Method for producing a compound having a 5-pentylfurfural (5-PF) structure and use thereof

Abstract

The present invention relates to novel 5-PF-amino acid derivatives (5-PF-AA); methods for producing 5-PF structural compounds or 5-PF-amino acid derivatives; and pharmaceutical compositions comprising 5-PF structural compounds or 5-PF-amino acid derivatives as active ingredients for inhibiting adipocyte differentiation or treating metabolic diseases.

Description

[Technical field]

[0001] The present invention relates to a method for producing a 5-pentylfurfural (5-PF) structure compound or a 5-PF-amino acid derivative (5-PF-AA) and a pharmaceutical composition containing the same as an active ingredient for inhibiting adipocyte differentiation or treating metabolic diseases. [Background technology]

[0002] Metabolic diseases such as diabetes include insulin-dependent diabetes and non-insulin-dependent diabetes, which differ in onset time, nature of the disease, severity and treatment methods. In the case of insulin-dependent diabetes, insulin secretion is absolutely insufficient, and therefore repeated and continuous injection of insulin is necessary. In the case of non-insulin-dependent diabetes, treatment methods such as blood glucose level control through diet and exercise therapy or use of medicines (e.g., oral hypoglycemic agents, insulin preparations, etc.) are available.

[0003] Sulfonylureas, biguanides, α-glucosidase inhibitors, etc. are used as hypoglycemic agents. Sulfonylureas act to promote insulin secretion, but can also cause side effects on the blood, gastrointestinal tract, cardiovascular system, kidneys, and skin, and can also lead to hypoglycemia. Biguanides act by inhibiting glucose production in the liver, but cause side effects on the gastrointestinal tract. Furthermore, α-glucosidase inhibitors cause side effects such as abdominal bloating, diarrhea, and liver tissue damage.

[0004] Not only hypoglycemic agents, but also medicines (e.g., insulin preparations, etc.) often cause side effects such as hypoglycemia, allergies, hyperlipidemia, polyuria, etc. Recently, in order to reduce these side effects, prevention or treatment of diabetes using foods or natural products has been actively researched (Korean Patent Publication No. 2005-0090867).

[0005] On the other hand, compounds extracted from Ceriporia lacerata have antibacterial and antifungal effects and are therefore used to treat vaginitis, nephritis, edema, etc. Compounds extracted from Ceriporia lacerata, such as 5-pentyl-2-furaldehyde, 5-(4-pentyl)-2-furaldehyde, and methyl-3-p-anisoleoxypropionide, are known to be nematicidal components. In particular, 5-pentyl-2-furaldehyde was reported to strongly inhibit melanin production in B16F10 cells without cytotoxicity (IC 50 = 8.4 μg / mL). However, until recently, this substance has not been used as a pharmaceutical composition for the treatment of metabolic diseases.

[0006] Furthermore, with regard to the method for producing 5-PF, in the existing synthesis method, it is known that 5-hydroxymethylfurfural (HMF) trifluoromethanesulfonic acid ester anhydride (triflate) is reacted using the Grignard substitution method at a reaction temperature of −78° C., which is an extremely low temperature environment ( WO2014 / 179156A1 ), but there are no examples of easily synthesizing 5-PF under a milder reaction environment, and no application examples such as the synthesis of various new derivatives through functionalization have been reported. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] KR Patent Publication 2005-0090867 [Non-patent literature]

[0008] [Non-Patent Document 1] J. Microbiol. Biotechnol. (2012), 22(6), 814-817 Summary of the Invention [Problem to be solved by the invention]

[0009] Under these circumstances, the present inventors have confirmed an improved method for producing a compound having a 5-PF structure, and further confirmed that a derivative combining 5-PF with an amine compound (e.g., 5-PF-amino acid (AA)) exerts a therapeutic effect on metabolic diseases through inhibition of adipocyte differentiation without side effects, thereby completing the present invention.

[0010] It is therefore an object of the present invention to provide an improved process for the preparation of compounds having the 5-PF structure.

[0011] Another object of the present invention is to provide novel 5-PF-AA derivatives that exert a therapeutic effect on metabolic diseases through inhibition of adipocyte differentiation.

[0012] A still further object of the present invention is to provide a pharmaceutical composition for inhibiting adipocyte differentiation or treating metabolic diseases comprising as an active ingredient 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma-ceutically acceptable salt thereof. [Means for solving the problem]

[0013] To achieve this object, one embodiment of the present invention provides a method for producing a compound containing a 5-pentylfurfural (5-PF) structure of the following formula 2, comprising: (i) a first step of adding butyllithium (BuLi) dissolved in hexane to a compound of the following formula 1 dissolved in an aprotic organic solvent to obtain a reaction product; and (ii) a second step of adding dimethylformamide (DMF) to the reaction product of the first step. [ka] [ka]

[0014] Another aspect of the present invention provides a 5-PF-amino acid (AA) derivative having the structure of Formula 3 or Formula 4 below; a hydrate or solvate thereof; or a pharma- ceutically acceptable salt thereof. [ka] [ka]

[0015] According to one embodiment of the present invention, in the above formula, R has the same meaning as defined in the Detailed Description of the Invention below.

[0016] Yet another aspect of the present invention provides a pharmaceutical composition for inhibiting adipocyte differentiation or treating a metabolic disease comprising as an active ingredient 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma-ceutically acceptable salt thereof.

[0017] Yet another aspect of the present invention provides a health functional food for improving a metabolic disease, comprising as an active ingredient 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma-ceutically acceptable salt thereof.

