CN107176914B

CN107176914B - GVS series compound and its use

Info

Publication number: CN107176914B
Application number: CN201610133063.3A
Authority: CN
Inventors: 段小群; 徐华强; 易伟; 胡增仁; 吴啸川
Original assignee: Zhejiang Xuchen Pharmaceutical Co ltd
Current assignee: Zhejiang Xuchen Pharmaceutical Co ltd
Priority date: 2016-03-09
Filing date: 2016-03-09
Publication date: 2022-06-28
Anticipated expiration: 2036-03-09
Also published as: CN107176914A; WO2017152570A1

Abstract

The invention provides a novel GVS series compound and application thereof, which have the function of obviously improving nonalcoholic fatty liver and fat accumulation. The medicine can (a) improve the effects of nonalcoholic liver inflammation and fat accumulation under the condition of maintaining normal body weight; (b) obviously reducing glutamic oxaloacetic transaminase (AST), glutamic pyruvic transaminase (ALT) and Malondialdehyde (MDA) in serum and simultaneously increasing the activity of superoxide dismutase (SOD); (c) reducing low density lipoprotein cholesterol (LDL-C), Triglycerides (TG), and increasing high density lipoprotein cholesterol (HDL-C); (d) increasing Adiponectin (ADP) content in serum and decreasing Fatty Acid Synthetase (FAS) activity.

Description

GVS series compound and its use

Technical Field

The invention belongs to the field of medicine application, and particularly relates to a GVS series compound and application thereof.

Background

Non-alcoholic fatty liver disease (NAFLD) refers to the clinical pathological syndrome characterized mainly by excessive fat deposition in liver cells caused by excluded alcohol and other definite liver damage factors, acquired metabolic stress liver injury closely related to insulin resistance and genetic susceptibility, including Simple Fatty Liver (SFL), non-alcoholic steatohepatitis (NASH) and related cirrhosis. With the global epidemic trend of obesity and related metabolic syndrome, the nonalcoholic fatty liver disease becomes an important cause of chronic liver disease in developed countries such as Europe and America and affluent areas of China, the prevalence rate of NAFLD of common adults is 10% -30%, wherein 10% -20% of NASH is NASH, and the incidence rate of cirrhosis of liver in 10 years of NASH is up to 25%.

Non-alcoholic fatty liver disease can directly cause decompensated liver cirrhosis, hepatocellular carcinoma and relapse of transplanted liver, can affect the progress of other chronic liver diseases, and is involved in the onset of type 2 diabetes and atherosclerosis. Therefore, prevention of further damage to the liver by early prevention and treatment of non-alcoholic fatty liver disease is of paramount importance. At present, medicines and health-care foods for preventing and treating the non-alcoholic fatty liver disease have various types, but have various problems, such as intolerance of patients, and various adverse reactions such as weight gain, fluid retention, cardiovascular disease and the like caused by the medicines. Therefore, there is an urgent need in the art to develop a novel drug for the prevention and treatment of non-alcoholic fatty liver disease.

Disclosure of Invention

The invention aims to provide a novel GVS series compound and application thereof.

In a first aspect of the present invention, there is provided a use of a compound represented by formula a, an optical isomer, a hydrate, a solvate, a prodrug thereof, or a pharmaceutically acceptable salt thereof, for preparing a medicament or a formulation for:

(1) treatment and/or prevention of fatty liver and fat accumulation;

(2) reducing the activity of aspartate Aminotransferase (AST) in the serum;

(3) reducing the activity of alanine Aminotransferase (ALT) in serum;

(4) reducing the level of Malondialdehyde (MDA) in serum;

(5) increasing the activity of superoxide dismutase (SOD);

(6) increasing the level of Adiponectin (ADP) in serum;

(7) reducing the activity of Fatty Acid Synthase (FAS);

(8) reducing the level of low density lipoprotein cholesterol (LDL-C);

(9) reducing the level of Triglycerides (TG); and/or

(10) Increasing the level of high density lipoprotein cholesterol (HDL-C);

in the formula (I), the compound is shown in the specification,

x is O or N;

R₁is-L-R_aWherein L is absent, or a divalent linking group selected from the group consisting of: - (CH)₂)m-、-(CR₄H) m-, m is 1, 2 or 3, R₄Is C₁-C₄Alkyl radical, C₁-C₄Haloalkyl, C₃-C₄A cycloalkyl group;

R_aselected from the group consisting of: substituted or unsubstituted C₁-C₁₀Alkyl, substituted or unsubstituted C₃-C₁₀Cycloalkyl, substituted or unsubstituted 4-20 membered heterocyclyl C containing 1-5 heteroatoms selected from N, S and O₁-C₁₀Alkyl, substituted or unsubstituted C₅-C₂₀Aryl, substituted or unsubstituted C₅-C₂₀Aryl radical C₁-C₁₀Alkyl, and substituted or unsubstituted C containing 1-5 heteroatoms selected from N, S and O₅-C₂₀A heteroaryl group;

the substituent is selected from the following group: c₁-C₁₀Alkyl radical, C₁-C₆Alkylcarbonyl, carboxyl, nitro and halogen;

R₂selected from the group consisting of: H. halogen, methyl, methoxy, carboxamido, nitro, trifluoromethyl and cyano;

R₃is one or more selected from the groupGroup (b): H. halogen, methyl, methoxy, carboxamido, nitro, trifluoromethyl, cyano, and 3, 4-cyclobutadienyl;

and, when X is O, the dotted line is none, i.e.

In another preferred example, the fatty liver is non-alcoholic fatty liver.

In another preferred example, the superoxide dismutase is serum superoxide dismutase.

In another preferred embodiment, the fatty acid synthase is a serum fatty acid synthase.

In another preferred embodiment, the drug or formulation has one or more properties selected from the group consisting of:

(a) weak activation ability to PPAR γ;

(b) strong binding ability to PPAR γ;

(c) weak preadipocyte 3T3-L1 ability to transform into adipocytes.

In another preferred embodiment, the "weak activating power" refers to the ratio of the measured value M1 of the test group to the reference value M0 of the control group (M1/M0) < 0.75, preferably < 0.6, more preferably < 0.5, more preferably < 0.4, most preferably < 0.3, wherein the compound of the control group is rosiglitazone.

In another preferred embodiment, the "strong binding capacity" refers to the ratio of the test group measured value C1 to the control group reference value C0 (C1/C0) > 0.9, preferably > 1, more preferably > 1.15, more preferably > 1.25, most preferably > 1.35, wherein the compound of the control group is rosiglitazone.

In another preferred embodiment, the "weak ability of preadipocytes 3T3-L1 to transform into adipocytes" means that the ratio of the measured value T1 of the test group to the reference value T0 of the control group (T1/T0) is less than 0.5, preferably less than 0.25, more preferably less than 0.1, more preferably less than 0.06, wherein the compound of the control group is rosiglitazone.

