CN107522657A - A kind of compound with the multiple agonist activities of PPAR and its preparation method and application - Google Patents

Abstract

The invention discloses a kind of compound with the multiple agonist activities of PPAR and its preparation method and application, the compound has chemical constitution shown in formula I：

Description

A compound with multiple agonistic activities of PPAR and its preparation method and application

technical field

The invention relates to a compound with multiple agonistic activities of PPAR and its preparation method and application, belonging to the technical field of chemistry and medicine.

Background technique

According to the World Health Organization, about 20%-25% of adults worldwide suffer from metabolic syndrome, 70%-80% of diabetic patients suffer from metabolic syndrome, and up to 86% of type II diabetic patients are diagnosed with metabolic syndrome. disorder. Studies have shown that: people's weight gain and inactive lifestyle can cause insulin resistance in fat cells, leading to more and more people suffering from metabolic syndrome (Diabetes&Metabolic Syndrome Clinical Research&Reviews[J],2015,9(2):124 -126). Metabolic syndrome is related to the disturbance of glucose metabolism in the body (Journal of Diabetes & Metabolic Disorders[J],2014,13(1):1-7), and is a risk factor leading to obesity, central obesity, insulin resistance, dyslipidemia and hypertension (Clinical Laboratory[J], 2013,59(3-4):369-374), and it is also related to other diseases such as periodontitis (Journal of Clinical Periodontology[J], 2013,40(6):599-606). It can be seen that metabolic syndrome has become a serious disease that threatens human health.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily of ligand-activated transcription factors, and there are three subtypes, namely: PPARα, β, γ, different PPARs subtypes Due to the specificity of their tissue distribution, activation ligands and co-cofactor recruitment, their functions are also different, and their functions are related to each other, but not completely the same. Studies have shown that: ①PPAR-α is mainly expressed in tissues such as liver, muscle, and macrophages (Circulation Research[J], 2000.87(6):516-521); PPAR-α affects liver lipid metabolism through ligand binding ( Journal of Molecular Graphics and Modeling[J],2014,51:27-36); the most classic drugs among PPAR-α synthetic agonists are fibrates, which are widely used clinically to treat hypertriglyceridemia . ②PPAR-β/δ is widely distributed in the body and is expressed in liver, kidney, skeletal muscle, abdominal fat and vascular smooth muscle cells (Biochimica Et Biophysica Acta Molecular Basis of Disease[J],2005,1740(2): 313-317); therefore, it cannot be associated with the exact clinical disease, so there is no PPAR-β/δ agonistic ligand drug in clinical practice; however, studies have found that activating PPAR-β/δ can improve lipid metabolism disorders and regulate cholesterol Outflow of protein, bone metabolism and insulin sensitivity etc. (The mechanism of protein synthesis[J],2002,53:409-435). ③PPAR-γ is highly expressed in adipocytes, spleen, adrenal gland, and colon, and lowly expressed in liver, skeletal muscle, kidney, blood vessels, and brain (Endocrinology[J], 1996,137:354-366), which is currently the most studied A mature drug target that plays an important regulatory role in the proliferation and differentiation of various cells and their insulin sensitivity.

In the treatment of metabolic syndrome, the hypoglycemic or lipid-lowering drugs currently used in clinical practice have a single effect and cannot regulate glucose and lipid metabolism at the same time. Studies have shown that dual or triple PPARs agonists can simultaneously act on both lipid metabolism and insulin, and can more effectively improve metabolic syndrome and provide a broader spectrum of therapeutic effects on metabolic syndrome (Cardiovascular Diabetology[J], 2005, 4(1):14). Therefore, screening compounds with dual or triple PPARs agonistic activity is of great value for the prevention and/or treatment of metabolic disorders.

Contents of the invention

In view of the above-mentioned problems in the prior art, the purpose of the present invention is to provide a compound with multiple agonistic activities of PPAR and its preparation method and application, and to screen a new drug for preventing and/or treating metabolic abnormalities.

