KR102206831B1 - Meroterpenoids and Their Use - Google Patents

Abstract

The present invention relates to a meroterpenoid-based compound, a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof that can be hydrolyzed in vivo, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition for preventing or treating osteoporosis comprising the same About.

Description

Merotepenoids and Their Use {Meroterpenoids and Their Use}

The present invention relates to a merotefenoi-based compound that can be used for the prevention and treatment of osteoporosis, a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof or a pharmaceutically acceptable salt thereof capable of being hydrolyzed in vivo, and a medicament containing the same, functional It relates to use as food, functional beverage and animal feed.

The bone tissue of the human body is a dynamic organ in which bone remodeling, such as bone formation by osteoblasts and bone resorption by osteoclasts, is constantly repeated throughout life. Bone tissue constitutes the cartilage and skeletal system, serves as support and muscle attachment through mechanical functions, protects living organs and bone marrow, and preserves calcium and phosphorus ions to maintain homeostasis. Bone tissue that functions as such is composed of cell substrates such as collagen and glycoproteins, and various types of cells such as osteoblasts, osteoclasts, and bone cells. Among these, osteoclasts are cells derived from hematopoietic stem cells and are responsible for the absorption of aged bone, and osteoblasts are cells derived from bone marrow stromal cells in the bone marrow and play a major role in bone formation.

Among them, mononuclear cells are differentiated into multinucleated osteoclasts by RANKL (receptor activator of nuclear factor-κB (RANK) ligand). In this differentiation process, RANKL outside of the cell binds to RANK and promotes the activity of mitogen-activated protein kinase (MAPK), which is a transcription factor called NF-κ that enters the nucleus and is involved in osteoclast differentiation. (tartrate-resistant acid phosphatase), MMP-9 (matrix metalloproteinase-9), c-Src tyrosine kinase, etc. can be increased by increasing the expression.Multinuclear osteoclasts formed by this process can produce mineralized bone. It serves to absorb. In addition, when RANKL binds to RANK, it promotes the activity of TRAF6 (tumor necrosis factor receptor-associated factor 6), thereby promoting the activity of transcription factors such as MAPK, NF-κAP-1, and NFATc1 (Biochem and Biophys.Res. Commun. 2003, 305, p211-214). Therefore, blocking the signaling pathway activated by RANKL is recognized as one of the therapeutic approaches for the treatment of bone diseases including osteoporosis.

Osteoporosis, which is a representative example of bone disease, is a disease caused by bone loss caused by loss of calcium in bone as the balance between bone resorption and remodeling is broken and the rate of bone resorption is accelerated. According to the literature, it is known that not only senile osteoporosis and postmenopausal osteoporosis due to lack of sex hormones, but also in young students these days due to lack of calcium intake due to unbalanced diet, causes osteoporosis. And those who take steroids or gastrointestinal drugs for a long time, those who have heavy alcohol, cigarettes, coffee, those who eat a lot of meat, those who do not exercise well, those who are small, those who work sedentary, those who have stomach surgery, low back pain, arthritis, people with muscle pain, People who lie down for a long time, people who feel tired well, etc. are at an increased risk of developing osteoporosis. However, the mechanism of action is not yet clear. Osteoporosis is known to cause various fractures that are easily caused by weakening of the bone rather than the symptoms itself, especially femur fractures, which limit long-term activities and cannot lead a healthy life, and consequently contribute to 15% of the deaths of the elderly. have.

A typical experimental method for developing a therapeutic agent for osteoporosis is to select a substance that primarily inhibits the activity of osteoclasts or increases the activity of osteoblasts, and then uses an animal showing symptoms similar to those of osteoporosis in humans to determine how much bone mass The efficacy of the drug is evaluated to the extent that it is recovered by and how much bone strength is recovered.

The causes of osteoporosis can be considered in various ways, but the most common is aging (aging) or postmenopausal hormonal imbalance in women, so the current treatment is mainly administered with estrogen, vitamin D, and calcitonin. However, the administration of hormones is not a safe treatment method because there is a risk of causing cancer (especially breast cancer, uterine cancer, etc.). Further, as a second-generation therapeutic agent, bisphosphonate is also being administered, but there is a problem that rebound occurs when the administration is stopped. In addition, although either method can delay the progression of osteoporosis, it has a disadvantage that it is difficult to regenerate the once reduced bone.

In addition, since it is necessary to control the balance between osteoclasts and osteoblasts in order to treat bone diseases, there are largely a bone resorption inhibitor and a bone formation stimulator as therapeutic agents for this. Among these, studies on stimulating agents for bone formation are actively progressing, but since strengthening bone density due to stimulating agents for bone formation does not necessarily mean a reduction in fractures, more studies are needed to be widely used clinically. In addition, as agents for stimulating bone formation, accelerators for activating osteoblasts and inhibitors for inhibiting bone resorption of osteoclasts are generally administered to patients for a long period of time, so it is desirable that they are less toxic and can be administered orally. Research is desperately needed.

Biochem. Biophys. Res. Commun. 2003, 305, p211-214

Accordingly, the present inventors have made diligent efforts to find a compound that is less toxic from natural products and can inhibit the differentiation of osteoclasts and promote the differentiation of osteoblasts. As a result, the meroterpenoid compounds remarkably inhibit the differentiation of osteoclasts. By promoting the differentiation of osteoblasts, it was confirmed that it can be used as a preventive and therapeutic agent for osteoporosis, and the invention was completed.

The present invention was intended to separate and purify secondary metabolic compounds that can be used as active ingredients such as medicines for preventing and treating osteoporosis, functional foods, functional beverages, and animal feed.

Accordingly, it is an object of the present invention to provide a use of a meroterpenoid-based compound as an active ingredient in medicine, functional food, functional beverage, and animal feed.

In addition, an object of the present invention is to provide a novel meroterpenoid compound.

In addition, an object of the present invention is to provide a fungal Penicillium rudallense strain that produces a meroterpenoid- based compound.

In order to solve the above problems, the present invention is a meroterpenoid compound represented by the following formula (1), a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof hydrolyzable in vivo, or a pharmaceutically acceptable salt thereof It provides a pharmaceutical composition for preventing or treating osteoporosis comprising as an active ingredient.

[Formula 1]

In Formula 1,

R ₁ is hydrogen, hydroxy or acetoxy;

는 하기 구조에서 선택되고;

Is selected from the following structure;

R ₅ is hydrogen, methoxy, acetoxy or hydroxy;

R ₆ is hydrogen or methyl.

In addition, the present invention comprises as an active ingredient a merotepenoid-based compound represented by Formula 1, a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof hydrolyzable in vivo, or a pharmaceutically acceptable salt thereof It provides a functional food supplement for preventing or improving osteoporosis, a functional beverage, a food additive and a composition for animal feed.

In addition, the present invention comprises as an active ingredient a merotepenoid-based compound represented by Formula 1, a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof hydrolyzable in vivo, or a pharmaceutically acceptable salt thereof It provides a composition that inhibits the differentiation of osteoclasts.

In addition, the present invention comprises as an active ingredient a merotepenoid-based compound represented by Formula 1, a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof hydrolyzable in vivo, or a pharmaceutically acceptable salt thereof It provides a composition that promotes the differentiation of osteoblasts.

In addition, the present invention provides a novel merotepenoid compound selected from the following, a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof hydrolyzable in vivo, or a pharmaceutically acceptable salt thereof.

