JP2008239538A - Suppressant of cell death derived from endoplasmic reticulum stress or oxidation stress - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pharmaceutical composition useful for suppressing cell death derived from endoplasmic reticulum stress and/or cell death derived from oxidation stress. <P>SOLUTION: A suppressant of cell death derived from endoplasmic reticulum stress and cell death derived from oxidation stress, containing a carbazole derivative represented by general formula (I) (wherein, R<SP>1</SP>is H or a 1-6C alkoxy; and R<SP>2</SP>, R<SP>3</SP>and R<SP>4</SP>are each H, a halogen, a 1-6C alkyl, a 1-6C alkyl halide, cyano or the like) or a pharmaceutically permissible salt thereof as an active ingredient, is provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a carbazole derivative and a pharmaceutical use thereof.

  In recent years, intracellular stress plays an important role in the onset of many diseases typified by changes that occur in the human body with aging, or cancer, heart disease, and stroke (so-called three major diseases). It has become clear.

  It has long been said that dysfunction of the mitochondria responsible for respiration among the organelles present in cells leads to cell death. However, recently, even when the endoplasmic reticulum, which is the biosynthesis site of secretory proteins, is damaged, it has been revealed that the endoplasmic reticulum cannot function properly, causing dysfunction or cell death. (Non-patent document 1, Non-patent document 2).

  The endoplasmic reticulum is a place where secretory proteins and membrane proteins are regularly folded to adjust their three-dimensional structure, and has various physiological functions as a reservoir of intracellular calcium and as a major organ of lipid metabolism. However, physicochemical stresses such as ischemia, hypoxia, heat shock, and gene mutations increase the number of unfolded proteins in the endoplasmic reticulum, causing dysfunction of the endoplasmic reticulum. It is known (Non-Patent Document 3).

  To counter this, the endoplasmic reticulum responds by protecting the accumulated protein by increasing the molecular chaperone inside it, reducing the inflow protein to reduce the load, and decomposing the protein. is doing. However, if this strong endoplasmic reticulum stress state continues, it has become clear that the cells cannot resist the stress and themselves select cell death (apoptosis) (Non-patent Document 4). .

  Such endoplasmic reticulum stress-derived cell death can be caused by cerebral ischemia or neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, polyglutamine disease, inflammatory neurological diseases such as multiple sclerosis, mental disorders such as manic depression, glaucoma Onset and progression of various diseases such as ocular diseases such as arteriosclerosis and ischemic heart disease, gastric ulcer, viral hepatitis, fatty liver, diabetes, diabetic complications, renal diseases such as glomerulonephritis and renal failure, and cancer (Non-patent document 5, Non-patent document 6, Non-patent document 7).

  For this reason, development of a system for controlling cell death derived from endoplasmic reticulum stress by suppressing these endoplasmic reticulum stresses is in progress.

  For example, dilinoleoylphosphatidylethanolamine (containing two linoleic acids as fatty acids) having a specific structure has been shown to have cell death induction inhibitory activity, particularly endoplasmic reticulum stress inhibitory activity, and contains this as an active ingredient A pharmaceutical composition has been proposed (Patent Document 1).

  It has also been shown that a polypeptide having a specific amino acid sequence has an endoplasmic reticulum stress-induced cell death inhibitory effect (Patent Document 2).

  Moreover, it has been shown that a fat-soluble extract component derived from Yamabushidatake has an endoplasmic reticulum stress-induced cell death inhibitory effect (Patent Document 3).

  However, the present situation is that a system, a compound, and the like disclosed in any patent document have not yet been able to obtain a sufficient effect on endoplasmic reticulum stress control. Therefore, development of a compound that is more effective and easily available is expected.

  On the other hand, oxidative stress is also known to cause various diseases such as neurodegenerative diseases, ischemic diseases, diabetes and the like as in endoplasmic reticulum stress (Non-Patent Document 8, Non-Patent Document 9, Non-Patent Documents). 10) Development of compounds that can effectively suppress oxidative stress-derived cell death is expected.

