KR102231203B1 - Composition for the prevention or treatment of depression comprising protein expression inhibitor and use thereof - Google Patents

Abstract

The present invention relates to a composition for preventing or treating depression, comprising a protein expression inhibitor and a use thereof. By inhibiting the protein expression of microglia, an excellent antidepressant effect due to the interaction between neurons and microglia can be exhibited. In addition, without a separate drug carrier, the composition of the present invention can pass through the blood-brain barrier only by single treatment, thereby exhibiting the excellent antidepressant effect.

Description

Composition for the prevention or treatment of depression comprising protein expression inhibitor and use thereof

The present invention relates to a composition for preventing or treating depression comprising a protein expression inhibitor and to a use thereof. More specifically, it relates to a composition capable of exhibiting an excellent preventive or therapeutic effect of depression, including a novel protein expression inhibitor, and a use thereof.

Depression is a syndrome characterized by a severe, long-lasting, depressed mood, which appears primarily as an emotional disorder. Symptoms include depressed mood, diminished conversation, slow thinking and behavior, and even suicidal attempts.

As a chronic psychiatric disorder, depression has become a troubling problem for medical health services in China due to a long course of treatment, slow onset of effects and a high rate of recurrence, disability and suicide.

According to "World Health Reports" published by the World Health Organization (WHO), depression became the fourth most common disease in the world, and depression was the second most common disease after heart disease in 2020. Will be, and accordingly become a serious problem for human health.

To date, the mechanism of action of antidepressants has not been clearly demonstrated. Drugs with a certain effect actually act on the synapses at the nerve endings and exert their healing effect by regulating the level of neurotransmitters in the synaptic gap. Biochemical studies in etiology have shown that depression is predominantly of five types of neurotransmitters: the central 5-hydroxytryptamine (5-HT), noradrenaline (NA), dopamine (DA), acetylcholine (Ach), and And γ-aminobutyric acid (GABA).

Antidepressants can be divided into two categories: early non-selective antidepressants and novel selective reuptake inhibitors.

Nonselective antidepressants mainly include monoamine oxidase inhibitors (MAOI) and tricyclic antidepressants (TCA); Selective reuptake inhibitors include selective 5-hydroxytryptamine (5-HT) reuptake inhibitors (SSRI), noradrenaline (NA) reuptake inhibitors (NRI), noradrenergic and specific 5-HT reuptake. It mainly includes inhibitors (NDRI), 5-HT and NA double reuptake inhibitors (SNRI), 5-HT re-uptake enhancers, and the like.

Early monoamine oxidase inhibitors and tricyclic antidepressants have serious side effects, and for subsequent selective NA reuptake inhibitors and selective 5-HT reuptake inhibitors, disadvantages such as slow onset, uncertain efficacy, etc., although fewer side effects are also present. exist.

Therefore, the effectiveness of all types of these drugs in the treatment of depression is not satisfactory.

(Patent Document 1) KR 10-2013-0099364 A1

An object of the present invention is to provide a composition for preventing or treating depression comprising a protein expression inhibitor and a use thereof.

Another object of the present invention is to provide a composition for preventing or treating depression comprising a protein expression inhibitor having an excellent antidepressant effect due to the interaction between neuro-microglia by inhibiting the protein expression of microglia, and a use thereof. will be.

Another object of the present invention is to provide a composition for preventing or treating depression, including a protein expression inhibitor capable of showing an excellent antidepressant effect by passing through the blood-brain barrier only by treatment alone, without a separate drug carrier, and a use thereof.

In order to achieve the above object, a composition for preventing or treating depression comprising a protein expression inhibitor according to an embodiment of the present invention comprises a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. Can be:

[Formula 1]

[Formula 2]

here,

A is a substituent represented by Chemical Formula 2 or Chemical Formula 3,

* Means the part to be joined,

L ₁ and L ₂ are the same as or different from each other, and each independently a single bond, a substituted or unsubstituted alkylene group having 1 to 10 carbon atoms and a substituted or unsubstituted arylene group having 6 to 10 carbon atoms,

X ₁ is selected from the group consisting of O, S and N (R _{7 ),}

Y ₁ and Y ₂ are the same as or different from each other, and each independently N or C (R ₈ ),

R ₁ to R ₈ are the same as or different from each other, and each independently hydrogen, deuterium, cyano group, nitro group, halogen group, hydroxy group, alkylthio group having 1 to 4 carbon atoms, alkyl group having 1 to 30 carbon atoms, 1 to 20 carbon atoms Cycloalkyl group, C2-C30 alkenyl group, C2-C24 alkynyl group, C7-C30 aralkyl group, C6-C30 aryl group, C5-C60 heteroaryl group, C6-C6 30 heteroarylalkyl group, C 1 to C 30 alkoxy group, C 1 to C 30 alkylamino group, C 6 to C 30 arylamino group, C 6 to C 30 aralkylamino group, C 2 to C 24 heteroarylamino group, C 1 It is selected from the group consisting of an alkylsilyl group of to 30, an arylsilyl group having 6 to 30 carbon atoms, and an aryloxy group having 6 to 30 carbon atoms, and may be combined with adjacent groups to form a substituted or unsubstituted ring,

