KR20090102145A - Composition comprising pyrazole derivatives for prevention and treatment of osteoporosis - Google Patents

Abstract

PURPOSE: A composition for preventing and treating osteoporosis, which contains pyrazole derivative is provided to ensure the effective activities for suppressing NADPH oxidase and RANKL. CONSTITUTION: A composition for preventing and treating osteoporosis comprises a pyrazole derivative. The pyrazole derivative is selected from a group of the chemical formulas 1 to 4. The pyrazole derivative suppressed NADPH oxidase activity and RNAKL.

Description

Composition comprising pyrazole derivatives for prevention and treatment of osteoporosis

The present invention relates to a composition for the prevention and treatment of osteoporosis comprising a pyrazole derivative having excellent NADPH oxidase and RANKL inhibitory activity.

Bone modeling and remodeling play an important role in bone development, growth and metabolism. Bone formation begins in the early stages and continues until the young adulthood, when the skeleton matures and the growth ends, forming the maximum bone mass in the 20s and early 30s. After about 30 years, the bone formation process is repeated to remove the bone and replenish it again. In this case, the bone formation and bone absorption are paired with each other to maintain balance. After this period, bone loss due to bone resorption cannot follow enough bone formation, resulting in bone loss of 0.3% to 0.5% per year, especially in women at the beginning of menopause, with significant bone loss of 2-3% per year. There is a loss.

Bone is largely composed of four cells: osteoblast, osteoclast, osteoclast, lining cell, and osteocyte. At this time, osteoblasts derived from bone marrow stromal cells lead to bone formation as differentiated cells synthesizing bone matrix, and osteoclasts derived from hematopoietic stem cells lead to bone resorption.

Osteoporosis is a condition in which bone mineralization is reduced due to the reduction of bone tissue, and thus the bone marrow cavity is widened. Bone mass is influenced by several factors, including genetics, nutrition, hormone changes, differences in exercise and lifestyle, and the causes of osteoporosis include old age, lack of exercise, low weight, smoking, low calcium diet, menopause, Ovarian ablation and the like are known. On the other hand, although there are individual differences, blacks have lower bone resorption levels than whites, resulting in higher bone mass, usually the highest in 14 to 18 years of age, and about 1% per year in old age. In particular, women after 30 years of bone reduction continues to progress, and by the hormonal changes, bone reduction rapidly progresses. In other words, estrogen concentration rapidly decreases at the end of menopause, in which a large amount of B-lymphocytes are generated, as in the case of IL-7 (interleukin-7), and the B cell precursor (pre-B) is added to the bone marrow. cells) accumulate, which increases the amount of IL-6, increasing the activity of osteoclasts, and ultimately decreases bone mass.

As such, osteoporosis is an unavoidable symptom for elderly people, especially postmenopausal women, although the degree of osteoporosis is increasing, and as the population ages in developed countries, interest in osteoporosis and its therapeutics is gradually increasing. It is also known that there is a market of about $ 130 billion related to the treatment of bone diseases around the world, which is expected to increase further. have. In Korea, the prevalence of osteoporosis is rapidly increasing as the average life expectancy reaches 80. Recently, a study of local residents showed that 4.5% of men and 19.8% of women had osteoporosis when standardized to the national population. It has been reported to have. This suggests that osteoporosis is more common than diabetes or cardiovascular disease, and osteoporosis is a very important health problem when estimating the pain or cost of treating patients who suffer from fractures.

To date, several substances have been developed for the treatment of osteoporosis. Among them, estrogen, which is most used as a therapeutic agent for osteoporosis, has not yet been tested for its actual efficacy and has to be taken continuously for life. Allendronate also has a problem that the effect is not clear, slow absorption in the digestive tract and inflammation of the stomach and esophagus mucosa. Calcium preparations are known to have fewer side effects and superior effects, but they are more preventive agents than therapeutic agents. Other vitamin D preparations, such as calcitonin, are known but have not been fully studied for their efficacy and side effects. Accordingly, there is a need for a new metabolic bone disease treatment agent having fewer side effects and excellent effects.

On the other hand, the fact that oxidative stress plays an important role in cell function began to be known in the early 1970s. Early studies were conducted mainly from the cytotoxic point of view (Boveris, A. and Chance, B., Biochem). J., 134: 707-716, 1973). In other words, oxidative stress acts directly on cells, causing lipid peroxidation of cell membranes, DNA damage, oxidation of intracellular lipids and proteins, poly (ADP-ribosylation), and depletion of energy in cells, thereby promoting cell death. Cause various diseases (Beckman, KB and Ames, BN, Physiol. Rev., 78: 547-581, 1998). However, subsequent studies suggest that oxidative stress not only has direct cytotoxicity, but also has various cellular functions such as acting as a secondary messenger in cells (Hensley, K. et al., Free Radic). Biol Med., 28: 1456-1462, 2000). In other words, the action of oxidative stress is not a simple cytotoxic effect, but by causing a variety of actions on a variety of cells can mean a direct or indirect impact on human health and disease.

