KR101481141B1 - Pharmaceutical Composition for Preventing or Treating Diabetes Containing Novel Compound Lobarstin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating diabetes, which contains a novel compound Lobarstin. More specifically, the present invention relates to a pharmaceutical composition for prevention or treatment of diabetes, which comprises an Stereocaulon The present invention relates to a novel pharmaceutical composition for preventing or treating diabetes comprising rovastine as an active ingredient and a functional food containing the compound. The Stereocaulon according to the present invention alpinum- derived rovarin is excellent in the inhibitory activity of protein tyrosine phosphatase 1B and has an excellent antidiabetic effect when applied to an animal model. Therefore, it can be effectively used for preventing or treating diabetes.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating diabetes, which comprises a novel compound lovastine,

The present invention relates to a pharmaceutical composition for preventing or treating diabetes, which comprises a novel compound lovastine , and more particularly, to a pharmaceutical composition for prevention or treatment of diabetes comprising Stereocaulon The present invention relates to a novel pharmaceutical composition for preventing or treating diabetes comprising rovastine as an active ingredient 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). The secondary metabolites produced by these lichens are mostly depside, depsidone and dibenzfurane, and these compounds are presumed to be related to the low growth rate of lichens (Kumar, KCS et al ., J. Nat . Prod ., 62: 817, 1999; Huneck, S., Naturwissenschaften , 86: 559, 1999). In addition, various biological activities of lichens metabolites including antibiotics, antimycobacteria, antiviral, analgesic and antipyretic effects have been confirmed by screening procedures (Ingolfsdottir, K., Phytochemistry , 61: 729, 2002; Kumar, KCS et al ., J. Nat . Prod ., 62: 817, 1999). Therefore, there is an increasing interest in the development of pharmaceutical products using the metabolites of lichen.

On the other hand, diabetes is a metabolic disorder syndrome including hyperglycemia caused by insulin action, insulin secretion or both of these defects, and is likely to be a future vascular complication. It can be roughly divided into type 1 diabetes and type 2 diabetes. Type 1 (insulin-dependent) diabetes is caused by the destruction of the beta cells of the islet by immune system and thus the absolute lack of insulin. Type 2 (non-insulin dependent) diabetes is secreted by insulin, It is caused by the inability to effectively utilize the body-secreted insulin. In the state of 'insulin resistance' where the cells of the body can not effectively work, the energy source of the body, especially sugar, is not well used and the energy required is insufficient. It is one of the chronic degenerative diseases that do not heal in the end.

The World Health Organization (WHO) and the United Nations (UN) will have about 246 million people worldwide with diabetes by the end of 2007, and deaths from diabetes are increasing year by year, Prevention of onset, strict blood glucose control and prevention of complications. In addition, the National Diabetes Association and the National Health Examination and Assessment Service have reported that in 2003, the total number of diabetic patients in Korea was 3.9 million, and that by 2030, the number of diabetic patients would reach 7.2 million and one per 7 Koreans . In particular, the rapid increase in medical expenses has been associated with an explosive increase in the number of diabetic patients, a continuous increase in complications due to diabetes, and an increase in the average life span of diabetic patients. While the life expectancy is extended due to the change of diet due to rapid economic development, chronic degenerative diseases such as diabetes are increasing.

In Korea, the features of type 2 diabetes are more than 99%, the incidence of type 1 diabetes is less than 1%, the number of reported cases of type 2 diabetes in foreign countries is about 90% It is different from being type 1 diabetes. The cause of diabetes is a complex intertwined of various factors. Important factors are hereditary (family history of about 20%), environment, age (about 60% between ages 40-49), obesity, , And stimulation by stress. Although the pathogenesis of diabetes is not yet elucidated in detail, except for some special diabetes (eg, MODY), it is difficult to find a coherent gene that is associated with multiple genetic causes. In other words, the onset of diabetes is complicated by various genes, and a large number of new genes are still being discovered.

Diabetes mellitus is caused by a variety of pathogenic mechanisms. Therefore, the treatment method must be varied. In addition, there is a need for a new treatment method because many of the conventional treatments do not have satisfactory effects. Research on diabetes mellitus has been actively conducted with the development of type 2 diabetes drugs, which account for more than 90% of diabetic patients (Table 1, 2).