When the R ₁ to R ₈ are substituted, hydrogen, deuterium, cyano group, nitro group, halogen group, hydroxy group, alkyl group having 1 to 30 carbon atoms, alkenyl group having 2 to 30 carbon atoms, alkynyl group having 2 to 24 carbon atoms, carbon number 2 to 30 heteroalkyl group, C 6 to C 30 aralkyl group, C 3 to C 20 cycloalkyl group, C 3 to C 20 heterocycloalkyl group, C 5 to C 30 aryl group, C 2 to C 30 heteroaryl group, C carbon number Substituted with a substituent selected from the group consisting of a 3 to 30 heteroarylalkyl group, a C 1 to C 30 alkoxy group, a C 1 to C 30 alkylsilyl group, a C 6 to C 30 arylsilyl group, and a C 6 to C 30 aryloxy group And, when substituted with a plurality of substituents, they are the same as or different from each other.

In the present specification, "halogen group" is fluorine, chlorine, bromine or iodine.

In the present invention, "alkyl" refers to a monovalent substituent derived from a linear or branched saturated hydrocarbon having 1 to 40 carbon atoms. Examples thereof include, but are not limited to, methyl, ethyl, propyl, isobutyl, sec-butyl, pentyl, iso-amyl, hexyl, and the like.

In the present invention, “alkenyl” refers to a monovalent substituent derived from a straight or branched unsaturated hydrocarbon having 2 to 40 carbon atoms having one or more carbon-carbon double bonds. Examples thereof include vinyl (vinyl), allyl (allyl), isopropenyl (isopropenyl), 2-butenyl (2-butenyl), and the like, but is not limited thereto.

In the present invention, “alkynyl” refers to a monovalent substituent derived from a straight or branched unsaturated hydrocarbon having 2 to 40 carbon atoms having at least one carbon-carbon triple bond. Examples thereof include, but are not limited to, ethynyl and 2-propynyl.

In the present invention, “aryl” refers to a monovalent substituent derived from an aromatic hydrocarbon having 6 to 60 carbon atoms in which a single ring or two or more rings are combined. In addition, a form in which two or more rings are simply attached to each other or condensed may be included. Examples of such aryl include, but are not limited to, phenyl, naphthyl, phenanthryl, anthryl, fluorenyl, and dimethylfluorenyl.

In the present invention, "heteroaryl" refers to a monovalent substituent derived from a monoheterocyclic or polyheterocyclic aromatic hydrocarbon having 6 to 30 carbon atoms. At this time, at least one carbon, preferably 1 to 3 carbons in the ring is substituted with a heteroatom such as N, O, S or Se. In addition, a form in which two or more rings are simply attached to each other or condensed may be included, and further, a form condensed with an aryl group may be included. Examples of such heteroaryl include 6-membered monocyclic rings such as pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl, phenoxathienyl, indolizinyl, indolyl ( indolyl), purinyl, quinolyl, benzothiazole, polycyclic rings such as carbazolyl and 2-furanyl, N-imidazolyl, 2-isoxazolyl , 2-pyridinyl, 2-pyrimidinyl, and the like, but are not limited thereto.

In the present invention, "aryloxy" is a monovalent substituent represented by RO-, and R means an aryl having 6 to 60 carbon atoms. Examples of such aryloxy include, but are not limited to, phenyloxy, naphthyloxy, and diphenyloxy.

In the present invention, "alkyloxy" is a monovalent substituent represented by R'O-, wherein R'refers to alkyl having 1 to 40 carbon atoms, and has a linear, branched, or cyclic structure It may include. Examples of the alkyloxy include, but are not limited to, methoxy, ethoxy, n-propoxy, 1-propoxy, t-butoxy, n-butoxy, pentoxy, and the like.

In the present invention, "alkoxy" may be a straight chain, branched chain or cyclic chain. Although the number of carbon atoms of the alkoxy is not particularly limited, it is preferably 1 to 20 carbon atoms. Specifically, methoxy, ethoxy, n-propoxy, isopropoxy, i-propyloxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, n-pentyloxy, neopentyloxy, Isopentyloxy, n-hexyloxy, 3,3-dimethylbutyloxy, 2-ethylbutyloxy, n-octyloxy, n-nonyloxy, n-decyloxy, benzyloxy, p-methylbenzyloxy, etc. May be, but is not limited thereto.

