Synthesis, 3r Ed., John Wiley and Sons (1999)； L. Fieser and M. Fieser, Fieser and Fieser，，s Reage ts for Organic Synthesis, John Wiley and Sons (1994)； 禾口 L. Paquette, ed.， Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995)中都有公开。
一些具有式 (I)所示母核结构的化合物也可自生物 （如动物或植物） 中提取、 分离和 纯化。 例如， 式 (III)化合物 （白桦酯醇） 是一类在自然界丰富存在的五环三萜类物质， 可以通过简单的分离方法 (分离得率在 30%以上)从桦树皮中进行大量的抽提制备。 用途
基于本发明人的新发现，本发明提供了具有式 （I) 所示的母核结构的化合物或其异 构体、 外消旋体、 药学上可接受的盐、 水合物或前体的用途， 用于制备防治代谢性疾病 的药物 (或组合物）。
所述的代谢性疾病是指新陈代谢不正常而引起的疾病。 代谢性疾病的病理基础是 糖， 脂肪和蛋白质代谢失常。 代谢性疾病虽然不会直接危及生命， 但却可以导致其它严 重威胁生命的疾病。 判断代谢性疾病的指标主要有： 血糖、 甘油三酯、 总胆固醇 (TC)、 不饱和游离脂肪酸 (NFFA)和尿酸； 上述指标超过正常水平， 则可患有代谢性疾病。 代谢 性疾病主要包括： 胰岛素抵抗、 高胰岛素血症、 糖尿病 (特别是 I I型糖尿病）、 糖耐量减 低、 肥胖、 高胆固醇血症、 脂质代谢异常、 动脉硬化、 冠心病、 高血压、 高尿酸血症和 痛风等，其临床主要表现为腹部肥胖、粥样硬化性血脂异常、血压升高、胰岛素抵抗 （伴 或不伴糖耐量异常） 以及栓塞和炎性反应状态等。
动脉粥样硬化症由胆固醇及胆固醇酯在动脉壁下沉积引起， 是导致冠心病和脑中风 的主要致病因素， 降低血液胆固醇水平是临床治疗该类疾病的主要手段。 脂肪酸和甘油 三酯代谢异常会直接引起胰岛素抵抗， 是导致 II型糖尿病的关键致病因素。在本发明的 具体实例中， 本发明人采用高胆固醇高脂肪饲料喂养小鼠的条件下， 白桦酯醇显著降低 小鼠体内血液和组织的脂质水平， 减少体重增加， 提高其胰岛素敏感性。 进一步， 在动 脉粥样硬化模型小鼠低密度脂蛋白受体 (LDLR)基因敲除小鼠中，检测到白桦酯醇能够有 效减小动脉粥样硬化斑块的大小， 并显著改善斑块的稳定性。
现有技术已经发现， SREBP是一类非常重要的转录因子， 它激活胆固醇、 脂肪酸和 甘油三酯等脂质的生物合成过程。 在本发明的具体实例中， 本发明所述的化合物的作用 机理是特异性地阻断 SREBP的成熟， 抑制其活性。 在细胞水平， 所述化合物能显著抑 制胆固醇、 脂肪酸和甘油三酯等脂质合成基因的表达， 减少脂质合成， 降低细胞内脂质 含量。 在动物个体水平上， 所述化合物表现为显著抑制高脂饮食引起的小鼠体重上升， 降低动物血液、 肝脏和脂肪等组织中的脂质水平， 提高肥胖小鼠对胰岛素的敏感性， 对 II糖尿病有明显的治疗作用。 同时， 所述化合物能够显著减少高胆固醇饮食诱导的动脉 内粥样硬化斑块的形成，并能有效增加斑块的稳定性，具有良好的抗动脉粥样硬化作用。
与目前临床广泛应用的一线降胆固醇药物 (如： 洛伐他汀)相比， 在给予相同剂量条 件下， 本发明的化合物具有与洛伐他汀相似甚至更好的疗效。 例如在本发明的具体实例 中证明： 1) 白桦酯醇能更显著地降低血液和组织中脂质水平； 2) 白桦酯醇能够抑制脂 肪酸和甘油三酯合成， 而他汀类药物则无此功效， 因此白桦酯醇能显著提高胰岛素敏感 性， 对 II型糖尿病有治疗作用； 3) 多数糖尿病患者并发有严重的动脉粥样硬化症， 与 目前常用的治疗糖尿病药物相比， 白桦酯醇不仅改善胰岛素的敏感性， 而且对动脉粥样 硬化斑块有很明显的疗效。 总之， 本发明的化合物通过全新的机制调控脂质代谢， 对 II 型糖尿病和动脉粥样硬化症等有显著疗效。 组合物
如本文所用， 术语 "本发明的组合物" 通常是药物组合物， 其含有式 （I) 所示母核 结构的化合物或其异构体、 外消旋体、 药学上可接受的盐、 水合物或前体作为防治代谢 性疾病的活性成分； 以及药学上可接受的载体或赋形剂。
本发明中， 术语 "含有" 表示各种成分可一起应用于本发明的混合物或组合物中。 因此， 术语 "主要由...组成" 和 "由 .. 且成"包含在术语 "含有" 中。
本发明中， "药学上可接受的"成分是适用于人和 /或动物而无过度不良副反应 (如毒 性、 剌激和变态反应)即有合理的效益 /风险比的物质。
本发明中， "药学上可接受的载体" 是用于将本发明的具有式 （I) 所示母核结构的 化合物或其异构体、 外消旋体、 药学上可接受的盐、 水合物或前体传送给动物或人的药 学上或食品上可接受的溶剂、 悬浮剂或赋形剂。 载体可以是液体或固体。 适用于本发明 的药学上可接受的载体包括（但并不限于）： 盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙醇、 及其组合。
本发明还提供了制备防治代谢性疾病的组合物的方法，包括使用具有式 (I)所示的母 核结构的化合物。 可将有效量的式 （I) 化合物与药学上可接受的载体混合获得本发明的 组合物， 活性成分在组合物中的重量比例例如可以是 0.0001-50 wt %； 较佳地可以是 0.001-20 wt %。
本发明的组合物也可以是一种中药或天然产物提取物，其中含有本发明的具有式 (I) 所示母核结构的化合物作为活性成分， 提取可以采用一些已知的方法。
本发明所述的药物组合物的剂型可以是多种多样的，只要是能够使活性成分有效地 到达哺乳动物患处的剂型都是可以的。 从易于制备和给药的立场看， 优选的药物组合物 是一种口服或注射的制剂。 比如可选自： 颗粒剂、 片剂、 胶囊剂、 溶液、 或悬浮液、 粉 末剂。 其中具有式 （I) 所示母核结构的化合物或其异构体、 外消旋体、 药学上可接受的 盐、 水合物或前体可以存在于适宜的固体或液体的载体或稀释液中。 本发明的组合物中 可以加入制备不同剂型时所需要的各种常规载体或辅料， 如填充剂、矫味剂、抗氧化剂、 香料、 色素、 润滑剂、 助流剂、 润湿剂、 乳化剂、 pH缓冲物质等。 这些添加剂都是本领 域人员所熟知的。 本发明还提供了一种防治代谢性疾病的方法， 包括步骤： 给需要的对象施用有效量 的式 （I) 化合物。 活性成分的给药量是治疗有效量。 当外用时， 本发明所述的化合物的 安全有效量通常约 0.1ng-100mg/kg体重； 较佳的约 lng-10mg/kg体重。 当然， 具体剂量 还应考虑给药途径、 用药者健康状况等因素， 这些都是熟练医师技能范围之内的。
此外， 本发明所述的化合物还可与其他活性成分或治疗剂 （如其它降血脂药物、 降 胆固醇药物、 糖尿病药物等） 一起使用。 本发明的主要优点在于：
(1) 首次揭示了一类新的 SREBP途径的抑制剂， 其在抑制 SREBP途径的同时， 不 会激活 LXR信号通路， 从而为动脉粥样硬化和 II型糖尿病等代谢性疾病的预防和治疗 提供新的途径。
(2) 本发明的化合物完全可以通过人工合成的方式生产， 成本低。
(3) 如白桦酯醇等化合物可以大量地从自然界中分离得到， 来源丰富， 毒副作用小， 成本低廉。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本发明 而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规 条件如 Sambrook等人，分子克隆：实验室指南 (New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建议的条件。 材料与方法
材料
辣根过氧化物酶结合的驴抗鼠和抗兔 IgG: 获自 Jackson免疫研究实验室。
甲羟戊酸 (mevalonate), 菲律宾菌素 (Filipin)获自 Sigma。
白桦酯醇及其类似物获自中国上海药谷生物公司。 白桦酯醇及其结构类似物的纯度 经气相色谱鉴定高于 99%。
甲基 - β -环化糊精（CDX): 获自 Cyclodextrin Technologies Development Inc.。
各类甾醇获自 Steraloids, Inc.。
荧光素酶分析用试剂盒及细胞裂解缓冲液获自 Pr0mega。
14C标记的乙酸盐获自 PerkinElmer。
去脂血清 (LPDS， d > 1.215 g/ml): 从新生小牛血清通过超离心获得。 蛋白免疫杂交用抗体
抗 T7的一抗获自 Novagen; 抗 Myc的一抗 (IgG-9E10)获自 Roche; 抗人
SREBP-l(IgG-H160)获自 Santa Cruz; 抗仓鼠 SREBP-2的一抗 (IgG-7D4)， 抗仓鼠 SCAP 的一抗 (IgG-9D5)， 抗 HMGCR的一抗 (IgG-A9)及抗人 SREBP-2 的一抗 (IgG-lD2)均常规 方法制备自小鼠杂交瘤细胞系 (ATCC); 抗 gp78， 抗 GFP及抗 HMGCR多克隆抗体均常 规方法获自兔子抗血清。 质粒
pCMV-Insig-l-Myc, pCMV-HMGCR-T7质粒信息参见文献 Sever et al.，
Insig-dependent ubiquitination and degradation of mammalian
3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol. J Biol Chem. 2003 Dec 26; 278(52):52479-90； pCMV-SCAP, pTK-BP2 质粒信息参见文献 Daisuke et al.， Three mutations in sterol-sensing domain of SCAP block interaction with insig and render SREBP cleavage insensitive to sterols Proc. Natl. Acad. Sci. U. S. A.
