KR20190114300A - RHOA inhibitor and use thereof - Google Patents

Abstract

The present invention relates to a RhoA inhibitor and a use thereof. More specifically, the present invention relates to the RhoA inhibitor having a structure of chemical formula 1 of the present invention or a pharmaceutically acceptable salt thereof, a pharmaceutical composition for preventing or treating diseases caused by RhoA expression or increased activity comprising the same as an active component, and a health functional food for preventing or alleviating diseases caused by RhoA expression or increased activity, comprising the same as the active component.

Description

RhoA inhibitor and use thereof

The present invention relates to a RhoA inhibitor and its use, and more particularly, to a RhoA inhibitor having a structure of Formula 1 or a pharmaceutically acceptable salt thereof, and a disease caused by an increase in RhoA expression or activity comprising the same as an active ingredient. The present invention relates to a preventive or therapeutic pharmaceutical composition, and a nutraceutical for preventing or improving a disease caused by an increase in RhoA expression or activity including the same as an active ingredient.

[Formula 1]

Ras homolog gene family member A (RhoA) is a small GTPase protein of the Rho family. The effects of RhoA activity are not all well known, but are mainly associated with cytoskeletal regulation, mainly actin stress fiber formation and actomyosin contraction. In humans, it is encoded by the RhoA gene. RhoA acts on several effectors. Among them, ROCK1 (Rho-associated, coiled-coil containing protein kinase 1) and DIAPH1 (Diaphanous Homologue 1) are best described. RhoA is considered an important regulatory factor, particularly in other functions such as cytoskeletal dynamics, transcription, cell cycle progression and cell transformation.

RhoA is associated with various diseases, and elevated expression of RhoA has been identified in malignant prostate cancer, gastric cancer and testicular cancer (Schmidt, Lucy J. (2012). Molecular Endocrinology. 26 (5): 716-735; Zhang S et al. Molecular Cancer Research. 7 (4): 570-580).

In addition, the present inventors confirmed that the RhoA protein is overexpressed in early gastric cancer (Hae Ryung Chang et al., Gut., 0: 1, 2014), and by using siRNA to suppress the expression of the RhoA gene or , RhoA inhibitors, it was confirmed that the tumor formation can be suppressed.

RhoA is also associated with chronic myeloid leukemia (CML). CML, a stem cell disease that causes bone marrow cells to function poorly, is associated with actin polymerization, and signal proteins such as RhoA regulate the polymerization of actin. Due to the difference in protein between normal and affected neutrophils, RhoA has become a key factor and further experiments have also demonstrated that the RhoA-inhibiting pathway prevents the overall growth of CML cells. As a result, RhoA has important potential as a therapeutic target for gene therapy techniques for CML therapy.

Known as a RhoA inhibitor, 'Rhosin' was synthesized in June 2012 from a researcher at Cincinnati Children's Hospital. Rosine has the function of inhibiting cancer proliferation and promoting neuronal regeneration, and specifically targets Rho GTPases to prevent the growth of cancer-related cells. Rosine inhibited the growth of cancer cells in a dose-dependent manner and has been shown to be effective in asthma and diabetes.

Against this background, the inventors have completed the present invention by designing a novel RhoA inhibitor that is more effective than Rosine by targeting RhoA and confirming its RhoA inhibitory activity.

The present invention has been made to solve the above problems, and an object of the present invention is to provide a novel RhoA inhibitor or a pharmaceutically acceptable salt thereof that inhibits RhoA more effectively than rosine.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease caused by RhoA expression or increased activity, including the RhoA inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

Still another object of the present invention is to provide a dietary supplement for preventing or ameliorating diseases caused by an increase in RhoA expression or activity including the RhoA inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to solve the above problems of the present invention, the present invention provides a RhoA inhibitor or a pharmaceutically acceptable salt thereof having the structure of Formula 1 below:

 [Formula 1]

In Chemical Formula 1,

R may be -S (= 0) ₂ R ¹ , -C (= 0) R ² or -C (= 0) R ³ R ² ,

R ¹ may be a phenyl substituted with unsubstituted or C _{1- 4} alkyl Beach,

R ² is unsubstituted or substituted by hydroxy, C _{1- 4} alkoxy, halo (halo), or a nitro-substituted phenyl; Phenyl fused with a 5- or 6-membered heterocyclic ring containing 1 or 2 heteroatoms selected from the group consisting of S, N and O; Biphenyl; benzyl; 5- or 6-membered monocyclic heteroaryl comprising one or two heteroatoms selected from the group consisting of S, N and O; Or 9- or 10-membered bicyclic heteroaryl including 1 to 4 heteroatoms selected from the group consisting of S, N and O,

R ³ is C _{1- 4} alkyl; C _{1- 4} alkoxy; C _{1- 4} alkylthio; Or a 5- or 6-membered heterocyclic group comprising one or two heteroatoms selected from the group consisting of S, N and O.