In a second aspect of the present invention, there is provided a compound represented by formula a, an optical isomer, a hydrate, a solvate, a prodrug thereof or a pharmaceutically acceptable salt thereof,

in the formula (I), the compound is shown in the specification,

x is O or N;

R₃is one or more groups selected from the group consisting of: H. halogen, methyl, methoxy, carboxamido, nitro, trifluoromethyl, cyano, and 3, 4-cyclobutadienyl;

and, when X is O, the dotted line is none, i.e.

In another preferred embodiment, when X is O, the compound of formula A is

In another preferred embodiment, when X is N, the compound of formula A is

In another preferred embodiment, R₁Selected from the group consisting of: substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted 4-10 membered heterocyclyl C containing 1-3 heteroatoms selected from N, S and O₁-C₆Alkyl, substituted or unsubstituted C₅-C₁₀Aryl, substituted or unsubstituted C₅-C₁₀Aryl radical C₂-C₆Alkyl, and substituted or unsubstituted 5-10 membered heteroaryl containing 1-3 heteroatoms selected from N, S and O.

In another preferred embodiment, R₁Selected from the group consisting of: benzyl, phenyl, pyridin-4-ylmethyl, pyridin-4-ylethyl, 1-phenylethyl, methoxycarbonylbenzyl, and carboxybenzyl.

In another preferred embodiment, the compound of formula a is selected from the group consisting of:

in a third aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) a compound of formula a according to the first and second aspects of the invention, an optical isomer, hydrate, solvate, prodrug thereof or a pharmaceutically acceptable salt thereof; and

(ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition comprises a therapeutically effective amount or a safe and effective amount of the compound of formula a, its optical isomer, hydrate, solvate, prodrug thereof or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the pharmaceutical composition contains 0.0001-99 wt% (preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of component (i), based on the total weight of the pharmaceutical composition.

In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating and/or preventing fatty liver.

In a fourth aspect of the present invention, there is provided a method for treating and/or preventing fatty liver, the method comprising: administering to a subject in need thereof a compound of formula a as described in the first and second aspects of the invention or a pharmaceutical composition as described in the third aspect of the invention.

In another preferred example, the object comprises a person.

In another preferred embodiment, the subject comprises a non-human mammal.

In another preferred embodiment, the non-human mammal includes a rodent, such as a mouse, rat.

In another preferred embodiment, the fatty liver is non-alcoholic fatty liver disease.

In another preferred embodiment, the dose is 10-10000 mg/kg/day, preferably 500-10000 mg/kg/day, more preferably 1000-10000 mg/kg/day.

In another preferred embodiment, the frequency of application is 1-5 times/day, preferably 1-2 times/day.

In another preferred embodiment, administration comprises one or more cycles, each cycle being from 2 to 30 days, preferably from 3 to 7 days.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the results of a test for GVS-12 targeting PPAR γ activity, (A) the chemical structural formula of GVS-12; (B) luciferase reporter gene assay using Cos-7 cells for PPAR γ activation by GVS-12 (n-3, ± SEM); (C) oil red O staining test; (D) DMSO (1 mu M) lipotrophy results in 3T3-L1 preadipocytes; (E) GVS-12(1 mu M) results in the lipotrophy of 3T3-L1 preadipocytes; (F) results of the lipotropism capacity of rosiglitazone (1 mu M) in 3T3-L1 preadipocytes; .

FIG. 2 shows the effect of compound GVS-12 on body weight in fatty liver rats^#P<0.05vs model set.

FIG. 3 shows the pathological assay of compound GVS-12 in nonalcoholic fatty liver rats.

FIG. 4 shows the effect of GVS-12 on AST, ALT, MDA, and SOD in serum of nonalcoholic fatty liver rats (Mean. + -. S.E.M) n ═ 10

P <0.05, P <0.01vs blank control group; # P <0.05, # P <0.01vs model group.

FIG. 5 shows the effect of GVS-12 on LDL-C, HDL-C, TG (Mean. + -. S.E.M) n ═ 10 in serum of rat with nonalcoholic fatty liver disease

P <0.05, P <0.01vs blank control group; # P <0.05, # P <0.01vs model group.

FIG. 6 shows the effect of compound GVS-12 on ADP and FAS in serum of nonalcoholic fatty liver rats (Mean. + -. S.E.M) n ═ 10

P <0.05, P <0.01vs blank control group; # P <0.05, # P <0.01vs model group.

Detailed Description

The present inventors have conducted extensive and intensive studies to synthesize a series of GVS compounds which are effective in agonizing PPAR γ receptor, do not significantly induce fat differentiation, and have an effect of improving nonalcoholic fatty liver and fat accumulation while maintaining normal body weight. The present invention has been completed based on this finding.

Description of the terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Radical definitions

The term "substituted or unsubstituted" as used herein means that the group may be unsubstituted or that H in the group is substituted with one or more (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, most preferably 1 to 2) substituents.

As used herein, the term "substituted" or "substituted" means that the group has one or more (preferably 1 to 6, more preferably 1 to 3) substituents selected from the group consisting of: c₁-C₆Alkylcarbonyl, carboxyl, nitro and halogen.

As used herein, the term "C₁-C₁₀Alkyl "means a straight or branched chain alkyl group having 1 to 10 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like; "C₁-C₃Alkyl "means a straight or branched chain alkyl group having 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, or the like.

As used herein, the term "C₃-C₁₀Cycloalkyl "refers to a cyclic alkyl group having 3 to 10 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or the like.

As used herein, the term "C₅-C₂₀Aryl "refers to a monovalent aromatic carbocyclic group of 5 to 20 (preferably 5-14) carbon atoms having a single ring (e.g., phenyl) or fused rings (e.g., naphthyl or anthracenyl) which may be non-aromatic if the point of attachment is at an aromatic carbon atom (e.g., 2-benzoxazolone, 2H-1, 4-benzoxazin-3 (4H) -one-7-yl, etc.). Preferred aryl radicalsIncluding phenyl and naphthyl. The term includes substituted and unsubstituted forms wherein the substituents are as defined above.

As used herein, the term "C₅-C₂₀Heteroaryl "refers to an aromatic group having 5 to 20 carbon atoms and 1 to 5 (preferably 1-3) heteroatoms selected from oxygen, nitrogen and sulfur, and such heteroaryl can be monocyclic (e.g., pyridyl or furyl) or fused rings (e.g., indolizinyl or benzothienyl), wherein the fused rings can be non-aromatic and/or contain one heteroatom, provided that the point of attachment is through an atom of the aromatic heteroaryl. Preferably, heteroaryl groups include pyridyl, pyrrolyl, indolyl, thienyl and furyl. The term includes substituted or unsubstituted heteroaryl groups.

