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  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
  • C07C217/54—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
  • C07C217/64—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with amino groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by carbon chains further substituted by singly-bound oxygen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
  • C07C217/54—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
  • C07C217/56—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with amino groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by carbon chains not further substituted by singly-bound oxygen atoms
  • C07C217/58—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with amino groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by carbon chains not further substituted by singly-bound oxygen atoms with amino groups and the six-membered aromatic ring, or the condensed ring system containing that ring, bound to the same carbon atom of the carbon chain
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  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C215/00—Compounds containing amino and hydroxy groups bound to the same carbon skeleton
  • C07C215/46—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
  • C07C215/48—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with amino groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by carbon chains not further substituted by hydroxy groups
  • C07C215/50—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with amino groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by carbon chains not further substituted by hydroxy groups with amino groups and the six-membered aromatic ring, or the condensed ring system containing that ring, bound to the same carbon atom of the carbon chain
• • 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
  • A61K31/13—Amines
  • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
• • 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
  • A61K31/13—Amines
  • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
  • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
• • 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
  • A61P3/08—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
  • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C209/00—Preparation of compounds containing amino groups bound to a carbon skeleton
  • C07C209/60—Preparation of compounds containing amino groups bound to a carbon skeleton by condensation or addition reactions, e.g. Mannich reaction, addition of ammonia or amines to alkenes or to alkynes or addition of compounds containing an active hydrogen atom to Schiff's bases, quinone imines, or aziranes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
  • C07C217/54—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
  • C07C217/64—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with amino groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by carbon chains further substituted by singly-bound oxygen atoms
  • C07C217/66—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with amino groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by carbon chains further substituted by singly-bound oxygen atoms with singly-bound oxygen atoms and six-membered aromatic rings bound to the same carbon atom of the carbon chain
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
  • C07C45/61—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups
  • C07C45/67—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton
  • C07C45/68—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C47/00—Compounds having —CHO groups
  • C07C47/52—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings
  • C07C47/575—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings containing ether groups, groups, groups, or groups

Definitions

• Novel compounds and pharmaceutical compositions for the prevention or treatment of obesity or metabolic syndrome comprising the same
• the present invention relates to novel compounds that can be usefully used for the prevention or treatment of obesity or metabolic syndrome and pharmaceutical compositions comprising the same.
• Obesity considered only a simple disease, has recently been classified as a disease.
• Obesity is a disease caused by a combination of various causes, including social, environmental, genetic, mental and endocrine causes.
• WHO World Health Organization
• the world's obese population has doubled since the 1980s.2 In the United States, about 34% of the total population was classified as obese in 2013. [3].
• the US government spends 10% of the nation's health care costs on obesity-related health care and campaigns every year to reduce the socioeconomic loss caused by obesity.
• the rise in obesity rates in Asian and African countries has also been prominent.
• According to the British medical journal Lancet there were about 90 million obese people in China in 2014 [4].
• the most effective treatment and prevention of obesity is to remove excess body fat, which is the causative agent, by using an in vivo system.
• body fat which is the causative agent
• Over-accumulated fat in tissues and blood is a major cause of various metabolic syndromes along with weight gain. Increased abdominal fat by high fat diet affects body fat and glucose metabolism and increases blood triglyceride levels.
• the increase in triglycerides in the blood causes the accumulation of triglycerides in various tissues.
• Liver disease is a representative metabolic syndrome in which fat accumulated in tissues is a direct cause of the disease. More than 5% of the accumulation of triglycerides in hepatocytes is defined as fatty liver and can develop into hepatitis [7]. Therefore, in order to prevent and treat liver disease, the accumulated fat in tissues Effective removal is probably the most effective way.
• the main use of drugs in preventing or treating obesity should be aimed at correcting metabolic abnormalities due to accumulated excess body fat, not simply weight loss, and ultimately metabolism such as diabetes and liver disease that can be caused by obesity. Focus should be on improving the prognosis of the syndrome.
