WO2008050600A1

WO2008050600A1 - Therapeutic or preventive agent for intractable disease based on oxidative stress-mediated cell death as molecular background

Info

Publication number: WO2008050600A1
Application number: PCT/JP2007/069619
Authority: WO
Inventors: Kazunori Tanaka; Joh-E Ikeda; Noriaki Hirayama; Takuya Kanno
Original assignee: Neugen Pharma Inc.
Priority date: 2006-10-25
Filing date: 2007-10-05
Publication date: 2008-05-02
Also published as: JPWO2008050600A1

Abstract

A therapeutic or preventive agent for an intractable disease based on oxidative stress-mediated cell death as a molecular background according to the invention contains a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. (1) In the formula, R1 represents a hydrogen atom, R2 represents a C6-C10 aryl group which may have a C1-C3 alkyl group as a substituent; or R1 and R2 may be combined with each other to form a tetrahydrofuranyl ring together with the adjacent oxygen atom; and R3 and R4 are independent of each other and represent a hydrogen atom, a pyridyl group or a C6-C10 aryl-C1-C3 alkyl group which may have a halogen atom as a substituent.

Description

Specification

Therapeutic or preventive agent for intractable diseases with molecular background of oxidative stress cell death

[0001] The present invention relates to a therapeutic or prophylactic agent for intractable diseases whose molecular background is oxidative stress cell death.

Background art

[0002] Amyotrophic lateral sclerosis (ALS), spinal muscular atrophy syndrome (SMA), Huntington's disease, Parkinson's disease, Alzheimer's disease, dementia after cerebrovascular disorder, and other dementia with neuronal loss A group of diseases involving the degeneration of nerve cell groups is collectively referred to as neurodegenerative diseases. Most neurodegenerative diseases have not yet been fundamentally treated, and there is a need for a search for treatment.

[0003] As an approach for treating neurodegenerative diseases, it is conceivable to administer a factor that suppresses degeneration of nerve cells. Administration of a factor that suppresses neurodegeneration is expected to have an advantageous effect on the treatment and prevention of such diseases. However, almost all of these factors that can actually be used as therapeutic drugs have been found! //!

[0004] As a factor that suppresses the degeneration of nerve cells, it is known that, for example, certain dopamine receptor agonists may have such an action. However, the causal relationship between dopamine antagonism and suppression of neuronal degeneration is unclear, and not all such dopamine receptor agonists have such effects.

[0005] As a serious modifier of spinal muscular atrophy (SMA), one of the intractable lower motor neurodegenerative diseases, a neuronal apoptosis inhibitor protein from human chromosome 5ql3.1 The (Neuronal Apoptosis Inhibitory Protein (NAIP)) gene has been isolated (Non-patent Document 1), and the entire amino acid sequence of NAIP and cDNA encoding NAIP have been isolated (Patent Document 1). In the process of searching for substances that increase NAIP production, certain dopamine receptor antagonists may increase NAIP production, which may actually suppress neurodegeneration. It has been found (Patent Document 2). The same -Like dopamine receptor antagonists are known to protect neurons and non-neuronal cells from apoptosis caused by oxidative stress, and to suppress neuronal cell death caused by ischemia! / Non-patent document 2).

[0006] Patent Document 1: Japanese Patent Laid-Open No. 11 116599

Patent Document 2: JP 2004-123562 A

Non-Patent Document 1: Roy M. et al. Cell (1995) 80, 167-178

Non-Patent Document 2: Okada Y. et al. Journal of Cerebral Blood Flow & Metabolism (2005) 25, 794-806

Disclosure of the invention

Problems to be solved by the invention

[0007] An object of the present invention is to provide a pharmaceutical composition useful for the treatment or prevention of intractable diseases having molecular background of oxidative stress cell death.

Means for solving the problem

[0008] As a result of intensive studies to find an effective substance that can be used as a therapeutic agent for refractory diseases with molecular background of oxidative stress cell death, the present inventors have found that a compound having a specific structure has an oxidative stress property. The present invention has been found to have the activity of remarkably suppressing cell death and to be effective as a therapeutic agent for refractory diseases derived from oxidative stress cell death such as neurodegenerative diseases ischemic heart disease. It came. That is, the present invention includes the following.

[0009] [1] The following general formula (1)

[Wherein, R ¹ represents a hydrogen atom, R ² represents a C6-C10 aryl group optionally having a C1-C3 alkyl group as a substituent; or R ¹ and R ² are bonded to each other; May form a tetrahydrofuranyl ring with an adjacent oxygen atom! /,

R ³ and R ⁴ each independently represent a hydrogen atom, a pyridyl group or a halogen atom as a substituent! /, May be! /, A C6_C10 aryl C1-C3 alkyl group. ] Compound represented by Or a pharmaceutically acceptable salt thereof as an active ingredient, a therapeutic or prophylactic agent for intractable diseases with molecular background of oxidative stress cell death.

