WO2019104748A1 - 化合物在制备药物中的用途 - Google Patents

化合物在制备药物中的用途 Download PDF

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WO2019104748A1
WO2019104748A1 PCT/CN2017/114645 CN2017114645W WO2019104748A1 WO 2019104748 A1 WO2019104748 A1 WO 2019104748A1 CN 2017114645 W CN2017114645 W CN 2017114645W WO 2019104748 A1 WO2019104748 A1 WO 2019104748A1
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compound
expression
gene
srebp
carbon atom
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PCT/CN2017/114645
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French (fr)
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饶燏
宋保亮
杨兴林
江世友
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清华大学
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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
    • 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
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • 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
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • 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
    • 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

Definitions

  • the present invention relates to the field of biomedicine, and in particular, to the use of a compound for the preparation of a medicament.
  • Metabolic diseases such as hyperlipidemia, obesity, atherosclerosis, cardiovascular and cerebrovascular diseases, and type 2 diabetes have become increasingly serious health problems in China and the world. It is known that the occurrence of these metabolic diseases is closely related to hyperlipidemia. Abnormal lipid metabolism, such as hypercholesterolemia, is the basis of atherosclerosis. Atherosclerosis is a major cause of many cardiovascular diseases, such as coronary heart disease, cerebral infarction, and peripheral vascular disease. High levels of fatty acids and triglycerides in the blood are the main causes of insulin resistance and type 2 diabetes. Therefore, lipid-lowering is an important indicator in the treatment and prevention of metabolic diseases.
  • SREBP transcription factor protein sterol response element binding protein
  • ER endoplasmic reticulum
  • SCAP SREBP cleavage-activating protein
  • S1P Site-1protease
  • S2P Site-2protease
  • Shear maturation of the SREBP protein is strictly regulated by the level of intracellular sterols (cholesterol or oxidized sterols such as 25-hydroxycholesterol).
  • intracellular sterols cholesterol or oxidized sterols 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.
  • the active form of SREBP in the nucleus increases, promoting cellular lipid synthesis.
  • mammalian cells have three different forms of SREBP: SREBP-1a, -1c and SREBP-2, which have the same shear-in nuclear regulatory characteristics, and their maturation processes are as described above.
  • SREBP-1a and -1c mainly regulate the expression of genes in the fatty acid synthesis pathway
  • SREBP-2 mainly regulates the expression of genes involved in the cholesterol synthesis pathway and low-density lipoprotein receptor gene expression.
  • SREBP shear maturation process was inhibited, which resulted in a significant down-regulation of lipid synthesis and a marked decrease in blood cholesterol and triglyceride levels, suggesting that SERBP pathway-specific inhibitors may As a potential lipid-lowering substance.
  • LXR is another type of steroid-regulated nuclear receptor factor that activates SREBP-1c and ultimately leads to hepatic necrosis and hypertriglyceridemia caused by fatty liver.
  • Is there a specific inhibitor of the new highly active SERBP pathway which specifically inhibits SERBP-1, SREBP-2 activation, and does not activate the LXR signaling pathway, which is hyperlipidemia, obesity, atherosclerosis
  • LXR signaling pathway which is hyperlipidemia, obesity, atherosclerosis
  • the inventors of the present application have proposed a compound of formula I or a stereoisomer, geometric isomer, tautomer, oxynitride, hydrate, solvate, metabolite, pharmaceutically acceptable salt or pre-form thereof.
  • the use of a drug in the preparation of a medicament The inventors have surprisingly found that the medicament can effectively prevent and/or treat metabolic diseases, effectively prevent and/or treat hyperlipemia, obesity, atherosclerosis, cardiovascular disease or type 2 diabetes.
  • the invention provides a compound of formula I or a stereoisomer, geometric isomer, tautomer, oxynitride, hydrate, solvate, metabolite, pharmaceutically thereof
  • the use of acceptable salts or prodrugs in the preparation of a medicament is for preventing and/or treating a metabolic disease
  • the carbon atom No. 3 is a single bond or a double bond between R 1 and
  • R 1 is selected from any one of the following groups, and R is an optionally substituted alkyl group:
  • R 1 is selected from any of the following groups:
  • R 2 is -OH, -F or -H
  • R 3 is selected from any of the following groups:
  • the carbon atom No. 5 and the carbon atom No. 6 are single or double bonds, or the carbon atoms of No. 5 and No. 6 carbon atoms together with the oxygen atom form a three-membered epoxy structure;
  • R 4 and R 5 are each independently selected from -OH, oxo, carbonyl, -F or -H;
  • R 6 and R 7 are each independently selected from -H or -OH, or the carbon atom of No. 8 and the carbon atom of No. 9 together with the oxygen atom form a three-membered ring.
  • R 8 is -H or methyl
  • the 22nd carbon atom and the 23rd carbon atom are single or double bonds, or the 22nd carbon atom and the 23rd carbon atom together with the oxygen atom form a ternary epoxy structure;
  • R 9 is an optionally substituted alkyl group, an optionally substituted alkenyl group, a hydroxyl group, an optionally substituted carbonyl group, an optionally substituted cycloalkyl group, an optionally substituted epoxy group, a phosphoric acid group, a sulfonic acid group or a carboxyl group.
  • R 9 is any one selected from the group consisting of:
  • n is independently an integer of 0 to 4, including an endpoint;
  • X is F, Cl, Br or I;
  • R' and R" are each independently a C 1-4 alkyl group.
  • the medicament can effectively prevent and/or treat a metabolic disease.
  • the above use may further include at least one of the following additional technical features:
  • the compound of formula I is any one of the following compounds:
  • the medicament can be further effective in preventing and/or treating a metabolic disease.
  • the metabolic disease is hyperlipidemia, obesity, atherosclerosis, cardiovascular or cerebrovascular disease or type 2 diabetes.
  • the medicament can effectively prevent and/or treat hyperlipemia, obesity, atherosclerosis, cardiovascular disease or type 2 diabetes.
  • the invention provides a compound of formula I or a stereoisomer, geometric isomer, tautomer, oxynitride, hydrate, solvate, metabolite, pharmaceutically thereof Use of an acceptable salt or prodrug in the preparation of a product that lowers fatty acid levels.
  • the product is effective to reduce fatty acid levels.
  • the above use may further include one of the following additional technical features:
  • the lowering of fatty acid levels is achieved by inhibiting the expression of the gene SREBP.
  • the product can further effectively reduce fatty acid levels.
  • the lowering of fatty acid levels is achieved by inhibiting the expression of the gene SREBP in Huh-7 cells or hepatic primary cells.
  • the product can further effectively reduce fatty acid levels.
  • the inhibiting the expression of the gene SREBP in Huh-7 cells or liver primary cells is achieved by at least one of the following:
  • the product can further effectively reduce fatty acid levels.
  • the invention provides a compound of formula I or a stereoisomer, geometric isomer, tautomer, oxynitride, hydrate, solvate, metabolite, pharmaceutically thereof Use of an acceptable salt or prodrug in the preparation of a cell model that inhibits the expression of the gene SREBP.
  • the cell model is effective to inhibit gene SREBP expression.
  • the invention provides a compound of formula I or a stereoisomer, geometric isomer, tautomer, oxynitride, hydrate, solvate, metabolite, pharmaceutically thereof Use of an acceptable salt or prodrug in the preparation of any of the following 1) to 3):
  • the product can effectively inhibit the increase in body weight and fat induced by a high-fat diet and/or decrease the total cholesterol and triglyceride content induced by a high-fat diet and/or increase the high-fat diet-induced obesity patient Glucose and insulin sensitivity.
  • the invention provides a compound of formula I or a stereoisomer, geometric isomer, tautomer, oxynitride, hydrate, solvate, metabolite, pharmaceutically thereof Use of an acceptable salt or prodrug in the preparation of an inhibitor that inhibits the SREBP signaling pathway.
  • the inhibitor is effective to inhibit the SREBP signaling pathway.
  • the above use may further include at least one of the following additional technical features:
  • the suppressing the SREBP signal path is achieved by at least one of the following:
  • the inhibitor may further effectively inhibit the SREBP signaling pathway.
  • the invention provides the use of a pharmaceutical composition for the preparation of a medicament.
  • the pharmaceutical composition comprises the compound of the above formula I or a stereoisomer, a geometric isomer, a tautomer, an oxynitride, a hydrate, a solvate, a metabolite,
  • a pharmaceutically acceptable salt or prodrug is used as an active ingredient for preventing and/or treating a metabolic disease.
  • the pharmaceutical composition can effectively prevent and/or treat a metabolic disease.
  • the above use may further include one of the following additional technical features:
  • the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutical composition can be further effective in preventing and/or treating a metabolic disease.
  • the invention provides a method of preventing and/or treating a metabolic disease.
  • the method comprises: administering to a patient a compound as described above or a stereoisomer, geometric isomer, tautomer, oxynitride, hydrate, solvate, metabolite, A pharmaceutically acceptable salt or prodrug or a pharmaceutical composition as described above.
  • the method is effective.
  • the above method may further comprise one of the following additional technical features:
  • the metabolic disease comprises hyperlipidemia or type 2 diabetes. According to an embodiment of the invention, the method is more effective.
  • the invention first provides a compound of formula I or a stereoisomer, geometric isomer, tautomer, oxynitride, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof.
  • the use in the preparation of a product for preventing and/or treating metabolic diseases such as hyperlipidemia and type 2 diabetes;
  • the carbon atom No. 3 and R 1 are a single bond or a double bond, and when it is a single bond, R 1 is selected from any of the following groups:
  • R is alkyl or substituted alkyl
  • R 1 is selected from any of the following groups:
  • R 2 is -OH, -F or -H
  • R 3 is selected from any of the following groups:
  • a carbon atom of No. 7, a carbon atom of No. 8, a carbon atom of No. 9 and a carbon atom of No. 11 are a single bond or a double bond;
  • R 4 and R 5 are selected from the group consisting of -OH, carbonyl, -F and -H;
  • R 6 and R 7 are both -H or -OH, or R 6 and R 7 together form a ternary with carbon number 8 and carbon number 9 Epoxy structure
  • R 8 is -H or methyl
  • R 9 is a substituted alkyl group, an alcohol, a ketone, an epoxy group, a phosphoric acid or a sulfonic acid, and is specifically selected from any of the following groups:
  • n is a number between 0 and 4;
  • X is -F, -Cl, -Br or -I;
  • R' and R" are each an alkyl group having 1 to 4 carbon atoms.
  • the compound of formula I according to the invention is specifically one of the following compounds:
  • the present invention verified the inhibitory effect of the compound on the activity of the SREBP reporter gene, and assayed in the Huh-7/SRE-Luc cell line (by expressing two reporter genes stably transfected in the human hepatoma cell line Huh-7 cells), The IC 50 values of the inhibition of the SREBP pathway by each compound were determined: compound 1G was 0.22 ⁇ M, compound 13A was 0.44 ⁇ M, compound 12C was 0.86 ⁇ M, compound 14A was 0.94 ⁇ M, compound 13C was 1.05 ⁇ M, and compound 14C was 1.11 ⁇ M.
  • Compound 5A was 1.23 ⁇ M
  • Compound 1C was 1.72 ⁇ M
  • Compound 4A was 2.07 ⁇ M
  • Compound 8A was 4.90 ⁇ M, demonstrating that the compounds according to the examples of the present invention have significant inhibitory effects on the SREBP reporter gene.
  • the present invention verified the expression of the gene SREBP in Huh-7 cells by the compound.
  • the compound 1G according to the embodiment of the present invention can significantly inhibit the SREBP target gene: SREBP-2 (NM_004599.35), HMGCR ( Expressions of NM_000859.2), HMGCS (NM_001098272.2) and SS (NM_004462).
  • SREBP-2 was down-regulated by approximately 40% and HMGCR, HMGCS and SS were down-regulated by approximately 80%. Simultaneously, it was demonstrated that the compound according to the examples of the present invention does not activate the expression of the LXR target gene, and has a significant down-regulation effect on SREBP-1c (NM_001321096.2), FASN (NM_004104.4) and SCD1 (NM_005063) involved in fatty acid synthesis.
  • the present invention verified the expression of the gene SREBP in the mouse liver primary cells by the compound, and the compound 1G according to the embodiment of the present invention can significantly inhibit the SREBP target gene: SREBP-2 (AF374267), HMGCR (by fluorescence real-time quantitative PCR). BF464069), HMGCS (BI143807) and SS (D29016).
  • SREBP-2 was down-regulated by approximately 40% and HMGCR, HMGCS and SS were down-regulated by approximately 50%.
  • the compounds according to the examples of the present invention do not activate the expression of the LXR target gene, and have a significant down-regulation effect on SREBP-1c, FASN and SCD1 involved in fatty acid synthesis.
  • the known LXR activator TO901317 can multiply the above-mentioned genes involved in fatty acid synthesis, which will lead to the formation of fatty liver.
  • a compound of formula I or a stereoisomer, geometric isomer, tautomer, oxynitride, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof, which prevents diet-induced obesity Increased body weight and fat in mice improved cholesterol and triglyceride levels in the blood and liver of mice and significantly increased insulin sensitivity in diet-induced obese mice.
  • a compound according to an embodiment of the present invention has a strong SREBP gene inhibitory activity, such as Compound 1G, which inhibits the SREBP gene pathway with an IC50 value of 0.22 ⁇ M.
  • SREBP gene inhibitory activity such as Compound 1G
  • IC50 value 0.22 ⁇ M.
  • real-time quantitative PCR detection revealed that 1G has a significant expression inhibitory effect on the SREBP target gene and does not activate LXR.
  • a compound according to an embodiment of the present invention can significantly inhibit the increase in body weight and fat of diet-induced obese mice by inhibiting the activity of the SREBP gene, improve cholesterol and triglyceride levels in blood and liver of mice, and increase diet-induced obese mice. Insulin sensitivity, which is significantly better at the animal level than the commercially available drug lovastatin.
  • Figure 1 is a graph showing the activity analysis of a compound according to an embodiment of the present invention using the SRE-Luc/GFP reporter system.
  • Figure 1 (A) is a schematic diagram of the SRE-Luc/GFP dual reporter system
  • Figure 1 (B) is the inhibitory activity of the compound on the SREBP pathway according to an embodiment of the present invention.
  • FIG. 2 is a diagram showing that the compound 1G inhibits the expression of the SREBP splicing into the nucleus and the SREBP target gene according to an embodiment of the present invention, wherein FIG. 2(A) is a Western blotting WB detection of 1G inhibition of SREBP protein shear processing, n-SREBP For the sheared activated form SREBP, pre-SREBP is the full-length form of SREBP, CHC is the internal reference protein; Figure 2 (B) is the quantitative analysis of the SREBP band in Figure 2 (A), and n-SREBP The ratio of /pre-SREBP represents the degree of SREBP processing; Figure 2 (C) shows the inhibition of 1G on the expression of SREBP target genes (SREBP-2, HMGCR, HMGCS, SS) by real-time quantitative PCR detection in Huh7 cells. Fig. 2(D) shows the inhibition of 1G on SREBP target gene expression by fluorescence real-time quantitative PCR in primary hepatocyte
  • FIG. 3 is a schematic diagram showing the expression of a compound 1G inactive LXR target gene according to an embodiment of the present invention, wherein FIG. 3(A) is a real-time quantitative PCR analysis of 1G to LXR target gene (SREBP-1c, ABCA1) in Huh7 cells. , ABCG8, FASN, SCD1) The effect of expression; Figure 3 (B) is the real-time quantitative PCR analysis of the effect of 1G on LXR target gene expression in primary liver cells.
  • FIG. 3(A) is a real-time quantitative PCR analysis of 1G to LXR target gene (SREBP-1c, ABCA1) in Huh7 cells. , ABCG8, FASN, SCD1) The effect of expression
  • Figure 3 (B) is the real-time quantitative PCR analysis of the effect of 1G on LXR target gene expression in primary liver cells.
  • FIG. 4 is a schematic diagram of Compound 1G specifically inhibiting the synthesis of cholesterol according to an embodiment of the present invention.
  • FIG. 4 is a schematic diagram showing that compound 1G significantly inhibits the increase in body weight and fat induced by a mouse high-fat and high-cholesterol diet according to an embodiment of the present invention
  • FIG. 5(A) is a schematic diagram showing inhibition of an increase in body weight of a mouse
  • FIG. 5(B) Schematic diagram of inhibition of mouse fat to body weight ratio.
  • Figure 5 is a schematic illustration of Compound 1G significantly reducing lipid levels in blood and liver of mice under high cholesterol and high fatty acid feeding conditions, wherein Figure 5 (A) is the level of total cholesterol (TC) in the blood, according to an embodiment of the present invention.
  • Schematic diagram of change Figure 5 (B) is a schematic diagram showing changes in total triglyceride (TG) levels in the blood
  • Figure 5 (C) is a graph showing changes in total cholesterol (TC) levels in the liver
  • Figure 5 (D) is a total of liver Schematic diagram of changes in triglyceride (TC) levels;
  • FIG. 6 is a schematic diagram of Compound 1G significantly increasing insulin sensitivity in mice according to an embodiment of the present invention, wherein FIG. 6(A) is a schematic diagram of glucose tolerance, and FIG. 6(B) is a schematic diagram of quantitative analysis of glucose tolerance test.
  • Fig. 6(C) is a schematic diagram of insulin resistance
  • Fig. 6(D) is a schematic diagram of quantitative analysis of insulin resistance test
  • Fig. 6(E) is a schematic diagram showing changes in glucose level in blood.
  • composition administered to a patient as described above or a stereoisomer, geometric isomer, interconversion thereof as used herein.
  • An isomer, an oxynitride, a hydrate, a solvate, a metabolite, a pharmaceutically acceptable salt or prodrug or a pharmaceutical composition as described above means that a predetermined amount of a substance is introduced into a patient by some suitable means.
  • the composition can be administered by any conventional route as long as it can reach the intended tissue.
  • the invention is not limited to these exemplary modes of administration.
  • oral administration the active ingredient of the orally administered composition should be coated or formulated to prevent its degradation in the stomach.
  • the compounds or pharmaceutical compositions of formula I of the invention may be administered using a particular device that delivers the active ingredient to the target cells.
  • the frequency and dosage of the pharmaceutical composition of the present invention can be determined by a number of relevant factors including the type of disease to be treated, the route of administration, the age, sex, weight and severity of the disease as well as the active ingredient. Type of drug.
  • therapeutically effective amount refers to an amount of a compound that is sufficient to significantly ameliorate certain symptoms associated with a disease or condition, that is, an amount that provides a therapeutic effect for a given condition and dosage regimen.
  • a therapeutically effective amount of a drug or compound does not require a cure for the disease or condition, but will provide a treatment for the disease or condition such that the onset of the disease or condition of the individual is delayed, prevented or prevented, or the symptoms of the disease or condition are alleviated, or the disease or The duration of the condition is altered, or for example the disease or condition becomes less severe, or the recovery is accelerated.
  • treatment is used to mean obtaining the desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing the disease and/or the adverse effects caused by the disease.
  • treatment encompasses the treatment of a disease in a mammal, particularly a human, including: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed; (b) inhibiting the disease; or (c) Relieve diseases, such as alleviating symptoms associated with the disease.
  • treatment encompasses any administration of a medicament or compound to an individual to treat, cure, ameliorate, ameliorate, ameliorate or inhibit the disease of the individual, including but not limited to administration of a compound or pharmaceutical composition of formula I described herein. Individuals in need.
  • the excipients include pharmaceutically acceptable excipients, lubricants, fillers, diluents, disintegrants, stabilizers, preservatives, emulsifiers, solubilizers, colorants well known in the formulation arts. , sweetener, made into tablets, pills, capsules, injections and other different dosage forms.