According to one preferred embodiment of the invention, the R ¹ can be an unsubstituted phenyl, R ² are unsubstituted or substituted by hydroxy, C _{1- 4} alkoxy, halo (halo), or a nitro-substituted phenyl; Phenyl fused to a 5-membered heterocyclic ring containing two heteroatoms O; Biphenyl; benzyl; 5- or 6-membered monocyclic heteroaryl containing one heteroatom O or N; Cyclic 9-membered bicyclic containing one heteroatom N and may be heteroaryl, R ³ is a C _{1- 4} alkyl, C _{1- 4} alkoxy, C _{1- 4} containing one alkyl thio or a hetero atom N 5-membered heterocyclic group.

According to another preferred embodiment of the present invention, R in Formula 1 may be any one selected from the group consisting of:

,

,

,

,

,

,

,

,

,

,

,

,

및

.

,

,

,

,

,

,

,

,

,

,

,

,

And

.

According to another preferred embodiment of the present invention, the RhoA inhibitor may specifically bind to the binding domain of the RhoA protein to inhibit the activity of the RhoA protein.

The present invention also provides a pharmaceutical composition for preventing or treating a disease caused by an increase in RhoA expression or activity comprising the above-described RhoA inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a dietary supplement for preventing or ameliorating a disease caused by an increase in RhoA expression or activity comprising the above-described RhoA inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

According to a preferred embodiment of the present invention, the disease caused by the increase in RhoA expression or activity may be one or more selected from the group consisting of cancer, chronic myeloid leukemia, diabetes mellitus and asthma.

According to another preferred embodiment of the present invention, the cancer may be one or more selected from the group consisting of gastric cancer, breast cancer, prostate cancer and testicular cancer.

According to another preferred embodiment of the present invention, the RhoA inhibitor or a pharmaceutically acceptable salt thereof may be administered at a concentration of 0.5 to 2.5 μM.

The present invention provides a novel RhoA inhibitor and a composition for preventing, ameliorating or treating a disease caused by an increase in RhoA expression or activity comprising the same as an active ingredient. Since the RhoA inhibitor of the present invention is more effective in inhibiting RhoA expression or activity than Rosine, which is a conventional RhoA inhibitor, it exhibits a remarkable effect in preventing, ameliorating or treating diseases caused by RhoA expression or increased activity, particularly gastric cancer. In addition, since the effect of inhibiting the survival rate of gastric cancer cells is excellent even at a relatively low concentration, even a small amount can effectively prevent, improve or treat gastric cancer.

Figure 1 graphically shows the cell viability of gastric cancer cell lines AGS, SNU668 and MKN1 according to the concentration of the JK-301 to JK-307 compound.
Figure 2 graphically shows the cell viability of gastric cancer cell lines SNU216, SNU620 and SNU601 according to the concentration of the JK-301 to JK-307 compound.
Figure 3 graphically shows the cell viability of gastric cancer cell lines AGS, SNU668 and MKN1 according to the concentration of JK-308 to JK-314 compounds.
Figure 4 graphically shows the cell viability of gastric cancer cell lines SNU216, SNU620 and SNU601 according to the concentration of JK-308 to JK-314 compounds.
Figure 5 graphically shows the RhoA inhibitory activity of JK-301 to JK-306 compounds in gastric cancer cell lines AGS, MKN1 and SNU601.
Figure 6 graphically shows the RhoA inhibitory activity of JK-301 to JK-306 compounds in gastric cancer cell lines SNU620, SNU668 and SNU216.
Figure 7 graphically shows the RhoA inhibitory activity of JK-307 to JK-314 compounds in gastric cancer cell lines AGS, MKN1 and SNU601.
8 is a graph showing the RhoA inhibitory activity of JK-307 to JK-314 compounds in gastric cancer cell lines SNU620, SNU668 and SNU216.
9 graphically shows the cell migration inhibitory activity of JK-312 compounds in gastric cancer cell lines AGS, SNU668, MKN1, SNU216 and SNU601.

Hereinafter, the present invention will be described in more detail.

As described above, the present inventors have completed the present invention by designing a novel RhoA inhibitor having superior RhoA inhibitory activity to rosin known as a RhoA inhibitor, and confirming its effect against gastric cancer cell lines.

The present invention provides a novel RhoA inhibitor and a composition for preventing, ameliorating or treating a disease caused by an increase in RhoA expression or activity comprising the same as an active ingredient. Since the RhoA inhibitor of the present invention is more effective in inhibiting RhoA expression or activity than Rosine, which is a conventional RhoA inhibitor, it exhibits a remarkable effect in preventing, ameliorating or treating diseases caused by RhoA expression or increased activity, particularly gastric cancer. In addition, since the effect of inhibiting the survival rate of gastric cancer cells is excellent even at a relatively low concentration, even a small amount can effectively prevent, improve or treat gastric cancer.

