KR102314995B1 - Pharmaceutical composition for preventing or treating epilepsy containing 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating epilepsy containing 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one as an active component. The present invention is very effective in alleviating hyperactivity, inhibiting neuronal activation, restoring cognitive ability, and slowing recovery from voltage-dependent sodium channel inactivation of a PTZ-induced seizure fry model, so that the pharmaceutical composition can be used for preventing or treating epilepsy.

Description

Prevention of epilepsy containing 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one as an active ingredient; or Therapeutic pharmaceutical composition TECHNICAL FIELD Pharmaceutical composition for preventing or treating epilepsy containing 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one as an active ingredient}

For prevention or treatment containing 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one as an active ingredient It relates to pharmaceutical compositions.

Seizure is a repetitive contraction of local or systemic muscles regardless of one's own will due to hyperexcitation of cells. Epilepsy (epilepsy) is a chronic disease in which seizures occur repeatedly, and although 1% of the world's population is epilepsy and feels discomfort and pain in daily life due to seizures, about 30% of patients suffer from existing anti- Other patients who do not respond to seizure drugs (ASDs) have to take two or more ASDs. For this reason, it is considered necessary to develop a new ASD (Zaccara et al., Pharmacological research 104(2016):38-48).

A female zebrafish lays 100 to 300 eggs at a time, and the fertilized egg develops very quickly, and most major organs and organs are formed 24 hours after fertilization. In addition, it is an experimental animal with various advantages to be used in the screening stage for new drug development because it is genetically identical to humans 70-80%. The ZC4H2 gene is one of the candidate genes related to X-linked intellectual disability, and the zc4h2 gene with a high homology of 94% exists in zebrafish. The expression of zc4h2 gene begins to be expressed throughout the embryonic embryo from 4 hours after fertilization, and is mainly expressed in the head and eyes as development progresses. Zebrafish zc4h2 KO fry were produced using TALEN genetic modification technology, and from the 4th day after fertilization, the bladder was not formed, so they could not balance and swim on their side, and seizure-like phenotypes such as abnormal excessive pectoral fin and jaw movements appeared (May, Melanie et al., Human molecular genetics 24.17 (2015): 4848-4861).

Pentylenetetrazole (PTZ) is an antagonist of the inhibitory neurotransmitter GABA, and is a compound that competitively binds to GABAA receptors and maintains the excited state of nerve cells. PTZ is one of the representative compounds used to induce seizures in several animal models because it dissolves very well in water, can be used in high concentrations, and is inexpensive. When PTZ is treated with zebrafish fry at a high concentration (5 mM), seizure-like behavior such as an increase in swimming distance, swimming duration, movement frequency, and speed can be observed. In addition, when PTZ is treated with zebrafish, cognitive decline, one of the complications of seizure patients, can be confirmed through the color-maze test. In mice, PTZ can also be used to induce acute seizures. When a high-dose PTZ is injected into mice, a variety of seizure-like behaviors can be observed, including partial or whole body convulsions within tens of seconds and tonic-clonic seizures (TCS) within 10 minutes.

In addition to behavioral characteristics, there are several indicators that can be used to determine epilepsy. Phosphorylated ERK1/2 protein (pERK), a representative marker of seizures, is a molecule found in activated neurons and is known to be overexpressed when seizures occur. This protein can be importantly used in the study of the relationship between the behavior of experimental animals and neurons (Houser et al., Neuroscience 156.1(2008):222-237). In addition, when a seizure occurs, electroencephalography (EEG), an electrical activity of the brain, also appears abnormally unstable, and the main method for diagnosing epilepsy is by repeatedly measuring the EEG. The voltage-gated Na+ channel of a neuron is rapidly activated when a stimulus is received, opens the channel, and induces depolarization by pouring out Na+ ions into the cell. Therefore, several ASDs that target the Na+ channel exist, such as valproate and carbamazepine. Neurotransmitters are secreted at the synapse of a neuron and bind to a receptor of an adjacent neuron to transmit information. Neurotransmitters are largely divided into excitatory and inhibitory, and among them, there are ASDs, such as phenobarbital and tiagabine, which have a mechanism to improve the transmission of GABA, which is an inhibitory neurotransmitter.

In the present invention, in order to develop a novel epilepsy treatment agent, effective substances were selected from 6566 species of the Korea Compound Bank library, and the antiepileptic efficacy was verified by various methods for the finally selected substances.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating epilepsy.

Another object of the present invention is to provide a functional food for preventing or improving epilepsy.

In order to achieve the above object,

The present invention provides a pharmaceutical composition for preventing or treating epilepsy comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

.

.

In addition, the present invention provides a health functional food for preventing or improving epilepsy containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

.

.

4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one according to the present invention comprising as an active ingredient The pharmaceutical composition for the prevention or treatment of epilepsy can suppress the hyperactivity of the jaw and pectoral fin of the fry in the PTZ-induced seizure fry model to a degree similar to that of retigabine used as an adjuvant treatment for epilepsy, as well as the movement of the seizure model. It was confirmed that there was an effect in reducing the distance, time, and number of prints.