Therefore, studies on the effects of oxidative stress have been actively conducted in many disease models. To date, studies on cardiovascular system including inflammatory diseases such as vasculitis, glomerulonephritis and systemic lupus erythematosus, ischemic heart disease, stroke and intestinal ischemia Diseases, neuro-muscular disorders such as Parkinson's disease, muscular dystrophy and dementia, numerous diseases associated with oxidative stress, including cancer, alcoholism, smoking-related diseases, AIDS, gastric ulcers and hypertension (McCord, JM, Am. J.). Med., 108: 652-659, 2000).

Recently, studies have reported that ROS is involved in bone metabolism (Darden, AG, et al., J. Bone Miner. Res., 11: 671-675, 1996; Yang, S., et al., J. Biol. Chem., 276: 5452-5458, 2001; Fraser, JH, et al., Bone 19: 223-226,1996; Yang, S., et al., Clacif.Tissue Int., 63: 346 -350, 1998). Moreover, bone remodeling is known through the relative action of osteoblasts that form bone and osteoclasts (OC) that resorb bone. Multinuclear osteoclasts have cell adhesion, proliferation, motility, and cell-cell contact to form multi-nucleated giant cells. And monocyte macrophage lineage of hematopoietic progenitor cells through a multistep process of terminal fusion. This process involves a receptor called the receptor activator of NF-κB (hereinafter abbreviated as "RANK") and a receptor activator of NF-κB ligand (hereinafter referred to as "RANKL"). RANKL, called "abbreviated", begins by binding, followed by activation of several signaling cascades. Activated signaling pathways include NF-κB, extracellular signal-regulated kinase (TNF) through a tumor necrosis factor receptor-associated factor 6 (abbreviated as "TRAF6"). Abbreviated "ERK"), c-Jun, N-terminal kinase (abbreviated "JNK") and p38 MAP kinase (p38 mitogen-activated protein kinase). This signaling phenomenon directly affects the differentiation and regulation of osteoclasts (Boyle, W.J., et al., Nature, 423: 337-342, 2003). Once the osteoclasts differentiate, bone resorption is promoted by ROS generated by NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase). Inhibitors of NADPH oxidase induce a decrease in ROS and bone resorption (Yang, S., et al., Clacif. Tissue Int., 63: 346-350, 1998). These results are consistent with the theory that the occurrence of ROS in osteoclasts depends on NADPH oxidase activity and is directly related to osteoclast function.

Accordingly, the present inventors focused on the development of a therapeutic agent for osteoporosis using a molecular mechanism that inhibits the activity of RANKL, and as a result of intensive studies, the pyrazole derivatives of the present invention have excellent NADPH oxidase and RANKL inhibitory activity. The present invention was completed by confirming that they can be used for the prevention or treatment of osteoporosis.

An object of the present invention relates to a composition for preventing and treating osteoporosis excellent in NADPH oxidase and RANKL inhibitory activity including a pyrazole derivative.

Another object of the present invention relates to a method for preventing and treating osteoporosis using the composition.

In one embodiment, the present invention provides a composition for preventing and treating osteoporosis excellent in NADPH oxidase and RANKL inhibitory activity including a pyrazole derivative.

As used herein, the term "osteoporosis" is a state in which the absolute amount of bone excluding the empty part such as the bone marrow is reduced in the whole bone, and may include senile and postmenopausal osteoporosis, endocrine osteoporosis, congenital osteoporosis, floating osteoporosis or traumatic osteoporosis. have.

The pyrazole derivatives of the present invention have a structure such as the following Chemical Formulas 1 to 4.

<Formula 1>

<Formula 2>

<Formula 3>

<Formula 4>

The pyrazole derivatives are not limited thereto, but may be synthesized by a cyclization reaction of hydrazine and β-ketoester, as shown in Scheme 1 below.

<Scheme 1>

The cyclization reaction is Korean Patent No. 726,672, Min-Sup Park et al. Synthetic Communications 2004, 34, 1541-1550; Hyun-Ja Park, et. al. Bioorganic & Medicinal Chemistry Letters 2005, 15, 3307-3312 can be prepared by a method known. Wherein R ¹ is pyridine, R ² is phenyl, methoxyphenyl, nitrophenyl, furanyl. Thus, for example, 2-hydrazinopyridine may be used as the hydrazine. In addition, the β-ketoester is for example ethyl benzoyl acetate, 3- (3-methoxyphenyl) -3-oxo-propionic acid ethyl ester, 3- (4-nitrophenyl) -3-oxo-propionic acid or Ethyl 3- (3-furyl) -3-oxopropanoate may be used. As the reaction solvent, ethanol or acetic acid is preferably used. The cyclization reaction is preferably carried out for 2 to 72 hours at a temperature of 100 to 130 ℃.