[2] The treatment according to [1], wherein the C6-C10 aryl group optionally having a C1-C3 alkyl group in R ² is a 2,4,6-trimethylphenyl group Or prophylactic agent. [3] The therapeutic or prophylactic agent according to [1] or [2], wherein either one of R ³ and R ⁴ is a hydrogen atom, and the other is a pyridyl group or a 2,5-dichlorophenylmethyl group. [4] The therapeutic or prophylactic agent according to [1], wherein the compound represented by the formula (1) is the following compound 1A or 1B;

[Wherein, R ⁵ and R ⁶ each independently has a C6-C10 aryl group or a C1-C6 alkyl group optionally having a halogen atom as a substituent! /, Moyo! / Indicates a pyrrolyl group. Or a pharmaceutically acceptable salt thereof as an active ingredient. A therapeutic or prophylactic agent for intractable diseases having molecular background of oxidative stress cell death.

[6] R ⁵ , R ⁶ may be! /, And either of them may have a halogen atom as a substituent! / The therapeutic or prophylactic agent according to [5], which is V, a phenyl group, and the other has a C1-C3 alkyl group as a substituent! /, May! /, A pyrrolinole group.

[7] The therapeutic or prophylactic agent according to [5], wherein the compound represented by the formula (2) is the following compound 2A or 2B;

The following general formula

[Wherein X represents an oxygen atom or a sulfur atom;

R ⁷ represents any group represented by the following formula;

Or a pharmaceutically acceptable salt thereof as an active ingredient. A therapeutic or prophylactic agent for intractable diseases with molecular background of oxidative stress cell death.

[9] The therapeutic or prophylactic agent according to [8], wherein the compound represented by the formula (3) is the following compound 3A or 3B;

(3B).

[10] A therapeutic or prophylactic agent for intractable diseases with oxidative stress cell death as a molecular background, comprising as an active ingredient any of the following compounds or pharmaceutically acceptable salts thereof;

[11] The therapeutic or prophylactic agent according to any one of [1] to [10], wherein the intractable disease whose molecular background is oxidative stress cell death is a neurodegenerative disease or an ischemic heart disease. [12] The neurodegenerative disease is associated with amyotrophic lateral sclerosis (ALS), spinal muscular atrophy syndrome (SMA), Huntington's disease, Parkinson's disease, Alzheimer's disease, dementia or neuronal loss after cerebrovascular disorder The therapeutic or prophylactic agent according to [11], which is dementia.

[13] The therapeutic or prophylactic agent according to [11], wherein the ischemic heart disease is angina or myocardial infarction.

[14] Further, the present invention provides an effective amount of [1] to [; 10] for patients who need treatment or prevention of intractable diseases whose molecular background is oxidative stress cell death. It also relates to a method for the treatment or prevention of refractory diseases with molecular background of oxidative stress cell death comprising the step of administering the described compound or a pharmaceutically acceptable salt thereof. [0010] [15] Further, the present invention relates to [1] to [; 10]! /, For producing a therapeutic or prophylactic agent for intractable diseases having molecular background of oxidative stress cell death. Or the pharmaceutically acceptable salts thereof.

[Embodiment of the Invention]

In this specification, a “Cl_Cn alkyl group” is a monovalent group derived by removing one arbitrary hydrogen atom from an aliphatic hydrocarbon, and has a heteroatom or an unsaturated carbon-carbon bond in the skeleton. Does not contain, has a subset of hydrocarbyl or hydrocarbon containing hydrogen and carbon atoms. Alkyl groups include linear or branched structures. The “C 1 -Cn alkyl group” indicates 1 to n carbon atoms, and the “C1_C6 alkyl group” indicates carbon atoms;!

Specifically, for example, methyl group, ethyl group, n propyl group, i propyl group, n butynol group, sec butyl group, t butyl group, isobutyl group, pentyl group, isopentyl group, 2, 3 dimethylpropyl Group, hexyl group, 2,3 dimethylhexyl group, 1,1 dimethylenopentyl group, heptyl group, octyl group and the like.

In the present specification, the “C6_Cn aryl group” means an aromatic hydrocarbon cyclic group having 6 to n carbon atoms. n is preferably 10. Specific examples include a phenyl group, a 1-naphthyl group, and a 2-naphthyl group. A phenyl group is preferred.

In the present specification, the “C6_Cn aryl group Cl—Cn alkyl group” is a group in which any hydrogen atom in the above-defined “C1-Cn alkyl group” is substituted with the above-mentioned definition “C6_Cn aryl group”. means.

In the present specification, the “phenyl Cl-Cn alkyl group” means a group in which any hydrogen atom in the above-defined “C1-Cn alkyl group” is substituted with the above-mentioned “phenyl group”.

In the present specification, the “halogen atom” means a fluorine atom, a chlorine atom, a bromine atom or a silicon atom.

