KR20120036026A - Pharmaceutical composition for preventing or treating diabetes or obesity containing novel compound lobarin - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing a novel compound, lobarin, is provided to suppress tyrosine phosphatase 1B and to ensure antidiabetic effect. CONSTITUTION: A novel compound, lobarin, is denoted by chemical formula 1. A method for preparing the compound of chemical formula 1 comprises: a step of extracting Stereocaulon alpinum with methanol; a step of eluting methanol solution by column chromatography; a step of eluting the eluted fraction by reverse-phase high performance liquid chromatography using a acetonitrile(CH_3CN) solution to prepare a fraction containing lobaric acid; a step of dissolving the fraction in solvent and a step of adding NaOH or KOH. A pharmaceutical composition for preventing or treating diabetes or obesity contains the compound of chemical formula 1 or pharmaceutically acceptable salt thereof as an active ingredient.

Description

Pharmaceutical Composition for Preventing or Treating Diabetes or Obesity Containing Novel Compound Lobarin

The present invention relates to a pharmaceutical composition for the prevention or treatment of diabetes or obesity containing the novel compound lobarin , more specifically, Stereocaulon (Antarctic lichen) alpinum ) relates to a pharmaceutical composition for the prevention or treatment of diabetes or obesity containing a novel compound lovarin synthesized from a compound isolated from the extract, and a functional food containing the compound.

Lichens are known to produce higher plants and other unique secondary metabolites (Ingolfsdottir, K., Phytochemistry , 61: 729, 2002), and the secondary metabolites produced by these lichens are mostly depside and depsidone ( depsidone) and dibenzfurane, and these compounds are believed to be associated with low growth rates of lichens (Kumar, KCS et. al ., J. Nat . Prod ., 62: 817, 1999; Huneck, S., Naturwissenschaften , 86: 559, 1999). In addition, various biological activities of lichen metabolites, including antibiotics, antimycobacterial, antiviral, analgesic and antipyretic activities, have been identified by the screening process (Ingolfsdottir, K., Phytochemistry , 61: 729, 2002; Kumar, KCS et al ., J. Nat . Prod ., 62: 817, 1999). Therefore, there is a growing interest in the development of drugs using metabolites of lichens.

On the other hand, diabetes is a metabolic disorder including hyperglycemia caused by a deficiency of insulin action, insulin secretion, or both, and is a disease with a high possibility of future vascular complications. Type 1 (insulin dependent) diabetes is caused by beta-cell destruction of the pancreatic islets due to immunity and the absolute deficiency of insulin. Type 2 (insulin-independent) diabetes is produced by insulin, but not in sufficient amounts. It is caused by the body's inability to utilize insulin secreted effectively. In the state of 'insulin resistance' in which the cells of the body do not work effectively, the energy source in the body, especially sugars, is not used well, so the necessary energy is insufficient. Accumulated and eventually discharged into the urine is one of the chronic degenerative diseases that do not heal fundamentally.

The World Health Organization (WHO) and the United Nations (UN) will have about 246 million people with diabetes worldwide by the end of 2007, and deaths from diabetes are increasing year by year. Emphasis is placed on prevention, strict blood sugar control and the prevention of complications. In addition, according to a survey by the Korean Diabetes Association and the Korea Health Insurance Review & Assessment Service, in 2003, there were 401 million diabetic patients in Korea, and in 2030, 7.2 million diabetic patients will be 1 per 7 people. . In particular, the rapid increase in medical expenses is closely related to the explosive increase in the number of diabetics, the continuous increase in complications due to diabetes, and the increase in the average life expectancy of diabetics. As life expectancy changes due to rapid economic development, life expectancy is increasing, while chronic degenerative diseases such as diabetes are increasing.

Types of diabetes mellitus in Korea account for more than 99%, and the incidence of type 1 diabetes is less than about 1%. Type 2 diabetes reported in foreign countries is about 90%, and about 10% It is different from type 1 diabetes. The causes of diabetes are intertwined with various factors, and important factors include heredity (approximately 20% of family history), environment, age (approximately 60% between 40-49 years old), obesity, reduced body resistance, drug abuse And stress stimulation. The pathogenesis of diabetes has not yet been elucidated, but with the exception of a few specific diabetes mellitus (eg, MODY), it is a disease that is caused by a multigenic cause. In other words, the onset of diabetes is complicated by a number of genes and a large number of new genes continue to be identified.

Diabetes mellitus is caused by a variety of mechanisms, so the treatment also has to vary, moreover, the existing conventional treatments alone do not see a satisfactory effect, so a new treatment is required. Diabetes treatment research has been actively developed with the focus on Type 2 diabetes treatment, which accounts for more than 90% of patients with diabetes (Tables 1 and 2).

