KR20190114500A - (-)-epigallocatechin gallate prodrugs , preparation method thereof and composition for mitochondrial biosis - Google Patents

Abstract

The present invention relates to an epigallocatechin gallate prodrug, a method for manufacturing the same, and a composition for promoting mitochondrial biosynthesis comprising the same. A compound represented by chemical formula (1) according to the present invention increases the activity of PGC-1α, AMPK, and SIRT1, known as mitochondrial biosynthesis regulators, increases the amount of mitochondrial DNA, and increases a NAD+/NADH ratio, a level of cytochrome c, the synthesis of ATP, and oxygen consumption, thereby confirming an effect of increasing the mitochondrial activity and biosynthesis. In addition, it is confirmed that the compound has an effect of having a high absorption rate and slow metabolism in vivo compared to an existing epigallocatechin gallate, thereby being useful for a purpose of preventing or treating diseases related to mitochondrial dysfunction.

Description

Epigallocatechin gallate prodrug, preparation method thereof, and composition for promoting mitochondrial biosynthesis comprising same {(-)-epigallocatechin gallate prodrugs, preparation method about and composition for mitochondrial biosis}

The present invention relates to an epigallocatechin gallate prodrug, a preparation method thereof, and a composition for promoting mitochondrial biosynthesis including the same.

Mitochondria are intracellular organelles present in most eukaryotic cells and have their own mitochondrial DNA (mtDNA), which is separated from nuclear DNA. The main function of mitochondria is to produce ATP, an intracellular energy source. ATP is produced in the electron transport system using NADH, FADH2, which is produced through the TCA circuit in the mitochondrial substrate. The ATP thus produced is used to drive various energy-required biosynthesis and various metabolic activities.

In addition, mitochondria store calcium ions in the substrate, which play an important role in intracellular signal transduction, and also supply the cytoplasm when necessary. In addition, it is known to play a role in regulating cell death, proliferation, metabolism, and the like. Moreover, mitochondrial DNA (mtDNA), unlike nuclear DNA of cells, does not have its own repair mechanism, and is relatively prone to damage because it lacks histone proteins that serve to protect DNA. The damage of mitochondrial DNA (mtDNA) is known to be closely related to the development of mitochondrial disease, leading to a decrease in the function of mitochondria, reducing the synthesis of ATP, an energy source necessary for cellular activities, and causes various diseases. .

Mitochondrial dysfunction has been associated with age-related degenerative diseases such as obesity, diabetes, metabolic syndrome, insulin resistance, cardiovascular disease, Parkinson's disease, Huntington's disease, ischemic brain disease (eg stroke), and dementia. . In other words, in these disease models, electron micrographs of mitochondria may be swelled or enlarged compared to normal, and structural changes such as loss of cristae structure may be observed, and the number of mitochondrial DNA (mtDNA) per cell may be reduced. A mutation of mtDNA was also observed. When monitoring the changes in mitochondrial function according to aging in humans and animals, the number of copies of mitochondrial DNA (mtDNA) and mtRNA decreased as aging progressed. Recently, it has been reported that substances identified as factors causing the onset of such neurodegenerative diseases exist in mitochondria or are closely related to mitochondrial dysfunction.

Mitochondria are also believed to be a central cause of ischemia-reperfusion (IR) damage. IR induces a change in the structure and biochemical function of the cardiac mitochondria, which results in a loss of mitochondrial ATP produced by the mitochondria, which prevents the heart from obtaining enough energy to contract. In addition, the activity of respiratory chain complex, NADH dehydratase activity, membrane potential loss, loss of ATP synthesis and increase of ATP hydrolysis, ion homeostasis, ROS formation, etc.

Endurance, such as running and swimming, increases skeletal muscle respiratory ability, which affects the mitochondrial electron transport chain, citric acid cycle, and enzymes associated with fatty acid oxidation. In addition, the size and number of mitochondria in skeletal muscle are increased by increasing the ratio of synthetic metabolism rather than the decomposition metabolism of mitochondrial protein. Therefore, the study of mitochondrial biosynthesis in skeletal muscle is very important in the improvement of exercise ability because the improvement of oxygen utilization in skeletal muscle mitochondria improves the ability of ATP production through oxidative phosphorylation.

Mitochondria are the core of the cellular energy system and also cause the body's oxidative stress, which is particularly sensitive to aging, resulting in large losses in quantity and quality. However, the process of mitochondrial biosynthesis, which increases the number and amount of mitochondria, is an excellent target for preventing and treating mitochondrial loss due to aging.

Epigallocatechin gallate ((-)-epigallocatechin gallate, (-)-EGCG) (hereinafter referred to as EGCG) is one of the representative catechin compounds included in green tea, which plays a key role in mitochondrial metabolism. It is known to regulate the activity of peroxisome proliferator-activated receptor gamma-coactivator-1alpha and SIRT1 (NAD-dependent protein deacetylase sirtuin 1).

However, EGCG has a problem of showing very low bioavailability due to its low in vivo absorption rate and fast metabolism, and therefore, a prodrug approach to overcome this problem is drawing attention.

Therefore, the present inventors completed the invention by synthesizing EGCG prodrugs incorporating promoiety at the 4 "-position of the EGCG D-ring and verifying the effect of inducing mitochondrial biosynthesis by these substances.

Korea Patent Registration No. 10-1822306

Seo, Y .; Kim, M. K .; Choo, H .; Chong, Y. Facile synthesis of 4 ″ -O-alkyl-(-)-EGCG derivatives through regioselective deacetylative alkylation. Synth. Commun. 2017, 47, 655-659

It is an object of the present invention to provide a compound represented by formula (1), or a pharmaceutically acceptable salt thereof.

Another object of the present invention to provide a method for preparing a compound represented by the formula (1).

Still another object of the present invention is to provide a composition for promoting mitochondrial biosynthesis comprising the compound represented by Formula 1, or a pharmaceutically acceptable salt thereof.

Another object of the present invention consists of a degenerative disease, a brain disease, a neurological disease, a heart disease, a liver disease, a kidney disease, a pancreatic disease and a muscle disease, including a compound represented by the formula (1), or a pharmaceutically acceptable salt thereof. It provides a pharmaceutical composition for preventing or treating a disease selected from the group.

In order to achieve the above object,

The present invention provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Formula 1]

(In the above formula 1,

,

또는

이고;R ^A is

,

or

ego;

R ¹ is H, or C _1-6 straight or branched alkoxy;

R ² is H, or C _1-6 straight or branched alkyl;

R ³ is C _1-6 straight or branched alkyl, or C _1-6 straight or branched alkoxy;

R ⁴ is C _1-6 straight or branched alkyl, C _1-6 straight or branched alkoxycarbonylC _1-6 straight or branched alkyl, or C _6-12 aryl C _1-6 straight or branched alkyl to be).

In addition, the present invention as shown in Scheme 1,

Compound 2 (Epigallocatechin gallate, EGCG) and excess acetic acid were added to the organic solvent, and reacted at 10-40 ° C. for 4-8 hours to obtain Compound 3, in which all of the hydroxyl groups of Compound 2 were acetylated ( Step 1); And

Compound 3, potassium carbonate (K 2 CO 3), potassium iodide (KI), and R ^A X (wherein X is halogen and R ^A is as defined in Scheme 1 below) are added to the organic solvent and 2- at 35-65 ° C. Reacting for 6 hours to obtain compound 1 (Step 2);

It provides a method for producing a compound represented by the formula (1) comprising a.

Scheme 1

(In Scheme 1,

R ^A is as defined in Formula 1 of claim 1).

Furthermore, the present invention provides a composition for promoting mitochondrial biosynthesis comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention is selected from the group consisting of a degenerative disease, brain disease, neurological disease, heart disease, liver disease, kidney disease, pancreatic disease and muscle disease comprising a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof It provides a pharmaceutical composition for the prevention or treatment of diseases.