[0018] Yet another aspect of the invention provides a method for treating or preventing a metabolic disease in an individual comprising administering 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma-ceutically acceptable salt thereof.

[0019] Yet another aspect of the present invention provides the use of 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma-ceutically acceptable salt thereof for inhibiting adipocyte differentiation or for treating or preventing a metabolic disease.

[0020] Yet another aspect of the present invention provides the use of 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma-ceutically acceptable salt thereof, for the manufacture of a medicament for inhibiting adipocyte differentiation or for the treatment or prevention of a metabolic disease. Effect of the Invention

[0021] The pharmaceutical composition comprising 5-PF or a 5-PF-AA derivative as an active ingredient according to the present invention contains 5-PF derived from a natural product and is therefore non-cytotoxic, and therefore unlikely to cause side effects; furthermore, it has excellent inhibitory effects on adipocyte differentiation and inflammation, and therefore can be used extremely effectively in the improvement, prevention or treatment of metabolic diseases such as obesity, adult diseases, diabetes, obese diabetes, vascular diabetic complications and non-alcoholic steatohepatitis (NASH).

[0022] Furthermore, the present invention exhibits functionality equal to or greater than that of existing drug doses at a lower dose compared to existing drug doses, and can be effectively used for improving, preventing or treating metabolic diseases. [Brief description of the drawings]

[0023] [Figure 1] The results of a comparative LC / MS analysis between (a) 5-PF obtained from a conventional Ceriporia lacera extract and (b) synthetically obtained 5-PF are shown.

[0024] [Diagram 2] (a) The results of a comparative analysis by 1H-NMR between 5-PF obtained from a conventional Ceriporia lacera extract and (b) 5-PF obtained synthetically, and (c) the results of 13C-NMR of the extracted 5-PF are shown.

[0025] [Diagram 3] The results of GC / MS analysis of synthetic 5-PF are shown below.

[0026] [Figure 4] The figures show the results of measuring cell activity when (a) 3T3-L1 adipocytes, (b) C2C12 muscle cells, (c) HUVEC human vascular epithelial cells, and (d) HACAT human skin cells were treated with 5-PF-glutamine (Q) (5-PF-Q), a derivative of 5-PF-AA.

[0027] [Diagram 5] 1 shows results confirming the inhibitory effect of 5-PF-Q on adipocyte differentiation.

[0028] [Figure 6] 1 shows results confirming the effect of 5-PF-Q on adiponectin expression.

[0029] [Figure 7] 1 shows results confirming the effect of 5-PF-Q on AMPK expression.

[0030] [Figure 8] 1 shows results confirming the effect of 5-PF-Q on inflammatory protein and cytokine expression.

[0031] [Figure 9] 1 shows results confirming the inhibitory effect of 5-PF-Q on fatty liver or steatosis via inhibition of adipocyte differentiation.

[0032] [Figure 10] 1 shows results confirming the effect of 5-PF-Q on glycated hemoglobin and insulin.

[0033] [Figure 11] 4 shows results confirming the effect of 5-PF-Q on TC, TG, ALP, AST and ALT.

[0034] [Figure 12-13] 1 shows results identifying changes in body weight and food intake over a 6-week period upon administration of 5-PF-Q to Ob / Ob mice groups A and B, respectively.

[0035] [Figure 14-15] 1 shows the results of confirming a decrease in blood glucose level when an OGTT test was performed after administration of 5-PF-Q to Ob / Ob mice of groups A and B, respectively.

[0036] [Figure 16]4 shows results confirming changes in liver indices (ALT, AST, ALP, LDH), kidney indices (CREA, BUN) and lipid indices (cholesterol, TG, LDL, HDL, LDH) in Ob / Ob mice groups treated with 5-PF-Q.

[0037] [Figure 17] 1 shows results ascertaining whether insulin resistance was improved in the group of Ob / Ob mice treated with 5-PF-Q. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Hereinafter, various experimental examples will be described in detail so that those skilled in the art can easily practice the present invention. However, the experimental examples may be embodied in various different forms and are not limited to the experimental examples described herein.

[0039] In this specification, when a moiety is described as "comprising" a certain component, this is not intended to exclude other components; rather, other components may be included unless expressly stated otherwise.

[0040] In this specification, unless otherwise indicated, the singular terms "a," "an," and "the" are to be construed as including the singular or the plural as required by context.

[0041] Furthermore, all numbers and expressions expressing quantities of ingredients, reaction conditions, and the like described herein should be construed as being modified in all instances by the term "about," unless otherwise indicated.

[0042] The terms first, second, etc. are used herein to describe various components, and the components are not limited by these terms. These terms are used only to distinguish multiple components from one another.

[0043] One embodiment of the present invention provides a method for producing a compound comprising a 5-pentylfurfural (5-PF) structure of formula 2, comprising: (i) a first step of adding butyllithium (BuLi) dissolved in hexane to a compound of formula 1 dissolved in an aprotic organic solvent to obtain a reaction product; and (ii) a second step of adding dimethylformamide (DMF) to the reaction product of the first step. [ka] [ka]

[0044] The compounds containing the above 5-PF structure include 5-PF-amino acid (AA) derivatives, hydrates or solvates thereof, or pharma- ceutically acceptable salts thereof.

[0045] In one embodiment of the present invention, the compound comprising the 5-PF structure may be a compound of formula 2 below. [ka]

[0046] In certain embodiments of the present invention, the aprotic organic solvent can be tetrahydrofuran (THF).