In addition, the effect of mitigating various phenomena on epilepsy of the PTZ-induced seizure fry model was confirmed through neural cell activation experiments and cognitive ability tests of the seizure model, and by slowing recovery from voltage-dependent sodium channel inactivation through electrochemical experiments. Since it was confirmed that it is effective in suppressing epilepsy, the pharmaceutical composition according to the present invention can be effectively used for preventing or treating epilepsy.

1a is a graph showing the results of the efficacy of alleviating pectoral fin movement in the epilepsy model (zc4h2 KO) performed in Experimental Example 2 according to the present invention.
Figure 1b is a graph showing the results for the efficacy of alleviating jaw movement in the epilepsy model (zc4h2 KO) performed in Experimental Example 2 according to the present invention.
FIG. 2A is a graph showing the distance traveled by an individual among the hyperactivity suppression test results for the PTZ-induced seizure model performed in Experimental Example 3 according to the present invention.
FIG. 2B is a graph showing the movement time of an individual among the hyperactivity suppression test results for the PTZ-induced seizure model performed in Experimental Example 3 according to the present invention.
FIG. 2c is a graph showing the number of times an individual moved among the hyperactivity suppression test results for the PTZ-induced seizure model performed in Experimental Example 3 according to the present invention.
FIG. 2D is a graph showing the average speed of an individual among the hyperactivity suppression test results for the PTZ-induced seizure model performed in Experimental Example 3 according to the present invention.
3A is a photograph showing the results of imaging and fluorescence intensity measurement with a fluorescence microscope in the nerve cell activation inhibition experiment for the PTZ-induced seizure model performed in Experimental Example 4 according to the present invention.
3B is a graph showing the average values of fluorescence intensity of the forebrain, cerebrum, cerebellum, and hindbrain in the nerve cell activation inhibition experiment for the PTZ-induced seizure model performed in Experimental Example 4 according to the present invention.
4 is a graph showing the color preference test results of zebrafish fry for the PTZ-induced seizure model performed in Experimental Example 5 according to the present invention.
5 is a graph showing the results of the power spectrum according to the electroencephalogram (EEG) of the adult zebrafish PTZ-induced seizure model performed in Experimental Example 6 according to the present invention.
6A is a graph showing the results of analyzing the change in the magnitude of the voltage-dependent sodium current induced by depolarizing voltage stimulation in the electrochemical experiment of Experimental Example 7 according to the present invention.
6B is a graph showing the analysis result of the membrane voltage-inactivation relationship of the voltage-dependent sodium channel in the electrochemical experiment of Experimental Example 7 according to the present invention.
6c is a graph showing the result of analyzing the action of the voltage-dependent sodium channel on the inactivation kinetics in the electrochemical experiment of Experimental Example 7 according to the present invention.
Figure 6d is a graph showing the results of analyzing the action on the recovery kinetics from the inactivation of the voltage-dependent sodium channel in the electrochemical experiment of Experimental Example 7 according to the present invention.
7a is a graph showing the results of quantitative analysis of the amounts of acetylcholine, choline, betaine, and serine in the brain in the neurotransmitter analysis experiment of Experimental Example 8 according to the present invention.
Figure 7b is a graph showing the results of quantitative analysis of the amount of GABA, glutamic acid, glutamine and the excitatory/inhibitory substance ratio of the brain in the neurotransmitter analysis experiment of Experimental Example 8 according to the present invention.
7c is a graph showing the results of quantitative analysis of the amounts of phenylalanine, tyrosine, dopamine, and norepinephrine in the brain in the neurotransmitter analysis experiment of Experimental Example 8 according to the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating epilepsy comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

.

.

In this case, the pharmaceutical composition may alleviate hyperactivity. In the present invention, it was confirmed that when the compound according to the present invention was treated in the epilepsy zebrafish model (zc4h2 KO), it was effective in alleviating hyperactivity of the pectoral fin and jaw ( FIGS. 1A and 1B ). In addition, it was confirmed that hyperactivity of the subject was suppressed in the zebrafish seizure model induced with pentylenetetrazol (PTZ) ( FIGS. 2a to 2d ).

In addition, the pharmaceutical composition may inhibit the activation of nerve cells. In the present invention, it was confirmed that neuronal activation was inhibited when the compound according to the present invention was treated in a zebrafish seizure model induced by pentylenetetrazol (PTZ). ) was confirmed through immunohischemistry for phosphorylated ERK1/2 protein (p-extracellular signal-regulated kinase-1/2, pERK1/2) ( FIGS. 3a and 3b ).

In addition, the pharmaceutical composition may reduce the power of EEG signals. In the present invention, it was confirmed that the power spectrum of EEG was effectively reduced when the compound according to the present invention was treated in a zebrafish seizure model induced with pentylenetetrazol (PTZ) ( FIG. 5 ).

In addition, the pharmaceutical composition can suppress the magnitude of the voltage-dependent sodium current, and facilitate the inactivation of the voltage-dependent sodium channel, as well as slow the recovery from the inactivation. The present invention confirmed the effect of suppressing the magnitude of voltage-dependent sodium current, promoting voltage-dependent sodium channel inactivation and inhibiting recovery from inactivation by applying the compound according to the present invention to rat hippocampal neurons ( FIGS. 6a to 6d ).