Domestic Diabetes Related Drug Development Status Company Name Development theme name Development stage Remarks Samjin Pharmaceutical Co., Ltd. Development of diabetes medicines from natural products Preclinical New drug development Differentiation of embryonic stem cells into pancreatic beta cells quest New drug development SK Corporation Diabetes Applications New drug development Yu Yu Co., Ltd. YYGG quest New drug development Yuhan Corporation Biochip Applications Improvement, diagnosis Chong Kun Dang Recombinant human insulin Phases Ⅲ Improved New Drug Neo-Ma Riljeong Product launch Improved New Drug Hanmi Pharmaceutical Co., Ltd. HM 80200 Development progress Raw drug

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

Insulin-like effects in the target tissues can be measured by a combination of two or more of the following: insulin-stimulating agents (pirogliride, linogliride, 2,4-diamino-5-cyanobromopridine, incretin, repaglinide, nateglinide), insulin resistance enhancer (Such as pirogliride, linogliride, dichloroacetate, insulin lispro, insulin aspart), grape sugar synthesis inhibitors (lipolysis inhibitors, carnitine transferase inhibitors, beta oxidation inhibitors), agents that delay carbohydrate uptake Many studies on amylin analogs (pramlintide) have been conducted.

Some of these are currently on the market, but many are still at an experimental stage or toxicology stage, which is still not well suited for human use. In particular, rapid - acting insulin secretagogues and insulin resistance modifiers considering biorhythm will be one of effective methods for the treatment of diabetes, and it is expected that such drug opening will be more active in the future.

In addition, studies on diabetes mellitus so far have been conducted for the past decade, assuming that insulin resistance is a cause of insulin receptor problems, and research is now shifting toward insulin signaling.

Diabetes Treatment Drug Development Status Mechanism of action Clinical stage The 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-Genetics, 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 Ⅳ inhibitor 2 One Bristol-Myers Squibb, Novatis Peroxisome proliferator activated receptor 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

The expression of PTP1B in adipocytes of obese and non-obese type 2 diabetic patients was 3 times and 5.5 times higher than that of the normal group, respectively And activity was reported to be 71% and 88%, respectively. Recently, it has been reported that mice with knock-out of protein tyrosine phosphatase-1B (PTP1B) exhibit increased sensitivity to insulin and resistance to high-fat diet. In addition, a number of recent studies suggest that a substance that inhibits the activity of PTP1B may overcome insulin resistance by increasing insulin sensitivity in target cells. In Korea, compound screening has been carried out randomly in order to develop PTP1B inhibitors from tens of thousands of compounds that have not yet been developed as drugs in Korea Compound Bank.

Leptin, on the other hand, is released from adipocytes into the blood, passes through the brain blood membrane, acts on receptors in the central nervous system, inhibits food ingestion, reduces body weight and promotes energy consumption. Thus, PTP1B is expected to regulate leptin activity itself, and PTP1B is expected to have a synergistic effect when used in combination with a leptin agonist (Koren, S., Best Pract . Res . Clin . Endocrinol . Metab ., 21: 621, 2007).

Therefore, the importance of inhibitors to PTP1B has been increasing in the development of obesity and obesity type diabetes, and there have been reports of leading substances discovered recently through hight throughput screening (HTS). There are no clinically successful studies on PTP-1B and the development of its inhibitors. However, as shown in Table 3, it is known that many research groups and companies are proceeding with interest.

PTP-1B inhibitor in development Mechanism Medicine 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 PTP1B inhibitor OC-86839 Ontogen preclinical selective non-peptide inhibitor of PTP1B PTP1B inhibitor Abbott preclinical phosphatase-1B inhibitor PTP1B inhibitor Array BioPharma preclinical PTP1B inhibitor PTP1B inhibitor Structural Bioinformatics preclinical orally-active selective PTP1B inhibitor PTP1B inhibitor Kaken Pharmaceutical preclinical orally-active PTPase inhibitor

However, most of the inhibitors of PTP1B have difficulties in their selectivity and bioavailability because they have been developed as nonhydrolyzable phosphotyrosine mimics targeting the active sites of positively charged PTP1B (Liu, S. et al. meat al ., J. Am . Chem . Soc ., 130: 17075, 2008).

The present inventors have made intensive efforts to develop a therapeutic agent effective for the treatment of diabetes and obesity. As a result, the present inventors have found that a compound newly synthesized from rovaric acid isolated from Stereocaulon alpinum extract, which is an Antarctic lichen, effectively inhibits PTP1B The present inventors have confirmed that the protein tyrosine dephosphorylase family selectively acts on PTP1B and exhibits an antidiabetic effect upon administration of a disease model animal. Thus, the present invention has been completed.