As used herein, the term "C₄-C₂₀Heterocyclyl "refers to a saturated, partially saturated, or unsaturated group (but not aromatic) having a single ring or fused rings (including bridged ring systems and spiro ring systems, having 4 to 20 carbon atoms and 1 to 4 heteroatoms selected from nitrogen, sulfur, or oxygen, in which fused ring systems one or more of the rings may be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through a non-aromatic ring.

As used herein, the term "halogen" refers to fluorine, chlorine, bromine, or iodine, preferably fluorine, chlorine, and bromine.

The term "halogenated" as used herein refers to a group substituted with the same or different one or more of the above-mentioned halogen atoms, which may be partially halogenated or fully halogenated, for example, trifluoromethyl, pentafluoroethyl, heptafluoroisopropyl, or the like.

The compounds of the present invention may contain one or more asymmetric centers and thus occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and individual diastereomers. Asymmetric centers that may be present depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers and all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds are included within the scope of the invention. The present invention includes all isomeric forms of the compounds.

Use of the compounds of the invention

The invention provides the use of a compound of formula a as described for the manufacture of a medicament or formulation for:

(1) treatment and/or prevention of fatty liver and fat accumulation;

(2) reducing the activity of aspartate Aminotransferase (AST) in the serum;

(3) reducing the activity of alanine Aminotransferase (ALT) in serum;

(4) reducing the level of Malondialdehyde (MDA) in serum;

(5) increasing the activity of superoxide dismutase (SOD);

(6) increasing the level of Adiponectin (ADP) in serum;

(7) reducing the activity of Fatty Acid Synthase (FAS);

(8) reducing the level of low density lipoprotein cholesterol (LDL-C);

(9) reducing the level of Triglycerides (TG); and/or

(10) Increasing the level of high density lipoprotein cholesterol (HDL-C).

Compounds of the invention

As used herein, "compound of the present invention", "compound of formula a", "compound of the GVS series", which are used interchangeably, refer to a compound of formula a or formula a-1 or formula a-2, an optical isomer, hydrate, solvate, prodrug, or pharmaceutically acceptable salt thereof.

The invention provides a compound shown in a formula A, an optical isomer, a hydrate, a solvate, a prodrug or a pharmaceutically acceptable salt thereof,

in the formula (I), the compound is shown in the specification,

x is O or N;

the substituents are selected from the following group: c₁-C₁₀Alkyl radical, C₁-C₆Alkylcarbonyl, carboxyl, nitro and halogen;

and, when X is O, the dotted line is none, i.e.

In another preferred embodiment, when X is O, the compound of formula A is

In another preferred embodiment, when X is N, the compound of formula A is

In another preferred embodiment, R₁Selected from the group consisting of: substituted or unsubstituted C₁-C₆An alkyl group,Substituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted 4-10 membered heterocyclyl C containing 1-3 heteroatoms selected from N, S and O₁-C₆Alkyl, substituted or unsubstituted C₅-C₁₀Aryl, substituted or unsubstituted C₅-C₁₀Aryl radical C₂-C₆Alkyl, and substituted or unsubstituted 5-10 membered heteroaryl containing 1-3 heteroatoms selected from N, S and O.

In another preferred embodiment, the compound of formula a is a compound prepared according to the embodiments of the present invention.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising a safe and effective amount of the active ingredient, and a pharmaceutically acceptable carrier.

The "active ingredient" of the present invention refers to the compound of general formula a, its optical isomer, hydrate, solvate, prodrug or pharmaceutically acceptable salt thereof.

The active ingredient and the pharmaceutical composition are used for preparing the medicine for treating and/or preventing the fatty liver.

"safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of active ingredient per dose, more preferably, 10-200mg of active ingredient per dose. Preferably, said "dose" is a tablet.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of being combined with the active ingredients of the present invention and with each other without significantly diminishing the efficacy of the active ingredient.

The compounds of the preferred embodiments of the present invention may be administered as the sole active agent or in combination with one or more other agents useful in the treatment of diabetes. The compounds of the preferred embodiments of the present invention are also effective in combination with known therapeutic agents, and combinations of presently known compounds with other therapeutic agents for the treatment of diabetes are within the scope of the preferred embodiments. One of ordinary skill in the art will be able to identify effective combinations of agents based on the particular nature of the drug and the disease involved. Such therapeutic agents for treating diabetes include (but are not limited to) the following: vinia, echol, etc. The compounds of the preferred embodiments are also effective when administered concurrently with a therapeutic agent for the treatment of diabetes.

In general, the compounds of the preferred embodiments will be administered in a therapeutically effective amount by any acceptable mode of administration of the agents having a similar effect. The actual amount of the compound (i.e., active ingredient) of the preferred embodiment will depend on a number of factors, such as the severity of the condition being treated, the age and relative health of the patient, the potency of the compound being administered, the route and form of administration, and other factors. The medicament may be administered multiple times a day, preferably once or twice a day. All of these factors are considered by the attending physician.

In the present invention, a therapeutically effective dose may generally be a total daily dose administered to a patient in one dose or in divided doses, e.g., from about 0.001 to about 1000 mg/kg body weight per day, preferably from about 1.0 to about 30 mg/kg body weight per day. A unit dose composition (Dosage unit composition) may include its Dosage factors to form a daily dose. The choice of dosage form depends on various factors, such as the mode of administration and the bioavailability of the drug substance. In general, the compounds of the preferred embodiments may be administered as pharmaceutical compositions by any of the following routes: oral, systemic (e.g., transdermal, intranasal, or by suppository), or parenteral (e.g., intramuscular, intravenous, or subcutaneous). The preferred mode of administration is oral, and convenient daily dosages may be adjusted to the bitter taste. The composition may take the form of a tablet, pill, capsule, semi-solid, powder, sustained release formulation, solution, suspension, elixir, aerosol, or any other suitable composition. Another preferred mode of administering the compounds of the preferred embodiments is by inhalation. This is an effective method of delivering therapeutic agents directly to the respiratory tract (see, e.g., U.S. patent No. 5,607,915).

Suitable pharmaceutically acceptable carriers or excipients include: such as treatment agents and drug delivery modifiers and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, sodium methyl cellulose, carboxymethyl cellulose, glucose, hydroxypropyl-B-cyclodextrin, polyvinylpyrrolidone, low melting waxes, ion exchange resins, and the like, and combinations of any two or more thereof. Liquid and semisolid excipients can be selected from glycerol, propylene glycol, water, ethanol, and various oils, including petroleum, animal oils, vegetable oils, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Preferred liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose solutions, and glycols. Other suitable pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences, Mack pub. Co., New Jersey (1991), which is incorporated herein by reference.

