KR20210071245A - Pharmaceutical composition for for Inhibiting or Treating for Sarcopenia or muscle atrophy - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, which comprises an indirubin derivative as an active component, for prevention or treatment of sarcopenia or muscle atrophy, and a method for treatment of sarcopenia or muscle atrophy by using the same. More specifically, the present invention relates to a novel use of an indirubin derivative in treating sarcopenia or muscle atrophy, wherein the pharmaceutical composition provided by the present invention can be advantageously used for effectively preventing or treating sarcopenia or muscle atrophy through the increase of myotube diameters, the suppression of myotube atrophy, the maintenance of ratios of myotubes by diameter, and the silencing of gene atrogine-1, which destroys muscle proteins.

Description

Pharmaceutical composition for the prevention or treatment of sarcopenia or muscular atrophy {Pharmaceutical composition for for Inhibiting or Treating for Sarcopenia or muscle atrophy}

The present invention relates to a pharmaceutical composition for preventing or treating sarcopenia or muscular atrophy comprising an indirubin derivative as an active ingredient, and a method for treating sarcopenia or muscular atrophy using the same.

Skeletal muscle accounts for more than 30% of body mass. This tissue plays a pivotal role in motor and metabolic homeostasis such as glucose consumption. Skeletal muscle wasting (muscle atrophy) can be caused by a number of factors, including immobility and aging (sarcopenia). Sarcopenia, a geriatric disease, is on the rise due to the proportional increase in the elderly (over 60 years old), which is expected to account for 29% of the population in 2050. A person's physical decline begins in their 30s and accelerates when they reach their 50s. Thus, the percentage of skeletal muscle mass continues to decrease and falls below 25% at the age of 75-80 years. About 36.5% of those over the age of 60 have sarcopenia, and this weakness significantly increases the mortality rate compared to those without sarcopenia. Current approaches to treating sarcopenia rely on exercise, nutritional supplements, and hormone therapy. However, for some people, the above therapy is not suitable due to the condition or non-compliance of the physical or disease. The exact molecular mechanisms responsible for age-related muscle loss have not been elucidated. Muscle loss associated with sarcopenia can be modeled in vitro using myotube diameter analysis and dexamethasone treatment. Muscle-specific E3 ubiquitin ligase (E3 ubiquitin ligase), atrogin-1 (atrogin-1) mediates protein ubiquitination and is a marker of protein synthesis. Overexpression of atrogine-1 decreases the diameter of myotube, and knockout of denovation opposite to atrogine-1 induces muscle loss. It is presented as a signal of a potential therapeutic among anti-sarcopenia drug candidates. Currently, there are no approved small molecule drug therapies to prevent the progression of sarcopenia. This small molecule approach has advantages in drug development compared to protein or oligonucleotide therapy, such as ease of administration, structural manipulation, dose optimization and cost-effectiveness.

So far, there has been no indirubin-based small molecule reported to prevent muscle loss by increasing the diameter of the root canal and preventing the atrophic effect of dexamethasone (Korean Patent No. KR 10-1997334 B1). This is in contrast to drug development approaches that repair or replace damaged or atrophied muscle fibers by inducing proliferation of muscle stem cells.

The present application describes the development of an indirubin-based small molecule to treat muscle loss associated with sarcopenia by reducing the expression of atrozine in atrophic muscle fibers.

The present inventors made intensive research efforts to derive novel compounds having therapeutic efficacy for sarcopenia or muscular atrophy. As a result, it was found that certain indirubin derivative compounds can effectively inhibit muscle loss by reducing the expression of atrozin-1, increasing the diameter of the root canal, and preventing the effect of atrophy of the root canal. The present invention has been completed.

Accordingly, an object of the present invention is to provide a novel compound having a therapeutic effect on sarcopenia or muscular atrophy, and a pharmaceutically acceptable salt, solvate or hydrate thereof.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of sarcopenia or muscular atrophy.

Another object of the present invention is to provide a method for preventing or treating sarcopenia or muscular atrophy using the composition.