The present invention provides a RhoA inhibitor or a pharmaceutically acceptable salt thereof having the structure of Formula 1:

 [Formula 1]

In Chemical Formula 1,

R is -S (= 0) ₂ R ¹ , -C (= 0) R ² or -C (= 0) R ³ R ² ,

R ¹ is phenyl unsubstituted or substituted with C _1-4 alkyl,

R ² is unsubstituted or substituted by hydroxy, C _{1- 4} alkoxy, halo (halo), or a nitro-substituted phenyl; Unsubstituted phenyl fused to an unsubstituted 5- or 6-membered heterocyclic ring comprising one or two heteroatoms selected from the group consisting of S, N and O; Biphenyl; benzyl; Unsubstituted 5- or 6-membered monocyclic heteroaryl comprising one or two heteroatoms selected from the group consisting of S, N and O; Or an unsubstituted 9- or 10-membered bicyclic heteroaryl comprising 1 to 4 heteroatoms selected from the group consisting of S, N and O,

R ³ is C _{1- 4} alkyl, C _{1- 4} alkoxy, C _{1- 4} alkylthio or S, N, and 5-or 6 membered heterocyclic containing one or two heteroatoms selected from the group consisting of O It may be a flag.

Unless otherwise specified, the term "alkyl" as used herein includes saturated monovalent hydrocarbon radicals having straight, branched or cyclic moieties. For example, alkyl radicals are methyl, ethyl, propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, n-pentyl, 3- (2-methyl) butyl, 2-pentyl, 2-methylbutyl, neopentyl, cyclopentyl, n-hexyl, 2-hexyl, 2-methylpentyl and cyclohexyl. Similarly, C _{1- 4} alkyl is 1, 2, 3 or 4 means a straight-chain or branched alkyl having carbon atoms and preferably methyl, ethyl, propyl, isopropyl, n- butyl, isobutyl, sec-butyl and t-butyl may be selected from the group consisting of.

As used herein, the term "halogen" means fluoro, chloro, bromo or iodo, unless otherwise specified. Preferred halogen groups include F, Cl and Br.

As used herein, the term "aryl" refers to an unsubstituted or substituted mono- or polycyclic ring system containing carbon ring atoms, unless otherwise specified, naphthyl, biphenyl, and the like. And a hydrocarbon ring having 6 to 12 carbon atoms.

The term "heteroaryl" also means a 5-12 membered aromatic radical comprising 1-4 heteroatoms selected from the group consisting of S, N and O, and a 5-6 membered monocyclic heteroaryl Radicals and bicyclic heteroaryl radicals formed by fusion with a benzene ring or a pyridine ring. For example, monocyclic heteroaryl is thiazole, pyrazole, oxazole, imidazole, pyrrole, furan, thiophene, isothiazole, isoxazole, triazole, thiadiazole, tetrazole, oxadiazole , Triazine, pyridine, pyridazine, pyrimidine, pyrazine and the like. In addition, bicyclic heteroaryl is benzothiazole, benzoxazole, benzimidazole, benzofuran, benzothiophene, benzisoxazole, indole, indolin, quinoline, isoquinoline, quinazoline, imidazopyridine, oxa Zolopyridine and the like.

The term "heterocycle" means a 3 to 12 membered mono- or poly-cyclic containing at least one, preferably 1 to 4, identical or different heteroatoms selected from the group consisting of heteroatoms S, N and O Ring, and does not include aromatic ring. For example, pyrrolidine, imidazoline, imidazolidine, pyrazoline, pyrazolidine, piperidine, morpholine, piperazine, tetrahydrofuran, tetrahydropyrimidin-2 (1H) -one, Imidazolidin-2-ones and the like. Preferably, the heterocycle may be a 5-6 membered mono-cyclic ring, for example pyrrolidine, morpholine, piperazine, tetrahydrofuran, piperidine, or dioxolane ) And the like.

As used herein, the term "alkoxy" includes O-alkyl groups unless otherwise noted. Wherein alkyl may be C _1-4 alkyl having 1 to 4 carbon atoms.

As used herein, the term "alkylthio" includes S-alkyl groups unless otherwise noted. Wherein alkyl may be C _1-4 alkyl having 1 to 4 carbon atoms.

In the RhoA inhibitory agent of the present invention, preferably, wherein R ¹ is unsubstituted phenyl, and may be, the R ² is unsubstituted or substituted by hydroxy, C _{1- 4} alkoxy, substituted by halo (halo), or nitro ring Phenyl; Phenyl fused to a 5-membered heterocyclic ring containing two heteroatoms O; Biphenyl; benzyl; 5- or 6-membered monocyclic heteroaryl containing one heteroatom O or N; 9-membered bicyclic heteroaryl containing one heteroatom N; R ³ may be an cyclic C _{1- 4} alkyl, C _{1- 4} alkoxy, C _{1- 4} 5- membered heteroaryl containing one to alkylthio, or N heteroatoms.