• the first goal should be a selective reduction of excess fat, without changing the overall metabolic action.
• the most effective method is to utilize an existing system for maintaining intracellular homeostasis.
• autophagy mechanisms exist that degrade or recycle components such as damaged organelles and proteins, using organs called lysosomes. Cells are activated when they are exposed to stress or under nutrition, and cells use these mechanisms to properly maintain the intracellular environment. Depending on the substrate to be degraded, it is classified into mitophagy, xenophagy and lipophagy [8].
• the lipophagy system is known to function to remove the accumulated fat granules in cells.
• Nature Journal collaborated with Mark J. Czaja and Ana Maria Cuervo.
• the lipophagy process can be controlled by a technical method, it can be used as an effective treatment to replace the existing obesity treatments that prevent fat accumulation by adjusting the feeding form and energy metabolism.
• the present inventors have made intensive efforts to develop the drug as described above. As a result, the present inventors have confirmed that the novel compound to be described later satisfies the above characteristics, and completed the present invention. ⁇ references ⁇
• the present invention is to provide a novel compound or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising the same, which can be usefully used for the prevention or treatment of obesity or metabolic syndrome.
• the present invention provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
• 3 ⁇ 4 is--(phenyl),.
• Seedlings 2 is hydrogen, or ⁇ 2 -, and (phenyl),
• both 3 ⁇ 4 and 3 ⁇ 4 are not benzyl.
• 3 ⁇ 4 it is - ⁇ 2 - (phenyl), and; 3 ⁇ 4 is hydrogen,- ⁇ 2 (3 ⁇ 4_ (phenyl),-(3 ⁇ 4 (3 ⁇ 4 ⁇ ⁇ (phenyl)), or-(3 ⁇ 4 [3 ⁇ 4 (3 ⁇ 4 (3 ⁇ 4_ (phenyl)).
• 3 ⁇ 4 and 3 ⁇ 4 are identical to each other. More preferably, 3 ⁇ 4 and
• 3 ⁇ 4 is- ⁇ 2 ⁇ 2- (phenyl), _ (3 ⁇ 4 (3 ⁇ 4 ⁇ ⁇ (phenyl), or- ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2- (phenyl).
• Representative examples of the compound represented by Formula 1 are as follows:
• the compound represented by Chemical Formula 1 may be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt.
• Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes.
• non-toxic organic acids such as dioate, aromatic acids, aliphatic and aromatic sulfonic acids, and organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid and the like.
• Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, eye Odide, Fluoride, Acetate, Propionate, Decanoate, Caprylate, Acrylate, Formate, Isobutyrate, Caprate, Heptanoate, Propiolate, Oxalate, Malonate, Succinate, Sube Latex, sebacate, fumarate, maleate, butyne-1,4-dioate, nucleic acid-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, Methoxybenzoate, Phthalate, Terephthalate, Benzenesulfonane , Toluenesulfonate, Chloro
• the acid addition salt may be prepared by a conventional method.
• a precipitate formed by dissolving a derivative of Formula 1 in an organic solvent such as methanol, ethanol, acetone, dichloromethane, acetonitrile, and the like may be added to an organic or inorganic acid . It may be prepared by filtration, drying, or by distillation under reduced pressure of the solvent and excess acid, followed by drying and crystallization in an organic solvent. Bases can also be used to make pharmaceutically acceptable metal salts.
• Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate.
• the metal salt is not a pharmaceutically suitable for preparing the sodium, potassium or calcium salt.
• Corresponding salts are also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate).
• the present invention includes not only the compound represented by Formula 1 and pharmaceutically acceptable salts thereof, but also solvates, optical isomers, hydrates, and the like that can be prepared therefrom.
• the present invention provides a method for producing a compound represented by the formula (1) as shown in Scheme 1 as an example.
