KR101742238B1 - Novel compounds, Composition Comprising the Same for Preventing and Treating Inflammatory Diseases - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a novel compound and a composition for preventing or treating an inflammatory disease containing the same as an active ingredient. More particularly, the present invention relates to a novel compound and a composition for preventing or treating an inflammatory disease comprising Aspergillus sp. Asperanin A, asperanin B, asperanin C, 5-prenyl-dihydrobarcocholine F and 5-prenyl-dihydrodoburumazine A, which have anti-inflammatory effects isolated from SF-5976, And a composition for preventing or treating an inflammatory disease containing the same. The present invention relates to an inflammatory disease comprising asperanin A, asperanin B, asperanin C, 5-prenyl-dihydrobarcocholine F or 5-prenyl-dihydrobromarin A according to the present invention as an active ingredient The composition for preventing or treating RA inhibits the proinflammatory cytokine and mediator production in RAW264.7 macrophages and BV2 microglia cells in association with the inflammatory reaction and thus can be usefully used for the prophylactic or therapeutic use of inflammatory diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel compound and a pharmaceutical composition for preventing or treating an inflammatory disease containing the same as an effective ingredient.

TECHNICAL FIELD The present invention relates to a novel compound and a composition for preventing or treating an inflammatory disease containing the same as an active ingredient. More particularly, the present invention relates to a novel compound and a composition for preventing or treating an inflammatory disease comprising Aspergillus sp. Asperanin A, asperanin B, asperanin C, 5-prenyl-dihydrobarcocholine F and 5-prenyl-dihydrodoburumazine A, which have anti-inflammatory effects isolated from SF-5976, And a composition for preventing or treating an inflammatory disease containing the same.

Inflammation is a useful protective response to injury or damage, which is designed to inhibit the deleterious effects of injury (Zhang, G et al., 2001. J. Clin. Invest, 107, 13-19). Macrophages and microglia are the major inflammatory and immune effector cells activated by bacterial lipopolysaccharides (LPS), interferon-gamma and proinflammatory cytokines. The activated cells are interleukin -1β, interleukin-6, tumor necrosis factor -α, nitrogen monoxide (NO), and prostaglandin _{E 2 (prostaglandin E 2; PGE} 2) and the cells of inflammation through the product and mediators of inflammatory cytokines such as And molecular pathophysiology, which are known to be involved in the onset of chronic diseases such as cardiovascular and neurological diseases as well as cancer and autoimmune diseases.

Nitric oxide (NO) is inducible by inflammatory molecules produced by nitric oxide synthase (iNOS), an excessive increase in iNOS activity or production of nitric oxide is the pathogenesis of various inflammatory diseases (McCartney-Francis et al., J. Exp, Med ., 178, 749754, 1993, Szabo et al., New Horiz , 3, 232, 1995). Since prostaglandin E ₂ (PGE ₂ ) synthesized by the cyclooxygenase-2 (COX-2) enzyme is an important mediator of inflammatory symptoms such as fever and pain (Samuelsson et al., Pharmacol. Rev. 59: 207224 , 2007; Simmons et al., Pharmacol. Rev. 56: 387437, 2004), inhibition of the production of these inflammatory mediators is useful in the treatment of various inflammatory diseases. Thus, inhibition of inflammatory cytokines and mediators may be therapeutic targets for the prevention of inflammation-related chronic diseases.

Marine microorganisms including bacteria, cyanobacteria, microalgae and fungi are important sources of new pharmacologically active secondary metabolites (Bugni and Ireland et al., Nat. Prod. Rep. 21: 143163, 2004) Is an abundant and promising source of novel antiviral, antiinflammatory, antimicrobial and anticancer agents (Bhadury et al., J. Ind. Microbiol. Biotechnol. 33: 325337, 2006). Recently, the present inventors have found that Aspergillus sp. SF-5976 reported that some dihydroisocoumarins exhibit anti-inflammatory effects on LPS-stimulated BV2 microglia (Kim et al., Nat. Prod . 2015, 78, 2948-2955).

Thus, the present inventors have found that marine fungus Aspergillus sp. Scale-up fermentation was performed from SF-5976 to obtain the physiologically active secondary metabolites Asperanin A, Asperanin B, Asperanin C, 5-prenyl-dihydrobarcocholine F and 5 -Prednyl-Dihydron Bromazine A was isolated and the anti-inflammatory effects of the above five compounds were confirmed on the inflammatory response induced by LPS in RAW 264.7 macrophages and BV2 microglia, thereby completing the present invention.

It is an object of the present invention to provide novel compounds having anti-inflammatory activity.

Another object of the present invention is to provide a pharmaceutical composition containing asperinin A, asperanin B, asperanin C, 5-prenyl-dihydrobarcocholine F or 5-prenyl-dihydrowromeum A as an active ingredient And to provide a composition for preventing and treating inflammatory diseases.

Another object of the present invention is to provide a pharmaceutical composition containing asperanin A, asperanin B, asperanin C, 5-prenyl-dihydrobarcocholine F or 5-prenyl-dihydrowromeum A as an active ingredient And to provide a functional food for preventing or improving an inflammatory disease.

In order to achieve the above object, the present invention provides the following compounds or a pharmaceutically acceptable salt thereof:

i) 6-hydroxy-2 - ((E) -1-hydroxybut-2-enyl) -7- (3-methylbut-2-enyl) chroman-5-carbaldehyde;

ii) 2,6-dihydroxy-7- (3-methylbut-2-enyl) chroman-5-carbaldehyde;

2-methylbut-3-ene-2-yl) - (3-methylbutyl) 1H-indol-3-yl) methyl) -6-methylpiperazine-2,5-dione; And

2- (2-methylbut-3-en-2-yl) - (2-methyl- 1H-indol-3-yl) methyl) -6-methylpiperazine-2,5-dione.