[0047] The molar concentration ratio of BuLi added in the first step to DMF added in the second step can be 1:1 to 1:2. Specifically, the molar concentration ratio of BuLi added in the first step to DMF added in the second step can be 1:1 to 1:1.8, 1:1 to 1:1.6, 1:1 to 1:1.5, or 1:1 to 1:1.4, but is not limited to these ratios.

[0048] In one embodiment of the present invention, the first and / or second step may be carried out at a temperature of -10° C. to 10° C. Specifically, the first step may be carried out at a temperature of -8° C. to 8° C., -7° C. to 7° C., -6° C. to 6° C., -5° C. to 5° C., -4° C. to 4° C., -3° C. to 3° C., -2° C. to 2° C., or -1° C. to 1° C., but the temperature is not limited to these values.

[0049] In certain embodiments of the invention, the method may further comprise an agitation step after the first step; and / or an agitation step after the second step.

[0050] In certain embodiments of the invention, the method may further comprise an extraction step with methyl tertiary butyl ether (MTBE) and a washing step with salt water after the second step.

[0051] Another aspect of the present invention is a 5-PF-amino acid (AA) derivative having the structure of Formula 3 below; a hydrate or solvate thereof; or a pharma- ceutically acceptable salt thereof: [ka] In the above formula 3, R is [ka] or R may be attached to the nitrogen atom adjacent to the carbon atom to which R is attached to form a pyrrolidine ring.

[0052] Yet another aspect of the present invention is a 5-PF-amino acid (AA) derivative having the structure of Formula 4 below; a hydrate or solvate thereof; or a pharma- ceutically acceptable salt thereof: [ka] In the above formula 4, R is [ka] or R may be attached to the nitrogen atom adjacent to the carbon atom to which R is attached to form a pyrrolidine ring.

[0053] In one embodiment of the present invention, the structure of formula 4, where R is attached to the nitrogen atom adjacent to the carbon atom to which it is attached to form a pyrrolidine ring, is the same as formula 5 shown below. [ka]

[0054] In one embodiment of the present invention, in the compound of formula 3 above, R is [ka] It can be, but is not limited to,

[0055] In one embodiment of the present invention, in the compound of formula 4 above, R is [ka] It can be, but is not limited to,

[0056] 5-PF or 5-PF-amino acid (AA) derivatives can inhibit the activity of at least one of α-amylase, α-glucosidase and lipase. α-amylase or α-glucosidase are carbohydrate decomposition enzymes, and lipase is a lipolysis enzyme. Medicines that can inhibit the activity of these enzymes have been developed for the management of blood glucose levels and improvement of lipid metabolism in patients with metabolic diseases such as diabetes or obesity (e.g., orlistat); however, due to various side effects and difficulties in synthesis, there is a demand to develop natural products with similar activity. The pharmaceutical composition of the present invention containing 5-PF or 5-PF-amino acid (AA) derivatives as active ingredients can be useful for use in the prevention, treatment and improvement of diabetes by directly inhibiting lipolysis.

[0057] 5-PF or 5-PF-amino acid (AA) derivatives may exert their effect by inhibiting the expression of at least one of CCAAT enhancer binding protein alpha (C / EBPα), CCAAT enhancer binding protein beta (C / EBPβ), peroxisome proliferator-activated receptor gamma (PPARγ), adiponectin and fatty acid binding protein 4 (FABP4), thereby increasing the expression of the glucose transporter type 4 (GLUT4) gene.

[0058] In particular, adipocyte differentiation is a complex process involving changes in morphology, hormone sensitivity, and gene expression. Some transcription factors have been shown to have important roles in adipocyte differentiation. The main transcription factors involved in adipocyte formation include C / EBPα and PPARγ. In particular, PPARγ increases the expression of lipogenic marker genes such as adiponectin and FABP4. In addition, GLUT4 is a glucose transporter gene, and when stimulated by insulin, GLUT4 in cells translocates to the plasma membrane to promote glucose transport. Thus, GLUT4 plays a role in lowering blood glucose levels by promoting the influx of blood glucose into cells. That is, the antidiabetic effect increases with the increase in the activity and expression of GLUT4, which is involved in glucose transport.

[0059] Yet another aspect of the present invention provides a pharmaceutical composition for inhibiting adipocyte differentiation or treating a metabolic disease comprising as an active ingredient 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma-ceutically acceptable salt thereof.

[0060] The pharma- ceutically acceptable salt of 5-PF or its 5-PF-amino acid (AA) derivative has low toxicity to the human body and does not inhibit the biological activity and physicochemical properties of 5-PF or 5-PF-amino acid (AA). In some embodiments, the pharma- ceutically acceptable salt includes, but is not limited to, pharma- ceutical useful free acid and acid addition salt of 5-PF or 5-PF-AA, alkali metal salt (e.g., sodium salt, etc.) and alkaline earth metal salt (e.g., calcium salt, etc.).

[0061] In some embodiments of the present invention, the acid addition salts may be salts with inorganic or organic acids. As inorganic acids, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, bromic acid, etc. may be used. Furthermore, as organic acids, acetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, fumaric acid, maleic acid, malonic acid, phthalic acid, succinic acid, lactic acid, citric acid, gluconic acid, tartaric acid, salicylic acid, malic acid, oxalic acid, benzoic acid, embonic acid, aspartic acid, glutamic acid, etc. may be used. These salts may be prepared by conventional methods. For example, these salts may be prepared by dissolving 5-PF or 5-PF-AA in a water-miscible solvent (e.g., methanol, ethanol, acetone, and 1,4-dioxane), adding the free acid or free base, and then crystallizing the resultant.