The compound with multiple agonistic activities of PPAR described in the present invention has the chemical structure shown in formula I:

A method for preparing the compound with PPAR multiple agonistic activity according to the present invention, comprising the steps of:

a) making p-hydroxybenzaldehyde react with p-methoxybenzyl chloride in the presence of a base to obtain a compound of formula II;

b) reacting 3-methoxy-4-bromoaniline with acetonitrile in the presence of a catalyst to obtain a compound of formula III;

c) reacting the compound of formula II with the compound of formula III in the presence of a base to obtain the compound of formula IV;

d) making the compound of formula IV undergo an addition reaction in the presence of a catalyst to obtain the compound of formula V;

e) making the compound of formula V undergo Suzuki coupling reaction with prenylboronic acid pinacol ester in the presence of a palladium catalyst and a base to obtain a compound of formula VI;

f) removing the PMB protecting group (i.e.: p-methoxybenzyl) from the compound of formula VI under acid catalysis to obtain the compound of formula I;

Its specific reaction scheme is as follows:

As a preferred solution, the base in step a) is an inorganic base, and the inorganic base is preferably a carbonate, such as potassium carbonate and sodium carbonate.

As a preferred solution, the reaction solvent in step a) is a non-alcoholic organic solvent, any one selected from dichloromethane, chloroform, acetonitrile, DMF, THF, preferably DMF.

As a preferred version, the molar ratio of p-hydroxybenzaldehyde to p-methoxybenzyl chloride in step a) is 1:1.

As a preferred solution, the reaction temperature in step a) is 0-30°C, more preferably 20-30°C.

As a preferred version, the catalyst in step b) is boron trichloride and aluminum trichloride.

As a further preferred solution, the specific operation of step b) is as follows: after dissolving 3-methoxy-4-bromoaniline in the reaction solvent, slowly add boron trichloride at 0-5°C, and then slowly Add aluminum trichloride and acetonitrile, after the addition is complete, react at room temperature for 10-60 minutes, then raise the temperature to 65-75°C and react until the reaction ends.

As a further preferred solution, the molar ratio of boron trichloride to aluminum trichloride is 1:1; the molar ratio of 3-methoxy-4-bromoaniline to aluminum trichloride is 1:1˜1:2.

As a preferred solution, the reaction solvent in step b) is dichloromethane or chloroform.

As a preferred solution, the molar ratio of 3-methoxy-4-bromoaniline to acetonitrile in step b) is 1:1˜1:3.

As a preferred solution, the base in step c) is an inorganic base, and the inorganic base is preferably sodium hydroxide, potassium hydroxide or lithium hydroxide.

As a preferred solution, the reaction solvent in step c) is an alcoholic organic solvent, such as methanol, ethanol, propanol and the like.

As a preferred solution, the reaction temperature in step c) is 50-70°C.

As a preferred solution, the molar ratio of the compound of formula II to the compound of formula III in step c) is 1:2-2:1.

As a preferred solution, the catalyst in step d) is a Lewis acid, and the Lewis acid is preferably antimony trichloride.

As a preferred solution, the reaction solvent in step d) is at least one of acetonitrile, DMF, DMSO, toluene, THF, and chloroform.

As a preferred solution, the reaction temperature in step d) is 55-85°C.

As a preferred solution, the palladium catalyst in step e) is selected from any one of Pd(dppf) ₂ Cl ₂ , PdCl ₂ (PPh ₃ ) ₂ , and Pd(PPh ₃ ) ₄ , preferably Pd(dppf) ₂ Cl ₂ .

As a preferred solution, the base in step e) is an inorganic base selected from any one of sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate and potassium acetate, preferably cesium carbonate.

As a preferred solution, the reaction solvent in step e) is at least one selected from DMF, DMSO, toluene, THF, dioxane, toluene, methanol, and ethanol.

As a preferred solution, the reaction temperature in step e) is 60-80°C.

As a preferred embodiment, the acid in step f) is p-toluenesulfonic acid.

As a preferred solution, the reaction temperature in step f) is 50-70°C.

At least one of the compound with PPAR multiple agonistic activity described in the present invention or its pharmaceutically acceptable salt, tautomer, stereoisomer, precursor compound, hydrate or solvate as the active ingredient , can be used for preparing medicine or health food for treating and/or preventing metabolic syndrome.

Further, the metabolic syndrome includes abnormal glucose metabolism and/or abnormal lipid metabolism.

Furthermore, the metabolic syndrome includes at least one disease among diabetes, obesity, hyperlipidemia and atherosclerosis.

In addition, the drugs of the present invention can be administered to patients by various routes of administration, including but not limited to oral, transdermal, intramuscular, subcutaneous and intravenous injection.

