WO2007141926A1

WO2007141926A1 - Extended amyloid beta production inhibitor

Info

Publication number: WO2007141926A1
Application number: PCT/JP2007/050903
Authority: WO
Inventors: Masayasu Okochi; Masatoshi Takeda; Shinji Tagami
Original assignee: Osaka University; Shionogi & Co., Ltd.
Priority date: 2006-06-02
Filing date: 2007-01-22
Publication date: 2007-12-13
Also published as: JP5135588B2; JPWO2007141926A1

Abstract

The invention relates to a pharmaceutical composition containing as an active ingredient a compound represented by the formula (I): (wherein R1 and R2 each independently represent aryloxy which may have a substituent or arylthio which may have a substituent, R3 represents a hydrogen atom or a lower alkyl group) or a pharmaceutically acceptable salt thereof or a solvate thereof, in particular, based on an extended amyloid β production inhibitory action of the compound, a pharmaceutical composition that can be effectively used for preventing or treating a disease associated with extended amyloid β, particularly Alzheimer’s disease. Further, the invention relates to a reagent for screening a substance capable of inhibiting the production of extended amyloid β.

Description

Specification

Prolonged amyloid 0 production inhibitor

Technical field

[0001] The present invention relates to a pharmaceutical composition having an inhibitory action on prolonged amyloid β production. The present invention also relates to a pharmaceutical thread and composition that can be effectively used for the prevention or treatment of diseases associated with prolonged amyloid ι8, particularly Alzheimer's disease, based on the inhibitory action on prolonged amyloid ι8 production. Furthermore, the present invention relates to the use of the compound having an inhibitory action on extended amyloid) 8 production other than the above-mentioned pharmaceuticals (for example, screening reagents and test agents).

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 is a representative of progressive neurodegenerative diseases accompanied by marked loss of nerve cells. A part of Argno-Ima disease (AD) is familial AD (FAD) with genetic mutations The majority is sporadic AD (SAD) with no family history. In both cases, amyloid β peptide deposited in the brain (hereinafter simply “単に β”) is a pathological finding characterized by senile plaques and neurofibrillary tangles caused by abnormal phosphorylation of tau protein. For this reason, it is considered that the increase in Α | 8 is deeply related to the pathology of Arno-Ima disease! / (Amyloid force scale hypothesis) (Non-patent document 1).

[0004] Α β, a major determinant of Alzheimer's disease, is a metabolite of β-amyloid precursor protein (iS APP), mainly 40, 42 or 43 amino acid residues (each | 8 40 ”,“ A | 8 42 ”and“ Α β 43 ”)). V, slippage is generated by β ΑΡΡ undergoing enzymatic cleavage by the action of β-secretase and γ-secretase. Α | 8 Ν-end is formed by β-secretase cleavage between | 8 メ methionine residue 596 and aspartate residue 597 (see, for example, Patent Document 1 or 2), and Α β-C terminus Is formed by cleaving the / 3-secretase cleaved j8 APP fragment at positions 38, 40 or 43 with γ-secretase (the γ-secretase For a review, see, for example, US Pat.

[0005] In normal individuals, 力 β40 is the dominant force that has two major Aβs, Αβ40 and Αβ42. However, in patients with familial Alzheimer's disease, Αβ42 is predominantly increased compared to normal, and in neuritic plaques found in lesions of Arno and Imah's disease, it is much higher than Α | 840 In proportion 8 | 8 42 exists. It has also been reported that A42 accumulates early and preferentially in soft tissue plaques, whereas Αβ40 is unrelated to the early deposition of amyloid plaques. For this reason, it is considered that Α | 842 plays a major role in amyloid plaque deposition in patients with familial Alzheimer's disease (see, for example, Non-Patent Documents 2 to 5).

[0006] As described above, Aβ, in particular, extended Αβ represented by Αβ42, is deeply involved in the pathogenesis and development of Alzheimer's disease, and therefore, a substance that suppresses the production of these extended Α. Is considered to be a preventive or therapeutic agent for Alzheimer's disease.

[0007] For example, Patent Document 4 describes reducing the production and / or level of A j842 produced by cleavage of j8 APP in certain nonsteroidal anti-inflammatory drugs (NSAIDS) force cell cultures. It has been suggested that potent drugs are effective in preventing, delaying or ameliorating the progression of Alzheimer's disease. However, a large amount of drug is required to significantly reduce the Aj842 level using these drugs, which causes serious gastrointestinal side effects such as hemorrhagic ulcers (Non-Patent Documents). 6).

[0008] Therefore, there is a need for a substance having an action of suppressing the production of extended Aβ by a mechanism different from that of nonsteroidal anti-inflammatory drugs (NSAIDS)! /

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

Non-Patent Document 2: Roher et al., 1993, Proc. Natl. Acad. Sci. USA 90: 10836

Non-Patent Document 3: Iwatasubo, T. et al., 1994 Neuron 13:45

Non-Patent Document 4: Yamaguchi et al., 1995, Amyloid Int. J. Clin. Invest. 2: 7-16

Non-Patent Document 5: Mann et al., 1996 Am. J. Pathol. 148: 1257

Non-Patent Document 6: Langman et al., 1994, Lancet 343: 1075-1078

Patent Document 1: US Pat. No. 6,440,698

Patent Document 2: US Pat. No. 5,744,346 Patent Document 3: US Patent Application No. 20020025540