The pharmaceutical composition has an effect of increasing the secretion of inhibitory neurotransmitter, at this time, the inhibitory neurotransmitter is not limited as long as it is an inhibitory neurotransmitter already known in the art, for example, serine, phenylalanine , tyrosine, dopamine, glutamine, acetylcholine, choline, glycine, betaine, glutamic acid, norepinephrine, serotonin, histamine and at least one selected from the group consisting of γ-aminobutyric acid. As a result of neurotransmitter analysis by processing the compound according to the present invention in a zebrafish model of PTZ-induced seizures, it was confirmed that the secretion of inhibitory neurotransmitters such as serine, phenylalanine, and dopamine in the brain significantly increased (Fig. 7a). to Figure 7c).

The pharmaceutical composition of the present invention may further include one or more active ingredients exhibiting the same or similar function in addition to the above active ingredients.

The pharmaceutical composition of the present invention may include a carrier, a diluent, an excipient or a mixture thereof commonly used in biological agents. Any pharmaceutically acceptable carrier may be used as long as it is suitable for delivering the composition in vivo. Specifically, the carrier is Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or mixtures thereof. In addition, conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed.

When formulating the composition, commonly used fillers, extenders, binders, wetting agents, disintegrants, diluents or excipients such as surfactants may be added.

The composition of the present invention may be formulated as an oral preparation or a parenteral preparation. Oral formulations may include solid formulations and liquid formulations. The solid preparation may be a tablet, pill, powder, granule, capsule, or troche, and the solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin, or a mixture thereof. In addition, the solid formulation may contain a lubricant, examples of which include magnesium stearate, talc, and the like. Meanwhile, the liquid formulation may be a suspension, an internal solution, an emulsion, or a syrup. In this case, the liquid formulation may include excipients such as wetting agents, sweetening agents, fragrances, and preservatives.

The parenteral formulation may include injections, suppositories, powders for respiratory inhalation, aerosols for sprays, powders and creams, and the like. The injection may include a sterile aqueous solution, a non-aqueous solution, a suspension solution, an emulsion, and the like. In this case, as the non-aqueous solvent or suspension solution, vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, or injectable esters such as ethyl oleate may be used.

The composition of the present invention may be administered orally or parenterally according to a desired method. Parenteral administration may include intraperitoneal, intrarectal, subcutaneous, intravenous, intramuscular or intrathoracic injection.

The composition may be administered in a pharmaceutically effective amount. This may vary depending on the type of disease, severity, drug activity, patient's sensitivity to the drug, administration time, administration route, treatment period, drugs used simultaneously, and the like. However, for a desirable effect, the amount of the active ingredient included in the pharmaceutical composition according to the present invention may be 0.0001 to 100 mg/kg, specifically 0.001 to 70 mg/kg. The administration may be once or several times a day.

The composition of the present invention may be administered alone or in combination with other therapeutic agents. When administered in combination, the administration may be sequential or simultaneous.

In addition, the present invention provides a health functional food for preventing or improving epilepsy containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

.

.

As used herein, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 of the Health Functional Food Act, and 'functionality' refers to the structure of the human body. and regulating nutrients for function or obtaining useful effects for health applications such as physiological action. On the other hand, health food refers to food that has an active health maintenance or promotion effect compared to general food, and health supplement refers to food for the purpose of health supplementation. can be mixed. The health functional food of the present invention can be prepared by a method commonly used in the art. It can be manufactured in various types of dosage forms, and unlike general drugs, it has the advantage of not having side effects that may occur during long-term administration of the drug using food as a raw material, and can be excellent in portability.

When manufacturing the health functional food, it can be prepared by adding raw materials and ingredients commonly added in the industry, and the type is not particularly limited. For example, it may include, as an additional component, various herbal extracts, food-logically acceptable food supplements or natural carbohydrates, such as conventional food, but is not limited thereto.

Hereinafter, Examples and Experimental Examples of the present invention will be specifically illustrated and described below. However, the Examples and Experimental Examples to be described below are merely illustrative of a part of the present invention, and are not limited thereto.

< Example 1> 4-(2- Chloro -4- Fluorobenzyl )-3-(2- Cyenyl )-1,2,4- oxadiazole -5(4 H Preparation of a pharmaceutical composition for the prevention or treatment of epilepsy through the synthesis of )-on

Compound name: 4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5( 4H )-one

4-(2-chloro-4-fluorobenzyl)-3-(2-thienyl)-1,2,4-oxadiazol-5(4H)-one

CAS No: 902873-60-3

Molecular Formula: C ₁₃ H ₈ ClFN ₂ O ₂ S

Molecular Weight: 310.73

full reaction scheme

Step 1: (Z)-N'- hydroxythiophene -2- Carboximideamide Preparation of ((Z)-N'-hydroxythiophene-2-carboximidamide)

Thiophene-2-carbonitrile (2.0 g, 18.32 mmol), hydroxyammonium hydrochloride (1.91 g, 27.48 mmol) and diisopropylethylamine (5.11 mL, 29.32 mmol) were placed in a reaction vessel and under reduced pressure in a vacuum state. Then, distilled EtOH (75 mL) was added under nitrogen gas, and the mixture was stirred for 4 hours and refluxed. Upon completion of the reaction, the solvent was removed under reduced pressure and extraction (AcOEt/H2O) was performed. Purified by column chromatography (AcOEt; Hexane = 2: 8, v/v) as a white solid (Z)-N'-hydroxythiophene-2-carboximidamide (2.379 g, 91.3) %) was obtained.