• the reaction is a reaction for preparing the compound represented by Chemical Formula 1 by reacting the compound represented by Chemical Formula 1-1 with the compound represented by Chemical Formula 1-2, and substantially consists of two steps. Specifically, the A compound represented by Chemical Formula 1-1 is reacted with a compound represented by Chemical Formula 1-2 to prepare an imine compound, and then reduced to prepare a compound represented by Chemical Formula 1.
• a solvent that may be used in the reaction, water, ethanol, tetrahydrofuran, dichloromethane, toluene, or acetonitrile may be used, but is not limited thereto.
• reaction temperature is not particularly limited, it may be carried out at 10 ° C to 90 ° C, preferably 10 ° C to 30 ° C or 60 ° C to 80 ° C.
• the reducing agent is not particularly limited, for example NaBH 4 may be used.
• a purification step may be included as necessary.
• the present invention provides a pharmaceutical composition for preventing or treating obesity or metabolic syndrome, including the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
• the compound according to the present invention As confirmed by the Oil red 0 staining method, the size of the fat granules accumulated by selectively degrading the lipid droplets (lipid droplets) accumulated in the adipocyte (adipocyte) is small Confirmed losing.
• the weight loss effect of the compound of the present invention in high-fat feed mice when injected intraperitoneally into the obesity-induced mice through high fat feed, it was statistically significant compared to the control drug-treated mice The level of weight loss effect was confirmed.
• Examples of the metabolic syndrome include any one selected from the group consisting of myocardial infarction, arteriosclerosis, hyperlipidemia, hypertension, cerebral infarction, cerebral hemorrhage, fatty liver, and type 2 diabetes.
• This metabolic syndrome is a major cause of obesity due to high-fat diet and reduced physical activity, and is accompanied by metabolic disorders such as increased body fat, increased blood pressure, and abnormal blood lipids. Therefore, treatment or prevention of obesity through the removal of excess body fat will be effective in improving the prognosis of metabolic syndrome.
• prevention of the present invention means any action that inhibits or delays the occurrence, spread, and recurrence of the above-mentioned diseases by administration of the pharmaceutical composition of the present invention
• treatment is the administration of the pharmaceutical composition of the present invention. It means all the actions that improve or beneficially change the symptoms of the disease
• the pharmaceutical composition according to the present invention can be formulated in oral or parenteral dosage form according to standard pharmaceutical practice.
• acceptable additives such as carriers, adjuvants or diluents, etc. Suitable carriers include, for example, physiological saline, polyethylene glycol, ethanol, vegetable oils and isopropyl myristate, and the like.
• Lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine but are not limited to these
• the compounds of the present invention may be dissolved in oils, propylene glycol or other solvents commonly used in the preparation of injectable solutions
• the compounds of the present invention may be formulated with ointments or creams for topical action.
• the preferred dosage of the compound represented by Formula 1 according to the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art.
• the compound represented by the formula (1) according to the present invention is 0.0001 to 100 per day , Preferably 0.001 to 100 / 13 ⁇ 4 (weight) 2019/190172 1 »(: 1 ⁇ 1 ⁇ 2019/003514
• Administration can be administered via oral or parenteral routes once or divided daily.
• the pharmaceutical composition according to the present invention may contain 0.001 to 99% by weight, preferably 0.01 to 60% by weight of the compound represented by the formula (1) according to the present invention or a pharmaceutically acceptable salt thereof.
• the pharmaceutical composition of the present invention can be administered to mammals including rats, mice, livestock and humans by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.
• the compound according to the present invention or a pharmaceutically acceptable salt thereof can be usefully used for the prevention or treatment of obesity or metabolic syndrome.
• Fig. 4 shows the ⁇ 31 fat cells (Fig. 4 and It is the result of confirming that the compounds of the present invention activate the lipophagy process by immunofluorescence staining in 0,2 liver cells (Fig. 8) and that the activated lipophagy process reduces the size and number of fat granules accumulated in the cells. .