Mori, K, Cell. 2000, 101, p 451-454 Oyadomari, S, and M. Mori, Cell Death Differ., 2004, Vol. 11, p 381-389 Takano K, Tabata Y, Kitao Y, Murakami R, Suzuki H, Yamada M, Iinuma M, Yoneda Y, Ogawa S, Hori O. Methoxyflavones protect cells against endoplasmic reticulum (ER) stress and neurotoxin. Am J. Physiol (Cell Physiol ) 2006 Sep 13; [Epub ahead of print] Hori O, Ichinoda F, Yamaguchi A, Tamatani T, Taniguchi M, Koyama Y, Katayama T, Tohyama M, Stern DM, Ozawa K, Kitao Y, Ogawa S. Role of Herp in the endoplasmic reticulum stress response. Genes Cells. 2004 May; 9 (5): 457-69. Masakazu Oyadomari, Experimental Medicine, Vol.23, No.18, 2005: 2778-83 Ryosuke Takahashi, Yoshitaka Tashiro, Experimental Medicine, Vol.23, No.18, 2005: 2789-94 Tadafumi Kato, Chihiro Kakiuchi, Ayako Hayashi, Kazuaki Kasahara, Experimental Medicine, Vol.23, No.18, 2005: 2795-98 Mikito Asanuma, Norio Ogawa, Separate Volume, History of Medicine, Oxidative Stress-The Forefront of Free Radical Medical Biology, p177-181 Masafumi Otsuka, Kazunari Komuro, Separate Volume, History of Medicine, Oxidative Stress-The Forefront of Free Radical Medical Biology, p189-192 Tomomi Takeda, Hiroshi Ihara, Hiroshi Seino, Separate Volume, History of Medicine, Oxidative Stress-The Forefront of Free Radical Medical Biology, p262-265 JP 2005-247728 A Japanese Patent Laying-Open No. 2005-082557 JP 2003-212790 A

  The present invention relates to a compound useful as an agent for suppressing endoplasmic reticulum stress-derived cell death or oxidative stress-derived cell death, or a pharmaceutically acceptable salt thereof, and endoplasmic reticulum stress-derived cell death or oxidative stress-derived cell death. It is an object of the present invention to provide a drug that suppresses the above.

  The present inventors have found an evaluation system using F9 Herp-deficient cells for the purpose of developing new drug discovery and functional foods by controlling these endoplasmic reticulum stress-derived cell deaths (JP-A-2005-245247). Issue gazette). Herp is a gene having a ubiquitin-like domain in the endoplasmic reticulum that is considered to be related to the removal of unnecessary proteins accumulated in the endoplasmic reticulum.

From the results of preliminary experiments using the above evaluation system, ER stress in the same cells was also observed in some antioxidants such as Dantrolene, α-tocopherol and β-carotene, which inhibit Ca ⁺⁺ efflux from the endoplasmic reticulum. Since the effect of suppressing cell death was recognized, about 300 types of compounds (100 types of natural products and 200 types of synthetic compounds) were actually screened.

  As a result, the inventors have found that a carbazole derivative having a specific structure has a strong endoplasmic reticulum stress-derived cell death inhibitory effect and / or oxidative stress-derived cell death inhibitory effect, and has completed the present invention.

That is, the present invention includes the following.
[1] The following general formula (I)
[Wherein, R ¹ represents a hydrogen atom or a C ₁ -C ₆ alkoxy group; R ² , R ³ and R ⁴ each independently represent a hydrogen atom, a halogen atom, a C ₁ -C ₆ alkyl group, C ₁ —C _{6 represents a} halogenated alkyl group or a cyano group. ]
An inhibitor of endoplasmic reticulum stress-derived cell death or oxidative stress-derived cell death, which comprises, as an active ingredient, a carbazole derivative represented by the formula: or a pharmaceutically acceptable salt thereof.
[2] The inhibitor according to [1], wherein R ³ is a hydrogen atom, a halogen atom, a C ₁ -C ₆ alkyl group, or a C ₁ -C ₆ halogenated alkyl group.
[3] The inhibitor according to [1] or [2], wherein R ⁴ is a hydrogen atom, a halogen atom, or a C ₁ -C ₆ alkyl group.
[4] The inhibitor according to [1] to [3], wherein the compound represented by the general formula (I) is any one selected from the following group;
(1) 1A;
9- (2-methylbenzyl) -6-methoxy-1,4-dimethylcarbazole,
(2) 1B;
9- (4-methylbenzyl) -6-methoxy-1,4-dimethylcarbazole,
(3) 1C;
9- (3-trifluoromethylbenzyl) -6-methoxy-1,4-dimethylcarbazole,
(4) 1D;
9- (3-cyanobenzyl) -1,4-dimethylcarbazole,
(5) 1E;
9- (3-cyanobenzyl) -6-methoxy-1,4-dimethylcarbazole,
(6) 1F;
9- (2,5-dimethylbenzyl) -6-methoxy-1,4-dimethylcarbazole,
(7) 1G;
9- (3,4-difluorobenzyl) -6-methoxy-1,4-dimethylcarbazole,
(8) 1H;
9- (2-iodobenzyl) -6-methoxy-1,4-dimethylcarbazole,
(9) 1I;
9- (4-trifluorobenzyl) -1,4-dimethylcarbazole, and (10) 1J;
9- (3-Bromobenzyl) -6-methoxy-1,4-dimethylcarbazole.
[5] A prophylactic or therapeutic agent for endoplasmic reticulum stress or oxidative stress-related disease comprising the inhibitor of endoplasmic reticulum stress-derived cell death or oxidative stress-derived cell death according to [1] to [4].
[6] The endoplasmic reticulum stress or oxidative stress-related disease is diabetes, diabetic complications, neurodegenerative disease, manic depression, glaucoma, gastric ulcer, fatty liver, viral hepatitis, chronic glomerulonephritis, renal failure, or allergic disease The preventive or therapeutic agent for endoplasmic reticulum stress or oxidative stress-related disease according to [5].
[7] 9- (4-trifluorobenzyl) -1,4-dimethylcarbazole, or a pharmaceutically acceptable salt thereof.
[8] The following general formula (I)
[Wherein, R ¹ represents a hydrogen atom or a C ₁ -C ₆ alkoxy group; R ² , R ³ and R ⁴ each independently represent a hydrogen atom, a halogen atom, a C ₁ -C ₆ alkyl group, C _1- C _{6 represents a} halogenated alkyl group or a cyano group. ]
Of endoplasmic reticulum stress or oxidative stress-related disease, comprising the step of administering a pharmaceutically effective amount of a carbazole derivative represented by: or a pharmaceutically acceptable salt thereof to a patient with endoplasmic reticulum stress or oxidative stress-related disease. Method of treatment.
[9] The following general formula (I) for preparing an inhibitor of endoplasmic reticulum stress-derived cell death or oxidative stress-derived cell death
[Wherein, R ¹ represents a hydrogen atom or a C ₁ -C ₆ alkoxy group; R ² , R ³ and R ⁴ each independently represent a hydrogen atom, a halogen atom, a C ₁ -C ₆ alkyl group, C _1- C _{6 represents a} halogenated alkyl group or a cyano group. Use of a compound represented by