1H NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H), 7.47 (dd, J = 3.6 Hz, J = 0.9 Hz, 1H), 7.41 (dd, J = 5.1 Hz, J = 0.9 Hz, 1H) ), 7.03 (dd, J = 5.1 Hz, J = 3.6 Hz, 1H), 5.92 (brs, 2H).

step 2: 3 -( thiophene -2-yl)-1,2,4- oxadiazole -5(4H)-one (3-( thiophen Preparation of -2-yl)-1,2,4-oxadiazol-5(4H)-one)

(Z)-N'-hydroxythiophene-2-carboximidamide (2.38 g, 16.73 mmol), CDI (N,N'-Carbonyldiimidazole) (3.26 g, 20.08 mmol) and DBU (1,8-Diazabicyclo) [5.4.0] undec-7-ene) (2.75 mL, 18.40 mmol) was placed in a reaction vessel, 1,4-dioxane (30 mL) was added to dissolve, and the mixture was stirred for 3 hours and refluxed. When the reaction was completed, pH = 2 was adjusted with 3 M HCl solution, and extraction (AcOEt / H 2 O) was performed 3 times. To obtain an organic layer, remove the solvent, dissolve in AcOEt/Hexane, recrystallize from DCM, and then white solid 3-(thiophen-2-yl)-1,2,4-oxadiazol-5(4H)-one (2.45 g, 87.1%).

1H NMR (400 MHz, DMSO) δ 13.13 (s, 1H), 7.93 (dd, J = 5.0, 1.1 Hz, 1H), 7.72 (dd, J = 3.7, 1.1 Hz, 1H), 7.29 (dd, J = 5.0, 3.8 Hz, 1H).

step 3: 4 -(2- Chloro -4- Fluorobenzyl )-3-( thiophene -2-yl)-1,2,4- oxadiazole -5(4H)-one (4-(2- chloro -4- fluorobenzyl )-3-( thiophen -2- yl )-1,2,4- oxadiazol Preparation of -5(4H)-one )

1- (bromomethyl) -2-chloro-4-fluorobenzene (558 mg, 2.50 mmol), 3- (thiophen-2-yl) -1,2,4-oxadiazole-5 (4H) -one (500 mg, 2.97 mmol) and potassium carbonate (411 mg, 2.97 mmol) were placed in a reaction vessel, and acetone (10 mL) was added to dissolve it, followed by reflux with stirring for 12 hours. Upon completion of the reaction, the solvent was removed under reduced pressure and extraction (AcOEt / H2O) was performed three times. Purified by column chromatography (AcOEt; Hexane = 2: 8, v/v) as a white solid 4-(2-chloro-4-fluorobenzyl)-3-(thiophen-2-yl) )-1,2,4-oxadiazol-5(4H)-one (530 mg, 57%) was obtained.

1 H NMR (400 Mhz, DMSO4-(2-chloro-4-fluorobenzyl)-3-(thiophen-2-yl)-1,2,4-oxadiazol-5(4H)-one): δ 7.90 (d, J = 4.96 Hz, 1H), 7.50 (dd, J = 11.44 Hz, 1H), 7.42 (d, J = 3.81 Hz, 1H), 7.29-7.33 (m, 1H), 7.198 (t, J = 8.39 Hz, 1H), 7.12-7.18 (m, 1H), 4.98 (s, 1H).

<Experimental Example 1> Screening

Compounds that inhibit seizure-like behavior were selected by reacting 6,566 representative compounds owned by the Korea Compound Bank to zebrafish fry showing seizure-like behavior.

The zc4h2 knock-out (KO) zebrafish, an epilepsy model showing seizure-like behavior, is a model in which the function of zc4h2 , a zebrafish homologous gene of the ZC4H2 gene, known as the causative gene of human Miles-Carpenter Syndrome, is suppressed. This zebrafish is characterized by behavior similar to human seizures, such as overall movements in the fry on the 5th day after fertilization, as well as hyperactivity in the jaw and pectoral fins (May et al., 2013. Hum. Mol. Genet.; Frints et al., 2019. Hum. Mutat.).

1st screening

For the primary screening, embryonic embryos obtained by crossing zc4h2 heterozygous adults were developed in an incubator at 28 °C until the 5th day after fertilization. On the 5th day after fertilization, approximately 75% of the individuals selected wild-type fry (hereinafter WT) and approximately 25% of the zc4h2 homozygous (hereinafter referred to as zc4h2 KO) fry, in which zc4h2 function was not inhibited, were selected, and then acclimatized to the laboratory environment in the morning. At 1 pm, two zc4h2 KO fry were dispensed into each well of a 96-well plate, and then acclimatized to the environment for 30 minutes. 6,566 representative compounds dissolved in about 5 mM in DMSO were diluted to 20 μM (1/250), and movements were observed and recorded through a microscope at 30, 60, and 120 minutes after exposure. As a negative control, 0.4% DMSO, and as a positive control, 5 μ concentration of retigabine were used.