In the present invention, "aralkyl" refers to an aryl-alkyl group in which aryl and alkyl are as described above. Preferred aralkyls include lower alkyl groups. Non-limiting examples of suitable aralkyl groups include benzyl, 2-phenethyl and naphthalenylmethyl. Bonding to the parent moiety is made through alkyl.

In the present invention, “arylamino group” refers to an amine substituted with an aryl group having 6 to 30 carbon atoms.

In the present invention, "alkylamino group" refers to an amine substituted with an alkyl group having 1 to 30 carbon atoms.

In the present invention, "aralkylamino group" refers to an amine substituted with an aryl-alkyl group having 6 to 30 carbon atoms.

In the present invention, “heteroarylamino group” refers to an aryl group having 6 to 30 carbon atoms and an amine group substituted with a heterocyclic group.

In the present invention, "heteroaralkyl group" refers to an aryl-alkyl group substituted with a heterocyclic group.

In the present invention, "cycloalkyl" refers to a monovalent substituent derived from a monocyclic or polycyclic non-aromatic hydrocarbon having 3 to 40 carbon atoms. Examples of such cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and adamantine.

In the present invention, “heterocycloalkyl” refers to a monovalent substituent derived from a non-aromatic hydrocarbon having 3 to 40 carbon atoms, and at least one carbon in the ring, preferably 1 to 3 carbons, is N, O, S or Se Is substituted with a hetero atom such as. Examples of such heterocycloalkyl include, but are not limited to, morpholine, piperazine, and the like.

In the present invention, “alkylsilyl” refers to silyl substituted with alkyl having 1 to 40 carbon atoms, and “arylsilyl” refers to silyl substituted with aryl having 6 to 60 carbon atoms.

In the present invention, "condensed ring" means a condensed aliphatic ring, a condensed aromatic ring, a condensed heteroaliphatic ring, a condensed heteroaromatic ring, or a combination thereof.

In the present invention, "adjacent 　 groups are bonded to each other to form a ring" means 　adjacent 　 groups are bonded to each other to form a substituted or unsubstituted aliphatic hydrocarbon ring; A substituted or unsubstituted aromatic hydrocarbon ring; Substituted or unsubstituted aliphatic heterocycle; Substituted or unsubstituted aromatic heterocycle; Or it means to form a condensed ring of these.

In the present invention, the "aliphatic hydrocarbon ring" refers to a ring that is not aromatic and consists of only carbon and hydrogen atoms.

Examples of the "aromatic hydrocarbon ring" in the present invention include a phenyl group, a naphthyl group, an anthracenyl group, and the like, but are not limited thereto.

In the present invention, "aliphatic heterocycle" refers to an aliphatic ring containing one or more heteroatoms.

In the present invention, "aromatic heterocycle" refers to an aromatic ring containing at least one heteroatom.

In the present invention, the aliphatic hydrocarbon ring, the aromatic hydrocarbon ring, the aliphatic heterocycle and the aromatic heterocycle may be monocyclic or polycyclic.

In the present invention, "substitution" means that the hydrogen atom bonded to the carbon atom of the compound is replaced with another substituent, and the position to be substituted is not limited as long as the position at which the hydrogen atom is substituted, that is, the position where the substituent can be substituted, and when two or more are substituted , Two or more substituents may be the same or different from each other. The substituent is hydrogen, deuterium, cyano group, nitro group, halogen group, hydroxy group, alkyl group having 1 to 30 carbon atoms, alkenyl group having 2 to 30 carbon atoms, alkynyl group having 2 to 24 carbon atoms, heteroalkyl group having 2 to 30 carbon atoms, carbon number Aralkyl group having 6 to 30, aryl group having 5 to 30 carbon atoms, heteroaryl group having 2 to 30 carbon atoms, heteroarylalkyl group having 3 to 30 carbon atoms, alkoxy group having 1 to 30 carbon atoms, alkylamino group having 1 to 30 carbon atoms, carbon number It may be substituted with one or more substituents selected from the group consisting of an arylamino group having 6 to 30, an aralkylamino group having 6 to 30 carbon atoms, and a hetero arylamino group having 2 to 24 carbon atoms, but is not limited to the above examples.

The present invention relates to a composition for preventing or treating depression comprising a protein expression inhibitor and to a use thereof, and by inhibiting protein expression in microglia, it is possible to exhibit excellent antidepressant effect due to the interaction between neuro-microglia. have.

In addition, it is possible to exhibit excellent antidepressant effect by passing through the blood-brain barrier only by treatment without a separate drug carrier.