• FIG. 5 shows that the elimination of fat granules does not occur by treating the compound of the present invention with 83 1011 ⁇ 11, which is an inhibitor of lipophagy, in order to verify that the compounds of the present invention have removed fat granules through the lipophage process. The result is confirmed. ,
• Figure 7 shows the results recorded by parking for each weight change by placebo and compound treatment of the present invention.
• Figure 8 is a graph showing the results of measuring the weight of each tissue to confirm that the weight loss effect caused by the treatment of the compound of the present invention is due to a decrease in fat tissue weight.
• FIG 9 is a graph comparing the weight of the adipose tissue reduced by the compound of the present invention.
• Figure 10 is a photograph comparing the actual appearance of each tissue removed to confirm that the weight of the fat tissue alone. In each picture, the left side treated vehi cle and the right side treated the compound of the present invention.
• 11 is a graph showing the proportion of body weight lost through the compound of the present invention.
• the results of height measurements were shown to compare the degree of development during the administration period.
• the results of a survey of feed intake were shown to determine whether the weight loss effect was due to a decrease in feed intake.
• Figure 12 is the results of the items collected and tested the blood of the placebo group and the compound administration group mice to determine the pathological state of blood lipid components, blood sugar, tissues of the weight loss effect of the compound of the present invention.
• Figure 13 is the result of observing the liver tissue of the mouse through the staining method in order to confirm the effect of removing the fat granules accumulated in the tissue by the compound of the present invention.
• White circular structures marked with yellow arrows indicate fat granules.
• Figure 14 is a result of observing the adipose tissue of the mouse via 3 ⁇ 4 £ staining to confirm that the size of the fat cells by the compound of the present invention. Structures marked with blue arrows and black and yellow dots represent fat cells in adipose tissue.
• Tissue immunochemical staining resulted in improved expression of 1X3 protein, a marker of lipase activity.
• Figure 16 is a result of observing the expression of macrophages, inflammatory cells through tissue immunochemical staining in order to observe the alleviating effect of the compound of the present invention in inflammatory symptoms in liver tissue caused by the accumulation of excess fat granules.
• Example 1 Preparation of 2- (3,4-diphenethoxybenzylamino) ethanol Hydrochloride Step 1) Preparation of 3,4-diphenethoxybenzaldehyde and 3-hydroxy-4-phenethoxybenzaldehyde
• Adipose precursor cells 3T3-L1 were purchased from the Bank of Korea Cell Line, 10% neonatal serum (NCS, Invitrogen Corporation, Auckland, New Zeal and) and high glucose DMEM (high glucose Dulbecco's modified Eagle's Medium, Sigma Co., St. Louis, M0, USA).
• adipose cells were condensed for 2 days with 100,000 cells / ml density of 3T3-L1 with 10% fetal bovine serum (FBS) and high glucose DMEM, followed by 0.5 mM IBMX ( Incubate for 2 days with 10% FBS / high glucose DMEM with 3-i sobytyl-l-methyl xanthine), 0.5! IM dexamethasone (dexamethasone) and 5 ug / ral insulin (MDI).
• FBS fetal bovine serum
• IBMX Incubate for 2 days with 10% FBS / high glucose DMEM with 3-i sobytyl-l-methyl xanthine
• IM dexamethasone diexamethasone
• MDI 5 ug / ral insulin
• the red circular structure shows the fat granules accumulated in the cells.
• the size and number of fat granules are decreased by the treatment of the compound according to the present invention.
• the compound according to the present invention was confirmed to have a fat granule removal effect in 3T3-L1 adipocytes.
• Experimental Example 2 Analysis of Lipogranucle Removal Effect of the Compound in Hep G2 Liver Cells by Oi l red 0 Staining
• Hep G2 cells were cultured in DMEM medium containing 10% fetal bovine serum (Hyclone, USA), 100 U / mL penicillin, and 100 mg / mL streptomycin (streptomycin, Hyclone, USA). Incubated for 24 hours after replacing with DMEM medium containing 1 mM oleic acid and 0.5 mM palmitic acid to induce intracellular fat accumulation when the cells were about 50% full, and synthesized according to the present invention. Each compound was replaced with DMEM medium containing 10 uM and further incubated for 24 hours.
