KR20080027191A - New benzoxazole derivative, process for the preparation thereof and pharmaceutical composition comprising the same - Google Patents

Abstract

A benzoxazole derivative is provided to show excellent 5-lipoxygenase inhibitory activity, reduce the degree of airway hypersensitivity, levels of cytokines IL-4, IL-5, and IL- 13, ova-specific IgE in serum, and degree of the peribronchial and perivascular inflammation, reduce the number of TRAP(+) MNCs statistically significantly, and significantly inhibit osteoclastogenesis in a co-culture system of bone marrow cells and osteoblasts, thereby being used for preventing and treating diseases induced by leukotriene, in particular, inflammatory diseases including asthma or osteoporosis. A method for preparing a benzoxazole derivative represented by the formula(1) comprises the steps of: (a) reacting 2-amino-4-methylphenol with 4-ethylphenyl isocyanate in an organic solvent to prepare an N-(2-hydroxy-5-methylphenyl)-N'-(4-ethylphenyl)thiourea compound; and (b) reacting the N-(2-hydroxy-5-methylphenyl)-N'-(4-ethylphenyl)thiourea compound with potassium superoxide to prepare 8-methyl-2-[N-(4-ethylphenyl)]aminobenzoxazole. A pharmaceutical composition for preventing and treating diseases induced by leukotriene comprises the benzoxazole derivative or a pharmaceutically acceptable salt thereof.

Description

Novel benzoxazole derivatives, preparation method thereof and pharmaceutical composition comprising the same

The present invention relates to a novel benzoxazole derivative, a preparation method thereof and a pharmaceutical composition comprising the same.

Leukotriene is an oxidized polyunsaturated fatty acid with a large number of biologically active substances that is metabolized in several steps from arachidonic acid, which is released from the cell membrane by calcium action, and is one of a group of inflammatory mediators that exhibit various biological activities. Biosynthesis as.

First, as a first step, the 5-lipoxygenase is generated an unstable epoxide intermediate, leukotriene A ₄ (LTA ₄₎ by the action of the arachidonic acid from, the LTA _4, respectively through the two paths leukotriene B _4, and leukotriene C ₄ , D ₄ , E ₄ .

Leukotriene B ₄ (LTB ₄ ) produced through the first pathway is a bioactive compound produced by hydration of the hydrolase of LTA ₄ . The compound exhibits chemotaxis to inflammatory cells such as polymorphonuclear leukocytes and causes degranulation and aggregation of inflammatory cells. In addition, leukotriene B ₄ increases vascular permeability to form edema.

Leukotriene C ₄ (LTC ₄ ), leukotriene D ₄ produced via the second pathway Among the (LTD ₄ ) and leukotriene E ₄ (LTE ₄ ), LTC ₄ is produced by the addition of glutathione to the epoxide of LTA ₄ by LTC ₄ synthase, and the metabolism of the peptide site of LTC ₄ is changed to LTD ₄ and LTE. ₄ is generated. These three leukotrienes are a major component of the slow reaction substance of hypersensitivity and affect urgent hypersensitivity. These compounds also cause smooth muscle contraction, increase mucus secretion and increase vascular permeation.

The biological effects of leukotriene play an important role in skin diseases such as asthma, cystic fibrosis, chronic bronchitis, gout, rheumatoid arthritis, bronchitis, allergic rhinitis, psoriasis, arthritis, inflammatory bowel disease, sepsis, It is known to be involved in various cardiopulmonary diseases including myocardial ischemia, cardiac hypersensitivity, cerebrovascular spasm, ischemia and the like.

Of these, asthma is a disease with the most reliable evidence that leukotriene plays a pivotal role, so the focus of most leukotrienes research is focused on this area. In particular, LTC ₄ and LTD ₄ have a very strong bronchial contraction effect on the human airway, which is 1000 times more powerful than histamine. These compounds strongly promote mucosal secretion and increase capillary permeability in the human airways. These results can be seen by showing a high level of leukotriene in the body fluids of asthma patients, and a close correlation between the content and the severity of the disease.