It is an object of the present invention to provide a stereoculo- alpinum extract of the present invention as an active ingredient. The present invention also provides a pharmaceutical composition and a functional food for preventing or treating diabetes.

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

[Chemical Formula 1]

Wherein R is selected from the group consisting of H, an alkyl group, an aryl group, an arylalkyl group, and an acyl group.

 The present invention provides compounds represented by the following formula (2): < EMI ID =

(2)

The present invention also provides a process for preparing a compound represented by the general formula (2), comprising the steps of:

(a) extracting Stereocaulon alpinum with methanol;

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

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

(d) dissolving the fraction containing rovaric acid in a solvent, adding NaOH or KOH thereto and stirring, and adding an acidic solution to terminate the reaction, and then obtaining the compound of formula (2).

The present invention also provides a pharmaceutical composition for preventing or treating diabetes mellitus comprising 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 comprising the compound represented by the above formula (1) as an active ingredient.

The new compound derived from Stereocaulon alpinum according to the present invention is excellent in the inhibitory activity of protein tyrosine phosphatase 1B and has an excellent antidiabetic effect when applied to an animal model. It is very useful because it can be effectively used for treatment.

Figure 1 shows a refinement of lobarstin by HPLC analysis.
Fig. 2 shows HRESIMS analysis results of lobarstin.
Figure 3 shows the ¹ H NMR spectrum (400 MHz, DMSO- d ₆ ) of lobarstin.
Figure 4 shows the ¹³ C NMR spectrum (400 MHz, DMSO- d ₆ ) of lobarstin.
Figure 5 shows COZY data (400 MHz, DMSO- d ₆ ) of lobarstin.
6 shows the HSQC data (400 MHz, DMSO- d ₆ ) of lobarstin.
Figure 7 shows HMBC data (400 MHz, DMSO- d ₆ ) of lobarstin.
Figure 8 shows the NOESY data (400 MHz, DMSO- d ₆ ) of lobarstin.
FIG. 9 is a graph showing the PTP1B inhibitory activity of lobarstin. FIG.
10 is a graph showing the inhibitory activity of lobarstin against PTP1B, PTPN2, PTPN5, PTPN6, PTPN7 and PTPN13 measured by absorbance at 620 nm.
11 is a graph showing changes in blood glucose after administration of intraperitoneal administration of lobarstin.
FIG. 12 is a graph showing blood glucose measurement after fasting for 6 hours according to intraperitoneal administration of lobarstin.
FIG. 13 is a graph showing a change in body weight according to the intraperitoneal administration of lobarstin. FIG.
14 is a glucose tolerance validation graph after 28 days of intraperitoneal administration of lobarstin.

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 general formula (2) was isolated from lobaric acid isolated from the extract of Stereocaulon alpinum and named it lobarstin.

(2)

The novel compound Lovastine represented by the above formula (2) can be preferably prepared by a method comprising the steps of: (a) extracting Stereocaulon alpinum with methanol; (b) eluting the Stereocaulon alpinum extract obtained in step (a) in an aqueous methanol solution using column chromatography; (c) eluting the fraction eluted in step (b) with an aqueous solution of acetonitrile (CH ₃ CN) using reverse phase high performance liquid chromatography to obtain a fraction containing lobaric acid; And (d) dissolving the fraction containing rovaric acid in a solvent, adding water, adding NaOH or KOH and stirring, adding an acidic solution to terminate the reaction, step.

In step (d), any acidic solution capable of neutralizing the aqueous solution may be used as the acidic solution. Preferably, the step (d) comprises dissolving the fraction containing rovaric acid in acetone, And the mixture was stirred for 10 to 20 minutes. The reaction was terminated by addition of HCl solution and the mixture was concentrated to obtain a methylene chloride dissolution layer through partition between methylene chloride and aqueous solution. To obtain a compound.

In one embodiment of the present invention, the lichen, Stereocaulon alpinum (Hedw.) GL Sm., Was deposited in the vicinity of Sejong Station (S 62 ° 13.3 ', W58 ° 47.0') in King George Island, (Barton Peninsular). Lobaric acid was extracted from the dried Stereocaulon alpinum with methanol for 24 hours, and then the solvent was distilled to obtain an extract. The extract was purified by flash column chromatography (silica gel column) packed with silica gel (C ₁₈ ) (v / v) methanol (MeOH) was added to the column, followed by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% Sequentially, and each fraction was obtained. Then, lobalic acid of the formula (3) was isolated from the fractions.

