WO2007125720A1 - Molecular chaperone expression inducer - Google Patents

Abstract

Disclosed is a pharmaceutical composition having an activity of inducing the expression of an endoplasmic reticulum chaperone, which can be used effectively for the prevention of the cell death caused by endoplasmic reticulum stress or for the prevention of occurrence or progression of a neurodegenerative disease or the amelioration of a neurodegenerative disease. The pharmaceutical composition comprises a compound represented by the formula (I), a pharmaceutically acceptable salt thereof, or a solvate of the compound or the salt, as an active ingredient. (I) wherein R1 and R2 independently represent a hydrogen atom, a halogen atom, a hydroxyl group, a lower alkyl group or a lower alkoxy group; Alk represents a lower alkylene chain; and X represents an oxygen atom or a sulfur atom.

Description

 Specification

 Molecular chaperone expression inducer

 Technical field

 [0001] The present invention relates to a pharmaceutical composition having an action of inducing expression of an endoplasmic reticulum molecular chaperone. The present invention also relates to a pharmaceutical composition effective for suppressing cell death due to endoplasmic reticulum stress, or a pharmaceutical composition effective as a preventive or therapeutic agent for neurodegenerative diseases based on the molecular chaperone expression inducing action.

 Background art

 [0002] With the aging of society, the number of patients with dementia such as Alzheimer's disease has increased dramatically. However, the molecular mechanism of the pathogenesis has not been clarified yet, and no effective treatment has been found.

 [0003] Alzheimer's disease and Parkinson's disease are representative of progressive neurodegenerative diseases accompanied by marked loss of neurons. A part of Alzheimer's disease (AD) is familial AD (FAD) with a genetic mutation. The majority are sporadic AD (SAD) without a family history. Both ADs are characterized by senile plaques composed of amyloid protein (A / 3) deposited in the brain and neurofibrillary tangles caused by abnormal phosphorylation of tau protein. For this reason, it is thought that an increase in amyloid protein (A / 3) is deeply related to the pathology of Alzheimer's disease (Amyloid cascade hypothesis) (Non-patent document 1). As a therapeutic agent for Alzheimer's disease, amyloid precursor A / 3 / γ-secretor inhibitor that lowers Aβ by suppressing the excision of amyloid protein (A) from protein (APP) and amyloid vaccine have been proposed and developed (non-patent literature) 2 and 3).

 [0004] In addition, in many of the neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, there is a common pathological condition that unfolded proteins are aggregated and accumulated in nerve cells. In recent years, this abnormal protein accumulation (this state is called “ER stress”) has been implicated in the development of various neurodegenerative diseases. (Non-Patent Documents 4 to 9).

[0005] In the endoplasmic reticulum, when a protein synthesized in the ribosome matures, a sugar chain is added. A folded three-dimensional structure is formed by a modification such as a sulfide bond. The protein correctly folded in the endoplasmic reticulum is transported to the gonores. The protein that fails to fold is retained in the endoplasmic reticulum until the correct conformation is achieved (endoplasmic reticulum stress). If the endoplasmic reticulum stress is excessive or persists for a long time, the cells die and die. However, the endoplasmic reticulum stress response (which is a defense mechanism to avoid such cell death)

Unfolding Protein Response (UPR) exists and contributes to the maintenance of cell survival. Such defense mechanisms can be roughly divided into the following three categories.

 [0006] (1) ER associated degraration ("ERAD")

 It is a protective mechanism that excretes abnormal proteins into the cytoplasm and degrades them by the ubiquitin 'proteasome system.

 [0007] (2) Molecular chaperone induction mechanism

 This mechanism induces chaperone molecules present in the endoplasmic reticulum at the transcriptional level to unwind abnormal proteins. When it is sensed that abnormal protein has accumulated, a signal is transmitted downstream to induce a molecular chaperone (also called “ER chaperone” or “ER molecular chaperone”). Molecular chaperone is an endoplasmic reticulum-specific molecule (folding enzyme) whose expression is induced by endoplasmic reticulum stress (Non-patent Documents 10 and 11), and is extremely important for folding of newly secreted proteins and membrane proteins in the endoplasmic reticulum. Playing a role. The sensor or transducer protein that induces the molecular chaperone is IRE1 a or A.

TF6 is known.

[0008] (3) Translation suppression mechanism

 When abnormal proteins accumulate in the endoplasmic reticulum, this mechanism promotes phosphorylation of the translation initiation factor (eIF2 a) and stops protein translation. This biological defense mechanism has sensor protein P

It is known that ERK is involved.

[0009] However, if cells are subjected to excessive endoplasmic reticulum stress, the molecular chaperone induction mechanism (endoplasmic reticulum stress response (UPR)) and the endoplasmic reticulum-related degradation mechanism (ERAD) cannot cope with each other. The living body actively chooses apoptosis (ER stress cell death) in order not to disturb the harmony with surrounding cells for stimulation.

[0010] By the way, for mammals, the molecular chaperone involved in the endoplasmic reticulum stress response is used. 8 glucose regulatory proteins (GRP), namely GRP78 / Bip, GRP94 / ERp99, ORP 150 / GRP170, ERp72, GRP58 / ERp60 / ERp61, calreticulin, protein disulfide isomerase (PDI) and FKBP13 Have been identified. Among them, GRP78 / Bip is a molecular chaperone that has been analyzed, and for the rat GRP78 gene, the CORE region located in the upstream region and the C1 region containing the CCAAT sequence have been shown to be important for transcriptional control ( Non-patent literature 12-: 14). Regarding yeast, the transcriptional control sequence (UPRE sequence: CAGNGTG) of the GRP78 gene of budding yeast has been clarified (Non-patent Document 15), and there is a sequence similar to the UPRE sequence in the upstream region of the human GRP78 gene. It is clear that there is.