Patent Document 4: US Patent Application No. 20020128319A1

Disclosure of the invention

Problems to be solved by the invention

[0009] The present invention relates to a compound having an action of suppressing the production of extended Aβ that causes Alzno and Imah's disease, an extended Α production inhibitor, and a disease caused by extended A (prolonged type). A | 8-related diseases), in particular, Alzheimer's disease. More specifically, an object of the present invention is to provide a pharmaceutical composition effective as an extended A j8 production inhibitor or an extended A | 8 related disease preventive or therapeutic agent, particularly as a preventive or therapeutic agent for Alzheimer's disease. And

[0010] The present invention also provides an extended A j8 production inhibitor, particularly an extended A j8 related disease, among test substances, using a compound having an action of suppressing the production of extended A as a standard reagent. It is an object of the present invention to provide a method (screening method) for selecting candidate compounds useful as an active ingredient of a prophylactic or therapeutic agent.

Means for solving the problem

[0011] The inventors of the present invention have made extensive studies to achieve the above object, and as a result, 3,5-bis (4-nitrophenoxy) benzoic acid represented by the general formula (I) and its analogs are excellent. It is found that these compounds have an inhibitory effect on prolonged Aβ production! /, And these compounds are used as inhibitors of prolonged 型 β production and diseases caused by the production of prolonged Αβ (prolonged Αβ We were convinced that it can be effectively used as a preventive or therapeutic agent for (beta-related diseases), particularly as an agent for preventing or treating Alzheimer's disease. In addition, the present inventors have confirmed that a potent compound has no anti-inflammatory effect, and the compound suppresses extended Α | 8 production by a mechanism different from NSAIDS, which is a concern with NSAIDS. We were convinced that it is useful as a prophylactic or therapeutic agent for prolonged Aβ-related diseases without worrying about side effects such as hemorrhagic ulcers. In addition, from this, the present inventors use the above-mentioned compound (I) as a standard substance (in other words, a positive control substance) having an extended A j8 production inhibitory action, from among test substances, It was determined that it was possible to select a substance (extended 抑制 β production inhibitory substance) that has an effect of suppressing the extension of Aβ production and can be an active ingredient of a prophylactic or therapeutic agent for extended Αβ-related diseases. [0012] The present invention has been completed on the basis of strong knowledge and has the following aspects:

Item 1.Formula (I):

[0013] [Chemical 1]

(Wherein R ¹ and R ² are each independently an aryloxy which may have a substituent or an optionally substituted aryloxy, and R ³ is a hydrogen atom or a lower group. An alkyl group)

Or a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.

[0015] Item 2. The pharmaceutical composition according to Item 1, which is an extended amyloid β production inhibitor.

Item 3.—In General Formula (I), substituent power of optionally substituted aryloxy or optionally substituted arylolthio nitro group, carboxy group, lower alkoxycarbo- Item 3. The pharmaceutical composition according to any one of Items 1 to 2, wherein a group force comprising a ru group, an acyl group, a sulfo group, a sulfino group, a sulfeno group, a lower alkyl sulfonyl group, and a cyan group force is also selected. .

Item 4. The pharmaceutical composition according to any one of Items 1 to 3, wherein, in the general formula (I), R ¹ and R ² are -trophenoxy groups, and R ³ is a hydrogen atom.

[0018] Item 5. The pharmaceutical composition according to any one of Items 1 to 4, which is a prophylactic or therapeutic agent for an extended amyloid β-related disease.

[0019] Item 6. The pharmaceutical composition according to Item 5, wherein the extended amyloid j8-related disease is Alzheimer's disease.

[0020] Item 7. A pharmaceutical composition described in item 5 or 6 is administered to a subject suffering from or likely to suffer from a prolonged amyloid β-related disease. Treatment or prevention methods. [0021] Item 8. Formula (I):

[0022] [Chemical 2]

(Wherein R ¹ and R ² are each independently an aryloxy which may have a substituent or an arylenethio which may have a substituent, and R ³ is a hydrogen atom or a lower group. An alkyl group)

Or a salt thereof, or a solvate thereof, a reagent used for screening an extended amyloid β production inhibitory substance from among test substances.

[0024] Item 9. The reagent according to Item 8, wherein the reagent is a positive control reagent in screening for an extended amyloid β production inhibitor.

[0025] Item 10. Examine the test substance for an inhibitory effect on prolonged amyloid β production,

Formula (I):

[0026] [Chemical 3]

A test substance having an inhibitory action higher than that of the extended Αβ production inhibitory compound or a salt thereof, or a solvate thereof, as an extended amyloid) 8 production inhibitory substance, A method of screening for an elongated amyloid β production inhibitor.

Item 11. An extended amyloid 13 production inhibitor according to Item 10, which has the following steps: Screening method:

(1) contacting a test substance with a cell capable of expressing and producing β-amyloid precursor protein (ι8-ΑΡΡ);

(2) a step of measuring the amount of Αβ42 produced by the cell force,

(3) The production amount of Αβ42 is changed to the above (1) and (2) using the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof or a solvate thereof instead of the test substance. The test substance that showed a production level of Αβ42 that was lower than the positive control value compared to the production amount of the control 8 | 842 (positive control value) obtained by the process of The process of selecting as a candidate substance for β production inhibitory substance.