(3)

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 extract of the present invention is eluted from the aqueous methanol solution using column chromatography and the eluted fractions are eluted with an aqueous acetonitrile (CH ₃ CN) aqueous solution using reversed phase high performance liquid chromatography. Then, the obtained rovaric acid was dissolved in acetone, and NaOH was added thereto. After stirring at room temperature and adding HCl solution to terminate the reaction, the reaction mixture was concentrated and then washed with methylene chloride and aqueous solution = 2) to obtain a methylene chloride dissolution layer to obtain robustine represented by the following formula (2).

(2)

In addition, other derivatives in which some alkyl groups have been modified from rovastine of formula (2) above will also fall within the scope of the present invention. In this regard, the present invention relates to compounds of formula (I)

[Chemical Formula 1]

Wherein R is selected from the group consisting of H, an alkyl group, an aryl group, an arylalkyl group, and an acyl group.

The alkyl group, aryl group, arylalkyl group, and acyl group may be, for example, 1 to 20 carbon atoms or 1 to 10 carbon atoms, and the alkyl group includes substituted and unsubstituted alkyl and cycloalkyl groups.

It is obvious to those skilled in the art to obtain the compound of formula (1) through the synthesis and modification of known compounds known in the art from rovastine of formula (2). For example, R of the compound of formula (I) can be various derivatives by changing the number of carbons and the bonding structure. For example, when R is a propyl chain, it is derived from the reaction with sodium pentanoate. It is possible to synthesize derivatives having different carbon numbers using sodium pentanoate, sodium butyrate, sodium propionate, and sodium hexanoate having different carbon numbers.

In the present invention, rovastine , a novel derivative of rovaric acid isolated from the extract of Stereocaulon alpinum, has excellent PTP1B activity inhibitory activity and thus is effective for preventing or treating diabetes. Accordingly, in one aspect, the present invention relates to a pharmaceutical composition for preventing or treating diabetes comprising rovastine or its pharmaceutically acceptable salt of Formula 2 as an active ingredient. In addition, the present invention relates to rovastine according to the present invention as a functional food containing the same as an active ingredient. In another aspect, the present invention relates to a functional food for preventing or improving diabetes comprising rovastine as an active ingredient.

In addition, the compound of the formula (I) or a pharmaceutically acceptable salt thereof having some alkyl groups as shown below, as well as rovastine of the formula (2), may exhibit the same or similar effects. Therefore, a pharmaceutical composition for preventing or treating diabetes, Can be provided as a functional food.

In an embodiment of the present invention, showed a very good PTP1B inhibitory effects as a result of measuring the inhibitory activity for PTP1B in the Rover sustaining, IC ₅₀ = 154.6 nM, this novel compound Rover sustaining the pharmaceutical treatment and prevention of diabetes It is confirmed that this is a possible substance.

In addition, in an embodiment of the present invention, the selectivity of rovastine to the protein tyrosine dephosphorylase family was examined. As a result, the most similar amino acid sequence and 3D structure to PTP1B, embrionic lethal, (PTPN2), which is known to be similar to the active site including the -phosphate binding site. These experimental results show that the present invention can be applied to the present invention Is a PTP1B inhibitor and is available for the treatment of type 2 diabetes mellitus.

In another embodiment of the present invention, rovastine is administered to db / db mouse, which is an animal model of disease, to measure changes in dietary intake, body weight change, blood glucose concentration, and blood insulin concentration to determine the correlation between body weight and blood glucose and insulin resistance And the antidiabetic effect was verified.

Meanwhile, the lovastine of Formula 2 used in the present invention may be in the form of a pharmaceutically acceptable salt. The pharmaceutically acceptable salts in the present invention can be prepared by conventional methods in the art and include, for example, salts with inorganic acids such as hydrochloric acid, hydrogen bromide, sulfuric acid, sodium hydrogenphosphate, phosphoric acid, Or with pharmaceutically acceptable acids such as formic, acetic, oxalic, benzoic, citric, tartaric, gluconic, gestic, fumaric, lactobionic, salicylic, or acetylsalicylic acid (aspirin) To form salts, or to react with alkali metal ions such as sodium, potassium or the like to form their metal salts, or to react with ammonium ions to form another type of pharmaceutically acceptable salt.

The pharmaceutical composition containing the compound according to the present invention can be administered orally or parenterally in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, And can be used as formulations. Examples of carriers, excipients and diluents that can be included in the composition including the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, sucrose), lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 60, cacao paper, laurin, glycerogelatin and the like can be used.

