KR102054392B1 - Protein tyrosine phosphatase 1B inhibitors from an Antarctic marine fungal Aspergillus sp. SF-5929 - Google Patents

Abstract

The present invention relates to a marine derived mold Aspergillus sp. derived novel compound. More specifically, the present invention relates to an Aspergillus sp. derived novel diphenol derivative, (±)-tylopilusin D, and a protein tyrosine phosphatase 1B inhibiting composition containing the (±)-tylopilusin D or a salt thereof as an active ingredient. The (±)-tylopilusin D according to the present invention effectively suppresses activity of PTP1B, thereby being useful for treatment of diseases related to the PTP1B such as cancer, diabetes, and obesity.

Description

Antarctic marine origin Aspergillus sp. SF-5929 Protein tyrosine phosphatase 1B inhibitors {Protein tyrosine phosphatase 1B inhibitors from an Antarctic marine fungal Aspergillus sp. SF-5929}

The present invention is marine fungi strain Aspergillus sp . It relates to a novel compound derived, more particularly Aspergillus sp . The present invention relates to a composition for inhibiting protein tyrosine phosphatase 1B containing the derived new diphenol derivative (±) -tylopilusin D and (±) -tylopilusin D as an active ingredient.

Protein tyrosine phosphatases (PTPs) are signaling enzymes that play an important role in the regulation of protein tyrosine phosphorylation levels and the regulation of many cellular processes (Zhang et al ., Expert Opin Investig Drug . 12: 223, 2003). Malfunctions of PTP activity are associated with several diseases such as diabetes / obesity, cancer, inflammation and autoimmune diseases (Bakke et al ., Semin Cell Dev Biol . 37: 58-65, 2015). Protein tyrosine phosphatase 1B (PTP1B) in PTP is known to be involved in cell survival, proliferation and migration, intercellular communication, metabolism, cytoskeletal tissue and energy consumption (Liu et al. , J Am Chem Soc . 130: 17075, 2008). The main function of PTP1B is to regulate leptin and insulin signaling, which plays a key role in regulating human metabolism. PTP1B has been validated by numerous biochemical and genetic studies as a therapeutic target for the treatment of human diseases such as diabetes / obesity, cancer, inflammation and autoimmune diseases. For example, PTP1B deficient mice have a significantly lower obesity rate and higher basal metabolism and higher energy expenditure, resulting in no dietary obesity (Koren et al., Best Pract Res Clin Endocrinol Metab. 21: 621, 2007 ). These results are consistent with the assumption that PTP1B is a major regulator of energy balance, insulin sensitivity and body fat accumulation in vivo, and suggests that inhibiting PTP1B may be a novel treatment for the treatment of type 2 diabetes and obesity.

It has also been demonstrated that PTP1B is not limited to metabolic regulation but also plays a role in other signaling pathways associated with inflammation and cancer. TNF-α was found to regulate PTP1B expression via NF-κB, and increased TNF-α expression was found to be associated with PTP1B expression under inflammatory conditions (Zabolotny et al., J Biol Chem. 283: 14230 , 2008). It has also been demonstrated that PTP1B plays an important role in breast cancer by inducing breast cancer with expression of PTP1B (Julien et al., Nat Genet . 39: 338, 2007). As biochemical and genetic evidence suggests that PTP1B is associated with a number of diseases, the need for PTP1B inhibition has emerged, and studies of synthetic, pharmaceutical and natural products have been conducted to develop PTP1B inhibitors (Combs, J.). Med Chem. 53: 2333, 2010; Thareja et al., Med Res Rev. 32: 459, 2012, Jiang et al., Acta pharmacologica Sin . 33: 1217, 2012, Zhou et al., Chem Biodivers . 14: e 1600462, 2017).

Accordingly, the present inventors have made intensive efforts to develop a natural substance having PTP1B inhibitory activity. As a result, the present inventors have isolated secondary metabolites having PTP1B inhibitory activity from the extract of Aspergillus niger SF-5929 derived from the Antarctic Ocean and completed the present invention.

An object of the present invention to provide a secondary metabolite having PTP1B inhibitory activity in the extract of Aspergillus niger SF-5929 from Antarctic ocean.

Another object of the present invention to provide a composition for inhibiting PTP1B containing the secondary metabolite as an active ingredient.

Another object of the present invention to provide a pharmaceutical composition for the treatment or prevention of PTP1B-related diseases containing the secondary metabolite as an active ingredient.