[0062] In certain embodiments of the invention, hydrates or solvates of 5-PF or 5-PF-amino acids (AA) can be prepared by known methods, preferably the hydrates or solvates are non-toxic and water soluble, and preferably the compounds are hydrates or solvates having from 1 to 5 molecules of water or alcohol solvent (especially ethanol, etc.) associated therewith.

[0063] In one embodiment of the present invention, the pharmaceutical composition for inhibiting adipocyte differentiation or treating metabolic disease may further comprise, in addition to 5-PF or 5-PF-amino acids (AA), a substance known to have an effect of improving metabolic disease (such as anti-obesity / anti-diabetes effect). The known substance may be, but is not limited to, an inhibitor of C / EBPα, C / EBPβ, PPARγ, adiponectin or FABP4; or a promoter of GLUT4.

[0064] In certain embodiments of the present invention, the pharmaceutical composition for inhibiting adipocyte differentiation or treating a metabolic disease may further comprise one or more pharma- ceutically acceptable additives selected from the group consisting of additives, lubricants, humectants, sweeteners, flavoring agents and preservatives.

[0065] In one embodiment of the present invention, the pharmaceutical composition for inhibiting adipocyte differentiation or treating metabolic disease can be formulated according to the conventional methods to be used. In particular, the pharmaceutical composition can be formulated by applying methods known in the art to provide a rapid, sustained or delayed release of the active ingredient after administration to a mammal.

[0066] In one embodiment of the present invention, the method of administering pharmaceutical compositions for inhibiting adipocyte differentiation or treating metabolic disease can be easily selected depending on the formulation, and can be administered orally or parenterally.For example, pharmaceutical compositions can be used via intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes, but the administration route is not limited thereto.Solid formulations for oral administration can include, but are not limited to, tablets, pills, soft or hard capsules, pills, powders and granules.On the other hand, formulation types for parenteral administration can include, but are not limited to, creams, lotions, ointments, pastes, liquids, patches and injections.

[0067] In certain embodiments of the present invention, the dosage of the pharmaceutical composition for inhibiting adipocyte differentiation or treating metabolic disease may vary depending on the patient's age, sex, weight, disease severity and route of administration, but generally, the dosage may be 0.5 mg / kg to 100 mg / kg, 0.5 mg / kg to 50 mg / kg, 0.5 mg / kg to 10 mg / kg or 0.5 mg / kg to 5 mg / kg administered in divided doses once to three times a day. However, the dosage does not limit the scope of the present invention in any way.

[0068] In some embodiments of the present invention, the dose of 5-PF or 5-PF-amino acid (AA) derivatives, hydrates or solvates thereof, or pharma- ceutically acceptable salts thereof may be 10 μg / mL or more, 20 μg / mL or more, 30 μg / mL or more, 40 μg / mL or more, or 50 μg / mL or more. Specifically, the dose may be, but is not limited to, 10 μg / mL to 140 μg / mL, 20 μg / mL to 130 μg / mL, 30 μg / mL to 120 μg / mL, 40 μg / mL to 110 μg / mL, or 50 μg / mL to 100 μg / mL.

[0069] In one embodiment of the present invention, the metabolic disease may be any selected from the group consisting of obesity, adult diseases, diabetes, obese diabetes, vascular diabetic complications, non-alcoholic steatohepatitis (NASH), dyslipidemia, arteriosclerosis, hyperlipidemia, hypercholesterolemia and cardiovascular disease.

[0070] Yet another aspect of the present invention provides a health functional food for improving a metabolic disease, comprising as an active ingredient 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma-ceutically acceptable salt thereof.

[0071] In one embodiment of the present invention, the health functional food may contain 5-PF or 5-PF-amino acids (AA); hydrates or solvates thereof; and / or pharma- ceutically acceptable salts thereof in an amount of 0.001 wt% to 100 wt%, 0.001 wt% to 50 wt%, 0.1 wt% to 100 wt%, 0.1 wt% to 50 wt%, or 0.1 wt% to 40 wt%, based on the total weight of the health functional food.

[0072] In one embodiment of the present invention, the health functional food may be in the form of powder, granules, tablets, capsules or beverages, and may further contain other natural or synthetic substances for health / function and additives for product formulation.

[0073] In one embodiment of the present invention, when the health functional food is a beverage, 5-PF or 5-PF-amino acids (AA) and / or their pharma- ceutically acceptable salts may be included in an amount of 0.001g-2g, 0.001g-1g, 0.01g-2g, 0.01g-1g, or 0.01g-0.1g based on 100mL of the health functional food. Furthermore, there is no particular limit to the liquid component, and various flavoring agents, natural carbohydrates, etc. may be included as additional components in the usual amounts as in conventional beverages.

[0074] Examples of the natural carbohydrates include monosaccharides, disaccharides such as glucose and fructose, polysaccharides such as maltose and sucrose, oligosaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the flavoring agent, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)), synthetic flavoring agents (saccharin, aspartame, etc.) may be used.

[0075] In addition, the health functional food for improving obesity / diabetes of the present invention may contain various nutritional supplements, vitamins, minerals (electrolytes), flavors including synthetic flavors and natural flavors, colorants and thickeners (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonizing agents for use in carbonated drinks, etc. In addition, the health functional food may contain natural fruit juice, fruit pulp for the production of fruit juice drinks and plant drinks. These ingredients may be used independently or in combination.

[0076] Health functional foods for improving metabolic diseases, comprising as an active ingredient 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma- ceutically acceptable salt thereof, may include, for example, various foods, beverages, gums, multivitamins, dietary supplements, and the like.