According to one aspect of the present invention, the present invention provides a compound represented by the following formula (1), a pharmaceutically acceptable salt, solvate or hydrate thereof:

[Formula 1]

In Formula 1,

R ₁ is hydrogen, halogen, or halogen-substituted or unsubstituted C ₁ -C ₄ alkoxy;

R ₂ is

또는

or

이며, 이때, n은 2 이상 4이하의 자연수이고,

In this case, n is a natural number of 2 or more and 4 or less,

R ₃ is hydrogen or halogen,

wherein R ₄ is

,

,

또는

or

이며,

is,

In this case, X is CH or N,

L is a single bond or NH,

R ₅ and R ₆ are each independently hydrogen; C ₁ -C ₄ alkyl; Or unsubstituted or C ₁ -C ₄ alkyl substituted piperidinyl (piperidinyl) or piperazinyl (piperazinyl),

R ₇ is hydrogen, -(CH ₂ )n-COOH, or -(CH ₂ )n-COOR ₁₀ (in this case, n=1 or more and 5 or less, R ₁₀ is hydrogen or C ₁ -C ₆ alkyl),

R ₈ is hydrogen or C ₁ -C ₆ alkyl, hydroxyalkyl, alkylaminoalkyl, or aminoalkyl,

R ₉ is C ₁ -C ₆ alkyl, hydroxyalkyl, alkylaminoalkyl, or aminoalkyl.

In one embodiment of the present invention, R ₂ of Formula 1 may be a compound selected from the group consisting of the following compounds, a pharmaceutically acceptable salt, solvate or hydrate thereof.

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

, ,

,

,

,

및

.

and

.

In another embodiment of the present invention, the compound represented by Formula 1 of the present invention may be any one selected from the group consisting of the following compounds:

4-(3-((((2Z,3E)-6'-bromo-2'-oxo-[2,3'-biindolinylidene]-3-ylidene)amino)oxy)propanoyl)piperazin-1-ium (compound 1), 4-(3-((((2Z,3E)-6'-bromo-2'-oxo-[2,3'-biindolinylidene]-3-ylidene)amino)oxy)propanamido)piperidin-1- ium (compound 2), and 4-((2-((((2Z,3E)-6'-Bromo-2'-oxo-[2,3'-biindolinylidene]-3-ylidene)amino)oxy)ethyl )sulfonyl)piperazin-1-ium (Compound 3).

According to another aspect of the present invention, the present invention provides (a) a compound represented by the following formula (1), a pharmaceutically acceptable salt, solvate or hydrate thereof; And (b) provides a pharmaceutical composition for the prevention or treatment of sarcopenia or muscular atrophy comprising a pharmaceutically acceptable carrier:

[Formula 1]

In Formula 1,

R ₁ is hydrogen, halogen, or halogen-substituted or unsubstituted C ₁ -C ₄ alkoxy;

R ₂ is

또는

or

이며, 이때, n은 2 이상 4이하의 자연수이고,

In this case, n is a natural number of 2 or more and 4 or less,

R ₃ is hydrogen or halogen,

wherein R ₄ is

,

,

또는

or

이며,

is,

In this case, X is CH or N,

L is a single bond or NH,

R ₅ and R ₆ are each independently hydrogen; C ₁ -C ₄ alkyl; Or unsubstituted or C ₁ -C ₄ alkyl substituted piperidinyl (piperidinyl) or piperazinyl (piperazinyl),

R ₇ is hydrogen, -(CH ₂ )n-COOH, or -(CH ₂ )n-COOR ₁₀ (in this case, n=1 or more and 5 or less, R ₁₀ is hydrogen or C ₁ -C ₆ alkyl),

R ₈ is hydrogen or C ₁ -C ₆ alkyl, hydroxyalkyl, alkylaminoalkyl, or aminoalkyl,

R ₉ is C ₁ -C ₆ alkyl, hydroxyalkyl, alkylaminoalkyl, or aminoalkyl.

In one embodiment of the present invention, R ₂ of Formula 1 may be a pharmaceutical composition, characterized in that it is selected from the group consisting of the following compounds:

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

, ,

,

,

,

및

.

and

.

In another embodiment of the present invention, the compound may be any one selected from the group consisting of the following compounds:

4-(3-((((2Z,3E)-6'-bromo-2'-oxo-[2,3'-biindolinylidene]-3-ylidene)amino)oxy)propanoyl)piperazin-1-ium (compound 1), 4-(3-((((2Z,3E)-6'-bromo-2'-oxo-[2,3'-biindolinylidene]-3-ylidene)amino)oxy)propanamido)piperidin-1- ium (compound 2), and 4-((2-((((2Z,3E)-6'-Bromo-2'-oxo-[2,3'-biindolinylidene]-3-ylidene)amino)oxy)ethyl )sulfonyl)piperazin-1-ium (Compound 3).