 [0011] As described above, the molecular chaperone induction mechanism (endoplasmic reticulum stress response (UPR)) and the endoplasmic reticulum-related degradation mechanism (ERAD) function effectively against endoplasmic reticulum stress. Therefore, if these biological defense functions are enhanced, it is possible to protect against excessive endoplasmic reticulum stress, and to avoid pathological conditions and diseases caused by endoplasmic reticulum stress, and neuronal cell death. Conceivable. From this point of view, various techniques for inducing molecular chaperone expression have been proposed, focusing on UPR.

 [0012] A representative compound among the compounds (I) according to the present invention is a known compound (for example, Non-Patent Document 16).

 Non-Patent Document 1: Hardy, J.A. & Selkoe, D.J., Science, 19, 353-356

 Non-Patent Document 2: Beher, D. & Graham, S. L., Expert. Opin. Investig. Drugs, 14, 1385-

1409 (2005)

 Non-Patent Document 3: Gilman, S. et al "Neurology, 10, 1553-1562 (2005)

 Non-Patent Document 4: Kitada, T. Et al., Nature 392, 605-608 (1998).

 Non-Patent Document 5: Imai, Y. et al "Cell 105, 891-902 (2001).

 Non-Patent Document 6: Nishitoh, H. et al., Genes Dev. 16, 1345-1355 (2002).

 Non-Patent Document 7: Katayama, T et al "Nat Cell Biolog, 8, 479-485 (1999).

 Non-patent literature 8: Katayama, T. et al "J. Biol. Chem. 276, 43446-43454 (2001) Non-patent literature 9: Yasuda, Y. et al" Biochem. Biophys. Res. Commun. 296, 313- 318 (2

002) Non-Patent Document 10: Kozutsumi, Y. et al., Nature, 332, 462-464 (1988)

 Non-Patent Document 11: Lee, A.S., Trends Biochem. ScL, 12, 20-23 (1987)

 Non-Patent Document 12: Resendez, E. et al., Mol. Cell. Biol., 8, 4579-4584 (1988)

 Non-Patent Document 13: Wooden, S.K.et al., Mol. Cell. Biol., 11,5612-5623 (1991)

 Non-Patent Document 14: Li.W.W. et al., Mol. Cell. Biol., 14, 5533-5546 (1994)

 Non-Patent Document 15: Mori. K. et al., Genes Cells, 1, 803-817 (1996)

 Non-Patent Document 16 Journal of the Chemical Society, Perkin Transactions 2 (2002), (2), 3

29-336

 Disclosure of the invention

 Problems to be solved by the invention

[0013] An object of the present invention is to focus on UPR as a biological defense mechanism against endoplasmic reticulum stress, and to provide a method for increasing the level of molecular chaperone particularly in nerve cells. More specifically, an object of the present invention is to provide a compound having an action of inducing molecular chaperone expression, and a pharmaceutical composition containing the compound as an active ingredient. In addition, the present invention provides a novel molecular chaperone expression inducer, a cell death inhibitor against endoplasmic reticulum stress, and a neurodegenerative disease therapeutic agent for the pharmaceutical composition based on the molecular chaperone expression inducing action of the pharmaceutical composition. The purpose is to provide usage. Means for solving the problem

[0014] The inventors of the present invention have made extensive studies to achieve the above object. As a result, the 1-phenyl-thiocyanatoalkane-1-one derivative (I) represented by the following formula was converted into a nerve cell. Is found to increase the expression level of molecular chaperone (GRP78 / Bip) and to develop resistance to endoplasmic reticulum stress. Further, the compound is administered to nerve cells to give molecular chaperone (GRP78 / Bip). It was confirmed that when the expression level of Bip) was increased, the production of amyloid protein (A) from amyloid precursor protein (APP) was inhibited and A production was suppressed. Furthermore, it was confirmed that the occurrence and extension of cerebral infarction caused by transient ischemia can be suppressed by administering the compound (I) into the ventricle. That is, the present inventors confirmed that the compound (I) functions as a molecular chaperone expression inducer and counters endoplasmic reticulum stress by promoting protein folding. I) are caused by endoplasmic reticulum stress. We were convinced that it is useful as an active ingredient for preventive or therapeutic agents for pathological conditions and diseases. This invention has been completed based on strong knowledge.

 That is, the present invention has the following aspects:

 Term 1.Formula (I):

[0016] [Chemical 1]

[In the formula, R ¹ and IT each independently represent a hydrogen atom, a halogen atom, a hydroxyl group, a lower alkyl group or a lower alkoxy group; Alk represents a lower alkylene chain; X represents an oxygen atom or a sulfur atom)

 Or a pharmaceutically acceptable salt thereof, or a solvate thereof, as an active ingredient. A pharmaceutical composition comprising: a compound represented by formula (1-phenyl-thiocyanatoalkane_1_one derivative):

 [0018] Item 2. Formula (Γ):

 [0019] [Chemical 2]

[0020] (wherein R ¹ 'and IT are both hydroxyl groups or fluorine atoms, or R ¹ chlorine atoms, and R ² ' is a hydrogen atom)

 Item 2. A pharmaceutical composition according to Item 1, comprising a compound represented by the above or a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.

[0021] Item 3. The pharmaceutical composition according to Item 1 or 2, which is a molecular chaperone expression inducer.

[0022] Item 4. The pharmaceutical composition according to Item 1 or 2, which is a cell death inhibitor against endoplasmic reticulum stress.

[0023] Item 5. The pharmaceutical composition according to Item 1 or 2, which is a preventive or therapeutic agent for a neurodegenerative disease.

[0024] Item 6. The neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or mild cognitive impairment Item 6. The pharmaceutical composition according to Item 5.