Current Status of Diabetes-Related Drug Development Company Name Development theme name Development stage Remarks Samjin Pharmaceutical Co., Ltd. Developed curator diabetes treatment with natural products Preclinical New Drug Development Differentiation from Embryonic Stem Cells to Pancreatic Beta Cells quest New Drug Development SK Corporation Diabetes Applications New Drug Development Youyu Co., Ltd. YYGG quest New Drug Development Yuhan Corporation Biochip Applications Improvement, diagnosis Chong Kun Dang Genetically modified human insulin Phases III Improved drug Neomarl Tablet Product Release Improved drug Hanmi Pharmaceutical Co., Ltd. HM 80200 Development Drug substance

Source: 2004-White Paper Pharmaceutical Industry, Korea Health Industry Development Institute, 2004, 12

Insulin secretagogues (pirogliride, linogliride, 2,4-diaminino-5-cyan-bromorididine, incretin, repaglinide, nateglinide), insulin enhancers (troglitazone), insulin resistance enhancers, and insulin-like effects in target tissues Indicating drugs (pirogliride, linogliride, dichloroacetate, insulin lispro, insulin aspart), glucosynthesis inhibitors (lipase inhibitors, carnitine transferase inhibitors, beta antioxidants), drugs that delay carbohydrate absorption (dietary fiber, alphaglucocyte) A lot of researches have been carried out on the (izase inhibitor), amylin analogue (pramlintide).

Some of these are currently on the market, but many are still in experimental or toxicological testing that are insufficient for human use. In particular, fast-acting insulin secretagogues and insulin resistance-improving agents considering biorhythms will be one of the effective methods for the treatment of diabetes mellitus.

In addition, studies on the pathogenesis of diabetes mellitus have been conducted for the past 10 years, presuming that the cause of insulin resistance may be a problem with the insulin receptor, and the direction of the research is shifting toward the insulin signaling system.

Development of New Drugs for Diabetes Treatment Mechanism of action Clinical stage Leading Company L Ⅲ Ⅱ I P α-Glucosidase inhibitor 3 One Bayer, Takeda, Chong Kun Dang Insulin agonist 13 5 11 4 27 Chiron, Eli Lilly, IDEAZymo-Genet ics, Aventis, Novo Nordisk, Akzo Nobel, Biobras, Alkermes, Merk KGaA Glucagon like peptide-1 agonist One 4 One 4 Amylin, Eli Lilly, Novo Nordisk, Restoragen, Zealand Pharmaceuticals β3-Adrenoreceptor agonist One 2 One Dainippon, Asahi Kasei, GlaxoSmithKline Dipeptidyl peptide Ⅳinhibitor 2 One Bristol-Myers Squibb, Novatis Peroxisome proliferator activated receptorα agonist 2 2 6 Novatis, Kyorin, BMS, GlaxoSmithKline Protein tyrosine phosphatase-1B inhibitor One 7 Wyeth, ISIS Pharmaceutical Leptin stimulator One One Amgen, Tularik Melanocortin-4 agonist One Neurocrine Biosciences AMPKstimulant 2 2 One One Andrx, Merck KGaA, Flamel Technologies Peroxisome proliferator activated receptor γagonist 3 4 9 GlaxoSmithKline, Samchundang, BMS, Japan Tabacco, Dr Reddy's, Kyorin

Source: Health Industry Technology Trends, "Recent Trends in Diabetes Treatment," 2003.

The activity of PTP-1B in adipocytes of obese type and non-obese type 2 diabetes mellitus was examined. It increased fold and activity was reported to appear 71%, 88%. In addition, recent experimental results showed that mice knocked out protein tyrosine phosphatase-1B (PTP1B) showed increased insulin sensitivity and resistance to high fat diet. In addition, according to a number of recently published studies, substances that inhibit the activity of PTP-1B may overcome insulin resistance by increasing insulin sensitivity in target cells. In Korea, randomized bulk screening is performed to develop inhibitors of PTP1B activity from tens of thousands of compounds not yet developed as drugs by the Korea Compound Bank.

On the other hand, leptin is released from the adipocytes into the blood and passes through the cerebral blood membrane and acts on receptors in the central nervous system, inhibits food intake, reduces weight, and promotes energy consumption. As new findings suggest that PTP1B modulates leptin activity itself, it is expected to have synergistic effects when PTP1B is used with leptin agonists (Koren, S., Best Pract . Res . Clin . Endocrinol . Metab ., 21: 621, 2007).

Therefore, the importance of inhibitors to PTP-1B is increasing in the development of treatment of obesity and obesity type diabetes, and there is a report on the leading substance recently discovered through the HTS (hight throughput screening). To date, there have been no clinically successful studies of PTP-1B and its inhibitors. However, as shown in Table 3, it is known that many research groups and companies are proceeding with interest.