As used herein, the term "sarcopenia" refers to a gradual weakening of muscle density and function, and the cause is known to cause progressive degeneration and destruction of motor neurons or muscle cells in the spinal nerve or diencephalon. In particular, muscle loss due to aging is called age-related sarcopenia.

As used herein, the term "muscle atrophy" refers to a generic name for a disease in which the muscles of the extremities are gradually atrophied in an almost symmetrical manner, and when cancer, aging, kidney disease, hereditary disease, and various chronic diseases are induced It may be accompanied by amyotrophic lateral sclerosis (Lou Gehrig's disease), and is represented by progressive progressive muscular atrophy of the spinal cord.

In the present invention, in the muscle atrophy model induced by dexamethasone, the effect of inhibiting the expression of atrozin-1 gene for increasing the diameter of the pharmaceutical composition, suppressing root canal atrophy, maintaining the ratio of root canals by diameter, and destroying muscle proteins, was confirmed. It was confirmed that it can be used as a therapeutic agent for sarcopenia or muscular atrophy.

As used herein, the term “treatment” refers to a compound of Formula 1 and a pharmaceutically acceptable salt, solvate or hydrate thereof; And it means any action of inhibiting or delaying sarcopenia or muscular atrophy by administration of a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

As used herein, the term “prevention” refers to a compound of Formula 1 and a pharmaceutically acceptable salt, solvate or hydrate thereof; And it refers to any action in which the symptoms of sarcopenia or muscular atrophy are improved or advantageously changed by administration of a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier in addition to the active ingredient. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity of the patient. can be Meanwhile, the dosage of the pharmaceutical composition of the present invention is preferably 1-1000 mg/kg (body weight) per day.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, it may be administered by topical application to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. . Considering that the pharmaceutical composition of the present invention is applied for the treatment of sarcopenia or muscular atrophy, the administration is preferably made by intravenous or oral administration.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. Or it can be prepared by introducing into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

The pharmaceutical composition of the present invention can be administered orally, and solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc., and these solid preparations contain at least one compound of the present invention. It is prepared by mixing one or more excipients, for example, starch, calcium carbonate, sucrose or lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, or syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. can

Formulations for oral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used.

Considering that the pharmaceutical composition of the present invention is a composition for preventing or treating sarcopenia or muscular atrophy, it may be preferably administered parenterally, for example, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration. can be administered using

The indirubin derivative of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid may be used. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids, and organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid and fumaric acid. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Toxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol lactate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the indirubin derivative of Formula 1 in an organic solvent, for example, methanol, ethanol, acetone, methylene chloride, acetonitrile, or the like, and adding an organic or inorganic acid to the precipitate. It can be prepared by filtration and drying, or by drying or crystallization in an organic solvent after distilling the solvent and excess acid under reduced pressure.

In addition, a pharmaceutically acceptable metal salt may be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. Also, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate). In addition, the present invention includes all possible solvates, hydrates, stereoisomers, etc. that can be prepared therefrom, as well as indirubin derivatives represented by Formula 1 and pharmaceutically acceptable salts thereof.

As another aspect, the present invention provides a method of treating sarcopenia or muscular atrophy comprising administering the pharmaceutical composition to an individual having sarcopenia or muscular atrophy in a pharmaceutically effective amount or an individual at risk of developing sarcopenia or muscular atrophy do.

As used herein, the term "individual" may include, without limitation, mammals, farmed fish, and the like, including mice, livestock, humans, etc. that are likely to or have developed sarcopenia or muscular atrophy.

The administration route of the pharmaceutical composition for the treatment of muscular atrophy or sarcopenia of the present invention may be administered through any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but depending on the intended route, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, intrapulmonary administration, rectal administration, etc. can be administered through However, since the pharmaceutical composition containing the compound of Formula 1 may be denatured by gastric acid during oral administration, the oral composition should be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the composition may be administered by any device capable of transporting the active agent to a target cell.

In another aspect, the present invention provides the use of the pharmaceutical composition in the manufacture of a medicament for the prevention or treatment of muscular atrophy or sarcopenia.