1 is a result of confirming the inhibition of protein phosphorylation in brain tissue by a compound administered to a mouse through intraperitoneal injection according to an embodiment of the present invention.
2 is a result of confirming the inactivity time by performing a forced swimming experiment and a tail hanging experiment with respect to the compound-treated group according to an embodiment of the present invention.
3 is a compound treated with a neuron (HT22, N2a), microglia (BV2), astrocytes (C8-D1A), glioblastoma cancer cells (U373MG, U87MG) according to an embodiment of the present invention, and protein This is the result of confirming the effect of inhibiting phosphorylation.
4 is a result of confirming the effect on the activity of antidepressant pathways AKT, GSK3β, ERK1/2 and CREB in microglia (BV2) and neuronal cells (HT22) according to an embodiment of the present invention.
5 is a result of confirming the effect on the activity of antidepressant pathways AKT, GSK3β, ERK1/2, and CREB in astrocytes (C8-D1A) and neurons (HT22) according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

Microglia is a type of glial cell system that exists in the central nervous system and functions to assist or protect nerve functions in the brain and spinal cord. In particular, the interaction of neuron-microglia is known as one of the representative mechanisms for maintaining the neuron-immune system normally, and regulating the activation state of the microglia is very important for maintaining the balance of the neuro-immune system.

The imbalance of the neuro-immune system is also the cause of several neuropsychiatric disorders such as Alzheimer's disease, Parkinson's disease, and major depressive disorder.

However, it is not known how the neuro-microglia interaction functions in these neuropsychiatric disorders. Signal transduction and transcriptional activator 3 are generally expressed by various cytokines and growth factors in immune cells, and are genes that play a role in causing cell activation and inflammatory reactions.

In previous studies, microglia-specific signal transduction and deletion of transcriptional activator 3 have been studied to maintain the balance of the neuroimmune system and alleviate symptoms through neuro-microglia interactions in neuropsychiatric disorders such as major depressive disorder. Performed.

First, microglia-specific signal transduction and transcriptional activator 3 deficient mouse models were made to inhibit microglia signal transduction and transcriptional activator 3 gene activity. Forced swimming, tail hanging, and sucrose preference were performed to confirm antidepressant behavior.

Microglia cells deficient in signal transduction and transcriptional activator 3 interacted with surrounding neurons to increase the secretion of macrophage colony-stimulating factor (M-CSF) from neurons.

Interacting neurons autocrine M-CSF to activate ERK1/2 and Akt/GSK3β, known as antidepressant signaling pathways, and increase brain-derived nerve growth factor (BDNF) expression.

The activation of ERK1/2, Akt/GSK3β and the function of BDNF protein in neurons is well known to increase the release of neurotransmitters, which is the basis for explaining the antidepressant-like behavior shown in the mouse model. something to do.

The present invention uses a portion where the antidepressant effect due to the interaction between neuro-microglia by inhibiting the expression of the signal transduction and transcriptional activator 3 protein of microglia cells, signal transduction and transcription to improve and treat depression It relates to a compound for inhibiting the activator 3 protein.

Conventional microglia signal transduction and dysfunction of transcriptional activator 3 regulated depression-related behavior through M-CSF-mediated neuronal activity, but the present invention relates to microglia-specific signal transduction and transcriptional activator 3 It shows that it can be a new treatment strategy that can overcome the limitations of existing depression treatments through function inhibition.

The blood-brain barrier (BBB), which blocks blood vessels and nerves in the brain, is famous for its low permeability, so it is often not possible to pass necessary drugs. In order to be a treatment for brain diseases, knowing whether it is absorbed through the blood-brain barrier is very important in developing drugs for the brain.

By confirming the inhibition of signal transduction and transcriptional activator 3 phosphorylation in the brain tissue of the animal model administered with the compound of the present invention, it was confirmed that the blood-brain barrier can be sufficiently passed by only treatment without the need for other carriers.

More specifically, a composition for preventing or treating depression comprising a protein expression inhibitor according to an embodiment of the present invention may include a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

[Formula 2]

here,

A is a substituent represented by Chemical Formula 2 or Chemical Formula 3,

* Means the part to be joined,

L ₁ and L ₂ are the same as or different from each other, and each independently a single bond, a substituted or unsubstituted alkylene group having 1 to 10 carbon atoms and a substituted or unsubstituted arylene group having 6 to 10 carbon atoms,

X ₁ is selected from the group consisting of O, S and N (R _{7 ),}

Y ₁ and Y ₂ are the same as or different from each other, and each independently N or C (R ₈ ),