[0017] In the present specification, the "refractory disease with oxidative stress cell death as a molecular background" is a refractory disease with oxidative stress cell death as a molecular background! / Neurodegenerative diseases, ischemic heart diseases, diabetes and the like.

“Molecular background” means “pathogenesis” and the cause of the disease or the course of the disease. Show over.

[0018] As used herein, "neurodegenerative disease" means a neurological disease caused by the gradual death of a specific group of nerve cells in the central nerve. Examples of neurodegenerative diseases include cranial nerve degenerative diseases. Such cranial neurodegenerative diseases include, for example, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy syndrome (SMA), Huntington's disease, Parkinson's disease, Alzheimer's disease, dementia after cerebrovascular disorder, and other This includes dementia with nerve loss.

[0019] In this specification, "ischemic heart disease" is a disease caused by ischemia caused by a decrease or interruption of blood supply to the heart muscle, such as angina pectoris and myocardial infarction. Can be mentioned.

[0020] "Pharmaceutically acceptable salt" means that the biological effectiveness and properties of the compound represented by the formula (1) are retained, and the strength, suitable non-toxic organic or inorganic acid or organic or It means a normal salt formed from an inorganic base.

Acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, as well as P-toluenesulfonic acid, salicylic acid, methanesulfonic acid, sulfur Those derived from organic acids such as acid, succinic acid, succinic acid, malic acid, lactic acid, fumaric acid are included.

Base addition salts include those derived from quaternary ammonium hydroxides, such as potassium hydroxide, sodium, ammonium, and tetramethylammonium hydroxide.

[0021] In addition, the compound used in the present invention may absorb moisture by adhering to the atmosphere and may be adsorbed water or become a hydrate. Such a hydrate Are also included in the salts of the present invention.

Furthermore, the compound used in the present invention may absorb a certain other solvent and become a solvate, and such a solvate is also encompassed in the salt of the present invention.

[0023] The compounds that can be used for the treatment or prevention of intractable diseases with molecular background of oxidative stress cell death according to the present invention will be described below.

The compound of the first embodiment used in the present invention is represented by the following general formula (1). (In this specification, the compound represented by the formula (1) may be referred to as the compound 1. In addition, the compound 1 may be represented by the formula ( It may be referred to as a compound represented by 1). The same applies to other formulas and compounds. )

[0025] In the formula, R ¹ represents a hydrogen atom, and R ² represents a C6-C10 aryl group which may have a C1-C3 alkyl group as a substituent. Alternatively, R ¹ and R ² may be bonded to each other to form a tetrahydrofuranyl ring with an adjacent oxygen atom.

[0026] The C1-C3 alkyl group of the C6-C10 aryl group that may have a C1-C3 alkyl group as a substituent in R ² is preferably a methyl group. The aryl group is preferably a phenyl group. The C6_C10 aryl group having a C1-C3 alkyl group as a substituent for R ² is more preferably a 2,4,6-trimethylphenyl group.

[0027] R ³ and R ⁴ each independently represent a hydrogen atom, a pyridyl group or a halogen atom as a substituent! /, Or may be a C6_C10 aryl C1-C3 alkyl group.

[0028] Preferably, one of R ³ and R ⁴ is a hydrogen atom, and the other is a pyridyl group or a 2,5-dichlorophenylmethyl group.

[0029] Specific examples of the compound represented by the formula (1) include the following compound 1A or 1B.

[0030] Compound of the second aspect of the present invention is a compound represented by the following general formula (2) _c

In the formula, R ⁵ and R ⁶ each independently have a C6-C10 aryl group or a C1-C6 alkyl group optionally having a halogen atom as a substituent! / , Even! / Represents a pyrrolyl group.

In the above R ⁶ , preferably, any one of them has a halogen atom as a substituent, V, may be! /, A phenyl group, and the other has a C1-C3 alkyl group as a substituent. ! /, Even! / ヽ pyrrolyl group.

[0033] Specific examples of the compound represented by the formula (2) include the following compound 2A or 2B.

The compound according to the third aspect of the present invention is a compound represented by the following general formula (3).

In the formula, X represents an oxygen atom or a sulfur atom. R ⁷ represents any group represented by the following formula. (In the formula, “(” indicates a bonding position.)

[0036] Specific examples of such a compound represented by the formula (3) include the following compound 3A or 3B.

(3B).

[0037] Further, examples of the compound according to another embodiment of the present invention include the following compounds 4A to 9A.

[0038] As the above compounds 1A, 1B, 2A, 2B, 3A, 3B, 4A, 5A, 6A, 7A, 8A, 9A, for example, commercially available products can be used. In addition, the ability to obtain compounds represented by formulas (1), (2), and (3) by appropriately converting functional groups and the like by a method commonly used in organic synthesis reactions using commercially available products as starting materials. Touch with S.

[0039] The compounds represented by the formulas (1), (2), (3), (4A) to (9A), and pharmaceutically acceptable salts thereof used in the present invention include the compounds listed above. All stereoisomers (eg, enantiomers, diastereomers (including cis and trans geometric isomers)), Semi-, and other mixtures are included.

