Novel lappaconitine derivatives and uses thereof

Abstract

The present invention relates to a novel lappaconitine derivative, a preparation method thereof and a pharmaceutical use utilizing its osteogenesis-promoting activity. The lappaconitine derivative induces differentiation of stem cells into osteoblasts, increases bone density if applied to an osteoporosis animal model, and induces osteogenesis in a fracture animal model, and thus can be effectively used to prevent, improve or treat osteoporosis and other bone-related diseases, as well as to treat non-disease fractures caused by physical trauma.

Description

Technical Field

[0001] The present invention relates to a novel lappaconitine derivative belonging to the diterpenoid alkaloid series, a preparation method thereof and a pharmaceutical use thereof utilizing its bone formation promoting activity. Background Art

[0002] Aconitin is one of the components of the traditional Chinese medicine Aconiti Lateralis Radix. It is a compound belonging to the alkaloid series and is a highly toxic substance. However, similar to the phenomenon that the toxicity of Aconiti Lateralis Radix is ​​reduced when it is decocted into a decoction, if the compound is directly heated, it will be converted into less toxic compounds such as benzoyl aconitine or aconitine through deacetylation or debenzoylation. Based on this principle, Aconiti Lateralis Radix (processed Aconiti Lateralis Radix or refined Aconiti Lateralis Radix) whose toxicity is reduced by heat treatment has been used as a medicinal material to reduce inflammation and pain in patients with neuralgia (Xu et al., J Ethnopharmacol 2006).

[0003] In addition, belonging to the diterpene alkaloid series such as aconitine, with similar chemical structures such as deoxyaconitine, mesoaconitine, mesoaconitine and lappaconitine, hundreds of various alkaloid compounds are being studied by semi-synthesis and structural confirmation, and their physiological activities have been revealed one after another (Turabekova et al., Environ Toxicol Pharmacol. 2008). For example, Shaanxi University of Science and Technology disclosed that the 2-acetylaminobenzoyl group located at the 4th carbon of lappaconitine was removed and connected to various cinnamon derivatives (cinnamic derivatives) to show anticancer activity. At the same university, various compounds were synthesized by changing the functional groups at the 4th, 8th and 9th positions (Liang Cheng et al., CN107540680A, published on January 5, 2018). Dr. Vladimirovich of Russia also confirmed the inflammatory effect of derivatives of various aromatic compounds connected at the 4th position of lappaconitine (SV Vladimirovich, WO 2017 / 209653A1).

[0004] Among many alkaloid compounds, lappaconitine is known to have multiple effects such as anti-arrhythmia, anti-inflammatory, antioxidant, anti-cancer and anti-epileptic (Wang et al., Chem Pharm Bull. 2009). In the process of studying the new pharmacological activity of lappaconitine, the present inventors prepared a compound with a new structure by hydrolyzing lappaconitine and found that the new compound promotes osteogenesis, thus completing the present invention. Summary of the invention

[0005] Problems to be solved by the invention

[0006] The object of the present invention is to provide a novel lappaconitine derivative having the function of promoting bone formation and treating osteoporosis, a preparation method thereof, and a pharmaceutical composition containing the same as an active ingredient for preventing, improving or treating bone diseases.

[0007] Solutions for solving problems

[0008] In order to achieve the above object, one aspect of the present invention provides a compound represented by the following Chemical Formula 1, a stereoisomer thereof, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof.

[0009]

Chemical formula 1

[0010]

[0011] According to a specific example of the present invention, in the above chemical formula 1, R 1 and R 2 Each is independently selected from the group consisting of hydrogen, C1-C6 alkyl, thioalkyl, arylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylarylalkyl, dihydroxyalkylarylalkyl, alkoxyalkyl, acyloxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxycarbonylaminoalkyl, acylaminoalkyl, arylsulfonamidoalkyl, allyl, dihydroxyalkylallyl, dioxolaneallyl, carbonylalkoxyalkyl (caralkoxyalkyl), methyl, ethyl, isopropyl, tert-butyl and phenyl. Wherein, "alk-" or "alkyl" is branched or linear, preferably C1-C3 alkyl, in which case the carbon chain can be arbitrarily, ether (-O-) bonded.

[0012] According to a specific example of the present invention, the compound of the above Chemical Formula 1 is (2aR, 2a1S, 3S, 4aS, 8S, 11S, 11bS, 12S)-6-ethyl-4a, 8-dihydroxy-3, 11-dimethoxy-tetradecahydro-8, 11a, 5-(epiethane [1, 1, 2] triyl) cyclopenta [7, 1] indeno [5, 4-b] azosine-1, 2-dione ((2aR, 2a1S, 3S, 4aS, 8S, 11S, 11bS, 12S)-6-ethyl-4a, 8-dihydroxy-3, 11-dimethoxy-tetradecahydro-8, 11a, 5-(epiethane [1, 1, 2] triyl) cyclopenta [7, 1] indeno [5, 4-b] azosine-1, 2-dione) (Chemical Formula 2).

[0013]

Chemical formula 2

[0014]

[0015] In the present invention, the compound of the above Chemical Formula 1 includes not only the compound represented by Chemical Formula 1 and pharmaceutically acceptable salts thereof, but also hydrates, solvates, stereoisomers and radioactive derivatives thereof.