PTP-1B inhibitor under development Mechanism Drug name Development company step Etc protein tyrosine phosphatase 1B inhibitor Ertiprotafib Wyeth phase Ⅱ Benzenepropanoic acid (discontinued) SIS-113718 ISIS Pharmaace uticals preclinical 2nd-generation antisense PTP-1B inhibitor OC-86839 Ontogen preclinical selective non-peptide inhibitor of PTP-1B PTP1B inhibitor Abbott preclinical phosphatase-1B inhibitor PTP1B inhibitor Array BioPharma preclinical PTP-1B inhibitor PTP1B inhibitor Structural Bioinformatics preclinical orally-active selective PTP-1B inhibitor PTP1B inhibitor Kaken Pharmaceutical preclinical orally-active PTPase inhibitor

Source: Pharmaproject, 2002

However, most of the inhibitors of PTP1B have been difficult to selectivity and bioavailability because they have been developed as non-hydrolyzable phosphotyrosine mimetics that target active sites of positively charged PTP1B (Liu, S. et al ., J. Am . Chem . Soc ., 130: 17075, 2008).

Accordingly, the present inventors have made intensive efforts to develop an effective therapeutic agent for the treatment of diabetes and obesity. As a result, the newly synthesized compound from the lovaric acid isolated from the extract of the stealocaulon alpinum, antarctic lichen, effectively inhibits PTP1B. After confirming the structure of the protein tyrosine dekinase family, it selectively acts only on PTP1B and shows antidiabetic effect upon administration of disease model animals, and completed the present invention.

An object of the present invention to provide a pharmaceutical composition and functional food for the prevention or treatment of diabetes or obesity containing a novel compound synthesized from a compound isolated from stereocaulon alpinum extract as an active ingredient.

In order to achieve the above object, the present invention provides a compound represented by the following formula (1)

[Formula 1]

.

.

The present invention also provides a method for preparing a compound represented by Formula 1, comprising the following steps:

(a) extracting Stereocaulon alpinum with methanol;

(b) Stereocaulon obtained in step (a) alpinum ) eluting the extract in an aqueous methanol solution using column chromatography;

(c) eluting the fraction eluted in step (b) with acetonitrile (CH ₃ CN) aqueous solution using reverse phase high performance liquid chromatography to obtain a fraction containing lobaric acid; And

(d) dissolving the fraction containing robaric acid in a solvent, stirring by adding NaOH or KOH, terminating the reaction by adding an acidic solution, and then obtaining the compound of Formula 1.

The present invention also provides a pharmaceutical composition for preventing or treating diabetes or obesity containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a functional food for preventing or improving diabetes or obesity containing the compound represented by Formula 1 as an active ingredient.

Stereocaulon according to the present invention ( Stereocaulon alpinum ) -derived new compound Lobarin has excellent inhibitory activity of protein tyrosine phosphatase 1B and has been shown to have excellent antidiabetic effect even when applied to animal models, and thus can be effectively used for the prevention or treatment of diabetes and obesity. .

1 shows the purification of lobarin by HPLC analysis.
Figure 2 shows the HRESIMS analysis of Lobarin (Lobarin).
3 shows a ¹ H NMR spectrum of Lobarin (400 MHz, DMSO- d ₆ ).
4 shows a ¹³ C NMR spectrum (400MHz, DMSO- d ₆ ) of Lobarin.
5 shows HSQC data (400MHz, DMSO- d ₆ ) of Lobarin.
6 shows HMBC data (400MHz, DMSO- d ₆ ) of Lobarin.
Figure 7 is a graph showing the PTP1B inhibitory activity of Lobarin (Lobarin).
8 is a graph showing the absorbance at 620 nm of inhibitory activity against PTP1B, PTPN2, PTPN5, PTPN6, PTPN7, and PTPN13 by Lobarin.
Figure 9 is a graph measuring the blood glucose change after intraperitoneal administration of Lobarin (Lobarin).
10 is a graph of blood glucose measurement results after 6 hours of fasting according to intraperitoneal administration of Lobarin.
11 is a graph measuring the weight change according to the abdominal administration of Lobarin (Lobarin).
12 is a graph measuring blood glucose change after 28 days of lobarin intraperitoneal administration.

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

In the present invention, a novel compound represented by the following Chemical Formula 1 was isolated from lobaric acid isolated from an extract of stereocaulon alpinum , and was named lobarin.

[Formula 1]

The novel compound Lobarin represented by Formula 1 may preferably be prepared by a method comprising the following steps: (a) Stereocaulon alpinum ) with methanol; (b) Stereocaulon obtained in step (a) alpinum ) eluting the extract in an aqueous methanol solution using column chromatography; (c) eluting the fraction eluted in step (b) with acetonitrile (CH ₃ CN) aqueous solution using reverse phase high performance liquid chromatography to obtain a fraction containing lobaric acid; And (d) dissolving the fraction containing robaric acid in a solvent, stirring by adding NaOH or KOH, terminating the reaction by adding an acidic solution, and then obtaining the compound of Chemical Formula 1.