The preferred dosage of the compound of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at a dose of 0.1 to 1000 mg / kg, preferably 1 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The functional food of the present invention can be used in the form of powder, granule, tablet, capsule or beverage, for example, various foods, candy, chocolate, beverage, gum, tea, vitamin complex and health supplement.

The compounds of the present invention can 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 may generally be 0.01 to 50% by weight, preferably 0.1 to 20% by weight, of the total food weight of the health functional food composition of the present invention, Can be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g, based on the total weight of the composition.

Such a health beverage composition of the present invention is not particularly limited to liquid ingredients except that it contains the compound of the present invention as an essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates as an additional ingredient have. Examples of natural carbohydrates include conventional saccharides such as monosaccharides such as glucose, fructose, etc., disaccharides such as maltose, sucrose, polysaccharides such as dextrin, cyclodextrins, etc., and xylitol , Sorbitol, and erythritol. As a flavoring agent, it is possible to advantageously use a natural flavoring agent (tautatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavorings (saccharin, aspartame, etc.) In general, the functional food of the present invention may contain various nutrients, vitamins, minerals (electrolytes), synthetic flavors, and natural flavors (Such as cheese and chocolate), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols and carbonated drinks. Etc. The functional food of the present invention may also contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. 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.

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.

The novel compound Lobarstin's  Produce

1-1: Lichen Stereo Cowlon Alpinum ( Stereocaulon alpinum ) Preparation of extract

The licorice Stereocaulon alpinum (Hedw.) GL Sm. Has been found in the Barton Peninsula around King Sejong Station (S 62 ° 13.3 ', W58 ° 47.0') in Antarctica on January, And it can be easily collected from the Barton Peninsula.

50 g of dried Stereocaulon alpinum was extracted twice with 1 L of methanol for 24 hours to obtain 3.6 g of methanol extract. The obtained extract was loaded on a flash column chromatography (5 x 25 cm) filled with silica gel (C ₁₈ ), and 10%, 20%, 30%, 40%, 50%, 60% Each fraction was obtained by stepwise concentration gradient with%, 80%, 90% and 100% (v / v) methanol (Methanol).

1-2: Lichen Stereo Cowlon Alpinum ( Stereocaulon alpinum ) Preparation of rovaric acid from the extract

204.6 mg of the fraction obtained by eluting with 80% methanol obtained in Example 1-1 was injected into flash column chromatography (2.5 x 30 cm) packed with silica gel (C ₁₈ ) and subjected to TLC analysis To obtain the eight major fractions obtained by the above procedure, 200 ml of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% Solution and 100% (v / v) methanol were injected into each fraction.

59 mg of the fraction eluted with 9% methanol was again injected into the semi-preparative reverse-phase HPLC, and then an aqueous acetonitrile (CH ₃ CN) solution containing 0.1% to the concentration giving 83% of the gradient elution over 30 minutes so as to separate the Rover acid (lobaric acid) of the formula 3 (22.9mg; t _R = 39 min).

(3)

1-3: Synthesis of novel compounds from Lobaric acid

50 mg of lobaric acid of Example 1-2 was dissolved in 50 mL of acetone, 50 mL of water was added, and the mixture was stirred. 0.25 mL of 2 N NaOH was added, stirred at room temperature for 15 minutes, and 0.5 mL of 1N HCl solution was added to terminate the reaction. The reaction mixture was concentrated and the methyl chloride solubilization layer was obtained by partitioning between methyl chloride and aqueous solution (pH = 2) and concentrated to obtain 50 mg of a novel compound of the formula (2), which was then purified by "Lobarstin" .

(2)

On the other hand, to increase the purity, reverse phase HPLC was performed using an Agilent Eclipse XDB-C18 column (4.6 x 150 mm, USA). The solvent system used was A line with 0.1% formic acid and acetonitrile (B line) with 0.1% formic acid. Initiation was carried out from acetonitrile 40% to 50% for 5 minutes, 50% to 80% for 15 minutes and 80% to 90% for 10 minutes, the final purity being 97.6% (FIG.

The novel compound Lobarstin's  Structure analysis

The molecular structure of robustin synthesized in Example 1 was identified by high resolution mass spectrometry (HRESIMS) and NMR spectroscopy. Anion analysis of HRESIMS was performed using a Q-TOF micro LC-MS / MS instrument (Waters, USA) and as shown in Figure 2, robustine showed a molecular ion peak at m / z 455.1708, Is the C ₂₅ H ₂₈ O ₈ molecular formula.