In the present specification, an “alkyl group” is a monovalent group derived by removing one arbitrary hydrogen atom from an aliphatic hydrocarbon, and contains a hetero atom or an unsaturated carbon-carbon bond in the skeleton. Without having a subset of hydrocarbyl or hydrocarbon containing hydrogen and carbon atoms. The alkyl group includes a linear or branched structure, and the “C ₁ -C ₆ alkyl group” means an alkyl group having 1 to 6 carbon atoms.

  Specifically, for example, methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, sec-butyl group, t-butyl group, isobutyl group, pentyl group, isopentyl group, 2,3- Examples thereof include a dimethylpropyl group, a hexyl group, a 2,3-dimethylhexyl group, a 1,1-dimethylpentyl group, a heptyl group, an octyl group, and the like, preferably a methyl group.

In the present specification, “alkoxy group” means an oxy group to which the above-defined “alkyl group” is bonded, and “C ₁ -C ₆ alkoxy group” means _{1 to 6} carbon atoms. Means an alkoxy group of

  Specifically, for example, specifically, for example, methoxy group, ethoxy group, 1-propyloxy group, 2-propyloxy group, 2-methyl-1-propyloxy group, 2-methyl-2-propyloxy group, 1-butyloxy group, 2-butyloxy group, 1-pentyloxy group, 2-pentyloxy group, 3-pentyloxy group, 2-methyl-1-butyloxy group, 3-methyl-1-butyloxy group, 2-methyl- Examples include 2-butyloxy group, 3-methyl-2-butyloxy group, 2,2-dimethyl-1-propyloxy group, and methoxy group is preferable.

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

In the present specification, the “halogenated alkyl group” refers to a group in which the same or different 1 to 5 “halogen atoms” are bonded to the “C ₁ -C ₆ alkyl group”.

  Specifically, for example, trifluoromethyl, trichloromethyl, difluoromethyl, dichloromethyl, dibromomethyl, fluoromethyl, 2,2,2-trifluoroethyl, 2,2,2-trichloroethyl, 2-bromoethyl, 2- A chloroethyl, 2-fluoroethyl, 2-iodoethyl, pentafluoroethyl, 3-chloropropyl, 4-fluorobutyl, 6-iodohexyl or 2,2-dibromoethyl group, preferably a trifluoromethyl group.

  Cell death includes apoptosis and necrosis. Apoptosis is called cell suicide, programmed cell death, or active cell death, and is cell death expressed by physiological factors or pathological factors. Morphologically, nuclei and cells shrink, but organelles in the cytoplasm such as mitochondria are normal and are not accompanied by inflammation. On the other hand, necrosis is called cell killing, accidental cell death, or passive cell death, and is cell death expressed by pathological factors. Morphologically, both whole cells and mitochondria are swollen and accompanied by inflammation. However, apoptosis and necrosis are not contradictory concepts, and apoptosis and necrosis may coexist depending on the stage in the same factor and the same cell.

  Cell death derived from endoplasmic reticulum stress has a form of apoptosis morphologically, but “endoplasmic reticulum stress-derived cell death” in the present invention is not necessarily limited to apoptosis, and is caused by endoplasmic reticulum stress. Including all cell deaths caused.