In addition, the present invention is a fungus Penicillium producing the meroterpenoid-based compound of Formula 2 rudallense strains are provided.

The meroterpenoid-based compound according to the present invention effectively inhibits the differentiation of osteoclasts, thereby inhibiting excessive bone resorption, effectively promoting the differentiation of osteoblasts, promoting new bone formation, and ultimately preventing and treating osteoporosis. It can be usefully used as an active ingredient of the composition.

Fig. 1-A picture showing the results of TRAP staining of a meroterpenoid-based compound and a graph showing the result of counting the number of cells that were positive for TRAP staining according to treatment with a merotepenoid-based compound
Figure 2-A graph showing the results of a toxicity test for osteoclasts of a meroterpenoid compound 157
Figure 3-A graph confirming the mRNA expression level of TRAP according to the daily treatment of the meroterpenoid compound 157 by RT-PCR
Figure 4-A graph confirming the mRNA expression level of osteoclast differentiation markers according to the treatment of meroterpenoid compound 157 by RT-PCR
Figure 5-Results of confirming the expression of NFATc1 protein according to the treatment of the meroterpenoid compound 157 by Western blot experiment
Figure 6-A picture showing the results of ALP staining according to the treatment of the meroterpenoid compound 157 (C; control, negative control)
Figure 7-Graph showing the results of the toxicity test of the meroterpenoid compound 157 against osteoblast cells
Figure 8-A graph confirming the mRNA expression level of ALP, an osteoblast differentiation marker, by RT-PCR after 6 days treatment of the meroterpenoid compound 157
Figure 9-A graph confirming the mRNA expression level of osteoblast differentiation markers according to the treatment of the meroterpenoid compound 157 for 3 days by RT-PCR
Figure 10-A graph confirming the mRNA expression level of osteoblast differentiation markers according to the treatment of the meroterpenoid compound 157 for 6 days by RT-PCR
11-micro-CT photographs confirming the pattern of reduction of bone loss in mouse femurs according to treatment with meroterpenoid compound 157
Figure 12-A graph showing the results of quantitatively measuring bones according to the treatment of meroterpenoid compound 157

The present invention relates to a meroterpenoid compound, a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof hydrolyzable in vivo, or a pharmaceutically acceptable salt thereof, a method for producing, separating and purifying the same from a mold, and the same It relates to use as a pharmaceutical, functional food, functional beverage and animal feed, including.

One aspect of the present invention is a meroterpenoid compound represented by the following formula (1), a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof hydrolyzable in vivo, or a pharmaceutically acceptable salt thereof as an active ingredient. It provides a pharmaceutical composition for preventing or treating osteoporosis comprising:

[Formula 1]

In Formula 1,

R ₁ is hydrogen, hydroxy or acetoxy;

는 하기 구조에서 선택되고;

Is selected from the following structure;

R ₅ is hydrogen, methoxy, acetoxy or hydroxy;

R ₆ is hydrogen or methyl.

In one embodiment, the meroterpenoid-based compound may be used for preventing or treating osteoporosis, as a functional food, a functional beverage, and an animal feed.

In one embodiment, the meroterpenoid-based compound may be represented by Formula 2:

[Formula 2]

In Chemical Formula 2,

R ₁ is hydrogen or hydroxy;

는 하기 구조에서 선택되고;

Is selected from the following structure;

R ₆ is hydrogen or methyl.

In one embodiment, the meroterpenoid-based compound of Formula 2 may be any one or more specifically selected from the following structures.

R ₁ is hydrogen or hydroxy; R ₆ is hydrogen or methyl.

In one embodiment, the meroterpenoid- based compound of Formula 2 is a fungus Penicillium rudallense Strain, specifically marine fungus Penicillium rudallense It can be obtained from an extract of a culture of the strain.

In one embodiment, the meroterpenoid- based compound of Formula 2 is marine fungus Penicillium rudallense It can be obtained by culturing the strain in a liquid nutrient medium and extracting and purifying the culture solution and the cells.

In one embodiment, the meroterpenoid-based compound may more specifically be any one or more selected from the following structures.

As used herein, the term "pharmaceutically acceptable salt" refers to a formulation of a compound that does not cause serious irritation to the organism to which the compound is administered, and does not impair the biological activity and physical properties of the compound. The pharmaceutically acceptable salt is an acid that forms a non-toxic acid addition salt containing a pharmaceutically acceptable anion, for example, an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc., and tartaric acid. , Formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, organic carboxylic acids such as salicylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid Acid addition salts formed by sulfonic acids, such as folic acid, are included. For example, pharmaceutically acceptable carboxylate salts include metal salts or alkaline earth metal salts formed of lithium, sodium, potassium, calcium, magnesium, etc., amino acid salts such as lysine, arginine, guanidine, dicyclohexylamine, N-methyl Organic salts such as -D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline and triethylamine, and the like.

As used herein, the term "osteoporosis" refers to a condition in which there is no abnormality in the structure of the bone and the amount of minerals and substrates constituting the bone is excessively reduced, resulting in a lot of small holes like a sponge in the bone, and it is soft and easily broken. It is also called'osteoporosis' or'osteoporosis'. The osteoporosis generally accompanies symptoms of a decrease in bone mass, that is, a decrease in bone density or a deterioration in bone tissue.

The osteoporosis may include, for example, primary osteoporosis such as osteoporosis following menopause, senile osteoporosis, and osteoporosis following ovariectomy in women; Secondary osteoporosis such as gluticosteroid-induced osteoporosis, hyperthyroidism osteoporosis fixation-induced osteoporosis, heparin-induced osteoporosis, immune suppression-induced osteoporosis, osteoporosis following renal failure, inflammatory osteoporosis, osteoporosis and rheumatoid osteoporosis according to Cushing syndrome; It may be any one or more of bone diseases such as, but is not limited thereto.

In the present specification, the term "prevention" refers to any action that suppresses or delays the onset of osteoporosis by administration of the composition of the present invention, and the term "treatment" in the present specification refers to osteoporosis by administration of the composition of the present invention. It refers to any action in which the symptoms caused by or are beneficially changed.

Preferably, the pharmaceutical composition of the present invention contains a meroterpenoid-based compound, a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof hydrolyzable in vivo, or a pharmaceutically acceptable salt thereof as an active ingredient. Effectively inhibits the expression of NFATc1, DC-STAMP, cathepsin K, and TRAP, which are known as osteoclast differentiation markers, and promotes the expression of OCL, OPN, Runx2 and ALP, which are known as osteoblast activation markers, It can be usefully used as a prophylactic or therapeutic agent for osteoporosis.

In a specific embodiment, in order to determine how the meroterpenoid-based compound affects the differentiation of osteoclasts, bone marrow cells in the femur and tibia of a 5-week-old male mouse were separated, cultured, and then Noid-based compounds were treated and the pattern of osteoclast formation was confirmed. As a result, it was confirmed that osteoclast formation decreased with increasing the treatment concentration of the meroterpenoid-based compound of the present invention, and in particular, at a concentration of 3 μM or more, the differentiation of osteoclasts was significantly reduced, thereby effectively inhibiting the formation of osteoclasts. There was (Fig. 1).

In addition, in another specific example, real-time PCR and western blot experiments were performed to confirm whether the meroterpenoid-based compound inhibits the expression of NFATc1, which plays a key role in osteoclast differentiation. As a result, it was confirmed that the expression of NFATc1 increased by RANKL at a concentration of 10 μM or more of the meroterpenoid compound was remarkably reduced (FIGS. 4 to 5 ).