As used herein, the term "pharmaceutically acceptable salt" refers to a non-toxic acid or alkaline earth metal salt of a compound of formula I. These salts can be prepared in situ during the final isolation and purification of the compounds of formula I or by reacting a suitable organic or inorganic acid or base, respectively, with a basic or acidic functional group. Representative salts include, but are not limited to: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthylsulfonate, oxalate, pamoate, pectate, thiocyanate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate and undecanoate. In addition, the nitrogen-containing basic groups may be quaternized with the following agents: alkyl halides such as methyl, ethyl, propyl, butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl and phenethyl bromide, and the like. Thus obtaining a water-soluble or oil-soluble or dispersible product. Examples of acids which may be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, sulphuric acid, phosphoric acid, and organic acids such as oxalic acid, maleic acid, methanesulphonic acid, succinic acid, citric acid. Base addition salts can be prepared in situ during the final isolation and purification of the compounds of formula I, or by reacting the carboxylic acid moiety with a suitable base (e.g., a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation) or ammonia, or an organic primary, secondary or tertiary amine, respectively. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, aluminum, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to: ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Other representative organic amines useful for the formation of base addition salts include diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

As used herein, the term "pharmaceutically acceptable prodrug" refers to a prodrug of the compound of those preferred embodiments, a compound that is rapidly converted in vivo to the parent compound represented by the general formula above, e.g., by hydrolysis in blood. A complete discussion is provided in "t.higuchi and v.stella, Pro-drugs as Novel Delivery Systems (Pro-drugs as Novel Delivery Systems), volume 14 of a.c.s.1. symposium Series" and "Edward b.roche eds," Bioreversible Carriers in Drug Design, american pharmaceutical association and Pergamon press, 1987, "both of which are incorporated herein by reference.

The main advantages of the invention are:

(1) the compound of the present invention is effective for preventing and/or treating fatty liver, and particularly, has an effect of improving non-alcoholic fatty liver and fat accumulation while maintaining normal body weight.

(2) Provides a compound with a novel structure and a general formula A.

(3) The compounds of the present invention are able to agonize PPAR γ receptors and do not significantly induce adipodifferentiation.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

The test materials and reagents used in the following examples are commercially available without specific reference.

Instrument and general method

The chemicals related to 4-hydroxybenzoic acid, benzyl bromide, benzylamine, etc. were purchased from sigma.

Solvents such as petroleum ether and ethyl acetate are available from Shanghai nationality.

Model Brukeram-400 and Varian Mercury plus-400 NMR spectrometers.

200-mesh 300-mesh column chromatography silica gel (Qingdao ocean chemical plant).

HSGF254TLC plates (Nicoti, chemical research institute).

All solvents were analytically pure.

Preparation examples

Example 1: synthesis of GVS-1:

step 1: p-hydroxybenzoic acid (1.38g, 10mmol) and benzylamine (1.08g, 10mmol)) Placing in 25mL round bottom flask, adding BOP condensing agent (5.3g,12mmol) and DMAP (1.46g,12mmol), adding dry DMF 20mL, stirring at room temperature, TLC tracing reaction, reacting for 3H, completely EA extracting water washing and concentrating to obtain intermediate 1, MS (EI) M/z 227.1[ M + H ]]⁺.

Step 2: intermediate 1(230mg,1mmol) and 3, 4-difluorobenzyl bromide (206mg,1mmol) were placed in a 25mL round-bottomed flask, and Cs was added₂CO₃(490mg,1.5mmol) in a flask, then adding dry DMF 8mL, stirring at room temperature, TLC tracing reaction, after 3h reaction complete, extracting with water/ethyl acetate until the aqueous phase is free of compound, combining organic phases washing twice with water, then washing with saturated NaCl solution, drying with anhydrous sodium sulfate, finally separating and purifying with silica gel column (petroleum ether/ethyl acetate 8:1) to obtain the target product. Yield 80% product was a white solid.¹H NMR (400MHz, chloroform-d) δ 7.79-7.73 (M,2H), 7.44-7.37 (M,1H),7.35(d, J ═ 4.0Hz,4H), 7.32-7.27 (M,1H), 7.25-7.21 (M,1H),7.14(M,1H), 6.99-6.92 (M,2H),6.38(t, J ═ 5.7Hz,1H),5.06(t, J ═ 0.7Hz,2H),4.63(d, J ═ 5.6Hz,2H), lcms (esi): M/z 370.1[ M + H, 2H ], (esi)]⁺.

Example 2: synthesis of GVS-2:

GVS-2 was prepared by substituting 3-fluoro-4-chlorobenzyl bromide for 3, 4-difluorobenzyl bromide as described for GVS-1 (85% yield).¹H NMR (400MHz, chloroform-d) δ 7.80-7.73 (M,2H),7.36(d, J ═ 4.4Hz,4H), 7.33-7.25 (M,2H), 7.25-7.10 (M,2H), 7.01-6.93 (M,2H),6.32(t, J ═ 5.4Hz,1H),5.05(s,2H),4.64(d, J ═ 5.6Hz,2H), lcms (esi): M/z 354.1[ M + H, 2H) ], and]⁺.

example 3: synthesis of GVS-3:

GVS-3 was prepared by preparing GVS-1 using 4-trifluoromethyl benzyl bromide instead of 3, 4-difluoro benzyl bromide (81% yield).¹H NMR (400MHz, chloroform-d) δ 7.80-7.74 (M,2H), 7.69-7.61 (M,2H),7.54(M,2H),7.35(d, J ═ 4.6Hz,4H), 7.33-7.27 (M,1H), 7.02-6.95 (M,2H),6.34(s,1H),5.17(s,2H),4.63(d, J ═ 5.6Hz,2H), lcms (esi): M/z 386.1[ M + H, 2H ], (M, 2H): lcm/z 386.1]⁺.

Example 4: synthesis of GVS-4:

step 1: preparation of intermediate 2 by 4-methylaminopyridine instead of benzylamine preparation of intermediate 1, LCMS (ESI) M/z 229.1[ M + H ]]⁺.

Step 2: GVS-4 was prepared as described for GVS-1 using intermediate 2 instead of intermediate 1 (79% yield).¹H NMR (400MHz, chloroform-d) δ 8.55(d, J ═ 4.7Hz,2H),7.79(d, J ═ 8.4Hz,2H),7.41(t, J ═ 7.8Hz,1H),7.25(s,2H), 7.18-7.10 (M,1H),6.98(d, J ═ 8.4Hz,2H),6.60(t, J ═ 6.1Hz,1H),5.08(s,2H),4.64(d, J ═ 6.0Hz,2H), lcms (esi): M/z 355.1[ M + H, 2H) ]]⁺.