Item 12. The screening method according to Item 11, further comprising the following steps:

(4) Candidate substances for prolonging amyloid β production inhibitor selected in step (3)! Then, investigate anti-inflammatory action, and select substances with no or little anti-inflammatory action as non-anti-inflammatory prolongation Screening as a candidate substance for a type amyloid β production inhibitory substance.

[0030] Item 13. The screening method according to any one of Items 10 to 12, wherein the extended amyloid β production inhibitor is an active ingredient of a prophylactic or therapeutic agent for extended amyloid β-related diseases.

[0031] Item 14. Formula (I):

[0032] [Chemical 4]

Or a salt thereof, or a solvate thereof, is applied to β-amyloid precursor protein or an amyloid precursor protein-expressing cell, and a method for inhibiting prolonged amyloid β production. The invention's effect

[0034] 3,5-bis (4-nitrophenoxy) benzoic acid and its analog (I) provided by the present invention have an excellent extended A production inhibitory action. Therefore, the compound (I) and the yarn and composition comprising the compound (I) as an active ingredient are effectively used as extended A production inhibitors, particularly A 42 production inhibitors, for various purposes such as reagents, test agents or medicines. be able to. As mentioned earlier, the onset and development of Alzheimer's disease is deeply related to the production and deposition of Aβ in the brain, especially in the central nerve cells, especially extended Αβ, typically Αβ42. It is known that The pharmaceutical composition comprising 3,5-bis (4-nitrophenoxy) benzoic acid and its analog (I) provided by the present invention as an active ingredient has an excellent elongation type Α | 8 production inhibitory action as described above. Therefore, it is useful as a preventive or ameliorating agent for the onset and progression of Arnno-Ima disease. Further, unlike NSAIDS, these compounds (I) are considered to be useful pharmaceutical compositions with few side effects because they have excellent effects in small amounts and do not have anti-inflammatory effects. BEST MODE FOR CARRYING OUT THE INVENTION

[0035] (1) Pharmaceutical yarn composition and its use

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

[0036] [Chemical 5]

(Wherein R ¹ and R ² are each independently an optionally substituted aryloxy or optionally substituted arylthio, and R ³ is a hydrogen atom or a lower group. An alkyl group)

Or a pharmaceutically acceptable salt thereof as an active ingredient.

[0038] In formula (I), in aryloxy or arylthio defined by R ¹ and R ² , examples of aryl groups include a phenol group, a naphthyl group, an anthryl group, and a phenanthryl group. Can do. A phenyl group is preferred. R ¹ and R ² are Any of oxy and arylo may be used. Aryloxy is preferable, and a phenoxy group is particularly preferable.

[0039] Further, the substituents defined by R ¹ and R ² as "having a substituent, may be aryloxy" and "optionally substituted arylothio" may be a nitro group , Carboxy group, lower alkoxycarbo group, acyl group, sulfo group, sulfino group, sulfino group, lower alkylsulfonyl group, cyano group, halogen atom, hydroxy group, lower alkoxy group, acyloxy group, amino group, acylamino group And a lower alkylamino group, a lower alkylthio group, a strong rubamoyl group, a lower alkyl strength rubamoyl group, an aryl and the like. Preferred are a nitro group, a carboxy group, a lower alkoxycarbonyl group, an acyl group, a sulfo group, a sulfino group, a sulfino group, a lower alkyl sulfol group, and a cyano group, and more preferred is a -tro group. The position of the substituent is not particularly limited. For example, when the aryl group is a phenol group, it is preferably a meta or para position, more preferably a para position with respect to the oxygen atom or sulfur atom bonded to the phenyl group.

In the above, the “lower alkyl group” means a linear or branched alkyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, and more preferably 1 to 3 carbon atoms. For example, methyl, ethyl, n propyl, isopropyl, n butyl, isobutyl, sec butyl, tert butyl, n pentyl, isopentyl, neopentyl, hexyl, isohexyl, n —Heptyl group, isoheptyl group, n-octyl group, isooctyl group, n-nonyl group, n-decyl group and the like can be mentioned. In addition, the “lower alkylsulfonyl group”, “lower alkoxy group”, “lower alkylamino group”, “lower alkylthio group”, “lower alkyl strength rubermoyl group” and “lower alkoxycarbonyl group” are the lower alkyl group moiety. Also, the same group as the above-mentioned “lower alkyl group” can be exemplified. A methyl group, an ethyl group and an n-propyl group are preferred.

[0041] "Acyl group" mentioned as a substituent includes an aliphatic acyl group having 1 to 10 carbon atoms, a carbon ring carbonyl group, and a heterocyclic carbo group. Specifically, formyl group, acetyl group, propionyl group, ptylyl group, isoptylyl group, valeryl group, bivaloyl group, hexanoyl group, attalyloyl group, propioroyl group, methacryloyl group, crotonol group, benzoyl group, cyclohexanecarboxyl Group, pyridine carbo group, francal Examples thereof include a ball group, a thiophene carbo group, a benzothiazole carbo ol group, a pyrazine carbonyl group, a piperidine carbo ol and the like. Examples of the acyl group of the “acyloxy group” and “acylamino group” include the same groups as the above “acyl group”.

Furthermore, examples of the “halogen atom” mentioned as a substituent include a chlorine atom, a fluorine atom, a fluorine atom and a bromine atom.

