WO2023191513A1 - Sugar-conjugated imidazoline derivative and use thereof - Google Patents

Abstract

The present invention relates to a composition comprising an imidazoline derivative and a sugar as active ingredients. According to the composition and a micelle according to an aspect, the solubility of the imidazoline derivative can be improved without using additives such as a surfactant, and thus, there is the effect of effectively using a drug in the prevention, amelioration or treatment of diseases. In addition, in the case of using the micelle, there is the effect of ameliorating side effects due to a drug by improving the selectivity of the drug for cells or tissues in need of drug treatment. In particular, by specifically inducing the apoptosis of senescent cells, the micelle can be effectively used in the prevention or treatment of aging-related diseases.

Description

Sugar-linked imidazoline derivatives and uses thereof

It relates to sugar-linked imidazoline derivatives and their uses.

In general, poorly soluble drugs, for example, imidazoline derivative compounds, contain hydrophobic portions in their structure and do not dissolve well in water, which often limits their practicality. These poorly soluble drugs cause severe pain to patients even when administered in a curable amount, and cannot be administered in large amounts due to excessive side effects. In order to use these poorly soluble drugs, additional substances must be added to resolve the poor solubility, but many cases have been reported in which use is restricted due to the toxicity of the added substances.

For example, there is a known method for systemic administration by improving water solubility using a solvent called Cremorphor EL, which is a mixture of polyoxyethylated castor oil and absolute ethanol. , Clinically, it has been reported that excessive administration of these solvents causes side effects such as cardiotoxicity, neurotoxicity, neuropathy, and hypersensitivity (Onetto, N. and Grechko J., Overview of taxol safety, J. Natl. Cancer Inst Monogr., 1993, 15: 131; Mazzo, D. and Denis, P., Compatibility of docetaxel and paclitaxel in intravenous solutions with polyvinyl chloride infusion materials, Am. J. Health Sys. Pharm., 1997, 54: 566; Gregory R. and Delisa, A.F., Paclitaxel: a new antitineoplastic agent for refractory ovarian cander, Clin. Pharm., 1993, 12: 401). In addition, methods such as emulsion, microemulsion, liposome, micelle, or PEGylation have been used, but have the disadvantage of requiring the use of additives such as surfactants.

Accordingly, in order to solve the above problem, the present inventors dramatically improved the solubility by combining an imidazoline derivative compound and a sugar, so that it can be effectively used to remove senescent cells without the use of additional additives, and can be used to remove nanocrystals in an aqueous solution without the use of surfactants. It was confirmed that particle micelles could be produced, and the present invention was completed.

One aspect is to provide a composition comprising a compound represented by Formula 1:

[Formula 1]

.

Another aspect is to provide micelles containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

Another aspect is to provide a pharmaceutical composition for preventing or treating aging-related diseases, which includes the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Another aspect is to provide a health functional food for preventing or improving aging-related diseases containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Another aspect is to provide a method for preventing or treating aging-related diseases, comprising administering an effective amount of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof.

Another aspect is to provide a method for improving aging comprising administering an effective amount of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof.

Another aspect is to provide a use of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof for use in the manufacture of a pharmaceutical agent for preventing or treating age-related diseases.

Another aspect is to provide a use of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof for use in health functional foods for preventing or treating age-related diseases.

One aspect provides a composition comprising a compound represented by Formula 1:

[Formula 1]

.

In Formula 1, R ₁ to R ₂ are each independently H, substituted or unsubstituted alkyl of C ₁ to C ₁₀ , substituted or unsubstituted alkenyl of C ₂ to C ₁₀ , or substituted C ₂ to C ₁₀ or unsubstituted alkynyl, substituted or unsubstituted alkoxy of C ₁ to C ₁₀ , or substituted or unsubstituted aryl; R ₃ is sugar or a derivative thereof; R ₄ is substituted or unsubstituted alkylene of C ₁ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ , substituted or unsubstituted alkynylene of C ₂ to C ₁₀ , or substituted or unsubstituted alkynylene. is an arylene; and X ₁ and X ₂ are each independently halogen.

As used herein, the term “substituted” refers to one or more substituents, such as hydroxy group, alkyl group, alkoxy group, mercapto group, cycloalkyl group, substituted cycloalkyl group, heterocyclic group, substituted heterocyclic group, aryl. group, substituted aryl group, heteroaryl group, substituted heteroaryl group, aryloxy group, substituted aryloxy group, halogen group, trifluoromethyl group, cyano group, nitro group, oxo group, amino group, amido group, aldehyde It means substituted with a group, acyl group, oxyacyl group, carboxyl group, carbamate group, sulfonyl group, sulfonamide, sulfuryl group, etc.

As used herein, the term “alkyl” refers to a saturated aliphatic hydrocarbon group containing 1 to 10 carbon atoms, such as 1 to 8, 1 to 6, or 1 to 4 carbon atoms, and the alkyl group is straight chain or Or it may be branched. Additionally, the alkyl includes cycloalkyl, heteroalkyl, and heterocycloalkyl. Examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-heptyl, or 2-ethylhexyl.

The alkyl may have one or more substituents, such as halo, phospho, cycloaliphatic (e.g., cycloalkyl or cycloalkenyl), heterocycloaliphatic (e.g., heterocycloalkyl or heterocycloalkenyl), aryl. , heteroaryl, alkoxy, aroyl, heteroaroyl, acyl (e.g., (aliphatic)carbonyl, (cycloaliphatic)carbonyl, or (heterocycloaliphatic)carbonyl), nitro, cyano, amido ( For example, (cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino, hetero aralkylcarbonylamino alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, arylaminocarbonyl, or heteroarylaminocarbonyl), amino (e.g., aliphatic amino, cycloaliphatic amino, or heterocycloaliphatic amino), sulfonyl (e.g., aliphatic-SO ₂ -), sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl, cycloaliphaticoxy. , heterocycloaliphaticoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroarylalkoxy, alkoxycarbonyl, alkylcarbonyloxy, or hydroxy. Substituted alkyl may be carboxyalkyl (e.g. HOOC-alkyl, alkoxycarbonylalkyl or alkylcarbonyloxyalkyl), cyanoalkyl, hydroxyalkyl, alkoxyalkyl, asylalkyl, aralkyl, (alkoxyaryl)alkyl, (sulfonylamino)alkyl (such as (alkyl-SO ₂ -amino)alkyl), aminoalkyl, amidoalkyl, (cycloaliphatic)alkyl, haloalkyl, amine cation, quaternary ammonium or quaternary phosphonium.

As used herein, the term “heteroalkyl” refers to alkyl consisting of a carbon atom and one or more heteroatoms selected from the group consisting of O, N, and S. The nitrogen and sulfur atoms can optionally be oxidized, and the heteroatoms O, N and/or S can be placed at any internal position of the heteroalkyl group.

As used herein, the term “alkenyl” refers to an aliphatic carbon group containing 2 to 10, for example, 2 to 8, 2 to 6, or 2 to 4 carbon atoms and one or more double bonds. Similar to alkyl groups, alkenyl groups can be straight chain or branched. Additionally, the alkenyl includes cycloalkenyl, heteroalkenyl, and cycloheteroalkenyl. The alkenyl may be, for example, allyl, isoprenyl, 2-butenyl, or 2-hexenyl, but is not limited thereto.

The alkenyl may have one or more substituents, such as halo, phospho, cycloaliphatic (e.g., cycloalkyl or cycloalkenyl), heterocycloaliphatic (e.g., heterocycloalkyl or heterocycloalkenyl), Aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, acyl (e.g., (aliphatic)carbonyl, (cycloaliphatic)carbonyl, or (heterocycloaliphatic)carbonyl), nitro, cyano, amido (For example, (cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino, Heteroaralkylcarbonylamino alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, arylaminocarbonyl, or heteroarylaminocarbonyl), amino (e.g., aliphatic amino, cycloaliphatic amino, heterocycloaliphatic amino, or aliphatic sulfonylamino), sulfonyl (e.g., alkyl-SO ₂ -, cycloaliphatic-SO ₂ -, or aryl-SO ₂ -), sulfinyl, sulfanyl, sulfoxy, urea , thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl, cycloaliphaticoxy, heterocycloaliphaticoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkoxy, alkoxycarbonyl, alkylcarbonyloxy, Or it may be substituted with hydroxy. Substituted alkenyls include cyanoalkenyl, alkoxyalkenyl, acylalkenyl, hydroxyalkenyl, aralkenyl, (alkoxyaryl)alkenyl, (sulfonylamino)alkenyl (e.g., (alkyl-SO ₂ -amino) alkenyl), aminoalkenyl, amidoalkenyl, (cycloaliphatic)alkenyl, or haloalkenyl.