In addition, recently, 5-lipoxygenase metabolites are produced in osteoblasts, thereby promoting bone resorption, and C433 cells, which are interstitial cells obtained from giant cell tumors, are used to produce 5-lipoxygenase metabolites. It has been reported to increase osteoclast number and promote activity, and the addition of synthetic LTB ₄ in bone tissue culture promotes bone resorption, and LTB _{4 is} also reported to cause bone resorption through osteoclast production both in vitro and in vivo. There is a bar. Thus, compounds that antagonize the LTB ₄ receptor are expected to affect bone tissue metabolic diseases, and there are many studies on them.

5-lipoxygenase, on the other hand, is restricted to neutrophils, eosinophils, basophils, monocytes, macrophages, mast cells, and B lymphocytes. To be distributed. On the other hand, LTA ₄ hydrolase and LTC ₄ synthase are widely distributed in more diverse tissues.

Therefore, in the case of drugs capable of selectively inhibiting the action of 5-lipoxygenase enzymes, the biosynthesis of leukotriene can be blocked more effectively in the early stages so that diseases caused by leukotriene, in particular inflammatory diseases or osteoporosis, including asthma, etc. It is considered to be useful for the prevention and treatment of

Looking at the recent research trend related to the present invention, a) inhibition of 5-lipoxygenase, b) inhibition of 5-lipoxygenase activating protein (FLAP), c) peptidyl leukotriene (LTD ₄ ) Many efforts are being made to develop receptor antagonists (eg montelukast, franlukast, zafirlukast), d) LTB ₄ receptor antagonists.

Recently, zileuton is a drug for the purpose of inhibiting 5-lipoxygenase, and in addition, E6080, A-61443, BW-755c, A-63162, MK-886, MK-591, WY-50,295, Substances such as ZD5138 are under development as 5-lipoxygenase inhibitors and are in clinical trials.

Gilluton, a drug synthesized by Abbott Laboratories, was received by the US Food and Drug Administration (FDA) in July 1997 and is the first commercially available drug to treat asthma with 5-lipoxygenase inhibitory activity. E6080 also inhibits the release of LTB ₄ and LTC ₄ as a potent 5-lipoxygenase inhibitor that is highly selective and orally administered by Eisai Co. Ltd. of Japan.

Therefore, it is thought that a therapeutic agent for inhibiting 5-lipoxygenase that effectively inhibits the biosynthesis of leukotriene is more effective in the treatment of inflammatory diseases or osteoporosis, including asthma, in which leukotriene plays a major role.

Accordingly, the present inventors have developed a pharmaceutical composition having 5-lipoxygenase inhibitory activity in Korean Patent Publication No. 10-0544901. In the present invention, a novel benzoxazole derivative having a particularly high inhibitory activity against 5-lipoxygenase was synthesized, and the compound has a degree of airway hypersensitivity, cytokine IL-4, IL-5, IL-13 concentration, and serum Ova-specific IgE and peribronchial and perivascular inflammation are significantly reduced, the number of TRAP (+) MNCs is statistically significantly reduced, and osteoclast formation in co-culture systems of bone marrow and osteoblasts Significant inhibition was found and the present invention was completed.

The present invention is to provide a novel benzoxazole derivative having a 5-lipoxygenase inhibitory activity, a preparation method thereof and a pharmaceutical composition for the prevention and treatment of diseases caused by leukotriene, including the same.

The present invention provides a benzoxazole derivative represented by the following formula (1).

The compounds of the present invention can be prepared with pharmaceutically acceptable salts and solvates according to methods conventional in the art.

As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and acid or alcohol (eg, glycol monomethylether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered. At this time, the free acid as may be used an organic acid and an inorganic acid, inorganic acid used may be hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc., organic acids include methanesulfonic acid, p - toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacic acid, galactu Lonic acid, glutamic acid, glutaric acid (glutaric acid), glucuronic acid (glucuronic acid), aspartic acid, ascorbic acid, carbonic acid, vanic acid, hydroiodic acid may be used, but is not limited thereto.

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

Pharmaceutically acceptable salts of the compound represented by Formula 1 include salts of acidic or basic groups which may be present in the compound of Formula 1, unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts, and the like. It can be prepared through the process.