NMR spectra of Robustin were measured by dissolving in DMSO-d ₆ solvent and then using JEOL ECP-400 spectrometer (JEOL, Japan). The chemical shift values were obtained from the chemical shift values of DMSO-d ₆ ? C /? H = 40.0 / 2.50 ppm). In the case of 1H-Detected heteronuclear single-quantum coherence (HSQC), ¹ J _CH = 140 Hz was set and HMBC (Heteronuclear Multiple-Bond Coherence) experiment was performed after setting ⁿ _{CH CH} = 8 Hz.

First, ¹ H NMR and ¹³ C NMR spectra show that ¹ H NMR and ¹³ C NMR spectra of rovastine are very similar to NMR spectra of Lobarin of formula (4), as shown in FIGS. 3 and 4 Respectively.

[Chemical Formula 4]

Therefore, the structure of rovastine has a very similar structure to that of rovarin, and it can be predicted that it is a compound produced through removal of water molecule from rovarin considering that the difference in molecular weight is 18 dalton. Comparing the NMR data of rovarin with the NMR data of rovastine reveals that sp ³ hybrid carbons exhibiting absorption peaks at the down field shifted observed in rovavin in the ¹³ C NMR spectrum (C-8 of rovastine , 106.3 ppm) and the absorption peak of the aliphatic methylene carbon disappeared and an absorption peak observed in the two double bond regions was observed instead. In addition, the absorption peak (6.00 ppm) of the magnetic field region in which the olefin group hydrogen, which was not present in the rovaline compound, was observed by ¹ H NMR, and this peak has a spin-spin coupling with the aliphatic methylene group. (Fig. 5). Thus, the structure of rovastine was predicted to be a compound in which double bonds were formed on carbon 8 and 9 while the tertiary alcohol functional group present in rovarin was removed through dehydration reaction. The structure of rovastine thus predicted was confirmed through the analysis of additional two-dimensional NMR spectroscopy, HSQC and HMBC (Table 4). The positions of peaks corresponding to each carbon and hydrogen of rovastine were confirmed through analysis of HSQC (FIG. 6) and HMBC data (FIG. 7), and in particular, those observed from H-5, 9, 10 and 11 in the robustin structure The HMBC correlations provided important data identifying the proposed robustin structure. Also, the geometrical steric structure of the double bonds formed at C-8 and C-9 was Z -type (cis-type), and was confirmed based on NOE correlation observation between H-5 and H-9 (FIG.

NMR Data for lobarstin (400 MHz, DMSO- d 6) Position δ _C [delta] _H , mult. ( J in Hz) HMBC ^a One 104.2 - - 2 157.4 - - 3 101.7 5.90, d (1.8) 1, 2, 4, 5, 7 4 166.4 - - 5 96.7 7.19, d (1.8) 1, 2, 3, 4, 8 6 143.4 - - 7 163.2 - - 8 144.9 - - 9 109.3 6.00, t (7.7) 6, 8 10 27.3 2.36, m 8, 9, 11, 12 11 21.9 1.52, m 9, 10, 12 12 3.63 0.96, t (7.0) 10, 11 One' 108.4 - - 2' 158.2 - - 3 ' 101.9 6.41, s 1 ', 2', 4 ', 5', 7 ' 4' 153.2 - - 5 ' 131.8 - - 6 ' 137.3 - - 7 ' 171.2 - - 8' 27.5 2.70, m / 2.52, m - 9 ' 29.5 1.39, m / 1.27, m - 10 ' 31.4 1.12, m - 11 ' 21.4 1.12, m - 12 ' 13.56 069, t (7.0) 10 ', 11' 4-OCH ₃ 56.3 3.79, s 4 4'-OH - 10.39, s 3 ', 4', 5 '

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

compound Lobarstin's PTP1B  Inhibitory activity assay

In order to analyze the protein tyrosine dephosphorylase-1B (PTP1B) inhibitory activity of rovastine, the enzymatic activity was measured spectroscopically. That is, a solution of compound rovastin (0.5 mg / ml) in PTP1B (Bionea, Korea), PTP1B buffer (20 mM Tris-HCl, pH 8.0, 0.75 mM NaCl, 0.5 mM EDTA, 5 mM? -Mercaptoethanol, 50% (PTyr1146) Insulin Receptor (1142-1153, Santa Cruz, USA) was added to each well and incubated at room temperature for 10 to 30 minutes. After incubation at room temperature for 10 minutes, the reaction with Lobarstin, the inhibitor of PTP1B, and the substrate was terminated. The absorbance at 620 nm was then measured using a Malachite Green-Molybdate Dye Solution (1142-1153, Santa Cruz, Respectively.