At this time, the acidic solution in step (d) may be used for all kinds of acidic solution that can neutralize the aqueous solution, preferably in step (d) after dissolving the fraction containing the robaric acid in acetone, NaOH After stirring, the reaction is terminated and concentrated by the addition of HCl solution to obtain a methylene chloride dissolving layer through the partition between methylene chloride and an aqueous solution and concentrated to obtain the compound of formula (1). .

In one embodiment of the invention, lichen stereocaulon ( Stereocaulon) alpinum (Hedw.) GL Sm.) was collected from Barton Peninsular around King Sejong Station (S 62 ° 13.3 ', W58 ° 47.0') on King George Island in Antarctica in January 2003. Lovaric acid was extracted with dried Stereocaulon alpinum with methanol for 24 hours, and then the solvent was distilled off to obtain an extract, and the extract was flash column chromatography filled with silica gel (C ₁₈ ). chromatography, 5 × 25 cm) and 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% (v / v) methanol (MeOH) Injection was carried out sequentially, and each fraction was obtained, and then lovaric acid of the following Chemical Formula 2 was separated from the fractions.

[Formula 2]

Rover rigs acid stereo cowl theory to the over (Stereocaulon alpinum) Alfie extracted with methanol, to obtain the stereo cowl Rhone Alfie over (Stereocaulon alpinum ) extracts can be obtained by eluting with an aqueous solution of acetonitrile (CH ₃ CN) using column chromatography, and eluted fractions using a reverse phase high performance liquid chromatography. After dissolving the obtained lobaric acid in acetone, the mixture was stirred at room temperature by adding NaOH, and the reaction mixture was terminated by adding HCl solution. The reaction mixture was concentrated, and then Methylene chloride and aqueous solution (pH = 2) to obtain a methylene chloride dissolving layer through partitioning to obtain Lobarin of the formula (1).

[Formula 1]

In the present invention, lobarin, a novel derivative of lobaric acid isolated from the extract of Stereocaulon alpinum, has a very excellent activity of inhibiting the activity of PTP1B, confirming that it is effective in preventing or treating diabetes or obesity. It was. Therefore, in one aspect, the present invention relates to a pharmaceutical composition for preventing or treating diabetes or obesity containing Lobarin of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. In addition, lobulin (Lobarin) according to the present invention can be provided as a functional food containing it as an active ingredient, and thus the present invention in another aspect, prevention of diabetes or obesity containing lobulin (Lobarin) as an active ingredient Or to a functional food for improvement.

In one embodiment of the present invention, as a result of measuring the inhibitory activity of Lobarin to PTP1B, IC ₅₀ = 149nM showed a very excellent PTP1B inhibitory effect, so that the new compound Lobarin (Pharmaceutical for diabetes and obesity) It was confirmed that the material can be treated and prevented.

In addition, in one embodiment of the present invention, as a result of examining the selectivity of the protein tyrosine dekinase family of lovarin, 2nd aryl having the most similar to PTP1B, embrionic lethal, PTP1B in the amino acid sequence and 3D structure It was confirmed that the active site including the -phosphate binding site selectively acts only on PTP1B among the family of protein tyrosine dekinase containing TC-PTP (PTPN2), which is known to be similar, and the experimental results are described in the present invention. It is suggested that lovarin, a compound according to the present invention, can be used as a PTP1B inhibitor for the treatment of heterogeneous diabetes.

In another embodiment of the present invention, the relationship between body weight, blood glucose, and insulin resistance by measuring the change in dietary intake, weight change, blood glucose concentration, and blood insulin concentration by administering lovarin to a disease animal model db / db mouse The antidiabetic effect was verified by confirming.

Meanwhile, Lobarin of Formula 1 used in the present invention may be in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts in the present invention can be prepared by conventional methods in the art, for example, salts with inorganic acids such as hydrochloric acid, hydrogen bromide, sulfuric acid, sodium hydrogen sulfate, phosphoric acid, carbonic acid, and the like. Or a pharmaceutically acceptable acid together with an organic acid such as formic acid, acetic acid, oxalic acid, benzoic acid, citric acid, tartaric acid, gluconic acid, gestyic acid, fumaric acid, lactobionic acid, salicylic acid, or acetylsalicylic acid (aspirin) Salts may be formed, or they may be reacted with alkali metal ions such as sodium or potassium to form their metal salts, or may be reacted with ammonium ions to form another form of a pharmaceutically acceptable salt.