  Further, in the present specification, “oxidative stress-derived cell death” includes all cell death caused by oxidative stress as in the case of the “endoplasmic reticulum stress-derived cell death”.

  As used herein, “endoplasmic reticulum stress-related disease” means all diseases caused by endoplasmic reticulum stress-derived cell death, such as cerebral ischemia or Alzheimer's disease, Parkinson's disease, polyglutamine disease. Neurodegenerative diseases such as, inflammatory neurological diseases such as multiple sclerosis, mental disorders such as manic depression, eye diseases such as glaucoma, arteriosclerosis and ischemic heart disease, gastric ulcer, viral hepatitis, fatty liver, diabetes, Examples include diabetic complications, glomerulonephritis, renal diseases such as renal failure, cancer, and the like.

  In the present specification, “oxidative stress-related disease” means all diseases caused by oxidative stress-derived cell death, and examples thereof include neurodegenerative diseases, ischemic heart diseases, diabetes and the like.

  In the present specification, the “pharmaceutically acceptable salt” is not particularly limited as long as it forms a salt with the compound according to the present invention and is pharmacologically acceptable. Organic acid salts, acidic amino acid salts and the like. Examples of inorganic acid salts include hydrochlorides, sulfates, nitrates, and phosphates. Examples of organic acid salts include acetates, succinates, fumarate salts, maleates, tartrate salts, and citric acids. Examples of the acidic amino acid salt include aspartate and glutamate.

  When the compound of the present specification is left in the atmosphere, it absorbs moisture and may be adsorbed water or become a hydrate, and such salts are also included in the present invention.

  Furthermore, solvates in which solvent molecules are coordinated with the compounds used herein are also encompassed in the salts of the present invention.

  Below, the compound which can be used for suppression of endoplasmic reticulum stress origin cell death or oxidative stress origin cell death concerning the present invention is explained.

  The compound used in the present invention is represented by the following general formula (I).

In the formula, R ¹ represents a hydrogen atom or a C ₁ -C ₆ alkoxy group, and R ² , R ³ and R ⁴ each independently represent a hydrogen atom, a halogen atom, a C ₁ -C ₆ alkyl group, or a C ₁ -C _{6 represents a} halogenated alkyl group or a cyano group.

The C ₁ -C ₆ alkoxy group for R ¹ is preferably a methoxy group. In addition, the halogen atom in R ² , R ³ and R ⁴ is preferably fluorine, bromine or iodine, and the C ₁ -C ₆ alkyl group is preferably a methyl group, and C ₁ -C ₆ halogenated. The alkyl group is preferably a trifluoromethyl group.

R ¹ is preferably a hydrogen atom or a methoxy group, and particularly preferably a hydrogen atom.

R ² is preferably a hydrogen atom, a fluorine atom, a bromine atom, a methyl group, a trifluoromethyl group, or a cyano group, and particularly preferably a cyano group.

R ³ is preferably a hydrogen atom, a fluorine atom, a methyl group, or a trifluoromethyl group, and particularly preferably a hydrogen atom.

R ⁴ is preferably a hydrogen atom, an iodine atom, or a methyl group, and particularly preferably a hydrogen atom.

Specific examples of such a compound represented by the formula (I) include the following compounds (1A) to (1J).
1A: 9- (2-methylbenzyl) -6-methoxy-1,4-dimethylcarbazole,
1B: 9- (4-methylbenzyl) -6-methoxy-1,4-dimethylcarbazole,
1C: 9- (3-trifluoromethylbenzyl) -6-methoxy-1,4-dimethylcarbazole,
1D: 9- (3-cyanobenzyl) -1,4-dimethylcarbazole,
1E: 9- (3-cyanobenzyl) -6-methoxy-1,4-dimethylcarbazole,
1F: 9- (2,5-dimethylbenzyl) -6-methoxy-1,4-dimethylcarbazole,
1G: 9- (3,4-difluorobenzyl) -6-methoxy-1,4-dimethylcarbazole,
1H: 9- (2-iodobenzyl) -6-methoxy-1,4-dimethylcarbazole,
1I: 9- (4-trifluorobenzyl) -1,4-dimethylcarbazole,
1J: 9- (3-bromobenzyl) -6-methoxy-1,4-dimethylcarbazole.

The compound represented by the compound represented by formula (I) used in the present invention is 1,4-dimethylcarbazole (produced by the method described in J. Chem. Soc, 1962, page 3482), or 6- Produced by reacting alkoxy-1,4-dimethylcarbazole (produced according to the method described in J. Chem. Soc, 1962, page 3482) and a substituted benzyl halide represented by the following formula II can do.
(In the formula, R ¹ , R ² and R ³ are as defined above, and X is a halogen atom.)