As used herein, the term “heteroalkenyl” refers to alkenyl consisting of a carbon atom and one or more heteroatoms selected from the group consisting of O, N, and S. The nitrogen and sulfur atoms can optionally be oxidized, and the heteroatoms O, N and/or S can be placed at any internal position of the heteroalkenyl group.

As used herein, the term “alkynyl” refers to an aliphatic carbon group containing 2 to 10 carbon atoms, such as 2 to 8, 2 to 6, or 2 to 4 carbon atoms, and having at least one triple bond; , the alkynyl group may be straight chain or branched. The alkynyl includes cycloalkynyl, heteroalkynyl, and cycloheteroalkynyl. For example, the alkynyl group can be propargyl or butynyl.

Alkynyl may have one or more substituents, such as aroyl, heteroaroyl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, nitro, carboxy, cyano, halo, hydride. Roxy, sulfo, mercapto, sulfanyl (e.g. aliphatic sulfanyl or cycloaliphatic sulfinyl), sulfinyl (e.g. aliphatic sulfinyl or cycloaliphatic sulfinyl), sulfonyl (e.g. aliphatic sulfinyl) SO ₂ -, aliphatic amino-SO ₂ -, or cycloaliphatic-SO ₂ -), amido (e.g. aminocarbonyl, alkylaminocarbonyl, alkylcarbonylamino, cycloalkylaminocarbonyl, heterocycloalkyl Aminocarbonyl, cycloalkylcarbonylamino, arylaminocarbonyl, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (cycloalkylalkyl)carbonylamino, heteroaralkylcarbonylamino , heteroarylcarbonylamino or heteroarylaminocarbonyl), urea, thiourea, sulfamoyl, sulfamide, alkoxycarbonyl, alkylcarbonyloxy, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, acyl (e.g. For example, (cycloaliphatic)carbonyl or (heterocycloaliphatic)carbonyl), amino (e.g. aliphatic amino), sulfoxy, oxo, carboxy, carbamoyl, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy. , or (heteroaryl)alkoxy.

As used herein, the term “heteroalkynyl” refers to alkynyl consisting of a carbon atom and one or more heteroatoms selected from the group consisting of O, N, and S. The nitrogen and sulfur atoms can optionally be oxidized, and the heteroatoms O, N and/or S can be placed at any internal position of the heteroalkynyl group.

As used herein, the term “alkoxy” refers to a group in which the hydrogen atom of hydroxyl is replaced with the alkyl, alkenyl, or alkynyl. The alkoxy may be a C ₁ to C ₁₀ alkoxy such as methoxy, ethoxy, propoxy, butoxy, and pentoxy.

As used herein, the term “aryl” refers to an aromatic ring system containing 6 to 16 ring atoms, and includes heteroaryl. The aryl group may be a monocyclic, bicyclic or tricyclic group, or may be connected by a bond to form a biaryl group. Typically, the aryl group may be phenyl, naphthyl, or biphenyl. In one embodiment, the aryl group may include an alkylene linkage to form an arylalkyl group, such as a benzyl group. The aryl group may have 6 to 12 ring members, such as phenyl, naphthyl, or biphenyl, and the aryl may be substituted or unsubstituted.

As used herein, the term “heteroaryl” refers to a monocyclic, fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where 1 to 5 of the atoms constituting the ring are Heteroatoms may be for example N, O or S. The heteroatoms may be oxidized and examples include, but are not limited to, N-oxide, -S(O)-, and -S(O) ₂ -. Heteroaryl groups can have any number of ring atoms, for example 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, It may contain 3 to 11, or 3 to 12 ring members. According to one embodiment, the heteroaryl group has 5 to 8 ring members and 1 to 4 heteroatoms, or 5 to 8 ring members and 1 to 3 heteroatoms, or 5 to 6 ring members and 1 to 4 heteroatoms. It may have heteroatoms, or 5 to 6 ring members and 1 to 3 heteroatoms. The heteroaryl group is pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4-, and 1,3, 5-isomer), thiophene, furan, thiazole, isothiazole, oxazole and isoxazole. The heteroaryl group can also be fused to an aromatic ring system, such as a phenyl ring, to form benzopyrroles such as indole and isoindole, benzopyridines such as quinoline and isoquinoline, benzopyrazine, benzopyrimidine, benzopyridazine such as phthalazine and cinine. Including, but not limited to, noline, benzothiophene, and benzofuran. Additionally, the heteroaryl group may include a heteroaryl ring linked by a bond, for example, bipyridine, and the heteroaryl group may be substituted or unsubstituted.

The heteroaryl group may be connected through any position on the ring. For example, pyrrole includes 1-, 2-, and 3-pyrrole; Pyridines include 2-, 3-, and 4-pyridines; Imidazoles include 1-, 2-, 4-, and 5-imidazole; Pyrazoles include 1-, 3-, 4- and 5-pyrazoles; Triazoles include 1-, 4-, and 5-triazoles; Tetrazoles include 1- and 5-tetrazole; Pyrimidines include 2-, 4-, 5-, and 6-pyrimidines; Pyridazines include 3- and 4-pyridazine; 1,2,3-triazines include 4- and 5-triazines; 1,2,4-triazines include 3-, 5- and 6-triazines; 1,3,5-triazine includes 2-triazine; Thiophenes include 2- and 3-thiophene; Furans include 2- and 3-furans; Thiazoles include 2-, 4-, and 5-thiazoles; Isothiazoles include 3-, 4-, and 5-isothiazoles; Oxazoles include 2-, 4- and 5-oxazole; Isoxazole includes 3-, 4- and 5-isoxazole; Indoles include 1-, 2-, and 3-indoles; Isoindoles include 1- and 2-isoindoles; Quinolines include 2-, 3-, and 4-quinolines; Isoquinolines include 1-, 3-, and 4-isoquinolines; Quinazolines include 2- and 4-quinazolines; Cinnolines include 3- and 4-cinnolines; Benzothiophenes include 2- and 3-benzothiophenes; Benzofurans may include 2- and 3-benzofurans.

As used herein, the term “sugar” refers to a common compound/composition (CH ₂ O) _n or a derivative thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. .

According to one embodiment, the sugar may be a monosaccharide or a disaccharide.

The monosaccharide may be triose, tetrose, pentose, hexose, heptose, octose, or nonose.

According to one embodiment, the monosaccharides include galactose, glucose, fructose, mannose, allose, altrose, gulose, and talose ( talose, psicose, sorbose, tagatose, idose, rhamnose, xylose, arabinose, fucose , glyceraldehyde, erythrose, threose, ribose, lyxose, dihydroxyacetone, erythrulose and ribulose. ) may be any one or more selected from the group consisting of

The disaccharide may be sucrose, lactulose, lactose, maltose, trehalose, or cellobiose.

As used herein, the term “sugar derivative” refers to a sugar in which one or more hydroxyl groups of the sugar have been modified with different substituents. For example, the sugar derivative may be one in which the hydroxyl group of the sugar is replaced with an alkoxy group.

In one embodiment, the sugar derivative may be a compound represented by the following formula (2):

[Formula 2]

.

In Formula 2, R ₄ is substituted or unsubstituted alkylene of C ₁ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ It may be substituted alkynylene, or substituted or unsubstituted arylene.

In one embodiment, the sugar derivative may be a compound represented by the following formula (3):

[Formula 3]

.

In Formula 3, R ₄ is substituted or unsubstituted alkylene of C ₁ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ It may be substituted alkynylene, or substituted or unsubstituted arylene.

In one embodiment, the sugar derivative may be a compound represented by the following formula (4):

[Formula 4]

.

In Formula 4, R ₄ is substituted or unsubstituted alkylene of C ₁ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ It may be substituted alkynylene, or substituted or unsubstituted arylene.

In one embodiment, the sugar derivative may be a compound represented by the following formula (5):

[Formula 5]

.

In Formula 5, R ₄ is substituted or unsubstituted alkylene of C ₁ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ It may be substituted alkynylene, or substituted or unsubstituted arylene.

In one embodiment, the sugar derivative may be a compound represented by the following formula (6):

[Formula 6]

.