In addition, the present invention

1) N- (2-hydroxy-5-methylphenyl) -N '-(4-ethylphenyl) thiourea compound was prepared by reacting 2-amino-4-methylphenol and 4-ethylphenyl isocyanate in an organic solvent. Steps, and

2) The N- (2-hydroxy-5-methylphenyl) -N '-(4-ethylphenyl) thiourea compound is reacted with potassium peroxide to give 8-methyl-2- [N- (4-ethylphenyl)] It provides a method for producing a benzoxazole derivative represented by the formula (1) comprising the step of preparing the amino benzoxazole.

2-amino-4-methylphenol used as a starting material in the reaction may be prepared by reducing 4-methyl-2-nitrophenol with Pd / C, or may be purchased commercially.

After dissolving 2-amino-4-methylphenol and 4-ethylphenyl isocyanate in an organic solvent in step 1) for about 24 hours, N- (2-hydroxy-5-methylphenyl) -N '-(4- Ethylphenyl) thiourea compounds are prepared.

At this time, the organic solvent used is methanol, ethanol, ether and the like, methanol is most preferred.

In step 2), potassium peroxide is preferably used in about 5 equivalents based on 1 equivalent of N- (2-hydroxy-5-methylphenyl) -N '-(4-ethylphenyl) thiourea compound.

The present invention also provides a pharmaceutical composition for the prevention and treatment of diseases caused by leukotriene, including the benzoxazole derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof.

Diseases caused by leukotriene include asthma, whooping cough, psoriasis, arthritis, inflammatory bowel disease, cystic fibrosis, chronic bronchitis, allergic rhinitis, gout, rheumatoid arthritis, sepsis, cardiomyopathy, heart hypersensitivity, cerebrovascular spasm, ischemia , Osteoporosis, breast cancer, pancreatic cancer or pain.

The benzoxazole derivative according to the present invention has an IC ₅₀ value of 0.12 μM in BMMC (Bone-marrow mast cell), and its inhibitory activity against 5-lipoxygenase is significantly superior to that of the conventional 5-lipoxygenase inhibitor.

In addition, the benzoxazole derivatives according to the present invention significantly increase the degree of airway hypersensitivity, cytokine IL-4, IL-5, IL-13 concentrations, serum Ova-specific IgE, and inflammation of the peribronchial and perivascular areas. Decrease.

In addition, the benzoxazole derivatives according to the present invention statistically significantly decreased the number of TRAP (+) MNCs as concentration increased (** p <0.01 compared to the control), and coexistence culture system of bone marrow cells and osteoblasts. Significantly inhibits osteoclastogenesis.

Accordingly, the benzoxazole derivatives according to the present invention can be usefully used for the prevention and treatment of diseases caused by leukotriene, especially inflammatory diseases or osteoporosis, including asthma and the like.

The composition of the present invention may contain one or more known active ingredients having a prophylactic and therapeutic effect of a disease caused by leukotriene together with the benzoxazole derivative of the formula (1).

The composition of the present invention can be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active 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, as necessary. And other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a 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 dosage of the compound of Formula 1 is about 10-1000 mg / kg, and more preferably administered once to several times a day.

The composition of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers for the prevention and treatment of diseases caused by leukotriene.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example  One  : 8- methyl -2- [N- (4- Ethylphenyl )] Of aminobenzoxazole  Produce

1.2-amino-4- Methylphenol

Pd / C was added to 4-methyl-2-nitrophenol as a catalyst, and then air was completely removed using a pump. Then, methanol was injected to dissolve the reagents, and a hydrogen balloon was attached to replace hydrogen. The mixture was stirred vigorously at room temperature for 5 hours, filtered to completely remove Pd / C, and the filtrate was concentrated under reduced pressure to obtain 2-amino-4-methylphenol.

2.N- (2-hydroxy-5- Methylphenyl )- N ' -(4- Ethylphenyl ) Thiourea

132.6 mg (0.812 mmol) of 4-ethylphenyl isothiocyanate was added to 100 mg (0.812 mmol) of 2-amino-4-methylphenol prepared in 1 above, and 25 ml of methanol was added thereto to completely dissolve and stirred for 24 hours. The precipitate formed over time was filtered under reduced pressure to obtain 100 mg (yield: 43%) of N- (2-hydroxy-5-methylphenyl) -N '-(4-ethylphenyl) thiourea as a brown powder.