The present invention also provides a composition for the prevention and treatment of inflammatory diseases, which comprises the following compound or a pharmaceutically acceptable salt thereof as an active ingredient:

i) 6-hydroxy-2 - ((E) -1-hydroxybut-2-enyl) -7- (3-methylbut-2-enyl) chroman-5-carbaldehyde;

ii) 2,6-dihydroxy-7- (3-methylbut-2-enyl) chroman-5-carbaldehyde;

2-methylbut-3-ene-2-yl) - (3-methylbutyl) 1H-indol-3-yl) methyl) -6-methylpiperazine-2,5-dione; 3-methylbutyl) -5- (3-methylbut-2-enyl) -2- (2-methylbut-3-en- 2- yl) -1H-indol-3-yl) methyl) -6-methylpiperazine-2,5-dione.

The present invention also provides a functional food for improving inflammation comprising, as an active ingredient, the following compound or a pharmaceutically acceptable salt thereof:

i) 6-hydroxy-2 - ((E) -1-hydroxybut-2-enyl) -7- (3-methylbut-2-enyl) chroman-5-carbaldehyde;

ii) 2,6-dihydroxy-7- (3-methylbut-2-enyl) chroman-5-carbaldehyde;

2-methylbut-3-ene-2-yl) - (3-methylbutyl) 1H-indol-3-yl) methyl) -6-methylpiperazine-2,5-dione; And

2- (2-methylbut-3-en-2-yl) - (2-methyl- 1H-indol-3-yl) methyl) -6-methylpiperazine-2,5-dione.

The compounds according to the invention, especially asperanin A, asperanin B, asperanin C, 5-prenyl-dihydrobarcocholine F and 5-prenyl-dihydrowromeamine A, Inhibits proinflammatory cytokine and mediator production in RAW264.7 macrophages and BV2 microglia, and thus can be usefully used for the prophylactic or therapeutic use of inflammatory diseases.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the results of a comparison of asperanin A (A), asperanin B (B), asperanin C (C), 5-prenyl-dihydrobarcocholine F and 5-prenyl- (D). ≪ / RTI >
Figure 2 shows the effect of compounds of the invention on iNOS and COX-2 protein expression in LPS-stimulated RAW 264.7 macrophages, including asperanin A (A), asperanin B (B), asperanin C (C), 5-prenyl-dihydrobarcocholine F (D) and 5-prenyl-dihydrowromeamine A (E).
Figure 3 shows the effect of compounds of the invention on the expression of iNOS and COX-2 protein in LPS-stimulated BV2 microglia cells, including asperanin A (A), asperanin B (B), asperanin C (C), 5-prenyl-dihydrobarcocholine F (D) and 5-prenyl-dihydrowromeamine A (E).

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 and the experimental methods described below are well known and commonly used in the art.

In the present invention, the marine fungus Aspergillus sp., Collected in Ross Sea (N 76 ° 06.256 ', E 169 ° 2.675'). A novel compound was isolated from SF-5976 and its structure was identified.

Thus, in one aspect, the present invention relates to a compound or a pharmaceutically acceptable salt thereof,

i)

2-enyl) -7- (3-methylbut-2-enyl) chroman-5-carbaldehyde (6-hydroxy- Ethyl] -7- (3-methylbut-2-enyl) chroman-5-carbaldehyde)

[Chemical Formula 1]

ii) Preparation of 2,6-dihydroxy-7- (3-methylbut-2-enyl) chroman-5-carbaldehyde (2,6-dihydroxy- Carbaldehyde)

(2)

iii)

3 - ((7- (3-chloro-2-hydroxy-3-methylbutyl) -5- (3-methylbut- 2-enyl) -2- (2-methylbut- 2-hydroxy-3-methylbutyl) -5- (3-methylbut-2 Yl) methyl) -6-methylpiperazin-2, 5-dione) (2-methyl-

(3)

or,

2- (2-methylbut-3-en-2-yl) - (2-methyl- Methylpiperazine-2,5-dione (3 - ((7- (2-hydroxy-3-methoxy- Yl) methyl) -6-methylpiperazin-2, 5-dione) (2-methyl-

[Chemical Formula 4]

In one embodiment of the present invention, Asperanin A represented by the following formula 1-1, i.e., (R) -6-hydroxy-2- ((S, E) -1-hydroxybut- ) - 7- (3-methylbut-2-enyl) chroman-5-carbaldehyde [(R) -6- (3-methylbut-2-enyl) chroman-5-carbaldehyde] was isolated and its structure was confirmed.

[Formula 1-1]

The Asperanin A of the present invention is a marine fungus Aspergillus sp., Which was collected at Ross Sea (N 76 ° 06.256 ', E 169 ° 2.675'). The compound having anti-inflammatory effect extracted from SF-5976 (Accession No .: KCTC 12950BP), which is a pale yellow amorphous solid, is a compound of molecular formula C ₁₉ H ₂₄ O ₄ and has a structure of formula 1-1 .

Also, analysis of ¹ H and ¹³ C NMR data of asperanin A showed that chaetopyranin and 2-hydroxybutyrate were present at the C-4 'except for the presence of a hydroxy group and a double bond at C-5' and C-6 ' It has been confirmed that it has a benzaldehyde structure similarly having 2- (1-hydroxy-but-2-enyl) -dihydropyran and 3-methylbut-2-enyl (prenyl) group.