The pharmaceutical compositions comprising the compounds according to the invention, respectively, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, external preparations, suppositories, and sterile injectable solutions according to conventional methods. Can be formulated and used. Carriers, excipients, and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose or lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 60, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds of the present invention depend 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, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.1 to 1000 µg / kg, preferably at 1 to 100 µg / kg. Administration may be administered once a day or may be divided orally. The dosage does not limit the scope of the invention in any aspect.

The functional food of the present invention includes various foods, candy, chocolate, beverages, gums, teas, vitamin complexes, health supplements, and the like, and can be used in the form of powders, granules, tablets, capsules or beverages.

The compounds of the present invention may be added to foods or beverages for the purpose of preventing diabetes and obesity. At this time, the amount of the compound in the food or beverage is generally added to the dietary supplement composition of the present invention to 0.01 to 50% by weight, preferably 0.1 to 20% by weight of the total food weight, the health beverage composition is 100 ml It can be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g as a reference.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the compound of the present invention as an essential ingredient in the indicated proportions, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of natural carbohydrates are monosaccharides such as glucose, fructose and the like, disaccharides such as maltose, sucrose and the like, conventional sugars such as polysaccharides such as dextrin, cyclodextrin and xylitol Sugar alcohols such as sorbitol and erythritol. As flavoring agents, natural flavoring agents (tauumatin, stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention, in addition to the above, the functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors. Flavoring agents, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonated drinks Carbonic acid used, etc. The functional food of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. It may be used either individually or in combination. It is common ratio of the additive is selected from the range of the composition per 100 parts by weight 0 to about 20 parts by weight of the present invention, but not so important.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

New compounds Lobarin Manufacture

1-1: lichen Stereo cowlon Alpinum ( Stereocaulon alpinum ) Preparation of extract

Lichen Stereocaulon alpinum (Hedw.) GL Sm.) Was collected at Barton Peninsular around King Sejong Station (S 62 ° 13.3 ', W58 ° 47.0') in King George Island, Antarctica, in January 2003. It is lichen that can be done.

Dried Stereocaulon alpinum ) 50 g was extracted twice with 1 L of methanol for 24 hours to obtain 3.6 g of methanol extract. The obtained extract was loaded into flash column chromatography (5 × 25 cm) filled with silica gel (C ₁₈ ), 10%, 20%, 30%, 40%, 50%, 60%, 70 Concentration gradients of%, 80%, 90% and 100% (v / v) methanol (MeOH) yielded each fraction.

1-2: lichen Stereo cowlon Alpinum ( Stereocaulon alpinum ) Extract from Lobaric acid Manufacture

204.6 mg of the fraction obtained by elution with 80% methanol obtained in Example 1-1 was again loaded into flash column chromatography (2.5 × 30 cm) filled with silica gel (C ₁₈ ), and analyzed by TLC analysis. 200 ml of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% of methanol (in CH ₂ Cl) to obtain the eight major fractions obtained, respectively. ₂ ) A solution and 100% (v / v) methanol were injected to give each fraction.

After injecting 59 mg of the fraction eluted with 9% methanol into semi-preparative reverse-phase HPLC, acetonitrile (CH ₃ CN) aqueous solution containing 0.1% formic acid was 75 to 83 Lobaric acid (22.9 mg; t _R = 39 min) was isolated by elution for at least 30 minutes with a gradient of%.

[Formula 2]

1-3: Lobaric acid  ( Lobaric acid Synthesis of New Compounds from

Dissolve 50 mg of lobaric acid in Example 1-2 in 5 mL of acetone, add 1 mL of 0.5 N NaOH, stir at room temperature for 5 minutes, and add 0.5 mL of 1N HCl solution. Terminated. The reaction mixture was concentrated to obtain a methylene chloride dissolving layer through concentration between Methylene chloride and aqueous solution (pH = 2) and concentrated to obtain 50 mg of a novel compound of Formula 1, which was referred to as "Lobarin". Named it.

[Formula 1]

On the other hand, to increase the purity was analyzed by reverse phase HPLC using an Agilent Eclipse XDB-C18 column (4.6 x 150mm, USA). The solvent system used was 0.1% formic acid mixed water (A Line) and 0.1% formic acid mixed acetonitrile (B line). The analysis was carried out with acetonitrile 40% to 50% for 5 minutes, 50% to 80% for 15 minutes, and 80% to 90% for 10 minutes with a final purity of 96.1% (Figure 1).

New compounds Lobarin Structural Analysis of

The molecular structure of Lobarin synthesized in Example 1 was characterized by high resolution mass spectrometry (HRESIMS) and NMR spectroscopy.