[0016] The above-mentioned "pharmaceutically acceptable salt" refers to a salt that does not cause excessive toxicity, irritation, allergic reaction, etc. within the scope of pure medical judgment, is suitable for use in contact with human and lower animal tissues, and does not adversely affect the biological activity and physicochemical properties of the parent compound. The above-mentioned pharmaceutically acceptable salts are well known in the art. For example, they are described in detail in the literature (SM Berge et al., J. Pharmaceutical Sciences, 66, 1, 1977). The salt can be prepared in the same reaction system during the final separation and purification of the compound of the present invention, or prepared by reacting with an inorganic base or an organic base separately. Suitable additional salt forms are, for example, alkali metal salts and alkaline earth metal salts (calcium salts, etc.) such as ammonium salts, lithium salts, sodium salts, potassium salts, magnesium salts, calcium salts, etc.; salts with organic bases, for example, primary, secondary, tertiary aliphatic and aromatic amines, for example, methylamine, ethylamine, propylamine, isopropylamine, four butylamine isomers, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinuclidine, pyridine, quinoline and isoquinoline, benzathine, N-methyl-D-glucamine, 2-amino-2-(hydroxymethyl)-1,3-propanediol, hydrabamine salts, and salts with amino acids such as arginine and lysine, etc.

[0017] In the present invention, the hydrate or solvate of the compound of the above Chemical Formula 1 can be prepared by conventional methods. For example, the basic compound of Chemical Formula 1 can be dissolved in a solvent such as water, methanol, ethanol, acetone, 1,4-dioxane, etc., and a free acid or free base is added, followed by crystallization or recrystallization.

[0018] In addition, the compound of the above chemical formula 1 may have one or more asymmetric centers, and for such compounds, enantiomers or diastereomers may exist. Therefore, in the compound of the present invention, each isomer or a mixture of these isomers is included. In addition, different isomers can be separated or split by conventional methods, or any isomer can be obtained by conventional synthesis methods or by stereospecific or asymmetric synthesis. In addition, the compound of the present invention includes radioactive derivatives of the compounds represented by the above chemical formula, and these radioactive compounds are useful in the field of biological research.

[0019] In the present invention, the compound of the above Chemical Formula 1 can be prepared by a method comprising the following steps:

[0020] Step (a), reacting lappaconitine with an oxidizing agent; and

[0021] Step (b) is to react the product of step (a) with an organic solvent in the presence of a base.

[0022] According to a specific example of the present invention, the lappaconitine in step (a) above may be lappaconitine hydrogen bromide. In this case, step (a) may further include a process of removing hydrogen bromide before reacting lappaconitine with an oxidant. For example, as shown in the following reaction formula 1, dichloromethane (CH 2 Cl 2 ) is a process for removing hydrogen bromide from lappaconitine hydrogen bromide.

[0023] [Reaction 1]

[0024]

[0025] Thereafter, as shown in the following reaction formula 2, the lappaconitine is oxidized by reacting with an oxidant to generate a lappaconitine derivative (LAD). The oxidant may be selected from phenyliodine diacetate (PhI(OAc)) dissolved in dimethylformamide (DMF). 2 or lead(II) acetate (Pb(CH 3 CO 2 ) 2 ), lead(II) acetate (Pb(CH 3 CO 2 ) 4 ), ozone and HIO 4 Composed of groups.

[0026] [Reaction 2]

[0027]

[0028] In the present invention, the synthesized lappaconitine derivative (LAD) can react with an organic solvent in the presence of a base to generate the compound of the above chemical formula 1. The above base can be selected from the group consisting of sodium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate and potassium hydroxide, and the above organic solvent can be an aliphatic alcohol or an alkoxy alcohol.

[0029] The above-mentioned aliphatic alcohol refers to a general formula CH 3 (CH 2 )nOH (n is 0 or a positive integer) represented by an alcohol, an alkoxy alcohol refers to an alcohol represented by the general formula CH 3 (CH 2 )nO(CH 2)nCH 3 (n is 0 or a positive integer independently of each other). For example, the aliphatic alcohol may specifically be methanol, ethanol, n-propanol, isopropanol, n-butanol, etc., and the alkoxy alcohol may be methoxymethanol, methoxyethanol, ethoxyethanol, etc.

[0030] For example, as shown in the following reaction formula 3, the above-mentioned lappaconitine derivative (LAD) can react with ethanol in the presence of sodium hydroxide to generate the compound (QG3030) of Chemical Formula 1.

[0031] [Reaction 3]

[0032]

[0033] In the present invention, the compound of Chemical Formula 1 synthesized by the above method can be separated and purified by conventional separation and purification processes, such as dilution and washing with an organic solvent, and then the organic layer is concentrated under reduced pressure for separation, and when necessary, it can be purified by column chromatography and recrystallization methods using various solvents.

[0034] In the process of studying the new pharmacological activities of lappaconitine with anti-inflammatory, antioxidant and anti-cancer effects, the inventors synthesized the novel compound of the above chemical formula 1 by hydrolyzing lappaconitine and confirmed that the compound has the activity of promoting bone formation.

[0035] Therefore, another aspect of the present invention provides a pharmaceutical composition for preventing or treating bone-related diseases, comprising the compound represented by the above Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an effective ingredient.

[0036] In the above pharmaceutical composition, the compound represented by the above Chemical Formula 1 or a pharmaceutically acceptable salt thereof includes a hydrate, a solvate, a stereoisomer and a radioactive derivative thereof.