In one specific embodiment of the present invention, it was confirmed that the compound of the present invention has an excellent ability to inhibit osteoclasts by RANKL in proportion to concentration.

In addition, the compound of the present invention was found that the average 50% inhibition of bone destruction caused by LPS injection, it was confirmed that the average of 64% inhibition of bone destruction by RANKL injection.

Compositions for the prevention and treatment of osteoporosis comprising the pyrazole derivatives of the present invention may further comprise a suitable carrier, excipient or diluent according to conventional methods.

Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Compositions comprising the pyrazole derivatives according to the invention are in the form of oral dosage forms, external preparations, suppositories or sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, respectively, according to conventional methods. Can be formulated and used.

Specifically, when formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ), Lactose, gelatin and the like can be mixed. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

As another aspect, the present invention relates to a method for preventing and treating osteoporosis using the composition. In one specific aspect, the method for preventing and treating osteoporosis of the present invention relates to a method for increasing the therapeutic effect of osteoporosis by administration. As used herein, the term "administration" means introducing a composition for preventing and treating osteoporosis of the present invention to a patient by any suitable method, and the route of administration of the composition for preventing and treating osteoporosis of the present invention can reach a target tissue. Administration can be by any common route as long as it is available. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, pulmonary administration, rectal administration, intranasal administration, intraperitoneal administration, intradural administration, but not limited thereto. . The composition for preventing and treating osteoporosis according to the present invention may be administered once or two or three times at regular time intervals.

The osteoporosis prevention and treatment composition according to the present invention may be appropriately administered according to various methods commonly known to those skilled in the art in consideration of the type, dosage form, and therapeutic effect of osteoporosis.

The following examples illustrate the invention in more detail. These examples are only for illustrating the present invention, but the scope of the present invention is not limited to the following examples.

The composition for preventing and treating osteoporosis comprising the pyrazole derivatives according to the present invention is excellent in NADPH oxidase and RANKL inhibitory activity and can be used for treating osteoporosis without any special side effects such as conventional therapeutics.

1 is a schematic of a general bone reconstitution process for bone absorption and production.

Figure 2 is a graph measuring the degree of differentiation and TRAP staining by treating osteoclast inhibition by RANKL by concentration.

Figure 3a is a photograph showing that the compound of the present invention inhibits the destruction of bones generated in mice by RANKL or LPS injection.

Figure 3b is a graph quantifying the degree of inhibition of the compound of the present invention to the bone disruption action generated in mice by RANKL or LPS injection.

Production Example : Pyrazole  Synthesis of Derivatives

Preparation of Formula 1 (3-phenyl-1- (pyridin-2-yl) -1H-pyrazol-5-ol)

Ethyl benzoyl acetate (1 equivalent) and 4 ml of ethanol were added to a round bottom flask, and 2-hydrazinopyridine (1.1 equivalent) was diluted in 3 ml of ethanol and slowly added dropwise at 0 ° C. Heated to reflux for 20 minutes. After distillation under reduced pressure to remove the solvent, the resulting solid was washed with hexane and ethyl acetate and dried in vacuo to give the title compound in 87% yield.

Preparation of Formula 2 (3- (3-methoxyphenyl) -1- (pyridin-2-yl) -1H-pyrazol-5-ol)

Into a round bottom flask, 3- (3-methoxyphenyl) -3-oxo-propionic acid ethyl ester (1 equivalent) and 4 ml of ethanol were added thereto, and 2-hydrazinopyridine (1.1 equivalent) was diluted in 3 ml of ethanol. It was slowly added dropwise at 0 ℃. Heated to reflux for 20 minutes. After distillation under reduced pressure to remove the solvent, the resulting solid was washed with hexane and ethyl acetate and dried in vacuo to give the target compound in a yield of 67%.

Preparation of Formula 3 (3- (4-nitrophenyl) -1- (pyridin-2-yl) -1H-pyrazol-5-ol)

In a round bottom flask, 3- (4-nitrophenyl) -3-oxo-propionic acid ethyl ester (1 equivalent) and 4 ml of ethanol were added thereto, and 2-hydrazinopyridine (1.1 equivalent) was diluted in 3 ml of ethanol. It was slowly added dropwise at 0 ° C. Heated to reflux for 20 minutes. After distillation under reduced pressure to remove the solvent, the resulting solid was washed with hexane and ethyl acetate and dried in vacuo to give the title compound in 92% yield.