The present invention relates to a treatment for sarcopenia or muscular atrophy, which is a novel use of an indirubin derivative, and the pharmaceutical composition provided in the present invention increases the diameter of the root canal, suppresses atrophy of the root canal, maintains the ratio of the root canal by diameter and destroys muscle protein It can be usefully used for effective prevention or treatment of sarcopenia or muscular atrophy through suppression of the expression of atrozin-1 gene.

1 relates to the effect of compounds 1 and 3 in a model of muscular atrophy induced by dexamethasone.
1A is a representative image of a mitotracker of a stained root canal. Scale bar represents 100 μm.
1B is a quantitative result of the average root canal diameter.
Figure 1c shows the number of root canals according to the tube diameter.
Figure 2 is a real-time qPCR-based mRNA expression analysis results of muscle E3 ligase atrogin-1 (E3 ligase atrogin-1) according to the treatment of compound 1 to confirm the molecular marker of muscle atrophy.
3 is a result of MTT cell proliferation assay for confirming the cytotoxicity of each concentration of the compound.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Preparation Example 1. Compound Synthesis

Compounds 1 to 3 were designed and synthesized.

1-1. Synthesis Schematic 1

Reagents and reaction conditions: (a) Indoxyl acetate, Na ₂ CO ₃ , methanol: water (2:1), RT, 3h (b) Hydroxylamine hydrochloride, pyridine, reflux, 6h

Isatin analogue 1a-d dissolved in MeOH was added to indoxyl acetate and stirred for 5 minutes. Next, anhydrous Na ₂ CO ₃ (2.5eq) was added and stirred at room temperature for 3 hours. After filtration by adding water, and washing several times with cold water, the product 2a-d substituted at position 5 was obtained in the form of a precipitate. Next, the obtained products were dissolved in pyridine (0.1M), hydroxylamine hydrochloride (5eq) was added, and refluxed for 6 hours. After the mixture is cooled and acidified with 1N HCl, the resulting precipitate is obtained by filtration and washing with water several times. Finally, it was purified through solidification using DCM and hexane to obtain final products 3a-d.

1-2. Synthesis Schematic 2

시약 및 반응조건: (a) Methyl 3-bromopropionate, TEA, DMF, 80^oC, 1h (b) aq 20% KOH, MeOH, RT, 1h (c) Piperazine, EDC, TEA, DMF, 60^oC, O/N (d) 4N HCl in dioxane, THF, 2h (e) 4-Amino-1-Boc-piperidine, EDC, TEA, DMF, 60^oC, O/N (f) TFA, DCM, RT, 2h (g) 4N HCl in dioxane, THF, 2h

Reagents and reaction conditions: (a) Methyl 3-bromopropionate, TEA, DMF, 80 ^o C, 1h (b) aq 20% KOH, MeOH, RT, 1h (c) Piperazine, EDC, TEA, DMF, 60 ^o C, O/N (d) 4N HCl in dioxane, THF, 2h (e) 4-Amino-1-Boc-piperidine, EDC, TEA, DMF, 60 ^o C, O/N (f) TFA, DCM, RT, 2h (g) 4N HCl in dioxane, THF, 2h

Methyl 3-bromopropionate is dissolved in DMF, and indirubin-3'-oxime 3a-d is added and stirred for 5 minutes. Next, TEA (2eq) was added and stirred at 80°C for 1 hour. The mixture is then cooled and neutralized with an aqueous _{NH 4 Cl solution.} The neutralized product is extracted using EA and concentrated by rotary evaporation. This extract is purified by column chromatography to obtain products 4a-d.

The obtained products 4a-d are dissolved in MeOH, aq 10% KOH is added and stirred for 1 hour. Then, MeOH in the mixture was removed using rotary evaporation and neutralized with 1N HCl aqueous solution. The neutralized product is extracted using EA and concentrated by rotary evaporation. The extract is purified by column chromatography to obtain products 5a-d.

The obtained product 5a-d was dissolved in DMF, and EDC (1.5eq) and Piperazine (10eq) were added thereto and stirred for 5 minutes. Next, TEA (2eq) was added and stirred at O/N at 60°C. The mixture is then cooled and neutralized with an aqueous _{NH 4 Cl solution.} The neutralized product is extracted using EA and concentrated by rotary evaporation. This extract is purified by column chromatography to obtain products 6a-d.