Compound represented by the formula (1) according to the present invention can increase the activity of PGC-1α, AMPK, SIRT1 known as mitochondrial biosynthesis regulators and increase the amount of mitochondrial DNA, NAD + / NADH ratio, cytochrome c level, ATP The synthesis and oxygen consumption were increased to confirm the effect of increasing the activity and biosynthesis of mitochondria, and also compared to the epigallocatechin gallate in vivo, the effect of higher absorption rate and slow metabolism, It may be useful for the purpose of preventing or treating related diseases.

1 shows (a) Comparative Compound 2 (EGCG) (b) Comparative Compound 3 (AcEGCG) (c) Example 3, (d) Example 6, (e) Example 7, (f) in Hepa1-6 cells. ) Example 9, (g) The change in Mitotracker fluorescence intensity according to Example 10 is shown.
Figure 2 shows the results of staining Hepa1-6 cells using ActinGreen 488 and Mitotracker ((a) Comparative Compound 2 (EGCG) (b) Comparative Compound 3 (AcEGCG) (c) Example 3, (d) Example 6, (e) Example 7, (f) Example 9, (g) Example 10).
Figure 3a shows the ratio of mitochondrial region density and cytoplasmic volume observed by Mitotracker and ActinGreen 488.
Figure 3b shows the ratio of mitochondrial DNA and nuclear DNA in Hepa1-6 cells.
4 shows changes in the levels of mitochondrial modulators by Comparative Compound 2 (EGCG), Comparative Compound 3 (AcEGCG), Example 3, Example 6, Example 7, Example 9 and Example 10 in Hepa1-6 cells. (A) PGC-1α, (b) p-AMPK, (c) SIRT1).
Figure 5 shows the change of oxidative phosphorylation related factors by Comparative Compound 2 (EGCG), Comparative Compound 3 (AcEGCG), Examples 6 and 7 in Hepa1-6 cells ((a) NAD + / NADH ratio, (b) degree of ATP synthesis, (c) amount of cytochrome c, and (d) oxygen consumption).

Hereinafter, the present invention will be described in detail.

The compound or a pharmaceutically acceptable salt thereof

The present invention provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Formula 1]

(In the above formula 1,

,

또는

이고;R ^A is

,

or

ego;

R ¹ is H, or C _1-6 straight or branched alkoxy;

R ² is H, or C _1-6 straight or branched alkyl;

R ³ is C _1-6 straight or branched alkyl, or C _1-6 straight or branched alkoxy;

R ⁴ is C _1-6 straight or branched alkyl, C _1-6 straight or branched alkoxycarbonylC _1-6 straight or branched alkyl, or C _6-12 aryl C _1-6 straight or branched alkyl to be).

Preferably,

,

또는

이고;R ^A is

,

or

ego;

R ¹ is H or C _1-3 straight or branched alkoxy;

R ² is H or C _1-3 straight or branched alkyl;

R ³ is C _1-4 straight or branched alkyl or C _1-4 straight or branched alkoxy;

R ⁴ is C _1-3 straight or branched alkyl, C _1-3 straight or branched alkoxycarbonylC _1-3 straight or branched alkyl, or C _{6-8 arylC 1-3} straight or branched alkyl .

More preferably,

,

또는

이고;R ^A is

,

or

ego;

R ¹ is H or methoxy;

R ² is H or methyl;

R ³ is t-butyl, t-butoxy, isopropoxy or ethoxy;

R ⁴ is methyl, methoxycarbonylethyl or phenylmethyl.

Preferred examples of the compound represented by Formula 1 according to the present invention include the following compound groups.

1) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (ethoxymethoxy) benzoyl) oxy) -chroman-2-yl ) Benzene-1,2,3-triyl triacetate;

2) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4-((2-methoxyethoxy) methoxy) -benzoyl) oxy) Chroman-2-yl) benzene-1,2,3-triyl triacetate;

3) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4-((pivaloyloxy) methoxy-benzoyl) oxy) chromen- 2-yl) benzene-1,2,3-triyl triacetate;

4) 5-((2R, 3R) -5,7-Diacetoxy-3- (3,5-diacetoxy-4-((isopropoxycarbonyloxy) methoxy) benzoyloxy) chroman- 2-yl) benzene-1,2,3-triyl triacetate;

5) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (1-((isopropoxycarbonyl) oxy) -ethoxy) Benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate;

6) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (1-((ethoxycarbonyl) oxy) ethoxy) -benzoyl ) Oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate;

7) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4-(((tert-butoxycarbonyl) oxy) -methoxy) benzoyl ) Oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate;

8) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (2-(((S) -1-methoxy-1-oxo) Propan-2-yl) amino) -2-oxoethoxy) benzoyl) oxy) chroman-2-yl) benzene-1,2,3-tritriacetate;

9) (S) -dimethyl 2- (2- (2,6-diacetoxy-4-((((2R, 3R) -5,7-diacetoxy-2- (3,4,5-tria) Cetoxyphenyl) chroman-3-yl) oxy) carbonyl) phenoxy) acetamido) pentanedioate; And

10) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (2-(((S) -1-methoxy-1-oxo) -3-phenylpropan-2-yl) amino) -2-oxoethoxy) benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate.

The compound represented by Chemical Formula 1 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 is useful. The expression pharmaceutically acceptable salt is a concentration that has a relatively nontoxic and harmless effect on the patient and that any side effects due to the salt do not degrade the beneficial efficacy of the base compound of formula 1, or Means inorganic addition salts. These salts may include inorganic acids and organic acids as free acids, hydrochloric acid, bromic acid, nitric acid, sulfuric acid, perchloric acid, phosphoric acid, and the like, and citric acid, acetic acid, lactic acid, maleic acid, and fumarine as organic acids. Acids, Gluconic Acid, Methanesulfonic Acid, Glyconic Acid, Succinic Acid, Tartaric Acid, Galluturonic Acid, Embonic Acid, Glutamic Acid, Aspartic Acid, Oxalic Acid, (D) or (L) Malic Acid, Maleic Acid, Methanesulphonic Acid, Ethene Sulfur Phonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid or malonic acid and the like can be used. These salts also include alkali metal salts (sodium salts, potassium salts, and the like), alkaline earth metal salts (calcium salts, magnesium salts, and the like) and the like. For example, acid addition salts include acetates, aspartates, benzates, besylates, bicarbonates / carbonates, bisulfates / sulfates, borates, camsylates, citrates, disylates, ecylates, formates, fumarates, Gluceptate, Gluconate, Glucuronate, Hexafluorophosphate, Hibenzate, Hydrochloride / Chloride, Hydrobromide / Bromide, Hydroiodide / Iodide, Isetionate, Lactate, Maleate, Mali Eate, malonate, mesylate, methyl sulfate, naphthylate, 2-naphsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, saccha Laterate, stearate, succinate, tartrate, tosylate, trifluoroacete , Aluminum, arginine, benzatin, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, zinc salts, and the like. Heavy hydrochloride or trifluoroacetate are preferred.

Acid addition salt according to the present invention is a conventional method, for example, a precipitate formed by dissolving a compound represented by the formula (1) in an organic solvent, for example methanol, ethanol, acetone, methylene chloride, acetonitrile and the like, and adding an organic or inorganic acid The solvent may be prepared by filtration, drying, or by distillation under reduced pressure of the solvent and excess acid, followed by drying or crystallization under an organic solvent.

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

Furthermore, the present invention includes not only the compound of Formula 1 and pharmaceutically acceptable salts thereof, but also possible solvates, hydrates, isomers, optical isomers, and the like that can be prepared therefrom.

Manufacturing method

As shown in Scheme 1 below,

Compound 2 (Epigallocatechin gallate, EGCG) and excess acetic acid were added to the organic solvent, and reacted at 10-40 ° C. for 4-8 hours to obtain Compound 3, in which all of the hydroxyl groups of Compound 2 were acetylated. (Step 1); And

Compound 3, potassium carbonate (K 2 CO 3), potassium iodide (KI), and R ^A X (wherein X is halogen and R ^A is as defined in Scheme 1 below) are added to the organic solvent, and 2 Reacting for 6 hours to obtain compound 1 (Step 2);

It provides a method for producing a compound represented by the formula (1) comprising a.