[0025] Item 7. A method for preventing or treating a neurodegenerative disease, comprising a step of administering the pharmaceutical composition according to Item 1 or 2 to a subject.

[0026] Item 8. The prevention or treatment method according to Item 7, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or mild cognitive impairment.

[0027] Item 9. A method for producing a preventive or therapeutic agent for a neurodegenerative disease:

[0028] [Chemical 3]

[In the formula, R ¹ and IT each independently represent a hydrogen atom, a halogen atom, a hydroxyl group, a lower alkyl group or a lower alkoxy group; Alk represents a lower alkylene chain; X represents an oxygen atom or a sulfur atom)

 Or a pharmaceutically acceptable salt thereof, or a solvate thereof (1 -phenyl-thiocyanatoalkane-1-one derivative) or a pharmaceutically acceptable salt thereof.

Item 10 · The above compound (1-phenyl-thiocyanatoalkane-1-one derivative) is represented by the following formula (

 1 '):

 [0031] [Chemical 4]

[0032] (wherein R ¹ 'and IT are both a hydroxyl group or a fluorine atom, or R ¹ force S is a chlorine atom, and R ² ' is a hydrogen atom)

 Item 12. The use according to Item 9, which is a compound represented by:

 The invention's effect

[0033] The 1-phenyl-thiocyanatoalkane-1-one derivative (I) and the pharmaceutical composition comprising this as an active ingredient provided by the present invention are molecular shunts involved in endoplasmic reticulum stress response (UPR). Pen Since it has an action of inducing Ron expression, it can be effectively used for the prevention and treatment of pathological conditions and diseases caused by excessive endoplasmic reticulum stress. That is, according to the present invention, various neurodegenerative diseases caused by excessive endoplasmic reticulum stress (Alzheimer's disease, Parkinson's disease, polygnoretamine disease, mild cognitive impairment, stroke, diabetic neuropathy, glaucoma, etc.) are effectively prevented or Can be treated.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0034] The pharmaceutical composition provided by the present invention has the following formula (I):

[0035] [Chemical 5]

[In the formula, R ¹ and R ² each independently represent a hydrogen atom, a halogen atom, a hydroxyl group, a lower alkyl group or a lower alkoxy group; Alk represents a lower alkylene chain; X represents an oxygen atom or a sulfur atom. )

 Or a pharmaceutically acceptable salt thereof as an active ingredient.

In the formula (I), examples of the halogen atom defined by R ¹ and R ² include a chlorine atom, a fluorine atom, an iodine atom, and a bromine atom. Preferred are a chlorine atom and a fluorine atom. Examples of the lower alkyl group include linear or branched alkyl groups having 1 to 6 carbon atoms. Specifically, methinole group, ethyl group, n-propyl group, isopropyl group, n_butyl group, isobutyl group, see-butyl group, ter-butynole group, pentyl group, 1,2-dimethylpropyl group, 1 , 1-dimethylpropyl group, 2,2-dimethylpropyl group, 2_ethylpropyl group, n_hexyl group, 1,2_dimethylbutyl group, 2,3_dimethylbutyl group, 1,3_dimethylbutyl group, Examples include 1_ethyl_2_methylpropyl group, 1_methyl_2_ethylpropyl group, and the like. Preferred is a linear or branched alkyl group having 1 to 4 carbon atoms, and more preferred is a methyl group. Further, examples of the lower alkoxy group include those having 1 to 6 carbon atoms, specifically, methoxyl group, ethoxyl group, propoxyl group, isopropoxyl group, butoxyl group, secondary butoxynole group and A tertiary butoxyl group can be exemplified. Preferably Toxyl group. R ¹ and R ² may be the same or different from each other.

 [0038] In the formula (I), examples of the lower alkylene chain defined by Alk include straight-chain or branched alkylene chains having 1 to 6 carbon atoms. Specific examples include a methylene chain, an ethylene chain, a propylene chain, a butylene chain, and an ethylmethylene chain. A methylene chain is preferred.

 [0039] In the formula (I), X may be either an oxygen atom or a sulfur atom, but is preferably an oxygen atom.

[0040] In the formula (I), the following formula bonded to the benzene ring:

[0041] [Chemical 6]

 \ ノ Alk— N

丫^S

 X

[0042] (wherein Alk and X are the same as above)

The position of the group represented by is not particularly limited, but is preferably a para position relative to the R ¹ group or the R ² group. More preferably, it is para to the R ¹ group.

[0043] In the formula (I), Alk is preferably a methylene chain. In this case, X is preferably an oxygen atom, R ¹ and R ² are the same or different, and a halogen atom (preferably Chlorine atom or fluorine atom), hydroxyl group, and hydrogen atom. Specific examples of such a compound include an α-thiocyanatotophenone compound having a substituent at the 3-position and / or 4-position represented by the following formula (Γ).

 [0044] Formula (Γ):

 [0045] [Chemical 7]

(Where R ¹ and R ² 'are both hydroxyl groups or fluorine atoms, or R ¹ ' is a chlorine atom and R ² 'is a hydrogen atom) In addition, the compound includes, specifically, to (3,4-dihydroxy-phenyl) -2-thiocyanatoethanone (molecular weight: 209.22) in which both R ¹ ′ and R ² ′ are hydroxyl groups; R ¹ And R "are both fluorine atoms 1- (3,4-difluoro-phenyl) -2-thiocyanatoethanone (molecular weight: 213.20); and R ¹ is a chlorine atom and IT is a hydrogen atom 1 -(4-Chloro-phenyl) -2-thiocyanato-ethanone (molecular weight: 211.67), preferably 1- (3,4-dihydroxy-phenyl) where R ¹ 'and R ² ' are both hydroxyl groups ) -2-Thiocyanato-ethanone (hereinafter also referred to as “BIX”).