99: 16672-16677。 pSRE-Luciferase 启动子区来自以
5'-GCGGGATCCCACAAAACAAAAAATATTTTTTTGG-3' (SEQ ID NO: 1)禾口
5'-GCCAAGCTTTCGCAGCCTCTGCCAGGCAGTGTCC-3 ' (SEQ ID NO: 2)作为弓 |物， PCR扩增人肝 cDNA获得人 LDLR启动子区 -588〜+92核苷酸序列（SEQ ID NO: 3), 并 通过 BamHI, Hindlll双酶切位点克隆接入载体 pGL3-Basic(购自 Promega；)， 获得表达质 粒 -Luciferase。
Figure imgf000015_0001
TGCGA (SEQ ID NO : 3) 培养基
培养基 A组分是 Dulbecco's改良的 Eagle's培养基， 含有 100单位 /ml青霉素和 lOO g/ml链霉素。 培养基 B组分是 Dulbecco's改良的 Eagle's培养基与 F12培养基等比 例混合， 含有 100单位 /ml青霉素和 100μ§/ιη1链霉素。培养基 C组分是培养基 Β再加上 5%FBS。培养基 D组分是培养基 C再加上 5 μ§/ιη1胆固醇， 1 mM甲羟戊酸钠，及 20 μΜ 油酸钠。 培养基 Ε组分是培养基 Β再加上 5% LPDS , 1 μΜ洛伐他汀加上 50 μΜ甲羟戊 酸钠。 细胞培养
CRL-1601 (McArdle RH7777大鼠肝癌细胞； 购自 ATCC)禾 P HuH7 (人肝癌细胞系； 购自 ATCC)单细胞层在 37°C和 5% C02中， 细胞生长在培养基 A (Dulbecco's 改良的 Eagle's 培养基， 含有 100单位 /ml青霉素和 lOO g/ml 链霉素;)再加上 10% FBS。 CHO-7 (CHO-K1细胞在去脂蛋白血清培养基中筛选得到的单克隆细胞； 购自 ATCC) 单细胞层 在 37°C和 5% C02中， 细胞生长在培养基 B (Dulbecco's 改良的 Eagle's 培养基与 F12 培养基等比例混合， 含有 100单位 /ml青霉素和 100μ§/ιη1 链霉素)再加上 5% FBS。 SRD-13A (SCAP 缺陷细胞株， 来自 CHO-7; 购自 ATCC) 单细胞层在 37°C和 5% C02 中， 细胞生长在培养基 D中。
Huh-7/SRE-Luc细胞株建立及其筛选候选物质的方法
将 pSRE-Luciferase与 pEGFP-Nl (Invitrogen)按照 5 : 1的比例共转染 Huh-7细胞 48小 时后给细胞换液， 换成培养基 A加 10% FBS， 700 g/ml G418。 此后每 2-3天换液一次， 2周后形成独立的单克隆。 挑取单克隆并鉴定其荧光素酶表达调控特征。 最终选取表达 量高且调控好的细胞株， 命名为 Huh-7/SRE-LUC。 鉴定荧光素酶活性采用的是荧光素酶 分析用试剂盒方法， 检测过程中， 绿荧光蛋白的量作为内参。
对候选物质进行筛选时， Huh-7/SRE-Luc细胞在含有去脂蛋白血清，他汀及微少量的 甲羟戊酸钠 (50 μΜ)的培养基中处理 16小时， 这一去除胆固醇的过程能够有效上调细胞 内源性活性形式的 SREBP。 给细胞换含有不同候选物质的培养基 (其中不同化合物作用 浓度在 0.5-5 μΜ范围内）， 培养 6小时后测定细胞荧光素酶活性变化。
SRD-13A细胞中瞬时转染实验
转染前一天将 SRD-13A细胞 (CHO-7细胞来源的 SCAP基因缺陷细胞株， 经体细胞 遗传学方法获得； CHO-7细胞获自 ATCC; SRD-13 Α细胞，参见 Rawson RB, DeBose-Boyd R, Goldstein JL, Brown MS. Failure to cleave sterol regulatory element-binding proteins (SREBPs) causes cholesterol auxotrophy in Chinese hamster ovary cells with genetic absence of SREBP cleavage-activating protein. J Biol Chem. 1999 Oct l;274(40):28549-56)按照 4 X 105每 60-mm培养皿的密度接种在培养基 C中。 24小时后给细胞换培养基 B并采用 FuGENE 6试剂 (Roche)进行转染。质粒 DNA总转染量是 3 μg每盘细胞， 不足用 pTK空 载或 pcDNA3C购自 Promega)补平。 转染 24小时后给细胞换培养基 E再加上 1% CDX。 37°C培养 1小时， PBS洗细胞两次， 换培养基 C再加上 醇或者白桦酯醇。 对照细胞加 DMSO (所有细胞中 DMSO终浓度为 0.1%)。 换液 6小时后， PBS洗细胞并收集细胞蛋 白， 下一步进行免疫杂交或免疫共沉淀实验。 蛋白免疫杂交实验
1. 收集细胞全蛋白裂解液
1) 收集细胞并将其重悬于预冷的 120 μΐ RIPA 裂解液 (50 mM Tris-HCl (pH 8.0), 150 mM NaCl/0.1% (w/v) SDS, 1.5% (w/v) NP40, 0.5% deoxycholate, 2 mM MgCl2) 中， 裂解液中需预先加入预冷的蛋白酶抑制剂 （10 g/ml leupeptin, 5 g/ml pepstatin A， 25 g/ml ALLN, 1 mM PMSF禾卩 10 μΜ MG132)。
2) 用 7号针头剪切 10次后， 12000rpm冷冻离心 10分钟， 收集上清即为细胞全蛋白 裂解液。 样品蛋白浓度根据 Lowry方法 (Bio-Rad)进行定量后， 加入 4XSDS上样缓冲液 (150 mM Tris-HCl (pH 6.8)， 12% SDS, 30% (v/v) 甘油， 0.02% (w/v) 溴酚蓝， 6% (v/v) 2- 巯基乙醇；)。
3) 100°C加热 10分钟后上样，进行 SDS聚丙烯酰胺凝胶电泳。转移到硝酸纤维素膜， 蛋白免疫杂交。 各个抗体的使用浓度： IgG-lD2， 3 g/ml; IgG-H160， 0.2 g/ml。
2. 收集细胞膜蛋白
1) 收集细胞并将其重悬于预冷的 500 μΐ 缓冲液 AC10mMHEPES/KOHCpH7.6;)，1.5 mMMgCl2， 10mMKCl， 5 mM EDTA, 5 mM EGTA, 250 mM Sucrose) 裂解液中需预 先加入预冷的蛋白酶抑制剂 （lO g/ml Leupeptin, 5
Figure imgf000017_0001
Pep statin A, 25 g/ml ALLN, 1 mMPMSF和 10 MMG132)。 用 7号针头剪切 30次后 1000 g， 4°C 离心 7分钟。
2) 沉淀重悬于 0.1 ml 缓冲液 C (20 mMHEPES/KOH， pH7.6; 2.5%(V/V) 甘油; 1.5mM MgCl2; 0.42 MNaCl; 1 mM EDTA; 1 mM EGTA) 裂解液中需预先加入预冷的 蛋白酶抑制剂 （10 g/ml Leupeptin, 5 g/ml Pepstatin A， 25 g/ml ALLN, 1 mM PMSF 和 10 MMG132;>4°C低速旋转 1 h 使缓冲液 C 充分作用于细胞，然后 105g4°C高速离 心 30 分钟。 上清即为膜蛋白。