In another specific embodiment, in order to determine how the meroterpenoid-based compound affects the differentiation of osteoblasts, after culturing C2C12 cells, the merotepenoid derivative was treated and the formation pattern of osteoblasts was confirmed. . As a result, it was confirmed that osteoblast formation increased with increasing the treatment concentration of the meroterpenoid-based compound, and in particular, the formation of osteoblast cells was significantly increased at a concentration of 10 μM or more (FIGS. 6 and 8).

In another specific embodiment, a confirmation experiment was performed to confirm whether the meroterpenoid-based compound promotes the expression of Runx2, which plays a key role in promoting osteoblast differentiation. As a result, it was confirmed that the expression of Runx2 was remarkably increased at a concentration of 10 μM or more of the meroterpenoid compound (FIGS. 9 to 10 ).

In another specific embodiment, the effect of reducing bone loss of the meroterpenoid-based compound was conducted in vivo using a mouse. As a result, it was confirmed that bone loss was reduced when 2 mg/kg or 4 mg/kg of the meroterpenoid compound was treated as compared to the control not treated with the meroterpenoid derivative (FIGS. 11 to 12 ).

As described above, the pharmaceutical composition containing the meroterpenoid-based compound of the present invention as an active ingredient inhibits the formation and differentiation of osteoclasts by inhibiting the expression of increased differentiation factors of osteoclasts, and expression of osteoblast differentiation factors. By promoting the formation and differentiation of osteoblasts, it was confirmed that it was possible to effectively treat osteoporotic diseases by ultimately controlling bone resorption by osteoclasts and promoting bone production by osteoblasts.

In another aspect of the present invention, a merotepenoid compound represented by Formula 1, a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof hydrolyzable in vivo, or a pharmaceutically acceptable salt thereof, as an active ingredient It provides a composition that inhibits the differentiation of osteoclasts, including.

In another aspect of the present invention, a merotepenoid compound represented by Formula 1, a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof hydrolyzable in vivo, or a pharmaceutically acceptable salt thereof, as an active ingredient It provides a composition that promotes the differentiation of osteoblasts comprising.

In another aspect of the present invention, a merotepenoid compound represented by Formula 1, a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof hydrolyzable in vivo, or a pharmaceutically acceptable salt thereof, as an active ingredient It provides a method of preventing or treating osteoporosis by administering a pharmaceutical composition comprising it to an individual in need thereof.

As used herein, the term "individual" refers to all animals including humans who have already developed or may develop osteoporosis, and specifically, may be mammals including humans, but is not limited thereto. In addition, in the present invention, the individual may be excluded from humans, but is not limited thereto. By administering the pharmaceutical composition of the present invention to an individual, there is an effect of effectively preventing and treating the disease.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to an individual by any suitable method, and the route of administration may be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. have.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment in medical treatment, and the effective dose level refers to the type and severity of the individual, and age. , Sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art. The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the degree of the disease, the form of the drug, the route of administration and the duration, and the total amount of daily dosage may be determined by the treating physician within the correct medical judgment range In general, an amount of 0.001 to 1000 mg/kg, preferably 0.05 to 200 mg/kg, and more preferably 0.1 to 100 mg/kg may be administered once or several times a day.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient. Pharmaceutically acceptable carriers can be used without limitation, those commonly used in this field as vehicles or media usable for pharmaceutical administration. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose. , Polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils, but are not limited thereto.

Each of the compositions of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions according to conventional methods. . Specifically, when formulated, it may be prepared using diluents or excipients such as fillers, weight agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. These solid preparations may be prepared by mixing at least one or more excipients, for example starch, calcium carbonate, sucrose, lactose, gelatin, and the like. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It can be prepared by adding various excipients, such as wetting agents, sweetening agents, fragrances, preservatives, and the like, in addition to oral liquids and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, 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 ethyloleate, and the like can be used. As a base for suppositories, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

The composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to a desired method, and the dosage is the patient's condition and weight, the degree of disease , It depends on the drug form, administration route and time, but may be appropriately selected by those skilled in the art.

In another aspect of the present invention, a merotepenoid compound represented by Formula 1, a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof hydrolyzable in vivo, or a pharmaceutically acceptable salt thereof, as an active ingredient It provides health functional foods for preventing or improving osteoporosis, including.

As used herein, the term "improvement" refers to any action that at least reduces the severity of a parameter associated with the condition being treated, for example a symptom.

There is no particular limitation on the type of the health functional food, and specific examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, There are teas, drinks, alcoholic beverages, vitamin complexes, and the like, and may include all health functional foods in the usual sense, and may include foods used as feed for animals.

The health functional foods include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, It may contain organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, it may contain flesh for the manufacture of natural fruit juice and fruit juice beverages and vegetable beverages. These components may be used independently or in combination.

In addition, the above health functional food may additionally contain food additives, and the suitability as a "food additive" shall be determined according to the General Regulations of the Food Additive Code approved by the Food and Drug Administration and general test methods, etc. It is judged according to standards and standards.

Items listed in the "Food Additives Code", for example, chemical synthetic products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as red pigment, licorice extract, crystalline cellulose, and guar gum, L- Mixed preparations, such as a sodium glutamate preparation, an alkali additive for noodles, a preservative preparation, and a tar color preparation, are mentioned.

In the process of manufacturing the health functional food, the content of the extract according to the present invention added to food including beverages may be appropriately added or subtracted as needed.

Another aspect of the present invention is a fungus Penicillium that produces the meroterpenoid-based compound of Formula 2 rudallense Provide strain.

In one embodiment, the fungus Penicillium rudallense The strain is the marine fungus Penicillium rudallense It may be a strain.

Another aspect of the invention is the fungus Penicillium It provides a method for producing, separating and purifying the meroterpenoid compound of Formula 2 from rudallense .

The meroterpenoid-based compound of Formula 2 is the fungus Penicillium rudallense Strain, specifically marine fungus Penicillium rudallense It can be produced and separated from the culture of the strain. The meroterpenoid-based compound of Formula 1 is the marine fungus Penicillium rudallense It can be obtained by culturing the strain in a liquid nutrient medium and extracting and purifying the culture solution and the cells.

In one embodiment, the meroterpenoid- based compound of Formula 2 is obtained by extracting the culture of the marine fungus Penicillium rudallense strain with acetone and methanol, evaporating the solvent under reduced pressure, and partitioning extraction with water and ethyl acetate. Separated from the residue obtained by evaporating the solvent of the acetate layer again, the residue was first purified by flash column chromatography on silica using an eluent in which chloroform and methanol were mixed stepwise, and then acetonitrile and water were mixed. Using the eluent that was stepwise differently, the meroterpenoid-based compound of Formula 2 may be obtained by secondary purification by C18 reverse phase high performance liquid chromatography.

In one embodiment, preferably, the residue is first purified by flash column chromatography on silica using an eluent consisting of 1 to 2% methanol and 98 to 99% chloroform, and the elution rate is based on 1 mL/min. As an eluent, a 65% aqueous acetonitrile solution is used as an eluent, followed by secondary purification by C18 reverse phase high performance liquid chromatography to obtain a meroterpenoid-based compound of Formula 2 at a retention time of 45 minutes.

Another aspect of the present invention provides a novel merotepenoid compound selected from the following, a hydrate thereof, a solvate thereof, a stereoisomer thereof, a precursor thereof hydrolyzable in vivo, or a pharmaceutically acceptable salt thereof:

The meroterpenoid-based compound of the above structure can be used for preventing or treating osteoporosis, as a functional food, a functional beverage, and an animal feed.