[0047] Of the above compound (I), representative compounds are known compounds described in Non-Patent Document 16, etc., and can be produced by known methods. In particular, thiothionatacetophenone compounds represented by the following formula (Γ) are commercially available (for example, Maybrige (UK), Chemst mark (France), etc.).

 [0048] These compounds can also be synthesized by the following method.

[0049] [Chemical 8]

 a

 (I)

[0050] (In the figure, Y represents a leaving group such as halogen, methanesulfonyloxy, p-toluenesulfonyloxy, etc., and ZSCN represents a metal or ammonium salt of thiocyanate. Also, R ¹ R ² , Alk, And X is as described above.)

 Specifically, compound a and thiocyanate (ZSCN) prepared by a known method are -20 ° C to 200 ° C, preferably 0 ° C to 100 ° C, in a solvent such as toluene, tetrahydrofuran and dimethylformamide. Compound (I) can be obtained by reacting at 0 ° C. for 0.5 to 24 hours, preferably 1 to 10 hours. In the case where a substituent such as a hydroxyl group hinders the reaction, it may be protected in advance at a necessary stage, and the protecting group may be removed after the reaction with thiocyanate.

[0051] These compounds may be either in free or salt form. Here, the salt is usually a pharmaceutically acceptable salt such as a salt with an inorganic base or an organic base. Can be mentioned. Examples of the inorganic base include alkali metals such as sodium and potassium; alkaline earth metals such as calcium and magnesium; aluminum and ammonium. Examples of organic bases include primary amines such as ethanolamine; secondary amines such as jetylamine, diethanolamine, dicyclohexylamine, N, N'-dibenzylethylenediamine, trimethylamine, and triethylamine. And tertiary amines such as pyridine, picoline and triethanolamine.

 [0052] The above compound may be a solvate of a free substance or a salt thereof. Here, solvates include hydrates.

 [0053] The compound (I) has an effect of inducing expression of a molecular chaperone gene (Experimental Example)

 1). Here, the molecular chaperone gene refers to the gene of a molecular chaperone that is localized in the lumen of the endoplasmic reticulum and plays an important role in folding secreted proteins and membrane proteins in the endoplasmic reticulum. For example, ORP150 gene (Ikeda, J. et al, Biochem. Biophys. Res. Common., 230, 94-99 (1997)), GRP94 gene (Maki. RGet al., Proc. Natl. Acad. Sci. USA, 87, 5658-5662 (1990)), GRP78 gene (Ting J. and Lee, AS DNA, 7, 275-286 (19 88)), calreticulin gene (Rokeach, LA. Et al., J. Immunol) 147, 3031-3039 (1991)), ERp72 gene, GRP58 gene, PDI gene, FKBP13 gene and the like. Preferred are GRP78 gene, ORP150 gene, and GRP94 gene that are highly expressed in nerve cells.

 [0054] The molecular chaperone expression-inducing action of compound (I) can be evaluated based on the amount of transcript (mRNA) or gene product (protein) of the molecular chaperone gene. Specifically, a transcript (mRNA) or gene product of a molecular chaperone gene in a cell (preferably a nerve cell) treated with the compound (I) by a usual method such as Northern blotting or Western blotting. When the amount of (protein) increases from that of untreated cells, the action of inducing molecular chaperone expression of the compound can be evaluated.

[0055] Therefore, the pharmaceutical composition of the present invention containing compound (I) as an active ingredient can be effectively used as a molecular chaperone expression inducer. In this case, the ratio of the compound (I) contained in the pharmaceutical composition may be any amount as long as the pharmaceutical composition has a molecular chaperone expression-inducing action, for example, 0.1 to 100 weights. % As appropriate You can. Preferably:! To 60% by weight, more preferably 3 to 40% by weight.

[0056] Further, compound (I) can relieve endoplasmic reticulum stress by folding proteins based on the molecular chaperone expression-inducing action and suppressing accumulation of abnormal proteins in the endoplasmic reticulum. It can suppress cell death caused by endoplasmic reticulum stress or enhance resistance to endoplasmic reticulum stress (see Experimental Example 2). The cell death inhibitory effect of compound (I) on endoplasmic reticulum stress is the ratio of viable cells when an endoplasmic reticulum stress inducer is allowed to act on cells in the presence of compound (I). It can be evaluated by comparing with the ratio of viable cells when an endoplasmic reticulum stress inducer is allowed to act on cells in the presence. As endoplasmic reticulum stress-inducing agents, tuna mycin (tunicamycin: Tm), which inhibits N-glycosinolation of proteins, calcium ionophore A23187, which depletes calcium storage, and thapsigargin, which inhibits calcium ATPase ( thapsigargin (Tg) is known. In this case, based on the increase of viable cells (inhibition of dead cells) due to the action of compound (I), the effect of compound (I) on cell death against endoplasmic reticulum stress can be evaluated.

 Therefore, the pharmaceutical composition of the present invention comprising compound (I) as an active ingredient can be effectively used as a cell death inhibitor against endoplasmic reticulum stress or a resistance enhancer against endoplasmic reticulum stress. In this case, the ratio of the compound (I) contained in the pharmaceutical composition may be any amount as long as the pharmaceutical composition has a cell death inhibitory action or a resistance enhancing action against endoplasmic reticulum stress. For example, it can be appropriately selected from the range of 0.1 to 100% by weight. Preferably:! To 60% by weight, more preferably 3 to 40% by weight.

 [0058] Furthermore, compound (I) relieves endoplasmic reticulum stress and suppresses the production of amyloid protein A involved in the onset and progression of neurodegenerative diseases such as Alzheimer's disease based on the expression-inducing action of molecular chaperones. (See Experimental Example 3).