Scheme 1

(In Scheme 1,

R ^A is as defined in Formula 1 of claim 1).

In one embodiment of the present invention, the organic solvent of step 1 or step 2 is pyridine, t-butanol, ethanol, tetrahydrofuran (THF), benzene, KOH / MeOH, MeOH, toluene, CH 2 Cl 2, hexane, dimethylform Amides (DMF), diisopropyl ether, diethyl ether, dioxane, dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), acetone and chlorobenzene may be used alone or in combination, preferably step 1 The organic solvent of may be used pyridine, the organic solvent of step 2 may be used acetone.

Mitochondrial biosynthesis promoting composition

The present invention provides a composition for promoting mitochondrial biosynthesis comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Formula 1]

(In the above formula 1,

,

또는

이고;R ^A is

,

or

ego;

R ¹ is H, or C _1-6 straight or branched alkoxy;

R ² is H, or C _1-6 straight or branched alkyl;

R ³ is C _1-6 straight or branched alkyl, or C _1-6 straight or branched alkoxy;

R ⁴ is C _1-6 straight or branched alkyl, C _1-6 straight or branched alkoxycarbonylC _1-6 straight or branched alkyl, or C _6-12 aryl C _1-6 straight or branched alkyl to be).

Preferably,

,

또는

이고;R ^A is

,

or

ego;

R ¹ is H or C _1-3 straight or branched alkoxy;

R ² is H or C _1-3 straight or branched alkyl;

R ³ is C _1-4 straight or branched alkyl or C _1-4 straight or branched alkoxy;

R ⁴ is C _1-3 straight or branched alkyl, C _1-3 straight or branched alkoxycarbonylC _1-3 straight or branched alkyl, or C _{6-8 arylC 1-3} straight or branched alkyl .

More preferably,

,

또는

이고;R ^A is

,

or

ego;

R ¹ is H or methoxy;

R ² is H or methyl;

R ³ is t-butyl, t-butoxy, isopropoxy or ethoxy;

R ⁴ is methyl, methoxycarbonylethyl or phenylmethyl.

Preferred examples of the compound represented by Formula 1 according to the present invention include the following compound groups.

1) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (ethoxymethoxy) benzoyl) oxy) -chroman-2-yl ) Benzene-1,2,3-triyl triacetate;

2) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4-((2-methoxyethoxy) methoxy) -benzoyl) oxy) Chroman-2-yl) benzene-1,2,3-triyl triacetate;

3) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4-((pivaloyloxy) methoxy-benzoyl) oxy) chromen- 2-yl) benzene-1,2,3-triyl triacetate;

4) 5-((2R, 3R) -5,7-Diacetoxy-3- (3,5-diacetoxy-4-((isopropoxycarbonyloxy) methoxy) benzoyloxy) chroman- 2-yl) benzene-1,2,3-triyl triacetate;

5) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (1-((isopropoxycarbonyl) oxy) -ethoxy) Benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate;

6) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (1-((ethoxycarbonyl) oxy) ethoxy) -benzoyl ) Oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate;

7) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4-(((tert-butoxycarbonyl) oxy) -methoxy) benzoyl ) Oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate;

8) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (2-(((S) -1-methoxy-1-oxo) Propan-2-yl) amino) -2-oxoethoxy) benzoyl) oxy) chroman-2-yl) benzene-1,2,3-tritriacetate;

9) (S) -dimethyl 2- (2- (2,6-diacetoxy-4-((((2R, 3R) -5,7-diacetoxy-2- (3,4,5-tria) Cetoxyphenyl) chroman-3-yl) oxy) carbonyl) phenoxy) acetamido) pentanedioate; And

10) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (2-(((S) -1-methoxy-1-oxo) -3-phenylpropan-2-yl) amino) -2-oxoethoxy) benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate.

The composition of the present invention induces AMP-activated protein kinase (AMPK) activation or increases the activity of PGC-1 (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) α or NRT-dependent deacetylase sirtuin-1 (SIRT1). Or increase the copy number of mitochondrial DNA.

In addition, the composition of the present invention can increase the activity and biosynthesis of mitochondria by increasing the NAD + / NADH ratio, the level of cytochrome c, the synthesis of ATP, and the oxygen consumption.

SIRT1 (sirtuin 1) and PGC1α (PPARγ coactivator 1α) may play an important role in mitochondria biogenesis. SIRT1 is a NAD-dependent deacetylase, which may increase the production of mitochondria. PGC1α is a nuclear protein of 90 kDa, which is a transcriptional coactivator that regulates genes involved in energy metabolism. PGC1α activated by SIRT1 may increase expression of genes involved in ATP production and mitochondrial production. In this regard, it was confirmed that the activity of SIRT1 (sirtuin 1) and PGC1α (PPARγ coactivator 1α) was increased as confirmed in the following examples.

In addition, as compared to the existing epigallocatechin gallate it can be useful for the purpose of preventing or treating diseases associated with reduced mitochondrial function as it confirms a high absorption rate and slow metabolism in vivo.

Pharmaceutical compositions for the prevention or treatment of diseases

The present invention is a disease selected from the group consisting of degenerative disease, brain disease, neurological disease, heart disease, liver disease, kidney disease, pancreatic disease and muscle disease, including a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof It provides a pharmaceutical composition for the prophylaxis or treatment of.

[Formula 1]

(In the above formula 1,

R ^A is as defined in Formula 1 of claim 1).

In one embodiment of the present invention, the disease comprising the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof is various degenerative diseases, brain diseases, neurological diseases, heart diseases, liver diseases associated with decreased activity of mitochondria , Kidney disease, pancreatic disease or muscle disease.

In one embodiment of the present invention, the degenerative disease is not particularly limited, but may be preferably degenerative arthritis, rheumatoid arthritis or osteoarthritis.

In addition, in one embodiment of the present invention, the brain disease is not particularly limited, but may be preferably dementia, Parkinson's disease, stroke, developmental delay, neuropsychiatric disorder, migraine, autism, mental retardation, seizures or stroke.

In addition, in one embodiment of the present invention, the neurological disease is not particularly limited, but preferably, the eyelids, optic nerve atrophy, strabismus, retinal pigmentosa, blindness, hearing loss, eye muscle paralysis, reflex action, syncope, nerves Pain or autonomic ataxia.

In addition, in one embodiment of the present invention, the heart disease is not particularly limited, but may preferably be a heart attack or cardiomyopathy.

In addition, in one embodiment of the present invention, the liver disease is not particularly limited, but may preferably be hypoglycemia or liver failure.

In addition, in one embodiment of the present invention, the kidney disease is not particularly limited, but may preferably be renal tubuloacidosis.

In addition, in one embodiment of the present invention, the pancreatic disease is not particularly limited, but may preferably be a pancreatic dysfunction or parathyroid deficiency.

In addition, in one embodiment of the present invention, the muscle disease is not particularly limited, but preferably may be irritable bowel syndrome, myalgia, muscular dystrophy, gastroesophageal reflux disease, hypotension, convulsions, movement disorders, constipation or diarrhea.

The compound of the present invention may be administered in various oral and parenteral dosage forms for clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, may be used. Are manufactured.

Solid form preparations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, which form at least one excipient such as starch, calcium carbonate, water, or the like. It is prepared by mixing cross, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, or syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.

In addition, the effective dosage of the compound of the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and is generally about 0.001 to 100 mg / kg / day, preferably Preferably 0.01-35 mg / kg / day. Based on an adult patient with a weight of 70 kg, it is generally 0.07 ~ 7000 mg / day, preferably 0.7 ~ 2500 mg / day, once a day at regular intervals depending on the judgment of the doctor or pharmacist Multiple doses may be administered.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited by the following examples.