Example 5: synthesis of GVS-5:

GVS-5 was prepared as described for GVS-2 using intermediate 2 instead of intermediate 1 (75% yield).¹H NMR (400MHz, chloroform-d) δ 8.52(d, J ═ 5.2Hz,2H),7.80(d, J ═ 8.4Hz,2H), 7.31-7.11 (M,4H),6.97(d, J ═ 8.4Hz,2H),6.78(d, J ═ 6.2Hz,1H),5.05(s,2H),4.62(d, J ═ 6.0Hz,2H), lcms (esi): M/z 371.1[ M + H]⁺.

Example 6: synthesis of GVS-6:

GVS-6 was prepared as described for GVS-3 using intermediate 2 instead of intermediate 1 (77% yield).¹H NMR (400MHz, chloroform-d) δ 8.59-8.51 (m,2H), 7.83-7.77 (m,2H),7.65(d, J ═ 8.1Hz,2H),7.58–7.52(m,2H),7.25–7.22(m,2H),7.04–6.96(m,2H),6.63(t,J＝6.0Hz,1H),5.17(s,2H),4.64(d,J＝6.0Hz,2H).LCMS(ESI):m/z 387.1[M+H]⁺.

Example 7: synthesis of GVS-7:

step 1: intermediate 3 was prepared by substituting 3-methylaminopyridine for benzylamine control for intermediate 1, LCMS (ESI) M/z 229.1[ M + H: (M + ESI)]⁺.

Step 2: GVS-7 was prepared as described for GVS-1 using intermediate 3 instead of intermediate 1 (81% yield).¹H NMR (400MHz, chloroform-d) δ 8.63-8.56 (M,1H),8.53(dd, J ═ 5.0,1.7Hz,1H), 7.79-7.74 (M,2H),7.71(d, J ═ 7.9Hz,1H),7.40(t, J ═ 7.8Hz,1H), 7.30-7.27 (M,1H),7.23(dd, J ═ 9.7,2.0Hz,1H), 7.16-7.12 (M,1H), 6.99-6.94 (M,2H),6.53(t, J ═ 5.9Hz,1H),5.07(s,2H),4.64(d, J ═ 5.9Hz,2H), s/z 355.1 (M + H) M/z 89m + H (M,1H), s/z (esi) ("M, M" + 355.1H]⁺.

Example 8: synthesis of GVS-8:

GVS-8 was prepared as described for GVS-2 using intermediate 3 instead of intermediate 1 (70% yield).¹H NMR (400MHz, chloroform-d) δ 8.58(d, J ═ 2.3Hz,1H),8.53(dd, J ═ 4.9,1.7Hz,1H), 7.83-7.73 (M,2H),7.71(dt, J ═ 7.9,2.0Hz,1H),7.40(t, J ═ 7.8Hz,1H), 7.30-7.20 (M,3H), 7.18-7.10 (M,1H), 7.02-6.91 (M,2H),6.53(t, J ═ 6.1Hz,1H),5.07(s,2H),4.64(d, J ═ 5.9Hz,2H), lcms esi (M/z 371.1[ M + H371.1 ], [ M + H]⁺.

Example 9: synthesis of GVS-9:

GVS-9 was prepared as described for GVS-3 using intermediate 3 instead of intermediate 1 (78% yield).¹H NMR (400MHz, chloroform-d) δ 8.49(dd, J ═ 17.4,3.8Hz,2H), 7.81-7.75 (M,2H),7.68(dd, J ═ 7.8,2.0Hz,1H), 7.66-7.60 (M,2H),7.52(d, J ═ 8.0Hz,2H),7.23(dd, J ═ 7.8,4.9Hz,1H),6.95(dt, J ═ 9.6,2.5Hz,3H),5.14(s,2H),4.59(dd, J ═ 6.0,2.8Hz,2H), lcms (esi): M/z 387.1[ M + H387.1 ]: M + H]⁺.

Example 10: synthesis of GVS-10:

step 1: intermediate 4 was prepared by substituting 3-methylaminofuran for benzylamine to prepare intermediate 1, LCMS (ESI) M/z 218.1[ M + H]⁺.

And 2, step: GVS-10 was prepared as described for GVS-1 using intermediate 4 instead of intermediate 1 (85% yield).¹H NMR (400MHz, chloroform-d) δ 8.12(dd, J ═ 1.8,0.7Hz,1H),7.65(dd, J ═ 8.6,1.8Hz,1H),7.38(dd, J ═ 1.8,0.9Hz,1H),7.23(dd, J ═ 8.7,0.8Hz,1H),7.16(d, J ═ 3.2Hz,1H),7.08(M,1H),6.63(dd, J ═ 3.3,0.8Hz,1H),6.46(t, J ═ 5.2Hz,1H), 6.37-6.26 (M,2H),5.29(d, J ═ 1.3Hz,2H),4.67 (esi, J ═ 5.5, 8H, 0.8H, 344.1H) ((M, 2H) ((M, 78 Hz, 78H) ((M, J]⁺.

Example 11: synthesis of GVS-11:

GVS-11 was prepared as described for GVS-2 using intermediate 4 instead of intermediate 1 (79% yield).¹H NMR (400MHz, chloroform-d) δ 7.79-7.70 (M,2H), 7.44-7.35 (M,2H),7.23(dd, J ═ 9.7,1.9Hz,1H),7.14(ddt, J ═ 8.3,2.1,0.9Hz,1H), 6.99-6.92 (M,2H), 6.38-6.31 (M,2H), 6.31-6.27 (M,1H),5.06(s,2H),4.62(d, J ═ 5.4Hz,2H), lcms (esi): M/z 360.1[ M + H ], (M + H) ]]⁺.

Example 12: synthesis of GVS-12:

GVS-12 was prepared as described for GVS-3 using intermediate 4 instead of intermediate 1 (82% yield).¹H NMR (400MHz, chloroform-d) δ 7.81-7.72 (M,2H),7.65(d, J ═ 8.0Hz,2H), 7.58-7.52 (M,2H),7.37(dd, J ═ 1.9,0.9Hz,1H), 7.01-6.95 (M,2H), 6.36-6.32 (M,2H),6.29(M,1H),5.16(s,2H), 4.66-4.59 (M,2H), lcms (esi): M/z 376.1[ M + H) ("M, 2H"), (esi]⁺.