[0059] The A β production inhibitory action of the compound (I) is such that the compound (I) is allowed to act on cells capable of expressing the amyloid precursor protein (ΑΡΡ), and the ratio of amyloid protein Α / 3 in the cells. Can be evaluated by comparing with the case where compound (I) is not allowed to act. In this case, based on the decrease in the amount of Aβ produced by the action of the compound (I), the action of inhibiting the formation of ββ can be evaluated for the compound (I). In addition, as shown in Experimental Example 3, Α β of compound (I) The production inhibitory action is based on the movement of cocoons from the site where the enzyme that cleaves A j3 from APP (γ-secretase) acts. Therefore, in place of the above-mentioned decrease in the amount of production, it is possible to make a clear interpretation of the action of inhibiting Aβ production by using a decrease in APP (mature type) or an increase in modified APP (immature type) as an index.

[0060] Therefore, the pharmaceutical composition of the present invention containing Compound (I) as an active ingredient can be effectively used as a preventive or therapeutic agent for neurodegenerative diseases. In this case, the harm IJ of compound (I) contained in the pharmaceutical composition is that the pharmaceutical composition is affected by endoplasmic reticulum stress based on the molecular chaperone expression inducing action, preferably the molecular chaperone expression inducing action. On the other hand, as long as it has a cell death inhibitory action and / or a Α / 3 production inhibitory action, it may be an appropriate amount, for example, from 0.1 to 100% by weight. Preferably it is 1-60 weight%, More preferably, it is 3-40 weight%.

[0061] It should be noted that the neurodegenerative diseases targeted by the present invention include diseases in which the onset involves ER stress or cell death due to ER stress. Examples include Alzheimer's disease, mild cognitive impairment (MCI), Parkinson's disease, stroke (cerebral infarction), polyglutamine disease, prion disease, diabetes, glaucoma and the like. Alzheimer's disease, mild cognitive impairment (MC1), and Parkinson's disease are preferable.

[0062] The pharmaceutical composition of the present invention is usually an effective amount of a compound (I) effective in inducing molecular chaperone expression, and thus in suppressing cell death and / or suppressing A production due to endoplasmic reticulum stress. ) And a pharmaceutically acceptable carrier or additive.

[0063] The administration method of the pharmaceutical composition of the present invention includes oral administration and parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration, transmucosal administration, transdermal administration, and rectal administration. Ability to do S.

 [0064] Preferred are oral administration and intravenous administration, and more preferred is oral administration. The pharmaceutical composition of the present invention can be prepared into various forms of preparations (dosage forms) depending on the strength and the administration method. Each formulation (dosage form) will be described below, but the dosage form used in the present invention is not limited to these, and various dosage forms usually used in the field of pharmaceutical preparations can be used.

[0065] The dosage forms for oral administration include powders, granules, capsules, pills, tablets, and elixirs. Examples include sills, suspensions, emulsions and syrups, and can be appropriately selected from these. In addition, these formulations can be modified such as sustained release, stabilization, easy disintegration, hard disintegration, entericity, easy absorption, and the like.

 [0066] In addition, dosage forms for intravenous administration, intramuscular administration, or subcutaneous administration include injections and infusions (including dry products for preparation at the time of use), which can be selected as appropriate. .

 [0067] In addition, dosage forms for transmucosal administration, transdermal administration, or rectal administration include mastication agents, sublingual agents, buccal agents, troches, ointments, patches, liquids, etc. You can choose here depending on the location. In addition, these formulations can be modified such as sustained release, stabilization, easy disintegration, difficulty disintegration, and easy absorption.

 [0068] The pharmaceutical composition of the present invention may contain pharmaceutically acceptable carriers and additives depending on the dosage form (oral administration or various parenteral administration dosage forms). Pharmaceutically acceptable carriers and additives include solvents, excipients, coating agents, bases, binders, lubricants, disintegrants, solubilizers, suspending agents, thickeners, emulsifiers. , Stabilizers, buffers, tonicity agents, soothing agents, preservatives, flavoring agents, fragrances, and coloring agents. The ability to list specific examples of pharmaceutically acceptable carriers and additives below The present invention is not limited to these.

 [0069] Examples of the solvent include purified water, sterilized purified water, water for injection, physiological saline, laccase oil, ethanol, glycerin and the like. Excipients include starches (for example, potato starch, wheat starch, corn starch), lactose, glucose, sucrose, crystalline cellulose, calcium sulfate, calcium carbonate, sodium bicarbonate, sodium chloride, talc, titanium oxide, Examples include trehalose and xylitol.

 [0070] Examples of the binder include starch and derivatives thereof, cellulose and derivatives thereof (for example, methinoresenorelose, ethinoresenorelose, hydroxypropenoresenorelose, strong noroxymethylcellulose), gelatin, sodium alginate, tragacanth, Examples thereof include natural polymer compounds such as gum arabic, synthetic polymer compounds such as polyvinylinolepyrrolidone and polybutal alcohol, dextrin, hydroxypropyl starch and the like.

[0071] Lubricants include light anhydrous carboxylic acid, stearic acid and its salts (eg, magnesium stearate), talc, waxes, wheat starch, macrogol, hydrogenated plants. Oil, sucrose fatty acid ester, polyethylene glycol, silicone oil and the like.

 [0072] Examples of the disintegrant include starch and derivatives thereof, agar, gelatin powder, sodium hydrogen carbonate, calcium carbonate, cellulose and derivatives thereof, hydroxypropyl starch, strength lupoxymethylcellulose and salts thereof, and cross-linked products thereof, low substitution type And hydroxypropylcellulose.