3) 在步骤 1)中 1,000 g 离心这一步得到的上清再次 105g， 4°C 离心 30 分钟后， 沉 淀用 0.1 ml SDS裂解缓冲液（10 mMTris-HCl， pH6.8; 100 mM NaCl; 1% SDS; 1 mM EDTA; I mMEGTA)重悬， 室温 lOOOrpm震荡摇匀 30分钟后即为细胞核蛋白。 蛋白免疫共沉淀方法
1) 收集细胞并将其重悬于预冷的 600 μΐ 缓冲液 Β (50 mMHEPES-KOH(pH7.4); 100 mM NaCl; 1.5 mM MgCl2; 0.1% (v/v) Nonidet P-40) 裂解液中需预先加入预冷的蛋白酶 抑制剂 （同上)。
2) 用 7号针头剪切 15次， 16,000 g， 4°C 离心 10 分钟。 每个样品上清中加入 60 μΐ 交联了 anti-Myc抗体的 beads, 4°C 旋转 2小时。 1000 g， 4°C 离心 3 分钟， 保留上清 样品， 弃去剩余上清。
3) 向沉淀的 beads中加入 1 ml 缓冲液 B， 于 4 旋转 5分钟后 4°C， 1000g离心 5 分钟， 弃去上清， 如此反复 3次。 4) 弃去上清， 每份样品中加入 100 μΐ的 2 X上样缓冲液， 100°C空气浴处理 10分钟。
5) 混匀， 室温 13200rpm离心 1分钟。 转移 90 μΐ上清到新的 1.5 ml的管子当中， 用 于 Western Blot检测。 同位素示踪胆固醇和脂肪酸合成实验方法
CRL-1601细胞培养在含有 10%去脂蛋白去脂肪酸血清， 1 μΜ洛伐他汀及微少量的 甲羟戊酸 (50 μΜ)的培养基 Α中同时加入不同浓度梯度的白桦酯醇处理细胞 16小时后， 给细胞换成含有 10%去脂蛋白去脂肪酸血清的培养基 A， 同时不同浓度梯度的白桦酯醇 的培养基，并向细胞中加入 14C标记的乙酸盐 (6 μ /ml)作为胆固醇和脂肪酸从头合成的 底物， 示踪细胞胆固醇和脂肪酸的合成， 2个小时后收集细胞。 采用有机溶剂抽提的方 法获得细胞胆固醇和脂肪酸组分， 通过薄层层析方法分离各个脂质组分， 然后利用放射 自显影方法分析不同条件下细胞胆固醇和脂肪酸的合成情况。 菲律宾 (Filipin)染色方法和尼罗红 (Nile-Red)染色方法
新鲜的 Filipin储液浓度是 5 mg/ml, 工作液是 0.5 mg/ml,稀释在无水乙醇中。用 4% 多聚甲醛 (paraformaldehyde, 稀释在 PBS中;)室温固定细胞 30分钟， 用 PBS 洗细胞 3 次， 然后用 0.5 g/ml 工作液避光处理细胞 30 分钟。 Filipin 信号用 Zeiss LSM 510 confocal microscope 双光子激光共聚焦显微镜观察， 激发光 720 nm。 新鲜的 Nile-Red 储液浓度是 0.5 mg/ml, 工作液浓度是 0.5 μ§/ιη1, 稀释在含有 10血清的 PBS中， 用 4% 多聚甲醛室温固定细胞后， 用 PBS 洗细胞 3次， 然后用 0.5 g/ml Nile-Red工作液避光 37°C处理细胞 10 分钟。 Nile-Red信号用 Zeiss LSM 510 confocal microscope观察， 激发 光 523 nm。 荧光定量
将对照细胞的平均信号强度定义为 1， 在每次实验里的每个实验组中， 任意选择 50 个细胞作为定量对象。 用 Image Pro Plus 5.02软件荧光定量。 荧光实时定量 PCR (Q-PCR)
1. RNA抽提与定量
1) 收集培养处理后的细胞或者动物组织， 动物组织等量取自 4-5只小鼠。
2) 裂解细胞。 每个 60mm培养皿中加入 1 ml Trizol 试剂 （Invitrogen)， 室温处理 10 分钟。
3) 将细胞裂解液转移到 1.5 ml Eppendorf 管中。
4) 每份样品加入 200 μΐ 的氯仿， 振荡低速混匀。
5) 4°C， 13200rpm离心 15分钟，转移 500μ1的上层水相至新的 1.5ml Eppendorf管中。 6) 每份样品中加入 500μ1异戊醇， 振荡低速混匀。
7) 4°C , 13200rpm离心 10分钟， 弃去上清， 每份样品中加入 lml的 70%乙醇， 颠倒 混匀。
8) 4°C , 13200rpm离心 10分钟， 弃去上清， 沉淀室温晾干， 加入适量的 DEPC处理 过的去离子水， 充分溶解。
9) 取适量 RNA稀释， 测定 260 nm的光吸收值， 计算样品浓度， 测定 260 nm与 280 nm光吸收值的比值， 计算样品纯度， 最终调整样品浓度为 1 μ§/μ1
2. 逆转录反应
cDNA合成采用 Promega公司的逆转录酶与缓冲液。每一体系含有 4 g的 RNA 0.5μβ 的 01igodT(T15Vl)， 终浓度各 0.4 mM的 dNTP
1) 每一体系中加入 4 g的 RNA 0.5 g的 01igodT(T15Vl)， 10.5μ1 DEPC 水， 混匀。
2) 70°C变性 5分钟， 冰上骤冷。
3) 每一反应体系中加入 3 μΐ DEPC 水， 5 μΐ 5 X MLV缓冲液， 1 μΐ dNTP (各 10 mM)， 1 μΐ M-MLV逆转录酶， 混匀。
4) 37°C反应 1小时。
5) 70°C延伸 10分钟。
3. 实时定量 PCR
Realtime PCR采用 20 μΐ体系。 使用 Takara公司的 HS Taq酶与缓冲液。 每一体系含 有 0.5 U HS Taq酶， 终浓度 0.5 X SYBR绿， 终浓度 1.5 mM MgCl2， 终浓度各 62.5 μΜ dNTP, 终浓度 0.6 μΜ引物， 1.5μ1模板 cDNA (逆转录产物稀释 4倍用）。
1) 将水， 缓冲液， dNTP, 镁离子， 引物， 模板与酶加入反应体系， 混匀。
2) 按照以下反应条件进行反应： a. 94 °C预变性 5分钟， b. 94°C变性 30秒， c. 60°C退 火 30秒， d. 72 °C延伸 30秒， e. b-d循环 40次， f. 72°C延伸 10分钟。
2) 根据荧光读数计算结果 (所用的读数系统为 Stmtagene Mx30005P™ QPCR
Systems)
本发明中荧光实时定量 PCR采用的部分引物序列见表 1。 上述所有反应都进行三次 独立的实验。
表 1
SEQ ID GeneBank 物种 基因 正向和反向引物序列 （5' 到 3')
NO: 登录号 小鼠 TGTGTCCGTCGTGGATCTGA 4
GAPDH M32599
(Mus musculus) CCTGCTTCACCACCTTCTTGAT 5
GGCCGAGATGTGCGAACT 6
SREBP-la 011480
TTGTTGATGAGCTGGAGCATGT 7
GGAGCCATGGATTGCACATT 8 Shimomura, I.等
SREBP-lc 9 (1997); J. Clin.