[0077] Yet another aspect of the invention provides a method for treating or preventing a metabolic disease in an individual comprising administering 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma-ceutically acceptable salt thereof.

[0078] Yet another aspect of the present invention may provide the use of 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma- ceutically acceptable salt thereof for inhibiting adipocyte differentiation or for treating or preventing a metabolic disease.

[0079] Yet another aspect of the present invention provides the use of 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma-ceutically acceptable salt thereof, for the manufacture of a medicament for inhibiting adipocyte differentiation or for the treatment or prevention of a metabolic disease.

[0080] Hereinafter, the present invention will be described in further detail in the following examples, which are intended to illustrate the present invention, but the scope of the present invention is not limited to these examples. EXAMPLES

[0081] Preparation Example 1. Organic synthesis method for 5-pentylfurfural (5-PF) or 5-PF-AA BuLi (1.1 equiv.) dissolved in hexane was added to 50 g of the following compound 1 dissolved in 500 mL of THF at 0° C. After stirring for 30 min, DMF (1.5 equiv.) was added at the same temperature and the reaction mixture was stirred overnight. After stirring overnight, the resultant was poured into 1M HCl in ice water, extracted with MTBE (200 mL×3), washed with brine (200 mL×3), and purified by NaCl. 2 SO 4 The concentrated crude product was distilled under reduced pressure (1 mm, 90° C.) to obtain pure compound 2. The synthesis amount was 31 g, and the yield indicated by NMR analysis was 95-99%. [ka]

[0082] In the existing synthesis method, HMF triflate was synthesized using the Grignard substitution method at an extremely low reaction temperature of -78°C, whereas in the present invention, the synthesis was carried out in a relatively mild reaction environment of 0°C.

[0083] Tertiary amides can be readily used to form ketones and aldehydes since they react only once with Grignard and organolithium reagents, and in this preparation DMF was used to introduce the formyl group.

[0084] Furthermore, novel derivatives can be prepared in a one-pot reaction of 5-PF with an amino acid (AA) buffer (e.g., glutamine) containing an amine group that can be easily linked to the aldehyde group of compound 2. The aldehyde group of compound 2 and the amine group of the amino acid sodium salt form a hydrogen bond between a ketone (acceptor) and an amide (donor), whereby the novel derivatives Compound 3 or 5-PF-AA group can be obtained by forming the next derivative Compound 4 via a furfurylamine covalent bond, and more than 20 novel derivatives can be obtained by combination of 20 types of amino acids (Green Chem., 2015, 17, 4151-4156).

[0085] Specifically, as shown in the following compound 4, 5-PF dissolved in ethanol was mixed with a buffer solution (pH 8.5) containing 10 mM L-glutamine (Q), which is one of the AA group, in a molar ratio of 1:1, and then reacted at 37°C for 1 hour to obtain 5-PF-Q. [ka]

[0086] Experimental example 1. 5-PF analysis method 1-1: Comparative analysis of 5-PF compounds obtained from Ceriporia extracts and synthetic 5-PF compounds via LC / MS A sample (a) of 5-PF compound obtained by ethyl acetate extraction of mycelium culture of Ceriporia lacerata disclosed in Korean Patent 10-1031605 and a sample (b) of 5-PF compound obtained via organic synthesis in Preparation Example 1 were comparatively analyzed (Figure 1).

[0087] Specifically, LC / MS was performed to compare the similarity between the extracted sample (a) and the synthetic sample (b). The bioactive compounds present in the Ceriporia mycelium culture were separated using a UHPLC analytical system. The analysis was performed using a Thermo Scientific Ultimate 3000 UHPLC system coupled to a tandem mass spectrometer (LCQ Fleet, Thermo Fisher Scientific, MA, USA) equipped with a SCINChrom C18G 100A column (100 mm × 2.1 mm, 1.8 μm, Scinco, Australia). The samples were eluted using a gradient system consisting of solvent A (0.1% formic acid in water, v / v) and solvent B (0.1% formic acid in acetonitrile, v / v). The gradient elution at a flow rate of 0.2 mL / min started at 5% B, increased to 100% B in 20 min, and changed to 5% B in 35 min, with a total run time of 40 min, including a column conditioning time of 5 min. The injection volume was 2 μL and the column temperature was set at 40 °C. Electrospray ionization of the analytes was used in positive mode. Mass spectra were collected in full scan mode over a mass range of 100 Da to 1,000 Da. MS / MS spectra were acquired at a collision energy of 25 ev. Split peak analysis showed that 5-PF was measured at 16.18 min and 16.07 min (slight differences in tR due to instrument conditions) and comparison by UV-vis spectroscopy mass spectrometry integrated into a Thermo Scientific Ultimate 3000 UHPLC system confirmed that the compounds in samples (a) and (b) were identical.

[0088] 1-2: 1 Comparative analysis of 5-PF compounds obtained from Ceriporia extracts and synthetic 5-PF compounds via H-NMR Ceriporia extract and the compound synthesized in Preparation 1 were compared by NMR spectroscopy. NMR data were obtained using a 700 MHz Bruker Avance III HD NMR spectrometer and a room temperature TBO probe. To correct for static magnetic field inhomogeneity, 3D and 1D shimming experiments were performed prior to NMR data acquisition, and data was obtained using a 1D nuclear Overhauser effect spectroscopy experiment with water suppression using 128k points and 128 scans.