[0043] R ¹ and R ² may be the same or different from each other, but are preferably the same group.

In the formula (I), the “lower alkyl group” defined by R ³ also has a carbon number of 1 as described above.

Means 10 linear or branched alkyl groups. A straight-chain or branched alkyl group having 16 carbon atoms is preferable, and an alkyl group having 13 carbon atoms is more preferable, specifically, a methyl group, an ethyl group, and an n-propyl group.

In formula (I), R ¹ and R ² are preferably both p--trophenoxy groups, and in this case, R ³ is preferably a hydrogen atom or a lower alkyl group. As such a compound, specifically, 3,5-bis (4-nitrophenoxy) benzoic acid [3,5-bis (4-nitrophenoxy) -benzoic acid] represented by the following formula (Γ) or a lower alkyl ester thereof: It is possible to raise.

Formula (Γ):

[0046] [Chemical 6]

[In the formula, R ³ represents a hydrogen atom or a lower alkyl group.]

The compound (Γ) is a known compound. For example, Gang Yang et al. (Macromole cules 1998, 31, 5964; Macromolecules 1999, 32, 2215-2220) and International Publication WO 01/02466 A1 (Example 4) ).

[0048] Examples of the method for producing compound (I) include the following methods.

[0049] [Chemical 7]

(la) (lb)

[Wherein, Z is an oxygen atom or a sulfur atom, Ar is an aryl which may have a substituent, R ³ represents a lower alkyl group, and Hal represents a halogen atom. Specifically, the compound (la) in which R ³ is a lower alkyl group is obtained by converting 3,5-dihydroxybenzoic acid or 3,5-dimercaptobenzoic acid lower alkyl ester (III) to copper or copper It can be produced by reacting with an aryl halide (II) in the presence or absence of a salt in the presence of a base. Here, the copper salt is a copper halide such as cuprous iodide or cuprous bromide, and the base is an alkali metal hydroxide or carbonate such as sodium hydroxide, Examples include potassium hydroxide, sodium carbonate and potassium carbonate. In addition, the compound (lb) in which R ³ ′ is substituted with hydrogen is obtained by converting the lower alkyl ester (la) obtained above in the presence of a base such as lithium hydroxide or sodium hydroxide, or sulfuric acid or hydrochloric acid. It can manufacture by hydrolyzing in acid presence, such as.

[0051] Although these compounds have a free or salt form, they may be misaligned. Here, examples of the salt include pharmaceutically acceptable salts such as salts with inorganic bases or organic bases. Examples of the inorganic base include alkali metals such as sodium and potassium; alkaline earth metals such as calcium and magnesium; aluminum and ammonia. 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] Further, the compound (I) targeted by the present invention may be a solvate of a free substance or a salt thereof. Here, solvates include hydrates.

In the present specification, “extended A | 8” means that the position opened by γ-secretase is 1 amino acid residue to 8 amino acid residues, preferably 1 amino acid residue to 3 It means amyloid j8 that is shifted to the C-terminal side by the amino acid residue. Specific examples include A | 8 42 and A | 8 43. It is.

[0054] The compound (I) has an effect of inhibiting the production of extended Aβ, specifically, the production of Αβ42 by γ-secretase degradation of j8 APP (Example 1). reference). The inhibitory effect of compound (I) on extended Αβ production can be evaluated based on measuring (quantifying) the amount of extended Αβ secreted from cells. Specifically, cells that express and produce ι8 ΑΡΡ are reacted with compound (I) in the presence of an appropriate solvent such as DMSO, and the amount of 42β42 produced secreted outside the cell for a certain period of time. It can be evaluated by measuring. The amount of A j842 produced can be measured by a conventional method such as ELISA or Western blotting, and the amount of Aj842 produced from the cells (preferably nerve cells) treated with compound (I) can be measured. When the amount of A j842 produced by untreated cells is lower, the Aβ42 production inhibitory action 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 an extended type β β production inhibitor. In this case, the ratio of the compound (I) contained in the pharmaceutical composition may be any amount as long as it has the effect of suppressing the production of extended cocoon, for example, 0.1 to: the range power of LOO wt% is also appropriately selected. can do. Preferably it is 1-60 weight%, More preferably, it is 3-40 weight%.

[0056] Further, Compound (I) is a disease caused by the production, secretion or deposition of extended A | 8 based on its inhibitory action on extended A | 8 (in the present invention, "prolonged amyloid β-related" It is possible to prevent the occurrence and development of (disease ”or“ prolonged epilepsy-related disease ”) and to alleviate the disease state. Therefore, the pharmaceutical composition of the present invention containing Compound (I) as an active ingredient can be effectively used as a prophylactic or therapeutic agent for prolonged Α | 8 related diseases.

[0057] Such extended type β-related diseases include, for example, Alzheimer type dementia (Alzheimer's disease, Alno, ima type 1 senile dementia, etc.), Down's syndrome, memory impairment, prion disease (Kreuzfeld 'Jakob disease, etc.), mild cognition Disorder (MCI), Dutch hereditary amyloid cerebral hemorrhage, cerebral amyloid angiopathy, other degenerative dementia such as vascular degenerative mixed dementia, dementia associated with Parkinson's disease, dementia associated with progressive supranuclear paralysis, cortical base Examples include dementia associated with nuclear degeneration, diffuse Lewy body Arno, Imah's disease, age-related macular degeneration, Parkinson's disease, and amyloid angiopathy. Among them, Alzheimer is preferable Type dementia, especially Alzheimer's disease.