[0040] In addition, the compounds represented by the formulas (1), (2), (3), (4A) to (9A) and pharmaceutically acceptable salts thereof used in the present invention have several tautomers. It may exist in sex forms, such as the enol and imine forms, the keto and enamine forms, and mixtures thereof. Tautomers exist in solution as a mixture of tautomeric sets. In solid form, one tautomer is usually predominant. The ability to describe one tautomer The present invention includes all tautomers of the compounds of the invention.

[0041] When these isomers exist, they can be isolated by a conventional method using the difference in physicochemical properties between the isomers. For example, when a racemate is present, it can be made into a stereopure isomer by a general optical resolution method, for example, a method in which a diastereomeric salt with an optically active acid such as tartaric acid is induced and optically resolved. . When a mixture of diastereomers is present, it can be separated by fractional crystallization and various types of chromatography (for example, thin-layer chromatography, column chromatography, gas chromatography, etc.).

[0042] When the compound used in the present invention is obtained in a free form, it can be converted into a salt or a hydrate or solvate thereof, which may be formed by the compound, according to a conventional method. .

When the compound used in the present invention is obtained as a salt, hydrate, or solvate of the compound, it can be converted into a free form of the compound according to a conventional method.

[0043] The compounds represented by the formulas (1), (2), (3), (4A) to (9A) and the pharmaceutically acceptable salts thereof used in the present invention are oxidative stress. It has the activity of suppressing sexual cell death. For this reason, the compounds represented by the formulas (1), (2), (3), (4A) to (9A) and pharmaceutically acceptable salts thereof are intractable due to the molecular background of oxidative stress cell death. It is used as a preventive or therapeutic agent for sexually transmitted diseases.

[0044] Examples of refractory diseases with molecular background of oxidative stress cell death include neurodegenerative diseases, ischemic heart diseases, diabetes and the like.

Examples of the neurodegenerative diseases include amyotrophic lateral sclerosis (ALS), spinal muscular atrophy syndrome (SMA), Huntington's disease, Parkinson's disease, Alzheimer's disease, cerebrovascular disease. This includes dementia after harm and other dementia with neurological deficits.

Examples of the ischemic heart disease include angina pectoris and myocardial infarction.

[0045] The present invention also relates to a method for the prevention or treatment of intractable diseases with molecular background of oxidative stress cell death, wherein a therapeutically effective amount of formula (1), (2 ), (3), (4A) to (9A) or a pharmaceutically acceptable salt thereof, which comprises administering a compound or a pharmaceutically acceptable salt thereof. Treatment or prevention of an intractable disease having oxidative stress cell death as a molecular background Also related to the method

Yes

[0046] Furthermore, the present invention provides formulas (1), (2), (3), (4A) for the manufacture of a preventive or therapeutic agent for intractable diseases having molecular background of oxidative stress cell death. It also relates to the use of a compound represented by ~ (9A) or a pharmaceutically acceptable salt thereof.

[0047] When the pharmaceutical composition of the present invention is used as a prophylactic or therapeutic agent for intractable diseases having molecular background of oxidative stress cell death, the administration method is oral, rectal, parenteral ( Intravenous, intramuscular, subcutaneous), intracisternal, intravaginal, intraperitoneal, intravesical, topical (infusion, powder, ointment, gel or cream) and inhalation (oral or nasal) Spray). The dosage forms include, for example, tablets, capsules, granules, powders, pills, aqueous and non-aqueous oral solutions and suspensions, and containers adapted for subdividing individual doses. Parenteral solution. The dosage form can also be adapted to various modes of administration including controlled release formulations such as subcutaneous implantation.

[0048] The above-mentioned preparation is produced by a known method using additives such as excipients, lubricants (coating agents), binders, disintegrants, stabilizers, flavoring agents, and diluents.

[0049] Examples of excipients include starches such as starch, potato starch, and corn starch, lactose, crystalline cellulose, calcium hydrogen phosphate, and the like.

[0050] Examples of the coating agent include ethyl cellulose and hydroxypropyl cellulose.

, Hydroxypropylmethylcellulose, shellac, talc, carnauba wax, paraffin, etc.

[0051] Examples of the binder include polybulurpyrrolidone, macrogol, and the same compounds as the excipient. [0052] Examples of the disintegrant include compounds similar to the above-mentioned excipients and chemically modified starch / celluloses such as croscarmellose sodium, sodium carboxymethyl starch, and crosslinked polybululpyrrolidone. .

[0053] As the stabilizer, for example, para-benzoic acid esters such as methyl paraben and propyl paraben; alcohols such as chlorobutanol, benzyl alcohol, and phenolic alcoholic alcohol; benzalkonium chloride; such as phenolic and talesol Mention may be made of phenols; thimerosal; dehydroacetic acid; and sorbic acid.