The dosage form of the medicine of the present invention is not limited, as long as it is the dosage form that can make the active ingredient reach the body effectively, including: tablet, sugar-coated tablet, film-coated tablet, enteric-coated tablet, capsule, hard capsule medicines, soft capsules, oral liquids, buccal preparations, granules, granules, pills, powders, ointments, elixirs, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays Agents, drops, patches, etc.; preferred oral dosage forms, such as: capsules, tablets, oral liquids, granules, pills, powders, elixirs, ointments, etc.

In addition to the main active ingredient, the medicine of the present invention may also contain a small amount of minor ingredients that do not affect the active ingredient and/or pharmaceutically acceptable carriers and necessary adjuvants for various preparations. For example, when the drug is in an oral dosage form, it may contain commonly used excipients, such as binders, fillers, diluents, tableting agents, lubricants, disintegrating agents, coloring agents, flavoring agents and wetting agents, as necessary Tablets may be coated when necessary. Suitable fillers include cellulose, mannitol, lactose and other similar fillers; suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycolate; suitable lubricants include, for example, starch magnesium stearate; suitable pharmaceutically acceptable wetting agents include sodium lauryl sulfate.

Definitions of terms in the present invention are as follows:

The term "pharmaceutically acceptable salt" refers to the salt formed of the compound and a pharmaceutically acceptable inorganic acid or organic acid, and the inorganic acid includes but not limited to: hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, Sulfuric acid; the organic acids include but not limited to: formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalene disulfonic acid, subacid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid , diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, niacin, iso Nicotinic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, and amino acids; said "pharmaceutically acceptable" means that it is applicable to humans without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, there are A substance with a reasonable benefit/risk ratio.

The term "tautomer" refers to isomers of functional groups resulting from the rapid movement of an atom in a molecule between two positions, for example: enol and the corresponding ketone.

The term "stereoisomer" refers to isomers produced by different arrangements of atoms in the molecule in space, for example: cis-trans isomers, enantiomers, conformational isomers and the like.

The term "precursor compound" refers to a compound that is inactive in vitro, but can be converted into the active ingredient of the present invention through metabolism or chemical reaction in vivo, thereby exerting its pharmacological effect.

Compared with the prior art, the present invention has the following significant beneficial effects:

The research results of the present invention show that the compound of formula I described in the present invention can significantly increase the transcriptional activity of PPARα, β and γ, and has multiple agonistic activities of PPARα, β, γ, and is expected to be used as an active ingredient in the preparation of preventive and/or therapeutic The medicine or health food for metabolic syndrome is particularly expected to be used in the preparation of medicine or health food for preventing and/or treating abnormal glucose metabolism and/or abnormal lipid metabolism, and has wide application prospects and significant medicinal value.

detailed description

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, usually follow the conventional conditions or the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Embodiment 1: the preparation of formula II compound

Add p-hydroxybenzaldehyde (3.7g, 30.0mmol) and potassium carbonate (12.4g, 90.0mmol) into DMF (40mL), stir and dissolve at room temperature, slowly drop p-methoxybenzyl chloride (3.5g, 30.0 mmol), react at room temperature (about 20-25° C.) for 12 hours, end the reaction, add saturated NaCl solution to quench the reaction, then add ethyl acetate for extraction until the extraction is complete, combine the organic phases, and dry the organic phases with anhydrous sodium sulfate. Suction filtration, concentrated under reduced pressure until no solution distilled off to obtain a white powder substance, which is the compound of formula II (named: 4-p-methoxybenzylhydroxy-benzaldehyde): 6.2g, 25.6mmol, molar yield was 85.3%.