In Formula 6, R ₄ is substituted or unsubstituted alkylene of C ₁ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ It may be substituted alkynylene, or substituted or unsubstituted arylene.

As used herein, the term “alkylene” refers to a divalent radical derived from an alkane, and may be, for example, -CH ₂ CH ₂ CH ₂ CH ₂ -. The alkylene includes cycloalkylene, heteroalkylene, and heterocycloalkylene.

In one embodiment, the heteroalkylene or heterocycloalkylene may include a carbon atom and one or more heteroatoms selected from the group consisting of O, N, and S.

As used herein, the term “alkenylene” refers to a divalent alkene having at least one carbon-carbon double bond, for example, -CH=CH-CH ₂ -. The alkenylene includes cycloalkenylene, heteroalkenylene, and heterocycloylkylene.

According to one embodiment, the heteroalkenylene or heterocycloalkenylene may include a carbon atom and one or more heteroatoms selected from the group consisting of O, N, and S.

As used herein, the term “alkynylene” refers to a functional group having at least one carbon-carbon triple bond, for example, -CH ₂ -C≡C-CH ₂ -CH ₂ -. The alkynylene includes cycloalkynylene, heteroalkynylene, and heterocycloalkynylene.

According to one embodiment, the heteroalkynylene or heterocycloalkynylene may include a carbon atom and one or more heteroatoms selected from the group consisting of O, N, and S.

As used herein, the term “arylene” generally refers to a divalent aromatic group having 6 to 14 carbon atoms. The arylene includes heteroarylene.

According to one embodiment, the heteroalkynylene or heterocycloalkynylene may include a carbon atom and one or more heteroatoms selected from the group consisting of O, N, and S.

More specifically, R ₄ is C ₁ -C ₆ alkyl; C ₁ -C ₂ alkoxy; -C=CHCOO-; -CON(CH ₂ -) ₂ -; -N-(C ₁ -C ₆ alkyl) ₂ -; -NHCH ₂ CH ₂ R ₅ -; -N(CH ₂ CH ₂ OH)CH ₂ CH ₂ O-; -N(CH ₃ )CH ₂ CH ₂ NCH ₂ - ; -N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )CH ₂ -; saturated 4-, 5- and 6-membered rings; and C ₁ -C ₆ alkyl, -C=OR ₆ -, C ₁ -C ₆ alkyl which may be substituted by hydroxy, C ₁ -C ₆ alkyl which may be substituted by -NH ₂ , C ₁ -C ₆ alkyl. from 5- and 6-membered saturated rings containing one or more heteroatoms selected from amino, -SO ₂ CH ₂ -, -O-, -CH ₂ C=OCH ₂ -, and S, N and O, substituted by is selected from saturated and unsaturated 5- and 6-membered rings containing one or more heteroatoms selected from S, N and O, and wherein R ₆ is H, hydroxy, C ₁ -C ₆ alkyl, -NH ₂ , amino substituted with C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl which may be substituted with hydroxy, and C ₁ -C ₆ alkyl which may be substituted with NH ₂ , R ₅ may be selected from -N(CH ₃ )CH ₃ , -NHCH ₂ CH ₂ NH ₂ , -NH ₂ , morpholinyl and piperazinyl.

R ₄ is morpholinyl, piperazinyl, piperidinyl, cyclopentyl, cyclohexyl, thiophenyl, isoxazolyl, and furanyl, C ₁ -C ₃ alkyl, C ₁ -C ₂ alkoxy, -C= OCH ₂ -, -SO ₂ CH ₂ -, -C=O-, oxo, -O-, -CH ₂ NH-, -C=OCH ₂ NH-, -C=OCH ₂ O-, -C=OC( OH)CH ₂ O-, -CH ₂ C(OH)-CH ₂ O-, -C=ON(CH ₂ -)CH ₂ -, -C=ONH- and -C=ON(CH ₃ )CH ₂ - Piperazinyl substituted with one or more groups selected from, -C=OC(CH3) ₂ - -CH2COCH ₃ -, -CH ₂ CHCH ₃ O-, -CH(CH ₃ )CH(OH)CH ₂ - and -CH ₂ CH ₂ O-.

As used herein, the term “halogen” means fluorine, chlorine, bromine, or iodine.

As used herein, the term “pharmaceutically acceptable” means that it is physiologically acceptable and does not usually cause gastrointestinal upset, allergic reactions such as dizziness, or similar reactions when administered to humans.

As used herein, the term “pharmaceutically acceptable salt” refers to a salt according to one aspect of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Salts of the parent compound can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Generally, these salts are prepared by reacting the free acid form of these compounds with a stoichiometric amount of an appropriate base, such as sodium, calcium, magnesium, or potassium, or by reacting the free base form of these compounds with a stoichiometric amount of an appropriate acid. It can be manufactured by: These reactions are typically carried out in water or in organic solvents or mixtures of the two. Generally, where practicable, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile may be used. The pharmaceutically acceptable salt includes both addition salts of acids or bases and their stereochemical isomeric forms, and may be, for example, addition salts of organic acids or inorganic acids. The salt includes any salt that maintains the activity of the parent compound in the subject of administration and does not cause undesirable effects, and is not particularly limited.

These salts include inorganic and organic salts, such as acetic acid, nitric acid, aspartic acid, sulfonic acid, sulfuric acid, maleic acid, glutamic acid, formic acid, succinic acid, phosphoric acid, phthalic acid, tannic acid, tartaric acid, and hydrobromic acid. , propionic acid, benzenesulfonic acid, benzoic acid, stearic acid, lactic acid, bicarboxylic acid, bisulfuric acid, bitartaric acid, oxalic acid, butyric acid, calcium idete, carbonic acid, chlorobenzoic acid, citric acid, idetic acid, toluene. Sulfonic acid, fumaric acid, gluceptic acid, esilinic acid, pamoic acid, gluconic acid, methylnitric acid, malonic acid, hydrochloric acid, hydroiodoic acid, hydroxynaphtholic acid, isethionic acid, lactobionic acid, bay. Delicic acid, mucinic acid, naphsilic acid, muconic acid, p-nitromethanesulfonic acid, hexamic acid, pantothenic acid, monohydrogenophosphate, dihydrogenophosphate, salicylic acid, sulfamic acid, sulfanilinic acid, and methanesulfonic acid. there is.

In addition, the salts include salts of alkali and alkaline earth metals such as ammonium salts, lithium salts, sodium salts, potassium salts, magnesium salts and calcium salts, for example, benzathine, N-methyl-D-glucamine, hydrochloric acid salts, etc. It includes salts with organic bases such as labamine salts, and salts with amino acids such as arginine and lysine. Additionally, the salt form may be converted to the free form by treatment with a suitable base or acid.

The imidazoline derivative compound represented by Formula 1 may be a compound selected from the group consisting of Formulas 7 to 10 below, or a pharmaceutically acceptable salt thereof.

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

The compound represented by Formula 1 may be an imidazoline derivative or a pharmaceutically acceptable salt thereof; And it may be obtained by combining sugar or a derivative thereof.

The imidazoline derivative may be one selected from the group consisting of Nutlin-1, Nutlin-2, or Nutlin-3, and the Nutlin-3 may be Nutlin-3a or Nutlin-3b, but is not limited thereto. .

The bond may be an imidazoline derivative or a pharmaceutically acceptable salt thereof; And the sugar or its derivative can be combined by reacting in the presence of a base and a solvent for 5 to 20 hours, for example, 10 to 20 hours, 15 to 20 hours, 15 to 19 hours, or 15 to 18 hours. The reaction can be performed at room temperature, and those skilled in the art can appropriately change the reaction time depending on the temperature.

The base is NaH, lithium diisopropylamide (LDA), 4-dimethylaminopyridine (DMAP), triethylamine (TEA), pyridine, ammonia, methylamine, ethylamine, propyl Amine, isopropylamine, dimethylamine, diethylamine, dipropylamine, diisopropylamine, trimethylamine, tripropylamine, triisopropylamine, aniline, methylaniline, dimethylaniline, pyridine, azazulolidine, benzyl It may be an amine, methylbenzylamine, dimethylbenzylamine, 2,6-lutidine, morpholine, piperidine, piperazine, proton-sponge, ammonium hydroxide, triethanolamine, ethanolamine, or trismile.