In order to achieve the above object, the present invention provides a novel diphenol derivative (±) -tylopilusin D having the structure of Formula 1:

The present invention also provides a composition for inhibiting protein tyrosine phosphatase 1B containing (±) -tylopilusin D or a salt thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for the treatment or prevention of diseases induced by PTP1B expression containing (±) -tylopilusin D or a salt thereof as an active ingredient.

The present invention also provides a composition for inhibiting protein tyrosine phosphatase 1B containing a compound having a formula of any one of Formulas 2 to 7 or a salt thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for the treatment or prevention of diseases induced by PTP1B expression containing a compound having a formula of any one of Formulas 2 to 7 or a salt thereof as an active ingredient.

Aspergillus sp. Biologically active secondary metabolites derived from SF-5929 can effectively inhibit the activity of PTP1B and can be used for the treatment of PTP1B related diseases such as cancer, diabetes, obesity.

1 is isolated from the present invention Aspergillus sp. The structural formulas of 11 bioactive secondary metabolites derived from SF-5929 are shown.
Figure 2 shows the inhibition of hydrolysis of p- NPP by PTP1B activity of (A) tylopilusin D (Compound 1 ), (B) funalenone (Compound 2 ) and (C) aurasperone F (Compound 5 ) isolated from the present invention. It is shown as a Lineweaver Burk plot. Data represent mean ± SD of 3 replicates.

Unless defined otherwise, 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, marine-derived fungus Aspergillus sp. Eleven physiologically active secondary metabolites derived from SF-5929 were isolated and identified as racemic compounds of novel diphenol derivatives in the secondary metabolites, and were named (±) -tylopilusin D. The structure of the (±) -tylopilusin D was confirmed by NMR and MS analysis, and the structure of the remaining 10 compounds was confirmed, and it was confirmed that the compound was previously discovered, and for the 11 compounds, protein tyrosine phos The inhibitory effect on the fatase 1B activity was evaluated to confirm that compounds 1, 2 and 5-7 inhibited PTP1B activity.

Thus, in one aspect, the present invention relates to a novel diphenol derivative (±) -tylopilusin D having the structure of formula (1).

[Formula 1]

In the present invention, marine-derived fungus Aspergillus sp. SF-5929 (KCTC13550BP) was cultured, and the obtained cells were subjected to a combination of chromatography steps using an EtOAc extract to isolate 11 compounds, of which new diphenol-based metabolites were isolated (± ) -tylopilusin D (Compound 1). In addition, 10 previously reported secondary metabolites (compounds 2-11) were obtained.

[Compound 1]-(±) -tylopilusin D

[Compound 2]

[Compound 3]

[Compound 4]

[Compound 5]

[Compound 6]

[Compound 7]

[Compound 8]

[Compound 9]

[Compound 10]

[Compound 11]

As a result of confirming the inhibitory effect of PTP1B on the isolated novel diphenol metabolite (±) -tylopilusin D (Compound 1), the IC ₅₀ value for PTP1B activity was 8.1 ± 0.4 μM, which showed a markedly high inhibitory activity. It was confirmed that (±) -tylopilusin D (Compound 1) could be used as a therapeutic agent for diseases caused by PTP1B expression.

Therefore, in another aspect, the present invention relates to a composition for inhibiting protein tyrosine phosphatase 1B containing (±) -tylopilusin D or a salt thereof as an active ingredient.

In another aspect, the present invention relates to a pharmaceutical composition for treating or preventing a disease induced by PTP1B expression containing (±) -tylopilusin D or a salt thereof as an active ingredient.

In the present invention, diseases induced by the expression of PTP1B include cancer, diabetes, obesity , breast cancer, rheumatoid arthritis , hypertension and the like.

In the present invention, marine-derived fungus Aspergillus sp. Among 11 metabolites isolated from SF-5929 (KCTC13550BP), PTP1B inhibitory activity was confirmed for the first time against compounds 2-7. Aromatic triple ring metabolites (Compound 2), naphtho-γ-pyrones (Compounds 3-6) and cyclic pentapeptide (Compound 7) showed potent PTP1B inhibitory activity in a concentration-dependent manner, and IC ₅₀ values ranged from 3.3 ± 0.2 to 7.9 ± 0.4 μM range.

Therefore, in another aspect, the present invention relates to a composition for inhibiting protein tyrosine phosphatase 1B containing a compound having a formula of any one of Formulas 2 to 7 or a salt thereof as an active ingredient:

In still another aspect, the present invention relates to a pharmaceutical composition for treating or preventing a disease induced by PTP1B expression containing a compound having a formula of any one of Formulas 2 to 7 or a salt thereof as an active ingredient.

