KR20240138992A - Pharmaceutical Composition for preventing or treating epilepsy - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating epilepsy, comprising a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, and to a use in preventing or treating epilepsy.
The compound of chemical formula 1 can effectively inhibit neuronal cell death, neuroinflammation, cell necrosis, and synaptic damage caused by epilepsy, and thus can be usefully used for preventing, improving, or treating epilepsy.

Description

{Pharmaceutical Composition for preventing or treating epilepsy}

The present invention relates to a pharmaceutical composition for preventing or treating epilepsy, comprising a compound of formula 1, or a pharmaceutically acceptable salt thereof, and a method for preventing or treating epilepsy.

Epilepsy is a chronic disease group in which symptoms such as seizures occur repeatedly due to disordered electrical activity caused by abnormal excitement of nerve cells despite the absence of physical abnormalities. It is known that 10% of the world's population experiences it.

In particular, temporal lobe epilepsy (TLE) is the most common epilepsy in adults and can be caused by sclerosis of the medial temporal lobe, particularly the hippocampus, tumors of the medial temporal lobe, or stroke.

The causes of most epilepsy have not yet been clearly identified, and there is currently no breakthrough treatment or cure for epilepsy. Temporal lobe epilepsy in particular is known to be difficult to treat with other antiepileptic drugs.

Therefore, a comprehensive understanding of the molecular biological mechanisms and drug development are required to prevent the onset of epilepsy and to control and prevent seizure recurrence.

Meanwhile, sudden neuronal excitation in the brain has been reported to contribute to hippocampal sclerosis in patients with epilepsy (Steve et al., 2014;Tai et al., 2018), and in animal models reproducing epilepsy, it has been shown that long-term seizures, called status epilepticus (SE), can induce multifaceted processes in the hippocampus, resulting in neuronal death characterized by apoptosis or necrosis (Sloviter et al., 1996;Sankar et al., 1998;Fujikawa et al., 2000b). In addition, neuroinflammation, including reactive astrocytosis and microglial activation, has been frequently observed in the hippocampus after the onset of epilepsy, and it has been reported that neuronal death and neuroinflammation can cause symptoms through complex mechanisms in neurological diseases such as epilepsy (Yu et al., 2021).

Accordingly, the inventors of the present invention investigated the effects of epileptic seizures on hippocampal neuron death and neuroinflammatory responses, and confirmed that the indole derivative compound of the present invention is effective in preventing, improving, or treating epilepsy, thereby completing the present invention.

International Patent Publication WO2009/025478

The purpose of the present invention is to provide a use of a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof for preventing, improving or treating epilepsy.

The present invention provides a pharmaceutical composition for preventing or treating epilepsy, comprising a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

In the above formula,

n is an integer from 1 to 3,

m is 0 or 1,

A represents phenyl,

R ¹ is hydrogen, or C ₁ -C ₆ -alkyl,

R ² represents hydrogen, halogen or C ₁ -C ₆ -alkoxy, or hydroxy-C _1- C ₆ -alkyl, -(CH ₂ ) _p CO ₂ R ⁷ , -NHR ⁸ , -N(H)S(O) ₂ R ⁷ or -NHC(O)R ⁷ , wherein p is an integer from 0 to 3, R ⁷ represents hydrogen or C ₁ -C ₃ -alkyl, R ⁸ represents C _1- C ₃ -alkylpiperidinyl, or C _1- C ₃ -alkylsulfonyl,

R ³ represents hydrogen, halogen, C ₁ -C ₆ -alkyl or phenyl, or represents -(CH ₂ ) _p -heterocycle, which is a 5 to 6-membered ring containing 1 or 2 heteroatoms selected from S, N and O atoms, wherein p is an integer from 0 to 3, provided that when m is 0, R ³ is phenyl,

R ⁴ is halogen, C ₁ -C ₆ -alkyl, hydroxy-C _1- C ₆ -alkyl, -O-phenyl, -(CH ₂ ) _p CO ₂ R ⁷ , -(CH ₂ ) p -heterocycle, wherein the heterocycle contains 1 or 2 heteroatoms selected from _S , N and O atoms and is a 5 to 6 membered ring, or proline-N-carbonyl, wherein p is an integer from 0 to 3, R ⁷ is as defined above,

R ⁵ is hydrogen, or C ₁ -C ₆ -alkyl,

R ⁶ represents C ₁ -C ₆ -alkyl, C ₃ -C ₆ -cycloalkyl, heterocycle or heterocyclyl-C ₁ -C ₆ -alkyl, wherein the heterocycle is a 3 to 8 membered ring containing 1 to 3 heteroatoms selected from S, N and O atoms, and R ⁶ may be substituted with C ₁ -C ₆ -alkylamine, hydroxy-C ₁ -C ₆ -alkyl, or C ₁ -C ₆ -alkylsulfonyl.

The present invention also provides a method for preventing or treating epilepsy, comprising a step of administering a compound of formula 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof in a pharmaceutically acceptable amount.

According to the composition or method according to the present invention, the compound of chemical formula 1 can prevent or treat hippocampal neuronal cell death caused by epilepsy in in vitro and in vivo models, prevent, improve or treat synaptic damage and neuroinflammation of neurons caused by the onset of epilepsy, and has an effect of inhibiting hippocampal necrosis, so the compound of chemical formula 1 of the present invention can be usefully used for the prevention or treatment of epilepsy-related diseases.