  This reaction is carried out in a solvent inert to the reaction (for example, benzene, chloroform, methylene chloride, tetrahydrofuran, N, N-dimethylformamide, dimethyl sulfoxide, water, etc., or a mixture thereof) or in the absence of a solvent with a basic compound ( For example, in the presence of pyridine, triethylamine, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium hydride) or in the presence of the basic compound and a phase transfer catalyst (such as tetrabutylammonium hydrogen sulfate), The reaction is carried out under ice cooling to stirring at the reflux temperature of the solvent used.

  The solvent and basic compound are appropriately selected depending on the type of the compound of formula II to be used. After completion of the reaction, when water is usually added, crystals precipitate and the desired product can be obtained by filtration. When crystals do not precipitate, extraction with an appropriate solvent (for example, benzene, chloroform, ethyl acetate, etc.) and treatment by a conventional method can give the desired product.

  When the compound used in the present invention is obtained as a free form, it can be converted into a salt which may be formed by the compound, or a hydrate or solvate thereof 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.

  Such a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof used in the present invention has an activity of suppressing endoplasmic reticulum stress-derived cell death or oxidative stress-derived cell death, It is used as an inhibitor of endoplasmic reticulum stress-derived cell death or oxidative stress-derived cell death, or a preventive or therapeutic agent for endoplasmic reticulum stress-related disease or oxidative stress-related disease.

  The present invention also includes the step of administering an effective amount of the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof to a patient having an endoplasmic reticulum stress-related disease or an oxidative stress-related disease. It also relates to a method for treating endoplasmic reticulum stress-related diseases or oxidative stress-related diseases.

  Furthermore, the present invention relates to the use of a compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof for the preparation of an inhibitor of endoplasmic reticulum stress-derived cell death or oxidative stress-derived cell death. Also related.

  When administering the pharmaceutical composition concerning this invention, the form is not specifically limited, Orally or parenterally may be sufficient by the method used normally. Specifically, for example, tablets, powders, granules, capsules, syrups, troches, inhalants, suppositories, injections, ointments, eye ointments, eye drops, nasal drops, ear drops, It can be formulated and administered as an agent such as a poultice or lotion.

  Excipients, binders, lubricants, colorants, flavoring agents, and, if necessary, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives Antioxidants and the like can be used, and they are generally formulated by blending ingredients used as raw materials for pharmaceutical preparations.

  In order to produce an oral preparation, for example, the compound according to the present invention or a pharmacologically acceptable salt and excipient thereof, and if necessary, a binder, a disintegrant, a lubricant, a coloring agent, a flavoring agent. After adding the agent, etc., it can be made into powders, fine granules, granules, tablets, coated tablets, capsules and the like by conventional methods.

  Examples of the excipient include lactose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose, silicon dioxide and the like.

  Examples of the binder include polyvinyl alcohol, polyvinyl ether, methylcellulose, ethylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, polypropylene glycol, polyoxyethylene block polymer, meglumine and the like. It is done.

  Examples of the disintegrant include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, carboxymethylcellulose / calcium and the like.

  Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, hydrogenated vegetable oil, and the like.

  Examples of the coloring agent include those permitted to be added to pharmaceuticals, and examples of the flavoring agent include cocoa powder, mint brain, aroma powder, mint oil, dragon brain, and cinnamon powder.

  Examples of the flavoring agent include sweeteners, acidulants, and fragrances that are commonly used.

  When producing liquid preparations such as syrups and injectable preparations, a compound according to the present invention or a pharmacologically acceptable salt thereof, a pH adjuster, a solubilizer, an isotonic agent, etc. Add solubilizers, stabilizers, etc., and formulate by conventional methods.

  The dosage of the pharmaceutical composition according to the present invention can be appropriately selected depending on the degree of symptoms, age, sex, body weight, dosage form / salt type, specific type of disease, and the like. For example, about 0.03-1000 mg, preferably 0.1-500 mg, more preferably 0.1-100 mg per day as a normal adult can be administered in 1 to several times a day. In this case, the dose is usually about 1 μg / kg-3000 μg / kg, preferably about 3 μg / kg-1000 μg / kg.

Another embodiment of the present invention is a novel compound having the following structural formula or a pharmaceutically acceptable salt thereof.
1I: 9- (4-trifluorobenzyl) -1,4-dimethylcarbazole

  EXAMPLES Hereinafter, although this invention is demonstrated using an Example, this invention is not limited to an Example at all.