Next, the obtained indirubin derivative 6a-d was dissolved in THF, 4N HCl in dioxane (6-18 eq) was added at 0° C., and stirred at room temperature for 2 hours. After confirming the completion of the reaction, the resulting precipitate was washed several times with DCM through filtration and dried to obtain a clean solid. Indirubin derivatives were obtained in the form of salts 7a-d.

The obtained product 5a-d was dissolved in DMF, EDC (1.5eq) and 4-Amino-1-Boc-piperidine (3eq) were added and stirred for 5 minutes. Next, TEA (2eq) was added and stirred at O/N at 60°C. The mixture is then cooled and neutralized with an aqueous _{NH 4 Cl solution.} The neutralized product is extracted using EA and concentrated by rotary evaporation. The obtained product was dissolved in DCM, added with TFA and stirred for 2 hours. Then, the mixture was neutralized with 1N aqueous NaOH solution, extracted using EA, and concentrated by rotary evaporation. This extract was purified by column chromatography to obtain products 8a-d.

Next, the obtained indirubin derivative 8a-d was dissolved in THF, 4N HCl in dioxane (6-18 eq) was added at 0° C., and stirred at room temperature for 2 hours. After confirming the completion of the reaction, the resulting precipitate was washed several times with DCM through filtration and dried to obtain a clean solid. Indirubin derivatives were obtained in the form of salts 9a-d.

Confirmation of compound synthesis

Product 7c: 4-(3-((((2Z,3E)-6'-bromo-2'-oxo-[2,3'-biindolinylidene]-3-ylidene)amino)oxy)propanoyl)piperazin-1- ium (compound 1)

¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 11.70 (s, 1H), 10.95 (s, 1H), 9.14 (br. s., 2H), 8.51 (d, J = 8.70 Hz, 1H), 8.10 (d, J = 7.79 Hz, 1H), 7.44 (d, J = 3.66 Hz, 2H), 7.17 (dd, J = 1.83, 8.24 Hz, 1H), 6.94 - 7.10 (m, 2H), 4.84 (t) , J = 6.41 Hz, 2H), 3.59 - 3.78 (m, 4H), 2.95 - 3.15 (m, 6H); MS (ESI): M ⁺ =497.83

Product 9c: 4-(3-((((2Z,3E)-6'-bromo-2'-oxo-[2,3'-biindolinylidene]-3-ylidene)amino)oxy)propanamido)piperidin-1- ium (compound 2)

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.64 - 11.75 (m, 1H), 10.89 - 10.98 (m, 1H), 8.56 - 8.76 (m, 2H), 8.48 - 8.55 (m, 1H), 8.25 - 8.33 (m, 1H), 8.02 - 8.13 (m, 1H), 7.37 - 7.49 (m, 2H), 7.12 - 7.22 (m, 1H), 6.97 - 7.09 (m, 2H), 4.74 - 4.88 (m, 2H), 3.79 - 3.92 (m, 1H), 3.13 - 3.24 (m, 2H), 2.82 - 2.99 (m, 2H), 2.63 - 2.76 (m, 2H), 1.74 - 1.88 (m, 2H), 1.46 - 1.62 (m, 2H)

1-3. Synthesis Schematic 3

시약 및 반응조건: (a) MsCl, TEA, DCM, RT, 1h (b) K₂CO₃, DMF, 60^oC, O/N (c) TFA, DCM, RT, 2h (d) 4N HCl in dioxane, THF, 2h

Reagents and reaction conditions: (a) MsCl, TEA, DCM, RT, 1h (b) K ₂ CO ₃ , DMF, 60 ^o C, O/N (c) TFA, DCM, RT, 2h (d) 4N HCl in dioxane, THF, 2h

Dissolve 1-BOC-4-(2-hydroxyethyl)piperazine in DCM, add MsCl (1.5eq) and TEA (1.5eq), and stir for 1 hour. Thereafter, the mixture _{was neutralized with an aqueous NH 4} Cl solution, extracted using EA, and concentrated by rotary evaporation. This extract is purified by column chromatography to obtain product 11.