Figure 1a is a microscopic image showing changes in degenerative neurons stained with Fluoro-Jade B (FJB) when treated with Example Compound 1 in a pilocarpine-induced epilepsy mouse model according to Experimental Example 1 (scale bar represents 200 μm).
Figure 1b is a graph showing the number of FJB-positive neurons in the hilus, CA1, and CA3 subfields of the hippocampus when treated with Example Compound 1 in a pilocarpine-induced epilepsy mouse model according to Experimental Example 1.
Figure 2a shows confocal images of the placebo treatment group and the groups treated with 10 μM and 30 μM of Example Compound 1 in the cell model induced with 0.9 mM [Mg ²⁺ ] _o or 0.1 mM [Mg ²⁺ ] _o according to Experimental Example 2. The red part shows MAP2, the green part shows PSD95, and the maximum z-stack of hippocampal neurons expressing them is shown.
Figure 2b is a graph showing the number of synaptic contacts distributed per 0.1 mm of a dendrite when treated with Example Compound 1 according to Experimental Example 2.
Figure 3a is a microscope image of immunohistochemical staining of Iba1 in the dentate gyrus (DG) when treated with Example Compound 1 according to Experimental Example 3, and the enlarged areas in each image are indicated by the rectangular areas in a, b, and c. The scale bar is 200 μm for the low-magnification images and 50 μm for the high-magnification images. The lower right graph is a graph showing the Iba1 immunoreactive area in the DG.
Figure 3b is a confocal image of the treatment with Example Compound 1 according to Experimental Example 3, where the green part is Iba1 and the blue part is DAPI, showing the maximum z-stack of hippocampal neurons expressing them. The lower graph is a graph showing the intensity of Iba1 expression in the dentate gyrus (DG).
Figure 3c is a microscope image of immunohistochemical staining of GFAP in the dentate gyrus (DG) when treated with Example Compound 1 according to Experimental Example 3, and the enlarged areas in each image are indicated by the rectangular areas in a, b, and c. The scale bar is 200 μm for the low-magnification images and 50 μm for the high-magnification images. The lower right graph is a graph showing the GFAP immunoreactive area in the DG.
Figure 3d is a confocal image of the treatment with Example Compound 1 according to Experimental Example 3, where the red portion is GFAP and the green portion is DAPI, showing the maximum z-stack of hippocampal neurons expressing them. The lower graph is a graph showing the intensity of GFAP expression in the dentate gyrus (DG).
Figure 4a shows the Western blot results when treating with Example Compound 1 according to Experimental Example 4, and Figure 4b is a graph showing the relative expression levels of MLKL/GAPDH.

The present invention is described in detail below.

Meanwhile, each description and embodiment disclosed in the present application can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present application fall within the scope of the present invention. In addition, the scope of the present invention cannot be said to be limited by the specific description described below.

When a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may have other components, unless otherwise specifically stated.

The present invention provides a pharmaceutical composition for preventing or treating epilepsy, comprising a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

In the above formula,

n is an integer from 1 to 3,

m is 0 or 1,

A represents phenyl,

R ¹ is hydrogen, or C ₁ -C ₆ -alkyl,

R ² represents hydrogen, halogen or C ₁ -C ₆ -alkoxy, or hydroxy-C _1- C ₆ -alkyl, -(CH ₂ ) _p CO ₂ R ⁷ , -NHR ⁸ , -N(H)S(O) ₂ R ⁷ or -NHC(O)R ⁷ , wherein p is an integer from 0 to 3, R ⁷ represents hydrogen or C ₁ -C ₃ -alkyl, R ⁸ represents C _1- C ₃ -alkylpiperidinyl, or C _1- C ₃ -alkylsulfonyl,

R ³ represents hydrogen, halogen, C ₁ -C ₆ -alkyl or phenyl, or represents -(CH ₂ ) _p -heterocycle, which is a 5 to 6-membered ring containing 1 or 2 heteroatoms selected from S, N and O atoms, wherein p is an integer from 0 to 3, provided that when m is 0, R ³ is phenyl,

R ⁴ is halogen, C ₁ -C ₆ -alkyl, hydroxy-C _1- C ₆ -alkyl, -O-phenyl, -(CH ₂ ) _p CO ₂ R ⁷ , -(CH ₂ ) p -heterocycle, wherein the heterocycle contains 1 or 2 heteroatoms selected from _S , N and O atoms and is a 5 to 6 membered ring, or proline-N-carbonyl, wherein p is an integer from 0 to 3, R ⁷ is as defined above,

R ⁵ is hydrogen, or C ₁ -C ₆ -alkyl,

R ⁶ represents C ₁ -C ₆ -alkyl, C ₃ -C ₆ -cycloalkyl, heterocycle or heterocyclyl-C ₁ -C ₆ -alkyl, wherein the heterocycle is a 3 to 8 membered ring containing 1 to 3 heteroatoms selected from S, N and O atoms, and R ⁶ may be substituted with C ₁ -C ₆ -alkylamine, hydroxy-C ₁ -C ₆ -alkyl, or C ₁ -C ₆ -alkylsulfonyl.

The compound of chemical formula 1 is a compound disclosed in International Patent Publication No. WO2009-025478, and is a substance known to exhibit preventive or therapeutic and improving effects on cell necrosis and related diseases.

The present invention has confirmed the effect of preventing, improving, and otherwise treating epilepsy by treating a compound of the above chemical formula 1 to a cell model and an animal model of epilepsy, thereby inhibiting hippocampal neuronal cell death and neuroinflammation, and hippocampal necrosis, and improving synaptic damage. Accordingly, the present invention has discovered a novel use of the compound of the above chemical formula 1.

The compound of the above chemical formula 1 of the present invention can be used in the form of a pharmaceutically acceptable salt thereof. In particular, the pharmaceutically acceptable salt may be an acid addition salt formed by a free acid. Here, the acid addition salt can be obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, and the like, non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids, and the like, organic acids such as trifluoroacetic acid, acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, and the like. Such pharmaceutically acceptable salts may include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphate chloride, bromides, iodides, fluorides, acetates, propionates, and the like.

The composition of the present invention may include the compound of formula 1, a pharmaceutically acceptable salt thereof, as well as all salts, isomers, hydrates and/or solvates that can be prepared by conventional methods.

In this specification, "isomer" may mean a compound of the present invention or a salt thereof having the same chemical formula or molecular formula but structurally or sterically different. Such isomers include structural isomers such as tautomers, isomers such as R or S isomers having an asymmetric carbon center, geometric isomers (trans, cis), and optical isomers (enantiomers). All of these isomers and mixtures thereof are also included in the scope of the present invention.

As used herein, "hydrate" may mean a compound of the present invention or a salt thereof, which contains a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. The hydrate of the compound represented by the chemical formula 1 of the present invention may contain a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. The hydrate may contain 1 equivalent or more, preferably, 1 to 5 equivalents of water. Such a hydrate may be prepared by crystallizing a compound represented by the chemical formula 1 of the present invention, an isomer thereof, or a pharmaceutically acceptable salt thereof, from water or a solvent containing water.

As used herein, the term "solvate" may mean a compound of the present invention or a salt thereof, which contains a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents therefor include solvents that are volatile, non-toxic, and/or suitable for administration to humans.