Most preferably, R ¹ may be phenyl, wherein R ² is phenyl substituted with hydroxy, methoxy, fluoro or nitro; Phenyl fused to dioxolane; Biphenyl; benzyl; Furyl; Pyridyl; May be benzimidazolyl, and R ³ may be methyl, methoxy, methylthio or pyrrolidinyl.

According to a preferred embodiment of the present invention, R in Formula 1 may be any one selected from the group consisting of:

,

,

,

,

,

,

,

,

,

,

,

,

및

.

,

,

,

,

,

,

,

,

,

,

,

,

And

.

According to another preferred embodiment of the present invention, the RhoA inhibitor may specifically bind to the binding domain of the RhoA protein to inhibit the activity of the RhoA protein.

In the present invention, RhoA is an abbreviation of Ras homolog geen family, member A, a small GTPase protein known to regulate actin cytoskeleton in the formation of stress fibers.

RhoA activates ROCK (RhoA kinase), which stimulates LIM kinase, then stimulates cofilin, effectively reconstituting the actin cytoskeleton of cells. In the case of neurons, activation of the RhoA pathway leads to growth cone collapse, which inhibits the growth and repair of nerve pathways and axons. Inhibition of the RhoA pathway by various factors causes some improved levels of nerve fiber myelin remodeling.

Known RhoA inhibitors include Rhosin, which is represented by the following formula (2).

[Formula 2]

The present inventors have prepared a novel RhoA inhibitor represented by the following formula (1) from the rosin.

[Formula 1]

The inventors of the previous study, as a novel RhoA inhibitor, developed the compound of formula 3, which was named JK-122.

[Formula 3]

In the present invention, for the development of activity and selectivity targeting RhoA, a novel RhoA inhibitor was designed using a hydrazide moiety that combines the structural properties of JK-122 and rosin as a backbone. A hit compound having a structure of was obtained.

[Formula 4]

For the synthesis of the target hydrazide, commercial benzhydrazide was used and reacted with the appropriate aldehyde in methanol or ethanol at room temperature (or heated) to give the corresponding final product. The reaction scheme is shown in Scheme 1 below.

Scheme 1

Reagents and reaction conditions: methanol or ethanol, reflux, 0.5-6 hours

인 화학식 5의 화합물을 합성하였고, 하기 화학식 5의 화합물을 기반으로 하여 최종적으로 화학식 1의 구조를 갖는 히트 화합물을 도출하였다.Ar in Chemical Formula 4 through Scheme 1

The compound of Formula 5 was synthesized, and a heat compound having a structure of Formula 1 was finally derived based on the compound of Formula 5.

[Formula 5]

The novel RhoA inhibitor of the present invention may be prepared by the general method described below, but is not limited thereto, and may be prepared by a manufacturing method commonly used in the art, in addition to the following manufacturing method.

General Synthetic Methods of Compounds of Formula 1

A mixture of 6- (benzo [d] [1,3] dioxol-5-yl) picolinaldehyde (1 mmol) and the appropriate hydrazide compound (1 mmol) dissolved in methanol or ethanol is added at reflux at room temperature or Heated and stirred for 0.5-4 h. The reaction process was monitored by TLC. After completion of the reaction, the contents were cooled to room temperature and poured into cold ice water (5 mL) with stirring. The solid was filtered and dried and purified by recrystallization using methanol or ethanol to give the hydrazide products JK-301 to JK-314.

(JK-301),

(JK-302),

(JK-303),

(JK-304),

(JK-305),

(JK-306),

(JK-307),

(JK-308),

(JK-309),

(JK-310),

(JK-311),

(JK-312),

(JK-313) 및

(JK-314).The 14 compounds of JK-301 to JK-314 obtained are those in which R in Formula 1 is substituted with the substituents described below in order:

(JK-301),

(JK-302),

(JK-303),

(JK-304),

(JK-305),

(JK-306),

(JK-307),

(JK-308),

(JK-309),

(JK-310),

(JK-311),

(JK-312),

(JK-313) and

(JK-314).

It was confirmed that the 14 compounds provided in the present invention can specifically bind to the binding domain of RhoA as in Rosine, and showed higher binding capacity than Rosine.