[0054] Examples of the flavoring agent include commonly used sweeteners, acidulants, and fragrances.

[0055] Further, as a solvent for producing a liquid agent, ethanol, phenol, black-and-white crezo monole, purified water, distilled water, or the like can be used.

[0056] Examples of surfactants or emulsifiers include polysorbate 80, polyoxynole 40 stearate, lauro macro gonole and the like.

[0057] When the pharmaceutical composition of the present invention is used as a prophylactic or therapeutic agent for intractable diseases having molecular background of oxidative stress cell death, use of the compound of the present invention or a pharmaceutically acceptable salt thereof The amount depends on symptoms, age, weight, relative health, presence of other medications, method of administration, etc. For example, for patients (warm-blooded animals, especially humans), generally effective amounts are preferably 0.1 to 1000 mg per kg body weight per day, more preferably 1 kg body weight per day in the case of oral agents as active ingredients. Per day; the daily dose is preferably in the range of 10 to 800 mg for normal weight adult patients. In the case of a parenteral preparation, it is preferably 0.1 to 1000 mg per kg body weight per day, more preferably 10 to 800 mg per kg body weight. This should be administered once or several times a day according to the symptoms.

All prior art documents cited in this specification are incorporated herein by reference.

Brief Description of Drawings

[0058] [FIG. 1] FIG. 1 is a graph showing the results of cell death inhibitory activity of compounds 1A, 1B, 2A, 2B, 3A and 3B by a cell survival test. In Figure 1, A is Compound 1A, B is Compound 1B, and C is Compound 2A and D show the results of Compound 2B, E shows the results of Compound 3A, and F shows the results of Compound 3B.

FIG. 2 is a graph showing the results of cell death inhibitory activity of compounds 4A, 5A, 6A, 7A, 8A and 9A by a cell survival test. In Figure 2, G is compound 4A, H is compound 5A, and I is compound

6A and J are the results of compound 7A, K is the compound 8A, and L is the result of compound 9A.

FIG. 3 is a graph showing the efficacy of Compound 1A in an ALS (S0D1 H46R) mouse model. The result of the K apian-Meier curve regarding the onset day of the control group 1 and the administration group 1 (compound 1A, 1 mg / kg / day oral administration) is shown.

FIG. 4 is a graph showing the efficacy of Compound 1A in an ALS (S0D1 H46R) mouse model. The result of the Kaplan-Meier curve regarding the onset day of the control group 1 and the administration group 2 (Compound 1A, lOmg / kg / day oral administration) is shown.

FIG. 5 shows the inhibitory effect of Compound 1A on neuronal cell death induced by 6-hydroxydopamine (6-OHDA) in dopaminergic human neuroblastoma cell line SH-SY5Y cells. It is a figure which shows the outline of this examination experiment.

FIG. 6 is a graph showing the cell death inhibitory effect of Compound 1A on 6-OHDA damage (cell death) in SH-SY5Y cells 5 days after induction of differentiation.

Example

[0059] The present invention will be described below with reference to examples, but the present invention is not limited to the examples.

[Example 1]

In this example, the cell death inhibitory activity of a candidate low molecular weight compound was examined by a cell viability test against cell death induced by oxidative stress.

[0061] [Reagents, instruments]

Sakai: HeLa cells

Medium: DMEM (SIGMA) (contains 10% FBS, 4 mM Glutamine, 100 μg / ml streptomycin, 100 U / ml penicillin G

Reagent 穎: Menadione (SIGMA), Dimethyl sulfoxide (DMSO) (SIGMA), AlamarBlue (Biosource)

[0062] Compounds: 1A, 1B, 2A, 2B, 3A, 3B, 4A, 5A, 6A, 7A, 8A, 9A

urn

6T9690 / .00Zdf / X3d LV 0090S0 / 800Z OAV Υ Υ Λ

V6

义

V8 νε z V9

What

V9

93 S

^S people

vz

T9690 / .00Zdf / X3d 81- 0090S0 / 800Z OAV , () deseatuS 057879 iAinxPlinm AN44

¾sf s¾5, y i: ^ [() () ucoetuooAe3taocatldNhlN4: 4: tIrdlllizl2lr-lllIlllll

, (I) oeceto OSS53630nPrinnK2-pf, yyyyy ()

^ #I: 2A

ppyyy ,, yy (() ()) () (d ± ao6teteo acetade Compound 8A:

(E)-2- (2-fluorobenzylthio)-6,7- dihydro- [l, 3,4] thiadiazolo [3,2-a] [l, 3] diaz in- 8 (5H) -one (LifeChemicals , F0597-0028)

Compound 9A:

[(2,5_dichlorophenyl) sulfonyl] [(8_methyl (4_hydroimidazo [l, 2_a] pyridin_2_yl)) methyl

] amine (LifeChemicals, F1837-0136)

[0063] Each compound was used after being dissolved or diluted with DMSO.