The solvent is dimethylacetamide (DMAc), dichloromethane (DCM), tetrahydrofuran (THF), dimethylformamide (DMF), acetonitrile (ACN), DMAP, water, Acetic acid, acetone, dioxane, benzene, 1-butanol, 2-butanol, tert-butyl alcohol, carbon tetrachloride, chloroform, cyclohexane, hexane, diethyl ether, dimethyl sulfoxide (DMSO), ethanol, ethyl acetate, It may be ethylene glycol, glycerin, heptane, pentane, pyridine, toluene, hydrochloric acid, and triethyl amine.

Another aspect provides micelles containing a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof:

[Formula 1]

.

In Formula 1, R ₁ to R ₂ are each independently H, substituted or unsubstituted alkyl of C ₁ to C ₁₀ , substituted or unsubstituted alkenyl of C ₂ to C ₁₀ , or substituted C ₂ to C ₁₀ or unsubstituted alkynyl, substituted or unsubstituted alkoxy of C ₁ to C ₁₀ , or substituted or unsubstituted aryl; R ₃ is sugar or a derivative thereof; R ₄ is substituted or unsubstituted alkylene of C ₁ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ , substituted or unsubstituted alkynylene of C ₂ to C ₁₀ , or substituted or unsubstituted alkynylene. is an arylene; and X ₁ and X ₂ are each independently halogen.

The compound represented by Formula 1 is as described above.

As used herein, the term “micelle” refers to an aggregate of amphiphilic molecules having an inner core and an outer surface in an aqueous medium, wherein most of the amphiphilic molecules have their hydrophobic portion forming the core and the hydrophilic portion forming the core. It is oriented to form an outer surface. The micelle according to the present invention is oriented so that the drug forms the hydrophobic part to form the core, and the hydrophilic peptide forms the hydrophilic part to form the outer surface.

According to one embodiment, the micelles can be prepared in an aqueous solution without using a surfactant. The micelles are prepared by dissolving the compound represented by Formula 1 in an organic solvent such as fluorinated alcohol, dimethylformamide, dimethyl sulfoxide, chlorinated hydrocarbon, hydrocarbon or alkyl alcohol, and then dissolving the compound represented by Formula 1 at 10°C to 30°C, for example 15°C. The micelles may be prepared by stirring at ℃ to 30℃, 15℃ to 25℃, or room temperature, and then performing artificial dialysis. The artificial dialysis may be performed for 1 to 5 days, for example, 1 to 4 days or 2 to 3 days.

The size of the micelles may be 50 nm to 200 nm. For example, it may be 50 nm to 200 nm, 50 nm to 150 nm, or 50 nm to 100 nm.

According to one embodiment, the zeta potential of the micelle may be -10 mv to -40 mv. For example, it may be -10 mv to -30 mv or -20 mv to -30 mv.

Another aspect provides a pharmaceutical composition for preventing or treating aging-related diseases, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof:

[Formula 1]

.

In Formula 1, R ₁ to R ₂ are each independently H, substituted or unsubstituted alkyl of C ₁ to C ₁₀ , substituted or unsubstituted alkenyl of C ₂ to C ₁₀ , or substituted C ₂ to C ₁₀ or unsubstituted alkynyl, substituted or unsubstituted alkoxy of C ₁ to C ₁₀ , or substituted or unsubstituted aryl; R ₃ is sugar or a derivative thereof; R ₄ is substituted or unsubstituted alkylene of C ₁ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ , substituted or unsubstituted alkynylene of C ₂ to C ₁₀ , or substituted or unsubstituted alkynylene. is an arylene; and X ₁ and X ₂ are each independently halogen.

The compound represented by Formula 1 is as described above.

In one embodiment, the compound of the composition may selectively induce apoptosis in mitochondria within senescent cells by overexpressing p53 and p21 compared to normal cells.

Apoptosis is a form of cell death controlled by genes, and is distinguished from necrosis, which is cell death caused by stimuli such as burns, bruises, or poisons, as necrosis or pathological death of cells. This refers to the death of cells, which begins with cells shrinking, then gaps are created between adjacent cells, and within the cells, DNA is regularly cut and fragmented.

The age-related diseases include cancer, cardiovascular disease or disorder in the elderly, inflammatory or autoimmune disease or disorder in the elderly, neurodegenerative disease in the elderly, metabolic disease in the elderly, pulmonary disease in the elderly, eye disease or disorder in the elderly, age-related disorders in the elderly, and dermatology in the elderly. It may be one or more selected from the group consisting of adverse diseases or disorders.

According to one embodiment, the cancer may be hematological cancer or solid cancer.

The blood cancer is selected from the group consisting of Acute Myeloid Leukemia, Acute Lymphoblastic Leukemia, Chronic Myelogenous Leukemia, Multiple Myeloma, and Lymphoma. However, it is not limited to this.

The solid cancers include breast cancer, colon cancer, head and neck cancer, lung cancer, stomach cancer, skin cancer, colon cancer, prostate cancer, bladder cancer, kidney cancer, rectal cancer, thyroid cancer, liver cancer, cervical cancer, skin cancer, rectal cancer, anal cancer, urethral cancer, ovarian cancer, and esophageal cancer. and pancreatic cancer, but is not limited thereto.

According to one embodiment, the elderly cardiovascular disease or disorder is atherosclerosis, angina pectoris, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary thrombosis, myocardial infarction, hypertension, aortic aneurysm, heart failure. It may be dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, cardiac load resistance, cardiac fibrosis, cerebral aneurysm, and stroke.

According to one embodiment, the geriatric inflammatory or autoimmune disease or disorder may be osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel disease, kyphosis, and intervertebral disc herniation.

According to one embodiment, the geriatric neurodegenerative disease may be Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, mild cognitive impairment, and motor neuron dysfunction.

According to one embodiment, the metabolic disease in the elderly may be diabetes, diabetic ulcer, metabolic syndrome, and obesity.

According to one embodiment, the elderly lung disease may be pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis, and age-related loss of lung function.

According to one embodiment, the elderly eye disease may be macular degeneration, glaucoma, cataract, presbyopia, and vision impairment.

In this specification, the term “macula” is the name of the central part of the retina, which is responsible for central vision in contrast to peripheral vision. “Macular degeneration” includes age-related macular degeneration, diabetic macular edema, cystic macular edema, macular hole, and macular telangiectasia.

As used herein, the term “age-related macular degeneration (AMD)” refers to progressive or multifactorial degeneration or atrophy of the retina, retinal pigment epithelium, choriocapillaris, etc. in adults in their 50s or older. It is a disease in which photoreceptor cells in the macular area are lost with aging, resulting in visual impairment.

The macular degeneration includes dry macular degeneration and wet macular degeneration. Dry age-related macular degeneration, which accounts for the majority of age-related macular degeneration, refers to lesions such as drusen, pigmentary abnormality, or atrophy of the retinal epithelium in the retina. It does not usually cause severe vision loss, but it can develop into a wet form. Wet age-related macular degeneration is a condition in which choroidal new blood vessels grow under the retina, causing exudate and hemorrhage in the macula, affecting central vision and, in severe cases, blindness.

According to one embodiment, the age-related disorder may be kidney disease, renal failure, weakness, hearing loss, muscle fatigue, skin condition, skin wound healing, liver fibrosis, pancreatic fibrosis, oral submucosal fibrosis, and sarcopenia.

According to one embodiment, the dermatological diseases include eczema, psoriasis, hyperpigmentation, nevus, rash, atopic dermatitis, urticaria, diseases and disorders related to photosensitivity or photoaging, wrinkles; pruritus; sensory impairment; eczematous rash; Eosinophilic dermatosis; reactive neutrophilic dermatosis; pemphigus; pemphigoid; immunobullous dermatosis; Fibrohistiocytic proliferation of the skin; Cutaneous lymphoma; and cutaneous lupus.

As used herein, the term “prevention” refers to partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or its secondary symptoms, preventing the acquisition or re-acquisition of a disease or disorder, or preventing the development or recurrence of a disease or disorder. A general term for reducing the risk of acquisition. The prevention refers to any action that suppresses or delays the occurrence of inflammation or inflammation-related diseases, disorders, or symptoms by administering the composition according to the present invention.

As used herein, the term “treatment” refers to any action that improves or beneficially changes a disease, disorder, or its accompanying symptoms.

As used herein, the term “therapeutic agent” or “pharmaceutical composition” refers to a molecule or compound that imparts some beneficial effect upon administration to a subject. Beneficial effects include enabling diagnostic decisions; Improvement of a disease, symptom, disorder or condition; Reducing or preventing the onset of a disease, symptom, disorder or condition; and generally includes responding to a disease, symptom, disorder or condition.