Example 13: synthesis of GVS-13:

indole-5-carboxylic acid methyl ester (0.2mmol), 3-fluoro, 4-chlorobenzyl bromide (0.22mmol), NaH (0.22mmol) and DMF (N, N-dimethylformamide) (2mL) were added to the reaction flask, stirred in ice bath until room temperature and TLC checked for completion of the reaction. 1mol/L hydrochloric acid (50ml) was added to the reaction flask, extracted 3 times with ethyl acetate, and the organic phases were combined. The organic phase was washed with 1mol/L hydrochloric acid and saturated NaCl 1 time each. Dried over anhydrous sodium sulfate overnight. And (4) removing the drying agent by suction filtration, and spin-drying to obtain a crude product. And then purified by silica gel column chromatography to obtain 1- (4-chloro-3-fluorobenzyl) -1H-indole-5-carboxylic acid methyl ester (B-1) (57mg,0.18mmol), yield 90%.

To the obtained methyl 1- (4-chloro-3-fluorobenzyl) -1H-indole-5-carboxylate (B-1) (0.18mmol), 4mol/L NaOH solution (8mL) was added, and the reaction was refluxed, stirred and checked by TLC to be complete. Adding 1mol/L hydrochloric acid solution into a reaction bottle to adjust the pH value to be acidic, extracting for 3 times by ethyl acetate, and combining organic phases. The organic phase was washed with 1mol/L hydrochloric acid and saturated NaCl 1 time each. Dried over anhydrous sodium sulfate overnight. And (4) filtering to remove the drying agent, and spin-drying to obtain a crude product. And separating and purifying by silica gel column chromatography to obtain hydrolysate 1- (4-chloro-3-fluorobenzyl) -1H-indole-5-carboxylic acid (B-2) (52mg,0.17mmol) with yield of 95%.

To the obtained 1- (4-chloro-3-fluorobenzyl) -1H-indole-5-carboxylic acid (B-2) (0.17mmol) were added benzylamine (0.19mmol), BOP (benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate) (0.34mmol), DMAP (4-dimethylaminopyridine) (0.34mmol) and DMF (N, N-dimethylformamide) (2mL), stirred at room temperature, and the reaction was detected by TLC to be complete. Adding 1mol/L hydrochloric acid into a reaction bottle(50ml), extracted 3 times with ethyl acetate and the organic phases combined. The organic phase was washed with 1mol/L hydrochloric acid and saturated NaCl 1 time each. Dried over anhydrous sodium sulfate overnight. And (4) removing the drying agent by suction filtration, and spin-drying to obtain a crude product. And then the condensation product N-benzyl-1- (4-chloro-3-fluorobenzyl) -1H-indole-5-formamide (GVS-13) (63mg,0.16mmol) is obtained after separation and purification by silica gel column chromatography, and the yield is 92%.¹HNMR(400MHz,DMSO):δ8.92(s,1H),8.19(s,1H),7.75–7.65(m,1H),7.62(d,1H,J＝3.2Hz),7.56–7.48(m,2H),7.32(s,2H),7.31(s,2H),7.27(dd,1H,J＝10.3,1.8Hz),7.24-7.20(m,1H),7.00(dd,1H,J＝8.3,1.4Hz),6.62(d,1H,J＝3.1Hz),5.49(s,2H),4.49(d,2H,J＝6.0Hz).

Example 14: synthesis of GVS-14:

GVS-14 was prepared according to the method for GVS-13 using aniline instead of benzylamine (91% yield).¹H NMR(400MHz,DMSO):10.13(s,1H),8.26(d,1H,J＝1.0Hz),7.83-7.77(m,2H),7.74(dd,1H,J＝8.6,1.5Hz),7.66(d,1H,J＝3.2Hz),7.59(d,1H,J＝8.7Hz),7.54(t,1H,J＝8.1Hz),7.34(t,2H,J＝7.9Hz),7.29(dd,1H,J＝10.2,1.5Hz),7.07(t,1H,J＝7.4Hz),7.01(d,1H,J＝7.8Hz),6.68(d,1H,J＝3.1Hz),5.52(s,2H).

Example 15: synthesis of GVS-15:

GVS-15 was prepared according to the method for GVS-13 using 4-aminomethylpyridine instead of benzylamine (93% yield).¹H NMR(400MHz,DMSO):δ9.01(t,1H,J＝5.9Hz),8.49(dd,2H,J＝4.5,1.5Hz),8.21(s,1H),7.70(dd,1H,J＝8.7,1.5Hz),7.63(d,1H,J＝3.2Hz),7.59–7.46(m,2H),7.30(d,2H,J＝5.9Hz),7.29–7.25(m,1H),7.07–6.94(m,1H),6.64(d,1H,J＝3.0Hz),5.49(s,2H),4.50(d,2H,J＝5.9Hz).

Example 16: synthesis of GVS-16:

GVS-16 was prepared according to the method for GVS-13 using 1-phenethylethylamine instead of benzylamine (92% yield).¹H NMR(400MHz,DMSO):δ8.66(d,1H,J＝8.1Hz),8.19(d,1H,J＝1.2Hz),7.67(dd,1H,J＝8.6,1.6Hz),7.61(d,1H,J＝3.2Hz),7.54-7.50(m,2H),7.41(s,1H),7.39(s,1H),7.31(t,2H,J＝7.6Hz),7.25(dd,1H,J＝10.3,1.7Hz),7.20(t,1H,J＝7.3Hz),6.99(d,1H,J＝8.3Hz),6.63(d,1H,J＝2.9Hz),5.48(s,2H),5.22-5.15(m,1H),1.48(d,3H,J＝7.1Hz).

Example 17: synthesis of GVS-17:

GVS-17 was prepared as described for GVS-13 using 3, 4-difluorobenzyl bromide instead of 3-fluoro-4-chlorobenzyl bromide (72% yield).¹H NMR(400MHz,DMSO-d₆)δ8.93(s,1H),8.21(s,1H),7.71(d,1H,J＝8.7Hz),7.62(d,1H,J＝3.2Hz),7.56(d,1H,J＝8.7Hz),7.42-7.28(m,6H),7.24-7.20(m,1H),7.09-6.96(m,1H),6.62(d,1H,J＝3.1Hz),5.46(s,2H),4.50(d,2H,J＝5.7Hz)；HRMS(ESI)m/z calcd for C₂₃H₁₈F₂N₂O[M+Na]⁺:399.1279,found:376.1387

Example 18: synthesis of GVS-18:

GVS-18 was prepared according to the method for synthesizing GVS-17 using 4-isopropylamine instead of benzylamine (yield 76%).¹H NMR(400MHz,DMSO-d₆)δ8.90(s,1H),8.22(s,1H),7.78-7.51(m,3H),7.39-7.29(m,2H),7.21(dd,4H,J＝32.7and 7.8Hz),7.03(s,1H),6.62(d,1H,J＝2.6Hz),5.45(s,2H),4.47(d,2H,J＝5.6Hz),2.83(t,1H,J＝6.8Hz),1.16(d,6H,J＝6.9Hz)；HRMS(ESI)m/z calcd for C₂₆H₂₄F₂N₂O[M+Na]⁺:441.1756,found:418.1857.