GGCCCGGGAAGTCACTGT
Invest.99, 838-845
GCGTTCTGGAGACCATGGA 10
SREBP-2 ACAAAGTTGCTCTGAAAACAAATC 1 1 AF374267
A
Figure imgf000020_0001
- -
Figure imgf000021_0001
Figure imgf000021_0002
εζο.οι/ΐΐοί OAV
Figure imgf000022_0001
其中， 细胞水平的实时荧光定量 PCR是在大鼠细胞系， 所用引物序列参考大鼠的弓 I 物列表； 动物水平的 PCR是在小鼠个体上进行的， 所用引物序列参考小鼠引物列表。 动物实验方法
所有实验用动物的饲养方法都按照上海实验动物中心的动物饲养及使用条例。
a) 出生 8周后的雄性 C57BL/6J 获自上海 Slaccas 实验动物中心。将小鼠随机分笼 后，给小鼠喂养基础饲料或高脂肪高胆固醇的饲料 (高脂肪高胆固醇饲料组成是在基础饲 料加 1.25% 胆固醇和 20%猪油)洛伐他汀和白桦酯醇的给药量是 30mg/kg/天， 给药方式 是灌胃。 每周统计小鼠的体重， 摄食量。 6周后采用 NMR(Bmker， Houston)方法统计分 析小鼠脂肪组织的含量。 随后将小鼠禁食 14小时， 取血液和各个器官组织。
b) 实验验证体内条件下白桦酯醇对小鼠胰岛素抵抗的作用效果： 小鼠随机分组后， 分别喂养基础饲料或者高脂肪高胆固醇饲料， 同时灌胃洛伐他汀或者白桦酯醇 （给药量 30mg/kg/天 ；)， 8周后进行胰岛素及血糖耐量的测试实验。
c) 实验分析白桦酯醇对小鼠动脉粥样硬化形成的作用效果： 取出生后 6周的 LDL 受体缺陷小鼠 (；与 C57BL/6J鼠反交 10次获得， 购自 Jackson Lab)。 喂养至 8周后将小鼠 随机分成两组， 每组 5只， 两组小鼠在喂养高脂肪高胆固醇饲料的同时分别灌胃生理盐 水或者白桦酯醇 （30mg/kg/天)， 灌胃 10周后， 取血液和各个器官组织进行实验。 小鼠血液及肝脏脂质水平测定
利用试剂盒 (Wako， Japan)测定血液总胆固醇总甘油三酯水平， 测定血液 LDL胆固 醇和 HDL胆固醇水平的方法参试剂盒手册 （Applygen, China)。 将肝组织匀浆后离心， 取上清测定总胆固醇和总甘油三酯水平， 蛋白定量方法是 Lowry定量法 CBIO-RAD)。 小鼠肝脏和脂肪组织的组织病理学分析
用 Tissue-Tek OCT cryostat molds (Leica)对小鼠肝脏组织进行切片前的包埋， -80 °C 预冷后切取 10-μιη厚度的切片， 采用 0.5%油红 0及苏木精染色细胞。 用 Paraffin对小 鼠脂肪组织进行切片前的包埋， 切取 5 μιη厚度切片置于多 L-赖氨酸包被的玻片后 hematoxylin-eosin染色。 对月旨肪细胞大小参照 Computer- Assisted Morphometric Analysis 方法进行定量。 葡萄糖耐量、 胰岛素耐受实验
C57BL/6对照组小鼠 (购自中国上海 Slaccas实验动物中心)喂养基础饲料， 处理组小 鼠喂养高脂肪高胆固醇饲料同时分别给予特定浓度的生理盐水、 洛伐他汀 (30mg/kg/天)、 白桦酯醇 (30mg/kg/天)， 8周后统计测试小鼠血糖耐量及胰岛素耐受情况。 测试前， 小鼠 禁食 12小时， 采取腹腔注射方式给小鼠注射 2 g/kg 葡萄糖， 或者 0.75 U/kg 胰岛素， 15， 30， 60及 120分钟后， 尾尖取血， 检测各时间点血液葡萄糖水平。 3天后， 再次检 测小鼠血液葡萄糖水平和血液胰岛素水平。 在动物水平上检测化合物白桦酯醇对小鼠动脉粥样硬化形成的作用效果
8周龄 LDLR-/-小鼠 (购自 Jackson Lab)分成 2组，在喂养高脂肪高胆固醇饲料条件下， 分别灌胃生理盐水和白桦酯醇 (30mg/kg/天；)， 10周后取小鼠主动脉， 固定后苏丹 IV染色 6分钟， 80%乙醇复染 3分钟。同时 10周后取小鼠主动脉近心脏处组织块， Tissue-Tek OCT cryostat molds包埋固定后进行组织切片得到 10-μιη厚度的组织切片，采用 0.5% 油红 0 及苏木精染色方法分析脂质沉积情况。 采用巨噬细胞和肌肉细胞免疫组化染色方法分析 主动脉近心脏处板块的稳定性变化， 最终染色结果通过计算机软件进行定量分析。 腺病毒包装和纯化
1 ) 293Α细胞 (；购自 ATCC；)， 消化后接种到 6孔板中， 接种量为 4χ 105/孔， 于 37°C 培养 24小时。
2 ) 利用 FuGENE HD(Roche)转染试剂， 将 Pad内切酶消化后的病毒表达载体转染 细胞， 每孔细胞转染 4 μ§质粒， 37°C培养 24小时。
3 ) 给细胞换上含有 1.25% 低熔点琼脂糖的膏状培养基， 室温冷却使凝固。 固体层 上加入 l ml液体培养基。 37 °C培养， 每隔 3天补加适量新鲜液体培养基， 连续培养约 10天， 直至观察到肉眼清晰可见的病毒噬斑。
4 ) 移液器枪尖穿剌固体培养层， 吸取空斑周围正在裂解的细胞碎片和空斑内的液 体， 室温和 -80°C反复冻融 3次后， 13,200 rpm， 室温离心 10分钟得到上清为第一代病 毒粒子。
5 ) 293A细胞， 消化后接种到 24孔板中， 接种量为 5 >< 104/孔， 于 37 °C培养 24小 时。
6 ) 取 4 ) 中第一代病毒粒子感染 5 ) 中细胞， 每孔加入 200 μΐ病毒上清， 37 °C培 养 6小时后， 补加 l ml新鲜培养基， 37 °C培养 4-7天， 观察到 70%细胞病变裂解时， 收 集细胞和培养基， 室温和 -80°C反复冻融后， 13,200 rpm， 室温离心 10分钟得到上清为 第二代病毒粒子。
7 ) 取第二代病毒粒子 200 μ1， 抽提病毒基因组 DNA， 用于鉴定基因型。 鉴定为复 制缺陷且包含目的基因片段的单克隆的剩余第二代病毒粒子将再次感染 293A细胞， 大 规模扩增。
8 ) 将 293A细胞铺于 30-40个 10 cm皿， 待细胞长满， 向每块板中加入合适滴度 (约 107-108 pfu/ml ) 的病毒上清 10ul， 继续培养， 待细胞完全病变后 （4一 7天） ， 向 每块皿中加入约 500 μΐ 10%的 ΝΡ-40以裂解细胞， 裂解液冻存于 -80 °C。
9 ) 提前 1天将 8 ) 中裂解液从 -80 °C冰箱中拿出， 室温 （或 4°C ) 融解。 收集整个 细胞裂解物， 12,000 rpm离心 10分钟， 弃细胞碎片， 收集上清。 每 100 ml上清加入 50 ml PEG8000 ( 20%PEG8000 , 2.5 M NaCl ) ， 冰上放置 1小时沉淀病毒。 12,000 rpm离 心上述混合物 20分钟， 弃上清， 将沉淀物悬浮在 10 ml密度为 1. 10 g/ml的 CsCl溶液中 (溶剂为 20mM Tris-HCl， PH8.0 ) ， 4 °C， 7000 rpm离心 5分钟， 取上清。
10 ) CsCl梯度的制备方法如下： 加入 2.0 ml密度为 1.40 g/ml的 CsCl溶液 （溶剂 同上），然后加入 3.0 ml密度为 1.30 g/ml的 CsCl溶液，再加入 5 ml的病毒悬浮液。20,000 rpm, 室温离心 2小时。