Embodiment 2: the preparation of formula III compound

Dissolve 3-methoxy-4-bromoaniline (6.0g, 30.0mmol) in dichloromethane (30mL), cool to 0°C in an ice bath, and slowly add BCl ₃ dichloromethane solution dropwise at 0°C (1.0mol/L, 33mL), keep stirring for half an hour, then slowly add AlCl ₃ (4.4g, 33.0mmol) and CH ₃ CN (3.2mL, 60.0mmol) at 0°C, Stir the reaction for half an hour, then raise it to 70°C and keep it warm for 12 hours, then end the reaction, add 2N HCL to quench the reaction after the reaction liquid is cooled (the reaction liquid will produce a lot of white bubbles during the process of adding hydrochloric acid, try to slow down the reaction in an ice bath at 0°C). Add), then stir at 70°C for 2 hours, after cooling to room temperature, add 1N NaHCO ₃ solution to adjust the solution to neutrality, then add dichloromethane for extraction until the extraction is complete, combine the organic phases, and dry the organic phases with anhydrous sodium sulfate , filtered with suction, concentrated under reduced pressure until no solution distilled off, and the residue was purified by silica gel column chromatography (cyclohexane/EA 50:1) to obtain a brown yellow powder, which is the compound of formula III (named: 2-amino-4 -Methoxy-5-bromoacetophenone): 3.7 g, 15.2 mmol, and the molar yield is 50.7%.

Tested:

¹ H NMR (300MHz, CDCl ₃ )δ7.85(s,1H,ArH),6.48(s,2H,NH2),6.10(s,1H,ArH),3.89(s,3H,ArOCH ₃ ),2.53( s,3H,COCH ₃ ).

Embodiment 3: the preparation of formula IV compound

Add the compound of formula II (1.7g, 6.5mmol) and the compound of formula III (1.2g, 5.0mmol) into absolute ethanol (20mL), then add solid sodium hydroxide (800.0mg, 20.0mmol), after the addition is complete, the temperature is raised Reaction at 60°C for 12 hours, the reaction was terminated, the reaction liquid was cooled to room temperature, filtered, the filter cake was washed with ethanol, and dried to obtain a yellow powder substance, which was the compound of formula IV (named: 1-(2'-amino-4 '-methoxy-5'-bromo)-3-(4'-methoxybenzylanisole)-2E-propen-1-one): 2.2 g, 4.66 mmol, and the molar yield is 93.2%.

Tested:

¹ H NMR (300MHz, CDCl ₃ ) δ8.00 (s, 1H, ArH), 7.70 (d, J = 15.3Hz, 1H, = H), 7.59 (d, J = 8.4Hz, 2H, ArH), 7.44 –7.33(m,3H,=H/ArH),6.96(dd,J=21.1,8.3Hz,4H,ArH),6.59(s,2H,NH2),6.13(s,1H,ArH),5.04(s , 2H, OCH ₂ ), 3.89 (s, 3H, ArOCH ₃ ), 3.82 (s, 3H, ArOCH ₃ ).

Embodiment 4: the preparation of formula V compound

Add the compound of formula IV (933.0mg, 1.99mmol) into anhydrous acetonitrile (15mL), then add antimony trichloride solid (136.8mg, 0.6mmol), after the addition is complete, raise the temperature to reflux state and react for 6 hours to end the reaction. The reaction solution was cooled to room temperature, filtered, and the filter cake was washed and dried to obtain a light yellow powder, which was the compound of formula V (named: 2,3-dihydro-2-(4'-methoxybenzyl Anisole)-6-bromo-7-methoxy-4H-1-quinolin-4-one): 495.0 mg, 1.06 mmol, the molar yield is 53.3%.

Tested:

¹ H NMR (400MHz, CDCl ₃ )δ8.04(s,1H,ArH),7.35(dd,J=8.5,5.7Hz,4H,ArH),6.95(dd,J=22.2,8.7Hz,4H,ArH ),6.13(s,1H,ArH),5.00(s,2H,OCH2),4.67(dd,J=13.6,3.9Hz,1H,H-2),4.54(s,1H,NH),3.87(s ,3H,OCH ₃ ),3.82(s,3H,OCH ₃ ),2.81(dd,J=16.3,13.5Hz,1H,H-3),2.69(dd,J=16.4,3.8Hz,1H,H- 3);

¹³ C NMR (125MHz, DMSO-d ₆ )δ190.0, 160.1, 158.9, 157.9, 153.4, 133.3, 130.4, 129.4, 128.9, 127.9, 114.8, 113.8, 112.7, 99.2, 98.2, 68.9, 56.2, 51.4, 55.5 .