Example 19: synthesis of GVS-19:

GVS-19 (79% yield) was prepared according to the method for GVS-17 synthesis using 2-aminomethylfuran instead of benzylamine.¹H NMR(400MHz,DMSO-d₆)δ8.82(t,1H,J＝7.6Hz),8.17(d,1H,J＝2.0Hz),7.67(dd,1H,J＝11.6and 2.4Hz),7.62(d,1H,J＝4.4Hz),7.57-7.53(m,2H),7.42-7.28(m,2H),7.05-7.00(m,1H),6.61(d,1H,J＝4.0Hz),6.39(dd,1H,J＝4.4and 2.4Hz),6.25(dd,1H,J＝4.4and 0.8Hz),5.45(s,2H),4.47(d,2H,J＝7.6Hz)；HRMS(ESI)m/z calcd for C₂₁H₁₆F₂N₂O₂[M+H]⁺:367.1254,found:366.1180.

Example 20: synthesis of GVS-20:

GVS-20 was prepared as synthetic GVS-13 using benzyl bromide instead of 3-fluoro-4-chlorobenzyl bromide (82% yield).¹H NMR (400MHz, chloroform-d) δ 8.13(dd, J ═ 1.8,0.7Hz,1H),7.64(dd, J ═ 8.6,1.8Hz,1H), 7.42-7.26 (M,9H),7.18(d, J ═ 3.2Hz,1H), 7.11-7.04 (M,2H),6.61(dd, J ═ 3.2,0.9Hz,1H),6.47(t, J ═ 5.7, 1H),5.33(s,2H),4.67(d, J ═ 5.6Hz,2H), lcms (esi) (M/z 341.2[ M + H): M/z 341.2[ M, J ═ 5.7Hz,1H ]]⁺.

Example 21: synthesis of GVS-21:

GVS-21 was prepared as described for GVS-13 using 2-bromomethylnaphthalene instead of 3-fluoro-4-chlorobenzyl bromide (69% yield).¹H NMR (400MHz, chloroform-d) δ 8.15(dd, J ═ 1.8,0.8Hz,1H), 7.85-7.68 (m,3H),7.63(dd, J ═ 8.6,1.8Hz,1H),7.52(s,1H), 7.49-7.42 (m,2H), 7.41-7.26 (m,6H), 7.25-7.18 (m,2H),6.64(dt, J ═ 3.2,0.8Hz,1H),6.40(d, J ═ 6 ═ d, J ═ 1.8Hz,1H), 6.85 (d, J ═ 6).0Hz,1H),5.50(s,2H),4.68(d,J＝5.6Hz,2H).LCMS(ESI):m/z 391.2[M+H]⁺.

Example 22: synthesis of GVS-22:

GVS-22 was prepared as described for GVS-18 (77% yield) using 4-trifluoromethyl benzyl bromide instead of 3, 4-difluorobenzyl bromide.¹H NMR(400MHz,DMSO-d₆)δ8.87(t,1H,J＝6.0Hz),8.20(s,1H),7.65(dd,4H,J＝23.7and 5.5Hz),7.49(d,1H,J＝8.6Hz),7.34(d,2H,J＝8.0Hz),7.21(dd,4H,J＝24.9and 8.1Hz),6.63(d,1H,J＝3.0Hz),5.59(s,2H),4.44(d,2H,J＝5.8Hz),2.93–2.75(m,1H),1.17(d,6H,J＝6.9Hz)；HRMS(ESI)m/z calcd for C₂₇H₂₅F₃N₂O[M+H]⁺:451.1987,found:450.1919.

In vitro Activity assay

Example 23: evaluation of PPAR γ activation ability of compound (luciferase activity assay):

cos-7 cells were purchased from ATCC, cultured in 10% FBS antibiotic-free DMEM, 37 ℃, 5% CO₂An incubator. Plasmid co-transfection (50ng full length hPPAR γ, 100ng PPAR γ, 5ng renilla luciferase plasmid) was performed according to the instructions of lipofectamine 2000(Invitrogen) when cells were seeded into 24-well plates in logarithmic growth phase and cells were fused to about 70%. After 24h, transfected cells were intervened with 1 μ M compound, respectively, with 1 μ M rosiglitazone as positive control and DMSO as negative control. Luciferase activity was assayed 24h after the intervention as described in the Reporter luciferase assay kits (Promega) protocol, with 3 independent test wells per group.

The results are shown in Table 1.

TABLE 1Luciferase method test the activating ability of compound GVS1-22 on PPAR γ

(Note: the activating ability is marked as + at 0.1-0.5 and as + +, at 0.5-1.0.)

Example 24: TR-FRET method test compounds for ability to bind to PPAR γ step:

1. compounds 1 to 22 were diluted to 1mM with DMSO. DMSO is used as a negative control, and rosiglitazone is used as a positive control.

2. The diluted compounds (1 to 22, rosiglitazone) were diluted again to 2. mu.M with TR-FRETBuffer.

3. Preparation of Fluormone with TR-FRET buffer as solvent^TMPan-PPAR Green solution(20nM)。

4. 20nM Tb anti-GST antibody and 4. mu.M PPAR γ -LBD protein were prepared using TR-FRET buffer as a solvent.

5. Mu.l of the step 2 solution, 10. mu.l of the step 3 solution and 10. mu.l of the step 4 solution were mixed in a 384-well plate and shaken for 6 hours. Read on a microplate reader.

The results are shown in Table 2.

TABLE 2TR-FRET method test Compounds GVS1-22 for their ability to bind to PPAR γ

(Note: binding capacity is marked as + at 0.1-0.5, as + +, at 0.5-0.8, and as + + +, at 0.8-1.5)

TABLE 3 ratio of activating capacity to binding capacity of some compounds

(Note: the ratio is marked as + at 0.1-0.5, as + +, at 0.5-1.0, and as + + +, at 1.0-5.0.)

Example 25: GVS compound induced fat differentiation capability test and Real-time PCR determination

3T3-L1 preadipocytes were purchased from ATCC and cultured in 10% FBS DMEM containing penicillin-streptomycin double antibody at 37 ℃ in 5% CO₂An incubator. Inoculating to culture plate, and adding inducing solution (10% FBS DMEM containing 0.5mmol/L IBMX (3-isobutyl-1-methylxanthine), 1. mu. mol/L DEX (dexamethasone), 850nmol/L insulin) 2d after confluence. After 72h, the medium was changed to 10% FBS high-glucose DMEM containing 850nmol/L insulin every 2 days. mu.M rosiglitazone was used as a positive control, DMSO was used as a negative control, and the sample group was 1. mu.M. The induction was started at 8d for oil red O staining and DAPI staining, and the differentiation rate of adipocytes was calculated by photographing with a microscope (OLYMPUS).