Anion analysis of HRESIMS was measured using a Q-TOF micro LC-MS / MS instrument (Waters, USA). As shown in FIG. 2, lovarin showed a molecular ion peak of m / z 473.1774, which was determined by Lobarin. It is shown that the molecular formula is C ₂₅ H ₃₀ O ₉ .

NMR spectra of Lobarin was measured using a JEOL ECP-400 spectrometer (JEOL, Japan) after dissolving Lobarin in DMSO-d6 solvent. The chemical shift value of -d 6 (δC / δH = 40.0 / 2.50 ppm) is indicated. HMQC (1H-Detected Heteronuclear Multiple-Quantum Coherence) was set to 1 JCH = 140 Hz and measured. HMBC (Heteronuclear Multiple-Bond Coherence) experiment was set to nJCH = 8 Hz. Mass spectrometry was measured using Q-TOF micro LC-MS / MS.

First, looking at the ¹ H NMR and ¹³ C NMR spectra, as shown in Figs. 3 and 4, ¹ H NMR and ¹³ C NMR spectra of Lobarin showed a very similar pattern and NMR spectra of the Rover acid (lobaric acid). Therefore, the structure of Lobarin is very similar to that of lobaric acid, and considering that the difference in molecular weight is 18 dalton, it can be predicted that the compound was produced through the hydration reaction from lobaric acid. Comparing the Lobaric acid of NMR data and Lobarin of the NMR data, especially ¹³ C NMR spectrum is ¹³ C peak for the ketone functional group were observed at lobaric acid disappeared from the place of was observed the ¹³ C peaks at 106.3 ppm of OH functional group in 1H NMR Proton peaks (7.65 ppm) were observed. Based on these differences in NMR data, Lobarin's structure shows that the ketone functionalities of lobaric acid are converted to oxygen anions by nucleophilic attack of hydroxyl groups, as shown in Equation 1, which causes nucleophilic addition reactions to adjacent neighboring ester functionalities. The ester group was predicted to degrade. The predicted structure was confirmed by analysis of additional two-dimensional NMR spectroscopy, HMQC and HMBC (Table 4).

Analysis of HMQC data (FIG. 5) and HMBC data (FIG. 6) confirmed the location of the peaks for each carbon and hydrogen in lobarin. These data were similar to the NMR measurements of lobaric acid. The HMBC correlations from the peak corresponding to the proton of the OH functional group (7.65 ppm) to the ¹³ C NMR peaks corresponding to the C-6, C-7, and C-8 positions also reveal the structure of Lobarin presented. Provided important data.

NMR Data for lobarin (400 MHz, DMSO- d ₆ ) Position δ _C δ _H , mult. ( J  in Hz) HMBC ^a One 107.1 - - 2 158.0 - - 3 101.7 5.91, d (1.8) 1, 2, 4, 5 4 166.8 - - 5 99.8 6.75, d (1.8) 3, 1, 4, 5 6 155.0 - - 7 165.6 - - 8 106.3 - - 9 38.7 1.97, m
2.07, m 8 10 25.7 1.14, m
1.25, m - 11 22.5 1.12, m
1.38, m - 12 14.3 0.82, br t 10, 11 One' 109.4 - - 2' 153.3 - - 3 ' 102.2 6.32, s 1 ', 5', 2 ', 4', 7 ' 4' 160.7 - - 5 ' 131.8 - - 6 ' 138.5 - - 7 ' 172.0 - - 8' 27.7 2.78, m - 9 ' 30.2 1.14, m
2.24, m - 10 ' 32.2 1.11, m
1.29, m - 11 ' 22.1 1.23, m
1.27, m - 12 ' 14.4 0.72, br s - 4-OCH ₃ 56.7 3.74, s 4 8-OH - 7.66, s 9, 8, 6

^a HMBC correlations, optimized for 8 Hz, are from proton (s) stated to the indicated carbon (s).

Lovarin  ( Lobarin )of PTP1B  Inhibitory Activity Assay

To analyze the inhibitory activity of Lobarin protein tyrosine dephosphorylase 1B (PTP1B), the enzyme activity was measured spectroscopically.

That is, compound lovarin in PTP1B (Bionia, Korea), PTP1B buffer solution (20 mM Tris-Hcl, pH 8.0, 0.75 mM NaCl, 0.5 mM EDTA, 5 mM β-mercaptoethanol, 50% Glycerol) at a concentration of 0.5 mg / ml (Lobarin) Add 0, 1, 3, 10, 30, 100, 300, 1000, 3000 nM and substrate [pTyr1146] Insulin Receptor (1142-1153, Santa Cruz, USA) and then react at room temperature for 10-30 minutes After the reaction with Malachite Green-Molybdate Dye Solution (1142-1153, Santa Cruz, USA) for 10 minutes at room temperature to terminate the reaction with PTP1B, the compound (Lovarin) and the substrate, the absorbance was measured at 620 nm. .