  • the articles used herein are used to refer to the articles of one or more than one (ie, at least one).
  • a component refers to one or more components, that is, there may be more than one component contemplated for use or use in embodiments of the embodiments.
  • Stereoisomer refers to a compound that has the same chemical structure but differs in the way the atoms or groups are spatially aligned. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotomers), geometric isomers (cis/trans) isomers, atropisomers, etc. .
  • “Chirality” is a molecule that has properties that cannot overlap with its mirror image; “non-chiral” refers to a molecule that can overlap with its mirror image.
  • Enantiomer refers to two isomers of a compound that are not superimposable but are mirror images of each other.
  • Diastereomer refers to a stereoisomer that has two or more centers of chirality and whose molecules are not mirror images of each other. Diastereomers have different physical properties such as melting point, boiling point, spectral properties and reactivity. The mixture of diastereomers can be separated by high resolution analytical procedures such as electrophoresis and chromatography, such as HPLC.
  • optically active compounds Many organic compounds exist in optically active forms, i.e., they have the ability to rotate a plane of plane polarized light.
  • the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule with respect to one or more of its chiral centers.
  • the prefixes d and l or (+) and (-) are symbols for specifying the rotation of plane polarized light caused by the compound, wherein (-) or l indicates that the compound is left-handed.
  • Compounds prefixed with (+) or d are dextrorotatory.
  • a particular stereoisomer is an enantiomer and a mixture of such isomers is referred to as a mixture of enantiomers.
  • a 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process.
  • any asymmetric atom (e.g., carbon, etc.) of the compounds disclosed herein may exist in racemic or enantiomerically enriched form, such as the (R)-, (S)- or (R, S)-configuration presence.
  • each asymmetric atom has at least 50% enantiomeric excess in the (R)- or (S)-configuration, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess.
  • the compounds of the invention may be one of the possible isomers or a mixture thereof
  • the complex for example in the form of a mixture of racemates and diastereomers (which depends on the number of asymmetric carbon atoms), is present.
  • Optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituent may be in the E or Z configuration; if the compound contains a disubstituted cycloalkyl group, the substituent of the cycloalkyl group may have a cis or trans configuration.
  • the resulting mixture of any stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, diastereomers, for example, by chromatography, depending on the difference in physicochemical properties of the components. Method and / or step crystallization.
  • racemate of any of the resulting end products or intermediates can be resolved into the optical antipodes by methods known to those skilled in the art by known methods, for example, by obtaining the diastereomeric salts thereof. Separation. Racemic products can also be separated by chiral chromatography, such as high performance liquid chromatography (HPLC) using a chiral adsorbent.
  • HPLC high performance liquid chromatography
  • enantiomers can be prepared by asymmetric synthesis, for example, see Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2 nd Ed. Robert) E.
  • tautomer or "tautomeric form” refers to structural isomers having different energies that are interconvertible by a low energy barrier. If tautomerism is possible (as in solution), the chemical equilibrium of the tautomers can be achieved.
  • proton tautomers also known as prototropic tautomers
  • Valence tautomers include interconversions by recombination of some bonding electrons.
  • keto-enol tautomerization is the interconversion of a pentane-2,4-dione and a 4-hydroxypent-3-en-2-one tautomer.
  • Another example of tautomerization is phenol-keto tautomerization.
  • a specific example of phenol-keto tautomerization is the interconversion of pyridin-4-ol and pyridine-4(1H)-one tautomers. All tautomeric forms of the compounds of the invention are within the scope of the invention unless otherwise indicated.
  • the compounds of the present invention may be optionally substituted with one or more substituents, such as the compounds of the above formula, or specific examples, subclasses, and inclusions of the present invention.
  • substituents such as the compounds of the above formula, or specific examples, subclasses, and inclusions of the present invention.
  • a class of compounds A class of compounds.
  • substituents such as the compounds of the above formula, or specific examples, subclasses, and inclusions of the present invention.
  • a class of compounds A class of compounds.
  • substituents such as the compounds of the above formula, or specific examples, subclasses, and inclusions of the present invention.
  • a class of compounds A class of compounds.
  • C 1 - 6 alkyl refers particularly to the disclosure independently methyl, ethyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, and C 6 alkyl.
  • linking substituents are described.
  • the Markush variable recited for that group is understood to be a linking group.
  • the definition of the Markush group for the variable is "alkyl” or "aryl”
  • the “alkyl” or “aryl” respectively represent the attached An alkylene group or an arylene group.
  • alkyl or "alkyl group” as used herein, denotes a saturated straight or branched monovalent hydrocarbon group containing from 1 to 20 carbon atoms, wherein the alkyl group may be optionally selected The ground is replaced by one or more substituents described herein. Unless otherwise specified, an alkyl group contains from 1 to 20 carbon atoms. In one embodiment, the alkyl group contains from 1 to 4 carbon atoms.
  • alkyl groups include, but are not limited to, methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), n-propyl (n-Pr, -CH 2 CH 2 CH 3 ), isopropyl (i-Pr, -CH(CH 3 ) 2 ), n-butyl (n-Bu, -CH 2 CH 2 CH 2 CH 3 ), isobutyl (i-Bu, -CH 2 CH) (CH 3 ) 2 ), sec-butyl (s-Bu, -CH(CH 3 )CH 2 CH 3 ), tert-butyl (t-Bu, -C(CH 3 ) 3 ), n-pentyl (-CH) 2 CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl (-CH(CH 2 CH 3 ) 2 ), 2-methyl -2-butyl (-C(CHCH
  • alkenyl denotes a straight or branched chain monovalent hydrocarbon radical containing from 2 to 15 carbon atoms, wherein there is at least one site of unsaturation, i.e., having a carbon-carbon sp 2 double bond, wherein the alkenyl group
  • the group may be optionally substituted with one or more substituents described herein, including the positioning of "cis” and “tans", or the positioning of "E” and "Z”.
  • alkynyl denotes a straight or branched chain monovalent hydrocarbon radical containing from 2 to 15 carbon atoms, wherein at least one site of unsaturation, i.e., has a carbon-carbon sp triple bond, wherein the alkynyl group It may be optionally substituted with one or more of the substituents described herein.
  • alkynyl groups include, but are not limited to, ethynyl (-C ⁇ CH), propargyl (-CH 2 C ⁇ CH), 1-propynyl (-C ⁇ C-CH 3 ), and the like. .
  • prodrug denotes a compound which is converted in vivo to a compound of formula (I). Such transformation is affected by the hydrolysis of the prodrug in the blood or by enzymatic conversion to the parent structure in the blood or tissue.
  • the prodrug-like compound of the present invention may be an ester.
  • the ester may be used as a prodrug such as a phenyl ester, an aliphatic (C 1-24 ) ester, an acyloxymethyl ester, or a carbonate. , carbamates and amino acid esters.
  • a compound of the invention comprises a hydroxyl group, i.e., it can be acylated to give a compound in the form of a prodrug.
  • Other prodrug forms include phosphates, such as those obtained by phosphorylation of a hydroxy group on the parent.
  • Metal product refers to a product obtained by metabolism of a specific compound or a salt thereof in vivo. Metabolites of a compound can be identified by techniques well known in the art, and the activity can be characterized by experimental methods as described herein. Such a product may be obtained by administering a compound by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage and the like. Accordingly, the invention includes metabolites of a compound, including metabolites produced by intimate contact of a compound of the invention with a mammal for a period of time.
  • the "pharmaceutically acceptable salt” as used in the present invention means an organic salt and an inorganic salt of the compound of the present invention.
  • Pharmaceutically acceptable salts are well known in the art, as described in the literature: SMBerge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19.
  • Salts formed by pharmaceutically acceptable non-toxic acids include, but are not limited to, mineral acid salts formed by reaction with amino groups, hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, And organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, or by other methods described in the literature, such as ion exchange These salts.
  • salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, disulfate, borate, butyrate, camphoric acid Salt, camphor sulfonate, cyclopentylpropionate, digluconate, lauryl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate Salt, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, Malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectate, persulphate, 3 -Phenylpropionate
  • Salts obtained by appropriate bases include the alkali metal, alkaline earth metal, ammonium and N + (C 1-4 alkyl) 4 salts.
  • the present invention also contemplates quaternary ammonium salts formed from any of the compounds comprising a group of N. Water soluble or oil soluble or dispersed products can be obtained by quaternization.
  • Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Pharmaceutically acceptable salts further comprise suitable amine cation nontoxic ammonium, quaternary ammonium, and the counterion, such as halide, hydroxide, carboxylate, sulfated, phosphorylated compounds, nitrate compounds, C 1 -8 sulfonate and aromatic sulfonate.
  • suitable amine cation nontoxic ammonium, quaternary ammonium, and the counterion such as halide, hydroxide, carboxylate, sulfated, phosphorylated compounds, nitrate compounds, C 1 -8 sulfonate and aromatic sulfonate.
  • Solvent-forming solvents include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol.
  • hydrate means that the solvent molecule is an association formed by water.
  • any disease or condition as used in the present invention refers to ameliorating a disease or condition (ie, slowing or preventing or alleviating the progression of a disease or at least one of its clinical symptoms).
  • “treating” refers to alleviating or ameliorating at least one physical parameter, including physical parameters that may not be perceived by the patient.
  • “treating” refers to modulating a disease or condition from the body (eg, stabilizing a detectable symptom) or physiologically (eg, stabilizing the body's parameters) or both.
  • “treating” refers to preventing or delaying the onset of a disease or condition, Occurred or worsened.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids, such as acetate, aspartate, benzoate, besylate, bromide/hydrobromide, bicarbonate/ Carbonate, hydrogen sulfate/sulfate, camphorsulfonate, chloride/hydrochloride, chlorophylline, citrate, ethanedisulfonate, fumarate, glucoheptonate, Portuguese Saccharate, glucuronate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, Malay Acid salt, malonate, mandelic acid salt, methanesulfonate, methyl sulfate, naphthoate, naphthalene sulfonate, nicotinate, nitrate, octadecanoate, oleate, oxalic acid Salt, palmitate
  • Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid. , ethanesulfonic acid, p-toluenesulfonic acid, sulfosalicylic acid, and the like.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic bases and organic bases.
  • Inorganic bases from which salts can be derived include, for example, ammonium salts and metals of Groups I to XII of the Periodic Table.
  • the salt is derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include the ammonium, potassium, sodium, calcium, and magnesium salts.
  • Organic bases from which salts can be derived include primary, secondary and tertiary amines, and substituted amines include naturally occurring substituted amines, cyclic amines, basic ion exchange resins and the like.
  • Certain organic amines include, for example, isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine. .
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, basic or acidic moiety by conventional chemical methods.
  • such salts can be obtained by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base such as a hydroxide, carbonate, bicarbonate, or the like of Na, Ca, Mg or K.
  • a suitable base such as a hydroxide, carbonate, bicarbonate, or the like of Na, Ca, Mg or K.
  • the free base form of these compounds is prepared by reaction with a stoichiometric amount of a suitable acid. This type of reaction is usually carried out in water or an organic solvent or a mixture of the two.
  • a non-aqueous medium such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile.
  • a non-aqueous medium such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile.
  • the compounds disclosed in the present invention may also be obtained in the form of their hydrates or in the form of their solvents (e.g., ethanol, DMSO, etc.) for their crystallization.
  • solvents e.g., ethanol, DMSO, etc.
  • the compounds disclosed herein may form solvates either intrinsically or by design with pharmaceutically acceptable solvents, including water; thus, the invention is intended to include both solvated and unsolvated forms.
  • any structural formula given by the present invention is also intended to indicate that these compounds are not isotopically enriched and isotopically enriched.
  • Isotopically enriched compounds have the structure depicted by the general formula given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number.
  • Exemplary isotopes that may be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 17 O , 18 O, 18 F, 31 P, 32 P, 35 S, 36 Cl and 125 I.
  • the compounds of the invention include isotopically enriched compounds of the invention, for example, those in which a radioisotope such as 3 H, 14 C and 18 F is present, or in which a non-radioactive isotope is present, such as 2 H and 13 C.
  • a radioisotope such as 3 H, 14 C and 18 F
  • a non-radioactive isotope such as 2 H and 13 C.
  • isotopically enriched compounds can be used for metabolic studies (using 14 C), reaction kinetic studies (using, for example, 2 H or 3 H), detection or imaging techniques such as positron emission tomography (PET) or including drugs or Single photon emission computed tomography (SPECT) of substrate tissue distribution assays, or may be used in patient radiation therapy.
  • 18 F enriched compounds are particularly desirable for PET or SPECT studies.
  • the isotopically enriched compound of formula (I) can be prepared by conventional techniques familiar to those skilled in the art or by the use of suitable isotopically labeled reagents in place of the previously used unlabeled reagents as described in the Examples and Preparations of the present invention.
  • isotopes particularly deuterium (i.e., 2 H or D)
  • substitution of heavier isotopes may provide certain therapeutic advantages resulting from higher metabolic stability. For example, increased in vivo half-life or reduced dose requirements or improved therapeutic index.
  • Isotopic enrichment factors can be used to define the concentration of such heavier isotopes, particularly ruthenium.
  • isotopic enrichment factor refers to the ratio between the isotope abundance and the natural abundance of a given isotope.
  • a substituent of a compound of the invention is designated as hydrazine
  • the compound has at least 3500 for each of the specified hydrazine atoms (52.5% of ruthenium incorporation at each of the specified ruthenium atoms), at least 4,000 (60% of ruthenium incorporation), At least 4,500 (67.5% of cerium incorporation), at least 5,000 (75% of cerium incorporation), at least 5,500 (82.5% of cerium incorporation), at least 6,000 (90% of cerium incorporation), at least 6333.3 (95%) Iridium enrichment factor with at least 6466.7 (97% cerium incorporation), at least 6600 (99% cerium incorporation) or at least 6633.3 (99.5% cerium incorporation).
  • the present invention can include pharmaceutically acceptable solvates wherein the solvent of crystallization may be isotopically substituted, for example D 2 O, acetone -d 6, DMSO-d 6 solvate of those.
  • the invention relates to an intermediate for the preparation of a compound encompassed by formula (I).
  • the invention relates to a process for the preparation, isolation and purification of a compound encompassed by formula (I).
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the invention, a pharmaceutically acceptable carrier, an excipient, a diluent, an adjuvant, a vehicle, or a combination thereof.
  • the pharmaceutical composition can be in the form of a liquid, solid, semi-solid, gel or spray.
  • “Combination” means a fixed combination in a single dosage unit form or a kit for a portion to be administered in combination, wherein the compound of the present disclosure and the combination partner can be administered separately at the same time or can be administered separately at certain intervals, in particular It is to make the joint partners show cooperation, such as synergy.
  • the terms "co-administered” or “co-administered” and the like as used herein are intended to encompass the administration of a selected combination partner to a single individual (eg, a patient) in need thereof, and is intended to include wherein the substance does not have to be administered by the same route of administration or simultaneously. Treatment plan.
  • pharmaceutical combination product denotes a product obtained by mixing or combining more than one active ingredient, and includes both a fixed combination of active ingredients and a non-fixed combination.
  • fixed combination means that the active ingredient, such as a compound of the present invention and a combination partner, is administered to a patient simultaneously in the form of a single entity or dosage.
  • non-fixed combination means that the active ingredient, such as a compound of the present invention and a combination partner, are administered to a patient as a separate entity simultaneously, together or without specific time constraints, wherein the administration provides a therapeutically effective level of both compounds in the patient. .
  • Wool sterol (50%, purchased from TCI, 100 mg) was dissolved in dichloromethane (30 mL), and added to the above solution in two portions of m-CPBA (85%, 28 mg) and NaHCO 3 (14 mg) at intervals of 3 h in an ice bath. Stir at room temperature overnight. The reaction solution was washed with a saturated aqueous solution of sodium hydrogencarbonate, dried over anhydrous sodium carbonate, and then evaporated to dryness, and the solvent was evaporated on a silica gel column ( petroleum ether: ethyl acetate 10:1) to give compound 1C (45 mg) Compound 1A (40 mg).
  • the compound (b) (100 mg) was dissolved in a mixture of 3 ml of DCM and 2 ml of DMF.
  • the reaction solution was washed with a saturated aqueous solution of sodium hydrogencarbonate, dried over anhydrous sodium carbonate, and then evaporated to dryness, and then the solvent was evaporated on a silica gel column ( petroleum ether: ethyl acetate 50:1) to give compound (c) (60 mg) ), the yield was 71%.
  • Huh-7 human hepatoma cell line; purchased from ATCC
  • Huh-7/SRE-Luc cells were cultured in a 37 ° C, 5% CO 2 incubator, and the cells were grown in medium A (DMEM, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin) plus 10% FBS.
  • CHO-7 monoclonal cells screened by CHO-K1 cells in delipoprotein serum medium; purchased from ATCC) were cultured in a 37 ° C, 5% CO 2 incubator, and cells were grown in medium B (DMEM and The F12 medium was mixed in equal proportions, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin) plus 5% FBS.
  • Example 22 Determination of SREBP reporter gene activity inhibition by a compound according to an embodiment of the invention
  • the following method utilizes the Huh-7/SRE-Luc cell line to determine the activity of a compound according to an embodiment of the invention to inhibit the SREBP signaling pathway.
  • the Huh-7/SRE-Luc cell line expresses two reporter genes by stable transfection in human hepatoma cell line Huh-7 cells: firefly with promoter having SREBP protein binding site and dependent on SREBP signaling activity
  • the luciferase SRE-Firefly Luciferase, and the green fluorescent protein EGFP which is continuously expressed as an internal reference, are shown in Figure 1 (A).
  • Huh-7/SRE-Luc cells were cultured in DMEM medium (containing 10% FBS, 100 units/ml P/S), and when the cell confluence reached 80%, 0.25% trypsin was used. EDTA) was digested and mixed, and then inoculated into a 24-well plate at 5 ⁇ 10 4 cells per well. After incubating for 24 hours at 37 ° C in a 5% CO 2 incubator, the medium was aspirated and washed 1 time with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 ).
  • PBS buffer 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 .
  • delipoprotein DMEM medium containing 10% delipoprotein serum, 1 ⁇ M lovastatin, 10 ⁇ M mevalonate, P/S
  • the SREBP signaling pathway is activated to a degree.
  • the medium was aspirated, 150 ⁇ l of cell lysate Reporter Lysis Buffer (Promega, E397A) was added to each well, and the 24-well plate was frozen in a -80 ° C refrigerator, and then thawed at room temperature for 1 time, followed by vortexing at 800 rpm. Shake on the spinner for 10 minutes to achieve full lysis of cells.
  • the compounds of the examples of the present invention have significant inhibitory effects on the SREBP reporter gene, wherein the most active compound 1G IC50 is 0.22 ⁇ M.
  • Example 23 a compound according to an embodiment of the invention inhibits shear processing ripening of SREBP
  • the following method was used to determine the inhibition of shear processing maturation of SREBP by a compound according to an embodiment of the invention.
  • the experimental method is briefly described as follows: CHO-7 cells were transferred to a 60 mm culture dish at 6 ⁇ 10 5 and cultured for 1 day. The medium was aspirated, washed once with PBS, PBS was removed, and delipidated DMEM medium containing different concentrations of compound (containing 10% delipoprotein and de-fatty acid serum, 1 ⁇ M lovastatin, 10 ⁇ M mevalonate, P/) was added. S) After 15 hours of treatment, MG132 (Cayman, 10012628) was added to a final concentration of 10 ⁇ M.