The compound of formula 1 according to the present invention can be used in the form of a pharmaceutically acceptable salt. As the salts, acid addition salts formed by various organic or inorganic acids that are pharmaceutically or physiologically acceptable are useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanediodes. Obtained from non-toxic organic acids such as acids, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide and iodide Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propionic acid, oxalic acid, malonic acid, succinic acid, suverate, sebacate, Fumarate, maleate, butyne-1,4-dioate, hexane-1,6-diionic acid, benzoic acid, chlorobenzoic acid, methylbenzoic acid, dinitro benzoic acid, hydroxy benzoate, methoxybenzoic acid, phthalic acid, terephthalate, Benzenesulfonic acid, toluenesulfonic acid, chlorobenzenesulfonic acid, xylenesulfonic acid, phenylacetic acid, phenylpropionic acid, phenyl Thirate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, tri It can be prepared using fluoroacetic acid or the like.

At this time, the acid addition salt according to the present invention is dissolved in a conventional method, for example, the compound of formula 1 in an excess of aqueous acid solution, and the salt is mixed with a water miscible organic solvent such as methanol, ethanol, acetone or aceto. It can be prepared by precipitation using nitrile. It may also be prepared by evaporating the solvent or excess acid from the mixture followed by drying, or by suction filtration of the precipitated salt.

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

Furthermore, amino acids can be used to make pharmaceutically acceptable salts. As an amino acid salt, it is pharmaceutically suitable to manufacture natural amino acids, such as glycine, alanine, phenylalanine, valine, lysine, glutamic acid, for example.

Compounds having the structure of Formula 1 of the present invention may be in the form of racemates, optical isomers, diastereomers, mixtures of optical isomers or mixtures of diastereomers.

As used herein, the term "optical isomer" refers to one of two stereoisomers that are mirror images that do not overlap with each other due to the presence of one or more chiral carbons.

The term "diastereomer" refers to a stereoisomer that is not an optical isomer, and occurs when two or more stereoisomers of the compound have different structures at one or more (but not all) of the equivalent chiral centers, and thus are mirror images of each other. This is not it.

The term "racemate" as used herein means that two stereoisomers, which are different stereo-structures, are equivalent and lack optical activity.

The present invention also provides a pharmaceutical composition for preventing or treating a disease caused by an increase in RhoA expression or activity comprising the above-described RhoA inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical composition of the present invention is used for the prevention or treatment of diseases caused by increased RhoA expression or activity, i.e., a disease requiring RhoA inhibition, for example, in a group consisting of cancer, chronic myeloid leukemia, diabetes mellitus and asthma. It may be one or more selected, but is not limited thereto.

At this time, the pharmaceutical composition of the present invention can be used without limitation in the prevention or treatment of all cancers caused by overexpression or excessive activity increase of RhoA, for example, selected from the group consisting of gastric cancer, breast cancer, prostate cancer and testicular cancer. It can be used for the prevention or treatment of more than one type of cancer.

The route of administration and mode of administration to which the pharmaceutical composition of the present invention is administered is not particularly limited and may be in accordance with any route of administration and mode of administration as long as the composition comprising the composition can reach the desired site of interest. Specifically, the composition may be administered through various routes, oral or parenteral, and non-limiting examples of the route of administration include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal What is administered through intralateral or inhalation etc. are mentioned.

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

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

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

In addition, the effective dosage of the compound represented by Formula 1 according to the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and generally about 0.001-100 mg / kg / day, preferably 0.01-35 mg / kg / day. Based on an adult patient weighing 70 kg, it is generally 0.07-7000 mg / day, preferably 0.7-2500 mg / day, once a day at regular intervals according to the judgment of the doctor or pharmacist. Multiple doses may be administered.

The present invention also provides a dietary supplement for preventing or ameliorating a disease caused by an increase in RhoA expression or activity comprising the above-mentioned RhoA inhibitor or a food acceptable salt thereof as an active ingredient.

Since the RhoA inhibitor and its RhoA inhibitory effect, which are included as an active ingredient in the health functional food of the present invention, are the same as described above, the description thereof is omitted.

The health functional food according to the present invention can be added to health functional foods such as foods and beverages for the purpose of preventing or improving diseases caused by RhoA expression or increased activity, that is, diseases requiring RhoA inhibition.

There is no particular limitation on the kind of food. Examples of foods to which the substance may be added include dairy products, various soups, drinks, meat, sausages, breads, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and the like includes all the health functional foods in the conventional sense.

The dietary supplement composition containing the compound of formula 1 according to the present invention or a food acceptable salt thereof as an active ingredient may be added to a food as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. Can be used. The mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement). In general, the amount of the composition in the dietary supplement may be added at 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

The nutraceutical composition of the present invention is not particularly limited to other ingredients except for containing the compound as essential ingredients in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 dietary food compositions of the present invention.

In addition to the above, the health functional food composition containing the compound of formula 1 of the present invention or a food acceptable salt thereof as an active ingredient is a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors. , Colorants and neutralizers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, etc. It may contain. In addition, the health functional food composition of the present invention may contain a flesh for preparing natural fruit juice and fruit juice beverage and vegetable beverage.