[0089] To compare the similarity between the extracted sample (a) and the synthetic sample (b), 1 H-NMR confirmed that the compounds in samples (a) and (b) were identical (Figure 2). Furthermore, the 5-PF structure was confirmed to be identical to that of the synthetic sample. 13 This was reconfirmed by the C-NMR results (c).

[0090] 1-3: GC / MS analysis of synthetic 5-PF compounds The compound synthesized in Preparation 1 above was analyzed by GC / MS. The 5-PF sample was injected with a GC-MS injector equipped with a SPME manual holder and analyzed with HS-SPME-GC-MS (Agilent 7000C GC-MS system (Agilent Technologies, Wilmington, DE, USA)). The capillary column used for GC-MS analysis was a DB-5MS column (30 m x 0.25 mm ID x 0.25 μm, Agilent Technologies, Wilmington, DE, USA). The oven temperature was first set at 60 °C for 5 min, and then increased to 250 °C at 3 °C / min. The sample injector and detector temperatures were set at 250 °C. The sample was injected by splitless method. Helium (He, 99.999%) was used as the carrier gas, and the flow rate was 1.0 mL / min. The ionization energy was 70ev, and the produced ions were analyzed with a full scan from m / z 30 to m / z 500. The analytes adsorbed on the SPME fiber were desorbed from the GC injector and injected into a GC capillary column for analysis. The results confirmed that the compounds were 5-PF molecules (Figure 3).

[0091] Example 1. Cytotoxicity / activity measurements (a) 3T3-L1 adipocytes (obtained from ATCC), (b) C2C12 muscle cells (obtained from ATCC), (c) HUVEC human vascular epithelial cells (obtained from ATCC), and (d) HACAT human skin cells (obtained from ATCC) were cultured at 1 × 10 4 The cells were cultured in high glucose medium (10% FBS, 1% penicillin / streptomycin) and 5-PF-Q samples were added at various concentrations (1-100 μg / mL) and cultured for 24 hours. The OD values ​​at 450 nm were then quantified using CCK8 solution and plotted (Figure 4). Sample-free culture medium (NC) and culture medium containing 1% ethanol (EtOH) were used as negative controls. As shown in Figure 4, it was confirmed that the treatment of various cell types with 5-PF-Q samples did not affect cell activity and cytotoxicity.

[0092] Example 2. Evaluation of the effects of induction and inhibition of adipocyte differentiation In experiments to confirm the inhibition of adipogenesis, 3T3-L1 (adipocyte precursor cells) were differentiated into adipocytes in DMEM high glucose medium (containing 10% calf serum, 1% penicillin / streptomycin) while simultaneously treating the samples. 3T3-L1 cells were cultured in 24-well plates and differentiation was performed according to the following flowchart. [Table 1]

[0093] To confirm adipocyte differentiation, the 24-well plate was washed twice with PBS, and then the cells were fixed with 4% paraformaldehyde for 15 min. The cells were washed twice with PBS, 60% isopropanol was added to it, and the cells were left at room temperature for 5 min. The 24-well plate was incubated at 4°C for 1 h at 25°C for 2 h at 25°C. 2After removal from the incubator, the cell distribution was checked by light microscopy (magnification: 40x) to ensure that 100% confluency had been reached, and then the medium was replaced with 1 mL / well of fresh culture medium. After 48 hours, the samples to be treated were diluted to different concentrations and replaced with 1 mL / well of differentiation-inducing medium (made by mixing DMEM high glucose medium with 10% FBS, 1% penicillin / streptomycin and MDI solution (0.5 mM IBMX, 1 μM dexamethasone, 10 μg / mL insulin)). After 72 hours, the samples to be treated were diluted to different concentrations with differentiation-inducing medium and replaced with 1 mL / well. Until the 9th day of differentiation, the samples to be treated in differentiation-inducing medium were diluted to different concentrations and replaced with 1 ml / well every 48 hours. 5-PF-Q was diluted at various concentrations and treated in the medium used from the start of differentiation to the completion of differentiation (day 9). The concentrations used were 10 μg / mL, 50 μg / mL, and 100 μg / mL, and 0.5 mM metformin (90 μg / mL) was used as a positive control.

[0094] For lipid staining, Oil Red O solution was added thereto, and the cells were stained for 15 minutes at room temperature, washed twice with PBS, and after microscopic observation, eluted with 100% isopropanol, and the absorbance was measured at 490 nm.

[0095] As a result of carrying out an experiment in which cells were treated with various concentrations of 5-PF-Q while inducing lipogenesis for 9 days, lipogenesis was inhibited in a concentration-dependent manner in the group treated with the 5-PF-Q sample (Figure 5). Compared to the metformin 90 μg / mL treatment group (i.e., the positive control group), the 5-PF-Q treatment group showed a tendency similar to metformin at the low concentration portion (10 μg / mL), while showing a higher lipogenesis inhibition than metformin at the high concentration portion (50 μg / mL and 100 μg / mL).

[0096] Example 3. Confirmation of effect on adiponectin expression The effect of 5-PF-Q on adiponectin expression, a marker related to adipocyte differentiation, was confirmed via Western blotting (Figure 6). Compared with the positive control group (i.e., treated with 90 μg / mL metformin), the expression rate was 3.5 times higher than that of metformin in the low concentration portion (10 μg / mL), and the expression of adiponectin was obviously inhibited in the high concentration portion (50 μg / mL and 100 μg / mL). It is speculated that peroxisome proliferator-activated receptor gamma (PPAR gamma), a major regulator of adiponectin expression, was dose-dependently regulated without toxicity or potential side effects in the high dose portion (50 μg / mL-100 μg / mL), and that 5-PF-Q administration could inhibit adipocyte differentiation even in the low dose portion (1 μg / mL-40 μg / mL), while simultaneously regulating metabolism-related pharmacological functionality.