As a result, as shown in Figure 7, the analysis of the inhibitory activity of Lobarin (PTP1B) against the PTP1B, IC ₅₀ = 149 nM showed excellent PTP1B inhibitory effect, concentration-dependently increased the inhibition rate diabetes mellitus And it was confirmed that it is a substance capable of pharmaceutical treatment and prevention of obesity.

Lovarin  ( Lobarin ) Protein Tyrosine  Kinase ( Protein tyrosin  Selectivity analysis for phophatase

In order to investigate the selectivity of the family of Lobarin protein tyrosine dephosphorylase enzyme, the inhibitory activity against PTP1B, PTPN2, PTPN5, PTPN6, PTPN7 and PTPN13 was examined spectroscopically by measuring enzyme activity.

First, 0.5 / concentration PTP1B, PTPN2, PTPN5, PTPN6, PTPN7, PTPN13 (Bionia, Korea) and protein tyrosine dephosphatase buffer solution (20 mM Tris-Hcl, pH 8.0, 0.75 mM NaCl, 0.5 mM EDTA, 5 mM Lobarin 0, 50, 100, 200 nM and substrate [pTyr1146] Insulin Receptor (1142-1153, Sigma, USA) were added to β-mercaptoethanol (50% Glycerol) and reacted at room temperature for 10-30 minutes. After Malachite Green-Molybdate Dye Solution (Sigma, USA) was added to react at room temperature for 10 minutes to terminate the reaction with the substrate and the absorbance was measured at 620 nm.

As a result of investigating the selectivity of the protein tyrosine dekinase family of Lobarin (Lobarin), as shown in Figure 8, Lobarin is IC ₅₀ for PTP1B At 200 nM, the inhibition rate was 52.2%, and there was no inhibitory activity against other families including TC-PTP (PTPN2).

TC-PTP (PTPN2), which is known to be the kinase most similar to PTP1B in amino acid sequence and 3D structure, is embrionic lethal, and has similar enzymatic properties to PTP1B and similar active site including 2nd aryl-phosphate binding site. . Thus, 757 substances have been registered as targets for PTP1B, but no compound has entered clinical trials with PTP1B as the main target, and only plant extracts that act on multiple targets, including PTP1B, have been released or are in clinical trials.

Thus, the above experimental results showing that the protein tyrosine dekinase family only selectively acts on PTP1B, suggests that the compound according to the present invention, Lobarin, can be used to treat heterologous diabetes as a PTP1B inhibitor.

Disease Model Animals  For compounds ( Lobarin Validation of)

5-1: PTP1B  Changes in blood glucose after intraperitoneal administration of an inhibitor

Based on preliminary, potent, toxicological and data studies for lovain, the dosage (expressed as test substance (mg) / weight of test animal (Kg)) was determined. Seven-week-old male db / db mice (type 2 diabetes model animals, C57 / BLKS / J-db / db, Korea Research Institute of Bioscience and Biotechnology) were treated with 200 μl of PBS as a control and 10 mg / kg of Lobarin as an experimental group. Intraperitoneally, blood glucose was measured twice a week.

The blood glucose level of 7-week-old male db / db mice (type 2 diabetes model animals, C57 / BLKS / J-db / db, Korea Research Institute of Bioscience and Biotechnology) was measured by the abdominal injection of lobarin. As shown in 9, the control group (n = 6) averaged 268 mg / dL on day 0, 381 mg / dL on day 3, 404 mg / dL on day 7, 432 mg / dL on day 10, 454 mg / dL on day 14, 17 A rapid increase in blood glucose was observed at 479 mg / dL per day and 482 mg / dL at 21 days. However, in the experimental group (n = 6) injected with lobarin 10 mg / kg (n = 6), the average was 0 days 267 mg / dL, 3 278 mg / dL per day, 298 mg / dL for 7 days, 315 mg / dL for 10 days, 352 mg / dL for 14 days, 379 mg / dL for 17 days, and 425 mg / dL for 21 days Could confirm.

5-2: Compound ( Lobarin ) After 6 hours fasting, observe blood glucose change

For more accurate antidiabetic effects on lobarin, PBS as a control in 7-week-old male db / db mice (type 2 diabetes model animals, C57 / BLKS / J-db / db, Korea Research Institute of Bioscience and Biotechnology) The blood glucose was measured twice a week after intraperitoneal injection of 200 μl, lovarin at 10 mg / kg daily in the experimental group. At this time, after fasting for 6 hours after the intraperitoneal injection, blood glucose was measured.