As a result, as shown in FIG. 9, inhibition activity of Robustin against PTP1B was analyzed. As a result, it was confirmed that PTP1B inhibitory effect was excellent at IC ₅₀ = 154.6 nM and that the inhibition rate was increased in a concentration- It was confirmed that it is possible to treat and prevent pharmacological treatment.

compound Lobarstin's  Protein tyrosine phosphorylase ( Protein tyrosine  phophatase)

In order to investigate the selectivity of rovastine to the family of protein tyrosine dephosphorylases, the inhibitory activities of PTP1B, PTPN2, PTPN5, PTPN6, PTPN7 and PTPN13 were investigated spectrophotometrically. First, 0.5 unit concentration of PTP1B, PTPN2, PTPN5, PTPN6, PTPN7, PTPN13 (Bioneer, Korea) and protein tyrosine dephosphate buffer solution (20 mM Tris-HCl, pH 8.0, 0.75 mM NaCl, 0.5 mM EDTA, Lobarstin 0, 50, 100, 200 nM and substrate [pTyr1146] Insulin Receptor (1142-1153, Sigma, USA) were added to the reaction mixture and reacted at room temperature for 10 to 30 minutes After the addition of Malachite Green-Molybdate Dye Solution (Sigma, USA) for 10 minutes at room temperature, the reaction with the substrate was terminated and the absorbance was measured at 620 nm.

The selectivity of Robustin to the protein tyrosine dephosphorylase family was investigated. As shown in Fig. 10, rovastine showed an IC ₅₀ for PTP1B At 200 nM, the inhibition rate was 47.96%, and it was found that there was no inhibitory activity against other family members including TC-PTP (PTPN2) (FIG. 10).

TC-PTP (PTPN2), which is most similar to PTP1B in amino acid sequence and 3D structure, is known to be an embrionic lethal and has similar enzyme properties to PTP1B and similar active sites including 2nd aryl-phosphate binding site . Therefore, although 757 substances are registered as targets for PTP1B, none of the compounds that have entered into clinical use with PTP1B as a main target, and only plant extracts that act on various targets including PTP1B have been released or in clinical status. Thus, the above experimental results showing that PTP1B selectively acts on PTP1B in the protein tyrosine dephosphorylase family suggests that rovastine, a compound according to the present invention, can be used as a PTP1B inhibitor in the treatment of type II diabetes mellitus.

compound Lobarstin's  diabetes Disease model animal  Validation of efficacy

5-1: Compound ( Lobarstin ) Was administered intraperitoneally to observe changes in blood glucose

The doses (expressed in mg of test substance / body weight (Kg) of the experimental animals) were determined based on preliminary experiments, efficacy experiments, toxicity tests and data on rovastine. In a 7-week-old male db / db mouse (type 2 diabetic model animal, C57 / BLKS / J-db / db, Korea Research Institute of Bioscience and Biotechnology), 200 袖 L of PBS was administered as a control group and rovastine 10 mg / , And blood glucose was measured twice a week.

The blood glucose changes due to intraperitoneal injection of rovastine in 7-week-old male db / db mice (type 2 diabetes model animal, C57 / BLKS / J-db / db, Korea Research Institute of Bioscience and Biotechnology) In the control group (n = 6), there was a rapid increase in blood glucose level at 271 mg / dL on day 0, 326 mg / dL on day 7, 479 mg / dL on day 14, and 486 mg / dL on day 21, (259 mg / dL on day 14, 267 mg / dL on day 14 and 242 mg / dL on day 21) in the experimental group (n = 6) injected with 10 mg / kg of Lobarstin (Fig. 11).

5-2: Compound ( Lobarstin ) Was intraperitoneally administered. After 6 hours of fasting,

To measure more accurately the antidiabetic effect on rovastine, 200 [mu] l of PBS as a control group was injected into 7-week-old male db / db mice (type 2 diabetes model animal, C57 / BLKS / J-db / db, Korea Research Institute of Bioscience) , Lobarstin (10 mg / kg) was intraperitoneally injected daily as a test group, and blood glucose was measured twice a week. At this time, after fasting for 6 hours after intraperitoneal injection, blood glucose was measured.