< Experimental example 2> epilepsy Model ( zc4h2 chin in KO) pectoral fin hyperactivity inhibition experiment

On the 5th day after fertilization, two WT and zc4h2 KO fry were dispensed into each well of a 96-well plate and acclimatized for 30 minutes. Thereafter, the compound of Example 1 was exposed to 5, 10, and 20 μM for 30 minutes (n=6), and then fixed in 5% methyl cellulose with the side facing up. The number of jaw movements was recorded by recording for 10 seconds after fixation, and is shown in FIG. 1A. In addition, after exposing the compound of Example 1 to 5, 10, and 20 μM for 30 minutes (n=6), the back side was facing up, and then fixed in 5% methyl cellulose. The number of pectoral fin movements was recorded by recording for 10 seconds after fixation, and is shown in FIG. 1B.

As shown in FIGS. 1A and 1B , the compound of Example 1 according to the present invention has very excellent ability to alleviate hyperactivity of the pectoral fin and jaw in the zebrafish fry model with epilepsy, and in particular, for epilepsy Compared with retigabine, an anticonvulsant drug used as an adjuvant treatment, when the compound of Example 1 was treated at 20 μM, the number of jaw movements was almost the same as retigabine. Through this, it can be seen that the compound of Example 1 of the present invention exhibits excellent hyperactivity inhibitory efficacy in a concentration-dependent manner.

< Experimental example 3> pentylenetetrazole ( Pentylenetetrazol , PTZ ) Hyperactivity inhibition experiment in induced seizure model

Common wild-type zebrafish embryos were developed in an incubator at 28° C. until the 5th day after fertilization. On the 5th day after fertilization, the fry were acclimatized to the laboratory environment during the morning, and at 1:00 PM, one individual was dispensed into each well of a 96-well plate, and then acclimatized to the environment for 30 minutes. There are a total of 8 experimental groups, specifically, DMSO-treated group, Example 1 compound 0.5, 1, and 2 μM treatment group in the PTZ untreated group, and DMSO-treated group in the 5 mM PTZ treatment group, Example 1 compound 0.5, 1, A 2 μM treatment group was prepared (n=10). At the same time as the experimental solution was processed, the movement was tracked for 30 minutes using DanioVision (Noldus, Netherlands), a zebrafish behavioral tracking device. The tracked data were calculated for each movement through EthoVsion software (Noldus, Netherlands), and are shown in FIGS. 2a to 2d. The calculated values were calculated through Excel (Microsoft, US). A detailed description and calculation method for each movement is as follows.

motion output

Hyperactivity: Movement with an average speed of the fry of 40 mm/s or more. Refers to hyperactivity similar to seizures.

Overall action: All movements regardless of the average speed of the fry.

Hyperactivity/Total Behavior Ratio: The ratio of hyperactivity to overall movement.

Evaluation criteria for hyperactivity

Distance Moved: The distance the object has moved in 30 minutes.

Movement Time: The amount of time the object actually moved in 30 minutes.

Number of Movements: The number of times the object actually moved in 30 minutes.

Average speed: The average speed during which the object is actually moving in 30 minutes.

As shown in FIG. 2 , it was found that the compound of Example 1 according to the present invention showed no effect on movement in a normal situation without PTZ, and showed concentration-dependent alleviation effect on hyperactivity in the PTZ-induced seizure model. In particular, the distance moved by the subject was reduced by about half when Example 1 was treated with 2 μM, and the movement time and number of movements was reduced to about 70% when Example 1 was treated with 2 μM. Thus, it can be seen that there is an excellent effect in alleviating hyperactivity.

<Experimental Example 4> Neuronal activation measurement experiment in PTZ-induced seizure model

Phosphorylated ERK1/2 protein (hereinafter, pERK) is a molecule found in activated neurons and is important for the study of the association between individual behavior and neuronal activation (Ji et al., 1999. Nat. Neurosci.; Xia et al. ., 1996. J. Neurosci.).

In order to verify the effectiveness of the compound of Example 1 in the PTZ-induced seizure model, we tried to measure the activation of nerve cells. In this experiment, the activation of nerve cells in zebrafish fry was verified through immunohistochemistry for the pERK protein.

On the fifth day after fertilization, the fry were acclimatized to the laboratory environment during the morning, and at 1 PM, 10 fry were dispensed into each well of a 12-well plate. There are four experimental groups, and specifically, DMSO, Example 1 compound 2 μM treatment group and 5 mM PTZ treatment group, DMSO, Example 1 compound 2 μM treatment group were prepared in the PTZ untreated group (n=10). After exposing each experimental solution to each well with zebrafish fry for 30 minutes, it was replaced with 4% paraformaldehyde solution and fixed overnight. The fixed object was washed with water three times at 15-minute intervals in phosphate buffered saline (hereinafter referred to as PBST) containing 0.1% tween-20, followed by 1% H ₂ O ₂ /3% KOH solution for bleaching pigment cells. was reacted for about 1 hour. The fry from which pigment cells were removed were washed 3 times in PBST at 15-minute intervals, replaced with a 150 mM TrisHCl (pH 9.0) solution, and reacted at 70° C. for 20 minutes, followed by washing twice with PBST for 5 minutes. It was replaced with a 0.05% Trypsin-EDTA solution and reacted at 4 °C for 45 minutes, and then washed three times with PBST for 15 minutes. After replacing with blocking solution (2% normal goat serum, 1% bovine serum albumin, 1% DMSO added to PBST), the reaction was carried out at room temperature for 1 hour, and the primary antibody, pERK (Cell Signaling, US), was added 1:500 was diluted in blocking solution and reacted at 4 °C overnight. After washing three times with PBST for 15 minutes, the secondary antibody (Invitrogen, US) was diluted 1:500 in blocking solution and reacted at room temperature for 4 hours. After washing three times with PBST for 15 minutes, it was fixed in 3% methyl cellulose, and imaging and fluorescence intensity were measured with a fluorescence microscope (BioTek, US), and are shown in FIG. 3A. In addition, the average values were compared by measuring the fluorescence intensity of the forebrain, tectum, cerebellum, and hindbrain in the images acquired in the dorsal direction of the fry.