 [0073] Examples of solubilizers include cyclodextrin, ethanol, propylene glycol, and polyethylene glycol. Examples of the suspending agent include sodium carboxymethyl cellulose, polypinyl pyrrolidone, gum arabic, tragacanth, sodium alginate, aluminum monostearate, citrate, and various surfactants.

 [0074] Examples of the thickening agent include sodium carboxymethylcellulose, polypinylpyrrolidone, methinoresenololose, hydroxypropino methenoresenorelose, polyvinylino oleconole, tragacanth, gum arabic, sodium alginate and the like.

 [0075] Examples of the emulsifier include gum arabic, cholesterol, tragacanth, methylcellulose, lecithin, various surfactants (for example, polyoxyl 40 stearate, sorbitan sesquioleate, polysorbate 80, sodium lauryl sulfate).

 [0076] Stabilizers include tocopherols, chelating agents (eg EDTA, thioglycolic acid), inert gases (eg nitrogen, carbon dioxide), reducing substances (eg sodium hydrogen sulfite, sodium thiosulfate, ascorbic acid, longgarit And the like.

 [0077] Examples of the buffer include sodium hydrogen phosphate, sodium acetate, sodium citrate, boric acid and the like.

 [0078] Examples of isotonic agents include sodium chloride, glucose and the like. Examples of soothing agents include local anesthetics (pro-in hydrochloride, lidocaine), pendyl alcohol, glucose, sorbitol, amino acids and the like.

 [0079] Examples of the corrigent include sucrose, saccharin, licorice extract, sorbitol, xylitol, dariserine and the like. Examples of the fragrances include spruce tincture and rose oil. Examples of the colorant include water-soluble food dyes and lake dyes.

[0080] Preservatives include benzoic acid and its salts, paraoxybenzoic acid esters, Examples include robutanol, reverse soap, benzyl alcohol, phenol, tiromesal, dehydroacetic acid, boric acid, and the like.

 [0081] Coating agents include sucrose, hydroxypropylcellulose (HPC), shellac, gelatin, glycerin, sonolebithonole, hydroxypropylmethylcellulose (HPMC), ethylcellulose, polybutylpyrrolidone (PVP), hydroxypropylmethylcellulose phthalate ( HPMCP), cellulose acetate phthalate (CAP), methyl methacrylate, monomethacrylic acid copolymer, and the polymers described above.

 [0082] Examples of the base include petrolatum, liquid paraffin, carnauba wax, beef tallow, hardened oil, paraffin, beeswax, vegetable oil, macrogol, macrogol fatty acid ester, stearic acid, sodium carboxymethylcellulose, bentonite, cacao butter, wittebuzole, Gelatin, stearyl alcohol, caroline lanolin, cetanol, light liquid paraffin, hydrophilic salmon, single ointment, white ointment, hydrophilic ointment, macrogol ointment, hard fat, oil-in-water emulsion base, water-in-oil emulsion Mention may be made of sexing agents.

[0083] It should be noted that a known drug delivery system (DDS) technique can be employed for each of the above dosage forms. As used herein, DDS preparations include sustained-release preparations, topical preparations (troches, buccal tablets, sublingual tablets, etc.), drug release control preparations, enteric preparations and gastric preparations, etc. It is a preparation with the optimal dosage form, taking into account the best availability and side effects.

 [0084] When the pharmaceutical composition of the present invention is used as a molecular chaperone expression inducer, a cell death inhibitor against endoplasmic reticulum stress, or a neurodegenerative disease preventive / therapeutic agent, its oral dose is compound (I). The range of 0.03 to 300 mg / kg in terms of the amount is more preferable, and more preferably 0.:! To 50 mgZkg. When administered intravenously, increase the dose so that the effective blood concentration of compound (I) is in the range of 0.2-50 8/111, preferably 0.3 S SO zg / mL. That power S.

 [0085] These dosages may vary depending on age, sex, body type, and the like.

[0086] The present invention also relates to a method for preventing and treating a neurodegenerative disease in a subject by administering the pharmaceutical composition of the present invention to the subject.

The neurodegenerative disease targeted here is, as described above, endoplasmic reticulum stress or Includes diseases involving cell death due to endoplasmic reticulum stress. Specific examples include Alzheimer's disease, mild cognitive impairment (MCI), Parkinson's disease, stroke (cerebral infarction), polyglutamine disease, prion disease, diabetes, glaucoma and the like. Alzheimer's disease, mild cognitive impairment (MC1), and Parkinson's disease are preferable.

 [0087] In addition to the patients suffering from the above-mentioned neurodegenerative diseases, the subjects targeted by the present invention include patients who may suffer from neurodegenerative diseases. Neurodegenerative diseases are caused by environmental factors, drugs, inheritance and aging. For this reason, people exposed to contaminated water, contaminated air or pesticides (patients), patients addicted to narcotics, patients who develop side-effect disorders due to drugs, and hereditary patients also have the above-mentioned neurodegeneration. Included in patients who may have the disease.

[0088] As described above, the administration method of the pharmaceutical composition to the subject is not particularly limited, and is oral administration, intravenous administration, intramuscular administration, subcutaneous administration, transmucosal administration, transdermal administration, and intrarectal administration. Parenteral administration such as administration can be mentioned. Oral administration and intravenous administration are preferred. The dose is 0.03 to 300 mg / kg, preferably 0.1 to 50 mg / kg, converted into the amount of compound (I) in the case of oral administration, and compound in the case of intravenous administration. Examples of the ratio (I) include an effective blood concentration in the range of 0.2 to 50/1 _§ / 111, preferably 0.5 to 20 _β g / mL.

 Example

 [0089] Hereinafter, the present invention will be described with reference to experimental examples, but the present invention is not limited to such experimental examples. In the following experimental examples, unless otherwise specified, genetic manipulation, cell culture, etc., Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocols in Molecular Biology ( reene Publishing Associates and Willey—Interscience).