[0037] According to a specific example of the present invention, the compound of the above chemical formula 1 promotes the differentiation of stem cells into osteoblasts, increases the expression of bone formation markers selected from the group consisting of RUNX2 (short stature-related transcription factor 2), BMP2 (bone morphogenetic protein 2) and osteocalcin, and can help bone formation. The above stem cells can use mesenchymal stem cells, hematopoietic stem cells, adipose stem cells, bone marrow stem cells, etc.

[0038] In the present invention, the above-mentioned bone disease can be selected from the group consisting of osteoporosis, bone fracture, rheumatoid arthritis, periodontitis, osteomalacia, osteopenia, bone atrophy, osteoarthritis, bone defect, osteolysis and osteonecrosis, preferably bone fracture and osteoporosis.

[0039] The term "fracture" used in this specification refers to a state in which bones supporting the body are broken due to traffic accidents, falls, industrial accidents and other physical stimulations.

[0040] The term "osteoporosis" used in this specification refers to a state in which the minerals (especially calcium) and matrix that constitute bones decrease, causing the microstructure of bone tissue to degenerate, ultimately increasing the risk of fractures. Osteoporosis can be divided into primary osteoporosis, such as postmenopausal osteoporosis and senile osteoporosis, and secondary osteoporosis caused by diseases that affect bone formation and reduction, drugs, drinking and smoking, etc.

[0041] In addition to the compound of the above chemical formula 1 as an effective ingredient, the pharmaceutical composition of the present invention may further include suitable carriers, excipients and diluents conventionally used in the preparation of medicaments. The carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0042] When formulating the pharmaceutical composition of the present invention, the commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants and surfactants are used for preparation. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations are prepared by mixing at least one or more excipients in the composition of the present invention, for example, the excipients are starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talcum powder are also used. Liquid preparations for oral administration are equivalent to suspensions, oral solutions, oils, syrups, etc., and in addition to the commonly used simple diluents water and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, oils, lyophilized preparations and suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used.

[0043] The pharmaceutical composition of the present invention can be administered orally or parenterally. The preferred dosage of the composition of the present invention varies according to the patient's condition and weight, the degree of the disease, the drug form, the route of administration and the time period, but can be appropriately selected by those skilled in the art. For example, the dosage of the compound of the above chemical formula 1 may vary according to the patient's age, weight, sex, form of administration, health status and degree of disease. For an adult patient weighing 70 kg, generally 0.01 mg to 5000 mg can be administered per day. The above dosage can be administered once a day to multiple times, and the above dosage does not limit the scope of the present invention in any way.

[0044] In addition, the present invention provides a method for treating bone diseases, comprising administering the pharmaceutical composition to an individual in need of treatment. The types of bone diseases and the dosage of the pharmaceutical composition are the same as those described in the pharmaceutical composition for preventing or treating the above-mentioned bone diseases.

[0045] In the present invention, when the pharmaceutical composition for preventing or treating bone diseases is orally administered, the pharmaceutical composition for oral administration may be a solid preparation, a semisolid preparation or a liquid preparation for oral administration. Solid preparations for oral administration include, for example, tablets, pills, hard or soft capsules, powders, fine granules, granules, solution or suspension reconstituted powders, lozenges, wafers, oral strips, dragees, and chewable gums, but are not limited thereto. Liquid preparations for oral administration include liquids, suspensions, emulsions, syrups, elixirs, spirits, aromatic waters, lemons, extracts, precipitants, tinctures, and medicated oils.

[0046] In the present invention, when the pharmaceutical composition for preventing or treating bone diseases is used as an injection, it can be directly injected into the bone disease site, and when formulated as an injection, it can include a non-toxic buffer solution isotonic with blood as a diluent, such as a phosphate buffer solution with a pH of 7.4, etc. In addition to the buffer solution, the injection may also contain other diluents or additives.

[0047] In addition, according to a specific example of the present invention, an excipient such as a collagen sponge can be mixed with the above-mentioned pharmaceutical composition and then transplanted into the fracture site to treat an individual.

[0048] Another aspect of the present invention provides a composition for inducing osteoblast differentiation in vitro, using the compound represented by the above Chemical Formula 1.

[0049] According to one specific example of the present invention, when mesenchymal stem cells are treated with the compound represented by the above Chemical Formula 1, the production of calcium and minerals increases, thereby promoting the differentiation of stem cells into osteoblasts.

[0050] Effects of the Invention

[0051] The lappaconitine derivatives of the present invention induce the differentiation of stem cells into osteoblasts, increase bone density when applied to an osteoporosis animal model, and promote bone formation when injected into the fracture site of a fracture animal model, which can promote the recovery of fracture trauma and induce bone formation. Therefore, they can be effectively used to prevent, improve or treat bone-related diseases, such as fractures and osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The structure of a lappaconitine derivative (QG3030) according to an example of the present invention is shown.

[0053] Figure 2aThe results are the results of confirming the levels of calcium (Alizarin Red) and mineral production (Von Kossa) after treating mesenchymal stem cells with a lappaconitine derivative (QG3030).

[0054] Figure 2b It is represented by a graph Figure 2a Figure 2 shows the staining results.