These components can be used independently or in combination. The ratio of such additives is not so important, but it is generally selected from the range of 0.1 to about 20 parts by weight per 100 parts by weight of the nutraceutical composition containing the compound of formula 1 of the present invention or a food acceptable salt thereof as an active ingredient. to be.

In addition, the present invention provides a method for treating a disease caused by increased RhoA expression or activity comprising administering to a subject in need thereof a pharmaceutically effective amount of a RhoA inhibitor having a structure of Formula 1 or a pharmaceutically acceptable salt thereof. Provide a method.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to exhibit and achieve efficacy and action for preventing, alleviating or treating a disease caused by increased RhoA expression or activity.

In the method of treatment of the present invention, the dosage, route of administration and dosage form of the RhoA inhibitor or a pharmaceutically acceptable salt thereof is as described above.

As used herein, the term "individual" means all animals, including rats, mice, livestock, etc., including humans, who may or may have a disease caused by increased RhoA expression or activity. Specifically, it may be a mammal including a human, but is not limited thereto.

The term "administration" refers to the introduction of a substance to an individual in a suitable manner. In particular, the RhoA inhibitor of the present invention or a pharmaceutically acceptable salt thereof may be administered to an individual alone, or may be administered concurrently, sequentially, or in reverse order with other therapeutic agents.

The method of treating a disease caused by the increase in RhoA expression or activity of the present invention may be combined with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.

According to another preferred embodiment of the present invention, the RhoA inhibitor, its pharmaceutically acceptable salt or its food acceptable salt may be administered at a concentration of 0.5 to 2.5 μM.

Hereinafter, the present invention will be described in detail with reference to a preferred embodiment so that those skilled in the art can easily practice the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.

Synthesis of Compound Having Structure of Formula 1 of the Present Invention

A mixture of 6- (benzo [d] [1,3] dioxol-5-yl) picolinaldehyde (1 mmol) and the appropriate hydrazide compound (1 mmol) dissolved in methanol or ethanol is added at reflux at room temperature or Heated and stirred for 0.5-4 h. The reaction process was monitored by TLC. After completion of the reaction, the contents were cooled to room temperature and poured into cold ice water (5 mL) with stirring. The solid was filtered and dried and purified by recrystallization using methanol or ethanol to give the hydrazide products JK-301 to JK-314.

(JK-301),

(JK-302),

(JK-303),

(JK-304),

(JK-305),

(JK-306),

(JK-307),

(JK-308),

(JK-309),

(JK-310),

(JK-311),

(JK-312),

(JK-313) 및

(JK-314).The 14 compounds of JK-301 to JK-314 obtained are those in which R in Formula 1 is substituted with the substituents described below in order:

(JK-301),

(JK-302),

(JK-303),

(JK-304),

(JK-305),

(JK-306),

(JK-307),

(JK-308),

(JK-309),

(JK-310),

(JK-311),

(JK-312),

(JK-313) and

(JK-314).

All reactions sensitive to air or moisture were carried out under a nitrogen atmosphere. Reagents were purchased from Aldrich and TCI. Unless otherwise specified, all anhydrous solvents were distilled on CaH ₂ , P ₂ O ₅ , or Na / benzophenone before the reaction. Analytical thin layer chromatography (TLC) was performed using commercially precoated TLC plates (Silica gel 60, F-254) purchased from Merck. Spots were detected under ultraviolet light (lamp at 254 nm) or by soaking in any of the following solutions and staining with carbonization: phosphomolybdic acid (PMA) dissolved in ethanol or potassium permanganate aqueous solution. Flash column chromatography was performed on silica gel 60 (0.040-0.063 mm, 230-400 mesh, Merck). Infrared spectra were recorded on an Agilent Cary670 instrument. ¹ H NMR spectra (CDCl ₃ , CD ₃ OD, D ₂ O or DMSO- d ₆ ) were recorded on Agilent 400-MR (400 MHz). Chemical shifts are reported in ppm (δ) for solvent peaks. ¹ H NMR data was reported as peak multiplicity: singlet was s; Doublet is d; The doublet of doublets is dd; The doublet of doublets of doublets is ddd; The triplet is t; Pseudo triplets are pseudo t; Broad singlet brs; And the polyline is m. Coupling constants were reported in Hertz. ¹³ C NMR spectra (CDCl ₃ , CD ₃ OD, D ₂ O or DMSO- d ₆ ) were recorded on Agilent 400-MR (100 MHz). Chemical shifts are reported in ppm (δ) for solvent peaks. Mass spectra were recorded on an ESI ⁺ source of methylene chloride or methanol.

(E) -N '-((6- ( benzo [d] [1,3] dioxol -5-yl) pyridin-2-yl) methylene) benzenesulfonohydrazide ( JK- 301) : ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 11.92 (s, 1H), 7.97 (s, 1H), 7.87 (m, 4H), 7.65 (m, 6H), 7.02 (d, J = 12.0 Hz, 1H) , 6.08 (s, 2 H).