[0058] In this case, the ratio of the compound (I) contained in the pharmaceutical composition may be any amount as long as it has an action of suppressing the production of extended Aβ, for example, 0.1 to: LOO weight % Can be selected as appropriate. Preferably it is 1-60 weight%, More preferably, it is 3-40 weight%.

[0059] The pharmaceutical composition of the present invention usually contains an effective amount of the compound (I) that suppresses prolonged Αβ production, specifically, an effective amount of the compound (I) that suppresses cleavage of β による by enzymatic degradation. In addition to pharmaceutically acceptable carriers or additives.

[0060] 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. be able to. Oral administration and intravenous administration are preferred, and oral administration is more preferred.

[0061] The pharmaceutical composition of the present invention can be prepared into various forms of preparations (dosage forms) according to the powerful 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 commonly used in the field of pharmaceutical preparations can be used.

[0062] Examples of dosage forms for oral administration include powders, granules, capsules, pills, tablets, elixirs, suspensions, emulsions, and syrups. can do. In addition, these formulations can be modified such as sustained release, stabilization, easy disintegration, hard disintegration, entericity, easy absorption, and the like.

[0063] In addition, dosage forms for intravenous administration, intramuscular administration, or subcutaneous administration include injections and infusions (including dry products prepared at the time of use), and can be appropriately selected.

[0064] In addition, dosage forms for transmucosal administration, transdermal administration, or rectal administration include mastication agents, sublingual agents, buccal agents, lozenges, ointments, patch agents, liquid agents, etc. You can choose here depending on the location. These preparations can also be modified such as sustained release, stabilization, easy disintegration, difficulty disintegration, and easy absorption.

[0065] The pharmaceutical composition of the present invention may contain pharmaceutically acceptable carriers and additives depending on the dosage form (oral or various parenteral dosage forms). Pharmacologically allowed Carriers and additives to be contained include solvents, excipients, coating agents, bases, binders, lubricants, disintegrants, solubilizers, suspending agents, thickeners, emulsifiers, stabilizers, Examples include buffering agents, isotonic agents, soothing agents, preservatives, flavoring agents, fragrances, and coloring agents. Specific examples of pharmaceutically acceptable carriers and additives are listed below, but the present invention is not limited thereto.

[0066] 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 (eg, potato starch, wheat starch, corn starch), lactose, glucose, sucrose, crystalline cellulose, calcium sulfate, calcium carbonate, sodium bicarbonate, sodium chloride salt, tark, oxidized Titanium, trehalose, xylitol and the like can be mentioned.

[0067] Examples of binders include starch and its derivatives, cellulose and its derivatives (for example, methinoresenorelose, ethinoresenorelose, hydroxypropinoresenorelose, canoleboxymethylcellulose), gelatin, sodium alginate, tragacanth, arabian Examples thereof include natural polymer compounds such as rubber, synthetic polymer compounds such as polyvinyl pyrrolidone and polybutyl alcohol, dextrin and hydroxypropyl starch.

[0068] Lubricants include light anhydrous carboxylic acid, stearic acid and its salts (eg, magnesium stearate), talc, waxes, wheat starch, macrogol, hydrogenated vegetable oil, sucrose fatty acid ester, polyethylene glycol, Examples include silicone oil.

[0069] Examples of the disintegrant include starch and derivatives thereof, agar, gelatin powder, sodium hydrogen carbonate, calcium carbonate, cellulose and derivatives thereof, hydroxypropyl starch, strength ruboxymethyl cellulose and salts thereof, and cross-linked products thereof, low substitution type And hydroxypropylcellulose.

[0070] Examples of solubilizers include cyclodextrin, ethanol, propylene glycol, and polyethylene glycol. Examples of the suspending agent include sodium carboxymethyl cellulose, polypyrrole pyrrolidone, gum arabic, tragacanth, sodium alginate, aluminum monostearate, citrate, and various surfactants.

[0071] Examples of the thickener include sodium carboxymethylcellulose, polypyrrole pyrrolidone, and methyl. Examples include cellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, tragacanth, gum arabic, and sodium alginate.

[0072] 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) and the like.

[0073] Stabilizers include tocopherols, chelating agents (eg EDTA, thioglycolic acid), inert gases (eg nitrogen, diacid-carbon), reducing substances (eg sodium bisulfite, sodium thiosulfate, ascorbine). Acid, longalite) and the like.

[0074] Examples of the buffer include sodium hydrogen phosphate, sodium acetate, sodium citrate, boric acid and the like.

[0075] Examples of the isotonic agent include sodium chloride salt and glucose. Examples of soothing agents include local anesthetics (pro-in hydrochloride, lidocaine), pendyl alcohol, glucose, sorbitol, amino acids and the like.

[0076] 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.

[0077] Examples of the preservative include benzoic acid and salts thereof, paraoxybenzoic acid esters, chlorobutanol, reverse soap, benzyl alcohol, phenol, tiromesal, dehydroacetic acid, boric acid, and the like.