As used herein, the term “alkyl” means an aliphatic hydrocarbon radical. An alkyl may be a “saturated alkyl” containing no alkenyl or alkynyl moiety, or an “unsaturated alkyl” containing at least one alkenyl or alkynyl moiety, and may have from 1 to 20 carbon atoms unless otherwise defined.

The term 'alkoxy', unless otherwise defined, means alkyl-oxy having 1 to 10 carbon atoms.

The term 'cycloalkyl', unless otherwise defined, means a saturated aliphatic 3- to 10-membered ring. Typical cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term 'heterocycle', unless otherwise defined, means a 3 to 10 membered ring, preferably a 4 to 8 membered ring, more preferably a 5 to 6 membered ring, which contains 1 to 3 heteroatoms selected from the group consisting of N, O and S, which may be fused with benzo or C ₃ -C ₈ cycloalkyl, and which is saturated or contains 1 or 2 double bonds. It can also be used interchangeably with the term 'heterocyclyl'. Examples of heterocycles include, but are not limited to, pyrroline, pyrrolidine, imidazoline, imidazolidine, pyrazoline, pyrazolidine, pyran, piperidine, morpholine, thiomorpholine, piperazine, hydrofuran, and the like.

Other terms and abbreviations used in this specification, unless otherwise defined, can be interpreted as having the meaning commonly understood by those skilled in the art to which the present invention belongs.

In one embodiment of the present invention, in the compound of formula 1,

R ³ represents hydrogen, halogen, or phenyl, or represents -(CH ₂ ) _p -heterocycle wherein the heterocycle is morpholino, piperazinonyl, wherein p is an integer from 0 to 1, provided that when m is 0, R ³ can be phenyl.

In one embodiment of the present invention, in the compound of formula 1,

R ⁴ is halogen, C ₁ -C ₃ -alkyl, hydroxy-C _1- C ₃ -alkyl, -O-phenyl, -(CH ₂ ) _p CO ₂ -ethyl, -(CH ₂ ) p -heterocycle wherein the heterocycle is thiomorpholino, _morpholino , piperazinonyl, or pyrrolidinyl, or proline-N-carbonyl, wherein p can be an integer from 0 to 1.

In one embodiment of the present invention, in the compound of formula 1,

R ⁵ is hydrogen, or C ₁ -C ₃ -alkyl,

R ⁶ represents C ₁ -C ₃ -alkyl, C ₃ -C ₆ -cycloalkyl, heterocycle or heterocyclyl-C ₁ -C ₃ -alkyl, wherein the heterocycle is tetrahydro-2H-pyran, or piperidinyl, and when R ⁶ is heterocycle or heterocyclyl-C ₁ -C ₃ -alkyl, it may be substituted with C ₁ -C ₆ -alkylamine, hydroxy-C ₁ -C ₆ -alkyl, or C ₁ -C ₆ -alkylsulfonyl.

In the present invention, examples of the compound of chemical formula 1 include compounds 1 to 33 listed in Table 1 below or pharmaceutically acceptable salts thereof.

In a preferred embodiment of the present invention, the compound of formula 1 may be a compound of formula 2 below.

[Chemical formula 2]

In the present invention, the compound of chemical formula 1 may suppress, prevent, or treat convulsions or seizures caused by epilepsy.

In the present invention, the compound of chemical formula 1 can reduce, inhibit, or improve the occurrence of hippocampal neuronal cell death, synaptic damage, neuroinflammation, and necrosis of neurons.

In particular, in the present invention, the compound of chemical formula 1 can prevent hippocampal neuronal cell death caused by epilepsy, prevent or improve synaptic damage and neuroinflammation of neurons caused by the onset of epilepsy, and inhibit hippocampal necrosis.

In one embodiment of the present invention, it was confirmed that the compound of chemical formula 1 has a protective effect against hippocampal neuronal cell death caused by epileptic seizures. (See Experimental Example 1)

In one embodiment of the present invention, it was confirmed that the compound of chemical formula 1 inhibits the expression of Iba-1 (ionized calcium binding adaptor molecule 1) and GFAP (glial fibrillary acidic protein) in hippocampal neurons. (See Experimental Example 2)

In one embodiment of the present invention, it was confirmed that the compound of chemical formula 1 can reduce the activity of microglia and astrocytes, and specifically, can reduce the activity of microglia and astrocytes activated by epilepsy. (See Experimental Example 3)

In one embodiment of the present invention, it was confirmed that the compound of chemical formula 1 can reduce the expression of MLKL (Mixed Lineage Kinase domain-Like pseudokinase) in hippocampal neurons. (See Experimental Example 4)

In the present invention, the epilepsy is status epilepticus, temporal lobe epilepsy, infantile convulsions, febrile seizure, benign familial neonatal convulsion, early myoclonic encephalopathy, benign infantile myoclonic epilepsy, severe myoclonic epilepsy of infancy, myoclonic atopic epilepsy, Lennox-Gastaut syndrome, childhood absence seizure epilepsy, myoclonic absence seizure epilepsy, benign partial epilepsy. It may be at least one selected from the group consisting of epilepsy, Lando-Crefter syndrome, Dravet syndrome, and Rasmussen syndrome, but is not limited thereto. Specifically, in the present invention, it may be status epilepticus or temporal lobe epilepsy.

In the present specification, the compound of formula 1 or a pharmaceutically acceptable salt thereof in the composition may be characterized in that it is included in a concentration of 0.01 μM to 1000 μM. According to one specific example, the concentration may be, but is not limited to, 0.1 μM to 500 μM, 1 μM to 500 μM, 1 μM to 300 μM, 5 μM to 300 μM, 5 μM to 150 μM, 5 μM to 120 μM, 10 μM to 100 μM, or 20 μM to 40 μM.

In this specification, “treatment” means stopping or delaying the progression of a disease when used on a subject exhibiting symptoms of the disease, and “prevention” means stopping or delaying the signs of the disease when used on a subject not exhibiting symptoms of the disease but at high risk of such.

In the present invention, the “pharmaceutical composition” may include a pharmaceutically acceptable carrier as needed together with the compound of the present invention.

The compound of chemical formula 1 according to the present invention can be administered in various oral and parenteral dosage forms during clinical administration, and when formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc., and these solid preparations are manufactured by mixing one or more compounds of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included.

Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, etc. Non-aqueous solutions and suspensions can be used, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can be used, such as witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerol, and gelatin.