(E) -N '-((6- ( benzo [d] [1,3] dioxol -5-yl) pyridin-2-yl) methylene) -4 -hydroxybenzohydrazide ( JK- 302) : ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 11.87 (s, 1H), 10.18 (s, 1H), 8.52 (s, 1H), 7.84-7.90 (m, 5H), 7.67 (s, 2H) , 7.04 (d, J = 8.0 μs, 1H), 6.89 (d, J = 8.0 μs, 2H), 6.10 (s, 2H).

(E) -N '-((6- ( benzo [d] [1,3] dioxol -5-yl) pyridin-2-yl) methylene) -4 -methoxybenzohydrazide ( JK- 303) : ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 11.94 (s, 1H), 8.54 (s, 1H), 7.93 (m, 5H), 7.68 (s, 2H), 7.07 (m, 3H), 6.10 (s, 2 H), 3.85 (s, 3 H).

(E) -N '-((6- ( benzo [d] [1,3] dioxol -5-yl) pyridin-2-yl) methylene) -4- fluorobenzohydrazide ( JK -304) : ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.09 (s, 1H), 8.54 (s, 1H), 7.89-8.03 (m, 5H), 7.67 (m, 2H), 7.40 (t, J = 8.0 Hz, 2H), 7.04 (d, J = 8.0 Hz, 1H), 6.10 (s, 2H).

(E) -N '-((6- ( benzo [d] [1,3] dioxol -5-yl) pyridin-2-yl) methylene) -3- nitrobenzohydrazide ( JK- 305) : ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.36 (s, 1H), 8.79 (s, 1H), 8.57 (s, 1H), 8.46 (d, J = 8.0 Hz, 1H), 8.40 (d, J = 8.0 μs, 1H), 7.84-7.93 (m, 4H), 7.68 (m, 2H), 7.04 (d, J = 8.0 μs, 1H), 6.10 (s, 2H).

(E) -N '- ((6- (benzo [d] [1,3] dioxol -5-yl) pyridin-2-yl) methylene) benzo [d] [1,3] dioxol -5- Carbohydrazide ( JK- 306) : ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 11.91 (s, 1H), 8.52 (s, 1H), 7.87-7.92 (m, 3H), 7.68 (m , 2H), 7.56 (d, J = 8.0 μs, 1H), 7.48 (s, 1H), 7.08 (d, J = 8.0 μs, 1H), 7.05 (d, J = 8.0 μs, 1H), 6.15 (s , 2H), 6.10 (s, 2H).

Gastric Cancer Cell Survival Assay

2-1. Cell culture

Human gastric tumor cell lines used in the present invention were used within 6 months after harvesting. Cell lines were stored in a 5% CO ₂ , 37 ° C environment. Specifically, human gastric tumor cell lines were AGS (ATCC, USA), MKN1 (RIKEN), SNU-668 (KCLB), SNU-620 (KCLB), SNU-601 (KCLB), and SNU-216 (KCLB). Incubated in RPMI-1640 (Invitrogen, USA) and 10% fetal bovine serum (Hyclone).

2-2. MTS Assay

The six gastric cancer cells of Example 2-1 were each seeded at 3 × 10 ³ / well in 96 well plates and exposed to various concentrations of JK-301 to JK-314 compounds for 72 hours. The cell survival effects of 14 compounds were 3-4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS ; Promega, USA) assay. IC ₅₀ values are defined as 50% cell growth inhibition compared to the control treated with DMSO. Values represent the average of triplicate wells from three independent experiments for each drug concentration.

As a result, as shown in FIGS. 1 to 4, all of the compounds of JK-301 to JK-314 exhibited superior gastric cancer cell inhibitory activity than rosine in a concentration-dependent manner for 6 gastric cancer cells.

RhoA  Active measurement

Active and GTP-binding RhoA was prepared according to the manufacturer's instructions. Measurements were made using the RhoA G-LISA Activity Assay Kit purchased from (USA). Specifically, JK-301 to JK-314 were treated to six gastric cancer cells of Example 2-1 for 48 hours. Protein lysates were collected for later analysis. Protein lysates were then tested on G-LISA plates using RhoA specific antibodies for detection of active RhoA captured according to the manufacturer's instructions. In all cases, the constitutively active RhoA protein provided in the G-LISA kit was used as a positive control to confirm the accuracy of the assay. The signal was read by measuring the absorbance at 490 nm using a micro plate reader.

As a result, as shown in Figs. 5 to 8, it was confirmed that the compounds of the 14 kinds of compounds, depending on the type of gastric cancer cell line, generally suppress RhoA activity.