1 1 ) 收集密度在 1.30 g/ml和 1.40 g/ml之间的病毒条带至透析袋中（透析袋使用前 用 10 mM的 EDTA-Na2煮沸 10分钟） 。 在透析缓冲液（50 g蔗糖， 10 ml 1 M Tris-HCl , pH8.0， 2 ml IM MgC 定容至 1 L ) 中， 4 °C透析 24小时， 中间换一次透析液。 收集病 毒， 测定病毒滴度 (具体测定方法见 Stratagene 病毒滴度定量试剂盒使用手册）。 腺病毒介导的小鼠肝脏 SCAP基因沉默
本发明人采用 AdEasyTM 腺病毒表达载体 (购自 Stratagene)系统介导小鼠肝脏 SCAP基因沉默。 具体做法如下：
小鼠 SCAP shRNA序列为（SEQ ID NO: 138)：
5 '-GATCCCCgatcgacatggtcaagtccTTCAAGAGAggacttgaccatgtcgatcTTTTTA-3 '
阴性对照 shRNA 序列为（SEQ ID NO: 139)：
5 '-GATCCCCttctccgaacgtgtcacgtTTCAAGAGAacgtgacacgttcggagaaTTTTTA-3 ' ;
1 ) 将上述 shRNA序列接入 pEGFP-Hl 载体 (购自 Clontech)的 BamHI/Hindlll位点 内。
2 ) 将 shRNA序列及其上游 HI启动子序列从前述载体中用 Xhol/Hindlll酶切后亚 克隆转接入 pShuttle 载体 (购自 Stratagene)的 Xhol/Hindlll位点内。
3 ) 利用 pAdEasy载体使发生重组， 得到腺病毒表达载体。
4 ) 采用 293A细胞包装腺病毒， 并氯化铯密度梯度离心方法进行浓缩纯化。
5 ) 采用病毒滴度试剂盒 (Stratagene)测定病毒滴度后， 按照每只小鼠 5 >< 108 pfu的感 染量对小鼠采用尾静脉注射， 注射体积不大于 200 μΐ为佳。
6 ) 5天后， 处死小鼠， 采集血液和肝脏组织进行后续鉴定实验。
II. 实施例
实施例 1、 白桦酯醇是 SREBP途径的特异性抑制剂
细胞核内活性形式的 SERBP通过与靶基因启动子区域的 醇调控元件 (SRE)结合启 动下游基因的表达， 进而促进脂质的生物合成 (Wang,X.等 （1993). Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. II. Purification and characterization. J. Biol. Chem. 268, 14497- 14504)。 本发明人构建了荧光素酶报告基 因系统， 该荧光素酶的启动子区包含一个甾醇调控元件 (SRE)， 在人的肝癌细胞系 Huh-7 细胞中稳定表达该荧光素酶， 最终得到 Huh-7/SRE-Luc细胞（图 1A)用作筛选 SREBP途 径的小分子抑制剂。 采用不同的候选化合物处理该细胞一定时间后， 收集细胞并检测其 荧光素酶活性的变化。 通过对大量的化合物进行筛选， 发现化合物白桦酯醇
(lup-20(29)-ene-3 , 28-diol)能够有效降低荧光素酶的活性（图 1B)。
本发明人比较了白桦酯醇和 25-羟胆固醇在抑制 SERBP方面的作用效果及特异性。
25-羟胆固醇有三个主要的效果： 抑制 SREBP-2剪切， 减少细胞核内活性形式的
SREBP-2(图 2A); 激活 LXR信号通路， 上调 SERBP-1转录， 组成性的上调细胞核内活 性形式的 SERBP-1(图 2A); 促进胆固醇合成途径 HMGCR蛋白的降解（图 2B)。 与 25-羟 胆固醇不同的是， 白桦酯醇能够同时抑制 SREBP-1 , SREBP-2的剪切（图 2A)， 但是不会 激活 LXR信号通路， 而且不促进 HMGCR蛋白降解 (图 2B)。 此外， 与内源性实验结果 一致的是， 采用瞬时转染方法检测白桦酯醇对外源性 SREBP-2的剪切调控， 结果显示， 白桦酯醇能完全的抑制外源性 SREBP-2的剪切入核， 并且这一抑制效果是 Insig蛋白依 赖性的（图 2C， 泳道 3-11)。 所以， 白桦酯醇这一化合物特异性的抑制 SREBP途径， 同 时不会激活 LXR信号通路， 不促进 HMGCR蛋白降解。 而且， 蛋白免疫共沉淀实验证 实， 白桦酯醇能促进 SCAP和 Insig蛋白的相互作用（图 2D， 泳道 4-5)， 该实验解释了白 桦酯醇的作用机制。
为进一步了解白桦酯醇的作用机制， 本发明人分析比较了 5种白桦酯醇的结构类似 物 (图 3A；)， 它们在结构上不同主要在于化合物 17位的基团， 白桦酯醇 17位的基团是甲 醇基 (-CH2OH)决定了它抑制 SREBP途径的作用效果， 其他化合物是甲醛基 (-CHO), 甲 羟基 (-COOH), 或者甲基 (-CH3)则无此活性。 此外原人参二醇) (20(S)-protopanaxadiol)原 人参三醇) (20(s)-protopanaxatriol)也不具有上述活性（图 3B；)。说明白桦酯醇作用效果特异 性取决于其结构特异性。 此外， 白桦酯醇特异性的抑制 SREBP转录因子剪切成熟， 抑 制细胞胆固醇合成降低细胞胆固醇水平， 在一定浓度条件下使细胞因为缺少胆固醇而死 亡， 向细胞补充胆固醇后可以使细胞存活。 因此， 其 17位上的羟基是其活性所必须的， -CHO, -COOH, 或者 -CH3都不再具有抑制 SREBP剪切的活性， 另外两种结构类似物原 人参二醇， 原人参三醇也不具有上述活性。
在没有外源胆固醇供给的情况下， 细胞内源性的胆固醇合成是细胞生长所必须的。 将 CHO-7细胞培养在含有去脂蛋白血清的培养基中， 该培养条件下细胞不能获得外源 胆固醇， 其生长所需的胆固醇来源于内源合成途径。 此时给予细胞不同浓度梯度的白桦 酯醇处理一段时间后， 随着化合物处理浓度的升高， 细胞死亡（图 3C， 上排；)。 如果在给 予白桦酯醇的同时向培养基中补加一定量的外源胆固醇则能够使细胞在较高的白桦酯 醇浓度下存活 (图 3C， 下排)， 说明白桦酯醇处理后细胞死亡的原因是胆固醇缺失引起， 白桦酯醇抑制了细胞内源性胆固醇的合成。 经验证白桦酯醇的结构类似物不具有上述特 性。 所以， 白桦酯醇 17位的基团甲醇基是其抑制 SERBP剪切， 抑制细胞胆固醇合成等 活性所必须的。 实施例 2、 白桦酯醇下调 SERBP靶基因表达并降低细胞脂质合成
由于 SREBP-2是其本身的靶基因（Sato, R.等（1996). Sterol-dependent transcriptional regulation of sterol regulatory element-binding protein-2. J. Biol. Chem. 277, 26461-26464) , 所以 SERBP-2的表达在白桦酯醇处理后下调大约 40%。 与此同时， 本发明人检测到其 他参与胆固醇合成途径的数十种 SREBP-2靶基因如 HMGCR, HMG-CoA合成酶