In another embodiment of the present invention, asperanin B, i.e., (R) -6-hydroxy-2- ((R, E) -1-hydroxybut- ) - 7- (3-methylbut-2-enyl) chroman-5-carbaldehyde [(R) -6- (3-methylbut-2-enyl) chroman-5-carbaldehyde] was isolated and its structure was confirmed.

[Formula 1-2]

The asperanin B of the present invention is a marine fungus Aspergillus sp. ≪ RTI ID = 0.0 > g. ≪ / RTI > collected from the Ross Sea (N 76 ° 06.256 ', E 169 ° 2.675' A compound having an anti-inflammatory effect extracted from SF-5976 (Accession No .: KCTC 12950BP), a pale yellow amorphous solid, a molecular formula of C ₁₉ H ₂₄ O ₄ and a spectrum similar to asperanin A, 2. ≪ / RTI >

Absolute alignment of asperanin A and asperanin B was determined by comparing the experimental and theoretical ECD spectra using a time-dependent density functional theory (TDDFT) method. The morphological distribution was performed under the Merk Molecular Force Field (MMFF) using Spartan'14 software, and the geometric optimization of the selected shape is performed at the B3LYP / 6-31 + G (dp) level in the Gaussian 09 package Respectively. The calculated ECD spectra were expressed at the CAM-B3LYP / TZVP level in MeCN with a conductor like polarizable continuum solvent model (CPCM), consistent with the experimental results, and the absolute arrangement of compound 1-1 as 3'R and 4 ' 'Absolute arrangement of compound 1-2 as R and 4'R (FIG. 1).

In another embodiment of the present invention, asperanin C, i.e., (R) -2,6-dihydroxy-7- (3-methylbut-2-enyl) chroman- 5-carbaldehyde [(R) -2,6-dihydroxy-7- (3-methylbut-2-enyl) chroman-5-carbaldehyde] was isolated and its structure was confirmed.

[Formula 2-1]

Asperanin C of the present invention is a marine fungus Aspergillus sp., Collected in the Ross Sea (N 76 ° 06.256 ', E 169 ° 2.675'). SF-5976 (Accession No .: KCTC 12950BP). It was found to be a pale yellow amorphous solid and a compound of molecular formula C ₁₅ H ₁₈ O ₄ having the structure of formula (2-1).

In addition, the absolute arrangement of asperanin C was determined based on empirical and measured ECD spectrum comparisons using the TDDFT method and shows the 3'R arrangement (FIG. 1).

In another embodiment of the present invention, 5-Prenyl-dihydrovariecolorin F, i.e., (3R, 6R) -3 - ((7 - ((S) -3-chloro-2-hydroxy-3-methylbutyl) -5- (3-methylbut-2-enyl) -2- 3-yl) methyl) -6-methylpiperazine-2,5-dione [(3R, 6R) -3 - ((7- ( Yl) methyl) -6-methylpiperazine-2,5 (3-methylbut-2-enyl) - Dione] was isolated and its structure confirmed.

[Formula 3-1]

The 5-prenyl-dihydrobarcocholine F of the present invention is a marine fungus Aspergillus sp. Collected in the Ross Sea (N 76 ° 06.256 ', E 169 ° 2.675'). SF-5976 (Accession No .: KCTC 12950BP), which is a white amorphous solid and has the structure of Formula 3-1 as a compound of molecular formula C ₂₉ H ₄₀ ClN ₃ O ₃ .

The overall structure of 5-prenyl-dihydrovariocolorin F was determined by detailed analysis of COZY and HMBC data. A modified Mosher's method was used with COZY and ¹ H NMR data to specify the 27 S sequence. Relative positioning at C-9 and C-12 was confirmed by the NOESY cross-peak of H-9 / H-12. Absolute arrays at C-9 and C-12 were deduced by comparing the experimental and theoretical ECD spectra using the TDDFT method. Two possibilities (4-p1: 9R and 12R 4-p2: 9S and 12S) were predicted at the CAM-B3LYP / SVP level in the MeCN CPCM model and the arrangement of the remaining positions was fixed at C-27, (FIG. 1).

In another embodiment of the present invention, 5-Prenyl-dihydrorubrumazine A, namely (3R, 6R) -3 - ((7- ( (S) -2-hydroxy-3-methoxy-3-methylbutyl) -5- (3-methylbut-2-enyl) -2- (2-methylbut- methyl-6-methylpiperazine-2,5-dione [(3R, 6R) -3 - ((7 - ((S) -2-hydroxy-3-methoxy- Yl) methyl) -6-methylpiperazine-2,5 (2-methylbut-2-enyl) - Dione] was isolated and its structure confirmed.

[Formula 4-1]

The 5-prenyl-dihydrobromarin A of the present invention is a marine fungus Aspergillus sp. Collected from the Ross Sea (N 76 ㅀ 06.256 ', E 169 ㅀ 2.675'). SF-5976: a compound having an anti-inflammatory effect extracted from (Accession No. KCTC 12950BP), a white amorphous solid, which was identified to have the structure of formula 4-1 with a compound of the molecular formula _{C 30 H 43 N 3 O 4} .

The relative arrangement at C-9 and C-12 is identical to 5-prenyl-dihydrobaroicholine F, and the absolute sequence at C-9 and C-12 is 5-prenyl-dihydrobaroicholine F Lt; RTI ID = 0.0 > 9R and 12R < / RTI >

The compounds of the present invention are useful in the treatment of marine fungi Aspergillus sp. SF-5976 (KCTC12950BP).

In the present invention, Aspergillus sp. The novel compounds extracted from SF-5976 inhibit the production of nitric oxide and PGE ₂ in RAW264.7 macrophages and BV2 microglia, and inhibit iNOS and COX-2 expression, thus being effective for the prevention or treatment of inflammatory diseases Respectively.