[Example 1]
Preparation of 9- (4-trifluorobenzyl) -1,4-dimethylcarbazole (Compound: 11) Under a nitrogen atmosphere, 0.46 g of 60% sodium hydride and 5 ml of dimethyl sulfoxide were stirred at 60 ° C. for 1 hour and cooled on ice. Thereafter, 12 ml of an anhydrous tetrahydrofuran solution of 2.25 g of 1,4-dimethylcarbazole was added and stirred at room temperature for 30 minutes. To this was added 15 ml of an anhydrous tetrahydrofuran solution of 2.33 g of 4-trifluorobenzyl chloride under ice-cooling, and the mixture was further stirred at room temperature for 12 hours. Water was added to the reaction solution, and the precipitated crystals were collected by filtration, washed with water, dried and recrystallized with benzene-n-hexane to obtain 3.58 g of the desired product.
mp: 113-115 ℃
¹ H NMR (300MHz, CDCl ₃ ): d 2.61 (3H, S), 2.90 (3H, s), 5.84 (2H, s), 6.95 (1H, d, J = 7 Hz), 7.04 (1H, d, J = 7 Hz), 7.08 (1H, d, J = 7 Hz), 7.24-7.49 (6H, m), 8.24 (1H, d, J = 7 Hz)

Other compounds described in Table 1 below (Compounds 1A to 1H, 1J) were also produced in the same manner as described above.

[Example 2]
Primary screening (F9 Herp deficient cells)
Genes Cells. 2004 May; 9 (5): 457-69 and Japanese Patent Application Laid-Open No. 2005-245247, F9 Herp-deficient cells were prepared and cultured. Next, as a preliminary experiment, the effect of various compounds including Dantrolene, α-tocopherol, and procol on the inhibitory effect of endoplasmic reticulum stress-derived cell death in F9 Herp-deficient cells was determined, and a compound that could be used as a positive control was determined. Next, the endoplasmic reticulum stress-derived cell death inhibitory effect in F9 Herp-deficient cells was measured for about 300 kinds of compounds (100 kinds of natural products and 200 kinds of synthetic compounds).

  As the positive control, Dantrolene, probucol, and tangeretin (Am J. Physiol (Cell Physiol) 2006 Sep 13), which was already reported by the applicants, were used. The specific protocol proceeded as follows.

(1) After 96-well or 24-well culture dishes are gelatin-coated, wild type F9 cells and Herp-deficient F9 cells are seeded.
(2) Culture is performed for 2 days until the cells occupy 50-60% of the culture area (DMEM medium + 20% FBS: Sigma, St. Louis, MO).
(3) After adding tunicamycin 0.8 μg / ml and a test substance to the cells at the same time and culturing for 48 hours, cell viability was measured by cell counting-8 assay (method for evaluating the number of viable cells: Dojindo Laboratories, Kumamoto ).

[Example 3]
Secondary screening (PC12 cells)
As a result of the primary screening, compounds with a cytoprotective effect higher than Dantrolene were measured for the same endoplasmic reticulum stress-derived cell death inhibitory effect and oxidative stress-derived cell death inhibitory effect using rat neuronal cell line PC12 cells. did.

  Tunicamycin (Tm: 0.75 μg / ml, 38 hr) was used as the endoplasmic reticulum stress induction method, and bionine sulfoximine (BSO, 1 mM, 38 hr) that depletes intracellular glutathione was used as the oxidative stress induction method.

  In order to induce oxidative stress and endoplasmic reticulum stress simultaneously, the cells were treated with tunicamycin (Tm: 0.75 μg / ml) and BSO (1 mM) for 24 hr. The test compound for determining the cytoprotective effect was administered simultaneously with each stress inducer. Cell viability was determined by MTT assay.

[Example 4]
Analysis of action on endoplasmic reticulum stress As a result of secondary screening, compounds that strongly recognized cytoprotective effect on endoplasmic reticulum stress were examined for the mechanism of action by the following method.

  PC12 cells were cultured for 15 hours in the presence of positive control Dantrolene or a test compound, and then treated with tunicamycin (Tm: 2 μg / ml, 6 hr) to induce endoplasmic reticulum stress. After RNA was extracted from the cells, Northern blot analysis was performed according to the method described in Genes Cells. 2004 May; 9 (5): 457-69, using a probe specific for GRP78, CHOP, and β-actin.

[Example 5]
Analysis of action against oxidative stress As a result of the secondary screening, the antioxidative action of the compounds that recognized the oxidative stress-derived cell death inhibitory effect was examined by the following method.

PC12 cells were cultured for 24 hours in the presence of positive control Dantrolene or a test compound, then treated with 1 mM BSO for 15 hr, and the degree of oxidative stress was measured using a fluorescent reagent H ₂ DCFDA (manufactured by molecular probes). In addition, the decrease (depletion) of intracellular glutathione amount by BSO administration was measured using a glutathione assay kit (manufactured by Cayman chemical).

〔result〕
In a primary screening using F9 Herp-deficient cells, a plurality of carbazole derivatives (compounds 1A to 1J) were found to have a strong endoplasmic reticulum stress-derived cell death inhibitory effect (FIGS. 1 and 2).