[0078] Coating agents include sucrose, hydroxypropylcellulose (HPC), shellac, gelatin, glycerin, sorbitol, hydroxypropylmethylcellulose (HPMC), ethylcellulose, polybutylpyrrolidone (PVP), hydroxypropylmethylcellulose phthalate ( HPMCP), cellulose acetate phthalate (CAP), methyl methacrylate, methacrylic acid copolymer, and the polymers described above.

[0079] Bases include petrolatum, liquid paraffin, carnauba wax, beef tallow, hydrogenated oil, norafin, beeswax, vegetable oil, macrogol, macrogol fatty acid ester, stearic acid, sodium carboxymethylcellulose, bentonite, cacao butter, witetbuzol , Gelatin, stearyl alcohol, hydrolanolin, cetanol, light liquid paraffin, hydrophilic , Simple ointment, white ointment, hydrophilic ointment, macrogol ointment, hard fat, oil-in-water emulsion base, water-in-oil emulsion glaze, and the like.

[0080] For each of the above dosage forms, a known drug delivery system (DDS) technique can be employed. The DDS preparations referred to in this specification include sustained release preparations, topical preparations (troches, buccal tablets, sublingual tablets, etc.), drug release control preparations, enteric preparations and gastric preparations, etc., administration routes, bioavailability First, it is a drug product in the optimal drug product form taking into account side effects.

[0081] The pharmaceutical composition of the present invention is a disease caused by production of extended A (disease related to A)

(Preferably Arno-Ima type 1 dementia, in particular Arno-Ima Ima), prevention of the onset and progression of extended A-related diseases in subjects who have or may be affected It is possible to delay or improve.

[0082] When the pharmaceutical composition of the present invention is used as a preventive or therapeutic agent for a disease caused by extended Aβ production (extended Αβ-related disease) (for example, a prophylactic or therapeutic agent for Alzheimer's disease) The dose is preferably in the range of 0.03 to 300 mgZkg body weight, more preferably 0.1 to 50 mgZkg body weight in terms of the amount of compound (I). In the case of intravenous administration, the dosage is such that the effective blood concentration of compound (I) is in the range of 0.2-50 / ζ ₈ Ζπ ^, more preferably 0.5-20 ^ g / mL. be able to.

[0083] It should be noted that these dosages may vary depending on age, sex, body type and the like.

[0084] (2) Screening method for extended Aβ production inhibitor and standard reagent used for the present invention The present invention also provides 3,5-bis (4-nitrophenoxy) benzoic acid represented by the above general formula (I) and its Using an analog (I compound (1)) or a salt thereof, or a solvate thereof as an extended amyloid ι8 production inhibitory standard (positive control reagent) Provided is a method for screening an extended type Αβ production inhibitory substance having an amyloid β production inhibitory action. The present invention also provides use of compound (I) or a salt thereof, or a solvate thereof as a standard reagent (positive control reagent) in the screening method.

[0085] The target compound (I) includes all the compounds (I) described in the above (1). Preferably, a lower alkyl of 3,5-bis (4-nitrophenoxy) benzoic acid represented by the following formula (Γ) is used. Alkyl ester, and more preferably 3,5-bis (4-nitrophenoxy) benzoic acid.

[0086] [Chemical 8] N ₂ 0

[In the formula, R ³ represents a hydrogen atom or a lower alkyl group.]

Herein, the lower alkyl group means a linear or branched alkyl group having 1 to LO, preferably 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms, as described above. A methyl group, an ethyl group, and an n-propyl group are preferable.

[0088] The salt of compound (I) is not particularly limited as long as it forms a salt with compound (I)! ,

. Preferable examples include the pharmaceutically acceptable salts described above, for example, salts with inorganic bases or organic bases. In addition, the solvate of compound (I) or a salt thereof includes a hydrate.

[0089] The screening method provided by the present invention is a method of selecting a substance having an inhibitory action on prolonged amyloid ι8 production from test substances. This method can also be used as a method for selecting a candidate substance that can be an active ingredient of a prophylactic or therapeutic agent for an extended amyloid β-related disease (particularly Alzheimer's disease).

[0090] Specifically, the method examines the extended Α production inhibitory action of the test substance, and from that, suppresses the extended A production of the compound (I) or salt, or a solvate thereof. It can be performed by selecting a test substance having an inhibitory action higher than the action as an extended amyloid | 8 production inhibitor. More specifically, the method can be performed by a method having the following steps (1) to (3):

(1) contacting a test substance with β amyloid precursor protein (jS-APP) or a cell capable of expressing and producing the protein;

(2) measuring the amount of / β-APP or A β 42 produced from the cells;

(3) The amount of Α42 produced above is controlled by the control A obtained by carrying out the steps (1) and (2) above using the compound (I) or salt or a solvate thereof instead of the test substance. | 8 42 births A process in which test substances that show a production amount of A | 844 lower than the positive control value in comparison with the amount of raw material (positive control value) are selected as candidate substances for prolonging amyloid β production.

[0091] Here, as the test substance, synthetic compounds, natural products (animals and plants, microorganisms, soil, etc.) or compositions or isolates obtained therefrom, and those obtained by genetic engineering techniques are particularly limited. And can be used widely.