EGCG Prodrug Manufacturing

The method for synthesizing the EGCG prodrugs of Examples 1 to 10 is as shown in the following scheme. Specifically, the preparation method of Example 1 was as follows, and the remaining compounds were synthesized by a similar method.

Scheme 1

(In Scheme 1,

Ac ₂ O is acetic anhydride, Pyr is pyrdine, X is halogen, and R ^A is as defined in Chemical Formula 1).

Step 1: 5-((((2 R , 3 R ) -5,7-diacetosyl-2- (3,4,5-triacetocyphenyl) chroman-3-yl) oxy) carbonyl) benzene-1,2,3-trile triacetate (EGCG peracetate, AcEGCG, Preparation of Compound 3)

To pyrdine (25 mL) add EGCG (Compound 2) (1 g, 2.2 mmol) and acetic anhydride (acetic anhydride, Ac ₂ O) (3.1 mL, 32.8 mmol) and stir at room temperature for 6 hours. After 6 hours, the mixture was concentrated under reduced pressure, and the concentrate was purified by silica gel column chromatography (hexane: acetone = 1: 1) to obtain Compound 3, which is in the form of a white powder, in 88% yield.

Step 2: 5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4- (ethoxymethoxy) benzoyl) oxy) -chroman-2- I) Preparation of Benzene-1,2,3-Trial Triacetate (Compound 1a)

Compound 3 (0.30 g, 0.38 mmol) was dissolved in acetone (5 mL), then potassium carbonate (K ₂ CO ₃ ) (0.09 g, 0.05 mmol) and potassium iodide (potassium iodide, KI) (0.13 g, 0.76 mmol). Then R ^A X (X is halogen and R ^A is as defined in Formula 1) is added and heated at 50 ^° C. for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, and then filtered through potassium carbonate with filter paper and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (hexane: acetone = 1: 1) to give 5-((2R, 3R) -5,7-diacetoxy-3-((3, 5-Diacetoxy-4- (ethoxymethoxy) benzoyl) oxy) -chroman-2-yl) benzene-1,2,3-trile triacetate was prepared.

Example 1 5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4- (ethoxymethoxy) benzoyl) oxy) -chroman- Preparation of 2-yl) benzene-1,2,3-triyl triacetate (Compound 1a)

¹ H NMR (500 MHz, Acetone-d6) δ 7.52 (s, 2H), 7.41 (s, 2H), 6.74 (d, J = 2.3 Hz, 1H), 6.61 (d, J = 2.3 Hz, 1H), 5.74 (br s, 1H), 5.52 (br s, 1H), 5.17 (d, J = 6.0 Hz, 1H), 5.15 (d, J = 6.0 Hz, 1H), 3.75 (q, J = 7.0 Hz, 2H ), 3.21 (dd, J = 18.0, 4.5 Hz, 1H), 3.08 (dd, J = 18.0, 1.3 Hz, 1H), 2.30 (s, 6H), 2.28 (s, 3H), 2.26 (s, 3H) , 2.25 (s, 3H), 2.24 (s, 6H), 1.17 (t, J = 7.1 Hz, 3H).

¹³ C NMR (100 MHz, Acetone-d6) δ 168.0, 167.6, 167.4, 166.9, 166.0, 163.1, 154.4, 149.7, 149.5, 145.7, 143.5, 143.2, 135.4, 134.2, 124.1, 121.9, 118.5, 109.6, 108.7, 107.2, 97.0, 76.0, 67.8, 64.6, 29.2, 25.1, 19.6, 19.4, 19.3, 19.1, 18.7, 13.9; HRMS (FAB) m / z Found: 811.2091 [M + H] ⁺ . Calcd for C ₃₉ H ₃₉ O ₁₉ : 811.2086 (Yield 15%).

Example 2 5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4-((2-methoxyethoxy) methoxy) -benzoyl Synthesis of oxy) chroman-2-yl) benzene-1,2,3-tritriacetate (1b)

¹ H NMR (400 MHz, Acetone-d6) δ 7.52 (s, 2H), 7.41 (s, 2H), 6.73 (d, J = 2.3 Hz, 1H), 6.60 (d, J = 2.3 Hz, 1H), 5.74 (br s, 1H), 5.53 (s, 1H), 5.19 (d, J = 5.9 Hz, 1H), 5.17 (d, J = 5.9 Hz, 1H), 3.83 (t, J = 4.8 Hz, 2H) , 3.50 (dd, J = 5.0, 3.8 Hz, 2H), 3.31 (s, 3H), 3.21 (dd, J = 18.0, 4.5 Hz, 1H), 3.08 (dd, J = 18.0, 1.3 Hz, 1H), 2.31 (s, 6H), 2.28 (s, 3H), 2.25 (s, 3H), 2.24 (s, 3H), 2.24 (s, 6H).

¹³ C NMR (100 MHz, Acetone-d6) δ 167.9, 167.6, 167.5, 166.9, 166.1, 163.1, 154.4, 149.7, 149.5, 145.7, 143.5, 143.2, 135.4, 134.2, 124.2, 121.9, 118.5, 109.6, 108.7, 107.2, 97.4, 76.0, 70.9, 68.4, 67.8, 57.4, 25.1, 19.6, 19.3, 19.2, 19.1, 18.6; HRMS (FAB) m / z Found: 841.2194 [M + H] ⁺ . Calcd for C ₄₀ H ₄₁ O ₂₀ : 841.2191 (Yield 15%)

Example 3 5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4-((pivaloyloxy) methoxy-benzoyl) oxy) Synthesis of Chroman-2-yl) benzene-1,2,3-tritriacetate (Compound 1c)

¹ H NMR (400 MHz, Acetone-d6) δ 7.56 (s, 2H), 7.42 (s, 2H), 6.74 (d, J = 2.2 Hz, 1H), 6.60 (d, J = 2.2 Hz, 1H), 5.75 (br s, 1H), 5.67 (d, J = 5.7 Hz, 1H), 5.65 (d, J = 5.7 Hz, 1H), 5.53 (s, 1H), 3.22 (dd, J = 18.0, 4.5 Hz, 1H), 3.09 (dd, J = 18.0, 1.2 Hz, 1H), 2.31 (s, 6H), 2.28 (s, 3H), 2.25 (s, 6H), 2.24 (s, 6H), 1.17 (s, 9H );

¹³ C NMR (100 MHz, Acetone-d6) δ 167.9, 167.5, 167.2, 166.9, 166.1, 163.1, 154.4, 149.7, 149.5, 144.4, 144.0, 143.8, 143.2, 135.4, 134.2, 125.4, 121.8, 118.5, 109.6, 108.7, 107.2, 87.2, 76.0, 68.0, 37.9, 25.8, 25.1, 19.6, 19.3, 19.2, 19.1, 18.6; HRMS (FAB) m / z Found: 867.2344 [M + H] ⁺ . Calcd for C ₄₂ H ₄₃ O ₂₀ : 867.2348 (Yield 5%).

Example 4 5-((2R, 3R) -5,7-Diacetoxy-3- (3,5-diacetoxy-4-((isopropoxycarbonyloxy) methoxy) benzoyloxy) Synthesis of Chroman-2-yl) benzene-1,2,3-tritriacetate (Compound 1d)

¹ H NMR (400 MHz, Acetone-d6) δ 7.58 (s, 2H), 7.42 (s, 2H), 6.76 (d, J = 2.2 Hz, 1H), 6.63 (d, J = 2.2 Hz, 1H), 5.76 (br s, 1H), 5.62 (d, J = 6.3 Hz, 1H), 5.60 (d, J = 6.3 Hz, 1H), 5.51 (s, 1H), 4.88 (septet, 6.3 Hz, 1H), 3.22 (dd, J = 17.8, 4.4 Hz, 1H), 3.10 (dd, J = 17.8, 1.0 Hz, 1H), 2.32 (s, 6H), 2.27 (s, 3H), 2.26 (s, 3H), 2.25 ( s, 6H), 2.24 (s, 3H), 1.29 (d, J = 6.2 Hz, 6H).