R ₁ to R ₈ are the same as or different from each other, and each independently hydrogen, deuterium, cyano group, nitro group, halogen group, hydroxy group, alkylthio group having 1 to 4 carbon atoms, alkyl group having 1 to 30 carbon atoms, 1 to 20 carbon atoms Cycloalkyl group, C2-C30 alkenyl group, C2-C24 alkynyl group, C7-C30 aralkyl group, C6-C30 aryl group, C5-C60 heteroaryl group, C6-C6 30 heteroarylalkyl group, C 1 to C 30 alkoxy group, C 1 to C 30 alkylamino group, C 6 to C 30 arylamino group, C 6 to C 30 aralkylamino group, C 2 to C 24 heteroarylamino group, C 1 It is selected from the group consisting of an alkylsilyl group of to 30, an arylsilyl group having 6 to 30 carbon atoms, and an aryloxy group having 6 to 30 carbon atoms, and may be combined with adjacent groups to form a substituted or unsubstituted ring,

When the R ₁ to R ₈ are substituted, hydrogen, deuterium, cyano group, nitro group, halogen group, hydroxy group, alkyl group having 1 to 30 carbon atoms, alkenyl group having 2 to 30 carbon atoms, alkynyl group having 2 to 24 carbon atoms, carbon number 2 to 30 heteroalkyl group, C 6 to C 30 aralkyl group, C 3 to C 20 cycloalkyl group, C 3 to C 20 heterocycloalkyl group, C 5 to C 30 aryl group, C 2 to C 30 heteroaryl group, C carbon number Substituted with a substituent selected from the group consisting of a 3 to 30 heteroarylalkyl group, a C 1 to C 30 alkoxy group, a C 1 to C 30 alkylsilyl group, a C 6 to C 30 arylsilyl group, and a C 6 to C 30 aryloxy group And, when substituted with a plurality of substituents, they are the same as or different from each other.

The L ₁ and L2 may be the same as or different from each other, and may each independently be a single bond or an alkylene group having 1 to 10 carbon atoms.

The Y ₁ and Y ₂ may be N.

X ₁ may be O or S.

The pharmaceutical composition for preventing or treating depression may include the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as one or two or more active ingredients.

The compound represented by Formula 1 may be selected from the group consisting of the following compounds:

A pharmaceutical composition for preventing or treating depression comprising a protein expression inhibitor according to another embodiment of the present invention may contain the compound in a pharmaceutically effective amount.

Compound synthesis

Preparation Example 1.3-((2,4-dichlorophenoxy)methyl-5-(trichloromethyl)-1,2,4-oxadiazole (3-((2,4-dichlorophenoxy)methyl)-5 Preparation of -(trichloromethyl)-1,2,4-oxadiazole; ODZ 10117)

<1-1> Preparation of 2-(2,4-dichlorophenoxy)acetonitrile

[Scheme 1]

2,4-dichlorophenol (1g, 6.10 mmol) was dissolved in dimethylformamide (8 mL), potassium carbonate (840 mg, 6.10 mmol) was added at room temperature, followed by bromoacetonitrile (0.44 mL, 6.10 mmol). ) Was dissolved in dimethylformamide (3 ml) and dropped slowly. After stirring at room temperature for 24 hours, water was added to the reaction mixture, followed by extraction with ethyl acetate. Thereafter, the organic layer was washed with a saturated aqueous sodium sulfate solution, dried with magnesium sulfate, the solvent was removed under reduced pressure, and then the target compound (1.18g, 95%) was obtained through silica gel column chromatography (n-hexane: ethyl acetate = 3: 1). Obtained.

<1-2> Preparation of 2-(2,4-dichlorophenoxy)-N'-hydroxyacetimidamide

[Scheme 2]

Triethylamine (2.70 mL, 19.52 mmol) was dissolved in 50% ethanol aqueous solution (21 mL), and then hydroxylamine hydrochloride (1.36g, 19.52 mmol) was added at room temperature. After stirring at room temperature for 5 minutes, the obtained 2-(2,4-dichlorophenoxy)acetonitrile was dissolved in ethanol (83 ml) and added thereto, followed by refluxing at 100° C. for 3 hours. Thereafter, the reaction mixture was diluted with water, filtered, and washed again with water to obtain the target compound (2.71 g, 77%).

¹ H NMR (500 MHz, CDCl3) δ4.58 (s, 2H), 4.85 (s, 2H), 6.53 (s, 1H), 6.96 (d, J=8.8 Hz,1H), 7.17 (dd, J= 2.3 Hz, 8.8 Hz, 1H), 7.37 (d, J=2.4 Hz, 1H)

<1-3> Preparation of 3-((2,4-dichlorophenoxy)methyl-5-(trichloromethyl)-1,2,4-oxadiazole (ODZ10117)

[Scheme 3]

The obtained 2-(2,4-dichlorophenoxy)-N'-hydroxyacetimidamide (100 mg, 0.43 mmol) and trichloroacetonitrile (0.040 ml, 0.43 mmol) were mixed with dimethylformamide (1 ml). After dissolving in, para-toluenesulphonic acid (41 mg, 0.22 mmol) and zinc chloride (30 mg, 0.22 mmol) were added. Then, the reaction compound was refluxed at 80° C. for 16 hours, and the organic layer was washed with sodium bicarbonate, dried over magnesium sulfate, and the solvent was removed under reduced pressure. Thereafter, the remaining residue was subjected to silica gel column chromatography (n-hexane: ethyl acetate = 10: 1) to obtain the target compound (81 mg, 53%).