¹ H NMR (Acetone-d _{6 ,} 400 MHz) δ 1.178 (t, J = 8.0 Hz, 3H), 2.586 (q, J = 8.0 Hz), 6.879 (m, 1H), 7.178-7.204 (m, 4H), 7,711 ~ 7.738 (m, 2H)

3. 8- methyl -2- [N- (4- Ethylphenyl )] Aminobenzoxazole

100 mg (0.35 mmol) of N- (2-hydroxy-5-methylphenyl) -N '-(4-ethylphenyl) thiourea prepared in 2 and 123.9 mg (1.75 mmol) of potassium peroxide were mixed. Then, 5 ml of acetonitrile was added to the mixture under nitrogen gas substitution. The mixture was reacted at room temperature for 12 hours with vigorous stirring, and then poured into cooling water and diluted. The mixture was extracted with dichloromethane, dried over MgSO ₄ , and the solvent was concentrated under reduced pressure to obtain 62.5 mg (yield: 71%) of white powder of 8-methyl-2- [N- (4-ethylphenyl)] aminobenzoxazole.

Melting Point: 184 ~ 186.2 ℃

¹ H NMR (Acetone-d _{6 ,} 400 MHz) δ 1.364 (t, J = 6.8 Hz, 3H), 2.383 (s, 3H), 4.041 (q, J = 6.8 Hz, 2H), 6.895-6.718 (m, 1H ), 6.933 ~ 6.965 (m, 2H), 7.199 ~ 7.219 (m, 2H), 7.722 ~ 7.762 (m, 2H), 9.210 (s, NH),

FABHRMS (m / z): 253.1345 (M ⁺ +1, C ₁₆ H ₁₇ N ₂ O requires 253.1341)

Experimental Example  One  5- Lipoxygenase  Inhibitory activity test

In order to determine the 5-lipoxygenase inhibitory activity of the benzoxazole derivative of Formula 1 according to the present invention, the following experiment was performed.

One. BMMC ( Bone - marrow mast cell Manufacturing

Two female BALB / c mice of 5-6 weeks were prepared, sterilized with 71% ethanol, and the leg bones were separated. After inserting a 25G needle into the center of the leg bone, the bone marrow was pushed out with 25 ml RPMI and centrifuged (1000 rpm, 5 mins) to discard the supernatant, followed by incubation with RPMI 1640/10% FBS. After a day or so, observed and passaged and confirmed 90% or more BMMC, the following experiment was performed.

2. Leukotrien C ₄ ( Leukotriene C ₄ ) formation Assay

After 3 weeks or more, the BMMC was transferred to a 50 ml falcon tube, followed by centrifugation (1000 rpm, 5 min, 4 ° C.) to discard the supernatant, and RPMI 1640 was added thereto. 50 μl supernatant samples, 50 μl LTC ₄ AChE tracer, 50 μl LTC ₄ immunoserum were aliquoted into the wells, and when diluted, EIA buffer was used. NSB wells were dispensed with 100 μl EIA buffer, 50 μl LTC ₄ AChE tracer and 50 μl LTC ₄ immunoserum. After incubation at room temperature for 16-20 hours, the cells were washed 5 times with 200 μl of wash buffer. 200 μl of Elman's reagent was added to each well and incubated for 60 minutes, and the plate was read at 405 nm.

IC ₅₀ values of benzoxazole derivatives used as active ingredients in the 5-lipoxygenase inhibitory pharmaceutical composition of Korean Patent Publication No. 10-0544901 are described below.

As a result of the experiment, the IC ₅₀ value of the benzoxazole derivative prepared in Example 1 was 0.12 μM, 7.91 higher than the IC ₅₀ value of the benzoxazole derivative described in Korean Patent Publication No. 10-0544901. It was confirmed that the inhibitory activity against 5-lipoxygenase was ˜236.4 times.