¹³ C NMR (100 MHz, Acetone-d6) δ 168.0, 167.6, 167.4, 167.0, 166.1, 163.0, 154.4, 153.0, 149.7, 149.5, 145.1, 143.9, 143.2, 135.4, 134.2, 125.7, 121.9, 118.5, 109.6, 108.7, 107.3, 90.5, 76.0, 72.0, 68.0, 29.3, 25.1, 20.5, 19.6, 19.3, 19.2, 18.7; HRMS (FAB) m / z Found: 869.2144 [M + H] ⁺ . Calcd for C ₄₁ H ₄₁ O ₂₁ : 869.2140 (Yield 25%).

Example 5 5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4- (1-((isopropoxycarbonyl) oxy) oxy)- Synthesis of ethoxy) benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate (1e)

¹ H NMR (400 MHz, Acetone-d6) δ 7.55 (d, J = 2.7 Hz, 2H), 7.41 (s, 2H), 6.74 (d, J = 2.1 Hz, 1H), 6.61 (d, J = 2.1 Hz, 1H), 6.17 (q, J = 5.4 Hz, 1H), 5.75 (br s, 1H), 5.52 (s, 1H), 4.71 (septet, J = 6.3 Hz, 1H), 3.21 (dd, J = 18.0, 4.5 Hz, 1H), 3.10 (d, J = 17.9 Hz, 1H), 2.33 (s, 6H), 2.28 (s, 3H), 2.25 (s, 3H), 2.24 (s, 6H), 2.23 ( s, 3H), 1.58 (d, J = 5.2 Hz, 3H), 1.19 (d, J = 6.2 Hz, 6H).

¹³ C NMR (100 MHz, Acetone-d6) δ 167.9, 167.5, 167.2, 166.9, 166.1, 163.1, 154.4, 152.6, 149.7, 149.5, 144.1, 143.2, 135.3, 129.5, 125.6, 121.7, 118.5, 113.3, 109.6, 108.7, 107.2, 98.9, 76.0, 71.6, 68.0, 54.2, 25.1, 20.4, 19.6, 19.3, 19.2, 19.1, 18.6; HRMS (FAB) m / z Found: 883.2300 [M + H] ⁺ . Calcd for C ₄₂ H ₄₃ O ₂₁ : 883.2297 (Yield 30%).

Example 6 5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4- (1-((ethoxycarbonyl) oxy) ethoxy Synthesis of ) -benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate (Compound 1f)

¹ H NMR (400 MHz, Acetone-d6) δ 7.55 (s, 2H), 7.41 (s, 2H), 6.74 (br s, 1H), 6.61 (br s, 1H), 6.18 (quintet, J = 5.5 Hz , 1H), 5.75 (br s, 1H), 5.23 (s, 1H), 4.10-4.06 (m, 2H), 3.21 (dd, J = 18.0, 4.4 Hz, 1H), 3.10 (d, J = 17.8 Hz , 1H), 2.32 (s, 6H), 2.27 (s, 3H), 2.25 (s, 6H), 2.24 (s, 3H), 2.23 (s, 3H), 1.58 (d, J = 5.3 Hz, 3H) , 1.22-1.19 (m, 3 H).

¹³ C NMR (100 MHz, Acetone-d6) δ 167.9. 167.5, 167.2, 166.9, 166.1, 163.0, 154.4, 153.1, 149.7, 149.5, 145.0, 144.1, 143.2, 135.4, 134.2, 125.6, 121.7, 118.5, 109.6, 108.7, 107.2, 99.1, 76.0, 68.0, 63.4, 25.1, 19.6, 19.3, 19.2, 19.1, 18.6, 12.9; HRMS (FAB) m / z Found 869.2144: [M + H] ⁺ . Calcd for C ₄₁ H ₄₁ O ₂₁ : 869.2140 (Yield 10%).

Example 7 5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4-(((tert-butoxycarbonyl) oxy) -meth Synthesis of oxy) benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate (compound 1 g)

¹ H NMR (400 MHz, Acetone-d6) δ7.56 (s, 2H), 7.41 (s, 2H), 6.74 (d, J = 2.1 Hz, 1H), 6.61 (d, J = 2.1 Hz, 1H) , 5.76 (br s, 1H), 5.57 (d, J = 6.4 Hz, 1H), 5.55 (d, J = 6.4 Hz, 1H), 5.52 (br s, 1H), 3.22 (dd, J = 18.0, 4.5 Hz, 1H), 3.09 (d, J = 17.8 Hz, 1H), 2.36 (s, 6H), 2.30 (s, 3H), 2.25 (s, 3H), 2.24 (s, 3H), 2.23 (s, 6H ), 1.47 (s, 9 H).

¹³ C NMR (100 MHz, Acetone-d6) δ 168.0, 167.6, 167.4, 167.0, 166.1, 163.0, 154.4, 151.7, 149.7, 149.5, 145.3, 143.8, 143.2, 135.4, 134.2, 125.6, 121.9, 118.5, 109.6, 108.7, 107.2, 90.2, 82.2, 76.0, 68.0, 26.4, 25.1, 19.6, 19.3, 19.2, 19.1, 18.6; HRMS (FAB) m / z Found: 883.2301 [M + H] ⁺ . Calcd for C ₄₂ H ₄₃ O ₂₁ : 883.2297 (Yield 22%).

Example 8 5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4- (2-(((S) -1-methoxy-) Synthesis of 1-oxopropan-2-yl) amino) -2-oxoethoxy) benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate (Compound 1h)

¹ H NMR (400 MHz, Acetone-d6) δ 7.56 (s, 2H), 7.42 (s, 2H), 6.75 (d, J = 2.2 Hz, 1H), 6.61 (d, J = 2.2 Hz, 1H), 5.74 (br s, 1H), 5.52 (s, 1H), 4.56 (q, J = 7.4 Hz, 1H), 4.51 (d, J = 14.6 Hz, 1H), 4.46 (d, J = 14.6 Hz, 1H) , 3.70 (s, 3H), 3.22 (dd, J = 18.0, 4.5 Hz, 1H), 3.09 (d, J = 16.9 Hz, 1H), 2.34 (s, 6H), 2.28 (s, 3H), 2.25 ( s, 3H), 2.24 (s, 9H), 1.43 (d, J = 7.3 Hz, 3H).

¹³ C NMR (100 MHz, Acetone-d6) δ 172.1, 168.0, 167.8, 167.6, 167.0, 166.4, 166.1, 163.0, 154.4, 149.7, 149.5, 146.1, 143.3, 143.2, 135.4, 134.2, 124.9, 122.0, 118.5, 109.6, 108.7, 107.3, 76.0, 71.0, 68.0, 51.1, 47.1, 25.1, 19.6, 19.4, 19.3, 19.1, 18.6, 16.5; HRMS (FAB) m / z Found: 896.2253 [M + H] ⁺ . Calcd for C ₄₂ H ₄₂ NO ₂₁ : 896.2249 (Yield 19%).

Example 9 (S) -Dimethyl 2- (2- (2,6-diacetoxy-4-((((2R, 3R) -5,7-diacetoxy-2- (3,4, Synthesis of 5-triacetoxyphenyl) chroman-3-yl) oxy) carbonyl) phenoxy) acetamido) pentanedioate (Compound 1i)

¹ H NMR (400 MHz, Acetone-d6) δ 7.58 (s, 2H), 7.44 (s, 2H), 6.76 (d, J = 2.3 Hz, 1H), 6.63 (d, J = 2.2 Hz, 1H), 5.75 (br s, 1H), 5.54 (s, 1H), 4.62-4.57 (m, 1H), 4.58 (d, J = 14.6 Hz, 1H), 4.49 (d, J = 14.6 Hz, 1H), 3.72 ( s, 3H), 3.61 (s, 3H), 3.23 (dd, J = 18.0, 4.5 Hz, 1H), 3.10 (dd, J = 18.0, 1.5 Hz, 1H), 2.46-2.42 (m, 2H), 2.36 (s, 6H), 2.29 (s, 3H), 2.27 (s, 3H), 2.26 (s, 9H), 2.08-2.04 (m, 2H).