In addition, the present invention provides a method for preventing or treating epilepsy, comprising a step of administering a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof in a pharmaceutically acceptable amount.

Furthermore, the present invention provides a use of the compound of formula 1 or a pharmaceutically acceptable salt thereof in the prevention or treatment of epilepsy. In the above prevention or treatment method and use, the description of the compound of formula 1, the pharmaceutically acceptable salt, epilepsy, and the prevention or treatment can be applied in the same manner as described for the pharmaceutical composition.

In this specification, "administration" means introducing a given substance into a human or animal by any appropriate method, and the route of administration of the preventive or therapeutic composition according to the present invention may be oral or parenteral administration through any general route as long as it can reach the target tissue.

In the present invention, the “subject” requiring the administration may include both mammals and non-mammals. Here, examples of the mammals may include, but are not limited to, non-human primates such as humans, chimpanzees or monkeys, and livestock animals such as cows, horses, sheep, etc.

In addition, the effective dosage for the human body of the compound of chemical formula 1 of the present invention may vary depending on the patient's age, body weight, sex, dosage form, health condition, and disease severity, and is generally about 0.001-100 mg/kg/day, and preferably 0.01-35 mg/kg/day. When based on an adult patient weighing 70 kg, it is generally 0.07-7000 mg/day, and preferably 0.7-2500 mg/day, and may be administered once or several times a day at regular intervals depending on the judgment of a doctor or pharmacist.

The numerical values set forth in this specification are to be interpreted as including an equivalent range unless otherwise specified.

Example

Hereinafter, the present invention will be described in detail by the following experimental examples. However, the following experimental examples are only intended to illustrate the present invention, and the content of the present invention is not limited by the following experimental examples. In addition, since these experimental examples are only intended to help understanding of the present invention, the scope of the present invention is not limited by them in any sense.

<Pilocarpine-induced epilepsy mouse model and preparation of its sample>

Status epilepticus (SE) was induced by pilocarpine injection as previously described (Jeong et al., 2011; Cho et al., 2019; Choi et al., 2021). Briefly, scopolamine methyl nitrate (2 mg/kg) and terbutaline hemisulfate (2 mg/kg) were intraperitoneally (i.p.) administered to 6-week-old C57BL/6 male mice, followed by pilocarpine hydrochloride (280 mg/kg) intraperitoneally 30 minutes later to induce status epilepticus. The severity and duration of behavioral seizures were observed for 3 hours.

Seizure stages were assessed according to the Racine scale (Racine, 1972): stage 1: facial clonus; stage 2: head nodding; stage 3: forelimb clonus; stage 4: rearing; stage 5: rearing and falling.

Only animal models showing Stage 3 to 5 were defined as status epilepticus (SE) and were used in this study. Three hours after induction of status epilepticus, diazepam (i.p.; 10 mg/kg) was administered to calm behavioral seizures, and all experimental animals were grown in an incubator (30°C) and fed food soaked in water to promote recovery. This was done until the mice could maintain a stable body temperature on their own.

After treatment with Example 1, the mice were deeply anesthetized with a mixture of ketamine (50 mg/ml) and xylazine (23.3 mg/ml) (4:0.5), and then transcardially perfused with normal saline. The brains were removed and divided into two halves, fixed with 4% paraformaldehyde (pH 7.4) for 3 days, and then cryoprotected with 30% sucrose solution (in 0.1 M PBS) for 3 days. The brains were then frozen in liquid nitrogen, and coronal sections (serial hippocampal coronal sections, 30 μm thick) of the divided brains were obtained using a cryostat. For the other half of the brain, the hippocampus was harvested, frozen in liquid nitrogen, and used for Western blotting.

<Primary cell culture>

Hippocampal neurons were obtained from 19-day-old rat fetuses and primary cultured. First, the mother rat was anesthetized with 16.5% urethane (0.8 ml/100 g), and hippocampal tissue from the fetus was obtained using HHSS (HEPES-buffered Hanks's salt solution) without Ca2 ⁺ and Mg2 ⁺ at pH 7.45. Dissected hippocampi were triturated with a flame-narrowed Pasteur pipette, and pure neurons were cultured in Neurobasal medium containing 2% B27 supplement, 0.25% Glutamax I, and penicillin/streptomycin/amphotericin B (100 U/ml, 100 μg/ml, and 0.025 μg/ml, respectively), or glial cultures were cultured in DMEM medium containing 10% FBS, 10% HS, and penicillin/streptomycin/amphotericin B (100 U/ml, 100 μg/ml, and 0.025 μg/ml, respectively).

The hippocampal neurons were seeded at 1.1 × 10 ⁵ /200 μl on 60 mm circular cover glasses coated with 0.2 mg/ml Matrigel (BD Bioscience). Thereafter, the neurons were cultured in a cell incubator under the conditions of 10% CO ₂ and 90% air, pH 7.4, and 37°C, with 75% of the culture medium replaced every 3 and 7 days.

<Treatment of Example Compounds>

1) 0.1mM [Mg ²⁺ ] _o Cell (in vitro) model induced by and treatment with compound 1

Low concentrations of Mg ²⁺ are known to induce seizures in brain slices and induce severe synaptic activity in brain cultures. Furthermore, exposure to high frequency spontaneous recurrent epileptiform discharges (HFRSEDs) in hippocampal neurons cultured in media containing reduced magnesium concentrations of 0.1 mM induced neurotoxicity. Therefore, a cell model of epilepsy was prepared by exposing primary cultured neurons to 0.1 mM [Mg ²⁺ ] _o .

In the pretreatment group, placebo, 10 μM, 30 μM, and 100 μM of Example Compound 1 were treated 1 hour before induction of epilepsy, and in the posttreatment group, placebo, 10 μM, 30 μM, and 100 μM of Example Compound 1 were treated 1 hour after induction of epilepsy, and fixed for 3 hours or 23 hours.

2) Pilocarpine-induced epilepsy mouse model and treatment with compound 1

In vivo experiments on pilocarpine-induced epilepsy mouse models were conducted by dividing the mice into three groups: placebo treatment group, 20 mg/kg, or 40 mg/kg of Example Compound 1 treatment group.

One hour after diazepam administration, each group was intraperitoneally administered a placebo or a drug solution dissolved in 5% glucose at 20 mg/kg or 40 mg/kg. The placebo or Example Compound 1 at the above doses was administered daily for 7 days after pilocarpine administration.