Experimental Example  2  Asthma inhibition effect measurement in vivo )

1. Asthma induction

Six week-old sterile, healthy female BALB / c mice (Korean Research Institute of Chemistry Technology, Daejeon, Korea) were bred in a laminar flow cabinet. BALB / c mice were divided into three groups and 6 animals were assigned to each group. Group 1 was sham-sensitization plus challenge with phosphate-buffered saline (PBS; ipNeb); Group 2 includes sensitization plus challenge with OVA (ovalbumin: Sigma A5503; Sigma, St. Louis, MO) (ipNeb); Group 3 was sensitization with OVA (ip) plus challenge with OVA (Neb) and samples (po). On the first day, 20 μg of ovalbumin / 4 mg of adjuvant aluminum hydroxide was injected into the intraperitoneal cavity, and on day 11, intraperitoneal injection of OVA / alum and 1% ovalbumin antigen in 20 minutes And 1% ovalbumin antigen was sprayed on days 21, 22 and 23. On day 25, the ovalbumin antigen was finally exposed in a spray form to cause inflammation. The drug was administered orally (50 mg / kg body weight / horse) daily from day 21 to day 25. Animal activity was assessed 24 hours after the last ovalbumin antigen challenge.

2. Measurement of airway hypersensitivity

Measurement of airway hypersensitivity was performed in a brometric plethysmographic chamber with all mice free and conscious after 24 hours of the last challenge. The baseline for airway hypersensitivity was the average of the measurements over 3 minutes, followed by 5 minutes of inhalation for 3 minutes at each concentration, gradually increasing the metacholine from 0 mg / ml to 30 mg / ml. The degree of each airway hypersensitivity was measured. The degree of airway hyperresponsiveness was expressed as an increased ratio of the enhanced pause (Penh) value (Penh _MCH ) measured after administration of each methacholine compared to the Penh value (Penh _PBS ) measured after inhalation of physiological saline. The formula for calculating the Penh value is represented by Equation 1 below. As a positive control group, zileuton was used.

Penh _PBS = [expiratory time (Te) / relaxation time (RT) -1] x [peak expiratory flow (PEF) / peak inspiratory flow (PIF)]

The results are shown in Table 1.

Methacholine Concentration (mg / ml) 0 5 10 20 30 Negative Control 0.656 1.376 2.418 2.878 3.277 Positive control group 0.567 1.673 2.344 2.588 2.866 Example 1 0.383 0.756 1.339 1.863 2.724

As shown in Table 1, the degree of airway hypersensitivity of the benzoxazole derivative according to the present invention was significantly reduced compared to the negative control group and the positive control group.

3. Cytokines IL -4, IL -5, IL -13 lesson In serum Ova Specific IgE Measure

24 hours after the airway hypersensitivity was measured, mice were sacrificed using an excess pentobarbital and tracheotomy was performed. The rib cage of the mouse was incised to expose the bronchus, the upper part of the trachea was dissected, and the catheter was carefully inserted before ligation. 0.5 ml of cold PBS was slowly injected into the bronchus-lung, and the collected bronchial alveolar lavage fluid (BALF) was collected and centrifuged while maintaining at 4 ° C. The supernatant was stored at -70 ° C. The concentrations of cytokines IL-4, IL-5 (BD Biosciences Pharmingen Inc., San Dieogo, Calif., USA), IL-13 (Pierce Endogen, Inc., Woburn, Massachusetts, USA) were measured in the supernatant of bronchoalveolar lavage fluid. Quantification and testing by ELISA method.

Plasma was obtained through cardiac puncture following tracheostomy. The concentration of Ova-specific IgE (BD Biosciences Pharmingen Inc., San Dieogo, Calif., USA) in serum was quantified and tested by ELISA method.

The measured concentrations of cytokines IL-4, IL-5, IL-13 are shown in Tables 2, 3 and 4, respectively.

Concentration of Cytokine IL-4 IL-4 concentration One 2 3 4 5 Average Standard Deviation Ovalbumin 422.03 476.73 316.4 424.01 470.2 421.872 64.1715 Gilton 274.43 321.74 358.3 266.8 204.9 285.234 58.2696 Example 1 147.6 234.42 218.2 154.17 237.3 198.34 43.9897

Concentration of Cytokine IL-5 IL-5 concentration One 2 3 4 5 Average Standard Deviation Ovalbumin 248.82 227.21 219.52 215.05 323 246.717 44.5694 Gilton 113.31 161.01 181.44 219.27 214.3 177.864 43.3198 Example 1 174.05 109.83 112.81 86.353 101.6 116.923 33.5457

Concentration of Cytokine IL-13 IL-13 concentration One 2 3 4 5 Average Standard Deviation Ovalbumin 128.45 123.16 105.09 191.87 165.9 142.89 35.1914 Gilton 124.37 101.3 83.84 85.45 104.3 99.842 16.4802 Example 1 75.23 48.19 41.873 80.2 76.02 64.303 17.8335

As shown in Table 2 to Table 4, the cytokine IL-4, IL-5, IL-13 concentration of the benzoxazole derivatives according to the present invention is significantly reduced compared to the negative control and positive control, Ova-specific in serum It can be seen that the enemy IgE is reduced.