(HMGCS), 禾 P Squalene Epoxidase (SE)等的表达在白桦酯醇处理后均显著下调（图 4A；)。 同时， 参与细胞脂肪酸合成途径的基因和酶类如 SREBP-l c， 脂肪酸合成酶 (FAS) , 和乙 酰辅酶 A羧化酶 a (ACC)等表达水平均被白桦酯醇显著下调（图 4B)。 此外， 与之前观察 到的结果一致的是： LXR的靶基因如 ABCG5和 ABCG8的表达不受白桦酯醇的影响（图 4C)。 与基因表达变化结果一致的是采用同位素示踪实验检测白桦酯醇处理后细胞胆固 醇和脂肪酸的从头合成过程 (图 4D-E)以及脂质染色试验方法分析细胞胆固醇和中性脂 质 (主要是胆固醇酯和甘油三酯； 图 4F-I)的量的变化。结果显示与对照组相比， 白桦酯醇 处理后细胞胆固醇和脂肪酸的从头合成过程被显著抑制， 细胞脂质水平有明显的降低。 总而言之， 该结果证实白桦酯醇能够抑制 SERBP途径， 下调胆固醇和脂肪酸生物合成， 降低细胞脂质水平。 实施例 3、 白桦酯醇逆转饮食诱导肥胖小鼠的体重增加， 改善小鼠血液、肝脏及脂肪 组织的脂质组成
接下来本发明人检测了白桦酯醇在动物体内的作用效果。 已知洛伐他汀是 HMGCR 的特异性抑制剂， 其对代谢性疾病的疗效已经被人们广泛接受。 本发明人比较了白桦酯 醇和洛伐他汀的生物学效果。 8周龄 C57BL/6J小鼠被随机分成 4组， 其中第 1组小鼠喂 养基础饲料 (Chow), 第 2-4组喂养高脂肪高胆固醇饲料 (WD)， 给料同时第 1组 2组小鼠 灌胃生理盐水， 第 3组 4组小鼠灌胃洛伐他汀 (30mg/kg/天)和白桦酯醇 (30mg/kg/天)。 6 周后， 统计分析小鼠的摄食量变化， 体重增加， 及各个脏器的比重变化。 结果显示与灌 胃生理盐水的对照组相比， 在食物摄取量没有明显变化 (图 5A)的情况下， 洛伐他汀或者 白桦酯醇均能显著降低小鼠的体重 (约降低 1 1%)及体重增加值 (约降低 40%)， 见表 2。所 有试验数据均为平均值 ± 标准差 *， p < 0.05 ; **， p < 0.01 为与喂养高脂肪高胆固醇 组小鼠的数据比较所得 (EXCEL, student test) 0
表 2、 白桦酯醇对小鼠摄食量， 体重， 器官重的影响
Chow WD WD +洛伐他汀 WD+白桦酯醇
n 8 7 8 6 食物摄取 (g) 2.5 ± 0.4 2.2 ± 0.5 2.3 ± 0.1 2.4 ± 0.2
体重 (g) 26.9 ± 1.6 30.9 ± 1.3 27.6 ± 1.1 * 27.4 ± 1.3 * 体重增加 (g) 4.3 ± 0.5 8.3 ± 0.6 5.1 ± 0.5 * 4.9 ± 0.4**
肝重 (g) 1.04 ± 0.17 1.23 ± 0.09 1.29 ± 0.07 1.34 ± 0.12 脾重 (g) 0.08 ± 0.02 0.09 ± 0.01 0.10 ± 0.02 0.08 ± 0.01 心重 (g) 0.12 ± 0.02 0.12 ± 0.02 0.12 ± 0.01 0.12 ± 0.01 肾重 (g) 0.15 ± 0.02 0.16 ± 0.01 0.17 ± 0.02 0.16 ± 0.01 脑重 (g) 0.42 ± 0.01 0.42 ± 0.02 0.39 ± 0.03 0.38士 0.06 核磁共振分光光度计分析小鼠的脂肪组织与肌肉组织的比值变化， 及脂肪组织与体 重的比值变化， 结果显示： 与对照组相比， 这两个比值在白桦酯醇处理后均下调了 35 -40% (图 5C-D)。
此外， 血清总胆固醇和总甘油三酯的水平在洛伐他汀和白桦酯醇处理后均显著低于 对照组 (图 5E-F)。 而且显而易见地， 白桦酯醇有效降低 LDL胆固醇含量同时升高 HDL 胆固醇含量， 这一效果与洛伐他汀相类似 (图 5G-H)。 在肝脏当中， 白桦酯醇和洛伐他汀 均能够下调总胆固醇水平， 但有趣的是， 只有白桦酯醇能够下调总甘油三酯水平， 下调 约 36% (图 5I-J) , 这显示了白桦酯醇相比他汀所具有的优势效果。
组织病理学分析结果显示： 白桦酯醇能够有效减小白色脂肪组织和褐色脂肪组织中 脂肪细胞的大小， 而洛伐他汀仅能够减小褐色脂肪组织中脂肪细胞的大小（图 5K)。 油红 0对组织切片染色结果表明白桦酯醇及洛伐他汀处理组小鼠的肝脏细胞中性脂质的含量 显著少于对照组 (图 5Κ)。这一结果与前面洛伐他汀和白桦酯醇降低肝脏脂质含量的结果 一致（图 5I-J)。 实施例 4、 白桦酯醇显著提高饮食诱导肥胖小鼠的胰岛素敏感性
已知脂肪酸和甘油三酯代谢异常是诱发胰岛素抵抗和 II型糖尿病的重要原因。由于 白桦酯醇能够有效降低血液和组织中包括脂肪酸和甘油三酯的脂质水平， 本发明人检测 了白桦酯醇对小鼠胰岛素敏感性的作用效果。 与喂养基础饲料的小鼠相比， 高胆固醇高 脂肪饮食诱导的肥胖小鼠表现出葡萄糖耐量和胰岛素耐受。 白桦酯醇或者洛伐他汀处理 后， 可以观察到小鼠的葡萄糖耐量和胰岛素耐受得到明显改善 (图 6A-D)。 进一步， 在饮 食诱导的肥胖小鼠中， 禁食引起的血液葡萄糖和胰岛素水平的升高在白桦酯醇处理组被 显著降低， 洛伐他汀则没有该效果 (图 6E-F)。 综合来讲， 上述数据说明白桦酯醇明显改 善饮食诱导肥胖鼠的胰岛素耐受情况。 实施例 5、 在动物体内， 白桦酯醇有效调控脂质代谢基因的表达变化
白桦酯醇对饮食诱导肥胖鼠肝脏和脂肪组织中基因表达调控特征可反映出该化合物 的有益效果。 白桦酯醇处理后小鼠肝脏中 SREBP-2 mRNA水平下调约 30% (图 7A)。 因 此， 该处理组小鼠肝脏中， 由 SREBP-2调控的参与细胞胆固醇合成途径的一系列基因和 酶类如 HMGCS , HMGCR和三十碳六烯合成酶 （squalene synthase , SS) 等均被下调约 3 1-65%(图 7 A)。同时白桦酯醇显著下调脂肪酸代谢相关基因如 SREBP- 1 c被下调约 32%， 其靶基因脂肪酸合成酶 FAS 被下调约 39%， SCD- 1被下调约 20%。 同时 Malic酶和 PPARa分别被上调约 3 1%， 59% (图 7B)。提示白桦酯醇通过降低细胞脂肪酸合成， 及促 进脂肪水解反应起到降低脂质水平的作用 (DUVal,C.等 (2007). PPARalpha and dyslipidemia. Biochim. Biophys. Acta 1771, 961 -971)。 肝酯酶 (HL)和载脂蛋白 ApoE的 mRNA水平在白桦酯醇处理后被上调 (图 7C)， 进一步促进了白桦酯醇降血脂效果
(Brown,R丄等 (2007). Lipases as modulators of atherosclerosis in murine models. Curr. Drug Targets. 8, 1307- 13 19.； Van Dijk,K.W.等（1999). Dissection of the complex role of apolipoprotein E in lipoprotein metabolism and atherosclerosis using mouse models. Curr. Atheroscler. Rep. 7, 101 - 107) 0 此外， 对于糖代谢相关的基因， 白桦酯醇显著增加了葡萄 糖 -6-磷酸脱氢酶的 mRNA水平，不影响 PEPCK, IRS- 1， IRS-2 ,或葡萄糖激酶 (Glucokinase) 等的表达变化 (图 7D)。