Embodiment 5: the preparation of formula VI compound

The compound of formula V (468.0 mg, 1.0 mmol) was dissolved in anhydrous dimethylformamide (15 mL), and a palladium catalyst Pd(dppf) ₂ Cl ₂ (Chinese name: [1,1'-bis(diphenyl Phosphine) ferrocene] palladium dichloride dichloromethane complex) solid (245.0mg, 0.3mmol) and cesium carbonate solid (977.5mg, 3.0mmol), the temperature of the reaction solution was raised to 70°C under anhydrous and oxygen-free conditions, Slowly add isopentenylboronic acid pinacol ester (also known as 3-methyl-2-butenylboronic acid pinacol ester, CAS#141550-13-2, 0.59mL, 2.5mmol), after the addition is completed, Insulate the reaction at 70°C for 12 hours, end the reaction, cool the reaction solution to room temperature, add saturated NaCl solution to quench the reaction, then add ethyl acetate to extract, and extract repeatedly until the extraction is complete, combine the organic phase, and dry the organic phase with anhydrous sodium sulfate , filtered with suction, concentrated under reduced pressure until no solution distilled off, and the residue was purified by silica gel column chromatography (cyclohexane/EA 20:1) to obtain a light yellow powder substance, which is the compound of formula VI (named: 2,3-di Hydrogen-2-(4'-methoxybenzylanisole)-6-prenyl-7-methoxy-4H-1-quinolin-4-one): 198.0mg, 0.43mmol, mol The yield was 43.0%.

Tested:

¹ H NMR (500MHz, CDCl ₃ )δ7.63(s,1H,ArH),7.35(dd,J=8.6,4.2Hz,4H,ArH),6.94(dd,J=24.0,8.6Hz,4H,ArH ), 6.07(s,1H,ArH),5.26(t,J=7.3Hz,1H,=H),4.99(s,2H,OCH ₂ ),4.64(dd,J=13.8,3.5Hz,1H,H -2),4.41(s,1H,NH),3.81(s,3H,OCH ₃ ),3.80(s,3H,OCH ₃ ),3.19(d,J=7.3Hz,2H,=CH ₂ ),2.78 (dd,J=16.1,13.9Hz,1H,H-3),2.65(dd,J=16.2,2.7Hz,1H,H-3),1.72(s,3H,=CH ₃ ),1.69(s, 3H,=CH ₃ );

¹³ C NMR (125MHz, Chloroform-d ₃ )δ191.6, 163.2, 159.0, 158.3, 151.8, 133.10, 132.0, 128.7, 128.3, 127.6, 127.3, 121.8, 121.6, 114.7, 113.6, 112.1, 95.7, 69 , 54.8, 45.8, 27.3, 25.4, 17.3.

Embodiment 6: the preparation of formula I compound

Add the compound of formula VI (110.0mg, 0.25mmol) into (THF:MeOH volume ratio 1:1) solvent system 4mL, then add p-toluenesulfonic acid solid (215.0mg, 1.25mmol), after the addition is complete, the reaction solution is heated to React at 60°C for 30 minutes to end the reaction, cool the reaction solution to room temperature, add saturated NaCl solution to quench the reaction, then add ethyl acetate for extraction, and extract repeatedly until the extraction is complete, combine the organic phases, and dry the organic phases with anhydrous sodium sulfate. Suction filtration, concentrated under reduced pressure until no solution distilled off, and the residue was purified by silica gel column chromatography to obtain a light yellow powder substance, which is the compound of formula I (named: 2,3-dihydro-2-(4'-hydroxyl Phenyl)-6-prenyl-7-methoxy-4H-1-quinolin-4-one): 18.0 mg, 0.05 mmol, the molar yield is 21.3%.

Tested:

¹ H NMR (500MHz, CD ₃ OD) δ7.47 (s, 1H, ArH), 7.31 (d, J = 8.5Hz, 2H, ArH), 6.81 (d, J = 8.5Hz, 2H, ArH), 6.31 (s,1H,ArH),5.29–5.22(m,1H,=H),4.60(dd,J=13.4,4.1Hz,1H,H-2),3.84(s,3H,OCH ₃ ),3.17( d,J=7.3Hz,2H,=CH ₂ ),2.76(dd,J=16.2,13.4Hz,1H,H-3),2.58(dd,J=16.2,4.1Hz,1H,H-3), 1.75(s,3H,=CH ₃ ), 1.70(s,3H,=CH ₃ );

¹³ C NMR (125MHz, CD ₃ OD) δ193.6, 164.3, 156.9, 154.3, 132.3, 131.8, 127.5, 126.7, 122.2, 120.8, 114.9, 111.2, 95.8, 57.5, 54.6, 45.5, 27.2, 24.5, 16.3;

ESI-MS LR(m/z): 338.2[M+H] ⁺ ; ESI-MS HR(m/z): 338.1761[M+H] ⁺ ; the molecular formula of the corresponding compound is C ₂₁ H ₂₃ NO ₃ .