In the present invention, diseases induced by the expression of PTP1B include cancer, diabetes, obesity , breast cancer, rheumatoid arthritis, hypertension and the like. .

In one embodiment of the present invention, the results of the kinetics analysis of the inhibitory activity of PTP1B of the compound, it was confirmed that compounds 1, 2 and 5 are non-competitive inhibitors of PTP1B, heterogeneous allosteric or active site of the enzyme Targeted noncompetitive PTP1B inhibitors are more likely to be developed as PTP1B survival-based drugs because the catalytic sites of PTP1B share the common structural motifs of other PTPs (Wiesmann et al. , Nat Struct Mol Biol. 11: 730-737, 2004; Liu et al. , J Am Chem Soc . 130: 17075-17084, 2008).

Carriers used in the pharmaceutical compositions of the present invention include pharmaceutically acceptable carriers, adjuvants and vehicles and are collectively referred to as “pharmaceutically acceptable carriers”. Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of the invention include, but are not limited to, ion exchange, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer materials (eg, Various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal Silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substrates, polyethylene glycols, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene-polyoxypropylene-blocking polymers, polyethylene glycols and wool, and the like.

The route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal.

Oral and parenteral administration is preferred. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intracapsular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques.

Pharmaceutical compositions may be in the form of sterile injectable preparations as sterile injectable aqueous or oily suspensions. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (eg, Tween 80) and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions (eg, solutions in 1,3-butanediol) in nontoxic parenterally acceptable diluents or solvents. Vehicles and solvents that may be used that are acceptable include mannitol, water, ring gel solution, and isotonic sodium chloride solution. In addition, sterile nonvolatile oils are conventionally employed as a solvent or suspending medium. For this purpose, any non-irritating oil may be used including synthetic mono or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in injection formulations as well as pharmaceutically acceptable natural oils (eg olive oil or castor oil), especially their polyoxyethylated ones.

The pharmaceutical compositions of the present invention may be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, and aqueous suspensions and solutions. For oral tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. Useful diluents for oral administration in a capsule form include lactose and dried corn starch. The active ingredient is combined with emulsifiers and suspending agents when the aqueous suspension is administered orally. If desired, sweetening and / or flavoring and / or coloring agents may be added.

The pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the compounds of the invention with suitable non-irritating excipients which are solid at room temperature but liquid at rectal temperature. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

Oral administration of the pharmaceutical compositions according to the invention is particularly useful when the desired treatment involves a site or organ that is easily accessible by topical application. When applied topically to the skin, the pharmaceutical composition should be formulated in a suitable ointment containing the active ingredient suspended or dissolved in the carrier. Carriers for topical administration of a compound of the present invention include, but are not limited to, mineral oil, liquid paraffin, white waselin, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition may be formulated in a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of the invention may also be applied topically to the lower intestine by rectal suppositories and also with suitable enema. Topically applied transdermal patches are also included in the present invention.

The pharmaceutical composition of the present invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmacy, and are prepared in saline using benzyl alcohol or other suitable preservatives, absorption accelerators to enhance bioavailability, fluorocarbons and / or other solubilizers or dispersants known in the art. It can be prepared as a solution.

The compounds of the present invention can be used in combination with conventional anti-inflammatory agents or in combination with matrix metalloprotease inhibitors, lipoxygenase inhibitors and inhibitors of cytokines other than IL-1β. The compounds of the present invention also provide immunomodulators (eg, bropyrimin, anti-human alpha interferon antibodies, IL-2, GM-CSF, methionine enkephalins, interferons to prevent or combat IL-1 mediated disease symptoms such as inflammation). Alpha, diethyldithiocarbamate, tumor necrosis factor, naltrexone and rEPO) or prostaglandin. When the compounds of the present invention are administered in combination with other therapeutic agents, they may be administered sequentially or simultaneously to the patient. Alternatively, the pharmaceutical composition according to the present invention may be made by mixing the compound of formula (1) with other therapeutic or prophylactic agents described above.