[0055] Figure 3a The results are the results of confirming the changes in RUNX2 expression after treating mesenchymal stem cells with different concentrations of a lappa luteolin derivative (QG3030) for a specific period of time.

[0056] Figure 3b The results are the results of confirming the changes in BMP-2 expression after treating mesenchymal stem cells with different concentrations of lappa luteolin derivatives (QG3030) for a specific period of time.

[0057] Figure 4a The results are the results of treating mesenchymal stem cells with different concentrations of lappa luteolin derivative (QG3030) for a specific period of time, and then confirming the changes in osteocalcin expression using fluorescence microscopy.

[0058] Figure 4b It is represented by a graph Figure 4a Figure 5. Graph of the fluorescence levels of .

[0059] Figure 5a The results are from the study of the phosphorylation levels of various phosphorylases after treating mesenchymal stem cells with a lappaconitine derivative (QG3030).

[0060] Figure 5b It is represented by a diagram Figure 5a Figure 3 Phosphorylation levels confirmed in Figure 3.

[0061] Figure 6 The results were obtained by co-treating mesenchymal stem cells with a lappaconitine derivative (QG3030) and various phosphorylase inhibitors to confirm calcium levels.

[0062] Figure 7a The results are the results of confirming the phosphorylated ERK (pERK) level after mesenchymal stem cells were treated with a lappaconitine derivative (QG3030) for 0 to 24 hours.

[0063] Figure 7b The results are as follows: the level of phosphorylated ERK (pERK) was confirmed after mesenchymal stem cells were treated with a lappaconitine derivative (QG3030) for 7 days.

[0064] Figure 7c The results are as follows: the level of phosphorylated ERK (pERK) was confirmed after mesenchymal stem cells were treated with a lappaconitine derivative (QG3030) for 14 days.

[0065] Figure 8a The results are the results of co-treatment of mesenchymal stem cells with a lappaconitine derivative (QG3030) and an ERK inhibitor (PD98059), and then confirming changes in RUNX2 expression using a fluorescence microscope.

[0066] Figure 8b It is represented by a graph Figure 8a Figure 5. Graph of the fluorescence levels of .

[0067] Figure 8c The results are from co-treatment of mesenchymal stem cells with a lappaconitine derivative (QG3030) and an ERK inhibitor (PD98059) to confirm the level of phosphorylated ERK (pERK).

[0068] Figure 9a The diagram shows the schedule of administration of the lappaconitine derivative (QG3030) to an osteoporosis animal model and the types of experimental groups.

[0069] Figure 9b This is a diagram showing the bone cross section of the femur after administering a lappaconitine derivative (QG3030) to an osteoporosis animal model.

[0070] Fig.9c This is a graph showing the bone density of the femur after administering a lappaconitine derivative (QG3030) to an osteoporosis animal model.

[0071] Fig.10a This graph shows the bone formation level after administering a lappaconitine derivative (QG3030) to an osteoporosis animal model.

[0072] Fig.10b This figure shows the expression level of RUNX2 after administering a lappaconitine derivative (QG3030) to an osteoporosis animal model.

[0073] Fig.11 A method for administering a lappaconitine derivative (QG3030) to an animal model of bone fracture is schematically shown.

[0074] Fig.12a The results are the results of confirming the bone cross-section of rat long bones after applying a lappaconitine derivative (QG3030) to a fracture animal model.

[0075] Figure 12b The results are the results of confirming the degree of osteogenesis in rat long bones after applying a lappaconitine derivative (QG3030) to a fracture animal model.

[0076] Fig.13 The results are as follows: after administering a lappaconitine derivative (QG3030) to a fracture animal model, the bone formation level (A) and collagen expression level (B) were confirmed. DETAILED DESCRIPTION

[0077] Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrating one or more specific examples, and the scope of the present invention is not limited to these examples.

[0078] Preparation Example: Preparation of QG3030

[0079] 1-1. Preparation of lappaconitine

[0080] Dichloromethane (500 ml) and sodium hydroxide solution (NaOH 10 g + water 100 g) were added to lappaconitine·hydrogen bromide (10.08 g, 0.017 mol), and the aqueous layer and the organic layer were separated using a separatory funnel. The separated organic layer was washed with water several times, dried over anhydrous magnesium sulfate, and then distilled to obtain the desired lappaconitine, with a yield of 89% (8.90 g).

[0081] 1 H-NMR (CDCl 3 ,400MHz)δ10.98(s,1H),8.60(d,J=8.4Hz,1H),7.86-7.83(m,1H),7.42(t,1H),6.95(t,1H),3.52-3.37(m,2H),3.36-3.35(m,2H),3.36( s,J=7.1Hz,3H),3.22(s,3H),3.13(s,3H),3.11-3.09(m,2H),2.60-2.46(m,10H),2.15(s,3H),2.14-1.73(m,6H),1.74-1.68(m,1H),1.74 -1.70(m,1H),1.06(t,J=6.8Hz,3H);

[0082] 13 C-NMR (CDCl 3 ,100MHz)δ169.02,167.39,141.6,134.34,131.07,122.31,120.19,115.76,90.10,84.62,84.13,82.89,78.57,75.56,61.4 8,57.89,56.52,56.10,55.50,50.85,49.86,48.97,48.51,47.61,44.76,36.28,31.83,26.77,26.20,25.53,24.12,13.53;

[0083] HRMS(ES + ):C 32 H44 N 2 O 8 m / z calculated: 585.3098 [M+H] + .