Example 7: Using dual luciferase reporter gene method to analyze the effect of the compound of formula I on the transcriptional activity of PPAR

The reporter gene detects the effect of the compound of formula I on the transcriptional activity of PPAR. The inventors used two plasmids, the full-length PPAR gene and the ligand binding domain (ligand binding domain, LBD), to detect the effect of the compound on the activity of nuclear receptor transcription factors; after transfecting it into 293T cells, after 24 hours of drug intervention, the effect of the compound was detected. Firefly luciferase activity; and jellyfish luciferase activity was used as a transfection efficiency control.

(1) 293T cell culture

The 293T cell line (human embryonic kidney cell line) was cultured in DMEM high glucose containing 10% calf serum and 1% double antibody in a 37°C, 5% CO ₂ incubator. The 293T cells in the logarithmic growth phase were plated in a 48-well plate at a cell density of 1×10 ⁵ -2×10 ⁵ cells/mL.

(2) Plasmid for transfection

pCMX-Gal-mPPARγLBD plasmid, PPARα-LBD plasmid, PPARβ-LBD plasmid, Gal4reportervector MH100×4-TK-Luc recombinant plasmid and renilla luciferase internal control plasmid; PPARγ full-length plasmid; PPARα full-length plasmid; PPARβ full-length plasmid.

References for the construction of the above plasmids: Biochemical and Biophysical Research Communications2006(348):571-578; Cell Metabolism.2(2005)239-249; J.Biol.Chem.272(1997)18779-1878; Cell 83(1995)803 -812.

(3) Transfection

Overnight, when the cells grew to 50-80% density, transfection was carried out. Prepare the transfection system with DMEM (serum-free and double-antibody-free): 10 μg of total plasmid per mL of DMEM, and 15 μL of transfection reagent-FuGENE HD [Roche], then vortex the transfection system, and incubate at room temperature After standing for 15 minutes, the transfection system was co-transfected into 293T cells, and cultured with complete medium (DMEM, 10% FBS, 1% double antibody) for 24 hours.

(4) Dosing intervention

After 24 h, the compound of formula I with different concentration gradients (0.01, 0.1, 0.3, 1, 3, 10, 30 μM) diluted with complete medium or different concentration gradients (0.001, 0.01, 0.1, 1 μM) diluted with complete medium were added. , 3, 10 μM) rosiglitazone (specific PPARγ agonist) or different concentration gradients of PPARα agonist fenofibrate (0.001, 0.01, 0.1, 1, 3, 10 μM) for intervention or different concentration gradients of PPARβ agonist GW7647 (0.001, 0.01, 0.1, 1, 3, 10 μM) was used for intervention.

(5) Cell treatment

① After 24 hours, wash the cells twice with PBS to remove the remaining cell culture medium;

② Add 65 μL of lysate to each well and shake on the shaker for 15 minutes. After the cells are completely lysed, transfer the cell lysate to a 1.5 mL centrifuge tube;

③Centrifuge the cell lysate at 1000rpm for 5min, take 10μL of the supernatant into a new centrifuge tube for testing.

(6) Determination and analysis of luciferase intensity

① Add 20 μl of LAR II solution [purchased from Promega], mix well, measure fluorescence, delay for 2 seconds, and read for 10 seconds. Transfection efficiency was calibrated using internal control Renilla luciferase activity. All transfection experiments were repeated at least three times independently, with at least 2 secondary wells for each experimental group.

②Bio-Tek, Synergy HT multifunctional microplate reader was used to detect the fluorescence intensity of fireflies and marine coelenterates. The expression intensity of firefly luciferase is expressed by the ratio of firefly fluorescence intensity and marine coelenterate fluorescence intensity, relative fluorescence intensity = firefly fluorescence intensity/marine coelenterate fluorescence intensity, that is, the relative expression activity of luciferase is mainly used to reflect the external drug Whether it affects the transcriptional activity of PPARs through functional combination with PPARs receptors.