4. Histological Analysis of Bronchus and Lungs

After fixation (0.8% formalin, 4% acetic acid) in the lung and bronchus of the mouse through intubation, lung parenchyma and bronchial tissue were collected, fixed in 10% neutral formaldehyde solution for 20 to 24 hours, and paraffin embedding fragments were prepared. It was. For histological examination, paraffin-embedded tissues were cut into 4 μm thickness using an ultrathin sectioner (Leica Microsystems Nussloch GmbH, Nussloch, Germany), followed by deparaffinization, and then stained with HE (hematoxylin; Sigma MHS-16 and eosin). , Sigma HT110-1-32) and Dako-mounting medium (Dakocytomation; Denmark Carpinteria CA) were stained.

Inflammation was objectively scored from 0 to 3 in the peribronchial and perivascular areas. In other words, 0 when no inflammatory cells are observed, 1 when colonies of inflammatory cells are observed, 1 when most of the bronchial or blood vessels form one to five cells thick, 2, Most of the bronchial or vascular periphery (more than five cells thick) form a score of 3, and the overall lung inflammation is defined as the average score of the bronchial and perivascular area. It was.

The results are shown in Table 5.

Ovalbumin Gilton Example 1 Average 2.225 1.835 1.2427 Deviation 0.224 0.195 0.232

As shown in Table 5, it can be seen that the degree of inflammation of the peribronchial and peripheral vessels of the benzoxazole derivative according to the present invention is significantly reduced.

Experimental Example  3  : Osteoclast differentiation inhibitory effect

In order to confirm the effect of the benzoxazole derivative of formula 1 according to the present invention on the formation and differentiation of osteoclasts, the following experiment was performed through co-culture.

1. Reagents and Laboratory Animals

Minimal essential medium alpha modification (α-MEM), fetal bovine serum (FBS), phenylsilin, streptomycin and trypsin-EDTA used for cell culture were purchased from Gibco-BRL. For osteoclast studies, 1,25 (OH) ₂ D ₃ , dexamethasone, Tartrate-resistant acid phosphatase staining kit, and other chemical reagents were purchased from Sigma-Aldrich. New ICR mice were purchased from Biolinks Co. (Korea) and 6-week old ddY mice were purchased from Charles River Japan Lab (Japan). The compound prepared in Example 1 was dissolved in DMSO and diluted in culture medium (containing 0.1% solvent).

2. Osteoclast formation assay Coexistence Culture )

Osteoclast formation index was determined by calculating TRAP-positive multinucleated osteoclasts (TRAP (+) MNCs). Bone marrow cells (2.5 × 10 ⁵ cells / cm 2) and calvaria-derived osteoblasts (4 × 10 ⁴ cells / cm 2) prepared in Example 1 (0.01, 0.1 and 1 μg / ml) And 1,25 (OH) ₂ D ₃ with α-MEM containing 10% FBS And incubated for a co-existence (10 ^-8 M) and 7 days in the presence of dexamethasone (10 ^-7 M). Bone marrow cells were isolated from the femora of 6-week-old ddY mice, and osteoblasts were isolated from the capillaries of neonatal ICR mice by sequential dipping with 0.2% collagenase. The medium was replaced with fresh medium every two days. On day 6, cells were fixed with 10% formaldehyde and stained with TRAP. The number of TRAP (+) MNCs containing more than 6-7 nuclei was calculated by observation with an electron microscope (ZEISS Axiovert 25, Switzerland).

The number of TRAP (+) MNCs is shown in Table 6, and the effect of the benzoxazole derivative of the present invention on osteoclast inhibitory activity is shown in FIG. 1.