• the red circular structure shows the fat granules accumulated in the cells.
• the compound according to the present invention Treatment shows that the size and number of fat granules decreases, thus the compounds according to the invention ⁇ 2 was confirmed to have an effect of removing fat granules in liver cells.
• Experimental Example 3 Analysis of Lipid Granulation Removal Effect of the Compound in 3T3-L1 Adipose Progenitors and Hep G2 Hepatocytes by B0DIPY Staining
• Adipose precursor cells 3T3-L1 were purchased from the Bank of Korea Cell Line, 10% neonatal serum (NCS, Invitrogen Corporation, Auckland, New Zealand) and high glucose DMEM (high glucose Dulbecco's modified Eagle's Medium, Sigma Co., St. Louis, MO, USA) and maintained.
• cells were condensed for 2 days with 100,000 cells / ml density of 3T3-L1 with 10% fetal bovine serum (FBS) and high glucose DMEM, followed by 0.5 mM IBMX ( Incubate for 2 days with 10% FBS / high glucose DMEM with 3-isobytyl-l-me1: hyl xanthine), 0.5!
• FBS fetal bovine serum
• IBMX Incubate for 2 days with 10% FBS / high glucose DMEM with 3-isobytyl-l-me1: hyl xanthine
• IM dexamethasone (dexamethasone) and 5 yg / ml insulin (MDI) and incubate 5 ug / ml insulin
• 10 days FBS / high glucose DMEM was added and cultured for another 6 days, followed by incubation for 10 days to differentiate into fat cells.
• the compounds were treated at a concentration of 10 y M each time the medium was changed.
• Adipose precursor cells 3T3-L1 were purchased from the Bank of Korea Cell Line, 10% neonatal serum (NCS, Invitrogen Corporation, Auckland, New Zeal and) and high glucose Dulbecco's modified Eagle's Medium, Sigma Co., St. Louis , M0, USA) and maintained.
• cells were condensed for 2 days with 100,000 cells / ml density of 3T3-L1 with 10% fetal bovine serum (FBS) and high glucose DMEM, followed by 0.5 mM IBMX ( 3-isobytyl-1-methyl xanthine), 0.5 uM dexamethasone (dexamethasone) and 10% FBS / high glucose DMEM with 5 ug / ml insulin (MDI) incubated for 2 days and 5 ug / ml insulin (I) After further incubation in 10% FBS / high glucose DMEM added only 4 days, 10% ⁇ S / high glucose DMEM alone was added to incubate for another 6 days, incubated for a total of 10 days to differentiate into fat cells.
• FBS fetal bovine serum
• IBMX 3-isobytyl-1-methyl xanthine
• dexamethasone 0.5 uM dexamethasone
• the cells were concentrated and cultured, and then each time the medium was changed, the compounds were treated at a concentration of 10 uM.
• Hep G2 liver cells were inoculated into a 24 well cell culture plate containing cover siip and incubated in DMEM medium containing 1 mM oleic acid and 0.5 mM palmitic acid for 24 hours to induce fat accumulation. Compounds were further incubated for 24 h with 10 uM treatment. Each cell was washed twice with PBS solution, fixed for 15 minutes using 4% paraformaldehyde solution, and blocked for 1 hour in PBS solution containing 2% seedlings. After blocking was completed, the reaction was performed with the primary antibody.
• the primary antibody reaction was performed overnight at 4 ° C using LC3 rabbi t polyclonal ant ibody (1: 300, Sigma-aldr i ch, USA). Cover sl ip after the completion of the primary antibody reaction was washed twice with PBS and the secondary antibody (1: 500, goat ant i rabbi t Alexa f lour 555, Thermo Fisher, US) was reacted at room temperature for 1 hour. After the completion of the secondary antibody reaction, washed twice with PBS, B0DIPY 493/503 was reacted at room temperature for 10 minutes and then mounted on slide glass using a mounting medium. The stained cells were imaged using a confocal microscope and the results are shown in FIG. 4. In FIG.