  • the cells in the culture dish were scraped off with a cell scraper, transferred to a 15 ml centrifuge tube, and placed on ice. After centrifugation at 1000 g for 5 minutes at 4 ° C, the medium was discarded, 1 ml of PBS buffer was added, and transferred to a 1.5 ml centrifuge tube, and centrifuged at 1000 g for 5 minutes at 4 ° C. The PBS was removed, and 300 ⁇ l of SDS Lysis Buffer (10 mM Tris-HCl (pH 7.6), 100 mM sodium chloride, 1% sodium dodecyl sulfate and protease inhibitor) was added, and the No. 7 needle was applied 15 times.
  • SDS Lysis Buffer 10 mM Tris-HCl (pH 7.6), 100 mM sodium chloride, 1% sodium dodecyl sulfate and protease inhibitor
  • Electrophoresis The protein was first introduced into the concentrated gel with a voltage of 85 volts for 30 minutes, and then the protein was allowed to enter the separation gel at 120 volts for 1 hour.
  • Transfer film After the electrophoresis is finished, the gel is taken out into the film transfer device in the order of filter paper, nitrocellulose film, gel, and filter paper, and the bubbles are removed, and the film is transferred at a constant pressure of 100 volts for 1.5 hours.
  • Fig. 2(A) the compound according to the examples of the present invention significantly reduced the formation of the nuclear form (n-SREBP) of SREBP accompanied by an increase in the precursor form SREBP (pre-SREBP).
  • Fig. 2(B) show that the compound according to the embodiment of the present invention is very active in inhibiting SREBP shear processing, and has an IC50 value of 0.046 ⁇ M.
  • Example 24 Determination of inhibitory activity of a compound according to an embodiment of the present invention on SREBP target gene expression
  • Huh-7 cells were transferred to a 60 mm culture dish at 6 x 10 5 and cultured for 24 hours. The medium was aspirated and washed once with PBS. The PBS was removed, and delipoprotein and de-fatty acid DMEM medium (containing 10% delipoprotein and de-fatty acid serum, 1 ⁇ M lovastatin, 10 ⁇ M mevalonate, P/S) containing different concentrations of compounds were added and treated for 16 hours.
  • delipoprotein and de-fatty acid DMEM medium containing 10% delipoprotein and de-fatty acid serum, 1 ⁇ M lovastatin, 10 ⁇ M mevalonate, P/S
  • the medium was aspirated, 1 ml of TRIzol (Sigma, T9424) was added, and the cells were lysed for 10 minutes at room temperature, and the cell lysate was transferred to a 1.5 ml Eppendorf tube, followed by extraction of mRNA, reverse transcription synthesis of cDNA, and real-time quantitative PCR.
  • TRIzol Sigma, T9424
  • RNA Take 2 ⁇ l of RNA into 98 ⁇ l of water, mix and measure the absorbance at 260 nm with a spectrophotometer (Eppendorf), calculate the sample concentration, determine the ratio of the absorbance at 260 nm to 280 nm, calculate the sample concentration, and finally adjust the sample concentration to 1 ⁇ g. / ⁇ l.
  • a spectrophotometer Eppendorf
  • cDNA synthesis was performed using Promega's M-MLV reverse transcriptase kit. Each 50 ⁇ l system contained 4 ⁇ g of RNA, 1 ⁇ g of OligodT, and a final concentration of 0.4 mM dNTPs.
  • Realtime PCR uses Shengyuan Bio's Sharpvue 2x Universal qPCR Master Mix reagent.
  • Sharpvue 2x Mix 10 ⁇ l, a primer with a final concentration of 0.5 ⁇ M, 2 ⁇ l of template cDNA (diluted 4 times for reverse transcription), and supplemented with water to 20 ⁇ l. After the reaction system is configured, mix and mix, and each sample has 3 duplicate wells.
  • the compound 1G according to the example of the present invention can significantly inhibit the expression of the SREBP target genes: SREBP-2, HMGCR, HMGCS and SS.
  • SREBP-2 was down-regulated by approximately 40%
  • HMGCR, HMGCS, and SS were down-regulated by approximately 80%.
  • Example 25 Determination of inhibitory activity of a compound according to an embodiment of the present invention on SREBP target gene expression in mouse hepatic primary cells
  • collagenase solution 100 ml was exchanged at the same rate for 0.15 mg/ml.
  • the collagenase digestion solution was: 137 mM NaCl, 5.4 mM KCl, 0.5 mM NaH2PO4, 0.4 mM Na 2 HPO 4 , 4.2 mM NaHCO 3 , 10 mM HEPES, pH 7.4. , 15 mg type I collagenase (Worthington Biochemical). Every 100 ml of solution.
  • a sieve having a pore size of 70 ⁇ m was centrifuged at 500 rpm for 1 minute. The supernatant was carefully aspirated, and the cells centrifuged to the bottom of the tube were added to an appropriate amount of DMEM medium (containing 10% FBS, P/S), counted, and inoculated at 1 ⁇ 10 6 cells per 60 mm dish.
  • DMEM medium containing 10% FBS, P/S
  • the results of the assay are shown in Fig. 2(D).
  • the compound 1G of the present invention significantly inhibited the expression of the SREBP target genes: SREBP-2, HMGCR, HMGCS and SS.
  • SREBP-2 was down-regulated by approximately 40%
  • HMGCR, HMGCS, and SS were down-regulated by approximately 50%.
  • the compounds of the examples of the present invention have significant inhibitory effects on the expression of SREBP target genes in primary hepatocytes, indicating the effect of lowering blood fat.
  • Example 26 a compound according to an embodiment of the invention does not activate LXR
  • the effect of the compound according to the examples of the present invention on LXR was determined in the same manner as in Example 3, and the following is briefly described as follows: in the human hepatoma cell line Huh7 cells, after adding delipoprotein and de-fatty acid DMEM medium containing different concentrations of compounds, , mRNA extraction, reverse transcription and real-time quantitative PCR analysis.
  • the primer sequences used in the fluorescent quantitative PCR in this example are shown in Table 5.
  • LXR target genes are involved in cholesterol efflux and fatty acid synthesis. Activation of LXR significantly upregulates SREBP-1c expression, leading to fatty liver formation and hypertriglyceridemia.
  • the results of the assay in Figure 3 (A) show that the compound according to an embodiment of the present invention does not activate the expression of the LXR target gene, and has a significant down-regulation effect on SREBP-1c, FASN and SCD1 involved in fatty acid synthesis. It is known that the LXR activator TO901317 (Sigma) significantly activates all LXR target genes.
  • This example demonstrates that a compound according to an embodiment of the present invention does not activate expression of an LXR target gene and has a significant inhibitory effect on a fatty acid synthesis-related gene.
  • Example 27 a compound according to an embodiment of the invention does not activate LXR in mouse hepatic primary cells
  • the effect of the compound according to the embodiment of the present invention on LXR was determined in mouse hepatic primary cells in the same manner as in Example 4, and the following is briefly described as follows: First, the mouse liver primary cells are isolated, and delipidated with different concentrations of compounds is added. After protein and de- fatty acid DMEM medium treatment, mRNA extraction, reverse transcription and real-time quantitative PCR analysis were performed. The primer sequences used in the fluorescent quantitative PCR in this example are shown in Table 6.
  • the results of the assay in Figure 3 (B) show that Compound 1G according to an embodiment of the present invention does not activate the expression of the LXR target gene, and has a significant down-regulation effect on SREBP-1c, FASN and SCD1 involved in fatty acid synthesis.
  • the known LXR activator TO901317 can multiply the above-mentioned genes involved in fatty acid synthesis, which will lead to the formation of fatty liver.
  • Example 28 a compound according to an embodiment of the invention prevents diet and induced increase in body weight and fat in obese mice
  • the above cell experiments show that the compounds according to the examples of the present invention strongly inhibit the shear processing of the SREBP protein, thereby inhibiting the expression of the SREBP target gene and inhibiting the synthesis of cholesterol and fatty acids.
  • the lipid lowering effect of the compound according to the examples of the present invention was analyzed.
  • the feeding methods of all the following mouse experiments were in accordance with the Animal Feeding and Use Regulations of the Experimental Animal Center of Wuhan University.
  • mice 8 week old C57BL/6J mice (purchased from Shanghai Lingchang Company) were randomly divided into 4 groups of 5 mice each. The first group of mice were fed a basal diet (Chow Diet, CD) while a normal saline was administered daily. Groups 2-4 were fed a diet of high-fat hypercholesterolemia (Western Diet, WD), also known as Western diet, containing 20% fat, 1.25% cholesterol, and 0.5% sodium cholate.
  • CD basal diet
  • a normal saline was administered daily.
  • Groups 2-4 were fed a diet of high-fat hypercholesterolemia (Western Diet, WD), also known as Western diet, containing 20% fat, 1.25% cholesterol, and 0.5% sodium cholate.
  • mice were given normal saline once a day;
  • Group 3 mice were administered lovastatin (Lovastatin, Lova) by intragastric administration at a dose of 60 mg/kg body weight/day;
  • group 4 mice were given Stomach Compound 1G according to an embodiment of the present invention, administered in an amount of 60 mg/kg body weight/day.
  • the system examined and analyzed various metabolic indicators of mice.
  • Fig. 4(A) (statistical one-way ANOVA test analysis, where * indicates p ⁇ 0.05, *** indicates p ⁇ 0.001), high fat and high cholesterol feed after 12 weeks of saline, saline
  • the control mice became obese and gained weight to 31 g.
  • the mice in the lovastatin-administered group weighed 27.5 g, while the mice in the compound 1G-administered group according to the examples of the present invention weighed 24.8 g, which was close to the body weight of the basal-feed group.
  • the above results demonstrate that the compound 1G according to the embodiment of the present invention has an increase in body weight of mice induced by inhibiting high-fat and high-cholesterol diet, has a weight-loss effect, and is more effective than lovastatin.
  • Example 29 a compound according to an embodiment of the invention improves cholesterol and triglyceride levels in blood and liver of mice
  • mice After the mice were sacrificed, blood was collected, allowed to stand at room temperature for 1 hour, centrifuged at 1500 g for 10 minutes at 4 ° C, and the supernatant was taken for subsequent measurement.
  • the contents of total cholesterol, triglyceride, glucose and free fatty acids in the blood were determined using the corresponding kits and determined according to the manufacturer's instructions (Shanghai Kehua Bioengineering Co., Ltd.).
  • mice were sacrificed by cervical dislocation after blood collection, the thoracic cavity was opened, the right atrial appendage was cut, and about 20 ml of PBS was perfused from the left ventricle. After perfusion, the liver was removed, and about 50 mg was weighed and the weight was recorded. The mixture was added to a 2 ml tissue disrupting tube containing 1.2 ml of chloroform-methanol (2:1) and a small amount of ceramic beads, and a homopolymer was used with Precellys 24 ( Bertin) was homogenized and crushed at 5500 rpm for 10 seconds for 3 times.