褐色脂肪组织在机体能量代谢中具有重要作用， 白桦酯醇能够显著促进褐色脂肪组 织中 Adiponectin, LPL及 PPAR-γ等基因的表达（图 7E)， 而已知这些基因参与一系列重 要生物学过程， 他们的上调具有抗糖尿病和抗炎症的效果 (Havel,P.J. (2002). Control of energy homeostasis and insulin action by adipocyte hormones: leptin, acylation stimulating protein, and adiponectin. Curr. Opin. Lipidol. 13, 51 -59； Tontonoz,P.等 (2008). Fat and beyond: the diverse biology of PP ARgamma. Annu. Rev. Biochem. 77, 289-3 12)。 总之， 白 桦酯醇调控小鼠肝脏和脂肪组织中一系列 SREBP靶基因的表达， 从而起到降低机体脂 质水平， 提高胰岛素敏感性的作用效果。 实施例 6、 白桦酯醇有效减少动脉粥样硬化斑块形成并增加斑块的稳定性
采用 LDLR基因敲除小鼠 (Ishibashi, S.等（1993). Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J. Clin. Invest 92, 883-893)作为研究对象检测白桦酯醇对动脉粥样硬化斑块形成的作用效 果。 结果显示白桦酯醇处理后， 大动脉处斑块的数量和大小显著低于对照组 (图 8A-D)。 需要指出的是， 与对照组相比， 主动脉弓和胸动脉处的斑块面积在白桦酯醇处理后分别 显著减小约 43%和 77% (图 8C-D)。在另一组实验中， 取小鼠主动脉弓动脉窦的横向切片 做脂质染色实验并进行定量， 结果显示： 与对照组相比， 白桦酯醇处理能显著减少斑块 的面积 (；图 8F-G)。
此外，本发明人通过免疫组化的实验方法检测斑块处巨噬细胞的标志分子 MOMA-2 和平滑肌细胞的标志分子 SMC-actin的分布和面积， 结果显示： 与对照组相比， 白桦酯 醇处理后巨噬细胞斑块的累积显著减少约 55% (图 8F， H) , 平滑肌细胞的量显著增加约 21% (图 8F， 1)。 表明 LDLR基因敲除小鼠在高胆固醇高脂肪饲养条件下， 白桦酯醇能 够显著抑制动脉粥样硬化斑块的形成， 并有效增加斑块的稳定性。 实施例 7、 白桦酯醇特异性结合 SCAP
本发明人推测白桦酯醇的直接靶蛋白是 SCAP蛋白， 为验证这一假说， 本发明人基 于白桦酯醇的结构特征， 合成其小分子探针并命名为化合物 1 (图 9A)。 化合物 1(光亲和探针， 获自华东师范大学)以白桦酯醇为主结构，在其侧链连接光激 活基团和叠氮基团， 当包含靶蛋白 SCAP的膜蛋白 (来源于 CHO-7细胞膜的蛋白混合物) 被探针分子标记之后，可收集蛋白样品，通过 Click Chemistry连接叠氮修饰的报告基团， 进行靶点检测、 富集和鉴定。 首先本发明人检测到化合物 1具有类似于白桦酯醇的生物 学活性 (图 9B)。 该化合物与细胞膜蛋白孵育后经 UV照射使化合物 1与其靶蛋白共价结 合。然后进行后续的 click 环加成反应，最后通过生物素 -亲和素反应使靶蛋白充分富集。
实验结果显示： 在 1和 3 μΜ 化合物 1存在的情况下 SCAP 与之特异性结合， 这一 结合作用在高浓度白桦酯醇存在的情况下受到显著抑制 (图 9C)作为阴性对照转铁蛋白 受体 (Transferrin Receptor)与探针分子结合很弱， 且不被白桦酯醇竞争性抑制。 上述结果 证实了白桦酯醇特异性结合 SCAP蛋白。 实施例 8、 白桦酯醇显著降低小鼠血液脂质水平是 SCAP蛋白依赖性的
为深入探讨白桦酯醇在个体水平的作用效果是否通过调控 SCAP-SREBP 途径， 本发 明人利用腺病毒介导的 RNA干扰实验使小鼠肝脏 SCAP沉默表达后， 检测肝脏基因表 达变化及小鼠血液脂质水平的变化。 腺病毒介导 SCAP shRNA 在小鼠肝脏表达后， 肝 脏 SCAP基因表达水平下调大于 50% (图 10A)， 表明基因沉默效果佳。 同时， 与前期结 果一致的是 shRNA对照组中， 白桦酯醇处理前后 SREBP-2、 HMGCS和 SS等脂质合成 途径基因表达有明显下调； SCAP shRNA处理组由于 SREBP无法正常入核， 这些基因 表达水平组成型下调，白桦酯醇处理后没有看到进一步下调效果 (图 10A)。小鼠血液 TC、 TG、 LDL-c和 HDL-c检测结果显示： shRNA对照组 (Ad-shControl)白桦酯醇处理前后血 液脂质水平变化与前期结果一致， 而 SCAP shRNA处理组 (Ad-shSCAP)白桦酯醇处理后 没有明显效果（图 10B-E)。
该结果提示白桦酯醇在动物体内是通过抑制 SCAP-SREBP途径起到降低脂质水平 的作用效果。 讨论
SREBP蛋白是调控胆固醇脂肪酸及其他脂质生物合成的关键转录因子。 本发明人构 建了一个细胞生物学的分析方法， 利用该方法高通量筛选特异性调控 SREBP途径的小 分子， 并鉴定出白桦酯醇是 SREBP途径的特异性抑制剂。 白桦酯醇通过促进 SCAP与 Insig的相互作用将 SREBP滞留在内质网， 从而抑制 SREBP剪切入核， 下调胆固醇和脂 肪酸合成途径的基因及酶类的表达， 抑制细胞脂质合成途径， 降低细胞脂质水平。 在饮 食诱导的肥胖的小鼠中， 白桦酯醇可降低动物机体总胆固醇和总甘油三酯水平， 提高胰 岛素敏感性。 更进一步地， 白桦酯醇在动脉粥样硬化模型小鼠 LDLR基因敲除小鼠中能 够有效抗动脉粥样硬化的形成。 作为 SREBP抑制剂， 在机体水平， 白桦酯醇具有多方 面的良好疗效， 这证明 SREBP途径可作为潜在的针对代谢性疾病的药物靶点。 尤其针 对于二型糖尿病和动脉粥样硬化症等。
在过去的几十年中， Brown-Goldstein实验室的工作已经揭示了 SREBP途径的调控特 征 (Goldstein, J.L.等 (2006). Protein sensors for membrane sterols. Cell 124, 35-46；
Brown, M. S. and GoldsteinJ.L. (2009). Cholesterol feedback: from Schoenheimer' s bottle to Scap' s MELADL. J. Lipid Res. 50 Suppl, S 15-S27) 0 他们的工作揭示出： 核内活性形式的 SREBP在启动下游靶基因表达后会快速通过 SCFFbw7介导的泛素化途径进行降解