Hereinafter, the present invention will be described in more detail through examples. These examples are for describing the present invention in more detail, and the scope of the present invention is not limited by these examples, and various modifications and changes may be made.

[Example 1] Extraction, purification, and identification of meroterpenoid compounds of formula (2)

The meroterpenoid compound of Chemical Formula 2 according to the present invention was isolated from the culture of the marine fungus Penicillium rudallense . Immediately after collecting the marine soil fungi for separating solid plate medium (PDA, Difco) one months from the fungus Penicillium grew after culturing up to The rudallense was isolated. Isolated Penicillium rudallense was cultured in a PDB liquid medium (seawater or fresh water) for one week, the culture and mycelia were extracted with acetone and methanol, the solvent was evaporated under reduced pressure, and the residual extract was partitioned and extracted with water and ethyl acetate. The solvent was evaporated from the partitioned extracted ethyl acetate layer under reduced pressure. The residue was first purified by flash column chromatography on silica using a solution obtained by stepwise mixing chloroform and methanol as an eluent. Methanol 1% and 2% fractions were eluted with 65% acetonitrile aqueous solution on C18 reverse phase high performance liquid chromatography (Luna 2 C18, particle diameter 5um, 250 × 10 mm, UV detector, elution rate 1 ml/min), retention time The solvent was removed from the eluate for 45 minutes under reduced pressure to obtain meroterpenoid compounds 151 to 161 in an amorphous solid state.

The molecular structures of the obtained novel merotepenoid compounds are ¹ H, ¹³ C, COSY (correlated spectroscopy), HSQC (heteronuclear single quantum coherence), HMBC (heteronuclear multiple-bond correlation), NOESY (nuclear overhauser effect spectroscopy). , ROESY (rotating-frame nuclear overhauser effect spectroscopy), CD (circular dichroism), etc. spectrometry), HRFABMS (high resolution fast-atom bombardment mass spectrometry), and other spectroscopic methods.

(a) meroterpenoid compound 151 (austalide H)

The following compound 151 (austalide H) is Penicillium It was separated and purified from rudallense according to the above method.

It was isolated as an amorphous solid, and the molecular formula was determined as C ₂₆ H ₃₆ O ₈ based on HRESIMS data. The maximum absorption wavelength (λ _max 223, 268 nm) of the UV spectra showed the presence of a phthalide substructure. Based on the 1D and 2D NMR spectra, a total of 7 methyl group signals were confirmed, including 1 aromatic methyl group, 2 methoxy groups, and 4 aliphatic groups. In addition, 4 methylene and 1 oxidized methylene were identified, 2 methine groups, 1 oxygenated methine, and 11 non-protonated carbons. carbon) was confirmed. It was confirmed that there were two ester carboxyl carbons at δ 176.5 and 171.9 of the ¹³ C NMR spectrum. The cyclic structure was confirmed through NMR data and HMBC signal of Compound 151, and the tetracyclic structure was determined by connecting the rings.

¹ H NMR (700 MHz, CD ₃ OD): δ 5.12 (s), 4.56 (br s), 4.05 (s), 3.68 (s), 3.06 (d, 18.5), 2.81 (dd, 18.5, 8.1), 2.47 (m), 2.43 (dd, 15.5, 2.4), 2.34 (m), 2.33 (m), 2.07 (s), 1.94 (dd, 15.5, 4.0), 1.83 (m), 1.78 (d, 8.1), 1.52 (d, 2.0), 1.45 (s), 1.24 (s), 1.42 (s), 0.98 (s); ¹³ C NMR (175 MHz, CD ₃ OD): δ 176.5, 171.9, 159.7, 156.5, 147.5, 108.4, 117.4, 116.0, 78.4, 76.8, 70.0, 62.3, 52.2, 51.9, 46.0, 42.1, 41.5, 35.9, 32.9 , 31.8, 30.1, 28.2, 21.8, 18.5, 10.8.

(b) meroterpenoid compound 152 (austalide W)

The following compound 152 (austalide W) is Penicillium rudallense It was separated and purified according to the above method.

It was separated as an amorphous solid, and the molecular formula was determined as C ₂₅ H ₃₂ O ₆ based on HRFABMS data. Except for the oxidized methine (δ _H 4.38, δ _C 68.5) and the methoxy group (δ _H 4.05, δ _C 62.3), the structural features of Compound 157 were the same. The IR spectra showed absorption at 1748 cm ^{- 1} and 1700 cm ^-1 , and from this, it was confirmed that an ester carbonyl group and a ketone group were present, respectively. Maximum absorption wavelength of UV spectra (λ _max 216, 267 nm) showed the presence of a phthalide substructure. The pentacyclic structure of Compound 152 was confirmed based on 1D and 2D NMR spectra. Through NOE, the relative configuration of H-13/H3-25, H-14/H3-25, H3-26/H3-27 is 11S*, 13R*, 14R*, 20S*, 21R* respectively. Confirmed.

¹ H NMR (700 MHz, CD ₃ OD): δ 5.23 (s), 4.38 (br s), 4.05 (s), 3.05 (d, 18.4), 2.90 (dd, 18.4, 8.1), 2.76 (ddd, 15.7 , 13.7, 6.4), 2.48 (dd, 15.4, 2.5), 2.30 (ddd, 15.7, 5.1, 2.9), 2.12 (ddd, 13.4, 7.0, 3.6), 2.09 (s), 2.00 (dd, 15.4, 3.8) , 1.72 (d, 8.1), 1.56 (br s), 1.52 (td, 13.4, 5.3), 1.36 (s), 1.24 (s), 1.17 (s), 1.06 (s); ¹³ C NMR (175 MHz, CD ₃ OD): δ 218.9, 171.8, 159.6, 156.7, 147.6, 117.2, 116.2, 108.6, 78.6, 69.9, 68.5, 62.3, 56.4, 49.6, 48.0, 47.3, 41.4, 38.5, 35.2 , 27.7, 26.7, 24.4, 19.0, 17.0, 10.8.

(c) meroterpenoid compound 153 (austalide L)

The following compound 153 (austalide L) is Penicillium rudallense It was separated and purified according to the above method.

It was separated as an amorphous white solid, and the molecular formula was determined as C ₂₅ H ₃₂ O ₆ based on HRESIMS data. The IR spectra showed absorption at 1700 cm ^-1 , confirming the existence of a carbonyl group in the six-membered ring. Maximum absorption wavelength of UV spectra (λ _max 221, 267 nm) showed the presence of a phthalide substructure. The pentacyclic structure of Compound 153 was confirmed based on 1D and 2D NMR spectra, and a total of 6 methyl group signals including 1 aromatic methyl group, 1 methoxy group, and 4 aliphatic groups. Was confirmed. In addition, 5 methylene and 1 oxidized methylene were identified, and 1 methine group and 12 non-protonated carbon were identified. ^{In the 13} C NMR spectrum of δ 169.3 and 216.2, it was confirmed that one ester carboxyl carbon and carbonyl carbon existed, respectively. Through the deuterium substitution experiment, it was confirmed that a hydroxy group was present in C-14, the cyclic structure was confirmed through the HMBC signal, and the structure was determined by connecting the pentacyclic structure.