[0092] The 13 APP-mutated and wild-type PS1 constitutively expressed HEK293 cells, jS APP Swedish mutated and PS1 L166P mutated HEK293 cells, and 13 APP Swedish mutated HEK293 cells, wild-type 13 APP constitutively expressed HEK293 cells, or HE K293 cells, CHO cells or HeLa cells that transiently express wild-type β APP or 13 APP Swedish mutations can be used. . Preferred are HE8293 cells with constitutive expression of j8 APP swedish mutation and wild type PS1, and HEK293 cells with constitutive expression of j8 APP Swedish mutation and PS1 L166P mutation.

[0093] In the step (1), the method of bringing a test substance into contact with a cell capable of expressing and producing j8-APP or / 3-APP is not particularly limited. However, when using a jS-APP-expressing cell, It is necessary that the cell and the test substance coexist at least under conditions that allow the cell to express and produce β-APP. For example, after treatment of the cells with methionine-depleted MEM, the cells are plated with a medium containing 450 Ci [35S] methionine / cysteine (Redivue Promix; Amersham), 5% dialyzed FCS (without amino acids), etc. Examples of the method include adding a test substance at a suitable concentration to the culture medium during the pulse period.

[0094] In the step (2), the method for measuring the amount of Aβ42 produced from 13-APP or j8-APP-expressing cells is as follows. A method of performing Western blotting can be mentioned.

[0095] It should be noted that the steps (1) and (2) are carried out using a compound (I) or salt having an inhibitory action on extended amyloid | 8 production, or a solvate thereof, instead of the test substance. In step (3), the production amount of A | 842 obtained by the above method is used as a control production amount of Α | 844 (positive control value) for evaluating the inhibitory action of the test substance on extended amyloid β production. [0096] Specifically, in step (3), the amount of A | 842 production obtained in (1) and (2) above using the test substance was compared with the amount of A | 842 production (positive) In contrast to the control value, a test substance that shows a production amount of Aβ42 lower than the positive control value is selected as a candidate substance for an extended amyloid β production inhibitory substance. That is, in the step (3), a test substance having a higher extended amyloid β-inhibitory action than a test substance having an extended amyloid β inhibitory activity using a compound (I) or a salt, or a solvate thereof as a positive standard reagent. This is a process of selecting as a candidate substance for β production inhibitory substance. Here, 3,5-bis (4-nitrophenoxy) benzoic acid is preferable as a positive standard reagent.

[0097] Compound (I) or a salt thereof or a solvate thereof is, as described above, its extended form A

It can be suitably used in the research field or the basic field in drug development as a positive standard reagent or the like in the method of screening for an extended type β β production inhibitor using the β production inhibitory action.

[0098] The reagent of the present invention may be compound (I) or a salt thereof, or a solvate thereof, and various compounds in addition to these compounds (I) and the like depending on the intended use. It may have a form of a composition in which a carrier or an additive is blended. In this case, the ratio of the compound (I) contained in such a composition may be any amount as long as it has an effect of suppressing the production of extended β-β. You can choose.

[0099] Compound (I) or a salt thereof, or a solvate thereof is used for the purpose of suppressing the production of extended Aβ, preferably Αβ42, produced from, for example, j8 APP. It can be used for a protein (j8 APP) or a cell that expresses it. Examples of cells expressing j8 APP include neurons, particularly neurons in the brain, and cells artificially constructed to express βAPP by gene manipulation.

[0100] Therefore, in the present invention, compound (I) or a salt thereof or a solvate thereof is applied to β APP or an expression cell thereof to give an extended 型 | 8, preferably A | 842 Includes methods of inhibiting production.

Example [0101] Hereinafter, the present invention will be described with reference to experimental examples, but the present invention is not limited to such experimental examples.

1. Pile, test and test compound

The anti-A j8 antibody 4G8 antibody was purchased from Signet Laboratories (Dedham, MA). Non-steroidal anti-inflammatory drugs (NSAIDSKsulindac sulfide, indomethacin, napr oxen), peroxisome proliferator activated receptor a fenofibrate, rarnesyl pyrophosphate ammonium salt (FPP), and geranylgeranyl pyrophosphate ammoni um salt ( (GGPP) is purchased from Sigma, and 3,5-bis (4-trophenoxy) benzoic acid (hereinafter referred to as “Comp ₀ und Wj” or “CW”) is purchased from Tokyo Chemical Industry Co., Ltd. did.

[0103] 2. mm m M

The jS APP Swedish mutation and wild-type PSl constitutively expressed HEK293 cells, and the j8 APP Swedish mutation and PSl L166P constitutively expressed HEK293 cells are well known in the literature (Okochi, M., Steiner, H., Fukumon, A., Tann, H., Tomita, T., Tanaka, T., Iwatsubo, T., Kuao, T., Taked a, M., and Haass, C. (2002) Embo J 21 (20), 5408-5416; and Steiner , H., Romig, H., Grim, MG, Philipp, U., Pesold, B., Citron, M., Baumeister, R., and Haass, C. (1999) J Biol Chem 274 (12), 7615 -7618). These two types of constitutively expressing cells were cultured in a medium supplemented with 200 μg / ml G418 and 200 μg / ml zeocin.