As a result, as shown in FIG. 12, in the control group (n = 6), an average of 134 mg / dL on day 0, 238 mg / dL on day 7, 350 mg / dL on day 14, and 479 mg / (N = 6), the mean dose was 134 mg / dL on day 0, 182 mg / dL on day 7, 162 mg / dL on day 14, and 204 mg on day 21 on the day of administration of 10 mg / kg of Lobarstin / dL, which was lower than that of the control group as in Example 5-1.

5-3: Compound ( Lobarstin ) Were weighed after intraperitoneal administration

200 μl of PBS was used as a control in 7-week-old male db / db mice (type 2 diabetic model animal, C57 / BLKS / J-db / db, Korea Research Institute of Bioscience and Biotechnology) and 10 mg / kg of Lobarstin To investigate the change in body weight after intraperitoneal injection and intraperitoneal injection every day, body weight was measured using an animal scales at regular intervals twice a week.

As a result, as shown in Fig. 13, the body weight of the control group (n = 6) was 37.79 g on day 0, 38.74 g on day 7, 38.68 g on day 14, 40.55 g on day 21, (n = 6), the mean body weight change was 37.11 g on day 0, 38.26 g on day 7, 38.56 g on day 14, and 40.90 g on day 21. This indicates that in type 2 diabetic animals treated with the compound (Lobarstin), the body weight was not significantly changed compared with the control group.

5-4: Compound ( Lobarstin ) 28 days after treatment Glucose tolerance test

The following experiments were performed to determine the intraperitoneal glucose tolerance test (IPGTT) in an animal model of the compound (Lobarstin).

20% DMSO was used as a control in 7-week-old male db / db mice (type 2 diabetes model animal, C57 / BLKS / J-db / db, Korea Research Institute of Bioscience and Biotechnology) After 28 days of intraperitoneal injection, glucose (500 ㎎ / ㎖, dose 200 ㎕) was injected by intraperitoneal injection after 16 hours of fasting without 20% DMSO and rovastine, Blood glucose changes were observed in blood samples taken from the tail vein at 60, 90 and 120 min.

As shown in Fig. 14, the glucose tolerance of the type 2 diabetic animals was 204 mg / dL at the time of administration of glucose (20% DMSO) (496 mg / dL), 60 minutes (542 mg / dL), 90 minutes (483 mg / dL) and 120 minutes (424 mg / dL) Lobarstin was administered at a dose of 152 mg / dL for 15 minutes, 227 mg / dL for 15 minutes, 307 mg / dL for 30 minutes, 205 mg / dL for 60 minutes, 162 mg / dL for 90 minutes, / dL, and it was confirmed that the blood glucose level was normalized by low blood glucose increase and fast blood glucose lowering due to drug concentration dependency (FIG. 14). The results of Examples 5-1 to 5-4 show that the novel compound (Lobarstin) according to the present invention has a very excellent antidiabetic effect.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

A compound represented by the following formula (1):
[Chemical Formula 1]

Here, R represents an alkyl group.
2. A compound according to claim 1, wherein said compound is represented by formula (2): < EMI ID =
(2)

A process for preparing a compound represented by the following formula (2), comprising the steps of:
(a) extracting Stereocaulon alpinum with methanol;
(b) eluting the Stereocaulon alpinum extract obtained in step (a) in an aqueous methanol solution using column chromatography;
(c) eluting the fraction eluted in step (b) with an aqueous solution of acetonitrile (CH ₃ CN) using reverse phase high performance liquid chromatography to obtain a fraction containing lobaric acid; And
(d) dissolving the fraction containing rovaric acid in a solvent, adding water, adding NaOH or KOH and stirring, adding an acidic solution to terminate the reaction, and then obtaining a compound of the following formula
(2)

.
The method according to claim 3, wherein the step (d) comprises dissolving the fraction containing rovaric acid in acetone, adding water, adding NaOH, stirring for 10 to 20 minutes, adding an HCl solution to terminate the reaction And concentrated to obtain a methylene chloride dissolution layer through partitioning between methylene chloride and an aqueous solution (pH = 2) to obtain a compound of formula (2)
(2)

.
A pharmaceutical composition for the prevention or treatment of diabetes comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

Here, R represents an alkyl group.
The pharmaceutical composition for preventing or treating diabetes according to claim 5, wherein the compound is represented by formula (2)
(2)

.
A functional food for preventing or improving diabetes comprising a compound represented by the following formula (1) as an active ingredient:
[Chemical Formula 1]

Here, R represents an alkyl group.
8. The diabetic prophylactic or ameliorative functional food according to claim 7, wherein the compound is represented by formula (2)
(2)

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