¹ H NMR (500 MHz, chloroform-d): δ 5.077 (s), 1.167 (s), 1.467 (ddd, 13.4, 4.0, 4.0), 1.747 (d, 12.9), 1.970-2.028 (m), 2.015 ( s), 2.075 (ddd, 13.4, 13.4, 4.0), 2.156 (ddd, 13.4, 13.4, 4.0), 2.254 (dd, 7.0, 1.9), 2.736-2.829 (m), 4.064 (s), 0.781 (s) , 1.092 (s), 1.128 (s); ¹³ C NMR (125 MHz, chloroform-d): δ 10.61, 18.04, 18.26, 21.62, 23.57, 24.19, 26.81, 33.06, 33.39. 33.3, 40.72, 41.23, 56.62, 61.85, 68.16, 76.12, 79.64, 107.23, 114.40, 115.84, 145.41, 155.29, 158.59, 169.35, 216.

(d) meroterpenoid compound 154 (13-deacetoxyaustalide I)

The following compound 154 (13-deacetoxyaustalide I) is Penicillium rudallense It was separated and purified according to the above method.

It was separated as an amorphous white solid, and the molecular formula was determined as C ₂₅ H ₃₂ O ₆ based on HRESIMS data. The IR spectra showed absorption at 1748 cm ^-1 , and it was confirmed that an ester carbonyl group was present. Maximum absorption wavelength of UV spectra (λ _max 221, 267 nm) showed the presence of a phthalide substructure. Based on the 1D and 2D NMR spectra, the pentacyclic structure of compound 154 was confirmed, and a total of 6 methyl group signals, including 1 aromatic methyl group, 1 methoxy group, and 4 aliphatic groups. Confirmed. In addition, 5 methylene and 1 oxidized methylene were identified, and 2 methine groups and 11 non-protonated carbons were identified. Δ of the ¹³ C NMR spectrum In 175.2 and 169.7, it was confirmed that two ester carboxyl carbons were present, the cyclic structure was confirmed through HMBC signal confirmation, and the structure was determined by connecting the pentacyclic structures.

¹ H NMR (500 MHz, chloroform-d): δ 5.12 (s), 2.24 (td, 2.7, 14.0), 1.66 (m), 1.92 (m), 1.54 (m), 1.84 (dd, 1.5, 13) , 2.67 (ddd, 2.7, 10.9, 15.7), 2.59 (ddd, 2.7, 8.5, 15.7), 1.95 (m), 1.68 (m), 1.54 (d, 8.1), 2.95 (d, 18.5), 2.82 (dd , 8.0, 18.5), 2.05 (s), 1.20 (s), 1.41 (s), 1.51 (s), 0.82 (s), 4.12 (s); ¹³ C NMR (125 MHz, chloroform-d): δ 169.9, 158.9, 155.4, 145.4, 115.8, 114.1, 107.1, 78.9, 76.5, 68.3, 61.8, 54.2, 48.1, 40.4, 38.8, 38.2, 38.1, 28.5, 27.3 , 27.1, 18.0, 17.9, 15.7, 14.3, 10.6.

(e) meroterpenoid compound 155 (austalide Q)

The following compound 155 (austalide Q) is Penicillium rudallense It was separated and purified according to the above method.

Separated as an amorphous white solid, the molecular formula was determined to be C ₂₆ H ₃₄ O ₇ based on HRESIMS data. Maximum absorption wavelength of UV spectra (λ _max 222, 268 nm) showed the presence of a phthalide substructure. The structure of Compound 155 was confirmed based on 1D and 2D NMR spectra, and a total of 6 methyl group signals were confirmed, including 1 aromatic methyl group, 2 methoxy groups, and 3 aliphatic groups. I did. In addition, 5 methylene and 1 oxidized methylene were identified, 2 methine groups, 1 oxygenated methine, and 11 non-protonated carbons. carbon) was confirmed. Δ of the ¹³ C NMR spectrum In 178.1 and 171.8, it was confirmed that two ester carboxyl carbons were present, and the cyclic structure was confirmed through NMR data and HMBC signal confirmation, and the tetracyclic structure was determined by connecting the rings.

¹ H NMR (700 MHz, CD ₃ OD): δ 5.22 (s), 5.05 (d, 1.9), 4.97 (s), 4.06 (s), 4.06 (br s), 3.68 (s), 2.94 (d, 18.4), 2.86 (dd, 18.4, 7.8), 2.42 (dd, 15.3, 2.3), 2.37 (m), 2.21 (d, 2.0), 2.09 (s), 2.03 (dd, 15.3, 4.1), 1.82 (d , 7.8), 1.66 (m), 1.26 (s), 1.91 (s), 0.87 (s); ¹³ C NMR (175 MHz, CD ₃ OD): δ 176.1, 171.8, 159.6, 156.7, 147.6, 147.0, 117.2, 116.2, 116.2, 108.6, 79.1, 72.1, 69.9, 62.3, 53.5, 52.2, 45.9, 40.7, 40.7 , 36.1, 29.8, 27.9, 26.5, 20.8, 18.9, 10.8.

(f) meroterpenoid compound 156 (austalide P)

The following compound 156 (austalide P) is Penicillium rudallense It was separated and purified according to the above method.

It was isolated as an amorphous white solid, and the molecular formula was determined to be C ₂₆ H ₃₆ O ₇ based on HRESIMS data. The IR spectra showed absorption at 1748 cm ^-1 , and it was confirmed that an ester carbonyl group was present. Maximum absorption wavelength of UV spectra (λ _max 223, 267 nm) showed the presence of a phthalide substructure. The structure of compound 156 was confirmed based on 1D and 2D NMR spectra, and a total of 7 methyl group signals were confirmed, including 1 aromatic methyl group, 2 methoxy groups, and 4 aliphatic groups. I did. In addition, 5 methylene and 1 oxidized methylene were identified, and 2 methine groups and 11 non-protonated carbons were identified. Δ of the ¹³ C NMR spectrum In 171.1 and 177.7, it was confirmed that there were two ester carboxyl carbons, and the cyclic structure was confirmed through NMR data and HMBC signal confirmation, and the tetracyclic structure was determined by connecting the rings.

¹ H NMR (500 MHz, Methanol-d): δ 5.21 (s), 1.61 (dt, 3.8, 14.3), 1.52 (t, 10.7), 1.83 (m), 2.14 (dd, 3.4, 13.7), 2.32 ( m), 2.60 (m), 1.80 (m), 2.42 (m), 1.70 (d, 8.1 ), 2.78 (dd, 8.1, 18.5), 3.03 (d, 18.6), 2.08 (s), 1.20 (s) , 1.19 (s), 5.05 (d, 1.8), 1.27 (s), 0.70 (s), 3.68 (s), 4.04 (s); ¹³ C NMR (150 MHz, Methanol-d): δ 177.7, 172.1, 160.0, 158.1, 148.0, 118.0, 117.3, 108.0, 78.3, 75.7, 69.8, 62.1, 52.0, 41.6, 41.2, 40.2, 40.1, 34.9, 33.2 , 30.0, 28.0, 27.9, 22.4, 19.5, 18.5, 10.6.

(g) meroterpenoid compound 157 (austalide K)

The following compound 157 (austalide K) is Penicillium rudallense It was separated and purified according to the above method.

It was separated as an amorphous solid, and the molecular formula was determined as C ₂₅ H ₃₂ O ₅ based on HRFABMS data. The IR spectra showed absorption at 1748 cm ^{- 1} and 1700 cm ^-1 , and from this, it was confirmed that an ester carbonyl group and a ketone group were present, respectively. Maximum absorption wavelength of UV spectra (λ _max 216, 267 nm) showed the presence of a phthalide substructure. Based on the 1D and 2D NMR spectra, the pentacyclic structure of Compound 157 was confirmed, and a total of 6 methyl group signals, including 1 aromatic methyl group, 1 methoxy group, and 4 aliphatic groups. Confirmed. In addition, 5 methylene and 1 oxidized methylene were identified, and 2 methine groups and 11 non-protonated carbons were identified. Δ of the ¹³ C NMR spectrum In 169.6 and 216.8, it was confirmed that ester carboxyl carbon and carbonyl carbon were present, respectively, and the cyclic structure was confirmed through HMBC signal confirmation, and the structure was determined by connecting the pentacyclic structure.

¹ H NMR (700 MHz, chloroform-d): δ 5.12 (s), 4.11 (s), 1.48 (d, m), 1.49 (m), 2.29 (dt, 14.1, 2.80), 1.66 (td, 14.1, 4.3), 2.10 (ddd, 11.7, 7.0, 3.8), 1.51 (m), 2.53 (ddd, 16.1, 11.7, 7.0), 2.41 (ddd, 16.1, 6.4, 3.8), 1.19 (s), 1.12 (s) , 1.03 (s), 1.81 (qd, 13.0, 3.5), 1.52 (m), 2.93 (d, 18.5), 2.81 (dd, 18.5, 8.1), 0.72 (s), 2.04 (s); ¹³ C NMR (175 MHz, chloroform-d): δ 216.8, 169.6, 158.8, 155.5, 145.7, 115.4, 114.6, 107.5, 76.3, 68.4, 62.1, 54.3, 47.4, 47.2, 39.9, 38.5, 37.8, 34.2, 27.2 , 26.8, 21.8, 19.2, 18.4, 14.3, 10.8.

(h) meroterpenoid compound 158 (austalide X)

The following compound 158 (austalide X) is Penicillium It was separated and purified from rudallense according to the above method.

It was isolated as an amorphous white solid, and the molecular formula was determined as C ₂₆ H ₃₄ O ₆ based on HRESIMS data. Maximum absorption wavelength of UV spectra (λ _max 223, 268 nm) showed the presence of a phthalide substructure. The structure of compound 158 was confirmed based on 1D and 2D NMR spectra, and a total of 6 methyl group signals were confirmed, including 1 aromatic methyl group, 2 methoxy groups, and 3 aliphatic groups. I did. In addition, 6 methylene and 1 oxidized methylene were identified, and 2 methine groups and 11 non-protonated carbons were identified. Δ of the ¹³ C NMR spectrum In 176.1 and 171.8, it was confirmed that two ester carboxyl carbons existed, and the cyclic structure was confirmed through NMR data and HMBC signal confirmation, and the tetracyclic structure was determined by connecting the rings.

¹ H NMR (700 MHz, CD ₃ OD): δ 5.22 (s), 4.90 (s), 4.70 (s), 4.05 (s), 3.68 (s), 2.90 (d, 18.3), 2.83 (dd, 18.3 , 7.9), 2.46 (ddd, 15.5, 11.3, 4.9), 2.36 (ddd, 15.5, 11.1, 6.6), 2.23 (m), 2.15 (dt, 16.2, 3.4), 2.10 (qd, 13.4, 3.6), 2.07 (s), 1.75 (s), 1.73 (m), 1.73 (m), 1.70 (m), 1.65 (m), 1.43 (ddt, 13.4, 4.2, 2.4), 1.22 (s), 0.59 (s); ¹³ C NMR (175 MHz, CD ₃ OD): δ 171.8, 160.4, 156.7, 148.5, 147.5, 117.1, 116.0, 114.6, 108.3, 78.2, 69.8, 62.3, 52.2, 50.9, 41.2, 40.3, 39.9, 34.6, 29.5 , 27.5, 25.1, 24.0, 19.0, 18.8, 10.7.

(i) meroterpenoid compound 159 (austalide V)

The following compound 159 (austalide V) is Penicillium It was separated and purified from rudallense according to the above method.

It was separated as an amorphous white solid, and the molecular formula was determined to be C ₂₄ H ₃₀ O ₅ based on HRFABMS data. Maximum absorption wavelength of UV spectra (λ _max 223, 267 nm) showed the presence of a phthalide substructure. Based on the 1D and 2D NMR spectra, one aromatic methyl group and four aliphatic methyl groups were identified. And 6 methylene and 2 methine were identified. Three aliphatic quaternary carbon, aromatic carbon, ketone carbonyl, and ester carbonyl groups were identified through the HSQC signal. Compound 159 was inferred from the molecular formula to have a total of 5 rings. Through COSY and HMBC data, oxidized benzylic methylene is linked to aromatic carbone and ester carbonyl carbon, and diastereotopic benzylic methylene at the diastereotopic benzylic site methylene) was connected to aromatic carbon, it was confirmed that it had a phthalide partial structure, and a pentacyclic structure was connected. The relative structure of compound 159 was determined as 11S*, 14R*, 20S*, and 21R* by analyzing ROESY spectrum, and the absolute structure of compound 159 was confirmed as 11S, 14R, 20S, and 21R through CD measurement.

¹ H NMR (700 MHz, CD ₃ OD): δ 5.22 (s), 2.89 (d, 18.3), 2.79 (dd, 18.3, 8.0), 2.56 (ddd, 16.2, 11.3, 7.2), 2.43 (ddd, 16.2 , 6.8, 3.9), 2.26 (dt, 13.9, 2.9), 2.10 (ddd, 10.1, 7.1, 3.9), 2.03 (s), 1.83 (qd, 12.8, 3.4), 1.75 (td, 13.9, 4.1), 1.64 (d, 8.0), 1.60 (m), 1.57 (m), 1.21 (s), 1.12 (s), 1.03 (s), 0.74 (s); ¹³ C NMR (175 MHz, CD ₃ OD): δ 219.7, 174.1, 160.7, 153.9, 145.5, 112.6, 111.0, 103.1, 77.7, 70.6, 55.0, 48.3, 47.9, 40.7, 39.3, 38.8, 35.1, 27.3, 27.2 , 22.1, 20.2, 18.7, 14.7, 10.6.

(j) meroterpenoid compound 160 (austalide P acid)

The following compound 160 (austalide P acid) is Penicillium It was separated and purified from rudallense according to the above method.

Separated as an amorphous white solid, the molecular formula was determined to be C ₂₅ H ₃₆ O ₇ based on HRESIMS data. Maximum absorption wavelength of UV spectra (λ _max 223, 268 nm) showed the presence of a phthalide substructure. The structure of Compound 160 was confirmed based on 1D and 2D NMR spectra, and a total of 6 methyl group signals were confirmed, including 1 aromatic methyl group, 1 methoxy group, and 4 aliphatic groups. I did. In addition, 5 methylene and 1 oxidized methylene were identified, and 2 methine groups and 11 non-protonated carbons were identified. Δ of the ¹³ C NMR spectrum In 172.0 and 184.4, it was confirmed that there were ester carboxyl carbon and carboxylic carbonyl carbon, respectively, and the cyclic structure was confirmed through NMR data and HMBC signal confirmation, and the tetracyclic structure by connecting the rings. Was determined.

¹ H NMR (700 MHz, CD ₃ OD): δ 5.22 (s), 4.02 (s), 3.05 (d, 18.5), 2.77 (dd, 18.5, 7.9), 2.43 (m), 2.42 (m), 2.18 (m), 2.12 (dt, 14.0, 3.0), 2.06 (s), 1.86 (m), 1.83 (m), 1.74 (d, 7.9), 1.63 (m), 1.60 (m), 1.54 (m), 1.30 (s), 1.21 (s), 1.21 (s), 0.69 (s); ¹³ C NMR (175 MHz, CD ₃ OD): δ 184.4, 172.0, 160.5, 156.5, 147.5, 117.4, 115.9, 108.2, 78.3, 76.0, 70.0, 62.2, 51.6, 42.9, 41.1, 40.4, 35.9, 33.3, 32.9 , 28.2, 27.8, 22.8, 19.7, 18.8, 10.7.

(k) meroterpenoid compound 161 (17S-dihydroaustalide K)

The following compound 161 (17S-dihydroaustalide K) is Penicillium rudallense It was separated and purified according to the above method.

It was separated as an amorphous white solid, and the molecular formula was determined to be C ₂₅ H ₃₄ O ₅ based on HRESIMS data. Maximum absorption wavelength of UV spectra (λ _max 223, 266 nm) showed the presence of a phthalide substructure. Based on the 1D and 2D NMR spectra, the pentacyclic structure of Compound 161 was confirmed, and a total of 6 methyl group signals, including 1 aromatic methyl group, 1 methoxy group, and 4 aliphatic groups. Confirmed. In addition, 5 methylene and 1 oxidized methylene were identified, 2 methine groups, 1 oxidized methine, and 10 non-protonated carbons. carbon) was confirmed. Δ of the ¹³ C NMR spectrum At 169.9, it was confirmed that there was ester carboxyl carbon, the cyclic structure was confirmed through HMBC signal confirmation, and the structure was determined by connecting the pentacyclic structure.

¹ H NMR (500 MHz, chloroform-d): δ 5.11 (s), 2.27 (m), 2.24 (m), 1.64 (m), 1.60 (m), 0.93 (d, 11.5), 3.24 (dd, 4.5 , 11.7), 1.57 (m), 1.68 (m), 1.91 (m), 0.88 (m), 1.39 (d, 8.8), 2.91 (d, 18.6), 2.73 (dd, 8.3, 18.6), 2.03 (s ), 1.16 (s), 0.78 (s), 1.03 (s), 0.61 (s), 4.10 (s); ¹³ C NMR (125 MHz, chloroform-d): δ 175.2, 169.7, 158.4, 155.5, 145.9, 115.3, 114.2, 107.6, 85.9, 76.1, 68.3, 62.2, 54.0, 47.5, 40.6, 39.5, 37.4, 32.5, 31.8 , 27.3, 25.8, 22.3, 18.7, 16.8, 10.7.

[Example 2] Culture and differentiation of osteoclasts

Bone marrow cells were obtained by separating the femur and tibia of a 5-week-old mouse, and washing the intraosseous space with a 1 cc syringe. The isolated bone marrow cells were cultured for 3 days in α-minimum essential medium (α-MEM) medium containing 10% FBS, antibiotics, and M-CSF (30 ng/ml). After 3 days, the attached cells were used as phagocytic cells (bone marrow macrophage, BMM). Phagocytic cells were cultured with the addition of M-CSF (30 ng/ml) and RANKL (10 ng/ml) and treated with a meroterpenoid-based compound at different concentrations. After 4 days, the cultured cells were stained with a TRAP (tartarate resistance acid phosphatase) solution (Sigma Aldrich, USA), and cells stained in red were regarded as osteoclasts.

[Example 3] TRAP (tartarate resistance acid phosphatase) staining and activity analysis

Multinuclear osteoclasts are fixed with 3.7% formalin for 10 minutes and stained red by treatment with TRAP solution when the permeability of the cell membrane is increased with 0.1% Triton X-100 for 10 minutes. The red-stained osteoclasts were recognized as osteoclasts only having three or more nuclei. TRAP activity was treated with TRAP buffer solution (100 mM sodium citrate pH 5.0, 50 mM sodium tartrate) containing 3 mM p-nitrophenyl phosphate (Sigma-Aldrich) in the osteoclasts stained with TRAP, and reacted at 37° C. for 5 minutes. After the reacted supernatant was transferred to a new plate, 0.1 N NaOH was added in an equal amount to check the absorbance at 405 nm.

As a result, as shown in FIG. 1, treatment with the meroterpenoid-based compounds (152, 153, 154, 156, 157, 159, 161) significantly reduced the number of red-stained osteoclasts while inhibiting TRAP activity in a concentration-dependent manner. It was confirmed that it was reduced to In particular, it was found that merotepenoid-based compounds (152, 153, 154, 156, 157, 159, 161) significantly reduced the TRAP activity at a concentration of 3 μM or more, thereby significantly reducing the number of osteoclasts.

Therefore, it was found that the meroterpenoid-based compound according to the present invention effectively inhibits the differentiation of osteoclasts and thus can be usefully used in bone diseases such as osteoporosis.

[Example 4] Cytotoxicity analysis

Phagocytic cells were cultured for 3 days in a 96-well plate at a density of 1 × 10 ⁴ cells/well with the addition of a meroterpenoid compound and M-CSF (30 ng/ml). After 3 days, 50 µl of the CCK solution was added to each well, and after incubation for 4 hours, the absorbance was confirmed at 450 nm using an ELISA reader (Molecular Devices, CA, USA).

As a result, as shown in FIG. 2, with respect to the 100% survival rate of the control group, which was not treated with anything, the cell survival rate did not significantly decrease even when the meroterpenoid compound 157 was treated by concentration. Therefore, it was confirmed that the meroterpenoid-based compound according to the present invention had little cytotoxicity.

[Example 5] RT-PCR analysis

RT-PCR analysis was performed to confirm the mRNA expression pattern of key differentiation markers involved in the differentiation process of osteoclasts, and specifically, the patterns of NFATc1, TRAP, DC-STAMP and Cathepsin K were confirmed. First, RANKL was treated to induce the differentiation of osteoclasts, and then a meroterpenoid-based compound was treated. RNA for conducting the experiment was isolated from the cells cultured in Example 2 with TRIzol (Invitrogen) solution according to the manufacturer's method. 1 μg of the isolated RNA was synthesized as cDNA using oligo dT primer, dNTP, buffer, dithiothreitol, RNase inhibitor, and Superscript II reverse transcriptase. The synthesized cDNA was obtained by real-time PCR amplification using SYBR green using primers.

As a result, RANKL, which induces the differentiation of osteoclasts, increased the expression of the TRAP gene as time passed, but it was confirmed that the meroterpenoid compound 157 decreased the expression of the TRAP gene (FIG. 3). In addition, when RANKL treatment, which induces osteoclast differentiation, increases the expression pattern of all markers, treatment with the meroterpenoid compound 157 according to the present invention significantly reduces the expression of the increased osteoclast differentiation markers. It could be confirmed (Fig. 4).

[Example 6] Western blot analysis

Western blot analysis was performed to confirm the protein expression pattern of NFATc1, a key differentiation marker involved in the differentiation process of osteoclasts, and through which pathway the meroterpenoid compounds act on the differentiation of osteoclasts. I did.

Specifically, the cells cultured in Example 2 were lysis buffer (50 mM tris-Cl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1% deoxycholate, and protease. inhibitors), and centrifugation (14,000 rpm) was performed to obtain pure protein. Protein was quantified using a DC Protein assay kit (BioRad, Hercules, CA, USA), and the same amount of protein was isolated on 10% SDS-polyacrilamide gel. The separated protein was transferred to a PVDF membrane (Amersham Biosciences) and the protein expression level was confirmed using an antibody.

As a result, as shown in Figure 5, the protein expression of NFATc1 was increased by the treatment of RANKL, it was confirmed that the treatment with a meroterpenoid-based compound, in particular compound 157, significantly decreased on the third day.

[Example 7] Culture and differentiation of osteoblasts

C2C12 cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium containing 10% FBS (fetal bovine serum), antibiotics, and α-minimum essential medium (α-MEM). After 1 day, cells were differentiated by adding 10% FBS and rh-BMP2 (100 ng/mL). And the meroterpenoid compound 157 was treated by concentration. The medium was changed every 3 days.

[Example 8] ALP (Alkaline phosphatase) staining and activity analysis

Osteoblasts were fixed with 10% formalin for 30 seconds and washed twice with PBS (phosphate-buffered saline). After washing with distilled water once more, it was stained in the dark using an ALP staining kit (Sigma-Aldrich).

As shown in FIG. 6, it was confirmed that more osteoblasts were generated in a concentration-dependent manner when the meroterpenoid-based compound 157 was treated for 4 days, compared to BMP-2 alone.

Therefore, it was found that the meroterpenoid-based compound according to the present invention effectively increases the differentiation of osteoblasts and thus can be usefully used in bone diseases such as osteoporosis.

[Example 9] Cytotoxicity analysis

C2C12 cells were cultured by adding a meroterpenoid compound in a 96-well plate at a density of 4 × 10 ³ cells/well. After 3 days, the absorbance was checked at 450 nm with an ELISA reader (Molecular Devices, CA, USA) using Cell Counting Kit-8 (Dojindo Molecular Technologies, ML).

As a result, as shown in FIG. 7, with respect to the 100% survival rate of the control group that was not treated with anything, the cell survival rate did not significantly decrease even when the meroterpenoid compound 157 was treated by concentration.

Therefore, it was confirmed that the meroterpenoid-based compound according to the present invention has little cytotoxicity.

[Example 10] RT-PCR analysis

RT-PCR analysis was performed to confirm the mRNA expression patterns of key differentiation markers involved in the differentiation process of osteoblasts, and specifically, gene expression patterns of ALP, Runx2, OCL and OPN were confirmed. In order to conduct the experiment, RNA was used as a TRIzol (Invitrogen) solution for cells treated with a meroterpenoid compound by concentration and cells treated with a meroterpenoid compound for 3 days and 6 days under the conditions of Example 7. It was separated according to the manufacturer's method. 1 μg of the isolated RNA was synthesized as cDNA using oligo dT primer, dNTP, buffer, dithiothreitol, RNase inhibitor, and Superscript II reverse transcriptase. The synthesized cDNA was obtained by real-time PCR amplification using SYBR green using primers.

As a result, expression of ALP, an osteoblast differentiation marker, on day 6 of treatment with meroterpenoid compound 157, and Runx2, OCL, and OPN, which are other differentiation markers on days 3 and 6 of treatment with meroterpenoid compound 157. It was confirmed that the expression of the gene was significantly increased (FIGS. 8 to 10 ).

[Example 11] in vivo experiments using mouse femoral bone model (LPS induced bone erosion experiment)

To induce bone erosion of the mouse femur, LPS (lipopolysaccharide; 5 mg/kg of body weight) was injected intraperitoneally into the mouse. The meroterpenoid-based compound 157 (2 or 4 mg/kg of body weight) according to the present invention was dissolved in 5% Kolliphor EL (in PBS (phosphate buffer saline)) and used. Mice in which born erosion was induced by LPS were administered intraperitoneally once daily for 7 days with the meroterpenoid compound 157. The control group was administered phosphate buffer saline (PBS). Mice were autopsied to measure the degree of bone loss 7 days after the administration of the merotepenoid compound 157 (n=6). The measurement of the bone loss of the femur was taken using micro-CT.

As a result, as shown in FIG. 11, the meroterpenoid-based compound 157 significantly reduced bone loss of the mouse femur by LPS at 2 mg/kg. In addition, it was confirmed that the merotepenoid-based compound tends to decrease more than 2 mg/kg at a dose of 4 mg/kg. That is, it was confirmed that the meroterpenoid-based compound according to the present invention reduced bone loss in a dose-dependent manner.

In addition, as shown in Fig. 12, the meroterpenoid-based compound 157 (1) recovered the decrease in bone mineral density caused by LPS induction by 3 times or more; (2) remarkably restored trabecular damage caused by LPS induction; (3) The reduction in bone volume caused by LPS induction was significantly restored by more than 3 times; (4) It was confirmed that the thickness of the cancellous bone was slightly recovered and increased.

Therefore, it was found that the merotepenoid-based compound according to the present invention effectively reduces bone loss of the mouse femur caused by LPS, and thus can be usefully used in bone diseases such as osteoporosis.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

Claims (9)

A pharmaceutical composition for preventing or treating osteoporosis comprising a merotepenoid compound represented by the following Formula 1, a hydrate thereof, a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

In Formula 1,
R ₁ is hydrogen, hydroxy or acetoxy;

Is selected from the following structure;

R ₅ is hydrogen, methoxy, acetoxy or hydroxy;
R ₆ is hydrogen or methyl. The method of claim 1,
The meroterpenoid-based compound is represented by the following formula (2), a pharmaceutical composition for preventing or treating osteoporosis:
[Formula 2]

In Chemical Formula 2,
R ₁ is hydrogen or hydroxy;

Is selected from the following structure;

R ₆ is hydrogen or methyl. The method of claim 2,
The meroterpenoid-based compound is any one or more selected from the following structures, a pharmaceutical composition for preventing or treating osteoporosis:

R ₁ is hydrogen or hydroxy; R ₆ is hydrogen or methyl. The method of claim 2,
The meroterpenoid-based compound is obtained from an extract of the fungus Penicillium rudallense, a pharmaceutical composition for preventing or treating osteoporosis. Functional food supplements, functional beverages, foods for preventing or improving osteoporosis comprising a merotepenoid compound represented by the following Formula 1, a hydrate thereof, a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient Composition for additives or animal feed.
[Formula 1]

In Formula 1,
R ₁ is hydrogen, hydroxy or acetoxy;

Is selected from the following structure;

R ₅ is hydrogen, methoxy, acetoxy or hydroxy;
R ₆ is hydrogen or methyl. A composition for inhibiting the differentiation of osteoclasts comprising a meroterpenoid compound represented by the following Formula 1, a hydrate thereof, a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

In Formula 1,
R ₁ is hydrogen, hydroxy or acetoxy;

Is selected from the following structure;

R ₅ is hydrogen, methoxy, acetoxy or hydroxy;
R ₆ is hydrogen or methyl. A composition for promoting the differentiation of osteoblasts comprising a meroterpenoid-based compound represented by Formula 1 below, a hydrate thereof, a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

In Formula 1,
R ₁ is hydrogen, hydroxy or acetoxy;

Is selected from the following structure;

R ₅ is hydrogen, methoxy, acetoxy or hydroxy;
R ₆ is hydrogen or methyl.
delete A meroterpenoid-based compound selected from the following, a hydrate thereof, a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

Images