 [0090] Further, in the following experimental examples, l_ (3,4-dihydroxy_phenyl) -2-thiocyanato-ethanone (BIX) was used as a representative example of compound (I).

 [0091] Experimental Example 1

As described below, neuroblastoma SK-N-SH cells were exposed to l-50 / i M of (3,4-dihydroxy-phenyl) -2-thiocyanato-ethanone (BIX) for 6 hours. Molecular chaperone induction The presence or absence of was measured by Northern blotting.

 [0092] (1) Transfusion

 SK-N-SH cells are grown to 80% confluence in 24-well plates, then using Lipofectamine 2000 reagent (Invitrogen), a reporter plasmid with the firefly luciferase gene under the control of the GRP78 / Bip promoter (0.2 μg) was transfected. Also. As a control, SK-N-SH cells were transfected using the control plasmid pRL-SV40 (0.02 μg) (Promega) having the Renill luciferase gene under the control of the SV40 enhancer and the promoter (control). cell). After 12 hours, each SK-N-SH cell was treated with 1-50 μΜ Μ or 300 ηΜ thapsigargin (Tg) (endoplasmic reticulum stress inducer) (control) for 6 hours.

 [0093] (2) RNA isolation and semi-quantitative RT-PCR analysis

Each of the above SK-N-SH cells was washed with phosphate buffered saline (PBS) and collected by centrifugation. Total RNA was isolated from cells using RNeasy kit (Qiagen Κ.Κ ·) according to the manual. The RNA concentration was measured with a spectrophotometer with a wavelength of 260 nm. After developing on a 1.0% denatured agarose gel, it was transferred to Immobilon-NY + membrane (Millopore). Using a random primer DNA labeling kit (TaKaRA), ³² P_ labeled cDNA probe prepared from GRP78 / Bip cDNA was hybridized, washed with 2xSSC, 0.1% SDS and 0.1xSSC, 0.1% SDS, then IP plate It was exposed on (Fuji Film) and analyzed with the BAS1800 system (Fuji Film).

 [0094] The results are shown in FIG. Figure 1 shows that BIX has the effect of inducing molecular chaperone gene expression in a concentration-dependent manner.

[0095] Experimental Example 2

Neuroblastoma SK-N-SH cells were cultured for 12 hours in the presence of 5 μΜ of BIX, and this was then added to the endoplasmic reticulum stress inducer Tsuyu forcemycin (Tm) at a rate of 0.5 μg / ml The SK-N-SH cells were transferred to fresh culture medium and further cultured for 12 to 48 hours, and the number of viable SK-N-SH cells was measured. The number of viable cells was measured by staining cells that had undergone apoptosis with Hoechst33258 staining and distinguishing them from viable cells. Specifically, the cultured cells were treated with 4% paraformaldehyde at 4 ° C for 30 minutes, and then with PBS for 5 minutes. Wash cells for a period of 20 minutes with PBS containing 100 μΜ Hoechst33258 (Wako Pure Chemical Industries, Ltd.) to stain cells that have undergone apoptosis, and the strength of apoptotic cells with nuclei fragmented by Hoechst33258 The number of viable cells was calculated. As a control experiment, SK-N-SH cells cultured in the absence of BIX for 12 hours were similarly treated with an endoplasmic reticulum stress inducer (Tm) to increase the number of surviving SK-N-SH cells. It was measured.

 [0096] The results are shown in FIG. In the figure, BIX → Tm is the result of the BIX treatment group (cultured in the presence of BIX and then in the presence of Tm), and Normal → Tm is the BIX non-treatment group (after culture in the absence of BIX, the presence of Tm The results of (cultured under) (control group) are shown. The results (Fig. 2) are shown as the ratio of the number of viable cells (cell viability) when the number of viable cells during the 12-hour Tm treatment in the control group is 1. As can be seen from FIG. 2, the cell viability of the BIX-treated group was higher than that of the non-treated group (control group) (inhibition of cell death) over time, 28, 36 and 48 hours. From this, it can be seen that BIX has an action of imparting resistance to endoplasmic reticulum stress to cells and suppressing cell death due to endoplasmic reticulum stress.

 [0097] Experimental Example 3

SY5Y / APPsw (a SY5Y cell with APP having a Swedish mutation was transformed into a parent cell line) was cultured in the presence of 0 to 10 μg / ml BIX for 8 hours. Next, this was washed with PBS, Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mM HEPES pH 7.6, lOOmM NaCl, 3 mM MgCl, 5 mM dithiothreitol, 0.1% protease inhibitor cock tail (Sigma It was added to) and lysed. Cell lysates were sonicated 3 times for 5 seconds on ice and centrifuged at 15000 μm for 5 minutes. The supernatant was transferred to a new tube and boiled for 5 minutes in SDS sample buffer. Apply to each lane of 10-15% SDS-PAGE so that the amount of protein is equivalent, and after electrophoresis, transfer to PVDF membrane, and the first antibody [anti-APP antibody (Chemicon, Temecula, CA, USA;, i ~ A β antibodies, 4G8 and 6E10 (bignet Pathology systems Inc., Dedham, MA, USA)]. The membrane was then washed with TBS / Tween_20, and the alkaline phosphatase-conjugated second antibody Incubated with ECL kit anti-rat / mouse antibody (Sigma) and corresponding bands were detected using Nitro Blue Tetrazolium (Sigma) and 5-Bromo-4-chloro-3-indoly phosphate (Roche). Anti-KDEL antibody is an enzyme that recognizes GRP78 / Bip. [0098] The results are shown in FIG. As can be seen from this figure, it was observed that the expression induction of molecular chaperone (Bip) was increased by BIX in a concentration-dependent manner, and the amount of secreted A (amyloid protein) decreased accordingly. It was also observed that APP, the precursor of amyloid protein, decreased in mature type and increased in immature type as the BIX concentration increased. Therefore, _BIX immatures APP by inducing a molecular chaperone (Bip), which makes APP resistant to Ίsecretase cleavage and suppresses A j3 production and secretion by ΑΡΡ cleavage. It is thought that.