The obtained product 11 was added to Indirubin-3'-oxime 3a-d dissolved in DMF and stirred for 5 minutes. Next, K ₂ CO ₃ (5eq) is added and stirred at O/N at 60°C. The mixture is then cooled and neutralized with an aqueous _{NH 4 Cl solution.} The neutralized product is extracted using EA and concentrated by rotary evaporation. The resulting mixture was dissolved in DCM, added with TFA and stirred for 2 hours. Then, the mixture was neutralized with 1N aqueous NaOH solution, extracted using EA, and concentrated by rotary evaporation. This extract is purified by column chromatography to obtain products 12a-d.

Next, the obtained indirubin derivative 12a-d was dissolved in THF, 4N HCl in dioxane (6-18 eq) was added at 0° C., and stirred at room temperature for 2 hours. After confirming that the reaction was complete, the precipitate was washed several times with DCM through filtration and dried to obtain a clean solid. Indirubin derivatives were obtained in the form of salts 13a-d.

Confirmation of compound synthesis

Product 13c: 4-((2-((((2Z,3E)-6'-Bromo-2'-oxo-[2,3'-biindolinylidene]-3-ylidene)amino)oxy)ethyl)sulfonyl)piperazin -1-ium (Compound 3)

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.80 (s, 1H), 9.07 - 9.26 (m, 2H), 8.63 (d, J = 8.47 Hz, 1H), 8.23 (d, J = 7.40 Hz, 1H), 7.34 - 7.53 (m, 3H), 7.18 (dd, J = 1.95, 8.36 Hz, 1H), 6.99 - 7.12 (m, 1H), 4.26 (t, J = 6.75 Hz, 2H), 3.60 (t) , J = 6.80 Hz, 2H), 3.37 - 3.49 (m, 4H), 3.03 - 3.20 (m, 4H) MS (ESI): M ⁺ =533.62

Experimental Example 1. Cell culture

C2C12 mouse myoblasts were cultured in DMEM, 10% FBS and 1% penicillin streptomycin (GM), and differentiation was induced with DMEM, 2% horse serum and 1% penicillin streptomycin (DM). To make myotubes, differentiation medium was treated for 72 hours and further treatment was performed after the differentiation procedure.

Experimental Example 2. MTT cell proliferation assay (MTT Cell proliferation assay)

The antiproliferative effect of chemicals was evaluated by MTT cell proliferation assay. C2C12 mouse myoblasts were seeded in 96-well plates and stabilized for 24 hours.

Chemicals were diluted in complete medium to the indicated concentrations and treated for 48 hours. 1X MTT (methylthiazolyldiphenyl-tetrazolium bromide, Sigma aldrich) was prepared as a serum-free medium and treated for 1 hour. The precipitate was dissolved in 50 μL DMSO and absorbance was measured at 570 nm using a microplate reader.

Experimental Example 3. In vitro dexamethasone ( Analysis of dexamethasone)-induced muscle atrophy

^{30x10 4} C2C12 cells stabilized in DMEM, 10% FBS and 1% penicillin streptomycin for 24 hours were seeded in 12 well plates. Differentiation was induced with 2% horse serum DMEM (Differentiation media, DM) for 72 h, followed by dexamethasone sodium phosphate (Santa cruz, SC-204715) at a concentration of 10 μM in DM at a concentration of 10 μM in 2% horse serum DMEM for additional 24 hours was treated, and the chemical was treated simultaneously with dexamethasone.

Experimental Example 4. Mitotracker staining

MitoTracker CMXRos (Invitrogen, M7512) and DAPI (Sigma, D9542) were added to myotubes treated with dexamethasone and chemicals at final concentrations of 50 nM and 1 μM, respectively. Cells were incubated for 30 min and then visualized by fluorescence microscopy (Leica DMI 3000B; Leica).

Experimental Example 5. Immunofluorescence staining

To measure the diameter of myotubes, the myosin heavy chain was stained with a specific antibody (Santa cruz SC-53095). 30x10 ⁴ C2C12 cells were seeded in 12 well plates and differentiated for 72 hours after stabilization for 24 hours. Differentiated myotubes were treated with individual drugs for 24 hours and fixed with 4% formaldehyde solution. Cells were permeabilized and blocked with 0.5% Triton-X and 1% BSA solutions, respectively. Myosin heavy chain antibody was exposed to O/N in a 1:50 ratio and conjugated with Alexa 488 (Thermo, A11001) for 1 h. The stained myotubes were confirmed with a fluorescence microscope (Leica DMI 3000B; Leica).