[0084] Test value (Found): 585.3176.

[0085] 1-2. Preparation of lappaconitine derivatives (LAD) from lappaconitine

[0086] In the case of phenyliodine diacetate (PhI(OAc) 2 (14.07 g, 0.044 mol) was dissolved in a solution of dimethylformamide (DMF, 150 mL), and lappaconitine (8.9 g, 0.015 mol) was slowly added and stirred for 10 minutes. After the reaction was completed, the solution was diluted with ethyl acetate (EA) and saturated sodium bicarbonate (NaHCO 3 ) aqueous solution to extract it. The organic layer was washed with water several times to remove dimethylformamide, dried over anhydrous magnesium sulfate, and distilled under reduced pressure. The crude product was separated by column chromatography (ether: ethyl acetate: hexane = 3:2:5) to obtain the desired lappaconitine derivative (lappaconitine derivative, LAD)(3S,6S,7S,9S,11S,16S)-1-ethyl-6,9,11-trimethoxy-8,13-dioxododecahydro-2H-3,6a,14-(epiethane[1,1,2]triyl)-7,10-methanocyclodeca[b]azosin-3(4H)-yl2-acetaminobenzoate, yield 25.7% (2.3 g).

[0087] 1 H-NMR (CDCl 3,400MHz)δ10.99(s,1H),8.62(d,J=8.4Hz,1H),7.89(d,J=9.6Hz,1H),7.47-7.43(m,1H),7.01-6.99(m,1H),3.88-3.64( m,4H),3.65(s,3H),3.41(s,3H),3.18(s,3H),2.90-2.25(5,4H),2.20(s,3H),2.19-1.80(m,4H),1.15(t,J=7.0Hz,3H);

[0088] 13 C-NMR (CDCl 3 ,100MHz)δ211.85,204.31,169.10,167.40,162.18,143.74,141.74,134.61,131.07,122.41,120.30,115.45,86.92,82.75,8 1.45,78.21,76.28,60.73,58.17,57.37,54.35,52.98,50.19,48.78,46.06,45.92,38.41,32.06,25.56,25.39,25.36,12.72;

[0089] HRMS(ES + ):C 32 H 42 N 2 O 8 m / z calculated: 583.3018 [M+H] + .

[0090] Test value (Found): 583.2941.

[0091] 1-3. Preparation of QG3030 from LAD

[0092] LAD (2.3 g), sodium hydroxide solution (NaOH 0.7 g + water 5.1 g) and ethanol (51 ml) were placed in a 100 ml three-necked round-bottom flask and refluxed for 1 hour. After the reaction was terminated, ethanol was distilled and column chromatography was performed with ethyl acetate to obtain the final compound QG3030 {(2aR, 2a1S, 3S, 4aS, 8S, 11S, 11bS, 12S)-6-ethyl-4a, 8-dihydroxy-3, 11-dimethoxy-tetradecahydro-8, 11a, 5-(ethan[1,1,2]triyl)cyclopenta[7,1]indeno[5,4-b]azooxazine-1,2-dione} with a yield of 62.5%.

[0093] 1H-NMR (DMSO-d 6 ,400MHz)δ8.88(s,1H),4.65(s,1H),4.35(s,1H),4.27-4.20 9m,1H),4.10-4.00(m,1H),3.60(s,1H),3.35(s,3H),3.20(s,3H),3.10-. 2.99(m,1H),2.85-2.19(m,6H),1.99-1.50(m,7H),1.05(t,J=7.0Hz,3H);

[0094] 13 C-NMR (CDCl 3 ,100MHz)δ201.20,149.88,148.78,83.25,79.92,79.50,67.87,62.74, 60.20,58.10,53.23,49.55,48.86,47.88,46.92,42.39,38.43,13.56;

[0095] HRMS(ES + ):C 22 H 31 NO 6 m / z calculated: 406.2151 [M+H] + .

[0096] Found: 406.2242; X-ray structure.

[0097] 1-4. Confirm the structure of QG3030

[0098] The QG3030 obtained in the above-mentioned Preparation Example 1-4 was recrystallized with water, and after obtaining appropriate crystals by X-ray diffraction (X-ray diffraction), the X-ray structure was confirmed using it (X-ray Diffractometer, R-AXISRAPID). The results are as follows Figure 1 And as shown in Table 1.

[0099]

Table 1

[0100]

[0101]

[0102] Experimental Example 1: Confirmation of the efficacy of QG3030 (in vitro)

[0103] 1-1. Confirmation of the osteoblast differentiation ability of QG3030 in MSCs

[0104] Osteoblasts involved in bone formation are differentiated from mesenchymal stem cells (MSCs) according to external stimuli and various transcription factors related to signal transduction (Garg et al., Orthop Surg. 2017). Therefore, it was tested whether QG3030 can induce human MSCs to differentiate into osteoblasts.

[0105] Normal human MSCs were purchased from ATCC in the United States and cultured using MSC-specific medium (Gibco). The cultured cells were separated by trypsin and centrifuged at 3 × 10 4 / well were inoculated into 24-well plates. The next day, the MSC culture medium was replaced with the experimental medium DMEM medium (DMEM / 10% FBS / penicillin / streptomycin), and the cells were treated with QG3030 (0.1 μM) alone or OIM (osteoblastic induction medium; StemPro osteogenic differentiation kit, Thermo Fisher Scientific). Thereafter, the cell culture medium was replaced with fresh DMEM medium every day, treated with QG3030 or OIM, and cultured for 3 weeks. After the culture was terminated, the cells were stained with Alizarin Red for calcium recognition and calcium salt staining solution (Von Kossa) for mineral recognition.