  Next, regarding the compounds 1C, 1D, 1E, 1I, and 1J, which had a cell death inhibitory effect of “++” or more in the primary screening, whether PC12 cells also have an endoplasmic reticulum stress-derived cell death inhibitory effect was examined ( Secondary screening, FIG. 3). As a result, 1D, 1I, and 1J (1D> 1I = 1J) were recognized to have a cytoprotective effect more than Dantrolene.

  Subsequently, the effects of 1D, 1H, 1J and Dantrolene on the endoplasmic reticulum stress were performed by comparing the stress response to the endoplasmic reticulum stress and the activation of the UPR.

  1D, 1H, 1J, or Dantrolene was added to PC12 cells and incubated for 15 hours (pretreatment). After that, endoplasmic reticulum stress was induced with tunicamycin (2 μg / ml, 6 hr). As a result, UPR target genes GRP78 and CHOP Expression decreased in the order of 1D> 1J = Dantrolene> 1H (FIG. 4).

  In the case of endoplasmic reticulum stress control, stress resistance is enhanced by activating UPR, which is a stress defense system, in advance, like a flavonoid, tangeretin (Takano K, Tabata Y, Kitao Y, Murakami). R, Suzuki H, Yamada M, Iinuma M, Yoneda Y, Ogawa S, Hori O. Methoxyflavones protect cells against endoplasmic reticulum (ER) stress and neurotoxin. Am J. Physiol (Cell Physiol) 2006 Sep 13) and endoplasmic reticulum environment There is a case where the endoplasmic reticulum stress itself is decreased (in this case, the activation of the UPR for the endoplasmic reticulum stress is suppressed as a result).

  From the above results, compounds 1D, 1J, and 1H are considered to fall under the latter, like Dantrolene, and improve endoplasmic reticulum environment relatively early after endoplasmic reticulum stress loading to reduce stress and suppress cell death. It was suggested that

  When 1D, which showed the strongest cytoprotective effect against endoplasmic reticulum stress, was examined for its cytoprotective action against oxidative stress, it was oxidized by depleting intracellular glutathione with butionine sulfoximine (BSO, 1 mM, 38 hr). When stress was induced, it showed a cytoprotective effect as in Dantrolene (FIG. 5A).

  At this time, intracellular glutathione was not recovered (FIG. 5B), but intracellular oxidative stress was decreased (FIG. 5C).

  When cells were treated with tunicamycin (Tm: 0.75 μg / ml) and BSO (1 mM) for 24 hr in order to induce oxidative stress and endoplasmic reticulum stress at the same time, both Dantrolene and 1D showed a cell death inhibitory effect (FIG. 6). ).

From the above results, it was found that the physiological activity of Compound 1D is very similar to Dantrolene that suppresses Ca ⁺⁺ efflux from the endoplasmic reticulum and contributes to the maintenance of the endoplasmic reticulum environment.

That is, it is considered that Compound 1D contributes to the control of endoplasmic reticulum stress and the control of oxidative stress by being involved in intracellular Ca ⁺⁺ control.

  ADVANTAGE OF THE INVENTION According to this invention, the pharmaceutical composition and functional food useful for suppression of endoplasmic reticulum stress origin cell death or oxidative stress origin cell death, or prevention or treatment of endoplasmic reticulum stress related disease or oxidative stress related disease are provided. .