  • the mixture was shaken and mixed at room temperature for 1-2 hours, then centrifuged at 13200 rpm for 10 minutes at room temperature. Pipette 1 ml of the supernatant and transfer to a new 1.5 ml centrifuge tube. Add 400 ⁇ l of double distilled water to shake and mix. Leave at room temperature for 10 minutes. Centrifuge for another 10 minutes at 13200 rpm. The upper aqueous phase was removed, and 500 ⁇ l of the lower organic phase was pipetted into a new 1.5 ml centrifuge tube, dried with nitrogen, dissolved in alcohol, and the total cholesterol and triglyceride content were determined in the same manner as above.
  • the compound 1G according to the embodiment of the present invention significantly lowers the total cholesterol and triglyceride content in the liver fat induced by the high fat and high cholesterol feed, and is more remarkable than lovastatin, and has an effect of lowering fatty liver.
  • Example 30 a compound according to an embodiment of the invention significantly increases insulin sensitivity in diet-induced obese mice
  • Glucose tolerance test After 12 weeks of dosing, the mice were fasted to starvation overnight before the start of the experiment. On day 2, each mouse was weighed and labeled, and D-glucose solution (2 g/kg body weight) was intraperitoneally injected according to the body weight of the mice. The glucose concentration in the blood of the tail vein of the mouse was measured by a blood glucose meter (Johnson) at 0, 30, 60, and 120 minutes after the injection.
  • Insulin tolerance test fasting starvation for 4 hours before the start of the experiment, intraperitoneal injection of insulin (0.7 U/kg) according to the body weight of the mice, and detection of glucose in the blood of the tail vein of the mouse by blood glucose meter at 0, 30, 60, and 120 minutes after the injection. concentration.

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Abstract

式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药在制备药物中的用途，所述药物用于预防和/或治疗代谢性疾病，

Description

化合物在制备药物中的用途
优先权信息
本申请请求2017年11月29日向中国国家知识产权局提交的、专利申请号为201711224892.3的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及生物医药领域，具体地，本发明涉及化合物在制备药物中的用途。
背景技术
高脂血症、肥胖症、动脉粥样硬化症、心脑血管疾病和2型糖尿病等代谢性疾病已经成为中国乃至全世界越来越严重的健康难题。已知这些代谢性疾病的发生与高脂血症有着密切的关系。脂质代谢异常如高胆固醇血症是动脉粥样硬化的病变基础。动脉粥样硬化又是许多心血管疾病的主要诱因，如冠心病、脑梗死、外周血管病等。血液中脂肪酸和甘油三酯过高是胰岛素抵抗及2型糖尿病的主要诱因。因此，在代谢性疾病的治疗和预防中都将降脂作为重要指标。
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甾醇反应元件结合蛋白(SREBP)。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-1protease(S1P)和Site-2protease(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甾醇(胆固醇或氧化型甾醇如25-羟胆固醇)水平的调控。细胞内甾醇水平过高时，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-1a、-1c和SREBP-2，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-1a、-1c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S1P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。LXR是另一类受甾醇调控的核受体因子，具有激活SREBP-1c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。
发明内容
本申请是基于发明人对以下事实和问题的发现和认识作出的：
是否存在新的高活性的SERBP途径的特异性抑制剂，从而使其特异性抑制SERBP-1、SREBP-2活化，同时不激活LXR信号通路，为高脂血症、肥胖症、动脉粥样硬化症、心脑血管疾病和2型糖尿病等代谢性疾病的预防和治疗提供新的途径。基于上述问题的发现， 本申请发明人提出了式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药在制备药物中的用途。发明人惊喜地发现，所述药物可以有效预防和/或治疗代谢性疾病，有效预防和/或治疗高脂血症、肥胖症、动脉粥样硬化症、心脑血管疾病或2型糖尿病。
在本发明的第一方面，本发明提出了式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药在制备药物中的用途。根据本发明的实施例，所述药物用于预防和/或治疗代谢性疾病；
Figure PCTCN2017114645-appb-000001
其中，3号碳原子与R1之间为单键或双键，
当3号碳原子与R1之间为单键时，R1选自如下基团中任一种，R为任选取代的烷基：
Figure PCTCN2017114645-appb-000002
当3号碳原子与R1之间为双键时，R1选自如下基团中任一种：
Figure PCTCN2017114645-appb-000003
R2为-OH、-F或-H；
R3选自如下基团中的任一种：
Figure PCTCN2017114645-appb-000004
5号碳原子和6号碳原子之间为单键或双键，或5号碳原子和6号碳原子与氧原子共同形成三元环氧结构；
7号碳原子、8号碳原子、9号碳原子和11号碳原子的相邻碳原子之间为单键或双键；
R4和R5分别独立地选自-OH、氧代、羰基、-F或-H；
8号碳原子和9号碳原子之间为单键时，R6和R7分别独立地选自-H或-OH，或8号碳原子和9号碳原子与氧原子共同形成三元环氧结构；
R8为-H或甲基；
22号碳原子和23号碳原子之间为单键或双键，或22号碳原子和23号碳原子与氧原子共同形成三元环氧结构；
R9为任选取代的烷基、任选取代的烯基、羟基、任选取代的羰基、任选取代的环烷基、任选取代的环氧基、磷酸基、磺酸基或羧基，可选地，R9为选自如下基团中的任一种：
Figure PCTCN2017114645-appb-000005
各式中，n分别独立地为0～4的整数，包括端点；X为F、Cl、Br或I；
R'和R”分别独立地为C1～4烷基。
根据本发明的实施例，所述药物可以有效预防和/或治疗代谢性疾病。
根据本发明的实施例，上述用途还可进一步包括如下附加技术特征至少之一：
根据本发明的实施例，式Ⅰ所示化合物为下述化合物中任一种：
Figure PCTCN2017114645-appb-000006
Figure PCTCN2017114645-appb-000007
Figure PCTCN2017114645-appb-000008
Figure PCTCN2017114645-appb-000009
根据本发明的实施例，所述药物可以进一步有效预防和/或治疗代谢性疾病。
根据本发明的实施例，所述代谢性疾病为高脂血症、肥胖症、动脉粥样硬化症、心脑血管疾病或2型糖尿病。根据本发明的实施例，所述药物可以有效预防和/或治疗高脂血症、肥胖症、动脉粥样硬化症、心脑血管疾病或2型糖尿病。
在本发明的第二方面，本发明提出了式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药在制备降低脂肪酸水平的产品中的用途。根据本发明的实施例，所述产品可以有效降低脂肪酸水平。
根据本发明的实施例，上述用途还可进一步包括如下附加技术特征之一：
根据本发明的实施例，所述降低脂肪酸水平是通过抑制基因SREBP的表达而实现的。根据本发明的实施例，所述产品可以进一步有效降低脂肪酸水平。
根据本发明的实施例，所述降低脂肪酸水平是通过抑制Huh-7细胞或肝原代细胞中基因SREBP的表达而实现的。根据本发明的实施例，所述产品可以进一步有效降低脂肪酸水平。
根据本发明的实施例，所述抑制Huh-7细胞或肝原代细胞中基因SREBP的表达是通过下列的至少之一而实现的：
1)抑制基因SREBP-1c的表达；
2)抑制基因SREBP-2的表达；
3)抑制基因HMGCS的表达；
4)抑制基因HMGCR的表达；
5)抑制基因SS的表达；
6)抑制基因FASN的表达；
7)抑制基因SCD1的表达。
根据本发明的实施例，所述产品可以进一步有效降低脂肪酸水平。
在本发明的第三方面，本发明提出了式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药在制备抑制基因SREBP表达的细胞模型中的用途。根据本发明的实施例，所述细胞模型可以有效抑制基因SREBP表达。
在本发明的第四方面，本发明提出了式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药在制备下述1)-3)中任一种产品中的用途：
1)抑制高脂饮食诱导的体重和脂肪增加的产品；
2)降低高脂饮食诱导的总胆固醇和甘油三酯的含量的产品；
3)提高高脂饮食诱导肥胖患者对葡萄糖和胰岛素的敏感性的产品。
根据本发明的实施例，所述产品可以有效抑制高脂饮食诱导的体重和脂肪增加和/或降低高脂饮食诱导的总胆固醇和甘油三酯的含量和/或提高高脂饮食诱导肥胖患者对葡萄糖和胰岛素的敏感性。
在本发明的第五方面，本发明提出了式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药在制备抑制SREBP信号通路的抑制剂中的用途。根据本发明的实施例，所述抑制剂可以有效抑制SREBP信号通路。
根据本发明的实施例，上述用途还可进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述抑制SREBP信号通路是通过下列至少之一而实现的：
1)抑制基因SREBP-1c的表达；
2)抑制基因SREBP-2的表达；
3)抑制基因HMGCS的表达；
4)抑制基因HMGCR的表达；
5)抑制基因SS的表达；
6)抑制基因FASN的表达；
7)抑制基因SCD1的表达。
根据本发明的实施例，所述抑制剂可以进一步有效抑制SREBP信号通路。
在本发明的第六方面，本发明提出了药物组合物在制备药物中的用途。根据本发明的实施例，所述药物组合物包括上述式I所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药作为有效成分，所述药物用于预防和/或治疗代谢性疾病。根据本发明的实施例，所述药物组合物可以有效预防和/或治疗代谢性疾病。
根据本发明的实施例，上述用途还可进一步包括如下附加技术特征之一：
根据本发明的实施例，所述药物组合物进一步包括药学上可接受的辅料。根据本发明的实施例，所述药物组合物可以进一步有效预防和/或治疗代谢性疾病。
在本发明的第七方面，本发明提出了一种预防和/或治疗代谢性疾病方法。根据本发明的实施例，所述方法包括：给予患者前面所述的化合或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药或前面所述的药物组合物。根据本发明的实施例，所述方法疗效好。
根据本发明的实施例，上述方法还可进一步包括如下附加技术特征之一：
根据本发明的实施例，所述代谢性疾病包括高脂血症或2型糖尿病。根据本发明的实施例，所述方法疗效更好。
本发明首先提供了式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药在制备预防和/或治疗高脂血症、2型糖尿病等代谢性疾病的产品中的应用；
Figure PCTCN2017114645-appb-000010
式Ⅰ中，3号碳原子与R1之间为单键或双键，当为单键时，R1选自如下基团中任一种：
Figure PCTCN2017114645-appb-000011
式中R为烷基或取代的烷基；
当为双键时，R1选自如下基团中任一种：
Figure PCTCN2017114645-appb-000012
R2为-OH、-F或-H；
R3选自如下基团中任一种：
Figure PCTCN2017114645-appb-000013
5号碳原子和6号碳原子之间为单键或双键，或5号碳原子和6号碳原子与氧原子形成三元环氧结构；
7号碳原子、8号碳原子、9号碳原子和11号碳原子相邻碳原子之间为单键或双键；
R4和R5选自-OH、羰基、-F和-H；
当8号碳原子和9号碳原子之间为单键时，R6和R7均为-H或-OH，或R6和R7共同与8号碳原子和9号碳原子形成三元环氧结构；
R8为-H或甲基；
22号碳原子和23号碳原子之间为单键或双键，当为单键时存在环氧结构；
R9为取代的烷基、醇、酮、环氧、磷酸或磺酸，具体选自如下基团中任一种：
Figure PCTCN2017114645-appb-000014
各式中，n均为0～4之间的数；X为-F、-Cl、-Br或-I；
R'和R”均为碳原子数为1～4的烷基。
本发明所涉及的式Ⅰ所示化合物具体为下述化合物中任一种：
Figure PCTCN2017114645-appb-000015
Figure PCTCN2017114645-appb-000016
Figure PCTCN2017114645-appb-000017
式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药用于抑制SREBP信号通路。本发明验证了化合物对SREBP报告基因活性的抑制作用，在Huh-7/SRE-Luc细胞系(通过在人肝癌细胞系Huh-7细胞中稳定转染表达了2个报告基因)中进行测定，并测定了各化合物对SREBP通 路抑制的IC50值：化合物1G为0.22μM、化合物13A为0.44μM、化合物12C为0.86μM、化合物14A为0.94μM、化合物13C为1.05μM、化合物14C为1.11μM、化合物5A为1.23μM、化合物1C为1.72μM、化合物4A为2.07μM、化合物8A为4.90μM，证明根据本发明实施例的化合物对SREBP报告基因均具有显著的抑制效果。
式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药能够抑制基因SREBP的表达。本发明验证了所述化合物对Huh-7细胞中基因SREBP的表达，经荧光实时定量PCR，根据本发明实施例的化合物1G能显著抑制SREBP靶基因：SREBP-2(NM_004599.35)、HMGCR(NM_000859.2)、HMGCS(NM_001098272.2)和SS(NM_004462)的表达。化合物1G在1μM时，SREBP-2下调了约40％，HMGCR、HMGCS和SS下调了约80％。同时试验证明根据本发明实施例的化合物不激活LXR靶基因的表达，对参与脂肪酸合成的SREBP-1c(NM_001321096.2)、FASN(NM_004104.4)和SCD1(NM_005063)有显著下调作用。本发明验证了所述化合物对小鼠肝原代细胞中基因SREBP的表达，经荧光实时定量PCR，根据本发明实施例的化合物1G能显著抑制SREBP靶基因：SREBP-2(AF374267)、HMGCR(BF464069)、HMGCS(BI143807)和SS(D29016)。化合物1G在1μM时，SREBP-2下调了约40％，HMGCR、HMGCS和SS下调了约50％。同时试验证明根据本发明实施例的化合物不激活LXR靶基因的表达，对参与脂肪酸合成的SREBP-1c、FASN和SCD1有显著下调作用。而已知的LXR激活剂TO901317能成倍地激活上述脂肪酸合成相关的基因，将导致脂肪肝的形成。
式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药能够阻止饮食诱导肥胖小鼠体重和脂肪的增加，改善小鼠血液和肝脏中胆固醇和甘油三酯水平并且可以显著提高饮食诱导肥胖小鼠的胰岛素敏感性。
1、根据本发明实施例的化合物有很强的SREBP基因抑制活性，如化合物1G，其抑制SREBP基因通路的IC50值为0.22μM。在原代肝细胞中，利用荧光实时定量PCR检测发现1G对SREBP靶基因有显著的的表达抑制作用，并且不会激活LXR。
2、根据本发明实施例的化合物通过抑制SREBP基因的活性可以显著阻止饮食诱导肥胖小鼠体重和脂肪的增加，改善小鼠血液和肝脏中胆固醇和甘油三酯水平并且提高饮食诱导肥胖小鼠的胰岛素敏感性，其在动物水平的效果要显著优于市售药物洛伐他汀。
附图说明
图1为利用SRE-Luc/GFP报告基因系统对根据本发明实施例的化合物进行活性分析的 示意图，其中，图1(A)为SRE-Luc/GFP双报告基因系统的示意图，图1(B)为根据本发明实施例的化合物对SREBP通路的抑制活性。
图2为根据本发明实施例的化合物1G抑制SREBP的剪切入核和SREBP靶基因的表达，其中图2(A)为蛋白质印迹WB检测1G对SREBP蛋白剪切加工的抑制作用，n-SREBP为剪切加工后的激活形式SREBP，pre-SREBP为全长形式的SREBP，CHC为内参蛋白；图2(B)为图2(A)中的SREBP条带进行定量分析，并以n-SREBP/pre-SREBP的比值代表SREBP加工的程度；图2(C)为在Huh7细胞中，利用荧光实时定量PCR检测分析1G对SREBP靶基因(SREBP-2，HMGCR，HMGCS，SS)表达的抑制作用；图2(D)为在原代肝细胞中，利用荧光实时定量PCR检测分析1G对SREBP靶基因表达的抑制作用。
图3为根据本发明实施例的化合物1G不激活LXR靶基因的表达的示意图，其中，图3(A)为在Huh7细胞中，荧光实时定量PCR分析1G对LXR靶基因(SREBP-1c，ABCA1，ABCG8，FASN，SCD1)表达的影响；图3(B)为在肝原代细胞中，荧光实时定量PCR分析1G对LXR靶基因表达的影响。
图4为根据本发明实施例的化合物1G特异地抑制胆固醇的合成的示意图。
图4为根据本发明实施例的化合物1G显著抑制小鼠高脂高胆固醇饲料诱导的体重和脂肪的增加的示意图，图5(A)为抑制小鼠体重的增加的示意图，图5(B)为抑制小鼠脂肪体重比的示意图。
图5为根据本发明实施例的化合物1G显著降低高胆固醇高脂肪酸饲养条件下的小鼠血液和肝脏的脂质水平的示意图，其中，图5(A)为血液中总胆固醇(TC)水平的变化示意图，图5(B)为血液中总甘油三酯(TG)水平的变化示意图，图5(C)为肝脏中总胆固醇(TC)水平的变化示意图，图5(D)为肝脏中总甘油三酯(TC)水平的变化示意图；以及
图6为根据本发明实施例的化合物1G显著提高小鼠的胰岛素敏感性的示意图，其中，图6(A)为葡萄糖耐受示意图，图6(B)为葡萄糖耐受实验的定量分析示意图，图6(C)为胰岛素耐受示意图，图6(D)为胰岛素耐受试验的定量分析示意图，图6(E)为血液中葡萄糖水平的变化示意图。
具体实施方式
下面详细描述本发明的实施例，下面描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
在本文中所使用的术语“给予患者前面所述的化合或其立体异构体、几何异构体、互变 异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药或前面所述的药物组合物”指将预定量的物质通过某种适合的方式引入病人。本发明的式I所述化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药或者药物组合物可以通过任何常见的途径被给药，只要它可以到达预期的组织。给药的各种方式是可以预期的，包括腹膜，静脉，肌肉，皮下，皮层，口服，局部，鼻腔，肺部和直肠，但是本发明不限于这些已举例的给药方式。然而，由于口服给药时，口服给药的组合物的活性成分应该被包被或被配制以防止其在胃部被降解。此外，本发明的式I所述化合物或者药物组合物可以使用将活性成分传送到靶细胞的特定器械来给药。
本发明的药物组合物的给药频率和剂量可以通过多个相关因素被确定，该因素包括要被治疗的疾病类型，给药途径，病人年龄，性别，体重和疾病的严重程度以及作为活性成分的药物类型。
术语“治疗有效量”是指化合物足以显著改善某些与疾病或病症相关的症状的量，也即为给定病症和给药方案提供治疗效果的量。治疗有效量的药物或化合物不需要治愈疾病或病症，但将为疾病或病症提供治疗，使得个体的疾病或病症的发作被延缓、阻止或预防，或者疾病或病症的症状得以缓解，或者疾病或病症的期限被改变，或者例如疾病或病症变得不严重，或者加速康复。
术语“治疗”用于指获得期望的药理学和/或生理学效果。所述效果就完全或部分预防疾病或其症状而言可以是预防性的，和/或就部分或完全治愈疾病和/或疾病导致的不良作用而言可以是治疗性的。本文使用的“治疗”涵盖哺乳动物、特别是人的疾病的治疗，包括：(a)在容易患病但是尚未确诊得病的个体中预防疾病或病症发生；(b)抑制疾病；或(c)缓解疾病，例如减轻与疾病相关的症状。本文使用的“治疗”涵盖将药物或化合物给予个体以治疗、治愈、缓解、改善、减轻或抑制个体的疾病的任何用药，包括但不限于将含本文所述式I化合物或药物组合物的给予有需要的个体。
根据本发明的实施例，所述辅料包括制剂领域公知的可药用的赋形剂、润滑剂、填充剂、稀释剂、崩解剂、稳定剂、防腐剂、乳化剂、助溶剂、着色剂、甜味剂，制成片剂、丸剂、胶囊剂、注射剂等不同剂型。
现在详细描述本发明的某些实施方案，其实例由随附的结构式和化学式说明。本发明意图涵盖所有的替代、修改和等同技术方案，它们均包括在如权利要求定义的本发明范围内。本领域技术人员应认识到，许多与本文所述类似或等同的方法和材料能够用于实践本发明。本发明绝不限于本文所述的方法和材料。在所结合的文献、专利和类似材料的一篇或多篇与本申请不同或相矛盾的情况下(包括但不限于所定义的术语、术语应用、所描述的技术，等等)，以本申请为准。
应进一步认识到，本发明的某些特征，为清楚可见，在多个独立的实施方案中进行了描述，但也可以在单个实施例中以组合形式提供。反之，本发明的各种特征，为简洁起见，在单个实施方案中进行了描述，但也可以单独或以任意适合的子组合提供。
除非另外说明，本发明所使用的所有科技术语具有与本发明所属领域技术人员的通常理解相同的含义。本发明涉及的所有专利和公开出版物通过引用方式整体并入本发明。
除非另外说明，应当应用本文所使用得下列定义。出于本发明的目的，化学元素与元素周期表CAS版，和《化学和物理手册》，第75版，1994一致。此外，有机化学一般原理可参考"Organic Chemistry",Thomas Sorrell,University Science Books,Sausalito:1999,和"March's Advanced Organic Chemistry”by Michael B.Smith and Jerry March,John Wiley&Sons,New York:2007中的描述，其全部内容通过引用并入本文。
除非另有说明或者上下文中有明显的冲突，本文所使用的冠词“一”、“一个(种)”和“所述”旨在包括“至少一个”或“一个或多个”。因此，本文所使用的这些冠词是指一个或多于一个(即至少一个)宾语的冠词。例如，“一组分”指一个或多个组分，即可能有多于一个的组分被考虑在所述实施方案的实施方式中采用或使用。
术语“包含”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。
“立体异构体”是指具有相同化学构造，但原子或基团在空间上排列方式不同的化合物。立体异构体包括对映异构体、非对映异构体、构象异构体(旋转异构体)、几何异构体(顺/反)异构体、阻转异构体，等等。
“手性”是具有与其镜像不能重叠性质的分子；而“非手性”是指与其镜像可以重叠的分子。
“对映异构体”是指一个化合物的两个不能重叠但互成镜像关系的异构体。
“非对映异构体”是指有两个或多个手性中心并且其分子不互为镜像的立体异构体。非对映异构体具有不同的物理性质，如熔点、沸点、光谱性质和反应性。非对映异构体混合物可通过高分辨分析操作如电泳和色谱，例如HPLC来分离。
本发明所使用的立体化学定义和规则一般遵循S.P.Parker，Ed.，McGraw-Hill Dictionary of Chemical Terms(1984)McGraw-Hill Book Company，New York；and Eliel，E.and Wilen，S.，“Stereochemistry of Organic Compounds”，John Wiley&Sons，Inc.，New York，1994。