Experimental Example 6. RNA analysis using qPCR

Total RNA from C2C12 myotubes was extracted using TRI solution (BIO Science Technology) according to the manufacturer's protocol. The extracted RNA was quantified with Nanodrop One (Thermo) and reacted with RT-PCR premix (BIONEER, K-2044) to make cDNA. For real-time PCR analysis, cDNA was mixed with SBR green master mix (Enzynomix) and detected with StepOnePlus real-time PCR system (Applied biosystems).

Example 1. Effect in dexamethasone-induced muscular atrophy model

In order to verify the effect of the composition of the present invention on muscle atrophy, the dexamethasone-induced muscle atrophy model, which is the most used model for this kind of study, was used. 10 μM of dexamethasone and the indicated concentrations of compounds were administered to the differentiated myotubes for 72 hours. Myotubes were visualized with a mitotracker indicating intracellular mitochondrial activity (FIG. 1A).

In the dexamethasone-induced muscle atrophy cell-based model, the changes in root canal diameter and root canal size distribution, which were reduced by dexamethasone alone treatment, were significantly alleviated by treatment with the compound of the present invention ( FIG. 1B ).

Summarizing the results, the diameter of the root canal was reduced by about 30% by treatment with dexamethasone alone, whereas 98% and 88% and 88 of normal myotubes (control without dexamethasone) were treated with 7C (Compound 1) and 13C (Compound 3). % level, respectively. Dexamethasone treatment alone changed the size distribution of root canals, increasing the number of relatively small (<15 μm) canals and decreasing large (> 30 μm) canals. However, the distribution of these small and large myotubes was normalized by treatment with compounds 1 and 3.

That is, according to the above results, it was confirmed that the compound of the present invention has an effect of alleviating muscle loss because it alleviates the decrease in diameter of the root canal and the change in the size distribution of the root canal in the desamethasone-treated muscle atrophy model.

Example 2. Modulating effect of muscle-specific E3 ligase atrogin-1 (E3 ligase atrogin-1)

In order to confirm the inhibitory effect of the muscle loss inducer, qPCR analysis was performed targeting the muscle-specific E3 ligase atrozin-1, which is one of the markers of muscle atrophy, based on the phenotypic effect of the compound. The results are shown in FIG. 2 .

As can be seen in FIG. 2 , the dexamethasone-treated group showed a rapid increase in atrozin-1, and it was confirmed that representative compound 1 suppressed the expression of atrozin-1 induced by dexamethasone in a dose-dependent manner.

Due to the above results, it was confirmed that the compound of the present invention can be useful as a therapeutic agent for sarcopenia by inhibiting the expression of E3 ligase atrozin-1, a sarcopenia inducer.

Example 3. Cytotoxicity analysis of C2C12 myoblast proliferation at myotube protective concentration

MTT cell proliferation assay was performed to confirm the chemical toxicity of the compound of the present invention. Inhibition of muscle loss was effective in both compounds (Compounds 1 and 3) at a concentration of 300 nM, and thus was evaluated in the range of less than 1 μM (Fig. 3).

As can be seen in FIG. 3 , compounds 1 and 3 were all non-cytotoxic at concentrations of 125, 250, and 500 nM, confirming the stability as a therapeutic agent for muscle loss.

As a result of the above, the present application has technical significance in identifying novel uses of previously unknown indirubin derivatives by confirming the therapeutic effect of indirubin derivatives on sarcopenia or muscular atrophy.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

A compound represented by the following formula (1), a pharmaceutically acceptable salt, solvate or hydrate thereof:
[Formula 1]

In Formula 1,
R ₁ is hydrogen, halogen, or halogen-substituted or unsubstituted C ₁ -C ₄ alkoxy;
R ₂ is

or

In this case, n is a natural number of 2 or more and 4 or less,
R ₃ is hydrogen or halogen,
wherein R ₄ is