In a preferred embodiment, the pharmaceutical composition for oral administration can be prepared by mixing the active ingredient with excipients in solid form and can be prepared in granule form for preparation in tablet or dragee form. Suitable excipients include sugar forms such as lactose, sucrose, mannitol and sorbitol, or cellulose such as starch, methyl cellulose, hydroxypropylmethyl-cellulose or sodium carboxymethylcellulose from corn, flour, rice, potatoes or other plants. Carbohydrates such as gums, including gum arabic, tagacan gum, or protein fillers such as gelatin and collagen. If necessary, disintegrants or solubilizers in the form of respective salts such as crosslinked polyvinylpyrrolidone, agar and alginic acid or sodium alginic acid can be added.

In a preferred embodiment, for parenteral administration, the pharmaceutical composition of the present invention can be prepared in an aqueous solution. Preferably, a physically suitable buffer such as Hanks' solution, Ringer's solution, or physically buffered saline may be used. Aqueous injection suspensions may add a substrate that can increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. In addition, suspensions of the active ingredient may be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or carriers include fatty acids such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Polycationic amino polymers can also be used as carriers. Optionally, the suspension may use a suitable stabilizer or agent to increase the solubility of the compound and to prepare a high concentration of solution.

Monoclonal antibodies in pharmaceutical compositions are adsorbed and unstable in glass containers such as vials and syringes, etc., and are easily deactivated by various physicochemical factors such as heat, pH and humidity. Thus, stabilizers, pH adjusters, buffers, solubilizers, surfactants and the like are added to formulate into stable forms. Examples of the stabilizer include amino acids such as glycine and alanine, sugars such as dextran 40 and mannose, sugar alcohols such as sorbitol, mannitol, and xyltol, and two or more kinds thereof may be used in combination. The amount of these stabilizers added is preferably 0.01 to 100 times, in particular 0.1 to 10 times the weight of the antibody. By adding these stabilizers, the storage stability of the liquid preparation or the lyophilized preparation can be improved. Examples of the buffer include phosphate buffers and citric acid buffers. The buffer adjusts the pH of the aqueous solution after redissolution of the liquid or lyophilized formulation and contributes to the stability and solubility of the antibody. As the addition amount of the buffer, for example, the amount of the liquid preparation or the freeze-dried preparation is preferably set to 1 to 10 mM relative to the yield. Polysorbate 20, pulluronic F-68, polyethyleneglycol, etc. can be used as surfactant, Especially preferably, polysorbate 80 is mentioned, You may use together 2 or more types.

As described above, polymer proteins such as antibodies are easily adsorbed to glass, resin, and the like, which are the materials of the container. Therefore, by adding a surfactant, adsorption of the antibody into the container after re-dissolution of the liquid or lyophilized agent can be prevented. have. As the addition amount of the surfactant, it is preferable to add 0.001 to 1.0% to the weight of water after re-dissolution of the liquid formulation or the lyophilized formulation. Although the above stabilizer, buffer, or adsorption agent can be added to the preparation of the antibody of the present invention, the osmotic pressure ratio that can be used as the osmotic pressure ratio is preferably 1 to 2, especially when used as a medical or animal medicine injection. Osmotic pressure ratio can be prepared by increasing or decreasing sodium chloride at the time of chemical | medicalization. The antibody content in the preparation can be appropriately adjusted according to the applied disease application route, and the dose of the humanized antibody to human is determined by the affinity of the antibody for human protein NSP, that is, the dissociation constant for human protein NSP (Kd value). Depends on the drug, the drug can be expressed even if the dose to humans is reduced by the high affinity (low Kd value).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1 Isolation and Identification of Marine-Derived Fungal Strains

Antarctic marine-derived fungus spergillus sp. SF-5929 (KCTC13550BP) was isolated from coral samples collected from the Antarctic Ross Sea (S 74-37.39 '; E 164-14.26'). 1 g of sample was mixed with 10 mL of sterile seawater and filtered. 0.1 mL of the filtered sample was plated in a PDA (poatato dextrose agar) medium containing seawater, and the plate was incubated at 25 ° C for 14 days. Strain colonies were isolated, passaged several times, and the final pure cultures were selected and stored at -70 ° C. The isolated fungal strain was named SF-5929 and was identified based on analysis of 28S ribosomal RNA (rRNA) sequences. GenBank searches using 28S rRNA gene of SF-5929 (GenBank Accession No. KY131802) were performed with Aspergillus niger (JN938942), A. japonicus (KC128815), A. flavus (JN938926) and A. oryzae (HQ825090), respectively 99.88%, 97.35 %, 97.23% and 97.23% sequence identity, indicating that Aspergillus sp. It was identified as SF-5929.