Therefore, the present invention provides, in a different aspect, a pharmaceutical composition comprising the aspirinin A, asperanin B, asperanin C, 5-prenyl-dihydrobarcocholine F or 5-prenyl- And to pharmaceutical compositions for the prevention and treatment of inflammatory diseases.

The inflammatory disease of the present invention is a group consisting of arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, dermatitis, periodontitis and gingivitis But it is not limited thereto.

The asperanin A, asperanin B, asperinin C, and 5-prenyl-dihydrodoburmagine A isolated in the present invention inhibited the expression of the inducible NO synthase (iNOS) in RAW 264.7 cells, Inhibits the production of nitrogen (NO), and aspenin A, asperanin B, asperanin C, 5-prenyl-dihydrobarcocholine F and 5-prenyl- Inhibit the production of nitrogen monoxide by inhibiting the expression of iNOS in the cells.

In addition, among the compounds of the present invention, asperanin A, asperanin B, asperinin C, and 5-prenyl-dihydrowromeamine A were detected in RAW 264.7 cells as cyclooxygenase-2 -2), thereby inhibiting the production of prostaglandin E2 (PGE2) and inhibiting the expression of asperanin A, asperanin B, asperanin C, 5-prenyl-dihydrobarericholine F and 5- Neil-dihydrobromazine A inhibits the production of PGE2 by inhibiting the expression of COX-2 in BV2 cells.

The compositions containing the compounds of the invention may additionally comprise suitable carriers, excipients or diluents according to conventional methods. 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.

The composition containing the compound of the present invention may be administered orally in the form of powders, pills, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, Or < / RTI >

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 composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may be appropriately determined depending on the health condition of the patient, , The excretion rate, the degree of disease, the type of drug, the time of administration, the route of administration, and the period of administration, 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 a daily dose of 0.001 to 1000 mg / kg, preferably 0.01 to 100 mg / kg. 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.

In addition, the composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of inflammatory diseases.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising asperanin A, asperanin B, asperanin C, 5-prenyl-dihydrobieriocolorin F or 5-prenyl-di The present invention relates to a food composition for improving inflammation containing Drawbrumarin A as an active ingredient.

The functional food of the present invention includes all forms such as nutritional supplement, health food, and food additives. These types of functional foods can be prepared in various forms according to conventional methods known in the art. For example, as a health food, the benzaldehyde or dioxopiperazine alkaloid of the present invention may be prepared in the form of tea, juice, and drink for drinking, granulated, encapsulated, and powdered. In addition, the functional food of the present invention can be used in a variety of forms such as beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruits, bottled, jam, marmalade, etc.), fish, meat and processed foods ), Breads and noodles (eg udon noodles, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, sugar, dairy products (eg butter, cheese etc.), edible vegetable oils, margarine, vegetable protein, And can be prepared by adding the benzaldehyde derivative or dioxapiperazine alkaloid derivative of the present invention to foods, frozen foods, various kinds of seasonings (e.g., soybean paste, soy sauce, sauce, etc.).

The definitions of the main terms used in the description of the present invention and the like are as follows.

As used herein, the term "inflammation" refers to a defense mechanism of biological tissues against a certain stimulus, and is a mechanism of biological defense to recover injury by various harmful stimuli to restore original state. Irritation of the inflammation, infection, or chemical and physical stimulation, and the process of inflammation can be divided into two types of acute and chronic inflammation. Acute inflammation is a short-term reaction within a few days. Plasma components and blood cells are involved in dephosphorylation by displaying a microcirculatory system. Chronic inflammation has a long duration, and tissue proliferation is seen.

As used herein, the term "nitric oxide" refers to a substance that increases the production amount of nitric oxide synthase upon inducing an intracellular inflammatory reaction. In the nervous system, nitric oxide is synthesized by the nervous system nitric oxide synthase (NOS) present in nerve cells. The synthesized nitric oxide increases the production of cGMP in brain cells, and thereby functions to store externally-recognized information for a long time. Nitric oxide (NO) is a free radical known to be involved in physiological and pathological processes. NO is synthesized by oxidation of L-arginine by nitric oxide synthase (Atkan, et al., 75: 639-653, 2004).

As used herein, "COX-2" is an enzyme involved in the production of prostaglandin, a protein related to an inflammatory reaction. An increase in intracellular COX-2 expression level may be an indicator of progress of the inflammatory reaction.

As used herein, "prostaglandin E ₂ (PGE ₂ )" is an inflammatory mediator produced at the site of inflammation by COX-2, called prostaglandin endoparcoxide synthase. PGE ₂ has been implicated in chronic inflammatory diseases including many cardiovascular diseases, arthritis, inflammatory bowel disease and chronic gastric ulcer (St-Onge, M. et al., Biochim. Biophys. Acta. 1771: 1235-1245, 2007 Et al., Annu. Rev. Med. 53: 35-57, 2002; Rocca, B. et al., Int. Immunopharmacol. 2: 603-630, 2002; Singh, VP et al., Pharmacology 72: 77-84, 2004).

[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.

Example 1: Marine fungi Aspergillus  sp. Identification and culture of SF-5976

From an unidentified marine organism collected from the Ross Sea (N 76 ° 06.256 ', E 169 ° 2.675'), Aspergillus sp. SF-5976 (accession number: KCTC 12950BP) was obtained and identified based on rRNA sequence analysis. The GenBank results retrieved with the 28S rRNA gene of SF-5976 (KR150444) showed 100% sequence similarity to A. pseudoglaucus (FR839678), A. cibarius (FR848828), and A. glaucus (JN938918) Not confirmed. The mold strain SF-5976 was cultured in a Petri dish (150 mm x 2 cm x 150 plate) with PDA supplemented with 3% NaCl for 20 days at 25 ° C.