[0064] Measuring instrument: CYTOFLUOR (registered trademark) Multi-Well Plate Reader Series 4000 (PerSpective Biosystems)

[Method of experiment]

1: Cell

HeLa cells used in this study were cultured in DMEM medium (SIGMA) containing 10% FBS, 4 mM Glutamine, lOO ^ g / ml streptomycin, lOOU / ml penicillin G.

[0066] 2: Cell viability test

HeLa cells (1.0 × 10 ⁶ cells / 75 cm ² flask (Iwaki, 3123-075)) are cultured in the above medium at 37 ° C overnight in the presence of 5% C 0, so that the final concentration is 40 The compound solution was added and further cultured for 24 hours. In this experiment, DMSO alone was used as the control experiment. DMSO and compound-treated HeLa cells were washed with the above medium, and after collection, the number of cells was counted and suspended in a 96-well plate (FALCON, 35-3072) so as to be 1 · 5 × 10 ⁴ cells / well The liquid was added.

[0067] After 6 hours of culture, add the oxidative stress agent Menadione to a final concentration of 0 \, 30 μ ^ Ο μ ^ 50 μ ^ 60 、, and 37 ° C in the presence of 5% CO. A 4-hour oxidative stress treatment was performed. A blank treated with the above medium containing 1% TritonX-100 instead of Menadione was used. After washing with the above medium, the medium is replaced with the medium containing 10% A1 讓 arBlue, and CYTOFLUOR (registered trademark) Mult Well Plate Reader Series 4000 within 15 to 20 hours under 37 ° C culture conditions in the presence of 5% CO. The number of viable cells was determined by measuring the excitation wavelength (530 nm) and emission wavelength (580 nm) using (Perspective Biosystems).

[0068] The results of the cell death inhibitory effect of the previous compound on Menadione, an oxidative stress agent It is shown in Fig.1 and Fig.2. The horizontal axis of the graph represents Menadione concentration, and the vertical axis represents cell viability. The numerical value on the vertical axis under each condition is shown as a relative value when the cell viability under the Menadione untreated (0 M) condition in the control experiment is defined as 100%.

[0069] [Result]

Hatching stress induction ¾ί Analysis of the inhibitory effect of candidate compounds on cell death

With respect to cell death induced by oxidative stress, the compounds 1 に関する, 1Β, 2Α, 2Β, 3Α, 3Β, 4Α, 5Α, 6Α, 7Α, 8Α, 9Α were analyzed for the cell death inhibitory effect. As shown in Fig. 1 and Fig. 2, the comparison with the survival rate of the control experiment treated with Menadione (black bar graph) shows that compounds 1A, 1B, 2A, 2B, 3A, 3B, 4A, 5A, 6A, 7A, Cells pretreated with 8A and 9A showed a significant survival rate (white bar graph) even after treatment with Menadione. This result indicates that the compounds 1A, 1B, 2A, 2B, 3A, 3B, 4A, 5A, 6A, 7A, 8A and 9A have an inhibitory effect on cell death induced by oxidative stress.

[0070] It has been suggested that oxidative stress is closely related to neurodegenerative diseases and ischemic heart disease (Mariani E. et al. Journal of Chromatography B (2005), 827, 65-75). In addition, a compound having an inhibitory effect on cell death induced by oxidative stress is effective in preventing neurodegenerative diseases and ischemic heart diseases!

[Example 2] Efficacy evaluation of Compound 1A in ALS (S0D1 H46R) mouse model

Amyotrophic lateral sclerosis (ALS) is a progressive, intractable motor neurodegenerative disease characterized by degeneration of the upper and lower motor nerves. As the molecular background of the onset and progression of ALS, a great deal of knowledge has been accumulated to support motor nerve degeneration and cell death due to oxidative stress and reactive oxygen species. In this example, for the purpose of ALS therapeutic drug development, of the 12 candidate low molecular weight compounds (1A, IB, 2A, 2B, 3A, 3B, 4A, 5A, 6A, 7A, 8A, 9A) Using Compound 1A, which showed the highest cell death inhibitory activity against cell death induced by the stress free radical inducer menadione, the efficacy of the compound in an ALS (S0D1 H46R) mouse model was examined.