As shown in the image of Figure 3a, it was confirmed with the naked eye that the compound of Example 1 according to the present invention reduced the amount of pERK protein in each brain part in the PTZ-induced seizure model. Even when comparing the average values of fluorescence intensity in each of the sciatic, cerebellar, and hindbrain, it was confirmed that the fluorescence intensity was significantly decreased by the compound of Example 1. Through this, it can be seen that the compound of Example 1 according to the present invention effectively alleviates neuronal activation in the PTZ-induced seizure model.

<Experimental Example 5> Zebrafish color preference test

In animal experiments, the color preference test is a behavioral evaluation method used as a tool for evaluation of learning and memory or decision making. Zebrafish also have the ability to discriminate colors, and it is known that the blue color is the highest in fry (Park et al., 2016. Mol. Cells.). Therefore, the color preference test of zebrafish fry through color maze can evaluate the cognitive ability of the individual.

On the fifth day after fertilization, the fry were acclimatized to the laboratory environment during the morning, and at 1 PM, 40 fry were transferred to the color maze (white, red, yellow, blue) for zebrafish fry. There are 4 experimental groups, and specifically, DMSO, Example 1 compound 2 μM treatment group and 5 mM PTZ treatment group, DMSO, Example 1 compound 2 μM treatment group were prepared in the PTZ untreated group (n=40). After recording an image while exposing each experimental solution to a color maze with zebrafish fry for 30 minutes, the recorded image is captured every 2 minutes, the number of fry staying in each color is recorded, and the average of the fry color preference was calculated and the results are shown in FIG. 4 .

As a result, as shown in FIG. 4, white preference decreased, yellow preference increased, red preference increased, and blue preference decreased in the group treated with PTZ and DMSO (in order of preference: red>blue>yellow>white) , when treated with the compound of Example 1 according to the present invention (preference order: blue>red>white>yellow), white, yellow, compared to the untreated group (preference order: blue>red>white>yellow) A similar trend was observed in both red and blue preferences. In particular, it can be seen that the blue color is alleviated in the PTZ-induced seizure model with reduced preference. Through this, it can be seen that when the compound of Example 1 according to the present invention is treated, the color discrimination ability can be recovered in the color preference evaluation, and thus it can be seen that there is an effect on the cognitive ability recovery.

< Experimental example 6> Eucharist zebrafish PTZ EEG potentials in an induced seizure model recordkeeping (electroencephalographphy, EEG) analysis

In the PTZ-induced seizure model of adult zebrafish, the following experiment was conducted to determine whether the EEG power spectrum decreased through electroencephalography analysis.

First, by measuring the weight of adult male zebrafish, 400 to 750 mg of individuals were selected, and there were 4 experimental groups, specifically, water in the PTZ untreated group, and water in the 1 mM compound of Example 1 and 15 mM PTZ administered group, Example 1 A group administered with 1 mM compound was prepared (n=4). In this experiment, after attaching an electrode to the head of an adult zebrafish non-invasively, the EEG signal of an individual was measured and calculated using the MP36 (BIOPAC Systems Inc., US) system (Cho et al., 2017. Sci. Rep.). For anesthesia of adult zebrafish, the subject was placed in a dedicated frame for the experiment, and the subject was anesthetized through 15 ppm external exposure of Eugenol, 8 ppm oral administration, and EEG was measured as a baseline for 10 minutes, each Depending on the experimental group, 4 mL of PTZ or a mixed solution of PTZ and Example 1 was orally administered for 2 minutes. EEG was measured for 25 minutes to calculate power spectrum increase/decrease data in the 0-16 Hz section through the following formula, and the results are shown in FIG. 5 .

[formula]

As a result, as can be seen in FIG. 5 , the relative power signal increased from about 10 AU to about 300 AU by PTZ treatment. It can be seen that 40 AU is reduced to about 87%. Through this, it was confirmed that the compound of Example 1 according to the present invention can effectively reduce the power spectrum for EEG in the adult zebrafish PTZ-induced seizure model.