Cell migration Assay

Based on the results of Examples 2 and 3, the cell migration assay was performed using the JK-312 compound having the highest activity. Specifically, the five gastric cancer cells of Example 2-1, namely AGS, SNU668, MKN1, SNU216, and SNU601 gastric cancer cells (2 × 10 ⁵ ), respectively, were prepared in a Boeden chamber (0.8 μm pore size, Corning, Corning, NY, USA). The bottom chamber was filled using medium containing FBS. The migrated cells were stained with a Diff-Quick kit (Thermo Fisher, Waltham, Mass., USA) and photographed with a reversed phase microscope. From each well the cells were counted in 3 microscopic fields and the percentage of migrated cells was measured by comparison with control cells.

As a result, as shown in Figure 9, it was confirmed that the migration inhibitory effect of six kinds of gastric cancer cells by JK-312 compound treatment is significantly superior to Rosine.

Claims (12)

A RhoA inhibitor having the structure of Formula 1 or a pharmaceutically acceptable salt thereof:
[Formula 1]

In Chemical Formula 1,
R is -S (= 0) ₂ R ¹ , -C (= 0) R ² or -C (= 0) R ³ R ² ,
R ¹ is phenyl unsubstituted or substituted with C _1-4 alkyl,
R ² is unsubstituted or substituted by hydroxy, C _{1- 4} alkoxy, halo (halo), or a nitro-substituted phenyl; Phenyl fused with a 5- or 6-membered heterocyclic ring containing 1 or 2 heteroatoms selected from the group consisting of S, N and O; Biphenyl; benzyl; 5- or 6-membered monocyclic heteroaryl comprising one or two heteroatoms selected from the group consisting of S, N and O; Or 9- or 10-membered bicyclic heteroaryl comprising 1 to 4 heteroatoms selected from the group consisting of S, N and O,
R ³ is C _{1- 4} alkyl; C _{1- 4} alkoxy; C _{1- 4} alkylthio; Or a 5- or 6-membered heterocyclic group comprising one or two heteroatoms selected from the group consisting of S, N and O. The method of claim 1,
R ¹ is unsubstituted phenyl,
R ² is unsubstituted or substituted by hydroxy, C _{1- 4} alkoxy, halo (halo), or a nitro-substituted phenyl; Phenyl fused to a 5-membered heterocyclic ring containing two heteroatoms O; Biphenyl; benzyl; 5- or 6-membered monocyclic heteroaryl containing one heteroatom O or N; 9-membered bicyclic heteroaryl containing one heteroatom N,
R ³ is C _{1- 4} alkyl, C _{1- 4} alkoxy, C _{1- 4} alkylthio or N heteroatom one five-membered heterocyclic group that features RhoA inhibitor or a pharmaceutically acceptable thereof, which comprises one possible salt. The RhoA inhibitor or a pharmaceutically acceptable salt thereof according to claim 1, wherein R in Formula 1 is any one selected from the group consisting of:

,

,

,

,

,

,

,

,

,

,

,

,

And

. The RhoA inhibitor or a pharmaceutically acceptable salt thereof according to claim 1, wherein the RhoA inhibitor or a pharmaceutically acceptable salt thereof is characterized by specifically binding to the binding domain of the RhoA protein to inhibit the activity of the RhoA protein. A pharmaceutical composition for preventing or treating a disease caused by RhoA expression or increased activity comprising the RhoA inhibitor of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof as an active ingredient. The method of claim 5, wherein the disease caused by increased RhoA expression or activity is at least one member selected from the group consisting of cancer, chronic myeloid leukemia, diabetes mellitus, and asthma. Or therapeutic pharmaceutical compositions. The pharmaceutical composition for preventing or treating a disease caused by RhoA expression or increased activity, characterized in that at least one selected from the group consisting of gastric cancer, breast cancer, prostate cancer and testicular cancer. The method of claim 5, wherein the RhoA inhibitor or a pharmaceutically acceptable salt thereof is administered at a concentration of 0.5 to 2.5 μM pharmaceutical composition for the prevention or treatment of diseases caused by RhoA expression or increased activity. A health functional food for preventing or ameliorating diseases caused by an increase in RhoA expression or activity comprising the RhoA inhibitor of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof as an active ingredient. 10. The method of claim 9, wherein the disease caused by increased RhoA expression or activity is at least one selected from the group consisting of cancer, chronic myeloid leukemia, diabetes mellitus and asthma. Or dietary supplements for improvement. The health functional food for preventing or ameliorating diseases caused by RhoA expression or increased activity, wherein the cancer is at least one selected from the group consisting of gastric cancer, breast cancer, prostate cancer and testicular cancer. The health functional food for preventing or ameliorating a disease caused by RhoA expression or increased activity, wherein the RhoA inhibitor or a pharmaceutically acceptable salt thereof is administered at a concentration of 0.5 to 2.5 μM.

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