¹ H NMR (300 MHz, CDCl3) δ5.27 (s, 2H), 7.01 (d, J=8.8 Hz, 1H),7.20 (dd, J=2.5 Hz, 8.7 Hz, 1H), 7.40 (d, J =2.6 Hz, 1H)

Preparation Example 2. 3-((2,4-dichlorophenoxy)methyl)-5-propoxy-1,2,4-oxadiazole (3-((2,4-dichlorophenoxy)methyl)-5-propoxy -1,2,4-oxadiazole; ODZ10181)

[Scheme 4]

3-((2,4-dichlorophenoxy)methyl-5-(trichloromethyl)-1,2, obtained after dissolving sodium propoxide (119 mg, 1.45 mmol) in dimethylformamide (10 ml) After adding 4-oxadiazole (350 mg, 0.97 mmol) at room temperature, the mixture was stirred for 10 minutes at room temperature, the reaction mixture was washed with a saturated aqueous sodium sulfate solution and brine, and the organic layer was dried over magnesium sulfate. Under the conditions, the solvent was removed, and the residue was subjected to silica gel column chromatography (n-hexane: ethyl acetate = 10: 1) to obtain the target compound (64 mg, 22%).

¹ H NMR (400 MHz, CDCl3) δ1.02 (t, J=7.4 Hz, 3H), 1.85 (qd, J=7.0 Hz, 14.1 Hz, 2H), 4.47 (t, J=6.6 Hz, 2H), 5.05 (s, 2H), 7.00 (d, J=8.8 Hz, 1H), 7.17 (dd, J=2.0 Hz, 8.8 Hz, 1H), 7.37 (d, J=1.9 Hz, 1H)

Preparation Example 3. 5-(Secondary-butoxy)-3-((2,4-dichlorophenoxy)methyl)-1,2,4-oxadiazole (5-(sec-butoxy)-3-(( Preparation of 2,4-dichlorophenoxy)methyl)-1,2,4-oxadiazole; ODZ8292)

[Scheme 5]

Sodium secondary-butoxide (613 mg, 6.37 mmol) was dissolved in dimethylformamide (20 ml), and the obtained 3-((2,4-dichlorophenoxy)methyl-5-(trichloromethyl)-1,2, 4-oxadiazole (462 mg, 1.27 mmol) was added at room temperature and stirred at room temperature for 10 minutes Thereafter, the resulting reaction mixture was washed with a saturated aqueous sodium sulfate solution and brine, and the organic layer extracted with ethyl acetate was dried over magnesium sulfate. Next, the solvent was removed under reduced pressure, and the residue was subjected to silica gel column chromatography (n-hexane: ethyl acetate = 15: 1) to obtain the target compound (78 mg, 19%).

¹ H NMR (400 MHz, CDCl3) δ 0.97 (t, J=7.4 Hz, 3H), 1.42 (d, J=6.2 Hz, 3H), 1.67-1.87 (m, 2H), 5.05 (s, 2H) , 7.00 (d, J=8.8 Hz, 1H), 7.17 (dd, J=2.0 Hz, 8.8 Hz, 1H), 7.37 (d, J=2.0 Hz, 1H)

Cell culture

Neurons (HT22, N2a), microglia (BV2), astrocytes (C8-D1A), glioblastoma cancer cells (U373MG, U87MG) were purchased from ATCC (Manassas, VA). _{Cells were cultured in a 37°C, 5% CO 2} incubator using Dulbecco's modified Eagle's medium (DMEM, Hyclone, Logan, UT) medium containing 10% FBS and 1% penicillin streptomycin. The cultured cells were dispensed in a 6-well plate in ^{a number of 2×10 5} wells and adhered for 24 hours, and then the compounds were treated at various concentrations for 3 hours.

Co-culture experiment

In order to investigate the effect of microglia or astrocytes on the activation of neurons, transwell culture (mixed culture) in which only secretory factors are exchanged without physical interference between cell groups was used, and for this purpose, micropores of 0.4 μm were used. Cell culture inserts (Falcon cell culture inserts, Corning, Durham, NC) were used. BV2 cells or C8D-1A cells were dispensed into a 6-well plate and HT22 cells were dispensed into a cell culture insert in ^{a number of 2×10 5} /well, respectively, and adhered for 24 hours. Performed.