Example 2: Extraction and Separation of Compound 1-11 from SF-5929 Strain

Aspergillus sp. SF-5929 was individually inoculated into 2 ml fungal strain culture medium in 10 Fernbach flasks containing 300 mL of PDA medium containing 3% NaCl (w / v) and incubated at 25 ° C. for 14 days. The cultured PDA medium was extracted with 4 L of EtOAc and 3.5 g of the dried crude extract were fractionated by reverse phase (RP) C ₁₈ flash column chromatography (4.5 x 30 cm) and then 400 mL of 20, 30, 40, Nine fractions (SF5929-1 to SF5929-9) were obtained through a stepwise gradient with an aqueous methanol solution of 50, 60, 70, 80 and 100% (v / v) MeOH. Fraction SF5929-4 is first separated into two fractions of SF5929-41 and SF5929-42 using Sephadex LH-20 (3x3.5 cm) as stationary phase and MeOH / H ₂ O 3/1 (v / v) solution as mobile phase It was. The separated fraction SF5929-41 was then chromatographed on RP C ₁₈ column (1.5 × ₃₀ cm) and eluted with MeOH / H ₂ O 2/3 (v / v) solution. The fraction SF5929-412 was then further separated over 19 minutes in RP C ₁₈ prep HPLC [45-68% MeOH in H ₂ O (0.1% HCOOH)] to give two metabolites, Compound 10 (3.3 mg, tR = 13 Min) and 11 (3.2 mg, tR = 18 min). Similarly, fraction SF5929-42 was fractionated using RP C18 CC (1.5 × 30 cm) and eluted with a mixture of MeOH (in H ₂ O) in water 2/3 (v / v) to metabolite compound 9 (23 mg, Rf = 0.25).

Fraction SF5929-5 was applied to an RP-C ₁₈ (2.0 × 30 cm) column and the column was eluted with MeOH / H ₂ O 2/3 (v / v) solution to give compound 1 (16 mg, Rf = 0.3) , Three different fractions, SF5929-51 to SF5929-54. Fraction 4 SF5929-54 was treated with a column of Sepadex LH-20, eluted with MeOH / H ₂ O 3/1 (v / v) solution, then further purified by RP C ₁₈ prep HPLC, gradient of MeOH Purification with (0.1% HCOOH) (35-60% over 24 minutes) gave Compound 4 (3.5 mg, tR = 18 minutes). Fraction SF5929-6 was separated into five sub-fractions from SF5929-61 to SF5929-65 by RP C ₁₈ column (2.5 × 35 cm) and eluted with 2/1 (v / v) solution of MeOH / H ₂ O MeOH. I was. The third subfraction SF5929-63 was applied to a Sephadex LH-20 column (2.5 x 35 cm) and eluted with MeOH / H ₂ O 3/1 (v / v) solution to give two metabolites, Compound 2 (27 mg). And Compound 6 (10 mg), and SF5929-631 in the remaining subfractions SF5929-631 and SF5929-634 were further purified by RP C18 column, eluting with MeOH / H ₂ O 2/1 (v / v) solution. Compound 8 (2.5 mg, Rf = 0.3) was obtained as a metabolite. In addition, the SF-2929-65 sub fraction was purified over 30 minutes with RP C18 prep HPLC [50-80% MeOH in H2O (0.1% HCOOH)] to afford compound 5 (8 mg, tR = 28 minutes). In addition, fractionation was carried out from SF5929-8 on RP C18 preparative HPLC [55-73% ACN in H ₂ O (0.1% HCOOH) to give Compound 7 (10 mg, tR = 12 minutes) and Compound 3 (6.5 mg, 20 minutes). Or more] was obtained.

Example 3: Structural Analysis of Compound 1-11 from SF-5929 Strain

Compound 1 obtained in Example 2 was identified by molecular formula C ₁₉ H ₁₆ O ₇ , based on the protonated molecule in m / z 357.0965 (calculation for C ₁₉ H ₁₇ O ₇ , 357.0974) in the HRESIMS data, and was colorless. As an oil of (±) -tyropilusin D (Tylopilusin D) (1) was named. As shown in Table 1, the 1 H NMR spectra of Compound 1 showed δH 6.69 (2H, d, J = 8.8 Hz, H), 6.78 (2H, d, J = 8.8 Hz, H-14 / 18) and 7.78 (2H , d, J = 8.8 Hz, H-8.8 Hz, H-2 / 6), showed signals for two sets of ortho-coupled aromatic protons, and appeared to consist of two para-phenyl rings.