许多有机化合物以光学活性形式存在，即它们具有使平面偏振光的平面发生旋转的能力。在描述光学活性化合物时，使用前缀D和L或R和S来表示分子关于其一个或多个手性中心的绝对构型。前缀d和l或(+)和(-)是用于指定化合物所致平面偏振光旋转的符号，其中(-)或l表示化合物是左旋的。前缀为(+)或d的化合物是右旋的。一种具体的立体异构体是对映异构体，这种异构体的混合物称作对映异构体混合物。对映异构体的50:50混合物称为外消旋混合物或外消旋体，当在化学反应或过程中没有立体选择性或立体特异性时，可出现这种情况。
本发明公开化合物的任何不对称原子(例如，碳等)都可以以外消旋或对映体富集的形式存在，例如(R)-、(S)-或(R,S)-构型形式存在。在某些实施方案中，各不对称原子在(R)-或(S)-构型方面具有至少50％对映体过量，至少60％对映体过量，至少70％对映体过量，至少80％对映体过量，至少90％对映体过量，至少95％对映体过量，或至少99％对映体过量。
依据起始物料和方法的选择，本发明化合物可以以可能的异构体中的一个或它们的混 合物，例如外消旋体和非对映异构体混合物(这取决于不对称碳原子的数量)的形式存在。光学活性的(R)-或(S)-异构体可使用手性合成子或手性试剂制备，或使用常规技术拆分。如果化合物含有一个双键，取代基可能为E或Z构型；如果化合物中含有二取代的环烷基，环烷基的取代基可能有顺式或反式构型。
所得的任何立体异构体的混合物可以依据组分物理化学性质上的差异被分离成纯的或基本纯的几何异构体，对映异构体，非对映异构体，例如，通过色谱法和/或分步结晶法。
可以用已知的方法将任何所得终产物或中间体的外消旋体通过本领域技术人员熟悉的方法拆分成光学对映体，如，通过对获得的其非对映异构的盐进行分离。外消旋的产物也可以通过手性色谱来分离，如，使用手性吸附剂的高效液相色谱(HPLC)。特别地，对映异构体可以通过不对称合成制备，例如，可参考Jacques,et al.,Enantiomers,Racemates and Resolutions(Wiley Interscience,New York,1981)；Principles of Asymmetric Synthesis(2nd Ed.Robert E.Gawley,Jeffrey Aubé,Elsevier,Oxford,UK,2012)；Eliel,E.L.Stereochemistry of Carbon Compounds(McGraw-Hill,NY,1962)；Wilen,S.H.Tables of Resolving Agents and Optical Resolutions p.268(E.L.Eliel,Ed.,Univ.of Notre Dame Press,Notre Dame,IN 1972)；Chiral Separation Techniques:APractical Approach(Subramanian,G.Ed.,Wiley-VCH Verlag GmbH&Co.KGaA,Weinheim,Germany,2007)。
术语“互变异构体”或“互变异构形式”是指具有不同能量的可通过低能垒(low energy barrier)互相转化的结构异构体。若互变异构是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(protontautomer)(也称为质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异构化。价键互变异构体(valence tautomer)包括通过一些成键电子的重组来进行的互相转化。酮-烯醇互变异构的具体实例是戊烷-2,4-二酮和4-羟基戊-3-烯-2-酮互变异构体的互变。互变异构的另一个实例是酚-酮互变异构。酚-酮互变异构的一个具体实例是吡啶-4-醇和吡啶-4(1H)-酮互变异构体的互变。除非另外指出，本发明化合物的所有互变异构体形式都在本发明的范围之内。
像本发明所描述的，本发明的化合物可以任选地被一个或多个取代基所取代，如上面的通式化合物，或者像实施例里面特殊的例子，子类，和本发明所包含的一类化合物。应了解“任选取代的”这个术语与“取代或非取代的”这个术语可以交换使用。一般而言，术语“取代的”表示所给结构中的一个或多个氢原子被具体取代基所取代。除非其他方面表明，一个任选的取代基团可以在基团各个可取代的位置进行取代。当所给出的结构式中不只一个位置能被选自具体基团的一个或多个取代基所取代，那么取代基可以相同或不同地在各个位置取代。
另外，需要说明的是，除非以其他方式明确指出，在本发明中所采用的描述方式“各…独立地为”与“…各自独立地为”和“…独立地为”可以互换，均应做广义理解，其既可以是指在不同基团中，相同符号之间所表达的具体选项之间互相不影响，也可以表示在相同的基团中，相同符号之间所表达的具体选项之间互相不影响。
在本说明书的各部分，本发明公开化合物的取代基按照基团种类或范围公开。特别指 出，本发明包括这些基团种类和范围的各个成员的每一个独立的次级组合。例如，术语“C1-6烷基”特别指独立公开的甲基、乙基、C3烷基、C4烷基、C5烷基和C6烷基。
在本发明的各部分，描述了连接取代基。当该结构清楚地需要连接基团时，针对该基团所列举的马库什变量应理解为连接基团。例如，如果该结构需要连接基团并且针对该变量的马库什基团定义列举了“烷基”或“芳基”，则应该理解，该“烷基”或“芳基”分别代表连接的亚烷基基团或亚芳基基团。
本发明使用的术语“烷基”或“烷基基团”，表示含有1至20个碳原子，饱和的直链或支链一价烃基基团，其中，所述烷基基团可以任选地被一个或多个本发明描述的取代基所取代。除非另外详细说明，烷基基团含有1-20个碳原子。在一实施方案中，烷基基团含有1-4个碳原子。
烷基基团的实例包含，但并不限于，甲基(Me、-CH3)，乙基(Et、-CH2CH3)，正丙基(n-Pr、-CH2CH2CH3)，异丙基(i-Pr、-CH(CH3)2)，正丁基(n-Bu、-CH2CH2CH2CH3)，异丁基(i-Bu、-CH2CH(CH3)2)，仲丁基(s-Bu、-CH(CH3)CH2CH3)，叔丁基(t-Bu、-C(CH3)3)，正戊基(-CH2CH2CH2CH2CH3)，2-戊基(-CH(CH3)CH2CH2CH3)，3-戊基(-CH(CH2CH3)2)，2-甲基-2-丁基(-C(CH3)2CH2CH3)，3-甲基-2-丁基(-CH(CH3)CH(CH3)2)，3-甲基-1-丁基(-CH2CH2CH(CH3)2)，2-甲基-1-丁基(-CH2CH(CH3)CH2CH3)，正己基(-CH2CH2CH2CH2CH2CH3)，2-己基(-CH(CH3)CH2CH2CH2CH3)，3-己基(-CH(CH2CH3)(CH2CH2CH3))，2-甲基-2-戊基(-C(CH3)2CH2CH2CH3)，3-甲基-2-戊基(-CH(CH3)CH(CH3)CH2CH3)，4-甲基-2-戊基(-CH(CH3)CH2CH(CH3)2)，3-甲基-3-戊基(-C(CH3)(CH2CH3)2)，2-甲基-3-戊基(-CH(CH2CH3)CH(CH3)2)，2,3-二甲基-2-丁基(-C(CH3)2CH(CH3)2)，3,3-二甲基-2-丁基(-CH(CH3)C(CH3)3)，正庚基，正辛基，等等。
术语“烯基”表示含有2-15个碳原子的直链或支链一价烃基，其中至少有一个不饱和位点，即有一个碳-碳sp2双键，其中，所述烯基基团可以任选地被一个或多个本发明所描述的取代基所取代，其包括“cis”和“tans”的定位，或者"E"和"Z"的定位。烯基基团的实例包括，但并不限于，乙烯基(-CH＝CH2)、烯丙基(-CH2CH＝CH2)等等。
术语“炔基”表示含有2-15个碳原子的直链或支链一价烃基，其中至少有一个不饱和位点，即有一个碳-碳sp三键，其中，所述炔基基团可以任选地被一个或多个本发明所描述的取代基所取代。炔基基团的实例包括，但并不限于，乙炔基(-C≡CH)、炔丙基(-CH2C≡CH)、1-丙炔基(-C≡C-CH3)等等。
本发明所使用的术语“前药”，代表一个化合物在体内转化为式(I)所示的化合物。这样的转化受前体药物在血液中水解或在血液或组织中经酶转化为母体结构的影响。本发明前体药物类化合物可以是酯，在现有的发明中酯可以作为前体药物的有苯酯类，脂肪族(C1-24)酯类，酰氧基甲基酯类，碳酸酯，氨基甲酸酯类和氨基酸酯类。例如本发明里的一个化合物包含羟基，即可以将其酰化得到前体药物形式的化合物。其他的前体药物形式包括磷酸酯，如这些磷酸酯类化合物是经母体上的羟基磷酸化得到的。关于前体药物完整的讨论可以参考以下文献：T.Higuchi and V.Stella,Pro-drugs as Novel Delivery Systems,Vol.14of the  A.C.S.Symposium Series,Edward B.Roche,ed.,Bioreversible Carriers in Drug Design,American Pharmaceutical Association and Pergamon Press,1987,J.Rautio et al.,Prodrugs:Design and Clinical Applications,Nature Review Drug Discovery,2008,7,255-270,and S.J.Hecker et al.,Prodrugs of Phosphates and Phosphonates,Journal of Medicinal Chemistry,2008,51,2328-2345。
“代谢产物”是指具体的化合物或其盐在体内通过代谢作用所得到的产物。一个化合物的代谢产物可以通过所属领域公知的技术来进行鉴定，其活性可以通过如本发明所描述的那样采用试验的方法进行表征。这样的产物可以是通过给药化合物经过氧化，还原，水解，酰氨化，脱酰氨作用，酯化，脱脂作用，酶裂解等等方法得到。相应地，本发明包括化合物的代谢产物，包括将本发明的化合物与哺乳动物充分接触一段时间所产生的代谢产物。
本发明所使用的“药学上可接受的盐”是指本发明的化合物的有机盐和无机盐。药学上可接受的盐在所属领域是为我们所熟知的，如文献：S.M.Berge et al.,describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences,1977,66:1-19.所记载的。药学上可接受的无毒的酸形成的盐包括，但并不限于，与氨基基团反应形成的无机酸盐有盐酸盐，氢溴酸盐，磷酸盐，硫酸盐，高氯酸盐，和有机酸盐如乙酸盐，草酸盐，马来酸盐，酒石酸盐，柠檬酸盐，琥珀酸盐，丙二酸盐，或通过书籍文献上所记载的其他方法如离子交换法来得到这些盐。其他药学上可接受的盐包括己二酸盐，藻酸盐，抗坏血酸盐，天冬氨酸盐，苯磺酸盐，苯甲酸盐，重硫酸盐，硼酸盐，丁酸盐，樟脑酸盐，樟脑磺酸盐，环戊基丙酸盐，二葡萄糖酸盐，十二烷基硫酸盐，乙磺酸盐，甲酸盐，反丁烯二酸盐，葡庚糖酸盐，甘油磷酸盐，葡萄糖酸盐，半硫酸盐，庚酸盐，己酸盐，氢碘酸盐，2-羟基-乙磺酸盐，乳糖醛酸盐，乳酸盐，月桂酸盐，月桂基硫酸盐，苹果酸盐，丙二酸盐，甲磺酸盐，2-萘磺酸盐，烟酸盐，硝酸盐，油酸盐，棕榈酸盐，扑酸盐，果胶酸盐，过硫酸盐，3-苯基丙酸盐，苦味酸盐，特戊酸盐，丙酸盐，硬脂酸盐，硫氰酸盐，对甲苯磺酸盐，十一酸盐，戊酸盐，等等。通过适当的碱得到的盐包括碱金属，碱土金属，铵和N+(C1-4烷基)4的盐。本发明也拟构思了任何所包含N的基团的化合物所形成的季铵盐。水溶性或油溶性或分散产物可以通过季铵化作用得到。碱金属或碱土金属盐包括钠，锂，钾，钙，镁，等等。药学上可接受的盐进一步包括适当的、无毒的铵，季铵盐和抗平衡离子形成的胺阳离子，如卤化物，氢氧化物，羧化物，硫酸化物，磷酸化物，硝酸化物，C1-8磺酸化物和芳香磺酸化物。
本发明的“溶剂化物”是指一个或多个溶剂分子与本发明的化合物所形成的缔合物。形成溶剂化物的溶剂包括，但并不限于，水，异丙醇，乙醇，甲醇，二甲亚砜，乙酸乙酯，乙酸和氨基乙醇。术语“水合物”是指溶剂分子是水所形成的缔合物。
如本发明所使用的术语“治疗”任何疾病或病症，在其中一些实施方案中指改善疾病或病症(即减缓或阻止或减轻疾病或其至少一种临床症状的发展)。在另一些实施方案中，“治疗”指缓和或改善至少一种身体参数，包括可能不为患者所察觉的身体参数。在另一些实施方案中，“治疗”指从身体上(例如稳定可察觉的症状)或生理学上(例如稳定身体的参数)或上述两方面调节疾病或病症。在另一些实施方案中，“治疗”指预防或延迟疾病或病症的发作、 发生或恶化。
可药用的酸加成盐可与无机酸和有机酸形成，例如乙酸盐、天冬氨酸盐、苯甲酸盐、苯磺酸盐、溴化物/氢溴酸盐、碳酸氢盐/碳酸盐、硫酸氢盐/硫酸盐、樟脑磺酸盐、氯化物/盐酸盐、氯茶碱盐、柠檬酸盐、乙二磺酸盐、富马酸盐、葡庚糖酸盐、葡糖酸盐、葡糖醛酸盐、马尿酸盐、氢碘酸盐/碘化物、羟乙基磺酸盐、乳酸盐、乳糖醛酸盐、月桂基硫酸盐、苹果酸盐、马来酸盐、丙二酸盐、扁桃酸盐、甲磺酸盐、甲基硫酸盐、萘甲酸盐、萘磺酸盐、烟酸盐、硝酸盐、十八酸盐、油酸盐、草酸盐、棕榈酸盐、扑酸盐、磷酸盐/磷酸氢盐/磷酸二氢盐、聚半乳糖酸盐、丙酸盐、硬脂酸盐、琥珀酸盐、磺基水杨酸盐、酒石酸盐、甲苯磺酸盐和三氟乙酸盐。
可以由其衍生得到盐的无机酸包括例如盐酸、氢溴酸、硫酸、硝酸、磷酸等。
可以由其衍生得到盐的有机酸包括例如乙酸、丙酸、羟基乙酸、草酸、马来酸、丙二酸、琥珀酸、富马酸、酒石酸、柠檬酸、苯甲酸、扁桃酸、甲磺酸、乙磺酸、对甲苯磺酸、磺基水杨酸等。
可药用碱加成盐可与无机碱和有机碱形成。
可以由其衍生得到盐的无机碱包括，例如铵盐和周期表的I族至XII族的金属。在某些实施方案中，该盐衍生自钠、钾、铵、钙、镁、铁、银、锌和铜；特别适合的盐包括铵、钾、钠、钙和镁盐。
可以由其衍生得到盐的有机碱包括伯胺、仲胺和叔胺，取代的胺包括天然存在的取代的胺、环状胺、碱性离子交换树脂等。某些有机胺包括，例如，异丙胺、苄星青霉素(benzathine)、胆碱盐(cholinate)、二乙醇胺、二乙胺、赖氨酸、葡甲胺(meglumine)、哌嗪和氨丁三醇。
本发明的可药用盐可以用常规化学方法由母体化合物、碱性或酸性部分来合成。一般而言，该类盐可以通过使这些化合物的游离酸形式与化学计量量的适宜碱(如Na、Ca、Mg或K的氢氧化物、碳酸盐、碳酸氢盐等)反应，或者通过使这些化合物的游离碱形式与化学计量量的适宜酸反应来进行制备。该类反应通常在水或有机溶剂或二者的混合物中进行。一般地，在适当的情况中，需要使用非水性介质如乙醚、乙酸乙酯、乙醇、异丙醇或乙腈。在例如“Remington′s Pharmaceutical Sciences”，第20版，Mack Publishing Company，Easton，Pa.，(1985)；和“药用盐手册：性质、选择和应用(Handbook of Pharmaceutical Salts：Properties，Selection，and Use)”，Stahl and Wermuth(Wiley-VCH，Weinheim，Germany，2002)中可找到另外一些适宜盐的列表。
另外，本发明公开的化合物，包括它们的盐，也可以以它们的水合物形式或包含其溶剂(例如乙醇、DMSO，等等)的形式得到，用于它们的结晶。本发明公开化合物可以与药学上可接受的溶剂(包括水)固有地或通过设计形成溶剂化物；因此，本发明旨在包括溶剂化的和未溶剂化的形式。
本发明给出的任何结构式也意欲表示这些化合物未被同位素富集的形式以及同位素富集的形式。同位素富集的化合物具有本发明给出的通式描绘的结构，除了一个或多个原子 被具有所选择原子量或质量数的原子替换。可引入本发明化合物中的示例性同位素包括氢、碳、氮、氧、磷、硫、氟和氯的同位素，如2H，3H，11C，13C，14C，15N，17O，18O，18F，31P，32P，35S，36Cl和125I。
另一方面，本发明所述化合物包括同位素富集的本发明所定义的化合物，例如，其中存在放射性同位素，如3H，14C和18F的那些化合物，或者其中存在非放射性同位素，如2H和13C。该类同位素富集的化合物可用于代谢研究(使用14C)、反应动力学研究(使用例如2H或3H)、检测或成像技术，如正电子发射断层扫描术(PET)或包括药物或底物组织分布测定的单光子发射计算机断层成像术(SPECT)，或可用于患者的放疗中。18F富集的化合物对PET或SPECT研究而言是特别理想的。同位素富集的式(I)所示化合物可以通过本领域技术人员熟悉的常规技术或本发明中的实施例和制备过程所描述使用合适的同位素标记试剂替代原来使用过的未标记试剂来制备。
此外，较重同位素特别是氘(即，2H或D)的取代可提供某些治疗优点，这些优点是由代谢稳定性更高带来的。例如，体内半衰期增加或剂量需求降低或治疗指数得到改善带来的。可以用同位素富集因子来定义该类较重同位素特别是氘的浓度。本发明所使用的术语“同位素富集因子”是指所指定同位素的同位素丰度和天然丰度之间的比例。如果本发明化合物的取代基被指定为氘，该化合物对各指定的氘原子而言具有至少3500(各指定氘原子处52.5％的氘掺入)、至少4000(60％的氘掺入)、至少4500(67.5％的氘掺入)，至少5000(75％的氘掺入)，至少5500(82.5％的氘掺入)、至少6000(90％的氘掺入)、至少6333.3(95％的氘掺入)、至少6466.7(97％的氘掺入)、至少6600(99％的氘掺入)或至少6633.3(99.5％的氘掺入)的同位素富集因子。本发明可药用的溶剂化物包括其中结晶溶剂可以是同位素取代的例如D2O、丙酮-d6、DMSO-d6的那些溶剂化物。
另一方面，本发明涉及制备式(I)所包含的化合物的中间体。
另一方面，本发明涉及式(I)所包含的化合物的制备、分离和纯化的方法。
另一方面，本发明提供一种药物组合物，所述药物组合物包含本发明化合物，药学上可接受的载体，赋形剂，稀释剂，辅剂，溶媒，或它们的组合。在一些实施方案，药物组合物可以是液体，固体，半固体，凝胶或喷雾剂型。
“联合”表示在单个剂量单位形式中的固定组合或用于组合施用的部分的药盒，其中本发明公开化合物和组合伴侣可以在同一时间独立施用或者可以在一定的时间间隔内分别施用，特别是使联合合伴侣表现出合作、例如协同作用。如本文所用的术语“共同给药”或“联合给药”等意欲囊括将所选的组合伴侣施用于需要其的单个个体(例如患者)，并且意欲包括其中物质不必通过相同施用途径或同时施用的治疗方案。如本文所用的术语“药物组合产品”表示将一种以上活性成分混合或组合所得到的产品，并且既包括活性成分的固定组合也包括非固定组合。术语“固定联合”表示活性成分如本发明公开化合物和组合伴侣以单一实体或剂量的形式同时施用于患者。术语“非固定联合”表示活性成分如本发明公开化合物和组合伙伴均作为单独实体同时、共同或无特定时间限制地先后施用于患者，其中该施用在患者体内提供了两种化合物的治疗有效水平。
一、化合物的合成
实施例1、化合物1C的合成
羊毛甾醇(50％，购于TCI，100mg)溶于二氯甲烷(30mL)，在冰浴下，m-CPBA(85％，28mg)和NaHCO3(14mg)间隔3h分两批加入到上述溶液中，室温搅拌过夜。反应液用饱和碳酸氢钠溶液洗涤，无水碳酸钠干燥，然后再旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯10：1)，得化合物1C(45mg)和化合物1A(40mg)。
化合物1C：1H-NMR(400MHz,CDCl3)δ(ppm)3.21(d,J＝8.0Hz,1H),2.69-2.67(m,1H),0.68(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.6,134.4,79.0,77.5,77.2,76.8,65.0,64.9,58.5,58.2,50.51,50.48,50.4,49.9,44.6,39.0,37.1,36.4,36.3,35.7,32.9,32.7,31.10.31.08,30.9,28.4,28.3,28.1,28.0,26.6,26.0,25.7,25.06,25.05,24.4,21.1,19.3,18.9,18.79,18.76,18.7.
实施例2、化合物1F的合成
羊毛甾醇(50％，购于TCI，220mg)溶于THF/H2O(20mL/5mL)，NBS(54mg)加入到反应液中，反应液在室温下搅拌2h。反应液加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯5：1)，得化合物1F(110mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)4.13-3.94(m,1H),3.25-3.21(m,1H),0.69(s,3H).
实施例3、化合物1G的合成
化合物1F(80mg)溶于无水THF(10mL)中，LiAlH4(38mg)加入到反应液中，反应液加热回流2h。冷却到室温，加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯5：1)，得化合物1G(40mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)3.25-3.22(m,1H),2.03-1.89(m,5H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.58,134.56,79.1,77.5,77.2,76.8,71.3,50.7,50.6,50.0,44.7,44.6,39.0,37.2,36.9,36.6,35.8,31.2,31.0,29.5,29.4,28.4,28.1,28.0,26.7,24.4,21.3,21.2,19.3,18.8,18.4,15.9,15.6.
实施例4、化合物1H的合成
1C(80mg)，醋酸酐(300uL)，DMAP(2mg)，吡啶(1ml)和二氯甲烷(10ml)加入到反应瓶中。室温搅拌6h。加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯20：1)，得化合物1H(80mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)4.51-4.47(m,1H),2.68(t,J＝6.4Hz,1H),0.69(s,3H)； 13C-NMR(100MHz,CDCl3)δ(ppm)171.2,134.5,134.4,81.0,77.5,77.2,76.8,65.1,64.9,58.5,58.3,50.6,50.5,50.4,49.9,44.6,37.9,37.0,36.5,36.3,35.4,32.9,32.7,31.07,31.05,30.9,28.4,28.3,28.0,26.5,26.1,25.7,25.09,25.07,24.4,24.3,21.5,21.1,19.3,18.9,18.8,18.7,18.2,16.7.
实施例5、化合物2F的合成
1D(80mg)，醋酸酐(300uL)，DMAP(2mg)，吡啶(1ml)和二氯甲烷(10ml)加入到反应瓶中。室温搅拌6h。加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯20：1)，得化合物2F(80mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)4.51-4.47(m,1H),3.36-3.27(m,1H),0.68(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)171.2,134.6,134.4,81.1,79.8,78.9,73.4,73.3,50.7,50.6,49.9,44.6,37.9,37.0,36.9,36.4,35.4,33.7,33.2,31.1,30.9,28.8,28.5,28.4,28.3,28.0,26.7,26.5,24.4,24.3,23.4,23.3,21.5,21.1,19.3,19.0,18.7,18.2,16.7,15.9.
实施例6、化合物3G的合成
1G(100mg)，PCC(100mg)和NaOAc(10mg)溶解到二氯甲烷中，室温搅拌1h。加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯20：1)，得化合物3G(90mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)2.56-2.40(m,2H),0.88(s,6H),0.70(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)217.9,135.47,133.28,71.17,51.4,50.6,50.0,47.5,44.6,44.5,37.0,36.9,36.6,36.2,34.7,31.1,31.0,29.5,29.4,28.3,26.5,26.3,24.4,21.4,21.2,19.6,18.8,16.0.
实施例7、化合物4A的合成
1G(50mg)溶于二氯甲烷(30mL)，在冰浴下，m-CPBA(85％，28mg)和NaHCO3(14mg)间隔3h分两批加入到上述溶液中，室温搅拌过夜。反应液用饱和碳酸氢钠溶液洗涤，无水碳酸钠干燥，然后再旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯5：1)，得化合物4A(45mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)3.21-3.19(m,1H),0.76-0.75(m,6H)13C-NMR(100MHz,CDCl3)δ(ppm)78.6,71.2,70.8,68.3,49.0,48.5,44.5,43.7,41.9,38.6,38.0,36.7,36.4,33.0,29.42,29.36,28.6,28.4,27.3,27.0,23.6,21.6,21.2,20.1,19.1,17.1,16.6,16.4,15.2.
实施例8、化合物4G的合成
将50mg化合物1E溶于无水5ml THF中。加入4ml 4M异丙基溴化镁的四氢呋喃溶液。TLC检测反应完成后，加水终止反应，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯5：1)，得化合物4G 17mg。
1H-NMR(400MHz,CDCl3)δ(ppm)3.33-3.32(m,1H),3.23(dd,J＝11.6Hz,J＝4.8Hz,1H),0.81(s,3H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.54,134.51,79.1,50.6,50.5,44.6,39.0,37.2,35.7,31.1,31.0,28.1,28.0,27.0,26.6,24.4,22.8,21.1,19.3,19.2,19.0,18.4,16.8,15.9,15.6,14.3.