<MTT assay>

Hippocampal neurons were seeded into 96-well plates and cultured under conditions of 10% _CO2 and 90% air, pH 7.4, and 37°C for 9 days.

The pretreatment group of Example Compound 1 was treated with Example Compound 1, then treated with MK-801 for 30 minutes, and then treated with neurobasal medium containing L-glutamine, 2% B27 supplement, 0.25% Glutamax I, penicillin/streptomycin/amphotericin B (100 U/ml, 100 μg/ml, 0.025 μg/ml) for 24 hours.

The post-treatment group of Example Compound 1 was treated with the neural basal medium, and then treated with Example Compound 1 1 hour later. After 24 hours, 20 μl of MTT solution (3-[4,5-dimethylthiazol-2-yl-2,5-dipheyltetrazolium bromide, 0.5 mg/ml) was treated and cultured in a cell incubator for 4 hours. The absorbance at 570 nm was measured using a microplate reader.

<Histologic assessments>

1) Immunocytochemistry

Primary hippocampal neurons were cultured and immunocytochemistry was performed 9 days later. First, the cells were washed three times with phosphate-buffered saline (PBS) and fixed in cold methanol stored at -20 °C. Then, the cells were washed three times with PBS and permeabilized with 0.3% Triton X-100 for 5 minutes. The cells were washed three times with PBS and blocked with 10% bovine serum albumin (BSA) for 1.5 hours at room temperature. After blocking, the primary antibody was incubated at 4 °C for 16 hours.

To identify synaptic contacts, MAP2 antibody (mouse anti-MAP2 (1:200 sigma-aldrich)) and PSD95 antibody (rabbit anti-PSD95 (1:200; Abcam)) were used.

To examine the morphological changes of neurons, Glia Fibrillary Acid Protein (GFAP) antibody (mouse anti-glial fibrillary acidic protein; 1:200; Millipore) and Iba1 antibody (rabbit anti-ionized calcium binding adaptor molecule 1; 1:1000; Wako) were used.

To visualize the immunostained neurons, the cells were incubated with secondary antibodies, Alexa Fluor 488 anti-rabbit IgG (1:500; Invitrogen) and Alexa Fluor 555 IgG antibody (1:500; Alexa Fluor 555 anti-mouse IgG (Invitrogen)), for 1.5 h at room temperature.

After washing three times with PBS, cell nuclei were stained with DAPI, and coverslips were mounted using VECTASHIELD Mounting Medium (Vector Laboratories, Inc., Burlingame, CA). Neurons stained with Alexa Fluor 488 (excitation, 488 nm; emission, > 519 nm)) and Alexa Fluor 555 (excitation, 555 nm; emission, > 565 nm)) were observed using a confocal microscope.

2) Immunohistochemistry

To evaluate neuronal apoptosis by pilocarpine-induced SE, Fluoro-Jade B (FJB) staining was performed (Choi et al., 2022). Brain hippocampal slices were placed on microscope slides and washed with distilled water for 3 min. The slides were then immersed in 0.06% KMnO ₄ solution for 7 min and incubated with 0.001% FJB (Biosensis, TR-150-FJB) solution diluted in 0.1% acetic acid for 30 min. The samples were washed with distilled water and dried in a 37 °C incubator for 1 h. The slides were then dehydrated with ethanol and xylene and treated with dibutyl phthalate polystyrene xylene mounting medium (DPX). After that, staining images were obtained with Lionheart FX (Biotek).

For reactive gliosis, immunohistochemical staining was performed using the free-floating method as previously reported (Brulet et al., 2017; Jang et al., 2019; Cho et al., 2020). Each section was first washed with phosphate-buffered saline (PBS) 0.01 M and then placed in a 3.5% H ₂ O ₂ solution for 15 min at room temperature to remove endogenous peroxidase activity. After washing again with 0.01 M PBS, the sections were blocked with 3% normal goat serum and 0.3% Tripton X-100 for 1 h, and the samples were incubated with Iba1 antibody (Iba1, 1:500, Wako, 019-19741) or GFAP antibody (1:1000, Millipore, MAB360) overnight at 4°C.

The next day, after further washing, the sections were incubated with peroxidase-conjugated secondary antibody for 2 h at room temperature, then developed with 0.05% 3,3'-diaminobenzidine (DAB), and the reaction was terminated with 0.05 M trizma buffer. Finally, they were mounted with DPX and observed under a light microscope (Olympus. BX51).

<Confocal imaging>

Primary hippocampal neurons were observed through a 60x objective using a confocal microscope (LSM 700, Carl Zeiss, Germany). A total of eight sections were obtained at 1 μm intervals along the z-axis of the cell using the z-stack imaging technique, and they were merged to obtain one image. Using an Ar ion laser, GFP can be excited at 488 nm and emits light at 519 nm (10 nm band pass), and RFP can be excited at 555 nm (HeNelaser) and emit light at 565 nm.

<Western Blot>

Western blotting was performed as described in a previous study (Choi et al., 2021). Briefly, hippocampi were lysed in ProEX™CETi lysis buffer (TransLab Biosciences), and the lysate was centrifuged to extract proteins from the supernatant. Each protein was separated on a 12% SDS-polyacrylamide gel, transferred to a PVDF membrane, washed with Tris-buffered saline containing 0.1% Tween-20 (TBST), and the membrane was incubated with 3% bovine serum albumin (BSA) in TBST for 1 h at room temperature. The membrane was then incubated with anti-MLKL antibody and left on a shaker at 4 °C overnight. Secondary anti-rabbit IgG (1:2,000, GeneTex) conjugated with horseradish peroxidase was added to the membrane and kept at room temperature for 2 h. Subsequently, bands were detected using an Enhanced chemiluminescent detector kit (Elpis Biotech, Inc.). For loading control, the membranes were stripped with Restore Western Blot Stripping buffer (Thermo Scientific) and reprobed with mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (1:2000, GeneTex) and visualized on ImageQuant LAS 4000 (Fujifilm), and the immunoreactivity of each band was relatively quantified using densitometry.

<Microscopic analysis and quantification>

Immunoreactive cells or FJB-positive cells were quantified using a previously published method (Choi et al., 2022). Each marker-positive cell was counted in every ^12th coronal section throughout the hippocampus, and the total number of FJB-expressing cells in each experimental animal was estimated by adding all the counts and multiplying by 24.