NMR data of (±) -tylopilusin D ( 1 ) location δ _C ^{a, c} δ _H ^{a, d} (mult, J in Hz) δ _C ^{b, c} δ _H ^{b, d} (mult, J in Hz) COZY ^a HMBC ^a One 124.0 - 125.2 - - 2 131.5 7.78 (d, 8.8) 132.0 7.87 (d, 9.0) 3 3, 4, 6, 7 3 115.7 6.78 (d, 8.8) 116.3 6.78 (d, 9.0) 2 1, 2, 4, 5 4 158.4 - 159.6 - 5 115.7 6.78 (d, 8.8) 116.3 6.78 (d, 9.0) 6 1, 3, 4, 6 6 131.5 7.78 (d, 8.8) 132.0 7.87 (d, 9.0) 5 2, 4, 5, 7 7 136.3 - 137.3 - 8 149.7 - 151.2 - 9 198.5 - 200.2 - 10 62.9 3.87 (s) 64.3 3.90 (s) 7, 9, 11, 12, 13, 14, 18. 11 83.2 - 84.7 - 12 172.9 - 174.6 - 13 124.7 - 125.8 - 14 130.8 6.90 (d, 8.8) 132.4 7.00 (d, 8.6) 15 10, 15, 16 15 115.1 6.69 (d, 8.8) 116.0 6.72 (d, 8.6) 14 13, 14, 16, 17 16 157.1 - 158.3 - 17 115.1 6.69 (d, 8.8) 116.0 6.72 (d, 8.6) 18 13, 15, 16, 18 18 130.8 6.90 (d, 8.8) 132.4 7.00 (d, 8.6) 17 10, 16, 17 19 52.1 3.13 (s) 52.8 3.22 (s) 12 4-OH - 9.84 (br) - 8-OH - 10.27 (br) - 11-OH - 6.41 (br) 7, 11, 12 16-OH - 9.38 (br) -

^a Recorded in DMSO- d ₆ , ^b Recorded in MeOH- d ₄ , ^c 100 MHz, ^d 400 MHz

In addition, signals corresponding to methoxy groups (δH 3.13) and sp ³ methine (δH 3.87) were observed. Four signals of δ H 6.41 (4-OH), 9.38 (16-OH), 9.84 (4-OH) and 10.27 (8-OH) were identified as hydroxy protons in the 1 H NMR spectrum. The ¹³ C and DEPT spectra in Table 1 were obtained from 19 carbon signals for 14 aromatic / olefinic carbons (two 1,4-double substituted aromatic rings and four substituted olefins), one at δC 52.2 (C-19). One methine group was shown at δC 62.9 (C-10) together with sp3 quaternary carbon and two carbonyl carbons at the methoxy group, (C-12) and 194.9 (C-9). These units occupy 11 degrees of unsaturation and indicate that there are additional rings in the molecule. DEPT data showed that 12 protons were bound to carbon, requiring four hydroxyl groups in the molecule. In view of this, the chemical shift data shows that three of the aromatic / olefinic carbons (δC 158.4, 149.8 and 157.1) are hydroxylated. After allocating all carbons having protons by analysis of HMQC data, the positions of the two p -hydroxyphenyl rings were identified based on the HMBC correlation of H-2 and C-6 and H-12 and C-16 It became. The presence of carbomethoxy units was confirmed by HMBC correlation of methoxy protons with C-12, C12 and C11 of methoxy protons bound, H-10 and C-11 and C-12 and 11-OH The location of C-7, C-11 and C-12 was determined based on the HMBC correlation. One of the p -hydroxyphenyl groups is linked to C-7 by HMBC correlation of H-6 and C-7, and the other is HMBC correlation of H-14 and C-10 and C-13 and C-14. To C-10. Since the linkage of C-1, -C-7 and -C-11 has already been established in the above, the remaining oxygenated olefin carbon C-8 is paired via C-7 and π-bond, and in this position, the ketone Only the position of carbon (C-9, δC 198.6) is left, and ketone carbon is located between C-8 and C-10 by HMBC correlation and chemical shift of H-10 and C-9. Finally, the overall structure of the established fire ◎ thing 1 is shown below.