In one embodiment, the composition may further include antioxidants, and the antioxidants include nicotinamide, anthocyanin, benzenediol abiethane diterpene, carnosine, carotenoid, xanthophyll, and carotenoids in saffron, cur Cuminoid, cyclopentenone prostaglandin, flavonoid, prenylflavonoid, retinoid, stilbenoid, uric acid, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B9, vitamin B12, vitamin C, vitamin E, selenium , zinc, inhibitors and scavengers of lipid peroxidation and its by-products, tirilazade, and analogs, derivatives, salts, and combinations thereof.

In addition, the composition may additionally contain a pharmaceutically effective amount of a bioactive ingredient or may contain one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutically effective amount refers to an amount sufficient to exhibit the desired physiological or pharmacological activity when the bioactive ingredient is administered to animals or humans. The pharmaceutically effective amount may vary appropriately depending on the age, weight, health status, gender, administration route, and treatment period of the administration subject.

The “biologically active ingredient” is a substance that can induce a desired biological or pharmacological effect by promoting or inhibiting physiological functions in the body of animals or humans, and is a chemical or biological substance or compound suitable for administration to animals or humans. It means (1) has a preventive effect on organic matter by preventing unwanted biological effects, such as preventing infection, and (2) alleviates conditions caused by disease, for example, alleviating pain or infection as a result of disease. , (3) can play a role in alleviating, reducing or completely eliminating disease from organic matter. Additionally, “the bioactive ingredient” may be used interchangeably with the term “therapeutic agent.”

The bioactive ingredient may be selected from the group consisting of proteins, anticancer agents, anti-inflammatory painkillers, antibiotics, antibacterial agents, hormones, genes, and vaccines, but is not limited thereto.

The pharmaceutical composition may be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants by methods known in the art, and such formulations may be oral or parenteral administration formulations. there is. The oral dosage form may be granules, powder, solution, tablet, capsule, dry syrup, or a combination thereof, and the parenteral dosage form may be an injection, but is not limited thereto.

The administration may be performed by methods known in the art. Administration may be administered directly to the subject by any means, such as, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. You can. The administration may be administered systemically or locally.

The subject may include, but is not limited to, mammals, such as humans, cattle, horses, pigs, dogs, sheep, goats, or cats. The subject may be an individual in need of improvement in conditions related to aging.

The therapeutically effective dosage of the pharmaceutical composition is 0.01 to 1000 mg per kg of body weight per day, for example, 0.1 to 500 mg or 0.1 to 300 mg, and can be administered in one to several divided doses. However, the therapeutically effective dosage or therapeutically effective amount may vary depending on the formulation method, administration method, administration time, and/or administration route of the pharmaceutical composition. In addition, the type and degree of reaction to be achieved by administration of the composition, type of subject to be administered, age, weight, general health condition, symptoms or degree of disease, gender, diet, excretion, simultaneous or different effects on the subject. It may vary depending on various factors, including drugs and other composition components used together, and similar factors well known in the pharmaceutical field, and a person skilled in the art can easily determine the effective dosage for the desired treatment. and can be prescribed.

Another aspect provides a health functional food for preventing or improving aging-related diseases containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.

In Formula 1, R ₁ to R ₂ are each independently H, substituted or unsubstituted alkyl of C ₁ to C ₁₀ , substituted or unsubstituted alkenyl of C ₂ to C ₁₀ , or substituted C ₂ to C ₁₀ or unsubstituted alkynyl, substituted or unsubstituted alkoxy of C ₁ to C ₁₀ , or substituted or unsubstituted aryl; R ₃ is sugar or a derivative thereof; R ₄ is substituted or unsubstituted alkylene of C ₁ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ , substituted or unsubstituted alkynylene of C ₂ to C ₁₀ , or substituted or unsubstituted alkynylene. is an arylene; and X ₁ and X ₂ are each independently halogen.

The compound represented by Formula 1 is as described above.

In one embodiment, the health functional food may further include the antioxidants described above.

In this specification, the term “health functional food” refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food, and nutrients for the structure and function of the human body. It means ingestion for the purpose of controlling health or obtaining useful health effects such as physiological effects.

When using the composition of the present invention as a food additive, the composition can be added as is or used with other foods or ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use.

The above “food additives” usually refer to small amounts intentionally added to food for the purpose of improving appearance, flavor, texture or storage, and improve the quality of food, improving preservation or palatability as well as nutritional value and It means using it for the purpose of increasing the practical value of food. As defined in Article 2, Paragraph 2 of the Food Sanitation Act, this may be a substance used by adding, mixing, infiltrating, or using other methods in manufacturing, processing, or preserving food.

There is no particular limitation on the type of food of the present invention. Examples of foods to which the composition of the present invention can be added include meat, sausages, breads, chocolates, candies, snacks, confectionery, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, There are drinks, alcoholic beverages, vitamin complexes, etc., and can include all foods in the conventional sense, and include foods used as feed for animals.

The health functional food of the present invention may contain common food additives, and its suitability as a food additive is determined in accordance with the general provisions and general test methods of the food additive code approved by the Food and Drug Administration, unless otherwise specified. Judgment is made according to specifications and standards.

In addition, the food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, and alcohol. , may contain carbonating agents used in carbonated beverages, etc. In addition, it may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks.

In addition, the food can be manufactured in dosage forms such as tablets, granules, powders, capsules, liquid solutions, and pills according to known manufacturing methods. Other than including the compound of the present invention as an active ingredient, there are no particular restrictions on other ingredients, and various common flavoring agents or natural carbohydrates may be included as additional ingredients.

In one embodiment, the health functional food in tablet form is prepared by granulating a mixture of the compound of Formula 1, which is the active ingredient of the present invention, with excipients, binders, disintegrants and other additives in a conventional manner, and then adding a lubricant. etc. can be put into compression molding, or the mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain flavoring agents, etc., if necessary.

The granule formulation can be prepared into granules by mixing the compound of Formula 1, which is the active ingredient of the present invention, with excipients, binders, disintegrants, etc., using a known method, and if necessary, flavoring agents and flavoring agents. It may contain etc.

Among the capsule formulations, hard capsules can be prepared by filling a regular hard capsule with a mixture of the compound of Formula 1, which is the active ingredient of the present invention, with additives such as excipients, and soft capsules can be prepared by filling a regular hard capsule with a mixture of the compound of Formula 1, which is the active ingredient of the present invention, with additives such as excipients. It can be prepared by filling a mixture of additives such as excipients into a capsule base such as gelatin, and may contain plasticizers such as glycerin or sorbitol, colorants, and preservatives, if necessary.

The ring formulation can be prepared by molding a mixture of the compound of Formula 1, which is the active ingredient of the present invention, and excipients, binders, disintegrants, etc., by a known method, and if necessary, can be added with sucrose or other skin-care agents. It can be peeled off, or the surface can be coated with a substance such as starch or talc.

According to the composition and micelle according to one aspect, the solubility of imidazoline derivatives is improved without the use of additives such as surfactants, and the drug can be effectively used to prevent, improve, or treat diseases. In addition, when using the micelles, there is an effect of improving drug-induced side effects by improving the selectivity of the drug for cells or tissues that require drug treatment.

Figure 1 is an image showing the degree of redispersion of N201-gal micelles in the aqueous solution prepared in Example 2.

Figure 2 is a graph confirming the critical micelle concentration (CMC) concentration of N201-gal micelles by comparing I ₃₃₅ /I ₃₃₂ values at each concentration.

Figure 3 is a TEM image of N201-gal micelles.

Figure 4 is a graph showing the DLS results of N201-gal micelles.

Figure 5 is a graph showing the DLS results of N201-gal micelles in artificial gastrointestinal fluid (SGF-SIF solution).

Figure 6 is a graph comparing the HLPC intensity results of N201-gal micelles in artificial gastrointestinal fluid (SGF-SIF solution) over time.

Figure 7 is an image showing the chemical structures of N101, N201, and N201-gal.

Figure 8 is an image showing a plot of the binding ratio to MDM2 according to the concentration of N101, N201, and N201-gal in logarithmic values.

Figure 9 is an image showing the protein electrophoresis results of SnC-ARPE19 cells treated with N101, N201, and N201-gal.

Figure 10 is a graph comparing the viability of cells treated with N201 and N201-gal.