实施例9、化合物4H的合成
将37mg化合物4A溶于3ml二氯甲烷，加入37mg PCC和4mg NaOAc。室温下搅拌2h。TLC检测反应完成后水洗，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离。将分离产物溶于乙醇，加入12mg盐酸羟胺和20mg醋酸钠。60℃搅拌2h。TLC检测反应完成后水洗，先用旋转蒸发仪蒸发除去大部分溶剂后，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱(乙酸乙酯：石油醚5：1)分离得到21mg化合物4H。1H-NMR(400MHz,CDCl3)δ(ppm)3.14-3.10(m,1H),1.00(d,J＝6Hz,3H),0.85(s,3H),0.70(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)166.4,71.3,70.7,68.3,49.0,48.5,44.5,43.7,43.1,40.0,38.1,36.7,36.4,32.9,32.1,29.44,29.38,28.6,27.6,26.9,23.9,22.8,21.7,21.2,20.1,19.1,17.4,17.2,17.0,16.4.
实施例10、化合物5A的合成
将40mg化合物1G溶于4ml吡啶中，加入12mg丁二酸酐和11mg DMAP。置于80℃下搅拌3h。TLC检测反应完成后用10％的盐酸洗去吡啶，并用乙酸乙酯萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱(二氯甲烷：甲醇20：1)分离得到31mg化合物5A。
1H-NMR(400MHz,CDCl3)δ(ppm)4.52(dd,J＝11.6Hz,J＝4.8Hz,1H),2.69-2.63(m,4H),0.68(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)172.0,170.7,134.6,134.3,81.6,71.4,60.6,50.6,49.9,44.6,44.5,38.0,37.0,36.8,36.6,35.3,31.1,30.9,29.5,29.4,29.3,29.1,28.5,28.4,28.0,26.5,24.4,24.2,21.3.21.2,21.1,19.3,18.8,18.2,16.7.
实施例11、化合物5H的合成
1G(80mg)，醋酸酐(300uL)，DMAP(2mg)，吡啶(1ml)和二氯甲烷(10ml)加入到反应瓶中。室温搅拌6h。加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯20：1)，得化合物5H(80mg)。
实施例12、化合物6C的合成
5H(30mg)，RuCl3·3H2O(1mg)和TBHP(in decane，5-6M)加入到2mL的环己烷中。室温反应6h。旋转蒸发仪旋干，所得产品采用硅胶柱分离(石油醚：乙酸乙酯5：1)，得 到的化合物溶于10％KOH的乙醇溶液中，回流3h，冷却至室温，加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯3：1)，得化合物6C(10mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)3.29-3.25(m,1H),2.89(d,J＝14.0Hz,1H),2.75(d,J＝14.0Hz,1H),2.60(d,J＝14.0Hz,1H),2.50-2.44(m,2H),0.80(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)202.6,202.4,151.9,150.8,77.8,71.2,51.8,50.3,49.3,49.1,47.5,44.4,39.9,39.0,36.6,36.3,34.2,32.3,29.5,29.4,28.0,27.8,27.5,26.0,21.1,18.7,17.7,17.0,15.6.
实施例13、化合物7A的合成
将30mg化合物4A溶于3ml THF中，加入100ul 40％的氢氟酸溶液，室温下搅拌4天。TLC检测反应完成后用饱和碳酸氢钠溶液洗，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱(乙酸乙酯：石油醚1：3)分离得到27mg化合物7A。
1H-NMR(400MHz,CDCl3)δ(ppm)5.47(s,br,1H),5.32(s,br,1H),3.24(dd,J＝11.2Hz,J＝4.4Hz,1H),0.56(s,3H)13C-NMR(100MHz,CDCl3)δ(ppm)146.0,142.8,120.3,116.5,79.1,71.2,51.2,50.5,49.2,44.5,43.9,38.8,38.0,37.5,36.8,36.4,35.8,31.6,29.5,29.4,28.3,28.1,27.9,25.7,23.1,22.9,21.3,18.6,15.9,15.8.
实施例14、化合物14A和14B的合成
Figure PCTCN2017114645-appb-000018
化合物(a)140mg溶于无水DCM(20mL)中，在冰浴条件下，氮气保护加入TBDMSOTf(291uL，4.0eq)，2，6-lutidine(187uL，5.0eq)。反应液在室温下搅拌，TLC检测反应至原料消失(2h)。旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯200：1)，得化合物(b)(190mg)，产率90％。
化合物(b)1H-NMR(400MHz,CDCl3)δ(ppm)4.62-4.60(m,1H),1.19(s,3H),1.18(s,3H),1.05(s,3H),1.00(s,3H),0.95(s,12H),0.86(s,12H),0.73(s,3H),0.17(s,3H),0.15(s,3H),0.07(s,6H)；13C-NMR(100MHz,CDCl3)δ(ppm)156.5,156.5,135.2,133.1,98.9,73.7,50.6,50.2,49.5,45.7,44.6,38.5,37.0,36.7,36.4,31.3,31.2,30.3,29.8,28.6,28.4,27.1,26.7,26.1,26.0,24.4,21.0,20.8,19.8,19.6,18.9,18.6,18.5,18.3,16.1,-1.88,-3.9,-4.5.
化合物(b)(100mg)溶于3ml的DCM和2ml的DMF的混合溶液，在冰浴条件下加入selectfluor(50mg，1.0eq)，反应液在室温下搅拌1h。反应液用饱和碳酸氢钠溶液洗涤，无水碳酸钠干燥，然后再旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯50：1)，得化合物(c)(60mg)，产率71％。
化合物(c)1H-NMR(400MHz,CDCl3)δ(ppm)5.34(ddd,J＝48.0Hz,J＝13.0Hz,J＝6.2Hz,1H),2.53-2.74(m,1H),1.31(s,3H),1.177(s,3H),1.170(s,3H),1.15(s,3H),1.11(s,3H),0.91(d,J＝6.4Hz,3H),0.89(s,3H),0.87(s,9H),0.71(s,3H),0.05(s,6H)；13C-NMR(100MHz,CDCl3)δ(ppm)210.2(d,J＝11.8Hz,1C),135.75,133.0,89.3(d,J＝184.9Hz,1C),73.6,52.1,50.4,49.9,48.5,45.6,44.6,43.7,43.6,38.1,38.0,36.9,36.5,30.8,30.2,29.8,28.3,26.1,26.0,24.6,24.4,21.6,21.4,20.9,20.1,19.0,18.8,18.2,15.8,-1.9.
化合物(c)(57mg)溶于DCM/MeOH(1mL/3mL)，室温下硼氢化钠(20mg，5eq)加入到反应液中，室温搅拌1h。反应液用饱和碳酸氢钠溶液洗涤，无水碳酸钠干燥，然后再旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯50：1)，得化合物(d)(30mg)，产率70％；化合物(e)(9mg)，产率20％。
化合物(d)1H-NMR(400MHz,CDCl3)δ(ppm)4.69-4.53(m,1H),3.29(t,J＝12.0Hz,1H),1.18(s,3H),1.17(s,3H),1.08(s,3H),1.06(s,3H),0.87(d,J＝6.4Hz,3H),0.88(s,3H),0.87(s,3H),0.85(s,9H),0.69(s,3H),0.06(s,6H).
化合物(e)1H-NMR(400MHz,CDCl3)δ(ppm)5.00-4.85(m,1H),3.67(d,J＝6.8Hz,1H),1.18(s,3H),1.17(s,3H),1.06(s,3H),1.04(s,3H),0.90(d,J＝6.4Hz,3H),0.897(s,3H),0.88(s,3H),0.85(s,9H),0.69(s,3H),0.06(s,6H).
化合物(d)(30mg)溶于乙腈/THF(1ml/1mL)，40％HF水溶液加入到反应液，50摄氏度搅拌过夜。反应液残留HF用氯化钙溶液中和，化合物用DCM萃取，无水碳酸钠干燥，然后再旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯10：1)， 得化合物(14A)(18mg)，产率75％。
化合物(14A)1H-NMR(400MHz,CDCl3)δ(ppm)4.66-4.49(m,1H),3.28(dd,J＝13.0Hz,J＝9.6Hz,1H),1.21(s,6H),1.07(s,3H),1.05(s,3H),0.90(d,J＝6.3Hz,3H),0.88(s,3H),0.86(s,3H),0.68(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.9,133.5,93.4(d,J＝165.6Hz,1C),81.0(d,J＝15.4Hz,1C),71.1,50.4,50.2,50.1,49.8,44.5,44.4,41.1,40.9,39.5,39.4,38.7,38.5,36.7,36.5,30.8,30.76,29.7,29.3,29.2,28.4,28.2,26.2,24.3,21.3,21.1,20.2,18.7,18.0,16.6,15.7.
化合物(d)(20mg)溶于乙腈/THF(1ml/1mL)，40％HF水溶液加入到反应液，50摄氏度搅拌过夜。反应液残留HF用氯化钙溶液中和，化合物用DCM萃取，无水碳酸钠干燥，然后再旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯10：1)，得化合物(14B)(14mg)，产率85％。
化合物(14B)1H-NMR(400MHz,CDCl3)δ(ppm)5.01-4.85(m,1H),3.67(d,J＝7.7Hz,1H),1.21(s,6H),1.05(s,3H),1.03(s,3H),0.92-0.88(m,9H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.7,133.9,91.5(d,J＝167.1Hz,1C),76.7,71.2,50.5,50.0,44.6,44.5,43.5,38.8,38.7,38.6,38.5,36.8,36.6,35.8,35.6,31.0,30.9,29.8,29.5,29.4,28.3,28.2,26.1,24.4,21.9,21.3,21.26,20.3,18.8,17.9,15.8.
实施例15、化合物15A的合成
Figure PCTCN2017114645-appb-000019
化合物(b)(380mg，0.56mmol)溶于DCM中，在冰浴条件下，将m-CPBA(1.0eq)与NaCO3(0.7eq)的DCM混悬液缓慢加入反应液中，搅拌20min，TLC检测反应完成，反应液中加入饱和碳酸氢钠溶液停止反应，用水洗涤，DCM萃取，无水硫酸钠干燥，旋转蒸发仪蒸干溶剂，所得产品用硅胶柱分离(石油醚：乙酸乙酯80：1)，得化合物(f)(290mg)， 产率90％。
化合物(f)1H-NMR(400MHz,CDCl3)δ(ppm)4.65-4.60(q,J1＝6Hz,J2＝12Hz,1H),1.17-1.16(d,6H),1.11(s,3H),1.08(s,3H),0.90(s,6H),0.71(s,3H),0.14(s,3H),0.02(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)214.08,135.16,133.69,73.64,71.62,52.11,50.45,49.86,48.19,46.85,45.62,44.60,37.87,36.87,36.52,32.02,30.91,30.84,30.18,29.74,29.45,28.29,26.21,25.96,25.94,24.97,24.34,22.78,21.84,21.55,20.92,20.07,19.31,19.17,18.81,18.71,18.19,15.82,14.50,-1.98,-4.50,-5.45.
化合物(f)(290mg，0.51mmol)溶于MeOH/DCM中，将NaBH4(10.0eq)缓慢加入反应液中，室温下搅拌过夜，TLC检测反应完成，旋转蒸发仪蒸干溶剂，所得产品用硅胶柱分离(石油醚：乙酸乙酯40：1)，得化合物(g)，全部用于下一步反应。
化合物(g)溶于乙腈/THF(1ml/1mL)，40％HF水溶液(100ul)加入到反应液，50摄氏度搅拌过夜。反应液残留HF用氯化钙溶液中和，化合物用DCM萃取，无水硫酸钠干燥，旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(DCM：MeOH 50：1)，得化合物(15A)(36mg)，两步产率15％。
化合物(15A)1H-NMR(400MHz,CD3OD)δ(ppm)3.66-3.60(m,1H),2.93-2.91(d,J＝9.6Hz,1H),1.05(s,3H),1.01(s,3H),0.93-0.91(d,3H),0.89(s,3H),0.82(s,3H),0.92(s,3H)；13C-NMR(100MHz,CD3OD)δ(ppm)135.48,135.14,83.97,71.26,69.66,51.49,51.41,50.61,45.38,44.98,44.75,40.02,38.91,37.70,37.39,31.88,31.57,29.11,28.99,28.95,28.91,27.18,24.55,21.97,21.84,20.58,19.12,19.09,17.07,16.18.
实施例16、化合物13A、13D、13F和13G的合成
Figure PCTCN2017114645-appb-000020
化合物(a)16g(40％，13.7mmol)溶于DCM中，前3h在室温下加入一半m-CPBA与NaHCO3，后3h在冰浴条件下加入剩下的m-CPBA，TLC检测反应完，反应液用饱和碳酸氢钠溶液洗涤，无水硫酸钠干燥，旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯80：1)，得化合物(b)，产率80％。
化合物(b)1.1g(2.4mmol)溶于乙醚中，HIO4加入反应液中，室温下搅拌20min，TLC检测反应完全，反应液用水洗涤，无水硫酸钠干燥，旋转蒸发仪蒸干溶剂，所得产品即化合物(c)可直接用于下一步反应。
化合物(c)200mg(0.4mmol)溶于DCM中，将前处理后的叶立徳试剂加入反应液中，氮气保护室温下搅拌4h，旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯50：1)，得化合物(d)(190mg)，产率90％。
化合物(d)1H-NMR(400MHz,CDCl3)δ(ppm)5.01-4.85(m,1H),3.67(d,J＝7.7Hz,1H),1.21(s,6H),1.05(s,3H),1.03(s,3H),0.92-0.88(m,9H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.7,133.9,91.5(d,J＝167.1Hz,1C),76.7,71.2,50.5,50.0,44.6,44.5,43.5,38.8,38.7,38.6,38.5,36.8,36.6,35.8,35.6,31.0,30.9,29.8,29.5,29.4,28.3,28.2,26.1,24.4,21.9,21.3,21.26,20.3,18.8,17.9,15.8.
化合物(d)100mg(0.2mmol)溶于乙醇/水(4ml/2ml)中，加入KOH(3.0eq)，120℃加热过夜，TLC检测反应完全，反应液用旋转蒸发仪蒸去乙醇，水相中滴加浓盐酸调至pH<7，过滤所得产品采用硅胶柱分离(石油醚：乙酸乙酯5：1)，得化合物13G(30mg)，产率33％。
化合物(13G)1H-NMR(400MHz,DMSO)δ(ppm)12.09(s,1H),6.63(s,1H),4.31-4.32(d,1H),3.00-2.97(dd,1H),1.98(s,3H),1.12(s,3H),0.96-0.90(m,9H),0.83(s,3H),0.69(s,3H)；13C-NMR(100MHz,DMSO)δ(ppm)168.81,141.99,134.30,133.48,127.38,76.72,50.01,49.75,49.34,44.02,38.50,36.52,35.74,35.16,34.50,30.48,30.37,28.07,27.65,27.54,25.97,24.94,23.99,20.47,18.96,18.30,17.85,15.79,15.52,12.10.
化合物(d)80mg(0.15mmol)溶于甲醇中，加入20％钯碳，在氢气中室温下搅拌过夜，TLC检测反应完全，反应液用硅藻土过滤，旋转蒸发仪蒸干后所得产品溶于乙醇/水(3ml/1.5ml)，加入KOH(3.0eq)，120℃加热过夜，TLC检测反应完全，反应液用旋转蒸发仪蒸去乙醇，水相中滴加浓盐酸调至PH<7，过滤所得产品采用硅胶柱分离(石油醚：乙酸乙酯5：1)，得化合物13F(20mg)，总产率29％。
化合物(13F)1H-NMR(400MHz,DMSO)δ(ppm)3.02-2.98(dd,1H),2.31-2.28(t,1H),1.98(s,3H),1.03-1.02(d,3H),0.91-0.89(d,3H),0.85-0.83(m,3H),0.69(s,3H)；
化合物(d)100mg(0.19mmol)溶于无水DCM，在冰浴中缓慢滴加DIBAL(5.0eq),在氮气条件下反应3h，反应液中缓慢滴加甲醇中和剩余DIBAL，旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯10：1)，得化合物(68mg)，产率85％。
化合物(13F)1H-NMR(400MHz,CDCl3)δ(ppm)5.41-5.38(t,1H),4.00(s,2H),3.25-3.21(dd,1H),1.06-0.98(m,9H),0.93-0.92(d,3H),0.87(s,3H),0.80(s,3H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.52,134.47,134.41,127.19,79.10,69.22,50.51,50.48,49.92,44.61,39.00,37.13,36.40,36.09,35.69,31.10,30.94,29.81,28.33,28.07,27.95,26.61,24.62,26.36,21.11,19.25,18.72,18.36,15.87,15.53,13.75.
化合物(13A)
1H-NMR(400MHz,CDCl3)δ(ppm)3.52-3.48(m,1H),3.44-3.39(m,1H),3.25-3.21(m,1H),0.68(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.5,79.1,68.7,68.5,50.6,50.5,49.9, 44.6,39.0,37.2,36.7,36.6,36.5,36.0,35.7,33.8,33.7,31.1,31.0,29.8,28.4,28.1,28.0,26.6,24.4,21.1,19.3,18.9,18.8,18.4,16.9,16.6,15.9,15.6
实施例17、化合物13D和13E的合成
Figure PCTCN2017114645-appb-000021
化合物(d)400mg(0.8mmol)溶于DCM中，将前处理后的叶立徳试剂加入反应液中，氮气保护室温下搅拌4h，旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯30：1)，得化合物(e)(436mg)，产率95％。
化合物(b)1H-NMR(400MHz,CDCl3)δ(ppm)5.01-4.85(m,1H),3.67(d,J＝7.7Hz,1H),1.21(s,6H),1.05(s,3H),1.03(s,3H),0.92-0.88(m,9H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.7,133.9,91.5(d,J＝167.1Hz,1C),76.7,71.2,50.5,50.0,44.6,44.5,43.5,38.8,38.7,38.6,38.5,36.8,36.6,35.8,35.6,31.0,30.9,29.8,29.5,29.4,28.3,28.2,26.1,24.4,21.9,21.3,21.26,20.3,18.8,17.9,15.8.
化合物(e)140mg(0.25mmol)溶于乙醇/水(4ml/2ml)中，加入KOH(3.0eq)，120℃加热过夜，TLC检测反应完全，反应液用旋转蒸发仪蒸去乙醇，水相中滴加浓盐酸调至pH<7，过滤所得产品采用硅胶柱分离(石油醚：乙酸乙酯5：1)，得化合物13E(E型，24.4mg)，产率22％。
化合物(13E)1H-NMR(400MHz,CDCl3)δ(ppm)6.83-6.76(td,1H),5.77-5.73(d,1H,J＝15.2Hz),4.33-4.31(d,1H),3.02-2.97(dd,1H),1.15-1.11(t,3H),0.97-0.88(m,9H),0.84(s,3H),0.70(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)167.12,149.37,134.29,133.47,121.64,76.71,66.97,50.00,49.77,49.33,44.02,38.49,36.50,35.58,35.15,34.09,30.35,28.30,28.07, 27,59,27.53,25.96,23.99,20.46,18.95,18.23,17.84,15.79,15.52.
化合物(e)50mg(0.09mmol)溶于甲醇中，加入20％钯碳，在氢气中室温下搅拌过夜，TLC检测反应完全，反应液用硅藻土过滤，旋转蒸发仪蒸干后所得产品溶于乙醇/水(3ml/1.5ml)，加入KOH(3.0eq)，120℃加热过夜，TLC检测反应完全，反应液用旋转蒸发仪蒸去乙醇，水相中滴加浓盐酸调至pH<7，过滤所得产品采用硅胶柱分离(石油醚：乙酸乙酯5：1)，得化合物13D(E型，10mg)，总产率25％。
化合物(13D)1H-NMR(400MHz,CDCl3)δ(ppm)4.32-4.31(d,1H),3.01-2.96(dd,1H),2.19-2.16(t,2H),1.14-1.09(t,3H),1.02-0.89(m,9H),0.85-0.83(m,7H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)174.50,133.53,76.75,50.04,49.90,49.36,43.99,38.52,36.54,35.71,35.36,35.19,33.70,30.52,30.38,28.10,27.70,27.56,26.00,25,16,24.94,24.03,20.50,18.98,18.49,17.87,15.82,15.55.