< Experimental example 7> rat Voltage-dependent sodium in hippocampal neurons Action analysis on pathways

In order to analyze the action on voltage-dependent sodium channels in rat hippocampal neurons, rats (Sprague Dawley system, 12-17 days old) were anesthetized with ketamine (50 mg/kg, intraperitoneal administration) and then cut and quickly The cerebrum was isolated and excised. Using a microslicer (vibratome 1000; Warner Instruments, Hamden, CT, USA) to prepare a tissue section containing the hippocampus with a thickness of 400 μm, the extracted cerebrum was prepared with 95% O ₂ and 5% at room temperature (21-24 ℃). Preserved for at least 1 hour in artificial cerebrospinal fluid (ACSF, in mM; 120 NaCl, 2 KCl, 1 KH ₂ PO ₄ , 26 NaHCO ₃ , 2 CaCl ₂ , 1 MgCl ₂ and 10 glucose) saturated with CO ₂ did. After transferring the tissue section containing the hippocampal CA3 region to a culture dish, under standard extracellular solution (in mM; 150 NaCl, 3 KCl, 1 2 CaCl ₂ , 1 MgCl ₂ , 10 glucose, 10 HEPES, pH 7.4 with Tris-base) Neurons in a single hippocampal CA3 region were isolated using a neuron mechanical separation device. These neurons were electrophysiologically recorded using the whole-cell patch clamp technique under an inverted microscope (TE2000, Nikon, Tokyo, Japan). used the 'Y-tube' so that the drug could be replaced quickly.

All electrophysiological recordings were performed using whole-cell patch clamp recording using a microcurrent amplifier (MultiClamp 700B; Molecular Devices; Union City, CA, USA). For recording electrodes, use a silica glass tube (1.5 mm outer diameter, 0.9 mm inner diameter; G-1.5; Narishige, Tokyo, Japan) with a dedicated electrode maker (P-97; Sutter Instrument Co., Novato, CA, USA). produced. The resistance of the prepared electrode was about 1 to 2 MΩ when the electrode inner solution was filled. The membrane current recorded during whole cell recording was filtered at 2 kHz and then digitally signaled at 10 kHz using an analog-to-digital signal converter (Digidata 1440A, Molecular Devices). was used and saved online to a computer. Series resistance was measured by applying a hyperpolarized voltage stimulus of 10 mV to all whole-cell recordings. Recording was stopped when there was a change of more than 15% in the series resistance value.

To measure the voltage-dependent sodium current, the membrane voltage was fixed at -100 mV. For the intracellular solution (electrode solution), ^{a solution containing Cs +} as the main cation (internal solution; in mM; 140 CsF, 10 CsCl, 2 EGTA, 10 Hepes, pH 7.2 with Tris-base) was used, and the extracellular solution was standard the extracellular fluid Cd ^{2 +} (100 μM) of voltage-dependent calcium channel inhibitors, excitatory glutamate receptors and inhibitory GABA _a receptor inhibitor, kynurenic acid (3 mM) and SR95531 (10 μM) was added. Voltage-dependent sodium currents were activated by depolarizing voltage stimulation from -100 mV to -20 mV. The experimental results are shown in FIGS. 6A to 6D .

As shown in Fig. 6a, it can be seen that the compound of Example 1 according to the present invention significantly suppresses the magnitude of the voltage-dependent sodium current induced by the depolarizing voltage stimulation.

Figure 6b shows the action of the compound of Example 1 on the voltage-inactivation relationship of the voltage-dependent sodium channel. For the membrane voltage-inactivation relationship, _{a trend line was established using the Boltzmann equation (I/I max} = 1/1 + exp[(V - V ₅₀ )/ k ]) and each index was estimated. Here, I _max is the magnitude of the maximum current, V ₅₀ is the magnitude of the voltage stimulus that induces a current magnitude of 50% of the magnitude of the maximum current, V is the magnitude of each voltage stimulus, and k means the slope of the curve. As shown in FIG. 6b , _{it can be seen that the compound of Example 1 shifts the V 50} value toward the hyperpolarization, and thus it can be seen that the voltage-dependent sodium channel is easily inactivated.

Figure 6c shows the action of the compound of Example 1 on the voltage-dependent inactivation kinetics of the sodium channel. To measure the inactivation kinetics, two consecutive voltage stimulations were applied with an interval of 20 ms, and the duration of the first voltage stimulation was changed from 2 ms to 8000 ms. Here, when the _{ratio of sodium current induced by the first (P 1} ) and second (P ₂ ) voltage stimulation (P2/P1 ratio) is expressed as a relationship with the duration of the first voltage stimulation, the The temporal relationship of the degree of inactivation can be expressed by the exponential formula (I(t) = A ₀ + A _fast x [exp(-t/τ _fast )] + A _slow x [exp(-t/τ _slow )]). have.

Here, I(t) is the magnitude of the sodium current at each duration of the first voltage stimulation, τfast and τslow are the time constants for inactivation, and A _fast and A _slow are the ratios at each time constant. As shown in FIG. 6c , it can be seen that the compound of Example 1 according to the present invention _{significantly reduces the fast} inactivation portion (τ fast ) among the inactivation kinetic components of the voltage-dependent sodium channel, which indicates that the compound of Example 1 significantly reduces the voltage This means that it accelerates the inactivation of the dependent sodium channel.