¹³ C NMR (100 MHz, Acetone-d6) δ 172.0, 171.2, 168.0, 167.8, 167.6, 167.0, 166.8, 166.1, 163.0, 154.4, 149.7, 149.5, 146.0, 143.3, 143.2, 135.4, 134.2, 124.8, 122.0, 118.5, 109.6, 108.7, 107.3, 76.0, 70.9, 68.0, 51.2, 50.8, 50.4, 29.1, 26.2, 25.1, 19.6, 19.4, 19.3, 19.1, 18.6; HRMS (FAB) m / z Found: 968.2459 [M + H] ⁺ . Calcd for C ₄₅ H ₄₆ NO ₂₃ : 968.2461 (32%).

Example 10 5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4- (2-(((S) -1-methoxy-) 1-oxo-3-phenylpropan-2-yl) amino) -2-oxoethoxy) benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate (Compound 1j) synthesis

¹ H NMR (400 MHz, Acetone-d6) δ 7.57 (s, 2H), 7.46 (s, 2H), 7.31-7.22 (m, 5H), 6.76 (d, J = 2.3 Hz, 1H), 6.63 (d , J = 2.3 Hz, 1H), 5.76 (br s, 1H), 5.54 (s, 1H), 4.74-4.70 (m, 1H), 4.54 (d, J = 14.6 Hz, 1H), 4.45 (d, J = 14.6 Hz, 1H), 3.23 (dd, J = 18.0, 4.5 Hz, 1H), 3.16-3.09 (m, 3H), 2.29 (s, 3H), 2.28 (s, 6H), 2.27 (s, 3H) , 2.26 (s, 3H), 2.25 (s, 6H), 1.42 (s, 9H).

¹³ C NMR (100 MHz, Acetone-d6) δ 169.7, 168.0, 167.7, 167.6, 167.0, 166.2, 166.1, 163.0, 154.4, 149.7, 149.5, 146.0, 143.3, 143.2, 136.3, 135.4, 134.2, 129.0, 127.8, 126.2, 124.8, 122.0, 118.5, 110.0, 108.7, 107.3, 76.0, 70.9, 68.0, 53.1, 37.0, 29.2, 26.7, 19.6, 19.4, 19.3, 19.1, 18.7; HRMS (FAB) m / z Found: 1014.3033 [M + H] ⁺ . Calcd for C ₅₁ H ₅₂ NO ₂₁ : 1014.3032 (Yield 40%).

Table 1 shows the substituents R ^A and the compound names of Examples 1 to 10.

Example R ^A Compound name One

5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4- (ethoxymethoxy) benzoyl) oxy) -chroman-2-yl) benzene-1,2,3-triyl triacetate 2

5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4-((2-methoxyethoxy) methoxy)-benzoyl) oxy) chroman-2-yl) benzene-1, 2,3-triyl triacetate 3

5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4-((pivaloyloxy) methoxy) -benzoyl) oxy) chroman-2-yl) benzene-1,2, 3-triyl triacetate 4

5-((2R, 3R) -5,7-diacetoxy-3- (3,5-diacetoxy-4-((isopropoxycarbonyloxy) methoxy) benzoyloxy) chroman-2-yl) benzene-1,2,3-triyl triacetate 5

5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4- (1-((isopropoxycarbonyl) oxy) -ethoxy) benzoyl) oxy) chroman-2-yl) benzene -1,2,3-triyl triacetate 6

5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4- (1-((ethoxycarbonyl) oxy) ethoxy) -benzoyl) oxy) chroman-2-yl) benzene -1,2,3-triyl triacetate 7

5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4-(((tert-butoxycarbonyl) oxy) -methoxy) benzoyl) oxy) chroman-2-yl) benzene -1,2,3-triyl triacetate 8

5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4- (2-(((S) -1-methoxy-1-oxopropan-2-yl) amino) -2-oxoethoxy) benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate 9

(S) -Dimethyl 2- (2- (2,6-diacetoxy-4-((((2R, 3R) -5,7-diacetoxy-2- (3,4,5-triacetoxyphenyl) chroman-3-yl ) oxy) carbonyl) phenoxy) acetamido) pentanedioate 10

5-((2R, 3R) -5,7-Diacetoxy-3-((3,5-diacetoxy-4- (2-(((S) -1-methoxy-1-oxo-3-phenylpropan-2- yl) amino) -2-oxoethoxy) benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate

Experimental Example 1 Mitochondrial Biogenesis Assay

<Experimental Example 1-1> Flow cytometry (fluorescence-activated cell sorting, FACS)

Hepa1-6 cells (1 × 10 ⁵ cells / well) were seeded in 6-well plates and 24 hours later cells were incubated with control or medium supplemented with EGCG prodrug. After 48 hours, mitochondria and cytoplasm were detected with MitoTracker ™ Deep Red and Calcein green AM, respectively.

Cells were stained for 30 min with 100 nM MitoTracker ™ Deep Red, 250 nM Calcein green AM. After staining, the cells were washed three times with serum free DMEM and the stained cells were harvested. Samples were centrifuged at 5 ° C. (5 min × 3300 × g) and resuspended in PBS. Stained cells were analyzed by flow cytometry (BD Biosciednces).

Some of the EGCG prodrugs according to the invention, in particular Example 3 (Compound 1c), Example 6 (Compound 1f), Example 7 (Compound 1g), Example 9 (Compound 1i), and Example 10 (Compound 1j) ) Promotes mitochondrial biosynthesis, as can be seen in the results of FIG. 1 where fluorescence of cells treated with Mitotracker dye is analyzed by flow cytometry. 1, the intensity of fluorescence emitted by the mitochondria is increased by staining with Mitotracker, which means that mitochondrial biosynthesis is promoted by EGCG prodrugs.

Experimental Example 1-2 MitoTracker Assay

Mitochondria, cytoplasm and nuclei were detected in Hepa1-6 cells by MitoTracker Deep Red FM (Thermo Fisher), ActinGreenTM 488 (Thermo Fisher) and Hoeschst 33342 (Thermo Fisher). Cells were seeded with 96-well clear bottom black plates (1 × 10 ⁴ cells / well) and 24 hours later, cells were incubated with control or medium supplemented with EGCG prodrug. After 48 hours, cells were washed twice with serum free DMEM and stained for 30 minutes with 100 nM Mitotracker Deep Red FM, 2 drops / ml ActinGreen ™ 488 and 50 mM Hoeschst 33342. After staining, cells were washed three times with serum free DMEM. Staining was performed using Cytation 5 imaging Multi-Mode Reader (BioTek instruments Inc., VT, USA; MitoTracker Deep Red FM, CY5 Filter cube excitation 628/640 nm, emission 685/640 nm; ActinGreenTM 488, GFP Filter cube excitation 469/435 nm , Emission 525/535 nm; Hoeschst 33342, DAIP Filter cube, excitation 377/350 nm, emission 447/450 nm).

Experimental Example 1-3 Analysis of Mitochondrial / Nuclear DNA Ratio by qRT-PCR

Hepa1-6 cells (1 × 10 ⁵ cells / well) were seeded in 6-well plates and 24 hours later cells were incubated with control or medium supplemented with EGCG prodrug. Genomic DNA (including both mitochondria and nuclear DNA) was isolated from cells (Geneall Biotechnology Co.) according to the manufacturer's instructions.