[0104] 3. Pulse-chase experiment

β APP Swedish mutation and wild-type PSl constitutive expression ΗΕΚ293 cells were treated with methionine blight MEM, and then plated over 450 Ci [35S] methionine / cystine (Redivue Pro mix; Amersham), 5% dialyzed FCS ( Metabolic labeling in medium containing no amino acids) and at the same time Compound W (100 μΜ) ^ fenofibrate (100 μΜ), farnesyl pyrophosphate ammonium salt (FPP) (100 μM), geranylgeranyl pyrophosphate ammonium salt (GG PP) (100 μM), naproxen (100 μM), indomethacin (100 μM) and sulfindac sulfide (100 / zM) were added to the culture solution at each concentration during the pulse period. As a control, 0.1% DMSO equal to the applied DMSO concentration of these compounds was used.

[0105] 4. Immunoprecipitation / autoradiography The collected culture supernatant was diluted to a final concentration of 50 mM Tris HC1 (pH 7.4), 1: 1000 amount of protease in hibitor cocktail (Sigma), 5 mM EDTA (pH 8.0). The culture supernatant was immunoprecipitated using 4G8 antibody. Proteins were separated by Tris-tricine gel or Tris-bicine SDS-PAGE. The gel was fixed with 50% methanol Zl0% acetic acid. Autoradiography was performed by drying the gel and performing a fluorographic analysis.

[0106] 5.Immunoprecipitation / MALDI-TOF MS analysis

The immunoprecipitated protein is eluted from the precipitation carrier using a trifluoroacetic acid / acetone / water mixture (mixing ratio 1:20:20) saturated with a-cyano-4-hydroxy cinnamic acid and centrifuged. A soluble fraction was obtained. This was dried on a stainless steel chamber for MALDI-TOF MS analysis, and mass spectrometry was performed by MALDI-TOF MS. The peak height and molecular weight obtained by mass spectrometry were corrected using angiotensin and insulin β chain (Sigma) as standards.

The results are shown in FIG. 1 and FIG.

[0108] As a result, among the test compounds, sulindac sulfide (100 μ μ), indomethacin (100 μΜ), and Compound W (100 μM) significantly increased the production and secretion of Aβ42 in endogenous PS-expressing cells. Decreased. Among these compounds, Compound W, which has a high inhibitory effect on A j842 production, markedly decreased the secretion of A | 842. In addition, as can be seen from FIG. 2, Compo und W showed Αβ43 production inhibitory action in addition to Αβ42 production inhibitory action.

[0109] Compound W was confirmed to have 0% and 11% inhibitory activity against COX-1 and COX-2 enzymes at 10 μΜ, respectively. Therefore, Compound W is considered to exhibit A | 8 production suppression action by a mechanism different from anti-inflammatory drugs such as non-steroid anti-inflammatory drugs (NSAIDS).

Brief Description of Drawings

[0110] [Figure 1] Figure showing the relative ratio of A | 842 secreted when endogenous PS-expressing cells were treated with various test compounds (Α | 8 42 quantity Z total A | 8 quantity) It is. * Indicates that the relative ratio is significantly different from control (using DMSO instead of test compound) (P 0.001).

[Figure 2] Constant expression of PS1 L166P mutation HEK293 cells were treated with Compound W (100 M). FIG. 6 is a graph showing changes in various types of A | 8 secreted by an immunoprecipitation / MALDI-TOF MS method. The up and down arrows indicate a clear increase and decrease, respectively, compared to the control (using DMSO instead of test compound).

Claims

The scope of the claims

(In the formula, R ¹ and R ² each independently have a substituent, may be aryloxy or may have aryl, and R ³ is a hydrogen atom or lower alkyl. Is the base)

2. The pharmaceutical composition according to claim 1, which is an extended amyloid β production inhibitor.

In general formula (I), optionally substituted aryloxy or optionally substituted arylothio substituent power nitro group, carboxy group, lower alkoxy carbo yl group, acyl group, sulfo group The pharmaceutical composition according to claim 1, wherein a group force including a group, a sulfino group, a sulfino group, a lower alkylsulfonyl group and a cyano group is also selected.

[4] The pharmaceutical composition according to claim 1, wherein in general formula (I), R ¹ and R ² are -trophenoxy groups, and R ³ is a hydrogen atom.

[5] The pharmaceutical composition according to claim 1, which is a prophylactic or therapeutic agent for an elongated amyloid j8-related disease.

6. The pharmaceutical composition according to claim 5, wherein the extended amyloid j8-related disease is Alzheimer's disease.

[7] A method for treating or preventing an extended amyloid j8-related disease, comprising administering the pharmaceutical composition according to claim 5 to a subject suffering from a force afflicted with an extended amyloid j8-related disease. .

[8] Formula (I): [Chemical 2]

R ¹

R ²ヽ COOR ³

Or a salt thereof, or a solvate thereof, for screening for an extended amyloid β production inhibitor.

Investigate the prolongation of amyloid ι8 production on the test substance,

Formula (I):

[Chemical 3]

A test substance having an inhibitory action higher than the extended amyloid β production inhibitory action of the compound or a salt thereof, or a solvate thereof, as an extended amyloid β production inhibitor, For screening type amyloid β production inhibitor.

10. The screening method according to claim 9, wherein the extended amyloid β production inhibitor is an active ingredient of a prophylactic or therapeutic agent for extended amyloid β-related diseases.

[11] Formula (I):

[Chemical 4]

Or a salt thereof, or a solvate thereof, is applied to β-amyloid precursor protein or an amyloid precursor protein-expressing cell, and a method for inhibiting prolonged amyloid β production.