[Reagent]

Compound lA: N- (4- (2-pyridyl) (l, 3-thiazcJ—2-yl)) — 2— (2,4,6-trimethylphenoxy) acetamid e (ENAMINE, T5252123) 〔animal〕

Cu / Zn-SODl gene transgenic mice with amyotrophic lateral sclerosis (ALS): ALS (SOD 1 H46R) mice (Aoki M. et al., Saishin Igaku (2002), Vol.57 (7), 1622-1627)

[Method of experiment]

Compound 1A was suspended in 0.5% Carboxymethyl cellulose-Na (Maruishi Pharmaceutical Co., Ltd., Japan) solution to prepare a 2 mg / mL administration solution. The lmg / kg and 10 mg / kg doses were calculated based on the body weight of ALS (SODl H46R) mice, and the volume of solution to be administered per mouse individual was 0.5 mL Carboxymethyl cellulose-Na solution. Prepared. Administration of ALS (SODl H46R) mice was started at 12 weeks of age (84 days after birth). The dose was administered once a day by oral administration using a gastric tube until daily death. ALS (SODl H46R) mouse population administered orally with lmg / kg dose of Compound 1 A administration group 1, ALS (SODl H46R) mouse population administered orally with Compound 1 A at 10 mg / kg dose 2 and ALS (SODl H46R) mice that were orally administered with 0.5% Carboxy methyl cellulose-Na solution alone were designated as Control Group 1. The evaluation of the onset of neurological symptoms in each treatment group was performed using the balance beam test (Tariq M. et al ·, Brain Res. Bull. (2005), 67, 161-168) (using a cylindrical bar, the length of the bar being 45 cm, The diameter was 0.9 cm and the floor force was 13 cm (height up to the bar). The time when the mouse's hind limb slipped when the mouse was walked on the bar was defined as the onset of neurological symptoms. The Kaplan-Meier method (SPSS 15.0J software) was used for the analysis, and a log-rank test determined that there was a statistically significant difference when P was 0.05.

[0074] [Result]

The in vivo efficacy of Compound 1A was verified using ALS (SODl H46R) mice. The onset date of the control group l (n = 5) to which 0.5% carboxymethyl cellulose-Na solution alone was orally administered was approximately 1 27.8 ± 2.2 days. On the other hand, the onset days of administration group l (n = 5) and administration group 2 (n = 5) to which compound 1A was orally administered were 131 · 0 ± 5 · 7 days and 134 · 8 ± 4 · 9 days, respectively. It was. The results of Kaplan-Meier curve for the onset date of control group 1 and administration group 1 are shown in FIG. 3, and the results of the Kaplan-Meier curve for the onset date of control group 1 and administration group 2 are shown in FIG. These results indicate that compared with control group 1, neurological symptoms were delayed about 4 days in administration group 1 administered compound 1A and about 7 days in administration group 2. Control group 1 and treatment group 1 There was a statistically significant difference between them (P 0.05), and there was also a statistically significant difference between control group 1 and treated group 2 (P 0.05). In other words, these results indicate that Compound 1A can be a therapeutic agent for amyotrophic lateral sclerosis (ALS)!

[Example 3] Dopaminergic human neuroblastoma cell line [0075] The effect of compound 1A on the cell death inhibitory effect on neuronal cell death induced by 6-hy droxydopamine (6-OHDA) in SH-SY5Y cells Consideration

Parkinson's disease (PD) is a selective degeneration of dopaminergic cells in the dense substratum of the midbrain. It is a degenerative disease. The etiology of PD is thought to be due to aging, oxidative stress due to free radicals, nitric oxide (NO), the excitatory amino acid glutamate, endogenous / exogenous neurotoxins, etc. . The neurotoxin 6-hy droxydopamine (6-OHDA), which is frequently used in in vitro and in vivo PD experimental models, is a highly reactive para-quinone and peroxidation as a metabolic intermediate in dopaminergic neurons. Produces hydrogen, causes cell dysfunction, and kills cells (Bove J. et al. NeuroRx (2005) 2, 484-49 4). This cell death is widely used as a model of dopamine neuron death observed in PD. Therefore, in this example, for the purpose of developing a PD therapeutic drug, the cell death inhibitory activity of Compound 1A against 6-OHDA injury was measured using dopaminergic human neuroblastoma cell line SH-SY5Y. It was examined by survival test.

[Reagent]

Compound lA: N- (4- (2-pyridyl) (l, 3-thiazcJ—2-yl)) — 2— (2,4,6-trimethylphenoxy) acetamid e (ENAMINE, T5252123), compound is DMSO Used after dissolution or dilution

Yes

[0077] Experimental method

(1) Cell

The dopaminergic human neuroblastoma cell line SH-SY5Y (obtained as CRL2266 from the American Type Culture Collection) used in this study is 10% FBS, 4 mM glutamine, 100 μg / ml streptomycin, lOOU / ml The cells were cultured in DMEM medium (Sigma) containing penicillin G at 37 ° C in the presence of 5% CO. (2) Cell viability test