The target genes were nuclear cystic fibrosis (CF) and mitochondrial nicotinamide adenine dinucleotide dehydrogenase-5 (ND-5). 10 ng of DNA was used as a template for nuclear DNA quantification, and 0.1 nm of DNA was used as a template for DNA quantification of mitochondria (CF forward primer: 5'-TGT TGT GAA GAC GAG CTG ATG TAA AG-3 ', CF reverse primer: 5'-TGC ATT AAA AGAG GAG CAT GTG TTG-3 ', ND-5 Forward primer: 5'-TGG ATG ATG GTA CGG ACG AA-3', ND-5 reverse primer: 5'-TGC GGT TAT AGA GGA TTG CTT GT-3 ').

qRT-PCR was performed using TOPrealTM qPCR 2X PreMIX SYBR Green with low ROX (enzynomics). PCR began with denaturation at 95 ° C. and enzyme activation for 35 seconds, followed by 45 cycles of denaturation at 95 ° C. for 10 seconds and annealing and elongation at 60 ° C. and 72 ° C. for 35 seconds, respectively. The linearity of the dissociation curves was analyzed using Roter-gene 6 software (Corbett Research). The sample was triple analyzed and the data analysis was based on cycle threshold (CT) measurements.

In order to quantitatively express the degree of mitochondrial biosynthesis activation by quantifying the intensity of mitotracker fluorescence, Hepa1-6 cells treated with EGCG prodrug were stained with Hoechester nuclear stain (blue), cytoplasmic dye ActinGreen 488 (green), and mitochondria. After treatment with the chromosome Mitotracker (red), each fluorescence was measured (see Fig. 2).

By comparing the change of mitochondrial fluorescence with nuclear and cytoplasmic fluorescence before and after EGCG prodrug treatment, the degree of mitochondrial biosynthesis by EGCG prodrug can be quantitatively verified. As shown in FIG. 3, Example 6 (Compound 1f) of EGCG prodrug ) And Example 7 (Compound 1g) can be confirmed that the effect of inducing mitochondrial biosynthesis in particular. EGCG prodrugs Example 6 (Compound 1f) and Example 7 (Compound 1g) increased mitochondrial biosynthesis by 1.5 and 1.4 times, respectively.

Experimental Example 2 Measurement of Protein Expression Related to Mitochondrial Biosynthesis

Experimental Example 2-1 Measurement of PCG-1α Expression

Hepa1-6 cells (1 × 10 ⁵ cells / well) were seeded in 6-well plates and 24 hours later cells were incubated with control or medium supplemented with EGCG prodrug. After 48 hours, cells were washed with cold PBS and scraped off. Centrifuge the sample at 4 ° C (5 min x 1500 x g) and lysis buffer (1 M HEPES, pH 7.9, 400 mM NaCl, 0.1 mM EDTA, 5%) containing protease inhibitors (Thermo Fisher) and PMSF (Thermo Fisher) Glycerol, 1 mM DTT). The sample was centrifuged at 4 ° C. (20 min × 16100 × g) and the protein concentration of the resulting protein lysate was determined by BCA analysis. Total cell lysates (20 μg / well) were separated by SDS-PAGE (Bio-Rad) and transferred to nitrocellulose membrane (Bio-Rad).

The membrane was blocked for 1 hour at 36 ° C. in 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST). The membrane was then incubated with TBST using mouse anti-PGC-1α (Millipore) for 24 hours at 4 ° C, and then horse anti-mouse IgG conjugated to horseradish peroxidase (1: 1000 in TBST, Cell Signaling Technology) for 1 hour. Incubated for

Bands were visualized in the order of enhanced chemiluminescence (ECO-rad), autoradiography, and quantified using a concentration meter (ChemiDocTM Imaging Systems, Bio-rad).

Experimental Example 2-2 AMPK Expression and Phosphorylation

Hepa1-6 cell protein was isolated as in Experimental Example 2-1. AMPK phosphorylation and total AMPK protein in threonine 172 were immunoblotted using the corresponding antibody (Cell Signaling Technology).

Experimental Example 2-3 SIRT1 Expression Measurement

Hepa1-6 cell protein was isolated as in Experimental Example 2-1. SIRT1 protein was immunoblotted using SIRT1 mouse monoclonal antibody (Cell Signaling Technology).

After verifying the effect of promoting mitochondrial biosynthesis by EGCG prodrug in Example 1, the relationship between the factors related to mitochondrial biosynthesis and EGCG prodrug was investigated. In particular, PGC1-α (peroxisome proliferator-activated receptor gamma-coactivator-1alpha) is known as a representative mitochondrial biosynthesis regulator, and AMPK (AMP-activated protein kinase) and SIRT1 (NAD-dependent protein deacetylase sirtuin 1) are PGC- It is known to regulate the function of PGC-1α by catalyzing the phosphorylation and deacetylation reaction of 1α. EGCG prodrugs Example 6 (Compound 1f) and Example 7 (Compound 1g) showed mitochondrial biosynthesis-inducing effects showed an effect of increasing the protein level of PGC-1α, AMPK, SIRT1 (see Figure 4)

Experimental Example 3 Oxidative Phosphorylation Assay

Experimental Example 3-1 NAD / NADH ratio assay

Hepa1-6 cells (1 × 10 ⁵ cells / well) were seeded in 6-well plates and 24 hours later cells were incubated with control or medium supplemented with EGCG prodrug. After 48 hours, cells were washed with cold PBS and scraped off. Samples were centrifuged (5 min x 1500 x g) at 4 ° C. Intracellular NAD / NADH ratio was used NAD / NADH assay kit (Abcam) according to the manufacturer's instructions. Absorbance (OD 450 nm) was measured 1-4 hours after NADH deveploper addition, and NAD total concentration and NADH concentration were calculated (NAD total, NADH concentration = [NAD total, NAD / Sv] * D, Sv = sample bolume added to the reaction well [uL], D = sample dilution factor).

Experimental Example 3-2 Cellular ATP Analysis

Hepa1-6 cells (1 × 10 ⁴ cells / well) were seeded in 96-well plates and 24 hours later cells were incubated with control or medium supplemented with EGCG prodrug for 48 hours. Cellular ATP analysis was performed according to manufacturer's instructions (Abcam).

Experimental Example 3-3 Cytochrome C ELISA Analysis

Hepa1-6 cells (1 × 10 ⁵ cells / well) were seeded in 6-well plates and 24 hours later cells were incubated with control or medium supplemented with EGCG prodrug. After 48 hours, cells were washed with cold PBS and scraped off. Centrifuge the sample at 4 ° C (5 min x 1500 x g) and lysis buffer (1 M HEPES, pH 7.9, 400 mM NaCl, 0.1 mM EDTA, 5%) containing protease inhibitors (Thermo Fisher) and PMSF (Thermo Fisher) Glycerol, 1 mM DTT). Cell lysates were obtained by centrifugation (20 min x 16100 x g) at 4 ° C. Cytochrome C ELISA analysis was performed according to the manufacturer's instructions.

Experimental Example 3-4 Oxygen Consumption Analysis

Hepa1-6 cells were seeded in 96-well clear bottom black plates (1 × 10 ⁴ cells / well) 24 hours later, and the cells were incubated with control or EGCG prodrug for 48 hours. Oxygen consumption analysis was performed according to the manufacturer's instructions (Enzo Life Sciences).

Mitochondria are organs that perform the oxidative phosphorylation process, the final stage of cellular respiration, and include an electron transport system and a chemical osmotic mechanism. Therefore, increased mitochondrial biosynthesis can be expected to promote the oxidative phosphorylation process, in particular the effect of EGCG prodrug on NAD + / NADH ratio, cytochrome c level, ATP synthesis, and oxygen consumption. Indeed, the present invention confirmed that EGCG prodrug Example 6 (Compound 1f) increased the NAD + / NADH ratio, the level of cytochrome c, the synthesis of ATP, and oxygen consumption by 2.2, 1.4, 1.5, and 2.1 times, respectively. (See Figure 5).

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown not in the above description but in particular in the claims, and all differences within the scope will be construed as being included in the present invention.