Example 2: Aspergillus  sp. Extraction and Separation of Compounds from SF-5976

IR spectra were recorded on a Varian 640 FT-IR spectrometer (Varian, Palo Alto, Calif., USA) with optical rotation of Jasco P-2000 (Jasco, Tokyo, Japan), UV spectra of Mecasys optizen pop spectrometer (Mecasys, Daejeon, Korea) (Waters, Manchester, UK) was used for the JASCO J-1100 spectropolarimeter, the Varian 500 MHz NMR spectrometer for the NMR spectrum, and the ESI-TOF-MS data for the CD spectra.

The strain culture of Example 1 was extracted with MeOH (3 X 2.0 L) at room temperature and evaporated in vacuo at 30 ° C. The residue was suspended in H ₂ O (1.0 L) and partitioned with EtOAc (3 × 1.0 L) to give an EtOAc-dissolving extract (2.6 g). The extract was further divided on silica gel into 16 fractions (F1-F16) with MPLC (n-hexane? HCl3? Cetone, 1: 1: 0 to 0: 0: 1, flow rate 35.0 mL / min) F5 (68.3 mg) was purified by preparative HPLC (MeCN-H2O, 1: 1 to 1: 0, flow rate 8.0 mL / min) to give asperanin A (compound 1, 1.4 mg), asperanin B 2, 2.9 mg) and asperanin C (compound 3, 1.0 mg). F12 (147.0 mg) was purified by preparative HPLC (MeCN-H2O, 1: 4 to 1: 0, flow rate 8.0 mL / min) to give 5-prenyl-dihydrovariecolorin F (compound 4, 3.4 mg) and 5-prenyldihydrorubrumazine A (Compound 5, 1.7 mg).

2-1: Asperanin A, light yellow amorphous solid

[?] ²² _D -7.0 (c 0.01, MeOH); UV (MeOH)? _Max (log?) 208 (4.0), 236 (3.7), 277 (3.6), 386 (3.3) nm; IR? _Max (ATR) 3426, 2920, 1641, 1436, 1297, 1195 cm ^-1 ; CD (c 0.5 mM, MeCN)?? -0.9 (219), +1.1 (232), +0.7 (240); ¹ H and ¹³ C NMR (CDCl ₃ , 500 and 125 MHz), see Table 1; ESIMS (negative) m / z 315 [M - H] ^??? ; HRESIMS m / z 315.1607 [MH] ^??? _{(calcd for C 19 H 23 O} 4, 315.1596).

2-2: Asperanin B, light yellow amorphous solid

[?] ²² _D -5.8 (c 0.01, MeOH); UV (MeOH)? _Max (log?) 206 (3.9), 233 (3.6), 277 (3.5), 386 (3.1) nm; IR? _Max (ATR) 3427, 2920, 1640, 1434, 1296, 1194 cm ^-1 ; CD (c 0.5 mM, MeCN)?? +0.8 (212), +1.7 (220), +0.7 (231), +1.2 (241); ¹ H and ¹³ C NMR (CDCl ₃ , 500 and 125 MHz), see Table 1; ESIMS (negative) m / z 315 [M ?? H] -; HRESIMS m / z 315.1598 [M ??? H] ^??? _{(calcd for C 19 H 23 O} 4, 315.1596).

2-3: Asperanic C, light yellow amorphous solid

[?] ²² _D -3.2 (c 0.01, MeOH); UV (MeOH)? _Max (log?) 208 (3.9), 234 (3.7) 276 (3.6) nm; IR? _Max (ATR) 3408, 2925, 1680, 1640, 1435, 1295, 1210 cm ^-1 ; CD (c 0.5 mM, MeCN)?? -1.6 (206), +0.3 (218), -0.1 (225), +0.9 (244); ¹ H and ¹³ C NMR (CDCl ₃ , 500 and 125 MHz), see Table 1; ESIMS (negative) m / z 261 [MH] ^- ; HRESIMS m / z 261.1136 [MH] ?? _{(calcd for C 15 H 17 O} 4, 261.1127).