The results are shown in Table 4.

TABLE 4GVS compound lipogenic differentiation ability

Example 26: function of GVS series compound in non-alcoholic fatty liver disease

1. Material

1.1 animals

Animal SPF grade SD male rats of 60 animals, weighing 180 + -200 g, were provided by the Guilin medical college SPF laboratory animal center.

1.2 drugs and reagents

The kit for synthesizing GVS series compounds (purity is more than 99.5%), glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST), Malondialdehyde (MDA) and superoxide dismutase (SOD) is purchased from Nanjing institute of bioengineering.

Low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), Triglyceride (TG) kit purchased from Beijing Kangtai clinical reagent Limited.

Adiponectin (ADP) and Fatty Acid Synthase (FAS) are available from Wuhan Irelet Biotech, Inc.

1.3 materials and instruments

H2050R desk refrigerated centrifuge, hunan instrument centrifuge instruments ltd;

an Epoch microplate reader, BioTek corporation, usa;

model 752 spectrophotometer, third analytical instrument factory, shanghai;

o1ymPus BX51 microscope, produced by olympus;

AUW220D electronic balance, sartorius, germany;

tanon electrophoresis apparatus, Shanghai Tianneng.

2. Method of producing a composite material

2.1 grouping and modeling

50 SD rats (body weight 180 ± 200 g) were randomly assigned to 5 groups (n ═ 10):

a blank control group;

a model group;

③ GVS-12 Low dose group (2 mg/kg);

fourthly, a middle-dose group (6mg/kg) of GVS-12;

GVS-12 high dose group (18 mg/kg).

Establishing a high fat diet induced non-alcoholic fatty liver disease rat model, and feeding the rat model with a blank group by using a common feed for 16 weeks; the model group and the administration group were fed with high fat diet (feed ratio: 82.5% normal diet + 10% lard + 5% egg yolk powder + 2% cholesterol + 0.5% sodium cholate) for 16 weeks, and administration was started at the fourth week, GVS-12 was administered by subcutaneous injection for 12 weeks, and the blank control group and the model group were administered with distilled water of the same volume.

2.2 detection of indicators

The operation is strictly carried out according to the requirements and specifications of the kit.

The kit for glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), Malondialdehyde (MDA) and superoxide dismutase (SOD) is purchased from Nanjing institute of bioengineering.

Low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), Total Cholesterol (TC), Triglyceride (TG) kits were purchased from Beijing Huakangtai clinical reagent, Inc.

Adiponectin (ADP) and Fatty Acid Synthase (FAS) kits are available from Wuhan Eleret Biotech, Inc.

2.3 pathological Observation

The same part of the liver of each rat is taken, fixed by 10% neutral formalin, dehydrated, embedded, sliced and dewaxed, and the improvement condition of GVS-12 on the model lipid deposition is visually detected by adopting oil red O staining. And (3) observing the fat accumulation degree and the inflammation degree of the liver of the rat by adopting HE (human immunodeficiency Virus) staining, and analyzing the pathological condition of the liver tissue by microscopic observation.

2.4 data processing

Statistical analysis was performed using SPSS13.0 software. The data are presented as Mean ± standard deviation (Mean ± s.e.m), and the Mean between groups is compared using one-way anova followed by Dunnett-t test, with P <0.05 as the significance difference criterion.

3. Results

(1) The effect of GVS-12 on body weight of non-alcoholic fatty liver rats was examined.

As can be seen from fig. 2, the body weight of the rats in the administration group was not significantly increased as compared with that in the model group.

(2) And (4) observation of pathological morphology, namely, adopting oil red O and HE staining to observe the degeneration degree and the inflammation degree of the rat.

As can be seen from fig. 3, the oil red O staining results showed that the fat of the model group had been significantly differentiated and deposited, while the fat of the administered group had significantly decreased in differentiation.

The HE staining result shows that the liver tissues of the model group have serious inflammatory injury and hepatic fibrosis, and the inflammatory injury of the administration group is gradually reduced and is continuously improved in a dose-dependent manner.

(3) The influence of GVS-12 on fatty liver rat liver function factors of glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), Malondialdehyde (MDA) and superoxide dismutase (SOD) is detected by a biochemical method.

As can be seen from FIG. 4, GVS-12 significantly decreased the activity and concentration of glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT) and Malondialdehyde (MDA) in serum, and significantly increased the activity of superoxide dismutase (SOD) compared to the model group;

(4) detecting the influence of GVS-12 on low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and Triglyceride (TG) of fat factors of rats with fatty liver by a biochemical method;

as shown in FIG. 5, GVS-12 decreased the low-density lipoprotein cholesterol (LDL-C), Triglyceride (TG) and increased the high-density lipoprotein cholesterol (HDL-C) content as compared to the model group.

(5) The effect of GVS-12 on Adiponectin (ADP) and Fatty Acid Synthase (FAS) in fatty liver rats was examined by ELISA.

As can be seen from FIG. 6, it was found that the Adiponectin (ADP) level in serum was increased and the Fatty Acid Synthase (FAS) activity was decreased as compared with the model group.

The results fully show that GVS-12 has the function of improving nonalcoholic liver inflammation and fat accumulation under the condition of maintaining normal body weight, and has good function of preventing and treating nonalcoholic fatty liver.

In conclusion, the GVS series compound with a brand-new mother nucleus structure has a structure different from that of rosiglitazone, is simple and easily available in raw materials, can bind and excite a PPAR gamma receptor, has the characteristic effect of no obvious induction of fat differentiation, and has the result equivalent to or even better than that of rosiglitazone.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Use of a compound of formula a, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament or formulation for:

treatment and/or prevention of fatty liver and fat accumulation; the fatty liver is non-alcoholic fatty liver;

wherein the compound of formula A is

2. The use according to claim 1, wherein the compound of formula a is selected from the group consisting of:

3. the use according to claim 1, wherein the compound of formula a is

4. The use according to claim 1, wherein the medicament or formulation has one or more properties selected from the group consisting of:

(a) weak activation ability to PPAR γ;

(b) strong binding ability to PPAR γ;

(c) weak ability of preadipocytes 3T3-L1 to transform into adipocytes.

5. A compound of formula A or a pharmaceutically acceptable salt thereof,

the compound of formula a is selected from the group consisting of:

6. a pharmaceutical composition comprising:

(i) a compound of formula a according to claim 5 or a pharmaceutically acceptable salt thereof; and

(ii) a pharmaceutically acceptable carrier.