实施例18、化合物12C的合成
Figure PCTCN2017114645-appb-000022
化合物(f)(215mg)溶于THF(10ml)中，搅拌条件下加入四氢锂铝(38mg，2.0eq)，反应在室温下搅拌6个小时。反应结束后，向反应液滴加甲醇，中和未反应的四氢锂铝，硅藻土过滤，然后用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(二氯甲烷：甲醇5：1)，得化合物12C，产率90％.
化合物14H 1H-NMR(400MHz,CDCl3)δ(ppm)3.64(m,t,J＝6.6Hz,2H),3.25-3.21(m,1H),1.00(s,3H),0.98(s,3H),0.87(s,3H),0.81(s,3H),0.68(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.53,79.13,63.25,50.57,50.53,49.94,44.59,39.02,37.15,36.51,36.31,35.72,33.03,31.11,30.97,28.36,28.10,27.98,26.63,26.35,26.28,24.40,21.14,19.28,18.84,18.39,15.88,15.56.
实施例19、化合物12D的合成
Figure PCTCN2017114645-appb-000023
化合物(g)(50mg)溶于DCM，DMP(50mg，1.2eq)加入到反应体系中，室温搅拌5小时，反应液用饱和碳酸氢钠溶液洗涤，无水碳酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯10：1)，得化合物(h)(43mg)，产率85％。1H-NMR(400MHz,CDCl3)δ(ppm)4.51-4.47(m,1H),2.60-2.43(m,2H),2.04(s,3H),1.38(s,6H),1.00(s,3H),0.91-0.88(m,12H),0.68(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)215.2,171.2,134.5,134.4,81.0,76.3,50.6,50.5,50.0,44.7,37.9,37.0,36.2,35.4,32.7,31.1,30.9,30.3,28.3,28.0,26.7,26.5,24.4,24.3,21.5,21.1,19.3,18.6,18.2,16.7,15.9.
化合物(h)(50mg)溶于10％氢氧化钾乙醇溶液(3mL)，反应在90℃反应两个小时，反应液用饱和碳酸氢钠溶液洗涤，DCM萃取，无水碳酸钠干燥有机相，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯5：1)，得化合物(12D)(45mg)，产率为89％。
1H-NMR(400MHz,CDCl3)δ(ppm)3.25-3.22(m,1H),2.57-2.48(m,2H),1.00(s,3H),0.98(s,3H),0.91-0.88(m,10H),0.81(s,3H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)215.1,134.6,134.5,79.1,76.4,50.60,50.57,50.0,44.7,39.1,37.2,36.3,35.8,32.8,32.1,31.2,31.0,30.4,29.9,29.5,28.3,28.1,26.72,26.67,24.4,22.8,21.2,19.3,18.6,18.4,15.9,15.6,14.3.
实施例20、化合物13B和13C的合成
Figure PCTCN2017114645-appb-000024
化合物(i)(200mg)溶于二乙胺(4mL)中，加入金属锂(100mg)，室温搅拌过夜。甲醇慢慢滴加到反应液中，直至没有气泡产生反应变澄清。所得溶液在旋转蒸发仪中除去甲醇二乙胺，所得产品用饱和碳酸氢钠洗涤，DCM萃取。无水碳酸钠干燥，然后再用旋转蒸 发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯5：1)，得化合物(13B)和(13C)，产率分别为25％和15％。
化合物13B：1H-NMR(400MHz,CDCl3)δ(ppm)3.19-3.16(m,1H),1.19(s,3H),1.01(s,3H),0.98(s,3H),0.91(s,3H),0.89(d,J＝6.3Hz,3H),0.79(s,3H),0.77(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)78.2,77.3,71.2,50.6,47.6,45.8,45.3,44.5,42.8,40.6,38.9,36.9,36.1,33.9,29.6,29.4,29.32,29.26,28.4,28.1,28.1,28.06,27.5,23.9,21.5,21.2,18.8,18.4,16.9,15.5,14.7.
化合物13C：1H-NMR(400MHz,CDCl3)δ(ppm)5.41(t,J＝3.7Hz,1H),3.28-3.24(m,1H),1.21(s,6H),1.17(s,3H)1.19(s,3H),1.01(s,3H),0.98(s,3H),0.91(s,3H),0.89(d,J＝6.3Hz,3H),0.79(s,3H),0.77(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)78.2,77.3,71.2,50.6,47.6,45.8,45.3,44.5,42.8,40.6,38.9,36.9,36.1,33.9,29.6,29.4,29.32,29.26,28.4,28.1,28.1,28.06,27.5,23.9,21.5,21.2,18.8,18.4,16.9,15.5,14.7.
实施例21、豆甾醇衍生物的合成
Figure PCTCN2017114645-appb-000025
将250mg豆甾醇溶于5ml吡啶中，加入116mg TsCl，8mg DMAP催化，室温下搅拌12h后，TLC监测反应完全。用饱和碳酸氢钠水溶液、二氯甲烷萃取，分离有机相，旋转蒸干，硅胶色谱柱分离纯化得到190mg化合物2.
1H-NMR(400MHz,CDCl3)δ(ppm)7.79(d,J＝8.24Hz,2H),7.32(d,J＝8.08Hz,2H),5.29-5.29(m,1H),5.14-5.10(m,1H),5.04-4.97(m,1H),2.46-2.39(m,4H),2.28-2.23(m,1H),2.04-1.91(m,4H),0.67(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)144.5,138.9,138.4,134.8,134.8,129.8,129.4,127.7,123.6,82.46,56.8,56.0,51.3,50.0,42.3,40.6,39.6,39.0,37.0,36.6,36.4,32.0,31.9,31.8,29.0,28.7,25.5,24.4,21.7,21.3,21.2,21.1,19.2,19.1,12.4,12.1.
将190mg化合物2溶于5ml甲醇中，加入190mg醋酸钾，加热回流3小时。TLC监测反应完全。用饱和碳酸氢钠水溶液、二氯甲烷萃取，分离有机相，旋转蒸干，硅胶色谱柱分离纯化得到109mg化合物3。
1H-NMR(400MHz,CDCl3)δ(ppm)5.18-5.12(m,1H),5.04-4.98(m,1H),3.32(s,3H),2.76(dd,J＝2.5Hz,J’＝2.5Hz,1H),0.74(s,3H),0.66(dd,J＝4.5,J’＝4.5Hz,1H),0.44(dd,J＝8.0Hz,J’＝5.0Hz,1H)；13C-NMR(100MHz,CDCl3)δ(ppm)138.4,129.2,82,4,56.7,56.6, 56.1,51.3,48.1,43.4,42.7,40.6,40.2,35.3,35.0,33.4,31.9,30.5,29.1,25.4,25.0,24.3,22.8,21.5,21.2,21.1,19.3,19.0,13.1,12.5,12.3.
Figure PCTCN2017114645-appb-000026
将200mg豆甾醇溶于7ml二氯甲烷中，于冰水浴条件下加入300mg间氯过氧酸(85％)和164mg碳酸氢钠，搅拌1小时后恢复至室温继续搅拌12小时，TLC监测反应完全。用饱和碳酸氢钠水溶液、二氯甲烷萃取，分离有机相，旋转蒸干，硅胶色谱柱分离纯化得到126mg化合物B7，其中包含4种异构体。
1H-NMR(400MHz,CDCl3)δ(ppm)0.61(m,3H),0.95(m,3H),1.05(s,3H),2.91(d,J＝4.3Hz,1H,),3.06(d,J＝2.1Hz,1H),3.61-3.78(m,1H),3.81-3.99(m,1H)；13C-NMR(100MHz,CDCl3)δ(ppm)12.01,12.15,12.53,12.66,16.08,16.36,16.45,19.51,19.59,19.68,20.38,20.75,20.80,20.98,21.07,24.42,24.27,27.04,27.94,28.91,29.29,29.46,30.03,31.17,32.56,35.01,38.89,39.01,39.34,39.35,39.96,42.70,42.81,42.81,42.83,48.43,48.91,53.26,55.91,56.58,56.61,58.74,59.41,59.48,62.26,62.42,63.28,65.96,68.74,11.90,12.61,16.25,17.22,19.75,20.33,22.09,22.14,24.60,27.19,29.94,32.72,37.41,38.70,42.33,51.44,51.53,53.62,55.97,56.22,63.18,63.8,69.48.
Figure PCTCN2017114645-appb-000027
将70mg化合物3溶于4ml二氯甲烷中，冰浴下加入33mg间氯过氧酸，15mg碳酸氢钠搅拌10分钟，然后允许恢复室温搅拌12小时。12小时后TLC监测反应完全，加入1ml水终止反应。用饱和碳酸氢钠水溶液、二氯甲烷萃取，分离有机相，旋转蒸干，硅胶色谱柱分离纯化得到42mg化合物7。
将化合物7溶于5ml1，4-二氧六环水溶液中(1，4-二氧六环：水＝4：1)，加入一水合对甲苯磺酸5mg，80℃下搅拌10小时，TLC监测反应完全，用饱和碳酸氢钠水溶液、二 氯甲烷萃取，分离有机相，旋转蒸干，硅胶色谱柱分离纯化得到20mg化合物8H。
1H-NMR(400MHz,CDCl3)δ(ppm)5.35(d,J＝4.80Hz,1H),3.56-3.50(m,1H),2.74(dd,J1＝7.04Hz,J2＝2.00Hz,1H),2.49(d,J＝3.56Hz,1H),0.68(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)140.93,121.72,71.92,62.28,56.50,53.56,50.21,48.45,42.78,42.41,39.70,38.86,37.37,36.63,32.00,31.78,29.29,28.09,24.70,21.17,21.00,20.34,19.70,19.54,16.36,12.63,11.99.
Figure PCTCN2017114645-appb-000028
将90.5mg化合物7溶于5ml二氯甲烷中，冰浴下加入120mg三氯化铝，90m四氢铝锂搅拌10分钟，然后置于80℃下搅拌2小时，TLC监测反应完全，加入1ml水终止反应。用饱和碳酸氢钠水溶液、二氯甲烷萃取，分离有机相，旋转蒸干，硅胶色谱柱分离纯化得到70mg化合物8。
将化合物7溶于5ml1，4-二氧六环水溶液中(1，4-二氧六环：水＝4：1)，加入一水合对甲苯磺酸16mg，80℃下搅拌10小时，TLC监测反应完全，用饱和碳酸氢钠水溶液、二氯甲烷萃取，分离有机相，旋转蒸干，硅胶色谱柱分离纯化得到30mg化合物8H。
1H-NMR(400MHz,CDCl3)δ(ppm)5.34(s,1H),3.91(t,J＝6.66Hz,1H),3.56-3.48(m,1H),2.32-2.19(m,2H),0.06(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)140.89,121.79,71.91,70.76,57.06,56.84,50.20,49.37,42.67,42.54,42.42,39.91,37.37,36.62,34.34,32.00,31.78,28.74,28.62,24.42,21.28,21.20,19.56,18.47,13.99,11.96.
Figure PCTCN2017114645-appb-000029
将106mg化合物4B溶于5ml THF中，于冰浴条件下缓慢加入90mg四氢铝锂，加完后允许恢复至室温并回流搅拌1.5小时，TLC监测反应完全，加入1ml水终止反应。用饱和碳酸氢钠水溶液、二氯甲烷萃取，分离有机相，旋转蒸干，硅胶色谱柱分离纯化得到90mg 化合物7H和13mg化合物8A。
化合物7H：1H-NMR(400MHz,CDCl3)δ(ppm)4.07-4.05(m,1H),2.71(d,J＝6.76Hz,1H),2.47(d,J＝8.68Hz,1H),1.97-1.81(m,5H),0.63(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)75.39,67.34,63.22,62.24,58.65,56.21,55.88,53.58,53.54,48.86,48.38,45.99,45.90,44.00,43.15,43.11,40.02,39.90,38.98,38.88,38.80,34.83,34.50,30.95,30.91,29.41,29.22,28.05,27.19,26.02,24.46,24.42,21.45,21.40.21.02,20.96,20.29,19.66,19.55,19.45,16.33,16.25,12.57,12,47,12.36,12.21.
化合物8A：1H-NMR(400MHz,CDCl3)δ(ppm)3.80(s,br,1H),3.69-3.60(m,1H),2.74(d,J＝6.12Hz,1H),2.49(d,J＝7.88Hz,1H),0.68-0.66(m,6H)；13C-NMR(100MHz,CDCl3)δ(ppm)72.15,71.85,67.49,63.25,62.78,58.71,57.16,56.31,55.91,54.44,54.36,48.93,48.45,47.52,46.00,43.17,40.18,39.97,39.80,39.04,38.76,38.67,35.54,34.54,34.37,31.65,31.03,30.94,30.53,29.75,29.48,29.30,27.22,26.06,24.62,24.23,21.49,21.21,21.09,20.31,20.06,19.58,19.51,18.49,16.42,15.93,12.53,12.39,12.24.
二、化合物的生物学评价
本测试例细胞培养用到的培养基组分及细胞培养条件：
Huh-7(人肝癌细胞系；购自ATCC)和Huh-7/SRE-Luc细胞在37℃、5％CO2培养箱中培养，细胞生长在培养基A(DMEM，100单位/ml青霉素，100μg/ml链霉素)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)细胞在37℃、5％CO2培养箱中培养，细胞生长在培养基B(DMEM与F12培养基等比例混合，100单位/ml青霉素，100μg/ml链霉素)加上5％FBS。
实施例22、根据本发明实施例的化合物对SREBP报告基因活性抑制的测定
以下方法运用Huh-7/SRE-Luc细胞系测定根据本发明实施例的化合物抑制SREBP信号通路的活性。Huh-7/SRE-Luc细胞系是通过在人肝癌细胞系Huh-7细胞中稳定转染表达了2个报告基因：分别为启动子具有SREBP蛋白结合位点并依赖于SREBP信号活性表达的萤火虫荧光素酶SRE-Firefly Luciferase，和持续表达作为内参的绿色荧光蛋白EGFP，示意图如图1(A)所示。
实验方法简述如下：将Huh-7/SRE-Luc细胞培养在DMEM培养基中(含10％FBS，100units/ml P/S)，待细胞汇合度达到80％时，用0.25％胰酶(EDTA)消化吹匀后以每孔5×104细胞接种到24孔板中。于37℃、5％CO2培养箱中培养24小时后，吸掉培养基，用PBS缓冲液(137mM NaCl，2.7mM KCl，10mM Na2HPO4，2mM KH2PO4)洗1遍。再加入配置有不同浓度化合物的去脂蛋白DMEM培养基(含10％去脂蛋白血清，1μM洛伐他汀，10μM甲羟戊酸，P/S)，在去脂蛋白培养条件下，有利于最大程度地激活SREBP信号通路。 培养16小时后，吸掉培养基，每孔加入150μl细胞裂解液Reporter Lysis Buffer(Promega，E397A)，将24孔板置于-80℃冰箱中冻结，再室温解冻1次，随后以800rpm在涡旋仪上振荡10分钟，达到充分裂解细胞的目的。
吸取100μl裂解液加入到96孔黑色酶标板中(PerkinElmer，6005329)，用EnVsion酶标仪(PerkinElmer)以激发光为485nm，发射光为535nm，测量绿色荧光蛋白EGFP的荧光强度。吸取20μl裂解液加入到96孔白色酶标板中(Greiner，655075)，并按照Promega公司的Luciferase Assay System试剂盒说明书，加入30μl的荧光素酶底物，立即将酶标板放入EnVision酶标仪中，600rpm振荡5秒混匀后，测量荧光的发光值。最后以荧光素酶的荧光发光值除以EGFP的荧光强度值的比值，作为反应SREBP信号活性的相对值，并以DMSO未处理组标准化为1。
对根据本发明实施例的化合物进行初步筛选时，采用3μM和10μM两个浓度，通过以上方法测定其抑制SREBP信号通路的活性，测得的对SREBP通路进行抑制的效果如图1(B)所示。
表1根据本发明实施例的化合物对SREBP信号通路的抑制活性筛选
Figure PCTCN2017114645-appb-000030
Figure PCTCN2017114645-appb-000031
随后对在初筛中活性较好的化合物进一步做更为详细的浓度梯度，同样通过上述方法测得的对SREBP通路进行抑制的半效抑制浓度IC50值如表2所示。
表2根据本发明实施例的化合物抑制SREBP报告基因活性的IC50
Figure PCTCN2017114645-appb-000032
Figure PCTCN2017114645-appb-000033
Figure PCTCN2017114645-appb-000034
由图1和表2中的数据可以看出，本发明实施例化合物对SREBP报告基因均具有显著的抑制效果，其中活性最强的化合物1G IC50为0.22μM。
实施例23、根据本发明实施例的化合物抑制SREBP的剪切加工成熟
以下方法用于测定根据本发明实施例的化合物抑制SREBP的剪切加工成熟。实验方法简述如下：将CHO-7细胞以6×105传到60mm培养皿中，培养1天后。吸掉培养基，PBS洗1遍，去掉PBS，加入含有不同浓度化合物的去脂蛋白DMEM培养基(含10％去脂蛋白和去脂肪酸血清，1μM洛伐他汀，10μM甲羟戊酸，P/S)，处理15小时后，再加入终浓度为10μM的MG132(Cayman，10012628)。处理1小时后，用细胞刮将培养皿中的细胞刮下，转移到15ml离心管中，并置于冰上。4℃1000g离心5分钟后，倒掉培养基，加入1ml PBS缓冲液，并转移至1.5ml离心管，4℃1000g离心5分钟。去掉PBS，加入300μl SDS Lysis Buffer(10mM Tris-HCl(pH 7.6)，100mM氯化钠，1％十二烷基硫酸钠和蛋白酶抑制剂)，过7号针头15次。13200rpm室温离心10分钟，转移270μl上清至新1.5ml离心管，并加入90μl 4xLoading Buffer(1.2％十二烷基硫酸钠，30％甘油，150mM Tris-HCl(pH 6.8)，0.6％β-巯基乙醇和适量溴酚蓝)，混匀后95度煮10分钟，储存于负20度冰箱中。