[0106] The staining results confirmed that QG3030 induced calcium and mineral production in human MSCs, similar to the positive control group OIM for osteoblast differentiation ( Figure 2a and Figure 2b ). This result indicates that QG3030 can differentiate MSCs into osteoblasts at the cellular level.

[0107] 1-2. Confirmation of changes in RUNX2 and BMP2 expression in MSCs

[0108] RUNX2 (short stature-related transcription factor 2; core binding factor, Cbfa1) is the most important transcriptional regulator in osteogenesis, and is known to be involved in osteoblast differentiation, matrix production and mineralization during osteogenesis (Bruderer M et al., Eur Cell Mater. 2014). In addition, RUNX2 itself can be regulated by BMP (bone morphogenetic protein), which is a type of cytokine (Sun J et al., Mol Med Rep. 2015). Therefore, it was confirmed whether QG3030 induces the expression of specific factors related to osteogenesis to induce osteogenesis.

[0109] MSCs were plated at 3 × 10 4 / well were seeded in a 24-well plate and cultured for 24 hours. From the next day, QG3030 was treated once a day at different concentrations (0.001 μM, 0.01 μM and 0.1 μM) for 7 and 14 days. After the experiment was terminated, MSCs were stained with RUNX2 and BMP-2 antibodies and observed by fluorescence microscopy.

[0110] The results show that QG3030 strongly induces the expression of RUNX2 and BMP-2 even at low concentrations (0.001 μM = 1 nM), and this trend becomes stronger with increasing concentrations ( Figure 3a and Figure 3b ). The results show at the molecular level that QG3030 can participate in osteogenesis by inducing human MSCs to differentiate into osteoblasts.

[0111] 1-3. Confirmation of changes in osteocalcin expression in MSCs

[0112] Osteocalcin is expressed only in osteoblasts (Lee et al., Cell 2007) and is used as a useful biomarker for osteogenesis (Bharadwaj et al., Osteoporosis International., 2009). Therefore, the effect of QG3030 on the expression of osteocalcin, an osteoblast-specific marker, was confirmed.

[0113] After the experiment was performed in the same manner as in Experimental Examples 1-3 above, MSCs were stained with an osteocalcin antibody and confirmed by fluorescence microscopy. As a result, even at a low concentration (0.001 μM), QG3030 strongly induced the expression of osteocalcin on day 7 of treatment, and its level was similar to that of the positive control group OIM treatment group ( Figure 4a and Figure 4b ).

[0114] 1-4. Confirmation of the mechanism of MSC differentiation into osteoblasts

[0115] To investigate the mechanism by which QG3030 differentiates MSCs into osteoblasts, the related signal transduction was investigated.

[0116] MSCs cultured in experimental DMEM medium were treated with QG3030 (1 μM) or control compound (DMSO) and cultured for 24 hours. After the cells were recovered, the experiment was performed according to the method specified in the phosphorylase antibody array kit (R&D Systems), which contains antibodies that specifically recognize the phosphorylated forms of 43 phosphorylases. Afterwards, the samples were analyzed using the ImageJ (NIH, USA) program.

[0117] The analysis results showed that QG3030 strongly increased the phosphorylation of signal transduction molecules such as ERK, Akt, and WNK1 compared with DMSO as a control group ( Figure 5a and Figure 5b ).

[0118] 1-5. Confirmation of the mechanism of MSC differentiation into osteoblasts

[0119] To further investigate the mechanism by which QG3030 differentiates MSCs into osteoblasts, experiments using phosphorylase inhibitors were performed.

[0120] After culturing MSCs, phosphorylase inhibitors (1 μM) and QG3030 (1 μM) were simultaneously treated, and the next day, the medium was replaced and phosphorylase inhibitors and QG3030 were simultaneously treated again. This operation was repeated for 3 weeks, and then MSCs were stained with Alizarin Red.

[0121] The results showed that the osteoblast differentiation ability of QG3030 was inhibited in the experimental group treated with inhibitors of ERK, P38 or Akt compared with the group treated with QG3030 alone ( Figure 6 ). The results indicate that QG3030 participates in the differentiation of MSCs into osteoblasts by activating the signal transduction of these enzymes.

[0122] 1-6. Confirmation of the effect on ERK phosphorylation in MSCs

[0123] According to existing studies, MAP-kinase signaling mechanisms including ERK and p38 play an important role in osteoblast differentiation (Greenblatt et al., MB, Annu Rev Cell Dev Biol. 2013). Consistent with existing studies, the results of Examples 1-5 above confirm that QG3030 has the potential to differentiate MSCs into osteoblasts via ERK and p38 MAP-kinase ( Figure 6 In addition, the results of Experimental Examples 1-4 confirmed the possibility that QG3030 differentiates into osteoblasts through phosphorylation of ERK rather than phosphorylation of p38 ( Figure 5a and Figure 5b ). Therefore, the level of ERK phosphorylation was confirmed over time after MSCs were treated with QG3030.