[Compound 1]-(±) -tylopilusin D

(+) -Tylopilusins A and B analogs of Compound 1 have been isolated from the fruiting bodies of Tylopilus eximius (Fukuda et al ., J Nat Prod. 75: 2228, 2012). The oxidized quaternary carbon of the (±) -tylopilusins A and B (ie, the C-10 position) is replaced by sp3 methine in compound 1. Since the NOESY data of Compound 1 cannot provide definitive evidence that can specify a relative arrangement, the relative arrangement of C-10 and C-11 of Compound 1 will result in a ¹ H chemical shift of 12-OCH ₃ (δH 3.13) (± ) -tylopilusins A and B compared to the ¹ H chemical shift was confirmed. The chemical shift of methoxy groups in (±) -tylopilusins A and B resonated abnormally in the up-field shifted region, indicating that the protons of the methoxy group are located on the shielding surface of the p -hydroxyphenyl ring attached to C-10. Means that. This carboxylated methoxy group and p at C-10 - means having - (cis relationship) (see the structural formula of compound 1) Relationship - cis with respect to the center-hydroxyphenyl group cyclopentanone ring (cyclopentanone ring).

The structures of other compounds isolated other than compound 1 were determined by analyzing the optical rotation values as well as NMR and MS data and comparing them with previously published data in the literature. TMC-256A1 (4) (Sakurai et al.,. J Antibiot. 55: 685-692, 2002), aurasperone F (5) (Bouras et al., Nat Prod Res . 19: 653-659, 2005), quinoline (2) (Inokoshi et al., J Antibiot. 52: 1095-1100, 1999), fonsecin (6) (Watanabe et al., Tetrahedron Lett. 40: 91-94, 1999), malformin A1 (7) (Kim et al., Biosci Biotechnol Biochem . 57, 240, 1993), carbonarone A (8) (Zhang et al., J Antibiot. 60: 153-157, 2007), pyranonigrin A (9) (Schlingmann et al., J Nat Prod. 70: 1180-1187, 2007), campyrone C (10) (Talontsi et al., Tetrahedron 69: 7147-7151, 2013 ) and campyrone A (11) (Talontsi et al., Tetrahedron 69: 7147-7151, 2013) .

[Compound 2]

[Compound 3]

[Compound 4]

[Compound 5]

[Compound 6]

[Compound 7]

[Compound 8]

[Compound 9]

[Compound 10]

[Compound 11]

Compound 2 is formed in an aromatic tricyclic system and has been reported to inhibit type I collagenase (MMP-1) and to have HIV-1 integrase inhibitory activity (Inokoshi et al., J Antibiot. 52 : 1095-1100, 1999; Shiomi et al., J Antibiot. 58: 65-68, 2005).

Among the isolated compounds, compounds 3 to 6 belong to the group of naphtho-γ-pyrone analogs which are considered to be rich in biological activity. Among these, rubrofusarin B (Compound 3) has an inhibitory effect on Escherichia coli (Ma et al., Nat Prod Res. 30: 1499-1503, 2016) and cytotoxic activity against colon cancer cell line SW1116 (Song et al., FEMS microbiol Lett. 241: 67-72, 2004).

TMC-256A1 (Compound 4) has been shown to inhibit IL-4 signaling (Sakurai et al., J Antibiot. 55: 685-692, 2002). Compound 4 also exhibits potent radical scavenging activity (Leutou et al., Arch Pharm Res . 39: 806-810, 2016).

Recently, an inhibitory effect of dimer naphthopinone (Compound 5) on COX-2 enzyme has been reported (Fang et al., Molecules. 21: E941, 2016).

In addition, various biological properties have been reported for malformin A1 (Compound 7), including cytotoxic effects on human cancer cell lines NCI-H460, MIA Pa Ca-2, MCF-7 and SF-268 (Zhan et al. ., Phytochemistry . 68: 368-372, 2007). Recently, malformin A1 (Compound 7) has been identified as a promising candidate for new virucide based on anti-tobacco mosaic virus (anti-TMV) properties (Herbert et al., Biochem Pharmacol. 48: 1211-1217., 1994). Compounds 8 and 9 contain p-pyron units, while compounds 10 and 11 contain α-pyron units. Compound 8 showed moderate cytotoxicity against K562 cells (Zhang et al., J Antibiot . 60: 153-157, 2007), compound 9 showed antioxidant activity (Tan et al., Int J Mol Sci . 16: 5750-5761, 2015). On the other hand, the biological activities of compounds 10 and 11 have not been reported yet.