,

or

is,
In this case, X is CH or N,
L is a single bond or NH,
R ₅ and R ₆ are each independently hydrogen; C ₁ -C ₄ alkyl; Or unsubstituted or C ₁ -C ₄ alkyl substituted piperidinyl (piperidinyl) or piperazinyl (piperazinyl),
R ₇ is hydrogen, -(CH ₂ )n-COOH, or -(CH ₂ )n-COOR ₁₀ (in this case, n=1 or more and 5 or less, R ₁₀ is hydrogen or C ₁ -C ₆ alkyl),
R ₈ is hydrogen or C ₁ -C ₆ alkyl, hydroxyalkyl, alkylaminoalkyl, or aminoalkyl,
R ₉ is C ₁ -C ₆ alkyl, hydroxyalkyl, alkylaminoalkyl, or aminoalkyl.
According to claim 1, wherein R ₂ Is

,

,

,

,

,

,

,

,

,

,

,

,

or

wherein the compound, a pharmaceutically acceptable salt, solvate or hydrate thereof.
4. The compound of claim 1, wherein the compound is 4-(3-((((2Z,3E)-6'-bromo-2'-oxo-[2,3'-biindolinylidene]-3-yl) den)amino)oxy)propanoyl)piperazin-1-ium (compound 1), 4-(3-((((2Z,3E)-6′-bromo-2′-oxo-[2,3] '-Biindolynylidene]-3-ylidene)amino)oxy)propanamido)piperidin-1-ium (compound 2) or 4-((2-((((2Z,3E)-6 '-Bromo-2'-oxo-[2,3'-biindolynylidene]-3-ylidene)amino)oxy)ethyl)sulfonyl)piperazin-1-ium (compound 3) , a compound, a pharmaceutically acceptable salt, solvate or hydrate thereof.
(a) a compound represented by the following formula (1), a pharmaceutically acceptable salt, solvate or hydrate thereof: (b) prevention or treatment of sarcopenia or muscle atrophy comprising a pharmaceutically acceptable carrier Pharmaceutical composition for:
[Formula 1]

In Formula 1,
R ₁ is hydrogen, halogen, or halogen-substituted or unsubstituted C ₁ -C ₄ alkoxy;
R ₂ is

or

In this case, n is a natural number of 2 or more and 4 or less,
R ₃ is hydrogen or halogen,
wherein R ₄ is

,

or

is,
In this case, X is CH or N,
L is a single bond or NH,
R ₅ and R ₆ are each independently hydrogen; C ₁ -C ₄ alkyl; Or unsubstituted or C ₁ -C ₄ alkyl substituted piperidinyl (piperidinyl) or piperazinyl (piperazinyl),
R ₇ is hydrogen, -(CH ₂ )n-COOH, or -(CH ₂ )n-COOR ₁₀ (in this case, n=1 or more and 5 or less, R ₁₀ is hydrogen or C ₁ -C ₆ alkyl),
R ₈ is hydrogen or C ₁ -C ₆ alkyl, hydroxyalkyl, alkylaminoalkyl, or aminoalkyl,
R ₉ is C ₁ -C ₆ alkyl, hydroxyalkyl, alkylaminoalkyl, or aminoalkyl.
5. The method of claim 4, wherein R ₂ is

,

,

,

,

,

,

,

,

,

,

,

,

or

which is, a pharmaceutical composition.
5. The compound of claim 4, wherein the compound is 4-(3-((((2Z,3E)-6'-bromo-2'-oxo-[2,3'-biindolinylidene]-3-yl) den)amino)oxy)propanoyl)piperazin-1-ium (compound 1), 4-(3-((((2Z,3E)-6′-bromo-2′-oxo-[2,3] '-Biindolynylidene]-3-ylidene)amino)oxy)propanamido)piperidin-1-ium (Compound 2), or 4-((2-((((2Z,3E)- 6'-Bromo-2'-oxo-[2,3'-biindolynylidene]-3-ylidene)amino)oxy)ethyl)sulfonyl)piperazin-1-ium (Compound 3) Phosphorus, a pharmaceutical composition.
5. The method of claim 4, wherein the composition exhibits an effect selected from the group consisting of an increase in root canal diameter, suppression of root canal atrophy, maintenance of the ratio of root canals by diameter, inhibition of expression of genes that destroy muscle proteins, and combinations thereof, pharmaceutical composition.
The pharmaceutical composition according to claim 7, wherein the gene that destroys the muscle protein is a gene encoding Atrogin-1.
A method for preventing or treating sarcopenia or muscular atrophy, comprising administering the pharmaceutical composition of any one of claims 4 to 8 to a subject other than a human in a pharmaceutically effective amount.

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