One location δ _C 隆_H HMBC ^a One 116.4, C 2 119.6, C 3 146.2, C 4 127.3, CH 6.97, s 2, 3, 6, 1 " 5 130.2, C 6 156.4, C 7 194.4, C 10.22, s 1, 6 One' 20.3, CH ₂ 3.16, m 2, 3, 2 ', 3' 3.00, m 2, 2 ' 2' 23.2, CH ₂ 2.06, m 2, 1 ' 1.74, m 1 ', 3' 3 ' 78.1, CH 3.81, ddd (8.0, 7.5, 2.0) 4' 75.1, CH 4.13, dd (8.0, 7.0) 3 ', 5', 6 ' 5 ' 128.8, CH 5.56, ddd (15.0, 7.0, 1.5) 7 ' 6 ' 130.9, CH 5.88, dq (15.0, 6.5) 4 ', 7' 7 ' 18.0, CH ₃ 1.77, d (6.0) 5 ', 6' One" 27.0, CH ₂ 3.29, d (7.5) 4, 5, 6, 2 ", 3 " 2" 121.0, CH 5.29, t (7.5) 4 ", 5 " 3 " 133.9, 4" 25.8, CH ₃ 1.76, s 2 ", 3 ", 5 " 5 " 17.8, CH ₃ 1.70, s 2 ", 3 ", 4 " OH-6 11.99, s 1, 5, 6 2 location δ _C 隆_H HMBC ^a One 116.5, C 2 119.7, C 3 146.7, C 4 127.3, CH 6.96, s 2, 3, 6, 1 " 5 129.7, C 6 156.3, C 7 194.4, C 10.22, s 1, 6 One' 20.5, CH ₂ 3.20, m 2, 3, 2 ', 3' 3.00, m 2, 2 ' 2' 21.3, CH ₂ 2.06, m 2, 1 ' 1.86, m 1 ', 3' 3 ' 78.1, CH 3.92, ddd (7.5, 3.5, 1.5) 4' 74.1, CH 4.34, dd (7.0, 3.5) 5 ' 128.5, CH 5.60, ddd (15.0, 7.0, 1.5) 7 ' 6 ' 130.0, CH 5.84, dq (15.0, 6.5) 4 ', 7' 7 ' 18.0, CH ₃ 1.76, d (6.0) 5 ', 6' One" 27.0, CH ₂ 3.29, d (7.5) 4, 5, 6, 2 ", " 2" 121.1, CH 5.29, t (7.5) 4 ", 5 " 3 " 133.8, 4" 25.8, CH ₃ 1.76, s 2 ", 3 ", 5 " 5 " 17.8, CH ₃ 1.70, s 2 ", 3 ", 4 " OH-6 11.98, s 1, 5, 6 3 location δ _C 隆_H HMBC ^a One 116.3, C 2 119.8, C 3 143.5, C 4 127.3, CH 6.92 s 2, 3, 6, 1 " 5 130.2, C 6 156.5, C 7 194.4, C 10.27 s 1, 6 One' 16.1, CH ₂ 3.15m 3.09 m 2' 26.3, CH ₂ 2.12 m 2.00, m 3 ' 90.2, CH 5.61, t (3.0) 4' 27.0, CH ₂ 3.29, d (7.5) 4, 5, 6, 2 " 5 ' 121.0, CH 5.28, t (7.0) 6 ' 133.9, C 7 ' 25.8, CH ₃ 1.75, s 2 ", 3 ", 5 " One" 17.8, CH ₃ 1.69, s 2 ", 3 ", 4 " 2" 3 " 4" 5 " OH-6 11.98, s 1, 5, 6 ^a HMBC correlations, optimized for 8 Hz.

2-4: 5-prenyl-dihydrobarcocholine F, white amorphous solid

[?] ²⁴ _D -41.5 (c 0.01, MeOH); UV (MeOH)? _Max (log?) 208 (3.7), 230 (3.8), 288 (3.2) nm; IR? _Max (ATR) 3347, 2925, 1674, 1457, 1373, 1327 cm- ¹ ; CD (c 0.5 mM, MeCN)?? -3.2 (210), +6.2 (225), -1.1 (240), +2.9 (252), +3.9 (272), -0.4 (301); ¹ H and ¹³ C NMR (CDCl ₃ , 500 and 125 MHz), see Table 2; ESIMS (positive) m / z 514 [M + H] < ⁺ >; ESIMS (negative) m / z 512 [MH] ^- , 514 [M + 2] H]; HRESIMS m / z 512.2665 [M ?? H] ^??? (calcd for C ₂₉ H ₃₉ ClN ₃ O ₃ , 512.2680).

2-5: 5-Prenyl-Dihydron Bromazine A, white amorphous solid

_{^{[α] 23 D -23.0 (c}} 0.01, MeOH); UV (MeOH)? _Max (log?) 207 (3.7), 228 (3.7), 279 (3.2) nm; IR? _Max (ATR) 3357, 2923, 1677, 1442, 1378, 1209 cm ^-1 ; CD (c 0.5 mM, MeCN)?? +0.5 (208), +3.9 (228), -2.8 (243), +1.7 (278), -1.4 (300); ¹ H and ¹³ C NMR (CDCl ₃ , 500 and 125 MHz), see Table 2; ESIMS (positive) m / z 510 [M + H] < ⁺ >; ESIMS (negative) m / z 508 [M - H] ^- ; HRESIMS m / z 508.3171 [M - H] - (calcd for C 30 H 42 N 3 O 4, 508.3175).