FIG. 1 is a diagram showing endoplasmic reticulum stress-derived cell death inhibitory activity in a primary screening using F9 Herp-deficient cells. In the figure, “+” is a kind of antioxidant, procol, “++” is Dantrolene, and “++” is a degree of cell inhibition of a kind of flavonoid, tangeretin (Am J. Physiol (Cell Physiol) 2006 Sep 13). Indicates that there is. FIG. 2 is a graph showing endoplasmic reticulum stress-derived cell death inhibitory activity in a primary screening using F9 Herp-deficient cells. In the figure, “+” on the upper side of the horizontal axis means the case where tunicamycin is added, “−” means the case where it is not added, and the lower side shows the concentration (μM) of the test compound. In addition, "-" in the lower part means the case where a test compound is not added. The vertical axis indicates the cell survival rate (%) of F9 Herp-deficient cells. FIG. 3 is a diagram showing endoplasmic reticulum stress-derived cell death inhibitory activity in secondary screening using PC12 cells. In the figure, “+” on the upper side of the horizontal axis means the case where tunicamycin is added, “−” means the case where it is not added, and the lower side shows the concentration (μM) of the test compound. In addition, "-" in the lower part means the case where a test compound is not added. The vertical axis indicates the cell viability (%) of PC12 cells. FIG. 4 is a diagram showing Northern blot analysis of RNA extracted from PC12 cells using probes specific to GRP78, CHOP, and β-actin. In the figure, “+” in the upper part means that tunicamycin is added, “−” means that no tunicamycin is added, and the lower part shows the concentration (μM) of the test compound. In addition, "-" in the lower part means the case where a test compound is not added. In the figure, “Dan” means Dantrolene. FIG. 5a is a diagram showing oxidative stress-derived cell death inhibitory activity using PC12 cells. In the figure, “+” on the upper side of the horizontal axis means the case where BSO is added, “−” means the case where it is not added, and the lower side shows the concentration (μM) of the test compound. In addition, "-" in the lower part means the case where a test compound is not added. The vertical axis indicates the cell viability (%) of PC12 cells. FIG. 5b is a graph showing the amount of intracellular glutathione by BSO administration. In the figure, “+” on the upper side of the horizontal axis means the case where BSO is added, “−” means the case where it is not added, and the lower side shows the concentration (μM) of the test compound. In addition, "-" in the lower part means the case where a test compound is not added. The vertical axis represents the amount of intracellular glutathione (nmol / μg). FIG. 5 c is a photograph in which the degree of oxidative stress in PC12 cells was measured using a fluorescent reagent H ₂ DCFDA (manufactured by molecular probes). In the figure, I is a control, II is a case where only BSO was added, III was a case where Dantrolene (30 μM) was treated with BSO (1 mM) for 24 hours, and IV was a compound 1D (30 μM) treated with BSO ( 1m) for 24 hours. FIG. 6 is a diagram showing endoplasmic reticulum stress and oxidative stress-derived cell death inhibitory activity using PC12 cells. In the figure, “+” in the upper part of the horizontal axis means that BSO is added, “−” means not added, and “+” in the middle part means that tunicamycin is added and “−” means that added. The lower case shows the concentration (μM) of the test compound. In addition, "-" in the lower part means the case where a test compound is not added. The vertical axis indicates the cell viability (%) of PC12 cells.

Claims (7)

The following general formula (I)
[Wherein, R ¹ represents a hydrogen atom or a C ₁ -C ₆ alkoxy group; R ² , R ³ and R ⁴ each independently represent a hydrogen atom, a halogen atom, a C ₁ -C ₆ alkyl group, C ₁ —C _{6 represents a} halogenated alkyl group or a cyano group. ]
An inhibitor of endoplasmic reticulum stress-derived cell death or oxidative stress-derived cell death, which comprises, as an active ingredient, a carbazole derivative represented by the formula: or a pharmaceutically acceptable salt thereof. The inhibitor according to claim 1, wherein R ³ is a hydrogen atom, a halogen atom, a C ₁ -C ₆ alkyl group, or a C ₁ -C ₆ halogenated alkyl group. The inhibitor according to claim 1 or 2, wherein R ⁴ is a hydrogen atom, a halogen atom, or a C ₁ -C ₆ alkyl group. The inhibitor according to claim 1, wherein the compound represented by the general formula (I) is any one selected from the following group;
(1) 1A;
9- (2-methylbenzyl) -6-methoxy-1,4-dimethylcarbazole,
(2) 1B;
9- (4-methylbenzyl) -6-methoxy-1,4-dimethylcarbazole,
(3) 1C;
9- (3-trifluoromethylbenzyl) -6-methoxy-1,4-dimethylcarbazole,
(4) 1D;
9- (3-cyanobenzyl) -1,4-dimethylcarbazole,
(5) 1E;
9- (3-cyanobenzyl) -6-methoxy-1,4-dimethylcarbazole,
(6) 1F;
9- (2,5-dimethylbenzyl) -6-methoxy-1,4-dimethylcarbazole,
(7) 1G;
9- (3,4-difluorobenzyl) -6-methoxy-1,4-dimethylcarbazole,
(8) 1H;
9- (2-iodobenzyl) -6-methoxy-1,4-dimethylcarbazole,
(9) 1I;
9- (4-trifluorobenzyl) -1,4-dimethylcarbazole, and (10) 1J;
9- (3-Bromobenzyl) -6-methoxy-1,4-dimethylcarbazole.   An agent for preventing or treating endoplasmic reticulum stress or oxidative stress-related disease, comprising the inhibitor of endoplasmic reticulum stress-derived cell death or oxidative stress-derived cell death according to claim 1.   The endoplasmic reticulum stress or oxidative stress-related disease is diabetes, diabetic complications, neurodegenerative disease, manic depression, glaucoma, gastric ulcer, fatty liver, viral hepatitis, chronic glomerulonephritis, renal failure, or allergic disease, The preventive or therapeutic agent for endoplasmic reticulum stress or an oxidative stress related disease of Claim 5.   9- (4-trifluorobenzyl) -1,4-dimethylcarbazole, or a pharmaceutically acceptable salt thereof.