Preparation of Formula 4 (3- (furan-2-yl) -1- (pyridin-2-yl) -1H-pyrazol-5-ol)

In a round bottom flask, ethyl 3- (3-furyl) -3-oxopropanoate (1.2 equiv) and 3 mL of acetic acid were added, and a solution of 2-hydrazinopyridine (1 equiv) diluted in 2 mL of acetic acid was added. After slowly dropwise addition, the mixture was heated to reflux at 130 ° C. for 72 hours. After distillation under reduced pressure to remove the solvent, the resulting solid was washed with hexane and dried to obtain the target compound in 66% yield.

Example  One: RANKL Osteoclasts by Inhibition  Experiment

Bone marrow cells harvested from mice 5 to 6 weeks old (C57BL / 6J) were placed in 48-well plates, and the macrophage differentiation factor hM-CSF (R & D system Inc.) at a concentration of 30 ng / ml. In differentiation medium (α-MEM (Invitrogen. Co.,), 10% (v / v) fetal bovine serum (FBS), 100 units / ml penicillin, 100 μg / ml streptomycin) for 4 days at 4 × 10 4 cells / well Macrophages derived from bone marrow cells were obtained by incubating at 37 ° C., 95% (v / v) air, 5% (v / v) CO ₂ . In addition to the hM-CSF at a concentration of 30 ng / ml, mouse RANKL, an osteoclast differentiation factor, was expressed in Escherichia coli, purified, purified, and added at a concentration of 100 ng / ml, followed by culturing in osteoclasts under the same conditions. Cell differentiation was induced. At this time, the compounds developed in the present invention were added at the concentrations of 2.5 μM, 5. μM, 10 μM and 20 μM, respectively, and the cells were cultured. During the experiment, the differentiation medium was exchanged every two days. As a result, it was confirmed that cell differentiation was inhibited in a concentration-dependent manner in the experimental group, unlike the control group in which normal differentiation into osteoclasts.

Figure 2 is a graph measuring the degree of differentiation and TRAP staining by treating the effect of the compound by concentration in the process of differentiating osteoclast progenitor cells from the bone marrow into osteoclasts by RANKL treatment. Figure 2 shows the results observed by optical microscopy stained with TRAP (tartrate resistant acid phosphatase) on the 5th day from the start of incubation with the addition of the compound. Specifically, the TRAP staining was performed by fixing the cells at room temperature for 15 minutes in 3.7% formaldehyde and washing twice with distilled water, followed by acetate, phosphate, Leukocyte (TRAP) kitTM (Sigma Co.) at the rate described in the instructions for use. 200 μl / well of a dye solution prepared by mixing Fast Gargnet GBC base, naphthol AS-BI phosphoric acid, sodium nitrite, and tartrate was added and reacted at 37 ° C. for 20 minutes.

As shown in Figure 2, it was confirmed that the compound of the present invention inhibits the osteoclasts in proportion to the concentration.

Example  2: RANKL  or LPS Inhibitory effect of the compounds according to the invention on bone destruction induced by

In order to examine the inhibitory effects of RANKL or LPS-induced bone destruction in bone loss mice model, three groups of control group (untreated group without any treatment) were placed on the cranial calvaria of mice at 7 weeks of age. Consisting of LPS (12.5 mg / kg) and RANKL (2 mg / kg) treated groups, 3 groups of experimental groups treated with 40 mg / kg of compound (untreated group treated with nothing, LPS (12.5 mg / kg) and A total of six groups of RANKL (consisting of 2 mg / kg) treatment groups were injected daily for 4 days. On day 5, the two cranial tubes were taken and their cross sections were examined by H & E staining. The degree of inhibition of bone destruction was analyzed using Image-pro Plus4.5 (Media Cybernetics, Inc.).

3A and 3B are photographs showing the degree of inhibition of the compound of the present invention and the extent to which the compound of the present invention inhibits bone destruction generated in mice by RANKL or LPS injection.

As shown in Figure 3, by administering the compound of the present invention it can be seen that the destruction of bone caused by LPS injection on average 50%, the average destruction of bone by RANKL injection is obtained an average 64% inhibition Could.

Claims (4)

A composition for preventing and treating osteoporosis comprising a pyrazole derivative selected from the group consisting of the following Chemical Formulas 1 to 4: <Formula 1> <Formula 2> <Formula 3> <Formula 4> The composition of claim 1, wherein the pyrazole derivatives inhibit NADPH oxidase activity. The composition of claim 1, wherein the pyrazole derivatives inhibit RANKL in osteoclasts. A method for preventing and treating osteoporosis using the composition of claim 1 or 3.