(Sundqvist,A. ^(2005). Control of lipid metabolism by phosphorylation-dependent degradation of the SREBP family of transcription factors by SCF(Fbw7). Cell Metab 1, 379-391)。这一调控过程的复杂性味着存在多种不同的策略可以使 SREBP失去或者降低 活性。 例如， 促进 SCAP与 Insig的相互作用， 抑制 S 1P或者 S2P蛋白酶活性， 促进核 内活性形式的 SREBP降解等。 在本发明中， 通过建立细胞水平的报告基因分析方法， 可以用它来筛选激活或者抑制 SREBP途径的小分子， 这些小分子的作用靶点可以作用 在上述调控过程中的任何一个步骤。 对白桦酯醇的筛选和鉴定的结果证实了本发明人构 建的这一系统的有效性。比较有趣的一点在于，白桦酯醇的作用机制是促进 SCAP与 Insig 的相互作用， 这也正是细胞内源性的调控因子如胆固醇或者 25-羟胆固醇等的调控机制。
25-羟胆固醇是多种已知的 SREBP抑制剂中效力最佳的一种，其效力是胆固醇的 100 倍以上， 而本发明人这里鉴定得到的白桦酯醇其效力与 25-羟胆固醇相当。 并且， 白桦 酯醇不激活 LXR信号通路， 不促进 HMGCR降解。 其高效力和高特异性的特点使其具 有显著降低胆固醇和脂肪酸， 显著改善机体的脂质代谢情况， 有效抗动脉粥样硬化和二 型糖尿病。
虽然胆固醇和 25-羟胆固醇都能够促进 SCAP与 Insig的相互作用， 但是两者的作用 机制是不同的。 胆固醇可以直接结合在 SCAP蛋白的甾醇感受域 (SSD)， 25-羟胆固醇结 合 Insig蛋白。 而白桦酯醇的直接靶点没有鉴定清楚， 考虑到白桦酯醇的结构类似于甾 醇， 而且它不促进 HMGCR降解， 所以白桦酯醇可能像胆固醇一样， 在生理条件下结合 SCAP。
他汀是目前被广泛应用于治疗高胆固醇症的一线药物。作为 HMGCR 的抑制剂， 他 汀有效抑制胆固醇合成， 同时， 肝脏中胆固醇合成受到抑制以后会上调 SREBP的表达， 上调 LDLR的表达促进 LDL的吸收。 通过这两种机制， 他汀有效的降低血清中胆固醇 的水平。 然而， 他汀活化 SREBP表达后将使肝脏胆固醇和脂肪酸合成途径的基因和酶 类的表达， 导致不良的效果 (Kita,T.等（1980). Feedback regulation of
3 -hydroxy-3 -methylglutaryl coenzyme A reductase in livers of mice treated with mevinolin, a competitive inhibitor of the reductase. J. Clin. Invest 66, 1094- 1 100； Singer,I.I.等 (1984). Hydroxymethylglutaryl-coenzyme A reductase-containing hepatocytes are distributed periportally in normal and mevinolin-treated rat livers. Proc. Natl. Acad. Sci. U. S. A 81, 5556-5560)。 与此不同的是， 白桦酯醇直接抑制 SREBP途径， 下调胆固醇和其他脂质的 生物合成。 小鼠在等剂量的洛伐他汀和白桦酯醇的给药条件下， 白桦酯醇表现出与他汀 相当甚至更佳的作用效果。 例如， 白桦酯醇降低小鼠血液， 肝脏， 褐色脂肪组织， 白色 脂肪组织中的脂质水平的效果佳于洛伐他汀。 而且白桦酯醇在改善胰岛素抵抗方面表现 出比洛伐他汀更好的效果。 原因在于白桦酯醇在抑制胆固醇合成的同时还有效抑制脂肪 酸甘油三酯的合成。
综上所述， 本发明人鉴定出白桦酯醇是 SREBP途径的特异性抑制剂， 可以显著降 低脂质水平， 提高胰岛素的敏感性， 抗动脉粥样硬化的形成。 以上实验数据支持一种观 点： 抑制 SREBP途径是一种有益的针对二型糖尿病和动脉粥样硬化症的策略。 白桦酯 醇可作为一种潜在的治疗或者控制代谢性疾病的药物组成。 在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引 用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域技术人员 可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所附权利要求书所限定 的范围。

Claims

权 利 要 求
1. 一种具有如式 I所示的母核结构的化合物或其药学上可接受的盐在制备预防或治 疗代谢性疾病的组合物中的用途：
Figure imgf000033_0001
2. 如权利要求 1所述的用途， 化合物具有如式 II所示的结构。
Figure imgf000033_0002
其中， R独立地选自： 氢、 羟基、 C1-C4垸基、 C2-C4链烯基、 C2-C4链炔基、 C1 -C4 烷氧基、 卤素。 如权利要求 2所 式 III所示的结构:
Figure imgf000033_0003
4. 如权利要求 1所述的用途， 其特征在于， 所述的代谢性疾病包括： II型糖尿病、 高脂 血症、 高胆固醇症、 脂肪肝、 胰岛素抵抗、 肥胖症、 动脉粥样硬化症、 冠心病、 中风、 心 肌梗塞等。
5.如权利要求 4所述的用途， 其特征在于， 所述高脂血症是包括但不仅限于动脉粥样 硬化症或 II型糖尿病。
6. 如权利要求 1所述的用途， 其特征在于， 所述的组合物还用于：
抑制 醇反应元件结合蛋白途径； 或
促进 SREBP切割激活蛋白和 Insig蛋白的相互作用； 或
结合 SREBP切割激活蛋白。 替换页 （细则第 26条)
7. 如权利要求 1所述的用途， 其特征在于， 所述的组合物还用于：
下调甾醇反应元件结合蛋白 -1， 甾醇反应元件结合蛋白 -2， β-羟 [基] -β-甲 [基]戊二酸 单酰辅酶 Α还原酶， β-羟 [基] -β-甲 [基]戊二酸单酰辅酶 Α合成酶，鲨烯环氧酶， SC4MOL, 24-脱氢胆固醇还原酶， FPPS, 7-脱氢胆固醇还原酶， 甲羟戊酸激酶， 羊毛固醇合成酶， FDFT1 , 低密度脂蛋白受体， 胰岛素诱导基因， 鲨烯合成酶， 脂肪酸合成酶， ATP-柠檬 酸裂解酶， 硬脂酰辅酶 A去饱和酶 -1， 硬脂酰辅酶 A去饱和酶 -2， 甘油 -3-磷酸转酰酶， 乙酰辅酶 A羧化酶 α， 脂肪酸去饱和酶 -1或脂肪酸去饱和酶 -2的表达。 8. 如权利要求 1所述的用途， 其特征在于， 所述的组合物还用于：
下调胆固醇、 甘油三酯或脂肪酸生物合成；
降低细胞脂质水平；
降低血液和肝脏的总胆固醇和总甘油三酯水平；
降低血液低密度脂蛋白胆固醇含量， 升高血液高密度脂蛋白胆固醇含量；
减小白色脂肪组织和褐色脂肪组织中脂肪细胞的大小；
改善胰岛素抵抗， 减低血糖， 或提髙胰岛素敏感性；
减少动脉粥样硬化斑块面积； 或
增加动脉粥样硬化斑块稳定性。 9. —种制备药物的方法， 所述的药物用于预防或治疗代谢性疾病， 其特征在于， 所 述方法包括： 将有效量的具有如式 I所示的母核结构的化合物或其药学上可接受的盐与 药学上可接受的载体混合：
Figure imgf000034_0001
10. 一种体外降低细胞胆固醇和脂肪酸生物合成的方法， 其特征在于， 对细胞施用具 有如式 I所示的母核结构的化合 盐：
Figure imgf000034_0002
- 33 - 替换页 （细则第 26条)
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