SH-SY5Y cells were seeded in a 96-well microplate (BD Falcon, PRIMARIA, Catalog # 353872) at a cell density of l.OxlO ⁴ cells / well, cultured for 24 hours, and then 10 M all-trans-retinoic acid (Wako) Differentiation was induced by culturing in the aforementioned medium containing 5 days. Differentiation-induced SH—SY5Y Itoda Tsuki-Bag compound with final concentration of 5〃 Μ, 1Μ, 5〃 Μ, 10〃 Μ, 20〃 Μ, 30〃〃, 40〃 Μ, 50 ^ 化合物1 Α was added and further cultured for 24 hours. In this example, a control experiment was conducted by adding only dimethyl sulfoxide (DMSO) (Sigma) instead of compound 1A. Final concentration power ^) Add 6_OHDA (Sigma, H4381) to M, 60M, 80M, 120M, 160M, and in the presence of 5% CO at 37 ° C for 4 hours. Processed. A blank (6-OHDA untreated group) was treated with the above medium containing 0.1% TritonX-100 instead of 6-OH DA. After the treatment, the medium was replaced with 10% AlamarBlue (Biosource), cultured for 14 hours at 37 ° C in the presence of 5% CO, and then used with CYTOFLUOR (registered trademark) Multi-Well Plate Reader Series 4000 (Perspective Biosystems). The number of viable cells was determined by measuring the excitation wavelength (530 nm) and the emission wavelength (580 nm). Figure 5 outlines the experimental method of this example. In addition, Fig. 6 shows the results of the cell death inhibitory effect of Compound 1A on 6-OHDA. The horizontal axis of the graph indicates 6-OHDA concentration (M), and the vertical axis indicates cell viability (%). The value on the vertical axis is the relative value when the cell viability under 6-OHDA untreated (0 M) conditions is taken as 100%.

[0078] [Result]

With respect to the cell death induced by 6-OHDA, the compound 1A was used to analyze the cell death inhibitory effect. Compared with the survival rate of the control experiment treated with 6-OHDA as shown in Fig. 6 (black bar graph), cells pretreated with Compound 1A showed a superior survival rate even after treatment with 6-OHDA. The cell death inhibitory effect was dependent on Compound 1A concentration. This result indicates that Compound 1A has an inhibitory effect on cell death of dopaminergic neurons induced by 6-OHDA. In other words, these results indicate that Compound 1A can be a therapeutic agent for Parkinson's disease.

Industrial applicability

[0079] According to the present invention, treatment or treatment of intractable diseases having molecular background of oxidative stress cell death. Provides a pharmaceutical composition useful for prevention.

Claims

The scope of the claims

The following general formula (1)

R ³ and R ⁴ each independently represent a hydrogen atom, a pyridyl group or a halogen atom as a substituent! /, May be! /, A C6_C10 aryl C1-C3 alkyl group. Or a pharmaceutically acceptable salt thereof as an active ingredient. A therapeutic or prophylactic agent for intractable diseases with molecular background of oxidative stress cell death.

[2] C6-C10 aryl group optionally having a C1-C3 alkyl group in R ² as a substituent

The therapeutic or prophylactic agent according to claim 1, which is a 1S 2, 4, 6-trimethylphenyl group.

[3] The therapeutic or prophylactic agent according to claim 1 or 2, wherein one of R ³ and R ⁴ is a hydrogen atom, and the other is a pyridyl group or a 2,5-dichlorophenylmethyl group.

[4] The therapeutic or prophylactic agent according to claim 1, wherein the compound represented by the formula (1) is the following compound 1A or 1B;

[5] The following general formula (2)

[6] In the above R ⁶ , either one is a phenyl group optionally having a halogen atom as a substituent, and the other is a pyrrolyl group optionally having a C1-C3 alkyl group as a substituent. The therapeutic or prophylactic agent according to claim 5, wherein

[7] The therapeutic or prophylactic agent according to claim 5, wherein the compound represented by the formula (2) is the following compound 2A or 2B;

N -C H

(2 B)

[8] The following general formula

[Wherein X represents an oxygen atom or a sulfur atom;

R ⁷ represents any group represented by the following formula;

[9] The therapeutic or prophylactic agent according to claim 8, wherein the compound represented by the formula (3) is the following compound 3A or 3B;

(3 B).

A therapeutic or prophylactic agent for intractable diseases having molecular background of oxidative stress cell death, comprising any of the following compounds or a pharmaceutically acceptable salt thereof as an active ingredient;

[11] The therapeutic or prophylactic agent according to any one of [1] to [10], wherein the intractable disease whose molecular background is oxidative stress cell death is a neurodegenerative disease or an ischemic heart disease.

[12] The neurodegenerative disease is amyotrophic lateral sclerosis (ALS), spinal muscular atrophy syndrome (SMA), Huntington's disease, Parkinson's disease, Alzheimer's disease, dementia or neuropathy after cerebrovascular disorder. The therapeutic or prophylactic agent according to claim 11, which is dementia accompanied by dropout.

[13] The therapeutic or prophylactic agent according to claim 11, wherein the ischemic heart disease is angina pectoris or myocardial infarction.

[14] An effective amount of the compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof to a patient in need of treatment or prevention of an intractable disease whose molecular background is oxidative stress cell death. A method for treating or preventing an intractable disease having molecular background of oxidative stress cell death, comprising a step of administering a salt obtained.

[15] The compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof for producing an agent for treating or preventing an intractable disease having molecular background of oxidative stress cell death. Use of.