Figure 11 is an image of the medium in which senescent cells isolated from 3D cartilage tissue were treated with 50 uM and 100 uM of N101, N201, and N201-gal drugs.

Figure 12 is an image comparing the staining results of SA-β-gal in retinal tissue when N201-gal was orally administered. G1 (Vehicle, n=6); G2 (Doxorubicin, n=7); G3 (N201g 10 mg/kg, n=6); G4 (N201g 25 mg/kg, n=8); G5 (N201g 50 mg/kg, n=9); G6 (N201g 100 mg/kg, n=2)

Figure 13 is a graph comparing the relative value of the intensity of images quantifying the staining results of SA-β-gal in retinal tissue when N201-gal was orally administered.

Figure 14 is an image comparing the staining results of p16 protein in retinal tissue when N201-gal was orally administered. G1 (Vehicle, n=3); G2 (Doxorubicin, n=4); G3 (N201g 10 mg/kg, n=4); G4 (N201g 25 mg/kg, n=3); G5 (N201g 50 mg/kg, n=3)

Figure 15 is an image comparing the staining results of p21 protein in retinal tissue when N201-gal was orally administered. G1 (Vehicle, n=3); G2 (Doxorubicin, n=3); G3 (N201g 10 mg/kg, n=4); G4 (N201g 25 mg/kg, n=3); G5 (N201g 50 mg/kg, n=3)

Figure 16 is a graph comparing and analyzing the number of p16 protein and double-stained nuclei in percentage compared to the total number of nuclei in retinal tissue when N201-gal was orally administered.

Figure 17 is a graph comparing and analyzing the number of p21 protein and double-stained nuclei in percentage compared to the total number of nuclei in retinal tissue when N201-gal was orally administered.

Figure 18 is an image comparing Mito-sox staining results in retinal tissue when N201-gal was orally administered. G1 (Vehicle, n=3); G2 (Doxorubicin, n=3); G3 (N201g 10 mg/kg, n=3); G4 (N201g 25 mg/kg, n=3); G5 (N201g 50 mg/kg, n=2)

Figure 19 is a graph comparing the relative value of the intensity of images quantifying the staining results of Mito-sox in retinal tissue when N201-gal was orally administered.

Figure 20 is an image comparing the staining results of retinal cell walls in retinal tissue when N201-gal was orally administered. G1 (Vehicle, n=6); G2 (Doxorubicin, n=6); G3 (N201g 10mg/kg, n=3); G4 (N201g 25mg/kg, n=6); G5 (N201g 50mg/kg, n=7); G6 (N201g 100mg/kg, n=3)

Figure 21 is a graph comparing the relative value of the intensity of images quantifying the area comprised by tight junctions of retinal pigment epithelial cells in retinal tissue when N201-gal is orally administered.

Figure 22 is an image comparing the staining results of p21 protein in retinal tissue when N201-gal was administered intravitreally. G1 (Vehicle, n=2); G2 (Doxorubicin, n=3); G3 (N201g 50ng/ul, n=3); G4 (N201g 100ng/ul, n=3); G5 (N201g 200ng/ul, n=4), scale bar; 50um

Figure 23 is a graph comparing and analyzing the number of p21 protein and double-stained nuclei in percentage compared to the total number of nuclei in retinal tissue when N201-gal was administered intravitreally.

This will be described in more detail through examples below. However, these examples are intended to illustrate one or more embodiments and the scope of the present invention is not limited to these examples.

Example 1. Preparation of N201-gal conjugate

An N201-gal conjugate was prepared in which a sugar was bound to an imidazoline derivative.

Specifically, the nutlin derivative (N201, 200 mg, 0.42 mmol) was incubated with AcGal-Br ((2S, 3R, 4R, 5S, 6S)- in the presence of DMAP (281.2 mg, 2.21 mmol)/DCM solution for 16 hours at room temperature. 2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate), 340.8 mg, 0.87 mmol). Additionally, 2 ml of ammonia solution was added to the reaction and stirred for 1 hour. Then, N201-gal was separated through silica column chromatography.

The reaction is shown in Scheme 1 below.

[Scheme 1]

¹ H-NMR (400mHz, MeOD-d ₄ ) δ7.52 (d,2H) 7,12 (d,2H), 7.08-6.99 (dd, 4H), 6.91(d,2H), 6.67(d,2H) ), 5.72(dd,2H), 5.49(d,1H), 4.71(m,1H), 4.15(t,1H), 3.86(s,3H), 3.81(m,1H), 3.70(m,3H) , 3.53(d,2H), 3.17(m,4H), 2.38(d,2H), 2.08(d,4H), 1.40-1.31(dd, 6H).

Example 2. Preparation of micelles of N201-gal

Prepared in Example 1 above N201-gal was made into a methanol solution (10 mM, 1 ml), diluted with 50 ml purified water, and freeze-dried for 3 days. As a result, the parent compound N201 was able to produce micelle-type nanoparticles with amphiphilic properties, and the results are shown in Figure 1.

Figure 1 is an image showing the degree of redispersion of N201-gal micelles in the aqueous solution prepared in Example 2.

As shown in Figure 1, it was confirmed that the parent compound N201 was completely dispersed at a concentration of 1 mM as a result of the binding of the hydrophilic sugar, which is about 1000 times more improved than the solubility (1 uM) of the parent compound N201.

More specifically, the following experiment was performed to confirm whether N201-gal micelles as described above were formed.

The N201-gal micelles freeze-dried as above were again made into a 1 mM solution, then diluted to concentrations of 0.2, 0.5, 2, 3, 5, 10, 20, 30, 100, 200, and 300 uM and added with 1 mM pyrene ( pyrene) is added dropwise. After stirring it overnight, the FL value was measured. The results are shown in Figure 2.

Figure 2 is a graph confirming the critical micelle concentration (CMC) concentration of N201-gal micelles by comparing I ₃₃₅ /I ₃₃₂ values at each concentration.

As shown in Figure 2, it was confirmed that the critical micelle concentration of N201-gal micelles was 10 uM: Fluorescence excitation 300 nm-350 nm, Emission 390 nm, Slit width Ex 2.5, Em 5.0.

These results indicate that N201-gal forms micelle-structured nanoparticles.

Example 3. Preparation of N301-gal

An N301-gal conjugate was prepared in which a sugar was bound to an imidazoline derivative.

Specifically, the nutlin derivative (N301, 200 mg, 0.42 mmol) was incubated with AcGal-NO2((2S,3R,4R,5S,6R)- in the presence of DMAP (431.4 mg, 2.21 mmol)/DCM solution for 16 hours at room temperature. 2-(acetoxymethyl)-6-(2-nitro-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate), 340.8 mg, 0.87 mmol). Afterwards, 1 ml of 1 M sodium tert-butoxide in THF was added and reacted for 10 minutes. Then, N301-gal was separated through silica column chromatography.

The reaction is shown in Scheme 2 below.

[Scheme 2]

¹ H-NMR (400mHz, MeOD-d ₄ ) δ 7.38(dd,1H), 7.28(m,2H), 7.14-7.03(m,6H), 6.99(d,2H), 6.52(d,1H), 6.45 (d,1H), 5.73(q,2H), 4.97(m,2H), 4.86(d,2H), 4.62(m,1H), 3.89(d,1H), 3.81(s,3H), 3.77 -3.68(m,3H), 3.56(dd,1H), 1.37(d,3H), 1.13(d,3H).

Experimental Example 1. Characteristics and stability analysis of N201-gal micelles

Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis experiments were performed to observe the shape, size, and stability of the N201-gal micelles prepared in Example 2.

The N201-gal micelles prepared in Example 2 above were dispersed in 100 uM purified water to prepare a solution. TEM images were taken by taking 8 μl of the solution, drying it on a TEM grid for about a day, performing uranyl staining, and removing the remaining uranyl staining solution after 5 minutes in the dark. The results are shown in Figure 3. The DLS Number measurement value was measured by taking 0.7 ml of the above solution, placing it in a DLS tube, and storing it at room temperature for 7 days. The results are shown in Figure 4.

Figure 3 is a TEM image of N201-gal micelles.

As shown in Figure 3, when N201-gal micelles were observed using TEM, it was confirmed that N201-gal micelle aggregates were formed with a particle size of 40 to 60 nm.

Figure 4 is a graph showing the DLS results of N201-gal micelles.

As shown in Figure 4, when N201-gal micelles were observed with DLS, the average size of N201-gal micelles was confirmed to be 50 to 80 nm, and it was confirmed that the micelles remained stable for 7 days in a dispersed state.