 [0099] Experimental Example 4

 BIX (5 or 20 xg / 2 z 1) dissolved in 10% DMSO was administered into the ventricle of mice (20-27 g, male, DDY mice) (obtained from SLC), and then one side middle cerebral artery ( MCA) was closed with an obturator and hypoxic conditions were caused artificially to cause endoplasmic reticulum stress. BIX was administered into the ventricles by inserting a needle through the skull based on the brain chart.

[0100] Twenty-four hours after the occlusion treatment, the degree of neurological damage can be scored and evaluated according to the following criteria from the behavior of the mouse.

 <Degree of neurological disorder>

 0 (normal): No neurological impairment

 1 (Mild): Extension disorder on right forefoot

 2 (Medium): There is circling on the contralateral side (turning behavior)

 3 (Severe): Inability to walk or rebounding disappeared.

 [0101] Next, the brain was decapitated and the brain was taken out, and the forebrain was divided into 5 mm intervals from the olfactory brain (see A in Fig. 4). Each section was stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) to identify the infarct site, recorded as an image using a digital camera, and infarcted using Image J. The ratio of area (area, volume) was quantified.

[0102] The ratio of brain edema can also be calculated according to the following equation.

[0103] [Equation 1]

 Percentage of brain edema (%) ==

 (Volume of infarcted area + volume of non-injured area on the same side-contralateral volume) Z contralateral volume X 100 (%)

[0104] As a control, BIX (5 xg / 2 z 1 or 20 z gZ2 x 1) was administered into the ventricle. In addition, the same amount of DMSO was administered and the test was conducted in the same manner as described above.

[0105] The results of TTC staining are shown in Fig. 4B. In FIG. 4B, the right side is the result of the BIX administration group, and the left side is the result of the BIX non-administration group (control group). The white area of the brain is where the infarction is occurring. The area of the infarct area for each section is shown in FIG. 5A, and the volume of the infarct area in the section 6 mm from the mouse olfactory brain is shown in FIG. 5B.

As can be seen from FIGS. 5A and 5B, in the BIX 20 μg administration group, a significant decrease in the infarct area was observed in the 6-mm region of the mouse olfactory brain force. A decrease in infarct area was also observed in the BIX 5 x g administration group.

 [0107] From the above, the compound (I) represented by BIX has an action of inducing molecular chaperone expression, and accordingly, suppresses cell death due to endoplasmic reticulum stress, and amyloid protein (A / It turns out that it has the effect | action which suppresses the production | generation of 3).

 Brief Description of Drawings

 [0108] [Figure l] Molecular chaperone by l- (3,4-dihydroxy-phenyl) -2-thiocyanato_ethanone (BIX)

 It is a result which shows the expression induction of (GRP78 / Bip) (Experimental example 1)

 [Fig. 2] Results showing the inhibitory effect of BIX on cell death induced by endoplasmic reticulum stress inducer (Tm) (Experiment 2)

 [Fig. 3] Results showing the inhibitory effect of BIX on amyloid protein (A) production (Experimental Example 3)

 [Fig. 4] In Experimental Example 4, A is a diagram showing the cutting pattern of the forebrain, and B is a diagram showing the result of TTC staining of the cut brain section.

 [Fig. 5] A is the area of the infarct area of each brain section, B is the volume of the infarct area in the 6 mm section from the olfactory brain, with and without BIX (5, 20 zg) administration (control) The comparison results are shown in the case of.

Claims

 The scope of the claims

(Wherein R ¹ and R ² are each independently a hydrogen atom, a halogen atom, a hydroxyl group, a lower alkyl group or a lower alkoxy group; Alk is a lower alkylene chain; X is an oxygen atom or a sulfur atom)  Or a pharmaceutically acceptable salt thereof, or a solvate thereof, as an active ingredient.  [2] Equation (Γ):  [Chemical 2]

(Wherein R ¹ ′ and IT are both a hydroxyl group or a fluorine atom, or R ¹ ′ is a chlorine atom and R ² ′ is a hydrogen atom)  2. The pharmaceutical composition according to claim 1, comprising a compound represented by the above or a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.  [3] The pharmaceutical composition according to claim 1, which is a molecular chaperone expression inducer. [4] The pharmaceutical composition according to claim 1, which is a cell death inhibitor against endoplasmic reticulum stress. [5] The pharmaceutical composition according to claim 1, which is a preventive or therapeutic agent for a neurodegenerative disease. 6. The pharmaceutical composition according to claim 5, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or mild cognitive impairment. [7] A method for preventing or treating a neurodegenerative disease, comprising a step of administering the pharmaceutical composition according to claim 1 or 2 to a subject. The method for prevention or treatment according to claim 7, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease or mild cognitive impairment.  Formula (I) for producing a preventive or therapeutic agent for neurodegenerative diseases  [Chemical 3] (I)

(Wherein R ¹ and R ² are each independently a hydrogen atom, halogen atom, hydroxyl group, lower alkyl group or lower alkoxy group; Alk is a lower alkylene chain; X means an oxygen atom or a sulfur atom)  Or a pharmaceutically acceptable salt thereof, or a solvate thereof [10] The above compound is represented by the following formula (Γ)  [Chemical 4]

(Where R ¹ and R ² 'are both hydroxyl groups or fluorine atoms, or R ¹ ' is a chlorine atom and R ² 'is a hydrogen atom)  The use according to claim 9, which is a compound represented by: