KR101329219B1 - New aesculin beta derivative, method for preparing the same and pharmaceutical composition comprising the same for preventing or treating inflammatory disease - Google Patents

Abstract

The present invention relates to a novel esculin beta derivative, a preparation method thereof and a pharmaceutical composition for preventing or treating inflammatory diseases containing the same as an active ingredient. The esculin beta derivatives according to the present invention are reacted with recombinant beta glucosidase derived from esculin, cellobiose and thermotoga neapolitana , so that the glucose portion of cellobiose reacts with the glucose portion of esculin. 1 → 3) -glycosidic bonds are formed to become aforesaid, so that the water-soluble and nitric oxide production inhibitory activity is better than that of esculin, and there is no toxicity in the body, so the anti-inflammatory effect is excellent. Therefore, the esculin beta derivatives according to the present invention can be usefully used for the prevention or treatment of inflammatory diseases.

Description

New aesculin beta derivative, method for preparing the same, and pharmaceutical composition for preventing or treating inflammatory disease containing the same as an active ingredient

The present invention relates to a novel esculin beta derivative, a preparation method thereof and a pharmaceutical composition for preventing or treating inflammatory diseases containing the same as an active ingredient.

Inflammation or salt is a local protective protective response to damage to biological tissues, in which blood components exit the tissues through the walls of blood vessels. Causes of inflammation include physical wounds, surgery, microbial infections, and oxidative stress. In particular, since free radicals such as peroxides are always produced in the body, oxidative stress in tissues is known as an important cause of inflammation.

Chemicals that act on the inflammatory process include histamine, kinin and prostaglandins. Histamine acts to bring more blood and lymph to damaged areas, and kinin relaxes muscle contractions to facilitate capillary blood transport and pain. Prostaglandins are synthesized when white blood cells enter the cell, causing pain and pain. Generates heat. If left untreated, the inflammation develops from inflammation to ulcers, and even worse, to cancer. Therefore, early treatment of inflammation is an important goal of disease treatment.

Inflammation may induce an anti-inflammatory response or an inflammatory response through the activation of two transcription factors. NF-kB is known as a transcription factor that induces inflammation, and Nrf2 is known as a transcription factor that induces anti-inflammatory. When NF-kB transcription factors are activated by oxidative stress or physical damage, enzymes and proteins such as COX-2 and inducible nitric oxide synthase (iNOS) related to inflammation are expressed. When these enzymes are expressed, prostaglandins or nitric oxides (NO) are produced, causing pain and fever at the site of inflammation. In particular, nitric oxide produced by iNOS causes cytotoxicity and tissue damage in animal cells and tissues. Thus, inhibition of nitric oxide production is known to potentially prevent septic shock in addition to inflammation. On the other hand, when the Nrf2 transcription factor is activated, enzymes and proteins such as catalase, quinone oxidoreductase 1 (NQO-1), and heme oxygenase-1 (HO-1), which are related to anti-inflammatory, are expressed, resulting in inflammatory reactions through the elimination of oxidative stress. Anti-inflammatory reactions occur. Therefore, most of the studies for preventing local injury due to inflammation focus on the inhibition of NF-kB transcription factor or activation of Nrf2 transcription factor.

In recent years, chronic inflammatory diseases such as arthritis, osteoporosis, asthma and psoriasis have increased rapidly. Therapeutic agents used in such chronic inflammatory diseases are mainly known steroid-based drugs or cox inhibitors. In particular, steroidal drugs, which are well known as anti-inflammatory drugs, are mainly used as ointments because they have a steroid nucleus and are well dissolved in fat. Steroid-based drugs are mainly absorbed into the sebaceous glands, enter the center of the cell nucleus through the cell membrane and bind to steroid receptors, thereby inducing anti-inflammatory responses. As a result, steroidal drugs lead to substances with potent anti-inflammatory effects, such as cortisone and hydrocortisone, in the adrenal cortex. However, long-term use of steroid drugs can cause a variety of side effects. In other words, long-term use of steroidal drugs lowers the function of the adrenal cortex, which produces natural steroids, resulting in atrophy of the adrenal gland and ultimately loss of adrenal function. In addition, steroidal drugs have the problem of inhibiting lymphocytes producing antibodies and eventually suppressing immune action.

Therefore, many researchers are studying phytochemicals, which are substances that prevent inflammation from natural resources, to prevent side effects such as immunosuppression and lowering of the adrenal cortex, but no effective anti-inflammatory agents have been developed. to be.

On the other hand, esculin (aesculetin 6-β-glucoside) is a derivative in which glucopyranoside is bound to esculin (aesculetin) extracted from the horse chestnut tree, and has an antioxidant and anti-inflammatory function represented by the following formula (1): It is known to have a fluorescent substance.

Esculin is a component of general medicine and cosmetics, and its function is similar to that of vitamin P, and is used for the purpose of functioning as an anti-inflammatory agent and strengthening of capillaries. In addition, bile esculin agar treated with ferric citrate and bile salts is esculin hydrolyzed and decomposed into esculin and glucose by enterococci, aerococcus and leukostock stocks. Retin reacts with iron citrate to form dark brown or black color, which is used for the identification and analysis of bacteria. This principle takes advantage of the ability to lose fluorescence when hydrolyzed esculin is hydrolyzed at 360 nm of the spectrophotometer. As a study related to esculin, Korean Patent No. 10-153478 discloses an esculin derivative, a preparation method thereof, and a pharmaceutical composition for cartilage protection comprising the same, and the Republic of Korea Patent Publication No. 10-1996-22498 No. Esculletin derivatives, methods for preparing the same, and pharmaceutical compositions for treating arthrosis comprising the same are described in Korean Patent Publication No. 10-517757, Esculetin derivatives or salts or hydrates thereof, and methods for preparing the same Is described.

As described above, studies on esculin derivatives are ongoing and reports on the anti-inflammatory efficacy of coumarin derivatives having a structure similar to that of esculin, but there is no research and development on esculin derivatives. . All.

Therefore, there is an urgent need for development of esculin derivatives having excellent anti-inflammatory activity and preventing side effects such as immunosuppression and lowering of the adrenal cortex.

While the present inventors are working to develop an esculin derivative having excellent anti-inflammatory activity, the glucose portion of cellobiose is reacted by reacting recombinant beta glucosidase derived from Thermotoga neapolitana with esculin, cellobiose and thermomoto . Escherichia beta derivatives were prepared by a sugar transfer reaction that forms β- (1 → 3) -glycosidic bonds in the glucose portion of esculin, and the esculin beta derivatives are more water soluble than esculin and inhibit the production of nitric oxide. It was confirmed that the activity is better, and there is no toxicity in the body, the anti-inflammatory effect is excellent, and completed the present invention.

Accordingly, the present invention is to provide a novel esculin beta derivative, a preparation method thereof and a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing the same as an active ingredient.

The present invention provides an esculin beta derivative having an anti-inflammatory activity represented by the following formula (2).

The esculin beta derivatives of formula (II) according to the invention can be used in the form of pharmaceutically acceptable salts, solvates, or hydrates. As the pharmaceutically acceptable salt, acid addition salts formed by pharmaceutically acceptable free acid are useful. The inorganic acid and organic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, fumaric acid, formic acid, propionic acid, oxalic acid, and trifluoric acid may be used as the organic acid. Roacetic acid, methanesulfonic acid, benzenesulfonic acid, maleic acid, benzoic acid, gluconic acid, glycolic acid, succinic acid, 4-morpholine ethanesulfonic acid, camposulfonic acid, 4-nitrobenzenesulfonic acid, hydroxy-O-sulfonic acid , 4-toluenesulfonic acid, caluturonic acid, emboic acid, glutamic acid, aspartic acid and the like can be used.

The acid addition salts according to the present invention are dissolved in conventional methods, for example, by dissolving the esculin beta derivatives of formula (2) in an excess of aqueous acid solution, which salts are water miscible organic solvents such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation using. It may also be prepared by evaporating the solvent or excess acid from the mixture and then drying or by suction filtration of the precipitated salt.

Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding silver salts are also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg silver nitrate).

In addition, the present invention is Esculin, cellobiose, and Thermotoga Neapolitana ( Thermotoga glucosidase derived from neapolitana ) was reacted at 70-90 ° C. for 30-100 minutes to allow the glucose portion of cellobiose to form β- (1 → 3) -glycosidic bonds to the glucose portion of esculin. It provides a method for producing an esculin beta derivative, characterized in that for producing the esculin beta derivative of Formula 2 through a transglycosylation reaction.

The beta glucosidase derived from thermomotoga neapolitana converts asparagine, the 291th amino acid of the beta glucosidase bg1A gene derived from neapolitana, into threonine through site-directed mutagenesis. As an enzyme, a recombinant beta glucosidase (N291T) with increased sugar transfer activity.

The esculin and cellobiose are used as acceptors and donors, respectively, and are included in 1% by weight of the total weight of the reaction mixture.

The compound prepared by the above method was purified through large-scale preparative HPLC, and the purified compound was subjected to chemical change of C-3 ′ in the glucose portion by TLC, ¹ H NMR and ¹³ C NMR analysis. Was significantly changed from 77.2ppm to 84ppm (+ 6.8ppm). It is believed that the glucose portion of cellobiose forms a β- (1 → 3) -glycosidic bond to the glucose portion of esculin. Thus, the purified compound was identified as a compound of formula 2 [β-D-glucopyranosyl- (1 → 3) -aesculin], which is a sugar transfer product of esculin.

In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing the esculin beta derivative of Formula 2 as an active ingredient.

Escurin beta derivatives according to the present invention is Esculin, cellobiose and thermomotor neapolitana ( Thermotoga Reacting beta glucosidase derived from neapolitana ) to form a β- (1 → 3) -glycosidic bond to the glucose portion of esculin by reacting the recombinant beta glucosidase derived from esculin, thereby making it more water-soluble and nitrogen monoxide than esculin. Has a superior anti-inflammatory effect and has no anti-toxic effect in the body. Therefore, the esculin beta derivatives according to the present invention can be usefully used for the prevention or treatment of inflammatory diseases.

The inflammatory diseases include dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, Psoriasis, arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay salt, periarthritis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, and the like.

The composition of the present invention may contain one or more known active ingredients having an anti-inflammatory effect together with the esculin beta derivative of formula (2).

The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dose of the esculin beta derivative of Formula 2 is about 10-20 mg / kg, preferably about 1-2 mg / kg, but may be added or subtracted according to clinical trial results, and administered once or several times a day. It is preferable.

The composition of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, drug treatment and biological response modifiers for the prophylactic or therapeutic effect of inflammatory diseases.

Escurin beta derivatives according to the present invention is more water-soluble and nitric monoxide production inhibitory activity than esculin, there is no toxicity in the body has excellent anti-inflammatory effect.

1 is a diagram showing the results of HPLC analysis of esculin beta derivatives according to the present invention.
Figure 2 is a diagram showing the results of TLC analysis of esculin beta derivatives according to the present invention.
Figure 3 is a diagram showing the cytotoxicity results of the esculin beta derivatives according to the present invention.
Figure 4 is a diagram showing the nitrite concentration of the esculin beta derivatives according to the present invention.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  One  : Recombinant Beta Glucosidase  Used Esculin  Preparation of Beta Derivatives

1. Recombinant Beta Glucosidase  Produce

Thermotoga Asparagine, the 291th amino acid of the β-glucosidase bg1A gene derived from neapolitana ), was converted to threonine through a site-directed mutagenesis to construct mutant N291T having increased glycosylation activity. Mutant N291T construction process has Nde and Hind as restriction sites in plasmid p6xHis119-bglAp6 bglA gene was based on 1.35 kb. N291T-F and N291T-R were designed as primers for mutation, and the two primers and chromosomal DNA of T. neapolitana were used for mutagenic PCR. 10 × Pfu reaction buffer, 2.5 mmol / L dNTP, 1.5 mmol / L MgCl ₂ , and Pfu 10 pmol of each designed primer and 10ng of plasmid p6xhis119 (Seoul National University, Korea) were mixed under reaction conditions containing Ultra HF DNA polymerase 2.5 units. PCR amplification conditions were predenaturated at 95 ° C for 30 seconds, followed by 1 minute denaturation at 95 ° C, 1 minute annealing at 55 ° C, and 6-minute extension at 68 ° C. Repeated. The mutated PCR product was restricted by Dpn restriction enzyme and then transformed into the E. coli MC1061 strain by Inoue heat-shock method. 10 μl of PCR product was dispensed into 200 μl of Inoue competent cells, gently mixed by pipetting and incubated for 30 minutes on ice. Then, after incubation for 30 minutes at 4 ℃ was subjected to a heat shock at 42 ℃ for 2 minutes, immediately put in ice for 2 minutes to cool. 800 μl of LB medium was added and shaken at 37 ° C. for 1 hour, followed by incubation of 100-200 μl in LB agar medium having a final concentration of 100 μg / ml of ampicillin antibiotics to select the transformed strain. .

2. Recombinant Beta Glucosidase  refine

The recombinant beta glucosidase mutant enzyme (N291T) at room temperature was purified via heat stack. Specifically, E. coli with the recombined p6xHis119-bg1Ap6 was incubated at 37 ° C. in a shaker for 18 hours and then centrifuged at 9,000 rpm for 30 minutes at 4 ° C. The cell solution obtained after centrifugation was suspended in 100 mM Tris-HCl buffer (pH 7.5), and the cells were disrupted by sonication. After cell disruption, the mixture was centrifuged at 16,600 rpm for 30 minutes at 4 ° C, and the supernatant was separated and heat-treated at 80 ° C for 30 minutes. After heat treatment, the supernatant was separated after centrifugation at 4 ° C. for 30 minutes at 16,600 rpm to remove denatured protein. The supernatant was filtered through a 0.45 μM filter and then purified through a nickel-nitrilotriacetic acid (Ni-NTA) column (Qiagen). Ni-NTA columns were washed twice with wash buffer [100 mM Tris-HCl buffer (pH 7.5), 100 mM imidazole] and with elution buffer [100 mM Tris-HCl buffer (pH 7.5), 250 mM imidazole]. The enzyme was purified. Dialysis was performed using Tris-HCl buffer (pH 7.5) to remove imidazole.

3. Recombinant Beta Glucosidase  Used Esculin  Preparation of Beta Derivatives

1% by weight of the escalin receptor, 1% by weight of the donor cellobiose, and 1.8 µg of the recombinant beta glucosidase enzyme (N291T) were added to 4 ml of 100 mM sodium phosphate buffer (pH 7.0), followed by mixing at 80 ° C. The reaction was carried out for 1 hour. After completion of the reaction, the reaction mixture was purified by large volume preparative high performance liquid chromatography (recycling preparative HPLC). Large-capacity preparative high-performance liquid chromatography was performed using a JAIGEL W-251 column (2 × 50 cm; JAI, Tokyo, Japan) and a RI-50 detector (JAI). Distilled water was used as the eluting solvent for elution, and the compound was purified by loading distilled water at a flow rate of 3.0 ml / min. The HPLC analysis results and the TLC analysis results of the purified compound are shown in FIGS. 1 and 2, respectively.

4. Structural Analysis of Purified Compounds

Exchange 3 times of the compound in the tablet 20㎎ 3 in D ₂ O, and dissolved in pure D ₂ O 0.5㎖ at 23 ℃. Next, ¹ H NMR and ¹³ C NMR were measured using a Varian Inova AS 500 MHz NMR spectrometer.

¹ H NMR and ¹³ C NMR analysis of the purified compound are shown in Table 1.

As shown in Table ^1, 1 H NMR of the purified compound and ¹³ C NMR analysis of the results was observed a signal 21 from the ¹³ C NMR, the chemical shift of C-3 'in the glucose portion is at 77.2ppm 84ppm ( +6.8 ppm) was confirmed to change significantly. It is believed that the glucose portion of cellobiose forms a β- (1 → 3) -glycosidic bond to the glucose portion of esculin. Therefore, the purified compound was identified as β-D-glucopyranosyl- (1 → 3) -aesculin, a sugar transfer product of esculin, and the structural formula is as follows.

Structural formula of β-D-glucopyranosyl- (1 → 3) -aesculin

Experimental Example  One  : ≪ / RTI & Esculin  Water Solubility Measurement of Beta Derivatives

In order to confirm the water solubility of the esculin beta derivative according to the present invention, the following experiment was performed.

Excess esculin and the esculin beta derivatives prepared in Example 1 were each placed in an Eppendorf tube and suspended by adding 1 ml of distilled water. Each suspension was sonicated at room temperature for 1 hour to dissolve excess esculin and esculin beta derivatives and then centrifuged at 10,000 rpm for 10 minutes. After centrifugation, the supernatant was filtered through a 0.45 μm filter, and the absorbance was measured at 238 nm through a UV spectrophotometer (Gene Quan pro UV / Vis spectrophotometer, UK).

The results are shown in Table 2.

compound receptivity Esculin 6.2mM Esculin Beta Derivatives of Example 1
[β-D-glucopyranosyl- (1 → 3) -aesculin] > 5400mM

As shown in Table 2, it was confirmed that the water solubility of the esculin beta derivative according to the present invention is increased by about 870 times than that of esculin.

Experimental Example  2  : ≪ / RTI & Esculin  Cytotoxicity of Beta Derivatives

In order to confirm the cytotoxicity of the esculin beta derivative according to the present invention, the following MTT assay was performed.

Specifically, RAW 264.7 cells (2.5 × 10 ⁵ cells / well), which are mouse macrophages, were seeded in a 24-well culture plate and incubated at 37 ° C. for 24 hours. After the culturing, 30 ~ 480μM concentration of esculin and the esculin beta derivative prepared in Example 1 was added to each well and incubated for 24 hours. After incubation, 5 [mu] l of MTT [3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, a yellow tetrazole] solution (5 mg / ml) was dispensed into each well and 37 ° C for 4 hours. Incubated at 5% CO ₂ . After incubation, the supernatant was removed and the formazan crystals produced in the surviving cells were dissolved in 150 μl of DMSO. Samples of each well were measured for absorbance at 575 nm using a microplate reader.

The results are shown in FIG.

As shown in Figure 3, it was confirmed that the esculin beta derivatives according to the present invention does not significantly inhibit cell growth.

Experimental Example  3  : ≪ / RTI & Esculin  Determination of Nitric Oxide Production by Beta Derivatives

In order to confirm the amount of nitrogen monoxide produced by the esculin beta derivative according to the present invention, the following experiment was performed.

Specifically, LPS (lipopoysaccharide) (2 µg / ml) was treated to rat macrophages (RAW 264.7) to induce an inflammatory response. When the inflammatory reaction occurs, the cells produce a lot of nitric oxide (NO). Inflammatory response-induced macrophages (RAW 264.7) were treated with 30 to 480 μM of esculin and the esculin beta derivatives prepared in Example 1, respectively, and inoculated into 24 well culture plates, followed by 24 hours at 37 ° C. Incubated for 2 hours. Culture medium was sampled to determine the concentration of nitrite, the nitric oxide production index. Nitrite concentration was measured by Griess reaction. In other words, 1% (w / v) sulfanilamide was dissolved in 5% (v / v) phosphoric acid in the same volume in the sampled supernatant of each well, and 0.1% (w / v) in distilled water. ) Naphthylethylene-diamine dihydrochloride dissolved] After treatment with 100μl and incubated for 10 minutes at room temperature. Nitrite concentration was then determined by measuring absorbance at 575 nm using an ELISA plate reader.

The results are shown in Fig.

As shown in Figure 4, the esculin beta derivative according to the present invention was confirmed that the concentration of nitrite is more reduced than that of esculin to further reduce the amount of nitrogen monoxide produced. Therefore, it can be seen that the esculin beta derivative according to the present invention has an excellent anti-inflammatory effect.

Examples of formulations for the composition of the present invention are illustrated below.

Formulation example  One  : Sanje  Produce

200 mg of esculin beta derivative of formula (2)

Lactose 100mg

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

Formulation example  2  : Preparation of Tablet

200 mg of esculin beta derivative of formula (2)

Corn Starch 100mg

Lactose 100mg

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

Formulation example  3  : Preparation of Capsule

200 mg of esculin beta derivative of formula (2)

Corn Starch 100mg

Lactose 100mg

Magnesium stearate 2 mg

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

Formulation example  4  : Preparation of Injection

200 mg of esculin beta derivative of formula (2)

Mannitol 100mg

Na ₂ HPO ₄ .12H ₂ O 2 mg

Appropriate sterile distilled water for injection

Injectables were prepared by mixing the above ingredients per ampoule (2 ml) according to the usual method for preparing injectables.

Claims (5)

delete Escurin, cellobiose, and thermotoga beta glucosidase from Thermotoga neapolitana were reacted at 70 to 90 ° C. for 30 to 100 minutes so that the glucose portion of cellobiose was reacted with the glucose portion of esculin. (1 → 3)-A method for producing an esculin beta derivative, characterized in that to produce an esculin beta derivative of the formula (2) through a transglycosylation reaction to form a glycosidic bond.
(2)

The site-directed mutagenesis of claim 2, wherein the beta glucosidase derived from thermomotor neapolitana is a 291 th amino acid of the beta glucosidase bg1A gene derived from neapolitana. The recombinant beta glucosidase (N291T) converted to threonine through, characterized in that the production method of the esculin beta derivatives. A pharmaceutical composition for preventing or treating pneumonia, comprising an esculin beta derivative represented by Formula 2 as an active ingredient.
(2)
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