One location δ _C 隆_H HMBC ^a 2 142.3, C 3 142.3, C 3a 129.2, C 4 115.8, CH 7.18, s 3, 3a, 6, 7a, 21 5 133.6, C 6 123.7, CH 6.76, s 4, 7a, 21, 26 7 122.0, C 7a 132.7, C 8 29.4, CH ₂ 3.65, dd (14.5, 3.5) 3.19, dd (14.5, 11.5) 2, 3, 3a, 9 9 54.6, CH 4.40, dd (11.5, 3.5) 10 168.4, C 12 50.8, CH 4.09, q (7.0) 13, 20 13 167.8, C 15  39.0, C 16 145.8, CH 6.12, dd (17.5, 10.5) 15 17 112.1, CH ₂ 5.15, d (17.5) 15, 16 5.14, d (10.5) 15, 16 18 28.0, CH ₃ 1.55, s 2, 15, 16, 19 19 27.9, CH ₃ 1.53, s 2, 15, 16, 18 20 19.9, CH ₃ 1.53, d (7.0) 12, 13 21 34.5, CH ₂ 3.39, m 5, 22, 23 22 124.4, CH 5.34, t (7.0) 23 131.7, C 24 17.9, CH ₃ 1.74, s 22, 23, 25 25 25.8, CH ₃ 1.73, s 22, 23, 24 26 35.6, CH ₂ 3.08, d (14.0) 6, 7, 7a, 28 2.92, dd (14.5, 9.0) 6, 7, 7a, 27 27  80.6, CH 3.91, d (8.5) 7 28  75.7, C 29 28.4, CH ₃ 1.74, s 27, 28, 30 30 26.9, CH ₃ 1.68, s 27, 28, 29 NH-1 9.22, s 2, 3, 3a NH-11 5.69, s 10, 12 NH-14 6.07, s 9, 13 2 location δ _C 隆_H HMBC ^a 2 142.4, C 3 103.6, C 3a 129.1, C 4 115.5, CH 7.16, s 3, 6, 7a, 21 5 133.3, C 6 123.5, CH 6.73, s 4, 7a, 21, 26 7 123.3, C 7a 132.8, C 8 29.4, CH ₂ 3.65, dd (15.0, 3.5) 3.19, dd (14.5, 11.5) 3, 3a 9  54.6, CH 4.41, dd (11.5, 3.5) 10 168.4, C 12  50.8, CH 4.09, q (7.0) 10, 20 13 167.7, C 15  39.0, C 16 146.0, CH 6.12, dd (17.5, 10.5) 15 17 111.9, CH ₂ 5.13, d (17.5) 15 5.11, d (10.5) 15 18 28.0, CH ₃ 1.54, s 2, 15, 16, 19 19 27.9, CH ₃ 1.52, s 2, 15, 16, 18 20 19.9, CH ₃ 1.53, d (7.0) 12, 13 21 34.6, CH ₂ 3.39, m 22 124.6, CH 5.35, t (7.0) 23 131.6, C 24 17.9, CH ₃ 1.74, s 22, 23, 25 25 25.8, CH ₃ 1.73, s 22, 23, 24 26 36.1, CH ₂ 2.92, dd (14.5, 8.5) 6, 27 2.84, d (14.5) 6, 7a 27  78.1, CH 3.79, d (9.0) 28  77.6, C 29 20.3, CH ₃ 1.28, s 27, 28, 30 30 18.5, CH ₃ 1.24, s 27, 28, 29 NH-1 9.84, s 3, 3a NH-11 5.67, s NH-14 5.85, s OCH3-28 49.2, CH ₃ 3.27, s 28 ^a HMBC correlations, optimized for 8 Hz.

Example 3: Preparation of MTPA Ester Derivatives of 5-prenyl-dihydrobarcocholine F and 5-prenyl-Dihydrowromeum A

1.2 mg of 5-prenyl-dihydrobarcocholine F and 5-prenyl-dihydrodorbornamine A in 600 μL of pyridine- ds were divided into two portions and transferred to a clean tube. Subsequently, MTPA (20 μL) (Sigma-Aldrich, St. Louis, Mo., USA) was added to the N ₂ gas stream , And the solution was carefully mixed. The tube was left at room temperature for 24 hours to complete the reaction to give a mono (S) -MTPA ester derivative (compound 4a) of 5-prenyl-dihydrovarycolorine F (compound 4). (R) -MTPA ester derivative (compound 4b) of Compound 4 was obtained by treatment of (S) - (+) -? - MTPA chloride. The same procedure was followed to give the (S) -MTPA ester and (R) -MTPA ester (compounds 5a and 5b) of 5-prenyl-dihydrodorbornamine A (compound 5). ¹ H NMR ¹ H NMR and COSY to obtain chemical shift spectra from.

3-1: 4- (S) -MTPA ester (Compound 4a)

^{1 H NMR (pyridine- d5, 500} MHz) δ H 6.20 (1H, m, H-27), 3.62 (1H, overlapped, H-26a), 3.43 (1H, overlapped, H-26b), 1.54 (3H, s, H-29), 1.51 (3H, s, H-30); ESIMS (positive) m / z 730 [M + H] < ⁺ >

3-2: 4- (R) -MTPA ester (compound 4b)

^{1 H NMR (pyridine- d5, 500} MHz) δ H 6.16 (1H, m, H-27), 3.65 (1H, overlapped, H-26a), 3.47 (1H, overlapped, H-26b), 1.49 (3H, s, H-29), 1.46 (3H, s, H-30); ESIMS (positive) m / z 730 [M + H] < ⁺ >.

3-3: 5- (S) -MTPA ester (Compound 5a)

^{1 H NMR (pyridine- d5, 500} MHz) δ H 5.95 (1H, m, H-27), 3.45 (1H, overlapped, H-26a), 3.18 (1H, overlapped, H-26b), 1.21 (3H, s, H-29), 1.19 (3H, s, H-30); ESIMS (positive) m / z 726 [M + H] < ⁺ >.

3-4: 5- (R) -MTPA ester (Compound 5b)

^{1 H NMR (pyridine- d5, 500} MHz) δ H 5.93 (1H, m, H-27), 3.49 (1H, overlapped, H-26a), 3.25 (1H, overlapped, H-26b), 1.07 (3H, s, H-29), 1.00 (3H, s, H-30); ESIMS (positive) m / z 726 [M + H] < ⁺ >.

Example 4: Cell culture and survival analysis

In this Example, the cytotoxicity of asperanin A, asperanin B, asperanin C, 5-prenyl-dihydrobarcocholine F and 5-prenyl-dihydrowromeum A was assayed by MTT assay And confirmed in RAW264.7 macrophages and BV2 microglia.

RAW 264.7 and BV2 cells were maintained at 5 × 10 ⁵ cells / mL concentration in DMEM medium with supplement and incubated at 37 ° C. in a humidified atmosphere of 5% CO ₂ . 50 mg / mL of MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) was added to 1 mL of each cell suspension, and the resulting prosum was dissolved in acidic 2-propanol. Was measured at 540 nm.