Number of TRAP (+) MNCs / well 1st test 2nd test Control 85 ± 4.8 119 ± 3.2 Example 1 Compound 0.01 µg / ml 49 ± 6.7 ** 78 ± 7.2 ** 0.1 µg / ml 18 ± 1.4 ** 41 ± 3.1 ** 1 µg / ml 8 ± 0.8 ** 18 ± 0.6 **

As shown in Table 6, the benzoxazole derivatives of the present invention significantly decreased the number of TRAP (+) MNCs with increasing concentration (** p <0.01 compared to the control).

In addition, as shown in Figure 1, the benzoxazole derivatives of the present invention exhibited osteoclast differentiation inhibitory activity of 39%, 73% and 88% at 0.01, 0.1 and 1 ㎍ / ㎖ concentration, respectively.

Accordingly, the benzoxazole derivatives of the present invention can be seen to be an excellent osteoclast differentiation inhibitor by significantly inhibiting osteoclastogenesis in the co-culture system of bone marrow cells and osteoblasts.

Examples of preparations for the compositions of the present invention are illustrated below.

Formulation example  One  : Preparation of Injection Solution

Injection solution containing 10 mg of the active ingredient was prepared by the following method.

1 g of Compound 1, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

The components of the injection solution are as follows.

1 g of compound of Formula 1

0.6 g sodium chloride

0.1 g of ascorbic acid

Distilled Water Determination

Formulation example  2  : Preparation of Syrup

Syrup containing the compound of Formula 1 as an active ingredient 2% (weight / volume) was prepared by the following method.

Compound 1, saccharin and sugars were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed thereto. Water was added to this mixture to 100 ml.

The components of the syrup are as follows.

2 g of a compound of formula 1

Saccharin 0.8 g

25.4 g per

Glycerin 8.0 g

0.04 g of spices

Ethanol 4.0 g

0.4 g of sorbic acid

Distilled Water Determination

Formulation example  3  : Preparation of Tablet

A tablet containing 15 mg of the active ingredient was prepared by the following method.

250 g of compound 1 was mixed with 175.9 g lactose, 180 g potato starch, and 32 g colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

The components of the tablet are as follows.

250 g of compound of Formula 1

Lactose 175.9 g

180 g potato starch

32 g of colloidal silicic acid

10% gelatin solution

Potato Starch 160 g

50 g of talc

5 g of magnesium stearate

The benzoxazole derivatives according to the present invention are excellent in inhibitory activity against 5-lipoxygenase, the degree of airway hypersensitivity, cytokine IL-4, IL-5, IL-13 concentration, serum Ova-specific IgE, And leukotriene by significantly reducing the degree of inflammation around the bronchi and perivascular vessels, statistically significantly reducing the number of TRAP (+) MNCs, and significantly inhibiting osteoclast formation in the co-culture system of bone marrow cells and osteoblasts. It can be usefully used for the prevention and treatment of diseases caused by, in particular inflammatory diseases, including asthma and the like.

1 is a diagram showing the effect of benzoxazole derivatives of the present invention on osteoclast inhibitory activity.

Claims (5)

Benzoxazole derivatives represented by the following formula (1). <Formula 1>

1) N- (2-hydroxy-5-methylphenyl) -N '-(4-ethylphenyl) thiourea compound was prepared by reacting 2-amino-4-methylphenol and 4-ethylphenyl isocyanate in an organic solvent. Steps, and 2) reacting the N- (2-hydroxy-5-methylphenyl) -N '-(4-ethylphenyl) thiourea compound with potassium peroxide to yield 8-methyl-2- [N- (4-ethylphenyl)] Method for producing a benzoxazole derivative of claim 1 comprising the step of preparing amino benzoxazole. Asthma, whooping cough, psoriasis, arthritis, inflammatory bowel disease, cystic fibrosis, chronic bronchitis, allergic rhinitis, gout, Pharmaceutical composition for the prevention and treatment of rheumatoid arthritis, sepsis, myocardial ischemia, cardiac hypersensitivity, cerebrovascular spasm, ischemia, osteoporosis, breast cancer, pancreatic cancer or pain. The pharmaceutical composition of claim 3, wherein the disease caused by leukotriene is asthma. The pharmaceutical composition of claim 3, wherein the disease caused by leukotriene is osteoporosis.

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