Experimental Example 2. Stability analysis in artificial gastrointestinal fluid (SGF-SIF solution)

The stability of N201-gal micelles prepared in Example 2 was confirmed in artificial gastrointestinal fluid (SGF-SIF solution).

First, simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to the method proposed by the United States Pharmacopeia. For artificial gastrointestinal fluid, dissolve 2.0 g of NaCl and 3.2 g of pepsin (hog stomach, Fluka 77163) in 500 ml of distilled water, add 7.0 ml of HCl (30%), make 1000 ml with distilled water, and adjust the pH to 1.2 with 1N HCl. Adjusted. For the artificial small intestine solution, dissolve 6.8 g of KH ₂ PO ₄ in 250 ml of distilled water, add 190 ml of 0.2 N NaOH and 400 ml of distilled water, then add 10 g of pancreatin (porcine pancrease, Sigma P-1500), and then add 0.2 N NaOH. After adjusting the pH to 7.5, the solution was adjusted to 1,000 ml with distilled water.

The following experiment was performed to confirm whether N201-gal micelles remained stably dispersed in artificial gastrointestinal fluid (SGF-SIF solution). The SGF condition was dissolved in 100 uM of artificial gastrointestinal fluid as above. The SGF-SIF conditions were dissolved at 1 mM in artificial gastrointestinal fluid (SGF) as above, and then changed to 100 uM artificial small intestinal fluid (SIF) as above 1 hour later. Afterwards, 0.7 ml of solution was taken from each and placed in a DLS tube and measured by number. The results are shown in Figure 5.

As shown in Figure 5, the average size of N201-gal micelles in artificial gastrointestinal fluid (SGF solution) was confirmed to be 40 to 50 nm, and the average size of N201-gal micelles in artificial gastrointestinal fluid (SGF-SIF solution) was It was confirmed that the size was 40 to 80 nm, and it was confirmed that the micelles remained stably dispersed even in artificial gastrointestinal fluid under acidic conditions.

Additionally, the following experiment was performed to confirm whether N201-gal micelles remained stable even after several hours. In the SGF_0h condition, the artificial gastrointestinal fluid (SGF) as described above was dissolved in 10 mM, then 10 uM was added and diluted to 1 mM. Afterwards, as time passed by 1 hour, the artificial small intestine fluid (SIF) was diluted to 1 mM and the high-performance liquid chromatography (HPLC) values were measured. The results are shown in Figure 6.

As shown in Figure 6, it was confirmed that the above stable dispersion state was maintained without chemical change for 5 hours.

Experimental Example 3. Confirmation of MDM2 binding ability and MDM2-p53 binding inhibition ability

The MDM2 protein binding ability and inhibitory ability against the MDM2-p53 complex of the N201-gal micelle prepared in Example 2 were confirmed.

Specifically, 12.5 uM FITC-p53 (FITC-LC-ETFSDLWKLL-NH2) and 100 nM MDM2 were subjected to a binding reaction at room temperature for 1 hour. Afterwards, 10uM N101, N201, and N201-gal were serially diluted with assay buffer in a 96 well plate (n=3), and a mixed sample of FITC-p53 and MDM2 was dispensed and reacted at room temperature for 2 hours. Fluorescence polarization (FP) signals were measured in a multi-microplate reader, and the results are shown in FIG. 8.

Additionally, ARPE19/ ^SnC -ARPE19 (8 Total protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit (PIERCE) according to the manufacturer's instructions. 20 ug of protein was heat denatured at 95°C for 10 minutes, resolved on a TGX SDS-PAGE gel, and blotted with a 0.45 μm PVDF membrane. Primary antibodies include anti-p53 (sc-126), anti-p21 (sc-817), anti-MDM2 (ab259265), anti-p16 (A0262), anti-HMGB1 (ab18256) and anti-GAPDH (sc-32233). ) was used, and then incubated with an appropriate secondary antibody. Protein concentration was measured by detection with SuperSignal West Pico plus ECL (# 34580, Pierce), and the results are shown in Figure 9. For reference, the chemical structures of nutlin derivatives N101, N201, and N201-gal are shown in Figure 7.

As shown in Figure 8, it was confirmed that even if sugar was attached to N201, the binding to MDM2 was maintained, and as shown in Figure 9, even if sugar was attached to N201, the intracellular p53 and p21 protein concentration increased. These results mean that sugar-bound N201 works well as an MDM2-p53 inhibitor.

Experimental Example 4. Comparison of aging cell death ability

The ability of N201-gal micelles prepared in Example 2 to kill senescent cells was compared and analyzed with N201.

Specifically, ARPE19/SnC-ARPE19 (8X10 ⁵ /60mm) cells were treated with N201 in DMSO (dimethyl sulfoxide) solution for 24 hours. After 24 hours, 10 μl CCK-8 solution (CK04, Cell counting kit-8) and 90 μl culture medium were added to the well, and cultured for 3 hours at 37°C in a humidified incubator with 5% CO2. The viability of the cells was measured using an ELISA reader (450 nm), and the comparison results are shown in Figure 10.

As shown in Figure 10, N201 was confirmed to have lower toxicity in normal cells and selectively remove senescent cells due to the attachment of the sugar group.

Experimental Example 5. Comparison of aging cell death ability in 3D cartilage tissue

The senescent cell killing ability of N201-gal micelles prepared in Example 2 was compared and analyzed with N201 in 3D cartilage tissue.

Cartilage cells were isolated from cartilage tissue removed from patients undergoing knee osteoarthritis surgery, and the cells were distributed in a circular 96 well plate at a number of ^4x105 cells. Using a centrifuge, centrifuge at 150 g for 10 minutes to allow the cells to clump together. The plate was placed in a CO ₂ incubator at 37°C and cultured for a week while changing the medium every two days. Senescent cells were treated with 50 uM and 100 uM N101, N201, and N201-gal drugs for 4 days, and senescent cells were stained using Senescence β-Galactosidase Staining Kit (#9860). The results are shown in Figure 11.

As shown in Figure 11, it was observed that N201-gal removed senescent cells to the same level as N101 and N201.

Experimental Example 6. Oral administration efficacy evaluation: confirmation of removal of senescent cells in the retina

When the N201-gal micelle prepared in Example 2 was orally administered, it was confirmed that senescent cells in the retina were removed in vivo.

First, doxorubicin (Dox), a senescent cell inducing substance, was used to induce senescent cells in mouse retinal tissue as follows. Under a light microscope (Olympus SZ51, Tokyo, Japan), a small hole was made in the limbus using a 30-gauge sterile needle (BD Science, San Jose, USA). A blunt 35-gauge Hamilton microsyringe (Hamilton Company, NV, USA) was then slowly inserted through the hole. 1 uL of 100 ng/μl Dox was injected under the retina of C57BL/6N or C57BL/6J mice, respectively. Afterwards, N201-gal was administered orally at doses of 10, 25, 50, and 100 mg/kg once a day for 7 days using a gastric tube.

Afterwards, the mouse was anesthetized with CO ₂ gas, and the eyes were immediately removed. The anterior chamber eye was dissected in cold PBS.

After carefully removing the retina from the enucleated eye, RPE/choroid/sclera complex tissue or sectioned retina was immediately stained with SA-β galactosidase (SA-β-gal) staining kit (BioVision, # K320, California, They were fixed for 20 min at room temperature with the fixative provided with the SA-β-gal staining kit (USA) and stained with the staining solution mix provided with the SA-β-gal staining kit according to the manufacturer's protocol. After SA-β-gal staining overnight at 37°C, the dark melanin pigment in the RPE/choroid was bleached, revealing SA-β-gal staining masked by melanin pigment in these tissues. For depigmentation of the RPE/choroid, the RPE/choroid/sclera composite tissue was immersed in 30% H ₂ O ₂ , incubated in a 55 °C heat block for 45 min, then rinsed with PBS, and micro-dissected under a light microscope (Olympus SZ51). It was held flat with pointed forceps and a surgical blade (World Precision Instruments, Florida, USA). Images of stained RPE/choroid flat mounts or sectioned retinas were taken using an inverted microscope (Leica Microsystems #DMi1, Wetzlar, Germany), and the results are shown in Figure 12.

In addition, in order to compare the intensity of the images with relative values, the gray value of β-galactosidase expressed in senescent cells was measured and analyzed with Image J, and the results are shown in FIG. 13.