Western blot analysis

After washing the cells with cold PBS, cell lysis buffer (1% Triton X-100, 50mM Tris-HCl (pH 7.4), 0.35M NaCl, 0.5% Nonidet P-40, 10% glycerol, 0.1% SDS, 1mM EDTA) , 1mM EGTA, 0.2mM Na ₃ VO ₄ , 1mM PMSF, 0.5mM NaF was dissolved using). After incubation on ice for 30 minutes, the dissolved solution was centrifuged at 13000 rpm for 15 minutes at 4° C. to remove insoluble debris, and only the solution containing protein was extracted.

Protein quantification was performed using a protein assay dye reagent (Bio-Rad, Hercules, CA, USA), and the same concentration of protein was separated by electrophoresis in SDS-polyacrylamide through quantification, and nitrocellulose Transferred to the membrane (GE Healthcare, Pittsburgh, PA).

The membrane was blocked for 1 hour at room temperature with TBS-T (Tris-buffered saline/0.05% Tween-20) solution containing 5% skim milk, and primary antibodies (p-STAT3, STAT3, p-Akt , Akt, P-GSK3β, GSK3β, p-ERK, ERK, pCREB, CREB, α-tubulin) at 4° C. for overnight.

This was washed three times for 10 minutes each using TBS-T, and a secondary antibody labeled with HRP (horeradish peroxidase) was reacted at room temperature for 1 hour. For visualization, an enhanced chemiluminescence system was used.

Experimental Example 1

Western blot analysis was performed to confirm whether the expression of the STAT3 phosphorylated protein was decreased compared to the control in the brain tissue of the mice administered with the compound via intraperitoneal injection. For quantitative comparison of p-STAT3 expression, total STAT3 and α-tubulin antibody as a housekeeping gene were used.

As shown in Figure 1, compared to the control group, the expression of p-STAT3 was significantly reduced in the brain tissue of the mice administered with the compound. Through the above results, it was confirmed that ODZ10017 can inhibit p-STAT3 expression by passing through the blood-brain barrier.

Experimental Example 2

In order to confirm the antidepressant effect of the compound, the mice forced swimming method and the mouse tail hanging test method were performed.

As shown in Figure 2, 10mg / kg administration of the compound significantly reduced the inactivity time compared to the control group. Administration of 30mg/kg of the compound significantly reduced inactivity time similar to that of imipramine 30mg/kg, an antidepressant. These results show the behavioral effects of the compounds to improve and treat depression.

Experimental Example 3

In in vitro experiments, it was confirmed that the treatment of the compound as an inhibitor of STAT3 activity was effective on neurons (HT22, N2a), microglia (BV2), astrocytes (C8-D1A), glioblastoma cancer cells (U373MG, U87MG) .

As shown in FIG. 3, the expression of p-STAT3 in all cells treated with the compound at each concentration (5, 10, 20, 30, 40 μM) for 3 hours was significantly decreased in a concentration gradient. As an inhibitor of STAT3 activity, it was demonstrated that Compound X is efficacious in various cells.

Experimental Example 4

In order to investigate the effect of inhibition of STAT3 activity on the activation of neurons through neuronal-microglia interactions in microglia cells, they were co-cultured through cell culture inserts.

After attaching BV2 cells to a 6-well plate and HT22 cells to a cell culture insert for 24 hours, co-culture was performed for 24 hours in a state treated with 20 μM compound. Subsequently, proteins were extracted from each cell and subjected to Western blot analysis.

As shown in FIG. 4, the expression of proteins of p-AKT, p-GSK3β, p-ERK1/2, and p-CREB, which are antidepressant pathways that control depression and mental illness, was increased in co-cultured HT22 cells by treatment with a compound. .

HT22 cells that were not co-cultured did not change the protein expression of p-AKT, p-GSK3β, p-ERK1/2, and p-CREB even after treatment with 20 μM compound for 24 hours.

Therefore, it was demonstrated that inhibition of STAT3 activity in microglia by compound treatment activates a protein exhibiting antidepressant effect in neurons through neuronal cell-microglia interaction.

Experimental Example 5

In order to investigate the effect of inhibition of STAT3 activity on the activation of neurons through neuronal-astrocytic interactions in astrocytes, they were cultured together through cell culture inserts.

After attaching C8-D1A cells to a 6-well plate and HT22 cells to a cell culture insert for 24 hours, co-culture was performed for 24 hours in a state treated with 20 μM compound. Subsequently, proteins were extracted from each cell and subjected to Western blot analysis.