Example 4: Analysis of PTP1B Inhibitory Activity of Compounds 1-11

In this example, the enzymatic activity of PTP1B (human, recombinant, ATGen Co., Ltd., Korea) was measured in a reaction mixture containing 2.0 mM EDTA, 5.0 mM DTT and 1 mM pNPP in 50 mM Bis-Tris (pH 6.0). It was. The reaction mixture was reacted for 30 minutes in a 37.5 ° C. water bath, and then 10N NaOH was added to terminate the reaction. The amount of pNPP produced was estimated by measuring absorbance at 405 nm, and the result was corrected for non-enzymatic hydrolysis of pNPP by subtracting the increase in absorbance at 405 nm obtained in the reaction mixture without PTP1B enzyme.

Ursolic acid was used as a positive control in the assay. (Na et al., Planta Me d. 72: 261-263, 2006). Diphenolic metabolite (Compound 1), aromatic tricyclic metabolite (Compound 2), naphtho-γ-pyrones (Compound 3-6) and cyclic pentapeptide (Compound 7) in test compounds 1-11 showed concentration-dependent potent PTP1B inhibitory activity. The IC50 values shown ranged from 3.3 ± 0.2 to 8.1 ± 0.4 μM. On the other hand, compounds 8-11 did not show any inhibitory activity at the 20 μM level. The PTP1B inhibitory activity of these metabolites is summarized in Table 2.

PTP1B inhibitory activity of compounds 1-11 Compounds PTP1B Inhibitory Effects ^a One 8.1 ± 0.4 2 6.1 ± 0.3 3 6.5 ± 0.3 4 5.1 ± 0.3 5 7.9 ± 0.4 6 3.3 ± 0.2 7 5.2 ± 0.5 8 > 20 9 > 20 10 > 20 11 > 20 Ursolic acid ^b 4.3 ± 0.2

^a IC ₅₀ values in μM. ^b a positive control.

Example 5 PTP1B Inhibitor Dynamic Analysis

For kinetics, the activity of PTP1B was tested in the presence or absence of compounds 1, 2 and 5 and reaction mixtures containing different concentrations of pNPP, a PTP1B substrate.

In order to clarify the properties of the PTP1B inhibitor, compounds 1, 2 and 5 were selected, and p-nitrophenylphosphate (p-NPP) was selected as the PTP1B substrate, and the reaction rate analysis was performed by treating p-NPP at a concentration of 0.25-4 mM. Was performed. PTP1B enzyme (1 mg / mL) was diluted to 20.0 μg / mL and test compound (0-10 μM) was incubated with PTP1B enzyme for 30 minutes. The initial rate was determined based on the increase in absorbance at 405 nm. Lineweaver-Burk plots were prepared for test compounds using data obtained from kinetic studies to determine Michaelis-Menten constants (Km) and maximum velocities (Vmax) of PTP1B (GeaphPad Prism 5.01).

As a result, as shown in FIG. 2, Compounds 1, 2 and 5 reduced the Vmax value but did not change the Km value and the titer of the test compound was crossed to the left of the 1 / V axis. This indicates that compounds 1, 2 and 5 inhibit PTP1B enzyme activity in a non-competitive manner. Inhibition constant (Ki) values of compounds 1, 2 and 5 were determined to be 4.0 μM, 3.1 μM and 4.3 μM, respectively. Therefore, Compounds 1, 2 and 5 analyzed through this Example act as non-competitive inhibitors of PTP1B, and the site where the inhibitor acts was different from the catalytic site of the PTP1B enzyme.

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

Novel diphenol derivatives (±) -tylopilusin D having the structure of Formula 1:
[Formula 1]

.
A composition for inhibiting protein tyrosine phosphatase 1B containing (±) -tylopilusin D or a salt thereof as an active ingredient.
A pharmaceutical composition for treating or preventing a disease induced by PTP1B expression containing (±) -tylopilusin D or a salt thereof according to claim 1 as an active ingredient.
The pharmaceutical composition of claim 3, wherein the disease induced by PTP1B expression is selected from the group consisting of cancer, diabetes, obesity , breast cancer, rheumatoid arthritis and hypertension.
A composition for inhibiting protein tyrosine phosphatase 1B containing a compound having a formula of any one of Formulas 2 to 7 or a salt thereof as an active ingredient:

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

.
[Formula 6]

[Formula 7]

.
A pharmaceutical composition for treating or preventing a disease induced by PTP1B expression containing a compound having a formula of any one of Formulas 2 to 7 or a salt thereof as an active ingredient:

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

.
[Formula 6]

[Formula 7]

.
The pharmaceutical composition of claim 6, wherein the disease induced by PTP1B expression is selected from the group consisting of cancer, diabetes, obesity , breast cancer, rheumatoid arthritis and hypertension.

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