Additionally, the same experiment as above was conducted using aging markers p16 and p21.

After separating the RPE and retina from the enucleated eye, the RPE/choroid/sclera composite tissue or sectioned retina was immediately fixed at room temperature for 20 minutes. Primary antibody staining, secondary antibody staining, and nuclear staining were performed while increasing cell membrane permeability and blocking non-specific reactions. Images of the stained nuclei were taken using a confocal microscope (40X, 60X), and the results are shown in Figures 14 and 15 .

In addition, in order to compare the intensity of the images as a relative value, the number of nuclei stained with senescent protein and double staining was compared and analyzed as a percentage relative to the total number of nuclei, and the results are shown in FIG. 13.

As shown in Figures 12 to 17, it was confirmed that when N201-gal was orally administered, senescent cells in the retina were removed even at 10 mg/kg.

Experimental Example 7. Evaluation of oral administration efficacy: Confirmation of reduction of inflammatory environment in the retina

Additionally, the inflammatory environment within the retina was confirmed.

The surrounding tissue was removed from the eye extracted in Experimental Example 6, and the RPE and retina were separated. The RPE/choroid/sclera complex tissue or sectioned retina was then stained with Mito-sox. Images of stained RPE/choroid flat mounts or sectioned retinas were taken using a confocal microscope (63X); The results are shown in Figure 18.

In addition, in order to compare the intensity of the images with relative values, the gray value of β-galactosidase expressed in senescent cells was measured and analyzed with Image J, and the results are shown in FIG. 19.

As shown in Figures 18 and 19, it was confirmed that when N201-gal was orally administered, the inflammatory environment within the retina was reduced even at 10 mg/kg.

Experimental Example 8. Evaluation of oral administration efficacy: Confirmation of alleviation of retinal tissue damage

Additionally, the retinal tissue alignment was confirmed.

After separating the RPE and retina from the eye extracted in Experimental Example 6, the RPE/choroid/sclera composite tissue or the sliced retina was fixed at room temperature for 20 minutes. Primary antibody staining, secondary antibody staining, and nuclear staining were performed while increasing cell membrane permeability and blocking non-specific reactions. Images of the stained nuclei were taken using a confocal microscope (40X, 60X), and the results are shown in Figure 20 .

In addition, in order to compare the intensity of the images as a relative value, the area comprised by the tight junctions of retinal pigment epithelial cells was measured and the cross-sectional area difference between groups was compared, and the results are shown in Figure 21.

As shown in Figures 20 and 21, it was confirmed that when N201-gal was orally administered, retinal tissue damage was alleviated even at 10 mg/kg.

Experimental Example 9. Evaluation of efficacy of intravitreal administration: confirmation of removal of senescent cells in the retina

When the N201-gal micelles prepared in Example 2 were administered into the vitreous cavity, it was confirmed that senescent cells in the retina were removed in vivo.

First, doxorubicin (Dox), a senescent cell inducing substance, was used to induce senescent cells in mouse retinal tissue as follows. Under a light microscope (Olympus SZ51, Tokyo, Japan), a small hole was made in the limbus using a 30-gauge sterile needle (BD Science, San Jose, USA). A blunt 35-gauge Hamilton microsyringe (Hamilton Company, NV, USA) was then slowly inserted through the hole. 1 uL of 100 ng/μl Dox was injected under the retina of C57BL/6N or C57BL/6J mice, respectively, to induce senescence in retinal tissue. After Dox was injected, 1 ul of N201-gal was administered into the vitreous cavity at concentrations of 50, 100, and 200 ng/ul on the 3rd day.

Then, the mouse was anesthetized with CO ₂ gas and the eyes were immediately removed. The anterior chamber eye was dissected in cold PBS.

After separating the RPE and retina from the eye enucleated as described above, the RPE/choroid/sclera composite tissue or sectioned retina was immediately fixed at room temperature for 20 minutes. Primary antibody staining, secondary antibody staining, and nuclear staining were performed while increasing cell membrane permeability and blocking non-specific reactions. Images of the stained nuclei were taken using a confocal microscope (40X, 60X), and the results are shown in Figure 23.

Additionally, in order to compare the intensity of the images as a relative value, the number of senescent protein and double-stained nuclei was compared and analyzed as a percentage relative to the total number of nuclei, and the results are shown in Figure 23.

As shown in Figures 22 and 23, it was confirmed that when N201-gal was administered intravitreally, senescent cells in the retina were removed even at 50 ng/ul.

Claims (20)

1. Compound represented by Formula 1 or a pharmaceutically acceptable salt thereof:

   [Formula 1]

   

   .

   In Formula 1, R ₁ to R ₂ are each independently H, substituted or unsubstituted alkyl of C ₁ to C ₁₀ , substituted or unsubstituted alkenyl of C ₂ to C ₁₀ , or substituted C ₂ to C ₁₀ or unsubstituted alkynyl, substituted or unsubstituted alkoxy of C ₁ to C ₁₀ , or substituted or unsubstituted aryl; R ₃ is sugar or a derivative thereof; R ₄ is substituted or unsubstituted alkylene of C ₁ to C ₁₀ , substituted or unsubstituted alkenylene of C ₂ to C ₁₀ , substituted or unsubstituted alkynylene of C ₂ to C ₁₀ , or substituted or unsubstituted alkynylene. is an arylene; and X ₁ and X ₂ are each independently halogen.
2. In claim 1,

   The compound or a pharmaceutically acceptable salt thereof, wherein the sugar is a monosaccharide or disaccharide.
3. In claim 2,

   The monosaccharides include galactose, glucose, fructose, mannose, allose, altrose, gulose, talose, and psicose ( psicose, sorbose, tagatose, idose, rhamnose, xylose, arabinose, fucose, glyceraldehyde ), erythrose, threose, ribose, lyxose, dihydroxyacetone, erythrulose and ribulose. Any one or more compounds or pharmaceutically acceptable salts thereof.
4. In claim 2,

   The disaccharide is a compound or Pharmaceutically acceptable salts thereof.
5. In claim 1,

   The imidazoline derivative compound represented by Formula 1 is one selected from the group consisting of Formulas 7 to 10 below, or a pharmaceutically acceptable salt thereof.

   [Formula 7]

   

   [Formula 8]

   

   [Formula 9]

   

   [Formula 10]

   
6. A micelle containing the compound of claim 1 or a pharmaceutically acceptable salt thereof.
7. In claim 6,

   The micelles have a size of 50 to 200 nm.
8. A pharmaceutical composition for preventing or treating aging-related diseases, comprising the compound of claim 1, a pharmaceutically acceptable salt thereof, or the micelle of claim 6 as an active ingredient.
9. In claim 8,

   A pharmaceutical composition, wherein the compound of the pharmaceutical composition induces apoptosis in mitochondria within senescent cells.
10. In claim 8,

    The age-related diseases include cancer, cardiovascular disease or disorder in the elderly, inflammatory or autoimmune disease or disorder in the elderly, neurodegenerative disease in the elderly, metabolic disease in the elderly, pulmonary disease in the elderly, eye disease or disorder in the elderly, age-related disorders in the elderly, and dermatology in the elderly. A pharmaceutical composition comprising one or more diseases or disorders selected from the group consisting of:
11. In claim 10,

    A pharmaceutical composition wherein the elderly eye disease or disorder is at least one selected from the group consisting of macular degeneration, glaucoma, cataracts, presbyopia, and vision impairment.
12. In claim 11,

    The pharmaceutical composition is a pharmaceutical composition that is administered orally.
13. In claim 11,

    The pharmaceutical composition is a pharmaceutical composition administered intraocularly.
14. In claim 10,

    A pharmaceutical composition wherein the elderly inflammatory disease or disorder is osteoarthritis.
15. A health functional food for improving aging, comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof or the micelle of claim 6 as an active ingredient.
16. In claim 15,

    The compound of the health functional food is a health functional food that induces apoptosis in mitochondria within aging cells.
17. A method for preventing or treating an age-related disease comprising administering an effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof or the micelle of claim 6 to an individual in need thereof.
18. A method for improving aging comprising administering an effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof or the micelle of claim 6 to a subject in need thereof.
19. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof or the micelle of claim 6 for use in the manufacture of a pharmaceutical agent for the prevention or treatment of age-related diseases.
20. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof or the micelle of claim 6 for use in health functional foods for the prevention or treatment of age-related diseases.

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