As a result, viability of cells treated with asperanin A, asperanin B, or asperinin C at a concentration of 40 μM or more for 24 hours was decreased, and 5-prenyl-dihydrobarcocholine F or 5- Viability of cells treated with prenyl-dihydroprotein B decreased at a concentration of 80 μM or more. Thus, in the following Examples, the maximum concentration of asperanin A, asperanin B, or asperanin C was 40 [mu] M, 5-prenyl-dihydrobarcocholine F or 5-prenyl-dihydrobromide The maximum concentration of A was limited to 80 μM.

Example 5: Effect on inhibition of nitrite and PGE2 production

To assess the anti-inflammatory activity of the compounds as measured by inhibition of the LPS-induced NO products, all isolated compounds were investigated in mouse macrophage-like cell line RAW 264.7 and immortalized rat brain macrophage BV2 cells . The nitrite concentration in the medium was measured using the Griess reaction as an indicator of the NO product. Each supernatant (100 μL) was mixed in the same volume as the grease reagent (Solution A: 222488; Solution B: S438081, Sigma-Aldrich) and the absorbance of the mixture was determined at 525 nm using an ELISA plate reader.

As a result, all of the compounds tested induced dose-dependent inhibition of NO production in both RAW264.7 and BV2 cells (Table 3).

In addition, asperanin A (compound 1), asperanin B (compound 2), asperanin C (compound 3), 5-prenyl-dihydrobariocolorin F (compound 4) - The effect of Dihydron Bromage A (Compound 5) on the PGE ₂ product was further evaluated. Pre-incubated for 3 hours at different concentrations of compounds 1-5, RAW 264.7 and BV2 cells were cultured in 24-well plates and stimulated with LPS for 24 hours. The supernatant was collected from the culture (100 μL) to determine the PGE ₂ concentration using an ELISA kit (R & D Systems, Minneapolis, MN, USA).

As a result, in the case of LPS-stimulated RAW 264.7 cells, Compounds 1-3 and 5 reduced the PGE ₂ product in a concentration dependent manner, and Compounds 1-5 inhibited the production of PGE ₂ in LPS-stimulated BV2 cells Table 4).

Example  5: iNOS  And COX -2 protein expression

The effect of Compound 1-5 isolated in Example 2 on iNOS and COX-2 protein expression was confirmed by Western blotting.

RAW 264.7 and BV2 cells were harvested and the cells were washed with PBS by centrifugation at 16,000 rpm for 15 minutes and the protease inhibitor mixture (0.1 mM PMSF, 5 mg / mL aprotinin, 5 mg / mL pepstadin (pH 7.4) containing 10 mM Tris-HCl buffer (pH 7.4, pepstatin A, and 1 mg / mL chymostatin). Protein concentration was measured with a Lowry protein assay kit (P5626, Sigma-Aldrich) and electrophoresed using 7.5% SDS-PAGE. And then transferred by electrophoresis with an ECL nitrocellulose membrane (Bio-Rad, Hercules, Calif., USA). (Amersham Pharmacia Biotech, Piscataway, NJ, USA), followed by blocking with 5% skim milk and then reacting with primary antibody (Santa Cruz Biotechnology, CA, USA) and horseradish peroxidase- ECL (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

RAW264.7 macrophages and BV2 microglia were pretreated with the indicated concentrations of compounds 1-5 for 3 hours followed by stimulation with LPS (1 [mu] g / mL) for 24 hours. As a result, Compounds 1-3 and 5 inhibited iNOS and COX-2 protein expression in LPS-stimulated RAW 264.7 cells (Fig. 2) and compounds 1-5 inhibited iNOS and COX-2 protein (Fig. 3).

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 present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Korea Biotechnology Research Institute KCTC12950BP 20151125

Claims (6)

A compound selected from the group consisting of the following groups:
2-enyl) -7- (3-methylbut-2-enyl) chroman-5-carbaldehyde ((E) -1-hydroxybut- -Enyl) -7- (3-methylbut-2-enyl) chroman-5-carbaldehyde);
ii) Preparation of 2,6-dihydroxy-7- (3-methylbut-2-enyl) chroman-5-carbaldehyde (2,6-dihydroxy- Carbaldehyde); And
iii) a pharmaceutically acceptable salt thereof.
The compound according to claim 1, selected from the group consisting of the following groups:
2-enyl) chroman-5-carbaldehyde ((R) -6- (3-methylbut- -Hydroxy-2 - ((S, E) -1-hydroxybut-2-enyl) -7- (3-methylbut-2-enyl) chroman-5-carbaldehyde);
ii) Preparation of (R) -6-hydroxy-2- ((R, E) -1-hydroxybut-2-enyl) 7- (3-methylbut- -Hydroxy-2 - ((R, E) -1-hydroxybut-2-enyl) -7- (3-methylbut-2-enyl) chroman-5-carbaldehyde);
iii) (R) -2,6-dihydroxy-7- (3-methylbut-2-enyl) chroman-5-carbaldehyde ((R) -2,6-dihydroxy- 7- - enyl) chroman-5-carbaldehyde); And
iv) a pharmaceutically acceptable salt thereof.
3. The method according to claim 1 or 2, wherein the compound is selected from the group consisting of marine fungi Aspergillus sp. SF-5976 (KCTC12950BP).
A pharmaceutical composition for preventing or treating an inflammatory disease containing the compound of claim 1 or 2 as an active ingredient.
5. The method of claim 4,
A pharmaceutical composition for the prevention or treatment of inflammatory diseases, which further comprises a pharmaceutically acceptable carrier, excipient or diluent.
A functional food for improving inflammation comprising the compound of any one of claims 1 and 2 as an active ingredient.

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