蛋白免疫印迹杂交(Western Blot)检测SREBP的剪切加工
1)上样：每个蛋白样品上样量30μl加于8％SDS-PAGE胶中的胶孔。
2)电泳：先用85伏电压30分钟让蛋白进入浓缩胶，随后120伏1小时让蛋白进入分离胶。
3)转膜：电泳结束后，取出凝胶按照滤纸、硝酸纤维素膜、凝胶、滤纸的顺序放入转膜装置中，并注意排除气泡，100伏恒压转膜1.5小时。
4)封闭：转膜结束后，将硝酸纤维素膜浸泡于含有5％脱脂奶粉的TBST缓冲液(20mM Tris-HCl(pH 7.4)，150mM氯化钠和0.05％Tween-20)中，室温摇床孵育1小时。
5)一抗孵育：封闭完成后，用TBST缓冲液洗膜3次。加入4μg/ml的SREBP2抗体(IgG-7D4，识别N-端仓鼠SREBP2的小鼠单克隆抗体)，室温孵育2小时或4度过夜。回收抗体，将膜用TBST洗4次，每次8分钟。
6)二抗孵育：弃去TBST，加入1:5000稀释的羊抗小鼠HRP偶联二抗，室温孵育1小时。TBST洗4次，每次8分钟。
7)显色拍照：将ECL发光底物覆盖于硝酸纤维素膜上，用保险膜封好，用LAS4000冷光/生物发光影像分析仪(Fujifilm)拍摄图片。
8)SREBP2剪切加工比例的定量分析，将得到的灰度图片用Image J(NIH)软件进行定量分析，将核形式SREBP除以前体形式SREBP即为SREBP剪切加工的比例，以未加药组为1。
测定结果如图2(A)所示，根据本发明实施例的化合物显著地减少了SREBP的核形式(n-SREBP)的形成，同时伴随前体形式SREBP(pre-SREBP)的增多。图2(B)定量分析结果显示根据本发明实施例的化合物抑制SREBP剪切加工的活性非常强，其IC50值为0.046μM。
实施例24、根据本发明实施例的化合物对SREBP靶基因表达的抑制活性的测定
以下方法用于测定根据本发明实施例的化合物对SREBP靶基因表达的抑制作用。简述如下：
将Huh-7细胞以6×105传到60mm培养皿中，培养24小时。吸掉培养基，PBS清洗1遍。去掉PBS，加入含有不同浓度化合物的去脂蛋白和去脂肪酸DMEM培养基(含10％去脂蛋白和去脂肪酸血清，1μM洛伐他汀，10μM甲羟戊酸，P/S)，处理16小时。吸去培养基，加入1ml TRIzol(Sigma，T9424)，室温放置10分钟，将细胞裂解液转移到1.5ml Eppendorf管中，随后用于mRNA的提取、逆转录合成cDNA以及荧光实时定量PCR。
1、mRNA的提取和定量
1)每1ml TRIzol中加入200μl的氯仿，涡旋仪上剧烈振荡混匀，室温静置15分钟。
2)4℃，13200rpm离心10分钟，转移500μl的上层水相至新的1.5ml Eppendorf管中。
3)每份样品中加入600μl异戊醇，来回颠倒混匀。
4)4℃，13200rpm离心10分钟，弃去上清，管底可见乳白色RNA沉淀。每份样品中加入1ml的70％乙醇(用DEPC处理过的去离子水稀释)，颠倒混匀。
5)4℃，13200rpm离心10分钟，弃去上清，沉淀室温晾干，加入30μl DEPC处理过的去离子水，充分溶解。
6)取2μl RNA到98μl水中，混匀后用分光光度计(Eppendorf)测定260nm的光吸收值，计算样品浓度，测定260nm与280nm光吸收值的比值，计算样品浓度，最终调整样品浓度为1μg/μl。
2、逆转录合成cDNA
cDNA合成采用Promega公司的M-MLV逆转录酶试剂盒。每50μl体系含有4μg的RNA，1μg的OligodT，终浓度各为0.4mM的dNTP。
1)每个0.2ml PCR管中，加入4μl浓度为1μg/μl的RNA，1μl 1μg/μl的OligodT， 15μl DEPC水，混匀。
2)70℃变性5分钟，冰上骤冷。
3)每个反应体系中加入17μl DEPC水，10μl 5×MLV缓冲液，1μl dNTP(各10mM)，2μl M-MLV逆转录酶，混匀。
4)37℃反应1小时。
5)70℃变性10分钟。
6)10℃降温5分钟，加入200μl水稀释cDNA。
3、荧光实时定量PCR
Realtime PCR采用盛元生物公司的Sharpvue 2x Universal qPCR Master Mix试剂。每20μl体系中，Sharpvue 2x Mix:10μl，终浓度为0.5μM的引物，2μl模板cDNA(逆转录产物稀释4倍用)，用水补至20μl。反应体系配置好后，混匀，每个样品3个复孔。在Stratagene Mx30005P实时荧光定量PCR仪上按照以下程序运行：a.95℃热激活5分钟，b.95℃变性30秒，c.60℃退火30秒，d.72℃延伸30秒，延伸结束后采集荧光信号，e.b-d循环40次，f.溶解曲线分析：95℃15秒，60℃1分钟，以1％的速度上升到95℃，95℃持续15秒，60℃上升到95℃的过程中采集荧光信号。运用比较Ct法计算mRNA的相对含量，并标准化以DMSO组为1。
本实施例中荧光定量PCR采用的引物序列见表3。
表3本实施例中荧光定量PCR所用引物的信息
Figure PCTCN2017114645-appb-000035
测定结果如图2(C)所示，根据本发明实施例的化合物1G能显著抑制SREBP靶基因：SREBP-2、HMGCR、HMGCS和SS的表达。1G在1μM时，SREBP-2下调了约40％，HMGCR、HMGCS和SS下调了约80％。
上述试验结果证明根据本发明实施例的化合物对SREBP靶基因的表达具有显著的抑制作用。
实施例25、小鼠肝原代细胞中，根据本发明实施例的化合物对SREBP靶基因表达的抑制活性的测定
小鼠肝原代细胞分离：
1)1％戊巴比妥钠麻醉小鼠后，迅速打开腹腔，找到下腔静脉，插入留置针，同时剪断肝门静脉，以7.5ml/分钟的流速灌流150ml，灌流液为：137mM NaCl，5.4mM KCl,0.5mM NaH2PO4，0.4mM Na2HPO4，4.2mM NaHCO3，0.5mM EGTA，5mM Glucose，pH 7.4。
2)换0.15mg/ml的胶原酶溶液同样速度灌流100ml，胶原酶消化液为：137mM NaCl，5.4mM KCl,0.5mM NaH2PO4，0.4mM Na2HPO4，4.2mM NaHCO3，10mM HEPES，pH 7.4，15mg I型胶原酶(Worthington Biochemical)。每100ml溶液。
3)小心将肝摘下并转移入培养皿，用镊子撕碎包膜，将肝组织匀碎，去除包膜。
4)过70μm孔径的筛网，500rpm离心1分钟。小心吸去上清，取离心至管底的细胞加入适量DMEM培养液(含10％FBS，P/S)，计数，按1x 106细胞每60mm培养皿接种培养。
5)4小时后换液，PBS洗1遍，加入含有不同浓度化合物的去脂蛋白和去脂肪酸DMEM培养基(含10％去脂蛋白和去脂肪酸血清，1μM洛伐他汀，10μM甲羟戊酸，P/S)，处理16小时后。吸去培养基，加入1ml TRIzol(Sigma，T9424)，随后用于mRNA的提取、逆转录合成cDNA以及荧光实时定量PCR，方法同测试例3。本实施例中荧光定量PCR采用的引物序列见表4。
表4本实施例中荧光定量PCR所用引物的信息
Figure PCTCN2017114645-appb-000036
Figure PCTCN2017114645-appb-000037
测定结果见图2(D)，利用分离得到的肝原代细胞，本发明中化合物1G显著抑制SREBP靶基因：SREBP-2、HMGCR、HMGCS和SS的表达。1G在1μM时，SREBP-2下调了约40％，HMGCR、HMGCS和SS下调了约50％。
本发明实施例化合物在原代肝细胞中，对SREBP靶基因的表达均具有显著的抑制作用，表明有降血脂的功效。
实施例26、根据本发明实施例的化合物不激活LXR
测定根据本发明实施例的化合物对LXR的影响，方法与实施例3相同，简述如下：在人肝癌细胞系Huh7细胞中，加入含有不同浓度化合物的去脂蛋白和去脂肪酸DMEM培养基处理后，进行mRNA的提取、逆转录和荧光实时定量PCR分析。本实施例中荧光定量PCR采用的引物序列见表5。
表5本实施例中荧光定量PCR所用引物的信息
Figure PCTCN2017114645-appb-000038
Figure PCTCN2017114645-appb-000039
LXR的靶基因参与了胆固醇的外排和脂肪酸的合成，激活LXR后显著上调SREBP-1c的表达，将导致脂肪肝的形成和高甘油三酯血症。图3(A)中的测定结果，显示根据本发明实施例的化合物不激活LXR靶基因的表达，对参与脂肪酸合成的SREBP-1c、FASN和SCD1有显著下调作用。而已知LXR激活剂TO901317(Sigma)显著地激活了所有的LXR靶基因。
本实施例证明根据本发明实施例的化合物不激活LXR靶基因的表达，且对脂肪酸合成相关基因具有显著的抑制作用。
实施例27、小鼠肝原代细胞中，根据本发明实施例的化合物不激活LXR
在小鼠肝原代细胞中，测定根据本发明实施例的化合物对LXR的影响，方法与实施例4相同，简述如下：首先分离小鼠肝原代细胞，加入含有不同浓度化合物的去脂蛋白和去脂肪酸DMEM培养基处理后，进行mRNA的提取、逆转录和荧光实时定量PCR分析。本实施例中荧光定量PCR采用的引物序列见表6。
表6本实施例中荧光定量PCR所用引物的信息
Figure PCTCN2017114645-appb-000040
Figure PCTCN2017114645-appb-000041
在肝原代细胞中，图3(B)中的测定结果显示根据本发明实施例的化合物1G不激活LXR靶基因的表达，对参与脂肪酸合成的SREBP-1c、FASN和SCD1有显著下调作用。而已知的LXR激活剂TO901317能成倍地激活上述脂肪酸合成相关的基因，将导致脂肪肝的形成。
上述试验结果证明本发明实施例化合物1G在原代肝细胞中，不激活LXR靶基因的表达，且对脂肪酸合成相关基因具有显著的抑制作用，不太可能导致脂肪肝的形成。
实施例28、根据本发明实施例的化合物阻止饮食诱导肥胖小鼠体重和脂肪的增加
上述细胞实验表明根据本发明实施例的化合物强烈抑制SREBP蛋白的剪切加工，进而抑制SREBP靶基因的表达，抑制胆固醇和脂肪酸的合成。接下来在小鼠动物水平，分析根据本发明实施例的化合物的降脂效果。以下所有小鼠实验的饲养方法都按照武汉大学实验动物中心的动物饲养及使用条例。
小鼠实验方法如下：8周龄C57BL/6J小鼠(购自上海灵畅公司)随机分为4组，每组5只小鼠。第1组小鼠喂养基础饲料(Chow Diet，CD)，同时每天灌胃一次生理盐水。第2-4组小鼠喂养高脂高胆固醇血症饲料(Western Diet，WD)，Western Diet也叫西方饮食饲料，含20％脂肪、1.25％胆固醇和0.5％胆酸钠。第2组小鼠同时每天灌胃生理盐水一次；第3组小鼠通过灌胃给药洛伐他汀(Lovastatin，Lova)，给药量为：60mg/kg体重/天；第4组小鼠灌胃根据本发明实施例的化合物1G，给药量：60mg/kg体重/天。灌胃给药12周后，系统检测分析小鼠的各项代谢指标。
结果如图4(A)所示(采用统计学的常规One-way ANOVA检验分析，其中*表示p<0.05，***表示p<0.001)，高脂高胆固醇饲料喂养12周后，生理盐水对照组小鼠变肥胖，体重增加到31g。洛伐他汀给药组小鼠体重为27.5g，而根据本发明实施例的化合物1G给药组小鼠体重为24.8g，和基础饲料组小鼠体重接近。以上结果说明根据本发明实施例的化合物1G具有抑制高脂高胆固醇饲料诱导的小鼠体重的增加，具有减肥效果，而且效果比洛伐他汀还显著。
高脂高胆固醇饲料诱导的肥胖，会导致脂肪的增加，通过摘取小鼠附睾垫白色脂肪并称重，将脂肪重量除以体重得到脂肪体重比。如图4(B)所示(采用统计学的常规One-way ANOVA检验分析，其中*表示p<0.05，**表示p<0.01，****表示p<0.0001。)，洛伐他汀轻 度减少脂肪体重比，而根据本发明实施例的化合物1G比洛伐他汀更为显著地降低脂肪体重比。
实施例29、根据本发明实施例的化合物改善小鼠血液和肝脏中胆固醇和甘油三酯水平
小鼠处死后，收集血液，室温放置1小时后，4℃1500g离心10分钟，取上清用于后续测定。血液中总胆固醇、甘油三酯、葡萄糖和游离脂肪酸的含量采用相应的试剂盒，并按照厂家说明书测定(上海科华生物工程股份有限公司)。如图5(A)和(B)所示(采用统计学的常规One-way ANOVA检验分析，其中*表示p<0.05，**表示p<0.01，****表示p<0.0001)，高脂高胆固醇饲料长期喂养后，小鼠血液中的总胆固醇和甘油三酯水平成倍的增加，根据本发明实施例的化合物1G给药后，总胆固醇和甘油三酯水平均显著的下降，而且下降的水平比洛伐他汀还要低。
小鼠肝脏中总胆固醇和甘油三酯的测定：小鼠采血后断颈处死，打开胸腔腹腔，剪开右心耳，从左心室灌流PBS约20ml。灌流后摘取肝脏，并剪下约50mg称重并记录下重量，加入到含有1.2ml氯仿-甲醇(2：1)以及少量陶瓷珠的2ml组织破碎管中，用Precellys 24组织匀质器(Bertin)中5500rpm匀浆破碎10秒，共3次。匀浆破碎后室温摇床旋转混匀1-2小时，然后室温13200rpm离心10分钟，吸取1ml上清转移到新1.5ml离心管中，加入400μl双蒸水震荡混匀，室温放置10分钟后，13200rpm再离心10分钟。去掉上层水相，吸取500μl下层有机相到新的1.5ml离心管中，用氮气吹干，酒精溶解，按照上面同样的方法测定总胆固醇和甘油三酯含量。
如图5(C)和(D)所示(采用统计学的常规One-way ANOVA检验分析，其中*表示p<0.05，***表示p<0.001，****表示p<0.0001)，根据本发明实施例的化合物1G显著降低了高脂肪高胆固醇饲料诱导的肝脂中总胆固醇和甘油三酯的含量，而且比洛伐他汀更为显著，具有降低脂肪肝的效果。
实施例30、根据本发明实施例的化合物显著提高饮食诱导肥胖小鼠的胰岛素敏感性
机体中脂肪酸和甘油三酯水平异常升高是诱发胰岛素抵抗和2型糖尿病的重要原因。由于根据本发明实施例的化合物1G能有效降低血液和肝脏中总胆固醇和甘油三酯的脂质水平，接下来检测了根据本发明实施例的化合物对小鼠胰岛素敏感性的作用效果。
葡萄糖耐受实验：小鼠给药12周后，实验开始前禁食饥饿过夜。第2天，对每只小鼠进行称重并做好标记，根据小鼠体重腹腔注射D-葡萄糖溶液(2g/kg体重)。注射后0、30、60、120分钟时利用血糖仪(强生)检测小鼠尾静脉血液中葡萄糖浓度。
胰岛素耐受实验：实验开始前禁食饥饿4小时，根据小鼠体重腹腔注射胰岛素(0.7U/kg)，注射后0、30、60、120分钟时利用血糖仪检测小鼠尾静脉血液中葡萄糖浓度。
如图6(A)-图6(D)所示(用统计学的常规One-way ANOVA检验分析，其中*表示 p<0.05，**表示p<0.01，****表示p<0.0001)，与正常基础饲料喂养的小鼠相比，高脂高胆固醇饲料诱导的肥胖小鼠具有明显的葡萄糖和胰岛素耐受。洛伐他汀具有轻微的改善作用，而根据本发明实施例的化合物1G显著的提高了饮食诱导肥胖小鼠的葡萄糖和胰岛素敏感性。同时如图6(E)所示，根据本发明实施例的化合物1G也能显著降低饮食诱导肥胖小鼠的血糖水平。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (10)

  1. 式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药在制备药物中的用途，所述药物用于预防和/或治疗代谢性疾病；
    Figure PCTCN2017114645-appb-100001
    其中，3号碳原子与R1之间为单键或双键，
    当3号碳原子与R1之间为单键时，R1选自如下基团中任一种，R为任选取代的烷基：
    Figure PCTCN2017114645-appb-100002
    当3号碳原子与R1之间为双键时，R1选自如下基团中任一种：
    Figure PCTCN2017114645-appb-100003
    R2为-OH、-F或-H；
    R3选自如下基团中的任一种：
    Figure PCTCN2017114645-appb-100004
    5号碳原子和6号碳原子之间为单键或双键，或5号碳原子和6号碳原子与氧原子共同形成三元环氧结构；
    7号碳原子、8号碳原子、9号碳原子和11号碳原子的相邻碳原子之间为单键或双键；
    R4和R5分别独立地选自-OH、氧代、羰基、-F或-H；
    8号碳原子和9号碳原子之间为单键时，R6和R7分别独立地选自-H或-OH，或8号碳原子和9号碳原子与氧原子共同形成三元环氧结构；
    R8为-H或甲基；
    22号碳原子和23号碳原子之间为单键或双键，或22号碳原子和23号碳原子与氧原子 共同形成三元环氧结构；
    R9为任选取代的烷基、任选取代的烯基、羟基、任选取代的羰基、任选取代的环烷基、任选取代的环氧基、磷酸基、磺酸基或羧基，可选地，R9为选自如下基团中的任一种：
    Figure PCTCN2017114645-appb-100005
    各式中，n分别独立地为0～4的整数，包括端点；X为F、Cl、Br或I；
    R'和R”分别独立地为C1～4烷基。
  2. 根据权利要求1所述的用途，其特征在于，式I所示化合物选自下列的任一种：
    Figure PCTCN2017114645-appb-100006
    Figure PCTCN2017114645-appb-100007
    Figure PCTCN2017114645-appb-100008
    Figure PCTCN2017114645-appb-100009
    任选地，所述代谢性疾病为高脂血症、肥胖症、动脉粥样硬化、心脑血管疾病或2型糖尿病。
  3. 式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药在制备降低脂肪酸水平的产品中的用途。
  4. 根据权利要求3所述的用途，其特征在于，所述降低脂肪酸水平是通过抑制基因SREBP的表达而实现的；
    任选地，所述降低脂肪酸水平是通过抑制Huh-7细胞或肝原代细胞中基因SREBP的表达而实现的。
  5. 根据权利要求4所述的用途，其特征在于：所述抑制Huh-7细胞或肝原代细胞中基因SREBP的表达是通过下列的至少之一而实现的：
    1)抑制基因SREBP-1c的表达；
    2)抑制基因SREBP-2的表达；
    3)抑制基因HMGCS的表达；
    4)抑制基因HMGCR的表达；
    5)抑制基因SS的表达；
    6)抑制基因FASN的表达；
    7)抑制基因SCD1的表达。
  6. 式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药在制备抑制基因SREBP表达的细胞模型中的用途。
  7. 式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药在制备下述1)-3)中任一种产品中的用途：
    1)抑制高脂饮食诱导的体重和脂肪增加的产品；
    2)降低高脂饮食诱导的总胆固醇和甘油三酯的含量的产品；
    3)提高高脂饮食诱导肥胖患者对葡萄糖和胰岛素的敏感性的产品。
  8. 式Ⅰ所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药在制备抑制SREBP信号通路的抑制剂中的用途；
    任选地，所述抑制SREBP信号通路是通过下列至少之一而实现的：
    1)抑制基因SREBP-1c的表达；
    2)抑制基因SREBP-2的表达；
    3)抑制基因HMGCS的表达；
    4)抑制基因HMGCR的表达；
    5)抑制基因SS的表达；
    6)抑制基因FASN的表达；
    7)抑制基因SCD1的表达。
  9. 药物组合物在制备药物中用途，其特征在于，所述药物组合物包括权利要求1～9中任一项式I所示化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药作为有效成分，所述药物用于预防和/或治疗代谢性疾病；
    任选地，所述药物组合物进一步包括药学上可接受的辅料。
  10. 一种预防和/或治疗代谢性疾病方法，其特征在于，包括：
    给予患者权利要求1～9中任一项所述的化合物或其立体异构体、几何异构体、互变异构体、氮氧化物、水合物、溶剂化物、代谢产物、药学上可接受的盐或前药或权利要求9所述的药物组合物；
    任选地，所述代谢性疾病为高脂血症、肥胖症、动脉粥样硬化、心脑血管疾病或2型糖尿病。
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