• the green circular structures show the fat granules accumulated in the cells, which can be confirmed to accumulate according to the differentiation process from 3T3-L1 adipocytes to adipocytes.
• the treatment of the compound of the present invention compared to the placebo-treated cells was found to increase the LC3 stained in red, which is a marker that can track the activity of lipophages.
• comparing the degree of overlap of the granules and LC3 can be seen that the degree of overlap significantly increased when the compound of the present invention is treated compared to the placebo-treated cells.
• the granules produced in 3T3-L1 adipocytes decreased in size and number through increased lipophagy action by the compound of the present invention.
• the green circular structures represent the fat granules accumulated in the cells, and the fat granule accumulation was induced as compared to the control group treated with BSA by treating the olei c aci d and palmi tic acid, which are one of the fatty acids. You can see that. After treatment with Olei c acid and palmi tic, when the compound according to the present invention was treated, it was confirmed that the LC3 stained with red, which is a marker that can track the activity of lipophages, was increased. In addition, superimposed images of fat granules stained with green and LC3 stained with red 2019/190172 1 »(: 1 ⁇ 1 ⁇ 2019/003514
• the image shows that the marker of lipophage, 1X3, is located on the surface of the fat granules. Therefore, it was found that the effect of reducing the number and size of fat granules exhibited by the compound of the present invention was mediated by the lipophage action.
• the lipophages together with the compound according to the present invention in the fat cells differentiated from the fat progenitor cells and accumulated the fat granules After treating the inhibitor of action: 88: 1011 ( ⁇ 11 at a concentration of 5), it was confirmed that the removal effect of the fat granules did not appear, and the results are shown in FIG. In Fig. 5, the red circular structures show fat granules and show that the fat granules reduced by the compounds of the invention do not decrease with treatment of 3 101117 ( ⁇ 11).
• mice 6-week-old wild type 057 6 mice were distributed from Seoul National University Experimental Animal Resource Research Center, and were allocated to the general feed group (1) and the high calorie diet group ( ⁇ !) by wireless assignment. After dividing into), the high fat intake group was further divided into a placebo treatment group and a treatment group of the compound of the present invention, and divided into three groups to simultaneously induce obesity and compound treatment. Through this, it was analyzed whether the compound can effectively prevent the obesity by removing excessive inflow of fat. Specifically, the mice were fed after a week of adaptation in the breeding cage, the water intake was freely, the feed was total 2019/190172 1 »(: 1 ⁇ 1 ⁇ 2019/003514
• the weight of the mice was measured three times a week, and 8 hours after the final administration to verify the effect of removing fat in the body After 24 hours, including fasting time, euthanasia was induced through an oversupply of ⁇ 2, and laparotomy was observed to observe changes in body fat.
• the results of the body fat removal effect according to the administration of the high fat feed and the compound according to the present invention, the weight change, the difference in feed intake and height difference are shown in FIGS. 6, 7, 8, 9, 10, and 11, respectively.
• mice of Experiment 5 were analyzed by immunohistochemical staining to determine whether the size reduction or removal of fat granules occurred in the liver.
• the staining solution used for immunohistochemical staining Purchased from All procedures were performed on 5,111 sections of liver tissue embedded in paraffin at room temperature. Antigen regeneration was performed on formalin-fixed tissue for 15 minutes at 1001: in a water bath prior to immunohistochemical staining. Intracellular endogenous peroxidase activity was blocked with hydrogen peroxidase. Primary antibodies were detected with HRP-binding polymers and developed with DAB. The slides were then counterstained with hematoxylin, dehydrated with multistage alcohol and mounted with mounting medium. The results are shown in FIG.

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• 2019
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