Claims (14)

A compound represented by formula (1) or a pharmaceutically acceptable salt thereof:
[Formula 1]

(In the above formula 1,
R ^A is

,

or

ego;
R ¹ is H, or C _1-6 straight or branched alkoxy;
R ² is H, or C _1-6 straight or branched alkyl;
R ³ is C _1-6 straight or branched alkyl, or C _1-6 straight or branched alkoxy;
R ⁴ is C _1-6 straight or branched alkyl, C _1-6 straight or branched alkoxycarbonylC _1-6 straight or branched alkyl, or C _6-12 aryl C _1-6 straight or branched alkyl to be). The method of claim 1,
R ^A is

,

or

ego;
R ¹ is H, or C _1-3 straight or branched alkoxy;
R ² is H, or C _1-3 straight or branched alkyl;
R ³ is C _1-4 straight or branched alkyl, or C _1-4 straight or branched alkoxy;
R ⁴ is C _1-3 straight or branched alkyl, C _1-3 straight or branched alkoxycarbonylC _1-3 straight or branched alkyl, or C _{6-8 arylC 1-3} straight or branched alkyl to be. The method of claim 1,
R ^A is

,

or

ego;
R ¹ is H or methoxy;
R ² is H or methyl;
R ³ is t-butyl, t-butoxy, isopropoxy or ethoxy;
R ⁴ is methyl, methoxycarbonylethyl or phenylmethyl. The method of claim 1,
Compound represented by Formula 1 is any one selected from the following compound group or a pharmaceutically acceptable salt thereof:
1) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (ethoxymethoxy) benzoyl) oxy) -chroman-2-yl ) Benzene-1,2,3-triyl triacetate;
2) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4-((2-methoxyethoxy) methoxy) -benzoyl) oxy) Chroman-2-yl) benzene-1,2,3-triyl triacetate;
3) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4-((pivaloyloxy) methoxy-benzoyl) oxy) chromen- 2-yl) benzene-1,2,3-triyl triacetate;
4) 5-((2R, 3R) -5,7-Diacetoxy-3- (3,5-diacetoxy-4-((isopropoxycarbonyloxy) methoxy) benzoyloxy) chroman- 2-yl) benzene-1,2,3-triyl triacetate;
5) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (1-((isopropoxycarbonyl) oxy) -ethoxy) Benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate;
6) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (1-((ethoxycarbonyl) oxy) ethoxy) -benzoyl ) Oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate;
7) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4-(((tert-butoxycarbonyl) oxy) -methoxy) benzoyl ) Oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate;
8) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (2-(((S) -1-methoxy-1-oxo) Propan-2-yl) amino) -2-oxoethoxy) benzoyl) oxy) chroman-2-yl) benzene-1,2,3-tritriacetate;
9) (S) -dimethyl 2- (2- (2,6-diacetoxy-4-((((2R, 3R) -5,7-diacetoxy-2- (3,4,5-tria) Cetoxyphenyl) chroman-3-yl) oxy) carbonyl) phenoxy) acetamido) pentanedioate; And
10) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (2-(((S) -1-methoxy-1-oxo) -3-phenylpropan-2-yl) amino) -2-oxoethoxy) benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate. As shown in Scheme 1 below,
Compound 2 (Epigallocatechin gallate, EGCG) and excess acetic acid were added to the organic solvent and reacted at 10-40 ° C. for 4-8 hours to obtain Compound 3 in which all of the hydroxyl groups of Compound 2 were acetylated ( Step 1); And
Compound 3, potassium carbonate (K 2 CO 3), potassium iodide (KI), and R ^A X (wherein X is halogen and R ^A is as defined in Scheme 1 below) are added to the organic solvent and 2- at 35-65 ° C. Reacting for 6 hours to obtain compound 1 (Step 2);
Method for preparing a compound represented by Formula 1 of claim 1 comprising:

Scheme 1

(In the above Reaction Scheme 1,
R ^A is as defined in Formula 1 of claim 1). The method of claim 5,
The organic solvent of step 1 or step 2 is pyridine, t-butanol, ethanol, tetrahydrofuran (THF), benzene, KOH / MeOH, MeOH, toluene, CH2Cl2, hexane, dimethylformamide (DMF), diisopropyl ether And diethyl ether, dioxane, dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), acetone and chlorobenzene. A composition for promoting mitochondrial biosynthesis comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof:
[Formula 1]

(In the above formula 1,
R ^A is

,

or

ego;
R ¹ is H, or C _1-6 straight or branched alkoxy;
R ² is H, or C _1-6 straight or branched alkyl;
R ³ is C _1-6 straight or branched alkyl, or C _1-6 straight or branched alkoxy;
R ⁴ is C _1-6 straight or branched alkyl, C _1-6 straight or branched alkoxycarbonylC _1-6 straight or branched alkyl, or C _6-12 aryl C _1-6 straight or branched alkyl to be). The method of claim 7, wherein
R ^A is

,

or

ego;
R ¹ is H, or C _1-3 straight or branched alkoxy;
R ² is H, or C _1-3 straight or branched alkyl;
R ³ is C _1-4 straight or branched alkyl, or C _1-4 straight or branched alkoxy;
R ⁴ is C _1-3 straight or branched alkyl, C _1-3 straight or branched alkoxycarbonylC _1-3 straight or branched alkyl, or C _{6-8 arylC 1-3} straight or branched alkyl to be. The method of claim 7, wherein
R ^A is

,

or

ego;
R ¹ is H or methoxy;
R ² is H or methyl;
R ³ is t-butyl, t-butoxy, isopropoxy or ethoxy;
R ⁴ is methyl, methoxycarbonylethyl or phenylmethyl. The method of claim 7, wherein
Compound represented by Formula 1 is any one selected from the following compound group:
1) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (ethoxymethoxy) benzoyl) oxy) -chroman-2-yl ) Benzene-1,2,3-triyl triacetate;
2) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4-((2-methoxyethoxy) methoxy) -benzoyl) oxy) Chroman-2-yl) benzene-1,2,3-triyl triacetate;
3) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4-((pivaloyloxy) methoxy-benzoyl) oxy) chromen- 2-yl) benzene-1,2,3-triyl triacetate;
4) 5-((2R, 3R) -5,7-Diacetoxy-3- (3,5-diacetoxy-4-((isopropoxycarbonyloxy) methoxy) benzoyloxy) chroman- 2-yl) benzene-1,2,3-triyl triacetate;
5) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (1-((isopropoxycarbonyl) oxy) -ethoxy) Benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate;
6) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (1-((ethoxycarbonyl) oxy) ethoxy) -benzoyl ) Oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate;
7) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4-(((tert-butoxycarbonyl) oxy) -methoxy) benzoyl ) Oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate;
8) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (2-(((S) -1-methoxy-1-oxo) Propan-2-yl) amino) -2-oxoethoxy) benzoyl) oxy) chroman-2-yl) benzene-1,2,3-tritriacetate;
9) (S) -dimethyl 2- (2- (2,6-diacetoxy-4-((((2R, 3R) -5,7-diacetoxy-2- (3,4,5-tria) Cetoxyphenyl) chroman-3-yl) oxy) carbonyl) phenoxy) acetamido) pentanedioate; And
10) 5-((2R, 3R) -5,7-diacetoxy-3-((3,5-diacetoxy-4- (2-(((S) -1-methoxy-1-oxo) -3-phenylpropan-2-yl) amino) -2-oxoethoxy) benzoyl) oxy) chroman-2-yl) benzene-1,2,3-triyl triacetate. The method of claim 7, wherein
The composition is characterized in that the induction of AMP-activated protein kinase (AMPK) activation. The method of claim 7, wherein
The composition is characterized in that for increasing the activity of PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) or SIRT1 (NAD-dependent deacetylase sirtuin-1). The method of claim 7, wherein
The composition is characterized in that for increasing the copy number of mitochondrial DNA (copy number). Prevention of a disease selected from the group consisting of degenerative disease, brain disease, neurological disease, heart disease, liver disease, kidney disease, pancreatic disease and muscle disease comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof: Therapeutic Pharmaceutical Compositions:
[Formula 1]

(In the above formula 1,
R ^A is as defined in Formula 1 of claim 1).

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