The CA1 and CA3 pyramidal cell layers (PCLs) were distinguished by PCL thickness and soma size. The hilus was defined as a triangular region connecting the endpoints of the upper and lower granule cell layers (GCLs), excluding the CA4 PCL.

For quantitative analysis of reactive gliosis, the ratio of Iba1- and GFAP-immunoreactivity in the dentate gyrus (DG) was analyzed using NIH ImageJ software. Specifically, the pixel intensity of the non-tissue area in each image was measured to establish the background intensity, and then the background intensity was used as the threshold for reactivity to measure Iba1- and GFAP-immunoreactivity. Finally, the percentage of marker immunoreactive area relative to the DG area value was calculated for all images, and the values were then averaged for each mouse.

Experimental Example 1. Evaluation of the effect of preventing epilepsy-induced hippocampal neuron death

The neuroprotective effect of Example Compound 1 was examined in a cell model induced by 0.1 mM [Mg ²⁺ ] _o . Primary cell cultured hippocampal neurons were treated with Example Compound 1 at concentrations of 10 μM, 30 μM, and 100 μM before exposure to 0.9 mM [Mg ²⁺ ] _o medium or 0.1 mM [Mg ²⁺ ] _o medium for 24 h (0.1 mM Mg pretreatment group), or after exposure to 0.1 mM [Mg ²⁺ ] _o medium for 24 h, Example Compound 1 was treated 1 h later at concentrations of 10 μM, 30 μM, and 100 μM (0.1 mM Mg posttreatment group).

It was measured by the MTT assay method, and the results are shown in Table 2. In Table 2, “0.9 mM Mg CON” is data that was integrated because there was no epilepsy induction in both the groups treated with Example Compound 1 (pre-treatment group, post-treatment group) before and after exposing the neurons to 0.9 mM [Mg ²⁺ ] _o medium. The data in Table 2 is the cell viability shown in each experimental group when the data of the 0.9 mM Mg CON placebo treatment group is considered as 100%.

Placebo group 10 μM 30 μM 100 μM 0.9 mM Mg CON 100 ± 1.0% 95 ± 2.6% 99.2 ± 1.7% 84.7 ± 0.7% 0.1 mM Mg pretreatment group 76.1 ± 1.6% 76.3 ± 2.4% 78.2 ± 1.4% 75.2 ± 5.3% 0.1 mM Mg post-treatment group 76.1 ± 1.6% 79.0 ± 2.5% 85.4 ± 1.6% 78.8 ± 2.3%

As shown in Table 2 above, the post-treatment group treated with Example Compound 1 in cells in which epilepsy was induced by treatment with 0.1 mM [Mg ²⁺ ] _o exhibited an effect of inhibiting neuronal cell death. In particular, treatment with 30 μM Example Compound 1 exhibited an excellent effect of inhibiting neuronal cell death.

In addition, the 0.1 mM Mg pretreatment group, in which Example Compound 1 was treated before exposing the neurons to 0.1 mM [Mg ²⁺ ] _o medium, also showed a neuronal cell death inhibition effect, although smaller than the post-treatment group. In “0.9 mM Mg CON” where epilepsy is not induced, cell viability slightly decreased due to treatment with Example Compound 1, but the 0.1 mM Mg pretreatment group increased cell viability or maintained it at a similar value, indicating that it is effective in cell survival.

Additionally, the ability of compound 1 to reduce excitotoxic hippocampal neuronal death was evaluated using a pilocarpine-induced SE mouse model by quantitative analysis of FJB-labeled cells in each hippocampus (dentate gyrus, CA1, CA3).

Placebo group Example compound 1
20 mg/kg Example compound 1
40 mg/kg Tooth and gums 15,807 ± 2,286 9,478 ± 3,324 10,508 ± 747 CA1 26,278 ± 6,382 24,480 ± 5,194 23,631 ± 6,121 CA3 32,479 ± 9,507 31,229 ± 6,779 48,052 ± 15,483

As shown in Table 3 and Fig. 1, the number of FJB-positive neurons in the dentate gyrus was significantly reduced in the compound 1 treatment group compared to the placebo treatment group. The number of FJB-positive neurons in CA1 and CA3 was slightly reduced or increased compared to the placebo treatment group. This suggests that the neuroprotective effect of compound 1 differs depending on the location in the hippocampus. In addition, it is shown that treatment with compound 1 has a significant neuroprotective effect in the dentate gyrus.

Experimental Example 2. Evaluation of synaptic protection effect after epileptic seizure

The effect of treatment with Example Compound 1 on synaptic contacts was evaluated in a cell model of epilepsy induced by 0.1 mM [Mg ²⁺ ] _o . Immunocytochemistry was performed using synaptic contacts with PSD95 (postsynaptic density protein 95), a marker of excitatory synapses. PSD95 and MAP2 antibodies were fluorescently labeled, respectively, and the number of synaptic contacts distributed per 0.1 mm of dendrites recognized by MAP2 was quantitatively measured. Hippocampal neurons were exposed to 0.1 mM [Mg ²⁺ ] _o medium, and treated with Example Compound 1 at concentrations of 10 μM and 30 μM after 1 hour. In Fig. 2 , “CON” represents a group in which neurons were exposed to 0.9 mM [Mg ²⁺ ] _o medium and then treated with placebo or Example Compound 1.

Placebo group 10 μM 30 μM Number of synaptic contacts 214.8 ± 17.1 242.3 ± 22.8 329.3 ± 23.3

As shown in Table 4 and Fig. 2b, the group treated with the compound of Example 1 showed a higher number of synaptic contacts compared to the placebo treatment group. Accordingly, it was confirmed that the synaptic damage caused by the treatment with 0.1 mM [Mg ²⁺ ] _o was significantly improved by the treatment with the compound of Example 1.

Experimental Example 3. Evaluation of the effect of reducing neuroinflammation after epileptic seizures

Immunohistochemistry for Iba1 (ionized calcium-binding adapter molecule 1) and GFAP (glial fibrillary acid protein) was performed in a pilocarpine-induced SE mouse model to evaluate the activation of astrocytes and microglia in the dentate gyrus (DG), and the results are presented in Tables 5 and 6 and Fig. 3.