[0097] Example 4. Confirmation of effect on AMPK expression To confirm the effect of 5-PF-Q on AMP-activated protein kinase (AMPK) expression, differentiated muscle cells C2C12 cells were treated with 10 μg / mL, 50 μg / mL and 100 μg / mL 5-PF-Q for 24 hours, respectively (10% gel, 20 μg PTN, 1° ab pAMPK 1:3,000, tAMPK 1:1,000 / 2° ab 1:4,000), as shown in Figure 7. AMPK expression was clearly observed in the positive control group (i.e., treated with 90 μg / mL metformin), confirming that 5-PF-Q also inhibited the activation of AMPK expression in a concentration-dependent manner (Figure 7).

[0098] Example 5. Confirmation of effects on expression of inflammation-inducing proteins and cytokines To determine the effect of 5-PF-Q on metabolic diseases such as diabetic complications that cause vascular inflammation, the expression rate of inflammation-related cytokines was measured (Figure 8). Vascular cells, HUVEC cells, were cultured, pretreated for 30 minutes, and then treated with TNF-α (10 ng / mL) for 4 hours. As a result of performing real-time PCR after RNA prep, the expression of increased adhesion molecules was decreased in a concentration-dependent manner in the TNF-α treatment group, and the expression level of intercellular adhesion molecule (ICAM) was slightly lower when treated with 5-PF-Q (50 μg / mL) compared to dexamethasone, the expression level of E-selectin was slightly lower when treated with 5-PF-Q (100 μg / mL), and in particular, the expression level of vascular cell adhesion molecule (VCAM) was lower when treated with 5-PF-Q (10 μg / mL) compared to treatment with dexamethasone.

[0099] To observe the ability of 5-PF-Q to inhibit NO, RAW 264.7 cells were cultured at 5 × 10 4 Cells / well were cultured and then treated with various concentrations of 5-PF-Q samples and LPS for 24 hours. The supernatants were obtained and Griess reagent assay was performed, and the OD values ​​were measured at 520 nm. The levels of NO increased by LPS were decreased in a concentration-dependent manner in the 5-PF-Q treated groups. It was confirmed that the expression level was slightly decreased at the low concentration portion (1-10 μg / mL), while the expression level was rapidly decreased at the high concentration portion (50 μg / mL and 100 μg / mL).

[0100] IL-6 and monocyte chemotactic protein-1 (MCP-1) expression levels were decreased in a 5-PF-Q concentration-dependent manner, with expression levels similar to or lower than those treated with 50 μg / mL and 100 μg / mL dexamethasone, whereas IL-1α and IL-8 expression levels were increased in a 5-PF-Q concentration-dependent manner, with expression levels similar to or lower than those treated with 10 μg / mL and 50 μg / mL dexamethasone.

[0101] Example 6. Evaluation of effects on inhibition of adipocyte differentiation in vivo and evaluation of biochemical effects As shown in Table 1 below, histopathological results according to lipogenesis were obtained through in vivo experiments in which 5-PF-Q was administered to a group of diabetic mice. [Table 2]

[0102] Upon sacrificing the mice, the percentage of hepatocytes with fat deposits was scored as follows in tissue examination after biopsy: less than 5%=0; 5%-33%=1; 33%-66%=2; and 66% or more=3. Thus, the 5-PF-Q-treated group showed significantly lower fat deposits compared with the control group and the Met-treated group (FIG. 9). Through these results, it was confirmed that 5-PF-Q may have the effect of preventing or treating steatosis or steatohepatitis in the liver through inhibition of adipocyte differentiation.

[0103] Furthermore, the biochemical blood results confirmed that 5-PF-Q tended to reduce the levels of glycated hemoglobin and insulin (Figure 10) and TG levels, but did not affect biochemical values ​​such as TC, ALP, AST, and ALT (Figure 11). These results indicate that 5-PF-Q is not toxic to the liver.

[0104] Example 7. Confirmation of antidiabetic effect in Ob / Ob mice To confirm the antidiabetic effects of 5-PF-Q, changes in body weight, food intake, reduction in blood glucose levels, toxicity, and insulin resistance following administration of 5-PF-Q were measured.

[0105] 7-1: Mouse body weight and food intake measurement Mice were divided into group A (body weight ≥ 35g) and group B (body weight ≤ 35g) based on body weight. According to the flow chart below, each group was assigned to receive metformin (200mg / kg) (positive control group) and 5-PF-Q (15mg / kg) (experimental group) orally for 3 weeks. After the 3-week treatment period, blood glucose levels were measured by performing an oral glucose tolerance test (OGTT), and the mice were sacrificed 3 weeks later. OGTT was also performed at the same time in the Ob / Ob vehicle group (negative control group) and non-diabetic C57BL / 6 mice not receiving any compound, and sacrificed. [Table 3]

[0106] As a result, when body weight and food intake were checked once a week for 6 weeks in groups A and B, no significant differences in body weight or food intake were found between the groups (Figures 12 and 13).

[0107] 7-2: Measurement of blood glucose levels using the mouse OGTT test Review of the results of OGTT performed in groups A and B classified in 7-1 above revealed that in group A, blood glucose levels declined in a mild curve in the Ob / Ob vehicle group (negative control group), but the blood glucose decline pattern was similar in the metformin-treated group (positive control group) and the 5-PF-Q-treated group (experimental group) (Figure 14).