Figure 6d shows the action of the compound of Example 1 on the kinetics of recovery from voltage-dependent sodium channel inactivation. To measure the recovery kinetics from inactivation, two consecutive voltage stimulations were applied, the duration of the first voltage stimulation was fixed at 500 ms, and the recovery duration until the second voltage stimulation was 1 ms to 5000 ms.

Here, when the ratio of sodium current induced by _{the first (P 1} ) and second (P _{2 ) voltage stimulation (P 2} /P ₁ ratio) is expressed as a relationship with the recovery time until the second voltage stimulation is applied , the temporal relationship of the degree of recovery from the voltage-dependent inactivation of sodium current is the exponential equation (I(t) = A ₀ + A _fast x [1 - exp(-t/τ _fast )] + A _slow x [1 - exp( -t/τ _slow )]). where I(t) is the magnitude of the sodium current at each duration of the first voltage stimulation, τfast and τslow are the recovery time constants from inactivation, and A _fast and A _slow are the ratios at each time constant. As shown in FIG. 6d , it can be seen that the compound of Example 1 significantly increases the _fast recovery part (τ fast ) among the recovery kinetic components from the inactivation of the voltage-dependent sodium channel, which indicates that the compound of Example 1 is the voltage-dependent sodium channel. means to slow recovery from inactivation of the pathway.

< Experimental example 8> Eucharist zebrafish PTZ Neurotransmitter analysis in the induced seizure model

Neurotransmitters secreted for signal transduction in neurons play a role in regulating potentials at synapses. Therefore, neurotransmitters in the adult zebrafish PTZ-induced seizure model were analyzed. 12 adult zebrafish were transferred to a 1L tank and 4 experimental groups were prepared. Specifically, in the PTZ untreated group, DMSO, Example 1 compound 2 compound 2 μM treatment group was prepared, and in the 1 mM PTZ treatment group, DMSO, Example 1 compound 2 μM treatment group was prepared (n=12). Each material was placed in a 1 L water bath for 24 hours, and brain tissue of adult zebrafish was isolated. Tertiary distilled water equivalent to 10 times the weight of brain tissue was added, homogenized for 5 seconds with an ultrasonic device (Ultrasonic processorVCX-130; Sonics & Materials, Inc., Newtown, CT), and then the same amount of methanol was added to transmit nerves in brain tissue The material was extracted. The supernatant was collected by centrifugation for 10 minutes, and each neurotransmitter was quantitatively analyzed by LC-MS/MS (Waters Xevo-tqs, USA), and the results are shown in FIG. 7 .

As a result, as shown in FIGS. 7a to 7c , when the compound of Example 1 was treated, serine ( FIG. 7a ), phenylalanine, tyrosine, dopamine ( FIG. 7b ), glutamine ( FIG. 7c ) in the brain compared to the DMSO control group It can be seen that the amount of is significantly increased. In particular, in the PTZ-treated group, which is an antagonist of the GABA A receptor, the amount of GABA of the inhibitory neurotransmitter significantly increased, but it was observed that DMSO was restored to the normal control level when the compound of Example 1 was additionally treated. As a result, the ratio of excitatory (GABA) / inhibitory (glutamic acid) neurotransmitters in the brain of the PTZ-induced seizure model was significantly significantly reduced, but the recovery to normal control levels of DMSO when additionally treated with Example 1 compound was observed. Through this, it can be seen that the compound of Example 1 is effectively involved in the signaling mechanism of the inhibitory neurotransmitter GABA in the brain.

As mentioned above, although the present invention has been described in detail through preferred examples and experimental examples, the scope of the present invention is not limited to specific examples, and should be interpreted by the appended claims. In addition, those skilled in the art should understand that many modifications and variations are possible without departing from the scope of the present invention.

Claims (12)

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

.
According to claim 1,
The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of epilepsy, characterized in that alleviating hyperactivity.
According to claim 1,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating epilepsy, characterized in that it inhibits the activation of nerve cells.
4. The method of claim 3,
A pharmaceutical composition for the prevention or treatment of epilepsy, characterized in that the activation of the nerve cells is induced by pentylenetetrazol.
4. The method of claim 3,
The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of epilepsy, characterized in that it reduces the amount of phosphorylated ERK1/2 protein (p-extracellular signal-regulated kinase-1/2, pERK1/2) in the brain.
According to claim 1,
The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of epilepsy, characterized in that it reduces the power (power) of the EEG signal.
According to claim 1,
The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of epilepsy, characterized in that it suppresses the magnitude of the voltage-dependent sodium current.
According to claim 1,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating epilepsy, characterized in that it promotes voltage-dependent sodium channel inactivation.
According to claim 1,
The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of epilepsy, characterized in that it slows recovery from inactivation of voltage-dependent sodium channels.
According to claim 1,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating epilepsy, characterized in that it increases the secretion of inhibitory neurotransmitters.
11. The method of claim 10,
The inhibitory neurotransmitter is 1 selected from the group consisting of serine, phenylalanine, tyrosine, dopamine, glutamine, acetylcholine, choline, glycine, betaine, glutamic acid, norepinephrine, serotonin, histamine and γ-aminobutyric acid A pharmaceutical composition for the prevention or treatment of epilepsy, characterized in that more than one species.
A health functional food for preventing or improving epilepsy containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

.

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