[0124] As a result, it was confirmed that the phosphorylated ERK (pERK) level increased significantly within 30 minutes of QG3030 treatment, and in particular, the phosphorylated ERK (pERK) level increased even after 24 hours of QG3030 treatment ( Figure 7a ).

[0125] In addition, in order to confirm whether QG3030 induces phosphorylation of ERK for a long time, MSCs were treated with different concentrations of QG3030 (0.001, 0.01 and 0.1 μM), and the level of phosphorylated ERK (pERK) was confirmed after 7 and 14 days. As a result, the level of phosphorylated ERK (pERK) increased significantly in all experimental groups treated with QG3030, especially at low concentrations, the level of phosphorylated ERK (pERK) increased more than that in the positive control group OIM. This trend continued until 14 days after QG3030 treatment, thus confirming that QG3030 can increase the phosphorylation of ERK for a long time ( Figure 7b and Figure 7c ).

[0126] This result suggests that the long-term activation of the ERK signaling mechanism by QG3030 may be the core mechanism of the osteoblast differentiation ability of QG3030.

[0127] 1-7. Confirmation of the effect of ERK inhibitor on QG3030-induced increase in RUNX2 expression

[0128] ERK induces phosphorylation of RUNX2 to increase transcriptional activity and can participate in stability to increase the amount of RUNX2 protein (Greenblatt et al., MB, Annu Rev Cell Dev Biol. 2013). Therefore, increasing ERK phosphorylation by QG3030 can induce the expression of RUNX2, which can participate in the differentiation of MSC into osteoblasts. To demonstrate this, the expression level of RUNX2 was confirmed after MSC was treated with QG3030 (0.1 μM) and RK inhibitor PD98059 (50 μM).

[0129] The results show that RUNX2 expression increased significantly within 12 hours of QG3030 treatment, but was significantly reduced when treated with the ERK inhibitor PD98059. This trend was the same even after 24 hours of QG3030 treatment ( Figure 8a and Figure 8b ).

[0130] Furthermore, as expected, QG3030 treatment increased the phosphorylation level of ERK in MSCs, which was inhibited by PD98059 treatment ( Figure 8c ).

[0131] These results indicate that QG3030-induced ERK activation is the main mechanism of osteogenic activity.

[0132] Experimental Example 2: Confirmation of the efficacy of QG3030 (in vivo)

[0133] 2.1. Confirmation of the therapeutic ability of QG3030 using an osteoporosis animal model

[0134] The osteoporosis therapeutic ability of QG3030 was confirmed in an ovariectomy (OVX) mouse animal model, which is commonly used as an osteoporosis animal model.

[0135] As a positive control group, teriparatide (Forteo; Eli Lilly), a parathyroid hormone (PTH)-type therapeutic agent that promotes bone formation, and Fosamax (Fosamax; Merck), a bone resorption inhibitor that belongs to the alendronate series, were subcutaneously administered. For the negative control group, water (H 2 O), QG3030 was orally administered at a concentration of 5 or 30 mg / kg ( Figure 9a The drug was administered daily for 10 weeks, and then bone mineral density (BMD) was measured.

[0136] The measurement results showed that the bone density in the QG3030-treated group increased significantly compared with the negative control group, demonstrating the therapeutic effect on osteoporosis. The effect of QG3030 was similar to that of thiramivir and significantly better than that of teriparatide ( Figure 9b and Fig.9c ).

[0137] In addition, tissue staining results showed that QG3030 induced osteogenesis in the femur of ovariectomized mice ( Fig.10a ), and confirmed a significant increase in the expression of RUNX2, a transcription factor involved in osteogenesis ( Fig.10b The experimental results are summarized in the following Table 2. In Table 2, BMD represents bone density, TV represents total volume, BV represents bone volume, Tb.Th represents trabecular thickness, and Tb.N represents trabecular number.

[0138]

Table 2

[0139]

[0140] 2.2. Confirmation of the therapeutic ability of QG3030 using a fracture animal model

[0141] Bone defects were induced in the long bones of Sprague Dawley rats by drilling holes with a diameter of 2 mm and implanted with excipients (collagen sponge, ) mixed with QG3030 (6, 30 and 150 μg / 10 μl) were implanted into the bone defect site to evaluate the bone regeneration efficacy ( Fig.11 At this time, BMP2 (bone morphogenetic protein-2) was treated in the positive control group, and DMSO was treated in the negative control group (Table 3).

[0142]

Table 3

[0143] Animal Group concentration Animal No. Sham control group 5 Fracture + DMSO Collagen sponge + DMSO (10uL) 5 Fracture + BMP2 Collagen sponge + BMP2 (1ug / 10uL) 5 Fracture+QG3030(low) Collagen sponge + QG3030 (6ug / 10ul) 5 Fracture+QG3030(middle) Collagen sponge + QG3030 (30ug / 10ul) 5 Fracture+QG3030(high) Collagen sponge + QG3030 (150ug / 10ul) 5

[0144] The results of micro-CT images read 4 weeks after transplantation showed that QG3030 showed similar bone regeneration ability as BMP2 at all concentrations ( Fig.12a ).