Placebo group Example compound 1
20 mg/kg Example compound 1
40 mg/kg Iba1-immunoreactive region 10.61 ± 1.40% 5.85 ± 0.66% 4.69 ± 0.74% GFAP-immunoreactive area 24.00 ± 2.43% 22.62 ± 2.35% 20.21 ± 1.23%

Figure 3a shows microscopic images after immunohistochemical staining of Iba1 in the dentate gyrus (DG), and hypertrophic activated microglia were identified in all groups.

As shown in the graphs in Table 5 and Fig. 3, the placebo treatment group showed high immunoreactivity to Iba1, indicating high astrocyte and microglia activation, whereas the 20 mg/kg and 40 mg/kg treatment groups of Example Compound 1 significantly reduced immunoreactivity to Iba1.

GFAP-immunoreactive area is a factor associated with seizure-induced reactive astrocytosis, and a decrease in GFAP-immunoreactive area was confirmed by treatment with Example Compound 1.

As can be seen in Fig. 3c, hypertrophic GFAP-expressing astrocytes were observed in the placebo-treated group (black arrows), but in the enlarged image of the compound 1-treated group, hypertrophic GFAP-expressing astrocytes were hardly found, and instead, arborescent or less hypertrophic and branched astrocytes were observed (white arrows). This means that the morphological change of astrocytes from an activated form after an epileptic seizure to an inactivated form was induced by the treatment of compound 1.

Additionally, we quantitatively measured the intensity of Iba-1 and GFAP using immunocytochemistry and confocal imaging in a cell model of epilepsy induced by 0.1 mM [Mg2 ⁺ ] _o in the dentate gyrus (DG) to determine the effect on the activation of microglia and astrocytes.

Placebo group 10 μM 30 μM Strength of Iba-1 4.31 ± 0.35 × 10 ⁵ 4.73 ± 0.56 × 10 ⁵ 2.78 ± 0.24 × 10 ⁵ Strength of GFAP 5.45 ± 0.5 × 10 ⁶ 5.03 ± 0.6 × 10 ⁶ 3.47 ± 0.54 × 10 ⁶

In Figures 3b and 3d, “CON” represents the group treated with placebo or example compound 1 after exposing the neurons to 0.9 mM [Mg ²⁺ ] _o medium.

As shown in Table 6 above and Fig. 3b, the intensity of Iba-1 increased due to exposure to 0.1 mM [Mg ²⁺ ] _o medium, but the intensity of Iba-1 significantly decreased by treatment with Example Compound 1, indicating that Example Compound 1 reduces the activity of microglial cells due to epilepsy induction.

In addition, as shown in Table 6 and Figure 3d, exposure to 0.1 mM [Mg ²⁺ ] _o medium increased the size of astrocytes and increased the intensity of GFAP, but treatment with Example Compound 1 decreased the size of astrocytes and the intensity of GFAP. This suggests that Example Compound 1 not only lowers the intensity of GFAP, but also reduces the morphological changes of astrocytes and maintains or restores the morphology. This suggests that treatment with the present Example Compound 1 can alleviate epilepsy-induced neuroinflammation in the dentate gyrus (DG).

Experimental Example 4. Evaluation of the effect of reducing MLKL expression level

The expression levels of MLKL, which is used as an indicator of hippocampal necrosis, were measured by Western blot analysis for the placebo treatment group, 20 mg/kg, and 40 mg/kg of Example Compound 1 treatment groups on day 7 of pilocarpine-induced epilepsy development in the pilocarpine-induced SE mouse model. GAPDH was used as a loading control, and the results are shown in Fig. 4 and Table 7.

Placebo group Example compound 1
20 mg/kg Example compound 1
40 mg/kg MLKL expression level 1.00 ± 0.15 0.67 ± 0.08 0.46 ± 0.12

As shown in Table 7, the treatment group of Example Compound 1 showed a significant decrease in MLKL expression compared to the placebo group. This suggests that subacute administration of Example Compound 1 after epileptic seizures can reduce hippocampal necrosis induced by seizures.

Claims (8)

A pharmaceutical composition for preventing or treating epilepsy, comprising a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