(7) Data analysis

The software SPSS16.0 was used for data analysis, the difference between different intervention groups was compared using one-way analysis of variance (ANOVA), p<0.05 was considered to be statistically significant in the difference between groups, and the software GraphPad Prism 6 was used to calculate the value of each compound intervention group _EC50 value.

(8) Experimental results

The experimental results are shown in Table 1 and Table 2.

Table 1 Transcriptional activity (activation%, 25μM) of different compounds on PPARs

compound PPAR-α-LBD PPAR-β-LBD PPAR-γ-LBD Fenofibric acid 12.13±3.42 - - GW7647 - 55.91±17.17 - Pioglitazone - - 41.52±4.16 Compound of formula I 9.04±1.43 4.98±0.79 35.45±7.49

Table 2 formula I compound to the half effective concentration value ( _EC50 value, μ M) of PPAR transcriptional activity

PPAR-α-LBD, EC ₅₀ (μM) PPAR-β-LBD, _EC50 (μM) PPAR-γ-LBD, EC ₅₀ (μM) 22.58±2.35 3.54±1.49 65.33±28.64

Combining Table 1 and Table 2, it can be seen that the compound of formula I described in the present invention exhibits significant agonistic activity on PPARα, β, and γ, and has multiple agonistic activities of PPARα, β, and γ.

Because existing studies have shown that: PPARs multiple agonists can simultaneously act on both lipid metabolism and insulin, can effectively improve metabolic syndrome, and provide a broader spectrum of therapeutic effects on metabolic syndrome, therefore, according to the present invention The compound of formula I or at least one of its pharmaceutically acceptable salts, tautomers, stereoisomers, precursor compounds, hydrates or solvates, as an active ingredient, can be used for the preparation of therapeutic and/or prophylactic The medicine or health food for metabolic syndrome is particularly expected to be used in the preparation of medicine and health food for preventing and/or treating abnormal glucose metabolism and/or abnormal lipid metabolism, and has wide application prospects and significant medicinal value.

Finally, it should be pointed out here that: the above are only some preferred embodiments of the present invention, and should not be interpreted as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the above-mentioned contents of the present invention. All belong to the protection scope of the present invention.

Claims (9)

1. A compound with multiple agonistic activities of PPAR, characterized in that it has a chemical structure shown in formula I: 2. A method for preparing a compound with PPAR multiple agonistic activity according to claim 1, comprising the steps of: a) making p-hydroxybenzaldehyde react with p-methoxybenzyl chloride in the presence of a base to obtain a compound of formula II; b) reacting 3-methoxy-4-bromoaniline with acetonitrile in the presence of a catalyst to obtain a compound of formula III; c) reacting the compound of formula II with the compound of formula III in the presence of a base to obtain the compound of formula IV; d) making the compound of formula IV undergo an addition reaction in the presence of a catalyst to obtain the compound of formula V; e) making the compound of formula V undergo Suzuki coupling reaction with prenylboronic acid pinacol ester in the presence of a palladium catalyst and a base to obtain a compound of formula VI; f) removing the PMB protecting group from the compound of formula VI under acid catalysis to obtain the compound of formula I; Its specific reaction scheme is as follows: 3. The method according to claim 2, characterized in that: the alkali in step a) is an inorganic alkali. 4. The method according to claim 2, characterized in that: the catalyst in step b) is boron trichloride and aluminum trichloride. 5. The method according to claim 2, characterized in that: the alkali in step c) is an inorganic base, and the catalyst in step d) is a Lewis acid. 6. The method according to claim 2, characterized in that: the palladium catalyst in step e) is selected from any one of Pd(dppf) ₂ Cl ₂ , PdCl ₂ (PPh ₃ ) ₂ and Pd(PPh ₃ ) ₄ Kind; The acid in step f) is p-toluenesulfonic acid. 7. The application of the compound with multiple agonistic activities of PPAR according to claim 1, characterized in that: the compound shown in formula I or its pharmaceutically acceptable salt, tautomer, stereoisomer, At least one of the precursor compound, hydrate or solvate is used as an active ingredient for preparing medicine or health food for treating and/or preventing metabolic syndrome. 8. The application according to claim 7, characterized in that: the metabolic syndrome includes abnormal glucose metabolism and/or abnormal lipid metabolism. 9. The application according to claim 8, characterized in that: the metabolic syndrome includes at least one disease among diabetes, obesity, hyperlipidemia and atherosclerosis.