[0145] In order to statistically quantify the bone regeneration ability of QG3030, the degree of bone formation was presented in a three-dimensional image and ROI (region of interest) was set. The bone volume ratio (bone volume / total volume) (BV / TV, %), trabecular thickness (trabecular thickness) (Tb.Th, mm), trabecular number (trabecular number) (Tb.N, 1 / mm) and trabecular separation (trabecular separation) (Tb.Sp, mm) parameters were used for analysis.

[0146] The results showed that the bone volume ratio BV / TV, an indicator of osteogenesis, increased at the same level in the BMP2-treated group (positive control group) and the QG3030-treated group (low, medium, and high concentrations) compared to the negative control group treated with DMSO ( Figure 12b ).

[0147] In addition, in the analysis of bone thickness Tb.Th, bone tissue number Tb.N, and bone tissue distance Tb.Sp for evaluating the bone quality of the formed bone tissue, in Tb.N and Tb.Sp other than Tb.Th, it was confirmed that high-quality bone tissue was formed in a statistically significant manner in the BMP2-treated group (positive control group) and the QG3030-treated group (low, medium, and high concentrations) compared with the DMSO-treated group ( Figure 12b ). Interestingly, although not statistically significant, QG3030 produced better quality bone compared to BMP2. Statistically significant values ​​were *<0.05, **<0.01, ***<0.001.

[0148] To demonstrate bone regeneration by QG3030 at the tissue level, tissue staining was performed for cells (H&E, hematoxylin & eosin), collagen (Masson's trichrome staining), type I collagen, and osteocalcin. The results showed that the above markers were expressed in the bone defect sites (indicated by solid lines) treated with QG3030 (low, medium, and high concentrations), and the effect was similar to that of BMP2 ( Fig.13 ).

[0149] The bone regeneration results of this experimental example prove that QG303O has the same excellent bone regeneration ability as BMP2.

[0150] The above results confirm that the QG3030 compound of the present invention induces the production of calcium and minerals in MSCs, increases the expression of RUNX2, BMP-2 and osteocalcin, and induces ERK activation, thereby inducing differentiation into osteoblasts. In addition, when applied to an osteoporosis animal model, it increases bone density and induces osteogenesis in a fracture animal model. Therefore, the QG3030 compound can be used not only for osteoporosis and other bone-related diseases, but also can be effectively used for the treatment of non-disease fractures caused by physical trauma.

[0151] Preparation Examples

[0152] On the other hand, the novel compound QG3030 according to the present invention can be formulated in various forms according to the purpose. The following is an example of a formulation method of a composition containing the novel compound QG3030 according to the present invention as an active ingredient, but the present invention is not limited thereto.

[0153] 1. Tablets (direct compression)

[0154] 5.0 mg of the active ingredient was sieved, mixed with 14.1 mg of lactose, 0.8 mg of crospovidone USNF and 0.1 mg of magnesium stearate and compressed into tablets.

[0155] 2. Tablets (wet granulation)

[0156] After sieving 5.0 mg of active ingredient, 16.0 mg of lactose and 4.0 mg of starch were mixed. After 0.3 mg of polysorbate 80 was dissolved in pure water, an appropriate amount of the solution was added to the above mixture to atomize it. After drying, the fine particles were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The fine particles were compressed into tablets.

[0157] 3. Powder and capsules

[0158] 5.0 mg of the active ingredient is sieved and mixed with 14.8 mg of lactose, 10.0 mg of polyvinyl pyrrolidone and 0.2 mg of magnesium stearate. The mixture is filled into a solid No. 5 gelatin capsule using a suitable device.

[0159] 4. Injection

[0160] By combining 100 mg of active ingredient, 180 mg of mannitol and 26 mg of Na 2 HPO 4 / H 2 O was dissolved in 2974 mg of distilled water to prepare injection.

Claims

1. A compound represented by the following chemical formula 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: 【Chemical formula 2】 2. A pharmaceutical composition for preventing or treating bone diseases, comprising a compound represented by the following chemical formula 2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient, 【Chemical formula 2】 3. The pharmaceutical composition for preventing or treating bone diseases according to claim 2, characterized in that: The bone disease is selected from the group consisting of osteoporosis, bone fracture, osteomalacia, osteopenia, bone atrophy, osteoarthritis, bone defect, osteolysis and osteonecrosis.

4. The pharmaceutical composition for preventing or treating bone diseases according to claim 2, characterized in that: The compound represented by the chemical formula 2 promotes differentiation of stem cells into osteoblasts.

5. The pharmaceutical composition for preventing or treating bone diseases according to claim 2, characterized in that: The compound represented by the Chemical Formula 2 increases the expression of an osteogenic marker selected from the group consisting of RUNX2, BMP2 and osteocalcin.

6. A method for synthesizing the compound according to claim 1, comprising: Step (a), reacting lappaconitine with an oxidant; as well as Step (b) is to react the product of step (a) with an organic solvent in the presence of a base.

7. The method for synthesizing the compound represented by Chemical Formula 2 according to claim 6, characterized in that: The oxidant of step (a) is selected from the group consisting of phenyl iodide diacetate, lead (II) acetate, lead (II) acetate, ozone and HIO4.

8. The method for synthesizing the compound represented by Chemical Formula 2 according to claim 6, characterized in that: The base in step (b) is selected from the group consisting of sodium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate and potassium hydroxide.

9. The method for synthesizing the compound represented by Chemical Formula 2 according to claim 6, characterized in that: The organic solvent in step (b) is an aliphatic alcohol.

Images