As shown in FIG. 5, the protein expression of p-AKT, p-GSK3β, p-ERK1/2, and p-CREB was not changed in HT22 cells co-cultured by treatment with the compound. It was demonstrated that inhibition of STAT3 activity in astrocytes by compound treatment did not affect the activation of neurons.

Experimental Example 6

The antidepressant effect of the ODZ10117 compound was confirmed through previous experiments. The effect of mixing ODZ10181 and ODZ8292 in the ODZ10117 compound was confirmed.

When mixing, the experiment was carried out by mixing the compounds of each 20 μM concentration in the following weight ratio.

Example 1 Example 2 Example 3 Example 4 ODZ10117 One One One One ODZ10181 - One - One ODZ8292 - - One One

(Unit weight part)

The mixed compounds for Examples 2 to 4 were administered to mice, and a quantitative experiment was performed on the degree of expression. In Example 1, it was confirmed that p-STAT3 expression was decreased as shown in FIG. 1, and in Examples 2 to 4, it was confirmed that the expression decreased to the same or similar degree.

The results of the forced swimming and tail hanging experiment of mice are shown in Table 2 below. The experimental results were used for comparison, and the result of Example 1 was set to the index 5, and relative evaluation was performed.

Example 1 Example 2 Example 3 Example 4 Forced swimming 5 6 7 5 Tail hanging 5 5 8 5

Judging from the above experimental results, compared to Example 1, Examples 2 and 4 did not show a significant difference in results, whereas Example 3 confirmed significant experimental results.

That is, it was confirmed that the inactivity time was significantly reduced when ODZ8292 was mixed and used as a complex compound compared to the case where only ODZ10117 was treated alone.

In addition, for Examples 2 to 4, it was confirmed whether there is an effect on neurons (HT22, N2a), microglia (BV2), astrocytes (C8-D1A), glioblastoma cancer cells (U373MG, U87MG).

Similar to the previous experimental results, compared to Example 1, Examples 2 and 4 confirmed the effect of reducing p-STAT3 expression in cells to a similar degree, but it was confirmed that it was significantly reduced in Example 3.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention defined in the following claims are also present. It belongs to the scope of rights of

Claims (7)

A protein expression inhibitor comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient
Pharmaceutical composition for preventing or treating depression comprising a protein expression inhibitor:
[Formula 1]

[Formula 2]

here,
A is a substituent represented by Chemical Formula 2,
* Means the part to be joined,
L ₁ is a single bond,
L ₂ is a methylene group,
X ₁ is O,
Y ₁ and Y ₂ are N,
R ₁ and R ₃ are -Cl,
R ₂ , R ₄ and R ₅ are H,
R ₆ is hydrogen, deuterium, cyano group, nitro group, halogen group, hydroxy group, alkylthio group having 1 to 4 carbon atoms, alkyl group having 1 to 30 carbon atoms, cycloalkyl group having 3 to 20 carbon atoms, alkenyl group having 2 to 30 carbon atoms, Alkynyl group having 2 to 24 carbon atoms, aralkyl group having 7 to 30 carbon atoms, aryl group having 6 to 30 carbon atoms, heteroaryl group having 5 to 60 nuclear atoms, heteroarylalkyl group having 6 to 30 carbon atoms, alkoxy having 1 to 30 carbon atoms Group, C1-C30 alkylamino group, C6-C30 arylamino group, C6-C30 aralkylamino group, C2-C24 hetero arylamino group, C1-C30 alkylsilyl group, C6-C30 It is selected from the group consisting of an arylsilyl group and an aryloxy group having 6 to 30 carbon atoms, and may be combined with an adjacent group to form a substituted or unsubstituted ring, and when the R ₆ is substituted, hydrogen, deuterium, cyano group , A nitro group, a halogen group, a hydroxy group, an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 30 carbon atoms, an alkynyl group having 2 to 24 carbon atoms, a heteroalkyl group having 2 to 30 carbon atoms, an aralkyl group having 6 to 30 carbon atoms, 3 carbon atoms To 20 cycloalkyl group, C 3 to C 20 heterocycloalkyl group, C 5 to C 30 aryl group, C 2 to C 30 heteroaryl group, C 3 to C 30 heteroarylalkyl group, C 1 to C 30 alkoxy group, C carbon number It is substituted with a substituent selected from the group consisting of an alkylsilyl group having 1 to 30, an arylsilyl group having 6 to 30 carbon atoms, and an aryloxy group having 6 to 30 carbon atoms, and when substituted with a plurality of substituents, they are the same or different from each other. delete delete delete delete The method of claim 1,
The compound represented by Formula 1 is selected from the group consisting of the following compounds
Composition for preventing or treating depression comprising a protein expression inhibitor:

delete

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