As a result, as shown in Figure 10, in the control group (n = 6) on average 0 days 151 mg / dL, 3 days 199 mg / dL, 7 days 262 mg / dL, 10 days 291 mg / dL, 14 days 397 mg / dL, 17 days at 455 mg / dL, 21 days at 483 mg / dL, blood glucose increased rapidly, but in the experimental group intraperitoneally injected with Lobarin 10 mg / kg (n = 6), on average 0 days 152 mg / dL, 3 days 141 mg / dL, 7 days 155 mg / dL, 10 days 198 mg / dL, 14 days 261 mg / dL, 17 days 287 mg / dL, 21 days 340 mg / dL, as in Example 5-1 Compared to the low blood sugar increase.

5-3: PTP1B  Observation of weight change after intraperitoneal administration of an inhibitor

Seven-week-old male db / db mice (type 2 diabetes model animals, C57 / BLKS / J-db / db, Korea Research Institute of Bioscience and Biotechnology) were treated with 200 μl of PBS as a control and 10 mg / kg of lovarin as experimental groups daily. Post mortem body weights were measured using animal scales at regular times twice a week to investigate weight changes after intraperitoneal injection.

As a result, as shown in Figure 11, in the control group (n = 6), the average 0 days 30.8 g, 3 days 35.1 g, 7 days 38.7 g, 10 days 39.4 g, 14 days 40.3 g, 17 days 41.1 g, 21 days 41.3 g of body weight was changed, and in the experimental group (n = 6) injected with lobarin 10mg / kg on average 0 days 29.1 g, 3 days 33.7 g, 7 days 37.5 g, 10 days 38.3 g, 14 39.3 g per day, 39.9 g for 17 days, and 40.3 g for 21 days.

This indicates that there was no significant change in weight in the type 2 diabetic animals treated with Lobarin compared to the control group.

5-4: Compound ( Lobarin ) Validation of glucose tolerance test after 28 days of treatment

In order to determine the intraperitoneal glucose tolerance test (IPGTT) in the animal model of the compound (Lobarin), the following experiment was performed.

Seven-week-old male db / db mice (type 2 diabetic model animals, C57 / BLKS / J-db / db, Korea Research Institute of Bioscience and Biotechnology) were treated with 20% DMSO as a control and 10 mg / kg of lovarin daily as experimental groups. After daily intraperitoneal injection, after fasting for 16 hours without administration of 20% DMSO and lovarin, respectively, glucose (500 mg / ml, administration volume 200 μl) was injected by intraperitoneal injection, followed by 0, 15, 30, 60 Blood glucose changes were observed in blood samples taken from the tail vein at 90 and 120 minutes.

As a result of measuring glucose tolerance according to subcutaneous glucose administration in type 2 diabetic animals, as shown in FIG. 12, in the control group (20% DMSO intraperitoneal administration), 0 minutes after administration of glucose 293 mg / dL and 15 minutes PTP1B activity while exhibiting 429 mg / dL, 30 minutes 557 mg / dL, 60 minutes 553 mg / dL, 90 minutes 539 mg / dL, 120 minutes 568 mg / dL, showing rapid trends in blood glucose elevation and very slow hypoglycemic behavior. In the experimental group administered with inhibitor 10 mg / kg of Lobarin, 0 minutes 153 mg / dL, 15 minutes 358 mg / dL, 30 minutes 436 mg / dL, 60 minutes 390 mg / dL, 90 minutes 335 mg / dL, 120 minutes Drug concentration dependence of 290 mg / dL confirmed the normalization of blood glucose through low and high blood glucose levels.

The results of Examples 1 to 4 of Example 5 show that the novel compound Lobarin according to the present invention has a very good antidiabetic effect.

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Unsigned

Claims (5)

Compound represented by the following formula (1)
[Formula 1]

.
Method for preparing a compound represented by the following formula (1) comprising the following steps:
(a) extracting Stereocaulon alpinum with methanol;
(b) Stereocaulon obtained in step (a) alpinum ) eluting the extract in an aqueous methanol solution using column chromatography;
(c) eluting the fraction eluted in step (b) with acetonitrile (CH ₃ CN) aqueous solution using reverse phase high performance liquid chromatography to obtain a fraction containing lobaric acid; And
(d) dissolving the fraction containing robaric acid in a solvent, stirring by adding NaOH or KOH, terminating the reaction by adding an acidic solution, and then obtaining a compound of Formula 1
[Formula 1]

.
The method of claim 2, wherein the step (d) dissolves the fraction containing robaric acid in acetone, and then stirred by adding NaOH, and adding HCl solution to terminate and concentrate the reaction, and then between methylene chloride and aqueous solution. Distributing a methylene chloride dissolving layer through distribution and concentrating to obtain a compound of formula
[Formula 1]

.
A pharmaceutical composition for preventing or treating diabetes mellitus or obesity containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

Functional food for the prevention or improvement of diabetes or obesity containing the compound represented by the formula (1) as an active ingredient
[Formula 1]

.

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