[0108] Similarly, in group B, the Ob / Ob vehicle group (negative control group) showed a gentle curve-like decrease in blood glucose levels, while the 5-PF-Q-treated group (experimental group) tended to further decrease blood glucose levels compared to the negative control group (Figure 15).

[0109] 7-3: Confirmation of toxicity in mouse liver As a result of blood tests performed on the mice experimented in 7-1 above, it was confirmed that there were no significant differences in ALT, AST, ALP and LDH (i.e., liver indexes), CREA and BUN (i.e., kidney indexes) and cholesterol, TG, LDL, HDL and LDH (i.e., lipid indexes) in the 5-PF-Q administration group (experimental group). These results confirm that 5-PF-Q is not toxic to the liver (Figure 16).

[0110] 7-4: Confirmation of insulin resistance in mice Compared with the Ob / Ob vehicle group (negative control group), both the 5-PF-Q-treated group (experimental group) and the metformin-treated group (positive control group) showed a tendency toward improved insulin resistance, and in particular, the degree of improvement in insulin resistance was greater when 5-PF-Q was administered compared with metformin. Meanwhile, no significant difference in C-peptide was observed in all treatment groups. These results confirm that 5-PF-Q helps improve insulin resistance (Figure 17).

Claims

1. A method for producing a compound containing a 5-pentylfurfural (5-PF) structure of the following formula 2: (i) a first step of adding a compound of the following formula 1 dissolved in an aprotic organic solvent to butyl lithium (BuLi) dissolved in hexane to obtain a reaction product; and (ii) a second step of adding dimethylformamide (DMF) to the reaction product of the first step; A method comprising: 【Chemistry 1】 【Chemistry 2】

2. The method of claim 1, wherein the compound containing the 5-PF structure is a compound of the following formula 2: 【Chemistry 3】

3. 2. The process of claim 1, wherein the aprotic organic solvent is tetrahydrofuran (THF).

4. 2. The method of claim 1, wherein the molar concentration ratio of BuLi added in the first step to DMF added in the second step is 1:1 to 1:

2.

5. 2. The process of claim 1, wherein the first and / or second steps are carried out at a temperature of from -10°C to 10°C.

6. 10. The method of claim 1, wherein the method further comprises stirring after the first step; and / or stirring after the second step.

7. 10. The method of claim 1, wherein the method further comprises extracting with methyl tertiary butyl ether (MTBE) and washing with brine after the second step.

8. A 5-PF-amino acid (AA) derivative having the structure of Formula 3 below; a hydrate or solvate thereof; or a pharma- ceutically acceptable salt thereof: 【Chemistry 4】 [In the above formula 3, R is 【Chemistry 5】 or R is bonded to the nitrogen atom adjacent to the carbon atom to which R is attached to form a pyrrolidine ring.

9. A 5-PF-amino acid (AA) derivative having the structure of Formula 4 below; a hydrate or solvate thereof; or a pharma- ceutically acceptable salt thereof: 【Chemistry 6】 [In the above formula 4, R is 【Chemistry 7】 or R is bonded to the nitrogen atom adjacent to the carbon atom to which R is attached to form a pyrrolidine ring.

10. In the compound of formula 3 above, R 【Chemistry 8】 9. The 5-PF-amino acid (AA) derivative of claim 8, which is:

11. In the compound of formula 4 above, R 【Chemistry 9】 10. The 5-PF-amino acid (AA) derivative of claim 9, which is:

12. A pharmaceutical composition for inhibiting adipocyte differentiation or treating a metabolic disease, comprising as an active ingredient 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma- ceutically acceptable salt thereof.

13. The pharmaceutical composition of claim 12, wherein the metabolic disease is any one selected from the group consisting of obesity, adult diseases, diabetes, obese diabetes, vascular diabetic complications, nonalcoholic steatohepatitis (NASH), dyslipidemia, arteriosclerosis, hyperlipidemia, hypercholesterolemia and cardiovascular disease.

14. A health food with health claims for improving a metabolic disease, comprising as an active ingredient 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma- ceutical acceptable salt thereof.

15. A method of treating or preventing a metabolic disease in an individual comprising administering 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma-ceutically acceptable salt thereof.

16. The method of claim 15, wherein the metabolic disease is any one selected from the group consisting of obesity, adult diseases, diabetes, obese diabetes, vascular diabetic complications, nonalcoholic steatohepatitis (NASH), dyslipidemia, arteriosclerosis, hyperlipidemia, hypercholesterolemia and cardiovascular disease.

17. Use of 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma- ceutically acceptable salt thereof for inhibiting adipocyte differentiation or for treating or preventing a metabolic disease.

18. The use of claim 17, wherein the metabolic disease is any one selected from the group consisting of obesity, adult diseases, diabetes, obese diabetes, vascular diabetic complications, nonalcoholic steatohepatitis (NASH), dyslipidemia, arteriosclerosis, hyperlipidemia, hypercholesterolemia and cardiovascular disease.

19. Use of 5-PF or a 5-PF-amino acid (AA) derivative; a hydrate or solvate thereof; or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for inhibiting adipocyte differentiation or for treating or preventing a metabolic disease.

20. The use of claim 19, wherein the metabolic disease is any one selected from the group consisting of obesity, adult diseases, diabetes, obese diabetes, vascular diabetic complications, nonalcoholic steatohepatitis (NASH), dyslipidemia, arteriosclerosis, hyperlipidemia, hypercholesterolemia and cardiovascular disease.

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