In the above formula,
n is an integer from 1 to 3,
m is 0 or 1,
A represents phenyl,
R ¹ is hydrogen, or C ₁ -C ₆ -alkyl,
R ² represents hydrogen, halogen or C ₁ -C ₆ -alkoxy, or hydroxy-C _1- C ₆ -alkyl, -(CH ₂ ) _p CO ₂ R ⁷ , -NHR ⁸ , -N(H)S(O) ₂ R ⁷ or -NHC(O)R ⁷ , wherein p is an integer from 0 to 3, R ⁷ represents hydrogen or C ₁ -C ₃ -alkyl, R ⁸ represents C _1- C ₃ -alkylpiperidinyl, or C _1- C ₃ -alkylsulfonyl,
R ³ represents hydrogen, halogen, C ₁ -C ₆ -alkyl or phenyl, or represents -(CH ₂ ) _p -heterocycle, which is a 5 to 6-membered ring containing 1 or 2 heteroatoms selected from S, N and O atoms, wherein p is an integer from 0 to 3, provided that when m is 0, R ³ is phenyl,
R ⁴ is halogen, C ₁ -C ₆ -alkyl, hydroxy-C _1- C ₆ -alkyl, -O-phenyl, -(CH ₂ ) _p CO ₂ R ⁷ , -(CH ₂ ) p -heterocycle, which is a 5 to 6 membered ring containing 1 or 2 heteroatoms selected from _S , N and O atoms, or proline-N-carbonyl, wherein p is an integer from 0 to 3, R ⁷ is as defined above,
R ⁵ is hydrogen, or C ₁ -C ₆ -alkyl,
R ⁶ represents C ₁ -C ₆ -alkyl, C ₃ -C ₆ -cycloalkyl, heterocycle or heterocyclyl-C ₁ -C ₆ -alkyl, wherein the heterocycle is a 3 to 8 membered ring containing 1 to 3 heteroatoms selected from S, N and O atoms, and R ⁶ may be substituted with C ₁ -C ₆ -alkylamine, hydroxy-C ₁ -C ₆ -alkyl, or C ₁ -C ₆ -alkylsulfonyl. In the first paragraph,
R ³ represents hydrogen, halogen, or phenyl, or represents -(CH ₂ ) _p -heterocycle wherein the heterocycle is morpholino, piperazinonyl, wherein p is an integer from 0 to 1, provided that when m is 0, R ³ is phenyl,
R ⁴ is halogen, C ₁ -C ₃ -alkyl, hydroxy-C _1- C ₃ -alkyl, -O-phenyl, -(CH ₂ ) _p CO ₂ -ethyl, -(CH ₂ ) p -heterocycle wherein the heterocycle is thiomorpholino, _morpholino , piperazinonyl, or pyrrolidinyl, or proline-N-carbonyl, wherein p is an integer from 0 to 1,
R ⁵ is hydrogen, or C ₁ -C ₃ -alkyl,
A pharmaceutical composition for preventing or treating epilepsy, wherein R ⁶ represents C ₁ -C ₃ -alkyl, C ₃ -C ₆ -cycloalkyl, heterocycle or heterocyclyl-C ₁ -C ₃ -alkyl, wherein the heterocycle is tetrahydro-2H-pyran, or piperidinyl, and when R ⁶ is heterocycle or heterocyclyl-C ₁ -C ₃ -alkyl, it may be substituted with C ₁ -C ₆ -alkylamine, hydroxy-C ₁ -C ₆ -alkyl, or C ₁ -C ₆ -alkylsulfonyl. In the first paragraph,
A pharmaceutical composition for preventing or treating epilepsy, characterized in that the compound of the above chemical formula 1 is any one selected from the following group of compounds.
<1>5-[(1,1-dioxido-4-thiomorpholinyl)methyl]-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine;<2> Ethyl 7-(cyclopentylamino)-2-phenyl-1H-indole-5-carboxylate; <3>(7-(cyclopentylamino)-2-phenyl-1H-indol-5-yl)methanol;<4>5-chloro-N,1-dimethyl-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine;<5>4-((7-(cyclopentylamino)-2-(3-fluorophenyl)-1H-indol-5-yl)methyl)piperazin-2-one;<6>4-((2-phenyl-7-(((tetrahydro-2H-pyran-4-yl)methyl)amino)-1H-indol-5-yl)methyl)thiomorpholine1,1-dioxide;<7>5-chloro-N-(1-methylpiperidin-4-yl)-2-phenyl-1H-indol-7-amine;<8>5-phenoxy-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine;<9>5-chloro-3-(morpholinomethyl)-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine;<10>2-(4-((5-fluoro-2-phenyl-1H-indol-7-yl)amino)piperidin-1-yl)ethan-1-ol;<11>N-(4-(5-chloro-7-(cyclopentylamino)-1H-indol-2-yl)phenyl)methanesulfonamide;<12>5-chloro-3-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine;<13>4-((2-(3-fluorophenyl)-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-5-yl)methyl)thiomorpholine1,1-dioxide;<14>5-chloro-N-cyclopentyl-2-(4-((1-methylpiperidin-4-yl)amino)phenyl)-1H-indol-7-amine;<15>4-((7-(isopentylamino)-2-(4-methoxyphenyl)-1H-indol-5-yl)methyl)thiomorpholine1,1-dioxide;<16>N-(4-(7-(cyclopentylamino)-5-((1,1-dioxidothiomorpholino)methyl)-1H-indol-2-yl)phenyl)acetamide;<17>4-((3-bromo-2-phenyl-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-5-yl)methyl)thiomorpholine1,1-dioxide;<18>4-((5-chloro-2-phenyl-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-3-yl)methyl)piperazin-2-one;<19>4-((7-(methyl(tetrahydro-2H-pyran-4-yl)amino)-2-phenyl-1H-indol-5-yl)methyl)thiomorpholine1,1-dioxide;<20>5-methyl-N-(1-(methylsulfonyl)piperidin-4-yl)-2-phenyl-1H-indol-7-amine;<21>N-(4-(5-(1,1-dioxidothiomorpholino)-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)acetamide;<22>4-((7-((1-(methylsulfonyl)piperidin-4-yl)amino)-2-phenyl-1H-indol-5-yl)methyl)thiomorpholine1,1-dioxide;<23>N ¹ -(5-chloro-2-phenyl-1H-indol-7-yl)-N ⁴ -methylcyclohexane-1,4-diamine; <24>Methyl 2-(3-(5-chloro-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)acetate; <25>(2-phenyl-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indole-5-carbonyl)-D-proline;<26>(3-(5-chloro-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)methanol;<27>N-cyclopentyl-2-phenyl-5-(2-(pyrrolidin-1-yl)ethyl)-1H-indol-7-amine;<28>Methyl2-(4-(5-chloro-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)acetate;<29>Methyl4-(5-chloro-7-(cyclopentylamino)-1H-indol-2-yl)benzoate;<30>2-(4-(5-chloro-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)ethan-1-ol;<31>3-Bromo-5-(morpholinomethyl)-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine;<32>4-((3-phenyl-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-5-yl)methyl)thiomorpholine1,1-dioxide; and <33>N-cyclopentyl-5-methyl-2-phenyl-1H-indol-7-amine. In the first paragraph,
A pharmaceutical composition for preventing or treating epilepsy, wherein the compound of chemical formula 1 is a compound of chemical formula 2 below.
[Chemical formula 2]
In the first paragraph,
The above epilepsies are status epilepticus, temporal lobe epilepsy, infantile convulsions, febrile seizure, benign familial neonatal convulsion, early myoclonic encephalopathy, benign infantile myoclonic epilepsy, severe myoclonic epilepsy of infancy, myoclonic atopic epilepsy, Lennox-Gastaut syndrome, childhood absence seizure epilepsy, myoclonic absence seizure epilepsy, benign partial epilepsy, A pharmaceutical composition for preventing or treating epilepsy, wherein the epilepsy is at least one selected from the group consisting of Lando-Crefter syndrome, Dravet syndrome and Rasmussen syndrome. In the first paragraph,
A pharmaceutical composition for preventing or treating epilepsy, wherein the compound of the above chemical formula 1 or a pharmaceutically acceptable salt thereof inhibits neuronal cell death in the dentate gyrus in the hippocampus. In the first paragraph,
A pharmaceutical composition for preventing or treating epilepsy, wherein the compound of the above chemical formula 1 or a pharmaceutically acceptable salt thereof inhibits the expression of Iba-1 (ionized calcium binding adaptor molecule 1) and GFAP (glial fibrillary acidic protein) in hippocampal neurons. In the first paragraph,
A pharmaceutical composition for preventing or treating epilepsy, wherein the compound of formula 1 or a pharmaceutically acceptable salt thereof is contained in a concentration of 5 μM to 120 μM.